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    • 专利摘要:
    Meyerozyma guilliermondii strain, composition and methods to promote growth and activate plant defenses. The present invention relates to a new strain of Meyerozyma guilliermondii that simultaneously has the ability to promote plant growth and the ability to control infections by phytopathogenic fungi. It also includes mutant strains and/or derivatives of this strain of M. guilliermondii, which maintain the same properties. It also refers to compositions that include said strain, its mutants and/or derived strains, for use as a biostimulant and/or biopesticide in plants, and to methods to promote growth and activate plant defenses that include the addition of the new strain. by M. guilliermondii. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2792777A1
    申请号:ES202030830
    申请日:2020-08-03
    公开日:2020-11-11
    发明作者:Garay Aránzazu Gómez；López Beatriz Pintos；Calderón Sergio Astudillo
    申请人:Universidad Complutense de Madrid；
    IPC主号:
    专利说明:

    [0002] STRAIN OF Meyerozyma guilliermondii, COMPOSITION AND METHODS TO PROMOTE GROWTH AND ACTIVATE PLANT DEFENSES
    [0004] TECHNICAL SECTOR
    [0006] The present invention is framed within the agricultural sector, in the field of control of fungal infections in plants and agri-food products, and the promotion of plant growth through biofertilizers. More specifically, it is related to the sectors of Plant Production, Phytopathology and the Food Industry, and refers to the application of yeasts as growth promoters, for the biocontrol of phytopathogenic fungi and as elicitors of compounds of agri-food interest.
    [0008] BACKGROUND OF THE INVENTION
    [0010] In the field of agriculture, during the last decades, substitutes for synthetic chemical products have been sought both to avoid infections in plants and food losses in harvested products, as well as to favor plant growth and enhance the increased plant production. In this sense, one of the fields of study has focused on the use of microorganisms with antifungal and / or antimicrobial capacity and both bacteria and useful yeasts have been obtained. Similarly, another group of microorganisms have proven useful as biofertilizers.
    [0012] Bacteria and yeasts with antifungal power have been described:
    [0014] US6960342B2 refers to a strain of Bacillus amyloliquefaciens with antifungal activity against a variety of fungi including different species of genera such as Alternaria, Curvularía, Fusarium, Sclemtium, etc.
    [0016] ES2519168T3 and ES2401004T3 describe the use of a Metschnikowia fructicola strain in the treatment of plants against unwanted microorganisms, citing species belonging, among others, to the Aspergillus, Penicillium or Fusarium genera .
    [0017] US6423310B1 refers to the biocontrol of postharvest diseases in plants by methods that include the use of antagonistic microorganisms belonging to, among others, the genera Candida, Cryptococcus, Pichia, Bulleromyces, Rhodotorula, etc.
    [0019] In Biological Control (2008) 47: 207-215, Chanchaichaovivat, A. et al. describe a strain of Pichia guilliermondii with postharvest antifungal capacity against Colletotrichum capsici.
    [0021] As for the solutions to increase plant growth, we can cite several documents:
    [0023] ES2147160B1 includes the description of two bacterial strains ( Bacillus pumilus and B. licheniformis) that produce plant growth regulating compounds, from the group of gibberellins.
    [0025] ES2594455T3 includes a strain of Glomus iranicum var. Tenuihypharum which, along with other components, is used as a biostimulant.
    [0027] In 2019, Xia et al. published a work in which they identified 740 cultivable fungal isolates that may be beneficial for the growth, health and performance of plants (Xia, Y. et al. Scientific Reports (2019) 9: 1669). Among them, they selected 6 strains with especially interesting capacities: Coniothyrium aleuritis isolate 42, Pichia guilliermondii isolate F15, Fusarium oxysporum strain NSF2, Fusarium proliferatum strain AF04, Aspergillus nidulans strain FH5, and Trichoderma spirale strain YIMPH30310.
    [0029] Basha, H. and Ramanujam, B. published in 2015 ( Biocontrol Science and Technology 25 (2): 185-206) a study on a strain of Pichia guilliermondii and a strain of Hanseniaspora uvarum with an interest in promoting growth and in the biocontrol, respectively.
    [0031] The interest in finding strains of microorganisms capable of improving the conditions of plants of agri-food interest, either against diseases or in terms of its performance, it is evident. However, so far, strains of bacteria or yeasts with specific capacities have been disclosed within the wide range of activities that are being investigated.
    [0033] EXPLANATION OF THE INVENTION
    [0035] Meyerozyma guilliermondii strain , composition and methods to promote growth and activate plant defenses.
    [0037] One aspect of the present invention refers to a Meyerozyma guilliermondii strain isolated in 2016 from vine wood without disease symptoms in a field of plants with tinder symptoms in La Rioja and deposited in the Spanish Collection of Type Cultures (CECT ), University of Valencia, UV Science Park, 46980 Paterna (Valencia), with reference number CECT13190, which comprises the sequence characterized by SEQ ID NO: 3.
