Fungal isolations

Fungal isolations. In 2016 and 2017, all the leaves, fruits and branches of mango with anthracnose symptoms collected from the six mango-grown provinces listed above in China were used for fungal isolation. A total of 205 Colletotrichum isolates were obtained from all the mango growing region investigated, and only 136 isolates selected for further study. The number of Colletotrichum isolations from each sampling site include at least one or more than one isolate. All these selected isolations representing the vast mango growing areas in China were subject to morphological and molecular analysis. All the selected Colletotrichum species, isolate number, GenBank number and their geographic origin were listed in Table 2. Isolated species in other genera include Alternaria, Phomopsis, Botryosphaeria, and Pestalotia were not researched further in this study.
Phylogenetic analyses. ITS sequence analysis was initially used to screen 136 Colletotrichum isolates (34 from Yunnan, 23 from Hainan, 23 from Guizhou, 13 from Sichuan, 16 from Guangdong and 27 from Fujian province). These isolates from different areas were then selected for sequencing and analyses of GAPDH, ACT, TUB2, and CHS-1 loci. All sequences generated in this study were firstly compared with NR database from NCBI-GenBank, and then submitted to Genbank (see Table 2). For phylogenetic analysis, all the selected sequences were finally aligned with a list of registered GenBank sequences for Colletotrichum species from varied hosts and country of origin (Table 3).
Combined all the sequences for 136 Colletotrichum isolates, ex-type or type strain from five loci including ACT, TUB2, CHS-1, GAPDH and ITS region, a phylogenetic tree was constructed and presented in Fig1. The results showed that 38 isolates were clustered with four C. asianum type strains (ICMP 18605, ICMP 18603, IMI 313839 and ICMP 18580) with 100% posterior probability; 38 isolates were aligned with eight C. siamense type strains with well-supported 100% probability; 32 isolates were placed with two C. fructicola type strains (ICMP 18727 and ICMP 18613) and one C. ignotum strain (CBS125397) supported with 100% probability. Five isolates clustered with type strains of C. karstii CBS118401, CBS 128524 and CBS 128500, with supporting rate of (100%); four isolates with type strain of C. endophytica CAUG28 obtained 99.6% support; three isolates clustered with type strains of C. scovillei CAUA1, CBS 126529 and CBS 120708 with supporting rate of 100%; isolates YN8-2 and YN31-4 with type strain of C. clivaie CBS 125375 were well-supported (100%); isolate HN42-2 with type strain of C. gigasporum CBS 101881 were well-supported (100%); isolate HN32-1 with type strain of C. tropicale CBS 124949 were well-supported (100%); isolate GZ14-G-1 with type strain of C. gloeosporioides IMI 356878 were well-supported (100%); isolate YN33-1 with type strain of C. liaoningense CAUOS2, isolate GZ10 with type strain of C. jiangxiense LC3463 obtained 100% support; HN23-5 with type strain of C. cordylinicola ICMP 18579 were well-supported (100%); GZ23-3 with type strain of C. musae ICMP 19119 were well-supported (100%).