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  • Research
  • Open Access

Identification, prevalence and pathogenicity of Colletotrichum species causing anthracnose of Capsicum annuum in Asia

IMA Fungus201910:8

https://doi.org/10.1186/s43008-019-0001-y

  • Received: 18 April 2019
  • Accepted: 25 April 2019
  • Published:

Abstract

Anthracnose of chili (Capsicum spp.) causes major production losses throughout Asia where chili plants are grown. A total of 260 Colletotrichum isolates, associated with necrotic lesions of chili leaves and fruit were collected from chili producing areas of Indonesia, Malaysia, Sri Lanka, Thailand and Taiwan. Colletotrichum truncatum was the most commonly isolated species from infected chili fruit and was readily identified by its falcate spores and abundant setae in the necrotic lesions. The other isolates consisted of straight conidia (cylindrical and fusiform) which were difficult to differentiate to species based on morphological characters. Taxonomic analysis of these straight conidia isolates based on multi-gene phylogenetic analyses (ITS, gapdh, chs-1, act, tub2, his3, ApMat, gs) revealed a further seven known Colletotrichum species, C. endophyticum, C. fructicola, C. karsti, C. plurivorum, C. scovillei, C. siamense and C. tropicale. In addition, three novel species are also described as C. javanense, C. makassarense and C. tainanense, associated with anthracnose of chili fruit in West Java (Indonesia); Makassar, South Sulawesi (Indonesia); and Tainan (Taiwan), respectively. Colletotrichum siamense is reported for the first time causing anthracnose of Capsicum annuum in Indonesia and Sri Lanka. This is also the first report of C. fructicola causing anthracnose of chili in Taiwan and Thailand and C. plurivorum in Malaysia and Thailand. Of the species with straight conidia, C. scovillei (acutatum complex), was the most prevalent throughout the surveyed countries, except for Sri Lanka from where this species was not isolated. Colletotrichum siamense (gloeosporioides complex) was also common in Indonesia, Sri Lanka and Thailand. Pathogenicity tests on chili fruit showed that C. javanense and C. scovillei were highly aggressive, especially when inoculated on non-wounded fruit, compared to all other species. The existence of new, highly aggressive exotic species, such as C. javanense, poses a biosecurity risk to production in countries which do not have adequate quarantine regulations to restrict the entry of exotic pathogens.

Keywords

  • Phylogenetics
  • Plant pathology
  • New taxa

INTRODUCTION

Colletotrichum is one of the most important genera of plant pathogenic fungi with many of the 200 plus species known to cause disease in plant crops worldwide (Udayanga et al. 2013; Marin-Felix et al. 2017). Colletotrichum species causing anthracnose are particularly important as post-harvest pathogens of fruit and vegetable crops growing in tropical and subtropical climates (Alahakoon et al. 1994; Rojas et al. 2010; Cannon et al. 2012; Damm et al. 2012a, 2012b; Udayanga et al. 2013; Shivas et al. 2016; De Silva et al. 2017a).

Chili (Capsicum spp.) is an important vegetable crop in tropical and subtropical climates and the fresh or dried fruit is a major culinary ingredient in many cuisines. Anthracnose is a major disease of chili fruit causing significant yield loss as well as reducing the marketability of the fruit. Anthracnose of chili has been shown to be caused by 24 Colletotrichum species (Mongkolporn and Taylor 2018) reported from many countries including Australia (De Silva et al. 2017a), Brazil (de Oliveira et al. 2017), China (Diao et al. 2017), India (Sharma and Shenoy 2014), Indonesia (Voorrips et al. 2004), Korea (Kim et al. 1999), Malaysia (Noor and Zakaria 2018), Sri Lanka (Ranathunge et al. 2009), Thailand (Than et al. 2008) and the USA (Harp et al. 2008).

Colletotrichum species causing anthracnose of chili in Asia were previously identified as C. acutatum (straight conidia with acute ends), C. gloeosporioides (straight conidia with obtuse ends) and C. truncatum (falcate conidia) (Than et al. 2008, Mongkolporn et al. 2010,). However, with the implementation of multigene phylogenetic analyses, C. acutatum was demonstrated to be a species complex (acutatum complex) composed of 34 closely related species (Marin-Felix et al. 2017), with seven species identified as causing anthracnose in chili (Mongkolporn and Taylor 2018). Similarly, C. gloeosporioides was shown to be a species complex of 38 closely related species (Marin-Felix et al. 2017), with nine species identified to cause anthracnose in chili (Mongkolporn and Taylor 2018). Morphological characters cannot differentiate many of the species with straight conidia, especially those in the acutatum and gloeosporioides complexes that are pathogens of chili, and thus multigene phylogenetic analyses are required for proper identification of these species (De Silva et al. 2017a).

The distribution of the Colletotrichum species that cause anthracnose of chili is quite variable across countries that produce this crop. For example, in Australia only six out of the 24 Colletotrichum pathogens of chili have been identified (De Silva et al. 2017a), in Thailand only three have been reported (Mongkolporn and Taylor 2018), and five from Malaysia (Noor and Zakaria 2018). In most of the previous reports the identification of species was based only on morphological data. Therefore, the status of the taxonomy of Colletotrichum spp. causing anthracnose in chili producing countries in Asia remains uncertain. Proper identification of these pathogens is important for mitigating the risk of incursion of new pathogens which if happens, may have devastating consequences for the local industries. In addition, accurate identification of the species is important for resistance breeding programs and in identifying the host-range of species. Several Colletotrichum species such as C. karsti, C. siamense and C. truncatum have broad host ranges (Cannon et al. 2012; Damm et al. 2012b). The main Colletotrichum species causing anthracnose in chili are known to be in the acutatum and gloeosporioides complexes. However, recently further species from the boninense and orchidearum complexes were implicated (Diao et al. 2017; Damm et al. 2019). Therefore, it is important to understand the taxonomy, diversity and pathogenicity of Colletotrichum species that infect chili and their distribution across countries.

The aims of the study were to (1) identify the Colletotrichum species with straight conidia (cylindrical and fusiform) causing anthracnose of chili in selected regions of Indonesia, Malaysia, Taiwan, Thailand and Sri Lanka, and (2) determine the pathogenicity of those species on chili.

MATERIALS AND METHODS

Isolates

A total of 260 isolates associated with anthracnose disease symptoms on chili fruit and leaves were collected from chili producing countries in Asia: Indonesia, Malaysia, Taiwan, Thailand, and Sri Lanka (Table 1). Type specimens and ex-type cultures are deposited in the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands (CBS), and in the University of Melbourne culture collection (UOM), Victoria, Australia.
Table 1

Collection sites and numbers of Colletotrichum isolates

Country and region

Number of isolates

Thailand

96

Chiang Mai

20

Chiang Rai

44

Kanchana Buri

4

Nakhon Pathom

7

Suphan Buri

12

Ratchaburi

7

Bangkok

2

Malaysia

12

Pahang

3

Johor

4

Kelantan

5

Sri Lanka

19

Kandy

5

Matara

14

Indonesia

113

Gowa

31

Soppeng

6

Jeneponto

45

Makassar

7

Maros

4

West Java (East West Seed Co. Indonesia)

20

Taiwan (World Vegetable Center collection)

20

Tainan

14

Taichung

1

Nantou

2

Pingtung

1

Ilan

1

Hsinchu

1

Figures in bold represent the total number of isolates from each country

Fungal isolates were established from lesions on infected fruit and leaves using two methods. Surface sterilised (~ 1% ai sodium hypochlorite for 5 min) infected tissue (0.5 cm2) was cultured on water agar (WA; Crous et al. 2009) and then after 2 to 3 d fungal hyphae were subcultured onto potato dextrose agar (PDA, Difco) and synthetic nutrient-poor agar (SNA, Nirenberg 1976) as described by De Silva et al. (2017a). Freshly collected fruit from field grown chili plants with typical anthracnose lesions was incubated for 1 to 2 d in a moist chamber until conidiomata appeared and then single spore isolation was performed according to Choi et al. (1999). Selected isolates were also cultured on oatmeal agar (OA; Crous et al. 2009) and malt extract agar (MEA) at 20 °C under near UV light with a 12 h photoperiod for 10 d. Cultures were isolated and maintained either at the AQIS quarantine laboratory at the University of Melbourne or the Evolutionary Pathology Laboratory at the Westerdijk Fungal Biodiversity Institute, Netherlands (CBS).

Morphology

Cultures grown on PDA at 27 °C were used for morphological analysis. Colony colour and texture were examined after 10 d, and colony growth rate calculated by measuring colony diameter 7 and 10 d after incubation.

Conidia from the conidiomata in culture were mounted in lactic acid and the length and width measured for 30 randomly selected conidia for each isolate, with the range and mean calculated. Size and shape of appressoria were determined on WA using a slide culture technique (Johnston and Jones 1997). Production of acervular conidiomata was observed on dried, sterilised chili peduncles inoculated with mycelia and incubated on WA and SNA media. Cultures were examined periodically for the development of perithecia. Ascospores were measured and described from perithecia squashed in lactic acid. Morphological characters were examined using a Leica DM6000 LED compound microscope with differential interference contrast (DIC) optics.

DNA extraction, PCR amplification and sequencing

The 260 Colletotrichum isolates were initially identified on the basis of culture characteristics on PDA (based on distinct morphotype groups), morphology of the spores, and/or internal transcribed spacer and intervening 5.8S nrDNA gene (ITS) sequence. A total of 115 isolates were identified as C. truncatum and the remaining 145 isolates with straight conidia were subsequently selected for multigene phylogenetic analyses. Genomic DNA was extracted from fresh mycelia grown on PDA using the DNeasy Plant Mini kit (QIAGEN, Australia), following the manufacturer’s instructions. DNA quality was assessed on a 1.4% (w/v) agarose gel, quantified by comparing with a known amount of Lambda DNA/HindIII marker (Invitrogen, Australia), diluted to 2 ng/μL and then stored at _20 °C until ready for PCR.

Isolates belonging to the acutatum complex were further analysed with partial gene sequences of five genomic loci: an intron sequence of the glyceraldehyde-3-phosphate dehydrogenase (gapdh), partial sequences of the chitin synthase 1 (chs-1), actin (act), beta-tubulin (tub2) and histone 3 (his3) genes. Isolates of the gloeosporioides complex were further analysed with chs-1, act, gapdh, tub2, Apn2–MAT1–2 intergenic spacer and partial mating type MAT1–2 gene (ApMat) and glutamine synthetase (gs) genes. Isolates belonging to the boninense and orchidearum complexes were further analysed with gapdh, tub2 and act genes. The genes were amplified and sequenced using the respective primer pairs for each gene region: ACT-512F + ACT-783R (act; Carbone and Kohn 1999), AMF1 + AMR1 (ApMat; Silva et al. 2012b), CHS-79F + CHS-345R (chs-1; Carbone and Kohn 1999), GDF1 + GDR1 (gapdh; Guerber et al. 2003), GSF1 + GSR1 (gs; Stephenson et al. 1997), CYLH3F + CYLH3R (his3; Crous et al. 2004a), ITS1 + ITS4 (ITS; White et al. 1990), and Btub2Fd + Btub4Rd (tub2; Woudenberg et al. 2009).

The PCR for each reaction was performed in a 2720 Thermal Cycler (Applied Biosystems) in a total volume of 25 μL, comprised of 1× PCR buffer, 0.2 mM dNTP, 0.4 μM of each primer, 2 mM MgCl2, 1 U Taq DNA polymerase (MangoTaq DNA polymerase; Bioline) and 6 ng template DNA and components were adjusted as required. PCR amplification protocols were performed as described by Damm et al. (2012a, 2012b) and Silva et al. (2012), except for the annealing temperatures which were adjusted to 55 °C for ITS, gapdh, tub2, 58 °C for act, 60 °C for gs and 62 °C for ApMat.

All PCR products were purified with the QIAquick PCR Purification kit (QIAGEN, Australia), according to manufacturer’s instructions. DNA sequence analysis of the PCR products was carried out at either the Australian Genome Research Facility (AGRF, Melbourne) or at the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands. The purified PCR products were sequenced in both forward and reverse directions, and the consensus sequences were obtained by alignment using Geneious Pro v. 11.1.4 (Kearse et al. 2012). The consensus sequences were deposited in GenBank (Table 2) and taxonomic novelties in MycoBank (Crous et al. 2004b). Sequences of each locus were assembled with MEGA v. 6 (Tamura et al. 2013). GenBank accession numbers of all the isolates used in the phylogenetic analyses are listed in Table 2.
Table 2

Strains of Colletotrichum species used in the phylogenetic analyses with details of host and location, and GenBank accession numbers of the sequences

Species

Accession No.1

Host/Substrate

Country

GenBank accession number

ITS

gapdh

chs-1

his3

act

tub2

ApMat

gs

Acutatum complex

C.abscissum

COAD 1877a

Citrus sinensis

Brazil

KP843126

KP843129

KP843132

KP843138

KP843141

KP843135

  

C. acutatum

CBS 112996, ATCC 56816, STE-U 5292a

Carica papaya

Australia

JQ005776

JQ948677

JQ005797

JQ005818

JQ005839

JQ005860

 

CBS 144.29

Capsicum annuum

Sri Lanka

JQ948401

JQ948732

JQ949062

JQ949392

JQ949722

JQ950052

C. australisinense

CGMCC 3.18886, GX1655a

Hevea brasiliensis

China

MG209623

MG241962

MG241981

MG241947

MG209645

  

C. bannaense

CGMCC 3.18887, YNWD31a

Hevea brasiliensis

China

MG209638

MG242006

MG241996

MG242002

MG209660

  

C. brisbanense

CBS 292.67, DPI 11711a

Capsicum annuum

Australia

JQ948291

JQ948621

JQ948952

JQ949282

JQ949612

JQ949942

C. cairnsense

BRIP 63642a, CBS 140847a

Capsicum annuum

Australia

KU923672

KU923704

KU923710

KU923722

KU923716

KU923688

C. chrysanthemi

CBS 126518, PD 84/520a

Carthamus sp., twisted stem

Netherlands

JQ948271

JQ948601

JQ948932

JQ949262

JQ94992

JQ949922

 C. cosmi

CBS 853.73, PD 73/856a

Cosmos sp., seed

Netherlands

JQ948274

JQ948604

JQ948935

JQ949265

JQ949595

JQ949925

C. costaricense

CBS 330.75a

Coffea arabica, cv. Typica, berry

Costa Rica

JQ948180

JQ948510

JQ948841

JQ949171

JQ949501

JQ949831

  

C. citri

CBS 134233a

C. aurantifolia shoot

China

KC293581

KC293741

KC293621

KC293661

  

C. cuscutae

IMI 304802a

Cuscuta sp.

