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  • Open Access

Biodiversity of Lecanosticta pine-needle blight pathogens suggests a Mesoamerican Centre of origin

IMA Fungus20191:2

https://doi.org/10.1186/s43008-019-0004-8

  • Received: 26 March 2019
  • Accepted: 3 April 2019
  • Published:

Abstract

Lecanosticta acicola causes the disease known as brown spot needle blight (BSNB), on Pinus species. The pathogen is thought to have a Central American centre of origin. This was based on the morphological variation between isolates believed to represent L. acicola from native Pinus spp. Two species of Lecanosticta, L. brevispora and L. guatemalensis, have recently been described from Mexico and Guatemala respectively based on morphology and sequence-derived phylogenetic inference. However, the putative native pathogen, L. acicola, was not found in those areas. In this study, the species diversity of a large collection of Lecanosticta isolates from Central America was considered. Phylogenetic analyses of the BT1, ITS, MS204, RPB2 and TEF1 gene regions revealed six species of Lecanosticta, four of which represented undescribed taxa. These are described here as Lecanosticta jani sp. nov. from Guatemala and Nicaragua, L. pharomachri sp. nov. from Guatemala and Honduras, L. tecunumanii sp. nov. from Guatemala and L. variabilis sp. nov. from Guatemala, Honduras, and Mexico. New host and country records were also found for the previously described L. brevispora and L. guatemalensis. Lecanosticta acicola was not found in any of the samples from Central America, and we hypothesize that it could be a northern hemisphere taxon. The high species diversity of Lecanosticta found in Mesoamerica suggests that this is a centre of diversity for the genus.

Keywords

  • Brown spot needle blight
  • Lecanosticta
  • Mesoamerica
  • Pinus pathogens
  • phylogeny

INTRODUCTION

Brown spot needle blight (BSNB) or Lecanosticta needle blight is an important needle disease on Pinus species. The disease is characterised by brown spots on necrotic yellow lesions at the points of infection and die-back of the needles from the apex, which often leads to premature defoliation (Ivory 1987). BSNB is caused by the fungal pathogen, Lecanosticta acicola (Siggers 1944). The fungus is a well-known pathogen in the USA and has also been recorded in Central America, Colombia, Europe as well as Asian countries including China, Japan and Korea. Lecanosticta acicola is regarded as an A2 quarantine pathogen in Europe and Colombia where it is present as well as an A1 quarantine pathogen in the rest of South America (COSAVE), Africa (IASPC) and the Eurasian Economic Union countries where it has yet to be recorded (https://gd.eppo.int/taxon/SCIRAC/categorization). Despite its quarantine status, L. acicola has been discovered in various new locations and on new hosts in Europe during the past decade (Jankovsky et al. 2009; Markovskaja et al. 2011; Anonymous 2012; Hintsteiner et al. 2012; Adamson et al. 2015; Janoušek et al. 2016; Ortíz de Urbina et al. 2017; Mullett et al. 2018; Cleary et al. 2019; Sadiković et al. 2019).

Siggers (1944) and Evans (1984) summarised the taxonomic and nomenclatural history of Lecanosticta acicola, which was complicated by the former system which allowed asexual and sexual morphs of the same species of fungi to be given separate scientific names (Kais 1971; Evans 1984). From 1972 to 2012, the name Mycosphaerella dearnessii was widely used for the causal agent of BSNB. It was, however, recently recognised that Mycosphaerella is polyphyletic and should be strictly used for fungi in Ramularia (Crous et al. 2007; Crous 2009). Following the One Fungus One Name (1F1N) convention (Hawksworth et al. 2011), the nomenclatural rules were changed in July 2011, and included in subsequent editions of the International Code of Nomenclature for algae, fungi, and plants (ICN) (Turland et al. 2018). Lecanosticta was taken up as the appropriate name, with L. acicola as type species of the genus (Crous et al. 2009a; Quaedvlieg et al. 2012).

Five species of Lecanosticta have been described: Lecanosticta acicola, L. brevispora, L. guatemalensis (Quaedvlieg et al. 2012), L. gloeospora (Evans 1984), and L. longispora (Marmolejo 2000). Lecanosticta acicola remains the best-known species and records suggest that it has a wide distribution in North and South America, Europe, and Asia (https://gd.eppo.int/taxon/SCIRAC/distribution). The remaining four species are known only from Mesoamerica (Evans 1984; Marmolejo 2000; Quaedvlieg et al. 2012). Lecanosticta gloeospora was described, based only on morphology, from disease symptoms on Pinus pseudostrobus from Iturbide, Nuevo León, Mexico (Evans 1984). It was subsequently reported on P. pseudostrobus collected in 1990 in Mexico (Marmolejo 2000). Lecanosticta longispora was originally described from Pinus culminicola in Nuevo León, Mexico, based on morphology (Marmolejo 2000). Quaedvlieg et al. (2012) redescribed and epitipified L. longispora based on DNA sequence and morphological data. Quaedvlieg et al. (2012) delineated Mycosphaerella species of quarantine significance in Europe, including isolates believed to be L. acicola from Central America. Those isolates were distinct taxa and were named L. brevispora and L. guatemalensis from Pinus sp. in Mexico and from P. oocarpa in Guatemala.

Names assigned to Lecanosticta species prior to 2012 were based only on morphological characteristics. Cryptic diversity in Lecanosticta is illustrated by L. guatemalensis (IMI281598), which was initially identified as L. acicola (Evans 1984; Quaedvlieg et al. 2012). Identifications made utilising only morphological characteristics should clearly be re-evaluated using DNA sequence data and phylogenetic inference.

Central America is believed to be the centre of origin of L. acicola. This hypothesis was first proposed by Evans (1984), when the fungus was isolated from native trees in pristine forests. In a recent phylogenetic study, high levels of diversity were found in the Translation Elongation 1-α gene region (TEF1) of isolates from Mexico and Guatemala (Janoušek et al. 2016). Furthermore, Central American isolates did not group in the same clade as isolates from Asia, Europe, and North America. Likewise, Janoušek et al. (2016) reported poor amplification of microsatellite regions that had been developed for L. acicola suggesting that the isolates could represent cryptic species. The present study emerged from an opportunity to collect pine needles infected with Lecanosticta spp. in Guatemala, Honduras and Nicaragua from 2010 to 2012. Specimens were identified based on DNA sequence comparisons and an attempt was made to confirm whether L. acicola occurs in Central America.

MATERIALS AND METHODS

Collections used in the study

Specimens prepared from ex-type cultures and other representatives of all known Lecanosticta species and closely related species (Quaedvlieg et al. 2012) were obtained from the culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands (CBS), and from the UK National Fungus Collection maintained by CABI Bioscience (Egham, UK: IMI). Living cultures or DNA of six isolates from Central America examined by Evans (1984), and believed to represent L. acicola, were also acquired from IMI (Table 1). Furthermore, isolates of Dothistroma septosporum, D. pini, Phaeophleospora eugenia, P. gregaria, and Amycosphaerella africana that represent genera in Mycosphaerellaceae closely related to Lecanosticta (Quaedvlieg et al. 2012) were included for comparative purposes. These cultures were obtained from CBS and the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI) in Pretoria, South Africa (Table 1).
Table 1

Details of isolates used in this study

Species

CMW numbera

Other collection numberb

Sampling site (Country, Region, Location)

Host

Collection date

Collector

GenBank accession numbersd

ITS

TEF1

BT1

MS204

RPB2

Amycosphaerella africana

45395

CBS 110843

South Africa, Western Cape Province, Pampoenvlei

Eucalyptus cladocalyx

Nov 1994

Crous PW

KF901702

JX901653

MK015047

MK015515

MK015290

A. africana

45396

CBS 680.95

South Africa, Western Cape Province, Stellenbosch mountain

E. viminalis

Oct 1994

Crous PW

AY626981

KF903117

MK015048

MK015516

MK015291

Dothistroma pini

10930

CBS 116485

USA, Michigan, Montcalm County, Crystal Lake

Pinus nigra

2001

Adams G, Barnes I

AY808301

AY808266

AY808196

NA

MK015292

D. pini

10951

CBS 116487

USA, Michigan, Montcalm County, Stanton

P. nigra

2001

Adams G, Barnes I

AY808302

AY808267

AY808197

NA

MK015293

D. septosporum

44656

CBS 140339

Russia, St. Petersburg, Park Sosnovka

P. sylvestris

Nov 2013

Drenkhan R, Musolin D, Adamson K

KU948400

MK015397

MK015049

MK015517

MK015294

D. septosporum

44657

CBS 141531

Russia, St. Petersburg, Park Sosnovka

P. sylvestris

Nov 2013

Drenkhan R, Musolin D, Adamson K

KU948401

MK015398

MK015050

MK015518

MK015295

Lecanosticta acicola

9985

CBS 871.95

France

P. radiata

Apr 1995

Morelet M

GU214663

MK015399

MK015051

MK015519

MK015296

L. acicola

45426

CBS133790

Lithuania

P. mugo

2009

Markovskaja S, Kacergius A, Treigiene A

HM367708

JX901645

MK015052

MK015520

MK015297

L. acicola

45427

CBS 133791

USA, New Hampshire, Blackwater

P. strobus

Jun 2011

Ostrofsky B

KC012999

KC013002

MK015053

MK015521

MK015298

L. acicola

45428

CBS 322.33

USA

P. palustris

Feb 1933

Siggers PV

MK015156

MK015400

MK015054

MK015522

MK015299

L. acicola

50541

 

Lithuania, Curonian Spit, Juodkrante

P. mugo

Sep 2014

Markovskaja S

MK015157

MK015401

MK015055

MK015523

MK015300

L. acicola

50542

 

Lithuania, Curonian Spit, Juodkrante

P. mugo

Sep 2014

Markovskaja S

MK015158

MK015402

MK015056

MK015524

MK015301

L. brevispora

- e

1A.N5S2

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015159

MK015403

L. brevispora

- e

1C.N1S3

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015160

MK015404

MK015057

NA

NA

L. brevispora

- e

1C.N5S4

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015161

MK015405

MK015058

MK015525

MK015302

L. brevispora

- e

1C.N6S2

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015162

MK015406

L. brevispora

- e

1D.N1S3

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015163

MK015407

MK015059

NA

NA

L. brevispora

- e

IB31.4a

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015164

MK015408

MK015060

MK015526

MK015303

L. brevispora

36894

 

Guatemala, Finca La Soledad (near Jalapa), Mataquescuintla

P. pseudostrobus

Oct 2010

Barnes I

MK015165

MK015409

MK015061

NA

MK015304

L. brevispora

37123

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015166

MK015410

NA

NA

MK015305

L. brevispora

42646

 

Honduras

P. oocarpa

MK015167

MK015411

MK015062

MK015527

MK015306

L. brevispora

42647

 

Guatemala, Lugar, La Soledad, Jalapa

P. oocarpa

Oct 2010

Barnes I

MK015168

MK015412

MK015063

MK015528

MK015307

L. brevispora

45424

CBS 133601

Mexico

Pinus sp.

