DNA polymorphisms and biocontrol of Bacillus antagonistic to citrus bacterial canker with indication of the interference of phyllosphere biofilms.

Tzu-Pi Huang,Chun-Han Chen,Bing-Fang Hwang,Kuo-Ching Tzeng,Ya-Huei Lee,Wen-Di Huang,Amy C L Wong,Kuan-Min Lu,Dean Der-Syh Tzeng

doi:10.1371/journal.pone.0042124

Abstract

Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a devastating disease resulting in significant crop losses in various citrus cultivars worldwide. A biocontrol agent has not been recommended for this disease. To explore the potential of bacilli native to Taiwan to control this disease, Bacillus species with a broad spectrum of antagonistic activity against various phytopathogens were isolated from plant potting mixes, organic compost and the rhizosphere soil. Seven strains TKS1-1, OF3-16, SP4-17, HSP1, WG6-14, TLB7-7, and WP8-12 showing superior antagonistic activity were chosen for biopesticide development. The genetic identity based on 16S rDNA sequences indicated that all seven native strains were close relatives of the B. subtilis group and appeared to be discrete from the B. cereus group. DNA polymorphisms in strains WG6-14, SP4-17, TKS1-1, and WP8-12, as revealed by repetitive sequence-based PCR with the BOXA1R primers were similar to each other, but different from those of the respective Bacillus type strains. However, molecular typing of the strains using either tDNA-intergenic spacer regions or 16S–23S intergenic transcribed spacer regions was unable to differentiate the strains at the species level. Strains TKS1-1 and WG6-14 attenuated symptom development of citrus bacterial canker, which was found to be correlated with a reduction in colonization and biofilm formation by X. axonopodis pv. citri on leaf surfaces. The application of a Bacillus strain TKS1-1 endospore formulation to the leaf surfaces of citrus reduced the incidence of citrus bacterial canker and could prevent development of the disease.

Highlights

Bacillus species are natural inhabitants of the phyllosphere [1] and rhizosphere [2]
Bacillus strains with a broad spectrum of antagonistic activity against various phytopathogens including Pythium aphanidermatum, Rhizoctonia solani AG4, Xanthomonas axonopodis pv. vesicatoria XVT12 and X. axonopodis pv. citri XW19 were isolated from plant potting mixes, organic compost and soil samples collected from the field
Seven of the 326 strains tested (HSP1, TKS1-1, OF3-16, SP4-17, WG6-14, TLB7-7, and WP8-12) that showed superior antagonistic activity, along with one other strain (NT-2 isolated from natto, a Japanese fermented soybean product), were used in this study

Summary

Introduction

Bacillus species are natural inhabitants of the phyllosphere [1] and rhizosphere [2]. They form endospores and various strains are capable of producing enzymes, antibiotics, proteins, vitamins or secondary metabolites that exhibit the ability to promote growth or induce defense mechanisms in animals and plants [3]. Bacillus species are important candidates for microbial control agents for plant diseases and pests [2,4,5,6], protectants for seeds [7], and probiotics [8]. Identification and selection of ‘generally recognized as safe’ (GRAS) organisms prior to the intensive development process required for biocontrol agents is recommended

Methods

Results

Conclusion