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Transcription factors (TFs) bind to the promoters of target genes to regulate gene expression in response to different stimuli. The functions and regulatory mechanisms of transcription factors (TFs) in Verticillium dahliae are, however, still largely unclear. This study showed that a C2H2-type zinc finger TF, VdCf2 (V. dahliae chorion transcription factor 2), plays key roles in V. dahliae growth, melanin production, and virulence. Transcriptome sequencing analysis showed that VdCf2 was involved in the regulation of expression of genes encoding secreted proteins, pathogen-host interaction (PHI) homologs, TFs, and G protein-coupled receptors (GPCRs). Furthermore, VdCf2 positively regulated the expression of VdPevD1 (VDAG_02735), a previously reported virulence factor. VdCf2 thus regulates the expression of several pathogenicity-related genes that also contribute to virulence in V. dahliae. VdCf2 also inhibited the transcription of the Vd276-280 gene cluster and interacted with two members encoding proteins (VDAG_07276 and VDAG_07278) in the gene cluster. IMPORTANCE Verticillium dahliae is an important soilborne phytopathogen which can ruinously attack numerous host plants and cause significant economic losses. Transcription factors (TFs) were reported to be involved in various biological processes, such as hyphal growth and virulence of pathogenic fungi. However, the functions and regulatory mechanisms of TFs in V. dahliae remain largely unclear. In this study, we identified a new transcription factor, VdCf2 (V. dahliae chorion transcription factor 2), based on previous transcriptome data, which participates in growth, melanin production, and virulence of V. dahliae. We provide evidence that VdCf2 regulates the expression of the pathogenicity-related gene VdPevD1 (VDAG_02735) and Vd276-280 gene cluster. VdCf2 also interacts with VDAG_07276 and VDAG_07278 in this gene cluster based on a yeast two-hybrid and bimolecular fluorescence complementation assay. These results revealed the regulatory mechanisms of a pivotal pathogenicity-related transcription factor, VdCf2 in V. dahliae.

Feruloyl esterase (ferulic acid esterase, FAE) is an essential component of many biological processes in both eukaryotes and prokaryotes. This research aimed to investigate the role of FAE and its regulation mechanism in plant immunity. We identified a secreted feruloyl esterase VdFAE from the hemibiotrophic plant pathogen Verticillium dahliae. VdFAE acted as an important virulence factor during V. dahliae infection, and triggered plant defence responses, including cell death in Nicotiana benthamiana. Deletion of VdFAE led to a decrease in the degradation of ethyl ferulate. VdFAE interacted with Gossypium hirsutum protein dihydroflavanol 4‐reductase (GhDFR), a positive regulator in plant innate immunity, and promoted the degradation of GhDFR. Furthermore, silencing of GhDFR led to reduced resistance of cotton plants against V. dahliae. The results suggested a fungal virulence strategy in which a fungal pathogen secretes FAE to interact with host DFR and interfere with plant immunity, thereby promoting infection. [ABSTRACT FROM AUTHOR]

Rhizosphere microbial communities are known to be related to plant health; using such an association for crop management requires a better understanding of this relationship. We investigated rhizosphere microbiomes associated with Verticillium wilt symptoms in two cotton cultivars. Microbial communities were profiled by amplicon sequencing, with the total bacterial and fungal DNA quantified by quantitative polymerase chain reaction based on the respective 16S and internal transcribed spacer primers. Although the level of V. dahliae inoculum was higher in the rhizosphere of diseased plants than in the healthy plants, such a difference explained only a small proportion of variation in wilt severities. Compared to healthy plants, the diseased plants had much higher total fungal/bacterial biomass ratio, as represented by quantified total fungal or bacterial DNA. The variability in the fungal/bacterial biomass ratio was much smaller than variability in either fungal or bacterial total biomass among samples within diseased or healthy plants. Diseased plants generally had lower bacterial alpha diversity in their rhizosphere, but such differences in the fungal alpha diversity depended on cultivars. There were large differences in both fungal and bacterial communities between diseased and healthy plants. Many rhizosphere microbial groups differed in their abundance between healthy and diseased plants. There was a decrease in arbuscular mycorrhizal fungi and an increase in several plant pathogen and saprophyte guilds in diseased plants. These findings suggested that V. dahliae infection of roots led to considerable changes in rhizosphere microbial communities, with large increases in saprophytic fungi and reduction in bacterial community. [ABSTRACT FROM AUTHOR]

