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Verticillium wilt caused by Verticillium dahliae is a notorious soil-borne fungal disease and seriously threatens the yield of economic crops worldwide. During host infection, V. dahliae secretes many effectors that manipulate host immunity, among which small cysteine-rich proteins (SCPs) play an important role. However, the exact roles of many SCPs from V. dahliae are unknown and varied. In this study, we show that the small cysteine-rich protein VdSCP23 inhibits cell necrosis in Nicotiana benthamiana leaves, as well as the reactive oxygen species (ROS) burst, electrolyte leakage and the expression of defense-related genes. VdSCP23 is mainly localized in the plant cell plasma membrane and nucleus, but its inhibition of immune responses was independent of its nuclear localization. Site-directed mutagenesis and peptide truncation showed that the inhibition function of VdSCP23 was independent of cysteine residues but was dependent on the N-glycosylation sites and the integrity of VdSCP23 protein structure. Deletion of VdSCP23 did not affect the growth and development of mycelia or conidial production in V. dahliae. Unexpectedly, VdSCP23 deletion strains still maintained their virulence for N. benthamiana, Gossypium hirsutum and Arabidopsis thaliana seedlings. This study demonstrates an important role for VdSCP23 in the inhibition of plant immune responses; however, it is not required for normal growth or virulence in V. dahliae.

Verticillium dahliae is a soilborne plant fungal pathogen that causes Verticillium wilt, a disease that reduces the yields of many economically important crops. Despite its worldwide distribution and harmful impacts, much remains unknown regarding how the numerous effectors of V. dahliae modulate plant immunity. Here, we identified the intracellular effector VdCE11 that induces cell death and defense responses in Nicotiana benthamiana to counter leaf pathogens such as Sclerotinia sclerotiorum and Botrytis cinerea. VdCE11 also contributes to the virulence of V. dahliae in cotton and Arabidopsis. Yeast two-hybrid library screening and immunoprecipitation revealed that VdCE11 interacts physically with the cotton aspartic protease GhAP1. GhAP1 and its Arabidopsis homolog AtAP1 are negative regulators of plant immunity, since disruption of either increased the resistance of cotton or Arabidopsis to V. dahliae. Further, VdCE11 plays a role in promoting the accumulation of the AP1 proteins and increasing its hydrolase activity. Taken together, these results indicate a novel mechanism regulating virulence whereby the secreted effector VdCE11 increases cotton susceptibility to V. dahliae by promoting the accumulation and activity of GhAP1. IMPORTANCE Verticclium dahliae is a plant fungal pathogen that causes a destructive vascular disease on a large number of plant hosts, resulting in great threat to agricultural production. In this study, we identified a V. dahliae effector VdCE11 that induces cell death and defense responses in Nicotiana benthamiana. Meanwhile, VdCE11 contributes to the virulence of V. dahliae in cotton and Arabidopsis. Yeast two-hybrid library screening and immunoprecipitation revealed that VdCE11 interacts physically with the cotton aspartic protease GhAP1. GhAP1 and its Arabidopsis homolog AtAP1 are negative regulators of plant immunity since disruption of either increased the resistance of cotton or Arabidopsis to V. dahliae. Further research showed that VdCE11 plays a role in promoting the accumulation of the AP1 proteins and increasing its hydrolase activity. These results suggested that a novel mechanism regulating virulence whereby VdCE11 increases susceptibility to V. dahliae by promoting the accumulation and activity of GhAP1 in the host.

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.

BackgroundVerticillium 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.ResultsNine 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.ConclusionsIn 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.

BackgroundDuring 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.ResultsWe 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.ConclusionsWe 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.

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.

Verticillium dahliae is a notorious soil-borne pathogen that enters hosts through the roots and proliferates in the plant water-conducting elements to cause Verticillium wilt. Historically, Verticillium wilt symptoms have been explained by vascular occlusion, due to the accumulation of mycelia and plant biomacromolecule aggregation, and also by phytotoxicity caused by pathogen-secreted toxins. Beyond the direct cytotoxicity of some members of the secretome, this review systematically discusses the roles of the V. dahliae secretome in vascular occlusion, including the deposition of polysaccharides as an outcome of plant cell wall destruction, the accumulation of fungal mycelia, and modulation of plant defence responses. By modulating plant defences and hormone levels, the secretome manipulates the vascular environment to induce Verticillium wilt. Thus, the secretome of V. dahliae colludes with plant defence responses to modulate Verticillium wilt symptoms, and thereby bridges the historical concepts of both toxin production by the pathogen and vascular occlusion as the cause of wilting symptoms.

