Fungal Diversity DOI 10.1007/s13225-014-0293-6

Endophytic species of Colletotrichum associated with mango in northeastern Brazil Willie A. S. Vieira & Sami J. Michereff & Marcos A. de Morais Jr & Kevin D. Hyde & Marcos P. S. Câmara

Received: 17 January 2014 / Accepted: 7 June 2014 # Mushroom Research Foundation 2014

Abstract Endophytic species of Colletotrichum associated with Mangifera indica (mango) are poorly understood. In this study, Colletotrichum species were isolated from mango in Pernambuco State, Brazil. There were significant differences in isolation frequencies of Colletotrichum species among sites and plant tissues. Mature leaf blades were colonized by most Colletotrichum isolates at the majority of sites. Partial sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of 97 Colletotrichum isolates were amplified as an initial measure of genetic diversity. Phylogenetic analysis with a subset of 22 isolates were performed based on a multilocus dataset (ACT, TUB2, CAL, CHS-1, GAPDH, ITS) followed by Apn2/MAT IGS sequence-analysis for isolates within the C. gloeosporioides species complex. Molecular analysis associated with phenotypic characteristics revealed six previously described species [C. asianum, C. cliviae, C. dianesei (syn. C. melanocaulon), C. fructicola, C. karstii and C. tropicale] and one new species. This new species is introduced as C. endomangiferae. All species isolated were pathogenic on mango fruits but varied in their virulence. There was no distribution pattern of species among sites and plant tissues, although C. asianum was the most prevalent species at all sites and in all plant tissues studied. Five previously reported Colletotrichum species causing anthracnose in W. A. S. Vieira : S. J. Michereff : M. P. S. Câmara (*) Departamento de Agronomia, Universidade Federal Rural de Pernambuco, 52171-900 Recife, Brazil e-mail: [email protected] M. A. de Morais Jr Departamento de Genética, Universidade Federal de Pernambuco, 50732-970 Recife, Brazil K. D. Hyde Institute of Excellence in Fungal Research, and School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand

mango fruits in northeastern Brazil were also recovered as endophytes. Keywords Anthracnose . Apn2/MAT IGS . Distribution . Endophytism . Phylogeny . Polyphasic taxonomy

Introduction Mango (Mangifera indica L.) is regarded as one of the world’s most important tropical and subtropical fruit crops, and hundreds of cultivars are known worldwide (Nelson 2008). Mango production is affected by a number of diseases at all stages of development (Ploetz 2003; Prakash 2004), and anthracnose, caused by species of Colletotrichum, is the most important disease of mango in Brazil (Santos Filho and Matos 2003). Colletotrichum is an important and widespread genus. Species are serious plant pathogens, and some are frequent endophytes of tropical plants (Cannon and Simmons 2002, Rodrigues and Petrini 1997, Suryanarayanan et al. 2002). Colletotrichum species are among the most commonly occurring pathogens and foliar endophytes of terrestrial plants and have been recorded from approximately 2,200 plant species (Farr and Rossman 2013). Endophytes infect living plant tissues without causing symptoms of disease (Petrini 1991). However, many fungal endophytes are related to biotrophic and necrotrophic plant pathogens (Delaye et al. 2013). Colletotrichum species are fungal pathogens that devastate crop plants worldwide. Host infection involves the differentiation of specialized cell types that are associated with penetration, growth inside living host cells (biotrophy) and tissue destruction (necrotrophy) (O’Connell et al. 2012) The quiescent infections occur during the pre-harvest stages, flowering and fruit development; however, infection is only evident post-harvest (Prusky 1996). Recent observations of endophytic fungi suggest asymptomatic

Persea americana

ICMP 18621

Coffea arabica Mangifera indica, endophyte in old limb Mangifera indica, endophyte in young vein Mangifera indica, endophyte in young vein Mangifera indica, endophyte in old limb Mangifera indica, endophyte in young limb Brachyglottis repanda Camellia sp. Crinum asiaticum var. sinicum

ICMP 18580*, CBS 130418

CMM 3736

CBS 128527, ICMP 18594* CBS 128547, ICMP 10338

CBS 123755, MAFF 305972*

USA

Cymbidium sp.

CBS 128503, ICMP 12936

CBS 123757, MAFF 306100

Solanum betaceum

CBS 128504, ICMP 12941*

C. constrictum

C. cymbidiicola

Citrus limon

CBS 129817, G1 CBS 129818, G2*

Mangifera indica, endophyte in old vein Mangifera indica, endophyte in young limb Mangifera indica, endophyte in young limb Mangifera indica, endophyte in young vein Passiflora edulis Passiflora edulis

C. colombiense

CMM 3808

CMM 3782

CMM 3746

CMM 3742

Clivia miniata

CSS1

C. cliviae

C. cliviae

Clivia miniata

CSSK4

C. brassicicola

Japan

New Zealand

New Zealand

Colombia Colombia

Brazil, São João

Brazil, Recife

Brazil, Aldeia

Brazil, Aldeia

China

China

Brazil New Zealand

CBS 128528, ICMP 18606, PAS10 CBS 101059, LYN 16331*

Passiflora edulis Brassica oleracea var. gemmifera

Brazil

Japan

New Zealand New Zealand

Brazil, Recife

Brazil, Aldeia

Brazil, Aldeia

Brazil, Aldeia

Brazil, Goiana

Thailand

Colombia Australia

New Zealand

New Zealand

CBS 128501, ICMP 18607, PAS12* Passiflora edulis

CMM 3793

CMM 3758

CMM 3747

CMM 3738

Hevea indica Mangifera indica

CBS 129826, CH1* IMI 313839, ICMP 18696

Aeschynomene virginica

Israel Japan

Location

C. brasiliense

C. beeveri C. boninense

C. annellatum C. asianum

Malus domestica

ICMP 17673*, ATCC 201874

ICMP 12071*

C. aeschynomenes

C. alienum

Persea americana Pyrus pyrifolia

ICMP 18608* ICMP 18686

Colletotrichum aenigma

Host

Culture accession No. 1

Species

JX010406

JQ005656 JX010384

JX010386

JX010411

JX010392

JX010389 JX010390

TUB2

FJ917506

JQ005743 JX009723

JX009657

JX009654

JX009721

JX009683 JX009684

CAL

JX009867

JQ005396 JX009753

JX009755

JX009882

JX009799

JX009774 JX009789

CHS-1

JX010053

JQ005309 JX009915

JX009959

JX010028

JX009930

JX010044 JX009913

GPDH

FJ972612

JQ005222 JX010192

JX010246

JX010251

JX010176

JX010244 JX010243

ITS

JQ005668 JQ005606

JQ005669

JQ005588

JQ005605 JQ005593

JQ005755 JQ005693

JQ005756

JQ005674

JQ005692 JQ005680

JQ005408 JQ005346

JQ005409

JQ005327

JQ005345 JQ005333

JQ005321 JQ005259

JQ005322

JQ005240

JQ005258 JQ005246

JQ005234 JQ005172

JQ005235

JQ005153

JQ005171 JQ005159

JQ005516

JQ005585

JQ005586

JQ005521 JQ005522

JQ005602

JQ005671

JQ005672

JQ005607 JQ005608

JQ005689

JQ005758

JQ005759

JQ005694 JQ005695

JQ005342

JQ005411

JQ005412

JQ005347 JQ005348

JQ005255

JQ005324

JQ005325

JQ005260 JQ005261

JQ005168

JQ005237

JQ005238

JQ005173 JQ005174

KC702910 KC992329 KC992360 KC598111 KC702943 KC702992

KC702907 KC992326 KC992357 KC598096 KC702940 KC702974

KC702909 KC992328 KC992359 KC598101 KC702942 KC702981

KC702908 KC992327 KC992358 KC598100 KC702941 KC702980

GU085861 GU085869 GU085864 GU085866 GU085868 GU109480

GQ856777 GQ849440 GQ849464 GQ856722 GQ856756 GQ485607

JQ005582 JQ005520

JQ005583

JQ005501

JQ005519 JQ005507

KC702893 KC702998 KC992347 KC598092 KC702930 KC702964

KC702898 KC703003 KC992352 KC598103 KC702935 KC702983

KC702897 KC703002 KC992351 KC598102 KC702934 KC702982

KC702896 KC703001 KC992350 KC598097 KC702933 KC702977

KC702901 KC703006 KC992355 KC598108 KC702938 KC702989

JX009584

JQ005570 JX009576

JX009552

JX009572

JX009483

JX009443 JX009519

ACT

GenBank Accession No. b

Table 1 Colletotrichum isolates included in the multi-gene sequence analysis, with details of culture collection, host, location, and GenBank accessions of the sequences generated

Fungal Diversity

Mangifera indica

CMM 4084

CMM 4085

CMM 3741

Diospyros australis Mangifera indica, endophyte in old limb Musa sapientum Musa sp.

