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Staphylococcus aureus is an important infectious factor in the food industry and hospital infections. Many methods are used for detecting bacteria but they are mostly time-consuming, poorly sensiti...

Proteases constitute a significant part of cell wall-degrading enzymes (CWDEs) produced by fungal biocontrol agents and particularly crucial in mycoparasitism of fungal phytopathogens. Plate-based screening methods are routinely used for screening protease-producing microorganisms including fungi. Skim milk agar (SMA) is one of the most popular media for the detection of protease producing bacteria. However, SMA is not efficient to test fast growing fungi, because it does not give an estimation of the actual amount of secreted protease produced by fungal inocula. In the current study, the efficacy of two modified plate-screening methods, including split-SMA (SSMA) and minimal medium supplemented with skim milk (MSMW) was assessed for detection of protease production by three representative fungal strains including Trichoderma longibrachiatum strain N, Beauveria bassiana strain B and Purpureocillium lilacinum strain PL. Protease production was revealed on the three tested media by the three strains. However, the halo diameter of the fungal strains (a proxy for protease production) was the smallest on SMA. Furthermore, protease production could not be detected for T. longibrachiatum strain N on SMA due to its fast growth; while it showed the highest protease activity on both modified media compared with the other strains. According to the result of this study, the SSMA medium is an easy and more accurate method compared with the two other different methods as it displays the actual amount of protease produced by fungal strains and therefore this method is recommended for quantitative and qualitative detection of protease production by slow and fast growing fungi.

Between March and September 2012, grapevines in vineyards of Meshginshahr county located in Ardabil province as well as Azarshahr, Ajab Shir, and Malekan counties located in East Azerbaijan province, north-western Iran represented general decline symptoms and in some cases black vascular discoloration. Annular or irregular discoloration, dark vascular streaking, and V-shaped unilateral canker or brown necrosis were observed in the pit and vascular tissues of the infected stems. These symptoms were accompanied with necrotic and wilted leaves. Nine fungal isolates having analogous morphological traits were recovered from symptomatic tissues on potato dextrose agar culture medium. Morphological and molecular characterizations confirmed the identity of all nine isolates as Kalmusia variispora (formerly known as Dendrothyrium variisporum). Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses were conducted for genetic diversity assessment of 13 K. variispora isolates from grapevine, one isolate from Rosa hybrida and one isolate from Pinus eldarica and the results indicated the existence of polymorphism among the isolates (77.27% and 81.25% for RAPD and ISSR, respectively). The clustering of RAPD and ISSR analyses revealed that there was no correlation between the isolates and their geographical as well as host origins. A pathogenicity assay confirmed K. variispora to be pathogenic on grapevine plants (cv. Keshmeshi) causing similar symptoms as those observed in naturally infected grapevines. Kalmusia variispora is associated with grapevine decline symptoms and grapevine trunk disease in Iran.

S. aureus is one of important causes of disease, food poisoning in humans and animals. The generally methods for detection of S. aureus is time consuming. Therefore, a new method is necessary for rapid, sensitive and specific diagnosis of S. aureus. In the present study, two probes and a Bio-barcode DNA were designed for detection of S. aureus (Protein A). Firstly, magnetic nanoparticle (MNPs) and gold nanoparticle (AuNPs) were synthesized at 80 °C and 100 °C, respectively. The AuNPs and the MNPs were functionalized with probe1, Bio-barcode DNA and probe2, respectively. Target DNA was added into the nanomaterial's system containing bio-barcode DNA-AuNPs-probe1 and probe2-MNPs to formed bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex. The bio-barcode DNA-AuNPs-probe1-target DNA-probe2-MNPs complex was separated with magnetic field. Finally, the bio-barcode DNA was released from surface of complex using DTT (0.8 M) and there was isolated of nanoparticles by magnetic field and centrifuge. The fluorescence intensity of bio-barcode DNA was measured in different concentrations of S. aureus (101 to 108 CFU mL−1) by fluorescence spectrophotometry. The results showed that standard curve was linearly from 102 to 107 CFU mL−1. Limit of detection of bio-barcode assay for both PBS and real samples was 86 CFU mL−1.

