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Carbon catabolite repression (CCR) is a common transcriptional regulatory mechanism that microorganisms use to efficiently utilize carbon nutrients, which is critical for the fitness of microorganisms and for pathogenic species to cause infection. Here, we characterized two CCR genes, VdCreA and VdCreC , in Verticillium dahliae that cause cotton Verticillium wilt disease. The VdCreA and VdCreC knockout mutants displayed slow growth with decreased conidiation and microsclerotium production and reduced virulence to cotton, suggesting that VdCreA and VdCreC are involved in growth and pathogenicity in V. dahliae . We further generated 36 highly reliable and stable ΔVdCreA and ΔVdCreC libraries to comprehensively explore the dynamic expression of genes and metabolites when grown under different carbon sources and CCR conditions. Based on the weighted gene co-expression network analysis (WGCNA) and correlation networks, VdCreA is co-expressed with a multitude of downregulated genes. These gene networks span multiple functional pathways, among which seven genes, including PYCR (pyrroline-5-carboxylate reductase), are potential target genes of VdCreA . Different carbon source conditions triggered entirely distinct gene regulatory networks, yet they exhibited similar changes in metabolic pathways. Six genes, including 6-phosphogluconolactonase and 2-ODGH (2-oxoglutarate dehydrogenase E1), may serve as hub genes in this process. Both VdCreA and VdCreC could comprehensively influence the expression of plant cell wall-degrading enzyme (PCWDE) genes, suggesting that they have a role in pathogenicity in V. dahliae . The integrated expression profiles of the genes and metabolites involved in the glycolysis/gluconeogenesis and pentose phosphate pathways showed that the two major sugar metabolism-related pathways were completely changed, and GADP (glyceraldehyde-3-phosphate) may be a pivotal factor for CCR under different carbon sources. All these results provide a more comprehensive perspective for further analyzing the role of Cre in CCR.

Dormant microsclerotia play a vital role in the survival and spread of Verticillium longisporum , as they can stay viable in the soil and maintain their infectivity for many years. In our previous work, we revealed that soil bacterial volatiles are a key inhibitory factor causing microsclerotia dormancy in the soil. In this study, we further demonstrate that root exudates collected from both host and non-host plants can effectively rescue microsclerotia from bacterial suppression and initiate germination. To identify the specific compounds in root exudates responsible for microsclerotia germination, we fractionated the collected root exudates into polar and non-polar compounds. Subsequently, we conducted comprehensive bioassays with each fraction on germination-suppressed microsclerotia. The result revealed a pivotal role of primary metabolites in root exudates, particularly glutamic acid, in triggering microsclerotia germination and overcoming bacterial inhibition. Moreover, our studies revealed a decrease in inhibitory bacterial volatile fatty acids when bacteria were cultured in the presence of root exudates or glutamic acid. This suggests a potential mechanism, by which root exudates set-off bacterial suppression on microsclerotia. Here, we reveal for the first time that plant root exudates, instead of directly inducing the germination of microsclerotia, enact a set-off effect by counteracting the suppressive impact of soil bacteria on the microsclerotia germination process. This nuanced interaction advances our understanding of the multifaceted dynamics governing microsclerotia dormancy and germination in the soil environment., Importance: Our research provides first-time insights into the crucial interaction between plant root exudates and soil bacteria in regulating the germination of Verticillium longisporum microsclerotia, a significant structure in the survival and proliferation of this soil-borne pathogen. We describe so far unknown mechanisms, which are key to understand how root infections on oilseed rape can occur. By pinpointing primary metabolites in root exudates as key factors in overcoming bacteria-induced dormancy and promote microsclerotia germination, our study highlights the potential for exploiting plant - as well as soil microbe-derived - compounds to control V. longisporum . This work underscores the importance of elucidating the nuanced interactions within the soil ecosystem to devise innovative strategies for managing root infective plant diseases, thereby contributing to the resilience and health of cropping systems., Competing Interests: The authors declare no conflict of interest.

