ENDOPHYTIC STRAIN OF CLONOSTACHYS ROSEA FOR BIOCONTROL OF PHYTOPATHOGENIC FUNGI
20230000087 · 2023-01-05
Inventors
Cpc classification
International classification
Abstract
The invention relates to a biocontroller product of phytopathogenic fungi, specifically to a biocontrol composition which comprises an endophytic strain of Clonostachys rosea R36.1, CChRGM 989 (entry), CChRGM 2905. Where this specific strain has the ability to control phytopathogenic fungi associated with wood diseases, especially vine wood such as Neofusicoccum parvum, Diplodia seriata and Phaeomoniella chlamydospora, among others.
And a method to prevent and control fungal diseases in plants that includes applying the biocontrol composition comprising the previously described Clonostachys rosea strain R36.1 CChRGM 2905 in a plant susceptible to developing a fungal infection.
Claims
1. A biocontrol composition for phytopathogenic fungi CHARACTERIZED in that it comprises Clonostachys rosea strain R36.1, CChRGM 2905 and an agronomically appropriate vehicle.
2. The biocontrol composition of claim 1, CHARACTERIZED in that it comprises a suspension of conidia of Clonostachys rosea strain R36.1 in a concentration between 10.sup.4 and 10.sup.9.
3. The biocontrol composition of claim 2, CHARACTERIZED in that the vehicle is selected from the group consisting of water, aqueous solutions, thick suspensions, granules and powders.
4. The biocontrol composition of claim 1, CHARACTERIZED in that the biocontrol composition additionally comprises other biocontroller microorganisms of the same species as another.
5. The biocontrol composition of claim 1, CHARACTERIZED in that the biocontrol composition additionally comprises additives selected from the group consisting of fertilizer, insecticide, fungicide, nematicide, surfactants, UV protection systems and mixtures thereof
6. A method to prevent and control fungal diseases in plants CHARACTERIZED in that it includes applying the biocontrol composition comprising Clonostachys rosea strain R36.1 CChRGM 2905 according to claim 1 in a plant susceptible to developing a fungal infection.
7. The method of claim 6, CHARACTERIZED in that plant disease is mediated by fungi of species such as Neofussicoccum parvum, Diplodia seriata and other species of the Family Botryosphaereacea, Phaeomoniella chlamydospora and Botrytis cinerea
8. The method of claim 6, CHARACTERIZED in that the plant is selected from the group consisting of flowers, ornamental plants, fruit vegetables, hydroponic crops, leafy vegetables and cabbage crops, pome fruits, deciduous trees, vines, citrus fruits, pines, stone fruits, nuts, grains and herbs.
9. The method of claim 8, CHARACTERIZED in that the plants to be treated are vines.
10. The method of claim 6, CHARACTERIZED in that the application is carried out by: spraying, liquid or dry application in furrows, soaking of plant material, on wounds of pruning cuts, direct incorporation into soils or mixtures of planting in greenhouse, pots, fields, granular formulations or granules, or direct treatment of seeds or propagating material or grafts.
11. The method of claim 6, CHARACTERIZED in that the biocontrol composition comprising Clonostachys rosea strain R36.1 CChRGM 2905 is applied in the form of a suspension comprising between 1×10.sup.4 to 1×10.sup.9 total conidia per milliliter.
Description
DESCRIPTION OF THE FIGURES
[0013]
[0014]
DESCRIPTION OF THE INVENTION
[0015] The invention relates to a biocontroller product of phytopathogenic fungi, specifically to a biocontrol composition comprising an endophytic strain of Clonostachys rosea R36.1, CChRGM 2905. The inventors have determined that this specific strain has the ability to control phytopathogenic fungi associated with wood diseases, especially vine wood diseases such as Neofusicoccum parvum, Diplodia seriata and Phaeomoniella chlamydospora, among others.
[0016] Inventors have found that this strain has an antibiotic capacity, and at the same time an ability to co-repress other fungi, so it would attack with more than one mechanism the pathogens present in the plant tissue.
[0017] For the expert in the technique it will be evident that the most efficient way to apply a strain of a biocontroller fungus is a composition with its propagation material, in this case the conidia.
[0018] The biocontrol composition of the invention comprises, in addition to the conidia of the strain of the invention, an appropriate vehicle, which is selected from the group consisting of water, aqueous solutions, thick suspensions, granules and powders. In addition, the composition of the invention may contain other biocontroller strains of the same species or another, and/or additives selected from the group consisting of fertilizer, insecticide, fungicide, nematicide surface-active substances, UV protection systems and mixtures thereof.
[0019] In a second aspect, the invention points to a method to prevent and control fungal diseases in plants which includes applying the biocontrol composition comprising the endophytic strain of Clonostachys rosea R36.1, CChRGM 2905, in a plant susceptible to developing a fungal infection; where plant disease is especially wood disease, especially the vine, where the pathogen belongs especially to species such as Neofusicoccum parvum, Diplodia seriata and other species of the Botryosphaereacea family, Phaeomoniella chlamydospora and Botrytis cinerea, for example. The plant to be protected is selected from the group consisting of flowers, ornamental plants, fruit vegetables, hydroponic crops, leafy vegetables and cabbage crops, pome fruits, deciduous trees, vines, citrus fruits, pines, stone fruits, nuts, grains and herbs. The plants to be treated are especially vines.
