Biodiversity Data Journal : Taxonomy & Inventories
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Taxonomy & Inventories
Additions to Neopestalotiopsis (Amphisphaeriales, Sporocadaceae) fungi: two new species and one new host record from China
expand article infoYu-Ke He‡,§,|, Qi Yang§, Ya-Ru Sun, Xiang-Yu Zeng§, Ruvishika S. Jayawardena, Kevin D. Hyde, Yong Wang§
‡ Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand
§ Department of Plant Pathology, Agriculture College, Guizhou University, Guiyang, 550025, China
| School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
Open Access

Abstract

Background

In this study, three Neopestalotiopsis taxa were identified, associated with leaves of Zingiber officinale, Elaeagnus pungens and Salacca zalacca.

New information

Based on morphology and multi–gene analyses of the internal transcribed spacer (ITS), beta-tubulin (TUB2) and translation elongation factor 1–alpha (TEF1), the five strains of Neopestalotiopsis represent two novel and one known species. They are introduced with descriptions, illustrations and notes herein.

Keywords

morphology, Pestalotiod, phylogeny, taxonomy, Zingiberaceae

Introduction

Pestalotiod fungi distribute commonly as saprobes, pathogens and endophytes, which can cause a variety of plant diseases (Huanaluek et al. 2021). Most of this fungal group lack sexual morphs and only 13 species can reproduce the sexual stage (Maharachchikumbura et al. 2011, Nozawa et al. 2017). Pestalotioid fungi are placed in Sporocadaceae (Amphisphaeriales) (Jayawardena et al. 2019, Wijayawardene et al. 2020). Based on the conidia pigment colour, conidiophores and molecular phylogeny, Neopestalotiopsis was segregated from the old Pestalotiopsis genus (Maharachchikumbura et al. 2014a). Neopestalotiopsis, Pestalotiopsis and Pseudopestalotiopsis differ from each other by the colour of the conidial three median cells (Maharachchikumbura et al. 2014a). However, the delimitation of species, only through phenotypic characteristics, is difficult (Maharachchikumbura et al. 2016), thus morphological and phylogenetic approaches should be combined to determine the new taxa. Seventy-two species of Neopestalotiopsis are recorded in Index Fungorum (2022), but only forty-one species of Neopestalotiopsis are accepted, based on molecular data (Jayawardena et al. 2019). In this paper, two new species and a new Chinese record of Neopestalotiopsis found on Zingiber officinale Rosc., Elaeagnus pungens Thunb. and Salacca zalacca (Gaertn.) Voss. in Zingiberaceae are described and illustrated.

Materials and methods

Sample collection and fungal strains isolation

Diseased fresh leaf samples were collected from Z. officinale, E. pungens and S. zalacca in Hainan Province, China in 2020. Fresh specimens were taken to the laboratory in paper envelopes. The strains were obtained using single spore isolation, following Senanayake (2020). Once the single spore germinated, it was transferred to potato dextrose agar (PDA) and cultured at room temperature (24°C).

Morphological description

Microscopic slides were prepared with lactic acid and examined using an Axioscope 5 with Axiocam 208 colour (ZEISS, Oberkohen, Germany) at 1000× magnification. The morphology of fungi was photographed by the camera. Photo–plates were made by Adobe Photoshop CS6, with the Tarosoft (R) Image Frame Work programme being used for measurements. Herbaria materials were deposited in the Herbarium of the Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). Cultures were deposited to the Culture Collection of the Department of Plant Pathology, Agriculture College, Guizhou University (GUCC) (Table 1). The taxonomic information of new species was submitted to MycoBank (http://www.mycobank.org).

Table 1.

GenBank accession numbers of molecular phylogenetic analyses. Ex–type isolates are labelled with superscript T. The new isolates are in bold.

Taxa

Strain number

Host

Country

ITS

TUB2

TEF1

Reference

Neopestalotiopsis acrostichi

MFLUCC 17–1754T

Acrostichum aureum

Thailand

MK764272

MK764338

MK764316

Norphanphoun et al. 2019

N. acrostichi

MFLUCC 17–1755

Acrostichum aureum

Thailand

MK764273

MK764339

MK764317

Norphanphoun et al. 2019

N. alpapicalis

MFLUCC 17–2544T

Rhyzophora mucronata

Thailand

MK357772

MK463545

MK463547

Kumar et al. 2019

N. aotearoa

CBS 367.54T

Canvas

New Zealand

KM199369

KM199454

KM199526

Maharachchikumbura et al. 2014b

N. asiatica

MFLUCC 12–0286T

unidentified tree

China

JX398983

JX399018

JX399049

Maharachchikumbura et al. 2012

N. australis

CBS 114159T

Telopea sp.