Verticilllium wilt of cotton is a devastating soil-borne disease, which is caused by Verticillium dahliae Kleb. Bacillus velezensis strain AL7 was isolated from cotton soil. This strain efficiently inhibited the growth of V. dahliae. But the mechanism of the biocontrol strain AL7 remains poorly understood. To understand the possible genetic determinants for biocontrol traits of this strain, we conducted phenotypic, phylogenetic and comparative genomics analysis. Phenotypic analysis showed that strain AL7 exhibited broad-spectrum antifungal activities. We determined that the whole genome sequence of B. velezensis AL7 is a single circular chromosome that is 3.89 Mb in size. The distribution of putative gene clusters that could benefit to biocontrol activities was found in the genome. Phylogenetic analysis of Bacillus strains by using single core-genome clearly placed strain AL7 into the B. velezensis. Meantime, we performed comparative analyses on four Bacillus strains and observed subtle differences in their genome sequences. In addition, comparative genomics analysis showed that the core genomes of B. velezensis are more abundant in genes relevant to secondary metabolism compared with B. subtilis strains. Single mutant in the biosynthetic genes of fengycin demonstrated the function of fengycin in the antagonistic activity of B. velezensis AL7. Here, we report a new biocontrol bacterium B. velezensis AL7 and fengycin contribute to the biocontrol efficacy of the strain. The results showed in the research further sustain the potential of B. velezensis AL7 for application in agriculture production and may be a worthy biocontrol strain for further studies., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The Negative on TATA-less (NOT) 2_3_5 domain proteins play key roles in mRNA metabolism and transcription regulation, but few comprehensive studies have focused on this protein family in plants. In our study, a total of 30 NOT2_3_5 genes were identified in four cotton genomes: Gossypium. arboretum , G. raimondii , G. hirsutum and G. barbadense. Phylogenetic analysis showed that all the NOT2_3_5 domain proteins were divided into two classes. The NOT2_3_5 genes were expanded frequently, and segmental duplication had significant effects in their expansion process. The cis -regulatory elements analysis of NOT2_3_5 promoter regions indicated that NOT2_3_5 domain proteins might participate in plant growth and development processes and responds to exogenous stimuli. Expression patterns demonstrated that all of the GhNOT2_3_5 genes were expressed in the majority of tissues and fiber development stages, and that these genes were induced by multiple stresses. Quantitative real-time PCR showed that GbNOT2_3_5 genes were up-regulated in response to verticillium wilt and the silencing of GbNOT2_3_5-3/8 and GbNOT2_3_5-4/9 led to more susceptibility to verticillium wilt than controls. Identification and analysis of the NOT2_3_5 protein family will be beneficial for further research on their biological functions.

Verticillium dahliae ( V. dahliae ) infects roots and colonizes the vascular vessels of host plants, significantly reducing the economic yield of cotton and other crops. In this study, the protein VdTHI20, which is involved in the thiamine biosynthesis pathway, was characterized by knocking out the corresponding VdTHI20 gene in V. dahliae via Agrobacterium tumefaciens -mediated transformation (ATMT). The deletion of VdTHI20 resulted in several phenotypic defects in vegetative growth and conidiation and in impaired virulence in tobacco seedlings. We show that VdTHI20 increases the tolerance of V. dahliae to UV damage. The impaired vegetative growth of ΔVdTHI20 mutant strains was restored by complementation with a functional copy of the VdTHI20 gene or by supplementation with additional thiamine. Furthermore, the root infection and colonization of the ΔVdTHI20 mutant strains were suppressed, as indicated by green fluorescent protein (GFP)-labelling under microscope observation. When the RNAi constructs of VdTHI20 were used to transform Nicotiana benthamiana, the transgenic lines expressing dsVdTHI20 showed elevated resistance to V. dahliae. Together, these results suggest that VdTHI20 plays a significant role in the pathogenicity of V. dahliae. In addition, the pathogenesis-related gene VdTHI20 exhibits potential for controlling V. dahliae in important crops., Competing Interests: The authors declare no conflict of interest.

Verticillium wilt caused by Verticillium alfalfae results in severe production losses in alfalfa crops and is a Class A quarantined disease in China. During 2015 to 2017, 365 alfalfa fields from 21 locations in six provinces were surveyed, and 45 fields from three closely located sites in Gansu, China were found to have alfalfa plants with symptoms typical of Verticillium wilt, with disease incidence of 12.6 to 53.6%. Isolates were identified to species using morphological characteristics and a maximum likelihood phylogeny of the concatenated partial sequences of actin, elongation factor, glyceraldehyde-3-phosphate dehydrogenase, and tryptophan synthase gene regions of Verticillium isolates. Isolation incidence was 93.9% from roots, 71.7% from stems, 66.1% from petioles, and 32.2% from leaves of field-infected plants, indicative of systemic disease and sporadic distribution of this pathogen. In greenhouse tests, the pathogen infected seedlings and colonized vascular tissues when inoculated on seeds, on root tips, in soil, or in injured, but not uninjured, aerial tissues, causing systemic symptoms like those in the field and significant losses. Pathogenicity testing also revealed that five locally grown perennial legumes (stylo, milkvetch, sainfoin, white clover, and red clover) could host V. alfalfae , with a high virulence to milkvetch, sainfoin, and stylo. This study confirmed that V. alfalfae has become established in some regions of Gansu, China and that is a risk to the alfalfa industry in China.

