A Brevundimonas sp. BWT19-19 and its application in controlling plant root-knot nematodes

Abstract

The present invention discloses a Brevundimonas sp. strain BWT19-19 and its application in controlling plant root-knot nematodes, belonging to the technical field of microbial pesticides and fertilizers. The Brevundimonas sp. strain BWT19-19 of the present invention is deposited in the Guangdong Microbial Culture Collection Center, with the deposit number GDMCC No: 62303; the Brevundimonas sp. BWT19-19 has an obvious lethal effect on tomato root-knot nematodes, and the lethal rate of the original fermentation broth reaches 100%, and the lethal rate of the 10-fold diluted solution reaches 76.51%. In the greenhouse pot experiment, the control effect of the BWT19-19 bacterial solution on tomato root-knot nematodes reaches 46.67%, and the control effect is excellent. In addition, the bacterial solution and fermentation broth of this strain can significantly increase the plant height and dry matter accumulation of tomato plants while controlling pests. Therefore, the Brevundimonas sp. BWT19-19 disclosed in the present invention has good application prospects in the control of plant root-knot nematodes and the promotion of plant growth.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial pesticides and fertilizers, and particularly relates to a Brevundimonas strain BWT19-19 and its application in controlling plant root-knot nematodes. Background Art

[0002] Root-knot nematodes (Meloidogyne spp.) are the most serious wide-host plant parasitic nematodes that harm plants, and can infect more than 2,000 plants including vegetables, melons, medicinal materials, flowers, and fruit trees. In recent years, with the increase in multiple cropping index, overuse of pesticides and fertilizers, and non-standard farming operations, the transmission speed of root-knot nematodes has accelerated. After infecting plants, root-knot nematodes will cause trauma to plant roots, making plants more susceptible to infection by pathogens such as bacterial wilt, root rot, and fusarium wilt, resulting in complex diseases. According to research reports, root-knot nematodes can cause a reduction in yield of some crops by 13% - 25%, and in severely affected plots, it can be as high as over 70%, or even result in a complete crop failure. Moreover, this nematode disease is a soil-borne disease, and once it occurs, it is very difficult to eradicate. Currently, the area of land affected by root-knot nematodes in China exceeds 20 million mu, and the agricultural economic losses caused by root-knot nematodes exceed 70 billion yuan. Root-knot nematodes have now become the second largest plant disease after fungal diseases.

[0003] Currently, the methods for controlling plant root-knot nematodes mainly include chemical control, physical control, biological control, agricultural measure control, and cultivation of resistant varieties, etc. Chemical agents are still the main means for controlling root-knot nematodes due to their quick effect and stable performance. However, the high cost of chemical agents, misuse, and abuse can cause harm to the environment, humans, and livestock, and many highly toxic products have now been prohibited or restricted from use. Physical control, agricultural measure control, and resistant breeding control also have many limitations. For example, cultivating resistant varieties and crop rotation with paddy fields can greatly improve the control effect on root-knot nematodes. However, the wide-host root-knot nematodes limit the number of resistant varieties, and the limitation of land resources in China makes it difficult to implement crop rotation. Physical control methods such as soil disinfection and ultraviolet irradiation are difficult to popularize in production practice due to time-consuming and laborious reasons. Therefore, it is urgent to find safer, more efficient, green, and environmentally friendly nematode control technical means from an ecological perspective.

[0004] Biological control uses beneficial organisms, their metabolites or active substances to inhibit, parasitize or directly poison nematodes, so it is non-toxic and harmless to non-target organisms and can overcome the defects brought by chemical agent control. Biological control has always been considered the most potential means for controlling root-knot nematodes and is an essential control method for the current green industry. At present, the biocontrol factors for controlling root-knot nematodes include fungi, bacteria, actinomycetes, predatory nematodes, viruses, etc. Among them, biocontrol fungi are the most widely used, such as Paecilomyces lilacinus, Verticillium chlamydosporium, Trichoderma harzianum, etc. However, fungi generally have the defects of long fermentation cycle, weak sporulation ability, short spore survival time, and the colonization ability of fungi in the soil is greatly affected by soil temperature and humidity, so the application effect is often unstable. Compared with fungi, bacteria have better affinity for plants, are more likely to colonize on plants after inoculation, and have a persistent and stable biocontrol effect. The fermentation level is also higher than that of fungi, which is an ideal biocontrol factor for controlling root-knot nematodes. However, there are not many high-efficiency biocontrol bacteria resources at present, and even fewer can be applied in practice. Therefore, it is necessary to continuously explore potential biocontrol bacterial strains to provide resources for exploring potential biological nematicidal preparations in the future. Summary of the Invention

[0005] To increase the resource library of high-efficiency biocontrol bacteria for root-knot nematodes, the present invention provides a strain of Brevundimonas sp. BWT19-19 and its application in controlling plant root-knot nematodes. The bacterial liquid and fermentation liquid of Brevundimonas sp. BWT19-19 have a significant control effect on plant root-knot nematodes, and at the same time, this strain has an obvious growth-promoting effect on host plants.

