Biodiversity Data Journal : Taxonomic Paper
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Taxonomic Paper
Ascodesmis rosicola sp. nov. and Talaromyces rosarhiza sp. nov., two endophytes from Rosa roxburghii in China
expand article infoHong Zhang‡,§, Tian-Peng Wei, Yu-Tao Mao|, Ming-Xia Ma§, Kai Ma§, Ying Shen§, Mei-Juan Zheng§, Wei-Yu Jia, Ming-Yan Luo, Yan Zeng, Yu-Lan Jiang, Guang-Can Tao§
‡ Department of Plant Pathology, College of Agriculture, Guizhou University, Guiyang, China
§ Guizhou Academy of Testing and Analysis, Guiyang, China
| Grain and Oil Quality Testing Center of Guiyang, Guiyang, China
Open Access

Abstract

Background

Rosa roxburghii Tratt., a deciduous shrub of the family Rosaceae, is usually used as food and medicinal materials and also cultivated as an ornamental. Plant endophytic fungi are a large class of microbial resources not fully researched, with great potential applications. Two strains of Ascodesmis and Talaromyces were isolated during a survey of biodiversity on endophytic fungi of R. roxburghii in China. Multigene phylogenetic analyses showed that each of the two fungi formed a distinct lineage and separated from known congeneric species and they are proposed as two novel taxa.

New information

Ascodesmis rosicola sp. nov. usually has one or two conspicuous simple or branched ridges extending to the majority of the ascospore surface and remarkably small asci, distinguishing it from the previously-described species in the genus Ascodesmis. Talaromyces rosarhiza sp. nov., of the section Talaromyces, is closely related to T. francoae. It differs from the latter by having both monoverticillate and biverticillate conidiophores, while those of T. francoae are biverticillate. Both novel endophytes are illustrated and described.

Keywords

multigene phylogenetics, morphology, new taxa, taxonomy, endophytic fungi

Introduction

Rosa roxburghii Tratt. is currently attracting significant attention for notably high vitamin C, superoxide dismutase and flavonoids (Xu et al. 2019). Vitamin C content of Gui Nong No. 5, one of the main cultivars, is 10–100 times higher than many other fruit and vegetable species, reaching 1000 mg/100 g (Xu et al. 2019). Rosa roxburghii is commonly used to treat diseases in traditional Chinese medicine, such as scurvy, type 2 diabetes mellitus and cacochylia (Wang et al. 2018, Wang et al. 2020). Its fruit is also widely applied in food and cosmetics for the development of functional products, due to diverse bioactive compounds with potential health-promoting properties (Yang et al. 2020). However, little is known about the endophytic fungi associated with this plant.

Endophytic fungi are phylogenetically diverse microorganisms that can colonise asymptomatically in various parts of living and healthy plants, ranging from the roots, stems and leaves to the flowers, fruits and seeds. To date, endophytes have been obtained from almost all plants investigated. Woody plants, in particular, may contain hundreds or thousands of fungal endophytes (Faeth and Fagan 2002, Jia et al. 2016). There are many endophytic fungi with various biological activities, such as growth promotion by producing plant hormones, enhancing host plant resistance to stresses through the production of bioactive compounds and facilitating the accumulation of medicinal ingredients, which is especially important for medicinal plants (Jia et al. 2016). Furthermore, they are also potential and inexhaustible bio-resources of many biologically-active compounds for medicinal applications (Abdel-Aziz et al. 2020). However, one should note that fungal endophytes may become pathogenic in the aging period of host plants or under specific environmental conditions (Park et al. 2017). Therefore, endophytic fungi play significant roles in agriculture and medicine.

Endophytic fungi belong to Ascomycota, Basidiomycota and Zygomycota, but Ascomycota is the predominant group compared to others (Gupta et al. 2020). During surveys of endophytic fungi associated with R. roxburghii, two strains of Ascodesmis and Talaromyces were collected in Guizhou Province (China). The genus Ascodesmis was introduced by Tieghem (1876), characterised by ascomata consisting of an unprotected bundle of asci only. Ascodesmis aurea and A. nigricans were considered to be the basic type of the Discomycetes (Brummelen 1981). Since 1981, no new species of Ascodesmis have been described worldwide. Ascospores are one of the most prominent morphologic features for species identification. Immature ascospores are hyaline and smooth before forming ornamentation. Upon maturity, the ascospores become pale to dark brown (Brummelen 1981, Kristiansen 2011).

