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Verticillium dahliae is a broad host-range pathogen that causes vascular wilt in plants. The Verticillium wilt disease severity assay on plants caused by V. dahliae mainly includes two methods, one is a plant disease grade classification based on disease severity statistics [namely the disease index (DI)], and the other is the V. dahliae biomass quantification in plants (namely the fungal biomass). In this study, the relationships of pathogenicity with the DI, pathogenicity with the biomass, and the correlation analysis of the DI and relative fungal biomass were analysed. The results showed that pathogenicity assessment of V. dahliae strains using the DI method was able to give an intuitive reflection of the pathogenic ability for defoliating and non-defoliating strains; moreover, the method of quantitative PCR for fungal biomass also had high repeatability and stability. As a whole, the correlation coefficient between the DI and fungal biomass values of 28 strains was 0.728, indicating that the two data sets were highly correlated; however, the correlation coefficients of the defoliating strains and non-defoliating strains were only 0.5384 and 0.4547, respectively. In conclusion, the correlation coefficient between the DI and the fungal biomass presented high consistency, which could provide some meaningful exploration for the more accurate pathogenicity identification of V. dahliae.

Abstract Background Verticillium wilt, caused by the fungus Verticillium dahliae, is a soil-borne vascular fungal disease, which has caused great losses to cotton yield and quality worldwide. The strain KRS010 was isolated from the seed of Verticillium wilt-resistant Gossypium hirsutum cultivar “Zhongzhimian No. 2.” Results The strain KRS010 has a broad-spectrum antifungal activity to various pathogenic fungi as Verticillium dahliae, Botrytis cinerea, Fusarium spp., Colletotrichum spp., and Magnaporthe oryzae, of which the inhibition rate of V. dahliae mycelial growth was 73.97% and 84.39% respectively through confrontation test and volatile organic compounds (VOCs) treatments. The strain was identified as Bacillus altitudinis by phylogenetic analysis based on complete genome sequences, and the strain physio-biochemical characteristics were detected, including growth-promoting ability and active enzymes. Moreover, the control efficiency of KRS010 against Verticillium wilt of cotton was 93.59%. After treatment with KRS010 culture, the biomass of V. dahliae was reduced. The biomass of V. dahliae in the control group (Vd991 alone) was 30.76-folds higher than that in the treatment group (KRS010+Vd991). From a molecular biological aspect, KRS010 could trigger plant immunity by inducing systemic resistance (ISR) activated by salicylic acid (SA) and jasmonic acid (JA) signaling pathways. Its extracellular metabolites and VOCs inhibited the melanin biosynthesis of V. dahliae. In addition, KRS010 had been characterized as the ability to promote plant growth. Conclusions This study indicated that B. altitudinis KRS010 is a beneficial microbe with a potential for controlling Verticillium wilt of cotton, as well as promoting plant growth.

Abstract Background Populations of the plant pathogenic fungus Verticillium dahliae display a complex and rich genetic diversity, yet the existence of sexual reproduction in the fungus remains contested. As pivotal genes, MAT genes play a crucial role in regulating cell differentiation, morphological development, and mating of compatible cells. However, the functions of the two mating type genes in V. dahliae, VdMAT1-1-1, and VdMAT1-2-1, remain poorly understood. Results In this study, we confirmed that the MAT loci in V. dahliae are highly conserved, including both VdMAT1-1-1 and VdMAT1-2-1 which share high collinearity. The conserved core transcription factor encoded by the two MAT loci may facilitate the regulation of pheromone precursor and pheromone receptor genes by directly binding to their promoter regions. Additionally, peptide activity assays demonstrated that the signal peptide of the pheromone VdPpg1 possessed secretory activity, while VdPpg2, lacked a predicted signal peptide. Chemotactic growth assays revealed that V. dahliae senses and grows towards the pheromones FO-a and FO-α of Fusarium oxysporum, as well as towards VdPpg2 of V. dahliae, but not in response to VdPpg1. The findings herein also revealed that VdMAT1-1-1 and VdMAT1-2-1 regulate vegetative growth, carbon source utilization, and resistance to stressors in V. dahliae, while negatively regulating virulence. Conclusions These findings underscore the potential roles of VdMAT1-1-1 and VdMAT1-2-1 in sexual reproduction and confirm their involvement in various asexual processes of V. dahliae, offering novel insights into the functions of mating type genes in this species.

