Application of trichoderma asperellum strain yn4 in combination with fungicides in the prevention and treatment of plant fungal diseases

Abstract

The application discloses application of Trichoderma asperellum strain YN4 and fungicides in prevention and treatment of plant fungal diseases. The application research shows that the Trichoderma asperellum strain YN4 has a good inhibitory effect on pepper anthracnose, rubber anthracnose, mango anthracnose, soybean anthracnose and the like. Further, compared with the single use of fungicides or the Trichoderma asperellum strain, the inhibitory effect of the combination of the two is better. Under certain conditions, the Trichoderma asperellum strain is combined with 10% difenconazole water dispersible granules, 70% thiophanate-methyl wettable powder or 450g / L prochloraz emulsion in water, and has a synergistic effect. Not only can the use amount of fungicides be greatly reduced, but also the inhibitory effect on plant pathogenic fungi can be further improved, the incidence can be reduced, the inhibition on pathogenic bacteria can be enhanced, the use and residue of chemical pesticides can be reduced, and technical support is provided for the selection of fungicides for pepper anthracnose and the like and the combination of bacteria and drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant disease control. More specifically, it relates to the application of Trichoderma asperellum YN4 strain and fungicide in the prevention and treatment of plant fungal diseases. BACKGROUND

[0002] According to statistics, the global food loss caused by diseases and pests can reach about 10% of the total amount (FAO data) every year. Chemical pesticides are still the main measure to prevent and control plant diseases and pests. However, the excessive use of chemical agents has led to the increase of disease and pest resistance, the serious pesticide residues in agricultural products, the destruction of soil environment, and the decrease of the number of beneficial natural enemies. Therefore, the use of biological control measures is one of the keys to reduce the use of chemical pesticides and achieve green control.

[0003] The use of agricultural control, physical control, biological control, ecological regulation, and scientific, reasonable and safe use of pesticides can effectively control crop diseases and pests, ensure the safety of crop production, the quality of agricultural products and the safety of agricultural ecological environment, and promote the purpose of increasing agricultural production and income.

[0004] Plant disease control is an important part of agricultural production. At present, it mainly relies on chemical means for prevention and control. However, due to the continuous and large-scale use of chemical pesticides, it threatens human health, pollutes the environment, is toxic to non-target organisms, and easily causes plant pathogens to develop resistance, which leads to various restrictions on its application. Biological pesticides can overcome the defects of chemical pesticides due to their high efficiency, low toxicity, and easy degradation, and have great development potential. This paper reviews the mechanism and application status of microbial fungicides for plant use, summarizes the current problems in the research and application of microbial fungicides for plant use, and looks forward to the future development trend. With people's increasing demand for environmental protection, microbial pesticides are undoubtedly one of the future directions of pesticide development.

[0005] Biological control is to screen and use beneficial microorganisms and make biological agents to prevent and control the occurrence of plant diseases and pests, so as to achieve the purpose of disease control. It is a new idea and method of green control of plant diseases and pests. Microbial agents have the characteristics of safety, sustainability, broad spectrum and green. The mechanism of microbial agents includes antibiosis, competition, hyperparasitism and induction of plant systemic resistance. Microbial agents can also promote plant growth and have yield-increasing effect on agricultural products. The use of microbial preparation can reduce the application amount of chemical fertilizers and pesticides by 30%-60%, increase the yield of crops by 5%-40% depending on different crops, and increase the resistance of plants to diseases and pests, thereby fundamentally reducing the use of chemical pesticides.

[0006] Biocontrol agents have a good application prospect due to small environmental impact and difficulty of pathogen resistance, but are limited by slow effect and environmental impact. Due to the problems of chemical pesticides and biocontrol agents, the existing research proposes an ideal measure of combining chemical pesticides and biocontrol agents to reduce the use amount of chemical pesticides and enhance the inhibition of pathogenic bacteria. Although this combination mode can achieve the prevention and treatment effect and slow down the generation of pathogen resistance, the chemical pesticides are easy to kill biocontrol agents, resulting in reduced efficiency of biocontrol agents. Therefore, in order to reduce the use amount of chemical pesticides, it is urgent to develop more and more efficient biocontrol agent-pesticide combination drug programs and means, which can prevent and treat plant diseases in a small amount and high efficiency. SUMMARY

[0007] The technical problem solved by the present application is to overcome the defects and deficiencies of the existing biological pesticides for preventing and treating pepper anthracnose and the like, to provide the application of Trichoderma asperellum YN4 and fungicides in preventing and treating plant fungal diseases, to provide a new scheme of drug-bacteria combination, to greatly reduce the use amount of chemical pesticides, and to have a good inhibitory effect on pepper anthracnose.

