Rhododendron delavayi Franch, a member of Ericaceae family, is globally famous for its garden flowers with significant ornamental value (Liu et al., 2020). In July 2020 and 2021, a disease survey of R. delavayi groves was conducted in Baili Azalea Forest Area (N27°10'-27°20', E 105°04'-106°04'). We arbitrarily selected an area with around 280 R. delavayi trees covering 2.5 hectares in R. delavayi grove where 20-35% of leaves showed symptoms of anthracnose. Typical symptoms included elliptical to irregularly shaped brown lesions on leaves and masses of black dots clustered on it. About 30 pieces of leaves with anthracnose lesions were collected. A few black dots were picked from the lesions with a sterilized needle, plated on water agar and incubated at 25℃ for 12 h to observe spore germination (Fang, 2007). Then the germinated spores were transferred onto PDA medium for further purification and morphological observation. Fourteen single-spore isolates with similar morphology were obtained. The surface of the colony was white or gray and spongy; the edge was smooth; and the back side was pinkish brown after 7 days of growth on PDA. Conidia were spindle-shaped, transparent, 11.1-16.6×3.6-4.9 μm (n=50). Appressorium from conidia was nearly ovate or proximate, brown or dark brown in color, 4.3-10.3 ×3.2-7.6 μm (n=50). These characteristics are consistent with Colletotrichum fioriniae reported by Shivas and Tan (2009). DNA was extracted from a representative isolate MYDJ12. The internal transcribed spacer region (ITS), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB), actin (ACT), and chitin synthase 1 (CHS-1) genes were amplified using primer pairs described by Damm et al. (2012). The sequences were deposited in GenBank with accession number MW692854 (ITS), MW727518 (GAPDH), MW727519 (TUB2), MW727520 (ACT), and MW727521 (CHS-1). BLASTN searches of the ITS, GADPH, TUB2, ACT and CHS-1 genes revealed 100% (540/540 nucleotides), 100% (254/254 nucleotides), 99.38% (4488/491 nucleotides), 98.77% (242/245 nucleotides) and 100% (282/282 nucleotides) homology with those of C. fioriniae CBS:128517T in GenBank (NR_111747, JQ948622, JQ949943, JQ949613 and JQ948953 respectively). The phylogenetic tree showed the isolate MYDJ12 to cluster with C. fioriniae CBS:128517T. Finally, two-year old R. delavayi plants (n=5) were inoculated by wounding with a syringe needle and placing 10 μL of spore suspension (106 spores per mL) of the isolate MYDJ12 on three leaves per plant. Control leaves were inoculated with sterile water. The experiment was conducted twice. Inoculated leaves were wrapped in parafilm tape and then the plants were placed in a greenhouse at 25°C with high relative humidity (90 to 95%). Seven days after incubation, brown lesions appeared, similar to those observed in the grove. Black dots clustered on the lesions after 15 days. Re-isolation was conducted 20 days after inoculation. From all the five inoculated plants, similar symptoms were observed, and the same pathogen was re-isolated. One of the isolates was selected for morphological observation and multi-gene (ITS, GAPDH, ACT, TUB2 and CHS-1) analysis indicated the reisolated fungus to be C. fioriniae. No fungal pathogens were isolated from mock inoculated plants. This study can provide effective management and useful information for the control of this disease on R. delavayi in Baili Azalea Forest Area. References: Damm, U., et al. 2012. Stud Mycol 73: 37. Fang, Z. D. 2007. Research Methods of Plant Diseases (Third edition). China Agriculture Press. Liu, J., et al. 2020. Mitochondrial DNA B 5:37. Shivas, R, G; Tan, Y, P. 2009. Fungal Divers 39:111.
Keywords: Anthracnose; Colletotrichum fioriniae; Rhododendron delavayi; disease.