endophytic compositions and methods for improving plant traits

专利摘要:
This invention relates to methods and materials for modulating the characteristics of a plant, in which said plant is heterologously arranged in an endophyte organism or in a plurality of endophyte organisms, or derived from a willing plant reproductive element, formheterologous, in an endophyte organism or a plurality of endophyte organisms.
公开号:BR112019018234A2
申请号:R112019018234-9
申请日:2017-12-01
公开日:2020-07-28
发明作者:Raymond Riley
申请人:Indigo Ag, Inc.；
IPC主号:

专利说明:

[0001] [0001] This request claims priority to Provisional Application No. 62 / 556,288, filed on September 8, 2017; Provisional Application No. 62 / 467,740, filed on March 6, 2017; Provisional Application No. 62 / 467,742, filed on March 6, 2017; Provisional Application No. 62 / 467,755, filed on March 6, 2017; Provisional Application No. 62 / 466,253, filed on March 2, 2017; Provisional Application No. 62 / 465,834, filed on March 2, 2017; Provisional Application No. 62 / 466,256, filed on March 2, 2017; Provisional Application No. 62 / 465,797, filed on March 1, 2017; Provisional Application No. 62 / 465,819, filed on March 1, 2017; and Provisional Order No. 62 / 465,798, filed on March 1, 2017, the disclosures of which are incorporated in this document as a reference in their entirety. SEQUENCE LISTING
[0002] [0002] The immediate order contains a Sequence Listing with 71 sequences that was submitted through EFS-Web and is incorporated by reference in this document in its entirety. Said ASCII copy, created on November 29, 2017, is called 39061_10101_Sequence_Listing.txt and is 54,920 bytes in size. FIELD OF THE INVENTION
[0003] [0003] The present invention relates to compositions and methods to improve the traces of plants, particularly plants important for human or animal consumption, for example rice (Oryza sativa and related varieties), soybeans (Glycine max and related varieties), wheat (Triticum aestivum and related varieties) and corn (Zea mays and related varieties). For example, this invention describes microbes that are capable of living within or arranged heterologously to a plant, and that can be used to impart enhanced traits to the plants with which they are or have been heterologously disposed. The disclosed invention also describes methods for improving the characteristics of plant elements by introducing microbes to the original plants. In addition, this invention also provides methods of treating plant elements with microbes that are able to live within a plant, particularly rice, soybeans, wheat and corn, to provide better yield and other agronomic characteristics to that plant. FUNDAMENTALS
[0004] [0004] According to the United Nations Food and Agricultural Organization, the world population will exceed 9.6 billion people by 2050, which will require significant improvements in agriculture to meet the growing demand for food. There is a need for improved agricultural vegetables that allow almost doubled demand for food production with fewer resources and more environmentally sustainable inputs for plants with improved responses to various abiotic stresses.
[0005] [0005] Currently, crop performance is optimized mainly through technologies aimed at the interaction between the genotype of the crops (eg plant breeding, genetically modified (GM) crops) and their surrounding environment (eg fertilizers, synthetic herbicides, pesticides). While these paradigms have helped to double global food production over the past fifty years, production yield rates have stagnated in many important crops and changes in climate have been linked to production instability as well as changing pest and disease pressures , leading to an urgent need for new solutions for crop improvement. In addition to their long regulatory and development timelines, public fears of GM crops and synthetic chemicals have challenged their use in many key countries and cultures, resulting in the lack of acceptance of many GM traits and the exclusion of GM crops and many synthetic chemicals. of some global markets. Thus, there is a significant need for innovative, effective, environmentally sustainable and publicly acceptable approaches to improve yield and other agronomically important characteristics of plants.
[0006] [0006] In this document, methods and compositions are provided to improve the agronomically important characteristics of plants, associating these plants with disclosed endophytes. SUMMARY OF THE INVENTION
[0007] [0007] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a corn plant comprising disposing, in a heterologous manner, of an endophyte organism to a plant element of corn in an amount effective to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group that consists of SEQ ID NO: 60, 61 and 62, in which the vegetable element of maize is the variety Stine 9734 or a variety closely related to it.
[0008] [0008] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62.
[0009] [0009] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an amount effective to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group that consists of SEQ ID NOs: 60, 61 and 62 and where the soybean plant is the selected variety of the group consisting of Pfister 38R25, Dairyland DSR1808R2Y, Stine 3920 and varieties closely related to these.
[0010] [0010] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an amount effective to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62.
[0011] [0011] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group that consists of SEQ ID NO: 60, 61 and 62, where the vegetable element of wheat is the variety selected from the group consisting of SDSU Focus, SDSU Select, and varieties closely related to these.
[0012] [0012] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant, comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62.
[0013] [0013] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant, comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62 and where the peanut plant is a variety selected from the group consisting of AT-9899, FloRun 107, Tamnut OL06, Georgia-06G and varieties closely related to these.
[0014] [0014] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length and root dry weight in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71.
[0015] [0015] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length and root dry weight in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, and in which the soybean is of the variety Stine 33E22 or a variety closely related to it is.
[0016] [0016] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant, comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71.
[0017] [0017] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant, comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, and the peanut plant is a variety selected from the group consisting of FloRun 107, Georgia-06G and varieties closely related to these.
[0018] [0018] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a rice plant, comprising disposing, in a heterologous manner, of an endophyte organism to a vegetable element of rice in an effective amount to increase the root length in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to a selected polynucleotide sequence of the group consisting of SEQ ID NOs: 63, 64 and 71.
[0019] [0019] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase the plant yield derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41.
[0020] [0020] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a canola plant, comprising disposing, in a heterologous manner, of an endophyte organism to a canola plant element in an effective amount to increase the plant yield derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 70, where the canola plant is the Brett Young 552 variety or a variety closely related to it.
[0021] [0021] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 65 and 66.
[0022] [0022] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism for a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus It curves and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 65 and 66, and in which the trait of agronomic importance is yield and the soybean plant is a variety selected from the group consisting of Pfister 38R25, Stine 3920 and varieties closely related to these.
[0023] [0023] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an amount effective to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 65 and 66.
[0024] [0024] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 65 and 66, in which the wheat plant is a variety selected from the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
[0025] [0025] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length, dry shoot biomass and yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44 and
[0026] [0026] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soy plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soy plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area,
[0027] [0027] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44 and 45.
[0028] [0028] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44 and 45, in which the wheat plant is the selected variety of the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
[0029] [0029] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a corn plant, comprising disposing, in a heterologous manner, of an endophyte organism to a plant element of corn in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67, where the corn plant is the Stine 9734 variety or a variety closely related to it.
[0030] [0030] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67.
[0031] [0031] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Cladosporium e comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NOs: 67, and in which the trait of agronomic importance is yield and the soybean plant is a variety selected from the group consisting of Pfister 38R25, Stine 3920 and varieties closely related to these.
[0032] [0032] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67.
[0033] [0033] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an amount effective to increase the plant yield derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NOs: 67, where the wheat plant is the selected variety of the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
[0034] [0034] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a canola plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a canola plant element in a amount effective to increase the yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Paecilomyces and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 69.
[0035] [0035] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a canola plant, comprising disposing, in a heterologous manner, of an endophyte organism to a canola plant element in an effective amount to increase the plant yield derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Paecilomyces and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 69, where the canola plant is variety NCC1015 or a variety closely related to it.
[0036] [0036] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism for a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Acremonium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51.
[0037] [0037] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Acremonium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, where the trait of agronomic importance is yield and yield The soybean plant is the Pfister 38R25 variety or a variety closely related to it.
[0038] [0038] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to enhance a trait of agronomic importance selected from the group that consists in decreasing the moisture of the grain and increasing the yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Acremonium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51.
[0039] [0039] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to enhance the trait of agronomic importance selected from the group that consists in decreasing the moisture of the grain and increasing the yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Acremonium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, where the wheat plant is the selected variety of the group which consists of SDSU Focus, SDSU Select and varieties closely related to these.
[0040] [0040] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant, comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Acremonium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, and the peanut plant is the Georgia-06G variety or a closely related variety.
[0041] [0041] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a rice plant, comprising disposing, in a heterologous manner, of an endophyte organism to a rice plant element in an effective amount to increase the dry shoot biomass in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51.
[0042] [0042] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising having, in a heterologous manner, a first endophyte organism and a second endophyte organism for a wheat plant element in an effective amount to improve a trait of agronomic importance selected from the group consisting of plant height, fresh root weight and fresh sprout weight in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the first endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, and the second endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a selected polynucleotide sequence none of the group consisting of SEQ ID NOs: 38, 39, 40 and 41.
[0043] [0043] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a rice plant, comprising disposing, in a heterologous manner, of a first endophyte organism and a second endophyte organism for a rice plant element in a amount effective to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the first endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, and the second endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41.
[0044] [0044] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an amount effective to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 68.
[0045] [0045] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soy plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soy plant element in an effective amount to increase the plant yield derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NOs: 68, where the soybean plant is the selected variety of the group consisting of Pfister 38R25, Stine 3920 and varieties closely related to these.
[0046] [0046] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an amount effective to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 68.
[0047] [0047] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an amount effective to increase the plant yield derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NOs: 68, where the wheat plant is the selected variety of the group consisting of SDSU Focus, SDSU Select and varieties closely related to them.
[0048] [0048] In one aspect, the invention provides a method of enhancing the trait of agronomic importance in a peanut plant, comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 68 and the peanut plant is the variety
[0049] [0049] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising having, in a heterologous manner, a first endophyte organism and a second endophyte organism for a soybean plant element in a amount effective to increase the dry root biomass in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the first endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97 % identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, the second endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, and the soybean plant is the Stine 33E22 variety or a va closely related
[0050] [0050] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a corn plant, comprising disposing, in a heterologous manner, of an endophyte organism to a corn plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59.
[0051] [0051] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soy plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soy plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length, dry shoot biomass and yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59.
[0052] [0052] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length, dry shoot biomass and yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59, in which the trait of agronomic importance is yield and the soybean plant is the variety Pfister 38R25 or a variety closely related to it.
[0053] [0053] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and
[0054] [0054] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59, where the wheat plant is the selected variety of the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
[0055] [0055] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a corn plant comprising disposing, in a heterologous manner, of an endophyte organism to a corn plant element in an amount effective to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group that consists of SEQ ID NO: 60, 61 and 62, in which the corn plant element is the variety Stine 9734 or a variety closely related to it, in which the plant element is a seed, optionally a modified seed.
[0056] [0056] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an amount effective to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62, in which the plant element is a seed, optionally a modified seed.
[0057] [0057] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase the plant yield derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97%
[0058] [0058] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant, comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62, in which the plant element is a seed, optionally a modified seed.
[0059] [0059] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism for a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length and root dry weight in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, in which the plant element is a seed, optionally a modified seed.
[0060] [0060] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant, comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, in which the plant element is a seed, optionally a modified seed.
[0061] [0061] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a rice plant, comprising disposing, in a heterologous manner, of an endophyte organism for a vegetable element of rice in an effective amount to increase the root length in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to a selected polynucleotide sequence from the group consisting of SEQ ID NOs: 63, 64 and 71, in which the plant element is a seed, optionally a modified seed.
[0062] [0062] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an amount effective to increase the plant yield derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41, in which the plant element is a seed, optionally a modified seed.
[0063] [0063] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a canola plant, comprising disposing, in a heterologous manner, of an endophyte organism to a canola plant element in an effective amount to increase the plant yield derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 70, where the canola plant is the Brett Young 552 variety or a variety closely related to it, where the plant element is a seed, optionally a modified seed.
[0064] [0064] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of the root area, root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 65 and 66, wherein the plant element is a seed, optionally a modified seed.
[0065] [0065] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 65 and 66, in which the plant element is a seed, optionally a modified seed.
[0066] [0066] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soy plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soy plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length, dry shoot biomass and yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44 and 45, in which the plant element is a seed, optionally a seed modified.
[0067] [0067] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, and 45, where the plant element is a seed, optionally a modified seed.
[0068] [0068] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a corn plant, comprising disposing, in a heterologous manner, of an endophyte organism to a plant element of corn in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67, where the corn plant is the variety Stine 9734 or a variety closely related to it, where the plant element is a seed, optionally a modified seed.
[0069] [0069] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67, wherein the plant element is a seed, optionally a modified seed.
[0070] [0070] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67, in that the plant element is a seed, optionally a modified seed.
[0071] [0071] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a canola plant, comprising disposing, in a heterologous manner, of an endophyte organism to a canola plant element in an effective amount to increase the plant yield derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Paecilomyces and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 69, wherein the plant element is a seed, optionally a modified seed.
[0072] [0072] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Acremonium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, where the plant element is a seed, optionally a seed modified.
[0073] [0073] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to enhance a trait of agronomic importance selected from the group that consists in decreasing the moisture of the grain and increasing the yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Acremonium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, where the plant element is a seed, optionally a seed modified.
[0074] [0074] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant, comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Acremonium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, and the peanut plant is the Georgia-06G variety or a closely related variety, where the plant element is a seed, optionally a modified seed .
[0075] [0075] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a rice plant, comprising disposing, in a heterologous manner, of an endophyte organism to a rice plant element in an effective amount to increase the dry shoot biomass in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, in which the plant element is a seed, optionally a modified seed.
[0076] [0076] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising having, in a heterologous manner, a first endophyte organism and a second endophyte organism for a wheat plant element in a effective amount to improve a trait of agronomic importance selected from the group consisting of plant height, fresh root weight and fresh sprout weight in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the the first endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, and the second endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a selected polynucleotide sequence that of the group consisting of SEQ ID NOs: 38, 39, 40 and 41, in which the plant element is a seed, optionally a modified seed.
[0077] [0077] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a rice plant, comprising having, in a heterologous manner, a first endophyte organism and a second endophyte organism for a rice plant element in a amount effective to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the first endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, and the second endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41, in which the plant element is a seed, opc a modified seed.
[0078] [0078] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soy plant comprising disposing, in a heterologous manner, of an endophyte organism to a soy plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 68, in that the plant element is a seed, optionally a modified seed.
[0079] [0079] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 68, in that the plant element is a seed, optionally a modified seed.
[0080] [0080] In one aspect, the invention provides a method of enhancing the trait of agronomic importance in a peanut plant, comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 68 and the The peanut plant is the variety AT-9899 or a variety closely related to it, in which the plant element is a seed, optionally a modified seed.
[0081] [0081] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising having, in a heterologous manner, a first endophyte organism and a second endophyte organism for a soybean plant element in a amount effective to increase the dry root biomass in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the first endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97 % identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, the second endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, and the soybean plant is the Stine 33E22 variety or a va closely related to this, in which the plant element is a seed, optionally a modified seed.
[0082] [0082] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a corn plant, comprising disposing, in a heterologous manner, of an endophyte organism to a corn plant element in an effective amount to increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, in which the plant element is a seed, optionally a modified seed.
[0083] [0083] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant, comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root area, root length, dry shoot biomass and yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, in which the element vegetable is a seed, optionally a modified seed.
[0084] [0084] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, in which the plant element is a seed, optionally a modified seed .
[0085] [0085] In some embodiments, any of the endophyte organisms is arranged heterologously to the plant element in a formulation, whose formulation additionally comprises one or more of the following: a stabilizer, a preservative, a carrier, a surfactant, fungicide, a nematicide, a bactericide, an insecticide or herbicide, or any combination thereof.
[0086] [0086] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a corn plant comprising disposing, in a heterologous manner, of an endophyte organism to a corn plant element in an effective amount to increase yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Exserohilum and comprises at least one 97%, at least 98%, at least 99% polynucleotide sequence, or at least 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 60, 61 and 62, in which the corn plant element is the variety Stine 9734 or a variety closely related to it, in which the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides.
[0087] [0087] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an amount effective to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 % or at least 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 60, 61 and 62, and where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0088] [0088] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 %, or at least 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 60, 61 and 62, where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides.
[0089] [0089] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Exserohilum and comprises at least one 97%, at least 98%, at least 99% polynucleotide sequence, or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 60, 61 and 62, in which the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400, or at least least 500 nucleotides.
[0090] [0090] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant comprising the heterologous disposition of an endophyte to a soybean plant element in an amount effective to improve a trait of selected agronomic importance from the group consisting of the root area, root length, and dry root weight in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64, and 71, where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0091] [0091] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 %, or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 63, 64 and 71, in which the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400 , or at least 500 nucleotides.
[0092] [0092] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a rice plant, comprising disposing, in a heterologous manner, of an endophyte organism to a rice plant element in an amount effective to increase the root length in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99%, or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, where the percentage of identity is determined over an alignment region of at least 100, 200, 300 , 400 or at least 500 nucleotides.
[0093] [0093] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 %, or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, and 41, and in which the percent identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides.
[0094] [0094] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a canola plant comprising disposing, in a heterologous manner, of an endophyte organism to a canola plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 % or 100% identical to SEQ ID NO: 70 and the canola plant is the Brett Young 552 variety or a variety closely related to it, where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0095] [0095] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an amount effective to improve a trait of agronomic importance selected from the group consisting of the root area, root length, and yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus It curves and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 65 and 66, where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0096] [0096] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an amount effective to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 %, or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 65 and 66, where the percent identity is determined over an alignment region of at least 100, 200, 300, 400, or at least least 500 nucleotides.
[0097] [0097] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an amount effective to improve a trait of agronomic importance selected from the group consisting of the root area, root length, dry shoot biomass and yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, and 45, where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0098] [0098] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 %, or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 42, 43, 44, and 45, and in which the percent identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides.
[0099] [0099] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a corn plant comprising disposing, in a heterologous manner, of an endophyte organism to a corn plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 % or 100% identical to SEQ ID NO: 67 and the corn plant is the variety Stine 9734 or a closely related variety, where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0100] [0100] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an amount effective to improve a trait of agronomic importance selected from the group consisting of the root area, root length, and yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 67, where the percentage of identity is determined over an alignment region of at least 100, 200 , 300, 400 or at least 500 nucleotides.
[0101] [0101] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an amount effective to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 %, or 100% identical to SEQ ID NO: 67, where the percent identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides.
[0102] [0102] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a canola plant comprising disposing, in a heterologous manner, of an endophyte organism to a canola plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus
[0103] [0103] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an amount effective to improve a trait of agronomic importance selected from the group consisting of root area, root length, and yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Acremonium e comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0104] [0104] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising the heterologous disposition of an endophyte to a wheat plant element in an amount effective to improve a trait of selected agronomic importance of the group that consists in decreasing the moisture of the grain and increasing the yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Acremonium and comprises at least one sequence polynucleotide of at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides.
[0105] [0105] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant comprising disposing, in a heterologous manner, of an endophyte organism to a peanut plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Acremonium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 % or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 46, 47, 48, 49, 50, and 51, and the peanut plant is the Georgia-06G variety or a closely related variety, in that the percent identity is determined over an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0106] [0106] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a rice plant comprising disposing, in a heterologous manner, of an endophyte organism to a rice plant element in an amount effective to increase biomass of dry shoot on the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Acremonium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, where the percent identity is determined over a hair alignment region at least 100, 200, 300, 400 or at least 500 nucleotides.
[0107] [0107] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant, comprising having, in a heterologous manner, a first endophyte organism and a second endophyte organism for a wheat plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of plant height, fresh root weight and fresh sprout weight in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the first endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99%, or 100% identical to a polynucleotide sequence selected from the group consisting of
[0108] [0108] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a rice plant, comprising the heterologous arrangement of a first endophyte organism and a second endophyte organism for a rice plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the first endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97%, at least 98 %, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, and the second endophyte organism is a member of the genus Coniochaeta and comprises at least at least one polynucleotide sequence at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41, where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0109] [0109] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant comprising disposing, in a heterologous manner, of an endophyte organism to a soybean plant element in an amount effective to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 %, or 100% identical to SEQ ID NO: 68, where the percent identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides.
[0110] [0110] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising disposing, in a heterologous manner, of an endophyte organism to a wheat plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 %, or 100% identical to SEQ ID NO: 68, where the percent identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides.
[0111] [0111] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a peanut plant comprising the heterologous disposition of an endophyte organism to a peanut plant element in an amount effective to increase yield in the derived plant the treated plant element in relation to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 68 and the peanut plant is the AT-9899 variety or a closely related variety, where the identified percentage is determined in an alignment region of at least 100, 200, 300, 400 or at least least 500 nucleotides.
[0112] [0112] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant comprising the heterologous arrangement of a first endophyte organism and a second endophyte organism for a soybean plant element in an amount effective for increase the dry root biomass in the plant derived from the treated plant element in relation to a plant derived from a reference plant element, in which the first endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% , at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, the second endophyte organism is a member of the genus Chaetomium and comprises at least one sequence polynucleotide at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, and the soybean plant is the Stine 33E22 variety or a closely related variety, where the percentage of identification is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0113] [0113] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a corn plant comprising disposing, in a heterologous manner, of an endophyte organism to a corn plant element in an effective amount to increase yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99 % or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, where the percentage of identification is determined in a hair alignment region at least 100, 200, 300, 400 or at least 500 nucleotides.
[0114] [0114] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a soybean plant comprising the heterologous disposition of an endophyte organism to a soybean plant element in an effective amount to enhance a trait of agronomic importance selected from the group consisting of the root area, root length, dry biomass and yield in the plant derived from the treated plant element in relation to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55 , 56, 57, 58 and 59, where the identified percentage is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0115] [0115] In one aspect, the invention provides a method of enhancing a trait of agronomic importance in a wheat plant comprising the heterologous disposition of an endophyte organism to a wheat plant element in an amount effective to enhance a trait of agronomic importance selected from the group consisting of root length and yield in the plant derived from the treated plant element in relation to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence of at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, in that the identified percentage is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0116] [0116] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Exserohilum and comprises at least one sequence polynucleotide at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62.
[0117] [0117] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the Epicoccum genus and comprises at least one sequence polynucleotide at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71.
[0118] [0118] In one aspect, the invention provides an agro-chemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Exserohilum and comprises at least one sequence polynucleotide at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62 further comprising a second endophyte organism, in which the second endophyte organism is of the genus Chaetomium and comprises at least one sequence of polynucleotides at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59.
[0119] [0119] In one aspect, the invention provides an agro-chemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Coniochaeta and comprises at least one sequence polynucleotide at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41.
[0120] [0120] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the Epicoccum genus and comprises at least one sequence polynucleotide at least 97% identical to SEQ ID NO: 70.
[0121] [0121] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Curvularia and comprises at least one sequence polynucleotide at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 65, and 66.
[0122] [0122] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Cladosporium and comprises at least one sequence polynucleotide at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68.
[0123] [0123] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Paecilomyces and comprises at least one sequence polynucleotide at least 97% identical to SEQ ID NO: 69.
[0124] [0124] In one aspect, the invention provides an agro-chemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Acremonium and comprises at least one sequence polynucleotide at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51.
[0125] [0125] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Cladosporium and comprises at least one sequence polynucleotide at 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68 and in which the microbial active ingredient further comprises a second endophyte organism, in which the second endophyte organism is of the genus Coniochaeta and comprises at least at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41.
[0126] [0126] In one aspect, the invention provides an agro-chemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Chaetomium and comprises at least one sequence polynucleotide at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59.