[0008] The present application aims to provide the application of Trichoderma asperellum YN4 strain and fungicides in preventing and / or treating plant fungal diseases.

[0009] Another object of the present application is to provide a medicament for preventing and / or treating plant fungal diseases.

[0010] Still another object of the present application is to provide a method for preventing and / or treating plant fungal diseases.

[0011] The above objects of the present application are achieved by the following technical solutions:

[0012] The application research shows that the Trichoderma asperellum YN4 strain has a good inhibitory effect on pathogenic fungi Colletotrichum scovillei, Colletotrichum siamense, Colletotrichum truncatum and / or Colletotrichum fructicola, and can be used for preventing and treating plant diseases caused by pathogenic fungi Colletotrichum scovillei, Colletotrichum siamense, Colletotrichum truncatum and / or Colletotrichum fructicola. Further combined use research with fungicides shows that compared with the use of fungicides and the Trichoderma asperellum strain alone, the combined use has a better inhibitory effect, and has a synergistic effect under certain conditions, which not only greatly reduces the use amount of fungicides, but also further improves the inhibitory effect on plant pathogenic fungi and reduces the incidence, and has a significant effect on preventing and treating plant fungal diseases.

[0013] Therefore, the application provides the application of the Trichoderma asperellum YN4 strain and fungicides in preventing and treating plant fungal diseases or in preparing products for preventing and / or treating plant fungal diseases.

[0014] Further, the plant fungal disease is a plant disease caused by pathogenic fungi Colletotrichum scovillei, Colletotrichum siamense, Colletotrichum truncatum and / or Colletotrichum fructicola.

[0015] Preferably, the plant fungal disease is pepper anthracnose, rubber anthracnose, mango anthracnose, soybean anthracnose, strawberry gray mold, tobacco anthracnose, soybean brown spot, tea tree anthracnose, rice brown spot and the like.

[0016] Further, the Trichoderma asperellum YN4 strain has been preserved in the Guangdong Microbial Culture Collection Center on October 22, 2021, and the preservation number is GDMCC NO.62007.

[0017] Preferably, the fungicide is 10% difenconazole water dispersible granules, 70% thiophanate-methyl wettable powder and 450g / L prochloraz emulsion in water. It is particularly pointed out that the fungicides used in the application are all conventional products purchased in the market.

[0018] The present application also provides a medicine for preventing and / or treating plant fungal diseases, which comprises the Trichoderma asperellum YN4 strain and a fungicide as effective components.

[0019] Preferably, the fungicide is a 10% difenconazole water dispersible granule with a concentration of 0.4-1.2 μg / mL; the fungicide is a 70% thiabendazole wettable powder with a concentration of 0.1-0.5 μg / mL; and the fungicide is a 450 g / L prochloraz emulsion in water with a concentration of 0.02-0.1 μg / mL.

[0020] The present application also provides a method for preventing and / or treating plant fungal diseases, which comprises the medicine as described above.

[0021] In particular, the present application shows that the Trichoderma asperellum YN4 strain and the fungicide have good inhibitory effects on the pathogenic fungi Colletotrichum scovillei, Colletotrichum siamense, Colletotrichum truncatum and Colletotrichum fructicola, and thus, by preventing and treating the pathogenic fungi, the present application can be used for preventing and treating plant diseases caused by the pathogenic fungi Colletotrichum scovillei, Colletotrichum siamense, Colletotrichum truncatum and Colletotrichum fructicola, such as pepper anthracnose, rubber anthracnose, mango anthracnose, soybean anthracnose, strawberry gray mold, tobacco anthracnose, soybean brown spot, tea anthracnose, rice brown spot and the like, and is not limited to the plant diseases provided in the present application, but can be used for preventing and treating any plant diseases caused by the pathogenic fungi.