The arms race between fungal pathogens and plant hosts involves recognition of fungal effectors to induce host immunity. Although various fungal effectors have been identified, the effector functions of ribonucleases are largely unknown. Herein, we identified a ribonuclease secreted by Verticillium dahliae (VdRTX1) that translocates into the plant nucleus to modulate immunity. The activity of VdRTX1 causes hypersensitive response (HR)-related cell death in Nicotiana benthamiana and cotton. VdRTX1 possesses a signal peptide but is unlikely to be an apoplastic effector because its nuclear localization in the plant is necessary for cell death induction. Knockout of VdRTX1 significantly enhanced V. dahliae virulence on tobacco while V. dahliae employs the known suppressor VdCBM1 to escape the immunity induced by VdRTX1. VdRTX1 homologs are widely distributed in fungi but transient expression of 24 homologs from other fungi did not yield cell death induction, suggesting that this function is specific to the VdRTX1 in V. dahliae. Expression of site-directed mutants of VdRTX1 in N. benthamiana leaves revealed conserved ligand-binding sites that are important for VdRTX1 function in inducing cell death. Thus, VdRTX1 functions as a unique HR-inducing effector in V. dahliae that contributes to the activation of plant immunity.

Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. However, these functions have not been assessed in Verticillium dahliae, a soil-borne fungal pathogen that causes devastating wilt diseases in many crops. The discovery and identity of novel lncRNAs and their association with virulence may contribute to an increased understanding of the regulation of virulence in V. dahliae. Here, we identified a total of 352 lncRNAs in V. dahliae. The lncRNAs were transcribed from all V. dahliae chromosomes, typically with shorter open reading frames, lower GC content, and fewer exons than protein-coding genes. In addition, 308 protein-coding genes located within 10 kb upstream and 10 kb downstream of lncRNAs were identified as neighboring genes, and which were considered as potential targets of lncRNA. These neighboring genes encode products involved in development, stress responses, and pathogenicity of V. dahliae, such as transcription factors (TF), kinase, and members of the secretome. Furthermore, 47 lncRNAs were significantly differentially expressed in V. dahliae following inoculation of susceptible cotton (Gossyoiumhisutum) cultivar Junmian No.1, suggesting that lncRNAs may be involved in the regulation of virulence in V. dahliae. Moreover, correlations in expression patterns between lncRNA and their neighboring genes were detected. Expression of lncRNA012077 and its neighboring gene was up-regulated 6 h following inoculation of cotton, while the expression of lncRNA007722 was down-regulated at 6 h but up-regulated at 24 h, in a pattern opposite to that of its neighboring gene. Overexpression of lncRNA012077 in wild-type strain (Vd991) enhanced its virulence on cotton while overexpression of lncRNA009491 reduced virulence. Identification of novel lncRNAs and their association with virulence may provide new targets for disease control.

Verticillium dahliae is a destructive soil-borne pathogen of many economically important dicots. The genetics of pathogenesis in V. dahliae has been extensively studied. Spt-Ada-Gcn5 acetyltransferase complex (SAGA) is an ATP-independent multifunctional chromatin remodeling complex that contributes to diverse transcriptional regulatory functions. As members of the core module in the SAGA complex in Saccharomyces cerevisiae, Ada1, together with Spt7 and Spt20, play an important role in maintaining the integrity of the complex. In this study, we identified homologs of the SAGA complex in V. dahliae and found that deletion of the Ada1 subunit (VdAda1) causes severe defects in the formation of conidia and microsclerotia, and in melanin biosynthesis and virulence. The effect of VdAda1 on histone acetylation in V. dahliae was confirmed by western blot analysis. The deletion of VdAda1 resulted in genome-wide alteration of the V. dahliae transcriptome, including genes encoding transcription factors and secreted proteins, suggesting its prominent role in the regulation of transcription and virulence. Overall, we demonstrated that VdAda1, a member of the SAGA complex, modulates multiple physiological processes by regulating global gene expression that impinge on virulence and survival in V. dahliae.

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.

BackgroundPlant 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.ResultsIn 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.ConclusionsUnder 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.