Oct 2009

Yanes-Morales M

JX901763

JX901649

MK015064

MK015529

MK015308

L. brevispora

46499

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015169

MK015413

L. brevispora

46500

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015170

MK015414

L. brevispora

46501

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015171

MK015415

MK015065

NA

NA

L. brevispora

46502

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015172

MK015416

L. brevispora

46503

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015173

MK015417

MK015066

MK015530

MK015309

L. brevispora

46504

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015174

MK015418

MK015067

MK015531

MK015310

L. brevispora

46505

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015175

MK015419

NA

NA

MK015311

L. brevispora

46506

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015176

MK015420

L. brevispora

46507

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015177

MK015421

L. brevispora

46508

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015178

MK015422

L. brevispora

46509

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015179

MK015423

L. brevispora

46510

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015180

MK015424

NA

NA

MK015312

L. brevispora

46511

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015181

MK015425

L. brevispora

46512

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015182

MK015426

L. brevispora

46807

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015183

MK015427

MK015068

MK015532

MK015313

L. brevispora

49291

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015184

MK015428

MK015069

NA

MK015314

L. brevispora

49292

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015185

MK015429

MK015070

MK015533

MK015315

L. brevispora

49293

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015186

NA

MK015071

MK015534

MK015316

L. brevispora

49294

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015187

NA

MK015072

MK015535

MK015317

L. brevispora

49295

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015188

NA

MK015073

MK015536

MK015318

L. brevispora

49296

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015189

MK015430

MK015074

MK015537

MK015319

L. brevispora

49297

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015190

MK015431

MK015075

MK015538

MK015320

L. brevispora

49298

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015191

MK015432

MK015076

MK015539

MK015321

L. brevispora

50523

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015192

MK015433

L. brevispora

50526

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015193

MK015434

MK015077

NA

NA

L. brevispora

50527

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015194

MK015435

NA

NA

MK015322

L. brevispora

50528

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015195

MK015436

MK015078

NA

NA

L. brevispora

50529

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015196

MK015437

L. brevispora

50530

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015197

MK015438

MK015079

MK015540

MK015323

L. brevispora

50531

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015198

MK015439

MK015080

MK015541

MK015324

L. brevispora

50532

 

Guatemala, Chimaltenango, Tecpán, Finca La Esperanza

P. pseudostrobus

Jun 2011

Barnes I

MK015199

MK015440

L. brevispora

51050

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015200

MK015441

NA

MK015542

MK015325

L. gloeospora c

42645

IMI 283812

Mexico, Nuevo León, Iturbide-Galeana

P. pseudostrobus

May 1983

Evans HC

KU948431

MK015442

MK015081

MK015543

MK015326

L. guatemalensis

- e

IB30/2d

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015201

MK015443

L. guatemalensis

- e

IB32/1a

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015202

MK015444

L. guatemalensis

- e

IB32/2e

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015203

MK015445

MK015082

NA

NA

L. guatemalensis

- e

IB35/2e

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015204

MK015446

MK015083

NA

NA

L. guatemalensis

- e

IB35/2j

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015205

MK015447

L. guatemalensis

- e

IB35/9a

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015206

MK015448

MK015084

NA

NA

L. guatemalensis c

 

IMI 275573

Honduras, Yoro

P. oocarpa

Oct 1980

Evans HC

MK015207

MK015449

NA

NA

NA

L. guatemalensis c

 

IMI 281563

Honduras

P. caribaea

May 1982

Evans HC

MK015208

NA

NA

NA

NA

L. guatemalensis c

 

IMI 281596

Nicaragua

P. tecunumanii

Nov 1981

Evans HC

MK015209

MK015450

NA

NA

NA

L. guatemalensis

- e

N3/1c

Nicaragua, Matagalpa

P. oocarpa

Jun 2011

Barnes I

MK015210

MK015451

MK015085

MK015544

MK015327

L. guatemalensis

36811

 

Guatemala, Jalapa, Finca Forestal Soledad

P. maximinoi

Oct 2010

Barnes I

MK015211

MK015452

MK015086

NA

MK015328

L. guatemalensis

36812

 

Guatemala, Coban, San Juan Chamelco

P. maximinoi

Oct 2010

Barnes I

MK015212

MK015453

MK015087

MK015545

MK015329

L. guatemalensis

37121

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015213

MK015454

L. guatemalensis

37122

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015214

MK015455

MK015088

MK015546

MK015330

L. guatemalensis

37124

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015215

MK015456

L. guatemalensis

37126

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015216

MK015457

MK015089

MK015547

MK015331

L. guatemalensis

37127

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015217

MK015458

L. guatemalensis c

42206

IMI 281598

Guatemala

P. oocarpa

1983

Evans HC

JX901764

JX901650

MK015090

MK015548

MK015332

L. guatemalensis

43890

 

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015218

MK015459

L. guatemalensis

43891

 

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015219

MK015460

MK015091

NA

NA

L. guatemalensis

43892

 

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015220

MK015461

MK015092

NA

NA

L. guatemalensis

43893

 

Guatemala, Chiquimula, San José la Arada

P. oocarpa

Oct 2010

Barnes I

MK015221

MK015462

MK015093

NA

NA

L. guatemalensis

43894

 

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015222

MK015463

MK015094

NA

NA

L. guatemalensis

43895

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015223

MK015464

MK015095

MK015549

MK015333

L. guatemalensis

45386

 

Nicaragua, Matagalpa

P. oocarpa

Jun 2011

Barnes I

MK015224

MK015465

L. guatemalensis

45387

 

Nicaragua, Matagalpa

P. oocarpa

Jun 2011

Barnes I

MK015225

MK015466

MK015096

MK015550

MK015334

L. guatemalensis

45391

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015226

MK015467

MK015097

NA

NA

L. guatemalensis

45392

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2011

Barnes I

MK015227

MK015468

MK015098

MK015551

MK015335

L. guatemalensis

45393

 

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015228

MK015469

L. guatemalensis

45394

 

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015229

NA

L. guatemalensis

46811

 

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015230

MK015470

MK015099

MK015552

MK015336

L. guatemalensis

46817

 

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015231

MK015471

MK015100

MK015553

MK015337

L. guatemalensis

46819

 

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015232

NA

L. guatemalensis

47108

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015233

MK015472

L. guatemalensis

49400

 

Nicaragua, Matagalpa

P. oocarpa

Jun 2011

Barnes I

MK015234

MK015473

MK015101

MK015554

MK015338

L. guatemalensis

49402

 

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015235

MK015474

MK015102

MK015555

MK015339

L. guatemalensis

51052

 

Guatemala, Chiquimula, San José la Arada

P. oocarpa

Oct 2010

Barnes I

MK015236

MK015475

MK015103

MK015556

MK015340

L. guatemalensis

51142

 

Nicaragua, Matagalpa

P. oocarpa

Jun 2011

Barnes I

MK015237

MK015476

MK015104

MK015557

MK015341

L. jani

- e

267.44.N1

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. tecunumanii

Sep 2012

Barnes I

MK015238

MK015477

MK015105

MK015558

MK015342

L. jani

- e

267.47.N1

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. tecunumanii

Sep 2012

Barnes I

MK015239

MK015478

MK015106

MK015559

MK015343

L. jani

- e

267.47.N2

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. tecunumanii

Sep 2012

Barnes I

MK015240

MK015479

MK015107

MK015560

MK015344

L. jani

- e

267.51.N2S1

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. tecunumanii

Sep 2012

Barnes I

MK015241

MK015480

NA

NA

MK015345

L. jani

- e

267.52.N1S1

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. tecunumanii

Sep 2012

Barnes I

MK015242

MK015481

MK015108

MK015561

MK015346

L. jani

- e

267.52.N2S1

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. tecunumanii

Sep 2012

Barnes I

MK015243

MK015482

MK015109

MK015562

MK015347

L. jani

- e

IB30/2b

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015244

MK015483

MK015110

MK015563

NA

L. jani

- e

IB35/3c

Guatemala, Chiquimula

P. oocarpa

Oct 2010

Barnes I

MK015245

MK015484

MK015111

MK015564

MK015348

L. jani

- e

IB13/2f

Guatemala

P. maximinoi

Oct 2010

Barnes I

MK015246

MK015485

MK015112

MK015565

MK015349

L. jani

- e

N3/2c

Nicaragua, Matagalpa

P. oocarpa

Jun 2011

Barnes I

MK015247

NA

MK015113

MK015566

MK015350

L. jani

36808

 

Guatemala, Jalapa, Finca Forestal Soledad

P. maximinoi

Oct 2010

Barnes I

MK015248

NA

MK015114

MK015567

MK015351

L. jani

36810

 

Guatemala, Jalapa, Finca Forestal Soledad

P. maximinoi

Oct 2010

Barnes I

MK015249

NA

MK015115

MK015568

MK015352

L. jani

37128

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015250

MK015486

MK015116

MK015569

MK015353

L. jani

38950

CBS 144446; PREM 62186

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015251

MK015487

MK015117

MK015570

MK015354

L. jani

38958

CBS 144456; PREM 62185

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015252

MK015488

MK015118

MK015571

MK015355

L. jani

38959

 

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015253

NA

NA

NA

NA

L. jani

38968

 

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015254

NA

MK015119

NA

NA

L. jani

45388

 

Guatemala

P. maximinoi

Oct 2010

Barnes I

MK015255

NA

MK015120

MK015573

MK015356

L. jani

45389

 

Guatemala

P. maximinoi

Oct 2010

Barnes I

MK015256

MK015489

MK015121

MK015574

MK015357

L. jani

47109

 

Guatemala

P. maximinoi

Oct 2010

Barnes I

MK015257

MK015490

MK015122

MK015575

MK015358

L. jani

48830

 

Nicaragua, Matagalpa

P. oocarpa

Jun 2011

Barnes I

MK015258

NA

MK015123

MK015576

MK015359

L. jani

48831

CBS 144447; PREM 62187

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015259

MK015491

MK015124

MK015577

MK015360

L. jani

49401

 

Guatemala

P. maximinoi

Oct 2010

Barnes I

MK015260

MK015492

NA

MK015578

MK015361

L. jani

51051

 

Guatemala

P. maximinoi

Oct 2010

Barnes I

MK015261

MK015493

MK015125

MK015579

MK015362

L. jani

51058

 

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. tecunumanii

Sep 2012

Barnes I

MK015262

MK015494

MK015126

MK015580

MK015363

L. jani

51059

 

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. tecunumanii

Sep 2012

Barnes I

MK015263

MK015495

MK015127

MK015581

MK015364

L. jani

51143

 

Nicaragua, Matagalpa

P. oocarpa

Jun 2011

Barnes I

MK015264

NA

MK015128

MK015582

MK015365

L. longispora

45429

CBS 133602

Mexico

Pinus sp.

