First Report of Lasiodiplodia theobromae Causing Dieback of Aquilaria sinensis in Taiwan.

Abstract

Incense trees (Aquilaria sinensis (Lour.) Gilg) belong to a plant family used for alternative medicine in China and the production of wood. In the summer of 2012, at a nursery in Niaosong district, Kaohsiung City, Taiwan, more than 30% of a total of 400 incense trees had dieback symptoms on twigs with leaves attached, leading to eventual death of the entire plant. Symptomatic twigs and trunk pieces from six trees were collected and discolored tissues were excised, surface sterilized in 0.5% sodium hypochlorite solution, rinsed in sterilized distilled water, dried on sterilized filter paper, and then placed in petri dishes containing 2% water agar (WA). The dishes were incubated at room temperature for 1 to 2 days to obtain fungal strains from diseased tissues. The hyphal tips from developing fungal colonies were transferred to potato dextrose agar (PDA, Difco) dishes and placed under UV light (12 h/day) at 30°C. The purified colonies were used as inoculum in the pathogenicity tests. Pathogenicity tests were performed on 2-month-old A. sinensis seedlings, each treatment had three plants. Each plant was wounded by removing bark of the twigs with a disinfected scalpel enough to place a mycelium plug (about 5 × 10 mm2) of 7-day-old fungal isolate on the wound. The inoculated area was wrapped with a wet paper towel and Parafilm. Control plants were treated with PDA plugs. The symptoms described above were observed on inoculated plants 4 to 8 days after inoculation whereas control plants did not show symptoms. Diseased twigs were cut and placed in a moist chamber 21 days after inoculation and conidia oozing from pycnidia were observed. The same fungal pathogen was reisolated from inoculated plants, but not from the control. To identify the pathogen, the fungus was cultured as described above. The colonies were initially white with green to gray aerial mycelium after 5 to 6 days and eventually turned darker. Immature conidia were hyaline and one-celled, but mature conidia were dark brown, two-celled, thin-walled, and oval-shaped with longitudinal striations. The average size of 100 conidia was 25.23 ± 1.97 × 13.09 ± 0.99 μm with a length/width ratio of 1.92. For the molecular identification, the internal transcribed spacer (ITS) region of ribosomal DNA was PCR amplified with primers ITS1 and ITS4 (2) and sequenced. The sequences were deposited in GenBank (Accession No. JX945583) and showed 99% identity to Lasiodiplodia theobromae (HM346871, GQ469929, and HQ315840). Hence, both morphological and molecular characteristics confirmed the pathogen as L. theobromae (Pat.) Griffon & Maubl (1). To the best of our knowledge, this is the first report of L. theobromae causing dieback on Incense tree. This disease threatens tree survival and may decrease the income of growers.