Submerged mycelial cultures of the ascomycete Colpoma&nbsp, 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).

I. Background: DNA extraction is an important step of any molecular experiment, in conjunction with the members of Actinomycetes, DNA couldn’t be quickly extracted by the usual methods of lysis. Due to the low efficiency of most of the conventional DNA extraction techniques, development of an effective techniques for DNA extraction of Actinobacteria in emergency case seems to be necessary. Since, most of the known DNA extraction techniques and commercial kits do not have sufficient efficiency in the extraction of DNA from a different group of Actinobacteria, the objective of this study was to improvement an efficient method from conventional methods for DNA extraction from Actinobacteria. II. Methods and Results: For this purpose, DNA extraction was performed by five methods (an improvedmethod, Invisorb Spin Plant Mini Kit, EZ-10 Spin Column, phenol-chloroform method and Kirby Bauer's method). To evaluate the quantity and quality of extracted genomic DNA, UV absorbance of all samples and efficiency of Polymerase Chain Reaction (PCR) were evaluated. Overall, the results showed the highest quantity of DNA (up to 4000 ng/µl) was obtained by employing introduced DNA extraction method, also yielding good quality. III. These results indicate the recently introduced improved method is more efficient for extraction of DNA from Actinobacteria for DDH (DNA–DNA hybridization) test and for those they require the high concentration of DNA.

In this study, strain Streptomyces sp. Act4Zk was isolated based on a method developed for the isolation of myxobacteria. Due to the low efficiency of the majority of conventional DNA extraction techniques, for molecular identification of the strain Streptomyces sp. Act4Zk, a new technique for DNA extraction of Actinobacteria was developed. In order to explore potential bioactivities of the strain, extracts of the fermented broth culture were prepared by an organic solvent (i.e. ethyl acetate) extraction method using. These ethyl acetate extracts were subjected to HPLC fractionation against standard micro-organisms, followed by LC/MS analysis. Based on morphological, physiological, biochemical and 16S rRNA gene sequence data, strain Streptomyces sp. Act4Zk is likely to be a new species of Streptomyces, close to Streptomyces genecies and Streptomyces roseolilacinus. Antimicrobial assay indicated high antifungal activity as well as antibacterial activity against Mycobacterium smegmatis and Gram-positive bacteria for the new strain. HPLC and LC/MS analyses of the extracts led to the identification of three different compounds and confirmed our hypothesis that the interesting species of the genus Streptomyces being a good producer of staurosporine and some derivatives.

Two new α-pyrone derivatives, udagawanones A (1) and B (2), along with the known compounds (Z)-4-hydroxy-3-(3-hydroxy-3-methylbut-1-en-1-yl)benzoic acid (3), isosclerone (4), cyclo-(L-Leu-L-Pro) (5), and cyclo-(L-Pro-L-Tyr) (6), were isolated from cultures of the endophyte Neurospora udagawae. Their structures were elucidated by extensive spectroscopic methods and single crystal X-ray diffraction. Both compounds feature oxidized functionalities at the C-2 position not previously observed in other tetrasubstituted α-pyrones from fungi. Compound 1 exhibited moderate antibacterial (vs. Staphylococcus aureus) and antifungal (vs. Rhodoturula glutinis) activities and cytotoxicity against KB3.1 cells.

Two new metabolites possessing the unusual 1-oxa-7-azaspiro[4.4]non-2- ene-4,6-dione core (2, 3) along with the recently described pseurotin A3 (1) were isolated from the pathogenic fungus Wilsonomyces carpophilus (previously named Stigmina carpophila). The producer organism was obtained from Prunus armeniaca collected in Iran and was identified by morphological and molecular phylogenetic methods. The structures of the isolated compounds were elucidated on the basis of extensive NMR spectroscopic analysis, high-resolution mass spectrometry and ECD analysis. The compounds were screened for their antimicrobial, cytotoxic, nematicidal and biofilm inhibition activities but, no significant effect was observed. To the best of our knowledge, this is the first report on the isolation of secondary metabolites produced by W. carpophilus.

