Dahliae Strain Research Articles

English

Verticillium dahliae is a causal agent of vascular wilt smoke-tree Cotinus coggygrial. The genetic transformation of V. dahliae has been carried out by Agrobacterium and polyethylene glycol-mediated transformation. Due to technical simplicity and efficiency, PEG-mediated transformation is the most commonly used method for genetic transformation in filamentous fungi. However, polyethylene glycol (PEG)-mediated transformation is still deficient in V. dahliae. In this study, we studied the main factors affecting the release of protoplasts from mycelia of V. dahliae such as lytic enzymes, enzymolysis buffers and incubation time. The optimal combination for protoplast preparation from smoke-tree wilt fungus V. dahliae can release 2.8 × 106 protoplasts/ml from fresh mycelia of smoke-tree wilt fungus. The majority of spheroplast contained nuclei and well regenerated. After PEG mediated transformation, green fluorescent protein (GFP) expressing V. dahliae strains were obtained. Therefore, this study provides a protocol suitable for protoplast preparation and transformation of smoke-tree wilt fungusV. dahliae.   Key words: Verticillium dahliae, smoke-tree wilt, protoplast preparation, polyethylene glycol (PEG) transformation

Evidence for Functional Diversification Within a Fungal NEP1-Like Protein Family

In this study, we functionally analyzed the gene family encoding necrosis- and ethylene-inducing-like proteins (NLP) of the vascular wilt pathogen Verticillium dahliae. We show that the composition of the NLP gene family varies little among V. dahliae isolates. The cytotoxic activity of NLP family members of a tomato-pathogenic V. dahliae strain was determined, demonstrating that only two of the seven NLP induced plant cell death. The genes encoding these cytotoxic NLP were found to be induced in V. dahliae upon colonization of tomato. Interestingly, targeted deletion of either of the two genes in V. dahliae significantly compromised virulence on tomato as well as on Arabidopsis plants, whereas deletion of only one of the two genes affected virulence on Nicotiana benthamiana. This could be attributed to differential induction of the two NLP genes in V. dahliae upon N. benthamiana colonization, revealing that the in planta induction of NLP genes varies between plant hosts. Intriguingly, one of the NLP genes appears to also affect vegetative growth and conidiospore production, because the corresponding deletion strain produced significantly fewer conidiospores and developed extensive aerial mycelium. In conclusion, we demonstrate that the expanded V. dahliae NLP family shows functional diversification, revealing not only differential cytotoxicity between family members but also that the cytotoxic NLP play a role in vegetative growth and asexual reproduction in addition to their contribution to virulence.

Genetic variability of tolerance to Verticillium albo-atrum and Verticillium dahliae in Medicago truncatula

Verticillium wilt caused by Verticillium albo-atrum and Verticillium dahliae is responsible for yield losses in many economically important crops. The capacity of pathogenic fungi to adapt to new hosts is a well-known threat to the durability of resistant crop varieties. In the present work, 25 Medicago truncatula genotypes from a core collection and six V. albo-atrum and V. dahliae strains were used to study the potential of non-host Verticillium strains isolated from different plant species to infect this legume plant and the plant's susceptibility to the pathogens. The experiment was designed as factorial with randomised complete blocks and with three repetitions. The wilt symptoms caused by V. albo-atrum and V. dahliae were scored on a disease index scale from 0 to 4, during 30 days after inoculation of 10-day-old plantlets. Disease severity was quantified by maximum symptom scores (MSS) and areas under the disease progress curves (AUDPC), which integrate the time course of symptom development. Highly significant differences were observed among plant genotypes and fungal strains, and their interaction was also significant. The correlation between MSS and AUDPC was 0.86. The most severe symptoms were caused by the alfalfa strain V. albo-atrum V31-2 and the least severe by V. dahliae JR2, as shown by mean values obtained on the 25 genotypes. M. truncatula genotype TN8.3 was the most susceptible genotype by mean values obtained with the six fungal strains, whereas F11013-3, F83005.9 and DZA45.6 were highly resistant to all strains studied. The results can be used to choose parents for studying the genetics of resistance in Medicago truncatula to Verticillium strains with different levels of aggressiveness.

