CN113678828B - Application of trimethylamine oxide dihydrate as synergist in bacillus biopesticide - Google Patents
Application of trimethylamine oxide dihydrate as synergist in bacillus biopesticide Download PDFInfo
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Abstract
The invention discloses application of trimethylamine oxide dihydrate as a synergist in bacillus biopesticides. The invention innovatively utilizes trimethylamine oxide dihydrate as a synergist of the bacillus biopesticide, and the trimethylamine oxide dihydrate can improve the stress resistance and colonization in the environment of the bacillus fungicide, thereby improving the stability and control effect of the biopesticide.
Description
Technical Field
The invention belongs to the technical field of pesticide biology, and particularly relates to application of trimethylamine oxide dihydrate as a synergist in bacillus biopesticides.
Background
The bacillus is the most important biocontrol bacterium type, and has the advantages of wide control spectrum, high control efficiency, low fermentation cost, strong environmental adaptability and the like. Therefore, most of the living microbial pesticides in the pesticide markets of China and even the world are bacilli, 118 varieties (8 months in 2021, Chinese pesticide information network http:// www.chinapesticide.org. cn) are registered and effective at present, and the microbial pesticides are widely applied to prevention and control of fungal and bacterial diseases of various crops. According to incomplete statistics, the reported and researched biocontrol potential bacteria of the bacillus are not less than 200 strains, so that more and more bacillus biological pesticides emerge in the future. Most of the Bacillus strains that have been commercialized or have commercial potential are Bacillus subtilis, followed by Bacillus cereus, Bacillus amyloliquefaciens, Bacillus methylotrophicus, Bacillus beleisi, and the like. In China, the bacillus subtilis accounts for more than 50% of registered bacillus type living biological pesticides. And the existing bacillus subtilis strains approved by Environmental Protection Agency (EPA) approved commercial or limited commercial production application in the united states have 13 strains, including BU1814, AFS032321, QST713, FZB24, IAB/BS03, RTI477, CBS 133255, FMCH002, CX-9060, D747, GB03 and MBI600, and are used for biological control of fungi and bacterial diseases of various field and commercial crops such as corn, soybean, wheat, potato and the like (NPIRS, http:// npirspulic. ceris. purdue. edu, up to 2 months 2020).
However, as a live microbial pesticide, bacillus-type biopesticides also have their inherent drawbacks. First, the control effect is unstable, and after the bacillus is introduced into a working environment, the bacillus must survive in the environment and successfully proliferate to maintain a certain population number to play a role. The natural environment is different from the laboratory with controllable conditions, and harmful factors such as sunlight, especially ultraviolet rays, high temperature, drought and the like are widely existed in the laboratory, so that the survival and proliferation of bacillus are not facilitated. These uncontrollable unfavorable factors make it impossible to achieve stable control of bacillus species. The same reason why most biocontrol bacteria have excellent performance in laboratories but cannot achieve ideal control effect in fields is that the biocontrol bacteria are not suitable for the field. In addition to the increased demands placed on strain performance, improvements and innovations in biocontrol agent technology are the most operational solutions to circumvent these adverse factor effects in the environment. However, the technology of biocontrol bacteria preparation is innovative for decades, is a short board of the biological pesticide industry, and cannot meet the industrial demand. The pesticide synergist is an important auxiliary agent in the pesticide preparation process, and can improve the control effect of the pesticide, reduce the pesticide cost, slow down the pesticide resistance and reduce the environmental pollution. The biological pesticide has higher requirement on the auxiliary agent, improves the application performance of the pesticide, has no toxicity and harm to the biological control bacteria, improves the stress resistance of the biological pesticide, and is beneficial to the colonization of the biological pesticide in the environment. At present, most of biopesticide synergists in the market are natural materials such as microbial fermentation products, extracts, biomass and the like, and not only can the components be uncontrollable, but also the ideal effect can not be realized. Therefore, there is a strong need in the biopesticide industry for highly efficient, stable biopesticide synergists.
The biological membrane (Biofilm) is a community structure formed by bacteria, is beneficial to resisting the influence of external adverse environment, and has important significance for preventing bacteria. After the bacillus is released into the field, especially in a humid environment such as a rice field, a biofilm structure can be generated, and the influence of adverse factors such as ultraviolet rays and high temperature on the survival and colonization of the bacillus is favorably reduced. Research shows that the earlier the stable and mature biological membrane structure is formed, the better the colonization of bacillus in the environment is, and the biological control effect is stably exerted.
