Molecular phylogeny, pathogenicity and toxigenicity of Fusarium oxysporum f. sp. lycopersici.

The present study aimed at the molecular characterization of pathogenic and non pathogenic F. oxysporum f. sp. lycopersici strains isolated from tomato. The causal agent isolated from symptomatic plants and soil samples was identified based on morphological and molecular analyses. Pathogenicity testing of 69 strains on five susceptible tomato varieties showed 45% of the strains were highly virulent and 30% were moderately virulent. Molecular analysis based on the fingerprints obtained through ISSR indicated the presence of wide genetic diversity among the strains. Phylogenetic analysis based on ITS sequences showed the presence of at least four evolutionary lineages of the pathogen. The clustering of F. oxysporum with non pathogenic isolates and with the members of other formae speciales indicated polyphyletic origin of F. oxysporum f. sp. lycopersici. Further analysis revealed intraspecies variability and nucleotide insertions or deletions in the ITS region among the strains in the study and the observed variations were found to be clade specific. The high genetic diversity in the pathogen population demands for development of effective resistance breeding programs in tomato. Among the pathogenic strains tested, toxigenic strains harbored the Fum1 gene clearly indicating that the strains infecting tomato crops have the potential to produce Fumonisin.

Polymorphism of Beauveria bassiana (Deuteromycota: Hyphomycetes) strains isolated from Ixodes ricinus (Acari: Ixodidae) in Moldova.

Polymorphism of 10 Beauveria bassiana strains, isolated from Ixodes ricinus in Moldova, was evaluated using traditional (morphological and cultural properties) and molecular (RAPD patterns and ITS sequences) methods. The isolates differed greatly in morphological and cultural features, such as color, consistence, and growth rate. Four RAPD-PCR markers were used to evaluate genetic diversity of the strains. Phylogenetic neighbor-joining analysis of RAPD patterns divided strains into 3 major clades. The ITS sequences of 8 strains were identical to those of known B. bassiana strains. Two subsets (1 and 2) different by one nucleotide change were found in the ITS1 region. One strain of subset 1 was different from known B. bassiana strains by possessing 2 point mutations in the ITS region. RAPD-based clustering correlated to ITS sequence and colony morphology-based grouping of the strains.

Molecular characterization of pathogenic Fusarium species in cucurbit plants from Kermanshah province, Iran.

Fusarium is one of the important phytopathogenic genera of microfungi causing serious losses on cucurbit plants in Kermanshah province, the largest area of cucurbits plantation in Iran. Therefore, the objectives in this study were to isolate and identify disease-causing Fusarium spp. from infected cucurbit plants, to ascertain their pathogenicity, and to determine their phylogenetic relationships. A total of 100 Fusarium isolates were obtained from diseased cucurbit plants collected from fields in different geographic regions in Kermanshah province, Iran. According to morphological characters, all isolates were identified as Fusarium oxysporum, Fusarium proliferatum, Fusarium equiseti, Fusarium semitectum and Fusarium solani. All isolates of the five Fusarium spp. were evaluated for their pathogenicity on healthy cucumber (Cucumis sativus) and honeydew melon (Cucumis melo) seedlings in the glasshouse. F. oxysporum caused damping-off in 20-35 days on both cucurbit seedlings tested. Typical stem rot symptoms were observed within 15 days after inoculation with F. solani on both seedlings. Based on the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) restriction fragment length polymorphism (RFLP) analysis, the five Fusarium species were divided into two major groups. In particular, isolates belonging to the F. solani species complex (FSSC) were separated into two RFLP types. Grouping among Fusarium strains derived from restriction analysis was in agreement with criteria used in morphological classification. Therefore, the PCR-ITS-RFLP method provides a simple and rapid procedure for the differentiation of Fusarium strains at species level. This is the first report on identification and pathogenicity of major plant pathogenic Fusarium spp. causing root and stem rot on cucurbits in Iran.

Occurrence, pathogenicity and distribution of Fusarium spp. in stored wheat seeds Kermanshah Province, Iran.

