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  • Article
  • Open Access

Novel primers improve species delimitation in Cercospora

IMA Fungus20189:902299

https://doi.org/10.5598/imafungus.2018.09.02.06

  • Received: 14 March 2018
  • Accepted: 11 September 2018
  • Published:

Abstract

The genus Cercospora includes many important plant pathogens that are commonly associated with leaf spot diseases on a wide range of cultivated and wild plant species. Due to the lack of useful morphological features and high levels of intraspecific variation, host plant association has long been a decisive criterion for species delimitation in Cercospora. Because several taxa have broader host ranges, reliance on host data in Cercospora taxonomy has proven problematic. Recent studies have revealed multi-gene DNA sequence data to be highly informative for species identification in Cercospora, especially when used in a concatenated alignment. In spite of this approach, however, several species complexes remained unresolved as no single gene proved informative enough to act as DNA barcoding locus for the genus. Therefore, the aims of the present study were firstly to improve species delimitation in the genus Cercospora by testing additional genes and primers on a broad set of species, and secondly to find the best DNA barcoding gene(s) for species delimitation. Novel primers were developed for tub2 and rpb2 to supplement previously published primers for these loci. To this end, 145 Cercospora isolates from the Iranian mycobiota together with 25 additional reference isolates preserved in the Westerdijk Fungal Biodiversity Institute were subjected to an eight-gene (ITS, tef1, actA, cmdA, his3, tub2, rpb2 and gapdh) analysis. Results from this study provided new insights into DNA barcoding in Cercospora, and revealed gapdh to be a promising gene for species delimitation when supplemented with cmdA, tef1 and tub2. The robust eight-gene phylogeny revealed several novel clades within the existing Cercospora species complexes, such as C. apii, C. armoraciae, C. beticola, C. cf. flagellaris and Cercospora sp. G. The C. apii s. lat. isolates are distributed over three clades, namely C. apii s. str, C. plantaginis and C. uwebrauniana sp. nov. The C. armoraciae s. lat. isolates are distributed over two clades, C. armoraciae s. str. and C. bizzozeriana. The C. beticola s. lat. isolates are distributed over two clades, namely C. beticola s. str. and C. gamsiana, which is newly described.

Key words

  • Bar codes
  • biodiversity
  • Cercospora apii complex
  • host specificity
  • multi-gene phylogeny
  • new taxa

Introduction

Fungi belonging to the genus Cercospora (Mycosphaerellaceae, Capnodiales) are common etiological agents of leaf spots, but some also cause necrotic lesions on flowers, fruits, bracts, seeds and pedicels of many woody and herbaceous plants in a range of climates worldwide (Ellis 1976, Crous & Braun 2003, Agrios 2005, Groenewald et al. 2013, Bakhshi et al. 2015a).

Cercospora is a species-rich genus of cercosporoid fungi that was established by Fresenius (1863) for passalora-like species with pluriseptate conidia. During the course of the next 100 years, the concept of Cercospora had been continuously widened (Saccardo 1880, Solheim 1930) and all kinds of superficially similar species, with or without conspicuous conidiogenous loci, with hyaline or pigmented conidia, formed singly or in chains, were assigned to this genus (Braun et al. 2013). In 1954, the genus was monographed by Chupp (1954), who treated 1419 Cercospora species while applying this broad generic concept. He also stated that species of Cercospora were generally host-specific and used this argument as the basis of formulating the concept that each plant host genus or family would have its own Cercospora species. The number of Cercospora species increased rapidly to more than 3000, which led Pollack (1987) to publish her annotated list of Cercospora names. Since the introduction of the genus, several attempts to split Cercospora s. lat. into smaller generic units have been made by applying a combination of characters such as conidiomatal structure, mycelium, conidiophores, conidiogenous cells, and conidia (e.g. Deighton 1973, 1979, 1983, Ellis 1971, 1976, Braun 1995, 1998). Crous & Braun (2003) published an annotated list of the names published in Cercospora and Passalora and used the structure of conidiogenous loci and hila as well as the absence or presence of pigmentation in conidiophores and conidia in their revision. They recognised 659 names in Cercospora, with a further 281 species names reduced to synonymy with C. apii s. lat., since they were morphologically not or barely distinguishable from C. apii s. str. Braun et al. (2013, 2014, 2015a, b, 2016) published a series of papers in a stepwise approach at plant family level in order to update the monograph of Cercospora and allied genera.

Scientific advances in DNA sequencing and supplementary software to store, share and compare the emerging molecular data have revolutionised the procedures underpinning the discovery and identification of fungal taxa, including the cercosporoid fungi (Crous & Groenewald 2005, Groenewald et al. 2013, Bakhshi et al. 2015a, Nguanhom et al. 2015, Guatimosim et al. 2017). Numerous molecular studies of Cercospora species have been conducted based on ITS nrDNA data as well as multi-gene approaches (Stewart et al. 1999, Crous et al. 2000, 2004b, 2009a, b, Goodwin et al. 2001, Tessmann et al. 2001, Pretorius et al. 2003, Groenewald et al. 2005, 2006, 2013, Montenegro-Calderón et al. 2011, Bakhshi et al. 2012b, 2015a, Nguanhom et al. 2015, Soares et al. 2015, Albu et al. 2016, Guatimosim et al. 2017, Guillin et al. 2017). A comprehensive and detailed molecular examination of Cercospora s. str. based on a multi-locus DNA sequence dataset of five genomic loci including the ITS (ITS1, 5.8S nrRNAgene and ITS2), together with parts of four protein coding genes, viz. translation elongation factor 1-alpha (tef1), actin (actA), calmodulin (cmdA) and histone H3 (his3) was conducted by Groenewald et al. (2013). The main conclusion of this study was that C. apii s. lat. could not be confirmed as a plurivorous monophyletic species, and that several lineages originally referred to C. apii s. lat., or considered close to this complex based on morphology (Crous & Braun 2003), were separated as distinct phylogenetic species. Hence, speciation within Cercospora s. str. is more complicated than formerly assumed, and far from being resolved. To date, multi-locus DNA sequence analyses combined with ecology, morphology and cultural characteristics, referred to as the Consolidated Species Concept (Quaedvlieg et al. 2014), proved the most effective method for the delimitation of Cercospora species (Groenewald et al. 2010, 2013).

At a higher taxonomic level, among the genera of cercosporoid fungi, the monophyly of Cercospora s. str. has until recently been tested based on phylogenetic association of taxa with the type species of Cercospora, C. apii (Groenewald et al. 2013, Bakhshi et al. 2015a, Braun & Crous 2016). Bakhshi et al. (2015b) recovered some cercospora-like isolates from Ammi majus, and in their subsequent multi-gene phylogenetic study (28S nrDNA, ITS, actA, tef1 and his3), elucidated these isolates to represent a new genus, Neocercospora, clustering in a clade in Mycosphaerellaceae apart from Cercospora s. str., suggesting that cercospora-like morphologies are not necessarily part of a single monophyletic genus. This finding led to the conclusion that identification and descriptions of new cercospora-like taxa should be avoided without support of molecular sequence data, not only at species but also at generic level.

Species of Cercospora are known to be widely distributed, occurring on a broad range of plant hosts in many climate zones of Iran (Bakhshi et al. 2012, Hesami et al. 2012, Pirnia et al. 2012), where the biodiversity of the genus has recently received much attention (Bakhshi et al. 2015a, b). The most inclusive study was that of Bakhshi et al. (2015a), who compared 161 Cercospora isolates, recovered from 74 host species from Iran based on DNA sequence data of five genomic loci (ITS, tef1, actA, cmdA and his3), host, cultural, and morphological data, revealing a rich species diversity. However, the problem concerning species delimitation in Cercospora due to the high level of conservation among DNA sequences of commonly used loci, (i.e. ITS, tef1, actA, cmdA, and his3), could not be resolved. Furthermore, cryptic clades in several species complexes remained unresolved in the five-gene phylogenetic tree, for example C. apii, C. armoraciae, C. cf. flagellaris, and Cercospora sp. G (Groenewald et al. 2013, Bakhshi et al. 2015a). Therefore, the aim of the present study was to assess three additional potential candidate gene regions including the partial β-tubulin (tub2) gene, part of the second largest subunit of RNA-polymerase II (rpb2) gene, and part of the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene, in order to firstly generate an eight-gene DNA dataset to resolve cryptic taxa within these species complexes, and secondly to identify the best barcoding gene(s) for species resolution in Cercospora.

Material and Methods

Specimens and isolates

A total of 170 strains, including 145 previously identified as Cercospora species in Bakhshi et al. (2015a), as well as 25 other related strains formerly identified by Groenewald et al. (2013), were studied. Isolates used in this study (Table 1) are maintained in the collection of the Westerdijk Fungal Biodiversity Institute (CBS), Utrecht, The Netherlands, the working collection of Pedro Crous (CPC; housed at CBS), the culture collection of the Iranian Research Institute of Plant Protection (IRAN C), Tehran, Iran, and the culture collection of Tabriz University (CCTU), Tabriz, Iran. Type material of the new species recognized is preserved in the Fungal Herbarium of the Iranian Research Institute of Plant Protection (IRAN F).
Table 1

Collection details and GenBank accession numbers of isolates included in this study. Ex-type isolates and newly generated sequences are highlighted in bold.

Species

Culture accession number (s) 1

Host

Host Family

Origion

Collector

GenBank accession numbers 2

ITS

tef1

actA

cmdA

his3

tub2

rpb2

gapdh

Cercospora althaeina

CCTU 1028

Althaea rosea

Malvaceae

Iran, Guilan, Sowme’eh Sara

M. Bakhshi

KJ886394

KJ886233

KJ885911

KJ885750

KJ886072

MH496336

MH511833

MH496166

 

CCTU 1001

Althaea rosea

Malvaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886392

KJ886231

KJ885909

KJ885748

KJ886070

MH496337

MH511834

MH496167

 

CCTU 1026

Althaea rosea

Malvaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886393

KJ886232

KJ885910

KJ885749

KJ886071

MH496338

MH511835

MH496168

 

CCTU 1152

Althaea rosea

Malvaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886396

KJ886235

KJ885913

KJ885752

KJ886074

MH496339

MH511836

MH496169

 

CBS 248.67; CPC 5117 (TYPE)

Althaea rosea

Malvaceae

Romania, Fundulea

O. Constantinescu

JX143530

JX143284

JX143038

JX142792

JX142546

MH496340

MH496170

 

CCTU 1194; IRAN 2674C

Malva sylvestris

Malvaceae

Iran, East Azerbaijan, Kaleybar

M. Arzanlou

KJ886397

KJ886236

KJ885914

KJ885753

KJ886075

MH496341

MH511837

MH496171

 

CCTU 1071

Malva sylvestris

Malvaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886395

KJ886234

KJ885912

KJ885751

KJ886073

MH496342

MH511838

MH496172

Cercospora apii

CBS 116455; CPC 11556 (TYPE)

Apium graveolens

Apiaceae

Germany, Heilbron

K. Schrameyer

AY840519

AY840486

AY840450

AY840417

AY840384

MH496343

MH496173

 

CBS 536.71;CPC 5087

Apium graveolens

Apiaceae

Romania, Bucuresti

O. Constantinescu

AY752133

AY752166

AY752194

AY752225

AY752256

MH496344

MH511839

MH496174

 

CCTU 1069

Cynanchum acutum

Apocynaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886410

KJ886249

KJ885927

KJ885766

KJ886088

MH496345

MH511840

MH496175

 

CCTU 1086; CBS 136037; IRAN 2655C

Cynanchum acutum

Apocynaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886411

KJ886250

KJ885928

KJ885767

KJ886089

MH496346

MH511841

MH496176

 

CCTU 1215

Cynanchum acutum

Apocynaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886412

KJ886251

KJ885929

KJ885768

KJ886090

MH496347

MH511842

MH496177

 

CCTU 1219; CBS 136155

Cynanchum acutum

Apocynaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886413

KJ886252

KJ885930

KJ885769

KJ886091

MH496348

MH511843

MH496178

 

CPC5112

Molucella laevis

Lamiaceae

New Zealand, Auckland

C.F. Hill

DQ233321

DQ233347

DQ233373

DQ233399

DQ233425

MH496349

MH511844

MH496179

 

CBS 110813; CPC 5110; 01-3

Molucella laevis

Lamiaceae

U.S.A., California

S.T. Koike

AY156918

DQ233345

DQ233371

DQ233397

DQ233423

MH496350

MH511845

MH496180

Cercospora armoraciae

CBS 250.67; CPC 5088 (TYPE)

Armoracia rusticana (= A. lapathifolia)

Brassicaceae

Romania, Fundulea

O. Constantinescu

JX143545

JX143299

JX143053

JX142807

JX142561

MH496351

MH496181

Cercospora beticola

CPC 12028

Beta vulgaris

Chenopodiaceae

Egypt

M. Hasem

DQ233336

DQ233362

DQ233388

DQ233414

DQ233437

MH496352

MH511846

MH496182

 

CPC 12029

Beta vulgaris

Chenopodiaceae

Egypt

M. Hasem

DQ233337

DQ233363

DQ233389

DQ233415

DQ233438

MH496353

MH511847

MH496183

 

CCTU 1135

Beta vulgaris

Chenopodiaceae

Iran, Guilan, Astara

M. Bakhshi

KJ886432

KJ886271

KJ885949

KJ885788

KJ886110

MH496354

MH511848

MH496184

 

CBS 116456; CPC 11557 (TYPE)

Beta vulgaris

Chenopodiaceae

Italy, Ravenna

V. Rossi

AY840527

AY840494

AY840458

AY840425

AY840392

MH496355

KT216555

MH496185

 

CCTU 1057; IRAN 2651C

Chenopodium sp.

Chenopodiaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886424

KJ886263

KJ885941

KJ885780

KJ886102

MH496356

MH511849

MH496186

 

CCTU 1065

Chenopodium sp.

Chenopodiaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886425

KJ886264

KJ885942

KJ885781

KJ886103

MH496357

MH511850

MH496187

 

CCTU 1087

Chenopodium sp.

Chenopodiaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886427

KJ886266

KJ885944

KJ885783

KJ886105

MH496358

MH511851

MH496188

 

CCTU 1089; CPC 24911

Plantago lanceolata

Plantaginaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886429

KJ886268

KJ885946

KJ885785

KJ886107

MH496359

MH511852

MH496189

 

CCTU 1108

Plantago lanceolata

Plantaginaceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886430

KJ886269

KJ885947

KJ885786

KJ886108

MH496360

MH511853

MH496190

 

CCTU 1088; CBS 138582

Sonchus asper

Asteraceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886428

KJ886267

KJ885945

KJ885784

KJ886106

MH496361

MH511854

MH496191

Cercospora bizzozeriana

CCTU 1013

?

?

Iran, East Azerbaijan, Mianeh

M. Torbati

KJ886414

KJ886253

KJ885931

KJ885770

KJ886092

MH496362

MH511855

MH496192

 

CCTU 1022; CBS 136028

?

?

Iran, East Azerbaijan, Mianeh

M. Torbati

KJ886415

KJ886254

KJ885932

KJ885771

KJ886093

MH496363

MH511856

MH496193

 

CCTU 1127; CBS 136133

Capparis spinosa

Capparidaceae

Iran, Khuzestan, Ahvaz

E. Mohammadian

KJ886420

KJ886259

KJ885937

KJ885776

KJ886098

MH496364

MH511857

MH496194

 

CCTU 1117; CBS 136132

Cardaria draba

Brassicaceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886418

KJ886257

KJ885935

KJ885774

KJ886096

MH496365

MH511858

MH496195

 

CCTU 1234

Cardaria draba

Brassicaceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886419

KJ886258

KJ885936

KJ885775

KJ886097

MH496366

MH511859

MH496196

 

CCTU 1107

?

?

Iran, Zanjan, Tarom

M. Bakhshi

KJ886417

KJ886256

KJ885934

KJ885773

KJ886095

MH496367

MH511860

MH496197

 

CBS 258.67; CPC 5061 (TYPE)

Cardaria draba

Brassicaceae

Romania, Fundulea

O. Constantinescu

JX143546

JX143300

JX143054

JX142808

JX142562

MH496368

MH496198

 

CBS 540.71; IMI 161110; CPC 5060

Cardaria draba

Brassicaceae

Romania, Hagieni

O. Constantinescu

JX143548

JX143302

JX143056

JX142810

JX142564

MH496369

MH496199

 

CCTU 1040; CBS 136131

Tanacetum balsamita

Asteraceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886416

KJ886255

KJ885933

KJ885772

KJ886094

MH496370

MH511861

MH496200

Cercospora chenopodii

CCTU 1060; IRAN 2652C

Chenopodium album

Chenopodiaceae

Iran, Guilan, Bandar-e Anzali

M. Bakhshi

KJ886438

KJ886277

KJ885955

KJ885794

KJ886116

MH496371

MH511862

MH496201

 

CCTU 1163

Chenopodium album

Chenopodiaceae

Iran, Guilan, Lahijan

M. Bakhshi

KJ886440

KJ886279

KJ885957

KJ885796

KJ886118

MH496372

MH511863

MH496202

 

CCTU 1033

Chenopodium album

Chenopodiaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886437

KJ886276

KJ885954

KJ885793

KJ886115

MH496373

MH511864

MH496203

Cercospora convolvulicola

CCTU 1083; CBS 136126 (TYPE)

Convolvulus arvensis

Convolvulaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886441

KJ886280

KJ885958

KJ885797

KJ886119

MH496374

MH511865

MH496204

 

CCTU 1083.2

Convolvulus arvensis

Convolvulaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886442

KJ886281

KJ885959

KJ885798

KJ886120

MH496375

MH511866

MH496205

Cercospora conyzae-canadensis

CCTU 1008

Conyza canadensis

Asteraceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886443

KJ886282

KJ885960

KJ885799

KJ886121

MH496376

MH511867

MH496206

 

CCTU 1119; CBS 135978 (TYPE)

Conyza canadensis

Asteraceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886445

KJ886284

KJ885962

KJ885801

KJ886123

MH496377

MH511868

MH496207

 

CCTU 1105; IRAN 2657C

Conyza canadensis

Asteraceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886444

KJ886283

KJ885961

KJ885800

KJ886122

MH496378

MH511869

MH496208

Cercospora cylindracea

CCTU 1016

Cichorium intybus

Asteraceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886446

KJ886285

KJ885963

KJ885802

KJ886124

MH496379

MH511870

MH496209

 

CCTU 1114

Cichorium intybus

Asteraceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886450

KJ886289

KJ885967

KJ885806

KJ886128

MH496380

MH511871

MH496210

 

CCTU 1081; CBS 138580; IRAN 2654C (TYPE)

Lactuca serriola

Asteraceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886449

KJ886288

KJ885966

KJ885805

KJ886127

MH496381

MH511872

MH496211

 

CCTU 1207

Lactuca serriola

Asteraceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886453

KJ886292

KJ885970

KJ885809

KJ886131

MH496382

MH511873

MH496212

 

CCTU 1044; CBS 136021

Lactuca serriola

Asteraceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886447

KJ886286

KJ885964

KJ885803

KJ886125

MH496383

MH511874

MH496213

 

CCTU 1183

Lactuca serriola

Asteraceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886451

KJ886290

KJ885968

KJ885807

KJ886129

MH496384

MH511875

MH496214

 

CCTU 1189

Lactuca serriola

Asteraceae

Iran, West Azerbaijan Khoy

M. Arzanlou

KJ886452

KJ886291

KJ885969

KJ885808

KJ886130

MH496385

MH511876

MH496215

 

CCTU 1049

Lactuca serriola

Asteraceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886448

KJ886287

KJ885965

KJ885804

KJ886126

MH496386

MH511877

MH496216

Cercospora cf. fagellaris clade 1

CPC 5441

Amaranthus sp.

