Characterization of fungi causing lesion blight on Papaver dubium in Iran.

Papaver dubium (common name, blindeyes, Papaveraceae) is widespread throughout Europe and America and is an important weed in western Iran. Since 2009, a blight disease has occurred in several areas in Hamedan Province, Iran, causing significant damage to plants of P. dubium. Small, yellow-brown lesions appeared on lower leaves and eventually expanded to the whole plant, resulting in necrosis and complete wilting. This study was conducted to identify the causal agent(s) of the blight disease on blindeyes plants. On the basis of cultural and microscopic characters, as well as representative DNA sequence data of the 5.8S rRNA (ITS), partial LSU rRNA partial ß-tubulin (TUB2), and partial G3PD genes, 82 isolates were identified as follows: Ascochyta pisi M. A. Libert. (42), Neodidymelliopsis longicolla L.W. Hou, Crous & L. Cai. (28), and Allophoma zantedeschiae (Dippen.) Q. Chen & L. Cai (12). Pathogenicity tests in the greenhouse showed that all the isolates of A. pisi and Neod. longicolla caused typical spots on inoculated blindeyes plants, and the fungi were successfully re-isolated from the symptomatic tissues. Based on the high isolation frequency for A. pisi, and the severity of symptoms induced by this species in pathogenicity tests, this fungus was indicated as the major pathogen causing the blight disease on blindeyes. It has potential as a biocontrol agent against P. dubium. To the best of our knowledge, Al. zantedeschiae and Neod. longicolla observed in this study are new taxa for the mycobiota of Iran.

Peptaibol profiles of Iranian Trichoderma isolates.

Five Iranian Trichoderma isolates from species T. viride, T. viridescens, T. asperellum, T. longibrachiatum and T. citrinoviride - selected from the Fungal Collection of the Bu Ali Sina University, Hamedan, Iran - were investigated for their peptaibol production. All examined isolates showed remarkable antibacterial activities during the screening of their extracts for peptaibol content with a Micrococcus luteus test culture. HPLC-ESI-IT MS was used for identification and elucidation of the amino acid sequences of peptaibols. The detected peptaibol compounds contain 20 or 18 amino acid residues and belong to the trichobrachin and trichotoxin groups of peptaibols, respectively. T. longibrachiatum and T. citrinoviride produced trichobrachins, while trichotoxins could be detected in T. viride, T. viridescens and T. asperellum. Out of 37 sequences detetermined, 26 proved to be new, yet undescribed compounds, while others were identified as previously reported trichotoxins (trichotoxin A-50s and T5D2) and trichobrachins (longibrachins AI, AII, AIII, BII and BIII). Compounds within the two groups of detected peptaibols differed from each other only by a single or just a few amino acid changes.

Changes in the expression of COI1, TIR1, and ERF1 genes and respective MiRNAs in Fusarium basal Rot-Stressed onion.

Fusarium oxysporum f.sp. cepae (FOC), as basal rot fungus, is the most detrimental pathogen causing a serious threat to onion productivity in the world. In this study, we first determined FOC tolerance in seven Iranian onion cultivars, two known international onions (Texas Early Grano and Sweet Yellow Spanish), and an Allium species related to the onion (Allium asarence) based on the infection severity. Then, a transcriptional screen was performed by comparing the transcript levels of some pathogen-responsive genes (ERF1, COI1, and TIR1) and their predicted miRNAs in the sensitive (Ghermeze Azarshahr Cv.) and the resistant (A. asarence) onions to determine key genes and their miRNAs involved in the defense responses of onions to FOC. From our results, a difference was found in the COI1 and ERF1 expression 48 h after inoculation with FOC as compared to the respective 24 and 72 h. It can be explained by either special mechanisms involved in raising energy consumption efficiency or the interactive effects of other genes in the jasmonic acid (JA) and ethylene (ET) signaling pathways. Moreover, expression analysis of the pathogen-responsive genes and their targeting miRNAs identified the miR-5629, which targets the COI1 gene as a likely key factor in conferring resistance in the FOC-resistant onion, i.e., A. asarence. However, exploring the function of the miRNA/target pair is highly recommended to deeply understand the effect of the miRNA/target pair-associated pathway in the control of A. asarense-FOC interaction.

Pathogenic potential and phytotoxic effects of Coniolariella gamsii Iran 2506C on Iranian knapweed (Centaurea depressa).