Verticillium wilt, caused by the fungal pathogen Verticillium dahliae, is the major cause of disease-related yield losses in cotton (Gossypium hirsutum). Despite these losses, the major cultivars of G. hirsutum remain highly susceptible to Verticillium wilt. The lack of understanding on the genetic basis for Verticillium wilt resistance may further hinder progress in deploying elite cultivars with proven resistance, such as the wilt resistant G. hirsutum cultivar Zhongzhimian No. 2. To help remedy this knowledge gap, we sequenced the whole genome of Zhongzhimian No. 2 and assembled it from a combination of PacBio long reads, Illumina short reads, and high-throughput chromosome conformation capture technologies. The final assembly of the genome was 2.33 Gb, encoding 95,327 predicted coding sequences. The GC content was 34.39% with 99.2% of the bases anchored to 26 pseudo-chromosomes that ranged from 53.8 to 127.7 Mb. This resource will help gain a detailed understanding of the genomic features governing high yield and Verticillium wilt resistance in this cultivar. Comparative genomics will be particularly helpful, since there are several published genomes of other Gossypium species. [Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

ABSTRACT Verticillium dahliae is a soilborne fungal pathogen that causes disease on many economically important crops. Based on the resistance or susceptibility of differential cultivars in tomato, isolates of V. dahliae are divided into three races. Avirulence (avr) genes within the genomes of the three races have also been identified. However, the functional role of the avr gene in race 3 isolates of V. dahliae has not been characterized. In this study, bioinformatics analysis showed that VdR3e, a cysteine-rich secreted protein encoded by the gene characterizing race 3 in V. dahliae, was likely obtained by horizontal gene transfer from the fungal genus Bipolaris. We demonstrate that VdR3e causes cell death by triggering multiple defense responses. In addition, VdR3e localized at the periphery of the plant cell and triggered immunity depending on its subcellular localization and the cell membrane receptor BAK1. Furthermore, VdR3e is a virulence factor and shows differential pathogenicity in race 3-resistant and -susceptible hosts. These results suggest that VdR3e is a virulence factor that can also interact with BAK1 as a pathogen-associated molecular pattern (PAMP) to trigger immune responses. IMPORTANCE Based on the gene-for-gene model, research on the function of avirulence genes and resistance genes has had an unparalleled impact on breeding for resistance in most crops against individual pathogens. The soilborne fungal pathogen, Verticillium dahliae, is a major pathogen on many economically important crops. Currently, avr genes of the three races in V. dahliae have been identified, but the function of avr gene representing race 3 has not been described. We investigated the characteristics of VdR3e-mediated immunity and demonstrated that VdR3e acts as a PAMP to activate a variety of plant defense responses and induce plant cell death. We also demonstrated that the role of VdR3e in pathogenicity was host dependent. This is the first study to describe the immune and virulence functions of the avr gene from race 3 in V. dahliae, and we provide support for the identification of genes mediating resistance against race 3.

Abstract Background During the disease cycle, plant pathogenic fungi exhibit a morphological transition between hyphal growth (the phase of active infection) and the production of long-term survival structures that remain dormant during “overwintering.” Verticillium dahliae is a major plant pathogen that produces heavily melanized microsclerotia (MS) that survive in the soil for 14 or more years. These MS are multicellular structures produced during the necrotrophic phase of the disease cycle. Polyketide synthases (PKSs) are responsible for catalyzing production of many secondary metabolites including melanin. While MS contribute to long-term survival, hyphal growth is key for infection and virulence, but the signaling mechanisms by which the pathogen maintains hyphal growth are unclear. Results We analyzed the VdPKSs that contain at least one conserved domain potentially involved in secondary metabolism (SM), and screened the effect of VdPKS deletions in the virulent strain AT13. Among the five VdPKSs whose deletion affected virulence on cotton, we found that VdPKS9 acted epistatically to the VdPKS1-associated melanin pathway to promote hyphal growth. The decreased hyphal growth in VdPKS9 mutants was accompanied by the up-regulation of melanin biosynthesis and MS formation. Overexpression of VdPKS9 transformed melanized hyphal-type (MH-type) into the albinistic hyaline hyphal-type (AH-type), and VdPKS9 was upregulated in the AH-type population, which also exhibited higher virulence than the MH-type. Conclusions We show that VdPKS9 is a powerful negative regulator of both melanin biosynthesis and MS formation in V. dahliae. These findings provide insight into the mechanism of how plant pathogens promote their virulence by the maintenance of vegetative hyphal growth during infection and colonization of plant hosts, and may provide novel targets for the control of melanin-producing filamentous fungi.

