Stemphylium persianum, A. Ahmadpour, Y. Ghosta, Z. Alavi & F. Alavi, 2024

Ahmadpour, Abdollah, Ghosta, Youbert, Alavi, Fatemeh, Alavi, Zahra, Karunarathna, Samantha C. & Tibpromma, Saowaluck, 2024, Stemphylium persianum sp. nov., (Pleosporales, Ascomycota) from Iran, Phytotaxa 650 (1), pp. 47-59 : 53-56

Stemphylium persianum A. Ahmadpour, Y. Ghosta, Z. Alavi & F. Alavi, sp. nov. Figs. 2 , 3

MycoBank number: MB851921

Etymology: The name refers to the old name of Iran, Persia, from which it was collected.

Holotype:— IRAN 18500 F

Description: —Associated with culms of Schoenoplectus sp. showing necrotic lesions. Sexual morph: Ascomata 200–360 × 150–300 μm (x̄ = 290 × 230 μm, n = 20), solitary, scattered, semi-immersed to immersed, becoming erumpent at maturity, globose to subglobose or oval, dark brown to black, ostiolate. Ostioles central, papillate, opening by a pore, filled with hyaline cells. Peridium 25–45 μm wide, composed of two layers, outer layer of heavily pigmented thick-walled cells of textura angularis, inner layer composed of light brown to hyaline thick-walled cells of textura angularis. Hamathecium 2–3 μm wide, cellular, septate, branched, dense pseudoparaphyses, and embedded in a gelatinous matrix. Asci 105–167 × 22–28 μm (x̄ = 140 × 25 μm, n = 20), 8-spored, bitunicate, fissitunicate, cylindrical to cylindric-subclavate, straight or curved, with short pedicel and a minute ocular chamber. Ascospores 24–38 × 13–18 μm (x̄ = 31 × 15 μm, n = 50), uni- to bi-seriate, partially overlapping, mostly ellipsoidal, tapering towards the rounded ends, muriform, with 3–6 transverse septa, 3–5 longitudinal septa, and 0–1 oblique septa, constricted at the septa, strongly constricted at the median septum, golden brown or brown, with mucilaginous sheath. Asexual morph: Sporulation abundant on PCA, from the erect conidiophores that arise directly from the surface or aerial hyphae. Hyphae are pale brown, septate, effuse, branched, 2–5 μm wide. Conidiophores 45–70 × 4–6 μm (x̄ = 55 × 5 μm, n = 20), macronematous, mononematous, simple, solitary, straight or flexuous, cylindrical, erect, septate, smooth-walled, brown, bearing 1–2 thickened, darkened, percurrent rejuvenation sites. Conidiogenous cells 6–10 × 5–7 μm (x̄ = 8 × 6 μm, n = 20), monotretic, integrated or terminal, swollen at apex, pale-brown. Conidia (35–)38–55(–60) × (10–)13– 20(–22) μm (x̄ = 47 × 17 μm, n = 50), solitary, acrogenous, straight or slightly curved, dark reddish brown, verrucose, oblong-ellipsoid or ovoid with pointed apex, occasionally rounded or subtruncate at the apex, rounded or subtruncate at the base, muriform, with 2–6 transverse septa, 2–6 longitudinal septa, and 1–2 oblique septa, constricted at multiple darkened transverse septa, without mucilaginous sheath.

Culture characteristics: ― Colonies on PDA reaching 46 mm diam. after seven days at 25 °C, with sparse white to grey aerial mycelia, olivaceous grey at the center, white at the margin; reverse pale brown. Colonies on V-8 A reaching 34 mm diam. after seven days at 25 °C, sparse white aerial mycelia, surface olivaceous grey to pale brown at the margin, white at the center; reverse white, pale brown at the center. Colonies on PCA reaching 40 mm diam. after seven days at 25 °C, smooth margin, surface white to pale olivaceous, floccose aerial mycelia; reverse buff, with pale olivaceous near the center. Ascomata formed on PCA containing culms of host plant after 2‒3 months at 25 ºC.

Specimens examined: ― IRAN, Golestan Province, Bandar-e Gaz County, on culm lesions of Schoenoplectus sp. ( Cyperaceae , Poales ), 30 September 2022, A. Ahmadpour , isolate IRAN 18500 F (holotype, dried culture; extype IRAN 4984 C) ; ibid. 30 September 2022, A. Ahmadpour, isolate FCCUU 1301. GenBank accession numbers: ITS‒rDNA: PP253963 and PP253964, gapdh: PP259104 and PP259105; cmdA: PP259106 and PP259107.