[0006] The first object of the present invention is to provide a strain of Brevundimonas sp. BWT19-19 with a preservation number of GDMCC No: 62303. The Brevundimonas sp. BWT19-19 is isolated from the soil environment. Its colonies are slightly yellow, round, with neat edges and opaque. The cell surface is smooth, moist and shiny, and is easy to pick up; its 16S rDNA gene sequence is as shown in SEQ ID NO.1.

[0007] The second object of the present invention is to provide a microbial preparation containing the bacterial liquid or fermentation liquid of the above-mentioned Brevundimonas sp. BWT19-19.

[0008] In a preferred example, the microbial preparation is the fermentation liquid of Brevundimonas sp. BWT19-19. The preparation of the fermentation liquid includes the following steps: inoculating Brevundimonas sp. BWT19-19 into NB liquid medium for culture; centrifuging the culture solution, taking the supernatant, and filtering and sterilizing to obtain the fermentation liquid of Brevundimonas sp. BWT19-19.

[0009] Preferably, the culture conditions are: 30 °C, 180 r·min-1 , culture for 24 - 36 h.

[0010] Preferably, the filtration and sterilization is carried out by filtering with a 0.22 μm microporous membrane for 2 - 3 times.

[0011] In another preferred example, the microbial agent is a bacterial liquid of Brevundimonas sp. BWT19 - 19, and the preparation of the bacterial liquid includes the following steps: inoculate Brevundimonas sp. BWT19 - 19 into NB liquid medium for culture; the obtained bacterial cells after centrifugation of the culture solution are diluted with sterile water to obtain the bacterial liquid of Brevundimonas sp. BWT19 - 19.

[0012] Preferably, the culture conditions are: 30 °C, 180 r·min -1 , culture for 24 - 36 h.

[0013] Preferably, for the dilution, the bacterial cells are diluted with sterile water to an OD of the bacterial liquid = 1.0.

[0014] The third object of the present invention is to provide the application of the above - mentioned Brevundimonas sp. BWT19 - 19 or microbial agent in the control of plant root - knot nematodes.

[0015] Preferably, the plant root - knot nematode is Meloidogyne incognita. The Meloidogyne incognita includes Meloidogyne incognita on tomatoes.

[0016] The fourth object of the present invention is to provide the application of the above - mentioned Brevundimonas sp. BWT19 - 19 or microbial agent in promoting the growth and development of host plants.

[0017] Preferably, the plant is tomato.

[0018] The fifth object of the present invention is to provide the application of the above - mentioned Brevundimonas sp. BWT19 - 19 or microbial agent in the preparation of pesticides or fertilizers.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The Brevundimonas sp. BWT19 - 19 screened by the present invention has an obvious lethal effect on Meloidogyne incognita on tomatoes. The lethal rate of the original fermentation broth is 100%, and the lethal rate of the 10 - fold diluted solution is 76.51%.

[0021] (2) In greenhouse pot experiments, the control effect of the bacterial liquid of Brevundimonas sp. BWT19 - 19 on Meloidogyne incognita on tomatoes reaches 46.67%, and the control effect is excellent.

[0022] (3) The bacterial liquid and fermentation broth of Brevundimonas sp. BWT19 - 19 can significantly increase the plant height and dry matter accumulation of tomato plants while controlling pests, and have an obvious plant growth - promoting effect.

[0023] Collection Instructions

[0024] The Brevundimonas sp. BWT19-19 of the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on March 18, 2022, with the address being 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Postal Code: 510070, and the deposit number is: GDMCCNo: 62303. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a colony morphology diagram of Brevundimonas BWT19-19 of the present invention on a plate.

[0026] Figure 2 This is a colony morphology diagram of the Brevundimonas BWT19-19 strain of the present invention.

