The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: intraspecific heterogeneity within the intergenic spacer region

The complete DNA sequence of the nuclear ribosomal RNA gene complex of Verticillium dahliae: Intraspecific heterogeneity within the intergenic spacer region. Fungal Genetics and Biology 29, 19-27. The complete sequence of the nuclear ribosomal DNA gene complex of the phytopathogenic fungus Verticillium dahliae has been determined. The tandemly repeated unit was 7216 bp long and appears to be the shortest rDNA cluster described so far among filamentous fungi. Primer pairs were designed for amplification of the region spanning half of the 28S subunit, the intergenic spacer (IGS), and the 5' end of 18S subunit of a number of Verticillium strains, isolated from various hosts and geographic origins. Great heterogeneity was detected in the amplified products of the IGS region resulting in fragments varying from 1.6 to 2.0 kb. The majority of Verticillium isolates were classified into two groups with 1.6- and 1.7-kb amplified products, respectively. The former group included 31 V. dahliae, 7 V. longisporum, and 1 V. albo-atrum isolates, whereas the latter included 10 V. dahliae and 1 V. albo-atrum isolates. Sequence analysis of representative PCR products of the above groups identified a "hot-spot" region harboring most of larger insertions, whereas most of the small changes were due to transitions and transversions. One V. longisporum isolate with a 2.0-kb PCR product contained 13 perfectly conserved tandem repeats of 39 bp long. The presence of similar incomplete sequences in the corresponding regions of V. dahliae, V. longisporum, and V. albo-atrum isolates revealed a particular standard motif of insertions in the IGS region of the genus and is discussed.     

Molecular analysis of Japanese isolates of Verticillium dahliae and V. albo-airum

Sixteen isolates of different pathogenicity groups of the plant pathogen Verticillium dahliae and four isolates of V. albo-atrum from Japan were analysed by means of an RAPD (random amplified polymorphic DNA) method using a PCR (polymerase chain reaction). Verticillium dahliae and V. albo-atrum could be distinguished by RAPD analysis. Four pathogenicity groups of V. dahliae could also be classified to a certain extent by this method. Similarities and differences in banding patterns obtained by RAPD may be a useful molecular tool in phylogenetic studies of the pathogenicity groups.     

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.     

DNA sequence analysis of conserved genes reveals hybridization events that increase genetic diversity in Verticillium dahliae

The hybrid origin of a Verticillium dahliae isolate belonging to the vegetative compatibility group (VCG) 3 is reported in this work. Moreover, new data supporting the hybrid origin of two V. dahliae var. longisporum (VDLSP) isolates are provided as well as information about putative parentals. Thus, isolates of VDLSP and V. dahliae VCG3 were found harboring multiple sequences of actin (Act), β-tubulin (β-tub), calmodulin (Cal) and histone 3 (H3) genes. Phylogenetic analysis of these sequences, the internal transcribed sequences (ITS-1 and ITS-2) of the rRNA genes and of a V. dahliae-specific sequence provided molecular evidences for the interspecific hybrid origin of those isolates. Sequence analysis suggests that some of VDLSP isolates may have resulted from hybridization events between a V. dahliae isolate of VCG1 and/or VCG4A and, probably, a closely related taxon to Verticillium alboatrum but not this one. Similarly, phylogenetic analysis and PCR markers indicated that a V. dahliae VCG3 isolate might have arisen from a hybridization event between a V. dahliae VCG1B isolate and as yet unidentified parent. This second parental probably does not belong to the Verticillium genus according to the gene sequences dissimilarities found between the VCG3 isolate and Verticillium spp. These results suggest an important role of parasexuality in diversity and evolution in the genus Verticillium and show that interspecific hybrids within this genus may not be rare in nature.     