Australia

KM199348

KM199432

KM199537

Maharachchikumbura et al. 2014b

N. brachiata

MFLUCC 17–1555T

Rhizophora apiculata

Thailand

MK764274

MK764340

MK764318

Norphanphoun et al. 2019

N. brasiliensis

COAD 2166

Psidium guajava

Brazil

MG686469

MG692400

MG692402

Bezerra et al. 2018

N. chiangmaiensis

MFLUCC 18–0113

Pandanaceae

Thailand

MH412725

MH388404

Tibpromma et al. 2018

N. chrysea

MFLUCC 12–0261T

dead leaves

China

JX398985

JX399020

JX399051

Maharachchikumbura et al. 2012

N. clavispora

MFLUCC 12–0281T

Magnolia sp.

China

JX398979

JX399014

JX399045

Maharachchikumbura et al. 2012

N. cocoes

MFLUCC 15–0152T

Cocos nucifera

Thailand

NR156312

KX789689

Norphanphoun et al. 2019

N. coffea–arabicae

HGUP4015T

Coffea arabica

China

KF412647

KF412641

KF412644

Song et al. 2013

N. cubana

CBS 600.96T

leaf litter

Cuba

KM199347

KM199438

KM199521

Maharachchikumbura et al. 2014b

N. dendrobii

MFLUCC 14–0106

Dendrobium cariniferum

Thailand

MK993571

MK975835

MK975829

Ma et al. 2019

N. egyptiaca

CBS 140162T

Mangifera indica

Egypt

KP943747

KP943746

KP943748

Crous et al. 2015

N. elaeagni

HGUP10002

HGUP10004

Elaeagnus pungens,

Elaeagnus pungens

China

China

MW930716

ON597079

MZ683391

ON595537

MZ203452

ON595535

This study

This study

N. ellipsospora

MFLUCC 12–0283T

dead plant material

China

JX398980

JX399016

JX399047

Maharachchikumbura et al. 2012

N. eucalypticola

CBS 264.37T

Eucalyptus globulus

KM199376

KM199431

KM199551

Maharachchikumbura et al. 2014b

N. foedans

CGMCC 3.9123T

unidentified mangrove plant

China

JX398987

JX399022

JX399053

Maharachchikumbura et al. 2012

N. formicidarum

CBS 362.72T

dead ant

Ghana

KM199358

KM199455

KM199517

Maharachchikumbura et al. 2014b

N. hadrolaeliae

COAD 2637T

Hadrolaelia jongheana

Brazil

MK454709

MK465120

MK465122

Freitas et al. 2019

N. haikouensis

SAUCC212271T

Ilexchinensis

China

OK087294

OK104870

OK104877

Zhang et al. 2022

N. honoluluana

N. hydeana

CBS 114495T

MFLUCC 20–0132

Telopea sp.

Artocarpus heterophyllus

USA

Thailand

KM199364

MW266069

KM199457

MW251119

KM199548

MW251129

Maharachchikumbura et al. 2014b

Huanaluek et al. 2021

N. iranensis

CBS 137768T

Fragaria × ananassa

Iran

KM074048

KM074057

KM074051

Song et al. 2013

N. javaensis

CBS 257.31T

Cocos nucifera

Indonesia

KM199357

KM199437

KM199543

Maharachchikumbura et al. 2014b

N. keteleeria

MFLUCC 13–0915T

Keteleeria pubescens

China

KJ503820

KJ503821

KJ503822

Song et al. 2013

N. magna

MFLUCC 12–0652T

Pteridium sp.

France

KF582795

KF582793

KF582791

Maharachchikumbura et al. 2012

N. macadamiae

BRIP 63740a

Macadamia sp.

Australia

KX186617

KX186656

KX186628

Akinsanmi et al. 2017

N. mesopotamica

CBS 336.86T

Pinus brutia

Iraq

KM199362

KM199441

KM199555

Maharachchikumbura et al. 2014b

N. musae

MFLUCC 15–0776T

Musa sp.

