INTRODUCTION

Paspalum dilatatum Poir. or dallisgrass is a perennial grass native to South America that has been introduced into tropical and subtropical areas as a common weed (https://www.cabi.org/isc/datasheet/38 953#tosummaryOfInvasiveness). The genus Claviceps Tul. includes 79 species (www.indexfungorum.org) which parasitize only the flowers of specific grasses causing ergot (Alderman et al. 1999). The sclerotia of many Claviceps species contain alkaloids (Blaney et al. 2000). Claviceps paspali F. Stevens & J.G. Hall causal agent of ergot is only known to colonize Paspalum grasses such as P. dilatatum, which are important as animal feed (http://toxinology.nilu.no/). This disease has most frequently been observed in the southeastern United States, Central and South America, parts of Europe and South Africa, as well as Australia and New Zealand (Evans & Gupta 2007). This fungus has been recorded on Brachiaria eruciformis by Esfandiari in 1948 from Mazandaran province of Iran. Also, C. microcephala (Wallr.) Wint. has been reported on Alopecurus sp. by Esfandiari (1948) from Kandovan, Iran. Claviceps purpurea (Fr.) Tul. has been identified on Secale cereal (Ershad 1995), Agropyron repens and Lolium perenne (Viennot-Bourgin 1958) from Iran.

Another fungus often found in association with ergot was identified as Cerebella sp. (Blaney et al. 2000). Cerebella Ces. is a black-colored saprophytic mold with a deeply invaginated surface and spherical shape that resembles a brain, and extensively colonizes the honeydew produced by Claviceps species. So, the presence of Cerebella should be considered only as a sign or indicator of possible ergot that must be confirmed by identification of actual fungal bodies or other structures of Claviceps spp. (Alderman et al. 1999). Blaney et al. (2000) study showed that sorghum ergot (Claviceps africana Freder., Mantle & De Milliano) contents were mature sclerotia free from floral parts of sorghum, and immature sclerotia with adhering floral parts and with or without attached black sporodochia of Cerebella. Also, Epicoccum andropogonis (Ces.) Schol-Schwarz(Cerebella andropogonis Ces.) conidiomata forming on the honeydew of C. paspali in the spikelet of grasses (Ryley et al., (n.d.) in Ergot fungi of Australia (http://collections.daff.qld.gov.au/web/key/ergotfungi/Media/Html/cerebella.html)). In the present study, fungi were isolated from P. dilatatum specimens with ergot symptoms and characterized based on morphological and molecular data.

MATERIALS and METHODS

Samples and fungal isolates

Paspalum dilatatum with ergot symptoms (Fig. 1) were collected from Rice Research Institute of Iran in Rasht, Guilan province in October 27, 2018. Fungal isolation and purification was conducted according to Ebrahimi & Fotouhifar (2016a). Dried specimens are maintained in the Fungal Reference Collection of the Iranian Research Institute of Plant Protection, Tehran, Iran (IRAN) (accession number: IRAN 17618F). Pure isolates were deposited in the Iranian Fungal Culture Collection (IRAN) at the Iranian Research Institute of Plant Protection, Tehran, Iran (accession number: IRAN 3738C).

Morphological characterization

Culture characteristics were described based on cultures of potato dextrose agar (PDA), oat meal agar (OA) and malt extract agar (MEA) media after 7 and 14 days incubation at 25 °C in dark and under near-UV light source (12 h light/12 h dark). Colony colors (surface and reverse) were assessedusing the color charts of Rayner (1970). Microscopic observations were based on the morphological characteristics of conidia and conidiophores on culture media and naturally infected host substrates. Measurements (n = 50) and microphotographs were taken from slides using an Olympus BH2 light microscope (Olympus, Japan).

Molecular characterization

DNA extraction was performed according to the method described in Ebrahimi et al. (2016). Extracted DNA was diluted in 50 µl distilled water and were kept at -20°C for future use. Molecular identification of the fungal isolate was performed based on ITS-rDNA sequence that was amplified using the ITS1/ITS4 primer pair (White et al. 1990). The reaction mixture and PCR condition for ITS was the same as described by Ebrahimi and Fotouhifar (2016b). PCR product of the ITS region was purified and directly sequenced in one direction with ITS1 primer, by Microsynth Company (Microsynth, Switzerland). After sequencing, sequences were manually edited with Chromas 2.4 software (Technelysium, Australia) and the edited sequence was saved in FASTA format.

