Fengycin produced by Bacillus subtilis NCD-2 is involved in suppression of clubroot on Chinese cabbage

Highlights

• Bacillus subtilis strain NCD-2 reduced clubroot disease severity by up to 50%.

• Fengycin contributed to cell lysis of P. brassicae resting spore.

• Fengycin defective mutant ΔfenC reduced suppression of clubroot.

• Fengycin production of B. subtilis NCD-2 is essential for clubroot control.

Abstract

Clubroot, caused by the obligate parasite Plasmodiophora brassicae, is one of the most important diseases of Brassicaceae family. Bacillus spp. that produce lipopeptide antibiotics are often used as biocontrol agents to suppress soilborne diseases. B. subtilis NCD-2 isolated from the cotton rhizosphere has been found to inhibit pathogenic fungi through the production of fengycin-type cyclopeptides. In this study, the biocontrol efficacy of B. subtilis NCD-2 against P. brassicae and the role of fengycin in disease suppression were evaluated on Chinese cabbage in glasshouse. B. subtilis NCD-2 reduced clubroot disease incidence and severity up to 47.7% and 51.6%, respectively, while the fengycin defective mutant ΔfenC and a PhoP/PhoR two-component regulatory system mutant, ΔphoP, were unable to prevent this disease. In vitro, P. brassicae resting spore cells were lysed when treated with standard fengycin and crude fengycin extracts of the wild type B. subtilis NCD-2, but not of its mutant ΔfenC. Moreover, the population dynamics of B. subtilis and P. brassicae was determined using quantitative real-time PCR (qPCR). The results were surprising that the wild type B. subtilis NCD-2 and its mutant ΔfenC colonized at similar rates in the rhizosphere, while levels of P. brassicae DNA in the rhizosphere and root tissue were reduced following B. subtilis NCD-2 treatment, but there was no effect of the mutant ΔfenC. Thus, the production of fengycins in B. subtilis NCD-2 may play a promising role in the suppression of P. brassicae in the rhizosphere and reduction of incidence of clubroot disease in Chinese cabbage.

Introduction

The obligate parasite, Plasmodiophora brassicae causes clubroot disease in Brassicaceae crops that results in 10–15% reductions in yield worldwide (Dixon, 2009). China is the leading producer of Chinese cabbage and it is cultivated annually on 2.67 million ha area of southwest, northeast and central China (Shen et al., 2009, Yang et al., 2011, Chai et al., 2014, Zhang et al., 2017). Numerous fields were reported for the increased in P. brassicae infection in recent years and the estimated yield losses were 20%–30% (Wang et al., 2011, Chai et al., 2014). Strategies for the management of clubroot in Brassicaceae crops include development of resistant cultivars (Diederichsen et al., 2009, Rahman et al., 2014), crop rotation (Wallenhammar, 1996), enhanced soil alkalinity (Bélec et al., 2004), and chemical treatment (Donald and Porter, 2009), along with biological control as a sustainable approach (Cheah et al., 2000, Peng et al., 2011). Use of biocontrol approach is more attractive because it reduces pressure chemical dosages and may provide durable protection if it can colonize on roots (Dixon, 2014, Peng et al., 2014).

The use of Bacillus strains as biopesticides has been favored, because their endospores tolerate adverse environmental conditions and are easily stored and transported in a marketable formulation (Tiago et al., 2004, Horikoshi, 2008). A number of Bacillus isolates have been identified as potential biocontrol agents against clubroot diseases (Chai et al., 2014). Bacillus subtilis QST713 (Senerade) is effective against canola clubroot under controlled environmental conditions in Canada (Peng et al., 2011). Accordingly, several Bacillus strains in China, including B. subtilis XF-1, B. subtilis Y10, B. amyloliquefaciens BS2004, B. pumilus YN201305, and B. subtilis YN201310, are effective against clubroot on Chinese cabbage (Xiong et al., 2009, Zhou et al., 2012, Wang et al., 2014, Du et al., 2015). Although, the mechanisms of Bacillus agents in clubroot control have not been fully determined, antibiosis is one of the most studied phenomena (Tao et al., 2011, Li et al., 2013). Bacillus spp. produce a variety of amphiphilic membrane-active biosurfactants and peptide antibiotics with potent antimicrobial activities, particularly the surfactin, iturin, and fengycin families of lipopeptide (Cawoy et al., 2015). Fengycin-type cyclopeptides are effective suppressors of crop diseases caused by pathogenic fungi and oomycetes, such as Fusarium graminearum, Monilinia laxa and M. fructicola, Verticillium dahliae, Rhizoctonia solani and Pythium aphanidermatum (Chan et al., 2009, Yánez-Mendizábal et al., 2012, Guo et al., 2014, Zouari et al., 2016). Fengycin-type cyclopeptides extracted from B. subtilis XF-1 have been shown to directly cleave resting spores in vitro, indicating a potential function of fengycin compounds of Bacillus-based suppression of clubroot disease (Li et al., 2013).

B. subtilis NCD-2 is a biocontrol agent commercially used in China against cotton Verticillium wilt (Guo et al., 2014). This bacterium produces at least four lipopeptides (surfactin, fengycin, bacillaene, and subtilosin); among these, fengycin is the major antifungal compound against Rhizoctonia solani and suppressing cotton damping-off disease (Guo et al., 2014). In this study, we tested the efficacy of B. subtilis NCD-2 and its fengycin-deficient mutants to control clubroot caused by P. brassicae to strengthen the role of fengycin in biocontrol of this economically important pathogen.