Phytopathogen xylanases play critical roles in pathogenesis, likely due to their ability to degrade plant structural barriers and manipulate host immunity. As an invader of plant xylem vessels, the fungus Verticillium dahliae is thought to deploy complex cell wall degrading enzymes. Comparative genomics analyses revealed that the V. dahliae genome encodes a family of six xylanases, each possessing a glycosyl hydrolase 11 domain, but the functions of these enzymes are undetermined. Characterizing gene deletion mutants revealed that only V. dahliae xylanase 4 (VdXyn4) degraded the plant cell wall and contributed to the virulence of V. dahliae. VdXyn4 displayed cytotoxic activity and induced a necrosis phenotype during the late stages of infection, leading to vein and petiole collapse that depended on the enzyme simultaneously localizing to nuclei and chloroplasts. The internalization of VdXyn4 was in conjunction with that of the plasma membrane complexLeucine-rich repeat (LRR)-receptor-like kinase suppressor of BIR1-1 (SOBIR1)/LRR-RLK BRI1-associated kinase-1 (BAK1), but we could not rule out the possibility that VdXyn4 may also act as an apoplastic effector. Immune signaling (in the SA-JA pathways) induced by VdXyn4 relative to that induced by known immunity effectors was substantially delayed. While cytotoxic activity could be partially suppressed by known effectors, they failed to impede necrosis in Nicotiana benthamiana. Thus, unlike typical effectors, cytotoxicity of VdXyn4 plays a crucial intracellular role at the late stages of V. dahliae infection and colonization, especially following pathogen entry into the xylem; this cytotoxic activity is likely conserved in the corresponding enzyme families in plant vascular pathogens.

The accumulation of reactive oxygen species (ROS) is a widespread defence mechanism in higher plants against pathogen attack and sometimes is the cause of cell death that facilitates attack by necrotrophic pathogens. Plant pathogens use superoxide dismutase (SOD) to scavenge ROS derived from their own metabolism or generated from host defence. The significance and roles of SODs in the vascular plant pathogen Verticillium dahliae are unclear. Our previous study showed a significant upregulation of Cu/Zn-SOD1 (VdSOD1) in cotton tissues following V. dahliae infection, suggesting that it may play a role in pathogen virulence. Here, we constructed VdSOD1 deletion mutants (ΔSOD1) and investigated its function in scavenging ROS and promoting pathogen virulence. ΔSOD1 had normal growth and conidiation but exhibited significantly higher sensitivity to the intracellular ROS generator menadione. Despite lacking a signal peptide, assays in vitro by western blot and in vivo by confocal microscopy revealed that secretion of VdSOD1 is dependent on the Golgi reassembly stacking protein (VdGRASP). Both menadione-treated ΔSOD1 and cotton roots infected with ΔSOD1 accumulated more O2- and less H2 O2 than with the wildtype strain. The absence of a functioning VdSOD1 significantly reduced symptom severity and pathogen colonization in both cotton and Nicotiana benthamiana. VdSOD1 is nonessential for growth or viability of V. dahliae, but is involved in the detoxification of both intracellular ROS and host-generated extracellular ROS, and contributes significantly to virulence in V. dahliae.

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.

Secreted small cysteine-rich proteins (SCPs) play a critical role in modulating host immunity in plant-pathogen interactions. Bioinformatic analyses showed that the fungal pathogen Verticillium dahliae encodes more than 100 VdSCPs, but their roles in host-pathogen interactions have not been fully characterized. Transient expression of 123 VdSCP-encoding genes in Nicotiana benthamiana identified three candidate genes involved in host-pathogen interactions. The expression of these three proteins, VdSCP27, VdSCP113, and VdSCP126, in N. benthamiana resulted in cell death accompanied by a reactive oxygen species burst, callose deposition, and induction of defence genes. The three VdSCPs mainly localized to the periphery of the cell. BAK1 and SOBIR1 (associated with receptor-like protein) were required for the immunity triggered by these three VdSCPs in N. benthamiana. Site-directed mutagenesis showed that cysteine residues that form disulphide bonds are essential for the functioning of VdSCP126, but not VdSCP27 and VdSCP113. VdSCP27, VdSCP113, and VdSCP126 individually are not essential for V. dahliae infection of N. benthamiana and Gossypium hirsutum, although there was a significant reduction of virulence on N. benthamiana and G. hirsutum when inoculated with the VdSCP27/VdSCP126 double deletion strain. These results illustrate that the SCPs play a critical role in the V. dahliae-plant interaction via an intrinsic virulence function and suppress immunity following infection.

The lettuce downy mildew pathogen, Bremia lactucae, is an obligate oomycete that causes extensive produce losses. Initial chlorotic symptoms that severely reduce the market value of the produce are followed by the appearance of white, downy sporulation on the abaxial side of the leaves. These spores become airborne and disseminate the pathogen. Controlling lettuce downy mildew has relied on repeated fungicide applications to prevent outbreaks. However, in addition to direct economic costs, heterogeneity and rapid adaptation of this pathogen to repeatedly applied fungicides has led to the development of fungicide-insensitivity in the pathogen. We deployed a quantitative PCR assay-based detection method using a species-specific DNA target for B. lactucae coupled with a spore trap system to measure airborne B. lactucae spore loads within three commercial fields that each contained experimental plots, designated EXP1 to EXP3. Based upon these measurements, when the spore load in the air reached a critical level (8.548 sporangia per m3 air), we advised whether or not to apply fungicides on a weekly basis within EXP1 to EXP3. This approach saved three sprays in EXP1, and one spray each in EXP2 and EXP3 without a significant increase in disease incidence. The reduction in fungicide applications to manage downy mildew can decrease lettuce production costs while slowing the development of fungicide resistance in B. lactucae by eliminating unnecessary fungicide applications.

