Introduction

Perilla frutescens, or shiso, is an herbaceous plant belonging to the Lamiaceae family and was originally cultivated throughout East and South-East Asia as a culinary herb, oil source and as a traditional medicine1,2. The plant produces a significant amount of volatile compounds3 that contribute to its distinctive odour, as well as a high amount of polyphenols with antioxidant activities4. Apart from direct consumption, P. frutescens is also used industrially, whereby oil from the seed may be used as a drying oil2. In Korea, perilla oil has consistently been the third highest domestically produced vegetable oil with a volume of more than 30,000 metric tonnes produced in 2015/165. In Japan, 6,708 tonnes of P. frutescens were reported to be grown for direct consumption, while a further 2,763 tonnes were grown for industrial uses in 20146. In the West, perilla has been grown as an ornamental plant since the Victorian Era, where it is known as the “beefsteak plant”7.

Fukui (1925) first reported an anthracnose disease of perilla in Japan and described the causal organism as Colletotrichum yoshinaoi Fukui8. In a study from Korea, the causal agents of perilla anthracnose were identified as C. gloeosporioides, C. coccodes, C. dematium and Glomerella cingulata, which had previously been regarded as the sexual morph of C. gloeosporioides9. In the study, the authors hypothesised the species described by Fukui to be a synonym of C. gloeosporioides, since C. gloeosporioides was more frequently isolated from infected plants9. However, none of these reports was confirmed by molecular data and the systematic position of C. yoshinaoi, which lacks a living type strain, is unknown.

More recently, C. destructivum was identified as being responsible for perilla anthracnose in Japan using ITS sequences10, while fungal strains causing anthracnose of Lamium amplexicaule (henbit), that also belongs to the Lamiaceae, were identified as C. higginsianum based on ITS sequences11. However, all previous reports were carried out before the epitypification of the respective species and the treatments of the respective species complexes12,13,14,15,16 and were based on morphology or ITS only.

With the advent of affordable high throughput genome sequencing technologies, the genomes of multiple members of the Colletotrichum genus of plant pathogenic fungi have been sequenced and released17,18,19,20,21,22,23,24,25,26,27. Sequenced genomes have included strains belonging to different species complexes, which comprise closely related species that are phylogenetically distinct from other members of the same genus. Members of the same species complex exhibit similarities in terms of their infection lifestyles and whole genome comparative analyses have revealed genomic adaptions that contribute to these differences17,18,28. For example, members of the Colletotrichum graminicola species complex which specifically infect graminiaceous hosts have reduced numbers of pectin-degrading enzymes17,18. Comparisons between different members of the same species complex, such as between C. sublineola and C graminicola, which infect sorghum and maize respectively, have also been performed; this led to the identification of genes that were not found to be conserved between different members of the same species complex, and which may contribute to adaptation to their specific host niches29.

The aims of this study were to characterise one of the causal agents of anthracnose of P. frutescens in Japan based on multi-locus sequence data and morphology. Further, we aimed to characterise the species at the molecular level by sequencing and assembly of its genome. In addition, we wanted to identify what types of genes show different conservation patterns between shiso-infecting Colletotrichum and close relatives in the Colletotrichum genus. In particular, we aimed to analyse the conservation patterns of genes encoding small, secreted proteins, since these may contribute to differences in infection outcomes.

Results

Multi-locus phylogenetic analysis

An initial BLASTn search of the NCBI non-redundant nucleotide database using the internal transcribed spacers (ITS) sequence from Colletotrichum strain JCM 31818 from P. frutescens as a query was conducted, revealing that seven of the top ten hits, differing by 7–8 mismatches, belong to the C. destructivum species complex (Supplementary Table S1). As strain MAFF 240106 was also isolated from P. frutescens and was previously identified as C. destructivum on the basis of its ITS sequence10, sequences from both strains were compared and found to be identical. In order to identify these strains to the species level, a phylogenetic tree based on ITS, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), actin (ACT) and beta-tubulin (TUB2) sequences was calculated and used for comparison of the strains from P. frutescens with all currently accepted species in the C. destructivum species complex (Supplementary Table S2). DNA sequences obtained from the MAFF Genebank project of several strains isolated from L. amplexicaule, a host from the same family as P. frutescens, which had previously been identified as C. higginsianum based on ITS sequences11, were also included (Supplementary Table S2).

In the multi-locus phylogenetic analysis, sequences were aligned, trimmed and then concatenated to generate a sequence alignment comprising 1,778 characters (gene boundaries ITS: 1–546, GAPDH: 547–740, CHS-1: 741–1,020, ACT: 1,021–1,275, TUB2: 1,276–1,778) from 94 isolates.

In maximum parsimony analyses, 1,318 characters were found to be constant, while 266 and 194 of the variable characters were found to be parsimony informative and uninformative respectively. The heuristic search yielded 64 equally most parsimonious trees (tree length: 659, consistency index (CI): 0.819, retention index (RI): 0.931, rescaled consistency index (RC): 0.763, homoplasy index (HI): 0.181). Analysis of the concatenated alignment as well as alignments of each individual gene indicated that the strains from P. frutescens are distinct from the other members of the C. destructivum species complex (Supplementary Figs 1–6) and may represent a separate species.

