Controlling and Defence‐related Mechanisms of Bacillus Strains Against Bacterial Leaf Blight of Rice

Abstract

Bacillus strains are broadly studied for their beneficial role in plant growth and biological control of plant disease and pest; however, little is known about their underlying mechanisms. In this study, we assessed the controlling and defence-related mechanisms of three Bacillus strains including rice seed-associated strain B. subtilis A15, rhizobacterial strains B. amyloliquefaciens D29 and B. methylotrophicus H8, all of which are against bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae. Results indicated that all three strains showed strong biofilm formation ability. The culture filtrates of each strain significantly suppressed the growth and biofilm formation of X. oryzae, while changes in bacterial cell morphology such as cell swell and severe cell wall alterations were observed through the transmission electron microscopy images. PCR analysis revealed that all three strains harbour the antimicrobial-associated genes that are responsible for biosynthesis of bacillomycin, fengycin, iturin and surfactin. Subsequent real-time qPCR analysis revealed the upregulated expression of fenD and srfAA genes in D29 and H8, and fenD and ituC genes in A15 during their in vitro interaction with X. oryzae. It suggests that the antibacterial mechanisms of the three strains may be at least partially associated with their ability to secrete corresponding lipopeptides. Interestingly, the applications of the three strains in greenhouse conditions were found to be effective in controlling the BLB disease, which was achieved through the activation of inducing systemic resistance resulted from the enhanced activities of defence-related enzymes. This is the first report of demonstration of the mode of antibacterial effect of Bacillus strains against X. oryzae. Overall, data from the current study provide valuable information for biological control of BLB disease in rice.

Halotolerant bacteria belonging to operational group Bacillus amyloliquefaciens in biocontrol of the rice brown stripe pathogen Acidovorax oryzae

“…On the other hand, the strong in vitro inhibitory effect of halotolerant bacteria against this rice bacterial pathogen in this study supports that metabolites-associated mechanisms play important role in inhibition of Bacillus against phytopathogenic bacteria (Yoshida et al 2001;Ongena and Jacques 2008;Elshakh et al 2016;Zhao et al 2017).…”

“…; Elshakh et al . ). In addition, culture filtrates were able to destroy cell morphology even after 4 h of contact time.…”

“…This revealed the importance of iturin in the antagonistic activity of halotolerant bacteria against Ao. Furthermore, it has been reported that many Bacillus species successfully colonize the roots of plants and contribute to inhibit the wide range of phytopathogens concomitantly (Islam et al 2015;Elshakh et al 2016). In addition, culture filtrates were able to destroy cell morphology even after 4 h of contact time.…”

“…The culture filtrates of halotolerant bacteria were prepared by incubating the antagonists in LB broth at 30°C for 48 h, and adjusting the OD 600 to 1·0 (about 10 8 CFU per ml) and then filtrating through 0·22‐ μ m filters twice, while bacterial contamination was excluded by incubating 100 μ l of culture filtrate on LB agar for 24 h. The inhibitory activity of culture filtrates (v/v; 50%) on the growth of Ao strains RS‐1 and RS‐2 were determined according to the method of Elshakh et al . () which was carried out using a sterilized polystyrene flat‐bottom 96‐well plate (Corning‐Costar Corp., Corning, NY) by measuring optical density at 600 nm (OD 600 ) using a SPECTRAmax ® PLUS384 Microplate Spectrophotometer (Molecular Devices Corporation). The effect of culture filtrates on motility of Ao strains RS‐1 and RS‐2 was determined as described by Masum et al .…”

“…After centrifugation, washing and dissolving in sterile water, the concentration of rice pathogenic bacteria was adjusted to OD 600 = 0Á6 (approximately~1 9 10 8 CFU per ml). The culture filtrates of halotolerant bacteria were prepared by incubating the antagonists in LB broth at 30°C for 48 h, and adjusting the OD 600 to 1Á0 (about 10 8 CFU per ml) and then filtrating through 0Á22-lm filters twice, while bacterial contamination was excluded by incubating 100 ll of culture filtrate on LB agar for 24 h. The inhibitory activity of culture filtrates (v/v; 50%) on the growth of Ao strains RS-1 and RS-2 were determined according to the method of Elshakh et al (2016) which was carried out using a sterilized polystyrene flat-bottom 96-well plate (Corning-Costar Corp., Corning, NY) by measuring optical density at 600 nm (OD 600 ) using a SPECTRAmax â PLUS384 Microplate Spectrophotometer (Molecular Devices Corporation). The effect of culture filtrates on motility of Ao strains RS-1 and RS-2 was determined as described by Masum et al (2017), which was performed by spotting pathogenic bacteria on the centre of each swimming LB plate (0Á3% agar) supplemented with culture filtrate of antagonistic bacteria and then measuring the colony diameter after 48 h of incubation at 30°C.…”