Colonization in cotton plants by a green fluorescent protein labelled strain of Verticillium dahliae

Verticillium wilt of cotton (Gossypium hirsutum) is a widespread and destructive disease caused by the soil-borne fungal pathogen Verticillium dahliae. In this study, a green fluorescent protein (GFP) labelled V. dahliae strain (TV7) was obtained by transforming gfp into defoliating strain V991. Strain TV7 was used to study infection and colonization of wilt resistant cotton cultivar Zhongzhimian KV1 and susceptible cultivar 861 with the aid of confocal laser scanning microscopy. The results showed that initial infection and colonization of V. dahliae in Zhongzhimian KV1 and 861 were similar. Conidia and hyphal colonies formed and penetrated in the root meristematic and elongation zones and in the conjunction of the lateral and main roots. The invaded conidia started to germinate by 2 hpi (hours post-inoculation), penetrated into the root cortex and vascular bundles, eventually colonized in the stem xylem vessels and grew restrictedly in the individual tracheae of both resistant and susceptible cultivars. Moreover, pathogen DNA could be detected by qPCR in roots and stems of both cultivars, but its content in the wilt susceptible cultivar 861 was much higher than that in the wilt resistant cultivar Zhongzhimian KV1. The results indicated that the resistant cultivar has ability to suppress V. dahliae reproduction.

Comparative expression analysis in susceptible and resistant Gossypium hirsutum responding to Verticillium dahliae infection by cDNA-AFLP

Verticillium wilt caused by the soil-borne fungus Verticillium dahliae (V. dahliae), is a devastating disease of cotton, leading to serious loss of lint yield worldwide. To study its resistance responses in both susceptible (Ejing No. 1) and resistant (NJ0703 and NJ0705) upland cotton cultivars (Gossypium hirsutum), cDNA-AFLP analysis was used to identify differentially expressed transcripts from resistant and susceptible cultivars that were infected with V. dahliae strain V991. A total of 83 transcript-derived fragments (TDFs) were obtained using 64 pairs of primer combinations. Interestingly, none of the differentially expressed fragments identified from susceptible cultivar Ejing No. 1 was found from resistant cultivars (NJ0703 and NJ0705). However, there were some similarities between NJ0703 (R) and NJ0705 (R), and 10 differentially expressed fragments were identified from both two resistant cultivars. The results indicated that the susceptible and resistant upland cottons responded differently to Verticillium infection. Moreover, the expression of transcripts was further validated through quantitative real-time PCR. Data showed that the activation of the transcripts was rapid and transient upon V. dahliae infection.

Cotton gene expression profiles in resistant Gossypium hirsutum cv. Zhongzhimian KV1 responding to Verticillium dahliae strain V991 infection

Verticillium wilt of cotton (Gossypium hirsutum) is a widespread and destructive disease that is caused by the soil-borne fungus pathogen Verticillium dahliae (V. dahliae). To study the molecular mechanism in wilt tolerance, suppression subtractive hybridization (SSH) and dot blot techniques were used to identify the specifically expressed genes in a superior wilt-resistant cotton cultivar (G. hirsutum cv. Zhongzhimian KV1) after inoculation with pathogen. cDNAs from the root tissues of Zhongzhimian KV1 inoculated with V. dahliae strain V991 or water mock were used to construct the libraries that contain 4800 clones. Based on the results from dot blot analysis, 147 clones were clearly induced by V. dahliae and selected from the SSH libraries for sequencing. A total of 92 up-regulated and 7 down-regulated non-redundant expressed sequences tags (ESTs) were identified as disease responsive genes and classified into 9 functional groups. Two important clues regarding wilt-resistant G. hirsutum were obtained from this study. One was Bet v 1 family; the other was UbI gene family that may play an important role in the defense reaction against Verticillium wilt. The result from real-time quantitative reverse transcription polymerase chain reaction showed that these genes were activated quickly and transiently after inoculation with V. dahliae.

