First Report of Bacterial Soft Rot and Blackleg on Potato Caused by Pectobacterium parmentieri in Hawaii

Abstract

Pectobacterium genus comprises numerous soft-causing bacteria affecting vegetable, ornamental, and fruit crops (Charkowski 2018; Ma et al. 2007). Potato is the major host for several Pectobacterium species including P. parmentieri, a newly described species formerly known as P. wasabiae (Khayi et al. 2016). During surveys conducted in January 2018 and January 2019 at Oahu, 67 infected potato samples exhibiting wilting, water-soaked regions with watery ooze, and darkened and necrotic basal stem symptoms extending upward were collected. Symptomatic stems and tubers were cut into 0.5- to 1-cm pieces and surface sterilized with 10% sodium hypochlorite solution for 30 s, followed by three rinses with sterile distilled water. After sterilization, each stem piece was macerated, streaked on crystal violet pectate (CVP) medium, and incubated at 26 ± 2°C for 48 h. Colonies, forming pits on the CVP medium, were restreaked on 2,3,5-triphenyl-tetrazolium chloride medium. DNA was isolated from infected plant tissues using the DNeasy Plant Mini Kit (Qiagen, Germantown, MA). Bacterial genomic DNA from purified cultures was isolated using a DNeasy Blood and Tissue Kit (Qiagen). End-point PCR was performed using the primers Pec.dnaA-F1 and Pec.dnaA-R1 (Dobhal et al. 2019) to amplify the dnaA gene; PCR products were enzymatically purified by adding 2 µl of ExoSAP-IT. Sequencing was performed at the GENEWIZ facility; both forward and reverse strands were aligned and manually edited using Geneious. The BLASTn results of the dnaA region displayed 99 to 100% identity and 100% query cover with all P. parmentieri strains sequences in the NCBI GenBank database. Eleven strains originating from the same field (growing multiple potato cultivars) but from different plants, namely PL30 (MN428432), PL32 (MN428433), PL67 (MN428434), PL70 (MN428435), PL71 (MN428436), PL72 (MN428437), PL74 (MN428438), PL75 (MN428439), PL123 (MN428440), PL124 (MN428441), and PL128 (MN428442), were identified as P. parmentieri. Three-week-old potato plants, grown from healthy tubers under temperature-controlled conditions, were artificially inoculated at three consecutive nodes starting from the base of the stem by injecting 100 µl of bacterial inoculum of PL70 (3.56 × 10⁸ CFU/ml) and PL72 (2.78 × 10⁸ CFU/ml); control plants were inoculated with 100 µl of sterile water, and the experiment was performed in triplicates with consistent results. After ∼72 h, blackleg and stem rot symptoms resembling those observed in the field were observed; no symptoms on control plants were observed. Bacteria from inoculated symptomatic stems were reisolated on CVP; pits were produced by both strains. To fulfill Koch’s postulates, DNA was isolated, amplified, and sequenced for the dnaA region; BLASTn results showed 100% identity with the original P. parmentieri strains. Additionally, a phylogenetic analysis based on the dnaA gene was conducted with the 11 P. parmentieri Hawaiian strains; all these strains were clustered together with 18 other P. parmentieri strains in the GenBank database, further confirming the identity and genetic relationship of these new P. parmentieri strains. This is the first report of P. parmentieri causing soft rot and blackleg disease on potato at Oahu, Hawaii. The presence of this pathogen along with the previous report of Dickeya dianthicola (Boluk and Arif 2019) outline the importance of exerting proper crop management and of accurate and efficient diagnostics to prevent its spread; it also warrants a statewide survey for pectolytic bacteria on vegetable crops.