Amaranthaceae

Fiji

C.F. Hill

JX143611

JX143370

JX143124

JX142878

JX142632

MH496387

MH511878

MH496217

 

CCTU 1159; CBS 136148

Arachis hypogaea

Fabaceae

Iran, Guilan, Lahijan

M. Bakhshi

KJ886493

KJ886332

KJ886010

KJ885849

KJ886171

MH496388

MH511879

MH496218

 

CCTU 1162; IRAN 2670C

Citrullus lanatus

Cucurbitaceae

Iran, Guilan, Lahijan

M. Bakhshi

KJ886496

KJ886335

KJ886013

KJ885852

KJ886174

MH496389

MH511880

MH496219

 

CBS 132653; CPC 10884

Dysphania ambrosioides (= Chenopodium ambrosioides)

Chenopodiaceae

South Korea, Jeju

H.D. Shin

JX143603

JX143361

JX143115

JX142869

JX142623

MH496390

MH511881

MH496220

 

CCTU 1007; CBS 136031

Hydrangea sp.

Hydrangeaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886456

KJ886295

KJ885973

KJ885812

KJ886134

MH496391

MH511882

MH496221

 

CCTU 1027; CBS 136034

Lepidium sativum

Brassicaceae

Iran, Guilan, Chamkhaleh

M. Bakhshi

KJ886459

KJ886298

KJ885976

KJ885815

KJ886137

MH496392

MH511883

MH496222

 

CCTU 1128; CBS 136141; IRAN 2661C

Phaseolus vulgaris

Fabaceae

Iran, Guilan, Astara

M. Bakhshi

KJ886476

KJ886315

KJ885993

KJ885832

KJ886154

MH496393

MH511884

MH496223

 

CCTU 1168; IRAN 2715C

Phaseolus vulgaris

Fabaceae

Iran, Guilan, Kiashahr

M. Bakhshi

KJ886499

KJ886338

KJ886016

KJ885855

KJ886177

MH496394

MH511885

MH496224

 

CPC 1051

Populus deltoides

Salicaceae

South Africa

P.W. Crous

AY260069

JX143367

JX143121

JX142875

JX142629

MH496395

MH511886

MH496225

 

CCTU 1171

Raphanus sativus

Brassicaceae

Iran, Guilan, Kiashahr

M. Bakhshi

KJ886500

KJ886339

KJ886017

KJ885856

KJ886178

MH496396

MH511887

MH496226

 

CCTU 1120

Raphanus sativus

Brassicaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886475

KJ886314

KJ885992

KJ885831

KJ886153

MH496397

MH511888

MH496227

 

CCTU 1031;CBS 136036; IRAN 2648C

Urtica dioica

Urticaceae

Iran, Guilan, Sowme’eh Sara

M. Bakhshi

KJ886461

KJ886300

KJ885978

KJ885817

KJ886139

MH496398

MH511889

MH496228

Cercospora cf. fagellaris clade 2

CCTU 1204

Abutilon theophrasti

Malvaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886505

KJ886344

KJ886022

KJ885861

KJ886183

MH496399

MH511890

MH496229

 

CCTU 1198; CBS 136151

Acer velutinum

Aceraceae

Iran, Mazandaran, Ramsar

M. Bakhshi

KJ886504

KJ886343

KJ886021

KJ885860

KJ886182

MH496400

MH511891

MH496230

 

CBS 132667; CPC 11643

Celosia argentea var. cristata (= C. cristata)

Amaranthaceae

South Korea, Hoengseong

H.D. Shin

JX143604

JX143362

JX143116

JX142870

JX142624

MH496401

MH511892

MH496231

 

CCTU 1115; CBS 136139; IRAN 2659C

Cercis siliquastrum

Caesalpinaceae

Iran, Guilan, Astara

M. Bakhshi

KJ886473

KJ886312

KJ885990

KJ885829

KJ886151

MH496402

MH511893

MH496232

 

CCTU 1195

Datura stramonium

Solanaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886503

KJ886342

KJ886020

KJ885859

KJ886181

MH496403

MH511894

MH496233

 

CCTU 1059; CBS 136136

Ecballium elaterium

Cucurbitaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886464

KJ886303

KJ885981

KJ885820

KJ886142

MH496404

MH511895

MH496234

 

CCTU 1216; IRAN 2717C

Ecballium elaterium

Cucurbitaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886510

KJ886349

KJ886027

KJ885866

KJ886188

MH496405

MH511896

MH496235

 

CCTU 1223; CBS 136154; IRAN 2683C

Eclipta prostrata

Asteraceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886512

KJ886351

KJ886029

KJ885868

KJ886190

MH496406

MH511897

MH496236

 

CCTU 1068

Xanthium spinosum

Asteraceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886466

KJ886305

KJ885983

KJ885822

KJ886144

MH496407

MH511898

MH496237

 

CCTU 1085

Xanthium strumarium

Asteraceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886471

KJ886310

KJ885988

KJ885827

KJ886149

MH496408

MH511899

MH496238

Cercospora cf. flagellaris clade 3

CCTU 1172

Oenothera biennis

Onagraceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886501

KJ886340

KJ886018

KJ885857

KJ886179

MH496409

MH511900

MH496239

 

CCTU 1154; CBS 136147

Abutilon theophrasti

Malvaceae

Iran, Guilan, Rasht

M. Bakhshi

KJ886489

KJ886328

KJ886006

KJ885845

KJ886167

MH496410

MH511901

MH496240

 

CCTU 1072; IRAN 2653C

Amaranthus blitoides

Amaranthaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886468

KJ886307

KJ885985

KJ885824

KJ886146

MH496411

MH511902

MH496241

 

CCTU 1064

Amaranthus retroflexus

Amaranthaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886465

KJ886304

KJ885982

KJ885821

KJ886143

MH496412

MH511903

MH496242

 

CCTU 1021; CBS 136033

Amaranthus retrolexus

Amaranthaceae

Iran, Guilan, Fuman

M. Bakhshi

KJ886458

KJ886297

KJ885975

KJ885814

KJ886136

MH496413

MH511904

MH496243

 

CCTU 1084; CBS 136156

Amaranthus sp.

Amaranthaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886470

KJ886309

KJ885987

KJ885826

KJ886148

MH496414

MH511905

MH496244

 

CCTU 1167; CBS 136150

Anubias sp.

Araceae

Iran, Guilan, Kiashahr

M. Bakhshi

KJ886498

KJ886337

KJ886015

KJ885854

KJ886176

MH496415

MH511906

MH496245

 

CBS 143.51;CPC 5055

Bromus sp.

Poaceae

M.D. Whitehead

JX143607

JX143365

JX143119

JX142873

JX142627

MH496416

MH511907

MH496246

 

CCTU 1150

Buxus microphylla

Buxaceae

Iran, Guilan, Fuman

M. Bakhshi

KJ886488

KJ886327

KJ886005

KJ885844

KJ886166

MH496417

MH511908

MH496247

 

CCTU 1140; CBS 136143; IRAN 2666C

Calendula officinalis

Asteraceae

Iran, Guilan, Astara

M. Bakhshi

KJ886481

KJ886320

KJ885998

KJ885837

KJ886159

MH496418

MH511909

MH496248

 

CBS 115482; A207 Bs+; CPC4410

Citrus sp.

Rutaceae

South Africa, Messina

M.C. Pretorius

AY260070

DQ835095

DQ835114

DQ835141

DQ835168

MH496419

MH511910

MH496249

 

CCTU 1029; CBS 136035; IRAN 2647C

Cucurbita maxima

Cucurbitaceae

Iran, Guilan, Rudsar

M. Bakhshi

KJ886460

KJ886299

KJ885977

KJ885816

KJ886138

MH496420

MH511911

MH496250

 

CCTU 1136

Cucurbita pepo

Cucurbitaceae

Iran, Guilan, Astara

M. Bakhshi

KJ886478

KJ886317

KJ885995

KJ885834

KJ886156

MH496421

MH511912

MH496251

 

CCTU 1143; CBS 136145

Datura stramonium

Solanaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886484

KJ886323

KJ886001

KJ885840

KJ886162

MH496422

MH511913

MH496252

 

CCTU 1209; CBS 136152

Glycine max

Fabaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886506

KJ886345

KJ886023

KJ885862

KJ886184

MH496423

MH511914

MH496253

 

CCTU 1210; IRAN 2679C

Glycine max

Fabaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886507

KJ886346

KJ886024

KJ885863

KJ886185

MH496424

MH511915

MH496254

 

CCTU 1211

Glycine max

Fabaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886508

KJ886347

KJ886025

KJ885864

KJ886186

MH496425

MH511916

MH496255

 

CCTU 1218; IRAN 2682C

Hibiscus trionum

Malvaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886511

KJ886350

KJ886028

KJ885867

KJ886189

MH496426

MH511917

MH496256

 

CCTU 1006; CBS 136030

Impatiens balsamina

Balsaminaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886455

KJ886294

KJ885972

KJ885811

KJ886133

MH496427

MH511918

MH496257

 

CCTU 1130; CBS 136142

Olea europaea

Oleaceae

Iran, Zanjan, Tarom

M. Torbati

KJ886477

KJ886316

KJ885994

KJ885833

KJ886155

MH496428

MH511919

MH496258

 

CCTU 1010; CBS 136032

Pelargonium hortorum

Geraniaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886457

KJ886296

KJ885974

KJ885813

KJ886135

MH496429

MH511920

MH496259

 

CCTU 1138; IRAN 2664C

Phaseolus vulgaris

Fabaceae

Iran, Guilan, Astara

M. Bakhshi

KJ886479

KJ886318

KJ885996

KJ885835

KJ886157

MH496430

MH511921

MH496260

 

CCTU 1139; IRAN 2665C

Phaseolus vulgaris

Fabaceae

Iran, Guilan, Astara

M. Bakhshi

KJ886480

KJ886319

KJ885997

KJ885836

KJ886158

MH496431

MH511922

MH496261

 

CCTU 1155.11

Phaseolus vulgaris

Fabaceae

Iran, Guilan, Fuman

M. Bakhshi

KJ886490

KJ886329

KJ886007

KJ885846

KJ886168

MH496432

MH511923

MH496262

 

CCTU 1161; IRAN 2669C

Phaseolus vulgaris

Fabaceae

Iran, Guilan, Lahijan

M. Bakhshi

KJ886495

KJ886334

KJ886012

KJ885851

KJ886173

MH496433

MH511924

MH496263

 

CCTU 1175; IRAN 2673C

Phaseolus vulgaris

Fabaceae

Iran, Guilan, Sowme’eh Sara

M. Bakhshi

KJ886502

KJ886341

KJ886019

KJ885858

KJ886180

MH496434

MH511925

MH496264

 

CCTU 1142; IRAN 2667C

Phaseolus vulgaris

Fabaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886483

KJ886322

KJ886000

KJ885839

KJ886161

MH496435

MH511926

MH496265

 

CCTU 1118; CBS 136140; IRAN 2660C

Populus deltoides

Salicaceae

Iran, Guilan, Astara

M. Bakhshi

KJ886474

KJ886313

KJ885991

KJ885830

KJ886152

MH496436

MH511927

MH496266

 

CCTU 1075

Raphanus sativus

Brassicaceae

Iran, Guilan, Sowme’eh Sara

M. Bakhshi

KJ886469

KJ886308

KJ885986

KJ885825

KJ886147

MH496437

MH511928

MH496267

 

CCTU 1212; CBS 136153; IRAN 2680C

Silybum marianum

Asteraceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886509

KJ886348

KJ886026

KJ885865

KJ886187

MH496438

MH511929

MH496268

 

CCTU 1141; CBS 136144

Tagetes patula

Asteraceae

Iran, Guilan, Rudsar

M. Bakhshi

KJ886482

KJ886321

KJ885999

KJ885838

KJ886160

MH496439

MH511930

MH496269

 

CCTU 1147

Urtica dioica

Urticaceae

Iran, Guilan, Masal

M. Bakhshi

KJ886486

KJ886325

KJ886003

KJ885842

KJ886164

MH496440

MH511931

MH496270

 

CCTU 1160; CBS 136149

Vicia faba

Fabaceae

Iran, Guilan, Astara

M. Bakhshi

KJ886494

KJ886333

KJ886011

KJ885850

KJ886172

MH496441

MH511932

MH496271

 

CCTU 1158; IRAN 2668C

Xanthium strumarium

Asteraceae

Iran, Guilan, Langarud

M. Bakhshi

KJ886492

KJ886331

KJ886009

KJ885848

KJ886170

MH496442

MH511933

MH496272

 

CCTU 1156

Xanthium strumarium

Asteraceae

Iran, Guilan, Rasht

M. Bakhshi

KJ886491

KJ886330

KJ886008

KJ885847

KJ886169

MH496443

MH511934

MH496273

 

CCTU 1005; IRAN 2644C

Xanthium strumarium

Asteraceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886454

KJ886293

KJ885971

KJ885810

KJ886132

MH496444

MH511935

MH496274

 

CCTU 1048; CBS 136029

Xanthium strumarium

Asteraceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886462

KJ886301

KJ885979

KJ885818

KJ886140

MH496445

MH511936

MH496275

Cercospora gamsiana

CBS 144962; CCTU 1074; CPC 24909 (TYPE)

Malva neglecta

Malvaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886426

KJ886265

KJ885943

KJ885782

KJ886104

MH496446

MH511937

MH496276

 

CCTU 1035

Malva sylvestris

Malvaceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886423

KJ886262

KJ885940

KJ885779

KJ886101

MH496447

MH511938

MH496277

 

CCTU 1109

Malva sylvestris

Malvaceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886431

KJ886270

KJ885948

KJ885787

KJ886109

MH496448

MH511939

MH496278

 

CCTU 1199; CBS 136128; IRAN 2675C

Rumex crispus

Polygonaceae

Iran, Mazandaran, Ramsar

M. Bakhshi

KJ886433

KJ886272

KJ885950

KJ885789

KJ886111

MH496449

MH511940

MH496279

 

CCTU 1205; CBS 136127; IRAN 2677C

Sesamum indicum

Pedaliaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886435

KJ886274

KJ885952

KJ885791

KJ886113

MH496450

MH511941

MH496280

 

CCTU 1208; IRAN 2678C

Sonchus sp.

Asteraceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886436

KJ886275

KJ885953

KJ885792

KJ886114

MH496451

MH511942

MH496281

Cercospora cf. gossypii

CCTU 1070; CBS 136137

Gossypium herbaceum

Malvaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886467

KJ886306

KJ885984

KJ885823

KJ886145

MH496452

MH511943

MH496282

 

CCTU 1055; IRAN 2650C

Hibiscus trionum

Malvaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886463

KJ886302

KJ885980

KJ885819

KJ886141

MH496453

MH511944

MH496283

Cercospora iranica

CCTU 1196; CBS 136123

Hydrangea sp.

Hydrangeaceae

Iran, Mazandaran, Ramsar

M. Bakhshi

KJ886515

KJ886354

KJ886032

KJ885871

KJ886193

MH496454

MH511945

MH496284

 

CCTU 1137; CBS 136124 (TYPE)

Vicia faba

Fabaceae

Iran, Guilan, Astara

M. Bakhshi

KJ886513

KJ886352

KJ886030

KJ885869

KJ886191

MH496455

MH511946

MH496285

Cercospora plantaginis

CCTU 1082; CBS 138728

Plantago lanceolata

Plantaginaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886402

KJ886241

KJ885919

KJ885758

KJ886080

MH496456

MH511947

MH496286

 

CCTU 1095

Plantago lanceolata

Plantaginaceae

Iran, East Azerbaijan, Horand

M. Bakhshi

KJ886403

KJ886242

KJ885920

KJ885759

KJ886081

MH496457

MH511948

MH496287

 

CCTU 1041;CPC24910

Plantago lanceolata

Plantaginaceae

Iran, Guilan, Chaboksar

M. Bakhshi

KJ886400

KJ886239

KJ885917

KJ885756

KJ886078

MH496458

MH511949

MH496288

 

CCTU 1179; IRAN 2716C

Plantago lanceolata

Plantaginaceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886404

KJ886243

KJ885921

KJ885760

KJ886082

MH496459

MH511950

MH496289

 

CCTU 1047

Plantago lanceolata

Plantaginaceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886401

KJ886240

KJ885918

KJ885757

KJ886079

MH496460

MH511951

MH496290

 

CBS 252.67; CPC 5084 (TYPE)

Plantago lanceolata

Plantaginaceae

Romania, Domnesti

O. Constantinescu

DQ233318

DQ233342

DQ233368

DQ233394

DQ233420

MH496461

MH496291

Cercospora pseudochenopodii

CCTU 1176

Chenopodium album

Chenopodiaceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886518

KJ886357

KJ886035

KJ885874

KJ886196

MH496462

MH511952

MH496292

 

CCTU 1045

Chenopodium sp.

Chenopodiaceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886517

KJ886356

KJ886034

KJ885873

KJ886195

MH496463

MH511953

MH496293

 

CCTU 1038; CBS 136022; IRAN 2649C (TYPE)

Chenopodium sp.

Chenopodiaceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886516

KJ886355

KJ886033

KJ885872

KJ886194

MH496464

MH511954

MH496294

Cercospora cf. richardiicola

CCTU 1004

Bidens tripartita

Asteraceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886519

KJ886358

KJ886036

KJ885875

KJ886197

MH496465

MH511955

MH496295

Cercospora rumicis

CCTU 1123

Rumex crispus

Polygonaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886521

KJ886360

KJ886038

KJ885877

KJ886199

MH496466

MH511956

MH496296

 

CCTU 1129; IRAN 2662C

Rumex crispus

Polygonaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886522

KJ886361

KJ886039

KJ885878

KJ886200

MH496467

MH511957

MH496297

 

CCTU 1121

Urtica dioica

Urticaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886520

KJ886359

KJ886037

KJ885876

KJ886198

MH496468

MH511958

MH496298

Cercospora solani

CCTU 1043; CBS 136038

Solanum nigrum

Solanaceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886523

KJ886362

KJ886040

KJ885879

KJ886201

MH496469

MH511959

MH496299

 

CCTU 1050

Solanum nigrum

Solanaceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886524

KJ886363

KJ886041

KJ885880

KJ886202

MH496470

MH511960

MH496300

Cercospora sorghicola

CCTU 1173; CBS 136448; IRAN 2672C (TYPE)

Sorghum halepense

Poaceae

Iran, Guilan, Kiashahr

M. Bakhshi

KJ886525

KJ886364

KJ886042

KJ885881

KJ886203

MH496471

MH511961

MH496301

Cercospora sp. G clade 1

CCTU 1197

Bidens tripartita

Asteraceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886540

KJ886379

KJ886057

KJ885896

KJ886218

MH496472

MH511962

MH496302

 

CCTU 1015; CBS 136024; IRAN 2645C

Plantago major

Plantaginaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886528

KJ886367

KJ886045

KJ885884

KJ886206

MH496473

MH511963

MH496303

 

CPC 5438

Salvia viscosa

Lamiaceae

New Zealand, Manurewa

C.F. Hill

JX143682

JX143442

JX143196

JX142950

JX142704

MH496474

MH496304

Cercospora sp. G clade 2

CCTU 1058

Helminthotheca echioides

Asteraceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886534

KJ886373

KJ886051

KJ885890

KJ886212

MH496475

MH511964

MH496305

 

CCTU 1090

Abutilon theophrasti

Malvaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886536

KJ886375

KJ886053

KJ885892

KJ886214

MH496476

MH511965

MH496306

 

CCTU 1079; CBS 136025

Amaranthus retroflexus

Amaranthaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886535

KJ886374

KJ886052

KJ885891

KJ886213

MH496477

MH511966

MH496307

 

CCTU 1054

Amaranthus sp.

Amaranthaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886533

KJ886372

KJ886050

KJ885889

KJ886211

MH496478

MH511967

MH496308

 

CCTU 1122

Amaranthus sp.

Amaranthaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886538

KJ886377

KJ886055

KJ885894

KJ886216

MH496479

MH511968

MH496309

 

CBS115518;CPC 5360

Bidens frondosa

Asteraceae

New Zealand, Kopuku

C.F. Hill

JX143681

JX143441

JX143195

JX142949

JX142703

MH496480

MH496310

 

CCTU 1030; CBS 136026

Bidens tripartita

Asteraceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886530

KJ886369

KJ886047

KJ885886

KJ886208

MH496481

MH511969

MH496311

 

CCTU 1002

Celosia cristata

Amaranthaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886527

KJ886366

KJ886044

KJ885883

KJ886205

MH496482

MH511970

MH496312

 

CCTU 1053; CBS 136027

Cichorium intybus

Asteraceae

Iran, Guilan, Sowme’eh Sara

M. Bakhshi

KJ886532

KJ886371

KJ886049

KJ885888

KJ886210

MH496483

MH511971

MH496313

 

CCTU 1144; CBS 136130

Cucurbita maxima

Cucurbitaceae

Iran, Guilan, Masal

M. Bakhshi

KJ886539

KJ886378

KJ886056

KJ885895

KJ886217

MH496484

MH511972

MH496314

 

CCTU 1046

Plantago major

Plantaginaceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886531

KJ886370

KJ886048

KJ885887

KJ886209

MH496485

MH511973

MH496315

 

CCTU 1116

Plantago major

Plantaginaceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886537

KJ886376

KJ886054

KJ885893

KJ886215

MH496486

MH511974

MH496316

 

CCTU 1020; CBS 136023

Sorghum halepense

Poaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886529

KJ886368

KJ886046

KJ885885

KJ886207

MH496487

MH511975

MH496317

Cercospora sp. T

CCTU 1148; CBS 136125

Coreopsis sp.

Asteraceae

Iran, Guilan, Rasht

M. Bakhshi

KJ886541

KJ886380

KJ886058

KJ885897

KJ886219

MH496488

MH511976

MH496318

Cercospora uwebrauniana

CCTU 1200; CBS 138581 (TYPE)

Heliotropium europaeum

Boraginaceae

Iran, Ardabil, Moghan

M. Bakhshi

KJ886408

KJ886247

KJ885925

KJ885764

KJ886086

MH496489

MH511977

MH496319

 

CCTU 1134

Heliotropium europaeum

Boraginaceae

Iran, Guilan, Astara

M. Bakhshi

KJ886407

KJ886246

KJ885924

KJ885763

KJ886085

MH496490

MH511978

MH496320

Cercospora violae

CCTU 1025; IRAN 2646C

Viola sp.

Violaceae

Iran, Mazandaran, Nowshahr

M. Bakhshi

KJ886543

KJ886382

KJ886060

KJ885899

KJ886221

MH496491

MH511979

MH496321

 

CBS 251.67; CPC 5079 (TYPE)

Viola tricolor

Violaceae

Romania, Cazanele Dunarii

O. Constantinescu

JX143737

JX143496

JX143250

JX143004

JX142758

MH496492

MH496322

Cercospora zebrina

CCTU 1039

Alhagi camelorum

Fabaceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886545

KJ886384

KJ886062

KJ885901

KJ886223

MH496493

MH511980

MH496323

 

CBS 108.22; CPC 5091

Medicago arabica (= M. maculata)

Fabaceae

 

E.F. Hopkins

JX143744

JX143503

JX143257

JX143011

JX142765

MH496494

MH496324

 

CCTU 1225

Medicago sativa

Fabaceae

Iran, East Azerbaijan, Marand

M. Bakhshi

KJ886550

KJ886389

KJ886067

KJ885906

KJ886228

MH496495

MH511981

MH496325

 

CCTU 1180

Medicago sativa

Fabaceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886547

KJ886386

KJ886064

KJ885903

KJ886225

MH496496

MH511982

MH496326

 

CCTU 1110; IRAN 2658C

Medicago sativa

Fabaceae

Iran, Zanjan, Tarom

M. Bakhshi

KJ886546

KJ886385

KJ886063

KJ885902

KJ886224

MH496497

MH511983

MH496327

 

CCTU 1012; CBS 136129

Medicago sp.

Fabaceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886544

KJ886383

KJ886061

KJ885900

KJ886222

MH496498

MH511984

MH496328

 

CCTU 1181

Trifolium repens

Fabaceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886548

KJ886387

KJ886065

KJ885904

KJ886226

MH496499

MH511985

MH496329

 

CBS 113070; CPC 5367

Trifolium repens

Fabaceae

New Zealand, Blockhouse Bay

C.F. Hill

JX143745

JX143507

JX143261

JX143015

JX142769

MH496500

MH496330

 

CBS 118790; IMI 262766;WA2030;WAC 7973

Trifolium subterraneum

Fabaceae

Australia

M.J. Barbetti

JX143748

JX143510

JX143264

JX143018

JX142772

MH496501

MH496331

 

CBS 129.39; CPC 5078

Trifolium subterraneum

Fabaceae

U.S.A., Wisconsim

JX143750

JX143512

JX143266

JX143020

JX142774

MH496502

MH496332

 

CCTU 1185

Vicia sp.

Fabaceae

Iran, West Azerbaijan, Khoy

M. Arzanlou

KJ886549

KJ886388

KJ886066

KJ885905

KJ886227

MH496503

MH511986

MH496333

 

CCTU 1239; CBS 135977

Vitis vinifera

Vitaceae

Iran, East Azerbaijan, Kaleybar

M. Arzanlou

KJ886551

KJ886390

KJ886068

KJ885907

KJ886229

MH496504

MH511987

MH496334

Cercospora cf. zinniae

CCTU 1003

Zinnia elegans

Asteraceae

Iran, Guilan, Talesh

M. Bakhshi

KJ886552

KJ886391

KJ886069

KJ885908

KJ886230

MH496505

MH511988

MH496335

1 CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; CCTU: Culture Collection of Tabriz University, Tabriz, Iran; CPC: Culture collection of Pedro Crous, housed at CBS; IMI: International Mycological Institute, CABI-Bioscience, Egham, Bakeham Lane, U.K.; IRAN: Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Tehran, Iran; WAC: Department of Agriculture Western Australia Plant Pathogen Collection, Perth, Australia.

2 ITS: internal transcribed spacers and intervening 5.8S nrDNA; tef1: partial translation elongation factor 1-alpha gene, actA: partial actin gene, cmdA: partial calmodulin gene, his3: partial histone H3 gene, tub2: partial beta-tubulin gene, rpb2: partial RNA polymerase II gene, gapdh: partial glyceraldehyde-3-phosphate dehydrogenase gene.

DNA extraction and PCR amplification

DNA samples comprised those previously extracted by Bakhshi et al. (2015a) and Groenewald et al. (2013). Three additional partial nuclear genes were targeted for PCR amplification and sequencing, namely, glyceraldehyde-3-phosphate dehydrogenase (gapdh), RNA polymerase II second largest subunit (rpb2), and β-tubulin (tub2), using corresponding primer sets (Table 2). PCR amplifications were performed in a total volume of 12.5 µL on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). The gapdh PCR mixture consisted of 5–10 ng genomic DNA, 1 × PCR buffer (Bioline, London), 2 mM MgCl2 (Bioline), 50 µM of each dNTP, 0.5 µL BSA (10 mg/ml) (Promega, Madison, WI), 0.28 µM of each primer and 0.5 units GoTaq® Flexi DNA polymerase (Promega). The tub2 PCR mixture contained 5–10 ng genomic DNA, 1 × PCR buffer, 2 mM MgCl2, 40 µM of each dNTP, 0 µL/0.5 µL BSA, 0.25 µM of each primer and 0.5 units GoTaq® Flexi DNA polymerase using respectively the BT-1F/BT-1R (this study) or T1 (O’Donnell & Cigelnik 1997)/β-Sandy-R (Stukenbrock et al. 2012) primer sets. The rpb2 gene was amplified in three parts with three primer sets. Part three was only amplified in some selected species in order to design a new reverse primer for amplification of part two. The rpb2 PCR mixtures using the fRPB2-5F (Liu et al. 1999)/fRPB2-414R (Quaedvlieg et al. 2011) primer set consisted of 5–10 ng genomic DNA, 1 × PCR buffer, 2 mM MgCl2, 40 µM of each dNTP, 0.5 µL BSA, 0.2 µM of each primer and 0.5 units GoTaq® Flexi DNA polymerase. The PCR mixtures using RPB2-C5F/RPB2-C8R (this study) and fRPB2-7cF/fRPB2-11aR primer sets (Liu et al. 1999) were the same as gapdh.
Table 2

Primercombinations used during this studyforamplification and sequencing.

Locus

Primer

Primer sequence 5′ to 3′

Annealing temperature (°C)

Orientation

Reference

Beta-tubulin (tub2)

T1

AAC ATG CGT GAG ATT GTAAGT

48

Forward

O’Donnell & Cigelnik 1997

 

β-Sandy-R

GCR CGN GGV ACR TAC TTG TT

48

Reverse

Stukenbrock et al. 2012

 

BT-1F

GTC CWC ACC GCC CCT GAT

56

Forward

This study

 

BT-1R

CTT GTT RCC RGAAGC CTR TGS

56

Reverse

This study

RNA polymerase II second largest subunit (rpb2)

fRPB2-5F

GAY GAY MGW GAT CAY TTY GG

47

Forward

Liu et al. 1999

 

fRPB2-414R

ACM ANN CCC CAR TGN GWR TTR TG

47

Reverse

Quaedvlieg et al. 2011

 

fRPB2-7cF

ATG GGY AAR CAA GCY ATG GG

49

Forward

Liu et al. 1999

 

fRPB2-11aR

GCR TGG ATC TTR TCR TCS ACC

49

Reverse

Liu et al. 1999

 

RPB2-C5F

TGGGGA GAY CAR AAR AAA GC

60→58→56

Forward

This study

 

RPB2-C8R

ACG GAA TCT TCC TGG TTG TA

60→58→56

Reverse

This study

Glyceraldehyde-3-phosphate dehydrogenase (gapdh)

Gpd1-LM

ATT GGC CGC ATC GTC TTC CGC AA

60→58→53

Forward

Myllys et al. 2002

 

Gpd2-LM

CCC ACT CGT TGT CGT ACC A

60→58→53

Reverse

Myllys et al. 2002

To obtain the partial tub2 and rpb2 (using the fRPB2-5F/fRPB2-414R and fRPB2-7cF/fRPB2-11aR primer sets) sequences, PCR amplification conditions were set as follows: an initial denaturation temperature of 94 °C for 3 min, followed by 40 (tub2) or 45 (rpb2) cycles of denaturation temperature of 94 °C for 30 s, primer annealing at the temperature stipulated in Table 2 for 30 s, primer extension at 72 °C for 45 s and a final extension step at 72 °C for 5 min.

A touchdown PCR protocol was used to amplify the partial gapdh (using the Gpd1-LM/Gpd2-LM primer set (Myllys et al. 2002)) and rpb2 (using the RPB2-C5F/RPB2-C8R primer set) sequences: initial denaturation (94 °C, 5 min), five amplification cycles (94 °C, 45 s; 60 °C, 45 s; 72 °C, 90 s), five amplification cycles (94 °C, 45 s; 58 °C, 45 s; 72 °C, 90 s), 30 amplification cycles (94 °C, 45 s; 53 °C (gapdh) or 56 °C (rpb2), 45 s; 72 °C, 90 s) and a final extension step (72 °C, 5 min). PCR products were visualised by electrophoresis using a 1.2 % agarose gel, stained with GelRed™ (Biotium, Hayward, CA) and viewed under ultra-violet light. Size estimates were made using a HyperLadder™ I molecular marker (Bioline).

Sequencing and phylogenetic analyses

The resulting PCR fragments were sequenced in both directions using the same primers used for amplification (Table 2) and the BigDye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems, Foster City, CA), following the manufacturer’s instructions. DNA sequencing amplicons were purified through Sephadex G-50 Superfine columns (SigmaAldrich, St Louis, MO) in 96-well MultiScreen HV plates (Millipore, Billerica, MA) as outlined by the manufacturer and analysed with an ABI Prism 3730xl Automated DNA analyser (Life Technologies Europe BV, Applied Biosystems™, Bleiswijk, The Netherlands).

The raw DNA sequences of tub2, gapdh and rpb2 were edited using MEGA v. 6 (Tamura et al. 2013) and forward and reverse sequences for each isolate were assembled manually to generate consensus sequences. Two parts of the rpb2 gene (part amplified with the fRPB2-5F/fRPB2-414R primer set + part amplified with the RPB2-C5F/RPB2-C8R primer set) were compiled manually using MEGA v. 6. The assembled consensus sequences were initially aligned with MEGA v. 6 and optimised with the multiple sequence alignment online interface of MAFFT using default settings (http://mafft.cbrc.jp/alignment/server/) (Katoh & Standley 2013), and adjusted manually where necessary. In addition, sequences of the same isolates corresponding to the ITS locus (including ITS1, 5.8S, ITS2), together with parts of four protein coding genes, viz. translation elongation factor 1-alpha (tef1), actin (actA), calmodulin (cmdA) and histone H3 (his3), were retrieved from the NCBIs GenBank nucleotide database and included in the analyses, after separate alignment as described above. Sequences of Cercospora sorghicola (CBS 136448 = IRAN 2672C) were used as outgroup. Evolutionary models for phylogenetic analyses were selected independently for each locus using MrModeltest v. 2.3 (Nylander 2004) under the Akaike Information Criterion (AIC) (Table 3). The individual alignments of the different loci were subsequently concatenated with Mesquite v. 2.75 (Maddison & Maddison 2011) prior to being subjected to a combined multi-gene analysis. Given the different sizes of the data partitions, they could not be properly used in statistical tests for (in) congruency. Phylogenetic reconstruction was performed using Bayesian inference (BI) Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.2.2 (Ronquist et al. 2012). Two simultaneous MCMC analyses, each consisting of four Markov chains, were run from random trees until the average standard deviation of split frequencies reached a value of 0.01, with trees saved every 100 generations and the heating parameter was set to 0.15. Burn-in phase was set to 25 % and the posterior probabilities (Rannala & Yang 1996) were calculated from the remaining trees. The resulting phylogenetic tree was generated with Geneious v. 5.6.7 (Drummond et al. 2012).
Table 3

Phylogenetic data and the substitution models used in the phylogenetic analysis, per locus. Abbreviations of loci follow Table 1.

Locus

ITS

tef1

actA

cmdA

his3

tub2

rpb2

gapdh

Number of characters

470

291

187

248

358

415

1229

869

Unique site patterns

16

75

48

66

63

105

259

231

Substitution model used

SYM-gamma

K80-gamma

K80-gamma

K80-gamma

HKY-gamma

GTR-gamma

GTR-gamma

GTR-I-gamma

Number of generations (n)

   

2 405 000

   

Total number of trees (n)

   

4 812

   

Sampled trees (n)

   

3 610

   

All new sequences generated in this study were deposited in NCBIs GenBank nucleotide database (www.ncbi.nlm.nih.gov; Table 1) and the alignment and phylogenetic trees in TreeBASE S22944 (www.TreeBASE.org).

Morphology

Morphological descriptions are based on structures from dried material. Diseased leaf tissues were viewed under a Nikon® SMZ1500 stereo-microscope and taxonomically informative morphological structures (stromata, conidiophores and conidia) were picked up from lesions with a sterile dissecting needle and mounted on glass slides in clear lactic acid. Structures were examined under a Nikon Eclipse 80i light microscope, and photographed using a mounted Nikon digital sight DS-f1 high definition colour camera.

Thirty measurements were made at ×1000 for each microscopic structure, and 95 % confidence intervals were derived for the measurements with extreme values given in parentheses. Colony macro-morphology on MEA was determined after 1 mo at 25 °C in the dark in duplicate and colony colour was described using the mycological colour charts of Rayner (1970). Nomenclatural novelties and descriptions were deposited in MycoBank (www.mycobank.org; Crous et al. 2004). The naming system for tentatively applied names used by Groenewald et al. (2013) and Bakhshi et al. (2015a) is continued in this manuscript to simplify comparison between the studies.

Identification of the best-performing DNA barcode

The dataset of the eight loci, ITS, tef1, actA, cmdA, his3, tub2, rpb2 and gapdh, was individually tested for two factors: Kimura-2-parameter (K2P) values (barcode gap) and molecular phylogenetic resolution (clade recovery). Inter- and intraspecific distances of eight loci were calculated for each single-locus sequence data alignment, using MEGA v. 6.0 with the Kimura-2-parameter distance values using the pairwise deletion model. Microsoft Excel 2010 was subsequently used to sort these distance values into distribution bins (from distance 0–0.1 with intervals of 0.01 between bins) and the frequency of entries for each individual bin was then plotted against the Kimura-2-parameterdistance of each bin.

