Colletotrichum acutatum KC05 isolated from an infected pepper fruit in Gangwon Province of South Korea was used in this study (Han et al., 2016). The isolate was identified based on combined sequence analyses of the nuclear ribosomal internal transcribed (ITS) region, β-tubulin (TUB2), actin (ACT), partial sequences of the chitin synthase 1 (CHS-1), an intron sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and histone 3 (HIS3) genes (Damm et al., 2012; da Silva et al., 2017). The six gene sequences of C. acutatum KC05 were obtained from Han et al. (2016), and the gene sequences of other Colletotrichum species (C. acutatum, C. scovillei, and C. simmondsii) were obtained from GenBank (Supplementary Table 1). Percentage sequence identities of the six genes were obtained from the National Center for Biotechnology Information database (NCBI) using nucleotide BLAST. A phylogenetic tree was generated using maximum likelihood analyses based on combined analyses of the six gene sequences of the selected Colletotrichum species.

Fungal conidia for protoplast generation were collected from a 10-day-old oatmeal medium agar (5% oatmeal and 2.5% agar) culture, inoculated into 100 ml complete medium broth (0.6% yeast extract, 0.6% casamino acids, and 1% sucrose), and cultured on a shaker at 150 rpm for 1 day at 25°C in the dark. The resultant mycelia were harvested onto a sterilized cheese cloth and washed twice with 20% sucrose. Lysing enzyme and driselase were suspended in 1 M NH₄Cl and sterilized through 20 μm pore filters (Sigma, St. Louis, MO, USA). To evaluate the enzymatic effects for protoplast generation, the washed mycelia were divided into 1,500 mg aliquots in 50 ml conical centrifuge tubes (SPL, Pocheon, Korea) and resuspended with 10 ml 1 M NH₄Cl containing different lytic enzymes. Then the suspension was incubated at 25°C with gentle shaking at 70 rpm, and protoplasts were counted using a hemocytometer. Protoplasts were pelleted by centrifugation at 5,000 rpm, washed, and suspended individually in 1.5 ml microcentrifuge tubes containing 200 μl 1× STC buffer (40% sucrose, 1 M Tris-HCl, and 1 M CaCl₂). A differential interference contrast microscope (Axio Imager.A2, Zeiss, Jena, Germany) was used to examine the shapes of protoplasts. Protoplasts were stored at −75°C. Experiments were conducted in triplicate and repeated three times. All data were processed using the SigmaStar statistical software package (SPSS Science, Chicago, IL, USA), and error bars represent 95% confidence intervals.

Hygromycin B (Millipore, Billerica, MA, USA) and geneticin (Gibco Life Technologies, Carlsbad, CA, USA) were used as selection antibiotics. Aliquots (200 μl) of protoplast suspension were transferred to 15 ml conical centrifuge tubes, mixed with an equal volume of PTC buffer (2× STC plus 40% PEG), incubated for 20 min at room temperature, and gently shaken in a rotary shaker for 5 h. Different concentrations of antibiotics and incubated protoplast aliquots were added to regeneration media (20% sucrose, 0.3% yeast extract, 0.3% casamino acids, 1% glucose, and 0.8% agar; autoclaved and cooled to 55°C) and poured into Petri dishes (90 mm in diameter). Plates were incubated at 25°C in the dark for 4 days.

