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    • 专利摘要:
    The present invention relates to a microbial composition or to a kit for the preparation thereof, which comprises at least one microbial strain that exhibits urease activity, preferably of the
    公开号:ES2555674A1
    申请号:ES201590038
    申请日:2013-03-15
    公开日:2016-01-07
    发明作者:Estefania HINAREJOS ESTEVE；Raquel DEL VAL BUEDO；Nuria TARANCON VALERA；Enrique RIQUELME TORRES
    申请人:Investig Y Aplic Biotecnologicas S L；INVESTIGACIONES Y APLICACIONES BIOTECNOLOGICAS SL；
    IPC主号:
    专利说明:

    USEFUL MICROBIAL COMPOSITION AGAINST NEMATODS OF VEGETABLE CROPS DESCRIPTION Invention Sector
    The present invention is framed in the field of biopesticides. More specifically, this invention relates to a set of microorganisms, which formulated in a carrier liquid maintains microbial viability, and which by its characteristics, promotes an environment that is unfavorable to phytopathogenic nematodes. Prior art
    Nematodes are the cause of the greatest damage to agriculture in the 10 tropical, subtropical and temperate regions around the world. Meloidogyne spp. It is the most significant genus of plant parasitic nematodes; its activity causes losses between 11% and 25% of crops in virtually all tropical regions.
    The nodules or gill-forming nematodes easily reach 15 harmful thresholds in a short time, if they encounter susceptible crops, such as tomatoes. They are so common in horticultural crops of subtropical and tropical climates, that they are sometimes taken as "representatives of phytopathogenic nematodes" in general (Luc et al., 1990). The nodules or gill-forming nematodes (Meloidogyne spp.) Are sedentary endoparasitic nematodes, therefore, the absence of host plant for prolonged periods, would tend to make them disappear.
    In general, favorable conditions for plant growth will also be favorable for the reproduction of Meloidogyne spp., Which is a problem for the population control of these parasites. Therefore, it is interesting to develop 25 compositions that are capable of controlling a plague of nematodes and simultaneously fertilize the soil or crop surface of a plant.
    It is known that PGPR (Plant Growth Promoting Rhizobacteria or bacteria that promote plant root growth), can improve plant growth and crop yield, in different ways, and through different
    mechanisms (Nelson, 2007). Some of the strains isolated by IAB, S.L. (Investigaciones y Aplicaciones Biotecnológica S.L.), considered PGPR, have been identified as biological control forms and alternatives to the use of pesticides, but without being related to nematode control. Specifically, Bacillus thuringiensis var. Kurstaki, strain IAB / BT / 01, Bacillus subtilis, strain CECT 7254, 5 Pseudomonas fluorescens, strain CECT 7255, Trichoderma harzianum, strain IAB / TH / 01 (Hinarejos, 2008).
    Under natural conditions, a plant is a potential host for several phytopathogenic microorganisms, among which relationships that can be of different nature are established. It could be expected that the root colonization of different 10 PGPR groups could establish, in relation to other phytopathogenic organisms, competitive relationships for occupying the same ecological niche, or simply some kind of interaction, that would improve host resistance, against attacks of phytoparasite nematodes. Any beneficial rhizospheric bacteria colonize the same tissues as sedentary phytoparasitic nematodes such as Meloidogyne spp .. At a priori, there must be some kind of relationship between phytoparasite nematodes and rhizospheric microorganisms.
    An example that there are interactions between nematodes and PGPR is the interaction between Rhizobium spp. and Bradyrhizobium spp. However, the interaction between them can be stimulating or inhibiting, both of the nodulation and nitrogen fixation, depending on the species of nematode that interacts. Another example is mycorrhizae, whose mechanisms to increase host tolerance to nematodes is the modification of root exudates and their influence on the orientation of nematodes towards it, as well as the hatching of eggs and the subsequent development of Nematodes inside the root. However, although the 25 root exudates of the host plants can inhibit the hatching of eggs or the infection process, repel and even kill some species of nematodes, they can also orient the nematodes towards the root and stimulate the juveniles so that They infect the plant.
    Likewise, it is known that chitinolytic fungi and bacteria that share the habitat of nematodes can maintain a certain biological balance, and somehow limit the proliferation of nematodes. In fact, bacterial endophyte microorganisms (PGPR) and fungi have been used for biocontrol of sedentary and migratory endoparasitic nematodes that attack banana and tomato
    (Hallman and Sikora, 1994; Hallman et al., 2001; Pocasangre et al., 2000). These studies revealed that these endophytic root microorganisms are capable of giving multiple points of vulnerability in the life cycle of nematodes, through the inhibition of penetration, reducing reproductive capacity and retarding the mobility of nematodes and hatching of the eggs. 5
    Several species of Bacillus, such as B. subtilis, B. megatherium and B. licheniformis, have demonstrated a suppressive effect of pathogenic fungi or bacterial populations of the soil. In addition, some strains of Bacillus spp. that interfere with the normal development of nematode populations. Specifically, Padgham and Sikora demonstrate the modes of action through which B. megaterium reduces damage 10 by Meloidogyne graminicola (Padgham and Sikora, 2007). It is also known that Bacillus thuringiensis produces a thermostable toxin that can be toxic to populations of Meloidogyne spp. and prevents or prevents nematode larvae from forming nodules in tomato roots (Sayre, 1980). On the other hand, Márquez and Fernández have reviewed a selection of Bacillus thuringiensis var kurstaki 15 strains with a nematicidal effect (Márquez and Fernández, 2006).
    Trichoderma spp. It has enzymatic action through chitinases, which degrade chitin, a component present in nematode eggs (Sharon et al., 2001). Pseudomonas fluorescens can also be an antagonist of different soil pathogens, through different mechanisms, such as the production of siderophores, which inhibit plant pathogens, through competition for iron, the emission of suppressive antibiotics of competing microorganisms, and through the chitinases and glucanases that cause cell lysis of microbial cells (Sharman et al. 2003).
    It is widely known that there are different PGPR microorganisms, both bacteria and fungi, with antagonistic and suppressive capacity of nematodes, including several of the genus Bacillus spp (B. subtilis, B. megatherium, B. licheniformis, B. thuringiensis ...), and which are also found naturally in healthy soil, also of interest from a nutritional and biological point of view. However, in the development of microbial compositions with any of these types of microorganisms, the maintenance of microbial viability is complicated, especially when there is more than one microorganism in its composition. The microbial compositions marketed so far for the treatment of nematodes comprise certain species and strains of said microorganisms.
    with other non-microbial components. In these compositions, the effect against the parasite is achieved by the action of the microorganism itself, without the use of non-microbial components related to an additional antiparasitic and / or nutritional effect (eg fertilizer) on the plant. However, although the non-microbial components used are associated with an increase in the viability of the 5 microorganisms, these compositions do not maintain the viability for long periods of time (less than one year).
    Bello et al. have described that the contents of urease and chitinase in the soil are inversely correlated with the number of nodules of M. arenaria (Bello et al., 1996). 10
    In general, Gram-positive (Gram +) bacilli are likely to use as a source of nitrogen, mineral nitrogen, particularly ammonia, nitrates, nitrites, and organic nitrogen molecules such as urea, amino acids, nitrogen bases and other low molecular weight compounds. .
    Of all the chemicals that can have nematicidal action, obtained by the activity of microorganisms in the decomposition of organic matter, ammonium has been the best studied. Although it is difficult to say that a single component is responsible for nematode mortality, the nematicidal activity of ammonia was recognized by Eno et al. (Eno et al., 1955), when they were carrying out a series of works on the use of anhydrous ammonia as a nitrogen fertilizer, by verifying that applied by injection at the concentration of 300-900 mg kg-1 of soil reduced nematode problems . Subsequent experiments with urea, which is converted to ammonium by the action of urease in the soil, show that it is a good nematicide if applied in amounts greater than 300 mg of N kg-1 of soil (Huebner et al., 1983) . 25
    On the other hand, in the search for alternatives to control nematodes, it has been shown that organic matter, with a C / N ratio between 8-20, has nematicidal activity without phytotoxic effect (Rodriguez-Kabana et al., 1987) , and that the incorporation of organic materials in the soil reduces the densities of nodule-forming nematodes (Muller and Gooch, 1982). In fact, 30 oilseed, sawdust and urea residues have been used with some success (Singh and Sitaramaiah, 1966, 1967; Sikora et al., 1973a). They have also been tested as soil amendments for the control of nematodes and other plant pathogens, materials with high nitrogen content
    which generate ammonia, which acts as a nematicide in the soil (Canullo et al., 1992b). In addition, Tenuta, Hobbs and Lazarovits have also studied the mechanisms associated with the control of pathogenic organisms with organic matter, indicating that it is associated with NH3, an effect that is maintained for 4 days in sandy soils. According to these authors, phytoparasitic nematodes are affected in 5 counts, by the use of urea and other nitrogen sources (Tenuta et al., 1997).
    Although the use of organic amendments for nematode control would reduce the population density of nematodes to a different degree, their use is limited by the large quantities needed (Luc et al., 1990). An example of this is the remarkable reduction in activity and mobility that was observed in the second state 10 of the juveniles of Meloidogyne spp. treated with organic emulsion extracts, suggesting that these substances are excreted by the decomposition of the amendments and have a nematostatic effect (Miano, 1999).
    There are products on the market whose composition is based on various strains of microorganisms extracted from the soil and nutritional elements that give it a high nitrogen content. Specifically, the microorganisms present (PGPRs of the species Bacillus thuringiensis, Bacillus circulans, Brachyrhizobium spp., Rhizobium spp. And Azotobacter) are a combination of fungi and bacteria with known nematicidal properties. However, this product is not capable of keeping the introduced microorganisms that it contains viable over time.
    Consequently, there is currently a need to prepare fertilizer compositions with action against nematodes where any microorganism is able to maintain its viability for long periods of time in the presence of the nutritional elements of the composition, and where the effectiveness of the product is given by 25 fertilizing elements and by the action of the microorganism in the formulation, not by the action of the microorganism in the phytopathogenic nematode.
    Description of the invention
    Based on the foregoing, the present invention proposes a new formulation viable from the microbiological point of view, from suitable PGPR and a non-phytotoxic carrier liquid containing urea, amino acids, nitrogen bases and / or other compounds of low molecular weight that serve the plant nutrients, and that by the
    The action of the microorganism in the formulation, once diluted in water, promotes an environment that is unfavorable to nematodes. This formulation produces a significant decrease in the attack of the nodule-forming nematodes (Meloydogine spp.) In tomato cultivation (Valencian Tomato), as evidenced by the results obtained by bioassay to evaluate the effect or type of interaction induced by the formulation when applied to the soil of a crop exposed to nematodes. In particular, said formulation comprises a rhizobacterium of the genus Bacillus spp., Specifically, subtilis species, selected from the cepario de IAB, SL (Investigaciones y Aplicaciones Biotecnológica SL), which is formulated in a carrier liquid, alone or in conjunction with other rhizobacteria, Maintains microbial viability for 10 years. In addition, it has been shown that by "in vitro" and bioassay tests, the formulation of the present invention produces a significant decrease in the attack of nodule-forming nematodes (Meloydogine spp ..) in tomato cultivation (Valencian Tomato), due to some existing effect or type of interaction. Said decrease is reflected through the measurement of the root nodulation index in the bioassay.
    The formulation of the invention is a fertilizer (providing nutritional values to the crop), and at the same time it is able to keep the microorganism viable for long periods of time. In addition, the action of the microorganism on the non-microbial components contained in the formulation itself manages to keep the nematode plague below a threshold, which allows the culture to be maintained in suitable growth conditions.
    The results obtained here, have allowed the design of the composition, which once tested in the field, has resulted in a product of notable commercial interest. 25
    Accordingly, a first aspect of the invention refers to a microbial composition for nematode control, useful for preventing and / or treating a nematode infestation in a plant, characterized in that it comprises:
    a) at least one microbial strain with urease activity capable of enzymatically transforming ammoniacal nitrogen to NH3, where preferably said strain is Bacillus spp.,
    b) a liquid fertilizer medium comprising:
    b.1) a source of amino acids, preferably a protein hydrolyzate, and more preferably a protein hydrolyzate of plant origin;
    b.2) a source of fulvic acids, preferably a source of potassium lignosulfate; 5
    b.3) a source of urea nitrogen, preferably urea;
    b.4) a source of nutrients, preferably molasses; Y
    b.5) water.
    The above microbial composition can be applied to treat and / or prevent a nematode infestation caused by a type of nodule-forming nematode (or 10 gills) in the root or roots of a plant. Preferably said nematode is an endoparasitic sedentary nematode belonging to a Meloidogyne species, and more preferably it is a Meloidogyne incognita species nematode. Phytoparasite nematodes, gill-forming peptides belonging to the genus Meloidogyne, are considered the most economically important worldwide 15 for the damage they cause, and are characterized by a significant reduction in yields and the large number of plant species that attack , which includes most vegetables, fruit, ornamental and weed flora. Among the plants that can commonly suffer this type of infestation, and therefore can be treated with the microbial composition described herein, there are, and without limitation, onion, asparagus, pepper, vine , carrot and other horticultural crops as usual as for example tomato (in the case of the test, Valencian tomato).
    To achieve the desired effect against the previously defined nematodes, the above microbial composition comprises at least one microorganism with urease activity, that is, a microorganism capable of enzymatically transforming urea nitrogen into ammonia (NH3), which is toxic to these nematodes. . In this way, the microorganism generates an unfavorable environment for the population of nematodes, which migrates to other areas, which can also favor the colonization of the root by said microorganism if it is of the rhizospheric type and / or by some another rhizospheric microorganism that is present in the microbial composition itself or in the farmland. Preferably, said microorganism with urease activity is a microbial strain of Bacillus spp., As
    are and without limitation, strains of the species B. subtilis, B. megatherium, B. thuringiensis and / or Bacillus licheniformis. More preferably, the microbial strain is a strain of Bacillus subtilis, and even more preferably an isolated strain of Bacillus subtilis IAB / BS03 deposited in the DSMZ German type culture collection with Accession Number DSM 24682, or a mutant of said strain, and also deposited in the 5 Spanish Type Crops Collection with access number CECT 7254, owned by the company that presents the present invention.
    Bacillus subtilis strain DSM 24682
    The following strain has been deposited on June 6, 2011, in the German Type Crops Collection (DSMZ), Inhoffenstraβe 7 B, 38124 Braunschweig 10 (Germany), by Ms. Estefanía Hinarejos Esteve, IAB, S.L. (Investigaciones y Aplicaciones Biotecnológica S.L.), Av. Paret del Patriarca 11-B, Ap.30, 46113 Moncada, Valencia (Spain).
    The deposit of the deposited strain whose reference is Bacillus subtilis var. subtilis designated as IAB / BS03, CECT 7254, was identified by DSMZ with access number 15 DSM 24682 once said International Authority for Deposit declared that said strain in question was viable, under the provisions of the Budapest Treaty on Recognition International Deposit of Microorganisms for Patent Procedure Purposes.
    The DSM 24682 strain of Bacillus subtilis, previously used as a biological control agent based on its ability to produce antibiotic substances against phytopathogenic fungi, in the present invention is capable of acting on the liquid fertilizing medium to transform ammoniacal nitrogen into NH3 , and thus generate an environment that is unfavorable for the nematode.
    In addition, as previously indicated, the previous strain has been deposited on March 25, 2007, in the Spanish Type Culture Collection (CECT), Building 3 CUE, Parc Scientific University of Valencia, Professor Agustín Escardino 9, Paterna, 46980 Valencia (SPAIN), by Raquel del Val Buedo del IAB, SL The deposit of the pure and viable strain was received by the CECT with the access number CECT 7253.
    In a preferred embodiment of the microbial composition defined above, the microbial strain with urease activity comprises at least one strain of Bacillus spp., And in a more preferred embodiment comprises a strain selected from at least
    one of the group consisting of: Bacillus subtilis strain (preferably Bacillus subtilis DSM 24682 microbial strain), Bacillus thuringiensis strain (preferably B. thuringiensis var. kurstaki), Bacillus megatherium strain, Bacillus licheniformis strain and any combination of previous. In an even more preferred embodiment of the above, the microbial strain of Bacillus spp. it comprises a strain of Bacillus subtilis with urease activity, and more preferred is strain B. subtilis DSM 24682. However, when the microbial composition comprises a strain of Bacillus subtilis with urease activity (eg, strain B. subtilis DSM 24682), said composition may further comprise at least one additional strain of Bacillus spp .. Thus, in preferred embodiments, the microbial composition in addition to the strain of 10 Bacillus subtilis with urease activity, which includes those microbial compositions with strain B. subtilis DSM 24682, comprises an additional microbial strain of Bacillus spp. Examples of additional microbial strains of Bacillus are, without limitation, some strain selected from at least one of the group consisting of: Bacillus thuringiensis strain (preferably B. thuringiensis var. kurstaki), 15 strain of Bacillus megatherium, strain of Bacillus licheniformis and any combination thereof. Even more preferred, a composition where the additional microbial strain comprises a combination of a strain of Bacillus thuringiensis (preferably B. thuringiensis var. Kurstaki), a strain of Bacillus megatherium and a strain of Bacillus licheniformis. In fact, in a preferred embodiment, the microbial composition comprises the strain with urease activity Bacillus subtilis DSM 24682, and in addition, the commercial strains Bacillus thuringiensis var. Kurstaki IAB / BT / 01, Bacillus megatherium CECT 7253 and Bacillus licheniformis CECT 7252.
    Bacillus subtilis DSM 24682 (CECT 7254), Bacillus thuringiensis var. Kurstaki IAB / BT / 01, Bacillus megatherium CECT 7253 and Bacillus licheniformis CECT 25 7252, mentioned above, are kept in the cepario of IAB, S.L. and they are currently commercial product, dehydrated and lyophilized. These rhizospheric bacteria have been identified by the Official Association of Weighers and Public Meters of Barcelona (COPMB) by molecular identification of the 16S rRNA sequence in the Genbank database. And in addition, the rhizobacteria Bacillus 30 subtilis DSM 24682 (CECT 7254), Bacillus megatherium CECT 7253 and Bacillus licheniformis CECT 7252, once identified, remain deposited in the Spanish Type Culture Collection (CECT) of Valencia, with the corresponding access numbers or CECT deposit indicated here, although in these cases such deposits were not made according to the Budapest Treaty. 35
    The strain of Bacillus thuringiensis var. Kurstaki is a commercially accessible strain of commercial reference IAB / BT / 01.
    The Bacillus megatherium strain with deposit number CECT 7253 has been deposited on March 30, 2007, in the Spanish Type Culture Collection (CECT), Building 3 CUE, Parc Cientific Universitat de Valencia, Professor Agustín Escardino 9, Paterna, 5 46980 Valencia (SPAIN), by Raquel del Val Buedo del IAB, SL. The deposit of the pure and viable strain was received by the CECT with the access number CECT 7253.
    The Bacillus licheniformis strain with deposit number CECT 7252 has been deposited on March 30, 2007, in the Spanish Type Crops Collection (CECT), Building 3 CUE, Parc Cientific Universitat de Valencia, Professor Agustín Escardino 9, Paterna, 10 46980 Valencia (SPAIN), by Raquel del Val Buedo del IAB, SL. The deposit of the pure and viable strain was received by the CECT with the access number CECT 7252.
    Advantageously, and due to the characteristic formulation of the fertilizing liquid medium, the microbial composition described in the present invention has a microbial viability of 80% for a minimum of at least one year, a percentage of viability difficult to maintain in other compositions known in the state of the art after more than 6 months after its preparation. In fact, said liquid fertilizing medium is capable of maintaining 100% microbial viability for at least 6 months, and at least 80% for one year, of different microorganisms such as different bacterial strains of Bacillus spp., Such as 20 per Examples include, among others, the strains of Bacillus subtilis, Bacillus thuringiensis, Bacillus megatherium and Bacillus licheniformis mentioned above. Likewise, the liquid fertilizing medium is capable of maintaining microbial viability at least 80% for 6 weeks of bacterial strains of Azotobacter vinelandii and Rhizobium leguminosarum. Other microorganisms of fungal nature, such as Saccharomyces cerevisiae, have a viability in the 100% fertilizing liquid medium for at least 24 hours.
    Characteristically, the liquid fertilizer, in addition to promoting an environment unfavorable to the phytopathogenic nematodes, is stable and is suitable for supporting the microbial base (the Bacillus strain or strains mentioned above, 30 and where appropriate, other microbial or microorganism strains additional fungi, for example as previously indicated), maintaining the viability of the microorganisms it contains. The liquid medium of fertilizing characteristics,
    It is presented as an interesting formulation, not only for its fertilizing characteristics, but for the characteristic of maintaining the microbial viability of different genera and microbial species, in isolation, or as a microbial consortium. Accordingly, the microbial composition of the invention may comprise other microorganisms that are viable in said formulation, preferably other PRPG 5 known as other Bacillus spp. Strains, for example Bacillus thuringiensis var. Kurstaki, Bacillus megatherium and / or Bacillus licheniformis. Therefore, the liquid fertilizer medium can be considered as a novel formulation of the present invention. The amounts of the ingredients of said liquid medium may vary in such a way that they maintain their stability characteristics and / or that they allow their usefulness to support said microbial base.
    Typically, the source of amino acids may be present in an amount of between 45% and 95% by weight with respect to the total volume of the composition, including between 60% to 80% by weight with respect to the total volume of the composition, including from 70% to 78% by weight with respect to the total volume of the composition. The term "source of amino acids" in the present invention refers to a material where amino acids can be found, not necessarily purified, and can be found as mixtures of amino acids. In this sense, the average specialist in the field can use as sources of amino acids, for example and not limited to, a protein hydrolyzate, amino acids obtained by fermentation and / or even amino acids obtained by synthesis. Without limitation, the source of amino acids can include amino acids such as lysine, alanine, histidine, cystine and cysteine, arginine, valine, hydroxyproline, methionine, aspartic acid, isoleucine, threonine, leucine, serine, tyrosine, glutamic acid, phenylalanine, proline, tryptophan, glycine and other similar amino acids. Preferably, the source of amino acids is of plant origin, comprising amino acids obtained from plant proteins, such as, for example, and without limitation, a vegetable protein hydrolyzate.
    The source of fulvic acids can typically be present in an amount of between 1% and 20% by weight with respect to the total volume of the composition, including from 5% to 15% by weight with respect to the total volume of the composition, including between 10% and 15% by weight with respect to the total volume of the composition. The term "source of fulvic acids" in the present invention refers to a material where known fulvic acids can be found.
    by the skilled artisan, such as lignosulfonic acid and / or any of its salts, preferably alkali cation salts such as potassium or sodium. In a preferred embodiment, the source of fulvic acids is potassium lignosulfonate.
    The source of urea nitrogen may typically be present in an amount of 5 between 1% and 20% by weight with respect to the total volume of the composition, including between 5% to 15% by weight with respect to the total volume of the composition, including between 8% to 12% by weight with respect to the total volume of the composition, including between 10% to 11% by weight with respect to the total volume of the composition. In the present invention, the term "source of ureic nitrogen" refers to a material where nitrogen of ureic origin can be found, such as urea (CO (NH2) 2), and Calurea (Ca (NO3) 2 · 4CO ( NH2) 2). Preferably, the source of urea nitrogen is urea.
    The term "source of nutrients" in the present invention refers to a material or substance that provides a varied mixture of nutrients that can be used by microorganisms, and which comprises nutritional compounds essential for the performance of the vital functions of microorganisms. such as, among others, sugars, minerals, trace elements, vitamins and / or amino acids. Thus, when at least one microorganism is added to the liquid fertilizing medium, the source of nutrients contributes to the maintenance, growth and / or development of said microorganism. The source of nutrients is typically present in an amount of between 1% and 10% by weight with respect to the total volume of the composition, including between 1% to 5% by weight with respect to the total volume of the composition, including from 2% to 4% by weight with respect to the total volume of the composition, including from 2% to 3% by weight with respect to the total volume of the composition. A preferred example of a nutrient source is molasses, preferably selected from the group consisting of: sugar cane molasses, beet molasses and any combination thereof.
    The term molasses, as understood in the present invention, refers to a thick liquid product derived from sugarcane and to a lesser extent from sugar beet, obtained from the remaining residue in the sugar extraction tanks. Nutritionally it has a very high content of carbohydrates, in addition to vitamins of group B and abundant minerals, among which iron, copper and magnesium stand out. Its water content is low.
    The water content of the microbial composition is present in an amount sufficient to complete 100% by weight (C.S.P.) with respect to the total volume of the composition. Depending on the amounts of the above components, water is present in the composition in an amount ranging from 0% to 52% by weight with respect to the total volume of the composition, including from 0% to a 29% 5 by weight with respect to the total volume of the composition, including from 0% to 25% by weight with respect to the total volume of the composition, including from 0% to 8% by weight with respect to the total volume of the composition.
    Unless otherwise specified, all percentages as used herein are by weight with respect to the total volume of the total composition (% w / v), 10 being equally understood as the weights expressed in grams per 100 milliliters of total composition. The numerical intervals as used herein are intended to include each number and subset of numbers within the range, whether specifically described or not. For example, a numerical range of 45% to 95% should be considered as supporting a range of 60% 15 to 80%, 70% to 78% and the like.
    In a preferred embodiment, the liquid fertilizer medium of a microbial composition as any of those defined above comprises:
    b.1) between 45% and 95%, preferably between 60% and 80%, by weight of the source of amino acids with respect to the total volume of composition, including both limits;
    b.2) between 1% and 20%, preferably between 5% and 15%, by weight of the source of fulvic acids with respect to the total volume of composition, including both limits;
    b.3) between 1% and 20%, preferably between 5% and 15% (and more preferably between 8% and 12%), by weight of the urea nitrogen source with respect to the total volume of composition, including both limits;
    b.4) between 1% and 10%, preferably between 1% and 5% (and more preferably between 2% and 4%), by weight of the source of nutrients with respect to the total volume of composition , including both limits; and 30