Thailand

NR156311

KX789686

KX789685

Li et al. 2016

N. natalensis

CBS 138.41T

Acacia mollissima

South Africa

NR156288

KM199466

KM199552

Maharachchikumbura et al. 2014b

N. pandanicola

KUMCC 17–0175

Pandanaceae

China

MH412720

MH388389

Tibpromma et al. 2018

N. pernambucana

GS 2014 RV01T

Vismia guianensis

Brazil

KJ792466

Maharachchikumbura et al. 2014b

N. petila

MFLUCC 17–1738T

Rhizophora mucronata

Thailand

MK764275

MK764341

MK764319

Norphanphoun et al. 2019

N. petila

MFLUCC 17–1737

Rhizophora mucronata

Thailand

MK764276

MK764342

MK764320

Norphanphoun et al. 2019

N. phangngaensis

MFLUCC 18–0119

Pandanaceae

Thailand

MH388354

MH412721

MH388390

Tibpromma et al. 2018

N. piceana

CBS 394.48T

Picea sp.

UK

KM199368

KM199453

KM199527

Maharachchikumbura et al. 2014b

N. piceana

CBS 254.32

Cocos nucifera

Indonesia

KM199372

KM199452

KM199529

Maharachchikumbura et al. 2014b

N. piceana

CBS 225.3

Mangifera indica

KM199371

KM199451

KM199535

Maharachchikumbura et al. 2014b

N. protearum

CBS 114178T

Leucospermum cuneiforme cv. Sunbird

Zimbabwe

JN712498

KM199463

KM199542

Maharachchikumbura et al. 2014b

N. protearum

CMM1357

KY549597

KY549632

KY549594

Maharachchikumbura et al. 2014b

N. rhizophorae

MFLUCC 17–1550T

Rhizophora mucronata

Thailand

MK764277

MK764343

MK764321

Norphanphoun et al. 2019

N. rhizophorae

MFLUCC 17–1551

Rhizophora mucronata

Thailand

MK764278

MK764344

MK764322

Norphanphoun et al. 2019

N. rosae

CBS 101057T

Rosa sp.

New Zealand

KM199359

KM199429

KM199523

Maharachchikumbura et al. 2014b

N. rosicola

CFCC 51992T

Rosa chinensis

China

KY885239

KY885245

KY885243

Jiang et al. 2018

N. rosicola

CFCC 51993

Rosa chinensis

China

KY885240

KY885246

KY885244

Jiang et al. 2018

N. samarangensis

MFLUCC 12–0233T

Syzygium samarangense

Thailand

JQ968609

JQ968610

JQ968611

Maharachchikumbura et al. 2012

N. samarangensis

HGUP10003

Salacca zalacca

China

MW930717

MZ683392

MZ540914

This study

N. saprophytica

MFLUCC 12–0282T

Magnolia sp.

