Biodiversity Data Journal : Taxonomic Paper
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Taxonomic Paper
Curvicladiella paphiopedili sp. nov. (Hypocreales, Nectriaceae), a new species of orchid (Paphiopedilum sp.) from Guizhou, China
expand article infoLian-chai Song‡,§, Lu Huang|, Ling-ling Liu, Yao Feng#, Li-li Wang§, Zuo-yi Liu¤
‡ College of Agriculture, Guizhou University, Guiyang, China
§ Scientific research administration office, Guizhou Academy of Agricultural Sciences, Guiyang, China
| Institute of Plant Protection, Guizhou Academy of Agricultural Sciences, Guiyang, China
¶ Guizhou Institute of Soil and Fertilizer, Guizhou Academy of Agricultural Sciences, Guiyang, China
# School of Ethnic Medicine, Guizhou Minzu University, Guiyang, China
¤ Guizhou Key Laboratory of Agricultural Biotechnology, Guizhou Academy of Agricultural Sciences, Guiyang, China
Open Access

Abstract

Background

An asexual fungus, collected from diseased leaves of Paphiopedilum sp. from Guizhou Province, China, and based on the phylogenetic analyses and morphological characters, it was identified as a new species in Curvicladiella. The genus Curvicladiella are recorded for the first time for China.

New information

The morphology of Curvicladiella paphiopedili sp. nov. is characterised by penicillate conidiophores with a stipe, dull, tapering towards the apex, the curved stipe extension and cylindrical conidia. In the phylogenetic analyses of combined cmdA, his3, ITS, LSU, tef1 and tub2 sequence data, this taxon was clustered as sister to Curvicladiella cignea within Nectriaceae.

Keywords

Curvicladiella, morphology, phylogeny, Paphiopedilum, taxonomy

Introduction

Nectriaceae (order Hypocreales) includes many important plant and human pathogens and some species have been used as biodegrading and biocontrol agents in industrial and commercial applications (Lombard et al. 2015). Based on molecular studies, many sexual genera in Nectriaceae were placed in Nectria sensu lato (Rehner and Samuels 1995, Rossman et al. 1999). However, Nectria sensu stricto is restricted to the type species N. cinnabarina (Tode) Fr et al. with tubercularia-like asexual morphs (Rossman 2000, Hirooka et al. 2012). A number of studies have treated taxonomic concepts within Nectriaceae, based on multi-gene phylogenetic inference (Lombard et al. 2010a, Lombard et al. 2010b, Chaverri et al. 2011, Gräfenhan et al. 2011, Schroers et al. 2011, Hirooka et al. 2012,Lombard and Crous 2012a, Lombard and Crous 2012b, Lombard et al. 2014a, Lombard et al. 2014b). Lombard et al. (2015) provided a phylogenetic backbone tree for Nectriaceae, based on combined sequence data of 10 gene regions. Curvicladiella is one of the genera in the Nectriaceae.

Decock and Crous (1998) established Curvicladium (as Curvicladiella) with C. cigneum (as Curvicladiella cignea) as the type species. The genus is distinct from morphologically-similar genera, such as Cylindrocladium Morgan, Cylindrocladiella Boesew, Gliocladiopsis Saksena, Falcocladium Silveira, Alfenas, Crous, Wingf and Xenocylindrocladium Decock, Hennebert, Crous by having cylindrical conidia and stipe extensions (Decock and Crous 1998). Curvicladiella cignea is the only species in the genus.

Based on the phylogenetic analyses and morphological characters, the fungus collected from diseased leaves of Paphiopedilum sp. is identified as a new species in Curvicladiella, the artificial infection test shows that it is a pathogen and the specific infection process has been described by Song et al. (Song et al. 2020). Paphiopedilum is known as“slipper orchids”, has a high ornamental value and can be used as household bonsai and garden plants(Luan et al. 2019).

Materials and methods

Sample collection and isolation

Diseased orchid leaves were collected from Guizhou Botanical Garden, Guizhou Province, China (in August 2019). The samples were brought to the laboratory in envelopes, photographed and identified. Pieces of leaves (5 × 5 mm), half of which were diseased and half healthy, were sterilised by 75% ethanol for 5–10 s, rinsed three times with sterilised distilled water, placed on potato dextrose agar (PDA) and incubated at 25°C for two days (Fang 2001). Mycelia were transferred to PDA and incubated for ten days at 25°C to obtain the pure cultures. The morphological characters of the fungi obtained from the diseased leaves collected in the field and cultured with PDA, and the fungi obtained from the diseased leaves after an artificial infection test were observed using a Nikon SMZ 745 stereomicroscope. Measurements were made using Image Frame Work.