[0127] [0127] In some embodiments, the formulation oil comprises rapeseed, NEEM or erucic acid, or comprises herbicidal or insecticidal properties. In some embodiments, the formulation surfactant is a non-ionic detergent, Tween 20 or Triton X-100. In some embodiments, the formulation polymer is Flo Rite®, DISCOTM or Kannar® Universal Wonder. In some embodiments, the microbial active ingredient of the formulation comprises a spore suspension, spray dried spores or whole cell broth. In some embodiments, the formulation further comprises one or more of the following items: fungicide, nematicide, bactericide, insecticide, herbicide, stabilizer, preservative, carrier, anti-complex agent or any combination thereof. In some embodiments, the formulation's endophyte organism is stable on the shelf
[0128] [0128] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Exserohilum and comprises at least one sequence polynucleotide at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62, where the identified percentage is determined in a region alignment of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0129] [0129] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Epicoccum and comprises at least one sequence polynucleotide at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, where the identified percentage is determined in a region alignment of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0130] [0130] In one aspect, the invention provides an agro-chemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Exserohilum and comprises at least one sequence polynucleotide by at least 97%, at least 98%, at least 99%, or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62 further comprising a second endophyte organism, in which the second endophyte organism is of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99%, or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, where the percentage of identification is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0131] [0131] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Coniochaeta and comprises at least one sequence polynucleotide at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41, in which the identified percentage is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0132] [0132] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Epicoccum and comprises at least one sequence polynucleotide at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 70, where the identified percentage is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0133] [0133] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Curvularia and comprises at least one sequence polynucleotide by at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 65, and 66, where the percentage identified is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0134] [0134] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Cladosporium and comprises at least one sequence polynucleotide at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68, where the identified percentage is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0135] [0135] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Paecilomyces and comprises at least one sequence polynucleotide at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 69, where the identified percentage is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0136] [0136] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Acremonium and comprises at least one sequence polynucleotide at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, where the percentage identified is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0137] [0137] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Cladosporium and comprises at least one sequence polynucleotide at 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68 and in which the microbial active ingredient further comprises a second endophyte organism, where the second endophyte organism is of the genus Coniochaeta and comprises at least one polynucleotide sequence in at least 97%, at least 98%, at least
[0138] [0138] In one aspect, the invention provides an agrochemically active microbial formulation comprising at least one oil, surfactant, polymer and a microbial active ingredient, wherein the microbial active ingredient comprises a first endophyte organism of the genus Chaetomium and comprises at least one sequence polynucleotide at least 97%, at least 98%, at least 99% or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59 , where the identified percentage is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0139] [0139] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism.
[0140] [0140] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte organism, wherein the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a sequence polynucleotide selected from the group consisting of SEQ ID NOs: 60, 61 and 62, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, in which the Vegetable element is a vegetable element of corn and the improved trait of agronomic importance is yield.
[0141] [0141] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, where the vegetable element is a vegetable element of soy.
[0142] [0142] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologically disposed endophyte organism, wherein the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a sequence polynucleotide selected from the group consisting of SEQ ID NOs: 60, 61 and 62, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, in which the Vegetable element is a variety of soy selected from the group consisting of: Pfister 38R25, Dairyland DSR1808R2Y, Stine 3920 and closely related varieties, and the enhanced trait of agronomic importance is yield.
[0143] [0143] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, where the vegetable element is a vegetable element of wheat and the improved trait of agronomic importance is yield.
[0144] [0144] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, where the vegetable element is a peanut vegetable element and the improved trait of agronomic importance is yield.
[0145] [0145] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, where plant element is a variety of peanuts selected from the group consisting of: AT9899, FloRun 107, Georgia-06G, Tamnut OL06 and closely related varieties.
[0146] [0146] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism.
[0147] [0147] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte organism, wherein the endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97% identical to a sequence polynucleotide selected from the group consisting of SEQ ID NOs: 63, 64 and 71, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, in which the Vegetable element is a vegetable element of soy and the enhanced trait of agronomic importance is selected from the group consisting of area, length and dry weight of the root.
[0148] [0148] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64, and 71, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism , in which the plant element is a variety of Stine 33E22 soybean or a closely related variety thereof.
[0149] [0149] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, where the vegetable element is a peanut vegetable element and the improved trait of agronomic importance is yield.
[0150] [0150] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, where the vegetable element is a peanut vegetable element is selected from the group consisting of the FloRun 107, Georgia-06G variety, and its closely related varieties and the improved trait of agronomic importance is yield.
[0151] [0151] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte organism, wherein the endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97% identical to a sequence polynucleotide selected from the group consisting of SEQ ID NOs: 63, 64 and 71, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that does not further understand the endophyte organism and comprises a second endophyte organism, in which the second endophyte organism is of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 52, 53, 54, 55, 56, 57, 58 and 59, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism.
[0152] [0152] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64, and 71, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism , in which the vegetable element is the soybean variety Stine 33E22 and the trait of agronomic importance is the dry root biomass.
[0153] [0153] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41, which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism .
[0154] [0154] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, in which the endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet understand the organism endophyte, in which the vegetable element is a vegetable element of wheat and a trait of agronomic importance is yield.
[0155] [0155] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 70, in which the said synthetic composition is able to provide an improved trait of agronomic importance compared to the reference plant element that does not additionally comprise the endophyte organism.
[0156] [0156] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 70, in which the said synthetic composition is able to provide an improved trait of agronomic importance compared to the reference plant element that does not additionally comprise the endophyte organism, in which the plant element is the Brett canola variety Young 5525 or a closely related variety
[0157] [0157] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 65, and 66, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that does not yet understand the endophyte organism.
[0158] [0158] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte organism, wherein the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a sequence polynucleotide selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 65 and 66, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet understand the endophyte organism, in which the plant element is a plant element of soy and the trait of agronomic importance is selected from the group consisting of area, length and root yield.
[0159] [0159] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte organism, wherein the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a sequence polynucleotide selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 65, and 66, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that it does not yet understand the endophyte organism, in which the plant element is a variety of soy selected from the group consisting of Pfister 38R25, Stine 3920 and their closely related varieties, and the trait of agronomic importance is selected from the group consisting of root area, length of root and yield.
[0160] [0160] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 65, and 66, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that it does not yet understand the endophyte organism, in which the vegetable element is a vegetable element of wheat and the trait of agronomic importance is yield.
[0161] [0161] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte organism, wherein the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a sequence polynucleotide selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 65 and 66, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet understand the endophyte organism, in which the vegetable element is a variety of wheat selected from the group consisting of SDSU Focus, SDSU Select and closely related varieties, and the trait of agronomic importance is yield.
[0162] [0162] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67, in which said synthetic composition is able to provide an improved trait of agronomic importance in comparison with the reference plant element that does not additionally comprise the endophyte organism.
[0163] [0163] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophytic organism, wherein the endophyte is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, in which the plant element is a soy plant element and the trace of agronomic importance is selected from the group consisting of root area, root length and yield.
[0164] [0164] In one aspect, the invention provides a synthetic composition comprising a plant element and an heterologously disposed endophyte organism, in which the endophyte organism is a member of the genus
[0165] [0165] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism.
[0166] [0166] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, in which the vegetable element is a vegetable element of corn and the trait of agronomic importance is yield.
[0167] [0167] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, in which the vegetable element is the Stine 9734 maize variety and the trait of agronomic importance is yield.
[0168] [0168] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte, in which the endophyte is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, in which the Vegetable element is a vegetable element of soybean and the trait of agronomic importance is selected from the group consisting of root area, root length and yield.
[0169] [0169] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte, in which the endophyte is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, in which the Vegetable element is a variety of soy selected from the group consisting of Pfister 38R25, Stine 3920 and closely related varieties and the trait of agronomic importance is yield.
[0170] [0170] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism,
[0171] [0171] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, in which the vegetable element is a variety of wheat selected from the group consisting of SDSU Focus, SDSU Select and closely related varieties and the trait of agronomic importance is yield.
[0172] [0172] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte, in which the endophyte is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to a sequence polynucleotide selected from the group consisting of SEQ ID NOs: 67 and 68, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, in which the endophyte organism comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 68, the plant element is a peanut of the AT9899 variety and the trait of agronomic importance is yield.
[0173] [0173] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Paecilomyces and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 69, in which the said synthetic composition is capable of providing an improved trait of agronomic importance in comparison with the reference plant element that does not additionally comprise the endophyte organism.
[0174] [0174] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Paecilomyces and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 69, where said synthetic composition is able to provide an improved trait of agronomic importance compared to the reference plant element that does not additionally comprise the endophyte organism, where the plant element is the canola variety NCC1015 and the trait of agronomic importance is yield.
[0175] [0175] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that does not yet understand the endophyte organism.
[0176] [0176] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that it does not yet comprise the endophyte organism, in which the plant element is a soy plant element and the feature of agronomic importance is dry shoot biomass.
[0177] [0177] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte organism, wherein the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, and in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not it also includes the endophyte organism, in which the vegetable element is a vegetable element of wheat and the trait of agronomic importance is moisture or grain yield.
[0178] [0178] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that it does not yet understand the endophyte organism, in which the vegetable element is the SDSU Select wheat variety and the trait of agronomic importance is yield.
[0179] [0179] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that does not yet understand the endophyte organism, in which the plant element is the Georgia-06G peanut variety and the trait of agronomic importance is yield.
[0180] [0180] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that does not yet understand the endophyte organism, in which the vegetable element is a vegetable element of rice and the trait of agronomic importance is dry shoot biomass.
[0181] [0181] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte organism, wherein the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, and in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a reference plant element that it does not yet comprise the endophyte organism and also comprises a second endophyte organism, in which the second endophyte organism is of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41, in which the synthetic composition is able to provide an improved trait of agronomic importance in comparison with m a plant reference element that does not yet comprise the endophyte organism.
[0182] [0182] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte organism, wherein the endophyte organism is a member of the Acremonium genus and comprises at least a polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, and in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not it also comprises the endophyte organism and also comprises a second endophyte organism, in which the second endophyte organism is of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38 , 39, 40 and 41, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference vegetable element that does not yet comprise the endophyte organism, where the vegetable element is a vegetable element of wheat and the trait of agronomic importance is selected from the group consisting of plant height, fresh root biomass and fresh sprout weight .
[0183] [0183] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte organism, wherein the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, and in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not it also comprises the endophyte organism and also comprises a second endophyte organism, in which the second endophyte organism is of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38 , 39, 40 and 41, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference vegetable element that does not yet comprise the endophyte organism, where the vegetable element is a vegetable element of rice and the trait of agronomic importance is yield.
[0184] [0184] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Chaetomium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to an element reference plant that does not yet comprise the endophyte organism.
[0185] [0185] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte, in which the endophyte is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a plant element of reference that does not yet understand the endophyte organism, in which the plant element is a soy plant element and the feature of agronomic importance is selected from the group consisting of root area, root length, dry shoot biomass and yield.
[0186] [0186] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte, in which the endophyte is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a plant element reference that does not yet comprise the endophyte organism, in which the plant element is a soybean variety selected from the group consisting of Pfister 38R25, Stine 3920 and closely related varieties and the trait of agronomic importance is yield.
[0187] [0187] In one aspect, the invention provides a synthetic composition comprising a plant element and a heterologously disposed endophyte organism, wherein the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to a plant element of reference that does not yet understand the endophyte organism, in which the vegetable element is a vegetable element of wheat and the trait of agronomic importance is the length or yield of the root.
[0188] [0188] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance compared to an element reference vegetable that does not yet comprise the endophyte organism, in which the vegetable element is a wheat variety selected from the group consisting of SDSU Focus, SDSU Select and closely related varieties and the trait of agronomic importance is yield.
[0189] [0189] In some embodiments, any of the synthetic compositions described here further comprises a vegetable element is a seed, optionally in which the seed is modified. In some modalities, in which the improved trait of agronomic importance is conferred under normal conditions of irrigation. In some embodiments, the plant element is placed on a substrate that promotes plant growth, optionally in the soil. In some embodiments, a plurality of said plant elements are placed on the soil in rows, with substantially equal spacing between each seed within each row.
[0190] [0190] In some embodiments, any of the synthetic compositions described herein further comprises a formulation comprising one or more of the following: stabilizer, preservative, carrier, surfactant, anti-complexing agent or any combination thereof and / or one or more of the following: fungicide, nematicide, bactericide, insecticide or herbicide. In some embodiments, any of the synthetic compositions described here is confined to an object selected from the group consisting of: bottle, flask, ampoule, package, vase, purse, bag, box, trash can, envelope, cardboard box, container, silo , shipping container, truck bed or case. In some embodiments, any of the synthetic compositions described herein is shelf stable.
[0191] [0191] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97%, at least minus 98%, at least 99%, or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62, where the synthetic composition is capable of providing an enhanced trait of agronomic importance in comparison with a plant reference element that does not yet comprise the endophyte organism, in which the percentage of identification is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0192] [0192] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97%, at least minus 98%, at least 99%, or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, in which the synthetic composition is capable of providing an enhanced trait of agronomic importance in comparison with a plant reference element that does not yet comprise the endophyte organism, in which the percentage of identification is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0193] [0193] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41, in which the said synthetic composition is capable of providing an enhanced trace of agronomic importance compared to the reference plant element that does not additionally comprise the endophyte organism, where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides .
[0194] [0194] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97%, at least minus 98%, at least 99%, or 100% identical to SEQ ID NOs: 70, where the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism , where the percentage of identification is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0195] [0195] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97%, at least minus 98%, at least 99%, or 100% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 65, and 66, where the synthetic composition is capable of providing a improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism, in which the percentage of identification is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0196] [0196] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 67, in which the said synthetic composition is capable of providing an improved trait of agronomic importance compared to the reference plant element that does not additionally comprise the endophyte organism, in which the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides.
[0197] [0197] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 67 and 68, in which the said synthetic composition is capable of providing an improved trait of agronomic importance in comparison with the reference plant element that does not further comprise the endophyte organism, in which the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides.
[0198] [0198] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Paecilomyces and comprises at least one polynucleotide sequence at least 97%, at least minus 98%, at least 99%, or 100% identical to SEQ ID NOs: 69, where the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism , where the percentage of identification is determined in an alignment region of at least 100, 200, 300, 400 or at least 500 nucleotides.
[0199] [0199] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Paecilomyces and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51, in which said synthetic composition is capable of provide an enhanced trait of agronomic importance compared to the reference plant element that does not additionally comprise the endophyte organism, where the percentage of identity is determined over an alignment region of at least 100, 200, 300, 400, or at least 500 nucleotides.
[0200] [0200] In one aspect, the invention provides a synthetic composition comprising a plant element and an endophyte organism arranged in a heterologous manner, wherein the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59, in which said composition synthetic is able to provide an enhanced trait of agronomic importance compared to the reference plant element that does not additionally comprise the endophyte organism, in which the percentage of identity is determined over an alignment region of at least 100, 200, 300 , 400, or at least 500 nucleotides. BRIEF DESCRIPTION OF THE FIGURES
[0201] [0201] Figure 1A shows exemplary rice plants grown as described in Example 29. The pot on the left contains untreated control rice plants. The pot on the right shows rice plants treated with MIC-68178.
[0202] [0202] Figure 2 shows the washed roots of exemplary rice plants grown as described in Example 29. The roots of untreated control rice plants are on the left. The roots of the rice plants treated with MIC-68178 are on the right.
[0203] [0203] Figure 3 shows exemplary soybean roots from soybean plants grown as described in Example 29. The roots of untreated control rice plants are on the left. The roots of the soybean plants treated with a combination of MIC-68178 and MIC-33414 are on the right.
[0204] [0204] Figure 4 shows the pedigree of the SDSU Focus wheat variety.
[0205] [0205] Figure 5 shows the pedigree of the SDSU Select wheat variety. DETAILED DESCRIPTION
[0206] [0206] As demonstrated in this document, agricultural plants are heterologously arranged in symbiotic microorganisms, called endophyte organisms, particularly bacteria and fungi, which contribute to the survival, performance and characteristics of plants.
[0207] [0207] This document describes endophyte organisms that are capable of living inside or otherwise heterologously arranged in plants to improve the characteristics of the plant. This document describes methods of using endophyte organisms that are heterologously arranged in plants to transmit new characteristics to a host plant, as well as distinguishing plant elements from the host plant. In some embodiments, the endophytic compositions are isolated and purified from sources of plants or fungi and arranged heterologously with a plant element to transmit improved agronomic potential and / or improved agronomic traits to the host plant. In some modalities, endophyte organisms that are able to live inside plants are isolated and purified from their native sources and arranged heterologously, for example, manually, mechanically or artificially combined, with a plant element, to confer a better agronomic potential and / or improved agronomic traits for the host plant or host plant elements. Such endophyte organisms that are capable of living within plants can be further manipulated or combined with additional elements before being combined with plant elements.
[0208] [0208] As described in this document, endophyte organisms can be robust derived from heterologous, homologous or manipulated sources, optionally cultivated, applied manually, mechanically or artificially applied heterologously to plant elements, for example, heterologically arranged and, as a result of manual application mechanical or artificial, they confer several beneficial properties. This is surprising, given the variability observed in the technique in isolating endophytic microbes and previous observations of inefficient colonization of plant pathogens from host plant tissues.
[0209] [0209] In part, the present invention provides preparations of endophyte organisms that are capable of living inside plants, and the creation of synthetic compositions of plant elements and / or seedlings with heterologously arranged endophyte organisms, and formulations comprising the compositions as well as the recognition that synthetic compositions have a variety of beneficial and unexpected properties present in agricultural plants and / or in populations of heterologous endophyte organisms. Beneficial properties include, but are not limited to, metabolism, transcript expression, proteomic changes, morphology, resilience to a variety of environmental stresses and any combination of such properties. The present invention also provides methods of using endophyte organisms described in this document to benefit the host plant with which they are heterologously arranged. Definitions
[0210] [0210] The terms used in the claims and specifications are defined as set out below, unless otherwise specified.
[0211] [0211] It should be noted that, as used in this specification and the appended claims, the singular forms "a" "an" and "o / a" include plural referents, unless the context clearly dictates otherwise.
[0212] [0212] An "endophyte organism" is an organism capable of living in a plant element (for example, rhizoplane or phyllosphere) or within a plant element, or on a physical surface close to a plant element, for example, the rhizosphere, or in a seed. A "beneficial" endophyte organism does not cause disease or harm the host plant. Endophyte organisms can occupy the intracellular or extracellular spaces of plant tissue, including leaves, stems, flowers, fruits, seeds or roots. An endophyte organism can be, for example, a bacterial or fungal organism and can confer a beneficial property on the host plant, such as an increase in yield, biomass, resistance or fitness. An endophyte organism can be a fungus or a bacterium. As used in this document, the term "microbe" is sometimes used to describe an endophyte organism.
[0213] [0213] A "population" of endophyte organisms, or an "endophyte population", refers to one or more endophyte organisms that share a common genetic derivation, for example, one or more propagules of a single endophyte organism, that is, endophyte organisms created from a single chosen colony. In some modalities, a population refers to endophyte organisms of identical taxonomy. In some cases, a population of endophyte organisms refers to one or more endophyte organisms of the same genus. In some cases, a population of endophyte organisms refers to one or more endophyte organisms from the same OTU.
[0214] [0214] A "plurality of endophytic organisms" means two or more types of endophytic entities, for example, bacteria or fungi, or combinations thereof. In some embodiments, the two or more types of endophytic entities are two or more individual endophytic organisms, regardless of genetic derivation or taxonomic relationship. In some embodiments, the two or more types of endophytic entities are two or more populations of endophyte organisms. In other embodiments, the two or more types of endophytic entities are two or more species of endophytic organisms. In still other modalities, the two or more types of endophyte entities are two or more genera of endophyte organisms. In still other modalities, the two or more types of endophyte entities are two or more families of endophyte organisms. In other embodiments, the two or more types of endophytic entities are two or more orders of endophyte organisms. In other embodiments, the two or more types of endophytic entities are two or more classes of endophyte organisms. In still other modalities, the two or more types of endophyte entities are two or more phyla of endophyte organisms. In some embodiments, a plurality refers to three or more endophyte organisms, distinct individual organisms or distinct members of different genetic derivations or taxa. In some embodiments, a plurality refers to four or more distinct individual endophytic organisms or distinct members of different genetic derivations or taxa. In some embodiments, a plurality refers to five or more, ten or more, or an even greater number of distinct individual endophytic organisms or distinct members of different genetic derivations or taxa. In some modalities, the term "consortium" or "consortium" can be used as a collective noun synonymous with "plurality", when describing more than one population, species, genus, family, order, class or phylum of endophyte organisms.
[0215] [0215] As used in this document, the term "microbe" or "microorganism" refers to any species or taxon of microorganism, including, but not limited to archaea, bacteria, microalgae, fungi (including mold and yeast species), mycoplasmas, microspores, nanobacteria, oomycetes and protozoa. In some embodiments, a microbe or microorganism is an endophyte organism, for example, a bacterial or fungal endophyte organism, which is capable of living inside a plant. In some embodiments, a microbe or microorganism comprises individual cells (eg, single-celled microorganisms) or more than one cell (eg, multicellular micro-organism). A "population of microorganisms" can therefore refer to multiple cells of a single microorganism, in which the cells share a common genetic derivation.
[0216] [0216] As used in this document, the term "bacterium" or "bacterium" refers in general to any prokaryotic organism, and can refer to an organism from Kingdom Eubacteria (Bacteria), Kingdom Archeobacteria (Archae), or both. In some cases, bacterial genera have been reassigned for various reasons (such as, but not limited to, the evolving field of complete genome sequencing), and it is understood that such nomenclature reassignments are within the scope of any claimed genus. For example, certain species of the genus Erwinia have been described in the literature as belonging to the genus Pantoea (Zhang, Y. & Qiu, S. Antonie van Leeuwenhoek (2015) 108: 1037).
[0217] [0217] The term 16S refers to the DNA sequence of a bacterial 16S ribosomal RNA (rRNA) sequence. Sequencing the 16S rRNA gene is a well-established method for studying the phylogeny and taxonomy of bacteria.
[0218] [0218] As used in this document, the term "fungus" or "fungus" refers in general to any organism in the Fungi Kingdom. The historical taxonomic classification of fungi is in accordance with the morphological presentation. From the mid-1800s, it was recognized that some fungi have a pleomorphic life cycle and that different nomenclature designations were being used for different forms of the same fungus. In 1981, the Sydney Congress of the International Mycological Association established rules for naming fungi according to their status as anamorph, teleomorph or holomorph (Taylor JW. One Fungus = One Name: DNA and fungal nomenclature twenty years after PCR. IMA Fungus 2 (2): 113-120. 2011.). With the development of genomic sequencing, it became evident that the taxonomic classification based on molecular phylogenetics did not align with the morphological nomenclature (Shenoy BD, Jeewon R, Hyde KD. Impact of DNA sequence-data on the taxonomy of anamorphic fungi. Fungal Diversity 26 (10) 1-54. 2007). As a result, in 2011, the International Botanical Congress adopted a resolution approving the International Nomenclature Code for Algae, Fungi and Plants (Melbourne Code) (International Nomenclature Code for Algae, Fungi and Plants (Code of Melbourne), adopted by the 18th International Botanical Congress of Melbourne, Australia, July 2011), with the stated result of designating “One Fungus = One Name” (Hawksworth DL. Managing and coping with names of pleomorphic fungi in a period of transition IMA Fungus 3 (1): 15-24. 2012.). However, systematic experts have not aligned themselves with the common nomenclature for all fungi, nor do all existing databases and information resources include updated taxonomies. As such, many fungi referred to in this document can be described by their anamorphic form but it is understood that, based on identical genomic sequencing, any pleomorphic state of this fungus can be considered the same organism. For example, the genus Alternaria is the anamorphic form of the Lewia teleomorphism (Kwasna H and Kosiak B. Lewia avenicola sp. Nov. And its Alternaria anamorph from oat grain, with a key to the species of Lewia. Mycol Res 2003; 107 (Pt 3): 371-6.), Therefore, both would be understood as the same organism with the same DNA sequence. For example, it is understood that the genus Acremonium is also reported in the literature as the genus Sarocladium, as well as the genus Tilachilidium (Summerbell RC, C. Gueidan, HJ. Schroers3, GS de Hoog, M. Starink, Y. Arocha Rosete, J. Guarro and JA Scott. Acremonium phylogenetic overview and revision of Gliomastix, Sarocladium, and Trichothecium. Studies in Mycology 68: 139–162. 2011.). For example, the genus Cladosporium is an anamorph of the teleomorphic genus Davidiella (Bensch K, Braun U, Groenewald JZ, Crous PW. The genus Cladosporium. Stud Mycol. June 15, 2012; 72 (1): 1–401.) And is understood to describe the same organism. In some cases, fungal genera have been reassigned for various reasons, and it is understood that such nomenclature reassignments are within the scope of any claimed genus.
[0219] [0219] "Internal Transcribed Spacer" (ITS) refers to the spacer DNA (non-coding DNA) located between the small subunit ribosomal RNA (rRNA) and large subunit rRNA (LSU) genes on the chromosome or the corresponding transcribed region on region transcribed in the polycistronic rRNA precursor transcript. Sequencing of ITS genes is a well-established method for studying the phylogeny and taxonomy of fungi. In some cases, the “Large SubUnit” (LSU) sequence is used to identify fungi. The genetic sequencing of LSU is a well-established method for the study of fungal phylogeny and taxonomy. Some endophytic fungal organisms can be described by an ITS sequence and some can be described by an LSU sequence. Both are understood to be equally descriptive and accurate in determining taxonomy.
[0220] [0220] As used in this document in relation to fungi and bacteria, the term "marker gene" refers to a conserved gene comprising sequence variation between related organisms, for example, a 16S polynucleotide sequence (for bacteria) or ITS (for fungi) of the organism, fusA gene or genomic regions, by which a microbe can be specifically identified and assigned to a taxonomic nomenclature. In some embodiments, the marker genes include, but are not limited to, actin, elongation factor G (fusA), tubulin, major subunit of RNA polymerase II (RPB1), long subunit rRNA gene (LSU), second major subunit of RNA polymerase II (RPB2), small subunit rRNA (SSU) gene, phosphoglycerate kinase, beta-tubulin and combinations thereof.
[0221] [0221] The terms "pathogen" and "pathogenic" in reference to a bacterium or fungus include any organism of that type that is capable of causing or affecting a disease, disorder or condition of a host comprising the organism.
[0222] [0222] A "spore" or a population of "spores" refers to bacteria or fungi that are generally viable, more resistant to environmental influences such as heat and bactericidal or fungicidal agents than other forms of the same bacteria or fungi, and typically capable of germination and growth. Bacteria and fungi that are "capable of forming spores" are those bacteria and fungi that comprise the genes and other skills necessary to produce spores under appropriate environmental conditions.
[0223] [0223] "Biomass" means the total mass or weight (fresh or dry), at any given moment, of a plant tissue, plant tissue, an entire plant or a population of plants. Biomass is generally given as weight per unit area. The term can also refer to all plants or species in the community (community biomass).
[0224] [0224] The term "isolated" is intended to refer specifically to an organism, cell, tissue, polynucleotide or polypeptide that is removed from its original source.
[0225] [0225] As used in this document, an isolated endophyte or microbe is an endophyte or microbe that has been removed from its natural environment. “Pure cultures” or “isolated cultures” are cultures in which the organisms present are only of a particular genus and species. This contrasts with "mixed cultures", which are cultures in which more than one genus and / or species of microorganism are present. As such, the term "isolated" does not necessarily reflect the extent to which the microbe has been purified. A "substantially pure culture" of the microbe refers to a culture that contains substantially no other endophyte or microbe organism than the desired endophyte or microbe. In other words, a substantially pure endophyte organism or microbial culture is substantially free of other contaminants, which may include microbial contaminants. Besides that,
