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  • Article
  • Open Access

Molecular phylogeny of Cytospora species associated with canker diseases of fruit and nut crops in California, with the descriptions of ten new species and one new combination

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IMA Fungus20189:902333

https://doi.org/10.5598/imafungus.2018.09.02.07

  • Received: 30 March 2018
  • Accepted: 12 September 2018
  • Published:

Abstract

Cytospora species are destructive canker and dieback pathogens of woody hosts in natural and agroecosystems around the world. In this genus, molecular identification has been limited due to the paucity of multi-locus sequence typing studies and the lack of sequence data from type specimens in public repositories, stalling robust phylogenetic reconstructions. In most cases a morphological species concept could not be applied due to the plasticity of characters and significant overlap of morphological features such as spore dimensions and fruiting body characters. In this study, we employed a molecular phylogenetic framework with the inclusion of four nuclear loci (ITS, translation elongation factor 1-alpha, actin, and beta-tubulin) to unveil the biodiversity and taxonomy of this understudied important genus of plant pathogens. Phylogenetic inferences based on 150 Californian isolates revealed 15 Cytospora species associated with branch and twig cankers and dieback of almond, apricot, cherry, cottonwood, olive, peach, pistachio, plum, pomegranate, and walnut trees in California. Of the 15 species recovered in this study, 10 are newly described and typified, in addition to one new combination. The pathogenic status of the newly described Cytospora species requires further investigation as most species were associated with severe dieback and decline of diverse and economically important fruit and nut crops in California.

Keywords

  • Cytosporaceae
  • Cytospora canker
  • Diaporthales
  • multigene phylogeny
  • new taxa
  • taxonomy

Introduction

The generic name Cytospora (Sordariomycetes, Diaporthales, Cytosporaceae) was introduced in 1818 and includes seemingly innocuous endophytes isolated from the bark, xylem, and leaves of asymptomatic woody plants (Spielman 1983, Bills 1996), saprobes that colonize and degrade the wood of dead or dying trees (Christensen 1940), and destructive canker pathogens (known as Cytospora-, Leucostoma-, Valsa-, or perennial canker) that cause dieback of more than 85 woody plant species (Sinclair et al. 1987, Adams et al. 2005, 2006). The chronic wood infections caused by Cytospora species can be devastating to stone fruit, pome fruit, and nut crops such as Prunus persica, P armeniaca, P. avium, Malus spp., and Juglans spp. (Biggs & Grove 2005, Wang et al. 2011, Fan et al. 2015a). Cytospora species mainly impact branches, but they can cause more destructive infections in the trunks and larger scaffolds, severely limiting the longevity and productivity of orchards (Biggs 1989, Chang et al. 1991).

To date, approximately 612 Cytospora species have been described according to Index Fungorum. Kirk et al. (2008) listed approximately 110 accepted Cytospora species, while all other species names were considered synonyms of previously described taxa or treated as non-Cytospora species before the one fungus = one name rule came into force in July 2011 (Hawksworth 2011). Therefore, all taxa including the former sexual and asexual morphs that no longer have nomenclatural priority should be considered in order to resolve nomenclatural issues in this group of challenging fungi. The asexual morph is commonly encountered in nature. The pycnidia arise in a stroma embedded in host tissues (Grove 1923), and possess either a single locule or a complex of invaginated walls producing labyrinthine locules with filamentous conidiophores which may be reduced to conidiogenous cells that bear hyaline, allantoid conidia (Adams et al. 2006). Pycnidia exude conidia in a yellow, orange to red polysaccharide matrix, a cirrus, via an ostiole (Adams et al. 2005, 2006). Conidia oozing from pycnidia embedded in dead or dying host cortical tissues during humid or wet conditions are considered the infectious propagules potentially initiating new infections; the role of ascospores has not been determined. Conidia are dispersed to new plant tissues by rain-splash, where they germinate and infect the host plant via cracks and wounds to the bark created by pruning wounds, leaf scars, insect injuries, winter-injured buds, twigs and bark, and breakage of shade-weakened twigs and branches (Tekauz & Patrick 1974, Biggs 1989). Bertrand & English (1976b) showed that Cytospora conidia were routinely trapped up to 76.8 m from the primary inoculum source after wind-blown rain in California, thus providing compelling evidence for Cytospora spore dispersal across large areas within orchards during times of inclement weather.

Species diagnosis in Cytospora has traditionally relied on morphological characters of pycnidia/perithecia (Grove 1923), including locule shape/organization and spore dimensions (Spielman 1985), as well as the arrangement of stromatic tissues (Adams et al. 2002). This morphological species approach is confounded by many examples of morphological character overlap among species and by the morphological plasticity of pycnidial locules which are affected by the host bark and cambium characteristics (Adams et al. 2002, Wang et al. 2011). Species diagnosis based on host association has also proven unreliable as several species of Cytospora have been recovered from multiple distantly related hosts, while a single host species can harbour more than one species of Cytospora (Adams et al. 2005, 2006, Wang et al. 2011, Fan et al. 2015a, b).

Défago (1935) questioned the utility of morphological characters in delimiting Cytospora species. Spielman (1985) reported that the asexual morph of Cytospora leucosperma was indistinguishable from that of many other species of Cytospora. Traditionally, sexual morphs of Cytospora were classified within several genera including Leucostoma, Valsa, Valsella, and Valseutypella. Tulasne & Tulasne (1863) postulated that Cytospora and Valsa were the asexual and sexual morphs of the same fungus. All these studies have highlighted the difficulty to properly disentangle taxa that share similar morphologies. Species identification based on molecular data could overcome these difficulties, which has been illustrated using ITS rDNA phylogenies (Adams et al. 2002, 2005, 2006). Recently, the use of the generic name Cytospora has been recommended for protection and use over Leucocytospora, Leucostoma, Valsa, Valsella, and Valseutypella (Rossman et al. 2015).

According to Norphanphoun et al. (2017) there are currently only 23 ex-type Cytospora species sequences deposited in GenBank. The majority of these sequences correspond to a single nuclear ribosomal gene region covering the ITS or the partial nuclear large ribosomal RNA subunit (nrLSU). Molecular data from type specimens are thus limited in public repositories and hamper abilities to properly circumscribe or identify taxa to the species-level in Cytospora. Recently, the utility of additional protein-coding loci, such as beta-tubulin, actin, and translation elongation factor 1-alpha, has been demonstrated for Cytospora sequence-based identification: more Cytospora species were recognized when using analyses including multiple protein-coding loci, relative to analyses relying on ITS only or combined ITS and nrLSU (Lawrence et al. 2017a).

Although Cytospora species are known pathogens of stone fruits and nut crops worldwide, the taxonomy and host distribution of Cytospora species occurring in California orchards are still elusive, with only C. leucostoma and/or C. cincta known to affect French prune (Bertrand & English 1976a), peach and nectarine (French 1989), and sweet cherry (Trouillas et al. 2012). California is the largest and most productive perennial agricultural area in North America, producing diverse fruit and nut crops which constitute potential hosts for Cytospora species. The objectives of this study were to examine the phylogenetic diversity of Cytospora species isolated from orchards exhibiting dieback and canker diseases in California. Our hypotheses were that new Cytospora species would be identified from a region and crops that have been under-examined, especially given the recent advances in molecular identification of fungi (Hibbett et al. 2016). We hypothesized also that distinct species of Cytospora would infect distinct crop species, as expected if host specificity would favour pathogen speciation (Giraud et al. 2006). Morphological characters in conjunction with multilocus phylogenetic analyses will afford us the first glimpse into the biodiversity of this important genus of canker pathogens.

Materials and Methods

Fruit and nut crop sampling and fungal isolation

Between 2010 and 2017, putative Cytospora species were isolated periodically from declining fruit and nut trees throughout the Central Valley region of California as part of the diagnosis activity of the co-operative extension laboratories at the Kearney Agricultural Research and Extension Centre, in the centre of major agricultural industries. Sampled trees expressed general symptoms and signs of canker diseases including branch dieback, leaf wilting, dead and split bark, sunken lesions on branches, internal wood discoloration, gumming on trunks and scaffold limbs, cracked bark revealing blackened tissues, and presence of pinhead-sized dark pycnidia erupting through the bark or exposed upon peeling the outer layer of the bark (Figs 13). Mass-hyphal isolates were recovered using 10–12 wood pieces (4 × 4 × 2 mm) per sample, cut from the margins of necrotic and apparently healthy wood, surface disinfested in 0.6 % sodium hypochlorite for 60 s, rinsed in two serial baths of sterile deionized water for 30 s, and plated on potato dextrose agar (PDA, Difco, Detroit, MI) dishes amended with tetracycline (1 mg L−1). A number of isolates were also collected directly from conidial masses exuding from freshly exposed pycnidia on declining branches. Masses of conidia were collected using a sterilized needle, placed into 1.5 mL tubes containing sterile water, and spread onto the surface of PDA Petri dishes. Petri dishes were incubated at 25 °C in the dark for up to 28 d. Isolates with morphological characters of Cytospora, namely colonies with uneven to highly uneven growth margins and thus lobate to highly lobate colony morphology, were hyphal-tip purified to fresh PDA dishes. In total, 150 isolates from symptomatic orchards and adjacent ornamental trees throughout the Central Valley of California were recovered in pure culture and used for phylogenetic and morphological analyses (Table 1). Representative cultures used in this study are permanently preserved in the collections of the Department of Plant Pathology at the Kearney Agricultural Research and Extension Centre of the University of California, Parlier, CA. The holotypes of the newly described species are preserved as dried cultures in BPI, with ex-type cultures deposited in CBS.
Fig. 1
Fig. 1

Signs and symptoms of Cytospora canker/dieback in various fruit and nut crops in California. A. Twig dieback in sweet cherry. B. Twig and scaffold branch dieback in French prune. C. Pimpled-bark indicating underlying asexual fruiting bodies in a sweet cherry branch affected with Cytospora canker. D. Below bark, asexual fruiting bodies associated with Cytospora canker of French prune. E–F. Cankers and wood discoloration associated with Cytospora canker of sweet cherry.

Fig. 2
Fig. 2

Signs and symptoms of Cytospora canker/dieback in various fruit and nut crops in California. A–B. Gumming and underlying elongated canker associated with Cytospora canker in almond. C. Cytospora associated cankers in olive twigs. D. Cankers and wood discoloration associated with Cytospora in pistachio. E. Conidial masses exuding from Cytospora asexual fruiting bodies in walnut.

Fig. 3
Fig. 3

Signs and symptoms of Cytospora canker/dieback in cottonwood and pomegranate hosts in California. A–B. Dead cottonwood tree parts on a roadside surrounding orchards and associated Cytospora asexual fruiting bodies erupting through the bark. C–D. Cytospora canker, wood discoloration and associated branch dieback in pomegranate.

Table 1

Cytospora species recovered from symptomatic hosts in California.

     

GenBank Accession No.