Sunflower yellow wilt is a widespread and destructive disease caused by the soil-borne pathogen Verticillium dahliae (V. dahliae). To better understand the pathogenesis mechanism of V. dahliae in sunflower, T-DNA insertion library was generated via Agrobacterium tumefaciens mediated transformation system (ATMT). Eight hundred positive transformants were obtained. Transformants varied in colony morphology, growth rate, conidia production and pathogenicity in sunflower compared to the wild type strain. A mutant, named VdGn3-L2, was chosen for further analysis based on its deprivation on microsclerotia formation. The flanking sequence of T-DNA insertion site of VdGn3-L2 was identified via hiTAIL-PCR, and the interrupted gene encoded an initiation-specific α-1, 6-mannosyltransferase, named as VdOCH1. The deletion mutant ΔVdOCH1 was impaired in certain characteristics such as fungal growth, conidia production, and microsclerotia formation. Also, ΔVdOCH1 mutants were more sensitive to the cell wall perturbing reagents, such as SDS and Congo red, lost their penetration ability through cellophane membrane, and exhibited dramatically decreased pathogenicity to sunflower. The impaired phenotypes could be restored to the wild type level by complementation of the deletion mutant with full-length VdOCH1 gene. In conclusion, VdOCH1, encoded α-1,6-mannosyltransferase, manipulating the biological characteristics, microsclerotia formation and pathogenic ability of V. dahliae in sunflower., (Copyright © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The conserved RNA interference mechanism (RNAi) in the fungal kingdom has become a focus of intense scientific investigation. The three catalytic core components, Dicer-like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RdRP), and their associated small interfering RNA molecules (siRNAs) have been identified and characterised in several fungal species. Recent studies have proposed that RNAi is a major contributor to the virulence of fungal pathogens as a result of so-called trans-kingdom RNA silencing. In the present study, we report on the existence of three core RNAi proteins in the pathogenic plant fungus Verticillium nonalfalfae, which is a soilborne plant pathogen that causes severe wilting disease in hops (Humulus lupulus L.). Two DCL proteins, two AGO proteins, and two RdRP proteins were identified, and their conserved RNAi domains were characterised. Our phylogeny results confirm the existing taxonomic relationships in the Ascomycete fungal phylum and show that the fungi of the Hypocreomycetidae subclass of the Sordariomycetes class have high amino acid sequence similarity. The expression analysis revealed a potential role of RNAi in the pathogenicity of the fungi, since all the RNAi genes were highly upregulated in the highly virulent isolate T2 and were also differentially expressed in the V. nonalfalfae-susceptible Celeia and V. nonalfalfae-resistant Wye Target cultivars.

Verticillium dahliae nuclear transcription factors Som1 and Vta3 can rescue adhesion in a FLO8-deficient Saccharomyces cerevisiae strain. Som1 and Vta3 induce the expression of the yeast FLO1 and FLO11 genes encoding adhesins. Som1 and Vta3 are sequentially required for root penetration and colonisation of the plant host by V. dahliae. The SOM1 and VTA3 genes were deleted and their functions in fungus-induced plant pathogenesis were studied using genetic, cell biology, proteomic and plant pathogenicity experiments. Som1 supports fungal adhesion and root penetration and is required earlier than Vta3 in the colonisation of plant root surfaces and tomato plant infection. Som1 controls septa positioning and the size of vacuoles, and subsequently hyphal development including aerial hyphae formation and normal hyphal branching. Som1 and Vta3 control conidiation, microsclerotia formation, and antagonise in oxidative stress responses. The molecular function of Som1 is conserved between the plant pathogen V. dahliae and the opportunistic human pathogen Aspergillus fumigatus. Som1 controls genes for initial steps of plant root penetration, adhesion, oxidative stress response and VTA3 expression to allow subsequent root colonisation. Both Som1 and Vta3 regulate developmental genetic networks required for conidiation, microsclerotia formation and pathogenicity of V. dahliae., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Verticillium insectorum J3 was isolated from a local lead-zinc deposit tailing, and its biosorption characteristics and reaction to the toxicities of different Pb(II) and Zn(II) concentrations were investigated. SEM, FTIR, a pH test and a desorption experiment were carried out to identify a possible mechanism. The biosorption of J3 presented an inhibition trend at low concentrations (25-75 mg L-1) and promotion at high concentrations (100-300 mg L-1). J3 absorbed Pb(II) prior to Zn(II) and produced alkaline substances, while mycelial and pellet morphology modifications were important for the removal of Pb(II) and Zn(II) under different stressful conditions (SEM results). Both intracellular accumulation and extracellular absorption may contribute to the removal of Pb(II) at lower concentrations (25-50 mg L-1), although mainly extracellular biosorption occurred at higher concentrations (75-300 mg L-1). However, Zn(II) bioaccumulation occurred at all concentrations assayed. Verticillium insectorum J3 may have evolved active defenses to alleviate the toxicity of heavy metals and proved to be a highly efficient biosorbent, especially for Pb(II) at high concentrations. This study is a useful reference for the development of biotreatment technologies to mitigate heavy metal waste., Competing Interests: Hunan Guozhen Environmental Technology Co., Ltd just provided support in the form of salaries for author [TL], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

Fiber flax (Linum usitatissimum L.), an important crop in Normandy (France), is increasingly affected by Verticillium wilt caused by the soilborne fungus Verticillium dahliae. This disease leads to nonnegligible yield losses and depreciated fibers that are consequently difficult to upgrade. Verticillium wilt is a major threat to a broad range of agriculture. In this study, susceptible fiber flax cultivar Adélie was infected by VdLu01 (isolated from fiber flax, this study) or green fluorescent protein-tagged VdLs17 (transformed and provided by the department of Plant Pathology, University of California, Davis). Between 3 and 4 weeks postinoculation, wilting symptoms on leaves were first observed, with acropetal growth during the following weeks. Pathogen development was tracked by confocal laser-scanning microscopy during the asymptomatic and symptomatic stages. First, conidia germination led to the development of hyphae on root epidermis; more particularly, on the zone of cell differentiation and around emerging lateral roots, while the zone of cell division and the root tip were free of the pathogen. At 3 days postinoculation, the zone of cell differentiation and lateral roots were embedded into a fungal mass. Swelling structures such as appressoria were observed at 1 week postinoculation. At 2 weeks postinoculation and onward, the pathogen had colonized xylem vessels in roots, followed by the stem and, finally, leaves during the symptomatic stage. Additionally, observations of infected plants after retting in the field revealed microsclerotia embedded inside the bast fiber bundle, thus potentially contributing to weakening of fiber. All of these results provide a global account of V. dahliae development when infecting fiber flax.

