Preparation And Application Of Microbial Agent For Repairing Continuous Cropping Obstacles Of Coptis Chinensis

BackgroundCoptis chinensis is a well-known medicinal plant.However,the continuous cropping obstacles that appeared during the planting process severely restricted the industrial development of the Coptis chinensis.Continuous cropping will cause serious soil-borne diseases,which will Inhibit the growth of crops and decrease yields.Allelochemicals secreted by Coptis chinensis itself are one of the important reasons for continuous cropping obstacles.Its accumulation in the soil year by year will change the structure of soil microbial community and inhibit the growth and development of Coptis chinensis.Therefore,finding a method to efficiently degrade allelochemicals to control continuous cropping obstacles has aroused widespread concern.Traditional physical or chemical control measures will have harmful effects on the ecosystem,which will lead to deterioration of soil properties,threats of water pollution,and high fragility and instability of crop production systems.It will further increase the burden of agricultural production.In order to alleviate the negative impact,biological control technology,with its high efficiency,low toxicity and environmental friendliness,is emerging as a necessity to maintain ecological and economic balance and sustainable development.The research of development and application in microbial agents at home and abroad is showing a rapid development trend.With the endless emergence of new formulations,the continuous innovation of related supporting production equipment and the wide expansion of the actual application area,the development of microbial agents in China has entered a new era.ObjectiveThis research aims to screen phenolic acid degrading bacteria from the rhizosphere soil of Coptis chinensis,Analyzing its biocontrol functions and optimizing the fermentation process.According to the relevant national standards of microbial agent,a composite microbial agent is prepared to promote the high-quality and high-quantity production of Coptis chinensis.Creating a new solution to alleviate the continuous cropping obstacles of Coptis chinensis.Method1.Separating the microorganisms in the rhizosphere soil of Coptis chinensis by different separation medium.Screening strains with phenolic acid degradation ability and carrying out physiological and biochemical index detection and molecular biological identification.Then,the degradation performance of the strain on various phenolic acids was detected by HPLC.2.In order to find out whether the phenolic acid-degrading bacteria have other growth-promoting effects.The Salkowski colorimetric method was used to determine the ability of the strains to secrete IAA.The molybdenum-antimony colorimetric method was used to determine its phosphorus dissolving effect.The competitive culture assay was used to test its ability to inhibit plant pathogenic bacteria and fungi.3.In order to explore the optimal fermentation process conditions for phenolic acid degrading bacteria.Based on the single factor experiments of culture medium,inoculation amount,solid-liquid ratio,fermentation time and p H,response surface analysis was used to optimize the fermentation conditions.4.Determining the antagonistic effect between strains by the inhibition zone method,and select the most suitable strain ratio based on the magnitude of the antagonistic effect.In order to improve the stability of the microbial agent.Screening the type and amount of carrier,wetting agent,dispersant and protectant by comparing the cost,suspension rate,wetting time,biocompatibility and other indicators.5.Measuring various indicators of wettable powder in accordance with relevant national standards,and testing the degradation effect of microbial agents on phenolic acid by simulating the actual soil environment.6.The continuous cropping soil of Coptis chinensis was used as the test material,and different concentrations of microbial agents were set for field trials.By measuring the growth of Coptis chinensis seedlings and the degradation of phenolic acid in soil,the actual application effect of the microbial agent in the continuous cropping process of Coptis chinensis was verified.Result1.Ten strains of phenolic acid degrading bacteria were screened from the soil of Coptis chinensis.Among them,one of the actinomycetes with higher ferulic acid and vanillic acid degradation efficiency was identified as S.griseoloalbus 3132.S.griseoloalbus 3132 and the phenolic acid degrading bacteria S.olivochromogenes A035 and the biocontrol bacteria Bacillus subtilis HL1 that have been isolated in the laboratory were tested for the degradation of ferulic acid and vanillic acid.The ferulic acid and vanillic acid can be completely degraded after the three strains are cultured for72 hours.And they also have a broad-spectrum degradation ability of cinnamic acid,p-coumaric acid,p-hydroxybenzoic acid,protocatechuic acid,benzoic acid and other phenolic acids.2.Strains can promote growth of host plants by dissolving calcium phosphate,synthesizing IAA,and inhibiting the growth of plant pathogenic bacteria.The test results