Trimethylamine oxide dihydrate (Trimethyamine-N-oxide dehydrate, CAS: 62637-93-8). The molecular formula is (CH)3)3NO•2H2O, which is widely used, is used as an oxidizing agent in the organic synthesis industry due to its weak oxidizing property, and can be used as a flavoring agent and an industrial adjuvant. The compound is naturally distributed widely in the muscle of marine teleosts and is believed to act as a protein stabilizer. In the pesticide industry, no application record can be found for the compound, and no application and related report in the production of biopesticides exist.
Disclosure of Invention
Aiming at the problems that the living biological pesticides of the bacillus are not easy to colonize in the working environment and the control effect is not high and unstable caused by the colonization in the working environment in the prior art, the invention aims to utilize trimethylamine oxide dihydrate as a synergist of the biological pesticides of the bacillus, and the trimethylamine oxide dihydrate can promote the colonization in the environment and improve the stability and the control effect of the biological bactericide by improving the stress resistance of the bacillus.
The application is realized by the following technical scheme:
the trimethylamine oxide dihydrate is used as a synergist.
Application of trimethylamine oxide dihydrate as synergist of bacillus biopesticide.
The trimethylamine oxide dihydrate is used as a synergist in the control of plant diseases by bacillus biological pesticides.
The application of trimethylamine oxide dihydrate as a synergist for promoting the growth of bacillus.
The application of trimethylamine oxide dihydrate as a synergist for promoting the improvement of the control effect of bacillus.
The application of trimethylamine oxide dihydrate as a synergist for promoting the formation of bacillus biofilms.
The Bacillus compound biopesticide is characterized by containing trimethylamine oxide dihydrate and Bacillus, wherein the Bacillus is Bacillus subtilis, Bacillus belief, Bacillus amyloliquefaciens, Bacillus cereus or other biocontrol bacteria in Bacillus (Bacillus).
Furthermore, the addition ratio m/m of the trimethylamine oxide dihydrate in the bacillus biological agent is 0.1-10%, and the bacillus contains 1-1000 hundred million spores/g.
The trimethylamine oxide dihydrate is soluble in water, can be directly added into various bacillus formulations without changing the existing preparation process as a biological pesticide additive, and can also be directly added into prepared working solution.
The toxicity of the trimethylamine oxide dihydrate to the bacillus is extremely low, and the growth of the bacillus is not adversely affected.
The addition ratio of the trimethylamine oxide dihydrate in the biological agents of the bacillus is 0.1-10% (m/m), the biocontrol effect of the biological pesticides of the bacillus can be obviously improved, and the biological activity is not inhibited.
The addition of trimethylamine oxide dihydrate in a certain proportion can prolong the lasting period of the bacillus biological agent.
The addition ratio of the trimethylamine oxide dihydrate in the biological agent of the bacillus is less than 0.1 percent (m/m) and more than 10 percent (m/m), so that the biological control effect of the biological pesticide of the bacillus is also improved.
The concentration of the trimethylamine oxide dihydrate in the working solution of the bacillus biological agent is 1-100 mg/l, so that the biocontrol effect of the bacillus biological pesticide is obviously improved, and the activity of bacteria is not inhibited.
The concentration of the trimethylamine oxide dihydrate in the working solution of the bacillus biopesticide is less than 1 mg/L and more than 100mg/L, so that the biocontrol effect of the bacillus biopesticide is improved.
The invention innovatively utilizes trimethylamine oxide dihydrate as a synergist of bacillus biopesticide, and the trimethylamine oxide dihydrate can enhance the stress resistance of the bacillus bactericide and promote the colonization in the environment so as to improve the stability and the prevention effect of the bactericide.
Drawings
FIG. 1 shows the effect of trimethylamine oxide dihydrate on the fermentation rate of Bacillus subtilis;
FIG. 2 is a graph showing the effect of trimethylamine oxide dihydrate on the colonization ability of Bacillus subtilis;
FIG. 3 is a graph showing the effect of trimethylamine oxide dihydrate on the formation of Bacillus subtilis biofilms.
Detailed Description
The present invention will be further described with reference to the following specific examples to better understand the technical solution of the present invention.
Example 1: synergistic effect of trimethylamine oxide dihydrate on prevention and treatment of rice sheath blight of bacillus subtilis
1. The test conditions are as follows: the test site is in the test field of the Chinese Rice institute, and the test crop is rice (Xiushui 134). The culture conditions were uniform in all test cells.