Soilborne plant pathogenic species in the fungal genus Verticillium cause destructive Verticillium wilt disease on economically important crops worldwide. Since R gene-mediated resistance is only effective against race 1 of V. dahliae, fortification of plant basal resistance along with cultural practices are essential to combat Verticillium wilts. Plant hormones involved in cell signaling impact defense responses and development, an understanding of which may provide useful solutions incorporating aspects of basal defense. In this review, we examine the current knowledge of the interplay between plant hormones, salicylic acid, jasmonic acid, ethylene, brassinosteroids, cytokinin, gibberellic acid, auxin, and nitric oxide, and the defense responses and signaling pathways that contribute to resistance and susceptibility in Verticillium-host interactions. Though we make connections where possible to non-model systems, the emphasis is placed on Arabidopsis-V. dahliae and V. longisporum interactions since much of the research on this interplay is focused on these systems. An understanding of hormone signaling in Verticillium-host interactions will help to determine the molecular basis of Verticillium wilt progression in the host and potentially provide insight on alternative approaches for disease management.

Verticillium dahliae is a soil-borne fungus that causes vascular wilt on numerous plants worldwide. The fungus survives in the soil for up to 14 years by producing melanized microsclerotia. The protective function of melanin in abiotic stresses is well documented. Here, we found that the V. dahliae tetraspan transmembrane protein VdSho1, a homolog of the Saccharomyces cerevisiae Sho1, acts as an osmosensor, and is required for plant penetration and melanin biosynthesis. The deletion mutant ΔSho1 was incubated on a cellophane membrane substrate that mimics the plant epidermis, revealing that the penetration of ΔSho1 strain was reduced compared to the wild-type strain. Furthermore, VdSho1 regulates melanin biosynthesis by a signalling mechanism requiring a kinase-kinase signalling module of Vst50-Vst11-Vst7. Strains, ΔVst50, ΔVst7 and ΔVst11 also displayed defective penetration and melanin production like the ΔSho1 strain. Defects in penetration and melanin production in ΔSho1 were restored by overexpression of Vst50, suggesting that Vst50 lies downstream of VdSho1 in the regulatory pathway governing penetration and melanin biosynthesis. Data analyses revealed that the transmembrane portion of VdSho1 was essential for both membrane penetration and melanin production. This study demonstrates that Vst50-Vst11-Vst7 module regulates VdSho1-mediated plant penetration and melanin production in V. dahliae, contributing to virulence.

Key messageTwo QTLs for resistance to lettuce drop, qLDR1.1 and qLDR5.1, were identified. Associated SNPs will be useful in breeding for lettuce drop and provide the foundation for future molecular analysis. Lettuce drop, caused by Sclerotinia minor and S. sclerotiorum, is an economically important disease of lettuce. The association of resistance to lettuce drop with the commercially undesirable trait of fast bolting has hindered the integration of host resistance in control of this disease. Eruption is a slow-bolting cultivar that exhibits a high level of resistance to lettuce drop. Eruption also is completely resistant to Verticillium wilt caused by race 1 of Verticillium dahliae. A recombinant inbred line population from the cross Reine des Glaces × Eruption was genotyped by sequencing and evaluated for lettuce drop and bolting in separate fields infested with either S. minor or V. dahliae. Two quantitative trait loci (QTLs) for lettuce drop resistance were consistently detected in at least two experiments, and two other QTLs were identified in another experiment; the alleles for resistance at all four QTLs originated from Eruption. A QTL for lettuce drop resistance on linkage group (LG) 5, qLDR5.1, was consistently detected in all experiments and explained 11 to 25% of phenotypic variation. On LG1, qLDR1.1 was detected in two experiments explaining 9 to 12% of the phenotypic variation. Three out of four resistance QTLs are distinct from QTLs for bolting; qLDR5.1 is pleiotropic or closely linked with a QTL for early bolting; however, the rate of bolting shows only a small effect on the variance in resistance observed at this locus. The SNP markers linked with these QTLs will be useful in breeding for resistance through marker-assisted selection.