The genus Talaromyces, within the order Eurotiales, was erected by Benjamin (1955) to accommodate teleomorphic Penicillium spp. The characteristic is soft cleistothecial ascomata, which are generally surrounded by multi-layered interwoven hyphae. This genus was redefined by Stolk and Samson (1972) and restricted to species producing only asci in chains. All gymnothecial ascomycetes that had the Penicillium state were included in Talaromyces by Pitt (1979). Later, phylogenetic research suggested that Talaromyces spp. and members of Penicillium subgenus Biverticillium belonged in a clade distinct from Penicillium sensu stricto (Houbraken and Samson 2011, Chen et al. 2016). Thereafter, all Penicillium subgenus Biverticillium spp. were transferred to Talaromyces according to the principle of nomenclatural priority and single name nomenclature (Samson et al. 2011). Subsequently, Talaromyces was subdivided into seven sections with eighty-eight species, based on multi-gene phylogenetic analyses, combined with morphological observations. These sections were Bacillispori, Helici, Islandici, Purpurei, Subinflati, Talaromyces and Trachyspermi (Yilmaz et al. 2014, Chen et al. 2016). Lately, a new section, section Tenues, was introduced by Sun et al. (2020) for T. tenuis. To date, this genus consists of more than 180 species, classified into eight sections.

In this study, we introduce Ascodesmis rosicola and Talaromyces rosarhiza as two novel taxa. Both of them are confirmed by multigene phylogeny and morphological characters.

Materials and methods

Sample collection

The wild-type R. roxburghii was collected from Guizhou Province, China (April and August 2020) (Table 1). Healthy tissues (roots and fruit) of R. roxburghii were collected randomly from the different sampling sites. All materials were sent to the laboratory immediately and stored in a refrigerator at 4°C. Each sample tissue was examined within 48 hours of collection (Ranjan et al. 2019).

Table 1.

Location charateristics and climate features of samples collection sites

Sampling site

Latitude and longitude

Altitude

(m)

Mean annual temperature

(℃)

Mean annual rainfall

(mm)

Mean annual sunshine hours (h)

Sampling time

Guiyang City,

Guizhou Province

27°4'50"N, 106°29'50"E

1184

15.3

1130

1235

22 April 2020

Liupanshui City,

Guizhou Province

25°52'52"N, 104°33'59"E

2047

15.2

1390

1593

4 August 2020

Isolation and culture

All tissues, especially roots, were cleaned under tap water for half an hour, rinsed with double-distilled water for 10 min,and dried under natural conditions. Then, they were cut into small pieces and transferred to the clean bench for surface disinfection. The margin of these pieces was trimmed off under aseptic conditions. All samples were surface-sterilised in 75% ethanol (1 min) and rinsed three times with sterile water. Subsequently, they were treated with 1% (w/v) aqueous sodium hyprochlorite (NaOCl) for several minutes (roots, 2 min; fruit, 1 min) and washed three times with sterile water again. After washing, the surface water of tissues was blotted with sterile filter papers. The potato dextrose agar (PDA, Shanghai Bio-way Technology Co., Ltd., China) medium had been supplemented with streptomycin sulphate (0.5 g/l) to avoid bacterial contamination. Each culture plate contained three segments. These plates were incubated at 28±1°C in a 12-h light:12-h dark photoperiod for 3 to 7 days. After several days, hyphal tips were subcultured into fresh PDA plates to obtain pure endophytic strains (Ranjan et al. 2019).

The effectiveness of surface sterilisation was monitored with three methods. First, the final rinsing water (0.1 ml) was coated on PDA plates (Rojas et al. 2020). The second way was that the surface-sterilised plant materials were rolled for 1 min and placed for 20 min on PDA plates and then removed (Li et al. 2016, Singh et al. 2017). In addition, during the isolation process, three open PDA plates were placed in the clean bench to confirm that colonies growing on isolated plates were produced from plant tissues and not contaminating fungi from the environment.

Morphology

Morphological identification was performed with PDA (Shanghai Bio-way Technology Co., Ltd., China), oatmeal agar (OA, Beijing Solarbio Science & Technology Co., Ltd., China), and malt-extract agar (MEA, Shanghai Bio-way Technology Co., Ltd., China). Macroscopic morphology, for example, growth rate, colony colour and pigmentation, were examined at 2-day intervals. Microscopic characteristics of ascospores or conidia were determined and recorded and spores of each strain were randomly selected for measurement (length and width) (Quezado et al. 2010), with the range and mean calculated. The colonies were observed under a stereomicroscope and morphological characters were confirmed by optical microscope (Olympus BX53, Japan).

