Camarosporium arezzoensis on Cytisus sp., an addition to sexual state of Camarosporium sensu stricto.

During a study of saprobic fungi from Bagno di Cetica Province, Italy, we collected a pleosporoid ascomycete on stems of Cytisus sp. In morphology, our collection is similar to Cucurbitaria species, but molecular analysis of SSU, LSU and ITS genes reveals it can be referred to Camarosporium. In this study we compare all other Cucurbitaria species from Cytisus sp. and based on both morphology and molecular data, we introduce our collection as a new species in Camarosporium viz. C. arezzoensis.

Introduction

The genus Camarosporium was introduced by Schulzer (1870) with Camarosporium quaternatum (Hazsl.) Schulz. as the type species. Index Fungorum (2015) lists 508 records as Camarosporium which was formerly recognised as asexual morphs in Botryosphaeriales, Cucurbitariaceae, Phaeosphaeriaceae and related genera (Kirk et al., 2008, Wijayawardene et al., 2012, Doilom et al., 2013, Hyde et al., 2013). However, Wijayawardene et al., 2014a, Wijayawardene et al., 2014b showed that Camarosporium sensu stricto belongs to Pleosporineae, Pleosporales and has cucurbitaria-like sexual morphs.

During our on-going studies, we found a new taxon with bitunicate asci and muriform ascospores which is morphologically similar with members in Cucurbitariaceae, Pleosporales (Doilom et al., 2013, Hyde et al., 2013). The blast results of small subunit rDNA (SSU), large subunit rDNA (LSU) and internal transcribed spacer (ITS) showed this taxon is related to Camarosporium sp. Thus we have carried out molecular analyses viz. maximum-parsimony (MP) and confirmed its placement in Pleosporineae, Pleosporales. As our new collection groups with Camarosporium sensu stricto, we introduce it as a new species of Camarosporium viz. C. arezzoensis.

Materials and methods

Sample collection and morphological study

Fresh fungal specimens were obtained from recent collections made in Italy. Morphological structures were examined under a Carl Zeiss microscopy GmbH (AxioCam ERC 5S) stereo microscope. To observe the fungal structures, ascomata were picked up and put into rehydrated water or lactoglycerol. For hand cross sections 5% KOH was added prior to examination. Microscopic fungal structures were mounted in water for observation under a Nikon ECLIPSE80i compound microscope and photographs were taken with a Cannon 550D digital camera fitted to the microscope. All micro morphologies were measured using Tarosoft® Image Framework program v.0.9.0.7.

Isolation

Single spore isolation was carried out following the method described in Chomnunti et al. (2014) on potato-dextrose agar (PDA). Germinated spores were transferred to fresh PDA media and incubated at 16 °C. Culture characteristics were observed after four weeks and these cultures were also used for molecular study. The specimens are deposited in the Mae Fah Luang University (MFLU) Herbarium, Chiang Rai, Thailand. Living cultures are deposited at the Mae Fah Luang University Culture Collection (MFLUCC) Chiang Rai, Thailand, Centraalbureau Voor Schimmelcultures, Netherlands (CBS) and International Collection of Microorganisms from Plants, New Zealand (ICMP).

DNA extraction, PCR amplification and sequencing

Mycelia grown on PDA media at 16 °C for four weeks were used for DNA extraction. Total DNA extraction was established by using a Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co., Ltd., Hangzhou, PR China). The concentration of DNA was determined using an ultraviolet spectrophotometer. PCR reactions were carried out according to Telle and Thines (2008) with the primers ITS1-F (Gardes and Bruns, 1993) and ITS4 (White et al., 1990) to amplify the complete internal transcribed spacer (ITS) region. Twenty micro litres (20 μl) of the reaction mixture contained 2 Mix 10 μl, ITS1-F 0.35 μl, ITS4 0.35 μl, 50 ng/μl DNA 0.6 μl, ddH2O 8.7 μl for each sample. The PCR programme was set according to Douanla et al. (2005) with the following modifications: an initial denaturation at 94 °C for 3 min, annealing at 55 °C for 45 s, and extension at 72 °C for 1 min, and a final elongation step of 7 min at 72 °C. To check the PCR products, 1% agarose gel electrophoresis (AGE) for 30 min at 220V was used. All PCR products were sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for purification and sequencing.

