CN110878272B - Bacillus subtilis ZWZ-19 for preventing and controlling rot stem nematode and application thereof - Google Patents
Bacillus subtilis ZWZ-19 for preventing and controlling rot stem nematode and application thereof Download PDFInfo
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Abstract
The invention relates to a Bacillus subtilis ZWZ-19 for preventing and controlling stem rot nematodes, belonging to the technical field of microbial pesticides. The strain ZWZ-19 related to the invention has been preserved in China general microbiological culture Collection center in 22 months 10 and 2019, and the preservation numbers are as follows: CGMCC No. 18718. Indoor tests show that the strain has good contact killing activity on the rot stem nematodes. The field test shows that after the ZWZ-19 strain fermentation liquor is used for seed dressing treatment of potato seeds, the strain has a good prevention effect on rot stem nematode disease, and meanwhile, the strain also has a certain yield increase effect.
Description
The technical field is as follows:
the invention belongs to the technical field of microbial pesticides, and particularly relates to a bacillus subtilis ZWZ-19 for preventing and controlling rot stem nematodes and application thereof.
The background art comprises the following steps:
the rot stem nematode (Ditylenchus destructor) is an important plant pathogenic nematode in the world, belongs to a parasitic nematode in migratory plants, and is an internationally important quarantine nematode. The host range of the nematode is wide, including more than 70 crops and weeds, and the nematode can also parasitize on hypha of soil fungi, the main hosts are potatoes and sweet potatoes, other common hosts comprise iris, tulip, calamus, beet, clover, dahlia, carrot, peanut, sunflower, soybean, tomato, tobacco, sugarcane, barley, wheat and the like, and various wild plants, and the rot stem nematode disease is a soil-borne disease which is extremely difficult to control. In recent years, with the continuous expansion of the areas of potatoes and sweet potatoes in China, the distribution of rot stem nematodes is more and more wide, mainly occurs in 12 provinces (regions) such as Hebei, Henan, Beijing, Shandong, Jiangsu, Anhui, Jilin, inner Mongolia and the like, and becomes the most serious disease in the sweet potato production region in North China; the yield of the damaged field can be reduced by 30-50 percent generally, and the yield can be reduced by more than 80 percent seriously, even the field is not harvested. Threatens the development of related industries such as potatoes, sweet potatoes and the like, and has very adverse effect on local economy. At present, chemical control is mainly used for preventing and treating rot stem nematode diseases, and most of used nematicides seriously pollute the environment and destroy the ecological balance. In recent years, with the rapid development of sustainable agriculture, people have turned the work of preventing and controlling nematode diseases to biological prevention and control.
The biological control factors of plant nematode diseases comprise fungi, bacteria, actinomycetes, viruses, protozoa and the like. Becker et al (1988) reported that Pseudomonas (Pseudomonas) and Bacillus (Bacillus) have control effects on Meloidogyne diseases. Liu jin Hui et al (2012) reported that Bacillus thuringiensis (Bacillus thuringiensis) strains have high virulence for sweetpotato stem nematodes. YaoSMIN (2018) reports that Bacillus megaterium (D45) strain fermentation liquor and volatile substances have nematicidal activity on potato stem rot nematodes. Therefore, the method further screens the high-activity biocontrol factors aiming at the diseases of the rot stem nematodes, accelerates the research and development and application of the microbial nematicide and has great significance for effective prevention and control of the microbial nematicide.
The invention content is as follows:
the invention screens biological control factors for stem rot nematodes (Ditylenchus destructor), and treats the nematodes by using fermentation liquor after conventional liquid fermentation of microbial strains so as to evaluate the contact killing effect of the nematodes.
The strain ZWZ-19 obtained by screening is identified as Bacillus subtilis, and the preservation unit is as follows: china general microbiological culture Collection center; address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing; the preservation date is as follows: 2019, 10, 22 months; number of deposit registration entry: CGMCC No. 18718.
The Bacillus subtilis ZWZ-19 has good contact killing effect on the stem rot nematodes in indoor experiments, and field experiments show that the strain has obvious control effect on the stem rot nematodes.
The Bacillus subtilis ZWZ-19 microbial inoculum can be used for seed dressing treatment during potato seeding.
The Bacillus subtilis ZWZ-19 strain is prepared by activating a plate and then performing liquid fermentation, and the specific method comprises the following steps:
1. plate activation
The formula of the culture medium is Luria-Bertani culture medium (LB):tryptone10g，Yeast extract5g, NaCL5g, agar 20g, and distilled water 1000 mL. The strain is inoculated on an LB plate and cultured for 2 d-3 d.