The efficacy of Verticillium leptobactrum isolate (HR1) was evaluated in the control of root-knot nematode and Fusarium wilt fungus under laboratory and greenhouse conditions. Five concentrations of V. leptobactrum (HR1) isolate were tested for their nematicidal and fungicidal activities against Meloidogyne javanica and Fusarium oxysporum f.sp. lycopersici in vitro. Laboratory trials showed that mycelium growth inhibition of Fusarium wilt fungus was correlated to the increase of the concentration of culture filtrate. All dilutions showed efficiency in reducing the growth of Fusarium oxysporum f.sp. lycopersici. The greatest nematicidal activity was observed at 50, 75, and 100% filtrate dilutions. The egg hatching percentage reached 42%, and the juvenile's corrected mortality registered 90% for the above treatments. In greenhouse experiment, the biocontrol agent fungus enhanced significantly tomato growth components (height and weight of plant and root). The multiplication rate of root-knot nematode and the Fusarium wilt disease incidence declined significantly with soil application of V. leptobactrum as with chemical treatments. The isolate HR1 was efficient to control wilt disease complex caused by M. javanica and Fusarium oxysporum f.sp. lycopersici.

Ferric reductases are integral membrane proteins involved in the reduction of environmental ferric iron into the biologically available ferrous iron. In the most overwhelming phytopathogenic fungus, Verticillium dahliae, these ferric reductase are not studied in details. In this study we explored the role of FreB gene (VDAG_06616) in the ferric reduction and virulence of V. dahliae by generating the knockout mutants (ΔFreB) and complementary strains (ΔFreB-C) using protoplast transformation. When cultured on media supplemented with FeSO 4 , FeCl 3 and no iron, ΔFreB exhibited significantly reduced growth and spore production especially on media with no iron. Transmembrane ferric reductase activity of ΔFreB was decreased up to 50% than wild type strains (Vd-wt). The activity was fully restored in ΔFreB-C. Meanwhile, the expression levels of other related genes (Frect-4, Frect-5, Frect-6 and Met) were obviously increased in ΔFreB. Compared with the Vd-wt and ΔFreB-C, ΔFreB-1 and ΔFreB-2 were impaired in colony diameter and spore number on different carbon sources (starch, sucrose, galactose and xylose). ΔFreB-1 and ΔFreB-2 were also highly sensitive to oxidative stress as revealed by the plate diffusion assay when 100 µM H 2 O 2 was applied to the fungal culture. When Nicotiana benthamiana plants were inoculated, ΔFreB exhibited less disease symptoms than Vd-wt and ΔFreB-C. In conclusion, the present findings not only indicate that FreB mediates the ferric metabolism and is required for the full virulence in V. dahliae, but would also accelerate future investigation to uncover the pathogenic mechanism of this fungus.

The vascular pathogen Verticillium dahliae infects the roots of plants to cause Verticillium wilt. The molecular mechanisms underlying V. dahliae virulence and host resistance remain elusive. Here, we demonstrate that a secretory protein, VdSCP41, functions as an intracellular effector that promotes V. dahliae virulence. The Arabidopsis master immune regulators CBP60g and SARD1 and cotton GhCBP60b are targeted by VdSCP41. VdSCP41 binds the C-terminal portion of CBP60g to inhibit its transcription factor activity. Further analyses reveal a transcription activation domain within CBP60g that is required for VdSCP41 targeting. Mutations in both CBP60g and SARD1 compromise Arabidopsis resistance against V. dahliae and partially impair VdSCP41-mediated virulence. Moreover, virus-induced silencing of GhCBP60b compromises cotton resistance to V. dahliae . This work uncovers a virulence strategy in which the V. dahliae secretory protein VdSCP41 directly targets plant transcription factors to inhibit immunity, and reveals CBP60g, SARD1 and GhCBP60b as crucial components governing V. dahliae resistance., Competing Interests: JQ, KW, LS, HX, SW, LL, SC, HG, JZ No competing interests declared, (© 2018, Qin et al.)

