Identification of the species Parastagonospora dactylidis on poaceous plants in Iran

INTRODUCTION

Parastagonospora (Berk.) Quaedvlieg, Verkley & Crous belonged to the class Dothideomycetes, order Pleosporales and family Phaeosphaeriaceae (Quaedvlieg et al. 2013). Some species of the genus Parastagonospora are responsible for significant crop losses in wheat, barley and rye worldwide (Quaedvlieg et al. 2013). Parastagonospora was first defined as Septoria (Sacc) (Weber 1922). Based on conidial morphology, Septoria and Stagonospora (Sacc.) were grouped as two different genera and Stagonospora was accepted as the correct description in place of Septoria. The conidia of Stagonospora species were more than 10 times smaller than those of Septoria (Sprague, 1950). Quaedvlieg et al. (2013) introduced and used a new genus Parastagonospora (Quaedvlieg, Verkley & Crous) instead of Stagonospora genus. Since January 2013, following new rules for naming of pleomorphic fungi outlined in the International Code of Nomenclature for Algae, Fungi and Plants (ICN) as the pleomorphic fungus may have only one name (Wingfield et al. 2012). Therefore, the name Parastagonospora has been established as the valid name instead of Phaeosphaeria(I. Miyake)(Quaedvlieg et al. 2013).

Today, there are many deficiencies and problems of morphological taxonomy that have been solved using molecular techniques. Because of the overlapping observed in the morphological systematic studies within the genus, molecular techniques based on multilocus DNA sequencing analysis have enabled researchers to solve these problems (Quaedvlieg et al. 2013). In the last two decades, the genus Parastagonospora was analyzed using different genes including mating-type loci (Bennett et al. 2003, Ueng et al. 2003), ITS sequences (Ueng et al. 1998), ß-tubulin gene (Malkus et al. 2005), ß-glucosidase gene (Reszka et al. 2005), RNA polymerase II (Arkadiusz et al. 2006), histidine synthase gene (Wang et al. 2007) and ß-xylosidase gene (McDonald et al. 2012).

So far, three species of the genus Parastagonosporaincluding P. nodorum (Berk.), P. avenae f. sp. avenaria (A.B. Frank) (Paa) and P. avenae f. sp. tritici 5 (A.B. Frank) (Pat5) have been reported based on molecular and morphological characteristics on poaceous plants in Iran (Ghaderi et al. 2017). Therefore, this study was conducted to determine otherspecies of Parastagonospora in association with poaceous plants in Iran. Species identification was done according to the morphology of anamorph and molecular confirmation using ITS and LSU sequences.

MATERIALS AND METHODS

Sampling and morphological characterization

During surveys from 2016 to 2017, symptomatic leaves and ears were collected from different poaceous species in the field from three provinces of Iran including Fars, Khuzestan and Kohgiluyeh and Boyerahmad, and taken to the laboratory. Fresh samples were photographed before collection to save distinctive characters using camera EOS 1300D. The specimens examined based on macro and micro-morphological characteristics.

For the isolation of causal agents, the diseased leaves and ears were cut into segments of 4–8 cm, surface-sterilized in 70% ethanol for 1 min, rinsed in sterile water and placed in glass slides with tape and kept under high humidity conditions until the pycnidia produced cirri containing pycnidiospores. Then, colonies were transferred into Petri dishes containing Yeast Sucrose Agar (YSA, 10 g.L^-1 Yeast Extract, 10 g.L^-1 sucrose, and 1.2% agar) amended with 50 μg of kanamycin. Plates were stored at 25 °C for five days and transferred to YSA medium for the next steps. To examine colony morphology, isolates were grown on YSA plate containing sterilized poaceous straws. Cultures were incubated at 20 °C and a 12-h photoperiod near ultraviolet light for pycnidia (300 nm <X< 400 nm), and intensities of 400 and 600 pW/cm² for 14–30 days to promote sporulation (Halama & Lacoste 1999). Parastagonospora species were identified using their morphological characters such as conidia and conidiomata morphology, pigmentation, and colony color and growth rate.

Mating type determination and fertility

Mating type idiomorphs for each isolate was determined using the mating type primers described by Bennett et al. 2003. Multiplex PCR amplifications were also performed as described previously (Sommerhalder et al. 2006). PCR amplifications were performed in 20 μl reactions containing 0.05 μM of each primer, 1 × Dream Taq Buffer (MBI Fermentas), 0.4 μM dNTPs (MBI Fermentas) and 0.5 units of Dream Taq DNA polymerase (MBI Fermentas). The PCR cycle parameters were: 2 min initial denaturant-
ion at 96 °C followed by 35 cycles at 96 °C for 30 s, annealing at 55 °C for 45 s, and extension at 72 °C for 1 min. A final 7 min extension was made at 72 °C. Negative controls were included in every PCR. The PCR products were separated on 1% agarose gel. The gels were stained with ethidium bromide and visually analyzed under UV light (GelDoc, Bio-Rad Laboratories).