Molecular phylogenetic analysis

BLAST searches of LSU, SSU and ITS sequence data were carried out to reveal the closest taxa to our strain in GenBank (http://www.ncbi.nlm.nih.gov/). Combined analyses of LSU, SSU and ITS dataset of the closest relatives in Coniothyriaceae, Cucurbitariaceae and Pleosporaceae were used to carry out phylogenetic analyses. Bioedit v.7.2.5 (Hall, 2004), ClustalW v.1.6 (Thompson et al., 1997) and MAFFT v.6 (Katoh et al., 2002, Katoh and Toh, 2008) online sequence alignment editor under the default settings (mafft.cbrc.jp/alignment/server/) were used for aligning the sequences separately for each gene region. The individual datasets were finally combined into one dataset and used PAUP v. 4.0b10 (Swofford, 2002) to perform maximum-parsimony (MP) analysis by bootstrap analysis with 10,000 replicates. All multiple, equally parsimonious trees were saved and descriptive tree statistics for parsimony consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI) were calculated. The robustness of the best parsimonious tree was estimated by a bootstrap (BT) value with 10,000 replicates, each with 10 replicates of random stepwise addition of taxa (Liu et al., 2011, Phookamsak et al., 2013), and the trees were figured in Treeview v.1.6.6.

Results

Phylogenetic analysis

The combined gene data set of SSU, ITS and LSU rDNA consists of 23 taxa including our strain of IT 791 (MFLUCC 14-0238) and the outgroup taxon Leptosphaeria doliolum (CBS 541.66). The dataset consists of 2092 characters including coded alignment gaps; 1835 are constant, and 114 are parsimony informative in the MP analysis. A best scoring tree is shown in Fig. 1. Bootstrap support (BS) values of MP (equal to or above 50% based on 10,000 replicates) are shown above branches (TL = 447, CI = 0.694, RI = 0.700, RC = 0.486, HI = 0.306). Our strain of MFLUCC 14-0238 belongs to the genus Camarosporium sensu stricto and were separated from representative species of the genus with a relatively higher bootstrap values as circumscribed by Wijayawardene et al. (2014b).

Figure 1

One of the most parsimonious trees generated with SSU, ITS and LSU rDNA combined data analysis. The tree is rooted with Leptosphaeria doliolum (CBS 541.66). Type and ex-type strains are in bold. Newly introduced species in red.

Taxonomy

Camarosporium arezzoensis Tibpromma, Wijayawardene, Camporesi & K.D. Hyde, sp. nov.

Index Fungorum Number: IF550877; Facesoffungi number: 00382

Etymology: Refers to the name of the province in Italy where the fungus was collected

Saprobic on decaying plant stems of Cytisus sp. Sexual morph: Ascomata 400–500 μm high, 450–550 μm diam. (=449 × 482 μm, n = 10), black, semi-immersed, scattered beneath the host periderm or on decorticated wood, fully or partly erumpent, globose, rough or hairy, with an ostiole. Ostiole central, short, slightly sunken, minute and inconspicuous at the surface, smooth, ostiolar canal filled with hyaline cells. Peridium 30–45 μm wide at the base, 35–70 μm wide in sides, thick, comprising 8–10 layers, outer layer heavily pigmented, thick-walled, comprising blackish to dark brown cells of textura angularis, inner layer composed of hyaline, thin-walled cells of textura angularis. Hamathecium comprising numerous, 5.5 μm (n = 40) wide, filamentous, branched septate, pseudoparaphyses. Asci 180–240 × 10–15 μm (=199 × 13 μm, n = 40), 8-spored, bitunicate, fissitunicate, cylindrical, short-pedicellate, apex rounded with a minute ocular chamber. Ascospores 19–28 × 9–15 μm (=26 × 12 μm, n = 50), partially overlapping, mostly ellipsoidal, muriform, with 5–7 transverse septa, with 4–6 longitudinal septa, constricted at the central septum, initially hyaline, becoming brown at maturity, with slightly paler ends, conical and narrowly rounded at the ends, not surrounded by a mucilaginous sheath.

