Application of Burkholderia gladioli in preventing and controlling crop diseases

Abstract

The present invention discloses the application of Burkholderia gladioli in preventing and controlling crop diseases. Specifically, it relates to the technical field of crop disease prevention and control. The bacteria of the Burkholderia gladioli species are cells of a bacterial strain with a preservation number of CGMCC NO. 24051 or its progeny strains or subcloned strains, and the crop diseases are selected from at least one of millet diseases, rice diseases, vegetable diseases, leguminous plant diseases, sugarcane diseases, tobacco diseases, cotton diseases, wheat diseases, and sesame diseases. The Burkholderia gladioli provided by the present invention has broad-spectrum crop disease prevention and control activity, especially for vegetable diseases, and has a control effect comparable to that of chemical drugs. It has the application prospect of preparing microbial agents, microbial fertilizers, or seed coating agents.

Description

Technical Field

[0001] The present invention relates to the technical field of crop disease control, and more particularly, to the application of Burkholderia gladioli in controlling crop diseases. Background Art

[0002] Downy mildew refers to a plant disease caused by downy mildew fungi. Downy mildew is an obligate parasite, and only a very small number of downy mildews can be artificially cultured, such as Sclerospora graminicola causing white blight of millet and Peronospora parasitica causing downy mildew of Chinese cabbage. This disease can occur at all stages from seedling to harvest, with adult plants being more severely affected. It mainly damages the leaves, developing from the base to the upper leaves. At the initial stage of the disease, light yellow nearly round to polygonal lesions are formed on the leaf surface, and angular leaf spot is likely to occur concurrently. When the air is humid, a downy mildew layer is produced on the back of the leaf, and sometimes it can spread to the leaf surface. In the later stage, the lesions wither and merge into patches, showing yellowish-brown color. When the disease is severe, all the outer leaves wither and die, similar to verticillium wilt.

[0003] Transmission routes and disease occurrence conditions: The pathogen overwinters by infecting seeds or winter lettuce in autumn and winter, and can also overwinter with oospores on diseased residues. It is mainly transmitted through air currents, watering, farming operations, and insects. The germination temperature of the pathogen spores is 6 - 10°C, and the suitable infection temperature is 15 - 17°C. Diseases are likely to occur when the planting density in the field is too high, watering is too early, too much, the soil humidity is high, and drainage is poor after planting. It is most likely to occur in late spring and early summer or during continuous rainy weather in autumn. When the disease is severe, it can cause a 20% - 40% yield loss.

[0004] Currently, agricultural control measures include treating planting holes with quicklime, burying or burning outside the field, chemical control (spraying with 500 - fold liquid of 75% chlorothalonil wettable powder, 500 - fold liquid of 58% metalaxyl - mancozeb wettable powder, or 800 - fold liquid of 69% dimethomorph - mancozeb wettable powder), or physical control (spraying 200 - fold liquid of high - fat film (emulsion) 2 weeks before the disease occurs). However, the above - mentioned methods have problems such as poor control effect and food safety issues of vegetables caused by pesticide residues.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide an application of Burkholderia gladioli in controlling crop diseases to solve the above - mentioned control problems.

[0007] The present invention is implemented as follows:

[0008] In a first aspect, the present invention provides the use of a bacterium of the species Burkholderia gladioli in controlling crop diseases. The bacterium of the species Burkholderia gladioli is a cell of a bacterial strain with the preservation number CGMCC NO. 24051 or its progeny strain or subcloned strain, and the crop diseases are selected from at least one of millet diseases, rice diseases, vegetable diseases, legume diseases, sugarcane diseases, tobacco diseases, cotton diseases, wheat diseases, and sesame diseases.

[0009] The preservation date of the bacterial strain CGMCC NO. 24051 is December 8, 2021, and the preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The proposed taxonomic name is: Burkholderia gladioli strain. The test result is survival.

