First Report of Lasiodiplodia gilanensis Causing Twig and Leaf Blight on Machilus thunbergii in Zhejiang Province of China

Machilus thunbergii Sieb. et Zucc. (Lauraceae) is a broad-leaved evergreen tree and an endangered species in warm-temperate and subtropical areas of China. It also has high ornamental, timber-use, and soil-conservation functions. In recent years, brown to dark-brown lesions were found on leaves or on twigs of M. thunbergii, which caused decay of the cortex. We first found this disease on about 30% of M. thunbergii trees in April 2017 in Zhejiang Tongling Mountain National Forest Park at Wencheng, Wenzhou, Zhejiang province. Symptomatic material from leaves and stems was surface sterilized in 75% ethanol for 10 s and then in 3% NaClO for 3 min, rinsed three times in sterilized distilled water, dried, transferred onto potato dextrose agar (PDA), and incubated at 25°C. Fourteen pure isolates from symptomatic tissues were obtained by single-spore isolations. These isolates formed colonies with abundant villous aerial hyphae. The colonies initially were gray and then turned to black. The strains had black, scattered, or clustered pycnidia and cylindrical conidiophores that occasionally branched. The conidia of the strains were fusiform, thick walled, 31.2 ± 2.5 μm long, and 16.7 ± 1.5 μm wide (length-to-width ratio = 1.9 ± 0.2), and with obtuse apices tapering to a truncate base. Initially, conidia were single celled and transparent, and then they became double celled and the melanin gradually increased. The morphology was similar to the description of Lasiodiplodia gilanensis Abdollahzadeh, Javadi & A.J.L. Phillips (Abdollahzadeh et al. 2010). Genomic DNA samples of the 14 isolates were extracted using the cetyl trimethylammonium bromide method. The β-tubulin (TB) gene was amplified by polymerase chain reaction assay using primers bt2a/bt2b, and a portion of the translation elongation factor 1-alpha (EF1-α) gene was amplified using primers EF-1-986/EF-728. The amplified DNA fragments were then sequenced, and the sequences of all 14 isolates were identical. The sequences of TB (GenBank accession no. MG372749) and EF1-α (GenBank accession no. MG372751) in the representative strain HNDNYB-1 were 100 and 100% identical to the TB sequence of L. gilanensis strain 3K59 (GenBank accession no. KF955888) and the EF1-α sequence of L. gilanensis strain IRAN1501C (GenBank accession no. GU945341), respectively (Abdollahzadeh et al. 2010; Chen et al. 2014). Morphological and molecular results confirmed the isolate as L. gilanensis. Five isolates were selected for pathogenicity tests. Healthy twigs and leaves of M. thunbergii were collected in the field and wounded with a sterilized needle. A 5-mm-diameter mycelial plug of each isolate was excised from the margin of a 4-day-old colony from a PDA plate and placed to the wounded twigs or leaves, and the twigs and leaves were treated similarly with uncolonized plugs as a control. All plants were maintained in an artificial climate chamber at 25°C and 90% relative humidity with a 12-h photoperiod per day. After 5 days, typical lesions developed on inoculated twigs and leaves, whereas control plants remained asymptomatic. The L. gilanensis strains were only reisolated from inoculated plants, and then they were confirmed via morphological observations and genomic DNA testing as identical to the original inoculated strains. L. gilanensis has previously been isolated on California pistachio and pomegranate (Chen et al. 2014; Úrbez-Torres et al. 2016), but to the best of our knowledge, this is the first report of this pathogen on M. thunbergii worldwide.