Complex microbial inoculant for preventing and treating tomato fusarium wilt and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant for preventing and treating tomato fusarium wilt and application of the complex microbial inoculant, and belongs to the field of plant disease prevention and treatment. The invention relates to a composite microbial agent, which comprises the following active ingredients: bacillus thuringiensis (Bacillus thuringiensis), bacillus pumilus (Bacillus pumilus) and streptomyces werraensis (Streptomyces werraensis), and is characterized in that the active ingredients of the composite microbial agent comprise bacillus thuringiensis (Bacillus thuringiensis), bacillus pumilus (Bacillus pumilus) and streptomyces werraensis (Streptomyces werraensis). The complex microbial inoculant provided by the invention is a bacterial combination. The action mechanisms of bacteria are relatively similar, growth of fusarium oxysporum can be jointly inhibited by means of competing nutrition and space, generating antibacterial substances and the like, synergism is easier in the action mechanisms, and the biological prevention and treatment effect can be quickly and effectively achieved. Meanwhile, the requirements of the bacterial combination on nutrition and environmental conditions are relatively consistent, the cost is relatively low, and the method is more suitable for large-scale industrial production and application.

Description

Technical Field

[0001] The invention relates to the field of plant disease prevention and treatment, in particular to a composite bacterial agent for preventing and treating tomato wilt and application thereof. Background Art

[0002] Tomato is one of the most important economic crops in the world and is widely grown because of its rich nutritional value. Tomato wilt caused by Fusarium oxysporum causes tomato seedlings to become sick and fruit yields to decrease. In severe cases, it can cause more than 30% agricultural losses. Fusarium oxysporum is mostly found in the soil and invades the vascular tissue of tomatoes through the roots, causing vascular blockage and hindering the transmission of nutrients. When the disease occurs, the tomato seedlings turn yellow and wilt from the bottom to the top until the whole plant withers and dies. At present, the main method for preventing and controlling tomato wilt is to take comprehensive measures such as planting disease-resistant varieties and chemical control methods.

[0003] Beneficial bacteria in the rhizosphere of plants (biocontrol bacteria) are important microbial control resources that can promote plant growth and development, prevent and control the infection of various pathogens, and colonize in plants or in the rhizosphere soil by interacting with plants without causing other plant diseases. Microbial agents can secrete a variety of active substances that inhibit plant pathogens, and on the other hand, they can produce a variety of growth-promoting substances such as IAA and siderophores. Composite agents are microbial preparations made from two or more non-antagonistic strains, which make up for the shortcomings of insufficient resistance of a single strain. At present, the development of new resistant plant varieties has a slow output rate due to the long research and development cycle and high input-output ratio; and the pesticides used in traditional chemical control often remain in the soil, which leads to the loss of soil fertility over time and reduces the reusability of land resources. Because biological control methods have the characteristics of environmental friendliness and higher biosafety, the development of microbial agents to replace traditional chemical pesticides is becoming increasingly important in crop production.

[0004] At present, in the field of microbial control of tomato wilt, most of the research focuses on developing a combination of bacteria and fungi. However, the interaction between bacteria and fungi is very complex, and different relationships such as competition and antagonism may occur. If the interaction between the two is not coordinated, the biological control effect may be unstable. Therefore, the development of different types of microbial combinations is of great significance for enriching the types of composite microbial agents and promoting the development of microbial control of tomato wilt. Summary of the invention

[0005] The purpose of the present invention is to provide a composite bacterial agent for preventing and controlling tomato wilt and its application, so as to solve the problems existing in the above-mentioned prior art. The composite bacterial agent provided by the present invention is a bacterial combination, and the combination of bacteria is more likely to cooperate in the mechanism of action, and can quickly and effectively exert a biological control effect.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a composite bacterial agent for preventing and treating tomato wilt. The effective ingredients include Bacillus thuringiensis, Bacillus pumilus and Streptomyces werraensis.

[0008] Furthermore, the composite bacterial agent is a liquid bacterial agent.

[0009] Furthermore, the liquid bacterial agent includes a composite bacterial liquid of the Bacillus thuringiensis, the Bacillus pumilus and the Streptomyces cereus.

[0010] Furthermore, the composite bacterial solution is prepared by mixing bacterial solutions of the Bacillus thuringiensis, the Bacillus pumilus and the Streptomyces cereus in a volume ratio of 2:1:2.

[0011] Furthermore, the effective viable counts in the bacterial liquids of the Bacillus thuringiensis, the Bacillus pumilus and the Streptomyces cereus are all 1.0×10 8 cfu / mL.