    [0039] This specification shows how M. guilliermondii CECT13190 favors plant growth, so one of its applications is as a biostimulant in plants and, in addition, it activates plant defenses, so that it can also be applied as a biopesticide.
    [0041] Indole-3-acetic acid (IAA), or auxin, is a promoter of growth and crop yield by inducing the formation and increase of root hairs that facilitate the uptake of nutrients. M. guilliermondii CECT13190 presents yields of 9.8 pg / mL of IAA. The production of indole acetic acid is observed in the absence of L-tryptophan (7.2 pg / mL), which shows that this yeast is capable of producing the hormone through a different pathway and, therefore, without the use of the amino acid as precursor. The concentrations are sufficient so that an increase in the growth of the plant is observed.
    [0043] On the other hand, the main nutrient in agriculture for plant growth is nitrogen; in fact, for a plant that has its water needs covered, the fundamental limiting factor is nitrogen. Therefore, the microorganisms capable of fixing it are a useful tool as biostimulants. The ability of M. guilliermondii CECT13190 to fix atmospheric nitrogen and, therefore, to transform it in NH4 +, it makes this nutrient available to the plant. This ability is considered a characteristic feature of Plant Growth Promoting Microorganisms. Furthermore, the nitrogenous nutritional regimen has an impact on the amino acid biosynthesis patterns that affect gene expression, including that of defense genes.
    [0045] Another essential nutrient for plants is phosphorus, which is very frequently found in the soil in an insoluble form. In this sense, microorganisms capable of solubilizing phosphorus are recognized as promoters of plant growth since soluble phosphates are absorbed by the plant and this improves its growth and productivity. The M. guilliermondii strain CECT13190 is also capable of solubilizing phosphorus.
    [0047] In addition, M. guilliermondii CECT13190 produces 3-methyl-1-butanol, a volatile organic compound known to exhibit activity as a plant growth promoter.
    [0049] M. guilliermondii CECT13190 also acts in the control of internal pathogens that grow and are transported through the vessels of plants and that, consequently, are difficult to treat with conventional treatments that, in addition, can be harmful to the environment (such as phytochemicals).
    [0051] Among the volatile organic compounds produced by M. guilliermondii CECT13190, significant amounts of hexamethyl cyclotriloxane, known for its activity against pathogens, phenyl ethyl alcohol, which slows fungal growth, and isogeraniol, a compound known as an antifungal, have been detected.
    [0053] In addition, the strain of the invention produces siderophores that behave as chelating agents, sequestering iron even in the presence of other metals and reducing it to Fe2 +, which favors its solubility and its availability as a nutrient, promoting plant growth, while promoting the biological control of fungi and bacteria pathogens for plants.
    [0055] M. guilliermondii CECT13190 reduces the production of superoxide dismutase (SOD). Thus, the transformation of reactive oxygen species (ROS) to hydrogen peroxide is reduced, which could translate into an increase in oxidative stress, inducing the expression of genes related to pathogenesis (PR).
    [0057] M. guilliermondii CECT13190 increases the production of oxalic acid in plants. Oxalic acid can act as an inhibitor of the SOD enzyme function, preventing the transformation of ROS to hydrogen peroxide, which could be translated into an increase in oxidative stress, inducing the expression of PR genes.
    [0059] Plants inoculated with M. guilliermondii CECT13190 present higher amounts of salicylic acid than plants that are not inoculated with the strain of the invention. Salicylic acid participates in several processes that promote plant growth, increasing crop productivity, and also in plant pathogen resistance.
    [0061] On the other hand, plants have a series of inducible mechanisms to defend themselves against pathogens, mechanisms based on the recognition of the pathogen, the transduction of signals and the consequent activation of the plant's defenses. Among the mechanisms that are activated in these situations, there is the involvement of PR proteins and two types of very active response are distinguished: acquired systemic resistance (RSA) and induced systemic resistance (RSI). The strain of the invention, when inoculated into a plant, induces both the expression of RSA genes and the expression of RSI genes.
    [0063] The invention also relates to mutant strains and / or derivatives of the M. guilliermondii CECT13190 strain that maintain their properties, that is:
    [0064] - production of IAA, even in the absence of L-tryptophan;
    [0065] - fixation of atmospheric nitrogen and transformation into NH4 +;
    [0066] - solubilization of phosphorus;
    [0067] - production of 3-methyl-1-butanol;
    [0068] - production of hexamethyl cyclotriloxane;
    [0069] - production of phenyl ethyl alcohol;
    [0070] - production of siderophores;
    [0071] - reduction of SOD production, which implies reduction of ROS transformation and induction of PR genes;
    [0072] - increased production of oxalic acid in the plants to which it is administered; - increase in the amount of salicylic acid in the plants to which it is administered; - induction of the expression of the RSA and RSI genes in the plants to which it is administered.
