J. Gen. Plant Pathol. 68 : 307-320 (2002)
Grouping of Colletotrichum Species in Japan Based on rDNA Sequences Jouji MORlWAKll*, Takao TSUKIBOSHl2and Toyozo SAT03
ABSTRACT Internal transcribed spacers (ITS) of the ribosomal RNA gene (rDNA) were sequenced for 236 isolates covering 26 Colletotrichum species collected in Japan. The Japanese isolates could be grouped into 20 ribosomal groups (RGs) based on the sequences of ITS1, correlating the species identified by the morphology. Colletotrichum gloeosporioides sensu lato separated into three RGs that were morphologically different. Colletotrichum destructivum, C. linicola and C. higginsianum were possibly conspecific. Colletotrichum dematium sensu lato including C. capsici and other species that produce falcate conidia except for graminicolous ones were separated into three RGs that were difficult to distinguish morphologically. In the phylogenetic study using ITS2 and the 285 rDNA domain 2 region, topologies compiled by neighbor-joining and maximum-parsimony methods were almost the same, reflecting the conidial morphology. The phylogenetic group 1(PG1) produced conidia with acute ends, e.g., C. acutatum, C. destructivum and C. graminicola; PG2 produced those with obtuse ends, e.g., C. gloeosporioides, and C. orbiculare. Colletotrichum theae-sinensis, which produced the smallest conidia, was grouped as PG3, far from other species, indicating it should not belong to Colletotrichum. Grouping and phylogenetic analysis using ribosomal DNA was an effective tool to classify and identify Colletotrichum species without using morphology.
(Received July 15, 2002 ; Accepted November 12, 2002) Key words : Colletotrichum, rDNA ITS regions, 285 rDNA, phylogenetic analysis, ribosomal group.
INTRODUCTION Colletotrichum species, belonging to Coelomycetes, are important fungi causing anthracnose of more than 1000 plant species. Although the number of species classified as Colletotrichum rose to several hundred, Arx') reduced them in 1957 to 11 species with 12 forms based on the morphology of conidia related to their perfect stages and pathogenicity. At present, S ~ t t o n ~has ~ , accepted ~~) 38 species, one variety and eight formae specialis based on taxonomic criteria for conidial and appressorial morphologies, cultural characteristics and pathogenicities. However, because these characteristics are simple and sometimes uncertain, the species criteria are somewhat ambiguous4). In addition, many species have not yet been examined using Sutton's criteria, and the number of the species could increase. The rDNA of Colletotrichum species have been analyzed to infer their phylogenetic distribution^^^,^^). Sher-
riff et ~ 1 . reported ~ ~ ) that the genus Colletotrichum was separated into two genetic groups that have different morphological characteristics based on phylogenetic analysis using rDNA ITS2 and 285 rDNA domain 2 (D2) regions. Sreenivasaprasad et ~ 1 . reported ~ ~ ' that in Colletotrichum species the ITS1 region (50.3%variable sites) has a greater degree of intra- and inter-specific divergence than ITS2 (12.4% variable sites), and molecular phylogenetic analysis based on rDNA ITS 1 sequence revealed that 18 species of Colletotrichum were separated into six phylogenetic groups that correlated approximately with the conidial morphology. Sherriff et ~ 1 . con~ ~ 1 firmed the distinction between C. graminicola from Zea and C. sublineolum from Sorghum and Rottboellia using ITS2 sequences. Bailey et aL2) analyzed the Colletotrichum species on Malvaceae using ITS2 and D2 sequences and separated them into two species, i.e. C. gloeosporioides from Gossypium and C. orbiculare from Sida, Lavatera and Malva. Latunde-Dada et al.'') suggested that the species hemibiotrophic to cowpea must not be
National Agricultural Research Center, Joetsu 943-0193, Japan National Institute for Agro-EnvironmentalSciences, Tsukuba 305-8604, Japan National Institute of Agobiological Sciences, Tsukuba 305-8602, Japan * Corresponding author (E-mail: moriwakia affrc.go.jp)
308
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considered as C. orbiculare but as a form of C. destructivum, based on ITS2 and D2 sequence data and morphology. Thus, rDNA ITS regions of many Colletotrichum species were analyzed, and molecular taxonomic research proceeded. However, the rDNA ITS regions of Colletotrichum isolates collected in Japan have not yet been analyzed. The purpose of the study is to classify Japanese Colletotrichum isolates based on the sequences of their rDNA regions, to compare them with the isolates from overseas and to clarify the phylogenetic relationships among Colletotrichum spp. in Japan.