[0022] The present application has the following advantages:

[0023] The application provides application of the Trichoderma asperellum YN4 strain and fungicides in prevention and / or treatment of plant fungal diseases, and researches show that the Trichoderma asperellum YN4 strain has a good inhibitory effect on plant diseases caused by pathogenic fungi such as Colletotrichum scovillei, Colletotrichum siamense, Colletotrichum truncatum and Colletotrichum fructicola, for example, pepper anthracnose, rubber anthracnose, mango anthracnose, soybean anthracnose and the like, further combined use of the Trichoderma asperellum YN4 strain with fungicides shows that, compared with the fungicides and the Trichoderma asperellum YN4 strain used alone, the combined use has a better inhibitory effect, and under certain conditions, the Trichoderma asperellum YN4 strain is combined with 10% difenconazole water dispersible granules, 70% thiabendazole wettable powder or 450g / L prochloraz emulsion in water, has a synergistic effect, can greatly reduce the use amount of fungicides, further improve the inhibitory effect on plant pathogenic fungi and reduce the incidence, and has a good inhibitory effect on pepper anthracnose, rubber anthracnose, mango anthracnose, soybean anthracnose and the like, and has a remarkable effect in prevention and treatment of plant fungal diseases.

[0024] The research of the application not only can enhance the inhibition rate on pathogenic bacteria, but also can reduce the use of chemical pesticides and reduce pesticide residues in the field, and provides data support for exploring the fungicide screening of pepper anthracnose and the combined use of bacteria and drugs. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Inhibition effect of endophytic Trichoderma asperellum YN4 on four kinds of anthracnose fungi (A is the inhibition effect of endophytic Trichoderma asperellum YN4 on the growth of mycelium of pepper anthracnose fungus (Colletotrichum scovillei), rubber anthracnose fungus (Colletotrichum siamense), soybean anthracnose fungus (Colletotrichum truncatum) and mango anthracnose fungus (Colletotrichum fructicola); B is the inhibition rate of endophytic Trichoderma asperellum YN4 on pepper anthracnose fungus (Colletotrichum scovillei), rubber anthracnose fungus (Colletotrichum siamense), soybean anthracnose fungus (Colletotrichum truncatum) and mango anthracnose fungus (Colletotrichum fructicola), and different letters in the figure represent significant difference analysis (p<0.05) by Duncan's new multiple range method.

[0026] Figure 2Inhibitory effect of Trichoderma asperellum YN4 combined with fungicides on Colletotrichum scovillei (A is the control group, B is 70% thiophanate-methyl wettable powder 5000 times liquid, C is 70% thiophanate-methyl wettable powder 5000 times liquid combined with Trichoderma asperellum, D is Trichoderma asperellum treatment group, E is 450g / L prochloraz emulsion in water 5000 times liquid, F is 450g / L prochloraz emulsion in water 5000 times liquid combined with Trichoderma asperellum). DETAILED DESCRIPTION

[0027] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0028] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0029] The Trichoderma asperellum YN4 strain used in the present application is the result of the previous research of the research group of the present application. The strain has been preserved in the Guangdong Microbial Culture Collection Center on October 22, 2021, with the preservation number GDMCC NO.62007, and is disclosed in patent CN 114410481 A.

[0030] Example 1 Inhibitory effect of Trichoderma asperellum YN4 on various pathogenic fungi

[0031] Test plant pathogenic fungi: Colletotrichum scovillei, Colletotrichum siamense, Colletotrichum truncatum, and Colletotrichum fructicola. The above plant pathogenic fungi strains are preserved in the laboratory.

[0032] The above plant pathogenic fungus strains are target strains, and the antagonistic effect of Trichoderma asperellum strain YN4 on four plant pathogenic fungi is determined by confrontation method. The Trichoderma and pathogenic fungi grown on PDA plates for 3-5 days are punched with a puncher with a diameter of about 5 mm at the edge of the colony to obtain a fungus cake. In a PDA plate with a diameter of about 9 cm, at a distance of 2 cm from the edge of the plate, one side is inoculated with Trichoderma, and the other side is inoculated with pathogenic fungi. This is the treatment group, and each pathogenic fungus is repeated three times. The blank control group is only inoculated with pathogenic fungus cake (d=6mm) at a distance of 2 cm from the edge. After inoculation, it is placed in a 28°C constant temperature incubator for culture. The colony growth is observed every day, and when the Trichoderma and pathogenic fungi colonies just meet, the distance from the inoculation point to the edge of the plate of the plant pathogenic fungi in the treatment group and the blank control group is recorded, and the inhibition rate is calculated according to the following formula.