    [0074] A second aspect of the invention is a composition that includes the M. guilliermondii CECT13190 strain, mutant strains and / or derivatives thereof that maintain the same properties, as biostimulants, promoters of plant development and growth, and / or as biopesticides. , stimulating the plant's defenses
    [0076] Another aspect of the invention refers to a composition that includes the M. guilliermondii CECT13190 strain, mutant strains and / or derivatives thereof that maintain the same properties, whose presentation can be solid, liquid or in gel.
    [0078] The invention also refers to a method to promote growth and activate plant defenses that includes the addition of M. guilliermondii CECT13190, mutant strains and / or derivatives thereof that maintain the same properties, or of the compositions as contain the plants to be treated. This addition can be made by any of the commonly used treatments, for example, including the strain or composition in the irrigation water, by root treatments in the nursery or by spraying.
    [0080] BRIEF DESCRIPTION OF THE DRAWINGS
    [0082] To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, a set of drawings is attached as an integral part of said description in which, with an illustrative and non-limiting nature, it has been represented the next:
    [0084] Figure 1 . Detail of the cucumber seedlings one week after sowing
    [0086] Figure 2. Representation of the leaf area, in cm2, of control cucumber plants (blue column) and cucumber plants treated with M. guilliermondii CECT13190 (green column).
    [0088] Figure 3. Experimental design to verify the applicability of M. guilliermondii CECT13190 as a preventive and / or curative treatment against Fusarium oxysporum f sp. Cucumerinum.
    [0089] Figure 4. Cucumber fusarium symptoms in 14-day post-emergence plants with different treatments.
    [0091] PREFERRED EMBODIMENT OF THE INVENTION
    [0093] The present invention is illustrated by the following examples which are not intended to be limiting of its scope.
    [0095] Example 1. Isolation of a strain of M. guillierm ondii CECT13190.
    [0096] The isolation was carried out from vine wood flakes, from samples taken in a private farm in La Rioja, in September 2016. For this, a superficial asepsis was carried out by washing with 1.5% sodium hypochlorite for 1 minute and two subsequent washes with sterile distilled water for one minute each. The wood flakes, approximately 0.5x0.5 cm2, were obtained in a laminar flow cabinet with a scalpel. The flakes were seeded in PDA medium ( Potato dextrose agar) in a 9 cm diameter Petri dish and kept at 22 ± 2 ° C, in the dark. After 48 hours, seeding was carried out in fresh PDA medium under the same conditions and this process was repeated until the axenic culture of M. guilliermondii CECT13190 was achieved.
    [0098] Example 2. Genetic identification of the strain of M. guillierm ondii CECT13190 The genetic identification of the strain of M. guilliermondii CECT13190 was carried out as follows:
    [0100] 2.1. DNA extraction
    [0101] DNA extraction was performed using the protocol described below. Yeast was grown in PDB medium ( Potato Dextrose Broth) for 72 hours at 33 ± 2 ° C. Subsequently, it was centrifuged at 10,000 rpm and the supernatant was decanted. 300 µL of extraction buffer (200 mM Tris HCl pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS) and 150 µL of 3 M sodium acetate pH 5.2 were added to the precipitate and incubated at - 20 ° C for 10 minutes. After this time, it was centrifuged for 5 minutes at 10,000 rpm and the supernatant was transferred to another tube where the same volume of isopropanol was added. It was incubated for 5 minutes at room temperature, after this time it was centrifuged at 10,000 rpm for 5 minutes. The precipitate was washed with 70% ethanol. Once dry, it was resuspended in 50 µL of TE (Tris-EDTA). DNA was stored at -20 ° C until use. The amount of DNA was quantified by spectrophotometry using absorbance at 260 nm (50 pg / ml of DNA has an OD of 260 = 1) and purity calculating the A260 / A280 ratio (1.7-1.9 corresponds to pure DNA) using a NanoDrop One (Thermo).
    [0103] 2.2. DNA amplification by PCR of the ITS region fragment ( Internal Transcribed Spacer region)
    [0104] PCR amplification was carried out in a Mastercycler Gradient thermal cycler (Eppendorf), in a final volume of 25 µl with the following mixture: 1x Taq polymerase buffer, 1.2mM MgCl2, 0.2mM dNTPs, 50 ng ITS1 primer; ITS450 ng primer; Taq DNA polymerase (Ecogen) 1 U and DNA 20 ng, using DNA extracted as explained in Example 2.1.
    [0106] The primers used, ITS1 and ITS4 (White, TJ, et al. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A guide to Methods and Applications. Innis, MA, DH Gelfand, JJ Sninsky , and TJ White (eds). Academic Press, San Diego, USA. pp: 315-322) characterized by SEQ ID NO: 1 and SEQ ID NO: 2, respectively, are universal primers, which are located in the 18s flanking regions and 28s, and which are used for the identification of fungal species by amplifying a fragment in the ITS region and 5.8s rDNA (400-650 bp). The amplification conditions were the following: 5 minutes 95 ° C, 40 cycles: 30 seconds 95 ° C, 30 seconds 55 ° C and 1 minute 72 ° C to finish with an extension of 10 minutes at 72 ° C.
