Biodiversity Data Journal : Taxonomic Paper
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Taxonomic Paper
Two new species of Cladosporium from leaf spots of Paris polyphylla in north-western Yunnan Province, China
expand article infoYue-Xin Xu‡,§, Hong-Wei Shen§,|,, Dan-Feng Bao§,|,#, Zong-Long Luo§, Hong-Yan Su§, Yu-E Hao
‡ College of Public Health, University of South China, Hengyang, China
§ College of Agriculture and Biological Sciences, Dali University, Dali, China
| Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand
¶ School of Science, Mae Fah Luang University, Chiang Rai, Thailand
# Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand
Open Access

Abstract

Background

During the survey of pathogenic fungi on medicinal plant leaves in Yunnan Province, China, two Cladosporium-like taxa were isolated from leaf spots of Paris polyphylla. Based on morphological characteristics and phylogenetic analysis of combined ITS, TEF1-α and ACT genes, two new species were discovered.

New information

Two new species Cladosporium yunnanensis and C. paris are introduced, the detailed descriptions and illustrations are provided. Morphology of the two new species is compared with other related Cladosporium species. This study widens the host diversity of the genus Cladosporium.

Keywords

asexual morph, Cladosporium, hyphomycetes, phylogeny, taxonomy

Introduction

Cladosporium is one of the largest and most heterogeneous genera of hyphomycetous fungi (Dugan et al. 2004). It was initially described by Persoon (1794) from rotten wood as Dematium herbarum Pers., which was later synonymised by Link (1816) as Cladosporium herbarum (Pers.: Fr.). Cladosporium is currently only known as the asexual morph, which is characterised by erect, straight or geniculate conidiophores, abundant branched acropetal chains of smooth to roughened dry conidia produced from mono- or polyblastic conidiogenous cells, the coronate structure of conidiogenous loci and conidial hila, consisting of a central convex dome surrounded by a raised periclinal rim (David 1997, Crous et al. 2007).

To clarify the relationship of species in the complex Cladosporium, subsequent researchers have been constantly revising this genus and the use of molecular analysis is necessary as well as morphological characters (David 1997, Dugan et al. 2004, Heuchert et al. 2005, Schubert 2005, Schubert et al. 2007, Crous et al. 2007, Sandoval-Denis et al. 2016, Bezerra et al. 2017, Bezerra et al. 2017, Abdollahzadeh et al. 2020). Some phylogenetic studies have proposed a multi-locus sequence analysis approach to clarify species diversity within the genus with internal spacers of the rDNA genes (ITS), translation elongation factor 1-α (TEF1-α) and actin (ACT) (Bensch et al. 2012, Bensch et al. 2015, Bensch et al. 2018, Tibpromma et al. 2019, Iturrieta-González et al. 2021, Zimowska et al. 2021). Based on the phylogenetic analyses and morphological features, about 237 species have been accepted within the genus, which are split into three species complexes, Cladosporium herbaum (Schubert et al. 2007), C. sphaerospermum (Zalar et al. 2007, Dugan et al. 2008) and C. cladosporioides (Bensch et al. 2010).

The species of Cladosporium are able to colonise a wide range of substrates and can be isolated in any natural or anthropogenically-affected environment (Flannigan et al. 2002, Bensch et al. 2010, Bensch et al. 2012, Bensch et al. 2018, Sandoval-Denis et al. 2015, Temperini et al. 2018, Chung et al. 2019). They are well known as plant pathogens, which may occur on leaves, stems and fruits on different plants, for example, Asparagaceae, Asteraceae, Fabaceae, Myrtaceae, Orchidaceae and Poaceae (Schubert 2005, Bensch et al. 2012, Bensch et al. 2015, Marin-Felix et al. 2017, Rosado et al. 2019). Besides, some species have been reported as pathogens of animals and humans, saprobes and endophytes and been also reported as hyperparasites on other fungi (Sandoval-Denis et al. 2015, Sandoval-Denis et al. 2016, Zhou et al. 2016, Velázquez-Jiménez et al. 2019). Furthermore, some species have shown the ability to produce medicinal compounds or their potential as biological agents to control plant diseases (Köhl et al. 2015, Khan et al. 2016, Adorisio et al. 2019).

