Antifungal Properties of Bio-AgNPs against D. pinodes and F. avenaceum Infection of Pea (Pisum sativum L.) Seedlings.

Ascochyta blight and Fusarium root rot are the most serious fungal diseases of pea, caused by D. pinodes and F. avenaceum, respectively. Due to the lack of fully resistant cultivars, we proposed the use of biologically synthesized silver nanoparticles (bio-AgNPs) as a novel protecting agent. In this study, we evaluated the antifungal properties and effectiveness of bio-AgNPs, in in vitro (poisoned food technique; resazurin assay) and in vivo (seedlings infection) experiments, against D. pinodes and F. avenaceum. Moreover, the effects of diseases on changes in the seedlings' metabolic profiles were analyzed. The MIC for spores of both fungi was 125 mg/L, and bio-AgNPs at 200 mg/L most effectively inhibited the mycelium growth of D. pinodes and F. avenaceum (by 45 and 26%, respectively, measured on the 14th day of incubation). The treatment of seedlings with bio-AgNPs or fungicides before inoculation prevented the development of infection. Bio-AgNPs at concentrations of 200 mg/L for D. pinodes and 100 mg/L for F. avenaceum effectively inhibited infections' spread. The comparison of changes in polar metabolites' profiles revealed disturbances in carbon and nitrogen metabolism in pea seedlings by both pathogenic fungi. The involvement of bio-AgNPs in the mobilization of plant metabolism in response to fungal infection is also discussed.

Molecular Diversity and Phylogeny Reconstruction of Genus Colobanthus (Caryophyllaceae) Based on Mitochondrial Gene Sequences.

Mitochondrial genomes have become an interesting object of evolutionary and systematic study both for animals and plants, including angiosperms. Although the framework of the angiosperm phylogeny was built on the information derived from chloroplast and nuclear genes, mitochondrial sequences also revealed their usefulness in solving the phylogenetic issues at different levels of plant systematics. Here, we report for the first time the complete sequences of 26 protein-coding genes of eight Colobanthus species (Caryophyllaceae). Of these, 23 of them represented core mitochondrial genes, which are directly associated with the primary function of that organelle, and the remaining three genes represented a facultative set of mitochondrial genes. Comparative analysis of the identified genes revealed a generally high degree of sequence conservation. The Ka/Ks ratio was <1 for most of the genes, which indicated purifying selection. Only for rps12 was Ka/Ks > 1 in all studied species, suggesting positive selection. We identified 146−165 potential RNA editing sites in genes of the studied species, which is lower than in most angiosperms. The reconstructed phylogeny based on mitochondrial genes was consistent with the taxonomic position of the studied species, showing the separate character of the family Caryophyllaceae and close relationships between all studied Colobanthus species, with C. lycopodioides sharing less similarity.

Prevalence of Fusarium fungi and Deoxynivalenol Levels in Winter Wheat Grain in Different Climatic Regions of Poland.

Fusarium head blight (FHB) caused by fungi of the genus Fusarium is one of the most dangerous crop diseases, which has a wide geographic distribution and causes severe economic losses in the production of major cereal species. The infection leads to the accumulation of mycotoxins in grains, which compromises its suitability for human and animal consumption. The study demonstrated that grain samples from warmer regions of Poland, including Sulejów and Tomaszów Boleslawicki (results differed across years of the study), were colonized mainly by F. graminearum and were most highly contaminated with deoxynivalenol (DON). Samples from Northeastern Poland, i.e., Ruska Wies, which is located in a cooler region, were characterized by a predominance of Fusarium species typical of the cold climate, i.e., Fusarium poae and Penicillium verrucosum. A Spearman's rank correlation analysis revealed that the severity of grain infection with F. avenaceum/F. tricinctum was affected by the mean daily temperature and high humidity in May, and the corresponding values of the correlation coefficient were determined at R = 0.54 and R = 0.50. Competitive interactions were observed between the F. avenaceum/F. tricinctum genotype and DON-producing F. culmorum and F. graminearum, because the severity of grain infections caused by these pathogens was bound by a negative correlation.

Characterization and phylogenetic analysis of the complete mitochondrial genome of the pathogenic fungus Ilyonectria destructans.