Oct 2009

Yanes-Morales M

JX901766

JX901651

MK015129

MK015583

MK015366

L. longispora

45430

CPC 17941

Mexico

Pinus sp.

Oct 2009

Yanes-Morales M

JX901765

JX901652

MK015130

MK015584

MK015367

L. pharomachri

- e

267.8A.N2S1

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015265

MK015496

NA

NA

MK015368

L. pharomachri

- e

267.12.N1S2

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015266

NA

NA

NA

MK015369

L. pharomachri

- e

267.30.MD.N1

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015267

NA

NA

NA

MK015370

L. pharomachri

- e

267.30.MD.N2

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015268

MK015497

MK015131

NA

MK015371

L. pharomachri

- e

267.30.N4

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015269

MK015498

MK015132

MK015585

MK015372

L. pharomachri

37132

 

Guatemala, Baja Verapaz, San Jerónimo, Salamá

P. tecunumanii

Oct 2010

Barnes I

MK015270

MK015499

MK015133

MK015586

MK015373

L. pharomachri

37133

 

Guatemala, Baja Verapaz, San Jerónimo, Salamá

P. tecunumanii

Oct 2010

Barnes I

MK015271

MK015500

MK015134

MK015587

MK015374

L. pharomachri

37134

 

Guatemala, Baja Verapaz, San Jerónimo, Salamá

P. tecunumanii

Oct 2010

Barnes I

MK015272

MK015501

MK015135

MK015588

MK015375

L. pharomachri

37136

CBS 144448; PREM 62188

Guatemala, Baja Verapaz, San Jerónimo, Salamá

P. tecunumanii

Oct 2010

Barnes I

MK015273

MK015502

MK015136

MK015589

MK015376

L. pharomachri

38947

CBS 144695;

PREM 62189

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015274

MK015503

MK015137

MK015590

MK015377

L. pharomachri

38974

CBS 144449; PREM 62190

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015275

MK015504

MK015138

MK015591

MK015378

L. pharomachri

38975

 

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015276

NA

NA

NA

NA

L. pharomachri

38976

 

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015277

MK015505

MK015139

NA

MK015379

L. pharomachri

46810

 

Honduras

P. oocarpa

MK015278

MK015506

MK015140

MK015592

MK015380

L. pharomachri

46813

 

Guatemala, Baja Verapaz, San Jerónimo, Salamá

P. tecunumanii

Oct 2010

Barnes I

MK015279

MK015507

MK015141

MK015593

MK015381

L. pharomachri

51053

 

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015280

NA

MK015142

NA

MK015382

L. pharomachri

51054

 

Guatemala, Jalapa, Finca La Soledad, Mataquescuintla

P. oocarpa

Sep 2012

Barnes I

MK015281

MK015508

NA

NA

MK015383

L. tecunumanii

46805

CBS 144450; PREM 62191

Guatemala, Baja Verapaz, San Jerónimo, Salamá

P. tecunumanii

Oct 2010

Barnes I

MK015282

MK015509

MK015143

MK015594

MK015384

L. tecunumanii

46812

CBS 144452; PREM 62193

Guatemala, Baja Verapaz, San Jerónimo, Salamá

P. tecunumanii

Oct 2010

Barnes I

MK015283

MK015510

MK015144

MK015595

MK015385

L. tecunumanii

49403

CBS 144451; PREM 62192

Guatemala, Baja Verapaz, San Jerónimo, Salamá

P. tecunumanii

Oct 2010

Barnes I

MK015284

MK015511

MK015145

MK015596

MK015386

L. variabilis

36809

CBS 144455; PREM 62195

Guatemala, Jalapa, Finca Forestal Soledad

P. maximinoi

Oct 2010

Barnes I

MK015285

MK015512

MK015146

MK015597

MK015387

L. variabilis

37125

CBS 144454; PREM 62194

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015286

KJ938446

MK015147

MK015598

MK015388

L. variabilis

37129

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015287

KJ938445

MK015148

MK015599

MK015389

L. variabilis c

42205

IMI 281561; CBS 144453; PREM 62196

Honduras, Santa Barbara, Lago Yojoa

P. caribaea

Oct 1980

Evans HC

MK015288

MK015513

MK015149

MK015600

MK015390

L. variabilis

45390

 

Guatemala, Alta Verapaz, Santa Cruz Verapaz, near Tactíc

P. oocarpa

Oct 2010

Barnes I

MK015289

MK015514

MK015150

MK015601

MK015391

L. variabilis

45425

CBS 133789

Mexico

Pinus sp.

Nov 2009

Yanez-Morales M

JX901762

JX901648

MK015151

MK015602

MK015392

Phaeophleospora eugeniae

45432

CPC15159

Brazil, Vicosa, Paraiso

Eugenia uniflora

Mar 2008

Alfenas AC

FJ493189

JX901667

MK015152

MK015603

NA

P. eugeniae

45433

CPC 15143

Brazil, Vicosa, Paraiso

E. uniflora

Mar 2008

Alfenas AC

FJ493188

JX901666

MK015153

MK015604

NA

P. gregaria

45434

CBS 111166

South Africa, Western Cape Province, de Hoop Nature Reserve

Eucalyptus cladocalyx

Sep 1995

Wood A

JX901773

JX901664

MK015154

MK015605

MK015393

P. gregaria

45435

CBS 114662

South Africa, Western Cape Province, Devon Valley, Stellenbosch

Eucalyptus sp.

Jun 1995

Crous PW

DQ302953

JX901654

MK015155

MK015606

MK015394

aCMW Culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa;

bCBS Culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, CPC Personal collection of Pedro Crous housed at CBS, IMI The UK National Fungus Collection, CABI Bioscience, Egham, UK, PREM The dried herbarium collection of the South African National Collection of Fungi, Mycology Unit, Biosystematics Division, Plant Protection Institute, Agricultural Research Council, Pretoria, South Africa

cCultures were collected by HC Evans in Central America

d‘-‘= was not amplified; ‘NA‘= amplification unsuccessful;

e = no viable culture available that could be submitted to CMW

Ex-type isolate of each species is indicated in bold

Pine needles, showing symptoms of brown spots or bands, were collected from Pinus species native to Central America from 2010 to 2012 in Guatemala, as well as from Honduras and Nicaragua in 2011 (Table 1). Conidiomata formed on the needles were aseptically excised, rolled onto 2% Dothistroma Sporulating Media (DSM: 5 g yeast extract (Biolab, Merck, Modderfontein, South Africa), 20 g malt extract (Biolab) and 15 g agar (BD Difco™, Sparks, MD) per litre of distilled water) with 100 mg/L streptomycin (Sigma-Aldrich, St Louis, MO) in order to release conidia from the conidiomata as described by Barnes et al. (2004). The isolated conidiomata were incubated for one to two days at 23 °C. The plates were examined using a dissection microscope and single germinating conidia were selected and replated onto 2% DSM. The single conidial isolates were grown for 4–6 wk. on a natural day light cycle, at 23 °C.

DNA extractions and sequencing

Fungal tissue was scraped from the surface of the cultures on 2% DSM with a sterile scalpel blade and lyophilized. The freeze-dried mycelium was homogenized using a Retsch MM301 mixer mill (Haan, Germany) and approximately 20 ng of the crushed mycelium was used as starting material for DNA extractions. DNA was extracted using a Zymo Research ZR Fungal/Bacterial DNA MiniPrep™ kit (Irvine, CA) and eluted into a final volume of 50 μl. The quality and quantity of the extracted DNA was determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA). DNA concentrations were diluted to 20 ng/μl working stock for polymerase chain reaction (PCR) amplifications and stored at − 20 °C until further use.

The nuclear rDNA region encompassing the internal transcribed spacers (ITS) 1 and 2, along with the 5.8S rDNA region was amplified using primers ITS1 and ITS4 (White et al. 1990) and a portion of the translation elongation factor 1-α gene (TEF1) using primers EF1-728F (Carbone and Kohn 1999) and EF2 (O’Donnell et al. 1998) for all the isolates. The Beta-tubulin-2 gene region (BT2) was amplified using the primer pair T1 (O’Donnell and Cigelnik 1997) and β-Sandy-R (Stukenbrock et al. 2012) or the primers Bt2A and Bt2B (Glass and Donaldson 1995). The Beta-tubulin-1 gene region (BT1) was amplified using primers Bt1A and Bt1B (Glass and Donaldson 1995), the RNA polymerase II second largest subunit (RPB2) gene region using primers RPB2-5f2 (Sung et al. 2007) and RPB2-7cR (Liu et al. 1999) and the guanine nucleotide-binding protein subunit beta (MS204) using primers MS204F.cerato and MS204R.cerato (Fourie et al. 2015).

PCR reactions for each of the six regions contained 20 ng DNA, 2.5 μl 10x PCR reaction buffer, 2.5 mM MgCl2, 400 nM of each primer, 200 μM of each dNTP and 1 U Faststart Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN). Reaction volumes were adjusted to 25 μl with sterile SABAX water (Adcock Ingram, Midrand, South Africa). PCR reactions were carried out on an Applied Biosystems® Veriti® 96 well Thermal cycler (Thermo Fisher Scientific, Waltham, MA). The cycling conditions for all six gene regions included an initial denaturation step at 95 °C for 4 min, 10 cycles consisting of 94 °C for 20 s (denaturation), a 45 s annealing step according to the primer pair annealing temperature (Table 2) and an elongation step of 45 s at 72 °C. This was followed by a further 25 cycles of 94 °C for 20 s, 45 s with a 5 s extension step per cycle at the indicated annealing temperature, a 72 °C extension for 45 s and a final step of 72 °C for 10 min. The annealing temperature was set at 56 °C for ITS, 52 °C for TEF1, 50 °C for BT1, 52 °C for BT2, 55 °C for MS204 and 56 °C for RPB2. To visualise amplified products, 5 μl PCR products were stained with 1 μl GelRed™ nucleic acid gel stain (Biotium, Fremont, CA) and separated on 2% SeaKem® LE agarose gel (Lonza, Rockland, ME) for 20 min at 100 V and viewed under a UV light using the GelDoc™ EZ Imager (BioRad, Hercules, CA). PCR products were cleaned with a 6.65% G-50 Sephadex solution (Sigma-Aldrich, St Louis, MO) following the manufacturer’s instructions using Centri-sep spin columns (Princeton Separations, Freehold, NJ).
Table 2

Primers used for PCR amplification and sequencing in this study

Locus

Primer name

Direction

Primer sequence 5′ to 3’

Annealing temperature used (°C)

Amplification success

Reference

BT1

Bt1a

Forward

TTC CCC CGT CTC CAC TTC TTC ATG

50

87.4%

Glass and Donaldson 1995

 

Bt1b

Reverse

GAC GAG ATC GTT CAT GTT GAA CTC

50

 

Glass and Donaldson 1995

BT2a

T1

Forward

AAC ATG CGT GAG ATT GTA AGT

52

O’Donnell and Cigelnik 1997

 