Fusarium is one of the most important pathogenic and toxigenic fungi widely distributed all over the world, including Iran. Fusarium species are found frequently in stored agriculture products especially wheat. The objective of this study was to identify Fusarium species associated with stored wheat seeds and their pathogenicity on root and head of wheat in Kermanshah, the leading province in wheat production in Iran. In this survey 75 seed samples of stored wheat were collected from 10 different regions during 2006-2008 and tested for the presence of Fusarium. Fusarium spp. were found in 51 (68%) of 75 samples. A total of 580 Fusarium strains were isolated, identified and preserved. All these strains belong to 20 Fusarium spp. according to morphological characters. Each conidial suspension of selected strains representing all species was evaluated for their pathogenicity on roots and spikes of healthy wheat var. Fallat in the greenhouse. F. graminearum, F. crookwellense, F. trichothecioides, F. culmorum and F. verticillioides were the most pathogenic to wheat's head. Foot rot assessment revealed that F. pseudograminearum and F. culmorum were the most damaging species. Of the Fusarium isolates, F. graminearum was the most prevalent followed by F. verticillioides and F. proliferatum. This is the first comprehensive report on identity and distribution of Fusarium spp. from stored wheat seeds in Iran while F. nelsonii was reported for the first time from wheat seeds in Iran.

Genetic variation, real-time PCR, metabolites and mycotoxins ofFusarium avenaceum and related species.

In this paper the latest studies dealing with genetic variation and mycotoxins ofF. avenaceum and related species are reviewed and compared to the data from chromatographic image analyses. Forty-three European strains ofFusarium avenaceum and related species were classified by chromatographic image analysis on full chromatographic matrices. The results were in most cases in agreement with those from morphological and molecular analyses and supported the separation betweenF. avenaceum, F. arthrosporioides andF. tricinctum and betweenF. avenaceum groups I and II. The mycotoxin profiles of the FinnishF. avenaceum, F. arthrosporioides andF tricinctum strains were very similar to each other. Moniliformin and enniatins were the main mycotoxins produced. A fluorogenic TaqMan PCR assay (qPCR) was used for the detection ofF. avenaceum/ F. arthrosporioides DNA in Finnish barley and wheat. The qPCR results obtained from grain samples were compared to mycotoxin levels. A correlation was found betweenF. avenaceum/F. arthrosporioides DNA and moniliformin (MON) and enniatin (ENNs) levels in barley. A correlation was also found between the combinedF. avenaceum/F. arthrosporioides/F. tricinctum contamination and MON and ENNs levels in barley in 2002, but not in 2003. This was probably due to the higher MON and ENNs levels in 2002 than in 2003. It was possible to use the DNA levels ofF. avenaceum/F. arthrosporioides to distinguish between most barley samples containing high amounts of MON and ENNs from those containing low levels of the mycotoxins.

Presence and concentrations of the Fusarium-related mycotoxins beauvericin, enniatins and moniliformin in finnish grain samples.

Fusarium mycotoxins beauvericin, enniatins (A, A1, B, B1) and moniliformin were analysed in 38 Finnish grain samples (14 wheat, 22 barley, one rye, one oats) harvested in 2001-02. The contaminating Fusarium species were identified with the primer-specific polymerase chain reaction as well as with morphological studies. All the studied mycotoxins were found in the samples. Enniatins B and B1 were detected in all samples, and enniatin A, enniatin A1, beauvericin and moniliformin in 74, 95, 95 and 74% of the samples, respectively. There were higher concentrations of the mycotoxins analysed in 2001 compared with 2002. The highest levels of mycotoxins were detected in samples harvested late in the autumn after a long rainy period. Fusarium avenaceum was the most abundant Fusarium species in Finland during both years (0-29.5%) measured as infected kernels. A significant correlation was found between F. avenaceum contamination level and the concentration levels of enniatins B and B1, as well as moniliformin.

Phylogenetic relationship of Fusarium langsethiae to Fusarium poae and Fusarium sporotrichioides as inferred by IGS, ITS, beta-tubulin sequences and UP-PCR hybridization analysis.

Fusarium langsethiae was recently described to accommodate "powdery" isolates of Fusarium poae, which morphologically resemble F. poae, but whose metabolite profile is similar to that of Fusarium sporotrichioides. In order to investigate the phylogenetic relationship of F. langsethiae to closely related species, we sequenced the internal transcribed spacer (ITS) regions 1 and 2 and part of the intergenic spacer (IGS) region of the rDNA cluster and part of the beta-tubulin gene from 109 strains of F. poae, F. sporotrichioides, F. langsethiae and Fusarium kyushuense from different geographic origin. Sequence analysis of ITS1 and 2 was unable to separate all F. sporotrichioides strains from F. langsethiae strains. Sequence analysis of beta-tubulin distinguished all four species, but it did not resolve the phylogenetic relationship between these two species. Sequence analysis of the IGS region distinguished the four species and led to a higher number of subgroups of the individual species, of which that of F. sporotrichioides var. minus isolates was even better supported than that of F. poae and F. langsethiae. Neighbor-joining and POY analyses of all combined sequences reliably separated all species studied, including F. langsethiae, clearly from F. sporotrichioides. The high intraspecific variability of the IGS sequences were found useful to group isolates according to their geographic origin. These results are in accordance with the results of the UP-PCR hybridization analysis. In summary, our data offer molecular support for the description of F. langsethiae as a new species in section Sporotrichiella.

