First Report of Black Dot Caused by Colletotrichum coccodes on Potato in the Tibet Autonomous Region of China.

Potato black dot causing by Colletotrichum coccodes is a common disease in potato throughout the world, infecting underground stems, tubers, roots and foliage. Potato is becoming the third main food crop produced on ~16,000 ha annually in the Tibet Autonomous Region of China, situated on the world's highest plateau. However, the disease causing by C. coccodes has not been reported in this region. During the disease survey in the Bailang County of Tibet in August, 2020, some potato plants cv. "JiZhang 12" with chlorosis and wilting of foliage were observed. The incidence of affected plants was 20%. Necrotic lesions were also observed on the basal stems of the affected plants. Three affected plants were collected for pathogen isolation and three isolates were obtained for further investigation. The colonies on potato dextrose agar (PDA) were initially white, turning gradually black with age and producing abundant black sclerotia. Conidia were cylindrical, hyaline, aseptate, guttulate, with average size of 13.80 to 18.55×4.94 to 5.35 μm for the three isolates in which 30 conidia each were measured. Such characteristics are similar to C. coccodes (Lees and Hilton, 2003). Mycelial growth rate was 0.69 to 0.74 cm/day at 25 oC over the three isolates. The three isolates were confirmed to be C. coccodes by species-specific PCR using primer set of Cc1NF1/Cc2NR1 producing 350 bp amplicons in the internal transcribed spacer (ITS) region according to Cullen et al. (2002). The Cc1NF1/Cc2NR1 sequences were identical for three isolates, therefore one sequence from isolate BL_JZ_J1 was submitted to the GenBank with accession number OM368349. Additional genes were also sequencing including the actin (ACT), chitin synthase l (CHS1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and another larger ITS region were also amplified from genomic DNA using primers sets ACT512F/ACT783R, CHS-79F/CHS-354R (Carbone and Kohn 1999), GDF1/GDR1 (Templeton et al. 1992) and ITS1/ITS4 (Glass and Donaldson 1995), respectively. Sequences obtained for those four regions were 216 bp, 218bp, 235bp and 576 bp, respectively. Each region in the three isolates were also identical, therefore one sequence for each region was submitted to the GenBank with accession number of OM417059, OM417060, OM417061, and OM349570, respectively, which had 100% similarity with C. coccodes of MN336525 (ACT), KU821274 (CHS1), KU821397 (GAPDH) and KU821175 (ITS), respectively. Phylogenetic analyses based on the concatenated sequences of those four loci showed that the BL_JZ_J1 was close to C. coccodes, a reference isolate of CPOS1 with the accession numbers of GQ856787 (ACT), GQ856723 (CHS1), GQ856744 (GAPDH) and GQ485588 (ITS) (Yang et al. 2009). Pathogenicity was confirmed by inoculating a conidia suspension (100 µl of 105 conidia/ml) on three stems of 6-week-old potato plant cv. 'Favorita' with an artificial wound generated by sterile toothpick for each isolate. An equal volume of sterile water was injected on the wound of three stems as a noninoculated control. Brownish necrotic lesions were observed on all inoculated stems 3 days post-inoculation under natural conditions, whereas control stems remained symptomless. Reisolation of the fungus from all inoculated symptomatic plants confirmed Koch's Postulates. To our knowledge, this is the first report of C. coccodes in potato in Tibet Autonomous Region of China. The finding of black dot in this region has important management implications for the growers since the pathogen can survive for long periods in the field both on potato debris and in soil.