β-Sandy-R

Reverse

GCR CGN GGV ACR TAC TTG TT

52

 

Stukenbrock et al. 2012

 

Bt2a

Forward

GGT AAC CAA ATC GGT GCT GCT TTC

52

Glass and Donaldson 1995

 

Bt2b

Reverse

ACC CTC AGT GTA GTG ACC CTT GGC

52

 

Glass and Donaldson 1995

TEF1

EF1-728F

Forward

CAT CGA GAA GTT CGA GAA GG

52

88.2%

Carbone and Kohn 1999

 

EF-2

Reverse

GGA RGT ACC AGT SAT CAT GTT

52

 

O’Donnell et al. 1998

ITS

ITS1

Forward

GAA GTA AAA GTC GTA ACA AGG

56

100%

White et al. 1990

 

ITS4

Reverse

TCC TCC GCT TAT TGA TAT GC

56

 

White et al. 1990

MS204

MS204F.cerato

Forward

AAG GGC ACC CTC GAG GGC CAC

55

71.7%

Fourie et al. 2015

 

MS204R.cerato

Reverse

GAT GGT RAC GGT GTT GAT GTA

55

 

Fourie et al. 2015

RPB2

RPB2-5f2

Forward

GGG GWG AYC AGA AGA AGG C

56

82.7%

Sung et al. 2007

 

fRPB2-7cR

Reverse

CCC ATR GCT TGY TTR CCC AT

56

 

Liu et al. 1999

aBT2 amplification success using all primer combinations was very low and abandoned

The concentrations of the cleaned PCR products were determined using a NanoDrop ND-1000 spectrophotometer and 60–100 ng of DNA and products were sequenced in both the forward and reverse direction using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) on an ABI PRISM 3500xl capillary auto sequencer (Thermo Fisher Scientific).

Forward and reverse sequences were aligned and consensus sequences generated in CLC Main workbench version 8.0 (CLC Bio, https://www.qiagenbioinformatics.com/products/clc-main-workbench/). All consensus sequences generated in this study were deposited in GenBank that is hosted by the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/genbank/) (Table 1).

Data analyses

Five datasets (BT1, ITS, MS204, RPB2 and TEF1) were generated and analysed individually. A partition homogeneity test (PHT) was performed with the software package PAUP* 4.0b10 (Swofford 2003) to test congruence between the five gene regions and a sixth dataset, where sequences were available for all five gene regions, was compiled and analysed. The BT1, ITS, MS204 and RPB2 datasets included all of the sequences generated in this study and additional sequences available from GenBank (Table 1). The TEF1 dataset included all of the sequence data generated in this study as well as additional sequences representing 14 different TEF1 haplotypes of L. acicola (including possible cryptic species) (Janoušek et al. 2016) that were downloaded from GenBank (Table 3). Sequences for all datasets were aligned with the online version of MAFFT Version 7 (Katoh and Standley 2013; http://mafft.cbrc.jp/alignment/server/) using default settings. Aligned data were imported into MEGA 7.0.14 (Kumar et al. 2016) and manually checked and adjusted.
Table 3

GenBank numbers of Lecanosticta acicola TEF1 haplotypes included in the TEF1 phylogenetic analysis (Fig. 2) as well as additional locations represented by the haplotypes

Species name assigned in this studya

GenBank Accession number

Country

State / Region

Location

Host

Date of collection

Collector / Supplier

Lecanosticta acicola

KJ938442

Japan

Shimane

Matsue, Hamanogi

Pinus thunbergii

Feb 2010

Suto Y

L. acicola

KJ938439

Mexico

Nuevo León

Iturbide, Bosque Escuela

Pinus halepensis

May 2010

Marmolejo JG

L. acicola

KJ938440

Mexico

Nuevo León

Iturbide, Bosque Escuela

Pinus halepensis

May 2010

Marmolejo JG

L. acicola

KJ938441

Mexico

Nuevo León

Iturbide, Bosque Escuela

Pinus halepensis

May 2010

Marmolejo JG

L. acicola

KJ938438

USA

Maine

York, Lyman

Pinus strobus

Jun 2011

Ostrofsky W

L. acicola

KJ938443

USA

Mississippi

Harrison County

Pinus palustris

Oct 2012

Bartlett B, Burdine C

L. acicola

KJ938444

USA

Mississippi

Harrison County

Pinus palustris

Oct 2012

Bartlett B, Burdine C

L. acicola

KJ938450

USA

Mississippi

Harrison County

Pinus palustris

Oct 2012

Bartlett B, Burdine C, Roberds J

L. acicola

KJ938451

USA

Mississippi

Harrison County

Pinus palustris

Oct 2012

Bartlett B, Burdine C

Lecanosticta variabilis

KJ938445

Guatemala

Alta Verapaz

Santa Cruz Verapaz, near Tactíc

Pinus oocarpa

Oct 2010

Barnes I

L. variabilis

KJ938446

Guatemala

Alta Verapaz

Santa Cruz Verapaz, near Tactíc

Pinus oocarpa

Oct 2012

Barnes I

L. variabilis

KJ938447

Mexico

Nuevo León

Piñal de los Amoles, Querétaro

Pinus sp.

2011

Kunte L

L. variabilis

KJ938448

Mexico

Nuevo León

Iturbide, Bosque Escuela

Pinus halepensis

May 2010

Marmolejo JG

L. variabilis

KJ938449

Mexico

Nuevo León

Galeana, Cerro del Potosí

Pinus arizonica var. stormiae

Apr 2010

Marmolejo JG

Countries, regions, locations and hosts represented by the above isolatesb

 

the same as KJ938438

Austria

Lower Austria

Hollenstein an der Ybbs

Pinus mugo

Oct 2004

Kirisits T, Barnes I

the same as KJ938438

Austria

Lower Austria

Opponitz

Pinus mugo

2010

Hintsteiner M

the same as KJ938438

Austria

Lower Austria

Saimannslehen

Pinus sp.

2010

Hintsteiner M

the same as KJ938438

Austria

Lower Austria

Sankt Gallen

Pinus mugo

2010

Hintsteiner M

the same as KJ938438

Austria

Lower Austria

Steyer, Pestalozzistraße

Pinus mugo

2010

Hintsteiner M

the same as KJ938438

Austria

Lower Austria

Waidehofen an der Ybbs

Pinus mugo

Aug 2010

Janoušek J

the same as KJ938438

Austria

Upper Austria

Gmunden

Pinus nigra

Jun 2012

Kirisits T

the same as KJ938438

Canada

Québec

Demers-Centre

Pinus strobus

Jun 2011

Harvey L

the same as KJ938438

Canada

Québec

Lake Aberdeen

Pinus strobus

Jun 2011

Harvey L

the same as KJ938438

Canada

Québec

Lake Pinseault

Pinus strobus

Jun 2011

Harvey L

the same as KJ938438

Canada

Québec

Montréal

Pinus mugo

Jun 2011

Harvey R

the same as KJ938438

Canada

Québec

Waltham

Pinus strobus

Jun 2011

Harvey L

the same as KJ938442

China

Fujie

 

Pinus elliottii

1988

Zheng-Yu H

the same as KJ938451

Colombia

Refocosta L-75

Villanueva, Casanare

Pinus caribaea

Mar 2011

Rodas C, Barnes I

the same as KJ938438

Croatia

 

Zadar

Pinus halapensis

Sep 2009

Diminic D

the same as KJ938438

Czech Republic

Southern Bohemia

Borkovická Blata

Pinus uncinata subsp. uliginosa

Oct 2011

Janoušek J

the same as KJ938438

Czech Republic

Southern Bohemia

Červená Blata

Pinus uncinata subsp. uliginosa

Aug 2009

Dvořák M, Janoušek J

the same as KJ938438

Estonia

Harju maakond

Tallin

Pinus ponderosa

Jul 2008

Cech T

the same as KJ938451

France

Pyrénées-Atlantiques

 

Pinus radiata

2012

Kersaudy E, Ioos R

the same as KJ938438

Germany

Bavaria

Grassau

Pinus mugo

2000

Blaschke FR, Wulf

the same as KJ938438

Germany

Bavaria

Murnau

Pinus mugo

Feb 2010

Nannig A

the same as KJ938438

Germany

Bavaria

Murnauer Filze

Pinus mugo

Nov 2011

Nannig A

the same as KJ938438

Germany

Bavaria

Pfrűhlmoos

Pinus mugo

Nov 2011

Nannig A

the same as KJ938438

Italy

Brecia

Gardone

Pinus mugo

Jun 2008

Cech T

the same as KJ938438

Lithuania

Klaipėdský kraj

Curonian Spit, Juodkrante

Pinus mugo

2010

Markovskaja S

the same as KJ938438

Slovenia

Upper Carniola

Bled

Pinus mugo

Jul 2009

Jurc D

the same as KJ938442

South Korea

Naju

Sanpo-myeon

Pinus thunbergii

2010

KACC, Seo ST

the same as KJ938451

Spain

Cantabria

San Sebastián de Garabandal

Pinus radiata

Oct 2012

Jankovský L, Janoušek J

the same as KJ938438

Switzerland

Canton St Gallen

Walensee

Pinus mugo

Oct 1999

Wulf

the same as KJ938438

USA

Maine

Androscoggin, Leeds

Pinus strobus

Jun 2011

Ostrofsky W

the same as KJ938438

USA

Maine

Piscataquis, Sangerville

Pinus strobus

Jun 2011

Weimer J

the same as KJ938438

USA

Maine

York, Lyman

Pinus strobus

Jun 2011

Ostrofsky W

the same as KJ938438

USA

Michigan

Wexford County, Springville Township

Pinus sylvestris

2011

Odonnell J

the same as KJ938444

USA

Mississippi

Harrison County

Pinus palustris

Oct 2012

Bartlett B, Burdine C, Roberds J

the same as KJ938438

USA

New Hampshire

Hillsboro, Fox State Park

Pinus strobus

Jun 2011

Weimer J

the same as KJ938438

USA

New Hampshire

Merrimack, Black Water Reserve

Pinus strobus

Jun 2011

Weimer J

the same as KJ938438

USA

New Hampshire

Merrimack, Hopkinton-Everett

Pinus strobus

Jun 2011

Weimer J

the same as KJ938438

USA

Vermont

Washington, Waterbury

Pinus strobus

Jun 2011

Lackey J

the same as KJ938438

USA

Vermont

Windsor, Bethel

Pinus strobus

Jul 2011

Munck I

the same as KJ938438

USA

Wisconsin

Merrillan

Pinus sylvestris

Apr 2010

Stanosz G

aLecanosticta variabilis was previously identified as L. acicola but is now defined as a new species

bInformation adapted from Janoušek et al. (2016), Table S1

Three separate analyses were performed for each of the six datasets: Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian inference (BI). The MP analysis were performed with the software package PAUP* 4.0b10 (Swofford 2003). Gaps were treated as a fifth character state. One thousand random stepwise addition heuristic searches were performed with tree-bisection-reconnection (TBR) as the branch-swapping algorithm. Uninformative characters were excluded and the consistency index (CI), homoplasy index (HI), rescaled consistency index (RC), retention index (RI) and tree length (TL) were determined for the resulting trees (Table 4). The confidence levels were estimated by performing 1000 bootstrap replicates.
Table 4

