A composite microbial agent containing Bacillus pumilus and Trichoderma viride and its application in biological control of citrus

Abstract

The present invention belongs to the technical field of crop control, and discloses a compound microbial agent containing Bacillus pumilus and Trichoderma viride and its application in biological control of citrus. The Bacillus pumilus is Bacillus pumilus ( Bacillus pumilus ), HBTG2356, and its preservation number is: CCTCC NO: M20232119. The Trichoderma viride is Trichoderma viride ( Trichoderma virens ), HBTG3206, and its preservation number is: CCTCC NO: M20232117. Compared with single strains, this compound strain has a synergistic effect on diseases such as citrus canker and citrus root-knot nematode, and has a certain application prospect in the prevention and control of citrus diseases.

Description

Background Art

[0001] Citrus (Citrus) belongs to the Rutaceae family and is a general term for mandarin orange, orange, grapefruit, tangerine, trifoliate orange, kumquat, etc. Due to the different varieties, climatic conditions, cultivation management habits and production levels in the main cultivation areas, post-harvest penicillium, canker, sand skin disease and nematode diseases occur seriously. In production, the prevention and control of citrus diseases mainly rely on chemical fungicides. However, the frequent use of chemical pesticides has caused disease resistance, environmental pollution and pesticide residues, which seriously endanger food and environmental safety. Microbial agents are not easy to develop resistance, have no pesticide residues and environmental pollution, have a regulatory effect on the structure of soil microbial communities, and inhibit the occurrence of diseases. Based on the above characteristics, microbial agents have been widely used in the prevention and control of crop diseases.

[0002] At present, there are few reports on the application of Bacillus pumilus and Trichoderma viride in citrus. Chen Jiaomei et al. reported that Bacillus pumilus YH1 has a good control effect on citrus canker. Yi Tuyong et al. reported that Bacillus pumilus Y2 has a certain control effect on citrus sand skin disease.

[0003] Citrus cultivation is mainly located in the acidic soil in the south. As a microorganism, Bacillus is not conducive to its survival and reproduction in acidic soil. Therefore, Bacillus used for citrus disease prevention and control must also have a certain tolerance to acid. Therefore, it is of great significance to screen out Bacillus that has a good prevention and control effect on citrus diseases and a certain tolerance to acidity.

[0004] The present invention relates to Bacillus pumilus. The applicant has submitted a patent application on March 14, 2024, with application number 2024102897829. The strain has good effects in preventing and treating canker disease and root-knot nematodes. Unlike Bacillus pumilus Y2 (CN 108384736 A) in the prior art, the Bacillus pumilus of the present invention has no significant preventive effect on sand skin disease. At the same time, since it is isolated from citrus, it can have good colonization ability in citrus leaves, roots and fruits, and has certain acid resistance, and has broad application prospects.

[0005] The applicant isolated a strain of Trichoderma viride from the rhizosphere soil of healthy citrus plants in a field with severe citrus nematode disease, and found that after compounding with the above-mentioned Bacillus brevis, it can be used to treat citrus canker and citrus root-knot nematode disease, and the effect is better than using a single bacterium. The composite bacterial agent has no cross-resistance with existing fungicides. Because it comes from the natural environment, it has the advantages of high efficiency, low toxicity, no environmental pollution, and not easy to produce drug resistance, and has broad application prospects. Summary of the invention

[0006] The object of the present invention is to provide a composite bacterial agent, which comprises Bacillus pumilus HBTG2356 and Trichoderma virens HBTG3206, and the deposit numbers are CCTCC NO: M20232119 and CCTCC NO: M20232117, respectively.

[0007] Another object of the present invention is to provide an application of the above-mentioned composite bacterial agent in the prevention and treatment of citrus diseases.

[0008] In order to achieve the above object, the present invention adopts the following technical measures:

[0009] The applicant isolated a Trichoderma strain from the rhizosphere soil of healthy citrus plants in a field with severe citrus nematode disease, which was identified as Trichoderma virens. It was sent to the China Center for Type Culture Collection for preservation on November 2, 2023, and the classification name is: Trichoderma virens HBTG 3206; the preservation number is CCTCC NO: M20232117; address: Wuhan University, Wuhan, China.

[0010] The colonies of Trichoderma viride HBTG3206 grew well in PDA medium, and the colonies were green ( Figure 1 ).