In addition, Bayesian analyses using the corresponding nucleotide substitution models (Table 3) were applied to each data partition to check the stability and robustness of each species clade (clade recovery) under the different loci (data not shown, trees deposited in TreeBASE S22944) (Table 4). The clade recovery and Kimura-2-parameter values for each locus were calculated after applying the consolidated species concept to the results of eight-gene phylogenetic tree.
Table 4

Summary of clade support (Bayesian posterior probabilities (PP) values) for each species and locus or combination of loci. Green cells represent the PP values of species which are supported as distinct species, purple cells represent the PP values of species which are indistinct from one other species; while white cells represent species which cannot be distinguished from several other species for the given locus or combination of loci. The K2P inter-/intraspecies variation ratio as well as the number of species in the three different coloured categories are indicated per locus below the table. Abbreviations of loci follow Table 1.

Locus/Loci

ITS

tef1

actA

cmdA

his3

tub2

rpb2

gapdh

All 8 loci

C. althaeina

 

1

0.91

 

0.79

1

0.97

0.7

1

C. apii

       

1

1

C. armoraciae

 

0.73

 

1

0.98

1

?

1

1

C. beticola

   

1

   

1

1

C. bizzozeriana

 

0.96

 

1

0.98

1

1

1

1

C. chenopodii

0.99

1

1

0.95

1

1

1

1

1

C. convolvulicola

  

1

0.99

   

1

1

C. conyzae-canadensis

 

0.96

1

0.92

1

1

1

0.57

1

C. cylindracea

 

1

0.9

 

0.99

0.98

0.97

0.75

1

C. cf. flagellaris clade 1

       

1

1

C. cf. flagellaris clade 2

       

1

1

C. cf. flagellaris clade 3

       

1

1

C. gamsiana

   

1

   

1

1

C. cf. gossypii

       

1

1

C. iranica

 

1

0.94

1

1

0.98

1

0.91

1

C. plantaginis

       

1

1

C. pseudochenopodii

 

1

1

0.95

1

0.99

1

1

1

C. cf. richardiicola

 

1

1

1

1

1

1

1

1

C. rumicis

 

0.99

  

1

 

1

1

1

C. solani

1

0.99

0.93

0.83

1

1

1

1

1

C. sorghicola

1

1

1

1

1

1

1

1

1

Cercospora sp. G clade 1

 

0.98

 

1

0.86

1

1

1

1

Cercospora sp. G clade 2

 

0.98

 

1

0.86

1

1

1

1

Cercospora sp. T

 

1

0.94

1

1

1

1

0.91

1

C. uwebrauniana

 

1

  

0.76

0.99

 

1

1

C. violae

 

0.97

1

0.93

0.94

 

1

1

1

C. zebrina

 

0.73

0.87

 

0.84

 

1

0.75

1

C. cf. zinniae

 

1

1

1

1

1

1

1

1

K2P inter-/intraspecies variation ratio

4

127

15

76

13

71

74

44

 

Number of distinct species

3

11

12

9

13

12

9

17

 

Number of two indistinct species

0

8

2

      

Number of unresolved species

25

9

14

11

9

12

10

0

 

Allele group designation

The isolates in each of the Cercospora species complexes, including C. apii, C. armoraciae, C. beticola, C. cf. flagellaris, and Cercospora sp. G, were compared using the individual alignments of the eight single loci in MEGA v. 6. Allele groups were established for each locus based on sequence identity, i.e. each sequence with one or more nucleotide difference from the other sequence was regarded as a different allele.

Results

DNA amplification and phylogenetic analysis

New primers were designed for rpb2 and tub2 in this study (Table 2) and proved to be effective for the selected Cercospora species. Approximately 400, 1000, and 1200 bp were obtained for tub2, gapdh and rpb2 loci, respectively. The final concatenated eight-locus alignment contained 169 ingroup taxa and a total of 4 099 characters including alignment gaps were processed. The gene boundaries were 1–470 bp for ITS, 475-765 bp for tef1, 770–956 bp for actA, 961–1 208 bp for cmdA, 1 213–1 570 bp for his3, 1 575–1 989 bp for tub2, 1 994–3 222 bp for rpb2, and 3 227–4 099 bp for gapdh. For the total alignment, 28 characters which were artificially introduced as spacers to separate the loci, were excluded from the phylogenetic analyses. The alignment contained 863 unique site patterns (Table 3).

The Bayesian analysis lasted 2 405 000 generations and generated 4 812 trees from which the first 1 202 trees (25 %), representing the burn-in phase of the analyses, were discarded, and the remaining trees (3 610) were used for calculating posterior probabilities (PP) values in the phylogenetic tree (50 % majority rule consensus tree) (Fig. 1).
Fig. 1
Fig. 1

Consensus phylogram (50 % majority rule) of 3 610 trees resulting from a Bayesian analysis of the combined eight-gene sequence alignment using MrBayes v. 3.2.2. The scale bar indicates 0.02 expected changes per site. Hosts and country of origin are indicated in green and black text, respectively. The tree was rooted to Cercospora sorghicola (isolate CBS 136448 = IRAN 2672C).

Taxonomy

Species delimitation in the genus Cercospora in this study follows the Consolidated Species Concept accepted in recent revisions of the taxonomy of cercosporoid fungi (e.g. Groenewald et al. 2013, Crous et al. 2013, Bakhshi et al. 2015a, Videira et al. 2017). Twenty-eight lineages of Cercospora were resolved based on the clustering and support in the Bayesian tree obtained from the combined ITS, tef1, actA, cmdA, his3, tub2, rpb2, and gapdh alignment (Fig. 1, Table 4). Ofthese, 15 species including C. althaeina, C. chenopodii, C. convolvulicola, C. conyzae-canadensis, C. cylindracea, C. iranica, C. pseudochenopodii, C. cf. richardiicola, C. rumicis, C. solani, C. sorghicola, Cercospora sp. T, C. violae, C. zebrina, and C. cf. zinnia, were the same as those also accepted before in the five-gene phylogenetic tree (ITS, tef1, actA, cmdA, and his3) (Bakhshi et al. 2015a). However, the eight-gene phylogenetic tree separated strains previously recognised as C. apii, C. armoraciae, C. beticola, C. cf. fagellaris, and Cercospora sp. G, based on five-gene phylogenetic tree (Groenewald et al. 2013, Bakhshi et al. 2015a) into at least three, two, two, four and two well-supported clades respectively (Fig. 1). Some of these clades are supported by the host range or morphological characters of the isolates and are therefore described as new below.

Cercospora apii complex

The 16 isolates previously recognised as C. apii based on five-gene phylogenetic tree (Groenewald et al. 2013, Bakhshi et al. 2015a) are assigned here to three lineages based on the eight-gene phylogenetic tree, host association, and morphology, including C. apii s. str., C. uwebrauniana sp. nov., and C. plantaginis (Fig. 1, part 2). The results of allele group designation for the isolates in this complex detected one, four, two, two, four, three, four and two allele groups for the ITS, tef1, actA, cmdA, his3, tub2, rpb2, and gapdh sequences, respectively (Table 5).
Table 5

Results from allele group designation per locus for Cercospora apii s. lat. isolates in this study. Abbreviations of loci and collection accession numbers follow Table 1.

Species

Culture accession number

Host

Allele group per locus

ITS

tef1

actA

cmdA

his3

tub2

rpb2

gapdh

C. apii s. str.

CCTU 1069

Cynanchum acutum

I

II

I

I

II

I

IV

I

 

CCTU 1086; CBS 136037; IRAN 2655C

Cynanchum acutum

I

II

I

I

II

I

I

I

 

CCTU 1215

Cynanchum acutum

I

II

I

I

II

I

I

I

 

CCTU 1219; CBS 136155

Cynanchum acutum

I

II

I

I

II

I

II

I

 

CBS536.71; CPC 5087

Apium graveolens

I

II

I

I

II

I

I

I

 

CBS 116455; CPC 11556 (TYPE)

Apium graveolens

I

 

I

I

 

I

 

I

 

CBS 110813; CPC5110

Molucella laevis

I

II

I

II

II

I

I

I

 

CPC5112

Molucella laevis

I

II

I

II

II

I

I

I

C. plantaginis

CCTU 1041; CPC24910

Plantago lanceolata

I

II

I

II

III

I

I

II

 

CCTU 1047

Plantago lanceolata

I

II

I

II

II

II

I

II

 

CCTU 1082; CBS 138728

Plantago lanceolata

I

II

I

II

III

II

I

II

 

CCTU 1095

Plantago lanceolata

I

II

I

II

III

II

I

II

 

CCTU 1179

Plantago lanceolata

I

II

 

II

II

I

III

II

 

CBS 252.67; CPC 5084 (TYPE)

Plantago lanceolata

I

III

 

II

III

II

II

C. uwebrauniana

CCTU 1134

Heliotropium europaeum

I

IV

I

II

IV

III

I

I

 

CCTU 1200; CBS 138581 (TYPE)

Heliotropium europaeum

I

IV

I

II

IV

III

I

I

Cercospora apii Fresen., Beitr. Mykol. 3: 91 (1863).

Sensu Groenewald et al., Phytopathology 95: 954 (2005).

(Fig. 2)
Fig. 2
Fig. 2

Cercospora apii (CBS 136037). A. Leafspots. B–C. Fasciculate conidiophores. D–H. Conidia. Bars = 10 µm.

Type: Germany: Oestrich, on Apium graveolens (Apiaceae), Fuckel, Fungi rhen. 117, in HAL (lectotype designated by Groenewald et al. 2005); Heilbronn, Landwirtschaftsamt, on A. graveolens, 10Aug. 2004, K. Schrameyer (CBS 116455 = CPC 11556 — epitype designated by Groenewald et al. 2005).

Description: Leaf spots amphigenous, distinct, circular to subcircular, 1–9 mm diam, white-grey in centre, surrounded by a dark purple-brown border. Mycelium internal. Caespituli amphigenous, brown. Conidiophores aggregated in moderately dense fascicles (4–15), arising from the upper cells of a well-developed brown stroma, to 50 µm wide; conidiophores brown, becoming pale brown towards the apex, 1–6-septate, straight to variously curved, unbranched, uniform in wide, (45–)80–95(–125) × 4–5.5 µm. Conidiogenous cells integrated, lateral or terminal, unbranched, brown, smooth, proliferating sympodially, 20–40 × 3.5–5 µm, multi-local; loci thickened, darkened, refractive, apical or lateral, 2–3.5 µm diam. Conidia solitary, smooth, obclavate-cylindrical to acicular, straight to slightly curved, hyaline, distinctly 3–9(–15)-septate, apex subacute or subobtusely rounded, base subtruncate to obconically truncate, (30–)65–80(–115) × 3–5 µm; hila thickened, darkened, refractive, 2–3.5 µm diam.

Note: This clade includes the ex-epitype strain of C. apii (isolate CBS 116455 = CPC 11556), therefore we fixed the application of C. apii s. str. to this clade.

Specimens examined: Germany: Heilbron, Landwirtschaftsamt, on A. graveolens, K. Schrameyer (CBS 116455 = CPC 11556 —ex-epitype culture). — Iran: Ardabil Province: Moghan, on leaves of Cynanchum acutum (Apocynaceae), Oct. 2011, M. Bakhshi (IRAN 17016F, IRAN 17017F, CCTU 1069, CCTU 1086 = IRAN 2655C = CBS 136037); Moghan, on leaves of C. acutum, Oct. 2012, M. Bakhshi (IRAN 17018F, IRAN 17019F, CCTU 1215, CCTU 1219 = CBS 136155). — New Zealand: Auckland, on M. laevis, C.F. Hill (CPC 5112). — Romania: Bucuresti, on A. graveolens, 2 Oct. 1969, O. Constantinescu (CBS 536.71 = CPC 5087). — USA: California: on Moluccella laevis (Lamiaceae), S.T. Koike (CBS 110813 = CPC 5110).

Cercospora plantaginis Sacc., Michelia 1: 267 (1878).

(Fig. 3)

Type: Italy: Selva, on Plantago lanceolata (Plantaginaceae), Sep. 1873, P.A. Saccardo (PAD, s.n. — holotype, according to Art. 9.1, Note 1). — Romania: Domnesti, on P. lanceolata, 3 Aug. 1965, O. Constantinescu (CBS 252.67 — epitype designated here, MBT 383093, preserved as a metabolically inactive culture).
Fig. 3
Fig. 3

Cercospora plantaginis (CPC 24910). A. Leafspots. B–C. Fasciculate conidiophores. D–J. Conidia. Bars = 10 µm.

Description: Leaf spots amphigenous, circular to subcircular, 1–4 mm diam, white to grey with distinct raised brown borders. Mycelium internal. Caespituli amphigenous, brown. Conidiophores aggregated in loose fascicles, arising from a moderately developed, intraepidermal and substomatal, dark brown stroma, to 30 µm diam; conidiophores brown at the base, becoming paler towards the apex, 2–10-septate, straight to geniculate-sinuous due to sympodial proliferation, simple, uniform in width, somewhat constricted at the proliferating point, (45–)60–85 × 4–5 µm. Conidiogenous cells integrated, terminal or lateral, pale brown to brown, proliferating sympodially, 8–25 × 3.5–5 µm, multi-local; loci distinctly thickened, darkened and somewhat refractive, apical or formed on shoulders caused by sympodial proliferation, 2–3 µm diam. Conidia solitary, subcylindrical, filiform to acicular, straight to mildly curved, hyaline, (40–)60–70(–105) × 2–3.5 µm, (4–)8–13(–17)-septate, with subobtuse to subacute apices and truncate bases; hila thickened, darkened, refractive, 1.5–2.5 µm diam.

Notes: Based on the results of the eight-gene phylogenetic tree, all isolates obtained from P. lanceolata from five different provinces in Iran together with a European isolate from this host plant, previously recognised as C. apii based on a five-gene phylogenetic tree (Groenewald et al. 2013, Bakhshi et al. 2015a), cluster separately from the other isolates in this clade (Fig. 1, part 2). Three species of Cercospora, including C. apii, C. pantoleuca and C. plantaginis, have been reported from Plantago (Crous & Braun 2003, https://nt.ars-grin.gov/fungaldatabases/). This species is morphologically close to C. plantaginis described from Italy on P. lanceolata (Chupp 1954). Since one European isolate from P. lanceolata in Romania (CBS 252.67 = CPC 5084) also resides in this clade, we designate an epitype here for this species, and fix the application of the name C. plantaginis to this clade.

Additional specimens examined: Iran: Guilan Province: Chaboksar, on P. lanceolata, Jul. 2012, M. Bakhshi (IRAN 17076F, CCTU 1041 = CPC 24910). Zanjan Province: Tarom, Pasar, on P. lanceolata, Sep. 2011, M. Bakhshi (IRAN 17078F, CCTU 1047). Ardabil Province: Moghan, on P. lanceolata, Sep. 2011, M. Bakhshi (CCTU 1082 = CBS 138728). East Azerbaijan Province: Arasbaran, Horand, on P. lanceolata, Oct. 2011, M. Bakhshi (CCTU 1095). West Azerbaijan Province: Khoy, Firouragh, on P. lanceolata, Sep. 2012, M. Arzanlou (IRAN 17077F, CCTU 1179 = IRAN 2716C).

Cercospora uwebrauniana M. Bakhshi & Crous, sp. nov.

MycoBank MB827521

(Fig. 4)
Fig. 4
Fig. 4

Cercospora uwebrauniana (CBS 138581). A. Leafspots. B–C. Fasciculate conidiophores. D–I. Conidia. Bars = 10 µm.

Etymology: Named in honourof Uwe Braun, who has published extensively on the genus Cercospora, and also provided a modern treatment for allied genera of Mycosphaerellaceae.

Diagnosis: Differs from C. taurica in the cylindrical conidia with truncate or subtruncate bases and somewhat shorter and wider conidia, (23–)38–48(–70) × 4.5–8 µm vs 40–110 × (2.5–)4–6(–7) µm in C. taurica.

Type: Iran: Ardabil Province: Moghan, on Heliotropium europaeum (Boraginaceae), Oct. 2012, M. Bakhshi (IRAN 16864F — holotype; CCTU 1200 = CBS 138581 — ex-type culture).

Description: Leaf spots distinct, circular to irregular, 3–10 mm, grey-brown to dark brown, surrounded by brown margin. Mycelium internal. Caespituli amphigenous, brown. Conidiophores in moderately dense fascicles, arising from the upper cells of a moderately developed, intraepidermal and substomatal, brown stroma, to 40 µm wide; conidiophores straight to slightly geniculate, pale brown to brown, unbranched, regular in width, (60–)115–145(–230) × 3.5–5.5 µm, 2–9-septate. Conidiogenous cells integrated, terminal, brown, proliferating sympodially, 15–35 × 3.5–5.5 µm, mostly mono-local, sometimes multi-local; loci distinctly thickened, darkened, refractive, apical or formed on the shoulders caused by geniculation, 2–3.5 µm. Conidia solitary, hyaline, subcylindrical to cylindrical, straight or slightly curved, truncate to subtruncate at the base, obtuse to rounded at the apex, (23–)38–48(–70) × 4.5–8 µm, (0–)3–4(–9)-septate; hila thickened, darkened, refractive, 1.5–3 µm diam.

Notes: Two isolates, obtained from H. europaeum in different provinces in Iran, clustered in a small clade within C. apiis. str. (Fig. 1, part 2). This independent clade is supported by tef1, his3 and tub2 from C. apii s. str. Morphologically, these two strains are completely distinct from their most closely related species in the phylogenetic tree, namely C. apii (conidia acicular, subacute or subobtusely rounded at the apex, (30–)65–80(–115) × 3–5 µm), C. beticola (conidia subacute to acute apex, (40–)90–140(–300) × 2–5 µm), C. gamsiana (conidia subobtuse at the apex, (27–)49–62(–100) × 2–4 µm) and C. plantaginis (conidia subobtuse to subacute apices, (40–)60–70(–105) × 2–3.5 µm), by the obtuse to rounded apex, wider and shorter conidia ((23–)38–48(–70) × 4.5–8 µm), and are regarded as a separate species, appearing to be confined to H. europaeum.

Presently, three species of Cercospora have been described from Heliotropium, C. apii, C. heliotropiicola, and C. taurica (Crous & Braun 2003, https://nt.ars-grin.gov/fungalda-tabases/). Cercospora uwebrauniana differs from C. taurica in the cylindrical conidia with truncate or subtruncate bases and somewhat shorter and wider conidia, (23–)38–48(–70) × 4.5–8 µm vs 40–110 × (2.5–)4–6(–7) µm in C. taurica (Braun 2002). In addition, C. taurica has obclavate-cylindrical conidia with obconically truncate bases and rather wider conidiophores, 4–9 µm diam (Braun 2002). Cercospora heliotropiicola is morphologically quite distinct from C. uwebrauniana in having acicular or subulate, much thinner (2–3 µm wide) and longer (to 300 µm long) conidia with subobtuse or acute apex (Pons & Sutton 1996).