Understanding how plant pathogenic fungi adapt to their hosts is of critical importance to securing optimal crop productivity. In response to pathogenic attack, plants produce reactive oxygen species (ROS) as part of a multipronged defense response. Pathogens, in turn, have evolved ROS scavenging mechanisms to undermine host defense. Thioredoxins (Trx) are highly conserved oxidoreductase enzymes with a dithiol-disulfide active site, and function as antioxidants to protect cells against free radicals, such as ROS. However, the roles of thioredoxins in Verticillium dahliae, an important vascular pathogen, are not clear. Through proteomics analyses, we identified a putative thioredoxin (VdTrx1) lacking a signal peptide. VdTrx1 was present in the exoproteome of V. dahliae cultured in the presence of host tissues, a finding that suggested that it plays a role in host-pathogen interactions. We constructed a VdTrx1 deletion mutant ΔVdTrx1 that exhibited significantly higher sensitivity to ROS stress, H2O2, and tert-butyl hydroperoxide (t-BOOH). In vivo assays by live-cell imaging and in vitro assays by western blotting revealed that while VdTrx1 lacking the signal peptide can be localized within V. dahliae cells, VdTrx1 can also be secreted unconventionally depending on VdVps36, a member of the ESCRT-II protein complex. The ΔVdTrx1 strain was unable to scavenge host-generated extracellular ROS fully during host invasion. Deletion of VdTrx1 resulted in higher intracellular ROS levels of V. dahliae mycelium, displayed impaired conidial production, and showed significantly reduced virulence on Gossypium hirsutum, and model plants, Arabidopsis thaliana and Nicotiana benthamiana. Thus, we conclude that VdTrx1 acts as a virulence factor in V. dahliae.

Abstract Background Verticillium dahliae is a fungal pathogen that causes a vascular wilt on many economically important crops. Common fungal extracellular membrane (CFEM) domain proteins including secreted types have been implicated in virulence, but their roles in this pathogen are still unknown. Results Nine secreted small cysteine-rich proteins (VdSCPs) with CFEM domains were identified by bioinformatic analyses and their differential suppression of host immune responses were evaluated. Two of these proteins, VdSCP76 and VdSCP77, localized to the plant plasma membrane owing to their signal peptides and mediated broad-spectrum suppression of all immune responses induced by typical effectors. Deletion of either VdSCP76 or VdSCP77 significantly reduced the virulence of V. dahliae on cotton. Furthermore, VdSCP76 and VdSCP77 suppressed host immunity through the potential iron binding site conserved in CFEM family members, characterized by an aspartic acid residue in seven VdSCPs (Asp-type) in contrast with an asparagine residue (Asn-type) in VdSCP76 and VdSCP77. V. dahliae isolates carrying the Asn-type CFEM members were more virulent on cotton than those carrying the Asp-type. Conclusions In the iron-insufficient xylem, V. dahliae is likely to employ the Asp-type CFEM members to chelate iron, and Asn-type CFEM members to suppress immunity, for successful colonization and propagation in host plants.

Plants produce reactive oxygen species (ROS) to defend pathogens. To counteract this attack, certain pathogens express superoxide dismutases (SODs) to scavenge host-derived ROS. However, the roles of SODs in Verticillium dahliae, an important vascular pathogen, are not clear. Our previous study has shown that a putative extracellular SOD (VdSOD5) of V. dahliae is significantly induced by culturing in cotton tissues, suggesting that VdSOD5 may play an important role in host–pathogen interactions and virulence. Here, we showed that VdSOD5 encoded a superoxide dismutase with a cofactor copper-binding site and a functional signal peptide that can conduct protein secretion in an invertase-mutated yeast strain. The mutations in VdSOD5 (ΔVdSOD5) did not change the normal vegetative growth and conidial production but reduced the virulence of V. dahliae on susceptible host cotton. Further studies showed that the transcription of VdSOD5 was significantly up-regulated during the early stage of infection, and the loss-of-function of VdSOD5 decreased culture filtrate and fungal tissue SOD activities of V. dahliae by 74 and 28%, respectively. Compared to the wild-type strain Vd991, the ΔVdSOD5 showed the same sensitivity to the intracellular ROS generator menadione. Furthermore, nitroblue tetrazolium (NBT) staining demonstrated that VdSOD5 functioned in the detoxification of superoxides generated by host roots during infection. These results suggest that VdSOD5 of V. dahliae is an important virulence factor, secreted out of cells to combat host-derived ROS.

