Fungal Diversity

, Volume 67, Issue 1, pp 21–125

One stop shop: backbones trees for important phytopathogenic genera: I (2014)

  • Kevin D. Hyde
  • R. Henrik Nilsson
  • S. Aisyah Alias
  • Hiran A. Ariyawansa
  • Jaime E. Blair
  • Lei Cai
  • Arthur W. A. M. de Cock
  • Asha J. Dissanayake
  • Sally L. Glockling
  • Ishani D. Goonasekara
  • Michał Gorczak
  • Matthias Hahn
  • Ruvishika S. Jayawardena
  • Jan A. L. van Kan
  • Matthew H. Laurence
  • C. André Lévesque
  • Xinghong Li
  • Jian-Kui Liu
  • Sajeewa S. N. Maharachchikumbura
  • Dimuthu S. Manamgoda
  • Frank N. Martin
  • Eric H. C. McKenzie
  • Alistair R. McTaggart
  • Peter E. Mortimer
  • Prakash V. R. Nair
  • Julia Pawłowska
  • Tara L. Rintoul
  • Roger G. Shivas
  • Christoffel F. J. Spies
  • Brett A. Summerell
  • Paul W. J. Taylor
  • Razak B. Terhem
  • Dhanushka Udayanga
  • Niloofar Vaghefi
  • Grit Walther
  • Mateusz Wilk
  • Marta Wrzosek
  • Jian-Chu Xu
  • JiYe Yan
  • Nan Zhou
Open Access
Article

DOI: 10.1007/s13225-014-0298-1

Cite this article as:
Hyde, K.D., Nilsson, R.H., Alias, S.A. et al. Fungal Diversity (2014) 67: 21. doi:10.1007/s13225-014-0298-1

Abstract

Many fungi are pathogenic on plants and cause significant damage in agriculture and forestry. They are also part of the natural ecosystem and may play a role in regulating plant numbers/density. Morphological identification and analysis of plant pathogenic fungi, while important, is often hampered by the scarcity of discriminatory taxonomic characters and the endophytic or inconspicuous nature of these fungi. Molecular (DNA sequence) data for plant pathogenic fungi have emerged as key information for diagnostic and classification studies, although hampered in part by non-standard laboratory practices and analytical methods. To facilitate current and future research, this study provides phylogenetic synopses for 25 groups of plant pathogenic fungi in the Ascomycota, Basidiomycota, Mucormycotina (Fungi), and Oomycota, using recent molecular data, up-to-date names, and the latest taxonomic insights. Lineage-specific laboratory protocols together with advice on their application, as well as general observations, are also provided. We hope to maintain updated backbone trees of these fungal lineages over time and to publish them jointly as new data emerge. Researchers of plant pathogenic fungi not covered by the present study are invited to join this future effort. Bipolaris, Botryosphaeriaceae, Botryosphaeria, Botrytis, Choanephora, Colletotrichum, Curvularia, Diaporthe, Diplodia, Dothiorella, Fusarium, Gilbertella, Lasiodiplodia, Mucor, Neofusicoccum, Pestalotiopsis, Phyllosticta, Phytophthora, Puccinia, Pyrenophora, Pythium, Rhizopus, Stagonosporopsis, Ustilago and Verticillium are dealt with in this paper.

Keywords

Ascomycota Basidiomycota Endophytes Mucormycotina Molecular identification Oomycota Plant pathogens Protozoa 
Contents and contributors (main contributors underlined)
  1. 1.

    BipolarisDS Manamgoda, KD Hyde

     
  2. 2.

    BotryosphaeriaceaeAJ Dissanayake, JK Liu, JY Yan, XH Li, KD Hyde

     
  3. 3.

    BotryosphaeriaAJ Dissanayake, JK Liu, JY Yan, XH Li, KD Hyde

     
  4. 4.

    BotrytisRB Terhem, M Hahn, JAL van Kan

     
  5. 5.

    ChoanephoraJ Pawłowska, G Walther, M Wilk, M Gorczak, M Wrzosek

     
  6. 6.

    ColletotrichumRS Jayawardena, DS Manamgoda, L Cai, XH Li, JY Yan, KD Hyde

     
  7. 7.

    CurvulariaDS Manamgoda, KD Hyde

     
  8. 8.

    DiaportheAJ Dissanayake, D Udayanga, JY Yan, XH Li, KD Hyde

     
  9. 9.

    DiplodiaAJ Dissanayake, JK Liu, JY Yan, XH Li, KD Hyde

     
  10. 10.

    DothiorellaJK Liu, KD Hyde

     
  11. 11.

    FusariumB Summerell, MH Laurence

     
  12. 12.

    GilbertellaJ Pawłowska, G Walther, M Wilk, M Gorczak, M Wrzosek

     
  13. 13.

    LasiodiplodiaJK Liu, KD Hyde

     
  14. 14.

    MucorJ Pawłowska, G Walther, M Wilk, M Gorczak, M Wrzosek

     
  15. 15.

    NeofusicoccumAJ Dissanayake, JK Liu, JY Yan, XH Li, KD Hyde

     
  16. 16.

    PestalotiopsisSSN Maharachchikumbura SA Alias KD Hyde

     
  17. 17.

    PhyllostictaN Zhou, L Cai

     
  18. 18.

    PhytophthoraF Martin, JE Blair

     
  19. 19.

    PucciniaAR McTaggart, RG Shivas

     
  20. 20.

    PyrenophoraHA Ariyawansa, KD Hyde

     
  21. 21.

    PythiumCFJ Spies, TL Rintoul, AWAM de Cock, SL Glockling, CA Lévesque

     
  22. 22.

    RhizopusM Gorczak, G Walther, J Pawłowska, M Wilk, M Wrzosek

     
  23. 23.

    StagonosporopsisN Vaghefi, PWJ Taylor

     
  24. 24.

    UstilagoAR McTaggart, RG Shivas

     
  25. 25.

    VerticilliumPWJ Taylor, PVR Nair

     

Introduction

Fungi form a large and heterogeneous eukaryotic kingdom with an estimated 1.5 million extant species. While all fungi are heterotrophic, a wide range of nutritional strategies is known in the kingdom. Most of the ca. 100,000 described species of fungi are associated with plants through interactions including symbiosis, endophytism, saprotrophy and parasitism (Stajich et al. 2009; Delaye et al. 2013; Persoh 2013; Hyde et al. 2013b). As plant parasites, fungi can cause significant economic loss with far-reaching social implications and consequences in agriculture, forestry and natural ecosystems (Fisher et al. 2012). They are also part of the natural ecosystem and play an important role in regulation of species (Hantsch et al. 2014).

The study of plant pathogenic fungi–their systematics, biology, and biological control–has a long and rich history (Udayanga et al. 2011; Maharachchikumbura et al. 2011; Manamgoda et al. 2011). The inconspicuous nature of most fungi makes their study difficult. For example, there are typically few discriminatory morphological characters, which often makes precise field- or laboratory-based identification problematic. Morphological characters that vary with the choice of host or environmental conditions form an additional, serious concern. Many species are difficult or impossible to keep alive in culture, which excludes them from physiological and often molecular tests that are available. The formation of sexual fruiting bodies is rarely observed in many plant pathogenic fungi, which has hampered their integration in the fungal tree of life, resulting in the proliferation of polyphyletic asexual genera. The biology of most plant pathogenic fungi remains poorly understood.

The last 25 years have witnessed the emergence of molecular data (DNA sequences) as a source of high fidelity information that has revolutionised mycology (Nilsson et al. 2014). DNA sequences offer a means by which to examine and compare fungi, independent of morphological plasticity, cultivability, and host-pathogen interactions. Since integration of molecular data in the study of plant pathogenic fungi in the early 1990s, there has been a much deeper understanding of the ecology, distribution, and systematics of plant pathogenic fungi (Bridge et al. 2005; Wingfield et al. 2012; Udayanga et al. 2013; Manamgoda et al. 2013). The use of molecular data in diagnostics and systematic studies is not without pitfalls and shortcomings that researchers must consider (Kang et al. 2010; Ko et al. 2011; Hyde et al. 2013a). Synonyms, homonyms, asexual-sexual relationships, ambiguous and misidentified specimens are rife in the plant pathology literature and public databases of DNA sequences, which posses an enormous challenge for the unwary. Equally challenging is the large number of unidentified and seemingly unidentifiable fungi and fungal sequences isolated from plants (Kõljalg et al. 2013; Unterseher et al. 2013). Certain plant pathogenic fungi require specialized extraction and PCR primers/protocols in order to amplify their DNA. Furthermore, the same genetic markers that give unparalleled phylogenetic resolution in some fungi may give none whatsoever in others. Many plant pathology studies focus on single species of fungi, and recent revisions or synopses at the generic or higher levels are lacking for the majority of plant pathogenic fungi.

The present study seeks to facilitate present and future studies of plant pathogenic fungi by providing phylogenetic backbone trees for as many groups of fungi as our expertise allowed. Our ambition is to synthesize all recent molecular data, recommendations on correct names, type material, geo/ecological observations, literature, and lineage-specific laboratory advice into a comprehensive, uniform molecular treatise for some of the largest and most widely encountered lineages of plant pathogenic fungi.

Material and methods

The phylogenetic analyses were performed based on up to date ex-type, ex-epitype or otherwise authentic sequence data available in GenBank as a concerted effort of the multiple contributors listed in authors section. By authentic sequence data we refer to those sequences used for names that are considered by the current working groups with the support of reliable publications in each genus as representative for each species. Sequences for the genes and genetic markers recommended for each genus were selected based on the current publications and have commonly been used for each of the genera (Table 1). The single gene sequence alignments were initially aligned with Clustal X and improved in MAFFTv. 7.017 (Katoh et al. 2002). Individual alignments were then concatenated and used to construct the backbone trees of each pathogenic genus listed. The phylogenetic analyses were performed for maximum parsimony in PAUP v. 4.0b10 (Swofford 2002), maximum likelihood in RAxML 7.4.2 Black Box or RAxMl GUI (Stamatakis 2006; Stamatakis et al. 2008), PhyML 3.0 (Guindon et al. 2010) or Bayesian inference in MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001) as specified in the legend of each phylogenetic tree. The trees used to represent each genus were analyzed by multiple contributors based on the selection of genes in given publications under each description.
Table 1

Gene regions and primers

Genus

Gene regions

Primers

Reference

Forward

Reverse

Bipolaris

ITS

ITS5

ITS4

White et al. 1990

GPDH

GPD1

GPD2

Berbee et al. 1999

Botryosphaeriaceae

ITS

ITS5

ITS4

White et al. (1990)

LSU

LROR

LR5

Vilgalys and Hester (1990)

SSU

NS1

NS4

White et al. (1990)

TEF

728F

986R

Carbone and Kohn (1999)

β- tubulin

BT2A

BT2B

Glass and Donaldson (1995)

Botryosphaeria

ITS

ITS5

ITS4

White et al. (1990)

LSU

LROR

LR5

Vilgalys and Hester (1990)

SSU

NS1

NS4

White et al. (1990)

TEF

728F

986R

Carbone and Kohn (1999)

β- tubulin

BT2A

BT2B

Glass and Donaldson (1995)

Botrytis

RPB2

RPB2for+

RPB2rev+

Staats et al. (2005)

HSP60

HSP60for+

HSP60rev+

Staats et al. (2005)

GPDH

G3PDHfor+

G3PDHrev+

Staats et al. (2005)

NEP1

NEP1(−207)for, NEP1for

NEP1revA, NEP1revB, NEP1(+1124)rev

Staats et al. (2007a, b)

NEP2

NEP2(−200)for, NEP2forD, NEP2forE, NEP2forF

NEP2(+1124)rev, NEP2revD, NEP2revE

Staats et al. (2007a, b)

Choanephora

ITS

V9G

LR3

de Hoog and Gerrits Van den Ende (1998)

Colletotrichum

ITS

ITS1-F

ITS4

Gardes and Bruns (1993)

GPDH

GDF

GDR

Templeton et al. 1992

CHS-1

CHS-79F

CHS-345R

Carbone and Kohn (1999)

HIS3

CYLH3F

CYLH3R

Crous et al. 2004

ACT

ACT-512F

ACT783R

Carbone and Kohn (1999)

β- tubulin

T1

T2

O’Donnell and Cigelnik 1997

ApMat

AM-F

AM-R

Silva et al. (2012)

Curvularia

ITS

ITS5

ITS4

White et al. 1990

GPDH

GPD1

GPD2

Berbee et al. 1999

Diplodia

ITS

ITS5

ITS4

White et al. (1990)

TEF

728F

986R

Carbone and Kohn (1999)

β- tubulin

BT2A

BT2B

Glass and Donaldson (1995)

Dothiorella

ITS

ITS5

ITS4

White et al. (1990)

TEF

728F

986R

Carbone and Kohn (1999)

β- tubulin

BT2A

BT2B

Glass and Donaldson (1995)

RPB2

   

Gilbertella

ITS

V9G:

LR3

de Hoog and Gerrits Van den Ende 1998

Lasiodiplodia

ITS

ITS5

ITS4

White et al. (1990)

TEF

728F

986R

Carbone and Kohn (1999)

β- tubulin

BT2A

BT2B

Glass and Donaldson (1995)

Mucor

LSU

NL1

1492R

O’Donnel 1993

Vilgalys and Hester 1990

Neofusicoccum

ITS

ITS5

ITS4

White et al. (1990)

LSU

LROR

LR5

Vilgalys and Hester (1990)

SSU

NS1

NS4

White et al. (1990)

TEF

728F

986R

Carbone and Kohn (1999)

β- tubulin

BT2A

BT2B

Glass and Donaldson (1995)

Pestalotiopsis

ITS

ITS5

ITS4

White et al. 1990

TEF

526F

1567R

Rehner 2001

β-tubulin

BT2A

BT2B

Glass and Donaldson 1995; O’Donnell and Cigelnik 1997

Phyllosticta

ITS

ITS1

ITS4

White et al. 1990

ACT

ACT512F

ACT783R

Carbone and Kohn 1999

TEF

EF1–728F

EF1–786R

Carbone and Kohn 1999

GPDH

GDF1

Gpd2–LM

Myllys et al. 2002; Guerber et al. 2003

Phytophthora

LSU

LROR-O (LSUFint)

LR6-O (LSURint)

Blair et al. (2008)

β-tubulin

Btub_F1

Btub_R1A

Blair et al. (2008)

cox2

FM35

Phy10b

Martin et al. (2014)

nad9

Nad9-F

Nad9-R

Blair et al. (2008)

rps10

Prv9-F

Prv9-R

Blair et al. (2008)

Puccinia

LSU

Rust 2INV

LR6

Aime (2006), Vilgalys and Hester (1990)

SSU

NS1

Rust 18SR

White et al. (1990), Aime (2006)

ITS

ITS5-u

ITS4

Pfunder et al. (2001); White et al. (1990)

Pyrenophora

ITS

ITS5

ITS4

White et al. 1990

LSU

LROR

LR5

Vilgalys and Hester (1990)

GPDH

GDF

GDR

Templeton et al. 1992

Pythium

5.8S, ITS2, LSU

Oom-up5.8S01

Un-lo28S1220

Man in ’t Veld et al. (2002), Bala et al. (2010a)

SSU, ITS1, 5.8S

NS1

Oom-lo5.8S47

White et al. (1990), Man in ’t Veld et al. (2002)

cox2

COX2F, FM35, FM82

COX2R, FM78_Pyt, FM52, FM83, Oom-cox1-lev-lo

Hudspeth et al. (2000), Martin (2000), Martin and Tooley (2003b), Robideau et al. (2011), Eggertson (2012)

β-tubulin

BtubF1A, Oom-Btub-up-415

Oom-Btub-lo-1401, BT-R2 (5′- CTTGATGTTGTTNGGRATCCACTC-3′)

Bilodeau et al. (2007), Blair et al. (2008), this study

Rhizopus

ITS

V9G

LR3

de Hoog and Gerrits Van den Ende 1998

Stagonosporopsis

ITS

V9G/ITS1

ITS4

V9G, de Hoog and Gerrits Van den Ende 1998

SSU

NS1

NS4

White et al. 1990

LSU

LROR

LR7

Rehner and Samuels 1994; Vilgalys and Hester 1990

CAL

CAL-228F

CAL-737R /CAL2Rd

Carbone and Kohn (1999), Quaedvlieg et al. (2011)

ACT

ACT-512F

ACT-783R

Carbone and Kohn (1999)

β-tubulin

BT2Fd

BT4R

Woudenberg et al. (2009)

Ustilago

ITS

ITS1F

ITS4

Gardes and Bruns (1993), White et al. (1990)

LSU

LROR

LR7

Vilgalys and Hester (1990)

Verticillium

ITS

ITS1-F

ITS4

Gardes and Bruns (1993), White et al. (1990)

TEF

VEFf

VEFr

Inderbitzin et al. (2011b)

ACT

VActF

VActR

Inderbitzin et al. (2011b)

GPDH

VGPDf2

VGPDr

Inderbitzin et al. (2011b)

TS

VTs3f

VTs3r

Inderbitzin et al. (2011b)

Table 2

Bipolaris. Details of the isolates used in the phylogenetic tree

Species

Isolate

Host

GenBank accession number

ITS

GPDH

EF

Bipolaris chloridis

CBS 242.77*

Chloris gayana

JN192372

JN600961

 

B. cynodontis

ICMP 6128*

Cynodon dactylon

JX256412

JX276427

JX266581

B. drechsleri

CBS 136207

Microstegium vimineum

KF500530

KF500533

 

B. drechsleri

CBS 136208

Microstegium vimineum

KF500532

KF500535

 

B. eleusines

8749C*

Eleusine indica

AF081451

AF081405

 

B. luttrellii

14643-1*

Dactyloctenium aegyptium

AF071350

AF081402

 

B. maydis

C5*

Zea mays

AF071325

AF081380

 

B. melinidis

BRIP 12898

Melinis minutiflora

JN601035

JN600972

 

B. microlenae

CBS 280.91

Microlaena stipoides

JN601032

JN600974

JN601017

B. oryzae

MFLUCC 100694*

Oryza sativa

JX256413

JX276428

JX266582

B. oryzae

MFLUCC 100716

O. sativa

JX256415

JX276429

JX266584

B. peregianensis

BRIP 12970

Cynodon dactylon

JN601034

JN600977

JN601022

B. sorghicola

MAFF511378*

Sorghum sudanense

AF071332

AF081387

 

B. sorokiniana

ICMP 6233a

Lolium perenne

JX256418

  

B. urochloae

DAOM 171970*

Urochloa panicoides

AF071334

AF081389

 

B. victoriae

CBS 174.57*

Avena sativa

JN601027

 

JN601005

Curvularia lunata

CBS 730.96

Unknown

JX256429

JX276441

JX266596

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 3

Botryosphaeriaceae. Details of the isolates used in the phylogenetic tree

Species

Isolate

ITS

β- tubulin

TEF

SSU

LSU

Barriopsis fusca

CBS 174.26*

EU673330

EU673109

EU673296

EU673182

DQ377857

B. iraniana

IRAN1448C*

KF766150

KF766127

FJ919652

KF766231

KF766318

Botryobambusa fusicoccum

MFLUCC 11-0143*

JX646792

JX646857

JX646826

JX646809

Botryosphaeria agaves

MFLUCC 11-0125*

JX646791

JX646841

JX646856

JX646825

JX646808

B. corticis

CBS 119047*

DQ299245

EU673107

EU017539

EU673175

EU673244

B. dothidea

CMW 8000*

AY236949

AY236927

AY236898

EU673173

AY928047

B. fusispora

MFLUCC 10-0098*

JX646789

JX646839

JX646854

JX646823

JX646806

Cophinforma eucalypti

MFLUCC 11-0425*

JX646800

JX646848

JX646865

JX646833

JX646817

Diplodia corticola

CBS 112549*

AY259100

DQ458853

AY573227

EU673206

AY928051

D. cupressi

CBS 168.87*

DQ458893

DQ458861

DQ458878

EU673209

EU673263

D. mutila

CBS 112553*

AY259093

DQ458850

AY573219

EU673213

AY928049

Dothiorella iberica

CBS 115041*

AY573202

EU673096

AY573222

EU673155

AY928053

D. sarmentorum

IMI 63581b*

AY573212

EU673102

AY573235

EU673158

AY928052

D. thailandica

MFLUCC11-0438*

JX646796

JX646844

JX646861

JX646829

JX646813

Endomelanconiopsis endophytica

CBS 120397*

KF766164

KF766131

EU683637

KF766249

EU683629

E. microspora

CBS 353.97*

KF766165

EU683636

KF766250

KF766330

Lasiodiplodia crassispora

CBS 118741*

DQ103550

EU673133

EU673303

EU673190

DQ377901

L. gonubiensis

CBS 115812*

DQ458892

DQ458860

DQ458877

EU673193

DQ377902

L. parva

CBS 494.78*

EF622084

EU673114

EF622064

EU673201

EU673258

L. pseudotheobromae

CBS 116459*

EF622077

EU673111

EF622057

EU673199

EU673256

L. theobromae

CBS 164.96*

AY640255

EU673110

AY640258

EU673196

EU673253

Macrophomina phaseolina

CBS 227.33*

KF766195

KF766422

KF766281

KF766364

Neodeightonia palmicola

MFLUCC 10-0822*

HQ199221

HQ199223

HQ199222

N. phoenicum

CBS 122528*

EU673340

EU673116

EU673309

EU673205

EU673261

N. subglobosa

MFLUCC11-0163*

JX646794

JX646842

JX646859

JX646811

Neofusicoccum luteum

CBS 110299*

AY259091

DQ458848

AY573217

EU673148

AY928043

N. mangiferae

CBS 118532*

AY615186

AY615173

DQ093220

EU673154

DQ377921

N. parvum

CMW 9081*

AY236943

AY236917

AY236888

EU673151

AY928045

Neoscytalidium dimidiatum

IP127881

AF258603

FM211167

EU144063

AF258603

DQ377925

N. hyalinum

CBS145.78*

KF531816

KF531796

KF531795

KF531815

DQ377922

N. novaehollandiae

WAC 12691*

EF585543

EF585574

EF585548

Phaeobotryon mamane

CPC 12440*

EU673332

EU673121

EU673298

EU673184

EU673248

Pseudofusicoccum adansoniae

WAC 12689*

EF585534

EF585567

EF585554

P. ardesiacum

CMW 26159*

KF766221

EU144075

KF766307

KF766387

P. kimberleyense

CMW 26156*

KF766222

EU144072

KF766308

KF766388

P. stromaticum

CMW13434*

KF766223

EU673094

KF766437

KF766309

KF766389

Spencermartinsia viticola

CBS 117009*

AY905554

EU673104

AY905559

EU673165

DQ377873

Sphaeropsis citrigena

ICMP 16812*

EU673328

EU673140

EU673294

EU673180

EU673246

S. eucalypticola

CBS 133993*

JX646802

JX646850

JX646867

JX646835

JX646819

S. porosa

CBS 110496*

AY343379

EU673130

AY343340

EU673179

DQ377894

S. visci

CBS 186.97*

EU673325

EU673128

EU673293

EU673178

DQ377868

Tiarosporella graminis var. karoo

CBS 118718

KF531828

KF531808

KF531807

KF531827

DQ377939

T. tritici

CBS 118719*

KF531830

KF531810

KF531809

KF531829

DQ377941

T. urbis-rosarum

CMW 36479*

JQ239408

JQ239382

JQ239395

JQ239421

Melanops tulasnei

CBS 116805*

FJ824769

KF766423

KF766474

KF766365

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 4

Botryosphaeria. Details of the ex-type and voucher isolates used in the phylogenetic tree

Species

Isolate

GenBank accession numbers

SSU

ITS

LSU

TEF

β-tubulin

Botryosphaeria agaves

CBS 133992*

JX646825

JX646825

JX646808

JX646856

JX646841

B. corticis

CBS 119047*

EU673175

DQ299245

EU673244

EU017539

EU673107

B. dothidea

CBS 115476*

EU673173

AY236949

AY928047

AY236898

AY236927

B. fabicerciana

CBS 127193*

N/A

HQ332197

N/A

HQ332213

N/A

B. fusispora

MFLUCC 10-0098*

JX646823

JX646789

JX646806

JX646854

JX646839

B. ramose

CBS 122069*

N/A

EU144055

N/A

EU144070

N/A

B. scharifii

CBS 124703*

N/A

JQ772020

N/A

JQ772057

N/A

Macrophomina phaseolina

CBS 227.33*

KF531823

KF531825

DQ377906

KF531804

KF531806

Type strains and voucher stains are bolded

Table 5

Botrytis. Details of the isolates used in the phylogenetic tree

Species

Isolate

Host

GenBank accession numbers

RPB2

HSP60

G3DPDH

NEP1

NEP2

Botrytis aclada

MUCL8415

Allium spp.

AJ745664

AJ716050

AJ704992

AM087059

AM087087

B. byssoidea

MUCL94

Allium spp.

AJ745670

AJ716059

AJ704998

AM087045

AM087079

B. calthae

MUCL1089

Caltha palustris

AJ745672

AJ716061

AJ705000

AM087031a

AM087088a

B. cinerea

MUCL87

>200 species

AJ745676

AJ716065

AJ705004

DQ211824a

DQ211825a

B. caroliniana

CB15*

Rubus fruticosus

JF811590

JF811587

JF811584

JF811593

NA

B. convoluta

MUCL11595

Iris spp.

AJ745680

AJ716069

AJ705008

AM087035

AM087062

B. croci

MUCL436

Crocus spp.

AJ745681

AJ716070

AJ705009

AM087047

AM087065

B. deweyae

CBS134649*

Hemerocallis spp.

HG799518

HG799519

HG799521

HG799527

HG799520

B. elliptica

BE9714

Lilium spp.

AJ745684

AJ716073

AJ705012

AM087049

AM087080

B. fabae

MUCL98

Vicia spp.

AJ745686

AJ716075

AJ705014

DQ211829

DQ211831

B. ficariarum

MUCL376

Ficaria verna

AJ745688

AJ716077

AJ705016

AM087056

AM087085a

B. fabiopsis

BC-2*

Vicia faba

EU514473

EU514482

EU519211

NA

NA

B. galanthina

MUCL435

Galanthus spp.

AJ745689

AJ716079

AJ705018

AM087057

AM087067a

B. gladiolorum

MUCL3865

Gladiolus spp.

AJ745692

AJ716081

AJ705020

AM087041

AM087072a

B. globosa

MUCL444

Allium ursinum

AJ745693

AJ716083

AJ705022

AM087044a

AM087071

B. hyacinthi

MUCL442

Hyacinthus spp.

AJ745696

AJ716085

AJ705024

AM087048

AM087066a

B. narcissicola

MUCL2120

Narcissus spp.

AJ745697

AJ716087

AJ705026

AM087046

AM087078

B. paeoniae

MUCL16084

Paeonia spp.

AJ745700

AJ716089

AJ705028

AM087033

AM087064a

B. pelargonii

CBS 497.50

Pelargonium spp.

AJ745662

AJ716046

AJ704990

AM087030

DQ211834a

B. polyblastis

CBS287.38

Narcissus spp.

AJ745702

AJ716091

AJ705030

AM087039

AM087074

B. porri

MUCL3234

Allium spp.

AJ745704

AJ716093

AJ705032

AM087060

AM087063

B. pseudocinerea

VD110

Vitis vinifera

Unpublished

Unpublished

Unpublished

NA

NA

B. ranunculi

CBS178.63

Ranunculus spp.

AJ745706

AJ716095

AJ705034

AM087054

AM087086

B. sinoallii

HMAS250008

Allium spp.

EU514479

EU514488

EU519217

NA

NA

B. sphaerosperma

MUCL21481

Allium triquetrum

AJ745708

AJ716096

AJ705035

AM087042

AM087068

B. squamosa

MUCL1107

Allium cepa

AJ745710

AJ716098

AJ705037

AM087052

AM087084

B. tulipae

BT9830

Tulipa spp.

AJ745713

AJ716102

AJ705041

AM087037

AM087077

Monilinia fructigena

9201

Stone fruit and pome fruit

AJ745715

AJ716047

AJ705043

NA

NA

Sclerotinia sclerotiorum

484

>400 species

AJ745716

AJ716048

AJ705044

NA

NA

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 6

Choanephora. Details of the isolates used in the phylogenetic tree

Species

Isolate

Host

GenBank no

Choanephora infundibilifera

CBS 153.51

JN206236

C. infundibilifera

CBS 155.51

JN206237

C. infundibilifera

CBS 155.58

JN206238

C. cucurbitarum

CBS 445.72

JN206234

C. cucurbitarum

CBS 178.76

Dead insect

JN206235

C. cucurbitarum

CBS 674.93

JN206233

C. cucurbitarum

CBS 120.25

JN206231

C. cucurbitarum

CBS 150.51

JN206232

Poitrasia circinans

CBS 153.58*

Soil

JN206239

P. circinans

CBS 647.70

Soil

JN206240

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 7

Colletotrichum. Details of the isolates used in the phylogenetic tree

Species

Isolate

GenBank Accession Number

ApMat

ITS

GPDH

CHS-1

HIS3

ACT

β-tubulin

C. acerbum*

CBS 128530

JQ948459

JQ948790

JQ949120

JQ949450

JQ949780

JQ950110

C. acutatum*

CBS112996

JQ005776

JQ948677

JQ005797

JQ005818

JQ005839

JQ005860

C. aenigma*

ICMP 18608

JX010244

JX010044

JX009774

JX009443

JX010389

C. aeschynomenes*

ICMP 17673

JX010176

JX009930

JX009799

JX009483

JX010392

C. agaves

CBS 118190

DQ286221

C. alatae*

ICMP 17919

JX010190

JX009990

JX009837

JX009471

JX010383

KC888932

C. alcorni*

IMI 176619

JX076858

      

C. alienum*

ICMP 12071

JX010251

JX010028

JX009882

JX009572

JX010411

KC888927

C. annellatum*

CBS 129826

JQ005222

JQ005309

JQ005396

JQ005483

JQ005570

JQ005656

C. anthrisci*

CBS 125334

GU227845

GU228237

GU228335

GU228041

GU227943

GU228139

C. aotearoa*

ICMP 18537

JX010205

JX010005

JX009853

JX009564

JX010420

KC888930

C. asianum*

ICMP 18580

FJ972612

JX010053

JX009867

JX009584

JX010406

FR718814

C. australe*

CBS116478

JQ948455

JQ948786

JQ949116

JQ949446

JQ949776

JQ950106

C. axonopodi

IMI 279189

EU554086

C. baltimorense*

SD11

JX076866

C. beeveri*

CBS 128527

JQ005171

JQ005258

JQ005345

JQ005432

JQ005519

JQ005605

C. bletillum*

CGMCC 3.15117

JX625178

KC843506

KC843542

JX625207

C. bidentis*

COAD 1020

KF178481

KF178506

KF148530

KF178554

KF178578

KF178602

 

C. boninense*

CBS 123755

JQ005153

JQ005240

JQ005327

JQ005414

JQ005501

JQ005588

C. brasiliense*

CBS 128501

JQ005235

JQ005322

JQ005409

JQ005496

JQ005583

JQ005669

C. brassicola*

CBS 101059

JQ005172

JQ005259

JQ005346

JQ005433

JQ005520

JQ005606

C. brevisporum*

BCC 38876

JQ247623

JQ247599

JQ247647

JQ247635

C. brisbanense*

CBS292.67

JQ948291

JQ948621

JQ948952

JQ949282

JQ949612

JQ949942

C. carthami*

SAPA100011

AB696998

AB696992

C. caudasporum*

CGMCC 3.15106

JX625162

KC843512

KC843526

JX625190

C. caudatum*

BPI423339

JX076860

C. cereale

CBS 129663

JQ005774

JQ005795

JQ005816

JQ005837

JQ005858

C.chlorophyti*

IMI 103806

GU227894

GU228286

GU228384

GU228090

GU227992

GU228188

C. chrysanthemi

IMI364540

JQ948273

JQ948603

JQ948934

JQ949264

JQ949594

JQ949924

Glomerella cingulata “f.sp. camelliae”

ICMP 10643

JX010224

JX009908

JX009891

JX009540

JX010436

C. circinans*

CBS 221.81

GU227855

GU228247

GU228345

GU228051

GU227953

GU228149

C. citri*

ZJUC41

KC293581

KC293741

KC293621

KC293661

C. citricola*

SXC151

KC293576

KC293736

KC293792

KC293616

KC293656

C. clidemia*

ICMP 18658

JX010265

JX009989

JX009877

JX009537

JX010438

KC888929

C.cliviae*

CBS 125375

JX519223

GQ856756

JX519232

JX519240

JX519249

C.coccodes

CBS 369.75

JQ005775

HM171673

JQ005796

JQ005817

JQ005838

JQ005859

C. coccodes

ITCC 6079

KC790652

C. colombiense*

CBS 129818

JQ005174

JQ005261

JQ005348

JQ005435

JQ005522

JQ005608

C. constrictum*

CBS 128504

JQ005238

JQ005325

JQ005412

JQ005499

JQ005586

JQ005672

C. cordylinicola*

ICMP 18579

JX010226

JX009975

JX009864

HM470234

JX010440

JQ899274

C. cosmi*

CBS 853.73

JQ948274

JQ948604

JQ948935

JQ948604

JQ949595

JQ949925

C. costaricense*

CBS 330.75

JQ948180

JQ948510

JQ948841

JQ949171

JQ949501

JQ949831

C. curcumae*

IMI 288937

GU227893

GU228285

GU228383

GU228089

GU227991

GU228187

C. cuscutae*

IMI 304802

JQ948195

JQ948525

JQ948856

JQ949186

JQ949516

JQ949846

C. cymbidiicola*

IMI 347923

JQ005166

JQ005253

JQ005340

JQ005427

JQ005514

JQ005600

C. dacrycarpi*

CBS 130241

JQ005236

JQ005323

JQ005410

JQ005497

JQ005584

JQ005670

C. dematium*

CBS 125.25

GU227819

GU228211

GU228309

GU228015

GU227917

GU228113

C. destructivum

CBS 149.34

AJ301942

JQ005785

JQ005806

JQ005827

JQ005848

C. dianensei*

CMM4083

KC329779

KC517194

KC517298

KC517254

KJ155461

C.dracaenophilum*

CBS 118199

JX519222

JX519230

JX519238

JX519247

C. duyunensis*

CGMCC 3.15105

JX625160

KC843515

KC843530

JX625187

C. echinochloae*

MAFF 511473

AB439811

C. eleusines*

MAFF 511155

JX519218

JX519226

JX519234

JX519243

C. endomagniferae*

CMM 3814

KC702994

KC702955

KC598113

KC702922

KC702922

KJ155453

C. endophytica*

LC0324

KC633854

KC832854

KF306258

C. endophytum*

CGMCC 3.15108

JX625177

KC843521

KC843533

JX625206

C. eremochloae*

CBS 129661

JX519220

JX519228

JX519236

JX519245

C. excelsum altitudum*

CGMCC 3.15130

HM751815

KC843502

KC843548

JX625211

C. falcatum

CBS 147945

JQ005772

JQ005793

JQ005814

JQ005835

JQ005856

C. fioriniae*

CBS 128517

JQ948292

JQ948622

JQ948953

JQ949283

JQ949613

JQ949943

C. fructi*

CBS 346.37

GU227844

GU228236

GU228334

GU228040

GU227942

GU228138

C. fructicola*

ICMP 18581

JX010165

JX010033

JX009866

FJ907426

JX010405

JQ807838

C. fructivorum*

Coll1414

JX145145

JX145196

C. fuscum

CBS 130.57

JQ005762

JQ005783

JQ005804

JQ005825

JQ005846

C. gigasporum*

MUCL 44947

AM982797

FN557442

C. gloeosporioides*

CBS 112999

JQ005152

JQ005239

JQ005326

JQ005413

JQ005500

JQ005587

JQ807843

C. godetiae*

CBS 133.44

JQ948402

JQ948733

JQ949063

JQ949393

JQ949723

JQ950053

C. graminicola*

CBS 130836

JQ005767

JQ005788

JQ005809

JQ005830

JQ005851

C. grevilleae*

CBS 132879

KC297078

KC297010

KC296987

KC297056

KC296941

KC297102

C. guajave*

IMI 350839

JQ948270

JQ948600

JQ948931

JQ949261

JQ949591

JQ949921

C. guizhouensis*

CGMCC 3.15112

JX625158

KC843507

KC843536

JX625185

C. hanaui*

MAFF 305404

JX519217

JX519225

JX519242

C. hemerocallidis*

CDLG5

JQ400005

JQ400012

JQ399998

JQ399991

JQ400019

C. higginsianum

IMI 349063

JQ005760

JQ005781

JQ005802

JQ005823

JQ005844

C. hippeastri*

CBS 125376

JQ005231

JQ005318

JQ005405

JQ005492

JQ005579

JQ005665

C. horii

ICMP 10492

GQ329690

GQ329681

JX009752

JX009438

JX010450

JQ807840

C. hsienjenchng

MAFF 243051

AB738855

AB738846

AB738847

AB738845

C. incanum*

ATCC 64682

KC110789

KC110807

KC110798

KC110825

KC110816

C. indonesiense*

CBS 127551

JQ948288

JQ948618

JQ948949

JQ949279

JQ949609

JQ949939

C. jacksonii*

MAFF 305460

JX519216

JX519224

JX519233

JX519241

C. jasiminigenum*

MFU 10-0273

HM131513

HM131499

HM131508

HM153770

C. johnstonii*

CBS 128532

JQ948444

JQ948775

JQ949105

JQ949435

JQ949765

JQ950095

C. kahawe*

ICMP17816

JX010231

JX010012

JX009813

JX009452

JX010444

JQ899282

C. kartsii*

CORCG6

HM585409

HM585391

HM582023

HM581995

HM585428

C. kinghornii*

CBS 198.35

JQ948454

JQ948785

JQ949115

JQ949445

JQ949775

JQ950105

C. lacticiphilum*

CBS 112989

JQ948289

JQ948619

JQ948950

JQ949280

JQ949610

JQ949940

C. lilii

CBS 109214

GU227810

GU228202

GU228300

GU228006

GU227908

GU228104

C. limetticola*

CBS 114.14

JQ948193

JQ948523

JQ948854

JQ949184

JQ949514

JQ949844

C. lindemuthianum*

CBS 144.31

JQ005779

JX546712

JQ005800

JQ005821

JQ005842

JQ005863

C. lineola*

CBS 125337

GU227829

GU228221

GU228319

GU228025

GU227927

GU228123

C. linicola

CBS 125337

GU227829

GU228221

GU228319

GU228025

GU227927

GU228123

C. liriopes*

CBS 119444

GU227804

GU228196

GU228294

GU228000

GU227902

GU228098

C. lupini

CBS 109225

JQ948155

JQ948485

JQ948816

JQ949146

JQ949476

JQ949806

C. melanocaulon*

Coll131

JX145313

C. malvarum*

CBS 527.97

KF178480

KF178504

KF178529

KF178553

KF178577

KF178601

 

