RNA interference core components identified and characterised in Verticillium nonalfalfae, a vascular wilt pathogenic plant fungi of hops.

The conserved RNA interference mechanism (RNAi) in the fungal kingdom has become a focus of intense scientific investigation. The three catalytic core components, Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RdRP), and their associated small interfering RNA molecules (siRNAs) have been identified and characterised in several fungal species. Recent studies have proposed that RNAi is a major contributor to the virulence of fungal pathogens as a result of so-called trans-kingdom RNA silencing. In the present study, we report on the existence of three core RNAi proteins in the pathogenic plant fungus Verticillium nonalfalfae, which is a soilborne plant pathogen that causes severe wilting disease in hops (Humulus lupulus L.). Two DCL proteins, two AGO proteins, and two RdRP proteins were identified, and their conserved RNAi domains were characterised. Our phylogeny results confirm the existing taxonomic relationships in the Ascomycete fungal phylum and show that the fungi of the Hypocreomycetidae subclass of the Sordariomycetes class have high amino acid sequence similarity. The expression analysis revealed a potential role of RNAi in the pathogenicity of the fungi, since all the RNAi genes were highly upregulated in the highly virulent isolate T2 and were also differentially expressed in the V. nonalfalfae-susceptible Celeia and V. nonalfalfae-resistant Wye Target cultivars.

Identification, Characterization, Pathogenicity, and Distribution of Verticillium alfalfae in Alfalfa Plants in China.

Verticillium wilt caused by Verticillium alfalfae results in severe production losses in alfalfa crops and is a Class A quarantined disease in China. During 2015 to 2017, 365 alfalfa fields from 21 locations in six provinces were surveyed, and 45 fields from three closely located sites in Gansu, China were found to have alfalfa plants with symptoms typical of Verticillium wilt, with disease incidence of 12.6 to 53.6%. Isolates were identified to species using morphological characteristics and a maximum likelihood phylogeny of the concatenated partial sequences of actin, elongation factor, glyceraldehyde-3-phosphate dehydrogenase, and tryptophan synthase gene regions of Verticillium isolates. Isolation incidence was 93.9% from roots, 71.7% from stems, 66.1% from petioles, and 32.2% from leaves of field-infected plants, indicative of systemic disease and sporadic distribution of this pathogen. In greenhouse tests, the pathogen infected seedlings and colonized vascular tissues when inoculated on seeds, on root tips, in soil, or in injured, but not uninjured, aerial tissues, causing systemic symptoms like those in the field and significant losses. Pathogenicity testing also revealed that five locally grown perennial legumes (stylo, milkvetch, sainfoin, white clover, and red clover) could host V. alfalfae, with a high virulence to milkvetch, sainfoin, and stylo. This study confirmed that V. alfalfae has become established in some regions of Gansu, China and that is a risk to the alfalfa industry in China.

Verticillium Wilt Caused by Verticillium dahliae and V. nonalfalfae in Potato in Northern China.

Potato (Solanum tuberosum L.) is one of the most important staple foods in many parts of the world including China. In recent years, Verticillium wilt has become a severe threat to potato production in China. During 2015 to 2016, 287 samples of symptomatic potato plants were collected from 15 counties in five provinces from northern China. One hundred and eighty-seven Verticillium-like colonies were isolated from these samples and identified to species based on cultural and morphological characteristics, and multigene phylogeny based on the partial sequences of actin (ACT), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS) genes. A consensus-rooted most parsimonious phylogenetic tree was generated from the data. One hundred and fifteen isolates comprising 61.5% of the total were identified as Verticillium dahliae, and the remaining 38.5% of the isolates were identified as V. nonalfalfae. V. dahliae was widely distributed in Shaanxi (84.1%), Inner Mongolia (76.7%), Gansu (12.8%), and Qinghai (100%, representing a single isolate). V. dahliae was not recovered from the samples in Ningxia. V. nonalfalfae dominated the collections from Gansu (87.2%) and Ningxia (100%) but was also recovered from Shaanxi (15.9%) and Inner Mongolia (23.3%) at lower frequencies. Neither V. albo-atrum nor V. alfalfae was recovered from the sampled areas. The V. nonalfalfae isolates were predominantly isolated from the samples collected from altitudes above 1,800 m, and in contrast, V. dahliae isolates were mainly recovered from fields sampled below 1,800 m. The optimum temperature for the colony growth of V. nonalfalfae was lower (20°C) than that for V. dahliae (25°C). Pathogenicity tests demonstrated that V. dahliae and V. nonalfalfae were both pathogens of potato Verticillium wilt, with V. dahliae isolates exhibiting higher virulence than V. nonalfalfae isolates regardless of the collection area of the species. This is the first documentation of V. nonalfalfae infecting S. tuberosum in China and the higher altitudes associated with infections of V. nonalfalfae anywhere in the world.