Genetic,morphological, and virulence characterization of the entomopathogenic fungus Verticillium lecanii

In order to clarify relationships among genetic diversity, virulence, and other characteristics of conidia, 46 isolates of Verticillium lecanii from various hosts and geographical locations were examined. The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of ribosomal DNA (rDNA), mitochondrial small subunit rDNA (mt-SrDNA) and beta-tubulin were analyzed by PCR-RFLP. PCR-single stranded conformational polymorphism (SSCP) was performed on regions of the mitochondrial large subunit rDNA, mt-SrDNA, beta-tubulin and histone 4. There were no relationships among the results of RFLP, SSCP, isolation source, and location. However, amplified product size of IGS did have relationships with conidia size and sporulation. Six isolates with 4.0-kb IGS products had large conidia dimensions, and yielded low numbers of conidia compared with other isolates. Three out of the six isolates were high virulence (over 90%) against green peach aphids. Furthermore, double-stranded RNA (dsRNA) was detected in 22 out of 35 V. lecanii isolates and related with the amplicon sizes of IGS, though not with virulence or isolation location. Isolates containing dsRNA were divided into six distinct types based on banding pattern. These data demonstrate the level of genetic diversity of V. lecanii, and suggest relations among the genetic properties and conidial morphology.     

The ascomycete Verticillium longisporum is a hybrid and a plant pathogen with an expanded host range

Hybridization plays a central role in plant evolution, but its overall importance in fungi is unknown. New plant pathogens are thought to arise by hybridization between formerly separated fungal species. Evolution of hybrid plant pathogens from non-pathogenic ancestors in the fungal-like protist Phytophthora has been demonstrated, but in fungi, the most important group of plant pathogens, there are few well-characterized examples of hybrids. We focused our attention on the hybrid and plant pathogen Verticillium longisporum, the causal agent of the Verticillium wilt disease in crucifer crops. In order to address questions related to the evolutionary origin of V. longisporum, we used phylogenetic analyses of seven nuclear loci and a dataset of 203 isolates of V. longisporum, V. dahliae and related species. We confirmed that V. longisporum was diploid, and originated three different times, involving four different lineages and three different parental species. All hybrids shared a common parent, species A1, that hybridized respectively with species D1, V. dahliae lineage D2 and V. dahliae lineage D3, to give rise to three different lineages of V. longisporum. Species A1 and species D1 constituted as yet unknown taxa. Verticillium longisporum likely originated recently, as each V. longisporum lineage was genetically homogenous, and comprised species A1 alleles that were identical across lineages.     

Interfamily transfer of tomato Ve1 mediates Verticillium resistance in Arabidopsis

Vascular wilts caused by soil-borne fungal species of the Verticillium genus are devastating plant diseases. The most common species, Verticillium dahliae and Verticillium albo-atrum, have broad host ranges and are notoriously difficult to control. Therefore, genetic resistance is the preferred method for disease control. Only from tomato (Solanum lycopersicum) has a Verticillium resistance locus been cloned, comprising the Ve1 gene that encodes a receptor-like protein-type cell surface receptor. Due to lack of a suitable model for receptor-like protein (RLP)-mediated resistance signaling in Arabidopsis (Arabidopsis thaliana), so far relatively little is known about RLP signaling in pathogen resistance. Here, we show that Ve1 remains fully functional after interfamily transfer to Arabidopsis and that Ve1-transgenic Arabidopsis is resistant to race 1 but not to race 2 strains of V. dahliae and V. albo-atrum, nor to the Brassicaceae-specific pathogen Verticillium longisporum. Furthermore, we show that signaling components utilized by Ve1 in Arabidopsis to establish Verticillium resistance overlap with those required in tomato and include SERK3/BAK1, EDS1, and NDR1, which strongly suggests that critical components for resistance signaling are conserved. We subsequently investigated the requirement of SERK family members for Ve1 resistance in Arabidopsis, revealing that SERK1 is required in addition to SERK3/BAK1. Using virus-induced gene silencing, the requirement of SERK1 for Ve1-mediated resistance was confirmed in tomato. Moreover, we show the requirement of SERK1 for resistance against the foliar fungal pathogen Cladosporium fulvum mediated by the RLP Cf-4. Our results demonstrate that Arabidopsis can be used as model to unravel the genetics of Ve1-mediated resistance.     