Stromata of a Daldinia species were collected from half-burnt sugarcane stalks in South India. Based on a combination of morphological and chemotaxonomic evidence, the species was identified as the first recent record of Daldinia sacchari. While it was impossible to obtain cultures from the ascospores, the stromata were subjected to DNA extraction and DNA sequencing and secondary metabolites were analyzed by high-performance liquid chromatography coupled with diode array and mass spectrometric detection (HPLC-DAD/MS). The fruiting body extract was subjected to preparative HPLC for the isolation of secondary metabolites. Two new cytochalasins, for which we propose the trivial names saccalasins A and B, were elucidated besides two known cytochalasins and binaphthalene tetrol (BNT) by NMR spectroscopy and mass spectrometry. Whereas sequencing of housekeeping genes from the stromatal DNA unfortunately failed, an ITS DNA sequence was obtained from this species for the first time and compared to those of related Hypoxylaceae in a phylogenetic tree. The results indicate a close relationship of D. sacchari to the D. eschscholtzii complex, from which cytochalasins are also known as predominant stromatal metabolites. Phylogenetic analyses based on the ITS-rDNA barcode (which can only discriminate the Hypoxylaceae and other Sordariomycetes into species groups, rather than serve as a means of genus or species discrimination) confirmed that D. sacchari belongs to the D. eschscholtzii species complex. However, as with the majority of tropical Hypoxylaceae species, the availability of cultures that can be used to generate DNA sequence data that are more conclusive than ITS will be imperative to further narrow down the phylogenetic affinities of these fungi.

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.

Togninia minima is the main fungal species associated with grapevine leaf stripe disease worldwide. This species is mainly known from its asexual state in nature; nevertheless, a biallelic heterothallic mating strategy has been confirmed for this species based on in vitro crossing studies. There are no data available on the incidence of an active sexual cycle within the populations of this species in many grapevine-producing countries as well as Iran. The possibility of a clandestine sexual cycle within the Iranian isolates of T. minima was evaluated by analysing the distribution and frequency of the mating-type alleles on a microspatial and a macrogeographical scales. Towards this aim, a total of 90 T. minima isolates were recovered from grapevines with esca disease from the vineyards in north and north-western Iran. A multiplex PCR method previously designed by authors was applied for simultaneous identification and determination of the mating-type alleles in T. minima populations. The results on the screening of mating-type alleles using multiplex PCR method revealed the mating-type identity of 77 isolates as Mat1-2 and 23 isolates as Mat1-1. Our results showed that both Mat1-1 and Mat1-2 isolates are present in a single vineyard and even on single vines. The distribution of mating-type alleles in the sampled area skewed from the 1 : 1 ratio (77 : 23); however, co-occurrence of both mating types in a single vineyard and even on single vines is suggestive for the presence of an active sexual cycle for T. minima in north-western Iran.

Two new tetracyclic polyketides with a spirocenter, simplicilones A (1) and B (2) were isolated from the broth-culture of the endophytic fungus Simplicilliumsubtropicum (SPC3) in the course of our screening for new bioactive secondary metabolites. This endophytoic fungus is naturally harboured in the fresh bark of the Cameroonian medicinal plant Duguetia staudtii (Engl. and Diels) Chatrou. The planar structures of the simplicilones were elucidated by MS and 1D as well as 2D NMR spectroscopic techniques. The relative configuration was assigned by NOESY experiments in conjunction with coupling constants, subsequently, the absolute configurations were assigned by the modified Mosher&rsquo, s method. The compounds showed weak cytotoxic effects against the cell line KB3.1 (in vitro cytotoxicity (IC50) = 25 &micro, g/mL for 1, 29 &micro, g/mL for 2), but were inactive against the tested Gram-positive and Gram-negative bacteria as well as fungi.