Analysis of the hybrid genomes of two field isolates of the soil-borne fungal species Verticillium longisporum.

BACKGROUND: Brassica plant species are attacked by a number of pathogens; among them, the ones with a soil-borne lifestyle have become increasingly important. Verticillium stem stripe caused by Verticillium longisporum is one example. This fungal species is thought to be of a hybrid origin, having a genome composed of combinations of lineages denominated A and D. In this study we report the draft genomes of 2 V. longisporum field isolates sequenced using the Illumina technology. Genomic characterization and lineage composition, followed by selected gene analysis to facilitate the comprehension of its genomic features and potential effector categories were performed. RESULTS: The draft genomes of 2 Verticillium longisporum single spore isolates (VL1 and VL2) have an estimated ungapped size of about 70 Mb. The total number of protein encoding genes identified in VL1 was 20,793, whereas 21,072 gene models were predicted in VL2. The predicted genome size, gene contents, including the gene families coding for carbohydrate active enzymes were almost double the numbers found in V. dahliae and V. albo-atrum. Single nucleotide polymorphisms (SNPs) were frequently distributed in the two genomes but the distribution of heterozygosity and depth was not independent. Further analysis of potential parental lineages suggests that the V. longisporum genome is composed of two parts, A1 and D1, where A1 is more ancient than the parental lineage genome D1, the latter being more closer related to V. dahliae. Presence of the mating-type genes MAT1-1-1 and MAT1-2-1 in the V. longisporum genomes were confirmed. However, the MAT genes in V. dahliae, V. albo-atrum and V. longisporum have experienced extensive nucleotide changes at least partly explaining the present asexual nature of these fungal species. CONCLUSIONS: The established draft genome of V. longisporum is comparatively large compared to other studied ascomycete fungi. Consequently, high numbers of genes were predicted in the two V. longisporum genomes, among them many secreted proteins and carbohydrate active enzyme (CAZy) encoding genes. The genome is composed of two parts, where one lineage is more ancient than the part being more closely related to V. dahliae. Dissimilar mating-type sequences were identified indicating possible ancient hybridization events.

Multilocus genotyping based species identification of entomopathogenic fungi of the genus Lecanicillium (=Verticillium lecanii s.l.).

Mitochondrial gene NADH dehydrogenase subunit 1 (nad1), ß-tubulin gene, and elongation factor 1-alpha (tef) were used to characterize and to identify 42 Lecanicillum spp. isolates (former complex species Verticillium lecanii Zimm. Viegas) and to study the phylogenetic relationships in this group. Within the isolates under investigation, Lecanicillium muscarium was the most common species (about 70% of all isolates, collected on the different hosts, predominantly on the insects from the order Hemiptera). Based on nad1 sequencing four main molecular haplotypes were revealed. All four haplotypes have Holarctic origin. Most of them were isolated in the Central part of Russia. One haplotype showed a specific association with the certain geographical area, limited to southwest Georgia and the Krasnodar Territory. For most strains their affiliation to species L. muscarium, L. longisporum, L. psalliotae, L. pissodes were confirmed by the phylogenetic tree, based on the combined sequences of nad1, ß-tub, and tef genes. Only five strains of haplotype C and strain F-2643 could not be identified to any present Lecanicillium species and their position remains ambiguous. Thus, the use of multilocus molecular approach based on these genes was useful to identify the Lecanicillium species. Inter-simple sequence repeat (ISSR) study evaluated a high diversity among the L. muscarium strains. The topology of the NJ-tree based on the ISSR-PCR markers has shown the genetic relationships with the support values 62-91% between L. muscarium isolates.