[0033]

[0034] The results are shown in Figure 1 As shown in Figure 1 A), the Trichoderma asperellum has good inhibition effect on the mycelial growth of the four Colletotrichum species, among which the inhibition effect of Trichoderma asperellum on C. siamense is the strongest, and the inhibition effect is significantly different compared with the confrontation effect of the other three Colletotrichum species, with an inhibition rate of 75.47%, while the mycelial growth inhibition rates of Trichoderma asperellum on C. scovillei, C. fructicola and C. truncatum are 59.21%, 68.75% and 65.91% respectively Figure 1 B).

[0035] Example 2 Inhibition effect of fungicides on Colletotrichum

[0036] The mycelial growth rate method is used to explore the mycelial growth inhibition effect of five fungicides (10% difenoconazole water dispersible granules, 70% thiabendazole wettable powder, 450g / L prochloraz water emulsion, 325g / L benzyl strobilurin suspension concentrate, 75% chlorothalonil wettable powder) on C. scovillei, C. siamense, C. truncatum and C. fructicola.

[0037] The 10% difenoconazole water dispersible granules, 70% thiophanate-methyl wettable powder, 450 g / L prochloraz emulsion, 325 g / L benzyl-pyraclostrobin suspension, and 75% chlorothalonil wettable powder were respectively prepared into working solutions with concentrations of 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, 2000 μg / mL; 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL; 8.125×10 -4 μg / mL, 1.625×10 -3 μg / mL, 3.25×10 -2 μg / mL, 6.25×10 -2 μg / mL, 1.25 μg / mL; 62.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL; 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, 2000 μg / mL.

[0038] The PDA medium was heated and melted, and then cooled to 55-65°C. 1 mL of the pesticide solution was added into 49 mL of the PDA medium, and then mixed thoroughly. The mixture was quickly poured into a sterile culture dish to prepare a toxin-containing medium with different concentrations of the pesticide. The control group was added with the same amount of sterile water. The mycelium blocks were cut from the edge of the colonies of the anthracnose bacteria which had been cultured in a 28°C incubator for 7 days, and then transferred to the PDA medium with 10% difenoconazole water dispersible granules, 70% thiophanate-methyl wettable powder, 450 g / L prochloraz emulsion, 325 g / L benzyl-pyraclostrobin suspension, and 75% chlorothalonil wettable powder with final concentrations of 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL; 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL; 1.625×10 -5 μg / mL, 3.25×10 -4 μg / mL, 6.5×10 -3 μg / mL, 1.25×10 -3 μg / mL, 2.5×10 -2 μg / mL; 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL; 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL. Each treatment was repeated three times, and then cultured in a 28°C constant temperature incubator in the dark until the control group was fully covered. The colony diameters were measured by the cross method, and the inhibition percentage was calculated according to the following formula.

[0039]

[0040] The virulence regression equation Y=a+bX was calculated with the concentration logarithm (X) and the probability value of the mycelium growth inhibition rate (Y), and the effective median concentration EC50 for inhibiting mycelium growth was calculated, and the specific results are shown in Table 1.

[0041] The mycelium growth virulence determination results of the tested fungicides on the four kinds of Colletotrichum are shown in Table 1, which shows that the five kinds of fungicides all have inhibitory effects on the mycelium growth of the four kinds of Colletotrichum, and the inhibition rate increases with the increase of the concentration. Among them, the inhibition effect of 450 g / L prochloraz emulsion in water on the four kinds of Colletotrichum is the strongest, and the EC50 value is less than 0.1.

[0042] Table 1 Inhibition of five kinds of fungicides on Trichoderma asperellum and Colletotrichum

[0043]

[0044] Example 3 Compatibility determination of fungicides and Trichoderma asperellum

[0045] In this test, the ratio of EC50 of the fungicide to Trichoderma asperellum to the EC50 of the fungicide to Colletotrichum is greater than 1 as the standard for screening fungicides that can be used in combination with Trichoderma asperellum. If the ratio of EC50 values is greater than 1, it is considered that the fungicide can be used in combination with Trichoderma asperellum.

