A taxonomic study on Stemphylium species associated with black (sooty) head mold of wheat and barley in Iran

INTRODUCTION

The genus Stemphylium Wallr. was established with S. botryosum as the type species in 1833 (Wallroth 1833). Percurrent proliferation of conidiophores and production of single muriform and pigmented conidia on swollen conidiogenous cell at the tip of conidiophores are the main morphological characteristics of the genus (Simmons 1969). Association between Stemphylium and a teleomorph state (previously known as Pleospora) or sclerotial bodies, has been established for a number of Stemphylium species (Câmara et al. 2002). The number of described Stemphylium species vary from 20 (Câmara et al. 2002) up to 150 (Wang & Zhang 2006), while a search of Index Fungorum (May 2017; http://www.indexfungorum.org) lists 160 unique names. Over the past decade, more than 10 new species have been described (Wang & Zhang 2006, 2009; Wang et al. 2009, 2010; Pei et al. 2009, 2010, 2011; YanFang et al. 2012; Deng et al. 2014; Crous et al. 2016).

Before the development of molecular approaches in fungal taxonomy, species delimitation and identification in this genus was based primarily on the morphological characteristics including conidial shape, size, septation, length/width ratio and ornamentation. However, overlapping of these characters among the species and their dependences on environmental conditions such as temperature and substrate type has resulted in a complexity of the genus taxonomy (Leach & Aragaki 1970; Hosen et al. 2009; Chowdhury et al. 2015; Subash & Saraswati 2016).

In recent years, several molecular–based studies have been conducted using the sequence data of the internal transcribed spacer (ITS) of nuclear rDNA, mitochondrial small subunit (mtSSU) of rDNA, translation elongation factor 1–alpha (TEF1–α), intergenic spacer between vmaA and vpsA and gene encoding glyceraldehyde–3–phosphate dehydrogenase (GPDH) to species delimitation and inferring phylogenetic relationship within the genus (Câmara et al. 2002; Pryor & Bigelow 2003; Kodsueb et al. 2006; Inderbitzin et al. 2009). However, sequence data were unable to distinguish some species that were clearly distinct by morphological characters which is necessary to combine morphological and molecular data to delimit species in this genus (Inderbitzin et al. 2009; Pei et al. 2011). 

According to Farr and Rossman (2017), more than 90 Stemphylium species have been isolated from different host plants around the world. Several species viz. S. alfalfae, S. botryosum, S. eturmiunum, S. globuliferum, S. herbarum, S. lotii, S. solani, and S. vesicarium have been known to be plant pathogens on important agricultural crops (Seaney 1973; Elis & Gibson 1975; Irwin 1984; Johanson & Lunden 1986; Simmons 1990; Aveling & Snyman 1993).

Black (sooty) head mold of wheat and barley is commonly in association with a diverse group of saprophytic or weakly parasitic fungi (Bockus et al. 2010). The typical symptom of black head mold in wheat and barley is the blackened appearances of mature or dead spikes under wet or humid weather conditions (Prescott et al. 1986). The presence of Stemphylium species with black head mold symptoms and grain discoloration of cereals have been reported in several publications (Zillinsky 1983; Prescott et al. 1986; Sisterna & Sarandon 2010; Hershman 2011; Zare 2013). 

The aim of this study was isolation and identification of Stemphylium species associated with the black (sooty) head mold of wheat and barley in different regions of Golestan, Alborz and Qazvin provinces in Iran using morphological characters and molecular phylogenetic data.

MATERIALS AND METHODS

Sampling and isolation of Fungi

Samples with characteristic symptoms of black head mold were randomly collected from different wheat and barley fields in Golestan, Alborz and Qazvin provinces during spring and summer of 2014 and 2015. Samples were air dried for 1–2 days and kept at room temperature until processed. Isolation of fungal isolates was performed using a moist chamber (blotter) method. The growing fungi with typical characteristics of genus Stemphylium were picked up directly with a fine sterile needle and transferred onto the new potato dextrose agar (PDA) plates. Pure cultures were obtained by using single spore and hyphal tip methods on 2% water agar (2% WA) and PDA media, respectively. Purified isolates were placed on PCA slants including 20 g white potato, 20 g carrot and 20 g agar per 1 liter of distilled water and then kept at 4 ̊C for further examination.

Morphological assessment

Morphological characters were assessed based on standardized condition suggested by Simmons (2001). Purified cultures were incubated at 23–25°C on Potato Carrot Agar (PCA) under cool/white fluorescent with 10/14 h light/dark photoperiod for 5–7 days. The Sellotape technique was used for slide preparation (Schubert et al. 2007) with 25% Lactic acid solution as mounting fluid. Macro– and micro–morphological features were recorded and compared with available literature. Pure cultures of all identified species were deposited in fungal culture collections of University of Tehran (UTFC) and Iranian Research Institute of Plant Protection (Table 1).