    [0108] 2.3. ITS fragment sequencing
    [0109] Subsequently, the PCR products were sequenced, using the ITS1 and ITS4 primers, characterized by SEQ ID NO: 1 and 2, at the Genomics Unit of the UCM. The sequences obtained were used for identification in the GenBank database using the BLAST algorithm ( Basic Local Alignment Search Tool). The sequence of the ITS fragment, characterized by SEQ ID NO: 3, showed 99.35% homology with 100 strains of M. guilliermondii.
    [0111] Example 3. Characterization of the strain of M. guillierm ondii CECT13190
    [0113] 3.1. Growth curve.
    [0114] To perform the growth curve, the new isolate was grown in test tubes with 5 mL of PDB medium for 24 hours at 28 ± 2 ° C while shaking (250 rpm). After 24 hours, the culture was transferred to a flask with 100 mL of PDB medium and incubated at 28 ± 2 ° C for 52 hours at 250 rpm. Samples were collected at 4 hour intervals. Growth was calculated by analyzing the turbidity of the medium, measuring the absorbance at 600 nm in a spectrophotometer (Genesys 10S UV-Vis; Thermo). The maximum growth was 9 x 107 colony forming units / ml at 48 hours.
    [0116] 3.2. Production of indole-3-acetic acid (IAA).
    [0117] To analyze the production of indole-3-acetic acid (IAA), the yeast was inoculated into flasks with 100 mL of Trypticasein Soy Broth medium (TSB) supplemented with L-tryptophan at different concentrations (1, 100, 200, 300, 400 and 500 pg / mL). The culture was kept on an orbital shaker at 150 rpm and 30 ± 2 ° C. Samples were taken at 24, 48, 72 and 96 hours. The samples were centrifuged at 10,000 rpm and to 1 mL of supernatant, 4 mL of Salkowsky's reagent (250 mL of distilled water, 150 mL of H2SO4 and 7.5 mL of 0.5 M FeCh-6 H2O) were added and stirred. The mixture was incubated at room temperature for 20 minutes and in the dark. After this time, the absorbance at 535 nm was measured. AIA quantification was carried out with a standard curve of known AIA concentrations (0-400 pg / mL).
    [0119] The highest production of IAA was obtained when the amount of the amino acid in the medium was 400 pg / mL, with productions of 9.8 pg / mL of IAA. The production of indole acetic acid was observed in the absence of L-tryptophan (7.2 pg / mL), which shows that this yeast is capable of producing the hormone through a different pathway and, therefore, without the use of the amino acid as precursor. The concentrations are sufficient so that an increase in the growth of the plant is observed.
    [0121] 3.3. Fixation of nitrogen.
    [0122] The nitrogen fixing capacity of the yeast was evaluated by the following protocol. M. guilliermondii was grown in PDB for 48 hours, 30 ± 2 ° C and 140 rpm on an orbital shaker. Then, 10 mL were centrifuged at 10,000 rpm for 5 minutes. The pellet was resuspended in a 0.8% NaCl solution. A 10 pL aliquot was inoculated into vials with NFb medium (malic acid 5g, K2HPO40.5g, MgSO4-7H2O 0.2g, NaCl 0.1g, CaCl-2H2O 0.02 g, Na2MoO4-2H2O 1g, MnSO4-H2O 1.175g, KOH 4g , 3.2-3.9 g agar, 4 ml of bromothymol blue solution (5g / l in 0.2 N KOH), 1000 ml distilled H2O, pH between 6.8-7) and incubated at 33 ± 2 ° C.
    [0123] After its incubation, the presence of a veil (the culture medium became translucent) with a size of 0.2 cm deep was observed in the culture tube with a diameter of 3 cm. The ability of M. guilliermondii CECT13190 to fix atmospheric nitrogen and, therefore, to transform it into NH4 +, allows this nutrient to remain available to the plant.
    [0125] 3.4. Solubilization of phosphates from the medium
    [0126] The ability of yeast to solubilize phosphates was also evaluated: it was cultivated at 33 ± 2 ° C in the dark for 96 hours on Pikovskaya medium (900 ml of solution A: 13 g dextrose, 2.5 g calcium phosphate, 0.5 g ammonium sulfate , 0.2 g 10 potassium chloride, 0.1 g magnesium sulfate, 1 ml traces of manganese sulfate (100 mg / L), 1 ml traces of iron sulfate (100 mg / L), 0.022 g bromocresol green, 15 g agar ) and 100 ml of solution B: 2.4 g tricalcium phosphate).
    [0128] The solubilization index (IS) of phosphates was calculated according to the following formula:
    [0129] IS = ((diameter of the colony diameter of the halo) / diameter of the colony).
    [0131] An IS of 1.84 ± 0.03 was obtained, which is found in the average of the values of other yeasts with the ability to solubilize phosphates.
