Endophytic species of Nigrospora from grasses and shrubs of Shadegan International Wetland, with new species and records from Iran.

The "Shadegan International Wetland" (SIW) is one of the wetlands internationally recognized in the Ramsar convention. The vegetation of this wetland ecosystem consists of mostly grasses and shrubs that host a large number of fungi including endophytes. In this study, Nigrospora isolates were obtained from healthy plants of this wetland and its surrounding salt marshes and identified based on morphological features and multilocus phylogenetic analyses based on three DNA loci, namely the internal transcribed spacer regions 1 and 2 including the intervening 5.8S nuclear ribosomal DNA (ITS), ß-tubulin (tub2), and elongation factor 1-α (tef1-α). Accordingly, the following Nigrospora species were identified: N. lacticolonia, N. oryzae, N. osmanthi, N. pernambucoensis and a novel taxon N. shadeganensis sp. nov., which is described and illustrated. To the best of our knowledge, 10 new hosts for Nigrospora species are here reported, namely Aeluropus lagopoides, Allenrolfea occidentalis, Anthoxanthum monticola, Arthrocnemum macrostachyum, Cressa cretica, Halocnemum strobilaceum, Seidlitzia rosmarinus, Suaeda vermiculata, Tamarix passerinoides, and Typha latifolia. Moreover, the species N. lacticolonia and N. pernambucoensis are new records for the mycobiota of Iran.

Nitrogen sources alter ligninase and cellulase activities of thermophilic fungi isolated from compost and vermicompost.

Fungi harboring lignocellulolytic activity accelerate the composting process of agricultural wastes; however, using thermophilic fungal isolates for this process has been paid little attention. Moreover, exogenous nitrogen sources may differently affect fungal lignocellulolytic activity. A total of 250 thermophilic fungi were isolated from local compost and vermicompost samples. First, the isolates were qualitative assayed for ligninase and cellulase activities using Congo red (CR) and carboxymethyl cellulose (CMC) as substrates, respectively. Then, twenty superior isolates harboring higher ligninase and cellulase activities were selected and quantitatively assayed for both enzymes in basic mineral (BM) liquid medium supplemented with the relevant substrates and nitrogen sources including (NH4)2SO4 (AS), NH4NO3 (AN), urea (U), AS + U (1:1), or AN + U (1:1) with final nitrogen concentration of 0.3 g/L. The highest ligninase activities of 99.94, 89.82, 95.42, 96.25, and 98.34% of CR decolorization were recorded in isolates VC85, VC94, VC85, C145, and VC85 in the presence of AS, U, AS + U, AN, and AN + U, respectively. Mean ligninase activity of 63.75% in superior isolates was achieved in the presence of AS and ranked the highest among other N compounds. The isolates C200 and C184 exhibited the highest cellulolytic activity in the presence of AS and AN + U by 8.8 and 6.5 U/ml, respectively. Mean cellulase activity of 3.90 U/mL was achieved in AN + U and ranked the highest among other N compounds. Molecular identification of twenty superior isolates confirmed that all of them are belonging to Aspergillus fumigatus group. Focusing on the highest ligninase activity of the isolate VC85 in the presence of AS, the combination can be recommended as a potential bio-accelerator for compost production.

Development of a Novel Diagnostic Tool for Cercospora Species Based on BOX-PCR System.

The genus Cercospora contains many devastating plant pathogens linked to leaf spot diseases afflicting various plants. Identification of Cercospora species based on morphology or host plant association has proven unreliable due to simple morphology and wide host range in many cases; hence, multi-gene DNA sequence data are essential for accurate species identification. Considering the complexity and cost involved in application of multi-locus DNA phylogenetic approaches for species delineation in Cercospora; rapid and cost-effective methods are urgently needed for species recognition. In this study, we applied rep-PCR (repetitive-sequence based polymerase chain reaction) fingerprinting methods referred to as BOX-PCR to differentiate species of Cercospora. Cluster analysis of the banding patterns of 52 Cercospora strains indicated the ability of BOX-PCR technique using BOXA1R primer to generate species-specific DNA fingerprints from all the tested strains. Since this technique was able to discriminate between all the 20 examined Cercospora species during this study, which corresponded well to the species identified based on multi-gene DNA sequence data, our findings revealed the efficiency of BOX-PCR system as a suitable complementary method for molecular identification of the genus Cercospora at species level.

