Colletotrichum species collection

A total of 352 single-spore cultures were isolated from 173 symptomatic samples. Eighty-eight isolates were subsequently selected for further determination on the basis of their origins, colony characteristics and conidial morphologies (Table 1).

Morphological and cultural characteristics

Eighty-eight isolates were classified into six morphological groups according to morphological and cultural characteristics. Group 1 included 23 isolates fitting the description of the C. gloeosporioides complex, and Group 2 included 16 isolates fitting the description of C. fructicola. In addition, Group 3 consisted of 32 isolates matching the description of C. truncatum, Group 4 had seven isolates fitting the description of the C. acutatum complex, and Group 5 consisted of six isolates matching the description of C. brevisporum. Group 6 contained four isolates that did not fit the description of any currently known Colletotrichum species. Group 3 (C. truncatum) was the predominant group, accounting for 36.4% of the total isolates. A summary of the morphological data for the Colletotrichum species in Groups 1–6 is presented in Table 2.

Table 2

Summary of morphological data for Colletotrichum isolates.

Group	Species	Colonies appearance	Growth rate (mm/day)	Conidia			Conidial appressoria			Characteristics of mycelial appressoria
				Length (μm)	Width (μm)	Shape	Length (μm)	Width (μm)	Characteristics
Group 1(23)†	Colletotrichum gloeosporioides, C. siamense	pale yellowish colonies, reverse white	5.6±1.2 ab* 4.7–6.7	16.5±2.0 c* 12.0–24.2	5.5±0.6 b* 3.8–7.3	Cylindrical	7.6±1.0 c* 5.5–9.9	5.9±0.6 b* 4.8–7.7	brown to dark black, ovoid to slightly irregular	light brown to brown, ovoid or slightly irregular to irregular
Group 2(16)	C. fructicola	white to black green	5.9±0.4ab 5.4–6.6	16.2±2.1 c 11.1–25	5.5±0.6 b 2.5–7.7	Cylindrical	7.9±0.6 bc 6.2–9.1	6.1±0.5 ab 4.4–7.2	brown to dark black, ovoid to slightly irregular	light brown to brown, ovoid, clavate and slightly irregular to irregular, smooth or slightly lobed
Group 3(32)	C. truncatum	pale grey to dark grey, reverse dark brown	4.5±0.5b 3.9–5.4	24.1±1.9 a 18.8–29.87	3.7±0.3 d 2.7–4.9	Falcate	7.8±1 bc 4.6–11.2	5.4±0.5 c 4.1–7	brown to dark black, ovoid to slightly irregular	brown to dark brown, ovate, ellipsoidal or slightly irregular to irregular
Group 4(7)	C. scovillei	white to pale orange, reverse pale orange	3.8±0.4c 3.3–4.2	11.7±2.3 d 8.2–17.2	3.7±0.3 d 3.1–4.6	Fusiform	6.0±1.2 d 4.2–9	4.8±0.5 d 3.5–6.0	Grey, globular in shape	light brown to brown, globose, ovate to slightly irregular
Group 5(6)	C. brevisporum	pale grey, reverse black	5.3±0.6 b 5.0–5.8	19.3±1.3 b 16.3–22.2	5.2±0.5 c 4.2–6.3	Long cylindrical	8.0±0.8 b 6.1–9.5	6.2±0.5 a 5–7.9	brown to dark black, ovoid to slightly irregular	brown to dark brown, sometimes black in the middle, ovoid or slightly irregular to irregular
Group 6(4)	C. sichuanensis	pale grey, reverse pale grey	6.1±0.4 a 5.5–6.7	16.7±1.2 c 14.2–19.1	6.3±0.4a 5.4–6.7	Cylindrical	11.1±1.7 a 8–14.2	6.3±2.0 a 4.3–11.0	brown to dark brown, irregular with a crenate edge	light brown to brown, ovoid or slightly irregular to irregular

Open in a separate window

^†The numbers shown in parentheses represent the number of isolates in each group.

