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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.

Genomics has emerged as a great tool in enhancing our understanding of the biology of Verticillium species and their interactions with the host plants. Through different genomic approaches, researchers have gained insights into genes, pathways and virulence factors that play crucial roles in both Verticillium pathogenesis and the defense responses of their host organisms. This review emphasizes the significance of genomics in uncovering the mechanisms that underlie pathogenicity, virulence, and host resistance in Verticillium fungi. Our goal is to summarize recent discoveries in Verticillium research highlighting progress made in comprehending the biology and interactions of Verticillium fungi. The integration of genomics into Verticillium studies has the potential to open avenues for developing strategies to control diseases and produce crop varieties resistant to verticillium, thereby offering sustainable solutions for enhancing agricultural productivity. [ABSTRACT FROM AUTHOR]

Verticillium longisporum is a vascular fungal pathogen of oilseed rape (OSR) that causes Verticillium stem striping. The pathogen segregates into three lineages originating from at least three hybridization events between four different Verticillium genomes, leading to the hybrid lineages A1/D1, A1/D2 and A1/D3. Although A1/D1 is considered the most relevant lineage in OSR, extensive studies addressing the geographical spread and phylogenetic relationships of Verticillium lineages are lacking. In the present study, the genetic diversity and relatedness of V. longisporum isolates from European and Canadian OSR fields were analysed. Based on genotyping by sequencing, genetic subgroups were identified and assigned to geographic origins and pathogenic traits. To assess the agronomic importance of different lineages, representative isolates were tested for pathogenicity and aggressiveness on OSR under greenhouse conditions. This study confirms that A1/D1 is the prevalent and most aggressive lineage of V. longisporum in European and Canadian OSR fields. Genetic clusters within the A1/D1 lineage were weakly correlated with geographic origin, but not with aggressiveness on winter OSR. Isolates from the United Kingdom, Latvia and Canada occurred only in a single and distinct subclade, indicating a more recent introduction, whereas isolates from locations where the disease has been known for a long time (France, Germany, Poland, Sweden) were less uniform and could be assigned to several subclades. Our results indicate that Verticillium stem striping in the United Kingdom, Latvia and Canada may have derived from separate and different single introductions of the pathogen in the recent past. [ABSTRACT FROM AUTHOR]

BACKGROUND: Melanin plays important roles in morphological development, survival, host-pathogen interactions and in the virulence of phytopathogenic fungi. In Verticillum dahliae, increases in melanin are recognized as markers of maturation of microsclerotia which ensures the long-term survival and stress tolerance, while decreases in melanin are correlated with increased hyphal growth in the host. The conserved upstream components of the VdCmr1-regulated pathway controlling melanin production in V. dahliae have been extensively identified, but the direct activators of this pathway are still unclear. RESULTS: We identified two genes encoding conserved C2H2-type zinc finger proteins VdZFP1 and VdZFP2 adjacent to VdPKS9, a gene encoding a negative regulator of both melanin biosynthesis and microsclerotia formation in V. dahliae. Both VdZFP1 and VdZFP2 were induced during microsclerotia development and were involved in melanin deposition. Their localization changed from cytoplasmic to nuclear in response to osmotic pressure. VdZFP1 and VdZFP2 act as modulators of microsclerotia melanization in V. dahliae, as confirmed by melanin biosynthesis inhibition and supplementation with the melanin pathway intermediate scytalone in albino strains. The results indicate that VdZFP1 and VdZFP2 participate in melanin biosynthesis by positively regulating VdCmr1. Based on the results obtained with yeast one- and two-hybrid (Y1H and Y2H) and bimolecular fluorescence complementation (BiFC) systems, we determined the melanin biosynthesis relies on the direct interactions among VdZFP1, VdZFP2 and VdCmr1, and these interactions occur on the cell walls of microsclerotia. Additionally, VdZFP1 and/or VdZFP2 mutants displayed increased sensitivity to stress factors rather than alterations in pathogenicity, reflecting the importance of melanin in stress tolerance of V. dahliae. CONCLUSIONS: Our results revealed that VdZFP1 and VdZFP2 positively regulate VdCmr1 to promote melanin deposition during microsclerotia development, providing novel insight into the regulation of melanin biosynthesis in V. dahliae.

Plant defense responses to the soil-borne fungus Verticillium longisporum causing stem stripe disease on oilseed rape (Brassica napus) are poorly understood. In this study, a population of recombinant inbred lines (RILs) using the Arabidopsis accessions Sei-0 and Can-0 was established. Composite interval mapping, transcriptome data, and T-DNA mutant screening identified the NITRATE/PEPTIDE TRANSPORTER FAMILY 5.12 (AtNPF5.12) gene as being associated with disease susceptibility in Can-0. Co-immunoprecipitation revealed interaction between AtNPF5.12 and the MAJOR LATEX PROTEIN family member AtMLP6, and fluorescence microscopy confirmed this interaction in the plasma membrane and endoplasmic reticulum. CRISPR/Cas9 technology was applied to mutate the NPF5.12 and MLP6 genes in B. napus. Elevated fungal growth in the npf5.12 mlp6 double mutant of both oilseed rape and Arabidopsis demonstrated the importance of these genes in defense against V. longisporum. Colonization of this fungus depends also on available nitrates in the host root. Accordingly, the negative effect of nitrate depletion on fungal growth was less pronounced in Atnpf5.12 plants with impaired nitrate transport. In addition, suberin staining revealed involvement of the NPF5.12 and MLP6 genes in suberin barrier formation. Together, these results demonstrate a dependency on multiple plant factors that leads to successful V. longisporum root infection. [ABSTRACT FROM AUTHOR]