The Arabidopsis thaliana DNA-binding protein AHL19 mediates verticillium wilt resistance

Verticillium spp. are destructive soilborne fungal pathogens that cause vascular wilt diseases in a wide range of plant species. Verticillium wilts are particularly notorious, and genetic resistance in crop plants is the most favorable means of disease control. In a gain-of-function screen using an activation-tagged Arabidopsis mutant collection, we identified four mutants, A1 to A4, which displayed enhanced resistance toward the vascular wilt species Verticillium dahliae, V. albo-atrum and V. longisporum but not to Fusarium oxysporum f. sp. raphani. Further testing revealed that mutant A2 displayed enhanced Ralstonia solanacearum resistance, while mutants A1 and A3 were more susceptible toward Pseudomonas syringae pv. tomato. Identification of the activation tag insertion site in the A1 mutant revealed an insertion in close proximity to the gene encoding AHL19, which was constitutively expressed in the mutant. AHL19 knock-out alleles were found to display enhanced Verticillium susceptibility whereas overexpression of AHL19 resulted in enhanced Verticillium resistance, showing that AHL19 acts as a positive regulator of plant defense.     

Presence of a 20 bp insertion/deletion in the ITS1 region of Verticillium lecanii

Polymerase chain reaction (PCR) amplification of the complete ribosomal RNA internally transcribed spacer (ITS) region of 36 isolates of Verticillium lecanii and related species gave a single 620 bp product in 31 isolates. Five isolates received as V. lecanii, however, gave a single product of 600 bp. Restriction fragment analysis of the PCR products from all isolates gave consistent patterns for the 31 isolates with a 620 bp product. The five isolates with the 600 bp product showed only minor discrepancies to these, generally related to the size of only one restriction fragment. The total ITS region was sequenced from 10 typical 620 bp isolates and one 600 bp isolate. Sequence variation between the isolates varied from 0 to 14.5%, and the 20 bp size discrepancy was found to relate to an insertion or deletion in the centre of the ITS1 region.     

Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium

Abstract Genomic DNA was extracted from seven species of Verticillium and digested with the restriction endonucleases Eco RI or Hae III. Hybridization with an homologous V. albo-atrum ribosomal RNA gene probe revealed restriction fragment length polymorphisms (RFLPs) which could differentiate V. lateritium, V. lecanii, V. nigrescens, V. nubilum and V. tricorpus . Digestion with Eco RI did not provide RFLPs which could distinguish between V. albo-atrum and V. dahliae . Digestion of genomic and mitochondrial DNA with Hae III showed distinctive patterns on ethidium bromide gels which allowed each species to be distinguished. Some intra-species variation in patterns occurred and a combination of mitochondrial and ribosomal RNA gene complex RFLPs has potential as an aid for the characterization of species and sub-species populations in the genes Verticillium .     

Characterization of Moroccan Isolates of Verticillium dahliaeKleb Using RAPD Markers

Isolates of Verticillium dahliae were sampled from different olive tree orchards in Morocco. These olive trees were located in different commercial culture locations in southern, central and northern Morocco. The isolates were characterized using genetic markers obtained after their DNA PCR amplification with random amplified polymorphic DNA (RAPD) primers. Among the 40 primers tested, 10 generated a total of 66 polymorphic fragments. Among the 38 isolates of V. dahliae tested, RAPD markers were successful in the characterization of groups based on their geographic origin. With the exception of one specific isolate, no correlation could be established among the isolates, based on the morphological appearance of the colony in culture.     

Cloning and characterization of the ribosomal RNA gene complex from the plant pathogen Verticillium albo-atrum

Abstract The DNA coding for the ribosomal RNA gene complex (rDNA) has been cloned from isolate 621P(PV1) of Verticillium albo-atrum which is pathogenic for hops ( Humulus lupulus ). The rDNA was mapped using a range of restriction enzymes. The functional units of the intergenic spacer (IGS), 18S, 5.8S and 25S regions were located by hybridization to specific gene probes from the rDNA complex of Aspergillus nidulans . The start points of the 18S and 5.8S regions were confirmed by partial sequencing. A genomic restriction enzyme map was found to be identical with the map of the cloned DNA. The rDNA repeat was 7.6 kb in length and this was used as an homologous probe to analyse the size of the repeat in 18 hop isolates of V. albo-atrum strains and in one isolate from alfalfa (Luc2). All of the isolates had a repeat size of 7.6 kb except for Luc2 where the rDNA complex was 8.4 kb.     

