Optimization Of Solid-state Fermentation Process And Development Of Biocontrol Agent From Streptomyces Microflavus AMYa-008

In this paper,an Actinomycete was firstly cultured in the laboratory.Plate colonies and strains cultivated in shaking flask were observed by microscopy an didentified by molecular biology.They were identified and named as Streptomyces microflavus AMYa-008.Subsequently,the antagonistic effect of plant disease bacteria was studied by using the plate confrontation method,and the results showed that Streptomyces microflavus AMYa-008 has a broad-spectrum inhibitory effect on eight common plant disease fungi.Based on the solid-state fermentation of Streptomyces microflavus AMYa-008 from cassava residue and other industrial,agricultural solid wastes,the single-factor and orthogonal test conditions were optimized,and the pilot scale-up experiment was finally conducted to determine Streptomyces microflavus AMYa-008's best solid-state fermentation medium formula and conditions,it includes the following components:cassava residue 89.85%,rice husk 10%,KH2PO4 0.05%,NaCl 0.05%,MgSO4 0.05%.The fermentation temperature is controlled at about 30?,the initial water content is around 50%,initial pH is 6.6-7,the material thickness is 5cm,the inoculation amount is 7.5%,the cultivation time is 4 days,and the spore yield is 4.8×10~9 cfu/g.The spore yield increased by 33.3%compared with the initial blank condition.The spore yield was 4.5 x 10~9 cfu/g after the pilot scale enlargement.Considering the cost of production,the spore yield was 4.2×10~9 cfu/g when the inoculation amount was 5%;in the pilot scale,the sporulation amount was 4.0×10~9 cfu/g.The survival rate of Streptomyces microflavus'spores is 75%,which compared with blank conditions increased by 30%after low temperature vacuum drying.Subsequently,the pot-grown biocontrol experiments were carried out using Streptomyces microflavus produced by solid-state fermentation.By means of the method of back-staining,the bio-accelerated effect on plants was verified,and the biocontrol potency and microbial community structure change analysis of the bacterial agent were performed.The results showed that the use of Streptomyces microflavus biological microbiological agents showed various degrees of biological control and promotion of growth in plant growth,among which B7 and D7 have the most significant effects.In the middle stage of plant growth,cucumber seedlings and wheat seedlings were increased by 33.68%and 7.73%in height compared with blank control,and respectively the degree of infection was light;at the end of plant growth,the lengths of the above ground and underground parts of cucumber seedlings,root diameter,leaf thickness,and above ground and underground dry weights were respectively increased by25.89%,62.15%,16.04%,22.29%,11.08%,and 55.56%.In addition to the root diameter,other indicators of wheat seedlings respectively increased by 6.83%,46.77%,5.16%,6.67%,and 25.95%compared with blank control.The above conclusions prove that the bio-control micro-ecological agent could effectively control the growth and reproduction of plant disease bacteria,and promote plant'growth and yield.Finally,the DGGE method was used to analyze the soil microbial community structure in potted plants with different levels of micro-ecological microbial agents and plant disease fungi.In both the cucumber and wheat pot experiments,the B7 and D7 experimental groups with a bacterial concentration of 3×10~7cfu/g achieved good biological control effects.In the cucumber pot test,compared with the control group B1 and experimental group B2 that only applied the pathogenic bacteria,the application of bacterial agents in the B7 experimental group respectively increased the diversity and richness of the bacterial community structure by 73.81%,167%and 12.31%,139.88%.The fungal community structure in the diversity and richness were lower than B1,B2 by 67.21%,66.6%,74.59%,180%.In the wheat experiment,the application of the bacterial agent in the D7 experimental group respectively increased the bacterial community structure by 9.62%,54.13%and 62.86%,84.86%in the diversity and richness compared with the control D1 and the disease-only D2 experimental group.The fungal flora structure in the diversity and richness were respectively reduced by 26.92%,60%and 58.46%,180%compared with D1 and D2.In summary,the addition of bio-control micro-ecological agent has affected the structure of soil microflora.In particular,it has shown a significant inhibitory effect on soil fungi.Among them,the biocontrol effect was the best when the number of viable cells was 3×10~7cfu/g.Within a certain range of microbial concentration,the biocides with high effective viable counts have better bio-accelerated effects.At the same time,it was further proved that the microbiological agent prepared by the solid-state fermentation of Streptomyces microflavus,which is based on cassava slag,rice husk and other industrial,agricultural solid wastes,could be used for the development of biocontrol micro-ecological fertilizers for plant disease fungi.It has a very broad application prospect in the field of plant disease control and other agricultural fields.In summary,this paper initially explored the solid fermentation process and spore preservation method of Streptomyces microflavus AMYa-008,which made it capable of sporulation and preservation for a longer time.This laid the foundation for the future development of Streptomyces microflavus into agricultural biocontrol agents.At the same time,the biocontrol micro-ecological probiotics developed can improve the plant's growth environment by changing the diversity and abundance of bacterial flora in the soil,and can prevent disease,protect seedlings,promote growth,and increase yield in agricultural production.As well as reducing the use of chemical fertilizers and other effects,it has broad prospects for development and application.