Additional specimen examined: Iran: Guilan Province: Astara, on H. europaeum, Jun. 2012, M. Bakhshi (IRAN 17096F, CCTU 1134).

Cercospora armoraciae complex

The 10 isolates previously recognised as C. armoraciae based on a five-gene phylogenetic tree (Groenewald et al. 2013, Bakhshi et al. 2015a) are assigned to two lineages here, based on the eight-gene phylogenetic tree, including C. armoraciae s. str. and C. bizzozeriana (Fig. 1, part 1). The results of allele group designation for the isolates in this complex revealed one, three, one, two, seven, three, three and two allele groups for the ITS, tef1, actA, cmdA, his3, tub2, rpb2 and gapdh sequences, respectively (Table 6).
Table 6

Results from allele group designation per locus for Cercospora armoraciae s. lat. isolates in this study. Abbreviations of loci and collection accession numbers follow Table 1.

Species

Culture accession number

Host

Allele group per locus

ITS

tef1

actA

cmdA

his3

tub2

rpb2

gapdh

C. armoraciae s. str.

CBS 250.67; CPC 5088 (TYPE)

Armoracia rusticana (= A. lapathifolia)

I

I

I

I

I

I

II

C. bizzozeriana

CCTU 1013

?

I

II

I

I

III

I

I

I

 

CCTU 1022; CBS 136028

?

I

II

I

I

III

I

I

I

 

CCTU 1040; CBS 136131

Tanacetum balsamita

I

III

I

II

VI

I

II

I

 

CCTU 1107

?

I

II

I

I

VII

I

I

I

 

CCTU 1117; CBS 136132

Cardaria draba

I

II

I

I

V

I

I

I

 

CCTU 1234

Cardaria draba

I

II

I

I

V

III

I

I

 

CCTU 1127; CBS 136133

Capparis spinosa

I

II

I

I

IV

 

III

I

 

CBS540.71; CPC 5060

Cardaria draba

I

II

I

I

II

I

I

 

CBS 258.67; CPC 5061 (TYPE)

Cardaria draba

I

II

I

I

II

I

I

Cercospora armoraciae Sacc., Nuovo Giorn. Bot. Ital. 8: 188 (1876).

Note: This clade includes the ex-type culture of C. armoraciae (CBS 250.67).

Cercospora bizzozeriana Sacc. & Berl., Malpighia 2: 248 (1888).

(Fig. 5)
Fig. 5
Fig. 5

Cercospora bizzozeriana (CBS 136132). A–B. Leafspots. C. Fasciculate conidiophores. D–J. Conidia. Bars = 10 µm.

Type: Italy: Padova, on Lepidium latifolium (Brassicaceae), (Berlese, Malpighia 1: tab. XIV, fig. 23, 1887 — lectotype, designated here, MBT 383343); Romania: Fundulea, on Cardaria draba, isol. by O. Constantinescu [deposited in the CBS culture collection in 1967] (CBS 258.67 — epitype designated here, MBT 383154, preserved as a metabolically inactive culture).

Notes: Type material of C. bizzozeriana is not preserved in Saccardo’s herbarium (see Gola 1930). Therefore, the original illustration published by Saccardo & Berlese (in Berlese 1888) is designated as lectotype (according to Art. 9.3 and 9.4). Berlese’s article “Fungi veneti novi vel critici” was split into several parts published in Malpighia 1 (1887) and 2 (1888). The description of C. bizzozeriana was published in vol 2, but with reference to tab. XIV, fig. 23 already issued in vol. 1.

Description: Leaf spots amphigenous, circular, 1–5 mm, white to white-grey with grey to black dots (stroma with conidiophores) and definite brown border. Mycelium internal. Caespituli amphigenous, brown. Conidiophores aggregated in dense fascicles, arising from a well-developed, brown stroma, to 75 µm diam; conidiophores brown, 1–5-septate, straight to geniculate-sinuous due to sympodial proliferation, simple, sometimes branched, uniform in width, sometimes constricted at the proliferating point, (30–)50–60(–80) × 4–7 µm. Conidiogenous cells integrated, terminal or lateral, pale brown to brown, proliferating sympodially, 10–25 × 3–6 µm, multi-local; loci distinctly thickened, darkened and somewhat refractive, apical, lateral or formed on shoulders caused by geniculation, 1.5–3 µm diam. Conidia solitary, obclavate-cylindrical, straight to slightly curved, hyaline, (20–)60–80(–125) × 3–6 µm, 2–10-septate, with obtuse apices and subtruncate or obconically truncate bases; hila thickened, darkened, refractive, 1.5–3 µm diam.

Notes: Isolates obtained from different host species including Tanacetum balsamita, Capparis spinosa and Cardaria draba clustered in a clade distinct from the ex-type isolate of C. armoraciae, and are regarded as a separate taxon. In addition, five isolates obtained from Car. draba (three from Iran and two from Romania) all cluster in this clade. Until now, three species of Cercospora are known from these host species, including C. bizzozeriana, C. chrysanthemi and C. capparis (Crous & Braun 2003, https://nt.ars-grin.gov/fungaldatabases/). Cercospora chrysanthemi is in the C. apii s. lat. complex (Crous & Braun 2003). Cercospora capparis differs from this species by the narrower (4–5.5 µm diam) conidiophores and 3–5 µm diam conidia (Chupp 1954). The species is morphologically close to C. bizzozeriana which was described from Italy on Car. draba (Chupp 1954). Since two European isolates from Car. draba in Romania also reside in this clade, we designate an epitype here (ex-epitype culture CBS 258.67 = CPC 5061) for this species, and fix the application of C. bizzozeriana to this clade.

Additional specimens examined: Iran: West Azerbaijan Province: Khoy, Firouragh, on leaves of Car. draba, Nov. 2011, M. Arzanlou (CCTU 1117 = CBS 136132); Khoy, Firouragh, on leaves of Car. draba, Oct. 2012, M. Arzanlou (IRAN 17027F, CCTU 1234). Zanjan Province: Tarom, Haroun Abad, on leaves of Tanacetum balsamita (Asteraceae), Sep. 2011, M. Bakhshi (IRAN 17029F, CCTU 1040 = CBS 136131); Tarom, Mamalan, Oct. 2011, M. Bakhshi (IRAN 17028F, CCTU 1107); Mianeh, Oct. 2012, M. Torbati (IRAN 17025F, IRAN 17026F, CCTU 1013, CCTU 1022 = CBS 136028). Khuzestan Province: Ahvaz, on leaves of Capparis spinosa (Capparidaceae), Dec. 2011, E. Mohammadian (CCTU 1127 = CBS 136133). — Romania: Hagieni, on Car. draba, O. Constantinescu (CBS 540.71 = IMI 161110 = CPC 5060).

Cercospora beticola complex

The 16 isolates previously recognised as C. beticola based on a five-gene phylogenetic analysis (Groenewald et al. 2013, Bakhshi et al. 2015a), are assigned to two lineages based on the eight-gene phylogenetic analysis (Fig. 1, part 2). One, one, one, one, one, two, three and four allele groups were distinguished for the ITS, tef1, actA, cmdA, his3, tub2, rpb2 and gapdh sequences, respectively (Table 7).
Table 7

Results from allele group designation per locus for Cercospora beticola s. lat. isolates in this study. Abbreviations of loci and collection accession numbers follow Table 1.

Species

Culture accession number

Host

Allele group per locus

ITS

tef1

actA

cmdA

his3

tub2

rpb2

gapdh

C. beticola

CCTU 1057; IRAN 2651C

Chenopodium sp.

I

I

I

I

I

I

II

III

 

CCTU 1065

Chenopodium sp.

I

I

I

I

I

I

II

II

 

CCTU 1087

Chenopodium sp.

I

I

I

I

I

I

II

III

 

CCTU 1088; CBS 138582

Sonchus asper

I

I

I

I

I

I

II

III

 

CCTU 1089; CPC24911

Plantago lanceolata

I

I

I

I

I

II

II

II

 

CCTU 1108

Plantago lanceolata

I

I

I

I

I

I

II

II

 

CBS 116456; CPC 11557 (TYPE)

Beta vulgaris

I

I

I

I

I

I

I

I

 

CCTU 1135

Beta vulgaris

I

I

I

I

I

I

II

III

 

CPC 12028

Beta vulgaris

I

I

I

I

I

I

II

III

 

CPC 12029

Beta vulgaris

I

I

I

I

I

I

II

III

C. gamsiana

CCTU 1035

Malva sylvestris

I

I

I

I

I

I

III

IV

 

CBS 144962; CCTU 1074; CPC 24909 (TYPE)

Malva neglecta

I

I

I

I

I

I

III

IV

 

CCTU 1109

Malva sylvestris

I

I

I

I

I

I

III

IV

 

CCTU 1199; CBS 136128; IRAN 2675C

Rumex crispus

I

I

I

I

I

I

II

IV

 

CCTU 1205; CBS 136127; IRAN 2677C

Sesamum indicum

I

I

I

I

I

I

II

IV

 

CCTU 1208; IRAN 2678C

Sonchus sp.

I

I

I

I

I

I

II

IV

Cercospora beticola Sacc., Nuovo Giorn. Bot. Ital. 8: 189 (1876).

Sensu Groenewald et al., Phytopathology 95: 954 (2005).

(Fig. 6)
Fig. 6
Fig. 6

Cercospora beticola (CCTU 1135). A. Leaf spots. B. Fasciculate conidiophores. C–G. Conidia. Bars = 10 µm.

Type: Italy: Vittorio (Treviso), on Beta vulgaris (Chenopodiaceae), Sep. 1897, P.A. Saccardo, Fungi ital. no. 197 (PAD — neotype designated by Groenewald et al. 2005); Ravenna, on B. vulgaris, 10 Jul. 2003, V. Rossi (CBS 116456 = CPC 11557 — epitype designated by Groenewald et al. 2005).

Description: Leaf spots amphigenous, distinct, circular to subcircular, 1–7 mm diam, white-grey, with grey dots (stroma with conidiophores), surrounded by distinct brown border. Mycelium internal. Caespituli amphigenous, brown. Conidiophores aggregated in loose to dense fascicles, emerging through stomatal openings or erumpent through the cuticle, arising from the upper cells of a moderately to well-developed brown stroma, to 110 µm diam; conidiophores brown, becoming paler towards apex, 2–8-septate, thick-walled, straight to geniculate-sinuous, unbranched, uniform in width, (30–)80–110(–185) × 4–5(–6) µm. Conidiogenous cells integrated, terminal or lateral, unbranched, brown, smooth, proliferating sympodially, 10–30 × 3.5–5.5 µm, mostly multi-local, sometimes mono-local; loci apical or formed on shoulders caused by geniculation, thickened, darkened, refractive, 1.5–2 µm diam. Conidia solitary, subcylindrical, filiform to acicular, straight to variously curved, hyaline, 3–15(–29)-septate, apex subacute to acute, base truncate to subtruncate, (40–)90–140(–300) × 2–5 µm; hila thickened, darkened, refractive, 1.5–2.5 µm diam.

Note: This clade includes the ex-epitype culture of C. beticola (CBS 116456 = CPC 11557), therefore we fixed the application of the name C. beticola s. str. to this clade.

Additional specimens examined: Egypt, on B. vulgaris, 15 Apr. 2004, M. Hasem (CPC 12028, CPC 12029). — Iran: Guilan Province: Talesh, Khotbeh Sara, on leaves of B. vulgaris, Jun. 2012, M. Bakhshi (IRAN 17020F, CCTU 1135). Zanjan Province: Tarom, Mamalan, on P. lanceolata, Oct. 2011, M. Bakhshi (IRAN 17023F, CCTU 1108). Ardabil Province: Moghan, on P. lanceolata, Oct. 2011, M. Bakhshi (CCTU 1089 = CPC 24911); Moghan, on Chenopodium sp. (Chenopodiaceae), Oct. 2011, M. Bakhshi (IRAN 17021F, IRAN 17022F, CCTU 1057 = IRAN 2651C, CCTU 1065, CCTU 1087); Moghan, on Sonchus asper (Asteraceae), Oct. 2011, M. Bakhshi (IRAN 17024F, CCTU 1088 = CBS 138582).

Cercospora gamsiana M. Bakhshi & Crous, sp. nov.

MycoBank MB827522

(Fig. 7)
Fig. 7
Fig. 7

Cercospora gamsiana (CPC 24909 = CBS 144962). A. Leafspots. B–C. Fasciculate conidiophores. D–H. Conidia. Bars = 10 µm.

Etymology: Dedicated to the recently deceased Walter Gams to honour his contribution to mycology.

Diagnosis: Morphologically distinct from species of the C. apii complex in the irregularly constricted, often conical and attenuated at the apex conidiophores, and conidia with long obconically truncate bases; sporulation is restricted to the terminal part of conidiophores.

Type: Iran: Ardabil Province: Moghan, on leaves of Malva neglecta (Malvaceae), Oct. 2011, M. Bakhshi (IRAN 17011F — holotype; CBS 144962 = CCTU 1074 = CPC 24909— ex-type culture).

Description: Leaf spots amphigenous, circular to irregular, 3–8 mm diam, grey to brown. Mycelium internal. Caespituli amphigenous, brown. Conidiophores aggregated in moderately dense fascicles, arising from a well-developed, intraepidermal and substomatal, brown stroma, to 45 µm diam; conidiophores pale brown, 1–5-septate, geniculate-sinuous, irregularly constricted, unbranched, moderately thin-walled, irregular in width, often conical and attenuated at the apex, sporulation is restricted at the terminal part of conidiophores, 45–60(–110) × 4–5 µm. Conidiogenous cells integrated, terminal, pale brown to olivaceous-brown, proliferating sympodially, 10–25 × 3.5–5 µm, uni- or multi-local; loci distinctly thickened, darkened and somewhat refractive, apical, circumspersed, 1.5–2 µm diam. Conidia solitary, subcylindrical to obclavate or somewhat narrowed towards the tip, straight to slightly curved, hyaline, thin-walled, (27–)49–62(–100) × 2–4 µm, distinctly 3–10-septate, subobtuse at the apex and long obconically truncate at the base; hila distinctly thickened, darkened, refractive, 1.5–2.5 µm diam.

Notes: Until now, 14 species names in Cercospora have been introduced from these host species, including C. apii, C. althaeina, C. beticola, C. hyalospora (C. apii s. lat. complex), C. malvarum (C. apii s. lat. complex), C. malvicola, C. sigesbeckiae, C. peckiana (C. apii s. lat. complex), C. rumicis, C. sonchi (C. apii s. lat. complex), C. sonchicola (C. apii s. lat. complex), C. sonchifolia, C. sesami (C. apii s. lat. complex), and C. sesamigena (Crous & Braun 2003, https://nt.ars-grin.gov/fungaldatabases/). Cercospora gamsiana is phylogenetically clearly distinguishable from C. apii, C. althaeina, C. beticola, C. sigesbeckiae and C. rumicis (Bakhshi et al. 2015a) (Fig. 1, part 2). It is morphologically well distinguished from species of the C. apii complex and other species of Cercospora by its irregularly constricted, thin-walled, often conical and attenuated at the apex conidiophores and, conidia with long obconically truncate bases; sporulation is restricted at the terminal part of conidiophores.

Additional specimens examined: Iran: Zanjan Province: Tarom, Zehtar Abad, on leaves of Malva sylvestris, Sep. 2011, M. Bakhshi (CCTU 1035); Tarom, Mamalan, on leaves of M. sylvestris, Oct. 2011, M. Bakhshi (CCTU 1109). Ardabil Province: Moghan, on leaves of Sonchus sp., Oct. 2012, M. Bakhshi (IRAN 17072F, CCTU 1208 = IRAN 2678C); Moghan, on leaves of Sesamum indicum (Pedaliaceae), Oct. 2012, M. Bakhshi (CCTU 1205 = IRAN 2677C = CBS 136127). Guilan Province: Ramsar, on leaves of Rumex crispus (Polygonaceae), Sep. 2012, M. Bakhshi (CCTU 1199 = IRAN 2675C = CBS 136128).

Cercospora cf. flagellaris complex

The 61 isolates previously recognised as C. cf. fagellaris based on a five-gene phylogenetic tree (Groenewald et al. 2013, Bakhshi et al. 2015a) cluster into at least four distinct phylogenetic clades based on the eight-gene phylogenetic tree including C. cf. gossypii, C. cf. fagellaris clades 1, 2 and 3 (Fig. 1, part 3). Three, four, six, seven, seven, seven, two and nine allele groups were distinguished for the ITS, tef1, actA, cmdA, his3, tub2, rpb2 and gapdh sequences, respectively (Table 8).
Table 8

Results from allele group designation per locus for Cercospora cf. flagellaris isolates in this study. Abbreviations of loci and collection accession numbers follow Table 1.

Species

Culture accession number

Host

Allele group per locus

ITS

tef1

actA

cmdA

his3

tub2

rpb2

gapdh

Cercospora cf. gossypii

CCTU 1055; IRAN 2650C

Hibiscus trionum

I

I

I

III

III

I

II

IV

CCTU 1070; CBS 136137

Gossypium herbaceum

I

I

I

III

III

I

II

IV

Cercospora cf. flagellaris clade1

CCTU 1007; CBS 136031

Hydrangea sp.

II

I

I

I

I

I

II

V

CCTU 1027; CBS 136034

Lepidium sativum

I

I

I

I

I

I

I

V

 

CCTU 1031; CBS 136036; IRAN 2648C

Urtica dioica

II

I

II

I

I

I

II

V

 

CCTU 1120

Raphanus sativus

II

I

I

I

I

I

II

V

 

CCTU 1128; CBS 136141; IRAN 2661C

Phaseolus vulgaris

II

I

I

I

I

I

II

V

 

CCTU 1159; CBS 136148

Arachis hypogaea

II

I

I

I

I

I

II

V

 

CCTU 1162; IRAN 2670C

Citrullus lanatus

II

I

I

I

I

I

I

V

 

CCTU 1168

Phaseolus vulgaris

II

I

I

II

I

I

 

V

 

CCTU 1171

Raphanus sativus

II

I

I

I

I

I

II

V

 

CPC 1051

Populus deltoides

II

I

III

II

I

I

II

V

 

CBS 132653; CPC 10884

Dysphania ambrosioides

II

I

I

III

VII

VI

II

VII

 

CPC 5441

Amaranthus sp.