Application of fungal phytotoxins is one of the possible solutions to reduce the use of chemical products in agricultural fields. Phytotoxic compounds isolated from phytopathogenic fungi provide a promising source of environmentally friendly herbicides. This paper focuses on the phytotoxic fungus Coniolariella gamsii Iran 2506C as a fungal pathogen against Iranian knapweed (Centaurea depressa) in the western Iran and investigate its phytotoxic constituents. The fungal pathogen was identified on the basis of morphological characteristics and confirmed by sequencing of the internal transcribed spacer (ITS) region and partial LSU rDNA gene. Pathogenicity tests were conducted on the weed seedlings and C. gamsii Iran 2506C isolate with high disease severity was selected for phytotoxin studies. Phytotoxic activity of the isolate was checked by screening the production of phytotoxins, which interestingly inhibited seed germination and seedling growth of Iranian knapweed as compared to wheat in the bioassays. The active metabolites were extracted from cell-free culture filtrate (CFCF) by ethyl acetate and separated by thin layer chromatography (TLC). The results indicated that two out of four spots had phytotoxicity with Rf values of 0.43 and 0.82 on the weed, whereas wheat was not sensitive in the bioassays. Using Gas Chromatography-Mass spectrometry (GC-MS), 3-Carene and Oleic acid were identified as the main constituents and quantified by a gas chromatographic method with 12.7 ± 0.03 and 2.9 ± 0.01 µg ml-1 respectively. This is the first finding presented on the phytotoxic effects of the active metabolites of C. gamsii Iran 2506C and highlights its herbicidal potential which can be used as a biocontrol agent of C. depressa.

Characteristics and health effects of potentially pathogenic bacterial aerosols from a municipal solid waste landfill site in Hamadan, Iran.

The aim of this study was to evaluate the potential pathogenic bacterial aerosols produced from the municipal solid waste landfill site and its health risk assessment in the Hamadan city at west of Iran. In this study, air samples were collected every month during spring and summer at six locations including the active zone, leachate collection pond, infectious waste landfill, upwind, closure landfill, and downwind using the Andersen impactor. Spatial and seasonal variations of the potential pathogenic bacterial aerosols were detected. Also, Health risk associated were estimated based on the average daily dose rates (ADD) of exposure by inhalation. The mean concentration of potentially pathogenic bacterial aerosols were 468.7 ± 140 CFU m- 3 1108.5 ± 136.9 CFU m- 3 detected in the active zone in spring and summer, respectively. Also, there was a significant relationship between meteorological parameters and bacterial concentration (p < 0.05). The predominant potential pathogenic bacterial identified in the spring were Proteus mirabilis, Streptococcus sp., and Pseudomonas sp., while in summer were Pseudomonas sp., Staphylococcus aureus, and Escherichia coli. The hazard quotient (HQ) in both seasons were less of 1. Bacteria were spread throughout the landfill space, but their maximum density was observed around the active zone and leachate collection pond. This study highlights the importance of exposure to potential pathogenic bacterial aerosols in the summer and its adverse effects, especially in the MSW landfill site active zone. Finally, controlled exposure can reduce the health hazard caused by the potential pathogenic bacterial aerosols.

Phytoremediation of petroleum-polluted soils: application of Polygonum aviculare and its root-associated (penetrated) fungal strains for bioremediation of petroleum-polluted soils.