[0012] The present invention also provides application of the composite bacterial agent in preventing and controlling plant diseases caused by Fusarium oxysporum and / or Botrytis cinerea.

[0013] Furthermore, the plant disease includes tomato wilt.

[0014] The invention also provides a method for preventing and controlling tomato wilt, comprising the step of applying the composite bacterial agent to tomato plants.

[0015] Furthermore, the application method includes root irrigation.

[0016] Furthermore, the tomato wilt is caused by Fusarium oxysporum.

[0017] The present invention discloses the following technical effects:

[0018] Compared with the microbial combination of bacteria and fungi in the prior art, the composite bacterial agent provided by the present invention is a bacterial combination. The action mechanism of the bacteria is relatively similar, and they can jointly inhibit the growth of wilt pathogens by competing for nutrition, space, and producing antibacterial substances. They are more likely to cooperate in the action mechanism and can quickly and effectively play a biological control role. At the same time, the requirements of the bacterial combination for nutrition and environmental conditions are relatively consistent, the cost is relatively low, and it is more suitable for large-scale industrial production and application. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] Fusarium oxysporum used in the present invention was donated by the Microbiology Laboratory of the Chinese Academy of Agricultural Sciences, and Botrytis cinerea was donated by the Microbiology Laboratory of Dalian University of Technology;

[0025] Bacillus thuringiensis and Bacillus pumilus used to prepare the compliant microbial agent are from the China Industrial Culture Collection Center, with strain numbers CICC21340 and CICC 22442 respectively; Streptomyces werraensis is from the China General Microbiological Culture Collection Center, with strain number CGMCC 4.1935, and the public can purchase it through the above-mentioned public channels.

[0026] The culture medium components used in the present invention are as follows:

[0027] NA medium (for culturing two types of Bacillus): beef extract 3.0 g / L, peptone 10.0 g / L, NaCl 5.0 g / L, agar 20 g / L, distilled water to 1000 mL, pH 7.4-7.6;

[0028] Yeast extract maltose agar medium (ISP2) (for culturing Streptomyces cereus): yeast powder 4 g / L, malt powder 10 g / L, glucose 4 g / L, agar 20 g / L, distilled water to 1000 mL, pH 7.2-7.3;

[0029] Fermentation medium: soybean powder 20g / L, corn starch 10g / L, glucose 10g / L, beef extract 3g / L, ammonium sulfate 2g / L, sodium chloride 2g / L, calcium carbonate 2g / L, magnesium sulfate 0.04g / L, potassium dihydrogen phosphate 0.04g / L, dipotassium hydrogen phosphate 0.04g / L, distilled water 1000mL, pH 7.2-7.4;

[0030] PDA medium (for culturing pathogenic fungi): 200 g / L potato, 20 g / L glucose, 20 g / L agar, distilled water to 1000 mL, natural pH.

[0031] Example

[0032] 1. Bacteria fermentation

[0033] Preparation of Bacillus thuringiensis and Bacillus pumilus fermentation broth:

[0034] Take the strain preserved in a glycerol tube, streak it onto NA medium, invert and culture it at 30℃ for 24h, take a single colony and inoculate it into 50mL NA medium, incubate it at 30℃, 180rpm for 24h, inoculate it into fermentation medium with an inoculum of 1% (v / v), ferment it at 30℃, 180rpm for 24h, and control the effective viable count of the bacterial solution to 1.0×10 8 cfu / mL or above.

[0035] Preparation of Streptomyces cereus fermentation broth:

[0036] The strains preserved in glycerol tubes were streaked onto ISP2 medium and incubated at 30°C for 3 days. The spores on the ISP2 solid medium were picked and inoculated into the liquid ISP2 medium. The medium was placed in a shaker and cultured at 30°C and 180 rpm for 3 days. The bacterial solution was inoculated into the fermentation medium at a 1% (v / v) inoculation ratio and cultured at 30°C and 180 rpm for 5 days. The effective viable count of the bacterial solution was controlled at 1.0×10 8 cfu / g or above.

[0037] 2. Preparation of composite bacterial agent

[0038] The bacterial liquid obtained by fermentation was prepared into 4 different composite bacterial agents according to the composition and proportion in Table 1.