[0027] Figure 3 This is a picture of tomato plants treated with the Brevundimonas BWT19-19 bacterial solution of the present invention, wherein the left picture is the Brevundimonas BWT19-19 bacterial solution treatment group, and the right picture is the sterile water control group.

[0028] Figure 4 This is a diagram of the root growth of tomato plants treated with the Brevundimonas BWT19-19 bacterial solution of the present invention, wherein the left figure is the Brevundimonas BWT19-19 bacterial solution treatment group, and the right figure is the sterile water control group. DETAILED DESCRIPTION

[0029] The following is a further description of the content of the present invention in combination with the implementation cases, but it is not intended to limit the present invention. Unless otherwise specified, the methods, reagents, instruments or materials used in the examples are conventional and can be obtained from commercial channels or public literature.

[0030] Example 1 Isolation, identification and preservation of Brevundimonas BWT19-19

[0031] 1. Isolation of strains: The strain BWT19-19 of the present invention was isolated from the soil where the root-knot nematodes were seriously infected in the tomato planting base in Xishuangbanna, Yunnan. The specific isolation steps are as follows: 10 g of soil sample was weighed and dissolved in 90 mL of sterile water, shaken at 30°C and 200 r / min for 30 min, and the soil sample was diluted with sterile water to 10 -2 -10 -6, then aspirate 100 μl of the above soil samples with different concentrations, and spread them on NA medium (formula: 10 g / L peptone, 3 g / L beef extract powder, 5 g / L sodium chloride, 15 g / L agar powder; preparation method: take 33 g of the above medium, add 1000 mL of deionized water, stir and heat until completely dissolved, adjust the pH of the solution to 7.2, dispense into Erlenmeyer flasks, autoclave at 121 °C for 15 min, and pour into disposable Petri dishes for standby). Repeat each concentration gradient 3 times, place them in an incubator at 30 °C and incubate in an inverted position for observation. Incubate for about 3 - 5 days. Wait until colonies grow out, pick colonies with different morphologies and colors and streak them for purification. Purify 2 - 3 times to obtain pure strains.

[0032] 2. Preservation of strains: For the already purified strains, according to the colony morphological characteristics, exclude the same strains, pick single colonies and transfer them to the corresponding slants respectively, and store them in a refrigerator at 4 °C for standby. At the same time, make a bacterial suspension with the bacteria using a 50% glycerol distilled water solution and store it in an ultra-low temperature freezer at -80 °C.

[0033] 3. Identification of strains:

[0034] (1) Morphological identification: Apparent identification of the strains is carried out according to colony morphology, cell body color, transparency, glossiness, etc. As Figure 1 and Figure 2 shown, the colonies of Brevundimonas sp. BWT19 - 19 on NA medium are slightly yellow, round, with neat edges and opaque. The cell surface is smooth, moist and shiny.

[0035] (2) Molecular identification: Extract the DNA of Brevundimonas sp. BWT19 - 19 strain according to the instructions of the OMEGA kit. Using the above DNA as a template, perform PCR amplification with the 16S rDNA universal primers 27F / 1492R. The reaction system is 25 μl; 12.5 μl of GreenTap Mix, 0.5 μl of each primer 27F / 1492R, 1 μl of template DNA, 10.5 μl of ddH2O. Reaction conditions: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s; annealing at 55 °C for 30 s; extension at 72 °C for 60 s, a total of 30 cycles; extension at 72 °C for 5 min. Sequence the above PCR products, and the sequencing results are as follows:

[0036]

[0037] Therefore, the Brevundimonas sp. BWT19-19 of the present invention was named Brevundimonas sp. BWT19-19, and was deposited with the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 18, 2022. The address is the 5th floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, with the postal code: 510070, and the deposit number is: GDMCC No: 62303.

[0038] Example 2 Preparation of microbial nematicide

[0039] Pick a single colony of Brevundimonas sp. BWT19-19 and inoculate it into NB liquid medium (10 g / L peptone, 3 g / L beef extract powder, 5 g / L sodium chloride; take 18 g of the above medium, add 1000 mL of deionized water, stir and heat until completely dissolved, adjust the pH of the solution to 7.2, dispense into Erlenmeyer flasks, and sterilize at 121 °C for 15 min before use), and culture at 30 °C and 180 r﹒min -1 , for 36 h. Then centrifuge at 10000 r﹒min -1 for 10 min. Take the supernatant and filter it through a 0.22 μm microporous membrane for sterilization 2-3 times to obtain the fermentation broth preparation of Brevundimonas sp. BWT19-19. The centrifuged cells were diluted with sterile water to a cell suspension (OD = 1.0) and stored in a refrigerator at 4 °C to obtain the cell suspension of Brevundimonas sp. BWT19-19.