triphosphates, 0.4 p M each primers, 1pl of templates, and 1.25 units of Taq polymerase (TaKaRa Co., Kyoto). The amplification was performed with a thermal cycler MP (TaKaRa Co., Kyoto). The cycle parameters were an initial denaturation at 95°C for 2min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72'C for 2 min, and a final extension for 5 min at 72°C. DNA sequencing DNA sequences of the PCR products were obtained using direct sequencing in a CEQ2000 sequencer (Beckman Coulter, Inc., CA). The sequence reactions were conducted using the Dye Terminator Cycle Sequencing Kit (Beckman Coulter, Inc.) MATERIALS AND METHODS following the manufacturer's protocol. Four primers, Fungal isolates ITS1, ITS4, NL1 and NL4 were used for the sequencThe 236 isolates of Colletotrichum ing635). species examined are shown in Table 1. Thirty-one isolates of C. acutatum from 17 host plants including Vitis, Phylogenetic analysis For phylogenetic analysis, ~~). Eustoma and Malus were used in the s t u d ~ ~ O 9Ninety rDNA ITS2 and D2 sequences of 20 Colletotrichum isolates selected from each ribosomal group (RG) were used isolates of C. gloeosporioides from host plants such as Vitis, Fragaria, Cucumis, Ranunculus, Matthiola, Caca(Tables 1 and 2). Two isolates of Pyricularia grisea were ~). to lia and Capsicum were also ~ s e d * , ' ~ , ~In' , ~addition also analyzed in the same methods as an outgroup. The these species, one to 15 isolates of C. capsici, C. caucomplete data set for analysis was obtained for each RG datum, C. circinans, C. coccodes, C. coffeanum, C. craswhere ITS2 was combined with D2 to produce an alignsipes, C. dematium, C. destructivum, C. falcatum, C. ment of 373 nucleotides. Multiple sequence alignment of fragariae, C. fuscum, C. graminicola, C. higginsianum, C. the data was initially carried out using the alignment lilii, C. lindemuthianum, C. linicola, C. musae, C. orbicusubroutines on CLUSTAL X32). The alignment of all sequences was checked visually. Phylogenetic trees were lare, C. sublineolum, C. theae-sinensis, C. trichellum, C. trifolii and C. truncatum from various hosts were used for obtained from the data by neighbor-joining (NJ) and phylogenetic analyses described 1ater7-9~16,21~27~29~31~33~34). maximum-parsimony (MP) methods. A tree showing the Some isolates, except for those with MAFF, CBS, IMI phylogenetic relatedness between the isolates was constructed from distance matrix values by the neighborand I F 0 numbers, were originally identified based on joining methodIg), using CLUSTAL X. The distances in their morphology by Sutton's criteriaz6). Unidentified Colletotrichum species from Hydrangea, Prunus and so on the ITS region were determined by Kimura's twowere also used for the study. Most cultures were supplied parameter model1o).Sites where gaps existed in any of from the MAFF (Ministry of Agriculture, Forestry and the sequences were excluded. A bootstrap analysis using Fisheries of Japan) Genebank and IF0 (Institute for 1000 resamples of the sequence data was carried out5). Fermentation, Osaka). Some were imported from CAB1 For parsimony analysis, the PAUP program version (Commonwealth Agricultural Bureaux International) 4b1OZ8)was used, and a heuristic search was performed Bioscience, UK and CBS (Centraalbureau voor Schimwith 100 repeats of random addition sequences with the melcultures, NED) with the permission of the Yokohama Stepwise-Addition Option and TBR swapping algorithm Plant Quarantine Office, Japan in 1999. in the Branch-Swapping Options. Confidence limits for the branches based on parsimony criteria were estimated DNA extraction and PCR of rDNA Each isolate was grown on potato dextrose agar (PDA, Eiken Chem. by bootstrap analysis of 1000 replicates, and a 50% Co., Tokyo) for 4 days at 25°C. Mycelia were homogemajority-rule consensus tree was generated. nized in a microtube using a silicon pick, and total DNA was extracted by the modified method of Rodriguez and RESULTS Y ~ d e r ' ~ The , ~ ~regions ). of rDNA repeat from the 3' end of the 18s rRNA gene to the 5' end of the 285 rRNA gene, Grouping based on rDNA sequence PCR products of about 1.2 kb were amplified from the including ITSl to 285 rDNA domain 2 (D2) region, were amplified by PCR with ITSl and NL4 primer^^,^^). The Colletotrichum species used. In results of the analysis on PCR amplification reactions were performed in a 50 p1 the sequences of 157 to 190 bp of rDNA ITSl region of mixture containing 50 mM KC1, 20 mM Tris-C1 (pH 8.4), 236 isolates, 20 ribosomal groups (RGs) could be identified with an intra-group similarity of more than 95%4) 2 mM MgC1, 200 mM each of the four deoxynucleotide
rDNA Grouping of Japanese Colletotrichum Species