    b.5) between 0% and 52% (preferably between 0% and 29%, and more preferably between 0% and 25%), by weight of water with respect to the total volume of composition, including both limits, such that the
    Total sum of the percentages by weight of all the components of the composition is 100%.
    In another preferred embodiment, the liquid fertilizer medium of the microbial composition as any of those defined above comprises:
    b.1) between 60% and 80%, preferably between 70% and 78%, by weight 5 of the amino acid source with respect to the total volume of composition,
    b.2) between 5% and 15%, preferably between 10% and 15%, by weight of the source of fulvic acids with respect to the total volume of composition,
    b.3) between 8% and 12%, preferably between 10% and 11%, by weight of the urea nitrogen source with respect to the total volume of composition, 10
    b.4) between 2% and 4%, preferably between 2% and 3%, by weight of the nutrient source with respect to the total volume of composition, and
    b.5) between 0% and 25%, preferably between 0% and 8%, by weight of water with respect to the total volume of composition, such that the total sum of the percentages by weight of all components of the composition 15 be 100%.
    And in a more preferred embodiment of the above, the liquid fertilizer means comprises:
    b.1) between 70% and 78%, preferably 70%, by weight of the source of amino acids with respect to the total volume of composition,
    b.2) between 10% and 15%, preferably 10%, by weight of the source of fulvic acids with respect to the total volume of composition,
    b.3) between 10% and 11%, preferably 10%, by weight of the urea nitrogen source with respect to the total volume of composition,
    b.4) between 2% and 3%, preferably 3%, by weight of the source of 25 nutrients with respect to the total volume of composition, and
    b.5) between 0% and 8% by weight of water with respect to the total volume of composition, such that the total sum of the percentages by weight of all components of the composition is 100%.
    And in an even more preferred embodiment of the above, the liquid fertilizer medium 30 comprises:
    b.1) 70% by weight of the source of amino acids, preferably of a
    vegetable protein hydrolyzate, with respect to the total volume of composition,
    b.2) 10% by weight of the source of fulvic acids, preferably potassium lignosulfate, with respect to the total volume of composition,
    b.3) 10% by weight of the urea nitrogen source, preferably urea, with respect to the total volume of composition,
    b.4) 3% by weight of the source of nutrients, preferably molasses, with respect to the total volume of composition, and
    b.5) a percentage by weight of water with respect to the total volume of composition (up to a maximum of 7%) so that the total sum of the percentages by weight of all the components of the composition is 100%. 10
    According to the present invention, the total microbial content of the microbial composition as any of those defined above, is between 103 and 109 colony forming units (cfu) per milliliter of composition. The term "colony forming units" (also referred to herein by its abbreviation cfu) refers to the number of spores or microbial cells capable of being viable. In preferred embodiments, the microbial compositions comprise a total microbial content of between 106 and 109 cfu per mL of composition. These compositions can be prepared from starting microbial strains of known concentration, for example, between 1010 and 1011 cfu / gram, such that in this case in the microbial composition the strain or 20 starting microbial strains are present in a amount comprised between 0.01% and 10% by weight with respect to the total volume of the composition. More preferably, the starting microbial strain or strains are present in the composition between 0.02% and 1% by weight of said microbial strain or strains with respect to the total volume of the composition. 25
    In a preferred embodiment, the microbial composition as any of those defined above comprises 0.02% by weight of the microbial strain of Bacillus spp. with urease activity Bacillus subtilis DSM 24682 with respect to the total volume of the composition. In a preferred embodiment of the above, in addition to strain B. subtilis DSM 24682, it comprises other microbial strains of Bacillus 30 spp. until reaching a total microbial content of 0.1% by weight of microbial strains with respect to the total volume of the composition, such as a composition comprising 0.02% by weight of B. subtilis (preferably B. subtilis DSM 24682), 0.02% by weight of B. licheniformis (preferably B.
    licheniformis CECT 7252), 0.02% by weight of B. megatherium (preferably B. megatherium CECT 7253) and 0.04% of B. thuringiensis (preferably B. thuringiensis var. kurstaki IAB / BT / 01), with with respect to the total volume of the composition.
    The above percentages of the strain or microbial strains, refer to the total weight of all strains of microorganisms present in the microbial composition with respect to the total volume of said composition, expressed as a percentage, and comprises the percentage by weight of the Bacillus strain capable of enzymatically transforming ammoniacal nitrogen to NH3 and also, where appropriate, the percentage by weight of the strain or additional microbial strains defined above.
    Hereinafter, all the microbial compositions defined above will be referred to as "microbial compositions of the invention", or simply "compositions of the invention."
    As demonstrated in in vitro assays, the microbial compositions of the invention, designed from suitable PGPR and a non-phytotoxic carrier liquid, are microbiologically viable and promote an unfavorable environment as desired. to the nematodes.
    On the other hand, tests have been carried out in vivo, by bioassay, to evaluate the efficacy of the microbial compositions of the invention. For the bioassay, tomato was used as a susceptible crop, using the variety, sensitive to nematode attacks, Valencian tomato. This crop grows in 20 optimal conditions for infection by phytopathogenic nematodes, and also ensures a reliable source of these nematodes, specifically Meloidogyne spp. Nematodes. In general, those favorable conditions for plant growth. They will also be favorable for the reproduction of Meloidogyne spp .. The results of the bioassay show that there is some effect or type of interaction that produces a significant decrease in the attack of the nodule-forming nematodes (Meloydogine spp.) in tomato cultivation (Tomato Valencian).
    Therefore, in a second aspect, the invention refers to the use of at least one of the microbial compositions of the invention to treat and / or prevent an infestation by a nematode in a plant crop.
    When the composition of the invention is applied to the soil of a plant or a crop of
    a plant with an infestation by at least one parasitic nodule-forming nematode, preferably Meloidogyne spp., produces a significant decrease in the attack of said nematodes that is reflected through a decrease in the measurement of the root nodulation index.
    Accordingly, a third aspect of the invention relates to a method for treating and / or preventing an infestation of a nematode (preferably Meloidogyne spp., And more preferably, Meloidogyne incognita) in a plant crop (preferably tomato, and more preferably, Valencian tomato) which comprises applying at least one of the microbial compositions of the invention defined herein to the soil or surface of said crop. 10
    The plants or plant cultures to which the microbial composition of the invention can be applied, to treat and / or prevent infestation by a nematode, are not particularly limited, which as mentioned above includes most of the vegetable, fruit, ornamental plants and weed flora. Examples of such plants may include, and are not limited to, cereals (eg, rice, barley, wheat, rye, oats, corn, etc.), vegetables and vegetables (soy, beans, fodder beans, peas , red beans, peanuts, cabbage, tomato, spinach, broccoli, lettuce, onion, scallion, paprika, eggplant, pepper, carrot, potato, sweet potato, cranberries, radish, lotus root, turnip, burdock, garlic, squash, cucumber, etc.), fruit / fruit (apples, citrus fruits, knobs, grapes, peaches, apricots, yellow peaches, bananas, strawberries, watermelon, melon, nuts, chestnuts, almonds, etc.), vegetable products for processing (cotton, hemp, beet, hops, sugar cane, sugar beet, olive, gum, coffee, tobacco, tea, etc.), grass plants (ball grass, sorghum, timotea grass, clover, alfalfa, etc.), lawns (grass , agrostis, etc.), ornamental plants, such as scent plants (lavender, rosemary, thyme, parsley, pepper, ginger, etc.), flowering plants (chrysanthemum, rose, carnation, orchid, etc.), garden trees (ginkgo, cherry, Japanese laurel, etc.) and forest trees (Abies sachalinensis, Picea jezoensis, pine, yours, cedar, cypress, etc.)
    In a preferred embodiment, the application to the soil of the microbial composition comprises:
    i) a first watering of the plant with an aqueous solution of the microbial composition, where said solution has a microbial concentration between 103 and 109 cfu per liter of solution, at a dose of
    irrigation between 10 and 30 liters of the aqueous solution per hectare of crop.
    In a more preferred embodiment of the above, in addition to the first irrigation, said application also comprises:
    ii) a second irrigation of the plant with an aqueous solution of the microbial composition as defined above in i), after a period of time between 10 and 20 days, with respect to the first irrigation.
    In preferred embodiments of any one of the methods described above in this aspect of the invention, the method is to treat and / or prevent an infestation of an endoparasite nodule-forming nematode of plants such as Meloidogyne spp., And more preferably, Meloidogyne incognita .
    In other preferred embodiments of the method this aspect of the invention, the crop plant on which any one of the above methods is applied, is tomato, and more preferably, Valencian tomato. fifteen
    A fourth aspect of the invention refers to a kit for preparing a microbial composition of the invention comprising at least one microbial strain with urease activity, as any of those defined above in the first aspect of the invention, and a liquid fertilizer, according to the liquid fertilizer media defined in any of the compositions of the invention defined above.
    Description of the figures
    FIGURE 1. Zucchini root, inoculum source of phytopathogenic nematodes for in vitro assays.
    FIGURE 2. Serial dilutions for calculating concentrations and viabilities. 25
    FIGURE 3. Image at 24h in the in vitro test, performed with 1 ml of the product at 1% v / v in 1 ml of nematode suspension.
    FIGURE 4. Image at 96 h in the in vitro test performed with 1 ml of the product at 1% v / v in 1 ml of nematode suspension.
    FIGURE 5. Equipment used in applications.
    FIGURE 6. First application.
    FIGURE 7. Root removal for evaluation.
    FIGURE 8. Prior evaluation with celery.
    FIGURE 9. Representation of the plot plan. 5
    FIGURE 10. General view of the plot where the test has been carried out to evaluate the efficacy of the microbial composition of the invention in the control of Meloidogyne sp. in tomato cultivation.
    FIGURE 11. Root nodulation indices (on a scale of 0 to 10) obtained with the different treatments evaluated: no treatment (1); treatment with the microbial composition of the invention (Bacillus spp. 108 cfu / mL) at an application dose of 10 L / ha (2); treatment with Quillay extract at 35% at an application dose of 10 L / ha (3); treatment with 24% fenamiphos at an application dose of 40 L / ha (4); treatment with the microbial composition of the invention (Bacillus spp. 108 cfu / mL) at an application dose of 10 L / ha combined with fermented Tagetes extract 15 applied at an application dose of 10 L / ha (5).
    FIGURE 12. Efficacy in the level of galling calculated by Abbott's formula (%) with the different treatments evaluated: treatment with the microbial composition of the invention (Bacillus spp. 108 cfu / mL) at an application dose of 10 L / ha (2); treatment with 35% Quillay extract at an application dose of 10 20 L / ha (3); treatment with 24% fenamiphos at an application dose of 40 L / ha (4); treatment with the microbial composition of the invention (Bacillus spp. 108 cfu / mL) at an application dose of 10 L / ha combined with fermented Tagetes extract applied at an application dose of 10 L / ha.
    FIGURE 13. Root status at the end of the test of untreated plants. 25
    FIGURE 14. State of the roots at the end of the test of the plants treated with the microbial composition of the invention.
    FIGURE 15. Root status at the end of the test of plants treated with Quillay extract.
    Bibliography
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    Bezooijen J. 2006. Revised version. Methods and techniques for nematology. University of Wageningen. 5
    Bridge J., S. L. J. Page, 1980. Estimation of root-knot nematodes infestation levels on roots using a rating chart. Tropical pest management 26: 296-298.
    Canullo, G. H .; R. Rodríguez-Kábana; J. W. Kloepper. 1992b. Changes in soil microflora associated with control of Sclerotium rolfsii by furfuraldehyde. Biocontrol Science and Technology 2, 159-169. 10
    Eno, C. F.; Blue WG; Good JM. 1955. The effect of anhydrous ammonia on nematodes, fungi, bacteria, and nitrification in some Florida soils. Proc. Soil Science Society of America 19, 55-58.
    Hallman, J., Sikora, R.A., 1994. Influence of Fusarium oxisporum, a mutualistic fungal endophyte, on Meloidogyne incognita infection of tomato. Zeit Pflanznkrank. 15 Pflanzenschutz. 101,475-481.
    Hallman, J., Quadt, H.A. Miller, W.G., Sikora R.A., Lindow, S.E., 2001. Endophytic colonization of plants by the biocontrol agent Rhizobium etli G12 in relation to Meloidogyne incognita infection. Phytopathology 91, 415-422.
    Hinarejos E, 2008. IAB, S.L. (Investigaciones y Aplicaciones Biotecnológica S.L.). twenty
    Huebner, R A; Rodríguez-Kábana R; Patterson RM. 1983. Hemicellulosic waste and urea for control of plant parasitic nematodes: effects on soil enzyme activities. Nematropica 13, 37-45.
    Luc M, Sikora R.A and Bridge J, 1990. Plant Parasitic Nematodes in Subtropical and Tropical agriculture. C.A.B. International Institute of Parasitology. p.240. 25
    Márquez Gutiérrez ME and Fernández Gonzálvez E, 2006. Selection of Bacillus thuringiensis strains with nematicidal effect Integrated Pest Management and Agroecology (Costa Rica) No. 78.
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    Moy, Terence I .; Anthony R. Ball, Zafia Anklesaria, Gabriele Casadei, Kim Lewis, and Frederick M. Ausubel * Contributed by Frederick M. Ausubel, 2006. Identification of 5 novel antimicrobials using a live-animal infection model.
    Muller, R. and Gooch, P.S., 1982. Organic reforms in nematode control. An exmination of the literature. Nematropica, 12: 319-326.
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    Padgham JL and Sikora R.A, 2007. Biological control potential and modes of action of Bacillus megatherium against Meloidogyne graminicola on rice. Crop Protection, Volume 26, Issue 7, July 2007, Pages 971-977. J.L.
    Pocasangre, L., Sikora, R.A., Vilich, V., Schuster, R.P., 2000. Survey of banana 15 endophytic fungi from Central America and screening for biological control of Radopholus similis. Act.Hort. 531,283-289.
    Riksen J, 2008. Laboratory of Nematology. Wageningen University and Research Center. www.nem.wur.nl/UK/Webshop/Products/Nematode+counting+dishes/. (Ref. Dr. Joost Riksen Joost.Riksen@wur.nl). twenty
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    Sayre, R.M. 1980. Biocontrol: Bacillus penetrans and related parasites of nematodes. Journal of Nematology 12: 261-270.
    Sharman, A., B.N. Johnson, A.K. Sharma and B.R. Glick 2003. Plant growth-promoting 25 bacterium Pseudomonas sp. strain GRP3 influences iron acquisition in mung bean (Vigna radiata L. Wilzeck). Soil Biology and Biochemistry. 35: 887-894.
    Sharon, E., M. Bar Eyal, A. Herrera Estrella, O. Kleifeld and Y. Spiegel. 2001. Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum.Phytopathology. 91: 687-693.
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    Sikora R.S. and Sitaramaiah K. 1973a. Control of root-knot throught organic and 10 inorganic soil amendments.3. Effect of rice husk and sugarcane bagasse. Haryana Journal of Horticultural Science, 2: 123-127.
    Tenuta, M .; S. Hobbs & G. Lazarovits, 1997. Mechanisms Associated with Disease Control by Organic Soil Amendments. pp. 4-1 - 4-3 in: 1997 Annual International Research Conference on Methyl Bromide Alternatives and Emission Reductions. San 15 Diego, USA. November 3 - 5, 1997.
    Zeck W M, 1971. A rating scheme for field evaluation of root-knot nematode infestation. Plazenshutz-Nacht. 24, 141-144.
    EXAMPLES
    The materials and methods that were used for the development of the present invention, as well as their embodiments, are detailed below. Such examples are included for illustrative purposes only and should not be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application. 25
    EXAMPLE 1: Design of the formulation and in vitro tests
    1.1. MATERIALS AND METHODS
    1.1.1. Identification of the rhizobacteria chosen
    A microbial feasibility study has been carried out on the microbial composition of the invention, with the following PGPR from the cepary of IAB, S.L .: Bacillus subtilis, strain CECT 7254 (B. subtilis DSM 24682).
    Said bacterium is maintained in the cepario of IAB, S.L. and it is currently a commercial product, dehydrated and lyophilized, so that the supply for routine tests was taken from determined and identified batches. Moreover, said bacterium is kept in active culture in agar at 4 ° C for routine matters, and frozen at -80 ° C in 30% glycerol for long-term storage. The identification of the rhizobacterium was carried out by the COPMB (Official College of Weighers and Public Meters of Barcelona) by molecular identification of sequence 10 of the 16S rRNA in the Genbank database. This rhizobacterium, once identified, also remains deposited in the Spanish Type Culture Collection of Valencia.
    1.1.2. Source of phytopathogenic nematodes.
    The inoculum for the in vitro tests was obtained from the zucchini roots of a naturally infected greenhouse, located in the Cooperativa del Perelló. Zucchini roots were left in tap water, in petri dish (as illustrated in Figure 1), and incubated for 24-48 hours at room temperature until the egg masses present hatched.
    1.1.3. Development of the liquid fertilizer medium (fertilizer formula). twenty
    A liquid fertilizer was designed, from urea (as a source of ureic nitrogen), potassium lignosulfonate (as a source of fulvic acids), hydrolyzed proteins (as a source of amino acids, obtained from vegetable protein) and molasses (as a source of nutrients). This liquid fertilizer was enriched with strain B. subtilis DSM 24682, resulting in a compound formula with different PGPR and a high content in organic matter, high total nitrogen content, and high ureic nitrogen content.
    To work the formula, the following raw materials were chosen:
    - AMINO ACIDS OBTAINED FROM VEGETABLE PROTEIN (contribution of organic matter and amino acids) 30
    - POTASSIC LIGNOSULFONATE (contribution of fulvic acids)
    - UREA (N ureic contribution)
    - MELAZA (organic source and nutrient intake)
    - WATER
    With these raw materials, the fertilizer formula was prepared in a final total volume of 250 ml, according to the weight / volume percentages (% w / v, expressed as percentages 5 of the concentration in g / mL) detailed below:
    FORMULA% p / v
    AMINO ACIDS ................................................. ............ 70
    POTASSIC LIGNOSULFONATE .................................. 10
    UREA. .................................................. ......................... 10 10
    MOLASSES................................................. ......................... 3
    WATER ................................................. ...................... CSP
    From this formula, before adding the microorganisms, the pH was measured at time 0 and at 24 hours, to verify that they are stable and suitable to support the microbial base, and that they do not undergo changes over time. fifteen
    A feasibility analysis was carried out on the fertilizer formula, according to the general analysis method described below. For this, the amount of microorganism per milliliter indicated in Table 3 was added first. Taking into account that in this study, a minimum product of 1010 cfu / g was used, an amount that varied between 0.1 to 0 was used , 2 grams / ml. However, depending on the 20 starting microbial concentrations, these amounts may vary.
    1.1.4. General microbial analysis method and calculation of microbial viability:
    For the determination of the microbial viability of each strain in the fertilizer formula prepared above, the following materials were used:
    - 10 ml threaded glass tubes of sterile saline solution, 25
    - sterile blue tips (1 ml),
    - 9 plates of culture medium corresponding to the microorganism to be evaluated;
    and a procedure consisting of the following steps was followed:
    1. Collect 1 ml of the liquid product of the fertilizer formula to be analyzed that contains a particular strain, where said strain is present in an initial theoretical (Cteoric) microbial concentration calculated according to the following mathematical expression:
    Ctheoric (cfu / ml) = [Mmicrobial × Cmicrobial heading] / Vformulation 5
    where Mmicrobial refers to the weight of the microbial inoculant expressed in grams, Cmicrobial heading refers to the starting microbial concentration of the microbial inoculant or pure culture of the microorganism expressed in cfu / g, and Vformulation refers to the volume on which the microorganism is placed expressed in milliliters. 10
    2. Dissolve the collected liquid product in a 10 ml thread tube of sterile saline (0.9% NaCl in water).
    3. Perform serial dilutions 1/10 in 10ml thread tubes of sterile saline solution to the theoretical concentration minus one unit (Figure 2), stirring 30 seconds each time with vortex. fifteen
    4. Plate 0.1 ml of the last three dilutions in triplicate on plate.
    5. Incubate at 27 ° C the time needed for each microorganism.
    6. Reading results by reading those plates containing between 5-50 grown colonies.
    7. Calculate the observed (or experimental) microbial concentration of fertilizer formula 20, expressed in colony forming units per milliliter (cfu / ml), according to the mathematical formula:
    Microbial concentration observed (cfu / ml) = (Xobserved * Yobserved) / 0.1
    where Xobserved is the number of colonies counted in the previous step, and Yobservado is the positive dilution factor (that is, the potency at which the microbial concentration is elevated, but with a + sign; for example for 10 (-6) the dilution factor in positive would be 6).
    8. Calculate the microbial viability (V) in the formulation, which is:
    - V = 100%, if Yteoric - Yobserved is less than or equal to zero,
    - V = (100 - R) * (1 + rX)%, if Yteoric - Yobserved is between 0 and 3,
    - V = Not Viable, if Yteoric - Yobserved is greater than 3;
    where:
    Yteoric is the exponent or positive factor, which should carry the microbial concentration, that is, if theoretically the concentration in cfu / ml should be 107 cfu / ml, the Yteoric is 7,
    Yobservado is the exponent or factor that comes out by count, that is, the concentration in cfu / ml that can be read on the plates,
    R is the percentage of loss of microbial viability calculated according to the mathematical formula:
    R = 10 * (Yteoric - Yobserved),
    rX is equal to 0.05 if Xtheoric-Xobserved is greater than or equal to 10, or rX is equal to 0 if Xtheoric-Xobserved is less than 10, Xtheoric being the number of colonies that theoretically had to be read on the plates, and Xobserved the number of 15 colonies that actually read on the plates.
    That is, in general a cfu / ml count was made, according to the usual laboratory procedures.
    The medium used was: Nutrient agar II (NUTRIENT AGAR II) for Bacillus spp. Strains, prepared according to the following general formula (for 1 L of medium), and finally adjusting the pH to 7.2:
    Meat extract ............................................... .. 1g
    Yeast Extract ............................................ 2g
    Tryptona ................................................. .............. 5g
    NaCl ................................................. .................... 5g 25
    Agar ................................................. .................. 20g
    Distilled water ................................................ ..... 1L
    The viability of the fertilizer formula has been determined, from the point of view
    microbial at 24 hours, at 6 weeks, at 6 months and at 12 months of preparation of the composition. The viabilities for all Bacillus spp. They were calculated globally, regardless of the species, since it is not possible, morphologically and visu, to study the microbial concentrations of the different species separately. 5
    1.1.4. Recovery method and nematode count.
    The simplest way to isolate nematodes from their host plant is to immerse the sample in water and select the nematodes under a microscope, although this is a tedious task, and can only be done if small samples need to be collected (Bezooijen, 2006). 10
    Live nematodes can be easily identified and studied in a preparation with water. In this type of preparation, certain structures, such as stiletto, lumen and excretory apparatus, can be seen more easily than in fixed and dead nematodes (Bezooijen, 2006).
    The procedure used to fix a nematode was to wash a 15-piece root with a 0.5% solution of sodium hypochlorite, and leave on a Petri dish, with 10 ml of tap water. Leave 24 hours and try a needle to extract the nematode.
    For the nematode count, a camera developed in the Department of Nematology of the University of Wageningen was used. The model contains a maximum capacity of 10 ml of suspension, with a grid of 7.5 by 3.5 mm and a total diameter of 6.3 cm. The slope of the circumference allows a complete view, and the grid is made on the back of the plate, so that the side on which the suspension is deposited can be easily cleaned. Because the available microscope is an optical microscope, and not dissection, this camera was reduced by means of a 3 cm diameter petri dish, in order to work with smaller volumes of water (2 ml), a field of reduced vision, and speed up counting.
    To perform the nematode count, the recommendations followed directly by Dr. Joost Riksen (Nematology Laboratory of the University of Wageningen, The Netherlands; www.nem.wur.nl/UK/Webshop/Products/Nematode+counting+dishes were used /; Ref. Dr. Joost
    Riksen –Joost.Riksen@wur.nl–), using the 4X magnification. For best results, proceed as follows (Bezooijen, 2006), but adjusted to the volume of our counting chamber (2 ml):
    - Collect the nematode suspension, extracted directly from the roots nodulated by nematodes (according to recommendations of Dr. Simon, professor at the University of Reading, United Kingdom, 2008 –Simon, 2008–). Once we have the suspension collected, in a 20 ml specimen, leave the specimens for 10 minutes.
    - Adjust the volume of the suspension to 10ml by adding or removing water.
    - Stir the suspension by blowing with a pipette, for 15 seconds. 10
    - Immediately take a 2 ml subsample of the suspension with a 2 ml graduated pipette and pass directly to the counting chamber.
    - Under the microscope, identify and have a hand counter.
    - Put the sample back in its place, and count 2 more times.
    - Calculate the average of specimens / ml. fifteen
    1.1.5. Methodology for the in vitro test of nematicide / nematostática activity.
    The nematicidal / nematicotic activity of the microbial composition and the filtrate of said composition was determined. To determine the effect on the nematodes, 1 ml of a dilution was taken independently:
    - 1% v / v of the microbial composition. twenty
    - 1% v / v of the filtrate, by 20 micron filter, of the microbial composition.
    - 1% v / v microbial composition left to ferment for 24 hours.
    These preparations were deposited in petri dishes, 3 cm in diameter, with 1 ml of deionized water, in which there was a suspension of nematodes counted by the previous methodology. The nematodes were counted on that same plate, at 0 h, 25 24 h and 96 h, using the Wageningen camera as a base, and counting them as dead when they did not move, testing with a fine needle, and / or when they presented
    a rigid aspect (Moy et al., 2006). In fact, there is a different morphology observed under the microscope between live and dead nematodes.
    1.2. Results of the formulation test.
    Table 1 shows the results of the pH readings in the fertilizer formula. 5
    Table 1. pH readings in the liquid fertilizer medium (fertilizer formula defined in 1.1.3).
     readings  Fertilizer formula
     pH at t 0h  4.47
     pH at t 24h  4.43
    According to this table, we can consider the pH as stable in the fertilizer formula.
    Table 2 shows the results obtained from the microbial viability readings of B. subtilis CECT 7254 at 24 hours. 10
    Table 2. Viability of strains B. subtilis CECT 7254 in the fertilizer formula at 24 hours.
     MICROORGANISMS IN THE FERTILIZING FORMULA  EXPECTED CONCENTRATION (cfu / ml) OBSERVED CONCENTRATION AT 24h (cfu / ml) VIABILITY (%)
     Bacillus subtilis CECT 7254  510 (7) 310 (8) 100
    Since the viability of all microorganisms was excellent in the fertilizer formula, it was chosen as valid and 100 ml were prepared with the following composition:
    COMPOSITION OF THE FERTILIZING FORMULA CHOSEN AS VALID:
    ELEMENTS% w / v
    AMINO ACIDS ................................................. ............ 70
    POTASSIC LIGNOSULFONATE .................................. 10 20
    UREA. .................................................. ......................... 10
    MOLASSES................................................. ......................... 3
    WATER ................................................. ..................... CSP
    On the composition of the above fertilizer formula, a number of microorganisms are added as indicated in Table 3, giving rise to the experimental microbial composition 5, whose microbial concentration is subsequently evaluated, at 6 weeks, at 6 months and at 12 months (Table 4) to check the viability of the microorganisms in said formula.
    Table 3. Microbial theoretical concentrations on the fertilizer formula according to the microorganism chosen. 10
     MICROORGANISM  QUANTITY% p / v INITIAL CONCENTRATION
     Bacillus subtilis CECT 7254  0.15 g / ml 110 (10) cfu / g

