Burkholderia sp. BWT1.3 antagonistic to multiple phytopathogens and its application

Abstract

The present invention belongs to the field of microbial technology, and particularly relates to a Burkholderia strain BWT1.3 that antagonizes multiple plant pathogenic bacteria and its applications. The Burkholderia strain BWT1.3 provided by the present invention is deposited in the Guangdong Provincial Culture Collection of Microorganisms, with the deposit number of GDMCC NO. 62714. Through plate confrontation studies, it is shown that this strain has significant inhibitory effects on various plant pathogenic bacteria such as Xanthomonas citri, Acidovorax avenae subsp. citrulli, Ralstonia solanacearum, Rhizoctonia solani, Fusarium oxysporum, and Colletotrichum musae, and can be used in plant biological control. At the same time, the bacterial liquid and fermentation broth of this strain have obvious lethal effects on Meloidogyne incognita and can be used in the control of plant root-knot nematode diseases. The Burkholderia strain BWT1.3 provided by the present invention not only has antagonistic effects on plant pathogenic bacteria, but also has good control effects on plant diseases and pests caused by pathogenic fungi and root-knot nematodes, and has extremely high utilization value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a Burkholderia BWT1.3 strain antagonizing multiple plant pathogens and an application thereof. Background Art

[0002] Soilborne diseases are a type of plant diseases caused by pathogens such as bacteria, fungi, and nematodes in the soil that infect the root system or stem base of crops under suitable conditions. They include root rot, bacterial wilt, damping-off, damping-off, blight, verticillium wilt, and root-knot nematode diseases. In recent years, due to the increase in the multiple cropping index and the irrational application of fertilizers and pesticides, the quality of cultivated soil has declined, the soil microbial flora has become unbalanced, the number of pathogens has increased sharply, and the pathogens have become more resistant to drugs, resulting in large-scale outbreaks of soilborne diseases in various agricultural planting areas. This has not only brought huge economic losses to agricultural production, but also seriously restricted the development of agriculture.

[0003] At present, there is no control agent or control method that can completely control the occurrence of soil-borne crop diseases. Commonly used methods include crop rotation, grafting, breeding of disease-resistant varieties and chemical agents. Although they can achieve certain results, they all have certain limitations. For example, breeding resistant varieties can only work for a period of time. As the genetic diversity of pathogens changes, the disease resistance of resistant varieties will decrease; crop rotation can only be achieved in a small area and is difficult to promote on a large scale; chemical agents can inhibit pathogens in a short period of time, but the use of chemical agents is often prone to problems such as drug residues, environmental pollution and accumulation of drug resistance, which does not meet the goal of green, healthy and sustainable development of agriculture. Therefore, the development of new, efficient, safe, green and environmentally friendly agents has important practical significance in the sustainable development of agriculture.

[0004] Biological control has broad application prospects in the prevention and control of soil-borne diseases due to its low cost, no pollution, low resistance and no residue, and will gradually replace traditional chemical control methods.

[0005] Biological control is a means of control that uses one or more microorganisms and some volatile substances, cell lytic enzymes and secondary metabolites they produce to inhibit and resist the growth of pathogens. At present, there are abundant biocontrol microbial resources for biological control, but most of them only target one or several target pathogens, and there is a lack of efficient biocontrol microbial resources for different target pathogens. Summary of the invention

[0006] In view of the current situation that there are relatively rich biocontrol microbial resources, but the vast majority are only effective against one or a few target pathogenic bacteria, lacking efficient biocontrol microbial resources for different target pathogenic microorganisms, the present invention provides a Burkholderia sp. BWT1.3 that antagonizes multiple plant pathogenic bacteria and its applications. The Burkholderia sp. BWT1.3 provided by the present invention not only has antagonistic effects against plant pathogenic bacteria, but also has good control effects on plant diseases and pests caused by pathogenic fungi and root-knot nematodes, and has extremely high utilization value.

[0007] In order to achieve the technical effects described in the present invention, the technical solution adopted by the present invention is as follows:

[0008] A strain of Burkholderia sp. BWT1.3, which is preserved in the Guangdong Microbial Culture Collection Center, with the preservation number GDMCC NO.62714, the preservation date is August 19, 2022, and the preservation address is Guangzhou, Guangdong Province, China.

[0009] The present invention also provides a biocontrol preparation, including one or several of the above-mentioned Burkholderia sp. BWT1.3, the culture of this strain, the metabolite of this strain, the supernatant separated from the culture of this strain, and the crude extract of the fermentation broth of this strain.

[0010] The present invention also provides a preparation method of the above-mentioned biocontrol preparation. The preparation process is as follows: After activating the strain BWT1.3, inoculate it into NB medium for fermentation culture, and mix the obtained culture, the metabolite of the strain, and / or the supernatant filtered and separated from its bacterial liquid and the extracted crude enzyme solution to obtain the product.