Five Tetramic Acid Derivatives Isolated from the Iranian Fungus Colpoma quercinum CCTU A372.

Submerged mycelial cultures of the ascomycete Colpoma quercinum CCTU A372 were found to produce five previously undescribed tetramic acids, for which we propose the trivial names colposetins A-C (1-3) and colpomenoic acids A and B (4 and 5), along with the known compounds penicillide (6) and monodictyphenone (7). The planar structures of 1-5 were determined by high-resolution electrospray ionization mass spectrometry (HR-ESIMS) and extensive 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Their absolute configurations were determined by the combination of electronic circular dischroism (ECD) analysis, J-based configurational analysis, and a rotating-frame Overhauser effect spectroscopy (ROESY) experiment. Colposetin B displayed weak antimicrobial activity against Bacillus subtilis and Mucor hiemalis (MIC 67 µg/mL).

Fungicolous Fusarium Species: Ecology, Diversity, Isolation, and Identification.

Fusarium species can have different lifestyles, including those of endophytes, parasites, or pathogens of plants, as well as pathogens or mutualists of animals. Fungicolous Fusarium species have been also reported in some studies, however, the information on the Fusarium interactions with other fungi is still unclear and the diversity of fungicolous Fusarium species is poorly known. In this study, we provide a survey of fungicolous Fusarium species and their hosts, and instructions for their isolation and identification. According to the survey, 80 fungicolous Fusarium isolates were reported associated with 36 host species and 32 fungal genera. The fungicolous isolates belong to 24 species grouped in nine species complexes (SC)-Fusarium chlamydosporum SC, Fusarium fujikuroi SC, F. heterosporum SC, F. lateritium SC, F. oxysporum SC, F. incarnatum-equiseti SC, F. sambucinum SC, F. solani SC (=Neocosmospora genus), and F. tricinctum SC. Fusarium associations with other fungi were predominantly necrotrophic. The prevalent fungal hosts for fungicolous Fusarium isolates were members of the sub-kingdom Dikarya, mostly microfungi. Other hosts belong to the sub-kingdom Mucoromyceta of the kingdom Fungi and to the phylum Oomycota (fungal-like organisms) of kingdom Straminipila. With this review, we hope to highlight the fungicolous associations of Fusarium, and to expand the understanding of the ecology and diversity of these fungi.

New species of Septoria associated with leaf spot diseases in Iran.

Species of Septoria are commonly associated with leaf spot diseases of a broad range of plant hosts worldwide. During our investigation of fungi associated with leaf spot diseases in northern and northwestern Iran, several Septoria isolates were recovered from symptomatic leaves on different herbaceous and woody plants in the Asteraceae, Betulaceae, and Salicaceae families. These isolates were studied by applying a polyphasic approach including morphological and cultural data and a multigene phylogeny using a combined data set of partial sequences of the 28S nuc rRNA gene (large subunit [28S]), internal transcribed spacer regions and intervening 5.8S nuc rRNA gene (ITS) of the nuc rDNA operon, actin (actA), translation elongation factor 1-α (tef1), calmodulin (cmdA), ß-tubulin (tub2), and DNA-directed RNA polymerase II second largest subunit (rpb2). Four novel species are proposed, namely, Septoria eclipticola on Eclipta prostrata, Septoria firouraghina on Cirsium arvense, Septoria guilanensis on Populus deltoides, and Septoria taleshana on Alnus subcordata. All species are illustrated, and their morphology and phylogenetic relationships with other Septoria species are discussed.

Cytotoxic, anti-biofilm and antimicrobial polyketides from the plant associated fungus Chaetosphaeronema achilleae.