Molecular Characterization of the Plant Pathogen Verticillium dahliae Kleb. Using RAPD-PCR and Sequencing of the 18SrRNA-Gene

Thirty-four isolates of Verticillium dahliae Kleb. from nine different genera of dicotyledonous host plants and a broad range of geographic regions were analysed genotypically, Random amplified polymorphic DNA (RAPD) markers were used for the estimation of the genetic variability within the species. Using four primers for the analysis, 79 distinct fragments were obtained. The derived phenogram clustered the isolates in two main groups; one consisted almost entirely of V. dahliae isolates from oilseed rape ( Brassica napus napus ), the other group comprised isolates from a wide range of host plants. No correlation between geographic location of the isolates and the RAPD-pattern was observed.
Sequencing of the gene for the 18SrRNA and calculation of the phylogenetic tree integrated the deuteromycetous fungus V. dahliae into the sexual system of the filamentous ascomycetes.     

Detection and genotyping of Giardia intestinalis isolates using intergenic spacers(IGS)-based PCR

Giardia intestinalis infections arise primarily from contaminated food or water. Zoonotic transmission is possible, and at least 7 major assemblages including 2 assemblages recovered from humans have been identified. The determination of the genotype of G. intestinalis is useful not only for assessing the correlation of clinical symptoms and genotypes, but also for finding the infection route and its causative agent in epidemiological studies. In this study, methods to identify the genotypes more specifically than the known 2 genotypes recovered from humans have been developed using the intergenic spacer (IGS) region of rDNA. The IGS region contains varying sequences and is thus suitable for comparing isolates once they are classified as the same strain. Genomic DNA was extracted from cysts isolated from the feces of 5 Chinese, 2 Laotians and 2 Koreans infected with G. intestinalis and the trophozoites of WB, K1, and GS strains cultured in the laboratory, respectively. The rDNA containing the IGS region was amplified by PCR and cloned. The nucleotide sequence of the 3' end of IGS region was determined and examined by multiple alignment and phylogenetic analysis. Based on the nucleotide sequence of the IGS region, 13 G. intestinalis isolates were classified to assemblages A and B, and assemblage A was subdivided into A1 and A2. Then, the primers specific to each assemblage were designed, and PCR was performed using those primers. It detected as little as 10 pg of DNA, and the PCR amplified products with the specific length to each assemblage (A1, 176 bp; A2, 261 bp; B, 319 bp) were found. The PCR specific to 3 assemblages of G. intestinalis did not react with other bacteria or protozoans, and it did not react with G. intestinalis isolates obtained from dogs and rats. It was thus confirmed that by applying this PCR method amplifying the IGS region, the detection of G. intestinalis and its genotyping can be determined simultaneously.     

WILT OF LUCERNE CAUSED BY SPECIES OF VERTICILLIUM

The incidence and symptoms of a disease of lucerne induced by Verticillium spp. are much less severe immediately after the crop has been first cut than later when the crop has matured. Infection becomes more marked as the season advances and the age of the ley increases, and cuts taken in the third harvest year are proving unprofitable.
In cross-inoculation studies the causal organisms of lucerne wilt, V. albo-atrum and V. dahliae prove to be only mild pathogens to hop, potato, tomato and antirrhinum, and are not pathogenic to sainfoin and clover. The last two may therefore be cultivated as fodder crops in those areas most severely affected by lucerne wilt, but the cultivation of potatoes after lucerne should be discouraged, particularly if lucerne is to be sown in future years, since the potatoes might build up a reservoir of inoculum of the lucerne pathogen. In studies of host-parasite relations it is shown that V. albo-atrum and V. dahliae from lucerne, V. albo-atrum from hop, V. tricorpus from tomato and V. nigrescens and V. nubilum from potato vary in their effects upon lucerne seedlings growing on agar media, V. albo-atrum from lucerne being, as in the field, the most virulent and the only one to reach the xylem vessels under the conditions of the experiments.
The dressings Agrosan-GN (containing mercury) and Fernasan (containing thiram) are toxic both to spores of Verticillium on lucerne seed coats and to infected plant material carried with seed, and although they are also toxic in vitro to Rhizobium mellelotus this did not prevent dressed seeds which had been inoculated with the bacterium from developing into healthy seedlings bearing normal root-nodules.     