Petroleum-polluted soils are a common disaster in many countries. Bioremediation of oil contamination in soils is based on the stimulation of petroleum-hydrocarbon-degrading fungal and microbial communities. A field study was conducted in a petroleum-contaminated site to find petroleum-resistant plants and their root-associated fungal strains for use in bioremediation of petroleum-polluted soils. Results and observations showed that the amounts of petroleum pollution in nonvegetated soils were several times higher than in vegetated soils. Plants collected from petroleum-polluted areas were identified using morphological characters. Results indicated that seven plant species were growing on the contaminated sites: Alhaji cameleron L. (Fabaceae), Amaranthus retroflexus L. var. retroflexus (Amaranthaceae), Convolvulus arvensis L. (Convolvulaceae), Chrozophora hierosolymitana Spreg. (Euphorbiaceae), Noea mucronata L. (Boraginaceae), Poa sp. (Poaceae), and Polygonum aviculare L. (Polygonaceae). The root-associated fungi of each plant were determined and results showed the presence of 11 species that associated with and also penetrated the roots of plants growing in the polluted areas. Altenaria sp. was common to all of the plants and the others had species-specific distribution within the plants. The largest numbers of fungal species (six) were determined for P. aviculare and Poa sp. in polluted areas. However, the variation of fungal strains in the plants collected from petroleum-polluted areas was greater than for nonpolluted ones. Culture of fungi in oil-contaminated media showed that all the studied fungi were resistant to low petroleum pollution (1% v/v) and a few species, especially Fusarium species, showed resistance to higher petroleum pollution (10% v/v) and may be suitable for bioremediation in highly polluted areas. Bioremediation tests with P. aviculare, with and without fungal strains, showed that application of both the plant and its root-associated fungal strains was more effective than of the plant and fungi separately, and Fusarium species were the most effective. Results indicated that fungal strains had the main role in bioremediation of petroleum-polluted soils, but plant roots enhanced the process.

Changes in Peptaibol Production of Trichoderma Species during In Vitro Antagonistic Interactions with Fungal Plant Pathogens.

Trichoderma species are widely used as biofungicides for the control of fungal plant pathogens. Several studies have been performed to identify the main genes and compounds involved in Trichoderma-plant-microbial pathogen cross-talks. However, there is not much information about the exact mechanism of this profitable interaction. Peptaibols secreted mainly by Trichoderma species are linear, 5-20 amino acid residue long, non-ribosomally synthesized peptides rich in α-amino isobutyric acid, which seem to be effective in Trichoderma-plant pathogenic fungus interactions. In the present study, reversed phase (RP) high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) mass spectrometry (MS) was used to detect peptaibol profiles of Trichoderma strains during interactions with fungal plant pathogens. MS investigations of the crude extracts deriving from in vitro confrontations of Trichodermaasperellum and T.longibrachiatum with different plant pathogenic fungi (Fusariummoniliforme, F.culmorum, F.graminearum, F.oxysporum species complex, Alternariasolani and Rhizoctoniasolani) were performed to get a better insight into the role of these non-ribosomal antimicrobial peptides. The results revealed an increase in the total amount of peptaibols produced during the interactions, as well as some differences in the peptaibol profiles between the confrontational and control tests. Detection of the expression level of the peptaibol synthetase tex1 by qRT-PCR showed a significant increase in T.asperellum/R.solani interaction in comparison to the control. In conclusion, the interaction with plant pathogens highly influenced the peptaibol production of the examined Trichoderma strains.

Antiproliferative and Antimicrobial Activities of Secondary Metabolites and Phylogenetic Study of Endophytic Trichoderma Species From Vinca Plants.

Endophytic fungi have been recognized as a potential source of bioactive secondary metabolites. The endophytic Trichoderma species were isolated from Vinca plants (Vinca major, Vinca herbacea, and Vinca minor), found in Iran and screened for antimicrobial and anti-proliferative activity. Based on morphological and phylogenetic analyses, four fungal species were identified: T. asperellum, T. brevicompactum, T. koningiopsis, and T. longibrachiatum. In addition, endophytic fungi bioactivity of methanol and ethyl acetate extracts (7.8-250 µgml-1) were assessed against a panel of pathogenic fungi and bacteria and IC80 was calculated. Data showed that both methanol and ethyl acetate extracts from all endophytic isolates had significant cytotoxic effects against the model target fungus Pyricularia oryzae. Further research indicated that they had significant antimicrobial bioactivity against the human pathogenic bacteria Staphylococcus aureus and Escherichia coli, and plant pathogenic bacteria Ralstonia solanacearum and Clavibacter michiganensis as well. According to the bioactivity results, crude ethyl acetate extract of T. koningiopsis VM115 isolate was determined for TLC and GC-MS analysis. An antifungal compound was isolated from ethyl acetate extract of T. koningiopsis VM115 based on bioassay guided fractionation. The 1H-NMR and 13C-NMR spectroscopic data showed that the compound was trichodermin, which exhibited strong fungicidal effects against P. oryzae, Aspergillus fumigatus, and Botrytis cinera with MICs of 31.25 µg ml-1 through in vitro antifungal tests. GC-MS analysis identified six classes of volatile compound produced by T. koningiopsis VM115 (alcohols, esters, pyrones (lactones), acids, furanes and lipids). 6-n-pentyl-6H-pyran-2-one (6PP) was identified as one of the most abundant metabolites in this research. These results indicate that the fungal endophytes from Vinca plants had antibacterial and cytotoxic activities; evidence that endophytes are a good source of biological activity and compounds. This work is the first report of Trichodermin production by T. koningiopsis species.

