WO2022191693A1

WO2022191693A1 - Mixtures and method for controlling phytopathogens that attack plant seeds during germination

Info

Publication number: WO2022191693A1
Application number: PCT/MX2021/000014
Authority: WO
Inventors: Luis Rodrigo BECERRA CARRANZA
Original assignee: Becerra Carranza Luis Rodrigo
Priority date: 2021-03-12
Filing date: 2021-03-16
Publication date: 2022-09-15
Also published as: MX2021003059A

Abstract

The present invention relates to mixtures for controlling phytopathogenic fungi that attack plant seeds during their germination, containing: Trichoderma harzianum strains as a biological control agent against various diseases caused by phytopathogenic fungi; Bacillus subtilis as a biological control agent for diseases caused by soil fungi and bacteria; Azospirillum brasilense as a nitrogen-fixing agent, making it available to the roots or radicles; Rhizophagus irregularis as a mycorrhizal symbiotic agent for giving greater tolerance to seedlings or plants born from seeds and/or grains treated with this fungus; and other agents of agronomic interest. A method for controlling phytopathogenic organisms that attack plant seeds during germination, which comprises applying a mixture in accordance with the present invention to plant seeds at the time of their germination.

Description

MIXTURES AND METHOD, FOR THE CONTROL OF PHYTOPATHOGENS THAT ATTACK VEGETABLE SEEDS DURING ITS GERMINATION

TECHNICAL FIELD OF THE INVENTION

The present invention is related to the technical field of Biotechnology and Agriculture, because it provides mixtures and a method for the control of phytopathogenic organisms that attack plant seeds during their germination.

BACKGROUND OF THE INVENTION

Resistance to fungicides, environmental contamination, toxicity and the natural existence of phytopathogenic microorganisms have motivated the search for other effective and non-harmful methods to combat this problem.

The Fusarium genus is a widely distributed pathogen worldwide (in more than 32 countries) and affects more than 80 commercially important crops. This fungus penetrates the plant through different routes and infects roots, stems and cobs. The pathogen produces various toxins in the tissue and kernels of the corn, which decreases its quality. Fumonisins are the main toxins excreted by the fungus. Leaf yellowing is one of the first symptoms. Lower leaves begin to lose turgor and hang down. In addition, this disease affects the vascular system, and when cutting the stem longitudinally we can see dark brown or brown spots. Before the plant matures, several leaves turn yellow and wilt, affecting fruit development and overall plant growth.

It lives in the soil and from there it is transmitted. It can survive up to three years and can lodge in crop residues or diseased live plants. It can also be transmitted or infest farms if contaminated seed is purchased. This pathogen is of warm climate, and develops optimally at 28°C. Similarly, soils with acidic pH and sandy texture are preferred by this pathogen, as well as moisture conditions in the soil. Among the most relevant ear rots are those induced by Fusarium species that, in addition to reducing yield, are the cause of deterioration and poor quality of the grains, and due to their ability to produce mycotoxins, they are also related to diseases in humans and animals that they consume them (Charmley et al., 1994; Jelinek et al., 1989; Macías and Peraza, 2008; Shurtleff, 1980; Sutton, 1982; Tuite et al., 1974).

The growing use of these products in biotechnology and environmental protection have strengthened the need to maintain microbial cultures in such a way that the properties that make them important, such as their contribution to seedling and root growth, as well as their fungicidal action, among others. profits remain stable, as can be seen from the following patent documents.