China

KM199345

KM199433

KM199538

Maharachchikumbura et al. 2012

N. sichuanensis

CFCC 54338 = SM15-1T

Castanea mollissima

China

MW166231

MW218524

MW199750

Jiang et al. 2021

N. sonneratae

MFLUCC 17–1745T

Sonneronata alba

Thailand

MK764279

MK764345

MK764323

Norphanphoun et al. 2019

N. sonneratae

MFLUCC 17–1744

Sonneronata alba

Thailand

MK764280

MK764346

MK764324

Norphanphoun et al. 2019

N. steyaertii

IMI 192475T

Eucalyptus viminalis

Australia

KF582796

KF582794

KF582792

Maharachchikumbura et al. 2012

N. surinamensis

CBS 450.74T

soil under Elaeis guineensis

Suriname

KM199351

KM199465

KM199518

Maharachchikumbura et al. 2014b

N. surinamensis

CBS 111494

Protea eximia

Zimbabwe

KM199462

KM199530

Maharachchikumbura et al. 2014b

N. thailandica

MFLUCC 17–1730T

Rhizophora mucronata

Thailand

MK764281

MK764347

MK764325

Norphanphoun et al. 2019

N. thailandica

MFLUCC 17–1731

Rhizophora mucronata

Thailand

MK764282

MK764348

MK764326

Norphanphoun et al. 2019

N. umbrinospora

MFLUCC 12–0285T

unidentified plant

China

JX398984

JX399019

JX399050

Maharachchikumbura et al. 2012

N. vitis

MFLUCC 15–1265T

Vitis vinifera

China

KU140694

KU140685

KU140676

Jayawardena et al. 2016

N. zimbabwana

CBS 111495T

Leucospermum cunciforme

Zimbabwe

JX556231

KM199456

KM199545

Norphanphoun et al. 2019

N. zingiberis

HGUP10001

HGUP10005

Zingiber officinale,

Zingiber officinale

China

China

MW930715

ON597078

MZ683390

ON595538

MZ683389

ON595536

This study

This study

Pestalotiopsis diversiseta

MFLUCC 12–0287T

dead plant material

China

NR120187

JX399040

JX399073

Maharachchikumbura et al. 2012

DNA extraction, PCR reaction and sequencing

The fresh mycelia were scraped off with a sterilised scalpel when colonies reached 80 mm in diameter. Genomic DNA was extracted using the Fungus Genomic DNA Extraction Kit (Biomiga GD2416), following the manufacturer’s instructions. Polymerase chain reactions (PCR) were performed in a 20 μl reaction volume: 1 μl of DNA template, 1 μl of each forward and reverse primers, 10 μl of 2× Bench TopTMTaq Master Mix and 7 μl double–distilled water (ddH2O). The partial internal transcribed spacer (ITS) rDNA was amplified with the primer pair ITS4/ITS5 (White et al. 1990), TEF1 was amplified with primers EF1–728f/EF2 (O’Donnell et al. 1998, Carbone and Kohn 1999) and the TUB2 was amplified with primers T1/Bt2b (Glass and Donaldson 1995, O'Donnell and Cigelnik 1997). PCR products were sequenced by using appropriate primers for amplification reactions by SinoGenoMax, Beijing. The obtained DNA sequences were submitted to GenBank to obtain their accession numbers (Table 1). DNA base differences on three loci between our strains and ex-type or representative strains of relative Neopestalotiopsis taxa are shown in Table 2.

Table 2.

The differences of DNA bases on different gene regions between our strains. Our strains are in bold.

Species

Strain number

ITS (1–568)

TEF1 (569–1520)

TUB2 (1521–1972)

Neopestalotiopsis elaeagni *

GUCC 21002

0

0

0

Neopestalotiopsis chrysea

MFLUCC 12–0261

1

27

2

Neopestalotiopsis umbrinospora

MFLUCC 12–0285

1

25

4

Neopestalotiopsis asiatica

MFLUCC 12–0286

1

19

5

Neopestalotiopsis zingiberis *

GUCC 21001

0

0

0

Neopestalotiopsis magna

MFLUCC 12–0652

16

3

35

Neopestalotiopsis samarangensis *

GUCC 21003

0

0

0

Neopestalotiopsis samarangensis

MFLUCC 12–0233

1

9

2

Phylogenetic analyses

The phylogeny was constructed by analyses from sequences of ITS, TEF1 and TUB2 sequence data. The fungal sequences were aligned by using the online version of MAFFT v. 7.307 (Katoh and Standley 2016) and edited by using the BioEdit programme (Hall 1999), using the SequenceMatrix 1.7.8 (Vaidya et al. 2011) to multi-source data merging. Ambiguous regions were excluded from analyses using AliView (Larsson 2014), gaps were treated as missing data and optimised manually with Pestalotiopsis diversiseta Maharachch. & K.D. Hyde (MFLUCC 12–0287) as the outgroup (Table 2). Combined analyses of ITS, TUB2 and TEF1 sequence data were performed. Phylogenetic analyses were constructed by Maximum Likelihood (ML), Maximum Parsimony (MP) and Bayesian Posterior Probability (BYPP) methods; they were carried out as detailed in Dissanayake et al. (2020).

Maximum Parsimony analysis was performed with PAUP v. 4.0b10 (Swofford 2002), 1000 bootstrap replicates, using heuristic search on random stepwise addition and tree bisection reconnection (TBR). Maxtrees was set to 5000. For each tree generated, consistency index (CI), retention index (RI), tree length (TL), rescaled consistency index (RC) and homoplasy index (HI) were calculated.

The Maximum Likelihood analysis was performed using the CIPRES Science Gateway web server RAxML–HPC BlackBox (Stamatakis 2014) and 1000 rapid bootstrap replicates were run with the GTR+GAMMA model of nucleotide evolution.