Dominica

JQ948195

JQ948525

JQ948856

JQ949186

JQ949516

JQ949846

  

C. fioriniae

CBS 128517a

Fiorinia sp.

USA

JQ948292

JQ948622

JQ948953

JQ949283

JQ949613

JQ949943

C. godetiae

CBS 133.44a

Clarkia hybrida, cv. Kelvon Glory, seed

Denmark

JQ948402

JQ948733

JQ949063

JQ949393

JQ949723

JQ950053

C. guajavae

IMI 350839, CPC 18893a

Psidium guajava, fruit

India

JQ948270

JQ948600

JQ948931

JQ949261

JQ949591

JQ949921

C. indonesiense

CBS 127551, CPC 14986a

Eucalyptus sp.

Indonesia

JQ948288

JQ948618

JQ948949

JQ949279

JQ949609

JQ949939

C. javanense

CBS 144963 a , UOM 1115, EWINDO 3

Capsicum annuum

Indonesia

MH846576

MH846572

MH846573

MH846571

MH846575

MH846574

C. laticiphilum

CBS 112989, IMI 383015a

Hevea brasiliensis

India

JQ948289

JQ948619

JQ948950

JQ949280

JQ949610

JQ949940

C. limetticola

CBS 114.14a

Citrus aurantifolia, young twig

USA, Florida

JQ948193

JQ948523

JQ948854

JQ949184

JQ949514

JQ949844

  

C. lupini

CBS 109225, BBA 70884a

Lupinus albus

Ukraine

JQ948155

JQ948485

JQ948816

JQ949146

JQ949476

JQ949806

C. melonis

CBS 159.84a

Cucumis melo

Brazil

JQ948194

JQ948524

JQ948855

JQ949185

JQ949515

JQ949845

  

C. nymphaeae

CBS 515.78a

Nymphaea alba, leaf spot

Netherlands

JQ948197

JQ948527

JQ948858

JQ949518

JQ949848

JQ949848

C. paranaense

CBS 134729a, CPC 20901

Malus domestica

Brazil

KC204992

KC205026

KC205043

KC205004

KC205077

KC205060

  

C. paxtonii

IMI 165753a, CPC 18868

Musa sp.

Saint Lucia

JQ948285

JQ948615

JQ948946

JQ949276

JQ949606

JQ949936

C. salicis

CBS 607 94a

Salix sp., leaf spot

Netherlands

JQ948460

JQ948791

JQ949121

JQ949451

JQ949781

JQ950111

C. scovillei

CBS 120708, HKUCC 10893, Mj6

Capsicum annuum

Thailand

JQ948269

JQ948599

JQ948930

JQ949260

JQ949590

JQ949920

 

CBS 126529, PD 94/921–3, BBA 70349a

Capsicum sp.

Indonesia

JQ948267

JQ948597

JQ948928

JQ949258

JQ949588

JQ949918

 

CPC 28551

Capsicum annuum

Thailand

MH618287

MH618361

MH686337

MH707595

MH645871

 

CPC 28552

Capsicum annuum

Thailand

MH618286

MH618362

MH686338

MH707594

MH645872

 

CPC 28577

Capsicum annuum

Indonesia

MH618295

MH618363

MH686339

MH707593

MH645873

 

CPC 28579

Capsicum annuum

Indonesia

MH618294

MH618364

MH686340

MH707592

MH645874

 

CPC 28591

Capsicum annuum

Indonesia

MH618293

MH618365

MH686341

MH707591

MH645875

 

CPC 28593

Capsicum annuum

Indonesia

MH618292

MH618366

MH686342

MH707590

MH645876

 

CPC 28599

Capsicum annuum

Indonesia

MH618291

MH618367

MH686343

MH707589

MH645877

 

CPC 28603

Capsicum annuum

Indonesia

MH618290

MH618368

MH686344

MH707588

MH645878

 

CPC 28615

Capsicum annuum

Indonesia

MH618289

MH618369

MH686345

MH707587

MH645879

 

CPC 28617

Capsicum annuum

Indonesia

MH618288

MH618370

MH686346

MH707586

MH645880

 

CPC 30197, Coll 1

Capsicum annuum

Indonesia

MH618268

MH618334

MH686347

MH707585

MH645881

 

CPC 30198, Coll 2

Capsicum annuum

Indonesia

MH618269

MH618335

MH686348

MH707584

MH645882

 

CPC 30199, Coll 3

Capsicum annuum

Indonesia

MH618270

MH618336

MH686349

MH707583

MH645883

 

CPC 30200, Coll 4

Capsicum annuum

Indonesia

MH618271

MH618337

MH686350

MH707582

MH645884

 

CPC 30201, Coll 5

Capsicum annuum

Indonesia

MH618272

MH618338

MH686351

MH707581

MH645885

 

CPC 30202, Coll 6

Capsicum annuum

Indonesia

MH618273

MH618339

MH686352

MH707580

MH645886

 

CPC 30205, Coll 9

Capsicum annuum

Indonesia

MH618274

MH618340

MH686353

MH707579

MH645887

 

CPC 30206, Coll 10

Capsicum annuum

Indonesia

MH618275

MH618341

MH686354

MH707578

MH645888

 

CPC 30215, Coll 19

Capsicum annuum

Indonesia

MH618276

MH618342

MH686355

MH707577

MH645889

 

CPC 30216, Coll 20

Capsicum annuum

Indonesia

MH618277

MH618343

MH686356

MH707576

MH645890

 

CPC 30217, Coll 21

Capsicum annuum

Indonesia

MH618278

MH618344

MH686357

MH707575

MH645891

 

CPC 30218, Coll 22

Capsicum annuum

Indonesia

MH618279

MH618345

MH686358

MH707574

MH645892

 

CPC 30219, Coll 23

Capsicum annuum

Indonesia

MH618280

MH618346

MH686359

MH707573

MH645893

 

CPC 30220, Coll 24

Capsicum annuum

Indonesia

MH618281

MH618347

MH686360

MH707572

MH645894

 

CPC 30229, Coll 33

Capsicum annuum

Thailand

MH618282

MH618348

MH686361

MH707571

MH645895

 

CPC 30230, Coll 34

Capsicum annuum

Thailand

MH618283

MH618349

MH686362

MH707570

MH645896

 

CPC 30231, Coll 35

Capsicum annuum

Thailand

MH618284

MH618350

MH686363

MH707569

MH645897

 

CPC 30232, Coll 36

Capsicum annuum

Thailand

MH618285

MH618351

MH686364

MH707568

MH645898

 

CPC 30239, Coll 153

Capsicum annuum

Taiwan

MH618299

MH836634

MH707528

MH707611

MH645855

MH635064

 

CPC 30240, Coll 329

Capsicum annuum

Taiwan

MH618300

MH836635

MH707529

MH707610

MH645856

MH635065

 

CPC 30241, Coll 524

Capsicum annuum

Taiwan

MH618301

MH836637

MH707530

MH707609

MH645857

MH635067

 

CPC 30242, Coll 683

Capsicum annuum

Taiwan

MH618302

MH836638

MH707531

MH707608

MH645858

MH635068

 

CPC 30243, Coll 1296

Capsicum annuum

Taiwan

MH618303

MH836639

MH707532

MH707607

MH645859

MH635069

 

CPC 30244, Coll 1297

Capsicum annuum

Taiwan

MH618304

MH836640

MH707533

MH707606

MH645860

MH635070

 

CPC 30246, Coll 1300

Capsicum annuum

Taiwan

MH618305

MH836641

MH707534

MH707605

MH645861

MH635071

 

CPC 30247, Coll 1301

Capsicum annuum

Taiwan

MH618306

MH836642

MH707535

MH707604

MH645862

MH635072

 

CPC 30248, Coll 1302

Capsicum annuum

Taiwan

MH618308

MH836643

MH707536

MH707603

MH645863

MH635073

 

CPC 30249, Coll 1303

Capsicum annuum

Taiwan

MH618307

MH836644

MH707537

MH707602

MH645864

MH635074

 

CPC 30250, Coll 1304

Capsicum annuum

Taiwan

MH618309

MH836645

MH707538

MH707601

MH645865

MH635075

 

CPC 30251, Coll 1306

Capsicum annuum

Taiwan

MH618310

MH836646

MH707539

MH707600

MH645866

MH635076

 

CPC 30252, Coll 141

Capsicum annuum

Taiwan

MH618311

MH836633

MH707540

MH707599

MH645867

MH635063

 

UOM 1101, 313

Capsicum annuum

Thailand

MH618256

MH618324

MH686324

MH707557

MH635089

MH635049

 

UOM 1102, 322

Capsicum annuum

Thailand

MH618259

MH618325

MH686325

MH707556

MH635090

MH635050

 

UOM 1103, 311

Capsicum annuum

Thailand

MH618255

MH618326

MH686326

MH707555

MH635091

MH635051

 

UOM 1104, 314

Capsicum annuum

Thailand

MH618257

MH618327

MH686327

MH707554

MH635092

MH635052

 

UOM 1105, MJ3

Capsicum annuum

Thailand

MH618264

MH618328

MH686328

MH707553

MH635093

MH635053

 

UOM 1106, MJ5

Capsicum annuum

Thailand

MH618265

MH618329

MH686329

MH707552

MH635094

MH635054

 

UOM 1107, MJ7

Capsicum annuum

Thailand

MH618266

MH618330

MH686330

MH707551

MH635095

MH635055

 

UOM 1108, MJ8

Capsicum annuum

Thailand

MH618267

MH618331

MH686331

MH707550

MH635096

MH635056

 

UOM 1109, 211

Capsicum annuum

Thailand

MH618254

MH618332

MH686332

MH707549

MH635097

MH635057

 

UOM 1110, 316

Capsicum annuum

Thailand

MH618258

MH618333

MH686333

MH707548

MH635098

MH635058

 

UOM 1111, GA1

Capsicum annuum

Thailand

MH618260

MH618357

MH686334

MH707547

MH635099

MH635059

 

UOM 1112, GA2

Capsicum annuum

Thailand

MH618261

MH618358

MH686335

MH707546

MH635100

MH635060

 

UOM 1113, GA3

Capsicum annuum

Thailand

MH618262

MH618359

MH707545

MH635101

MH635061

 

UOM 1114, GA5

Capsicum annuum

Thailand

MH618263

MH618360

MH686336

MH707544

MH635102

MH635062

 

UOM 1140, F59

Capsicum annuum

Malaysia

MH618316

MH618355

MH686322

MH707559

MH635087

MH635047

 

UOM 1141, A15

Capsicum annuum

Malaysia

MH618313

MH618356

MH686323

MH707558

MH635088

MH635048

 

UOM 1142, Coll 1307

Capsicum annuum

Taiwan

MH618298

MH836647

MH707542

MH707597

MH645869

MH635077

 

UOM 1143, Coll 1311

Capsicum annuum

Taiwan

MH618296

MH836648

MH707543

MH707596

MH645870

MH635078

 

UOM 1144, EWINDO 2

Capsicum annuum

Indonesia

MH587232

MH618317

MH686314

MH707567

MH635079

MH836628

 

UOM 1145, EWINDO 8

Capsicum annuum

Indonesia

MH587231

MH618318

MH686315

MH707566

MH635080

MH836629

 

UOM 1146, EWINDO 10

Capsicum annuum

Indonesia

MH587233

MH618319

MH686316

MH707565

MH635081

MH836630

 

UOM 1147, EWINDO 14

Capsicum annuum

Indonesia

MH587234

MH618320

MH686317

MH707564

MH635082

MH836631

 

UOM 1148, EWINDO 15

Capsicum annuum

Indonesia

MH587235

MH618321

MH686318

MH707563

MH635083

MH836632

 

UOM 1149, Coll 365

Capsicum annuum

Taiwan

MH618297

MH836636

MH707541

MH707598

MH645868

MH635066

 

UOM 1150, 4–46-3D

Capsicum annuum

Malaysia

MH618312

MH618352

MH686319

MH707562

MH635084

MH635044

 

UOM 1151, E15

Capsicum annuum

Malaysia

MH618314

MH618353

MH686320

MH707561

MH635085

MH635045

 

UOM 1152, E16

Capsicum annuum

Malaysia

MH618315

MH618354

MH686321

MH707560

MH635086

MH635046

C. simmondsii

CBS 122122a

Carica papaya, fruit

Australia

JQ948276

JQ948606

JQ948937

JQ949267

JQ949597

JQ949927

C. sloanei

IMI 364297, CPC 18929a

Theobroma cacao, leaf

Malaysia

JQ948287

JQ948617

JQ948948

JQ949278

JQ949608

JQ949938

C. tamarilloi

CBS 129814, T.A.6a

Solanum betaceum, fruit

Colombia

JQ948184

JQ948514

JQ948845

JQ949175

JQ949505

JQ949835

C. walleri

CBS 125472, BMT(HL)19a

Coffea sp., leaf tissue

Vietnam

JQ948275

JQ948605

JQ948936

JQ949266

JQ949596

JQ949926

Boninense complex

C. annellatum

CBS 129826a

Hevea brasiliensis, leaf

Colombia

JQ005222

JQ005309

JQ005396

JQ005570

JQ005656

C. beeveri

CBS 128527, ICMP 18594a

Brachyglottis repanda

New Zealand

JQ005171

JQ005258

JQ005345

JQ005519

JQ005605

 C. boninense

CBS 123755a, MAFF 305972

Crinum asiaticum var. sinicum

Japan

JQ005153

JQ005240

JQ005327

JQ005501

JQ005588

C. brasiliense

CBS 128501a, ICMP 18607, PAS12

Passiflora edulis, fruit anthracnose

Brazil

JQ005235

JQ005322

JQ005409

JQ005583

JQ005669

C. brassicicola

CBS 101059a, LYN 16331

Brassica oleracea var. gemmifera, leaf spot

New Zealand

JQ005172

JQ005259

JQ005346

JQ005520

JQ005606

C. constrictum

CBS 128504a, ICMP 12941

Citrus limon, fruit rot

New Zealand

JQ005238

JQ005325

JQ005412

JQ005586

JQ005672

C. karsti

CAUOS1

Capsicum sp.

