Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR

Abstract

Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale.

Signal Integration in Quorum Sensing Enables Cross-Species Induction of Virulence in Pectobacterium wasabiae

“…Different pathogen species are often isolated from diseased tubers within the same affected crops (33) and the same potato tubers (34). Therefore, it is plausible that multispecies lesions can occur in nature.…”

Bacterial Wilt of Solanaceous Crops: Diagnosis, Diversity and Management

“…Molecular based detection of R. solanacearum causing bacterial wilt of tomato, chilli, brinjal and potato has been done by using specific primers Y2 and OLII and it also distinguished R. solanacearum from other saprophytic bacteria grown on TTC medium (Singh et al, 2010(Singh et al, , 2014a. Various modifications have been made to improve the sensitivity of a single round PCR, viz., enrichment in semi-selective SMSA broth prior to PCR (Elphinstone et al, 1996;Ranjan et al, 2016;Singh et al, 2014a) or real time PCR called as BIO-PCR (Ozakman and Schaad, 2003) as well as double round PCR as nested-PCR (Khakvar et al, 2008). Grover et al (2009) detected R. solanacearum as low as 1 cfu/ml of within 8 h including DNA isolation.…”

“…Multiplex polymerase chain reaction (PCR) protocol was developed and validated simultaneous detection of R. solanacearum (bacterial wilt) and E. carotovora subsp. carotovora (soft rot disease) from potato tubers (Ranjan et al, 2016). Another multiplex real-time PCR method was developed by Sebasstien et al (2014) to detect simultaneously Clavibacter michiganensis subsp.…”

screening of Antibacterial Activity of Different Secondary Metabolites from Streptomcyces Against Potato Brown Rot and Soft Rot Diseases

“…carotovorum), cause soft rot and brown rot and diseases not only in the field but also during storage. To keep these diseases from infecting potato crops, and to limit production loss, early detection of these bacterial pathogens is critical [22].…”