Lignin metabolism has a central role in the resistance of cotton to the wilt fungus Verticillium dahliae as revealed by RNA-Seq-dependent transcriptional analysis and histochemistry.

The incompatible pathosystem between resistant cotton (Gossypium barbadense cv. 7124) and Verticillium dahliae strain V991 was used to study the cotton transcriptome changes after pathogen inoculation by RNA-Seq. Of 32 774 genes detected by mapping the tags to assembly cotton contigs, 3442 defence-responsive genes were identified. Gene cluster analyses and functional assignments of differentially expressed genes indicated a significant transcriptional complexity. Quantitative real-time PCR (qPCR) was performed on selected genes with different expression levels and functional assignments to demonstrate the utility of RNA-Seq for gene expression profiles during the cotton defence response. Detailed elucidation of responses of leucine-rich repeat receptor-like kinases (LRR-RLKs), phytohormone signalling-related genes, and transcription factors described the interplay of signals that allowed the plant to fine-tune defence responses. On the basis of global gene regulation of phenylpropanoid metabolism-related genes, phenylpropanoid metabolism was deduced to be involved in the cotton defence response. A closer look at the expression of these genes, enzyme activity, and lignin levels revealed differences between resistant and susceptible cotton plants. Both types of plants showed an increased level of expression of lignin synthesis-related genes and increased phenylalanine-ammonia lyase (PAL) and peroxidase (POD) enzyme activity after inoculation with V. dahliae, but the increase was greater and faster in the resistant line. Histochemical analysis of lignin revealed that the resistant cotton not only retains its vascular structure, but also accumulates high levels of lignin. Furthermore, quantitative analysis demonstrated increased lignification and cross-linking of lignin in resistant cotton stems. Overall, a critical role for lignin was believed to contribute to the resistance of cotton to disease.

The purification and characterization of a novel hypersensitive-like response-inducing elicitor from Verticillium dahliae that induces resistance responses in tobacco

PevD1, a novel protein elicitor from the pathogenic cotton verticillium wilt fungus, Verticillium dahliae, induced a hypersensitive response in tobacco plants. In this paper, the elicitor was purified and analyzed using de novo sequencing. The protein-encoding pevD1 gene consists of a 468-bp open reading frame that produces a polypeptide of 155 amino acids, with a theoretical molecular weight of 16.23 kDa. The sequence of elicitor protein PevD1 was matched to the genomic sequence (GenBank accession no. ABJE 01000445.1) of a putative protein from V. dahliae strain vdls.17, but a function had not yet been reported. The pevD1 gene was expressed in Escherichia coli, and the recombinant protein was characterized for its ability to confer systemic acquired resistance to tobacco mosaic virus (TMV). Recombinant PevD1-treated plants exhibited enhanced systemic resistance compared to control, including a significant reduction in the number and size of TMV lesions on tobacco leaves. The elicitor protein-induced hydrogen peroxide production, extracellular-medium alkalization, callose deposition, phenolics metabolism, and lignin synthesis in tobacco. Our results demonstrate that elicitor-PevD1 triggers defense responses in intact tobacco plants.

Symptom development, pathogen isolation and Real-Time QPCR quantification as factors for evaluating the resistance of olive cultivars to Verticillium pathotypes