Pure cultures were deposited in Guizhou Culture Collection (GZCC) Guizhou, China and Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand. Herbarium specimens were deposited in the Guizhou Academy of Agricultural Sciences (GZAAS), Guiyang, China and the Herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand.

DNA extraction, PCR amplification and sequencing

The fungal mycelia were scraped from the pure culture growing on PDA for ten days at 25ºC. DNA was extracted using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, China). Six gene regions, the 28S large subunit rDNA (LSU), calmodulin (cmdA), histone H3 (his3), internal transcribed spacer region and intervening 5.8S nrRNA gene (ITS), translation elongation factor 1-alpha (tef1) and β-tubulin (tub2) gene were amplified by the primer pairs LR0R and LR5(Vilgalys and Hester 1990, Rehner and Samuels 1994), CAL-228F and CAL2Rd (Carbone and Kohn 1999, Groenewald et al. 2013), CYLH3F and CYLH3R (Crous et al. 2004), ITS5 and ITS4 (White et al. 1990), EF1-728F and EF2 (O'Donnell et al. 1998, Carbone and Kohn 1999), T1 and CYLTUB1R (O'Donnell and Cigelnik 1997, Crous et al. 2004), respectively. Polymerase chain reaction (PCR) was carried out in 25 µl reaction volume containing 12.5 µl 2 × PCR Master Mix (Sangon Biotech, China), 9.5 µl ddH2O, 1µl of each primer and 1µl DNA template. The PCR products were examined by using 1.2% agarose electrophoresis gel, stained with ethidium bromide and were purified and sequenced by Sangon Biotech (Shanghai) Co. Ltd, China. The nucleotide sequences were submitted in GenBank.

Phylogenetic analyses

Phylogenetic analyses were performed using combined sequence data with six gene regions, LSU, cmdA, his3, ITS, tef1 and tub2. Related strains of Curvicladiella (Table 1) were referred to Lombard et al. (2015). Sequences were obtained from GenBank. The sequences were aligned using the online multiple alignment programme MAFFT v.7 (http://mafft.cbrc.jp/alignment/server) (Standley 2013). The alignments were checked visually and optimised manually by using BioEdit v. 7.2.6.1.

Table 1.

Taxa or selected taxa used in this study and their GenBank accession numbers. The type species have T as superscript and the newly-generated sequences have been highlighted in bold.