    [0133] 3.5. Volatile organic compound analysis.
    [0134] Using GC-MS (gas chromatography - mass spectrometry), an analysis of the major volatile organic compounds produced by M. guilliermondii CECT13190 was performed. The analyzes were carried out at the chromatography research support center of the Complutense University of Madrid.
    [0136] Among the volatile organic compounds produced by yeast and detected using this technique, the following stand out:
    [0137] - 3-methyl 1-butanol, which shows activity as a plant growth promoter (31 ± 2% of the total volatile compounds produced by yeast);
    [0138] - hexamethyl cyclotriloxane, with activity against pathogens (0.01 ± 0.01% of the total volatile compounds produced by yeast);
    [0139] - phenyl ethyl alcohol, which slows down fungal growth (8.51 ± 2.35% of the total volatile compounds produced by yeast);
    [0140] - iso geraniol, known as an antifungal (0.03 ± 0.02% of the total volatile compounds produced by yeast).
    [0142] 3.6. Production of siderophores.
    [0143] The determination of the production of siderophores was qualitatively measured in CAS medium ( chromne azurol S) agar. The composition of the CAS reagent was: 1 mM CAS, 10 mM FeCl3-6H2O (1 mM stock) diluted in a solution of 10 mM HCl and 2 mM of N, N-cetyl trimethyl ammonium bromide (CTAB). The yeasts were inoculated into the medium and incubated at 33 ± 2 ° C for 96 hours. The medium changed color from blue to yellow, orange or pink, which is interpreted as positive in the formation of siderophores.
    [0145] Example 4. Effects of the strain of M. gu illie rm ond ii CECT13190 on plant growth
    [0147] The effect of M. guilliermondii CECT13190 on the size of the plants was studied. For this, cucumber plants were obtained from commercial seeds and they were grown in seedbeds with coconut fiber substrate (EC 95 mS / m, pH 6.2) under controlled conditions (Photoperiod 16/8, 22 ± 1 ° C) . Twenty days after sowing, they were randomly distributed into two groups of 24 plants, each one located in a separate mini-greenhouse. The plants were then inoculated. One greenhouse was inoculated with M. guilliermondii CECT13190 and the other greenhouse was kept as a control (the experiment was repeated three times). In figure 1, a detail of cucumber seedlings one week after sowing is shown. In the upper right corner is the control plant, the other three plants correspond to the treatment with M. guilliermondii CECT13190.
    [0149] Figure 2 shows the average foliar area of cucumber plants in control treatment (C) and inoculated with M. guilliermondii CECT13190. The measurements were made from photographs of the leaves using a contrast technique with the ImageJ program. Those plants treated with M. guilliermondii CECT13190 were found to have leaf area / leaf values (14 ± 1 cm2) significantly higher than the control (10 ± 0.5 cm2). Similarly, the total number of leaves also reached maximum values in these plants.
    [0151] The state of development of the plants was determined according to the extended version of the BBCH scale. An advance in the phenology of plants treated with M.
    [0152] guilliermondii CECT13190 compared to controls in the number of true leaves (growth stage 1, foliar development, code 1. on the BBCH scale, number of true leaves) and in advance of flowering (growth stage 6, flowering, code 61 , on the BBCH scale) with one more leaf and a 17% advance of flowering.
    [0154] Example 5. M. guillierm ondii CECT13190 as an eliciter of defenses in plants
    [0156] The ability of M. guilliermondii CECT13190 as an elicitator of defenses in cucumber and vine plants was analyzed.
    [0158] In cucumber: cucumber plants were obtained from commercial seeds and were grown in seedbeds with coconut fiber substrate (EC 95 mS / m, pH 6.2) under controlled conditions (16/8 photoperiod, 22 ± 1 ° C) . At 20 days post-sowing, they were randomly distributed into three groups of 24 plants, each one located in a separate greenhouse. The plants were then inoculated as described below.
    [0160] On vine: glass jars (17x25.5x7cm) and vermiculite were used, autoclaved at 121 ° C for 20 minutes. Vine twigs (certified tempranillo grafts, RJ51 / 110R-E35 clone) were subjected to asepsis (70% ethanol for 5 minutes and 4% bleach for 10 minutes, after which, three washes with sterile distilled water for 5 minutes) ). The varetas were watered, every other week, with filtered water and Hoagland medium No. 2 in a 1:10 dilution. The plants were randomly divided into three groups of 24 plants. When the varetas were already fully sprouted in State 13 according to the BBCH Scale Guide for the vine (Lorenz, DH; KW Eichhorn; H. Bleiholder; R. Klose; U. Meier; E. Weber (1994). Weinrebe (Vitis vinifera L. ssp. Vinifera) ". Vitic. Enol. Sci. 49: 66-70), the plants were inoculated as described below.
    [0162] In figure 3, a diagram of the experiments designed to verify this characteristic of the strain is shown.
