Mycologia Iranica
Volume:6 Issue: 2, Summer Autumn 2019

Pythium plurisporium was originally isolated from the roots of bentgrass (Agrostis palustris). It is characterized by the production of multiple oospores in oogonium, which mostly has pedicellated stalk and swollen elements below its stalk. There are not many reports of the occurrence of this species in the literature. Recently, a report of recovering P. plurisporium isolates from Iran has been presented. Nevertheless, the re-examination of the isolates referring to P. plurisporium using morphological identification as well as multiple gene genealogies, using both nuclear (ITS and Btub) and mitochondrial (cox2) loci, arises the question about the existence of intraspecific phenotypic variation within this species. A revision of morphological characteristics among isolates assigned to P. plurisporium is discussed in the present paper.

The role of extracellular enzymes in mycoparasitism of Trichoderma spp. have been demonstrated in several cases. Trichoderma spp . produces several chitinolytic enzymes and this fungus is known as a powerful antagonist against Macrophomiona phaseolina, the causal agent of charcoal rot disease of soybean. In this study, two–dimensional protein pattern analysis and chitinaseactivity evaluation were performed for an Iranian Trichoderma koningii strain (NAS–K1) and its generated mutants were measured to indicate the potential role of endochitinases (N–acetylglucosam–inidase (NAG–I and NAG–II)) in its mycoparasitism. The results of chitinase activity assay using chitin and cell walls of M. phaseolina as substrates showed that the mutant isolates have significantly more enzyme activity compared to the wild type strain. The specific endochitinase enzyme activity in the mutant NAS–K1M25 was increased to approximately 2.5 times more and secreted three times more endochitinase than that of the wild type strain. This superior mutant showed up to 65% growth inhabitation against M. phaseolina in dual culture test (five times more than the wild type strain). Moreover, this strain showed sharper spots belong to endochitinase, and N–acetylglucosaminidase (I & II) presented in SDS–PAGE and 2D electrophoresis. Overall, induced mutation by gamma irradiation could be a useful method to develop such superior mutants, and the mutant NAS–K1M25 could be used as a potential biological control agent candidate for plant disease management programs of M. phaseolina. However, more detailed fermentation, formulation and field trial studies should be performed to finalize its biocontrol potentials.

Ascochyta blight caused by Ascochyta lentis is one of the most important diseases of lentil in the world. This disease has been reported in various areas in Iran causing serious damages every year. Currently, the use of resistant cultivars is considered the most effective and environmentally sustainable strategy for disease control. This study was carried out to determine the genetic diversity of A. lentis populations. For this purpose, samples were collected from eight different regions of Ilam and Kermanshah provinces including: Ilam, Ayvan, Malekshahi, Sirvan, Chardavol, Islamabad, Gilan-e Gharb, Sarpul-e Zahab. After isolation, purification and morphological identification, genetic variation of 94 A. lentis isolates were assessed using three pairs of SSR primers. Based on molecular analysis, 34 alleles were observed among the populations, and average number of alleles was 1.688. The amount of marker index (MI) in the ArA03T primers was the highest. The results of AMOVA analysis showed 92% variability within populations and 8% diversity among the populations. Total gene diversity (Ht) and gene diversities between populations (Hs) were estimated 0.315 and 0.269, respectively. Gene diversity attributable to differentiate among population (Gst) was 0.146, while gene flow (Nm) was 2.903. Cluster analysis based on UPGMA method showed the lowest genetic distance between Ayvan and Sarpol-e Zahab, afterward Ilam and the highest genetic distance between Islamabad and seven remaining populations. This is the first report and comprehensive survey of A. lentis pathogen genetic diversity in Iranian lentil fields. Results of this study can be useful in breeding programs with the aim of producing A. lentis resistant cultivars and developing effective methods for disease control.