C. melonis*

CBS 159.84

JQ948194

JQ948524

JQ948855

JQ949185

JQ949515

JQ949845

C. metake

NBRC 8974

AB738859

C. miscanthi*

MAFF 510857

JX519221

JX519229

JX519237

JX519246

C. musae*

ICMP19119

JX010146

JX010050

JX009896

JX009433

HQ596280

KC888926

C. murrayae*

GZAAS5.09506

JQ247633

JQ247609

JQ247657

JQ247644

C. navitas*

CBS 125086

JQ005769

JQ005790

JQ005811

JQ005832

JQ005853

C. nicholsonii*

MAFF 511115

JQ005770

JQ005791

JQ005812

JQ005833

JQ005854

C. nigrum*

CBS 169.49

JX546838

JX546742

JX546693

JX546791

JX546646

JX546885

C. novae-zelandiae*

CBS 128505

JQ005228

JQ005315

JQ005402

JQ005489

JQ005576

JQ005662

C. nupharicola*

ICMP 18187

JX010187

JX009972

JX009835

JX009437

JX010398

JX145319

C. nymphaeae*

CBS 515.78

JQ948197

JQ948527

JQ948858

JQ949188

JQ949518

JQ949848

C. ochracea*

CGMCC 3.15104

JX625156

KC843513

KC843527

JX625183

C. oncidii*

CBS 129828

JQ005169

JQ005256

JQ005343

JQ005430

JQ005517

JQ005603

C. orbiculare*

CBS 570.97

KF178466

KF178490

KF178515

KF178539

KF178563

KF178587

C.orchidophilum*

CBS 632.80

JQ948151

JQ948481

JQ948812

JQ949142

JQ949472

JQ949802

C. parsonsiae*

CBS 128525

JQ005233

JQ005320

JQ005407

JQ005494

JQ005581

JQ005667

C. paspali*

MAFF 305403

JX519219

JX519227

JX519235

JX519244

C. paxtonii*

IMI 165753

JQ948285

JQ948615

JQ948946

JQ949276

JQ949606

JQ949936

C. petchii*

CBS 378.94

JQ005223

JQ005310

JQ005397

JQ005484

JQ005571

JQ005657

C.phaseolorum

CBS 157.36

GU227896

GU228288

GU228386

GU228092

GU227994

GU228190

C. phormii*

CBS 118194

JQ948446

JQ948777

JQ949107

JQ949437

JQ949767

JQ950097

C. phyllanthi*

CBS 175.67

JQ005221

JQ005308

JQ005395

JQ005482

JQ005569

JQ005655

C. proteae

CBS132882

KC297079

KC297009

KC296986

KC297045

KC296940

KC297101

C.pseudoacutatum*

CBS 436.77

JQ948480

JQ948811

JQ949141

JQ949471

JQ949801

JQ950131

C. psidii

ICMP 19120

JX010219

JX009967

JX009901

JX009515

JX010443

C. pyricola*

CBS 128531

JQ948445

JQ948776

JQ949106

JQ949436

JQ949766

JQ950096

C. queenslandium*

ICMP 1778

JX010276

JX009934

JX009899

JX009447

JX010414

KC888928

C. rhexiae*

Coll 1026

JX145128

JX145179

JX145290

C. rhombiforme*

CBS 129953

JQ948457

JQ948788

JQ949118

JQ949448

JQ949778

JQ950108

C. rusci*

CBS 119206

GU227818

GU228210

GU228308

GU228014

GU227916

GU228112

C. salicis*

CBS 607.94

JQ948460

JQ948791

JQ949121

JQ949451

JQ949781

JQ950111

C. salsolae*

ICMP 19051

JX010242

JX009916

JX009863

JX009562

JX010403

KC888925

C. sansevieriae

MAFF 239721

AB212991

C. scovillei*

CBS 126529

JQ948267

JQ948597

JQ948928

JQ949258

JQ949588

JQ949918

C. siamense*

ICMP 18578

JX010171

JX009924

JX009865

FJ907423

JX010404

JQ899289

C. sidae*

CBS 504.97

KF178472

KF178497

KF178521

KF178545

KF178569

KF178593

C. simmondsii*

CBS 122122

JQ948276

JQ948606

JQ948937

JQ949267

JQ949597

JQ949927

C. sloanei*

IMI 364297

JQ948287

JQ948617

JQ948948

JQ949278

JQ949608

JQ949938

C. somersetense*

JAC 11-11

JX076862

C. spaethianum*

CBS 167.49

GU227847

GU228239

GU228337

GU228043

GU227945

GU228141

C. spinaceae

CBS 128.57

GU227847

GU228239

GU228337

GU228043

GU227945

GU228141

C. spinosum*

CBS 515.97

KF178474

KF178498

KF178523

KF178547

KF178571

KF178595

C. sublineola*

CBS 131301

JQ005771

JQ005792

JQ005813

JQ005834

JQ005855

C. syzygicola*

DNCL021

KF242094

KF242156

KF157801

KF254880

C. tabacum

CBS 161.53

JQ005763

JQ005784

JQ005805

JQ005826

JQ005847

C. tamarilloi*

CBS 129814

JQ948184

JQ948514

JQ948845

JQ949175

JQ949505

JQ949835

C. tanaceti*

CBS 132693

JX218243

JX218238

JX218233

C. tebeestii*

CBS 522.97

KF178473

KF178505

KF178522

KF178546

KF178570

KF178594

C. temperatum*

Coll883

JX145159

JX145211

JX145298

C. thailandicum*

MFUCC110113

JN050242

JN050231

JN050220

JN050248

C. theobromicola

ICMP 18649

JX010294

JX010006

JX009869

JX009444

JX010447

KC790726

C. ti*

ICMP 4832

JX010269

JX009952

JX009898

JX009520

JX010442

C. tofieldiae

CBS 495.85

GU227801

GU228193

GU228291

GU227997

GU227899

GU228095

C. torulosum*

CBS 128544

JQ005164

JQ005251

JQ005338

JQ005425

JQ005512

JQ005598

C.trichellum

CBS 217.64

GU227812

GU228204

GU228302

GU228008

GU227910

GU228106

C. trifolii*

CBS 158.83

KF178478

KF178502

KF178527

KF178551

KF178575

KF178599

C. tropicale*

ICMP18653

JX010264

JX010007

JX009870

JX009489

JX010407

KC790728

C.tropicicola*

BCC 38877

JN050240

JN050229

  

JN050218

JN050246

C. truncatum*

CBS 151.35

GU227862

GU228254

GU228352

GU228058

GU227960

GU228156

C. verruculosm*

IMI 45525

GU227806

GU228198

GU228296

GU228002

GU227904

GU228100

C. viniferum*

GZAAS5.08601

JN412804

JN412798

JN412795

JN412813

C. viniferum

GZAAS5.08608

KJ623242

C. walleri*

CBS 125472

JQ948275

JQ948605

JQ948936

JQ949266

JQ949596

JQ949926

C. xanthorrhoeae*

ICMP 17903

JX010261

JX009927

JX009823

JX009478

JX010448

KC790689

C. yunnanense*

CGMCC AS3.9167

EF369490

JX519231

JX519239

JX519248

C. zoysia*

MAFF 238573

JX076871

Ex-Type (ex-epitype) strains are bolded and marked with an * and authentic stains are bolded

Table 8

Details of the isolates used in the phylogenetic tree

Species

Code

Host

Gene bank accession numbers

ITS

GPDH

TEF

Curvularia affinis

DAOM 46365

 

AF071335

AF081390

 

C. alcornii

MFLUCC10703*

Zea mays

JX256420

JX276433

JX266589

MFLUCC10705

Panicum sp.

JX256421

JX276434

JX266590

C. australiensis

CBS 172.57

Oryza sativa

JN601026

JN601036

JN601003

C. clavata

ICMP 103444

Lawn

JX256444

JX276455

 

C. cymbopogonis

88109-1

 

AF071351

AF081403

 

C. ellisii

CBS 193.62*

Air

JN192375

JN600963

JN601007

C. gladioli

ICMP 6160

Gladiolus sp.

JX256426

JX276438

JX266595

C. graminicola

BRIP 23186

 

JN192376

JN600964

JN601008

C. gudauskasii

DAOM165085

 

AF071338

AF081393

 

C. hawaiiensis

BRIP 15933

Chloris gayana

JN601028

JN600965

JN601009

BRIP 10972

Chloris gayana

JN192377

JN600968

JN601012

C. heteropogonis

CBS 284.91*

Heteropogon contortus

JN192379

JN600969

JN601013

C. intermedia

8797-1

 

AF071327

AF081383

 

C. lunata

CBS 730.96*

Human lung biopsy

JX256429

JX276441

JX266596

C. ovariicola

CBS 470.90*

Eragrostis interrupta

JN601031

JN600976

JN601020

C. perotidis

CBS 7846-2

Perotis rara

AF071320

AF081374

 

C. ravenelii

BRIP 13165*

Sporobolus fertilis

JN192386

JN600978

JN601024

C. spicifera

CBS 274.52

Soil

JN192387

JN600979

JN601023

C. trifolii

ICMP 6149

Setaria glauca

JX256434

JX276457

JX266600

C. tripogonis

BRIP 12375*

Dactyloctenii aeygeptii

JN192388

JN600980

JX266600

C. tuberculata

CBS 146.63*

Zea mays

JN192374

JN601037

JX266599

C. verrucosa

MAFF235540

Triticum aestivum

AB444667

AF081388

 

Alternaria alternata

EGS 34.0160*

 

AF017346

AF081400

 

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 9

Diaporthe. Details of the isolates used in the phylogenetic tree

Species

Isolate

Host

GeneBank accession numbers

ITS

β-tubulin

EF 1-α

CAL

Diaporthe acaciigena

CBS 129521*

Acacia retinodes

KC343005

KC343973

KC343731

KC343247

D. alleghaniensis

CBS 495.72*

Betula alleghaniensis

KC343007

KC343975

KC343733

KC343249

D. alnea

CBS 146.46*

Alnus sp.

KC343008

KC343976

KC343734

KC343250

D. ambigua

CBS 114015*

Pyrus communis

KC343010

KC343978

KC343736

KC343252

D. ampelina

CBS 114016*

Vitis vinifera

AF230751

JX275452

AY745056

AY230751

D. amygdali

CBS 126679*

Prunus dulcis

KC343022

KC343990

AY343748

KC343264

D. anacardii

CBS 720.97*

Anacardium ocidentale

KC343024

KC343992

KC343750

KC343266

D. angelicae

CBS 111592*

Heracleum sphondylium

KC343027

KC343995

KC343753

KC343269

D. aquatica

IFRDCC 3051*

JQ797437

D. arecae

CBS 161.64*

Areca catechu

KC343032

KC344000

KC343758

KC343274

D. arengae

CBS 114979*

Arenga engleri

KC343034

KC344002

KC343760

KC343276

D. aspalathi

CBS 117169*

Aspalathus linearis

KC343036

KC344004

KC343762

KC343278

D. australafricana

CBS 111886*

Vitis vinifera

KC343038

KC344006

KC343764

KC343280

D. beilharziae

BRIP 54792*

Indigofera australis

JX862529

KF170921

JX862535

D. bicincta

CBS 121004*

Juglans sp.

KC343134

KC344102

KC343860

KC343376

D. brasiliensis

CBS 133183*

Aspidosperma tomentosum

KC343042

KC344010

KC343768

KC343284

D. caulivora

CBS 127268*

Glycine max

KC343045

KC344013

KC343771

KC343287

D. celastrina

CBS 139.27*

Celastrus sp

KC343047

KC344015

KC343773

KC343289

D. citri

CBS 135422*

Citrus sp.

KC843311

KC843187

KC843071

KC843157

D. citriasiana

ZJUD 30*

JQ954645

KC357459

JQ954663

KC357491

D. citrichinensis

ZJUD 34*

JQ954648

 

JQ954666

KC357494

D. crotalariae

CBS 162.33*

Crotalaria spectabilis

KC343056

KC344024

KC343782

KC343298

D. cuppatea

CBS 117499*

Aspalathus linearis

KC343057

KC344025

KC343783

KC343299

D. cynaroidis

CBS 122676*

Protea cynaroides

KC343058

KC344026

KC343784

KC343300

D. cytosporella

FAU461*

Citrus limon

KC843307

KC843221

KC843116

KC843141

D. endophytica

CBS 133811*

Schinus terebinthifolius

KC343065

KC343065

KC343791

KC343307

D. eres

AR5193*

Ulmus Sp.

KJ210529

KJ420799

KJ210550

KJ434999

P. cotoneastri

CBS 439.82*

Cotoneaster sp.

KC343090

KC344058

KC343816

KC343332

D. fraxini-angustifoliae

BRIP 54781*

Fraxinus angustifolia

JX862528

KF170920

JX862534

D. foeniculina

CBS 123208*

Foeniculum vulgare

KC343104

KC344072

KC343830

KC343346

D. foeniculina

CBS 123209*

Foeniculum vulgare

KC343105

KC344073

KC343831

KC343347

D. foeniculina

CBS 187.27 *

Camellia sinensis

KC343107

KC344075

KC343833

KC343349

D. ganjae

CBS 180.91*

Cannabis sativa

KC343112

KC344080

KC343838

KC343354

D. gulyae

BRIP 54025*

Helianthus annuus

JF431299

JN645803

D. helianthi

CBS 592.81*

Helianthus annuus

KC343115

KC344083

KC343841

KC343357

D. helicis

AR5211*

Hedera helix

KJ210538

KJ420828

KJ210559

KJ435043

D. hickoriae

CBS 145.26*

Carya glabra

KC343118

KC344086

KC343844

KC343360

D. hongkongensis

CBS 115448*

Dichroa febrífuga

KC343119

KC344087

KC343845

KC343361

D. inconspicua

CBS 133813*

Maytenus ilicifolia

KC343123

KC344091

KC343849

KC343365

D. infecunda

CBS 133812*

Schinus terebinthifolius

KC343126

KC344094

KC343852

KC343852

D. kochmanii

BRIP 54033*

Helianthus annuus

JF431295

JN645809

D. kongii

BRIP 54031*

Helianthus annuus

JF431301

JN645797

D. longispora

CBS 194.36*

Ribes sp.

KC343135

KC344103

KC343861

KC343377

D. lusitanicae

CBS 123212*

Foeniculum vulgare

KC343136

KC344104

KC343862

KC343378

D. mayteni

CBS 133185*

Maytenus ilicifolia

KC343139

KC344107

KC343865

KC343381

D. melonis

CBS 507.78 *

Glycine soja

KC343141

KC344109

KC343867

KC343383

D. musigena

CBS 129519*

Musa sp.

KC343143

KC344111

KC343869

KC343385

D. neoarctii

CBS 109490*

Ambrosia trifida

KC343145

KC344113

KC343871

KC343387

D. nothofagi

BRIP 54801*

Nothofagus cunninghamii

JX862530

KF170922

JX862536

D. novem

CBS 127270*

Glycine max

KC343155

KC344123

KC343881

KC343397

D. oxe

CBS 133186*

Maytenus ilicifolia

KC343164

KC344132

KC343890

KC343406

D. paranensis

CBS 133184*

Maytenus ilicifolia

KC343171

KC344139

KC343897

KC343413

D. pascoei

BRIP 54847*

Persea americana

JX862532

KF170924

JX862538

D. perjuncta

CBS 109745*

Ulmus glabra

KC343172

KC344140

KC343898

KC343414

D. pseudomangiferae

CBS 101339*

Mangifera indica

KC343181

KC344149

KC343907

KC343423

D. pseudophoenicicola

CBS 462.69*

Mangifera indica

KC343183

KC344151

KC343909

KC343425

D. psoraleae

CBS 136412*

Psoralea pinnata

KF777158

KF777251

KF777245

D. psoraleae-pinnatae

CBS 136413

Psoralea pinnata

KF777159

KF777252

D. pterocarpi

MFLUCC 10-0571*

JQ619899

JX275460

JX275416

JX197451

D. pterocarpicola

MFLUCC 10-0580*

JQ619887

JX275441

JX275403

JX197433

D. pulla

CBS 338.89*

Hedera helix

KC343152

KC344120

KC343878

KC343394

D. raonikayaporum

CBS 133182*

Spondias mombin

KC343188

KC344156

KC343914

KC343430

D. rudis

CBS 109291*

Laburnum anagyroides

KC843331

KC843177

KC843090

KC843146

D. rudis

CBS 113201*

Vitis vinifera

KC343234

KC344202

KC343960

KC343476

D. saccarata

CBS 116311*

Protea repens

KC343190

KC344158

KC343916

KC343432

D. salicicola

BRIP 54825*

Salix purpurea

JX862531

JX862531

JX862537

D. schini

CBS 133181*

Schinus terebinthifolius

KC343191

KC344159

KC343917

KC343433

D. sclerotioides

CBS 296.67*

Cucumis sativus

KC343193

KC344161

KC343919

KC343435

D. siamensis

MFLUCC 10-0573a*

JQ619879

JX275429

JX275393

D. terebinthifolii

CBS 133180*

Schinus terebinthifolius

KC343216

KC344184

KC343942

KC343458

D. thunbergii

MFLUCC 10-0576*

JQ619893

JX275449

JX275409

JX197440

D. toxica

CBS 534.93*

Lupinus angustifolius

KC343220

KC344188

KC343946

KC343462

Diaporthella corylina

CBS 121124*

Corylus sp.

KC343004

KC343972

KC343730

KC343246

P. lithocarpus

CGMCC 3.15175*

KC153104

KC153095

P. mahothocarpus

CGMCC 3.15181*

KC153096

KC153087

P. ternstroemia

CGMCC 3.15183*

KC153098

KC153089

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 10

Diplodia. Details of the isolates used in the phylogenetic tree

Species

Isolate no.

Host

GenBank

ITS

TEF

β-tubulin

Diplodia africana

CBS 120835*

Prunus persica

EF445343

EF445382

D. agrifolia

CBS 132777*

Quercus agrifolia

JN693507

JQ517317

JQ411459

D. alatafructa

CBS 124931*

Pterocarpus angolensis

FJ888460

FJ888444

D. allocellula

CBS 130408*

Acacia karroo

JQ239397

JQ239384

JQ239378

D. bulgarica

CBS 124254*

Malus sylvestris

GQ923853

GQ923821

D. corticola

CBS 112549*

Quercus suber

AY259100

AY573227

DQ458853

D. cupressi

CBS 168.87*

Cupressus sempervirens

DQ458893

DQ458878

DQ458861

D. fraxini

CBS 136010*

Fraxinus angustifolia

KF307700

KF318747

D. intermedia

CBS 124462*

Malus sylvestris

GQ923858

GQ923826

D. malorum

CBS 124130*

Malus sylvestris

GQ923865

GQ923833

D. mutila

CBS 112553*

Vitis vinifera

AY259093

AY573219

DQ458850

D. olivarum

CBS 121887*

Olea europaea

EU392302

EU392279

HQ660079

D. sapinea

CBS 393.84*

Pinus nigra

DQ458895

DQ458880

D. pseudoseriata

CBS 124906*

Blepharocalyx salicifolius

EU080927

EU863181

D. quercivora

CBS 133852*

Quercus canariensis

JX894205

JX894229

D. rosulata

CBS 116470*

Prunus africana

EU430265

EU430267

EU673132

D. scrobiculata

CBS 109944*

Pinus greggii

DQ458899

DQ458884

AY624258

D. seriata

CBS 112555*

Vitis vinifera

AY259093

AY573219

DQ458856

D. subglobosa

CBS 124133*

Lonicera nigra

GQ923856

GQ923824

D. tsugae

CBS 418.64*

Tsuga heterophylla

DQ458888

DQ458873

DQ458855

Lasiodiplodia theobromae

CBS 164.96*

Fruit along coral reef coast

AY640255

AY640258

EU673110

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 11

Dothiorella. Details of the isolates used in the phylogenetic tree

Species name1

Strain no.2

Host

ITS

TEF

Dothiorella americana

CBS 128309*

Vitis sp.

HQ288218

HQ288262

D. americana

CBS 128310

Vitis sp.

HQ288219

HQ288263

D. brevicollis

CBS 130411*

Acacia karroo

JQ239403

JQ239390

D. brevicollis

CBS 130412

Acacia karroo

JQ239404

JQ239391

D. casuarinae

CBS 120688*

Casuarina sp.

DQ846773

DQ875331

D. casuarinae

CBS 120690

Casuarina sp.

DQ846774

DQ875333

D. dulcispinae

CBS 130413*

Acacia karroo

JQ239400

JQ239387

D. dulcispinae

CBS 130414

Acacia karroo

JQ239401

JQ239388

D. dulcispinae

CBS 130415

Acacia karroo

JQ239402

JQ239389

D. iberica

CBS 115041*

Quercus ilex

AY573202

AY573222

D. iberica

CBS 113188

Quercus ilex

AY573198

EU673278

D. iberica

CAA 005

Quercus ilex

EU673312

EU673279

D. iranica

IRAN1587C*

Olea europaea

KC898231

KC898214

D. longicollis

CBS 122068*

Lysiphyllum cunninghamii

EU144054

EU144069

D. longicollis

CBS 122067

Lysiphyllum cunninghamii

EU144052

EU144067

D. moneti

MUCC 505*

Acacia rostellifera

EF591920

EF591971

D. moneti

MUCC 507

Acacia rostellifera

EF591922

EF591973

D. parva

IRAN1579C*

Corylus avellana

KC898234

KC898217

D. parva

IRAN1585C

Corylus avellana

KC898235

KC898218

D. pretoriensis

CBS 130404*

Acacia karroo

JQ239405

JQ239392

D. pretoriensis

CBS 130403

Acacia karroo

JQ239406

JQ239393

D. prunicola

IRAN1541*

Prunus dulcis

EU673313

EU673280

D. Santali

MUCC 509*

Santalum acuminatum

EF591924

EF591975

D. santali

MUCC 508

Santalum acuminatum

EF591923

EF591974

D. sarmentorum

IMI 63581b*

Ulmus sp.

AY573212

AY573235

D. sarmentorum

CBS 115038

Malus pumila

AY573206

AY573223

D. sempervirentis

IRAN1581C

Cupressus sempervirens

KC898237

KC898220

D. sempervirentis

IRAN1583C*

Cupressus sempervirens

KC898236

KC898219

D. striata

ICMP16819

Citrus sinensis

EU673320

EU673287

D. striata*

ICMP16824*

Citrus sinensis

EU673321

EU673288

D. thailandica

MFLUCC 11-0438*

Unknown

JX646796

JX646861

D. thripsita

BRIP 51876*

Acacia harpophylla

FJ824738

 

D. uruguayensis

CBS 124908*

Hexalamis edulis

EU080923

EU863180

D. vidmadera

DAR78992*

Vitis vinifera

EU768874

EU768881

D. vidmadera

DAR78993

Vitis vinifera

EU768876

EU768882

D. vidmadera

DAR78994

Vitis vinifera

EU768877

EU768883

Spencermartinsia viticola

CBS 117009*

Vitis vinifera

AY905554

AY905559

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 12

Fusarium. Details of the isolates used in the phylogenetic tree

Species

Isolate

GenBank accession numbers

RPB1

RPB2

Fusarium falciforme

NRRL 43529

JX171541

JX171653

F. solani

NRRL 45880

JX171543

JX171655

Fusarium sp.

NRRL 22436

JX171497

JX171610

F. ambrosium

NRRL 20438

JX171470

JX171584

F. phaseoli

NRRL 22276

JX171495

JX171608

F. virguliforme

NRRL 31041

JX171530

JX171643

Fusarium sp.

NRRL 22632

JX171501

JX171614

Fusarium sp.

NRRL 13444

JX171454

JX171568

Fusarium sp.

NRRL 28578

JX171526

JX171639

Fusarium sp.

NRRL 13338

JX171447

JX171561

F. aywerte

NRRL 25410

JX171513

JX171626

F. longipes

NRRL 13368

JX171448

JX171562

F. longipes

NRRL 13374

JX171450

JX171564

F. longipes

NRRL 20723

JX171483

JX171596

Fusarium cf. compactum

NRRL 13829

JX171460

JX171574

Fusarium sp.

NRRL 31008

JX171529

JX171642

F. sambucinum

NRRL 22187

JX171493

JX171606

F. venenatum

NRRL 22196

JX171494

JX171607

F. poae

NRRL 13714

JX171458

JX171572

F. sporotrichioides

NRRL 3299

JX171444

JX171558

F. langsethiae

NRRL 54940

JX171550

JX171662

F. armeniacum

NRRL 6227

JX171446

JX171560

F. asiaticum

NRRL 13818

JX171459

JX171573

F. graminearum

NRRL 31084

JX171531

JX171644

F. culmorum

NRRL 25475

JX171515

JX171628

F. pseudograminearum

NRRL 28062

JX171524

JX171637

F. equiseti

NRRL 13402

JX171452

JX171566

F. lacertarum

NRRL 20423

JX171567

JX171581

F. equiseti

NRRL 20697

JX171481

JX171595

Fusarium sp.

NRRL 26417

JX171522

JX171635

Fusarium sp.

NRRL 32175

JX171532

JX171645

F. subglutinans

NRRL 22016

JX171486

JX171599

F. circinatum

NRRL 25331

JX171510

JX171623

F. guttiforme

NRRL 22945

JX171505

JX171618

F. fujikuroi

NRRL 13566

JX171456

JX171570

F. proliferatum

NRRL 22944

JX171504

JX171617

F. mangiferae

NRRL 25226

JX171509

JX171622

F. sacchari

NRRL 13999

JX171466

JX171580

F. verticillioides

NRRL 20956

JX171485

JX171598

F. thapsinum

NRRL 22045

JX171487

JX171600

F. xylarioides

NRRL 25486

JX171517

JX171630

Fusarium sp.

NRRL 52700

JX171544

JX171656

F. nisikadoi

NRRL 25179

JX171507

JX171620

F. miscanthi

NRRL 26231

JX171521

JX171634

F. gaditjirrii

NRRL 45417

JX171542

JX171654

F. lyarnte

NRRL 54252

JX171549

JX171661

F. commune

NRRL 28387

JX171525

JX171638

F. inflexum

NRRL 20433

JX171469

JX171583

F. oxysporum

NRRL 25387

JX171512

JX171625

F. oxysporum

NRRL 34936

JX171533

JX171646

F. foetens

NRRL 38302

JX171540

JX171652

Fusarium sp.

NRRL 25184

JX171508

JX171621

F. redolens

NRRL 22901

JX171503

JX171616

F. hostae

NRRL 29889

JX171527

JX171640

Fusarium sp.

RBG 5116

KJ716216

HQ646395

F. burgessii

RBG 5319

KJ716217

HQ646392

F. beomiforme

NRRL 25174

JX171506

JX171619

F. concolor

NRRL 13459

JX171455

JX171569

F. anguioides

NRRL 25385

JX171511

JX171624

Fusarium sp.

NRRL 25533

JX171518

JX171631

F. babinda

NRRL 25539

JX171519

JX171632

Fusarium sp.

NRRL 22566

JX171500

JX171613

F. torulosum

NRRL 22748

JX171502

JX171615

F. flocciferum

NRRL 25473

JX171514

JX171627

F. tricinctum

NRRL 25481

JX171516

JX171629

F. nurragi

NRRL 36452

JX171538

JX171650

F. heterosporum

NRRL 20693

JX171480

JX171594

F. buharicum

NRRL 13371

JX171449

JX171563

F. sublunatum

NRRL 13384

JX171451

JX171565

F. lateritium

NRRL 13622

JX171457

JX171571

F. sarcochroum

NRRL 20472

JX171472

JX171586

F. stilbioides

NRRL 20429

JX171468

JX171582

Fusarium sp.

NRRL 54149

JX171548

JX171660

F. dimerum

NRRL 20691

JX171478

JX171592

F. lunatum

NRRL 36168

JX171536

JX171648

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 13

Gilbertella. Details of the isolates used in the phylogenetic tree

Species

Isolate

Host

GenBank no

Gilbertella persicaria

CBS 190.32*

Prunus persica

HM999958

G. persicaria

CBS 785.97

JN206218

G. persicaria

CBS 442.64

JN206219

G. persicaria

CBS 325.71A

Saccharum officinarum

JN206220

G. persicaria

CBS 403.51

JN206221

G. persicaria

CBS 246.59

Trickling filter plant system

JN206222

G. persicaria

CBS 421.77

Soil

JN206223

G. persicaria

CBS 532.77

Dung of mouse

JN206224

G. persicaria

CBS 325.71D

Wood

JN206225

G. persicaria

CBS 565.91

Dung of swine

JN206226

Choanephora cucurbitarum

CBS 120.25

JN206231

C. cucurbitarum

CBS 150.51

JN206232

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 14

Lasiodiplodia. Details of the isolates used in the phylogenetic tree

Species name1

Strain no.2

Host

ITS

TEF

Diplodia mutila

CBS 112553*

Vitis vinifera

AY259093

AY573219

Lasiodiplodia brasiliense

CMM 4015*

Mangifera indica

JX464063

JX464049

L. brasiliense

CMM 2320

Mangifera indica

KC484814

KC481544

L. brasiliense

CMM 2319

Mangifera indica

KC484798

KC481529

L. brasiliense

CMM 2314

Mangifera indica

KC484813

KC481543

L. citricola

CBS 124707*

Citrus sp.

GU945354

GU945340

L. citricola

CBS 124706

Citrus sp.

GU945353

GU945339

L. crassispora

CBS 118741*

Santalum album

DQ103550

EU673303

L. crassispora

WAC 12534

Eucalyptus urophylla

DQ103551

DQ103558

L. egyptiacae

CBS 130992*

Mangifera indica

JN814397

JN814424

L. egyptiacae

BOT 29

Mangifera indica

JN814401

JN814428

L. euphorbicola

CMM3609*

Jatropha curcas

KF234543

KF226689

L. euphorbicola

CMM3652

Jatropha curcas

KF234554

KF226715

L. gilanensis

CBS 124704*

Unknown

GU945351

GU945342

L. gilanensis

CBS 124705

Unknown

GU945352

GU945341

L. gonubiensis

CBS 115812*

Syzigium cordatum

AY639595

DQ103566

L. gonubiensis

CBS 116355

Syzigium cordatum

AY639594

DQ103567

L. hormozganensis

CBS 124709*

Olea sp.

GU945355

GU945343

L. hormozganensis

CBS 124708

Mangifera indica

GU945356

GU945344

L. iraniensis

CBS 124710*

Salvadora persica

GU945346

GU945334

L. jatrophicola

CMM3610

Jatropha curcas

KF234544

KF226690

L. lignicola

MFLUCC 11-0435*

Unknown

JX646797

JX646862

L. lignicola

MFLUCC 11-0656

Unknown

JX646798

JX646863

L. macrospora

CMM3833*

Jatropha curcas

KF234557

KF226718

L. mahajangana

CBS 124927*

Terminalia catappa

FJ900597

FJ900643

L. mahajangana

CBS 124925

Terminalia catappa

FJ900595

FJ900641

L. margaritacea

CBS 122519*

Adansonia gibbosa

EU144050

EU144065

L. margaritacea

CBS 122065

Adansonia gibbosa

EU144051

EU144066

L. marypalme

CMM 2275*

Carica papaya

KC484843

KC481567

L. marypalme

CMM 2274

Carica papaya

KC484841

KC481565

L. marypalme

CMM 2272

Carica papaya

KC484842

KC481566

L. marypalme

CMM 2271

Carica papaya

KC484844

KC481568

L. missouriana

CBS 128311*

Vitis vinifera

HQ288225

HQ288267

L. missouriana

CBS 128312

Vitis vinifera

HQ288226

HQ288268

L. parva

CBS 456.78*

Cassava-field soil

EF622083

EF622063

L. parva

CBS 494.78

Cassava-field soil

EF622084

EF622064

L. plurivora

CBS 120832*

Prunus salicina

EF445362

EF445395

L. plurivora

CBS 121103

Prunus salicina

AY343482

EF445396

L. pseudotheobromae

CBS 116459*

Gmelina arborea

EF622077

EF622057

L. pseudotheobromae

CBS 447.62

Citrus aurantium

EF622081

EF622060

L. rubropurpurea

CBS 118740*

Eucalyptus grandis

DQ103553

EU673304

L. rubropurpurea

WAC 12536

Eucalyptus grandis

DQ103554

DQ103572

L. subglobose

CMM3872*

Jatropha curcas

KF234558

KF226721

L. subglobosa

CMM4046

Jatropha curcas

KF234560

KF226723

L. theobromae

CBS 164.96*

Fruit on coral reef coast

AY640255

AY640258

L. theobromae

CBS 111530

Unknown

EF622074

EF622054

L. venezuelensis

CBS 118739*

Acacia mangium

DQ103547

EU673305

L. venezuelensis

WAC 12540

Acacia mangium

DQ103548

DQ103569

L. viticola

CBS 128313*

Vitis vinifera

HQ288227

HQ288269

L. viticola

CBS 128315

Vitis vinifera

HQ288228

HQ288270

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 15

Mucor. Details of the isolates used in the phylogenetic tree

Species

Isolate

Country of collection

GenBank accession no

Mucor abundans

CBS 521.66

Germany

JN206457

M. aligarensis

CBS 993.70*

UK

JN206461

M. amphibiorum

CBS 763.74*

Germany

HM849688

M. ardhlaengiktus

CBS 210.80*

India

JN206504

M.azygosporus

CBS 292.63*

USA

JN206497

M. bacilliformis

CBS 251.53*

USA

JN206451

M. bainieri

CBS 293.63*

India

JN206424

M. brunneogriseus1

CBS 129.41

Netherlands

 

M. circinelloides f. circinelloides

CBS 195.68*

Netherlands

HM849680

M. circinelloides f. griseocyanus

CBS 116.08

Norway

JN206421

M. circinelloides f. janssenii

CBS 205.68*

South Africa

JN206425

M.circinelloides f. lusitanicus

CBS 968.68

JN206419

M. ctenidius

CBS 293.66

USA

JN206417

M. durus

CBS 156.51*

Ukraine

JN206456

M. endophyticus

CBS 385.95*

China

JN206448

M. exponens

CBS 141.20*

Germany

JN206441

M. falcatus

CBS 251.35*

Germany

JN206509

M. flavus

CBS 234.35*

Germany

JN206468

M. fuscus

CBS 282.78

France

JN206442

M.fusiformis

CBS 336.68*

Finland

JN206447

M. genevensis

CBS 114.08*

Switzerland

JN206435

M.gigasporus

CBS 566.91*

China

JN206494

M. guiliermondii

CBS 174.27*

Russia

JN206475

M.heterogamus

CBS 405.58*

JN206487

M. hiemalis f. corticola

CBS 362.68

Norway

JN206449

M. hiemalis f. hiemalis

CBS 201.65*

USA

HM849683

M. inaequisporus

CBS 255.36*

Ghana

JN206502

M. indicus

CBS 226.29*

Switzerland

HM849690

M.irregularis

CBS 103.93

India

HM849684

M. japonicus

CBS 154.69*

Russia

JN206446

M. lanceolatus

CBS 638.74

France

JN206443

M. laxorrhizus

CBS 143.85*

United Kingdom

JN206444

M. luteus1

CBS 243.35*

Germany

 

M. megalocarpus

CBS 215.27*

France

JN206489

M. microsporus1

CBS 204.28

France

 

M. minutes

CBS 586.67

India

JN206463

M. moelleri

CBS 444.65*

USA

HM849682

M. mousanensis

CBS 999.70*

India

JN206434

M. mucedo

CBS 640.67*

Netherlands

HM849687

M. multiplex

CBS 110662*

China

JN206484

M. nederlandicus

CBS 735.70

JN206503

M. odoratus

CBS 130.41*

Denmark

JN206495

M. parviseptatus

CBS 417.77

Australia

JN206453

M. piriformis

CBS 169.25*

HM849681

M. plasmaticus

CBS 275.49

Netherlands

JN206483

M. plumbeus

CBS 634.74

Germany

HM849677

M. prayagensis

CBS 652.78

India

JN206498

M. racemosus f. racemosus

CBS 260.68*

Switzerland

HM849676

M. racemosus f. sphaerosporus

CBS 115.08*

Norway

JN206433

M. ramosissimus

CBS 135.65*

Uruguay

HM849678

M. saturninus

CBS 974.68*

Netherlands

JN206458

M. silvaticus

CBS 249.35*

Denmark

JN206455

M. strictus

CBS 100.66

Austria

JN206477

M. ucrainicus

CBS 674.88

Ukraine

JN206507

M. variisporus

CBS 837.70*

India

JN206508

M. zonatus

CBS 148.69*

Germany

JN206454

M. zychae

CBS 416.67*

India

JN206505

Backusella lamprospora

CBS 195.28

USA

JN206530

B. grandis

CBS 186.87*

India

JN206527

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 16

Neofusicoccum. Details of the isolates used in the phylogenetic tree

Species

Isolate

GenBank accession numbers

SSU

ITS

LSU

TEF

β-tubulin

Neofusicoccum andinum

CBS 117453*

N/A

AY693976

N/A

AY693977

N/A

N. arbuti

CBS 116131*

KF531814

AY819720

DQ377915

KF531792

KF531792

N. australe

CMW 6837*

N/A

AY339262

N/A

AY339270

AY339254

N. batangarum

CBS 124924*

N/A

FJ900607

N/A

FJ900653

FJ900634

N. cordaticola

CBS 123634*

N/A

EU821898

N/A

EU821868

EU821838

N. corticosae

CBS 120081*

N/A

DQ923533

N/A

N/A

N/A

N. eucalypticola

CBS 115679*

N/A

AY615141

N/A

AY615133

AY615125

N. grevilleae

CBS 129518*

N/A

JF951137

JF951157

N/A

N/A

N. kwambonambiense

CBS 123639*

N/A

EU821900

N/A

EU821870

EU821840

N. luteum

CBS 110299*

EU673148

AY259091

AY928043

AY573217

DQ458848

N. macroclavatum

CBS 118223*

N/A

DQ093196

N/A

DQ093217

DQ093206

N. mangiferae

CBS 118532*

EU673154

AY615186

DQ377921

DQ093220

AY615173

N. mediterraneum

CBS 121718*

N/A

GU251176

N/A

GU251308

GU251836

N. nonquaesitum

CBS 126655*

N/A

GU251163

N/A

GU251295

GU251823

N. occulatum

CBS 128008*

N/A

EU301030

N/A

EU339509

EU339472

N. parvum

CMW 9081*

EU673151

AY236943

AY928045

AY236888

AY236917

N. pennatisporum

WAC 13153*

N/A

EF591925

EF591942

EF591976

EF591959

N. protearum

CBS 114176*

N/A

AF452539

N/A

N/A

N/A

N. ribis

CBS 115475*

N/A

AY236935

N/A

AY236877

AY236906

N. umdonicola

CBS 123645*

N/A

EU821904

N/A

EU821874

EU821844

N. viticlavatum

CBS 112878*

N/A

AY343381

N/A

AY343342

N/A

N. vitifusiforme

CBS 110887*

N/A

AY343383

N/A

AY343343

N/A

Spencermartinsia viticola

CBS 117009*

EU673165

AY905554

DQ377873

AY905559

EU673104

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 17

Pestalotiopsis. Details of the isolates used in the phylogenetic tree

Species

Isolates

Host

GenBank accession number

ITS

β -tubulin

TEF

Pestalotiopsis adusta

ICMP6088*

On refrigerator door PVC gasket

JX399006

JX399037

JX399070

P. adusta

MFLUCC10-146

Syzygium sp.