Bayesian Posterior Probability analyses were performed by MrModeltest v.2.3 (Nylander et al. 2004) and MrBayes 3.2 (Ronquist et al. 2012) with the Markov Chain Monte Carlo (MCMC) method. The GTR model was selected as the best model for the TUB2. The MCMC runs were launched with four chains starting from random tree topology between 1,000,000–5,000,000 generations and sampling every 100 generations. The first 5000 samples were excluded as burn–in.

Genealogical Concordance Phylogenetic Species Recognition (GCPSR) analysis

The phi-test incorporated in the SplitsTree software (Huson 1998, Huson and Bryant 2006) was used to test signals of recombination as described by Quaedvlieg et al. (2014). The evolutionary independence was revealed, using the GCPSR concept for the Neopestalotiopsis dataset with relevant taxa. In the pairwise homoplasy index (PHI), if a value below 0.05 was obtained, it provided evidence for the presence of significant recombination within a dataset. The test is proven to be a robust calculation and no previous knowledge about population history, recombination rate, mutation rate and rate heterogeneity across sites is necessary (Bruen et al. 2006).

Taxon treatments

Neopestalotiopsis elaeagni Y.K. He & Yong Wang bis, sp. nov.

Material   Download as CSV 
Holotype:
  1. scientificName:
    Neopestalotiopsis elaeagni
    ; order:
    Amphisphaeriales
    ; family:
    Sporocadaceae
    ; genus:
    Neopestalotiopsis
    ; country:
    China
    ; stateProvince:
    Hainan
    ; locality:
    Haikou City, Leiqiong Haikou Volcano Cluster World Geopark
    ; verbatimCoordinates:
    109°39’ E, 20°13’ N
    ; recordedBy:
    Yu-ke He
    ; identifiedBy:
    Yu-ke He
    ; dateIdentified:
    2020
    ; collectionID:
    HGUP 10002
    ; occurrenceID:
    GUCC 21002

Description

Associated with the leaf blight of Elaeagnus pungens Thunb. Disease symptom: A large irregular scab on the leaves of E. pungens, light brown, edges dark brown to reddish-brown. The boundary of the scab was not obvious. There were many black, small and punctuate conidia on the scab. Sexual morph: not observed. Asexual morph (Fig. 2): Conidiomata dark, punctiform, scattered on the host scab, 110‒300 μm (n = 40), releasing black conidia. Conidiophores discrete to lageniform, hyaline, smooth– and thin–walled, 8‒13 × 2‒3 μm. Conidia 19‒25 × 4.5‒7 μm, fusiform to clavate, straight to slightly curved, 4–septate; basal cell obconic with a truncate base, hyaline or pale brown, smooth– and thin–walled, 3.5‒5 μm long; three median cells 12‒15 μm long, versicoloured, dark brown to light brown, septa and periclinal walls darker than the rest of the cell; second cell brown, 3.5‒5.5 μm long; third cell brown, 3‒5.5 μm long; fourth cell light brown 3.5‒5 μm long; apical cell 3‒5.5 μm long, hyaline, conic to acute, with 1–3 tubular appendages inserted at different loci, but in the same crest at the apex of the apical cell, unbranched, flexuous, 13‒30 μm long; most conidia have tubular appendages or single appendage in the basal cell, hyaline, unbranched, centric, 5‒7.5 μm long.

Figure 1.  

Consensus phylogram of 1,000 trees resulting from an RAxML analysis of the (ITS+TUB2+TEF1) alignment of the analysed Neopestalotiopsis sequences. Pestalotiopsis diversiseta (MFLUCC 12–0287) is used as the outgroup taxon. The MP bootstrap values ≥ 50%, ML bootstraps ≥ 70% and Bayesian posterior probabilities ≥ 0.90 (MPBS/MLBS/PPBY) are given at the nodes. New collections obtained in this study are in red.

Figure 2.  

Neopestalotiopsis elaeagni (Specimen code: HGUP 10002). a–c Appearance on host surface; d Colony top view and reverse view; e–f Conidiomata on PDA; g Conidiogenous cells; h–j Conidia. Scale bars: a–b = 10 mm, c = 1 mm, e–f = 500 μm, g–j = 20 μm.

Culture characteristics: Colonies on PDA medium reaching 5‒5.5 cm diam. After 10 d at 24℃, the mycelium white, cottony, odourless, soft, without exudate and round with regular edges. Under the surface of hyphal layer, releasing conidia in a black, slimy mass. The reverse side of the culture dish is smooth and light yellow.