Detection of Verticillium species in Swedish soils using real-time PCR.

Verticillium species are soilborne plant pathogens, responsible for big yield losses worldwide. Here, we report improved procedures to generate DNA from Verticillium species imbedded in farm soils. Using new genomic sequence information, primers for V. dahliae, V. albo-atrum, V. tricorpus, and V. longisporum were designed. In a survey of 429 samples from intensively farmed soil of two Swedish regions, only V. dahliae and V. longisporum were identified. A bias towards V. longisporum (40%) was seen in the south, whereas V. dahliae was more frequent in the western region (19%). Analyses of soil and leaf samples from 20 sugar beet fields, where foliar wilting had been observed, revealed V. dahliae DNA in all leaf and soil samples and V. longisporum in 18 soil samples, illustrating host choice and longevity of the V. longisporum microsclerotia. This study demonstrates the applicability of new molecular diagnostic tools that are important for growers of variable crops.

The Emerging British Verticillium longisporum Population Consists of Aggressive Brassica Pathogens.

Verticillium longisporum is an economically important fungal pathogen of brassicaceous crops that originated from at least three hybridization events between different Verticillium spp., leading to the hybrid lineages A1/D1, A1/D2, and A1/D3. Isolates of lineage A1/D1 generally cause stem striping on oilseed rape (Brassica napus), which has recently been reported for the first time to occur in the United Kingdom. Intriguingly, the emerging U.K. population is distinct from the north-central European stem striping population. Little is known about the pathogenicity of the newly emerged U.K. population; hence, pathogenicity tests were executed to compare British isolates to previously characterized reference strains. In addition to the model plant Arabidopsis thaliana, the pathogenicity of four British isolates was assessed on four cultivars of three Brassica crop species: oilseed rape (Quartz and Incentive), cauliflower (Clapton), and Chinese cabbage (Hilton). To this end, vascular discoloration of the roots, plant biomass accumulations, and fungal stem colonization upon isolate infection were evaluated. The British isolates appeared to be remarkably aggressive, because plant biomass was significantly affected and severe vascular discoloration was observed. The British isolates were successful stem colonizers and the extent of fungal colonization negatively correlated with plant biomass of cauliflower and Quartz oilseed rape. However, in Quartz, the fungal colonization of A1/D1 isolates was significantly lower than that of the virulent reference isolate from lineage A1/D3, PD589. Moreover, despite levels of stem colonization similar to those of A1/D1 strains, PD589 did not cause significant disease on Incentive. Thus, A1/D1 isolates, including British isolates, are aggressive oilseed rape pathogens despite limited colonization levels in comparison with a virulent A1/D3 isolate.

Protoplast transformation as a potential platform for exploring gene function in Verticillium dahliae.

