Pyrenophora lolii , a new species for Iranian mycobiota

INTRODUCTION

 Avena sativa L. is an annual grass (Dimberg et al. 1996) and placed in the family Poaceae and its wild ancestor is A. sterilis which is endemic to Iran, Iraq, and Turkey. Oat has been cultivated for centuries worldwide (Zhou et al. 1999) for its grain as food and fodder, as well as for medicine (Coffman 1977). Avena sativa is infected by two species of fungal pathogens of the genus Pyrenophora; P. avenae causing leaf blotch and black leaf spot, and P. lolii causing leaf spot (Mehta 2001). Species in the genus Pyrenophora (anamorph: Drechslera), cause disease on Poaceous plants (Zhang & Berbee 2001) and is placed in the family Pleosporaceae (Berbee 1996). Some species of this genus are agents of destructive diseases on Poaceaeincluding barley and wheat (Ariyawansa et al. 2014). In previous studies, phylogenetic analysis of Pyrenophora was carried out based on sequence data of ITS, GPDH, RPB2, nrSSU and nrLSU DNA regions (Ariyawansa et al. 2014, Zhang & Berbee 2001). 

Pyrenophora lolii cause leaf spot on grass (Jones 2013) and is found in different parts of the world including Sweden, New Zealand, England and Wales (Jones 2013, Lam 1984). Pyrenophora lolii infect Festuca spp., Lolium spp., Dactylis spp., Avena sativa (Oat) and Triticum aestivum (Wilkins 1973, Gönner et al. 1993, Tonin et al. 2015, Jones 2013) but it is more common on Lolium spp. Yield loss of this fungus in mixed infection with D. catenaria on Lolium perenne can reach up to 15 %. (Jones 2013). The goal of this study was to characterize and identify a new-found Pyrenophora species infecting oat in Iran, using morphological characterization and phylogenetic analyses based on GPDH and ITS sequences.

MATERIALS AND METHODS

Sampling and morphological characterization

Infected A. sativa leaves exhibiting leaf spot symptoms were collected from margins of barley fields during the spring of 2016 in the Golestan province located in the Northeast of Iran (Sadabad village), at an altitude of 160 m above sea level. After sterilization with 1% sodium hypochlorite solution, infected leaves were transferred on 2% water agar and incubated at
20 ^°C and with 12 h darkness and 12 h NUV light (Akhavan et al. 2016). After 10 days, spores emerged on the surface of leaves and these were used for morphological identification. The morphological characteristics of the specimen are summarized in Table 1.

Pathogenicity test

To assess the pathogenicity of the isolated fungal spores, A. sativa plants were grown in the greenhouse under 11 h light and 13 h darkness at 20 ^°C. For sporulation of P. lolii, it was cultured on Potato Dextrose Agar, where after cubes of 6-day-old colony margin were transferred to 2% WA (water agar) and incubated at 12 h NUV light and 12 h darkness at 20 ^°C. For plant inoculation, the concentration of spore suspension was adjusted to 5 × 10⁵ conidia per mL and sprayed on plant leaves at the third to fourth leaf stage (Akhavan et al. 2016). After inoculation, pots were covered by a plastic bag for 24 h in order to create a 100% humidity condition. The two days following inoculation, leaves were surveyed daily. When symptom developed after 10 days, infected leaves were cultured on 2% WA under 12 h NUV light and 12 h darkness at 20 ^°C for re-isolation (Akhavan et al. 2016).

DNA extraction, PCR amplification

For DNA extraction, one isolate was cultured on PDB (Potato Dextrose Broth) and incubated at 20 ^°C and with 12 h darkness and 12 h NUV light (Akhavan et al. 2016). After 10 days, DNA was extracted using the CTAB method (Murray & Thompson 1980). For molecular identification, ITS region and gpd gene were amplified and sequenced.

For amplification of the ITS1 - 5.8S – ITS2 region, forward primer ITS1 5'-TCCGTAGGTGAACCTGCG G-3' and reverse primer ITS4 5'-TCCTCCGCTTATT GATATGC-3' were used (White et al. 1990) and for amplification of the gpd gene, forward primer gpd1 5'-CAACGGCTTCGGTCGCATTG-3' and reverse primer gpd2 5'-GCCAAGCAGTTGGTTGTGC-3' were used (Berbee et al. 1999).

Reaction conditions for both ITS region and gpd gene consisted of an initial denaturation step at 95 °C for 3 min, followed by 30 cycles of 30 s at 95 °C, 30 s at 58 °C, and 1 min at 72 °C, followed by an extra extension at 72 °C for 7 min. PCR analysis was performed in a reaction mixture containing 10 µL master mix, 6 µL H2O, 2 µL DNA (3 ng/µL), 20 pmol of each of the forward and reverse primers, and final volume was 20 µL.

The sequencing was done by Sanger sequencing. For the ITS region, the sequenced fragment was approximately 560 bp including the internal transcribed spacer 1, partial sequence, 5.8 S gene and internal transcribed spacer 2, complete sequence; and 28 S ribosomal DNA gene, partial sequence. The gpd1/2 primers amplified a 600 bp fragment including 417 bp of the coding region and 180 bp of two introns. ITS and gpd sequences were deposited in NCBI with accession numbers of MN420825 and MN433343, respectively.

