Optimization Of High-density Fermentation System And Formulation Development Of Bacillus Altitudinis ST15

Bacterial leaf streak caused by Xanthomonas oryzae pv.oryzicola,which usually causes 10%-20%yield reduction in rice,or more than 50%in severe cases.It can cause rice yield reduction or even extinction.Currently,the control of this disease mainly relies on chemical pesticides,while Bacillus spp.has the advantages of broad antimicrobial spectrum,strong resistance,low toxic side effects and environmental friendliness,which is a class of beneficial microorganisms with potential for application development.In the previous study,our laboratory screened and obtained a strain of Bacillus altitudinis ST15 with independent intellectual property rights,which has strong antagonistic activity against Xanthomonas oryzae.Based on the previous experiments,the medium composition and culture conditions were optimized by single-factor tests and orthogonal design test.The final formulation of ST15 high-density fermentation medium was obtained:yeast powder 6 g/L,fructose 10 g/L,soybean powder 3 g/L,MnSO₄0.20 g/L;and the optimal culture conditions:initial pH 8.0,temperature 26℃,filling volume 30 mL（with 250 mL conical flask）,inoculum volume 10%,and fermentation time 60 h.After optimization,the average viable bacteria count reached6.42×10⁹CFU/mL,the budding cell count reached 4.56×10⁹CFU/mL,and the budding cell conversion rate reached 68%.At the same time,the fermentation level of ST15 reached up to 1.07×10¹⁰CFU/mL and the budding cell conversion rate reached up to more than 76%through the 5L fermenter for scaling up the experiment.In addition,the molecular mechanism of ST15 cell production was initially explored by qRT-PCR.The relative expression of the genes spo0B,abr B,sig F and sig E related to bud cell formation was up-regulated at 12 h and 24 h with the addition of 0.2 g/L MnSO₄,indicating that Mn²⁺could promote ST15 bud cell formation.In this study,we used different dilutions of ST15 fermentation solution to irrigate rice at the tiller stage（variety Jia You Zhongke No.1）and found that ST15fermentation solution had certain pro-growth effects on root length,root weight and plant height of rice,especially at the dilution of 5 times,the effect was most significant.At the same time,the ST15 fermentation solution was sprayed on the rice leaves,and the colonization of ST15 on the leaves was observed by scanning electron microscopy,and the test results showed that the strain ST15 could still colonize on the leaves in large quantities after 30 days.In addition,in this study,the optimal combination of ST15 suspension formulation:2.5%M-150,0.1%xanthan gum,1.0%nipagin methyl ester and 3.0%glycol was initially obtained through single-factor and orthogonal design tests to screen and combine p H,wetting and dispersing agents,thickening agents,preservatives and antifreeze agents,using the number of viable bacteria and suspension rate as measurement indexes.The combination has good biocompatibility,good dispersibility,good pouring,good indexes and qualified cold storage.This study can provide an important basis for the subsequent commercialization and development of ST15 strain,and also meets the requirements of pesticide formulations to develop in the direction of environmental protection and safety,and has good development prospects.