Verticillium wilt caused by Verticillium dahliae is a common soil-borne disease worldwide, affecting many economically important crop species. Soil microbes can influence plant disease development. We investigated rhizosphere and endosphere microbiomes in relation to cotton cultivars with differential susceptibility to Verticillium wilt. Soil samples from nine cotton cultivars were assessed for the density of V. dahliae microsclerotia; plants were assessed for disease development. We used amplicon sequencing to profile both bacterial and fungal communities. Unlike wilt severity, wilt inoculum density did not differ significantly among resistant and susceptible cultivars. Overall, there were no significant association of alpha diversity indices with wilt susceptibility. In contrast, there were clear differences in the overall rhizosphere and endosphere microbial communities, particularly bacteria, between resistant and susceptible cultivars. Many rhizosphere and endosphere microbial groups differed in their relative abundance between resistant and susceptible cultivars. These operational taxonomic units included several well-known taxonomy groups containing beneficial microbes, such as Bacillales, Pseudomonadales, Rhizobiales, and Trichoderma , which were higher in their relative abundance in resistant cultivars. Greenhouse studies with sterilized soil supported that beneficial microbes in the rhizosphere contribute to reduced wilt development. These findings suggested that specific rhizosphere and endosphere microbes may contribute to cotton resistance to V. dahliae. [ABSTRACT FROM AUTHOR]

Verticillium dahliae infection of strawberry (Fragaria x ananassa) is a major cause of disease-induced wilting in soil-grown strawberries across the world. To understand what components of the pathogen are affecting disease expression, the presence of the known effector VdAve1 was screened in a sample of Verticillium dahliae isolates. Isolates from strawberry were found to contain VdAve1 and were divided into two major clades, based upon their vegetative compatibility groups (VCG); no UK strawberry isolates contained VdAve1. VC clade was strongly related to their virulence levels. VdAve1-containing isolates pathogenic on strawberry were found in both clades, in contrast to some recently published findings. On strawberry, VdAve1-containing isolates had significantly higher virulence during early infection, which diminished in significance as the infection progressed. Transformation of a virulent non-VdAve1 containing isolate, with VdAve1 was found neither to increase nor decrease virulence when inoculated on a susceptible strawberry cultivar. There are therefore virulence factors that are epistatic to VdAve1 and potentially multiple independent routes to high virulence on strawberry in V. dahliae lineages. Genome sequencing a subset of isolates across the two VCGs revealed that isolates were differentiated at the whole genome level and contained multiple changes in putative effector content, indicating that different clonal VCGs may have evolved different strategies for infecting strawberry, leading to different virulence levels in pathogenicity tests. It is therefore important to consider both clonal lineage and effector complement as the adaptive potential of each lineage will differ, even if they contain the same race determining effector. [ABSTRACT FROM AUTHOR]

Verticillium dahliae is the causal agent of strawberry wilt. A microencapsulated terpene product containing cineole, camphor and borneol, digestate from anaerobic digestion, and BioFence™ derived from a mustard-based defatted seedmeal were tested for their suppressive activity against V. dahliae . First, naturally infested soil was amended with microencapsulated terpene, lavender waste pellet and BioFence™ (pellet) in a laboratory test to assess the efficacy against V. dahliae . Next, mini-field-plot experiments were conducted to evaluate the efficacy of individual and combined use of terpene, BioFence™ (liquid) and digestate against V. dahliae ; sterile distilled water treatment and untreated control were also included. In the laboratory test, all treatments significantly reduced V. dahliae densities, with the control efficacy ranging from 27% (BioFence™) to 69% (lavender waste pellet). Although the lowest (1×) rate of terpene treatment resulted in a much lower control efficacy (35%) than the other two higher rates (3× – 55%; 9× – 53%), these differences were not statistically significant. In the field mini-plot trials, all treatments led to significant reductions in the V. dahliae density, with the efficacy ranging from 50% (digestate) to 78% (combined three-product treatment), irrespective of the initial wilt level. There were no significant differences in all comparisons of pairwise treatments except between digestate and combined three-product treatment. For the combined two or three-product treatments, the observed efficacy was significantly less than the expected efficacy on the assumption of Bliss independence. Furthermore, there were no significant differences between the observed efficacy of combined treatments and the best single component product efficacy. Although the observed efficacy for the combined three-product treatment was consistently higher than the best single component across replicate plots, such a difference was not statistically significant. The results indicate the value of these alternative treatments in practice but these are not likely to reduce V. dahliae inoculum sufficiently to eliminate the risk of strawberry wilt and question the value of combined treatments. [ABSTRACT FROM AUTHOR]