    [0164] Group 1. Control (C): plants watered with filtered tap water.
    [0166] Group 2. Fusarium (P): inoculation with Fusarium oxysporum f sp. cucumerinum (in cucumber) and Fusarium equiseti (on vine). The plants were watered with a mixture of spores and mycelium.
    [0168] Group 3. M. guilliermondii CECT13190 (CECT13190): inoculation with M. guilliermondii CECT13190: 10% yeast (4ml of PDB medium with the yeast grown for 48h under stirring at 120rpm together with 36mL of filtered water).
    [0170] After inoculation, all plants were watered with filtered water for 20 days. Subsequently, a second inoculation was carried out. For this, the previous groups were subdivided and treated as indicated:
    [0172] Group 1a. Control (C): plants watered with filtered tap water.
    [0173] Group 1b. Control Fusarium (CP): plants watered with filtered tap water and, after 20 days, inoculated with Fusarium.
    [0175] Group 2a. Fusarium (P): plants previously inoculated with Fusarium sp., Were continued to be watered with filtered tap water.
    [0176] Group 2b: Fusarium + M. guilliermondii CECT13190 (P CECT13190): plants initially inoculated with Fusarium sp. and, after 20 days, inoculated with 10% M. guilliermondii CECT13190 (as described above).
    [0178] Group 3a. M. guilliermondii CECT13190 (CECT13190): inoculation with M. guilliermondii CECT13190 at 10%.
    [0179] Group 3b. M. guilliermondii CECT13190 Fusarium (CECT13190 P): plants initially inoculated with M. guilliermondii CECT13190 and, after 20 days, inoculated with Fusarium.
    [0181] In figure 4, an example of results obtained in cucumber plants is shown. The symptoms of cucumber fusariosis ( F. oxysporum f sp. Cucumerinum) are represented in plants 14 days post-emergence of the treatments: C (control), M (irrigated with M. guilliermondii strain CECT13190), F (irrigated with F. oxysporum f sp. cucumerinum), CF (control plants one week and then inoculated with F. oxysporum f sp. cucumerinum), MF (preventive treatment: one week with M. guilliermondii strain CECT13190 and then inoculated with F. oxysporum f sp. cucumerinum) and FM (curative treatment: one week with F. oxysporum f sp. cucumerinum and then irrigated with M. guilliermondii strain CECT13190); healthy plants are represented in green, live plants with symptoms in yellow, and dead plants in red.
    [0183] Example 6. Meyerozyma guillierm ondii CECT13190 as an activator of the defenses of grapevine plants, both via RSA (Systemic Acquired Resistance) and RSI (Systemic Induced Resistance).
    [0185] 6.1 Production of superoxide dismutase (SOD)
    [0186] 250mg of plant tissue were taken (in triplicate) from each of the inoculation tests described in Figures 3 and 4, previously treated with liquid nitrogen. The tissue was introduced into 2 ml Eppendorf tubes with 1.5mL of 0.05M potassium phosphate. The samples were centrifuged at 10,000rpm for 15 minutes. The supernatant, 200 ^ L, was collected and 3mL of the reaction mixture (2.35 ml of Na-P buffer 50 mM, 0.2 ml of EDTA Na20.66 mM, 0.3 ml of L-Methionine 10 mM, 0.1 ml NBT 33 ^ M and 0.05 ml of 0.0033 mM riboflavin). 200 µL of 3mL reaction mixture extraction buffer was used as blank and kept in the dark for 10 minutes. The control sample contained 200 µL of 3mL reaction mix extraction buffer and was incubated for 10 minutes exposed to strong light. After incubation, the absorbance at wavelength 560 nm was measured.
    [0188] The highest amount of SOD was produced in the plants that had received Control treatment. The least amount, in the treatment with the pathogen Fusarium sp. (10% less than in the control) and intermediate values were detected in the plants with crossed inoculations Fusarium sp. + Meyerozyma guilliermondii CECT13190 and for plants inoculated only with Meyerozyma guilliermondii CECT13190 (5% less than in the control). Thus, the transformation of ROS to hydrogen peroxide is reduced, which would translate into an increase in oxidative stress, inducing the transformation of the NPR1 oligomer to its monomeric form and, therefore, activating the expression of PR 3 and 5 (P -1,3-glucanase, osmotin and thaumatin, respectively.
    [0190] 6.2 Oxalic Acid Levels
    [0191] The oxalic acid levels in the vine twigs were measured with the Oxalic Acid Colorimetric Assay Kit MAK179-1KT (Sigma Aldrich). A higher quantity (an increase of 60%) of oxalic acid was detected in the rods inoculated with Meyerozyma guilliermondii CECT13190. Oxalic acid can act as an inhibitor of function of the SOD enzyme, avoiding the transformation of ROS to hydrogen peroxide. In this way, an increase in oxidative stress would be occurring, inducing the transformation of the oligomer of NPR1 to its monomeric form and, therefore, activating the expression of PR 3 and 5 (P-1,3-glucanase; osmotin and thaumatin , respectively).