China

KP890103

KP890134

KP890118

KP890126

KP890110

 

CAUOS7

Capsicum sp.

China

KP890108

KP890139

KP890124

KP890132

KP890116

 

CBS 125468

Coffea sp., berry tissue

Vietman

JQ005197

JQ005284

JQ005371

JQ005545

JQ005631

 

CBS 127595

Musa banksii

Australia

JQ005178

JQ005265

JQ005352

JQ005526

JQ005612

 

CBS 129815, T.A.7

Solanum betaceum, fruit

Colombia

JQ005187

JQ005274

JQ005361

JQ005535

JQ005621

 

CBS 129834

Musa sp.

Mexico

JQ005176

JQ005263

JQ005350

JQ005524

JQ005610

 

CBS 129927

Anthurium sp.

Thailand

JQ005206

JQ005293

JQ005380

JQ005554

JQ005640

 

CBS 128545, ICMP 18587

Capsicum annuum

New Zealand

JQ005207

JQ005294

JQ005381

JQ005555

JQ005641

 

CBS 128548, ICMP 18589

Solanum lycopersicum

New Zealand

JQ005205

JQ005292

JQ005379

JQ005553

JQ005639

 

CBS 132134, CGMCC 3.14194a

Vanda sp.

China

HM585409

HM585391

HM581995

HM585428

 

CPC 28553

Capsicum annuum

Indonesia

MH844440

MH844444

MH844456

MH844449

MH844448

 

CPC 28554

Capsicum annuum

Indonesia

MH844439

MH844443

MH844455

MH844450

MH844447

 

CPC 28601

Capsicum annuum

Indonesia

MH844438

MH844442

MH844454

MH844451

MH844446

 

CPC 28602

Capsicum annuum

Indonesia

MH844437

MH844441

MH844453

MH844452

MH844445

 

GM44 L01a

Annona muricata

Colombia

KC512141

KC506413

KC512162

KC512183

KC512204

C. petchii

CBS 378.94a

Dracaena marginata

Italy

JQ005223

JQ005310

JQ005397

JQ005571

JQ005657

C. phyllanthi

CBS 175.67a, MACS 271

Phyllanthus acidus

India

JQ005221

JQ005308

JQ005395

JQ005569

JQ005655

Truncatum complex

C. truncatum

CBS 151.35

Phaseolus lunatus

USA

GU227862

GU228254

GU228352

GU227960

GU228156

Gloeosporioides complex

C. aenigma

ICMP 18608a

Persea americana

Israel

JX010244

JX010044

JX009774

JX009443

JX010389

KM360143

JX010078

 

ICMP 18686

Pyrus pyrifolia

Japan

JX010243

JX009913

JX009789

JX009519

JX010390

JX010079

C. aeschynomenes

ICMP 17673a, ATCC 201874

Aeschynomene virginica

USA

JX010176

JX009930

JX009799

JX009483

JX010392

KM360145

JX010081

C. alatae

CBS 304.67a, ICMP 17919

Dioscorea alata

India

JX010190

JX009990

JX009837

JX009471

JX010383

KC888932

JX010065

C. alienum

ICMP 12071a

Malus domestica

New Zealand

JX010251

JX010028

JX009882

JX009572

JX010411

KM360144

JX010101

 

ICMP 18621

Persea americana

New Zealand

JX010246

JX009959

JX009755

JX009552

JX010386

JX010075

C. aotearoa

ICMP 18537a

Coprosma sp.

New Zealand

JX010205

JX010005

JX009853

JX009564

JX010420

KC888930

JX010113

C. asianum

ICMP 18580a, CBS 130418

Coffea arabica

Thailand

FJ972612

JX010053

JX009867

JX009584

JX010406

FR718814

JX010096

 

IMI 313839, ICMP 18696

Mangifera indica

Australia

JX010192

JX009915

JX009753

JX009576

JX010384

JX010073

C. camelliae

CGMCC 3.14925, LC1364a

Camellia sinensis

China

KJ955081

KJ954782

KJ954363

KJ955230

KJ954497

KJ954932

C. changpingense

MFLUCC 150022a

Fragaria ananassa

China

KP683152

KP852469

KP852449

KP683093

KP852490

C. chrysophilum

CMM 4268a, URM 7362

Musa sp.

Brazil

KX094252

KX094183

KX094083

KX093982

KX094285

C. clidemiae

ICMP 18706a

Clidemia hirta

USA, Hawaii

JX010265

JX009989

JX009877

JX009537

JX010438

KC888929

JX010129

C. conoides

CAUG17a

Capsicum annuum

China

KP890168

KP890162

KP890156

KP890144

KP890174

C. cordylinicola

MFLUCC 090551a, ICMP 18579

Cordyline fruticosa

Thailand

JX010226

JX009975

JX009864

HM470235

JX010440

JQ899274

JX010122

C. endophyticum

CAUG28

Capsicum annuum

China

KP145441

KP145413

KP145385

KP145329

KP145469

 

DNCL075

Unknown wild fruit

Thailand

KF242123

KF242181

KF157827

KF254857

KF242154

 

LC0324a

Pennisetum purpureum

Thailand

KC633854

KC832854

KF306258

 

UOM 1137, F5-2D

Capsicum annuum

Thailand

MH728809

MH707467

MH805853

MH781483

MH846566

MH728828

MH748267

C. fructicola

I TCC 6270

Mangifera indica

India

KC790774

KC888935

KC790663

KC790909

KC790713

 

ICMP 18581a, CBS 130416

Coffea arabica

Thailand

JX010165

JX010033

JX009866

FJ907426

JX010405

JQ807838

JX010095

 

LC2923, LF130

Camellia sinensis

China

KJ955083

KJ954784

KJ954365

KJ955232

KJ954499

KJ954934

 

CPC 28644

Capsicum annuum

Thailand

MH728811

MH707465

MH805851

MH781481

MH846564

MH728830

MH748265

 

CPC 28645

Capsicum annuum

Thailand

MH728810

MH707466

MH805852

MH781482

MH846565

MH728829

MH748266

 

UOM 1138, CPC 30253, Coll 853

Capsicum annuum

Taiwan

MH728817

MH707463

MH805846

MH781476

MH846559

MH728835

MH748260

 

UOM 1139, Coll 1318

Capsicum annuum

Taiwan

MH728808

MH707468

MH805854

MH781484

MH846567

MH728827

MH748268

C. gloeosporioides

IMI 356878, ICMP 17821, CBS 112999a

Citrus sinensis

Italy

JX010152

JX010056

JX009818

JX009531

JX010445

JQ807843

JX010085

C. grevilleae

CBS 132879, CPC 15481a

Grevillea sp.

Italy

KC297078

KC297010

KC296987

KC296941

KC297102

KC297033

C. grossum

CGMCC3.17614, CAUG7a

Capsicum sp.

China

KP890165

KP890159

KP890153

KP890141

KP890171

MG826120

C. hebeiense

MFLUCC13 0726a, JZB330028

Vitis vinifera

China

KF156863

KF377495

KF289008

KF377532

KF288975

C. helleniense

CBS 142418a, CPC 26844

Poncirus trifoliata

Greece

KY856446

KY856270

KY856186

KY856019

KY856528

C. henanense

LC3030, CGMCC 3.17354a

Camellia sinensis

China

KJ955109

KJ954810

KM023257

KJ955257

KJ954524

KJ954960

C. horii

ICMP 10492, MTCC 10841a

Diospyros kaki

Japan

GQ329690

GQ329681

JX009752

JX009438

JX010450

JQ807840

JX010137

C. hystricis

CBS 142411a, CPC 28153

Citrus hystrix

Italy

KY856450

KY856274

KY856190

KY856023

KY856532

C. jiangxiense

CGMCC 3.17363a

Camellia sinensis

China

KJ955201

KJ954902

 

KJ954471

KJ955348

KJ954607

KJ955051

C. kahawae subsp. kahawae

IMI 319418, ICMP 17816a

Coffea arabica

Kenya

JX010231

JX010012

JX009813

JX009452

JX010444

JQ894579

JX010130

C. makassarense

CPC 28555

Capsicum annuum

Indonesia

MH728816

MH728822

MH805847

MH781477

MH846560

MH728834

MH748261

 

CPC 28556

Capsicum annuum

Indonesia

MH728815

MH728821

MH805848

MH781478

MH846561

MH728833

MH748262

 

CBS 143664 a , CPC 28612

Capsicum annuum

Indonesia

MH728812

MH728820

MH805850

MH781480

MH846563

MH728831

MH748264

C. musae

CBS 116870, ICMP 19119, MTCC 11349a

Musa sp.

USA

JX010146

JX010050

JX009896

JX009433

HQ596280

KC888926

JX010103

 

CMM 4458

Musa sp.

Brazil

KX094249

KX094191

KX094080

 

KX093967

KX094292

C. nupharicola

CBS 469.96, ICMP 17938

Nuphar lutea subsp. polysepala

USA

JX010189

JX009936

JX009834

JX009486

JX010397

JX010087

 

CBS 470.96, ICMP 18187a

Nuphar lutea subsp. polysepala

USA

JX010187

JX009972

JX009835

JX009437

JX010398

JX145319

JX010088

C. perseae

GA100a

Persea americana

Israel

KX620308

KX620242

 

KX620145

KX620341

KX620177

KX620275

C. proteae

CBS 132882a

Proteaceae

South Africa

KC297079

KC297009

KC296986

 

KC296940

KC297101

  

C. psidii

CBS 145.29, ICMP 19120a

Psidium sp.

Italy

JX010219

JX009967

JX009901

JX009515

JX010443

KC888931

JX010133

C. queenslandicum

ICMP 1778a

Carica papaya

Australia

JX010276

JX009934

JX009899

JX009447

JX010414

KC888928

JX010104

 

ICMP 18705

Coffea sp.

Fiji

JX010185

JX010036

JX009890

JX009490

JX010412

 

JX010102

 

BRIP 63695

Capsicum annuum

Australia

      

KU923727

KU923737

C. salsolae

CBS 119296, ICMP 18693

Glycine max (inoculated)

Hungary

JX010241

JX009917

JX009791

JX009559

 

ICMP 19051a

Salsola tragus

Hungary

JX010242

JX009916

JX009863

JX009562

JX010403

KC888925

JX010093

C. siamense

CPC 28609

Capsicum annuum

Indonesia

MH728813

MH713886

MH748242

 

CPC 30209, UOM 13

Capsicum annuum

Indonesia

MH707471

MH707452

MH805834

MH781464

MH846547

MH713897

MH748231

 

CPC 30210, UOM 14

Capsicum annuum

Indonesia

MH707472

MH707453

MH805835

MH781465

MH846548

MH713896

MH748232

 

CPC 30211, UOM15

Capsicum annuum

Indonesia

MH707473

MH707454

MH805836

MH781466

MH846549

MH713895

MH748233

 

CPC 30212, UOM 16

Capsicum annuum

Indonesia

MH707474

MH707455

MH805837

MH781467

MH846550

MH713894

MH748234

 

CPC 30221, UOM 25

Capsicum annuum

Thailand

MH707475

MH707456

MH805838

MH781468

MH846551

MH713893

MH748235

 

CPC 30222, UOM26

Capsicum annuum

Thailand

MH707476

MH707457

MH805839

MH781469

MH846552

MH713892

MH748236

 

CPC 30223, UOM27

Capsicum annuum

Thailand

MH707477

MH707458

MH805840

MH781470

MH846553

MH713891

MH748237

 

CPC 30233, UOM37

Capsicum annuum

Indonesia

MH707478

MH707459

MH805841

MH781471

MH846554

MH713890

MH748238

 

CPC 30234, UOM38

Capsicum annuum

Indonesia

MH707479

MH707460

MH805842

MH781472

MH846555

MH713889

MH748239

 

CPC 30235, UOM39

Capsicum annuum

Indonesia

MH707480

MH707461

MH805843

MH781473

MH846556

MH713888

MH748240

 

CPC 30236, UOM40

Capsicum annuum

Indonesia

MH707481

MH707462

MH805844

MH781474

MH846557

MH713887

MH748241

 

UOM 1116

Capsicum annuum

Sri Lanka

MH707495

MH713872

MH748256

 

UOM 1117

Capsicum annuum

Sri Lanka

MH707496

MH713871

MH748257

 

UOM 1118

Capsicum annuum

Sri Lanka

MH707497

MH713870

MH748258

 

UOM 1124, F1-3A

Capsicum annuum

Thailand

MH707482

MH713885

MH748243

 

UOM 1125, F7-3B

Capsicum annuum

Thailand

MH707488

MH713879

MH748249

 

UOM 1126, F4-1C

Capsicum annuum

Thailand

MH707484

MH713883

MH748245

 

UOM 1127, F5-1A

Capsicum annuum

Thailand

MH707485

MH713882

MH748246

 

UOM 1128, F7-1B

Capsicum annuum

Thailand

MH707487

MH713880

MH748248

 

UOM 1129, F5-4A

Capsicum annuum

Thailand

MH707486

MH713881

MH748247

 

UOM 1130, F1-3C

Capsicum annuum

Thailand

MH707483

MH713884

MH748244

 

UOM 1131, F7-4A

Capsicum annuum

Thailand

MH707489

MH713878

MH748250

 

UOM 1132, RC1

Capsicum annuum

Thailand

MH707490

MH713877

MH748251

 

UOM 1133, RC2

Capsicum annuum

Thailand

MH707491

MH713876

MH748252

 

UOM 1134, RC3

Capsicum annuum

Thailand

MH707492

MH713875

MH748253

 

UOM 1135, RC4

Capsicum annuum

Thailand

MH707493

MH713874

MH748254

 

UOM 1136, RC5

Capsicum annuum

Thailand

MH707494

MH713873

MH748255

 

IMI 82267, CPC 16808

Vitis sp.