^*The mean difference is significant at the 0.05 level; the values with same letter in a column do not significantly differ according to Duncan’s multiple range test.

Colony characteristics (Fig. 2): Distinct morphology on potato dextrose agar (PDA) was observed in each group after 7 days. The isolates from Group 1 produced pale yellowish colonies, with sparse white aerial mycelia. The reverse side of the colonies was white, and many bright orange conidial masses were observed near the inoculum point. The colonies produced by Group 2 isolates varied from white to black-green on PDA, with dense grey aerial mycelia and a few bright orange conidial masses near the inoculum point. The colonies produced by Group 3 isolates varied from pale grey to dark grey, with dense pale grey aerial mycelia and small black granules over the entire surface. The reverse side of the colonies was dark brown, and a few pale yellow conidial masses were observed near the inoculum point. The colonies produced by Group 4 isolates varied from white to pale orange, with dense white aerial mycelia, and the reverse side of the colonies was pale orange. Isolates belonging to Group 5 produced dark grey colonies with sparse grey aerial mycelia. The reverse side of the colonies was grey, and a few bright orange conidial masses were observed near the inoculum point, as well as some spots scattered over the colony surface. Lastly, the isolates from Group 6 produced pale grey colonies, with sparse white aerial mycelia. The colonies from Group 3, 4 and 5 were stable and unique, and the colonies from Group 2 were significantly different compared with those from the other groups under stable culture conditions.

Open in a separate window

Figure 2

Morphology and cultural characteristics of Colletotrichum spp. from pepper anthracnose.

(A) Upper view of a colony on PDA; (B) reverse view of colony on PDA; (C) micrographs of conidia of Colletotrichum spp.; (D) micrographs of conidial appressoria of Colletotrichum spp.; (E) micrographs of mycelial appressoria of Colletotrichum spp.; (F) conidiophores. Scale bars=10μm for (C–E); 20μm for (F).

Growth rate (Table 2): Group 4 exhibited a significantly different growth rate compared with the other five groups (P=0.05). The isolates from Group 6 (6.1±0.4mm/day) grew the fastest, followed by those from Group 1 (5.6±1.2mm/day), Group 2 (5.9±0.4mm/day), Group 5 (5.3±0.6mm/day), Group 3 (4.5±0.5mm/day) and Group 4 (3.8±0.4mm/day).

Conidial morphology (Table 2 and Fig. 2): The following four types of conidia were observed: cylindrical (observed in Groups 1, 2 and 6), falcate (Group 3), fusiform (Group 4) and long cylindrical (Group 5). The conidial widths of Group 6 were significantly different from those of Groups 1 and 2; however, all of these groups had cylindrical conidia with obtuse to slightly rounded ends. The conidia produced by the Group 3 isolates were falcate, with gradual tapering towards each end. Group 4 produced fusiform conidia, whereas Group 5 produced long and cylindrical conidia, with obtuse to slightly rounded ends. The differences in the conidial shapes of Groups 3, 4 and 5 were very significant, allowing these groups to be easily distinguished from one another. Almost all of the conidia were aseptate, but they often developed a septum after germinating and forming appressoria.

Conidial appressorium morphology (Table 2 and Fig. 2): There was little distinction among the groups in terms of the sizes and shapes of conidial appressoria, except for Groups 4 and 6, which exhibited significant differences compared with the other groups. The conidial appressoria of Groups 1, 2, 3 and 5 varied from ovoid to slightly irregular in shape and from brown to dark black in colour. Group 4 produced grey, globular and smaller conidial appressoria. Most of the conidial appressoria produced by Group 6 were irregular and pale brown to dark brown, with a crenate edge.