[0011] A composite bacterial agent comprises Bacillus pumilus HBTG2356 and Trichoderma virens HBTG3206, and the deposit numbers are CCTCC NO: M20232119 and CCTCC NO: M20232117 respectively.

[0012] In the composite bacterial agent described above, preferably, the ratio of the effective bacterial concentration of Trichoderma viride to that of Bacillus pumilus is 0.5-1:0.5-1.

[0013] In the composite bacterial agent described above, preferably, the ratio of the effective bacterial concentration of Trichoderma viride to that of Bacillus pumilus is 1:1.

[0014] The protection scope of the present invention also includes:

[0015] Application of the above composite bacterial agent in the prevention and treatment of citrus diseases;

[0016] Application of the composite bacterial agent in the preparation of microbial preparations for preventing and controlling citrus diseases;

[0017] The citrus diseases mentioned above include: citrus canker and citrus nematode disease;

[0018] The above-mentioned application, when used for preventing and controlling citrus root-knot nematode disease, is to irrigate the roots of citrus with the above-mentioned composite bacterial agent; when used for citrus canker disease, is to spray the above-mentioned composite bacterial agent on the leaves and fruits of citrus.

[0019] In the above-mentioned application, the microbial preparation is in the form of a powder or a liquid preparation.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) The present invention provides a composite bacterial agent containing Bacillus pumilus and Trichoderma viride. The Trichoderma viride provided by the present invention has the function of resisting nematodes and various fungi. After the Trichoderma viride is compounded with Bacillus pumilus HBTG2356, the two have a synergistic effect, and the anti-nematode and antibacterial abilities are enhanced.

[0022] (2) Compared with the currently used chemical synthetic pesticides, since it comes from the natural environment, it has the advantages of high efficiency, low toxicity, no pollution to the environment, and is not easy to produce drug resistance.

[0023] (3) The Bacillus pumilus and Trichoderma virens screened by the present invention also have the characteristics of colonizing in the root system of citrus, and can provide long-term protection for citrus. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Colony morphology of HBTG3206 strain.

[0025] Figure 2 Spore morphology of HBTG3206 strain.

[0026] Figure 3 Genetic tree of HBTG3206

[0027] Figure 4 Schematic diagram of the antibacterial activity of HBTG3206. DETAILED DESCRIPTION

[0028] In order to better explain the present invention, the main contents of the present invention are further illustrated below in conjunction with specific examples, but the contents of the present invention are not limited to the following examples. The technical solutions of the present invention, if not otherwise specified, are conventional techniques in the art, and the reagents or materials, if not otherwise specified, are all derived from commercial channels.

[0029] Regarding the physiological and biochemical properties of Bacillus pumilus HBTG2356 involved in the present invention, the results of plate antibacterial experiments and the results of field experiments, etc., reference may be made to CN118109360A.

[0030] Embodiment 1:

[0031] Acquisition of Trichoderma virens HBTG3206:

[0032] The applicant isolated a Trichoderma strain from the rhizosphere soil of healthy citrus plants in a field with severe citrus nematode disease, which was identified as Trichoderma virens ( Figure 1 , Figure 2 and Figure 3 ), was sent to the China Center for Type Culture Collection for preservation on November 2, 2023, classified and named: Trichoderma virens HBTG 3206; the preservation number is CCTCCNO: M20232117; address: Wuhan University, Wuhan, China.

[0033] In the present invention, Trichoderma virens HBTG 3206 or HBTG3206 for short.

[0034] Morphological characteristics: Colony green ( Figure 1 ).

[0035] Embodiment 2:

[0036] Trichoderma virens HBTG3206 fermentation (the percentages in the following culture medium formula are all mass fractions):

[0037] Fermentation medium: bran 33%, soybean meal 10%, sucrose 5%, potassium sulfate 2%, and the rest is water, and the above percentages are all mass fractions.

[0038] The inoculation amount is 5%, the fermentation time is 5 days, the fermentation temperature is 30°C, and the fermentation is completed and dried. The spore count is 8 billion spores / g.

[0039] Embodiment 3:

[0040] In vitro antibacterial activity of Trichoderma virens HBTG3206

[0041] The tested strains were Pestalotiopsis theae, Botrytis cinerea, Fusarium oxysporum, Phytophthora capsici, Sclerotinia sclerotiorum, Rhizoctonia solani, Colletotrichum gloeosporioides and Penicillium italicum.