The plant parasitic nematode species Pratylenchus penetrans has been known to form a synergistic relationship with the fungal plant pathogen Verticillium dahliae in potato cropping systems across North America. The results of this interaction can be devastating; with plants suffering from choloris, wilting, and premature death or complete yield loss in infected fields. Many studies have been conducted in order to understand how this symbiotic connection is occurring; whether that be from competition within the soil microbial community, through the development of amendment-induced suppressive soils, or from other interactions caused by nematode activity. While the mechanisms behind the hostile interaction between V. dahliae and P. penetrans are undetermined, the purpose of this review is to compile recent developments relating to this symbiotic relationship, the effects the soil microbial community has on said relationship, as well as identify potential gaps in research. [ABSTRACT FROM AUTHOR]

Verticillium stripe, caused by Verticillium longisporum, is an emerging disease of canola (Brassica napus) in Canada. Studies were conducted to assess the impact of pH on both the growth of V. longisporum and its virulence on the canola host. Fungal growth was assessed by measuring the colony diameter following 14 and 21 days of incubation on potato dextrose agar at varying pH levels (4.7, 5.5, 6.5, 7.4, or 8.6). The results indicated that colonies of V. longisporum were approximately 16% greater in diameter at pH 7.4 and 8.6 compared with those at pH 5.5. The impact of pH on disease development at the seedling stage was investigated using a semi-hydroponic system with different pH levels of 4.4, 5.4, 6.3, 7.5, and 8.4 in half-strength Hoagland's solution. Verticillium stripe was most severe at pH 7.5 and 8.4 after a 10-day period in the semi-hydroponic system. In a second inoculation experiment, canola seedlings previously inoculated with the fungus were transplanted into potting mix amended to four pH levels (5.6, 6.4, 7.2, and 7.8). The transplants were cultivated under greenhouse conditions and evaluated for Verticillium stripe severity at plant maturity. Disease severity was greatest at pH 7.8. This is the first study on the effects of pH on V. longisporum in canola. It suggests a substantial risk of increased disease severity and yield losses due to Verticillium stripe in regions with neutral to slightly alkaline soils. [ABSTRACT FROM AUTHOR]

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.

BACKGROUND: The extracellular space between the cell wall and plasma membrane is a battlefield in plant-pathogen interactions. Within this space, the pathogen employs its secretome to attack the host in a variety of ways, including immunity manipulation. However, the role of the plant secretome is rarely studied for its role in disease resistance. RESULTS: Here, we examined the secretome of Verticillium wilt-resistant Gossypium hirsutum cultivar Zhongzhimian No.2 (ZZM2, encoding 95,327 predicted coding sequences) to determine its role in disease resistance against the wilt causal agent, Verticillium dahliae. Bioinformatics-driven analyses showed that the ZZM2 genome encodes 2085 secreted proteins and that these display disequilibrium in their distribution among the chromosomes. The cotton secretome displayed differences in the abundance of certain amino acid residues as compared to the remaining encoded proteins due to the localization of these putative proteins in the extracellular space. The secretome analysis revealed conservation for an allotetraploid genome, which nevertheless exhibited variation among orthologs and comparable unique genes between the two sub-genomes. Secretome annotation strongly suggested its involvement in extracellular stress responses (hydrolase activity, oxidoreductase activity, and extracellular region, etc.), thus contributing to resistance against the V. dahliae infection. Furthermore, the defense response genes (immunity marker NbHIN1, salicylic acid marker NbPR1, and jasmonic acid marker NbLOX4) were activated to varying degrees when Nicotina benthamiana leaves were agro-infiltrated with 28 randomly selected members, suggesting that the secretome plays an important role in the immunity response. Finally, gene silencing assays of 11 members from 13 selected candidates in ZZM2 displayed higher susceptibility to V. dahliae, suggesting that the secretome members confer the Verticillium wilt resistance in cotton. CONCLUSIONS: Our data demonstrate that the cotton secretome plays an important role in Verticillium wilt resistance, facilitating the development of the resistance gene markers and increasing the understanding of the mechanisms regulating disease resistance.

Genomics has emerged as a great tool in enhancing our understanding of the biology of Verticillium species and their interactions with the host plants. Through different genomic approaches, researchers have gained insights into genes, pathways and virulence factors that play crucial roles in both Verticillium pathogenesis and the defense responses of their host organisms. This review emphasizes the significance of genomics in uncovering the mechanisms that underlie pathogenicity, virulence, and host resistance in Verticillium fungi. Our goal is to summarize recent discoveries in Verticillium research highlighting progress made in comprehending the biology and interactions of Verticillium fungi. The integration of genomics into Verticillium studies has the potential to open avenues for developing strategies to control diseases and produce crop varieties resistant to verticillium, thereby offering sustainable solutions for enhancing agricultural productivity.

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.

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.