CBS 127597, BRIP 29085a

CMM 3797

IMI 52264, ICMP 17817

CBS 116870*, ICMP 19119

C. musae

Jasminum sambac Annona cherimola

ICMP 19118* CBS 128500, ICMP 18585

C. jasmini-sambac C. karstii

Diospyros kaki Hymenocallis americana

ICMP 10492*

ICMP 18642*

C. horii

Hippeastrum sp. Hippeastrum vittatum

IMI 356878*, ICMP 17821, CBS 112999 CBS 241.78, IMI 304052 CBS 125376, CSSG1*

CMM 3811

C. hymenocallidis

C. hippeastri

C. gloeosporioides

CBS 238.49, ICMP 17921

C. fructicola

CMM 3770

Ficus edulis Mangifera indica, endophyte in young vein Mangifera indica, endophyte in young limb Mangifera indica, endophyte in old limb Citrus sinensi

ICMP 18581*, CBS 130416 CBS 125397 (*), ICMP 18646

CMM 3814*

CMM 3740

Mangifera indica, endophyte in young limb Mangifera indica, endophyte in young limb Coffea arabica Tetragastris panamensis

Mangifera indica

CMM 4083

C. fructicola

C. endomangiferae

Mangifera indica

CMM 4082

CMM 3784

CMM 3779

Mangifera indica, endophyte in old limb Mangifera indica, endophyte in old limb Mangifera indica, endophyte in old vein Mangifera indica

CMM 3777

C. dianesei

Cymbidium sp. Dacrycarpus dacrydioides

IMI 347923*

CBS 130241, ICMP 19107*

Host

Culture accession No. 1

C. dacrycarpi

Species

Table 1 (continued)

USA

Kenya

Brazil, São João

Australia

Vietnam New Zealand

China

Japan

Netherlands China

Italy

Brazil, São João

Brazil, Recife

Brazil, Aldeia

Germany

Thailand Panama

Brazil, São João

Brazil, Aldeia

Brazil, Assu Valley

Brazil, São Francisco Valley Brazil, São Francisco Valley Brazil, Assu Valley

Brazil, Recife

Brazil, Recife

Brazil, Recife

New Zealand

Australia

Location

JQ005670

JQ005600

TUB2

JQ005757

JQ005687

CAL

JQ005410

JQ005340

CHS-1

JQ005323

JQ005253

GPDH

JQ005236

JQ005166

ITS

JX010400

JX010405 JX010409

JX009671

FJ917508 JX009674

JX009839

JX009866 JX009874

JX009923

JX010033 JX010032

JX010181

JX010165 JX010173

JX010450

JQ005666 JQ005665

JX010445

JQ005638

JQ005725

JX009713 JQ005723

JX009709

JX009604

JQ005753 JQ005752

JX009731

JQ005378

JX009895 JQ005376

JQ005232 JQ005231

JX010152

JQ005291

JQ005204

HM131497 HM131511 JQ005289 JQ005202

JX010278

GQ329681 GQ329690

JQ005319 JQ005318

JX010056

GQ856730 JX010019

JX009752

JQ005406 JQ005405

JX009818

JX009433

JX009432

JX009689 HQ596280 JX009742

JX010395

JX009896

JX009815

JX010050

JX010015

JX010146

JX010142

KC702920 KC992339 KC992370 KC598109 KC702953 KC702990

JQ005552

HM131507 JX010415 JQ005550 JQ005636

GQ856775 JX010410

JX009438

JQ005580 JQ005579

JX009531

KC702919 KC992338 KC992369 KC598112 KC702952 KC702993

KC702915 KC992334 KC992365 KC598093 KC702948 KC702966

KC702916 KC992335 KC992366 KC598099 KC702949 KC702979

JX009495

FJ907426 JX009581

KC702922 KC702922 KC992372 KC598113 KC702955 KC702994

KC702921 KC702921 KC992371 KC598098 KC702954 KC702978

KC533740 KC517275 KC517230 KJ155471 KC517196 KC329813

KC517307 KC517273 KC517228 KJ155470 KC517201 KC329811

KC517298 KC517254 KC517209 KJ155469 KC517194 KC329779

KC517296 KC517249 KC517204 KJ155468 KC517157 KC329774

KC702914 KC992333 KC992364 KC992382 KC702947 KC702976

KC702912 KC992331 KC992362 KC992380 KC702945 KC702971

KC702911 KC992330 KC992361 KC992379 KC702944 KC702970

JQ005584

JQ005514

ACT

GenBank Accession No. b

Fungal Diversity

Mangifera indica, endophyte in old vein Mangifera indica, endophyte in old limb Mangifera indica, endophyte in young vein Mangifera indica, endophyte in young vein

CMM 3767

CMM 3787

CMM 3783

CMM 3780

Theobroma cacao

CBS 124949*, ICMP 18653

Passiflora edulis Litchi chinensis

MAFF 239933, ICMP 18672

Brazil, Recife

Brazil, Recife

Brazil, Recife

Brazil, Aldeia

Panama

Japan

New Zealand

Panama New Zealand

USA

Thailand

Fiji Hungary

Australia

India

Italy

New Zealand Italy

Germany

Germany

USA USA

New Zealand

New Zealand

Location

JX010407

JX010396

JQ005597

JX010447 JQ005599

JX010393

JX010404

JX010412 JX010403

JX010414

JQ005655

JQ005658

JQ005667 JQ005657

JQ005604

JQ005603

JX010397 JX010397

JQ005663

JQ005662

TUB2

JX009719

JX009722

JQ005684

JX009591 JQ005686

JX009703

FJ917505

JX009694 JX009696

JX009691

JQ005742

JQ005745

JQ005754 JQ005744

JQ005691

JQ005690

JX009661 JX009661

JQ005750

JQ005749

CAL

JX009870

JX009826

JQ005337

JX009869 JQ005339

JX009805

JX009865

JX009890 JX009863

JX009899

JQ005395

JQ005398

JQ005407 JQ005397

JQ005344

JQ005343

JX009834 JX009834

JQ005403

JQ005402

CHS-1

JX010007

JX010020

JQ005250

JX010006 JQ005252

JX010051

JX009924

JX010036 JX009916

JX009934

JQ005308

JQ005311

JQ005320 JQ005310

JQ005257

JQ005256

JX009936 JX009936

JQ005316

JQ005315

GPDH

JX010264

JX010275

JQ005163

JX010294 JQ005165

JX010162

JX010171

JX010185 JX010242

JX010276

JQ005221

JQ005224

JQ005233 JQ005223

JQ005170

JQ005169

JX010189 JX010189

JQ005229

JQ005228

ITS

KC702923 KC992340 KC992376 KC992383 KC702956 KC702962

KC702927 KC992344 KC992375 KC992387 KC702958 KC702975

KC702926 KC992343 KC992374 KC992386 KC702961 KC702972

KC702928 KC992345 KC992378 KC992388 KC702960 KC702985

JX009489

JX009480

JQ005511

JX009444 JQ005513

JX009506

FJ907423

JX009490 JX009562

JX009447

JQ005569

JQ005572

JQ005581 JQ005571

JQ005518

JQ005517

JX009486 JX009486

JQ005577

JQ005576

ACT

GenBank Accession No. b

ATCC American Type Culture Collection, Virginia, USA; CBS Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CGMCC China General Microbiological Culture Collection, Beijing, China; CMM Culture Collection of Phythopathogenic Fung “Prof. Maria Menezes”, Recife, Brazil; DAR Queensland Plant Pathology Herbarium (Australia), Queensland, Australia; ICMP International Collection of Micro-organisms from Plants, Landcare Research, New Zealand; IMI International Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, U.K.; MAFF: MAFF Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Japan; MFLU Mae Fah Luang University Herbarium, Thailand; NBRC Biological Resource Center, National Institute of Technology and Evaluation, Chiba, Japan. * ex-type or ex-epitype. 2 ACT: actin; TUB-2: partial ß-tubulin (tub2); CAL: calmoudulin; CHS: chitin synthase; GAPDH: glyceraldehydes-3-phosphate dehydrogenase; ITS: partial rDNA-ITS region. Sequences derived in this study are emphasized in bold

1

C. tropicale

CBS 102667

Malus domestica

ICMP 17795 Theobroma cacao Solanum melongena

Coffea arabica

ICMP 18578*, CBS 130417

C. siamense

CBS 124945*, ICMP 18649 CBS 128544, ICMP 18586*

Coffea sp. Salsola tragus

ICMP 18705 ICMP 19051*

C. salsolae

C. theobromicola C. torulosum

Carica papaya

ICMP 1778*

C. queenslandicum

Dracaena marginata Phyllanthus acidus

CBS 379.94

CBS 175.67, MACS 271*

C. phyllanthi

Parsonsia capsularis Dracaena marginata

CBS 128525, ICMP 18590* CBS 378.94*

Oncidium sp.

CBS 130242

C. parsoniae C. petchii

Oncidium sp.

