First report of Pectobacterium polaris causing aerial stem rot of potato in Mexico.

In Mexico, potato (Solanum tuberosum L.) is one of the most important vegetable crops for local consumption and industry. More than 1.8 million tons of potatoes are produced annually, of which the state of Sinaloa contributes with 21.5% (SIAP. 2022). In January 2020, potato plants (cv. FL1867) showing aerial stem rot symptoms were observed in a commercial field from the Santa Rosa Valley, in Northern Sinaloa with an incidence of 36%. Putative pectolytic bacteria showing pitting on crystal violet pectate (CVP) plates were restreaked and purified onto Nutritive Agar (NA) medium at 28°C. Four independent isolates were obtained (L25F-83, L25F-105, L25F-115, and L25F-125) from four symptomatic stems with biochemical and morphological characteristics related to Pectobacterium, such as catalase positive, oxidase negative, pectinolytic activity, Gram-negative and non-fluorescent in B-King medium. Bacterial gDNA was used for amplification and sequencing of two housekeeping genes (dnaX and leuS) (Portier et al. 2019). The nucleotide sequence identity between our isolates was 100% with both housekeeping genes (dnaX, OP376536-OP376539 and leuS, OP376540-OP376543). The BLASTn analysis of dnaX gene shared 98.98% and 99.19% identity with two soft-rot-causing bacterial strains NIBIO1006T (CP017481) and NIBIO1392 (CP017482) of Pectobacterium polaris, respectively; and with leuS gene shared 99.56% identity with P. polaris type strain NIBIO1006T. To further validate the identification, two strains, S5 (isolate L25F-105) and S6 (L25F-125), were selected for whole genome sequencing (WGS). The ANI values for closely related species were calculated using the Orthologous Average Nucleotide Identity (Ortho-ANI) Software Tool (OAT) (Lee et al. 2016). The Type (Strain) Genome Server (TYGS) was used for accurate genome-based taxonomy (https://tygs.dsmz.de) (Meier-Kolthoff et al. 2019). The genomes of P. polaris strains S5 (4811345 pb, GC=52%, AULSZ000000000) and S6 (4809754 pb, GC=52%, JAULTA000000000) revealed 96.86% and 96.07% Ortho-ANI and 73.6% and 66.5% dDDH with P. polaris type strain NIBIO1006T and P. parvum strain CFBP8630, respectively. The MLSA was performed on concatenated complete sequences of dnaX (OR470476, OR470477), leuS (OR470484, OR470485), recA (OR470488, OR470488), acnA (OR470474, OR470475), gapA (OR470478, OR470479), gyrA (OR470480, OR470481), icdA (OR470482, OR470483), proA (OR470486, OR470487), and rpoA genes (OR470490, OR470491). The consensus tree, constructed using the maximum likelihood method (MEGA 7.0), clustered strains S5 and S6 with P. polaris strains NIBIO1006T and NIBIO1392. The four isolates resulted pathogenic in tuber slices and potato seedlings (cv. Fianna) 24 and 72 h, respectively, after being inoculated with 30 µL bacterial suspension (1 X 108 CFU/ml) and incubated at 28 °C and 85% relative humidity. Bacterial colonies were reisolated from the affected tissue and identified with the same PCR primers as described above. Accordingly, P. polaris isolates S5 and S6, fulfill Koch's postulates for aerial stem rot of potato. To our knowledge, this is the first report of P. polaris causing aerial stem rot of potato in Mexico. This bacterium could be a significant threat to the local potato producers; therefore, an accurate and sensitive method of detection and epidemiological studies are needed to support an effective disease diagnosis and management program.