First Report of Pectobacterium brasiliense Causing Bacterial Soft Rot and Blackleg Diseases of Potato in Hawaii

Pectobacterium brasiliense, first described in Brazil (Duarte et al. 2004), and recently elevated to the species level (Portier et al. 2019), has emerged as one of the most destructive bacterial species in the heterogenous Pectobacterium genus, which causes numerous diseases including blackleg of potato (Arizala and Arif 2019; Li et al. 2019). In January 2019, 67 potato plants showing characteristic symptoms of blackleg, stem rot, and leaf necrosis were observed in a field on Oahu, Hawaii. Affected tissues (light brown, macerated, and water-soaked stem sections) were cut into 1-cm pieces, followed by surface sterilization using 0.6% sodium hypochlorite for 30 s and then three successive rinses with distilled water. The tissues were ground and streaked on plates containing crystal violet pectate medium. Plates were incubated for 48 h at 26 ± 2°C; colonies producing pits were restreaked and purified on dextrose peptone agar. Later, the bacterial DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen). The Pec.dnaA-F1 and Pec.dnaA-R1 primer set (Dobhal et al. 2020) was used to amplify the dnaA gene, and PCR products were treated using the ExoSAP-IT kit (Affymetrix) and sent for sequencing at the GENEWIZ facility using both forward and reverse primers. Both strands were aligned and manually corrected for errors; the consensus sequence of each bacterial strain was analyzed with the NCBI BLASTn tool. Eleven strains isolated from different infected plants were confirmed as P. brasiliense with 100% query cover and 99 to 100% identity. After performing a phylogenetic analysis using the dnaA sequences representing different Pectobacterium sp., all 11 strains grouped within the clade of P. brasiliense. The dnaA consensus sequences were submitted to the GenBank database under the accession numbers MN428420 to MN428430 for the strains PL68, PL69, PL76, PL78, PL79, PL80, PL81, PL82, PL107, PL108, and PL109. To fulfill Koch’s postulates, a pathogenicity test was performed by injecting 100 µl of a bacterial suspension of strains PL108 (3.6 × 10⁸ CFU/ml) and PL109 (3.8 × 10⁸ CFU/ml) into the stem of healthy potato seedlings (3 weeks old); control plants were inoculated with 100 µl of distilled water. The experiment was performed in three replicates in a temperature-controlled greenhouse. Four days postinoculation, all inoculated seedlings (except the controls) showed typical symptoms of stem darkening, maceration, and watery ooze that resembled those manifested by the naturally infected plants on the field. Bacteria were reisolated from symptomatic tissues, and the dnaA region was resequenced. Sequence analysis confirmed the identity of these strains as P. brasiliense. This is the first report of P. brasiliense on potato in Hawaii. This along with first reports of other pectolytic soft rot bacteria associated with potato in Hawaii (Arizala et al. 2020; Boluk and Arif 2019) indicates a need for conducting further state-wide surveys of these vegetable crops.