Biodiversity Data Journal : Taxonomic Paper
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Taxonomic Paper
One new species and one new record of Zasmidium in China
expand article infoYuan-Yan An, Xiang-Yu Zeng‡,§,|, Kun Geng, Kevin David Hyde§,|, Yong Wang
‡ Department of Plant Pathology, Agriculture College, Guizhou University, Guiyang, China
§ Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, Thailand
| School of Science, Mae Fah Luang University, Chiang Rai, Thailand
¶ Guiyang plant protection and inspection station, Guiyang, China
Open Access

Abstract

Background

Two hyphomycetous species were collected from leaves of Smilax china (Liliales, Smilacaceae) and Cremastra appendiculata (Asparagales, Orchidaceae). ITS barcoding indicated that they belong to the genus Zasmidium.

New information

Morphological data in combination with molecular phylogenetic analyses based on ITS, LSU and rpb2 confirmed that our Chinese strains represented a new species, Zasmidium liboense and a new record of Z. citri-griseum.

Keywords

one new species, asexual morph, Dothideomycetes, Mycosphaerellaceae, taxonomy

Introduction

The fungi of southern Asian are extremely diverse (Hyde et al. 2018, Cheek et al. 2020). During a survey of fungal diversity in ornamental plants in south-western China from 2017 to 2019, more than 2000 strains were obtained, which represented asexual morphs of both Ascomycota and Basidiomycota. Some new taxa were previously described by our research group as pathogens or endophytes (e.g. Liang et al. 2018, Long et al. 2019, Sun et al. 2020, Wijesinghe et al. 2020, Zhang et al. 2020).

The genus Zasmidium was established by Fries (1849) with Z. cellare (Pers.) Fr. as the type species. It is currently placed in the order Capnodiales within the Dothideomycetes (Hongsanan et al. 2020, Wijayawardene et al. 2020). Arzanlou et al. (2007) showed that Zasmidium was the oldest name for Stenella-like hyphomycetes within Mycosphaerellaceae, which are characterised by conidiogenous loci and conidia with truncate hila (Bensch et al. 2012). Hence, many former Stenella species were transferred to Zasmidium (Braun et al. 2010, Kamal 2010). Up to now, the number of accepted species in the genus is about 150 (Wijayawardene et al. 2020).

In this paper, we report on Zasmidium species found on medicinal plants in China. One new species (Zasmidium liboense) and one new Chinese record (Z. citri-griseum) are reported, based on evidence from morphology and molecular phylogeny.

Materials and methods

Samples collection and fungal strains isolation

The samples were collected in Xishuangbanna City, Yunnan Province, China. In order to obtain pure cultures, diseased leaf pieces of Smilax china (Liliales, Smilacaceae) and Cremastra appendiculata (Asparagales, Orchidaceae) were surface-disinfected following the method of Zhang et al. (2020). The strains were isolated using the single-spore method (Chomnunti et al. 2014). Colonies growing from single spores were transferred to potato-dextrose agar (PDA) and incubated at room temperature (28ºC). The holotype was deposited in the Herbarium of Department of Plant Pathology, Agricultural College, Guizhou University (HGUP). The ex-type cultures were deposited in the Culture Collection at the Department of Plant Pathology, Agriculture College, Guizhou University, P.R. China (GUCC) and the Mae Fah Luang University Culture Collection (MFLUCC) in Thailand.

Morphological description

Morphological culture characters were recorded after 2–3 weeks of growth on PDA. Microscopic slides were prepared in lactophenol. Light microscopy observations were made using a BX53 compound microscopy (Olympus, Tokyo, Japan) at 1000× magnification. The morphology was observed using a compound microscope (OLYMPUS BX53) showing all necessary details of morphology and ontogeny of reproductive propagules. Measurements were made of 30 structures for conidia, hila and conidiophores. The new species name was submitted to MycoBank (www.mycobank.org).

DNA extraction, PCR amplification and sequencing

Fungal cultures were grown on PDA at 28°C. When the whole Petri-dish (90 mm diam.) was nearly covered, fresh mycelia were scraped from the surface with sterilised scalpels. Genomic DNA was extracted using Fungus Genomic DNA Extraction Kit (Biomiga #GD2416, San Diego, California, USA) and following the manufacturer's instructions. PCR amplification of the internal transcribed spacer (ITS) region and the large subunit (LSU) of the ribosomal RNA gene was performed in a 25-μl reaction volume system as in Liang et al. (2018). Primers V9G and ITS4 (White et al. 1990, de Hoog and van den Ende 1998) were used to amplify the ITS and LSU1Fd and LR5 for the LSU (Vilgalys and Hester 1990, Crous et al. 2009). In addition, one protein-coding gene fragment, RNA polymerase II second largest subunit (rpb2), was amplified with the primers fRPB2-5F and fRPB2-7cR (Liu et al. 1999). Purification and sequencing of the PCR amplicons were undertaken by SinoGenoMax (Beijing, China). The resulting DNA sequences were submitted to NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and their accession numbers are provided in Table 1.

