Morphology and multigene phylogeny of Talaromyces amyrossmaniae, a new synnematous species belonging to the section Trachyspermi from India

Kunhiraman C. Rajeshkumar; Neriman Yilmaz; Sayali D. Marathe; Keith A. Seifert

doi:10.3897/mycokeys.45.32549

Abstract

A new Talaromyces species, T. amyrossmaniae, isolated from decaying fruit and litter of Terminalia bellerica, is described and illustrated. On the natural substrate, the new species produces determinate synnemata, with a well-defined, vivid orange red to orange red cylindrical stipe, and a greyish green capitulum. Conidiophores are typically biverticillate, or sometimes have subterminal branches, with acerose phialides that produce globose to subglobose, smooth to slightly roughened conidia. Multigene phylogenetic analyses based on the internal transcribed spacer region (ITS), and partial sequences of β-tubulin (BenA), calmodulin (CaM), and DNA directed RNA polymerase second large subunit (RPB2) genes, along with morphological characterization, revealed that these isolates are distinct and form a unique lineage of Talaromyces in section Trachyspermi, closely allied to T. aerius, T. albobiverticillius, T. heiheensis, T. erythromellis, and T. solicola. The new species T. amyrossmaniae is the first species in section Trachyspermi with determinate synnemata.

Keywords

BenA , CaM , conidial fungi, RPB2 , synnemata, Trichocomaceae , Western Ghats

Introduction

The genus Talaromyces was described as a teleomorph-based holomorph genus (Benjamin 1955). It is characterized by cleistothecial ascomata that have a soft hyphal exterior giving them a yellow, cream, pink or reddish coloration; its anamorphs are predominantly biverticillate or rarely terverticillate conidiophores with acerose phialides with a narrow mouth (Samson et al. 2011, Yilmaz et al. 2014). Conventionally, species of Talaromyces were linked with Penicillium, Paecilomyces, Geosmithia, and Merimbla anamorphs (Pitt 1980, Pitt et al. 2000). Primary phylogenetic studies of Talaromyces spp. revealed that they form a distinct clade that includes species formerly classified in Penicillium subgenus Biverticillium, separate from Eupenicillium and Penicillium spp. in other subgenera (LoBuglio et al. 1993; Seifert et al. 1993; Berbee et al. 1995; Peterson 2000; Heredia et al. 2001; Seifert et al. 2004). As redefined following the new single name provision of the International Code of Nomenclature of algae, fungi and plants (ICN), Talaromyces was expanded to include asexual species formerly included in Penicillium subgenus Biverticillium (Samson et al. 2011; Visagie and Jacobs 2012; Visagie et al. 2012; Yilmaz et al. 2012, 2014). The landmark multigene phylogeny of Penicillium and allied genera by Houbraken and Samson (2011) segregated the prevailing concept of the family Trichocomaceae into three families, Aspergillaceae, Thermoascaceae, and Trichocomaceae. Talaromyces sensu stricto is presently classified in the Trichocomaceae along with Thermomyces, Sagenomella, Rasamsonia, and Trichocoma. The molecular taxonomy and nomenclature Talaromyces were comprehensively revised in the recent past (Houbraken and Samson 2011; Samson et al. 2011; Seifert et al. 2012; Visagie and Jacobs 2012; Visagie et al. 2012; Yilmaz et al. 2012; Yilmaz et al. 2014). Yilmaz et al. (2014) resolved the phylogenetic positioning of Talaromyces species using a polyphasic taxonomic concept and placing 88 accepted species in seven well-defined sections, namely Bacillispori, Helici, Islandici, Purpurei, Subinflati, Talaromyces, and Trachyspermi. Subsequent to the monograph by Yilmaz et al. (2014), 54 new Talaromyces species have been described from all over the world (Visagie et al. 2015; Chen et al. 2016; Luo et al. 2016; Crous et al. 2016; Romero et al. 2016; Wang et al. 2016; Wang et al. 2016; Yilmaz et al. 2016a, b; Crous et al. 2017; Guevara-Suarez et al. 2017; Peterson and Jurjević 2017; Wang et al. 2017; Barbosa et al. 2018; Su and Niu 2018; Jiang et al. 2018; Varriale et al. 2018).

During the 2009 monsoon season, routine surveys were conducted to explore microfungal diversity in natural forests of Lingmala waterfalls area (17.9218N; 73.6870E) of Mahabaleshwar, northern Western Ghats, India. A previously undescribed synnema-forming fungus with penicillate conidiophores and phialidic conidiogenous cells was collected from decaying fruits and litter of Terminalia bellerica (Combretaceae) fallen onto the ground near the Lingmala waterfalls. The fungus was isolated into pure culture on different culture media, microscopic characters were recorded and its classification studied using phylogenetic analysis of aligned DNA sequences from the nuclear ribosomal ITS region and BenA, CaM, and RPB2 partial gene sequences. This paper aims to resolve the taxonomy and phylogeny of this synnematous species, which is shown to represent a new species in Talaromyces section Trachyspermi, here named T. amyrossmaniae.

Materials and methods

Isolation

Conidia were removed from synnemata directly from the surface of fallen fruits under a Nikon stereomicroscope (model SMZ1500 with Digital camera; Nikon, Tokyo, Japan) and placed on malt extract agar (MEA) media containing the antibiotic Streptomycin sulphate (100 mg/L) CMS220-5G (HIMEDIA Laboratories Pvt. Ltd, Mumbai, India). Methods and media used for examining colony characters, inoculating and incubating cultures, and microscopic examination followed those of Visagie et al. (2014), with the addition of Oatmeal Agar (OA), and Potato Dextrose Agar (PDA), with incubation occurring in a Bio Multi Incubator (Model LH-30-8CT, Japan). Herbarium specimens were deposited in the Ajrekar Mycological Herbarium (AMH); cultures were accessioned and preserved in the National Fungal Culture Collection of India (NFCCI; WDCM-932), Agharkar Research Institute, Pune, India. Reference and ex-type strains used in this study are listed in Table 1.

