First report of pectobacterium parmentieri and pectobacterium polaris causing potato blackleg disease in Punjab, Pakistan

Abstract

Blackleg has become one of the most important bacterial diseases of potato (Solanum tuberosum) worldwide (McNally et al. 2017). Recent advancements in the classification of blackleg-causing bacteria such as Dickeya species have encouraged scientists to characterize the overall diversity of blackleg-related pathogens (Jiang et al. 2016). During 2014 to 2017, several potato samples exhibiting blackleg disease were collected from 35 farmer fields with disease incidence of ~12% in Okara and Chiniot districts of Punjab (Pakistan). Pathogens were isolated by homogenizing 1-cm sections of surface-sterilized symptomatic tissues (treated with 70% ethanol for 40 s) in 0.8% NaCl solution. Suspensions were plated on crystal violet pectate (CVP) agar medium and incubated for 48 h at 28ºC (Helias et al. 2012). Bacterial colonies producing pitting on CVP were further purified on nutrient agar medium. DNA of five representative bacterial isolates from both districts was extracted, and polymerase chain reaction (PCR) amplifications were performed using gapA primers (gapA-7-F and gapA-938-R) (Cigna et al. 2017) followed by sequencing. DNA sequence alignment and a phylogenetic tree of gapA barcodes generated using computer software MEGA7 showed that the isolates SS90, SS91, and SS92 (GenBank accessions MH188895 to MH188897) clustered with the type strain Pectobacterium parmentieri RNS 08.42.1AT (CP015749), and the isolates SS28 and SS94 (MH188893 and MH188894) clustered with the type strain Pectobacterium polaris NIBIO1006T (CP017481). To further validate the identification, one strain from each species (P. parmentieri SS90 and P. polaris SS28) was selected for genome sequencing. The nucleotide sequences of five housekeeping genes (acnA, gyrB, gapA, mtlD, and recA) (GenBank accession nos. MH188883 to MH188887 for P. parmentieri SS90, and MH188888 to MH188892 for P. polaris SS28) were concatenated for a robust multilocus sequence analysis with the reference strains from the genus Pectobacterium and Dickeya available in NCBI. CLC Genomics Workbench version 10.1.1 was used to determine the phylogenetic relationship among these strains, which showed the same results as gapA gene analysis. Pathogenicity of the five strains (SS90, SS91, SS92, SS28, and SS94) was evaluated on potato tubers and plants (cv. Santé Bacterial suspension (108 CFU/ml) in 0.8% NaCl solution or plain 0.8% NaCl solution (negative control) was used to inoculate disease-free surface-sterilized potato tubers and 2-week-old greenhouse-grown potato plants. Potato tubers were inoculated with 10 µl of inoculum and placed at 24ºC for 4 days in an incubator. Potato plants grown in pots were inoculated by stem injection (100 µl of inoculum) and kept at 24 to 26ºC under greenhouse conditions. All the tested bacterial isolates, but not the negative controls, produced typical soft rot symptoms 8 to 10 days postinoculation. Bacterial strains were reisolated from the diseased tissues of inoculated potato tubers and plants and were identified by PCR and DNA sequencing as described above to fulfill Koch's postulates. These results confirmed, for the first time, the presence of P. parmentieri and P. polaris in potato fields of Pakistan, therefore demanding improved crop breeding, better disease management, and strict quarantine practices for controlling potato blackleg disease in the country.