Etymology

elaeagni, in reference to the host genus (Elaeagnus) from which it was isolated.

Notes

Phylogenetically, the new species is sister to Neopestalotiopsis chrysea (MFLUCC 12–0261), Neopestalotiopsis umbrinospora (MFLUCC 12–0285) and Neopestalotiopsis asiatica (MFLUCC 12–0286). However, N. elaeagni differed from N. chrysea by having shorter apical appendage (N. elaeagni: 13‒30 μm vs. N. chrysea: 22‒30 μm), differed from N. umbrinospora by having smaller conidia and shorter apical appendage (Conidia: N. elaeagni: 19‒25 × 4.5‒7 μm vs. N. umbrinospora: 19‒29 × 6‒8 μm; apical appendage length: N. elaeagni: 13‒30 μm vs. N. umbrinospora: 22‒35 μm) and differed from N. asiatica by having shorter apical appendage (N. elaeagni: 13‒30 μm vs. N. asiatica: 20‒30 μm) (Maharachchikumbura et al. 2012) (Table 3). According to the PHI analysis, our dataset showed a 1.0 value indicating no significant genetic recombination between our newly-introduced Neopestalotiopsis strains with other related taxa. Combined with morphology, phylogenetic analysis and PHI test results and we propose N. elaeagni as a novel species.

Table 3.

Comparison of conidia of Neopestalotiopsis species related to this study. Our strains are in bold.

Species

Strain

Conidial size (μm)

Apical appendages

Basal appendage

Length (μm)

Number

Length (μm)

N. chrysea

MFLUCC 12–0261

20‒24 × 5.5‒7

3

22‒30

3–6

N. umbrinospora

MFLUCC 12–0285

19‒29 × 6‒8

1–3

22–35

5–7

N. asiatica

MFLUCC 12–0286

20‒26 × 5‒7

2–4

20–30

4–8

N. elaeagni *

GUCC 21002

19‒25 × 4.5‒7

1–3

13‒30

5‒7.5

GUCC 21006

N. magna

MFLUCC 12–0652

42‒46 × 9.5–12

2–4

16–26

11–15

N. zingiber *

GUCC 21001

21‒31 × 6‒9.5

1‒3

12‒15

0‒6

GUCC 21007

Neopestalotiopsis zingiberis Y.K. He & Yong Wang bis, sp. nov.

Material   Download as CSV 
Holotype:
  1. scientificName:
    Neopestalotiopsis zingiberis
    ; order:
    Amphisphaeriales
    ; family:
    Sporocadaceae
    ; genus:
    Neopestalotiopsis
    ; country:
    China
    ; stateProvince:
    Hainan
    ; locality:
    Haikou City, Wuzhishan Nature Reserve
    ; verbatimCoordinates:
    109°32’ E, 18°48’ N
    ; recordedBy:
    Yu-ke He
    ; identifiedBy:
    Yu-ke He
    ; dateIdentified:
    2020
    ; collectionID:
    HGUP 10001
    ; occurrenceID:
    GUCC 21001

Description

Associated with leaf blight of Zingiber officinale Rosc. Disease symptom: A long oval to irregular, ring-like scab, light brown, edge reddish-brown, slightly sunken on adaxial surface. The boundary of the scab is obvious, with a narrow yellow halo around the scab. There are many black, small and punctuate conidia on the scab. Sexual state: unknown. Asexual morph (Fig. 3): Conidiomata is dark, oblate, scattered on the host scab, 104‒202 μm. Conidiophores discrete to lageniform, hyaline, smooth– and thin–walled, annellidicae, 12‒25 × 3‒6 μm (n = 40). Conidia 21‒31 × 6‒9.5 μm, fusiform to clavate, straight to slightly, 4–septate; basal cell obconic with a truncate base, hyaline or pale brown, smooth– and thin–walled, 3‒6 μm long; three median cells 15‒19 μm long, septa and periclinal walls darker than rest of the cell, versicoloured, wall rugose; second cell brown, 4‒6 μm long; third cell brown, 4‒7 μm long; fourth cell light brown 4‒6 μm long; apical cell 3‒5 μm long, hyaline, conic to acute, with 1‒3 tubular appendages insert at different loci, but in the same crest at the apex of the apical cell, unbranched, flexuous, 12‒15 μm long; most spores have no tubular appendages or single appendage, unbranched, centric, 0‒6 μm long.