Brazil

      

KP703783

KP703698

 

ICMP 18575,HKUCC 10884

Capsicum annuum

Thailand

JX010256

JX010059

JX009785

JX009455

JX010404

KP703769

JX010094

 

ICMP 18578a, CBS 130417

Coffea arabica

Thailand

JX010171

JX009924

JX009865

 

FJ907423

JX010404

 

JX010094

 

LC0144, PE004–1

Coffea sp.

China, Yunnan

      

KP703785

KP703700

 

LC0148, PE007–1

Camellia sp.

China, Yunnan

      

KJ954494

KJ954929

C. siamense (syn. C. communis)

NK24, MTCC 11599

Mangifera indica

India

      

JQ894582

 

C. siamense (syn. C. endomangiferae)

CMM 3814a

Mangifera indica

Brazil

KC702994

KC702955

KC598113

 

KC702922

KM404170

KJ155453

 

C. siamense (syn. C. dianesei)

CMM 4083

Mangifera indica

Brazil

      

KX094304

KX094216

 

CMM 4085a

Mangifera indica

Brazil

      

KX094306

KX094218

C. siamense (syn. C. hymenocallidis)

CBS 125378, ICMP 18642, LC0043a

Hymenocallis americana

China

JX010278

JX010019

GQ856730

JX009441

JX010410

JQ899283

JX010100

 

CBS 112983, CPC 2291

Protea cynaroides

Zimbabwe

KC297065

KC297007

KC296984

KC296929

KC297100

KP703761

KC297030

 

CBS 113199. CPC 2290

Protea cynaroides

Zimbabwe

KC297066

KC297008

KC296985

KC296930

KC297090

KP703763

KC297031

 

CBS 116868

Musa sp.

India; Southern India

KC566815

KC566669

KC566382

KC566961

KP703429

KP703764

KP703679

C. siamense (syn. C. jasmini-sambac)

CBS 130420a, ICMP 19118

Jasminum sambac

Vietnam

HM131511

HM131497

JX009895

HM131507

JX010415

JQ807841

JX010105

 

CPC 16135, WTS9

Persea americana

South Africa

KP703760

KP703678

KC566375

KC566954

KP703597

KP703845

KP703760

C. siamense (syn. C. melanocaulon)

CBS 133251, coll131, BPI 884113a

Vaccinium macrocarpon

USA

JX145313

KP703685

C. siamense (syn. C. murrayae)

CBS 133239, GZAAS5.09506a

Murraya sp.

China

KP703770

JQ247621

C. syzygicola

DNCL021 MFLUCC 100624

Syzygium samarangense

Thailand

KF242094

KF242156

KF157801

KF254880

C. tainanense

CBS 143666 a , CPC 30245, UOM 1120, Coll 1298

Capsicum annuum

Taiwan

MH728818

MH728823

MH805845

MH781475

MH846558

MH728836

MH748259

 

UOM 1119, Coll 1290

Capsicum annuum

Taiwan

MH728805

MH728819

MH805857

MH781487

MH846570

MH728824

MH748271

C. theobromicola

MTCC 11350, CBS 124945, ICMP 18649a

Theobroma cacao

Panama

JX010294

JX010006

JX009869

JX009444

JX010447

KC790726

JX010139

C. ti

ICMP 4832a

Cordyline sp.

New Zealand

JX010269

JX009952

JX009520

JX010442

KM360146

JX010123

C. tropicale

CBS 124943, ICMP 18651

Annona muricata

Panama

JX010277

JX010014

JX009868

JX009570

KC790728

 

CBS 124946

Unknown

Brazil

KC566806

KC566660

KC566373

KC566952

KC566228

 

CBS 124949, ICMP 18653, MTCC 11371a

Theobroma cacao

Panama

JX010264

JX010007

JX009870

JX009489

JX010407

KC790728

JX010097

 

CMM 4071

Mangifera indica

Brazil

KC329785

KC517181

KC533726

KC517258

 

CMM 4243

Musa sp.

Brazil

KU213603

KU213601

KU213600

KU213596

KU213604

 

CPC 16260

Unknown

Brazil

KC566807

KC566661

KC566374

KC566953

KC566229

 

GM04-L01

Annona muricata

Colombia

KC512125

KC506397

KC512146

KC512167

KC512188

 

GM33-L01

Annona muricata

Colombia

KC512128

KC506400

KC512149

KC512170

KC512191

 

CPC 28607

Capsicum annuum

Indonesia

MH728814

MH707464

MH805849

MH781479

MH846562

MH728832

MH748263

 

UOM 1002

Capsicum annuum

Indonesia

MH728807

MH707469

MH805855

MH781485

MH846568

MH728826

MH748269

 

UOM 1003

Capsicum annuum

Indonesia

MH728806

MH707470

MH805856

MH781486

MH846569

MH728825

MH748270

C. viniferum

GZAAS 5.08601a

Vitis vinifera

China

JN412804

JN412798

JN412795

JN412813

JN412787

 

CAUG27

Capsicum sp.

China

KP145440

KP145412

KP145356

KP145384

KP145468

C. wuxiense

CGMCC 3.17894a

Camellia sinensis

China

KU251591

KU252045

KU251939

KU251672

KU252200

KU251722

KU252101

C. xanthorrhoeae

BRIP 45094, ICMP 17903, CBS 127831a

Xanthorrhoea preissii

Australia

JX010261

JX009927

JX009823

JX009478

JX010448

KC790689

JX010138

Orchidearum complex

C. cattleyicola

CBS 170.49a

Cattleya sp.

Belgium

MG600758

MG600819

MG600963

MG601025

  

C. cliviicola

CBS 125375a

Clivia miniata

China

MG600733

MG600795

MG600939

MG601000

 

CSSK4

Clivia miniata

China

GQ485607

GQ856756

GQ856777

GQ849440

 

CSSS1

Clivia miniata

China

GU109479

GU085868

GU085861

GU085869

 

CSSS2

Clivia miniata

China

GU109480

GU085868

GU085862

GU085870

C. dracaenophilum

CBS 118199a

Dracaena sanderana

China

JX519222

JX546707

JX519238

JX519247

C. musicola

CBS 132885a

Musa sp.

Mexico

MG600736

MG600798

MG600942

MG601003

C. orchidearum

CBS 135131a

Dendrobium nobile

Netherlands

MG600738

MG600800

MG600944

MG601005

 C. piperis

IMI 71397, CPC 21195a

Piper nigrum

Malaysia

MG600760

MG600820

MG600964

MG601027

  

C. plurivorum

CBS 125474a

Coffea sp.

Vietnam

MG600718

MG600781

MG600925

MG600985

 

CBS 132443

Coffea sp.

Vietnam

MG600717

MG600780

MG600924

MG600984

 

CMM 3742

Mangifera indica

Brazil

KC702980

KC702941

KC702908

KC992327

 

CMM 3746

Mangifera indica

Brazil

KC702981

KC702942

KC702909

KC992328

 

CORCG2

Cymbidium

hookerianum

China

HM585397

HM585380

HM581985

HM585422

 

CPC 28638

Capsicum annuum, leaf

Thailand

MH805810

MH805816

MH805828

MH805824

 

CPC 28639

Capsicum annuum, leaf

Thailand

MH805811

MH805817

MH805829

MH805825

 

LJTJ 16

Capsicum annuum

China

KP748207

KP823786

KP823739

KP823851

 

LJTJ 22

Capsicum annuum

China

KP748213

KP823792

KP823740

KP823852

 

LJTJ 30

Capsicum annuum

China

KP748221

KP823800

KP823741

KP823853

 

UOM 1004

Capsicum annuum

Thailand

MH805812

MH805818

MH805830

MH805824

 

UOM 1005

Capsicum annuum

Thailand

MH805813

MH805819

MH805831

MH805825

 

UOM 1006

Capsicum annuum

Thailand

MH805814

MH805820

MH805832

MH805826

 

UOM 1153, M2

Capsicum annuum

Malayasia

MH805815

MH805821

MH805827

C. sojae

CAUOS5

Capsicum sp.

China

KP890107

KP890138

KP890114

 

ATCC 62257a

Glycine max

USA

MG600749

MG600810

MG600954

MG601016

C. vittalense

CBS 181.82a

Theobroma cacao

India

MG600734

MG600796

MG600940

MG601001

1ATCC American Type Culture Collection, BBA Culture collection of the Biologische Bundesanstalt fur Land- und Forstwirtschaft, Berlin, Germany, BRIP Queensland Plant Pathology Herbarium, Australia, CPC Culture collection of P.W. Crous, housed at Westerdijk Fungal Biodiversity Institute, CBS Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, CGMCC China, General Microbiological Culture Collection, Beijing, China, DPI Department of Primary Industries, HKUCC The University of Hong Kong Culture Collection, Hong Kong, China, ICMP International Collection of Microorganisms from Plants, Landcare Research, Auckland, New Zealand, IMI Culture collection of CABI Europe UK Centre, Egham, UK, LC Working collection of Lei Cai, housed at CAS, China, LF Working collection of Fang Liu, housed at CAS, China, MFLUCC Mae Fah Luang University Culture Collection, ChiangRai, Thailand, NBRC NITE Biological Resource Center, Chiba, Japan, PD Plantenziektenkundige Dienst Wageningen, Netherlands, UOM University of Melbourne culture collection, Victoria, Australia, ZJUD Diaporthe strains in Zhejiang University, China. Cultures indicated with an asterisk (a) are ex-type cultures

Isolates and accession numbers in bold represents the isolates used in this study

Phylogenetic analyses

Gene sequences of each isolate were examined using Geneious Pro v. 11.1.4, aligned by CLUSTALW2 (Larkin et al. 2007) and edited manually where necessary. ITS and tub2 sequences of selected isolates representing all the species complexes were analysed to determine to which clade each isolate belonged, and an initial phylogenetic tree was produced with a maximum likelihood analysis (ML) as implemented in MEGA v. 6 with 1000 bootstrap replicates (data not shown). For isolates from the acutatum complex, concatenated datasets were generated comprising ITS, chs-1, act, gapdh, his3 and tub2 gene sequences. For isolates from the gloeosporioides complex, two concatenated datasets were generated comprising ITS, chs-1, act, gapdh and tub2 gene sequences, and comprising ApMat and gs gene sequences. For isolates from the boninense and orchidearum complexes concatenated datasets were generated comprising ITS, gapdh, act and tub2 gene sequences. Selected reference or ex-type strains from each complex (Table 2) were included in the analyses (Damm et al. 2012b, 2019; Marin-Felix et al. 2017; Weir et al. 2012).

Further phylogenetic analyses were performed using MrBayes v. 3.2.6 (Ronquist et al. 2012) for Bayesian inference analyses (BI), and PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 (Swofford 2003) for parsimony analyses. For BI analyses, the best nucleotide substitution model for each locus was determined by MrModeltest v. 2.3 (Nylander 2004) (Table 3), and eight simultaneous MCMC chains were run for 1 bn generations. Trees were sampled every 100 generations for the acutatum, boninense and orchidearum complexes, and every 1000 generations for the gloeosporioides complex 2-gene alignment and every 10 generations for the gloeosporioides complex 5-gene alignment. The heating parameter was set to 0.2 and analyses stopped once the average standard deviation of split frequencies was below 0.01. The first 25% of trees, representing the burn-in phase of the analyses, were discarded and the remaining trees in each analysis were used to calculate posterior probabilities. The generated 50% majority rule consensus tree was viewed in TreeView v. 1.6.6 (Page 1996). A maximum parsimony (MP) analysis was performed on the multilocus alignments as well as for each gene separately with PAUP v. 4.0b10 (Swofford 2003) using the heuristic search option with 100 random sequence additions and tree bisection and reconstruction (TBR) as the branch-swapping algorithm. Gaps were treated as new character states and missing data as missing characters. Bootstrap support values were calculated based on 1000 bootstrap replicates. Statistical measures calculated included tree length (TL), consistency index (CI), retention index (RI) and rescaled consistency index (RC) (Table 3). Alignments and tree files are deposited in TreeBASE (accession https://www.treebase.org/treebase-web/home.html; study S23829).
Table 3

Statistical information of the different phylogenetic analyses performed on each Colletotrichum complex

Dataset

Parameters and statistics of the Bayesian analyses

Total number of generations run

Substitution models (Number of Unique site patterns)

Number of trees used in consensus

ITS

gapdh

tub2

act

chs-1

his3

ApMat

gs

acutatum complex

HKY + I (108)

SYM + G (151)

GTR + G (134)

GTR + G (86)

K80 + I (54)

GTR + G (96)

  

45,602

3,040,000

boninense complex

HKY + I (42)

HKY (130)

HKY + G (111)

HKY + G (94)

HKY + G (55)

   

12,002

80,000

gloeosporioides complex, 2-gene

      

HKY + G (520)

GTR + G (432)

442,502

29,500,000

gloeosporioides complex, 5-gene

SYM + I (73)

HKY + G (163)

SYM + I (180)

HKY + I (85)

K80 + G (55)

   

102,752

685,000

orchidearum complex

GTR + I (34)

HKY (55)

HKY (90)

HKY (42)

    

4128

275,000

 

Statistics of the parsimony analyses

 
 

Number of strains (incl. Outgroup(s))

Number of included characters

Number of parsimony-informative characters

Number of parsimony-uninformative characters

Number of constant characters

Tree Length (TL)

Consistency index (CI)

Retention index (RI)

Rescaled consistency index (RC)

Number of equally most parsimonious trees saved

acutatum complex

100

2210

282

438

1490

1190

0.76

0.79

0.6

1000

boninense complex

24

1743

189

343

1211

776

0.87

0.79

0.68

3

gloeosporioides complex, 2-gene

92

1715

559

539

617

2003

0.73

0.88

0.64

161

gloeosporioides complex, 5-gene

85

1724

306

222

1196

926

0.71

0.857

0.610

1000

orchidearum complex

26

1417

72

282

1063

411

0.92

0.85

0.78

284

Pathogenicity assay

Pathogenicity tests on chili fruit were conducted using only Colletotrichum isolates with straight conidia as previous studies had extensively studied the pathogenicity of C. truncatum in chili (Mongkolporn et al. 2010, Ranathunge et al. 2012). There were 15 representative isolates of C. scovillei from Indonesia, Thailand and Taiwan, 10 isolates of C. siamense from Indonesia and Thailand, and one isolate each from the other eight species with straight conidia. Detached mature red chili fruits (Capsicum annuum genotype Bangchang) were used for the pathogenicity assay as described by De Silva et al. (2017a). Pathogenicity of each isolate was tested with both non-wound and wound inoculation methods. Three replicate fruits were tested per isolate while experiments were carried out three times.