[0011] The present invention also provides an application of the above-mentioned Burkholderia sp. BWT1.3 in the preparation of drugs for preventing and treating plant pathogenic bacteria and plant pathogenic fungal diseases.

[0012] Preferably, the plant pathogenic bacteria include Xanthomonas citrisubsp. citri (Xcc, citrus canker), Acidovorax avenaesubsp. Citrulli (cantaloupe angular leaf spot), Ralstonia solanacearum (tomato bacterial wilt), etc.

[0013] Preferably, the plant pathogenic fungi include Rhizoctonia solani (damping-off), Fusarium oxysporum (fusarium wilt), and Colletotrichum musae (banana anthracnose).

[0014] The present invention also provides an application of the Burkholderia sp. BWT1.3 in the preparation of a drug for preventing and controlling Meloidogyne incognita.

[0015] The present invention also provides an application of the Burkholderia sp. BWT1.3 in the preparation of a medicament for plant diseases and insect pests.

[0016] The Burkholderia sp. BWT1.3 provided by the present invention is isolated from a soil environment, and its gene sequence information is shown as SEQ ID NO.1. The colony is round, with a neat edge and is opaque. The surface of the bacterial cells is smooth, moist and shiny, and is easy to pick up. After culturing for 24 hours, the bacterial cells are yellow, and after culturing for 48 hours, the color of the bacterial cells changes from yellow to purple-red. The Burkholderia sp. BWT1.3 of the present invention is preserved in the Guangdong Microbial Culture Collection Center, the preservation date is August 19, 2022, and the preservation number is GDMCC NO.62714.

[0017]

[0018] The bacterial liquid and fermentation broth of Burkholderia sp. BWT1.3 of the present invention have obvious lethal effects on Meloidogyne incognita. After 10-fold dilution, the lethality rate against second-instar larvae can reach 83.90%, and the lethal effect is significant. This strain can also be used for the control of plant root-knot nematode diseases. Therefore, in the process of preparing drugs according to the present invention, in addition to directly using the Burkholderia sp. BWT1.3 strain, its bacterial liquid, bacterial cells, fermentation broth, etc. can also be used.

[0019] Compared with the prior art, Burkholderia sp. BWT1.3 provided by the present invention has the following advantages: Through plate confrontation studies, it has been shown that this strain has significant inhibitory effects on various plant pathogenic bacteria such as Xanthomonas citri subsp. citri (Xcc, citrus canker), Acidovorax avenae subsp. citrulli (Hami melon angular leaf spot), Ralstonia solanacearum (tomato bacterial wilt), Rhizoctonia solani (damping-off), Fusarium oxysporum (fusarium wilt), and Colletotrichum musae (banana anthracnose), and can be used in plant biological control. At the same time, the bacterial liquid and fermentation broth of this strain have obvious lethal effects on Meloidogyne incognita. After 10-fold dilution, the lethality rate against second-instar larvae can reach 83.90%, and the lethal effect is significant. This strain can also be used for the control of plant root-knot nematode diseases. Burkholderia sp. BWT1.3 provided by the present invention not only has an antagonistic effect on plant pathogenic bacteria, but also has good control effects on plant diseases and pests caused by pathogenic fungi and root-knot nematodes, so it has good development and utilization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a colony morphology diagram of Burkholderia sp. BWT1.3 on a plate in the embodiment of the present invention;

[0021] Figure 2 It is a single colony morphology diagram of Burkholderia sp. BWT1.3 strain cultured for 48 hours in the embodiment of the present invention;

[0022] Figure 3 It is an effect diagram of the inhibitory effect of Burkholderia sp. BWT1.3 on Fusarium oxysporum;

[0023] Figure 4 It is an effect diagram of the inhibitory effect of Burkholderia sp. BWT1.3 on Rhizoctonia solani;

[0024] Figure 5 Effect diagram of the inhibitory effect of Burkholderia sp. BWT1.3 on Colletotrichum musae

[0025] Figure 6 Effect diagrams of the inhibitory effects of Burkholderia sp. BWT1.3 on Xanthomonas citrisubsp. citri (Xcc), Ralstonia solanacearum, and Acidovorax avenae subsp. citrulli Specific implementation manners

[0026] The present invention will be further explained below in conjunction with specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions identical or similar to the present invention are within the protection scope of the present invention. For those not specifying specific techniques or conditions in this embodiment, operations are carried out according to conventional technical methods and the content of the instrument instructions in the art; for those reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0027] The formula of the NA medium is 10 g / L peptone, 3 g / L beef extract powder, 5 g / L sodium chloride, and 15 g / L agar powder; the preparation method is as follows: 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 it into Erlenmeyer flasks, sterilize it at 121 °C under high pressure for 15 min, and pour it into disposable petri dishes for standby after completion; the NB medium is the NA medium without agar powder.