showed that after S.olivochromogenes A035 was cultured on inorganic phosphorus solid medium for 7 days,a clear transparent circle could be observed.After7 days of cultivation in inorganic phosphorus liquid medium,the phosphorus content in the supernatant reached the maximum of 496μg/m L.When S.griseoloalbus 3132 was cultured to 18 days,the content of IAA in the medium reached a peak of 16.2μg/m L,and when S.olivochromogenes A035 was cultured to 15 days,the maximum IAA content was 10.8μg/m L.The fermentation broths of S.olivochromogenes A035 and Bacillus subtilis HL1 both have a certain inhibitory effect on the growth of Alternaria alternata.S.olivochromogenes A035 has an inhibitory rate of 21.6%.The inhibitory rate of Bacillus subtilis HL1 to Alternaria alternata reached 67.0%,and its inhibitory rate to Fusarium solani was 11.8%.3.Oats,soybean meal powder and soybean powder were selected as low-value raw materials for solid-state fermentation of S.olivochromogenes A035,S.griseoloalbus3132,and Bacillus subtilis HL1.The single factor experiment was used to select the solid-liquid ratio,inoculation amount and p H interval that were beneficial to fermentation,and the number of viable bacteria was used as the response value to conduct a response surface optimization experiment.The optimal solid-state fermentation parameters of S.olivochromogenes A035 were as follows:solid-liquid ratio was 1:1.5,the inoculation amount was 15%,and the p H was 7.0.After optimization,the number of viable bacteria reached 8.28×10⁸CFU/g,and the efficiency was increased by61.4%.The optimal solid-state fermentation parameters of S.griseoloalbus 3132 were as follows:solid-state ratio was 1:1.8,inoculation amount was 15%,p H was 7.4,the number of viable bacteria after optimization reached 3.08×10⁹CFU/g,and the efficiency was increased by 22.2%.The optimal solid-state fermentation parameters of Bacillus subtilis HL1 were as follows:solid-state ratio was 1:2.5,the inoculation amount was 10%,p H was 7.4,the number of viable bacteria after optimization reached1.65×10¹⁰CFU/g,and the efficiency increased by 19.5%.4.The above-mentioned three kinds of bacteria were screened for the most suitable strain ratio by antagonism test.The results showed that when the ratio of S.olivochromogenes A035 to S.griseoloalbus 3132 was 1:1,obvious bacteriostatic zone was not appeared.It was indicated that there was no antagonistic effect.When the ratio of S.olivochromogenes A035 to Bacillus subtilis HL1 was 1:1,an obvious inhibition zone appeared around the colony.It was indicated that there was an antagonistic effect between the strains.After adjusting the ratio to 1.5:1,the zone of inhibition disappeared.The most suitable strain ratio is A035:3132:HL1=1.5:1:1.By optimizing the carrier,protective agent and dispersant and other auxiliary agents,the best formula of the microbial agent was as follows:kaolin was used as the carrier to adsorb the bacterial agent fermentation filtrate to obtain the original microbial agent powder,the wetting agent SDBS accounts for 4.8%,and the dispersant STPP accounts for 3.2%.The protective agent sodium alginate accounts for 1%.5.The number of viable bacteria of the wettable powder can reach 2.55×10⁹CFU/g,the rate of miscellaneous bacteria was 0%,the p H was 7.3,the fineness was 98.1%,the drying loss was 2.5%,the wetting time was 48.0 s,and the suspension rate was 85.7%,which was in line with the country standard.The ferulic acid has been completely degraded after 20 days of microbial agent treatment in a simulated soil environment.6.The field test results in half a year showed that the 10-fold dilution of the compound microbial agent significantly promoted the root length,plant height,fresh weight and dry weight of Coptis chinensis seedlings,which increased by 34.9%,12.5%,26.2%,and 80.4%respectively compared with the control group.The content of various phenolic acids in the soil under different concentrations of microbial agents was tested.The results showed that the composite microbial agents could effectively degrade the phenolic acid substances accumulated in the soil.Among them,the degradation rate under the treatment of 10-fold dilution of the microbial agents could reach more than54.2%.ConclusionThe above results indicated that phenolic acid-degrading bacteria were widespread in the continuous cropping soil of Coptis chinensis.S.olivochromogenes A035,S.griseoloalbus 3132,and Bacillus subtilis HL1 have a broad-spectrum phenolic acid degradation function.In this study,the fermentation process of microbial agents was optimized.Through single factor and response surface optimization experiments,the best preparation method of microbial agents was obtained,and field experiments verified that it has a significant degradation effect on the phenolic acid in the continuous cropping soil of Coptis chinensis,so as to promote the growth of Coptis chinensis.In this study,multiple strains of phenolic acid-degrading bacteria were screened,and a composite microbial agent for repairing the continuous cropping obstacle of Coptis chinensis was prepared.Providing new ideas and methods for solving the continuous cropping obstacles of Coptis chinensis.