[0226] [0226] A "host plant" includes any plant, particularly a plant of agronomic importance, within which or on which a microbe, such as an endophyte organism, is heterologously disposed. As used in this document, a microbe is said to colonize a plant, plant element or seed, when it can exist as an endophyte organism in relation to a plant or plant element during at least part of the life cycle of the plant or microbe. In some embodiments, an endophyte organism is said to "colonize" a plant or plant element when it can be stably detected within the plant or plant element over a period of time, such as one or more days, weeks, months or years. Some of the compositions and methods described in this document involve a plurality of microbes in an amount effective to colonize a plant.
[0227] [0227] A "non-host target" means an organism or chemical compound that is altered in some way after coming into contact with a host plant that comprises an endophyte organism, as a result of a property conferred on the host plant by the endophyte organism.
[0228] [0228] As used herein, a nucleic acid has "homology" or is "homologous" to a second nucleic acid if the nucleic acid sequence has a sequence similar to the second nucleic acid sequence. The terms "identity", "percent identity", "percent sequence identity" or "identical" in the context of nucleic acid sequences refer to the nucleotides in the two sequences which are the same when aligned for maximum match. There are different algorithms known in the art that can be used to measure nucleotide sequence identity. The identity of the nucleotide sequence can be measured by local or global alignment, preferably implementing an optimal local or optimal global alignment algorithm. For example, a global alignment can be generated using an implementation of the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CD (1970) Journal of Molecular Biology. 48 (3): 443-53). For example, a local alignment can be generated using an implementation of the Smith-Waterman algorithm (Smith TF & Waterman, MS (1981) Journal of Molecular Biology. 147 (1): 195-197). Optimal global alignments using the Needleman-Wunsch algorithm and optimal local alignments using the Smith-Waterman algorithm are implemented in USEARCH, for example, USEARCH version v8.1.1756_i86osx32.
[0229] [0229] A gap is a region of an alignment in which one sequence does not align with a position in the other alignment sequence. In global alignments, terminal gaps are discarded before identity is calculated. For local and global alignments, internal gaps are counted as differences. A terminal gap is a region that begins at the end of a sequence in an alignment where the nucleotide at the end position of that sequence does not correspond to a nucleotide position in the other sequence in the alignment and extends to all contiguous positions in that sequence where the nucleotides in that sequence do not correspond to a nucleotide position in the other alignment sequence. An internal gap is a gap in an alignment that is flanked at the 3 'and 5' ends by positions where the aligned strings are identical.
[0230] [0230] In some embodiments, the nucleic acid sequence to be aligned is a complete gene. In some embodiments, the nucleic acid sequence to be aligned is a gene fragment. In some embodiments, the nucleic acid sequence to be aligned is an intergenic sequence. In a preferred embodiment, homology inference from a sequence alignment is made where the alignment region is at least 85% of the length of the query sequence.
[0231] [0231] The term "substantial homology" or "substantial similarity", when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 76%, 80%, 85%, or at least about 90%, or at least about 95%, 96%, at least 97%, 98%, 99% or 100 % of alignment positions, where the alignment region is at least about 50%, 60%, 70%, 75%, 85% or at least about 90%, or at least about 95%, 96%, 97 %, 98%, 99% or 100% of the query string length. In a preferred embodiment, the alignment region contains at least 100 positions, including any internal gaps. In some embodiments, the alignment region comprises at least 100 nucleotides from the query sequence. In some embodiments, the alignment region comprises at least 200 nucleotides from the query sequence. In some embodiments, the alignment region comprises at least 300 nucleotides from the query sequence. In some embodiments, the alignment region comprises at least 400 nucleotides from the query sequence. In some embodiments, the alignment region comprises at least 500 nucleotides from the query sequence. In some modalities, the query sequence is selected from the group consisting of SEQ ID NOs: 38-59.
[0232] [0232] As used in this document, the terms "operational taxonomic unit", "OTU", "taxon", "hierarchical cluster" and "cluster" are used interchangeably. An operational taxon unit (OTU) refers to a group of one or more organisms that comprises a node in a cluster tree. The level of a grouping is determined by its hierarchical order. In some modalities, an OTU is a group provisionally considered to be a valid taxon for purposes of phylogenetic analysis. In another modality, an OTU is any of the existing taxonomic units under study. In yet another modality, an OTU receives a name and a classification. For example, an OTU can represent a domain, a subdomain, a kingdom, a sub-kingdom, a phylum, a sub-phylum, a class, a subclass, an order, a sub-order, a family, a subfamily, a genus, a subgenus or a species. In some modalities, OTUs can represent one or more organisms from the kingdoms Eubacteria, Bacteria, Protista or Fungi, at any level of a hierarchical order. In some modalities, an OTU represents a prokaryotic or fungal order.
[0233] [0233] In some embodiments, the present invention contemplates synthetic compositions comprising the combination of a plant element, seedlings or whole plants and an endophytic population, in which the endophytic population is "heterologically disposed". In some embodiments, one or more endophyte organisms of the synthetic composition are arranged heterologously or manually, artificially inoculated or arranged in a plant element, seedlings, plants or in a plant growth medium or in one or more treatment formulas so that the endophyte organism exists on or in said plant element, seedling, plant, plant growth medium or treatment formulation in a manner not found in nature before the application of one or more endophyte organisms, for example, said combination that is not found in nature. In some modalities, such a way is contemplated to be selected from the group consisting of: the presence of the endophyte organism; presence of the endophyte organism in a different number of cells, concentration or quantity; the presence of the endophyte organism in a plant element, tissue, cell type or other different physical location in the plant; the presence of the endophyte organism in different periods of time, for example, stage of development of the plant or plant element, time of day, season season and combinations of these. In some embodiments, one or more endophyte organisms of a synthetic composition are arranged heterologously when one or more endophyte organisms are artificially inoculated, for example, they are manually or mechanically inoculated, or artificially applied, for example, manually or mechanically applied, to an element different plant or a different stage of development than that with which one or more endophyte organisms are found naturally or in a concentration, number or quantity greater than that which is found naturally in or on said plant element, seedling or plant. In some embodiments, "heterologously disposed" refers to the relationship between the endophyte organism and the inoculated host plant compared to the type of host plant with which said endophyte organism is normally associated. In one example, the endophyte organisms used in a synthetic composition can be obtained from an individual plant different from the same variety as the inoculated host plant for which it becomes heterologous, a plant of a different variety, but of the same genus and species, plant of a different cultivar or plant of a different genus.
[0234] [0234] An "inoculated" plant or plant element was artificially introduced into a heterologous endophytic organism at some point during the growth or development of the plant or plant element (including vegetative or reproductive phases). In some embodiments, the heterologous endophyte organism is transiently or permanently incorporated into the plant or plant element and is detectable using methods known in the art or described in this document. In some embodiments, a seed is inoculated with an endophyte organism by manual or mechanical contact with the seed with a formulation comprising said endophyte organism, which is detectable in or on the seed. In some embodiments, a plant is said to be inoculated with an endophyte organism if it is grown from a reproductive element (for example, a seed) that has been contacted manually or mechanically with a formulation comprising said endophyte organism, which is subsequently detectable in the plant. In some embodiments, a plant is said to be inoculated with an endophyte organism if any or more of its plant elements (for example, leaf, stem or root) are contacted manually or mechanically with a formulation comprising said endophyte organism, which it is subsequently detectable in the same plant element that was originally contacted with the said formulation or in a different plant element of that plant. The term "inoculation" can also refer to the manual or mechanical contact of an endophytic population with any substance, which is detectable in or on that substance after contact with the endophyte organism. In one example, said substance is soil or another plant growth medium. In another example, said substance is a storage medium such as glycerol. In some cases, "inoculation" may refer to the contact of a population of endophyte organisms with a living non-plant organism, for example, but not limited to an insect or fungus.
[0235] [0235] The term “isolation” is a comparative term and refers to organisms that are genetically identical, but that may differ in treatment. In one example, two genetically identical corn plant embryos can be separated into two different groups, one receiving a treatment (such as transformation with a heterologous polynucleotide, to create a genetically modified plant) and a control, for example, reference, which does not receive such treatment. Any phenotypic differences between the two groups can therefore be attributed only to the treatment and not to any inheritance of the genetic makeup of the plant. In another example, two genetically identical soybeans can be treated with a formulation, one that introduces an endophytic composition and one that does not. Any phenotypic differences between the plants derived (for example, grown or obtained from) of these seeds can be attributed to the treatment with endophyte organisms, thus forming a comparison of isolines.
[0236] [0236] Likewise, the term "reference agricultural plant" means an agricultural plant of the same species, variety or cultivar, for which a treatment, formulation, composition or preparation of an endophyte organism as described in this document is not administered / contacted. A reference agricultural plant, therefore, is identical to the treated plant with the exception of the presence of the endophyte and can serve as a control to detect the effects of the endophyte organism that is conferred on the plant. In some embodiments, the term "reference isolation plant" is used in this document to describe a reference plant that is genetically identical and subject to the same conditions, that is, a control plant, to the treated plant.
[0237] [0237] A "reference environment" refers to the environment, treatment or condition of the plant on which a measurement is made. For example, the production of a compound in a plant arranged heterologously to an endophyte organism can be measured in a water stress reference environment and compared with the levels of the compound in a reference agricultural plant under the same water stress conditions. Alternatively, the levels of a compound in plants heterologously arranged for an endophyte organism and reference agricultural plant can be measured under identical stress-free conditions.
[0238] [0238] A "plant element" is intended to refer generically to an entire plant or plant component, including, but not limited to, tissues, parts and cell types of the plant. A plant element is preferably one of the following: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, dermal tissue, seed, leaf, root, bud, stem, flower, fruit, stalk, bulb, tuber, corm, keikis, bud. As used in this document, a "plant element" is synonymous with a "portion" of a plant and refers to any part of the plant, and can include different tissues and / or organs and can be used interchangeably with the term " fabric "throughout this document.
[0239] [0239] Similarly, a "plant reproductive element" is intended to refer generically to any part of a plant that is capable of initiating other plants through sexual or asexual reproduction of that plant, for example, but not limited to : seed, seedling, root, bud, cut, graft, graft, stalk, bulb, tuber, corm, keikis or bud.
[0240] [0240] A "progeny seed", as used in this document, refers to the seed produced by a host plant that has been inoculated with, or heterologously disposed of, an endophyte organism. For example, in the present invention, a seed, plant element or whole plant may become heterologously disposed to an endophyte organism, and the plant that is grown from said seed, or plant that is grown in heterologous association with the said endophyte organism, can itself produce progeny seeds that comprise altered nutritional composition compared to seeds obtained from plants that have not been grown from a plant element arranged heterologously to an endophyte organism or obtained from a parental (host) plant that has become heterologously disposed to an endophyte organism at some point in its life cycle. In the general sense, the phrase “progeny seed” can be interpreted as representing any plant propagation unit produced by the host plant that is capable of becoming another individual of that same plant species.
[0241] [0241] A "population" of plants refers to more than one plant, which are of the same taxonomic category, typically of the same species, and usually also share a common genetic derivation.
[0242] [0242] As used in this document, an “agricultural seed” is a seed used to grow a plant typically used in agriculture (an “agricultural plant”). The seeds can be from a monocot plant and can be planted for the production of an agricultural product, for example, animal feed, food, fibers, fuels, industrial uses, etc. As used in this document, an agricultural seed is a seed that is prepared for planting, for example, on farms for cultivation.
[0243] [0243] “Agricultural plants” or “plants of agronomic importance include plants that are grown by humans for food, feed, fiber,
[0244] [0244] A "closely related variety" comprises a common genetic derivation with a variety of plants. In some embodiments, an closely related variety has at least one lineage of dual origin in common with the plant variety. In some embodiments, a closely related variety has at least one parental lineage in common with the plant variety. In some modalities, a closely related variety has at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, 99.99% of the same SNPs detected in the plant variety. In some embodiments, an closely related variety has at least 1, at least 2, at least 3, at least 4, at least 5, at least 7, at least 8, at least 9, at least 10, at least at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 or more of the same SNPs detected in the plant variety. In some embodiments, an closely related variety has at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200 , at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or 100 or more of the same SNPs detected in the plant variety. In some embodiments, a closely related variety has at least 1000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 7,000, at least 8,000, at least 9,000 or 10,000 or more than same SNPs detected in the plant variety. In some embodiments, a closely related variety has at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 60,000, at least 70,000, at least 80,000, at least 90,000 or 100,000 or more of those same SNPs detected in the plant variety.
[0245] [0245] A "synthetic composition" comprises one or more endophyte organisms combined by human effort with a heterologously arranged plant element or a treatment formulation, said combination that is not found in nature. In some embodiments, the term "synthetic composition" means one or more plant elements or formulation components combined by human effort with an isolated and purified endophyte organism composition. In some embodiments, said purified endophyte organism composition is mechanically or manually applied, artificially inoculated or disposed in a plant element in a way that is not found or in the plant element prior to application of the purified endophyte organism composition, for example, to said combination or association that is not found in nature.
[0246] [0246] In some embodiments, "synthetic composition" used to refer to a treatment formulation comprising an isolated and purified population of endophyte organisms arranged heterologously to a plant element. In some embodiments, "synthetic composition" refers to a purified population of endophyte organisms in a treatment formulation comprising additional compositions with which said endophyte organisms are not found in nature.
[0247] [0247] A "treatment formulation" refers to a mixture of chemicals that facilitate the stability, storage and / or application of endophyte organism compositions. The treatment formulations can comprise any one or more agents such as: surfactant, a buffer, an adhesion promoter, a microbial stabilizer, a fungicide, an anti-complex agent, a herbicide, a nematicide, an insecticide, a plant growth regulator , a rodenticide, a desiccant, a nutrient, an excipient, a wetting agent, a salt.
[0248] [0248] In some embodiments, an “agriculturally compatible carrier” can be used to formulate an agricultural formulation or other composition that includes a purified endophyte organism preparation. As used in this document, an "agricultural-compatible carrier" refers to any material, other than water, that can be added to a plant element without causing or having an adverse effect on the plant element (for example, by reducing the germination of plants). seeds) or the plant that grows from the plant element or similar.
[0249] [0249] “Plant health” is demonstrated by the presence or improvement of a trait of agronomic importance found in a plant or plant element compared to a reference plant or plant element. The compositions and methods described in this document can provide an enhanced "agronomic trait" or "agronomic importance trait" for a host plant, which include, but are not limited to disease resistance, drought tolerance, heat tolerance, cold tolerance , salinity tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, yield enhancement, health improvement, enhancement stamina, enhancement of growth, enhancement of photosynthetic capacity, enhancement of nutrition, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, increased root area, improved root architecture, modulation metabolite, modulation of the proteome, increased weight of the seed, altered composition of seed carbohydrates, altered composition of seed carbohydrates, altered composition of seed oil, altered seed protein altered composition of seed nutrients and combinations of these, in comparison with the reference plant derived from a seed without said seed treatment formulation.
[0250] [0250] In some modalities, a treatment is arranged in a heterologous way in a plant element in an effective amount to improve a trait of agronomic importance. In some modalities, treatments capable of improving plant health are applied in an effective amount to improve a trait of agronomic importance or tolerance by at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 3%, between 3% and 5%, at least 5%, between 5% and 10%, at least 10%, between 10% and 15%, for example, at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60 %, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%,
[0251] [0251] In some modalities, an improvement in a trait of agronomic importance is measured by the "gain rate". The victory rate is the proportion of repetitions in which the treatment shows an improvement in a trait of agronomic importance in relation to the reference replicas. In some embodiments, the replicas are individual plants. In some modalities, replicates are plots, for example, plots replicated within a randomized, complete, field test. In some modalities, replicas are field trials conducted in different geographies.
[0252] [0252] In some modalities, the endophyte organism is able to improve a trait of agronomic importance in concentrations detected in the treated plant element or at least 10 ^ 2 CFU or spores per plant element, between 10 ^ 2 and 10 ^ 3 CFU or spores per plant element, about 10 ^ 3 CFU or spores per plant element, between 10 ^ 3 and 10 ^ 4 CFU or spores per plant element, about 10 ^ 4 CFU or spores per plant element, or between 10 ^ 4 , of about 10 ^ 5 UFC or spores per plant element, at least 10 ^ 5 UFC or spores per plant element, between 10 ^ 5 and 10 ^ 6 UFC or spores per plant element, about 10 ^ 6 UFC or spores per plant element, between 10 ^ 6 and 10 ^ 7 CFU or spores per plant element, about 10 ^ 7 CFU or spores per plant element, between 10 ^ 7 and 10 ^ 8 CFU or spores per plant element, about 10 ^ 8 CFU or spores per plant element, or even greater than 10 ^ 8 CFU or spores per plant element. In some embodiments, the plant element is a seed.
[0253] [0253] The term "characteristic of nutritional quality" includes any measurable parameter of a seed that directly or indirectly influences the value (nutritional or economic) of that seed, for example, but not limited to: proteins, fats, carbohydrates, ash , moisture, fiber and calories. In some cases, “trait of nutritional quality” is synonymous with “trait of nutritional quality” or “trait of nutritional quality of seeds”, and can refer to any composition of the associated plant element, more particularly compositions providing benefit to other organisms that consume or use the said plant element. As used in this document, “oil” and “fat” are used interchangeably.
[0254] [0254] An increased "seed yield" can refer to any increase in weight, size or abundance of seeds or fruits per unit of measure, for example, per plant, by number of plants, by mass of plants, per acre planted , per acre collected. In some modalities, seed yield is reported as pounds or bushels of seeds produced per collected acre. The terms seed and grain are used interchangeably in this document. The yield can also refer to the recovery of a particular component of a plant tissue by processing, for example, the amount of oil that can be extracted per unit of seed. Typically, the particular characteristic is designated when referring to increased yield, for example, increased seed yield or increased oil yield. Where the characteristic is not specified, it can be assumed that the yield refers to the seed yield and the terms can be used interchangeably.
[0255] [0255] As used in this document, the terms “low water condition” and “dry condition”, or “little water” and “dry” can be used interchangeably. For example, a method or composition for improving a plant's ability to grow in dry conditions means the same as its ability to grow in conditions with little water. In such cases, it can be said that the plant has improved tolerance to water stress.
[0256] [0256] As used in this document, the terms "normal watering" and "well watered" are used interchangeably to describe a plant grown under typical growing conditions without water restrictions.
[0257] [0257] In addition, "altered metabolic function" or "altered enzyme function" may include, but is not limited to: altered auxin production, altered nitrogen fixation, altered production of an antimicrobial compound, altered production of a siderophore, alteration of the phosphate solubilization mineral, altered production of a cellulase, altered production of a chitinase, altered production of a xylanase, altered production of acetoin, altered use of a carbon source.
[0258] [0258] "Nutrient" or "seed nutrient" refers to any composition of the associated plant element, more particularly compositions that provide benefit to other organisms that consume or use said plant element.
[0259] [0259] “Potential for agronomic traits” means the ability of a plant element to exhibit a phenotype, preferably an improved agronomic trait, at some point in its life cycle, or to transport that phenotype to another plant element to which it is associated in same plant. For example, a plant element may comprise an endophyte organism that will provide benefit to the leaf tissue of a plant from which the plant element is grown; in such a case, the plant element comprising that endophyte organism has the potential for an agronomic trait for a particular phenotype (for example, increased biomass in the plant) even if the plant element itself does not exhibit that phenotype.
[0260] [0260] In some cases, the present invention contemplates the use of compositions that are "compatible" with agricultural chemicals, including, but not limited to, a fungicide, an anti-complex compound, a bactericide, a virucide, a herbicide, a nematicide, a parasiticide, a pesticide or any other agent widely used in agriculture that has the effect of killing or otherwise interfering with the optimal growth of another organism. As used in this document, a composition is "compatible" with an agricultural chemical when the organism is modified, such as by genetic modification, for example, contains a transgene that confers resistance to an herbicide, or is adapted to grow or survive the concentration of the agricultural chemical used in agriculture. For example, an endophyte organism on the surface of a plant element is compatible with the fungicide metalaxyl if it is able to survive the concentrations that are applied on the surface of the plant element.
[0261] [0261] As used in this document, a “colony forming unit” (“UFC”) is used as a measure of viable microorganisms in a sample. A CFU is an individual viable cell capable of forming a visible colony in a solid medium whose individual cells are derived by cell division of a parental cell. In some embodiments, a cell is a spore of fungi.
[0262] [0262] The terms "decreased", "less", "slower" and "increased", "faster", "enhanced", "greater" as used in this document refer to a decrease or increase in a characteristic of the plant element treated with an endophyte organism or resulting plant compared to an untreated plant element or resulting plant. For example, a decrease in a characteristic can be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, between 5% and 10%, at least 10%, between 10 % and 20%, at least 15%, at least 20%, between 20% and 30%, at least 25%, at least 30%, between 30% and 40%, at least 35%, at least 40%, between 40% and 50%, at least 45%, at least 50%, between 50% and 60%, at least about 60%, between 60% and 70%, between 70% and 80%, at least 75%, at least at least about 80%, between 80% and 90%, at least about 90%, between 90% and 100%, at least 100%, between 100% and 200%, at least 200%, at least about 300% , at least about 400% or more below the untreated control and an increase can be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, between 5% and 10 %, at least 10%, between 10% and 20%, at least 15%, at least 20%, between 20% and 30%, at least 25%, at least 30%, between 30% and 40%, at least 35%, at least 40%, between 40% and 50%, at least 45%, by at least 50%, between 50% and 60%, at least about 60%, between 60% and 70%, between 70% and 80%, at least 75%, at least about 80%, between 80% and 90 %, at least about 90%, between 90% and 100%, at least 100%, between 100% and 200%, at least 200%, at least about 300%, at least about 400% or more untreated control.
[0263] [0263] As used in this document, a microbe or plant or plant element is "modified" when it comprises an artificially introduced genetic or epigenetic "modification". In some embodiments, the modification is introduced by a genome manipulation technology. In some embodiments, the modification is introduced by a targeted nuclease. In some embodiments, targeted nucleases include, but are not limited to, nuclease with transcriptional activator effectors (TALEN), zinc finger nuclease (ZNF), short intergrated regulated interspatial palindromic repetitions (CRISPR), CRISPR / Cas9, CRISPR / CPF1 and combinations of these. In some embodiments, the modification is an epigenetic modification. In some embodiments, the modification is introduced by treatment with a DNA methyltransferase inhibitor, such as 5-azacytidine, or a histone deacetylase inhibitor, such as 2-amino-7-methoxy-3H-phenoxazin-3-one. In some embodiments, the modification is introduced through tissue culture. In some embodiments, a microbe or modified plant or plant element comprises a transgene. Endophytic Compositions
[0264] [0264] The endophyte organisms described in this document provide several unexpected and significant advantages to agricultural plants over other plant-associated microbes, as demonstrated in the Examples.
[0265] [0265] New compositions of endophytes are described here. In some modalities, the endophyte organism is selected from Table 4. In some modalities, the endophyte organism is selected from Table 6.
[0266] [0266] In some embodiments, the endophyte organism comprises a polynucleotide sequence that is at least 97% identical to at least one selected sequence from the group consisting of SEQ ID NOs: 67. In some embodiments, the endophyte organism comprises a polynucleotide sequence which is between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, at least 99.5% identical or 100% identical to at least one sequence selected from the group that consists of SEQ ID NOs: 67.
[0267] [0267] In some embodiments, the endophyte organism comprises a polynucleotide sequence that is at least 97% identical to at least one selected sequence from the group consisting of SEQ ID NOs: 68. In some embodiments, the endophyte organism comprises a polynucleotide sequence which is between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, at least 99.5% identical or 100% identical to at least one sequence selected from the group that consists of SEQ ID NOs: 68.
[0268] [0268] In some embodiments, the endophyte organism comprises a polynucleotide sequence that is at least 97% identical to at least one selected sequence from the group consisting of SEQ ID NOs: 69. In some embodiments, the endophyte organism comprises a polynucleotide sequence which is between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, at least 99.5% identical or 100% identical to at least one sequence selected from the group that consists of SEQ ID NOs: 69.
[0269] [0269] In some embodiments, the endophyte organism comprises a polynucleotide sequence that is at least 97% identical to at least one selected sequence from the group consisting of SEQ ID NOs: 70. In some embodiments, the endophyte organism comprises a polynucleotide sequence which is between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, at least 99.5% identical or 100% identical to at least one sequence selected from the group that consists of SEQ ID NOs: 70.
[0270] [0270] In some embodiments, the endophyte organism comprises a polynucleotide sequence that is at least 97% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 65 and 66. In some embodiments, the endophyte organism comprises a polynucleotide sequence that is between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, at least 99.5% identical or 100% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 65 and 66. In some embodiments, the endophyte organism comprises at least two polynucleotide sequences that are at least 97% identical to at least two sequences selected from the group consisting of SEQ ID NOs: 65 and 66. In some embodiments, the endophyte organism comprises at least two polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, or at least 99.5 % identical or 1 00% identical to at least two sequences selected from the group consisting of SEQ ID NOs: 65 and 66.
[0271] [0271] In some embodiments, the endophyte organism comprises a polynucleotide sequence that is at least 97% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71. In some embodiments, the endophyte organism comprises a polynucleotide sequence that is between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, at least 99.5% identical or 100% identical to at least one selected sequence within the group consisting of SEQ ID NOs: 63, 64 and 71. In some embodiments, the endophyte organism comprises at least two polynucleotide sequences that are at least 97% identical to at least two sequences selected from the group consisting of SEQ ID NOs: 63.64 and 71. In some embodiments, the endophyte organism comprises at least two polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, or at least 99.5% idê identical or 100% identical to at least two sequences selected from the group consisting of SEQ ID NOs: 63, 64 and 71. In some embodiments, the endophyte organism comprises at least three polynucleotide sequences that are at least 97% identical to at least three sequences selected from the group consisting of SEQ ID NOs: 63, 64 and 71. In some embodiments, the endophyte organism comprises at least three polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98 , 0% identical and 99.5% identical, or at least 99.5% identical or 100% identical to at least three sequences selected from the group consisting of SEQ ID NOs: 63.64 and 71.
[0272] [0272] In some embodiments, the endophyte organism comprises a polynucleotide sequence that is at least 97% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62. In some embodiments, the endophyte organism comprises a polynucleotide sequence that is between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, at least 99.5% identical or 100% identical to at least one selected sequence within the group consisting of SEQ ID NOs: 60, 61 and 62. In some embodiments, the endophyte organism comprises at least two polynucleotide sequences that are at least 97% identical to at least two sequences selected from the group consisting of SEQ ID NOs: 60, 61 and 62. In some embodiments, the endophyte organism comprises at least two polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, or at least 99.5% id emphasis or 100% identical to at least two sequences selected from the group consisting of SEQ ID NOs: 60, 61 and 62. In some embodiments, the endophyte organism comprises at least three polynucleotide sequences that are at least 97% identical to at least three sequences selected from the group consisting of SEQ ID NOs: 60, 61 and 62. In some embodiments, the endophyte organism comprises at least three polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98 , 0% identical and 99.5% identical, or at least 99.5% identical or 100% identical to at least three sequences selected from the group consisting of SEQ ID NOs: 60, 61 and 62.
[0273] [0273] In some embodiments, the endophyte organism comprises a polynucleotide sequence that is at least 97% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and
[0274] [0274] In some embodiments, the endophyte organism comprises a polynucleotide sequence that is at least 97% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, and
[0275] [0275] In some embodiments, the endophyte organism comprises a polynucleotide sequence that is at least 97% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51. In some embodiments, the endophyte organism comprises a polynucleotide sequence that is between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, at least 99.5% identical or 100% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51. In some embodiments, the endophyte organism comprises at least two polynucleotide sequences that are at least 97% identical at least two sequences selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51. In some embodiments, the endophyte organism comprises at least two polynucleotide sequences that are between 97% and 98% at least 98% identical, between 98.0% identical and 99.5% identical, or at least 99.5% identical or 100% identical to at least two sequences selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51. In some modalities , the endophyte organism comprises at least three polynucleotide sequences that are at least 97% identical to at least three sequences selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51. In some embodiments, the endophyte organism comprises at least three polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, or at least 99.5% identical or 100% identical at least three sequences selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51. In some embodiments, the endophyte organism comprises at least four polynucleotide sequences that are at least 97% identical to at least four sequences selected from the group consisting of SEQ ID N Os: 46, 47, 48, 49, 50, and 51. In some embodiments, the endophyte organism comprises at least four polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, or at least 99.5% identical or 100% identical to at least four sequences selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51. In some modalities, the endophyte organism comprises at least five polynucleotide sequences that are at least 97% identical to at least five sequences selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and 51. In some modalities , the endophyte organism comprises at least five polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, or at least 99.5% identical or 100% identical to at least five sequences selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50, and
[0276] [0276] In some embodiments, the endophyte organism comprises a polynucleotide sequence that is at least 97% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 , and 59. In some embodiments, the endophyte organism comprises a polynucleotide sequence that is between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, at least 99.5% identical or 100% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59. In some embodiments, the endophyte organism comprises at least two sequences polynucleotides that are at least 97% identical to at least two sequences selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59. In some embodiments, the endophyte organism comprises at least at least two polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, or at least 99.5% identical or 100% identical to at least two sequences selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56 , 57, 58, and 59. In some embodiments, the endophyte organism comprises at least three polynucleotide sequences that are at least 97% identical to at least three sequences selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59. In some embodiments, the endophyte organism comprises at least three polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5 % identical, or at least 99.5% identical or 100% identical to at least three sequences selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59. In some modalities, the endophyte organism comprises at least four polynucleotide sequences that are at least 97% identical to at least four selected sequences ionized among the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59. In some embodiments, the endophyte organism comprises at least four polynucleotide sequences that are between 97% and 98% identical , at least 98% identical, between 98.0% identical and 99.5% identical, or at least 99.5% identical or 100% identical to at least four sequences selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59. In some embodiments, the endophyte organism comprises at least five polynucleotide sequences that are at least 97% identical to at least five sequences selected from the group consisting of SEQ ID NOs : 52, 53, 54, 55, 56, 57, 58, and 59. In some embodiments, the endophyte organism comprises at least five polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98, 0% identical and 99.5% identical, or at least 99.5% identical or 100% identical to at least five strings selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59. In some embodiments, the endophyte organism comprises at least six polynucleotide sequences that are at least 97% identical to at least at least six sequences selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58, and 59. In some embodiments, the endophyte organism comprises at least six polynucleotide sequences that are between 97% and 98% identical, at least 98% identical, between 98.0% identical and 99.5% identical, or at least 99.5% identical or 100% identical to at least six sequences selected from the group consisting of SEQ ID NOs : 52, 53, 54, 55, 56, 57, 58, and 59. In some embodiments, the endophyte organism comprises at least seven polynucleotide sequences that are at least 97% identical to at least seven sequences selected from the group consisting of the SEQ