Species Colletotrichum acutatum
C. capsici
C. caudatum
C. circinans
Table 1. Colletotrichum isolates used in this study Source plants Isolatesa,b) Mol. Identification" Anemone coronaria MAFF 306488 C. acutatum MAFF 306507 C. acutatum MAFF 306509 C. acutatum MAFF 306172 Annona sp. C. acutatum Castanopsis cuspidata var. sieboldii MAFF 238654 C. acutatum MAFF 238655 C. acutatum MAFF 305596 Eriobotrya japonica C. acutatum MAFF 306406 C. acutatum MAFF 306408 C. acutatum MAFF 306410 C. acutatum Eustoma grandiflorum MAFF 238652 C. acutatum MAFF 238653 C. acutatum MAFF 306247 C. acutatum Fragaria X ananassa MAFF 306282* C. acutatum MAFF 306543 Malus pumila var. domestica C. acutatum MAFF 306544 C. acutatum MAFF 306546 C. acutatum MAFF 306547 C. acutatum MAFF 306548 C. acutatum MAFF 306549 C. acutatum K1 C. acutatum Prunus armeniaca MAFF 306528 C. acutatum MAFF 306489 Prunus domestica C. acutatum MAFF 306503 C. acutatum Prunus mume MAFF 306526 C. acutatum MAFF 306527 C. acutatum MAFF 306430 Prunus persica C. acutatum MAFF 306522 C. acutatum MAFF 306523 C. acutatum MAFF 306524 C. acutatum MAFF 306525 C. acutatum Pyrus communis var. sativa MAFF 306520 C. acutatum MAFF 306521 C. acutatum MAFF 306545 Rumex japonicus C. acutatum Vitis sp. MAFF 238647 C. acutatum MAFF 238710 Raphanus sativus var. hortensis C. dematium MAFF 238711 C. dematium MAFF 238714 C. dematium MAFF 238704 C. dematium MAFF 238705 C. dematium MAFF 238706 C. dematium 1-98 Agrostis sp. ND 1-99 ND Imperata cylindrica MAFF 305700* ND Zoysia tenuifolia MAFF 238575 ND AnHH12 ND Allium cepa MAFF 237304* C. circinans MAFF 238640 C. circinans Allium jistulosum: porrum group MAFF 237488 C. dematium MNN-6 C. dematium MNC-106 Allium tuberosum C. dematium MNC-3 C. dematium MNC-6 C. dematium
309
RGd) References 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
9 9 9 9 9 9 19 19 19 19 19 10 10 9 9 9 9 9
30 30
36
31 31
310
JGPP
C. coccodes
C. coffeanum
C. crassipes C. dematium
C. destructivum
C. falcatum C. fragariae C. fuscum C. gloeosporioides
Table 1. (continued) MAFF 712049 MAFF 237459 MAFF 238645 MAFF 238646 MAFF 712038 MAFF 238435 Patrinia scabiosifolia PC1 MAFF 425347 Phaseolus vulgaris MAFF 238560 Physalis alkekengi MAFF 238695 Solanum melongena MAFF 712102* MAFF 238719 Solanum tuberosum MAFF 305593 Coffea sp. MAFF 305995 MAFF 306161 MAFF 410253* Aucuba japonica MAFF 238702 Crinum gigas MAFF 306552 Fagopyrum esculentum MAFF 238700 Ipomoea batatas MAFF 305982 Passiflora edulis radish3 Raphanus sativus var. hortensis IMI-080025* Peperomia sp. Dianthus sp. IMI-288810* MAFF 410037 Robinia pseudoacacia MAFF 511453* Trifolium pratense MAFF 305426 Saccharum oficinarum MAFF 306170* MAFF 306299 MAFF 744017 Fragaria X ananassa MAFF 238340 Nemesia strumosa NC1 NC2 MAFF 306008 Abelmoschus esculentus MAFF 305997 Annona muricata MAFF 306293 Annona squamosa Aucuba japonica Gck95-1 MAFF 305791 Averrhoa carambola MAFF 306085 MG1 Cacalia delphiniifolia MAFF 238698 Capsicum annuum PC1 MAFF 306096 Capsicum annuum var. annuum MAFF 238697 Carica papaya MAFF 305787 MAFF 305789 MAFF 306160 MAFF 306173 MAFF 306304 MAFF 306203 Casimiroa edulis MAFF 306533 Citrus sp. MAFF 306534 MAFF 306204 Clivia minata MAFF 306205
Capsicum annuum Lycopersicon esculentum
C. coccodes C. coccodes C. coccodes C. coccodes C. coccodes C. destructivum C. destructivum C. coccodes C. destructivum C. coccodes C. coccodes C. coccodes C. gloeosporioides C. gloeosporioides C. gloeosporioides ND C. dematium C. capsici or C. truncatum ND C. capsici or C. truncatum C. dematium C. capsici or C. truncatum C. dematium C. destructivum C. destructivum ND ND ND C. gloeosporioides C. destructivum C. destructivum C. destructivum C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. acutatum C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides
2 2 2 2 2 3 3 2 3 2 2 2 4 4 4 8 9 12 11 12 9 12 9 3 3 18 18 18 4 3 3 3 6 4 4 4 6 4 4 4 4 1 6 4 4 4 4 4 4 4 4 5 5
33 33 16
21 8 21
8 27 27
34 34 34 8 21
21 8
8 8
311
rDNA Grouping of Japanese Colletotrichum Species
Table 1. (continued)
C. gloeosporioides
Crinum gigas
Cucumis melo
Cyclamen persicum Cymbidium sp. Diospyros kaki
Erythrophleum indica Fagopyrum esculentum Ficus benjamina Fragaria X ananassa
Gladiolus sp. Glycine m m Gossypium sp. Hydrangea sp. Juglans sp. Liriodendron tulipifera Lycopersicun esculentum Malus pumila var. domestica
Mangifera indica
Matthiola incana Paeonia albiflora Passiflora edulis
Percea americana Peucedanum boninense Phalaenopsis sp. Prunus persica
