Screening of Bacillus thuringiensis strains to identify new potential biocontrol agents against Sclerotinia sclerotiorum and Plutella xylostella in Brassica campestris L.

Highlights

• A pool of one hundred and sixty-one B. thuringiensis strains was screened.

• Six strains could protect B. campestris L. from S. sclerotiorum and P. xylostella.

• Bt triggered ISR by simultaneously activating SA, JA, and ET signaling pathway.

• Multifunctionality of Bt promote its use in controlling of pests and diseases.

Abstract

Sclerotiniose, caused by Sclerotinia sclerotiorum (Lib.) deBary, is a destructive disease of Brassica campestris L. Injuries caused by insect pests, such as Plutella xylostella (L.), increase the occurrence of this disease. One hundred and sixty-one Bacillus thuringiensis strains from the Bacillus Genetic Stock Center were screened to identify strains that might protect B. campestris from both plant diseases and insect pests in this study. Challenge-inoculation assays showed seventeen B. thuringiensis strains effectively suppressed S. sclerotiorum growth by inducing systemic resistance in B. campestris, and six of these strains exhibited high insecticidal activity against P. xylostella. In addition, B. thuringiensis elicited a strong hypersensitive response in B. campestris leaves and triggered systemic signals that were transferred from treated roots and leaves to untreated leaves. Quantitative real-time PCR and transcriptome sequencing showed genes involved in salicylic acid, ethylene, and jasmonic acid signaling and in brassinosteroid synthesis pathways were upregulated. The phenotypic and genotypic diversity of B. thuringiensis makes it an effective biocontrol agent for simultaneously protecting B. campestris from sclerotiniose and P. xylostella.

Introduction

Bacillus thuringiensis (Bt), belonging to the Bacillus cereus sensu lato group, is a well-known entomopathogenic bacterium that produces different insecticidal proteins, such as crystal proteins during the sporulation phase of growth and vegetative insecticidal proteins during the vegetative phase. Due to its excellent insecticidal activity against lepidopteran (Xue et al., 2008), coleopteran (Bi et al., 2015), and dipteran (Zhang et al., 2013) pests, Bt has been the most widely used biopesticide worldwide (Jouzani et al., 2017, Schnepf et al., 1998, Wang et al., 2018b). Pan-genome analysis showed that neither the plasmid nor the chromosome of Bt was conserved (Wang et al., 2017b). Due to the high ecological, phenotypic, and genotypic diversity of Bt, several of its characteristics have gradually been identified, including its capacities to promote plant growth (Cherif-Silini et al., 2016, Ortiz et al., 2015), synthesize metal nanoparticles (Najitha Banu et al., 2014), and degrade persistent chemical pesticides (Ferreira et al., 2016). Some Bt strains were reported to produce chitinase (Shrestha et al., 2015), bacteriocins (Jeong et al., 2016), fengycin (Kim et al., 2004), and volatile compounds (Zheng et al., 2013) that directly antagonize plant pathogens. Furthermore, the Bt serovars fukuokaensis B88-82 and sotto RG1-6 suppress bacterial wilt disease in tomato by induced systemic resistance (ISR) against Ralstonia solanacearum (Hyakumachi et al., 2013, Takahashi et al., 2014).

Plants and microorganisms have formed mutually beneficial relationships via long-term evolutionary processes. Most plant-associated Bacillus strains are plant growth-promoting rhizobacteria that provide new possibilities for sustainable agriculture. Moreover, they provide plants with resistance to a vast array of fungal and bacterial phytopathogens through several mechanisms. Some Bacillus strains compete for nutrients to prevent the pathogen from growing rapidly (Garbaye, 1991, Lawrence, 1989), while others produce effective antagonistic compounds such as volatiles (Chaurasia et al., 2005, Shanshan et al., 2018) and lipopeptides (Cawoy et al., 2015, Falardeau et al., 2013) that inhibit pathogen growth. Moreover, some Bacillus strains induce systemic resistance to protect plants from pathogenic infections (Choudhary and Johri, 2009). Salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play crucial roles in plant defense (Robert-Seilaniantz et al., 2007). Induced resistance to pathogens in plants includes systemic acquired resistance (SAR) and ISR (Vallad and Goodman, 2004). The SA-dependent signaling pathway is involved in SAR, while the JA and ET-dependent signaling pathways are involved in ISR (Glazebrook, 2001, Pieterse et al., 1998). Bacillus cereus AR156 and Bacillus amyloliquefaciens SQR9 induce systemic resistance against Pseudomonas syringae pv. tomato DC3000 via SA- and JA/ET-dependent signaling pathways (Niu et al., 2011, Wu et al., 2018a). The Bt serovar fukuokaensis B88-82 induces systemic resistance against R. solanacearum in tomato plants via the co-activation of the SA- and ET-dependent signaling pathways and suppression of the JA-dependent signaling pathway (Takahashi et al., 2014).

Brassica campestris L. is mainly distributed in the western high-altitude region of China, occupying a cultivation area of about 200,000 ha (Du et al., 2018). Sclerotinia sclerotiorum (Lib.) deBary is the causal agent of sclerotiniose, one of the three main diseases of Brassica species. This soil-borne plant fugal pathogen causes plant stem rot in fields and several of its virulence-related factors have been identified, including oxalic acid (Cessna et al., 2000), secreted protein SsSSVP1 (Lyu et al., 2016), and transcription factor SsFKH1 (Fan et al., 2017). Benzimidazole and dicarboximide were used to control sclerotiniose in China (Zhang et al., 2012), but S. sclerotiorum populations developed resistance to these fungicides. Moreover, feeding injuries caused by lepidopteran larvae, such as diamondback moth Plutella xylostella (L.), which is a major foliar pest of Brassica spp. vegetable crops, increase the infection frequency of S. sclerotiorum (Dillard and Cobb, 1995). Therefore, a method that could simultaneously control P. xylostella and S. sclerotiorum could improve the control efficiency of sclerotiniose. The present study aimed to find candidate Bt strains that could simultaneously protect B. campestris from the disease caused by S. sclerotiorum by ISR, and control lepidopteran insect pests, such as P. xylostella. We screened 161 Bt strains from the Bacillus Genetic Stock Center (BGSC) collection to determine their ISR activity and insecticidal activity against P. xylostella. The signaling pathway involved in Bt-mediated ISR was analyzed by transcriptome sequencing, and expression patterns of SA-responsive WRKY70 gene and the JA/ET-responsive HEL and PDF1.2 gene were determined by quantitative real-time PCR.