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.
Highlights
Fusarium wilt of banana (FWB) caused by Fusarium oxysporum f. sp. cubense, especially Tropical Race 4 (TR4), is one of the most destructive diseases affecting the crop (Ghag et al, 2015; Presti et al, 2015; Carvalhais et al, 2019)
Colonies glowed an obvious red color under the fluorescence microscope, and the red E. coli cells could still be visualized even after being cultured in the liquid. This verified that red fluorescent protein (RFP)-labeled cells can be observed under the fluorescence microscope (Figure 2A), indicating that the plasmid pYP69 can be expressed in E. coli
The results showed that the growth of RFP-YN0904, RFP-YN1282-2, RFP-WBN06, and RFP-N67 was consistent to WT strains, indicating that the plasmid pYP69 had no significant effect on the growth of these three B. velezensis
Summary
Fusarium wilt of banana (FWB) caused by Fusarium oxysporum f. sp. cubense, especially Tropical Race 4 (TR4), is one of the most destructive diseases affecting the crop (Ghag et al, 2015; Presti et al, 2015; Carvalhais et al, 2019). Due to its characteristic of surviving in the soil for decades, once the pathogen is introduced into the soil, the infected banana orchard cannot be used for growing susceptible banana cultivars, which seriously affects the sustainable development of the banana industry, as there are few proven TR4-resistant cultivars (Ploetz, 2006, 2015). Crop rotation and intercropping have been used to reduce the infections and inoculum levels (Nadarajah et al, 2016). Few TR4-resistant banana cultivars have been bred and popularized (Daniells, 2011). Even those cultivars still have to adapt to local cultivation practices and conditions. The spread of TR4 has led to an increase in research on biological control and biocontrol agents (BCAs) in suppressing the pathogen (Nadarajah et al, 2016; Bubici et al, 2019; Damodaran et al, 2020)
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