Abstract

This study aimed to improve the screening method for the selection of Bacillus biocontrol agents against crown gall disease. The relationship between the strain biocontrol ability and their in vitro studied traits was investigated to identify the most important factors to be considered for the selection of effective biocontrol agents. In fact, previous selection procedure relying only on in vitro antibacterial activity was shown to be not suitable in some cases. A direct plant-protection strategy was performed to screen the 32 Bacillus biocontrol agent candidates. Moreover, potential in vitro biocontrol traits were investigated including biofilm formation, motility, hemolytic activity, detection of lipopeptide biosynthetic genes (sfp, ituC and bmyB) and production of antibacterial compounds. The obtained results indicated high correlations of the efficiency of the biocontrol with the reduction of gall weight (p = 0.000) and the antibacterial activity in vitro (p = 0.000). Moreover, there was strong correlations of the efficiency of the biocontrol (p = 0.004) and the reduction in gall weight (p = 0.000) with the presence of the bmyB gene. This gene directs the synthesis of the lipopeptide bacillomycin belonging to the iturinic family of lipopeptides. These results were also confirmed by the two-way hierarchical cluster analysis and the correspondence analysis showing the relatedness of these four variables. According to the obtained results a new screening procedure of Bacillus biocontrol agents against crown gall disease could be advanced consisting on two step selection procedure. The first consists on selecting strains with high antibacterial activity in vitro or those harbouring the bmyB gene. Further selection has to be performed on tomato plants in vivo. Moreover, based on the results of the biocontrol assay, five potent strains exhibiting high biocontrol abilities were selected. They were identified as Bacillus subtilis or Bacillus amyloliquefaciens. These strains were found to produce either surfactin or surfactin and iturin lipopeptides. In conclusion, our study presented a new and effective method to evaluate the biocontrol ability of antagonistic Bacillus strains against crown gall disease that could increase the efficiency of screening method of biocontrol agents. Besides, the selected strains could be used as novel biocontrol agents against pathogenic Agrobacterium tumefaciens strains.

Highlights

  • Agrobacterium tumefaciens is the causal agent of the crown gall, one of the most economically important diseases of crops (Kennedy and Alcorn, 1980)

  • All isolates were subjected to antibacterial activity testing against A. tumefaciens strain C58

  • Thirty strains were found to form haloes of Agrobacterium cell lysis around the antagonist growth zone indicating the production of antibacterial compounds

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Summary

Introduction

Agrobacterium tumefaciens is the causal agent of the crown gall, one of the most economically important diseases of crops (Kennedy and Alcorn, 1980). In nurseries, affected plants must be culled and discarded This disease is difficult to control by the use of chemical pesticides and can spread to other plants . Two commercially biocontrol agents were developed to control crown gall disease These were Agrobacterium rhizogenes strain K84 and its derivative K1026 producing the agrocin 84, an antibiotic like product, with specific antibacterial activity toward Agrobacterium strains. These biocontrol agents failed to control tumour development on grapes and some other economically important plants (Otten et al, 2008). Bacillus strains were reported to be efficient in reducing gall formation on grapevine and on tomato plants (Eastwell et al, 2006; Gupta and Khosla, 2007; Rhouma et al, 2008; Hammami et al, 2009; Ben Abdallah et al, 2015; Frikha-Gargouri et al, 2017)

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