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

• Published in: Frontiers in microbiology Vol. 12; p. 754918
• Main Authors: He, Ping, Li, Shu, Xu, Shengtao, Fan, Huacai, Wang, Yongfen, Zhou, Wei, Fu, Gang, Han, Guangyu, Wang, Yun-Yue, Zheng, Si-Jun
• Format: Journal Article
• Language: English
• Published: Frontiers Media S.A 13-10-2021
• Subjects: Bacillus interaction with TR4; Bacillus spp; biocontrol; electro-transformation; Microbiology; RFP-labeled Bacillus
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2021.754918

Bacillus
spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by
Bacillus subtilis
,
Bacillus velezensis
, and
Bacillus amyloliquefaciens
of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one
B. subtilis
strain, three
B. velezensis
strains, and three
B. amyloliquefaciens
strains that are proven to significantly inhibit TR4
in vitro
, 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
600
of bacteria concentration=0.7, plasmid concentration=50ng/μl, plasmid volume=2μl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory
Bacillus
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.
In vivo
colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that
Bacillus
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
B. subtilis
,
B. velezensis
, and
B. amyloliquefaciens
can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.