 

Species

Isolate a

Host

Geographic origin

ITS

ACT1

TEF1

TUB2

Cytospora amydgali

LH356

Prunus dulcis

Yolo Co., California, USA

MG971852

MG972001

MG971658

MG971717

C. amydgali

LH357/CBS 144233

Prunus dulcis

Yolo Co., California, USA

MG971853

MG972002

MG971659

MG971718

C. californica

9C-24/CBS 144234

Juglans regia

Lake Co., California, USA

MG971935

MG972083

MG971645

C. californica

KARE264

Pistacia vera

Kern Co., California, USA

MG971920

MG972069

MG971630

MG971780

C. californica

KARE265

Pistacia vera

Kern Co., California, USA

MG971914

MG972064

MG971624

MG971776

C. californica

KARE303

Pistacia vera

Kern Co., California, USA

MG971913

MG972063

MG971623

MG971775

C. californica

KARE324

Pistacia vera

Kern Co., California, USA

MG971911

MG972061

MG971621

MG971773

C. californica

KARE325

Pistacia vera

Kern Co., California, USA

MG971918

MG972067

MG971628

C. californica

KARE326

Pistacia vera

Kern Co., California, USA

MG971919

MG972068

MG971629

C. californica

KARE1091

Pistacia vera

Kern Co., California, USA

MG971946

MG972096

MG971662

MG971790

C. californica

KARE1104

Prunus dulcis

Fresno Co., California, USA

MG971928

MG972077

MG971638

MG971783

C. californica

KARE1107

Prunus dulcis

Fresno Co., California, USA

MG971929

MG972078

MG971639

C. californica

KARE166

Prunus dulcis

Fresno Co., California, USA

MG971916

MG972093

MG971626

MG971778

C. californica

KARE197

Prunus dulcis

Fresno Co., California, USA

MG971932

MG972081

MG971642

MG971786

C. californica

KARE198

Prunus dulcis

Fresno Co., California, USA

MG971915

MG972065

MG971625

MG971777

C. californica

KARE1105

Prunus dulcis

Fresno Co., California, USA

MG971947

MG972097

MG971663

MG971791

C. californica

KARE1106

Prunus dulcis

Fresno Co., California, USA

MG971948

MG972094

MG971647

MG971788

C. californica

KARE1377

Prunus dulcis

Glenn Co., California, USA

MG971933

MG972057

MG971643

MG971787

C. californica

KARE1191

Prunus dulcis

Glenn Co., California, USA

MG971945

MG972095

MG971661

MG971789

C. californica

KARE884

Prunus dulcis

San Joaquin Co., California, USA

MG971925

MG972074

MG971635

C. californica

KARE894

Prunus dulcis

San Joaquin Co., California, USA

MG971927

MG972076

MG971637

C. californica

KARE895

Prunus dulcis

San Joaquin Co., California, USA

MG971926

MG972075

MG971636

C. californica

KARE896

Prunus dulcis

San Joaquin Co., California, USA

MG971936

MG972084

MG971646

C. californica

KARE902

Prunus dulcis

San Joaquin Co., California, USA

MG971924

MG972073

MG971634

MG971782

C. californica

KARE903

Prunus dulcis

San Joaquin Co., California, USA

MG971922

MG972071

MG971632

MG971781

C. californica

KARE904

Prunus dulcis

San Joaquin Co., California, USA

MG971923

MG972072

MG971633

C. californica

KARE905

Prunus dulcis

San Joaquin Co., California, USA

MG971921

MG972070

MG971631

C. californica

KARE62

Prunus dulcis

Stanislaus Co., California, USA

MG971912

MG972062

MG971622

MG971774

C. californica

KARE883

Prunus dulcis

Stanislaus Co., California, USA

MG971934

MG972082

MG971644

C. californica

KARE93

Prunus dulcis

Stanislaus Co., California, USA

MG971930

MG972079

MG971640

MG971784

C. californica

KARE94

Prunus dulcis

Stanislaus Co., California, USA

MG971931

MG972080

MG971641

MG971785

C. californica

KARE99

Prunus dulcis

Stanislaus Co., California, USA

MG971917

MG972066

MG971627

MG971779

C. chrysosperma

9E-33/CBS 144242

Camellia

Fresno Co., California, USA

MG971892

MG972041

MG971602

MG971758

C. eucalypti

KARE1585/CBS 144241

Prunus dulcis

Merced Co., California, USA

MG971907

MG972056

MG971617

MG971772

C. eucalypti

KARE888

Prunus dulcis

San Joaquin Co., California, USA

MG971909

MG972059

MG971619

C. eucalypti

KARE889

Prunus dulcis

San Joaquin Co., California, USA

MG971908

MG972058

MG971618

C. eucalypti

KARE890

Prunus dulcis

San Joaquin Co., California, USA

MG971906

MG972055

MG971616

C. eucalypti

7G-62

Sequoiadendron giganteum

Fresno Co., California, USA

MG971910

MG972060

MG971620

C. granati

6F-45/CBS 144237

Punica granatum

Tulare Co., California, USA

MG971799

MG971949

MG971514

MG971664

C. joaquinensis

9E-95

Juglans regia

Tulare Co., California, USA

MG971896

MG972045

MG971606

MG971762

C. joaquinensis

9E-44

Pistacia vera

Fresno Co., California, USA

MG971897

MG972046

MG971607

MG971763

C. joaquinensis

KARE195

Pistacia vera

Kern Co., California, USA

MG971894

MG972043

MG971604

MG971760

C. joaquinensis

KARE231

Pistacia vera

Kern Co., California, USA

MG971893

MG972042

MG971603

MG971759

C. joaquinensis

KARE975/CBS 144235

Populus deltoides

San Joaquin Co., California, USA

MG971895

MG972044

MG971605

MG971761

C. longispora

10F-57/CBS 144236

Prunus domestica

Glenn Co., California, USA

MG971905

MG972054

MG971615

MG971764

C. oleicola

KARE1021/CBS 144248

Olea europaea

San Joaquin Co., California, USA

MG971944

MG972098

MG971660

MG971752

C. parakantschavelii

KARE974/CBS 144243

Populus deltoides

San Joaquin Co., California, USA

MG971898

MG972047

MG971608

MG971765

C. parakantschavelii

KARE966

Populus fremontii

Yolo Co., California, USA

MG971903

MG972052

MG971613

MG971770

C. parakantschavelii

KARE967

Populus fremontii

Yolo Co., California, USA

MG971901

MG972050

MG971611

MG971768

C. parakantschavelii

KARE968

Populus fremontii

Yolo Co., California, USA

MG971900

MG972049

MG971610

MG971767

C. parakantschavelii

KARE969

Populus fremontii

Yolo Co., California, USA

MG971904

MG972053

MG971614

MG971771

C. parakantschavelii

KARE970

Populus fremontii

Yolo Co., California, USA

MG971902

MG972051

MG971612

MG971769

C. parakantschavelii

KARE971

Populus fremontii

Yolo Co., California, USA

MG971899

MG972048

MG971609

MG971766

C. parapistaciae

KARE232

Pistacia vera

Kern Co., California, USA

MG971807

MG971957

MG971522

MG971672

C. parapistaciae

KARE268

Pistacia vera

Kern Co., California, USA

MG971806

MG971956

MG971521

MG971671

C. parapistaciae

KARE269

Pistacia vera

Kern Co., California, USA

MG971805

MG971955

MG971520

MG971670

C. parapistaciae

KARE270/CBS 144506

Pistacia vera

Kern Co., California, USA

MG971804

MG971954

MG971519

MG971669

C. pistaciae

KARE441

Pistacia vera

Merced Co., California, USA

MG971800

MG971950

MG971515

MG971665

C. pistaciae

KARE442

Pistacia vera

Merced Co., California, USA

MG971803

MG971953

MG971518

MG971668

C. pistaciae

KARE443/CBS 144238

Pistacia vera

Merced Co., California, USA

MG971802

MG971952

MG971517

MG971667

C. pistaciae

KARE444

Pistacia vera

Merced Co., California, USA

MG971801

MG971951

MG971516

MG971666

C. plurivora

8C-55

Juglans regia

Butte Co., California, USA

MG971871

MG972020

MG971582

MG971736

C. plurivora

9F-01

Juglans regia

Glenn Co., California, USA

MG971873

MG972022

MG971584

MG971738

C. plurivora

9F-03

Juglans regia

Glenn Co., California, USA

MG971865

MG972014

MG971576

MG971730

C. plurivora

11I-89

Juglans regia

Sutter Co., California, USA

MG971855

MG972004

MG971566

MG971720

C. plurivora

9F-08

Juglans regia

Tehama Co., California, USA

MG971884

MG972033

MG971594

MG971749

C. plurivora

KARE1452/CBS 144239

Olea europaea

San Joaquin Co., California, USA

MG971861

MG972010

MG971572

MG971726

C. plurivora

9E-42

Pistacia vera

Colusa Co., California, USA

MG971870

MG972019

MG971581

MG971735

C. plurivora

9E-86

Prunus domestica

Sutter Co., California, USA

MG971869

MG972018

MG971580

MG971734

C. plurivora

11I-87

Prunus domestica

Sutter Co., California, USA

MG971872

MG972021

MG971583

MG971737

C. plurivora

8J-57

Prunus domestica

Tehama Co., California, USA

MG971854

MG972003

MG971565

MG971719

C. plurivora

11I-19

Prunus domestica

Tulare Co., California, USA

MG971866

MG972015

MG971577

MG971731

C. plurivora

11I-20

Prunus domestica

Tulare Co., California, USA

MG971868

MG972017

MG971579

MG971733

C. plurivora

11I-21

Prunus domestica

Tulare Co., California, USA

MG971867

MG972016

MG971578

MG971732

C. plurivora

KARE486

Prunus domestica

Tulare Co., California, USA

MG971879

MG972028

MG971655

MG971744

C. plurivora

KARE487

Prunus domestica

Tulare Co., California, USA

MG971878

MG972027

MG971589

MG971743

C. plurivora

9D-71

Prunus domestica

Yuba Co., California, USA

MG971858

MG972007

MG971569

MG971723

C. plurivora

9D-72

Prunus domestica

Yuba Co., California, USA

MG971857

MG972006

MG971568

MG971722

C. plurivora

KARE1518

Prunus dulcis

Kern Co., California, USA

MG971875

MG972024

MG971586

MG971740

C. plurivora

KARE1519

Prunus dulcis

Kern Co., California, USA

MG971876

MG972025

MG971587

MG971741

C. plurivora

KARE50

Prunus dulcis

Fresno Co., California, USA

MG971877

MG972026

MG971588

MG971742

C. plurivora

KARE1449

Prunus dulcis

Kern Co., California, USA

MG971859

MG972008

MG971570

MG971724

C. plurivora

KARE1450

Prunus dulcis

Kern Co., California, USA

MG971860

MG972009

MG971571

MG971725

C. plurivora

KARE91

Prunus dulcis

Stanislaus Co., California, USA

MG971862

MG972011

MG971573

MG971727

C. plurivora

6F-18

Prunus persica

Contra Costa Co., California, USA

MG971874

MG972023

MG971585

MG971739

C. plurivora

KARE79

Prunus persica

Fresno Co., California, USA

MG971882

MG972031

MG971592

MG971747

C. plurivora

KARE80

Prunus persica

Fresno Co., California, USA

MG971883

MG972032

MG971593

MG971748

C. plurivora

KARE81

Prunus persica

Fresno Co., California, USA

MG971881

MG972030

MG971591

MG971746

C. plurivora

KARE82

Prunus persica

Fresno Co., California, USA

MG971880

MG972029

MG971590

MG971745

C. plurivora

5L-29

Prunus domestica

Fresno Co., California, USA

MG971856

MG972005

MG971567

MG971721

C. plurivora

KARE1536

Prunus domestica

Glenn Co., California, USA

MG971886

MG972035

MG971596

MG971751

C. plurivora

KARE1537

Prunus domestica

Glenn Co., California, USA

MG971864

MG972013

MG971575

MG971729

C. plurivora

KARE1538

Prunus domestica

Glenn Co., California, USA

MG971863

MG972012

MG971574

MG971728

C. plurivora

KARE1539

Prunus domestica

Glenn Co., California, USA

MG971885

MG972034

MG971595

MG971750

C. populicola

KARE973/CBS 144240

Populus deltoides

San Joaquin Co., California, USA

MG971891

MG972040

MG971601

MG971757

C. punicae

7C-09

Punica granatum

Fresno Co., California, USA

MG971939

MG972087

MG971650

MG971794

C. punicae

7C-10

Punica granatum

Fresno Co., California, USA

MG971937

MG972085

MG971648

MG971792

C. punicae

7C-11

Punica granatum

Fresno Co., California, USA

