High-throughput Screening Of Antagonistic Bacteria Against Soft Rot Pathogens Based On Droplet Microfluidic

As one of the destructive diseases of crops,soft rot causes huge losses to agricultural production and economy every year,and has become one of the important factors restricting the sustainable development of agriculture.In the early stage of the laboratory,the pathogen of soft rot,Erwinia carotovora Ecc15,was isolated from susceptible konjac plants.In this study,antagonistic microorganisms with antibacterial activity were screened from soil communities.Traditional antagonistic bacteria isolation techniques usually have the characteristics of high labor intensity,high cost and low efficiency.Droplet microfluidic technology has developed into a useful tool for high throughput experiments in various research fields,including microbiology,due to its high accuracy,high throughput and low cost.Therefore,in our study,we applied droplet microfluidic technology to the screening of Ecc15 antagonists based on the Droplet Entrapping Microfluidic Cell-sorter（DREM Cell）,a droplet microfluidic platform.The main contents include:evaluation of DREM Cell culture performance and exploration of microbial resources,construction of a high throughput screening system for Ecc15 antagonistic bacteria based on microdroplets,application of the high throughput screening system,and mutation breeding of isolated antagonistic bacteria.The results were as follows:（1）Using CESE culture medium to cultivate soil microorganisms,the cultural performance of DREM Cell was evaluated by comparing the diversity of soil microorganisms cultured in microdroplet culture and agar plate culture.The results of PCA,α-diversity index and sample rarefaction curve showed that the bacteria diversity obtained by droplet culture was higher compared with the traditional agar plate culture method,and the bacterial composition of the two culture methods was significantly different.OTU analysis results found that more than 95%of the OTUs in the droplet samples belonged to the rare biosphere,and the relative abundance of these rare microorganisms in the original soil samples was less than 0.01%.Our results indicated that droplet culture has great potential in revealing the rare biosphere in complex microbial communities,and that droplet culture is a potential tool for recovering highly diverse microbial species from environmental samples.（2）The GFP-Ecc15 as the reporter strain,Bacillus velezensis and E.coli as antagonistic and non-antagonistic bacteria,respectively,the fluorescence signals of the two bacteria co-culturing with the reporter strain were verified in a microwell plate.The results showed that the fluorescence values between the two bacteria differed by 10～20 times.In the droplet microfluidic screening model,the oil pressure/water pressure for stable droplet formation was 320/360 mbar,the droplet diameter was 30μm,the rate was 2,400 Hz/s,and the concentration of bacterial solution of antagonistic and non-antagonistic bacteria was OD₆₀₀=0.03 and OD₆₀₀=0.01,respectively.The incubation time of droplet was 4～6 h,and the concentration of GFP-Ecc15 picoinjection was OD₆₀₀=0.4.The high-throughput screening system was tested and repeated for three times.The average enrichment rate of antagonistic bacteria reached 148 times.The results showed that the droplet microfluidic platform can enrich bacteria with inhibitory effects on Ecc15.（3）The high-throughput screening system was applied to soil microbial community.After a round of screening,microorganisms with inhibitory effect on Ecc15 were enriched from 3×10⁶droplet libraries,and 32 bacteria strains with antibacterial activity were obtained.All antagonistic bacteria were identified as 3phyla,6 families and 9 genera,among which D-62 strain had the strongest antibacterial activity.Thirteen strains of antagonistic bacteria were isolated and screened by agar plate,all of which belonged to Bacillus spp.Compared with the traditional agar plate screening system,the screening efficiency of droplet microfluidic platform was increased by about 3×10³times,and the reagent consumption of culture medium was reduced by nearly 1.2×10⁷times.（4）Strain D-62 was identified as Bacillus safensis by multigene sequencing.The mutant with high antibacterial activity was screened from the mutant library of B.safensis D-62 by combining atmospheric and room temperature plasma（ARTP）mutagenesis technology and droplet microfluidic high-throughput screening technology.The results indicated that the mutant R1-15 with significantly enhanced antibacterial activity was obtained from the 1×10⁵cell library,and the antibacterial activity of the aseptic fermentation cultured in a deep-well plate against Ecc15 was62%higher than that of the wild-type strain.The shake flask re-screening results showed that the diameter of the inhibition zone of the R1-15 mutant against Ecc15reached 26.15±0.29 mm,which was 42.82%higher than the original strain（18.31±0.64 mm）.After ten generations of subculture,the mutant R1-15 showed good genetic stability.