In the summer of 2012, an outbreak of a newly discovered root and basal stalk rot of wild rice (Zizania palustris L.) cv. Franklin was observed in a 16-ha field in Big Valley, Lassen County, California (GPS coordinates 41°08′41.93″ N 121°10′07.49″ W). Infected plants exhibiting rot and dieback of roots and stalks were in various stages of decline, including death. Symptomatic stem and root tissues from affected plants were surface disinfected in 1% NaOCl for 90 s and placed on PARP agar plates, which were then incubated at 25°C in the dark for 1 week. Hyphal tips were used to start and maintain the organism in pure cultures. Isolates were transferred into petri plates with water and sterilized blades of turfgrass for the production of hyphae and reproductive structures. Isolates had coenocytic hyphae and produced zoospores 20 to 30 μm in diameter outside of sporangia (75 to 160 × 46 to 110 μm) from a naked mass of protoplasm, unlike from a vesicle, which is characteristic of Pythium spp. (2). Based on these morphological features, the isolate was tentatively identified as a Pythiogeton sp. Total genomic DNA was extracted from mycelia using the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). The internal transcribed spacers (ITS) 1 and 2 flanking the 5.8S rRNA regions were amplified by PCR and sequenced using universal ITS5 and ITS4 primers. A BLAST search of the 855-bp sequences revealed 98% similarity with a sequence of P. ramosum isolate Pg-164 (GenBank Accession No. JQ610190.1). The 21 nucleotide differences suggest that the isolate from wild rice may be an unreported species. The sequences were submitted to GenBank (KF719169). To fulfill Koch's postulates and confirm pathogenicity, 100 wild rice seeds were surface disinfected in 1% NaOCl for 90 s and placed in a 500 ml sterile pot with 250 g of autoclaved sand. Three 5 mm-diameter disks from the margin of a 7-day-old culture growing on PARP were placed in each of five pots. As a control, three 5 mm-diameter disks from a non-inoculated PARP plate were placed in five different pots, and five pots with autoclaved sand were not inoculated. All pots were kept in a randomized complete block design at 25°C for 14 days under a 14-h photoperiod. The pathogenicity test was repeated three times. After 14 days, the inoculated plants in all tests developed root and basal stalk rot, consistent with the symptoms observed on diseased wild rice in the field. The Pythiogeton sp. was consistently re-isolated on PARP from symptomatic plants but not from control plants. The non-inoculated wild rice plants remained asymptomatic. DNA sequences of the ITS region of the re-isolated Pythiogeton sp. revealed 100% identity with the isolate from the field. There have been reports of P. zeae on corn in Korea and P. zizaniae on water bamboo in Taiwan (1,2,3). This is the first report of a Pythiogeton sp. on wild rice.
References: (1) P. J. Ann et al. Mycologia 98:116, 2006. (2) J. Huang et al. Mycoscience 54:130, 2013. (3) H. J. Jee et al. Mycologia 92:522, 2000.
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