[0277] [0277] In some modalities, the endophyte is an Exserohilum rostrata. In some embodiments, the endophyte is a Curvularia spicifera. In some modalities, the endophyte is a protuberant Curvularia. In some embodiments, the endophyte is an Acremonium alternatum. In some embodiments, the endophyte is a Cladosporium oxysporum. In some modalities, the endophyte is a Chaetomium globosum. In some embodiments, the endophyte is an Epicoccum nigrum. In some modalities, the endophyte is a Paecilomyces inflatus. In some modalities, the endophyte is from the Coniochaeta prunicola taxonomy.
[0278] [0278] In some cases, the endophyte organism, or one or more components thereof, is of monoclonal origin, providing high genetic uniformity of the population of endophyte organisms in an agricultural formulation or within a plant element or synthetic combination with the organism endophyte.
[0279] [0279] In some embodiments, the endophyte organism can be grown in a culture medium or it can be adapted to culture in a culture medium.
[0280] [0280] The synthetic compositions provided in this document are preferably stable. The endophyte organism can be stable in storage, where at least 0.01% of CFU are viable after storage in desiccated form (ie, moisture content of 30% or less) for 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10 or more than 10 weeks at 4 or room temperature. Optionally, a long-lasting formulation is in a dry formulation, in a powder formulation or in a lyophilized formulation. In some embodiments, the formulation is formulated to provide stability for the population of endophyte organisms. In one embodiment, the formulation is substantially stable at temperatures between about -20 ° C and about 50 ° C for at least about 1, 2, 3, 4, 5 or 6 days, or 1, 2, 3 or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or one or more years. In another modality,
[0281] [0281] It is contemplated that synthetic methods and compositions can be used to improve a characteristic of agronomic importance to a plant.
[0282] [0282] The methods described in this document can also be used with transgenic plants comprising one or more exogenous transgenes, for example, to produce additional trait benefits conferred by the newly introduced endophytic microbes.
[0283] [0283] For example, the endophyte can provide an improved benefit or tolerance to a plant that is at least 3%, between 3% and 5%, at least 5%, between 5% and 10%, at least 10%, between 10% and 15%, for example, at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40% , between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%, between 150% and 200%, at least 200%, between 200% and 300%, at least 300% or more, when compared to uninoculated plants grown under the same conditions.
[0284] [0284] In one embodiment, it is contemplated that the plant of the present invention is rice (Oryza spp.), In particular O. sativa and O. glaberrima, and members of the main subspecies of O. sativa japonica, javanica and indica. In one embodiment, it is contemplated that the plant of the present invention is the Rex rice variety and its closely related varieties. In some embodiments, the present invention contemplates the use of endophyte organisms that can confer a trace of agronomic benefit on a rice plant element or rice plant to which it is heterologously disposed.
[0285] [0285] In one embodiment, it is contemplated that the plant of the present invention is maize (Zea spp.), In particular Zea mays ssp, such as Zea mays indenata, Zea mays indurata, Zea mays amylacea, Zea mays saccharata and Zea mays everta . In one embodiment, it is contemplated that the plant of the present invention is the Stine 9734 maize variety and closely related varieties. In some embodiments, the present invention contemplates the use of endophyte organisms that can confer a trace of agronomic benefit on a corn plant element or corn plant to which it is heterologously disposed.
[0286] [0286] In one embodiment, it is contemplated that the plant of the present invention is wheat (Triticum spp.), Including the species T. aestivum and T. durum. In one embodiment, it is contemplated that the plant of the present invention is hard winter red (HRW), hard spring red (HRS), hard white (HW), durum, soft white (SW) or soft winter red (SRW). In one embodiment, it is contemplated that the plant of the present invention is the wheat variety SDSU Focus, SDSU Select, and its closely related varieties. In some embodiments, the present invention contemplates the use of endophyte organisms that can confer a trace of agronomic benefit on a wheat plant element or wheat plant to which it is heterologously disposed.
[0287] [0287] In one embodiment, it is contemplated that the plant of the present invention is soy (Glycine max). In one embodiment, it is contemplated that the plant of the present invention is the soybean variety Dairyland DSR1808R2Y, Pfister 38R25, Stine 3920, Stine 33E22 or closely related varieties.
[0288] [0288] In some embodiments, the present invention contemplates the use of endophyte organisms that can confer a trace of agronomic benefit on a soy plant element or soy plant to which it is heterologously disposed.
[0289] [0289] In one embodiment, it is contemplated that the plant of the present invention is peanut (Arachis hypogaea). In one embodiment, it is contemplated that the plant of the present invention is the AT9899, FloRun 107, Georgia-06G, Tamnut OL06 peanut variety or closely related varieties. In some embodiments, the present invention contemplates the use of endophyte organisms that may confer a trace of agronomic benefit on a peanut plant element or peanut plant to which it is heterologously disposed.
[0290] [0290] In one embodiment, it is contemplated that the plant of the present invention is a member of the genus Brassica. In one embodiment, it is contemplated that the plant of the present invention is Brassica napus. In one embodiment, it is contemplated that the plant of the present invention is a cultivar of Brassica napus with low content of erucic acid and low content of glucosinolate. In one embodiment, it is contemplated that the plant of the present invention is canola. In one embodiment, it is contemplated that the plant of the present invention is the Brett Young 5525, NCC1015 canola variety, or closely related varieties. In some embodiments, the present invention contemplates the use of endophyte organisms that can confer a trace of agronomic benefit on a canola plant element or canola plant to which it is heterologously disposed.
[0291] [0291] In some cases, the endophyte organisms described in this document are able to move from one type of tissue to another. For example, the detection and isolation of endophyte organisms of the present invention in mature plant tissues after coating a plant element on the outside demonstrates its ability to move from the plant element to the vegetative tissues of a maturing plant. Therefore, in some modalities, the endophyte population is able to move from outside the plant element to the vegetative tissues of a plant. In some modalities, the endophyte that is coated on the plant element of a plant is capable, after the germination of the plant element in a vegetative state, to be located in a different tissue of the plant. For example, endophytes may be able to locate any of the plant’s tissues, including: the root, adventitious root, seminal root, radicular, bud, leaf, flower, bud (tassel), tassel, meristem, pollen, pistil, ovaries, stamen, fruit, stolon, rhizome, nodule, tuber, trichome, guard cells, hydatode, petal, sepal, gluma, raque, vascular exchange, phloem and xylem. In one embodiment, the endophyte is able to locate itself in the root and / or hairs of the plant. In another modality, the endophyte is able to locate itself in photosynthetic tissues, for example, leaves and shoots of the plant. In other cases, the endophyte is located in the vascular tissues of the plant, for example, in the xylem and phloem. In yet another modality, the endophyte is able to locate itself in the reproductive tissues (flower, pollen, pistil, ovary, stamen, fruit) of the plant. In another modality, the endophyte is able to locate itself in the root, shoots, leaves and reproductive tissues of the plant. In yet another embodiment, the endophyte colonizes a tissue from the plant's fruit or plant element. In yet another modality, the endophyte is able to colonize the plant so that it is present on the surface of the plant (that is, its presence is detectable present outside the plant or in the plant's episphere). In yet other modalities, the endophyte is able to locate substantially in all or all tissues of the plant. In certain embodiments, the endophyte is not located at the root of a plant. In other cases, the endophyte is not located in the photosynthetic tissues of the plant.
[0292] [0292] In some cases, endophytes are able to replicate within the host plant and colonize the plant.
[0293] [0293] The endophyte populations described in this document are capable of colonizing a host plant. Successful colonization can be confirmed by detecting the presence of the endophytic population within the plant. For example, after applying the endophyte to plant elements, high endophyte titers can be detected in the roots and shoots of plants that germinate from plant elements. Detecting the presence of the endophyte within the plant can be performed by measuring the viability of the endophyte after sterilizing the surface of the plant or plant element: colonization of the endophyte results in an internal location of the endophyte, making it resistant to sterilization conditions the surface. The presence and quantity of endophytes can also be established using other means known in the art, for example, immunofluorescence microscopy using antibodies specific to microbes or fluorescence in situ hybridization. Alternatively, specific nucleic acid probes that recognize conserved sequences from an endophyte can be employed to amplify a region, for example by quantitative PCR, and correlated with CFUs through a standard curve.
[0294] [0294] In another embodiment, the endophyte is heterologously disposed, for example, on the surface of a reproductive element of an agricultural plant, in an amount effective to be detectable in the mature agricultural plant. In some modalities, the endophyte is heterologously disposed in an effective amount to be detectable in an amount of at least about 100 UFC, between 100 and 200 UFC, at least about 200 UFC, between 200 and 300 UFC, at least about 300 UFC , between 300 and 400 UFC, at least about 500 UFC, between 500 and
[0295] [0295] In some cases, the endophyte is able to colonize certain plant elements or types of plant tissues. In one embodiment, the endophyte is heterologously disposed in the plant element or changes in an effective amount to be detectable within a target tissue of the mature agricultural plant selected from a fruit, seed, leaf, root or part of it. For example, the endophyte can be detected in an amount of at least about 100 UFC, at least about 200 UFC, at least about 300 UFC, at least about 500 UFC, at least about 1,000 UFC, at least about 3000 UFC, at least about 10,000 UFC, at least about 30,000 UFC, at least about 100,000 UFC or more, in the target tissue of the mature agricultural plant. Endophytic organisms compatible with agrochemicals
[0296] [0296] In certain modalities, the endophyte is selected based on its compatibility with commonly used agrochemicals. As described in this document, plants, particularly agricultural plants, can be treated with a wide range of agrochemicals, including fungicides, biocides (anti-complex agents), herbicides, insecticides, nematicides, rodenticides, bactericides, virucides, fertilizers and other agents.
[0297] [0297] In some modalities, endophytes exhibit tolerance to an agrochemical selected from the group consisting of: Aeris®, Avicta® DuoCot 202, Cruiser®, Syntenta CCB® (A), Clariva®, Albaugh, Dynasty®, Apron®, Maxim®, Gaucho®, Provoke® ST, Syngenta CCB®, Trilex®, WG Purple, WG Silver, Azoxystrobin, Carboxine, Diphenoconazole, Fludioxonil, fluxpyroxade, Ipconazole, Mefenoxam, Metalaxyl, Miclobutanil, Penflufen, Tyraclobobone, Pyraclobobone, , Triadimenol (Baytan®), Trifloxystrobin, Triticonazole, Tolclofos-methyl, PCNB, Abamectina, Chlorpyrifos, Clotianidina, Imidacloprid, Thiametoxam, Thiodicarb.
[0298] [0298] In some cases, it may be important for the endophyte to be compatible with agrochemicals, particularly those with anti-complex properties, in order to persist in the plant, although, as described in this document, there are many of these anti-complex agents that do not penetrate the plant, at least less in a concentration sufficient to interfere with the endophyte. Therefore, when a systemic anti-complex agent is used in the plant, the compatibility of the endophyte to be inoculated with such agents will be an important criterion.
[0299] [0299] In one embodiment, natural isolates of endophytes that are compatible with agrochemicals can be used to inoculate plants according to the methods described in this document. For example, endophytes that are compatible with anti-complex agents used in agriculture can be isolated by placing an endophyte culture in a Petri dish comprising an effective concentration of the anti-complex agent and isolating endophyte colonies compatible with the anti-complex agent. In another embodiment, an endophyte that is compatible with an anti-complex agent is used for the methods described in this document.
[0300] [0300] Endophytes compatible with bactericides can also be isolated by selection in a liquid medium. The culture of endophytes can be plated in Petri dishes without any form of mutagenesis; alternatively, endophytes can be mutagenized using any means known in the art. For example, endophyte cultures may be exposed to UV light, gamma irradiation or chemical mutagenic agents, such as ethyl methanesulfonate (EMS), ethidium bromide (EtBr) dichlorvos (DDVP, methyl methane sulfon (MMS), triethylphosphate (TEP), trimethylphosphate (TMP)) nitrous acid or DNA base analogs, prior to selection in media comprising fungicides. Finally, when the mechanism of action of a specific bactericide is known, the target gene can be mutated specifically (by gene deletion, gene replacement, site-directed mutagenesis, etc.) to generate an endophyte that is resilient against that chemical in private. Note that the methods described above can be used to isolate endophytes that are compatible with both bacteriostatic and bactericidal compounds.
[0301] [0301] It will also be appreciated by one skilled in the art that a plant can be exposed to various types of anti-complex compounds, simultaneously or in succession, for example, at different stages of plant growth. Where the target plant is likely to be exposed to multiple anti-complex agents, an endophyte that is compatible with many or all of these agrochemicals can be used to inoculate the plant. An endophyte that is compatible with several agents can be isolated, for example, by serial selection. An endophyte compatible with the first agent can be isolated as described above (with or without previous mutagenesis). A culture of the resulting endophyte can then be selected for its ability to grow in liquid or solid media comprising the second agent (again, with or without prior mutagenesis). Colonies isolated from the second selection are then tested to confirm their compatibility for both agents.
[0302] [0302] Likewise, endophytes that are compatible with biocides (including herbicides such as glyphosate or anti-complex compounds, whether bacteriostatic or bactericidal) that are employed in agriculture can be isolated using methods similar to those described to isolate compatible endophytes. In some modalities, mutagenesis of the endophyte population can be performed before selection with an anti-complex agent. In another modality, the selection is performed in the population of endophytes without previous mutagenesis. In yet another embodiment, serial selection is carried out on an endophyte: the endophyte is first selected for compatibility with a first anti-complex agent. The isolated compatible endophyte is then cultured and selected for compatibility with the second anti-complex agent. Any colony so isolated is tested for compatibility with each or both of the anti-complex agents to confirm compatibility with these two agents.
[0303] [0303] Compatibility with an antimicrobial agent can be determined by several means known in the art, including comparison of the minimum inhibitory concentration of unmodified and modified endophytes. In some embodiments, the present invention discloses an isolated modified endophyte, in which the endophyte is modified to exhibit at least 3 times more, for example, at least 5 times more, between 5 and 10 times more, at least 10 times more , between 10 and 20 times more, at least 20 times more, between 20 and 30 times more, at least 30 times more or more minimum inhibitory concentration to an antimicrobial agent when compared to the unmodified endophyte.
[0304] [0304] In a specific modality, endophytes with improved compatibility with the herbicide glyphosate are disclosed in this document. In some embodiments, the endophyte has a doubling time in growth medium comprising at least 1 mM glyphosate, for example, between 1 mM and 2 mM glyphosate, at least 2 mM glyphosate, between 2 mM and 5 mM glyphosate at least 5 mM glyphosate, between 5 mM and 10 mM glyphosate, at least 10 mM glyphosate, between 10 mM and 15 mM glyphosate, at least 15 mM glyphosate or more, which is not more than 250% , between 250% and 100%, for example, not more than 200%, between 200% and 175%, not more than 175%, between 175% and 150%, not more than 150%, between 150% and 125%, or no more than 125%, of the endophyte doubling time in the same growth medium that does not comprise any glyphosate. In a particular embodiment, the endophyte has a doubling time in the growth medium comprising 5 mM glyphosate that is not more than 150% of the doubling time of the endophyte in the same growth medium that does not comprise glyphosate.
[0305] [0305] In another embodiment, the endophyte has a doubling time in a plant tissue comprising at least 10 ppm glyphosate, between 10 and 15 ppm, for example, at least 15 ppm glyphosate, between 15 and 10 ppm, at least at least 20 ppm glyphosate, between 20 and 30 ppm, at least 30 ppm glyphosate, between 30 and 40 ppm, at least 40 ppm glyphosate or more, which is not more than 250%, between 250% and 200%, for example, no more than 200%, between 200% and 175%, no more than 175%, between 175% and 150%, no more than 150%, between 150% and 125%, or no more than 125%, of doubling time of the endophyte in a reference plant tissue that does not comprise glyphosate. In a particular embodiment, the endophyte has a doubling time in a plant tissue comprising 40 ppm glyphosate which is not more than 150% of the doubling time of the endophyte in a reference plant tissue that does not comprise any glyphosate.
[0306] [0306] The selection process described above can be repeated to identify endophyte isolates that are compatible with a multiplicity of agents.
[0307] [0307] Candidate isolates can be tested to ensure that selection for compatibility with agrochemicals does not result in loss of the desired bioactivity. Endophyte isolates that are compatible with commonly used agents can be selected as described above. The resulting compatible endophyte can be compared with the parental endophyte in plants in terms of their ability to promote germination.
[0308] [0308] Endophytes compatible with agrochemicals generated as described above can be detected in samples. For example, when a transgene was introduced to make the endophyte compatible with the agrochemical (s), the transgene can be used as a target gene for PCR amplification and detection. In addition, when point mutations or deletions for a portion of a specific gene or several genes result in compatibility with the agrochemical (s), single point mutations can also be detected by PCR or other means known in the art. Such methods allow the detection of the endophyte, even if it is no longer viable. Thus, plant commodity products produced using the endophytes compatible with agrochemical products described in this document can be easily identified using these and related nucleic acid detection methods. Beneficial Attributes of Synthetic Compositions of Plant Elements and Endophyte Organisms
[0309] [0309] The present invention contemplates the establishment of a relationship between a symbiont and a plant element. In some modalities, the association of endophytes results in a detectable change in the plant element or in the entire plant. The detectable change can be an improvement in several agronomic traits (for example, improved general health, increased response to biotic or abiotic stresses or increased properties of the plant or a plant element, including fruits and grains). Alternatively, the detectable change can be a physiological or biological change that can be measured by methods known in the art. Detectable changes are described in more detail in the sections below. As used in this document, an endophyte is considered to have conferred an improved agricultural trait, regardless of whether the improved trait arose from the plant, the endophyte or the combined action between the plant and the endophyte. Therefore, for example, if a beneficial hormone or chemical is produced by the plant or the endophyte, for purposes, the endophyte will be considered to have conferred an improved agricultural trait to the host plant, compared to an isolation plant that has not been heterologously willing to said endophyte.
[0310] [0310] In some modalities, provided in this document, there are methods to produce a plant element from a plant with a heritably altered trait. The trait of the plant can be changed without known genetic modification of the plant's genome, and comprises the following steps. First, a preparation of an isolated endophyte that is heterologously disposed to the plant's plant element is provided and optionally processed to produce an endophyte formulation. The endophyte formulation is then contacted with the plant. The plants are then left to sow and the seeds are collected. Improved general health
[0311] [0311] Also described in this document are plants and plant fields that are heterologously disposed to beneficial endophytes, in such a way that the general fitness, productivity or health of the plant or a portion of it, is maintained, increased and / or improved over a period of time. Improvement in the overall health of the plant can be assessed using various physiological parameters including, but not limited to height, total biomass, root and / or sprout biomass, emergence, seed germination, seedling survival, photosynthetic efficiency, transpiration rate , number of seeds / fruits or mass, grain yield, leaf chlorophyll content, photosynthetic rate, root length or any combination of these. Drought tolerance
[0312] [0312] In some cases, a plant resulting from seeds or other plant elements treated with an endophyte may show a physiological change, as compensation for the stress-induced reduction in photosynthetic activity. Fv / Fm tests whether or not plant stress affects photosystem II in an adapted dark state. Fv / Fm is one of the most used chlorophyll fluorescence measurement parameters. The Fv / Fm test is designed to allow the maximum amount of light energy to follow the fluorescence path. It compares the pre-photosynthetic fluorescent state of dark-adapted leaves, called minimal fluorescence, or Fo, to the maximum fluorescence called Fm. At maximum fluorescence, the maximum number of reaction centers was reduced or closed by a saturating light source. In general, the greater the plant stress, the fewer open reaction centers available and the Fv / Fm ratio is reduced. Fv / Fm is a measurement protocol that works for many types of plant stress. For example, there would be a difference in Fv / Fm after exposure of a treated endophytic plant that had been subjected to heat shock or dry conditions, compared to a corresponding control, a genetically identical plant that does not contain endophytes grown under the same conditions . In some cases, the inoculated plant as disclosed in this document may exhibit an increased change in photosynthetic activity ΔFv (ΔFv / Fm) after treatment by heat shock or water stress, for example 1, 2, 3, 4, 5, 6, 7 days or more after treatment by thermal shock or water stress, or until the cessation of photosynthesis, in comparison with the corresponding control plant of similar stage of development, but which does not include endophytes. For example, a plant with an endophyte capable of imparting heat and / or drought tolerance may exhibit an ΔFv / Fm of about 0.1 to about 0.8 after exposure to thermal or dry shock or an ΔFv / Fm interval of about 0.03 to about 0.8 in one day, or in 1, 2, 3, 4, 5, 6, 7 or more than 7 days after heat shock or dry treatment or until photosynthesis ceases. In some embodiments, the stress-induced reductions in photosynthetic activity can be offset by at least about 0.25% (for example, at least about 0.5%, between 0.5% and 1%, at least about 1%, between 1% and 2%, at least about 2%, between 2% and 3%, at least about 3%, between 3% and 5%, at least about 5%, between 5% and 10 %, at least about 8%, at least about 10%, between 10% and 15%, at least about 15%, between 15% and 20%, at least about 20%, between 20% and 25% at least about 25%, between 25% and 30%, at least about 30%, between 30% and 40%, at least about 40%, between 40% and 50%, at least about 50%, between 50% and 60%, at least about 60%, between 60% and 75%, at least about 75%, between 75% and 80%, at least about 80%, between 80% and 85%, at least about 85%, between 85% and 90%, at least about 90%, between 90% and 95%, at least about 95%, between 95% and 99%, at least about 99% or at least minus 100%) compared to the decrease of photosynthetic activity in a corresponding reference agricultural plant after thermal shock conditions. The significance of the difference between inoculated and reference agricultural plants can be established by demonstrating statistical significance, for example, p <0.05 with an appropriate parametric or nonparametric statistic, for example, chi-square test, Student's t test, Mann-Whitney test or F test based on the assumption or known facts that the inoculated plant and the reference agricultural plant have identical or almost identical genomes (comparison of isolines).
[0313] [0313] In some embodiments, plants comprise endophytes capable of increasing heat and / or drought tolerance in sufficient quantity, such that increased growth or improved wilting recovery is observed under heat or drought stress conditions. For example, an endophyte population described in this document may be present in sufficient quantity in a plant, resulting in increased growth compared to a plant that does not contain endophytes, when grown under drought or thermal shock conditions, or following such conditions. The increase in tolerance to heat and / or drought can be evaluated with physiological parameters, including, among others, height increase, general biomass, biomass of roots and / or shoots, seed germination, seedling survival, photosynthetic efficiency, rate transpiration rate, number of seeds / fruits or mass, grain or fruit yield of plants, leaf chlorophyll content, photosynthetic rate, root length, wilting recovery, turgor pressure or any combination of these, compared to an agricultural plant of reference grown under similar conditions. For example, the endophyte may provide an improved benefit or tolerance to a plant that is at least 3%, between 3% and 5%, at least 5%, between 5% and 10%, at least 10%, between 10% and 15%, for example, at least 15%, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40 % and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 75%, at least 75%, between 75% and 100%, at least 100%, between 100% and 150%, at least 150%, between 150% and 200%, at least 200%, between 200% and 300%, at least 300% or more, when compared to uninoculated plants grown under the same conditions.
[0314] [0314] In various modalities, the endophytes arranged heterologously in the plant can confer several benefits to the plant, including but not limited to: thermal tolerance, herbicide tolerance, drought resistance, insect resistance, fungus resistance, virus resistance, resistance bacteria, male sterility, cold tolerance, salt tolerance, increased yield, greater efficiency in the use of nutrients, increased efficiency in the use of nitrogen, increased protein content, increased fermentable carbohydrate content, reduced lignin content , increased antioxidant content, increased water use efficiency, increased germination, increased biomass, altered root-to-sprout biomass ratio, improved soil water retention or a combination of these. A difference between the inoculated plant (for example, a plant to which one or more endophytes were heterologously disposed) and a reference agricultural plant can also be measured using other methods known in the art. Formulations for Agricultural Use
[0315] [0315] The endophyte populations described in this document are intended to be useful in the improvement of agricultural plants and, as such, can be formulated with other compositions as part of a carrier compatible with agriculture. It is considered that such carriers may include, but are not limited to: seed treatment, root washing, seedling soaking, leaf application, soil inoculum, furrow application, side coverage application, soil pretreatment, inoculation by wound, drip irrigation, vector mediation by means of a pollinator, injection, osmoconditioning, hydroponics, aquaponics, aeroponics. The carrier composition with endophyte populations can be prepared for agricultural application as a liquid, solid or gaseous formulation. Application to the plant can be achieved, for example, as a powder for surface deposition on plant leaves, as a spray for the entire plant or selected plant element, as part of a drip in the soil or roots, or as a coating on the element before planting. Such examples are intended to be illustrative and not to limit the scope of the invention.
[0316] [0316] The formulation useful for these modalities generally and typically includes at least one member selected from the group consisting of a buffer, a "tachifier", a microbial stabilizer, a fungicide, an anti-complex agent, a herbicide, a nematicide, an insecticide, a bactericide, a virucide, a plant growth regulator, a rodenticide, a desiccant and a nutrient.
[0317] [0317] The carrier can be a solid carrier or liquid carrier, and in various forms including microspheres, powders, emulsions and the like. The carrier may be one or more of a number of carriers that impart a variety of properties, such as greater stability, wettability or dispersibility. Wetting agents, such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or a combination thereof, can be included in a composition of the invention. Water-in-oil emulsions can also be used to formulate a composition that includes the purified population (see, for example, US Patent No. 7,485,451). Suitable formulations that can be prepared include wettable powders, granules, gels, agar strips or pellets, thickeners, biopolymers and the like, microencapsulated particles and the like, liquids such as aqueous fluids, aqueous suspensions, water-in-oil emulsions etc. The formulation may include grain or legume products, for example, ground grains or beans, broths or flour derived from grains or beans, starch, sugar or oil.
[0318] [0318] In some modalities, the agricultural carrier can be soil or a plant growth medium. Other agricultural carriers that can be used include water, fertilizers, herbal oils, humectants or combinations of these. Alternatively, the agricultural carrier can be a solid, such as diatomaceous earth, clay, silica, alginate, clay, bentonite, vermiculite, seed pods, other plant and animal products, or combinations, including granules, pellets or suspensions. Mixtures of any of the above mentioned ingredients are also contemplated as carriers, such as, but not limited to pesta (flour and kaolin clay), agar or pellets based on clay, sand or clay flour etc. The formulations can include food sources for cultivated organisms, such as barley, rice, wheat or other biological materials, such as seeds, plant elements, sugarcane bagasse, bark or stalks from grain processing, ground plant material or wood from the waste of a work, sawdust or small paper, fabric or wood recycling fibers. Other suitable formulations will be known to those skilled in the art.
[0319] [0319] In one embodiment, the formulation can include a tachifier or adherent. Such agents are useful for combining the complex population of the invention with carriers that may contain other compounds (e.g., non-biological control agents), to yield a coating composition. Such compositions help to create coatings around the plant or plant element to maintain contact between the endophyte and other agents with the plant or plant element. In some modalities, the adherents are selected from the group consisting of: alginate, gums, starches, lecithins, formononetine, polyvinyl alcohol, alkaline formononetinate, hesperetin, polyvinyl acetate, cephalins, arabic gum, xanthan gum, carrageenan, PGA, other biopolymers , Mineral Oil, Polyethylene Glycol (PEG), Polyvinylpyrrolidone (PVP),
[0320] [0320] It is also contemplated that the formulation may also comprise an anti-caking agent.
[0321] [0321] The formulation can also contain a surfactant, humectant, emulsifier, stabilizer or defoamer. Non-limiting examples of surfactants include nitrogen-surfactant mixtures such as Prefer 28 (Cenex), Surf-N (USA), Inhance (Brandt), P-28 (Wilfarm) and Patrol (Helena); esterified seed oils include Sun-It II (AmCy), MSO (UAP), Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); and organo-silicone surfactants include Silwet L77 (UAP), Silicine (Terra), Dyne-Amic (Helena), Cinica (Helena), Sylgard 309 (Wilbur-Ellis) and Century (Precision), polysorbate 20, polysorbate 80, Tween 20, Tween 80, Scattics, Alktest TW20, Canarcel, Peogabsorb 80, Triton X-100, Conco NI, Dowfax 9N, Igebapl CO, Makon, Neutronyx 600, Nonipol NO, Plytergent B, Renex 600, Solar NO, Sterox, Serfonic N , T-DET-N, Tergitol NP, Triton N, IGEPAL CA-630, Nonident P-40, Pluronic. In some modalities, the surfactant is present in a concentration between 0.01% v / v and 10% v / v .. In another modality, the surfactant is present in a concentration between 0.1% v / v and 1% v / v. An example of an antifoam agent would be Antifoam-C.
[0322] [0322] In certain cases, the formulation includes a microbial stabilizer. Such an agent can include a desiccant. As used herein, a "desiccant" can include any compound or mixture of compounds that can be classified as desiccant, regardless of whether the compound or compounds are used in concentrations that actually have a drying effect on the liquid inoculant. Such desiccants are ideally compatible with the population used and should promote the ability of the endophytic population to survive application to seeds and survive desiccation. Examples of suitable desiccants include one or more of trehalose, sucrose, glycerol and methylene glycol. Other suitable desiccants include, but are not limited to, non-reducing sugars and sugar alcohols (for example, mannitol or sorbitol). The amount of desiccant introduced into the formulation can vary from about 5% to about 50% by weight / volume, for example, between about 10% to about 40%, between about 15% and about 35%, or between about 20% and about 30%. In some embodiments, components of a sugar-based microbial stabilizer include, but are not limited to, glucose, sucrose, polyvinylpyrrolidone K 30 (PVP30K), mineral oil, soy lecithin, peptone, monopotassium phosphate (KH2PO4) and dipotassium phosphate (K2HPO4 ). In an alternative embodiment, the components of a sugar-free microbial stabilizer include, but are not limited to, polyvinylpyrrolidone K 30 (PVP30K), polyvinylpyrrolidone / vinyl acetate (PVP-VA), soy lecithin, peptone, mineral oil, hydroxypropyl guar (HP-Guar), monopotassium phosphate (KH2PO4) and dipotassium phosphate (K2HPO4). The components of exemplary microbial stabilizers for use with the invention described in this document are shown in Table 1 and Table 2. Table 1. Exemplary Sugar-Based Microbial Stabilizer Component Percentage (%), by weight Glucose 11.4 Sucrose 11.4 PVP30K 2.8 Mineral Oil 5.7 Soy lecithin 0.3 Peptone 11.4 KH2PO4 0.78 K2HPO4 0.99 Non-chlorinated water 55 Table 2: Microbial Stabilizer Not Based on Exemplary Sugar Component Percentage (%), by weight PVP30K 3.8 PVP-VA 3.8 Soy lecithin 0.4 Peptone 15.4 Mineral oil 6.0 HP-Guar 0.2 KH2PO4 0.96 K2HP04 1.23 Non-chlorinated water 68
[0323] [0323] In some cases, it is advantageous that the formulation contains agents such as a fungicide, an anti-complex agent, a herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, a bactericide, a virucide or a nutrient . Such agents are ideally compatible with the agricultural plant element or the seedling on which the formulation is applied (for example, it should not be harmful to the growth or health of the plant). In addition, the agent is ideally one that does not cause safety concerns for human, animal or industrial use (for example, without safety problems, or the compost is sufficiently labile for the plant-derived plant product to contain negligible amounts of the compost) .
[0324] [0324] In liquid form, for example, solutions or suspensions, endophytic populations can be mixed or suspended in water or in aqueous solutions. Suitable diluents or liquid carriers include water, aqueous solutions, petroleum distillates or other liquid carriers.
[0325] [0325] Solid compositions can be prepared by dispersing the endophyte populations of the invention in a suitably divided solid carrier, such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, fuller soil, pasteurized soil and the like. When these formulations are used as wettable powders, biologically compatible dispersing agents, such as emulsifying agents and non-ionic, anionic, amphoteric or cationic dispersants, can be used.
[0326] [0326] In some cases, a flowable polymer, also known as a plantability polymer, such as Flo Rite® for example, Flo-Rite® 1706 (BASF, Ludwigshafen, Germany). In some embodiments, a polymer of fluidity or plantability is DISCOTM AG (Incotec, Enkhuizen, Netherlands). In some embodiments, a polymer of fluidity or plantability is Kannar® Universal Wonder (Kannar Earth Science, Ltd., Buford, GA).
[0327] [0327] The solid carriers used in the formulation include, for example, mineral carriers, such as kaolin clay, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acidic white soil, vermiculite and perlite and inorganic salts such as ammonium sulphate, phosphate of ammonium, ammonium nitrate, urea, ammonium chloride and calcium carbonate. In addition, fine organic powders, such as wheat flour, wheat bran and rice bran, can be used. Liquid carriers include vegetable oils such as soybean oil, neem oil, cottonseed oil and other compositions such as glycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, etc.
[0328] [0328] In one embodiment, the formulation is ideally suited for coating a population of endophytes in plant elements. The endophyte populations described in the present invention are capable of imparting many fitness benefits to host plants. The ability to confer such benefits by coating populations on the surface of plant elements has many potential advantages, particularly when used on a commercial (agricultural) scale.
[0329] [0329] The endophyte populations in this document can be combined with one or more of the agents described above to produce a formulation suitable for combination with an agricultural plant element, seedling or other plant element. Endophyte populations can be obtained from growing in culture, for example, using a synthetic growth medium. In addition, endophytes can be grown in a solid medium, for example in Petri dishes, scraped and suspended in preparation. Endophytes at different growth stages can be used. For example, endophytes in delayed phase, initial log phase, half log phase, final log phase, stationary phase, early death phase or death phase can be used. Endophytic spores can be used for the present invention, for example, but not limited to: arthropospores, sporangiospores, conidia, clamadospores, pycnidiospores, endospores, zoospores.
[0330] [0330] Formulations comprising endophyte populations typically contain between about 0.1 and 95% by weight, for example, between about 1% and 90%, between about 3% and 75%, between about 5% and 60%, between about 10% and 50% in wet weight of the population. It is preferred that the formulation contains at least about 10 ^ 3 CFU per ml of formulation, for example, at least about 10 ^ 4, at least about 10 ^ 5, at least about 10 ^ 6, at least about 10 ^ 7 CFU, at least about 10 ^ 8 CFU per ml of formulation. It is preferable that the formulation is applied to the plant element at about 10 ^ 2 UFC / seed, between 10 ^ 2 and 10 ^ 3 UFC, at least about 10 ^ 3 UFC, between 10 ^ 3 and 10 ^ 4 UFC, at least about 10 ^ 4 UFC, between 10 ^ 4 and 10 ^ 5 UFC, at least about 10 ^ 5 UFC, between 10 ^ 5 and 10 ^ 6 UFC, at least about 10 ^ 6 UFC, between 10 ^ 6 and 10 ^ 7
