First Report of <i>Clausena lansium</i> Anthracnose Caused by <i>Colletotrichum siamense</i> in China

Clausena lansium (Lour.) Skeels is a tropical and subtropical plant species, it is not only a fruit but also has medicinal value. Its cultivated areas are mainly distributed in Guangdong, Guangxi, Fujian, and Hainan provinces in China. A severe anthracnose disease was observed during surveys of Clausena lansium disease in Yanfeng town of Hainan province in June 2024. The acreage of plantation was 2 acres. The percentage of symptomatic plants was about 70%. Symptoms appeared both on leaves and young shoots. Lesions appeared as brown point-sized at first on the leaf, and then enlarged to dark brown irregular lesions lined by a yellow halo. Diseased leaves became distorted and wrinkled at last. On young shoots, lesions appeared as water-soaked at first, and then enlarged to dark brown and sunken, ultimately leading to dieback. Five symptomatic leaves were sampled from different trees for pathogen isolation. 15 small pieces (5 mm) of necrotic tissue were removed from the border between symptomatic and healthy tissue, surface sterilized for 1 min in 1.5% NaOCl, washed three times with sterile distilled water, and plated onto potato dextrose agar (PDA). The plates were incubated at 28°C for five days. We obtained 10 Colletotrichum isolates with similar colony morphology. For a pure culture of the isolate, spore masses were picked off with a sterilized wire loop and streaked on the surface of water agar. After incubation overnight at 25 °C, single germinated spores were picked up with a sterile needle and transferred to PDA. Identification of the isolate was based on morphological as well as molecular characterisation. Colony characters and microscopic morphology characteristics of isolates were observed after growth on PDA at 28°C under dark for 7 days. The colonies on PDA were white at first, and then turned dark gray, cottony, with orange conidiomata, reverse dark gray to black at the center. Conidiophores formed on a cushion of medium brown roundish cells. Conidiophores were hyaline, Conidiogenous cells were cylindrical to ampulliform, straight to flexuous, 33-51 μm. Conidia were hyaline, aseptate, cylindrical with round ends, smooth-walled, guttulate, 9.9-19.9× 4.7-6.0 μm (mean ± SD = 12.8 ± 2.8 × 5.4 ± 0.4 μm, n=50). Genomic DNA of the 10 isolates was extracted from the culture, and DNA sequencing of an intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), actin (ACT), chitin synthase 1 (CHS-1), and the internal transcribed spacer (ITS) was conducted to accurately identify the species. ITS, TUB2 and ACT sequences from the ten isolates shared 100% identity, GAPDH and CHS-1 shared 99.6% identity. Sequences of the isolate named HPCS1 were deposited in GenBank (ITS Accession Nos. PQ113778; TUB2 Accession Nos: PQ133411; GAPDH Accession Nos: PQ137484; ACT Accession Nos: PQ782238, CHS-1 Accession Nos: PQ782239, respectively). ITS (513bp) of the Clausena lansium isolate HPCS1 were 100%, and GAPDH (232bp/234bp), ACT (256bp/257bp), CHS-1 (215bp/216bp) and TUB2 (419bp/420bp) were more than 99% similar with the Colletotrichum siamense type strain CBS130417 (Prihastuti et al. 2009) (GenBank Accession Nos. JX010171, JX009924, and JX010404) (Weir et al. 2012). In the phylogenetic analysis, the 10 isolates formed a monophyletic group and clustered within a clade with the ex-type isolate of Colletotrichum siamense (Sharma et al. 2015; Liu et al. 2016). Based on morphological and multilocus phylogeny, the Clausena lansium isolates were identified as C. siamense. To confirm pathogenicity, a conidial suspension (1 × 106 conidia/ml) of HPCS1 isolate was prepared by harvesting conidia from 10-day-old cultures growing on PDA and 5 mL was sprayed onto the healthy young shoots with tender leaves without wounding. All 9 young shoots inoculated were covered with plastic bags for 48 h to maintain high humidity and incubated at 28°C (Lin et al. 2020). After 5 days, symptoms were observed on the C. lansium leaves and were similar to those observed on the naturally infected plants. No symptoms were observed on the control leaves (inoculated with sterilized water in the same way). The pathogen was re-isolated from symptomatic tissues, but not from controls. These re-isolates matched the morphological and molecular characteristics of the original isolates, fulfilling Koch's postulates. C. siamense was described from Coffea arabica in Thailand (Prihastuti et al. 2009). In China, Colletotrichum scovillei was reported as a pathogen causing anthracnose on C. lansium (Lin et al. 2020). To our knowledge, this is the first report of C. lansium anthracnose causing by C. siamense.