[0040] Example 3 Toxic effect of Brevundimonas sp. BWT19-19 fermentation broth on Meloidogyne incognita

[0041] Obtaining of test Meloidogyne incognita: Wash the severely diseased tomato roots with tap water, then wash them 2-3 times with sterile water, dry the surface moisture with absorbent paper, cut the tomato roots into small pieces of 1-2 cm with scissors, then put them into a conical flask, add 0.5% sodium hypochlorite solution, shake vigorously for 3 min, pour the suspension into a set of sieves with 20 meshes, 60 meshes, 100 meshes, 300 meshes, and 500 meshes in turn, repeatedly wash with tap water, collect the eggs on the 500-mesh sieve, suspend the collected eggs with sterile water, place them in a culture dish, and incubate at 27 °C for 4 d to obtain a large number of second-stage larvae.

[0042] In a sterile 24-well cell culture plate, add 1 mL of the fermentation broth prepared in Example 2 to each well in the experimental group, add an equal amount of sterilized NB medium to the control group, add 100 μl of the second-stage larva suspension (about 50 nematodes) to each well, place it in a constant temperature incubator at 27 °C for culture, observe the death of nematodes after 24 h and 48 h of culture, and calculate the corrected mortality rate of nematodes. Each treatment was repeated 4 times, and the results were averaged.

[0043] Corrected mortality rate = (Survival rate of the control group - Survival rate of the treatment group) × 100 / Survival rate of the control group.

[0044] As can be seen from Table 1, after the root-knot nematodes were treated with the fermentation broth of Brevundimonas sp. BWT19-19 and its 10-fold dilution, the corrected mortality rates of the root-knot nematodes reached 100% and 76.51%, indicating that the fermentation broth of Brevundimonas sp. BWT19-19 can effectively kill the second-stage larvae of root-knot nematodes.

[0045] Table 1 Toxicity of the fermentation broth of Brevundimonas sp. BWT19-19 against root-knot nematodes

[0046]

[0047] Example 4 Greenhouse control efficacy test of the fermentation broth and bacterial solution of Brevundimonas sp. BWT19-19

[0048] The preparation methods of the fermentation broth and bacterial solution of Brevundimonas sp. BWT19-19 were the same as those in Example 2, and the method for obtaining the second-stage larvae of root-knot nematodes was the same as that in Example 3.

[0049] Tomato seedling transplantation: Remove the tomato seedlings with three true leaves and one heart from the seedling-raising substrate, wash the roots, and transplant them into flower pots containing 200 g / pot of soil (soil to sand volume ratio = 2:1). Water until the soil moisture content is about 80%, and cultivate for 3 days for standby.

[0050] The specific implementation steps of the pot experiment are as follows: Use a pipette to take 5 mL of the second-stage larva suspension with a density of about 400 larvae / mL and evenly inject it around the rhizosphere of the tomato seedlings cultivated for 3 days. About 2000 second-stage larvae are inoculated per plant, and continue to cultivate for 3 days. Then, 20 mL / plant of the tomato seedlings are inoculated with the fermentation broth of Brevundimonas sp. BWT19-19 or the bacterial solution of Brevundimonas sp. BWT19-19 as the experimental group, and the control group is inoculated with an equal amount of NB sterilized medium or an equal amount of sterile water. Each group of experiments is repeated 3 times, and each repetition has 10 tomato seedlings. After cultivating for 15 days, 30 mL / plant of the tomato seedlings are inoculated with the fermentation broth and bacterial solution of Brevundimonas sp. BWT19-19 for the second time, and the control is also inoculated with an equal amount of NB sterilized medium and sterile water correspondingly. The fermentation broth and bacterial solution of Brevundimonas sp. BWT19-19 are inoculated twice during the entire experimental period, and other management is carried out according to the high-quality cultivation management measures of tomatoes.

[0051] Disease index and control efficacy: 35 days after inoculating nematodes, the number of tomato root knots, disease index, and relative control efficacy were statistically analyzed using the agricultural grading standard of Liu Zhongliang (Liu Zhongliang, Gao Junjie, et al. Treatment of mushroom residue compost and its control efficacy against tomato root-knot nematode disease [J]. Northern Horticulture, 2020(06): 49-54.). The relative control efficacy was calculated according to the method of control efficacy in the literature.