To confirm this, maximum likelihood and Bayesian phylogenetic analyses were carried out. The best model for phylogenetic analysis of ACT, CHS-1, GAPDH, ITS and TUB2 was calculated as HKY + G, K80, HKY + I, K80 + I + G and K80 + I, respectively. The consensus tree obtained from Bayesian analysis of the multi-locus alignment showed the strains from P. frutescens form a distinct clade on a long branch with a Bayesian posterior probability value of 1.00 (Fig. 1), while the strains from L. amplexicaule (MAFF 244502, 244503) clustered with C. higginsianum, confirming their identities as C. higginsianum strains. In each consensus tree of individual loci generated by Bayesian analysis (Supplementary Figs 7–11), the strains obtained from P. frutescens formed a distinct clade within the C. destructivum species complex with Bayesian posterior probability values above 0.9. However, the position of this clade containing isolates from P. frutescens differed depending on the locus. The topologies of the ML trees calculated from the single and multi-locus alignments were consistent with the results from the Bayesian analyses (Supplementary Figs 12–17).

Figure 1
figure 1

Multi-locus phylogenetic tree based on ITS, GAPDH, CHS-1, ACT and TUB2 sequences of the Colletotrichum destructivum species complex using Colletotrichum boninense MAFF 305972 as an outgroup. Values at the nodes are Bayesian posterior probability values above 50%. C. shisoi strains are highlighted in blue. C. higginsianum strains isolated from Lamium amplexicaule are highlighted in red. Ex-type cultures are marked with an asterisk and in bold. Branches with double-bars are truncated two-fold.

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Taxonomy

Based on the DNA sequence data, the Colletotrichum species from P. frutescens was found to be distinct from other species in the C. destructivum species complex and therefore described as a new species below.

Colletotrichum shisoi

P. Gan, A. Tsushima, M. Kawaradani, Damm & K. Shirasu sp. nov., Mycobank MB 828333, Fig. 2.

Figure 2
figure 2

Colletotrichum shisoi (ex-holotype strain JCM 31818). (a) 10-day old culture on PDA. (b–d) Conidia from P. frutescens leaves, PDA and SNA, respectively. (e–j) Conidiophores on Anthriscus sylvestris stems. (k–l) Conidiomata on A. sylvestris stems. (m). Appressoria formed on OA. (n–o) Appressoria formed on SNA. (p–q) Appressoria formed on A. sylvestris stems. (r–s) Setae formed on P. frutescens leaves. (t–u) Trypan blue stained C. shisoi growing in perilla leaves at 40 hpi. (v–w) Biotrophic hyphae at 60 hpi in infected P. frutescens leaves. (x) Symptoms on P. frutescens leaves at two weeks after inoculation with C. shisoi. Scale bar of (b) applies to (e–j); scale bar of (m) applies to (n–s) and scale bar for (t) applies to (u–w).

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Etymology

Refers to the host from which the species was isolated, Perilla frutescens var. crispa, commonly known as shiso.

Sexual morph not observed. Asexual morph on synthetic nutrient-poor agar (SNA). Vegetative hyphae 3–5.5 μm, average (av.) ± standard deviation (SD): 4.0 ± 1.0 µm diam, hyaline, smooth-walled, septate and branched. Chlamydospores not observed. Conidiomata absent, conidiophores formed directly on conidiogenous cells. Setae not observed Conidiogenous cells hyaline to pale brown, smooth-walled, septate, cylindrical. Conidia hyaline, smooth-walled, aseptate, cylindrical, straight, with both ends round or with one end slightly tapered, 13–22.5 µm × 3–5 µm, av. ± SD: 16.5 ± 2.5 µm diam × 4.0 ± 0.5 µm, L/W ratio: 4.5. Appressoria single, olivaceous black, smooth-walled, obovoid to clavate with truncate base, 5.5–10 µm × 4–7.5 µm, av. ± SD: 7.5 ± 1.0 µm diam × 6.0 ± 1.0 µm, L/W ratio: 1.5.

Asexual morph on oatmeal agar (OA). Vegetative hyphae 1.5–4.0 μm diam, hyaline, smooth-walled, septate and branched. av. ± SD: 2.9 ± 0.5 µm diam. Setae unbranched, medium brown, smooth-walled, 1–2 septate, 29.5–76.0 µm long, tip rounded. Conidia 12–23 µm × 3.5–5.5 µm, av. ± SD: 16.0 ± 3.0 × 4.5 µm ± 0.5 µm, L/W ratio: 3.5. Appressoria single or in groups, greenish olivaceous to olivaceous buff, smooth-walled, globose to subglobose, 5.5–11 µm × 4.5–10 µm, av. ± SD: 8 ± 1.0 µm diam × 6.5 ± 1.0 µm, L/W ratio: 1.0.

Asexual morph on PDA. Vegetative hyphae 2.0–7.0 μm diam, hyaline, smooth-walled, septate and branched. av. ± SD: 4.0 ± 1.0 µm diam. Conidia 15.0–27.0 µm × 3.0–5.0 µm, av. ± SD: 17.0 ± 2.0 µm × 4.0 ± 0.0 µm, L/W ratio: 4.0. Setae unbranched, medium brown, smooth-walled, 1–2-septate, 23.5–78.0 µm in length, tip rounded. Appressoria 7.0–11.0 µm × 5.0–8.0 µm, av. ± SD: 9.0 ± 1.0 µm × 7 ± 1.0 µm, L/W ratio: 1.0.