JX399007

JX399038

JX399071

P. anacardiacearum

IFRDCC2397*

Mangifera indica

KC247154

KC247155

KC247156

P. asiatica

MFLUCC12-0286*

Unidentified tree

JX398983

JX399018

JX399049

P. camelliae

MFLUCC12-0277*

Camellia japonica

JX399010

JX399041

JX399074

P. camelliae

MFLUCC12-0278

Camellia japonica

JX399011

JX399042

JX399075

P. chrysea

MFLUCC12-0261*

Dead plant

JX398985

JX399020

JX399051

P. chrysea

MFLUCC12-0262

Dead plant

JX398986

JX399021

JX399052

P. clavata

MFLUCC12-0268*

Buxus sp.

JX398990

JX399025

JX399056

P. clavispora

MFLUCC12-0280

Magnolia sp.

JX398978

JX399013

JX399044

P. clavispora

MFLUCC12-0281*

Magnolia sp.

JX398979

JX399014

JX399045

P. coffeae–arabicae

HGUP4015*

Coffeae arabica

KF412647

KF412641

KF412644

P. coffeae–arabicae

HGUP4019

Coffeae arabica

KF412649

KF412643

KF412646

P. diversiseta

MFLUCC12-0287*

Rhododendron sp.

JX399009

JX399040

JX399073

P. ellipsospora

MFLUCC12-0283*

Dead plant

JX398980

JX399016

JX399047

P. ellipsospora

MFLUCC12-0284

Dead plant

JX398981

JX399015

JX399046

P. ericacearum

IFRDCC2439*

Rhododendron delavayi

KC537807

KC537821

KC537814

P. foedans

CGMCC3.9178

Neodypsis decaryi

JX398989

JX399024

JX399055

P. foedans

CGMCC3.9123*

Mangrove leaves

JX398987

JX399022

JX399053

P. foedans

CGMCC3.9202

Calliandra haematocephala

JX398988

JX399023

JX399054

P. furcata

MFLUCC12-0054*

Camellia sinensis

JQ683724

JQ683708

JQ683740

P. gaultheria

IFRD411-014*

Gaultheria forrestii

KC537805

KC537819

KC537812

P. inflexa

MFLUCC12-0270*

Unidentified tree

JX399008

JX399039

JX399072

P. intermedia

MFLUCC12-0259*

Unidentified tree

JX398993

JX399028

JX399059

P. licualacola*

HGUP4057*

Licuala grandis

KC436006

KC481683

KC481684

P. linearis

MFLUCC12-0271*

Trachelospermum sp.

JX398992

JX399027

JX399058

P. magna

MFLUCC12-652*

Pteridium sp.

KF582795

KF582793

KF582791

P. rhododendri

IFRDCC2399*

Rhododendron sinogrande

KC537804

KC537818

KC537811

P. rhodomyrtus

HGUP4230*

Rhodomyrtus tomentosa

KF412648

KF412642

KF412645

P. rosea

MFLUCC12-0258*

Pinus sp.

JX399005

JX399036

JX399069

P. samarangensis

MFLUCC12-0233*

Syzygium samarangense

JQ968609

JQ968610

JQ968611

P. saprophyta

MFLUCC12-0282*

Litsea rotundifolia

JX398982

JX399017

JX399048

P. simitheae

MFLUCC12-0121*

Pandanus odoratissimus

KJ503812

KJ503815

KJ503818

P. simitheae

MFLUCC12-0125

Pandanus odoratissimus

KJ503813

KJ503816

KJ503819

P. shorea

MFLUCC12-0314*

Shorea obtuse

KJ503811

KJ503814

KJ503817

P. steyaertii

IMI192475*

Eucalyptus viminalis

KF582796

KF582794

KF582792

P. theae

MFLUCC12-0055*

Camellia sinensis

JQ683727

JQ683711

JQ683743

P. theae

SC011

Camellia sinensis

JQ683726

JQ683710

JQ683742

P. trachicarpicola

MFLUCC12-0263

Unidentified tree

JX399000

JX399031

JX399064

P. trachicarpicola

MFLUCC12-0264

Chrysophyllum sp.

JX399004

JX399035

JX399068

P. trachicarpicola

MFLUCC12-0265

Schima sp.

JX399003

JX399034

JX399067

P. trachicarpicola

MFLUCC12-0266

Sympolocos sp.

JX399002

JX399033

JX399066

P. trachicarpicola

MFLUCC12-0267

Unidentified tree

JX399001

JX399032

JX399065

P. trachicarpicola

IFRDCC2403

Podocarpus macrophyllus

KC537809

KC537823

KC537816

P. trachicarpicola

OP068*

Trachycarpus fortunei

JQ845947

JQ845945

JQ845946

P. umberspora

MFLUCC12-0285*

Unidentified tree

JX398984

JX399019

JX399050

P. unicolor

MFLUCC12-0275

Unidentified tree

JX398998

JX399029

JX399063

P. unicolor

MFLUCC12-0276*

Rhododendron sp.

JX398999

JX399030

P. verruculosa

MFLUCC12-0274*

Rhododendron sp.

JX398996

JX399061

Seiridium sp.

SD096

JQ683725

JQ683709

JQ683741

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 18

Phyllosticta. Details of the voucher and extype isolates used in the phylogenetic tree

Species

Strain no.1

Host

Locality

GenBank accession number2

ITS

ACT

TEF

GPDH

Botryosphaeria obtusa

CMW8232

Conifers

South Africa

AY972105

AY972111

DQ280419

 

Guignardia alliacea

MUCC0014*

Allium fistulosum

Japan

AB454263

   

G. bidwellii

NBRC9757

Parthenocissus tricuspidata

Japan

AB095510

   

G. gaultheriae

CBS447.70*

Gaultheria humifusa

USA

JN692543

JN692519

JN692531

JN692508

G. mangiferae

IMI260.576*

Manifera indica

India

JF261459

JF343641

JF261501

JF343748

G. philoprina

CBS447.68*

Taxus baccata

Netherlands

AF312014

   

G. vaccinii

CBS126.22*

Oxycoccus macrocarpos

USA

FJ538353

   

Phyllosticta abieticola

CBS112067*

Abies concolor

Canada

KF170306

KF289238

  

P. aloeicola

CPC21020*

Aloe ferox

South Africa

KF154280

KF289311

KF289193

KF289124

P. ampelicida

ATCC200578*

Vitis riparia

USA

KC193586

KC193581

 

KC193584

P. ardisiicola

NBRC102261*

Ardisia crenata

Japan

AB454274

   

P. aspidistricola

NBRC102244*

Aspidistra elatior

Japan

AB454260

   

P. beaumarisii

CBS535.87 = IMI 298910 *

Muehlenbekia adpressa

Australia

AY042927

KF306232

KF289170

KF289074

P. bifrenariae

CBS128855*

Bifrenaria harrisoniae

Brazil

JF343565

JF343649

JF343586

JF343744

P. brazilianiae

CBS126270*

Mangifera indica

Brazil

JF343572

JF343656

JF343593

JF343758

P. capitalensis

CBS128856*

Stanhopea sp.

Brazil

JF261465

JF343647

JF261507

JF343776

 

CPC16592

Citrus limon

Argentina

KF206187

KF289273

KF289178

KF289092

P. cavendishii

BRIP554196*

Musa cv. Formosana

Taiwan

JQ743562

   

P. citriasiana

CBS 120486*

Citrus maxima

Thailand

FJ538360

FJ538476

FJ538418

JF343686

P. citribraziliensis

CBS100098*

Citrus limon

Brazil

FJ538352

FJ538468

FJ538410

JF343691

P. citricarpa

CBS127454*

Citrus limon

Australia

JF343583

JF343667

JF343604

JF343771

 

CPC16603

Citrus limon

Uruguay

KF170295

KF289274

KF289213

KF289147

P. citrichinaensis

ZJUCC200956*

Citrus reticulata

China

JN791620

JN791533

JN791459

 

P. citrimaxima

CBS136059*

Citrus maxima

Thailand

KF170304

KF289300

KF289222

KF289157

P. concentrica

CBS 937.7*

Hedera helix

Italy

FJ538350

KF289257

FJ538408

JF411745

P. cordylinophila

CPC20261*

Cordyline fruticosa

Thailand

KF170287

KF289295

KF289172

KF289076

P. cornicola

CBS111639

Cornus florida

USA

KF170307

KF289234

  

P. cussoniae

CBS136060*

Cussonia sp.

South Africa

JF343578

JF343662

JF343599

JF343764

P. ericarum

CBS132534*

Erica gracilis

South Africa

KF206170

KF289291

KF289227

KF289162

P. fallopiae

NBRC102266*

Fallopia japonica

Japan

AB454307

   

P. foliorum

CBS 447.68

Taxus baccata

Netherlands

KF170309

KF289247

KF289201

KF289132

P. hamamelidis

MUCC149

Hamamelis japonica

Japan

KF170289

KF289309

  

P. hostae

CGMCC3.14355*

Hosta plantaginea

China

JN692535

JN692511

JN692523

JN692503

P. hubeiensis

CGMCC3.14986*

Viburnum odoratissimim

China

JX025037

JX025032

JX025042

JX025027

P. hymenocallidicola

CBS 131309*

Hymenocallis littoralis

Australia

JQ044423

KF289242

KF289211

KF289142

P. hypoglossi

CBS 434.92*

Ruscus aculeatus

Italy

FJ538367

FJ538483

FJ538425

JF343695

P. ilicis-aquifolii

CGMCC3.14358*

Ilex aquifolium

China

JN692538

JN692514

JN692526

 

P. kerriae

NBRC102251*

Kerria japonica

Japan

AB454266

   

P. leucothoicola

CBS136073*

Leucothoe catesbaei

Japan

AB454370

KF289310

  

P. ligustricola

MUCC0024*

Ligustrum obtusifolium

Japan

AB454269

AB704212

  

P. maculate

CPC18347*

Musa cv. Goly-goly pot-pot

Australia

JQ743570

   

P. mangifera-indica

CPC20264*

Mangifera indica

Thailand

KF170305

KF289296

KF289190

KF289121

P. minima

CBS 585.84*

Acer rubrum

USA

KF206176

KF289249

KF289204

KF289135

P. musarum

BRIP55434*

Hill banana

India

JQ743584

   

P. musicola

CBS123405*

Musa acuminata

Thailand

FJ538334

FJ538450

FJ538392

 

P. neopyrolae

CPC21879*

Pyrola asarifolia

Japan

AB454318

AB704233

  

P. owaniana

CBS776.97*

Brabejum stellatifolium

South Africa

FJ538368

KF289254

FJ538426

JF343767

P. pachysandricola

MUCC0124*

Pachysandra terminalis

Japan

AB454317

AB704232

  

P. parthenocissi

CBS111645*

Parthenocissus quinquefolia

USA

EU683672

JN692518

JN692530

 

P. paxistimae

CBS112527*

Paxistima mysinites

USA

KF206172

KF289239

KF289209

KF289140

P. philoprina

CBS616.72

Ilex aquifolium

Germany

KF154279

KF289251

KF289205

KF289136

P. podocarpi

CBS111647

Podocarpus lanceolata

South Africa

KF154276

KF289235

KF289232

KF289168

P. podocarpicola

CBS728.79*

Podocarpus maki

USA

KF206173

KF289252

KF289203

KF289134

P. pseudotsugae

CBS111649

Pseudotsuga menziesii

USA

KF154277

KF289236

KF289231

KF289167

P. rhaphiolepidis

MUCC0432*

Rhaphiolepis indica

Japan

AB454349

AB704242

  

P. schimae

CGMCC3.14354*

Schima superb

China

JN692534

JN692510

JN692522

JN692506

P. speewahensis

BRIP58044

Orchids

northern Australia

KF017269

 

KF017268

 

P. spinarum

CBS292.90

Chamaecyparis pisifera

France

JF343585

JF343669

JF343606

JF343773

P. styracicola

CGMCC3.14985*

Styrax grandiflorus

China

JX025040

JX025035

JX025045

JX025030

P. telopeae

CBS777.97*

Telopea speciosissima

Tasmania

KF206205

KF289255

KF289210

KF289141

P. vaccinii

ATCC46255*

Vaccinium macrocarpon

USA

KC193585

KC193580

KC193582

KC193583

P. vacciniicola

CPC18590*

Vaccinium macrocarpum

USA

KF170312

KF289287

KF289229

KF289165

P. yuccae

CBS117136

Yucca elephantipes

New Zealand

JN692541

JN692517

JN692529

JN692507

Ex-type strains are bolded and marked with an * and voucher stains are bolded

Table 19

Pythium. Strain numbers, host information and GenBank accession numbers for species included in Fig. 21

Species

Isolate

Host

GenBank accession numbers

SSU

ITS

LSU

cox2

β- tubulin

Lagena radicicola

DAOM BR89-12

Triticum aestivum

KJ716869

KJ716869

KJ716869

KJ595434

N/A

Lagenidium giganteum

CBS 580.84

Mosquito larva

KJ716868

KJ716868

KJ716868

KJ595392

KJ595516

Lagenidium myophilum

ATCC 6680

Pandalus borealis

AB284577

AB285498

AB285220

AF290311

N/A

Lagenidium sp. PWL-2010e

DAOM 242348

Soil nematode

KJ716871

KJ716871

KJ716871

KJ595438

KJ595561

Lagenidium sp. PWL-2010f

CBS 127284

Soil nematode

HQ343198

HQ111472

HQ395652

HQ605945

N/A

Lagenidium sp. PWL-2010h

CBS 127285

Soil nematode

HQ343197

HQ111470

HQ395651

HQ660435

N/A

Lagenidium sp. PWL-2010i

CBS 127283

Soil nematode

HQ343199

HQ111471

HQ395653

HQ680580

N/A

Lagenidium sp. SLG-2014a

DAOM 242886

Soil nematode

N/A

KJ716872

KJ716872

KJ595442

KJ595565

Lagenidium sp. SLG-2014a

LEV6103

Soil nematode

KJ716870

KJ716870

KJ716870

KJ595441

KJ595564

Lagenidium sp. SLG-2014b

LEV6562

Oedogonium sp.

KJ716873

KJ716873

N/A

KJ595443

KJ595566

Phytophthora capsici

P1319

Capsicum annuum

JN635215

FJ801727

EU079741

GU221958

EU079737

Phytophthora cinnamomi

P8495

Beaucamea sp.

JN635088

FJ802007

EU079953

GU221971

EU079949

Phytopythium sindhum

DAOM 238986

Soil (Musa sp.)

HQ643396

HQ643396

HQ643396

KJ595436

KJ595559

Pilasporangium apinafurcum

UZ300

Soil

N/A

AB458660

AB458651

AB458820

N/A

Pilasporangium apinafurcum

UZ301

Soil

N/A

AB458657

AB458652

AB458818

N/A

Pythiogeton zeae

ATCC MYA-862

Zea mays

N/A

HQ643405

HQ665310

N/A

N/A

Pythium abappressorium

CBS 110198

Triticum aestivum

HQ643408

HQ643408

HQ643408

KJ595409

KJ595533

P. acanthicum

CBS 377.34

Solanum tuberosum

AY598617

AY598617

AY598617

KJ595380

KJ595504

P. acanthophoron

CBS 337.29 (AUTH)

Ananas sativus

AY598711

AY598711

AY598711

KJ595376

KJ595500

P. acrogynum

CBS 549.88 (AUTH)

Soil (Spinacia oleracea)

N/A

AY598638

AY598638

AB362324

KJ595458

P. adhaerens

CBS 520.74

Soil

AY598619

AY598619

AY598619

KJ595386

KJ595510

P. afertile

LEV2066

Turf grass

N/A

HQ643416

HQ643416

KJ595440

KJ595563

P. amasculinum

CBS 552.88 (AUTH)

soil (vegetable garden)

AY598671

AY598671

AY598671

KJ595390

KJ595514

P. anandrum

CBS 285.31

Rheum rhaponticum

AY598650

AY598650

AY598650

AB362328

KJ595450

P. angustatum

CBS 522.74 (VdPN)

Soil

AY598623

AY598623

AY598623

KJ595387

KJ595511

P. aphanidermatum

CBS 118.80

Unknown

AY598622

AY598622

AY598622

KJ595344

KJ595472

P. apiculatum

CBS 120945

soil (Vitis sp.)

HQ643443

HQ643443

HQ643443

KJ595422

KJ595547

P. apleroticum

CBS 772.81

Nymphyoides peltata

AY598631

AY598631

AY598631

KJ595400

KJ595524

P. aquatile

CBS 215.80

Soil

AY598632

AY598632

AY598632

KJ595355

KJ595481

P. aristosporum

CBS 263.38

Triticum aestivum

AY598627

AY598627

AY598627

AB507410

DQ071297

P. arrhenomanes

CBS 324.62 (VdPN)

Zea mays

AKXY02050628

AY598628

AY598628

AKXY02053172

KJ595451

P. attrantheridium

DAOM 230386

Prunus serotina

HQ643476

HQ643476

HQ643476

AB512889

AB512822

P. boreale

CBS 551.88

Soil

AY598662

AY598662

AY598662

EF408876

EF408882

P. buismaniae

CBS 288.31

Linum usitatissimum

AY598659

AY598659

AY598659

KJ595368

KJ595493

P. camurandrum

CBS 124059

Hordeum vulgare

GQ244426

GQ244426

GQ244426

KJ595433

KJ595558

P. canariense

CBS 112353

Soil

HQ643482

HQ643482

HQ665069

JX397983

JX397969

P. capillosum

CBS 222.94

Soil

AY598635

AY598635

AY598635

KJ595360

KJ595485

P. carbonicum

CBS 112544

Soil (spoil heap)

HQ643373

HQ643373

HQ643373

AB690678

KJ595464

P. carolinianum

CBS 122659

soil

N/A

HQ643484

HQ665111

KJ595427

KJ595551

P. catenulatum

CBS 842.68 (VdPN)

Turf grass

AY598675

AY598675

AY598675

KJ595404

KJ595528

P. caudatum

CBS 584.85

Xiphinema rivesi

HQ643136

HQ643136

HQ665277

AF290309

KJ595459

P. cederbergense

CBS 133716

Aspalathus linearis

N/A

JQ412768

KJ716864

JQ412805

JQ412781

P. chamaehyphon

CBS 259.30 (AUTH)

Carica papaya

AY598666

AY598666

AY598666

AB257280

KJ595448

P. chondricola

CBS 203.85

Chondrus crispus

N/A

AY598620

AY598620

KJ595354

KJ595480

P. citrinum

CBS 119171

Soil (Vitis sp.)

HQ643375

HQ643375

HQ643375

AB690679

KJ595465

P. coloratum

CBS 154.64

Soil (tree nursery)

AY598633

AY598633

AY598633

KJ595346

KJ595474

P. conidiophorum

CBS 223.88

Soil

AY598629

AY598629

AY598629

KJ595361

KJ595486

P. contiguanum

CBS 221.94

Soil (salt marsh)

HQ643514

HQ643514

HQ665162

KJ595358

KJ595483

P. cryptoirregulare

CBS 118731

Euphorbia pulcherrima

HQ643515

HQ643515

HQ643515

GU071763

GU071888

P. cucurbitacearum

CBS 748.96

Unknown

AY598667

AY598667

AY598667

AB690680

KJ595460

P. cylindrosporum

CBS 218.94

Soil

AY598643

AY598643

AY598643

GU071762

GU071877

P. cystogenes

CBS 675.85

Vicia faba

HQ643518

HQ643518

HQ643518

KJ595396

KJ595520

P. debaryanum

CBS 752.96

Tulipa sp.

AY598704

AY598704

AY598704

KJ595399

KJ595523

P. delawarense

CBS 123040

Glycine max

KF853241

EU339312

KF853240

KJ595430

KJ595555

P. deliense

CBS 314.33

Nicotiana tabacum

AY598674

AY598674

AY598674

KJ595372

KJ595497

P. diclinum

CBS 664.79

Beta vulgaris

N/A

AY598690

HQ665282

KJ595394

KJ595518

P. dimorphum

CBS 406.72

Pinus taeda

AY598651

AY598651

AY598651

AB362331

KJ595454

P. dissimile

CBS 155.64

Pinus radiata

AY598681

AY598681

AY598681

KJ595347

KJ595475

P. dissotocum

CBS 166.68 (VdPN)

Triticum aestivum

AY598634

AY598634

AY598634

KJ595351

KJ595479

P. echinulatum

CBS 281.64 (VdPN)

Soil (forest nursery)

AY598639

AY598639

AY598639

AB362327

KJ595449

P. emineosum

CBS 124057

Juniperus communis

N/A

GQ244427

GQ244427

KJ595432

KJ595557

P. erinaceum

CBS 505.80

Soil

N/A

AY598694

HQ665243

AB362326

KJ595456

P. flevoense

CBS 234.72

Soil

AY598691

AY598691

AY598691

KJ595363

KJ595488

P. folliculosum

CBS 220.94

Soil

AY598676

AY598676

HQ665160

N/A

N/A

P. glomeratum

CBS 122644

Soil

N/A

HQ643542

HQ665097

KJ595424

KJ595548

P. graminicola

CBS 327.62

Saccharum officinarum

AY598625

AY598625

AY598625

AF196593

KJ595452

P. grandisporangium

CBS 286.79

Decaying leaf (Zostera marina)

AY598692

AY598692

AY598692

KJ595367

KJ595492

P. helicandrum

CBS 393.54 (AUTH)

Rumex acetosella

AY598653

AY598653

AY598653

AB362329

KJ595453

P. helicoides

CBS 286.31 (AUTH)

Phaseolus vulgaris

AY598665

AY598665

AY598665

DQ071377

AB511994

P. heterothallicum

CBS 450.67

Soil (Sambucus)

AY598654

AY598654

AY598654

AB512919

AB512850

P. hydnosporum

CBS 253.60 (VdPN)

Unknown

AY598672

AY598672

AY598672

KJ595364

KJ595489

P. hypogynum

CBS 234.94

Soil

AY598693

AY598693

AY598693

AB362325

KJ595447

P. inflatum

CBS 168.68 (VdPN)

Saccharum officinarum

AY598626

AY598626

AY598626

KJ595352

N/A

P. insidiosum

CBS 574.85

Equus ferus

AF289981

AY598637

AY598637

KJ595391

KJ595515

P. intermedium

CBS 266.38 (VdPN)

Agrostis stolonifera

AY598647

AY598647

AY598647

AB507410

AB512836

P. irregulare

CBS 250.28

Phaseolus vulgaris

AY598702

AY598702

AY598702

GU071760

GU071886

P. iwayamai

CBS 132417

Poa annua

AKYA02013211, AKYA02014361, AKYA02012602

AKYA02012602, AKYA02013659

AKYA02013659, AKYA02016578, AKYA02016542

AKYA02009930, AKYA02012077

AKYA02004337

P. iwayamai

CBS 156.64 (VdPN)

Soil (Pinus sp.)

AY598648

AY598648

AY598648

JX397979

JX397965

P. kashmirense

CBS 122908

Soil

HQ643671

HQ643671

HQ643671

KJ595429

KJ595553

P. kunmingense

CBS 550.88

Soil (Vicia faba)

AY598700

AY598700

HQ665259

KJ595389

KJ595513

P. litorale

CBS 118360

Soil (Phragmites australis)

HQ643386

HQ643386

HQ643386

KJ595418

KJ595543

P. longandrum

CBS 112355

Soil

HQ643679

HQ643679

HQ665071

KJ595413

KJ595538

P. longisporangium

CBS 122646

Soil (Vitis sp.)

N/A

HQ643680

HQ665099

KJ595426

KJ595550

P. lucens

CBS 113342

Triticum

HQ643681

HQ643681

HQ643681

KJ595415

KJ595540

P. lutarium

CBS 222.88

Soil

HQ643682

HQ643682

HQ665163

KJ595359

KJ595484

P. lycopersici

CBS 122909

Soil (Lycopersicum esculentum)

N/A

HQ643683

HQ665119

KJ595343

KJ595554

P. macrosporum

CBS 574.80

Flower bulb

AY598646

AY598646

AY598646

AB512916

AB512842

P. mamillatum

CBS 251.28 (VdPN)

Beta vulgaris

AY598703

AY598703

HQ665173

AB362325

AB512844

P. marinum

CBS 750.96

Soil

N/A

AY598689

AY598689

KJ595398

KJ595522

P. marsipium

CBS 773.81

Nymphyoides peltata

N/A

AY598699

HQ665297

KJ595401

KJ595525

P. mastophorum

CBS 375.72 (VdPN)

Apium graveolens

AY598661

AY598661

AY598661

KJ595378

KJ595502

P. megacarpum

CBS 112351

Soil (Vitis sp.)

HQ643388

HQ643388

HQ643388

AB690665

KJ595536

P. megalacanthum

CBS 101356

Chrysanthemum

N/A

HQ643693

KJ716865

KJ595435

N/A

P. mercuriale

CBS 122443

Macadamia integrifolia

KF853243

DQ916363

KF853236

AB690666

KJ595466

P. middletonii

CBS 528.74 (VdPN)

Soil

N/A

AY598640

AY598640

AB362318

KJ595457

P. minus

CBS 226.88

Soil

HQ643696

HQ643696

HQ665168

AB362320

KJ595446

P. monospermum

CBS 158.73 (VdPN)

Soil

HQ643697

HQ643697

HQ643697

KJ595350

KJ595478

P. montanum

CBS 111349

Soil (Picea abies)

HQ643389

HQ643389

HQ643389

KJ595410

KJ595534

P. multisporum

CBS 470.50

Soil

AY598641

AY598641

AY598641

AB362319

KJ595455

P. myriotylum

CBS 254.70

Arachis hypogaea

AY598678

AY598678

AY598678

KJ595365

KJ595490

P. nagaii

CBS 779.96

Soil

AY598705

AY598705

AY598705

KJ595402

KJ595526

P. nodosum

CBS 102274

Soil

N/A

HQ643709

HQ665055

KJ595407

KJ595531

P. nunn

CBS 808.96

Soil

AY598709

AY598709

AY598709

AF196609

DQ071325

P. oedochilum

CBS 292.37 (AUTH)

Unknown

AY598664

AY598664

AY598664

AB108011

EF408883

P. okanoganense

CBS 315.81

Triticum aestivum

AY598649

AY598649

AY598649

KJ595373

KJ595498

P. oligandrum

CBS 382.34 (VdPN)

Viola sp.

AY598618

AY598618

AY598618

KJ595381

KJ595505

P. oopapillum

CBS 124053

Cucumis sativus

N/A

FJ655174

FJ655174

KJ595431

KJ595556

P. ornacarpum

CBS 112350

Soil

HQ643721

HQ643721

HQ643721

KJ595411

KJ595535

P. ornamentatum

CBS 122665

Soil

N/A

HQ643722

HQ665117

KJ595428

KJ595552

P. orthogonon

CBS 376.72

Zea mays

AY598710

AY598710

HQ665221

KJ595379

KJ595503

P. ostracodes

CBS 768.73 (VdPN)

Soil

AY598663

AY598663

AY598663

AB690668

EF408880

P. pachycaule

CBS 227.88

Soil

AY598687

AY598687

HQ665169

KJ595362

KJ595487

P. paddicum

CBS 698.83

Triticum and Hordeum

AY598707

AY598707

AY598707

JX397982

JX397968

P. paroecandrum

CBS 157.64 (VdPN)

Soil

AY598644

AY598644

AY598644

DQ071391

DQ071332

P. parvum

CBS 225.88

Soil

AY598697

AY598697

AY598697

AB362322

KJ595445

P. pectinolyticum

CBS 122643

Soil

HQ643739

HQ643739

HQ643739

N/A

KJ595469

P. periilum

CBS 169.68 (VdPN)

Soil

AY598683

AY598683

HQ665141

N/A

KJ595444

P. periplocum

CBS 289.31

Citrullus vulgaris

AY598670

AY598670

AY598670

KJ595369

KJ595494

P. perplexum

CBS 674.85

Vicia faba

AY598658

AY598658

AY598658

KJ595395

KJ595519

P. phragmitis

CBS 117104

Soil (Phragmites australis)

HQ643746

HQ643746

HQ665081

AJ890351

EU152854

P. pleroticum

CBS 776.81

Nymphyoides peltata

AY598642

AY598642

AY598642

AB362321

KJ595461

P. plurisporium

CBS 100530

Agrostis

AY598684

AY598684

AY598684

KJ595405

KJ595529

P. polare

CBS 118203

Sanionia uncinata

KJ716858

AB299390

KJ716859

KJ595417

KJ595542

P. polymastum

CBS 811.70 (VdPN)

Lactuca sativa

AY598660

AY598660

AY598660

KJ595403

KJ595527

P. porphyrae

CBS 369.79 (VdPN)

Porphyra yezoensis

AY598673

AY598673

AY598673

KJ595377

KJ595501

P. prolatum

CBS 845.68

Rhododendron sp.

AY598652

AY598652

AY598652

AB362330

KJ595462

P. pyrilobum

CBS 158.64

Pinus radiata

AY598636

AY598636

AY598636

KJ595349

KJ595477

P. radiosum

CBS 217.94

Soil

N/A

AY598695

HQ665156

KJ595356

N/A

P. recalcitrans

CBS 122440

Soil (Vitis vinifera)

N/A

DQ357833

KJ716861

KJ595423

EF195143

P. rhizo-oryzae

CBS 119169

Soil

HQ643757

HQ643757

HQ643757

KJ595420

KJ595545

P. rhizosaccharum

CBS 112356

Soil (Saccharum officinarum)

N/A

HQ643760

HQ665072

AB362323

KJ595463

P. rostratifingens

CBS 115464

Soil (Malus sp.)

HQ643761

HQ643761

HQ643761

KJ595416

KJ595541

P. rostratum

CBS 533.74

Soil

AY598696

AY598696

AY598696

KJ595388

KJ595512

P. salpingophorum

CBS 471.50 (VdPN)

Lupinus angustifolius

AY598630

AY598630

AY598630

KJ595384

KJ595508

P. schmitthenneri

CBS 129726

Glycine max

N/A

JF836869

KJ716862

JF895530

KJ595470

P. scleroteichum

CBS 294.37 (AUTH)

Ipomoea batatas

AY598680

AY598680

AY598680

KJ595370

KJ595495

P. segnitium

CBS 112354

Soil

HQ643772

HQ643772

HQ643772

KJ595412

KJ595537

P. selbyi

CBS 129728

Zea mays

N/A

JF836871

KJ716863

JF895532

KJ595471

P. senticosum

CBS 122490

Soil (forest)

HQ643773

HQ643773

HQ643773

AB362317

KJ595467

P. solare

CBS 119359

Phaseolus vulgaris

N/A

EF688275

KJ716860

KJ595421

KJ595546

P. sp.

CBS 113341

Soil

KF853244

KF853244

KF853244

KJ595414

KJ595539

P. sp. “jasmonium”

CBS 101876

Arabidopsis thaliana

HQ643778

HQ643778

HQ643778

KJ595406

KJ595530

P. sp. rooibos 2

STE-U 7549

Aspalathus linearis

N/A

JQ412770

KJ716867

JQ412783

JQ412807

P. sp. rooibos 2

STE-U 7550

Aspalathus linearis

N/A

JQ412777

N/A

JQ412813

JQ412789

P. spiculum

CBS 122645

Soil (Vitis sp.)

KF853242

KF853242

KF853242

KJ595425

KJ595549

P. spinosum

CBS 275.67 (VdPN)

Compost

AY598701

AY598701

AY598701

KJ595366

KJ595491

P. splendens

CBS 462.48 (VdPN)

Unknown

AY598655

AY598655

AY598655

AB512921

AB512852

P. stipitatum

DAOM 240293

Soil

N/A

KJ716866

KJ716866

KJ595437

KJ595560

P. sukuiense

CBS 110030

Soil

N/A

HQ643836

HQ665059

KJ595408

KJ595532

P. sulcatum

CBS 603.73

Daucus carota

AY598682

AY598682

HQ665281

KJ595393

KJ595517

P. sylvaticum

CBS 453.67

Soil

AY598645

AY598645

AY598645

KJ595383

KJ595507

P. takayamanum

CBS 122491

Soil

HQ643854

HQ643854

HQ643854

AB362315

KJ595468

P. tardicrescens

LEV1534

Turf grass

N/A

HQ643855

HQ643855

KJ595439

KJ595562

P. torulosum

CBS 316.33 (VdPN)

Grass

AY598624

AY598624

AY598624

KJ595374

KJ595499

P. tracheiphilum

CBS 323.65

Lactuca sativa

N/A

AY598677

HQ665207

KJ595375

N/A

P. ultimumvar.sporangiiferum

CBS 219.65

Chenopodium album

AKYB02045405

AY598656

AY598656

KJ595357

KJ595482

P. ultimumvar.ultimum

CBS 398.51

Lepidium sativum

AY598657

AY598657

AY598657

KJ595382

KJ595506

P. uncinulatum

CBS 518.77

Lactuca sativa

AY598712

AY598712

AY598712

KJ595385

KJ595509

P. undulatum

CBS 157.69 (VdPN)

Soil (Pinus sp.)

AY598708

AY598708

AY598708

KJ595348

KJ595476

P. vanterpoolii

CBS 295.37

Triticum aestivum

AY598685

AY598685

AY598685

KJ595371

KJ595496

P. vexans

CBS 119.80 (VdPN)

Soil

HQ643400

HQ643400

HQ643400

GU133518

EF426556

P. viniferum

CBS 119168

Soil (Vitis sp.)