 

III

V

III

IV

III

I

VI

Cercospora cf. flagellaris clade 2

CCTU 1059; CBS 136136

Ecballium elaterium

II

I

I

I

I

I

 

VIII

CCTU 1068

Xanthium spinosum

II

IV

III

III

III

I

II

VIII

 

CCTU 1085

Xanthium strumarium

II

I

I

III

V

V

II

VIII

 

CCTU 1115; CBS 136139; IRAN 2659C

Cercis siliquastrum

II

I

I

I

I

I

I

VIII

 

CCTU 1195

Datura stramonium

I

I

I

I

I

I

II

IX

 

CCTU 1198; CBS 136151

Acer velutinum

II

I

I

II

I

I

II

IX

 

CCTU 1204

Abutilon theophrasti

I

I

I

II

I

I

II

IX

 

CCTU 1216

Ecballium elaterium

II

I

I

I

I

I

II

VIII

 

CCTU 1223; CBS 136154; IRAN 2683C

Eclipta prostrata

I

I

I

II

I

I

II

VIII

 

CBS 132667; CPC 11643

Celosia argentea var. cristata (≡ C. cristata)

III

I

VI

III

III

I

II

VIII

Cercospora cf. flagellaris clade 3

CCTU 1005; IRAN 2644C

Xanthium strumarium

II

I

II

I

I

I

II

I

 

CCTU 1006; CBS 136030

Impatiens balsamina

II

I

I

I

I

II

II

I

 

CCTU 1010; CBS 136032

Pelargonium hortorum

II

I

I

I

I

I

I

I

 

CCTU 1021;CBS 136033

Amaranthus retroflexus

II

I

I

I

I

I

I

II

 

CCTU 1029; CBS 136035; IRAN 2647C

Cucurbita maxima

I

I

I

VI

I

I

I

II

 

CCTU 1048; CBS 136029

Xanthium strumarium

II

I

I

I

I

I

I

I

 

CCTU 1064

Amaranthus retroflexus

II

I

II

I

I

I

II

II

 

CCTU 1072; IRAN 2653C

Amaranthus blitoides

II

I

I

I

I

I

II

I

 

CCTU 1075

Raphanus sativus

I

I

I

VII

I

II

II

I

 

CCTU 1084; CBS 136156

Amaranthus sp.

II

I

I

I

I

II

I

I

 

CCTU 1118; CBS 136140; IRAN 2660C

Populus deltoides

II

I

II

II

I

I

I

II

 

CCTU 1130; CBS 136142

Olea europaea

II

I

I

I

I

I

II

I

 

CCTU 1136

Cucurbita pepo

II

I

I

I

I

I

II

I

 

CCTU 1138; IRAN 2664C

Phaseolus vulgaris

I

I

I

II

I

I

II

II

 

CCTU 1139; IRAN 2665C

Phaseolus vulgaris

I

I

II

I

I

I

II

I

 

CCTU 1140; CBS 136143; IRAN 2666C

Calendula officinalis

II

I

II

I

I

I

II

II

 

CCTU 1141;CBS 136144

Tagetes patula

I

I

II

I

I

VII

II

II

 

CCTU 1142; IRAN 2667C

Phaseolus vulgaris

II

II

III

IV

II

I

I

I

 

CCTU 1143; CBS 136145

Datura stramonium

II

I

I

I

I

I

I

I

 

CCTU 1147

Urtica dioica

I

I

I

II

I

I

I

II

 

CCTU 1150

Buxus microphylla

II

I

I

II

I

I

II

II

 

CCTU 1154; CBS 136147

Abutilon theophrasti

II

I

I

I

I

I

I

II

 

CCTU 1155.11

Phaseolus vulgaris

II

I

I

I

I

I

I

I

 

CCTU 1156

Xanthium strumarium

II

I

II

II

I

I

I

II

 

CCTU 1158; IRAN 2668C

Xanthium strumarium

II

I

I

I

I

I

I

I

 

CCTU 1160; CBS 136149

Vicia faba

II

I

I

I

I

I

II

I

 

CCTU 1161; IRAN 2669C

Phaseolus vulgaris

II

I

I

I

I

I

II

I

 

CCTU 1167; CBS 136150

Anubias sp.

II

I

II

I

I

I

I

I

 

CCTU 1172

Oenothera biennis

I

I

I

V

I

I

I

I

 

CCTU 1175; IRAN 2673C

Phaseolus vulgaris

II

I

I

II

I

I

I

I

 

CCTU 1209; CBS 136152

Glycine max

II

I

II

I

I

I

II

I

 

CCTU 1210; IRAN 2679C

Glycine max

II

I

II

I

I

I

II

II

 

CCTU 1211

Glycine max

II

I

I

I

I

I

I

I

 

CCTU 1212; CBS 136153; IRAN 2680C

Silybum marianum

II

I

I

I

I

I

II

II

 

CCTU 1218; IRAN 2682C

Hibiscus trionum

I

I

I

I

I

I

I

I

 

CBS 115482; CPC4410

Citrus sp.

II

I

IV

III

VI

IV

I

III

 

CBS 143.51;CPC5055

Bromus sp.

II

I

I

I

I

IV

II

I

Cercospora cf. gossypii Lall et al., Indian Phytopath. 14: 116 (1962) [“1961”].

(Fig. 8)
Fig. 8
Fig. 8

Cercospora cf. gossypii (CBS 136137). A. Leaf spots. B–C. Fasciculate conidiophores. D–F. Conidia. Bars = 10 µm.

Description: Leaf spots amphigenous, circular to subcircular, 1–4 mm diam, with grey-brown centre and purple-brown margins. Mycelium internal. Caespituli amphigenous, brown. Conidiophores aggregated in dense fascicles, arising from the upper cells of a well-developed, intraepidermal and substomatal, brown stroma, to 65 µm diam; conidiophores pale brown to brown, simple, rarely branched, 1–4-septate, straight or flexuous caused by sympodial proliferation, almost uniform in width, often constricted at proliferating point, (35–)60–75(–110) × 4–5 µm. Conidiogenous cells terminal or integrated, pale brown, smooth, proliferating sympodially, 10–45 × 3.5–5 µm, multi-local; loci thickened, darkened, refractive, apical, lateral, circumspersed, 1.5–2.5 µm diam. Conidia solitary, smooth, subcylindrical to obclavate, straight or mildly curved, successively tapering towards the apex, hyaline, 1–7-septate, apex subacute to subobtuse, base truncate to short obconically truncate, (30–)65–90(–160) × 2–4 µm; hila distinctly thickened, darkened, refractive, 1–2 µm diam.

Notes: This clade includes two isolates obtained from G. herbaceum and Hib. trionum, both in the Malvaceae (Fig. 1, part 3). Cercospora althaeina, C. fagopyri, C. malayensis (C. apii s. lat.), C. gossypii, C. gossypiicola, C. gossypina and C. lhuillieri (C. apii s. lat.) are six Cercospora species which have been reported until now on Gossypium and Hibiscus host genera (Crous & Braun 2003, https://nt.ars-grin.gov/fungaldatabases/). This species is phylogenetically distinct from C. althaeina (Fig. 1) and C. fagopyri (Groenewald et al. 2013, Bakhshi et al. 2015a). Cercospora gossypina is distinguished from this species in that it induces wider leaf spots (0.5–10 mm), and has unbranched, longer and wider conidiophores (75–250 × 4–6.5 µm) (Hsieh & Goh 1990). Cercospora malayensis is distinguished from C. cf. gossypii in that it has elliptical, yellow to tan leaf spots; unbranched, 1–8-septate conidiophores and mostly terminal conidiogenous cells and somewhat longer conidia (50–270 × 2.5–4 µm) (Little 1987). Cercospora gossypiicola (Narayan et al. 2001) and C. lhuillieri (Montegut 1967) resemble C. apii (with acicular conidia), but are different. They do not have stromata, and form less conidiophores per fascicle. The description of C. gossypii (Lall et al. 1961) is rather close to this taxon. The type of C. gossypii is from India. Thus, fresh material is needed from India to resolve the application of the name C. gossypii.

Specimens examined: Iran: Ardabil Province: Moghan, on Gossypium herbaceum (Malvaceae), Oct. 2011, M. Bakhshi (IRAN 17073F, CCTU 1070 = CBS 136137); Moghan, on Hibiscus trionum (Malvaceae), Oct. 2011, M. Bakhshi (IRAN 17074F, CCTU 1055 = IRAN 2650C).

Cercospora cf. flagellaris Ellis & G. Martin, Am. Nat. 16: 1003 (1882).

Clade 1; Clade 2; Clade 3

In view of the overlap between the morphological characters of these three clades, we provide a single over-arching description here.

Description: Mycelium internal. Caespituli amphigenous, brown. Conidiophores aggregated in loose to dense fascicles, arising from a weakly to well-developed, intraepidermal and substomatal, brown stroma; conidiophores pale brown to brown, 2–18-septate, straight, sinuous to distinctly geniculate, flexuous, simple, unbranched or rarely branched, uniform or irregular in width, sometimes constricted at septa and proliferating point, (75–)130–165(–300) × 4–5.5 µm in clade 1; (30–)80–120(–210) × 3.5–5.5 µm in clade 2; (25–)60–95(–230) × 3.5–5.5 µm in clade 3. Conidiogenous cells integrated, terminal, proliferating sympodially, mono- or multi-local; loci thickened, darkened, apical, lateral or circumspersed, 1.5–2.5 µm diam. Conidia solitary, hyaline, subcylindrical, filiform to obclavate, straight to slightly curved, with truncate to obconically truncate base and subacute to subobtuse apices, (60–)125–170(–300) × 3–5 µm, 5–20-septate in clade 1; (25–)60–95(–260) × 2.5–4.5 µm, (2–)8–11(–25)-septate in clade 2; (30–)100–155(–320) × 2–5 µm, (2–)10–14(–28)-septate in clade 3; hila distinctly thickened, darkened, refractive, 1–2 µm diam.

Notes: Screening the remaining isolates of C. cf. flagellaris, with three more genomic loci in this study (tub2, rpb2 and gapdh), clusters them into at least three distinct clades in the eight-gene phylogenetic tree (Fig. 1, part 3); clade 1 is sister to C. cf. gossypii; clade 3 is sister to C. convolvulicola and clade 2 is sister to the clade including C. cf. flagellaris clade 3 and C. convolvulicola. However, there is a high level of variation in morphological characteristics between different isolates of these three clades. In addition, several isolates originating from diverse hosts and families reside in these three clades and there is also overlap between host ranges among them. Different names can therefore be applied to these clades, and therefore we prefer to simply regard them as distinct phylogenetic species for now. To resolve their taxonomy, fresh collections authentic for the names (based on host and country) need to be recollected and included in future studies.

Cercospora cf. flagellaris Clade 1

Specimens examined: Fiji: on Amaranthus sp. (Amaranthaceae), C.F. Hill (CPC 5441). — Iran: Guilan Province: Talesh, Khotbeh Sara, on leaves of Phaseolus vulgaris (Fabaceae), Oct. 2012, M. Bakhshi (CCTU 1128 = IRAN 2661C = CBS 136141); Talesh, Jamakuh, on leaves of Raphanus sativus (Brassicaceae), Nov. 2011, M. Bakhshi (IRAN 17042F, CCTU 1120); Talesh, Dulbin, on Hydrangea sp. (Hydrangeaceae), Jul. 2011, M. Bakhshi (IRAN 17039F, CCTU 1007 = CBS 136031). Guilan Province: Kiashahr, on leaves of Ph. vulgaris, Aug. 2012, M. Bakhshi (CCTU 1168 = IRAN 2715C); Kiashahr, on leaves of R. sativus, Aug. 2012, M. Bakhshi (IRAN 17041F, CCTU 1171); Kiashahr, on leaves of Arachis hypogea (Fabaceae), Aug. 2012, M. Bakhshi (CCTU 1159 = CBS 136148); Sowme’eh Sara, Dowgur, on leaves of Urtica dioica (Urticaceae), Jun. 2012, M. Bakhshi (IRAN 17043F, CCTU 1031 = IRAN 2648C = CBS 136036); Chamkhaleh, on leaves of Lepidium sativum (Brassicaceae), Jun. 2012, M. Bakhshi (IRAN 17040F, CCTU 1027 = CBS 136034); Lahijan, Rudboneh, on leaves of Citrullus lanatus (Cucurbitaceae), Aug. 2012, M. Bakhshi (IRAN 17038F, CCTU 1162 = IRAN 2670C). — South Africa: Limpopo Province: Messina, 30 Apr. 1995, on Populus deltoides (Salicaceae), P.W. Crous (CPC 1051). — South Korea: Jeju, on Dysphania ambrosioides (syn. Chenopodium ambrosioides) (Chenopodiaceae), 12 Nov. 2003, H.D. Shin (CBS 132653 = CPC 10884) (as C. chenopodii-ambrosioidis).

Cercospora cf. flagellaris Clade 2

Specimens examined: Iran: Ardabil Province: Moghan, on leaves of Xanthium spinosum (Astraceae), Oct. 2011, M. Bakhshi (IRAN 17049F, CCTU 1068); Moghan, on leaves of Xanthium strumarium (Asteraceae), Oct. 2011, M. Bakhshi (IRAN 17050F, CCTU 1085); Moghan, on leaves of Ecballium elaterium (Cucurbitaceae), Oct. 2011, M. Bakhshi (IRAN 17047F, CCTU 1059 = CBS 136136); Moghan, on leaves of E. elaterium, Oct. 2012, M. Bakhshi (IRAN 17048F, CCTU 1216 = IRAN 2717C); Moghan, on leaves of Abutilon theophrasti (Malvaceae), Oct. 2012, M. Bakhshi (IRAN 17044F, CCTU 1204). Guilan Province: Astara, on leaves of Cercis siliquastrum (Caesalpinaceae), Oct. 2012, M. Bakhshi (CCTU 1115 = IRAN 2659C = CBS 136139); Talesh, Khotbeh Sara, on leaves of Eclipta prostrata (Astraceae), Oct. 2012, M. Bakhshi (CCTU 1223 = IRAN 2683C = CBS 136154); Talesh, on leaves of Datura stramonium (Solanaceae), Oct. 2012, M. Bakhshi (IRAN 17046F, CCTU 1195). Mazandaran Province: Ramsar, on leaves of Acer velutinum (Aceraceae), Sep. 2012, M. Bakhshi (IRAN 17045F, CCTU 1198 = CBS 136151). — South Korea: Hoengseong, on Celosia argentea var. cristata (syn. C. cristata) (Amaranthaceae), 11 Oct. 2004, H.D. Shin (CBS 132667 = CPC 11643).

Cercospora cf. flagellaris Clade 3

Specimens examined: Iran: Guilan Province: Rudsar, on leaves of Cucurbita maxima (Cucurbitaceae), Oct. 2012, M. Bakhshi (CCTU 1029 = IRAN 2647C = CBS 136035); Rudsar, Korjehposht, on leaves of Tagetes patula (Asteraceae), Aug. 2012, M. Bakhshi (IRAN 17065F, CCTU 1141 = CBS 136144); Talesh, Khotbeh Sara, on leaves of Cucurbita pepo (Cucurbitaceae), Jun. 2012, M. Bakhshi (CCTU 1136); Khotbeh Sara, on leaves of Vicia faba (Fabaceae), Oct. 2012, M. Bakhshi (IRAN 17067F, CCTU 1160 = CBS 136149); Khotbeh Sara, on leaves of Calendula officinalis (Asteraceae), Jun. 2012, M. Bakhshi (IRAN 17058F, CCTU 1140 = IRAN 2666C = CBS 136143); Talesh, Khalif Abad, on Ph. vulgaris, Jul. 2012, M. Bakhshi (CCTU 1142 = IRAN 2667C); Talesh, Dulbin, on leaves of X. strumarium, Jul. 2011, M. Bakhshi (IRAN 17069F, CCTU 1005 = IRAN 2644C); Dulbin, on leaves of Impatiens balsamina (Balsaminaceae), Jul. 2011, M. Bakhshi (IRAN 17062F, CCTU 1006 = CBS 136030); Dulbin, on leaves of Pelargonium hortorum (Geraniaceae), Aug. 2011, M. Bakhshi (CCTU 1010 = CBS 136032); Talesh, Jowkandan, on leaves of Po. deltoides, Oct. 2012, M. Bakhshi (CCTU 1118 = IRAN 2660C = CBS 136140); Talesh, Jowkandan, on leaves of Oenothera biennis (Onagraceae), Oct. 2012, M. Bakhshi (IRAN 17051F, CCTU 1172); Talesh, on leaves of D. stramonium, Oct. 2012, M. Bakhshi (IRAN 17059F, CCTU 1143 = CBS 136145); Guilan Province: Astara, Chubar, on leaves of Ph. vulgaris, Jun. 2012, M. Bakhshi (CCTU 1138 = IRAN 2664C, CCTU 1139 = IRAN 2665C); Rasht, Khomam, on leaves of X. strumarium, Aug. 2012, M. Bakhshi (IRAN 17068F, CCTU 1156); Khomam, on leaves of Ab. theophrasti, Aug. 2012, M. Bakhshi (IRAN 17052F, CCTU 1154 = CBS 136147); Langarud, Otaqvar, on leaves of X. strumarium, Aug. 2012, M. Bakhshi (CCTU 1158 = IRAN 2668C); Lahijan, Rudboneh, on leaves of Ph. vulgaris, Aug. 2012, M. Bakhshi (CCTU 1161 = IRAN 2669C); Guilan Province: Fuman, on leaves of Ph. vulgaris, Aug. 2012, M. Bakhshi (CCTU 1155.11); Fuman, on leaves of Buxus microphylla (Buxaceae), Jul. 2012, M. Bakhshi (IRAN 17057F, CCTU 1150); Fuman, on leaves of Amaranthus retroflexus, Sep. 2011, M. Bakhshi (IRAN 17054F, CCTU 1021 = CBS 136033); Sowme’eh Sara, Dowgur, on leaves of Ph. vulgaris, Aug. 2012, M. Bakhshi (CCTU 1175 = IRAN 2673C); Sowme’eh Sara, Bahambar, on leaves of R. sativus, Aug. 2012, M. Bakhshi (IRAN 17063F, CCTU 1075); Kiashahr, on leaves of Anubias sp. (Araceae), Oct. 2012, M. Bakhshi (IRAN 17056F, CCTU 1167 = CBS 136150); Masal, on leaves of U. dioica, Aug. 2012, M. Bakhshi (IRAN 17066F, CCTU 1147). Zanjan Province: Tarom, Pasar, on leaves of X. strumarium, Sep. 2011, M. Bakhshi (IRAN 17070F, CCTU 1048 = CBS 136029); Tarom, on leaves of Olea europaea (Oleaceae), Nov. 2011, M. Torbati (CCTU 1130 = CBS 136142). Ardabil Province: Moghan, on leaves of Silybum marianum (Astraceae), Oct. 2012, M. Bakhshi (IRAN 17064F, CCTU 1212 = IRAN 2680C = CBS 136153); Moghan, on leaves of A. retroflexus, Oct. 2011, M. Bakhshi (IRAN 17053F, CCTU 1064); Moghan, on leaves of Amaranthus sp., Oct. 2011, M. Bakhshi (IRAN 17055F, CCTU 1084 = CBS 136156); Moghan, on leaves of Amaranthus blitoides, Oct. 2011, M. Bakhshi (CCTU 1072 = IRAN 2653C); Moghan, on leaves of Glycine max (Fabaceae), Oct. 2012, M. Bakhshi (IRAN 17060F, CCTU 1209 = CBS 136152, CCTU 1210 = IRAN 2679C, CCTU 1211); Moghan, on leaves of Hib. trionum, Oct. 2012, M. Bakhshi (IRAN 17061F, CCTU 1218 = IRAN 2682C). — South Africa: Limpopo Province: Messina, on Citrus sp. (Rutaceae), M.C. Pretorius (CBS 115482 = CPC 4410). Unknown, on Bromus sp. (Poaceae), M.D. Whitehead (CBS 143.51 = CPC 5055).