[0010] The inventor isolated a bacterium from vegetable leaves in the field. After indoor and outdoor verification, it was found that the bacterium could effectively control crop diseases, especially having a good control effect on vegetable diseases. Therefore, this bacterium is expected to become a biocontrol agent for crop diseases in the future. After the inventor extracted the genome of the target bacterium and performed 16S rDNA sequence analysis, the sequence of one of the biocontrol bacteria (shown below) had a homology of 99.86% with Burkholderia gladioli strain 6BC with the accession number MK474976.1 in GenBank, confirming its taxonomic name as Burkholderia gladioli strain, named Burkholderia gladioli FB0002.

[0011] It should be noted that the above "control" means that after applying Burkholderia gladioli, any of the following phenomena exhibited by the crop pathogens are within the protection scope of the present invention: the number of pathogens decreases, the types of pathogens become fewer, or the growth of pathogens is inhibited and no longer grows, etc. The elimination of crop disease spots or the appearance of signs of elimination, the reduction of withering phenomena, and the restoration of the leaf color to the normal color are equivalent to the above positive control effects.

[0012] In a preferred embodiment of the application of the present invention, the crop diseases include but are not limited to white rust, downy mildew, late blight, root rot, or stem rot. They include but are not limited to diseases caused by bacteria and / or fungi or soil-borne diseases.

[0013] In a preferred embodiment of the application of the present invention, the millet diseases are selected from millet white rust; the rice diseases are selected from rice downy mildew; the vegetable diseases are selected from at least one of downy mildew of green vegetables, downy mildew of Chinese cabbage, downy mildew of cucumber, downy mildew of lettuce, late blight of potato, late blight of tomato, root rot of tomato, and root rot of Chinese onion.

[0014] The leguminous plant diseases are selected from at least one of downy mildew of soybean, stem rot of soybean, stem rot of adzuki bean, and stem rot of peanut.

[0015] The sugarcane disease is selected from downy mildew of sugarcane;

[0016] The tobacco diseases are selected from at least one of downy mildew of tobacco and stem rot of tobacco leaves;

[0017] The wheat disease is selected from root rot of wheat;

[0018] The sesame disease is selected from stem rot of sesame;

[0019] The cotton disease is selected from stem rot of cotton.

[0020] In a preferred embodiment of the application of the present invention, Burkholderia gladioli is made into a microbial agent, microbial fertilizer or seed coating agent for use.

[0021] The microbial agent is obtained by fermentation culture of Burkholderia gladioli.

[0022] The microbial agent is added to a carrier to prepare a microbial fertilizer. The carrier includes but is not limited to corncob, peat and charcoal.

[0023] For example, the seed coating agent also includes a coating carrier, a slow-release agent, a film-forming agent, a water-retaining agent, a solubilizing and dispersing agent, a stabilizer and a warning dye, and the seed coating is carried out to improve the germination rate or stress resistance of the seeds.

[0024] In a preferred embodiment of the application of the present invention, the microbial agent is used in the form of a powder, granule, liquid, emulsion or suspension.

[0025] In a preferred embodiment of the application of the present invention, the microbial agent is in the form of a wettable powder or water dispersible granule. The above-mentioned powder, granule, wettable powder and water dispersible granule can be microbial powder prepared by vacuum freeze drying or spray drying, and microbial granules. When in use, the microbial powder is dissolved.

[0026] In a second aspect, the present invention also provides a microbial agent, microbial fertilizer or seed coating agent containing Burkholderia gladioli and its progeny strains, and Burkholderia gladioli is a bacterial strain with a preservation number of CGMCC NO.24051.

[0027] For example, the seed coating agent also includes a coating carrier, a slow-release agent, a film-forming agent, a water-retaining agent, a solubilizing and dispersing agent, a stabilizer and a warning dye, and the seed coating is carried out to improve the germination rate or stress resistance of the seeds.