MG971942

MG972090

MG971653

MG971797

C. punicae

5A-80/CBS 144244

Punica granatum

Madera Co., California, USA

MG971943

MG972091

MG971654

MG971798

C. punicae

5A-81

Punica granatum

Madera Co., California, USA

MG971938

MG972086

MG971649

MG971793

C. punicae

5A-82

Punica granatum

Madera Co., California, USA

MG971941

MG972089

MG971652

MG971796

C. punicae

7C-33

Punica granatum

Stanislaus Co., California, USA

MG971940

MG972088

MG971651

MG971795

C. sorbicola

KARE1451

Olea europaea

Kings Co., California, USA

MG971850

MG971999

MG971563

MG971715

C. sorbicola

5D-48

Prunus armeniaca

Fresno Co., California, USA

MG971817

MG971967

MG971532

MG971682

C. sorbicola

KARE626

Prunus avium

Contra Costa Co., California, USA

MG971829

MG971979

MG971544

MG971694

C. sorbicola

KARE876

Prunus avium

Contra Costa Co., California, USA

MG971826

MG971976

MG971541

MG971691

C. sorbicola

KARE158

Prunus avium

Fresno Co., California, USA

MG971847

MG971996

MG971560

MG971712

C. sorbicola

KARE162

Prunus avium

Fresno Co., California, USA

MG971846

MG971995

MG971559

MG971711

C. sorbicola

3G-09

Prunus avium

Kern Co., California, USA

MG971838

MG971988

MG971656

MG971703

C. sorbicola

KARE1241

Prunus avium

Kings Co., California, USA

MG971851

MG972000

MG971564

MG971716

C. sorbicola

KARE612

Prunus avium

Sacramento Co., California, USA

MG971822

MG971972

MG971537

MG971687

C. sorbicola

KARE623

Prunus avium

Sacramento Co., California, USA

MG971809

MG971959

MG971524

MG971674

C. sorbicola

KARE882

Prunus avium

Sacramento Co., California, USA

MG971836

MG971986

MG971551

MG971701

C. sorbicola

5D-42

Prunus avium

San Benito Co., California, USA

MG971841

MG971991

MG971555

MG971706

C. sorbicola

5D-44

Prunus avium

San Benito Co., California, USA

MG971840

MG971990

MG971554

MG971705

C. sorbicola

KARE615

Prunus avium

San Joaquin Co., California, USA

MG971819

MG971969

MG971534

MG971684

C. sorbicola

KARE617

Prunus avium

San Joaquin Co., California, USA

MG971815

MG971965

MG971530

MG971680

C. sorbicola

KARE618

Prunus avium

San Joaquin Co., California, USA

MG971814

MG971964

MG971529

MG971679

C. sorbicola

KARE619

Prunus avium

San Joaquin Co., California, USA

MG971813

MG971963

MG971528

MG971678

C. sorbicola

KARE621

Prunus avium

San Joaquin Co., California, USA

MG971811

MG971961

MG971526

MG971676

C. sorbicola

KARE622

Prunus avium

San Joaquin Co., California, USA

MG971810

MG971960

MG971525

MG971675

C. sorbicola

KARE624

Prunus avium

San Joaquin Co., California, USA

MG971808

MG971958

MG971523

MG971673

C. sorbicola

KARE625

Prunus avium

San Joaquin Co., California, USA

MG971830

MG971980

MG971545

MG971695

C. sorbicola

KARE877

Prunus avium

San Joaquin Co., California, USA

MG971825

MG971975

MG971540

MG971690

C. sorbicola

KARE879

Prunus avium

San Joaquin Co., California, USA

MG971823

MG971973

MG971538

MG971688

C. sorbicola

KARE881

Prunus avium

San Joaquin Co., California, USA

MG971837

MG971987

MG971552

MG971702

C. sorbicola

KARE589

Prunus avium

Yolo Co., California, USA

MG971848

MG971997

MG971561

MG971713

C. sorbicola

KARE590

Prunus avium

Yolo Co., California, USA

MG971845

MG971994

MG971558

MG971710

C. sorbicola

KARE613

Prunus avium

Yolo Co., California, USA

MG971821

MG971971

MG971536

MG971686

C. sorbicola

KARE614

Prunus avium

Yolo Co., California, USA

MG971820

MG971970

MG971535

MG971685

C. sorbicola

KARE616

Prunus avium

Yolo Co., California, USA

MG971816

MG971966

MG971531

MG971681

C. sorbicola

KARE620

Prunus avium

Yolo Co., California, USA

MG971812

MG971962

MG971527

MG971677

C. sorbicola

KARE874

Prunus avium

Yolo Co., California, USA

MG971828

MG971978

MG971543

MG971693

C. sorbicola

KARE875

Prunus avium

Yolo Co., California, USA

MG971827

MG971977

MG971542

MG971692

C. sorbicola

KARE878

Prunus avium

Yolo Co., California, USA

MG971824

MG971974

MG971539

MG971689

C. sorbicola

4L-58

Prunus domestica

Yuba Co., California, USA

MG971839

MG971989

MG971553

MG971704

C. sorbicola

KARE59

Prunus dulcis

Fresno Co., California, USA

MG971849

MG971998

MG971562

MG971714

C. sorbicola

KARE78

Prunus dulcis

Fresno Co., California, USA

MG971844

MG971993

MG971557

MG971709

C. sorbicola

KARE226

Prunus dulcis

Stanislaus Co. California, USA

MG971835

MG971985

MG971550

MG971700

C. sorbicola

KARE227

Prunus dulcis

Stanislaus Co. California, USA

MG971834

MG971984

MG971549

MG971699

C. sorbicola

KARE228/CBS 144245

Prunus dulcis

Stanislaus Co. California, USA

MG971833

MG971983

MG971548

MG971698

C. sorbicola

KARE249

Prunus dulcis

Stanislaus Co. California, USA

MG971832

MG971982

MG971547

MG971697

C. sorbicola

KARE251

Prunus dulcis

Stanislaus Co. California, USA

MG971831

MG971981

MG971546

MG971696

C. sorbicola

KARE92

Prunus dulcis

Stanislaus Co. California, USA

MG971843

MG972092

MG971657

MG971708

C. sorbicola

KARE83

Prunus persica

Fresno Co., California, USA

MG971842

MG971992

MG971556

MG971707

C. sorbicola

9C-89

Prunus persica

Merced Co., California, USA

MG971818

MG971968

MG971533

MG971683

aIsolates in bold represent type specimens.

DNA extraction, sequencing, and phylogenetic analyses

Total genomic DNA was isolated from mycelium scraped with a sterile scalpel from the surface of 14-day-old cultures using the DNeasy Plant kit (Qiagen, Valencia, CA), following the manufacturer’s instructions. All PCR reactions utilized AccuPower™ PCR Premix (Bioneer, Alameda, CA), following the manufacturer’s instructions. Amplification of rDNA, including the intervening ITS regions and 5.8S rDNA (ITS1–5.8S-ITS2), using the primer set ITS5 and ITS4 followed the protocol of White et al. (1990). Amplification of translation elongation factor 1-α (TEF1) fragments utilized the primer set EF1-688F and EF1-1251R (Alves et al. 2008), beta-tubulin (TUB2) utilized primers Bt1a and Bt1b (Glass & Donaldson 1995), and actin (ACT1) utilized primers ACT-512F and ACT-783R (Carbone & Kohn 1999), with a slightly modified PCR program for TUB2 and ACT1 [initial denaturation (94 °C, 5 min) followed by 35 cycles of denaturation (94 °C, 30 s), annealing (58 °C for TUB2 and 63 °C for ACT1, 30 s), extension (72 °C, 60 s), and a final extension (72 °C, 10 min)]. PCR amplification of the TUB2 locus for some Californian Cytospora isolates (described below) was attempted at different annealing temperatures (50–60 °C). PCR products were visualized on a 1.5 % agarose gel (120 V for 25 min) stained with GelRed® (Biotium, Fremont, CA), following the manufacturer’s instructions, to confirm presence and size of amplicons, purified via Exonuclease I and recombinant Shrimp Alkaline Phosphatase (Affymetrix, Santa Clara, CA), and sequenced bidirectionally via BigDye® Terminator v. 3.1 Cycle Sequencing Kit (Thermo Fischer Scientific, Waltham, MA) on an ABI 3730 Capillary Electrophoresis Genetic Analyzer (College of Biological Sciences Sequencing Facility, University of California, Davis).

Forward and reverse nucleotide sequences were assembled, proofread, and edited in Sequencher v. 5 (Gene Codes Corporation, Ann Arbor, MI) and deposited in GenBank (Table 1). Homologous sequences with high similarity from ex-type and non-type Cytospora isolates were included for phylogenetic reference utilizing the BLASTn function in NCBI and extensive literature review (Table 2). Multiple sequence alignments were performed in MEGA v. 6 (Tamura et al. 2013) and manually adjusted where necessary in Mesquite v. 3.10 (Maddison & Maddison 2016). Alignments were submitted to TreeBASE under accession number S22195. Phylogenetic analyses were performed for each individual locus and for a four-gene concatenated dataset. Each dataset was analyzed using two different optimality search criteria, maximum parsimony (MP) and maximum likelihood (ML), in MEGA v. 6 (Tamura et al. 2013). For MP analyses, heuristic searches with 1000 random sequence additions were implemented with the Tree-Bisection-Reconnection algorithm, gaps were treated as missing data. Bootstrap analyses with 1000 pseudoreplicates were used to estimate branch support. For ML analyses, MEGA was used to infer a model of nucleotide substitution for each dataset, using the Akaike Information Criterion (AIC). All ML analyses utilized the Nearest-Neighbor-Interchange heuristic method and branch support was determined by 1000 bootstrap pseudoreplicates. Sequences of Diaporthe ampelina isolate Wolf912 and D. benedicti isolate SBen914 (Diaporthales, Diaporthaceae) (Lawrence et al. 2015) served as the outgroup taxa in all analyses.
Table 2

Fungal isolates used in this study and GenBank accession numbers.

     

GenBank Accession

 

Species

Isolate a

Host

Geographic origin

ITS

ACT1 b

TEF1

TUB2 b

Cytospora abyssinica

CMW 10181

Eucalyptus globulus

Wondo Genet, Ethiopia

AY347353

C. ampulliformis

MFLUCC 16-0629

Acer platanoides

Russia

KY417727

KY417693

C. atrocirrhata

CFCC 89615

Jugulans regia

Xining, Qinghai, China

KF225610

C. austromontana

CMW 6735

Eucalyptus pauciflora

NSW, Australia

AY347361

C. berberidis

CFCC 89927

Berberis dasystachyum

Qinghai Province, China

KP340985

C. berkeleyi

StanfordT3

Eucalyptus globulus

Palo Alto, California, USA

AY347350

C. brevispora

CBS 116811

Eucalyptus grandis ×-tereticornis

Tchittanga, Republic of Congo

AF192315

C. carbonacea

CFCC 50055

Ulmus pumila

Qiqihar, Heilongjiang, China

KP281262

KP310851

C. cedri

CBS 196.50

Unknown host

Italy

AF192311

JX438575

C. centrivillosa

MFLUCC 16-1206

Sorbus domestica

Italy

MF190122

C. chrysosperma

CFCC 89619

Juglans regia

Yinchuan, Ningxia, China

KF225614

KF498677

C. cincta

LP47

Prunus armeniaca

Michigan, USA

AF191169

C. cinereostroma

CMW 5700

Eucalyptus globulus

Chile

AY347377

C. cotini

MFLUCC 14-1050

Cotinus coggygria

Russia

KX430142

C. curvata

MFLUCC 15-0865

Salix alba

Russia

KY417728

KY417694

C. davidiana

CXY1350

Populus davidiana

China

KM034870

C. diatrypelloidea

CMW 8549

Eucalyptus globulus

Orbost, Victoria, Australia

AY347368

C. disciformis

CMW 6509

Eucalyptus grandis

Uruguay

AY347374

C. donetzica

MFLUCC 16-0574

Rosa sp.

Russia

KY417731

KY417697

C. elaeagni

CFCC 89632

Elaeagnus angustifolia

Guyuan, Ningxia, China

KF765676

C. eriobotryae

IMI 136523

Eriobotrya japonica

Saharanpur, India

AY347327

C. erumpens

MFLUCC 16-0580

Salix xfragilis

Russia

KY417733

KY417699

C. eucalypticola

ATCC 96150

Eucalyptus nitens

Tasmainia, Australia

AY347358

C. eucalyptina

CMW5882

Eucalyptus grandis

Cali, Colombia

AY347375

C. eugeniae

CBS 118569

Eugenia sp.