Verticillium species represent economically important phytopathogenic fungi with bacteria as natural rhizosphere antagonists. Growth inhibition patterns of Verticillium in different media were compared to saprophytic Aspergillus strains and were significantly more pronounced in various co-cultivations with different Pseudomonas strains. The Brassica napus rhizosphere bacterium Pseudomonas fluorescens DSM8569 is able to inhibit growth of rapeseed (Verticillium longisporum) or tomato (Verticillium dahliae) pathogens without the potential for phenazine or 2,4-diacetylphloroglucinol (DAPG) mycotoxin biosynthesis. Bacterial inhibition of Verticillium growth remained even after the removal of pseudomonads from co-cultures. Fungal growth response in the presence of the bacterium is independent of the fungal control genes of secondary metabolism LAE1 and CSN5. The phenazine producer P. fluorescens 2-79 (P_phen) inhibits Verticillium growth especially on high glucose solid agar surfaces. Additional phenazine-independent mechanisms in the same strain are able to reduce fungal surface growth in the presence of pectin and amino acids. The DAPG-producing Pseudomonas protegens CHA0 (P_DAPG), which can also produce hydrogen cyanide or pyoluteorin, has an additional inhibitory potential on fungal growth, which is independent of these antifungal compounds, but which requires the bacterial GacA/GacS control system. This translational two-component system is present in many Gram-negative bacteria and coordinates the production of multiple secondary metabolites. Our data suggest that pseudomonads pursue different media-dependent strategies that inhibit fungal growth. Metabolites such as phenazines are able to completely inhibit fungal surface growth in the presence of glucose, whereas GacA/GacS controlled inhibitors provide the same fungal growth effect on pectin/amino acid agar.

Two naturally infested Verticillium wilt-conducive soils from the Salinas Valley of coastal California were amended with disease-suppressive broccoli residue or crab meal amendments, and changes to the soil prokaryote community were monitored using Illumina sequencing of a 16S ribosomal RNA gene library generated from 160 bulk soil samples. The experiment was run in a greenhouse, twice, with eggplant as the Verticillium wilt-susceptible host. Disease suppression, plant height, soil microsclerotia density, and soil chitinase activity were assessed at the conclusion of each experiment. In soil with high microsclerotia density, all amendments significantly reduced Verticillium wilt severity and microsclerotia density, and increased soil chitinase activity. Plant height was increased only in the broccoli-containing treatments. In total, 8,790 error-corrected sequence variants representing 1,917,893 different sequences were included in the analyses. The treatments had a significant impact on the soil microbiome community structure but measures of α diversity did not vary between treatments. Community structure correlated with disease score, plant height, microsclerotia density, and soil chitinase activity, suggesting that the prokaryote community may affect the disease-related response variables or vice versa. Similarly, the abundance of 107 sequence variants correlated with disease-related response variables, which included variants from genera with known antagonists of filamentous fungal plant pathogens, such as Pseudomonas and Streptomyces. Overall, genera with antifungal antagonists were more abundant in amended soils than unamended soils, and constituted up to 8.9% of all sequences in broccoli+crabmeal-amended soil. This study demonstrates that substrate-mediated shifts in soil prokaryote communities are associated with the transition of Verticillium wilt-conducive soils to Verticillium wilt-suppressive soils, and suggests that soils likely harbor numerous additional antagonists of fungal plant pathogens that contribute to the biological suppression of plant disease.

Plant pathogens of the genus Verticillium pose a threat to many important crops worldwide. They are soil-borne fungi which invade the plant systemically, causing wilt symptoms. We functionally characterized the APSES family transcription factor Vst1 in two Verticillium species, V. dahliae and V. nonalfalfae, which produce microsclerotia and melanized hyphae as resistant structures, respectively. We found that, in V. dahliae Δvst1 strains, microsclerotium biogenesis stalled after an initial swelling of hyphal cells and cultures were never pigmented. In V. nonalfalfae Δvst1, melanized hyphae were also absent. These results suggest that Vst1 controls melanin biosynthesis independent of its role in morphogenesis. The absence of vst1 also had a great impact on sporulation in both species, affecting the generation of the characteristic verticillate conidiophore structure and sporulation rates in liquid medium. In contrast with these key roles in development, Vst1 activity was dispensable for virulence. We performed a microarray analysis comparing global transcription patterns of wild-type and Δvst1 in V. dahliae. G-protein/cyclic adenosine monophosphate (G-protein/cAMP) signalling and mitogen-activated protein kinase (MAPK) cascades are known to regulate fungal morphogenesis and virulence. The microarray analysis revealed a negative interaction of Vst1 with G-protein/cAMP signalling and a positive interaction with MAPK signalling. This analysis also identified Rho signalling as a potential regulator of morphogenesis in V. dahliae, positively interacting with Vst1. Furthermore, it exposed the association of secondary metabolism and development in this species, identifying Vst1 as a potential co-regulator of both processes. Characterization of the putative Vst1 targets identified in this study will aid in the dissection of specific aspects of development., (© 2016 BSPP AND JOHN WILEY & SONS LTD.)