THE EFFECTS OF NITROGEN SUPPLY UPON THE VERTICILLIUM WILT OF ANTIRRHINUM

Isolates of Verticillium albo-atrum, V. dahliae and V. nigrescens grown on media adequately supplied with sodium nitrate induced wilt rather more rapidly in antirrhinum plants growing in soils with a normal and an excessive amount of nitrogen than in plants in nitrogen-deficient soil, though plants became diseased in all soils. Similarly treated isolates of V. nubilum and V. tricorpus induced a greater incidence of wilt in plants in soil supplied with heavy dressings of organic nitrogenous fertilizer than in plants in soil deficient in nitrogen, although V. tricorpus from a medium containing much sodium nitrate, in contrast to V. nubilum , was pathogenic to plants in such deficient soil. The ability of the nitrogen-starved isolates to penetrate the host plant was significantly diminished, and even when wound-inoculated into stems their effect upon the host was much reduced.     

Successful strategy for the selection of new strawberry-associated rhizobacteria antagonistic to Verticillium wilt

In order to isolate and characterize new strawberry-associated bacteria antagonistic to the soil-borne pathogenic fungus Verticillium dahliae Kleb., rhizobacterial populations from two different strawberry species, Greenish Strawberry (Fragaria viridis) and Garden Strawberry (F. x ananassa) obtained after plating onto King's B and glycerol-arginine agar, were screened for in vitro antagonism toward V. dahliae. The proportion of isolates with antifungal activity determined in in vitro assay against V. dahliae was higher for the Garden Strawberry than for the Greenish Strawberry. From 300 isolates, 20 isolates with strong antifungal activity were selected characterized by physiological profiling and molecular fingerprinting methods. Diversity among the isolates was characterized with molecular fingerprints using amplified ribosomal DNA restriction analysis (ARDRA) and the more discriminating BOX-PCR fingerprint method. The physiological profiles were well correlated with molecular fingerprinting pattern analysis. Significant reduction of Verticillium wilt by bacterial dipping bath treatment was shown in the greenhouse and in fields naturally infested by V. dahliae. The relative increase of yield ranged from 117% (Streptomyces albidoflavus S1) to 344% (Pseudomonas fluorescens P10) in greenhouse trials, and 113% (Streptomyces albidoflavus S1) to 247% (Pseudomonas fluorescens P6) in field trials. Evaluation resulted in the selection of three effective biocontrol agents (Pseudomonas fluorescens P6, P10, and Streptomyces diastatochromogenes S9) antagonistic to the Verticillium wilt pathogen.     

WILT OF LUCERNE CAUSED BY SPECIES OF VERTICILLIUM

A wilt disease of lucerne caused by species of Verticillium is described: at twenty-eight disease areas in England and Wales the pathogen was V. albo-atrum , while at one site it was V. dahliae . Both pathogens form superficial conidia on the basal regions of infected stems. It is shown that V. albo-atrum is introduced into a new area in contaminated plant material harvested with the seed from an infected crop. Rapid secondary spread of the disease follows the dissemination of spores from infected stems, and by contact of these and transported fragments of diseased tissues with the wounded surfaces of recently cut lucerne plants. It is recommended that seed should be collected only from healthy crops and that machinery, footwear, etc. should be disinfected before leaving a site of infection.
Manurial trials showed that the incidence of wilt induced by V. albo-atrum was very severe under all soil conditions tested, whereas V. dahliae is a virulent pathogen only to plants in soil rich in superphosphate. Generally the more vigorous the growth of the lucerne–in soils rich in potash and hoof and horn–the more rapid is both the onset of wilt and the resulting secondary spread of the disease throughout the crop.