Data were analysed using the Mixed Procedure in SAS v. 9.4 by fitting the linear mixed model:

Yijkl = μ + Si + Ij(Si) + Rk + RkSi + RkIj(Si) + eijkl

where μ is the grand mean, Si is the fixed species effect, and Rk, Rk*Si, Rk*Ij(Si) and eijkl are respectively the random effects of replicate, replicate by species interaction, replicate by isolate within species interaction, and error. Separate analyses were done for wound and non-wound data as preliminary analysis showed there was significant species by wound interaction. Least squared means were estimated for each species and t-test carried out between each pair of means.

RESULTS

Isolates

The Colletotrichum isolates with falcate conidia and ITS sequences matching to those of the ex-type of C. truncatum were the most common (n = 115), representing 44% of all isolates. Colletotrichum truncatum was found in the collections from Indonesia, Malaysia, Sri Lanka and Thailand (Fig. 6). Colletotrichum truncatum isolates were not included in the collection from the World Vegetable Center in Taiwan as only the species with straight conidia were selected for identification. The remaining 56% of isolates (n = 145) were of species with straight conidia that mostly belonged to the acutatum and gloeosporioides complexes.

Phylogenetic analyses

Acutatum complex

For the 69 isolates and 29 reference species in the acutatum complex, the phylogenetic analysis of the combined data sets using six genes (ITS, tub2, gapdh, chs-1, act and his3) with C. boninense (CBS 123755) as the outgroup comprised 100 isolates including the outgroup and 2315 characters including the alignment gaps and excluded characters. The Bayesian analysis of this alignment, based on 629 unique site patterns (ITS: 108, tub2: 134, gapdh: 151, act: 86, chs-1: 54 and his3: 96) lasted 3,040,000 generations, resulting in 60,802 total trees of which 45,602 trees were used to calculate the posterior probabilities. The parsimony analysis yielded the maximum of 1000 equally most parsimonious trees. Bootstrap support values of the MP analysis (MP > 49%) and the BI posterior probabilities (PP > 0.90) were plotted at the nodes (Fig. 1). Overall, the species clades recognised received similar support values, although the association between species did not always receive similar support, e.g. the node linking C. paranaense and C. melonis (MP < 50% / PP = 0.99). The phylogenetic analyses of the acutatum complex identified C. scovillei as the most prevalent species in Indonesia, Malaysia, Thailand and Taiwan. However, C. scovillei was not isolated from Sri Lanka. In addition, an isolate from Java in Indonesia (UOM 1115) clustered related to C. brisbanense (96% BS/1 PP; Fig. 1).
Fig. 1
Fig. 1

First of 1000 equally most parsimonious trees obtained from a heuristic search of the combined ITS, tub2, gapdh, chs-1, his3 and act sequence alignment of the Colletotrichum isolates in the acutatum complex. The parsimony bootstrap support values (MP > 49%) and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (MP/PP). The tree was rooted to C. boninense (CBS 123755). The bar indicates 40 changes. Coloured blocks are used to indicate clades containing isolates from chili

Boninense complex

For the four isolates and 10 reference species in the boninense complex the phylogenetic analyses of the combined data sets using five genes (ITS, gapdh, tub2, act and chs-1) with C. truncatum (CBS 151.35) as the outgroup comprised 24 isolates and 1867 characters including the alignment gaps and excluded characters (Fig. 2). The Bayesian analysis of the combined alignment, based on 432 unique site patterns (ITS: 42, gapdh: 130, tub2: 111, act: 94 and chs-1: 55) lasted 80,000 generations, resulting in 16,002 total trees of which 12,002 trees were used to calculate the posterior probabilities. The parsimony analysis yielded three equally most parsimonious trees. Bootstrap support values of the MP analysis (MP > 49%) and the BI posterior probabilities (PP > 0.90) were plotted at the nodes (Fig. 2). Overall, the nodes received similar support values, except for the subclustering of strains CBS 128545, CBS 128548 and CBS 129927 in the C. karsti clade (MP 67% / PP = 0.98).The phylogenetic analyses of the boninense complex identified the most prevalent species as C. karsti occurring only in Indonesia.
Fig. 2
Fig. 2

First of three equally most parsimonious trees obtained from a heuristic search of the combined ITS, tub2, gapdh, chs-1 sequence alignment of the Colletotrichum isolates in the boninense complex. The parsimony bootstrap support values (MP > 49%) and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (MP/PP). The tree was rooted to C. truncatum (CBS 151.35). The bar indicates 30 changes. Coloured blocks are used to indicate clades containing isolates from chili

Gloeosporioides complex

For the 42 isolates and the 41 reference species in the gloeosporioides complex, two phylogenetic trees were constructed, one from the ApMat and gs sequence alignment and the second from the ITS, gapdh, act, tub2, chs-1 sequence alignment (Figs. 3 and 4). The analyses using the 5-gene alignment with C. theobromicola CBS 124945 as the outgroup (Fig. 4) comprised 85 isolates including the outgroup and 1863 characters including the alignment gaps and excluded characters. The Bayesian analysis of the combined alignment, based on 556 unique site patterns (ITS: 73, gapdh: 163, act: 85, tub2: 180, chs-1: 55) lasted 685,000 generations, resulting in 137,002 total trees of which 102,752 trees were used to calculate the posterior probabilities. The parsimony analysis yielded the maximum of 1000 equally most parsimonious trees. Bootstrap support values of the MP analysis (MP > 49%) and the BI posterior probabilities (PP > 0.90) were plotted at the nodes (Fig. 4). Overall, the species clades recognised in this study received similar support values, except for the C. siamense clade (MP < 50% / PP < 0.91) and the C. fructicola clade (MP 57% / PP = 0.99).
Fig. 3
Fig. 3

Phylogenetic analysis of Colletotrichum isolates in the gloeosporioides complex based on a 50% majority rule consensus tree derived from Bayesian analysis of the ApMat and gs regions. The parsimony bootstrap support values (MP > 49%) and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (MP/PP). The tree was rooted to C. theobromicola (CBS 124945). The bar indicates 0.02 expected changes per site. Coloured blocks are used to indicate clades containing isolates from chili

Fig. 4
Fig. 4

Phylogenetic analysis of Colletotrichum isolates in the gloeosporioides complex based on a 50% majority rule consensus tree derived from Bayesian analysis of the combined the ITS, tub2, gapdh, chs-1 and act sequence. The parsimony bootstrap support values (MP > 49%) and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (MP/PP). The tree was rooted to C. theobromicola (CBS 124945). The scale bar indicates 0.009 expected changes per site. Coloured blocks are used to indicate clades containing isolates from chili

The analysis using the ApMat and gs sequence alignment comprised of 92 isolates with C. theobromicola CBS 124945 as the outgroup (Fig. 3) and 1824 characters including the alignment gaps and excluded characters. The Bayesian analysis of the combined alignment, based on 952 unique site patterns (ApMat: 520, gs: 432) lasted 29,500,000 generations, resulting in 590,002 total trees, of which 442,502 trees were used to calculate the posterior probabilities. The parsimony analysis yielded 161 equally most parsimonious trees. Bootstrap support values of the MP analysis (MP > 49%) and the BI posterior probabilities (PP > 0.90) were plotted at the nodes (Fig. 3). Overall, the species clades recognised in this study received similar support values, except for the C. siamense clade (MP = 92% / PP < 0.91).

Phylogenetic analyses of the gloeosporioides species complex identified 69% (29) of the chili fruit isolates as C. siamense. In the 2-gene tree a distinct subclade within the C. siamense clade formed with 100% bootstrap support which contained isolates from Chiang Mai in Thailand, and Gowa and Jeneponto in South Sulawesi of Indonesia (Fig. 3). These isolates from Indonesia and Thailand had very distinct sequences compared to the ex-type reference C. siamense strain (CBS 130417) with 28 bp difference in the gs gene and 25 bp difference in the ApMat gene. A significant sub-clade formed within C. siamense with full (100% BS/1 PP) support values in the 2-gene tree. The same isolates in the 5-gene tree did not show the same level of difference but showed a strong similarity between the C. siamense isolates. In the 2-gene tree there were also significant subclades of isolates associated with different geographical regions, in particular the distinct subclade of the Sri Lankan isolates (UOM 1116, UOM 1117, UOM 1118) from Kandy and the separate subclade of Thai isolates from Ratchaburi (UOM 1132, UOM 1133, UOM 1134).

Other species identified in the gloeosporioides complex included C. endophyticum and C. fructicola from Thailand, C. fructicola and C. tainanense sp. nov. from Taiwan, and C. tropicale and C. makassarense sp. nov. from Indonesia. Most of the identified species including the two new species were supported in distinct clades with significant bootstrap values in both the 5-gene and 2-gene trees (Figs. 3 and 4). However, due to a lack of sequence data of the ApMat gene for some reference strains, it was difficult to provide a good support for placement of some species such as C. endophyticum in the 2-gene trees. Three isolates (CPC 28607, UOM 1002, UOM 1003) collected from the Makassar region in Indonesia showed a close relationship to the reference species C. tropicale in the ApMat and gs tree (Fig. 3). Individual gene trees of ITS, act, tub2, chs-1 loci (data not shown) also supported these isolates as C. tropicale. Nevertheless, in the 5-gene tree a separate sub clade was formed with full support (100% BS/1 PP) different to the C. tropicale reference species (Fig. 4). In both trees, two isolates (UOM 1120, UOM 1119) collected from Tainan in Taiwan formed a significant distinct clade with full support (100% BS/1 PP) separate from C. salsolae.

Orchidearum complex

For the six isolates and nine reference species in the orchidearum complex the phylogenetic analysis of the combined data sets using four genes (ITS, gapdh, tub2 and act) with C dracaenophilum (CBS 118199) as the outgroup comprised 26 isolates and 1543 characters including the alignment gaps and excluded characters. The Bayesian analysis of the combined alignment, based on 221 unique site patterns (ITS: 34, gapdh: 55, act: 42, tub2: 90) lasted 275,000 generations, resulting in 5502 total trees of which 4128 trees were used to calculate the posterior probabilities. The parsimony analysis yielded 284 equally most parsimonious trees. Bootstrap support values of the MP analysis (MP > 49%) and the BI posterior probabilities (PP > 0.90) were plotted at the nodes (Fig. 5). Overall, the species clades recognised in this study received similar support values, except for the C. plurivorum clade (MP = 64% / PP <  1) and the C. cliviicola clade (MP = 87% / PP <  1).
Fig. 5
Fig. 5

First of 284 equally most parsimonious trees obtained from a heuristic search of the combined ITS, tub2, gapdh, chs-1, and act sequence alignment of the Colletotrichum isolates in the orchidearum complex. The parsimony bootstrap support values (MP > 49%) and Bayesian posterior probabilities (PP > 0.90) are displayed at the nodes (MP/PP). The tree was rooted to C. dracaenophilum (CBS 118199). The scale bar indicates 20 changes. Coloured blocks are used to indicate clades containing isolates from chili

The phylogenetic analyses identified the isolates from Thailand and Malaysia as C. plurivorum. Five isolates collected from Thailand, including three taken from infected chili leaves from Chiang Rai and another two isolates collected from infected green chili fruit from Bangkok, formed a poorly supported subclade within C. plurivorum (Fig. 5).

TAXONOMY

Morphological observations and phylogenetic data of the straight conidia species clearly identified three novel species, two from Indonesia and one from Taiwan. Detailed morphological descriptions are provided below for all the Colletotrichum species associated with chili anthracnose (Table 4).
Table 4

Morphological characteristics of Colletotrichum species causing anthracnose of chili

Species

Conidiogenous cells length (μm)

Conidia length (μm)

Conidia width (μm)

Appressoria (μm)

C. endophyticum

12–21 × 3–4

(10.4–)12.5–13(−14.5)

(3–)4.5–5(−6.3)

(10.5–)12(−15) × (3–)4.5(−10)

C. fructicola

7–17.5

(10.5–)12.5–13(−18.5)

(3–) 4–5.5(−6.5)

C. javanense

7–17.5

(11.5–)13.5–14(−15.8)

(2.4–)3.5–4(−4.3)

(6–)8.2(−11.3) × (4.2–)5.6(−7.5)

C. karsti

(11.6–)12.5–13(−15.7)

(3–)4–5.2(−6.5)

6–12.5 × 3.5–8.2

C. makassarense

7–25 × 3–4

(11–)13–15(−17)

(4–)4.5–5

(6–)8(−10.5) × (4–)3.5(−8.6)

C. plurivorum

26–48 × 3–4

(13.7–)14–16(− 18.3)

(3.8–)5(−5.6)

(10.5–)12(−23) × (3.5–)5.5(− 11.5)

C. scovillei

7–17.5

(5.5–)9.5–10(−12)

(2.4–)3(−3.8)

(4–)5.5(−12.5) × (3.5–)4.5–5(− 6.5)

C. siamense

6.5–16

(13–)14(− 15.5)

(3–)4.2(−5.3)

(4.5–)7.5(− 10) × (3.5–)3(−5.5)

C. tainanense

(16–)17–18(−22)

(4.5–)5

(6.5–)10.3(− 14.3) × (6.2–)5.2(−9.5)

C. tropicale

7–15 × 3.5–4.5

(13–)14–16(− 17)

(3.5–)4–5(− 6)

Colletotrichum javanense D.D. De Silva, P.W. Crous & P.W.J. Taylor, sp. nov. MycoBank MB826936.