For formation of reproductive form (pseudothecia) of Parastagonospora, based on mating type idiomorphs, isolates grown on 2% WA containing sterilized wheat straws cultures at 10 °C and a 12-h photoperiod near ultraviolet light for pseudothecia (300 nm 400 nm), and intensities of 400 and 600 pW/cm² for 30–45 days to promote sporulation (Halama & Lacoste 1991).

DNA extraction

Single colonies were transferred to flasks containing 50 ml Yeast Sucrose Broth (YSB, 10g.L^-1 Yeast Extract, 10g.L^-1 sucrose) and grown on an orbital shaker for 7 days at 120 rpm at 18 °C. Mycelia were harvested using sterile gauze and rinsed with sterile distilled water. Dried mycelia were crushed into fine powder in liquid nitrogen. Total genomic DNA was isolated using a modified CTAB (cetyltrimethylammonium bromide) technique. Approximately 100 mg mycelium powder was suspended in 900 μL of extraction buffer (100 mM Tris- HCl, 100 mM EDTA, 250 mM NaCl), and then 100 μL of 10% N-Lauroylsarcosine (Sigma-Aldrich, Germany) was added. The suspension was incubated at 60 °C for 60 each tube, chloroform/isoamyl alcohol (24:1) was added. After mixing, the tubes were centrifuged for 10 min at 13000 × g. Upper phase of the suspension was transferred to a new 1.5 ml tube and the equal volume of isopropanol was added where the DNA was precipitated at –20 °C. To precipitate DNA, tubes were centrifuged for 10 min at 13000 × g then the supernatant was discarded gently. The sediment was washed with about 100 ml of 75% ethanol and DNA was precipitated. The pellet was diluted in 100 ml of deionized double-distilled sterile water (Murray & Thompson 1980).

Phylogenetic analysis

The primer pairs LROR-LR5 (Vilgalys and Hester 1990) and ITS4-ITS5 (White et al. 1990) were used to amplify a segment of the large subunit rDNA (LSU) and the internal transcribed spacers (ITS) respectively. PCR amplifications were performed in 20 μl reactions containing 0.05 μM of each primer, 1 × Dream Taq Buffer (MBI Fermentas), 0.4 μM dNTPs (MBI Fermentas) and 0.5 units of Dream Taq DNA polymerase (MBI Fermentas). The PCR cycle parameters were: 2 min initial denaturation at 96°C followed by 35 cycles of 96°C for 30 s, annealing at 57°C for 45 s, and extension at 72°C for 1 min. A final 10 min extension was applied at 72°C. Finally, the quantity and quality of PCR products were evaluated and visualized on 1.2% agarose gel. The gels were stained with ethidium bromide and visually analyzed under UV light (GelDoc, Bio-Rad Laboratories). The sequencing of PCR products were carried at Macrogen, Korea.

The consensus regions of the LSU and ITS-rDNA loci were blasted separately against the NCBI’s GenBank sequence database using Megablast to identify their closest neighbors.  LSU and ITS-rDNA sequences were obtained from GenBank mostly following previous literature (Liu et al. 2015) and new sequences described in this manuscript are listed in Table 1.

Multiple sequences were aligned using Bioedit v. 7.0.9 (Hall, 1999) and MAFFT v.7 online interface using default settings (http://mafft. cbrc.jp/alignment /server/) (Katoh & Standley 2013). The alignments were checked visually and improved manually wherever necessary. Maximum likelihood (ML) methods were used to determine the phylogenetic relationship among isolates using MEGA v. 6 software (Molecular Evolutionary Genetics Analysis) (Tamura et al. 2007). Maximum likelihood bootstrap (BS) values (Reeb et al. 2004) and Bayesian posterior probabilities (PP) values using BEAUti and BEAST v1.6.1 software (Drummond & Rambaut 2007) were used as criteria for strongly supported clades. An isolate of Pyrenophora tritici-repentis was used as an outgroup for phylogenetic analyses because P. tritici-repentis is closely related to the Parastagonospoa species complex (McDonald et al. 2012).

RESULTS AND DISCUSSION

The complete ITS region (949bp) and a segment of the large subunit rDNA (LSU) (869bp) sequences were concatenated using BEAUti and BEAST v1.6. for phylogenetic analysis of 32  Parastagonospora isolates. The best scoring RAxML tree was chosen as the backbone tree and is shown in Fig 1. A maximum likelihood tree was inferred by combined analysis of two loci (Fig 2). Based on the phylogenetic analyses, the twelve isolates from various plants in the present investigation grouped with P. dactylidis (Li et al. 2015) in a well-supported clade (ML-BS: 93%, PP: 100%). In fact, phylogenetic analysis confirmed the morphological identification. In the present research, based on morphological characters, all isolates belonged to Parastagonospora dactylidis which is described below:

