First Report of Tip Blight of Pinus tabulaeformis Caused by Sphaeropsis sapinea in China.

Pinus tabulaeformis is native to China and South East Asia, and used in afforestation and landscaping (Wang et al. 2010). Tip blight was first observed in P. tabulaeformis in 2004 in a forests of Hebei Province. The disease spread rapidly in recent years, and reached 70% incidence in 2014. The fungus mainly infects the shoots and needles of trees. At first, affected needles turn yellow or reddish and dry. From July to August, spherical black pycnidia appear on the shoots and at the bases of diseased needles, buried or half-buried under the epidermis. Later, the pycnidia emerge. In the final stage infected shoots and needles turn straw-colored to gray-white and take on a plexiform, withered shape, but they do not fall off. Diseased shoots were cut into 1-cm pieces, surface-sterilized (75% ethanol for 30 s, 0.1% HgCl2 for 3 min and rinsed 3 times in sterile water), and then plated onto potato dextrose agar (PDA) and corn meal agar (CMA) at 25°C. The resulting colonies were initially white with a smooth margin and vigorous cottony aerial hyphae. Then, 4 to 7 days later, the medium was stained dark green from pigment secreted from the colony. The colony turned dark gray 10 days later. No pycnidia formed on PDA after 30 days. However, on CMA at 25°C in light, pycnidia formed at 7 days and matured at 50 days. The pycnidia were black, spherical, 0.7 mm to 1.3 mm in length, and half-buried in the medium. Conidia were, light brown, single-celled or double-celled, 24.2 to 29.1 × 9.2 to 11.1 µm (avg. 26.5 × 10.7 µm). Genomic DNA was extracted from a culture on PDA. The complete ITS rDNA region was amplified by PCR with primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (Yu et al. 2010), and deposited at GenBank (Accession No. KU571453). The BLAST analysis indicated 99% identity with Sphaeropsis sapinea (syn. Diplodia pinea) (KF766159, AY156721, and AY159247). By phylogenetic analysis (MEGA 5.0), the gene sequence was placed in a cluster (>90% bootstrap value) with other S. sapinea using the neighbor-joining algorithm. The pathogen isolated from the diseased plants was identified as S. sapinea based on morphological characteristics and phylogenetic analysis. Twenty healthy 1-year-old shoots were inoculated by spraying with a conidial suspension (106 conidial/ml). Symptoms resembling those on the original diseased plants appeared 10 days after inoculation, whereas control plants remained asymptomatic. The fungi reisolated from the inoculated plants were morphologically identical to those originally isolated from the diseased plants. Sphaeropsis sapinea has a worldwide distribution on many pine species (Flowers et al. 2001), but this is the first report of tip blight of P. tabulaeformis caused byS. sapinea in the forests of China. Previous reports in China showed that several pines including P. tabulaeformis were susceptible to artificial inoculation, but this is the first report of the pathogen from forests in China. This report lays a foundation for further studies and development of effective control strategies. © 2016 The American Phytopathological Society.