Figure 6 Etymology: Named after Java, the island in Indonesia where the species was collected.
Fig. 6
Fig. 6

Colletotrichum javanense (CBS 144963). a Colony on PDA. b Reverse side of the colony on PDA. c Conidioma on PDA. d-e Conidiophores and conidia. f Chlamydospores. g–j Appressoria. k Conidia. Bars = 10 μm

Diagnosis: Colletotrichum javanense differs from C. brisbanense in forming distinct chlamydospores and acervular conidiomata on all the media tested. In contrast, C. brisbanense only produced Conidiomata on Anthriscus stem, but no basal cells observed. In addition, C. javanense grows faster than C. brisbanense (C. javanense on OA, MEA and SNA 60, 55 and 66 mm diam in 7 d, respectively, C. brisbanense on OA and SNA 18.5, 20 mm diam in 7 d, respectively). Colletotrichum javanense is phylogenetically distinct to C. brisbanense with both species being different in the sequences of chs-1, and most effectively with gapdh (7 bp difference) and his3 (4 bp difference).

Type: Indonesia: West Java, Purwakata regency, on fruit of Capsicum annuum, Dec. 2014, F. Fitriyah (CBS H-144963 – holotype; CBS 144963 = UOM 1115 = EWINDO 3 – ex-type cultures).

Description: Sexual morph not observed. Asexual morph on PDA. Vegetative mycelium 1–5 μm diam, hyaline, smooth-walled, septate, branched hyphae. Chlamydospores globose or elongate, pale brown, smooth-walled, 5–25 × 3–8 μm. Conidiomata acervular, setae not observed. Conidiophores hyaline, septate, branched. Conidiogenous cells hyaline, cylindrical or ampulliform, 7–17.5 μm, apex 1–3 μm diam. Conidia hyaline, aseptate, smooth-walled, mostly fusiform, one end rounded, the other end acute, or both ends acute (11.5–)13.5–14(− 16) × (2.5–) 4(− 4.5) μm. Conidia in mass yellow to orange colour. Appressoria single or in loose groups, medium brown, smooth-walled, subglobose or elliptical, with entire or undulate margin, (6–)8(− 11.5) × (4–)6(− 7.5) μm.

Asexual morph on SNA. Vegetative mycelium 1–7 μm diam, hyaline, smooth-walled, septate, branched hyphae. Chlamydospores globose or elongate, pale brown, smooth-walled, 4.5–28 × 4–8 μm. Conidiomata acervular, setae not observed. Conidiophores hyaline, septate, branched, 20–35 μm long. Conidiogenous cells hyaline, cylindrical or ampulliform, 5–20 μm, apex, 1–3 μm diam. Conidia hyaline, aseptate, smooth-walled, cylindrical with both ends acute or one end round and one end acute, (13.5–)16.5(− 24) × (2.5–) 3(− 4.5) μm. Conidia in mass with yellow to orange colour.

Culture characteristics: Colonies on PDA 48–54 mm diam in 7 d (6.5–7.5 mm/d), flat with entire margin; surface covered with grey to olive-green short aerial mycelium, margin white to light grey, reverse mostly cream whitish, olivaceous grey to black in the centre. Yellow to orange acervular conidiomata. Colonies on SNA were 60–66 mm diam in 7 d (8–9.5 mm/d), flat with entire margin, hyaline to pale brown, surface covered with short grey aerial mycelium, reverse same colours. Orange acervular conidiomata at the centre of the culture. Colonies on OA were 55–60 mm diam in 7 d (7.8–8.5 mm/d), flat with entire margin; surface covered with cream to grey short aerial mycelium, margin white, reverse mostly light orange, with brown pigments. Orange acervular conidiomata. Colonies on MEA surface pale grey short aerial mycelium, reverse light orange.

Notes: The closest match in a blastn search with the gapdh sequence was GenBank JQ948617, C. sloanei strain IMI 364297 with 98% identity (4 bp differences), while the closest matches with the his3 sequence with 99% identity (2 bp differences) were GenBank JQ949279 C. indonesiense strain CBS 127551 and GenBank KJ947248 C. guajavae isolate OBP19.

Colletotrichum makassarense D.D. De Silva, P.W. Crous & P.W.J. Taylor, sp. nov. MycoBank MB827691.

Figure 7 Etymology: Named after Makassar, the city in South Sulawesi, Indonesia, where the species was collected.
Fig. 7
Fig. 7

Colletotrichum makassarense (CBS 143664). a Colony on PDA. b Reverse side of the colony on PDA. c Conidiomata. d–e Appressoria. f Setae, g, h, j Conidiophores and conidia. i Conidia. Bars = 10 μm

Diagnosis: Colletotrichum makassarense is phylogenetically closely related to C. tropicale. Sequence data from ITS could not separate the two species, but they can be distinguished by all other genes tested and most effectively using ApMat (22 bp differences) and gs (18 bp differences) sequence data.

Type: Indonesia: Makassar, from fruit lesion of Capsicum annuum, 7 Jun. 2015, P.W.J. Taylor & A. Nasruddin (CBS H-143664 – holotype; CBS 143664 = CPC 28612 – ex-type cultures).

Description: Sexual morph not observed. Asexual morph on OA. Vegetative mycelium consisting of hyaline, smooth-walled, septate, branched hyphae, 2–3 μm diam. Chlamydospores not observed. Setae present, medium brown, 40–55 × 3–5 μm, 2–3-septate, tapering to acute apices. Conidiomata acervular, 100–200 μm diam, with orange conidial masses. Conidiophores subcylindrical, flexuous, 1–4-septate, hyaline, smooth, branched, 15–45 × 3–4 μm. Conidiogenous cells subcylindrical, hyaline, smooth, phialidic with periclinal thickening, 7–25 × 3–4 μm. Conidia hyaline, smooth, aseptate, subcylindrical, straight, apex obtuse, tapering at base to protruding truncate hilum, 1 μm diam, prominently guttulate, (11–)13–15(− 17) × (4–)4.5–5 μm. Appressoria solitary, medium brown, smooth-walled, subglobose, ellipsoidal to obovate, entire margin, (6–)8.0(− 10.5) × (4–)3.5(− 8.5) μm.

Culture characteristics: Colonies on PDA 45 mm diam after 7 d (6.5 mm/d), colonies flat, with moderate aerial mycelium, on OA surface smoke-grey. On PDA surface smoke-grey, reverse olivaceous grey. On MEA surface dirty white, reverse ochreous.

Notes: The closest match in a blastn search with the ApMat sequence was GenBank KU923732, C. queenslandicum strain AUS22 with a 98% identity (16 bp differences), while the closest match with the gs sequence with 99% identity (7 bp differences) was GenBank KJ947286 C. siamense isolate OBP24. The best matches with the gapdh sequence were GenBank KX578784 C. siamense (99% identity, 3 bp differences) and GenBank KU221347 C. queenslandicum (99%, identity, 3 bp differences).

Colletotrichum tainanense D.D. De Silva, P.W. Crous & P.W.J. Taylor, sp. nov. MycoBank MB827692.

Figure 8 Etymology: Named after Tainan, the city in Taiwan where the species was collected.
Fig. 8
Fig. 8

Colletotrichum tainanense (CBS 143666). a Colony on PDA. b Reverse side of the colony on PDA. c Conidia. d-e Conidiophores and Conidia. f Melanised hypae g-j Appressoria. Bars = 10 μm

Diagnosis: Colletotrichum tainanense differs from its closest phylogenetic neighbour C. salsolae in the culture characteristics on PDA, sparse aerial mycelium and pale mouse-grey surface mycelium, whereas C. salsolae produces a layer of acervuli-like structures with deep orange brown conidial masses and numerous setae. The two species are separable using all the genes tested except for ITS and most effectively with gapdh (7 bp difference), tub2 (6 bp difference) and act (5 bp difference) sequences. There is only one bp difference in the chs-1 sequence between the two species.

Type: Taiwan: Tainan: on fruit of Capsicum annuum, Aug. 2014, Z.M. Sheu (CBS H-143666 – holotype; CBS 143666 = CPC 30245 = UOM 1120 = Coll 1298 – ex-type cultures).

Description: Sexual morph not observed. Asexual morph on PDA. Vegetative mycelium branched, hyaline, smooth-walled, septate, hyphae 2–3 μm diam, melanised with time. A single conidioma found on a PDA plate, sterile on SNA, MEA, and OA. Chlamydospores and setae not observed. Conidiophores subcylindrical, flexuous, 1–2-septate, hyaline, smooth to pale brown, branched. Conidiogenous cells subcylindrical, hyaline, smooth, phialidic with periclinal thickening. Conidia hyaline, smooth, aseptate, subcylindrical to subclavate, straight or slightly curved, apex obtuse, tapering at base to protruding truncate hilum, 1.5–2 μm diam, prominently guttulate, (16–)17–18(− 22) × (4.5–)5 μm. Appressoria single or in loose groups, often narrow-cylindric, medium to dark brown, often tapering towards apex, the edge entire or undulate sometimes irregularly lobed (6.5–)10.5(− 14.5) × (6–)5(− 9.5) μm.

Culture characteristics: Colonies on PDA 45 mm diam after 7 d (6.5 mm/d), colonies flat, with moderate aerial mycelium. On OA surface pale mouse-grey. On PDA surface pale mouse-grey, reverse mouse-grey. On MEA surface pale mouse-grey, reverse olivaceous grey.

Notes: The closest match in a blastn search with the gapdh sequence with 99% identity (2 bp difference) was GenBank KC790761 Colletotrichum sp. strain MTCC 9664 while the closest match with the act sequence with 99% identity (2 bp difference) was GenBank KY995522 C. siamense strain LJDY1–2. The closest match with the tub2 sequence with 99% identity (7 bp difference) was GenBank MF143931 C. siamense strain 31-B-1.

Colletotrichum endophyticum Manamgoda et al., Fung. Diversity 61: 112 (2013); as ‘endophytica’.

Description: Colonies on PDA 42 mm diam after 7 d (5.5 mm/d), pale orange to white aerial mycelium; reverse pale white to orange and black at the centre and numerous orange conidiomata scattered over the surface. Chlamydospores not observed. Conidiomata present, conidiophores formed directly on hyphae. Setae present, moderately brown, 47–95 × 3–6 μm, 3–4-septate, tapering acute apices. Conidiophores hyaline, smooth-walled and unbranched. Conidiogenous cells hyaline, smooth-walled, aseptate, subcylindrical, 12–21 × 3–4 μm. Conidia hyaline, smooth-walled, aseptate, straight, cylindrical with two ends obtuse, (10.5–)12.5–13(− 14.5) × (3–)4.5–5(− 6.5) μm. Appressoria single or in loose groups, brown, slightly lobed, (10.5–)12(− 15) × (3–)4.5(− 10) μm.

Notes: Colletotrichum endophyticum was first described as a grass endophyte of Pennisetum purpureum from northern Thailand (Manamgoda et al. 2013). Later, it was reported from several other host species including Capsicum in China (Diao et al. 2017). The length of conidia of the isolate from Thailand (UOM 1137) was slightly shorter than that of the ex-type (LC0324) of C. endophyticum (conidia 13–19(− 21) × (3.5–)4.5–5.5 μm).

Material examined: Thailand: Kanchanaburi: from fruit lesion of Capsicum annuum, 2010, P.W.J. Taylor & O. Mongkolporn (culture UOM 1137 = F5-2D).

Colletotrichum fructicola Prihast. et al., Fung. Diversity 39: 158 (2009).

Description: Colonies on PDA 65 mm diam after 7 d (8.5–11 mm/d), flat with entire edge, aerial mycelium dense, cottony, pale grey to white aerial mycelium and numerous black stroma scattered over the surface, grey in the centre, white at the margin; reverse greyish green. Chlamydospores not observed. Conidiomata acervular, Setae was observed, brown, smooth-walled, 1–2-septate, 60 μm long, tapering acute apices. Conidiophores hyaline, septate, branched. Conidiogenous cells hyaline, cylindrical or ampulliform, 7–17.5 μm. Conidia hyaline, aseptate, smooth-walled, cylindrical, both ends obtuse, (10.5–)12.5–13(− 18.5) × (3–) 4–5.5(− 6.5). Appressoria not observed.

Notes: The sexual morph of these isolates was not observed in culture. Conidial length of isolate CPC 28644 was slightly longer than that of the ex-type (ICMP 18581 = BPD-I16) of C. fructicola (conidia 9.7–14 × 3–4.3 μm, x = 11.53 ± 1.03 × 3.55 ± 0.32 μm; Prihastuti et al. 2009).

Material examined: Thailand: Chiang Mai: from fruit lesion of Capsicum annuum, 7 Jun. 2015, P.W.J. Taylor & O. Mongkolporn (cultures CPC 28644 and CPC 28645). Taiwan: Cyonglin, Hsinchu, from fruit lesion of mature red fruit of Capsicum sp. (sweet pepper), 22 Apr. 2015, Z.M. Sheu (culture UOM 1139 = coll 1318); Nantou, Renai, from fruit lesion of green fruit of Capsicum sp. (sweet pepper), 4 Sep. 2008, Z.M. Sheu & C. Wang (culture UOM 1138 = coll-853).

Colletotrichum karsti You L. Yang et al. Cryptogamie, Mycologie 32: 241 (2011); as ‘karstii’.

Description: Colonies on PDA 65 mm diam after 7 d (6.5–10.5 mm/d), flat with entire edge, orange to white aerial mycelium and numerous orange conidial masses scattered over the surface, white at the margin; reverse yellow to orange. Chlamydospores not observed. Conidiomata acervular, setae were observed, brown, smooth-walled, 2–4-septate, 60 μm long, base submerged, tapered towards apex, tip mostly acute. Conidiophores hyaline, septate, branched, (10.5–)12–38(− 47.5) × (3–)4–5.5(− 6.5) μm. Conidiogenous cells hyaline, cylindrical or ampulliform, 7–15.5 μm. Conidia hyaline, aseptate, smooth-walled, short, cylindrical, both ends obtuse or one end slightly acute or truncate at the base, (11.5–)12.5–13(− 15.5) × (3–)4–5(− 6.5) μm. Appressoria single or in loose groups, brown, subglobose, circular outline, 6–12.5 × 3.5–8 μm.