C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides sc1 C. gloeosporioides MAFF 306302 C. gloeosporioides MAFF 306300 C. gloeosporioides GM2-3 C. gloeosporioides MAFF 306009 C. gloeosporioides MAFF 410045 C. destructivum MAFF 306539 C. gloeosporioides MAFF 238699 C. gloeosporioides MAFF 306007 C. gloeosporioides MAFF 238043 C. gloeosporioides MAFF 305145 C. acutatum MAFF 306538 C. gloeosporioides MAFF 305594 C. gloeosporioides MAFF 306122 C. gloeosporioides MAFF 306229 C. gloeosporioides MAFF 306303 C. gloeosporioides SN-A C. gloeosporioides SN-B C. gloeosporioides MAFF 306239 C. gloeosporioides MAFF 238696 C. gloeosporioides MAFF 305750 C. gloeosporioides MAFF 305751 C. gloeosporioides MAFF 305752 C. gloeosporioides MAFF 305973 C. gloeosporioides MAFF 305974* C. gloeosporioides MAFF 306305 C. gloeosporioides MAFF 305996 C. gloeosporioides MAFF 306084 C. gloeosporioides IF05952 C. acutatum IF06446 C. acutatum MAFF 306537 C. eloeosvorioides
MAFF 305972 MAFF 306094 MAFF 306099 MAFF 306162 MAFF 238657 MAFF 238658 MAFF 238659 MAFF 238660 960822-2 MAFF 305998 MG1013 MAFF 306100* MAFF 306540 MAFF 306429 911001-2 MAFF 306171 MAFF 306553 MAFF 306086 GcM96-2 GcS95-1 MAFF 305913* MG1004 MG1015
5 5 4 5 4
4 4 4 4 5 4 5 4 4 4 4 4 4 4 4 4 4 4 4
8 21 21 21 12 12 12 12 12 8
4 6 6 4 3 4 4 6 4 1
4 4 4 4 4 4 4 4 4 4 6 4 5 6 4 4 4 1 1
4
8
8 8
8 8 8 8 8 8 8
312
JGPP
C. gloeosporioides
Prunus persica Ranunculus sp. Robinia pseudoacacia Ruscus aculeatus Stylosanthes guianensis Vigna radiata Vitis vinifera Vitis sp.
C. graminicola
Table 1. (continued) MAFF 306541 MAFF 305144 MAFF 238701 MAFF 306536 MAFF 306412
Unknown Agrostis stolonifera Avena sativa Dactylis glomerata
Digitaria ciliaris Echinochloa utilis
C. lilii C. lindemuthianum C. linicola
Holcus lanatus Lolium perenne Paspalum notatum Polypogon fugax Zea mays Brassica pekinensis Brassica rapa Matthiola incana Raphanus sativus var. hortensis Unknown Rohdea japonica Phaseolus vulgaris Linum usitatissimum
C. musae
Musa sapientum
5
C. higginsianum
C. orbiculare
Carthamus tinctorius Cucumis melo
C. sublineolum
Cucumis sativus Gerbera sp. Sorghum bicolor
C. theae-sinensis
Camellia sinensis
SG1 MAFF 306157 MAFF 238648 15-1 MAFF 238649 MAFF 238650 MAFF 305140 MAFF 305141 MAFF 305142 910409-1 MAFF 236902 MAFF 305075 MAFF 305371* MAFF 305150 MAFF 511140 OGAnthracnose MAFF 305404 DC1-1 MAFF 305460 MAFF 305439* MAFF 305432 PRAnthracnose MAFF 305403 MAFF 305429 MAFF 511343* MAFF 305970 MAFF 305635 MAFF 238563 MAFF 238562 IF06182 MAFF 238703 MAFF 305390 CBS 172.51 IMI-103844 MAFF 305595 MAFF 305790 MAFF 306174 MCOl MAFF 306518* MAFF 306519 MAFF 726522 MAFF 306589 MAFF 305360* MAFF 305361 Sbl-2 Sb2-1 MAFF 238240 MAFF 238241 MAFF 238242
C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. orbiculare C. gloeosporioides C. gloeosporioides C. gloeosporioides C. gloeosporioides C. acutatum C. gloeosporioides C. acutatum C. gloeosporioides C. graminicola C. graminicola C. graminicola C. graminicola C. graminicola C. graminicola ND ND ND ND C. graminicola C. graminicola C. graminicola C. graminicola C. graminicola C. destructivum C. destructivum C. destructivum C. destructivum C. destructivum C. dematium C. orbiculare C. destructivum C. destructivum C. gloeosporioides C. gloeosporioides C. gloeosporioides C. orbiculare C. orbiculare C. orbiculare C. orbiculare C. orbiculare C. sublineolum C. sublineolum C. sublineolum C. sublineolum ND ND ND
4 4 4 4 4 4 7 4 4 4 4 1 4 1 4 15 15 15 15 15 15 16 16 16 16 15 15 15 15 14 3 3 3 3 3 9 7 3 3 4 4 4 7 7 7 7 7 17 17 17 17 20 20 20
36 36 36
21 7 7 29 27 27 8
9 9
rDNA Grouping of Japanese Colletotrichum Species
313
Table 1. (continued) MAFF 238243 ND 20 MAFF 238244 ND 20 20 MAFF 752003* ND Hedera rhombea C. trichellum 13 MAFF 237991 C. trichellum 13 MAFF 237992 C. trichellum 13 MAFF 237993 C. trichellum 13 MAFF 410046* C. trichellum Medicago sativa C. trifolii 7 MAFF 510847 C. orbiculare Trifolium pratense 7 MAFF 510896 C. orbiculare 12 C. truncatum Glycine m u MAFF 305754 C. capsici or C. truncatum Phaseolus vulgaris 11 MAFF 425349 ND Vigna radiata 11 MAFF 305748* ND 12 Voandzeia subterranea MAFF 306411 C. capsici or C. truncatum Colletotrichum sp. Brassica campestris 12 MAFF 238716 C. capsici or C. truncatum Cattleya sp. 5 MAFF 238641 C. gloeosporioides 5 Dendrobium kingianum MAFF 238642 C. gloeosporioides 12 Diffenbachia picta MAFF 238717 C. capsici or C. truncatum 12 Glycine max Falcon C. capsici or C. truncatum 12 Hippeastrum sp. MAFF 238718 C. capsici or C. truncatum 3 Hydrangea sp. MAFF 238561 C. destructivum 5 Prunus mume MAFF 238656 C. gloeosporioides 5 Apr2-1 C. gloeosporioides Oryza sativa Pyricularia grisea MAFF 235006* P. oryzae MAFF 235063* P. orvzae MAFF, Genebank, Ministry of Agriculture, Forestry and Fisheries, Japan; IFO, Institute for Fermentation, Osaka; CBS, Centraalbureau voor Schimmelcultures, NED; IMI, International Mycological Institute, supplied from Commonwealth Agricultural Bureaux International Bioscience, UK. Asterisks indicate the isolates used for the phylogenetic analysis. The matching species of Colletotrichum recorded in DNA Data Bank of Japan; ND, Not determined. RG, Ribosomal group based on ITSl sequence. C. theae-sinensis