CBS 129828*

C. oncidii

Nuphar lutea subsp. polysepala Nuphar lutea subsp. polysepala

CBS 469.96, ICMP 17938 CBS 470.96*, ICMP 18187

Citrus sp. (grapefruit)

CBS 130240, ICMP 12064

C. nupharicola

Capsicum annuum

CBS 128505, ICMP 12944*

C. novae-zelandiae

Host

Culture accession No. 1

Species

Table 1 (continued)

Fungal Diversity

Fungal Diversity

colonization is a balance of antagonism between the pathogen and the host (Devaraju and Satish 2010). Several endophytic species of Colletotrichum have been reported from a single host (Tao et al. 2013, Manamgoda et al. 2013). For example, 17 Colletotrichum species, including seven new species were reported from Bletilla ochracea Schltr. (Tao et al. 2013). Three species from the C. gloeosporioides species complex were associated with tropical grasses and one new species was introduced based on multi-locus phylogenetic analysis (Manamgoda et al. 2013). Although there have been many studies on the genus Colletotrichum, understanding of endophytic species remains very limited (Tao et al. 2013). Species circumscription and identification of Colletotrichum species has historically been based on host range, symptoms of infection on particular hosts, and a suite of morphological characters (Hyde 2009a, b). However, the use of these conventional taxonomic characters in Colletotrichum has failed to support the development of robust species concepts because of their plasticity (Hyde et al. 2009b; Rojas et al. 2010). Cai et al. (2009) proposed that identification, epitypification, or description of new Colletotrichum species should be based on a multi-gene phylogeny in conjunction with recognizable distinctive phenotypic characters, such as morphology, pathogenicity and cultural characteristics. Molecular data are essential for the accurate identification of Colletotrichum species and the current number of accepted species supported by molecular data is likely to rise with future studies (Hyde et al. 2009a, b; Cai et al. 2011a, b; Weir et al. 2012; Cannon et al. 2012). Endophytic species of Colletotrichum associated with mango are understudied. Endophytism is a part of the life cycle of many pathogens and can act as a survival strategy (Brown et al. 1998; Wikee et al. 2013). It is possible that reported species causing anthracnose on mango fruits are also found as endophytes in different areas and plant organs. This has been shown in the case of Phyllosticta capitalensis, which is a widespread endophyte of many hosts and a weak pathogen of some plant genera such as Citrus, Ficus, Ophiopogon and Punica (Glienke et al. 2011; Wang et al. 2012; Wikee et al. 2013). Similarly, Diaporthe (Phomopsis) species have often been reported as plant pathogens, non-pathogenic endophytes, or saprobes commonly isolated from a wide range of hosts (Gomes et al. 2013; Udayanga et al. 2011, 2012). The objectives of this study are to determine: (1) the isolation frequency of Colletotrichum from mango at different sites and in different plant parts; 2) which Colletotrichum species occur as endophytes in mango trees based on phenotypic and molecular data, with a description of a new species; 3) the pathogenicity and the virulence of endophytic isolates on mango fruits; 4) the Colletotrichum species prevalence at different sites and in different plant parts.

Material and methods Sampling and fungal isolation Healthy tissues from non-commercial mature mango plants (>10 years old) were sampled from five different sites in Pernambuco State: Recife (Site I and Site II), Goiana (Site III), Aldeia (Site IV) and São João (Site V). Site I was located in an urban area, while the other sites comprised forest fragments. Samples were collected between January and June 2011. Twelve randomly selected plants were sampled from each site. Two young and two mature leaves, one stem fragment (with cork layer), and one mature inflorescence were sampled from each plant. All samples were washed in running water prior to surface sterilization. Five discs (6 mm diam) without veins were cut from leaf blades using a sterile cork borer to isolate endophytes from leaf blades,. For isolation from veins, five segments approximately 6×2 mm were cut close to the veins with a flamed scalpe (one petiole segment, two segments with midrib, two segments with lateral vein). Five segments approximately 6 mm×2 mm (length x width) were cut from internal tissues of stems and inflorescences using a flamed scalpel. All leaf segments were surface sterilized in 75 % ethanol for 1 min and 1.5 % sodium hypochlorite for 2 min. The stem and inflorescence fragments were surface sterilized in 75 % ethanol for 10 s and 1.5 % sodium hypochlorite for 10 s. Sterilized segments were rinsed in sterilized distilled water and then dried on sterilized paper. This protocol was found to be the optimum procedure for isolating Colletotrichum spp. endophytes from mango based on a pilot experiment. Fragments (maximum 2) were evenly spaced in Petri dishes (9 cm diam.) containing potato-dextrose-agar (PDA) medium (Acumedia, Lansing, USA) amended with chloramphenicol (25 mg l−1) to suppress bacterial growth, and incubated at 25ºC with a 12-hour cycle of dark and fluorescent light for 15 days. The fungi growing out of the segments during the incubation period were recorded as endophytes, transferred to tubes containing PDA and Table 2 Nucleotide substitution models used in phylogenetic analyses Gene

C. gloeosporioides complex

C. boninense complex and C. cliviae

ACT TUB2 CAL CHS-1 GPDH ITS Apn2/MAT IGS

HKY+G K80+G TIM1ef+G TIM2ef+G HKY+G TIM2+I+G K80

HKY+G K80+G TrN+G K80+I HKY+G TrNef+G

Fungal Diversity Table 3 Colletotrichum isolates included in ML analysis of Apn2/MAT IGS sequence-data, with details of culture collection, host, location and GenBank accessions of the sequences generated Species

Culture accession No. 1

Host

Location

Apn2/MAT IGS

Colletotrichum asianum

CBS 130418* CMM 3736 CMM 3738 CMM 3747

Coffea arabica Mangifera indica Mangifera indica Mangifera indica

Thailand Brazil Brazil Brazil

FR718814 KJ155450 KJ155449 KJ155447

CMM 3758 CMM 3793 GM 021 GM 414 GM595 LC 0036 LC 0037 MFLU 090233 CMM 3777 CMM 3779 CMM 3784 CMM 4082 CMM 4083 CMM 4084 CMM 4085 Coll 131* GM 057 GM 172

Mangifera indica Mangifera indica Mangifera indica Mangifera indica Mangifera indica Coffea arabica Coffea arabica Coffea arabica Mangifera indica Mangifera indica Mangifera indica Mangifera indica Mangifera indica Mangifera indica Mangifera indica Vaccinium macrocarpon Mangifera indica Mangifera indica

Brazil Brazil India India India Thailand Thailand Thailand Brazil Brazil Brazil Brazil Brazil Brazil Brazil USA India India

KJ155448 KJ155451 JQ894557 JQ894537 JQ894554 JQ899287 JQ899285 FR718814 KJ155458 KJ155459 KJ155457 KJ155460 KJ155461 KJ155462 KJ155463 JX145313 JQ894551 JQ894562

CMM 3740 CMM 3814* 1087 CBS 130416* CMM 3741

Mangifera indica Mangifera indica Theobroma cacao Coffea arabica

Brazil Brazil Panama Thailand

KJ155452 KJ155453 GU994438 JQ807838

CMM 3770 CMM 3811 Coll 1126 GM 567 ICMP 18646 LC 0032 CBS 95397 ICMP 18642* LC 0043 ICMP 10492* ICMP 19118* LC 0921 ICMP 19119* LC 0084

Mangifera indica Mangifera indica Mangifera indica Vaccinium macrocarpon Mangifera indica Tetragastris panamensis Coffea arabica Citrus sinensis Hymenocallis americana Hymenocallis americana Diosptyos kaki Jasminum sambac Jasminum sambac Musa sp. Musa sp

Brazil Brazil Brazil USA India Panama Thailand Italy China China Japan Vietnam Vietnam USA USA

KJ155455 KJ155454 KJ155456 JX145315 JQ894576 JQ807839 JQ899291 JQ807843 JQ807842 JQ899283 JQ807840 JQ807841 JQ899273 KC888926 JQ899271

LC 0872 LC 0962 LC 0963 CBS 46996* CBS 472 Coll 878 Coll 922

Musa sp Musa sp Musa sp Nuphar lutea Nimphaea odorata Nuphar advena Nuphar lutea

Thailand Thailand Thailand USA USA USA USA

JQ899270 JQ899269 JQ899268 JX145319 JX145320 JX145323 JX145318

C. dianesei

C. dianesei (syn. C. melanocaulon)

C. endomangiferae C. fructicola

C. gloeosporioides C. hymenocallidis C. horii C. jasmini-sambac C. musae

C. nupharicola

Fungal Diversity Table 3 (continued) Species

Culture accession No. 1

Host

Location

Apn2/MAT IGS

C. quuenslandicum C. salsolae C. siamense s. s.

ICMP 1778 ICMP 19051 BPDI 2

Carica papaya Salsola tragus Coffea arabica

Australia Hungary Thailand

KC888928 KC888925 FR718813

ICMP 18578* LC 0034 LC 0035 5101 7423 CMM 3767 CMM 3780 CMM 3783 CMM 3787 Coll 918 E 406 ICMP 18653*

Coffea arabica Coffea arabica Coffea arabica Theobroma cacao Theobroma cacao Mangifera indica Mangifera indica Mangifera indica Mangifera indica Terpsichore taxifolia Pentagonia macrophyla Theobroma cacao

Thailand Thailand Thailand Panama Panama Brazil Brazil Brazil Brazil Puertorico Panama Panama

JQ899289 JQ899289 JQ899288 GU994425 GU994423 KJ155464 KJ155467 KJ155465 KJ155466 JX145307 GU994424 KC790728

C. tropicale

1 CBS Culture Collection of the Centraalbureau voor Schimmelcultures, Fungal Biodiversity centre, Utrecht, The Netherlands; ICMP International Collection of Microorganisms from Plants, Landcare Research, Auckland, New Zealand; CMM Culture Collection of Phythopathogenic Fung “Prof. Maria Menezes”, Recife, Brazil. * ex-type or ex-epitype. Sequences derived in this study are emphasized in bold

incubated as described above. The fungi were identified following sporulation. One hundred sixty-nine fungi were morphologically identified as Colletotrichum spp. (Sutton 1980) and 97 isolates were used in the present study (one isolate for each plant part sampled). Single spore cultures were obtained using the procedure described by Goh (1999). Pure cultures were stored in sterilized water in Eppendorf tubes at 5 °C and stock cultures were stored on PDA slants at 5 °C in the dark. The isolates were deposited in the culture collection of Universidade Federal Rural de Pernambuco, Coleção de Fungos Fitopatogênicos “Professora Maria Menezes” (CMM), Recife, Pernambuco, Brazil.