An integrated taxonomic study of Fusarium langsethiae, Fusarium poae and Fusarium sporotrichioides based on the use of composite datasets.

An integrated systematic study was carried out to clarify the taxonomical position and relationship of Fusarium langsethiae to other taxa within the Fusarium section Sporotrichiella. Strains of this species were compared with strains of the closely related species Fusarium poae and Fusarium sporotrichioides using a composite dataset. This set consisted of DNA sequences derived from the ribosomal internal transcribed spacer (ITS) regions, partial sequences of the ribosomal intergenic spacer (IGS) region, the beta-tubulin and translation elongation factor-1 alpha (EF-1alpha) genes, AFLP fingerprints, chromatographic data on secondary metabolites and morphological data and growth characteristics. From these combined data, a consensus matrix was calculated by taking the mean of all pairwise distances between single isolates over all separate datasets. The consensus matrix was used as the basis for the construction of a UPGMA dendrogram and a multidimensional scaling, both of which revealed a clear separation of the three taxa. Partial IGS, EF-1alpha and beta-tubulin sequence-as well as chromatography-and AFLP-derived similarities turned out to be comparably consistent, while ITS sequence- and morphology-derived similarity matrices were rather divergent.

Species and strain identification of the predatory mite Euseius finlandicus by RAPD-PCR and ITS sequences.

The variation within and between Finnish Euseius finlandicus populations was investigated by RAPD-PCR and ITS sequence analyses. Resin DNA extraction was found to be a suitable method for samples of single mites used in PCR. The banding patterns from 24 RAPD primers and 10 primer pairs were very similar and reproducible in all specimens of the predatory mite studied. However, the E. finlandicus K-strain could be distinguished from organophosphate-resistant predatory mites (R-strain), since almost all of them produced a 1,400 bp RAPD-PCR product, which was missing or very rare in other strains studied. Another RAPD band of ca. 680 bp was in turn much more common in other mites of E. finlandicus than in the K-strain mites. Mite specific primers were designed and used to follow the survival of the R-strain released on apple trees. The 680 bp band obtained with specific primers was specific to the species E. finlandicus mites studied, including those that had been negative with RAPD primers. The 1,400 bp specific primers could be used as a marker for following the survival of R-strain mites on apple trees. At the species level it was possible to distinguish adults and eggs of E. finlandicus from Anthoseius rhenanus and Phytonemus pallidus by RAPD-PCR. In addition, a band at 480bp was found to correspond to DNA of the predatory mite Phytoseius macropilis, when both specific primer pairs were used together. It was not possible to amplify the ITS region of E. finlandicus rDNA using several primer pairs that work in other mites and aphids. However, a basidiomycete rDNA sequence was amplified with one of these ITS primer pairs in K-strain mites. Finally, it was found that fungal rDNA-specific primers amplified an ITS region of ca. 650 bp in several strains of E. finlandicus. Internal primers, designed to amplify the central part of the 650 bp product, successfully amplified this product from all the mites.

Microtubule cytoskeleton in hyphal growth. Response to nocodazole in a sensitive and a tolerant strain of the homobasidiomycete Schizophyllum commune.

In the wild-type strains of the homobasidiomycete Schizophyllum commune microtubules were totally depolymerized by low concentrations of nocodazole, while high concentrations of benomyl only modified the structure of microtubule cytoskeleton. In the nocodazole-tolerant mutant strain NT30 the microtubule cytoskeleton remained partly functional at a nocodazole concentration which demolished the microtubules in the wild-type strains. The continuation of apical growth for several hours in the wild-type strain without cytoplasmic microtubules indicated that microtubules are not the major elements in hyphal extension growth. However, the irregular branching of the treated apical cells both in the nocodazole-sensitive and -tolerant strain suggested that an intact microtubule cytoskeleton is needed for maintaining the direct extension of the leading hyphae at the colony edge. In the nocodazole-sensitive strain growth in the absence of polymerized microtubules frequently led to the death of the apical cells even when the drug was removed. In the tolerant strain the nuclear divisions continued in spite of nocodazole, but the uninucleate hyphal compartments became multinucleate. This probably resulted from poor segregation of nuclei and septation of hyphae at telophase, which indicated that these processes might be dependent on proper polymerization of cytoplasmic microtubules in higher fungi. The different electrophoretic mobility of the beta-tubulin from the NT30 strain and its parental strains suggested that the tolerance of the NT30 to nocodazole could be due to a mutation in a beta-tubulin encoding gene.