During the investigation of pathogenic fungi on leaf spots of medicinal plants in Yunnan Province, China, two new species Cladosporium yunnanensis and C. paris were identified, based on morphology and multi-gene phylogenetic analysis. Full descriptions, illustrations and update of the phylogenetic backbone tree for Cladosporium are provided as well.

Materials and methods

Isolation and morphological examination

Leaf specimens with disease symptoms of cultivated Paris polyphylla were collected from Dali, Yunnan Province, China in October and November 2020 and taken back to the laboratory in an envelope. The leaves were kept at 4°C in Zip-lock plastic bags before they were processed in the laboratory. Single spore isolations were made onto potato dextrose agar (PDA). After 8–10 hours, a single germinating conidia was transferred aseptically to a new PDA plate to obtain cultures and grow at 20–25°C in daylight (Chomnunti et al. 2014).

The cultures are deposited in Kunming Institute of Botany, Chinese Academy of Sciences (KUNCC) and China General Microbiological Culture Collection Center (CGMCC). Cultures are deposited at the Herbarium of Cryptogams Kunming Institute of Botany Academia Sinica (Herb. HKAS). Facesoffungi and Index Fungorum numbers were obtained as in Jayasiri et al. (2015) and Index Fungorum.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from fresh mycelium grown on PDA at room temperature (25°C). The TreliefTM Plant Genomic DNA Kit (TSP101) was used to extract DNA according to the manufacturer’s instructions. ITS, TEF1-α and ACT gene regions were amplified using the primer pairs ITS1/ITS4, EF1-728F/EF1-986R and ACT–512F/ACT–783R. The final volume of the PCR reaction was 25 µl and contained 12.5 µl of 2 × Power Taq PCR MasterMix (a premix and ready to use solution, including 0.1 Units/µl Taq DNA Polymerase, 500 µM dNTP Mixture each (dATP, dCTP, dGTP, dTTP), 20 mM Tris–HCl pH 8.3, 100 mM KCl, 3 mM MgCl2, stabiliser and enhancer), 1 μl of each primer (10 μM), 1 µl genomic DNA extract and 9.5 µl deionised water. The PCR thermal cycle programme for ITS, TEF1-α and ACT amplification was as follows: initial denaturation of 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 56°C for 50 seconds, elongation at 72°C for 1 minute and the final extension at 72°C for 10 minutes. PCR products were purified using minicolumns, purification resin and buffer according to the manufacturer’s protocols (Amershamproduct code: 27–9602–01). The sequencing works were carried by Tsingke Biological Engineering Technology and Services Co., Ltd (Yunnan, P.R. China).

Phylogenetic analysis

Sequence data for relevant strains were downloaded from GenBank following latest publications (Freitas 2018, Iturrieta-González et al. 2021, Zimowska et al. 2021). The sequences aligned using MAFFTv.7 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013) and optimised manually when needed. The aligned dataset was analysed by Maximum Likelihood (ML) and Bayesian Inference (BI).

Maximum Likelihood analysis was performed by using RAxMLGUI v.1.3 (Silvestro and Michalak 2012). The optimal ML tree search was conducted with 1,000 separate runs using the default algorithm of the programme from a random starting tree for each run. The final tree was selected amongst suboptimal trees from each run by comparing the likelihood scores using the GTR+GAMMA substitution model. Maximum Likelihood bootstrap values equal to or greater than 70% were given as the first set of numbers above the nodes in the resulting ML tree (Fig. 1).

Figure 1.  

Maximum Likelihood (ML) tree obtained from the combined analysis of ITS, TEF1-α and ACT sequences of 161 strains from Cladosporium. The tree is rooted with Toxicocladosporium irritans (CBS 185.58) and T. protearum (CBS 126499). Numbers on the branches represent ML bootstrap support values (MLBS) ≥70%, followed by Bayesian posterior probabilities (PP) ≥ 0.95, lower values are indicated as “-”. Names of species newly described are indicated in red and ex-type strains and reference specimens are indicated in bold. Branch lengths are proportional to distance.