Geographic distribution of phylogenetic species of the Fusarium graminearum species complex and their 8-ketotrichothecene chemotypes on wheat spikes in Iran.

Isolates of the Fusarium graminearum species complex (FGSC, n = 446) were collected from wheat spikes from northern and western regions of Iran with a history of Fusarium head blight (FHB) occurrences. The trichothecene mycotoxin genotypes/chemotypes, the associated phylogenetic species, and geographical distribution of these isolates were analyzed. Two phylogenetic species, Fusarium asiaticum and F. graminearum, were identified and were found to belong to sequence characterized amplified region (SCAR) groups V and I. Isolates from F. asiaticum species lineage 6 were within SCAR group V, whereas F. graminearum species lineage 7 were of SCAR group I. Of the 446 isolates assayed, 274 were F. asiaticum species predominantly of the nivalenol (NIV) genotype, while other isolates were either deoxynivalenol (DON) plus 3-acetyldeoxynivalenol (3-AcDON) or DON plus 15-acetyldeoxynivalenol (15-AcDON) genotype. Based on Tri7 gene sequences, a new subpopulation of 15-AcDON producers was observed among F. asiaticum strains in which 11-bp repeats were absent in the Tri7 sequences. The trichothecene chemotype was confirmed and quantified by high-performance liquid chromatography (HPLC) in 46 FGSC isolates. Isolates produced NIV (33.4-108.2 µg/g) and DON (64.7-473.6 µg/g) plus either 3-AcDON (51.4-142.4 µg/g) or 15-AcDON (24.1-99.3 µg/g). Among FGSC isolates, F. asiaticum produced the highest levels of trichothecenes. Using BIOCLIM based on the climate data of 20-year during 1994-2014, modelling geographical distribution of FGSC showed that F. asiaticum was restricted to warmer and humid areas with a median value of mean annual temperature of about 17.5 °C and annual rainfall of 658 mm, respectively (P < 0.05). In contrast, F. graminearum (only 15-AcDON producers) was restricted to cooler and drier areas, with a median value of the mean annual temperature of 14.4 °C and an annual rainfall of 384 mm, respectively (P < 0.05). Based on climate parameters at anthesis, the recorded distribution of F. graminearum and F. asiaticum was similar to that based on BIOCLIM parameters. Therefore, geographic differences on the wheat-growing areas in Iran have had a significant effect on distribution of FGSC and their trichothecene chemotypes.

Nucleotide polymorphisms and protein structure changes in the Fg16 gene of Fusarium graminearum sensu stricto.

Fusarium graminearum is one of the most important causes of wheat scab in different parts of the world. This fungus is able to produce widespread trichothecene mycotoxins such as nivalenol (NIV) and deoxynivalenol (DON) which are harmful for both human and animals. The Fg16 target is located in chromosome 1 of the F. graminearum genome coding for a hypothetical protein whose function is not yet known. The Fg16 gene is involved in lipid biosynthesis and leads to sexual development during colonization in wheat stalks. This gene is used to detect F. graminearum and determine the lineage of F. graminearum complex species. In the present study, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and DNA sequencing methods were employed in screening for genetic variation in 172 F. graminearum s.s. isolates. The PCR reaction forced the amplification of 410-bp fragments of Fg16. Two single nucleotide polymorphisms (T82C and A352T) and one amino acid exchange (C65S) with three patterns (TA/TA, CT/CT and TA/CT genotypes) were found in the Fg16 gene fragment. Two haplotypes, 1A and 1B, were identified within F. graminearum s.s. populations in northern and western regions of Iran. Two different secondary structures of protein were predicted for CT/CT and TA/CT genotypes of Fg16 gene. The average diversity levels detected were relatively high (He: 0.3238; Heu: 0.334; Ho: 0.2894; mean PIC: 0.514; mean Shannon's information index: 0.4132; mean number of alleles per locus: 1.473). On the basis of the obtained results, it was revealed that the Fg16 gene had a high degree of polymorphism that can be considered for future control programming strategies and thus the associations between the SSCP patterns with different traits of F. graminearum such as wheat colonization, perithecium formation on stalk tissues and lineage discrimination should be investigated.