Patent document WO2019155252 (A1) describes a first isolated bacterial strain of the genus Bacillus, a simple species deposited on March 15, 2017 in the DSMZ with the access number DSM32459: strain Bacillus simplex VMC10/70, which is gram positive and it is preferably characterized by rod-shaped cells and can grow at 4 to 40°C. The strain may use at least one of the carbon sources selected from: arabinose, glucose, fructose, mannose, mannitol, malate, N-acetylglucosamine, maltose, sucrose, trehalose, fucose, potassium gluconate, malic acid, phenylacetic acid, acid adipic and any combination thereof. It also refers to an additional isolate that is a member of the genus Paenibacillus, polymyxa species deposited on March 15, 2017 in the DSMZ with the accession number DSM32460 and that has the following name Paenibacillus polymyxa strain VMC10/96), which is gram-positive and is preferably characterized by rod-shaped cells. Said document also proposes a mixture of both strains. According to a preferred embodiment of the invention, the strain(s) of Bacillus simplex and/or Paenibacillus polymyxa are used in combination with other microorganisms, preferably selected from: bacteria, preferably PGPR (or rhizobacteria), yeasts, mycorrhizae , mushrooms, any derivative as described above and any combination thereof. The PGPR of interest that can be used in combination with the strain(s) of the invention is preferably selected from: Aeromonas rivuli, Agromyces fucosus, Bacillus spp. The yeasts of interest that can be used in combination with the strain(s) of the invention are preferably selected from: Candida spp., Candida tropicalis, Saccharomyces spp., Saccharomyces bayanus, Saccharomyces bouiardii, Saccharomyces cerevisiae, Saccharomyces exiguous, Saccharomyces pastorianus, Saccharomyces pombe, and their combinations. The mycorrhizae of interest that can be used in combination with the strain(s) of the invention are preferably selected from: Glomus spp., Rhizophagus spp., Septoglomus spp., Funneliformis spp., and combinations thereof. The fungi of interest that can be used in combination with the strain(s) of the invention are preferably selected from: Trichoderma spp., Trichoderma atroviride, Trichoderma viride, Trichoderma afroharzianum, Paecilomyces spp., Beauveria bassiana., Metarhizium spp. ., Lecanicillium lecanii, Penicillium spp., Aspergillus spp., Conythyrium minitans, Pythium spp, and combinations thereof. It may comprise biostimulant; source of various nutritional elements, etc.

For its part, patent document WO2019135972 (A1) provides isolated microbial strains capable of improving crop yield, as well as compositions comprising the isolated strains and methods for using the isolated strains. A first aspect of this document is the Bacillus isolate having deposit accession number NRRL B-67533 (B. megaterium MON 205235) or NRRL B-67534 (B. megaterium MON 205620). A second aspect is a biologically pure culture of Bacillus megaterium MON 205235 (NRRL B-67533) or Bacillus megaterium MON 205620 (NRRL B-67534). A third aspect is an inoculant composition comprising Bacillus megaterium MON 205235 (NRRL B-67533) and/or Bacillus megaterium MON 205620 (NRRL B-67534) in an agriculturally acceptable carrier. In some embodiments, the inoculant composition comprises one or more stabilizing compounds, one or more pesticides, one or more lipo-chitooligosaccharides, one or more chitooligosaccharides, one or more chitinous compounds, one or more flavonoids and/or one or more additional microorganisms. A fourth aspect is a coated plant propagation material comprising a plant propagation material and a coating covering at least a portion of an outer surface of the plant propagation material, said coating comprising a composition inoculant of the present disclosure. A fifth aspect is a kit comprising a coated plant propagation material according to the present disclosure and a container that houses the coated plant propagation material.

In both documents WO2019155252 (A1) and WO2019135972 (A1) they propose mixtures that comprise isolated strains of microorganisms, however, these mixtures in turn combine them with any type of substance, from commercial microorganisms, to excipients, so said strains are complex and also its purpose is to promote the growth of vegetable plants and not to control phytopathogenic fungi, mainly.

Currently, it is common to isolate strains of microorganisms for different purposes. Due to the endemic characteristics of geographical regions, countries and/or territories, strategies are required to use microorganisms with activity and/or specific and efficient functions in said regions, countries and/or territories, where they are going to be applied, as an advantage for the use of such isolated microorganisms.

That is why, in order to contribute to the solution of the aforementioned drawbacks, mixtures and a method have been developed for the control of phytopathogenic organisms that attack plant seeds during their germination.

The characteristic details of the present invention are clearly shown in the following description, examples and accompanying figures, in an illustrative manner to demonstrate its conception and some of its preferred embodiments. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph where the number of plants with Fusarium spp. Figure 2 is a comparative graph of the percentage of plague of Fusarium sp. against seed in its natural genetic state

DETAILED DESCRIPTION OF THE INVENTION The present invention refers to a mixture for the control of phytopathogenic fungi that attack vegetable seeds during their germination, which comprises: i) an isolated strain of Trichoderma harzianum, as a biological control agent against various diseases caused by phytopathogenic fungi; ii) an isolated strain of Bacillus subtilis, as a biological control agent for diseases caused by fungi and soil bacteria;

iii) an isolated strain of Azospirillum brasilense, as a nitrogen fixing agent (N) and making it available for the roots or radicles; and iv) an efficient amount of a strain of Rhizophagus irregularis, as a mycorrhizal symbiotic agent to provide greater tolerance to the seedlings or plants that are born from the seeds and/or grains treated with this fungus.

One embodiment of the mixture of the present invention is when the Trichoderma harzianum strain has the characteristics of the strain deposited under accession number CM-CNRG TB174. One embodiment of the mixture according to the present invention is when the Bacillus subtilis strain has the characteristics of the strain deposited under accession number CM-CNRG TB175. Another embodiment of the mixture in question is when the Azospirillum brasilense strain has the characteristics of the strain deposited with accession number CM-CNRG TB176.

