Mycologia Iranica
Volume:9 Issue: 1, Winter and Spring 2022

The genus Globisporangium is a newly described taxon that has been recently separated from Pythium sensu lato. Although not many studies focused on isolating species assigned to this genus from Iran, some comprehensive studies showed that Globisporangium is an important genus with vast distribution in this part of the world. Even rare species assigned to Globisporangium have also been found in the country. Despite the importance of this genus, accurate identification and classification of Globisporangium is quite challenging worldwide. Morphological identification of Globisporangium is quite difficult due to the lack of identification keys, overlapping of some morphological features, the existence of species complexes, pleomorphism, and the absence of certain structures in some species. Furthermore, there is no universal DNA barcode for Globisporangium species yet, and most species cannot be delimitated using only one or two loci for the phylogenetic analyses. Besides, some studies in Iran do not include molecular investigations to support their morphological identification or make it possible to reidentify the reported species. Having no accurate checklist of the current species in the country also adds up to the problem. This review focuses on the current systematics of Globisporangium species in Iran, emphasizing the challenges in the morphological and molecular identification of the species in the country; it also proposes and discusses some solutions to resolve these problems.

Pyriculariaceae family has newly been established several new genera and species. Indeed, some species originally described Pyricularia subsequently have been synonymized or transferred to other genera. In this study an overview of taxonomical and phylogenetic findings, symptomology of Pseudopyricularia species and an identification key to Pseudopyricularia species by morphological criteria are provided.

The present paper is concerned with description of taxonomic diversity and study of aspects of ecology of water-borne microfungal biota of Sanjay Gandhi National Park (SGNP). The study area is home to freshwater streams, lakes and a saline creek. The study resulted in 28 isolates of water fungi obtained from 20 water samples. After morpho-molecular analysis, a total of 22 species were documented under 5 genera. Aspergillus was the dominant genus with 12 species, whereas Aspergillus flavus and A. terreus were dominant species, each represented by 3 isolates. Gini-Simpson’s index was 0.9439, Shannon’s index was 2.9978. Pielou’s evenness index was 0.9698, causing observed species richness (22) to be greater than true diversity, calculated as the effective number of species (20).

Rice is one of the most important crops in Iran and the rice blast caused by Pyricularia oryzae causes significant losses in rice fields during outbreak season. At the beginning of the disease cycle, the primary inoculum imposes severe damage to plants as it leads to plant weakness. The damage will shortly become significant as the second wave of inoculum spreads. This study aimed to determine the occurrence and severity of blast disease on some native Iranian cultivars and estimate the number of conidia produced on second leaves. Pyricularia oryzae Cavara isolates were collected from rice leaves from the north of Iran. One out of 123 isolates was inoculated on seven Iranian native cultivars named Hashemi, Tarom Mahali, Sang Tarom, Binam, Champa, Alikazemi, and Domsiyah at the four-leaf stage. Lesions on leaves were scored and the percentage of the spotted area was calculated by software Image J1.52a. Various lesion types and a range of diseased areas varying from 30% recorded in Hashemi to 55% in Tarom Mahali were observed. Under optimum conditions, the sporulation potential was calculated. Isolate produced different amounts of spores on different cultivars; Hashemi showed the highest and Tarom Mahali showed the lowest sporulation with 63 and 11 spores per mm2 of spotted area. The result indicated that the resistance threshold of the native cultivars could be different but not sufficient. Additionally, to estimate the damage on each cultivar on farms, we need to investigate the sporulation potential of the pathogen through time in each cultivar

In summer 2020, bean plants (Phaseolus vulgaris L.) with symptoms of wilting, chlorosis, drying and the formation of numerous microsclerotia in the stem were detected from Ghachsaran fields, Kohgiluyeh & Boyer-Ahmad provinces of Iran. Infected samples of stem were taken to the laboratory, cut into small sections, sterilized, washed in sterile water, dried on sterile paper towels, and cultured on Potato Dextrose Agar (PDA) amended with 50 µg of kanamycin to prevent bacterial contamination. Isolates were purified by hyphal tip technique. Four isolates from infected stems tissue were recovered. Fungal isolates were identified based on morphological characteristics and molecular data of tef1-α gene. According to the morphological and phylogenetic analysis, the isolates were identified as Macrophomina vaccinii. This is the first report of M. vaccinii, in Iran

In the spring of 2021, leaf spot disease symptoms were observed in the Garden Croton (Codiaeum variegatum) plants in one of the ornamental plant greenhouses of Mahallat County, Markazi Province, Iran. To identify the causal agent of the disease, infected plant leaves sampled and then were transferred to the mycology laboratory and nine fungal isolates were recovered. The fungal isolates were characterized based on the morphological features and molecular data using the combined sequences of the internal transcribed spacer (ITS) regions and the translation elongation factor 1-alpha (tef) gene for a representative isolate UT2022. According to the morphology and phylogenetic analysis, the isolate UT2022 was identified as Pestalotiopsis trachycarpicola. The pathogenicity of the isolate UT2022 was performed on healthy and growing leaves of the C. variegatum plants. Inoculated leaves showed leaf spot symptoms 14 days after the inoculation, while the leaves of the control plants were symptomless. To complete Koch’s postulate, P. trachycarpicola was re-isolated from the newly produced leaf spots. Based on the bibliography, P. trachycarpicola is reported as a new taxon for fungi of Iran, as well as P. trachycarpicola is reported as the causal agent of the leaf spot symptom of the C. variegatum in the world.

