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First Report of Clonostachys rhizophaga as a Pathogen of Water Chestnut (Eleocharisdulcis) in Anhui Province of China

Yang, X., Zhang, A.-F., Gu, C.-Y., Zang, H.-Y., Chen, Y.
Plant disease 2019 v.103 no.1 pp. 151
Clonostachys, DNA primers, Dendrocalamus giganteus, Eleocharis dulcis, Pinus canariensis, agar, bamboos, chickpeas, chlorosis, conidia, conidiophores, cotton, culture media, disease control, fungi, genes, growth chambers, internal transcribed spacers, mycelium, necrosis, pathogenicity, pathogens, plant growth, polymerase chain reaction, sodium hypochlorite, stems, subtropics, tubulin, villages, China
Water chestnut, Eleocharis dulcis (Burm. f.) Trin., is a sedge native to China and is widely grown in the world, especially in the tropical and subtropical regions. In September 2017, black spots were observed on about 80% of the water chestnuts in several villages of Wuwei County, Anhui Province. The diseased water chestnuts showed longitudinal chlorosis streaks with black spots on the stem surface and internal vascular necrosis. To isolate the pathogen, the diseased stem segments (2 mm in length) were surface sterilized with 1% NaClO for 1min, rinsed twice for 30 s with sterile deionized water, and then incubated on water agar medium at 25°C in a growth chamber for 3 days. Then a mycelial plug was transferred to potato dextrose agar (PDA) medium and incubated for 2 weeks in the growth chamber at 25°C. Representative isolates formed off-white mycelia, dimorphic, Verticillium-like (primary) or penicillate (secondary) conidiophores, and ovoidal to elongated, slightly curved or asymmetrical, hyaline conidia about 5 to 8.8 μm long and 2.5 to 3.9 μm wide (more than 50 conidia were measured),with laterally displaced hilum. To identify the eight isolates, partial fragments of internal transcribed spacer (ITS) and β-tubulin (tub2) gene were amplified according to the method of Abang et al. (2009). A 525-bp ITS fragment (GenBank accession no. MG872331) was amplified from the ITS for all eight isolates with universal primers ITS1/ITS4, showing 100% similarity to the Clonostachys rhizophaga strain CML2312 (GenBank accession no. KC806275) and CML2310 (GenBank accession no. KC806272). A 592-bp fragment of tub2 (GenBank accession no. MH325937) was amplified with the primers bt2a (5′-AACATGCGTGAGATTGTAAGT-3′) and bt2b (5′-TCTGGATGTTGTTGGGAATCC-3′) and showed 100% similarity to the C. rhizophaga strain 1019 (GenBank accession no. FJ593883). To verify the pathogenicity of these isolates, a 5-mm-diameter mycelial plug was cut from 2-week-old colonies grown on PDA for each of the isolates and inoculated on the punctured stem of healthy water chestnuts. The inoculation site was covered with wet cotton and wrapped with Parafilm. The control plants were treated by the same method but using sterile PDA plugs of the same size. All the treatments were incubated in a plant growth chamber at 25°C with a 12-h photoperiod. Two weeks after inoculation, symptoms on all inoculated stems were identical to those described above with a dark area surrounded by a yellow halo on the stem surface and internal browning, whereas the control plants did not show any symptoms. Furthermore, the fungus was reisolated from all the inoculated stems and identified to be C. rhizophaga as described above, fulfilling Koch’s postulates. This pathogen has been reported to infect other plants, such as chickpea (Abang et al. 2009), giant bamboo (Dendrocalamus giganteus) (Zazzerini and Quaglia 2010), and Canary lsland pine (Pinus canariensis) (Piontelli and Giusiano 2003); however, to our knowledge, this is the first report of C. rhizophaga infecting water chestnut plants in China. This disease is an emerging problem in Anhui Province, and it is necessary to develop new disease management practices to sustain commercial production in this region.