Taxa Isolate numbers GenBank Accession numbers
LSU CMDA HIS3 ITS TEF1 TUB2
Aquanectria penicillioides CBS 257.54 KM231613 KM231275 KM231743 KM231865 KM232000
Aquanectria submersa CBS 394.62 T KM231612 KM231458 HQ897796 KM231999
Calonectria brassicae CBS 111869 GQ280698 GQ267382 DQ190720 GQ280576 FJ918567 AF232857
Calonectria ilicicola CBS 190.50 T GQ280727 AY725764 AY725676 GQ280605 AY725726 AY725631
Calonectria naviculata CBS 101121 T GQ280722 GQ267399 GQ267252 GQ280600 GQ267317 GQ267211
Campylocarpon fasciculare CBS 112613 T HM364313 KM231297 JF735502 AY677301 JF735691 AY677221
Campylocarpon pseudofasciculare CBS 112679 T HM364314 KM231298 JF735503 AY677306 JF735692 AY677214
Corallonectria jatrophae CBS 913.96 T KM231611 KM231273 KM231457 KC479758 KM231863 KC479787
Curvicladiella cignea CBS 101411 JQ666075 KM231285 KM231459 KM231744 KM231866 KM232001
Curvicladiella cignea CBS 109168 JQ666074 KM231286 KM231460 KM231745 KM231868 KM232003
Curvicladiella cignea CBS 109167 T AY793431 KM231287 KM231461 AF220973 KM231867 KM232002
Curvicladiella paphiopedili MFLUCC 20-0110 T MT279199 MT294104 MT294105 MT279198 MT294103 MT294102
Curvicladiella paphiopedili GZCC22-0001 OM899803 OM903885
Cylindrocarpostylus gregarius CBS 101074 KM231614 KM231291 KM231746 KM231869 KM232004
Cylindrocarpostylus gregarius CBS 101072 T JQ666084 KM231292 KM231747 KM231870 KM232005
Cylindrocarpostylus gregarius CBS 101073 JQ666083 KM231293 KM231465 KM231748 KM231871 KM232006
Cylindrocladiella camelliae CPC 234 T JN099249 KM231280 AY793509 AF220952 JN099087 AY793471
Cylindrocladiella lageniformis CBS 340.92 T JN099165 KM231279 AY793520 AF220959 JN099003 AY793481
Cylindrocladiella parva CBS 114524 T JN099171 KM231281 AY793526 AF220964 JN099009 AY793486
Dematiocladium celtidis CBS 115994 T AY793438 KM231274 AY793430 KM231864
Gliocephalotrichum bulbilium CBS 242.62 T AY489732 KM231283 KF513326 KM231892 DQ377831
Gliocephalotrichum cylindrosporum CBS 902.70T JQ666077 KM231284 KF513353 DQ366705 KF513408 DQ377841
Gliocephalotrichum longibrachium CBS 126571 T KM231686 KM231282 KF513367 DQ278422 KF513435 DQ377835
Gliocladiopsis irregularis CBS 755.97 T JQ666082 KM231278 JQ666023 AF220977 KF513449 JQ666133
Gliocladiopsis pseudotenuis CBS 116074 T JQ666080 KM231277 JQ666030 AF220981 JQ666099 JQ666140
Gliocladiopsis sagariensis CBS 199.55 T JQ666078 KM231276 JQ666031 JQ666063 JQ666106 JQ666141
Penicillifer bipapillatus CBS 420.88 T KM231608 KM231270 KM231454 KM231740 KM231860 KM231996
Penicillifer diparietisporus CBS 376.59 T KM231609 KM231271 KM231455 KM231741 KM231861 KM231997
Penicillifer penicilliferi CBS 423.88 T KM231607 KM231269 KM231453 KM231739 KM231859 KM231995
Penicillifer pulcher CBS 560.67 T KM231610 KM231272 KM231456 KM231742 KM231862 KM231998
Rugonectria neobalansae CBS 125120 HM364322 KM231294 KM231466 KM231750 KM231874 HM352869
Rugonectria rugulosa CBS 129158 JF832761 KM231295 KM231467 JF832661 KM231872 JF832911
Rugonectria rugulosa CBS 126565 KM231615 KM231296 KM231468 KM231749 KM231873 KM232007
Thelonectria discophora CBS 125153 HM364307 KM231327 KM231489 HM364294 KM231897 HM352860
Thelonectria olida CBS 215.67 T HM364317 KM231325 KM231487 AY677293 HM364345 KM232024
Thelonectria trachosa CBS 112467 T HM364312 KM231326 KM231488 AY677297 KM231896 AY677258
Xenocylindrocladium guianense CBS 112179 T JQ666073 KM231289 KM231463 AF317348 KM231895 AF320197
Xenocylindrocladium serpens CBS 128439 T KM231688 KM231290 KM231464 AF220982 KM231894 AF320196
Xenocylindrocladium subverticillatum CBS 113660 T KM231687 KM231288 KM231462 AF317347 KM231893 AF320196
T Ex-type and ex-epitype cultures. CBS: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; CPC: P.W. Crous collection.

Maximum Likelihood (ML) analysis was performed using RaxmlGUI 1.3.1 (Silvestro and Michalak 2012). The optimal RAxML tree search was conducted with 1000 bootstrap replicates and the default algorithm was used from a random starting tree for each replicate. The final tree was selected from amongst suboptimal trees from each replicate by comparing likelihood scores under the GTR+GAMMA substitution model.

Bayesian analyses were carried out using MrBayes 3.2 (Huelsenbeck 2012). MrModeltest 2.2 was used to choose the best-fit evolutionary model (Nylander 2004). Posterior probabilities (PP) (Rannala and Yang 1996, Zhaxybayeva and Gogarten 2002) were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.2. Six simultaneous Markov chains were run for 10 million generations and trees were sampled every 1000th generation. The temperature values were lowered to 0.15, burn-in was set to 0.25 and the run was automatically stopped as soon as the average standard deviation of split frequencies reached below 0.01.