    Table 4. Feasibility of the fertilizer formula designed with microorganisms of Bacillus spp. at 6 weeks, at 6 months and at 12 months.
     WEATHER  EXPECTED CONCENTRATION (theoretical) OBSERVED CONCENTRATION (6 weeks) VIABILITY (%) OF Bacillus spp. (*)
     6 weeks  310 (8) cfu / ml 110 (8) cfu / ml 100%
     6 months  110 (8) cfu / ml 110 (6) cfu / ml 80%
     12 months  110 (8) cfu / ml 2.110 (6) cfu / ml 80%
    (*) Refers to the viability of the Bacillus subtilis strain CECT 7254.
    Taking into account that the initial (theoretical) total Bacillus concentration was between 1 · 10 (8) -1 · 10 (9) cfu / ml, and that the total Bacillus concentration observed at 6 months is 1 · 10 (6) cfu / ml, this implies a viability between 70% and 80%. The results show that the microorganisms Bacillus spp. (Bacillus subtilis CECT 7254) are capable of being viable for 6 months and one year in the fertilizer formula. twenty
    1.3. Results of in vitro tests.
    Next, in Tables 5–7 the results in triplicate of the in vitro test of nematicide / nematostática activity obtained for the above fertilizer formula comprising the microorganisms of the species Bacillus subtilis CECT 7254 (DSM 24682) are presented. 5
    Table 5. Reading of live and dead nematodes at time 0, 24 and 96 hours with 1 ml of the 1% v / v fermented product and filtered with a 20 µm filter, in 1 ml of nematode suspension.
     Half nematodes  Weather
     0 hours  24 hours 96 hours
     alive  315 95 129
     dead  0 11 19