[0020] The application of the composition of the invention is carried out by any method available in the technique, such as: spraying, liquid or dry application in furrows, soaking of plant material, on wounds of pruning cuts, direct incorporation into soils or planting mixtures in greenhouses, pots, fields, granular formulations or granules, or direct treatment of seeds or propagating material or grafts.
[0021] Where the composition of the invention is preferentially applied in the form of a suspension comprising between 1×10.sup.4 to 1×10.sup.9 total conidia per milliliter.
[0022] The composition may contain other formulation adjuvants, such as surface-active substances, UV protection systems, adherents, dispersants, disintegrators, wetting agents, among others.
APPLICATION EXAMPLES
Example 1. Obtaining a Composition of the Invention
[0023] The strain of the invention was isolated from the interior of vine roots, identified by ITS as Clonostachys rosea and deposited under the deposit number CChRGM 2905 in the Chilean Collection of Microbial Genetic Resources.
[0024] Liquid culture media were evaluated for the production of conidia by the strain. An inoculum of 1×10.sup.4 of the strain of the invention was added to conical tubes with 30 mL of PDb (potato broth dextrose, 24 gL.sup.−1), MEb (broth malt extract 15 gL.sup.−1), MEb with addition of sodium (MEb modified with 0, 85% NaCl). The samples were incubated at 25° C. with a stirring of 170 rpm at an angle of 45° with the caps loose for 10 days. This experiment was repeated 5 times.
[0025] Samples were collected from each tube in duplicate and the conidia were counted with a Neubauer chamber.
[0026] The production of C. rosea R36.1 conidia was higher in the PDb broth medium, followed by MEb. The conidial concentration obtained in PDb (1.4×10.sup.8 conidia mL.sup.−1), was 7 times higher than the best conidial production in any of the other media evaluated.
[0027] With the conidia produced under any of the conditions indicated, the formulation of the invention can be obtained at a concentration of 1×10.sup.6 conidia mL.sup.−1 and an agronomically appropriate vehicle such as water.
Example 2. Effect of Clonostachys rosea R36.1 on the Growth of GTD Fungi in Pruning Material
[0028] An in plantae trial was conducted to evaluate the fungal antagonist effect on GTD fungal growth. Internode sections of the annual vine pruning material (rods) were cut into 4.5 cm lengths and then autoclaved for 25 minutes at 121° C.
[0029] Inoculation with Clonostachys rosea was carried out by adding 40 μl of fresh conidia suspension (1×10.sup.6 mL.sup.−1 conidia) of antagonist ensuring that the suspension covered all the rods by capillarity. As a control, in parallel experiments tebuconazole was applied (recommended field dose of 60 ml/100 L) or sterile distilled water with the same procedure.
[0030] This experiment was carried out 5 times. The rods of annual material were incubated in individual wet chambers for 24 hours.
[0031] Then, we proceeded to challenge N. parvum and D. seriata with the pathogen, for which 10 μl of suspension of mixture of conidia and fresh mycelium of each of the pathogens were inoculated separately at the same end of each rod where it had been inoculated previously with the strain of the invention; and were immediately placed in a horizontal position, avoiding the diffusion of the suspension by the plant material.
[0032] The rods with the treatment and pathogen were incubated in wet chambers for 7 days. Subsequently, the surface of the pruned vine material was disinfected by rubbing with 70% ethanol. With a sterile hot scalpel, the bark and 0.5 cm from the ends of each rod were removed. Small sections of plant material located at 0.5 and 2.5 cm from the pathogen's inoculation point were collected and cultured in individual PDA (potato dextrose agar) plates at 25° C. for 7 days. In this way, it was possible to evaluate the progress of each pathogen through the pruning material with the 3 treatments.
[0033] To assess the viability of the pathogen's conidia and mycelium suspension, 10 μl of the solution was inoculated on one side of the piece of wood as described above and that was immediately processed to obtain small pieces at 0.5 and 2.5 cm from the pathogen's inoculation point. Each piece was grown in PDA at 25° C. for 7 days.
[0034] The presence of pathogens in solid culture was evaluated under a microscope.
[0035] The antagonist strain of the invention C. rosea R36.1 was recovered in all samples co-inoculated with pathogens after 7 days. The results obtained are shown in
[0036] Additionally, bioassays were also performed on natural pruning material without a sterilization process. Sections of 4.5 cm in length were inoculated in their entirety with 40 uL of fresh conidia of C. rosea R36.1 (1×10.sup.6 conidia mL.sup.−1). The fungicide Tebuconazole (recommended field dose of 60 ml/100 L) was used as a control, as well as sterile distilled water under the same procedure.