DNA extraction and PCR amplification

Total genomic DNA was extracted from single conidium cultures grown on 90 mm PDA petri plates according to Zhong & Steffenson (2001). The ITS–rDNA region and part of gelyceraldehyde–3–phosphate dehydrogenase (GPDH) gene were amplified with the primer pairs ITS5/ITS4 (White et al. 1990) and gpd1/gpd2 (Berbee 1999), respectively. Each PCR mixture contained 10 μM of each primer, eight μL of a ready master mix (Taq 2X Master Mix Red 1.5 Mm, Amplicon Company, Denmark) and about 10 ng of template DNA in a final volume of 25 μL. Conditions for PCR amplification of the ITS–rDNA region, consisted of an initial denaturation for 4 min at 95 °C followed by 35 cycles of 30 s denaturation at 95 °C, 30 s annealing at 56 °C and 60 s extension at 72 °C followed by a final extension step for 6 min at 72 °C. Part of GPDH gene was amplified using a touchdown (TD) PCR method (Korbie & Mattick 2008) of an initial denaturation for 90 s at 95 °C and then, a cycle of 60 s denaturation at 95 °C, 60 s annealing at 62 °C and 60 s extension at 72 °C, followed by 10 cycles with a 62–57 °C annealing temperature (annealing temperature decreased 0.5 °C per cycle) and 25 cycles with a 57 °C annealing temperature and a final extension for 5 min at 72 °C. The PCR products were purified and sequenced by Macrogen Corporation (South Korea). The newly generated sequences of ITS and GPDH in this study were submitted to GenBank (Table 1).

Phylogenetic analysis

The newly generated sequences were edited in BioEdit v. 7.2.5 (Hall 1999) and supplemented with sequences retrieved from GenBank (Table 1). Multiple sequence alignments were generated with MAFFT v. 7.304 (Katoh & Standley 2013), checked visually and improved manually where necessary. Neighbor Joining (NJ) and Maximum Parsimony (MP) analyses were performed using the ITS and GPDH combined datasets in PAUP 4.0 (Swofford 2002). The best fit model, general time reversible model (GTR) incorporating invariant sites (I) and gamma–distribution rate (G), for NJ analysis was selected by Akaike Information Criterion (AIC) in Mr.Modeltest 2.3 (Nylander 2008). MP analysis was done by using heuristic searches with 1000 random sequence additions and branch swapping with tree–bisection–reconnection (TBR) algorithm and gaps treated as missing data. The bootstrap values with 1000 replicates were performed to determine branch support. Sequences of Alternaria alternata (CBS 916.96), Curvularia australis (Turgan 77139) and Bipolaris sorokiniana (Tinline A20) were used as out–groups. The generated trees were observed in TreeView v. 1.6.6 (Page 1996).

RESULTS and DISSCUSION

A total of thirty–two isolates with Stemphylium characteristics were collected from black (sooty) head mold symptoms of wheat and barley in different regions of Golestan, Alborz and Qazvin provinces. Based on the combination of morphological characteristics and sequence data obtained from ITS–rDNA region and GPDH locus, four species viz. Stemphylium alfalfae, S. eturmiunum, S. lycii, and S. vesicarium were identified. Among the identified species, S. vesicarium and S. alfalfae were isolated more frequently than other species with 17 and nine isolates, respectively. The less frequently isolated species were S. lycii (four isolates) and S. eturmiunum (two isolates). Based on the available literature, all identified species are reported for the first time as Stemphylium species associated with black head mold of wheat and barley. Stemphylium eturmiunum and S. lycii are new records to the mycobiota of Iran. These two species, as well as S. alfalfae and S. vesicarium are described here alphabetically.

PCR amplification of ITS–rDNA region and a part of GPDH gene was generated DNA fragments about 550–570 and 570–590 bp, respectively. The BLAST searches of partial GPDH sequences showed a higher number of variable sites than the ITS sequences within the Stemphylium species. The alignment of ITS–GPDH sequence data matrix for 36 taxa was included a total of 1041 characters. The results showed that 718 characters were constant, 139 characters were variable and parsimony uninformative and 184 characters were parsimony informative. All examined characters were unordered and had equal weight. Phylogenetic analyses of ITS and GPDH combined dataset using MP and NJ methods resulted in phylogenetic trees with the same topologies. MP analysis using ITS and GPDH combined datasets yielded 24 most parsimonious trees (CI = 0.748, RI = 0.772, HI = 0.252). One of the most parsimonious trees was selected and the bootstrap values of MP and NJ analysis are shown at the nodes (Fig. 1). Two of identified species S. alfalfae and S. vesicarium clustered with those of S. alfalfae (EGS 36–088), S. herbarum (EGS 36–138.2), S. sedicola (EGS 48–095), S. tomatonis (EGS 29–089) and S. vesicarium (EGS 37–067) with 89/94% (MP/NJ) bootstrap supports. Results of previous studies have revealed that the species in this clade have nearly identical ITS and GPDH sequences, however their identification should be based on the morphological characters (Câmara et al. 2002; Inderbitzin et al. 2009). Isolates JS5–2, JS5–A and BT2–5 were well clustered with those of S. eturmiunum (EGS 29–099) (95/99% MP/NJ bootstrap supports) and S. lycii (CBS 125241) (100/100% MP/NJ bootstrap supports), respectively (Fig. 1).