Bayesian analysis was conducted with MrBayes v.3.1.2 (Ronquist and Huelsenbeck 2003) to evaluate posterior probabilities (Rannala and Yang 1996) by Markov Chain Monte Carlo sampling (MCMC). The best-fit models of evolution were estimated by MrModeltest v.2.2 (Nylander 2004). ITS selected the SYM+I+G model with inverse gamma-distributed rate in Bayesian analyses. TEF1-α and ACT selected the GTR+I+G model with inverse gamma-distributed rate in Bayesian analyses. The robustness of ML analyses was evaluated by bootstrap support (MLBS). The parameter settings, used in these analyses, were two simultaneous runs of 10,000,000 generations and four Markov chains, sampled every 1,000 generations. The 50% majority rule consensus tree and posterior probability values (PP) were calculated after discarding the first 25% of the samples. A PP value of ≥ 0.95 was considered significant (Hespanhol et al. 2019).

The phylogenetic trees were viewed and optimised in FigTree v.1.2.2 (Rambaut and Drummond 2008) and edited further using Microsoft Office PowerPoint. Newly-generated sequences in this study were deposited in GenBank (Table 1).

Table 1.

Isolates and sequences used in this study (newly-generated sequences are indicated with a “*”, strains isolated from the holotype and reference specimens are indicated in bold).