DNA extraction, PCR amplification and sequencing

DNA was extracted from fresh mycelia grown on PDA for 7 to 10 days using the Fungal gDNA Isolation Kit (BW-GD2416, Biomiga, China), following the manufacturer’s instructions. The products were stored at -20°C until polymerase chain reaction (PCR) was performed. PCR amplifications were conducted on a T100 Thermal Cycler (BIO-RAD, USA). According to manufacturer’s instructions in a total volume of 25 μLl, including 12.5 µl 2X SanTaq PCR Mix (the mixture of MgCl2, dNTP, Taq DNA Polymerase, PCR buffer and PCR enhancer solution), 9.5 µl double-distilled water, 1.0 µl each of forward and reverse primers (0.05–1 µmol/l) and 1.0 µl DNA sample (1–10 ng/µl). The forward and reverse PCR reaction primers are shown in Table 2. The ITS, LSU, BenA, CaM and RPB2 genes were amplified as previously mentioned by White et al. (1990), Vilgalys and Hester (1990), Glass and Donaldson (1995), Hong et al. (2006) and Liu et al. (1999). The amplified PCR products were directed to Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, P. R. China), a commercial sequencing provider.

Table 2.

The best-fit evolutionary models in the phylogenetic analyses.

Genus

Phylogenetic analysis

Model

ITS (ITS4/ITS5)

LSU

(LR0R/LR5)

BenA

(Bt2a/Bt2b)

CaM

(Cmd5/Cmd6)

RPB2

(fRPB2-5F/

fRPB2-7cR)

Ascodesmis

ML analysis

TNe+I+G4

TIM3+F+I+G4

-

-

TNe+I+G4

BI analysis

GTR+F+I

GTR+F+I+G4

-

-

SYM+I+G4

Talaromyces

ML analysis

TNe+R3

-

TIM2e+I+G4

K2P+I+G4

K2P+I+G4

BI analysis

GTR+F+I+G4

-

GTR+F+I+G4

SYM+I+G4

SYM+I+G4

Phylogenetic analyses

Forward and reverse sequences were utilised to create consensus sequences by BioEdit v. 7.0.9.0 (Hall 1999) and BLASTn searched in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify the genus-level taxonomic status. Phylogenetic analyses were performed, based on the sequences generated in this study and recently published data (Hansen et al. 2013, Vu et al. 2019, Schoch et al. 2009, Hansen et al. 2005, Lindemann et al. 2019, Chen et al. 2016, Manoch et al. 2013, Sun et al. 2020, Visagie et al. 2015, Wang et al. 2016, Wang et al. 2017, Yilmaz et al. 2012, Yilmaz et al. 2014, Yilmaz et al. 2016), which were downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) (Table 3). The multiple alignments were automatically generated using MAFFT v. 7 webserver (https://mafft.cbrc.jp/alignment/server/) (Katoh et al. 2019). Alignments were also adjusted manually when needed in BioEdit v. 7.0.9.0 (Hall 1999) and concatenated in PhyloSuite v. 1.2.2 (Zhang et al. 2020).

Table 3.

Taxa used in this study and their corresponding GenBank accession numbers.

Species

Strain no.

GenBank accession no.

ITS

LSU

BenA

CaM

RPB2

Ascodesmis microscopica

CBS 275.80

MH861263

MH873032

-

-

-

A. nigricans

CBS 389.68 T

-

DQ168335

-

-

JX943761

A. sphaerospora

CBS 125.61 T

MH857994

MH869550

-

-

-

A. rosicola sp. nov.

GUCC 190035.1 T

MZ221601

MZ221605

-

-

MZ333139

A. rosicola sp. nov.

GUCC 190204.1

MZ221602

MZ221606

-

-

MZ333140

Boubovia nicholsonii

KH.03.65 (FH)

-

DQ220395

-

-

JX943755

Eleutherascus lectardii

CBS 626.71 T

MH860289

MH872042

-

-

-

E. lectardii

AFTOL-ID 933

-

DQ470966

-

-

DQ470918

E. peruvianus

CBS 101.75 T

-

DQ220330

-

-

JX943760

E. peruvianus

CBS 104.77

MH861030

MH872802

-

-

-

Geopyxis vulcanalis

KH.04.37

(FH, dupl. DBG)

-

KC012680

-

-

JX943770

Lasiobolus ciliatus

KS-94-005 (C)

