Dalbergia odorifera, a New Host of Colletotrichum tropicale Causing Anthracnose in China

Dalbergia odorifera T. Chen (Family: Fabaceae) is an endangered, wild and second class key protected tree species in China. It has been cultivated widely in south China because of its valuable rosewood and pharmacological uses. During January and September 2022, anthracnose was observed on D. odorifera in Chikan, Zhanjiang, Guangdong (N21°15' 9.4" E110°22' 25.9"). All of the D. odorifera plants were infected and the leaf disease incidence was above 30%. The lesions were 0.35 to 0.65 cm in size, greyish-white, and gradually expanded into brown black circular spots surrounded by a yellowish halo. Later, the centers of spots were white with dark-brown borders. In severe cases, the leaves turned yellow, withered and fell off. To isolate the pathogen, about 5×5 mm tissues at the disease-health junction of spots were sterilized with 75% ethanol for 30s and 3% hydrogen peroxide for 3 min, rinsed with sterile water three times, and plated onto PDA for incubation at 25-28℃ in the dark. Isolates were obtained by picking the tip of hypha growing from the tissues, further purified by coating the diluted spore suspension to obtain single-spore isolates. Colonies with abundant aerial mycelium were white to light gray, 4.0 to 5.0 cm in diameter after 5 days at 25 to 28℃ in the dark. Conidiogenous cells were hyaline, cylindrical and monoblastic. Conidia were unicellular, subcylindrical, hyaline, guttulate, 3.5 to 5.6×11.1 to 16.8 (av. 4.6±0.48 × 14.0±1.25) μm (n>50). Appressoria were dark brown subglobose, clavate, fusiform, occasionally lobed, terminal, 7.8 to 16.3×4.6 to 8.9 μm. No swollen cells and setae were observed. To further confirm identity of the fungus, partial regions of six genetic loci of isolate ACCC 35246 (stored in Agricultural Culture Collection of China) were amplified and sequenced: actin (ACT), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GPDH), histone (HIS), beta-tubulin (TUB) and internal transcribed spacer (ITS) utilizing the primer pairs (Damm et al. 2012; Weir et al. 2012; White et al 1990) (OP314900-OP314904 and OP269660, respectively). Based on a BLAST analysis, the six sequences were about 99% identical to those of Colletotrichum tropicale holotype strain CBS124949 (ACT: JX009489 270/272; CHS: JX009870 295/299; GPDH: JX010007 274/279; HIS: KY856395 638/670; ITS: JX010264 546/548 and TUB: JX010407 494/497, respectively) (Weir et al. 2012). Phylogenetic analyses, using a combined dataset of ACT, CHS, GPDH, ITS and TUB were carried out in MEGA X using the maximum likelihood method, placed the fungus within the C. tropicale clade. Based on morphogical and molecular analyses, the fungus was identified as C. tropicale (Rojas et al 2010). To test the pathogenicity, five healthy leaves of 1 year-old potted plants were stab-wounded with a sterile needle and inoculated with 10 µL of spore suspension (105 conidia/mL). Another five healthy leaves were inoculated with sterile water as the control. Symptoms expressed by the inoculated leaves, 10 days after inoculation, were the same as those of the diseased plants, while the non-inoculated leaves remained asymptomatic. The same fungus based on morphogical and molecular criteria was re-isolated from the spots. As an endophyte or plant pathogen, C. tropicale inhabits a wide host range of plant species belonging to 31 genera in 25 families (Farr and Rossman 2022), D. odorifera is a new host of C. tropicale causing anthracnose in China. This work will be helpful for the diagnosis and control of this disease.