Species

Strain number

GenBank Accession Numbers

ITS

TEF1-α

ACT

Cladosporium acalyphae

CBS 125982

NR_119835

HM148235

HM148481

C. aciculare

CBS 140488

KT600411

KT600509

KT600607

C. aerium

DTO 323-G7

MF472899

MF473326

MF473749

C. aggregatocicatricatum

CBS 140493

NR_152300

KT600547

KT600645

C. alboflavescens

UTHSC DI-13-225

LN834420

LN834516

LN834604

C. allicinum

CBS 121624

NR_152266

EF679425

EF679502

C. allicinum

UTHSC DI-13-176

LN834354

LN834450

LN834538

C. allii

CBS 101.81

JN906977

JN906983

JN906996

C. angulosum

COAD 2500

MK253346

MK293786

MK249989

C. angustiherbarum

CBS 140479

NR_152286

KT600475

KT600574

C. angustisporum

CBS 125983

NR_111530

HM148236

HM148482

C. angustiterminale

CBS 140480

NR_152287

KT600476

KT600575

C. antarcticum

CBS 690.92

NR_121332

EF679405

EF679484

C. anthropophilum

CPC 22393

MF472922

MF473349

MF473772

C. aphidis

CBS 132182

JN906978

JN906984

JN906997

C. arenosum

CHFC-EA 566

MN879328

MN890011

MN890008

C. arthropodii

CBS 124043

NR_120011

JN906985

JN906998

C. asperulatum

CBS 126340

NR_119836

HM148239

HM148485

C. australiense

CBS 125984

NR_119837

HM148240

HM148486

C. austroafricanum

CBS 140481

NR_152288

KT600478

KT600577

C. austrohemisphaericum

CBS 140482

KT600382

KT600479

KT600578

C. basiinflatum

CBS 822.84

NR_111531

HM148241

HM148487

C. caprifimosum

FMR 16532

LR813198

LR813210

LR813205

C. chalastosporoides

CBS 125985

NR_119838

HM148242

HM148488

C. chasmanthicola

CPC 21300

NR_152307

KY646227

KY646224

C. chubutense

CBS 124457

NR_119728

FJ936161

FJ936165

C. cladosporioides

CBS 112388

NR_119839

HM148244

HM148490

C. cladosporioides

CBS 113738

HM148004

HM148245

HM148491

C. colocasiae

CBS 386.64

NR_119840

HM148310

HM148555

C. colocasiae

CBS 119542

HM148066

HM148309

HM148554

C. colombiae

CBS 274.80B

NR_119729

FJ936163

FJ936166

C. coprophilum

FMR 16164

LR813201

LR813213

LR813207

C. crousii

CBS 140686

LN834431

LN834527

LN834615

C. cucumerinum

CBS 171.52

NR_119841

HM148316

HM148561

C. cucumerinum

CBS 176.54

HM148078

HM148322

HM148567

C. cycadicola

CPC 17251

KJ869122

KJ869236

KJ869227

C. delicatulum

CBS 126344

MH863920

HM148325

HM148570

C. dominicanum

CBS 119415

DQ780353

JN906986

EF101368

C. echinulatum

CBS 123191

JN906980

JN906987

JN906999

C. europaeum

FP-027-A9

MH102078

MH102121

MH102068

C. exasperatum

CBS 125986

NR_119843

HM148334

HM148579

C. exile

CBS 125987

NR_111532

HM148335

HM148580

C. fildesense

F09-T12-1

JX845290

MN233633

MN233632

C. flabelliforme

CBS 126345

NR_119844

HM148336

HM148581

C. flavovirens

UTHSC DI-13-273

LN834440

LN834536

LN834624

C. floccosum

CBS 140463

LN834416

LN834512

LN834600

C. funiculosum

CBS 122129

NR_119845

HM148338

HM148583

C. funiculosum

CBS 122128

HM148093

HM148337

HM148582

C. fuscoviride

FMR 16385

LR813200

LR813212

LR813206

C. fusiforme

CBS 119414

DQ780388

JN906988

EF101372

C. gamsianum

CBS 125989

NR_111533

HM148339

HM148584

C. globisporum

CBS 812.96

NR_111534

HM148340

HM148585

C. grevilleae

CBS 114271

NR_119960

JF770472

JF770473

C. halotolerans

CBS 119416

DQ780364

JN906989

EF101397

C. herbaroides

CBS 121626

NR_119655

EF679432

EF679509

C. herbarum

CBS 121621

NR_119656

EF679440

EF679516

C. hillianum

CBS 125988

NR_119846

HM148341

HM148586

C. inversicolor

CBS 401.80

NR_111535

HM148345

HM148590

C. ipereniae

CBS 140483

NR_152290

KT600491

KT600589

C. iranicum

CBS 126346

NR_111536

HM148354

HM148599

C. iridis

CBS 138.40

NR_111271

EF679447

EF679523

C. kenpeggii

CPC 19248

KY646222

KY646228

KY646225

C. langeronii

CBS 189.54

DQ780379

JN906990

EF101357

C. lentulum

FMR 16288

LR813203

LR813215

LR813209

C. licheniphilum

CBS 125990

NR_119847

HM148355

HM148600

C. limoniforme

CBS 140484

KT600397

KT600494

KT600592

C. longicatenatum

CBS 140485

NR_152291

KT600500

KT600598

C. longissimum

CBS 300.96

DQ780352

EU570259