Figure 3.  

Neopestalotiopsis zingiberis (Specimen code: HGUP 10001). a–b Appearance on host surface; c Colony top view and reverse view; d–e Mycelium; f Conidiogenous cells; g–i Conidia. Scale bars: a =10 mm, b = 1 mm, d–e = 200 μm, f–i = 20 μm.

Culture characteristics: Colonies on PDA medium reaching 8‒9 cm diam. after 15 d at 24℃, the mycelium is yellowish or white, soft and round with irregular edges. Under the surface of hyphal layer, releasing conidia in a black, slimy mass. Dark brown pigment is deposited on the bottom of the Petri dish.

Etymology

zingiberis, in reference to the host genus (Zingiber) from which it was isolated.

Notes

Neopestalotiopsis zingiberis (GUCC 21001) formed a distinct clade and sistered to Neopestalotiopsis magna (MFLUCC 12–0652) (Fig. 1). Morphologically, conidia of N. zingiberis (21‒31 × 6‒9.5 μm) are smaller than N. magna (42‒46 × 9.5–12 μm) and also differed by having branched, flexuous apical tubular appendages (Maharachchikumbura et al. 2014a) (Table 3). Thus, we propose N. zingiberis as a novel taxon.

Neopestalotiopsis samarangensis (Maharachch. & K.D. Hyde)

Nomenclature

Neopestalotiopsis samarangensis (Maharachch. & K.D. Hyde) Maharachch., K.D. Hyde & Crous in Maharachchikumbura, Hyde, Groenewald, Xu & Crous, Stud. Mycol. 79: 147 (2014)

Material   Download as CSV 
  1. scientificName:
    Neopestalotiopsis samarangensis
    ; order:
    Amphisphaeriales
    ; family:
    Sporocadaceae
    ; genus:
    Neopestalotiopsis
    ; country:
    China
    ; stateProvince:
    Hainan
    ; locality:
    Haikou City, Xinglong Tropical Botanical Garden
    ; verbatimCoordinates:
    110°11’ E, 18°44’ N
    ; recordedBy:
    Yu-ke He
    ; identifiedBy:
    Yu-ke He
    ; dateIdentified:
    2020
    ; collectionID:
    HGUP 10003
    ; occurrenceID:
    GUCC 21003

Description

Associated with leaf spots of Salacca zalacca (Gaertn.) Voss. Disease symptom: a small oval scab, ring-like, the inner ring is light brown to dark brown and the outer ring is light brown, the boundary is obvious, dark brown. A few black, small, isolated and punctuate conidia irregularly distributed on the scab. Sexual state: unknown. Asexual morph (Fig. 4): Conidiomata is dark, oblate, scattered on the host scab, 70‒180 μm. Conidiophores discrete to lageniform or globular, hyaline, smooth– and thin–walled, simple and short. Conidia 18‒23 × 6‒7.5 μm, fusiform to clavate, straight to slightly, 4–septate; basal cell obconic with a truncate base, hyaline or pale brown, smooth– and thin–walled, 3.5‒5 μm long; three median cells 12.5‒15 μm long, light brown or hyaline, septa and periclinal walls darker than rest of the cell, wall rugose; second cell 4.5‒5.5 μm long; third cell 4‒5.5 μm long; fourth cell 5‒6 μm long; apical cell 3‒4.5 μm long, hyaline, conic to acute, with 1–2 tubular appendages inserted at different loci, but in the same crest at the apex of the apical cell, unbranched, flexuous, 12‒20 μm long. The spores have tubular appendages or single appendage, unbranched, centric, 3.5‒6 μm long.

Figure 4.  

Neopestalotiopsis samarangensis (Specimen code: HGUP 10003). a–b Appearance on host surface; c Colony top view and reverse view; d–e Conidiomata on PDA; f Conidiogenous cells; g–j Conidia. Scale bars: a = 10 mm, b = 1 mm, d–e = 500 μm, f–j = 20 μm.

Culture characteristics: Colonies on PDA medium reaching 4.5–5 cm diam. After 9 d at 24℃, odourless, without exudates, with black dots in the centre (conidiomata), the mycelium is white, soft and round with regular edges; reverse yellow to white. Under the surface of hyphal layer, releasing many conidia in a black, slimy mass.