Table 1.

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Accession numbers for fungal strains and strains used for the phylogenetic analysis.

Species	Collection no.	Substrate and origin	GenBank accession no.
Species	Collection no.	Substrate and origin	ITS	BenA	CaM	RPB2
T. aerius	CBS 140611^T	Indoor air, China	KU866647	KU866835	KU866731	KU866991
T. albobiverticillius	CBS 133440^T	Decaying leaves of a broad leaved tree, Taiwan	HQ605705	KF114778	KJ885258	KM023310
T. albobiverticillius	CBS 140498	Air from HVAC system, China	KR855658	KR855648	KR855653	KR855663
*T. amyrossmaniae*	NFCCI 1919^T	*Fallen decaying fruits of Terminalia bellerica* (Combretaceae), Maharashtra, India**	MH909062	MH909064	MH909068	MH909066
*T. amyrossmaniae*	NFCCI 2351	*Fallen decaying fruits of Terminalia bellerica* (Combretaceae), Maharashtra, India**	MH909063	MH909065	MH909069	MH909067
T. assiutensis	CBS 147.78^T	Soil, Egypt	JN899323	KJ865720	KJ885260	KM023305
T. assiutensis	CBS 645.80	Gossypium, India	JN899334	KF114802	*	*
T. atroroseus	CBS 133442^T	House dust, South Africa	KF114747	KF114789	KJ775418	KM023288
T. atroroseus	CBS 133449	Mouse dung, Denmark	KF114744	KF114788	*	*
T. austrocalifornicus	CBS 644.95^T	Soil, USA	JN899357	KJ865732	KJ885261	*
T. brasiliensis	CBS 142493^T	Honey of Melipona scutellaris; Recife, Pernambuco, Brazil	MF278323	LT855560	LT855563	LT855566
T. convolutus	CBS 100537^T	Soil, Nepal	JN899330	KF114773	*	JN121414
T. diversus	CBS 320.48^T	Leather, USA	KJ865740	KJ865723	KJ885268	KM023285
T. diversus	DTO 244-E6	House dust, New Zealand	KJ775712	KJ775205	*	*
T. erythromellis	CBS 644.80^T	Soil from creek bank, New South Wales	JN899383	HQ156945	KJ885270	KM023290
T. heiheensis	HMAS 248789^T	Rotten wood, China	KX447526	KX447525	KX447532	KX447529
T. minioluteus	CBS 642.68^T	Unknown	JN899346	KF114799	KJ885273	JF417443
	CBS 270.35	Zea mays, USA	KM066172	KM066129	*	*
	CBS 137.84	Fruit damaged by insect, Spain	KM066171	KF114798	*	*
T. minnesotensis	CBS 142381^T	Human ear, USA	LT558966	LT559083	LT795604	LT795605
T. solicola	DAOM 241015^T	Soil, South Africa	FJ160264	GU385731	KJ885279	KM023295
T. solicola	CBS 133446	Soil, South Africa	KF114730	KF114775	*	*
T. systylus	BAFCcult3419^T	Soil, Argentina	KP026917	KR233838	KR233837	*
T. trachyspermus	CBS 373.48^T	Unknown, USA	JN899354	KF114803	KJ885281	JF417432
T. trachyspermus	CBS 118437	Soil, Morocco	KM066169	KM066127	*	*
T. ucrainicus	CBS 162.67^T	Unknown	JN899394	KF114771	KJ885282	KM023289
T. ucrainicus	CBS 127.64	Soil treated with cyanimide, Germany (ex-type of T. ohiensis)	KM066173	KF114772	*	*
T. udagawae	CBS 579.72^T	Soil, Japan	JN899350	KF114796	KX961260	*

Morphology

Colony characters were recorded after 7 d of incubation on various media, including Czapek yeast autolysate agar (CYA), Blakeslee’s (1915) malt extract agar (MEAbl), yeast extract sucrose agar (YES), oatmeal agar (OA), and creatine sucrose agar (CREA). Bacto malt extract was used for MEAbl. Media preparation, inoculations, incubation conditions, and microscopic preparations followed the recommendations by Visagie et al. (2014). Colour codes and names used in descriptions are from Kornerup and Wanscher (1967). Microscopic observations were made with an Olympus (Model CX-41, Japan) dissecting microscope and Zeiss (AXIO Imager 2, Germany) compound microscope equipped with Nikon Digital sight DS-Fi1 and AxioCam MRc5 cameras driven by AxioVision Rel 4.8 software (AXIO Imager 2, Germany).

DNA extraction, amplification, and phylogenetic analyses

Colonies were grown on MEAbl plates, and genomic DNA was extracted following the rapid salt extraction method of Aljanabi and Martinez (1997). The ITS regions was amplified using primer pairs ITS5 and ITS4 (White et al. 1990). For the amplification of RPB2 gene region, primer pairs RPB2-5F and RPB2-7cR (Liu et al. 1999) were used with touch-up PCR conditions: 5 cycles with annealing temperature 48 °C followed by 5 cycles at 50 °C and final 25 cycles at 52 °C. The partial BenA gene was amplified with primer pair Bt2a and Bt2b (Glass and Donaldson 1995) with 50 °C as annealing temperature. The partial CaM gene was amplified using primer pair CF1M and CF4 (Hubka et al. 2014) subjected to 32 cycles under the following temperature regime: first cycle at 95 °C for 3 min, 55 °C for 30 seconds, and 72 °C for 1 min; followed by 30 cycles at 95 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 1 min; and a final cycle at 95 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 10 min. PCR products were purified with StrataPrep PCR Purification Kit (Agilent Technologies, TX, USA) and sequenced using the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Sequencing reactions were run on a ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems, USA).