Culture characteristics: on PDA reaching 2 cm diam. after 4 weeks at 16 °C, later with dense mycelium, circular, rough margin white at first, iron-grey after 6 weeks, reverse cinnamon, flat on the surface, without aerial mycelium. Hyphae septate, branched, hyaline, thin (see Fig. 2).

Figure 2

Camerosporium arezzoensis (holotype). (a) Ascomata on host substrate. (b) Section of ascoma. (c) Section of peridium. (d) Light brown hyphae around ascomata. (e) Pseudoparaphyses. (f–i) Asci. (j–n) Ascospores. Scale bars: b = 200 μm, c = 50 μm, d–i = 20 μm, j–n = 10 μm.

Material examined: ITALY, Arezzo Province, Bagno di Cetica, on stems of Cytisus sp., 1 October 2012, Erio Camporesi IT791 (MFLU14-0636, holotype), extype living cultures, MFLUCC 14-0238, CBS, ICMP (see Table 1).

Table 1

Strains used in this study (Type and ex-type strains are in bold, the new taxon is indicated with an asterisk).

Taxon	Culture collection number	GenBank Accession number
SSU	ITS	LSU
Alternaria alternata	EN24	–	FJ809940	–
Camarosporium aloes	CPC 21572	–	KF777142	KF777198
Camarosporium clematidis	MFLUCC 13-0336	KJ589414	KJ562213	KJ562188
Camarosporium elongata	AFTOL-ID 1568	DQ678009	–	DQ678061
Camarosporium elongata	MFLUCC 14-0260	–	–	KJ724249
Camarosporium arezzoensis∗	MFLUCC 14-0238	KP120928	KP120926	KP120927
Camarosporium quaternatum	CBS 483.95	GU296141	–	GU301806
Camarosporium robinium	MFLUCC 13-0527	KJ589415	KJ562214	KJ589412
Camarosporium spartii	MFLUCC 13-0548	KJ589416	–	KJ589413
Cochliobolus heterostrophus	ATCC 64121	–	JX094779	JX094789
Coniothyrium palmarum	CBS 400.71	EU754054	AY720708	JX681084
Decorospora gaudefroyi	CBS 332.63	AF394542	AF394541	–
Leptosphaeria doliolum	CBS 541.66	–	JF740206	JF740284
Pleospora herbarum	CBS 191.86	GU238232	–	GU238160
Pleospora typhicola	CBS 132.69	JF740105	–	JF740325
Pyrenophora phaeocomes	AFTOL-ID283	–	DQ491507	DQ499596
Pyrenophora tritici-repentis	DAOM 226213	–	JN943670	JN940071
Cucurbitaria berberidis	CBS 363.93	GQ387545	JF740191	GQ387606
Cucurbitaria berberidis	MFLUCC 11-0387	KC506800	–	KC506796
Cucurbitaria berberidis	MFLUCC 11-0386	KC506799	–	KC506795
Pyrenochaeta nobilis	CBS 407.76	EU754107	–	EU754206
Pyrenochaetopsis decipiens	CBS 343.85	GQ387563	–	GQ387624
Pyrenochaeta quercina	CBS 115095	GQ387558	–	GQ387619

Notes: Mirza (1968) and Ellis and Ellis (1985) have listed Cucurbitaria cytisi Mirza, Cucurbitaria laburni (Pers.) De Not., Cucurbitaria obducens (Schumach.) Petr. and Camarosporium spartii (Nees ex Fr.) Ces. & De Not. on Cytisus sp. We compared our collection with those species (Table 2). Molecular data analysis confirms our stain groups with C. quaternatum Schulzer (Schulzer, 1870), the type species of Camarosporium and other Camarosporium spp. C. arezzoensis however, differs in having 180–240 × 10–15 μm asci and 19–28 × 9–15 μm brown ascospores. Our new species should be considered as Camarosporium sensu stricto and it is not congeneric with Cucurbitaria sensu stricto (Cucurbitariaceae) (Fig. 1).