Cercospora sp. G complex

The 16 isolates previously recognised as Cercospora sp. G based on a five-gene phylogenetic tree (Groenewald et al. 2013, Bakhshi et al. 2015a) cluster into two distinct phylogenetic clades based on the eight-gene phylogenetic tree (Fig. 1, part 1). One, four, one, two, two, two, three and two allele groups were detected for the ITS, tef1, actA, cmdA, his3, tub2, rpb2 and gapdh sequences, respectively (Table 9).
Table 9

Results from allele group designation per locus for Cercospora sp. G isolates in this study. Abbreviations of loci and collection accession numbers follow Table 1.

Species

Culture accession number

Host

Allele group per locus

ITS

tef1

actA

cmdA

his3

tub2

rpb2

gapdh

Cercospora sp. G clade 1

CCTU 1015; CBS 136024; IRAN 2645C

Plantago major

I

IV

I

I

I

I

III

II

 

CCTU 1197

Bidens tripartita

I

IV

I

I

I

I

I

II

 

CPC 5438

Salvia viscosa

I

II

I

I

II

II

II

Cercospora sp. G clade 2

CCTU 1002

Celosia cristata

I

I

I

I

I

I

I

I

CCTU 1020; CBS 136023

Sorghum halepense

I

I

I

I

I

I

II

I

 

CCTU 1030; CBS 136026

Bidens tripartita

I

III

I

I

II

I

I

I

 

CCTU 1046

Plantago major

I

I

I

I

I

I

I

I

 

CCTU 1053; CBS 136027

Cichorium intybus

I

III

I

I

II

I

I

I

 

CCTU 1054

Amaranthus sp.

I

I

I

I

I

I

I

I

 

CCTU 1058

Helminthotheca echioides

I

I

I

I

I

I

I

I

 

CCTU 1079; CBS 136025

Amaranthus retrofexus

I

I

I

I

I

I

I

I

 

CCTU 1090

Abutilon theophrasti

I

I

I

I

I

I

I

I

 

CCTU 1116

Plantago major

I

I

I

I

I

I

I

I

 

CCTU 1122

Amaranthus sp.

I

I

I

I

I

I

I

I

 

CCTU 1144; CBS 136130

Cucurbita maxima

I

I

I

II

I

I

I

I

 

CBS 115518; CPC 5360

Bidens frondosa

I

IV

I

I

I

I

I

Cercospora sp. G Clade 1

Description: Mycelium internal. Caespituli amphigenous, brown. Conidiophores aggregated in loose fascicles, arising from a moderately developed, intraepidermal and substomatal, brown stroma, to 35 µm diam; conidiophores pale brown to brown, 2–11-septate, straight to flexuous, simple, unbranched, uniform in width, (55–)110–150(–260) × 3.5–5 µm. Conidiogenous cells integrated, terminal, proliferating sympodially, mono- and multi-local; loci thickened, darkened, apical or formed on shoulders caused by sympodial proliferation, 1.5–2.5 µm diam. Conidia solitary, hyaline, subcylindrical, filiform to obclavate, straight to slightly curved, with truncate to obconically truncate base and subacute to subobtuse apices, (40–)75–100(–165) × 2–4 µm, 4–15-septate; hila distinctly thickened, darkened, refractive, 1–2 µm diam.

Specimens examined: Iran: Guilan Province: Talesh, Dulbin, on leaves of Plantago major, Jul. 2011, M. Bakhshi (IRAN 17085F, CCTU 1015 = IRAN 2645C = CBS 136024); Talesh, Kishonben, on leaves of Bidens tripartita (Asteraceae), Sept. 2012, M. Bakhshi (IRAN 17084F, CCTU 1197). — New Zealand: Manurewa, on Salvia viscosa (Lamiaceae), C.F. Hill (CPC 5438) (as C. salviicola).

Cercospora sp. G Clade 2

Description: Mycelium internal. Caespituli amphigenous, brown. Conidiophores aggregated in loose to dense fascicles, arising from a weakly to well-developed, intraepidermal and substomatal, brown stroma, to 50 µm diam; conidiophores pale brown to brown, 3–11-septate, straight to flexuous, simple, unbranched, uniform in width, (30–)65–105(–240) × 2.5–5 µm. Conidiogenous cells integrated, terminal, proliferating sympodially, 10–30 × 2.5–5 µm, mono- or multi-local; loci distinctly thickened, darkened and somewhat refractive, apical or formed on shoulders caused by sympodial proliferation, 1.5–2.5 µm diam. Conidia solitary, subcylindrical, filiform to obclavate, straight to slightly curved, hyaline, (25–)75–110(–200) × 3.5–5.5 µm, (3–)8–15(–20)-septate, with subacute to subobtuse apices and truncate to obconically truncate bases; hila thickened, darkened, refractive, 1–2 µm diam.

Notes: Isolates of Cercospora sp. G clustered in two distinct clades with high posterior probability in the eight-gene phylogenetic tree (Fig. 1, part 1). However, several isolates from diverse host families cluster in these two clades, to which different names can be applied. Moreover, there is also overlap between host ranges of the two clades. On the other hand, there is no morphological basis to divide them into two distinct species. Based on the gene loci screened in the present study, we were unable to resolve the taxonomy of these isolates and for now prefer to treat them as unresolved phylogenetic species. As with C. cf. fagellaris, in order to resolve their taxonomy, fresh collections from the same host and country as the original material need to be recollected and included in future studies.

Specimens examined: Iran: Zanjan Province: Tarom, Pasar, on leaves of P. major, Oct. 2011, M. Bakhshi (CCTU 1046); Tarom, Pasar, on leaves of P. major, Nov. 2011, M. Bakhshi (IRAN 17093F, CCTU 1116). Guilan Province: Talesh, Kishonben, on leaves of Bi. tripartita, Oct. 2012, M. Bakhshi (IRAN 17091F, CCTU 1030 = CBS 136026); Talesh, on leaves of Sorghum halepense (Poaceae), Sep. 2011, M. Bakhshi (IRAN 17094F, CCTU 1020 = CBS 136023); Talesh, Dolbin, on leaves of Celosia cristata, Jul. 2011, M. Bakhshi (IRAN 17092F, CCTU 1002). Ardabil Province: Moghan, on leaves of A. retrofexus, Oct. 2011, M. Bakhshi (IRAN 17088F, CCTU 1079 = CBS 136025); Moghan, on leaves of Amaranthus sp., Oct. 2011, M. Bakhshi (IRAN 17089F, CCTU 1054); Moghan, on leaves of Helminthotheca echioides (Asteraceae), Oct. 2011, M. Bakhshi (IRAN 17086F, CCTU 1058); Moghan, on leaves of Ab. theophrasti, Oct. 2012, M. Bakhshi (IRAN 17087F, CCTU 1090). Guilan Province: Talesh, Jamakuh, on leaves of Amaranthus sp., Nov. 2011, M. Bakhshi (IRAN 17090F, CCTU 1122); Masal, on leaves of Cu. maxima, Jul. 2012, M. Bakhshi (CCTU 1144 = CBS 136130); Sowme’eh Sara, Dowgur, on leaves of Cichorium intybus (Asteraceae), Jun. 2012, M. Bakhshi (CCTU 1053 = CBS 136027). — New Zealand: Kopuku, on Bidens frondosa (Asteraceae), C.F. Hill (CBS 115518 = CPC 5360).

Identification of the best-performing DNA barcode

Kimura-2-parameter values

The Kimura-2-parameter distribution graphs (Fig. 9) visualise the inter- and intraspecific distances per locus corresponding to the barcoding gap (Hebert et al. 2003, Schoch et al. 2012). A useful barcoding locus should have no overlap between the inter- and intraspecific K2P distances and generally should have an average interspecific distance that is at least ten times as high as the average intraspecific distance of that locus (Quaedvlieg et al. 2012, Verkley et al. 2013, Stielow et al. 2015).
Fig. 9
Fig. 9

Frequency distributions of the Kimura-2-parameter distance (barcoding gap) for the eight loci.

The eight tested loci showed varying degrees of overlap in their K2P distribution between inter- and intraspecific variation graphs (Fig. 9). In this dataset, the average interspecific variation in ITS dataset was very low (0.002) compared to its intraspecific variation (0.0005), leading to a very low inter-to intraspecific variation ratios of 4:1 for this locus (Fig. 9, Table 4). This low ratio is far below the recommended 10:1 ratio, indicating a general lack of natural variation within the ITS locus, making it ill-suited for effective identification of the individual species of Cercospora. Due to the presence of introns in the seven protein coding loci, these genes provide much higher interspecific variation than the more conserved ITS locus. These protein coding genes had K2P inter- to intraspecific variation ratios of 127:1 for tef1, 76:1 for cmdA, 74:1 for rpb2, 71:1 for tub2, 44:1 for gapdh, 15:1 for actA and 13:1 for his3 (Table 4), making them all suitable for reliable species resolution of Cercospora spp. As the tef1, cmdA, rpb2, tub2 and gapdh have the largest barcoding gap, these loci should give the highest species resolution. However, all of these genes do have overlap between the inter- and intraspecific K2P distances (as is evident in the graphs of Fig. 9), suggesting that no one of them can serve as a single ideal barcoding locus for Cercospora spp.

Molecular phylogenetic resolution (clade recovery)

Based on the results of the individual gene tree assessments, no single gene region was found which could reliably distinguish all species, and occurrences of the same sequence(s) shared between multiple species were observed in each locus.

The ITS phylogeny had low resolution and was only able to distinguish C. chenopodii, C. solani and C. sorghicola from the other included species. The remaining loci had different levels of resolution. The gapdh region was more effective and could resolve 61 % of 28 lineages, whereas his3, tub2, actA, tef1, cmdA and rpb2 had respectively 48, 43, 43, 39, 32 and 32 % clade recovery. Based on the gapdh region, we were able to distinguish 17 of the 28 species clades, including C. althaeina, C. armoraciae, C. bizzozeriana, C. chenopodii, C. conyzae-canadensis, C. cf. fagellaris clade 1, C. cf. fagellaris clade 2, C. cf. gossypii, C. pseudochenopodii, C. cf. richardiicola, C. rumicis, C. solani, C. sorghicola, Cercospora sp. G clade 1, Cercospora sp. G clade 2, C. violae and C. cf. zinnia; whereas, 13 species clades including C. althaeina, C. chenopodii, C. conyzae-canadensis, C. cylindracea, C. pseudochenopodii, C. cf. richardiicola, C. rumicis, C. solani, C. sorghicola, C. uwebrauniana, C. violae, C. zebrina and C. cf. zinniae were distinguished in the his3 phylogeny; 12 species clades including C. althaeina, C. chenopodii, C. conyzaecanadensis, C. cylindracea, C. iranica, C. pseudochenopodii, C. cf. richardiicola, C. solani, C. sorghicola, Cercospora sp. T, C. uwebrauniana and C. cf. zinniae were distinguished in the tub2 phylogeny; 12 species clades including C. althaeina, C. chenopodii, C. convolvulicola, C. conyzae-canadensis, C. cylindracea, C. pseudochenopodii, C. cf. richardiicola, C. solani, C. sorghicola, C. violae, C. zebrina and C. cf. zinniae were distinguished in the actA phylogeny; 11 species clades including C. bizzozeriana, C. chenopodii, C. conyzae-canadensis, C. pseudochenopodii, C. cf. richardiicola, C. rumicis, C. solani, C. sorghicola, C. uwebrauniana, C. violae and C. cf. zinniae were distinguished in the tef1 phylogeny; nine species clades including C. convolvulicola, C. conyzae-canadensis, C. iranica, C. cf. richardiicola, C. solani, C. sorghicola, Cercospora sp. T, C. violae and C. cf. zinniae were distinguished in the cmdA phylogeny; and nine species clades including C. bizzozeriana, C. chenopodii, C. conyzae-canadensis, C. pseudochenopodii, C. cf. richardiicola, C. solani, C. sorghicola, C. zebrina and C. cf. zinniae were distinguished in the rpb2 phylogeny.

Therefore, the gapdh phylogeny displayed a high resolution and had the highest clade recovery and was responsible for resolving most of the cryptic taxa within C. apii, C. armoraciae, C. beticola, Cercospora sp. G, and C. cf. fagellaris.

Discussion

In this study, we re-assessed species of the genus Cercospora using a combined approach based on the evaluation of an eight-gene molecular DNA sequence dataset, host, and morphological data (in those cases where morphological variation was present). In recent years, the rapid advance of molecular techniques has brought about the possibility of a more precise species delimitation and a better consideration of the evolution of fungi. It is well-known that many fungal taxa based on morphology or on sequence data of the commonly used fungal barcode ITS region of the nrDNA operon (Schoch et al. 2012) hide cryptic species complexes when molecular data from multiple gene regions are considered (Lombard et al. 2010, Cabral et al. 2012, Crous et al. 2013, Groenewald et al. 2013, Quaedvlieg et al. 2013, Woudenberg et al. 2013). This is most likely an underestimation for many fungal taxa. For instance, the Colletotrichum acutatum species complex, once considered to be a single species, has been shown to include at least 31 cryptic taxa (Damm et al. 2012). In the present study, phylogenetic inference also revealed cryptic species complexes that could not be distinguished based on geography, host association, morphology, or ITS sequence data alone.

Before this study, Groenewald et al. (2013) and Bakhshi et al. (2015a) inferred phylogenies of Cercospora based on sequence data of five genomic loci (ITS, tef1, actA, cmdA and his3). Their results showed the importance of all five loci in a combined analysis for Cercospora taxonomy (Groenewald et al. 2013, Bakhshi et al. 2015a). Despite this, the sequences of these five loci were too conserved in Cercospora, and it was not possible to identify a single gene as the best DNA barcoding locus. In addition, several species complexes remained unresolved (Groenewald et al. 2013, Bakhshi et al. 2015a). To overcome these deficiencies, three more potential candidate gene regions, tub2, rpb2, and gapdh, were amplified and sequenced for Cercospora isolates previously investigated by Bakhshi et al. (2015a) and some related reference isolates investigated by Groenewald et al. (2013). Phylogenetic performance of the eight loci (ITS, tef1, actA, cmdA, his3, tub2, rpb2 and gapdh) were assessed based on the inter-/intraspecific distance ratio and clade recovery. With the final classification presented here, none of the genes we analysed provides an effective barcode on its own across the entire genus. However, gapdh emerged as a strong candidate for improved species delimitation in Cercospora and provides better insight, especially into species complexes. Groenewald et al. (2013) evaluated this gene in the Cercospora sp. Q species complex and their results also showed high variation in this gene. The performance of gapdh in species delimitation has been also reported in other fungal groups, including Alternaria (Woudenberg et al. 2013) and the Colletotrichum gloeosporioides species complex (Weir et al. 2012). Additionally, when using the gapdh gene, cmdA sequences are crucial to distinguish some species of Cercospora. We therefore recommend gapdh as the gene for species delimitation in Cercospora. However, it needs to be combined with cmdA, tef1 and tub2 to obtain a robust species identification. In addition, data from the ITS, actA, rpb2, and his3, have been useful, and were at times necessary, to provide clear evidence of multi-gene phylogenetic concordance to separate cryptic species.

The amplification of gapdh with the available primers was not, however, easy, and we need to design new primer sets for gapdh in Cercospora derived from the sequences generated. On the other hand, lack of ex-type or reliable sequences in public databases is a serious problem in the accurate molecular identification of Cercospora species, and it is essential to also amplify at least the gapdh and tub2 genes for all of the reference isolates used by Groenewald et al. (2013) in thefuture.

One of the main goals of this project was to generate an eight-gene DNA dataset for species of the genus Cercospora. In this regard, one of the achievements of this research was that the sequencing of additional loci revealed new clades within some taxa which were found to actually represented a species complex (in the eight-gene phylogenetic tree) rather than a single species, while the five-gene phylogenetic tree (Bakhshi et al. 2015a) was unable to resolve them. The phylogenetic tree based on the combined eight-gene dataset resolved at least four, three, two, two and two well-supported clades respectively within the species complexes C. cf. flagellaris, C. apii, C. beticola, C. armoraciae, and Cercospora sp. G.

Some of the species revealed by the eight-gene phylogeny in this study can be distinguished based on their morphology or host range. For example, as explained in the notes for C. uwebrauniana, characteristics of the conidia in this species, which clustered in the C. apii complex based on the five-gene phylogenetic tree (see Bakhshi et al. 2015a), are clearly distinguishable from those of C. apii. However, some species cannot be separated using morphological characters. For instance, the C. cf. flagellaris species complex included at least three distinct clades and there is considerable overlap between morphological features and host ranges of the clades 1, 2, and 3. In addition, pursuant to high levels of intraspecific variation in these three clades, the distinction between these clades is only possible based on molecular data. It is conceivable that some members of these three clades represent new species, yet to be described. This is also true for the Cercospora sp. G species complex.

Another problem arises because many morphological features change according to the host plant and different weather conditions. Such differences in morphological characters under different conditions have also been seen in other groups of fungi, such as Colletotrichum species (Weir et al. 2012). Because we do not yet have access to sequence data of most species of the Cercospora, we have chosen to consider these clades as different clades of C. cf. flagellaris and Cercospora sp. G rather than introduce new species names. Recent molecular studies on the Cercospora species associated with Cercospora leaf blight and purple seed stain on soybean, have revealed several Cercospora species, including C. cf. flagellaris as one of the most important agents (Bakhshi et al. 2015a, Soares et al. 2015, Albu et al. 2016). In this regard, Guillin et al. (2017) studied the genetic entanglement between Cercospora species infecting soybean and provided evidence that revealed interspecific gene flow played a significant role in the evolutionary dynamics of Cercospora species. Taking into consideration the shared host range that exists between different clades of C. cf. flagellaris, our data also provide more support for this hypothesis.