Mycelial appressorium morphology (Table 2 and Fig. 2): The mycelial appressoria produced by the isolates of Groups 1 and 2 varied from ovoid, clavate and slightly irregular to irregular, smooth or slightly lobed, and they were light brown to brown in colour. The appressoria of Group 3 ranged from ovate, ellipsoidal or slightly irregular to irregular in shape, and they were smooth or lobate and brown to dark brown. The appressoria produced by Group 4 were globose or ovate to slightly irregular, and they were light brown to brown and smaller in size than those of the other groups. In addition, the appressoria produced by Group 5 varied from ovoid, clavate or slightly irregular to irregular in shape. They were smooth or slightly lobed and brown to dark brown and were sometimes black in the middle. Further, the appressoria of Group 6 were ellipsoidal or irregular, smooth or slightly lobed to strongly lobed, solitary or in chains, and light brown to brown in colour.

Conidiophores (Fig. 2): The conidiophores of all groups were hyaline to pale brown, simple or septate, rarely branched, and smooth walled. Four types of conidiophores were observed: (i) nearly cylindrical, but narrower towards the end (as observed in Groups 1 and 2); (ii) cylindrical, with a truncate top (Groups 3 and 5); (iii) shortly clavate, nearly hyaline, with a cylindrical base, and obviously inflated, with gradually tapering towards the top (Group 4); and (iv) frequently produced by mycelia, cylindrical, with swollen ends (oblong) and slight narrowing in some areas (Group 6).

Setae: All isolates from Groups 3 and 5 and some isolates from Group 1 produced setae; in contrast, the isolates from all the other groups rarely produced setae. The setae were commonly smooth, septate, and light brown to dark brown in colour, base cylindrical to conical, and sometimes slightly inflated, and the tips were acute to roundish. No obvious differences in setal characteristics (shape and dimensions) were found among the different groups when grown on PDA.

Sclerotia and Ascomata: Most Group 5 isolates steadily produced a large amount of black solids that appeared similar to sclerotia and were round to irregular and semi-immersed. Conidial masses and setae sometimes formed on the black solids. On PDA, Group 6 isolates always produced ascomata in clusters, which were brown and globose to near globose and possessed a neck. The isolates from the other groups rarely produced ascomata, even in host tissues.

Phylogenetic analysis

A phylogram generated based on the GAPDH gene region revealed 5 primary clades (i.e., C. truncatum, C. brevisporum, C. gloeosporioides sensu lato, C. acutatum sensu lato and one unknown species (Colletotrichum sp.)) (Fig. 3).

Open in a separate window

Figure 3

A neighbour-joining tree based on partial GAPDH gene sequences from 88 Colletotrichum isolates.

Parsimony bootstrap values of more than 50% are shown at the nodes. Isolates selected for subsequent phylogenetic analyses are highlighted in red.

Fifty-two representative isolates were chosen from the morphological groups for molecular analysis, including 25 isolates from the C. gloeosporioides complex, 10 C. truncatum isolates, seven C. acutatum sensu lato isolates, six C. brevisporum isolates and four Colletotrichum sp. isolates.

Multi-locus phylogenetic analysis was conducted among 87 strains, with Monilochaetes infuscans (CBS 869.96) used as an outgroup (Table 3). The dataset for five genes (ITS, TUB2, ACT, GAPDH and CAL) contained 2,155 characters, including alignment gaps, of which 997 characters were parsimony-informative, 321 were parsimony-uninformative, and 837 were constant. This parsimony analysis resulted in the most parsimonious tree (TL=2800, CI=0.7257, RI=0.9541, RC=0.6924, and HI=0.2743). The phylogram showed that the 52 pepper anthracnose isolates belonged to seven distinct clades. The isolates from Group 2 clustered with C. fructicola, those from Group 3 clustered with C. truncatum, those from Group 4 clustered with C. scovillei, and those from Group 5 clustered with C. brevisporum. The Group 1 isolates grouped with two clades; 4 isolates clustered with C. gloeosporioides, and the remaining isolates clustered with C. siamense (Fig. 4). After combining two phylograms (Figs 3 and ​and4),4), 8 and 16 strains were found to belong to C. gloeosporioides sensu stricto and C. siamense, respectively. The isolates from Group 6 were from an unknown species (Colletotrichum sp.). The submission number of the sequence alignment in TreeBASE is 18832.