HQ643956

HQ643956

HQ643956

KJ595419

KJ595544

P. violae

CBS 132.37

Viola tricolor

AY598717

AY598717

AY598717

KJ595345

KJ595473

P. violae

CBS 159.64 (VdPN)

Soil

AY598706

AY598706

AY598706

JX397980

JX397966

P. violae

CBS 178.86

Daucus carota

AY598715

AY598715

HQ665143

KJ595353

N/A

P. volutum

CBS 699.83

Triticum and Hordeum

AY598686

AY598686

AY598686

KJ595397

KJ595521

P. zingiberis

CBS 216.82

Zingiber mioga

N/A

AY598679

HQ665155

N/A

N/A

Species names of type strains (including ex-type, type, neotype, holotype, isotype, and paratype material), authentic strains (AUTH), and strains used by Van der Plaats-Niterink (1981) for descriptions (VdPN) are indicated in bold. Details regarding amplification and sequencing are included in GenBank records for sequence data generated de novo for this analysis

Table 20

Pyrenophora. Details of the isolates used in the phylogenetic tree

Species

Isolate

GenBank accession numbers

ITS

LSU

GPDH

Drechslera andersenii

CBS 258.80

AY004804

 

AY004835

D. andersenii

CBS 967.87

AY004805

  

D. andersenii

DAOM 229292

JN943646

JN940084

 

D. avenae

CBS 189.29

AY004795

 

AY004827

D. avenae

CBS 279.31

AY004796

 

AY004828

D. biseptata

DAOM 208987

AY004786

 

AY004817

D. biseptata

CBS 308.69

JN712464

JN712530

AY004819

D. biseptata

CBS 599.7

AY004787

 

AY004818

D. biseptata

CBS 108940

AY004788

  

D. campanulata

BRIP15927

AF163058

  

D. catenaria

DAOM 63665A

AY004802

 

AY004833

D. catenaria

CBS 191.29

AY004803

 

AY004834

D. dactylidis

DAOM 92161

AY004781

 

AY004812

D. dematioidea

CBS 108963

AY004789

JN712532

AY004820

D. dematioidea

DAOM 229295

JN943648

JN940094

 

D. dematioidea

CBS 108962

JN712465

JN712531

 

D.dematioidea

CBS 108962

AY004790

JN712531

AY004821

Drechslera dictyoides

DAOM 63666

AY004806

JN940080

AY004836

D. erythrospila

CBS 108941

AY004782

 

AY004813

D. erythrospila

DAOM 55122

AY004783

 

AY004814

D. fugax

CBS 509.77

AY004791

 

AY004822

D.nobleae

CBS 259.80

AY004792

 

AY004823

D. nobleae

DAOM 229296

JN943647

JN940095

 

D. nobleae

CBS 966.87

AY004793

 

AY004824

D. nobleae

CBS 316.69

AY004794

 

AY004825

D. phlei

CBS 315.69

AY004807

 

AY004837

D. phlei

DAOM 225627

JN943656

JN940077

 

D. poae

DAOM 145373

AY004801

JN940082

AY004832

D. poae

DAOM 169240

JN943651

  

D. siccans

DAOM 115701

AY004797

JN940078

 

D. siccans

DAOM 115702

AY004799

  

Drechslerasp.

DAOM126766

AY004800

 

AY004831

Drechslerasp.

DAOM126772

AY004784

 

AY004815

Drechslerasp

CBS313.69

AY004785

 

AY004816

D. triseptata

NZ6120

AF163059

  

Pleospora herbarum

CBS 191.86*

DQ491516

DQ247804

AY316969

Pyrenophora bromi

DAOM 127414

AY004809

JN940074

AY004839

P. chaetomioides

DAOM 208989

AF081445

JN940091

AF081371

P. dictyoides

DAOM 75616

JN943654

JN940079

 

P. japonica

DAOM 169286

AF071347

 

AF081369

P. lolii

CBS 318.69

AY004798

 

AY004829

P. phaeocomes

DAOM 222769

JN943649

DQ499596

 

P. semeniperda

DAOM 213153

AF081446

JN940089

AY004826

P. tetrarrhenae

DAOM 171966

JN943663

JN940090

 

P. tritici-repentis

DAOM 226213

JN943670

AY544672

AF081370

P. tritici-repentis

DAOM 208990

AF071348

JN940071

AY004838

P. tritici-repentis

DAOM 107224

AY004808

DQ384097

 

P. graminea

11

Y10748

  

P. teres

PM2

Y08746

 

AY004830

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 21

Puccinia. Details of the isolates used in the phylogenetic tree

Species

Isolate

Host

GenBank accession no.

LSU

SSU

Aecidium kalanchoe

BPI 843633

Kalanchoe blossfeldiana

AY4631631

DQ3545242

Allodus podophylli

BPI 842277

Podophyllum peltatum

DQ3545432

DQ3545442

Caeoma torreyae

ECS 553

Torreya californica

AF5221833

AY1232844

Cumminsiella mirabilissima

BPI 871101

Mahonia aquifolium

DQ3545312

DQ3545302

Helicobasidium purpureum

CBS324.47

Not provided

AY8851683

D856483

Dietelia portoricensis

BPI 844288

Mikania micrantha

DQ3545162

AY1254144

Miyagia pseudosphaeria

BPI 842230

Sonchus oleraceus

DQ3545172

AY1254114

Pileolaria toxicodendri

BPI 871761

Toxicodendron sp.

DQ3239245

AY1233144

Prospodium lippiae

BPI 843901

Aloysia plystachya

DQ3545552

DQ8310246

P. tuberculatum

BRIP 57630

Lantana camara

KJ3961957

KJ3961967

Puccinia caricis

BPI 871515

Grossularia sp.

DQ3545142

DQ3545152

P. convolvuli

BPI 871465

Calystegia sepium

DQ3545122

DQ3545112

P. coronata

 

Rhamnus cathartica

DQ3545262

DQ3545252

P. dampierae

BRIP 57724

Dampiera linearis

KF6906888

NA

P. gilgiana

BRIP 57719

Lechenaultia linarioides

KF6906918

NA

P. graminis

NA

Not provided

AF5221779

NA

P. haemodori

BRIP 56965

Anigozanthus sp.

KF690692

NA

P. hemerocallidis

BPI 843967

Hemerocallis sp.

DQ3545192

DQ3545182

P. hordei

BPI 871109

Poaceae

DQ3545272

DQ4152782

P. lagenophorae

BRIP 57563

Emilia sonchifolia

KF6906968

NA

P. menthae

BPI 871110

Cunila origanoides

DQ3545132

AY1233154

P. psidii

BRIP 57991

Melaleuca leucadendra

KF3184437

KF3184557

P. poarum

NA

Tussilago sp.

DQ8310286

DQ8310296

P. polysora

BPI 863756

Zea mays

GU05802410

NA

P. saccardoi

BRIP 57725

Scaevola spinescens

KF6907018

NA

P. smilacis

BPI 871784

Smilax rotundifolia

DQ3545332

DQ3545322

P. stylidii

BRIP 60107

Stylidium armeria

KJ6222148

NA

P. ursiniae

BRIP 57993

Ursinia anthemoides

KF6907058

NA

P. violae

BPI 842321

Viola cucullata

DQ3545092

DQ3545082

P. xanthosiae

BRIP 57729

Xanthosia rotundifolia

KF690706

NA

Pucciniosira solani

NA

Solanum aphyodendron

EU85113711

NA

Uromyces appendiculatus

NA

Phaseolus vulgaris

AY7457043

DQ3545102

U. ari-triphylli

BPI 871111

Arisaena triphyllum

DQ3545292

DQ3545282

U. scaevolae

BRIP 60113

Selliera radicans

KJ6222138

NA

U. viciae-fabae

NA

Pisum sp.

AY7456953

NA

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 22

Rhizopus. Details of the isolates used in the phylogenetic tree

Species

Isolate

Host/source

GenBank accession no.

Rhizopus arrhizus

CBS111231

JN206338

R. arrhizus

CBS544.80

Sorghum malt

JN206337

R. arrhizus

CBS120.12

AB181318

R. arrhizus

IFO5438

DQ641276

R. arrhizus

CBS112.07

JN206323

R. arrhizus

CBS146.90

Homo sapiens

JN206324

R. arrhizus

NRRL1469*

DQ641279

R. microsporus

CBS357.93

Tempeh

JN206343

R. microsporus

CBS631.82

Bread

JN206344

R. microsporus

CBS536.80

Sorghum malt

HM999971

R. microsporus

AS3.1145

DQ641305

R. microsporus

CBS337.62

JN206362

R. microsporus

CBS699.68*

Soil

HM999970

R. homothallicus

CBS336.62*

Soil

HM999968

R. homothallicus

CBS111232

JN206365

R. caespitosus

CBS427.87*

HM999965

R. caespitosus

33515

AF115730

R. schipperae

CBS138.95*

Homo sapiens

HM999969

Syzygites megalocarpus

CBS108947

Amanita rubescens

JN206370

   

JN206371

Sporodiniella umbellate

CBS195.77

Umbonia

JN206372

R. stolonifer

CBS389.95*

DQ641318

Rhizopus sp. ‘stolonifer’

CBS442.74

Coffee-ground

JN206367

R. stolonifer

AFTOL-ID632

AY997085

R. sexualis

CBS336.39*

Fragaria

AB113017

R. americanus

CBS340.62*

Air

HM999967

R. lyococcus

CBS319.35

AB100449

R. lyococcus

CBS117.43

Hordeum vulgare

JN206375

R. lyococcus

JCM5589*

DQ641319

Backusella sp.

CBS538.80

Medicago sativa

HM999964

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 23

Stagonosporopsis. Details of the isolates used in the phylogenetic tree

Species name

Strain no.1

Host

GenBank accession number

LSU

SSU

ITS

β- tubulin

ACT

CAL

Stagonosporopsis actaeae*

CBS 106.96; PD 94/1318

Actaea spicata

GU238166

QBank

GU237734

GU237671

JN251974

S. ajacis*

CBS 177.93; PD 90/115

Delphinium sp.

GU238168

QBank

GU237791

GU237673

JN251962

QBank

S. andigena

CBS 101.80; PD 75/909;

       

IMI 386090

Solanum sp.

GU238169

GU238233

GU237714

GU237674

JN251958

QBank

 

S. artemisiicola

CBS 102636; PD 73/1409

Artemisia dracunculus

GU238171

QBank

GU237728

GU237676

JN251971

QBank

S. astragali

CBS 178.25; MUCL 9915

Astragalus sp.

GU238172

QBank

GU237792

GU237677

JN251963

QBank

S. caricae

CBS 248.90

Carica papaya

GU238175

QBank

GU237807

GU237680

JN251969

QBank

S. chrysanthemi

CBS 500.63; MUCL 8090

Chrysanthemum indicum

GU238190

QBank

GU237871

GU237695

JN251973

QBank

S. crystalliniformis*

CBS 713.85; ATCC 76027; PD 83/826

Lycopersicon esculentum

GU238178

QBank

GU237903

GU237683

JN251960

QBank

S. cucurbitacearum

CBS 133.96; PD 79/127

Cucurbita sp.

GU238181

GU238234

GU237780

GU237686

JN251968

QBank

S. dennisii

CBS 631.68; PD 68/147

Solidago floribunda

GU238182

GU238235

GU237899

GU237687

QBank

QBank

S. dorenboschii*

CBS 426.90; IMI 386093; PD 86/551

Physostegia virginiana

GU238185

QBank

GU237862

GU237690

JN251980

QBank

S. heliopsidis

CBS 109182; PD 74/231

Heliopsis patula

GU238186

QBank

GU237747

GU237691

QBank

QBank

S. hortensis

CBS 104.42

Unknown

GU238198

QBank

GU237730

GU237703

QBank

QBank

S. inoxydabilis*

CBS 425.90; PD 81/520

Chrysanthemum parthenii

GU238188

QBank

GU237861

GU237693

JN251972

QBank

S. loticola*

CBS 562.81; ICMP 6884

Lotus pedunculatus

GU238192

QBank

GU237890

GU237697

JN251978

QBank

S. lupini

CBS 101494; PD 98/5247

Lupinus albus

GU238194

QBank

GU237724

GU237699

QBank

QBank

S. oculo-hominis*

CBS 634.92; IMI 193307

Homo sapiens

GU238196

QBank

GU237901

GU237701

JN251976

QBank

S. rudbeckiae

CBS 109180; PD 79/175

Rudbeckia bicolor

GU238197

QBank

GU237745

GU237702

QBank

QBank

S.tanaceti*

CBS 131484; TAS 1

Tanacetum cinerariifolium

JQ897461

JQ897481

JQ897496

JQ897512

S. trachelii

CBS 379.91; PD 77/675

Campanula isophylla

GU238173

QBank

GU237850

GU237678

JN251977

QBank

S. valerianellae

CBS 329.67; PD 66/302

Valerianella locusta var. oleracea

GU238201

QBank

GU237832

GU237706

JN251965

QBank

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 24

Ustilago. Details of the isolates used in the phylogenetic tree

Species

Isolate

Host

Marker/GenBank accession no.

ITS

LSU

Anomalomyces panici

BRIP 46421

Panicum trachyrachis

DQ4593481

DQ4593471

Anthracocystis destruens

Ust. Exs. 472

Panicum miliaceum

AY3449762

AY7470773

Langdonia aristidae

BRIP 52755

Aristida hygrometrica

HQ0130964

NA

Macalpinomyces eriachnes

56573 (M)

Eriachne aristidea

AY7400373

AY7400903

M. mackinlayi

BRIP 52549

Eulalia mackinlayi

GU0148174

HQ0131314

M. neglectus

RB 2056 (TUB)

Setaria pumila

AY7400562

AY7401092

M. simplex

56577 (M)

Loudetia simplex

AY7401523

NA

M. spermophorus

HUV 13634

Eragrostis ferruginea

AY7401713

NA

 

BRIP 51858

Sporobolus australasicus

NA

HQ0131304

M. viridans

BRIP 49133

Sporobolus actinocladus

HQ0130894

HQ0131254

Melanopsichium pennsylvanicum

HUV 17548

Polygonum glabrum

AY7400403

AY7400933

Moesziomyces bullatus

Ust. Exs. 833

Paspalum distichum

AY7401533

AY7401533

Sporisorium aegyptiacum

Ust. Exs. 756

Schismus arabicus

AY3449702

AY7401293

S. sorghi

MP 2036a (USJ)

Sorgum bicolor

AY7400213

AF0098725

S. spinulosum

HMAS 193085

Capillipedium parviflorum

GU1391726

GU1391716

Stollia ewartii

BRIP 51818

Sarga timorense

HQ0130874

HQ0131274

Triodiomyces altilis

Ust. Exs. 418

Triodia pungens

AY7401663

NA

 

BRIP 52543

Triodia sp.

NA

HQ0131364

T. triodiae

HUV 17662

Triodia microstachya

AY7400743

AY7401263

Tubisorus pachycarpus

HUV 21891

Mnesithea rottboellioides

JN8717187

JN8717177

Ustilago affinis

MP 692

Stenotaphrum secundatum

AY3449952

AF1335813

U. austro-africana

56516 (M)

Enneapogon cenchroides

AY7400613

AY7401153

U. avenae

DB 559 (TUB)

Avena barbata

AY3449972

AF4539333

U. bouriqueti

56517 (M)

Stenotaphrum dimidiatum

AY7401672

NA

U. bromivora

HUV 19322

Bromus catharticus

AY7400643

AY7401183

U. bullata

MP 2363 (TUB)

Bromus diandrus

AY3449982

AF4539353

U. calamagrostidis

56518 (M)

Calamagrostis epigeios

AY7400653

AY7401193

U. crameri

Ust. Exs. 995

Setaria italica

AY3449992

AY7401433

U. curta

Ust. Exs. 1090

Tripogon loliiformis

AY7401653

HQ0131234

U. cynodontis

MP 1838

Cynodon dactylon

AY3450002

AF0098813

U. davisii

HUV 19252

Glyceria multiflora

AY7401693

NA

U. drakensbergiana

56523 (M)

Digitaria tricholaenoides

AY7401702

 

U. echinata

Ust. Exs. 540

Phalaris arundinacea

AY3450012

AY7401443

U. esculenta

Ust. Exs. 590

Zizania latifolia

AY3450022

AF4539373

U. filiformis

RB 3011 (TUB)

Glyceria fluitans

AY7400663

AY7401203

U. hordei

Ust. Exs. 784

Hordeum vulgare

AY3450032

AF4539433

U. ixophori

MP 2194

Ixophorus unisetus

AY7400672

AY7401212

U. maydis

RB 3093

Zea mays

AY3450042

NA

 

NA

Zea mays

NA

AF4539388

U. nuda

HUV 17782

Hordeum leporinum

AY7400693

AJ2361393

U. pamirica

Ust. Exs. 789

Bromus gracillimus

AY3450052

AY7401453

U. schmidtiae

BRIP 51848

Enneapogon sp.

HQ0131214

HQ0131294

U. schroeteriana

Ust. Exs. 887

Paspalum paniculatum

AY3450062

AY7401463

U. sparsa

Ust. Exs. 892

Dactyloctenium radulans

AY3450082

NA

U. sporoboli-indici

BRIP 39706

Sporobolous pyramidalis

AY7727369

NA

U. striiformis

HUV 18286

Alopecurus pratensis

AY7401723

DQ87537510

U. syntherismae

Ust. Exs. 998

Digitaria ternata

AY7400713

AY7401233

U. tragana

56562 (M)

Tragus berteronianus

AY7400723

AY7401243

U. tritici

NA

Triticum aestivum

AF13542411

NA

U. trichophora

56564 (M)

Echinochloa colona

AY3450093

AY7401483

U. turcomanica

HUV 23

Eremopyrum distans

AY3450113

AF4539363

U. vetiveriae

HUV 17954

Vetiveria zizanioides

AY3450113

AF4539373

U. xerochloae

Ust. Exs. 1000

Xerochloa imberbis

AY3450123

AF4539383

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Table 25

Verticillium. Details of the isolates used in the phylogenetic tree

Species

Isolate

Host

GenBank accession number

ITS

ACT

EF

GPD

TS

V. dahliae

PD322*

Lettuce

HQ206718

HQ206718

HQ414624

HQ414719

HQ414909

V. alfalfae

PD489*

Alfalfae

JN187971

JN188097

JN188225

JN188161

JN188033

V. nubilum

PD742*

Soil

JN188011

JN188139

JN188267

JN188203

JN188075

V. isaacii

PD660*

Lettuce

HQ206873

HQ206985

HQ414688

HQ414783

HQ414973

V. nonalfalfae

PD592*

Irish Potato

JN187973

JN188099

JN188227

JN188163

JN188035

V. albo-atrum

PD747*

Potato Soil

JN188016

JN188144

JN188272

JN188208

JN188080

V. zaregamsianum

PD736*

Lettuce

JN188005

JN188133

JN188261

JN188197

JN188069

V. tricorpus

PD690*

Garden Tomato

JN187993

JN188121

JN188249

JN188185

JN188057

V. klebahnii

PD401*

Lettuce

JN187967

JN188093

JN188221

JN188157

JN188029

V. longisporum

PD687* Allele D2

Horseradish

 

HQ206994

HQ414697

HQ414792

HQ414982

V. longisporum

PD687* Allele A1

Horseradish

 

HQ206993

HQ414696

HQ414791

HQ414981

Ex-type (ex-epitype) strains are bolded and marked with an * and voucher stains are bolded

Backbone tree for important phytopathogens

Condensed synopses are provided for 25 important plant pathogenic group or genera. Each synopsis includes notes on background, species identifications and numbers, molecular phylogeny, recommended genetic markers, tables of species and the latest phylogenetic trees. We have not been able to include all important phytopathogenic genera (e.g. Alternaria, powdery mildews), but intend to update or add these in future publications. Interested parties should contact the corresponding author.

Bipolaris

Background

The genus Bipolaris belongs to the family Pleosporaceae of the Pleosporales in Dothideomycetes (Ascomycota). Bipolaris was introduced by Shoemaker (1959) and typified with B. maydis. Bipolaris species are pathogens, saprobes or endophytes mostly associated with grasses including cultivated cereals. Some species are important plant pathogens. The Bengal famine in 1943 was caused by B. oryzae and caused 90 % of crop losses in India as well as the loss of 1.5 million human lives (Scheffer 1997). In the 1970s, around 19 million metric tons of wheat were destroyed in the USA due to southern corn leaf blight caused by B. maydis. Bipolaris sorokiniana causes southern leaf blotch, seedling blight and crown rot. Bipolaris sorokiniana was confirmed as the most economically important foliar pathogen in warm areas by the conference “Wheat for the national warm areas” held in Brazil in 1990. Bipolaris species have also been recorded from other plant families such as Alliaceae, Anacardiaceae, Araceae, Euphorbiaceae, Fabaceae, Malvaceae, Rutaceae and Zingiberaceae (Manamgoda et al. 2011).

Species identification and numbers

Bipolaris species were formerly described in Helminthosporium, however, species associated with grasses were morphologically distinct from H. velutinum, the type species (Luttrell 1963; Ellis 1971; Alcorn 1988). In several taxonomic refinements, these graminicolous Helminthosporium species were segregated into four genera; Bipolaris, Curvularia, Drechslera and Exserohilum (Sivanesan 1987). Later Subramanian and Jain (1966) placed all Bipolaris species in Drechslera, but this transfer was not accepted by later authors (Sivanesan 1987; Alcorn 1988). After molecular data became available, Drechslera was shown to be a phylogenetically different genus from Bipolaris (Berbee et al. 1999). The sexual state of Bipolaris is Cochliobolus (Drechsler 1934). Cochliobolus is the older name but conservation of the name Bipolaris over Cochliobolus has been proposed to avoid numerous name changes and Bipolaris is the most common name among plant pathologists (Manamgoda et al. 2012a; Rossman et al. 2013).

Morphology-based classification of Bipolaris species is challenging as the asexual state has overlapping conidia and conidiophore dimensions (Sivanesan 1987). A few Bipolaris species are known to be host-specific, while most of the other species are generalists (Manamgoda et al. 2011). However, some of the host-specific species are known only from limited collections. Therefore, the information on host-specificity may change with further collections. Interspecific compatibility can be observed between some taxa. For example, successful hybridization leading to ascospore production has been reported between B. zeicola and B. victoriae (Nelson 1960a, b) as well as between B. maydis and B. oryzae (Alcorn 1988). However, the latter species are definitively distinct phylogenetic species and also they are commonly recorded pathogens, causing different symptoms on their respective hosts. Identification of Bipolaris species using morphological and biological species concepts is not always correct and it is essential to use molecular tools in identifying species. Lack of DNA sequences from type material/ex-type cultures (or other authentic material) in public sequence databases is a problematic issue regarding the molecular identification of the Bipolaris species (Nilsson et al. 2014). Currently there are 118 Bipolaris names listed in Index Fungorum (2014), but nine of them do not belong to this genus based on phylogenetic evidence.

Molecular phylogeny

The first phylogenetic analysis for Bipolaris with its sister genus Curvularia was carried out by Berbee et al. (1999) and Goh et al. (1998) using a combined ITS and GPDH analysis. These studies showed that Bipolaris species cluster in two clades. Combined ITS, GPDH, EF and LSU phylogenetic analysis for Bipolaris and Curvularia by Manamgoda et al. (2012a) showed that Bipolaris and Curvularia cluster into two major clades. Nine Bipolaris species clustered with the generic type, Curvularia lunata Boedijn, while other species of Bipolaris clustered with the generic type, Bipolaris maydis. Accordingly, the nine Bipolaris species were moved to Curvularia, and Bipolaris was maintained as a distinct genus based on the generic type and those species that clustered with it. In this section we provide a backbone tree (Table 2, Fig. 1) for Bipolaris using combined ITS and GPDH sequence data.
Fig. 1

Phylogram generated from parsimony analysis based on combined ITS and GPDH sequenced data of Bipolaris. Bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Curvularia lunata

Recommended genetic markers

GPDH is the best single genetic marker for the genus Bipolaris (Manamgoda et al. 2012a). Combined ITS, EF and GPDH can resolve almost all species of Bipolaris currently known from sequence data (Manamgoda et al. 2012a).

Botryosphaeriaceae

The family Botryosphaeriaceae is classified in the order Botryosphaeriales of the Dothideomycetes (Ascomycota). Members of the fungal family Botryosphaeriaceae were described in the 1820’s as species of Sphaeria (Fr.) (Crous et al. 2006; Schoch et al. 2006). There have subsequently been various treatments of the family. von Arx and Müller (1954) included 15 genera, but later reduced it to 14 genera (von Arx and Müller 1975). Barr (1987) included only nine genera, which are mostly different from those of von Arx and Müller (1954). Hawksworth et al. (1995) listed five genera. Lumbsch and Huhndorf (2010) included 11 genera, while Hyde et al. (2011) and Wijayawardene et al. (2012) listed 16 genera. Liu et al. (2012) included 17 genera in the family based on molecular data and examination of generic types. Species of Botryosphaeriaceae range in habit from saprobic to parasitic or endophytic (Smith et al. 1996; Denman et al. 2000; Phillips et al. 2006; Slippers and Wingfield 2007; Huang et al. 2008; Pérez et al. 2010; Ghimire et al. 2011; González and Tello 2011). Members are cosmopolitan in distribution and occur on a wide range of monocotyledonous, dicotyledonous and gymnosperm hosts; on woody branches, herbaceous leaves, stems and culms of grasses; and on twigs and in the thalli of lichens (Barr 1987; Denman et al. 2000; Mohali et al. 2007; Lazzizera et al. 2008; Marincowitz et al. 2008).

Species identification and numbers

Currently, more than 2,000 species names are linked to Botryosphaeriaceae, including sexual and asexual states of Diplodia, Botryosphaeria, Fusicoccum, Dothiorella, Lasiodiplodia and Sphaeropsis. Identification to genus and species is presently undergoing major revision and it is likely that many older names will not be used in modern treatments. Identification of species in Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia and Neofusicoccum are dealt separately under this family entry.

Molecular phylogeny

Recent advances in DNA-based molecular techniques have begun to provide efficient tools to characterize the presence and identity of species of the Botryosphaeriaceae (Slippers and Wingfield 2007). Studies applying these tools are revealing significantly greater diversity on some hosts than was previously realized. Recent studies on the taxonomy of Botryosphaeria have employed molecular methods to reveal phylogenetic relationships among species (Jacobs and Rehner 1998) and to resolve species complexes (Denman et al. 2003; Alves et al. 2004; Phillips et al. 2005). Two major clades corresponding to species with Diplodia and Fusicoccum asexual morphs were revealed based on ITS phylogenies (Jacobs and Rehner 1998; Denman et al. 2003). Later studies, including additional species and a larger suite of genetic markers, supported this grouping (Zhou and Stanosz 2001; Alves et al. 2004; Slippers et al. 2004d). Lasiodiplodia has been treated as a distinct genus from Diplodia by many authors due to its distinct phylogeny (usually ITS or EF-1α) and morphology (striated or smooth conidia and presence or absence of pseudoparaphyses). Pavlic et al. (2004) employed morphological and phylogenetic data to separate Lasiodiplodia from Diplodia. The value of the intron-dominated sequences of the ITS, β-tubulin and TEF markers (on which most previous studies were based) to infer phylogenetic relationships across the diversity of the genus is, however, unclear. The more conserved mtSSU data have, for example, suggested that B. dothidea and B. corticis (Demaree and Wilcox) are unrelated to Fusicoccum (Zhou and Stanosz 2001) even though they are typically assigned to this genus.

Most taxonomic studies on Botryosphaeriaceae using molecular data have employed ITS rDNA phylogenies, but this single marker can underestimate the species diversity among closely related or cryptic species. Multiple gene sequence concordance phylogenies have therefore been applied to identify cryptic or previously overlooked species of Botryosphaeriaceae (Slippers et al. 2004a, b, c; Burgess et al. 2005; Phillips et al. 2005). As the elongation fctor 1- alpha (TEF) gene is consistently more variable than the ITS rDNA region in these fungi, most commonly data from TEF have been combined with ITS sequence data. Unfortunately no single genetic region is sufficient to distinguish all species, because not all single nucleotide polymorphisms (SNPs) represent restriction sites, especially between some closely related species.

The Botryosphaeriaceae has been separated into numerous distinct genera (Crous et al. 2006; Liu et al. 2012). A natural classification is needed for a more stable and accurate taxonomic framework and this will strongly influence the understanding of the ecology of the Botryosphaeriaceae. In this part we provide a tree to the genera of Botryosphaeriaceae (Table 3, Fig. 2) and deal with the important genera Botryosphaeria, Diplodia, Dothiorella, Lasiodiplodia and Neofusicoccum in the following parts.
Fig. 2

Phylogram generated from parsimony analysis based on combined SSU, LSU, TEF, β-tubulin and ITS sequence data of Botryosphaeriaceae. Parsimony bootstrap support values greater than 50 % and Bayesian posterior probabilities greater than 0.5 are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The scale bar indicates ten changes. The tree is rooted with Melanops tulasnei CBS 116805

Fig. 3

Phylogram generated from parsimony analysis based on combined ITS, TEF, β- tubulin, LSU and SSU sequenced data of Botryosphaeria. Parsimony bootstrap support values greater than 50 % and Bayesian posterior probabilities greater than 0.5 are indicated near the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Macrophomina phaseolina CBS 227.33

Recommended genetic markers

  • LSU, SSU, β-tubulin and ITS–generic level

  • TEF–species level

LSU has been shown to be suitable for distinguishing many ascomycetes at the generic level due to its relatively conserved nature (Crous et al. 2006; Schoch et al. 2006; Hibbett et al. 2007). The study of Liu et al. (2012) suggested that the combined TEF and β- tubulin gene analysis is best for delimiting genera of Botryosphaeriaceae. It has also been recommended that the RPB2 gene should be considered in similar combined analyses of genus and species levels of Botryosphaeriaceae (Pavlic et al. 2009a, b).

Botryosphaeria

Background

The genus Botryosphaeria (Botryosphaeriaceae) was introduced by Cesati and de Notaris (1863), amended by Saccardo (1877), and is based on the type species Botryosphaeria dothidea (Barr 1972; Slippers et al. 2004c). Species in Botryosphaeria were described largely on the basis of the morphology of their ascomata and host associations, and this has led to a proliferation of names. von Arx and Müller (1954) examined 183 taxa of Botryosphaeriales and reduced them to 11 species, with extensive synonymies under B. dothidea and B. quercuum, together with nine new combinations. In later studies these synonymies were not always accepted (Shoemaker 1964; Sivanesan 1984; Slippers et al. 2004a). Slippers et al. (2004b) epitypified the type species Botryosphaeria dothidea based on morphology and phylogeny (combined ITS, TEF and β-tubulin analysis) and this enabled a better resolution of species. Species of Botryosphaeria occur on a wide range of monocotyledonous, dicotyledonous and gymnosperm hosts, on woody branches, herbaceous leaves and grasses (Barr 1987). The life styles may be saprobic, parasitic and endophytic (Smith et al. 1996; Denman et al. 2000), and species can cause die-back and canker diseases of numerous woody hosts (von Arx 1987). Species in the genus Botryosphaeria have hyaline to dark ascospores, multiloculate ascomata, and a wide range of asexual morphs that typically lack a mucoid sheath and apical appendage.

Species identification and numbers

More than 18 asexual genera have been associated with Botryosphaeria. A phylogenetic study based on part of the 28S ribosomal DNA gene together with morphological characters revealed that Botryosphaeria comprises several distinct lineages, each comprising individual genera (Crous et al. 2006). In that study, only B. dothidea and B. corticis were retained in Botryosphaeria, while most species were reduced to synonymy under Diplodia (conidia mostly ovoid, pigmented, thick-walled), or Fusicoccum (conidia mostly fusoid, hyaline, thin-walled). Studies have also linked Botryosphaeria to species with pigmented, septate ascospores and Dothiorella asexual morphs, or Fusicoccum asexual morphs with Dichomera synanamorphs. More recently B. agaves (which has been epitypified), B. fusispora (Liu et al. 2012), and B. schariffi (Abdollahzadeh et al. 2013) were described in the genus Botryosphaeria, while B. fabicerciana was illustrated from Eucalyptus sp. in southern China (Chen et al. 2011). Phylogenetically, B. fabicerciana is closely related to B. corticis, B. dothidea, B schariffi and B. ramosa. The present phylogenetic analysis was performed based on up to date holotype or ex-epitype sequence data available in GenBank (Table 4).

Molecular phylogeny

Recent studies on the taxonomy of Botryosphaeria have employed molecular methods to reveal phylogenetic relationships among species (Jacobs and Rehner 1998) and to resolve species complexes (Smith and Stanosz 2001; Phillips et al. 2002, 2005; Denman et al. 2003; Alves et al. 2004; Slippers et al. 2004c). Studies including additional species and a larger suite of DNA-based markers supported this grouping (Zhou and Stanosz 2001; Alves et al. 2004; Slippers et al. 2004c). Based on combined ITS and TEF sequence data seven species are currently recognised in Botryosphaeria (Phillips et al. 2013). The phylogenetic tree constructed with holotype or ex-epitype sequences is presented in Fig. 3.
Fig. 4

Phylogram generated from Maximum likelihood analysis based on combined sequences of G3PDH, HSP60 and RPB2 from 28 recognized Botrytis species. Bootstrap support values greater than 50 % are indicated above/below the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Monilinia fructigena and Sclerotinia sclerotiorum

Fig. 5

Phylogram generated from Maximum likelihood analysis based on ITS sequenced data of Choanephora. Bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold

Fig. 6

Phylogram generated from parsimony analysis based on combined ITS, GADPH, CHS-1, ACT, HIS and β- tubulin data of Colletotrichum. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Monilochaetes infuscans CBS 869.96

Fig. 7

Phylogram generated from parsimony analysis based on combined ITS, GADPH, CHS-1, ACT, HIS and β- tubulin sequenced data of Colletotrichum acutatum complex. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with C. orchidophilum

Fig. 8

Phylogram generated from parsimony analysis based on combined ITS, GADPH, CHS-1, ACT, HIS and β- tubulin sequenced data of Colletotrichum gloeosporioides complex. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with C. coccodes ITCC6079

Recommended genetic markers

  • LSU, SSU and ITS–generic level

  • β-tubulin and TEF–species level

Botrytis

Background

Erected by Micheli in 1729, the genus Botrytis is one of the first described genera of fungi. Persoon (1801) designated five species under the binomial system of Linnaeus, validated the genus, and included one of Micheli’s species, B. cinerea, so named by Von Haller (1771). The genus name refers to the structure of the macroconidia, which rise and form clusters with the shape of grape bunches: ‘botryose’. Botrytis is the asexual stage of Botryotinia. The Botrytis community has in its recent meeting (Italy, 23–28 June 2013) unanimously recommended the exclusive use of the asexual name Botrytis over Botryotinia, the name of the sexual stage, since Botrytis is historically the oldest name and it is commonly used by plant pathologists, breeders and growers. In line with this recommendation, a list of generic names of fungi for protection under the International Code of Nomenclature has included this genus under the name Botrytis and not Botryotinia (Kirk et al. 2013). We therefore follow this recommendation in this paper and use Botrytis. Species of the genus Botrytis infect >250 host species, including major greenhouse and field crops such as tomato, grape, strawberry, onion and ornamentals such as rose, lily, and tulip (Staats et al. 2005). Most Botrytis species are necrotrophic pathogens that (are able to) kill the host tissue during infection. Interestingly, an endophytic species (B. deweyae) has recently been discovered, which under appropriate conditions can cause ‘spring sickness’ in ornamental Hemerocallis (daylily) hybrids (Grant-Downton et al. 2014). Botrytis cinerea is the best-studied species in the genus (Williamson et al. 2007) and was recently elected as the second most important plant pathogenic fungal species (Dean et al. 2012).

In the asexual state, Botrytis produces different tissues including mycelia, macroconidia, microconidia, and sclerotia. Macroconidia are ellipsoidal to obovoid shape and rise from conidiophore branches into botryose clusters. They are pale brown and range in size from 9–23 × 8–15 μm. Microconidia are more sphaerical and much smaller than macroconidia (about 1 μm), and function as male spermatia (Groves and Loveland 1953; Faretra et al. 1988; Beever and Parkes 1993; Fukumori et al. 2004). Sclerotia are irregularly hemispherical, convex and normally have a concave surface. They are usually black, with sizes ranging between 1 and 10 mm (Whetzel 1945), and function as survival structures during winter and serve as maternal parent in the production of apothecia.

The sexual state forms fruiting bodies called apothecia: a cup- or open saucer-shaped ascoma at the top of a stalk, that acts as a platform to discharge ascospores from the ascus. Botrytis apothecia vary in size depending on the species, between 1 and 25 mm high and 1–6 mm diam. (Hennebert and Groves 1963; Bergquist and Lorbeer 1972). Apothecia are brown and become darker when mature (Hennebert and Groves 1963; Bergquist and Lorbeer 1972; Faretra and Antonacci 1987). Generally multiple apothecia can develop on a single sclerotium. Mature apothecia normally can be observed 2 months after fertilization (Faretra et al. 1988; Hennebert and Groves 1963; Van Der Vlugt-Bergmans et al. 1993). In the genus Botrytis, both homothallic and heterothallic reproductive lifestyles have been reported. Homothallic (self-fertile) species can undergo sexual reproduction and form apothecia and generate progeny in the absence of a mating partner, e.g. B. porri and B. globosa (Buchwald 1953; Elliott 1964). By contrast, heterothallic (self-sterile, self-incompatible) species require isolates with compatible mating types in order to complete the sexual cycle. B. cinerea is considered a typical heterothallic fungus (Elliott 1964; Faretra et al. 1988). Mating is controlled by the mating type locus with two alleles, MAT1-1 and MAT1-2 (Faretra et al. 1988), each carrying two distinct, non-homologous genes (Amselem et al. 2011).

Species identification and numbers

Approximately half of the Botrytis species are named after the host that they are derived from (listed in Table 5). One hybrid species, B. allii which originated from hybridization between B. byssoidea and B. aclada (Nielsen and Yohalem 2001; Yohalem et al. 2003) could not be placed in the phylogeny (Staats et al. 2005) and was omitted from Table 3. The genus Botrytis predominantly comprises narrow host range pathogens that infect a single, or a few (often related) host species. There are two exceptions to this rule: B. cinerea can infect more than 200 host species (Jarvis 1977), and B. pseudocinerea has been isolated from several unrelated host species (Fournier et al. 2005; Leroch et al. 2013).