Asexual morph on autoclaved Anthriscus stem. Conidiomata conidiophores formed on hyaline cells. Setae not observed. Conidiophores hyaline, smooth-walled, simple. Conidiogenous cells hyaline, smooth-walled, straight with round ends or slightly tapered at one end. Conidia hyaline, smooth-walled, aseptate, cylindrical, straight to slightly curved, with both ends straight or with one end tapered 15.5–28.5 µm × 3.0–5.0 µm, av. ± SD: 19.0 ± 3.0 µm × 4.0 ± 0.5 µm, L/W ratio: 5.0. Appressoria single or in loose groups, globose to subglobose, occasionally with an irregular in shape, 3.0–11.0 µm × 3.5–7.5 µm, av. ± SD: 6.5 ± 2.0 µm × 5.0 ± 1.0 µm, L/W ratio: 1.0.

Asexual morph, infection structures and symptoms in/on leaves of Perilla frutescens: Lesions on leaves small, elliptical or irregular, appearing on cotyledons and fully developed leaves, gradually enlarging and becoming dark brown. Acervuli observed forming on lesions under conditions of high humidity. Setae unbranched, medium brown, smooth-walled, 1–4-septate, 77.5–45.5 µm long and 3–8.5 µm diameter in the base, base ampulliform or cylindrical, tip rounded. Conidia hyaline, smooth-walled, aseptate, cylindrical, straight to very slightly curved, with ends round or with one end tapered, 11.0–21.5 µm × 3.0–5.0 µm, av. ± SD: 17.0 ± 2.0 µm × 4.0 ± 0.5 µm, L/W ratio: 4.5. Appressoria single or in loose groups, 4.0–8.0 µm × 3.0–6.0 µm, av. ± SD: 6.0 ± 1.0 µm × 4.5 ± 1.5 µm, L/W ratio: 1.5. Intracellular hyphae in detached Perilla frutescens leaves: bulbous, hyphae observed within penetrated cells from 40 hpi.

Culture characteristics

Colonies on SNA at 25 °C flat with entire margin, hyaline, filter paper and Anthriscus stem partly covered with salmon and dark chestnut acervuli. Whitish aerial mycelia on medium. Reverse same colors. Growth rate 34–39 mm in 7 d (48–51.5 mm 10 d). Colonies on OA flat, radially striate with lobate edge, reverse the same, Growth rate 36.5–37.5 mm in 7 d (51.5–53 mm 10 d), olivaceous brown to brick, with white aerial hyphae at the edge, colonies of strain MAFF 240106 differ in forming radial crinkles, Colonies on PDA. Flat, olivaceous brown to light orange, hyaline at the edge. Growth rate 46.5–51 mm in 7 d (68–71 mm 10 d). Conidia in mass saffron.

Materials examined

JAPAN, Osaka, Ibaraki City from lesions of cultivated Perilla frutescens var. crispa cv. Aka-shiso, collection date 1 August 2006, collected by M. Kawaradani (TNS-F-40462 holotype, culture ex-holotype JCM 31818); JAPAN, Osaka, Ibaraki City from lesions of cultivated Perilla frutescens var. crispa cv. Aka-shiso, collection date July 2006, collected by M. Kawaradani (MAFF 240106). MAFF 240106 was characterised as being able to infect green and red shiso; as well as egoma varieties of Perilla frutescens from Japan, with strongest symptoms on red shiso10.

Notes: Colletotrichum shisoi is only known from P. frutescens plants in Japan. It belongs to the C. destructivum species complex and can be identified by its ITS, ACT, CHS-1, GAPDH, TUB2 sequences. Fukui (1925) reported a new anthracnose disease of P. frutescens in Japan caused by C. yoshinaoi8. Kim et al. (2001) regarded the name C. yoshinaoi as invalid because both a Latin diagnosis and the indication of a type is lacking9. However, a Latin diagnosis was only required between 1 January 1935 and 31 December 2011, and an indication of a type is only mandatory from 1 January 1958 (Art. 37.1)30; C. yoshinaoi is therefore validly described. Conidia of C. yoshinaoi were described as being oval with round ends and sometimes slightly curved, measuring 15–17 × 4–5 µm with an L/W ratio = 4, which is overlapping with C. shisoi. However, setae of C. yoshinaoi measure 40–50 × 3 µm and are sometimes slightly curved and appressoria are round (corresponding to L/W ratio of 1) and about 6 µm diam, while setae of C. shisoi are larger, measuring 45.5–77.5 × 3–8.5 µm and are straight and appressoria of C. shisoi on the host plant measure 4–8 µm × 3–6 µm with a L/W ratio of 1.5. Moreover, C. yoshinaoi infects stems causing early defoliation and inhibits fruiting and was never observed on leaves8, whereas C. shisoi infects cotyledons and fully developed leaves. Therefore, Kawaradani (2008) did not regard strain MAFF 240106 (included in this study as C. shisoi) as C. yoshinaoi but identified it as C. destructivum. Consequently, we describe the species in the C. destructivum complex infecting perilla leaves as a new species, instead of epitypifying C. yoshinaoi.

Another Colletotrichum species, C. perillae, causes a similar disease as C. yoshinaoi on stems and pedicels of P. ocymoides in the Primorskaya and Ussurskaya Oblast, an area in Russia close to Japan. This species forms acervuli with straight, cylindrical conidia with rounded ends, measuring 18–22 × 4.5–6 µm and straight to flexuous, aseptate, olivaceous setae becoming paler towards the tip, measuring 43–48 × 4–5 µm31. Apart from the fact that the disease caused is different, the conidia of C. perillae are larger than those of both C. shisoi (and C. yoshinaoi) and setae are shorter and possibly also differ in septation from those of C. shisoi. This species was also described without a Latin diagnosis, but before 1 January 1935; the name is therefore invalid (Art. 39.1)32.