Symptoms of infection okra plants (Abelmoschus esculentus L.) with Verticillium wilt were recorded during the summer of 2021 in Beni Suef Governorate specifically in Nasser, Beba, and El-Wasta Counties. The symptoms appeared on the external parts of plants, specifically through the alteration of color in the leaves of infected shoots, which transition from a normal color to a light green or yellow hue, had V-shaped chlorotic lesions, loss their turgidity and eventually desiccation, plant stunting and vascular discoloration. The presence of V. tricorpus on okra has not been previously documented. The isolated fungus was identified on the basis of morphological characteristics and molecular traits by utilizing sequencing techniques applied to the internal transcribed space (ITS region) of the ribosomal RNA gene. The analysis of the fungal sequencing data revealed a substantial genetic similarity and close ties between the fungus in question and V. tricorpus by 99.24–100% identity and 97–100% coverage with several strains of V. tricorpus, including the type strain CBS447.54 (NR_126128). The obtained sequences were submitted to the GenBank database and assigned the accession number MZ936483. Pathogenicity test confirmed that V. tricorpus had pathogenic properties, as shown by the appearance of disease symptoms that were similar to those previously noted on okra plants in the surveyed regions. This observation represents the initial documentation of V. tricorpus in relation to the occurrence of Verticillium wilt disease in okra. However, there is currently no information of host resistance to this disease among Egyptian okra cultivars, although it is essential for sustainable disease management. Our study indicate that cultivar of Balady green is more resistance to Verticillium wilt while the Iranian red cv. is more sensitive to infection. [ABSTRACT FROM AUTHOR]

Verticillium longisporum (Vl43) is a soilborne hemibiotrophic fungal pathogen causing stem striping on oilseed rape (OSR) and severe yield losses. Breeding for resistant varieties is the most promising approach to control this disease. Here, we report the identification of Hva22c as a novel susceptibility factor and its potential for improving OSR resistance. Hva22c is a member of the Hva22 gene family, originally described for barley (Hordeum vulgare). Several Hva22 members have been located at the endoplasmic reticulum. Hva22c is up-regulated in response to Vl43 in both Arabidopsis and OSR. We demonstrate that knock-out of Hva22c in OSR by CRISPR/Cas9 and its homolog in Arabidopsis by T-DNA insertion reduced plants' susceptibility to Vl43 infection and impaired the development of disease symptoms. To understand the underlying mechanism, we analysed transcriptomic data from infected and non-infected roots of hva22c knock-out and wild type plants. We identified a homozygous mutant with frame-shifts in all four BnHva22c loci displaying a vastly altered transcriptional landscape at 6 dpi. Significantly, a large set of genes was suppressed under mock conditions including genes related to the endomembrane systems. Among the up-regulated genes we found several defense-related and phytohormone-responsive genes when comparing mutant to the wild type. These results demonstrate that Hva22c is functionally required for a fully compatible plant-fungus interaction. Its loss of function reduces plant susceptibility, most likely due to endoplasmatic reticulum and Golgi dysfunction accompanied by additionally activated defense responses. These findings can help improve OSR resistance to V. longisporum infection. [ABSTRACT FROM AUTHOR]

Five stems of rapeseed with abundant black microsclerotia were collected from Huangyuan County of Qinghai Province, China, and fungal isolates were obtained from the stems. They were identified based on morphology, molecular features and specific PCR detection. The results showed that the 10 fungal isolates belonged to Verticillium longisporum lineage A1/D3. One of the 10 isolates (HW7-1) was tested for virulence on three species of rapeseed, including B. napus Zhongshuang 9, B. rapa Qingyou 9 and B. juncea Tayou 2 by conidia inoculation of HW7-1 on roots of young seedlings. Control seedlings were inoculated with V. dahliae conidia or water alone. The seedlings of these treatments were transplanted in culture mix and incubated in a growth chamber (20 ℃). Results suggested that the control seedlings of three cultivars appeared quite healthy, while the seedlings inoculated with HW7-1 turned yellowing leaves, seedling stunting or even death after 22 days post-inoculation. V. longisporum was re-isolated from he yellow leaves, thus fulfilling Koch's postulates. Moreover, compared to the control treatments, inoculation with HW7-1 caused flowering delay and seed yield reduction on Tayou 2 with production of microsclerotia on the stems. To our knowledge, this is the first report of V. longisporum lineage A1/D3 on rapeseed in northwestern China. [ABSTRACT FROM AUTHOR]

The experiments were carried out on 5 different bioproducts for control of Verticillium longisporum in oilseed rape. For in vitro trials, greenhouse trials and field trials, the bioproducts used were based on bacteria (Bacillus amyloliquefaciens, Pseudomonas veronii) and fungi (Pythium oligandrum, Trichoderma asperellum, Coniothyrium minitans). In in vitro trials, some products (based on Trichoderma asperellum) showed a 100% inhibition effect on the pathogen for a whole trial period of 45 days. The greenhouse trial showed significant differences in bioproducts effectiveness (P < 0.00723) compared to control. Three bioproducts (based on Coniothyrium minitans, Trichoderma asperellum and Pseudomonas veronii) showed a 100% inhibition effect against the pathogen. In field trials conducted in 3 locations, there were some differences in yield, which can be important for growers and practice use. The highest yield (19.1% higher than the control) was achieved with a bioproduct based on Trichoderma asperellum. In trials, it is possible to see that there are promising results that can be used for further testing. [ABSTRACT FROM AUTHOR]

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.

The darkling beetle, also known as the rust-red flour beetle Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a major pest that destroys grains and flour. It is also commonly used for research on ethology and food safety. The use of entomopathogenic fungi (Beauveria bassiana, Metarhizium anisopliae, and Verticillium lecanii) and chemical insecticides to control T. castaneum has been limited due to concerns about environmental sustainability. As a result, there is a pressing need to find environmentally friendly insecticides against this pest. This study evaluates the effects of entomopathogenic fungi and an extract from the green shoots of Arta, Calligonum comosum L'Hér., on adult T. castaneum. The fungi used in this study were isolated from soil in Egypt, while the plant extract samples were collected from natural vegetation in southern Sinai, Egypt. Different concentrations of the ethanolic plant extract (50, 75, and 100%) were applied to T. castaneum, and the effects were observed every 3 hours over a 24 hrs. period. The entomopathogenic fungi proved to be effective against adults of T. castaneum, B. bassiana, and V. lecanii causing 86.67% and 60.0% mortality, respectively, after seven days of infection at a concentration labeled as (C3). M. anisopliae, on the other hand, resulted in 100% mortality after seven days when infecting the (C3) concentration. The shoot extract of Arta, C. comosum, also exhibited effectiveness, with total mortality rates recorded after 48, 72, and 72 hrs. corresponding to concentrations of 100, 75, and 50, respectively. This study concludes that both the entomopathogenic fungi and C. comosum extract contain bioactive compounds that are toxic to T. castaneum. [ABSTRACT FROM AUTHOR]