Sequence alignment and phylogenetic analysis

Reference sequences of Talaromyces section Trachyspermi were downloaded from GenBank and aligned in MAFFT v. 7.305b (Katoh and Standley 2013) with the newly generated sequences. Alignments were manually adjusted in Geneious as needed. A Maximum Likelihood analysis was done in IQtree v. 1.6 (Nguyen et al. 2015) after selecting the most suitable substitution model with the Modelfinder (Kalyaanamoorthy) algorithm built into the software. The trees were visualized in Figtree v. 1.4.3 ((http://tree.bio.ed.ac.uk/software/figtree) and edited for publication in Affinity Designer v. 1.6.1 (Serif Europe Ltd, UK). The new DNA sequences were deposited in GenBank (Table 1).

Results

Phylogenetic analyses

The phylogenetic analysis showed that the new species described below as Talaromyces amyrossmaniae belongs to section Trachyspermi. The relationships of the new species with accepted species and its genetic coherence and phylogenetic consistency were analysed with single concatenated sequence datasets based on four loci (ITS, BenA, CaM and RPB2). The length of the data sets were 540 bp for ITS, 379 bp for BenA, 550 bp for CaM, and 851 bp for RPB2 loci. The best fitting models for the ITS analysis were TPM2u+F+I+G4 for ITS, TIM2e+G4 for BenA, K2P+I+G4 for CaM, and K2P+I+G4 for RPB2. All trees were rooted with T. pinophilus (CBS 631.66). The single gene trees and the multigene phylogram are shown in Figures 1, 2.

Figure 1.

Maximum likelihood (ITS) phylogenetic trees of ITS region, BenA, CaM and RPB2 genes of strains belong Talaromyces section Trachyspermi. Talaromyces pinophilus (CBS 631.66^T) was chosen as outgroup. Bootstrap values above 70% are indicated. Purple names indicate T. amyrossmaniae strains. ^T: ex-type.

Figure 2.

Maximum likelihood (ITS) combined phylogenetic trees using ITS region, BenA, CaM and RPB2 genes of strains belong Talaromyces section Trachyspermi. Talaromyces pinophilus (CBS 631.66^T) was chosen as outgroup. Bootstrap values above 70% are indicated. Purple names indicate T. amyrossmaniae strains. ^T: ex-type.

Because of the limited resolution of the official fungal DNA barcode, the ITS (Schoch et al. 2012), in the Trichocomaceae, BenA was proposed as the secondary DNA barcode for Talaromyces (Yilmaz et al. 2014). The overall tree topologies of ITS and BenA phylogenies had relatively consistent association of species. However, the type species of section Trachyspermi, T. trachyspermus was well separated from T. assiutensis in the BenA analysis whereas strains of the two species were intermixed in the ITS analysis. Talaromyces ucraicinus was consistently a sister clade to T. trachyspermus and T. assiutensis. Our proposed new species, T. amyrossmaniae, was distinguished from other species both by ITS and other markers (Figs 1, 2). It is consistently included in a major clade along with T. aerius, T. albobiverticillius, T. erythromellis, T. heiheensis, and T. solicola in the ITS analysis. As with the ITS, in the concatenated phylogeny and RPB2 analyses, T. amyrossmaniae clustered with T. albobiverticillius, T. heiheensis, T. erythromellis, T. aerius, and T. solicola (Figs 1, 2). However, in the BenA phylogeny, T. amyrossmaniae was segregated from that major-clade. Talaromyces amyrossmaniae is clustered with T. austrocalifornicus in the CaM analyses (Fig. 1). Yilmaz et al. (2014) mentioned that amplification of CaM is difficult in section Trachyspermi. CaM data could not be analyzed critically because the new sequences generated through Sanger sequencing (ABI PRISM 3100 Genetic Analyzer) contained a homopolymer stretch of around 30 bp after base 320, resulting in poor quality and short sequences, even after many attempts using modified PCR conditions and primers.

Morphology

In this study, we introduce one new species, Talaromyces amyrossmaniae belonging to section Trachyspermi. Strains conform with the general morphological characters of this section. Talaromyces amyrossmaniae was compared with its close relatives, with the distinguishing characters mentioned in the note after the species description. Also, Table 2 compares the new species with the closely allied species in section Trachyspermi. The main character that differentiates T. amyrossmaniae from other synnemata-producing species in the genus Talaromyces is the length of the synnemata. Talaromyces amyrossmaniae has the shortest synnemata (up to 150 µm). A synopsis of comparative morphology and growth rate of synnema producing species of Talaromyces is given in Table 3.

Table 2.

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Comparative morphology of Talaromyces section Trachyspermi.