[0331] [0331] In another embodiment, the invention provides a substantially uniform population of synthetic compositions comprising plant elements (PEs), comprising two or more PEs comprising the endophytic population, as described in this document, above. Substantial uniformity can be determined in several ways. In some cases, at least 10%, between 10% and 20%, for example, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40 % and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 90%, at least 90%, between 90% and 95%, at least 95% or more of the PEs in the population, comprise the endophytic population in an amount effective to colonize a plant or plants derived from said PEs when arranged on the surface of the PEs. In other cases, at least 10%, between 10% and 20%, for example, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40 % and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 90%, at least 90%, between 90% and 95%, at least 95% or more of the plant elements of the population, contain at least 1, between 10 and 10, 10, between 10 and 100, or 100 UFC on the surface of the plant element or per gram of plant element, for example, between 100 and 200 UFC, at least 200 UFC, between 200 and 300 UFC, at least 300 UFC, between 300 and 1,000 UFC, at least 1,000 UFC, between 1,000 and 3,000 UFC, at least 3,000 UFC, between 3,000 and 10,000 UFC, at least 10,000 UFC, between 10,000 and 30,000 UFC, at least
[0332] [0332] In a particular embodiment, the population of the plant elements is packed in a bag or container suitable for commercial sale. This bag contains a unit weight or count of plant elements comprising the endophytic population as described in this document and further comprising a label. In one embodiment, the bag or container contains at least 100 plant elements, between 100 and 1,000 plant elements, 1,000 plant elements, between 1,000 and 5,000 plant elements, for example, at least 5,000 plant elements, between 5,000 and 10,000 plant elements, at least 10,000 plant elements, between 10,000 and 20,000 plant elements, at least 20,000 plant elements, between 20,000 and
[0333] [0333] In some cases, a subpopulation of seeds comprising the endophytic population is still selected based on the increase in uniformity, for example, based on the uniformity of the microbial population. For example, elements of individual plants from groups collected from individual ears, individual plants, individual plots of land (representing plants inoculated on the same day) or individual fields can be tested for uniformity of microbial density, and only those groups that meet specifications
[0334] [0334] The methods described in this document may also comprise a validation step. The validation step may involve, for example, the growth of some plant elements collected from the inoculated plants on mature agricultural plants, and testing these individual plants for uniformity. This validation step can be performed on individual seeds collected from ears, individual plants, individual plots of land (representing plants inoculated on the same day) or individual fields, and tested as described above to identify groups that meet the required specifications.
[0335] [0335] In some embodiments, the methods described in this document include planting a synthetic composition described in this document. Suitable planters include a fertilizer and / or air seeder used in agricultural operations to apply particulate materials including one or more of the following, seeds, fertilizers and / or inoculants to the soil during the planting operation. Fertilizer / seeder devices may include a tool bar that has openers for engaging the soil, behind which a wheeled cart is towed that includes one or more containment tanks or associated containers and measuring means, respectively, to contain and measure , from them, particulate materials.
[0336] [0336] In certain embodiments, a composition described in this document can be in the form of a liquid, a suspension, a solid or a powder (wettable powder or dry powder). In another embodiment, a composition can be in the form of a seed coating. Compositions in liquid, suspension or powder form (e.g. wettable powder) may be suitable for coating plant elements. When used to coat plant elements, the composition can be applied to the plant elements and left to dry. In embodiments where the composition is a powder (for example, a wettable powder), it may be necessary to add a liquid, such as water, to the powder, prior to application to a seed.
[0337] [0337] In yet another embodiment, the methods may include introducing into the soil an inoculum from one or more of the endophyte populations described in this document. Such methods may include introducing into the soil one or more of the compositions described in this document. The inoculum (s) or compositions can be introduced into the soil according to methods known to those skilled in the art. Non-limiting examples include furrow introduction, spraying, seed coating, foliar introduction, etc. In a specific embodiment, the introduction step comprises the introduction of the inoculum or compositions described in this document.
[0338] [0338] In one embodiment, plant elements can be treated with the composition (s) described in this document in several ways, but preferably by spraying or dripping. Spray and drip treatment can be conducted by formulating compositions described in this document and spraying or dripping the composition (s) onto (one) seed (s) using a continuous treatment system (which is calibrated to apply treatment to a predefined rate in proportion to the continuous seed flow), such as a type of attendant drum. Group systems, in which a predetermined lot size of seeds and composition (s), as described in this document, are delivered in a mixer, can also be employed.
[0339] [0339] In another modality, the treatment involves the coating of plant elements. One of these processes involves coating the inside wall of a round container with the composition (s) described in this document, adding plant elements and then rotating the container to make the plant elements come into contact with the wall and the composition (s), a process known in the art as "container coating". Plant elements can be coated by combinations of coating methods. Immersion typically involves the use of liquid forms of the described compositions. For example, plant elements can be soaked for about 1 minute to about 24 hours (for example, for at least 1 min, between 1 and 5 min, 5 min, between 5 and 10 min, 10 min, between 10 and 20 min, 20 min, between 20 and 40 min, 40 min, between 40 and 80 min, 80 min, between 80 min and 3 h, 3 h, between 3 am and 6 h, 6 h, between 6 am and 12 h, 12 h, between 12 h and 24 h, 24 h). Plant Population / Agricultural Fields
[0340] [0340] One of the main focuses of the efforts to improve crops has been to select varieties with traits that provide, in addition to greater return, greater homogeneity and uniformity. If on the one hand inbreeding can produce plants with substantial genetic identity, heterogeneity in relation to plant height, flowering time and seed time, there are impediments that remain to obtain a homogeneous field of plants. The inevitable plant-to-plant variability is caused by a number of factors, including environmental conditions and irregular management practices. Another possible source of variability may, in some cases, be due to the heterogeneity of the endophytic population that inhabits the plants. By providing endophytic populations to the plant's reproductive elements, the resulting plants generated by the germination of the plant's reproductive elements have a more consistent collection of endophytes and, therefore, are expected to produce a more uniform plant population.
[0341] [0341] Therefore, in another embodiment, the invention provides a substantially uniform population of plants. The population can include at least 5 plants, between 5 and 10 plants, at least 10 plants, between 10 and 100 plants, for example, at least 100 plants, between 100 and 300 plants, at least 300 plants, between 300 and 1,000 plants at least 1,000 plants, between 1,000 and
[0342] [0342] The uniformity of plants can be measured in several different ways. In some modalities, there is an increased uniformity in relation to the endophytes within the plant population. For example, in some embodiments, a substantial portion of the plant population, for example at least 10%, between 10% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 70%, at least 70%, between 70% and 75% at least 75%, between 75% and 80%, at least 80%, between 80% and 90%, at least 90%, between 90% and 95%, at least 95% or more of the elements or plants in a population contain a limit number of a population of endophytes. The limit number can be at least 10 UFC, between 10 and 100 UFC, at least 100 UFC, between 100 and 300 UFC, for example, at least 300 UFC, between 300 and 1,000 UFC, at least 1,000 UFC, between 1,000 and 3,000 UFC, at least 3,000 UFC, between 3,000 and 10,000 UFC, at least 10,000 UFC, between 10,000 and 30,000 UFC, at least 30,000 UFC, between 30,000 and 100,000 UFC, at least 100,000 UFC or more, on the plant or part of the plant . Alternatively, in a substantial portion of the plant population, for example, I push at least 1%, between 1% and 10%, at least 10%, between 10% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 90%, at least 90%, between 90% and 95%, at least 95% or more of the plants in the population, the endophyte population supplied to the seed or seedling represents at least 0.1%, between 0.1% and 1% at least 1%, between 1% and 5%, at least 5%, between 5% and 10%, at least 10%, between 10% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40 % and 50%, at least 50%, between 50% and 60%, at least 60%, between 60% and 70%, at least 70%, between 70% and 80%, at least 80%, between 80% and 90%, at least 90%, between 90% and 95%, at least 95%, between 95% and 99%, at least 99%, between 99% and 100%, or 100% of the total endophyte population in the plant / seed.
[0343] [0343] In one embodiment, there is increased genetic uniformity of a substantial proportion or of all detectable endophytes within the rate, genus or species of a component relative to an uninoculated control. This increased uniformity may be a result of the endophyte being of monoclonal origin or derived from a population that comprises a more uniform sequence of the genome and plasmid repertoire than that which would be present in the population of endophytes a plant that derives its endophyte community mainly through assimilation of various soil symbionts.
[0344] [0344] In another modality, there is an increased uniformity in relation to a physiological parameter of plants within the population. In some cases, there may be an increased uniformity of plant height when compared to a population of reference agricultural plants grown under the same conditions. For example, there may be a reduction in standard deviation in plant height in the population of at least 5%, between 5% and 10%, for example, at least 10%, between 10% and 15%, minus 15%, between 15 % and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50%, between 50% and 60%, at least 60% or more, when compared to a population of reference agricultural plants grown under the same conditions. In other cases, there may be a reduction in the standard deviation in the flowering time of the plants in the population of at least 5%, between 5% and 10%, for example, at least 10%, between 10% and 15%, at least 15 %, between 15% and 20%, at least 20%, between 20% and 30%, at least 30%, between 30% and 40%, at least 40%, between 40% and 50%, at least 50% , between 50% and 60%, at least 60% or more, when compared to a population of reference agricultural plants grown under the same conditions. Vegetable Commodity Products
[0345] [0345] The present invention provides a plant commodity product, as well as methods for producing a plant commodity product, which is derived from a plant. As used herein, a "plant commodity product" refers to any composition or product that is made up of material derived from a plant, seed, plant cell or plant element of the present invention. Commodity plant products can be sold to consumers and can be viable or non-viable. Non-viable commodity products include, but are not limited to, plant elements and unviable grains; processed seeds, seed parts and plant elements; dehydrated vegetable tissue, frozen vegetable tissue and processed vegetable tissue; seeds and plant elements processed for animal feed for terrestrial and / or aquatic consumption of animals, oil, bran, flour, flakes, bran, fibers, paper, tea, coffee, silage, crushed whole grains and any other food for human use or animal consumption, such as fruit or another edible portion of the plant; and biomass and fuels; and raw material in the industry.
[0346] [0346] The industrial uses of oils derived from agricultural plants described in this document include ingredients for paints, plastics, fibers, detergents, cosmetics, lubricants and biodiesel fuel. Vegetable oils can be separated, interesterified, sulfurized, epoxidized,
[0347] [0347] As described in this document, populations of purified endophytes and synthetic compositions comprising them (for example, formulations) can be used to confer beneficial traits to the host plant.
[0348] [0348] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. Each patent application, magazine article, citation and other references are incorporated into this document by reference in its entirety, as if each had been incorporated by reference individually.
[0349] [0349] The isolation and cultivation of endophytic microbes from agricultural plants was carried out using methods well known in the art. DNA was extracted from the milled tissues using the DNeasy DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The endophytes were characterized by the sequences of genomic regions, these sequences are listed in Table 4. The primers that amplify genomic regions of the endophytes of the present invention are listed in Table 3. Table 3. Primer sequences useful in the identification of microbes of the present invention Primers Genomic locus 27f (5'- AGAGTTTGATYMTGGCTCAG- 3 ') (SEQ ID NO: 16S
[0350] [0350] The fungal endophytic organisms of the present invention can be identified by the sequence of one or more of the following sites: long subunit rRNA (LSU), small subunit rRNA (SSU), largest subunit of RNA polymerase II (RPB1), second larger subunit of RNA polymerase II (RPB2), beta-tubulin, actin, phosphoglycerate kinase (PGK). PCR amplification of the largest RNA polymerase I (RPB1) subunit using primer sequences RPB1-Af (SEQ ID NO: 21) and RPB1-Cr (SEQ ID NO: 22) is described in Cendejas-Bueno E, Kolecka A , Alastruey- Izquierdo A, et al. Reclassification of the Candida haemulonii Complex as Candida haemulonii (C. haemulonii Group I), C. duobushaemulonii sp. nov. (C. haemulonii Group II) and C. haemulonii var. vulnera var. nov.: Three multiresistant human pathogenic yeasts. Journal of Clinical Microbiology. 2012; 50 (11): 3641-3651. PCR amplification of the second largest RNA polymerase II (RPB2) subunit using the primer sequences fRPB2-5F (SEQ ID NO: 26) and fRPB2-7.1R (SEQ ID NO: 25) is described in Riess K, Oberwinkler F , Bauer R, Garnica S. High genetic diversity at the regional scale and possible speciation in Sebacina epigaea and S. incrustans. BMC Evolutionary Biology. 2013; 13: 102. doi: 10.1186 / 1471-2148-13-102. PCR amplification of beta-tubulin using Btub2Fd (SEQ ID NO: 30) and Btub4Rd (SEQ ID NO: 31) primer sequences is described in Aveskamp et al. (2009) DNA phylogeny reveals polyphyly of Phoma section Peyronellaea and multiple taxonomic novelties Mycologia, 101 (3): 363–382. PCR amplification of LSU using primer sequences LR0R (SEQ ID NO: 23) and LR5 (SEQ ID NO: 24) is described in Stielow JB, Lévesque CA, Seifert KA, et al. One fungus, which genes Development and assessment of universal primers for potential secondary fungal DNA barcodes. Persoonia: Molecular Phylogeny and Evolution of Fungi. 2015; 35: 242-263. doi: 10.3767 / 003158515X689135. PCR amplification of SSU using primer sequences SR1R (SEQ ID NO: 29) and NS4 (SEQ ID NO: 28) is described in Zhu et al. (2016) Helminthosporium velutinum and H. aquaticum sp. nov. from aquatic habitats in Yunnan Province, China. Phytotaxa 253 (3): 179–190. PCR amplification of Actin using primer sequences ACT512f (SEQ ID NO: 17) and ACT783r (SEQ ID NO: 18) is described in Carbone, I.
[0351] [0351] MIC-68390 can be identified by one or more of the following exemplary sequences: RPB2 sequence (SEQ ID NO: 61). MIC-68178 can be identified by one or more of the following exemplary sequences: beta-tubulin sequence (SEQ ID NO: 64). MIC-07010 can be identified by one or more of the following exemplary sequences: phosphoglycerate kinase sequence (SEQ ID NO: 66). MIC-96038 can be identified by one or more of the following exemplary sequences: actin sequence (SEQ ID NO: 47), beta-tubulin sequence (SEQ ID NO: 48), RPB2 sequence (SEQ ID NO: 49), sequence RPB1 (SEQ ID NO: 50). MIC-33414 can be identified by one or more of the following exemplary sequences: its actin sequence (SEQ ID NO: 53), RPB1 sequence (SEQ ID NO: 55), beta-tubulin sequence (SEQ ID NO: 58), LSU sequence (SEQ ID NO: 54), SSU sequence (SEQ ID NO: 56), SSU sequence (SEQ ID NO: 57). MIC-31593 can be identified by one or more of the following exemplary sequences: its RPB2 sequence (SEQ ID NO: 43), beta-tubulin sequence (SEQ ID NO: 44). Exemplary LSU and SSU sequences from MIC-19994 are SEQ ID NO: 39 and SEQ ID NO: 40, respectively. Example 3: Isolation and identification of bacterial and fungal endophytes using the ITS sequence
[0352] [0352] The classification of the fungal strain using ITS sequences was made by the following methodology.
[0353] [0353] Total genomic DNA was extracted from individual fungi isolates using the DNeasy Plant Mini Kit (Qiagen, Germantown, MD). Polymerase Chain Reaction (PCR) was used to amplify a genomic region that includes the nuclear ribosomal internal transcript spacers (ITS) using a pair of ITS_1 (5'-CTTGGTCATTTAGAGGAAGTAA-3 ') primers (SEQ ID NO: 5) and LR5 (5'-TCCTGAGGGAAACTTCG-3 ') (SEQ ID NO: 8). Each reaction mixture of
[0354] [0354] Taxonomic classifications were assigned to strings using the highest probability of attribution based on the results of industry standard taxonomic classification tools: LCA (performs USEARCH (Edgar, RC (2010) Bioinformtics. 26 (19): 2460-2461 ) with the search_global option and then, for all the best match results, returns the lowest taxonomic classification shared by all the best results of a query), SPINGO (Allard et al. (2015) BMC Bioinformtics. 16: 324 ), and UTAX (Edgar, RC, 2016), using the trainset 1 WARCUP Fungal ITS (Deshpande et al. (2016) Mycologia 108 (1): 1-5) and UNITE (Koljalg et al. (2013) Molecular Ecology, 22: 5271-5277). The combinations of classifier and database listed in Table 5 were used to assign taxonomy to fungal sequences. Table 5: The combinations of classifier and database used to classify ITS strings Classifier LCA UNITE database, Fungal ITS trainset 07/04/2014 RDP UNITE, Fungal ITS trainset 07/04/2014 WARCUP, Fungal ITS trainset 1
[0355] [0355] MIC-68390 can be identified by the sequence of its ITS sequence (SEQ ID NO: 60). MIC-68178 can be identified by the sequence of its ITS sequence (SEQ ID NO: 63). MIC-07010 can be identified by the sequence of its ITS sequence (SEQ ID NO: 65). MIC-31593 can be identified by the sequence of its ITS sequence (SEQ ID NO: 42). MIC-48747 can be identified by the sequence of its ITS sequence (SEQ ID NO: 67). MIC-96038 can be identified by the sequence of its ITS sequence (SEQ ID NO: 46). MIC-50414 can be identified by the sequence of its ITS sequence (SEQ ID NO: 68). MIC-33414 can be identified by the sequence of its ITS sequence (SEQ ID NO: 52). MIC-85555 can be identified by the sequence of its ITS sequence (SEQ ID NO: 70). MIC-50989 can be identified by the sequence of its ITS sequence (SEQ ID NO: 69). Example 4. Evaluation of Improved Plant Characteristics, Seedling Vigor
[0356] [0356] Seed preparation: The quality of the soybean seed lot was evaluated by testing the germination of 100 seeds. The seeds were placed, 8 seeds per Petri dish, on filter paper in Petri dishes, 12 ml of water were added to each dish and the dishes incubated for 3 days at 24C. The germination percentage was greater than 95%. A thousand soybean seeds were then sterilized on the surface by co-incubation with chlorine gas in a 20 x 30 cm container placed in a chemical extractor for 16 hours. The germination percentage of 50 seeds, per sterilization batch, was tested as above and confirmed as greater than 95%.
[0357] [0357] Preparation and heterologous disposition of endophytes: The spore solutions were made by rinsing and scraping the spores of the inclined agar that have been growing for about 1 month. The washing was done with 0.05% Silwet. Solutions were passed through Miracloth to filter mycelia. Spores per ml were counted under a microscope using a hemocytometer. The stock suspension was then diluted to 10 ^ 6 spores / ml using water. 3 of spore suspensions per soybean seed were used (~ 10 ^ 3 CFU / seed are obtained). Control treatments were prepared by adding equivalent volumes of sterile water to the seeds.
[0358] [0358] Seedling vigor test: Two rolled pieces of germination paper were placed in a sterile glass jar with 50 mL of sterile water and then removed when completely saturated. Then the papers were separated and the inoculated seeds were placed at intervals of approximately 1 cm along the length of a sheet of moist germinated paper, at least 2.5 cm from the top of the paper and 3.8 cm from the margin of the paper. The second sheet was placed on top of the soybean seeds and the paper and layered seeds were rolled without precision in a tube shape. Each tube was secured with a rubber band around its middle and placed in a single sterile glass bottle and covered with a lid. For each treatment, three bottles with 15 seeds per bottle were prepared. The position of the vials with the growth chamber was randomized. The flasks were incubated at 60% relative humidity and 22 ° C day, 18 ° C at night with 12 hours of light and 12 hours of darkness for 4 days, then the lids were removed and the flasks were incubated for another 7 days. Then, the germinated soybean seedlings were weighed and photographed and the root length and root surface area were scored as follows.
[0359] [0359] Dirt, excess water, seeds and other debris have been removed from the seedlings to allow an accurate sweeping of the roots. The individual seedlings were arranged in transparent plastic trays and the trays were arranged on an Epson Expression 11000XL scanner (Epson America, Inc., Long Beach CA). The roots were arranged manually to reduce the amount of overlap. For root measurements, shoots were removed if the shape of the bud caused it to overlap the roots.
[0360] [0360] The WinRHIZO Arabidopsis Pro2016a software version (Regents Instruments, Quebec, Canada) was used with the following acquisition settings: 4000 dpi grayscale image, "speed priority", "overlapping" ( overlap) (1 object), Root Morphology ("Root Morphology"): Precision ("Precision") (default), Crossing Detection ("Crossover Detection") (normal). The scanning area was defined as the maximum area of the scanner. When scanning was completed, the root area was selected and the root length and root surface area were measured.
[0361] [0361] Statistical analysis was performed using R (R Core Team, 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. R-project.org/). The results are summarized in Tables 7 and 8. Table 7. Root traits of soybean seedlings treated and not treated with endophytes. Area%% Area Length Deviation Deviation Length of the root Standard, Standard root, that of the root of Medium length Medium Medium difference area MIC ID from root to root NT NT Control and untreated (NT) 79.4 32 , 5 8.9 4.1 0.0 0.0 MIC- 68178 81.2 34.6 9.8 3.4 2.2 9.6 MIC- 07010 94.1 29.5 10.5 3.2 18.5 17.7
[0362] [0362] Seed preparation: The quality of the corn seed lot is evaluated first for germination by transferring 100 seeds and with 3.5 mL of water to a Petri dish coated with filter paper. The seeds are incubated for 3 days at 24C and to ensure that the germination percentage is greater than 95%. A thousand corn seeds are then sterilized on the surface by co-incubation with chlorine gas in a 20 x 30 cm container in a chemical extractor for 12 hours. The germination percentage of 50 seeds, per sterilization batch, is tested as above and confirmed as greater than 95%.
[0363] [0363] Optional reagent preparation: 7.5% PEG 6000 (Calbiochem, San Diego, CA) is prepared by adding 75 g of PEG to 1000 mL of water and then stirred on a hot plate until PEG be completely dissolved. The solution is then autoclaved.
[0364] [0364] Preparation and heterologous disposition of endophytes: The spore solutions are made by rinsing and scraping the spores of the inclined agar that have been growing for about 1 month. Washing is done with 0.05% Silwet. Solutions are passed through Miracloth to filter mycelia. Spores per ml are counted under a microscope using a hemocytometer. The stock suspension is then diluted to 10 ^ 6 spores / ml using water. 3 of spore suspensions are used per corn seed (~ 10 ^ 3 CFU / seed are obtained). Control treatments are prepared by adding equivalent volumes of sterile water to the seeds.
[0365] [0365] Seedling vigor test: 25 ml of sterile water (or, optionally, 25 ml of PEG solution, as prepared above) are added to each CygTM germination bag (Mega International, Newport, MN) and placed in the holder of scholarships (Mega International, Newport, MN). Sterile forceps are used to place corn seeds prepared as above in all other perforations in the germination pouch. The seeds are fitted with each perforation to ensure that they do not change when moving the bags. Before and between treatments, the forceps are sterilized using alcohol and flame and the workspace is removed with 70% ethanol. For each treatment, three bags with 15 seeds per bag are prepared. The germination supports with germination bags are placed in plastic vats and covered with perforated plastic to prevent drying. The vats are incubated at 60% relative humidity and at 22 ° C a day, 18 ° C at night with 12 hours of light and 12 hours of darkness for 6 days to allow germination and root length growth. The placement of bags inside shelves and shelves / vats inside the growth chamber is random to minimize the positional effect. At the end of 6 days, the corn seeds are scored manually for germination, sprout length and root.
[0366] [0366] Statistical analysis is performed using R (R Core Team, 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. R-project.org/). Wheat seedling vigor test
[0367] [0367] Seed preparation: The batch of wheat seeds was first evaluated for germination by transferring 100 seeds and with 8 mL of water to a Petri dish coated with filter paper. The seeds were incubated for 3 days at 24C and the germination percentage was greater than 95%. The wheat seeds were then sterilized on the surface by co-incubation with chlorine gas in a 20 x 30 cm container in a chemical extractor for 12 hours. The germination percentage of 50 seeds, per sterilization batch, was tested as above and confirmed as greater than 95%.
[0368] [0368] Reagent preparation: 7.5% polyethylene glycol (PEG) was prepared by adding 75 g of PEG to 1000 ml of water and then stirring them on a hot plate until the PEG is completely dissolved. The solution was then autoclaved.
[0369] [0369] Preparation and heterologous disposition of endophytes: The spore solutions were made by rinsing and scraping the spores of the inclined agar that had been growing for about 1 month. The washing was done with 0.05% Silwet. Solutions were passed through Miracloth to filter mycelia. Spores per ml were counted under a microscope using a hemocytometer. The stock suspension was then diluted to 10 ^ 6 spores / ml using water. 3 l of spore suspensions were used per wheat seed (~ 10 ^ 3 CFU / seed were obtained). Seeds and spores were combined with a 50 mL falcon tube and shaken gently for 5 to 10 seconds until completely coated. Control treatments were prepared by adding equivalent volumes of sterile water to the seeds.
[0370] [0370] Seedling vigor test: Petri dishes were prepared by adding four sheets of heavy sterile seed germination paper, then adding 50 mL of PEG solution as prepared above to each plate, allowing the liquid completely immerse in all the leaves. The sheets were positioned and then folded so that the back of the board and a side wall were covered, two sheets were then removed and placed on a sterile surface. Along the edge of the plate in front of the covered side wall, 15 inoculated wheat seeds were placed evenly at least an inch from the top of the plate and half an inch from the sides. The seeds were placed with the smooth side up and with the tip pointed at the side wall of the plate covered with germination paper. The seeds were covered by the two reserved leaves, and the moist layers of paper were smoothed to remove air bubbles and trap the seeds, and the cover was replaced. For each treatment, at least three plates with 15 seeds per plate were prepared. The plates were then randomly distributed in piles of 8 to 12 plates and a seedless plate was placed on top. The cells were incubated at 60% relative humidity and 22 ° C day, 18 ° C night with 12 hours of light and 12 hours of darkness for 24 hours, then each plate was turned to a semi-vertical position with the side wall covered with paper in the background. The plates were incubated for another 5 days, then the wheat seeds were manually marked for germination, sprout and root length.
[0371] [0371] Statistical analysis was performed using R (R Core Team, 2016. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. R-project.org/). The results are summarized in
[0372] [0372] Strains can be grown by the methods described herein and methods well known in the art. Preparation of fungal biomass Method 1
[0373] [0373] The biomass for MIC-19994 and MIC-96038 was produced growing for two weeks in liquid medium (PDB). The resulting biomass was homogenized by sonication (50% amplitude for 30 seconds) or in a FastPrep-24 (MP Biomedicals, Santa Ana, CA, USA) set at 4.5 m / s for 30 seconds. Method 2 The spore solutions were made by washing and scraping the spores from the inclined agar, which have grown for about 1 month. The washing was done with 0.05% Silwet. Solutions were passed through Miracloth to filter mycelia. Spores per ml were counted under a microscope using a hemocytometer. The stock suspension was then diluted to 10 ^ 6 spores / ml using water. 3 of spore suspensions per seed were used (~ 10 ^ 3 CFU / seed are obtained). Control treatments were prepared by adding equivalent volumes of sterile water to the seeds. Method 3
[0374] [0374] Preparation: The molasses broth was prepared by dissolving 30 g of molasses and 5 g of yeast extract per liter of deionized water in an autoclavable and autoclavable container (15 psi, 121 ° C) for 45 minutes. The dextrose agar (PDA) plates were prepared by dissolving 39.0 g powdered PDA per liter of deionized water in an autoclavable and autoclavable container (15 psi, 121 ° C) for 45 min. The agar was allowed to cool to 50-60 ° C, before pouring them into sterile Petri dishes (30 ml per 90 mm plate).
[0375] [0375] Liquid biomass: All equipment and consumables have been completely sterilized and the procedures performed in a biosafety cabinet. The inoculant is prepared by placing 1 buffer of cryopreserved material on a fresh PDA plate, sealing the plate with Parafilm® and incubating it at room temperature in the dark for 5 to 10 days. Then, the ~ 5x5 mm plugs were cut from the PDA plates and the 10-12 plugs were transferred to vials containing the sterile molasses broth, then covered, fixed on a shaker and incubated for at least 10 days with shaking at ~ 130 rpm. Then, the culture was placed in a blender for 5 seconds and 1 mL of the mixture was centrifuged and the supernatant was discarded and the pellet resuspended in 0.5 mL of phosphate buffered saline (PBS) 1x to generate inoculum.
[0376] [0376] Dry biomass: All equipment and consumables were completely sterilized and the procedures performed in a biosafety cabinet. The inoculant is prepared by placing 1 buffer of cryopreserved material on a fresh PDA plate, sealing the plate with Parafilm® and incubating it at room temperature in the dark for 5 to 10 days. Then, the ~ 5x5 mm plugs were cut from the PDA plates and the 10-12 plugs were transferred to vials containing the sterile molasses broth, then covered, fixed on a shaker and incubated for at least 10 days with shaking at ~ 130 rpm. Under sterile conditions, the liquid culture was carefully decanted using sterile 150 mm filter paper in a sterile Buchner funnel over a sterile flask. After all the liquid had passed through the funnel, the sediment was washed with sterile water until the filtrate was clean. When dry, the sediment was transferred to a drying cabinet and dried until it became brittle. The sediment was then ground into fine powder and the sample used to generate CFU counts. Seed inoculation
[0377] [0377] Unless otherwise specified, the inoculum was added to the seeds to achieve a targeted dose of 10 ^ 4 CFU. Where low, medium and high doses are indicated, and otherwise unspecified, a high dose is 10 ^ 5 CFU / seed, a medium dose is 10 ^ 4 CFU / seed and a low dose 10 ^ 3 CFU / seed. The seeds were agitated to disperse the inoculum evenly in the seeds. Formulation control treatments were prepared using equivalent volumes of PBS. The seeds were allowed to dry for approximately 2 minutes, then 2 fluid ounces were added per kg of Flo-Rite® 1706 seed (BASF, Ludwigshafen, Germany) and the seeds were agitated to disperse the powder evenly in the seeds. Example 6. Evaluation of Improved Plant Characteristics: Greenhouse Rice
[0378] [0378] Rice seeds of the Rex variety have been treated with fungicidal treatment and commercial insecticide CruiserMaxx® Rice (Syngenta, Basel, Switzerland). The seeds were arranged heterologously with the endophyte formulations and the formulation control (without any endophyte), as described in Example 28, untreated seeds (lack of formulation and endophyte) were also planted. The loaded dose of each microbe is shown in Table 42. The MIC-68178 / MIC-33414 endophytic treatment was co-inoculated with MIC-68178 and MIC-33414. In the co-inoculation treatment, MIC-68178 represented 17.45% of the loaded dose, and MIC-33414 represented 82.55% of the loaded dose. Table 10. Loaded doses (CFU per seed) of soybean seeds treated with endophytes.
[0379] [0379] Each pot was filled with Cahaba / Wickham type, fine sandy soil and sand and two seeds were sown evenly spaced in each pot. Ten pots were planted per treatment / control. The experimental design required a completely random pattern of each treatment within each block / replica. The plants were watered twice a day. After the appearance of true leaves, the plants were fertilized weekly at 250 PPM N using Peter's Peat-Lite 20-1020 water-soluble fertilizer.
[0380] [0380] On day 7, the pots were diluted to 1 seedling / pot. The root tissue was harvested from the experiment six weeks after planting and the soil was washed from the roots. The tissues of each plant were placed in an unlined paper bag. The fabric was dried in an oven at 85 ° C for 3 days. After being completely dry, the root biomass of individual plants was weighed and recorded.
[0381] [0381] MIC-68178 demonstrated a 51.5% increase in dry root weight compared to untreated control, with more than 80% confidence by Bayesian analysis. Table 11. Percentage difference in dry root weight in the Rex rice variety under greenhouse conditions Variety of Rex rice% difference Untreated control, dry root weight Untreated control 0 Formulation control 52.0 MIC-68178 51.5 Soy
[0382] [0382] The sandy clay was mixed in the proportion of 60% and 40% of mortar. Before mixing, both planting media were sieved through a 3/8 ”square steel mesh to remove larger particles and debris. Soya seeds of the Stine 33E22 variety were treated with fungicide treatment and commercial insecticide CruiserMaxx® Soy (Syngenta, Basel, Switzerland). The seeds were arranged heterologously with the endophyte formulations and the formulation control (without any endophyte), as described in Example 28, untreated seeds (lack of formulation and endophyte) were also planted.
[0383] [0383] Each pot was filled with 1000 ml of soil, watered with 225 ml of water and one seed sown per pot. Ten pots were planted per treatment / control. The experimental design required a completely random pattern of each treatment within each block / replica. The environmental conditions were defined in the 12h photoperiod, at a temperature of 23/20 ° C during the day / night and the light intensity was defined at 550 μMol m -2 s-1. After planting, the seeds were watered to maintain approximately 75% of the soil's capacity.
[0384] [0384] The root tissue was harvested from the experiment three weeks after planting and the soil was washed from the roots. The tissues of each plant were placed in an unlined paper bag. The fabric was dried in an oven at 85 ° C for 3 days. After being completely dry, the root biomass of individual plants was weighed and recorded.
[0385] [0385] The roots treated with the endophyte organism MIC-68178 demonstrated an increase of 16.9% in dry root weight compared to the untreated control, but with less than 80% confidence by Bayesian analysis. The roots treated with the endophyte organism MIC-33414 demonstrated a 23.5% decrease in dry root weight compared to the untreated control, but with less than 80% confidence by Bayesian analysis. However, the endophyte organism formulation comprising MIC-68178 and MIC-33414 demonstrated an increase in dry root weight of 36.8% compared to the untreated control, with more than 80% confidence by Bayesian analysis. A photo taken before drying the exemplary untreated control and the soy roots treated with MIC-68178 and MIC-33414 is shown in Figure 3. Table 12. Percentage difference in dry root weight in the Stine 33E22 soybean variety under conditions greenhouse. Soy variety Stine 33E22% difference Untreated control, dry root weight Untreated control 0 Formulation control 7.4 MIC-68178 16.9 MIC-68178 / MIC-33414 36.8 MIC-33414 -23.5 Example 7: Cultivation of Plants Treated with Endophytes in the Experiment with Greenhouse 1 Wheat
[0386] [0386] A sandy loam and commercial potting soil (Farfard®, Agawam, MA) were used in this experiment. The sandy clay was mixed in the proportion of 60% clay and 40% mortar sand (Northeast Nursery, Peabody, MA). Before mixing, both planting media were sieved through a 3/8 ”square steel mesh to remove larger particles and debris. Wheat seeds were treated with commercial fungicide and insecticide treatment. The seeds were arranged heterologously with the endophyte formulations and the formulation control (without any endophyte), as described in Example 5, untreated seeds (lack of formulation and endophyte) were also planted. Endophyte treatments were applied to the seeds in three target doses: high (10 ^ 5 CFU / seed), medium (10 ^ 4 CFU / seed), low (10 ^ 3 CFU / seed). Each pot was filled with 600 ml of its respective soil, watered with 200 ml of water, and then nine seeds were sown evenly in each pot (in a 3x3 pattern). The soil was then covered over the seeds (estimated average planting depth of 1 inch) and an additional 110 mL of water was added to moisten the overlapping soil substrate. The experimental design required a completely random pattern of each treatment within each block / replica. The environmental conditions were defined in the 12h photoperiod, at a temperature of 22 / 18ºC during the day / night and the light intensity was defined at 650 μMol m-2 s-1. After planting, the seeds were watered to maintain approximately 80% of the soil's capacity.
[0387] [0387] The emergence of wheat seedlings was recorded on days 4, 5 and 7 after planting, with days 4 and 5 representing the percentage of early emergence and day 7 representing the percentage of final emergence. On day 7, all pots were diluted to 3 seedlings / pot. The above-ground tissue was harvested from the experiment three weeks after planting. The tissues of the individual treatments (vases) were collected and placed in an unlined paper bag. All fabrics were dried in an oven at 85 ° C for 3 days. After being completely dry, the shoot biomass of the individual treatment repetitions (pots) was weighed and recorded.
[0388] [0388] The co-cultivated MIC-96038, MIC-96038 / MIC-19994 and MIC-19994 treatments increased the biomass of dry plant sprouts in the medium dose. Treatment with MIC-19994 resulted in a 6% increase (p <0.05) in dry shoot biomass at 10 ^ 4 CFU / seed. Table 13. Dry wheat sprout biomass treated with endophytes
