First report of anthracnose caused by Colletotrichum siamense on Illicium verum in China.

Aniseed (Illicium verum) is a woody spice tree that has been grown in China for a long time. Anthracnose is an important disease of aniseed, which can cause severe leaf drop. In Sep. 2020, severe anthracnose was observed in Shanglin (23°35'5"N, 108°19'51"E), Nanning, Guangxi in China, and the incidence was 85%. The symptoms at the early stage were small, round and watery, then became larger and gradually turned brown. The acervuli would appear at the later stage, and contain many conidia. Leaves with disease were randomly sampled from 10 plants, and were cut into small rectangular pieces of 0.5×1 cm, and disinfected with 75% alcohol 1 min, with 0.1% HgCl2 3 min. After washing with sterile water 3 times, they were placed onto potato dextrose agar (PDA) medium and incubated at 25°C for 5 days. The average colony growth rate was 11.85 mm/d in 7 days. The colony was white or light gray in the initial stage, with dense aerial mycelium, and the central mycelium of the colony was dark grey in the later stage. Conidia were colorless, single spore, smooth, cylindrical, both ends obtuse, with an average size of 14.95 ± 0.97 µm × 5.46 ± 0.44 µm (n = 100). The conidial appressorium was oval or club-shaped, brown, margin intact, with an average size of 7.83 ± 1.21 µm × 5.82 ± 0.58µm (n = 50). Three strains GXNN02, GXNN03 and GXNN05 were selected for further study. Primer pairs T1/ßt2b, ACT512/ACT783, GDF/GDR, CHS1-79F / CHS1-354R and ITS1/ITS4 (Weir et al. 2012) were used to amplify tubulin (TUB), actin (ACT), 3-phosphate glyceraldehyde dehydrogenase (GAPDH), chitinase (CHS1) and the internal transcribed spacers of rDNA (ITS) respectively. BLASTn searches showed that the TUB (ON619861-63 ), ACT (ON619852-54), GAPDH (ON619855-57), CHS1 (ON619858-60) and ITS (ON573028-30) sequences had the highest similarity to Colletotrichum siamense with up to 99% (699/702, 676/679, 699/702) identity for TUB (JX010404.1); 99% (281/282, 253/254, 249/250) identity for ACT (JX009518.1); 99% (275/277, 275/277, 239/241) identity for GAPDH (JX009924.1); 99% (296/299, 296/299, 259/262) identity for CHS1 (JX009865.1); up to 99% (527/530, 485/487, 527/530) identity for ITS (JX010171.1) of ex-type ICMP 18578. A ML tree was constructed by combining 5 sequenced loci, and three isolates clustered in the C. siamense clade with 94% bootstrap support. Therefore, combined with the morphological characteristics, the pathogens were identified as C. siamense. In a pathogenicity test, these three isolates were tested on 9 healthy aniseed seedlings with at least 10 leaves, and 3 seedlings as control. The leaves were surface disinfected with 75% alcohol, and then wiped with sterilized water three times. Holes were made near the edge of the leaves and were sprayed with conidial solution (6×106 spores/mL) in test groups, and use sterilized water as control. Then the leaves were sealed inside a plastic bag for 48 h to retain moisture. Brown spot and black acervuli, similar to the symptoms in the field, were observed on the leaves in test groups within 10-15 days. No symptoms were observed on the negative control leaves. The pathogens were reisolated from the treated infected leaves and were identified as C. siamense, thus fulfilling Koch's postulates. The pathogenicity test was confirmed by repeating in triplicate. The isolation frequency of C. siamense in our samples was 82.50%. To our knowledge, this is the first report of C. siamense in China. Further research on the occurrence of the disease will help prevent the spread of the disease.