LAUSR

In August 2020, anthracnose lesions were observed on fruits of Juglans regia and J. sigillata in walnut orchards, in Yijun (Shaanxi Province) and Nanhua (Yunnan Province) counties, China. Symptoms on walnut fruits first appeared as small necrotic spots that rapidly enlarged into subcircular or irregular sunken black lesions (Fig. 1a, b). Sixty diseased walnut fruits (30 fruits of J. regia and J. sigillata, respectively) were randomly sampled from six orchards (10-15 ha each orchard, three orchards were selected in each county) with severe anthracnose (incidence rate of fruit anthracnose is over 60% in the orchard.) in two counties. Twenty-six single spore isolates were obtained from diseased fruits as described by Cai et al. (2009). After seven days, isolates formed grey to milky white colony with abundant aerial hyphae on the upper surface of colony, and milky white to light olive on the back of PDA (Fig. 1c). Conidiogenous cells were hyaline, smooth-walled, and cylindrical to clavate (Fig. 1d). Conidia were smooth-walled, aseptate, cylindrical to fusiform, with both ends acute or one end round and one end slightly acute (Fig. 1e), and ranged in size from 15.5-24.3×4.9-8.1 µm (n=30). Appressoria were brown to medium brown, clavate to elliptical, with the edge entire or undulate (Fig. 1f), and ranged in size from 8.0-27.6×4.7-13.7μm (n=30). The morphological characteristics of 26 isolates were similar to those of the species complex Colletotrichum acutatum (Damm et al. 2012). Six representative isolates were randomly selected (three isolates for each province) for molecular analysis. The ribosomal internal transcribed spacers (ITS) (White et al. 1990), beta-tubulin (TUB2) (Glass and Donaldson 1995), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Templeton et al.1992) and chitin synthase 1 (CHS-1) (Carbone and Kohn 1999) genes were amplified and sequenced. Sequences of 6 of 26 isolates were submitted to GenBank (Accession Nos: ITS: MT799938-MT799943, TUB: MT816321-MT816326, GAPDH:MT816327-MT816332, CHS-1: MT816333-816338). Multi-locus phylogenetic analyses revealed that six isolates clustered together with Colletotrichum godetiae ex-type culture isolates CBS133.44 and CBS130251, and the bootstrap support value was 100% (Fig.2). The pathogenicity of two representative isolates (CFCC54247 and CFCC54244) was tested using healthy fruits of the " J. regia cv. Xiangling" and " J. sigillata cv. Yangbi" varieties. Forty sterilized fruits (20 fruits were inoculated with CFCC54247, and 20 fruits with CFCC54244) were wounded by puncturing with a sterile needle through walnut pericarp and inoculated in the wound site with 10 μl of conidial suspension (106 conidia/ml) from seven day old colonies grown on PDA at 25℃. Twenty wounded fruits were inoculated with sterile water as control. Inoculated and control fruits were incubated in containers at 25℃ in a 12/12h light/dark cycle. The experiment was repeated three times. Anthracnose symptoms (Fig. 1g-h) were observed in all inoculated fruits after 12 days, whereas controls showed no symptoms. Fungal isolates from inoculated diseased fruits showed the same morphological and molecular characteristics as the isolates obtained in this study, confirming Koch's postulates. To our knowledge, this is the first report of C. godetiae causing anthracnose on the two walnut species in China. The result will be helpful for providing a basis for further research on the control of the disease.