    Table 6. Reading of live and dead nematodes at time 0, 24 and 96 hours with 1 ml of the product at 1% v / v and filtered with a 20 µm filter in 1 ml of nematode suspension.
     Half nematodes  Weather
     0  24 hours 96 hours
     alive  299 76 106
     dead  0 4 7
    Table 7. Reading of live and dead nematodes at t 0, 24 and 96 h with 1 ml of the product at 1% v / v in 1 ml of nematode suspension.
     Half nematodes  Weather
     0 hours  24 hours 96 hours
     alive  328 0 ALL
     dead  0 All in general Not detectable
    In Table 7, it is observed that at 24 hours there is a great reduction in the activity and / or mobility of the nematodes (Figure 3), while at 96 hours (Figure 15 4), a high activity of those same nematodes.
    1.4. Final design of the product used to evaluate by bioassay
    A nematicidal microbial composition is prepared according to the percentage composition presented in Table 8, with a microbial concentration in said composition of 1 × 10 7 cfu / ml.
    Table 8. Percentage composition of the prepared nematicide formula. 5
     ELEMENTS  % p / v
     HYDROLYZED PROTEINS  70
     POTASSIC LIGNOSULFONATE  10
     UREA  10
     MOLASSES  3
     MICROORGANISMS Bacillus subtilis CECT 7254 (0.1% w / v)  0.1
     WATER  C.S.P.
    Said microbial composition useful for nematode control is a product of microbial, liquid and viable base, result of the selection of beneficial microorganisms or PGPR, developed in a matrix that provides macro and micro nutrients and maintains microbial viability. On the other hand, it is a microbial product that does not generate resistance and does not produce waste. When applied to the soil, the 10 microorganisms initiate a colonization process mainly in the root zone of the plants. The colonization process will last 3 to 5 weeks (depending on the application dose, soil type, fertility, humidity and temperature). Once the colonies have been established, the soil will have a great variety of microorganisms characteristic of a fertile soil, in which the 15 microorganisms can act directly and indirectly in the fixation and nitrification of atmospheric nitrogen, in the fixation, mineralization and Absorption of fertilizers and other nutrients from the soil, whether organic, mineral or synthetic, acting as root and soil regenerators. In turn, the product provides an unfavorable environment for nematodes. twenty
    Preferably, the application of the microbial composition of the invention is carried out via soil by localized irrigation of 1 to 2 applications, with intervals of 10 to 20 days. For this, the microbial composition of the invention is applied at a dose.
    between 10 and 20 liters per hectare (L x ha or L / ha).
    The purpose of the microbial composition prepared here is the application of the formula in a subsequent in vivo assay to keep Meloidogyne spp. below the economic damage threshold.
    In conclusion to the results obtained in the in vitro tests, and as demonstrated in said tests, the chosen formulation exerts a great reduction in the activity and / or mobility of Meloidogyne spp. at 24 hours, suggesting that the liquid may have some nematostatic effect, since at 96 hours, those same nematodes return to high activity and / or mobility.
    EXAMPLE 2: Efficacy of the microbial composition of the invention in control 10 of Meloydogine sp. in tomato cultivation.
    2.1. Summary
    A test was carried out on protected tomato, coded as SRS09-003-106NE. The objective of the test was to determine the efficacy and nematicidal selectivity of a microbial composition of the invention as defined in Table 8 of Example 1 (Bacillus spp, 1 × 108 CFU / ml), applied 3 times through drip irrigation In a sandy soil. It was tested against Meloidogyne spp. at a single dose (10 l p.f / ha - product lines per hectare, L / ha -) alone and combined with a previous application of fermented Tagetes extract (Extr. Ferm. Tagetes 100%). The standard products were phenamiphos (capsule suspension –CS, capsule suspension–) in 20-24% aqueous solution applied at a dose of 40 l pf / ha (L / ha) and Quillay extract (soluble concentrate) in aqueous solution at 35% applied at a dose of 10 l pf / ha (L / ha).
    For the development of the trial, a commercial plot of protected tomato cv. Valencian in El Perelló (Valencia); The ground was sandy. A randomized block design was performed, with 4 repetitions per treatment. The 25 elementary plots were 10.8 m2, with 22 floors / plot.
    For the Quillay extract and the microbial composition of the invention (Bacillus spp, 1 × 108 CFU / ml) three applications were made, while for the phenamiphos, only one was made. All applications were made with a motorized backpack, directly coupled to drip irrigation as illustrated in Figure 5, with a 30
    working pressure of 1.5 atm and 5000 l / ha of broth volume (except for applications D and E, where 6000 l / ha were required).
    The first application [May 22, 2009 (application A)] was carried out in a phenological state of 14 BBCH (4th sheet fully deployed), as seen in Figure 6, and the last [June 19, 2009 (application E)] in a phenological status of 51 BBCH (first 5 visible inflorescence). An evaluation of the level of clumping (on 06/17/2009) was carried out by removing the roots (Figure 7). Before the start of the trial, another evaluation of celery had been performed (Figure 8), in order to determine the areas of infestation and make an appropriate block distribution. Possible symptoms of phytotoxicity were also evaluated. Efficacy was calculated using Abbott's formula.
    The analysis of the results obtained, whose untreated data are collected in the Tables shown below, was performed with the ARM Revision 7.4.2 software, from Gylling Data Management. The data were analyzed using the analysis of variance (ANOVA) of the non-transformed values and of the transformed values 15 when the Barlett test indicated it. If the transformation did not improve the homogeneity assumptions of the variances, the original values were maintained, and therefore, significant differences (if any) should be interpreted with caution. The probability of occurrence of significant differences between treatments was calculated as the value of the probability F (Treatment Prob (F)). The Student-Newman-20 Keuls test (S-N-K) was applied when significant differences were found. The comparison of means was analyzed only when AOV Treatment P (F) was significant at the selected level. The results obtained were indicated by letters (mean values with different letters indicate significant differences according to the SNK test at a 95% confidence interval. When the data was transformed, the 25 letters were included in this transformed column. The results observed were:
    - Population levels were average during the trial, reaching the roots of untreated plants an index of 5 (on a scale of 0-10) at the end of the trial (approx. 2 months after transplantation).
    - The rest of the treatments showed similar values. The microbial composition 30 of the invention (COMP.) Alone and in combination with fermented Tagetes extract (COMP. + EXT. TAG.) Showed the lowest binding values, followed by the chemical standard phenamiphos (PHEN.). The highest index was observed
    for the biological standard of Quillay extract (QL). No statistical differences were found.
    - The best efficacy results were obtained with the chemical standard, closely followed by the microbial composition of the experimental invention, both alone (COMP.) And combined with fermented Tagetes extract (COMP. + 5 EXT. TAG.). The biological standard of Quillay extract (QL) obtained the lowest efficacy value. No statistical differences were found.
    - No symptoms of phytotoxicity were observed in any treatment.
    - No handling problems of the experimental product were observed.
    2.2. Experimental treatments 10
    Table 9 collects all the data referring to the different treatments (treatments 1 to 5) performed for the tests carried out in Example 2. Next, in Table 9, the data presented in it is indicated in more detail.
     Table 9
     Trt No.  Type Treatment Name Form Conc Form Unit Form Type
     one  CHK Untreated Check
     2  INSE COMP. 10000000 BIOEN / ML SL
     3  INSE QL 35% SL
     4  INSE PHEN. 24% CS
     5  INSE COMP. + EXTR. TAG 10000000 BIOEN / ML SL
     fifteen
     Table 9. (Cont.)
     Trt No.  Rate Rate Unit Other Rate Other Rate Unit Appl Code
     one
     2  10 L / HA ACE
     3  10 L / HA 3500 g A / ha ACE
     4  40 L / HA 9600 g A / ha A
     5  10 L / HA ACE
    Additional Treatment Information
    Type (“Type”)
    CHK = Check or Untreated (“Untreated”)
    INSE = Insecticide or Nematicide
    Treatment Name
    Untreated Check = Not treated 5
    COMP., 10000000, BIOEN / ML, SL = Bacillus spp | 108 CFU / ml
    QL, 35%, SL = Quillay Extract
    PHEN., 24%, CS = Fenamiphos 24% | PHI (pre-harvest interval) 60 days tomat., Cuc., Mel; 30 days pepper
    COMP. + EXTR. TAG., 10000000, BIOEN / ML, SL = Bacillus spp + Extr. Ferm. 10 Tagetes | 10,000 CFU / g + 100%
    Formulation units (“Form Unit”)
    BIOEN / ML = Biological Entities per milliliter of product (“Biological Entities per milliliter product”)
    % = Percentage of Active Ingredient in the product formulated in relation to 15 weight / weight,% AW / W (“Percent Active Ingredient in formulated product on a weight / weight basis; same as% AW / W”.)
    Type of Composition (“Form Type”)
    SL = Soluble Concentrate; Clear to opalescent liquid to be applied as a solution of the active ingredient after dissolution in water. The liquid may contain water-insoluble elements of the composition ("A clear to opalescent liquid to be applied as a solution of the active ingredient after dilution in water. The liquid may contain water insoluble formulants").
    CS = Capsule suspension (“Capsule suspension”); Stable suspension of capsules in a fluid, normally intended for dissolution in water prior to use (“A stable suspension of capsules in a fluid, normally intended for dilution with water before use”).
    Application Rate Unit (“Rate Unit”)
    L / HA = Liters of Product per Hectare (“Liters Product per Hectare” (US = QT / A); Q
    Other Unit of the Application Rate (“Other Rate Unit”) 30
    g A / ha = Grams of Active Ingredient per Hectare (“Grams Active Ingredient per Hectare”)
    Application mode (“Application Directions” or Appl. Code):
    A = at transplanting
    B = 7 days after A (“7 days after A”) 35
    C = 15 days after A (“15 days after A”)
    D = 7 days after B (“7 days after B”)
    E = 15 days after C (“15 days after C”)
    Repeats (“Replications”): 4, Untreated treatments: 1, Reference treatment number: 3, Performed under good laboratory practice guidelines [GLP] / guidelines Good 5 experimental [GEP] (“Conduct under GLP / GEP”): Yes - GEP with no protection, Design (“Design”): Randomized Complete Block, treatment (“Treatment units”): Treated plot size (“Dry Form. Unit”):%, Width of the treated soil (“Treated plot size Width”) ): 3 meters, 10 Length of the treated land size (“Treated plot size Length”): 8 meters, Application volume (“Application volume”): 5000 l / ha, Mix size (“Mix size”): 2 liters, Format definitions (“Format definitions”): G-All7.DEF, G-All7.FRM
    2.3. Site Description (“Site Description”)
    2.3.1. Trial location. fifteen
    El Perelló (Valencia), Spain. It is known that the selected plot has good nematode infestation since the previous culture of celery was infected. The previous evaluation and distribution of the plot was made according to its initial infestation.
    2.3.2. Guidelines of Good Experimental Practices (GEP) of the trial. twenty
    Table 10
     Guideline (“Guideline”) Description (“Description”)
     one.  CEB Méthode 44 Efficacy of nematicides against Meloidogyne sp. of tomato (“Efficacité de nématicides contre Meloidogyne sp. of tomato”)
     2.  PP1 / 152 (2) Design and analysis of efficacy evaluation trials
     3.  PP1 / 181 (3) Mode of operation and reporting in tests of
     effectiveness evaluation (“Conduct and reporting of efficacy evaluation trials”)
    2.3.3. Crop Description ("Crop Description").
    Cultivation 1 ("Crop 1"): Transplanted tomato. LYPXP Lycopersicon is., Transplanted
    Variety (“Variety”): Valencian Description (“Description”): Protected (“Protected”)
    BBCH Scale (“BBCH Scale”): BVSO Planting Date (May / 21/2009)
    Planting Method: “Transplanted by hand (“ TRANSPLANTED - HAND ”)
    Row spacing, Unit (“Row Spacing, Unit”): 1.2 m
    Row spacing, unit (“Spacing Within Row, Unit”): 0.4 m 10
    2.3.4. Description of the pest ("Pest Description").
    Pest Type 1 (Pest 1 Type): I Code (“Code”): MELGSP Meloidogyne sp.
    Common Name: Root-knot eelworms (Root-knot eelworms)
    2.3.5. Location and Design ("Site and Design").
    Plot Width, Unit: 1.2 m
    Plot Length, Unit: 9 M
    Type of site (“Site Type”): Greenhouse (“GREENHOUSE”)
    Type of crop (“Tillage Type”): Conventional (“CONVENTIONAL-TILL”) 20
    Repeats (“Replications”): 4
    Design of the studio (“Study Design”): Randomized Complete Block
    Land drainage (“Soil Drainage”): Good (G, “Good”)
    Figure 9 shows the arrangement or distribution on the plot of the different 25 repetitions (4 repetitions per treatment) of each of the treatments performed (treatments 1 to 5), as described in Table 11. Figure 10 is a general image of the land or plot when the test was performed.
    Table 11
     Trt Treatment Rate  Plot No. By Rep.
     No. Name Rate Unit  1 2 3 4
     1 Untreated Check  103 201 303 404
     2 COMP. 10 L / HA  105 205 304 405
     3 QL 10 L / HA  101 203 302 403
     4 PHEN 40 L / HA  102 202 301 402
     5 COMP. + 10 L / HA EXTR. TAG 10 L / HA  104 204 305 401
    Prior to the trial on Valencian tomato an evaluation of the nodulation of the celery root was made (Figure 8) to identify areas of infestation in the field and make an appropriate distribution by blocks.
    2.3.6. Description of the land (“Soil Description”). 5
    Texture ("Texture"): Sand ("SAND")
    Fertilization Level (“Fert. Level”): Good (“GOOD”)
    2.3.7. Moisture and Weather Conditions.
    Global Moisture Conditions: NORMAL
    Nearest weather station (“Closest Weather Station”): Polinya del Xuquer 10 Distance (”Distance”): 5 km
    Table 12 shows the meteorological data (“Weather Data”) referring to the values of minimum, maximum and average temperatures (in ºC), relative humidity and rainfall (in mm) recorded during the period of time in which the test of example 2 was performed. 15
    Table 12
     Date  Temp. Mean (ºC) Temp. Max. (ºC) Temp. Min. (ºC) Hum. Rel. (%) Precip. (mm)
     05/21/2009  19.9 25.91 14.41 75.5 0
     05/22/2009  20.04 24.15 16.44 81.9 0
     05/23/2009  21.76 28.48 17.74 71.5 0
     05/24/2009  20.59 23.94 17.55 81.1 0
     05/25/2009  20.72 25 15.39 73 0
     05/26/2009  21.21 27.94 14.73 60.94 0
     05/27/2009  18.54 23.04 12.77 65.48 0
     05/28/2009  19.58 27.28 11.26 53.34 0
     05/29/2009  19.48 25.45 12.77 59.98 0
     05/30/2009  20.66 26.44 13.82 65.05 0
     05/31/2009  19.54 26.13 16.04 82.2 3.8
     06/01/2009  20.16 25.2 14.41 76.3 0.2
     06/02/2009  20.68 25.98 15.26 78.4 0
     06/03/2009  21.71 25.91 16.31 77.1 0
     06/04/2009  22.11 26.11 18.07 75.8 0
    Table 12. (Cont.)
     Date  Temp. Mean (ºC) Temp. Max. (ºC) Temp. Min. (ºC) Relative Humidity (%) Precip. (mm)
     06/05/2009  24.43 32.32 18.07 49.82 0
     06/06/2009  22.62 29.12 15.32 41.72 1
     06/07/2009  21.37 28.6 15.13 49.73 0
     06/08/2009  22.74 28.2 14.87 42.53 0
     06/09/2009  22.79 30.16 14.99 46.98 0
     06/10/2009  23.54 30.88 15.85 51.55 0
     06/11/2009  23.35 30.36 15.59 63.5 0
     06/12/2009  22.48 27.36 17.62 70.1 0
     06/13/2009  23.07 28.53 17.56 71.8 0
     06/14/2009  23.57 29.97 17.55 69.27 0
     06/15/2009  24.06 29.37 19.06 66.92 0
     06/16/2009  23.34 26.11 19.25 76.2 0
     06/17/2009  23.55 29.24 18.27 71.3 0
     06/18/2009  24.05 29.27 18.53 70 0
     06/19/2009  24.82 31.22 20.17 63.65 0
     06/20/2009  23.53 27.8 18.34 73.7 0
     06/21/2009  22.63 25.45 19.45 70.6 0
     06/22/2009  23.87 28.2 19.32 70.8 0
     06/23/2009  22.99 27.94 17.42 74 0
     06/24/2009  24.38 34.81 18.47 72 0
     06/25/2009  24.84 30.07 19.97 71.1 0
     06/26/2009  25.73 34.48 19.84 56.91 0
     06/27/2009  23.67 27.93 18.67 73.4 0
     06/28/2009  24.74 31.21 19.38 68.41 0
     06/29/2009  23.88 28.91 17.55 75.6 0
     06/30/2009  24.66 30.42 18.93 72.1 0
     07/01/2009  25.77 33.5 20.24 67.91 0
     07/02/2009  26.06 32.39 21.09 75.2 0
     07/03/2009  25.59 29.95 21.35 74.6 0
     07/04/2009  25.77 32.78 19.45 70.4 0
     07/05/2009  26.1 31.14 21.41 73.3 0
     07/06/2009  26.62 31.07 22.85 74.3 0
     07/07/2009  25.82 29.11 23.51 77.6 0
     07/08/2009  22.72 24.94 20.5 87.6 25.2
     07/09/2009  24.06 27.49 21.68 82.3 0
     07/10/2009  25.64 29.77 21.81 78.4 0
     07/11/2009  26.1 29.05 23.11 75 0