PCR amplification size, phylogenetic data and the substitution models used in the phylogenetic analysis for each gene region and for the combined datasets

 

ITS

TEF1

BT1

MS204

RPB2

Combined datasets

Approximate amplicon size (bp)

550

520

420

760

940

Number of taxa analysed

153

147

111

91

105

76

Aligned characters (bp)

734

586

440

785

929

3344

Number of parsimony-uninformative characters

621

143

357

519

538

2438

Number of parsimony-informative characters

114

423

82

266

371

1121

Number of trees retained

108

396

1

2448

420

100

Consistency index

0.865

0.499

0.739

0.791

0.738

0.607

Homoplasy index

0.135

0.501

0.261

0.209

0.262

0.393

Rescaled consistency index

0.850

0.459

0.703

0.748

0.696

0.555

Retention index

0.982

0.919

0.951

0.946

0.943

0.914

Tree Length

163

1675

138

546

722

2642

Substitution model

TPM2uf + G

GTR + G

GTR + G

TVM + G

TrN + G

GTR + G

In order to determine the ML and BI, the best fit substitution model for each of the data sets were determined using jModelTest 0.1.1 (Posada 2008). Maximum likelihood analysis was performed with the program PhyML 3.0 (Guindon et al. 2010). The confidence levels were estimated with 1000 bootstrap replicates.

MrBayes 3.1.2 (Ronquist et al. 2012) was used to determine the BI for each data set by applying the Markov Chain Monte Carlo (MCMC) method. For each dataset, four independent MCMC chains were randomly started and run for six million generations, applying the best substitution model determined by jModelTest 0.1.1. Trees were sampled every 100 generations. Burn-in values were determined using Tracer 1.6 (Rambaut et al. 2014) by comparing the log likelihoods. Trees sampled in the burn-in phase were discarded. The remaining trees were used to construct majority rule consensus trees and to determine posterior probabilities for the tree topology.

Morphological characterization

Cultures were grown on 2% Malt Extract Agar (MEA), Oatmeal Agar (OA) and Potato Dextrose Agar (PDA) (Crous et al. 2009b; Quaedvlieg et al. 2012) at 20 °C for 2 wk. in darkness in order to examine the morphology and colour of the cultures of each species. Cultures on MEA were used for microscopic measurements of the conidiophores, conidiogenous cells and conidia. Slides were mounted in SABAX water (Adcock Ingram, Midrand, South Africa) for microscopy and examined using a Zeiss Axioskop 2 Plus compound microscope (Zeiss, Oberkochen, Germany). Photographic images were captured with a Nikon DS-Ri2 camera with the NIS Element BR v4.3 software package (Nikon, Tokyo, Japan). Up to 50 measurements of each morphologically characteristic structure was taken for each ex-type isolate and ten measurements were made for each of the paratypes examined. The mean, standard deviation, minimum and maximum were calculated for each morphological structure and the measurements presented as (minimum–) (mean – standard deviation) – (mean + standard deviation) (−maximum) for the conidia and conidiogenous cells. For the conidiophores, the maximum observed length was indicated together with the width as (minimum–) (mean) (−maximum).
Table 5

Specimens for which the morphology was examined for the description of Lecanosticta jani, L. pharomachri, L. tecunumanii and L. variabilis

Species

CMW numbera

Status of specimen

Herbarium specimenb

Ex-type isolatesc

Lecanosticta jani

CMW 38950d

Paratype

PREM 62186

CBS 144446

 

CMW 38958d

Holotype

PREM 62185

CBS 144456

CMW 48831e

Paratype

PREM 62187

CBS 144447

CMW 51058d

Additional material examined

 

CMW 51059d

Additional material examined

CMW 51143e

Additional material examined

CMW47109e

Additional material examined

Lecanosticta pharomachri

CMW 37136

Holotype

PREM 62188

CBS 144448

 

CMW 38947

Paratype

PREM 62189

CBS 144695

CMW 38974

Paratype

PREM 62190

CBS 144449

CMW 38976

Additional material examined

 

CMW 51053

Additional material examined

CMW 51054

Additional material examined

Lecanosticta tecunumanii

CMW 46805

Holotype

PREM 62191

CBS 144450

 

CMW 46812

Paratype

PREM 62193

CBS 144452

CMW 49403

Paratype

PREM 62192

CBS 144451

Lecanosticta variabilis

CMW 42205

Holotype

PREM 62196

CBS 144453, IMI 281561

 

CMW 37125

Paratype

PREM 62194

CBS 144454

CMW 36809

Paratype

PREM 62195

CBS 144455

CMW 45425

Additional material examined

CBS H-21112

CBS 133789

CMW 37129

Additional material examined

 

aCMW Culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa; bThe herbarium deposits are dried cultures that serve as holotype and paratype specimens. PREM = The dried herbarium collection of the South African National Collection of Fungi, Mycology Unit, Biosystematics Division, Plant protection Institute, Agricultural Research Council, Pretoria, South Africa; cThe ex-type cultures are living cultures linked to the holotype and paratype specimens. CBS = The culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; IMI = The UK National Fungus Collection maintained by CABI Bioscience, Egham, UK; d Lecanosticta jani cultures with the Type 2 morphology; e Lecanosticta jani cultures with the Type 1 morphology

Temperature requirements for growth in culture was studied on representative isolates selected for each of the novel species. Four by four millimeter blocks of each culture were plated, in triplicate, onto the centres of 2% MEA plates per temperature (10, 15, 20, 25, and 30 °C) and incubated in darkness. The diameters of each colony were recorded weekly along perpendicular axes for 4 wk. The colour and shape of each colony was recorded after 2 wk. of growth at 20 °C. Culture colour was determined using Rayner’s colour chart (Rayner 1970).

Accession of cultures and types

Holotype specimens of the new species, which are dried cultures, are deposited in the National Mycological Herbarium in Pretoria (PREM). Cultures are deposited in the culture collection (CBS) of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, and ex-type cultures, as well as all other isolates included in this study, are maintained in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI) in Pretoria, South Africa (Table 5).

RESULTS

Fungal collections

Twenty-six isolates or DNA samples were obtained from culture collections to include in the study. An additional 127 isolates of putative Lecanosticta species were obtained from symptomatic needles collected from 36 different trees in Guatemala, Nicaragua and Honduras (Table 1). In Guatemala, 22 isolates were obtained from Pinus oocarpa, P. maximinoi, and P. tecunumanii needles that were collected in the Alta Verapaz District, 16 isolates were obtained from P. oocarpa needles collected in Chiquimula, 35 isolates from P. pseudostrobus needles collected in the Chimaltenango District in the Tecpán Municipality, eight isolates from P. tecunumanii needles collected in the Baja Verapaz District, 29 isolates from P. tecunumanii and P. oocarpa needles collected in the Jalapa District, and seven isolates from P. maximinoi needles in Coban and other regions (Table 1). Two isolates were obtained from P. oocarpa needles collected in Honduras and eight isolates were made from P. oocarpa needles collected in Matagalpa, Nicaragua.

DNA extraction and sequencing

The ITS and TEF1 regions were sequenced for all 153 isolates obtained and the BT1, MS204 and RPB2 regions were sequenced for 127 representatives of all monophyletic groups identified in the generated ITS and TEF1 phylogenetic trees. The selected representatives included all of the closely related Mycosphaerellaceae isolates, all the isolates that did not group with known Lecanosticta species, and a selection of isolates that grouped with known Lecanosticta species (Table 1). PCR fragments of approximately 550 bp were generated for ITS, 520 bp for TEF1, 420 bp for BT1, 760 bp for MS204 and 940 bp for RPB2. The amplification success of the TEF1, BT1, MS204 and RPB2 gene regions varied for the isolates that were selected and the amplification success rate of TEF1 was 88.2%, BT1 was 87.4%, MS204 was 71.7 and 82.7% for the RPB2 region (Table 2). The BT2 region did not amplify well across species of Lecanosticta. The amplification success rate and subsequent sequencing of the BT2 region using the T1 and β-Sandy-R primer pair, as well as Bt2a and Bt2b was very poor and further analysis of the BT2 region was abandoned.

Phylogenetic analyses

For the analyses, the datasets of the ITS region consisted of 153 taxa with 734 aligned nucleotides including gaps; the TEF1 dataset consisted of 147 taxa with 586 aligned nucleotides, the BT1 dataset consisted of 111 taxa with 440 aligned nucleotides; the MS204 dataset consisted of 91 taxa with 785 aligned nucleotides, and the RPB2 dataset consisted of 105 taxa with 929 aligned nucleotides, all including gaps. The PHT test yielded a P value = 0.01 and therefore the five datasets were considered incongruent. However, it was previously argued that a P value > 0.01 did not reduce phylogenetic accuracy (Cunningham 1997) and a combined phylogenetic tree representing the five gene regions ITS, TEF1, BT1, MS204 and RPB2 was constructed for presentation purposes (Fig. 1). The combined dataset consisted of 76 taxa with 3344 aligned nucleotides including gaps. Constant characters, parsimony-uninformative and informative characters, the consistency index (CI), homoplasy index (HI), rescaled consistency index (RC), retention index (RI) and tree length (TL) values for the maximum parsimony analyses are indicated in Table 4. For the parsimony analyses, 108 trees were retained for ITS, 396 for TEF1, 1 for BT1, 2448 for MS204 and 420 for RPB2. The best fit substitution models for ML and BI were selected by Akaike Information Criterion (AIC) and are indicated in Table 4. A 10% burn-in value was selected in the BI analysis for each of the data matrices for each of the analyses. Because the MP, ML and BI analysis all resulted in similar tree topologies, the ML trees were selected and chosen for presentation (Figs. 1 and 2, Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3 and Additional file 4: Figure S4).
Fig. 1
Fig. 1

Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the combined data of the ITS, TEF1, BT1, MS204 and RPB2 gene regions. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma septosporum was used as the outgroup taxa. The indicated clades are referred to in the text. All represented type species are indicated in bold and with a “T”

Fig. 2
Fig. 2

Maximum likelihood tree representing the five known and four novel species of Lecanosticta generated from the TEF1 region. MP bootstrap support (> 70%) are indicated first, followed by ML bootstrap values (MP/ML, * = insignificant value). Bold branches indicate BI values > than 0.95. Dothistroma species were used as the outgroup taxa. All represented type species are indicated in bold and with a “T”. Clades indicated on the left correspond with the clades in Fig. 1. Within the L. jani clade a “∆” next to the isolate indicates that the isolate either exhibits Type 2 morphology and groups with Subclade 1, or, exhibits Type 1 morphology and groups with Subclade 2

Phylogenetic analyses of the combined dataset (Fig. 1), ITS (Additional file 1: Figure S1), TEF1 (Fig. 2) and MS204 (Additional file 3: Figure S3) consistently grouped the isolates sequenced in this study into seven distinct clades. The clades in Fig. 2 and Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3 and Additional file 4: Figure S4 are labelled according to the clades assigned in Fig. 1. In the case of RPB2 (Additional file 4: Figure S4) Clades 1–4, and 7 were also present but Clades 5 and 6 were not distinct from each other for this particular gene region. In the case of BT1 (Additional file 2: Figure S2), Clades 3, 5 and 6 could not be distinguished from each other. None of the isolates grouped with the types of L. gloeospora or L. longispora.