Fungi have been subjected to genetic engineering in various ways. Agrobacterium tumefaciens-mediated transformation (AtMT) is an important method for the genetic manipulation of different fungal species. Here, gene transfer to Trichoderma viridescens was performed and optimized using A. tumefaciens strain pSDM2315. Also, the effect of different temperatures on the growth and conidiation rates of the wild-type and transformed fungi was investigated. The results indicated that the best conditions for maximum transformation in T. viridescens were the combination of one day of incubation, 28˚C, pH 5.0, and a concentration of 107conidia mL-1. The results of gene transfer and stable expression of transgenes were confirmed using sequential culture in selective media and PCR.Moreover, the mycelial growth of transformed fungi at different temperatures did not show an obvious difference from the wild-type, but the mutants produced different numbers of conidia. This indicates the potential of AtMT for functional mutagenesis and physiological studies in T. viridescens.

During a survey of leaf spots on tropical trees, symptomatic leaf tissue of mango trees was collected from several sites in the Hormozgan, and Sistan and Baluchestan provinces. Isolations were performed on potato dextrose agar (PDA) and wet paper media at 25 °C. As a result, several fungal species were obtained, that three isolates belonging to Bartalinia and four of Beltrania. Phylogeny based on DNA sequences of the large subunit of the ribosomal RNA (LSU) and internal transcribed spacer (ITS-rDNA) regions combined with morphological criteria revealed the species of Bartalinia pini and Beltrania rhombica. To our knowledge, both species, B. pini and Be. rhombica are new species for the Funga of Iran.

Fusarium wilt, root and crown rot caused by Fusarium oxysporum f. sp. ciceris, (FOC) is the highly significant soil-borne disease of chickpea in the Kurdistan province of Iran. The distribution of pathogenic races of FOC in Kurdistan province was determined during this research. Infected plant samples were collected from 42 fields in the chickpea production area of the Kurdistan province. The causative microorganism of the disease was isolated and purified from each sample, and then FOC isolates were identified by morphological characters. After the pathogenicity test and evaluation of pathogenic variability on the susceptible cultivar Kaka, the DNA extraction, the molecular identification of species, and races of pathogenic isolates were performed using FOC-specific linked primers. Among the collected isolates, 37 were identified as Fusarium oxysporum f. sp. ciceris. Molecular identification of races using SCAR-linked markers (1B/C, 0, 2,3,4,5, 6, and 1A) revealed that 28 out of 37 isolates belonged to race 0, and other isolates belonged to race 1B/C. There was no relationship between the prevalence of races and their geographical distribution. Identification of the races is crucial for the evaluation of resistance and the development of new commercial cultivars. The application of resistant cultivars is a fundamental approach for the integrated management of the Fusarium wilt, root, and crown rot for durable chickpea production

In spring 2019, melon plants (Cucumis melo CV. Janna) with collapse and decline symptoms were collected from fields in Dashti county, Bushehr province in southern Iran. Twelve morphologically similar isolates from infected root and crown tissues were recovered. Fungal isolates were identified based on morphological characteristics and molecular data of the internal transcribed spacer (ITS-rDNA) region. According to the morphological and phylogenetic analysis, the isolates were identified as Plectosphaerella cucumerina. The pathogenicity test was performed on healthy seedlings of melon plants (CVs. Kharboze-Mashhadi and Shahde-Shiraz). Three weeks after inoculation, the melon seedlings showed root rot, necrosis and wilting symptoms then eventually death. To complete the Koch's postulate, P. cucumerina was re-isolated from inoculated plants. This is the first report of P. cucumerina causing decline on melon in Iran.

Large flowered pelargonium (Pelargonium grandiflorum) is a perspective flowering crop which is distributed around the world. The area of its application is quite wide: from room floriculture to design the gardens and parks. Observation of leaf spot symptoms on this plant, which was collected from Alborz province (Karaj) motivated us to find the causal agent(s) of the disease. So, the symptomatic parts were cultured on the PDA medium after surface sterilization. Two fungal colonies were appeared on the culture medium. They were identified as Alternaria alternata and Botrytis cinerea according to the morphological characterizations. Molecular study using the gapdh for Alternaria and ITS regions and rpb2 gene for Botrytis confirmed the result of the morphological identification. In the pathogenicity tests, the same spots on inoculated plants with Alternaria and the same spots plus gray mold symptoms and fungal body on leaves, buds and stems of the inoculated plants with Botrytis were another confirmation. Based on our knowledge, this is the first report of these two fungal species on the Pelargonium grandiflorum in Iran.

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).