First report of Colletotrichum fructicola causing anthracnose on Carya cathayensis Sarg. in China.

Carya cathayensis Sarg. (Chinese hickory) is one of the important economic forest plants, mainly distributed in Zhejiang and Anhui provinces in China. In September 2020, leaf spot disease occurred on 90% C. cathayensis in a 2.6 km2 plantation with 500 hickorys in Shangshu Village (30°26'N, 119°32'E), Huzhou, Zhejiang, China. Symptoms initially appeared as small brown spots. Later, the spots became dark brown, and joined into irregular shapes. Twenty diseased leaves with typical symptoms were collected and used to isolate the pathogen. The leaf tissues (5 × 5 mm) at junction of diseased and healthy portion were cut and surface-sterilized with 75% ethanol for 15 s, 0.1% NaClO for 2 min, and rinsed 3 times in sterile water, then placed on potato dextrose agar (PDA) plates and incubated at 25°C in the darkness for 3 days. Eight isolates with similar morphological characterizations were obtained after pure cultures by transferring hyphal tips. The colony growing on PDA for 7 days was circular, dense, white cotton-like hyphae, and light gray-black hyphae can be seen inside. The conidia were cylindrical, aseptate, hyaline, with rounded ends, and 12.5 to 20.0 × 5.0 to 7.5 µm (n = 50). The appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and were in the range of 6.4 to 10.2 × 5.0 to 6.7 µm (n = 50). The morphologies of the isolates were consistent with the genus description of Colletotrichum (Fuentes-Aragón et al. 2018; Liu et al. 2015). The internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), chitin synthase (CHS-1), beta-tubulin 2 (TUB2) and actin (ACT) genes were amplified from genomic DNA for the isolates using the primers described by Weir et al. (2012). The sequences of eight isolates were consistent and the representative isolate CFZJ-64 were deposited in GenBank under the following accession numbers: ITS, OK145563; ACT, OK216738; CAL, OK216739; CHS-1, OK216740; GAPDH, OK216741; and TUB2, OK216742. A phylogenetic tree was generated by combining ITS, ACT, CAL, CHS-1, TUB2, and GAPDH sequences in MEGA11. Three representative isolates CFZJ-42, CFZJ-53 and CFZJ-64 clustered in the C. fructicola clade with 90% bootstrap support. Based on morphological characteristics and phylogenetic analysis, the isolates were identified as C. fructicola. To confirm pathogenicity, 9 detached healthy leaves and 9 healthy leaves on 3-year-old C. cathayensis seedlings were inoculated with conidial suspension of each isolate (20 µL, 1 × 106 conidia/mL). The control leaves were treated with distilled water (20 µL). Each tested leaf was covered with a clean ziplock bag and incubated for 48h at about 27°C, and 14h photoperiod. After five days, 7 of 8 isolates caused on all detached leaves or part of the leaves on the seedlings developed lesions similar to those observed in the field, whereas controls were asymptomatic. The same fungus was re-isolated from all the diseased leaves and identified by sequencing, confirming Koch's postulates. As far as we know, this is the first report of C. fructicola causing anthracnose on C. cathayensis. This study not only expands the knowledge on this important pathogen of C. cathayensis in China, but also provides the foundation to further investigate the biology, epidemiology, and control of the disease.