Ilyonectria destructans is a pathogenic fungus causing root rot and other symptoms on trees and many crops. This paper analyses the mitochondrial genome of I. destructans and compares it with other published Nectriaceae mitogenomes. The I. destructans mitogenome appears as a circular DNA molecule of 42,895 bp and an overall GC content of 28.23%. It contains 28 protein-coding genes (15 core protein genes and 13 free-standing ORFs), two rRNAs and 27 tRNAs. The gene content and order were found to be conserved in the mitogenome of I. destructans and other Nectriaceae, although the genome size varies because of the variation in the number and length of intergenic regions and introns. For most core protein-coding genes in Nectriaceae species, Ka/Ks < 1 indicates purifying selection. Among some Nectriaceae representatives, only the rps3 gene was found under positive selection. Phylogenetic analyses based on nucleotide sequences of 15 protein-coding genes divided 45 Hypocreales species into six major clades matching the families Bionectriaceae, Cordycipitaceae, Clavicipitaceae, Ophiocordycipitaceae, Hypocreaceae and Nectriaceae. I. destructans appeared as a sister species to unidentified Ilyonectia sp., closely related to C. ilicicola, N. cinnabarina and a clad of ten Fusarium species and G. moniliformis. The complete mitogenome of I. destructans reported in the current paper will facilitate the study of epidemiology, biology, genetic diversity of the species and the evolution of family Nectriace and the Hypocreales order.

Evolutionary dynamics of the chloroplast genome sequences of six Colobanthus species.

The complete plastome sequences of six species were sequenced to better understand the evolutionary relationships and mutation patterns in the chloroplast genome of the genus Colobanthus. The length of the chloroplast genome sequences of C. acicularis, C. affinis, C. lycopodioides, C. nivicola, C. pulvinatus and C. subulatus ranged from 151,050 to 151,462 bp. The quadripartite circular structure of these genome sequences has the same overall organization and gene content with 73 protein-coding genes, 30 tRNA genes, four rRNA genes and five conserved chloroplast open reading frames. A total of 153 repeat sequences were revealed. Forward repeats were dominant, whereas complementary repeats were found only in C. pulvinatus. The mononucleotide SSRs composed of A/T units were most common, and hexanucleotide SSRs were detected least often. Eleven highly variable regions which could be utilized as potential markers for phylogeny reconstruction, species identification or phylogeography were identified within Colobanthus chloroplast genomes. Seventy-three protein-coding genes were used in phylogenetic analyses. Reconstructed phylogeny was consistent with the systematic position of the studied species, and the representatives of the same genus were grouped in one clade. All studied Colobanthus species formed a single group and C. lycopodioides was least similar to the remaining species.

rps3 as a Candidate Mitochondrial Gene for the Molecular Identification of Species from the Colletotrichum acutatum Species Complex.

Colletotrichum species form one of the most economically significant groups of pathogenic fungi and lead to significant losses in the production of major crops-in particular, fruits, vegetables, ornamental plants, shrubs, and trees. Members of the genus Colletotrichum cause anthracnose disease in many plants. Due to their considerable variation, these fungi have been widely investigated in genetic studies as model organisms. Here, we report the complete mitochondrial genome sequences of four Colletotrichum species (C. fioriniae, C. lupini, C. salicis, and C. tamarilloi). The reported circular mitogenomes range from 30,020 (C. fioriniae) to 36,554 bp (C. lupini) in size and have identical sets of genes, including 15 protein-coding genes, two ribosomal RNA genes, and 29 tRNA genes. All four mitogenomes are characterized by a rather poor repetitive sequence content with only forward repeat representatives and a low number of microsatellites. The topology of the phylogenetic tree reflects the systematic positions of the studied species, with representatives of each Colletotrichum species complex gathered in one clade. A comparative analysis reveals consistency in the gene composition and order of Colletotrichum mitogenomes, although some highly divergent regions are also identified, like the rps3 gene which appears as a source of potential diagnostic markers for all studied Colletotrichum species.

The complete chloroplast genome of Colobanthus apetalus (Labill.) Druce: genome organization and comparison with related species.