Verticillium longisporum is a soilborne fungal vascular pathogen of Brassica crops. Three hybrid lineages originating from three independent hybridization events of four haploid Verticillium parents are known. V. longisporum lineage A1/D2 has only been found in horseradish and previous pathogenicity tests have shown that it is non‐aggressive on oilseed rape (Brassica napus). The aim of this study was to investigate the interaction of the non‐aggressive lineage A1/D2 with oilseed rape in comparison with the aggressive A1/D1 lineage. Although A1/D2 did not cause severe disease symptoms, the study revealed restricted root infection and systemic colonization by this lineage, as assessed by quantitative PCR of fungal DNA. Assessment of root surface colonization by confocal laser microscopy indicated early sporulation, disorganized loosely attached hyphal growth and hyphal coiling of A1/D2. By contrast, A1/D1 showed organized hyphal growth tight to the root surface, as well as hyphal swellings at points of penetration. Vascular colonization was studied with transmission electron microscopy, which revealed less vessel colonization, higher intensity of vascular responses and lower plant cell wall degradation in A1/D2‐treated plants in comparison with A1/D1. Vascular responses consisted of strong coating of vessel walls and fungal hyphae with an electron‐dense material, indicating higher intensity of plant defence in comparison with A1/D1. The results, for the first time, demonstrate stem colonization and thus a basic level of pathogenic interaction of non‐aggressive strains of A1/D2 with oilseed rape associated with a lack of severe disease symptoms, which indicate tolerance in oilseed rape to lineage A1/D2. [ABSTRACT FROM AUTHOR]

The plant parasitic nematode species Pratylenchus penetrans has been known to form a synergistic relationship with the fungal plant pathogen Verticillium dahliae in potato cropping systems across North America. The results of this interaction can be devastating; with plants suffering from choloris, wilting, and premature death or complete yield loss in infected fields. Many studies have been conducted in order to understand how this symbiotic connection is occurring; whether that be from competition within the soil microbial community, through the development of amendment-induced suppressive soils, or from other interactions caused by nematode activity. While the mechanisms behind the hostile interaction between V. dahliae and P. penetrans are undetermined, the purpose of this review is to compile recent developments relating to this symbiotic relationship, the effects the soil microbial community has on said relationship, as well as identify potential gaps in research.

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.

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.

Restructuring tilth layers (RTL) is a tillage method that exchanges the 0-20 and 20-40 cm soil layers that can be applied during cotton cultivation to increase cotton yield, eliminate weeds and alleviate severe disease, including Verticillium wilt. However, the mechanism by which RTL inhibits Verticillium wilt is unclear. Therefore, we investigated the distribution of microbial communities after rotary tillage (CK) and RTL treatments to identify the reasons for the reduction of Verticillium wilt in cotton fields subjected to RTL. Illumina high-throughput sequencing was used to sequence the bacterial and fungal genes. The disease incidence and severity of Verticillium wilt decreased by 28.57% and 42.64%, respectively, after RTL. Moreover, RTL significantly enhanced bacterial richness and evenness at 20-40 cm and -reduced the differences in fungal evenness and richness between soil depths of 0-20 and 20-40 cm. The number of Verticillium dahliae decreased, while the relative abundance of biocontrol bacteria such as Bacillus and Pseudoxanthomonas increased significantly following RTL. Overall RTL improved bacterial diversity, decreased the number of Verticillium dahliae and increased the relative abundance of biocontrol bacteria, which may have suppressed the occurrence of Verticillium wilt in cotton fields. [ABSTRACT FROM AUTHOR]

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Phospholipase A2 (PLA2) is a lipolytic enzyme that hydrolyses phospholipids in the cell membrane. In the present study, we investigated the role of secreted PLA2 (VlsPLA2) in Verticillium longisporum, a fungal phytopathogen that mostly infects plants belonging to the Brassicaceae family, causing severe annual yield loss worldwide. Expression of the VlsPLA2 gene, which encodes active PLA2, is highly induced during the interaction of the fungus with the host plant Brassica napus. Heterologous expression of VlsPLA2 in Nicotiana benthamiana resulted in increased synthesis of certain phospholipids compared to plants in which enzymatically inactive PLA2 was expressed (VlsPLA2ΔCD). Moreover, VlsPLA2 suppresses the hypersensitive response triggered by the Cf4/Avr4 complex, thereby suppressing the chitin‐induced reactive oxygen species burst. VlsPLA2‐overexpressing V. longisporum strains showed increased virulence in Arabidopsis plants, and transcriptomic analysis of this fungal strain revealed that the induction of the gene contributed to increased virulence. VlsPLA2 was initially localized to the host nucleus and then translocated to the chloroplasts at later time points. In addition, VlsPLA2 bound to the vesicle‐associated membrane protein A (VAMPA) and was transported to the nuclear membrane. In the nucleus, VlsPLA2 caused major alterations in the expression levels of genes encoding transcription factors and subtilisin‐like proteases, which play a role in plant immunity. In conclusion, our study showed that VlsPLA2 acts as a virulence factor, possibly by hydrolysing host nuclear envelope phospholipids, which, through a signal transduction cascade, may suppress basal plant immune responses. [ABSTRACT FROM AUTHOR]