[0389] [0389] A sandy clay growth substrate was mixed in the greenhouse and consisted of 60% clay and 40% sand mortar (Northeast Nursery, Peabody, MA). Before mixing, the clay was sieved through a 3/8 ”square mesh sieve to remove larger particles and debris. The soybean seeds were treated with fungicide treatment and commercial insecticide CruiserMaxx® Vibrance (Syngenta, Basel, Switzerland) according to the manufacturer's instructions. The seeds were arranged heterologously with the endophyte formulations and the formulation control (without any endophyte), as described in Example 11, untreated seeds (lack of formulation and endophyte) were also planted. Endophyte treatments were applied to the seeds in three target doses: high (10 ^ 5 CFU / seed), medium (10 ^ 4 CFU / seed), low (10 ^ 3 CFU / seed). Each pot was filled with 600 ml of its respective soil, watered with 200 ml of water, and then nine seeds were sown evenly in each pot (in a 3x3 pattern). The soil was then covered over the seeds (estimated average planting depth of 1 inch) and an additional 110 mL of water was added to moisten the overlapping soil substrate. The experimental design required a completely random pattern of each treatment within each block / replica. The environmental conditions were defined in the 12h photoperiod, at a temperature of 22 / 18C during the day / night and the light intensity was defined at 650 μMol m -2 s-1. After planting, seeds grown under normal irrigation conditions were watered to maintain approximately 80% of soil capacity and seeds grown under dry conditions were watered to maintain approximately 40% of soil capacity. The above-ground tissue was harvested from the experiment three weeks after planting. The tissues of the individual treatments (vases) were collected and placed in an unlined paper bag. All fabrics were dried in an oven at 85 ° C for 3 days. After being completely dry, the shoot biomass of the individual treatment repetitions (pots) was weighed and recorded. Table 14. Endophyte treatments associated with increased dry shoot biomass under normal irrigation conditions compared to endophyte-treated soy formulation Normal irrigation Limited irrigation Weight% Dry weight% dry (g) change (g) change in relation to the control of formulation control formulation MIC-96038 1,987 7.3% 1,145 -0,72% MIC-96038 / MIC- 1,945 5,07% 1,155 0,17% 19994 MIC-19994 1,937 4.64% 1.106 -4.11% Rice
[0390] [0390] A sandy clay growth substrate was mixed in the greenhouse and consisted of 60% sieved clay and 40% sand mortar (Northeast Nursery, Peabody, MA). Before mixing, the clay was sieved through a 3/8 inch square mesh sieve to remove larger particles and debris.
[0391] [0391] Seeds treated with endophytes and control seeds (without endophytes) prepared in Example 26 were planted in a growth room experiment. For each treatment or control, 20 replicates were prepared as follows. Each pot was filled with 600 mL of soil, watered with 200 mL of water, and then six seeds were sown evenly in two rows (in a 2x3 pattern). The soil was then covered over the seeds (estimated average planting depth of 0.5 inches) and an additional 100 mL of water was added to moisten the overlapping substrate. The experimental design required a completely random pattern of each treatment within each block / replica. The environmental conditions were defined in the 12h photoperiod, at a temperature of 22 / 18C during the day / night and the light intensity was defined at 650 μMol m-2 s-1. After planting, the seeds were watered with 200 mL of water on alternate days. The pots were diluted to 3 seedlings approximately 9 days after planting.
[0392] [0392] The emergency was recorded on days 4, 5, 7, 8 and 9 days after planting, with days 4 and 5 representing the percentage of early emergence and day 9 representing the percentage of final emergence. The above-ground tissue was harvested from the experiment three weeks after planting. The tissues of the individual treatments (vases) were collected and placed in an unlined paper bag. All fabrics were dried in an oven at 85 ° C for 3 days. After being completely dry, the shoot biomass of the individual treatment repetitions (pots) was weighed and recorded. Table 15. The average dry biomass (g) of seeds treated with Flo Rite® Biomass Desv Error% vs% vs the standard dryness Treatment Dose control Control of standard mean of the untreated formulation (g) is shown. Control 0.03 0.107 0.00417 0 6.82 untreated 23 Control of 0.02 0.1002 0.0038 -6.38 0 formulation 94 0.05 Low 0.1053 0.01211 -1.61 5.09 42 0.02 MIC-68178 Medium 0.1195 0.00588 11.61 19.21 63 0.02 High 0.0967 0.00513 -9.67 -3.51 29
[0393] [0393] Endophyte compositions were generated comprising an oil rich in erucic acid, a nonionic surfactant and a plantability polymer. MIC-31593 and MIC-33414 were prepared in three different formulations (A_2, B_2, C_2) and MIC-96038 was prepared in formulation A_1, described in Table 15. Table 17. Components of endophyte compositions, volumes per 50 g of seed Volume 0.5% Flo Rite® Formulation Oil oil volume (mL) (mL) Triton X-
[0394] [0394] Endophyte inoculants, as prepared in Example 5, were applied to wheat seeds that had not been previously treated with chemical insecticide or fungicide. The seeds treated with endophytes were prepared in 5 target doses: 10 ^ 1, 10 ^ 2, 10 ^ 3, 10 ^ 4, 10 ^ 5 CFU / seed. Ten biological replicas each were planted for each treatment and control condition (without endophytes) in individual containers containing commercial potting media.
[0395] [0395] The emergency was registered at 7 days after planting and the plant height (cm) punctuated at 7, 14, 21 and 28 days after planting. The above and below ground tissue was harvested from the experiment four weeks after planting. The fresh weight of the roots and shoots was recorded and then the samples were dried in an oven at 85 ° C for 3 days. After being completely dry, the root and shoot biomass was weighed and recorded. Table 18. Percentage of germination, height and weight of plants in the wheat formulation controls treated with endophytes. DAP = Days after planting MIC-MIC- MIC- MIC- MIC- 96038 / MI 96038 / MIC 96038 / MIC Contr 96038 / MIC- 96038 / MIC- C-19994, -19994, -19994, ole 19994, 10 19994 , 10 ^ 2 10 ^ 3 10 ^ 4 10 ^ 5 form UFC / semp UFC / semp UFC / sem UFC / semp UFC / seme action between te% of 7 DBH Germ 100 100 100 100 90 90 Height of the 7 DBH plant (cm) 10.4 11.1 10.8 11.2 11 11.6 Height 14 of the DBH 15.3 16.2 17.1 17.2 17.2 17.7 plant (cm) Height of the 21 plant DBH (cm) 16.1 17.2 17.3 17.8 17.2 18.5 Height of the 28 plant DBH (cm) 16.29 17.21 17.66 18 17.767 18.74 Fresh Weight Weight of (g ) Root (g) 0.367 0.419 0.41 0.437 0.413 0.429 Fresh Weight Superi (g) or (g) 0.21 0.2406 0.253 0.265 0.276 0.267 Fresh Weight Total Weight (g) (g) 0.577 0.6596 0.663 0.702 0.699 0.696 Dry Weight Root weight (g) 0.0627 0.0729 0.0713 0.0756 0.07 0.074 Dry Weight Superi (g) or (g) 0.0582 0.0688 0.0695 0 , 0733 0.071 0.074 Dry Weight Total Weight (g) (g) 0.1209 0.1417 0.1408 0.1489 0.141 0.149
[0396] [0396] Endophyte inoculants, as prepared in Example 4, were applied to rice seeds that had not been previously treated with chemical insecticide or fungicide. The seeds treated with endophytes were prepared in 5 target doses: 10 ^ 1, 10 ^ 2, 10 ^ 3, 10 ^ 4, 10 ^ 5 CFU / seed. Ten biological replicas each were planted for each treatment and control condition (without endophytes) in individual containers containing commercial potting media.
[0397] [0397] The emergency was registered at 7 days after planting and the plant height (cm) punctuated at 7, 14, 21 and 28 days after planting. The above and below ground tissue was harvested from the experiment four weeks after planting. The fresh weight of the roots and shoots was recorded and then the samples were dried in an oven at 85 ° C for 3 days. After completely dry, the root and shoot biomass was weighed and recorded. Table 20. Percentage of germination, height and weight of plants of rice formulation controls treated with endophytes. DAP = Days after planting MIC- MIC- 96038 / M MIC- 96038 / MIC MIC- IC- MIC- Control 96038 / MIC -19994, 96038 / MIC- 19994, 96038 / MIC- and -19994, 10 10 ^ 2 19994, 10 ^ 3 10 ^ 4 19994, 10 ^ 5 form UFC / seed UFC / seed UFC / seed UFC / se UFC / seed action n e ntly and% 7 Germ DAP 90 100 100 100 80 90 Plant height 7 (cm) DBH 7.1 7.1 7.7 7.7 7.3 7.3 Height of the plant 14 (cm) DBH 11.7 11.5 12.9 13.1 14 12.6 Height of the plant 21 (cm) DBH 14.1 13.5 14.9 15.2 15.7 14.4
[0398] [0398] A sandy clay growth substrate was mixed in the greenhouse and consisted of 60% clay and 40% sand mortar (Northeast Nursery, Peabody, MA). Before mixing, the clay was sieved through a 3/8 ”square mesh sieve to remove larger particles and debris. The soybean seeds were treated with fungicide treatment and commercial insecticide CruiserMaxx® Vibrance (Syngenta, Basel, Switzerland) according to the manufacturer's instructions. The seeds were arranged heterologously with the endophyte formulations and the formulation control (without any endophyte), as described in Example 14, untreated seeds (lack of formulation and endophyte) were also planted. Each pot was filled with 600 ml of its respective soil, watered with 200 ml of water, and then nine seeds were sown evenly in each pot (in a 3x3 pattern). The soil was then covered over the seeds (estimated average planting depth of 1 inch) and an additional 110 mL of water was added to moisten the overlapping soil substrate. The experimental design required a completely random pattern of each treatment within each block / replica. The environmental conditions were defined in the 12h photoperiod, at a temperature of 22 / 18C during the day / night and the light intensity was defined at 650 μMol m -2 s-1. After planting, the seeds were watered to maintain approximately 80% of the soil's capacity. The above-ground tissue was harvested from the experiment three weeks after planting. The tissues of the individual treatments (vases) were collected and placed in an unlined paper bag. All fabrics were dried in an oven at 85 ° C for 3 days. After being completely dry, the shoot biomass of the individual treatment repetitions (pots) was weighed and recorded. Results
[0399] [0399] All formulations tested on soy resulted in a less than 5% change in dry shoot biomass. Formulation C2 was neutral (-0.09%). Formulations A2 and A4 had a slight negative effect on dry shoot biomass (-3.57% and -1.8%, respectively). The difference between the formulation that had a slight beneficial effect (B_2) and a slight negative effect (A_2) was in the oil concentrators and Triton X-100. MIC-31593 had the best overall performance with the formulations tested in that experiment; the greatest effect was observed with "B_2 low dose", "A2 medium dose" and "C_2 low dose", in that order, with an increase in dry shoot biomass of 6.9%, 6.87% and 5.26% ; respectively, compared to control.
[0400] [0400] A sandy clay growth substrate was mixed in the greenhouse and consisted of 60% clay and 40% sand mortar (Northeast Nursery, Peabody, MA). Before mixing, the clay was sieved through a 3/8 ”square mesh sieve to remove larger particles and debris. The soybean seeds were treated with fungicide treatment and commercial insecticide CruiserMaxx® Vibrance (Syngenta, Basel, Switzerland) according to the manufacturer's instructions. The seeds were arranged heterologously with the endophyte formulations and the formulation control (without any endophyte), as described in Example 11, untreated seeds (lack of formulation and endophyte) were also planted. The treatments with endophytes were applied to the seeds in two target doses: high (10 ^ 5 CFU / seed), medium (10 ^ 4 CFU / seed). Each pot was filled with 600 ml of its respective soil, watered with 200 ml of water, and then nine seeds were sown evenly in each pot (in a 3x3 pattern). The soil was then covered over the seeds (estimated average planting depth of 1 inch) and an additional 110 mL of water was added to moisten the overlapping soil substrate. The experimental design required a completely random pattern of each treatment within each block / replica. The environmental conditions were defined in the 12h photoperiod, at a temperature of 22 / 18C during the day / night and the light intensity was defined at 650 μMol m-2 s-1. After planting, seeds grown under normal irrigation conditions were watered to maintain approximately 80% of soil capacity and seeds grown under dry conditions were watered to maintain approximately 40% of soil capacity. The above-ground tissue was harvested from the experiment three weeks after planting. The tissues of the individual treatments (vases) were collected and placed in an unlined paper bag. All fabrics were dried in an oven at 85 ° C for 3 days. After being completely dry, the shoot biomass of the individual treatment repetitions (pots) was weighed and recorded. Table 23. Dry biomass of sprout of wheat seedlings treated with endophytes grown in sandy clay soil under normal conditions Biomass% change% change in Dry treatment in relation to the average (g) untreated formulation Formulation control 1,852 - 3.46 0 Untreated control 1.918 0 3.59 MIC-96038 10 ^ 4 1.987 3.58 7.3 MIC-96038 10 ^ 5 1.843 -3.91 -0.46 MIC-96038 / MIC-19994 1.945 1, 43 5.07 10 ^ 4 MIC-96038 / MIC-19994 1.885 -1.72 1.8 10 ^ 5 MIC-19994 10 ^ 4 1.937 1.01 4.64 MIC-19994 10 ^ 5 1.915 -0.14 3 , 44 Example 12. Fungal Biomass Preparation Methods and Seed Treatment for Field Experiments Preparation of fungal endophytes
[0401] [0401] Preparation of molasses broth and potato dextrose agar: The molasses broth was prepared by dissolving 30 g of molasses and 5 g of yeast extract per liter of deionized water in an autoclavable container and autoclaving it (15 psi, 121 ° C) for 45 min. Potato dextrose agar (PDA) plates were prepared by dissolving 39.0 g of powdered PDA per liter of deionized water in an autoclavable container and autoclaving it (15 psi, 121 ° C) for 45 min. The agar was allowed to cool to 50-60 ° C, before pouring it into sterile petri dishes (30 ml per 90 mm plate).
[0402] [0402] Liquid biomass: All equipment and consumables were completely sterilized and the procedures performed in a biosafety cabinet. The inoculant is prepared by placing 1 buffer of cryopreserved material on a fresh PDA plate, sealing the plate with Parafilm® and incubating it at room temperature in the dark for 5 to 10 days. Then, the ~ 5x5 mm plugs were cut from the PDA plates and the 10-12 plugs were transferred to vials containing the sterile molasses broth, then covered, fixed on a shaker and incubated for at least 10 days with shaking at ~ 130 rpm. Then, the culture was placed in a blender for 5 seconds and 1 mL of the mixture was centrifuged and the supernatant was discarded and the pellet resuspended in 0.5 mL of phosphate buffered saline (PBS) 1x to generate inoculum.
[0403] [0403] Dry biomass: All equipment and consumables have been completely sterilized and the procedures performed in a biosafety cabinet. The inoculant is prepared by placing 1 buffer of cryopreserved material on a fresh PDA plate, sealing the plate with Parafilm® and incubating it at room temperature in the dark for 5 to 10 days. Then, the ~ 5x5 mm plugs were cut from the PDA plates and the 10-12 plugs were transferred to vials containing the sterile molasses broth, then covered, fixed on a shaker and incubated for at least 10 days with shaking at ~ 130 rpm. Under sterile conditions, the liquid culture was carefully decanted using sterile 150 mm filter paper in a sterile Buchner funnel over a sterile flask. After all the liquid had passed through the funnel, the sediment was washed with sterile water until the filtrate was clean. When dry, the sediment was transferred to a drying cabinet and dried until it became brittle. The sediment was then ground into fine powder and the sample used to generate CFU counts. Preparation of sodium alginate and talc for seed treatments
[0404] [0404] A 2% weight / volume sodium alginate solution for the seed coatings is prepared by the following method. An Erlenmeyer flask is filled with the appropriate volume of deionized water and heated to 50 degrees Celsius on a hot plate with stirring using a stir bar. The appropriate mass of sodium alginate powder for the desired final concentration solution is slowly added until it dissolves. The solution is autoclaved at 121 degrees Celsius at 15 PSI for 30 minutes to sterilize.
[0405] [0405] Talcum powder for powdered seed coatings is prepared by the following method. The talcum powder is aliquoted in Ziploc bags or 50 mL falcon tubes, and autoclaved in a dry cycle (121 degrees Celsius at 15 PSI for 30 minutes) to sterilize.
[0406] [0406] The seeds were arranged heterologously to each endophyte according to the following treatment protocol.
[0407] [0407] Liquid formulation: The 2% sodium alginate solution prepared above added to the seeds at a rate of 15 ml per kg of seeds. The liquid fungal culture as prepared in Example 10 was added to the seeds at a rate of 8.3 ml per kg of seeds. Control treatments were prepared using equivalent volumes of sterile broth. The seeds were then stirred to disperse the solution evenly over the seeds.
[0408] [0408] Then, 12.5 g of talcum powder per kg of seed was added and the seeds were agitated to uniformly disperse the powder in the seeds. Then, 17 ml per kg of Flo-Rite® 1706 seed (BASF, Ludwigshafen, Germany) was added and the seeds were agitated to disperse the powder evenly in the seeds. The final concentration of endophyte was estimated to be at least 10 ^ 4 CFU. The treated seeds were allowed to dry overnight in a well-ventilated space before planting.
[0409] [0409] Dry formulation: The 2% sodium alginate solution prepared above was added to the seeds at a rate of 20 ml per kg of seeds. Equal parts of the prepared fungus powder and the talc prepared above were mixed. The solution is applied to the prepared seeds so that an equivalent of 12.5 g of fungus powder is applied per kg of seeds. Control treatments were prepared using equivalent volumes of talc. The seeds were then stirred to disperse the solution evenly over the seeds.
[0410] [0410] Then, 17 ml per kg of Flo-Rite® 1706 seed (BASF, Ludwigshafen, Germany) was added and the seeds were agitated to disperse the powder evenly in the seeds. The final concentration of endophyte was estimated to be at least 10 ^ 4 CFU. The treated seeds were allowed to dry overnight in a well-ventilated space before planting. Heterologous disposition of endophytes in soybean or peanut seeds
[0411] [0411] The seeds were arranged heterologously to each endophyte according to the following treatment protocol.
[0412] [0412] Liquid formulation: The 2% sodium alginate solution prepared in Example 10 was added to the seeds at a rate of 8.3 ml per kg of seeds. The liquid fungal culture as prepared in Example 9 was added to the seeds at a rate of 8.3 ml per kg of seeds. Control treatments were prepared using equivalent volumes of sterile broth. The seeds were then stirred to disperse the solution evenly over the seeds.
[0413] [0413] Then, 15 g per kg of seed of the talcum powder prepared in Example 10 was added and the seeds were agitated to disperse the powder evenly in the seeds. Then, 13.3 ml per kg of Flo-Rite® 1706 seed (BASF, Ludwigshafen, Germany) was added and the seeds were agitated to disperse the powder evenly in the seeds. The final concentration of endophyte was estimated to be at least 10 ^ 4 CFU. The treated seeds were allowed to dry overnight in a well-ventilated space before planting.
[0414] [0414] Dry formulation: The 2% sodium alginate solution prepared in Example 10 was added to the seeds at a rate of 16.6 ml per kg of seeds. Equal parts of the fungus powder prepared in Example 9 and the talc prepared in Example 10 were mixed. The solution was applied so that an equivalent of 10 g of fungal powder was applied per kg of seeds. Control treatments were prepared using equivalent volumes of talc. The seeds were then stirred to disperse the solution evenly over the seeds.
[0415] [0415] Next, 13.3 ml per kg of FloRite® 1706 seed (BASF, Ludwigshafen, Germany) was added and the seeds were agitated to disperse the powder evenly in the seeds. The final concentration of endophyte was estimated to be at least 10 ^ 4 CFU. The treated seeds were allowed to dry overnight in a well-ventilated space before planting. Heterologous disposition of endophytes in corn seeds
[0416] [0416] The seeds were arranged heterologously to each endophyte according to the following treatment protocol.
[0417] [0417] Dry formulation: The 2% sodium alginate solution prepared as above was added to the seeds at a rate of 23 ml per kg of seeds. Equal parts of the fungus and talcum powder as prepared above were mixed. The solution was applied so that an equivalent of 10 g of fungal powder was applied per kg of seeds. Control treatments were prepared using equivalent volumes of talc. The seeds were then stirred to disperse the solution evenly over the seeds.
[0418] [0418] Then, 16.6 ml per kg of FloRite® 1706 seed (BASF, Ludwigshafen, Germany) were added and the seeds were agitated to disperse the powder evenly in the seeds. The final concentration of endophyte was estimated to be at least 10 ^ 4 CFU. The treated seeds were allowed to dry overnight in a well-ventilated space before planting. Heterologous disposition of endophytes in rice
[0419] [0419] The seeds were treated with the commercial fungicide and insecticide product Sativa® IMF MAX (Nufarm Americas, Alsip, IL) according to the manufacturer's instructions (3.4 oz / cwt). The chemically treated rice seeds were arranged heterologically for each endophyte according to one of two different formulation protocols (Formulation Protocol A, Formulation Protocol B). A corresponding seed formulation control was also prepared, without any endophyte, for each type of formulation. Additional seeds without any formulation and endophyte were planted as an untreated baseline control. Formulation A included only 0.05% silwet and microbial diluent preparations. Formulation B included the same diluent and Flo Rite® 1706 seed plantability polymer applied to 2.0 oz / cwt of seeds by the manufacturer. Microbe and polymer were applied sequentially.
[0420] [0420] For endophytes formulated by Formulation Protocol A, microbial preparations were applied to the seeds at a rate of 1 µL / seed and the seeds were agitated for at least 20 seconds to disperse the microbe.
[0421] [0421] For endophytes formulated by Formulation Protocol B, microbial preparations were applied to the seeds at a rate of 1 µL / seed. Then, the Flo Rite® 1706 plantability polymer was applied to the seeds at the manufacturer's suggestion (2.0 oz / cwt of seed) and stirred for 20 seconds to disperse the polymer. Example 13: Cultivation of Plants Treated with Endophytes in Field Experiment 1 Seed yield test under field conditions, soybean
[0422] [0422] Field trials were conducted under non-irrigated conditions (dry land) in multiple locations, preferably in several geographic regions. The seeds were prepared with the endophyte formulations, as described in Example 12 and untreated seeds (lack of formulation and endophyte) were also planted. At the end of the field trial, using treatment with endophytes and control treatment plants, the batches were harvested using a 5-foot survey combination and the yield was calculated by the on-board computer. Only the two rows in the middle of the four rows were harvested to show border effects. The seeds were sown in rows regularly spaced in the soil, with a density of
[0423] [0423] At the end of the field trial, using treatment with endophytes and control treatment plants, the batches were harvested by machine with a combination of 5-foot survey and the yield was calculated by the on-board computer. Only the two rows in the middle of the four rows were harvested to prevent edge effects. Table 24. Yield of treated and untreated endophyte-treated soy and formulation controls, Field Experiment 1 Rendim Rendim Rendim Rendim Rendim Contr Control between then and then oil and then 1 location 2 location 3 location 4 overall do not formulate (bu / ac) (bu / ac) (bu / ac) (bu / ac) (bu / ac) treatment ∆ o∆%% general general Condition Stress Stress Good Pressure water Hydro severe herbal moisture, some weed pressure weed s Days 9 13 28 28 between treatment and planting Control 27.7 68.4 61 41.6 49.7 -1.4 and untreated Control 27.1 68.9 64.8 42, 5 50.4 1.4 0 and formulation MIC- 27.9 70.2 62.1 48 52.1 4.8 3.4 31593 MIC- 27 69.5 63.4 43.4 50.8 2 , 2 0.8 96038 MIC- 27.9 72 67.2 43.6 52.1 4.8 3.4 33414 Average 25.7 68.1 62.5 45.1 50.4 of the test seeds in field conditions, wheat
[0424] [0424] Field trials were conducted under non-irrigated conditions (dry land) in multiple locations, preferably in several geographic regions. Wheat seeds were treated with commercial fungicide and insecticide treatment. The seeds were arranged heterologously with the endophyte formulations described above in Example 12 and untreated seeds (lack of formulation and endophyte) were also planted. The seeds were sown in rows regularly spaced in the soil, with a density of 1.2 million seeds / acres. At each site, at least 3 replicated lots were planted for each treatment with endophyte or control in a randomized complete block design. Each lot consisted of seven 15.24 m (40 ft) rows.
[0425] [0425] At the end of the field trial, employing treatment with endophytes and control treatment plants, the batches were harvested using a 5-foot survey combination and the yield was calculated by the on-board computer. Table 25. Average yield per endophyte-treated spring wheat and formulation control and untreated control, Field Experiment 1
[0426] [0426] The seeds were prepared with the endophyte formulations and the formulation control (without any endophytes) as described in Example 12. The seeds were sown with a grain drill sized drill at a planting rate of 45 pounds per acre . Four replicated lots of 30x175 feet were planted by endophyte treatment or control in a randomized complete block design. Plots were flooded, weeds and insects were controlled with standard local practices.
[0427] [0427] At the end of the field trial, using treatment with endophytes and control treatment plants, the batches were harvested by machine with a 5-foot survey combination and the yield was calculated by the on-board computer. Table 27. Yield of treated and untreated endophyte rice and controls, Field Experiment 1% Moisture Weight Yield Yield% of grain emergence (lb / ground change) (lb / lot) (bushel / acre) ) Control Treatment 65.3 19.33 43.95 16.405 119.6 0 Control MIC- 67.8 18.88 41.33 17.165 125.9 5.27 96038 + MIC- 19994 Example 14: Cultivation of Plants Treated with Endophytes in Field Experiment 2 Seed yield test in field conditions, wheat
[0428] [0428] Field trials were conducted under non-irrigated conditions (dry land). Two varieties of spring wheat seeds were treated with commercial fungicide and insecticide treatment. The seeds were arranged heterologously with the endophyte formulations and the formulation control (without any endophyte), as described in Example 12, untreated seeds (lack of formulation and endophyte) were also planted. The seeds were sown in rows regularly spaced in the soil, with a density of 1.2 million seeds / acres. At each site, replicated lots were planted for each treatment with endophyte or control in a randomized complete block design. Each lot consisted of seven 15.24 m (40 ft) rows.
[0429] [0429] At the end of the field trial, employing treatment with endophytes and control treatment plants, the batches were harvested by machine with a 5-foot survey combination and the grain yield and percent moisture were calculated by the board.
[0430] [0430] Field trials were conducted in 2016, under non-irrigated conditions (dry land). Wheat seeds were treated with commercial fungicide and insecticide treatment. The seeds were arranged heterologously with the endophyte formulations and the formulation control (without any endophyte), as described in Example 12, untreated seeds (lack of formulation and endophyte) were also planted. The seeds were sown in rows regularly spaced in the soil, with a density of 1.2 million seeds / acres. At each site, replicated lots were planted for each treatment with endophyte or control in a randomized complete block design. Each lot consisted of seven 15.24 m (40 ft) rows.
[0431] [0431] At the end of the field trial, using treatment with endophytes and control treatment plants, the batches were harvested by machine with a combination of 5-foot survey and the yield was calculated by the on-board computer. Table 32. Yield per wheat site treated with endophyte and untreated control and formulation control, field experiment 3 Contr Varieda Varieda Varieda Varieda In Control ole 1, 2, 3, 4, general, and non-yielding rendim rendim rendim rendim formula treatment then ent then ent tion% ∆% o∆% (bu / ac) (bu / ac) (bu / ac) (bu / ac) (bu / ac) general general Control and not 27.9 27.4 39.8 43.2 34.6 -3.0 treated Control and 31.2 28.2 39.0 44.2 35.7 3.1 formulation MIC- 32.5 23.5 42, 6 43.4 35.5 2.7 -0.4 96038 MIC- 31.2 26.9 40.4 43.9 35.6 3.0 -0.1 19994 Example 16. Evaluation of Improved Plant Characteristics: Field Conditions Seed yield test under field conditions, wheat
[0432] [0432] Field trials were conducted under non-irrigated conditions (dry land) in multiple locations, preferably in several geographic regions. Wheat seeds were treated with commercial fungicide and insecticide treatment. The seeds were arranged heterologously with the dry endophyte formulations as described in Example 12 and untreated seeds (lack of formulation and endophyte) were also planted. The seeds were sown in rows regularly spaced in the soil, with a density of 1.2 million seeds / acres. At each site, at least 3 replicated lots were planted for each treatment with endophyte or control in a randomized complete block design. Each lot consisted of seven 15.24 m (40 ft) rows.
[0433] [0433] At the end of the field trial, using treatment with endophytes and control treatment plants, the batches were harvested by machine with a combination of 5-foot survey and the yield was calculated by the on-board computer.
[0434] [0434] Endophyte treatments, each comprising one of the following microbes: MIC-68390, MIC-68178, MIC-07010, MIC-31593, MIC-48747, MIC-96038, MIC-50414 or MIC-33414, resulted in average yield increases of 7-16% in the SDSU Focus wheat variety. Endophyte treatments, each comprising one of the following microbes: MIC-68390, MIC-68178, MIC-07010, MIC-31593, MIC-48747, MIC-96038, MIC-50414 or MIC-33414, resulted in average increases in 14-22% yield in the SDSU Select wheat variety. Table 33: Average yield of wheat treated with endophytes in field trials SDSU Focus, Variety SDSU Select, Variety 3 4% of Yield difference Yield mean difference of non-average from treated (BU / acre) treated (BU / acre) Untreated control 36.9 0 37.7 0% MIC-68390 39.3 7% 45.0 19% MIC-68178 40.9 11% 46.1 22% MIC-07010 41.1 11% 43.9 16 % MIC-31593 42.1 14% 44.3 18% MIC-48747 42.8 16% 44.1 17% MIC-96038 42.6 15% 43.4 15% MIC-50414 40.0 8% 45, 7 21% MIC-33414 41.6 13% 42.9 14% Seed yield test under field conditions, maize
[0435] [0435] Field trials were conducted in multiple locations, preferably in several geographic regions. The plots of land were not irrigated (dry land) or were maintained with less than ideal irrigation at a rate that aimed at a reduction of approximately 25% in yield. The seeds were prepared with the endophyte (dry) formulations and the formulation control (dry, without any endophyte), as described in Example 12, untreated seeds (lack of formulation and endophyte) were also planted. The seeds were sown in rows spaced regularly in the soil at planting densities typical for each region. At each site, 3 replicated lots were planted by endophyte treatment or control in a randomized complete block design. Each lot consisted of four rows of 15.24 m (40 feet), each separated by 76.2 cm (30 inches).
[0436] [0436] At the end of the field trial, using treatment with endophytes and control treatment plants, the batches were harvested by machine with a combination of 5-foot survey and the yield was calculated by the on-board computer. Only the two rows in the middle of the four rows were harvested to prevent edge effects.
[0437] [0437] Endophyte treatments, each comprising one of the following microbes: MIC-68390, MIC-48747, or MIC-33414, resulted in average yield increases of 0.9-1.5% compared to formulation control and in average increases in yield of 1.0-1.6% compared to the untreated control, in the variety of Stine 9734 corn. Table 34: Average yield of the variety of Stine 9734 corn treated with endophytes in Stine 9734 field trials, Variety 2 Yield% of average difference difference (BU / acre) of control of untreated formulation control Untreated 185.5 0.0% Control of 0.0% formulation (dry) 185.7 MIC-68390 187, 4 0.9% 1.0% MIC-48747 187.7 1.1% 1.2% MIC-33414 188.5 1.5% 1.6% Seed yield test under field conditions, soybean
[0438] [0438] Field trials were conducted under non-irrigated conditions (dry land) in multiple locations, preferably in several geographic regions. The seeds were prepared with the endophyte formulations, as described in Example 12 and untreated seeds (lack of formulation and endophyte) were also planted. MIC-68178 and MIC-33414 were formulated with the dry formulation; MIC-68390, MIC-07010, MIC-31593, MIC-48747, MIC-96038, and MIC-50414 were formulated with the liquid formulation.
[0439] [0439] The seeds were sown in rows regularly spaced in the soil, with a density of 40,000 seeds / acres. At each site, at least 3 replicated lots were planted by endophyte treatment or control in a randomized complete block design. Each lot consisted of four rows of 15.24 m (40 feet), each separated by 76.2 cm (30 inches).
[0440] [0440] At the end of the field trial, using treatment with endophytes and control treatment plants, the batches were harvested by machine with a combination of 5-foot survey and the yield was calculated by the on-board computer. Only the two rows in the middle of the four rows were harvested to prevent edge effects.
[0441] [0441] Endophyte treatments, each comprising one of the following microbes: MIC-68390 MIC-31593, or MIC-33414, resulted in average yield increases of 1.5-6.2% in the Dairyland DSR1808R2Y variety. Endophyte treatments, each comprising one of the following microbes: MIC-68390, MIC-31593, or MIC-33414, resulted in average yield increases of 1.5-6.2% in the Dairyland DSR1808R2Y variety. Endophyte treatments, each comprising one of the following microbes: MIC-68390, MIC-68178, MIC-07010, MIC-31593, MIC-48747, MIC-96038, MIC-50414, or MIC-33414, resulted in average increases yield of 2.5-15% in the Pfister 38R25 soybean variety. Endophyte treatments, each comprising one of the following microbes: MIC-68390, MIC-68178, MIC-07010, MIC-31593, MIC-48747, MIC-50414, or MIC-33414, resulted in average increases in yield of 1 , 1- 6% in the Stine 3920 soybean variety. Table 35: Average yield of the Dairyland DSR1808R2Y soybean variety treated with endophytes in Dairyland DSR1808R2Y field trials, Variety 1 Average yield% difference (pounds / acre) untreated control Control not 33.9 treated 0.0% MIC- 35.1 68390 3.5% MIC- 34.4 31593 1.5% MIC- 36.0 33414 6.2%
[0442] [0442] Field trials were conducted in multiple locations, preferably in several geographic regions. The plots of land were not irrigated (dry land) or were maintained with less than ideal irrigation at a rate that aimed at a reduction of approximately 25% in yield. Canola seeds were treated with commercial fungicide and insecticide treatment. The seeds were prepared with liquid endophyte formulations and the control of the liquid formulation (without any endophyte), as described in Example 12 and untreated seeds (lack of formulation and endophyte) were also planted. At each site, at least 3 replicated lots were planted for each treatment with endophyte or control in a randomized complete block design.
[0443] [0443] At the end of the field trial, using treatment with endophytes and control treatment plants, the batches were harvested by machine with a combination of 5-foot survey and the yield was calculated by the on-board computer.
[0444] [0444] Endophyte treatments comprising MIC-85555 resulted in an average yield increase of 0.3% over the formulation control and an average increase in yield by 0.2% over the untreated control, in the variety of canola Brett Young 5525. Endophyte treatments comprising MIC-50989 resulted in an average yield increase of 8.2% compared to the formulation control and an average increase in yield by 10.3% compared to the untreated control, in variety of canola NCC1015. Table 38. Average yield of the Brett Young 5525 canola variety treated with endophytes in Brett Young 5525 field trials, Variety 1 Yield% of mean difference difference (pounds / acre) formulation control untreated control 1111, 0 0.0% Formulation control (liquid) 1109.5 0.0% MIC-85555 1112.7 0.3% 0.2% Table 39. Average yield of NCC1015 canola variety treated with endophytes in NCC1015 field trials , Variety 2
[0445] [0445] Field trials were conducted in multiple locations, preferably in several geographic regions. The plots of land were not irrigated (dry land) or were maintained with less than ideal irrigation at a rate that aimed at a reduction of approximately 25% in yield. Peanut seeds were treated with commercial fungicide and insecticide treatment. The seeds were prepared with the endophyte formulations and the formulation control (without any endophyte), as described in Example 12 and untreated seeds (lack of formulation and endophyte) were also planted. MIC-68390 was formulated with dry and liquid formulation; MIC-50414, MIC-68178, and MIC-96038 were formulated with the liquid formulation. At each site, at least 3 replicated lots were planted for each treatment with endophyte or control in a randomized complete block design.
[0446] [0446] At the end of the field trial, using treatment with endophytes and control treatment plants, the batches were harvested by machine with a 5-foot survey combination and the yield was calculated by the on-board computer.
[0447] [0447] In dry formulation, treatments with endophytes comprising MIC-68390 resulted in an average yield increase of 9.1% compared to the formulation control and an average increase in yield by 0.7% compared to the untreated control. , in the AT-9899 peanut variety. In the liquid formulation, endophyte treatments comprising MIC-50414 resulted in an average increase in yield of 3.8% over the control of the formulation and an average increase in yield of 0.7% over the untreated control, in peanut variety AT-9899. In the liquid formulation, treatments with endophytes, each comprising one of the following microbes: MIC-68390 or MIC-68178, resulted in an average yield increase of 4.1-4.5% compared to formulation control and an increase in average yield of 10.0-10.5% compared to untreated control, in the FloRun 107 peanut variety. However, in dry formulations, treatments with endophytes comprising MIC-68390 resulted in an average yield decrease of 6 , 3% in the FloRun 107 peanut variety. In the liquid formulation, endophyte treatments, each comprising one of the following microbes: MIC-68390, MIC-68178, or MIC-96038, resulted in an average yield increase of 0, 2- 3.6% in relation to the formulation control and an average increase with yield of 6.4-10.0% in relation to the untreated control, in the Georgia-06G peanut variety.
[0448] [0448] Phylogenomic analysis of entire genomic sequences of endophytes can be used to identify distinctive sequence variants. Gene sets suitable for phylogenomic analysis, as well as methods for identifying them, are well known in the art, for example Floutas et al. (2012) The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science, 336 (6089): 1715-9. It hurts:
[0449] [0449] Analysis of the complete genome of endophytes can be used to identify genes whose presence, absence or supra or underrepresentation ("differential abundance") is associated with desirable phenotypes. To identify genes with differential abundance in the genome of an endophyte of interest, predicted protein sequences from the endophyte genomes and closely related species are compared in a peer-to-peer comparison with everyone (for example, using BLAST), followed by of clustering protein sequences based on alignment scores (for example, using MCL: Enright AJ, Van Dongen S., Ouzounis CA An efficient algorithm for large-scale detection of protein families. Nucleic Acids Research 30 (7): 1575 -1584 (2002)). Additional software tools useful for this analysis are well known in the art and include OMA, OrthoMCL and TribeMCL (Roth AC, Gonnet GH, Dessimoz C. Algorithm of OMA for large-scale orthology inference. BMC Bioinformatics. 2008; 9: 518. doi: 10.1186 / 1471-2105-9-518, Enright AJ, Kunin V, Ouzounis CA. Protein families and TRIBES in genome sequence space.Nucleic Acids Res. 2003; 31 (15): 4632-8 .; Chen F, Mackey AJ, Vermunt JK, Roos DS. Assessing performance of orthology detection strategies applied to eukaryotic genomes. PLoS One. 2007; 2 (4): e383.). Protein clusters are consulted to identify clusters with differential abundance of proteins derived from endophytes with desirable phenotypes. The proteins in these clusters define the unique properties of these endophytes, and the abundance of genes encoding these proteins can be used to identify endophytes of the present invention.
[0450] [0450] Having illustrated and described the principles of the present invention, it should be evident to those skilled in the art that the invention can be modified in the arrangement and details, without deviating from such principles. It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other modalities, advantages and modifications are within the scope of the following claims.

