First Report of Pectobacterium parvum Causing Diseases on Potato in Inner Mongolia, China

Soft rot enterobacterial plant pathogens Pectobacterium spp. and Dickeya spp. have caused devastating blackleg, aerial stem rot, and soft rot of potato tubers (Charkowski 2018). In August 2021, potato plants (cv. Xisen6# or Maiken1) with blackleg or aerial stem rot symptoms were observed in two commercial fields in Xinghe County, Ulanqab City, Inner Mongolia Autonomous Region, China. The plants were wilted, and the crown stem showed gradual degradation and browning. The disease incidence was around 3 to 7% and 20 to 25% in Xinghe Zhangyou Village (33 ha) and Bianjia Village (7 ha), respectively. Pathogens were isolated on crystal violet pectate agar (CVP) plates (Ge et al. 2018). Briefly, symptomatic stem tissues were surface sterilized in 75% ethanol, ground, then serial dilutions were cultured on CVP plates (Handique et al. 2022). The plates were incubated at 28oC for 2 days. Pure colonies of Pectobacterium spp. were obtained from the pits of CVP plates and sequenced for identification using the universal 16S rRNA gene primers 27F/1492R (Monciardini et al. 2002). Results of the comparison of 16S sequences against NCBI GenBank showed 100% sequence identity to P. parvum FN20211T (CP087392.1) type strain for the three colonies designated as ZRIMU1006 (1542/1542 bp), ZRIMU1019 (1542/1542 bp), and ZRIMU1020 (1542/1542 bp). Sequences were deposited under accession numbers OP941529, OP941525, OP941526, respectively. Additionally, six housekeeping gene sequences were used to confirm identification at the species level and were uploaded to GenBank: fusA (OP793177, OP793171, OP793172), gapA (OP793221, OP793216, OP793217), gyrB (OP793265, OP793259, OP793260), infB (OP793310, OP793304, OP793305), pgi (OP793355, OP793349, OP793350), and rplB (OP793400, OP793394, OP793395). Phylogenetic trees constructed using the MEGA X program of concatenated sequences of the housekeeping genes sequences show that the three isolates grouped with P. parvam FN20211T, confirming that they are P. parvam. Pathogenicity tests for stem rot were done by injecting a bacterial suspension into potato seedlings (cv. Favorita) grown from seed tubers. The tubers were planted in perlite potting mix and 3 weeks after emergence, a 100 μl bacterial suspension (105 CFU/ml) or sterile phosphate-buffered solution was injected at the stem base. The bacterial injection experiment was performed twice in a greenhouse with five plants inoculated per bacterial strain. Plants were covered with plastic bags to maintain 100% humidity at 25°C for 2 days. Seven days after inoculation, the inoculated area of the seedlings had rotted or turned black, while the controls remained symptomless. Symptomatic tissues from each strain were processed as above and placed on CVP plates to reisolate the Pectobacterium spp. 16S rRNA sequence analysis confirmed the bacteria to be similar in sequence to the inoculated strains, thus completing Koch's postulates. Soft rot development was performed by adding bacterial suspension (100 μl, 105 CFU/ml) on tuber slices. The infected tubers rotted, while the controls were symptomless. The vacuum infiltration method on tuber that is used to test pathogens for blackleg did not result in the development of blackleg symptoms. Briefly, five tubers were inoculated with the pathogen by vacuum infiltration and planted in potting mix. The plants showed wilting after emergence, but no blackleg symptoms were observed. Recently, multiple new Pectobacterium species including P. parmentieri, P. polaris, and P. punjabense, were identified to cause potato disease in different provinces of China (Cao et al. 2021; Handique et al. 2022). In China, P. parvum was first isolated from Brassica, and in the year 2022, it was reported to cause aerial stem-rot on potato in Hebei province in China (Wang et al. 2022). Inner Mongolia is a major potato-seed-producing province and the incidence of new strains of Pectobacterium in the province causing aerial stem rot and soft rot of tuber might cause a reduction in seed production. This report will draw attention to the management of P. parvum by the seed-producing companies in Inner Mongolia which distributes the seed throughout China.