The taxonomic classification and nomenclature in Botrytis have rarely been comprehensively reviewed. Morphological descriptions of most species have been published in the 19th and first half of the 20th century in separate papers, many of which are not easily accessible. The most recent taxonomic compilation of the genus is in a monograph by Jarvis (1977), which also lists ~25 excluded or doubtful species, and briefly describes the historical debates between mycologists and the confusion in classification of Botrytis species. Morphological features were often inadequate to distinguish species and the variability among isolates of the same species further complicated the situation (Jarvis 1977). Recent studies have identified B. cinerea and B. pseudocinerea as species that are very similar in morphology, yet recognized as distinct taxa that diverged several million years ago (Walker et al. 2011). Even more puzzling, the morphology and narrow host range of B. fabae separate this species clearly from B. cinerea and B. pseudocinerea, but phylogenetic studies revealed it to be a sister species of B. cinerea (see below). These examples illustrate the limitations of morphological characters for Botrytis species identification.

Molecular phylogeny

Holst-Jensen et al. (1998) were the first to use nuclear ribosomal ITS sequences to infer a phylogeny of the family Sclerotiniaceae, including several members of the genus Botrytis. The relationships among many Botrytis species could not be resolved because of the limited number of informative characters, however the study permitted the conclusion that Botryotinia asexual morphs along with Botrytis sexual morphs constitute a monophyletic lineage (Holst-Jensen et al. 1998). The phylogeny of the Sclerotiniaceae was further refined by Andrew et al. (2012) using three protein-coding genes: calmodulin, glyceraldehyde 3-phosphate dehydrogenase G3PDH and heat shock protein HSP60.

Staats et al. (2005) performed a comprehensive phylogenetic analysis of the genus Botrytis, at that time comprising 22 recognized species and one hybrid. Using three protein-coding genes (G3PDH, HSP60 and the DNA-dependent RNA polymerase subunit II gene RPB2), they corroborated the morphological and host plant-based classification of Botrytis spp. and divided the genus into two (rather widely separated) clades. Clade I contained species that only infect eudicot plants, while Clade II contained species that can infect either eudicotyledonous or monocotyledonous plants. The use of the same three genes facilitated the discovery of Botrytis sinoallii, a new species infecting Allium spp., and its distinction from other Botrytis spp. infecting the same hosts (Zhang et al. 2010b); B. fabiopsis, a new species infecting broad bean, very distant from B. fabae (Zhang et al. 2010a); and B. caroliniana, a new species infecting blackberry (Li et al. 2012).

Two genes, encoding phytotoxic proteins NEP1 and NEP2, were shown to provide higher resolution in distinguishing species in the genus Botrytis because they seem to be the subject of higher evolutionary rates than the housekeeping genes G3PDH, HSP60 and RPB2 (Staats et al. 2007a). The NEP1 and NEP2 genes were shown to have evolved under positive selection which suggested a role of these proteins in the infection process (Staats et al. 2007a). One might therefore infer that such genes cannot serve as neutral phylogenetic markers. Functional analysis in B. cinerea and B. elliptica using targeted knockout mutants failed to reveal a role of NEP genes in virulence of these two species (Staats et al. 2007b; Cuesta Arenas et al. 2010), which would lend support to considering these genes as neutral markers and adequate tools in phylogeny.

The studies by Staats et al. (2005) revealed incongruence between the phylogenies of Botrytis spp. and their hosts. Species infecting the same host clustered in different (sub) clades, e.g. B. aclada, B. squamosa, B. porri, B. byssoidea and B. sinoallii all infecting Allium. Conversely, closely related species can infect very different hosts, e.g. B. elliptica infecting the monocotyledonous host Lilium and B. ficariarum infecting the dicotyledonous host Ficaria (Staats et al. 2005). More recently, similar incongruence has been reported for newly described species, e.g. B. fabiopsis infecting Vicia faba is very distant from B. fabae infecting the same host (Zhang et al. 2010a), and B. caroliniana infecting blackberries and strawberries is very distant from B. cinerea (Li et al. 2012).

Recently, Khan et al. (2013) combined data from ITS and IGS regions with the G3PHD gene, with the aim of improving molecular identification of Botrytis species that cause neck rot disease on onion. ITS and IGS regions were insufficiently informative to distinguish B. allii and B. byssoidea. The sequences of ITS and IGS for B. allii and B. byssoidea confirmed that they have a close relationship, but G3PDH sequences of several B. allii isolates were clearly distinct, some clustering with B. aclada and others clustering with B. byssoidea (Khan et al. 2013), as might be expected for a hybrid species.

Sequence analysis of the G3PDH and β-tubulin genes amplified from herbarium specimens of Botrytis collected from grey mould-infected apple (deposited in 1932) enabled O’Gorman et al. (2008) to corroborate the existence of B. mali, a species that had been published (Ruehle 1931), but by lack of description was considered doubtful.

Figure 4 shows a maximum likelihood tree of Botrytis spp., based on concatenated sequences of parts of the three genes G3PDH, HSP60 and RPB2 (amplified using primers defined by Staats et al. (2005). Five species described after publication of the phylogeny by Staats et al. (2005), i.e. B. caroliniana, B. deweyae, B. fabiopsis, B. pseudocinerea and B. sinoallii, clearly cluster within the genus and are genuine Botrytis species. Botrytis mali could not be included in the tree due to lack of sequences for the HSP60 and RPB2 genes. Based on G3PDH and ß-tubulin sequences it would cluster with B. paeoniae (O’Gorman et al. 2008).

The Botrytis cinerea species complex

The Botrytis ‘dicot’ clade I consists of B. cinerea, B. pelargonii, B. fabae, B. pseudocinerea and B. calthae. Molecular data do not fully support a separation between B. pelargonii and B. cinerea (Staats et al. 2005, 2007a; Plesken et al. 2014), and the existence of B. pelargonii as a separate species is therefore doubtful. As mentioned above, B. cinerea and B. pseudocinerea are morphologically very similar yet phylogenetically more distant from each other than B. cinerea and B. fabae. All genes tested so far place B. calthae as most remote to all other clade I species.

Botrytis cinerea not only has a broad host range, but also shows considerable phenotypic variability in vegetative growth, conidiation and sclerotium formation (Kerssies et al. 1997; Martinez et al. 2003; Schumacher et al. 2013). Numerous studies have documented a similar variability in genotypic characters, such as amplified restriction length polymorphism, detection of transposable elements and microsatellite heterogeneity. Recently, B. cinerea strains have been described that produce bikaverin, a reddish pigment. These strains contain an intact bikaverin biosynthesis gene cluster (presumably acquired by horizontal gene transfer from Fusarium), which is partially deleted and nonfunctional in most non-bikaverin producing B. cinerea strains (Campbell et al. 2012; Schumacher et al. 2013

A subdivision of B. cinerea into genetically distinct groups has proved to be difficult. Analysis of the presence or absence of two types of transposable elements, named Boty (Diolez et al. 1995) and Flipper (Levis et al. 1997), was adopted as a tool to divide isolates into four transposon types, Transposa (isolates having both elements), Vacuma (isolates having neither element), Boty and Flipper (Giraud et al. 1997, 1999). This classification led to the discovery of B. pseudocinerea, which is usually Vacuma, but the transposon-based classification turned out to be of limited use since B. cinerea populations appear to consist of mixtures of different transposon types. Intriguingly, predominance of a certain type appears to be influenced by the host. While on grapes, strawberries and tomatoes, Transposa types are predominant, whereas B. cinerea populations from kiwi and apples are dominated by Vacuma types (Esterio et al. 2011; Johnston et al. 2013; Muñoz et al. 2002; Samuel et al. 2012; M. Hahn, unpublished). Reasons for this observation are unknown.

Evidence for genetic differentiation of B. cinerea populations with different host preference was obtained with microsatellite markers. In France, isolates from grapes and blackberries were shown to be divergent, indicating limited gene flow between populations on these host plants (Fournier and Giraud 2008). A recent study on grey mould isolates from fungicide-treated strawberry fields revealed the existence of a predominant B. cinerea genotype, named group S, that is closely related to but distinct from the common genotype of B. cinerea (Leroch et al. 2013). Sequencing of the highly polymorphic MRR1 gene revealed that group S isolates show more than 4 % divergence from B. cinerea strains B05.10 and T4, which have MRR1 genes with 99.9 % identity. Further sequencing of HSP60 and NEP2, and of two FUNYBASE genes that are suitable for phylogenetic studies (Marthey et al. 2008), partially supported the genetic separation of group S isolates (Johnston et al. 2013; Leroch et al. 2013). Genome sequencing of several B. cinerea and group S strains, and the analysis of additional polymorphic genes in isolates collected from various host plants in different countries, revealed at least two subclades that could be separated from the common B. cinerea genotype (Plesken and Hahn, unpublished). In fungicide-treated strawberry fields group S isolates dominated, whereas grapes were infected almost exclusively by common B. cinerea genotypes. These data, together with those of putative new endophytic Botrytis taxa that grouped close to B. cinerea (Shipunov et al. 2008), support the idea that B. cinerea represents a species complex, comprising genetically and phenotypically distinct groups.

Recommended genetic markers

G3PDH, RPB2 and HSP60—placement within the Sclerotiniaceae and the ascomycetes

NEP1 and NEP2—for higher resolution within the genus Botrytis,

The NEP1 and NEP2 genes are under positive selection (Staats et al. 2007a) and potentially influence interactions with the host plants. The NEP genes should therefore be used with caution.

Research is ongoing to identify a set of highly polymorphic genes that better resolve the phylogeny of taxa in clade I (Hahn et al., unpublished). It remains to be established whether those gene are equally useful for resolving the clade II species, and whether universal primers can be designed before these genes can be employed to infer a comprehensive phylogeny of the entire genus.

Choanephora

Background

The genus Choanephora belongs to family Choanephoraceae in the order Mucorales (former Zygomycota). The genus was introduced by Currey (1873) for C. cunninghamii, to replace the generic name of his newly described species Cunninghamia infundibulifera, as Cunninghamia already existed as a genus of conifers. Because the specific epithet could not be retained, Choanephora cunninghamia remained invalid, based on the same type as Cunninghamia infundibulifera. The proper name Choanephora infundibulifera was validly published by Saccardo (1891), so the correct authorship of the species is “(Currey) Sacc.” It is also the type species of the genus. Choanephora was monographed by Hesseltine (1953), Milko and Beljakova (1970) and Kirk (1984). Currently the genus is classified within the family Choanephoraceae which can be distinguished by the presence of a persistent sporangium wall that ruptures at preformed sutures. It is furthermore placed in the subfamily Choanephoroideae, which is characterized by the presence of apposed suspensors and smooth zygospores (Hoffmann et al. 2013).

Both species of the genus can grow as saprobes, but they frequently become plant pathogens causing various leaf and fruit rots and blights and are commonly reported from a wide range of plant hosts, including angiosperms (monocotyledons and dicotyledons) and gymnosperms (Farr and Rossman 2014). Their distribution is worldwide, however, disease development is more common in tropical and subtropical regions characterized by high temperatures and humidity. Choanephora cucurbitarum is the causal agent of fruit and blossom rot of various cucurbits, e.g. yellow crookneck squash (Kucharek and Simone 1983). This species is also known from crop plants such as green beans (McMillan 1972), garden peas (Oikawa et al. 1986), and okra (El-Sayed and El-Sajed 2013) and is reported as an agent of wet rot of Mesembryanthemum crystallinum in hydroponic greenhouse culture in Japan (Kagiwada et al. 2010). It is very common during rainy summers in the southeastern United States and globally in other regions with similar climates. Recently it was isolated also from cultivated Hyoscyamus muticus in Japan (Abdel-Motaal et al. 2010) and Withania in India (Saroj et al. 2012). Choanephora often attacks tissues that have been damaged mechanically by insects or otherwise; plants that are poorly adapted to a hot humid climate are particularly prone to infection by the genus. The general appearance of Choanephora rot is similar to that of blights caused by other Mucorales representatives. Signs of infections on fruits or leaves include water-soaked, necrotic lesions, which progress rapidly under wet conditions. As the fungus begins to produce spores, affected tissues become dark grey-brown and hairy. This specific appearance results from the tall sporangiophores that produce a cluster of brown, one-spored sporangiola at their tips (Turkensteen 1979).

Species identification and numbers

Although more than ten species (and many varieties) have been described within this genus, only two species (viz. Choanephora infundibulifera and Choanephora cucurbitarum) were finally recognized in a monograph of the genus (Kirk 1984). These two species can be distinguished by shape and ornamentation of indehiscent sporangiola. C. cucurbitarum produces ellipsoid sporangiola, which are usually distinctly longitudinally striate, whereas C. infundibulifera forms subglobose to obovoid sporangiola with usually smooth or faint striate ornamentation. The remaining species were synonymized under these taxa (e.g. C. mandshurica is currently a synonym of C. cucurbitarum) or were moved to other genera (e.g. C. persicaria is a synonym of Gilbertella persicaria). Choanephora circinans with its two varieties (C. circinans var. indica and C. circinans var. prolifera) were moved by Kirk (1984) to Poitrasia. Poitrasia was established for those species belonging to the family Choanephoraceae that do not form dehiscent or indehiscent sporangiola (Kirk 1984). Although Poitrasia is primarily a soil-borne genus, it has been isolated from Equisteum arvense (Rai 1990). Recent molecular studies confirmed the taxonomic position of Poitrasia proposed by Kirk (1984).

Molecular phylogeny

All Choanephora strains available in CBS culture collection (three strains of C. infundibulifera and five strains of C. cucurbitarum) have been sequenced for their ITS sequences and included in molecular analysis by Walther et al. (2013). These studies showed that the universal fungal DNA barcoding marker–the ITS region (Schoch et al. 2012)–is sufficient for Choanephora species identification (Table 6, Fig. 5). Multigene phylogenetic analysis including representatives of this genus was performed by Hoffmann et al. (2013).
Fig. 9

Phylogram generated from parsimony analysis based on combined ITS and GPDH sequenced data of Curvularia. Parsimony bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The scale bar indicates ten changes. The tree is rooted with Alternaria alternata

Recommended genetic markers

  • The internal transcribed spacer (ITS)–generic and species level

  • The large and small subunits (LSU and SSU) of nrDNA–placement within the Mucorales order, higher-level phylogeny

  • The partial actin gene (ACT) and the partial translation elongation factor 1-alpha gene (TEF)–higher-level phylogeny

Colletotrichum

Background

The genus Colletotrichum was introduced by Corda (1831) and belongs to the family Glomerellaceae (Glomerellales, Ascomycota). Colletotrichum is a coelomycetous phytopathogenic genus with a Glomerella sexual state that includes a number of important pathogens causing diseases of crops and fruits worldwide (Cai et al. 2009; Cannon et al. 2012; Doyle et al. 2013). Colletotrichum species have furthermore been recorded as endophytes in angiosperms, conifers, ferns, lichens and grasses (Hofstetter et al. 2012; Damm et al. 2012b; Cannon et al. 2012; McKenzie et al. 2009; Petrini et al. 1990; Manamgoda et al. 2013; Tao et al. 2013). This genus was voted the eighth most important group of plant pathogenic fungi in the world, based on perceived scientific and economic importance (Dean et al. 2012). Colletotrichum species commonly cause anthracnose resulting in sunken necrotic lesions on leaves, stems, flowers and fruits of numerous hosts, including important crops (Lenne 2002; Waller et al. 2002; Agrios 2005; Cai et al. 2009; Than et al. 2008; Peng et al. 2012; Doyle et al. 2013). It is therefore important to plant health disease practitioners, quarantine personnel and plant breeders to know what species infect which crops (Huang et al. 2013b; Lima et al. 2013; Giaretta et al. 2010; Sangeetha and Rawal 2010; Liu et al. 2009a; Akinbode and Ikotun 2008; Adegbite and Amusa 2008; Peres et al. 2002). Therefore, having a rigid and stable taxonomy for the identification of Colletotrichum species is a significant practical concern (Shenoy et al. 2007). Identification of Colletotrichum species has been difficult due to the lack of reliable morphological features and confused, ambiguous species boundaries (Hyde et al. 2009a, b; Cai et al. 2009). Difficulties in recognising Colletotrichum species has resulted from having a few and variable morphological characters, widespread host ranges and pathogenicity, lost specimens or type specimens in poor condition and incorrectly named sequences in NCBI (Freeman et al. 2000; Du et al. 2005; Thaung 2008; Crouch et al. 2009a, b; Damm et al. 2009; Cai et al. 2009).

Colletotrichum species are extensively studied as model organisms for research in genetics (Cannon et al. 2012). The pathogenicity genes of C. higginsianum were discovered by random mutagenesis (Huser et al. 2009). Genomes and transcriptomes of C. higginsianum and C. graminicola were studied through the use of two different infection strategies by O’Connell et al. (2012). Work on the genetics of pathogenicity in the C. orbiculare species aggregate led to transformation of pathogenic strains to endophytic forms (Cannon et al. 2012). Gene manipulation techniques such as Agrobacterium tumefacien-mediated transformation or protoplast transformation were established (Tsuji et al. 2003). Peroxisome biogenesis genes, PEX6 and PEX13 were identified and their pathogenesis was functionally analyzed (Fujihara et al. 2010). The importance of the pexophagy factor ATG26 for apressorium formation was discovered by Asakura et al. (2009). Whole genomes of C. higginsianum and C. graminicola have been sequenced (O’Connell et al. 2012). Correct species identification is essential in plant pathogenic genera. In order to have effective measures to prevent the unwanted entry of diseases in to a country, the plant pathologists should be able to name the Colletotrichum species confidently. Therefore, pathologists need to be able to clarify and identify the species of Colletotrichum using the wide genetic variation among the taxa (Cannon et al. 2000).

Species identification and numbers

Colletotrichum species have been traditionally named after their hosts. The history of naming Colletotrichum species has been reviewed in several key papers (Cannon et al. 2008, 2012; Hyde et al. 2009a). Cai et al. (2009) outlined the recent polyphasic protocols for species identification: A total of 25 Colletotrichum species have been epitypified, one has been neotypified and three have been lectotypified (Cannon et al. 2008; Damm et al. 2009, 2012a, b, 2013; Doyle et al. 2013; Liu et al. 2011a, b, 2013; Su et al. 2011; Weir and Johnston 2010; Weir et al. 2012). Significant changes to the understanding of Colletotrichum species took place with incorporation of these polyphasic approaches, especially the use of multi-marker phylogenetic analysis, classification and knowledge of species complexes, as well as epitypifications for many species (Cai et al. 2009; Cannon et al. 2012; Damm et al. 2012a, b, 2013; Doyle et al. 2013; Su et al. 2011; Weir et al. 2012). Cannon et al. (2012) studied nearly all presently sequenced species in the genus using a six-gene analysis, and revealed at least nine clades; 119 species previously thought to be well circumscribed proved to be polyphyletic. Colletotrichum gloeosporioides (Cannon et al. 2008; Phoulivong et al. 2010a, b; Weir et al. 2012), C. acutatum (Marcelino et al. 2008; Shivas and Tan 2009; Damm et al. 2012a), C. boninense (Moriwaki et al. 2003; Yang et al. 2009; Damm et al. 2012b), C. orbiculare (Damm et al. 2013) form important species complexes within Colletotrichum and well-resolved among all the nine clades. Further studies in the C. gloeosporioides species complex led to identification of C. murrayae (Peng et al. 2012), C. viniferum (Peng et al. 2013), C. citricola (Huang et al. 2013b), C. fructivorum (Doyle et al. 2013), C. melanocaulon (Doyle et al. 2013), C. temperatum (Doyle et al. 2013), C. endophyticta (Manamgoda et al. 2013) and C. syzygicola (Udayanga et al. 2013). Tao et al. (2013) introduced seven new species; four species belonging to the graminicola clade, two species belonging to the spaethianum clade and one singleton species. Damm et al. (2013) resolved C. orbiculare and introduced four new species. Crouch (2014) introduced a new species complex, C. caudatum, with five new species found on warm-season grasses, characterized by the conidial apex reducing into a filiform appendage. The current numbers of species recognised in the genus are listed in Table 7.

Molecular phylogeny

Some species such as Colletotrichum gloeosporioides were defined using ITS sequence data, but the outcome was not good partially due to prolific misidentification in GenBank and because ITS does not resolve Colletotrichum species well. In Colletotrichum, species definitions based on ITS sequence data, the “universal” DNA barcoding marker for fungal species has proved unsatisfactory (Du et al. 2005; Crouch et al. 2009b; García et al. 2009; Cannon et al. 2012; Doyle et al. 2013; Gunjan et al. 2013). Comparison of a phylogenetic tree of Colletotrichum species derived from ITS sequence alone and one generated from multi-marker data confirms that ITS resolves major clades well, although it does not reflect their higher-order relationships accurately in all cases (Cannon et al. 2012). Cannon et al. (2012) suggested that a robust sequence-based identification system for Colletotrichum species must therefore use an alternative molecular marker or a combination of markers. Damm et al. (2012a) indicated that the most diagnostic markers are β-tubulin and GPDH. β-tubulin performed marginally better than GPDH due to a larger overall number of base pair differences, but even so, some clades differed only by one base pair in the β-tubulin alignment. As single genes that were used are not efficient to differentiate the species, Cai et al. (2009) suggested using multiple markers. Cannon et al. (2012), Weir et al. (2012), and Damm et al. (2012a, b) used several genetic markers: actin (act), chitin synthase (chs1 β-tubulin and ITS which revealed that Colletotrichum comprises nine major clades as well as a number of small clusters and singleton species. Many recent studies used multimarker phylogeny including actin (act), chitin synthase (chs1), β-tubulin, calmodulin (cal), glyceraldehydes-3-phosphate dehydogenase (gadph), histamine (HIS3), glutamine synthetase (GS), DNA lyase (apn2), intergenic region of apn2 and MAT1-2-1 genes (ApMat) (Weir et al. 2012; Damm et al. 2012a, b; Cannon et al. 2012; Peng et al. 2012; Doyle et al. 2013; Gunjan et al. 2013) to understand the phylogenetic divergence of Colletotrichum species. There is, however, no agreement among mycologists as to which genetic markers should be used (Doyle et al. 2013; Gunjan et al. 2013). Silva et al. (2012) stressed the need to use ‘powerful genes’ such as ApMat and Apn25L. The Apmat marker provides better resolution as compared to the genetic markers used by Weir et al. (2012), Silva et al. (2012), Doyle et al. (2013) and Gunjan et al. (2013). Up to now it has been a better gene-marker for resolving species within C. gloeosporioides species complex (Doyle et al. 2013; Gunjan et al. 2013). Only ITS sequences are available for several species of Colletotrichum showing the need of sequencing the other important gene regions and those species were not included in this analysis. Here we present an analysis using six genetic markers for all the Colletotrichum species that are accepted (Fig. 6) and for the C. acutatum species complex (Fig. 7). Figure 8 presents the analysis of C. gloeosporioides species complex using the apmat gene. The whole genomes of several species of Colletotrichum have been sequenced, such that it is now possible to carry out whole-genome analysis, and compare this with single gene analysis to establish a gene (or gene combinations) that can really resolve species in the genus.
Fig. 10

Phylogram generated from parsimony analysis based on combined ITS, EF1-α, β- tubulin, and CAL sequenced data of Diaporthe. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Diaporthella corylina CBS 121124

Fig. 11

Phylogram generated from parsimony analysis based on combined ITS, TEF and β- tubulin sequenced data of Diplodia. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Lasiodiplodia theobromae CBS 164.96

Fig. 12

Phylogram generated from parsimony analysis based on combined ITS and TEF sequenced data of Dothiorella. Parsimony bootstrap support values greater than 50 % are indicated above the nodes, and branches with Bayesian posterior probabilities greater than 0.95 are given in bold. The ex-type (ex-epitype) and voucher strains are in bold. The scale bar indicates ten changes. The tree is rooted with Spencermartinsia viticola CBS 117009

Recommended genetic markers

  • ITS alone will not resolve species in the genus, but it can separate taxa to species complexes. Multigene analysis using the following genes has been recommended for a backbone tree for species of Colletotrichum:

  • GPDH–Glyceraldehyde-3-phosphate dehydrogenase- resolves to species level, more accurate.

  • β-tubulin–Beta-tubulin resolves to species level

  • ApMat–Intergenic region of apn2 and MAT1-2-1 genes can resolve within the C. gloeosporioides complex

  • GS–glutamine synthetase–CHS-1. HIS3–Histone3 and ACT–Actin–Placement within the genus and also some species-level delineation.

These marker combinations can resolve the phylogenetic positions of most species in the genus. GPDH alone can delineate the majority of species. However, research is ongoing to identify better genetic markers to resolve the phylogenetic position of many species of Colletotrichum.

Curvularia

Background

Curvularia is a dematiaceous hyphomycete genus in the family Pleosporaceae, Pleosporales, Dothideomycetes (Ascomycota) (Boedijn 1933). It is typified by C. lunata. Curvularia species have been recorded as saprobes and also plant, human and animal pathogens. Bipolaris and Curvularia species are associated with Cochliobolus sexual states (Sivanesan 1987). Curvularia species are found as plant pathogens especially associated with the family Poaceae. Species such as C. lunata, C. tuberculata and C. trifolii cause leaf spots and leaf blights of some cereal crops such as maize, rice and horticultural crops such as Bermuda grasses and turf grasses (de Luna et al. 2002). The most frequent human and animal pathogens within the genus are C. aeria, C. geniculata, C. lunata, C. inaequalis, C. verrucosa and C. borreriae. These species cause keratitis, sinusitis, cutaneous and subcutaneous infections, peritonitis, onychomycosis, endocarditis, endophthalmitis, cerebral phaeohyphomycosis, and allergic bronchopulmonary as well as disseminated disease (da Cunha et al. 2013).

Species identification and numbers

Curvularia is morphologically characterized by its dark mycelium, geniculate conidiophores with sympodial, tretic conidiogenous cells, conidia with smooth to slightly verrucose wall and several false septa (distosepta). Morphological species identification of Curvularia species is challenging as many species have morphological similarities and have overlapping conidial dimensions. Most of the clinical isolates and common plant pathogens of Curvularia are recorded as C. lunata, which was recently epitypified (Manamgoda et al. 2012a). Following phylogenetic assessments, it was revealed that most of the sequences named as C. lunata in GenBank are incorrectly identified (Cai et al. 2011; da Cunha et al. 2013). Ellis (1971) and Sivanesan (1987) described 37 species in the genus Curvularia and currently there are 122 species epithets in Index Fungorum.

Molecular phylogeny

Phylogenetic recognition is crucial for species identification in Curvularia. Former morphological identifications do not correlate with the phylogeny (Manamgoda et al. 2012a, b). Combined ITS and GPDH analysis for Curvularia and its sister genus Bipolaris by Berbee et al. (1999) revealed that some Bipolaris species cluster within the genus Curvularia. Curvularia was therefore redefined by Manamgoda et al. (2012a) based on a combined phylogenetic analysis of ITS, GPDH, TEF and LSU. Nine Bipolaris species clustering within Curvularia were transferred and their nomenclature redefined (Manamgoda et al. 2012a). Lack of ex-type cultures and epitypifications form limitations for phylogenetic species recognition. In this paper we present a phylogenetic tree with combined ITS and GPDH sequences obtained from available type material and voucher cultures (Table 8, Fig. 9). This can be used as a backbone in the identification of Curvularia species.
Fig. 13

Fusarium The single most parsimonious tree inferred from a combined RPB1 and RPB2 dataset indicating the phylogenetic relationships among species complexes in the genus Fusarium. Branches with bootstrap intervals greater than 70 % and Bayesian posterior probabilities greater than 0.95 are indicated in bold. The NRRL (Agricultural Research Service Culture Collection, Peoria, Illinois USA) and RBG (Royal Botanic Gardens Trust Culture Collection, Sydney, New South Wales, Australia) numbers are indicated for all reference taxa

Recommended genetic markers

  • GPDH is the best single genetic marker for the genus Bipolaris (Manamgoda et al. 2012a). It is recommended to use a combination of ITS and GPDH. Another useful gene is TEF.

Diaporthe

Background

Diaporthe (=Phomopsis) is a cosmopolitan genus of fungi comprised of endophytes, plant pathogens, and saprobes occurring on a wide range of annual and perennial hosts, including economically important crops (Uecker 1988; Farr and Rossman 2014; Udayanga et al. 2011). The genus belongs to class Sordariomycetes, order Diaporthales and the family Diaporthaceae, typified by the species Diaporthe eres Nitschke (Wehmeyer 1933). With the change to one scientific name for fungi (McNeill et al. 2012), Diaporthe has priority, being the older generic name compared to Phomopsis. Many species are able to colonise diverse hosts as opportunists; some species are host specific and multiple species can even co-occur on the same host (Mostert et al. 2001; Farr et al. 2002a; Crous and Groenewald 2005). Species of Diaporthe cause cankers, diebacks, root rots, fruit rots, leaf spots, blights and wilts on a wide range of plant host including some economically important hosts and have been the subject of considerable phytopathological research. Examples of diseases on major crops include Diaporthe/Phomopsis complex causing soybean seed decay, pod and stem blight and cankers, sunflower stem canker (D. helianthi), dead arm of grapevines (D. ampelina) and melanose in Citrus (D. citri) (Van Niekerk et al. 2005; Santos et al. 2011; Thompson et al. 2011; Udayanga et al. 2014a, b). In addition, several species of Diaporthe are known from clinical reports of immuno-compromised patients, although these pathogens are only provisionally identified to species level (Garcia-Reyne et al. 2011; Mattei et al. 2013). Diaporthe comprises a major component of endophytes in tropical and temperate trees, and several species have been used in secondary metabolite research (Isaka et al. 2001; Li et al. 2010a, b; Kaul et al. 2012).

Species identification and numbers

The Genealogical Concordance Phylogenetic Species Recognition (GCPSR) has been applied in the genus Diaporthe to define the species boundaries in recent studies (Udayanga et al. 2012b; Gomes et al. 2013; Tan et al. 2012). Therefore species delimitation is currently based on DNA sequence data and comparison of morphological characters (Santos and Phillips 2009; Santos et al. 2010; Diogo et al. 2010; Udayanga et al. 2014a, b). Although the genus Diaporthe has received much attention, few phylogenetic studies have thus far been conducted; hence the taxonomy of some of the species in this genus is still uncertain including many of the common plant pathogens. Index Fungorum lists 892 Diaporthe names and 983 Phomopsis names whereas MycoBank (2014) lists 919 Diaporthe names and 1,040 Phomopsis names. However, the names available in the literature are mostly applied based on host association and morphology except fewer species described in last two decades based on DNA sequence data. Ex-type cultures are available for less than 100 species known despite the large number of species listed in databases and literature. The delimitation of species within the genus Diaporthe improved once DNA sequence data were incorporated (Castlebury and Mengistu 2006; Van Rensburg et al. 2006; Santos et al. 2010; Udayanga et al. 2012b, 2014a, b), since this facilitates obtaining detailed insight into complex evolutionary relationships.

Molecular phylogeny

Since the first molecular phylogenetic study in Diaporthe (Rehner and Uecker 1994), rDNA ITS, partial sequences of translation elongation factor 1-α (TEF) and mating type genes (MAT 1-1-1/1-2-1) have commonly been used in molecular phylogenetic studies in this genus (Van Niekerk et al. 2005; Van Rensburg et al. 2006; Santos et al. 2010; Udayanga et al. 2011; Sun et al. 2012). Udayanga et al. (2012a) used ITS, TEF, β- tubulin and CAL genes with a selected set of ex-type cultures and additional isolates to infer the phylogeny of the genus. In a parallel study, a multi-marker phylogeny was effectively used to describe novel species in Diaporthe based on fresh collections from Thailand (Udayanga et al. 2012b). Gomes et al. (2013) used a Brazilian collection of isolates and existing ex-type cultures for a combined phylogenetic analysis of five genetic markers which included ITS, TEF, β- tubulin, CAL and HIS. They introduced several novel taxa from Brazilian collections from medicinal plants with one epitype for Diaporthe anarcardi from Kenya. Udayanga et al. (2014a, b) revisited the Diaporthe species associated with Citrus worldwide with comprehensive assessment of the genes including ITS, TEF, β- tubulin, CAL and ACT. The study revisited several important phytopathogens including D. citri, D. cytosporella, D. forniculina and D. rudis, with the epitypes designated with modern descriptions. The clarification of D. foeniculaina and D. rudis revealed the potential extensive host association of some species.

Udayanga et al. (2014a) further emphasized that ITS alone can cause much confusion in defining closely related taxa, which has also been noted by several previous researchers regarding closely related species in Diaporthe (Farr et al. 2002a, b; Murali et al. 2006; Santos et al. 2010). The variation of ITS sequences can result in superfluous, multiple terminal branches in combined analyses, even when other gene regions do not support these distinctions (Udayanga et al. 2014a, b). The TEF gene is informative when it comes to clarifying species limits in Diaporthe (Table 9, Fig. 10).
Fig. 14

Phylogram generated from Maximum likelihood analysis based on ITS sequenced data of Gilbertella. Bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold

Recommendations

ITS and TEF are recommended for preliminary identification of the species (Castlebury et al. 2001; Castlebury 2005; Santos and Phillips 2009; Santos et al. 2010). ITS, TEF, β- tubulin, CAL, HIS and ACT should be used in combined analysis (selection of 4–5 genes), with recommended primers in relevant publications (Udayanga et al. 2012b, 2014a, b; Gomes et al. 2013).

Diplodia

Background

Species of Diplodia (Botryosphaeriaceae) are endophytes, pathogens, or saprobes associated with cankers, dieback and fruit rot (Crous et al. 2006; Slippers and Wingfield 2007) in a wide range of hosts of agricultural and forestry importance (Farr and Rossman 2014). Cryptic speciation is common in the genus Diplodia, which makes species identification difficult if only based on morphological characters (Phillips et al. 2012, 2013). Denman et al. (2000) suggested that Lasiodiplodia could be a synonym of Diplodia, however recent studies accepted them as distinct genera (Pavlic et al. 2004; Burgess et al. 2006; Damm et al. 2007; Alves et al. 2008).

The genus Diplodia was introduced by Montagne (1834) with concepts altering over the years and has been regarded as including species with dark brown, 1-septate conidia (Phillips et al. 2005). Diplodia is defined by having uni or multilocular conidiomata lined with conidiogenous cells that form hyaline, aseptate, thick-walled conidia at their tips (Phillips et al. 2005). Diplodia mutila is the type species of Diplodia (Montagne 1834; Fries 1849), however, there are no living cultures linked to the holotype. As this has severely hampered studies on taxonomy and phylogeny of Diplodia, Alves et al. (2004) provided a detailed description of this species based on one isolate from grapevines in Portugal (CBS 112553).

Species identification and numbers

Diplodia is a large genus and a search in MycoBank (2014) revealed 1,317 names. Species in Diplodia were described, often based on host association, which later resulted in a proliferation of species names. According to Slippers et al. (2004d), host is not of primary importance in species differentiation, thus, many of the names in Diplodia are likely to be synonyms.

Based on DNA sequence data (single or multimarker) and minor differences in conidial morphology, there are currently about 20 Diplodia species (de Wet et al. 2003; Alves et al. 2004, 2006; Gure et al. 2005; Damm et al. 2007; Lazzizera et al. 2008; Pérez et al. 2010; Jami et al. 2012; Phillips et al. 2012, 2013; Linaldeddu et al. 2013; Lynch et al. 2013). The phylogenetic analysis was performed based on up to date holotype or ex-epitype sequence data available in GenBank (Table 10).

Molecular phylogeny

Studies on the taxonomy and phylogeny of Diplodia were hampered by a lack of an ex-type culture linked to the generic type, D. mutila. A collection of D. mutila from Populus with an ex-type culture was designated as epitype by Alves et al. (2014). They obtained a large collection of Diplodia strains from ash and other woody hosts showing V-shaped cankers and branch dieback. These strains were identified based on morphological characters and DNA sequence data. Since 2003 several new species have been described in Diplodia and these species were recognized mainly from DNA sequence data. Diplodia scrobiculata was differentiated from D. sapinea on the basis of multiple gene genealogies inferred from six protein coding genes and six microsatellite loci (de Wet et al. 2003). Diplodia africana (Damm et al. 2007), D. olivarum (Lazzizera et al. 2008) and D. cupressi (Alves et al. 2006) have been differentiated from D. mutila on the basis of formation of distinct clades in phylogenies based on ITS and TEF sequence data and due to their unique conidial morphology (Phillips et al. 2012).

Combined morphological and phylogenetic analyses of DNA sequence data from ITS and TEF (Alves et al. 2014) showed that the Fraxinus isolates from Italy, Netherlands, Portugal and Spain belong to three distinct species namely Diplodia fraxini, D. mutila and D. subglobosa. The phylogenetic tree constructed with holotype or ex-epitype sequences is presented in Fig. 11.

Recommended genetic markers

  • LSU and SSU–generic level

  • ITS, TEF and β-tubulin–species level

ITS, TEF and β-tubulin are the common genetic markers used in identification of Diplodia species. Combined ITS and TEF genes provide satisfactory resolution for resolving species.

Dothiorella

Background

Dothiorella (Botryosphaeriaceae) was proposed by Saccardo in 1880 (Crous and Palm 1999) with D. pyrenophora as the generic type. The delimitation of the genus has been in a state of flux since it was introduced, and detailed explanations of its taxonomy have been given by Sutton (1977), Crous and Palm (1999) and Phillips et al. (2008, 2013). Crous and Palm (1999) examined the holotype of D. pyrenophora and synonymised Dothiorella under Diplodia based on a broad morphological concept of Diplodia. That treatment was followed by Denman et al. (2000), Zhou and Stanosz (2001) and Slippers et al. (2004a). Phillips et al. (2005) re-examined the type of D. pyrenophora and found that the conidia become brown and 1-septate when they are still attached to the conidiogenous cells, while in Diplodia the conidia are hyaline and become dark and septate only after discharge from the conidiomata. Crous et al. (2006) confirmed these morphological differences by re-examining types of both Diplodia and Dothiorella. The sexual state of the species is rarely found in nature and no sexual morph was formed in culture for any of the species, except for D. sarmentorum and D. iberica. Therefore, differentiation of species is mostly derived based on the asexual morphs and cultural characteristics.