EF101385

C. lycoperdinum

CBS 126347

MH863923

HM148356

HM148601

C. lycoperdinum

CBS 574.78C

HM148115

HM148359

HM148604

C. macrocarpum

CBS 121623

NR_119657

EF679453

EF679529

C. macrocarpum

UTHSC DI-13-191

LN834379

LN834475

LN834563

C. magnoliigena

MFLUCC 18-1559

MK347813

MK340864

-

C. montecillanum

CBS 140486

NR_152292

KT600504

KT600602

C. montecillanum

CPC 15605

KT600407

KT600505

KT600603

C. myrtacearum

CBS 126349

MH863925

HM148360

HM148605

C. myrtacearum

CBS 126350

NR_119849

HM148361

HM148606

C. needhamense

Z-1866

MF473142

MF473570

MF473991

C. neopsychrotolerans

CGMCC3.18031

KX938383

KX938400

KX938366

C. ossifragi

CBS 842.91

NR_121333

EF679459

EF679535

C. oxysporum

CBS 125991

NR_152267

HM148362

HM148607

C. oxysporum

CBS 126351

MH863927

HM148363

HM148608

C. paracladosporioides

CBS 171.54

NR_119850

HM148364

HM148609

C. paralimoniforme

CGMCC3.18103

KX938392

KX938409

KX938375

C. paralimoniforme

CGMCC3.18104

KX938393

KX938410

KX938376

C. parapenidielloides

CBS 140487

NR_152293

KT600508

KT600606

C. parasubtilissimum

CPC 22396

MF473171

MF473594

MF474019

C. paris sp. nov.*

KUN HKAS 121701*

OK338503*

OL825681*

OL466938*

C. penidielloide

CBS 140489

KT600412

KT600510

KT600608

C. perangustum

CBS 125996

NR_119851

HM148365

HM148610

C. phaenocomae

CBS 128769

NR_119950

JF499875

JF499881

C. phlei

CBS 358.69

NR_120013

JN906991

JN907000

C. phyllactiniicola

CBS 126355

NR_111537

HM148397

HM148642

C. phyllophilum

CBS 125992

NR_111538

HM148398

HM148643

C. pini-ponderosae

CBS 124456

NR_119730

FJ936164

FJ936167

C. prolongatum

CGMCC3.18036

KX938394

KX938411

KX938377

C. pseudiridis

CBS 116463

NR_111272

EF679461

EF679537

C. pseudochalastosporoides

CBS 140490

NR_152296

KT600513

KT600611

C. pseudocladosporioides

CBS 125993

NR_119852

HM148402

HM148647

C. pseudotenellum

FMR 16231

LR813145

LR813196

LR813146

C. psychrotolerans

CBS 119412

DQ780386

JN906992

EF101365

C. puris

COAD 2494

MK253338

MK293778

MK249981

C. puyae

CBS 274.80A

NR_152298

KT600516

KT600614

C. ramotenellum

CBS 121628

NR_119658

EF679462

EF679538

C. rectoides

CBS 125994

NR_111539

HM148438

HM148683

C. rectoides

CBS 126357

MH863933

HM148439

HM148684

C. rhusicola

CBS 140492

NR_152299

KT600539

KT600637

C. rubrum

CMG 28

MN053018

MN066644

MN066639

C. ruguloflabelliform

CBS 140494

KT600458

KT600557

KT600655

C. rugulovarians

CBS 140495

KT600459

KT600558

KT600656

C. salinae

CBS 119413

DQ780374

JN906993

EF101390

C. scabrellum

CBS 126358

NR_119853

HM148440

HM148685

C. silenes

CBS 109082

NR_111270

EF679429

EF679506

C. sinense

CBS 143363

MF473252

MF473675

MF474102

C. sinuatum

CGMCC3.18096

KX938385

KX938402

KX938368

C. sinuosum

CBS 121629

NR_119659

EF679464

EF679540

C. soldanellae

CPC 13153

NR_120014

JN906994

JN907001

C. sp.

UTHSC DI-13-227

LN834422

LN834518

LN834606

C. sp.

UTHSC DI-13-245

LN834429

LN834525

LN834613

C. sp.

UTHSC DI-13-265

LN834435

LN834531

LN834619

C. sp.

UTHSC DI-13-218

LN834418

LN834514

LN834602

C. sp.

UTHSC DI-13-210

LN834414

LN834510

LN834598

C. sphaerospermum

CBS 193.54

NR_111222

EU570261

EU570269

C. spinulosum

CBS 119907

NR_119660

EF679466

EF679542

C. subcinereum

UTHSC DI-13-257

NR_148193

LN834529

LN834617

C. subinflatum

UTHSC DI-13-189

LN834391

LN834487

LN834575

C. subinflatum

CBS 121630

NR_119661

EF679467

EF679543

C. submersum

FMR 17264

LR813144

LR813197

LR813195

C. subtilissimum

CBS 113754

NR_111273

EF679475

EF679551

C. subtilissimum

CBS 113753

EF679396

EF679474

EF679550

C. subuliforme

CBS 126500

NR_119854

HM148441

HM148686

C. subuliforme

CPC 15833

KT600453

KT600552

KT600650

C. succulentum

CBS 140466

LN834434

LN834530

LN834618

C. tenellum

CBS 121634

NR_119662

EF679479

EF679555

C. tenellum

CPC 22410

MF473280

MF473703

MF474130

C. tenellum

CPC 12051

EF679400

EF679478

EF679554

C. tenellum

