Brown leaf spot of Cycas debaoensis Caused by Colletotrichum siamense in Sichuan, China

Cycas debaoensis Y. C. Zhong et C. J. Chen is an endemic species in China that is listed among China's national key preserved wild plants (Class I) (Xie et al. 2005). It is mainly distributed in south China (Guangxi, Guizhou, and other regions). In April 2017, a new leaf disease of C. debaoensis was found in Chengdu (30°35'32″ N; 104°05'11″E) in China with an incidence over 40%. Symptoms on C. debaoensis initially appeared as brown necrotic lesions on the margin or in the center of leaves. The lesions then enlarged gradually and developed into brown spots, necrotic lesions with dark brown margins. Many small and black dots were observed on necrotic lesions. Eventually, the diseased leaves withered and died. Ten samples were collected and surface-sterilized by 3% NaClO and 75% ehanol respectively for 60s and 90s, rinsed with autoclaved distilled water and then blot-dried with autoclaved paper towels. Five isolates from diseased leaves with similar morphology were isolated from single spores. Morphological characteristics were recorded from pure cultures grown on potato dextrose agar (PDA) incubated at 25°C for 3-9 days. Initially, the colonies grown on PDA were white, then, became pale gray with concentric zones and greenish black beneath. Conidia were single-celled, smooth-walled, straight, colorless, cylindrical with both ends bluntly rounded,13.0-16.5 × 4.7-5.8 μm in size (n = 100 spores). For molecular identification, the genomic DNA of the isolates was extracted using a DNeasyTM Plant Mini Kit (Qiagen). The internal transcribed spacer (ITS) (ITS1/ITS4 White et al., 1990), β-tubulin (TUB2) (BT2A/BT2B (O'Donnell et al., 1997)), actin (ACT) (ACT512F/ACT (Carbone & Kohn, 1999)), calmodulin (CAL) (CL1C/CL2C (Weir et al., 2012)), mating type protein and chitin synthase (CHS-1) (CHS-1) (CHS-9 79F/CHS-345R (Carbone & Kohn, 1999)) were amplified. BLAST results indicated that the ITS, TUB2, ACT, CAL, CHS-1 sequences (GenBank MN305712, MN605072, MT478663, MT465591 and MT478664) showed 99-100% identity with C. siamense sequences at NCBI (GenBank JF710564, MK341542, MK855094, MH351155 and MK471373). The Phylogenetic tree inferred from the combined dataesets (TEF, TUB and ACT) show that the isolate belongs to C. siamense clade with a credibility value of 99%. Two-year-old potted plants of C. debaoensis (10 plants) were used for pathogenicity test. On each plant, 5 leaves were sprayed with a conidial suspension (1 × 106 conidia/ml) on both sides of the leaves. Autocl