First report of Colletotrichum siamense causing leaf spot on Macropanax rosthornii in China

Macropanax rosthornii (Harms) C. Y. Wu ex Hoo is an evergreen broadleaf species cultivated in subtropical China as an ornamental (Liang et al. 2015). In August 2020, leaf spot symptoms were observed on the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E), Jiangxi province, China. The early symptoms were small spots on the edge or tip of the leaves. The spots gradually expanded and became grayish brown with reddish egdes, eventually developing large irregular lesions. The disease incidence was estimated at 45%. Leaf pieces (5 × 5 mm) from the lesion borders were surface disinfested in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water (Li et al. 2023). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark. Three representative single-spore isolates (DS-2, DS-3, and DS-5) were used for morphological studies and phylogenetic analyses. Colonies on PDA of the three isolates were white-to-gray with cottony mycelia. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 14.3-18.1 ×4.3-6.9 µm (15.8 ± 1.1 × 5.3 ± 0.2 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.6-9.4 × 4.5-6.9 µm (7.7 ± 0.3 × 5.5 ± 0.2 µm, n=100). Morphological features were similar to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, calmodulin (CAL), actin (ACT), β-tubulin 2 (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chitin synthase (CHS-1) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, CL1/CL2, ACT-512F/ACT-783R, T1/Bt2b, GDF/GDR and CHS-79F/CHS-354R (Weir et al. 2012), respectively. Sequences were deposited in GenBank under nos. OL895315 - OL895316 (ITS), OL830190 - OL830192 (ACT), OL830181 - OL830183 (GAPDH), OL830178 - OL830180 (TUB2), OL830184 - OL830186 (CHS-1), and OL830187 - OL830189 (CAL). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed DS-2, DS-3, and DS-5 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. Pathogenicity of the three isolates was verified on six 5-year-old Macropanax rosthornii plants, which were grown in the field. Three healthy leaves per plant were wounded using a sterile needle (Φ=0.5 mm) and inoculated with a 20-μL conidial suspension per leaf (106 conidia/mL). Another six control plants were treated with sterile water. Eighteen leaves were used for the pathogenicity test of three isolates. All leaves were covered with plastic bags to maintain humidity for 2 days. The inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic after 8 days. The fungi were consistently reisolated only from the inoculated and symptomatic leaves, fulfilling Koch's postulates. C. siamense can cause leaf diseases in a variety of hosts, including Liriodendron chinense × tulipifera (Zhu et al. 2019), Salix matsudana (Zhang et al. 2021), Carya illinoinensis (Zhuo et al. 2023). However, this is the first report of C. siamense infecting Macropanax rosthornii in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.