[0042] The slant strain was transferred to a PDA plate for activation and cultured at 30°C for 6 days. The pathogen and Trichoderma aspergillus HBTG3206 were placed at both ends of the culture medium, 3 cm apart. At the same time, only the pathogen was inoculated as a control. The experiment was repeated three times and cultured in a dark incubator at 25°C for 3 days. The colony radius of the pathogen pointing to Trichoderma was measured. Each setting in the experiment was repeated three times. The colony diameters of each treatment and control were counted, and the inhibition rate was calculated.

[0043] Inhibition rate = [(control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter)] × 100%

[0044] The in vitro antibacterial test showed that HBTG3206 had good antibacterial activity against Pseudomonas aeruginosa, Botrytis cinerea, Penicillium italicum, Sclerotinia sclerotiorum, Rhizoctonia solani and Colletotrichum gloeosporioides, but had poor antibacterial effect against Fusarium oxysporum and Phytophthora capsici. Figure 4 , Table 1).

[0045] Table 1 Antibacterial activity of HBTG3206 against pathogenic bacteria

[0046] Pathogens Antibacterial rate (%) Pestalotiopsis theae 63.88 Botrytis cinerea 56.79 Fusarium oxysporum 11.97 Phytophthora capsici 18.75 Penicillium italicum 68.42 Sclerotinia sclerotiorum 71.33 Rhizoctonia solani 55.00 Colletotrichum gloeosporioides 73.75

[0047] Embodiment 3:

[0048] Colonization ability of HBTG3206 in citrus roots:

[0049] The resistance of Trichoderma viride HBTG3206 was screened in different carbendazim-containing media, and the carbendazim-resistant strain 5 μg·mL was obtained. -1 The mutant strain (except for the resistance to carbendazim, the other physiological and biochemical characteristics of the strain were not significantly different from those of the native strain) can be grown in the presence of 5 μg mL -1 The carbendazim grew normally in PDA.

[0050] The resistant mutant HBTG3206 was fermented according to Example 9, and the spore count was adjusted to 100 million cfu / mL with clean water. The roots of citrus seedlings and pear seedlings (control plants) were irrigated with an application rate of 10L / plant. The roots were collected after 30 days, the surface was disinfected with 75%, and rinsed with sterile water 3 times. After drying, 1g of tea tree root and walnut root were weighed, and then 1mL of sterile water was added and ground in a mortar. After standing, the supernatant was aspirated, diluted by gradient dilution method, and applied to a 5μg·mL -1 The colonization ability of HBTG3206 on citrus and pear roots was determined by counting the plates with carbendazim and incubating at 30°C for 24 h.

[0051] The experiment showed that HBTG3206 had a stronger colonization ability in citrus tree roots, with a colonization rate of 7.7×10 5cfu / g, while in walnut root it was 2.9×10 2 cfu / g, and this colonization ability is more conducive to its biological control effect on citrus trees.

[0052] Embodiment 4:

[0053] Effects of single and combined bacterial agents on controlling citrus root-knot nematodes

[0054] The field test was conducted in Xinfeng County, Ganzhou City, Jiangxi Province. The citrus varieties tested were 5-year-old shatangju. The HBTG2356 fermentation liquid (8 billion cfu / ml) prepared according to CN118109360A, the HBTG3206 powder (8 billion spores / g) prepared in the above embodiment, and the composite bacterial agent (the effective bacterial concentration of the composite bacterial agent is 8 billion cfu / ml) prepared according to the effective bacterial concentration of 1:1 were diluted 200 times and irrigated the roots. Each plant was irrigated with 15L of root, and the roots were irrigated again after an interval of 15d, for a total of two root irrigations. The control was irrigated with clear water, and each treatment was repeated 3 times, with 5 trees in each repeat. The root knot index of each treatment was counted at 3 months and 5 months, and the prevention effect was calculated.

[0055] The occurrence of the disease is counted according to the disease classification standard: Level 0, no root knots; Level 1, root knots account for 1% to 10% of the whole root system; Level 2, root knots account for 11% to 25% of the whole root system; Level 3, root knots account for 26% to 50% of the whole root system; Level 4, root knots account for 51% to 75% of the whole root system; Level 5, root knots account for 69% to 90% of the whole root system; Level 6: root knots account for 91% to 100% of the whole root system.