BACKGROUND: Large efforts have focused on screening for genes involved in the virulence and pathogenicity of Verticillium dahliae, a destructive fungal pathogen of numerous plant species that is difficult to control once the plant is infected. Although Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used for gene screening, a quick and easy method has been needed to facilitate transformation. RESULTS: High-quality protoplasts, with excellent regeneration efficiency (65 %) in TB3 broth (yeast extract 30 g, casamino acids 30 g and 200g sucrose in 1L H20), were generated using driselase (Sigma D-9515) and transformed with the GFP plasmid or linear GFP cassette using PEG or electroporation. PEG-mediated transformation yielded 600 transformants per microgram DNA for the linear GFP cassette and 250 for the GFP plasmid; electroporation resulted in 29 transformants per microgram DNA for the linear GFP cassette and 24 for the GFP plasmid. To determine whether short interfering RNAs (siRNAs) can be delivered to the protoplasts and used for silencing genes, we targeted the GFP gene of Vd-GFP (V. dahliae GFP strain obtained in this study) by delivering one of four different siRNAs-19-nt duplex with 2-nt 3' overhangs (siRNA-gfp1, siRNA-gfp2, siRNA-gfp3 and siRNA-gfp4)-into the Vd-GFP protoplasts using PEG-mediated transformation. Up to 100 % silencing of GFP was obtained with siRNA-gfp4; the other siRNAs were less effective (up to 10 % silencing). Verticillium transcription activator of adhesion (Vta2) gene of V. dahliae was also silenced with four siRNAs (siRNA-vta1, siRNA-vta2, siRNA-vta3 and siRNA-vta4) independently and together using the same approach; siRNA-vta1 had the highest silencing efficiency as assessed by colony diameter and quantitative real time PCR (qRT-PCR) analysis. CONCLUSION: Our quick, easy transformation method can be used to investigate the function of genes involved in growth, virulence and pathogenicity of V. dahliae.

Verticillium longisporum, the invisible threat to oilseed rape and other brassicaceous plant hosts.

INTRODUCTION: The causal agents of Verticillium wilts are globally distributed pathogens that cause significant crop losses every year. Most Verticillium wilts are caused by V. dahliae, which is pathogenic on a broad range of plant hosts, whereas other pathogenic Verticillium species have more restricted host ranges. In contrast, V. longisporum appears to prefer brassicaceous plants and poses an increasing problem to oilseed rape production. TAXONOMY: Kingdom Fungi; Phylum Ascomycota; Class Sordariomycetes; Subclass Hypocreomycetida; Family Plectosphaerellaceae; genus Verticillium. DISEASE SYMPTOMS: Dark unilateral stripes appear on the stems of apparently healthy looking oilseed rape plants at the end of the growing season. Microsclerotia are subsequently formed in the stem cortex beneath the epidermis. GENOME: Verticillium longisporum is the only non-haploid species in the Verticillium genus, as it is an amphidiploid hybrid that carries almost twice as much genetic material as the other Verticillium species as a result of interspecific hybridization. DISEASE MANAGEMENT: There is no effective fungicide treatment to control Verticillium diseases, and resistance breeding is the preferred strategy for disease management. However, only a few Verticillium wilt resistance genes have been identified, and monogenic resistance against V. longisporum has not yet been found. Quantitative resistance exists mainly in the Brassica C-genome of parental cabbage lines and may be introgressed in oilseed rape breeding lines. COMMON NAME: Oilseed rape colonized by V. longisporum does not develop wilting symptoms, and therefore the common name of Verticillium wilt is unsuitable for this crop. Therefore, we propose 'Verticillium stem striping' as the common name for Verticillium infections of oilseed rape.

Globally invading populations of the fungal plant pathogen Verticillium dahliae are dominated by multiple divergent lineages.

The spread of aggressive fungal pathogens into previously non-endemic regions is a major threat to plant health and food security. Analyses of the spatial and genetic structure of plant pathogens offer valuable insights into their origin, dispersal mechanisms and evolution, and have been useful to develop successful disease management strategies. Here, we elucidated the genetic diversity, population structure and demographic history of worldwide invasion of the ascomycete Verticillium dahliae, a soil-borne pathogen, using a global collection of 1100 isolates from multiple plant hosts and countries. Seven well-differentiated genetic clusters were revealed through discriminant analysis of principal components (DAPC), but no strong associations between these clusters and host/geographic origin of isolates were found. Analyses of clonal evolutionary relationships among multilocus genotypes with the eBURST algorithm and analyses of genetic distances revealed that genetic clusters represented several ancient evolutionary lineages with broad geographic distribution and wide host range. Comparison of different scenarios of demographic history using approximate Bayesian computations revealed the branching order among the different genetic clusters and lineages. The different lineages may represent incipient species, and this raises questions with respect to their evolutionary origin and the factors allowing their maintenance in the same areas and same hosts without evidence of admixture between them. Based on the above findings and the biology of V. dahliae, we conclude that anthropogenic movement has played an important role in spreading V. dahliae lineages. Our findings have implications for the development of management strategies such as quarantine measures and crop resistance breeding.