Notes: The four isolates identified from Indonesia in the boninense complex produced distinct short conidia compared to the ex-epitype culture of C. karsti (14.5–17 × 5–6.5 μm; Yang et al. 2011). However, high variability of conidia size between different strains of C. karsti were reported by Damm et al. (2012a, 2012b) where the conidia measurements of CBS 129833 were (11.5–)12.5–14(− 14.5) × (5–)5.5–6(− 6.5) μm, mean ± SD = 13.1 ± 0.7 × 5.8 ± 0.4 μm; and CBS 111998 had a conidium length up to 18.5 μm, L/W ratio = 2.8. These isolates did not form a sexual morph in culture. Although these four isolates formed a fully supported (100% BS/1 PP) subclade within C. karsti, all the individual gene trees (data not shown) did not provide significant support to justify introducing a cryptic species for these isolates.

Material examined: Indonesia: Jeneponto, from fruit lesions of Capsicum annuum, 7 Jun. 2015, P.W.J. Taylor & A. Nasruddin (cultures CPC 28553, CPC 28554); from fruit lesion of mature red fruit and peduncle lesions of Capsicum sp. Jun. 2015, P.W.J. Taylor & A. Nasruddin (cultures CPC 28601, CPC 28602).

Colletotrichum plurivorum Damm et al., Stud. Mycol. 92: 31 (2019).

Description: Colonies on PDA 63 mm diam after 7 d (8 mm/d), grey to dark brown aerial mycelium; reverse grey to light brown with yellow-orange in the centre with abundant acervular conidiomata that ooze pale orange conidial masses at the centre, Chlamydospores not observed. Conidiomata present (near the inoculation point), conidiophores formed directly on hyphae. Sclerotia present. Setae present, medium brown, 94–125 × 4–6 μm, 1–4-septate, tapering towards acute apices, often with a constriction at the apex. Conidiophores hyaline to pale brown, smooth-walled, septate and branched. Conidiogenous cells hyaline, smooth-walled, aseptate, subcylindrical, straight to gently curved, 26–48 × 3–4 μm, phialidic, periclinal thickening conspicuous. Conidia hyaline, smooth-walled, aseptate, straight, cylindrical with two ends obtuse or one end slightly acute, (13.5–)14–16(− 18.5) × (4–)5(− 6.5) μm. Appressoria single or in loose groups, medium brown, irregular in shape, crenate or lobed outline, (10.5–)12(− 23) × (3.5–)5.5(− 11.5) μm. Sexual morph on PDA. Ascomata perithecia, formed after 2 wk., solitary, semi-immersed or immersed in the agar medium, nonstromatic, subspherical to ovoid, ostiolate, glabrous, medium brown, 96–130 × 160–200 μm. Peridium 10–12.5 μm thick, composed of pale to medium brown flattened angular cells 3.5–10 μm diam. Ascogenous hyphae hyaline, smooth-walled, delicate, rarely visible. Interascal tissue not observed. Asci unitunicate, 8-spored, cylindrical, tapering to apex and base, smooth-walled, 51–65 × 9.5–13 μm, the base truncate. Ascospores biseriately arranged, aseptate, hyaline, smooth-walled, fusiform, slightly curved, base rounded, apex acute or rounded, (13.5–)15–18(− 22) × 5–6(− 6.5) μm,

Notes: The conidial length of the isolates examined (CPC 28638, CPC 28639) was variable and fell within the range of the ex-type isolate (CBS 125474) of C. plurivorum (15–17 × 5.5 μm; Damm et al. 2019).

Material examined: Thailand: Chiang Rai, from leaf lesions of Capsicum annuum, 7 Jun. 2015, P.W.J. Taylor (cultures CPC 28638 and CPC 28639); Bangkok, restaurant in Phaya Thai area, infected tissue of Capsicum sp. Jun. 2015, P.W.J. Taylor (culture UOM 1004).

Colletotrichum scovillei Damm et al., Stud. Mycol. 73: 100 (2012).

Decription: Colonies on PDA 20–38 mm diam after 7 d (5–6.2 mm/d), flat with entire margin; surface covered with short light pink to orange aerial mycelium, turn grey with time, margin whitish to pale pink, reverse rosy buff, olivaceous grey to brown-grey in the centre; reverse orange to salmon, dark at the centre. Chlamydospores not observed. Conidiomata acervular, setae not observed, Conidiophores hyaline, septate, branched. Conidiogenous cells hyaline, cylindrical or ampulliform, 7–17.5 μm, apex 1–3 μm diam. Conidia hyaline, aseptate, smooth-walled, mostely fusiform, one end rounded, one end acute, (5.5–)9.5–10(− 12) × (2.5–)3(− 4). Conidia in mass with salmon to orange colour. Appressoria single or in loose groups, medium brown, ovoid, entire to crenate margin, (4–)5.5(− 12.5) × (3.5–)4.5–5(− 6.5) μm.

Notes: The majority of isolates identified as C. scovillei had similar spore shape and spore sizes, compared to the type specimen (10.5–)12.5–15(− 16.5) × (3–)3.5–4(− 4.5) μm, described by Damm et al. (2012a, 2012b). However, some isolates had varying colony colour, different colony growth rates and small differences in spore measurements.

Material examined: Indonesia: Gowa, from fruit lesions of Capsicum annuum, 7 Jun. 2015, P.W.J. Taylor & A. Nasruddin (cultures CPC 28577 and CPC 28579); West Java: from fruit lesion of Capsicum annuum, Dec. 2014, F. Fitriyah, UOM 1146/ EWINDO 10. Thailand: Chiang Mai: from fruit lesions of Capsicum sp. 2008, O. Mongkolporn (cultures UOM 1101/313, UOM 1111).

Colletotrichum siamense Prihast. et al., Fung. Diversity 39: 98 (2009)

Description: Colonies on PDA 79 mm diam in 7 d (5.5–6 mm/d). Pale yellow-white, grey, dense cottony aerial mycelium with orange acervular conidiomata at the centre; reverse pale yellowish. Chlamydospores not observed. Conidiomata acervular, conidiophores formed on a cushion of roundish and medium brown cells. Setae not observed. Conidiophores hyaline, branched. Conidiogenous cells hyaline, cylindrical to ampulliform, 6.5–16 μm. Conidia hyaline, aseptate, smooth-walled, fusiform to cylindrical, both ends bluntly rounded, (13–)14(− 15.5) × (3–)4(− 5.5) μm. Appressoria dark brown, solitary, circular, entire to crenate margin, (4.5–)7.5(− 10) × (3.5–)3(− 5.5) μm.

Notes: Colletotrichum siamense isolates from different countries showed variation of morphological characters, in growth rates and culture morphology on PDA. Representative conidial measurements for isolates representing different subclades in the phylogenetic trees (Figs. 2, 3) are: CPC 30233 (Gowa, Indonesia), 12.5–17 × 2.5–5.5 μm; UOM 1132 (Ratchaburi, Thailand) 9.5–14.5 × 3.5–5 μm; UOM 1126/ F4-1C (Kanchana Buri, Thailand) 12–15 × 5–7 μm; UOM 1116 (Kandy, Sri Lanka) 10.5–16.5 × 3.5–5.5. These morphological characters within a subclade were highly consistent within each country. The species was described by Prihastuti et al. (2009); conidia of the ex-holotype specimen (ICMP 18578/ BDP-I2) were reported as 7–18.3 × 3–4.3 μm (x = 10.18 ± 1.74 × 3.46 ± 0.36), which encompasses the range observed in our isolates. This species was reported to be biologically and geographically diverse, and is found on many hosts across several tropical and subtropical regions (Weir et al. 2012).

Material examined: Indonesia: Gowa, from fruit lesion of Capsicum annuum, 7 Jun. 2015, P.W.J. Taylor & A. Nasruddin (culture CPC 30233); Jeneponto, from fruit lesion of Capsicum sp. 7 Jun. 2015, P.W.J. Taylor & A. Nasruddin (culture CPC 30209). Thailand: Ratchaburi, from fruit lesion of Capsicum sp., Jan. 2010, P.W.J. Taylor & O. Mongkolporn (culture UOM 1132); Kanchanaburi, from fruit lesion of Capsicum sp. Jan. 2010, P.W.J. Taylor & O. Mongkolporn (culture UOM 1126 = F4-1C). Sri Lanka: Kandy, from fruit lesion of Capsicum sp. Sep. 2013, D.D. De Silva & N. Ranathunge (culture UOM 1116).

Colletotrichum tropicale E.I. Rojas et al., Mycologia 102: 1331 (2010)

Description: Colonies on PDA 45 mm diam in 7 d (6.5 mm/d). Colonies flat, spreading, with moderate aerial mycelium, On OA surface smoke grey. On PDA surface olivaceous grey to smoke grey, reverse olivaceous grey, numerous orange conidiomata scattered over the surface. On MEA surface dirty white, reverse ochreous. Asexual morph on OA. Vegetative mycelium consisting of hyaline to pale brown, smooth-walled, septate, branched, 2–2.5 μm diam hyphae. Chlamydospores not observed. Setae rare (only two seen), straight, medium brown, finely verruculose, 2–3-septate, to 120 μm long, apex subobtusely rounded. Conidiomata acervular, 150–250 μm diam, with orange conidial mass. Conidiophores subcylindrical, flexuous, 1–3-septate, hyaline, smooth, branched, 15–25 × 3.5–4.5 μm. Conidiogenous cells subcylindrical, hyaline, smooth, phialidic with periclinal thickening, 7–15 × 3.5–4.5 μm. Conidia hyaline, smooth, aseptate, subcylindrical, straight, apex obtuse, tapering at base to protruding truncate hilum, 1.5–2 μm diam, prominently guttulate, (13–)14–16(− 17) × (3.5–)4–5(− 6) μm. Appressoria not observed. Sexual morph not observed.

Material examined: Indonesia, Makassar, from fruit lesion of Capsicum annuum, 7 Jun. 2015, P.W.J. Taylor & A. Nasruddin (culture CPC 28607).

Prevalence of sampled Colletotrichum species

Overall, C. truncatum was the most prevalent species (44%) isolated from infected chili fruit (Fig. 9) and was readily identified by its falcate spores and abundant setae in the necrotic lesions. Of the species with straight conidia, C. scovillei (acutatum complex), was the most common species throughout the surveyed countries (35%), except for Sri Lanka where this species was not isolated. Colletotrichum siamense (gloeosporioides complex) was the next most common species that occurred in Thailand, Sri Lanka and Indonesia (11%). The remaining species were represented by fewer than 10% of the total number of isolates.
Fig. 9
Fig. 9

Prevalence of Colletotrichum species as a percentage of the total isolates collected in all regions and in Indonesia and Thailand specifically

In Indonesia, C. scovillei was isolated from infected chili fruit in all surveyed regencies of South Sulawesi, except in Makassar, and in the field trial site in West Java. Colletotrichum siamense was also isolated from throughout the region, from Gowa, Jeneponto and Makassar. The two new species, C. makassarense and C. javanense were isolated from Makassar and West Java, respectively.

In Thailand, C. scovillei was isolated mostly from the northern provinces of Chiang Mai and Chiang Rai, but was also obtained from infected chili fruit in a trial site of Kasetsart University in Nakhon Pathom. The Western provinces had a high incidence of C. siamense and one isolate of C. endophyticum was collected from Kanchana Buri. Colletotrichum plurivorum was isolated from chili leaves with necrotic lesions in Chiang Rai and from necrotic lesions of chili fruit found in a restaurant in Phaya Thai area of Bangkok. In addition, C. fructicola was identified from fruit collected from Chiang Mai.

In Taiwan, an isolate was identified as a new species, C. tainanense, collected from infected chili fruit in the Tainan province, and C. fructicola was identified from fruit in Hsinchu and Nantou, Taiwan. Colletotrichum plurivorum was also identified from a fruit collected in Johor, Malaysia.

Pathogenicity

All the Colletotrichum species caused anthracnose symptoms on wounded fruit, but there were significant differences in severity of the symptoms. On wounded fruit, isolates of C. scovillei and C. javanense showed the highest disease severity, producing large, necrotic lesions with mean lesion sizes 15.6–20.3 mm (Table 5). On non-wounded fruits, all species were less pathogenic, with mean lesion sizes less than 10 mm. Colletotrichum makassarense, C. tropicale and C. plurivorum, produced only very small lesions or no visible symptoms (mean lesion size < 1 mm) 10 d after inoculation of non-wounded fruits. Colletotrichum javanense and C. scovillei isolates were the most pathogenic in non-wounded fruit and produced lesions with mean sizes of 9.4 and 9.1 mm respectively. Lesions caused by C. scovillei were significantly larger than those caused by all other species except for C. javanense in wounded fruit and C. endophyticum in unwounded fruit. Significance of the pairwise t-tests is strongly dependent on the number of isolates of each species sampled, so the groupings shown in Table 5 do not simply change with the magnitude of the difference of the means; a large difference may not be significant if there are small sample sizes for both species while a smaller difference may be significant. In particular, comparisons involving C. javanense and C. endophyticum are very imprecise as only one isolate of each species was tested.
Table 5

Mean lesion size of symptoms caused by Colletotrichum species with straight conidia on inoculated mature red fruit of Capsicum annuum cv. Bangchang

Wound inoculation

Non-wound inoculation

Species

Least squares mean mm

Standard Error

t-groupinga

Species

Least squares mean mm

Standard Error

t-groupinga

C. scovillei

20.3

0.09

a

C. javanense

9.4

0.28

ab

C. javanense

15.6

0.35

ab

C. scovillei

9.1

0.07

a

C. siamense

9.7

0.11

bc

C. fructicola

3.6

0.2

bc

C. karsti

9.4

0.2

bcd

C. endophyticum

2.8

0.29

bc

C. fructicola

7.8

0.25

be

C. karsti

2

0.16

c

C. tainanense

6.9

0.25

be

C. tainanense

1.5

0.2

c

C. makassarense

6.4

0.18

ce

C. siamense

1.3

0.09

c

C. plurivorum

5.3

0.16

de

C. makassarense

<  1.0

0.14

c

C. endophyticum

4.4

0.35

ce

C. tropicale

<  1.0

0.16

c

C. tropicale

4.1

0.2

de

C. plurivorum

<  1.0

0.13

c

control

0

0.35

e

control

0

0.29

c

aPairwise t-tests between species least square means, significant differences at alpha = 0.05 level are indicated by different letters

DISCUSSION

Colletotrichum isolates collected from infected fruit and leaf tissue of chili plants from Thailand, Indonesia, Taiwan, Sri Lanka, and Malaysia were allocated to different species complexes with 11 Colletotrichum species being identified and three new species described.