Trichothecene production by Trichoderma brevicompactum.

Trichoderma brevicompactum, T. viride, T. harzianum, T. atroviride, T. longibrachiatum, T. erinaceum, T. citrinoviride, and Hypocrea lutea were screened for production of trichothecenes after growth on one or several solid and liquid media. Trichothecenes were detected by liquid chromatography combined with online UV/vis spectroscopy and electrospray high-resolution mass spectrometry. T. brevicompactum produced trichodermin and/or harzianum A on all media investigated, with liquid media yielding the largest amounts. Detection of octa-2Z,4E,6E-trienedioic acid in the harzianum-A-producing strains indicated that harzianum A was synthesized directly by esterification of trichodermol with octa-2Z,4E,6E-trienedioic acid. Both the T. viride strain from which trichodermin was originally isolated and the T. harzianum strain from which harzianum A was originally isolated were shown to belong to T. brevicompactum based on four independent criteria: metabolite profiles, micromorphology, macromorphology on yeast extract sucrose agar and potato dextrose agar, and DNA sequences of the ITS1/ITS2 regions of the nuclear ribosomal DNA.

Trichoderma brevicompactum sp. nov.

Trichoderma brevicompactum, a new species, was isolated from soil or tree bark in North, Central and South America, including the Caribbean Islands, and southwestern and southeastern Asia. Morphological and physiological characters, the internal transcribed spacer regions of the rDNA cluster (ITS1-5.8SrDNA-ITS2) and partial sequences of translation elongation factor 1-alpha (tef1) are described. Trichoderma brevicompactum is characterized by a pachybasium-type morphology, morphologically resembling other small-spored species referable to Trichoderma section Pachybasium but with essentially subglobose conidia. It is most closely related phylogenetically to Hypocrea lutea, from which it differs in morphological and physiological characters.

Genetically closely related but phenotypically divergent Trichoderma species cause green mold disease in oyster mushroom farms worldwide.

The worldwide commercial production of the oyster mushroom Pleurotus ostreatus is currently threatened by massive attacks of green mold disease. Using an integrated approach to species recognition comprising analyses of morphological and physiological characters and application of the genealogical concordance of multiple phylogenetic markers (internal transcribed spacer 1 [ITS1] and ITS2 sequences; partial sequences of tef1 and chi18-5), we determined that the causal agents of this disease were two genetically closely related, but phenotypically strongly different, species of Trichoderma, which have been recently described as Trichoderma pleurotum and Trichoderma pleuroticola. They belong to the Harzianum clade of Hypocrea/Trichoderma which also includes Trichoderma aggressivum, the causative agent of green mold disease of Agaricus. Both species have been found on cultivated Pleurotus and its substratum in Europe, Iran, and South Korea, but T. pleuroticola has also been isolated from soil and wood in Canada, the United States, Europe, Iran, and New Zealand. T. pleuroticola displays pachybasium-like morphological characteristics typical of its neighbors in the Harzianum clade, whereas T. pleurotum is characterized by a gliocladium-like conidiophore morphology which is uncharacteristic of the Harzianum clade. Phenotype MicroArrays revealed the generally impaired growth of T. pleurotum on numerous carbon sources readily assimilated by T. pleuroticola and T. aggressivum. In contrast, the Phenotype MicroArray profile of T. pleuroticola is very similar to that of T. aggressivum, which is suggestive of a close genetic relationship. In vitro confrontation reactions with Agaricus bisporus revealed that the antagonistic potential of the two new species against this mushroom is perhaps equal to T. aggressivum. The P. ostreatus confrontation assays showed that T. pleuroticola has the highest affinity to overgrow mushroom mycelium among the green mold species. We conclude that the evolutionary pathway of T. pleuroticola could be in parallel to other saprotrophic and mycoparasitic species from the Harzianum clade and that this species poses the highest infection risk for mushroom farms, whereas T. pleurotum could be specialized for an ecological niche connected to components of Pleurotus substrata in cultivation. A DNA BarCode for identification of these species based on ITS1 and ITS2 sequences has been provided and integrated in the main database for Hypocrea/Trichoderma (www.ISTH.info).