[0039] Table 1 Composition of composite bacterial agent

[0040]

[0041] 2. The inhibitory effect of compound microbial agents on various pathogenic fungi

[0042] 7mm cakes of Fusarium oxysporum and Botrytis cinerea were placed in the center of the PDA medium, and holes were punched with a sterile puncher at a distance of 2.5cm with the cake as the center. The composite bacterial agent was added for confrontation test (the bacterial agents were all diluted 10 times), and only the pathogens were inoculated on the PDA medium as a control. Cultured at 28℃, the length and width of the pathogen colony were measured after 7 days, and the inhibition rate of the composite bacterial agent on the pathogen was calculated. Inhibition rate = (DB) / D×100%, where D represents the diameter of the pathogen colony in the control group; B represents the width of the pathogen colony in the treatment group. Each treatment was repeated 3 times, and the average value of the 3 repetitions was taken.

[0043] The inhibition results of the composite bacterial agent on Fusarium oxysporum and Botrytis cinerea are shown in Table 2.

[0044] Table 2 Inhibition rate of composite bacterial agent on Fusarium oxysporum and Botrytis cinerea

[0045]

[0046] As shown in Table 1, the composite bacterial agent composed of two or more of the three bacteria Bacillus thuringiensis, Bacillus pumilus and Streptomyces cereus can produce a certain inhibitory effect on Fusarium oxysporum and Botrytis cinerea. Among them, the combination of Bacillus thuringiensis fermentation broth, Bacillus pumilus fermentation broth and Streptomyces cereus fermentation broth is significantly better than other combinations.

[0047] 3. The control effect of compound fungi and their proportions on tomato wilt

[0048] Select tomato seeds of uniform size, soak them in 28°C warm water for 4 h, rinse them with sterile water for 1 min, soak them in 75% ethanol for 1 min, wash them with sterile water three times, soak them in 10% sodium hypochlorite for 10 min, wash them with sterile water 5 times, and evenly place them on a culture dish with sterile filter paper. After the roots grow 1-2 cm, select seeds with uniform growth and sow them in a culture dish containing spores of Fusarium oxysporum (1×10 5 CFU / g soil) sterilized by high-pressure steam (1 tree / hole), inoculate the compound bacterial agent diluted 10 times by root dipping and root irrigation, treat 20 plants in each group, and repeat three times. The control group was treated with no bacterial agent. The soil is covered to a depth of about 1cm, water is applied in time, and culture is placed in a greenhouse with 16 hours of light. After 28 days of culture, the severity of the disease and the prevention effect are calculated according to the severity grading standard of tomato wilt disease. The severity grading standard of tomato wilt disease is as follows:

[0049] Level 0: healthy plants, no symptoms, normal growth of roots, stems and leaves;

[0050] Level 1: 1-2 leaves are obviously yellowing;

[0051] Level 2: 3-4 true leaves turn yellow, and the leaves wilt and droop;

[0052] Level 3: 5-6 true leaves turn yellow or wither and droop;

[0053] Level 4: The whole plant wilts severely and even dies.

[0054] Disease severity (DSW) = [∑ (number of diseased plants at each level × representative value of disease severity at each level) / (highest representative value × total number of plants surveyed)] × 100%;

[0055] Preventive effect = [(control disease index - treatment disease index) / control disease index] × 100%.

[0056] The volume ratio of each bacterial fermentation broth in the composite bacterial agent and the corresponding protective effect are shown in Table 3.

[0057] Table 3 Different ratios of composite microbial agents and their control effects

[0058]

[0059] Note: “Su” stands for Bacillus thuringiensis, “Duan” stands for Bacillus brevis, and “Wei” stands for Streptomyces cereus; 1-7 are fermentation liquid treatment groups, and 8 is the control group.

[0060] It can be seen from the data in Table 3 that when the ratio of Bacillus thuringiensis: Bacillus pumilus: Streptomyces cereus in the fermentation broth is (1-2):1:2 (v / v / v), the control effect of the composite bacterial agent on tomato wilt is significantly better than that of composite bacterial agents with other ratios. Among them, when Bacillus thuringiensis: Bacillus pumilus: Streptomyces cereus is 2:1:2 (v / v / v), the control effect of the composite bacterial agent is as high as 77.46%, which is the best effect.

[0061] 4. Determination of the control and treatment ability of composite microbial agents on tomato wilt

[0062] Use a cork puncher to punch out a 7 mm diameter Fusarium oxysporum pathogenic fungus block, inoculate it into PDA solid culture medium, and invert and culture at 28°C for 8-10 days until the mycelium fills the culture dish. Use 10 mL of sterile distilled water to scrape the spores, filter the mycelium through four layers of gauze, and count them under an optical microscope using a hemocytometer to calculate the concentration of Fusarium oxysporum pathogenic fungus spores. Use sterile distilled water to adjust the spore concentration to 1×10 6 cfu / mL.