Forty-two of the isolates from Central America grouped in Clade 1 based on the ITS analysis (Additional file 1: Figure S1) and were identified as Lecanosticta brevispora. This was the most common species identified from the Central American collection and most isolates were from Chimaltenango on Pinus pseudostrobus. The pathogen was also isolated from P. oocarpa needles near Jalapa as well as near Tactíc in Guatemala and in Honduras. This clade was well supported for all five of the gene regions analysed.

Twenty-seven isolates grouped into Clade 2 in the ITS analyses (Additional file 1: Figure S1) and represent an undescribed species. Clade 2 resolved into two subclades in the five gene analyses. Subclade 1 was mostly isolated from Chiquimula and Alta Verapaz in Guatemala on P. oocarpa, P. maximinoi and P. tecunumanii as well as from P. oocarpa in Nicaragua. Isolates collected in Jalapa in Guatemala mostly grouped into Subclade 2. However, the topology of isolates CMW 47109 (Subclade 1 on Additional file 1: Figure S1, Additional file 3: Figure S3, Additional file 4: Figure S4; Subclade 2 on Fig. 2), CMW 51059 (Subclade 1 on Additional file 1: Figure S1, Additional file 3: Figure S3, Additional file 4: Figure S4), IB30.2b (Subclade 1 on Additional file 1: Figure S1, Additional file 3: Figure S3; Subclade 2 on Fig. 2) and IB30.2b (Subclade 1 on Additional file 1: Figure S1, Additional file 3: Figure S3, Additional file 4: Figure S4; Subclade 2 on Fig. 2) changed in the two subclades depending on the gene region analysed (Fig. 2, Additional file 1: Figure S1, Additional file 3: Figure S3, Additional file 4: Figure S4). Furthermore, the two subclades were not well supported for the BT1 gene region. Therefore, the two subclades are treated here as representing a single species.

Clade 3 also represented an undescribed Lecanosticta species. This clade included 11 isolates from P. oocarpa in Jalapa, Guatemala, one isolate from P. oocarpa in Honduras, as well as five isolates collected from Baja Verapaz in Guatemala on P. tecunumanii. This clade had high bootstrap support for TEF1, MS204 and RPB2 but was not well supported in the ITS and BT1 gene regions. Three isolates collected from different needles on a single P. tecunumanii tree in Baja Verapaz in Guatemala grouped together in Clade 4 and represent another undescribed species. With the exception of BT1, Clade 4 was statistically well supported in all the gene regions that were analysed.

Clade 5 accommodated sequences representing nine of the 14 known TEF1 haplotypes of L. acicola identified by Janoušek et al. (2016). These TEF1 haplotypes represent isolates collected from North America (Canada, USA, and Mexico), South America (Colombia), Europe (Spain, France, Switzerland, Slovenia, Lithuania, Italy, Germany, Estonia, Czech Republic, Croatia, and Austria) and Asia (South Korea, Japan, and China) (Table 3). This clade was clearly distinct from other clades in the ITS, TEF1, BT1 and MS204 phylogenetic analysis and statistically well supported in the ITS, TEF1, and MS204 analyses. Clade 5 included the ex-type of L. acicola and therefore is that species. None of the isolates from Central America obtained in the present study grouped with this clade in any of the gene regions analysed.

The remaining five assigned L. acicola TEF1 haplotypes considered by Janoušek et al. (2016), grouped together in Clade 6. This was together with an isolate obtained from P. caribaea in Honduras collected in 1983 (Evans 1984), four isolates obtained in the present study from Guatemala on P. oocarpa and P. maximinoi, and an isolate previously identified as L. acicola from Mexico on an unknown Pinus species (Quaedvlieg et al. 2012). In the present study, Clade 6 is treated as a novel taxon. The ITS, TEF1, BT1 and MS204 gene regions clearly distinguish Clades 5 and 6, however, RPB2 was not effective in resolving these two groups.

The second most abundant species collected in this study was Lecanosticta guatemalensis, represented by Clade 7 in the phylogenetic analyses. This clade was well supported in all five gene regions that were analysed. A total of 37 isolates from our collection grouped together with L. guatemalensis based in the ITS and TEF1 analyses. Lecanosticta guatemalensis was identified on P. maximinoi and P. oocarpa in various regions of Guatemala, as well as on P. oocarpa in Nicaragua. Isolates that had previously been collected in Nicaragua and Honduras and that were identified as L. acicola by Evans (1984) based on morphological characteristics also grouped with L. guatemalensis in the present study.

TAXONOMY

Using phylogenetic analyses, 51 of the Lecanosticta isolates obtained from Guatemala, Honduras and Nicaragua, one isolate obtained from CBS, and one isolate obtained from IMI, were found to include four undescribed species. These are described below as follows:

Lecanosticta jani van der Nest, M.J. Wingf. & I. Barnes, sp. nov.

MycoBank MB 826875. (Fig. 3)

Etymology: The name is derived from Janus, the Roman god of gates and doorways having two faces or sides, and refers to the variable culture morphology ranging from light pink and fluffy to dark olive green and mucoid.

Diagnosis: Lecanosticta jani can be distinguished from the closely related L. brevispora by the distinct globose basal cells on the conidiophores that are mostly observed on MEA.

Type: Guatemala: Jalapa, Finca la Soledad, Mataquescuintla, on needles of Pinus oocarpa, 20 Sept 2012, I. Barnes (PREM 62185 – holotype; CMW 38958 = CBS 144456 – ex-type culture).

Description: Sexual morph unknown. Conidiomata isabelline to vinaceous brown on MEA. Conidiophores subcylindrical, often with a swollen globose basal cell, densely aggregated, honey to hyaline, smooth to verruculose, unbranched or branched at base, often encased in a yellow to light brown mucoid sheath, to 82 μm in length, 4.5–7.0 μm diam. Conidiogenous cells terminal, integrated, subcylindrical, honey to hyaline, smooth to verruculose, proliferating several times percurrently with visible annelations near apex, septate or aseptate, (8.5–)16.5(− 24.0) × (3.0–)4.5(− 6.5) μm. Conidia solitary, sub-cylindrical to narrowly fusoid-ellipsoidal, with subobtusely rounded apex, base truncate, brown, verruculose, frequently with mucoid sheath, two distinct sizes with conidial type one more abundant than conidial type two. Conidial type 1: 1–2-septate, base (1.5–)2.0–2.5(− 3.5) μm diam, (9.5–)14.5–21.5(− 30.0) × (2.0–)2.5–3.5(− 4.0) μm. Conidial type 2: 1–3-septate, base (1.5–)2.0–2.5(− 3.0) μm diam, (26.5–)30.5–37.0(− 38.0) × (2.0–)2.5–3.0(− 3.5) μm.

Culture characteristics: Colonies with two distinct morphologies. One type (Type 1), flat to somewhat erumpent, spreading with flat to fluffy aerial mycelium. A second type (Type 2) erumpent, mucoid and shiny, with irregular form and undulate to filiform edges. On MEA, the surface of Type 1 isolates pale to rosy vinaceous, reverse flesh to peach coloured. Type 2 isolates citrine to isabelline, reverse olivaceous to fuscuous black (Fig. 3). On PDA, Type 1 surface rosy vinaceous to peach in centre with dark brown edge, isabelline in reverse. Type 2, surface dark olivaceous with fuscious black centres and tufts of isabelline mycelium at edges, dark isabelline in reverse. On OA, Type 1 surface dirty white to pale vinaceous, fluffy mycelia to flat growth. Type 2 surface flat with smooth edge, fuscious black in centre at the point of inoculation with light apricot surrounding mycelium. Growth characteristics: optimal growth temperature for Type 1 isolates 25 °C, after 4 wk., colonies at 10, 15, 20, 25 and 30 °C reached maximum of 10.5, 22, 32, 32 and 10 mm respectively, with mean growth rate of 2.1, 5.1, 6.9, 7 and 1.8 mm / wk. respectively. Type 2 isolates optimal growth temperature 20 °C, after 4 wk., colonies at 10, 15, 20, 25 and 30 °C reached maximum of 12.5, 17, 29.5, 22 and 4.5 mm, with mean growth of 2.1, 3.3, 5.5, 5 and 1 mm / wk. respectively.
Fig. 3
Fig. 3

Lecanosticta jani (CMW38958; CMW38950; CMW48831; CMW47109; CMW51058; CMW51143) a-b Two wk. old colonies on MEA. A represents Type 1 colonies (CMW38950) and B represents Type 2 colonies (CMW48831). c-h Conidiogenous cells giving rise to conidia on MEA, with swollen globose basal cells of the conidiophores in E, F and H as well as annelations (see arrow) in G. i-k Swollen conidiogenous cells and conidia on MEA. Note endospore formation and germination in I. l Conidia on MEA. Bars: K = 50 μm; C-F and H-L = 10 μm; G = 5 μm

Notes: Lecanosticta jani resolved in a distinct clade (Clade 2, Figs. 1 and 2, Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3 and Additional file 4: Figure S4) based on all five gene regions considered. This clade divides into two subclades that were mostly represented by isolates obtained from Alta Verapaz and Chiquimula in Guatemala as well as in Nicaragua in subclade 1 and isolates obtained from Jalapa in Guatemala in subclade 2. Jalapa isolates all had the Type 2 morphology and the dark colour was associated with conidial production. Type 1 isolates produced few spores after 2 wk. The optimal growth temperature and growth rates were different for the two isolate types. However, the topology of some isolates changed between the two subclades depending on the gene region that is analysed and therefore the subclades are treated as one species. The morphological variation suggests that the two types could represent two ecotypes.

Additional material examined: Guatemala: Alta Verapaz, Santa Cruz Verapaz, near Tactíc, on needles of Pinus oocarpa, 21 Oct 2010, I. Barnes (culture CMW47109); loc. cit. I. Barnes (PREM 62187; CMW 48831 = CBS 144447 – culture); Jalapa, Finca la Soledad, Mataquescuintla, on needles of Pinus oocarpa, 20 Sept 2012, I. Barnes (PREM 62186, CMW 38950 = CBS 144446 – culture); Jalapa, Finca la Soledad, Mataquescuintla, on needles of Pinus tecunumanii, 20 Sept 2012, I. Barnes (cultures CMW 51058, CMW 51059). -Nicaragua: Matagalpa, on needles of Pinus oocarpa, 20 June 2011, I. Barnes (culture CMW 51143).