[0028] The seeds are seeds of the above-mentioned crops, including but not limited to leguminous plants, millet, rice, vegetables, sugarcane, tobacco, cotton, wheat and sesame.

[0029] In a preferred embodiment of the application of the present invention, the form of the bacterial agent, the bacterial fertilizer or the seed coating agent is powder, granule, liquid, emulsion or suspension.

[0030] In a preferred embodiment of the application of the present invention, the form of the bacterial agent, the bacterial fertilizer or the seed coating agent is wettable powder or water dispersible granule.

[0031] In a third aspect, the present invention also provides an application of Burkholderia gladioli and its progeny strains in the preparation of products for controlling crop diseases. The products are bacterial agents, bacterial fertilizers prepared from the bacterial agents or seed coating agents. The Burkholderia gladioli is a bacterial strain with the preservation number of CGMCC NO. 24051. The crop diseases are selected from at least one of millet diseases, rice diseases, vegetable diseases, leguminous plant diseases, sugarcane diseases, tobacco diseases, cotton diseases, wheat diseases and sesame diseases.

[0032] In an alternative embodiment, the crop diseases are white blight, downy mildew, late blight, root rot or stem rot.

[0033] In an alternative embodiment, the millet diseases are selected from millet white blight; the rice diseases are selected from rice downy mildew; the vegetable diseases are selected from at least one of downy mildew of green vegetables, downy mildew of Chinese cabbage, downy mildew of cucumber, downy mildew of lettuce, late blight of potato, late blight of tomato, root rot of tomato and root rot of Chinese onion;

[0034] The leguminous plant diseases are selected from at least one of downy mildew of soybean, stem rot of soybean, stem rot of adzuki bean and stem rot of peanut;

[0035] The sugarcane diseases are selected from sugarcane downy mildew;

[0036] The tobacco diseases are selected from at least one of tobacco downy mildew and stem rot of tobacco leaves;

[0037] The wheat diseases are selected from wheat root rot;

[0038] The sesame diseases are selected from sesame stem rot;

[0039] The cotton diseases are selected from cotton stem rot.

[0040] The present invention has the following beneficial effects:

[0041] (1) The Burkholderia gladioli provided by the present invention has broad-spectrum crop disease control activity.

[0042] (2) The Burkholderia gladioli has a control effect equivalent to that of chemical drugs especially on vegetable diseases.

[0043] (3) The fermentation and culture method of Burkholderia gladioli is simple and has strong adaptability, and it is expected to become a biocontrol agent for diseases of vegetables and other crops in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings.

[0045] Figure 1 It is a field test diagram;

[0046] Figure 2 It is a streak plate diagram;

[0047] Figure 3 It is a phylogenetic tree. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] Reference will now be provided in detail to embodiments of the present invention, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0049] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of those of ordinary skill in the art. Such techniques are well explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J.E. Coligan et al., eds., 1991), each of which is hereby expressly incorporated by reference.

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are followed. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained by commercial purchase.

[0051] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0052] Example 1

[0053] This example provides a method for isolating Burkholderia gladioli.

[0054] The separation method is as follows:

[0055] The green vegetable leaf samples for separating biocontrol bacteria were collected from a healthy green vegetable garden in Fengxian District, Shanghai. Weigh 10 g of green vegetable leaf samples. Under sterile conditions, place the leaves in a triangular flask containing 100 ml of sterile normal saline (0.9%), and place it on a shaker at 28 °C and 200 rpm for 30 min to prepare a dilution with a dilution factor of 10 -1 . Take 1 mL of the 10 -1 dilution and add it to a culture flask containing 9 mL of normal saline, and mix well to prepare a dilution with a dilution factor of 10 -2 . Subsequently, serially dilute it to 10 -5 . Take 100 μL of the dilutions (the dilution factors are: 10 -2 , 10 -3 , 10 -4 and 10 -5 ) and spread them on LB solid plates. Set 3 replicates for each dilution factor and culture at 28 °C for 48 h to isolate various bacteria. Pick the bacteria with obvious differences in colony size, shape, color and other phenotypic appearances, and obtain purified single colonies by the streak plate method for standby.