Table 1.

Taxa used for molecular phylogenetic analyses and their GenBank accession numbers. (T) = ex-type strain.

Species name Strain number GenBank Accession numbers
LSU ITS rpb2
Zasmidium angulare CBS 132094(T) = CPC 19042 = GA2 27B1a JQ622096 JQ622088 MF951690
Zasmidium anthuriicola CBS 118742(T) FJ839662 FJ839626 MF951691
Zasmidium arcuatum CBS 113477(T) EU041836 EU041779 MF951692
Zasmidium aucklandicum CPC 13569 MF951280 MF951409 MF951733
Zasmidium biverticillatum CBS 335.36 EU041853 EU041796
Zasmidium cellare CBS 146.36N(T) = ATCC 36951 = IFO4862 = IMI 044943 = LCP 52.402 = LSHBBB274 = MUCL 10089 EU041878 EU041821 MF951693
Zasmidium cerophillum CBS 103.59(T) of Acrotheca cerophila = MUCL10034 GU214485 EU041798 MF951694
Zasmidium citri-griseum CBS 122455 = CPC 15289 = X126 KF902151 KF901792 MF951695
GUCC 1507.3 MT712179 MT683372 MT700485
Zasmidium commune CBS 142530(T) KY979820.1 NR_156003.1
Zasmidium corymbiae CBS 145047(T) NG_066279.1 NR_161118.1 MK047534.1
Zasmidium daviesiae CBS 116002 = VPRI 31767 FJ839669 FJ839633 MF951698
Zasmidium ducassei BRIP 53367(T) NR_164517.1
Zasmidium elaeocarpi CBS 142187(T) = CPC 16642 MF951263 MF951398 MF951699
Zasmidium eucalypticola CBS 142186(T) = CPC 15149 MF951265 MF951400 MF951701
Zasmidium eucalyptorum CBS 118500(T) = CPC 11174 MF951266 KF901652 MF951702
Zasmidium fructicola CBS 139625(T) = CPC 24487 = ZJUM 80 KP895922 KP896052 MF951703
Zasmidium fructigenum CBS 139626(T) = CPC 24471 = ZJUM 36 KP895926 KP896056 MF951704
Zasmidium grevilleae CBS 124107(T) = CPC 14761 FJ839670 FJ839634 MF951705
Zasmidium gupoyu CBS 122099 = RoKi 3022 MF951267 MF951401 MF951706
Zasmidium hakeae CBS 142185(T) = CPC 15577 MF951268 MF951402 MF951707
Zasmidium hakeicola CBS 144590(T) NG_066335.1 NR_163384.1 MK442687.1
Zasmidium indonesianum CBS 139627(T) = CPC 15300 KF902086 KF901739 MF951710
Zasmidium iteae CBS 113094(T) = RoKi 1279 MF951271 MF951405 MF951711
Zasmidium liboense sp. nov. GUCC 1720.2 MT712180 MT683373 MT700486
Zasmidium lonicericola CBS 125008(T) of Cladosporium lonicericola = CPC11671 KF251787 KF251283 MF951712
Zasmidium musae CBS 121384 = CIRAD 41 = X877 MF951272 EU514292 MF951713
Zasmidium musae-banksii CBS 121710(T) = X1100 EU041852 EU041795 MF951716
Zasmidium musicola CBS 122479(T) = X1019 MF951275 EU514294 MF951717
Zasmidium musigenum CBS 190.63 = MUCL 9557 EU041857 EU041800 MF951718
Zasmidium nocoxi CBS 125009(T) = CPC 14044 KF251788 KF251284 MF951719
Zasmidium pitospori CBS 122274 = ICMP 17098 MF951276 MF951406 MF951720
Zasmidium podocarpi CBS 142529 KY979821.1 NR_156004.1
Zasmidium proteacearum CBS 116003 = VPRI 31812 FJ839671 FJ839635 MF951721
Zasmidium pseudoparkii CBS 110999(T) = CPC 1087 JF700965 DQ303023 MF951723
Zasmidium pseudotsugae

