First Report of Rugonectria rugulosa Causing Dieback of Falcataria moluccana in China

Falcataria moluccana is a fast-growing and valuable tropical tree species for forest plantations in southern China. From June 2015 to November 2016, a dieback disease was observed on 2- or 3-year-old F. moluccana in Boluo county, Guangdong, China. The incidence of the disease reached up to 20%. The symptoms observed were initially red-brown lesions on the trunk, chlorosis of the leaves on some branches and then a gradual defoliation, aggregates of red-orange perithecia on the bark, and finally the death of the trees. Diseased tissues were cut into 4-mm² pieces and surface disinfected with 70% ethanol for 30 s followed by 3% NaClO for 5 min, rinsed with distilled water, plated onto potato dextrose agar (PDA), and then incubated for 7 days at 25°C. Colonies appeared whitish to yellowish tint, sparse to denser, and the reverse was first beige and then turned into brown. Perithecia were globose to subglobose, conspicuously warted, and orange-red to reddish brown. Asci were cylindrical to clavate and were eight-spored. Ascospores were ellipsoidal to oblong, hyaline, uniseptate, and measuring 9.9 to 12.6 × 2.9 to 4.9 µm (n = 100). Macroconidia were fusiform with tapering ends, curved, hyaline, six- to seven-septate, and 49 to 61.2 × 5.8 to 7.4 μm (n = 100). Microconidia were ovoid to cylindrical with rounded ends, hyaline, zero- to one-septate, and 7 to 10 × 2.6 to 2.8 μm (n = 100). The internal transcribed spacer rDNA (ITS), β-tubulin 2 (TUB2), and translation elongation factor-1α (TEF) genes were amplified with the primer pairs ITS1/4, Bt2a/2b, and EF-728/986 (Glass and Donaldson 1995; White et al. 1990), respectively. BLAST analysis of the sequences (accession nos. MG991752 to 53, MG991754 to 55, and MG991748 to 49 for ITS, TUB2, and TEF, respectively) showed 99 to 100% identity with Rugonectria rugulosa. Based on the morphological and molecular characteristics, the isolates were identified as R. rugulosa and deposited in the specimen room in the Department of Plant Pathology, South China Agricultural University. To complete Koch’s postulates, pathogenicity tests were conducted on 6-month-old F. moluccana seedlings (clone TL16). The stems were wounded using a 5-mm sterile cork borer, and 7-day-old mycelium plugs of isolates (BL1610 and HD1509) were inoculated into the holes, respectively, and covered with moistened cotton and Parafilm. Sterile PDA plugs were placed into the wounds of control seedlings. Fifteen healthy seedlings were inoculated with isolates or PDA plugs (n = 5 plants/treatment). All plants were grown under greenhouse conditions (28°C, 16/8-h day/night at 80% relative humidity). The seedlings inoculated by the isolates showed brown lesions, fallen leaves, and dry branches, whereas the controls remained asymptomatic. R. rugulosa was successfully reisolated from the lesions but not from the control wounds and was identified by the methods described above. Nectria rugulosa (= R. rugulosa) has been reported to be associated with quick decline of macadamia trees in Hawaii (Ko 2009). The occurrence and spread of R. rugulosa in growing areas of F. moluccana have caused severe economic losses. This is the first report of dieback disease of F. moluccana caused by R. rugulosa worldwide. The identification of dieback disease of F. moluccana should be helpful in developing effective control strategies.