    [0193] 6.3 Salicylic Acid Levels
    [0194] The levels of salicylic acid in situ in the leaves were measured in the apical leaves of each stem, which were immersed in a solution of TiO2 nanoparticles for 24 hours while stirring at 120rpm. The percentage of leaf area covered by brown spots was measured using the ImageJ program. Plants inoculated with Meyerozyma guilliermondii CECT13190 showed a 2.5-fold increase in the amount of salicylic acid compared to control plants. The expression of PRs genes is regulated by the presence of salicylic acid, consequently increasing resistance to fungal pathogens. The presence of salicylic acid increases the activity of oxidative stress and the amount of antioxidant compounds in the cytoplasm, causing the conversion of the oligomer NPR1 to monomer. This monomer is able to enter the nucleus of cells, where it is phosphorylated. The fact that it is phosphorylated and in the presence of salicylic acid, allows its interaction with SUMO-3, a type of ubiquitin that increases the association between NPR1 and the transcription factor TGA and with polymerase, while reducing it with the factor transcriptional WRKY (which, in the absence of salicylic acid, acts as a transcriptional repressor). With this, the transcription of SAR genes is carried out.
    [0196] 6.4 Expression of different genes that encode PRs and key enzymes of ethylene synthesis by real-time quantitative PCR (RT-qPCR).
    [0198] 6.4.1 Extraction, purification and quantification of Total RNA
    [0199] The plant material was frozen with liquid nitrogen and pulverized. 0.3 grams of each sample were macerated in 2mL Eppendorf, with 1 mL of extraction buffer [2% (w / v) CTAB, 2% (w / v) PVP-40, 300 mM Tris-HCl pH 8, 0, 1.4 M NaCl, 25 mM EDTA pH 8.0, 0.005% (w / v) spermidine, 2% (v / v) p-mercaptoethanol], preheated to 65 ° C. The mixture was incubated at 65 ° C for 10 minutes with shaking every 2 min. They were centrifuged for 5 min at 12,000 xg, and an equal volume of chloroform: isoamyl alcohol (24: 1 v / v) was added to the supernatant at 4 ° C. They were then vortexed for 1 minute and centrifuged for 10 min at 12,000 x g. The aqueous phase was collected superior in new tubes and the selective precipitation of the RNA was proceeded, adding an isovolume of LiCl 4M and incubating overnight at 4 ° C. Subsequently, it was centrifuged at 12000 x g for 15 min at 4 ° C. The precipitate was washed 3 times with cold 70% ethanol (v / v), after drying it was resuspended in 50 pL of RNAse-free water. The purity and concentration of the RNA was quantified using a Nanodrop ND-100 spectrophotometer (NanoDrop® Technologies).
    [0201] Treatment with recombinant DNase I allowed genomic DNA to be eliminated, following the manufacturer's instructions (InvitrogenTM): a volume of 50 µl of reaction mixture with RNase-free water, 10 µg RNA, 5 µl incubation buffer and 1 µl DNase recombinant (RNase-free) 2 U / pl was incubated at 37 ° C for 30 minutes; 5pl of DNAse inactivation reagenty were added and incubated at room temperature for 2 minutes; it was centrifuged at 10,000 xg, the RNA being quantified in a Nanodrop ND-100 spectrophotometer (NanoDrop® Technologies).
    [0203] 6.4.2 Synthesis of complementary DNA (cDNA).
    [0204] The cDNA synthesis was performed using the NZY FirstStrand cDNA Synthesis kit from NZYtech, following the manufacturer's instructions. The reaction mixture was prepared in a final volume of 20 µl in RNase-free water, 10 µl NZYRT 2x Master Mix, 2 µl NZYRT Enzyme Mix and 1 µg DNA-free RNA, incubated at 25 ° C for 10 min and reverse transcription was carried out at 50 ° C for 20 min. Transcriptase was inactivated at 85 ° C for 5 minutes. 1 µl of NZY RNAse H ( Escherichia coli) was added and the samples were incubated at 37 ° C for 20 min. 30 pL of autoclaved MilliQ RNAase-free water was added. The stock concentration of cDNA was prepared to be 0.20 pg / pL.
    [0206] 6.4.3 Study of gene expression (quantitative real-time PCR)
    [0207] The study of the relative expression of the genes that encode: the nonexpresser of PR genes 1 ( NPR1) (using oligonucleotides published in Le Henanff, G., et al. (2009) was carried out. "Characterization of Vitis vinifera NPR1 homologs involved in the regulation of pathogenesis-related gene expression. ”BMC Plant Biology, 9 (1), 54); thaumatin ( VviTLS1), osmotin ( VviOsmo), class I chitinase ( Vcchitb), p-1, Class I 3-glucanase ( Vcgns1), 1-amino-cyclopropane-carboxylate (ACC) synthase ( VviACS1) and ACC oxidase ( VviACO1) (using oligonucleotides designed by the POSTBIOTEC group of ICTAN-CSIC), characterized by SEQ ID NO: 4-19 (Table 1). Expression was quantified by NZY qPCR Green Master Mix (NZYtech) using manufacturer's specifications. The reactions were carried out in multiwell plates in an iCycler iQ (Bio-Rad) thermal cycler and the results were processed using iQ5 2.0 ( Standard Edition Optical System Software V2.0, Bio-Rad).