2. And (3) experimental design: the experiment designed 7 processes in total, each process set 3 replicates, each replicate cell area being 22 square meters.
(1) The dosage of the bacillus subtilis H15810 hundred million spores per gram WP is 100 g/mu;
(2) 5 percent of trimethylamine oxide dihydrate WP dosage is 100 g/mu;
(3) the using amount of bacillus subtilis H15810 hundred million spores/gram and 0.1 percent of trimethylamine oxide dihydrate WP is 100 grams/mu;
(4) the using amount of the bacillus subtilis H15810 hundred million spores per gram and 1 percent of trimethylamine oxide dehydrate WP is 100 grams per mu;
(5) the using amount of bacillus subtilis H15810 hundred million spores/gram and 5 percent trimethylamine oxide dihydrate WP is 100 grams/mu;
(6) the using amount of bacillus subtilis H15810 hundred million spores/gram and 10 percent trimethylamine oxide dihydrate WP is 100 grams/mu;
(7) and (5) clear water control.
3. Administration and investigation
The pesticide is applied at the tillering stage respectively, the water consumption is 40 kg/mu, and the pesticide is sprayed on the middle and lower parts of the stalks and the leaf sheaths of the rice by adopting an electric sprayer in a coarse mist mode. The national standard GB/T17980.20-2000 bactericide for preventing and treating the rice sheath blight disease is adopted, the occurrence condition of the rice sheath blight disease is investigated 10 days and 20 days after the pesticide is applied, and the prevention effect is counted.
4. Test results
The test results are shown in table 1, and the effect of improving the control effect of the bacillus subtilis is obvious after 0.1-10% of trimethylamine oxide dihydrate is added no matter 10 days or 20 days after the application. The trimethylamine oxide dihydrate hardly has any effect on rice sheath blight, which shows that the trimethylamine oxide dihydrate plays a role of a synergist.
From the synergistic effect, the synergistic effect is 8.37-27.55% after 10 days, and is more obvious after 20 days, and reaches 17.11-37.41%.
Under the condition of no existence of trimethylamine dioxide dihydrate 20 days after the application, the control effect of the bacillus subtilis is rapidly reduced to 40.9%, and although the control effect is reduced to a certain degree in each treatment with the trimethylamine dioxide dihydrate, the reduction range is obviously lower, which shows that the trimethylamine dioxide dihydrate can prolong the lasting period of the bacillus subtilis.
TABLE 1 synergistic Effect of trimethylamine oxide dihydrate on Bacillus subtilis for controlling rice sheath blight
Example 2: synergistic effect of trimethylamine oxide dihydrate on preventing and treating rice blast of bacillus
1. The test conditions are as follows: the test site is developed in the Lin' an rice blast test base of the Chinese Rice research institute, and the test crop is rice (Nanjing 46). The culture conditions were uniform in all test cells.
2. And (3) experimental design: the experiment designed 10 treatments in total, each treatment setting 3 replicates, each replicate cell area 25 square meters.
(1) 5 percent of trimethylamine oxide dihydrate WP dosage is 100 g/mu;
(2) the dosage of the bacillus subtilis H15810 hundred million spores per gram WP is 100 g/mu;
(3) the using amount of the bacillus subtilis H15810 hundred million spores/gram and 5 percent of trimethylamine oxide dehydrate WP is 100 grams/mu;
(4) the dosage of the Bacillus belgii W168210 billion spores/gram WP is 100 g/mu;
(5) the dosage of the Bacillus beleisi W168210 hundred million spores/g and 5 percent trimethylamine oxide dihydrate WP is 100 g/mu;
(6) the dosage of the bacillus cereus is 100 g/mu per gram of WP in 20 hundred million spores;
(7) the dosage of the bacillus cereus is 20 hundred million spores/gram and 5 percent of trimethylamine oxide dihydrate WP is 100 grams/mu;
(8) the dosage of bacillus amyloliquefaciens B102100 hundred million spores per gram WP is 100 g/mu;
(9) bacillus amyloliquefaciens B102100 hundred million spores/gram and 5 percent of trimethylamine oxide dihydrate WP dosage of 100 grams/mu;
(10) and (5) clear water control.
3. Administration and investigation
The pesticide is applied once in the crevasse period and the spike aligning period respectively, and the water consumption is 40 kilograms per mu, and the pesticide is sprayed by an electric sprayer in a fine mist mode. The national standard GB/T17980.19-2000 bactericide for preventing and treating leaf diseases of rice is adopted, the occurrence condition of neck blast is investigated when the diseases are stable, and the prevention effect is counted.