Diversity and biotransformative potential of endophytic fungi associated with the medicinal plant Kadsura angustifolia.

This study investigated the diversity and host component-transforming activity of endophytic fungi in medicinal plant Kadsura angustifolia. A total of 426 isolates obtained were grouped into 42 taxa belonging to Fungi Imperfecti (65.96%), Ascomycota (27.00%), Zygomycota (1.64%), Basidiomycota (0.47%) and Mycelia Sterilia (4.93%). The abundance, richness, and species composition of endophytic assemblages were significantly dependent on the tissue and the sampling site. Many phytopathogenic species associated with healthy K. angustifolia were found prevalent. Among them, Verticillium dahliae was dominant with 16.43% abundance. From 134 morphospecies selected, 39 showed remarkable biocatalytic activity and were further identified as species belonging to the genera Colletotrichum, Eupenicillium, Fusarium, Hypoxylon, Penicillium, Phomopsis, Trametes, Trichoderma, Umbelopsis, Verticillium and Xylaria on the basis of the sequence analysis of the internal transcribed spacer (ITS1-5.8S-ITS2). The results obtained in this work show that K. angustifolia is an interesting reservoir of pathogenic fungal species, and could be a community model for further ecological and evolutionary studies. Additionally, the converting potency screening of some endophytic fungi from this specific medicinal plant may provide an interesting niche on the search for novel biocatalysts.

The endophyte Verticillium Vt305 protects cauliflower against Verticillium wilt.

AIMS: To investigate the interaction between cauliflower and the isolate VerticilliumVt305, obtained from a field suppressive to Verticillium wilt of cauliflower, and to evaluate the ability of VerticilliumVt305 to control Verticillium wilt of cauliflower caused by V. longisporum. METHODS AND RESULTS: Single and combined inoculations of VerticilliumVt305 and V. longisporum were performed on cauliflower seedlings. Symptom development was evaluated, and fungal colonization was measured in the roots, hypocotyl and stem with real-time PCR. No symptoms were observed after single inoculation of VerticilliumVt305, although it colonized the plant tissues. Pre-inoculation of VerticilliumVt305 reduced symptom development and colonization of plant tissues by V. longisporum. CONCLUSIONS: VerticilliumVt305 is an endophyte on cauliflower plants and showed effective biological control of V. longisporum in controlled conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work can contribute to the development of a sustainable control measure of V. longisporum in Brassicaceae hosts, which is currently not available. Additionally, this study provides evidence for the different roles of Verticillium species present in the agro-ecosystem.

Verticillium systematics and evolution: how confusion impedes Verticillium wilt management and how to resolve it.

Verticillium wilts are important vascular wilt diseases that affect many crops and ornamentals in different regions of the world. Verticillium wilts are caused by members of the ascomycete genus Verticillium, a small group of 10 species that are related to the agents of anthracnose caused by Colletotrichum species. Verticillium has a long and complicated taxonomic history with controversies about species boundaries and long overlooked cryptic species, which confused and limited our knowledge of the biology of individual species. We first review the taxonomic history of Verticillium, provide an update and explanation of the current system of classification and compile host range and geographic distribution data for individual species from internal transcribed spacer (ITS) GenBank records. Using Verticillium as an example, we show that species names are a poor vehicle for archiving and retrieving information, and that species identifications should always be backed up by DNA sequence data and DNA extracts that are made publicly available. If such a system were made a prerequisite for publication, all species identifications could be evaluated retroactively, and our knowledge of the biology of individual species would be immune from taxonomic changes, controversy and misidentification. Adoption of this system would improve quarantine practices and the management of diseases caused by various plant pathogens.