Lecanosticta pharomachri van der Nest, M.J. Wingf. & I. Barnes, sp. nov.

MycoBank MB 826876. (Fig. 4)

Etymology: The epithet refers to the Resplendid Quetzal (Pharomachrus mocinno), which is the national bird of Guatemala and the spirit bird/companion of Tecún Umán; a Guatemalan legend.

Diagnosis: Lecanosticta pharomachri is distinguished from the other taxa in the genus by all five gene regions investigated but especially by sequences of TEF1, MS204 or RPB2. Conidia are also larger than those of L. guatemalensis and similar to L. acicola but differ from these species in that the conidia are frequently surrounded by a thick mucoid sheath and are mostly straight.

Type: Guatemala: Baja Verapaz, San Jerónimo, Salamá, on needles of Pinus tecunumanii, Nov 2010, I. Barnes (PREM 62188 – holotype; CMW 37136 = CBS 144448 – ex-type cultures).

Description: Sexual morph not observed. Conidiomata dark vinaceous brown on MEA. Conidiophores subcylindrical to cylindrical, densely aggregated, vinaceous brown to hyaline, smooth to verruculose, unbranched or branched at base, often encased in a light brown mucoid sheath, to 45 μm in length, 2.5–4.0 μm diam. Conidiogenous cells terminal, integrated, subcylindrical to cylindrical, luteus brown to hyaline, smooth to verruculose, surrounded by mucilage, holoblastic, proliferating several times percurrently with visible annelations near apex, septate or aseptate, (6.5–)9.5–13.5(− 16.0) × (1.5–)2.0–2.5(− 3.0) μm. Conidia released in a greenish olivaceous to honey mass, solitary, straight to slightly curved, cylindrical, with subobtusely rounded apex, base truncate, guttulate, hyaline to light brown, verruculose, frequently with thick mucoid sheath, 1–3-septate, base (1.5–)2.0–3.0(− 3.5) μm diam, (21.0)25.0–34.0(− 49.0) × (2.5–)3.0–4.0(− 5.0) μm. Germ tubes observed between conidia as well as conidial budding - secondary conidia sometimes produced from apical cell, 0–2-septate.

Culture characteristics: Colonies flat to erumpent, form irregular with undulate edge, spreading with fluffy aerial mycelium at centers. On MEA, surface apricot to cinnamon with isabelline and rosy buff mycelial mat at centers, reverse isabelline to dark brick in centre with cinnamon to apricot edges. On PDA, surfaces rosy to pale vinaceous with light isabelline to greenish white edges, reverse isabelline with cream edges. On OA, surface dirty white to isabelline to dark brown, fluffy mycelium to flat growth. Growth characteristics: optimal growth temperature 20 °C, after 4 wk., colonies at 10, 15, 20, 25, and 30 °C reaching a maximum of 9, 17, 18.5, 18.5 and 8.5 mm diam, with mean growth rates of 1.9, 3.6, 4.6, 4.4, and 1.9 mm / wk. respectively.

Notes: Some of the isolates, including the ex-type strain, produced a luteus exudate that diffused into MEA after 4–6 wk. Conjugation tubes were reported previously in L. acicola cultures as well as in needles (Siggers 1950; Crosby 1966). Conjugation tubes were also observed in this species (Fig. 4g) in the present study. Endospores as described by Crosby (1966) were also observed in some conidia.
Fig. 4
Fig. 4

Lecanosticta pharomachri (CMW 37136; CMW38947). a, b Two wk. old colonies on MEA. c-e Conidiogenous cells giving rise to conidia on MEA. f, g Conjugation tube formation between conidia as well as conidia bearing smaller conidial cells. h-j Variation in conidia on MEA. Bars: D, F-H and J = 10 μm; C, E and I = 5 μm

Additional material examined: Guatemala: Jalapa, Finca la Soledad, Mataquescuintla, on needles of Pinus oocarpa, 20 Sept 2012, I. Barnes (cultures CMW 38976, CMW 51053 and CMW 51054); loc. cit., I. Barnes (PREM 62189; CMW 38947 = CBS 144695 – culture; PREM 62190, CMW 38974 = CBS 144449 – culture).

Lecanosticta tecunumanii van der Nest, M.J. Wingf. & I. Barnes, sp. nov.

MycoBank MB 826877. (Fig. 5)

Etymology: Name refers to the Guatemalan legend, Tecún Umán, and Pinus tecunumanii, the host plant from which the holotype was collected.

Diagnosis: Lecanosticta tecunumanii is distinguished from the other taxa by the ITS, TEF1, MS204 and RPB2 gene regions. Morphologically, it is distinct in having only 1-septate conidia after 2 wk. of incubation on MEA, but 2-septate and 3-septate conidia are occasionally observed in older cultures.

Type: Guatemala: Baja Verapaz, San Jerónimo, Salamá, on needles of Pinus tecunumanii, Oct 2011, I. Barnes (PREM 62191 – holotype; CMW 46805 = CBS 144450 – ex-type cultures).

Description: Sexual morph not observed. Conidiomata isabelline to visaceous brown on MEA. Conidiophores cylindrical, densely aggregated, hyaline to pale yellow-brown, smooth to slightly verruculose, unbranched or branched at base, to 120 μm in length, 2.0–5.0 μm diam. Conidiogenous cells terminal or indeterminate, integrated or discrete, cylindrical, hyaline to honey, smooth to verruculose, proliferating several times percurrently with visible annelations near apex or micronematous, septate or aseptate, (5.0–)7.0–14.5(− 15.5) × (1.5–)2.0–2.5(− 3.0) μm. Micronematous cells (6–)10.5–18.5(− 27.0) × (2.0–)2.0–2.5(− 3.0) μm. Conidia solitary, straight to slightly curved, subcylindrical to fusiform, with subobtusely rounded or sharply pointed apex, base truncate, guttulate, smooth to granulate, hyaline to cream buff to light brown, occasionally enclosed in mucoid sheath, 1-septate, base (1.5–)1.5–2.0(− 2.0) μm diam., (14.5–)16.0–21.0(− 24.0) × (2.0–)2.5–3.0(− 3.5) μm.

Culture characteristics: Colonies somewhat erumpent, spreading with flat to fluffy aerial mycelium. On MEA, surface olivaceous to isabelline with rosy buff mycelial tufts, reverse isabelline. On PDA, surface rosy vinaceous to peach in centre with a dark brown edge, isabelline in reverse. On OA, surface dirty white to pale vinaceous, fluffy mycelia to flat peach growth. Growth characteristics: optimal growth temperature 25 °C, after 4 wk., colonies at 10, 15, 20, 25, and 30 °C reached maximum of 9, 15.5, 24, 24, and 4.5 mm, with mean growth of 2.2, 3.8, 5.3, 5.7, and 1.1 mm / wk. respectively.

Notes: Micronematous conidiogenesis (Fig. 5E - F), observed more frequently than distinct conidiophores in culture.
Fig. 5
Fig. 5

Lecanosticta tecunumanii (CMW46805; CMW46812). a Two wk. old colony on MEA. b-d Conidiogenous cells giving rise to conidia on MEA. e-f Micronematous conidiogenesis observed on MEA with conidia. g-h Uniseptate conidia with or without a mucoid sheath observed on MEA. Bars: B-G = 10 μm; H = 5 μm

Additional material examined: Guatemala: Baja Verapaz, San Jerónimo, Salamá, on needles of Pinus tecunumanii, Oct 2011, I. Barnes (PREM 62192, CMW 49403 = CBS 144451 – culture; PREM 62193, CMW 46812 = CBS 144452 – culture).

Lecanosticta variabilis van der Nest, M.J. Wingf. & I. Barnes, sp. nov.

MycoBank MB 826878. (Fig. 6)
Fig. 6
Fig. 6

Lecanosticta variabilis (CMW42205; CMW37125). a Colony on MEA with luteus exudate diffusing into medium. b-c Conidiogenous cells giving rise to conidia on MEA. d-h Various conidial shapes and sizes on MEA. f Germinating conidia on MEA. g-h Swollen conidial cells giving rise to smaller conidia. i Conjugation tube formation between two conidia. j Conidium disintegrating on MEA. Bars: B-C, F-I = 10 μm; E, J = 5 μm; D = 2,5 μm

Etymology: The epithet refers to the variable size and shape of the conidia.

Diagnosis: Lecanosticta variabilis is distinguished from the closely related species, L. acicola, by either ITS, TEF1 or MS204. Morphologically, it is distinguished from other species with the exception of L. acicola by the diffusion of sulphur-yellow to cinnamon metabolite into PDA and a luteus to sienna coloured metabolite produced on MEA within 2 wk. This species also has smaller conidia than those of L. acicola.

Type: Honduras: Santa Barbara, on needles of Pinus caribaea, 1980, H.C. Evans, (PREM 62196 – holotype; CMW 42205 = IMI 281561 = CBS 144453 – ex-type culture).

Description: Sexual state not observed. Conidiomata olivaceous to vinaceous brown on MEA. Conidiophores cylindrical, extending in densely aggregated palisade, hyaline to honey to pale vinaceous brown, smooth to verruculose, unbranched or branched at base, septate or aseptate, often encased in granular yellow to light brown mucoid sheath, length up to 60 μm, 2.0–5.0 μm diam. Conidiogenous cells terminal, integrated, subcylindrical to cylindrical, hyaline to light brown, smooth to verruculose, proliferating several times percurrently with visible annelations near apex, septate or aseptate, (4.5–)5.5–10.5(− 12.0) × (1.5–)2.0–3.5(− 5.0) μm. Conidia three different conidial types. All three types solitary, smooth to verruculose, subhyaline to honey to light brown, often enclosed in granular light luteus mucoid sheath. Type 1 straight to strongly curved, subcylindrical to cylindrical, subobtusely rounded apex, truncate, 1–4-septate, base (1.5–)2.0–2.5(− 3.0) μm diam. (22–)25.0–34.0(− 43.0) × (2.0–)2.5–3.0(− 3.5) μm. Type 2 slightly curved, cylindrical with both apex and base rounded, 0–2-septate, (14.5–)15.5–19.5(− 22.0) × (2.0–)2.5–3.0(− 3.5) μm. Type 3 buds from larger conidia (see notes) or from conidiogenous cells, hyaline, fusiform to cylindrical with subobtusely rounded apex and base, 0–1-septate, (10.0–)11.0–14.0(− 15.5) × (2.0–)2.0–2.5(− 3.0) μm.