Camellia sinensis
(Table 2). The nucleotide sequence similarity in the ITSl regions among internal RGs of Colletotrichum species are shown in Table 3. The similarities were 70.0 to 95.9%, and each RG could be definitely differentiated (Table 3). Thirty-six isolates of C. acututum and four of C. gloeosporioides were designated as RG1 based on the sequence of ITSl and matched C. acutatum 232923 in DDBJ database (Tables 1 and 2). Intra-group similarity was 99.4%, and only the 32nd base in 181bp was different. The sequences of nine among 12 isolates of C. coccodes matched C. coccodes 232933 and were designated as RG2 with an intra-RG similarity of 100%. Two isolates of C. destructivum, five of C. higginsianum, two of C. linicola, three of C. coccodes, two of C. fuscum, and two of Colletotrichum sp. from Hydrangea were grouped, and the intra-group similarity, including gaps, was less than six bases (96.8%). This matched C. destructivum 232940 and was designated as RG3. The internal similarities between RG2 and 3 were 93.1%. All the species in RG 1-3 produced straight conidia, tapered at each end. The ITSl sequences of 66 isolates of C. gloeosporioides,
three of C. coffeanum, one of C. fragariae and three of C. musae were approximately consistent, and the differences, including gaps, were less than six bases. This group matched C. gloeosporioides 232955 and was designated as RG4. Twelve isolates of C. gloeosporioides from Crinum, Passiflora and so on could be grouped, and the differences of sequence including gaps were less than six bases. The group was close to C. gloeosporioides A5301974 and defined as RG5. The group of eight isolates of C. gloeosporioides from Glycine, Passiflora and so on with the differences less than seven bases were defined as RG6, matching C. gloeosporioides AJ301979. Five isolates of C. orbiculare, two of C. trifolii, one of C. lindemuthianum and one of C. gloeosporioides from Vigna could be designated as RG7 and matched C. orbiculare 232998. The sequence of one isolate of C. crussipes from Aucuba did not match those in DDBJ, so we defined this as RG8. RGs 4-8 include Colletotrichum species that produce straight conidia with an obtuse apex. The ITSl sequences of three isolates of C. dematium, six of C. capsici and five of C. circinbns were consistent
JGPP
314
with a difference only in the 97th base. The results matched C. dematium 232938 and were designated as RG9. The ITSl sequences of two isolates of C. circinans from Allium matched C. circinans A5301955 and were designated as RG10. The sequences of two isolates of C. truncatum from Vigna and Phaseolus and one of C. dematium from Ipomoea did not match any data in DDBJ were as RG11. Three isolates of C. dematium, two of C. truncatum and four of Colletotrichum species from Dieffenbachia, Brassica and so on could be grouped with a difference only in the 69th base. These matched C. capsici 232927 and C. truncatum A5301945 and were designated as RG12. Four isolates of C. trichellum were consistent with a difference only in the 138th base, matching C. trichellum 233002 and designated as RG13. RGs 9 to 13 include species producing lunate to falcate conidia, except for graminicolous species. The isolates of graminicolous species producing falcate conidia were divided into six RGs of RG14 to RG19.
RG 1 2 3
4 5 6 7
8 9 10 11 12 13 14 15 16 17 18 19 20
Internal similarities of ITSl sequence among these RGs were 92.2 to 95.9% (Table 3). The isolate of C. graminicola from Zea matched C. graminicola 232981 and was designated as RG14. Ten isolates of C. graminicola from Avena, Dactylis and so on could be grouped as RGl5, the sequences of which were consistent with differences in less than six bases and matched C. graminicola AF289224. The other four isolates of C. graminicola from Panicum and Digitaria were approximately consistent with differences in five bases including gaps. This group did not match any data in DDBJ and were defined as RG16. Four isolates of C. sublineolum, which matched C. sublineolum 232999, were designated as RG17. The sequences of three isolates of C. falcatum, designated as RG18, were consistent with a difference only in the 126th base, but did not match the sequences in DDBJ. Five isolates of C. caudatum could be grouped into RG19 with differences in less than four bases in ITSl region, but they did not match those in DDBJ.