Open in a separate window

Figure 4

Phylogram generated from maximum parsimony analysis based on alignment of ITS, TUB2, ACT, GADPH and CAL gene sequences, showing the phylogenetic relationships of Colletotrichum species causing anthracnose disease in Capsicum annuum from Sichuan Province, China.

Parsimony bootstrap values of more than 50% are shown at the nodes. Isolates from this study are shown in bold. The tree is rooted with Monilochaetes infuscans.

Taxonomy

Colletotrichum sichuanensis G.S. Gong & F.L. Liu, sp. nov. (Fig. 5).

Open in a separate window

Figure 5

Colletotrichum sichuanensis (from holotype).

(a,b) Colonies on PDA at 7 days, upper (a) and reverse (b); (c,d) conidia; (e) conidiogenous cells; (f,g) conidial appressoria; (h–j) mycelial appressoria; (k) ascomata on PDA; (l) peridium; (m–o) asci; (p,q) ascospores. Scale bars: c, d, f–j, p, q=10μm; e, l, m, o=20μm; n=40μm

MycoBank: MB 815288.

Etymology: sichuanensis, in reference to the province where the type was found.

Description: Colonies on PDA at first white, later becoming pale grey and reverse pale grey, with a maximum diameter of 68.7mm over 5 days at 28°C and a growth rate of 6.1–6.4mm/day (=6.3±0.1, n=5). Aerial mycelium white and sparse, with the frequent absence of conidial masses. Ascomata nearly always present in clusters on PDA. Conidiophores generated from mycelia are nearly hyaline, branched, and cylindrical, with slightly swollen ends, simple or occasionally branched. Conidia common on mycelia, one-celled, smooth-walled, hyaline, and cylindrical, with obtuse to slightly rounded ends, 15.0–18.9×5.4–6.5μm (=16.9±1.0×6.2±0.3, n=30). Conidial appressoria light brown to dark brown, slightly irregular to irregular, crenate or lobed, 8.1–12.4×5.4–8.8μm (=10.2±1.0×6.8±0.8, n=30). Appressoria in slide culture light brown to brown, ellipsoidal or irregular, smooth or slightly lobed to strongly lobed, solitary or in chains, 6.4–20.2×4.8–9.8μm (=11.5±3.0×7.0±1.1, n=30). Setae absent.

Teleomorph: Glomerella sp.

Ascomata, light brown to brown, globose to subglobose, with a neck, arranged in clusters. Peridium of textura angularis, thick-walled. Asci 30.8–61.6×7.4–13.8μm (=47.5±8.0×9.5±1.5, n=30), unitunicate, thin-walled, and clavate. Ascospores 10.2–23.3×3.9–6.8μm (=17.5±2.6×5.4±0.8, n=30), one-celled, hyaline, and slightly curved to curved, with obtuse to slightly rounded ends.

Holotype: Baoxing, Yaan City, Sichuan Province, China, on fruit of Capsicum annuum, 5 September 2013, coll. G. S. Gong (holotype living culture LJTJ30). A living culture (strain LJTJ30) was deposited at the Department of Plant Pathology of Sichuan Agricultural University. Known distribution: Sichuan Province, China.

Additional examined specimens: Jiangyou, Mianyang City, Sichuan Province, China, on Capsicum annuum fruit, 26 July 2013, coll. G. S. Gong (holotype living culture LJTJ3); Yuechi, Guangan City, Sichuan Province, China, on Capsicum annuum fruit, 27 August 2013, coll. F. L. Liu (holotype living culture LJTJ16); and Wenjiang, Chengdu City, Sichuan Province, China, on Capsicum annuum fruit, 3 July 2013, coll. F. L. Liu (holotype living culture LJTJ22). A living culture (strain LJTJ3, LJTJ16 and LJTJ22) was deposited at the Department of Plant Pathology at Sichuan Agricultural University.

