Michelia macclurei Dandy is an excellent timber and ornamental tree native to South China (Lan et al. 2010). In May 2020, a leaf spot disease of M. macclurei was found on the campus of Jiangxi Agricultural University (N28°45'56″, E115°50'21″). Approximately 25% (9 out of 35) of 32-year-old M. macclurei trees showed the leaf spot disease. On average, 40% of the leaves per individual tree were affected. The symptoms began as small dark brown lesions formed along the leaf margins and tips. The lesions' center was sunken with a dark brown border as the disease developed. Thirty pieces (5 × 5 mm) from the lesion margins were surface sterilized in 70% ethanol (30 s), then in 3% NaOCl (1 min), and finally rinsed three times with sterile water. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation. Sixteen strains with similar morphological characterizations were isolated, and three representative isolates (HX-1, HX-2, HX-3) were used for morphological and molecular characterization. The three isolates were white, cottony, and light gray on the reverse, producing dark-green pigmentation near the center. The conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 12.8-17.5 × 4.5-5.7 µm (14.7 ± 1.2 × 4.8 ± 0.2 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, and ranged from 5.9-8.8 × 4.4-6.7 µm (7.1 ± 0.6 × 5.6 ± 0.6 µm, n=100). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin2 (TUB2) were sequenced using the primers ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R, CL1/CL2, CHS-79F/CHS-345R, GDF/GDR, and T1/Bt2b, respectively (Weir et al. 2012). The sequences were deposited into GenBank (Accession Nos.: MZ323328, MZ323329, MW581269 for ITS, MZ889002, MZ889003, MW661166 for ACT, MZ889004, MZ889005, MW661167 for CAL, MZ889006, MZ889007, MW661168 for CHS-1, MZ889008, MZ889009, MW661169 for GAPDH, MZ889010, MZ889011, MW661170 for TUB2). A maximum likelihood and Bayesian posterior probability-based analyses using IQ-tree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, ACT, CAL, CHS-1, GAPDH, and TUB2 ) placed three isolates in the clade of Colletotrichum siamense Prihastuti, L. Cai & K. D. Hyde. Based on the morphological characteristics and molecular data, three isolates were identified as C. siamense (Fu et al. 2019).The pathogenicity of each isolate was tested on potted 2-yr-old seedlings of M. macclurei grown in a greenhouse at 25 ℃, 70% relative humidity with a 12-h photoperiod. Twenty healthy leaves on 10 M. macclurei plants were inoculated with 10 μL of spore suspension (106 conidia/mL). Another 20 healthy leaves were inoculated with sterile water as the control. All leaves were wounded with a sterile needle (Φ=0.5 mm). The resulting symptoms were similar to those on the original infected plants, whereas the control leaves remained asymptomatic for 8 days after inoculation. C. siamense was consistently re-isolated only from the diseased leaves, fulfilling Koch's postulates. C. siamense can cause leaf diseases in a variety of hosts, such as Salix matsudana (Zhang et al. 2021), Liriodendron chinense [Hemsl.] Sarg. × tulipifera L. (Zhu et al. 2019) and Magnolia grandiflora (Zhou et al. 2022). This is the first report of C. siamense associated with leaf spot disease on M. macclurei in China, and its potential threat should be evaluated in the future. These results will help to develop effective strategies for appropriately managing this newly emerging disease.
Keywords: Michelia macclurei; coelomycete; identification; multi-gene phylogeny; pathogen.