Endophytic fungus, Radulidium subulatum from Phragmites australis in Iran

INTRODUCTION

Endophytic fungi are diverse group of plant symbionts that live locally and sometimes systematically within plant tissues without causing symptoms of disease (Porras-Alfaro & Bayman 2011). There are several studies reporting the beneficial features of the fungal endophytic groups (González-Teuber 2016). The important ecological roles of endophytes in the issue of environmental adaptation of their hosts can result in increasing resistance against pathogens and herbivores (Clay & Schardl 2002), assisting the plants for mineral nutrition uptake (Kipfer et al. 2011), mitigating the effects of water stress (Atala et al. 2012) and affecting plant responses towards other biotic and abiotic stresses (Clay & Schardl 2002, Rodriguez et al. 2008, Cheplick & Faeth 2009, Soares et al. 2015). Symbiotic relationships can have a direct or indirect impact on the structure, function and composition of plant communities, the expansion of host niches (Rudgers et al. 2015) and food webs and ecosystem processes (Aschehoug et al. 2014).

Common reed (Phragmites australis (Cav.) Trin. Ex Steud.) is a perennial grass found in most habitats throughout the world. The microbial endophytic communities of P. australis have been examined in several previous studies (Wirsel et al. 2001, Li et al. 2012, Angelini et al. 2012, Fischer & Rodriguez 2013, Kim et al. 2014, Hipol & Cuevas 2014, Sim et al. 2015, Sauvêtre & Schröder 2015, Soares et al. 2015) and thought to play a key role in aiding plant establishment in marginal and high stress habitats. It has been suggested that success in invasion of this plant is related to some factors including plant ecological capability to tolerate and grow in a wide range of soil salinity and fertility levels, its wide dispersion efficiency (Meyerson et al.  2012), high genetic diversity (Fer & Hroudova 2009), and microbial symbiosis (Kowalski et al. 2015). These plants have also been shown to tolerate heavy metals as well as other stresses (Bonanno & Giudice 2010). The ability of P. australis to cope with salt stress plays a crucial role in colonization of coastal habitats (Achenbach & Brix 2014), although it is unclear how much the plant’s microbiome can influence the resistance to salt stress. According to our knowledge, there is no comprehensive study of endophytic fungi from common reed plants in Iran. Radulidium is an ascomycetous fungal genus that was described by Arzanlou et al. (2007) as a new taxon. Radulidium has been isolated and identified from a limited but divers types of the substrates/hosts including Epichloë typhina, an unknown Poaceae plant, Puccinia allii, Phragmites australis, incubator for cell cultures and Lasioptera arundinis (Arzanlou et al. 2007), so far. The aim of this study was morphological and molecular identification of Radulidium isolates which were isolated during the years 2017-2018 as endophytic fungi from P. australis plants grown in different natural habitats in several Northern provinces of Iran.

MATERIALS and METHODS

Sample collection

Numerous samplings were conducted from healthy Phragmites australis plants (without disease symptoms) grown in some areas in the southern parts of the Caspian Sea and saline soils around the Lake Urmia, during the summer and autumn of 2017 and 2018. At each sampling site, whole plants (1.5–3.0 m height) with undamaged leaves, roots and rhizomes were randomly collected and transferred to the laboratory in polythene bags and processed for the isolation of endophytic fungi in the same day or within a maximum of 48 hours using standard techniques (Rodriguez et al., 2008). Briefly, samples were washed in running tap water to remove dust and debris for 15 min, dried in the air and then cut into 1 cm segments. For surface sterilization, the plant slices were soaked in 70% ethanol for 1 min, then in sodium hypochlorite (3% available chlorine) for 2 min for leaf and stem and 3 min for root segments. Disinfection processes continued in 70% ethanol for 1 min and subsequently, the samples rinsed 5 times with autoclaved distilled water. The plant tissues were placed on the petri dishes containing potato dextrose agar (PDA, Merck, Darmstadt, Germany) and 2% water agar (WA, 2 %) media supplemented with chloramphenicol (20 μg/ml) and streptomycin (10 μg/ml). Petri dishes were kept at 20–25 °C in the darkness for 7–21 days. WA plates supplemented with chloramphenicol (20 μg/L) were inoculated with fungal tissues that had formed on WA and PDA plates. Single spore or hyphal tips isolates were prepared on PDA. Pure cultures were deposited at the Mycology Laboratory (UTFC …) of College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran (Table 1).

Morphological characterization

Morphological characterization was performed according to Arzanlou et al. (2007). Cultural growth rates and morphology were recorded from colonies grown on malt extract agar (MEA) for 14 days at 24 °C in the darkness and colony colors were determined according to Rayner (1970). Microscopic observations were conducted from colonies cultivated on MEA and OA (oatmeal agar, Gams et al., 2007). Structures were mounted in lacto-phenol cotton blue, and 30 measurements (× 1000 magnification) determined wherever possible.

DNA extraction and PCR amplification

Genomic DNA was extracted from colonies grown on PDA (25 °C for 7–10 days) using a standard phenol-chloroform extraction protocol (Sambrook & Russell, 2001). Amplification of the internal transcribed spacer (ITS) region of rDNA was performed using the ITS1 and ITS4 primers (White et al., 1990). Polymerase chain reaction was performed in a Corbett Thermocycler (Corbett Research, Australia) in a total volume of 25 μL. The PCR mixture contained 10–15 ng genomic DNA, 0.2 μM of each primer, 1 × Taq PCR buffer, 2 mM MgCl₂, 20 μM of each dNTP, 0.75 μl DMSO and 0.25 U Smart Taq DNA Polymerase (CinnaGen Co, Iran). The PCR program was as follows: an initial step of 5 min at 98 °C, 35 cycles of 10 s at 98 °C, 30 s at 60 °C and 30 s at 72 °C, followed by 10 min at 72 °C. The PCR products were sequenced by Bioneer Co. (South Korea) with the amplifying primers.