Several reports are available on species diversity of yeasts on grape berries in different grapevine producing countries, including Iran. However, there is a paucity of knowledge on species diversity of yeasts on post-harvest table grapes worldwide. Hence, this study was performed to explore the species diversity of epiphytic yeasts on post-harvest table grapes in markets of Tabriz, northwest Iran. Towards this aim, 120 grape samples, mostly Keshmesh, Shahani, Gezeluzum and Shast-arous cultivars, were purchased from selected main markets in Tabriz and subjected to yeast isolation. Total number of 180 epiphytic yeast isolates were recovered. The isolates were preliminary grouped based on the morphological characteristics and DNA fingerprinting profiles using MSP-PCR fingerprinting technique. The D1/D2 domain of the 26S rDNA was amplified and sequenced for one or two isolates representing each fingerprinting group. Totally, 20 isolates were sequenced and the phylogeny inferred from sequence data of D1/D2 region revealed a rich diversity of yeast species on post-harvest table grape berries. Sixteen yeast species belonging to both ascomycetes and basidiomycetes were identified. The majority of identified yeast species (75%) belonged to ascomycetes. Aureobasidium pullulans, Hanseniaspora uvarum and Metschnikowia sinensis are reported as the most frequently isolated yeasts. In this study, Clavispora lusitaniae and Cyberlindnera fabianii are newly reported on grape berries worldwide and C. lusitaniae, C. fabianii, Wickerhamomyces anomalus and Yamadazyma mexicana represent new records for the mycobiota of Iran.

Pyrenophora lolii causes leaf spot on grasses including Festuca spp., Lolium spp., Dactylis spp., Avena sativa and wheat (Triticum aestivum). Infected oat leaves (Avena sativa) showing leaf spot symptoms were collected from the margin of barley fields in Golestan province of Iran during the spring of 2016. A morphological examination of the Pyrenophora specimen was carried out using light microscopy. Inoculation of oat leaves with the isolates of Pyrenophora lolii in greenhouse induced leaf spot on leaves. In order to confirm the morphological identification, sequences of glyceradehyde-3-phosphate dehydrogenase (gpd) gene and Internal transcribed spacer (ITS) regions were amplified using gpd1/2 and ITS1/4 primers, respectively. The phylogenetic analysis based on these sequences showed that the isolated Pyrenophora specimen clustered together with sequences of P. lolii. Based on result of morphological examination and phylogenetic analysis, it was concluded that the causal agent of leaf spot of A. sativa (oat) was P. lolii.

During the study of fungal species associated with canker and dieback diseases of walnut trees (Juglans regia L.) in East Azerbaijan province, Iran, eight fungal isolates with similar characteristics resembling asexual stage of the genus Juglanconis were isolated from trees showing canker, dieback, and dead skin symptoms in Osko and Horand regions. Conidiomata on the host tissue were acervular and stromatic, 1 – 2.5 mm diam, blackish, scattered or confluent, covered with black conidial masses when mature. Conidiophores were unbranched or rarely branched at the base. Conidiogenous cells were cylindrical and annellidic. Conidia elliptic to ovate, truncate with distinct scar at the base, thick-walled and with distinct ornamentation on the inner side of the wall consisting of irregular confluent verrucae, hyaline when immature, brown to blackish when mature, covered with gelatinous sheath, (11)14–16 (–18) × (15–) 22–25 (–28) µm. Fungal isolates were identified as J. juglandina based on morphological characteristics and host association. Identification of the species was further confirmed by sequence analysis of the elongation factor (tef1-α) gene. Present study is the first report of J. juglandina for the mycobiota of Iran.