4. The test results are shown in Table 2.
TABLE 2 synergistic effect of trimethylamine oxide dihydrate on Bacillus subtilis for preventing and controlling rice blast
The test results are shown in table 2, and the control effect of the bacillus on rice blast is obviously improved by adding 5% of trimethylamine oxide dihydrate, wherein the control effect of the bacillus on bacillus beleisis W1682 is most obviously enhanced and reaches 27.20%. The synergistic effect on other three kinds of bacillus, namely bacillus subtilis, bacillus cereus and bacillus amyloliquefaciens, respectively reaches 11.54 percent, 22.06 percent and 18.38 percent, and the result shows that the trimethylamine oxide dihydrate shows excellent synergistic effect on the disease prevention effect of various bacillus.
Example 3: synergistic effect of trimethylamine oxide dihydrate on bacillus for preventing and treating rice false smut
1. The test conditions are as follows: the test site is developed in the Fuyang test base of the Chinese Rice research institute, and the tested crop is a rice indica-japonica hybrid rice variety (Yongyou 12). The culture conditions were uniform in all test cells.
2. And (3) experimental design: the experiment designed 10 treatments in total, each treatment setting 3 replicates, each replicate cell area 25 square meters.
(1) The dosage of Bacillus beleisi W168210 hundred million spores/gram WP is 100 g/mu;
(2) oxidizing trimethylamine dihydrate to the working concentration of 100 mg/L;
(3) the Bacillus belgii W168210 million spores/gram dosage is 100 g/mu, and after being diluted into working solution, trimethylamine oxide dihydrate is added to 1 mg/L;
(4) the Bacillus belgii W168210 million spores/gram dosage is 100 g/mu, and after being diluted into working solution, trimethylamine oxide dihydrate is added to 10 mg/L;
(5) the Bacillus belgii W168210 million spores/gram dosage is 100 g/mu, and after being diluted into working solution, trimethylamine oxide dihydrate is added to 100 mg/L;
(6) and (5) clear water control.
3. Application and investigation
Applying the pesticide once respectively 5-7 days before the crevasse period and the full spike period, and spraying the pesticide by adopting an electric sprayer and fine mist according to the water consumption of 40 kilograms per mu. The national standard NYT 1464.54-2014 pesticide field efficacy test criterion part 54 is adopted: the bactericide is used for preventing and treating false smut of rice, investigating the occurrence condition of the false smut when the disease is stable, and counting the prevention effect.
4. The test results are shown in Table 3.
TABLE 3 synergistic effect of trimethylamine oxide dihydrate on Bacillus beilesensis for prevention and treatment of Ustilaginoidea virens
The test results are shown in Table 3, and the addition of 1-100 mg/L trimethylamine oxide dihydrate to the working solution can improve the control effect of Bacillus belgii W1682 by 9.67-18.87%. The result shows that the trimethylamine oxide dihydrate can be directly added into the working solution of the bacillus, and the control effect can be obviously improved.
Example 4: influence of trimethylamine oxide dihydrate on fermentation status of bacillus
NB medium was prepared in 50mL portions per 250mL Erlenmeyer flask (Corning), and trimethylamine oxide dihydrate was added to NB medium to prepare gradients of 0, 0.2, 2, 20, 200 and 400mgL -13 bottles were prepared for each concentration. Each flask was inoculated with 50. mu.L of 50NTU of H158 seed solution. After incubation at 28 ℃ for 12h at 200rpm, turbidity was measured with a nephelometer (2100N, USA Hash). The relative turbidity value was calculated by the following formula.
The results are shown in fig. 1, and it can be seen that the concentration of trimethylamine oxide dihydrate is increased to some extent compared with the turbidity of the control at five concentrations of 0.2, 2, 20, 200 and 400 mg/L, which indicates that the trimethylamine oxide dihydrate has a certain growth promoting effect on bacillus subtilis. Although this growth promoting effect is not significant, it can be at least shown that trimethylamine oxide dihydrate does not exhibit toxicity to Bacillus subtilis even at a high concentration of 400 mg/L.