International audience; Fungal stromata were recently discovered in association with charcoal and burnt soil aggregates during an archaeological survey in the Châtillon-sur-Seine forest massif. The wood and soil in the samples were dated to the medieval period (between 738 and 1411 AD). Light microscopy and scanning electron microscopy revealed that a few of the stromatal fragments still contained ascospores. Their macromorphological characters were described and secondary metabolite profiles were generated using high performance liquid chromatography with diode array and mass spectrometric detection (HPLC-DAD/MS). The combination of these two data lines then allowed species identification. Most of the fragments were assigned to Hypoxylon fragiforme, the type species of the Hypoxylaceae (Xylariales). Two further species whose stromata grew on the fossil charcoal could be tentatively identified as Jackrogersella cohaerens and (more tentatively) as Hypoxylon vogesiacum. These three species are still commonly encountered in the forests of Central Europe today. Furthermore, the HPLC-HRMS data of H. fragiforme suggested the presence of unknown azaphilone dimers and of further new pigments. These archaeological compounds were compared to fresh stromata of H. fragiforme collected in Germany and subjected to the same analytical protocol. While the major components in both samples were identified as the known mitorubrin type azaphilones and orsellinic acid, the chemical structures of seven novel complex azaphilone pigments, for which we propose the trivial names rutilins C-D and fragirubrins A-E, were elucidated using spectral methods (NMR and CD spectroscopy, high resolution mass spectrometry). It appears that these pigments had indeed persisted for millennia in the fossil stromata.

Trunk diseases are potential threats for the grapevine industry owing to the worldwide incidence and economic impact of the diseases. Several fungal groups are known to be involved in these diseases. In a survey on grapevine trunk diseases in northern Iran, Cytospora isolates were repeatedly recovered from vines showing decline symptoms. The symptoms appeared as pale brown to brown streaks in longitudinal cuts of shoots. The morphological and cultural characteristics of the isolates were in agreement with the description of Cytospora chrysosperma. Sequence data of the ITS-rDNA region was used to further confirm the identity of the species. Phylogenetic analysis based on the sequence data obtained in this study and the sequences from GenBank, confirmed the morphological identification. Our isolates were clustered together with C. chrysosperma isolates known from other woody host plant species. The pathogenicity assay on detached shoots of grapevines induced the same symptoms as was observed in field conditions. Although, C. chrysosperma is known from several woody hosts in Iran, the occurrence of this species on grapevines showing decline symptoms is new. The economic impact, distribution, and degree of involvement of C. chrysosperma in decline of vines in other regions of Iran remains to be studied.

During 2014–2016, symptoms and signs of a powdery mildew disease were observed on the leaves and twigs of sour cherry (Prunus cerasus) in orchards from Firouragh in the Khoy region (West Azarbaijan province) of Iran. Young leaves and twigs were covered with a powdery growth consisting of mycelium and the asexual reproductive structures of a powdery mildew species. Affected leaves were curled upward and smaller than uninfected leaves, while affected twigs were stunted and often defoliated. The fungus was identified as belonging in the genus Podosphaera, based on morphological characteristics of its asexual state. Chasmothecia were not observed. The PCR amplification and sequencing of its ITS-rDNA revealed that the causal agent was Podosphaera clandestina. This is the first record on the occurrence of P. clandestina on sour cherry in Iran.

During surveys on grapevines trunk diseases in vineyards at different localities in East and West Azerbaijan and Ardabil provinces, Iran, wood samples (two, three year-old cordons) were collected from grapevine showing decline symptoms, including leaf yellowing and necrosis, stunted growth, internal wood necrosis, black vascular discolouration. Nine fungal isolates with similar morphology were recovered from grapevines with decline symptoms in five different areas, which one-third of the isolates were recovered as the sole fungal agents. The identity of fungal isolates was determined as Quambalaria cyanescens based on the combination of cultural and morphological characteristics and DNA phylogeny. Pathogenicity trials based on excised shoot assay and potted grapevine under greenhouse conditions confirmed Q. cyanescens being pathogenic on grapevines; the isolates induced symptoms similar to those observed in naturally infected grapevines. Our findings confirm Q. cyanescens as a new fungal trunk pathogen of grapevine. Members of the genus Quambalaria are known to cause leaf spot, shoot blight and canker disease on Eucalyptus and its relative Corymbia. The current study is the first regarding pathogenicity of the Q. cyanescens on woody hosts; in previous studies Q. cyanescens has proven to be non-pathogenic to Corymbia spp. The host range, extent of geographical distribution and economic significance of this new pathogen on grapevine remains to be studied.