CPC 22291

MF473279

MF473702

MF474129

C. tenellum

CPC 22290

MF473278

MF473701

MF474128

C. tenuissimum

CBS 125995

NR_119855

HM148442

HM148687

C. tianshanense

CGMCC3.18033

KX938381

KX938398

KX938364

C. tuberosum

UTHSC DI-13-219

LN834419

LN834515

LN834603

C. uredinicola

CPC 5390

AY251071

HM148467

HM148712

C. uwebrauniana

DTO 072-D8

MF473306

MF473729

MF474156

C. uwebraunianum

DTO 305-H9

MF473307

MF473730

MF474157

C. variabile

CBS 121635

NR_119663

EF679481

EF679557

C. varians

CBS 126362

NR_119856

HM148470

HM148715

C. velox

CBS 119417

DQ780361

JN906995

EF101388

C. verrucocladosporioides

CBS 126363

NR_111540

HM148472

HM148717

C. verruculosum

CGMCC3.18099

KX938388

KX938405

KX938371

C. verruculosum

CGMCC3.18100

KX938389

KX938406

KX938372

C. versiforme

CBS 140491

NR_152297

KT600515

KT600613

C. vicinum

CPC 22316

MF473311

MF473734

MF474161

C. vignae

CBS 121.25

HM148227

HM148473

HM148718

C. welwitschiicola

CPC 18648

NR_152308

KY646229

KY646226

C. westerdijkiae

CBS 113746

HM148061

HM148303

HM148548

C. wyomingense

CPC 22310

MF473315

MF473738

MF474165

C. xanthochromaticum

CBS 126364

HM148122

HM148366

HM148611

C. xantochromaticum

CBS 140691

LN834415

LN834511

LN834599

C. xylophilum

CBS 125997

NR_111541

HM148476

HM148721

C. xylophilum

CBS 113749

HM148228

HM148474

HM148719

C. yunnanensis sp. nov.*

KUN HKAS 121704*

OK338502*

OL825680*

OL466937*

Toxicocladosporium irritans

CBS 185.58

NR_152316

-

LT821375

Toxicocladosporium protearum

CBS 126499

NR_152321

-

LT821379

Taxon treatments

Cladosporium yunnanensis H.W. Shen, Y.X. Xu, H.Y. Su & Z.L. Luo, sp. nov.

Material    Download as CSV 
Holotype:
  1. scientificName:
    Cladosporium yunnanensis
    ; kingdom:
    Fungi
    ; phylum:
    Ascomycota
    ; class:
    Dothideomycetes
    ; order:
    Capnodiales
    ; family:
    Cladosporiaceae
    ; genus:
    Cladosporium
    ; locationRemarks:
    China, Yunnan Province, Dali, on diseased leaves of Paris polyphylla, 2 October 2020
    ; day:
    2020
    ; habitat:
    leaf spots of Paris polyphylla
    ; recordedBy:
    Yue-Xin Xu
    ; collectionID:
    1CL JD 5-1-4
    ; collectionCode:
    Y-23

Description

Asexual morph: hyphomycetous (Fig. 2). Mycelium superficial and immersed, composed of septate, branched, subhyaline, smooth-walled, 1–3 μm wide. Conidiophores macronematous, 127–190 × 4–6 μm (x̄ = 158.2 × 5.1 μm, n = 15), solitary or in small loose groups, erect to slightly flexuous, non-nodulose, sometimes subnodulose at the uppermost apex, unbranched, 0–6 septate, sometimes slightly constricted at septa, pale brown, smooth, sometimes somewhat irregularly rough-walled or verruculose. Conidiogenous cells terminal and intercalary, loci crowded at the apex forming clusters of pronounced scars, 1–2 conidiogenous loci formed at about the same level, loci often situated at lateral shoulders due to sympodial proliferation, loci 1–2 μm diam. Conidia solitary or in short unbranched chains, straight to slightly curved, cylindrical-oblong, 7–19 × 5–7 μm (x̄ = 13.2 × 5.7 μm, n = 30), 0–3 septate, sometimes slightly constricted at the septa, pale to pale medium olivaceous-brown. Sexual morph: Undetermined.

Figure 2.  

Cladosporium yunnanensis (KUN-HKAS 121704, holotype). a Colonies; b-c Conidiophores; d-g Conidiogenous cells with conidia; h-m Conidia; n Germinating conidium; o Culture on PDA from above and reverse. Scale bars: b-d = 20 μm; e-k = 15 μm; l = 30 μm.

Culture characteristics: Colonies on PDA attaining 25 mm diam. after 7 d, 45 mm diam. after 14 d and covering the whole Petridish after 30 d, dark green to olive green, velvety, furrowed; reverse dark green to black.

Material examined: China, Yunnan Province, Dali, on diseased leaves of Paris polyphylla, 2 October 2020, Y.X. Xu, Y-23. (KUN-HKAS 121704, holotype), ex-type living culture CGMCC 3.20622 = KUNCC 21-10712

Etymology

yunnanensis” refers to Yunnan Province, China, where the species was collected.