A further embodiment of said mixture is when the Rhizophagus irregularis strain has the characteristics of the commercial strain BTJ RI-19.

In a preferred embodiment of the mixture according to the present invention, it may also comprise at least: one pesticidal agent; a nematicidal agent; a root development stimulating agent; a mixture of enzyme cofactors; a mix of nutrients for vegetables; a mixture of free amino acids, among other substances of interest.

The pesticidal agent, for example, can be a mixture of 50 to 95% spinosyn A, and 5 to 50% spinosyn D.

An example of the nematicity agent is a seed extract of Ricinus spp.; preferring Ricinus communis L; and even more preferable that said Ricinus communis L. seed extract has an amount of oil of 40%.

The agent that stimulates root development can be, by way of example, indolebutyric acid.

The mixture of enzyme cofactors is preferred to comprise at least: thiamine and pyridoxine.

As for the mixture of plant nutrients, it is preferable to contain at least: MgS04, FeSC, NaaMoC, ZnSC, NH4NO3, and/or CaCh.

For the free amino acid mixture, it is desired that it contains at least: proline, glycine, glutamic acid, arginine, lysine, cysteine, and/or tryptophan. One more object of the present invention is a method for the control of phytopathogenic organisms that attack plant seeds during their germination, characterized by, comprising, applying a mixture in accordance with any of the preceding claims, to seeds at the time of their germination. germination; where the phytopathogenic organisms to control are, at least, fungi and insects. Fungi include those of the genus Fusarium spp.

The plant seeds that can be treated with this method according to the present invention can be those of the Poaceae family, preferably of the Zea genus; and more preferably of the species Zea mays L.

EXAMPLE

The following example is included to illustrate the design and some preferred embodiments of the invention, therefore, it should not be considered as limiting the scope of protection of the invention in question.

Example 1. Evaluation of several mixtures of the present invention (Maya Fusarium) in the control of Fusarium spp.

To find out if the mixtures of the present invention really control phytopathogenic organisms in seeds in their germination stage, a trial was carried out from April 21 to 28, 2020, in a plot located in Ocotlán, Jalisco, Mexico.

1. Yellow corn seeds (Zea mays L.) were used and for the general analysis of the study carried out on corn seeds, we considered the following points: a. Yellow corn was used b. Lot 3: parental seed 2.- The seeds were previously inoculated with the fungus Fusarium spp. strain RVP FO-20 and a control without inoculum, as well as 14 different treatments plus two control seeds, one inoculated with Fusarium spp and the other without inoculum, all treatments were applied to the inoculated seed, see Table 1.

3.- The evaluation process was carried out as follows:

A statistical analysis was made taking into account the different variables, the batch to be used, the number of treatments to be evaluated and the number of repetitions, so that the study was statistically representative. What led us to obtain that each treatment would be done 2 repetitions with 50 seeds each, which yielded the following data: a. Lot 3: 14 treatments, 1 control without inoculum plus a control with inoculum, is equal to a total of 16 variants with 100 seeds, each equivalent to 1600 seeds. b. Total studied population of 1,600 seeds

50 seeds were placed on a piece of paper, previously identified and moistened, which were potted and placed in a plastic bag also previously identified. These were taken to an incubator with the following conditions:

A. Temperature: 30 ± 1°C

b. Relative humidity: 25 ± 2%

C. 7 days of incubation

R1 : It was incubated from April 21-27, 2020.

R2: It was incubated from April 22-28, 2020.

This was done with the purpose of accelerating the growth of the seed, as well as subjecting it to constant stress, and the values thrown were the crudest that could be obtained, this given that in the field the seed is not subjected to so much stress ( high temperature all the time, the humidity percentage also fluctuates in the field) so from the results below analyzed, it is expected that in the field they will be better due to what has been described above. Protocol aligned to the standards established by ISTA, for the evaluation of seeds. Another important fact to mention is that applying condition A and B if the seed has some type of fungus problem (in addition to the Fusarium inoculum, both are forced to grow and express themselves so that it is noticeable; since what fungi ask of us for its growth it is water (moisture), temperature and a food source (seed).

4.- The data shown is an average of the repetitions carried out in the study.

The basic seed treatment should include a fungicide and an insecticide, such as deltamethrin, pirimiphosmethyl, carbocaptan, thiram. The insecticide is effective for storage pests and the fungicide is effective for fungal control at the time of sowing in the seed germination process. In figures 1 and 2 it can be seen that the Maya fusarium formulation had the best results, compared to the other treatments. Negative values represented better control of Fusarium spp.