The resulting trees of Maximum Likelihood and Bayesian were visualised with Fig Tree v.1.4.0. The layouts were undertaken using Microsoft Powerpoint 2010 and Adobe Photoshop CS6.

Taxon treatment

Curvicladiella paphiopedili Lian-Chai Song,Jing Yang, Zuo-Yi Liu, 2019, sp. nov.

Materials    Download as CSV 
Holotype:
  1. scientificName:
    Curvicladiella paphiopedili
    ; class:
    Sordariomycetes
    ; order:
    Hypocreales
    ; family:
    Nectriaceae
    ; genus:
    Curvicladiella
    ; locationRemarks:
    China, Gui Zhou Province, Guiyang City, Guizhou Botanical Garden, 26°37'N, 106°43'E, 13 August 2019
    ; habitat:
    Terrestrial
    ; fieldNotes:
    diseased leaves of Paphiopedilum sp.
    ; recordNumber:
    zwy-dl4-2
    ; recordedBy:
    Lian Chai Song
    ; type:
    Stilllmage
    ; language:
    English
    ; collectionID:
    MFLU 20-0203
Isotype:
  1. scientificName:
    Curvicladiella paphiopedili
    ; class:
    Sordariomycetes
    ; order:
    Hypocreales
    ; family:
    Nectriaceae
    ; genus:
    Curvicladiella
    ; locationRemarks:
    China, Gui Zhou Province, Guiyang City, Guizhou Botanical Garden, 26°37'N, 106°43'E, 13 August 2019
    ; habitat:
    Terrestrial
    ; fieldNotes:
    diseased leaves of Paphiopedilum sp.
    ; recordNumber:
    zwy-dl4-2
    ; recordedBy:
    Lian Chai Song
    ; type:
    Stilllmage
    ; language:
    English
    ; collectionID:
    GZAAS 19-2061

Description

The characters of pathogenic fungi on the leaves were identified through an artificial infection test. Asexual morph: Conidiomata white, scattered, hairy. Conidiophores straight to flexuous, consisting of a stipe bearing a penicillate arrangement of fertile branches, stipe septate, hyaline, smooth; stipe extensions septate, straight or curved, dull and tapering towards the apex, 128.5–549.9 µm long, (x̄= 288.1 µm, n = 20). The primary branches of conidiogenous apparatus aseptate, 9.3–17.5 × 2.6–3.7 μm; secondary branches aseptate, 9.9–19.1 × 2.5–3.9 μm; tertiary branches aseptate, 9.5–17.6 × 2.6–3.7 μm; quaternary and additional branches (–6) aseptate, 11–16.3 × 2.5–3.9 μm, each terminal branch producing 2–4 phialides; phialides doliiform to reniform, hyaline, aseptate, apex with minute periclinal thickening and inconspicuous collarette. Conidia cylindrical, rounded at both ends, straight, 1-septate, hyaline, (30.5–) 31.2–37.2 (–42.0) × (2.6–) 2.9–3.5 (–3.9) µm, (x̄= 34.2 × 3.2 µm, n = 20) (Fig. 1). Sexual morph: not observed.

Figure 1.  

Curvicladiella paphiopedili. a The diseased leaves were withered; b, c Conidiomata; d–g Stipes extension and conidiogenous cells; h–j Conidiogenous cells and conidiophores; k–n Conidia. Scale bars: d–g=50 µm, h–j=20 µm, k–n=10 µm.

The characters of fungus obtained from the diseased leaves collected in the field that were cultured with PDA: after 10 days at 25°C on PDA, colonies reached 47 mm in diam. Beige to pale yellow colony on the surface, brown in reverse with irregular margins, extensive sporulation on the medium surface. Conidiophores straight to flexuous, consisting of a stipe bearing a penicillate arrangement of fertile branches, stipe extensions septate, straight or slightly flexuous, 104.4–153.0 µm long, (x̄= 128.7 µm, n = 10). The primary branches of conidiogenous apparatus aseptate, 8.9–17.8 × 2.7–3.4 μm; secondary branches aseptate, 7.8–14.0 × 2.5–5.9 μm; tertiary branches aseptate, 8.9–17.7 × 2.3–3.5 μm; quaternary and additional branches (–6) aseptate, 9.3–16.7 × 2.3–3.7 μm, each terminal branch producing 2–4 phialides; phialides doliiform to reniform, hyaline, aseptate, apex with minute periclinal thickening and inconspicuous collarette. Conidia cylindrical, rounded at both ends, straight, 1-septate, hyaline, (38.5–) 45.2–56.6 (–63.2) × (2.2–) 2.9–4.2 (–4.9) µm, (x̄= 50.9 × 3.5 µm, n = 40). Chlamydospores thick-walled, ellipsoidal or sphaeropedunculate, brown to hyaline, (9.0–) 11.9–20.7 (–23.1) × (8.1–) 8.9–12.8 (–15.4) µm, (x̄= 16.3 × 10.8 µm, n = 20) (Fig. 2).

