The type-III effectors-based multiplex PCR for detection of Xanthomonas campestris pv. campestris causing black rot disease in crucifer crops

The black rot disease in crucifer crops is caused by Xanthomonas campestris pv. campestris (Xcc) which drastically reduces the productivity of crops. Three Xcc races, such as races 1, 4, and 6, have been identified from India that possess nine avr genes, or type-III effectors (T3Es). Here, we used three T3Es—avrXccC, avrBs1, and avrGf1 to identify Xcc from bacterial DNA, bacterial suspensions, Xcc-infected seeds, and the sap of the infected leaves using multiplex PCR. The T3Es were amplified using gene-specific primers with gDNA of Xcc. Then, the multiplex PCR was optimized and amplified T3Es using the sap of black rot-infected cauliflower leaves. Further, this method amplified T3Es from artificially infected seeds (1–100%) of cauliflower and from Xcc colonies (0.1–100%) grown on nutrient agar medium. The primer specificity of T3E genes elucidates that these are specifically detected in all Indian Xcc strains and races, while no bands were observed with other unrelated bacteria, such as X. euvesicatoria, X. oryzae pv. oryzae, Pseudomonas fluorescens, Ralstonia solanacearum, Bacillus subtilis, and B. amyloliquefaciens. Further, this PCR possesses high sensitivity and amplifies T3E genes using up to 0.01 ng Xcc DNA. The high specificity and sensitivity of T3Es-based multiplex PCR make it a potential method and can be used to amplify Xcc from various templates, such as purified DNA, Xcc-infected seeds and leaves, crude extracts, etc., without the need to extract plant or bacterial DNA.