Cytotoxic, antimicrobial and antiviral secondary metabolites produced by the plant pathogenic fungus Cytospora sp. CCTU A309.

Chemical analysis of extracts from cultures of the plant pathogenic fungus Cytospora sp. strain CCTU A309 collected in Iran led to the isolation of two previously unreported heptanedioic acid derivatives namely (2R,3S) 2-hydroxy-3-phenyl-4-oxoheptanedioic acid (1) and (2S,3S) 2-hydroxy-3-phenyl-4-oxoheptanedioic acid (2) as diastereomers, four previously undescribed prenylated p-terphenyl quinones 3-6 in addition to five known metabolites. Their structures were elucidated on the basis of extensive spectroscopic analysis and high-resolution mass spectrometry. For metabolites 1 and 2, the absolute configurations at C-2 were deduced from comparison of the 1H NMR difference of their (S)- and (R)-phenylglycine methyl ester derivatives while the relative configurations were tentatively assigned by a J-based analysis and confirmed by comparison of 13C chemical shifts to literature data. The isolated compounds were tested for their cytotoxic, antimicrobial (including biofilm inhibition), antiviral, and nematicidal activities. While only moderate antimicrobial effects were observed, the terphenyl quinone derivatives 3-6 and leucomelone (10) exhibited significant cytotoxicity against the mouse fibroblast L929 and cervix carcinoma KB-3-1 cell lines with IC50 values ranging from 2.4 to 26 µg/mL. Furthermore, metabolites 4-6 showed interesting antiviral activity against hepatitis C virus (HCV).

Novel primers improve species delimitation in Cercospora.

The genus Cercospora includes many important plant pathogens that are commonly associated with leaf spot diseases on a wide range of cultivated and wild plant species. Due to the lack of useful morphological features and high levels of intraspecific variation, host plant association has long been a decisive criterion for species delimitation in Cercospora. Because several taxa have broader host ranges, reliance on host data in Cercospora taxonomy has proven problematic. Recent studies have revealed multi-gene DNA sequence data to be highly informative for species identification in Cercospora, especially when used in a concatenated alignment. In spite of this approach, however, several species complexes remained unresolved as no single gene proved informative enough to act as DNA barcoding locus for the genus. Therefore, the aims of the present study were firstly to improve species delimitation in the genus Cercospora by testing additional genes and primers on a broad set of species, and secondly to find the best DNA barcoding gene(s) for species delimitation. Novel primers were developed for tub2 and rpb2 to supplement previously published primers for these loci. To this end, 145 Cercospora isolates from the Iranian mycobiota together with 25 additional reference isolates preserved in the Westerdijk Fungal Biodiversity Institute were subjected to an eight-gene (ITS, tef1, actA, cmdA, his3, tub2, rpb2 and gapdh) analysis. Results from this study provided new insights into DNA barcoding in Cercospora, and revealed gapdh to be a promising gene for species delimitation when supplemented with cmdA, tef1 and tub2. The robust eight-gene phylogeny revealed several novel clades within the existing Cercospora species complexes, such as C. apii, C. armoraciae, C. beticola, C. cf. flagellaris and Cercospora sp. G. The C. apii s. lat. isolates are distributed over three clades, namely C. apii s. str., C. plantaginis and C. uwebrauniana sp. nov. The C. armoraciae s. lat. isolates are distributed over two clades, C. armoraciae s. str. and C. bizzozeriana. The C. beticola s. lat. isolates are distributed over two clades, namely C. beticola s. str. and C. gamsiana, which is newly described.

Tolerance to heavy metals in filamentous fungi isolated from contaminated mining soils in the Zanjan Province, Iran.

In the present study the population of fungi inhabiting metal contaminated soil samples from lead-zinc mining area was investigated, and their tolerance and biosorption capacity towards Cd, Pb, Zn and Cu were evaluated. Fungal species, including Acremonium persicinum, Penicillium simplicissimum, Seimatosporium pistaciae, Trichoderma harzianum, Alternaria chlamydosporigena and Fusarium verticillioides were isolated. Fungal tolerance was determined by measuring of the "Minimum Inhibitory Concentration", after exposure to increasing concentrations of heavy metal chlorides. Among the test fungi, Trichoderma harzianum was the most tolerant against Cd, Pb and Cu. The Heavy metal content in the fungal biomass was quantified after combustion. The calculated total sorption of heavy metals showed that Acremonium persicinum and Penicillium simplicissimum had the highest accumulation of Zn and Cu, respectively. The data presented in this study should help to use the accumulation potential of some of those fungal species examined for bioremediation of contaminated soils.