Verticillium dahliae is a notorious pathogen that causes vascular wilt disease in numerous plant species worldwide. The fungus produces melanized microsclerotia, which helps it survive adverse environmental conditions that it may encounter within its hosts and in the soil. Previously, we determined that the high osmolarity glycerol (HOG) pathway is involved in the environmental stress response of V. dahliae. In this study, we investigated the function of VdMsn2, a homologue of the yeast C 2 H 2 transcription factor Msn2, which is predicted to function as a downstream player in the HOG pathway. Disruption of VdMsn2 has a discernible effect on hyphal growth and septation, but not on diverse stresses including hyperosmotic stresses and cell wall inhibitory agents. Furthermore, we show that VdMsn2 deletion mutants produce significantly more microsclerotia than the wild-type and exhibit attenuated virulence to smoke trees because of poor penetration. Taken together, our findings suggest that VdMsn2 controls hyphal growth, microsclerotia formation, and virulence but does not significantly contribute to stress responses in V. dahliae., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The metallic complexes μ-chloro-μ-[2,5-bis (2-pyridyl)-1,3,4-thiadiazole] aqua chlorocopper (II) dichlorocopper (II) (abbreviated 2PTH-Cu 2 -Cl 4 ); aquabis [2,5-bis (2-pyridyl)-1,3,4-thiadiazole-κ 2 N 2 ,N 3 ] (trifluoromethane-sulfonato-κO) copper(II) trifluoro metrhanesulfonate (2PTH-Cu-tF) and bis[(2,5-bis(pyridine-2-yl)-1,3,4-thiadiazole-di-azido copper(II)] (2PTH-Cu-Az) were compared for their antimicrobial activities in vitro, and their aptitude to control Verticillium wilt and crown gall diseases development of tomato in the greenhouse. Results showed that the complex 2PTH-Cu-Az inhibited drastically the growth of V. dahliae in vitro. 2PTH-Cu 2 -Cl 4 and 2PTH-Cu-tF did not display any noticeable antimicrobial activity in vitro against all of the pathogens tested. However, in planta evaluation revealed that the three complexes protected tomato against crown gall similarly. They also reduced Verticillium wilt disease severity, although the complex 2PTH-Cu-Az was the most efficient. When compared to other complexes, 2PTH-Cu-Az triggered only a weak oxidative burst as revealed by H 2 O 2 measurement and the activity of ascorbate peroxidase and catalase. These results suggest that the superiority of 2PTH-Cu-Az against V. dahliae rely on its direct antifungal activity and its ability to modulate H 2 O 2 accumulation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The aims of this study were to assess the potential suppressive effects of different olive mill composts on Verticillium wilt and to elucidate the suppressive mechanisms. To this end, four olive mill composts from different crop areas with two maturation levels were selected. After conducting the Verticillium wilt bioassays in cotton, the suppressive effect was observed in only one compost. Compost maturation level did not affect disease development. The standardized area under the disease progress curve and microsclerotia concentration were associated with low API-ZYM enzymatic diversity, β-glucosidase activity, pH, and high electrical conductivity (EC). To assess the nature of suppressiveness in the suppressive compost, additional bioassays were performed with three treated compost-amended growing media (N-supplemented, autoclaved, and heat treated at 60°C for 6 days). Suppressiveness was partially reduced with heat treatments, where N-acetyl-β-glucosaminidase activity disappeared. In this compost, high oligotrophic actinomycete populations were associated with disease reduction. Therefore, plant growth media amended with different olive mill composts do not always show suppressiveness against Verticillium wilt. Enzymatic diversity, β-glucosidase activity, pH, and EC may be sufficient to predict where olive mill compost plant growth media will be effective in reducing Verticillium wilt and microsclerotia concentration. General and specific suppressiveness are involved in the mechanism of compost suppression.

To identify key genes expressed in Verticillium dahliae in early stages of infection of cotton roots, spore suspensions of eight V. dahliae isolates with different virulence levels were induced by cotton roots and genes expressed in these isolates during the early stages of infection were profiled. A gene that was differentially expressed between highly and less virulent strains was identified. Cloning and bioinformatics analysis of the gene suggested that it belongs to the putative Gβ-like/RACK1 protein family, and has seven WD40 domains. Targeted deletion of the gene revealed that it controls a number of growth-related phenotypes, including conidia and microsclerotia production, normal spore germination and hyphal development. RACK1 is a component of eukaryotic ribosomes, and here we found by qRT-PCR that disruption of RACK1 in V. dahliae (designated VdRACK1) significantly altered the transcriptional levels of other ribosomal proteins, suggesting possible global effects of VdRACK1 deletion on the protein translation of other genes. VdRACK1-null mutants lost the ability to penetrate intact cotton roots. However, the mutant strain was able to infect root-wounded cotton plants and, intriguingly, resulted in a hypervirulent phenotype, implicating a role for VdRACK1 in the restriction of rampant growth within the plant., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A fungus with broad spectrum antifungal activity was isolated from the soil in Qinling Mountain, Shaanxi Province, in China. The fungus was identified as Purpureocillium lilacinum based on ITS rDNA gene analysis. The strain, coded as QLP12, showed high inhibition activity on fungal mycelium growth in vitro , especially to Mucor piriformis , Trichothecium roseum , Rhizoctonia solani, and Verticillium dahliae , and its potential for biocontrol efficacy of eggplant. Verticillium wilt disease caused by Verticillium dahliae among 10 fungal species tested was explored. In greenhouse experiments, QLP12 showed an excellent growth-promoting effect on eggplant seed germination (76.7%), bud growth (79.4%), chlorophyll content (47.83%), root activity (182.02%), and so on. QLP12 can colonize the eggplant interior and also develop in rhizosphere soil. In greenhouse, the incidence of Verticillium wilt decreased by 83.82% with pretreated QLP12 fermentation broth in the soil. In the field, QLP12 showed prominent biocontrol effects on Verticillium wilt by reducing the disease index over the whole growth period, a decline of 40.1%. This study showed that the strain QLP12 is not only an effective biocontrol agent for controlling Verticillium wilt of eggplant, but also a plant growth-promoting fungus that deserves to be further developed., Competing Interests: The authors have no conflict of interests to declare.