[0046] The ratio of EC50 values = EC50 of the fungicide to Trichoderma asperellum / EC50 of the fungicide to the pathogen

[0047] The mycelium growth rate method was used to determine the inhibition of fungicides on Trichoderma asperellum. Each test agent was prepared into a stock solution according to the needs, and each stock solution was diluted with sterile water to 5 different gradient concentrations: 10% fluquinconazole water dispersible granules 2.5-40 μg / mL, 70% thiabendazole wettable powder 0.625-10 μg / mL, 450 g / L prochloraz emulsion in water 0.063-1 μg / mL, 325 g / L benzyl strobilurin suspension concentrate 0.625-10 μg / mL, and 75% bercanthal wettable powder 0.063-1 μg / mL. 1 mL of the diluted fungicide solution was added to 49 mL of sterilized PDA to prepare a PDA plate containing the fungicide, and Trichoderma asperellum was inoculated after activation culture (28°C, 7d). The PDA plate without fungicide was used as a blank control. Each concentration was repeated 6 times, and the test was repeated 3 times. After the PDA plate was inverted in a 28°C constant temperature incubator for 7 days, the colony diameter was measured, and the inhibition rate (%) was calculated.

[0048] The statistical results are shown in Table 1 above, which shows that the five fungicides have the EC50 values of C. acuminata from large to small in the order of 10% difenoconazole water dispersible granules, 70% thiabendazole wettable powder, 325g / L benzyl pyraclostrobin suspension concentrate, 75% chlorothalonil wettable powder and 450g / L prochloraz emulsion in water. Among them, the inhibition effect of 10% difenoconazole water dispersible granules on C. scovillei and C. siamense is always greater than that on C. acuminata, and the EC50 value ratio is >1. 10% difenoconazole water dispersible granules and C. acuminata can be combined to control C. scovillei and C. siamense. Similarly, 70% thiabendazole wettable powder can be combined with C. acuminata to control C. scovillei, C. siamense and C. fructicola, and 450g / L prochloraz emulsion in water can be combined with C. acuminata to control the four kinds of C. scovillei.

[0049] Example 4 Inhibition effect of fungicides combined with C. acuminata on C. acuminata

[0050] The compatibility of three fungicides, 10% difenoconazole water dispersible granules, 70% thiabendazole wettable powder and 450g / L prochloraz emulsion in water, with C. acuminata was better, and the PDA containing the fungicides was prepared by adding the fungicides to the PDA to a final concentration of 0.4-2 μg / mL, 0.1-0.5 μg / mL and 0.02-0.1 μg / mL, respectively. The agar plugs of C. acuminata and pathogenic fungi, which were cultured at 28°C for 7 days, were cut by a puncher, and the agar plugs of C. acuminata and pathogenic fungi were inoculated at the same time (Φ=6mm, the distance between the agar plugs was 3cm). The PDA containing the fungicides was used as the treatment control, and the same amount of sterile water was added as the blank control. The treatment was repeated 3 times, and the colony radius of the control pathogenic fungi and C. acuminata and the colony radius of the treated pathogenic fungi and C. acuminata were measured after 7 days of incubation at 28°C in the dark. The inhibition rate was calculated according to the following formula.

[0051]

[0052] The evaluation test method of the synergistic effect of the two is the synergistic coefficient method, and the calculation formula is as follows:

[0053] Synergistic coefficient (S) = (R Bt -R BCK ) / (R Tt -R TCK )

[0054] wherein: R TCK and RBCK R represents the control colony radius of Trichoderma echinosporum and Trichoderma anthracis, respectively; Tt and R Bt These represent the radii of opposing colonies of Trichoderma and Anthrax, respectively.

[0055] If S≥1.5, it indicates that the combined use of Trichoderma and fungicide has a synergistic effect;

[0056] 1≤S<1.5 indicates that the combined use of Trichoderma and fungicide has an additive effect;

[0057] S < 1 indicates that the combined use of Trichoderma and fungicide has an antagonistic effect.

[0058] The inhibitory effect of the combined use of fungicides and *Trichoderma echinococcus* on the growth of *C. siamense* mycelia was determined using the confrontation culture method. The inhibition results are shown in Tables 2, 3, and 4. Among them, the combined use of 10% difenoconazole water-dispersible granules and *Trichoderma echinococcus* showed differences in inhibition of *C. siamense* mycelia due to the differences in the inhibitory effects of the fungicide and *Trichoderma* on *C. siamense* mycelia, as shown in Table 2. The results showed that the combined use of 10% difenoconazole water-dispersible granules and *Trichoderma echinococcus* had the best inhibitory effect on *C. siamense* mycelia, with an inhibition rate of 73.33%–83.02%. When the concentration of the fungicide was 0.4–1.2 μg / mL, the inhibition rate of both *C. siamense* mycelia increased; at concentrations of 1.2–2 μg / mL, the inhibition rate decreased.