Genome-wide identification, phylogeny and expression profile of vesicle fusion components in Verticillium dahliae.

Vesicular trafficking plays a crucial role in protein localization and movement, signal transduction, and multiple developmental processes in eukaryotic cells. Vesicle fusion is the final and key step in vesicle-mediated trafficking and mainly relies on SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the regulators including SM (Sec1/Munc18) family proteins, Rab GTPases and exocyst subunits. Verticillium dahliae is a widespread soil fungus that causes disruptive vascular diseases on a wide range of plants. To date, no genes involved in vesicular fusion process have been identified and characterized in V. dahliae. The recent publication of the draft genome sequence of V. dahliae allowed us to conduct a genome-wide identification, phylogeny and expression profile of genes encoding vesicular fusion components. Using compared genomics and phylogenetic methods, we identified 44 genes encoding vesicle fusion components in the V. dahliae genome. According to the structural features of their encoded proteins, the 44 V. dahliae genes were classified into 22 SNAREs (6 Qa-, 4 Qb-, 6 Qc-, 1 Qbc- and 5 R-types), 4 SM family proteins, 10 Rab GTPases and 8 exocyst proteins. Based on phylogeny and motif constitution analysis, orthologs of vesicle fusion component in filamentous fungi were generally clustered together into the same subclasses with well-supported bootstrap values. Analysis of the expression profiles of these genes indicated that many of them are significantly differentially expressed during vegetative growth and microsclerotia formation in V. dahliae. The analysis show that many components of vesicle fusion are well conserved in filamentous fungi and indicate that vesicle fusion plays a critical role in microsclerotia formation of smoke tree wilt fungus V. dahliae. The genome-wide identification and expression analysis of components involved in vesicle fusion should facilitate research in this gene family and give new insights toward elucidating their functions in growth, development and pathogenesis of V. dahliae.

Identification and Differentiation of Verticillium Species and V. longisporum Lineages by Simplex and Multiplex PCR Assays.

Accurate species identification is essential for effective plant disease management, but is challenging in fungi including Verticillium sensu stricto (Ascomycota, Sordariomycetes, Plectosphaerellaceae), a small genus of ten species that includes important plant pathogens. Here we present fifteen PCR assays for the identification of all recognized Verticillium species and the three lineages of the diploid hybrid V. longisporum. The assays were based on DNA sequence data from the ribosomal internal transcribed spacer region, and coding and non-coding regions of actin, elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase and tryptophan synthase genes. The eleven single target (simplex) PCR assays resulted in amplicons of diagnostic size for V. alfalfae, V. albo-atrum, V. dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii, V. nonalfalfae, V. nubilum, V. tricorpus, V. zaregamsianum, and Species A1 and Species D1, the two undescribed ancestors of V. longisporum. The four multiple target (multiplex) PCR assays simultaneously differentiated the species or lineages within the following four groups: Verticillium albo-atrum, V. alfalfae and V. nonalfalfae; Verticillium dahliae and V. longisporum lineages A1/D1, A1/D2 and A1/D3; Verticillium dahliae including V. longisporum lineage A1/D3, V. isaacii, V. klebahnii and V. tricorpus; Verticillium isaacii, V. klebahnii and V. tricorpus. Since V. dahliae is a parent of two of the three lineages of the diploid hybrid V. longisporum, no simplex PCR assay is able to differentiate V. dahliae from all V. longisporum lineages. PCR assays were tested with fungal DNA extracts from pure cultures, and were not evaluated for detection and quantification of Verticillium species from plant or soil samples. The DNA sequence alignments are provided and can be used for the design of additional primers.

