Phenotypic and molecular characterization of Chaetopyrena penicillata from Iran with description of a hyphomycete synanomorph

During a survey of fungi associated with Russian olive fruit rot in Northern Iran, a coelomycete fungus with setose pycnidia was isolated from symptomatic fruits. The fungus was identified as Chaetopyrena penicillata based on morphological characteristics and sequence data of ITS and LSU rDNA. A hyphomycete synanamorph, which was observed in pure cultures of this species for the first time, is described. The morphology and phylogeny of Chaetopyrena penicillata is discussed

Rapid and Easy Modified Plate-based Screening Methods for Quantitative and Qualitative Detection of Protease Production by Fungi

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

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

3openInternationalInternational coauthor/editorStrawberry 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.openKarimi, Kaivan; Arzanlou, Mahdi; Pertot, IlariaKarimi, K.; Arzanlou, M.; Pertot, I

Two novel <i>Aspergillus </i>species from hypersaline soils of The National Park of Lake Urmia, Iran

Two novel Aspergillus species, one belonging to the section Terrei and the other to section Flavipedes, were isolated from hypersaline soils of The National Park of Lake Urmia (Iran) and are here described as Aspergillus iranicus and Aspergillus urmiensis. A polyphasic taxonomic approach comprising extrolite profiles, phenotypic characters and molecular data (beta-tubulin, calmodulin and ribosomal polymerase II second largest subunit gene sequences) was applied to determine their novel taxonomic status. Aspergillus iranicus (CBS 139561T) is phylogenetically related to A. carneus, A. niveus, A. allahabadii and A. neoindicus, and it can be differentiated from those species by a unique extrolite pattern (citrinin, gregatins, and a terrequinone) and its conidial colour. Aspergillus urmiensis (CBS 139558T) shares a most recent common ancestor with A. templicola. The former species produces globose vesicles, and those of A. templicola are predominantly elongate. The Aspergillus urmiensis isolates produce several uncharacterized extrolites. Two other strains obtained during this study reside in a clade, together with the type strain of A. movilensis (CCF 4410T), and are identified accordingly. Based on the phylogenetic data presented in this study, A. frequens is reduced to synonymy with A. micronesiensis and A. mangaliensis is considered to be a synonym of A. templicola

Molecular Diagnostics in the Mycosphaerella Leaf Spot Disease Complex of Banana and for Radopholus similis

Mycosphaerella leaf spots and nematodes threaten banana cultivation worldwide. The Mycosphaerella disease complex 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, since their symptoms and life cycles are rather similar. Diagnosing these diseases and the respective causal agents is based on the presence of host symptoms and fungal fruiting structures, but is time consuming and not conducive to preventive management. In the present study, we developed rapid and robust species-specific diagnostic tools to detect and quantify M. fijiensis, M. musicola and M. eumusae. Conventional species-specific PCR primers were developed based on the actin gene that detected as little as 100, 1 and 10 pg/µl DNA from, respectively, M. fijiensis, M. musicola and M. eumusae. Furthermore, TaqMan real-time quantitative PCR assays that were developed based on the ß-tubulin gene detected quantities as low as 1 pg/µl DNA of each species from pure cultures and 1.6 pg/µl DNA/mg of M. fijiensis from dry leaf tissue. The efficacy of the tests was validated using naturally infected banana leaves. Similar technology has been used to develop a quantitative PCR assay for the banana burrowing nematode, Radopholus similis, which is currently being validate

Multiplex PCR for specific identification and determination of mating type in Togninia minima (anamorphic Phaeoacremonium aleophilum), a causal agent of esca disease of grapevine

Togninia minima is one of the fungi involved in esca disease of grapevine, worldwide. It has a biallelic heterothallic mating system. A multiplex PCR test was developed that can detect the species as well as the mating type. A T. minima-specific primer set, with expected amplicon size of 500 bp, was designed based on β-tubulin gene sequences. A previously designed degenerate primer set (NcHMG1 and NcHMG2) was successfully used to amplify a fragment of approximately 300 bp from the Mat1-2 gene of T. minima. The obtained sequence showed substantial homology to the Mat1-2 gene sequences of other related ascomycetes. A more specific primer set, with expected amplicon size of 230 bp, was designed based on the same Mat1-2 gene sequence. The specificity of the new primer set was verified on DNA extracted from a set of Phaeoacremonium and other fungal species frequently occurring on grapevine. Both primer sets were combined in a multiplex PCR for the simultaneous identification and determination of mating types of T. minima. A 500 bp amplicon was obtained from all available T. minima isolates and none from the other Phaeoacremonium spp. A 230 bp amplicon confirmed T. minima isolates that have the Mat1-2 allele. The species-specific β-tubulin-based primer set served as an internal control to confirm that the PCR reaction with the mating type primer set had worked properly. The efficacy of the multiplex test was evaluated on 31 isolates of T. minima from different vineyards in the Azarshahr region (East Azerbaijan province, Iran). Isolates of both mating types were found from the sampled areas; however, Mat1-2 isolates were more frequent than Mat1-1 isolates (19:12). This multiplex PCR assay developed can facilitate rapid screening of mating types in populations of T. minima

