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Occurrence and characterization of a new red-pigmented variant of Curtobacterium flaccumfaciens, the causal agent of bacterial wilt of edible dry beans in Iran

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Abstract

A number of crop diseases are emerging at an alarming rate worldwide. Bacterial wilt of dry beans, caused by Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), is one of them. In Iran, this disease was first reported in 2012, which, since then, has rapidly spread across the major dry bean growing areas of the country causing severe yield losses. Previously, only two colony variants (yellow and orange) of the pathogen had been described from Iran in association with bacterial wilt of dry beans. In this study, we describe a new red-pigmented variant of Cff, isolated from dry bean seeds stored in seed banks of Khomein Bean Research Station, the major seed supplier in the region. Because Cff is a quarantine pathogen in Iran and elsewhere, with a potential threat for dry bean productions, more knowledge about the biology of this pathogen and epidemiology of the disease it causes are a prerequisite for the development of effective disease management strategies. Within this framework, we performed phenotypic and genetic characterization of the red-pigmented variant of the pathogen, in comparison with previously isolated yellow and orange variants, including pathogenicity, host range, bacteriocin production and genetic diversity. Our results showed a similar host range of different Cff variants although they differed in their aggressiveness. Yellow and orange variants of the pathogen were more aggressive on cowpea and common bean, respectively while the red variant showed the same level of aggressiveness on both hosts. Orange- and red-pigmented strains were separated from yellow-pigmented strains in the phylogeny of gyrB sequences. All orange- or red-pigmented strains were clustered in a separate branch from yellow-pigmented strains, except strain CffK31, in phylogeny based on rpoB sequences. In BOX-PCR analysis, Cff strains used in this study were clustered in two distinct genetic groups, with yellow variants of the pathogen separated from the orange and red variants. Overall, our results provide evidence of a remarkable diversity of Cff in Iran, which needs further in-depth investigation.

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Acknowledgments

We thank the following institutions: Negin Bazre Pars co. in Khomein County, Markazi province for providing common bean seeds (cvs. Sadri, Derakhshan, Dorsa), Plant Improvement Institute (SPII) in Karaj, Alborz province for providing soybean seeds (cv. Katool), and Agricultural and Natural Resources Research Center of Safiabad, in Khuzestan province for supplying mung bean (cvs. L173 and Partow) and cowpea (cvs. L1057 and Mashhad) seeds. Financial support for this study was provided by Shiraz University.

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Authors and Affiliations

  1. Department of Plant Protection, College of Agriculture, Shiraz University, Shiraz, 71441-65186, Iran

    Ebrahim Osdaghi, S. Mohsen Taghavi, Habiballah Hamzehzarghani & Amal Fazliarab

  2. Panhandle Research & Extension Center, University of Nebraska, 4502 Ave. I., Scottsbluff, NE, 69361, USA

    Robert M. Harveson

  3. INRA, UAR 1240 Eco-Innov, BP 01, 78850, Thiverval-Grignon, France

    Jay Ram Lamichhane

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  1. Ebrahim Osdaghi
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Correspondence to Ebrahim Osdaghi.

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Table S1

Twenty plant-associated bacterial strains used in this study as bacteriocin indicator strains. The grey color scale shows the differences in sensitivity patterns among the indicator strains to the bacteriocins of yellow- (20Y), red- (50R), and orange- (80O) pigmented variants of Curtobacterium flaccumfaciens pv. flaccumfaciens. The sensitivity groups show the statistically different groups generated in Duncan grou** analysis (Fig. 2) (DOCX 14 kb)

Fig. S1

Production of bacteriocin by Curtobacterium flaccumfaciens pv. flaccumfaciens (called as producer strains) against different plant pathogenic bacteria (called as indicator strains). Clear zones appeared around the colonies of producer strains indicate inhibition after 3 days post incubation. a: strain 80O vs Pectobacterium carotovorum subsp. carotovorum, b: strain 20Y vs Pseudomonas fluorescens (CHAO), c: strain 80O vs Rhodococcus fascians, and d: strain 20Y vs Agrobacterium tumefaciens (JPG 757 kb)

Fig. S2

Agarose gel of PCR products obtained with the primer pairs CF4/CF5 (198 bp; a) and CffFOR2/CffREV4 (306 bp; b). Lanes: 1-20: bacterial strains as numbered in Table 1. 21: Xanthomonas axonopodis pv. phaseoli strain Araxa1, 22: 100 bp DNA Ladder. All the Curtobacterium flaccumfaciens strains were detected with the primer pairs, except strains CffG105 and Cmmeg20 which were isolated from tomato and eggplant, respectively (JPG 276 kb)

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Osdaghi, E., Taghavi, S.M., Hamzehzarghani, H. et al. Occurrence and characterization of a new red-pigmented variant of Curtobacterium flaccumfaciens, the causal agent of bacterial wilt of edible dry beans in Iran. Eur J Plant Pathol 146, 129–145 (2016). https://doi.org/10.1007/s10658-016-0900-3

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