Species	Conidiophore branching	Conidia ornamentation	Conidial shape	Conidial size (µm)
T. aerius	Biverticillate, minor proportion with subterminal branches	Smooth	Ellipsoidal	2–3.5 (–4.5) × 2–3
T. albobiverticillius	Biverticillate, minor proportion with subterminal branches	Smooth to finely roughened	Globose to subglobose	2–3.5 (–4) × 1.5–2.5
*T. amyrossmaniae*	Biverticillate, minor proportion with subterminal branches	Smooth to finely roughened	Globose or subglobose	2.5–4 (–6) × 2.5–3.5 (–8)
T. assiutensis	Mono to biverticillate	Smooth	Ovoidal to ellipsoidal	2–4 × 1.5–2.5
T. atroroseus	Biverticillate, minor proportion with subterminal branches	Finely roughened to rough	Ellipsoidal	2–3.5 × 1.5–2.5
T. austrocalifornicus	Biverticillate	Smooth	Subglobose	1.5–3 × 1.5–2.5
T. brasiliensis	Biverticillate	Finely roughened	Globose	2 × 3
T. convolutus	Mono to biverticillate	Smooth	Ellipsoidal	(2–) 3–4 × 1.5–2 (–3)
T. diversus	Biverticillate, minor proportion with subterminal branches	Smooth to finely roughened	Subglobose to ellipsoidal	2–3 (–5) × 2–3 (–3.5)
T. erythromellis	Biverticillate having symmetrical subterminal branches,	Smooth	Subglobose to ellipsoidal	2–3.5 × 1.5–2.5
T. heiheensis	Biverticillate with subterminal branches, minor proportionquaterverticillate	Smooth	Subglobose to ellipsoidal	2.5–3 × 2–2.5
T. minioluteus	Biverticillate	Smooth	Ellipsoidal	2.5–4 × 1.5–2.5
T. minnesotensis	Biverticillate	Smooth	Ellipsoidal	2.5–3.5 × 2–3
T. solicola	Biverticillate	Rough	Globose to subglobose	2–3.5 × 2–2.5
T. systylus	Biverticillate	Rough	Globose	3.5 × 4
T. trachyspermus	Mono to biverticillate	Smooth	Ellipsoidal	2–3.5 (–5) × 1.5–2.5
T. ucrainicus	Mono to biverticillate	Smooth	Broadly ellipsoidal to ovoidal	2–4 (–5) × 1.5–2.5 (–3)
T. udagawae	Biverticillate	Smooth	Subglobose to ellipsoidal	3–4 × 2–3

Table 3.

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Synopsis of comparative morphology and growth rate of synnema producing species of Talaromyces.

Species	Section	Synnemata			Growth rates (mm)
Species	Section	Shape	Time of production	Height (µm)	Acid production on CREA	CYA 25 °C	CYA 37 °C	MEA 25 °C
T. amyrossmaniae ^a	Trachyspermi	Determinate	Prolonged	Up to 150	Absent	4–6	No growth	12–14
T. calidicanius ^b	Talaromyces	Determinate	Prolonged	Up to 6000	Moderate	27–30	No growth	47–48
T. cecidicola ^b	Purpurei	Determinate	Prolonged	Up to 1250	Absent	33–34	No growth	37–38
T. choloroloma ^b	Purpurei	Determinate	Prolonged	Up to 1200	Weak to moderate	40–45	No growth	45–48
T. coalescens ^b	Purpurei	Determinate	Prolonged	Up to 1200	Very weak	32–34	2–4	43–45
T. dendriticus ^b	Purpurei	Determinate	Prolonged	Up to 5000	Absent	23–26	5–6	35–36
T. duclauxii ^b	Talaromyces	Indeterminate	After 7d	Up to 5000	Weak	25–27	3–4	48–50
T. flavovirens ^b	Talaromyces	Determinate, covered or masked by yellow mycelial covering	Prolonged	Up to 750	Absent	19–20	5–6	37–38
T. palmae ^b	Subinflati	Indeterminate	Prolonged	Up to 8000	Weak	20–25	No growth	22–26
T. panamensis ^b	Talaromyces	Determinate, cone shaped and often sterile	After 7d	Up to 6800	Strong	23–24	No growth	28–30
T. pittii ^b	Purpurei	Determinate, phototropic	Prolonged	Up to 1000	Absent	34–36	No growth	42–44
T. pseudostromaticus ^b	Purpurei	Determinate	Prolonged	Up to 8000	Absent	25–34	No growth	38–43
T. ramulosus ^b	Purpurei	Determinate	Prolonged	Up to 500	Absent	32–40	5–8	45–48
T. systylus ^c	Trachyspermi	Indeterminate	Prolonged	Up to 4000	Good	14–18	16–19	18–21

Taxonomy

Talaromyces amyrossmaniae Rajeshkumar, Yilmaz & Seifert, sp. nov.

MycoBank No: 518601

Figure 3

Etymology

Latin, named after Dr Amy Y. Rossman, in honour of her career as a research leader in Systematic Mycology and Microbiology, USDA ARS, Beltsville, Maryland, USA.

Diagnosis

Synnemata abundant in nature, determinate, 90–120 µm tall, with an unbranched stalk 10–35 µm wide, base wider, up to 50–60 µm. Synnema stipe orange red or vivid orange red, capitulum terminal, compact, globose with conidiophores and a powdery grey-green conidial mass in closely packed, split columns. On MEAbl synnemata produced after 2 weeks incubation, up to 120 µm long. Conidiophores biverticillate, with sometime terverticilate sub-branches. Acerose phialides producing smooth to slightly roughened globose to subglobose conidia. Restricted growth on all media, acid production absent on CREA.

In: Talaromyces section Trachyspermi.

Type

INDIA, Maharashtra, Mahabaleshwar, Lingmala falls; isolated from fallen decaying fruits and litter of Terminalia bellerica (Combretaceae), 9 June 2009, isolated by K.C.Rajeshkumar, holotype: AMH 9330, extype: NFCCI 1919, other culture NFCCI 2351.