rapssd

EF114704 EF114687
Zasmidium pseudovespa CBS 121159(T) = AC0466 KF901836 MF951407 MF951724
Zasmidium queenslandicum CBS 122475(T) = X1084 MF951277 EU514295 MF951725
Zasmidium scaevolicola CBS 127009(T) = CPC 17344 KF251789 KF251285 MF951726
Zasmidium schini CBS 142188(T) = CPC 19516 MF951278 MF951408 MF951727
Zasmidium sp. CBS 118494 = CPC 11004 MF951279 DQ303039 MF951728
Zasmidium strelitziae CBS 121711(T) = X1029 EU041860 EU041803 MF951729
Zasmidium suregadae P36 KC677939.1 KC677914.1
Zasmidium syzygii CBS 133580(T) = CPC 19792 KC005798 KC005777 MF951730
Zasmidium thailandicum CBS 145027(T) NG_066342.1 NR_164463.1 _
Zasmidium tsugae ratstk EF114705 EF114688
Zasmidium velutinum CBS 101948(T) = CPC 2262 EU041838 EU041781 MF951731
Zasmidium xenoparkii CBS 111185(T) = CPC 1300 JF700966 DQ303028 MF951732
Nothopericoniella perseamacranthae CBS 122097 = RoKi 2995 GU452682 MF951354 MF951583

Phylogenetic analyses

Our newly-generated sequences were aligned by locus with ex-type and other representative sequences of Zasmidium species, which were downloaded from GenBank (Table 1). Alignments were made using the online version of MAFFT v. 7.307 (Katoh and Standley 2016) and manually improved, where necessary, using MEGA v. 6.06 (Tamura et al. 2013). Mesquite v. 2.75 (Maddison 2008) was used to concatenate the aligned sequences of the different loci. Ambiguous regions were excluded from analyses using AliView (Larsson 2014) and gaps were treated as missing data. DNA base differences of different gene loci between our strains and ex-type or representative strains of relative taxa are shown in Table 2. The alignment document is available in TreeBASE under the study ID 27250.

Table 2.

The DNA base differences between our Chinese strains and related taxa in the three gene regions. Asterisks (*) denote our material.

Species

Strain number

ITS (1-480 bp)

LSU

(481-1254 bp)

RPB2

(1255-2355 bp)

Zasmidium citri-griseum *

GUCC 1507.3

-

-

-

Zasmidium citri-griseum

CBS 146.36

8

1

0

Zasmidium suregadae

P36

6

0

/

Zasmidium anthuriicola

CBS 118742T

6

0

52

Species

Strain number

ITS

(1-475 bp)

LSU

(476-1297 bp)

RPB2

(1298-2141 bp)

Zasmidium liboense sp. nov.*

GUCC 1720.2

-

-

-

Zasmidium cellare

CBS 122455NT

23

19

72

Maximum Parsimony (MP) analyses were performed in PAUP v. 4.0b10 (Swofford 2002), using the heuristic search option with 1,000 random sequence addition replicates and tree bisection-reconnection (TBR) as the branch swapping algorithm. Maxtrees was set at 10,000. The Tree Length (TL), Consistency Indices (CI), Retention Indices (RI), Rescaled Consistency Indices (RC) and Homoplasy Index (HI) were calculated for each tree generated.

Maximum Likelihood (ML) was inferred using the IQ-tree (Nguyen et al. 2015, Chernomor et al. 2016) under the Edge-linked partition model. Bootstrapping was done under 10000 ultrafast replicates (Hoang et al. 2018). ModelFinder (Kalyaanamoorthy et al. 2017) was used to select the best-fit partition model (Edge-linked) using BIC criterion. The best-fit model, according to BIC was: ITS: TNe+I+G4, LSU: TN+F+I+G4, rpb2: TN+F+I+G4.

For Bayesian Inference (BI), GTR+I+G was selected as the best model for all three loci (ITS, LSU and rpb2) as determined by MrModeltest v2 (Nylander 2004). BI analysis was undertaken using MrBayes v. 3.2.6 (Ronquist et al. 2012). Six Markov Chain Monte Carlo runs were launched with random starting trees for 1,000,000 generations and sampling every 1,000 generations. The first 25% resulting trees were discarded as burn-in.

Taxon treatments

Zasmidium liboense Y.Y. An, Yong Wang bis & K.D. Hyde, sp. nov.