Example 5: influence of trimethylamine oxide dihydrate on colonization ability of bacillus subtilis
The H158 strain concentration was adjusted to 1X 108cfu/mL, adding trimethylamine oxide dihydrate to final concentration of 100mg/L, and spraying on the surface of rice plant (Yongyou 12 tillering final stage) without adding waterAdding trimethylamine oxide dihydrate to directly spray the bacterial suspension as a control. Leaf specimens were collected at 0 (natural evaporation of the sprayed liquid 2h, etc.) 1, 3, 6, 10, 15 and 21d after application, respectively. Extracting DNA, developing qPCR according to methods published by J Broadsergnsen and Leser, synthesizing probes and primers by Shanghai Boshang biology company, adopting GoTaq Probe qPCR Master Mix by quantitative PCR kit Promega and 7500 system by quantitative PCR platform, calculating the amount of Bacillus subtilis (cfu g) contained in unit mass of leaves by CT value-1）。
As a result, as shown in FIG. 2, the amount of Bacillus subtilis H158 strain remaining was greatly reduced in the first day after spraying regardless of the addition of trimethylamine oxide dihydrate. In the first 3 days, whether the addition of trimethylamine oxide dihydrate is not large in the retention amount on the surfaces of the bacillus subtilis rice plants. However, the retention amount of the bacillus subtilis strain is greatly higher than that of the control by adding the trimethylamine oxide dihydrate from the sixth day, and even the retention amount of the bacillus subtilis strain treated by adding the trimethylamine oxide dihydrate by 21 days is more than thousand times that of the bacillus subtilis strain without the control. The results show that trimethylamine oxide dihydrate can promote the colonization of the bacillus subtilis strain on the surface of rice, and the method is a main mechanism for greatly improving the control effect of bacillus and prolonging the lasting period of bacillus.
Example 6: effect of trimethylamine oxide dihydrate on formation of Bacillus subtilis biofilm
Adding trimethylamine oxide dihydrate into MSGG culture medium to final concentration of 0, 50 and 100mg/L, inoculating H158 to NB culture medium, culturing at 37 ℃ to logarithmic phase, diluting to 50NTU, inoculating MSGG culture medium containing trimethylamine oxide dihydrate with different concentration according to the proportion of 1%, adding 96-hole culture plate (200 mu L per hole for quantitative research), and culturing in dark at 28 ℃. Biofilm formation on MSGG medium was observed at 24, 36, 48, 72, 96 and 120h of culture, respectively, and quantitative studies were performed on the formed biofilms according to the following methods: removing culture medium by suction with a row gun, adding 200 μ L of 0.1% crystal violet solution (Shanghai leaf organism) for staining for 20min, removing crystal violet by suction, washing with 200 μ L of PBS buffer solution twice, decolorizing with 200 μ L of anhydrous ethanol for 20min, measuring absorbance at 581nm with a TECAN microplate reader, and quantifying the biofilm according to the absorbance.
The result of the effect of trimethylamine oxide dihydrate on the formation of the bacillus subtilis biofilm is shown in fig. 3, and the effect of the presence or absence of the trimethylamine oxide dihydrate on the formation of the bacillus subtilis biofilm is little in 24 hours; at 36h and 48h, trimethylamine oxide dihydrate with different concentrations shows a promoting effect on the formation of a bacillus subtilis biofilm; the effect of trimethylamine oxide dihydrate at 72h and 96h on biofilm formation was not evident. From the results, trimethylamine oxide dihydrate can promote the earlier biofilm formation of Bacillus subtilis. As can be seen from FIG. 2, the reduction in bacterial load is most pronounced at the beginning of the administration of Bacillus. The biological membrane is an important structure of the bacillus for resisting external adverse factors, and the earlier the stable and mature biological membrane structure is formed, the more beneficial the colonization of the bacillus in the environment is, and the biological control effect is stably exerted. This may be the main mechanism of synergy of trimethylamine oxide dihydrate with bacillus.
Claims (7)
1. Application of trimethylamine oxide dihydrate as synergist of bacillus biopesticide.
2. The trimethylamine oxide dihydrate is used as a synergist in the control of plant diseases by bacillus biological pesticides.
3. The application of trimethylamine oxide dihydrate as a synergist for promoting the growth of bacillus.
4. The application of trimethylamine oxide dihydrate as a synergist for promoting the improvement of the control effect of bacillus.
5. The trimethylamine oxide dihydrate is used as a synergist for promoting the formation of the bacillus biofilm.
6. The use according to claim 1, wherein the trimethylamine oxide dihydrate is added to the bacillus biopharmaceutical in a ratio m/m of 0.1% to 10%.
7. The use of claim 1, wherein the concentration of trimethylamine oxide dihydrate in the bacillus-type biological agent working solution is 1-100 mg/l.
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