NONRACE-SPECIFIC DISEASE RESISTANCE (NDR1) is a widely characterized gene that plays a key role in defense against multiple bacterial, fungal, oomycete and nematode plant pathogens. NDR1 is required for activation of resistance by multiple NB and LRR-containing (NLR) protein immune sensors and contributes to basal defense. The role of NDR1 in positively regulating salicylic acid (SA)-mediated plant defense responses is well documented. However, ndr1-1 plants flower earlier and show accelerated development in comparison to wild type (WT) Arabidopsis plants, indicating that NDR1 is a negative regulator of flowering and growth. Exogenous application of gibberellic acid (GA) further accelerates the early flowering phenotype in ndr1-1 plants, while the GA biosynthesis inhibitor paclobutrazol attenuated the early flowering phenotype of ndr1-1, but not to WT levels, suggesting partial resistance to paclobutrazol and enhanced GA response in ndr1-1 plants. Mass spectroscopy analyses confirmed that ndr1-1 plants have 30-40% higher levels of GA3 and GA4, while expression of various GA metabolic genes and major flowering regulatory genes is also altered in the ndr1-1 mutant. Taken together this study provides evidence of crosstalk between the ndr1-1-mediated defense and GA-regulated developmental programs in plants.

Following publication of the original article [1], the author reported a processing error in Figure 5. This has been corrected in the original article.

BackgroundVerticillium wilt caused by the fungus Verticillium dahliae race 1 is among the top disease concerns for lettuce in the Salinas and Pajaro Valleys of coastal central California. Resistance of lettuce against V. dahliae race 1 was previously mapped to the single dominant Verticillium resistance 1 (Vr1) locus. Lines of tomato resistant to race 1 are known to contain the closely linked Ve1 and Ve2 genes that encode receptor-like proteins with extracellular leucine-rich repeats; the Ve1 and Ve2 proteins act antagonistically to provide resistance against V. dahliae race 1. The Vr1 locus in lettuce contains a cluster of several genes with sequence similarity to the tomato Ve genes. We used genome sequencing and/or PCR screening along with pathogenicity assays of 152 accessions of lettuce to investigate allelic diversity and its relationship to race 1 resistance in lettuce.ResultsThis approach identified a total of four Ve genes: LsVe1, LsVe2, LsVe3, and LsVe4. The majority of accessions, however, contained a combination of only three of these LsVe genes clustered on chromosomal linkage group 9 (within ~ 25 kb in the resistant cultivar La Brillante and within ~ 127 kb in the susceptible cultivar Salinas).ConclusionsA single allele, LsVe1L, was present in all resistant accessions and absent in all susceptible accessions. This allele can be used as a molecular marker for V. dahliae race 1 resistance in lettuce. A PCR assay for rapid detection of race 1 resistance in lettuce was designed based on nucleotide polymorphisms. Application of this assay allows identification of resistant genotypes in early stages of plant development or at seed-level without time- and labor-intensive testing in the field.

Improving genetic resistance is a preferred method to manage Verticillium wilt of cotton and other hosts. Identifying host resistance is difficult because of the dearth of resistance genes against this pathogen. Previously, a novel candidate gene involved in Verticillium wilt resistance was identified by a genome-wide association study using a panel of Gossypium hirsutum accessions. In this study, we cloned the candidate resistance gene from cotton that encodes a protein sharing homology with the TIR-NBS-LRR receptor-like defence protein DSC1 in Arabidopsis thaliana (hereafter named GhDSC1). GhDSC1 expressed at higher levels in response to Verticillium wilt and jasmonic acid (JA) treatment in resistant cotton cultivars as compared to susceptible cultivars and its product was localized to nucleus. The transfer of GhDSC1 to Arabidopsis conferred Verticillium resistance in an A. thaliana dsc1 mutant. This resistance response was associated with reactive oxygen species (ROS) accumulation and increased expression of JA-signalling-related genes. Furthermore, the expression of GhDSC1 in response to Verticillium wilt and JA signalling in A. thaliana displayed expression patterns similar to GhCAMTA3 in cotton under identical conditions, suggesting a coordinated DSC1 and CAMTA3 response in A. thaliana to Verticillium wilt. Analyses of GhDSC1 sequence polymorphism revealed a single nucleotide polymorphism (SNP) difference between resistant and susceptible cotton accessions, within the P-loop motif encoded by GhDSC1. This SNP difference causes ineffective activation of defence response in susceptible cultivars. These results demonstrated that GhDSC1 confers Verticillium resistance in the model plant system of A. thaliana, and therefore represents a suitable candidate for the genetic engineering of Verticillium wilt resistance in cotton.