Colletotrichum overall colonization The Isolation Frequency (IF) of Colletotrichum for each type of plant tissue (stem, inflorescence, young and mature leaf blades, young and mature veins) was calculated using the formula: IF%=(Nc/Nt)×100, where Nc is the number of fragments colonized by Colletotrichum from each tissue, and Nt is the total number of fragments from each site examined (Hata et al. 2002). The null hypothesis of no difference in isolation frequency was compared using the test for two proportions (Z test) in STATISTIX v. 9.0 (Analytical Software, Tallahassee, USA).

Table 4 Isolation frequency (IF%) of endophytic Colletotrichum in mango Plant tissue/site

Site I

Site II

Site III

Site IV

Site V

Stem Inflorescence Young leaf blade

0,28 b 0,00 b 0,56 ab

1,14 bcd 0e 1,43 bc

0d 0d 4,19 abc

0,29 bcde 1,14 abcd 2,57 a

0e 0,57 bcde 1,71 bc

Young vein Old leaf blade Old vein Total

0,28 b 1,97 a 0,28 b 3,38 E

0,57 bcde 4,29 a 1,71 bcd 9,14 BC

7,42 a 5,16 ab 4,19 abc 20,97 A

0,57 abcde 1,43 ab 1,14 abc 7,14 BCD

2 ab 4,29 a 1,43 bcd 10,00 B

The proportions difference is significant at the 0.05 level; values with same letter (row=uppercase, column=lowercase) based on Z test do not differ significantly

Fungal Diversity

DNA extraction, PCR amplification, DNA sequencing, and phylogenetic analysis of the GAPDH gene region Isolates were grown on potato dextrose (PD) broth for 7 days and mycelia were separated from the culture medium by filtration. Genomic DNA was extracted using a AxyPrep Multisource Genomic DNA Miniprep Kit (Axygen®) according to the manufacturer protocol. DNA concentrations were estimated visually in agarose gel by comparing band intensity with a 1 kb DNA ladder (Axygen). The partial sequence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene region of all strains was amplified (Templeton et al. 1992) as an initial measure to select representative isolates for a following multigene phylogenetic analysis. Polymerase chain reaction (PCR) was done to amplify this region using the primers-pair GDF1 and GDR1 and PCR conditions were used as described in Templeton et al. (1992). The PCR amplifications were performed in a 25 μl mixture containing 10.5 μl of PCR-grade water, 1 μl of DNA template, 1 μl of each primer (10 μM), and 12.5 μl of PCR Master Mix (2×) (Thermo Scientific, Waltham, USA). The PCR products were purified using the AxyPrep PCR Cleanup Kit (Axygen) following the manufacturer’s instructions. DNA sequencing was performed at the Sequencing Platform LABCEN/CCB in the Universidade Federal de Pernambuco, Recife, Brazil. Sequences were assembled using the Staden Package (Staden et al. 1998). An alignment comprising 97 sequences of the GAPDH gene region was generated with Clustal W implemented in MEGA v. 5 (Tamura et al. 2011) and manually adjusted to maximize sequence similarity. A distance tree using the Neighbor Joining (NJ) algorithm (Saitou and Nei 1987) was built in MEGA v. 5 to identify distinct haplotypes among the sequences. Sequences were compared with the NCBI sequence database using the BLAST algorithm as an approximate identification and to find reference strains to use in the phylogenetic analysis.

inference (BI). ML inference was carried out using PhyML v. 3.0 (Guindon et al. 2010). The analyses were performed using an input tree generated by BIONJ, and parameters of the HKY+I+G model of nucleotide substitution estimated with jModelTest. PhyML bootstrap trees (1000 replicates) were constructed using the same parameters as the individual ML tree. Bayesian phylogenetic estimates were inferred with MrBayes v. 3.2.1 (Ronquist et al. 2012) using the best-fit models of nucleotide substitution selected by jModelTest v. 0.1.1 (Posada 2008) for each loci with the Bayesian Information Criterion (BIC) (Table 2). The multiple nucleotide sequence alignments of all loci were concatenated and six simultaneous Markov chains were run for 106 generations with samples taken from the posterior every 1000 generations. Convergence of all parameters was checked using the Tracer program (Rambaut and Drummond 2007) and the first 25 % of generations were discarded as burn-in. Sequences of Colletotrichum ex-type strains from GenBank were included in the analyses (Table 1). Forty-five isolates were used in the first analysis including isolates of the C. gloeosporioides species complex. Some species recently described were not included in this analysis (C. fructivorum V. Doyle et al., C. melanocaulon V. Doyle et al., C. rhexiae Ellis & Everh. and C. temperatum V. Doyle et al.) (Doyle et al. 2013) due to an incomplete gene dataset. Colletotrichum horii strain ICMP 10492 was used as an outgroup. To elucidate the identity of one isolate as C. karstii and four isolates as C. cliviae, a second analysis using 36 isolates was carried out. Colletotrichum gloeosporioides strain CBS 953.97 was used as an outgroup in this analysis. Sequences generated in this study were deposited in GenBank (Table 1). Representative isolates of the Colletotrichum species obtained in this study were deposited in the Culture Collection of Phytopathogenic Fungi “Prof. Maria Menezes” (CMM) at the Universidade Federal Rural de Pernambuco, Recife, Pernambuco, Brazil.

Multilocus phylogeny Apn2/MAT IGS-based phylogenetic analysis Based on the GAPDH NJ tree, a subset of 22 isolates representing the range of genetic diversity (Table 1) were selected for further analysis using sequences from actin (ACT), β-tubulin (TUB2), calmodulin (CAL), chitin synthase (CHS-1) and GPDH genes and the rDNA ITS (ITS) region. PCR amplification was carried out with cycling parameters and primers as specified in Prihastuti et al. (2009) and Yang et al. (2009) and processed for DNA sequencing as described above. Independent gene trees were inferred under the Maximum Likelihood (ML) criterion and by Bayesian

The seventeen Colletotrichum isolates belonging to the C. gloeosporioides species complex were selected for Apn2/MAT IGS sequencing and analysis. PCR amplification was carried out with cycling parameters and primers as specified in Doyle et al. (2013). This analysis was carried out to identify species in this complex with special emphasis on species included in C. siamense sensu lato. Apn2/MAT IGS sequences of 42 isolates of the C. gloeosporioides species complex retrieved from NCBI-GenBank, and sequences of four isolates previously identified as C. dianesei Lima et al.

Fungal Diversity CMM3747 H10 CMM3737 CMM3765 CMM3791 CMM3733 CMM3776 CMM3792 CMM3766 CMM3749 CMM3786 CMM3757 CMM3773 CMM3788 CMM3771 CMM3795 CMM3750 CMM3760 CMM3775 CMM3743 CMM3754 CMM3738 CMM3739 CMM3745 CMM3751 CMM3759 H9 CMM3816 CMM3756 CMM3762 CMM3763 CMM3764 CMM3769 CMM3720 CMM3721 CMM3722 CMM3723 CMM3724 CMM3725 CMM3726 CMM3727 CMM3728 CMM3731 CMM3732 58 CMM3778 CMM3785 CMM3744 CMM3748 CMM3730 CMM3752 CMM3796 CMM3793 CMM3803 CMM3810 CMM3809 CMM3801 CMM3799 CMM3789 CMM3753 CMM3755 CMM3790 62 CMM3729 CMM3758 H8 99 CMM3761 CMM3734 CMM3736 CMM3798 CMM3802 CMM3800 CMM3805 CMM3807 CMM3813 CMM3815 CMM3772 CMM3781 64 CMM3784 H5 64 CMM3779 CMM3777 CMM3740 H6 62 CMM3814 CMM3787 86 CMM3794 H1 CMM3767 CMM3783 65 CMM3774 H2 96 CMM3780 CMM3812 CMM3735 63 CMM3770 CMM3768 H7 CMM3811 CMM3806 CMM3804 CMM3741 CMM3782 100 CMM3746 H3 CMM3808 CMM3742 CMM3797 H4

Fig. 1 A Neighbor Joining tree based on partial GAPDH gene sequences, depicting the terminal diversity of 97 Colletotrichum strains isolated as endophytes from Mangifera indica from Brazil. Terminal branches are labeled (H1—H10) according to GAPDH haplotypes. Isolates selected for the subsequent phylogenetic analyses are emphasized in red. The tree was rooted with the sequence from branch H4. The scale bar indicates the number of substitutions per nucleotide position