From extracts of the plant associated fungus Chaetosphaeronema achilleae collected in Iran, a previously unreported isoindolinone named chaetosisoindolinone (1) and a previously undescribed indanone named chaetosindanone (2) were isolated in addition to five known metabolites, 2-(2-acetyl-3,5-dihydroxyphenyl) acetic acid (3), vulculic acid (4), 2-(2-acetyl-3-hydroxy-5-methoxyphenyl)acetic acid (5), curvulin (6), and curvulol (7). Their structures were elucidated on the basis of extensive spectroscopic analysis and high-resolution mass spectrometry. The isolated compounds were tested for their antimicrobial, anti-biofilm, and nematicidal activities. Compound 2 exhibited cytotoxicity against the human breast adenocarcinoma MCF-7 cells with an IC50 value of 1.5 µg/mL. Furthermore, compounds 4 and 7 almost completely inhibited biofilm formation in Staphylococcus aureus at 256 µg/mL. Weak antimicrobial activities were also observed for some of the isolated compounds against Mucor hiemalis, Rhodoturula glutinis, Chromobacterium violaceum, and Staphylococcus aureus.

Weeds as Potential Inoculum Reservoir for Colletotrichum nymphaeae Causing Strawberry Anthracnose in Iran and Rep-PCR Fingerprinting as Useful Marker to Differentiate C. acutatum Complex on Strawberry.

Strawberry anthracnose caused by Colletotrichum spp. is considered one of the most serious and destructive disease of strawberry worldwide. Weeds, as possible hosts of the pathogen, could have a role as potential inoculum reservoir. To prove this hypothesis, symptomless weeds were collected in strawberry fields showing anthracnose symptoms in Iran. Ten isolates with Colletotrichum-like colonies were recovered from symptomless Amaranthus viridis L., Convolvulus arvensis L., Fumaria officinalis L., Lactuca serriola L., and Sonchus oleraceus L. plants. The isolates were identified as C. nymphaeae, based on a combination of morphological and sequence data of TUB and GADPH genes. This identification was further validated using Rep-PCR fingerprinting analysis, which produces species-specific DNA fingerprints and unveils inter and intra variation of the species examined in this study. Moreover, rep-PCR marker was used to reveal accurate taxonomic position of Colletorichum spp. causing strawberry anthracnose belonging to the C. acutatum complex, including C. acutatum sensu stricto, C. fiorinae, C. godetiae, C. nymphaeae, C. salicis, and C. simmondsii. The C. nymphaeae isolates originating from symptomless weeds confirmed their pathogenicity on detached strawberry, proving that weeds in strawberry field may have a role as reservoir of inoculum. However, further studies are necessary to quantify their actual contribution to anthracnose epidemics in strawberry fields.

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.

Tydeus shabestariensis sp. nov. and description of the male of Neopronematus sepasgosariani (Acari: Tydeoidea), with a key to the Iranian species of Tydeus.

A new species of the family Tydeidae from Iran, Tydeus shabestariensis sp. nov., is described from adult males and females collected from soil in apple orchards. A key to species of Tydeus of Iran, based on females, is provided. The male of Neopronematus sepasgosariani (Tydeoidea: Iolinidae) is also described and illustrated for the first time.

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.

Rapid identification of Fusarium graminearum species complex using Rolling Circle Amplification (RCA).

Rolling Circle Amplification (RCA) of DNA is a sensitive and cost effective method for the rapid identification of pathogenic fungi without the need for sequencing. Amplification products can be visualized on 1% agarose gel to verify the specificity of probe-template binding or directly by adding fluorescent dyes. Fusarium Head Blight (FHB) is currently the world's largest threat to the production of cereal crops with the production of a range of mycotoxins as an additional risk. We designed sets of RCA padlock probes based on polymorphisms in the elongation factor 1-α (EF-1α) gene to detect the dominant FHB species, comprising lineages of the Fusarium graminearum species complex (FGSC). The method also enabled the identification of species of the Fusarium oxysporum (FOSC), the Fusarium incarnatum-equiseti (FIESC), and the Fusarium tricinctum (FTSC) species complexes, and used strains from the CBS culture collection as reference. Subsequently probes were applied to characterize isolates from wheat and wild grasses, and inoculated wheat kernels. The RCA assays successfully amplified DNA of the target fungi, both in environmental samples and in the contaminated wheat samples, while no cross reactivity was observed with uncontaminated wheat or related Fusarium species. As RCA does not require expensive instrumentation, the technique has a good potential for local and point of care screening for toxigenic Fusarium species in cereals.

Evolutionary dynamics of mating-type loci of Mycosphaerella spp. occurring on banana.