Abstract Background Plant pathogens and their hosts undergo adaptive changes in managed agricultural ecosystems, by overcoming host resistance, but the underlying genetic adaptations are difficult to determine in natural settings. Verticillium dahliae is a fungal pathogen that causes Verticillium wilt on many economically important crops including lettuce. We assessed the dynamics of changes in the V. dahliae genome under selection in a long-term field experiment. Results In this study, a field was fumigated before the Verticillium dahliae race 1 strain (VdLs.16) was introduced. A derivative 145-strain population was collected over a 6-year period from this field in which a seggregating population of lettuce derived from Vr1/vr1 parents were evaluated. We de novo sequenced the parental genome of VdLs.16 strain and resequenced the derivative strains to analyze the genetic variations that accumulate over time in the field cropped with lettuce. Population genomics analyses identified 2769 single-nucleotide polymorphisms (SNPs) and 750 insertion/deletions (In-Dels) in the 145 isolates compared with the parental genome. Sequence divergence was identified in the coding sequence regions of 378 genes and in the putative promoter regions of 604 genes. Five-hundred and nine SNPs/In-Dels were identified as fixed. The SNPs and In-Dels were significantly enriched in the transposon-rich, gene-sparse regions, and in those genes with functional roles in signaling and transcriptional regulation. Conclusions Under the managed ecosystem continuously cropped to lettuce, the local adaptation of V. dahliae evolves at a whole genome scale to accumulate SNPs/In-Dels nonrandomly in hypervariable regions that encode components of signal transduction and transcriptional regulation.

ABSTRACT Verticillium dahliae is a widespread soilborne fungus that causes Verticillium wilt on numerous economically important plant species. In tomato, until now, three races have been characterized based on the response of differential cultivars to V. dahliae, but the genetic basis of race divergence in V. dahliae remains undetermined. To investigate the genetic basis of race divergence, we sequenced the genomes of two race 2 strains and four race 3 strains for comparative analyses with two known race 1 genomes. The genetic basis of race divergence was described by the pathogenicity-related genes among the three races, orthologue analyses, and genomic structural variations. Global comparative genomics showed that chromosomal rearrangements are not the only source of race divergence and that race 3 should be split into two genotypes based on orthologue clustering. Lineage-specific regions (LSRs), frequently observed between genomes of the three races, encode several predicted secreted proteins that potentially function as suppressors of immunity triggered by known effectors. These likely contribute to the virulence of the three races. Two genes in particular that can act as markers for race 2 and race 3 (VdR2e and VdR3e, respectively) contribute to virulence on tomato, and the latter acts as an avirulence factor of race 3. We elucidated the genetic basis of race divergence through global comparative genomics and identified secreted proteins in LSRs that could potentially play critical roles in the differential virulence among the races in V. dahliae. IMPORTANCE Deciphering the gene-for-gene relationships during host-pathogen interactions is the basis of modern plant resistance breeding. In the Verticillium dahliae-tomato pathosystem, two races (races 1 and 2) and their corresponding avirulence (Avr) genes have been identified, but strains that lack these two Avr genes exist in nature. In this system, race 3 has been described, but the corresponding Avr gene has not been identified. We de novo-sequenced genomes of six strains and identified secreted proteins within the lineage-specific regions (LSRs) distributed among the genomes of the three races that could potentially function as manipulators of host immunity. One of the LSR genes, VdR3e, was confirmed as the Avr gene for race 3. The results indicate that differences in transcriptional regulation may contribute to race differentiation. This is the first study to describe these differences and elucidate roles of secreted proteins in LSRs that play roles in race differentiation.

Verticillium dahliae is a soil-borne plant pathogenic fungus that causes Verticillium wilt on hundreds of dicotyledonous plant species. V. dahliae is considered an asexually (clonal) reproducing fungus, although both mating type idiomorphs (MAT1-1 and MAT1-2) are present, and is heterothallic. Most of the available information on V. dahliae strains, including their biology, pathology, and genomics comes from studies on isolates with the MAT1-2 idiomorph, and thus little information is available on the MAT1-1 V. dahliae strains in the literature. We therefore evaluated the growth responses of MAT1-1 and MAT1-2 V. dahliae strains to various stimuli. Growth rates and melanin production in response to increased temperature, alkaline pH, light, and H2O2 stress were higher in the MAT1-2 strains than in the MAT1-1 strains. In addition, the MAT1-2 strains showed an enhanced ability to degrade complex polysaccharides, especially starch, pectin, and cellulose. Furthermore, several MAT1-2 strains from both potato and sunflower showed increased virulence on their original hosts, relative to their MAT1-1 counterparts. Thus, compared to MAT1-1 strains, MAT1-2 strains derive their potentially greater fitness from an increased capacity to adapt to their environment and exhibit higher virulence. These competitive advantages might explain the current abundance of MAT1-2 strains relative to MAT1-1 strains in the agricultural and sylvicultural ecosystems, and this study provides the baseline information on the two mating idiomorphs to study sexual reproduction in V. dahliae under natural and laboratory conditions.