-

DQ167411

-

-

-

L. papillatus

KH.08.30

-

KC012687

-

-

JX943758

Pseudocoprotus catenipilus

ALV9774

MH846260

MH846258

-

-

MH844626

P. catenipilus

ALV14334

MH846261

MH846259

-

-

-

Pseudombrophila theioleuca

DHP 3498 (FH)

-

KC012696

-

-

JX943756

Pulvinula constellatio

KH.03.64 (FH)

-

DQ062987

-

-

JX943773

Pu. convexella

KH.01.20 (C)

-

DQ062986

-

-

JX943772

Talaromyces assiutensis

CBS 147.78 T

JN899323

-

KJ865720

KJ885260

KM023305

T. bacillisporus

CBS 296.48 T= IMI 040045 = NRRL 1025

KM066182

-

AY753368

KJ885262

JF417425

T. calidicanius

CBS 112002 T

JN899319

-

HQ156944

KF741934

KM023311

T. cecidicola

CBS 101419 T= DAOM 233329

AY787844

-

FJ753295

KJ885287

KM023309

T. cinnabarinus

CBS 267.72 T= NHL 2673

JN899376

-

AY753377

KJ885256

JN121477

T. coalescens

CBS 103.83 T

JN899366

-

JX091390

KJ885267

KM023277

T. diversiformis

CBS 141931 T= CGMCC3.18204 = DTO 317-E3

KX961215

-

KX961216

KX961259

KX961274

T. duclauxii

CBS 322.48 T= IMI 040044 = MUCL 28672 = NRRL 1030

JN899342

-

JX091384

KF741955

JN121491

T. euchlorocarpius

PF 1203 = DTO 176-I3 T= DTO 176-I4

AB176617

-

KJ865733

KJ885271

KM023303

T. flavus

CBS 310.38 T= IMI 197477 = NRRL 2098

JN899360

-

JX494302

KF741949

JF417426

T. francoae

CBS 113134 T= IBT 23221 = DTO 056-D9

KX011510

-

KX011489

KX011501

MN969188

T. helicus

CBS 335.48 T= DSM 3705 = IMI 040593 = NRRL 2106

JN899359

-

KJ865725

KJ885289

KM023273

T. intermedius

CBS 152.65 T= BDUN 267 = IFO 31752 = IMI 100874

JN899332

-

JX091387

KJ885290

KX961282

T. islandicus

CBS 338.48 T= IMI 040042 = MUCL 31324 = NRRL 1036

KF984885

-

KF984655

KF984780

KF985018

T. kendrickii

CBS 136666 T = DTO 273-F4 = IBT 13593

KF741987

-

KF741921

KF741967

MN969158

T. mangshanicus

CGMCC 3.18013

KX447531

-

KX447530

KX447528

KX447527

T. marneffei

CBS 388.87 T = ATCC 18224= CBS 334.59 = IMI 068794ii = IMI 068794iii

JN899344

-

JX091389

KF741958

KM023283

T. mimosinus

CBS 659.80 T = FRR 1875 = IMI 223991

JN899338

-

KJ865726

KJ885272

MN969149

T. neorugulosus

CBS 140623 T= CGMCC3.18215 = DTO 318-A8

KU866659

-

KU866846

KU866743

KU867003

T. panamensis

CBS 128.89 T= IMI 297546

JN899362

-

HQ156948

KF741936

KM023284

T. purpureus

CBS 475.71 T= FRR 1731 = IMI 181546

JN899328

-

GU385739

KJ885292

JN121522

T. purpurogenus

CBS 286.36 T= IMI 091926

JN899372

-

JX315639

KF741947

JX315709

T. qii

AS3.15414 T= CBS 139515

KP765384

-

KP765380

KP765382

MN969164

T. resinae

AS 3.4387 = CBS 324.83 T= DTO 027-G5

MT079858

-

MN969442

MT066184

MN969221

T. reverso-olivaceus

CBS 140672 T= CGMCC3.18195 = DTO 317-C3

KU866646

-

KU866834

KU866730

KU866990

T. rosarhiza sp. nov.

GUCC 190040.1 T

MZ221603

-

MZ333143

MZ333137

MZ333141

T. rosarhiza sp. nov.

GUCC 197011.1

MZ221604

-

MZ333144

MZ333138

MZ33314

T. rugulosus

CBS 371.48 T= IMI 040041 = MUCL 31201 = NRRL 1045

KF984834

-

KF984575

KF984702

KF984925

T. stipitatus

CBS 375.48 T= NRRL 1006 = IMI 39805

JN899348

-

KM111288

KF741957

KM023280

T. subinflatus

CBS 652.95 T= IBT 17520

JN899397

-

KJ865737

KJ885280

KM023308

T. tenuis

CBS 141840 T = DTO 340-G9

MN864275

-

MN863344

MN863321

MN863333

T. thailandensis

CBS 133147 T= KUFC 3399

JX898041

-

JX494294

KF741940

KM023307

T. trachyspermus

CBS 373.48 T = IMI 040043