Table 2

Comparison of our strain with the morphologically similar species in Mirza (1968).

Name	Ascomata	Peridium	Hypostoma	Asci	Ascospore
Camarosporium arezzoensis (In this study)	Black, semi-immersed, scattered beneath the host periderm or on decorticated wood, fully or partly erumpent, globose, rough or hairy, with an ostiole	Thick, comprising 8–10 layers, outer layer heavily pigmented, thick-walled, comprising blackish to dark brown cells of textura angularis, inner layer composed of hyaline, thin-walled cells of textura angularis	Comprising numerous, filamentous, branched septate, pseudoparaphyses	8-spored, bitunicate, fissitunicate, cylindrical, short-pedicellate, apex rounded with a minute ocular chamber	Partially overlapped, mostly ellipsoidal, muriform, with 5–7 transverse septa, with 4–6 longitudinal septa, constricted at the central septum, initially hyaline, becoming brown at maturity, with slightly paler ends, conical and narrowly rounded at the ends, not surrounded by a mucilaginous sheath
Cucurbitaria ahmadi	Erumpent, globose to subglobose or obovate, papilla bearing a comparatively wide ostiole	Uniform on sides, made up of dark-brown polygonal cells	Well developed, light-brown densely interwoven hyphae	Long stipitate, 4–8 spores, spore overlapped uniseriately or biseriately	Golden-brown, 3–7 transverse septa, one longitudinal septum
Cucurbitaria ononidis	Globose to subglobose, forming a slight depression bearing ostiole, papilla lacking	Slightly rough surface sometimes provided with hair-like structures	Poorly developed, a subiculum of dark-brown	Short stipitate, 4–8 spores, spore overlapped uniseriately	Brown, 5–9 transverse septa, 1–3 longitudinal septa
Cucurbitaria elaeagni	Erumpent, globose to subglobose	Slightly rough surface, made up of elongated polygonal cells, hyaline	Well developed, brown	Long stipitate, 4–8 spores, spore overlapped uniseriately or biseriately	Golden to dull brown, 5–7 transverse septa, up to 2 longitudinal septa

Discussion

Pleosporales is the largest order of Dothideomycetes (Kirk et al., 2008) and several studies have been carried out using multi-gene phylogeny, providing the groundwork towards a natural classification of the class (Nelsen et al., 2009, Nelsen et al., 2011, Schoch et al., 2009, Boonmee et al., 2011, Boonmee et al., 2012, Boonmee et al., 2014, Chomnunti et al., 2011, Chomnunti et al., 2014, Liu et al., 2011, Liu et al., 2012, Zhang et al., 2011, Zhang et al., 2012, Hyde et al., 2013, Wijayawardene et al., 2014c). Schoch et al. (2009) recognised the suborders Pleosporinae and Massarinae in Pleosporales and Zhang et al. (2012) confirmed it in their molecular data analyses. In their molecular data analyses, Wijayawardene et al., 2014a, Wijayawardene et al., 2014b, Wijayawardene et al., 2014c showed that Camarosporium sensu stricto clusters as a distinct phylogenetic lineage in Pleosporinae. In our molecular data analyses (Fig. 1) we also show Camarosporium sensu stricto is not related to Cucurbitariaceae, Pleosporaceae or/and Leptosphaeriaceae.