The soil-borne fungus Verticillium dahliae, the most notorious plant pathogen of the Verticillium genus, causes vascular wilts in a wide variety of economically important crops. The molecular mechanism of V. dahliae pathogenesis remains largely elusive. Here, we identify a small ubiquitin-like modifier (SUMO)-specific protease (VdUlpB) from V. dahliae, and find that VdUlpB facilitates V. dahliae virulence by deconjugating SUMO from V. dahliae enolase (VdEno). We identify five lysine residues (K96, K254, K259, K313 and K434) that mediate VdEno SUMOylation, and SUMOylated VdEno preferentially localized in nucleus where it functions as a transcription repressor to inhibit the expression of an effector VdSCP8. Importantly, VdUlpB mediates deSUMOylation of VdEno facilitates its cytoplasmic distribution, which allows it to function as a glycolytic enzyme. Our study reveals a sophisticated pathogenic mechanism of VdUlpB-mediated enolase deSUMOylation, which fortifies glycolytic pathway for growth and contributes to V. dahliae virulence through derepressing the expression of an effector. Verticillium dahliae, a soil-borne fungal pathogen, causes vascular wilt in a wide variety of economically important crops. This study reveals a sophisticated pathogenic mechanism of VdUlpB-deSUMOylated enolase to facilate fungal virulence by derepressing the expression of the effector VdSCP8. [ABSTRACT FROM AUTHOR]

Metarhizium brunneum is a soil-borne fungal entomopathogen that can be associated with plant roots. Previous studies have demonstrated that root colonization by beneficial fungi can directly affect soil-borne pathogens through competition and antibiosis and can activate a systemic response in plants, resulting in a primed state for a faster and/or stronger response to stressors. However, the mechanisms by which Metarhizium inoculation ameliorates symptoms caused by plant pathogens are not well known. This study evaluated the ability of M. brunneum to protect oilseed rape (Brassica napus L.) plants against the soil-borne pathogen Verticillium longisporum and investigated whether the observed effects are a result of direct interaction and/or plant-mediated effects. In vitro and greenhouse experiments were conducted to measure fungal colonization of the rhizosphere and plant tissues, and targeted gene expression analysis was used to evaluate the plant response. The results show that M. brunneum delayed pathogen colonization of plant root tissues, resulting in decreased disease symptoms. Direct competition and antibiosis were found to be part of the mechanisms, as M. brunneum growth was stimulated by the pathogen and inhibited the in vitro growth of V. longisporum. Additionally, M. brunneum changed the plant response to the pathogen by locally activating key defense hormones in the salicylic acid (SA) and abscisic acid (ABA) pathways. Using a split-root setup, it was demonstrated that there is a plant-mediated effect, as improved plant growth and decreased disease symptoms were observed when M. brunneum was in the systemic compartment. Moreover, a stronger systemic induction of the gene PR1 suggested a priming effect, involving the SA pathway. Overall, this study sheds light on the mechanisms underlying the protective effects of M. brunneum against soil-borne pathogens in oilseed rape plants, highlighting the potential of this fungal entomopathogen as a biocontrol agent in sustainable agriculture. [ABSTRACT FROM AUTHOR]

Verticillium wilt affects potato fields in Manitoba where 80% of the fields are planted to the moderately susceptible cultivar, Russet Burbank. An accurate determination of Verticillium dahliae inoculum in soil is critical for disease management. In this study, we investigated the presence of microsclerotia-producing Verticillium species in potato fields in Manitoba and compared published quantitative real-time PCR assays for V. dahliae, V. tricorpus and V. longisporum against a traditional plating method. Selected real-time PCR assays could differentiate and quantify the major microsclerotia-producing species, V. dahliae, V. tricorpus and V. longisporum. Results showed that the presence of V. tricorpus caused an overestimation of V. dahliae propagule density when using the plating method. As a result, Verticillium wilt severity was negatively related to cfu from traditional plating, while positive with the amounts of V. dahliae genomic DNA in soils. [ABSTRACT FROM AUTHOR]

Host resistance is an environmentally and economically sustainable disease management strategy that may be especially beneficial to small-scale and organic growers for whom other management tools such as synthetic pesticides are too costly or not permitted. In western North Carolina, the demand for vine-ripened tomatoes (Solanum lycopersicum L.) from local and organic farms has led to the development of cultivars bred for resistance to geographically relevant diseases, including late blight of tomato, which causes rapid defoliation and lesions on fruit. Grafting tomato plants has the potential to increase plant vigor and yield; however, this effect is known to be dependent on multiple factors, including scion and geographic location. In this study, we evaluated the yield response of one determinate ('Mountain Gem') and four indeterminate ('Mountain Heritage', 'Mountain Girl', 'Mountain Rouge', and 'NC10291') late blight-resistant tomato cultivars, grafted on tomato rootstock 'Maxifort' or nongrafted, on a commercial farm and at two research facilities in western North Carolina. Yield of marketable fruit from grafted plants was greater than that from nongrafted plants at one location (P 5 0.008); however, yield response of each cultivar, grafted or not grafted, differed by location. Yield was consistently greater from 'Mountain Gem' plants than other cultivars, and grafted 'Mountain Gem' plants had greater yields later in the season at two locations than nongrafted plants. Because of the late planting date intended to expose cultivars to the late blight pathogen, the full yield potential of the indeterminate cultivars was not realized at all locations. Disease severity caused by Verticillium wilt (Verticillium dahliae Kleb., Verticillium albo-atrum Reinke & Berthold) was lowest for cultivar Mountain Heritage at two of three locations. Results from this study emphasize the importance of conducting evaluations of grafted tomato plants at multiple locations, including on farm, to optimize the benefits associated with their use. [ABSTRACT FROM AUTHOR]