Figure 2.  

Curvicladiella paphiopedili. (MFLU 20-0203, holotype) a Paphiopedilum diseased leaf in the field; b The healthy leaves diseased after inoculating the mycelial PDA plug of Curvicladiella paphiopedili ; c The healthy leaves did not become infected after being inoculated with free PDA plug as control; d Colonies on PDA producing conidia masses; e–j Conidiophores, conidiogenous cells and stipes extension; k Chlamydosporae; l, m Conidia; n, o Culture on PDA; (n) from above, (o) from below. Scale bars: e–g=50 µm, h–k=20 µm, l, m=10 µm.

Material: ex-type living culture, MFLUCC 20-0110.

Etymology

Refers to the genus name Paphiopedilum.

Analysis

Phylogenetic analyses

The final alignment consists of the new species and the fungus obtained from the diseased leaves after use of the new species to infect the heathy Paphiopedilum and other genera of the families Nectriaceae. Additionally, the alignment of combined cmdA, his3, ITS, LSU, tef1 and tub2 sequence data comprised a total of 3877 characters with gaps (734bp for cmdA, 529bp for his3, 616bp for ITS, 840bp for LSU, 548bp for tef1 and 610bp for tub2). The dataset comprised 39 taxa with Campylocarpon fasciculare and C. pseudofasciculare as the outgroup taxa. The best scoring RAxML tree is shown in Fig. 3, with the Bayesian tree (not shown) having a similar topology with the ML tree. Curvicladiella paphiopedili was clustered as sister taxon to C. cignea within Nectriaceae with high support (99/1.00) (Fig. 3).

Figure 3.  

The RAxML tree, based on analysis of cmdA, his3, ITS, LSU, tef1 and tub2 sequences data. Bootstrap support values for ML and Bayesian greater than 75% and 0.95 were given near nodes, respectively. The tree was rooted with Campylocarpon fasciculare and Campylocarpon pseudofasciculare. The new isolate are shown in bold.

Discussion

Morphologically, Curvicladiella paphiopedili is similar to species in Calonectria, Cylindrocladium and Xenocylindrocladium, but distinct in having ellipsoidal or sphaeropedunculate chlamydospores (Fig. 2k), dull, tapering towards the apex (Fig. 1d and e, Fig. 2e–g) and curved extension stipes (Fig. 1f and g), without obpyriform, ovoid, ellipsoidal or sphaeropedunculate vesicles (Lombard et al. 2010a, Pham et al. 2019) or coiled stipes (Decock et al. 1997). The morphology of Curvicladiella paphiopedili is different from the type species Curvicladiella cignea in the size of stipe extensions and conidia, without the swollen cell below the apical septum; on the other hand, the stipe extensions of Curvicladiella cignea is curved obviously, while Curvicladiella paphiopedili is not. The stipe extensions of Curvicladiella paphiopedili are 128.5–549.9 µm long, the Curvicladiella cignea is 110-200 µm long. The conidia of Curvicladiella paphiopedili are (38.5–) 45.2–56.6 (–63.2) × (2.2–) 2.9–4.2 (–4.9) µm, the Curvicladiella cignea are (28–) 33–36 (–38) × 2.5–3µm. In the phylogenetic analyses, the two taxa of Curvicladiella formed a well-supported monoclade and Curvicladiella aphiopedili represented a distinct lineage (Fig. 3).

Acknowledgements

This research is supported by the following projects: National Natural Science Foundation of China (31660011),Guizhou Graduate Research Funding:Taxonomic and Phylogenetic studies of microfungi from Bamboo in Guizhou (Qianjiaohe YJSCXJH [2019] 022). Lian-chai Song thanks Jing Yang for guiding the experiments and modifing the articles.

References