Species-specific PCR primers for detection of Xanthomonas vesicatoria

Abstract

This study presents the development of a PCR method for specific detection of Xanthomonas vesicatoria, causal agent of bacterial spot of tomato and pepper. Primer pair XV1F and XV1R was designed based on partial DNA sequence of the catalytic subunit of the ATP synthase gene atpD and tested using a collection of various bacterial pathogens mainly for tomato and pepper. After optimization of the PCR conditions, the 365 bp long fragment was only produced in X. vesicatoria samples. Therefore, the primers and protocol allow rapid and species-specific detection of X. vesicatoria.

Introduction

Gram-negative bacterium Xanthomonas vesicatoria (ex Doidge, 1920) Vauterin et al. is the causal agent of bacterial spot of tomato (Lycopersicon esculentum Mill.) and pepper (Capsicum annuum L.). The disease causes considerable losses in productivity and fruit quality especially in areas of warmer and humid climate (Jones et al., 1998; Al-Dahmani et al., 2003). There are four phenotypically and genotypically distinct Xanthomonas species causing the bacterial spot of tomato and pepper – Xanthomonas euvesicatoria Jones et al., 2006 (group A, which contains most of the strains previously known as Xanthomonas campestris pv. vesicatoria or Xanthomonas axonopodis pv. vesicatoria), X. vesicatoria (group B), Xanthomonas perforans Jones et al., 2006 (group C) and Xanthomonas gardneri (ex Sutic, 1957) Jones et al., 2006 (group D). While strains belonging to groups A, B and D are pathogenic on tomato and pepper, group C strains are pathogenic on tomato only. Also geographic distribution varies among the groups. Groups A and B are distributed worldwide, whereas distribution (and resulting economical importance) of groups C and D seems to be limited (Jones et al., 2004).

Considering the economical losses of tomato and pepper production caused by X. vesicatoria and limited options of the pathogen suppression in later stages of the disease, rapid and accurate detection of the pathogen is vital for the tomato and pepper production (Al-Dahmani et al., 2003). Biochemical tests, serological assays, metabolic profiling, immunological techniques and flow cytometry are all used for identification of the pathogen. These methods are however laborious, time-consuming and results are sometimes inaccurate (Park et al., 2009). Although some PCR primer sequences for detection of the bacterial spot on tomato and pepper have been published (Leite et al., 1994; Obradovic et al., 2004; Moretti et al., 2009; Park et al., 2009), none is capable of accurate X. vesicatoria (group B) detection. In this study we present sequences of a new primer pair for specific detection of X. vesicatoria (group B) designed from partial DNA sequences of the catalytic subunit of the ATP synthase (atpD) gene. The atpD gene is a well characterized housekeeping gene with high degree of sequence conservation (Christensen and Olsen, 1998) which makes it suitable for design of specific primers (Tankouo-Sandjong et al., 2008; Anbazhagan et al., 2010). Also this gene is frequently used for multilocus sequence analysis (MLSA) –(Fargier et al., 2011).