Tanzania

AY347344

C. fraxinigena

MFLUCC 14-0868

Fraxinus ornus

Italy

MF190133

C. fugax

CBS 203.42

Salix sp.

Switzerland

AY347323

C. gigalocus

HMBF155

Juglans regia

Xining, Qinghai, China

KF225609

C. gigaspora

CFCC 89634

Salix psammophila

China

KF765671

KU711000

C. hippophaes

CFCC 89639

Hippophae rhamnoides

Gannan, Gansu, China

KF765681

C. junipericola

MFLUCC 17-0882

Juniperus communis

Italy

MF190125

C. kantschavelii

287-2

Populus deltoides

Iran

EF447367

C. leucosperma

CBS 191.42

Taxus baccata

Switzerland

AY347330

C. longiostiolata

MFLUCC 16-0628

Salix ×fragilis

Russia

KY417734

C. melnikii

MFLUCC 15-0851

Malus domestica

Russia

KY417735

KY417701

C. multicollis

CBS 105.89

Quercus ilex subsp. rotundifolia

Spain

DQ243803

C. myrtagena

HiloTibl

Tibouchina urvilleana

Hilo, Hawaii, USA

AY347363

C. nitschkii

CMW 10180

Eucalyptus globulus

Wondo Genet, Ethiopia

AY347356

C. nivea

CFCC 89642

Salix psammophila

Yulin, Shaanxi, China

KF765684

KU711006

C. notastroma

Cottonwood 16

Populus tremuloides

Colorado, USA

JX438631

C. palmoides

CXY1276

Cotinus coggygria

Beijing, Xiangshan, China

JN402990

C. parakantschavelii

MFLUCC 15-0857

Populus xsibirica

Russia

KY417738

KY417704

C. parasitica

MFLUCC 15-0507

Malus domestica

Russia

KY417740

KY417706

C. paratranslucens

MFLUCC 15-0506

Populus alba var. bolleana

Russia

KY417741

KY417707

C. personate

CBS 117.67

Rhododendron ponticum

Netherlands

AY347331

C. pinastri

CBS 194.42

Abies alba

Switzerland

AY347328

C. pini

CBS 197.42

Pinus sylvestris

Switzerland

AY347332

C. populina

CFCC 89644

Salix psammophila

Yulin, Shaanxi, China

KF765686

C. pruinopsis

CFCC 50034

Ulmus pumila

Harbin, Heilongjiang, China

KP281259

C. pruinosa

CBS 118555

Olea europaea v. africana

South Africa

DQ243790

C. punicae

CBS 199.50

Punica granatum

Turkey

JX438622

JX438568

C. quercicola

MFLUCC 14-0867

Quercus sp.

Italy

MF190129

C. ribis

CFCC 50026

Ulmus pumila

Yulin, Shaanxi, China

KP281267

C. rosae

MFLUCC 14-0845

Rosa canina

Italy

MF190131

C. rosarum

218

Rosa canina

Iran

EF447387

   

C. rostrata

Ls251

Salix cupularis

Gansu, China

KC313890

JX438568

C. rusanovii

MFLUCC 15-0854

Salix babylonica

Russia

KY417744

KY417710

C. sacculus

CFCC 89624

Juglans regia

Gannan, Gansu, China

KF225615

KP310860

C. salicacearum

MFLUCC 15-0509

Salix alba

Russia

KY417745

KY417711

C. salicicola

MFLUCC 15-0866

Salix alba

Russia

KU982635

KU982637

C. salicina

MFLUCC 15-0862

Salix alba

Russia

KY417750

KY417716

C. schulzeri

CBS 118570

Malus domestica

Michigan, USA

DQ243802

C. sequioae

CBS 116815

Sequoia sempervirens

California, USA

AY347340

C. sibiraeae

CFCC 50045

Sibiraea angustata

Gannan, Gansu, China

KP340987

C. sophorae

CFCC 89598

Sophora japonica

China

KR045654

KU711018

KU710941

KR045695

C. sophoricola

CFCC 89595

Sophora japonica var. pendula

Gannan, Gansu, China

KC880148

C. sorbi

MFLUCC 16-0631

Sorbus aucuparia

Russia

KY417752

KY417718

C. sorbicola

MFLUCC 16-0584

Acer pseudoplatanus

Russia

KY417755

KY417721

C. spiraeae

CFCC 50049

Spiraea salicirolia

Gansu, China

MG707859

MG708196

  

C. tanaitica

MFLUCC 14-1057

Betula pubescens

Russia

KT459411

KT459413

C. translucens

CBS 152.42

Salix sp.

St. Moritz, Switzerland

AF191182

C. ulmi

MFLUCC 15-0863

Ulmus minor

Russia

KY417759

KY417725

C. valsoidea

CMW 4309

Eucalyptus grandis

Sumatra, Indonesia

AF192312

C. variostromatica

CMW 6766

Eucalyptus globulus

Orbost, Victoria, Australia

AY347366

C. vinacea

CBS 141585

Vitis interspecific hybrid ‘Vidat

New Hampshire, USA

KX256256

KX256277

C. viticola

CBS 141586

Vitis vinifera ‘Cabernet Franc’

Connecticut, USA

KX256239

KX256260

Diaporthe ampelina

Wolf912

Vitis vinifera ‘Thompson seedless’

Solano Co., California, USA

KM669964

JGI

KM669820

JGI

Diaporthe benedicti

SBen914

Salix sp.

San Benito Co., California, USA

KM669929

KM669785

Leucostoma parapersoonii

CBS 116845

Pyrus serotina

Michigan, USA

AF191181

   

Valsa sordida

CBS 197.50

Populus tremula

United Kingdom

AY347322

aIsolates in bold represent type specimens.

bJGI Represents sequences that were retreived from the JGI Mycocosm genome portal.

Morphology

Mycelial plugs (5 mm diam) were taken from the margin of selected, actively growing cultures based on preliminary phylogenetic results and transferred to triplicate 90 mm diam Petri dishes containing 2 % PDA and incubated in the dark at 25 °C for 14 d. Radial growth was measured after 7 d by taking two measurements perpendicular to each other. Assessments of colony colour (Rayner 1970) and morphology were made after 14 d. Pycnidia were induced on corticated cherry wood embedded in PDA medium. Cherry cuttings (approx. 1 cm diam) were collected and cut into 5 cm sections. Sections were placed in glass Petri dishes and autoclaved twice, 24 h apart, at 122 °C for 25 min. Autoclaved wood sections were placed in 90 mm diam plastic Petri dishes, two sections per dish, and PDA was poured to embed them. A mycelial plug from an actively growing culture was placed between the two wood sections in each dish, one isolate per dish. Petri dishes were incubated at room temperature under natural photoperiod in August 2017, and pycnidial formation was monitored weekly for four weeks. Morphological characterization of the pycnidia (n = 20) included the diameter, presence/absence of a conceptacle, and colour using a binocular Leica MZ95 dissecting microscope (Leica microsystems CMS, Wetzlar, Germany). Pycnidial locular arrangements were assessed by transversely sectioning pycnidia by hand with a razor blade and observing as above. Conidial dimensions (n = 30) and conidiogenous cells (n = 20) were measured at ×1000 from approximately 28-day-old cultures by producing a pycnidial squash mount that was crushed in a sterile 50 % glycerol solution (no stain was applied, thus the natural pigments of each species was preserved) and observed with a Leica DM500B microscope (Leica microsystems CMS, Wetzlar, Germany). Morphological measurements are represented by the mean as a range depicting the standard deviation in the centre with minima and maxima in parentheses, respectively, in the species descriptions and taxonomy section below.

Results

Disease symptoms, hosts, and distribution

In total, 92 samples were obtained from symptomatic trees in 70 orchards of various fruit and nut crops including almond (Prunus dulcis), apricot (Prunus armeniaca), cherry (Prunus avium), olive (Olea europaea), peach (Prunus persica), pistachio (Pistacia vera), pomegranate (Punica granatum), prune (Prunus domestica), walnut (Juglans regia), and woody ornamentals such cottonwoods (Populus deltoides and P. fremontii), camellia (Camellia sp.) and sequoia (Sequoiadendron giganteum). Cankers and accompanying branch and twig dieback were the most common symptoms associated with Cytospora species. Trees expressing Cytospora cankers were observed in Butte, Colusa, Contra Costa, Fresno, Glenn, Kern, Kings, Lake, Madera, Merced, Sacramento, San Benito, San Joaquin, Stanislaus, Sutter, Tehama, Tulare, Yolo, and Yuba counties in California. Dieback symptoms were most obvious during the warm summer months, although putative infections might have occurred during the rainy winter and early spring seasons in California. Symptoms of Cytospora canker includes bark lesions with dead phloem and cambium, discoloration of the xylem, wood necrosis and gumming occurring at the canker margin. Cankers were often depressed or sunken, eventually causing splitting of the bark or girdling of branches. Cankers were most commonly associated with pruning wounds, sunburn, and oil injuries. A single French prune orchard in Yuba County, where the grower re-planted trees to fill the gaps from trees killed by Cytospora canker, showed 92 % Cytospora infection of pruning cuts made to select the main scaffolds of the newly planted trees. Wood cankers expressed as wedge shaped to irregularly shaped vascular discolorations of the xylem tissue below the affected bark area. Eventually, pycnidia occurred just beneath the periderm giving the bark a pimpled appearance diagnostic of Cytospora infection. Removing the periderm generally exposed numerous, solitary and scattered pycnidia. Erumpent pycnidia eventually ruptured the bark outermost layers exposing white (characteristic in branches of French prune) apical discs above the cankered area or in the dead branches and twigs. Spore tendrils consisting of conidial masses (cirrus) exuding from pycnidia generally were visible in the orchards following spring rains. Signs and symptoms of Cytospora associated cankers in various fruit and nut host plants are illustrated in Figs 13.

Phylogenetic analyses

For ML analyses, the best-fit model of nucleotide evolution was deduced based on the AIC (K2+G+I for both ACT1 and TEF1, HKY+G for TUB2, and ITS and combined analyses both utilized GTR+G+I). PCR amplification of the ITS region generated 497–527 bp fragments and the alignment of 229 ITS sequences resulted in a 604-character dataset (350 characters were constant, 74 characters were parsimony-uninformative, and 180 characters were parsimony informative (30 %)). MP analyses produced a single most parsimonious tree of 973 steps and a consistency index (CI), retention index (RI), and rescaled consistency index (RC) of 0.4193, 0.8813, and 0.3692, respectively. PCR amplification of the TEF1 locus generated 588–664 bp fragments and the alignment of 161 TEF1 sequences resulted in a 799-character dataset (313 characters were constant, 124 characters were parsimony-uninformative, and 362 characters were parsimony informative (45 %)). MP analyses produced four equally most parsimonious trees of 1411 steps and a CI, RI, and RC of 0.5506, 0.9470, and 0.5227, respectively. PCR amplification of the TUB2 locus was problematic for 14 isolates, which reside in sister clades as described below, nevertheless PCR amplification of the TUB2 locus generated 527–554 bp fragments and the alignment of 136 TUB2 sequences resulted in a 575-character dataset (428 characters were constant, 28 characters were parsimony-uninformative, and 119 characters were parsimony informative (21 %)). MP analyses produced four equally most parsimonious trees of 350 steps and a CI, RI, and RC of 0.6171, 0.9485, and 0.5834, respectively. PCR amplification of the ACT1 locus generated 280–298 bp fragments and the alignment of 184 ACT1 sequences resulted in a 365-character dataset (149 characters were constant, 74 characters were parsimony-uninformative, and 142 characters were parsimony informative (39 %)). MP analyses produced a single most parsimonious tree of 585 steps and a CI, RI, and RC of 0.4825, 0.9308, and 0.4836, respectively. The analysis of individual datasets yielded similar trees that only differed in the order of species divergences and varying levels of clade support (ITS, Fig. S1; TEF1, Fig. S2; TUB2, Fig. S3; and ACT1, Fig. S4).