0.01

C. gloeosporioides sensu lato

C. cliviae C. karstii

Fungal Diversity

(Lima et al. 2013) generated in this study were included in this analysis (Table 3). A multiple sequence alignment of 63 taxa was used for phylogenetic reconstruction. The ML and BI analysis were carried out as described above. Colletotrichum horii strain ICMP 10492 was used as outgroup. Morphological studies of Colletotrichum species For studies of colony and conidial morphology, a 4-mmdiameter mycelial plug from the growing margin of a 5-dayold colony was placed in the centre of a 90-mm-diameter PDA plate, and four replicates of each isolate were incubated at 25 °C with continuous fluorescent light (24 h). Discs of filter paper were placed onto the culture media surface to induce the conidiomata development. Colony diameter was measured daily for 4 days to calculate the mycelial growth rate (mm day−1). Colony aspect and size, color of the conidial masses, as well as shape and size of conidia harvested from the cultures were recorded (Than et al. 2008a) after 7 days. The colony colour was evaluated using the mycological colour chart (Rayner 1970). Conidia were mounted in 100 % lactic acid and digital images recorded with a Samsung SDC415 camera (Samsung Co., Seoul, Korea) on an Olympus BX41 microscope (Olympus Co., Tokyo, Japan). The length and width of 30 conidia per isolate were measured with the Motic Image Plus v. 2.0 image analysis software (Motic Group Co., Beijing, China). For the new Colletotrichum species, appressoria were produced using a slide culture technique in which 10 mm2 blocks of PDA were placed in an empty Petri dish. The edge of the agar was inoculated with spores taken from a sporulating culture and a sterile cover slip was placed over the inoculated agar (Johnston and Jones 1997). Mean and standard errors of the conidial and appressorial measurements were calculated. One-way analysis of variance (ANOVA) was conducted to determine the significance of differences in growth rates of Colletotrichum species, and means were compared by Fisher’s least significant difference (LSD) test at the 5 % significance level using STATISTIX v. 9.0 (Analytical Software, Tallahassee, USA). Pathogenicity and virulence on fruits All isolates were tested for pathogenicity/virulence on Mangifera indica. The inoculation was carried out on Mango fruits (cv. Tommy Atkins) that were not treated with fungicides at stage three of maturation (Assis 2004). Mangos were washed in running water, surface disinfected in 70 % ethanol for 1 min and 1 % NaOCl for 5 min, then rinsed in sterile distilled water. The fruits were placed on plastic trays on top of four paper towels wetted with distilled water to increase humidity. Each fruit was placed on a sterilized Petri dish to avoid direct contact with water. Fruits were wounded at

two points by pricking the surface with four sterile pins to a depth of 3 mm. The fruits were inoculated using the colonized agar plug method since part of the isolates did not produce conidia in culture. A mycelial plug (5 mm in diameter) removed from the margin of a 5-day-old PDA culture was placed on the wound. A non-colonized agar plug was used as the control. The trays were enclosed in plastic bags and incubated at 25 °C in the dark. The plastic bags and paper towels were removed after 48 h and the fruits were kept at the same temperature. Isolate virulence was assessed by measuring lesion length 8 days after inoculation. Three replicate experiments were arranged in a completely randomized design with two replicates (fruits) per treatment (isolate). Differences in virulence caused by Colletotrichum species was determined by a one-way ANOVA and means were compared by LSD test at the 5 % significance level using STATISTIX. Prevalence of Colletotrichum species To determinate the prevalence of Colletotrichum species in plant tissues and sampling sites, the Isolation Rate (IR) was calculated for each species with the formula, IR%=(Cx/Ct)× 100, where Cx is the number of isolates of the same species, and Ct is the total number of isolates per plant tissue or sampling site. The overall IR was calculated using Ct value equal to the total number of isolates across plant tissues and sampling sites.

Results Isolation of endophytic Colletotrichum strains A total of 1715 samples comprising 300 stem, and young and mature leaf blades and veins, and 215 inflorescence pieces were evaluated for endophytes. At least one fungal taxon was isolated from 86.5 % of the samples. Among the colonized fragments, the genus Colletotrichum was present in 169 samples. The frequency of isolation of Colletotrichum strains was above 3 % at all sites, with sites I and III having significantly

Fig. 2 A maximum likelihood tree based on a multilocus dataset (ACT,„ TUB2, CAL, CHS-1, GAPDH and ITS) inferred using PhyML showing phylogenetic affinities of 17 Colletotrichum strains isolated as endophytes from Mangifera indica from Brazil with other members of C. gloeosporioides species complex (strains of the ‘Musae clade’ sensu Weir et al. 2012). Colletotrichum horii strain ICMP 10492 was designated as outgroup. Bootstrap support values above 50 % (left) and Bayesian posterior probability values >95 % (right) are shown at the nodes. Ex-type and ex-epitype strains are emphasised in bold, and isolates from this study are emphasized in red. The scale bar indicates the number of substitutions per nucleotide position

Fungal Diversity

CBS 124949 Theobroma Panama

100/99

CMM 3767 Mangifera Brazil 100/100 61/99

CMM 3787 Mangifera Brazil CMM3783 Mangifera Brazil

83/100

Colletotrichum tropicale

CMM3780 Mangifera Brazil ICMP 18672 Litchi Japan

77/100 83/ -

ICMP 17673 Aeschynomene USA

C. aeschynomenes

CMM 4085 Mangifera Brazil 100/100

83/100

C. dianesei

CMM 3779 Mangifera Brazil

83/100

CMM 3777 Mangifera Brazil

61/-

CMM 3784 Mangifera Brazil CBS 130417 Coffea Thailand ICMP 18642 Hymenocallis China 64/100

91/100

ICMP 19118 Jasminum Vietnam 99/100

CMM 3814 Mangifera Brazil CMM 3740 Mangifera Brazil

99/100

ICMP 18608 Persea Israel ICMP 18686 Pyrus Japan 100/100

C. siamense C. hymenocallidis

C. siamense sensu lato

93/100

CMM 4082 Mangifera Brazil CMM 4083 Mangifera Brazil CMM 4084 Mangifera Brazil

C. jasmini-sambac

Colletotrichum sp. C. aenigma

CBS 116870 Musa USA ICMP 17817 Musa Kenya

C. musae

68/99

ICMP 12071 Malus New Zealand

78/100

ICMP 18621 Persea New Zealand

C. alienum

CMM 3811 Mangifera Brazil CMM 3770 Mangifera Brazil

97/100 97/100

ICMP 17921 Ficus Germany CMM 3741 Mangifera Brazil

74/ -

99/100 88/99

CBS 130416 Coffea Thailand ICMP 18646 Tetragastris Panama 100/100

63/98

CBS 47096 Nuphar USA CBS 46996 Nuphar USA ICMP 19051 Salsola Hungary

ICMP 1778 Carica Australia

97/100

ICMP 18705 Coffea Figi

79/99

100/100

C. fructicola

C. nupharicola

C. salsolae C. queenslandicum

CMM 3793 Mangifera Brazil ICMP 18696 Mangifera Australia CBS 130418 Coffea Thailand CMM 3758 Mangifera Brazil

C. asianum

CMM 3747 Mangifera Brazil CMM 3736 Mangifera Brazil CMM 3738 Mangifera Brazil CBS 953.97 Citrus Italy ICMP 10492 Diospryos Japan 0.006

C. gloeosporioides C. horii

Fungal Diversity

higher and lower isolation frequencies than all other sites, respectively. The isolation frequency of Colletotrichum in plant organs differed significantly within the five sites. The highest isolation frequencies were recorded from mature leaf blades in Sites I, II and V, from young leaf blades in Site IV, and from young veins in Site III. The lowest isolation frequencies were from inflorescences in Site II, from stems in Site V, and inflorescences and stems in Site III (Table 4). The isolation frequencies at sites differed significantly. Sites I and III had the lowest and highest isolation frequencies, respectively. Phylogenetic analyses The initial analysis of the partial GAPDH sequences of the 97 isolates revealed 10 haplotypes (H1–H10) (Fig. 1). Based on similarity to sequences in the NCBI sequence database, eight belong to the C. gloeosporioides species complex, all of them within the “musae clade” (sensu Weir et al. 2012). Haplotype 3 (H3) and haplotype 4 (H4) had a high degree of similarity (99 %) with sequences of type species of C. cliviae and C. karstii, respectively. Twenty-two isolates representing all haplotypes were randomly selected and used in a multilocus analysis. The selected haplotypes in the C. gloeosporioides species complex were further analyzed using sequence data from six loci. The sequences were concatenated to form an aligned supermatrix of 2316 characters. The locus boundaries in the alignment were: ACT: 1–213, TUB2: 214–619, CAL: 620– 1221, CHS-1: 1222–1518, GAPDH: 1519–1768, ITS: 1769– 2316. The endophyte isolates from the present study belong to five clades. Five isolates clustered with C. asianum, three isolates with C. dianesei and four isolates with C. tropicale, with all clades well supported in both analyses. Three isolates belong to the C. fructicola clade with a posterior probability <95 %, but well supported in ML analysis (99 %). One clade of two isolates with support >99 % in both analysis did not cluster with any known species, indicating that this clade might represent a new species (Fig. 2). Sequences of the Apn2/MAT IGS region were used to identify species within C. siamense sensu lato with 63 nucleotide sequences representing the diversity within the complex. The final alignment comprised 746 characters. The ML tree with the best score is shown in Fig. 3. The five clades observed in the multilocus analysis were recovered in the Apn2/MAT IGS analysis, all of them well supported in both BI and ML analyses. The clade containing the new putative Colletotrichum species was identified as a distinct novel lineage within C. siamense sensu lato. All isolates that clustered with C. dianesei in the multilocus analysis (including the extype) also clustered with the ex-type strain of C. melanocaulon Doyle et al. (2013) in the Apn2/MAT IGS

analysis. We propose to synonymize both species under C. dianesei since this species was described earlier. The second analysis included sequences from the C. boninense species complex and C. cliviae. The alignment comprised 2124 bp and the gene boundaries were as follows: ACT: 1–250, TUB2: 251–693, CAL: 694–1118, CHS-1: 1119–1335, GAPDH: 1336–1594, ITS: 1595–2124. One isolate clustered with the ex-type strain of C. karstii and four isolates grouped with the ex-type strain of C. cliviae. Both clades were inferred with support ≥99 % in both analyses (Fig. 4). Diversity analyses of the 97 isolates of Colletotrichum spp. show the following haplotype distribution within the species identified in the phylogenetic analyses: 72 isolates of C. asianum with haplotypes H8 (1), H9 (21) and H10 (50); 4 isolates of C. cliviae, all with haplotype H3; 6 isolates of C. fructicola, all with haplotype H7; 1 C. karstii isolate with haplotype H4; 4 isolates of C. dianesei 4 with haplotype H5; 6 isolates of C. tropicale with haplotypes H1 (3) and H2 (3); and 2 isolates of Colletotrichum sp. with haplotype H6. Morphological and cultural characterization Differences in colony characteristics among the Colletotrichum isolates allowed them to be differentiated into nine morphological types (Fig. 5). The species of Colletotrichum found in this study presented differences in conidial size and growth rates congruent with the data of the previously described species (Table 5). Taxonomy Colletotrichum endomangiferae W.A.S. Vieira, M.P.S. Camara & S.J. Michereff sp. nov. (Fig. 6a–f) MycoBank: MB 808814 Etymology: based on the name of the host plant (Mangifera indica) where this species was isolated from as an endophyte. Description: colonies on PDA at first white, becoming dark grey, reverse dark grey, growth rate at 25 ºC 13.89– 16.57 mm day−1 (x = 15.50±1.16, n=3). Aerial mycelium grey, felt-like, without conidial masses. Sclerotia absent.