Verticillium dahliae is a broad host-range pathogen that causes vascular wilts in plants. Interactions between three hosts and specific V. dahliae genotypes result in severe defoliation. The underlying mechanisms of defoliation are unresolved. Genome resequencing, gene deletion and complementation, gene expression analysis, sequence divergence, defoliating phenotype identification, virulence analysis, and quantification of V. dahliae secondary metabolites were performed. Population genomics previously revealed that G-LSR2 was horizontally transferred from the fungus Fusarium oxysporum f. sp. vasinfectum to V. dahliae and is exclusively found in the genomes of defoliating (D) strains. Deletion of seven genes within G-LSR2, designated as VdDf genes, produced the nondefoliation phenotype on cotton, olive, and okra but complementation of two genes restored the defoliation phenotype. Genes VdDf5 and VdDf6 associated with defoliation shared homology with polyketide synthases involved in secondary metabolism, whereas VdDf7 shared homology with proteins involved in the biosynthesis of N-lauroylethanolamine (N-acylethanolamine (NAE) 12:0), a compound that induces defoliation. NAE overbiosynthesis by D strains also appears to disrupt NAE metabolism in cotton by inducing overexpression of fatty acid amide hydrolase. The VdDfs modulate the synthesis and overproduction of secondary metabolites, such as NAE 12:0, that cause defoliation either by altering abscisic acid sensitivity, hormone disruption, or sensitivity to the pathogen.

Verticillium dahliae is an important soil-borne fungal pathogen that causes great yield losses in many cash crops. Effectors of this fungus are known to regulate plant immunity but the mechanism much remains unclear. A glycine-rich nuclear effector, VdCE51, was able to suppress immune responses in tobacco against Botrytis cinerea and Sclerotinia sclerotiorum. This effector was a required factor for full virulence of V. dahliae, and its nuclear localization was a requisite for suppressing plant immunity. The thioredoxin GhTRXH2, identified as a positive regulator of plant immunity, was a host target of VdCE51. Our findings show a virulence regulating mechanism whereby the secreted nuclear effector VdCE51 interferes with the transcription of PR genes, and the SA signaling pathway by inhibiting the accumulation of GhTRXH2, thus suppressing plant immunity. [ABSTRACT FROM AUTHOR]

Wilt caused by Verticillium dahliae significantly reduces cotton yields, as host resistance in commercially cultivated Gossypium species is lacking. Understanding the molecular basis of disease resistance in non-commercial Gossypium species could galvanize the development of Verticillium wilt resistance in cultivated species. Nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins play a central role in plant defence against pathogens. In this study, we focused on the relationship between a locus enriched with eight NBS-LRR genes and Verticillium wilt resistance in G. barbadense. Independent virus-induced gene silencing of each of the eight NBS-LRR genes in G. barbadense cultivar Hai 7124 revealed that silencing of GbaNA1 alone compromised the resistance of G. barbadense to V. dahliae isolate Vd991. In cultivar Hai 7124, GbaNA1 could be induced by V. dahliae isolate Vd991 and by ethylene, jasmonic acid and salicylic acid. Nuclear protein localization of GbaNA1 was demonstrated by transient expression. Sequencing of the GbaNA1 orthologue in nine G. hirsutum accessions revealed that all carried a non-functional allele, caused by a premature peptide truncation. In addition, all 10 G. barbadense and nine G. hirsutum accessions tested carried a full-length (∼1140 amino acids) homologue of the V. dahliae race 1 resistance gene Gbve1, although some sequence polymorphisms were observed. Verticillium dahliae Vd991 is a non-race 1 isolate that lacks the Ave1 gene. Thus, the resistance imparted by GbaNA1 appears to be mediated by a mechanism distinct from recognition of the fungal effector Ave1.

Fungal transcription factors (TFs) implicated in the regulation of virulence gene expression have been identified in a number of plant pathogens. In Verticillium dahliae, despite its agricultural importance, few regulators of transcription have been characterized. In this study, a T-DNA insertion mutant with significantly reduced virulence towards cotton was identified. The T-DNA was traced to VdFTF1, a gene encoding a TF containing a Fungal_trans domain. Transient expression in onion epidermal cells indicated that VdFTF1 is localized to the nucleus. The VdFTF1-deletion strains displayed normal vegetative growth, mycelial pigmentation and conidial morphology, but exhibited significantly reduced virulence on cotton, suggesting that VdFTF1 is required exclusively for pathogenesis. Comparisons of global transcription patterns of wild-type and VdFTF1-deletion strains indicated that VdFTF1 affected the expression of 802 genes, 233 of which were associated with catalytic processes. These genes encoded 69 potentially secreted proteins, 43 of which contained a carbohydrate enzyme domain known to participate in pathogenesis during infection of cotton. Targeted gene deletion of one VdFTF1-regulated gene resulted in significantly impaired vascular colonization, as measured by quantitative polymerase chain reaction, as well as aggressiveness and symptom severity in cotton. In conclusion, VdFTF1, which encodes a TF containing a Fungal_trans domain, regulates the gene expression of plant cell wall degradation enzymes in V. dahliae, which are required for full virulence on cotton.