The multi-locus dataset consisted of 2334 characters (1242 characters were constant, 293 characters were parsimony-uninformative, and 799 characters were parsimony informative (34 %)). MP analysis produced a single most parsimonious tree of 3434 steps and a CI, RI, and RC of 0.4947, 0.9253, and 0.4589, respectively. MP and ML analyses revealed that 150 Californian Cytospora isolates were divided into 15 species, five of which have been described previously (C. chrysosperma, C. parakantschavelli, C. punicae, C. sorbicola, and Valsa eucalypti) and 10 of which are not associated with a type or non-type isolate with DNA sequence data and thus represent novel phylogenetic species (Fig. 4). Descriptions of all species and taxonomic proposals are provided in the species descriptions and taxonomy section below.
Fig. 4
Fig. 4

The single most parsimonious tree generated from maximum parsimony analysis of the four-gene (ITS, TEF1, TUB2, and ACT1) combined dataset. Numbers in front and after the slash represent parsimony and likelihood bootstrap values from 1000 replicates, respectively. Values represented by an asterisk were less than 70 % for the bootstrap analyses. Ex-type isolates are indicated in bold. Bar indicates the number of nucleotide changes.

Taxonomy

Morphological comparisons coupled with multi-locus phylogenetic analyses (MP and ML) of the combined four-gene dataset identified 10 distinct and strongly supported lineages for which no apparent species names exist. Thus, we propose the following new species names and a new combination to properly circumscribe these unique taxa. Additionally, two previously described species are described from North America for the first time.

Cytospora amygdali D.P. Lawr., L.A. Holland & Trouillas, sp. nov.

MycoBank MB824274

(Figs 45)
Fig. 5
Fig. 5

Cytospora amygdali (ex-holotype culture CBS 144233). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Conidiophores and filamentous conidiogenous cells. D. Conidia. E. Pycnidia. Bars C–D = 10 µm; E = 500 µm.

Etymology: The name refers to the host, almond.

Diagnosis: Cytospora amygdali can be distinguished from the phylogenetically closely related C. plurivora by the production of large robust conidia and solitary pycnidia in culture.

Type: USA: California: Yolo County, isolated from wood canker of Prunus dulcis, 3 Mar. 2016, L.A. Holland LH357 (BPI 910650 [dried culture] — holotype; CBS 144233 — ex-holotype culture).

Description: Conidiomata on PDA pycnidial, solitary, globose to subglobose, without conceptacle, mouse-grey in centre with white to off-white surface hyphae, (455−)570–690(−850) µm diam (n = 20), with 1–2 internal locules. Conidiophores hyaline, smooth-walled, reduced to single monoblastic straight filamentous conidiogenous cells (5.5−)5.9–7.1(−7.5) × (1.0−)0.9–1.1(−1.0) µm (n = 20), that are wider at the base and taper towards apex. Conidia abundant, relatively large with wide girth, single, hyaline, eguttulate, aseptate, allantoid, (6.0−)6.2–7.0(−7.0) × (1.5−)1.6–1.8(−2.0) µm (n = 30). No sexual morph observed.

Culture characteristics: Colonies after 7 d at 25 °C on PDA average 57 mm, medium-growing, slightly dentate, off-white outer margin, and cinnamon-colored inner margin with centre of the colony becoming dark mouse-grey with age. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: Yolo County (California, USA).

Host: Prunus dulcis.

Notes: Based on the phylogenetic inference obtained in this study, C. plurivora is the closest relative to C. amygdali, albeit without significant bootstrap support. Cytospora amygdali produces larger conidia, (6.0−)6.2–7.0(−7.0) × (1.5−)1.6–1.8(−2.0) µm, in terms of both length and width and pycnidia are always solitary, contrary to smaller conidia, (3.5−)3.8–4.4(−4.5) × (1.0−)0.9–1.1(−1.5) µm, and aggregated pycnidia produced by C. plurivora.

Cytospora californica D.P. Lawr., L.A. Holland & Trouillas, sp. nov.

MycoBank MB824275

(Figs 4 and 6)
Fig. 6
Fig. 6

Cytospora californica (ex-holotype culture CBS 144234). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 1 mm; D = 10 µm; E = 5 µm.

Etymology: The name refers to the geographical region, California, from where this fungus was first isolated.

Diagnosis: Cytospora californica can be distinguished from the species C. eucalypti by the former producing, on average, shorter conidia (C. californica (4.0−)4.5–5.5(−6.0) × (1.0−)1.2–1.6(−1.5) µm vs. C. eucalypti (5.0−)5.4–6.5(− 7.5) × (1.0−)1.2–1.6(−2.0) µm) and slower growth rate (C. californica 58.8 mm in 7 d vs. C. eucalypti 85 mm in 7 d) and pattern in culture (C. californica produces two distinct margins in culture, with the internal margin darker than the peripheral margin, while C. eucalypti generally produces a homogenous pattern in culture).

Type: USA: California: Lake County, isolated from wood canker of Juglans regia, 14 Mar. 2014, T.J. Michailides 9C-24 (BPI 910651 [dried culture] — holotype; CBS 144234 — ex-holotype culture).

Description: Conidiomata on PDA pycnidial, mostly solitary, rarely aggregated, globose to subglobose, without conceptacle, white, (1255−)1356–1834(−2100) µm diam (n = 20), with multiple internal locules with shared invaginated walls. Conidiophores hyaline, smooth-walled, reduced to 3–4 monoblastic branching filamentous conidiogenous cells (5.0−)5.9–7.9(−9.0) × (1.0−)1.1–1.5(−1.5) µm (n = 20) that taper towards the apex. Conidia abundant, single, hyaline to brown, eguttulate, aseptate, allantoid, (4.0−)4.5–5.5(−6.5) × (1.0−)1.2–1.6(−2.0) µm (n = 30). No sexual morph observed.

Culture characteristics: Colonies after 7 d at 25 °C on PDA average 58.8 mm, medium-growing, margin mostly smooth with some unevenness, with short aerial tufts giving a cottony appearance, margin white to off-white with buff centre. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: Glenn, Fresno, Kern, Lake, San Joaquin, and Stanislaus Counties (California, USA).

Hosts: Juglans regia, Pistacia vera, and Prunus dulcis.

Notes: Based on the phylogenetic inference obtained in this study, C. eucalypti (syn. Valsa eucalypti) is the closest relative to C. californica. Most micro-morphological observations between the two species overlap, however the colony growth rate of C. californica is much slower (58.8 mm in 7 d) than that of C. eucalypti (85 mm in 7 d), and C. californica produces, on average, shorter conidia (4.0−)4.5–5.5(−6.5) than C. eucalypti (5.0−)5.4–6.5(−7.5). Amplification of the TUB2 locus using the primers Bt1a/Bt1b was problematic for this taxon. Several different annealing temperatures were attempted (annealing temperature ranging from 50–60 °C) with marginal success as only 19 out of 30 C. californica isolates produced a reliable TUB2 PCR amplicon.

Cytospora chrysosperma (Pers.) Fr., Syst. Mycol. 2(2): 542 (1823); nom. sanct.

Basionym: Sphaeria chrysosperma Pers., Neues Mag. Bot. 1: 82 (1794).

Synonyms: Naemaspora chrysosperma (Pers.) Pers., Obs. Mycol. 1: 80 (1796). Naemaspora populina Spreng., Fl. Hal.: 354 (1806).

(Figs 4 and 7)
Fig. 7
Fig. 7

Cytospora chrysosperma (CBS 144242). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 1 mm; D = 20 µm; E = 10 µm.

Description: Conidiomata on PDA pycnidial, mostly solitary, sometimes aggregated, globose to subglobose, without conceptacle, grey with off-white surface hyphae, (960−)1119–1681(−2070) µm diam (n = 20), with multiple internal locules with shared invaginated walls. Conidiophores some straight, some reduced to branching filamentous conidiogenous cells that taper towards the apex (7.0−)7.2–8.8(−10.0) × (1.0−)1.1–1.3(−1.5) µm (n = 20). Conidia abundant, single, hyaline to light brown, eguttulate, aseptate, allantoid, small, (3.0−)3.0–3.6(−4.0) × (1.0−)0.9–1.1(−1.0) µm (n = 30). No sexual morph observed.

Culture characteristics: Colony of C. chrysosperma isolate 9E-33, 90 mm diam in 7 d at 25 °C on PDA, fast-growing, off-white to cream with short aerial tufts giving a cottony appearance, aerial hyphae becoming darker with age. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: China, Germany, Iran, The Netherlands, South Africa, Switzerland, the UK, and USA (Fresno County, California).

Hosts: The USDA Fungus-Host Distribution Database (https://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm lists more than 260 host records for C. chrysosperma, therefore a limited list is provided here: Crataegus azarolus, Ficus carica, Juglans regia, Ligustrum latifolium, Malus pumila, Morus alba, Olea sativa, Persica vulgaris, Prunus armeniaca, Prunus domestica, Robinia pseudoacacia, Salicaceae, Sophora japonica, Thuja orientalis, Triticum, Ulmus, and Vitis vinifera.

Notes: Based on the phylogenetic inference obtained in this study, C. chrysosperma is sister to the clade that contains C. joaquinensis, C. longiostiolata, C. melnikii, C. populicola, C. rostrata, C. salicacearum, and C. salicina. Cytospora chrysosperma is the type species of the genus, and CFCC 89600 is an ex-type strain of the species (Fan et al. 2015) and our isolate 9E-33 clusters strongly with that strain.

Specimen examined: USA, California: Fresno County, isolated from shoot of Camellia sp., 21 May 2014, T.J. Michailides 9E-33 (BPI 910652 [dried culture]; CBS 144242).

Cytospora eucalypti (Cooke & Harkn.) D.P. Lawr., L.A. Holland & Trouillas, comb. nov.

MycoBank MB824284

(Figs 4 and 8)
Fig. 8
Fig. 8

Cytospora eucalypti (CBS 144241). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidia. E. Conidiophores and filamentous conidiogenous cells. Bars C = 1 mm; D–E = 10 µm.

Basionym: Valsa eucalypti Cooke & Harkn., Grevillea 9: 51 (1881).

Synonyms: Engizostoma eucalypti (Cooke & Harkn.) Kuntze, Rev. Gen. Plant. 3(2): 474 (1884).

Valsa eucalypti var. myrti Rolland, Bull. Soc. Mycol. France 21: 22 (1905).

Leucostoma sequoiae Bonar, Mycologia 20: 295 (1928).

Type: USA: California: on dead branches of Eucalyptus globulus 1880, Cooke & Harkness (UM 15128, MSC 11471 — isotypes).

Description: Conidiomata on PDA pycnidial, mostly solitary, rarely aggregated, globose, without conceptacle, dark black-grey, appearing dry, (990−)1268–1742(−2060) µm diam (n = 20), with multiple internal locules with shared invaginated walls. Conidiophores short, reduced to branching filamentous conidiogenous cells tapering toward apices (5.5−)8.1–11.1(− 11.5) × (1.0−)1.3–2.1(−2.5) µm (n = 20). Conidia abundant, relatively large, single, hyaline, eguttulate, aseptate, allantoid, (5.0−)5.4–6.5(−7.5) × (1.0−)1.2–1.6(−2.0) µm (n = 50). No sexual morph observed.

Culture characteristics: Colonies after 7 d at 25 °C on PDA average 85 mm, fast-growing, buff to honey with short aerial tufts giving a cottony appearance, aerial hyphae becoming darker with age. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: Fresno, Marin, Merced, San Joaquin, and Santa Clara Counties (California, USA).

Hosts: Eucalyptus globulus, Eucalyptus paniculata, Eucalyptus sp., Prunus dulcis, Sequoia sempervirens, and Sequoiadendron gigateum.