Species identification and numbers

As members of Botryosphaeriaceae, species of Dothiorella are known as endophytes, pathogens and saprobes in association with various woody plants, and species in Dothiorella were mostly described based on host association, much as for other members of Botryosphaeriaceae. This led to the introduction of many species names, and there are 368 epithets for Dothiorella in Index Fungorum (2014) and 393 species names in MycoBank (2014). Slippers et al. (2013) suggested that host association should not be considered an important factor in species definition of the Botryosphaeriaceae, therefore most of these names are likely synonyms. There are 19 described species with available cultures, and with the exception of D. sarmentorum all have been described after 2005. The phylogenetic analysis was performed based on up to date holotype or ex-epitype sequence data available in GenBank (Table 11).

Molecular phylogeny

Phillips et al. (2005) broadened the concept of Botryosphaeria and included Dothiorella in Botryosphaeria based on ITS analysis. Crous et al. (2006) recognised ten lineages within Botryosphaeriaceae corresponding to different genera based on phylogenetic analysis of 28S rDNA, and the three species D. iberica, D. sarmentorum and D. viticola formed a clade within Botryosphaeriaceae. These were assigned to Dothidotthia. Subsequently, Phillips et al. (2008) showed that Do. symphoricarpa (the type species of Dothidotthia) belongs in a distinct family within the Pleosporales, while D. sarmentorum, D. iberica and D. viticola fall within two separate genera in the Botryosphaeriaceae and a new genus, Spencermartinsia was introduced to accommodate D. viticola. Phillips et al. (2013) listed all cultures of available Dothiorella species, and provided a key to species, as well as a phylogenetic tree. Abdollahzadeh et al. (2014) introduced five new Dothiorella species which were associated with woody plants in Iran, New Zealand, Portugal and Spain. The phylogenetic tree constructed with holotype or ex-epitype sequences is presented in Fig. 12.

Recommended genetic markers

  • ITS–placement within the Botryosphaeriaceae (the generic level), and also some specific delineation.

  • TEF–the generic level and inter-specific delineation.

  • β-tubulin–inter-specific delineation.

Slippers et al. (2013) suggested that all of the known species of Dothiorella in culture can be separated based solely on ITS, but bootstrap support values for some of the internal nodes are quite low. Due to the studies on the other members of Botryosphaeriaceae, therefore, we strongly recommend that it is necessary to combine ITS and TEF (or intended β-tubulin gene) when molecular studies are carried out on Dothiorella.

Fusarium

Background

The genus Fusarium was described by Link (1809) and later became a sanctioned name (Fries 1821). It is based on the type species Fusarium sambucinum (Nirenberg 1995). Species in Fusarium were described largely on the basis of the morphology of the canoe shaped septate conidia produced by most species as well as the shape and formation of other asexual spores Leslie and Summerell 2006). The sexual morphs (ascospores produced in perithecia) have played little role in the differentiation of most species as they are rare, if produced at all (Seifert 2001). Fusarium includes a number of species that are very important plant pathogens, some that are potent producers of an array of mycotoxins and several species or species complexes that are involved in diseases of humans (Leslie and Summerell 2006). There are also many species that are apparently endophytic in plants as well as species that are saprobes in soil and in organic matter.

Two species, F. graminearum and F. oxysporum, were included in an assessment of the top 10 fungal plant pathogens by Dean et al. (2012). Fusarium graminearum is the cause of head blight of wheat (Windels 2000), and F. oxysporum causes wilt diseases in a range of crops including bananas, tomatoes and other vegetables as well as cotton (Beckman 1987). Other species of Fusarium cause stalk and cob rots in maize and sorghum, canker diseases in woody plants and root and crown diseases across a vast spectrum of plant species (Summerell et al. 2011). Species of Fusarium produce a very large number of secondary metabolites, but two toxin groups, trichothecenes and fumonisins, are particularly detrimental to livestock and humans (through consumption) and as such are heavily regulated in many parts of the world (Desjardins 2005). As a result of the importance of these diseases, the genus is one of the most heavily researched of all genera of fungi and an enormous body of work on all facets of its biology exists (Leslie and Summerell 2006).

Several sexual morph genera are associated with Fusarium, the most important of which is Gibberella (Desjardins 2003). Most Fusarium species, particularly the plant pathogenic species, have a Gibberella sexual morph. Other sexual morph genera include Albonectria, Haematonectria and Neocosmospora as well as a number of other generic names (Gräfenhan et al. 2011). With the changes to the International Code of Nomenclature for Algae, Fungi and Plants providing the opportunity to have a single name for fungi of this nature there has been a strong consensus amongst the community of researchers working on Fusarium that this name be used for all the fungi in the so-called terminal Fusarium clade (Geiser et al.2013). The end result of this is that species of Fusarium such as F. solani, F. decemcellulare and F. dimerum are included with species with Gibberella sexual morphs in the current generic definition of Fusarium (Geiser et al. 2013).

Species identification and numbers

It is difficult to accurately quantify the number of extant, currently recognized species of Fusarium. Over 1,500 names are listed in MycoBank; Leslie and Summerell (2006) documented 72 species, although this was not intended as a monograph, and many of species have been described in the intervening period (e.g. Jacobs et al. 2010; Laurence et al. 2011; Schroers et al. 2009; Walsh et al. 2010). Recent investigations into a number of important species (e.g. F. graminearum, F. incarnatum, F. oxysporum, F. solani) have provided evidence that they are complexes of phylogenetically distinct lineages that have been, or will eventually be described as species (Aoki et al. 2005; O’Donnell et al. 2004, 2008, 2009).

Molecular phylogeny

There has been substantial work on understanding the phylogenetic relationships within Fusarium, and in defining generic boundaries (e.g. Geiser et al. 2013; O’Donnell et al. 2013). This has provided refined concepts for several important plant pathogenic species (e.g. F. graminearum, F. pseudograminearum, F. subglutinans, F. verticillioides) and it has also shown that several important plant pathogens (especially F. oxysporum and F. solani) are in fact species complexes (Laurence et al. 2014; O’Donnell et al. 2008). A genus-wide phylogeny was inferred using the RNA polymerase largest subunit (RPB1) and RNA polymerase second largest subunit (RPB2) (O’Donnell et al. 2013), as these genes are very informative from a phylogenetic perspective across the whole genus (Table 12, Fig. 13)
Fig. 15

Phylogram generated from parsimony analysis based on combined ITS and TEF sequenced data of Lasiodiplodia. Parsimony bootstrap support values greater than 50 % are indicated above the nodes, and branches with Bayesian posterior probabilities greater than 0.95 are given in bold. The ex-type (ex-epitype) and voucher strains are in bold. The scale bar indicates ten changes. The tree is rooted with Diplodia mutila CBS 112553

Recommended genetic markers

The recommended and most frequently used gene for identification of species of Fusarium is the translation elongation factor 1α gene (TEF) and this is generally used for routine identifications, effectively performing a DNA barcoding function, and forms a significant component of the FUSARIUM-ID database (http://isolate.fusariumdb.org/; Geiser et al. 2004). This database provides a similar facility to GenBank but is based on sequences from accurately identified and validated cultures held in reference collections (Geiser et al. 2004). Using a standard approach (Summerell et al. 2003), sequencing the TEF gene and comparing the sequence with the FUSARIUM-ID database makes it possible to rapidly and accurately identify most pathogenic Fusarium species. The ITS region is less informative in Fusarium from both a barcoding and phylogenetic perspective and as a result it has not been used extensively. This is primarily because there are nonorthologous copies of ITS2 that are incongruent with species phylogenies derived from other unlinked loci in species of economic importance {O’Donnell and Cigelnik (1997) #1278}. As a consequence it is not recommended that ITS be used for differentiation or identification of Fusarium species (Summerell et al. 2003).

Gilbertella

Background

The monotypic genus Gilbertella belongs to the family Choanephoraceae and subfamily Gilbertelloideae (Mucorales, former Zygomycota). It was established by Hesseltine (1960) for species described earlier as Choanephora persicaria by Eddy (1925), and consequently the type species of the genus is Gilbertella persicaria. Benny (1991) proposed a new family, Gilbertellaceae to accommodate this genus. Currently, the genus belongs to the family Choanephoraceae and subfamily Gilbertelloideae that can be distinguished from Choanephoroideae (Voigt and Kirk 2012) by ornamented zygospores and opposed suspensors (Voigt 2012). Although G. persicaria was originally described as Choanephora persicaria (Eddy 1925), its separate position within the family has been confirmed in several studies (Papp et al. 2003; Hoffmann et al. 2013).

In tropical and subtropical regions Gilbertella is a common postharvest pathogen, causing rots of pears (Mehrotra 1963a), peaches (Hesseltine 1960; Mehrotra 1963b; Ginting et al. 1996) and tomatoes (Mehrotra 1966). It was reported by Butler et al. (1960) and Hesseltine (1960) from mulberry (Morus sp.) in USA. It was also recently isolated from pitaya fruits (Hylocereus undatus, Cactaceae) in Japan (Taba et al. 2011) and China (Guo et al. 2012).

Species identification and numbers

Currently, Gilbertella persicaria is the only species within the genus. Although another species–Gilbertella hainanensis–has been described (Cheng and Hu 1965), after recent molecular studies of its ITS sequence, it is not currently recognized as a separate species (Walther et al. 2013). Two varieties of G. persicaria have been described: G. persicaria var. persicaria and G. persicaria var. indica, however only the former was accepted in the monograph published by Benny (1991).

Gilbertella persicaria produces sporangia with a persistent wall that ruptures at preformed sutures in two halves. Ellipsoid, smooth-walled, hyaline sporangiospores with polar appendages are released in droplet of fluid. Light brown ornamented zygospores are formed on opposed suspensors (Hesseltine 1960). Examination of morphology is usually enough for correct species identification. Moreover, the morphological identification may be easily confirmed by ITS sequencing (Table 13, Fig. 14).

Molecular phylogeny

The phylogenetic relationships based on the complete ITS region of Gilbertella representatives and related Mucorales taxa was completed by Papp et al. (2003). All Gilbertella cultures available in the CBS culture collection have been sequenced for their ITS region and were included in a molecular analysis by Walther et al. (2013). These studies showed that the universal fungal DNA barcoding marker–the ITS region (Schoch et al. 2012)–is sufficient for Gilbertella species identification (Fig. 16). The multi-marker phylogenetic analysis including representatives of this genus performed by Hoffmann et al. (2013), confirmed a distinct, well-supported position of Gilbertella within Choanephoraceae family.
Fig. 16

Maximum likelihood tree based on partial LSU sequences for Mucor species and main groups within the genus. Detailed phylogenetic trees for each group may be found in Walther et al. (2013)

Recommended genetic markers

  • The internal transcribed spacer (ITS) region–generic and species level

  • The large and small subunits (LSU and SSU) of nrDNA–placement within the Mucorales order, higher-level phylogeny

Lasiodiplodia

Background

Lasiodiplodia (Botryosphaeriaceae) was introduced by Ellis in 1894 with L. tubericola as the type species. Clendenin (1896) provided a description of the genus and the species, but did not refer to any type or other specimens of the genus or species. Pavlic et al. (2004) could not locate the types, nor find any specimens from the original hosts or origins, but gave a clear concept of the genus and the type. A new status for the type species of Lasiodiplodia has been proposed by Phillips et al. (2013) and they designated CBS 164.96 as ex-neotype culture, and deposited a dried specimen as neotype with convincing reasons, although this specimen was collected from an unidentified fruit in Papua New Guinea, whereas the type was collected in Ecuador on cocoa plant. Twenty new species have been described since 2004; however the generic application of the name, L. theobromae, has not been resolved.

Species identification and numbers

Lasiodiplodia differs from Diplodia species in having striations on the conidia, and differs from Neodeightonia as Lasiodiplodia has conidiomatal paraphyses. Barriopsis differs as it has unique striate conidia, with the striations present on immature, hyaline conidia. A sexual morph has been reported for L. theobromae, which has been linked to Botryodiplodia rhodina (Cooke) Arx, but this link has not been unequivocally proven (Alves et al. 2008; Phillips et al. 2008). Phillips et al. (2013) transferred Auerswaldia lignicola (Liu et al. 2012) to Lasiodiplodia, and this is the only species where the asexual morph and sexual have been definitively linked. There are 30 epithets of Lasiodiplodia recorded in Index Fungorum (2014) and 32 species names in MycoBank (March 2014), and 24 species are currently kept in culture. Species can be differentiated based on conidial morphology (especially dimensions) and morphology of the paraphyses. The phylogenetic analysis was performed based on up to date holotype or ex-epitype sequence data available in GenBank (Table 14).

Molecular phylogeny

Denman et al. (2000) suggested that Lasiodiplodia could be a possible synonym of Diplodia based on the ITS data analysis. However, phylogenetic studies by Zhou and Stanosz (2001), Slippers et al. (2004a) and Phillips et al. (2008) show that it clusters separately from Diplodia. As more genes and molecular data have become available, more complex sections within Botryosphaeriaceae have been resolved. By combining TEF and β-tubulin genes with ITS, Phillips et al. (2005, 2008) reinstated the genus Neodeightonia in the Diplodia/Lasiodiplodia complex and also showed that the latter asexual genera are morphologically and phylogenetically distinct. Most of the known species with available cultures have been described based on at least two genetic markers (ITS, TEF/ β-tubulin). The phylogenetic tree constructed with holotype or ex-epitype sequences is presented in Fig. 15.

Recommended genetic markers

  • ITS–placement within the Botryosphaeriaceae (the generic level), and also some species-level delineation.

  • TEF–generic level and inter-specific delineation.

  • β-tubulin–generic level and inter-specific delineation, mostly for inter-specific delineation.

In most cases, a combination of ITS and TEF will separate all species and a minimal requirement for Lasiodiplodia species separation. However, for some groups, such as L. theobromae, β-tubulin is needed.

Mucor

Background

The genus Mucor belongs to the Mucoraceae, which is the largest and the most diverse family within Mucorales (former Zygomycota; Hoffmann et al. 2013). It was described by Fresenius (1850). The type species of the genus is Mucor mucedo, although the name Mucor had been used long before also by other authors to describe species currently classified as Rhizopus stolonifer (syn. Mucor mucedo L. 1753 or Mucor mucedo (Tode) Pers. 1801).

There has been no comprehensive molecular phylogenetic study in the genus Mucor and consequently its taxonomy is still widely based on morphological characters. Mucor representatives produce nonapophysate sporangia arising directly from the substrate and they do not form stolons. Rhizoids were also considered to be absent in Mucor, but it is now known that they can be produced under certain conditions (Walther et al. 2013).

Mucor representatives are saprotrophs that can be found mainly in soil or on plant debris. They are also known as postharvest plant pathogens, e.g. M. mucedo (Moline and Millner 1981) and M. piriformis (Michailides and Spotts 1990a). In case of peach and nectarine rots, Michailides and Spotts (1990b), Spotts (1990) and Michailides et al. (1992) regarded flies (especially Drosophila melanogaster) and nitidulid beetles (Carpophilus hemipterus and C. freemani) as effective vectors. Mucor rot symptoms include softening of juicy decayed tissue, often with a sweet odour, lesions with a sharp margin and eventually developing of grey mycelium with sporangia. Mucor isolated from several different plant hosts, angiosperms and gymnosperms, monocots as well as dicotyledons. USDA Fungus-Host Database reports 375 cases of Mucor infections from plants from approximately 40 countries in Europe, Central and South-East Asia, Australia, Africa, and North and South America (Farr and Rossman 2014). Mucor circinelloides causes rots in tomatoes (Smith et al. 1976), mangoes (Johnson 2008), yam (Amusa et al. 2003) and peaches (Restuccia et al. 2006). Mucor hiemalis can be pathogenic on guavas (Kunimoto et al. 1977), carrots and cassava (Snowdon 1991). Mucor piriformis is a destructive pathogen of fresh strawberries (Snowdon 1990; Pitt and Hocking 2009) and a major cause of rotting of cold-stored pears, apples, peaches, nectarines and tomatoes (Smith et al. 1979; Bertrand and Saulie-Carter 1980; Michailides and Spotts 1986; Michailides 1991; Mari et al. 2000; Pitt and Hocking 2009; Ukeh and Chiejina 2012), plums (Børve and Vangdal 2007), sweet persimmons (Kwon et al. 2004) and yams (Amusa and Baiyewu 1999; Iwama 2006). Mucor piriformis may infect the stem, calyx or wounds on the skin of fruits (Michailides and Spotts 1990a, b). Mucor mucedo was reported as important postharvest pathogen of strawberries (Dennis and Davis 1977), and from tomatoes (Moline and Kuti 1984). Mucor racemosus was noted causing soft rot of cherry tomato fruits in Korea (Kwon and Hong 2005). Some Mucor species (e.g. M. circinelloides) are also human opportunistic pathogens, especially dangerous to immunodeficient patients (Walther et al. 2013).

Species identification and numbers

The last extensive studies of the genus Mucor (Schipper 1973, 1975) are from the pre-molecular era. Based on morphological features and mating experiments Schipper (1976, 1978) recognized 39 species, 4 varieties and 11 formae. In the following years further species were described (e.g. Watanabe 1994; Zalar et al. 1997). Molecular phylogenetic analyses of the entire Mucorales revealed the polyphyly of the genus (Voigt and Wöstemeyer 2001; O’Donnell et al. 2001). The study of Walther et al. (2013) on the genetic diversity within the Mucorales based on sequences of the nuclear ribosomal internal transcribed spacer region (ITS) and the large ribosomal subunit (LSU) strongly supported the polyphyly of Mucor. The genus was split into several morphological groups differing in the size of the sporangia and the branching mode of the sporangiophores that are widely in agreement with the intrageneric classification of Schipper (1973). However, ?in molecular analyses these groups are intermingled by other sporangia-forming genera such as Pilaira und Pirella and sporangiola-forming genera such as Ellisomyces, Chaetocladium, Helicostylum and Thamnidium (Walther et al. 2013). The position of the Mycotyphaceae and the Choanephoraceae in relation to the Mucoraceae is still not resolved (Hoffmann et al. 2013).

Recently, the introduction of new species or changes of the taxonomic status were supported by sequence analyses of the ITS and/or rDNA genes (Jacobs and Botha 2008; Budziszewska et al. 2010; Álvarez et al. 2011; Madden et al. 2011). Several studies on certain species or species complexes (Li et al. 2011; Lu et al. 2013) or a particular ecological group (Hermet et al. 2012) used multi-marker approaches for phylogenetic species recognition in the genus Mucor. However, a comprehensive study on the entire genus is still lacking. As a consequence, species and even generic boundaries are still unclear for Mucor. Currently 58 species are recognised within the genus (Walther et al. 2013) (Table 15, Fig. 16).

Molecular phylogeny

The ITS region allows identification to species level for most mucoralean representatives (Walther et al. 2013). Detailed molecular species identification is currently not possible for species complexes such as M. circinelloides or M. flavus because of unclear species boundaries (Walther et al. 2013).

Along with the ITS region for species identification, the LSU (e.g. Fig. 16, Álvarez et al. 2011) or the SSU (e.g. Budziszewska et al. 2010) genes have frequently been used in molecular phylogenetic analyses of Mucor because the ITS is too variable to be confidently aligned across the entire genus (Walther et al. 2013). In addition, the RNA polymerase subunit gene (rpb1) was successfully used for multi-marker studies at the species level (Li et al. 2011; Hermet et al. 2012; Lu et al. 2013). Hermet et al. (2012) also used the fragment of a mini-chromosome maintenance protein (MCM7) and of the 20 S rRNA accumulation protein (tsr1). The multi-marker analysis of the entire Mucorales including representatives of genus Mucor by Hoffmann et al. (2013) were based on partial genes of actin and the translation elongation factor 1-alpha in addition to the rRNA genes.
Fig. 17

Phylogram generated from parsimony analysis based on combined ITS, TEF, β- tubulin, LSU and SSU sequenced data of Neofusicoccum. Parsimony bootstrap support values and Bayesian posterior probabilities greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The scale bar indicates ten changes. The tree is rooted with Spencermartinsia viticola CBS 117009

Recommended genetic markers

  • The internal transcribed spacer (ITS)–genus and species level

  • The RNA polymerase II largest subunit gene (RPB1)–species level

  • The large and small subunits (LSU and SSU) of nrDNA–placement within the Mucorales order, higher-level phylogeny

  • The mini-chromosome maintenance proteins gene (MCM7–higher-level phylogeny)

Neofusicoccum

Background

Pennycook and Samuels (1985) listed Fusicoccum parvum as the asexual morph when they described Botryosphaeria parvum. Neofusicoccum was introduced by Crous et al. (2006) for species that have an asexual morph that occurs with a “Dichomera” like synanamorph (morphologically similar, but phylogenetically distinct from Botryosphaeria). They suggested the name as it provides more information of the morphological state.

Species identification and numbers

On the basis of conidial dimensions and pigmentation, pigment production in media and ITS sequence data, 22 species are currently recognized in Neofusicoccum, although some of these characters have recently been questioned (Abdollahzadeh et al. 2013). Four new species, N. batangarum, N. cordaticola, N. kwambonambiense and N. umdonicola were identified in this complex based on congruence between genealogies of multiple genes (Pavlic et al. 2009a, b; Begoude et al. 2010). Though many species of Neofusicoccum are morphologically similar and can be very difficult to distinguish from one another, an attempt has been made to differentiate all species in a key by Phillips et al. (2013) (Table 16).

Molecular phylogeny

Crous et al. (2006) proposed new combinations for 13 species based on the sequence data from cultures. Based on DNA sequence data for five nuclear markers, Pavlic et al. (2009a, b) identified three new species of Neofusicoccum within the N. parvum/N. ribis species complex in South Africa. N. batangarum was described from Terminalia catappa by Begoude et al. (2010). Analysis of TEF, β-tubulin and LSU gene sequences (Alves et al. 2008; Abdollahzadeh et al. 2010) and Genealogical Sorting Index (GSI) has been used to resolve the asexual morph of Neofusicoccum (Sakalidis et al. 2011) (Fig. 17).

Recommended genetic markers

  • LSU, SSU and ITS–genus level

  • β-tubulin and TEF–species level

Common genetic markers that are used for the identification of Botryosphaeriaceae species are ITS, TEF, β- tubulin, LSU and SSU. Recent studies have shown that the combination of TEF, ITS and β- tubulin is sufficient to characterize species in this lineage. However, even when using only the TEF gene, it is possible to identify distinct species. The unavailability of the TEF sequence of several type species makes species identification using molecular phylogeny problematic. Therefore, in future research, it is recommended to use the combination of TEF, ITS and β- tubulin for better species level delimitation.

Pestalotiopsis

Background

Pestalotiopsis is an appendage-bearing conidial asexual coelomycetous genus in the family Amphisphaeriaceae, Xylariales, Sordariomycetes (Ascomycota) (Barr 1975; Kang et al. 1998) that is common in tropical and temperate ecosystems (Maharachchikumbura et al. 2011, 2012). The sexual state is Pestalosphaeria and only 13 species are known as compared to the asexual state (253 species names). Species of Pestalotiopsis cause a variety of disease in plants, including canker lesions, shoot dieback, leaf spots, needle blight, tip blight, grey blight, scabby canker, severe chlorosis, fruit rots and leaf spots (Espinoza et al. 2008; Maharachchikumbura et al. 2013a, b; Tagne and Mathur 2001). Species belonging to the genus Pestalotiopsis are thought to be a rich source for bioprospecting, and chemical exploration of endophytic Pestalotiopsis species is on the increase (Aly et al. 2010; Xu et al. 2010, 2014). Pestalotiopsis species have been recorded as saprobes where they are recyclers of dead plant material (Maharachchikumbura et al. 2012) and are also known to cause human and animal infections (Pestalotiopsis clavispora) (Monden et al. 2013).

Most Pestalotiopsis names in the literature are described based on host association. However, molecular data have shown that the genus needs revision (Maharachchikumbura et al. 2011, 2012; Zhang et al. 2013c), and many of the traditional species may be spurious. There are also numerous cryptic species, very few distinct species, species with wide host ranges, those with cosmopolitan distribution and some species being opportunistic pathogens. This calls for critical re-examination of the genus, using both morphological studies and a multi-marker phylogeny based on ex-type and ex-epitype cultures (Maharachchikumbura et al. 2012, 2013c).

Species identification and numbers

According to Index Fungorum (2014) there are 253 Pestalotiopsis names, while in MycoBank (2014) there are 264 names. The reason for the large number of names is historical and may not reflect the actual number of species (Jeewon et al. 2004). Kohlmeyer and Kohlmeyer (2001) described P. juncestris, which was isolated from the host Juncus roemerianus; this species is morphologically similar to P. versicolor and several other species of Pestalotiopsis, but the taxon was described as a new species based on the host occurrence. However, recent molecular data have shown that host association and geographical location is less informative for distinguishing taxa (Jeewon et al. 2004; Hu et al. 2007). Isolation of endophytic Pestalotiopsis strains for bioprospecting for new biochemical compounds has shown that the same species can be found in a range of hosts. It has been shown that most of the key conidial characters used in species level separation are not stable and vary with host range, generation, culture and other environmental conditions (Hu et al. 2007). Furthermore, the arrangement of species by Steyaert (1949) and Guba (1961) in various coloured groupings is problematic because this character has been shown to be variable within a species (Liu et al. 2010). Thus, most species in the above arrangements may be confused and many species are probably synonyms. Therefore, most of the species recorded in checklists and the literature may not reflect what actually occurs. Thus, many names assigned to Pestalotiopsis probably lack any accurate taxonomic basis, leaving the taxonomy of the genus markedly confused. Until 1990, phylogenetic understanding of the taxonomy associated with Pestalotiopsis and allied genera was based mainly on conidial characters (Steyaert 1949; Guba 1961; Nag Rag 1993), conidiogenesis (Sutton 1980) and sexual state association (Barr 1975). More recently, some new species have been introduced based on host occurrence, plus morphological and molecular data (Maharachchikumbura et al. 2012, 2013a, b; Strobel et al. 2000). Furthermore, currently only 36 Pestalotiopsis species have either ex-type or ex-epitype sequences.

Molecular phylogeny

Recently, many Pestalotiopsis species have been defined using ITS sequence data, however, there are only a few type cultures available for Pestalotiopsis. For example, Pestalotiopsis clavispora, P. disseminata, P. microspora, P. neglecta, P. photiniae, P. theae, P. virgatula and P. vismiae have numerous ITS sequences in GenBank. However, in phylogenetic studies all these species scattered throughout the phylogram and there appears to be no living ex-type strain for any of these species (Maharachchikumbura et al. 2011). Therefore it is unwise to use GenBank sequences to represent any of these names. Rapid development in molecular phylogeny has had a great impact on Pestalotiopsis taxonomy. For example, random amplification of polymorphic DNA (RAPD) can be used to detect genetic diversity in the genus (Tejesvi et al. 2007). Watanabe et al. (2012) evaluated the use of the ITS2 region and showed that it is conserved at the level of secondary structure rather than the level of primary sequence, which can be used for classification of the Pestalotiopsis. Hu et al. (2007) showed that the ITS region is less informative than the β-tubulin gene in differentiating endophytic species of Pestalotiopsis in Pinus armandii and Ribes spp. A combination of β-tubulin and ITS gave improved phylogenetic resolution, and they suggested that at least two genetic markers should be used to resolve the phylogeny of species of Pestalotiopsis. However, Liu et al. (2010) disagreed with above statement concerning the ITS region as being less informative when compared to the β-tubulin region. They indicated that alignment of the ITS region can be a useful character in grouping Pestalotiopsis to different types of pigmentation, which can be used as a key character for the phylogeny of the species. In order to select suitable markers for better species resolution, Maharachchikumbura et al. (2012) analyzed a combined ACT, β-tubulin, CAL, GPDH, GS, ITS, LSU, RPB 1, SSU and TEF dataset. They compared the morphological data versus the sequence data from each gene to establish which characters satisfactorily resolve the species. They narrowed down the 10 gene regions to three most applicable regions (ITS, β-tubulin and TEF), which were tested individually and in combination, to evaluate the differences between species. The species sequenced with ITS had a high PCR and sequence success rate and β-tubulin and TEF gene regions proved to be favourable taxonomic markers for Pestalotiopsis since they resolved the taxonomic relationships of most species studied. Further, TEF had better PCR amplification success rates and was found to be superior to β-tubulin. TEF is therefore a powerful tool to resolve lineages within Pestalotiopsis. Because of the better PCR and sequencing success rate and fewer difficulties with alignment, editing and better resolution, the TEF gene appears to be a very good molecular marker for phylogenetic investigation of Pestalotiopsis. Furthermore, a combination of ITS, β-tubulin and TEF gene data gave the best resolution as compared to any single marker (Table 17, Fig. 18). In addition to the above three markers, the authors also tested LSU, SSU, ACT and GPDH (low resolution), GS and RPB1 (cannot be synthesized using available primers or multiple copies) and CAL (species resolution is high, PCR success rate low).
Fig. 18

Strict consensus combined (ITS + β-tubulin + TEF) tree from Bayesian analysis of the analyzed Pestalotiopsis. Thickened lines indicate Bayesian posterior probabilities (PP) of 100 %. Strain accession numbers (ex-type are in bold) are followed by the species name. The scale bar represents the expected changes per site. The tree is rooted to Seiridium spp. (D96)

Recommended genetic markers

  • The large subunits of nrDNA (LSU)–placement within the Amphisphaeriaceae (generic level)

  • The internal transcribed spacer (ITS), β-tubulin and TEF–species level (as outlined in Maharachchikumbura et al. 2012)

Phyllosticta

Background

Phyllosticta is an important plant pathogenic genus with coelomycetes asexual states. It was previously placed in Botryosphaeriaceae, Botryosphaeriales, Dothideomycetes, Ascomycota. However following phylogenetic analysis, Wikee et al. (2013c) placed this genus in Phyllostictaceae which is sister to the Botryosphaeriaceae. Phyllosticta species are known to cause leaf spots and various fruit diseases worldwide on a diverse range of hosts including some economically important crops and ornamentals such as citrus, banana, apple, grapes, cranberry, orchids, mai dong and maple (Uchida and Aragaki 1980; Paul and Blackburn 1986; Baayen et al. 2002; McManus 1998; Olatinwo et al. 2003; Paul et al. 2005; Liu et al. 2009b; Wikee et al. 2011, 2012; Shivas et al. 2013b). Some species such as P. capitalensis are endophytes and weak pathogens (Baayen et al. 2002; Glienke et al. 2011; Wikee et al. 2013a), while others such as P. cocoicola are saprobes (Punithalingam 1974; Taylor and Hyde 2003). Phyllosticta species have been also used as bio-control agents and produce novel bioactive metabolites such as phyllostine and phyllostoxin (Yan et al. 2011; Evidente et al. 2008a, b; Wikee et al. 2011, 2013b).

The sexual state of Phyllosticta was named Guignardia which comprises 353 records in MycoBank (Hyde 1995; Crous et al. 1996; Hyde et al. 2010). Phyllosticta species have sometimes been named in Leptodothiorella after their spermatial state (Van der Aa 1973). Most species of Phyllosticta and Guignardia have been described independently, and only a few Phyllosticta species have been linked to their Guignardia sexual morphs (Wulandari et al. 2010). On the other hand, the host ranges of many diseases are poorly understood (Van der Aa and Vanev 2002; Wikee et al. 2011). It has been recommended that Phyllosticta which is the older, more commonly used and more species-rich, should have priority over Guignardia (Zhang et al. 2013a, b, c; Wikee et al. 2013c).

Phyllosticta species have been historically indentified based on morphology, culture characters as well as host association, which has resulted in several taxonomic revisions (Van der Aa 1973; Van der Aa and Vanev 2002). Fresh collections and future molecular analyses should help resolve species relationships (Hyde et al. 2010). Phylogenetic analysis has been routinely used in species identification, in combination with morphological characters (Crous and Groenewald 2005; Hyde et al. 2010; Wikee et al. 2013c). To create a stable and workable taxonomy, neo- or epitypification are required for many species of Phyllosticta (Hyde et al. 2010; Wikee et al. 2013c).

Species identification and numbers

The genus Phyllosticta was first introduced as the generic name for Sphaeria lichenoides by Persoon (1818). Desmazieres (1847) re-defined Phyllosticta, in which he did not restrict the genus to species with one-celled conidia. Consequently, many fungi with one-celled or septate conidia were named as Phyllosticta (Desmazieres 1847; Van der Aa 1973). Saccardo (1878) however, restricted Phyllosticta to species with one-celled conidia, and after that Phyllosticta was further restricted to leaf inhabiting species (Saccardo 1878, 1884; Van der Aa 1973; Petrak and Sydow (1927) published a compilation of Phyllosticta names, and gave extensive descriptions of 28 species. Van der Aa (1973) proposed a morphological identification criterion for the genus and detailed 46 Phyllosticta species based mostly on material collected in Europe and North America. The genus was revised by Van der Aa and Vanev (2002) and they accepted 141 species. The currently used generic circumscription of Phyllosticta is: “pycnidia globose, subglobose or tympaniform, conidiogenous cells holoblastic, with percurrent proliferation, conidia hyaline, 1-celled, ovoid, overate, ellipsoid, short cylindrical, or globose to subglobose, usually bearing a slime layer and an apical appendage” (Van der Aa 1973; Van der Aa and Vanev 2002). During 2002–2014, about 30 new species were described (Motohashi et al. 2008; Wulandari et al. 2009, 2010; Glienke et al. 2011; Wang et al. 2012; Su and Cai 2012; Wong et al. 2012; Wikee et al. 2012, 2013c; Zhang et al. 2013b; Shivas et al. 2013b), with the currently accepted species possibly being more than 171. Unfortunately, molecular data are currently available for about only 69 species (Table 18).

Molecular phylogeny

Phylogenetic analysis has become a standard approach in fungal identification and has been well applied in several other coelomycetous genera such as Colletotrichum (Cai et al. 2009; Crouch et al. 2009b, c; Hyde et al. 2009a, b) and Phoma (Aveskamp et al. 2008, 2010; de Gruyter et al. 2010). Recent reports on Phyllosticta have shown that molecular phylogenetic tools have significantly improved species identification and delimitation; similarly it has improved the resolution in species complexes (Wulandari et al. 2009; Glienke et al. 2011; Wicht et al. 2012).

Baayen et al. (2002) evaluated the P. citricarpa sensu lato from Citrus and associated hosts based on ITS sequence analysis and found that two phylogenetically distinct groups existed: a slowly growing pathogenic group and morphologically similar but fast-growing, non-pathogenic group which latter proved to be P. capitalensis. Wicht et al. (2012) used a polyphasic approach including morphological, molecular and proteomic techniques to analyze samples of G. bidwellii collected from grapevine cultivars and ornamental plants of various geographic origins, and showed that P. ampelicida isolated from grapevine cultivars should be split into two species.

Recent studies have provided clear phylogenetic relationships in the group. These efforts primarily used intron-dominated genes (ITS, ACT, TEF), and highly conserved gene coding regions (LSU, GPDH) that can recognize cryptic species in traditionally morphologically circumscribed species complexes, e.g. P. citricarpa on citrus, P. musarum on banana, P. vaccinii on Vaccinium, G. philoprina on Rhododendron, Hedera, Ilex, Magnolia and Taxus (Glienke et al. 2011; Wang et al. 2012; Wulandari et al. 2009; Wikee et al. 2013c, a, b, c; Wong et al. 2012; Zhang et al. 2013a) (Fig. 19).
Fig. 19

Phylogram generated from parsimony analysis based on combined ITS, TEF, GPDH and ACT sequenced data of Phyllosticta. Parsimony bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold

Recommendations

  • The large and small subunits of nrDNA (LSU and SSU)–placement within the ascomycetes (generic and family level)

  • The internal transcribed spacer (ITS)–generic level

  • Combined ITS, TEF, GPDH and ACT–inter-specific delineation

Phytophthora

Background

While resembling Eumycotan fungi with the production of hyphae, the genus is placed in the kingdom Straminipila, class Oomycetes, order Peronosporales, and family Peronosporacae. The type species is P. infestans described by de Bary in 1876. Since this time over 128 species have been described, many of which are important plant pathogens capable of significantly impacting agricultural production and natural ecosystems. Some species have a rather narrow host range (P. infestans, P. lateralis, P. sojae) while others are capable of infecting a wide range of plant host species (P. cinnamomi, P. nicotianae, P. ramorum). From a historical perspective, most investigations on the genus have focused on the impact of the genus on agricultural production systems, however, more recently there has been an increased interest in investigating the role this genus plays in natural ecosystems as exemplified by the number of publications concerning species such as P. ramorum and P. alni, as well as the description of many new species recovered from environmental sampling (Martin et al. 2012).

Although Phytophthora species resemble Eumycotan fungi with the production of hyphae, evolutionarily they are more closely related to chromophyte algae and plats than to Eumycotan fungi (Wainright et al. 1993). They have cell walls that are primarily cellulose rather than chitin as observed in fungi and they are incapable of synthesizing β-hydroxysterols (which are required for synthesis of hormones regulating sexual reproduction). In addition, Oomycetes are diploid throughout their life cycle in contrast to most true fungi.