Phylogenetic analysis

The programs ChromasPro 1.34 and EditSeq, parts of the DNA Lasergene (DNAstar, Madison, WI) software package, were used for viewing, editing and assembling the sequences.

Table 1. Radulidium isolates obtained from Phragmites australis, and characterized in this study.

RESULTS and DISCUSSION

Characterization of the species

A total of 21 isolates of Radulidium were isolated as endophytes from leaves, stems and roots of healthy P. australis plants showing no disease symptoms from six different geographical areas at four Northern provinces of Iran: East Azerbaijan, Guilan, Mazandaran and Golestan (Table 1). Investigating colonies and morphological characteristics of the isolates showed that all isolates belonged to only one morphotype with similar or identical characteristics. Based on the detailed morphological studies, it became clear that all isolates belong to the R. sublutum species. To confirm morphological identification, two isolates were selected for molecular identification by sequencing of ITS-rDNA genomic region. A BLAST search of the resulting sequences of N1P10S3 and N1P10S4 isolates showed 99.77% similarity to R. subulatum CBS 405.76 ex-type strain (EU041845; Arzanlou et al., 2007). Analysis of ITS-rDNA sequences of two selected isolates confirmed morphological identification and could resolve the identity of the R. subulatum isolates, totally in concordance of morphological characters. To the best of our knowledge, this is the first report of R. subulatum from P. austrialis to the mycobiota of Iran.

Phylogeny

The ITS sequences of N1P10S3 and N1P10S4 isolates were combined and aligned with the eight reference sequences of three taxa from GenBank. The alignment consisted of 542 characters including alignment gaps, of which 360 were constant, 185 were variable and 108 were parsimony-uninformative. Maximum parsimony analysis of the parsimony-informative characters resulted in three most parsimonious trees with a consistency index of 0.91, the retention index of 0.91 and composite index of 0.85 (Fig. 1). NJ analysis resulted in the trees with the same topology as the MP trees. The MP tree is shown with bootstrap values of both analyses at the nodes (Fig. 1). Phylogenetic analyses based on ITS-rDNA sequence (Fig. 1) placed the examined isolates N1P10S3 and N1P10S4 with reference strains of R. subulatum (including ex-type strain CBS405.76) in a strongly supported monophyletic clade with 74% bootstrap support. Furthermore, all isolates of the R. subulatum were well separated from the ex-type strain of the close species R. epichloes. Therefore, the molecular analysis based on ITS-rDNA sequence confirmed that the isolates belong to the R. subulatum.

Radulidium subulatum (de Hoog) Arzanlou, W. Gams & Crous, Studies in Mycology 58: 89 (2007)

Basionym: Ramichloridium subulatum de Hoog, Studies in Mycology 15: 83. 1977.

Cultural characteristics: Colonies on MEA rather fast growing, reaching 50 mm diam after 14 d at 24 °C, with entire but vague margin, velvety, floccose near the margin, centrally with fertile hyphal bundles up to 10 mm high, about 2 mm diam; mycelium whitish, later becoming greyish brown; reverse grey, zonate. 

Morphology on MEA: Submerged hyphae hyaline, thin-walled, 1–2.5 µm wide; aerial hyphae brownish. Conidiogenous cells arising laterally from vegetative hyphae, pale brown, smooth, thick-walled, sometimes without a basal septum, cylindrical to aculeate, tapering gradually towards the apex, widest at the base, 25–40 × 2–3 µm; proliferating sympodially, forming a pale brown rachis, with densely crowded, prominent, blunt conidium-bearing denticles, with pale brown apex. Conidia solitary, sub-hyaline, thin-walled, smooth, ellipsoidal to almost clavate, 5–6.5 × 1.5–2 µm, with a slightly pigmented, non-refractive hilum, about 1 µm diam (Fig. 2).

These morphological characters were completely accorded to the characteristics that examined and stablished by Arzanlou et al. (2007) in specimens examined: Czech Republic, on Phragmites australis, ex-type culture CBS 405.76; Czech Republic, from Lasioptera arundinis, CBS 101010.

Specimen examined. UTFC S77, UTFC S150, UTFC 187, UTFC S21-1, UTFC S21-2, UTFC S21-3, UTFC N1P8R6, UTFC N1P4S6, UTFC N1P4S5, UTFC N1P3S1, UTFC N1P2S2, UTFC N1P9S4, UTFC N1P10S3, UTFC N2P8R1, UTFC N2P8L1, UTFC N2P7L3, UTFC N2P6R1, UTFC N2P5S1, UTFC N2P4S4, UTFC N2P1S4 and UTFC N2P6R4.

Fig. 2. Radulidium subulatum (N1P10S3). a. Colony on MEA, 7 d growth. b. Colony on PDA, 14 d growth. c-g. Macronematous conidiophores with sympodially proliferating conidiogenous cells, resulting in a conidium-bearing rachis. Scale bars: c-e = 10 μm; f–g = 50 μm.