Biodiversity study of endophytic fungi associated with two Quercus species in Iran

Aim of study: In this study, frequency and diversity of fungal endophyte communities inhabiting twigs and branches of apparently healthy Q. macranthera and Q. brantii in East Azerbaijan and Lorestan provinces of Iran is presented.Area of study: East Azerbaijan and Lorestan provinces in Iran.Materials and methods: Culturable fungal endophytes were recovered from wood tissues using routine technique for isolation of fungal endophytes. The identity of fungal isolates were determined based on morphological characteristics and sequences data of ITS-rDNA region and Beta-tubulin gene. Frequency and diversity among fungal communities were analyzed using chi-square test and biodiversity indices.Main results: The highest frequency and diversity was detected for fungal endophyte community recovered from Q. macranthera and East Azerbaijan province. The assemblage of endophytic fungi characterized in this study in healthy tissues of oak trees indicates that some of the fungi are possible latent pathogens such as Biscogniauxia mediterranea with 18.28% frequency followed by Alternaria alternata and Trichothecium roseum respectively. Two fungal taxa of Pyronema domesticum and Valsa persoonii are reported for the first time in Iran. Overall, the results of this study show that the plant species and growth location influence frequency and diversity of culturable fungal endophytic communities of Quercus in Iran.Keywords: Quercus macranthera, Quercus brantii, Fungal endophytes, Molecular identification.Abbreviations used: CBS (Centraal Bureau voor Schimmelcultures); CCTU (Culture Collection of University of Tabriz); GTR (General Time Reversible); HKY (Hasegawa Kishino Yano); ITS-rDNA (Internal Transcribed Space); km (kilometer) ; PDA (Potato Dextrose Agar); TUB (Tubulin)

Plant tonic, a plant-derived bioactive natural product, exhibits antifungal activity against rice blast disease

The tendency towards application of natural products and botanical extracts as safer antimicrobial agents against plant pathogens has recently been increased. Plant Tonic9 (EOX-SOV) is an environmentally friendly product and by its application there is no concern of resistance as it is with conventional pesticides. The goal of the present research was to determine the effect of application of this product against Magnaporthe oryzae, the causal agent of rice blast. The efficacy of plant tonic against M. oryzae was evaluated through in vitro and in vivo experiments. Under in vitro conditions, application of plant tonic at all rates (2, 3, and 4 mL/L) could significantly inhibit the mycelial growth and conidial germination of fungus with the highest inhibition (83.63% and 95.15%, respectively) recorded by the rate of 4 mL. Plant tonic treatment (3 and 4 mL) was more effective than fungicide treatment (propiconazole 25% EC (0.1%); 250 ppm) to inhibit mycelial growth and conidial germination of M. oryzae. Under in vivo conditions, plant tonic application (4 mL) was also the most effective treatment and resulted in a significant reduction (57.12%) of the area under the disease progress curve (AUDPC) value as compared with the control. Application of plant tonic also caused increased accumulation of phenolic compounds and higher activity of peroxidase and polyphenol oxidase enzymes than the control. The maximum amount of phenolic compounds (0.49 mg Gallic acid equivalent/g leaf fresh weight) and the highest activity of the enzymes (1.24 and 7.85 Units/mL for peroxidase and polyphenol oxidase, respectively) were observed in plants treated with plant tonic (4 mL) and challenged with M. oryzae as compared with other treatments. No phytotoxicity was observed in plant tonic treated rice plants when compared with the control. Results of the present study confirmed the beneficial effects of plant tonic in controlling rice blast disease. Therefore, its application may help to develop appropriate management strategies and provide with the opportunity to have cleaner and safer environment for agriculture

Allicin from garlic inhibits the biofilm formation and urease activity of Proteus mirabilis in vitro

Several virulence factors contribute to the pathogenesis of Proteus mirabilis. This study determined the inhibitory effects of allicin on urease, hemolysin and biofilm of P. mirabilis ATCC 12453 and its antimicrobial activity against 20 clinical isolates of P. mirabilis. Allicin did not inhibit hemolysin, whereas it did inhibit relative urease activity in both pre-lysed (half-maximum inhibitory concentration, IC50 = 4.15 μg) and intact cells (IC50 = 21 μg) in a concentration-dependent manner. Allicin at sub-minimum inhibitory concentrations (2-32 μg mL-1) showed no significant effects on the growth of the bacteria (P&gt;0.05), but it reduced biofilm development in a concentration-dependent manner (P&lt;0.001).A higher concentration of allicin was needed to inhibit the established biofilms. Using the microdilution technique, the MIC90 and MBC90 values of allicin against P. mirabilis isolates were determined to be 128 and 512 μg mL-1, respectively.The results suggest that allicin could have clinical applications in controlling P. mirabilis infections. © FEMS 2015. All rights reserved