Verticillium dahliae populations from mint and potato are genetically divergent with predominant haplotypes.

In total, 286 Verticillium dahliae isolates from mint, potato, and other hosts and substrates were characterized for mating type, vegetative compatibility group (VCG), and multilocus microsatellite haplotype to determine population genetic structure among populations infecting mint and potato. Populations from mint and potato fit a clonal reproductive model, with all isolates a single mating type (MAT1-2) and multiple occurrences of the same haplotypes. Haplotype H02 represented 88% of mint isolates and was primarily VCG2B, while haplotype H04 represented 70% of potato isolates and was primarily VCG4A. Haplotypes H02 and H04 typically caused severe disease on mint and potato, respectively, in greenhouse assays regardless of host origin. Principal coordinate analysis and analysis of molecular variance indicated that mint and potato populations were significantly genetically diverged (P = 0.02), and identification of private alleles and estimation of migration rates suggested restricted gene flow. Migration was detected between infected potato plants and seed tubers, infested tare soil, and field soils. Genetic differentiation of V. dahliae from mint and potato may be due to the occurrence of a single mating type and differences in VCG. Populations of V. dahliae in potato and mint were characterized by the presence of aggressive, clonally reproducing haplotypes which are widely distributed in commercial mint and potato production.

Transposable elements in phytopathogenic Verticillium spp.: insights into genome evolution and inter- and intra-specific diversification.

BACKGROUND: Verticillium dahliae (Vd) and Verticillium albo-atrum (Va) are cosmopolitan soil fungi causing very disruptive vascular diseases on a wide range of crop plants. To date, no sexual stage has been identified in either microorganism suggesting that somatic mutation is a major force in generating genetic diversity. Whole genome comparative analysis of the recently sequenced strains VdLs.17 and VaMs.102 revealed that non-random insertions of transposable elements (TEs) have contributed to the generation of four lineage-specific (LS) regions in VdLs.17. RESULTS: We present here a detailed analysis of Class I retrotransposons and Class II "cut-and-paste" DNA elements detected in the sequenced Verticillium genomes. We report also of their distribution in other Vd and Va isolates from various geographic origins. In VdLs.17, we identified and characterized 56 complete retrotransposons of the Gypsy-, Copia- and LINE-like types, as well as 34 full-length elements of the "cut-and-paste" superfamilies Tc1/mariner, Activator and Mutator. While Copia and Tc1/mariner were present in multiple identical copies, Activator and Mutator sequences were highly divergent. Most elements comprised complete ORFs, had matching ESTs and showed active transcription in response to stress treatment. Noticeably, we found evidences of repeat-induced point mutation (RIP) only in some of the Gypsy retroelements. While Copia-, Gypsy- and Tc1/mariner-like transposons were prominent, a large variation in presence of the other types of mobile elements was detected in the other Verticillium spp. strains surveyed. In particular, neither complete nor defective "cut-and-paste" TEs were found in VaMs.102. CONCLUSIONS: Copia-, Gypsy- and Tc1/mariner-like transposons are the most wide-spread TEs in the phytopathogens V. dahliae and V. albo-atrum. In VdLs.17, we identified several retroelements and "cut-and-paste" transposons still potentially active. Some of these elements have undergone diversification and subsequent selective amplification after introgression into the fungal genome. Others, such as the ripped Copias, have been potentially acquired by horizontal transfer. The observed biased TE insertion in gene-rich regions within an individual genome (VdLs.17) and the "patchy" distribution among different strains point to the mobile elements as major generators of Verticillium intra- and inter-specific genomic variation.

Development of an assay for rapid detection and quantification of Verticillium dahliae in soil.

