First Report of <i>Pythium ultimum</i> Causing Pythium Leak Disease in Potatoes in Colorado

Potatoes are among the most important vegetable crops globally and in the United States. The San Luis Valley (SLV), CO, produces about 95% of Colorado's potatoes ranking as the second fresh potato producer nationwide (Daniel & Chikh-Ali, 2024). During 2023 and 2024 growing seasons, potato tubers of Soraya and Caribou Russet, respectively, showing symptoms similar to those of Pythium leak, were collected from two potato storage facilities in the SLV. The incidence of infection was 50-60%, and the storage conditions at these facilities included a temperature of approximately 8°C and relative humidity ranging from 85-90%. Symptoms included spongy, wet internal rot in the tubers. Upon cutting the tubers, affected tissues were distinctly separated by a dark boundary and turned black immediately or within 10-20 minutes. To identify the causal agent, tissue samples, approximately 10 mm² in size, were surface sterilized in 70% ethanol for 30 seconds, rinsed twice with sterilized water, air-dried, and placed onto potato dextrose agar (PDA) medium. Five plates were incubated in the dark at 25°C for one week until the white, dense colonies were observed. Microscopic examination revealed aseptate hyphae measuring 3.1 to 7.2 μm in diameter and forming sporangia as complexes of swollen hyphal branches measuring 22.6 × 21.4 µm (n=10). The oomycete produced terminal, smooth, globose oogonia with an average diameter of about 16.2 μm (n=10) and antheridia measuring 13.6 × 9.1 μm (n=10). The morphological characteristics of the five isolates collected matched those of Pythium ultimum (Watanabe, 2002). For molecular identification, conventional PCR and qPCR assays developed by Cullen et al. (2007) were used to identify P. ultimum in all DNA extracts collected from symptomatic tubers and PDA cultures. The primer pair Pu1F1/Pu2R1 amplified the internal transcribed spacer (ITS) region of rDNA, and an expected fragment of 309 bp was produced from symptomatic tubers and isolated hyphae but not from the healthy tubers. The symptomatic tubers and isolated hyphae were positive in the qPCR using the primer-probe set 92F/166R/16T (Cullen et al., 2007). To further confirm the morphological and molecular identification, the cytochrome oxidase II (cox II) gene was amplified using the primer pair FM58/FM66, generating a 588 bp amplicon (Villa et al., 2006). The ITS region sequence and cox II gene sequence were submitted to GenBank under accession numbers PQ897936 and PV009300, respectively. BLASTn searches showed that the ITS, and cox II genes were 100% identical to the corresponding sequences of P. ultimum, with GenBank accession nos. KU210609 and GU138663, respectively. A pathogenicity experiment was conducted to fulfill Koch's postulates by inoculating ten tubers each of the cvs. Soraya and Russet Norkotah-278 with a spore suspension (10⁴ spores/mL, 50 µl/tuber) from two isolates collected during the 2023 and 2024 harvests. Each tuber was inoculated by creating a hole at both ends, then incubated at 25°C with approximately 90-95% relative humidity for one week to facilitate infection and symptoms development. Symptoms comparable to those in the field appeared in the inoculated tubers after one week, while no symptoms were observed in ten control tubers injected with sterile distilled water. After re-isolating the pathogen, its identity was confirmed using morphological and aforementioned PCR and qPCR. Pythium leak is a serious storage disease of potatoes and can cause substantial losses. To effectively manage and control this disease, integrated strategies should be adopted to reduce the impact of this destructive pathogen on potato crops. To the best of our knowledge, this is the first documented case of P. ultimum causing potato Pythium leak in Colorado.