JN899354

-

KF114803

KJ885281

JF417432

T. tzapotlensis

NRRL 35203 T

KX946902

-

KX946884

KX946893

KX946922

T. viridis

CBS 114.72 T= ATCC 22467 = NRRL 5575

AF285782

-

JX494310

KF741935

JN121430

Trichocoma paradoxa

CBS 247.57, CBS 103.73, CBS 788.83 T

MH860643

-

JF417469

JF417506

JN121417

New species are marked in bold; T: indicates type or ex-type strains.

Maximum Likelihood (ML) and Bayesian Inference (BI) methods were used to construct the phylogenetic trees. The best-fit partition models were inferred for the independent sequence datasets using ModelFinder (Kalyaanamoorthy et al. 2017) integrated into PhyloSuite (Zhang et al. 2020) and the results were used for ML and BI (Table 2). ML phylogenies were conducted using IQ-TREE (Nguyen et al. 2014) under the edge-linked partition model for 10,000 ultrafast (Minh et al. 2013) bootstraps. Bootstrap support (BS) values were evaluated with 1000 replicates. BI phylogenies were calculated using MrBayes 3.2.6 (Ronquist et al. 2012) under the partition model (2 parallel runs, 2,000,000 generations), sampling every 1000 generations, the initial 25% of sampled data were discarded as burn-in and the remaining samples were used to generate the majority consensus tree and estimate the posterior probabilities (PP) (Maharachchikumbura et al. 2015). Phylograms were visualised in FigTree v.1.4.3 (Rambaut 2014) with bootstrap values (BS/PP) above or below the nodes and reorganised in Adobe Illustrator CC 2019.

Taxon treatments

Ascodesmis rosicola H. Zhang & Y. L. Jiang, sp. nov.

Materials    Download as CSV 
Holotype:
  1. scientificName:
    Ascodesmis rosicola
    ; kingdom:
    Fungi
    ; phylum:
    Ascomycota
    ; class:
    Pezizomycetes
    ; order:
    Pezizales
    ; family:
    Ascodesmidaceae
    ; genus:
    Ascodesmis
    ; country:
    China
    ; stateProvince:
    Guizhou
    ; locality:
    Guiyang
    ; catalogNumber:
    GUCC 190035.1
    ; recordedBy:
    Hong Zhang
    ; identifiedBy:
    Hong Zhang
    ; dateIdentified:
    2021
Other material:
  1. scientificName:
    Ascodesmis rosicola
    ; kingdom:
    Fungi
    ; phylum:
    Ascomycota
    ; class:
    Pezizomycetes
    ; order:
    Pezizales
    ; family:
    Ascodesmidaceae
    ; genus:
    Ascodesmis
    ; country:
    China
    ; stateProvince:
    Guizhou
    ; locality:
    Guiyang
    ; catalogNumber:
    GUCC 190204.1
    ; recordedBy:
    Hong Zhang
    ; identifiedBy:
    Hong Zhang
    ; dateIdentified:
    2021

Description

Endophytic fungi of R. roxburghii. Asexual morph not observed. Sexual morph (Fig. 1): Apothecia gregarious, superficial, sessile, 200–320 µm diameter, first hyaline, becoming brownish upon maturity. Excipulum absent. Asci broadly clavate or obovoid, with a short broad stalk or a broad base, 30.5–50.0 × 12.5–21.0 µm (av. = 38.0 × 19.0 µm, n = 30), 6–8-spored. Ascospores irregularly arranged, ellipsoid to perfectly spherical (length/breadth ratio 1.01–1.69, av. 1.32), at first hyaline, brownish upon maturity, 5.0–13.0 × 4.5–10.0 µm (av. = 10.5 × 8.0 µm, n = 30), at first smooth, later ornamented with a very variable pattern of isolated warts and spines, occasionally with a few fine connectives and one or two very obvious simple or branched ridges extending over the major part of the ascospore surface, very few without ridge. Paraphyses septate, simple and hyaline, 3.5–6.5 µm thick.