Material    Download as CSV 
Holotype:
  1. scientificName:
    Zasmidium liboense
    ; kingdom:
    Fungi
    ; class:
    Dothideomycetes
    ; order:
    Capnodiales
    ; family:
    Mycosphaerellaceae
    ; genus:
    Zasmidium
    ; country:
    China
    ; stateProvince:
    Guizhou
    ; locality:
    Gold Thread Cave of Libo county
    ; catalogNumber:
    HGUP 1720.2
    ; recordedBy:
    Wang Yong
    ; identifiedBy:
    Yuan-Yan An
    ; dateIdentified:
    2020
    ; type:
    ex-type cultures GUCC 1070.2; MFLUCC 20-0139
    ; language:
    en

Description

Pathogenic on the leaf spot of Smilax china (Liliales, Smilacaceae). Lesions on the upper leaf surface, scattered, distinct, irregular, rather large, the maximum length of the spot more than 20 mm, the edges of the disease reddish-brown, the centre dead to greyish-white, on the lower leaf surface similar. Colonies on PDA 10–15 mm diam. in 2 weeks, with an even, dark coffee margin. Mycelium composed of hyaline and pale brown to dark blackish-brown hyphae, verruculose, septate, branching, uniform in width, 2.5 μm. Conidiophores arising from hyphae, pale olivaceous brown to pale blackish-brown, finely verruculose, straight or slightly curved, dendritic rugged or rugose on the surface 20–350 × 1.5–3.5 μm. Conidiogenous cells integrated, apical, polyblastic, proliferating sympodially, with rim-like conidiogenous loci, thickened and darkened, located apically and lateraly as in a short rachis 1.5–2.5 μm diam. Conidia solitary, occasionally catenate, pale blackish-brown to pale olivaceous brown, verruculose, ellipsoidal, cylindrical to obclavate, base obconically truncate and apex rounded, straight or curved, 6–21 × 2–4 μm, 0–1-septate, sometimes constricted at septa, with hila thickened and darkened, 1–1.5 μm diam. (Fig. 1)

Figure 1.  

Zasmidium liboense (GUCC 1720.2). a. Leaf spot symptoms on the host; b. Culture on PDA; c, d. hyphae and conidiophores on PDA; e–m. Conidiophores and conidia; n, o. Conidiogenous cells and conidia; p. Conidia. Scale bars: c, d = 50 µm, e–j = 10 µm, k = 5 µm, l–p = 10 µm.

MycoBank Number: 

MB836278

Etymology

In reference to the location (Libo county, Guizhou Province), where the holotype was isolated.

Zasmidium citri-griseum (F.E. Fisher) U. Braun & Crous, IMA Fungus 5 (2): 337 (2014)

Material    Download as CSV 
  1. scientificName:
    Zasmidium citri-griseum
    ; kingdom:
    Fungi
    ; class:
    Dothideomycetes
    ; order:
    Capnodiales
    ; family:
    Mycosphaerellaceae
    ; genus:
    Zasmidium
    ; country:
    China
    ; stateProvince:
    YunNan
    ; locality:
    Xishuangbanna Dai Autonomous Prefecture
    ; catalogNumber:
    HGUP 1507.3
    ; recordedBy:
    An Yuan-Yan
    ; identifiedBy:
    Yuan-Yan An
    ; dateIdentified:
    2020
    ; language:
    en
    ; occurrenceID:
    living culture GUCC 1507.3 and MFLUCC 20-0138

Description

Pathogenic on leaf spot of Cremastra appendiculata (Asparagales, Orchidaceae). Lesions on the upper leaf surface, scattered to confluent, distinct, angular, spots elliptic to suborbicular, reddish-brown to dark brown, 2–5 mm, on the lower leaf surface similar. Colonies on PDA 10–15 mm diam. in 2 weeks, with an even, dark-brown margin. Surface fold, with gully shape. Conidiophores arising singly as lateral branches of superficial hyphae, semi-macronematous to macronematous, mononematous, erect to flexuous, sometimes curved, unbranched, thick-walled, non-smooth surface, 1–3-septate, cylindrical, geniculate, brown, 35–135 × 5–7 µm. Conidiogenous cells integrated, terminal, 5–10 µm long, sympodial, polyblastic, cylindrical, geniculate, scars slightly thickened and darkened, 0.5–1.5 µm in diam. Conidia solitary in simple or occasionally branched chains, short to long cylindrical, some ends swollen straight to somewhat curved, 8–41 × 2–5 µm, unseptate, light brown, thin-walled, verruculose, 2–3.5 µm wide. (Fig. 2)

Figure 2.  

Zasmidium citri-griseum (GUCC 1507.3) a. Leaf spot symptoms on host; b. Culture on PDA; c. Hyphae and conidiophores on PDA culture; d–h. Conidiophores, conidiogenous cells and conidia; i. Conidia; j, k. Conidiogenous cells and conidia; l–p. Conidia. Scale bars: c = 100 µm, d–g =10 µm, h=5 µm, i, j = 10 µm, k–p = 5 µm.