Verticillium wilt is the most serious olive disease in the Mediterranean countries and worldwide. The most effective control strategy is the use of resistant cultivars. However, limited information is available about the level and source of resistance in most of the olive cultivars and there are no published data using microsclerotia, the resting structures of Verticillium dahliae, as the infective inoculum. In the present study, we correlated symptomatology and the presence of the fungus along with the DNA relative amount (molecules μl−1) of a defoliating (D) and a non-defoliating (ND) V. dahliae strain in the susceptible cv. Amfissis and the tolerant cvs Kalamon and Koroneiki, as quantified by the Real-Time QPCR technology. The viability of the pathogen in the plant tissues was confirmed by isolating the fungus on PDA plates, while symptom assessment proved the correlation between the DNA relative amount of V. dahliae in plant tissues and cultivar susceptibility. It was further demonstrated that the D and ND strains were present at a significantly higher level in cv. Amfissis than in cvs Kalamon and Koroneiki. It was finally observed that the relative amount of the pathogen in roots was lower than in stems and shoots and declined in plant tissues over time. These data constitute a valuable contribution in evaluating resistance of olive cultivars or olive root-stocks to V. dahliae pathotypes.

Mode of action of a non-pathogenic Fusarium oxysporum strain against Verticillium dahliae using Real Time QPCR analysis and biomarker transformation

Verticillium wilt is a devastating disease of a wide range of herbaceous and woody plant hosts. It is incited by the soilborne fungus Verticillium dahliae. Management strategies are mainly focused on preventive measures. In a previous study, the efficacy of a non-pathogenic Fusarium oxysporum strain, designated as F2, isolated from a suppressive compost amendment, has been reported to reduce Verticillium wilt symptom development in eggplants under greenhouse and field conditions; in addition, antibiosis or parasitism were ruled out by using a dual culture test. In the present study, we investigated the mode of action of F2 against V. dahliae. For this purpose, the F2 and V. dahliae strains were transformed with the EGFP and DsRed2 reporter genes respectively, so as to visualize their presence on the root surface of eggplants. In addition, the ramification of both fungi into the plant vascular system was monitored by Real Time QPCR analysis. It was shown that F2 colonizes the root surface along the intercellular junctions excluding V. dahliae from the same ecological niche. In parallel, QPCR analysis showed that application of F2 reduces the levels of V. dahliae vascular colonization along with the disease severity. In a split root experiment it was demonstrated that F2 does not trigger the defense mechanisms of eggplants against V. dahliae. Therefore, it seems that competition for space or nutrients on the root surface are the main mechanism of action of F2 against V. dahliae.

Isolation and characterization of resistance and defense gene analogs in cotton (Gossypium barbadense L.)

Plant disease resistance gene (R gene) and defense response gene encode some conserved motifs. In the present work, a PCR strategy was used to clone resistance gene analogs (RGAs) and defense gene analogs (DGAs) from Sea-island cotton variety Hai7124 using oligonucleotide primers based on the nucleotide-binding site (NBS) and serine/threonine kinase (STK) in the R-gene and pathogenesis-related proteins of class 2 (PR2) of defense response gene. 79 NBS sequences, 21 STK sequences and 11 DGAs were cloned from disease-resistance cotton. Phylogenic analysis of 79 NBS-RGAs and NBS-RGAs nucleotide sequences of cotton already deposited in GenBank identified one new sub-cluster. The deduced amino acid sequences of NBS-RGAs and STK-RGAs were divided into two distinct groups respectively: Toll/Interleukin-1 receptor (TIR) group and non-TIR group, A group and B group. The expression of RGAs and DGAs having consecutive open reading frame (ORF) was also investigated and it was found that 6 NBS-RGAs and 1 STK-RGA were induced, and 1 DGA was up-regulated by infection of Verticillium dahliae strain VD8. 4 TIR-NBS-RGAs and 4 non-TIR-NBS-RGAs were arbitrarily used as probes for Southern-blotting. There existed 2-10 blotted bands. In addition, since three non-TIR-NBS-RGAs have the same hybridization pattern, we conjecture that these three RGAs form a cluster distribution in the genome.