Background: The antifungal properties of the nickel(II) complex bis(azido-κN)bis[2,5-bis(pyridin-2-yl)-1,3,4-thiadiazole-κ 2 N 2 ,N 3 ]nickel(II) [NiL 2 (N 3 ) 2 ] and its parental ligand 2,5-bis(pyridin-2-yl)-1,3,4-thiadiazole were examined to evaluate their ability to protect tomato plants against Verticillium dahliae. Our main objectives were to determine their effects on the in vitro growth of the pathogen, and their aptitude for controlling verticillium wilt and activating plant defence responses in the greenhouse., Results: NiL 2 (N 3 ) 2 exhibited in vitro an elevated inhibition of radial growth of three strains of the pathogen. According to the strain, the EC 50 values ranged from 10 to 29 µg mL -1 for NiL 2 (N 3 ) 2 . In the greenhouse, it induced an elevated protection against V. dahliae when it was applied twice as foliar sprays at 50 µg mL -1 . It reduced the leaf alteration index by 85% and vessel browning by 96%. In addition, its protective ability was associated with the accumulation of H 2 O 2 and the activation of total phenolic content, as well as potentiation of the activity of peroxidase and polyphenol oxidase., Conclusion: These results demonstrated that the coordination of the ligand with Ni associated with the azide as a coligand resulted in an improvement in its biological activity by both inhibiting the growth of V. dahliae and activating plant defence responses. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

The fungus Verticillium dahliae causes vascular wilt disease on many plant species, including economically important crop and ornamental plants worldwide. It produces darkly pigmented resting structures known as microsclerotia, which are able to survive for up to 14years in soil, and represent one of the defining characteristics of this species. The pigment produced in V. dahliae is dihydroxynaphthalene (DHN)-melanin, a form of melanin common among fungi and named so for the intermediary of this melanin biosynthetic pathway. In this study, we characterized the function of the V. dahliae Vayg1 gene, whose homologs were involved in melanin biosynthesis in Exophiala dermatitidis (Wayg1) and Aspergillus fumigatus (Aayg1), by deletion and complementation of the gene and co-incubating deletion mutant with wild-type strain. Results showed that melanin production and microsclerotial formation in deletion mutants are inhibited. The Vayg1 deletion mutant also exhibited reduced pathogenicity. These results showed that Vayg1 is necessary for melanin and microsclerotium production, and we may thus hypothesize that the Vayg1 product may catalyze two different precursors, one of which is essential for DHN melanin production and the other one is involved in a signal network for microsclerotial formation in V. dahliae., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study explores the biotechnological potential for plant production of twelve psychrotolerant yeasts strains from Northwest-Patagonia. These strains were isolated from different substrates associated with Nothofagus sp. in native forests and Vaccinium sp. in a commercial plantation. Yeasts characterization was performed using in vitro assays to evaluate the production of auxin-like compounds and siderophores, ability to solubilize inorganic phosphate and to reduce common plant pathogen growth. Strain YF8.3 identified as Aureobasidium pullullans was the main producer of auxin-like and siderophores compounds. Phosphate solubilization was a characteristic observed by strains L8.12 and CRUB1775 identified as Holtermaniella takashimae and Candida maritima, respectively. Different yeast strains were able to inhibit the growth of Verticillium dahliae PPRI5569 and Pythium aphanidermatum PPRI 9009, but they all failed to inhibit the growth of Fusarium oxysporum PPRI5457. The present study, suggests that yeasts present in different environments in Northwestern-Patagonian have physiological in vitro features which may influence plant growth. These results are promising for the developing of biological products based on Patagonian yeasts for plant production in cold-temperate regions., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: The optimization of a simple protocol for the mass production of viable microsclerotia (MS) of Verticillium spp., even for recalcitrant isolates, to the inoculation of olive cuttings., Method and Results: Four Verticillium spp. isolates were characterized by growth rate and morphology. Then, the production ability and the viability of MS over time were assessed in seven solid culture media and five aqueous media. The best culture medium, according to the quantity and the quality (size) of the MS produced, was the alkaline-modified sodium polipectate (AMSP) aqueous medium. The MS viability was higher in peat moss substrates. Finally, the MS obtained in this work were infective causing 100% incidence of Verticillium wilt (VW) disease in inoculated olive plants., Conclusion: This study demonstrates that the modified sodium polipectate medium amended with 0·1% agar is the most suitable for the production of MS of Verticillium dahliae isolates that have lost the ability to produce MS in standard culture media., Significance and Impact of the Study: Mass production of MS for artificial infestation of soil is critical to the study of epidemiological and control aspects of the VW. To overcome the failure in the production of MS in recalcitrant isolates, a culture media was optimized and a successful plant inoculation experiment was carried out with artificial MS., (© 2016 The Society for Applied Microbiology.)