An excellent overview of the ecology, biology and taxonomy of the genus (although missing more recently described species) can be found in Erwin and Ribeiro (1996), a review of the recent taxonomic status in Kroon et al. (2012) and an overview of the genus, including molecular identification and diagnostics, in Martin et al. (2012). There are several publically available databases that provide a wealth of up to date information on the genus, along with sequences useful for species identification via BLAST analysis, including the Phytophthora Database (www.phytophthoradb.org), Phytophthora ID (www.phytophthora-id.org) and Q-Bank (www.q-bank.eu). Cline et al. (2008) have published an online list of Phytophthora spp. with a hyperlink for each species to the USDA SMML database that includes host range, distribution and supporting literature.

Species identification and numbers

A complicating factor when trying to identify Phytophthora species or investigate phylogenetic relationships is hybridization among distinct evolutionary lineages. While this does not appear to be a common occurrence, several stable hybrid species have been identified, e.g. P. andina (Goss et al. 2011; Blair et al. 2012); P. alni (Brasier et al. 1999); P. x pelgrandis (Nirenberg et al. 2009); P. x serindipita (Man in ’t Veld et al. 2012) as well as hybrid clade 1 species recovered from the field (Man in ’t Veld et al. 1998, 2007; Hurtado-Gonzales et al. 2009; Bonants et al. 2000). While conducting a detailed evaluation of clade 6 Phytophthora spp. from natural ecosystems in Australia, Burgess et al. (2010) observed ‘hybrid swarms’ that contained mixtures of parent, offspring, and intermediate isolates with high tendencies for back-crossing and out crossing. The authors’ concluded that the presence of such hybrid swarms was indicative of sexual and somatic hybridization events; the high proportion of these variant isolates within the population also suggested that these hybridization events were not uncommon. Recently four interspecific hybrid clade 6 species have been recovered from riparian ecosystems in Australia and South Africa that reflect outcrossing between P. amnicola, P. thermophila and P. taxon PgChlamydo (Nagel et al. 2013). Additional putative interspecific hybrids from riparian ecosystems in Australia were reported by Hüberli et al. (2013). Hybridization is a topic that requires a more detailed investigation as it could have a profound influence on gene flow among species and the evolution of new species with an expanded host range that could impact agricultural and natural ecosystems (as observed with P. alni).

Traditional classification to species level has been based on morphological characterization of reproductive structures (reviewed in Martin et al. 2012). This includes the sporangium (asexual) and oospore (sexual) as well as the production of chlamydospores (asexual structure not produced by all species). Important features of the sporangium include their dimensions (length and breadth), shape, thickening at the terminus (papilla), length of stalk (pedicle), whether or not the sporangium can be easily dislodged from the sporangiophore (caducity), and proliferation of sporangia (internal, external or nested).

The sexual reproductive structures consist of the antheridium and oogonium (paternal and maternal gametangia, respectively) and are produced when cultures are grown on the appropriate sterol-containing medium. Their fusion leads to the formation of an oogonium that matures into a thick-walled resting structure referred to as an oospore. While most species are homothallic and form oospores in single culture, there are heterothallic species where pairing with opposite mating types is essential to stimulate production of sexual reproductive structures. Since Phytophthora is sexually dimorphic (an isolate of a heterothallic species can function either as the maternal or paternal parent depending on the isolate it is paired with) it is advisable to pair self-sterile isolates with two tester isolates of opposite mating type. While the use of tester isolates of the same species is advisable, isolates of other heterothallic species (such as P. cryptogea or P. cambivora) may also be used. Characteristics such as the diameter of the oogonium and oospore, thickness of the oospore wall, whether or not the oospore fills the oogonium (plerotic), ornamentation on the oogonial wall, and mode of attachment of the antheridium are useful for species classification.

In an effort to simplify isolate identification and establish groupings of isolates for comparison of morphological features (but not phylogenetic relationships), Waterhouse (1963) introduced the concept of morphological groups I through VI based on a number of characteristics, and is still useful today. Unfortunately a dichotomous key that includes recently described species is not available for identification of isolates but there are several recent efforts to simplify morphological identification of species, including a manual for identification of 60 species of Phytophthora by integration of a dichotomous key with a DNA fingerprinting technique based on PCR-single strand conformational polymorphism (SSCP) (Gallegly and Hong 2008). A LUCID key for identification of 55 common Phytophthora spp. is available (Ristaino 2011) and an expanded LUCID key including most described species should be available on a dedicated website in the near future (G. Abad and Y. Balci, personal communication). A tabular presentation of morphological features enabling comparison among 117 species may be found in Martin et al. (2012; a downloadable file of the table alone is available on the journal website).

In 1999 the number of described species in the genus Phytophthora was approximately 55 (Brasier 2007) but since then there has been a significant increase., Brasier (2007) reported a doubling in number to 105 described species, with this number recently increasing to 117 (Martin et al. 2012). Additional species have recently been described; P. lacustris (Nechwatal et al. 2012) P. pluvialis (Reeser et al. 2013), P. mississippiae (Yang et al. 2013), P. cichorii, P. dauci and P. lactucae (Bertier et al. 2013), P. pisi (Heyman et al. 2013), P. stricta and P. macilentosa (Yang et al. 2014) and the hybrid species P. x serendipita and P. x pelgrandis (Man in ’t Veld et al. 2012), bringing the total to at least 128 described species. With the number of provisional species names used in the literature, and research efforts to evaluate the distribution of this genus in natural ecosystem, this number is likely to continue to increase in the future.

Molecular phylogeny

Historically the genus Phytophthora has been placed in the Pythialeswith Pythium and related genera but more recent phylogenetic analysis with the large (LSU) or small (SSU) rDNA sequences or cox2 gene has indicated a closer affiliation with downy mildews and white rusts (Albugo.) in the Peronosporales (Beakes and Sekimoto 2009; Thines et al. 2009). However, additional multigene analyses with a larger number of downy mildew species is needed to better characterize this relationship and the placement of Phytophthora spp. in clade 9 and 10 (Blair et al. 2008). The relationship between the Peronosporales and Pythium (Pythiales) needs clarification as well. A new genus, Phytopythium, was erected to accommodate an inconsistency between taxonomic and phylogenetic grouping for certain “intermediate” Pythium species (Bala et al. 2010), and it is likely that additional taxonomic revisions of the Peronosporomycetidae will be needed to fully resolve taxonomic conflicts.

Early efforts to understand phylogenetic relationships in Phytophthora focused on the use of the nuclear encoded rDNA, primarily the ITS region (Förster et al. 2000; Cooke and Duncan 1997; Crawford et al. 1996). Cooke et al. (2000) published the first comprehensive phylogenetic analysis of the genus using the ITS region to examine the phylogeny of 50 species. Most isolates grouped within eight primary clades (numbered 1 to 8) with several other species placed in two additional clades (clades 9 and 10). Kroon et al. (2004) expanded this analysis using two nuclear (translation elongation factor 1α, β-tubulin) and two mitochondrial (cox1 and nad1) genes. While in general the results were congruent with those reported by Cooke et al. (2000), there were some notable differences in the grouping of some species. Subsequent analysis by Blair et al. (2008) using seven nuclear genes (60S ribosomal protein L10, ß-tubulin, enolase, heat shock protein 90, large subunit rDNA, TigA gene fusion and translation elongation factor 1α) representing 8.1 kb of sequence data for 82 Phytophthora spp. clarified these differences. This larger, multi-marker analysis supported the observations of Cooke et al. (2000) with eight main clades plus two additional closely affiliated clades (clades 9 and 10) as the sister clades to the rest of the genus. More recently, Martin et al. (2014) expanded on this analysis by adding four mitochondrial genes (cox2, nad9, rps10 and secY) and additional species. The resulting phylogeny from this 11-marker analysis (10,828 bp per isolate) was similar to the prior observations of Blair et al. (2008) and subsequent analysis indicated that similar results could be obtained when using only five markers (LSU, β-tubulin, cox2, nad9 and rps10).

While the ITS region may be useful for species identification (see below), length variation among species makes it impossible to construct an unambiguous alignment across the entire genus, thus hampering the utility of this marker for phylogenetic analysis. Likewise, the translation elongation factor 1α has been used for phylogenetic analysis, but recent analysis of Phytophthora genomic data indicates that the gene is duplicated; divergence among duplicates may complicate phylogenetic interpretations of species evolution (J. E. Blair, unpublished).

While the above noted phylogenetic analyses have provided insight into the broader evolutionary relationships within the genus, there is still ambiguity when examining some closely related species and species complexes. Significant progress has been made with the clarification of the P. megasperma complex and other clade six species (Brasier et al. 2003; Durán et al. 2008; Hansen et al. 2009; Jung et al. 2011a, b) but there are still several provisional species awaiting more comprehensive analysis (for example, P. taxon PgChlamydo, P. taxon raspberry, P. taxon canalensis, P. taxon erwinii, P. taxon hungarica, P. taxon oregonsis and P. taxon paludosa). While there have been advances in understanding the relationships among some clade 2 species, there is need for additional analysis to clarify species complexes such as P. citricola and P. citrophthora. One clade 8 species complex where phylogenetic resolution has been elusive is P. cryptogea and the closely related species P. drechsleri. The multigene analysis of Mostowfizadeh-Ghalamfarsa et al. (2010) confirmed that while P. drechsleri was monophyletic, the P. cryptogea complex formed three well-defined phylogenetic groups with group I closely affiliated with P. erythroseptica and group II and III as a separate clade (group III isolates have been reported as the provisional species, P. sp. kelmania; Martin et al. 2014). Some isolates were placed intermediate between groups II and III and exhibited a greater amount of heterozygosity than the other isolates, suggesting possible outcrossing between these groups. Using a parsimony-based ancestral recombination graph and genealogies inferred from the β-tubulin and translation elongation factor 1-α genes from greenhouse recovered isolates, Olson et al. (2011) suggested that divergence between P. cryptogea and P. drechsleri was recent and that speciation is still in progress.

In addition to the choice of markers to use for phylogenetic analysis, another important consideration is the type of analysis used for estimating phylogenetic relationships or for the description of new species. While traditional methods of phylogenetic analysis (maximum likelihood, neighbour-joining, Bayesian) have adequately described relationships among most species, they have been unable to fully resolve the deeper relationships among the ten Phytophthora clades or among related genera. A recent study by Martin et al. (2014) used a novel variation of a multispecies coalescent approach to evaluate the ten clades; in general support was higher than that observed in the phylogenetic analysis for the recovered relationships, but the position of certain clades (Clade 3 and the unique grouping of P. sp. ohioensis and P. quercina) remained ambiguous. Here we present an analysis using a more powerful and complex Bayesian method (Drummond et al. 2012) with five genetic markers (Fig. 20), and recover strong support for basal relationships among the clades that are quite similar to the 11-marker study of Martin et al. (2014). Newer phylogenetic methods may allow for more complex modelling of the evolutionary process, however they are still sensitive to the accuracy of a priori information provided by the user. Additional studies will be needed to provide more basic information on the tempo of molecular evolution within this group.
Fig. 20

Bayesian analysis of phylogenetic relationships within Phytophthora. Asterisks on nodes indicate posterior probabilities greater than 0.95 (95 %) generated from an analysis of five genes (nuclear LSU and β-tubulin; mitochondrial cox2, nad9, rps10). Evolutionary rates were estimated under a GTR + I + G model for nuclear markers and an HKY + I + G model for mitochondrial gene; each marker was treated as a separate partition. The analysis was run twice with 50 million generations under a strict clock model in BEAST v1.7.5. A 20 % burn in was removed before the maximum clade credibility tree was constructed. Ex-type isolates are shown in bold. Separate isolate numbers are shown for those few species that did not have sequence data available for both nuclear and mitochondrial genes from a single isolate

The description of new species is also an area were traditional phylogenetic methods may not accurately describe species relatedness. Aside from morphological characterization, recent species descriptions typically contain molecular evidence from one or a few genetic markers (primarily ITS and perhaps cox1 or 2). However, as described above, alignment ambiguity and the presence of intraspecific polymorphisms can seriously impact the recovered phylogeny; recent hybridization events and incomplete lineage sorting of ancestral polymorphisms also violate the assumptions made by traditional phylogenetic methods. The use of coalescent-based approaches to estimate species trees from a collection of gene trees has been gaining popularity among many other taxonomic groups, but has seen little attention in Phytophthora or oomycete research in general. The recent description of P. pisi (Heyman et al. 2013) employed a multispecies coalescent approach, which confirmed the individual analyses of ITS and cox2 data. In addition, a recent study of the hybrid species P. andina (Blair et al. 2012) used several coalescent methods to determine the likely parental lineages of this species, one of which was clearly P. infestans. In the future, the use of more complex phylogenetic methods as well as coalescent-based approaches will be needed to clarify relationships at both ends of the spectrum, from deep basal nodes to recently evolved and potentially interbreeding species complexes.

A common observation among all phylogenetic studies is there is no consistent correlation between phylogenetic grouping and morphological features (Cooke et al. 2000; Kroon et al. 2004, 2012; Blair et al. 2008; Martin et al. 2014). While there is some correlation with sporangial type (clade 4, 5, and 10 have primarily papillate sporangia while clade 3 has primarily semipapillate sporangia and clades 6, 7, and 9 primarily nonpapillate sporangia), other clades show combinations of these features (clade 1, 2 and 8). Characteristics such as oogonial ornamentation, heterothallism, and mode of antheridial attachment are all polyphyletic.

Because of the large number of species, intraspecific variation of some morphological features, and overlapping morphology among closely related species, traditional methods of species identification can be challenging and require some level of expertise to be effective. The use of molecular criteria has simplified this task and provides a tool for delineating distinct taxa within morphologically similar species complexes. The most accurate molecular method for species identification is sequence analysis of specific markers. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (rDNA) has been widely used and a large number of sequences are currently available in public databases. However, this marker may not be ideal for the identification of all species, especially those that are closely related. For example, many clade 1C species (P. infestans, P. mirabilis) cannot be distinguished using this marker alone, nor can P. fragariae and P. rubi. More recently a portion of the cox1 gene, along with the ITS region, have been proposed as the markers to use in the Barcode of Life Database (www.boldsystems.org) and representative sequences for all described and some provisional species have been deposited (Robideau et al. 2011).

Several nuclear (60S ribosomal protein L10, β-tubulin, enolase, heat shock protein 90, large subunit rRNA, TigA gene fusion, translation elongation factor 1α; (Blair et al. 2008; Kroon et al. 2004; Villa et al. 2006)) and mitochondrial (cox1, nad1, cox2, nad9, rps10 and secY; (Kroon et al. 2004; Martin 2008; Martin and Tooley 2003a, b; Martin et al. 2014) markers have been sequenced for phylogenetic analysis of Phytophthora and can also be used for species identification. Background information for amplification and sequencing of many of these markers, as well as the capability for BLAST searches against a curated database for isolate identification, may be found at the Phytophthora Database (www.phytophthoradb.org). A dataset for ITS and cox1 and 2 spacer sequences is also available at Phytophthora ID ((Grünwald et al. 2011), www.phytophthora-id.org) and sequence data for several markers (ITS, β-tubulin, elongation factor 1 alpha, and cox1), along with pictures of morphological features, may be found at Q-Bank (www.q-bank.eu).

There are several caveats to consider when using BLAST analysis to identify isolates to species level to prevent misidentification (Kang et al. 2010; Nilsson et al. 2012). BLAST scores are dependent on the length of the aligned sequences as well as the level of sequence identity; instances where high levels of sequence identity occur for only a portion of the target sequence may result in incorrect species identification. Also, it is common to encounter situations where scores are similar among multiple species, making it difficult to draw conclusions about an isolate’s identity (this can be especially problematic for isolates within or related to species complexes). In addition, the use of markers known to contain intraspecific polymorphisms may lead to inaccurate species identifications due to potentially lower similarities among closely related sequences. Heterozygosity in nuclear markers may also complicate identification efforts; while the presence of distinct alleles may indicate outcrossing (as Phytophthora is a known diploid), heterozygosity may also result from hybridization events between distinct lineages (as described above). Phylogenetic analysis of several markers is therefore suggested to confirm species identification, especially when working with species complexes. Additional gel based techniques, such as PCR-RFLP, SSCP, random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLP) and simple sequence repeat (SSR) analysis, for species identification and population analysis are reviewed in Martin et al. (2012).

Recommended genetic markers

The following genetic markers have been found to amplify well across all species and provided a similar level of phylogenetic resolution as a concatenated dataset of seven nuclear and four mitochondrial genes (Martin et al. 2014). Information on amplification and sequencing primers for these genes may be found at the Phytophthora Database (www.phytophthoradb.org).

Nuclear genes–LSU, β-tubulin

Mitochondrial–cox2, nad9, rps10

Phytophthora Data

Sequence alignments of the seven nuclear and four mitochondrial markers used in Martin et al. (2014) and Fig. 20 may be downloaded at TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S14595). A table with additional information on isolates used in the analysis may be found in Martin et al. (2014) with GenBank accession numbers listed in the supplementary material of this citation. These sequences can also be downloaded from the Phytophthora Database (www.phytophthoradb.org).

Pythium

Background

Pythium is classified as belonging to the family Pythiaceae sensu lato (s.l.), order Peronosporales s.l., class Peronosporomycetes, phylum Oomycota, and kingdom Straminipila (Beakes et al. 2014). Although many species are considered to be saprobes, the genus is known primarily for its parasitic interactions with plants. Several species also parasitize algae (green and red), fungi, other oomycetes, nematodes, insects, crustaceans, and fish. One species, P. insidiosum, is the causal agent of pythiosis in mammals, including humans (Van der Plaats-Niterink 1981; de Cock et al. 1987). Plant pathogenic Pythium species often target young below-ground plant parts such as fine roots, germinating seeds and emerging growth, resulting in damping-off, root rot and poor crop stands with stunted plants and reduced yield. Some species can also cause fruit rot, and at least one species, P. vexans, has been associated with trunk cankers of rubber trees (Van der Plaats-Niterink 1981; Zeng et al. 2005). Although some species have a limited host range, such as P. arrhenomanes that seems to be exclusively associated with gramineous crops, species like P. aphanidermatum, P. irregulare and P. ultimum are known for being highly virulent on an extensive range of plant hosts (Van der Plaats-Niterink 1981). However, not all Pythium species have a negative impact on the plants they are associated with. Besides saprobes, others can benefit plants by acting as biocontrol agents that parasitize pathogenic fungi and/or induce host resistance, e.g. Pythium oligandrum (Benhamou et al. 1997). Other species of Pythium have also been reported to stimulate plant growth (Mazzola et al. 2002). Recent genome sequencing of six Pythium species found high levels of variation in the number of CRN (“Crinkler”) effectors found in the different species, possibly suggesting species-specific infection strategies (Adhikari et al. 2013) that may contribute to the range of interactions of Pythium species with their hosts. Such species-specific host-interactions along with the ubiquitous nature of the genus in soils all over the world make accurate species identification necessary to facilitate disease diagnosis and management.

Debates regarding possible genera within Pythium were initially sparked by differences in sporangial morphology. Based on these characters some of the novel genera that have been proposed are Nematosporangium (for species with filamentous zoosporangia), Rheosporangium (species with lobulate zoosporangia), and Sphaerosporangium (species with ovoid, spherical or citriform sporangia) (Schröter 1897; Sparrow 1931). The legitimacies of these genera have been questioned for various reasons (Sideris 1931a; Sparrow 1932; Van der Plaats-Niterink 1981), and aside from some attempts at transferring Pythium species to Nematosporangium (Jaczewski and Jaczewski 1931; Sideris 1931b) the scientific community has stuck with the generic classification of Pythium versus these genera. As molecular taxonomy became a more popular approach to studying systematics, the paraphyletic nature of Pythium became apparent and the debate on splitting the genus was rekindled. Early sequence-based phylogenies provided strong arguments for P. vexans to be part of a separate genus (Briard et al. 1995; Cooke et al. 2000). The ITS and 28S phylogenies of Lévesque and de Cock (2004) divided Pythium into 11 clades (A-K) of which clade K (including P. vexans) is more closely related to Phytophthora than to the rest of the Pythium clades (Villa et al. 2006). A new genus, Phytopythium, was subsequently erected to include all clade K species, with Phytopythium sindhum as type species (Bala et al. 2010), although the official transfer of all clade K Pythium species to Phytopythium has not yet been published. The remaining ten clades (A-J) can be divided into two groups: species with filamentous zoosporangia (clades A-D) and species with globose zoosporangia (clades E-J) (Lévesque and de Cock 2004), calling to mind early suggestions of splitting the genus based on this character (Schröter 1893; Sparrow 1931). This division is echoed to varying degrees by phylogenies of the 28S rRNA, ITS, cytochrome c oxidase subunits 1 and 2 (cox1 and cox2), and β-tubulin, although the different gene trees are often incongruent and support for internal nodes low or absent (Martin 2000; Riethmüller et al. 2002; Villa et al. 2006; Hulvey et al. 2010; Robideau et al. 2011). Despite the shortcomings of these gene regions, Uzuhashi et al. (2010) used 28S and cox2 phylogenies to divide Pythium into five genera: Pythium (clades A-D), Globisporangium (clades E-G, I and J), Elongisporangium (clade H), Ovatisporangium (clade K, syn. Phytopythium), and Pilasporangium (distinct from any of the aforementioned 11 clades). Although this division is more or less in agreement with previous phylogenetic studies, it is problematic with regards to a lack of bootstrap support for the Pythium and Globisporangium clades, and the relationship and distinction between Elongisporangium and Globisporangium is not resolved with support (Fig. 21, Uzuhashi et al. 2010). Additionally, the genera Pythiogeton and Lagena seem to be phylogenetically situated within, or closely related to Pythium emend Uzuhashi, Tojo and Kakishima (Fig. 21, Huang et al. 2013a), so even this revised version of Pythium is paraphyletic. For these reasons investigators have generally been slow to adopt the proposed genera. Following this trend references to “Pythium” in this manuscript refer to Pythium s.l. (i.e. Pythium Pringsheim) unless stated otherwise.
Fig. 21

Maximum likelihood phylogeny of Pythium s.l. and related genera based on the concatenated 18S rRNA, ITS, 28S rRNA, cytochrome c oxidase subunit 2 (cox2), and β-tubulin regions. Bootstrap support values below 60 % are not indicated. Strains in bold typeface represent type-derived material, authentic strains or strains used by Van der Plaats-Niterink (1981) for descriptions. The 11 clades (A–K) of Lévesque and de Cock (2004) and the genera erected by Bala et al. (2010) and Uzuhashi et al. (2010) are indicated on the right along with related taxa such as Phytophthora, Lagenidium, Lagena, and Pythiogeton

Species identification and numbers

A combined list of Pythium species in MycoBank (2014) and Index Fungorum (2014) includes a total of 328 names of which several are either synonyms, orthographic variants or varieties that are rarely referred to and are possibly synonyms of other species (i.e. all varieties excluding varieties of P. ultimum). Excluding such cases along with putative synonyms based on cox1 and ITS sequence homology as identified by Robideau et al. (2011) leaves more or less 230 species of Pythium. Undoubtedly this number still includes species that should be synonymized and/or transferred to genera other than Pythium (Van der Plaats-Niterink 1981; Dick 1990, 2001), but for now this should serve as a rough estimate of the number of actual Pythium species discovered to date. Of these species 152 (66 %) are known to be represented by sequence(s) in GenBank, including at least 123 (53 %) species for which type-material, ex-type strains or strains described by Van der Plaats-Niterink (1981) were used to generate sequence data (Table 19, Fig. 21).

Identification of Pythium isolates to the species level is generally straightforward when comparing both ITS and cox1 sequences to that of ex-type, authentic or other reliable representative strains. For this purpose the sequences generated by Lévesque and de Cock (2004) and Robideau et al. (2011) are excellent resources. Using only the ITS region would more often than not allow suitably accurate species identification, but some species are indistinguishable using ITS and require cox1 sequences for further identification (see Text S1A of Robideau et al. 2011). Several other species are indistinguishable even when both ITS and cox1 sequences are compared (see Text S1B of Robideau et al. 2011), and many of these should probably be formally synonymized pending more thorough investigations with multiple hypervariable genes. This approach should also resolve species complexes found in the group formed by P. irregulare, P. paroecandrum, P. cylindrosporum, P. cryptoirregulare and P. mamillatum (Barr et al. 1997; Matsumoto et al. 2000; Garzón et al. 2007; Spies et al. 2011a), the varieties of P. ultimum (Barr et al. 1996), and the P. vexans and P. cucurbitacearum group (Spies et al. 2011b). Some species epithets have been applied to multiple phylogenetic species due to imprecise species descriptions and/or misidentifications. Examples of these include P. iwayamai, P. okanoganense and P. violae (Lévesque and de Cock 2004; McLeod et al. 2009; Bahramisharif et al. 2013). Mislabelling or contamination of reference strains and/or data cause similar problems, as illustrated by the case of P. terrestre (published as “terrestris”) of which the holotype strain ITS sequence published with the description suggests phylogenetic placement in clade E (Paul 2002), while the ITS and cox1 sequences generated for the ex-type strain available from the Centraal Bureau voor Schimmelcultures (CBS) suggests phylogenetic placement in clade F (Robideau et al. 2011). Species identification within genetically diverse species complexes (see Text S1C of Robideau et al. 2011 for a partial list) can also be tricky, more due to uncertain species boundaries than due to the ineffectiveness of ITS and/or cox1 as barcoding regions. The onus is on the investigator to keep such issues in mind when identifying strains to the species level and to consider the identification in context of the taxonomic history of the species and its closest relatives.

Molecular phylogeny

The first molecular phylogenies of Pythium were inferred from sequences of the 28S, ITS, and cox2 regions respectively, and although each analysis included only a few species, the observed variation merited speculation regarding the polyphyletic nature of Pythium at least for the ITS and 28S phylogenies (Briard et al. 1995; Cooke et al. 2000; Martin 2000). The first study to provide an extensive DNA sequence based phylogeny of Pythium was that of Lévesque and de Cock (2004) who sequenced the 28S region of 51 species and complete ITS region (ITS1-5.8S rRNA-ITS2) region of 116 species. Although a two-marker phylogeny of the ITS-28S region was presented by Lévesque and de Cock (2004), these markers are adjacent multi-copy markers that might not accurately represent the evolutionary relationships in Pythium. Villa et al. (2006) used multiple markers (ITS, cox2, β-tubulin) in individual phylogenetic analyses with 39 species and confirmed previous suggestions of an intermediate evolutionary position of clade K species between Pythium and Phytophthora, but also suggested that clade H species (represented by Phytophthora undulataPythium undulatum) occupy a similar intermediate position, which contrasted the position of this clade nestled among clades E, F, G, I, and J as suggested by Lévesque and de Cock (2004). The multi-marker phylogeny (18S-ITS-28S, cox2 and β-tubulin) of 152 Pythium species and some related taxa presented here confirms the association of clade K with Phytophthora, but fails to provide support for the evolutionary association of clade H with any of the other recognized groups within Pythium (Fig. 21). Furthermore, organisms such as the obligate root pathogen Lagena radicicola and strains resembling Lagenidium form an unresolved cluster of taxa related to clade C (Fig. 21). In itself this phylogenetic placement of the genus Lagena necessitates a further taxonomic revision of the genus Pythium that can only be achieved once the internal nodes of the Pythium phylogeny have been resolved with support. Despite the fact that the phylogeny in Fig. 21 represents the most extensive sampling of taxa and genetic markers in a multi-marker phylogeny of Pythium to date, it still fails to achieve this goal. Phylogenetic markers additional or alternative to those currently used in Pythium systematics are needed to resolve these issues and elucidate taxon boundaries.

Recommended genetic markers

  • The 18S (small subunit, SSU) and 28S (large subunit, LSU) nuclear rRNA genes–generic level phylogenies within Pythium s.l.

  • The internal transcribed spacers (ITS including ITS1, 5.8S rRNA, and ITS2), cytochrome c oxidase subunit 2 (cox2)–sub-generic, inter- and intra-specific level phylogenies

  • ITS and cox1–non-phylogenetic species identification

Mitochondrial regions such as cox1 and cox2 should be used with consideration of the fact that they mainly reflect evolution of maternal lineages and can produce incongruent phylogenies. This is especially true for cox1, which is why this region was not included in Fig. 21. The β-tubulin region has also been used to a limited extent in Pythium phylogenies (Villa et al. 2006; Belbahri et al. 2008; Spies et al. 2011a, b). Although this region fails to resolve Pythium into the genera observed when using the dataset from Fig. 21 (data not shown) and has limited power in resolving species-level phylogenies (Spies et al. 2011a, b), it amplifies and sequences well for most Pythium species and is an easy resource for use in concatenated datasets (e.g. Bahramisharif et al. 2013; Fig. 21).

Pyrenophora

Background

Pyrenophora represents a genus of plant pathogenic fungi associated with a wide variety of substrates. Fries (1849) list the genus as Pyrenophora typified with Pyrenophora phaeocomes. The genus Pyrenophora clusters in the suborder Pleosporineae of the family Pleosporaceae (Berbee 1996; Zhang and Berbee 2001; Hyde et al. 2013a, b; Zhang et al. 2012; Ariyawansa et al. 2014). Recent studies using multi-gene analysis and some coupled with morphology have provided the groundwork for classification of species in Pyrenophora (Berbee 1996; Zhang and Berbee 2001; Hyde et al. 2013a, b; Zhang et al. 2012).

Pyrenophora has been linked to asexual morphs in Drechslera. Pyrenophora species are important plant pathogens as well as saprobes. Many species cause disease on their graminicolous hosts and are usually present in their asexual state (Drechslera) (Zhang and Berbee 2001). Species of Pyrenophora are serious plant pathogens (Zhang and Berbee 2001). Pyrenophora teres (Drechslera teres) is a necrotrophic pathogen of economically important crops, such as barley (Gupta and Loughman 2001; Kingsland 1991). Pyrenophora graminea (Drechslera graminea) causes barley stripe resulting in significant yield losses (Tekauz 1983, 1990). Pyrenophora graminea lives within barley kernels as mycelium, and when seeds germinate, hyphae enter the seedling through the coleorrhiza, causing a systemic infection (Platenkamp 1976; Porta-Puglia et al. 1986). Pyrenophora tritici-repentis causes tan spot of wheat (Lamari and Bernier 1989) which occurs in all the major wheat-growing areas of the world and causes 3 to 50 % yield losses (Ballance et al. 1996). Its prevalence has increased recently.

Some Pyrenophora species have been used as biocontrol agents. Bromus tectorum is a dominant winter annual weed in cold deserts of the western United States (Meyer et al. 2007). Together with other annual brome grasses it has invaded many ecosystems of the western United States creating near-monocultures in which the native vegetation cannot compete (Meyer et al. 2007). Pyrenophora semeniperda has be used as a biocontrol agent to kill the dormant seeds of Bromus tectorum (Meyer et al. 2007). Several studies have assessed chemical production by Pyrenophora species. A new phytotoxic sesquiterpenoid penta-2,4-dienoic acid (pyrenophoric acid) was isolated from solid wheat seed culture of P. semeniperda.

Species identification and numbers

Pyrenophora is characterized by immersed, erumpent to nearly superficial ascomata, indefinite pseudoparaphyses, clavate to saccate asci usually with a large apical ring, and muriform terete ascospores. Morphologically, the terete ascospores of Pyrenophora can be easily distinguished from Clathrospora and Platyspora. The indefinite pseudoparaphyses and smaller ascospores of Pyrenophora can be clearly separated from those of Pleospora (Sivanesan 1984). Pyrenophora species can easy be distinguished from species in Cochliobolus and Setosphaeria on the basis of the shape, septation and colour of the ascospores (Zhang and Berbee 2001). Drechslera species were initially categorized in Helminthosporium on the basis of their dark colour, transversely septate conidia and a graminicolous habitat (Shoemaker 1959). Consequently, graminicolous Helminthosporium species were segregated into three genera, Bipolaris, Drechslera, and Exserohilum, defined based on their association with their sexual states Cochliobolus, Pyrenophora, or Setosphaeria, respectively (Zhang and Berbee 2001). Currently 198 species of Pyrenophora and 135 species of Drechslera are listed in Index Fungorum (2014).

Molecular phylogeny

Rapid identification of diseases caused by Pyrenophora has been determined via different DNA markers. Identification of molecular genetic markers in Pyrenophora teres f. teres associated with low virulence on ‘Harbin’ barley was assessed by random amplified polymorphic DNA (RAPD) (Weiland et al. 1999) and five RAPD markers were obtained that were associated in coupling with low virulence. The data suggested that the RAPD technique can be used to tag genetic determinants for virulence in P. teres f. teres (Weiland et al. 1999). Specific polymerase chain reaction (PCR) primers were developed from amplified fragment length polymorphism (AFLP) fragments of P. teres, in order to distinguish the two forms, P. teres f. teres (which cause net form blotch on barley leaves) and P. teres f. maculata (which causes spot form); the two forms are morphologically very similar in culture (Leisova et al. 2005). The PCR assay was certified with 60 samples of Pyrenophora species. The amplification with four designed PCR primer pairs provided P. teres form-specific products. No cross-reaction was observed with DNA of several other species, such as P. tritici-repentis and P. graminea (Leisova et al. 2005). Pyrenophora graminea is the causal agent of barley leaf stripe disease (Mokrani et al. 2012). Two leaf stripe isolates PgSy3 (exhibiting high virulence on the barley cultivar ‘Arabi Abiad’) and PgSy1 (exhibiting low virulence on Arabi Abiad), were mated and 63 progeny were isolated and phenotyped for the reaction on Arabi Abiad (Mokrani et al. 2012). From 96 AFLP markers, three AFLP markers, E37M50-400, E35M59-100 and E38M47-800 were linked to the virulence locus VHv1 in isolate PgSy3. Lubna et al. (2012) suggested that the three markers are closely linked to VHv1 and are unique to isolates carrying the virulence locus. Pecchia et al. (1998) developed an efficient PCR protocol for amplification of the IGS region in P. graminea and to characterize this region by restriction fragment analysis. During the study based on the length of the IGS-PCR product, ca. 3.8 or 4.4 kb, two groups of isolates were identified from six cultures i.e. I3/88 (Italy; CBS 100862), I7/88 (Italy; CBS100861), 60/93 (Austria; CBS 100866), I10/95 (Tunisia; CBS 100863), I28/95 (Tunisia; CBS 100864), I33/95 (Tunisia; CBS 100865). The RFLP patterns of isolates obtained with the 6-base cutting enzymes ApaI, BglII, DraI, EcoRV, HindIII and SacI were similar within each group and different between the two groups (Pecchia et al. 1998). Restriction patterns of IGS-PCR products digested with the 4-base cutting enzyme AluI were polymorphic among isolates in spite of their IGS-PCR product length (Pecchia et al. 1998).

Molecular studies of Pyrenophora/Drechslera species have detailed the taxonomic placement of the genus. Initially the 18S rRNA gene was used for the classification of Pyrenophora/Drechslera and related genera (Berbee 1996). Phylogenetic analysis based on 18S rRNA showed Pyrenophora to cluster within the Pleosporaceae (Zhang and Berbee 2001) rather than in Pyrenophoraceae (Zhang and Berbee 2001). Later, phylogenetic analysis of the ITS and gdp data showed that Pyrenophora is monophyletic (Zhang and Berbee 2001), and the asexual state Drechslera clustered with their predicted sexual relatives (Table 20, Fig. 22).
Fig. 22

Phylogram generated from parsimony analysis based on combined of ITS, gdp and LSU sequenced data of Pyrenophora. Parsimony bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Pleospora herbarum CBS 276.37

Recommended genetic markers

  • Large small subunits of nrDNA (LSU)–generic level

  • ITS and gdp–inter-specific delineation

Based on our phylogeny, we observed that gdp gives high resolution compared to ITS and LSU, such that it can be readily used to determine the placement of Pyrenophora species.

Puccinia

Background

Puccinia is the type genus of the family Pucciniaceae in the order of rust fungi, Pucciniales (Basidiomycota). Puccinia has approximately 4,000 named species (Kirk et al. 2008), and is a widespread genus of plant pathogens that has shaped history. For example, Puccinia graminis, the type species of Puccinia, was investigated as a biological warfare agent in the cold war (Line and Griffith 2001). It was the impetus for breeding wheat cultivars resistant to disease that started the Green Revolution, lead by 1970 Nobel Laureate, Norman Borlaug (Zeyen et al. 2014). Epidemics of stem rust of wheat caused by P. graminis remain a threat with the emergence of races such as Ug99 (Singh et al. 2011). Other species of Puccinia are also serious pathogens of grasses (Poaceae), including P. coronata and P. striiformis (Kirk et al. 2008). Rusts of Asteraceae, e.g., P. helianthi, and rusts of Fabaceae in the closely related genus Uromyces, e.g., U. viciae-fabae, U. appendiculatus and U. ciceris-arietini, are important pathogens of cultivated fodder and food crops.

