Fengycin produced by Bacillus subtilis NCD-2 is involved in suppression of clubroot on 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.
Section snippets
Bacterial cultures and pathogen inoculum
Bacterial strains and plasmids are enlisted in Table 1. Bacillus strains NCD-2 along with its two fengycin-deficient mutants ΔfenC and ΔphoP were used in this study. Former mutant is unable to produce fengycin due to deletion of biosynthetic gene fenC (Guo et al., 2014), while the later mutant produced lower level of fengycin than the wild type because its regulatory gene phoP of the two-component system PhoP/PhoR was disrupted (Dong et al., 2014).
Bacillus stock cultures from −80 °C freezer
Effect of fengycin production of B. subtilis NCD-2 on disease severity
Prior to treatment of P. brassicae-infected soil with Bacillus, we observed highest disease severity index (90.4) and incidence (97.8). Following the application of the wild type strain B. subtilis NCD-2, DSI and incidence reduced to 43.7 and 51.1, respectively (Fig. 1AB). However, application of mutants ΔfenC or ΔphoP had no significant effect on disease severity and disease incidence (Fig. 1AB), although the roots in soil treated with mutant ΔfenC were less severely galled (DSI 82.2). Galls
Discussion
In this study, we provided genetic evidence of the speculation that the fengycin of B. subtilis NCD-2 was involved in suppression of clubroot caused by P. brassicae on Chinese cabbage under greenhouse conditions. Fengycin extract from the wild type NCD-2, but not from mutant ΔfenC, caused resting spore cell lysis, which is consistent with previous in vitro research that fengycin-type cyclopeptides extracted from B. subtilis XF-1 directly cleave resting spores (Li et al., 2013). Such parallel
Declaration of Competing Interest
The authors declare there is no conflicts of interest regarding the publication of this paper.
Acknowledgments
This work was supported by grants from Special Fund for Agro-scientific Research in the Public Interest (201503109, 201003092), National Key Research and Development Program (2017YFD0201106), and the National Natural Science Foundation of China (31572045, 31872020).
References (55)
- et al.
The PhoR/PhoP two-component system regulates fengycin production in Bacillus subtilis NCD-2 under low-phosphate conditions
J. Integr. Agric.
(2018) - et al.
Fengycin produced by Bacillus subtilis NCD-2 plays a major role in biocontrol of cotton seedling damping-off disease
Microbiol. Res.
(2014) - et al.
Bacillus lipopeptides: versatile weapons for plant disease biocontrol
Trends Microbiol.
(2008) - et al.
Effects of root exudates and pH on Plasmodiophora brassicae resting spore germination and infection of canola (Brassica napus L.) root hairs
Crop Prot.
(2013) - et al.
Biocontrol activity of the endophytic Bacillus amyloliquefaciens strain CEIZ-11 against Pythium aphanidermatum and purification of its bioactive compounds
Biol. Control
(2016) - et al.
Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production
Plant Physiol.
(2004) - et al.
Liming and calcium cyanamid for clubroot control in cauliflower
Acta Hortic.
(2004) - et al.
Peptidoglycan metabolism is controlled by the WalRK (YycFG) and PhoPR two-component systems in phosphate-limited Bacillus subtilis cells
Mol. Microbiol.
(2010) - et al.
Molecular detection of Plasmodiophora brassicae, causal agent of clubroot of crucifers, in plant and soil
Plant Dis.
(2007) - et al.
A technique for the extraction and purification of viable Plasmodiophora brassicae resting spores from host root tissue
Mycologia
(1994)
Lipopeptides as main ingredients for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens
Microb. Biotechnol.
Research status of clubroot (Plasmodiophora brassicae) on cruciferous crops in China
Can. J. Plant Pathol.
Identification of lipopeptide antibiotics of a Bacillus subtilis isolate and their control of Fusarium graminearum diseases in maize and wheat
Biocontrol
Biological control of clubroot on cauliflower with Trichoderma and Streptomyces spp.
New Zealand Plant Prot.
Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects
Appl. Environ. Microbiol.