Pathogenicity tests

Three-week-old intact P. frutescens plants spray-inoculated with Colletotrichum shisoi JCM 31818 displayed typical symptoms of anthracnose lesions two weeks after inoculation while mock inoculated plants showed no symptoms (Fig. 3a). Symptoms were similar to symptoms of perilla anthracnose observed in nurseries of cultivated Aka-shiso P. frutescens plants previously reported by Kawaradani et al.10. Infected plants had smaller leaves than mock inoculated plants (Fig. 3a). These differences were reproduced in three independent experiments. The same fungus was consistently re-isolated from lesions of inoculated plants.

Figure 3
figure 3

Pathogenicity of Colletotrichum shisoi. (a) Perilla frutescens var. crispa Aka-shiso leaves inoculated with C. shisoi (top) compared to mock inoculated leaves (bottom) at two weeks post inoculation. Scale bar = 1 cm. The same results were observed in three independent experiments. (b) Boxplots comparing area sizes of lesions formed on leaves of Arabidopsis thaliana ecotypes after inoculations with C. shisoi (Cs) and C. higginsianum (Ch). The distributions of lesion areas were found to be significantly different between A. thaliana leaves inoculated with C. shisoi compared to leaves inoculated with C. higginsianum in all three A. thaliana ecotypes according to Mann-Whitney U tests (P-values < 0.01). Significant differences were detected in two independent experiments.

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As C. shisoi is closely related to the Arabidopsis thaliana-infecting species C. higginsianum, we tested if it can infect A. thaliana. C. shisoi did not form lesions on A. thaliana ecotypes Bay-0 and Ws-0 but could form lesions on Ler-0, although to a lesser extent than C. higginsianum (Fig. 3b). The distributions of lesion areas were found to be significantly different between C. shisoi and C. higginsianum with P-values < 0.01 according to Mann-Whitney U tests in all three ecotypes.

Genome sequence analysis

The genome size of C. shisoi was estimated, according to k-mer analysis, to be 58.6 Mb and sequenced to 603 × coverage. A total of 36,350 contigs were assembled from the 100 bp paired-end libraries with an N50 value of 7,997. These were then assembled into 20,745 scaffolds with N50 of 9,321 bp (Table 1). According to BUSCO analysis, 98.3% of 3,725 sordariomycete conserved proteins could be identified as complete sequences within the assembly, with an additional 0.8% found to be fragmented, indicating coverage of most of the gene coding space (Table 1). A total of 11,848 genes were predicted in the C. shisoi genome. The number of genes encoded is consistent with the gene numbers predicted in other Colletotrichum species (Fig. 4a), whose numbers range from 16,287 genes (C. gloeosporioides) to 10,419 (C. chlorophyti).

Table 1 Genome assembly statistics.
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Figure 4
figure 4

Conservation of all predicted proteins in selected Colletotrichum species. (a) Maximum likelihood phylogeny based on 128,141 characters from 254 eukaryotic conserved single copy protein orthologs identified by BUSCO analysis in different members of the Colletotrichum genus, showing the relationship between C. shisoi and other Colletotrichum species complex members. Predicted gene numbers are indicated by the bars on the right. Fusarium oxysporum was used as an outgroup. Numbers at the nodes represent percent support values out of 500 bootstrap replicates. (b) UpSet plot showing conservation of protein orthogroups in C. graminicola (GRM), C. incanum (INC), C. tofieldiae (TOF), C. higginsianum (HIG) and C. shisoi (SHI). Box plots show the distributions of orthogroup protein length means. Bars represent number of orthogroups showing specific conservation patterns with numbers also given above each bar. Numbers in brackets show number of proteins that belong to orthogroups according to their respective conservation pattern. (c) Bar chart showing GO terms significantly enriched among C. shisoi genes present only in the C. destructivum species complex (C. shisoi and C. higginsianum). FDR: False discovery rate of hypergeometric test after Benjamini-Hochberg multiple testing correction.

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Conservation of proteins amongst Colletotrichum species