[0056] The colony morphology diagram is referred to Figure 2 as shown.

[0057] The purified biocontrol strain was subjected to 16S rDNA sequence analysis. The sequence of one biocontrol bacterium (as shown in SEQ ID NO.1) had 99.86% homology with Burkholderia gladioli strain 6BC with the accession number MK474976.1 in GenBank. The phylogenetic tree is referred to Figure 3 as shown, confirming that its taxonomic name is Burkholderia gladioli strain, named Burkholderia gladioli FB0002.

[0058] Example 2

[0059] In this example, the field biocontrol effect of Burkholderia gladioli was verified. The experimental method is as follows: In the field plots, the green vegetable samples were equally divided into a positive control group, an FB0002 treatment group and a water control group. The green vegetable samples in the experimental groups were evenly sprayed with Burkholderia gladioli FB0002 solution with OD 600nm = 2.0, the water control group was sprayed with an equal amount of water, and the positive control group was sprayed with the chemical pesticide Infinito. The downy mildew occurred naturally. Observe the lesion area of the leaves.

[0060] Field test investigation method:

[0061] Five-point sampling is carried out along the diagonal of each plot, with 5 plants at each point. All leaves are surveyed, and the grading is based on the percentage of the lesion area on each leaf accounting for the entire leaf area.

[0062] Grading method

[0063] Grade 0: No lesions;

[0064] Grade 1: The lesion area accounts for less than 5% of the entire leaf area;

[0065] Grade 3: The lesion area accounts for 6%-10% of the entire leaf area;

[0066] Grade 5: The lesion area accounts for 11%-20% of the entire leaf area;

[0067] Grade 7: The lesion area accounts for 21%-50% of the entire leaf area;

[0068] Grade 9: The lesion area accounts for more than 51% of the entire leaf area.

[0069] Field trial results:

[0070]

[0071] The field trial results are referred to Figure 1 As shown, the results show that the biocontrol bacteria isolated in Example 1 have a comprehensive control effect close to that of commercially available chemical pesticides in the field application for controlling downy mildew of Chinese cabbage.

[0072] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of a bacterium of the species Burkholderia gladioli ( Burkholderia gladioli strain ) in the control of crop diseases, characterized in that The bacterium of the Burkholderia gladioli species is a bacterial strain with the preservation number of CGMCC NO.24051, and the crop disease is downy mildew of Chinese cabbage.

2. The application according to claim 1, characterized in that, The Burkholderia gladioli is made into a microbial agent, microbial fertilizer or seed coating agent for use.

3. The application according to claim 2, characterized in that, The microbial agent is used in the form of powder, granule, liquid, emulsion or suspension.

4. The application according to claim 2, characterized in that The microbial agent is in the form of wettable powder or water dispersible granule.

5. A bacterial agent, bacterial fertilizer or seed coating agent containing Burkholderia gladioli, characterized in that, The Burkholderia gladioli is a bacterial strain with the preservation number of CGMCC NO.24051.

6. The microbial agent, microbial fertilizer or seed coating agent according to claim 5, characterized in that, The microbial agent, microbial fertilizer or seed coating agent is in the form of powder, granule, liquid, emulsion or suspension.

7. The microbial agent, microbial fertilizer or seed coating agent according to claim 5, characterized in that, The microbial agent, microbial fertilizer or seed coating agent is in the form of wettable powder or water dispersible granule.

8. Use of Burkholderia gladioli in the preparation of a product for controlling crop diseases, characterized in that, The product is a microbial agent, a microbial fertilizer prepared from the microbial agent or a seed coating agent. The Burkholderia gladioli is a bacterial strain with the preservation number of CGMCC NO.24051; the crop disease is downy mildew of Chinese cabbage.

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