Gene sequences: ex-holotype MH909062(ITS), MH909064(BenA), MH909068(CaM), MH909066(RPB2).

Description

Colony diameter, 7 d (mm): CYA 4–6; CYA 37 °C no growth; MEAbl 12–14; YES 5–7; DG18 4–5; OA 10–13; CREA 3–5.

Colony characters: CYA 25 °C, 7 d: colonies low, plane; margins low, entire (< 1 mm); mycelia white; no germination; sporulation absent; soluble pigmentation absent; exudates absent; reverse Yellowish white (4A2). MEAbl 25 °C, 7 d: colonies low, slightly raised, synnemata present; margins low, entire (1 mm); mycelia white; texture velvety; sporulation dense (except margins); conidia en masse Dull green (27D4–27E4); soluble pigmentation yellow; exudates orange to reddish orange small droplets; reverse Hazel brown (6E6) at center fading into Light brown (6D8) to Light yellow (3A5). YES 25 °C, 7 d: colonies slightly raised, sulcate, sunken at center; margins low, entire (< 1 mm); mycelia pale pinkish red, with and appearance of Pastel red (7A4–7A5); texture floccose; sporulation absent; soluble pigmentation absent; exudates absent; reverse Light brown (7D6). DG18 25 °C, 7 d: colonies slightly raised, sulcate, sunken at center; margins low, entire (1 mm); mycelia pale yellow; texture floccose; sporulation moderately dense at center, margins absent; conidia en masse Greyish green to Dull green (26C4–26D4); soluble pigmentation absent; exudates absent; reverse Brownish orange to Brownish yellow (5C6) in the center, fading into Light yellow (4A5). OA 25 °C, 7 d: colonies low, plane; margins low, entire (< 1 mm); mycelia white; texture velvety; sporulation moderately dense at center, margins absent; conidia en masse Greyish green (27C4–27D4); soluble pigmentation dark red; exudates absent; reverse Brown (7E8) in the centre, fading into Copper red (7C8). CREA 25 °C, 7 d: acid production absent.

Figure 3.

Talaromyces amyrossmaniae (NFCCI 1919) A Colonies on CYA, MEAbl (obverse and reverse), Colonies obverse on YES, OA, DG18, CREAB Synnemata on Terminalia bellerica fruit in nature C Synnema formation on MEAbl after 14 d at 25 °C D–F Biverticillate penicilli G Biverticillate penicilli with subterminal branches H Conidia. Scale bar: 10 µm.

Micromorphology

Determinate synnemata formed after 2 weeks on MEAbl up to 80–150 µm long. Conidiophores biverticillate with a minor proportion having subterminal branches; stipes smooth walled 80–120 × 3–4 µm; extra branches up to 30 µm long; metulae three to six, divergent, 10–13 × 2.5–3 µm; phialides acerose, three to six per metulae, 12–15 (–18) × 2–3 µm; conidia smooth, globose to subglobose, 2.5–4 × 2.5–3.5 µm. Sometimes it produces large-sized conidia up to 6–8 µm. Ascomata not observed.

Discussion

In our study, a novel Talaromyces species, T. amyrossmaniae is described based on two isolates from decaying fruits and litter of Terminalia bellerica (Combretaceae). We used ITS, BenA, CaM and RPB2 sequences to apply genealogical concordance phylogenetic species recognition (GCPSR; Taylor et al. 2000) to delineate the species, and a multigene phylogenetic analysis to place T. amyrossmaniae in Talaromyces section Trachyspermi. Talaromyces section Trachyspermi (as ‘trachyspermus’) was introduced by Yaguchi et al. (1996) based on ubiquinone systems, overriding the traditional morphology based classification of Talaromyces. Yilmaz et al. (2014) applied multigene phylogenies and morphology to redefine classification of Talaromyces and divided the genus into seven sections. They noted that Talaromyces section Trachyspermi includes species with generally biverticillate penicilli with acerose phialides and when ascomata are produced, they are creamish white or yellow. Colonies generally grow restrictedly on CYA, YES, CREA and DG18; some species have colonies with abundant red pigments.

Morphologically, T. amyrossmaniae resembles the other species of section Trachyspermi and produces dark orange to red pigmentation on MEAbl, restricted growth on MEAbl, CYA, DG18, YES and CREA, symmetrical biverticillate penicilli with a minor proportion having sub-terminal branches, and acerose phialides that form globose to subglobose, smooth to slightly roughened conidia. Although, synnematous Talaromyces species also are found in section Purpurei, section Talaromyces, section Trachyspermi, and section Subinflati, T. amyrossmaniae is the first species in section Trachyspermi with determinate synnemata that are seen on fallen decaying fruits in nature and also on MEAbl after 7–14 d of incubation at 25 °C. Talaromyces systylus is another synnema producer in section Trachyspermi, but it produces indeterminate synnemata up to 4000 µm and grows at 37 °C (Romero et al. 2016). Talaromyces amyrossmaniae has the shortest synnemata in Talaromyces and it grows very restrictedly compared to the other synnema producing species on CYA and MEAbl. With these key characters, it is easy to distinguish the new species from the other synnemata producing species of Talaromyces.

Based on ITS, BenA, CaM, and RPB2 phylogenies, T. amyrossmaniae is part of the same clade as T. austrocalifornicus, T. convolutus, T. heiheensis, T. aerius, T. solicola, T. albobiverticillius, and T. erythromellis; however, it can be distinguished from all of these species by having determinate synnemata in nature and by differences in colony growth characteristics. Also, T. amyrossmaniae forms predominant concentric rings of synnemata on the different media used in our studies and even forms synnemata in vitro on MEAbl.