Colobanthus apetalus is a member of the genus Colobanthus, one of the 86 genera of the large family Caryophyllaceae which groups annual and perennial herbs (rarely shrubs) that are widely distributed around the globe, mainly in the Holarctic. The genus Colobanthus consists of 25 species, including Colobanthus quitensis, an extremophile plant native to the maritime Antarctic. Complete chloroplast (cp) genomes are useful for phylogenetic studies and species identification. In this study, next-generation sequencing (NGS) was used to identify the cp genome of C. apetalus. The complete cp genome of C. apetalus has the length of 151,228 bp, 36.65% GC content, and a quadripartite structure with a large single copy (LSC) of 83,380 bp and a small single copy (SSC) of 17,206 bp separated by inverted repeats (IRs) of 25,321 bp. The cp genome contains 131 genes, including 112 unique genes and 19 genes which are duplicated in the IRs. The group of 112 unique genes features 73 protein-coding genes, 30 tRNA genes, four rRNA genes and five conserved chloroplast open reading frames (ORFs). A total of 12 forward repeats, 10 palindromic repeats, five reverse repeats and three complementary repeats were detected. In addition, a simple sequence repeat (SSR) analysis revealed 41 (mono-, di-, tri-, tetra-, penta- and hexanucleotide) SSRs, most of which were AT-rich. A detailed comparison of C. apetalus and C. quitensis cp genomes revealed identical gene content and order. A phylogenetic tree was built based on the sequences of 76 protein-coding genes that are shared by the eleven sequenced representatives of Caryophyllaceae and C. apetalus, and it revealed that C. apetalus and C. quitensis form a clade that is closely related to Silene species and Agrostemma githago. Moreover, the genus Silene appeared as a polymorphic taxon. The results of this study expand our knowledge about the evolution and molecular biology of Caryophyllaceae.

The complete mitogenome of Mycosphaerella pinodes (Ascomycota, Mycosphaerellaceae).

In this study, the complete mitochondrial genome of plant pathogenic fungus, Mycosphaerella pinodes, was sequenced. The nucleotide composition of the genome is: 36.0% of A, 15.0% of C, 14.6% of G and 34.5% of T. The mitochondrial genome is 55 973 bp in length and consists of 11 protein-coding genes, two ribosomal RNAs and 25 tRNA genes. The mitogenome analysis of M. pinodes provide a molecular basis for further studies on molecular systematics and evolutionary dynamics of Ascomycetes fungi especially belonging to Dothideomycetes.

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.

PCR approach based on the esyn1 gene for the detection of potential enniatin-producing Fusarium species.

Fusarium head blight (FHB) is a disease of small-grain cereals and corn caused by a complex of fungal species of the genus Fusarium. The disease reduces the yield and quality of seeds and results in the accumulation of various mycotoxins which cause a variety of toxic effects on humans and livestock. Beauvericin (BEA) and enniatins (ENs) are a group of toxins with antimicrobial, insecticidal and phytotoxic activities produced mainly by F. avenaceum, F. poae and F. tricinctum. In this study, primer sets were designed that were targeted to esyn1 gene homologs encoding multifunctional enzyme enniatin synthetase. Primers used in multiplex PCR amplified products from the FHB species reported to produce (ENs) and/or BEA. The use of the marker developed on asymptomatic wheat seed samples originating from Northern and Southern Poland demonstrated that all samples were positive for the presence of potential enniatin-producing Fusarium species.

Development of PCR assay based on ITS2 rDNA polymorphism for the detection and differentiation of Fusarium sporotrichioides.

A polymerase chain reaction assay was developed for detection of Fusarium sporotrichioides, a plant pathogen in many parts of the world. Based on small nucleotide differences in ITS2 (Internal Transcribed Spacer) rDNA of our local isolate of F. sporotrichioides (Accession No. AY510069) and other isolates found in NCBI/GeneBank database, species specific primer FspITS2K was selected. Primer pair FspITS2K and P28SL amplified a fragment of 288 bp containing a portion of ITS2 and 28S rDNA of all the F. sporotrichioides isolates tested, originated from different hosts and regions of the world but did not amplify any other species of Fusarium and plant's DNA. To use the PCR assay in seed health testing, a protocol was setup for the rapid and effective preparations of fungal DNA from wheat seeds. The method developed may be useful for the rapid detection and identification of F. sporotrichioides both from culture and from plant tissue.