Notes: The species name Cytospora eucalypti has been applied in the past (Sharma et al. 1985), however no type was indicated and this appeared in a research report that was not effectively published, so the name was not validly published (Adams et al. 2005). The Californian isolates cluster strongly with an isolate named as Valsa eucalypti (CBS 116815 from Sequoia sempervirens) which also clusters with isolates collected from Eucalyptus in California (Adams et al. 2005, 2006). Based on the phylogenetic inference obtained in this study, C. eucalypti is sister to C. californica. Most morphological observations between the two species overlap, however, the colony growth rate of C. eucalypti is much faster (85 mm in 7 d) than that of C. californica (58.8 mm in 7 d), and C. eucalypti produces, on average, longer conidia than C. californica (4.0−)4.5–5.5(−6.0). Amplification of the TUB2 locus using the primers Bt1a/Bt1b was problematic. Several different annealing temperatures were attempted with little success as only one out of five isolates produced a reliable TUB2 amplicon. Similar TUB2 PCR failures were encountered with the sister species C. californica, suggesting apomorphic nucleotide substitution(s) in these primer site(s).

Specimen examined: USA: California: Merced County, isolated from wood canker of Prunus dulcis, 28 Sep. 2016, F.P. Trouillas KARE1585 (BPI 910653 [dried culture]; CBS 144241).

Cytospora granati D.P. Lawr., L.A. Holland & Trouillas, sp. nov.

MycoBank MB824278

(Figs 4 and 9)
Fig. 9
Fig. 9

Cytospora granati (ex-holotype culture CBS 144237). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidia. E. Conidiophores and filamentous conidiogenous cells. Bars C = 1 mm; D–E = 10 µm.

Etymology: The name refers to the host, Punica granatum, from which this fungus was first isolated.

Diagnosis: Cytospora granati can be distinguished from C. eucalypticola by the former producing, on average, longer and wider conidia.

Type: USA: California: Tulare County, isolated from wood canker of Punica granatum, 29 Jul. 2011, T.J. Michailides 6F-45 (BPI 910654 [dried culture] — holotype; CBS 144237 — ex-holotype culture).

Description: Conidiomata on PDA pycnidial, mostly solitary, sometimes aggregated, globose, conical to discoid, with yellow-coloured conidial exudate, without conceptacle, off-white to light-grey, (610−)673–897(−975) µm diam (n = 20), with a single internal locule. Conidiophores reduced to straight filamentous conidiogenous cells (16.0−)19.3–23.5(−26.5) × (2.0−)3.7–4.1(−5.0) µm (n = 20). Conidia copious, single, hyaline to light brown, aseptate, allantoid (4.0−)4.1–4.5(−5.0) × (1.0−)1.1–1.3(−1.5) µm (n = 30). No sexual morph observed.

Culture characteristics: Colonies after 7 d at 25 °C on PDA average 87.3 mm, fast-growing, white to buff, raised mixed olivaceous white colony centre with flat colony expansion throughout with buff margin in mature colonies. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: Tulare County (California, USA).

Host: Punica granatum.

Notes: Based on the phylogenetic inference obtained in this study, C. granati resides in a clade that contains Cytospora species isolated from Eucalyptus in Australia (C. austromontana, C. diatrypelloidea, and C. eucalypticola), California (C. berkeleyi), Chile (C. cinereostroma), and Uruguay (C. disciformis), and from Pistacia vera in California (C. parapistaciae and C. pistaciae). This study identified two distantly related Cytospora species recovered from symptomatic pomegranate trees. Cytospora granati is easily distinguished from C. punicae by differences in pycnidial sizes (C. granati pycnidia (610−)673–897(−975) µm are almost twice as large, on average, as compared to C. punicae (210−)237–383(−490) µm)), the much faster colony growth rate (C. granati (87.3 mm in 7 d) than C. punicae (64.7 mm in 7 d)), and colony colour/morphology (C. granati produces a white to buff colony while C. punicae produces a characteristic dark red colony).

Cytospora joaquinensis D.P. Lawr., L.A. Holland & Trouillas, sp. nov.

MycoBank MB824276

(Figs 4 and 10)
Fig. 10
Fig. 10

Cytospora joaquinensis (ex-holotype culture CBS 144235). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidium. D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 1 mm; D = 20 µm; E = 10 µm.

Etymology: The name refers to the San Joaquin Valley of California where the species was found.

Diagnosis: Cytospora joaquinensis can be distinguished from the related C. melnikii, C. salicacearum, and C. salicina as C. joaquinensis produces, on average, longer conidia.

Type: USA: California: San Joaquin County, isolated from wood canker of Populus deltoides, 21 Apr. 2016, F.P. Trouillas KARE975 (BPI 910655 [dried culture] — holotype; CBS 144235 — ex-holotype culture).

Description: Conidiomata on PDA pycnidial, mostly solitary, rarely aggregated, most with yellow conidial exudate, globose, no conceptacle, black-grey with off-white surface hyphae, (970−)1097–1533(−1760) µm diam (n = 20), with multiple internal locules with shared invaginated walls. Conidiophores reduced to mostly straight unbranched filamentous conidiogenous cells (6.5−)7.7–10.1(−13.5) × (1.0−)1.1–1.3(− 1.5) µm (n = 20). Conidia abundant, single, hyaline to light brown, eguttulate, aseptate, allantoid, (5.0−)5.1–5.7(−6.0) × (1.0−)1.1–1.3(−1.5) µm (n = 30). No sexual morph observed.

Culture characteristics: Colonies after 7 d at 25 °C on PDA average 86.7 mm, fast-growing, buff-coloured with short aerial tufts giving a cottony appearance, aerial hyphae becoming darker with age, centre becoming honey-coloured that extends to a white margin. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: Fresno, Kern, San Joaquin, and Tulare Counties (California, USA).

Hosts: Juglans regia, Pistacia vera, and Populus deltoides.

Notes: Based on the phylogenetic inference obtained in this study, C. melnikii, C. salicacearum, and C. salicina are the closest relatives to C. joaquinensis. Conidia of C. joaquinensis (5.0−)5.1–5.7(−6.0) × (1.0−)1.1–1.3(−1.5), on average, are longer than C. melnikii (3.1−)4.5–5 × 1–1.2(−1.3) µm, C. salicacearum (3.6−)4.9–6.4 × 0.9–1(−1.3) µm, and C. salicina (3.6−)4.2–4.7 × 1–1.1(−1.3) µm (Norphanphoun et al. 2017).

Cytospora longispora D.P. Lawr., L.A. Holland & Trouillas, sp. nov.

MycoBank MB824277

(Figs 4 and 11)
Fig. 11
Fig. 11

Cytospora longispora (ex-holotype culture CBS 144236). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidia. E. Conidiophores and filamentous conidiogenous cells. Bars C = 1 mm; D = 10 µm; E = 20 µm.

Etymology: The name refers to the exceptionally long conidia of this species.

Diagnosis: Unique mosaic colony morphology and conidia that are relatively long (6.0−)6.6–7.4(−7.5) × (1.0−)1.1–1.4(− 1.5) µm as compared to most other Cytospora species.

Type: USA: California: Glenn County, isolated from wood canker of Prunus domestica, 22 Oct. 2014, T.J. Michailides 10F-57 (BPI 910656 [dried culture] — holotype; CBS 144236 — ex-holotype culture).

Description: Conidiomata on PDA pycnidial, solitary, sometimes aggregated, many with cream-coloured conidial exudate, globose, no conceptacle, (805−)827–1393(−1635) µm (n = 20), with a single internal locule. Conidiophores smooth-walled, straight, reduced to filamentous conidiogenous cells (6.5−)7.9–10.9(−11.5) × (1.0−)1.0–1.4(− 1.5) µm (n = 20). Conidia long, abundant, single, hyaline, eguttulate, aseptate, allantoid, (6.0−)6.6–7.4(−7.5) × (1.0−) 1.1–1.4(−1.5) µm (n = 30). No sexual morph observed.

Culture characteristics: Colonies after 7 d at 25 °C on PDA average 67.3 mm, medium-growing, white to buff with short aerial tufts giving a cottony appearance in the centre, radially growing hyphae submerged, hyphae becoming darker with age. Outer margin a mosaic of sienna and amber with dark patches and a buff margin. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: Glenn County (California, USA).

Host: Prunus domestica.

Notes: Based on the phylogenetic inference obtained in this study, C. longispora clusters in a strongly supported clade that contains C. ampulliformis, C. cotini, C. personata, C. ribis, C. rosarum, C. tanaitica, and C. ulmi. Conidia of all relatives are, on average, much shorter than C. longispora, with the exception of the recently described C. ampulliformis which produces larger conidia to 9 µm in length (Norphanphoun et al. 2017).

Cytospora oleicola D.P. Lawr., L.A. Holland & Trouillas, sp. nov.

MycoBank MB824279

(Figs 4 and 12)
Fig. 12
Fig. 12

Cytospora oleicola (ex-holotype culture CBS 144248). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 500 µm; D = 20 µm; E = 10 µm.

Etymology: The name refers to the host Olea and -cola for inhabitor.

Diagnosis: Conidia of C. oleicola are wider and longer, on average, as compared to the closely related C. pruinosa.

Type: USA: California: San Joaquin County, isolated from twig canker of Olea europaea, 19 Apr. 2016, F.P. Trouillas KARE1021 (BPI 910657 [dried culture] — holotype; CBS 144248 — ex-holotype culture).

Description: Conidiomata on PDA pycnidial, mostly solitary, rarely aggregated, globose, light mouse-grey to almost black (640−)715–1185(−1545) µm diam (n = 20), with a single internal locule. Conidiophores straight, reduced to branching filamentous conidiogenous cells (6.5−)7.5–9.3(−12.5) × (1.0−)1.0–1.6(−2.0) µm (n = 20). Conidia abundant, single, hyaline to light brown, eguttulate, aseptate, allantoid, relatively large (5.5−)5.9–6.5(−7.0) × (1.5−)1.5–1.7(−2.0) µm (n = 30). No sexual morph observed.

Culture characteristics: Colonies after 7 d at 25 °C on PDA average 63.7 mm, medium-growing, white to off-white with sparse aerial tufts, peripheral hyphae submerged, hyphae becoming buff with age. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: San Joaquin County (California, USA). Host: Olea europaea.

Notes: Based on the phylogenetic inference obtained in this study, C. pruinosa (isolated from Olea europaea var. africana in South Africa) is the closest relative to C. oleicola. Conidia of C. oleicola (5.5−)5.9–6.5(−7.0) × (1.5−)1.5–1.7(−2.0) µm are, on average, larger in terms of both length and width than conidia of C. pruinosa (5–6 × 1.2 µm; Adams et al. 2006).

Cytospora parakantschavelli Norphanph. et al., Mycosphere 8: 1 (2017).

(Figs 4 and 13)
Fig. 13
Fig. 13

Cytospora parakantschavelii (CBS 144243). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Conidia. D. Conidiophores and filamentous conidiogenous cells. E. Pycnidia. Bars C = 20 µm; D = 10 µm; E = 1 mm.

Type: Russia: on branches and twigs of Populus ×sibirica 12 May 2015, T. Bulgakov (MFLUCC 15-2094 — holotype).

Description: Conidiomata in PDA pycnidial, mostly solitary, rarely aggregated, globose, without conceptacle, black-grey with off-white surface hyphae, (1215−)1381–2099(−2600) µm diam (n = 20), with a single internal locule. Conidiophores straight, slender, then branching into 3–4 conidiogenous cells (6.0−)6.9–9.5(−9.5) × (1.0−)1.1–1.5(−2.0) µm (n = 20). Conidia abundant, single, hyaline to light brown, eguttulate, aseptate, allantoid, (5.5−)6.0–7.0(−7.5) × (1.0−)1.2–1.6(−1.5) µm (n = 30). No sexual morph observed.

Culture characteristics: Colony of C. parakantschavelli isolate KARE974 70 mm diam in 7 d at 25 °C on PDA, fast-growing, off-white with cream centre with short aerial tufts giving a cottony appearance, peripheral hyphae submerged, aerial hyphae becoming darker with age. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: Rostov Region, Russia and San Joaquin and Yolo Counties (California, USA).

Hosts: Populus deltoides, Populus freemontii, Populus × sibirica, and Pyrus pyraster.

Notes: Based on the phylogenetic inference obtained in this study, C. salicicola and C. kantschavelli are the closest relatives to C. parakantschavellii. The name C. parakantschavellii was recently introduced by Norphanphoun et al. (2017) from Populus and Pyrus in Russia.