Plant pathogens migrate to new regions through human activities such as trade, where they may establish themselves and cause disease on agriculturally important crops. Verticillium wilt of lettuce, caused by Verticillium dahliae, is a soilborne fungus that was introduced to coastal California via infested spinach seeds. It has caused significant losses for lettuce growers. Once introduced, Verticillium wilt could be managed by fumigating with methyl bromide and chloropicrin, but this option is no longer available. Growers can also manage the disease by planting broccoli or not planting spinach. These control options require long-term investments for future gain. Verticillium wilt can also be prevented or controlled by testing and providing spinach seeds with little or no V. dahliae infestation. However, seed companies have been reluctant to test or clean spinach seeds, as spinach crops are not affected by Verticillium wilt. Thus, available control options are affected by externalities. Renters and other producers with short time horizons will not undertake long-term investments and seed companies do not take into account the effect of their decision not to test on lettuce producers. We review the literature on the economics of managing crop disease; discuss the economics of managing Verticillium wilt; and review the recent research on the externalities that arise with short-term growers, and between seed companies and growers due to Verticillium wilt. An externality arises whenever the actions of one individual or firm affects the payoffs to another individual or firm not involved in a specific transaction. These externalities have important implications for the management of Verticillium wilt and, more broadly, for the management of migratory pathogens and the diseases they cause in agriculture in general. This review is of interest to policy-makers, the producers, marketers, seed companies, and researchers.

Verticillium wilt caused by Verticillium dahliae results in severe losses in cotton, and is economically the most destructive disease of this crop. Improving genetic resistance is the cleanest and least expensive option to manage Verticillium wilt. Previously, we identified the island cotton NBS-LRR-encoding gene GbaNA1 that confers resistance to the highly virulent V. dahliae isolate Vd991. In this study, we expressed cotton GbaNA1 in the heterologous system of Arabidopsis thaliana and investigated the defense response mediated by GbaNA1 following inoculations with V. dahliae. Heterologous expression of GbaNA1 conferred Verticillium wilt resistance in A. thaliana. Moreover, overexpression of GbaNA1 enabled recovery of the resistance phenotype of A. thaliana mutants that had lost the function of GbaNA1 ortholog gene. Investigations of the defense response in A. thaliana showed that the reactive oxygen species (ROS) production and the expression of genes associated with the ethylene signaling pathway were enhanced significantly following overexpression of GbaNA1. Intriguingly, overexpression of the GbaNA1 ortholog from Gossypium hirsutum (GhNA1) in A. thaliana did not induce the defense response of ROS production due to the premature termination of GhNA1, which lacks the encoded NB-ARC and LRR motifs. GbaNA1 therefore confers Verticillium wilt resistance in A. thaliana by the activation of ROS production and ethylene signaling. These results demonstrate the functional conservation of the NBS-LRR-encoding GbaNA1 in a heterologous system, and the mechanism of this resistance, both of which may prove valuable in incorporating GbaNA1-mediated resistance into other plant species.

Verticillium dahliae isolates are most virulent on the host from which they were originally isolated. Mechanisms underlying these dominant host adaptations are currently unknown. We sequenced the genome of V. dahliae Vd991, which is highly virulent on its original host, cotton, and performed comparisons with the reference genomes of JR2 (from tomato) and VdLs.17 (from lettuce). Pathogenicity-related factor prediction, orthology and multigene family classification, transcriptome analyses, phylogenetic analyses, and pathogenicity experiments were performed. The Vd991 genome harbored several exclusive, lineage-specific (LS) genes within LS regions (LSRs). Deletion mutants of the seven genes within one LSR (G-LSR2) in Vd991 were less virulent only on cotton. Integration of G-LSR2 genes individually into JR2 and VdLs.17 resulted in significantly enhanced virulence on cotton but did not affect virulence on tomato or lettuce. Transcription levels of the seven LS genes in Vd991 were higher during the early stages of cotton infection, as compared with other hosts. Phylogenetic analyses suggested that G-LSR2 was acquired from Fusarium oxysporum f. sp. vasinfectum through horizontal gene transfer. Our results provide evidence that horizontal gene transfer from Fusarium to Vd991 contributed significantly to its adaptation to cotton and may represent a significant mechanism in the evolution of an asexual plant pathogen.

