Whole-Genome Re-Alignment Facilitates Development of Specific Molecular Markers for Races 1 and 4 of Xanthomonas campestris pv. campestris, the Cause of Black Rot Disease in Brassica oleracea.

Mehede Hassan Rubel,Ill-Sup Nou,Arif Hasan Khan Robin,Hoy-Taek Kim,Sathishkumar Natarajan,Joana G Vicente,Jong-In Park

doi:10.3390/ijms18122523

Mehede Hassan Rubel, Ill-Sup Nou + Show 5 more

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https://doi.org/10.3390/ijms18122523

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Abstract

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is a seed borne disease of Brassicaceae. Eleven pathogenic races have been identified based on the phenotype interaction pattern of differential brassica cultivars inoculated with different strains. Race 1 and 4 are the two most frequent races found in Brassica oleracea crops. In this study, a PCR molecular diagnostic tool was developed for the identification of Xcc races 1 and 4 of this pathogen. Whole genomic sequences of races 1, 3, 4 and 9 and sequences of three other Xanthomonas pathovars/species (X. campestris pv. incanae (Xci), X. campestris pv. raphani (Xcr) and X. euvesicatoria (Xev) were aligned to identify variable regions among races. To develop specific markers for races 1 and 4, primers were developed from a region where sequences were dissimilar in other races. Sequence-characterized amplified regions (SCAR) and insertion or deletion of bases (InDel) were used to develop each specific set of primers. The specificity of the selected primers was confirmed by PCR tests using genomic DNA of seven different Xcc races, two strains of X. campestris pathovars and other species of bacteria. Bacterial samples of the races 1 and 4 isolates were collected from artificially inoculated cabbage leaves to conduct bio-PCR. Bio-PCR successfully detected the two Xcc isolates. By using our race-specific markers, a potential race 1 strain from the existing Korean Xcc collection was identified. The Xcc race 1 and 4-specific markers developed in this study are novel and can potentially be used for rapid detection of Xcc races through PCR.

Highlights

A seed-borne disease caused by Xanthomonas campestris pv. campestris (Xcc), is one of the most important diseases of plants of the Brassicaceae family [1]
No race specific markers have been reported for detecting any of the Xcc races
We tested this method in cabbage leaves inoculated with seven different races. This approach was found very effective and accurate. These results suggested that the Polymerase Chain Reaction (PCR)-based technique can be used directly to detect and identify Xcc races 1 and 4 pathogens in infected cabbage leaf samples without isolating the bacteria from the infected leaves

Summary

Introduction

A seed-borne disease caused by Xanthomonas campestris pv. campestris (Xcc), is one of the most important diseases of plants of the Brassicaceae family [1]. Black rot is a global problem which can reduce more than fifty percent yield under severe attacks in favorable conditions [2]. This pathogen is found all over the world, showing diversity in different countries and provinces of the same country [3,4,5,6]. The pathogen enters the vascular tissues mainly through the hydathodes, and through wounded tissues and stomata and is able to develop disease symptoms systemically This disease is favored by warm, humid conditions and can spread rapidly by rain dispersal and irrigation water [8]. Crop debris and cruciferous weeds are potential inoculum sources of this disease

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