Table 2. Ribosomal groups (RGs) of Colletotrichum species and their matching in DDBJ Length of Maximum Accession number Main .isolates DDBJ matchese) ITSl (bp) differences in RG in DDBJ 181 1 C. acutatum c. acutatum 232923 174 0 C. coccodes 232933 C. coccodes 181-5 232940 6 C. destructivum C. destructivum C. higginsianum C. linicola C. fuscum 171-3 232955 6 C. gloeosporioides C. gloeosporioides 190 A5301974 6 C. gloeosporioides C. gloeosporioides 174 7 C. gloeosporioides AJ301979 C. gloeosporioides 159-162 232998 6 C. orbiculare C. orbiculare C. trifolii C. lindemuthianum 169 C. crassipes 1 174 C. dematium C. dematium 232938 C. capsici 172 0 C. circinas A5301955 C. circinm 182 0 C. truncatum C. dematium 1 177 C. dematium C. capsici 232937 AJ301945 C. truncatum C. truncatum 174 or 175 1 233002 C. trichellum C. trichellum 181 C. graminicola 232981 C. graminicola 176 or 178 AF289224 6 C. graminicola C. graminicola 176 or 179 5 C. graminicola 232999 185 0 C. sublineolum C. sublineolum 176 1 C. falcatum 169 4 C. caudatum 157 or 159 2 C. theae-sinensis -
a) DDBJ, DNA Data Bank of Japan.
References 27 27
27
27 Not published
Not published 27 -
27
Not published -
27
Not published 27 27
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1 2
91.7 90.3 88.0 84.8 83.4 92.6 88.5 91.2 91.7 89.9 84.3 90.3 90.3 90.8 90.3 89.9 85.3 91.7 72.4 86.6
1 -
93.1 93.5 91.7 81.6 91.2 92.2 94.5 94.0 89.9 90.8 95.4 92.2 93.5 92.2 93.5 89.4 92.6 73.3 87.6
-
2
91.7 88.9 80.2 91.7 90.3 95.4 92.6 90.8 90.3 94.5 92.2 94.5 92.6 93.5 89.4 92.6 72.4 87.6
-
3
92.2 82.5 91.7 89.4 93.5 91.7 90.3 90.8 92.2 93.5 91.7 92.6 93.5 89.4 91.7 74.2 86.6
-
4
81.1 91.2 89.9 91.2 90.3 86.6 90.3 90.8 90.3 89.9 90.8 90.8 88.5 88.9 70.5 86.2
-
5
86.6 84.8 82.9 82.5 81.1 81.6 82.5 83.4 83.9 82.5 83.9 81.6 82.9 70.0 83.9
-
6
93.5 93.5 91.7 92.6 90.3 91.2 93.1 91.2 92.2 91.7 91.7 94.5 75.6 86.2
-
7
93.5 92.6 88.5 87.6 89.9 91.7 90.8 91.7 91.7 92.2 93.1 73.7 85.7
-
8
95.9 91.2 91.2 94.5 94.9 94.9 93.1 95.9 90.3 94.5 73.7 87.6
-
9
a) Similarities were calculated from 208 sites, multiple aligned including gaps. b) OG, outgroup, the isolates of Pyriculariu grisea.
13 14 15 16 17 18 19 20 OGb)
11 12
3 4 5 6 7 8 9 10
Isolates
MAFF 306282 MAFF 712102 MAFF 511453 MAFF 305913 MAFF 306100 MAFF 305974 MAFF 306518 MAFF 410253 IMI-288810 MAFF 237304 MAFF 305748 IMI-080025 MAFF 410046 MAFF 511343 MAFF 305371 MAFF 305439 MAFF 305360 MAFF 306170 MAFF 305700 MAFF 752003 MAFF 235006
RG
89.4 90.8 94.0 92.6 94.9 91.7 94.0 90.8 93.1 73.7 88.0
-
10
88.5 89.4 89.4 88.5 90.8 87.6 86.2 90.8 72.4 85.3
-
11
90.8 89.4 90.3 90.3 90.3 88.9 88.5 73.7 87.1
-
12
91.7 94.0 91.7 93.1 90.8 90.8 72.4 88.9
-
13
93.1 94.5 95.9 89.4 94.0 71.4 86.6
-
14
92.2 94.9 89.9 92.6 71.4 86.6
-
15
Table 3. Internal ribosomal group (RG) similarities (%)‘) of ITS1 region
94.5 90.3 94.0 72.4 87.1
-
16
18
19
20
89.9 94.0 90.3 71.4 71.4 76.0 86.6 86.6 86.2 83.9
-
17
-
OGb)
85
316
JGPP
An exception was six isolates of C. theae-sinensis that produce the smallest conidia of 4-6 p m 10ng13J8),separating from the others with an ITS similarity of 70.7 to 76%, although the intra-group similarity was consistent with two base differences including gaps. We defined them as
RG20. Phylogenetic analysis In results of the phylogenetic analysis of the combined
ITS2 and D2 sequences of Colletotrichum species from each RGs by N J and MP methods, almost the same
MAFF 305371 RGI 5 I
C. destructivum MAFF511453 RG3
96
1
I!