Phylogenetic analyses

To assess the phylogenetic position of the Iranian Pyrenophora specimens on A. sativa, both their ITS and gpd sequences were compared with Pyrenophora ITS and gpd sequences available in GenBank. The ITS and gpd sequences of the Pyrenophora specimen were blasted using blast analyses (https://blast.ncbi.nlm. nih.gov/Blast.cgi) (2020). Alignment of sequences for both ITS and gpd was conducted by using MEGA7 and the align by clustal W option (Kumar et al. 2016) and the result of alignment were used for phylogenetic analyses utilizing a maximum likelihood (ML) method based on the Tamura-Nei model (Tamura & Nei 1993) and neighbor-joining (Nj)  method (Saitou & Nei 1987). The analysis was conducted with MEGA 7 using the bootstrap method option with 1,000 replicates (Kumar et al. 2016). The tree was drawn by using integrated sequences of gpd and ITS and rooted with Pleospora alfalfae.

RESULTS

Morphological characterization

The results of the morphological analysis of the specimen are included in Table 1 and depicted in Fig.1.

Pyrenophora lolii Dovaston, Transactions of the British Mycological Society 31 (3-4): 251 (1948) MycoBank MB 290353

Symptom on the infected oat leaves was an ellipsoid brown spot form. Red-brown, long and narrow conidiophores usually arise singly. They were enlarged at base. A group of 7-20 conidia grow at the top of conidiophores. The length of the conidiophores was 120-140 µm and the base of conidiophore was enlarged, globose, 10-20 µmwide. Cylindrical and straight conidia are 10-20 µm thick, 48-110 µm long and nearly round at both ends, pale to yellowish-brown, often having 3- 8 pseudosepta. The length and width of each cell of conidium is 10 to 20 µm. The diameter of the colony reaches to 7 cm after 5 days on PDA. The color of the colony is dark brown with the grey aerial mycelium developing later.

Specimens examined. Iran, Golestan province, Sadabad village, on Avena sativa, 15 April 2016, collected by A. Vasighzadeh.

The morphology of specimen on Avena sativa from Iran agrees with the description of P. lolii on Lolium perenne (Shoemaker 1962) and it is shown in Table 1.

Pathogenicity test

Inoculation of the Pyrenophora specimens on A. sativa leaves induced brown pin-like symptoms after 4 days.

This symptom developed after 10 days as oval and brown leaf spot with chlorotic margin (Fig. 1). By culturing of the symptomatic leaves on 2% WA, fungal spores emerged on the surface of the culture medium.

Phylogenetic analyses

After the blast analysis in NCBI, some of the sequences of P. lolii which were identical to the Pyrenophora specimen sequence, included in the alignment. The ITS sequence of Pyrenophora specimen in this study was 100% similar to previously published P. lolii sequences which were included in the phylogenetic analysis. The gpd sequence of P. lolii in this study was compared to gpd sequences of four isolates of P. lolii and was also 100% similar to sequences of three isolates of P. lolii. We only identified a one-nucleotide difference between Pyrenophora specimen sequence and Pyrenophora lolli strain HMCI which the sequence differentiation was 0.19 %. The trees obtaining from neighbor-joining (data not shown) and maximum likelihood methods based on gpd and ITS sequences showed the same tree topologies.

Based on phylogenetic analysis of integrated sequences of ITS and gpd, we found that the Pyrenophora specimen on A. sativa clustered with P. lolii isolates which had been isolated from  L. multiflorum (Fig 2.). The P. lolii isolates on L. multiflorum and Avena sativa formed a sister clade with P. chaetomioides and P. avenae which is a known pathogen of A. sativa (bootstrap values 100% for both methods). Pyrenophora lolii was distant to a monophyletic lineage on barley, wheat, and bromegrass (P. teres, P. graminea, P. tritici-repentis and P. bromi).

DISCUSSION

In previous studies, the connection between Pyrenophora and Drechslera which are teleomorph and anamorph, respectively, was proved based on ITS and gpd data (Zhang & Berbee 2001). For instance, ITS and gpd sequences of P. lolii and its related anamorph, D. siccans, were identical (Zhang & Berbee 2001). In this study, as it was excepted, Pyrenophora spp. and its related anamorph, Drechslera spp., clustered together in the phylogenetic tree. 

In previous studies, P. avenae, P. chaetomioides, and P. lolii which are related to D. avenae, D. avenacae and D. siccans anamorphs,were proposed to be one taxon based on numerical taxonomy (Ibrahim & Threlfall 1966) but the molecular study based on the ITS and gpd data showed that P. lolii formed a sister group with a clade comprising P. chaetomioidesand P. avenae and they could not be considered as one monophyletic lineage (Zhang and Berbee 2001). This result was similar to the phylogenic analyses of ITS and gpd sequences of these taxa in this study. According to the phylogenetic analysis based on ITS and gpd data, the Pyrenophora specimen on Avena sativa in this study grouped with P. lolii. This species is closely related to P. chaetomioides and P. avenae which is host similarity among these species. Results of phylogenetic analysis of both sequences of the Pyrenophora specimen agree with the morphological characterization and the specimen was identified as P. lolii. Several species of Pyrenophora/Drechslera were found in Iran. Some of these species are destructive pathogens on wheat and barley such as P. tritici-repentis, P. graminea and P. teres (Ershad 2009). This studyis the first record of P. lolii on Avena sativa in Iran.