Nineteen loci were selected from 10 of 27 different scaffolds (Han et al., 2016). For double-joint PCR and Southern blots, genomic DNA was extracted using a quick DNA extraction method (Chi et al., 2009). Gene knock-out constructs were generated using double-joint PCR (Park et al., 2014). Primer sequences used in this study are listed in Supplementary Table 2. The hygromycin phosphotransferase gene (HPH) cassette was amplified using the primers HPH_F (5′-GGCTTGGCTGGAGCTAGTGGAGG-3′) and HPH_R (5′-CTCCGGAGCTGACATCGACACCAAC-3′) from pBCATPH (Kim et al., 2009), and approximately 1.4 kb of two flanking regions of target loci were amplified using the primers 5F/R and 3F/R. The two fragments of flanking regions and the HPH cassette were fused using double-joint PCR, and the final construct was amplified using the primers NF and NR. Five micrograms of the construct haboring hygromycin resistance gene cassettes were introduced into 200 μl C. scovillei KC05 protoplasts (1 × 10⁷ protoplasts/ml) using PEG-mediated transformation (Park et al., 2014; Sweigard et al., 1992). Transformed protoplasts and hygromycin B (200 μg/ml) were mixed with 100-ml regeneration media (autoclaved and cooled to 55°C) and poured into 90-mm Petri dishes. The number of colonies growing on the regeneration media was counted 4 days after transformation, and PCR screening was performed using the primers SF/SR. Southern blot was performed according to standard procedures to determine the number of copies of the transformed construct (Shin et al., 2014).

Colletotrichum acutatum is a species complex with many members (Baroncelli et al., 2017). The previously identified strain C. acutatum KC05 (Han et al., 2016) was used in the present study to confirm the identity in C. acutatum sensu lato. Gene sequences of ITS, TUB2, ACT, CHS-1, GAPDH, and HIS3, which are mainly used in species classification (Caires et al., 2014), were analyzed in C. acutatum KC05, for comparison with those in C. scovillei CBS 126529, C. simmondsii CBS 122122, C. fioriniae CBS 128517, C. acutatum CBS 112996, C. graminicola CBS 112999, C. higginsianum IMI 349063, C. gloeosporioides CBS 112999, and C. orbiculare CBS 104-T. This analysis revealed that the ITS, TUB2, ACT, GAPDH, and HIS3 gene sequences of C. scovillei KC05 had 100% identity matches with C. scovillei CBS 126529, and the CHS-1 sequence exhibited 99% identity matches (Table 1). In the five multigene analyses, C. scovillei KC05 showed a higher match (> 90% identity) with members in C. acutatum sensu lato, C. scovillei, C. simmondsii, C. fioriniae, and C. acutatum, compared to the other species, C. graminicola, C. higginsianum, C. gloeosporioides, and C. orbiculare. A phylogenetic tree was subsequently constructed using the multigene sequences to analyze the genetic relationship among species.

As expected, C. scovillei KC05 was grouped in a clade with the members of C. acutatum sensu lato, C. scovillei, C. simmondsii, C. fioriniae, and C. acutatum, in which C. scovillei KC05 was almost identical to the reference strain C. scovillei CBS 126529 (Fig. 1). This result is consistent with that of Oo et al. (2017) and further indicates that C. acutatum KC05 is C. scovillei.

As a step toward understanding pepper anthracnose disease, we optimized a transformation system of C. scovillei KC05. For protoplast generation of C. scovillei KC05, two commonly available enzymes, lysing enzyme and driselase, were tested on C. scovillei KC05 mycelia. At 1 h after incubation of C. scovillei KC05 mycelia with lysing enzyme, 6.0 × 10⁴ protoplasts/ml were released with 0.5% lysing enzyme (Fig. 2). The addition of more enzyme enhanced protoplast yields. Averages of 13.0 × 10⁴ and 17.6 × 10⁴ protoplasts/ml were released from C. scovillei KC05 mycelia with 1% and 2% lysing enzyme, respectively. Longer incubation times also enhanced protoplast yields. At 3 h after incubation with 0.5% lysing enzyme, 75 × 10⁴ protoplasts/ml were released, which was 12.5-fold higher than after 1 h incubation. However, prolonged incubation for more than 3 h resulted in protoplast cell lysis; only 33.3 × 10⁴ protoplasts/ml were released from mycelia at 5 h after incubation, which was approximately 2.2-fold lower than at 3 h incubation. Driselase released lower numbers of protoplasts compared to lysing enzyme. The maximum release of protoplasts with driselase was obtained with 0.8% driselase at 3 h incubation, which released approximately 58 × 10⁴ protoplasts/ml. A combination of the two enzymes released more protoplasts than just one enzyme alone; 2% lysing enzyme and 0.8% driselase with 3 h incubation produced the maximum release of protoplasts, 860 × 10⁴ protoplasts/ml.