Table 1. Species used for phylogenetic analyses. Newly generated sequences are in bold.

^** This species appears in GenBank as Bipolaris australis. It has synonymized with Curvularia australis according to Manamgoda et al. (2014).

 Fig. 1. One of the most parsimonious trees generated from Maximum Parsimony analysis based on the ITS–rDNA and GPDH combined datasets. The bootstrap values (>50%) of MP and NJ analysis are shown at the nodes (MP/NJ). Isolates in bold were identified in present study. Alternaria alternata (CBS 916.96), Curvularia australis (Turgan 77139) and Bipolaris sorokiniana (Tinline A20) are used as outgroups.

Stemphylium alfalfae E.G. Simmons, Sydowia 38: 292 (1986) [1985] (Fig. 2a–f)

Colonies on PCA reached to 60 mm in diameter after seven days. They were flat and gray at center and creamy at margin with distinct olive to light brown concentric growth zones. Mycelia were superficial and composed of branched, septate, pale brown and smooth–walled hyphae. Sporulation was abundant on PCA, mostly from superficial hyphae and to a lesser extends from aerial hyphae. Conidiophores were straight or curved, pale brown, septate and reached to 110 μm in length. Conidiogenous cells swollen at the apex and were brown, 6–7 μm in wide and occasionally with 1–3 apical proliferations. Conidia developed singly at the apex of each conidiophore and were pale brown with darker septa. They were minutely verrucose, cylindrical, spherical, oblong to ellipsoidal, rounded at the apex and the base, mostly with 1–3 transverse septa, 2–4 longitudinal septa and 1–3 oblique septa, usually with distinct constriction at the median septum, L/W= 1.25–3.5 and 18–38 × 8–20 μm (Fig. 2b–d).

Ascomata formed abundantly on PCA after seven days and matured after four weeks. They were dark brown, thick–walled, spherical to subspherical. Asci were bitunicate, hyaline, straight or curved, 8–spored and 100–225 × 27–33 μm. Ascospores were oblong spherical, pale brown with darkened septa, 6–7 transverse septa, 5–7 longitudinal septa and 35–37 × 12–17 μm (Fig. 2e–f).

Specimens examined. Iran, Golestan province, Fazel Abad, on wheat head, May 2014, A. Poursafar, FA6–5 (UTFC 816) and FA6–10; Golestan province, Gorgan, on wheat head, May 2014, A. Poursafar, G10–7; Golestan province, Ali Abad–e Katul, on wheat head, May 2014, A. Poursafar, Al6–11; Alborz Province, Nazar Abad, on wheat head, June 2015, A. Poursafar, NZA1–A and NZA1–6; Alborz Province, Mohammad Shahr, on wheat head, June 2015, A. Poursafar, MHA5–8.

Based on ITS and GPDH sequence analyses, Stemphylium alfalfae is clustered with those of S. herbarum, S. sedicola, S. tomatonis and S. vesicarium (Fig. 1). Furthermore, it is morphologically close to S. vesicarium and S. tomatonis. According to Câmara et al. (2002) and Inderbitzin et al. (2009), ITS and GPDH sequences of mentioned species are nearly identical and their identification is dependent on morphological characteristics of the asexual and sexual states.

Stemphylium alfalfae was first described on alfalfa plants (Medicago sativa L.) in Western Australia (Simmons 1985). In Iran, this species was recently reported from alfalfa plants in Hamedan province (Bagherabadi et al. 2015), in which the authors erroneously referred to the first report of S. alfalfae in Iran according to Ershad (2009). When this reference was searched, no citation of S. alfalfae was observed and we found only a citation indicating the isolation of S. botryosum (Tel. Pleospora tarda) from alfalfa in Mollasani, Ahwaz (Mohajer–Shojai & Ebrahimi 1969). So as it was not described previously in Iran, we have provided a full description of this species here.

 Fig. 2. Stemphylium alfalfae (UTFC 816). a. Colony on PCA; b––d. Conidiophores and conidia; e––f. Asci and ascospores.

Fig. 3. Stemphylium eturmiunum (IRAN 2600C). a. Colony on PCA; b. Fascicle of hyphae bearing short conidiogenous branches with conidia; c–d. Conidiophores and conidia; e–f. Asci and ascospores.

Stemphylium eturmiunum E.G. Simmons, Harvard Papers in Botany 6 (1): 204 (2001) (Fig. 3a–f)

Colonies on PCA after seven days reached to 55 mm diam. They were flat, pale olive to light brown without distinct concentric growth zones. Mycelia were superficial or submerged, superficial mycelia composed of branched, septate, pale brown, smooth walled hyphae and 4–6 μm in wide. Sporulation were abundant on PCA, predominantly from short conidiogenous branches of hyphae that arise singly or in fascicles from substrate. Aerial axis hyphae commonly formed and reached to 1 mm or more long, bearing a large number of short up to 50 μm in length and pale brown conidiophores with a tip cell slightly swollen (5–8 μm wide).