[0056] Root knot index = ∑ (number of plants at each level × level) / (total number of plants surveyed × the highest representative level) × 100.

[0057] Control effect = (control root knot index - treatment root knot index) / control root knot index × 100%.

[0058] The test results show that the composite bacterial agent has a good control effect on citrus root-knot nematodes, which is better than a single bacterial agent and has a significant synergistic effect. The control effect reached 63.77% after 3 months and 60.54% after 5 months (Table 2).

[0059] Table 2 Field efficacy of HBTG2356 fungicide against citrus root-knot nematodes

[0060]

[0061] Embodiment 5:

[0062] Single bacteria and compound bacteria to control citrus canker

[0063] The field test was conducted in Xinfeng County, Ganzhou City, Jiangxi Province. The citrus varieties tested were 5-year-old shatangju. The HBTG2356 fermentation liquid (8 billion cfu / ml) prepared according to CN118109360A, the HBTG3206 powder (8 billion spores / g) prepared in the above embodiment, and the composite bacterial agent prepared according to the effective bacterial concentration of 1:1 (the effective bacterial concentration of the composite bacterial agent was 8 billion cfu / ml) were diluted 100 times, and the leaves and fruits were sprayed, and the second spray was performed at an interval of 7d. The drug was applied 3 times in total, and water was sprayed as a control. The field plots were arranged in random blocks, with an area of ​​2 plants in each treatment plot, and repeated 3 times.

[0064] After the control group is fully infected with the disease, the disease index is investigated and the prevention effect is calculated. Two trees are investigated in each plot, and samples are taken from each tree at five points: east, west, south, north, and center. Two summer shoots are randomly investigated at each point, and the number of leaves at each level on the summer shoots is recorded.

[0065] The leaf (fruit) grading method is as follows: Grade 0: disease-free; Grade 1: 1-5 lesions on each leaf (fruit); Grade 3: 6-10 lesions on each leaf (fruit); Grade 5: 11-15 lesions on each leaf (fruit); Grade 7: 16-20 lesions on each leaf (fruit); Grade 9: 21 or more lesions on each leaf (fruit).

[0066] Disease index (%) = ∑ (number of diseased leaves (fruits) at each level × level) / (total number of leaves (fruits) surveyed × the highest representative level) × 100.

[0067] Control effect = (control disease index - treatment disease index) / control root knot index × 100%

[0068] The test results showed that spraying the composite bacterial agent had a good control effect on citrus canker, with a prevention rate of 65.56%, which was better than that of a single bacterial agent and had a significant synergistic effect (Table 3).

[0069] Table 3. Compound bacterial agents for the prevention and control of citrus canker

[0070] deal with Disease index Prevention effect (%) HBTG2356 13.76 66.40 HBTG3206 15.87 61.25 Compound Bacterial Agent 10.51 74.33 Clear water control 40.95 -

Claims

1. A composite bacterial agent, comprising Bacillus pumilus ( Bacillus pumilus ) HBTG2356 and Trichoderma virens ( Trichoderma virens ) HBTG3206, the deposit numbers are CCTCC NO: M20232119 and CCTCC NO: M20232117 respectively.

2. The composite bacterial agent according to claim 1, characterized in that: The ratio of the effective bacterial concentration of Trichoderma viride to that of Bacillus pumilus is 0.5-1:0.5-1.

3. The composite bacterial agent according to claim 1, characterized in that: The ratio of the effective bacterial concentrations of Trichoderma viride and Bacillus pumilus is 1:

1.

4. Use of the composite bacterial agent according to claim 1 in the prevention and treatment of citrus diseases, wherein the citrus diseases are citrus canker and / or citrus root-knot nematode disease.

5. Use of the composite bacterial agent according to claim 1 in the preparation of a microbial preparation for preventing and controlling citrus diseases, wherein the citrus diseases are citrus canker and / or citrus root-knot nematode disease.

6. The use according to claim 4, wherein the application method is: when used for preventing and controlling citrus root-knot nematode disease, the composite bacterial agent according to claim 1 is used to irrigate the roots of citrus; when used for citrus canker disease, the composite bacterial agent according to claim 1 is sprayed on the leaves and fruits of citrus.

7. The use according to claim 5, wherein the microbial preparation is in the form of a powder or a liquid preparation.

Images