Sample collection and fungal isolation.
In 2016 and 2017, mango leaves, fruits and branches with anthracnose symptoms were collected from six provinces (Hainan, Yunnan, Sichuan, Guangdong, Guizhou and Fujian) in China, covering over an area about 1950 km by 800 km. Mango orchards were sampled with minimum 2 km distance. Lesion margins on mango leaves and fruits were cut to 3 mm X 3 mm in size, surface sterilized with ethanol (75%) for 15 s, sodium hypochlorite 1% (vol/vol) for 2 min, followed by rinsing three times in sterile distilled water for 30 s. Isolated fungus were cultured on potato dextrose agar (PDA) plates and kept in 25°C for incubation. Fungal growth was examined daily for 14 days. Fungal isolates were stored on PDA slants at 4°C in the dark.Morphological characterization. All isolates were cultured on PDA plates at 25°C under fluorescent light continuously for 5 to 7 days.
Mycelial plugs (6 mm diam) were cut from colony margins and placed in the center of each Petri dish (90 mm diam.) with four replications. Color of the colony and culture diameters (two perpendicular directions) were recorded after 3, 5, 7 and 10 days at 25°C. Hyphal growth rate (cm/day) was calculated based on colony diameters. Conidial production, shape and the size of conidia were examined up to three weeks on PDA plates, in which isolates were kept at 25 °C for incubation. Conidia of 120 per isolate were measured for length and width. Conidial appressoria were formed after conidia had germinated on glass slides placed in moisture plates overlaid with sterilized absorbent paper.
Mycelial appressoria were produced using a slide culture technique described by Cai et al. (2009). PDA plugs (1cm X 1 cm) were placed in an empty Petri dish, and the edge of the agar was inoculated with conidial spores and a sterile cover slip was placed over the inoculated agar. After 7 days, the shape and size of the appressoria formed on the underside of the cover slip were recorded. For each isolate, at least 30 appressoria were measured.
Photos were taken under a Nikon Eclipse Ni-E microscope.DNA isolation, PCR, and sequencing. Isolates were cultured on PDA in Petri plates overlaid with cellophane for 7 days at 25°C. Mycelia were harvested with a sterilized spatula, and genomic DNA of the isolates was extracted using cetyltrimethyl ammonium bromide method (Taylor et al.
1993). All strains in this study were subjected to analysis of the rDNA-ITS (ITS), partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH), partial actin (ACT), partial ?-tubulin (TUB2), and partial chitin synthase (CHS-1) genomic regions (Table 1). PCR amplification of GAPDH was done using the primers GDF1 and GDR1 (Templeton et al. 1992), and CHS-1 with CHS79F and CHS354R (Carbone and Kohn 1999).
The partial ACT, TUB2 and ITS regions were amplified with the primers-pair ACT512F and ACT783R (Carbone and Kohn 1999), T1 and Bt2b (Glass and Donaldson 1995), and ITS1 and ITS4 (White et al. 1990), respectively (also see Table 1). All PCR reactions were conducted in 50-?l volumes containing 1× PCR buffer, 0.2 mM concentrations of each dNTP, 4 mM MgCl2, 0.5 ?M concentrations of each primer, 0.
5 units Taq DNA polymerase (Takara), and 1 ?l of template DNA (20 ng/?l). The PCR program for GAPDH, ACT, TUB2 included a denaturation step at 94°C for 2 min, followed by 35 cycles at 94°C for 45 s, 60°C for 45 s, 72°C for 1 min and a final cycle at 72°C for 10 min. The PCR program for the ITS region included a 2 min denaturing step at 94°C followed by 34 cycles at 94°C for 1 min, 55°C for 30 s, 72°C for 1 min and a final cycle of 10 min at 72°C. The amplification products were sent to Shanghai Sangon Company for purification and sequencing. Phylogenetic analyses. DNA sequences generated with forward and reverse primers were analyzed to obtain consensus sequences using DNAMAN version 7.
0. Sequences of mango Colletotrichum isolates obtained from China were submitted to Genbank (Table2). Sequences were compared by BLAST (Altschul et al. 1990) against the NCBI NR database.
Sequences from extype or ex-epitype isolates of Colletotrichum species from GenBank were selected for the phylogenetic analyses (Table 3). Sequence alignments for each locus and multi loci were made with ClustalX v. 1.83. Phylogenetic trees were constructed using MrBayes software version 3.2.
6 (Ronquist et al., 2012). Nucleotide substitution models were generated using jmodeltest-2.1.7 (Darriba et al.
, 2012), and the GTR+I+G model with gamma-distributed rate was selected for constructing the phylogenic tree. Four runs with four chains for 50,000,000 generations were set up and the first 25% of generations were discarded as burn-in. The analyses were sampled every 1000 generations, which were stopped when the average standard deviation of split frequencies was below 0.01.