Furthermore, we found that all of the isolates of C. apii obtained from Plantago lanceolata from different localities clustered in clade 2 of this species in the eight-gene phylogenetic tree. Additionally, isolates of C. beticola and C. apii which intermix with P. lanceolata, had a common allele in gapdh. Thus, it seems that the gapdh gene might play a role in pathogenicity or host range, and has the potential to reflect this phylogenetically; however, that remains to be tested.

This study emphasises the complex nature of the evolutionary pathways that have been traversed within the genus Cercospora. Speciation has taken place much more prolifically than had previously been suspected in this genus, and it seems likely that the C. apii sensu Crous & Braun (2003) species complex is still rapidly evolving. The emergence of new species is doubtlessly encouraged by the opportunities for mixing gene pools that are provided by modern global agricultural practices, and indiscriminate use of fungicides combined with imperfect phytosanitary regulation.

The present study provides the first eight-gene phylogenetic overview of Cercospora species. We hope that this dataset will provide a stable platform to accommodate the numerous undescribed species that still await description, and the recollection and epitypification of already named species. Moreover, it seems that Cercospora should still be subjected to a more detailed analysis based on yet additional gene loci to provide a more vigorous phylogenetic basis for species delimitation.

Declarations

Acknowledgements

We gratefully acknowledge the Iran National Science Foundation (INSF), Research Deputy of the Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Westerdijk Fungal Biodiversity Institute, Research Deputy of the University of Tabriz, and Studienstiftung für mykologische Systematik und Ökologie for financial support. We thank Mieke Starink-Willemse, for her invaluable technical support and assistance in sequencing of some of the isolates. Uwe Braun (Martin Luther University, Halle, Germany) is thanked for comments pertaining to typification of several of the Cercospora spp. studied here.

This 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.

Authors’ Affiliations

(1)
Department of Botany, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), P.O. Box 19395-1454, Tehran, Iran
(2)
Plant Protection Department, Faculty of Agriculture, University of Tabriz, P.O. Box 5166614766, Tabriz, Iran
(3)
Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
(4)
Department of Genetics, Biochemistry and Microbiology, Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, 0002, South Africa
(5)
Wageningen University and Research Centre (WUR), Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands

References

  1. Agrios GN (2005) Plant Pathology. 5th edn. New York: Academic Press.Google Scholar
  2. Albu S, Schneider R, Price P, Doyle V (2016) Cercospora cf. flagellaris and Cercospora cf. sigesbeckiae are associated with Cercospora leaf blight and purple seed stain on soybean in North America. Phytopathology 106: 1376–85.View ArticleGoogle Scholar
  3. Bakhshi M, Arzanlou M, Babai-Ahari A (2012a) Comprehensive check list of Cercosporoid fungi from Iran. Plant Pathology and Quarantine 2: 44–55.View ArticleGoogle Scholar
  4. Bakhshi M, Arzanlou M, Babai-Ahari A (2012b) Morphological and molecular characterization of Cercospora zebrina from black bindweed in Iran. Plant Pathology and Quarantine 2: 125–130.View ArticleGoogle Scholar
  5. Bakhshi M, Arzanlou M, Babai-ahari A, Groenewald JZ, Braun U, Crous PW (2015a) Application of the consolidated species concept to Cercospora spp. from Iran. Persoonia 34: 65–86.View ArticleGoogle Scholar
  6. Bakhshi M, Arzanlou M, Babai-Ahari A, Groenewald JZ, Crous PW (2015b) Is morphology in Cercospora a reliable reflection of generic affinity? Phytotaxa 213: 22–34.View ArticleGoogle Scholar
  7. Berlese AN (1888) Fungi Veneti novi vel critici. Malpighia 2. 241–250.Google Scholar
  8. Braun U (2002) Miscellaneous notes on some micromycetes (II). Schlechtendalia 8: 33–38.Google Scholar
  9. Braun U, Crous PW (2016) (2415) Proposal to conserve the name Cercospora (Ascomycota: Mycosphaerellaceae) with a conserved type. Taxon 65: 185.View ArticleGoogle Scholar
  10. Braun U, Crous PW, Nakashima C (2014) Cercosporoid fungi (Mycosphaerellaceae) 2. Species on monocots (Acoraceae to Xyridaceae, excluding Poaceae). MA Fungus 5: 203–390.View ArticleGoogle Scholar
  11. Braun U, Crous PW, Nakashima C (2015a) Cercosporoid fungi (Mycosphaerellaceae) 3. Species on monocots [Poaceae], true grasses). IMA Fungus 6: 25–97.View ArticleGoogle Scholar
  12. Braun U, Crous PW, Nakashima C (2015b) Cercosporoid fungi (Mycosphaerellaceae) 4. Species on dicots [Acanthaceae] to Amaranthaceae). IMA Fungus 6: 373–469.View ArticleGoogle Scholar
  13. Braun U, Crous PW, Nakashima C (2016) Cercosporoid fungi (Mycosphaerellaceae) 5. Species on dicots [Anacardiaceae] to Annonaceae). IMA Fungus 7: 161–216.View ArticleGoogle Scholar
  14. Braun U, Nakashima C, Crous PW (2013) Cercosporoid fungi (Mycosphaerellaceae) 1. Species on other fungi, Pteridophyta and Gymnospermae. IMA Fungus 4: 265–345.View ArticleGoogle Scholar
  15. Cabrai A, Rego C, Nascimento T, Oliveira H, Groenewald JZ, Crous PW (2012) Multi-gene analysis and morphology reveal novel Ilyonectria species associated with black foot disease of grapevines. Fungal Biology 116: 62–80.View ArticleGoogle Scholar
  16. Chupp C (1954) A Monograph of the Fungus Genus Cercospora. Ithaca, NY: C. Chupp.Google Scholar
  17. Crous PW, Aptroot A, Kang JC, Braun U, Wingfield MJ (2000) The genus Mycosphaerella and its anamorphs. Studies in Mycology 45: 107–121.Google Scholar
  18. Crous PW, Braun U (2003) Mycosphaerella and its Anamorphs: 1. Names published in Cercospora and Passalora. [CBS Biodiversity Series no. 1.] Utrecht: Centraalbureau voor Schimmelcultures.Google Scholar
  19. Crous PW, Braun U, Hunter GC, Wingfield MJ, Verkley GJM, et al. (2013) Phylogenetic lineages in Pseudocercospora. Studies in Mycology 75: 37–114.View ArticleGoogle Scholar
  20. Crous PW, Gams W, Stalpers JA, Robert V, Stegehuis G (2004a) MycoBank: an online initiative to launch mycology into the 21st century. Studies in Mycology 50: 19–22.Google Scholar
  21. Crous PW, Groenewald JZ (2005) Hosts, species and genotypes: opinions versus data. Australasian Plant Pathology 34: 463–470.View ArticleGoogle Scholar
  22. Crous PW, Groenewald JZ, Mansilla JP, Hunter GC, Wingfield MJ (2004b) Phylogenetic reassessment of Mycosphaerella spp. and their anamorphs occurring on Eucalyptus. Studies in Mycology 50: 195–214.Google Scholar
  23. Crous PW, Schoch CL, Hyde KD, Wood AR, Gueidan C, et al. (2009a) Phylogenetic lineages in the Capnodiales. Studies in Mycology 64: 17–47.View ArticleGoogle Scholar
  24. Crous PW, Summerell BA, Carnegie AJ, Wingfield MJ, Hunter GC, et al. (2009b) Unravelling Mycosphaerella: do you believe in genera? Persoonia 23: 99–118.View ArticleGoogle Scholar
  25. Damm U, Cannon PF, Woudenberg JHC, Johnston PR, Weir BS, et al. (2012) The Colletotrichum boninense species complex. Studies in Mycology 73: 1–36.View ArticleGoogle Scholar
  26. Deighton FC (1973) Studies on Cercospora and allied genera. IV. Cercosporella Sacc, Pseudocercosporella gen. nov. and Pseudocercosporidium gen. nov. Mycological Papers 133: 1–62.Google Scholar
  27. Deighton FC (1979) Studies on Cercospora and allied genera VII. New species and redispositions. Mycological Papers 144: 1–156.Google Scholar
  28. Deighton FC (1983) Studies on Cercospora and allied genera. VIII. Further notes on Cercoseptoria and some new species and redispositions. Mycological Papers 151: 1–13.Google Scholar
  29. Drummond A, Ashton B, Buxton S, Cheung M, Cooper A, et al. (2012) Geneious. Version 5.6, https://doi.org/www.geneious.com.Google Scholar
  30. Ellis MB (1971) Dematiaceous Hyphomycetes. Kew: Commonwealth Mycological Institute.Google Scholar
  31. Ellis MB (1976) More Dematiaceous Hyphomycetes. Kew: Commonwealth Mycological Institute.Google Scholar
  32. Fresenius G (1863) Beiträge zur Mykologie. Vol. 3. Frankfurt: H.L. Brönner.Google Scholar
  33. Gola G (1930) L’Erbario Micologico di P A. Saccardo. Catalogo. Tipografia Editrice Antoniana, Padova.Google Scholar
  34. Goodwin SB, Dunkle LD, Zismann VL (2001) Phylogenetic analysis of Cercospora and Mycosphaerella based on the internal transcribed spacer region of ribosomal DNA. Phytopathology 91: 648–658.View ArticleGoogle Scholar
  35. Groenewald JZ, Groenewald M, Braun U, Crous PW (2010) Cercospora speciation and host range. In: Cercospora Leaf Spot of Sugar Beet and related species (Lartey RT, Weiland J, Panella L, Crous PW & Windeis C, eds): 21–37. St Paul, MN: American Phytopathological Society Press.Google Scholar
  36. Groenewald JZ, Nakashima C, Nishikawa J, Shin HD, Park JH, et al. (2013) Species concepts in Cercospora: spotting the weeds among the roses. Studies in Mycology 75: 115–170.View ArticleGoogle Scholar
  37. Groenewald M, Groenewald JZ, Braun U, Crous PW (2006) Host range of Cercospora apii and C. beticola and description of C. apiicola, a novel species from celery. Mycologia 98: 275–285.View ArticleGoogle Scholar
  38. Groenewald M, Groenewald JZ, Crous PW (2005) Distinct species exist within the Cercospora apii morphotype. Phytopathology 95: 951–959.View ArticleGoogle Scholar
  39. Guillin EA, de Oliveira LO, Grijalba PE, Gottlieb AM (2017) Genetic entanglement between Cercospora species associating soybean purple seed stain. Mycological Progress 16: 593–603.View ArticleGoogle Scholar
  40. Guatimosim E, Schwartsburd PB, Barreto RW, Crous PW (2017) Novel fungi from an ancient niche: cercosporoid and related sexual morphs on ferns. Persoonia 37: 106–41.View ArticleGoogle Scholar
  41. Hebert PDN, Cywinska A, Ball SL, Dewaard JR (2003) Biological identifications through DNA barcodes. Proceedings of the Royal Society of London B 270: 313–321.View ArticleGoogle Scholar
  42. Hesami S, Khodaparast S, Zare R (2012) New reports on Cercospora and Pseudocercospora from Guilan province. Rostaniha 13: 95–100.Google Scholar
  43. Hsieh W-H, Goh T-K (1990) Cercospora and Similar Fungi from Taiwan. Taiwan, Taipei: Maw Chang Book Company.Google Scholar
  44. Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772–780.View ArticleGoogle Scholar
  45. Lall G, Gill HS, Munjal RL (1961) Some Cercospora species from India-V. Indian Phytopathology 14: 115–119.Google Scholar
  46. Lartey R, Caesar-TonThat T, Caesar A, ShelverW, Sol N, Bergman J (2005) Safflower: a new host of Cercospora beticola. Plant Disease 89: 797–801.View ArticleGoogle Scholar
  47. Little S (1987) Cercospora malayensis. CMI Descriptions of Pathogenic Fungi and Bacteria 916: 1–2.Google Scholar
  48. Liu YJ, Whelen S, Hall BD (1999) Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit. Molecular Biology and Evolution 16: 1799–1808.View ArticleGoogle Scholar
  49. Lombard L, Crous PW, Wingfield BD, Wingfield MJ (2010) Species concepts in Calonectria (Cylindrocladium). Studies in Mycology 66: 1–13.View ArticleGoogle Scholar
  50. Maddison WP, Maddison DR (2011) Mesquite: a modular system for evolutionary analysis. Version 2.75. https://doi.org/mesquiteproject.org.Google Scholar
  51. Montegut J (1967) ’Etude syst’ematique des principales espèces. Coton et Fibres Tropicales 22: 451.Google Scholar
  52. Montenegro-Calder’on JG, Mart’inez-’Alvarez JA, Vieyra-Hern’andez MT, Rangel-Macias LI, Razzo-Soria T, et al. (2011) Molecular identification of two strains of Cercospora rodmanii isolated from water hyacinth present in Yuriria lagoon, Guanajuato, Mexico and identification of new hosts for several other strains. Fungal Biology 115: 1151–1162.View ArticleGoogle Scholar
  53. Myllys L, Stenroos S, Thell A (2002) New genes for phylogenetic studies of lichenized fungi: glyceraldehyde-3-phosphate dehydrogenase and beta-tubulin genes. Lichenologist 34: 237–246.View ArticleGoogle Scholar
  54. Narayan S, Kharwar RN, Singh RK (2001) Some novel taxa of hyphomycete genus Cercospora causing foliar spots in India. Indian Phytopathology 54: 351–357.Google Scholar
  55. Nylander JAA (2004) MrModeltest. Version 2.0. Program distributed by the author. Evolutionary Biology Centre, Uppsala University Uppsala, Sweden.Google Scholar
  56. O’Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution 7. 103–116.View ArticleGoogle Scholar
  57. Pirnia M, Zare R, Zamanizadeh HR, Khodaparast A (2012) New records of cercosporoid hyphomycetes from Iran. Mycotaxon 120: 157–169.View ArticleGoogle Scholar
  58. Pollack FG (1987) An annotated compilation of Cercospora names. Mycological Memoirs 12: 1–212.Google Scholar
  59. Pretorius MC, Crous PW, Groenewald JZ, Braun U (2003) Phylogeny of some cercosporoid fungi from Citrus. Sydowia 55: 286–305.Google Scholar
  60. Pons N, Sutton BC (1996) Cercospora and similar fungi on Heliotro-pium weeds. Mycological Research 100: 815–820.View ArticleGoogle Scholar
  61. Quaedvlieg W, Binder M, Groenewald JZ, Summerell BA, Carnegie AJ, et al. (2014) Introducing the Consolidated Species Concept to resolve species in the Teratosphaeriaceae. Persoonia 33: 1–40.View ArticleGoogle Scholar
  62. Quaedvlieg W, Groenewald JZ, De Jes’us Y’añez-Morales M, Crous PW (2012) DNA barcoding of Mycosphaerella species of quarantine importance to Europe. Persoonia 29: 101–115.View ArticleGoogle Scholar
  63. Quaedvlieg W, Kema G, Groenewald JZ, Verkley GJM, Seifbarghi S, et al. (2011) Zymoseptoria gen. nov: a new genus to accommodate septoria-like species occurring on graminicolous hosts. Persoonia 26: 57–69.View ArticleGoogle Scholar
  64. Quaedvlieg W, Verkley GJM, Shin H-D, Barreto RW, Altenas AC, et al. (2013) Sizing up Septoria. Studies in Mycology 75: 307–390.View ArticleGoogle Scholar
  65. Rannala B, Yang Z (1996) Probability distribution of molecular evolutionary trees: a new method of phylogenetic inference. Journal of Molecular Evolution 43: 304–311.View ArticleGoogle Scholar
  66. Rayner RW (1970) A Mycological Colour Chart. Kew: Commonwealth Mycological Institute.Google Scholar
  67. Ronquist F, Teslenko M, Van der Mark P, Ayres DL, Darling A, et al. (2012) MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542.View ArticleGoogle Scholar
  68. Saccardo PA (1880) Conspectus generum fungorum italiae inferiorum nempe ad Sphaeropsideae, Melanconieas et Hyphomyceteas pertinentium systemate sporologico dispositorum. Michelia 2 1–38.Google Scholar
  69. Schoch CL, Seifert KA, HuhndorfS, Robert V, Spouge JL, et al. (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences, USA 109: 6241–6246.View ArticleGoogle Scholar
  70. Soares APG, Guillin EA, Borges LL, Da Silva AC, De Almeida ’AM, et al. (2015) More Cercospora species infect soybeans across the Americas than meets the eye. PloS One 10: e0133495.View ArticleGoogle Scholar
  71. Solheim WG (1930) Morphological studies of the genus Cercospora. Illinois Biological Monographs 12: 1–85.Google Scholar
  72. Stewart EL, Liu Z, Crous PW, Szabo LJ (1999) Phylogenetic relation-ships among some cercosporoid anamorphs of Mycosphaerella based on rDNA sequence analysis. Mycological Research 103: 1491–1499.View ArticleGoogle Scholar
  73. Stielow JB, L’evesque CA, Seifert KA, Meyer W, Irinyi L, et al. (2015) One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes. Persoonia 35: 242–263.View ArticleGoogle Scholar
  74. Stukenbrock EH, Quaedvlieg W, Javan-Nikhah M, Zala M, Crous PW, McDonald BA (2012) Zymoseptoria ardabiliae and Z. pseudotritici, two progenitor species of the Septoria tritici leaf blotch fungus Z. tritici (synonym: Mycosphaerella graminicola). Mycologia 104: 1397–1407.View ArticleGoogle Scholar
  75. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution 30: 2725–2729.PubMedGoogle Scholar
  76. Tessmann DJ, Charudattan R, Kistler HC, Rosskopf EN (2001) A molecular characterization of Cercospora species pathogenic to water hyacinth and emendation of C. piaropi. Mycologia 93: 323–334.View ArticleGoogle Scholar
  77. Verkley GJM, Quaedvlieg W, Shin H-D, Crous PW (2013) A new approach to species delimitation in Septoria. Studies in Mycology 75: 213–305.View ArticleGoogle Scholar
  78. Videira SIR, Groenewald JZ, Nakashima C, Braun U, Barreto RW, et al. (2017) Mycosphaerellaceae - chaos or clarity? Studies in Mycology 87: 257–421.View ArticleGoogle Scholar
  79. Weir BS, Johnston PR, Damm U (2012) The Colletotrichum gloeosporioides species complex. Studies in Mycology 73: 115–180.View ArticleGoogle Scholar
  80. Woudenberg JHC, Groenewald JZ, Binder M, Crous PW (2013) Alternaria redefined. Studies in Mycology 75: 171–212.View ArticleGoogle Scholar

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