[0059] The synergistic coefficient method was used to evaluate the inhibitory effect of combined Trichoderma and fungicide on anthracnose mycelia. When Trichoderma echinococcus was used in combination with 10% difenoconazole water-dispersible granules to control *C. siamense*, an additive effect was observed within the experimental range. However, antagonistic effects may have been observed at high concentrations. Similarly, when 10% difenoconazole water-dispersible granules were used in combination to control *C. scovillei*, an antagonistic effect was observed within the experimental range.

[0060] Table 2. Inhibition and synergistic effects of combined use of Trichoderma and 10% difenoconazole water-dispersible granules on Anthracnose mycelia.

[0061]

[0062]

[0063] The combined use of 70% thiophanate-methyl WP and T. spinosum had the best inhibitory effect on C. siamense mycelium, with an inhibition rate of 87.36%-90.57%. The combined use of 70% thiophanate-methyl WP and T. spinosum had an inhibition rate of 76.31%-82.22% and 83.14%-88.29% on C. scovillei and C. fructicola mycelium, respectively, at 0.1-0.5 μg / mL, as shown in Table 3.

[0064] The synergistic coefficient method was used to evaluate the inhibitory effect of the combined use of T. spinosum and fungicides on C. siamense mycelium. The combined use of 70% thiophanate-methyl WP and T. spinosum had an additive effect at low concentrations and a synergistic effect at high concentrations. The combined use of T. spinosum and 70% thiophanate-methyl WP had an additive effect on C. fructicola. However, the combined use of T. spinosum and 70% thiophanate-methyl WP had an antagonistic effect on C. scovillei.

[0065] Table 3 Inhibition and synergistic effect of the combined use of T. spinosum and 70% thiophanate-methyl WP on C. siamense mycelium

[0066]

[0067]

[0068] The combined use of 450 g / L prochloraz EW and T. spinosum had the best inhibitory effect on C. siamense mycelium, with an inhibition rate of 82.12%-86.54%. The combined use of 450 g / L prochloraz EW and T. spinosum had an inhibition rate of 65.22%-73.91%, 73.98%-80.49%, and 79.56%-84.62% on C. scovillei, C. truncatum, and C. fructicola mycelium, respectively, at 0.02-0.1 μg / mL, as shown in Table 4.

[0069] The synergistic coefficient method was used to evaluate the mycelial growth inhibition of Colletotrichum spp. by Trichoderma spp. combined with fungicides. The combination of T. asperellum and 450 g / L prochloraz emulsifiable concentrate (EC) showed additive effect on C. fructicola and C. siamense at 0.02-0.1 μg / mL. The combination of T. asperellum and 450 g / L prochloraz EC showed antagonistic effect on C. truncatum at low concentration, but showed additive effect at 0.1 μg / mL. The combination of T. asperellum and 450 g / L prochloraz EC showed antagonistic effect on C. scovillei at all concentrations.

[0070] Table 4 Mycelial growth inhibition and synergistic effect of Trichoderma spp. combined with 450 g / L prochloraz EC on Colletotrichum spp.

[0071]

[0072]

[0073] Example 5 In vitro fruit method for determining the mycelial growth inhibition of Colletotrichum scovillei by fungicides combined with T. asperellum

[0074] T. asperellum cultured at 28°C for 7 days was cut into six pieces of mycelium with a size of about 5 mm x 5 mm using a needle, and then transferred into a 250 mL conical flask containing 150 mL of Czapek's liquid medium (CB). The flask was incubated at 28°C with shaking at 120 r / min for 7 days. The mycelium was filtered with sterile gauze, and the spore suspension of T. asperellum was diluted to 1 x 10 8 spores / mL. The spore suspension of C. scovillei was diluted to 3 x 10 6 spores / mL in the same way. Each fungicide was diluted to a final concentration of 1000x, 2000x, and 5000x.