Notes

Phylogenetically, isolated GUCC 21003 clustered with the ex-type strain of N. samarangensis (MFLUCC 12‒0233). In morphology, our strain is very similar to N. samarangensis (Maharachchikumbura et al. 2013). A comparison of DNA bases (Table 2) demonstrated that the differences between these two strains are minute. Therefore, we concluded that they are the same species, but occurring on different hosts (N. samarangensis GUCC 21003 on leaf of Salacca zalacca vs. N. samarangensis MFLUCC 12-0233 on Syzygium samarangense).

Analysis

Phylogenetic analysis

The final concatenated alignment comprised 1809 characters including 65 taxa. The combined dataset contained 1352 constant, 253 parsimony uninformative and 204 parsimony informative characters. According to different optimisation criteria, the tree topology was similar, so the individual datasets were congruent and could be combined. There were two equally parsimonious trees from MP analysis and we chose the best one to show the topology (Fig. 1) (TL = 855, CI = 0.680, RI = 0.651, RC = 0.442, HI = 0.320). Neopestalotiopsis elaeagni (GUCC 21002) is a sister taxon of N. chrysea and N. umbrinospora with high support (MP-BS = 90%/96% ML-BS = 96% BYPP = 0.98). Neopestalotiopsis zingiberis (GUCC 21001) is a sister taxon of N. magna (MFLUCC 12–0652) only with high BI support (PP = 0.96). GUCC 21003 was closer to the ex-type strain of N. samarangensis (MFLUCC 12-0233T) with high BI support (PP = 0.98). The base-pair differences amongst the three new collections are listed in Table 2. It showed that N. elaeagni (GUCC 21002), N. chrysea (MFLUCC 12-0261), N. umbrinospora (MFLUCC 12-0285) and N. asiatica (MFLUCC 12-0286), differ by only one character difference in the ITS region, 19-27 characters in TEF1 and 2-5 characters in TUB2. Between N. zingiberis (GUCC 21001) and N. magna (MFLUCC 12-0652), there were 16 character differences in the ITS region, three characters in TEF1 and 35 characters in TUB2. Between N. samarangensis (GUCC 21003) and N. samarangensis (MFLUCC 12-0233), there was only one character difference in the ITS, nine characters in TEF1 and two characters in TUB2.

Discussion

In this study, we describe two new species and one new host record from China, namely Neopestalotiopsis elaeagni, N. zingiberis and N. samarangensis, based on morphological and phylogenetic analyses. For the morphology, we chose several indicators for the classification of Neopestalotiopsis, such as the size of conidia, the number and length of apical appendages and the basal appendage length (Maharachchikumbura et al. 2014a). For the phylogeny, we found that the different gene segments can distinguish the different inter-species relationships in Neopestalotiopsis (Table 2). However, some differences lacked significant variation to clearly distinguish the species of Neopestalotiopsis, such as the length and colour of the three median cells, the number of basal appendages and the ITS sequence data of N. elaeagni and N. chrysea. Therefore, we needed to combine the morphology and the phylogeny data to identify the new species.

China has reported 55 fungal diseases on 10 species of Zingiberaceae, including new diseases (Qi and Jiang 1994). However, most of the species have been identified, based on morphology alone. Most studies focused on the secondary products of fungi in Zingiberaceae and little research has been done on the diversity of fungi in Zingiberaceae (Taechowisan et al. 2003, Ginting et al. 2013, Anisha and Radhakrishnan 2017, Gupta et al. 2022). Neopestalotiopsis have been found on many different hosts and plant families (Maharachchikumbura et al. 2014b, Hyde et al. 2020), but few species have been found on Zingiberaceae in China. Therefore, in future work, comprehensive studies on Zingiberaceous Neopestalotiopsis will result in many more species being described in China.

We were unable to conduct the pathogenicity test in this research, although the N. elaeagni and N. zingiberis were isolated from the leaf spots. On the future work, similar to other relevant fields in mycology, it is necessary to identify the pathogenic taxa to the species level (Jayawardena et al. 2021), as it can help us to prevent diseases caused by them and reduce economic losses.

Acknowledgements

The following projects supported the research: National Natural Science Foundation of China (No. 31972222, 31560489), Program of Introducing Talents of Discipline to Universities of China (111 Program, D20023), Talent project of Guizhou Science and Technology Cooperation Platform ([2017]5788–5, [2019]5641 and [2020]5001) and Guizhou Science, Technology Department International Cooperation Basic project ([2018]5806).

References