Genetic diversity of Verticillium dahliae isolates from olive trees in Algeria

Verticillium wilt of olive trees (Olea europaea L.), a wilt caused by the soil-borne fungus Verticillium dahliae (Kleb), is one of the most serious diseases in Algerian olive groves. To assess the pathogenic and genetic diversity of olive-infecting V. dahliae populations in Algeria, orchards from the two main olive-producing regions (north-western Algeria and Kabylia) were sampled and 27 V. dahliae isolates were recovered. For purposes of comparison, V. dahliae strains from France and Syria were added to the analysis. By means of PCR primers that specifically discriminate between defoliating (D) and non-defoliating (ND) V. dahliae pathotypes it was shown that all V. dahliae isolates belonged to the ND pathotype. The amount of genetic variation between the 43 isolates was assessed by random amplification of polymorphic DNA (RAPD). A total of 16 RAPD haplotypes were found on the basis of the presence or absence of 25 polymorphic DNA fragments. Genotypic diversity between the 27 Algerian isolates was low, with two RAPD haplotypes accounting for 70% of all isolates. Genotypic diversity was however greater between isolates from Kabylia than between isolates from north-western Algeria. Cluster analysis showed that most of the Algerian V. dahliae isolates grouped together with the French and Syrian isolates. On the basis of their ability to form heterokaryons with each other, a subset of 25 olive-pathogenic isolates was grouped into a single vegetative compatibility group (VCG). These results suggest that the olive-infecting V. dahliae populations in Algeria show limited diversity and that caution should be taken to prevent introduction of the D pathotype.

RAPD-PCR identification of Verticillium dahliae isolates with differential pathogenicity on cotton

Strains of the fungal wilt pathogen Verticillium dahliae cause considerable economic losses in field crops throughout the world. We have investigated the use of molecular genetic techniques for the identification and classification of strains infecting cotton plants (Gossypium spp.) in the major production regions of Australia. The amplification, sequencing, and restriction digestion of specific ribosomal DNA sequences proved to be diagnostic for the genus/species, but was incapable of differentiating between individual isolates with quite diverse morphologies or origins. RAPD-PCR analysis using a subset of 13 informative decamer primers, however, did reveal significant differences between the isolates and enabled the genetic similarity of the V. dahliae strains to be estimated. RAPD-PCR fingerprints were shown to be reproducible following in planta culture of V. dahliae isolates and could be a valuable tool for the identification and epidemiological study of fungal populations. Although fungal isolates were found to elicit a range of reactions during pathogenicity testing on 5 cotton cultivars, comparison of the RAPD-PCR banding patterns did not suggest a strong correlation between molecular genetic and virulence characteristics.

Relation between sensitivity to terpenoid phytoalexins and virulence to cotton of Verticillium dahliae strains

The terpenoid phytoalexins hemigossypol (HG), methoxyhemigossypol (MHG), desoxyhemigossypol (dHG), and desoxymethoxyhemigossypol (dMHG) from Verticillium dahliae-infected, wilt-resistant Seabrook Sea Island cotton were tested at pH 6.3–7.5 in liquid nutrient media for toxicity to two defoliating and two nondefoliating isolates of V. dahliae. Terpenoid concentrations of 20, 40, 50, and 90 μg/ml of dHG, HG, dMHG, and MHG, respectively, were required to kill uniformly mycelia of all four isolates of V. dahliae. Using this bioassay, differential sensitivity to terpenoid phytoalexins does not appear to be a factor in the differences in virulence of the defoliating and nondefoliating isolates of V. dahliae on cotton.

Influence of verticillium wilt resistant and susceptible potato genotypes on populations of antagonistic rhizosphere and rhizoplane bacteria and free nitrogen fixers

Verticillium wilt resistant A 66 107-51 and susceptible Russet Burbank potatoes differentially influenced populations of rhizosphere and rhizoplane bacteria. Although differences in total bacteria between the two genotypes were not significantly different, selective differences were evident. These included increases in bacteria antagonistic in vitro toward Verticillium dahliae strain RB 5, and bacteria capable of fixing nitrogen that were more commonly associated with the wilt-resistant potato. Bacteria that were antagonistic to V. dahliae were predominantly Bacillus spp. Other antagonists were species of Pseudomonas, Gluconobacter, Flavobacterium, and Streptomyces. Nitrogen-fixing bacteria were Azotobacter and Azomonas spp. The suppression of Verticillium wilt in Russet Burbank during the growing season following the planting of A 66 107-51 may, in part, be explained by the above findings.