    [0209] Table 1. Oligonucleotides used in the study of Example 6.4.3.
    [0214] Amplification reactions were carried out in a final volume of 20 µl containing 10 µl of NZY qPCR Green Master Mix, 2 µl of each oligonucleotide and 4 µl of cDNA. The amplification protocol was: 5 ° C 2 min, 95 ° C 10 min, followed by 40 cycles at 95 ° C 20 s, 55-60 ° C, finally, the reaction was kept at 4 ° C. At the end of the PCR, a dissociation curve was developed in a temperature gradient from 65 to 95 ° C, with plate readings every 0.2 ° C, to evaluate the presence of oligonucleotide dimers. Three biological replicates were carried out with their two technical repetitions for each sample and each of the genes. The ubiquitin gene (NCBI reference number: EE253706) from V. vinifera was used as an internal reference gene to normalize the values of amplification (using the oligonucleotides characterized by SEQ ID NO: 18-19). The relative expression of the gene was calculated following the method of Livak and Schmittgen (Livak, KJ and Schmittgen, TD 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 ( -Delta Delta C ( T)) method. Methods 25: 402-408), in which the Ct (threshold cycle) of each tested gene and the reference gene (ACt = Ct of the gene of interest-Ct of the reference gene) in each sample were directly compared, and subsequently The ACt of the test samples were compared with respect to the reference sample (AACt = ACt of each sample -ACt of the calibrator sample or control samples), through the following formula: 2 -AACt.
    [0216] Plants inoculated with M. guilliermondii CECT13190 showed a higher expression of NPR1, which leads to a higher expression of pathogenesis-related proteins (PR).
    [0218] Three types of protein families related to pathogenesis were expressed in grapevine plants treated with M. guilliermondii CECT13190: thaumatin (PR-5), p-1,3-glucanase (PR-2), chitinase type I (PR- 3) and osmotin (PR-5). Thaumatin and osmotin have been identified as PRs whose function is to increase the permeability of the fungal membrane, which ends up causing its rupture.
    [0220] RSA (Systemic Acquired Resistance) genes are transcribed, which transcribe for the PR families 1, 2 (P-1,3-glucanase) and 5 (thaumatin and osmotin).
    [0222] Meyerozyma guilliermondii CECT13190 inoculated in the plant caused a high expression of the genes for ethylene biosynthesis (ACS1 and ACO1). The presence of ethylene induces the activation of the PR-5 (thaumatin and osmotin) and PR-3 (type I chitinase) family. Therefore, the PR-5 family is not only regulated by the presence of salicylic acid, it is also regulated by the internal production of ethylene, that is, the RSI (Systemic Response Induced).
    权利要求:
    Claims (6)
    [1]
    1. Meyerozyma guilliermondii strain deposited in the Spanish Collection of Type Cultures with deposit number CECT13190 that comprises the sequence characterized by SEQ ID NO: 3.
    [2]
    2. Mutant strains and / or derived from the Meyerozyma guilliermondii strain deposited in the Spanish Collection of Type Cultures with deposit number CECT13190 and that maintain the same properties as said strain.
    [3]
    3. Composition comprising the Meyerozyma guilliermondii strain defined in claim 1 and / or the mutant and / or derived strains defined in claim 2, for use as a biostimulant and / or biopesticide in plants.
    [4]
    Composition according to claim 3, the presentation of which can be liquid, solid or gel.
    [5]
    5. Method to promote growth and activate plant defenses that includes the addition of the M. guilliermondii strain CECT13190 defined in claim 1, and / or of mutants and / or derivatives defined in claim 2, or of the compositions defined in claims 3-4, to said plants.
    [6]
    6. Method according to claim 5, wherein the addition is carried out by incorporation of the M. guilliermondii strain CECT13190 defined in claim 1, and / or of mutants and / or derivatives defined in claim 2, or of the compositions defined in claims 2-3 to irrigation water.
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    同族专利:
    公开号 | 公开日
    WO2022029350A1|2022-02-10|
    ES2792777B2|2021-05-21|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US5780023A|1988-04-04|1998-07-14|The United States Of America As Represented By The Secretary Of Agriculture|Inhibiting plant pathogens with an antagonistic microorganism|
    CN107937285A|2017-11-23|2018-04-20|青岛农业大学|Prevent the biocontrol microorganisms Pichia guilliermondii Y 1 and its biological prevention and control agent of Verticil-Veined Diseases of Apple Fruit|
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