权利要求:
Claims (1)
[1]
1. Method of improving the trait of agronomic importance in a corn plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a corn plant element in an effective amount to increase the yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62, where the vegetable element of corn is the variety Stine 9734 or a variety closely related to it.
2. Method of improving a trait of agronomic importance in a soybean plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a soybean plant element in an amount effective to increase the yield in the derived plant the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62.
3. Method according to claim 2, characterized by the fact that the soybean plant is a variety selected from the group consisting of Pfister 38R25, Dairyland DSR1808R2Y, Stine 3920, and varieties closely related to these.
4. Method for improving a trait of agronomic importance in a wheat plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a wheat plant element in an amount effective to increase the yield in the derived plant the treated plant element related to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of
SEQ ID NOs: 60, 61 and 62.
5. Method, according to claim 4, characterized by the fact that the wheat plant is a variety selected from the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
6. Method of improving a trait of agronomic importance in a peanut plant, characterized by the fact that it comprises, in a heterologous way, an endophyte organism for a peanut plant element in an effective amount to increase the yield in the derived plant the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62.
7. Method according to claim 6, characterized by the fact that the peanut plant is a variety selected from the group consisting of AT-9899, FloRun 107, Tamnut OL06, Georgia-06G and varieties closely related to these.
8. Method of improving a trait of agronomic importance in a soybean plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a soybean plant element in an effective amount to improve a trait of agronomic importance selected from the group consisting of root area, root length and root dry weight in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71.
9. Method according to claim 8, characterized by the fact that soy is a Stine 33E22 variety or a variety closely related to it.
10. Method for improving a trait of agronomic importance in a peanut plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a peanut plant element in an effective amount to increase the yield in the derived plant the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71.
11. Method according to claim 10, characterized by the fact that the peanut plant is a variety selected from the group consisting of FloRun 107, Georgia-06G and varieties closely related to these.
12. Method of improving a trait of agronomic importance in a rice plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a rice plant element in an amount effective to increase the length of the root in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of in SEQ ID NOs: 63, 64 and 71.
13. Method for improving a trait of agronomic importance in a wheat plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a wheat plant element in an effective amount to increase the yield in the derived plant the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41.
14. Method for improving the trait of agronomic importance in a canola plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a canola plant element in an effective amount to increase the yield in the plant derived from the treated plant element related to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 70, in which the plant canola is the Brett Young 552 variety or a variety closely related to it.
15. Method for improving a trait of agronomic importance in a soybean plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a soybean plant element in an effective amount to improve a trait of agronomic importance selected from the group consisting of root area, root length and plant yield derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 65 and 66.
16. Method, according to claim 15, characterized by the fact that the trait of economic importance is yield and the soybean plant is a variety selected from the group consisting of Pfister 38R25, Stine 3920 and varieties closely related to these.
17. Method for improving a trait of agronomic importance in a wheat plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a wheat plant element in an effective amount to increase the yield in the derived plant the treated plant element related to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 65 and 66.
18. Method according to claim 17, characterized by the fact that the wheat plant is a variety selected from the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
19. Method of improving a trait of agronomic importance in a soybean plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a soybean plant element in an effective amount to improve a trait of agronomic importance selected from the group consisting of root area, root length, dry shoot biomass and yield derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44 and
45.
20. Method, according to claim 19, characterized by the fact that the trait of economic importance is yield and the soybean plant is a variety selected from the group consisting of Pfister 38R25, Stine 3920 and varieties closely related to these.
21. Method of improving a trait of agronomic importance in a wheat plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a wheat plant element in an effective amount to increase the yield in the derived plant the treated plant element related to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44 and 45.
22. Method according to claim 21, characterized by the fact that the wheat plant is a variety selected from the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
23. Method for improving the trait of agronomic importance in a corn plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a corn plant element in an effective amount to increase the yield in the plant derived from the treated plant element related to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus
Cladosporium e comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67, in which the corn plant element is the Stine 9734 variety or an closely related variety.
24. Method of improving a trait of agronomic importance in a soybean plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a soybean plant element in an effective amount to improve a trait of agronomic importance selected from the group consisting of root area, root length and plant yield derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67.
25. Method, according to claim 24, characterized by the fact that the trait of economic importance is the yield and the soybean plant is a variety selected from the group consisting of Pfister 38R25, Stine 3920 and varieties closely related to these.
26. Method of improving a trait of agronomic importance in a wheat plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a wheat plant element in an effective amount to increase the yield in the derived plant the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67.
27. Method according to claim 26, characterized by the fact that the wheat plant is a variety selected from the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
28. Method of enhancing a trait of agronomic importance in a canola plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a canola plant element in an amount effective to increase the yield in the derived plant the treated plant element related to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Paecilomyces and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 69.
29. Method according to claim 28, characterized by the fact that the canola plant is variety NCC1015 or a variety closely related to it.
30. Method for improving a trait of agronomic importance in a soybean plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a soybean plant element in an effective amount to improve a trait of agronomic importance selected from the group consisting of root area, root length and plant yield derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51.
31. Method, according to claim 30, characterized by the fact that the trait of agronomic importance is yield and the soybean plant is variety Pfister 38R25 or a variety closely related to it.
32. Method for improving a trait of agronomic importance in a wheat plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a vegetable element of wheat in an effective amount to improve a trait of agronomic importance selected from the group consisting of root area, root length and plant yield derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51.
33. Method according to claim 32, characterized by the fact that the wheat plant is a variety selected from the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
34. Method of enhancing a trait of agronomic importance in a peanut plant, characterized by the fact that it comprises, in a heterologous way, an endophyte organism for a peanut plant element in an effective amount to increase the yield in the derived plant the treated plant element relative to a plant derived from a reference plant element, where: a. where the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51 and b . the peanut plant is a Georgia-06G variety or a closely related variety.
35. Method for improving a trait of agronomic importance in a rice plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a rice plant element in an amount effective to increase the dry shoot biomass in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group that consists of SEQ ID NOs: 46, 47, 48, 49, 50 and 51.
36. Method of improving a trait of agronomic importance in a wheat plant, characterized by the fact that it comprises, in a heterologous way, a first endophyte organism and a second endophyte organism for a wheat plant element in an effective amount for enhance a trait of agronomic importance selected from the group consisting of plant height, fresh root weight and fresh sprout weight in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which: a. the first endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of
SEQ ID NOs: 46, 47, 48, 49, 50 and 51, and b. the second endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41.
37. Method of enhancing a trait of agronomic importance in a rice plant, characterized by the fact that it comprises, in a heterologous way, a first endophyte organism and a second endophyte organism for a rice plant element in an effective amount for increase the yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, where: a. the first endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, and b . the second endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41.
38. Method of enhancing a trait of agronomic importance in a soybean plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a soybean plant element in an effective amount to increase the yield in the derived plant the treated plant element related to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 68.
39. Method according to claim 38, characterized by the fact that the soybean plant is a variety selected from the group consisting of Pfister 38R25, Stine 3920 and varieties closely related to these.
40. Method of enhancing a trait of agronomic importance in a wheat plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a wheat plant element in an effective amount to increase the yield in the derived plant the treated plant element related to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 68.
41. Method according to claim 40, characterized by the fact that the wheat plant is a variety selected from the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
42. Method of enhancing the trait of agronomic importance in a peanut plant, characterized by the fact that it comprises, in a heterologous way, an endophyte organism for a peanut plant element in an effective amount to increase the yield in the plant derived from the treated plant element related to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 68 and the peanut plant is the AT-9899 variety or a variety closely related to it.
43. Method for improving a trait of agronomic importance in a soybean plant, characterized by the fact that it comprises having, in a heterologous way, a first endophyte organism and a second endophyte organism for a soybean plant element in an effective amount for increase the dry root biomass in the plant derived from the treated plant element relative to a plant derived from a reference plant element, where: a. the first endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, b. the second endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59,
ç. and the soybean plant is variety Stine 33E22 or a variety closely related to it.
44. Method of improving a trait of agronomic importance in a corn plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a corn plant element in an amount effective to increase the yield in the derived plant the treated plant element related to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59.
45. Method for improving a trait of agronomic importance in a soybean plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a soybean plant element in an effective amount to improve a trait of agronomic importance selected from the group consisting of root area, root length, dry shoot biomass and yield on the plant derived from the treated plant element relative to a plant derived from a reference plant element, where the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59.
46. Method, according to claim 45, characterized by the fact that the trait of agronomic importance is yield and the soybean plant is the variety Pfister 38R25 or a variety closely related to it.
47. Method of enhancing a trait of agronomic importance in a wheat plant, characterized by the fact that it comprises having, in a heterologous way, an endophyte organism for a wheat plant element in an effective amount to improve a trait of agronomic importance selected from the group consisting of root length and yield in the plant derived from the treated plant element relative to a plant derived from a reference plant element, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59.
48. Method according to claim 47, characterized by the fact that the wheat plant is a variety selected from the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
49. Method according to any one of claims 1, 2, 4, 6, 8, 10, 12, 13, 14, 15, 17, 19, 21, 23, 24, 26, 28, 30, 32, 34 , 35, 36, 37, 38, 40, 42, 43, 44, 45 and 47, characterized by the fact that the plant element is a seed.
50. Method according to claim 49, characterized by the fact that the seed is modified.
51. Method according to any one of claims 1, 2, 4, 6, 8, 10, 12, 13, 14, 15, 17, 19, 21, 23, 24, 26, 28, 30, 32, 34 , 35, 36, 37, 38, 40, 42, 43, 44, 45 and 47, characterized by the fact that the endophyte organism is heterologously disposed to the plant element in the formulation, said formulation further comprising one or more of the following: a stabilizer, a preservative, a carrier, a surfactant, a fungicide, a nematicide, a bactericide, an insecticide or herbicide or any combination of these.
52. The method of any one of claims 1, 2, 4, 6, 8, 10, 12, 13, 14, 15, 17, 19, 21, 23, 24, 26, 28, 30, 32, 34 , 35, 36, 37, 38, 40, 42, 43, 44, 45 and 47, characterized by the fact that the identity percentage is at least 98%.
53. The method of any one of claims 1, 2, 4, 6, 8, 10, 12, 13, 14, 15, 17, 19, 21, 23, 24, 26, 28, 30, 32, 34 , 35, 36, 37, 38, 40, 42, 43, 44, 45 and 47, characterized by the fact that the identity percentage is at least 99%.
54. Method according to any one of claims 1, 2, 4, 6, 8, 10, 12, 13, 14, 15, 17, 19, 21, 23, 24, 26, 28, 30, 32, 34 , 35, 36, 37, 38, 40, 42, 43, 44, 45 and 47, characterized by the fact that the identity percentage is 100%.
55. Method according to any one of claims 1, 2, 4, 6, 8, 10, 12, 13, 14, 15, 17, 19, 21, 23, 24, 26, 28, 30, 32, 34 , 35, 36, 37, 38, 40, 42, 43,
44, 45 and 47, characterized by the fact that the percentage of identity is determined over an alignment region of at least 100 nucleotides.
56. The method of any one of claims 1, 2, 4, 6, 8, 10, 12, 13, 14, 15, 17, 19, 21, 23, 24, 26, 28, 30, 32, 34 , 35, 36, 37, 38, 40, 42, 43, 44, 45 and 47, characterized by the fact that the percentage of identity is determined over an alignment region of at least 200 nucleotides.
57. Method according to any one of claims 1, 2, 4, 6, 8, 10, 12, 13, 14, 15, 17, 19, 21, 23, 24, 26, 28, 30, 32, 34 , 35, 36, 37, 38, 40, 42, 43, 44, 45 and 47, characterized by the fact that the percentage of identity is determined over an alignment region of at least 300 nucleotides.
58. Method according to any one of claims 1, 2, 4, 6, 8, 10, 12, 13, 14, 15, 17, 19, 21, 23, 24, 26, 28, 30, 32, 34 , 35, 36, 37, 38, 40, 42, 43, 44, 45 and 47, characterized by the fact that the percentage of identity is determined over an alignment region of at least 400 nucleotides.
59. Method according to any one of claims 1, 2, 4, 6, 8, 10, 12, 13, 14, 15, 17, 19, 21, 23, 24, 26, 28, 30, 32, 34 , 35, 36, 37, 38, 40, 42, 43, 44, 45 and 47, characterized by the fact that the percentage of identity is determined over an alignment region of at least 500 nucleotides.
60. Agrochemically active microbial formulation, characterized by the fact that it comprises at least one oil, surfactant, polymer and a microbial active ingredient, in which the microbial active ingredient comprises a first endophyte organism of the genus Exserohilumm and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 60, 61 and 62.
61. Agrochemically active microbial formulation, characterized by the fact that it comprises at least one oil, surfactant, polymer and a microbial active ingredient, in which the microbial active ingredient comprises a first endophyte organism of the genus Epicoccum and comprises at least one polynucleotide sequence 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71.
62. Agrochemically active microbial formulation according to claim 60, characterized by the fact that it further comprises a second endophyte organism, in which the second endophyte organism is of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to one polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59.
63. Agrochemically active microbial formulation, characterized by the fact that it comprises at least one oil, surfactant, polymer and a microbial active ingredient, in which the microbial active ingredient comprises the first endophyte organism of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and
41.
64. Agrochemically active microbial formulation, characterized by the fact that it comprises at least one oil, surfactant, polymer and a microbial active ingredient, in which the microbial active ingredient comprises a first endophyte organism of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 70.
65. Agrochemically active microbial formulation, characterized by the fact that it comprises at least one oil, surfactant, polymer and a microbial active ingredient, in which the microbial active ingredient comprises a first endophyte organism of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44, 45, 65 and 66.
66. Agrochemically active microbial formulation, characterized by the fact that it comprises at least one oil, surfactant, polymer and a microbial active ingredient, in which the microbial active ingredient comprises a first endophyte organism of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 67 and 68.
67. Agrochemically active microbial formulation, characterized by the fact that it comprises at least one oil, surfactant, polymer and a microbial active ingredient, in which the microbial active ingredient comprises a first endophyte organism of the genus Paecilomyces and comprises at least one polynucleotide sequence 97% identical to SEQ ID NO:
69.
68. Agrochemically active microbial formulation, characterized by the fact that it comprises at least one oil, surfactant, polymer and a microbial active ingredient, in which the microbial active ingredient comprises a first endophyte organism of the genus Acremonium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51.
69. Agrochemically active microbial formulation according to claim 66, characterized by the fact that the microbial active ingredient further comprises a second endophyte organism, in which the second endophyte organism is of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97 % identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and
41.
70. Agrochemically active microbial formulation, characterized by the fact that it comprises at least one oil, surfactant, polymer and a microbial active ingredient, in which the microbial active ingredient comprises a first endophyte organism of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59.
71. Formulation according to any of claims 60-70, characterized in that it is oil is rapeseed or NEEM.
72. Formulation according to any one of claims 60-70, characterized in that the oil comprises erucic acid.
73. Formulation according to any one of claims 60-70, characterized in that the oil comprises herbicidal or insecticidal properties.
74. Formulation according to any one of claims 60-70,
characterized by the fact that the surfactant is a non-ionic detergent.
75. Formulation according to any of claims 60-70, characterized in that the surfactant is Tween 20 or Triton X-100.
76. Formulation according to any of claims 60-70, characterized by the fact that the polymer is FloRite®, DISCOTMor Kannar® Universal Wonder.
77. Formulation according to any of claims 60-70, characterized in that the microbial active ingredient comprises a spore suspension.
78. Formulation according to any one of claims 60-70, characterized in that the microbial active ingredient comprises spray-dried spores.
79. Formulation according to any one of claims 60-70, characterized in that the microbial active ingredient comprises whole cell broth.
80. Formulation according to any one of claims 60-70, characterized in that it further comprises one of the following: fungicide, nematicide, bactericide, insecticide or herbicide.
81. Formulation according to any one of claims 60-70, characterized in that it further comprises one of the following: stabilizer, preservative, carrier, anti-complex agent or any combination thereof.
82. Formulation according to any of claims 60-70, characterized by the fact that the endophyte organism is self-stable.
83. Formulation according to any of claims 60-70, characterized by the fact that the identity percentage is at least 98%.
84. Formulation according to any of claims 60-70, characterized by the fact that the percentage of identity is at least 99%.
85. Formulation according to any of claims 60-70, characterized by the fact that the identity percentage is 100%.
86. Formulation according to any one of claims 60-70, characterized in that the percentage of identity is determined over an alignment region of at least 100 nucleotides.
87. Formulation according to any of claims 60-70, characterized by the fact that the percentage of identity is determined over an alignment region of at least 200 nucleotides.
88. Formulation according to any of claims 60-70, characterized by the fact that the percentage of identity is determined over an alignment region of at least 300 nucleotides.
89. Formulation according to any of claims 60-70, characterized in that the percentage of identity is determined over an alignment region of at least 400 nucleotides.
90. Formulation according to any of claims 60-70, characterized in that the percentage of identity is determined over an alignment region of at least 500 nucleotides.
91. Synthetic composition characterized by the fact that it comprises a plant element and an endophyte organism arranged in a heterologous manner, in which the endophyte organism is a member of the genus Exserohilum and comprises at least one polynucleotide sequence at least 97% identical to a selected polynucleotide sequence from the group consisting of SEQ ID NOs: 60, 61 and 62, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism.
92. Synthetic composition according to claim 91, characterized by the fact that the vegetable element is a vegetable element of corn and the improved feature of agronomic importance is yield.
93. Synthetic composition according to claim 91, characterized by the fact that the vegetable element is a vegetable element of soy.
94. Synthetic composition according to claim 93, characterized by the fact that the vegetable element is a variety of soy selected from the group consisting of: Pfister 38R25, Dairyland DSR1808R2Y, Stine 3920 and varieties closely related to these and the improved trait of agronomic importance is yield.
95. Synthetic composition according to claim 91, characterized by the fact that the vegetable element is a vegetable element of wheat and the improved trait of agronomic importance is yield.
96. Synthetic composition according to claim 91, characterized by the fact that the vegetable element is a peanut vegetable element and the improved feature of agronomic importance is yield.
97. Synthetic composition according to claim 91, characterized by the fact that the vegetable element is a variety of peanuts selected from the group consisting of: AT9899, FloRun 107, Georgia-06G, Tamnut OL06, and varieties closely related to these .
98. Synthetic composition, characterized by the fact that it comprises a plant element and an endophyte organism arranged in a heterologous way, in which the endophyte organism is a member of the Epicoccum genus and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 63, 64 and 71, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism.
99. Synthetic composition according to claim 98, characterized by the fact that the plant element is a soy plant element and the enhanced feature of agronomic importance is selected from the group consisting of root area, root length and dry weight from the root.
100. Synthetic composition according to claim 98, characterized by the fact that the vegetable element is a variety of Stine 33E22 soybeans or a variety closely related to it.
101. Synthetic composition according to claim 98, characterized by the fact that the vegetable element is a peanut vegetable element and the enhanced feature of agronomic importance is yield.
102. Synthetic composition according to claim 101, characterized by the fact that the peanut vegetable element is selected from the group consisting of the FloRun 107, Georgia-06G variety, and varieties closely related to these.
103. Synthetic composition according to claim 98, characterized by the fact that it further comprises a second endophyte organism, in which the second endophyte organism is of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 52, 53, 54, 55, 56, 57, 58 and 59, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet understand the endophyte organism.
104. Synthetic composition according to claim 99, characterized by the fact that the vegetable element is the Stine 33E22 soybean variety and the improved trait of agronomic importance is dry root biomass.
105. Synthetic composition characterized by the fact that it comprises a plant element and an endophyte organism arranged in a heterologous way, in which the endophyte organism is a member of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a selected polynucleotide sequence from the group consisting of SEQ ID NOs: 38, 39, 40 and 41, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism.
106. Synthetic composition, according to claim 105, characterized by the fact that the vegetable element is a vegetable element of wheat and the trait of agronomic importance is yield.
107. Synthetic composition characterized by the fact that it comprises a plant element and an endophyte organism arranged in a heterologous way, in which the endophyte organism is a member of the genus Epicoccum and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 70, in which said synthetic composition is capable of providing a trait of enhanced agronomic importance compared to the reference plant element that does not additionally comprise the endophyte organism.
108. Synthetic composition according to claim 107,
characterized by the fact that the vegetable element is the Brett Young 5525 canola variety or a closely related variety.
109. Synthetic composition characterized by the fact that it comprises a plant element and an endophyte organism arranged in a heterologous way, in which the endophyte organism is a member of the genus Curvularia and comprises at least one polynucleotide sequence at least 97% identical to a selected polynucleotide sequence of the group consisting of SEQ ID NOs: 42, 43, 44, 45, 65 and 66, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet understand the organism endophyte.
110. Synthetic composition according to claim 109, characterized by the fact that the vegetable element is a soy vegetable element and the feature of agronomic importance is selected from the group consisting of root area, root length and yield.
111. Synthetic composition according to claim 110, characterized by the fact that the vegetable element is a variety of soy selected from the group consisting of Pfister 38R25, Stine 3920 and varieties closely related to these.
112. Synthetic composition according to claim 109, characterized by the fact that the vegetable element is a vegetable element of wheat and the feature of agronomic importance is yield.
113. Synthetic composition according to claim 112, characterized by the fact that the vegetable element is a variety of wheat selected from the group consisting of SDSU Focus, SDSU Select and varieties closely related to them.
114. Synthetic composition characterized by the fact that it comprises a plant element and an endophyte organism arranged in a heterologous manner, in which the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 67, in which said synthetic composition is capable of providing a trait of enhanced agronomic importance compared to the reference plant element that does not additionally comprise the endophyte organism.
115. Synthetic composition according to claim 114, characterized by the fact that the plant element is a soy plant element and the feature of agronomic importance is selected from the group consisting of root area, root length and yield.
116. Synthetic composition according to claim 115, characterized by the fact that the vegetable element is a variety of soy selected from the group consisting of Pfister 38R25, Stine 3920 and varieties closely related to these.
117. Synthetic composition characterized by the fact that it comprises a plant element and an endophyte organism arranged in a heterologous manner, in which the endophyte organism is a member of the genus Cladosporium and comprises at least one polynucleotide sequence at least 97% identical to a selected polynucleotide sequence of the group consisting of SEQ ID NOs: 67 and 68, in which the synthetic composition is capable of providing an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism.
118. Synthetic composition according to claim 117, characterized by the fact that the vegetable element is a vegetable element of corn and the feature of agronomic importance is yield.
119. Synthetic composition according to claim 118, characterized by the fact that the vegetable element is the Stine corn variety
9734.
120. Synthetic composition according to claim 117, characterized by the fact that the vegetable element is a soy vegetable element and the feature of agronomic importance is selected from the group consisting of root area, root length and yield.
121. Synthetic composition according to claim 120, characterized by the fact that the vegetable element is a variety of soy selected from the group consisting of Pfister 38R25, Stine 3920 and varieties closely related to these and the feature of agronomic importance is the Yield.
122. Synthetic composition according to claim 117,
characterized by the fact that the vegetable element is a vegetable element of wheat and the trait of agronomic importance is yield.
123. Synthetic composition according to claim 122, characterized by the fact that the vegetable element is a variety of wheat selected from the group consisting of SDSU Focus, SDSU Select and varieties closely related to these.
124. Synthetic composition according to claim 117, characterized by the fact that the endophyte organism comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 68, the plant element is a peanut of the AT9899 variety and the dash of agronomic importance is yield.
125. Synthetic composition characterized by the fact that it comprises a plant element and an endophyte organism arranged in a heterologous manner, in which the endophyte organism is a member of the genus Paecilomyces and comprises at least one polynucleotide sequence at least 97% identical to SEQ ID NO: 69, in which said synthetic composition is able to provide a trait of improved agronomic importance compared to the reference plant element that does not additionally comprise the endophyte organism.
126. Synthetic composition according to claim 125, characterized by the fact that the vegetable element is the canola variety NCC1015 and the trait of agronomic importance is yield.
127. Synthetic composition characterized by the fact that it comprises a plant element and an endophyte organism arranged in a heterologous way, in which the endophyte organism is a member of the Acremonium genus and comprises at least one polynucleotide sequence at least 97% identical to a selected polynucleotide sequence of the group consisting of SEQ ID NOs: 46, 47, 48, 49, 50 and 51, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet understand the organism endophyte.
128. Synthetic composition according to claim 127, characterized by the fact that the plant element is a soy plant element and the feature of agronomic importance is the dry shoot biomass.
129. Synthetic composition according to claim 127, characterized by the fact that the vegetable element is a vegetable element of wheat and the feature of agronomic importance is the moisture of the grain or the yield.
130. Synthetic composition according to claim 129, characterized by the fact that the vegetable element is the SDSU Select wheat variety and the trait of agronomic importance is yield.
131. Synthetic composition according to claim 127, characterized by the fact that the vegetable element is the Georgia-06G peanut variety and the trait of agronomic importance is yield.
132. Synthetic composition according to claim 127, characterized by the fact that the vegetable element is a vegetable element of rice and the feature of agronomic importance is the dry shoot biomass.
133. Synthetic composition according to claim 127, characterized by the fact that it further comprises a second endophyte organism, in which the second endophyte organism is of the genus Coniochaeta and comprises at least one polynucleotide sequence at least 97% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism.
134. Synthetic composition according to claim 133, characterized by the fact that the vegetable element is a vegetable element of wheat and the feature of agronomic importance is selected from the group consisting of plant height, fresh root biomass and weight of the fresh sprout.
135. Synthetic composition according to claim 133, characterized by the fact that the vegetable element is a vegetable element of rice and the feature of agronomic importance is yield.
136. Synthetic composition characterized by the fact that it comprises a plant element and an endophyte organism arranged in a heterologous way, in which the endophyte organism is a member of the genus Chaetomium and comprises at least one polynucleotide sequence at least 97% identical to a selected polynucleotide sequence of the group consisting of SEQ ID NOs: 52, 53, 54,
55, 56, 57, 58 and 59, in which the synthetic composition is able to provide an improved trait of agronomic importance compared to a reference plant element that does not yet comprise the endophyte organism.
137. Synthetic composition according to claim 136, characterized by the fact that the vegetable element is a soy vegetable element and the feature of agronomic importance is selected from the group consisting of root area, root length, sprout biomass dry and yield.
138. Synthetic composition according to claim 137, characterized by the fact that the vegetable element is a variety of soy selected from the group consisting of Pfister 38R25, Stine 3920 and varieties closely related to these and the trait of agronomic importance is the Yield.
139. Synthetic composition according to claim 136, characterized by the fact that the vegetable element is a vegetable element of wheat and the feature of agronomic importance is the length of the root or yield.
140. Synthetic composition according to claim 139, characterized by the fact that the vegetable element is a variety of wheat selected from the group consisting of SDSU Focus, SDSU Select and varieties closely related to these and the trait of agronomic importance is the Yield.
141. Synthetic composition according to any one of claims 91-140, characterized by the fact that the enhanced feature of agronomic importance is conferred under normal irrigation conditions.
142. Synthetic composition according to any one of claims 91-140, characterized by the fact that the plant element is a seed.
143. Synthetic composition according to claim 142, characterized by the fact that the seed is modified.
144. Synthetic composition according to any of claims 91-140, characterized by the fact that the plant element is placed on a substrate that promotes plant growth.
145. Synthetic composition according to claim 144,
characterized by the fact that the substrate that promotes plant growth is the soil.
146. Plurality of synthetic compositions according to claim 145, characterized by the fact that a plurality of said plant elements are placed in the soil in rows, with substantially equal spacing between each seed within each row.
147. Synthetic composition according to any one of claims 91-140, characterized in that it further comprises a formulation comprising one or more of the following: stabilizer, preservative, carrier, surfactant, anti-complex agent or any combination thereof.
148. Synthetic composition according to any one of claims 91-140, characterized in that it further comprises a formulation comprising one or more of the following: fungicide, nematicide, bactericide, insecticide or herbicide.
149. Synthetic composition according to any one of claims 91-140, characterized by the fact that said compositions are confined to an object selected from the group consisting of: bottle, vial, ampoule, package, container, bag, box, can, envelope, cardboard box, container, silo, shipping container, floor of the body or crate.
150. Synthetic composition according to any one of claims 91-140, characterized by the fact that the combinations are self-stable.
151. Synthetic composition according to any one of claims 91-140, characterized by the fact that the identity percentage is at least 98%.
152. Synthetic composition according to any of claims 91-140, characterized by the fact that the identity percentage is at least 98%.
153. Synthetic composition according to any one of claims 91-140, characterized by the fact that the identity percentage is 100%.
154. Synthetic composition according to any one of claims 91-140, characterized by the fact that the percentage of identity is determined over an alignment region of at least 100 nucleotides.
155. Synthetic composition according to any one of claims 91-140, characterized by the fact that the percentage of identity is determined over an alignment region of at least 200 nucleotides.
156. Synthetic composition according to any one of claims 91-140, characterized by the fact that the percentage of identity is determined over an alignment region of at least 300 nucleotides.
157. Synthetic composition according to any one of claims 91-140, characterized by the fact that the percentage of identity is determined over an alignment region of at least 400 nucleotides.
158. Synthetic composition according to any one of claims 91-140, characterized by the fact that the percentage of identity is determined over an alignment region of at least 500 nucleotides.