The conservation of genes between C. shisoi and four closely related and sequenced Colletotrichum species was assessed (Figs 4 and 5). The four that were chosen for comparisons were C. higginsianum, which has a chromosome-level assembly, publicly available annotations and also belongs to the C. destructivum species complex, C. incanum and C. tofieldiae, from the C. spaethianum species complex, and C. graminicola from the C. graminicola species complex. Members of the C. spaethianum and the C. graminicola species complexes were selected since they are closely related to the C. destructivum species complex (Fig. 4a). Among the five species assessed, C. higginsianum encodes the greatest number of predicted genes (14,651 genes). The number of predicted proteins for the other species were closer to the number of genes in C. shisoi with 11,436, 12,501 and 12,006 proteins predicted in C. incanum, C. tofieldiae and C. graminicola, respectively (Fig. 4a). A total of 11,914 orthogroups with two or more proteins were identified (Fig. 4b). Of these, 7,950 groups (74.0% proteins from C. shisoi, 63.0% from C. higginsianum, 78.3% from C. incanum, 74.2% from C. tofieldiae and 73.7% from C. graminicola) were conserved in all five species (Supplementary Tables S3 and S4). From this analysis, all C. shisoi genes could be classified into an orthogroup with a related sequence in one of the four other species or in the same genome. Only one orthogroup was predicted to be C. shisoi-specific. This orthogroup consisted of seven proteins annotated as MFS transporter proteins. Similarly, all C. higginsianum proteins were classified into an orthogroup with only two orthogroups found to be specific to C. higginsianum, one consisting of 13 ABC transporter genes and the second, consisting of 8 secondary metabolite regulator laeA protein-encoding genes. As expected from their close evolutionary relationship, C. shisoi and C. higginsianum were found to share an additional 2,585 orthogroups consisting of 23.4% proteins from C. shisoi and 20.8% proteins from C. higginsianum, including 1,026 orthogroups (8.7% proteins from C. shisoi and 7.4% proteins from C. higginsianum), which are present only in these two members of the C. destructivum clade. Proteins of C shisoi from the C. destructivum-specific orthogroups were significantly enriched for Gene Ontology (GO) terms involved in methyltransferase and protein kinase activity (FDR < 0.05) (Fig. 4c). In contrast, C. tofieldiae and C. incanum, which both belong to the Colletotrichum spaethianum clade, share only 97 orthogroups, consisting of 1.3% proteins from C. incanum and 0.9% proteins from C. tofieldiae, which were specific to these two members of the C. spaethianum clade.

Figure 5
figure 5

Conservation of secreted proteins in Colletotrichum species. (a) UpSet plot showing conservation of secreted protein orthogroups in C. graminicola (GRM), C. incanum (INC), C. tofieldiae (TOF), C. higginsianum (HIG) and C. shisoi (SHI). Box plots show the distributions of orthogroup protein length means. Bars represent number of orthogroups showing specific conservation patterns with numbers also given above each bar. Numbers in brackets show number of proteins that belong to orthogroups according to their respective conservation pattern. (b) Scatterplot showing orthogroup protein length means relative to the mean rate of pairwise non-synonymous mutations (dN)/synonymous mutations (dS) in each orthogroup.

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Conservation of secreted proteins

As secreted proteins are known to be important for infection, their conservation between the five species was also assessed (Fig. 5a, Supplementary Table S5). A total of 1,360 secreted protein orthogroups were identified. Of these, 540 orthogroups (39.7%) were found to be conserved in all five species. These included 48.4% proteins out of the 1,274 predicted secreted proteins from C. shisoi and 40.6% proteins of the 1,644 predicted secreted proteins from C. higginsianum (Supplementary Table S5). A further 154 secreted protein orthogroups, were identified as specific to the two C. destructivum clade members. In contrast, 28 secreted protein orthogroups were identified as being C. spaethianum clade-specific. No GO terms were found to be significantly associated with C. shisoi secreted proteins that were in C. destructivum clade-specific orthogroups. Since effector proteins tend to be small, secreted proteins under positive selection, we plotted the average lengths of orthogroups consisting of secreted proteins (Fig. 5a) to investigate if there was a relationship between conservation pattern and orthogroup protein length. All 2,186 C. destructivum clade-specific proteins (Fig. 4b) were found to be shorter than proteins belonging to orthogroups that were conserved in the five Colletotrichum species. Further, of particular interest to this study, the C. destructivum clade-specific secreted proteins (Fig. 5a) were found to be also under higher rates of positive selection, with higher rates of non-synonymous to synonymous mutations (dN/dS), compared to secreted proteins that were conserved in all five tested Colletotrichum species (Fig. 5b). In contrast, this was not observed among C. spaethianum clade-specific secreted proteins (Fig. 5b). No species-specific groups were identified amongst the secreted protein orthogroups in all five species, indicating that species-specific sequences did not belong to multi-gene families. A total of 846 secreted proteins, consisting of 216 proteins from C. graminicola, 128 proteins from C. incanum, 112 proteins from C. tofieldae, 225 proteins from C. higginsianum and 135 proteins from C. shisoi were not assigned to any orthogroup, and were found to be species-specific (Supplementary Table S5).

Discussion

Colletotrichum species can infect a wide range of plants. In this study, we identified a new species in the C. destructivum clade that infects the commercially important oil crop P. frutescens. Previously, species of the C. gloeosporioides clade, C. gloeosporioides, C. dematium and C. coccodes were reported as pathogens of P. frutescens in Korea by morphological examination of isolates9, and C. destructivum was identified as a pathogen of P. frutescens in Japan based on ITS sequences10. In this study, a multi-locus phylogenetic analysis showed that strains from P. frutescens previously identified as C. destructivum, are genetically distinct from other known species of the C. destructivum species complex, and were thus described as a new species, C. shisoi. Since well-studied species of the C. destructivum species complex have previously been confused with C. coccodes, C. gloeosporioides and Glomerella cingulata12, the strains from P. frutescens in Korea9 identified as these species could also represent C. shisoi and their re-examination may be warranted.