Among the ca. 120 to 160 genera of rust fungi (Cummins and Hiratsuka 2003; Kirk et al. 2008), Puccinia is readily recognized by the two-celled teliospores and the shape of the spermogonia (Cummins and Hiratsuka 2003). Uromyces with one-celled teliospores is typically differentiated from Puccinia, although some species of Puccinia have both one-celled (mesospores) and two-celled teliospores, e.g., P. lagenophorae. Teliospore morphology is homoplasious, and Puccinia and Uromyces were polyphyletic in systematic studies based on the LSU and SSU regions of nuclear ribosomal DNA (Maier et al. 2007; Aime 2006), and the two nuclear genes: elongation factor and β- tubulin (Van der Merwe et al. 2007). Some rust fungi have teliospores morphologically similar to Puccinia, but are not closely related or have an uncertain systematic position. For example, Allodus podophylli has two-celled teliospores convergent with Puccinia. A systematic analysis based on the nLSU and nSSU regions of rDNA determined Allodus and Puccinia were unrelated (Minnis et al. 2012). Puccinia psidii, which spread from South America to much of the Pacific region and South Africa, now infects 30 genera of Myrtaceae out of its natural host range (Pegg et al. 2013). It has two-celled teliospores, but its placement within the Pucciniales is unknown. Phylogenetic analyses of the nLSU and nSSU (Pegg et al. 2013) and the protein coding gene beta-tubulin (Van Der Merwe et al. 2008) indicated that P. psidii was sister to the Pucciniaceae. Several families and genera of rust fungi are polyphyletic, namely the Raveneliaceae, Phakopsoraceae and Pucciniaceae. These polyphyletic families and genera await resolution by molecular phylogenetic analyses.

Species identification and numbers

Rust fungi are usually considered host specific (Cummins and Hiratsuka 2003), although some, e.g., Puccinia psidii and P. lagenophorae, infect multiple host genera (McTaggart et al. 2014; Pegg et al. 2013). Some species of rust fungi are heteroecious, requiring two hosts in different families to complete their life cycle, e.g., P. graminis on Triticum (Poaceae) and Berberis (Berberidaceae).

Rust fungi have a complicated life cycle with up to five spore states (Cummins and Hiratsuka 2003). Consequently, up to three names have been proposed for the same taxon based on different life cycle stages. To add to the confusion, there are two systems of terminology that describe these spore states, one based on morphology (Laundon 1967), and the other on ontogeny (Arthur and Kern 1926; Cummins and Hiratsuka 2003; Hiratsuka 1973). These systems of terminology were summarised by Hennen and Hennen (2000).

Species of rust fungi are often identified on the basis of their host specificity, and monographs were organised by plant family (Sydow and Sydow 1904; McAlpine 1906; Cummins 1971, 1978). Morphological characters of the teliospores and urediniospores, such as size, apex shape and wall thickness, ornamentation, and germ pore position and number, are useful for species identification.

Molecular diagnostic tools have been developed for some species of Puccinia based on the ITS region of rDNA, e.g., P. coronata (Beirn et al. 2011; Pfunder et al. 2001), P. kuehnii (Glynn et al. 2010) and P. psidii (Langrell et al. 2008). The ITS region has successfully distinguished phylogenetic species in Uromyces (Barilli et al. 2011) and it was used in combination with TEF to resolve the taxonomy of P. melampodii (Seier et al. 2009). However, the ITS region was polymorphic in Puccinia lagenophorae (Littlefield et al. 2005; Scholler et al. 2011), and Morin et al. (2009) discovered a paralagous copy of the ITS region, which may have resulted from a hybridization event. A paralagous copy of the ITS region was also reported in P. kuehnii in the study by Virtudazo et al. (2001). Polymorphisms and paralogous copies are caveats for studies based on the ITS region in rust fungi.

Molecular phylogeny

Large-scale systematic studies of rust fungi have focused mainly on the SSU and LSU regions of rDNA (Aime 2006; Beenken et al. 2012; Dixon et al. 2010; Maier et al. 2003, 2007; Minnis et al. 2012; Wingfield et al. 2004; Yun et al. 2011) (Table 21). Protein coding genes such as beta-tubulin (Morin et al. 2009; Van der Merwe et al. 2007, 2008) and elongation factor (TEF) (Seier et al. 2009; Van der Merwe et al. 2007) were successfully used at the family, genus and species level in rust fungi, although beta-tubulin required cloning rather than direct sequencing of PCR product. Liu et al. (2013) included ITS, beta-tubulin, ribosomal polymerase subunit 2 (RPB2) and cytochrome c oxidase subunit 1 (COI) in a systematic study to resolve the P. coronata species complex. They discussed the difficulty of PCR amplification of older herbarium specimens, and that DNA repair was successful in some cases. Vialle et al. (2009) compared mitochondrial genes to rDNA markers in two genera of rusts, Chrysomyxa and Melampsora. They found rDNA had better species resolution than mitochondrial genes. Mitochondrial genes were since used in studies of the genera Chrysomyxa (Feau et al. 2011) and Dasyspora (Beenken et al. 2012), but have not yet been used for Puccinia.

Recommended genetic markers

  • The large subunit of nrDNA (LSU)–is useful for genus and species level identification of all rust fungi

  • The internal transcribed spacer (ITS)–is useful for species level identification, but may contain polymorphic sites and paralagous copies. Rust specific primers are recommended.

Rusts are obligate biotrophs and difficult to maintain in pure culture, which has posed a challenge for DNA extraction (Aime 2006). This is reflected by the relatively few species of Puccinia represented in GenBank, for example, there are ~110 species of Puccinia represented by the ITS and LSU regions of rDNA. This is less than 3 % of the estimated 4,000 species of Puccinia (Kirk et al. 2008). Reliance on molecular identification for some species of Puccinia is not recommended. For example, McTaggart et al. (2014) determined that several species of Puccinia on different plant families in Australia had near-identical ITS and LSU rDNA sequences (Fig. 23Puccinia).
Fig. 23

Puccinia. Phylogram obtained from a ML search in RAxML with the SSU and LSU regions of nrDNA. Bootstrap values (≥70 %) from a ML search with 1,000 replicates above nodes; posterior probabilities (≥0.95) from Bayesian inference below nodes. Puccinia and Uromyces are polyphyletic, and genera such as Cumminsiella, Dieteila, Miyagia and Pucciniosira are paraphyletic. The LSU region is not sufficient to distinguish closely related taxa in Australia as seen in the P. lagenophorae clade

Rhizopus

Background

Rhizopus is a genus of cosmopolitan saprotrophic fungi, currently included in the family Rhizopodaceae within the Mucorales (former Zygomycota; Hoffmann et al. 2013). Many Rhizopus species are common postharvest pathogens, causing fruit rots, and spoilage of crops, vegetables and wide range of stored foods (Pitt and Hocking 2009; Ray and Ravi 2005; Shtienberg 1997). Some species of this genus (e.g. R. arrhizus, R. microsporus and R. stolonifer) may also cause head rot disease in sunflowers (Yildirim et al. 2010). Among all Rhizopus species, R. arrhizus (syn. R. oryzae), and R. stolonifer are of particular importance, taking into account the frequency of isolation records (Farr and Rossman 2014). Extremely fast growth rates and abundant production of early maturing dry sporangiospores by Rhizopus species facilitate rapid spread of infection (Pitt and Hocking 2009). According to USDA Fungus-Host Database (Farr and Rossman 2014), Rhizopus species have been isolated from a wide range of plant taxa, both angiosperms and gymnosperms. Several members of the genus, among them R. arrhizus and R. microsporus are reported to cause human mucormycoses (Pitt and Hocking 2009), mostly in immunocompromised patients (Roden et al. 2005; Pitt and Hocking 2009; Chakrabarti et al. 2010; Skiada et al. 2011). Nevertheless, Rhizopus species are used by humans. Fermentation process of several kinds of Asian food and beverage strongly depends on Rhizopus strains (Henkel 2005; Nout and Aidoo 2010).

Species identification and numbers

Identification of Rhizopus species was traditionally based on the complexity of rhizoids, the length of the sporangiophores and the size of the sporangia along with the ability to grow in certain temperatures. In their revision, Schipper and Stalpers (1984) recognized five species in three major complexes. Later several new species and varieties were described (e.g. Ellis 1985; Schipper and Samson 1994). Following a comprehensive morphological revision, Zheng et al. (2007) recognized ten species and seven varieties. Molecular analyses (Abe et al. 2006, 2010; Hoffmann et al. 2013; Walther et al. 2013) supported the three complexes defined by Schipper and Stalpers (1984), but revealed that Rhizopus is paraphyletic containing Sporodiniella umbellata and Syzygites megalocarpus (Hoffmann et al. 2013; Walther et al. 2013). Based on molecular phylogenetic analyses several species were recognized to represent synonyms: e.g. Amylomyces rouxii is now treated as synonymous with R. arrhizus (Abe et al. 2006), R. reflexus was recognized as a synonym of R. lyococcus (Liou et al. 2007), and R. azygosporus was revealed to be conspecific with R. microsporus (Abe et al. 2006). Dolatabadi et al. (2014b) showed that the morphologically defined varieties of R. microsporus are not recognized in multi-marker phylogenies and consequently they reduced the varieties to synonyms. Abe et al. (2007) revealed that strains of R. arrhizus (as R. oryzae) split into producers of lactic acid and producers of fumaric and malic acid and that these two groups were molecular phylogenetically distinct. As a consequence, the authors treated fumaric-malic acid producers as a separate species, R. delemar, formerly regarded as a variety by Zheng et al. (2007). Gryganskyi et al. (2010) supported this concept by molecular phylogenetic studies based on several markers including mating type genes. In agreement with the previous studies, Dolatabadi et al. (2014a) recognized two phylogenetic species. However, they treated them as varieties of a single biological species because of the formation of zygospores between strains of the arrhizus- and strains of the delemar-group, the lack of differences in morphology and ecology and the small genetic distance between the two groups compared to the remaining species in Rhizopus. Variety tonkinensis, a third variety besides var. arrhizus and var. delemar, was recognized morphologically (Zheng et al. 2007) and through the use of short tandem repeat motives of IGS rDNA sequences (Liu et al. 2008), but it has not come out as a separate lineage in molecular phylogenetic studies (Walther et al. 2013; Dolatabadi et al. 2014a) and is regarded as doubtful. Abe et al. (2010) consider R. americanus and R. sexualis as varieties of R. stolonifer, while other authors (e.g. Zheng et al. 2007) recognize them as separate species. However, the large genetic distances of the ITS region among these taxa (Walther et al. 2013) rather suggest separate species. In the ITS trees of Walther et al. (2013), the strains morphologically defined as R. stolonifer form two distinctly separated groups suggesting the existence of an undescribed species. Currently seven species are accepted in Rhizopus: R. americanus, R. arrhizus including var. arrhizus and var. delemar, R. homothallicus, R. lyococcus, R. microsporus, R. sexualis, and R. stolonifer (Table 22).

Molecular phylogeny

The marker of choice for species identification in the genus Rhizopus is the ITS region (Walther et al. 2013) that can also distinguish the two varieties of R. arrhizus: var. arrhizus and var. delemar (Fig. 24). For the three species R. americanus, R. sexualis and R. stolonifer, sequencing of the ITS is often hampered by extended poly-A- and poly-T-regions but the large subunit of the ribosomal DNA (LSU) can be sequenced for species identification in these cases because it can also resolve these species (Walther et al. 2013). In case of R. americanus, multiple different ITS sequences within one strain were found, which should be considered in molecular identification (Liu et al. 2007; Abe et al. 2010).
Fig. 24

Phylogram generated from Maximum likelihood analysis based on ITS sequenced data of Rhizopus. Bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold

Several molecular markers have been applied for phylogenetic inference in this genus by using general fungal primers: actin (Abe et al. 2007, 2010; Dolatabadi et al. 2014a, b), ITS (Abe et al. 2006, 2007, 2010; Gryganskyi et al. 2010; Walther et al. 2013; Dolatabadi et al. 2014a, b), LSU (Abe et al. 2006; Liou et al. 2007; Walther et al. 2013; Dolatabadi et al. 2014a, b,), orotidine-5’-monophosphate decarboxylase gene (pyrG gene) (Liu et al. 2007), rpb1 (RNA polymerase II largest subunit gene) (Dolatabadi et al. 2014a), SSU (small subunit of the ribosomal DNA gene) (Abe et al. 2006), and tef (translation elongation factor gene) (Abe et al. 2007, 2010; Dolatabadi et al. 2014a, b). For R. arrhizus s.l., specific primers were designed for the rpb2 (RNA polymerase II second largest subunit gene) and the RNA helicase and the TP transporter gene of the mating locus by Gryganskyi et al. (2010) as well as for the lactate dehydrogenase B by Abe et al. (2007).

The tef marker cannot be recommended for phylogenetic studies because the gene is found in several different copies at least in R. arrhizus; these copies typically differ in the third base of numerous codons of this marker (Dolatabadi et al. 2014a). In the multi-marker study of Dolatabadi et al. (2014a), the rpb1 was the most variable gene.

Recommended genetic markers

  • The internal transcribed spacer (ITS)–generic and species level

  • The RNA polymerase II largest subunit gene (RPB1)–generic and species level

  • The large and small subunits of nrDNA (LSU and SSU)–placement within the Mucorales order, higher-level phylogeny

  • The partial actin gene (ACT)–higher-level phylogeny

Stagonosporopsis

Background

Stagonosporopsis is a coelomycetous genus in Didymellaceae (de Gruyter et al. 2009), accommodating several important phytopathogenic species, some of which have well-described sexual forms in Didymella (Diedicke 1912; Aveskamp et al. 2010). Many Stagonosporopsis species are considered serious quarantine organisms in many parts of the world. Some species have a global distribution. Stagonosporopsis andigena, the cause of black blight of potato (Turkensteen 1978), and S. crystalliniformis, a destructive pathogen of tomato and potato (Loerakker et al. 1986; Noordeloos et al. 1993), have only been reported in the Andes region, and thus listed as A1 quarantine organisms (EPPO 2014). Stagonosporopsis chrysanthemi and S. inoxydabilis are the cause of ray (flower) blight of Asteraceae (Stevens 1907; Van der Aa et al. 1990; Vaghefi et al. 2012), and A2 quarantine organisms (EPPO 2014) (listed as Didymella ligulicola). In Australia, S. tanaceti is known as the causal agent of ray blight of pyrethrum, capable of causing complete yield loss (Pethybridge et al. 2008). Stagonosporopsis cucurbitacearum (sexual state Didymella bryoniae) is a destructive seed-borne pathogen of Cucurbitaceae worldwide, causing gummy stem blight and black fruit rot (Punithalingam and Holliday 1972; Lee et al. 1984; Zitter and Kyle 1992). Stagonosporopsis species have also been reported from other plant families including Amaranthaceae, Campanulaceae, Caryophyllaceae, Fabaceae, Lamiaceae, Ranunculaceae, and Valerianaceae. The only species not isolated from a plant substrate is S. oculo-hominis, which was reported from human corneal ulcer in the USA (Punithalingam 1976).

Species identification and numbers

Stagonosporopsis was originally separated from Ascochyta on the basis of occasional formation of multi-septate (Stagonospora-like) conidia (Diedicke 1912). No type material was specified by Diedicke (1912) such that the first species combination described, S. actaeae, was interpreted as the generic type by some authors (Boerema et al. 1997, 2004). However, S. boltshauseri, currently known as S. hortensis (Boerema and Verhoeven 1979), was designated as the lectotype by Clements and Shear (1931).

In vitro, S. hortensis predominantly produces non-septate Phoma-like conidia, resembling those of Boeremia exigua var. exigua, while a few larger septate conidia can occasionally be found. In vivo, however, S. hortensis can be distinguished from B. exigua by predominance of one-septate (Ascochyta-like) conidia and occasional occurrence of two- or multi-septate (Stagonospora-like) spores. It is thus not a typical Ascochyta or Stagonospora, both of which produce septate conidia both in vivo and in vitro, and was classified under the genus Stagonosporopsis (Boerema and Verhoeven 1979).

Boerema et al. (1997, 1999) described multiple Stagonosporopsis spp. to be synanamorphs for several Phoma species in section Heterospora. The characteristic of section Heterospora is the in vivo production of distinctly large conidia (ascochytoid /stagonosporoid) in addition to relatively small (phomoid) conidia. The large conidial phenotypes may be dominant in vivo, hence described as Stagonosporopsis synanamorphs (Boerema et al. 1997, 1999, 2004).

Recent phylogenetic delineation of Phoma and allied genera placed the presumed Stagonosporopsis types in the family Didymellaceae (de Gruyter et al. 2009), and an emended description of the genus was proposed (Aveskamp et al. 2010). Some of the heterosporous Phoma species with known Stagonosporopsis synanamorphs were retrieved outside the Stagonosporopsis clade. On the other hand, many species from sections Heterospora, Phoma and Phyllostictoides, for which no records of a Stagonosporopsis synanamorph had been made, clustered with Stagonosporopsis spp. This indicated that the connection of Stagonosporopsis with heterosporous Phoma species was not justified. It also suggested that presence of Stagonospora-like spores is not a reliable criterion for identification of Stagonosporopsis species. Stagonosporopsis dorenboschiae, S. loticola, and S. ajacis lack the Stagonospora-like spores and any further features than a plain, globose pycnidium, and aseptate, hyaline conidia (Aveskamp et al. 2010). Due to unreliability of morphological characters, phylogenetic species recognition is essential for identification of Stagonosporopsis species.

Stagonosporopsis in its original description by Diedicke (1912) accommodated seven species, and currently more than 40 species are linked to this genus (data from MycoBank and Index Fungorum). However, only 21 Stagonosporopsis species have thus far been recognised based on multi-gene phylogenies (Table 23) (Aveskamp et al. 2010; Vaghefi et al. 2012). The phylogenetic reassessment of Didymellaceae (Aveskamp et al. 2010) included only those Stagonosporopsis species that had been designated as Phoma synanmorphs by Boerema et al. (1997, 1999). Molecular data for multiple other Stagonosporopsis species are still lacking and, therefore, the taxonomy of the genus Stagonosporopsis remains to be comprehensively reviewed.

Molecular phylogeny

Few phylogenetic analyses of Stagonosporopsis species are available (Pethybridge et al. 2004; Aveskamp et al. 2010; de Gruyter et al. 2012; Vaghefi et al. 2012, 2014), with the most comprehensive analysis being the three-marker phylogeny performed by Aveskamp et al. (2010). The phylogeny of combined sequences of large subunit nrDNA (LSU), the internal transcribed spacers and the 5.8 S nrRNA (ITS), and β-tubulin regions resulted in the recognition of 19 species (Aveskamp et al. 2010). Phylogenies based on the partial actin (ACT) sequence were later found to be congruent with the LSU- ITS- β- tubulin phylogeny (de Gruyter et al. 2012; Vaghefi et al. 2012). A four-marker phylogeny of the Stagonosporopsis spp. for which these DNA sequence data are available is shown (Fig. 25).
Fig. 25

Phylogram generated from Maximum likelihood analysis based on combined LSU, ITS, β- tubulin and ACT sequenced data of Stagonosporopsis. Bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Phoma herbarum CBS 615.75

Recommended genetic markers

  • The internal transcribed spacer (ITS)–family/generic level

  • β- tubulin and ACT–inter-specific delineation

A high level of infra-specific variation has been recorded for calmodulin (CAL) in Phoma-like species, however, it may be difficult to amplify in some Stagonosposopsis species, and requires optimization using different degenerate primers (Aveskamp et al. 2009, 2010; Vaghefi et al. 2012). Thus use of β- tubulin and ACT is suggested as they will give sufficient distinction between species, and are easier to amplify.

Ustilago

Background

Ustilago is the largest genus of the Ustilaginaceae in the order of smut fungi, Ustilaginales, with about 200 currently accepted species (Vánky 2013). Ustilago and related genera contain many important plant pathogens that destroy the inflorescence or culms of grasses (Poaceae) (Vánky 2011). Some agriculturally important pathogens of grain and edible crops are U. tritici on wheat (Triticum), U. hordei on barley (Hordeum) and U. maydis on corn (Zea mays). Species of Ustilago have been used as model organisms for the study of plant disease pathways and mating types (Andrews et al. 2000; Bakkeren et al. 2008; Kellner et al. 2011), as well as for studies in the co-evolution of pathogens with their hosts (Begerow et al. 2004). The genomes of U. maydis and U. hordei were released in 2003 and 2012, respectively (Kamper et al. 2006; Laurie et al. 2012).

Ustilago was until recently a catch-all genus for smut fungi on a diversity of host families, including the Carophyllaceae, Cyperaceae, Poaceae, Polygonaceae, Restionaceae, and Tilliaceae (McTaggart et al. 2012b). Closely related genera were not easily distinguished from Ustilago by morphology, and formed a complex (Stoll et al. 2003, 2005). Subsequent systematic studies reserved Ustilago s. lat. for species that infected Poaceae, with Ustilago s. str. restricted to the tribe Pooideae (McTaggart et al. 2012a; Stoll et al. 2005). Soral morphology and host range were later found to be synapomorphic character states for the smut genera Anthracocystis, Langdonia, Sporisorium, Stollia and Triodiomyces, which were differentiated from Ustilago (McTaggart et al. 2012c). Melanopsichium is closely related to Ustilago, and appears to have jumped hosts from Poaceae to Polygonaceae (Begerow et al. 2004; Stoll et al. 2005).

Species identification and numbers

The diversity of smuts in the Ustilaginaceae on Poaceae encompasses over 530 species (Vánky 2011). Cryptic species are certain to be revealed when species complexes, e.g., Macalpinomyces eriachnes, are investigated. Vánky (2011) recognised approximately 170 species of Ustilago, which were delimited by host and spore morphology. It is likely the species number of Ustilago will decrease when generic concepts are resolved in the Ustilaginaceae. Species currently recognized as Ustilago will be transferred to new or other genera delimited by sorus morphology and host range. For example, U. maydis does not fit the concept of Ustilago s. str. and warrants transfer to the earliest valid genus, Mycosarcoma, when these closely related genera are resolved (McTaggart et al. 2012a; Stoll et al. 2005; Vánky and Lutz 2011; Piepenbring et al. 2002) (Table 24).

Molecular phylogeny

Relationships between Ustilago and closely related genera are still unresolved, and Ustilago is polyphyletic (Fig. 26Ustilago). Systematic studies based on the nLSU or ITS regions of rDNA have assigned taxa within these closely related genera (Shivas et al. 2013a; Vánky and Lutz 2011; McTaggart et al. 2012c). Nuclear genes (EF1α, GPDH, RPB1 and RPB2), another ribosomal gene (SSU) and mating loci were explored as markers for the evolution of smut fungi in the Ustilaginaceae (Kellner et al. 2011; McTaggart et al. 2012a; Munkacsi et al. 2007). At this stage, these markers are not as widely used as ITS and LSU, which are recommended for species identification and generic placement, respectively.
Fig. 26

Phylogram generated from ML search in RA × ML based on combined ITS and LSU sequenced data of Ustilago. Bootstrap support values greater than 70 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold

Recommended genetic markers

  • The large subunit (LSU) of nrDNA–generic level

  • The internal transcribed spacer (ITS) of nrDNA–species level

Verticillium

Background

Verticillium belongs in the family Plectosphaerellaceae of the Ascomycota. Verticillium species are soilborne, vascular, fungal plant pathogens that cause Verticillium wilt disease in many important agricultural crops throughout the world (Pegg and Brady 2002). Based on susceptibility, 410 plant species that include nearly 80 plant genera have been recorded as being infected by Verticillium species (Pegg and Brady 2002). Correct species identification is important for determining the ecological roles of Verticillium species and for diagnosing disease. Sexual stages have not been identified for Verticillium species although mating type idiomorphs MAT1-1 and MAT1-2 have been identified in separate isolates of V. dahliae, V. albo-atrum, V. longisporum, V. alfalfa and V. nonalfalfae, indicating that these species are potentially heterothallic (Inderbitzin et al. 2011a, b; Usami et al. 2009).

Species identification and numbers

The genus Verticillium sensu stricto refers to a monophyletic group of plant pathogens comprising V. dahliae as the type of Verticillium (Gams et al. 2005). The genus can be identified based on its distinct ‘verticillate conidiophores’ with flask-shaped conidiophores arranged in whorls attached along a main axis that comprise the spore forming cells (Pegg and Brady 2002). The genus Verticillium has a long taxonomic history and approximately 190 species were originally classified by Zare et al. (2004). Recently Inderbitzin et al. (2011a) used four-marker phylogenetic analysis to identify ten Verticillium species.

Earlier studies identified Verticillium species primarily on the basis of morphology and sub-specific groups by virulence and aggressiveness on various hosts (Rowe 1995). Variation in conidial morphology of Verticillium species is minor and thus cannot be used to separate species (Rowe 1995). Resting structure morphology has been the major morphological character used to differentiate species of Verticillium.

Verticillium albo-atrum and V. dahliae are the most important plant pathogenic species. Verticillium albo-atrum was first described in Germany, 1879, by Reinke and Berthold as the causal agent of potato wilt. The resting structures identified from the diseased plant tissue were brown-pigmented hyphae which were described as ‘Dauermycelien’. Later this pigmented hyphae was termed dark ‘resting mycelium’ which had only transverse walls and no lateral budding (Isaac 1949). No microsclerotia were produced by V. albo-atrum.

Verticillium dahliae was first isolated by Klebahn in 1913 from wilting Dahlia. The isolate produces smaller and oval to elongate microsclerotia as a resting structure from budding hyphae, but not dark resting mycelium (Smith 1965). Verticillium tricorpus forms large and irregular microsclerotia with melanised hyphae and chlamydospores (hence the prefix “tri”). Moreover, V. tricorpus often produces yellow colonies on PDA upon first isolation (Goud et al. 2003). Verticillium nubilum produces only rounded to elongate chlamydospores, individually or in chains (Inderbitzin et al. 2011a). Verticillium longisporum refers to the species proposed by Karapapa et al. (1997) that infected hosts in the family Brassicaceae. Isolates of this species produce microsclerotia which are rounded to elongate with relatively long conidia, and nearly double the nuclear DNA content (Inderbitzin et al. 2011a).

Molecular techniques have been used in the characterisation and identification of Verticillium species for both species identification and phylogenetic comparisons (Collins et al. 2003; Collado-Romero et al. 2008). Using restriction fragment length polymorphism (RFLP) analysis, Typas et al. (1992) reported that mitochondrial DNA of Verticillium species were distinctive and easily differentiated V. albo-atrum (from alfalfa) from other V. albo-atrum isolates. Carder and Barbara (1991) used RFLP analysis to differentiate V. dahliae from all isolates of V. albo-atrum and found intraspecific variation within V. dahliae isolates. Subsequently, Okoli et al. (1993) probed Southern blots derived from 17 isolates of V. dahliae with 71 random genomic clones from V. dahliae and found that 15 isolates fitted clearly into two RFLP groups designated A and B. Although these groups correlated with isozyme patterns they did not show any correlation with host plant or geographic origin. Random amplified polymorphic DNA (RAPD) markers clearly differentiated 15 V. albo-atrum potato isolates from 20 alfalfa V. albo-atrum isolates and found that these two groups were genetically distinct (Barasubiye et al. 1995). Komatsu et al. (2001) used repetitive extragenic palindromic polymerase chain reaction (REP-PCR) and RAPD markers to show that V. dahliae isolates from potato were similar in genetic background, regardless of geographic origin.

In North America, characterization of vegetative compatibility groups (VCGs have the ability to undergo hyphal anastomosis with other isolates) using molecular markers confirmed that VCG 4A isolates of V. dahliae were more highly virulent than VCG 4B isolates (Dobinson et al. 2000). Molecular characterization of VCGs has been determined in many other crops (Collado-Romero et al. 2006, 2009; Dobinson et al. 1998).

Molecular phylogeny

Nazar et al. (1991) found only five nucleotide differences between V. dahliae and V. albo-atrum on the basis of the non-conserved ITS region (ITS 1 and ITS 2) of rDNA. Robb et al. (1993) reported 17 nucleotide differences between V. dahliae and V. tricorpus and 12 between V. albo-atrum and V. tricorpus (Moukhamedov et al. 1994). Phylogenetic analysis of the complete intergenic spacer (IGS) region of the nuclear ribosomal RNA (rDNA) and the β-tubulin gene showed distinct groups comprising isolates of V. albo-atrum, V. tricorpus, and V. dahliae from cruciferous and noncruciferous hosts (Qin et al. 2006).

Fahleson et al. (2004) studied three different markers (mitochondrial cytochrome b gene (cob), the mitochondrial small subunit rRNA gene (rns) and the nuclear ITS2 region) sequences from five plant pathogenic isolates of Verticillium and found five monophyletic groups corresponding to the Verticillium species. In addition, V. tricorpus displayed a closer relationship to V. albo-atrum, V. dahliae and V. longisporum. But V. nigrescens was distantly related to the other species. Based on nuclear large subunit ribosomal DNA (LSU) and ITS sequences, Zare et al. (2007) proposed Gibellulopsis as a genus to accommodate V. nigrescens.

Recent molecular phylogenetic studies by Inderbitzin et al. (2011a) using four gene sequences viz actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase, divided Verticillium into two separate groups, corresponding to the production of yellow pigment in culture (clade Flavexudans), or the lack of yellow pigment (clade Flavnonexudans). The species Verticillium albo-atrum, V. tricorpus, V. zaregamsianum, V. isaacii and V. klebahnii were placed in the Flavexudans clade of which the latter two species were morphologically indistinguishable from V. tricorpus. The species Verticillium dahliae, V. nubilum, V. longisporum, V. alfalfae and V. nonalfalfae were placed in the clade Flavnonexudans (Inderbitzin et al. 2011a). Interestingly, V. longisporum which is a diploid hybrid had alleles in different clades including the V. dahliae clade thus reflecting the ancestral origin of the hybrid. According to Inderbitzin et al. (2011b), each V. longisporum isolate contained two alleles at each locus with allele A1 being present in all isolates in addition to alleles D1, D2 or D3. Therefore, according to Inderbitzin et al. (2011a), V. longisporum should remain a polyphyletic species.

The phylogenetic tree of the ten species adopted by Inderbitzin et al. (2011a) did not include the ribosomal internal transcribed spacer region ITS, because V. longisporum isolates only had one ITS allele consistent with all other Verticillium species and hence this gene sequence could not retrace the evolution of the species (Inderbitzin et al. 2011b). Nevertheless, neither the four gene phylogenetic analysis nor the single ITS phylogenetic tree were able to differentiate V. longisporum alleles D2 and D3 from V. dahliae (Inderbitzin et al. 2011b).

In contrast to the above results, a four gene phylogenetic tree composed of only the type isolates (Fig. 27) failed to differentiate V. isaacii from V. klebahnii; while V. alfalfa was identical to V. nonalfalfae; and V. dahliae was identical to V. longisporum allele D2. Nevertheless, the phylogenetic tree based only on ITS (Fig. 28) provided better discrimination to differentiate V. isaacii from V. klebahnii, and V. alfalfa from V. nonalfalfae, albeit with weak bootstrap supports.
Fig. 27

Phylogram generated from parsimony analysis based on combined ACT, TEF, GPD and ITS sequenced data of Verticillium. Parsimony bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Gibellulopsis nigrescens

Fig. 28

Phylogram generated from parsimony analysis based on ITS sequenced data of Verticillium. Parsimony bootstrap support values greater than 50 % are indicated above the nodes. The ex-type (ex-epitype) and voucher strains are in bold. The tree is rooted with Gibellulopsis nigrescens

Another anomaly with the four gene phylogenetic tree based on only type isolates was that V. nubilum claded with the yellow pigment forming Flavexudans species whereas in the tree by Inderbitzin et al. (2011a), V. nubilum claded with the Flavnonexudans species. Nevertheless, the phylogenetic tree based only on ITS (Fig. 28) placed V. nubilum in the Flavnonexudans species group. In fact V. nubilum does not produce yellow pigment in culture, such that it is better placed in the Flavnonexudans species group.

Recommended genetic markers

Most of the ten Verticillium species can be identified using the ITS sequences of the type isolates (Table 25, Fig. 28) however, strong bootstrap support is provided for most clades using four gene sequences (Table 25, Fig. 27).
  • Internal transcribed spacer (ITS)–species level

  • Actin (ACT)–generic/species level

  • Elongation factor 1-alpha (EF)–generic/ species level

  • Glyceraldehyde-3-phosphate dehydrogenase (GPD)–generic/ species level

  • Tryptophan synthase (TS)–generic/ species level

Discussion

The present effort is far from exhaustive, and the selection of fungal lineages reflects the backgrounds of the authors rather than degree of pathogenicity or economic impact of the underlying fungi. Indeed, several of the groups covered are pathogens on plants that are used neither in agriculture nor forestry. Furthermore, the fact that a group is addressed in the present study should not be taken to mean that no further discoveries or insights in the group are likely to emerge; the opposite is certain to be true for all of the groups studied here. Knowledge of phytopathogenic fungi accumulates at a high pace, and we hope that the readers will use this study as a starting point in their pursuit. Towards that end, we aim to maintain rich, updated backbone trees of as many groups of plant pathogenic fungi as we can. These will be published as a joint paper on an annual or biennial basis as new data are produced. Researchers who can cover any group not presently covered–or improve on any of the groups that are covered already–are warmly invited to take part in this effort by contacting the corresponding author.

As one of the pursuits of this effort, we have attempted to address the question of which genes and genetic markers that will provide the highest phylogenetic/taxonomic resolution in various groups of plant pathogenic fungi. These differ markedly among groups. At the same time, for someone examining a sample of an unknown phytopathology-related fungus, the choice of initial genetic markers is easy. The ITS region–the formal fungal barcode–is the most commonly sequenced marker in mycology, such that a rich array of reference sequences is available. Although the ITS region will not always provide resolution at the species level, it will nearly always provide enough resolution to support assignment of the species to at least the level of subgenus/species complex. This information is likely to be enough for many applications; for others, it makes it much easier to make an informed choice of what genes to sequence next. However, researchers sometimes recover fungal ITS sequences that are not easily fitted into the corpus of reference ITS sequences. The next most sequenced marker in mycology is the nuclear ribosomal large subunit (nLSU; Begerow et al. 2010), which is significantly more conserved than the ITS region and offers resolution at the genus to order level. The nLSU is something of the mainstay of large-scale phylogenetic inference in fungi (Blackwell et al. 2006), and nearly all fungal nLSU sequences can be assigned to at least the ordinal level. For unknown samples, we thus advice researchers to sequence the ITS and nLSU regions as a first step.

Fungal plant pathogens attract the attention of numerous scientific and applied fields, including mycology, botany, agriculture, horticulture, silviculture, and medicine. In many cases this attention will centre on establishing, or ruling out, a pathogenic nature of specific fungal samples; and in many cases, such efforts will be based on molecular data. Molecular identification of fungi–DNA barcoding–has a long and rich history but was only recently formalized (Bruns et al. 1990; Schoch et al. 2012). Indeed, many parts of its realization still loom on the horizon. For instance, central barcoding resources and databases of wide acceptance in the mycological community are largely lacking. Most researchers, when processing newly generated fungal sequences, turn to GenBank (Benson et al. 2014) for sequence identification. Many entries in GenBank suffer from technical complications or low-resolution annotations, but efforts to standardize and improve on the data and level of metadata given are under way (Nilsson et al. 2014; Schoch et al. 2014). The largest database focusing on the formal fungal barcoding region–ITS–is UNITE (Kõljalg et al. 2013). Sharing data with GenBank, UNITE serves as the provider of reference fungal ITS datasets for a long range of applications and downstream uses. The results of the present effort–in particular, the sequences from type material–are being implemented in UNITE for all its diverse uses and for subsequent distribution to GenBank. We hope that this will lead to increased scientific resolution for researchers recovering any of the fungal lineages treated in this study.

The heterogeneous user base of data pertaining to phytopathogenic fungi suggests that many users of data pertaining to phytopathogenic fungi will not be–and cannot expected to be–up to date on recent developments in mycology, systematics, or the use of molecular data in biology. It is thus largely up to mycologists to provide the scientific community with as accurate and easily interpreted information on fungi and phytopathological fungal species as possible. The mycological community lives up to that expectation with various degrees of success. Improvement is particularly needed in the public sequence databases, where many researchers routinely submit phytopathologically relevant fungal sequences without any notion of taxonomic affiliation, host association, or country of collection (notably “Uncultured fungus”). Such sequences will be excluded from, or treated only superficially in, most research efforts and sequence comparisons, leading to reduced scientific resolution and explanatory power. We urge mycologists with a phytopathological inclination–indeed, with any inclination–to set good examples in this regard by providing rich, reliable annotations for their sequences. Guidelines on how to establish the integrity and improve the wide usefulness of fungal sequence data are readily available for consideration (Nilsson et al. 2012; Hyde et al. 2013a, b; Schoch et al. 2014). We similarly hope that all mycologists, when describing new species, will make it a habit to bundle at least one DNA sequence–starting with the ITS region–with the description (cf. Seifert and Rossman 2010). This will help others to interpret the name and will go a long way to make it available to the general scientific audience. Enclosing molecular data with species descriptions is not required by the current nomenclatural code governing fungi (McNeill et al. 2012), but we feel that this is a good opportunity for mycology to show its progressive nature. In a time where mycology finds it increasingly hard to compete for funding with disciplines deemed more cutting-edge, mycologists should make every effort to propagate their results and findings to the widest audience possible.

Acknowledgments

We would like to thank the CGIAR Research Program 1.2–Humidtropics: Integrated systems for the humid tropics, for partially funding this work. Kevin D. Hyde thanks the Chinese Academy of Sciences, project number 2013T2S0030, for the award of Visiting Professorship for Senior International Scientists at Kunming Institute of Botany. Thank to Plant Germplasm and Genomics Center in Germplasm Bank of Wild Species. Sajeewa Maharachchikumbura thanks the National Research Council of Thailand (grant for Pestalotiopsis No: 55201020008) and Mae Fah Luang University (grant for Pestalotiopsis No: 55101020004) for financial support. S.A. Alias thanks the University of Malaya for grant number RU006H-2014 entitled “diversity and importance of fungal mangrove disease”. Financial support to Julia Pawłowska and Marta Wrzosek was partially provided by the Polish Ministry of Science and Higher Education (MNiSW), grant no. NN303_548839. Henrik Nilsson acknowledges financial support from FORMAS (215-2011-498).