Parastagonospora dactylidis W.J. Li, Camporesi, D.J. Bhat & K.D. Hyde, Mycosphere 6 (6): 691 (2015)

Specimens examined. IRAN, Fars province, Noorabad, on leaves of Phalaris arundinacea, 3 May 2016, F. Ghaderi; IRAN, Fars province, Noorabad, on leaves of Bromus hordeaceus, 3 May 2016; IRAN, Golestan province, Aliabad, on ears of Aegilops tauschii. 20 April 2017. Colonies on YSA, reaching 17 mm after 2 weeks at 25 ℃, with dense, white, flat, aerial mycelium, with rounded, smooth, margins. There were partial differences in colonies color on YSA (Fig. 1. a-b). Conidiomata pycnidial, brown to black, subglobose to lenticular, immersed, formed mostly on sterilized wheat straws, exuding pale pink conidial cirrhus, 100–130μm in diameter, with a single ostiole, circular, papillate 14–17 μm wide (Fig. 1. c-f). Conidia hyaline, smooth, thin-walled, fusiform, curved, rounded at both ends, slightly narrower at the base, 3-septate, 21–42 × 4–5.3 μm (Fig. 1. g-h).

This fungus is diallelic heterothallic. Sampled isolates from different places Iran, has indicated both Mat1-1 and Mat1-2. So, the sexual phase formation is related to both of mating type idiomorphs. Pseudothecia formation of P. dactylidis is obtained in vitro on sterilized straws under strict conditions of light and temperature after one month. Ascocarps in longitudinal rows immersed on sterilized straws were subepidermal, globose, glabrous, 120–150 μm wide and 136–162 μm high, but we did not observe ascus and ascospores within pseudothecia.

Notes. Parastagonospora dactylidis is closely related to P. minima. However, these two species can be easily distinguished based on conidia morphology. P. dactylidis has fusiform conidia with a slightly narrower base, and distinctly granular cytoplasm, whereas P. minima has subcylindrical conidia which are wider in the basal half, and narrow at the apex. In addition, the conidiomata and conidia of P. dactylidis are larger and longer than those of P. minima (conidiomata: 40–70 μm high, 50–100 μm diam., conidia: 20–28 × 3.5–4.5 μm). Furthermore, in the present study, we also clarified the phylogenetic positions of P. dactylidis and P. minima.

The morphological characters cannot always provide all the necessary data to define a species; therefore, DNA data is useful for precise identification of the fungal species. The taxonomy of Parastagonospora is extremely complicated and unreliable (Quaedvlieg et al. 2013), hence identification of the species of this genus based on morphological characters is unclear, and specific morphological features to describe and identify Parastagonospora species are rather limited so that, further research is required to resolve this issue (Quaedvlieg et al. 2013). Overall, the genus Parastagonospora is pleomorphic and propagate through sexual or asexual reproduction in field, but it is almost difficult to produce both of sporulating structures (asexual morph adjacent to the sexual morph) on artificial media in laboratory that could help distinguish species of the genus Parastagonospora (Quaedvlieg et al. 2013, Shoemaker and Babcock, 1989).

In this study, none of the isolates produced pseudothecia when grown alone. The sexual stage is induced in the laboratory and controlled by mating type idiomorphs (Bennett et al. 2003). But pseudothecia formation obtained in vitro and we could not observe ascus and ascospores within pseudothecia to distinguish species.

Consequently, DNA sequence analysis and phylogenetic inference can irrefutably establish asexual and sexual morph connections, for example, the Mycosphaerella arbuticola / Septoria unedonis, Phaeosphaeria papaya / Phaeoseptoria oryzae, Leptosphaeria maculans / Plenodomus lingam, and also provide phylogenetic placements for asexual taxa within the modern taxonomic classification schemes (Quaedvlieg et al. 2013, Ariyawansa et al. 2015).

In the present report, ML consensus tree was supported by Bayesian posterior probabilities and bootstrap values. We could separate the morphological species for genus Parastagonospora using combined gene (LSU and ITS). In fact, sequence data together with morphology are used to delimit and propose one new species for the mycobiota of Iran that is named P. dactylidis. The correlation between phylogenetic and morphological species are in correspondence with phylogenetic analyses by Li et al. (2015) examining the relationship among species of Parastagonospora spp. They carried out a revision of Phaeosphaeriaceae based on multigene (LSU and ITS) sequence analyses coupled with morphological data and introduced the new species Parastagonospora dactylidis, P. minima, P. italica, P. uniseptata and P. allouniseptata in Italy.

In this research, P. dactilitis is reported for the first time from Iran that identified on Phalaris arundinacea, Bromus hordeaceus, and Aegilops tauschii where all of the recognized hosts are new to the world.

ACKNOWLEDGMENTS

We gratefully acknowledge Yasouj University (Yasouj, Iran) for financial assistance.