Diversity of culturable fungi inhabiting petroleum-contaminated soils in Southern Iran.

The present study was aimed at characterising species diversity of fungi inhabiting petroleum-contaminated soils of oil fields in a southern region of Iran. Two different techniques were used for fungal isolation including enrichment on atmospheres of phenolic hydrocarbons and crude oil as substrate. Phylogenetic analysis of the internal transcribed spacer of ribosomal DNA was used for taxonomic identification with additional information from the ß-tubulin gene for selected taxa. Overall, 183 strains from 14 genera and five orders were obtained: Pleosporales (Alternaria, Curvularia, Stemphylium, Ulocladium), Chaetothyriales (Exophiala), Eurotiales (Aspergillus), Hypocreales (Acremonium, Emericellopsis, Sarocladium, Stachybotrys, Fusarium, Trichoderma, Beauveria), and Capnodiales (Cladosporium). The most frequently isolated strains belonged to the genera Alternaria, Exophiala and Aspergillus. The crude oil substrate was the most successful isolation method, and among the four hydrocarbon enrichments, toluene substrate yielded the highest number of strains. Enrichment on xylene and benzene also yielded herpotrichiellaceous and other filamentous fungi.

Characterization of basidiomycetous yeasts in hypersaline soils of the Urmia Lake National Park, Iran.

Urmia Lake, located in northwest Iran, is an oligotrophic and extremely hypersaline habitat that supports diverse forms of life. Owing to its unique biodiversity and special environmental conditions, Urmia Lake National Park has been designated as one of the biosphere reserves by UNESCO. This study was aimed to characterize basidiomycetous yeasts in hypersaline soils surrounding the Urmia Lake National Park using a polyphasic combination of molecular and physiological data. Soil samples were collected from eight sites in Lake Basin and six islands insides the lake. Yeast strains were identified by sequencing the D1/D2 domains of the 26S rRNA gene. When D1/D2 domain sequencing did not resolve the identity of the species, strain identification was obtained by ITS 1 & 2 sequencing. Twenty-one species belonging to the genera Cystobasidium, Holtermanniella, Naganishia, Rhodotorula, Saitozyma, Solicoccozyma, Tausonia, Vanrija, and Vishniacozyma were identified. Solicoccozyma aeria represented the dominant species. The ability of isolates to grow at 10 and 15 % of NaCl was checked; about two-thirds of the strains grew at 10 %, while about 13 % of the isolates grew in medium with 15 % NaCl. this study is the first study on the culturable yeast diversity in hypersaline soils surrounding an Asian lake.

Pythium kandovanense sp. nov., a fungus-like eukaryotic micro-organism (Stramenopila, Pythiales) isolated from snow-covered ryegrass leaves.

Pythiumkandovanense sp. nov. (ex-type culture CCTU 1813T = OPU 1626T = CBS 139567T) is a novel oomycete species isolated from Lolium perenne with snow rot symptoms in a natural grassland in East-Azarbaijan province, Iran. Phylogenetic analyses based on sequence data from internal transcribed spacer (ITS)-rDNA, coxI and coxII mitochondrial genes clustered our isolates in Pythium group E as a unique, well supported clade. Pythium kandovanense sp. nov. is phylogenetically and morphologically distinct from the other closely related species in this clade, namely Pythium rostratifingens and Pythium rostratum. Pythium kandovanense sp. nov. can be distinguished from these two species by its cylindrical sporangia and lower temperatures for optimum and maximum growth rate. The development of zoospores released through a shorter discharge tube is an additional morphological feature which can be used to differentiate Pythium kandovanense sp. nov. from Pythium rostratifingens. Laboratory inoculation tests demonstrated the pathogenicity of Pythium kandovanense sp. nov. to L. perenne under wet cold (0-3 °C) conditions.