Tomasz Kulik

Improving ITS sequence data for identification of plant pathogenic fungi

SummaryPlant pathogenic fungi are a large and diverse assemblage of eukaryotes with substantial impacts on natural ecosystems and human endeavours. These taxa often have complex and poorly understood life cycles, lack observable, discriminatory morphological characters, and may not be amenable to in vitro culturing. As a result, species identification is frequently difficult. Molecular (DNA sequence) data have emerged as crucial information for the taxonomic identification of plant pathogenic fungi, with the nuclear ribosomal internal transcribed spacer (ITS) region being the most popular marker. However, international nucleotide sequence databases are accumulating numerous sequences of compromised or low-resolution taxonomic annotations and substandard technical quality, making their use in the molecular identification of plant pathogenic fungi problematic. Here we report on a concerted effort to identify high-quality reference sequences for various plant pathogenic fungi and to re-annotate incorrectly or insufficiently annotated public ITS sequences from these fungal lineages. A third objective was to enrich the sequences with geographical and ecological metadata. The results – a total of 31,954 changes – are incorporated in and made available through the UNITE database for molecular identification of fungi (http://unite.ut.ee), including standalone FASTA files of sequence data for local BLAST searches, use in the next-generation sequencing analysis platforms QIIME and mothur, and related applications. The present initiative is just a beginning to cover the wide spectrum of plant pathogenic fungi, and we invite all researchers with pertinent expertise to join the annotation effort.

Detection ofFusarium tricinctum from cereal grain using PCR assay

Contamination of cereals with mycotoxins produced byFusarium is a worldwide problem requiring rapid and sensitive detection methods. This paper describes the development of a PCR protocol facilitating the detection ofF. tricinctum, which belongs to the FHB (Fusarium Head Blight) complex responsible for contamination of cereal grains with enniatins and moniliformin. Sequence alignment of partial IGS rDNA revealed a single nucleotide polymorphism, which was used to design primers differentiatingF. tricinctum from other members of the FHB complex. The specificity of the assay was tested on 68 isolates belonging to 21Fusarium species originating from different parts of the world and hosts/substrates. Positive PCR results were obtained from all 12F. tricinctum isolates tested; however, unexpected amplicons were amplified from the templates ofF. acuminatum (CBS 618.87) andF. nurragi (CBS 393.96). No cross reactivity was found with any otherFusarium species tested.The PCR assay was tested on 24 asymptomatic wheat seed samples originating from Northern Poland and resulted in 13 positive samples, of which 11 samples were contaminated with moniliformin and/or antibiotic Y.

Plant lignans inhibit growth and trichothecene biosynthesis in Fusarium graminearum

Lignans are a group of diphenolic compounds with anticancer and antioxidant properties which are present in various grains, although their effect on toxigenic fungi has been poorly examined to date. In this study, the impact of the plant lignans pinoresinol and secoisolariciresinol on growth and trichothecene biosynthesis by five Fusarium graminearum strains of different chemotypes was examined in vitro. Both tested lignans exhibited radial growth inhibition against the fungal strains. RT‐qPCR analyses of tri4, tri5 and tri11 genes encoding the first steps of the trichothecene biosynthesis pathway revealed a decrease in tri mRNA levels in lignan‐treated fungal cultures. Correspondingly, decreased accumulation of toxins in lignan‐treated cultures was confirmed by GC‐MS analysis. This is the first study to demonstrate the inhibitory effect of both pinoresinol and secoisolariciresinol on growth and trichothecene biosynthesis in F. graminearum.

Development of TaqMan assays for the quantitative detection of Fusarium avenaceum/Fusarium tricinctum and Fusarium poae esyn1 genotypes from cereal grain

Fungi of the genus Fusarium are important plant pathogens and contaminants of cereal grains producing different types of mycotoxins. Enniatins are a group of mycotoxins with ionophoric properties frequently detected in North European grains. Within the Fusarium complex responsible for grain infection, Fusarium avenaceum, Fusarium poae and Fusarium tricinctum are the most potential enniatins producers. This study presents the development of two quantitative TaqMan MGB (Minor Groove Binder) assays for the specific quantification of F. avenaceum/F. tricinctum and F. poae esyn1 genotypes, respectively. Two sets of genotype-specific primers/probes were designed on the basis of esyn1 gene homologues encoding multifunctional enzyme enniatin synthetase. The specificity of the assays developed has been tested successfully on 111 Fusarium isolates from different geographical origins. The detection limits for F. avenaceum/F. tricinctum esyn1 genotype and F. poae genotype were 19 and 0.3 pg, respectively. The application of the assays developed on asymptomatic wheat grain samples revealed significant positive correlations between the enniatins levels and the amount of F. avenaceum/F. tricinctum esyn1 genotype (R=0.61) and F. poae esyn1 genotype (R=0.42).