Cysteine-rich receptor-like kinases (CRKs) are a large subfamily of plant receptor-like kinases that play a critical role in disease resistance in plants. However, knowledge about the CRK gene family in cotton and its function against Verticillium wilt (VW), a destructive disease caused by Verticillium dahliae that significantly reduces cotton yields is lacking. In this study, we identified a total of 30 typical CRKs in a Gossypium barbadense genome (GbCRKs). Eleven of these (>30%) are located on the A06 and D06 chromosomes, and 18 consisted of 9 paralogous pairs encoded in the A and D subgenomes. Phylogenetic analysis showed that the GbCRKs could be classified into four broad groups, the expansion of which has probably been driven by tandem duplication. Gene expression profiling of the GbCRKs in resistant and susceptible cotton cultivars revealed that a phylogenetic cluster of nine of the GbCRK genes were up-regulated in response to V. dahliae infection. Virus-induced gene silencing of each of these nine GbCRKs independently revealed that the silencing of GbCRK18 was sufficient to compromise VW resistance in G. barbadense. GbCRK18 expression could be induced by V. dahliae infection or jasmonic acid, and displayed plasma membrane localization. Therefore, our expression analyses indicated that the CRK gene family is differentially regulated in response to Verticillium infection, while gene silencing experiments revealed that GbCRK18 in particular confers VW resistance in G. barbadense.

Fusarium oxysporum species complex (FOSC) isolates were obtained from celery with symptoms of Fusarium yellows between 1993 and 2013 primarily in California. Virulence tests and a two-gene dataset from 174 isolates indicated that virulent isolates collected before 2013 were a highly clonal population of F. oxysporum f. sp. apii race 2. In 2013, new highly virulent clonal isolates, designated race 4, were discovered in production fields in Camarillo, California. Long-read Illumina data were used to analyze 16 isolates: six race 2, one of each from races 1, 3, and 4, and seven genetically diverse FOSC that were isolated from symptomatic celery but are nonpathogenic on this host. Analyses of a 10-gene dataset comprising 38 kb indicated that F. oxysporum f. sp. apii is polyphyletic; race 2 is nested within clade 3, whereas the evolutionary origins of races 1, 3, and 4 are within clade 2. Based on 6,898 single nucleotide polymorphisms from the core FOSC genome, race 3 and the new highly virulent race 4 are highly similar with Nei's Da = 0.0019, suggesting that F. oxysporum f. sp. apii race 4 evolved from race 3. Next generation sequences were used to develop PCR primers that allow rapid diagnosis of races 2 and 4 in planta.

Verticillium dahliae is a soilborne fungus that is introduced to the soil via infested spinach seeds and that causes lettuce to be afflicted with Verticillium wilt. This disease has spread rapidly through the Salinas Valley, the prime lettuce production region of California. Verticillium wilt can be prevented or controlled by the grower by fumigating, planting broccoli, or not planting spinach. Because these control options require long-term investment for future gain, renters might not take the steps needed to control Verticillium wilt. Verticillium wilt can also be prevented or controlled by a spinach seed company through testing and cleaning the spinach seeds. However, seed companies are unwilling to test or clean spinach seeds, as they are not affected by this disease. We discuss our research on the externalities that arise with renters, and between seed companies and growers, due to Verticillium wilt. These externalities have important implications for the management of Verticillium wilt in particular, and for the management of diseases in agriculture in general.

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

Downy mildew of spinach (Spinacia oleracea), caused by Peronospora effusa, is a production constraint on production worldwide, including in California, where the majority of U.S. spinach is grown. The aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay for detection of airborne inoculum of P. effusa in California. Among oomycete ribosomal DNA (rDNA) sequences examined for assay development, the highest nucleotide sequence identity was observed between rDNA sequences of P. effusa and P. schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris subsp. vulgaris). Single-nucleotide polymorphisms were detected between P. effusa and P. schachtii in the 18S rDNA regions for design of P. effusa- and P. schachtii-specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both P. effusa and P. schachtii rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of P. effusa from impaction spore trap samples collected from spinach production fields. The rDNA copy numbers of P. effusa were, on average, ≈3,300-fold higher from trap samples collected near an infected field compared with those levels recorded at a site without a nearby spinach field. In combination with disease-conducive weather forecasting, application of the assays may be helpful to time fungicide applications for disease management.

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

A 5-year project to facilitate the adoption of strawberry production systems that do not use methyl bromide initially focused on fumigant alternatives and resulted in increased use of barrier films that reduce fumigant emissions. The focus shifted in year 3 to evaluating and demonstrating nonfumigant alternatives: soilless production, biofumigation, anaerobic soil disinfestation (ASD) and disinfestation with steam. In the 2010–2011 strawberry production season, fruit yields on substrates were comparable to fruit yields using conventional methods. Anaerobic soil disinfestation and steam disinfestation also resulted in fruit yields that were comparable to those produced using conventionally fumigated soils. Additional work is in progress to evaluate their efficacy in larger-scale production systems in different strawberry production districts in California.