[0028] The formula of the PDA medium is 300 g / L potato extract leaching powder, 20 g / L glucose, 15 g / L agar powder, and 0.1 g / L chloramphenicol, and the preparation method is the same as that of the NA medium;

[0029] Example 1 Isolation, identification, and preservation of Burkholderia sp. BWT1.3

[0030] 1. Isolation of Burkholderia sp. BWT1.3 strain: The BWT1.3 strain was isolated from the rhizosphere soil with a severe outbreak of root-knot nematodes in Hainan Hami melons. The isolation steps are as follows: Weigh 10 g of rhizosphere soil sample and dissolve it in 90 mL of sterile water. Shake it at 30 °C and 200 r / min for 30 min, and sequentially dilute the soil sample with sterile water to 10 -2 ~10 -6, 100 μL of soil samples with different concentrations were taken and spread on NA medium respectively. Each concentration gradient was repeated 3 times, and then incubated in an inverted position in an incubator at 30 °C for observation. After culturing for about 1 - 3 days, colonies with different morphologies and colors were picked and streaked for purification. The purification was carried out 2 - 3 times to obtain pure strains.

[0031] 2. Identification of Burkholderia sp. strain BWT1.3: The strain was identified apparently according to colony morphology, cell body color, transparency, glossiness, etc. As Figure 1 shown, the colonies of Burkholderia sp. BWT1.3 on NA medium were round, with neat edges and opaque. The cell body surface was smooth, moist and shiny, and was easy to pick up. After culturing for 24 hours, the cell body was yellow, and after culturing for 48 hours, the cell body changed from yellow to purplish red ( Figure 2 ). Through 16sDNA sequencing analysis, the strain of the present invention had the highest similarity with the sequence of LRSZR63 Burkholderia sp., which was 99.93%. Combining with the morphological characteristics of the strain, the strain was identified as Burkholderia sp. and named Burkholderia sp. BWT1.3.

[0032] 3. Preservation of Burkholderia sp. strain BWT1.3: The identified single colony was picked and transferred to the corresponding test tube slant for storage in a 4 °C refrigerator for standby. At the same time, the cell bodies were made into cell suspensions with 50% glycerol distilled water solution and stored in a -80 °C ultra-low temperature refrigerator.

[0033] Example 2 Toxic killing effect of the fermentation broth of Burkholderia sp. BWT1.3 on Meloidogyne incognita 1. Test process: The single colony of Burkholderia sp. BWT1.3 was picked and inoculated into NB liquid medium, and cultured at 30 °C and 180 r / min for 36 h. After completion, it was centrifuged at 10000 r / min for 10 min, and the supernatant was filtered through a 0.22 μm microporous filter membrane for sterilization 2 - 3 times. In a sterile 24-well cell culture plate, 1 mL of the filtered and sterilized fermentation stock solution or 10-fold diluted fermentation broth was added to each well in the experimental group, and an equal volume of filtered and sterilized NB liquid medium was added to the control group. 100 μL of the second-instar larva suspension (about 50 nematodes) was added to each well, and it was cultured in a constant temperature incubator at 27 °C for 24 h and 48 h. The death situation of the nematodes was observed, and the corrected mortality rate of the nematodes was calculated. Each treatment was repeated 4 times, and the results were averaged.

[0034] Corrected mortality rate = (Survival rate of the control group - Survival rate of the treatment group) × 100 / Survival rate of the control group 2. Test results: The specific test results are shown in Table 1.

[0035] Table 1 Toxic killing situation of the fermentation broth of Burkholderia sp. BWT1.3 on root-knot nematodes

[0036] Treatment Corrected mortality at 24 h (%) Corrected mortality at 48 h (%) Stock solution 100 100 10-fold diluted solution 80.58 83.90

[0037] As can be seen from Table 1, after the root-knot nematodes were treated with the fermentation broth of Burkholderia sp. BWT1.3 and its 10-fold dilution, the corrected mortality rates reached 100% and 83.90% respectively, indicating that Burkholderia sp. BWT1.3 can effectively kill the second-stage larvae of root-knot nematodes.

[0038] Example 3 Inhibitory effect of Burkholderia sp. BWT1.3 on pathogenic fungi

[0039] 1. Test procedure: Strain activation and culture: Fusarium oxysporum, Rhizoctonia solani, and Colletotrichum musae strains were respectively inoculated onto PDA medium plates for activation. After the mycelia covered the plates, sterilized punchers were used to evenly punch out circular agar plugs with a diameter of 8 mm from the outer edge of the colonies for standby. Burkholderia sp. BWT1.3 was streaked onto an NA medium plate for activation and culture for 24 h for standby.