Cadophora was described with C. fastigiata Lagerb. & Melin as the type species of this dematiaceous hyphomycetous genus that produces solitary phialides with distinct hyaline, flared collarettes (Lagerberg et al. 1927). The generic name Cadophora was suggested by Gams (2000) for phialophora-like species with affinities to the Dermateaceae in the Helotiales. Cadophora species are primarily isolated from living plants, as pathogens or root colonizers, and produce melanized, septate hyphae that aptly placed them among the fungi labeling dark septate endophytes (DSEs) (Jumpponen & Trappe 1998; Zijlstra et al. 2005).Some samples were collected in May 2016, from Ferula ovina and Ferula felabelliloba (Apiaceae) in Zoshk highlands of Khorasan Razavi province, Iran (36°26 12.0 N 59°11 51.6 E) by Zahra Tazik. Isolation for obtaining endophytic fungi was done according to the method described by Hallmann et al.(Hallmann et al. 2007) with minor modifications. The fresh and disease-free root samples were washed with running tap water and allowed to dry. Then, the obtained samples were cut into pieces of 0.5–1 cm and root pieces were placed in 75% ethanol for 1 min followed by diluted sodium hypochlorite solution 4% for 3 min and finally, in ethanol 75% for 30s. The samples were washed in distilled water after sterilization, placed on filter paper in sterile conditions, and allowed to dry. The root parts were placed on potato dextrose agar (PDA; Merck, Germany) and malt extract agar (MEA; Merck, Germany) containing streptomycin (20 μg/mL) and chloramphenicol (30 μg/mL). The cultures were then incubated at 25-30 °C for 7-28 days. Hyphal tips of fungi emerging out of the root tissues, were picked and grown on PDA, oatmeal agar (OA; 30 g boiled and filtered oat flakes, 15 g agar, 1 L distilled water) and 2% MEA. For sporulation, corn meal agar (CMA; Merck, Germany) and OA media were used, and finally the microscopic slides of fungal isolates were prepared by staining with lactophenol cotton-blue (Vainio et al. 1998) and were examined under a light microscope (Olympus, Tokyo, Japan). The field emission scanning electron microscopy micrograph was taken using FESEM (TESCAN BRNO-Mira3 LMU, 2014, Germany) in the secondary electron imaging (SE) mode. The microscope was operated at 10 kV acceleration voltage, 1.8 kV extraction voltage and a working distance of 5.92 mm. The fungal isolate was deposited in the Fungarium of the Iranian Research Institute of Plant Protection, Tehran, Iran (IRAN) with voucher number: IRAN 3316C. The fungal genomic DNA was extracted using DenaZist Asia fungal DNA isolation kit according to the manufacturer’s instructions. The PCR amplifycation was performed in a total volume of 25 μL reaction containing 1 ng DNA template, 10 pM of each primer, 10 μL of Taq DNA Polymerase Master Mix RED (Amplicon, Odense, Denmark). Partial sequences of the internal described spacer regions with the 5.8S nuclear ribosomal RNA gene (ITS) were amplified using the ITS5 and ITS4 primers (White et al. 1990). Target regions within the large subunit of the nuclear ribosomal RNA gene cluster (LSU) and translation elongation factor 1-alpha (TEF1-α) genes were amplified using primer pairs LRORF/LR5R (Vilgalys & Hester 1990) and 983F/ EfgrR (Rehner & Buckley 2005), respectively. The PCR products were analyzed in 1.5% agarose gel electrophoresis with 1x Tris-Boric acid-EDTA buffer (TBE) and sent to Macrogen Korea for sequencing. The obtained sequences were subsequently analyzed using the BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and closely similar sequences obtained from the National Centre of Biological Information (NCBI) database. The number of sixteen reference strains of Cadophoraincluding our isolates and Cudoniella clavus, as outgroup, were chosen for phylogenetic analyses. The TEF1-α gene was also sequenced (MK512743), but was not used for phylogenetic analyses because comparable sequences of related species were not available. Therefore, we used concatenated data sets of ITS and LSU regions to maximize taxon coverage in our phylogenetic analyses. The Bayesian tree was generated with MrBayes version 3.1.2 (Ronquist & Huelsenbeck 2003) (Fig. 1). The obtained sequences were deposited in GenBank (NCBI) (accession numbers: MF186880, MK275617 (ITS), MH400226 (LSU), MK512748 (TEF1). According to BLASTn analysis, our ITS sequences were 98% similar to Cadophora luteo-olivacea CBS141.41 with a 90% similarity to the LSU sequence of the same species, indicating that our isolates belong to Cadophora genus.