M. C. Fan, Department of Plant Protection, Fengshan Tropical Horticultural Experiment Branch, Taiwan Agricultural Research Institute, Kaohsiung 83052, Taiwan, R.O.C.;
C. C. Huang, Department of Tropical Fruit Trees, Fengshan Tropical Horticultural Experiment Branch, Taiwan Agricultural Research Institute, Kaohsiung 83052, Taiwan, R.O.C.; and
J. S. Huang,
S. F. Tsai,
H. C. Yeh, and
C. F. Hong, Department of Plant Protection, Fengshan Tropical Horticultural Experiment Branch, Taiwan Agricultural Research Institute, Kaohsiung 83052, Taiwan, R.O.C.
Wax apple (Syzygium samarangense Merr. & Perry, syn. Eugenia javanica Lam.) belongs to the Myrtaceae family is an important economical tree fruit in Taiwan. The total production acreage of wax apple was 5,266 ha in which more than 77% were located in Pingtung County, southern Taiwan, in 2012. Since the winter of 2010, symptoms of withering leaves and cracking branches on wax apple trees were observed in some orchards in Nanjhou and Linbian Townships, Pingtung County. Diseased trees declined gradually and resulted in reduced fruit production. On the bark of diseased twigs and branches, black conidiamata with yellowish orange conidia were usually observed. For diagnosis, tissues from symptomatic branches were excised, surface sterilized with 0.5% sodium hypochlorite, and placed on 2% water agar in petri dishes. A total of four identical fungal isolates were obtained and maintained on potato dextrose agar (PDA). To fulfill Koch's postulates, three twigs of a wax apple tree were wounded with scalpel and inoculated with each of the four isolates, one tree per isolate. A 7-day-old hyphal mat (about 7 × 18 mm) of each fungal isolate was attached on the wound, wrapped with a wet absorbent cotton and Parafilm, and then covered with a layer of aluminum foil. For the control, the twigs of a wax apple tree were inoculated with PDA plugs. The pathogenicity test was repeated once. After 30 days, withering leaves and cracking twigs were observed on inoculated twigs and the same pathogen was reisolated. Conversely, all of the non-inoculated plants remained healthy. Identification of the pathogen was conducted using its morphological, physiological, and molecular characteristics. On malt extract agar, the colony was floccose and white with hazel hues. The optimal temperature for the mycelial growth was 30°C. Conidia were hyaline, and oblong, with the average size of 4.7 ± 0.6 × 2.7 ± 0.2 μm (100 conidia). Ascostromata were semi-immersed in the bark with fusoid asci, eight ascospores per ascus. Ascospores were hyaline, 2-celled, and tapered in both ends, with the average length of 6.8 ± 0.7 × 2.4 ± 0.3 μm (100 ascospores). For molecular identification, the internal transcribed spacer (ITS) of ribosomal DNA and β-tubulin genes was amplified using the ITS1/ITS4 (3), Bt1a/Bt1b, and Bt2a/Bt2b (1) primer pairs. The gene sequences were deposited in GenBank (Accessions KC792616, KC792617, KC792618, and KC792619 for the ITS region; KC792620, KC792621, KC792622, and KC792623 for Bt1 region, and KC812732, KC812733, KC812734, and KC812735 for Bt2 region) and showed 99 to 100% identity to the Chrysoporthe deuterocubensis isolate CMW12745 (DQ368764 for ITS region; GQ290183 for Bt1 region, and DQ368781 for Bt2 region). In addition, the Bt1 region of the β-tubulin gene consisted of two restriction sites for AvaI and one restriction site for HindIII. This is identical to the description of C. deuterocubensis, a cryptic species in C. cubensis, by Van Der Merwe et al. (2). According to these results, the pathogen was identified as C. deuterocubensis Gryzenh. & M. J. Wingf. To the best of our knowledge, this is the first report of canker disease caused by C. deuterocubensis on S. samarangense in Taiwan.
References: (1) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (2) N. A. Van Der Merwe et al. Fungal Biol. 114:966, 2010. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.