Morphological and cultural characterization

Mycelial discs (5mm diameter) were collected from actively growing areas near the growing edges of 5-day-old cultures, transferred to PDA and incubated at 27°C in the dark for 10 days. Five replicates were employed. The colony diameter was recorded each day from two perpendicular cross-sections, and the colony characteristics were also recorded.

The sizes and shapes of conidia, asci and ascospores from each culture were recorded. The lengths and widths of 30 conidia, asci and ascospores were measured for each isolate.

Conidial appressoria were induced according to the method of Yang et al.²⁷.

Mycelial appressoria were produced using an improved slide culture technique, as described by Sutton⁵¹ and Cai et al.²⁵. One hundred microlitres of hot water agar (WA) was placed on a sterile slide. Mycelial plugs of approximately 2mm in diameter were inoculated onto one-third of the WA and then incubated in a Petri dish with wet filter paper at 27°C. After 5–7 days, agar pieces containing the inoculated plugs were gently removed with a scalpel, and the shapes and sizes of the appressoria that formed along the WA were then recorded.

Samples for microscopy were prepared using clear water or lactic acid and observed with a Carl Zeiss Axio Imager Z2 microscope(Germany) or a Nikon Eclipse 80i microscope(Japan) using differential interference contrast (DIC) illumination.

DNA extraction

Fifty-two representative isolates were chosen according to the morphological and cultural characteristics and incubated on PDA at 27°C for 7–10 days. Mycelia were scraped from the colony surface using a sterile medicine spoon. Total genomic DNA was extracted from the isolates using a modified protocol, as outlined by Guo et al.⁵².

PCR amplification and DNA sequencing

As an initial analysis of genetic diversity, the glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene was amplified from the isolates in this study with the primers GDF/GDR⁵³. Fifty-two isolates representing wide ranges of genetic diversity and geographic origins were selected for further investigation.

The nuclear rDNA ITS region and the β-tubulin (TUB2), partial actin (ACT) and calmodulin (CAL) genes were amplified from 52 representative isolates using the primers ITS1/ITS4^54,55, Bt2a/Bt2b⁵⁶, ACT512F/ACT783R⁵⁷ and CL1/CL2A⁵⁸, respectively. PCR was performed under the conditions described by Prihastuti et al.²⁶.

The amplifications were performed in a 40μl mixture containing 17μl ddH₂O, 20μl 2×PCR MasterMix (TIANGEN Co., China), 1μl DNA template (30–50ng/μl), and 1μl of each primer (10μM). DNA sequencing was performed by Sangon Biotech Co., Ltd. (Shanghai, China).

Phylogenetic analysis

Alignment of the GAPDH genes of all of the isolates was performed using Clustal X⁵⁹. MEGA v. 5 was used to build a distance tree with the neighbour-joining (NJ) algorithm. The sequences were compared with those in the NCBI sequence database using the BLAST algorithm for approximate identification.

The sequences of the 52 isolates and the reference sequences obtained from GenBank (Table 3) were aligned using Clustal X. Then, a phylogenetic tree was constructed with the combined ITS, TUB2, ACT, GAPDH and CAL dataset.

Parsimony trees were inferred by PAUP v4.0b10 using a heuristic search option with 1,000 random sequence additions⁶⁰. All gaps were treated as missing data. Max trees were unlimited, zero-length branches were collapsed, and all multiple parsimonious trees were saved. Clade stability was assessed by bootstrap (BT) analysis with 1,000 replicates. In addition, descriptive tree statistics, such as parsimony (Tree Length [TL], Consistency Index [CI], Retention Index [RI], Related Consistency Index [RC] and Homoplasy Index [HI]), were calculated.

This study was funded by the Two-Way Support Project of Sichuan Agricultural University. We are grateful to our team at the Crop Disease Laboratory for helping to collect the samples.