SUMMARY: Plasma membrane represents a critical battleground between plants and attacking microbes. Necrosis‐and‐ethylene‐inducing peptide 1 (Nep1)‐like proteins (NLPs), cytolytic toxins produced by some bacterial, fungal and oomycete species, are able to target on lipid membranes by binding eudicot plant‐specific sphingolipids (glycosylinositol phosphorylceramide) and form transient small pores, causing membrane leakage and subsequent cell death. NLP‐producing phytopathogens are a big threat to agriculture worldwide. However, whether there are R proteins/enzymes that counteract the toxicity of NLPs in plants remains largely unknown. Here we show that cotton produces a peroxisome‐localized enzyme lysophospholipase, GhLPL2. Upon Verticillium dahliae attack, GhLPL2 accumulates on the membrane and binds to V. dahliae secreted NLP, VdNLP1, to block its contribution to virulence. A higher level of lysophospholipase in cells is required to neutralize VdNLP1 toxicity and induce immunity‐related genes expression, meanwhile maintaining normal growth of cotton plants, revealing the role of GhLPL2 protein in balancing resistance to V. dahliae and growth. Intriguingly, GhLPL2 silencing cotton plants also display high resistance to V. dahliae, but show severe dwarfing phenotype and developmental defects, suggesting GhLPL2 is an essential gene in cotton. GhLPL2 silencing results in lysophosphatidylinositol over‐accumulation and decreased glycometabolism, leading to a lack of carbon sources required for plants and pathogens to survive. Furthermore, lysophospholipases from several other crops also interact with VdNLP1, implying that blocking NLP virulence by lysophospholipase may be a common strategy in plants. Our work demonstrates that overexpressing lysophospholipase encoding genes have great potential for breeding crops with high resistance against NLP‐producing microbial pathogens. Significance Statement: NLP‐producing phytopathogens are a big threat to agriculture worldwide. There is no report on R proteins/enzymes that counteract the toxicity of NLPs in plants. This study reports that cotton uses a peroxisome‐localized enzyme, lysophospholipase, to neutralize NLP toxin secreted by Verticillium dahliae, a soil‐borne fungal pathogen that causes vascular disease in a broad range host, thus enhancing plant resistance to wilt disease caused by this pathogen. [ABSTRACT FROM AUTHOR]

The verticillium fungus has become a widespread threat to olive fields around the world in recent years. The accurate and early detection of the disease at scale could support solving the problem. In this paper, we use the YOLO version 5 model to detect verticillium fungus in olive trees using aerial RGB imagery captured by unmanned aerial vehicles. The aim of our paper is to compare different architectures of the model and evaluate their performance on this task. The architectures are evaluated at two different input sizes each through the most widely used metrics for object detection and classification tasks (precision, recall, mAP@0.5 and mAP@0.5:0.95). Our results show that the YOLOv5 algorithm is able to deliver good results in detecting olive trees and predicting their status, with the different architectures having different strengths and weaknesses. [ABSTRACT FROM AUTHOR]

Priming plants with beneficial microbes can establish rapid and robust resistance against numerous pathogens. Here, compelling evidence is provided that the treatment of rapeseed plants with Trichoderma harzianum OMG16 and Bacillus velezensis FZB42 induces defence activation against Verticillium longisporum infection. The relative expressions of the JA biosynthesis genes LOX2 and OPR3, the ET biosynthesis genes ACS2 and ACO4 and the SA biosynthesis and signalling genes ICS1 and PR1 were analysed separately in leaf, stem and root tissues using qRT-PCR. To successfully colonize rapeseed roots, the V. longisporum strain 43 pathogen suppressed the biosynthesis of JA, ET and SA hormones in non-primed plants. Priming led to fast and strong systemic responses of JA, ET and SA biosynthesis and signalling gene expression in each leaf, stem and root tissue. Moreover, the quantification of plant hormones via UHPLC-MS analysis revealed a 1.7- and 2.6-fold increase in endogenous JA and SA in shoots of primed plants, respectively. In roots, endogenous JA and SA levels increased up to 3.9- and 2.3-fold in Vl43-infected primed plants compared to non-primed plants, respectively. Taken together, these data indicate that microbial priming stimulates rapeseed defence responses against Verticillium infection and presumably transduces defence signals from the root to the upper parts of the plant via phytohormone signalling. [ABSTRACT FROM AUTHOR]

Understanding which carbon or nitrogen sources the competing fungal species prefer is pivotal for specific biotechnological applications. However, this is not straightforward, as each strain sometimes behaves differently under the experimental conditions adopted. To analyse the trophic overlap of two species, it is necessary to refine diagnostic techniques and exclude variables that may interfere with the measurements. A protocol for establishing the suitability of chromogenic dyes in the analysis of filamentous fungi with phenotype microarrays is described here. The research goal was to determine the most suitable redox dye indicator and its optimal concentration that reacts quantitatively to the respiratory activity of both Verticillium spp. and Trichoderma spp. isolates in the presence of a nitrogen source. The commercial BiologTM Redox Dye Mixes D, E, and F and also TTC (2,3,5-Triphenyltetrazolium chloride), INT (2-(4-iodophenyl)- 3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride), and MTT solutions were evaluated in selected concentrations. Both their nutritive and toxic effects were quantified. Results showed that 1% “F” and 0.5% “D” BiologTM dyes were appropriate for the functional evaluation of isolates belonging to the Verticillium and Trichoderma genera. They can be used in BiologTM PM-nitrogen plate respiratory assays for a comprehensive functional characterisation of these organisms. [ABSTRACT FROM AUTHOR]