Additional specimen examined: USA, California: San Joaquin County, isolated from wood canker of Prunus dulcis, 21 Apr. 2016, F.P. Trouillas KARE974 (BPI 910658 [dried culture]; CBS 144243).

Cytospora parapistaciae D.P. Lawr., L.A. Holland & Trouillas, sp. nov.

MycoBank MB824280

(Figs 4 and 14)
Fig. 14
Fig. 14

Cytospora parapistaciae (ex-holotype culture CBS 144506). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 500 µm; D–E = 10 µm.

Etymology: The name refers to the phylogenetic position of this fungus in relation to the sister taxon C. pistaciae.

Diagnosis: Cytospora parapistaciae is readily distinguished from C. pistaciae based on pycnidial shape (mostly solitary submerged vs. globose aggregated) and conidiogenous cells (single straight cells vs. 3–4 branching cells).

Type: USA: California: Kern County, isolated from wood canker of Pistacia vera, 26 June 2015, M.T. Nouri KARE270 (BPI 910659 [dried culture] — holotype; CBS 144506 — ex-holotype culture).

Description: Conidiomata on PDA pycnidial, mostly solitary, rarely aggregated, submerged to partially submerged, without conceptacle, black-grey, (335−)390–550(−590) µm diam (n = 20), with a single internal locule. Conidiophores hyaline, reduced to straight, slender, filamentous conidiogenous cells (7.0−)7.6–9.6(−11.0) × (1.0−)1.2–1.6(−2.0) µm (n = 20). Conidia abundant, single, hyaline to light brown, eguttulate, aseptate, allantoid, small, (3.0−)3.5–4.3(−4.5) × (1.0−)0.9–1.1(−1.5) µm (n = 30). No sexual morph observed.

Culture characteristics: Colonies after 7 d at 25 °C on PDA average 87.3 mm, fast-growing, buff to honey with short aerial tufts giving a cottony appearance, aerial hyphae very dense becoming darker buff to honey with white margin with age. Hyphae hyaline to light brown, smooth, straight, branched, and septate.

Distribution: Kern County (California, USA).

Host: Pistacia vera.

Notes: Based on the phylogenetic inference obtained in this study, C. pistaciae is the closest relative of C. parapistaciae, both of which originated from pistachio cankers in two separate counties in California.

Cytospora pistaciae D.P. Lawr., L.A. Holland & Trouillas, sp. nov.

MycoBank MB824281

(Figs 4 and 15)
Fig. 15
Fig. 15

Cytospora pistaciae (ex-holotype culture CBS 144238). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 1 mm; D = 10 µm; E = 20 µm.

Etymology: The name refers to the host, Pistacia vera, from which this fungus was first isolated.

Diagnosis: Cytospora pistaciae is readily distinguished from C. parapistaciae based on pycnidial shape (globose aggregated vs. mostly solitary submerged) and conidiogenous cells (3–4 branching cells vs. single straight cells).

Type: USA: California: Merced County, isolated from wood canker of Pistacia vera, 14 Oct. 2015, F.P. Trouillas KARE443 (BPI 910660 [dried culture] — holotype; CBS 144238 — ex-holotype culture).

Description: Conidiomata on PDA pycnidial, solitary to regularly aggregated, globose, without conceptacle, light mouse-grey, (975−)1196–2184(−2655) µm diam (n = 20), with a single internal locule. Conidiophores straight, reduced to 3–4 branching filamentous conidiogenous cells (5.5−) 7.1–8.9(−10.0) × (1.0−)1.1–1.5(−2.0) µm (n = 20). Conidia abundant, single, hyaline, eguttulate, aseptate, allantoid, (3.5−)4.0–4.8(−5.5) × (1.0−)1.1–1.3(−1.5) µm (n = 30). No sexual morph observed.

Culture characteristics: Colonies after 7 d at 25 °C on PDA average 87.3 mm, fast-growing, buff becoming honey with short aerial tufts giving a cottony appearance, peripheral hyphae submerged, aerial hyphae becoming darker with age. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: Merced County (California, USA).

Host: Pistacia vera.

Notes: Based on the phylogenetic inference obtained in this study, C. parapistaciae is the closest relative of C. pistaciae.

Cytospora plurivora D.P. Lawr., L.A. Holland & Trouillas, sp. nov.

MycoBank MB824282

(Figs 4 and 16)
Fig. 16
Fig. 16

Cytospora plurivora (ex-holotype culture CBS 144239). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Conidiophores and filamentous conidiogenous cells. D. Conidia. E. Pycnidia. Bars C = 20 µm; D = 10 µm; E = 1 mm.

Etymology: The name refers to the plethora of hosts this fungus was routinely isolated from.

Diagnosis: Cytospora plurivora is distinguished from C. amygdali and C. erumpens in the smaller conidia in terms of length and width.

Type: USA: California: San Joaquin County, isolated from twig lesions of Olea europaea, 24 June 2016, F.P. Trouillas KARE1452 (BPI 910661 [dried culture] — holotype; CBS 144239 — ex-holotype culture).

Description: Conidiomata on PDA pycnidial, large, some solitary, many gregarious, globose to extended globose, no conceptacle, black-grey with off-white surface hyphae, (1110−)1152–1968(−2745) µm diam (n = 20), with a single internal locule. Conidiophores reduced to single, straight, slender, filamentous conidiogenous cells (7.0−)7.7–10.0(− 11.0) × (1.0−)1.0–1.4(−1.5) µm (n = 20). Conidia abundant, single, hyaline to dark brown, eguttulate, aseptate, allantoid, (3.5−)3.8–4.4(−4.5) × (1.0−)0.9–1.1(−1.5) µm (n = 30). No sexual morph observed.

Culture characteristics: Colonies after 7 d at 25 °C on PDA average 82 mm, fast-growing, uneven lobate growth margin, off-white to cream with short aerial tufts giving a cottony appearance, aerial hyphae becoming light brown with age. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: Butte, Colusa, Contra Costa, Fresno, Glenn, Kern, San Joaquin, Stanislaus, Sutter, Tehama, Tulare, and Yuba Counties (California, USA).

Hosts: Juglans regia, Olea europaea, Pistacia vera, Prunus domestica, Prunus dulcis, and Prunus persica.

Notes: Based on the phylogenetic inference obtained in this study, C. amygdali is the closest species to C. plurivora, albeit with no statistical support. Cytospora plurivora is the most genetically diverse clade identified in this study which in part is likely due to its incidence on many different fruit and nut crop hosts throughout California.

Cytospora populicola D.P. Lawr., L.A. Holland & Trouillas, sp. nov.

MycoBank MB824283

(Figs 4 and 17)
Fig. 17
Fig. 17

Cytospora populicola (ex-holotype culture CBS 144240). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 500 µm; D-E = 10 µm.

Etymology: The name refers to the host Populus and -cola for inhabitor.

Diagnosis: Cytospora populicola is distinguished from C. longiostiolata and C. rostrata in the shorter conidia than C. longiostiolata and larger conidia than C. rostrata, respectively.

Type: USA: California: San Joaquin County, isolated from wood canker of Populus deltoides, 21 Apr. 2016, F.P. Trouillas KARE973 (BPI 910662 [dried culture] — holotype; CBS 144240 — ex-holotype culture).

Description: Conidiomata on PDA pycnidial, mostly solitary, rarely aggregated, some with yellow conidial exudate, globose to conical, without conceptacle, black-grey, (1015−) 1210–2210(−2735) µm diam (n = 20), with a single internal locule. Conidiophores reduced to 3–4 filamentous branching conidiogenous cells tapering toward apices (5.5−)6.1–8.1(−10.0) × (1.0−)1.5–1.9(−2.0) µm (n = 20). Conidia abundant, single, hyaline, eguttulate, aseptate, allantoid, (4.5−)4.7–5.3(−5.5) × (1.0−)1.1–1.4(−1.5) µm (n = 30). No sexual morph observed.

Culture characteristics: Colonies after 7 d at 25 °C on PDA average 87.3 mm, medium-growing with uneven margin expansion, off-white with short aerial tufts giving a cottony appearance, aerial hyphae becoming cream-coloured with age. Hyphae hyaline, smooth, straight, branched, and septate. Distribution: San Joaquin County (California, USA).

Host: Populus deltoides.

Notes: Based on the phylogenetic inference obtained in this study, C. longiostiolata and C. rostrata, both isolated from Salix, are the closest species to C. populicola. Conidia of C. populicola are, on average, larger than those of C. rostrata 3.6–4.8 × 1.0–1.6 µm (av. 4.1 × 1.4 µm) and smaller than those of C. longiostiolata (3.9)5.4–6.6 × 1.0–1.2(−1.5) (av. 5.5 × 1.3 µm).

Cytospora punicae Sacc., Michelia 1: 367 (1878); as ‘punica’.

Figs 4 and 18.
Fig. 18
Fig. 18

Cytospora punicae (CBS 144244). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 500 µm; D = 20 µm; E = 10 µm.

Description: Conidiomata on PDA pycnidial, gregarious, globose to subglobose, no conceptacle, black-grey with off-white surface hyphae, (210−)237–383(−490) µm diam (n = 20), with multiple internal locules with shared invaginated walls. Conidiophores single, straight, filamentous conidiogenous cells (5.5−)5.8–8.6(−9.5) × (1.0−)1.1–1.4(−2.0) µm (n = 20). Conidia abundant, single, hyaline to light brown, eguttulate, aseptate, allantoid, (3.5−)3.8–4.6(−5.0) × (0.5−)0.8–1.0(−1.0) µm (n = 30). No sexual morph observed.

Culture characteristics: Colony of C. punicae isolate 5A–80 64.7 mm diam in 7 d at 25 °C on PDA. Medium-growing, dark red becoming lighter with age. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: Fresno, Madera, and Stanislaus Counties (California, USA), Cyprus, Greece, Iran, South Africa, and Turkey.

Host: Punica granatum.

Notes: Based on the phylogenetic inference obtained in this study, C. myrtagena is the closest species to C. punicae. Only two species of Cytospora are known from pomegranate (C. granati and C. punicae) and these can be distinguished by the diagnostic red hyphae/colony of C. punicae in culture. The colony growth of Cytospora punicae is also much slower (64.7 mm in 7 d) compared to C. granati (87.3 mm in 7 d).

Specimen examined: USA: California: Madera County, isolated from wood canker of Punica granatum, 21 July 2010, T.J. Michailides 5A-80 (BPI 910663 [dried culture]; CBS 144244).

Cytospora sorbicola Norphanph. et al., Mycosphere 8: 1 (2017).

Figs 4 and 19.
Fig. 19
Fig. 19

Cytospora sorbicola (CBS 144245). A. Seven-day-old PDA culture. B. Fourteen-day-old PDA culture. C. Pycnidia. D. Conidiophores and filamentous conidiogenous cells. E. Conidia. Bars C = 1 mm; D = 20 µm; E = 5 µm.

Type: Russia: on dead and dying branches of Acer pseudoplatanus 18 June 2015, T. Bulgakov (MFLUCC 152203 — holotype).

Description: Conidiomata on PDA pycnidial, mostly solitary, sometimes aggregated, globose, without conceptacle, mouse-grey, (1020−)1220–1900(−2420) µm diam (n = 20) with 1–2 locules. Conidiophores branched, reduced to filamentous conidiogenous cells that taper towards the apices (4.5−)6.4–9.6(−10.0) × (1.0−)1.0–1.4(−2.0) µm (n = 20). Conidia abundant, single, hyaline to light brown, eguttulate, aseptate, allantoid, (3.5−)4.0–4.6(−4.5) × (1.0−)0.9–1.1(−1.0) µm (n = 30). No sexual morph observed.

Culture characteristics: Colony of C. sorbicola isolate KARE228 81.7 mm diam in 7 d at 25 °C on PDA, fast-growing, off-white to cream with general lack of aerial hyphae, colony darkens with age. Hyphae hyaline, smooth, straight, branched, and septate.

Distribution: Contra Costa, Fresno, Kings, Merced, Sacramento, San Benito, San Joaquin, Stanislaus, Yolo, and

Yuba Counties (California, USA), and Rostov Region, Russia.