Status and perspectives of clubroot resistance breeding in crucifer crops
J. Plant Growth Regul.
Clubroot (Plasmodiophora brassicae Woronin) – an agricultural and biological challenge worldwide
Can. J. Plant Pathol.
The occurrence and economic impact of Plasmodiophora brassicae and clubroot disease
J. Plant Growth Regul.
Integrated control of clubroot
J. Plant Growth Regul.
Regulation of fengycin biosynthase by regulator PhoP in the Bacillus subtilis strain NCD-2
Acta Phytopathol. Sin.
Colonization of GFP-tagged endophytic Bacillus subtilis Y10 in Chinese cabbage
Chinese J. Ecol.
Specific polymerase chain reaction primers for the detection of Plasmodiophora brassicae in soil and water
Phytopathology
PhoR/PhoP two component regulatory system affects biocontrol capability of Bacillus subtilis NCD-2
Genet. Mol. Biol.
Past, present and future of extremophiles
Extremophiles
The signal-transduction network for Pho regulation in Bacillus subtilis
Mol. Microbiol.
Evidence that the biofungicide Serenade (Bacillus subtilis) suppresses clubroot on canola via antibiosis and induced host resistance
Phytopathology
Mechanisms of the biofungicide Serenade (Bacillus subtilis QST713) in suppressing clubroot
Biocontrol Sci. Tech.
Cited by (25)
Surfactin and fengycin contribute differentially to the biological activity of Bacillus subtilis NCD-2 against cotton verticillium wilt
2022, Biological ControlCitation Excerpt :Fengycin has strong antifungal activity, especially against filamentous fungi (Kulimushi et al., 2017). A previous study showed that fengycin was a major component in strain NCD-2 acting against the growth of Botrytis cinerea, Rhizoctonia solani and Plasmodiophora brassicae (Guo et al., 2014; Guo et al., 2019; Dong et al., 2013). In this study, the antifungal activities of lipopeptides from the wild-type strain NCD-2 and mutant strains were evaluated against the growth of V. dahliae.
Induced biosynthesis of fengycin and surfactin in a strain of Bacillus amyloliquefaciens with oomyceticidal activity on zoospores of Phytophthora capsici
2022, Revista Argentina de MicrobiologiaQuantification of Pseudomonas protegens FD6 and Bacillus subtilis NCD-2 in soil and the wheat rhizosphere and suppression of root pathogenic Rhizoctonia solani AG-8
2021, Biological ControlCitation Excerpt :Culture-dependent quantification of B. subtilis NCD-2 CFUs in soil were determined by suspending 0.5 g of soil in 50 ml of SDD water, mixed (1 h at 120 rpm), serially diluted (1:10 in SDD) and 100 µl aliquots of each dilution spread onto 10 replicate TSA plates. NCD-2 CFUs from wheat roots were determined according to the method of Guo et al. (2019), with the exception that 0.1 g of soil-free wheat roots were processed in 10 ml SDD water and serial dilutions were plated on TSA. Soil and root CFUs with the same phenotype as strain NCD-2 were counted after 8–10 h incubation at 50 °C.
Biocontrol arsenals of bacterial endophyte: An imminent triumph against clubroot disease
2020, Microbiological ResearchCitation Excerpt :Gao and Xu (2014) combined the heat treatment with application of BCA cocktail (B. megaterium, Clostridium tyrobutyricum, and Saccharomyces cerevisiae) which reduced the clubroot infection upto 91.7 %. Guo et al. (2019) assessed the aptitude of B. subtilis NCD-2 isolated from cotton rhizosphere to control clubroot in Chinese cabbage, under greenhouse condition disease severity index and incidence reduced to 43.7 and 53.5 as compared to untreated 90.4 and 97.8, respectively. The strain also inhibited resting spore germination in vitro, this commendable antagonistic effect against pathogen was regarded to fengycin production as fengycin deficient mutant had declined control effect and lowered inhibition of spore germination.
Lipopeptides from Bacillus: unveiling biotechnological prospects—sources, properties, and diverse applications
2024, Brazilian Journal of Microbiology