For phylogenetic analysis, reference sequences of the homologous regions of Epicoccum species obtained from GenBank, NCBI (isolates information are provided in Table 1) and then the sequences were aligned with Clustal W (Thompson et al. 1994). Mycosphaerella rabiei [accession no. KY788119 (Table 1)] was used as an out-group taxon. Neighbor joining (NJ) analysis (Saitou & Nei 1987) and Maximum likelihood (ML) analysis (Felsenstein 1973) was performed by heuristic search with Mega 7 (Kumar et al. 2016). Bootstrap analysis (Felsenstein 1985) of the ML tree was performed on 1000 replicates. The sequence was deposited in GenBank (NCBI) with accession number MN757870.

RESULTS and DISCUSSION

Fungal isolates

Different fungal isolates such as Alternaria species (mostly) were identified. Five isolates isolate were identified as Epicoccum based on morphological features, and one isolate surveyed based on molecular data and identified as E. andropogonis, which has been reported as saprophytic fungus on Claviceps sp.

Morphology

Culture characteristics- Colonies on OA, PDA and MEA reached 43, 43 and 28 mm in diameter, respectively, after seven days incubation at 25 °C in 12 h dark and 12 h under near-UV light source. Colonies (three replicates) after 14 days on OA were flat, margin regular, with sparse white aerial mycelia, rosy buff to brick color in both side (Fig. 2a). Colonies on PDA margin irregular, aerial mycelia floccose, rust in center (with white dots) to olivaceous grey and cinnamon near to margin; reverse dark brown at center and cinnamon at margin (Fig. 2b). Colonies on MEA margin snaggy, covered by floccose aerial mycelia with some rust dots, vinaceous buff color; reverse black with an umber margin (Fig. 2c).

Fig. 1. Paspalum dilatatum specimens. a. with ergot symptoms, b. ergot sclerotium with brain like surface.

Table 1. Strains used in the phylogenetic analysis of Epicoccum species.

Morphology on host

Sclerotia of ergot were globular in shape, 2.5–5 (3.9) mm in diameter, black in color and irregularly roughened on the surface that resembles a brain (Fig. 1b) which was according to description of C. paspali by Brown (1916).

Unfortunately, none of sclerotia were grown in vitro condition to investigate the morphological features and molecular data of the fungus. Based on the ergot symptoms on P. dilatatum spikelets and host, and previous similar studies, the causal agent of the ergot symptoms is Claviceps cf. paspali. This fungus has been recorded on Brachiaria eruciformis by Esfandiari (1948) from Iran. This is the first record of ergot on Paspalum dilatatum in Iran.

Shape, color and size of conidia of E. andropogonis grown on host substrate (ergot) (8–20 (14.75) × 12–24 (19.8) μm) were similar to the cultured conidia on OA in vitro condition.

There was not any description of E. andropogonis on culture media among literatures to compare with our results in this study. Nevertheless, morphology of conidia in vitro was similar to the characteristics of conidia on host substrate, and it was according to the description of this species provided by Ryley et al. (n.d.) in Ergot fungi of Australia (http://collections. daff.qld.gov.au/web/key/ergotfungi/Media/Html/cerebella.html). This is the first record of E. andropogonis in Iran.

Specimen examined. Iran, Guilan province, Rasht, Rice Research Institute of Iran, on Paspalum dilatatum spikelet. S. Hatami Rad, L. Ebrahimi & H. Shahbazi, 27 Oct. 2018. Herbarium accession number: IRAN 17618F).

Fig. 2. Epicoccum andropogonis. Colony on a. OA, b. PDA, and c. MEA medium, d-e. Conidia formed on OA. — Scale bars = 10 μm.

Fig. 3. Neighbor-joining (NJ) tree based on aligned sequences of ITS region of 19 isolates of Epicoccum and Mycosphaerella rabiei KY788119 as out-group generated in MEGA 7. Bootstrap values (1000 replicates) indicated at the nodes. The scale bar indicates nucleotide substitution in NJ analysis, values ≥50 % are shown above/below the branches.