First Report of Pectobacterium peruviense as the Causal Pathogen of Blackleg and Soft Rot in Solanum tuberosum cv. Superchola in Cañar, Ecuador

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Potato (Solanum tuberosum) is grown in Ecuador's Andean region at altitudes from 2,200 to 3,600 m.a.s.l., and the most commercially significant cultivar is Superchola, which constitutes 60% of total production, covering around 11,000 hectares (MAG, s.f. 2022). From November 2022 to January 2023, dark, water-soaked lesions progressing upwards from the base of the stems were observed on Superchola potato plants in Cañar, Ecuador (2°23'56.4''S 78°59'13.2''W). Approximately 20 plants in a hectare showed symptoms. Symptomatic stems from five plants were collected, surface-disinfected with 2% sodium hypochlorite, followed by 70% ethanol, and thoroughly rinsed with sterile distilled water. Plant tissue sections (1 cm²) were homogenized in 10 mM MgCl₂, serially diluted, and plated on Tryptic Soy Agar (TSA). After 48 hours of incubation at 28°C, creamy-white, yellowish, and white round colonies appeared. Streak plating was performed, yielding 14 purified bacterial isolates named M1 to M14. For the pathogenicity tests, each purified isolate was inoculated into healthy potato tubers and stem pieces (5 cm) using the prick inoculation method described by Ma et al. (2018) with modifications. Surface-sterilized tubers and stems were wounded using autoclaved toothpicks, into which 5µl of a bacterial suspension, carrying ~1 x 106 CFU/ml of bacteria per strain, was deposited into a 5 mm deep wound. Three independent replicates were conducted. Only one isolate, designated as M3, presented maceration on potato tubers, and dark, watered-soaked lesions appeared on the stem 48 h post-inoculation with strain. Other bacterial isolates showed no symptoms in either stems or tubers. Pathogenicity tests were confirmed on healthy, 4-week-old potato plants grown in a 50% perlite and 50% peat moss substrate. Inoculations were done using two methods: (1) toothpick piercing, where 5 µl of saline solution containing ~1 x 106 CFU/ml of bacteria was applied to a wound approximately 5mm deep (Ma et al. 2018), and (2) immersion of the plant foliage in a bacterial suspension of 1x 108 CFU/ml in saline solution for 10 min Controls used bacteria-free toothpicks (wounded) and sterile 10 mM MgCl₂ solution (unwounded). Plants were enclosed in plastic containers to maintain high humidity and grown at 15/25°C (day/night) with a 12 h photoperiod. Five days post-inoculation, blackleg symptoms were observed in the stems of the infected area, which turned black and rotten (wounded and unwounded). In all cases, negative controls remained symptomless. To complete Koch's postulates, bacteria were reisolated from symptomatic potato stems and showed the same morphology. To identify the isolate M3, whole genome sequencing using Oxford Nanopore technology was performed. Three marker genes were extracted, and a 5,734-bp sequence was concatenated from dnaX (2073 bp), recA (1,074bp), and leuS (2,583bp) (GenBank accession nos. PQ177849, PQ177847 and PQ177848, respectively). The concatenated sequences of M3 and type strains were used to construct a multilocus Bayesian inference phylogenetic tree using BEAST version 1.8.4. Isolate M3 grouped with Pectobacterium peruviense, showing a 100% sequence identity with the type strain P. peruviense IFB5232(Drummond et al. 2012; Portier et al. 2019; Toth et al. 2021; Waleron et al. 2018). To our knowledge, this is the first report of P. peruviense causing blackleg and soft rot in potato plants in Ecuador. Further research is essential, but this information could assist in monitoring the pathogen's spread and developing disease management strategies.

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