[0037] After 24 hours, the pruning material was inoculated with 10 μl of suspension of conidia mixture and fresh mycelium of N. parvum and D. seriata separately at the same end of each rod where the strain of the invention had previously been inoculated; and was immediately placed in a horizontal position, avoiding the diffusion of the suspension by the plant material. The pruning material was incubated in wet chambers for 7 days.
[0038] After incubation, the surface of the sections was disinfected with 70% ethanol. Using a sterile hot scalpel, the bark and 0.5 cm from the ends of each rod were removed. Small sections of plant material located at 0.5 and 2.5 cm from the pathogen's inoculation point were collected and grown in individual PDA (potato dextrose agar) plates at 25° C. for 7 days for the advancement of each pathogen through the pruning material.
[0039] As a control, the viability of the suspension of conidia and mycelium of the pathogen was evaluated. Ten μl of the suspension of each pathogen was inoculated on one side of the piece of wood as described above and that was immediately processed to obtain small pieces at 0.5 and 2.5 cm from the inoculation point. Each piece was grown in PDA at 25° C. for 7 days. The presence of pathogens in solid culture was evaluated under a microscope. The results are shown in
[0040] The pathogens were able to colonize the entire piece in 7 days when they were not previously treated. As can be seen in the figure, the strain of the invention completely inhibited the development of the pathogen N. parvum, while the fungicide Tebuconazole was effective only at 2.5 cm from the point of inoculation. As for the other pathogen evaluated, D. seriata, 100% inhibition is observed in the section furthest from the inoculation point (2.5 cm) and 90% inhibition at 0.5 cm from the inoculation point. Conversely, Tebuconazole showed no inhibition of the growth of the pathogen D. seriata at 0.5 cm, and only 40% inhibition at 2.5 cm.
[0041] These results demonstrate that the strain of the invention is able by itself to inhibit the growth of pathogens in plant material.
Example 3. Evaluation of the Antagonistic Mechanism of the Strain of the Invention
[0042] To assess the mode of action of C. rosea strain R36.1, microscopic observations were made of the mycelium of the pathogenic fungi and the strain of the invention confronted in the area of interaction in petri dishes where a disc of agar of 5 mm of pathogen culture was placed on one side of a Petri dish with PDA (39 g L.sup.−1; Difco) or AA (water agar 20 gL.sup.−1) and on the opposite side a disc of agar of 5 mm containing la cultivated strain of the invention was placed. The plates were incubated at 25° C. until the mycelium of both fungi was in contact or formed an inhibition halo.
[0043] When the endophytic C. rosea strain of the invention was confronted with the pathogens D. seriata or N. parvum, a halo of 15 to 20 mm surrounding the antagonist colony prevented the pathogen from growing larger and reaching the hyphae of Clonostachys.
[0044] In another trial, plates with PDA (39 g L.sup.−1; Difco) with the surface covered with cellulose paper where a 5 mm mycelium agar disc of the antagonist was inoculated. After 7 days of incubation at 25° C., the paper was removed along with the C. rosea mycelium and a disc of mycelium agar of the same size as the pathogen in the center of the plate was inoculated on the same plate. The potential antibiotic compound from C. rosea showed inhibition over 47.2% of the growth of D. seriata and 50.1% of that of N. parvum.
[0045] Based on this result, the inventors deduce that what inhibits the growth of the pathogen, is an antibiotic compound secreted to the medium by the strain of the invention.
[0046] However, once the pathogen made contact with the hyphae of the antagonist, it was possible to observe under an optical microscope, a coiling of the hyphae of C. rosea over those of the pathogen at various points. Based on the literature and what was observed under the microscope, the inventors deduce that the coiling is given by the ability of the strain to mycoparasitize the pathogen. This, since the coiling is the step prior to the penetration of the hyphae of the pathogen, which generates cell death.
Example 4. Comparative Advantages of the Strain of the Invention
[0047] In order to demonstrate the advantages as an antagonist of the strain of the invention with respect to other biocontroller microorganisms, se evaluated in agar the ability to antagonize and stop the growth of phytopathogens D. seriata and N. parvum of several known biocontrol agents: Purpureocillium sp., Chaetomium sp., Trichoderma sp., Epicoccum sp., and three strains of C. rosea among which is the strain of the invention.
[0048] For which a 5 mm mycelium disc of each antagonist obtained from the growth edge of the colony was positioned 1 cm from the edge of a plate with diluted PDA (19 g L.sup.−1; Difco). At the opposite end, a mycelium disc of the same size of D. seriata or N. parvum was placed. The plates were left incubating for 14 days at 25° C. Subsequently, the area of the colony of the pathogen was measured. This trial was conducted in fivefold. The results are shown in
[0049] After this time, it was possible to observe that the strain of the invention generated an inhibition of the growth of both pathogens of 98%, on average while the other strains of C. rosea evaluated and the other microorganisms evaluated Purpureocillium sp., Chaetomium sp., Trichoderma sp. and Epicoccum sp. presented much lower inhibitions than this, between 10% and 80% approximately, as shown in
[0050] These examples should be considered as illustrative and not limiting to the present invention, which is fully defined in the accompanying claims.