ABSTRACT Verticillium dahliae is responsible for Verticillium wilt on a wide range of hosts, including strawberry, on which low soil inoculum densities can cause significant crop loss. Determination of inoculum density is currently done by soil plating but this can take 6 to 8 weeks to complete and delay the grower's ability to make planting decisions. To provide a faster means for estimating pathogen populations in the soil, a multiplexed TaqMan real-time polymerase chain reaction (PCR) assay based on the ribosomal DNA (rDNA) intergenic spacer (IGS) was developed for V. dahliae. The assay was specific for V. dahliae and included an internal control for evaluation of inhibition due to the presence of PCR inhibitors in DNA extracted from soil samples. An excellent correlation was observed in regression analysis (R(2) = 0.96) between real-time PCR results and inoculum densities determined by soil plating in a range of field soils with pathogen densities as low as 1 to 2 microsclerotia/g of soil. Variation in copy number of the rDNA was also evaluated among isolates by SYBR Green real-time PCR amplification of the V. dahliae-specific amplicon compared with amplification of several single-copy genes and was estimated to range from ≈24 to 73 copies per haploid genome, which translated into possible differences in results among isolates of ≈1.8 cycle thresholds. Analysis of the variation in results of V. dahliae quantification among extractions of the same soil sample indicated that assaying four replicate DNA extractions for each field sample would provide accurate results. A TaqMan assay also was developed to help identify colonies of V. tricorpus on soil plates.

Characterization, cloning, and heterologous expression of a subtilisin-like serine protease gene VlPr1 from Verticillium lecanii.

The entomopathogenic fungus Verticillium lecanii is a well-known biocontrol agent. V. lecanii produces subtilisin-like serine protease (Pr1), which is important in the biological control activity of some insect pests by degrading insect cuticles. In this study, a subtilisin-like serine protease gene VlPr1 was cloned from the fungus and the VlPr1 protein was expressed in Escherichia coli. The VlPr1 gene contains an open reading frame (ORF) interrupted by three short introns, and encodes a protein of 379 amino acids. Protein sequence analysis revealed high homology with subtilisin serine proteases. The molecular mass of the protease was 38 kDa, and the serine protease exhibited its maximal activity at 40°C and pH 9.0. Protease activity was also affected by Mg(2+) and Ca(2+) concentration. The protease showed inhibitory activity against several plant pathogens, especially towards Fusarium moniliforme.

Phylogenetics and taxonomy of the fungal vascular wilt pathogen Verticillium, with the descriptions of five new species.

Knowledge of pathogen biology and genetic diversity is a cornerstone of effective disease management, and accurate identification of the pathogen is a foundation of pathogen biology. Species names provide an ideal framework for storage and retrieval of relevant information, a system that is contingent on a clear understanding of species boundaries and consistent species identification. Verticillium, a genus of ascomycete fungi, contains important plant pathogens whose species boundaries have been ill defined. Using phylogenetic analyses, morphological investigations and comparisons to herbarium material and the literature, we established a taxonomic framework for Verticillium comprising ten species, five of which are new to science. We used a collection of 74 isolates representing much of the diversity of Verticillium, and phylogenetic analyses based on the ribosomal internal transcribed spacer region (ITS), partial sequences of the protein coding genes actin (ACT), elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD) and tryptophan synthase (TS). Combined analyses of the ACT, EF, GPD and TS datasets recognized two major groups within Verticillium, Clade Flavexudans and Clade Flavnonexudans, reflecting the respective production and absence of yellow hyphal pigments. Clade Flavexudans comprised V. albo-atrum and V. tricorpus as well as the new species V. zaregamsianum, V. isaacii and V. klebahnii, of which the latter two were morphologically indistinguishable from V. tricorpus but may differ in pathogenicity. Clade Flavnonexudans comprised V. nubilum, V. dahliae and V. longisporum, as well as the two new species V. alfalfae and V. nonalfalfae, which resembled the distantly related V. albo-atrum in morphology. Apart from the diploid hybrid V. longisporum, each of the ten species corresponded to a single clade in the phylogenetic tree comprising just one ex-type strain, thereby establishing a direct link to a name tied to a herbarium specimen. A morphology-based key is provided for identification to species or species groups.