Several species of the genus Alternaria are involved in leaf spot disease of sunflower, with Alternaria alternata being the dominant species responsible for this disease in Iran and many other sunflower-producing areas, worldwide. The disease causes a progressive destruction of the photosynthetic apparatus, resulting in annual yield loss. The routine disease management strategies are not effective for disease control; hence, alternative measures for disease management are of great interest. In the present study, the efficacy of Trichoderma harzianum T22 and Trichoderma sp. on biological control of the causal agent was evaluated under laboratory conditions. The effect of Trichoderma isolates on dry weight (DW) and radial growth (RG) rate of A. alternata was evaluated using dual culture, volatile and non-volatile cellular metabolites. The results obtained in this study revealed that in both Trichoderma isolates, non-volatile cellular metabolites had the highest inhibitory effect on DW and RG rate of the cau...

Phaeoacremonium spp. are known as the main Hyphomycetes associated with esca disease symptoms on grapevines worldwide. The role of other woody hosts in proximity of vineyards as a possible inoculum source for the causal agents of grapevine esca disease mainly remains unknown. The aim of this study was to investigate the occurrence of Phaeoacremonium spp. on pome and stone fruit with trunk diseases symptoms in northwestern parts of Iran. For this purpose, wood trees samples were collected from apple and apricot trees with trunk disease symptoms. Isolation was made using routine plant pathology methods. Based on the morphological and cultural characteristics, Phaeoacremonium aleophilum and Phaeoacremonium iranianum were identified from apple trees and P. aleophilum was recovered from apricot trees with trunk disease symptoms. A phylogenetic analysis based on the sequence data from β-tubulin gene further confirmed the identity of the species as P. aleophilum and P. iranianum. The results of inoculation assay...

In recent years, grapevine trunk diseases have gained growing attentions due to worldwide incidence of the disease. In a survey on fungal agents associated with grapevine trunk diseases in northern Iran, wood samples were collected from grapevines having the symptoms of declination. Isolation was made using routine plant pathology methods. A coelomycetous fungus with appendage-bearing conidia was recovered from symptomatic tissues. Based on morphological and cultural characteristics, the causal agent of the disease was identified as Truncatella angustata. The identity of the species was further confirmed by sequence data of internal transcribed spacer-rDNA region. A phylogeny inferred using sequence data obtained in this study, together with the sequences from GenBank, clustered our isolates together with T. angustata known from other host plant species. Pathogenicity tests performed on detached shoots of grapevines led to the same symptoms as observed in field conditions. This is first study on the pathogenicity of T. angustata on grapevine in Iran and first report on the occurrence of T. angustata on grapevine in Iran.

Summary Boxelder (Acer negundo) is planted as ornamental and shade tree in many countries including Iran. During July 2016, boxelder trees in parks and streets of different localities in East Azarbaijan province, Iran were found to be heavily infected with powdery mildew. Whitish powdery fungal mass developed mainly on the lower leaf surface. The infected young leaves became bubbled, curled and crinkled, giving affected plants an unpleasant appearance. During September, scattered, dark brown, spherical chasmothecia were produced on the lower leaf surface. The causal agent was identified as Sawadaea negundinis based on a combination of morphological characteristics of sexual and asexual morphs and sequence data of the rDNA internal transcribed spacer region (ITS1-5.8S-ITS2). This study provides the first report on the occurrence of S. negundinis on boxelder in Iran.