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法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-11-30| B06W| Patent application suspended after preliminary examination (for patents with searches from other patent authorities) [chapter 6.23 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762465819P| true| 2017-03-01|2017-03-01|

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US201762465798P| true| 2017-03-01|2017-03-01|

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US201762465797P| true| 2017-03-01|2017-03-01|

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US62/465,798|2017-03-01|

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US62/465,797|2017-03-01|

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US62/465,819|2017-03-01|

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US201762466256P| true| 2017-03-02|2017-03-02|

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US201762465834P| true| 2017-03-02|2017-03-02|

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US201762466253P| true| 2017-03-02|2017-03-02|

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US62/466,253|2017-03-02|

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US62/466,256|2017-03-02|

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US62/465,834|2017-03-02|

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US201762467742P| true| 2017-03-06|2017-03-06|

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US201762467755P| true| 2017-03-06|2017-03-06|

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US201762467740P| true| 2017-03-06|2017-03-06|

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US62/467,755|2017-03-06|

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US62/467,740|2017-03-06|

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US62/467,742|2017-03-06|

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US201762556288P| true| 2017-09-08|2017-09-08|

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US62/556,288|2017-09-08|

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PCT/US2017/064361|WO2018160245A1|2017-03-01|2017-12-01|Endophyte compositions and methods for improvement of plant traits|

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