[0075] Fresh and vigorous pepper fruits (Yanyanhongchaotian) were selected, and the T. asperellum suspension was sprayed evenly onto the surface of the fruits. The pepper stems were wrapped with cotton to keep the fruits moist, and then placed in a preservation box lined with filter paper. After drying, the fruits were incubated at 28°C for 1 day, and then sprayed with different concentrations of the fungicide solution. The fruits were dried, and then inoculated with the spore suspension of C. scovillei (3 x 10 6 spores / mL) on the surface without wounding. The above test was performed with water treatment, and the fruits treated with the fungicide solution and the T. asperellum suspension alone as controls. Each treatment had 15 pepper fruits, and the test was repeated three times. All the fruits were incubated at 28°C in a dark incubator for 7 days, and the incidence of pepper fruits was calculated.

[0076] The statistical results of the incidence of pepper fruit caused by the combination of Trichoderma asperellum and fungicides are shown in Table 5, which shows that the relative control effects of the two fungicides alone are lower than those of the combination of Trichoderma asperellum and fungicides, and the combination of the two significantly improves the inhibition of the pepper anthracnose fungus by the fungicides, showing synergistic effect. The disease sample is as follows Figure 2 It is shown that the control group shows a large range of spread of the anthracnose fungus, the disease spot is concave and shows water spot, and the rot is accompanied by orange conidial pileups Figure 2 a) After the combination of 70% thiophanate-methyl wettable powder 5000 times liquid and Trichoderma asperellum, Figure 2 b) The disease spot of the anthracnose fungus is significantly reduced compared with the disease spot of the fungicide alone Figure 2 c) and a large range of disease spot spread is not formed. The disease situation of the use of Trichoderma asperellum suspension alone Figure 2 d) and the use of 450g / L prochloraz emulsion in water 5000 times liquid alone Figure 2 d) is similar, and the disease situation of the combination of 450g / L prochloraz emulsion in water 5000 times liquid and Trichoderma asperellum Figure 2 e) is better than that of the fungicide alone Figure 2 f) of the same concentration, only black wounds are formed, and no concave annular anthracnose disease spots are formed.

[0077] Table 5 In vitro fruit method for determining the inhibition effect of the combination of Trichoderma asperellum and fungicides on the pepper anthracnose fungus

[0078]

[0079] The relative control effect of the combination of 70% thiophanate-methyl wettable powder 5000 times liquid and Trichoderma asperellum reaches 81.53%, which is equivalent to the control effect of 70% thiophanate-methyl wettable powder 2000 times liquid alone, and is 1.66 times higher than the relative control effect of the fungicide alone. The relative control effect of the combination of 450g / L prochloraz emulsion in water 5000 times liquid and Trichoderma asperellum reaches 88.05%, which is 1.4 times of the relative control effect of the fungicide alone, and has the same control effect as the use of 450g / L prochloraz emulsion in water 2000 times liquid alone. After the combination of 450g / L prochloraz emulsion in water 2000 times liquid and Trichoderma asperellum, the pepper fruits do not get sick, and the occurrence of pepper anthracnose can be completely inhibited. When the two fungicides are diluted to 1000 times liquid, the pepper fruits do not get sick at all, and the relative control effect reaches 100%.

[0080] The results of the present application show that compared with the use of fungicides and Trichoderma asperellum alone, the combination of the two has better inhibition effect, has synergistic effect under certain conditions, and the combination of 450g / L prochloraz emulsion in water and Trichoderma asperellum can control four kinds of anthracnose fungi.

[0081] The in vitro test of the combination of 70% thiophanate-methyl WP and 450 g / L prochloraz EW with Trichoderma asperellum for controlling pepper anthracnose showed that the combination had better control effect when the two fungicides were reduced by 2.5 times. Among them, the combination of 450 g / L prochloraz EW 5000 times liquid and Trichoderma asperellum was better than the use of the same concentration of fungicide alone, only forming black wounds, not forming concave wheel-shaped anthracnose lesions. The combination of 450 g / L prochloraz EW 2000 times liquid and Trichoderma asperellum did not cause the pepper fruit to be diseased, and could completely inhibit the occurrence of pepper anthracnose. Compared with the recommended use amount of 1000-1500 times liquid, it is estimated that the use amount of pesticide per mu in the field test can be reduced by 50%, which is 7.5 g less than the recommended use amount in the field. As shown in Table 6, the combination of 5000 times liquid 70% thiophanate-methyl WP and Trichoderma asperellum had a control effect of 81.53%, and it was estimated that the use amount of pesticide per mu in the field test could be reduced by 96.25% at most, which was 77 g less than the recommended use amount in the field. The combination of Trichoderma asperellum can effectively control the spread and spread of pepper anthracnose.