Figure 1.  

Ascodesmis rosicola (GUCC 190035.1, holotype). A-F On PDA, MEA and OA, respectively in 14 days at 28°C (A, C and E from above, B, D and F from below); G Sexual morph on SNA; H Paraphyses; I-K Apothecium and asci; L-Q Ascospores. Scale bars: G = 0.5 mm, H-K = 20 μm, L-Q = 10 μm.

Cultural characteristics: Description based on GUCC 190035.1. On PDA, reaching 6.0–7.5 cm in diameter after 14 days of cultivation in dark at 28°C, superficial, margin irregular, white, with abundant aerial mycelium, uneven, centre and margin uplifted; reverse yellowish. On MEA, 28°C: medium sparse, circular, flat. Mycelium white, cottony, margin regular. On OA, 28°C: radial, aerial mycelium sparse, pale white, growth regular. On MEA and OA, reaching 85 mm in diameter under the same conditions.

Etymology

The name refers to the host plant, Rosa roxburghii, from which this fungus was isolated.

Notes

Ascodesmis rosicola is introduced as a new species, based on morphological characteristic and phylogenetic analysis. The genus Ascodesmis, established by Tieghem (1876), presently contains 13 species (Trivedi et al. 1973, Brummelen 1981, Currah 1986), but these species have no available sequence data, except A. microscopica, A. nigricans and A. sphaerospora. In the phylogenetic analyses (Fig. 2), using the combined ITS, LSU and RPB2 sequence data of Pezizales, A. rosicola is related to Ascodesmis spp. The two A. rosicola isolates clustered with good support (BS = 94, PP = 1) and placed in a distinct clade, albeit with moderate support (BS = 61, PP = 0.92). This species shows some similarity to A. porcina in having ascospores with a conspicuously simple or branched ridge extending over the most part of the spore surface, which is well separated from other Ascodesmis spp. However, A. rosicola differs from A. porcin by its smaller asci (30.5–50.0 × 12.5–21.0 µm vs. 65–80 (–90) × 20–30 (–35) µm) and smooth surface of young ascospores (those of A. porcin are ornamented) (Brummelen 1981). Moreover, the hosts of A. rosicola and A. porcin (from the dung of donkey, goat, peccary, pig and rat) (Brummelen 1981) were notably different.

Figure 2.  

Phylogram generated from Maximum Likelihood analysis, based on combined ITS, LSU and RPB2 sequence data. Geopyxis vulcanalis (KH.04.37 (FH, dupl. DBG)) is used as the outgroup taxa. Bootstrap support values for ML greater than 50% and Bayesian posterior probabilities greater than 0.90 are given near nodes (BS/PP), respectively. The novel taxon is indicated in bold and black. T: type or ex-type.

Talaromyces rosarhiza H. Zhang & Y. L. Jiang, sp. nov.

Materials    Download as CSV 
Holotype:
  1. scientificName:
    Talaromyces rosarhiza
    ; kingdom:
    Fungi
    ; phylum:
    Ascomycota
    ; class:
    Eurotiomycetes
    ; order:
    Eurotiales
    ; family:
    Trichocomaceae
    ; genus:
    Talaromyces
    ; country:
    China
    ; stateProvince:
    Guizhou
    ; locality:
    Guiyang
    ; catalogNumber:
    GUCC 190040.1
    ; recordedBy:
    Hong Zhang
    ; identifiedBy:
    Hong Zhang
    ; dateIdentified:
    2021
Other material:
  1. scientificName:
    Talaromyces rosarhiza
    ; kingdom:
    Fungi
    ; phylum:
    Ascomycota
    ; class:
    Eurotiomycetes
    ; order:
    Eurotiales
    ; family:
    Trichocomaceae
    ; genus:
    Talaromyces
    ; country:
    China
    ; stateProvince:
    Guizhou
    ; locality:
    Guiyang
    ; catalogNumber:
    GUCC 197011.1
    ; recordedBy:
    Hong Zhang
    ; identifiedBy:
    Hong Zhang
    ; dateIdentified:
    2021

Description

Endophyte of R. roxburghii. Sexual morph not observed. Asexual morph (Fig. 3): Mycelium superficial, pale brown, septate, branched. Conidiophores monoverticillate and biverticillate, a minor proportion terverticillate, macronematous, mostly straight, smooth, branched, thick-walled. For biverticillate conidiophores, metulae 2–3, appressed or divergent, 6.0–14.5 × 1.5–3.0 µm (av. = 10.5 × 2.5 µm, n = 30); phialides ampulliform, tapering into very thin neck, 2–3 per metula, 6.5–15.0 × 1.5–3.5 µm (av. = 10.0 × 2.5 µm, n = 30). For monoverticillate ones, phialides 1–6, ampulliform, tapering into very thin neck, 10.5–16.0 × 2.5–4.0 µm (av. = 12.5 × 3.0 µm, n = 30), conidia subglobose to ellipsoidal, smooth-walled, 2.5–4.0 × 2.0–3.0 µm (av. = 3.0 × 2.5 µm, n = 30).

