Enhancement of antifungal activity of Burkholderia pyrrocinia JK‐SH007 genetically modified with Bacillus subtilis Chi113 gene

Abstract

Previous studies showed that Burkholderia pyrrocinia JK-SH007 is a potential biocontrol agent of poplar canker disease. In this report, B. pyrrocinia JK-SH007 was genetically modified by introducing the Bacillus subtilis Chi113 gene to enhance its antifungal activity. The green fluorescence of B. pyrrocinia JK-SH007E1 can be detected because of its transformation with plasmid pHKT2-Chi113 containing a gfp reporter gene. Real-time quantitative PCR (qPCR) showed that the B. subtilis Chi113 gene was highly expressed in B. pyrrocinia JK-SH007E1 at mRNA level. The chitinase activity and the antifungal activity of B. pyrrocinia JK-SH007E1 were significantly increased, and the clear halo was visible on the colloidal chitin agar plate. In addition, the constructed recombinant plasmid pHKT2-Chi113 was stably maintained for at least 100 generations in the absence of antibiotic selection in B. pyrrocinia JK-SH007E1. In western blot analysis, the Chi113-GFP fusion protein was detected in B. pyrrocinia JK-SH007E1 using the GFP polyclonal antibody. In conclusion, the B. subtilis Chi113 gene was successfully transformed into B. pyrrocinia JK-SH007 and was expressed both at mRNA level and protein level. Moreover, the antifungal activity of B. pyrrocinia JK-SH007 was improved through this genetic modification. 1 Amp r , ampicillin resistance; Tp r , trimethoprim resistance. Engineered B. pyrrocinia JK-SH007 633

Effects of genetically modified Burkholderia pyrrocinia JK‐SH007E1 on soil microbial community in poplar rhizosphere

“…Experts argue that the risk of genetically engineered microorganisms on environment can be reduced to a minimum as long as standard operating procedures developed for genetic engineering and the corresponding laws and regulations are strictly followed (Davison, ; Han et al., ). In previous work by this group, it was confirmed that B. pyrrocinia isolate JK‐SH007 was an effective biocontrol agent for poplar canker disease (Ren, Li, Liu, Ye, & Wu, ), and the engineered bacterium B. pyrrocinia JK‐SH007E1 with foreign chitinase gene was constructed (He et al., ). The risks posed by the genetically engineered isolate of B. pyrrocinia JK‐SH007E1 required evaluation prior to release into the natural environment.…”

“…In previous work by this group, it was confirmed that B. pyrrocinia isolate JK-SH007 was an effective biocontrol agent for poplar canker disease (Ren, Li, Liu, Ye, & Wu, 2016), and the engineered bacterium B. pyrrocinia JK-SH007E1 with foreign chitinase gene was constructed (He et al, 2016). The risks posed by the genetically engineered isolate of B. pyrrocinia JK-SH007E1 required evaluation prior to release into the natural environment.…”

“…Burkholderia pyrrocinia JK-SH007 (strain number: CCTCC M209028), in genomovar IX of the Burkholderia cepacia complex, was isolated from a poplar stem (Ren et al, 2011) and genetically engineered to produce strain JK-SH007E1 in previous work (He et al, 2016). Both the JK-SH007 and genetically engineered JK-SH007E1 strains were subcultured twice from frozen stock onto Luria Bertani agar (LB; 1% tryptone, 0.5% yeast extract, 0.15% sodium chloride, 2% agar, pH 7.0.).…”

“…Ren, Ye, Liu, Xu, and Wu () showed that a bacterium endophytic in poplar, Burkholderia pyrrocinia JK‐SH007, had growth‐promoting effects and decreased the incidence of cankers caused by C. chrysosperma , F. aesculi and P. macrospora , suggesting the potential of this bacterium as a biocontrol agent. Subsequently, to improve its biocontrol potential to poplar canker disease, He, Ye, Ren, Huang, and Wu () genetically modified JK‐SH007 by introducing the Bacillus subtilis Chi113 gene via vector pHKT2. The engineered bacterium JK‐SH007E1 has a higher antifungal activity than the wild‐type JK‐SH007.…”

Optimization of Constitutive Promoters Using a Promoter-Trapping Vector in Burkholderia pyrrocinia JK-SH007

“…Burkholderia pyrrocinia JK-SH007, belonging to Bcc, is a strain that excels in promoting plant growth, inhibiting fungal growth [13][14][15], and degrading natural and synthetic contaminants such as the production of siderophores to degrade Fe(III) to usable Fe(II) [14]. Accordingly, engineered bacteria with enhanced antagonistic fungal resistance [16] and insect resistance [17] have been obtained via genetic modification. These two engineered B. pyrrocinia JK-SH007derived strains that we constructed utilize an inducible promoter, namely, the λ phage transcriptional promoter (PRPL), to express the chitinase gene [16] and the cry218 gene, encoding the insecticidal crystal protein Cry1Ac [17].…”