First report of Neofusicoccum parvum causing stem canker and necrosis of Phoebe sheareri in Zhejiang province, China

Phoebe sheareri is a popular species established in Zhejiang province of China for timber. During surveys in June 2016, severe canker problems were observed in one plantation of P. sheareri in Kaihua county, Zhejiang. The disease started with black fusiform necrosis (8 to 15 mm long, 3 to 5 mm wide) on the green bark of shoots, and the cankers could be found on branches and even the main stem (with diameter up to 10 cm). These cankers can girdle stems, resulting in the death of plant tissues above. Ten stem sections containing the junction between healthy and necrotic xylem from five symptomatic trees were collected. Sections of symptomatic xylem were surface sterilized in 5% sodium hypochlorite water solution and placed on potato dextrose agar (PDA) at 25°C in the dark. After 3 days, seven isolates were generated by subculturing single hyphal tips onto fresh PDA medium and grown for 7 days in the dark. All seven isolates initially developed white aerial mycelium that later turned dark gray. After 2 weeks, black globular pycnidia could be found. The fusiform hyaline conidia were aseptate, with thin walls, and measured 20.9 ± 2.2 × 5.1 ± 0.4 μm. Three isolates (KHZJZ1 to KHZJZ3, respectively) were selected randomly for molecular characterization. The rDNA internal transcribed spacer (ITS), partial β-tubulin (tub2), and translation elongation factor 1-α (tef1) gene regions were amplified by polymerase chain reaction using the primer pairs ITS1/ITS4, Bt2a/Bt2b, and EF1-728F/EF1-986R, respectively (Alves et al. 2008; Glass and Donaldson 1995; White et al. 1990). Gene sequences of all three fungal isolates were identical. The ITS, tub2, and tef1 sequences were deposited in GenBank (accession nos. MK131330, MK139576, and MK139575, respectively). A local BLASTn search revealed that the ITS, tub2, and tef1 sequences shared 99, 99, and 100% identity, respectively, to corresponding sequences of ex-type isolate Neofusicoccum parvum strain CMW9081 (accession nos. AY236943, AY236917, and AY236888, respectively). Pathogenicity tests were conducted by wounding stems of 2-year-old P. sheareri seedlings with a 5-mm-diameter cork borer. Wounds were inoculated with PDA plugs excised from 7-day-old cultures of the N. parvum isolate (KHZJZ1). Stems inoculated with sterile PDA plugs acted as controls. The inoculated stem wounds were wrapped with Parafilm, and the seedlings were kept under natural conditions grown in the field (18 to 30°C). There were five replicates of each treatment. After 21 days, the N. parvum-inoculated stems had developed black necrotic lesions (11 ± 5 mm long) on the bark, whereas the control plants remained asymptomatic. The fungus was reisolated from diseased plant tissue but not from the controls, thus fulfilling Koch’s postulates. N. parvum has previously been reported as the causal agent of canker and die-back of Eucalyptus (Iturritxa et al. 2011), stem blight of blueberry (Koike et al. 2014), and canker of other woody plants (Jayakumar et al. 2011). To our knowledge, this is the first report of N. parvum causing canker of P. sheareri, which is becoming increasingly important in timber plantations in south China.