2. Liquid fermentation culture
Wherein the formula of the liquid fermentation medium is as follows: in terms of weight percentage, the weight percentage of the active carbon is,tryptone1％-2％，Yeast extract0.5-0.6 percent of NaCl0.5-0.6 percent of the total weight of the composition, and the balance of distilled water, wherein the pH value is 6.8-7.0.
Inoculating the strain into 250mL triangular flask (100 mL per bottle) liquid culture medium, at 25-28 deg.C, and rotating at 150 r.min with shaking table -1 ～180r·min -1 And fermenting for 2 d-3 d.
The specific implementation mode is as follows:
in order to further illustrate the invention in more detail, but not to limit it, the following examples are given.
Example 1: activation and fermentation of Bacillus subtilis ZWZ-19
Firstly, the Bacillus subtilis ZWZ-19 strain is subjected to plate activation. The formula of the culture medium is Luria-Bertani culture medium (LB):tryptone10g，Yeast extract5g, NaCl10g, agar 20g, and distilled water 1000 mL. The strain was inoculated on an LB plate and cultured for 2 d.
Then inoculating the strain into a 250mL triangular flask (100 mL per flask) liquid culture medium, wherein the formula of the liquid fermentation culture medium is as follows: in terms of weight percentage, the weight percentage of the composite material,tryptone1％～2％，Yeast extract0.5％～0.6％，NaCl 05% -0.6%, the balance being distilled water, the pH being 6.8-7.0. The fermentation temperature is 25-28 ℃, the rotating speed of a shaking table is 150 r.min -1 ～180r·min -1 And the fermentation time is 2 d-3 d.
Example 2: indoor contact killing test of Bacillus subtilis ZWZ-19 on rot stem nematode
1. Preparation of test preparations
Preparing the fermentation liquor of the bacillus subtilis ZWZ-19 according to the liquid fermentation culture method for later use.
2. Preparation of test nematode suspensions
Rot stem nematode (Ditylenchus destructor): separating the rot stem nematodes from the sweet potatoes or potatoes by adopting a modified Bermann funnel method, transferring the separated nematodes to a 10ml centrifuge tube by using a straw, adding 35% of sucrose solution, and centrifuging at 3500r/min for 2 min: taking surface layer nematode suspension, transferring the surface layer nematode suspension into sterile water, and centrifuging for 5min at 4000 r/min; discarding the supernatant, sterilizing with 0.5% sodium hypochlorite for 1min, and centrifuging at 4000r/min for 5 min; discarding supernatant, adding into mixed solution of 0.5% streptomycin sulfate and 0.5% penicillin, sterilizing for 6min, centrifuging, sucking out disinfectant, washing with sterile water for 3 times, and storing at 4 deg.C.
3. Test method
3.1 centrifuging the fermentation liquid of the bacillus subtilis ZWZ-19 at 6000r/min for 5min, and taking the supernatant. The fermentation liquor is sequentially diluted by 2, 6, 10 and 20 times and the fermentation stock solution by using sterile water to form 5 concentration gradients. 1.5ml of fermentation liquid of each strain is added into a 12-hole cell culture plate by using a pipette, and then about 50 pieces of the rot stem nematodes are placed into the cell culture plate. Each treatment was repeated 3 times, with sterile water as a control. And finally, placing the mixture into a polyethylene bag, fixing the mixture by using a rubber band, placing the mixture into an incubator at 25 ℃, and recording the death number of the nematodes after 24 hours, 48 hours and 72 hours.
3.2 evaluation of contact Effect
4. Test results
The measurement result shows that: after the stock solution is used for treating the rot stem nematodes for 24 hours, 48 hours and 72 hours, the corrected lethality rates of the rot stem nematodes reach 59.51%, 70.54% and 86.94% respectively; after the 2-time dilution treatment of the rot stem nematodes is carried out for 24 hours, 48 hours and 72 hours, the corrected lethality rates of the rot stem nematodes reach 44.72%, 67.73% and 80.11% respectively; after the treatment of the rot stem nematode for 24 hours, 48 hours and 72 hours by 6 times of dilution, the corrected lethality rates of the rot stem nematode reach 35.33%, 60.71% and 78.99% respectively; after the treatment of the rot stem nematode for 24 hours, 48 hours and 72 hours by diluting 10 times, the corrected lethality rates of the rot stem nematode reach 27.01 percent, 35.37 percent and 46.50 percent respectively; the corrected lethality rates of the putrescent stem nematodes after being diluted by 20 times and treated for 24h, 48h and 72h reach 26.21%, 30.48% and 44.03% respectively.