In order to characterise C. shisoi and to allow comparisons to other members of the Colletotrichum genus at the molecular level, the genome of C. shisoi was sequenced and assembled. The size of the C. shisoi assembly is 69.7 Mb, which is larger than those of other sequenced members in the C. destructivum species complex, including C. higginsianum33 (50.72 Mb), C. lentis21 (56.1 Mb) and, most recently, C. tanaceti26 (57.9 Mb). These sizes are closer to the genome size of C. shisoi estimated by k-mer analysis (58.6 Mb). A genome assembly size may deviate from k-mer estimates due to high levels of repeats or heterozygosity34. Members of the C. destructivum clade are known to be haploid pathogens that propagate asexually and thus heterozygosity is unlikely12. Further, BUSCO analysis of conserved genes reveals that only 0.1% of the conserved coding sequences present in the genome are duplicated, indicating that the genome is not heterozygous and that the gene coding regions at least are likely not to be duplicated. This is consistent with the predicted number of genes (11,848) in C. shisoi, which is within the range of other sequenced members of the C. destructivum species complex including the recently published genomes of C. lentis21 (11,436), C. tanaceti26 (12,172) and another isolate of C. higginsianum25 (MAFF 305635-RFP), which has 12,915 protein coding genes. It is noted that the genome assembly of C. shisoi generated in this study was sequenced using only short reads and is highly fragmented, with more than half of the scaffolds (11,424 scaffolds totalling 5.73 Mb) being less or equal to 1 kb in length. An earlier version of the genome of its close relative, C. higginsianum, which was assembled using a combination of Illumina GAII, Roche 454 and Sanger Fosmid reads, also suffered from fragmentation (10,269 scaffolds)18, possibly due to the abundance of transposable element-rich genomic regions. Since then, a chromosome level assembly for C. higginsianum has been generated using a combination of PacBio long reads and optical mapping data33. The C. shisoi genome may similarly be better resolved by adopting a similar sequencing strategy.

Comparison of the genomes of C. shisoi and C. higginsianum showed that the majority of C. shisoi genes (89%) have orthologous sequences in C. higginsianum. The latter species was previously reported11 to infect L. amplexicaule, a plant belonging to the Lamiaceae family. Both pathogens appear to adopt similar infection strategies. C. shisoi was observed to form bulbous intracellular hyphae within infected epidermal cells in early infection of P. frutescens leaves. These hyphae are morphologically similar to the primary, biotrophic hyphae formed by C. higginsianum infecting A. thaliana leaves35. Further, as in the case of C. higginsianum-infected A. thaliana plants, necrotic lesions formed later in infection. Taken together, these observations suggest that C. shisoi also adopts a hemibiotrophic infection strategy, as do other members of the C. destructivum complex36.

Previously, genus-wide analyses including members from the C. orbiculare, C. acutatum, C. graminicola, C. gloeosporioides and C. destructivum species complexes of Colletotrichum revealed that C. higginsianum has the highest number of lineage-specific genes amongst the genomes tested2. However, at the time, C. higginsianum was the only C. destructivum species complex member whose genome had been sequenced. Further, the assembly was highly fragmented, leading to the possibility that gene numbers were inflated. Since then, a chromosome level assembly for C. higginsianum has been published33 and our results indicate that C. higginsianum and C. shisoi do indeed have a high number of orthogroups specific to these two C. destructivum species complex members. The C. destructivum clade-specific genes were significantly enriched in kinases, indicating the presence of C. destructivum clade-specific signalling pathways. It is interesting to note that C. spaethianum clade members, despite their close phylogenetic relationship to the C. destructivum species complex, do not exhibit the same expansion in lineage-specific genes.

Secreted proteins that were specific to the two members of the C. destructivum species complex analysed in this study were also found to be subject to higher rates of diversifying selection than secreted proteins that were identified as C. spaethianum clade-specific. C. shisoi is less virulent on A. thaliana compared to C. higginsianum, forming significantly smaller lesions or no lesions at all on the accessions tested. Interestingly, the more distantly related strains from the C. spaethianum species complex, C. incanum and C. tofieldiae, have both been shown to infect A. thaliana plants17,37, indicating that components required for successful invasion of A. thaliana were possibly present in the ancestor of the C. destructivum and C. spaethianum clades. Given that small, secreted proteins known as effectors, which are important for manipulation of hosts, are often under diversifying selection to avoid recognition by specific host immune components38, the C. destructivum clade-specific secreted proteins could be candidate effectors involved in infection of host plants and their diversification may have resulted in the differences in the observed infection outcomes of C. higginsianum and C. shisoi.

Finally, Perilla frutescens produces a range of antimicrobial compounds and has been characterised by transcriptomic and metabolomic analyses39. The examination of the genome of its pathogen, C. shisoi, will provide insights into the mechanisms of this pathogen to overcome host defence and thus enable the development of better control strategies.

Materials and Methods

Isolates

The strains studied here originate from leaves of Perilla frutescens with anthracnose symptoms that had been collected in August 2006 and July 2006 from a perilla seedling bed in Ibaraki city, Osaka, Japan as previously described10. As described by Kawaradani et al. (2016), the seedling bed was located in the shaded part of a southwestern-facing mountain slope. Leaves showing symptoms were surface sterilised with sterile water and incubated on PDA plates containing 100 ppm streptomycin at 25 °C10. Isolates were isolated by hyphal tipping10. The holotype of the new species was deposited in the mycological herbarium of the National Museum of Nature and Science (TNS-F-40462), Tsukuba, Japan and the ex-type culture in the Japan Collection of Microorganisms (JCM 31818), Tsukuba, Japan. Isolates were stored as glycerol stocks at −80 °C and revived by incubation on PDA at 24 °C in the dark prior to experiments.

Phylogenetic analyses

Sequences of ACT, CHS-1, GAPDH, ITS and TUB2 were identified from the JCM 31818 assembly by BLASTn searches with sequences from C. higginsianum IMI 349063 and selecting sequence regions with the lowest E-values. The ITS sequence was used to query the NCBI non-redundant nucleotide database using default BLASTn settings to identify closely related fungal species.

