PhoR/PhoP two component regulatory system affects biocontrol capability of Bacillus subtilis NCD-2

Qinggang Guo,Shezeng Li,Xiuyun Lu,Ping Ma,Baoqing Li

doi:10.1590/s1415-47572010005000032

Abstract

The Bacillus subtilis strain NCD-2 is an important biocontrol agent against cotton verticillium wilt and cotton sore shin in the field, which are caused by Verticillium dahliae Kleb and Rhizoctonia solani Kuhn, respectively. A mutant of strain NCD-2, designated M216, with decreased antagonism to V. dahliae and R. solani, was selected by mini-Tn10 mutagenesis and in vitro virulence screening. The inserted gene in the mutant was cloned and identified as the phoR gene, which encodes a sensor kinase in the PhoP/PhoR two-component system. Compared to the wild-type strain, the APase activities of the mutant was decreased significantly when cultured in low phosphate medium, but no obvious difference was observed when cultured in high phosphate medium. The mutant also grew more slowly on organic phosphate agar and lost its phosphatidylcholine-solubilizing ability. The suppression of cotton seedling damping-off in vivo and colonization of the rhizosphere of cotton also decreased in the mutant strain when compared with the wild type strain. All of these characteristics could be partially restored by complementation of the phoR gene in the M216 mutant.

Highlights

Cotton is one of the major crops in China as well as in the world
In order to clone the functional gene associated with antifungal activity from the NCD-2 strain, a transposon mutant library of B. subtilis NCD-2 was constructed with mini-Tn10, whereby more than 4000 CmrEms colonies were selected and screened for antibiosis activities against V. dahliae mycelial growth
In B. subtilis, more than 30 genes were classified as belonging to the pho regulon and being controlled by the PhoR/PhoP system (Martin, 2004), and by genome-wide transcriptional analysis increasingly more genes were identified as belonging to the pho regulon (Allenby et al, 2005), to our knowledge, no antibiotics have been identified as being controlled by this two-component system in B. subtilis

Summary

Introduction

Verticillium wilt, caused by Verticillium dahliae Kleb is a devastating disease in cotton, causing heavy economic losses globally (Tjamos et al, 2000). This disease is difficult to control due to the absence of resistant varieties, the long viability of the resting structures, and the inability of fungicides to inhibit the pathogen once inside the xylem (Fradin and Thomma, 2006). Research on alternative management strategies for cotton verticillium wilt control is important. One alternative is the use of antagonistic microorganisms which can suppress soil-borne pathogens in the rhizosphere of cotton (Weller et al, 2002; Tjamos et al, 2004). The mechanisms by which B. subtilis reduces plant diseases include antagonism of fungal pathogens by competing for niche and nutriments (Handelsman and Stabb, 1996), by producing fungitoxic compounds (Toure et al, 2004; Leclere et al, 2005), and stimulating the defensive capacities of the host plant (Ongena et al, 2004)

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Results

Conclusion