The shapes of released protoplasts were spherical, and their average size was 8.82 ± 0.06 μm in diameter after 3 h incubation in the 2% lysing enzyme and 0.8% driselase mixture (Fig. 3). After 5 h incubation in the same mixture, the average size of protoplasts was 11.09 ± 0.07 μm, much larger than at 3 h incubation. Noticeably, lysed protoplasts were observed after 5 h incubation. These results indicate that the combination of lysing enzyme and driselase at 3 h incubation is most effective for protoplast generation from mycelia of C. scovillei.

The sensitivity of C. scovillei KC05 protoplasts to antibiotics was assessed to select a positive transformation marker and to determine the minimal concentration of antibiotics required. Hygromycin B and geneticin were used as the selective antibiotics, as both have been widely used for fungal transformation. Results indicated that hygromycin B completely inhibited the growth of C. scovillei KC05 protoplasts at a concentration of 200 μg/ml (Fig. 4). However, at lower concentrations (50 and 100 μg/ml), protoplast growth was not completely inhibited. Compared to hygromycin B, protoplasts were more resistant to geneticin; protoplast growth was not completely inhibited at concentrations of either 200 or 400 μg/ml. However, 800 μg/ml geneticin was able to completely inhibit protoplast growth. Based on these results, different concentrations of the two antibiotics were used in the selection of transformants of C. scovillei KC05.

To better understand the transformation efficiency of C. scovillei KC05, a total of 19 loci were randomly selected from the KC05 genome (Han et al., 2016). These loci were scattered throughout 10 (out of 27) different scaffolds, which covered 49.9 Mb, equivalent to 84.1% of the whole genome (Fig. 5). For the homology-dependent replacement of a targeted gene, knock-out DNA constructs were generated for the 19 loci using PCR. Protoplasts of KC05 were transformed with DNA of each knock-out construct (Figs. 5 and 6A). Transformed protoplasts were grown on regenaration media in 90-mm Petri dishes supplemented with hygromycin B. Many transformants were regenerated at 4 days after transformation, and 45-138 transformants were subsequently selected to simply screen putative knock-out transformants using a PCR method (Table 2). The PCR screening was performed with the primers SF/SR using genomic DNA extracted from the transformants (Fig. 6B). Two bands at 1.7 and 3.9 kb were amplified within many transformants (Fig. 6B), as illustrated in Fig. 6A. These results suggest that the targeted gene remained intact and that the DNA constructs were ectopically integrated in the genome. However, the detection of only the 1.7 kb band in several transformants suggests possible knock-out candidates via a homology-dependent replacement event. By measuring one band in transformants for the 19 loci, we obtained a variable number of positive candidates, ranging from 5 to 26 (Table 2). For example, 24 of 81 transformants were positively detected as knock-out candidates for the CSP_010985 locus, while 8 of 111 transformants were positive in the PCR screening for CSP_006969. The average number of candidates for knock-out mutants was 17.1% for the 19 loci tested in the PCR screening. To confirm the target gene-specific replacement in knock-out transformants, Southern blot analyses were performed with transformants. As illustrated in Fig. 6A, genomic DNA of transformants was digested using NdeI and then hybridized with the indicated probe. The analyses revealed that 4.0 kb NdeI bands were only detected in knock-out mutants, while 6.7 and 2 kb bands originated from transformants with ectopic integration and the wild-type copy of the gene, respectively (Fig. 6C). Knock-out mutants were obtained for different sizes of the 19 targeted loci (Table 2).