Distribution

China, Yunnan Province, Dali, on diseased leaves of Paris polyphylla

Notes

Based on the multi-locus phylogenetic analysis (Fig. 1), Cladosporium yunnanensis grouped in a well-supported clade, together with C. cladosporioides and C. magnoliigena. However, the genetic distance allows it to be considered a distinct species within the clade (Fig. 1). Morphologically, C. yunnanensis has much shorter conidiophores than C. cladosporioides (up to 190 μm vs. up to 350 μm), but longer than C. magnoliigena (up to 190 μm vs. up to 150 μm). Moreover, the new species differs from C. cladosporioides by the smaller conidiogenous cells (7–19 × 5–7 μm vs. 4–18 × 2–5 μm), but larger than C. magnoliigena (7–19 × 5–7 μm vs. 4–18 × 2–5 μm) (Bensch et al. 2012, Jayasiri et al. 2019). The BLAST analysis of TEF1-α and ACT shows that C. yunnanensis (KUN-HKAS 121704) is different from C. cladosporioides (CBS 112388) by 16 and 10 nucleotide differences, respectively and the comparison of TEF1-α between C. yunnanensis (KUN-HKAS 121704) and C. magnoliigena (CBS 140463) reveals 33 nucleotide differences.

Cladosporium paris H.W. Shen, Y.X. Xu, H.Y. Su & Z.L. Luo, sp. nov.

Material    Download as CSV 
Holotype:
  1. scientificName:
    Cladosporium paris
    ; kingdom:
    Fungi
    ; phylum:
    Ascomycota
    ; class:
    Dothideomycetes
    ; order:
    Capnodiales
    ; family:
    Cladosporiaceae
    ; genus:
    Cladosporium
    ; locationRemarks:
    China, Yunnan Province, Dali, on diseased leaves of Paris polyphylla
    ; year:
    2020
    ; habitat:
    leaf spots of Paris polyphylla
    ; recordedBy:
    Yue-Xin Xu
    ; collectionID:
    2CL JD 18-2-1
    ; collectionCode:
    Y-27

Description

Asexual morph: hyphomycetous (Fig. 3). Mycelium immersed and superficial, composed of septate, constricted at septa, unbranched, subhyaline, smooth hyphae, 2–6 μm wide. Conidiophores macronematous, 209–285 × 5–8 μm (x̄ = 246.9 × 6.5 μm, n = 15), solitary or in small fascicles, erect to slightly flexuous, sometimes slightly geniculate, non-nodulose, sometimes subnodulose at the uppermost apex, unbranched, 0–6 septate, sometimes slightly constricted at septa, pale to olivaceous-brown, smooth or almost so. Conidiogenous cells cylindrical, sometimes geniculate-sinuous, proliferation of sympodia with up to 5 conidiogenous loci, often crowded at the apex. Conidia 13–21 × 7–12 μm (x̄ = 17 × 9.7 μm, n = 30), solitary or catenate, usually in simple chains, broadly ellipsoid-ovoid, rather pale, pale olivaceous or olivaceous-brown, verruculose, ends usually broadly rounded. Sexual morph: Undetermined.

Figure 3.  

Cladosporium paris (KUN-HKAS 121701, holotype). a Colonies on leaves; b,c Conidiophores; d Conidiophore with conidium; e,f Conidiogenous cells with conidia; g-l Conidia; m Germinating conidium; n,o Culture on PDA from above and reverse. Scale bars: b-d = 50 μm; e,f = 30 μm; g-m = 20 μm.

Culture characteristics: Colonies on PDA attaining 21 mm diam. after 7 d, 40 mm diam. after 14 d and covering the whole Petridish after 30 d, radially folded, furrowed, margin irregularly undulate; reverse olivaceous grey.

Material examined: China, Yunnan Province, Dali, on diseased leaves of Paris polyphylla, 16 October 2020, Y.X. Xu, Y-27. (KUN-HKAS 121701, holotype), ex-type living culture CGMCC 3.20623 = KUNCC 21-10713.

Etymology

paris” refers to the host genus, Paris.