Verticillium wilt, caused by Verticillium dahliae, challenges olive cultivation and an Integrated Disease Management (IDM) approach is the best-suited tool to combat it. Since 1998, an IDM strategy in an orchard (called Granon, Spain) of the susceptible cv. Picual was conducted by increasing planting density with moderately resistant cv. Frantoio, chemical weed control, and replanting of dead olives with cv. Frantoio following soil solarization. The Verticillium wilt epidemic in Granon orchard was compared to the epidemic in a non-IDM orchard (called Ancla, Spain) with plowed soil and dead Picual olives replanted with the same cultivar. Field evaluations (2012-2013) showed an incidence and severity of the disease as Picual-Ancla > Picual-Granon > Frantoio-Granon. The spatiotemporal dynamics of the Verticillium epidemics from 1998 to 2010 were monitored with digital images using SIG. The annual tree mortalities were 5.6% for Picual olives in Ancla orchard, and 3.1 and 0.7% for Picual and Frantoio olives in Granon orchard, respectively. There was a negative relationship between the mortality of olive trees (%) by the pathogen and the height (m) above sea level. The annual mortality of cv. Picual olives was positively correlated with spring rainfalls. The Index of Dispersion and beta-binomial distribution showed aggregation of Verticillium-dead olives. In conclusion, this IDM strategy considerably reduced the disease in comparison with traditional agronomic practices.

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.

Above-ground material of members of the mint family is commercially distilled to extract essential oils, which are then formulated into a myriad of consumer products. Most of the research aimed at characterizing the processes involved in the formation of terpenoid oil constituents has focused on leaves. We now demonstrate, by investigating three mint species, peppermint (Mentha×piperita L.), spearmint (Mentha spicata L.) and horsemint (Mentha longifolia (L.) Huds.; accessions CMEN 585 and CMEN 584), that other organs - namely stems, rhizomes and roots - also emit volatiles and that the terpenoid volatile composition of these organs can vary substantially from that of leaves, supporting the notion that substantial, currently underappreciated, chemical diversity exists. Differences in volatile quantities released by plants whose roots had been dipped in a Verticillium dahliae-spore suspension (experimental) or dipped in water (controls) were evident: increases of some volatiles in the root headspace of mint species that are susceptible to Verticillium wilt disease (peppermint and M. longifolia CMEN 584) were detected, while the quantities of certain volatiles decreased in rhizomes of species that show resistance to the disease (spearmint and M. longifolia CMEN 585). To address the genetic and biochemical basis underlying chemical diversity, we took advantage of the newly sequenced M. longifolia CMEN 585 genome to identify candidate genes putatively coding for monoterpene synthases (MTSs), the enzymes that catalyze the first committed step in the biosynthesis of monoterpenoid volatiles. The functions of these genes were established by heterologous expression in Escherichia coli, purification of the corresponding recombinant proteins, and enzyme assays, thereby establishing the existence of MTSs with activities to convert a common substrate, geranyl diphosphate, to (+)-α-terpineol, 1,8-cineole, γ-terpinene, and (-)-bornyl diphosphate, but were not active with other potential substrates. In conjunction with previously described MTSs that catalyze the formation of (-)-β-pinene and (-)-limonene, the product profiles of the MTSs identified here can explain the generation of all major monoterpene skeletons represented in the volatiles released by different mint organs. [ABSTRACT FROM AUTHOR]

Verticillium wilt is a major threat to many crops, among them alfalfa (Medicago sativa). The model plant Medicago truncatula, a close relative of alfalfa was used to study the genetic control of resistance towards a new Verticillium alfalfae isolate. The accidental introduction of pathogen strains through global trade is a threat to crop production and such new strains might also be better adapted to global warming. Isolates of V. alfalfae were obtained from alfalfa fields in Iran and characterized. The Iranian isolate AF1 was used in a genome-wide association study (GWAS) involving 242 accessions from the Mediterranean region. Root inoculations were performed with conidia at 25°C and symptoms were scored regularly. Maximum Symptom Score and Area under Disease Progess Curve were computed as phenotypic traits to be used in GWAS and for comparison to a previous study with French isolate V31.2 at 20°C. This comparison showed high correlation with a shift to higher susceptibility, and similar geographical distribution of resistant and susceptible accessions to AF1 at 25°C, with resistant accessions mainly in the western part. GWAS revealed 30 significant SNPs linked to resistance towards isolate AF1. None of them were common to the previous study with isolate V31.2 at 20°C. To confirm these loci, the expression of nine underlying genes was studied. All genes were induced in roots following inoculation, in susceptible and resistant plants. However, in resistant plants induction was higher and lasted longer. Taken together, the use of a new pathogen strain and a shift in temperature revealed a completely different genetic control compared to a previous study that demonstrated the existence of two major QTLs. These results can be useful for Medicago breeding programs to obtain varieties better adapted to future conditions. [ABSTRACT FROM AUTHOR]