Figure 3.  

Talaromyces rosarhiza (GUCC 190040.1, holotype). A-F On PDA, OA and MEA, respectively in 14 days at 28°C (A, C and E from above; B, D and F from below); G-M Asexual morph on OA (G-L Conidiophores; M Conidia). Scale bars: G-I = 20 μm, J-M = 10 μm.

Cultural characteristics: Description based on GUCC 190040.1. On PDA, reaching 50 mm in diameter after 14 days of cultivation in dark at 28°C; moderately deep, slightly sulcate, flat; margin entire, mycelium white. On OA, reaching 42 mm in diameter under the same conditions, low, flat; margin low, entire; mycelium white; velvety; abundant sporulation; conidia en masse dark olive green. On MEA 28°C, 14 days: reaching 12 mm; raised; margin low, flat, entire; mycelium white; floccose to velvety; abundant sporulation, conidia en masse dull green; soluble pigments absent; exudates absent; reverse greyish orange.

Etymology

The word "rosarhiza" originated from “rosa” referring to the host plant, Rosa roxburghii and “rhiza” referring to root, from which this fungus was isolated.

Notes

Talaromyces rosarhiza is described as a new species, based on morphology and phylogenetic analyses. Phylogenetic analysis (Fig. 4) was carried out using combined ITS, BenA, CaM and RPB2 sequence data. Clustered together with T. francoae, T. kendrickii, T. mangshanicus, T. qii and T. thailandensis and belonged to section Talaromyces. The two T. rosarhiza isolates clustered with strong support (BS = 100, PP = 1) and closely related to T. francoae, but located in a distinct clade with good support (BS = 90, PP = 1). Detailed morphological differences between T. rosarhiza and its related taxa are summarised in Table 4 (Yilmaz et al. 2016, Visagie et al. 2015, Wang et al. 2017, Wang et al. 2016, Manoch et al. 2013). Talaromyces rosarhiza can be distinguished from T. francoae by its monoverticillate and biverticillate conidiophores (while T. francoae biverticillate), number of metulae per verticil (2–3 vs. 3–6), number of phialides per metulae (2–3 vs. 3–6), conidia shape (ellipsoidal vs. globose) and conidial wall (smooth vs. verrucose, rough) (Yilmaz et al. 2016).

Table 4.

Morphological comparisons between Talaromyces rosarhiza and its allies.

Species

Conidiophores pattern

No. of metulae per verticil

Metulae size (μm)

No. of phialides per metulae

Phialides size (μm)

Conidia shape

Conidial walls

Conidia size (μm)

T. rosarhiza sp. nov.

Monoverticillate and biverticillate

2–3

6.0–14.5 × 2.0–3.0

2-3

6.5–15.0 × 1.5–3.5

Ellipsoidal

Smooth

2.5–3.5 × 2.0 –3.0

T. francoae

Biverticillate

3-6

8–13 × 2.5–4.5

3–6

8.5–12 × 2.5–4

Globose

Verrucose, rough

2.5–4 × 2.5–4

T. kendrickii

Biverticillate

3–8

10–13 × 3–4

3–5

9–12 × 2.5– 3.5

Subglobose

Roughened

2.5–3 × 2.5–3

T. mangshanicus

Biverticillate

3–6

11–13.5 × 4–5

3–6

10.5–13.5 × 3.5–4.5

Subglobose to ellipsoidal

Echinulate

4.5–5.5 × 4–5

T. qii

Biverticillate

4–6

7–11 × 2.5–3

2–4

7–9×2–2.5

Ovoid to subglobose

Echinulate

3–3.5

T. thailandensis

Biverticillate

3–5

7.2–10.9 × 2.2–3.4

3–7

11.5–13.7 × 1.5–2.4

Subglobose to ellipsoidal

Smooth

1.8–2.4 × 1.7 –2.3

Figure 4.  

