First report of potato pink rot caused by Phytophthora erythroseptica in Colorado.

Pink rot, caused by the oomycete Phytophthora erythroseptica, is a serious disease that may cause substantial losses to potato growers. In 2023, high infection rates of pink rot were noticed in the San Luis Valley (SLV), Colorado around harvest time and in storage with several growers losing complete storage pins to pink rot. In September 2023, tubers with pink rot symptoms were collected from two grower fields of Russet Norkotah (~2-3% infection) and Canela Russet (~5% infection) near the San Luis Valley Research Center at harvest time. Infected tubers showed dark discoloration on the surface at the stem end. Upon cutting, affected tissue developed salmon pink color about 20-30 min later (Fig. 1). DNA was extracted from two symptomatic tubers from each cultivar and healthy tubers using DNeasy PowerSoil Pro Kit (Qiagen, MD, USA) according to the manufacturer's instructions. The conventional PCR and qPCR assays developed by Cullen et al. (2007) were used to detect P. erythroseptica. In the conventional PCR, the primer pair Pery2F1/R1 produced expected bands of 135bp from symptomatic tubers but not from the healthy tubers. The symptomatic tubers were positive in the qPCR using the primer-probe set 99F, 177R, and 133T (Cullen et al., 2007). P. erythroseptica was isolated by culturing specimens of infected tissues on PARP selective media (Jeffers and Martin, 1986). A week later colonies of white color grew on the media. These colonies were confirmed to be of P. erythroseptica by PCR as mentioned above. The colonies of two isolates were transferred to water agar media, then three days later a mycelium tip was excised and cultured on PDA media for single isolation. On PDA media the colonies showed chrysanthemum pattern. Four days later DNA was extracted from the two isolates, RN296-1 and Canela-2, and tested positive for P. erythroseptica by the above-mentioned PCR. PDA agar plugs of two isolates were used to inoculate surface sterilized potato tubers. The isolate SLV-2023-Canela2 was inoculated on tubers of Russet Norkotah and Reveille Russet, 10 each, while10 tubers of Russet Norkotah were inoculated with the isolate SLV-2023-RN296-1. Ten tubers of Russet Norkotah were inoculated by sterilized plugs of PDA for negative controls. Tubers were left at room temperature for a week. All tubers of Russet Norkotah inoculated with SLV-2023-Canela2 and RN296-4 developed pink rot symptoms and tested positive for P. erythroseptica by PCR, while only 9 tubers of Reveille Russet inoculated with SLV-2023-Canela2 developed symptoms and were positive to P. erythroseptica by PCR. No symptoms were observed on the mock inoculated tubers and no P. erythroseptica was detected by the PCR. P. erythroseptica was isolated again from Russet Norkotah tubers inoculated with RN296-1 on PDA media and tested positive for P. erythroseptica by PCR one week later. To further confirm the identity, the ITS region was amplified using the primers ITS-1 and ITS-4 (White et al., 1990) from DNA extracted from inoculated Russet Norkotah tubers with SLV-2023-Canela2 isolate. PCR product was purified using QIAquick Gel Extraction Kit (Qiagen, MD, USA)and cloned using TOPO TA Cloning Kit for Sequencing (Invitrogen, MD, USA). Four clones were submitted for sequencing. The 846 nt sequence of the ITS was submitted to the GenBank (accession number PP587391) shared 99.76% identity with the P. erythroseptica strain BBA 62683 (KJ755119). P. erythroseptica was identified previously using morphological tools from tubers with symptoms resembling pink rot collected in the SLV (Tyler et al., 2002). However, in that report neither Koch's Postulates, nor molecular identification were conducted, hence, the current study presents the first molecular report of pink rot in the SLV, Colorado. Rapidly changing climate and increased temperature around harvest time may lead to an increase in the incidence of pink rot, hence effective management strategies need to be developed and implemented.