Many species of Talaromyces have been recorded as saprophytes, endophytes, and human pathogens from different geoclimatic regions and microhabitats across India. Most importantly, Talaromyces marneffei is a potentially pathogenic thermally dimorphic fungus causing systemic mycosis in HIV-infected patients; its dissemination was thoroughly studied from Manipur state of India (Singh et al. 1999; Ranjana et al. 2002). Recent studies on endophytic T. pinophilus isolated from the rhizomes of Curcuma amada from Karnataka revealed the production and partial characterization of L-asparaginase (Krishnapura and Belur 2016). Likewise, endophytic T. radicus, isolated from Catharanthus roseus, produces vincristine and vinblastine and was studied for induce apoptotic cell death (Palem et al. 2015). Talaromyces flavus is also recorded as an endophytic fungi isolated from ethno-medicinal plants in the sacred forests of Meghalaya having antimicrobial and antioxidant activity (Bhagobaty and Joshi 2012). Devi et al. (2014) reported a marine strain of T. verruculosus, from Andhra Pradesh, as a potent polyhydroxybutyrate degrader. Similarly, the stress-tolerant soil fungus T. funiculosus, isolated from the neem rhizosphere, was identified as a potential strain for phosphate solubilization (Kanseet al. 2015). Talaromyces flavus isolated from paddy rhizosphere of Darjeeling Hills exhibited phosphate solubilizing activity in vitro and positively influenced the growth of Oryza sativa, Cicer arietinum, and Vigna radiata under greenhouse conditions (Chakraborty et al. 2011). A keratin degrading strain of T. trachyspermus was isolated from the grounds of a gelatin factory in Jabalpur, Madhya Pradesh, and digested human hair in stationary culture (Rajak et al. 1991). Talaromyces trachyspermus was reported as a soil saprophyte in paddy fields of Orissa (Dutta and Ghosh 1965). Species identifications of these Talaromyces strains were mostly based on micro- and macro morphological characters in these studies. Because such approaches often underestimate species diversity, adoption of a polyphasic approach to authenticate such identifications will increase the number of Talaromyces species known from different eco-geographic zones of India. Further investigation is also needed to study the ecological importance of these species.

Acknowledgements

KC Rajeshkumar thanks SERB, Department of Science and Technology, Government of India for providing financial support under the project YSS/2015/001590; Dr Amy Y. Rossman for invaluable support and guidance in mycology and Dr K.M. Paknikar (Director, ARI) provided facilities and motivation in our work.

References

Aljanabi SM, Martinez I (1997) Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Research 25: 4692–4693. https://doi.org/10.1093/nar/25.22.4692

Barbosa RN, Bezerra JD, Souza-Motta CM, Frisvad JC, Samson RA, Oliveira NT, Houbraken J (2018) New Penicillium and Talaromyces species from honey, pollen and nests of stingless bees. Antonie van Leeuwenhoek 13: 1–30. https://doi.org/10.1007/s10482-018-1081

Benjamin CR (1955) Ascocarps of Aspergillus and Penicillium. Mycologia 47: 669–687. https://doi.org/10.2307/3755578

Berbee ML, Yoshimura A, Sojiyamaj Taylor JW (1995) Is Penicillium monophyletic? An evaluation of phylogeny in the family Trichochomaceae 18S, 5.8S and ITS ribosomal DNA sequence data. Mycologia 87: 201–222. https://doi.org/10.2307/3760907

Bhagobaty RK, Joshi SR (2012) Antimicrobial and antioxidant activity of endophytic fungi isolated from ethnomedicinal plants of the “Sacred forests” of Meghalaya, India. Mikologia Lekarska 19: 5–11.

Blakeslee AF (1915) Lindner’s roll tube method of separation cultures. Phytopathology 5: 68–69.

Chakraborty BN, Chakraborty U, Sunar K, Dey PL (2011) RAPD profile and rDNA sequence analysis of Talaromyces flavus and Trichoderma species. Indian Journal of Biotechnology 10: 487–495.

Chen AJ, Sun BD, Houbraken J, Frisvad JC, Yilmaz N, Zhou YG, Samson RA (2016) New Talaromyces species from indoor environments in China. Studies in Mycology 84: 119–144. https://doi.org/10.1016/j.simyco.2016.11.003

Crous PW, Wingfield MJ, Burgess TI, Hardy GS, Crane C, Barrett S, Cano-Lira JF, Le Roux JJ, Thangavel R, Guarro J, Stchigel AM (2016) Fungal Planet description sheets 469–557. Persoonia 37: 252–253. https://doi.org/10.3767/003158516X694499

Crous PW, Wingfield MJ, Burgess TI, Carnegie AJ, Hardy GS, Smith D, Summerell BA, Cano-Lira JF, Guarro J, Houbraken J, Lombard L (2017) Fungal Planet description sheets 625–715. Persoonia 39: 460–461. https://doi.org/10.3767/persoonia.2017.39.11

Devi SS, Sreenivasulu Y, Rao KV (2014) Talaromyces verruculosus, a novel marine fungi as a potent polyhydroxybutyrate degrader. Research Journal of Pharmacy and Technology 7: 433–438.

Dutta BG, Ghosh GR (1965) Soil fungi from Orissa (India) IV. Soil fungi of paddy fields. Mycopathologia et Mycologia Applicata 25: 316–322. https://doi.org/10.1007/BF02049919

Glass NL, Donaldson GC (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61: 1323–1330.