Fig. 3 A maximum likelihood tree based on Apn2/MAT IGS dataset„ inferred using PhyML showing phylogenetic affinities of 17 Colletotrichum strains isolated as endophye from Mangifera indica from Brazil with other members of C. gloeosporioides species complex (strains of the ‘Musae clade’sensu Weir et al. 2012). Colletotrichum horii strain ICMP 10492 was designated as outgroup. Bootstrap support values above 50 % (left) and Bayesian posterior probability values >95 % (right) are shown at the nodes. Ex-type and ex-epitype strains are emphasised in bold, and isolates from this study are emphasized in red. The scale bar indicates the number of substitutions per nucleotide position

Fungal Diversity

C. siamense sensu lato

CMM 4083 Mangifera Brazil CMM 4082 Mangifera Brazil GM 172 Mangifera India (syn. C. melanocaulon) CMM 3777 Mangifera Brazil CMM 3779 Mangifera Brazil Colletotrichum dianesei CMM 3784 Mangifera Brazil (syn. C. melanocaulon) CMM 4085 Mangifera Brazil Coll 131 Vaccinium USA (syn. C. melanocaulon) 86/98 GM 057 Mangifera India (syn. C. melanocaulon) CMM 4084 Mangifera Brazil 100/100 ICMP 18578 Coffea Thailand LC 0035 Coffea Thailand C. siamense BPDI 2 Coffea Thailand LC 0034 Coffea Thailand 85/- 97/100 100/100 CMM 3814* Mangifera Brazil C. endomangiferae sp. nov. CMM 3740 Mangifera Brazil ICMP 19118Jasminum Vietnam 99/100 C. jasmini-sambac LC 0921 Jasminum Vietnam 92/100 ICMP 18642 Hymenocallis China C. hymenocallidis LC 0043 Hymenocallis China 100/100 C. salsolae ICMP 19051 Salsola Hungary C. queenslandicum ICMP 1778 Carica Australia CMM 3767 Mangifera Brazil 5101 Theobroma Panama E 406 Pentagonia Panama 7423 Theobroma Panama C. tropicale CMM 3787 Mangifera Brazil CMM3783 Mangifera Brazil 100/100 CMM3780 Mangifera Brazil Coll 918 Terpsichore Puertorico ICMP 18653 Theobroma Panama GM 595 Mangifera India 65/98 GM 414 Mangifera India GM 021 Mangifera India

98/100

99/100 65/99 100/100

CMM 3793 Mangifera Brazil CMM 3736 Mangifera Brazil CMM 3747 Mangifera Brazil CMM 3758 Mangifera Brazil CMM 3738 Mangifera Brazil

LC 0036 Coffea Thailand MFLU 090233 Coffea Thailand LC 0037 Coffea Thailand CBS 130418 Coffea Thailand 94/100 LC 0963 Musa Thailand 100/100 LC 0084 Musa USA ICMP 19119 Musa USA 67/100 LC 0872 Musa Thailand 67/LC 0962 Musa Thailand -/100

1087 Theobroma Panama ICMP 18646 Tetragastris Panama LC 0032 Coffea Thailand 80/CBS 130416 Coffea Thailand 60/100 GM 567 Mangifera India Coll 1126 Vaccinium USA CMM 3741 Mangifera Brazil 100/100 CMM 3770 Mangifera Brazil 68/99 CMM 3811 Mangifera Brazil 75/99 CBS 472 Nymphaea USA CBS 46996 Nuphar USA Coll 922 Nuphar USA 88/100 Coll 878 Nuphar USA CBS 953.97 Citrus Italy ICMP 10492 Diospryos Japan

C. asianum

C. musae

61/99

100/100

0.02

C. fructicola

C. nupharicola C. gloeosporioides C. horii

Fungal Diversity Fig. 4 A maximum likelihood tree based on a multilocus dataset (ACT, TUB2, CAL, CHS-1, GAPDH and ITS) inferred using PhyML showing phylogenetic affinities of 5 Colletotrichum strains isolated as endophye from Mangifera indica from Brazil with other members of C. boninense species complex and C. cliviae strains. Colletotrichum gloeosporioides strain CBS 953.97 was designated as outgroup. Bootstrap support values above 50 % (left) and Bayesian posterior probability values >95 % (right) are shown at the nodes. Ex-type and ex-epitype strains are emphasised in bold, and isolates from this study are emphasized in red. The scale bar indicates the number of substitutions per nucleotide position

CBS 128500 Annona New Zealand BRIP 29085a Dyospyros Australia 95/99 CMM 3797 Mangifera Brazil 100/100 CBS 175.678 Phyllanthus India 99/CBS 129826 Hevea Colombia 100/100 CBS 378.94 Dracaena Italy 82/CBS 379.94 Dracaena Italy 100/100 CBS 128505 Capsicum New Zealand CBS 130240 Citrus New Zealand 89/100 CBS 123757 Cymbidium Japan 74/IMI 347923 Cymbidium Australia 99/100 100/100 CBS 129828 Oncidium Germany 99/- CBS 130242 Oncidium Germany 100/100 CBS 128547 Camellia New Zealand 91/CBS 123755 Crinum Japan 100/100 CBS 128544 Solanum New Zealand CBS 102667 Passiflora New Zealand 99/100 CBS 129817 Passiflora Colombia 100/100 65/96 CBS 129818 Passiflora Colombia CBS 128527 Brachyglottis New Zealand CBS 101059 Brassica New Zealand 100/100 100/100 CBS 128501 Passiflora Brasil 83/96 CBS 128528 Passiflora Brasil CBS 128525 Parsonia New Zealand 100/100 CBS 241.78 Hippeastrum Netherlands CBS 125376 Hippeastrum China 100/100 99/100

C. karstii

99/100

98/100

C. phyllanthi C. annellatum C. petchii C. novae-zelandiae C. cymbidiicola C. oncidii C. boninense C. torulosum C. colombiense C. beeveri C. brassicicola C. brasiliense C. parsoniae C. hippeastri

CBS 128504 Citrus New Zealand CBS 128503 Solanum New Zealand CBS 1302418 Dacrycarpus New Zealand 95/- CSSK4 Clivia China CSSS1 Clivia China 100/100 100/100

99/100

99/100 96/100

CBS 953.97 Citrus Italy

C. constrictum C. dacrycarpi

CMM 3742 Mangifera Brazil CMM 3808 Mangifera Brazil CMM 3782 Mangifera Brazil CMM 3746 Mangifera Brazil

C. cliviae

C. gloeosporioides

0.04

Acervuli in filter paper, dark brown, present in culture. Setae in filter paper, abundant, long, dark brown, smooth-walled, four or more septa, base conical, tip±acute. Conidiophores hyaline, smooth-walled to verruculose, aseptate, unbranched. Conidiogenous cells hyaline, smooth-walled, cylindrical to ampulliform, often extending to form new conidiogenous loci. Conidia 14.5–19.9×4.5–6.20 μm (x = 16.44±1.16×5.17± 0.50, n=30), common in mycelium, one-celled, smoothwalled, hyaline, cylindrical with rounded ends, sometimes oblong, contents appearing granular. Appressoria in slide cultures, single or in groups of 2, medium to dark brown, smooth-walled, cylindrical, sometimes clavate with undulate margin, 7.1–9.8×5.8–7.7 μm (x = 8.44± 0.61×6.66±0.57, n=30). Sexual morph: not produced in culture. Holotype: BRAZIL, Pernambuco, São João, endophyte isolated from young leaf blade of Mangifera indica, 5 June 2011, Coll. W. A. S. Vieira (holotype living culture CMM 3814; ex-type in URM 7196; isotype in MFLU and ex-type culture in MFLUCC).