Notes: —Morphological characteristics and phylogenetic analyses showed that our isolates belong to Stemphylium ( Fig. 1 ). Based on multi-gene phylogeny, our isolates formed a well-separated lineage (MLBS/MPBS/BIPP = 100/100/1) and has close affinities with S. halophilum and S. lycii ( Fig. 1 ). However, our isolates ( S. persianum ) can be distinguished from S. halophilum by shorter conidiophores (up to 70 μm vs. up to 100 μm), longer conidia (35–60 μm vs. 26–41 μm), more longitudinal septa in conidia (2–6 vs. 1–3) and shorter ascospores (24–38 μm vs. 34–46 μm) ( Webster 1984). Stemphylium persianum can also be distinguished from S. lycii based on longer conidia (35–60 μm vs. 23–31 μm) and more transverse septa in conidia (2–6 vs. 1–3) ( Pei et al. 2011). Also, a comparison of nucleotide differences in ITS‒rDNA, gapdh and cmdA indicates that S. persianum ( IRAN 4984C) differs from S. halophilum (CBS 337.73) by 4/491 bp (0.81%) in ITS‒rDNA, 6/540 bp (1.11%) in gapdh and 7/609 bp (1.14%) in cmdA, and from S. lycii (CBS 125241) by 5/489 bp (1.02%, with one gap (0 %)) in ITS‒rDNA, 3/540 bp (0.55%) in gapdh and 3/609 bp (0.49%) in cmdA.

Discussion

Stemphylium species identification has mostly relied on the variations of morphological characteristics of conidiophores, conidia, and ascospores, and to some extent on host affinity ( Simmons 1969, 1989, 2001, 2004, Woudenberg et al. 2017, Brahmanage et al. 2019, Marin-Felix et al. 2019). However, more than one Stemphylium species have been reported from a single plant, S. botryosum , S. truncatulae , and S. vesicarium on alfalfa ( Chaisrisook et al. 1995, Woudenberg et al. 2017, Marin-Felix et al. 2019), S. botryosum , S. gracilariae , S. lycopersici , S. eturmiunum , S. lycii , S. simmonsii , S. solani , and S. vesicarium on tomato ( Woudenberg et al. 2017, Marin-Felix et al. 2019, Bessadat et al. 2022), S. eturmiunum , S. vesicarium , and S. waikerieanum on onion ( Woudenberg et al. 2017, Marin-Felix et al. 2019), and S. lycopersici , and S. vesicarium on asparagus ( Woudenberg et al. 2017, Marin-Felix et al. 2019) in different geographical locations. Their higher variability of morphological characteristics in different culture conditions and their overlapping among species has made it difficult to accurately identify species boundaries ( Leach & Aragaki 1970, Câmara et al. 2002, Hosen et al. 2009, Woudenberg et al. 2017, Brahmanage et al. 2019, Marin-Felix et al. 2019). Molecular tools have been widely used for accurate differentiation among Stemphylium species. In a recent revision of Stemphylium species, the combination of sequences obtained from ITS‒rDNA, cmdA, and gapdh genomic regions provided the highest resolution for phylogenetic analyses ( Woudenberg et al. 2017).

In the continuation of our studies, aiming to the isolation and identification of fungal species associated with the plants in the families of Cyperaceae and Juncaceae in Iran ( Ahmadpour et al. 2021), several isolates belonging to Stemphylium were isolated. Based on morphological and multi-gene phylogenetic analyses, the two isolates represented a new species of Stemphylium , described herein as S. persianum sp. nov. The isolates were obtained from culms of Schoenoplectus sp. , a member of the Cyperaceae family. Currently, 11 species of Stemphylium have been reported in Iran. However, none of these species have been found on plants in the Cyperaceae family ( Ershad 2009, Poursafar et al. 2016, 2018, Karimzadeh & Fotouhifar 2021). It is important to note that most of the identified Stemphylium species have only been identified based on their morphological characteristics ( Ershad 2009). To date, S. paludiscirpi is the only species reported from the plants in the family Cyperaceae ( Scirpus sp. ) in the USA ( Simmons 2001, Woudenberg et al. 2017, Farr et al. 2024).

Considering the rich diversity of plant species in the family of Cyperaceae in the mainland of Iran and the higher biodiversity of microfungi associated with these plants, more Stemphylium species are expected to be discovered in Iran. More studies are needed to elucidate the diversity of neglected Stemphylium species associated with these plants in Iran and aboard, and assess their pathogenicity and host range.

Figures

FIGURE 1. Phylogenetic tree inferred from maximum likelihood (ML) of combined dataset of ITS‒rDNA, gapdh and cmdA of Stemphylium species. The tree was rooted to Alternaria alternata (GV14-634a1) and newly identified strains are in red boldface. The maximum likelihood and maximum parsimony (MP) bootstrap support (MLBS/MPBS) values ≥60% and Bayesian posterior probabilities (BIPP) ≥0.90 are given at the nodes.The scale bar indicates the number of nucleotide substitutions. T, ET, HT and NT indicate ex-type, exepitype, holotype and ex-neotype strains, respectively.

FIGURE 2. Sexual morph of Stemphylium persianum (IRAN 4984C). a‒e. Ascomata on PCA medium containing culms of host plant; f. Vertical section through an ascoma; g‒h. Peridium; i‒m.Asci; n. Ascospores. Scale bars: d‒e = 100 μm, f‒n = 20 μm.

FIGURE 3. Asexual morph of Stemphylium persianum (IRAN 4984C). a‒b. Symptoms on Schoenoplectus sp.; c‒e. Colonies (front and reverse) on PDA (c), PCA (d) and V‒8A (e) culture media after seven days; f. Sporulation pattern on PCA medium; g–j. Conidiophores and conidia; k. Conidia. Scale bars: f‒k = 20 μm.