Analysis

Our final concatenated alignment included 2407 characters , viz. (ITS: 1–537, LSU: 538–1354, rpb2: 1355–2407), of which 645 were parsimony-informative characters. MP inference resulted in two equally-parsimonious trees (TL = 3705, CI = 0.35, RC = 0.59, HI = 0.20, RC = 0.65) and one of them was selected to show the topology (Fig. 3). Forty-eight Zasmidium strains clustered together as a clade, which received maximum support from both ML and BI analyses. Zasmidium liboense sp. nov. (GUCC 1720.2) was retrieved as sister taxon of Z. cellare (Pers.) Fr. (CBS 146.36) with high support (94 MP/89 ML/1.00 PP). Strain GUCC1507.3 grouped with a lineage consisting of Z. anthuriicola (U. Braun & C.F. Hill) Crous & U. Braun, Z. citri-griseum (F.E. Fisher) U. Braun & Crous and Z. suregadae Phengs., K.D. Hyde & U. Braun, with high support (99 MP/99 ML/1.00 PP). It was placed sister to Z. citri-griseum (CBS 122455) (86 ML).

Figure 3.  

MP phylogeny of Zasmidium reconstructed from a three-locus dataset (ITS, LSU, rpb2). MP and ML bootstraps > 70 and BI posterior probabilities (PP) > 0.9 are placed close to topological nodes and separated by “/”. Nothopericoniella perseamacranthae (CBS 122097) was selected as outgroup.

A comparison of DNA bases (Table 2) demonstrated that, between Z. liboense (GUCC 1720.2) and Z. cellara (CBS 146.36), there were 23 bp differences in the ITS region, 19 in the LSU and 72 in the rpb2. The ITS sequence of GUCC 1507.3 differed in 8 bp from Z. citri-griseum (CBS 122455). Their LSU sequences were identical, whereas a single base pair difference was found in the rpb2.

Discussion

Our phylogenetic analyses pointed out that Zasmidium liboense is different from Z. cellare. Morphologically, both species can be separated as well (Arzanlou et al. 2007). Zasmidium liboense produces conidia with 0-1 septa, whereas those of Z. cellare possess 0–1(–4) septa. Conidia of Z. cellare arise terminally or laterally and are subhyaline, whereas those of Z. liboense arise terminally and are pale blackish-brown to pale olivaceous brown. In addition, conidia of Z. liboense (6–21 × 2–4 μm) generally have larger dimensions compared to those of Z. cellare (6–9 × 1.8–2.5 µm). We also compared micro-morphology of our new taxon with 74 Zasmidium species, for which no sequence data are available. Most of those, 64 species, produce larger conidia with more septa compared to Z. liboense. In addition, Z. araliae, Z. cerophilum and Z. litseae all possess smaller conidia than Z. liboense. The conidia of Z. capparacearum, Z. eriolobi, Z. gahniicola and Z. mitellae have more septa compared to those of Z. liboense. Zasmidium clusiae produces conidia with basal unthickened hilum, whereas the conidia of Z. liboense, Z. deightonianum and Z. oxycocci have slightly or obviously thickened conidia. For these three species, the length of conidiophores can be used to distinguish amongst species: Z. liboense (20–350 μm), Z. deightonianum (10–30 μm) and Z. oxycocci (50–100 μm).

For strain GUCC 1507.3, our molecular phylogenetic data (Fig. 3, Table 2) showed that it was very close to Z. anthuriicola, Z. citri-griseum and Z. suregadae. Rbp2 sequences of strain GUCC 1507.3 and Z. citri-griseum (CBS 122455) are identical, but different from Z. anthuriicola (CBS 118742). Currently, no rpb2 sequences are available for Z. suregadae. Morphological comparison shows that conidia of our material (GUCC 1507.3) agree with the description of Z. citri-griseum; they are somewhat shorter although still in the range of measurements [Z. citri-griseum: 6–70(–120) × 2–4.5 μm] and they are aseptate [Z. citri-griseum: 0–1–(–6)-septate] (Braun et al. 2014). Zasmidium citri-griseum is one of the few species of Zasmidium with a wider host range. Braun et al. (2014) listed hosts in the Fabaceae, Musaceae and Rutaceae. The host of our Chinese material, Cremastra appendiculata (Orchidaceae), represents a new host family for this fungus.

Acknowledgements

This research was supported by the following sources: 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 and [2019]5641), Guizhou Science, Technology Department of International Cooperation Base Project ([2018]5806) and Guizhou Science and Technology Innovation Talent Team Project ([2020]5001).

References