In the present study, attempts were made to develop tolerant lines of tomato cv Solan Lalima against the pathogen Phytophthora nicotianae var parasitica causing buckeye rot, using in vitro selection approach. The highest callus survival and maximum shoot regeneration were attained on MS medium fortified with 1.0 mg/l 2,4-D and 0.5 mg/l Kn for cotyledon explants and 0.5 mg/l NAA with 1.0 mg/l BAP for leaf explants respectively. The proliferated callus was further exposed to selection pressure containing MS medium enriched with fungal culture filtrate (25%) that revealed 18.32 per cent mean callus survival and 34.59 per cent average shoot induction. The screened putative tomato tolerant somaclones against the targeted pathogen might be further subjected to molecular and agronomic evaluations including multi-location trials. Therefore, the strategy can be utilized as a promising selection method to generate disease-resistant plants of this commercially important crop. [ABSTRACT FROM AUTHOR]

The genus Verticillium is a group of ascomycete fungi that includes several pathogenic plant species. In 2011, a new taxonomic classification, proposed by Inderbitzin and coworkers (2011), re-defined the genus as Verticillium sensu stricto. The objective of our study was the re-classification of the fungal species held in the culture collection in the Slovenian Institute of Hop Research and Brewing in accordance with the newly established taxonomy. With the PCR marker system proposed by Inderbitzin and coworkers in 2011, we re-classified 88 Verticillium isolates out of the 105 samples that are held in the institute's bank, which were obtained from different geographic locations in Europe, North America, and Japan, and from different host plants, including alfalfa, cotton, hop, olive, potato, and tomato. However, the PCR marker for the V. dahliae identification proved to be less specific, and it resulted in the positive amplification of Gibellulopsis nigrescens, V. isaacii, and V. longisporum. To enable the accurate distinction of the fungi, the SSR and LAMP markers were added to the analyses. The 12 newly identified SSR markers, which were used in simplex PCR reactions or in combination, enabled the accurate identification of all included Verticillium isolates and could potentially be used as biomarkers for rapid and easy species identification. [ABSTRACT FROM AUTHOR]

Background: Nowadays, instead of chemicals, using microbial pesticides is very important and significant to protect the environment. Verticillium lecanii (Z), the entomopathogenic fungus, is widely used for management of many pests. The aim of this work is to determine whether the small leafhopper, Empoasca flavescens F. (Homoptera: Cicadellidae), can be exterminated only by biopesticide—V. lecanii or not. Results: After spraying with diluted suspension of V. lecanii spores on the small leafhopper, E. flavescens, the effects of infection and reinfection in accordance with the time, spore density and growth stage of the small leafhoppers were studied. When E. flavescens was sprayed with over 1.25 × 108 spores/ml of diluted suspension of fungal spores indoors, the reduction % was over 80% after 5 days and the decrease of imagoes was higher than that of larvae. Outdoors, E. flavescens was reduced by 50% for 3 years after treatment, while the reduction % was about 90% by reinfection year by year. Conclusion: Ninety percentages of E. flavescens on peach trees can be effectively exterminated in 2 years by spraying the diluted suspension of V. lecanii spore, and no chemical pesticides are needed for the control of E. flavescens in practice. [ABSTRACT FROM AUTHOR]

The olive (Olea europaea L.) is an ancient crop of great importance in the Mediterranean basin due to the production of olive oil and table olives, which are important sources of fat and have benefits for human health. This crop is expanding and increasing its production worldwide and five olive genomes have recently been sequenced, representing a wild olive and important cultivars in terms of olive oil production, intensive agriculture, and adaptation to the East Asian climate. However, few bioinformatic and genomic resources are available to assist olive research and breeding, and there are no platforms to query olive gene expression data. Here, we present OliveAtlas, an interactive gene expression atlas for olive with multiple bioinformatics tools and visualization methods, enabling multiple gene comparison, replicate inspection, gene set enrichment, and data downloading. It contains 70 RNA-seq experiments, organized in 10 data sets representing the main olive plant organs, the pollen germination and pollen tube elongation process, and the response to a collection of biotic and abiotic stresses, among other experimental conditions. OliveAtlas is a web tool based on easyGDB with expression data based on the 'Picual' genome reference and gene annotation. [ABSTRACT FROM AUTHOR]

Host resistance is an environmentally and economically sustainable disease management strategy that may be especially beneficial to small-scale and organic growers for whom other management tools such as synthetic pesticides are too costly or not permitted. In western North Carolina, the demand for vine-ripened tomatoes (Solanum lycopersicum L.) from local and organic farms has led to the development of cultivars bred for resistance to geographically relevant diseases, including late blight of tomato, which causes rapid defoliation and lesions on fruit. Grafting tomato plants has the potential to increase plant vigor and yield; however, this effect is known to be dependent on multiple factors, including scion and geographic location. In this study, we evaluated the yield response of one determinate (‘Mountain Gem’) and four indeterminate (‘Mountain Heritage’, ‘Mountain Girl’, ‘Mountain Rouge’, and ‘NC10291’) late blight–resistant tomato cultivars, grafted on tomato rootstock ‘Maxifort’ or nongrafted, on a commercial farm and at two research facilities in western North Carolina. Yield of marketable fruit from grafted plants was greater than that from nongrafted plants at one location (P = 0.008); however, yield response of each cultivar, grafted or not grafted, differed by location. Yield was consistently greater from ‘Mountain Gem’ plants than other cultivars, and grafted ‘Mountain Gem’ plants had greater yields later in the season at two locations than nongrafted plants. Because of the late planting date intended to expose cultivars to the late blight pathogen, the full yield potential of the indeterminate cultivars was not realized at all locations. Disease severity caused by Verticillium wilt (Verticillium dahliae Kleb., Verticillium albo-atrum Reinke & Berthold) was lowest for cultivar Mountain Heritage at two of three locations. Results from this study emphasize the importance of conducting evaluations of grafted tomato plants at multiple locations, including on farm, to optimize the benefits associated with their use.

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.

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.