Hosts: Acer pseudoplatanus, Cotonoeaster melanocarpus, Prunus armeniaca, P. avium, P. cerasus, P. domestica, P. dulcis, P. persica, and Sorbaronia mitschurinii.

Notes: Based on the phylogenetic inference obtained in this study, C. donetzica is the closest species to C. sorbicola. The C. sorbicola isolates collected in this study display some host affiliation with cherry, clustering strongly in the MP analysis and no support in the ML analysis. The level of support for the California-only C. sorbicola isolates and differences in morphology suggests that they may represent a distinct lineage sister to C. sorbicola collected in Russia. Additional data such as TEF1 and TUB2 from the Russian type of C. sorbicola will help answer this question.

Additional specimen examined: USA, California: Stanislaus County, isolated from bark canker of Prunus dulcis, 15 July 2015, M.T. Nouri KARE228 (BPI 910664 [dried culture]; CBS 144245).

Discussion

This manuscript presents a comprehensive molecular phylogenetic overview of Cytospora species currently known from culture, which was initiated due to a high incidence of Cytospora species associated with canker symptoms across diverse orchard crops in California. All Cytospora species known from culture and linked to publicly-available molecular data were considered for phylogenetic analyses in this study. The lack of ex-type cultures or sequence data for many species names makes it difficult to assess many older species names, especially for those described only by morphology. We deposited ten new ex-type specimens with two different public fungal biodiversity repositories, the Mycology and Nematology Genetic Diversity and Biology Laboratory in Beltsville, MD (BPI), and the Westerdijk Fungal Biodiversity Institute in The Netherlands (CBS), in conjunction with molecular data in GenBank, in order to strengthen and stabilize the taxonomy of Cytospora and to aid in the identification of Cytospora species via DNA sequence data in future studies by other mycologists and plant pathologists.

Cytospora species are ubiquitous, important pathogens of many woody hosts causing cankers, dieback and mortality of forest and urban trees (Adams et al. 2005, 2006, Worrall et al. 2010) and of many economically important crops including Juglans, Malus, Prunus, and Vitis (Biggs & Grove 2005, Wang et al. 2011, Fan et al. 2015a, Lawrence et al. 2017a). Results from this study unveiled 15 species of Cytospora from infected orchard crops and adjacent ornamentals in the Central Valley of California. These species include the previously described taxa C. chrysosperma, C. parakantschavelli, C. punicae, and C. sorbicola and 10 previously undescribed taxa names which are newly introduced: C. amygdali, C. californica, C. granati, C. joaquinensis, C. longispora, C. oleicola, C. parapistaciae, C. pistaciae, C. plurivora, and C. populicola, and a new combination, C. eucalypti. All species were strongly supported by both DNA sequence data and morphological observations. This study reports C. parakantschavelii and C. sorbicola for the first time in North America, including new host records for each species, Populus deltoides and P. freemontii for C. parakantschavelii and Olea europaea, Prunus avium, P. domestica, P. dulcis, and P. persica for C. sorbicola. Our Californian Cytospora eucalypti (syn. Valsa eucalypti) isolates cluster strongly with an isolate from the coastal redwoods (Sequoia sempervirens) reported in Adams et al. (2005), which also clusters strongly with isolates previously referred to as Valsa eucalypti, isolated from four species of Eucalyptus in California (Adams et al. 2006). This study expanded the known host range of C. eucalypti to include Prunus dulcis and Sequoiadendron gigateum (giant sequoia) in California.

The utility of asexual morph characters for species recognition has been questioned in Cytospora. Locule morphology seems to be influenced by the depth in the bark at which the pycnidia form, with variations from unilocular cytosporoid when formed deep in the bark to rosette cytosporoid when formed near the bark surface (Adams et al. 2005). Also, asexual morphs that form in nature can vary considerably from those forming in culture, and these morphological characters are not necessarily taxonomically informative (Adams et al. 2005). Considering that sexual morphs are rarely found in nature, the use of sexual morph morphology in species diagnosis has been limited. Furthermore, both ascospores and conidia of many Cytospora species are of similar shapes (single, allantoid, and aseptate) and sizes (4–8 × 1–2 µm) thus complicating morphological separation of distinct lineages (Adams et al. 2002, 2005, Wang et al. 2011). In this study, we found the morphological characteristics of the conidia were indistinguishable among most species, with similar dimensions among the examined species; most asexual morph characters were not taxonomically informative.

The genus Cytospora includes both generalist pathogens (i.e. C. chrysosperma with 265 host records; USDA Fungus-Host Distribution Database, https://nt.ars-grin.gov/fungaldatabases/fungushost/fungushost.cfm) and specialist pathogens (i.e. C. punicae with only one host record in the same USDA Database). As such, host associations do not appear to constitute an appropriate criterion for species recognition, as previously discussed (Adams et al. 2005, 2006). In this study, host association was not found to be taxonomically informative as many Cytospora species were recovered from multiple hosts. However, our work highlighted a few instances of close host associations. Prior to this study, C. punicae had been reported causing wood canker on pomegranate trees in California, Cyprus, and Iran (Peduto Hand et al. 2014, Samouel & Kanetis 2016, Mahdikhani & Davoodi 2017), pomegranate collar rot in Greece (Palavouzis et al. 2015), and pomegranate fruit rot in South Africa (Venter et al. 2017). Cytospora punicae was only recovered from pomegranate trees in this study, supporting this species as host specific despite a wide geographical distribution. Pomegranate trees harboured a second species, C. granati, which was only recovered from this host. Both C. punicae and C. granati have similar conidial shapes and dimensions, but the species have distinct pycnidial shapes and sizes and colony morphologies. Thus, host association paired with morphological observations may have utility when examining Cytospora species on pomegranate. In contrast, C. sorbicola was isolated from six hosts (almond, apricot, cherry, olive, peach, and plum) and these hosts typically harboured more than one Cytospora species. Within the C. sorbicola clade, a subclade strongly supported by parsimony analysis (86 %) but showing low support by likelihood analysis (<70 %) contained isolates that originated almost exclusively from cherry. These findings suggest some level of genetic divergence for C. sorbicola isolates from cherry, which could indicate some host specialization in these isolates; a preliminary step towards reproductive isolation and ecological speciation (Giraud et al. 2010).

Given the variability, plasticity, and complexity of morphological characters in the genus (e.g. stromatal arrangement in the host tissues, locular arrangement within pycnidia, locule division into chambers, independent or shared locular walls), previous studies have advocated the use of molecular data to accurately identify Cytospora species (Adams et al. 2002, 2005, 2006). In this study, we used molecular phylogenetic analyses of four loci (ITS+TUB2+TEF1+ACT1), not only to identify species but also to provide reference data for future phylogenetic studies. Before this study, most Cytospora sequences deposited in GenBank consisted of ITS. While ITS is the primary marker for fungal barcoding (Schoch et al. 2012), in some fungal groups, ITS has insufficient power for species recognition whereas protein-coding genes can be more informative sequence regions for species delineation (O’Donnell et al. 2015, Lawrence et al. 2017b). For instance, analyses of TEF1 sequence data provided more discriminatory power than ITS in delineating two recently described Cytospora species occurring on grapevine, C. vinacea and C. viticola (Lawrence et al. 2017a). In other xylophilous fungi, ‘secondary barcodes’ such as TUB2, TEF1, and histone 3 (HIS) can also be preferable based on their ability to delineate closely related or cryptic species and on the availability of sequence data for ex-type specimens. For example TUB2 is the preferred marker for identification of fungi in the Togniniaceae (i.e. Phaeoacremonium minimum) and TEF1 is the preferred marker for the Botryosphaeriaceae (i.e. Neofusicoccum parvum) and Diaporthales (which includes Cytosporaceae) (Lawrence et al. 2017b), especially for closely related or cryptic species. In agreement with previous studies (Adams et al. 2002, 2005), our findings revealed that ITS has sufficient power to discriminate the 15 Cytospora species reported from orchard crops in California. However, based on comparisons of clade support values of each locus used in this study, it appears that TEF1 is the preferential locus to use for Cytospora identification as it was able to strongly support all 15 lineages in this study. Moreover, in our study, 362/799 (45 %) of the aligned nucleotide positions in TEF1 and 142/365 (39 %) in ACT1 were parsimony informative, whereas only 119/575 (21 %) and 180/604 (30 %) were parsimony informative in TUB2 and ITS, respectively. Therefore, a DNA-based approach utilizing several gene regions (in order of priority: TEF1, ACT1, ITS, and TUB2 using the primer pairs in this study) would be the best method to resolve Cytospora species concepts, especially when morphological characters and host occurrences may be misleading due to significant overlap.

Until the present study, the diversity of Cytospora species affecting perennial crops in California has been largely overlooked and underestimated. Historically, two species, C. cincta and C. leucostoma, have been associated with Cytospora canker of stone fruits and pome fruits in North America (Bertrand & English 1976b, Biggs 1989, Biggs & Grove 2005). Surprisingly, we did not isolate either species in this study, suggesting that C. cincta and C. leucostoma were originally misidentified as the causal agents of Cytospora canker of stone fruits and pome fruits in California. Our findings suggest that many species of Cytospora are involved in the decline of fruit and nut crops in California, and they do not include either C. cincta nor C. leucostoma. The main putative causal agents of Cytospora canker of stone fruits (apricot, cherry, peach, and prune) in California included C. plurivora and C. sorbicola. Similarly, the main putative causal agents of Cytospora canker of nut crops (almond, pistachio, and walnut) in California included C. amygdali, C. californica, C. eucalypti, C. joaquinensis, C. parapistaciae, C. pistaciae, C. plurivora, and C. sorbicola. Three species were associated with Cytospora canker of Populus trees, C. joaquinensis, C. parakantschvelii, and C. populicola. Cytospora joaquinensis was also associated with cankers in pistachio and walnut, suggesting that cross infections occur between orchards and adjacent ornamentals and vice versa. Three species were associated with Cytospora canker of olive (C. oleicola, C. plurivora, and C. sorbicola) with the two latter species also collected from other hosts. Two species were exclusively associated with Cytospora canker of pomegranate (C. granati and C. punicae). These results strongly suggest the need for additional research concerning the epidemiology of Cytospora species that cause Cytospora canker in fruit and nut crops and proximal ornamentals in the diverse agricultural areas of the Central Valley of California.

Research on Cytospora canker of stone fruits had received broad attention before the advent of molecular identification of fungi, focusing on seasonal activities of pathogenic species (Bertrand & English 1976a), spore production (Bertrand & English 1976b), etiology, epidemiology and host resistance (Biggs 1989). According to our findings, pathogenicity studies should now be conducted to elucidate the role of the newly described Cytospora species in the fruit and nut crops in California. The large diversity of species revealed in this study also suggests that management of Cytospora canker needs to be re-evaluated following accurate molecular identification to determine the main pathogenic species involved within each crop. Control of Cytospora diseases is difficult and focusing management efforts against the most aggressive encountered Cytospora species will be essential. The genus Cytospora represents a good example of a fungal group where morphological features are extremely complex and not necessarily informative from a taxonomic standpoint, which could in part explain why in North America only two species were previously considered the main causal agents of Cytospora canker of perennial crops. This study constitutes a further step towards a sequence-based description of fungal species in an important group of plant pathogens, revealing a large species richness, providing type specimens associated with molecular data for new taxa, detailed morphological descriptions, and some evidence for appropriate selection of loci for molecular typing. Furthermore, this study provides a firm foundation for future pathogenicity, ecological, and epidemiological studies to better help manage canker diseases in perennial crops infected by Cytospora species.

Declarations

Acknowlegements

This manuscript is dedicated to the 200-year-old generic name Cytospora. We thank the California Cherry Board, the California Pistachio Research Board and the Almond Board of California for financial support. We thank also Francesca Peduto-Hand for supplying images of Cytospora canker of pomegranate.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Department of Plant Pathology, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA
(2)
Department of Plant Pathology, University of California, Davis, USA
(3)
Kearney Agricultural Research and Extension Centre, Parlier, CA 93648, USA

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