Known distribution: Pernambuco, Brazil. Additional specimens examined: Brazil, Pernambuco, Aldeia, endophyte on Mangifera indica young leaf blade, 18 February 2011, Coll. W.A.S. Vieira, CMM3740. Note: this species is closely related to C. hymenocallidis and C. jasmine-sambac. All three species are nested within C. siamense sensu lato. Colletotrichum endomangiferae has longer conidia (x = 16.44 μm) when compared with C. hymenocallidis (x = 8.5 μm) (Yang et al. 2009) and C. jasmine-sambac (x = 14 μm) (Wikee et al. 2011). Colletotrichum dianesei, a species reported to cause anthracnose in mango (Lima et al. 2013), is also included in the C. siamense sensu lato clade. The conidial length and growth rate (x = 12.06 μm; x = 8.01 mm day−1, respectively) of Colletotrichum dianesei is distinct from C. endomangiferae (x = 16.44 μm; 12.97 mm day − 1 ). Another species, C. mangiferae Kelkar (Kelkar and Rao 1963), reported isolated from mango leaves, but no DNA sequence data are available for this species. Conidial dimensions of C. mangiferae (10–13.2×3.3–5 μm) are smaller than C. endomangiferae

Fungal Diversity

1.a

1.b

2.a

2.b

3.a

3.b

4.a

4.b

5.a

5.b

6.a

6.b

7.a

7.b

8.a

8.b

9.a

9.b

Fig. 5 Morphotypes (1 to 9) of isolates of Colletotrichum endophytes from mango. Colony characteristics: Plates in A aerial view, reverse view in B

(14.5–19.9×4.5–6.20 μm). Colletotrichum endomangiferae is distinguished using ACT, Apn2/MAT IGS, TUB2, CAL and GAPDH sequences. Based on the Apn2/MAT IGS sequences, C. endomangiferae differs from C. hymenocallidis in

10 nucleotide positions, and from C. jasmine-sambac in 7 nucleotide positions. Colletotrichum dianesei N.B. Lima, M.P.S. Câmara & S.J. Michereff., Fungal Divers 61:75–88. 2013.

Table 5 Summary of phenotypic data of Colletotrichum isolates Colletotrichum species

Conidia

Growth rate (mm day−1)

Length (μm)

Width (μm)

Shape

C. asianum

14.84±1.79 (12.31–18.56)

5.06±0.43 (4.33–6.33)

Cylindrical 12.97±1.03d (4.24–10.92)

C. cliviae

17.01±1.12 (15.94–18.18)

5.19±0.18 (5.06–5.40)

Cylindrical 9.22±3.54bc (5.64–12.82)

13.96±0.96 (13.12–15.01) C. endomangiferae 15.22±1.72 (14.01–16.44)

4.67±0.26 (4.43–4.95) 4.82±0.50 (4.46–5.17)

Cylindrical 7.47±2.20 cd (4.63–10.01) Cylindrical 12.97±3.58a (10.44–15.50)

C. fructicola

13.70±1.98 (9.97–15.08)

5.23±0.46 (4.52–5.69)

Cylindrical 10.63±2.48b (6.95–13.14)

C. karstii

14.20±0.91 (12.30–15.80)

5.49±0.27 (4.90–5.90)

Cylindrical 9.69±1.20b (8.34–10.66)

C. tropicale

12.85±0.34 (12.41–13.45)

4.59±0.45 (4.14–5.43)

Cylindrical 9.94±0.91b (8.71–11.15)

C. dianesei

a

Morphotypes are presented in Fig. 4

Colony characteristics

Morphotypesa

Tufted, sometimes cottony, aerial mycelia white to mouse grey, reverse olivaceous to dark mouse grey Cottony, aerial mycelia pale mouse grey, reverse pale purplish grey to dark mouse grey at center Cottony, aerial mycelia salmon and reverse buff Cottony or felt-like, aerial mycelia white to violaceous grey, reverse olivaceous to dark mouse grey Felt-like, aerial mycelia pale purplish grey, reverse ochreous, with abundant orange sporulation masses Cottony, aerial mycelia pale purplish grey, reverse greysh sepia to dark mouse grey at center, and ochreous sporulation mass Cottony or felt-like, aerial mycelia pale mouse grey to fuscuous black, reverse pale purplish grey dark mouse grey, sometimes with orange sporulation mass

1; 2

6

3 2; 4

7

5

6; 8; 9

Fungal Diversity

= Colletotrichum melanocaulon V. Doyle, P.V. Oudem. & S.A. Rehner. PLoS One 8:e62394. 2013. Colletotrichum melanocaulon was described by Doyle et al. (2013) using sequence data of Apn2, Apn2/MAT IGS, TUB-2 and ITS regions (published: 6 May 2013) and is here placed in synonymy with C. dianesei (published online: 4 May 2013). Lima et al. (2013) described C. dianesei using a multi-locus phylogenetic approach with the ACT, TUB-2, CAL, GPDH, GS and ITS genes. Since the molecular data of C. dianesei were not available in a public database at the time of

submission, and different genes were used in the phylogenetic analysis, Doyle et al. (2013) introduced C. melanocaulon as a new species. We sequenced the Apn2/MAT IGS region for some isolates described as C. dianesei (including the ex-type) and included them in our analysis. The phylogenetic tree generated showed that the ex-type of C. melanocaulon nested with the ex-type of C. dianesei in a well-supported clade. Morphological characteristics such as conidial length and width, and mycelial growth rate are similar for both species.

Fig. 6 Colletotrichum endomangiferae (from holotype). Upper a and reverse b sides of cultures on PDA 7 days after inoculation. c, Conidiophores. d, Setae; e, Conidiogenous cells. f, Appressoria. g, Conidia. Scale bars =10 μm

a

c

f

b

d

e

g

Fungal Diversity 25

Pathogenicity and virulence in fruits

a

Lesion lenght (mm)

20

With the inoculation methods used, all isolates tested were pathogenic, resulting in fruit infection 8 days following inoculation. Fruit symptoms in all species were similar, with sunken, prominent, dark brown to black lesions. The negative control did not develop symptoms and differed statistically from the all other treatments. Colletotrichum karstii was significantly (P≤0.05) less virunlent than all other species. Colletotrichum asianum and C. endomangiferae produced the longest lesions (Fig. 7)

a

ab ab

15

b b

10 c

5

d

0

Prevalence of Colletotrichum species Colletotrichum asianum was the most prevalent species (74 %). The isolation rate of the other species varied from 8 % to 1 % (Fig. 8). Five species were isolated from each of Sites II, III and V and two species from Sites I and IV. Colletotrichum asianum was most prevalent at all sites, with the highest isolation rate (94 %) at Site IV and the lowest (47 %) at Site II (Fig. 9). Colletotrichum asianum was the most abundant species in all tissues evaluated, with an isolation rate above 65 %. The isolation rate of C. asianum in inflorescences and stems was 100 %. This data reveals low species diversity in this plant tissue. In contrast, five different Colletotrichum species were isolated from the young vein and mature leaf blade samples. Colletotrichum karstii was restricted to mature leaf blades and C. endomangiferae was restricted to young leaf blades. None of the other species were restricted to one specific tissue (Fig. 10).

Discussion This study characterizes endophytic Colletotrichum species associated with mango plants in Brazil. Endophytic Colletotrichum species have previously been isolated with a relatively high frequency from various tropical and temperate fruit trees and forests, and from medicinal plants (Cannon and Simmons 2002; Guo et al. 2000; Hanada et al. 2010; Hata et al. 2002; Huang et al. 2008; Kumar and Hyde 2004; Lu et al. 2004; Osono 2008; Photita et al. 2001; Prihastuti et al. 2009; Rojas et al. 2010). In this study Colletotrichum species were present with high IF values for foliar tissues (leaf blade and vein) at all sites. In contrast, the IF values of this genus were very low from stems and inflorescences. The comparison of IF values between different studies is problematic, due to differences in isolation protocols, particularly in the size of plant fragments plated (Cannon and Simmons, 2002), surface sterilization method employed, and the culture media used. This was observed during a

Colletotrichum species

Fig. 7 Mean lesion lengths (mm) caused by Colletotrichum species endophytes in mango, 8 days after inoculation with agar plug onto wounded fruits of Tommy Atkins cultivar. Fruits inoculated with a noncolonized agar plug was used as the control. Bars above columns are the standard error of the mean. Columns with same letter do not differ significantly according to Fisher’s LSD test (P≤0.05)

preliminary experiment conducted to evaluate different sterilization methods and culture media for isolation of endophytic Colletotrichum from mango tissues. In this assay, a high inhibition of Colletotrichum growth was observed when lignocellulose agar medium - LCA (Miura and Kudo 1970) and PDA media amended with bengal rose (30 mg l−1) were used. A high level of inhibition of endophytic fungi was also observed when the sterilization time in sodium hypochlorite exceeded 2 min (unpublished data). Similar to other recent systematic studies of Colletotrichum (Cai et al. 2009; Damm et al. 2009, 2012a, b; Rojas et al. 2010; Liu et al. 2011; Weir et al. 2012), the use of multilocus phylogenetics in conjunction with recognizable phenotypic characters was useful for identifying the majority of species associated with mango. However, the Apn2/MAT IGS region alone provided better phylogenetic resolution than the sixgene concatenated dataset to identify species in the C. gloeosporioides species complex as suggested by Silva et al. (2012), Doyle et al. (2013) and Sharma et al. (2013). Their result show the phylogenetic power of the Apn2/MAT IGS sequences in resolving species, and shows this marker as

4.1

6,2%

4,1%

C. asianum 1,0%

C. clivae 2.1

C. endomangiferae

4.1

C. fructicola 74,2%

C. karstii C. dianesei C. tropicale

Fig. 8 Overall Isolation Rate (IR%) of Colletotrichum species isolated as endophytes from mango

Fungal Diversity Fig. 9 Isolation Rate (IR%) of Colletotrichum species isolated as endophytes from mango from each sampled areas

Site III

Site II

Site I

6% 6%

18%

22%

47%

24%

78%

3% 3%

81%

6% 6%

Site V

Site IV 6%

5%

5%

19% 67%

94% 5%

C. asianum C. fructicola C. tropicale

C. endomangiferae C. melanocaulon

high level of fixed polymorphism among species in the C. gloeosporioides species complex (Silva et al. 2012), and a low level of intra-specific variation found in our analysis. In

a strong candidate for barcoding at least within the C. gloeosporioides species complex. The best phylogenetic resolution in the Apn2/MAT IGS analysis is explainable by a