Multilocus Phylogenetics Show High Intraspecific Variability within Fusarium avenaceum

Fusarium avenaceum is a common soil saprophyte and plant pathogen of a variety of hosts worldwide. This pathogen is often involved in the crown rot and head blight of cereals that affects grain yield and quality. F. avenaceum contaminates grain with enniatins more than any species, and they are often detected at the highest prevalence among fusarial toxins in certain geographic areas. We studied intraspecific variability of F. avenaceum based on partial sequences of elongation factor-1 alpha, enniatin synthase, intergenic spacer of rDNA, arylamine N-acetyltransferase and RNA polymerase II data sets. The phylogenetic analyses incorporated a collection of 63 F. avenaceum isolates of various origin among which 41 were associated with wheat. Analyses of the multilocus sequence (MLS) data indicated a high level of genetic variation within the isolates studied with no significant linkage disequilibrium. Correspondingly, maximum parsimony analyses of both MLS and individual data sets showed lack of clear phylogenetic structure within F. avenaceum in relation to host (wheat) and geographic origin. Lack of host specialization indicates no host selective pressure in driving F. avenaceum evolution, while no geographic lineage structure indicates widespread distribution of genotypes that resulted in nullifying the effects of geographic isolation on the evolution of this species. Moreover, significant incongruence between all individual tree topologies and little clonality is consistent with frequent recombination within F. avenaceum.

Quantification of Fusarium poae DNA and associated mycotoxins in asymptomatically contaminated wheat

Recent surveys have identified increased predominance of Fusarium poae causing FHB (Fusarium Head Blight) of wheat in Europe. Several studies revealed a correlation between levels of F. poae DNA and nivalenol (NIV) and enniatins (ENNs) in highly contaminated cereal grain. In this study, F. poae specific TaqMan assays and mycotoxin analysis were performed on 48 asymptomatically contaminated wheat grain samples obtained from six different locations in northern Poland in 2006 and 2007. TaqMan assays revealed the presence of F. poae DNA in all samples analyzed, however the amounts of target DNA between the samples differed. Mycotoxin analysis revealed the presence of 13 toxins in the grain analyzed, however only ENN B and B1 were detected at quantifiable concentrations. A significant positive correlation was revealed between F. poae DNA (R=0.75) and monthly mean rainfalls recorded in May (a month before wheat anthesis) in both years. Additionally, a significant positive correlation was found between levels of ENN B+B1 (R=0.49) and rainfalls in May, however, no correlation was found between the quantity F. poae DNA and the level of ENN B+B1. This paper confirms the increasing importance of F. poae in the FHB complex of wheat in Poland.

New taxonomical arrangement of the traditionally conceived genera Orthotrichum and Ulota (Orthotrichaceae, Bryophyta)

Abstract The traditionally conceived genera Orthotrichum Hedw. and Ulota F.Weber are here reclassified into six genera, Orthotrichum, Dorcadion Lindb., Nyholmiella Holmen & E.Warncke, Pulvigera Plášek, Sawicki & Ochyra, Plenogemma Plášek, Sawicki & Ochyra, and Ulota, based on morphological differences and partially on molecular evidence. The genus Pulvigera includes P. lyellii (Hook. & Taylor) Plášek, Sawicki & Ochyra (Orthotrichum lyellii Hook. & Taylor) which was selected as its generitype. The genus Plenogemma includes P. phyllantha (Brid.) Plášek, Sawicki & Ochyra (Ulota phyllantha Brid.) which was selected as its generitype.

The complete mitochondrial genome of the epiphytic moss Orthotrichum speciosum

Abstract The mitogenome of the Orthotrichum speciousum (GenBank accession number KM288416) has a total length of 104,747 bp and consist of 40 protein-coding genes, 3 ribosomal RNA (rRNA) and 24 transfer RNA. The gene order is identical to other known moss mitogenomes.

ToxGen: an improved reference database for the identification of type B-trichothecene genotypes in Fusarium

Type B trichothecenes, which pose a serious hazard to consumer health, occur worldwide in grains. These mycotoxins are produced mainly by three different trichothecene genotypes/chemotypes: 3ADON (3-acetyldeoxynivalenol), 15ADON (15-acetyldeoxynivalenol) and NIV (nivalenol), named after these three major mycotoxin compounds. Correct identification of these genotypes is elementary for all studies relating to population surveys, fungal ecology and mycotoxicology. Trichothecene producers exhibit enormous strain-dependent chemical diversity, which may result in variation in levels of the genotype’s determining toxin and in the production of low to high amounts of atypical compounds. New high-throughput DNA-sequencing technologies promise to boost the diagnostics of mycotoxin genotypes. However, this requires a reference database containing a satisfactory taxonomic sampling of sequences showing high correlation to actually produced chemotypes. We believe that one of the most pressing current challenges of such a database is the linking of molecular identification with chemical diversity of the strains, as well as other metadata. In this study, we use the Tri12 gene involved in mycotoxin biosynthesis for identification of Tri genotypes through sequence comparison. Tri12 sequences from a range of geographically diverse fungal strains comprising 22 Fusarium species were stored in the ToxGen database, which covers descriptive and up-to-date annotations such as indication on Tri genotype and chemotype of the strains, chemical diversity, information on trichothecene-inducing host, substrate or media, geographical locality, and most recent taxonomic affiliations. The present initiative bridges the gap between the demands of comprehensive studies on trichothecene producers and the existing nucleotide sequence databases, which lack toxicological and other auxiliary data. We invite researchers working in the fields of fungal taxonomy, epidemiology and mycotoxicology to join the freely available annotation effort.

The complete mitochondrial genome of the cryptic species C of Aneura pinguis

Abstract The structure of the Aneura pinguis mitochondrial genome (GenBank accession no. NC_026901) is similar to that of closely related Metzgeriales species: it has a total length of 165 603 bp, the base composition of the mitogenome is the following: A (26.2%), C(23.6%), G(23.8%), and T(26.4%). The A. piguis mitochondrial genome contains 69 genes. A complete mitochondrial genome sequence of A. pinguis will help better to understand mitogenome structure and content among Metzgeriales order.