    Table 12. (Cont.)
     Date  Temp. Mean (ºC) Temp. Max. (ºC) Temp. Min. (ºC) Relative Humidity (%) Precip. (mm)
     07/12/2009  26.45 32.12 21.03 72.6 0
     07/13/2009  25.52 29.89 21.94 75.1 0
     07/14/2009  25.17 29.57 21.09 80.8 0
     07/15/2009  25.42 29.57 20.96 78.3 0
     07/16/2009  26.01 30.48 22.07 80.5 0
     07/17/2009  27.75 36.96 22.39 49.26 0
     07/18/2009  22.02 26.38 15.52 53.37 0
     07/19/2009  22.94 27.94 17.29 68.63 0
     07/20/2009  23.55 28.33 19 74.6 0
     07/21/2009  24.38 28.46 20.04 79 0
     07/22/2009  26.25 30.09 23.05 80.4 0
     07/23/2009  28.85 39.05 20.95 57.2 0
     07/24/2009  28.02 34.93 21.35 54.82 0
     07/25/2009  24.97 28.07 20.03 76.3 0
     07/26/2009  25.05 29.77 19.78 69.65 0
     07/27/2009  25.82 30.81 20.63 74 0
     07/28/2009  25.18 28.72 21.02 73 0
     07/29/2009  26.91 35.98 18.99 71 0
     07/30/2009  25.67 28.99 21.09 77.2 0
     07/31/2009  25.57 29.24 20.83 75 0
    2.3.8. Application Description (“Application Description”): Table 13.
    Table 13
     A B C
     Application Date (“Application Date”):  May / 22/2009 May / 29/2009 Jun / 05/2009
     Time of Day:  Morning Morning Morning
     Application Method (“Application Method”):  DRIP DRIP DRIP
     Application Regulation (“Application Timing”):  POSPOS POSPOS POSPOS
     Application Place (“Application Placement”):  BANSOI BANSOI BANSOI
     Applied by (“Applied By”):  MB MB MB
     Air Temperature (“Air Temperature”):  22.5 ºC 25.5 ºC 24.8 ºC
     % Relative Humidity (“% Relative Humidity”):  82 42 80
     Wind Speed (“Wind Velocity”):  0 KPH 0 KPH 0 KPH
     Dew Presence (Y / N):  N N N
     Soil Temperature:  20.1 ºC 20 ºC 22 ºC
     Soil Moisture (“Soil Moisture”):  ADEQUATE ADEQUATE ADEQUATE
     % Cloud cover (“% Cloud Cover”):  70 0 0