Culture characteristics: Colonies flat to somewhat erumpent, spreading, with sparse aerial mycelium, surface folded, with smooth, lobate margins. On MEA, surface isabelline with patches of pale luteus to dark olivaceous green, reverse olivaceous to fuscous black. Mucoid yellow to peach to yellow-green exudate present. Luteus to sienna coloured metabolite diffusing into medium. On PDA, surface isabelline in centre, rosy buff in outer region, dark olivacous-brown on edges and isabelline in reverse. Sulphur yellow to cinnamon coloured metabolite diffuses into media. On OA, surface dirty white with diffuse umber outer region. Growth characteristics: optimal growth temperature 25 °C, after 4 wk., colonies at 10, 15, 20, 25 and 30 °C reached maximum of 11.5, 21, 31, 31.5 and 22.5 mm, with mean growth of 2.2, 4.5, 6.1, 6.9 and 3.6 mm / wk. respectively.

Notes: The cells in the conidia often swell and break off, forming endospores as described in L. acicola (Siggers 1950; Crosby 1966; Evans 1984). Secondary conidia were commonly produced in cultures of this species, similar to those previously described for L. acicola specimens examined directly from needles (Evans 1984).

Additional material examined: Guatemala: Alta Verapaz, Santa Cruz Verapaz, near Tactíc, on needles of Pinus oocarpa, 21 Oct 2010, I. Barnes (PREM 62194, CMW 37125 = CBS 144454 – culture); loc. cit., I. Barnes (culture CMW 37129); Jalapa, Finca Forestal Soledad, on needles of Pinus maximinoi, 21 Oct 2010, I. Barnes (PREM 62195, CMW 36809 = CBS 144455 – culture). –Mexico: on needles of a Pinus sp., 30 Nov 2009, M. de Jesús Yáñez-Morales (CBS H-21112; culture CMW45425 = CPC 17822 = CBS 133789);

DISCUSSION

Four novel species of Lecanostica from infected pine needles collected in Central America are reported and named as L. jani, L. pharomachri, L. tecunumanii, and L. variabilis. There are now nine species described in the genus and these can be distinguished based on a phylogenetic inference for multiple gene regions. The two previously described species, L. brevispora and L. guatemalensis, were also found in this study and they provide new host and country records. The well-known pine pathogen, L. acicola, was not found on any of the samples collected from five Pinus spp. in seven regions of Central America considered in this study. This suggests that the species is not native in that region.

Results of the present study support the view of Quaedvlieg et al. (2012) that a combination of the ITS and TEF1 should be used as barcoding loci to distinguish between species of Lecanosticta and other closely related species. Additionally, statistically well supported clades were obtained in this study using the MS204 gene region. However, genus-specific primers should ideally be designed to increase the amplification success rate for this gene region in Lecanosticta. Although the BT2 gene was also proposed as a possible barcoding region that could be used to distinguish between Lecanosticta species and other species of Mycosphaerellaceae (Quaedvlieg et al. 2012), it amplified poorly in the present study. The BT1 gene region distinguished most of the species, but not L. pharomachri and L. variabilis and provided low statistical support at all nodes.

The results of this study support the view of Evans (1984) that Lecanostica species are comprised of morphotypes or ecotypes. Based on phylogenetic analyses, we were able to define lineages for species also supported by morphological characteristics. The TEF1 sequences were highly variable but several well supported clades and subclades were observed within species (Fig. 2). These clades possibly represent additional new species but we lacked sufficient cultures and support to describe them. The clade with the most diversity in terms of unique TEF1 haplotypes, Clade 1, was L. brevispora (represented by 22.1% of TEF1 haplotypes in the genus) and this species was also represented by the largest number of isolates. High haplotype diversity was observed in the L. jani (16.1% of TEF1 haplotypes) and L. pharomachri (10.3% of TEF1 haplotypes) clades and different lineages were observed in the L. acicola (13.2% of TEF1 haplotypes), L. guatemalensis (17.6% of TEF1 haplotypes), and L. variabilis (13.2% of TEF1 haplotypes) clades. The other gene regions, especially MS204 and RPB2 were also highly variable in terms of distinguishing haplotypes. RPB2 is however, not recommended to distinguish between L. acicola and L. variabilis as these two species form paraphyletic groups in the tree for this gene region.

The paleo-geographic region that includes Mexico and extends into Central America is regarded as one of three centres of diversity of Pinus species (Farjon 1996). Pine needles that were sampled from Central America in this study were symptomatic but serious disease was not observed. This suggests that Lecanosticta species have co-speciated with their native pine hosts in this region. Of the nine known species, L. gloeospora and L. longispora have been identified only in Mexico and L. brevispora and L. variabilis have been identified in both Mexico and Central America. Lecanosticta guatemalensis, L. jani, L. pharomachri and L. tecunumanii are currently known only from Central America.

Lecanosticta acicola has been redefined in this study. All isolates from Central America that had previously been identified as L. acicola, based on morphological characteristics, are now treated as different species. This is based on newly available DNA sequence data and phylogenetic analyses emerging from this study as well as that of Quaedvlieg et al. (2012). L. acicola is, however, still considered as present in Mexico.

Based on TEF1 analyses, L. acicola resolves in three lineages. Janoušek et al. (2016) used microsatellites to show that a lineage of L. acicola from the northern USA was introduced into Central and Northern Europe, and a lineage from the southern USA was introduced into France, Spain, and Colombia. Similarly, Huang et al. (1995) reported that L. acicola was introduced into China from the southern part of the USA. Our analyses of the TEF1 sequences of isolates from the northern parts of the USA, Lithuania, and a representative sequence for Central and Northern Europe and Canada (KJ938438, Table 3), formed one distinct lineage with L. acicola (Fig. 2). All isolates from the southern parts of the USA, as well as representative sequences for Asia, France, Spain, and Colombia (Table 3), formed a second distinct lineage in the clade accommodating L. acicola (Fig. 2). The third lineage included only isolates from Mexico, which suggests that isolates in this lineage have remained in their area of origin and have not been introduced elsewhere. Because this Mexican lineage had strong bootstrap support separating it from the other two lineages, it could represent a further new species. Only TEF1 data are currently available for the Mexican collections (downloaded from GenBank) and other gene regions would need to be sequenced and analysed to determine whether this really represents a further novel taxon.

Evans (1984) first speculated that Central America could be the centre of origin of Lecanosticta. The phylogenetic analyses conducted in the present study showed that there is a high diversity of species and lineages for this genus in Central America, which supports Evans’ hypothesis. This is the first study where all known species of Lecanosticta have been delineated based on DNA sequence data and phylogenetic analysis, and it has led to the recognition of additional new taxa from Central America and Mexico. Eight of the nine species of Lecanosticta have been reported only from this region, and our results consequently represent strong support for a Mesoamerican Lecanosticta centre of diversity and likely origin. Population genetic analyses for the most common of these species will serve to provide additional support for this hypothesis.

CONCLUSIONS

Phylogenetic inference based on DNA sequence data including new collections from Mexico and Central America revealed four novel species and reaffirmed the identity of the five previously described taxa. The most important of these species is the well-known pine pathogen L. acicola that was redefined as a North American taxon and for which at least three distinct lineages can be distinguished using the TEF1 gene region. New regions of occurrence and host range emerged for Lecanosticta spp. with eight of the nine species occurring in Mesoamerica. This suggests that Mesoamerica is the most likely centre of origin for Lecanosticta. Lecanosticta acicola was best known as the causal agent of the important brown spot needle blight of Pinus palustris in the southeastern USA but it has more recently spread within the USA and Europe where it has become an increasingly important pathogen of numerous Pinus spp. The other species of Lecanosticta, including those newly described, are of unknown importance but it seems likely that some of them could pose a threat to Pinus spp. if they were introduced into new environments in the future. The fact that various Mesoamerican Pinus spp. are increasingly being used for plantation development in the Southern Hemisphere implies that extreme caution should be applied not to introduce Lecanosticta spp. together with germplasm needed for future planting programmes.

Abbreviations

1F1N: 

One Fungus One Name

AIC: 

Akaike Information Criterion

BI: 

Bayesian inference

BSNB: 

Brown spot needle blight

BT1: 

Beta-tubulin-1 gene region

BT2: 

Beta-tubulin-2 gene region

CA: 

California

CBS: 

The culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands

CI: 

Consistency index

CMW: 

The culture collection of the Forestry and Agricultural Biotechnology Institute

COSAVE: 

El Comité de Sanidad Vegetal

CPC: 

Personal collection of Pedro Crous housed at CBS

DSM: 

Dothistroma Sporulating Media

FABI: 

Forestry and Agricultural Biotechnology Institute

HI: 

Homoplasy index

IASPC: 

Inter-African Phytosanitary Council

ICN: 

International Code of Nomenclature for algae, fungi, and plants

IMI: 

The UK National Fungus Collection maintained by CABI Bioscience, Egham, UK

ITS: 

Internal transcribed spacers

MA: 

Massachusetts

MB: 

MycoBank

MCMC: 

Markov Chain Monte Carlo

MD: 

Maryland

ME: 

Maine

MEA: 

Malt Extract Agar

ML: 

Maximum likelihood

MO: 

Missouri

MP: 

Maximum parsimony

MS204

The guanine nucleotide-binding protein subunit beta

NCBI: 

National Centre for Biotechnology Information

NJ: 

New Jersey

OA: 

Oatmeal Agar

PCR: 

Polymerase chain reaction

PDA: 

Potato Dextrose Agar

PHT: 

Partition homogeneity test

PREM: 

The dried herbarium collection of the South African National Collection of Fungi

RC: 

Rescaled consistency index

RI: 

Retention index

RPB2: 

RNA polymerase II second largest subunit

TBR: 

Tree-bisection-reconnection

TEF1: 

Translation elongation factor 1-α gene

TL: 

Tree length

Declarations

Acknowledgements

We thank Jeff Garnas and Elmer Gutierrez from Camcore for their assistance in collecting pine needle samples. We also wish to thank Josef Janoušek and Yves du Toit for their assistance in isolating Lecanosticta spp. from the infected pine needles.

Funding

This project was financed by the National Research Foundation of South Africa (Thuthuka Grant no 80670, and Grant no 95875) as well as by members of the Tree Protection Cooperative Program (TPCP). AvdN was supported by a Scarce Skills Doctoral Scholarship (no 89086) provided by the National Research Foundation of South Africa. The NRF acknowledge that opinions, findings, conclusions and/or recommendations expressed in any publication generated by the NRF supported research are that of the author(s), and that the NRF accepts no liability whatsoever in this regard. The NRF had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

All data generated in this study are included in this published article and its supplementary files. The datasets analysed are available from the corresponding author on reasonable request.

Authors’ contributions

Acquisition of sample material was performed by PO and IB. Fungal isolations were done by IB. Data collection and all analyses were performed by AvdN. Funding acquisition was done by IB and MJW. IB and MJW supervised the project. AvdN wrote the original draft, and review and editing was performed by AvdN, IB MJW and PO. All authors read and approved the 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)
Forestry and Agricultural Biotechnology Institute (FABI), Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Pretoria, 0002, South Africa
(2)
Instituto Nacional de Bosques (INAB), Guatemala City, Guatemala

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