Sequences for MAFF 240106 ITS, GAPDH, CHS-1, ACT and TUB2 were amplified using the primer pairs ITS-1F40 + ITS-441, GDF1 + GDR142, CHS-354R + CHS-79F, ACT-512F + ACT-783R43 and T144 + Bt-2b45. PCR was carried out in a thermocycler using 2 × PCR Taq polymerase mix (Promega) at 95 °C for 3 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min and a final extension step at 72 °C for 5 min. Phylogenetic trees were calculated as previously described46. Sequences of each locus (Supplementary Table S2) were aligned in MAFFT v7.21547 using the auto setting and trimmed using trimAl v1.2rev5948 using the automated1 setting. Maximum parsimony analyses were carried out with PAUP* (Phylogenetic Analysis Using Parsimony) version 4.0a (build 165)49 using a heuristic search of 100 random sequence additions with tree bisection and reconstruction (TBR) as the branch-swapping algorithm. All sites were treated as unordered and equally weighted with gaps treated as missing data. A total of 1,000 bootstrap replicates using the same settings were carried out to determine support for the trees. To determine the best model for analyses, jModelTest250 was run on alignments with BIC criterion. For single locus trees, maximum likelihood trees were calculated using RAxML-ng using the specified jModelTest2 model with 1,000 bootstrap replicates. For Bayesian inference phylogenies based on single loci were calculated twice using MrBayes (v3.2.1) with 5 × 106 generations, sampling every 1,000 generations. Under these settings, the average standard deviation of split frequencies was found to be 0.006037 for ACT, 0.007120 for CHS-1, 0.005246 for GAPDH, 0.007322 for ITS and 0.006178 for TUB2. For multi-locus sequence analysis, the trimmed alignments were concatenated and then a maximum likelihood tree was calculated with RAxML-ng51 using the specified jModelTest2 model for each partition with 1,000 bootstrap replicates. Bayesian inference of the concatenated alignment was calculated twice using MrBayes (v3.2.1)52 with 5 × 106 generations, sampling every 1,000 generations. Under these settings, the standard deviation of split frequencies was 0.004981 and performance scale reduction factors were close to 1.000 for all tested parameters. The first 25% generations were discarded as burnin. Phylogenetic trees were generated for individual loci using the calculated jModelTest2 models as well as for the concatenated alignment using C. boninense as an outgroup.

For the genus-wide maximum likelihood tree of selected Colletotrichum species, BUSCO was run on the genome assemblies of C. trifolii (RYZW01000000), C. sidae (QAPF01000000), C. orbiculare (AMCV02000000) and C. spinosum (QAPG01000000)24, C. fructicola (ANPB00000000.1)19, C. gloeosporioides (QFRH00000000)27, C. chlorophyti (MPGH01000000)53, C. orchidophilum (MJBS00000000.1)54, C. salicis (JFFI00000000.1), C. fioriniae (JARH00000000.1), C. simondsii (JFBX00000000.1), C. nymphaea (JEMN00000000.1)28, C. sublineola (JMSE00000000.1)29, C. graminicola (ACOD00000000.1)18, C. incanum (JTLR01000000)17, C. tofieldiae (LFIV01000000)37, C. lentis (NWBT01000000)21, C. tanaceti (PJEX00000000)26, C. higginsianum (LTAN01000000)33 and Fusarium oxysporum (GCF_000149955.1)55 to identify highly conserved, single copy eukaryote genes (eukaryote_odb9). Sequences of orthologs from 254 single copy genes that were identified as non-duplicated in all the tested genomes were aligned using MAFFT and trimmed using trimAl as described above. Modeltest-ng v0.1.5 (https://github.com/ddarriba/modeltest) was run to determine the best model for amino acid substitutions under BIC criterion (Supplementary Table S6). Sequences for all 254 single copy genes were concatenated and RAxML-ng was used to estimate the maximum likelihood phylogeny using the modeltest-ng specified best model for each partition with 500 bootstrap replicates.

The generated trees were visualised in FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

Morphological characterisation

Culture morphology was assessed on PDA (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), oatmeal agar (OA, Difco) and synthetic nutrient-poor agar (SNA56) plates. Autoclaved filter paper and double-autoclaved stems of Anthriscus sylvestris were placed on the surface of SNA plates. Plates were inoculated with 8-mm-diameter mycelia-grown agar plugs from the edge of actively growing cultures. Cultures were incubated at 24 °C under near UV light with 12 h photoperiod for 10 d. Colony colours were rated after 7 d using the Rayner colour chart57. Growth rates were estimated after 7 and after 10 d. Structures were observed after 10 d. For formation of appressoria, conidial suspensions were incubated on SNA or OA coated glass slides with a plastic cover slip in 100% humidity at 24 °C in the dark for 24 h. Fungal structures were observed using a Leica M165FC dissecting microscope (DM) and an Olympus BX51 microscope with differential interference contrast (DIC) optics. Samples for DIC were mounted directly in lactic acid. Measurements were made for at least 30 structures each.