Spatial patterns of pathogen inoculum in field soils and the resulting patterns of disease may reflect the underlying mechanisms of pathogen dispersal. This knowledge can be used to design more efficient sampling schemes for assessing diseases. Spatial patterns of Verticillium dahliae microsclerotia were characterized in commercial cotton fields through quadrat and point sampling in 1994 and 2013, respectively. Furthermore, cotton plants with wilt symptoms, caused by V. dahliae, were assessed in six commercial cotton fields in 2013. Soil samples were assayed for the density of microsclerotia (expressed as CFU g-1 of soil) using a wet-sieving plating method and a real-time quantitative PCR method for the 1994 and 2013 study, respectively. The estimated inoculum threshold for causing wilt development on individual plants varied with the three fields: ca. 1.6 CFU g-1 of soil for one field, and 7.2 CFU g-1 of soil for the other two. Both quadrat and point sampling spatial analyses showed that aggregation of V. dahliae inoculum in soils was usually not detected beyond 1.0 m. Similarly, the spatial patterns of wilted cotton plants indicated that spatial aggregation of diseased plants were only observed below the scale of 1.0 m in six commercial cotton plantations. Therefore, spatial aggregation of both V. dahliae inoculum and cotton plants with wilt symptoms is not likely to be detected above the scale of 1.0 m for most commercial cotton plantations. When designing schemes for assessing wilt inoculum and wilt development, this scale needs to be taken into consideration. [ABSTRACT FROM AUTHOR]

Verticillium dahliae is a fungal pathogen causing Verticillium wilt on a range of economically important crops. Microsclerotia are its main survival and dormancy structures and serve as the primary inoculum on many hosts. Studies were conducted to determine the effect of temperature (5 to 50°C), pH (2 to 12) and nutrient regimes on microsclerotia germination. The optimal condition for microsclerotium germination was 20°C with pH 8.0 whereas nutrient regimes had no significant effect on its germination. The whole genome wide expression profiles during microsclerotium germination were characterized using the Illumina sequencing technology. Approximately 7.4 million of 21-nt cDNA tags were sequenced in the cDNA libraries derived from germinated and non-germinated microsclerotia. About 3.9% and 2.3% of the unique tags were up-regulated and down-regulated at least five-fold, respectively, in the germinated microsclerotia compared with the non-germinated microsclerotia. A total of 1654 genes showing differential expression were identified. Genes that are likely to have played important roles in microsclerotium germination include those encoding G-protein coupled receptor, lipase/esterase, cyclopentanone 1,2-monooxygenase, H(+)/hexose cotransporter 1, fungal Zn(2)-Cys(6) binuclear cluster domain, thymus-specific serine protease, glucan 1,3-beta-glucosidase, and alcohol dehydrogenase. These genes were mainly up-regulated or down-regulated only in germinated microsclerotia, compared with non-germinated microsclerotia. The differential expression of genes was confirmed by qRT-PCR analysis of 20 randomly selected genes from the 40 most differentially expressed genes. [ABSTRACT FROM AUTHOR]

The fungus Verticillium dahliae causes vascular wilt disease on many plant species, including economically important crop and ornamental plants worldwide. It produces darkly pigmented resting structures known as microsclerotia, which are able to survive for up to 14 years in soil, and represent one of the defining characteristics of this species. The pigment produced in V. dahliae is dihydroxynaphthalene (DHN)-melanin, a form of melanin common among fungi and named so for the intermediary of this melanin biosynthetic pathway. In this study, we characterized the function of the V. dahliae Vayg1 gene, whose homologs were involved in melanin biosynthesis in Exophiala dermatitidis ( Wayg1 ) and Aspergillus fumigatus ( Aayg1 ), by deletion and complementation of the gene and co-incubating deletion mutant with wild-type strain. Results showed that melanin production and microsclerotial formation in deletion mutants are inhibited. The Vayg1 deletion mutant also exhibited reduced pathogenicity. These results showed that Vayg1 is necessary for melanin and microsclerotium production, and we may thus hypothesize that the Vayg1 product may catalyze two different precursors, one of which is essential for DHN melanin production and the other one is involved in a signal network for microsclerotial formation in V. dahliae . [ABSTRACT FROM AUTHOR]