[0063] Tomato seeds of the same size were selected, soaked in warm water at 28℃ for 4h, rinsed with sterile water for 1min, soaked in 75% ethanol for 1min, and washed with sterile water three times; soaked in 10% sodium hypochlorite for 10min, washed with sterile water 5 times, and evenly placed on a culture dish with sterile filter paper. After the root length was 1-2cm, seeds with the same growth were selected and sown in soil sterilized with high-pressure steam (1 tree / hole). Treatment group 1: first root inoculated with a 10-fold diluted composite bacterial agent, and then inoculated with pathogens 2d later; treatment group 2: first inoculated with pathogens, and then inoculated with a 10-fold diluted composite bacterial agent 2d later; treatment group 3: only add composite bacterial agent; treatment group 4: only inoculate pathogens; blank group: only add water. In treatment groups 1 to 3, the composite bacterial agent is the formula of group 5 in Table 3. The changes in the aboveground and underground total phenol content of each group within 72h were measured; the disease index, prevention effect, and the length of the aboveground and underground parts of each group were measured after 28d. The results are shown in Tables 4 and 5.

[0064] Table 4 Total phenol content in different treatment groups

[0065]

[0066] Table 5 Prevention effect of different treatment groups

[0067] Plant height (cm) Root length (cm) Diseased plants (%) Prevention effect (%) Treatment Group 1 <![CDATA[10.24±1.3495 c ]]> <![CDATA[7.36±0.9904 ab ]]> <![CDATA[11.67±0.0072 a ]]> 79.65 Treatment Group 2 <![CDATA[9.1±1.2421 d ]]> <![CDATA[6.7±1.0823 bc ]]> <![CDATA[12.5±0.0125 b ]]> 78.2 Treatment Group 3 <![CDATA[8.46±1.0140 d ]]> <![CDATA[5.98±1.0702 c ]]> <![CDATA[57.34±0.0100 b ]]> / Treatment Group 4 <![CDATA[13.90±1.6333 b ]]> <![CDATA[6.93±1.4120 b ]]> / / Blank group 5 <![CDATA[16.81±2.0191 a ]]> <![CDATA[8.13±1.1567 a ]]> / /

[0068] In plants, phenols have a phenolic hydroxyl structure that has the ability to scavenge free radicals, thereby improving the antioxidant capacity of plant tissues. The increase in phenolic substances will enhance the resistance of plants to Fusarium oxysporum, thereby alleviating the development of wilt. As shown in Table 4, polyphenols in the roots, stems, and leaves of tomatoes infected with Fusarium oxysporum treated with a composite bacterial agent accumulated to varying degrees. Therefore, it is speculated that tomatoes can respond to Fusarium oxysporum stress by regulating the accumulation and metabolism of total phenols to improve the total antioxidant capacity.

[0069] The data in Table 5 show that the effect of adding the compound bacterial agent in advance is better than that of adding the compound bacterial agent later, indicating that the compound bacterial agent has a better preventive effect on tomato wilt.

[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A composite bacterial agent for preventing and treating tomato wilt, characterized in that: The active ingredients include Bacillus thuringiensis, Bacillus pumilus and Streptomyces werraensis.

2. The composite bacterial agent according to claim 1, characterized in that The composite bacterial agent is a liquid bacterial agent.

3. The composite bacterial agent according to claim 2, characterized in that: The liquid bacterial agent includes a composite bacterial liquid of the Bacillus thuringiensis, the Bacillus pumilus and the Streptomyces cereus.

4. The composite bacterial agent according to claim 3, characterized in that: The composite bacterial liquid is prepared by mixing the bacterial liquids of the Bacillus thuringiensis, the Bacillus pumilus and the Streptomyces cereus in a volume ratio of 2:1:

2.

5. The composite bacterial agent according to claim 4, characterized in that: The effective viable bacterial counts in the bacterial liquids of the Bacillus thuringiensis, the Bacillus pumilus and the Streptomyces cereus were all 1.0×10 8 cfu / mL.

6. Use of the composite bacterial agent according to any one of claims 1 to 5 in preventing and controlling plant diseases caused by Fusarium oxysporum and / or Botrytis cinerea.

7. The use according to claim 6, characterized in that: The plant diseases include tomato wilt.

8. A method for preventing and controlling tomato wilt, characterized in that: The method comprises the step of applying the composite bacterial agent described in any one of claims 1 to 5 to tomato plants.

9. The method according to claim 8, characterized in that The application method includes root irrigation.

10. The method according to claim 8, characterized in that The tomato wilt is caused by Fusarium oxysporum.

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