Colletotrichum truncatum was the most prevalent species of Colletotrichum causing anthracnose of chili in Asia, which supports previous reports of C. truncatum being widely distributed on chili fruit throughout Asia, Australia, and South America (Sharma et al. 2014; De Silva et al. 2017a; Diao et al. 2017; Mongkolporn and Taylor 2018). Colletotrichum truncatum has a broad host range infecting many crop species (https://nt.ars-grin.gov/fungaldatabases/; Ranathunge and Hewa Bajjamage 2016).

The remaining 56% of isolates with straight conidia belonged to the gloeosporioides, acutatum, boninense and orchidearum species complexes. Of these, C. scovillei, in the acutatum complex, was the most prominent species (35% of the total isolates) and was distributed in Indonesia, Malaysia, Thailand and Taiwan. However, C. scovillei was not identified in the survey of Sri Lanka, possibly because of the small number of samples assessed. Previous studies have also reported the predominance of C. scovillei in Asia and Brazil (Diao et al. 2017, Mongkolporn and Taylor 2018), but this species has not been identified in Australia (De Silva et al. 2017a) and hence remains an important biosecurity threat to the Australian capsicum/chili industry. Further surveys are required to confirm the presence of C. scovillei in Australia and Sri Lanka.

Colletotrichum siamense, in the gloeosporioides complex, was also prominent (11% of total isolates) in causing anthracnose of chili fruit in Indonesia, Sri Lanka and Thailand, but was not detected in Malaysia and Taiwan. Nevertheless, Noor and Zakaria (2018) reported the identification of C. siamense in Malaysia possibly due to a larger sampling size of infected chili across states in Malaysia. Within the C. siamense clade, small subclades were observed that coincided with different geographical regions from where the isolates were collected. Colletotrichum siamense has been reported to infect chili in Asia, Australia, Brazil, and is a common pathogen of many other plant species (Weir et al. 2012; James et al. 2014; Sharma and Shenoy 2014; Liu et al. 2016a, 2016b; de Oliveira et al. 2017; De Silva et al. 2017a; Diao et al. 2017; Suwannarat et al. 2017).

Colletotrichum siamense isolates from different countries appeared to show different morphological characters with varying growth rates and culture morphologies. This variability in morphological characters indicated that this taxon has high intra-specific diversity. The combined gene analyses of ApMat and gs sequences also supported the distinction of subclades within the C. siamense clade. In the last few years, there has been significant debate on whether C. siamense s. lat. should be separated into different species groups within the gloeosporioides complex, with the number of accepted species ranging from one to seven (Weir et al. 2012; Udayanga et al. 2013). However, a recent case study concluded that C. siamense s. lat. was a single species rather than a species complex as no independent evolutionary lineages were found within this species (Liu et al. 2016a).

Colletotrichum tropicale is reported for the first time as causing anthracnose in Capsicum in Indonesia. Rojas et al. (2010) noted that C. tropicale was initially isolated from a wide range of hosts in forests in tropical America, from rotting fruit and as a leaf endophyte. Silva et al. (2017) recently reported C. tropicale causing chili anthracnose in Brazil. Colletotrichum tropicale was also reported from Japan, Panama, Thailand, and from other host species (Mongkolporn and Taylor 2018).

Colletotrichum fructicola is reported for the first time causing chili anthracnose in Thailand and Taiwan. Colletotrichum fructicola was previously reported to cause anthracnose in chili from India and China (Sharma and Shenoy 2014; Diao et al. 2017). Prihastuti et al. (2009) originally isolated C. fructicola from coffee berries in Thailand, and then C. fructicola was reported as a leaf endophyte from several plants in South America (Weir et al. 2012; Vieira et al. 2014) Colletotrichum fructicola has a wide host range and was reported by Weir et al. (2012) as a biologically and geographically diverse species.

All the species in the gloeosporioides complex were identified using combined multi-locus gene analyses, based on the ITS, gapdh, chs-1, act and tub2 genes, which showed higher diversity on chili than those in the acutatum, boninense and orchidearum species complexes. Phylogenetic tree provided good resolution of the species with high support values, which supported the species boundaries and identified the novel species.

Phylogenetic trees built from ApMat and gs gene sequences had similar topologies to the multigene phylogenetic tree confirming that the ApMat and gs loci were highly informative and that they distinguished most species in the gloeosporioides species complex (Silva et al. 2012; Sharma et al. 2013; Liu et al. 2015). However, some recent new species (Diao et al. 2017; Marin-Felix et al. 2017) were unable to be placed in the ApMat and gs phylogenetic tree due to the absence of the ApMat gene sequence data. Although there were reports that the gs gene alone is not a good marker for differentiating C. siamense isolates (Weir et al. 2012), these data showed multiple base pair differences in gene sequences of the gs loci of C. siamense species similar to the ApMat locus. In addition, the lack of noticeable subclading in C. siamense in the 5-gene tree compared to the ApMat and gs tree, confirmed that the ApMat and gs loci were more informative than the other five gene loci. The ApMat gene has been shown previously to improve the systematics of the gloeosporioides species complex, providing complementary phylogenetic information compared to other loci (Silva et al. 2012). Liu et al. (2015) also applied the ApMAT gene in a more recent molecular phylogenetic analyses of the species in this complex and discussed the merit of using ApMat and ApMat in combination with gs to resolve the phylogeny.

Although four isolates from Indonesia were identified as C. karsti in the boninense complex, they formed a subclade within the C. karsti species clade and had different conidial sizes to the ex-type strain of C. karsti (Yang et al. 2011), suggesting that these might be a new species. However, sufficient phylogenetic support was not observed in all the individual gene trees to justify the introduction of a novel species. Besides, Damm et al. (2012a, 2012b) reported that the conidium size of C. karsti was quite variable. Colletotrichum karsti has been reported from China and India to cause anthracnose disease in Capsicum spp. (Liu et al. 2016b; Saini et al. 2016; Diao et al. 2017). Colletotrichum karsti has the widest known host range and distribution of all species in the boninense complex (Damm et al. 2012b). Most of the C. karsti strains had been isolated as endophytes but a few were derived from diseased plant tissues. This species has mostly been isolated from dicotyledonous plants, but some have occurred on monocotyledonous families, especially Orchidaceae and Musaceae (Damm et al. 2012b).

Colletotrichum plurivorum was identified for the first time causing anthracnose in Thailand and Malaysia. The five C. plurivorum isolates from chili in Thailand formed a distinct subclade with high support values within the C. plurivorum subclade, and separated from C. cliviicola (syn. C. cliviae, Damm et al. 2019). In addition, the C. plurivorum isolates formed a characteristic sexual morph in culture, which was not reported for C. cliviicola (Yang et al. 2009).

Recently, Damm et al. (2019) resolved the taxonomic placement of several Colletotrichum strains which did not belong to any of the accepted species complexes and assigned them to three new species complexes including the orchidearum complex. Recent studies in China and Brazil also identified multiple species belonging to these complexes, including C. brevisporum, C. cliviicola, C. liaoningense, and C. plurivorum that caused anthracnose disease in chili (Liu et al. 2016b; De Silva et al. 2017b; Diao et al. 2017). Colletotrichum plurivorum belongs to the orchidearum complex with many isolates reported to have a large host range (Damm et al. 2019). The type specimen was described as new from Coffea in Vietnam (Nguyen et al. 2010). Colletotrichum plurivorum was originally described as C. sichuanensis from Capsicum annuum in the Sichuan Province of China (Liu et al. 2016b). However, the name was invalid, because no holotype specimen was cited (Mongkolporn and Taylor 2018; Damm et al. 2019).

Pathogenicity tests of Colletotrichum spp. from chili showed that while all the species were pathogenic on chili fruits after wounding the fruit surface, most produced a low level of infection on non-wounded fruit. This illustrates the importance of the cuticle acting as a barrier to infection by Colletotrichum spp. (Auyong et al. 2015) and emphasises the need for informed and standardised inoculation techniques in pathogenicity assays. Some species such as C. tropicale, C. makassarense and C. plurivorum which produced a low level of infection in the assays on non-wounded fruit, may have a predominantly endophytic lifestyle then switch to a necrotrophic life style to complete their life-cycle (De Silva et al. 2017b). However, further pathogenicity tests on different chili cultivars and at different fruit maturity stages are necessary to comprehensively evaluate their pathogenicity. Pathogenicity testing of C. plurivorum on chili leaves and fruits showed that the isolates collected from Chiang Rai and Malaysia could infect leaves but not fruit (results not shown) and suggested they might be specialised leaf pathogens. In contrast, two isolates of C. plurivorum from Bangkok did not infect leaves but did infect wounded fruits. These results demonstrate the pathogenic variation that can exist within a single species.

Mongkolporn et al. (2010) identified pathotypes of C. truncatum, C. scovillei (as C. acutatum) and C. siamense (as C. gloeosporioides) within isolates of each species from Thailand. Pathotypes were identified by inoculating wounded fruit of Capsicum baccatum and C. chinense genotypes. All the isolates identified as C. gloeosporioides and C. acutatum in Mongkolporn et al. (2010) were subsequently re-identified as C. siamense and C. scovillei, respectively except for isolate UOM 1137 (F5-2D), which was identified as C. endophyticum. The isolate UOM 1137 was pathogenic in both the wound and non-wound bioassays, and was classified in the most virulent C. siamense pathotype group (PCg1-R) in Mongkolporn et al. (2010). This contrasts with the study by Manamgoda et al. (2013) where C. endophyticum was described as an endophyte of Pennisetum purpureum. The severity of infection in chili may indicate that Capsicum annuum was the preferred host for C. endophyticum and P. purpureum was a less favoured host, where the pathogen infected but existed in an endophytic lifestyle. In addition, isolate UOM 1137 also has shorter spores than the type isolate of C. endophyticum, thus further isolates of this species need to be collected from chili plants and P. purpureum in Thailand to confirm taxonomy and pathogenicity.

CONCLUSIONS

Multigene phylogenetic analyses of Colletotrichum species causing anthracnose disease of Capsicum in Asia showed high species diversity with the identification of 11 different Colletotrichum species, including three novel species. Although C. siamense has been reported as infecting many plant species before, this was the first report of C. siamense causing anthracnose in chili in Indonesia and Sri Lanka. This was also the first report of C. fructicola infecting chili in Thailand and Taiwan. In addition, all three novel species were new additions to the Colletotrichum species causing anthracnose in chili. More surveys in countries in Asia and Oceania need to be conducted to identify the diversity and prevalence of species causing chili anthracnose. Understanding of the taxonomy and the pathogenicity of Colletotrichum spp. has great significance to fruit and vegetable industries, where there are serious biosecurity implications of incursion by exotic pathogens.

Abbreviations

AGRF: 

Australian Genome Research Facility

BI: 

Bayesian inference analyses

CBS: 

Westerdijk Fungal Biodiversity Institute, The Netherlands

CI: 

Consistency index

DIC: 

Differential interference contrast

DNA: 

Deoxyribonucleic acid

MCMC: 

Markov chain Monte Carlo algorithm

MEA: 

Malt extract agar

ML: 

Maximum likelihood

MP: 

Maximum parsimony

OA: 

Oatmeal agar

PAUP: 

Phylogenetic analysis using parsimony

PCR: 

Polymerase chain reaction

PDA: 

Potato dextrose agar

PP: 

Posterior probabilities

RC: 

Rescaled consistency index

RI: 

Retention index

SNA: 

Synthetic nutrient-poor agar

TBR : 

Tree bisection and reconstruction

TL: 

Tree length

UOM: 

University of Melbourne

WA: 

Water agar

Declarations

Acknowledgements

We wish to thank World Vegetable Center AVDRC, Taiwan; Ratchadawan Cheewangkoon, Faculty of Agriculture, Chiang Mai University, Thailand and Nalika Ranathunge, Faculty of Agriculture, University of Ruhuna, Sri Lanka for their contribution in sample collections. Ulrike Damm is thanked for guidance and helpful suggestions to species identification.We also thank Arien Van Iperen and Mieke Starink-Willemse from the Westerdijk Fungal Biodiversity Institute for technical assistance. DD de Silva gratefully acknowledges the financial support from the Melbourne International Research Scholarship (MIRS) and Melbourne International Fee Remission Scholarship (MIFRS) awarded by the University of Melbourne.

Adherence to national and international regulations

The importation and use of isolates adhered to the regulations related to National Plant Health and Quarantine, and the Nagaoya Protocol to the Convention on Biological Diversity.

Funding

The research was not supported by any external grants, the student and project were supported by the University of Melbourne and the Westerdijk Fungal Biodiversity Institute.

Availability of data and materials

Alignments and tree files generated during the current study are available in the TreeBASE (accession https://www.treebase.org/treebase-web/home.html; study S23829). All sequence data are available in NCBI Genbank following the accession numbers in the manuscript.

Authors’ contributions

DD wrote the manuscript, made a substantial contribution to the conception of the study, analysed the isolates, interpreted the sequence data and performed the pathogenicity assays and microscopy. JG assisted in the analyses of the data and advised on the interpretation of the taxonomy. PC contributed to microscopic examination of fungal material, advised on the interpretation of the taxonomy, and was a major contributor in writing the manuscript. PA assisted in the analysing and interpretation of the pathogenicity statistical data, and was a major contributor in writing the manuscript. AN contributed to the collection of isolates. OM contributed to the collection of isolates. PT made a substantial contribution to the conception of the study, and was a major contributor in writing the manuscript and collection of isolates. All the authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC, 3010, Australia
(2)
Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584, CT, Utrecht, The Netherlands
(3)
Faculty of Science, The University of Melbourne, Parkville, VIC, 3010, Australia
(4)
Department of Plant Pest & Disease, Universitas Hasanuddin, Makassar, Indonesia
(5)
Department of Horticulture, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, Thailand

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Copyright

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