    Table 13. (Cont.)
     FROM
     Application Date (“Application Date”):  Jun / 12/2009 Jun / 19/2009
     Time of Day:  Morning morning
     Application Method (“Application Method”):  DRIP DRIP
     Application Regulation (“Application Timing”):  POSPOS POSPOS
     Application Place (“Application Placement”):  BANSOI BANSOI
     Applied by (“Applied By”):  MB MB
     Air Temperature (“Air Temperature”):  29 ºC 27 ºC
     % Relative Humidity (“% Relative Humidity”):  65 73
     Wind Speed (“Wind Velocity”):  0 KPH 0 KPH
     Dew Presence (Y / N):  N N
     Soil Temperature:  25 ºC 24 ºC
     Soil Moisture (“Soil Moisture”):  ADEQUATE ADEQUATE
     % Cloud cover (“% Cloud Cover”):  0 0
    2.3.9. Crop Stage At Each Application (Table 14): Table 14
    Table 14. 5
     A B C
     Crop 1 Code, BBCH Scale (“Crop 1 Code, BBCH Scale”):  LYPXP BVSO LYPXP BVSO LYPXP BVSO
     Stage Scale Used:  BBCH BBCH BBCH
     Most of the stage, percentage (“Stage Majority, Percent”):  14 100 16 100 19 100



