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

Abstract

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 (OD600 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.

A smooth vetch (Vicia villosa var.) strain endogenous to the broad-spectrum antagonist Bacillus siamensis JSZ06 alleviates banana wilt disease

“…From August to October 2023, the effectiveness of the fermentation solution of the antagonistic strain against banana wilt was assessed at 70% relative humidity and a standard room temperature of 28°C in a controlled environment. He et al (2021) contributed the overexpression procedure of the fluorescent green protein gene Foc TR4 (GFP-Foc TR4). One seedling of histocultured 'Baxi' banana (AAA) from the seedling bag was transplanted into a plastic pot (5 cm × 8 cm) containing 2 kg of sterile soil (Fan et al, 2021).…”

Antifungal Potential of Bacillus subtilis subsp. inaquosorum RLS76 for Management of Fusarium Wilt Disease of Bt- Cotton

“…Some rhizobacteria exhibit plant growth-promoting (PGPR) and biocontrol properties, making them promising candidates for managing plant diseases. Researchers identified different microorganisms such as Bacillus sp., 6,7 Pseudomonas sp., [8][9][10] Serratia sp., 8,11 and Trichoderma sp., [12][13][14][15] which would be potential candidates for management of plant diseases instead of chemical fungicides.…”

Banana disease-suppressive soil drives Bacillus assembled to defense Fusarium wilt of banana

“…At present, all the banana main producing areas have suffered from this disease in China (Li , 2019). The banana industry has been seriously threatened by this pathogen, and it severely restricts banana production in China (He et al, 2021;Li et al, 2021). As a perennial crop irrigated with large amounts of water and fertilizer, the reduction of organic matter and acidification of the soil has gradually become serious, which changed the soil microbial diversity (Wang et al, 2015;Fu et al, 2016;Shen et al, 2019).…”