Fig. 10 Isolation Rate (IR%) of Colletotrichum species isolated as endophytes from mango from each plant tissues

C. cliviae C. karstii

Inflorescense

Stem

Young leaf blade 10% 10%

100%

100%

Young vein

18% 65%

7%

9%

7% 11%

70%

Old vein

Old leaf blade 4%

12%

10%

5% 71%

9% 77%

6%

C. asianum C. fructicola C. tropicale

C. cliviae C. karstii

C. endomangiferae C. dianesei

Fungal Diversity

contrast with the Apn2/MAT IGS tree, the multilocus tree shows more variability within the mains clades. Based on Apn2/MAT IGS phylogenetic analysis, we identified two distinct lineages associated with endophytes of mango within the C. siamense species complex, including C. dianesei and the new species C. endomangiferae. C. dianesei was described by Lima et al. (2013) but was not published at the time the manuscript describing C. melanocaulon was submitted and gene sequences were not available in a public database. Furthermore, different genes were used to describe these species. In the present study, we were able to compare Apn2/MAT IGS sequences of both species, and the analysis showed that these two species are synonyms. This situation shows the necessity to define which genes should be used to establish species of Colletotrichum (Sharma et al., 2013, Doyle et al., 2013). Sequence diversity at the Apn2/MAT IGS locus provides better insight into the diversity of terminal clades than the other loci that have traditionally been used in studies of Colletotrichum diversity, as observed in recent studies (Rojas et al., 2010; Silva et al. 2012; Doyle et al. 2013; Sharma et al., 2013). Diversity can be observed in the minor clades within the species with a broad host range and/or broad geographic distribution, as with C. asianum and C. fructicola. In our study, the C. asianum clade contained three subclades: one subclade containing Brazilian strains endophytic in M. indica; a second subclade with Indian strains from M. indica; and another with Thai strains from Coffea sp. (including the ex-type). As observed by Silva et al. (2012), the Apn2/MAT IGS region is a promising marker for species distribution studies in some species of the C. gloeosporioides complex. All Colletotrichum species isolated in this study were pathogenic to mango fruits, and differed significantly in virulence (P<0.05). Despite this difference, this result may not accurately reflect the true virulence potential. Endophytic Colletotrichum isolates may be opportunistic pathogens and further research is needed to determine the pathogenicity according to natural infections rather than artificial inoculations (Prihastuti et al. 2009). Based on the various definitions of the term “endophyte”, the genus Colletotrichum may have an endophytic lifestyle (true endophytes being those in which the colonization never results in visible disease symptoms; Mostert et al. 2000) or as a part of the life cycle (endophytism as an infection strategy; Wilson 1995). In this study there was no obvious correlation in the distribution of Colletotrichum species across different sites or plant tissues. The most relevant result of this study was that C. asianum was the most prevalent species in all sampled areas and plant tissues, showing that the species is not restricted to a specific area or plant tissue. Since the number of isolates of other Colletotrichum species was small, it is not possible to accurately infer their geographic distribution and tissue specificity.

The prevalence of Colletotrichum species in our study can be compared with the results obtained by Lima et al. (2013) who found five Colletotrichum species associated with mango anthracnose in northeastern Brazil. Colletotrichum dianesei was the most frequently isolated species with 51.1 % all the isolates, followed by C. asianum with 27.7 %. In the present study, 4.1 % of Colletotrichum endophyte isolates were C. dianesei while 74.2 % of all endophyte isolates were C. asianum. The differences in the frequencies of these two species might be related to the fact that the isolates in Lima et al. (2013) were obtained from tissues showing typical anthracnose symptoms. In addition, their isolates were collected in commercial fruit growing areas with regular applications of agrochemicals. The majority of C. asianum isolates wer found in orchards without fungicide applications, while C. dianesei was restricted to orchards that had been sprayed with methyl benzimidazole carbamates (MBC), demethylation inhibitor (DMI) or other fungicides. Colletotrichum asianum was originally described as a species associated with coffee berries in Thailand. Epiphytic and endophytic strains were isolated both from apparently healthy and symptomatic berries (Prihastuti et al. 2009). This species was reported causing anthracnose in Mangifera indica in Australia, Colombia, Japan, Panama, the Philippines (Weir et al. 2012), and recently in Brazil (Lima et al. 2013). Since C. asianum was isolated as an endophyte in high frequency and it was also associated with mango fruits showing typical anthracnose symptoms, it is possible that C. asianum is an opportunistic or facultative pathogen. Colletotrchum cliviae was reported causing anthracnose of Clivia miniata in China (Yang et al. 2009). This is the first report of C. cliviae associated with another host outside of Asia. Colletotrichum dianesei was described causing anthracnose in mango fruits (Lima et al. 2013). This species was also described from North America, as C. melanocaulon, causing stem canker in Vaccinium macrocarpon (Doyle et al. 2013). Due to its low occurrence as an endophyte, we speculate that endophytism is not a common survival strategy in the life cycle of this species. Colletotrichum fructicola was originally reported causing coffee berry disease in Thailand (Prihastuti et al. 2009) and was recently reported as one of the species causing mango fruit anthracnose in Brazil (Lima et al. 2013). It was also found as a leaf endophyte in several plant species in Central America (syn. C. ignotum, Rojas et al. 2010). Colletotrichum fructicola has a broad geographical and host distribution. The species is presently known from Coffea arabica (Thailand), Dioscorea (Nigeria), Ficus (Germany), Fragaria×ananassa and Malus domestica (USA), Limonium (Israel), Malus domestica (Brazil), Persea americana (Australia), Pyrus pyrifolia (Japan), Theobroma and Tetragastris (Panama) (Rojas et al. 2010), Citrus (Peng et al. 2012; Huang et al. 2013), Vitis

Fungal Diversity

(China) (Peng et al. 2013), M. indica (Brazil) (Lima et al. 2013) and Vaccinium macrocarpon, V. corymbosum, and Rhexia virginica (USA) (Doyle et al., 2013).. Recently, it was reported as an endophyte of tropical grasses (Manamgoda et al. 2013). Colletotrichum karstii was reported from mango in Australia (Damm et al. 2012a), and recently in Brazil (Lima et al. 2013). In Brazil, it was also reported causing anthracnose on Passiflora edulis fruits (Damm et al. 2012a). This species was isolated as an endophyte from Musa acuminata in Thailand (Photita et al. 2005) and was also reported in China and the United States on several orchids (Yang et al. 2011; Jadrane et al. 2012) and from Citrus leaves (Peng et al. 2012). Recently, Tao et al. (2013) reported the species as an endophyte of Bletilla ochracea (Orchidaceae). Colletotrichum karstii occurs on many host plants and is the most common and geographically diverse species in the C. boninense complex (Damm et al. 2012a). Colletotrichum tropicale was described by Rojas et al. (2010) and reported in association with Theobroma cacao, Annona muricata, Persea americana, Trichilia tuberculata, Tetragastris panamensis, Pentagonia macrophylla, Virola surinamensis, Cordia olliodora and Zamia obliqua in Panamá. Recently it was reported from Terpsichore taxifolia from Puerto Rico (Doyle et al., 2013),causing mango anthracnose in Brazil (Lima et al. 2013), and isolated as endophyte from the tropical grass Pennisetum purpureum (Manamgoda et al. 2013). The five species reported as causing anthracnose of mango in Brazil were also isolated as endophytes. This indicates that endophytism plays an important role in the life cycle of Colletotrichum species. This hypothesis is corroborated by Delaye et al. (2013), who analyzed 5.8S rRNA sequence data to elucidate the phylogenetic relationships among endophytes, biotrophic, and necrotrophic pathogens. They concluded that fungi could switch between an endophytic and a necrotrophic lifestyle at the evolutionary and even the ecological timescale. The literature indicates both lifestyles occur in many plant pathogenic genera of fungi: Alternaria (Huang et al. 2009; Park et al. 2012; Yu et al. 2011), Colletotrichum (Photita et al. 2005; Promputtha et al. 2007; Than et al. 2008a, 2008b; Yan et al. 2011; Choi et al. 2012; Glenn and Bodri 2012; Delaye et al. 2013), Diaporthe (Gomes et al. 2013), Diplodia (Álvarez-Loayza et al. 2011), Exserohilum (Lin et al. 2011; Loro et al. 2012), Lasiodiplodia (Sakalidis et al. 2011) and Phyllosticta (Glienke et al. 2011; Wang et al. 2012; Wikee et al. 2013). Although the species reported as causing anthracnose in mango fruits from northeastern Brazil are also found as endophytes in this region, particular attention should be given to the management of C. asianum and C. dianesei. These two species are associated with mango anthracnose and are the most virulent (Lima et al. 2013). Colletotrichum asianum

is a relatively common pathogen and endophyte, and is widely distributed worldwide on several hosts (Weir et al. 2012, Lima et al. 2013). Colletotrichum dianesei is a prevalent species causing mango anthracnose in Brazil (Lima et al. 2013). Since these two species are the most virulent on mango fruit in Brazil, it would be appropriate to develop disease management strategies that can control both species. Acknowledgments We are grateful to Sequencing PlatformLABCEN/ CCB in the Universidade Federal de Pernambuco for use its facilities. This work was financed by Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuc (FACEPE) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). MPS Câmara and SJ Michereff also acknowledge the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) research fellowship. KD Hyde thanks to NRCT of Thailand – Colletotrichum 54201020003 for support.

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