    Table 14. (Cont.)
     FROM
     Crop 1 Code, BBCH Scale (“Crop 1 Code, BBCH Scale”):  LYPXP BVSO LYPXP BVSO
     Stage Scale Used:  BBCH BBCH
     Most of the stage, percentage (“Stage Majority, Percent”):  23 100 51 100
    2.3.10. Application Equipment: Table 15
    Table 15
     A B C D E
     Application equipment (“Appl. Equipment”):  MARU-08-1 MARU-07-1 MARU-07-1 MARU-07-1 MARU-07-1
     Operating Pressure (“Operating Pressure”):  1.5 ATM 1.5 ATM 1.5 ATM 1.5 ATM 1.5 ATM
     Vehicle (“Carrier”):  Water ("WATER") Water ("WATER") Water ("WATER") Water ("WATER") Water ("WATER")
     Spray Volume:  5000 L / HA 5000 L / HA 5000 L / HA 6000 L / HA 6000 L / HA
     Mix Size (“Mix Size”):  2 2 2 2 2
     pH (“Spray pH”):  7 7 7 7 7
     Propellant (“Propellant”):  Pump (“PUMP”) Pump (“PUMP”) Pump (“PUMP”) Pump (“PUMP”) Pump (“PUMP”)
     Tank mix (Y / N) (“Tank Mix”):  N N N N N
    Applications of the experimental microbial composition of the invention (COMP.) And Quillay extract (QL) were made in the last 10–15 minutes of the irrigation cycle. The next irrigation is delayed to the maximum to prevent washing of the product. This application was made on wet soil.
    In accordance with the indications on the application of the fenamiphos (PHEN., 5 phenamiphos) in drip or strip irrigation, taking into account that its dose is for mass treatment, the dose of application of the fenamiphos was adjusted to the actual area treated.
    2.4. Results
    2.4.1. Standardized Summary: Table 16. 10
     Table 16
     Pest Code  MELGSP MELGSP
     Crop Code  LYPES LYPES
     Part rated (“Part Rated”)  ROOT C ROOT C
     Rating Date (“Rating Date”)  Jul / 17/2009 Jul / 17/2009
     Type of data rated (“Rating Data Type”)  DAMNEM CONTRO
     Rating Unit (“Rating Unit”)  0-10% UNCK
     Sample Size (“Sample Size”)  10 10
     Sample Size Unit  PLANT PLANT
     Footnote Number (Footnote Number)  eleven
     Trt-Eval Interval  28 DA-E 28 DA-E
     ARM Action Codes     TAB [2]
     Trt Treatme Rate No. Name Rate Unit  2. 3
     1 Untreated Check  5.0 0.0
     2 COMP. 10 L / HA  3.5 30.0
     3 QL 10 L / HA  5.2 7.0
     4 PHEN 40 L / HA  4.0 30.5
     5 COMP. + 10 L / HA EXTR. TAG 10 L / HA  3.6 26.8
     Table 16. (cont.)
     Standard Deviation  1.41 20.61
     CV  33.27 109.21
     Grand mean  4.23 18.87
     Bartlett's X2  8,424 2,481
     P (Bartlett's X2)  0.077 0.479
     Replicate F  0.826 0.207
     Replicate Prob (F)  0.5046 0.8899
     Treatment F  1,279 1,927
     Treatment Prob (F)  0.3317 0.1706
    Means followed by same letter do not significantly differ ”] (P = .05, LSD)
    Comparisons of the means were carried out only when the P (F) AOV treatment is significant in comparison with the OSL mean ["Mean comparisons performed only when AOV Treatment P (F) is significant at mean comparison OSL"]. 5
    Pest Code
    MELGSP, = Meloidogyne sp. |
    Crop Code
    LYPES, BVSO, = Lycopersicon esculentum
    Part considered (“Part Rated”) 10
    ROOT = Root
    C = The crop is considered in part (“Crop is Part Rated”)
    Type of data rated (“Rating Data Type”)
    DAMNEM = DAMAGES - NEMATODE ("DAMAGE - NEMATODE")
    CONTRO = CONTROL / BURNDOWN or KNOCKDOWN 15
    Rating Unit (“Rating Unit”)
    0-10 = 0-10 INDEX / SCALE
    % UNCK = PERCENT OF UNTREATED CHECK
    Sample Size Unit
    PLANT = Plant ("Plant")
    ARM Action Codes
    TAB [2] = Abbott (% of Untreated -% of Untreated -) [2]
    Footnote Number (Footnote Number)
    Footnote No. 1 (“Footnote 1”): Root nodulation index on a 0-10 5 scale (“Root gall Index in a 0-10 scale”).
    Footnote 2 ("Footnote 2"): Abbott efficacy (%) on root nodulation [Abbott efficacy (%) on root galling].
    2.4.2. Raw Data (Tables 17): Tables 17 to 21
     Table 17
     Pest Code  MELGSP MELGSP
     Crop Code  APUGV LYPES
     Part Rated  ROOT C ROOT C
     Rating Date  May / 06/2009 Jul / 17/2009
     Rating Data Type  DAMNEM DAMNEM
     Rating Unit  0-10 0-10
     Sample Size  10 10
     Sample Size Unit  PLANT PLANT
     Footnote Number  eleven
     Trt-Eval Interval  -16 DA-A 28 DA-E
     ARM Action Codes
     Trt No.  Sub Plot
     one  103 1 2 3
     2 3 3
     3 2 6
     4 3 3
     5 2 5
     6 2 3
     7 4 2
     8 2 6
     9 2 3
     10 2 4
     one  201 1 4 5
     2 2 7
     3 3 5
     4 2 5
     5 2 8
     6 2 5
     7 2 9
     8 4 6
     9 4 7
     10 3 6

     Table 17 (Cont.)
     Pest Code  MELGSP MELGSP
     Crop Code  APUGV LYPES
     Part Rated  ROOT C ROOT C
     Rating Date  May / 06/2009 Jul / 17/2009
     Rating Data Type  DAMNEM DAMNEM
     Rating Unit  0-10 0-10
     Sample Size  10 10
     Sample Size Unit  PLANT PLANT
     Footnote Number  eleven
     Trt-Eval Interval  -16 DA-A 28 DA-E
     ARM Action Codes
     Trt No.  Sub Plot
     one  303 1 2 6
     2 2 4
     3 2 7
     4 3 3
     5 3 4
     6 2 4
     7 3 4
     8 2 6
     9 4 7
     10 2 8
     one  404 1 0 5
     2 1 4
     3 0 4
     4 1 3
     5 1 3
     6 2 5
     7 3 5
     8 2 6
     9 1 4
     10 3 5

     Table 18
     Pest Code  MELGSP MELGSP
     Crop Code  APUGV LYPES
     Part Rated  ROOT C ROOT C
     Rating Date  May / 06/2009 Jul / 17/2009
     Rating Data Type  DAMNEM DAMNEM
     Rating Unit  0-10 0-10
     Sample Size  10 10
     Sample Size Unit  PLANT PLANT
     Footnote Number  eleven
     Trt-Eval Interval  -16 DA-A 28 DA-E
     ARM Action Codes
     Trt No.  Sub Plot
     2  105 1 1 3
     2 2 3
     3 2 2
     4 2 2
     5 1 1
     6 2 4
     7 2 4
     8 1 2
     9 4 1
     10 3 2
     2  205 1  2 2
     2 1 1
     3 2 5
     4 1 3
     5 0 2
     6 0 1
     7 1 2
     8 1 5
     9 2 2
     10 2 3

     Table 18 (Cont.)
     Pest Code  MELGSP MELGSP
     Crop Code  APUGV LYPES
     Part Rated  ROOT C ROOT C
     Rating Date  May / 06/2009 Jul / 17/2009
     Rating Data Type  DAMNEM DAMNEM
     Rating Unit  0-10 0-10
     Sample Size  10 10
     Sample Size Unit  PLANT PLANT
     Footnote Number  eleven
     Trt-Eval Interval  -16 DA-A 28 DA-E
     ARM Action Codes
     Trt No.  Sub Plot
     2  304 1 1 1
     2 3 5
     3 2 2
     4 2 6
     5 3 2
     6 2 3
     7 4 5
     8 2 7
     9 0 3
     10 2 6
     2  405 1 3 0
     2 3 7
     3 3 5
     4 5 6
     5 3 6
     6 4 8
     7 7 3
     8 3 3
     9 3 6
     10 2 6

     Table 19
     Pest Code  MELGSP MELGSP
     Crop Code  APUGV LYPES
     Part Rated  ROOT C ROOT C
     Rating Date  May / 06/2009 Jul / 17/2009
     Rating Data Type  DAMNEM DAMNEM
     Rating Unit  0-10 0-10
     Sample Size  10 10
     Sample Size Unit  PLANT PLANT
     Footnote Number  eleven
     Trt-Eval Interval  -16 DA-A 28 DA-E
     ARM Action Codes
     Trt No.  Sub Plot
     3  101 1 2 6
     2 2 8
     3 4 7
     4 2 6
     5 0 7
     6 2 5
     7 2 5
     8 4 5
     9 6 4
     10 2 3
     3  203 1 2 5
     2 3 7
     3 3 4
     4 2 3
     5 2 3
     6 5 6
     7 3 6
     8 2 8
     9 2 4
     10 2 4