‘i
C.cocwdes MAFF712102 RG2 C. dematium lMl-288810 RG9
-C. gloeosporioides
MAFF 306100 RG5 C. gloeosporioides MAFF 305913 RG4
C
C. crassipes MAFF 410253 RG8
2
C. gloeosporioides MAFF 305974 RG6
II
C.orbiculare MAFF 306518 RG7
C. theae-sinensis MAFF 752003 RG20 Pyricuiaria grisea MAFF 235006
0.02
I Pyricuiaria grisea MAFF 235063
Fig. 1. Tree illustrating relatedness of Colletotrichum species, based on neighbor-joininganalysis of the ITS2 and 285 rDNA domain 2 regions. Percentages of neighbor-joining analysis of 1000 bootstrapped data sets that support specific branches are indicated at the respective nodes. Bootstrap values greater than 50% are shown. Pyricularia grisea were used to root the tree. Bar=distance correspondingto two base changes per 100 nucleotide positions. Numbers at side of tree indicate phylogenetic groups (PGs).
rDNA Grouping of Japanese Colletotrichum Species
topologies were obtained by both methods (Figs. 1and 2). In the N J analysis, three phylogenetic groups (PG) separated, such as PG1 that produced conidia with an acute end, PG2 with blunt-end conidia and PG3 of C. theae-sinensis with small conidia. Colletotrichum species without C. theae-sinensis showed monophyly with 100%bootstrap support. Colletotrichum theae-sinensis (RG20) was significantly far from the other Colletotrichum species with a
branch length of 0.235, as opposed to 0.010 to 0.052 in other Colletotrichum species and 0.124 in the outgroup. Graminicolous species of RG14 to 19 made a single clade, but bootstrap value was low at 42%. Colletotrichum acutatum (RGl) and C. destructiuum made a clade with a high bootstrap value, 96%. In MP analysis, a heuristic search produced three tree islands containing trees of 346, 346 and 347 steps with
-
I' 100
I
MAFF 51 1343 RG14
C. graminicola MAFF 305439 RG16 C. falcatum MAFF 306170 RG18
C. sublineolum MAFF 305360 RG17 84
J
317
C. acutatum MAFF 306282 RGI
C. gloeosporioides MAFF 305913 RG4 C. crassipes MAFF 410253 RG8 C. gloeosporioides MAFF 306100 RG5 C. gloeosporioides MAFF 305974 RG6
C. orbiculare MAFF 306518 RG7 C. theae-sinensis MAFF752003 RG20
I
Pyricularia grisea MAFF 235006 Pyricularia grisea MAFF 235063
Fig. 2. A 50% majority-rule consensus tree of seven most parsimonious trees generated using the sequences of ITS2 and 28s rDNA domain 2 regions for 20 RGs of Colletotrichurn and Pyricularia grisea used as the outgroup. Numbers above the branches are the bootstrap values indicating the frequencies with which a given branch appeared in 1000 replicatiohs. Bootstrap values greater than 50% are shown. Numbers at side of tree indicate phylogenetic groups (PGs).
318
JGPP
base attenuated3sZ6), so the four species may be conspecific. However, Sreenivasaprasad et aLZ7) reported that an isolate of C. fuscum was similar to C. gloeosporioides based on ITS1. The isolates of C. gloeosporioides sensu lato separated into four RGs, RG4 to RG7, with the isolates of C. musae, C. fragariae, C. coffeanum, C. orbiculare, C. lindemuthianum and C. trifolii. Most isolates of C. gloeosporioides and all of C. fragariae, C. coffeanum and C. musae were designated as RG4, that had cylindrical to oblong conidiaz6).The morphological characteristics of RG4 coincide with the original description of C. gloeosporioides. Eight isolates of C. gloeosporioides and four of Colletotrichum sp. were RG5, that had broad, cylindrical conidia with scar (data not shown). This group should be a new species as we have re p ~ r te d '~Eight ) . of C. gloeosporioides were RG6, that had oblong conidia and made ascospores DISCUSSION in perithecia on PDA (data not shown). All isolates of C. orbiculare, C. lindemuthianum and C. trifolii and one of The 236 isolates of Colletotrichum species in Japan could be grouped into 20 RGs by rDNA ITSl sequences, C. gloeosporioides were grouped as RG7 (C. orbiculare), that shared a low mycelial growth ratez6)and conidial reflecting the identification of species based on morphologies. Each RG was identified molecularly based on the septation at germinationzz).Colletotrichum crassipes desmatching with DDBJ sequence data for each species ignated as RG8 was defined clearly and made a clade with (Table 1).Because the deposited name of the isolates was C. gloeosporioides RG4, but bootstrap supports were low. sometimes mismatched with the molecular identification, The isolates of C. dematium sensu lato separated into there may have been incorrect identifications. Further five RGs of RG9 to RG13 with the isolates of C. truncatum, C. trichellum, C. circinans and C. capsici. RG9 (C. analyses are necessary to confirm the species name in future. dematiurn) included the isolates deposited as C. dematium and C. capsici. Colletotrichum dematium and C. capsici All the isolates of C. acutatum were grouped as RG1. have often been confused, because both species produce Four isolates that were isolated and deposited as C. similar falcate conidia and are isolated from many kind of gloeosporioides in the 1950s before the description of C. plantsz6).Even if the isolates had the characteristics of C. a c u t a t ~ mwere ~ ~ ) also identified molecularly as