Guevara-Suarez M, Sutton DA, Gené J, García D, Wiederhold N, Guarro J, Cano‐Lira JF (2017) Four new species of Talaromyces from clinical sources. Mycoses 60: 651–662. https://doi.org/10.1111/myc.12640

Heredia G, Rayes M, Aria RM, Bills GF (2001) Talaromyces ocotl sp. nov., and observation on T. rotundus from conifer forest soils of Veracruz State, Mexico. Mycologia 93: 528–540. https://doi.org/10.2307/3761738

Houbraken J, Samson R A (2011) Phylogeny of Penicillium and the segregation of Trichocomaceae into three families. Studies in Mycology 70: 1–51. https://doi.org/10.3114/sim.2011.70.01

Hubka V, Lyskova P, Frisvad JC, Peterson SW, Skorepova M, Kolarik M (2014) Aspergillus pragensis sp. nov. discovered during molecular reidentification of clinical isolates belonging to Aspergillus section Candidi. Medical Mycology 52: 565–576. https://doi.org/10.1093/mmy/myu022

Jiang XZ, Yu ZD, Ruan YM, Wang L (2018) . Three new species of Talaromyces sect. Talaromyces discovered from soil in China. Scientific Reports 8: 4932. https://doi.org/10.1038/s41598-018-23370-x

Kanse OS, Whitelaw-Weckert M, Kadam TA, Bhosale HJ (2015) Phosphate solubilization by stress-tolerant soil fungus Talaromyces funiculosus SLS8 isolated from the Neem rhizosphere. Annals of Microbiology 65: 85–93. https://doi.org/10.1007/s13213-014-0839-6

Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30: 772–780. https://doi.org/10.1093/molbev/mst010

Kornerup A, Wanscher JH (1967) Methuen Handbook of Colour (2^nd edn). Methuen, London.

Krishnapura PR, Belur PD (2016) Partial purification and characterization of l-asparaginase from an endophytic Talaromyces pinophilus isolated from the rhizomes of Curcuma amada. Journal of Molecular Catalysis B: Enzymatic 124: 83–91. https://doi.org/10.1016/j.molcatb.2015.12.007

Liu YJ, Whelen S, Hall BD (1999) Phylogenetic relationships among ascomycetes: Evidence from an RNA polymerse II subunit. Molecular Biology and Evolution 16: 1799–1808. https://doi.org/10.1093/oxfordjournals.molbev.a026092

LoBuglio KF, Pitt JI, Taylor JW (1993) Phylogenetic analysis of two ribosomal DNA regions indicates multiple independent losses of a sexual Talaromyces state among asexual Penicillium species in subgenus Biverticillium. Mycologia 85: 592–604. https://doi.org/10.2307/3760506

Luo Y, Lu X, Bi W, Liu F, Gao W (2016) Talaromyces rubrifaciens, a new species discovered from heating, ventilation and air conditioning systems in China. Mycologia 108: 773–779. https://doi.org/10.3852/15-233

Nguyen LT, Schmidt HA, Haeseler A Von, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32: 268–274. https://doi.org/10.1093/molbev/msu300

Palem PP, Kuriakose GC, Jayabaskaran C (2015) An endophytic fungus, Talaromyces radicus, isolated from Catharanthus roseus, produces vincristine and vinblastine, which induce apoptotic cell death. PloS One 10: e0144476. https://doi.org/10.1371/journal.pone.0144476

Peterson SW (2000) Phylogenic analysis of Penicillium species based on ITS and LSU-rDNA nucleotide sequences. In: Samson RA, Pitt JI (Eds) Integration of modern taxonomic methods for Penicillium and Aspergillus. The Netherlands: Harwood Academic Publishers, 163–178.

Peterson SW, Jurjević Ž (2017) New species of Talaromyces isolated from maize, indoor air, and other substrates. Mycologia 109: 537–556. https://doi.org/10.1080/00275514.2017.1369339

Pitt JI (1979) The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press, London, 634 pp.

Pitt JI, Samson RA, Frisvad JC (2000) List of accepted species and their synonyms in the family Trichocomaceae. In: Samson RA, Pitt JI (Eds) Integration of modern taxonomic methods for Penicillium and Aspergillus classiﬁcation.Harwood Academic Press, Amsterdam, 9–79.

Rajak RC, Parwekar S, Malviya H, Hasija SK (1991) Keratin degradation by fungi isolated from the grounds of a gelatin factory in Jabalpur, India. Mycopathologia 114: 83–87. https://doi.org/10.1007/BF00436426

Ranjana KH, Priyokumar K, Singh TJ, Gupta CC, Sharmila L (2002) Disseminated Penicillium marneffei infection among HIV-infected patients in Manipur state, India. Journal of Infection 45: 268–271. https://doi.org/10.1053/jinf.2002.1062

Romero SM, Romero AI, Barrera V, Comerio R (2016) Talaromyces systylus, a new synnematous species from Argentinean semiarid soil. Nova Hedwigia 102: 241–256. https://doi.org/10.1127/nova_hedwigia/2015/0306

Samson RA, Yilmaz N, Houbraken J, Spierenburg H, Seifert KA, Peterson SW, Varga J, Frisvad JC (2011) Phylogeny and nomenclature of the genus Talaromyces and taxa accommodated in Penicillium subgenus Biverticillium. Studies in Mycology 70: 159–184. https://doi.org/10.3114/sim.2011.70.04

Schoch CL, Seifert KA, Huhndorf S, Robert V, Spouge JL, Levesque CA, Chen W, Bolchacova E, Voigt K, Crous PW, Miller AN (2012) Nuclear ribosomal internal transcribed spacer (ITS) region as universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United States of America 109: 6241–6246. https://doi.org/10.1073/pnas.1117018109

Seifert KA, Frisvad JC, Houbraken J, Llimona X, Peterson SW, Samson RA, Visagie CM (2012) (2051) Proposal to conserve the name Talaromyces over Lasioderma (Ascomycota). Taxon 61: 461–462.