TABLE 1 contact killing Activity of Bacillus subtilis ZWZ-19 against Stem rot nematodes
Example 3: bacillus subtilis ZWZ-19 field test for preventing and treating rot stem nematode disease
1. Preparation of test preparations
Preparing the fermentation liquor of the bacillus subtilis ZWZ-19 according to the liquid fermentation culture method for later use.
2. Seed for test
Potato variety: feuretat, his 6, supplied by inner mongolia seifeng specie ltd.
3. Disease grading standard and disease index calculation of rot stem nematode
The disease index grading standard of the rot stem nematodes is referred to table 2;
the disease index is [ Σ (number of diseased potato blocks at each stage × representative value at relative stage)/(total number of investigated plants × 4) ] × 100.
TABLE 2 grading Standard of Heterodera rot disease
Note: reference segment jade seal, Wu gang, etc
4. Test method
The potatoes are planted in the soil naturally occurring in the field, and the test field is a continuous cropping potato field. The inter-field cells are designed into 3 treatments, 6 times of repetition and 8 cells in total, and are arranged in random block groups, wherein each cell is set to be 1m in ridge width and 12m in cell area 2 (3m x 1m x 4m), the plant spacing is 25cm, four ridges are planted in each plot, about 12 plants are planted in each ridge, about 50 plants are planted in each ridge, and protection rows with the width of 1-2m are arranged on the periphery of the test area. And (3) taking potatoes which are not subjected to suspension treatment and conventional seed dressing as a control, taking bacterial suspension seed dressing as treatment, after the potatoes are harvested, randomly taking 30 potatoes in each cell for investigation, and calculating the morbidity, disease index and yield.
5. Test results
After the potato blocks are mixed with the seed by the fermentation liquor of the bacillus subtilis ZWZ-19, compared with a control, the incidence rate and disease index of the potato rot stem nematode disease are obviously reduced, and the yield is obviously improved. After the variety Fiurestat is treated by dressing seeds, the morbidity and the disease index are respectively 37.44 percent and 14.24 (the contrast is 96.29 percent and 30.64 percent), and the prevention effect reaches 58.94 percent; after the seed dressing treatment of the variety Hissen No. 6, the morbidity and the disease index are respectively 30.55 percent and 10.27 percent (the contrast is 52.80 percent and 20.98 percent), and the control effect reaches 51.04 percent. After the bacillus subtilis ZWZ-19 is treated by dressing seeds, the yield of the Fiuroreta and the yield of the Hissen No. 6 are respectively improved by 23.82 percent and 18.10 percent.
TABLE 3 field control of rot-stem nematodes by Bacillus subtilis ZWZ-19
Claims (4)
1. A bacillus subtilis ZWZ-19 for preventing and controlling stem rot nematodes is characterized in that: the strain is Bacillus subtilis, which has been preserved in China general microbiological culture Collection center (CGMCC) 22.10.2019 with the preservation number of CGMCC No. 18718.
2. The biocontrol microbial inoculum prepared from the bacillus subtilis ZWZ-19 for controlling the stem rot nematode as claimed in claim 1.
3. The method for preparing the biocontrol microbial inoculum as set forth in claim 2: 1) inoculating Bacillus subtilis ZWZ-19 strain on LB plate, culturing for 2 d-3 d, the culture medium formula: 10g of tryptone, 5g of yeast extract, 5g of NaCl, 20g of agar and 1000mL of distilled water;
2) transferring the strain into a 250mL triangular flask, wherein each flask contains 100mL of liquid culture medium, and the rotating speed of a shaking table is 150 r.min at 25-28 DEG C -1 ～180r·min -1 Liquid fermentation culture for 2 d-3 d, wherein the formula of the liquid fermentation culture medium is as follows: 1 to 2 percent of tryptone, 0.5 to 0.6 percent of yeast extract, 0.5 to 0.6 percent of NaCl, and the balance of distilled water, wherein the pH value is 6.8 to 7.0.
4. Use of the biocontrol microbial inoculum of claim 2 for the control of necrotizing stemona disease.
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Non-Patent Citations (2)
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| The purL gene of Bacillus subtilis is associated with nematicidal activity;Yanfei Xia 等;《FEMS Microbiol Lett》;20110721;第322卷;表3、第100页右栏第1段 * |
| 枯草芽孢杆菌挥发产物的杀线虫活性评价及成分鉴定;刘玮玮 等;《植物病理学报》;20091231;第39卷(第3期);第304-309页 * |
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