C. acutacapsici, produced appressoria in chain (data not tum. The isolates with pink to reddish colonies and named C. acutatum f. sp. chromogen~m~) could not be separated and were pathogenic to Capsicum, they were molecularly from the gray ones, indicating that the intraspecific taxon identified as C. dematium as Sreenivasaprasad et ~ 1 . ~ ~ ) also reported. The confusion between these species should was not supported by ITSl sequence homology. RGl made a clade with C. destructivum (RG3) with high be resolved in the future. The isolates of C. circinans and bootstrap value, in which conidia of both RGs are straight C. trichellum were grouped as RGlO and 13, respectively, and tapered at each end (Figs. 1 and 2). However, C. with some exceptions. Colletotrichum truncatum was separated into RGll and 12. Perhaps RG 11 should be a coccodes (RG2) with almost similar conidia had separated. Colletotrichum coccodes produces s ~ l e r o t i a ' * ~ , ~ ~new , ~ ~species ). because it did not match any data of DDBJ. The isolates of graminicolous Colletotrichum were Whereas, C. destructivum frequently produces aggregations of dark mycelia3). The morphological boundary divided into six RGs, RG14 to RG19. Isolates of C. between C. coccodes and C. destructivum is thus unclear. graminicola were grouped into RGs 14-16. Interestingly, revealed ) that C. destructivum Sreenivasaprasad et d Z 7 all the isolates from C3 gramineous plants were grouped as RG15 based on the ITSl sequence homology. RG14 (RG3) could not be confidently separated (98% homology) included those from Zea and RG16 from Digitaria and from C. linicola based on ITSl sequence divergence. In results of our analysis using ITS1, C. destructivum, a Echinochloa, C4 gramineous plants. RG 15 and 16 may possibly be described as new species, because they are pathogen of the Leguminosae such as Medicago and different in conidial morphologies from C. graminicola T r i f ~ l i u m ' , ~was ) , grouped into RG3 with C. higginsianum, a pathogen on cruciferous plants ; C. linicola, pathogenic (data not shown). The isolates grouped in RGs 17, 18 and 19 coincided with morphologies or DDBJ sequence data of on Linum; and C. fuscum on Nemesia. Their conidial shapes were very similar, straight to slightly curved and C. sublineolum, C. falcatum and C. caudatum, respective-
115 parsimony-informative characters. The islands with the smallest tree length contained seven trees with a consistency index (CI) of 0.6693, retention index (RI) of 0.6467, a re-scaled consistency index (RC) of 0.4329 and homoplasy index (HI) of 0.3307. A 50% majority-rule consensus of these trees with bootstrap supports is shown in Fig. 2. Graminicolous species were separated, and C. graminicola (RG14, 16), C. sublineolum, C. falcatum and C. caudatum made a clade except for C. graminicola (RG15). C. acutatum and C. destructivum formed a clade (84% bootstrap support). The internal supports for nodes except for C. orbiculare and C. theae-sinensis are low, making it difficult to determine the relationships among Colletotrichum species, but it is clear that C. theaesinensis is far distant from the others.
rDNA Grouping of Japanese Colletotrichum Species
ly. In results of phylogenetic analyses, C4 gramineous anthracnose pathogen, RG14, 16, 17, 18 and 19, made a clade with 70% bootstrap supports in N J tree and 42% in MP tree (Figs. 1 and 2). It may indicate that the graminicolous Colletotrichum species have same origin in their evolution. RG20, the group of C. theae-sinensis producing particularly small conidia was far distant from the other RGs of Colletotrichum species rather than the outgroup, Pyricularia grisea. This distance means C. theae-sinensis does not belong to Colletotrichum and should be transferred to another genus in the future. In results of phylogenetic analysis of the Japanese Colletotrichum isolates, three PGs were recognized to produce different type of conidia. This report is the first that Colletotrichum species that produce acute and blunt end conidia separate into different phylogenetic groups and showing that phylogenetic analysis is a useful tool for the Colletotrichum taxonomy. Grouping and phylogenetic analysis using ribosomal DNA revealed a new frame of Colletotrichum species as described. The morphology of conidia and appressoria, cultural characteristics, pathogenicity and molecular characteristics of each RG should be studied and the species then classified to more distinguishably reflect their phylogeny. ACKNOWLEDGMENTS We thank Dr. H. Horie and Mr. J. Takeuchi of the Tokyo Metropolitan Agricultural Experiment Station, Japan, Mr. S. Uematsu of the Chiba Horticultural Experiment Station, Japan, Mr. K. Tomioka of the National Agricultural Research Center for Western Region, Japan, Mr. H. Kanno of the Miyagi Prefectural Horticultural Station, Japan, Mr. H. Ohkubo of the National Institute of Livestock and Grassland Science, Japan, Ms. A. Tanaka of the Institute for Green Science, Japan and Mr. K. Takano of the Toyama Agricultural Research Center for providing Colletotrichum isolates.
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