Relationship between DNA Content and Spore Volume in Sixteen Isolates of Verticillium lecanii and Two New Diploids of V. dahliae (= V. dahliae var. longisporum Stark)

Summary: Sixteen wild-type isolates of Verticillium lecanii from different insects and non-insect hosts, soils and locations exhibited a wide range in spore volumes, from 2.18 μm3 to 18.25 μm3, as recorded by Coulter counter analysis. A cold hydrolysis technique for Feulgen DNA microdensitometry indicated that all the isolates were haploid. Two wild-type strains of V. dahliae, from rape and sugarbeet from Sweden, were shown to be stable diploids by the criteria of conidial volume and relative DNA content. After treatment with p-fluorophenylalanine, both V. dahliae strains produced haploid segregants with half the conidial volume and relative DNA content of the original parents. Both diploid strains are regarded as new records for V. dahliae var. longisporum (Stark).

Acriflavine-induced hyaline variants of Verticillium albo-atrum and V. dahliae

Colonies developing from conidia of Verticillium albo-atrum and V. dahliae strains taken at 2, 4 and 6 weeks from agar media containing 10 −3 and 10 −4 m acriflavine (originally inoculated with conidia), showed a 10- to 20-fold increase in the percentage of strains lacking darkly pigmented resting structures. Conidial analysis over the next two spore generations in the absence of the drug, showed that the majority of presumptive hyaline variants eventually formed some dark pigment (‘partially hyaline’) and only 2% of the variants were stable. Of these partially hyaline strains 90–96% remained more or less stable over three spore generations. Similar results were obtained using mycelial block transfers on acriflavine medium and also when experiments were done using liquid media or agar well methods. In the latter the yield of hyaline variants was lower. One of the strains tested had larger spores and a two-fold increase in survival on acriflavine medium. On 6 × 10 −5 m and 5 × 10 −5 m acriflavine, acrA1 (resistant) and acrA + (sensitive) colonies segregated from this strain suggesting a heterozygous diploid (acrA1/acrA+). The effects of acriflavine on these fungi are: (1) a genetic effect increasing the incidence of hyaline variants, and (2) a physiological delay in the development of dark resting structures. All stable hyaline variants tested showed increased linear growth rate in the absence of acriflavine, compared with wild type. The results are discussed with particular reference to the mechanisms involved in the production of spontaneous and acriflavine-induced hyaline variants in these fungi.

Hybridization of verticillium albo-atrum and Verticillium dahliae

Four monoconidial haploid wild-type Verticillium isolates obtained from different geographical locations were studied. Two of these had dark mycelium ( Verticillium albo-atrum Reinke & Berth.), and two had microsclerotia (Verticillium dahliae Kleb). Auxotrophic mutants were obtained from each isolate, and complementary auxotrophs were combined in attempts to synthesize heterozygous diploids. No diploids were recovered from combinations of auxotrophs derived from the two V. dahliae strains, but frequent diploidization occurred in combinations of auxotrophs derived from the two V. albo-atrum strains and also in combinations of auxotrophs derived from the V. dahliae and V. albo-atrum strains. The intraspecific diploids ( V. albo-atrum × V. albo-atrum ) showed frequent haploidization and unrestricted genetic recombination. Interspecific diploids showed infrequent haploidization and restricted genetic recombination. The frequency of haploidization in these was apparently increased by growing them on medium containing the fungicide benlate, but these induced haploids also had a limited range of genotypes. The restricted genetic recombination in the interspecific diploids is interpreted as resulting from non-homology between the V. albo-atrum and V. dahliae genomes, and the results are discussed in relation to the taxonomie status of dark mycelial and microsclerotial Verticillium strains.