[0040] Determination of inhibitory effect: The activated pathogen agar plugs were respectively inoculated 25 mm away from the edge of the PDA medium plate, and the activated Burkholderia sp. BWT1.3 strain was inoculated about 50 mm opposite to it. Without inoculating Burkholderia sp. BWT1.3 was used as the control, with 4 replicates, and they were placed in an incubator at 28 °C for culture. Fusarium oxysporum was cultured for 5 d, Colletotrichum musae was cultured for 3 d, and Rhizoctonia solani was cultured for 7 d. After the culture was completed, the growth radius of the pathogenic fungi between the edge of the agar plug and the center of Burkholderia sp. BWT1.3 was measured, denoted as the growth radius of the treatment group; the growth radius of the pathogenic fungi in the control group was denoted as the growth radius of the control group. Finally, according to the following formula, the inhibition rate was calculated.

[0041] Inhibition rate (%) = (control growth radius - treatment growth radius) / control growth radius × 100

[0042] 2. Test results: From Figure 3 、 4 、5 (in Figures 3 - 5 , A is the control group, and B is the experimental group), Burkholderia sp. BWT1.3 has obvious inhibitory effects on Fusarium oxysporum, Rhizoctonia solani, and Colletotrichum musae, but there are differences in the inhibition degrees. The inhibitory effect on Rhizoctonia solani is the strongest, with an inhibition rate of 83.05%. Followed by Fusarium oxysporum, with an inhibition rate of 60.24%, and the inhibition rate on Colletotrichum musae is 38.71%.

[0043] Example 4 Antagonistic effect of Burkholderia sp. BWT1.3 on pathogenic bacteria

[0044] 1. Test procedure: Strain activation and culture: Xanthomonas citri subsp. citri, Acidovorax avenae subsp. citrulli, Ralstonia solanacearum, and Burkholderia sp. BWT1.3 were respectively inoculated onto NA plates for activation and fermented and cultured in NB (NA medium without agar powder) for 36 h.

[0045] Preparation of bacterial suspension and fermentation broth: Dilute the bacterial solution of the pathogen into a mother liquor with OD = 1.0, and then dilute it to 10 -3 for standby; The preparation method of the fermentation broth of Burkholderia sp. BWT1.3 is the same as that in Example 2.

[0046] Antagonistic experiment: Take 100 μL of the diluted bacterial solution of the pathogen and evenly coat it on the NA solid medium. Use a blue pipette tip to punch holes on the plate, and then take 70 μL of the fermentation broth of Burkholderia sp. BWT1.3 into the holes. Incubate at 30 °C for 1 - 3 d, observe the size of the inhibition zone, and take pictures to record the results.

[0047] 2. Test results: The specific test results are as Figure 6 shown, Figure 6 in which, A is the inhibition result of Xanthomonas citri, B is the inhibition result of Acidovorax avenae subsp. citrulli, and C is the inhibition result of Ralstonia solanacearum; It can be Figure 6 seen that the fermentation broth of Burkholderia sp. BWT1.3 shows good inhibitory effects on Xanthomonas citri, Acidovorax avenae subsp. citrulli, and Ralstonia solanacearum, and obvious inhibition zones are formed by the fermentation broth on each inhibition plate.

[0048] Finally, it should be noted that the above embodiments are only illustrative of the principles, properties and effects of the present invention, and are not used to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A strain of Burkholderia ( Burkholderia sp.) BWT1.3, characterized in that This strain is deposited in the Guangdong Microbial Culture Collection Center under the deposit number of GDMCC NO.62714.

2. A biocontrol agent, characterized in that, Including Burkholderia BWT1.3 described in claim 1.

3. A preparation method of the biocontrol agent according to claim 2, characterized in that, The preparation process is as follows: After activating strain BWT1.3, inoculate it into NB medium for fermentation culture to obtain the culture.

4. Use of Burkholderia sp. BWT1.3 as described in claim 1 in the preparation of drugs against Xanthomonas citri subsp. citri (Xcc), Acidovorax avenae subsp. citrulli, Ralstonia solanacearum, Xanthomonas citri , Acidovorax avenae subsp. citrulli, Ralstonia solanacearum, Acidovorax avenae Acidovorax avenae subsp. citrulli, Ralstonia solanacearum Ralstonia solanacearum, Rhizoctonia solani) Rhizoctonia solani, Fusarium oxysporum Fusarium oxysporum, Colletotrichum musae Colletotrichum musae, and Colletotrichum musae.

5. Use of Burkholderia BWT1.3 as described in claim 1 in controlling Meloidogyne incognita.

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