Table 1. Treatments submitted to evaluate the control of Fusarium spp. of the blends of the present invention.

Table 1...

LITERATURE CITED Charmley, L. L., A. Rosenberg, and L. H. Trenholm. 1994. Factors responsible for economic losses due to Fusarium mycotoxin contamination of grains, food, and feedstuffs. In Mycotoxins in grain: compounds other than aflatoxin, D. J. Miller and L. H. Trenholm (eds.). Eagan, Minn. p. 471-486.

Jelinek, C.F., A.E. Pohland, and G.E. Wood. 1989. Review of mycotoxin contamination. Journal Association Official Analytical Chemistry 72:223-

230.

Maclas Cervantes, J. and S. Peraza Medina. 2008. Maize diseases in northern Sinaloa. Agronet. www.agronet.com. mx/articles.cgi?Action=Viewhistory&Article=3&Type=A; 31.03-2008. Shurtleff, M.C. 1980. Compendium of corn diseases. American Phytopathological Society, St. Paul, Minn. 105 pages

Sutton, J. C. 1982. Epidemiology of wheat head blight and maize ear rot caused by Fusarium graminearum. Canadian Journal Plant Pathology 4:195-209.

Tuite, J., G. Sharner, G. Rambo, J. Foster, and R.W. Caldwell. 1974. The Gibberella ear rot epidemics of corn in Indiana in 1965 and 1972. Cereal Science

Today 19: 238-241.

Claims

1. A mixture for the control of phytopathogenic fungi that attack vegetable seeds during their germination, characterized by, comprising: i) an isolated strain of Trichoderma harzianum, as a biological control agent against various diseases caused by phytopathogenic fungi; ii) an isolated strain of Bacillus subtilis, as a biological control agent for diseases caused by fungi and soil bacteria; iii) an isolated strain of Azospirillum brasilense, as a nitrogen fixing agent (N) and making it available for the roots or radicles; and iv) a strain of Rhizophagus irregularis, as a mycorrhizal symbiotic agent to provide greater tolerance to the seedlings or plants that are born from the seeds and/or grains treated with this fungus.

2. The mixture of the preceding claim, wherein the strain of Trichoderma harzianum has the characteristics of the strain deposited with accession number CM-CNRG TB174.

3. The mixture of claim 1, wherein the strain of Bacillus subtilis has the characteristics of the strain deposited with accession number CM-CNRG TB175.

4. The mixture according to claim 1, wherein the Azospirillum brasilense strain has the characteristics of the strain deposited with the accession number CM-CNRG TB176.

5. The mixture according to claim 1, wherein the Rhizophagus irregularis strain has the characteristics of the commercial strain BTJ

IR-19.

6. The mixture according to any of the preceding claims, characterized by, further comprising: at least one pesticidal agent; a nematicidal agent; a root development stimulating agent; a mixture of enzyme cofactors; a mix of nutrients for vegetables; and/or a mixture of free amino acids.

7. The mixture of the preceding claim, wherein the pesticidal agent is a mixture of 50 to 95% spinosyn A, and 5 to 50% spinosyn D.

8. The mixture of claim 6, wherein the nematicity agent is an extract of Ricinus spp.

9. The mixture of the preceding claim, where the extract is from the seeds of Ricinus communis L.

10. The mixture according to the preceding claim, wherein the Ricinus communis L. seed extract contains an amount of oil of 40%.

11. The mixture according to claim 6, wherein the root development stimulating agent is indolebutyric acid.

12. The mixture according to claim 6, wherein the enzyme cofactor mixture comprises at least: thiamine and pyridoxine;

13. The mixture according to claim 6, wherein the plant nutrient mixture contains at least: MgSC>4, FeSC>4, Na2Mo04, ZnSC, NH4NO3, and/or CaCI ₂ .

14. The mixture according to claim 6, wherein the mixture of free amino acids contains at least: proline, glycine, glutamic acid, arginine, lysine, cysteine, and/or tryptophan.

15. A method for the control of phytopathogenic organisms that attack plant seeds during their germination, characterized by, comprising, applying a mixture according to any of the preceding claims, to plant seeds at the time of their germination.

16. The method of the preceding claim, wherein the phytopathogenic organisms are fungi and insects.

17. The method of the preceding claim, wherein the fungi are of the genus Fusarium spp.

18. The method according to claim 15, wherein the plant seeds are from the Poaceae family

19. The method of the preceding claim, wherein the plant seeds are of the Zea genus.

20. The method of the preceding claim, where the vegetable seeds are from Zea mays L.