Seifert KA, Frisvad JC, McLean MA (1993) Penicillium kananaskense, a new species from Alberta soil. Canadian Journal of Botany 72: 20–24. https://doi.org/10.1139/b94-004

Seifert KA, Hoekstra ES, Frisvad JC, Louis-Seize G (2004) Penicillium cecedicola, a new species on cynipid insect galls on Quercus pacifica in the western United States. Studies in Mycology 50: 517–523.

Singh PN, Ranjana K, Singh YI, Singh KP, Sharma SS, Kulachandra M, Nabakumar Y, Chakrabarti A, Padhye AA, Kaufman L, Ajello L (1999) Indigenous disseminated Penicillium marneffei infection in the State of Manipur, India: Report of four autochthonous cases. Journal of Clinical Microbiology 37: 2699–2702.

Su L, Niu YC (2018) Multilocus phylogenetic analysis of Talaromyces species isolated from curcurbit plants in China and description of two new species, T. curcurbitiradicus and T. endophyticus. Mycologia 110(2): 375–386. https://doi.org/10.1080/00275514.2018.1432221

Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC (2000) Phylogenetic species recognition and species concepts in fungi. Fungal Genetics and Biology 31: 21–32. https://doi.org/10.1006/fgbi.2000.1228

Varriale S, Houbraken J, Granchi Z, Pepe O, Cerullo G, Ventorino V, Chin-A-Woeng T, Meijer M, Riley R, Grigoriev IV, Henrissat B, de Vries RP, Faraco V (2018) Talaromyces borbonicus sp. nov., a novel fungus from biodegraded Arundo donax with potential abilities in lignocellulose conversion. Mycologia 27: 1–9. https://doi.org/10.1080/00275514.2018.1456835

Visagie CM, Houbraken J, Frisvad JC, Hong S-B, Klaassen CH, Perrone G, Seifert KA, Varga J, Yaguchi T, Samson RA (2014) Identification and nomenclature of the genus Penicillium. Studies in Mycology 78: 343–371. https://doi.org/10.1016/j.simyco.2014.09.001

Visagie CM, Jacobs K (2012) Three new additions to the genus Talaromyces isolated from Atlantis sandveld fynbos soils. Persoonia 28: 14–24. https://doi.org/10.3767/003158512X632455

Visagie CM, Llimona X, Vila J, Louis-Seize G, Seifert KA (2012) Phylogenetic relationships and the newly discovered sexual state of Talaromyces ﬂavovirens, comb. nov. Mycotaxon 122: 399–411. https://doi.org/10.5248/122.399

Visagie CM, Yilmaz N, Frisvad JC, Houbraken J, Seifert KA, Samson RA, Jacobs K (2015) Five new Talaromyces species with ampulliform-like phialides and globose rough walled conidia resembling T. verruculosus. Mycoscience 56: 486–502. https://doi.org/10.1016/j.myc.2015.02.005

Wang QM, Zhang YH, Wang B, Wang L (2016) Talaromyces neofusisporus and T. qii, two new species of section Talaromyces isolated from plant leaves in Tibet, China. Scientific Reports 6: 18622. https://doi.org/10.1038/srep18622

Wang XC, Chen K, Xia YW, Wang L, Li T, Zhuang WY (2016) A new species of Talaromyces (Trichocomaceae) from the Xisha Islands, Hainan, China. Phytotaxa 267: 187–200. https://doi.org/10.11646/phytotaxa.267.3.2

Wang XC, Chen K, Qin WT, Zhuang WY (2017) Talaromyces heiheensis and T. mangshanicus, two new species from China. Mycological Progress 16: 73–81. https://doi.org/10.1007/s11557-016-1251-3

White TJ, Bruns T, Lee J, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR protocols: a guide to methods and applications.Academic Press, San Diego, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1

Yaguchi T, Someya A, Udagawa SI (1996) A reappraisal of intrageneric classification of Talaromyces based on the ubiquinone systems. Mycoscience 37: 55–60. https://doi.org/10.1007/BF02461457

Yilmaz N, Houbraken J, Hoekstra ES, Frisvad JC, Visagie CM, Samson RA (2012) Delimitation and characterization of Talaromyces purpurogenus and related species. Persoonia 29: 39–54. https://doi.org/10.3767/003158512X659500

Yilmaz N, López-Quintero CA, Vasco-Palacios AM, Frisvad JC, Theelen B, Boekhout T, Samson RA, Houbraken J (2016a) Four novel Talaromyces species isolated from leaf litter from Colombian Amazon rain forests. Mycological Progress 15: 1041–1056. https://doi.org/10.1007/s11557-016-1227-3

Yilmaz N, Visagie CM, Frisvad JC, Houbraken J, Jacobs K, Samson RA (2016b) Taxonomic re-evaluation of species in Talaromyces section Islandici, using a polyphasic approach. Persoonia 36: 37–56. https://doi.org/10.3767/003158516X688270

Yilmaz N, Visagie CM, Houbraken J, Frisvad JC, Samson RA (2014) Polyphasic taxonomy of the genus Talaromyces. Studies in Mycology 78: 175–341. https://doi.org/10.1016/j.simyco.2014.08.001