[0082] Table 6 Reduction of fungicide use amount (compared with the recommended use amount of pesticide)

[0083]

[0084] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are included in the protection scope of the present application.

Claims

1. Trichoderma acicularis ( Trichoderma asperellum YN4 strain in the control of pathogenic fungi Colletotrichum scovillei and / or Colletotrichum fructicola Applications of *Trichoderma hygroscopicum* YN4 strain in plant diseases caused by its pathogenic fungi; the strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 22, 2021, with accession number GDMCC NO. 62007.

2. Trichoderma acicularis ( Trichoderma asperellum The application of YN4 strain and fungicide in the prevention and / or treatment of plant fungal diseases is characterized by, The fungal disease is Colletotrichum siamense The bactericide is 10% difenoconazole water-dispersible granules with a concentration of 1.2 μg / mL; the Trichoderma echinocandi YN4 strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on October 22, 2021, with the accession number GDMCC NO. 62007.

3. Trichoderma acicularis ( Trichoderma asperellum The application of YN4 strain and fungicide in the prevention and / or treatment of plant fungal diseases is characterized by, The fungal disease is Colletotrichum siamense The bactericide used is 70% thiophanate-methyl wettable powder with a concentration of 0.1~0.5 μg / mL, or 450 g / L prochloraz emulsion with a concentration of 0.02~0.1 μg / mL. The *Trichoderma echinococcus* strain YN4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 22, 2021, with accession number GDMCC NO. 62007.

4. Trichoderma acicularis ( Trichoderma asperellum The application of the YN4 strain and fungicide in the preparation of products for the prevention and / or treatment of plant fungal diseases is characterized by, The fungal disease is Colletotrichum siamense The bactericide is 10% difenoconazole water-dispersible granules with a concentration of 1.2 μg / mL; the Trichoderma echinocandi YN4 strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on October 22, 2021, with the accession number GDMCC NO. 62007.

5. Trichoderma acicularis ( Trichoderma asperellum The application of the YN4 strain and fungicide in the preparation of products for the prevention and / or treatment of plant fungal diseases is characterized by, The fungal disease is Colletotrichum siamense The bactericide used is 70% thiophanate-methyl wettable powder with a concentration of 0.1~0.5 μg / mL, or 450 g / L prochloraz emulsion with a concentration of 0.02~0.1 μg / mL. The *Trichoderma echinococcus* strain YN4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 22, 2021, with accession number GDMCC NO. 62007.

6. An agent for the prevention and / or treatment of plant fungal diseases, characterized in that, The agent uses Trichoderma echinocandes strain YN4 and a fungicide as active ingredients; the fungicide is 10% difenoconazole water-dispersible granules with a concentration of 1.2 μg / mL; or the fungicide is 70% thiophanate-methyl wettable powder with a concentration of 0.4~0.5 μg / mL; the Trichoderma echinocandes strain YN4 was deposited at the Guangdong Provincial Microbial Culture Collection Center on October 22, 2021, with the accession number GDMCC NO. 62007.

7. A method for preventing and / or treating plant fungal diseases, characterized in that, Control is achieved using a chemical agent; the agent contains *Trichoderma echinosporum* strain YN4 and a fungicide as active ingredients; the fungicide is 10% difenoconazole water-dispersible granules with a concentration of 1.2 μg / mL; the fungal disease is... Colletotrichum siamense The *Trichoderma echinococcus* strain YN4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 22, 2021, with accession number GDMCC NO. 62007.

8. A method for preventing and / or treating plant fungal diseases, characterized in that, Control is achieved using chemical agents; the agents contain *Trichoderma echinococcus* strain YN4 and a fungicide as active ingredients; the fungicide is 70% thiophanate-methyl wettable powder at a concentration of 0.1–0.5 μg / mL, or 450 g / L prochloraz emulsifiable concentrate at a concentration of 0.02–0.1 μg / mL; the fungal disease is… Colletotrichum siamense ; The *Trichoderma echinococcus* strain YN4 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 22, 2021, with accession number GDMCC NO. 62007.

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