Pathogenicity tests and observation of the infection process

Perilla plants (Aka Chirimen Shiso, Takii & Co., Ltd.) were cultivated on sterile soil in 12 h white light/12 h dark at 24 °C for three weeks before infections. For observations of trypan blue-stained invasive hyphae, approximately 100 µL of a conidia suspension of strain JCM 31818 (1 × 105 conidia/mL) was dropped onto the abaxial side of each detached perilla leaf. Then, pieces of 1.5 cm × 1 cm nylon mesh with 100 µm pores were placed on to conidial droplets to ensure even distribution of the conidial suspension on the surface of the leaf. Inoculated leaves were incubated in petri dishes for 40 and 60 hpi (hours post inoculation) at 24 °C under white light with 12 h light/12 h dark photoperiod and 100% humidity in a plastic dish with autoclaved filter paper moistened by sterilised water. For trypan blue staining, infected perilla leaves were boiled in 1 ml of alcoholic lactophenol (ethanol: phenol: glycerol: lactic acid: water (4:1:1:1:1, v/v/v/v/v)) containing 0.1 mg/ml trypan blue for 10 min at 95 °C and left overnight at room temperature. Boiled leaves were destained with chloral hydrate solution (500 g chloral hydrate + 200 ml water) overnight. Stained leaves were mounted in 60% glycerol solution and observed with DIC under a microscope. Further, for observation of setae, conidia and appressoria in/on P. frutescens leaves, intact three-week-old plants grown on sterile soil were sprayed with 5 × 105 conidia/mL conidial solution. Leaves were detached just prior to observation and mounted directly in lactic acid before DIC imaging and measurements. For pathogenicity tests, intact three-week-old plants grown on sterile soil were sprayed with 5 × 105 conidia/mL conidial solution. As negative controls, mock-treated plants were sprayed with water. Plants were maintained at 24 °C in 12 h white light/12 h dark conditions in 100% relative humidity and assessed for anthracnose lesions at two weeks post-inoculation. At least four plants were tested for each treatment in three independent experiments.

For pathogenicity tests of A. thaliana, the first three fully expanded leaves from four-week-old plants grown under short day conditions (8 h light/16 h dark) at 21 °C were inoculated 5 × 105 conidia/ml conidial suspensions of strain JCM 31818 from perilla or C. higginsianum strain MAFF 305635 from Brassica rapa. Each leaf was inoculated with one 5 μl droplet of prepared conidial suspension. Infected plants were maintained at 100% humidity under the same light and growth conditions as perilla plants. Images of infected leaves were captured 6 d after inoculation using a Canon EOS-M camera and lesion areas were determined using ImageJ58. Experiments were repeated twice using eight plants per ecotype per experiment. Lesion area size distributions were tested for significant differences using Mann-Witney U tests. For both P. frutescens and A. thaliana pathogenicity tests, leaves were only detached from intact plants just prior to imaging.

DNA extraction and genome sequencing

For sequencing and assembly of the JCM 31818 genome, genomic DNA was extracted and sequenced as previously described17. In brief, PD broth (BD Biosciences, Franklin Lakes, NJ, USA) was inoculated with hyphae from a growing colony. After incubating for 3 d at 24 °C under dark conditions, the mycelium was harvested and ground in liquid nitrogen and then the genomic DNA was extracted using CTAB buffer and 100/G genomic tips (QIAgen, Hilden, Germany) as previously described53. DNA from MAFF 240106 was extracted using the QIAgen genomic DNeasy kit according to the manufacturer’s instructions. Two differently sized insert libraries, 150 bp and 500 bp, were prepared using the Illumina TruSeq PCR-free DNA sample prep kit (Illumina) and sequenced to generate 100 bp paired-end reads with an Illumina HiSeq 2000 sequencing system (RIKEN Omics Science Center, Yokohama, Japan).

Genome assembly and annotation

Low quality reads were trimmed using TrimGalore wrapper with cutadapt (v1.2.1) and fastqc (v0.11.7). Sequences were assembled using Megahit59 followed by scaffolding using the SSPACE-Standard-3.0 scaffolder (Baseclear). The assembly was assessed using quast v4.560 and BUSCO v3.0 using the sordariomyceta_odb9 dataset61. The size of the genome was estimated by kmer analysis using jellyfish v1.1462 as previously described63. Genes were predicted with the MAKER v 2 pipeline64 after optimizing Augustus v 3.365 gene model parameters by running the BUSCO pipeline61 with the--long option to identify C. shisoi homologs of 3,659 sordariomycete conserved proteins; training Genemark-ES (v3.51)66 on the C. shisoi genome using the option to run the program using the branch point model for fungal gene predictions; and including proteins from C. higginsianum67 as additional evidence for gene model support.

Orthogroup identification

All predicted proteins from C. graminicola18, C. higginsianum67, C. incanum17 and C. tofieldiae37,68 were analysed using OrthoFinder v 2.2.669 with the default settings. For identification of secreted protein orthogroups, Deeploc v1.0 was utilised to predict the localisations of proteins from each species70. Then, OrthoFinder was run on the predicted secreted proteins to identify orthogroups within the secreted protein sequences. For analysis of dN/dS values of secreted protein-encoding gene sequences, the genes of secreted proteins grouped together by OrthoFinder were aligned using PRANK71 to produce codon alignments using default settings. Codon alignments were then analysed using the yn00 model72 implemented in the PAML suite of programs73. Conservation plots were drawn using the UpsetR package74 in R75. GO terms were assigned to C. shisoi sequences using Trinotate76 and enrichment of GO terms in selected groups was tested using the hypergeometric test in the GOstats package77 and applying the Benjamini-Hochberg multiple test correction on P-values using R.