The devastating Sigatoka disease complex of banana is primarily caused by three closely related heterothallic fungi belonging to the genus Mycosphaerella: M. fijiensis, M. musicola, and M. eumusae. Previous phylogenetic work showing common ancestry led us to analyze the mating-type loci of these Mycosphaerella species occurring on banana. We reasoned that this might provide better insight into the evolutionary history of these species. PCR and chromosome-walking approaches were used to clone the mating-type loci of M. musicola and M. eumusae. Sequences were compared to the published mating-type loci of M. fijiensis and other Mycosphaerella spp., and a novel organization of the MAT loci was found. The mating-type loci of the examined Mycosphaerella species are expanded, containing two additional Mycosphaerella-specific genes in a unique genomic organization. The proteins encoded by these novel genes show a higher interspecies than intraspecies homology. Moreover, M. fijiensis, M. musicola, and M. eumusae contain two additional mating-type-like loci, containing parts of both MAT1-1-1 and MAT1-2-1. The data indicate that M. fijiensis, M. musicola, and M. eumusae share an ancestor in which a fusion event occurred between MAT1-1-1 and MAT1-2-1 sequences and in which additional genes became incorporated into the idiomorph. The new genes incorporated have since then evolved independently in the MAT1-1 and MAT1-2 loci. Thus, these data are an example of the evolutionary dynamics of fungal MAT loci in general and show the great flexibility of the MAT loci of Mycosphaerella species in particular.

The Ascomycota tree of life: a phylum-wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits.

We present a 6-gene, 420-species maximum-likelihood phylogeny of Ascomycota, the largest phylum of Fungi. This analysis is the most taxonomically complete to date with species sampled from all 15 currently circumscribed classes. A number of superclass-level nodes that have previously evaded resolution and were unnamed in classifications of the Fungi are resolved for the first time. Based on the 6-gene phylogeny we conducted a phylogenetic informativeness analysis of all 6 genes and a series of ancestral character state reconstructions that focused on morphology of sporocarps, ascus dehiscence, and evolution of nutritional modes and ecologies. A gene-by-gene assessment of phylogenetic informativeness yielded higher levels of informativeness for protein genes (RPB1, RPB2, and TEF1) as compared with the ribosomal genes, which have been the standard bearer in fungal systematics. Our reconstruction of sporocarp characters is consistent with 2 origins for multicellular sexual reproductive structures in Ascomycota, once in the common ancestor of Pezizomycotina and once in the common ancestor of Neolectomycetes. This first report of dual origins of ascomycete sporocarps highlights the complicated nature of assessing homology of morphological traits across Fungi. Furthermore, ancestral reconstruction supports an open sporocarp with an exposed hymenium (apothecium) as the primitive morphology for Pezizomycotina with multiple derivations of the partially (perithecia) or completely enclosed (cleistothecia) sporocarps. Ascus dehiscence is most informative at the class level within Pezizomycotina with most superclass nodes reconstructed equivocally. Character-state reconstructions support a terrestrial, saprobic ecology as ancestral. In contrast to previous studies, these analyses support multiple origins of lichenization events with the loss of lichenization as less frequent and limited to terminal, closely related species.

Molecular diagnostics for the sigatoka disease complex of banana.

ABSTRACT The Sigatoka disease complex of banana involves three related ascomycetous fungi, Mycosphaerella fijiensis, M. musicola, and M. eumusae. The exact distribution of these three species and their disease epidemiology remain unclear, because their symptoms and life cycles are rather similar. Disease diagnosis in the Mycosphaerella complex of banana is based on the presence of host symptoms and fungal fruiting structures, which hamper preventive management strategies. In the present study, we have developed rapid and robust species-specific molecular-based diagnostic tools for detection and quantification of M. fijiensis, M. musicola, and M. eumusae. Conventional species-specific polymerase chain reaction (PCR) primers were developed based on the actin gene that detected DNA at as little as 100, 1, and 10 pg/mul from M. fijiensis, M. musicola, and M. eumusae, respectively. Furthermore, TaqMan real-time quantitative PCR assays were developed based on the beta-tubulin gene and detected quantities of DNA as low as 1 pg/mul for each Mycosphaerella sp. from pure cultures and DNA at 1.6 pg/mul per milligram of dry leaf tissue for M. fijiensis that was validated using naturally infected banana leaves.