Phylogram generated from Maximum Likelihood analysis, based on combined ITS, BenA, CaM and RPB2 sequence data. Trichocoma paradoxa (CBS 788.83) is used as the outgroup taxa. Bootstrap support values for ML greater than 50% and Bayesian posterior probabilities greater than 0.90 are given near nodes (BS/PP), respectively. The new taxon is indicated in bold and black. T: type or ex-type.

Analysis

Phylogenetic analyses

Ascodesmis rosicola: For the genus Ascodesmis, only three species, A. microscopica, A. nigricans and A. sphaerospora, have available sequence data. Therefore, phylogenetic relationships were deduced using sequence data of Pezizales. Nineteen strains were included in the combined analyses (ITS, LSU and RPB2) which comprised 1999 characters (561 for ITS, 819 for LSU, 619 for RPB2). The tree topology of the ML analysis was similar to the BI analysis. Two new strains, GUCC 190035.1 and GUCC 190204.1, were related to Ascodesmis spp., clustered with good support (BS = 94, PP = 1) and formed an independent moderately-supported clade (BS = 61, PP = 0.92) (Fig. 2).

Talaromyces rosarhiza: Phylogenetic relationships were investigated using sequence data of Talaromyces. Thirty-six strains were included in the combined analyses (ITS, BenA, CaM and RPB2) which comprised 2549 characters (541 for ITS, 570 for BenA, 647 for CaM, 791 for RPB2). The tree topology of ML analysis was similar to the BI analysis. Two new strains, GUCC 190040.1 and GUCC 197011.1, clustered with strong support (BS = 100, PP = 1) and formed a group with five species of Talaromyces that had been reported and belonged to section Talaromyces. Talaromyces rosarhiza was closely related to T. francoae, but placed in a distinct clade with high bootstrap support (BS = 90) and posterior probability (PP = 1) (Fig. 4).

Discussion

In this study, A. rosicola and T. rosarhiza, two new endophytes isolated from R. roxburghii in China, are proposed and described. Multigene phylogenetic analyses revealed that A. rosicola was phylogenetically close to A. nigricans and A. sphaerospora and was placed in a distinct clade with moderate support. However, combined with morphological characters, the novel species is confirmed. Similarly, based on morphology and phylogenetic analyses, T. rosarhiza is suggested as a new species.

Although there are 13 species of Ascodesmis listed in Mycobank (https://www.mycobank.org/), A. aurea and A. hawaiiensis are considered dubious species due to the lack of adequate description and material (Brummelen 1981). In addition, A. caninus and A. reticulata are two synonyms of A. microscopica and A. echinulata is regarded as a synonym of A. nigricans (Brummelen 1981). They are rarely collected, possibly because of their size, which hardly exceeds 0.5 mm in diameter or they are really rare (Kristiansen 2011).

Talaromyces, another genus in this study, is important in biotechnology, medicine and the food industry (Yilmaz et al. 2014). Talaromyces spp. are isolated from assorted substrates around the world (Yilmaz et al. 2016), including soil, plant, air, animals, food, dust, human and dung. Notably, many pathogenic fungi of Talaromyces have been shown to possess potential biological activities. For example, T. albobiverticillius causes post-harvest fruit rot on pomegranate (Mincuzzi et al. 2017),but it has been shown to possess anti-inflammatory properties (Bai et al. 2020). Talaromyces pinophilus, as a pathogen, has been reported to cause post-harvest rot of sugar beet (Beta vulgaris) (Haque and Parvin 2020); however, this fungus is also a promising biocontrol agent, able to inhibit Pythium and Rhizoctonia-induced damping-off of cucumber (Kazerooni et al. 2019). Thus, further investigations on the pathogenicity and biological activity of T. rosarhiza are needed.

In the present study, a total of 127 strains of endophytic fungi were successfully isolated from R. roxburghii and categorized into six classes (Sordariomycetes, Dothideomycetes, Eurotiomycetes, Pezizomycetes, Leotiomycetes and Agaricomycetes), of which strains GUCC 190035.1, GUCC 190204.1, GUCC 190040.1 and GUCC 197011.1 were identified as two new taxa. As known, besides agriculture and medicine, many endophytic fungi may play significant roles in shaping and maintaining the balance of microbial communities in plants (Su et al. 2016). Therefore, further research is also necessary for the ecological significance of A. rosicola and T. rosarhiza.

Acknowledgements

This work is supported by the following projects: the National Natural Science Foundation of China (No. 32060009) and the Guizhou Provincial Science and Technology Projects ([2020]1Y043 and [2018]5404).

References