Our combined LSU, SSU and ITS analyses show that our stain clusters with C. quaternatum, the type species of Camarosporium, with high bootstrap support 71% (Fig. 1). Recently introduced species of Camarosporium have been treated as host-specific (Wijayawardene et al. 2014b), but it is essential to re-collect and carry out generic revision. There are about 500 species epithets of Camarosporium and Cucurbitaria in Index Fungorum (2015) but most of the species lack good illustrations and descriptions, thus it is difficult to compare all the species with our collection. However, Mirza (1968) has accepted only 28 species based on morphological characteristics. We have compared our collection with accepted species in Mirza (1968) which have closer morphologies with our collection i.e. Cucurbitaria ahmadi Mirza, Cucurbitaria ononidis Massenot and Cucurbitaria elaeagni Mirza. (Table. 2). Furthermore, we compared the morphology of C. cytisi, C. laburni, C. obducens and C. spartii on Cytisus sp. (Mirza, 1968, Ellis and Ellis, 1985) with our strain (Table 3). Our collection has narrowly fusiform didymosporous ascospores, with mostly ellipsoidal, 5–7 transversely septate, with 4–6 vertical septa, constricted at the central septum, with 1–2 longitudinal septa, with acute ends constricted at the septum.

Table 3

Comparison of Cucurbitaria species on Cytisus sp.

Characters	Cucurbitaria cytisi (Mirza, 1968)	Cucurbitaria laburni (Pers.) De Not. 1862	Cucurbitaria obducens (Schumach.) Petr. 1927	Cucurbitaria spartii (Nees ex Fr.) Ces. & De Not. 1863	Camarosporium arezzoensis MFLUCC 14-0238
Fruiting bodies (Ascomata)	Pseudothecia 300–700 μm, gregarious in groups of 2–8, erumpent, papilla	Pseudothecia 500–700 μm, black, papillate, usually in large groups seated on a black hyphalsubiculum	Pseudothecia 300–500 μm, black, papillate, usually in large groups seated on a black hyphalsubiculum	Pseudothecia300–700 × 350–610 μm diam., black or blackish brown, erumpent in clusters seated on a scanty brown subiculum	Pseudothecia 450 × 480 μm, black, semi-immersed, scattered beneath the host
Peridium	Prominently rough 55–100 μm	Prominently rough 60–100 μm	Prominently rough up to 130 μm	Prominently rough 75–160 μm	Prominently rough 30–70 μm
Asci	140–200 × 13–15 μm	156–260 × 11–16 μm	100–160 × 17–22 μm	140–200 × 13–15.5 μm	180–240 × 10–15 μm
Spore	Dark- to light-golden brown, 18–26 × 7,5–10 μm, muriform, 3 to 7 transverse septa, constricted at the central septum, longitudinal septa 1 or continuos or dis-continuos	Golden brown, 25–35 × 9–15 μm, muriform, 5 to 7 transverse septa, constricted at the central septum, 1 to 2 longitudinal septa	Olive brown, 21–30 × 8.5–13 μm, muriform, 3 to 7 transverse septa, usually 5–7 transverse septa, constricted at the central septum, 1 to 2 longitudinal septa	Golden brown, 25–30 × 11–12 μm, muriform, 5 to 7 transverse septa, constricted at the central septum, with 1 longitudinal septa	Brown 19–28 × 9–15 μm, muriform, mostly ellipsoidal, 5–7 transversely septate, with 4–6 vertical septa, constricted at the central septum, with 1–2 longitudinal septa
Host species (Cytisus sp.)	C. pendulinus,C. scoparius,C. sessilifolius	C. alpinss,C. laburnum,C. radiatus	C. scoparius	C. capitatus,C. scoparius,Cytisus sp.	Cytisus sp.
Country	Portugal, Spain, France, Italy, Sweden	Germany, England, Italy, Switzerland	Spain	Germany, Portugal, Spain, Sweden	Italy
References	Mirza, 1968, Ellis and Ellis, 1985	Mirza, 1968, Ellis and Ellis, 1985	Mirza, 1968, Ellis and Ellis, 1985	Mirza, 1968, Ellis and Ellis, 1985	This study

In this study we used morphology and phylogenetic analyses for the identification of our collection. Thus it is important to carry out molecular analyses to confirm the taxonomic and phylogenetic placement. According to the morphological and phylogenetic analysis results, we introduce our taxon (MFLUCC 14-0238) as a new species of Camarosporium sensu stricto. Other Cucurbitaria spp. should be recollected and subjected to morphological and molecular analyses as Camarosporium sensu stricto has cucurbitaria-like sexual states (Wijayawardene et al., 2014a, Wijayawardene et al., 2014b).