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

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

Macrophomina phaseolina associated with grapevine decline in Iran

Grapevines exhibiting general decline symptoms were observed in July 2012 in Arbatan, a region in Marand county, north-western Iran. Leaf necrosis symptoms were also observed in affected grapevines. Dark brown necrotic lesions and wood discoloration were observed in the vascular tissues of affected cordons. Fungal isolates, all with similar cultural and morphological features, were obtained from symptomatic tissues. Morphological characteristics indicated that the isolates were Macrophomina phaseolina. Sequence analysis of the elongation factor (EF-1α) gene from isolates confirmed this identification, with 100% similarity to the reference M. phaseolina sequences obtained from GenBank. Pathogenicity assays of two M. phaseolina isolates on 2-year-old potted vines (cv. ‘Keshmeshi’) demonstrated that M. phaseolina was pathogenic on grapevine. This study confirmed the association of M. phaseolina with grapevine decline symptoms, and represents the first report of this fungus associated with grapevine trunk diseases in Iran

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)

Uneven distribution of mating type alleles in Iranian populations of Cercospora beticola, the causal agent of Cercospora leaf spot disease of sugar beet

Cercospora beticola, the causal agent of Cercospora leaf spot disease on sugar beet, is thought to be exclusively asexual because no teleomorph has yet been found. The possibility of a clandestine sexual cycle in the Iranian population of Cercospora beticola was evaluated by analyzing the distribution and frequency of the mating type alleles on a microspatial and a macrogeographical scale. A total of 89 single-conidial Cercospora beticola isolates were obtained from sugar beet fields in the Moghan, the Talesh and the Khoy regions. The isolates were identifed using a Cercospora beticola-specifc primer set in a PCR assay. A multiplex PCR method using previously designed mating type primers was used to study the distribution and the frequency of the mating type alleles. All isolates showed either the 805-bp fragment or the 442-bp fragment of the MAT1-1 and MAT1-2 genes, but no isolate had both fragments. The distribution of the mating type genes in the sampled areas was uneven. From three sugar beet fields sampled in the Moghan region, two fields had only MAT1-1 isolates; while in the third field all isolates had only the MAT1-2 allele. In the Talesh region only MAT1-1 isolates occurred, and in the Khoy region the mating type alleles were uniformly distributed amongst the isolates. The skewed distribution of mating type alleles in Northwestern Iran was in line with the lack of a sexual cycle for this species and may also indicate that sugar fields in the Moghan region were infected by C. beticola populations of different origins

Biological and molecular characterisation of Pilidium lythri, an emerging strawberry pathogen in Iran

Strawberry production is hampered by numerous biotic factors including fungal pathogens. Symptoms of dark brown necrotic lesions on fruits, stems and leaves were observed in a survey of strawberry fields in the Kurdistan province of Iran. Symptomatic plant tissues were collected and several fungal isolates were recovered from these tissues. Based on a combination of morphological characteristics and sequence data for ITS and LSU ribosomal DNA, the isolates were identified as Pilidium lythri (previously named P. concavum or Hainesia lythri). A pathogenicity assay confirmed that all new isolates induced symptoms resembling those observed in the field, as well as two P. lythri isolates from olive included in the assay; no significant disease incidence or severity differences were detected between isolates. This is the first report of P. lythri on strawberry in Iran. Genetic diversity between Pilidium lythri isolates from strawberry (18 isolates) and olive (two isolates) was evaluated using RAPD and M13 markers. No polymorphism was detected within and among the isolates, indicating limited genetic variability probably due to lack of recombination events and/or recent introduction. Given recent outbreaks and the presence of the pathogen in most strawberry growing areas in the Kurdistan province, quickly applied and appropriate management programmes are necessary to prevent spread of the disease

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

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