AUTHOR=He Ping , Li Shu , Xu Shengtao , Fan Huacai , Wang Yongfen , Zhou Wei , Fu Gang , Han Guangyu , Wang Yun-Yue , Zheng Si-Jun 

TITLE=Monitoring Tritrophic Biocontrol Interactions Between Bacillus spp., Fusarium oxysporum f. sp. cubense, Tropical Race 4, and Banana Plants in vivo Based on Fluorescent Transformation System

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.754918

DOI=10.3389/fmicb.2021.754918

ISSN=1664-302X

ABSTRACT=<p><italic>Bacillus</italic> spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by <italic>Bacillus subtilis</italic>, <italic>Bacillus velezensis</italic>, and <italic>Bacillus amyloliquefaciens</italic> of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one <italic>B. subtilis</italic> strain, three <italic>B. velezensis</italic> strains, and three <italic>B. amyloliquefaciens</italic> strains that are proven to significantly inhibit TR4 <italic>in vitro</italic>, optimized the genetic transformation conditions and explored their colonization process in banana plants. The results showed that we successfully constructed an optimized fluorescent electro-transformation system (OD<sub>600</sub> of bacteria concentration=0.7, plasmid concentration=50ng/μl, plasmid volume=2μl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory <italic>Bacillus</italic> spp. strains. The red fluorescent protein (RFP)-labeled strains were shown to have high stability with a plasmid-retention frequency above 98%, where bacterial growth rates and TR4 inhibition are unaffected by fluorescent plasmid insertion. <italic>In vivo</italic> colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that <italic>Bacillus</italic> spp. can colonize the internal cells of banana plantlets roots. Further, fluorescent observation by LSCM showed these RFP-labeled bacteria exhibit chemotaxis (chemotaxis ratio was 1.85±0.04) toward green fluorescent protein (GFP)-labeled TR4 hyphae in banana plants. We conclude that <italic>B. subtilis</italic>, <italic>B. velezensis</italic>, and <italic>B. amyloliquefaciens</italic> can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.</p>