|The Narcissus flower (Narcissus tazetta L., Amaryllidaceae) is one of the most important decorative flowers in Iran. This plant hosts a large number of endophytic and pathogenic fungi (Farr & Rossman 2022). During 2020-2021, N. tazetta plants growing in the natural resource areas of Behbahan in Khuzestan province (southwestern Iran) were visually inspected for disease symptoms. A typical brown spot on narcissus leaves was observed, which was collected for isolation of the potential fungal pathogen. The leaves were cut into approx. 5 mm pieces at the healthy and symptomatic margin. The pieces were then surface disinfected for 60–90 seconds in 1% sodium hypochlorite (NaOCl) and washed three times with sterilised distilled water, followed by drying on sterilised filter paper. Disinfected leaf pieces were plated on potato dextrose agar medium (PDA, potato extract 200–400 g L−1, sucrose 10 g L−1, agar 12 g L−1) supplemented with 30 mgL−1 of streptomycin and incubated at 25°C until ten days. The fungal hyphae growing from the leaf pieces were subcultured on PDA and then purified on water agar (WA) using the hyphal tipping method. Five morphologically identical phoma-like strains were isolated, and two of them (SCUA-Ba-NB2 and SCUA-Ba-NB24 isolates) were used for further morphological and molecular analyses. |
Morphological characteristics were determined from cultures grown on oatmeal agar (OA, oatmeal 30–60 g L−1, agar 12 g L−1) after ten days of incubation at 25 °C under a photoperiod of 12 h. Colonies on OA grew to a diameter of 60–72 mm (mean = 65 mm) after seven days of incubation at 25 °C ± 0.5.; circular with filiform margin, pale olivaceous-grey with darker margin, with aerial mycelium that was dense and cottony. Conidiomata were pycnidial, globose to sub-globose, pale brown to brown, immersed in the agar or superficial, 1-3-ostiolate, 117.5-313.5 × 107-293 μm, 95% confidence limits = 166.5-211.5 × 152-193.5 μm, (x ± SD = 189 ± 50 × 172.5 ± 46.5 μm, n =50). The pycnidial wall was pseudoparenchymatous, composed of isodiametric angular cells, 3–5 layered, brown, with age becoming darker. Conidiogenous cells were hyaline, ampulliform, and phialidic. Conidia were hyaline, smooth- and thin-walled, ellipsoid, 0-septate, with rounded ends, 4.-6.5 × 2.5-3.5 μm, 95% confidence limits = 5.1-5.6 × 3.1-3.3 μm, (x ± SD = 5.4 ± 0.5 × 3.2 ± 0.2 μm, n =40). Chlamydospores were unicellular or multicellular, globose to subglobose, solitary or in the chain, intercalary or terminal, and brown to dark brown (Fig. 1).
For molecular identification, the mycelial biomass of each strain produced on PDA was harvested by a sterile glass slide and powdered in liquid nitrogen. DNA was isolated according to a chloroform- and phenol-based organic method described by Mehrabi-Koushki et al. (2018). The internal transcribed spacer regions 1 and 2 including the intervening 5.8S nuclear ribosomal DNA (ITS) and a partial sequence of the β-tubulin gene (tub2) were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990) and Btub2Fd/ Btub4Rd (Woudenberg et al. 2009), respectively. PCR amplification and DNA analyses were performed by following methods described by Safi et al. (2021). Phylogenetic analyses were performed using reference sequences from related species of the strains under survey (Table 1). A combined ITS-tub2 DNA matrix was made, and then a two-locus maximum likelihood (ML) tree was constructed in the raxmlGUI 2.0 beta program (Edler et al. 2020) using the following options: general time-reversible (GTR) model of evolution, a gamma-distributed rate variation (G) and thorough bootstrapping analysis with 1000 replicate (MLBS). Maximum parsimony (MP) analysis was performed using MEGA 7 software (Tamura et al. 2013) with 1000 pseudo-sampling in bootstrapping analysis. Bayesian analysis (BI) was performed by MrBayes v.3.2.6 program (Ronquist et al. 2012) and using the GTR + G + I model for both loci, estimated by jModelTest 2 (Darriba et al. 2012). The BI and MP analyses showed a similar tree topology to that obtained in the ML analysis.
In phylogenetic tree (Fig. 2), both isolates (SCUA-Ba-NB2 and SCUA-Ba-NB24) clustered with the type strain of Didymella prosopidis (Crous & A.R. Wood) L.W. Hou, L. Cai & Crous (CBS 136414) in a moderately-supported clade (MLBS 76%, MPBS 95%, BPP 0.79). The ITS (accession numbers; OP821092 and OP821093) and tub2 (accession numbers; OP828921 and OP828922) sequences are deposited in GenBank.
According to both phylogenetic and morphological analyses, Iranian isolates were identified as D. prosopidis. This is the first record of D. prosopidis for mycobiota of Iran. This species was originally isolated from diseased stems of Prosopis sp. in South Africa and introduced as Peyronellaea prosopidis Crous & A.R. Wood (Crous et al. 2013). Later, Hou et al. (2020) recombined this species with Didymella (Hou et al. 2020). The genus Didymella is a fungus belonging to the Didymellaceae family and contains several pathogenic species mainly distributed in the field and ornamental crops as well as in wild plants (Chen et al. 2015, Ahmadpour et al. 2021, 2022). Many Didymella species are also saprobes that are commonly found in living or dead tissues of herbaceous and wooden plants (Chen et al. 2015); some species also act as mutualistic endophytes with some plant species (Rayner 1922). In this study, D. prosopidis was isolated from Narcissus tazetta showing leaf spot symptoms. So far, no other species from the family Didymellaceae has been reported from this genus, except Didymella curtisii (Berk.) Qian Chen & L. Cai in Armenia, Australia, and Poland (Boerema et al. 2004, Farr & Rossman 2022).