Distribution

China, Yunnan Province, Dali, on diseased leaves of Paris polyphylla

Notes

Phylogenetic analysis showed that Cladosporium paris is closely related to C. floccosum (Fig. 1). Morphologically, our new isolate is distinguished from C. floccosum by its longer conidiophores (up to 285 μm vs. up to 100 μm) and larger conidiogenous cells (13–21 × 7–12 μm vs. 8–15 × 6–8.5 μm). In addition, conidia of C. paris are 0–3 septate, while C. floccosum are 0–1 septate (Sandoval-Denis et al. 2016). A comparison of the TEF1-α and ACT between C. paris (KUN-HKAS 121701) and C. floccosum (CBS 140463) reveals 3 and 16 nucleotide differences, respectively, which indicates that they are distinct taxa.

Analysis

Phylogenetic analysis

The combined ITS, TEF1-α and ACT dataset consisted of 161 sequences representing all genera of the Cladosporium with Toxicocladosporium irritans (CBS 185.58) and T. protearum (CBS 126499) as outgroup taxa. The best scoring RaxML tree with the final ML optimisation likelihood value of -24601.202740 is shown here (Fig. 1). The alignment comprised 1297 characters including gaps. The matrix had 775 distinct alignment patterns, with 15.38% undetermined characters or gaps. Estimated base frequencies were as follows: A = 0.228337, C = 0.293636, G = 0.250484, T = 0.227544; substitution rates AC = 1.726214, AG = 3.618770, AT = 1.752951, CG = 1.098108, CT = 5.802327, GT = 1.000000; Tree-Length = 7.357731.

Phylogenetic analyses of combined ITS, TEF1-α and ACT sequence data showed that the two new isolates of Cladosporium yunnanensis (KUN-HKAS 121704) and C. paris (KUN-HKAS 121701) grouped with members of Cladosporium. Cladosporium yunnanensis (KUN-HKAS 121704) clustered with C. cladosporiordes (CBS 112388 and CBS 113738) and C. magnoliigena (MFLUCC 18-1559), but in an independent lineage with significant bootstrap (86 ML/1.00 PP). Cladosporium paris (KUN-HKAS 121701) formed a distinct lineage and sister to C. floccosum (CBS 140463) and basal to the genus with highly-supported value (94 ML/0.98 PP).

Discussion

In our study, based on the typical morphological features (Schubert et al. 2007, Zalar et al. 2007, Dugan et al. 2008, Bensch et al. 2010), Cladosporium yunnanensis and C. paris belong to the C. cladosporioides and C. herbarum species complex, respectively. The ITS sequences of the two new species are identical under the common barcode for fungi as previously reported studies for many other Cladosporium species (Bensch et al. 2010, Bensch et al. 2012, Marin-Felix et al. 2017). Therefore, multi-gene phylogenetic analysis (ITS, TEF1-α and ACT) can further prove the taxonomy of the two species in Cladosporium, which is consistent with the result by morphological features.

Cladosporium species are found as the dominant fungal genera in indoor and outdoor environments and are also important as saprobes and endophytes which have been screened from grains, fruits and chilled meat (Fradkin et al. 1987, Bullerman 2003, Hassan et al. 2021). However, Cladosporium yunnanensis and C. paris have been isolated from leaves of Paris polyphylla in Yunnan Province, China for the first time. Studies indicate that investigation on new hosts for fungi diversity would lead to the discovery of new fungal species and expand species resources (Hyde et al. 2018, Hyde et al. 2020). Certain Cladosporium species have been reported as producers of mycotoxin and to cause fungal allergies, particularly rhinitis and asthma. (Horner et al. 1995, Kurup 2003, Matheson et al. 2005, Simon-Nobbe et al. 2008, Mercier et al. 2013, Alwatban et al. 2014 , Segers et al. 2015). Both new species are isolated from diseased spots on plant leaves and many species of this genus are reported as plant pathogens, so they also have the potential to cause plant diseases. To determine whether these fungi are plant pathogens or have long-term adverse reactions on human health, pathogenicity determination and secondary metabolites of Cladosporium can be the focus of our future research.

Acknowledgements

This study is supported by the National Natural Science Foundation of China (Project ID: 31970021) and the Fungal Diversity Conservation and Utilization Innovation Team of Dali University (ZKLX2019213). Yue-Xin Xu thanks Rui Gu and Zheng-Quan Zhang for the help on collecting samples. We are grateful to and thank Ga-Heng Li and Hong-Yan Liu for their help on isolation, morphological examination, DNA extraction and PCR amplification.

References