[0052] As can be seen from Table 2, after tomato plants were irrigated with the bacterial suspension or fermentation broth of Brevundimonas sp. BWT19-19, the number of root knots and disease index of the plants were significantly lower than those treated with sterile water. In terms of the number of root knots, the number of root knots treated with the bacterial suspension of Brevundimonas sp. BWT19-19 decreased by 36.49% compared with the treatment with sterile water, and the fermentation broth treatment decreased by 24.73% compared with the treatment with NB sterilized medium, and the difference between treatments was significant. After irrigation with the bacterial suspension and fermentation broth of Brevundimonas sp. BWT19-19, the relative control effects on tomato root-knot nematodes reached 46.67% and 25.00%, indicating that the bacterial suspension and fermentation broth of Brevundimonas sp. BWT19-19 had good control effects on tomato root-knot nematodes.

[0053] Table 2 Incidence of tomato plants in each group 35 days after inoculation

[0054]

[0055] Note: Different letters in the same column indicate significant differences between treatments (p < 0.05).

[0056] Example 5 Growth-promoting effects of the fermentation broth and bacterial suspension of Brevundimonas sp. BWT19-19 on tomato seedlings

[0057] Referring to the pot experiment method and steps in Example 4, 25 days after inoculating the bacterial suspension, indicators such as the plant height, stem diameter, fresh and dry weights of the tomato plants were measured respectively.

[0058] As can be seen from Table 3, irrigation with the bacterial suspension and fermentation broth of Brevundimonas sp. BWT19-19 significantly affected the growth and development and dry matter accumulation of tomato plants. In terms of irrigation with the bacterial suspension, after tomato plants were irrigated with the bacterial suspension of Brevundimonas sp. BWT19-19, the plant height, stem diameter, dry weight of roots and dry weight of plants were significantly better than those treated with sterile water, and each index increased by 23.51%, 13.84%, 32.50% and 15.63% respectively compared with the treatment with sterile water, and the difference between treatments was significant. The plant height and dry weight of roots of tomatoes treated with the fermentation broth were also significantly better than those treated with NB sterilized medium. The growth conditions of the treated tomato plants and roots are shown in Figure 3 and Figure 4 . It shows that the bacterial suspension and fermentation broth of Brevundimonas sp. BWT19-19 have significant growth-promoting effects on tomato plants.

[0059] Table 3 Growth-promoting effects of the fermentation broth and bacterial suspension of Brevundimonas sp. BWT19-19 on tomato plants

[0060]

[0061] Note: Different letters in the same column indicate significant differences between treatments (p < 0.05).

[0062] The above embodiments have elaborated in detail different implementation processes of the present invention, but the implementation manners of the present invention are not limited thereto. Those of ordinary skill in the art can achieve the purpose of the present invention based on the content disclosed in the present invention.

[0063] For those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can still be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A Brevundimonas sp. BWT19-19 with a deposit number of GDMCC No: 62303. Brevundimonas ​ 2. A microbial preparation, characterized in that, contain the bacterial liquid or fermentation broth of Brevundimonas sp. BWT19-19 described in claim 1; The preparation of the fermentation broth comprises the following steps: inoculating Brevundimonas sp. BWT19-19 into NB liquid medium for culture; centrifuging the culture solution, taking the supernatant, and filtering and sterilizing to obtain the fermentation broth of Brevundimonas sp. BWT19-19; The preparation of the bacterial liquid comprises the following steps: inoculating Brevundimonas sp. BWT19-19 into NB liquid medium for culture; diluting the thalli obtained after centrifuging the culture solution with sterile water to obtain the bacterial liquid of Brevundimonas sp. BWT19-19.

3. Application of Brevundimonas sp. BWT19-19 described in claim 1 or the microbial preparation described in claim 2 in the control of plant root-knot nematodes, wherein the plant root-knot nematode is Meloidogyne incognita.

4. Application of Brevundimonas sp. BWT19-19 described in claim 1 or any one of the microbial preparations described in claim 2 in promoting the growth and development of host plants, wherein the host plant is tomato.

5. Application of Brevundimonas sp. BWT19-19 described in claim 1 or the microbial preparation described in claim 2 in the preparation of pesticides or fertilizers.

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