     Table 19 (Cont.)
     Pest Code  MELGSP MELGSP
     Crop Code  APUGV LYPES
     Part Rated  ROOT C ROOT C
     Rating Date  May / 06/2009 Jul / 17/2009
     Rating Data Type  DAMNEM DAMNEM
     Rating Unit  0-10 0-10
     Sample Size  10 10
     Sample Size Unit  PLANT PLANT
     Footnote Number  eleven
     Trt-Eval Interval  -16 DA-A 28 DA-E
     ARM Action Codes
     Trt No.  Sub Plot
     3  302 1 3 4
     2 0 5
     3 2 3
     4 1 3
     5 2 4
     6 1 6
     7 3 7
     8 2 6
     9 1 4
     10 2 7
     3  403 1 2  3
     2 2 6
     3 2 7
     4 2 2
     5 0 6
     6 1 7
     7 0 7
     8 1 6
     9 1 6
     10 2 3

     Table 20
     Pest Code  MELGSP MELGSP
     Crop Code  APUGV LYPES
     Part Rated  ROOT C ROOT C
     Rating Date  May / 06/2009 Jul / 17/2009
     Rating Data Type  DAMNEM DAMNEM
     Rating Unit  0-10 0-10
     Sample Size  10 10
     Sample Size Unit  PLANT PLANT
     Footnote Number  eleven
     Trt-Eval Interval  -16 DA-A 28 DA-E
     ARM Action Codes
     Trt No.  Sub Plot
     4  102 1 2 2
     2 2 3
     3 2 2
     4 3 4
     5 2 2
     6 3 2
     7 2 0
     8 3 2
     9 3 4
     10 2 3
     4  202 1 1 8
     2 4 7
     3 3 2
     4 2 6
     5 1 5
     6 3 5
     7 1 5
     8 3 3
     9 2 3
     10 5 4

     Table 20 (Cont.)
     Pest Code  MELGSP MELGSP
     Crop Code  APUGV LYPES
     Part Rated  ROOT C ROOT C
     Rating Date  May / 06/2009 Jul / 17/2009
     Rating Data Type  DAMNEM DAMNEM
     Rating Unit  0-10 0-10
     Sample Size  10 10
     Sample Size Unit  PLANT PLANT
     Footnote Number  eleven
     Trt-Eval Interval  -16 DA-A 28 DA-E
     ARM Action Codes
     Trt No.  Sub Plot
     4  301 1  4 4
     2 3 6
     3 5 6
     4 4 7
     5 4 9
     6 2 8
     7 3 9
     8 4 2
     9 4 8
     10 2 10
     4  402 1 2 8
     2 2 2
     3 1 1
     4 1 0
     5 2 2
     6 2 0
     7 1 0
     8 1 0
     9 2 2
     10 1 2

     Table 21
     Pest Code  MELGSP MELGSP
     Crop Code  APUGV LYPES
     Part Rated  ROOT C ROOT C
     Rating Date  May / 06/2009 Jul / 17/2009
     Rating Data Type  DAMNEM DAMNEM
     Rating Unit  0-10 0-10
     Sample Size  10 10
     Sample Size Unit  PLANT PLANT
     Footnote Number  eleven
     Trt-Eval Interval  -16 DA-A 28 DA-E
     ARM Action Codes
     Trt No.  Sub Plot
     5  104 1 1 4
     2 2 3
     3 1 1
     4 2 1
     5 3 2
     6 3 9
     7 2 2
     8 1 5
     9 2 3
     10 2 5
     5  204 1 3 7
     2 2 5
     3 4 2
     4 4 2
     5 5 2
     6 5 2
     7 5 8
     8 3 6
     9 7 8
     10 7 7

     Table 21 (Cont.)
     Pest Code  MELGSP MELGSP
     Crop Code  APUGV LYPES
     Part Rated  ROOT C ROOT C
     Rating Date  May / 06/2009 Jul / 17/2009
     Rating Data Type  DAMNEM DAMNEM
     Rating Unit  0-10 0-10
     Sample Size  10 10
     Sample Size Unit  PLANT PLANT
     Footnote Number  eleven
     Trt-Eval Interval  -16 DA-A 28 DA-E
     ARM Action Codes
     Trt No.  Sub Plot
     5  305 1 2 2
     2 2 3
     3 1 0
     4 2 6
     5 1 2
     6 1 0
     7 1 0
     8 2 2
     9 2 3
     10 2 5
     5  401 1 2 1
     2. 3. 4
     3. 4. 5
     4 3 1
     5 4 5
     6 3 3
     7 2 4
     8 2 2
     9 2 5
     10 3 5
    2.5 Discussion
    2.5.1. Root grinding (Figures 11 and 13 to 15).
    Population levels of the parasite were average during the trial, reaching the roots of untreated plants an index of 5 [Figure 11, column (1); Figure 13] on a scale of 0-10 at the end of the trial (approx. 2 months after transplantation). 5
    The treatments tested showed similar values. The microbial composition of the invention alone (COMP., Column (2) of Figure 11) and combined with fermented Tagetes extract (COMP. + EXT. TAG., Column (5) of Figure 11 and Figure 14) showed the lower clumping values (3.5 and 3.6, respectively), followed by the phenamiphos chemical standard (PHEN., column (4) of Figure 11) with an index 10 of 4.0. The highest index was observed for the biological standard of Quillay extract (QL), with a gall index of 5.2 [Figure 11, column (3); and Figure 15]. No statistical differences were found.
    2.5.2. Efficacy (Figure 12)
    When the root agglomeration efficacy was evaluated, the best results were obtained with the chemical standard phenamiphos (PHEN., 30.5% efficacy), followed closely by the microbial composition of the experimental invention, both alone. (COMP.) As combined with fermented Tagetes extract (COMP. + EXT. TAG.), With 30% and 26.8% efficiency, respectively. The biological standard of
    Quillay extract (QL) obtained the lowest efficacy value. No statistical differences were found.
    2.5.3. Other relevant results
    No symptoms of phytotoxicity were observed in any treatment, nor were management problems of the microbial composition of the invention observed. 5
    2.6. conclusion
    In the test of Example 2 performed, with a mean population of nematodes, a composition of the invention (COMP.) As described in Table 8 of Example 1 showed a control in the population of nematodes comparable to that obtained with a chemical standard. such as phenamiphos (PHEN.), and better than when using Quillay extract (QL). In addition, it was found that the composition of the previous invention, applied three times at a dose of 10 liters per hectare, was safe for cultivation. Also, the application of a fermented Tagetes extract (EXT. TAG.) Prior to the application of the composition of the invention did not improve the results obtained with said composition. fifteen
    权利要求:
    Claims (21)
    [1]

    1. Microbial composition useful for preventing and / or treating a nematode infestation in a plant, characterized in that it comprises:
    a) at least one microbial strain with urease activity; 5
    b) a liquid fertilizer medium comprising:
    b.1) a source of amino acids,
    b.2) a source of fulvic acids,
    b.3) a source of urea nitrogen,
    b.4) a source of nutrients, 10
    b.5) water,
    and because it has a microbial viability of at least 80% for at least one year.
    [2]
    2. Microbial composition according to claim 1, wherein the microbial strain is Bacillus spp. fifteen
    [3]
    3. Microbial composition according to any one of claims 1 or 2, wherein the microbial strain comprises a Bacillus subtilis strain.
    [4]
    4. Microbial composition according to any one of claims 1 to 3, wherein the microbial strain comprises Bacillus subtilis DSM 24682.
    [5]
    5. Microbial composition according to claim 4 which, in addition to the strain of 20 Bacillus subtilis, comprises an additional microbial strain of Bacillus spp. which is selected from at least one of the group consisting of: Bacillus thuringiensis strain, Bacillus megatherium strain, Bacillus licheniformis strain and any combination thereof.
    [6]
    6. Microbial composition according to any one of claims 1 to 5, wherein the liquid fertilizing medium comprises:
    b.1) between 45% and 95% by weight of the source of amino acids with respect to the total volume of composition,
    b.2) between 1% and 20% by weight of the source of fulvic acids with respect to
    to the total volume of composition,
    b.3) between 1% and 20% by weight of the urea nitrogen source with respect to the total volume of composition,
    b.4) between 1% and 10% by weight of the nutrient source with respect to the total volume of composition, and 5
    b.5) between 0% and 52% by weight of water with respect to the total volume of composition, such that the total sum of the percentages by weight of all components of the composition is 100%.
    [7]
    7. Microbial composition according to any one of claims 1 to 6, wherein the liquid fertilizing medium comprises:
    b.1) between 60% and 80% by weight of the source of amino acids with respect to the total volume of composition,
    b.2) between 5% and 15% by weight of the source of fulvic acids with respect to the total volume of composition,
    b.3) between 8% and 12% by weight of the urea nitrogen source with respect to the total volume of composition,
    b.4) between 2% and 4% by weight of the nutrient source with respect to the total volume of composition, and
    b.5) between 0% and 25% by weight of water with respect to the total volume of composition, such that the total sum of the percentages by weight of all the components of the composition is 100%.
    [8]
    8. Microbial composition according to any one of claims 1 to 7, characterized in that the amino acid source is of plant origin.
    [9]
    9. Microbial composition according to any one of claims 1 to 7, characterized in that the source of amino acids is a vegetable protein hydrolyzate. 25
    [10]
    10. Microbial composition according to any one of claims 1 to 9, characterized in that the source of fulvic acids is a salt of lignosulfonic acid.
    [11]
    11. Microbial composition according to claim 10, characterized in that the source of fulvic acids is potassium lignosulfonate.
    [12]
    12. Microbial composition according to one of claims 1 to 11, characterized in that the urea nitrogen source is urea.
    [13]
    13. Microbial composition according to one of claims 1 to 12, characterized in that the source of nutrients is molasses.
    [14]
    14. Microbial composition according to one of claims 1 to 13, characterized in that the microbial strain or strains are in an amount comprised between 103 and 109 colony forming units per milliliter of composition. 5
    [15]
    15. Use of a composition of any one of claims 1 to 14 to treat and / or prevent a nematode infestation in a plant crop.
    [16]
    16. A method for treating and / or preventing an infestation by a nematode in a crop of a plant comprising applying to the surface of said crop a microbial composition of any one of claims 1 to 14. 10
    [17]
    17. The method according to claim 16, characterized in that said application comprises:
    i. a first irrigation of the plant with an aqueous solution of the microbial composition, where said solution has a microbial concentration between 103 and 109 cfu per liter of solution, at an irrigation dose 15 comprised between 10 and 30 liters of the aqueous solution per hectare of crop.
    [18]
    18. A method according to claim 17, characterized in that, in addition to the first irrigation, said application also comprises:
    ii. a second irrigation of the plant with an aqueous solution of the microbial composition 20 as defined in i for the first irrigation, after a period of time between 10 and 20 days, with respect to the first irrigation.
    [19]
    19. A method according to any one of claims 17 to 18, characterized in that the nematode is Meloidogyne spp.
    [20]
    20. A method according to any one of claims 17 to 19, characterized in that the plant is tomato.
    [21]
    21. A kit comprising a microbial strain with urease activity and a fertilizer liquid, as defined in any one of claims 1 to 14.
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    同族专利:
    公开号 | 公开日
    ES2490395A1|2014-09-03|
    ES2555674B1|2016-10-11|
    ES2490395B1|2015-06-16|
    WO2014118401A1|2014-08-07|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    BRPI0603879B1|2006-08-29|2018-02-06|Empresa Brasileira De Pesquisa Agropecuária - Embrapa|BACILLUS-BASED COMPOSITION spp. AND RELATED GENDERS AND THEIR USE IN PEST CONTROL|
    BRPI0604602A|2006-10-13|2008-05-27|Marcus Macedo Cazarre|use of rhizobacteria of the genus bacillus subtilis and bacillus licheniformis in the control of soil phytonmatodes|
    ES2610359T3|2010-08-10|2017-04-27|Chr. Hansen A/S|Nematicidal composition comprising Bacillus subtilis and Bacillus licheniformis|
    WO2012037352A2|2010-09-15|2012-03-22|Microbes, Inc.|Methods and compositions for reducing pathogens in soil and improving plant growth|EP3240403B1|2014-12-29|2019-11-13|FMC Corporation|Microbial compositions and methods of use for benefiting plant growth and treating plant disease|
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