First Report of Colletotrichum aenigma Causing Walnut Anthracnose in China

English/Persian walnut (Juglans regia L.) is grown as an economically valuable crop in temperate and subtropical regions. In August of 2018, serious fruit anthracnose, with brown to black circular or subcircular or irregular sunken lesions (Fig.1A), occurred on walnut trees ("Xiangling" and "lvling") in 33 ha., 23 ha. and 20 ha. orchards in Lincheng and Neiqiu county, in Xingtai, Hebei, China. Diseased fruits were observed on 41% (19,000 trees), 31% (13,300 trees) and 34% (11,400 trees) walnut trees. Diseased leaves, with circular or irregular brown to gray sunken lesions, were observed on 2% (19,000 trees), 2% (13,300 trees) and 1% (11,400 trees) walnut trees. From each orchard, 25 diseased fruits and leaves were collected, respectively. Twenty-one single spore isolates were obtained from fruits of three orchards and none from leaves as described by Cai et al. (2009). Six representative isolates 1811-1, 1811-4, 1811-7, 1811-8, 1811-11 and 1811-18, two from each orchard, were selected for further study. Colonies on PDA grew 11.8 mm d-1 at 25℃ under a 12/12 h light/dark cycle for 7 d. The upper side of colonies was milky (Fig.1 B), and reverse side was dark brown to brownish yellow. A few acervuli were observed on colonies. Conidiogenous cells were cylindrical to clavate, 10.6-29.7 × 3.1-5.3 μm (mean=21.3 × 4.0 μm, n=30) (Fig.1F). Setae were not observed. Conidia were smooth-walled, aseptate, straight or slightly distorted, cylindrical with one end slightly acute or broadly rounded ends, and 16.6-21.6 × 6.0-7.5 μm (mean=19.2 × 6.7 μm, n=30) (Fig.1 C). Appressoria were mostly irregular in outline, deeply lobed or lightly lobed, gray brown to dark brown, 8.3-16.6 × 7.1-14.5 μm (mean=12.5 × 9.7 μm, n=30) (Fig.1 D-E). Microscopic features were similar to the description of C. aenigma (Weir et al. 2012). To further identify isolates, the ribosomal internal transcribed spacers (ITS), β-tubulin 2 (TUB2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS) and chitin synthase (CHS-1) loci of representative isolates were amplified using ITS4/ITS5, Bt2a/Bt2b, CL1/CL2, GDF1/GDR1, GSF1/GSR1 and CHS-79F/CHS-345R primers (Prihastuti et al. 2009; Carbone & Kohn 1999). Sequences of representative isolate 1811-1 were submitted to GenBank (ITS: MN893316, TUB: MN893317, CAL: MN893312, GAPDH: MN893314, GS: MN893315, CHS-1: MN893313). Maximum likehood analysis of sequences of representative isolates and reference sequences of Colletotrichum spp. from GenBank revealed that six isolates clustered together with C. aenigma ex-type culture ICMP18608, and the bootstrap value was 100% (Fig.2). Pathogenicity tests were conducted on walnut fruit as described by Wang et al. (2017, 2018) and Cai et al. (2009). 10 wounded and 10 nonwounded fruits ("Xiangling", 35 mm diameter) were inoculated with isolates 1811-1, 1811-7 and 1811-11 conidial suspension (106 spore/mL) obtained from 10 d colonies grown on PDA at 25℃, respectively. 10 wounded and 10 nonwounded fruits were inoculated with sterile water. Inoculated and control fruits were incubated in containers at 25℃ in a 12/12 h light/dark cycle. After 10 days, necrotic lesions were observed in all inoculated fruits. The pathogen C. aenigma was reisolated from all inoculated fruits but not from control fruits. To our knowledge, this is the first report of C. aenigma causing walnut anthracnose in China. It is urgent to control walnut anthracnose caused by different species of Colletotrichum.