Verticillium wilt, caused by the Verticillium dahliae phytopathogen, is a devastating disease affecting many economically important crops. Previous studies have shown that the exoproteome of V. dahliae plays a significant role in this pathogenic process, but the components and mechanisms that underlie this remain unclear. In this study, the exoproteome of V. dahliae was induced in a cotton-containing C’zapek-Dox (CCD) medium and quantified using the high-throughput isobaric tag technique for relative and absolute quantification (iTRAQ). Results showed that the abundance of 271 secreted proteins was affected by the CCD medium, of which 172 contain typical signal peptides generally produced by the Golgi/endoplasmic reticulum (ER). These enhanced abundance proteins were predominantly enriched in carbohydrate hydrolases; 126 were classified as carbohydrate-active (CAZymes) and almost all were significantly up-regulated in the CCD medium. Results showed that CAZymes proteins 30 and 22 participate in pectin and cellulose degradation pathways, corresponding with the transcription levels of several genes encoded plant cell wall degradation enzyme activated significantly during cotton infection. In addition, targeted deletion of two pectin lyase genes (VdPL3.1 and VdPL3.3) impaired wilt virulence to cotton. This study demonstrates that the V. dahliae exoproteome plays a crucial role in the development of symptoms of wilting and necrosis, predominantly via the pathogenic mechanisms of plant cell wall degradation as part of host plant infection.

Endophytes play important roles in promoting plant growth and controlling plant diseases. Verticillium wilt is a vascular wilt disease caused by Verticillium dahliae, a widely distributed soilbome pathogen that causes significant economic losses on cotton each year. In this study, an endophyte KRS015, isolated from the seed of the Verticillium wilt-resistant Gossypium hirsutum 'Zhongzhimian No. 2', was identified as Bacillus subtilis by morphological, phylogenetic, physiological, and biochemical analyses. The volatile organic compounds (VOCs) produced by KRS015 or its cell-free fermentation extract had significant antagonistic effects on various pathogenic fungi, including V. dahliae. KRS015 reduced Verticillium wilt index and colonization of V. dahliae in treated cotton seedlings significantly; the disease reduction rate was ~62%. KRS015 also promoted plant growth, potentially mediated by the growth-related cotton genes GhACL5 and GhCPD-3. The cell-free fermentation extract of KRS015 triggered a hypersensitivity response, including reactive oxygen species (ROS) and expression of resistance-related plant genes. VOCs from KRS015 also inhibited germination of conidia and the mycelial growth of V. dahliae, and were mediated by growth and development-related genes such as VdHapX, VdMcml, Vdpf, and Veil. These results suggest that KRS015 is a potential agent for controlling Verticillium wilt and promoting growth of cotton. [ABSTRACT FROM AUTHOR]

Though Verticillium dahliae is an asexually reproducing fungus, it is considered heterothallic owing to the presence of only one of the two mating-type idiomorphs (MAT1-1 or MAT1-2) in individual isolates. But sexual reproduction has never been observed either in nature or in the laboratory. All of the genomic information in the literature thus far has therefore come from studies on isolates carrying only the MAT1-2 idiomorph. Herein, we sequenced and compared high-quality reference genomes of MAT1-1 strain S011 and MAT1-2 strain S023 obtained from the same sunflower field. The two genomic sequences displayed high synteny, and encoded similar number genes, a similarity especially notable among pathogenicity-related genes. Homolog analysis between these two genomes revealed that 80% of encoded genes are highly conserved (95% identity and coverage), but only 20% of the single copy genes were identical. These novel genome resources will support the analysis of the structure and function of the two idiomorphs and provide valuable tools to elucidate the evolution and potential mechanisms of sexual reproduction in V. dahliae. [ABSTRACT FROM AUTHOR]