The fungus Verticillium dahliae has gained worldwide notoriety as a destructive plant pathogen, causing vascular wilt diseases on diverse plant species. V. dahliae produces melanized resting bodies, known as microsclerotia, which can survive for 15 years in the soil, and are thus critically important in its disease cycle. However, the molecular mechanisms that underpin microsclerotia formation, survival, and germination remain poorly understood. In this study, we observed that deletion of VdHog1 (ΔVdHog1), encoding a homolog of a high-osmolarity glycerol (HOG) response mitogen-activated protein kinase, displayed decreased numbers of melanized microsclerotia in culture, heightened sensitivity to hyperosmotic stress, and increased resistance to the fungicide fludioxonil. Through RNA-Seq analysis, we identified 221 genes differentially expressed in the ΔVdHog1 strain. Interestingly, the expression levels of genes involved in melanin biosynthesis, as well as the hydrophobin gene VDH1, involved in the early stage of microsclerotia formation, were significantly decreased in the ΔVdHog1 strains relative to the wild-type expression levels. The ΔVdHog1 strains exhibited decreased virulence relative to the wild type strain on smoke tree seedlings. These results indicate that VdHog1 regulates hyperosmotic stress responses in V. dahliae, and establishes the Hog1-mediated pathway as a target to further probe the up- and downstream processes that regulate asexual development in this fungus., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Fusarium and Verticillium wilts, two of the most important diseases in cotton, pose serious threats to cotton production. Here we introduced a novel antimicrobial protein Hcm1, which comprised harpin protein from Xanthomonas oryzae pv. oryzicola (Xoc), and the chimeric protein, cecropin A-melittin, into cotton. The transgenic cotton lines with stable Hcm1 expression showed a higher resistance to Verticillium and Fusarium wilts both in greenhouse and field trials compared to controls. Hcm1 enabled the transgenic cotton to produced a microscopic hypersensitive response (micro-HR), reactive oxygen species (ROS) burst, and caused the activation of pathogenesis-related (PR) genes in response to biotic stress, indicating that the transgenic cotton was in a primed state and ready to protect the host from pathogenic infection. Simultaneously, Hcm1 protein inhibited the growth of Verticillium dahliae (V. dahliae) and Fusarium oxysporum (F. oxysporum) in vitro. The spread of fungal biomass was also inhibited in vivo since the V. dahliae biomass was decreased dramatically in transgenic cotton plants after inoculation with V. dahliae. Together, these results demonstrate that Hcm1 could activate innate immunity and inhibit the growth of V. dahliae and F. oxysporum to protect cotton against Verticillium and Fusarium wilts.

In fungal cells, a phosphate (Pi) responsive signaling and metabolism (PHO) pathway regulates Pi-homeostasis. NUC-2/PHO81 and its homologs are one of the most important components in the regulation pathway. In soil-borne phytopathogenic fungus Verticillium dahliae, we identified a Neurospora crassa nuc-2 homolog gene VdNUC-2. VdNUC-2 is composed of 1,018 amino acids, and is highly conserved in tested filamentous fungi. Under conditions of Pi-starvation, compared with the wild-type strain and ectopic complementation strains, the VdNUC-2 knocked out mutants exhibited reduced radial growth, decreased production of conidia and microsclerotia, and were more sensitive to hydrogen peroxide stress. The virulence of VdNUC-2 defective mutants was significantly compromised, and that was unable to be restored by exogenous application of extra Pi. Additionally, the deletion mutants of VdNUC-1, a key transcription factor gene positively controlled by VdNUC-2 in the PHO pathway, showed the similar cultural phenotypes as VdNUC-2 mutants when both of them grew in Pi-limited conditions. However, the virulence of VdNUC-1 mutants was comparable to the wild-type strain. These evidences indicated that the virulence reduction in VdNUC-2 mutants is not due to the interruptions in the PHO pathway or the disturbance of Pi-homeostasis in V. dahliae cytoplasm. VdNUC-2 is not only a crucial gene in the PHO pathway in V. dahliae, but also is required for the full virulence during host-infection.

To overcome the challenges met with gene deletion in the plant pathogen Verticillium dahliae, a mutant strain with impaired non-homologous end joining DNA repair was generated to improve targeted gene replacement frequencies. A V. dahliae 991 ΔVdku70 null mutant strain was generated using Agrobacterium tumefaciens-mediated transformation. Despite having impaired non-homologous end joining DNA repair function, the ΔVdku70 strain exhibited normal growth, reproduction capability, and pathogenicity when compared with the wild-type strain. When the ΔVdku70 strain was used to delete 2-oxoglutarate dehydrogenase E2, ferric reductase transmembrane component 3 precursor, and ferric reductase transmembrane component 6 genes, gene replacement frequencies ranged between 22.8 and 34.7% compared with 0.3 and 0.5 % in the wild-type strain. The ΔVdku70 strain will be a valuable tool to generate deletion strains when studying factors that underlie virulence and pathogenesis in this filamentous fungus.

A limited number of fungi can cause wilting disease in plants through colonization of the vascular system, the most well-known being Verticillium dahliae and Fusarium oxysporum. Like all pathogenic microorganisms, vascular wilt fungi secrete proteins during host colonization. Whole-genome sequencing and proteomics screens have identified many of these proteins, including small, usually cysteine-rich proteins, necrosis-inducing proteins and enzymes. Gene deletion experiments have provided evidence that some of these proteins are required for pathogenicity, while the role of other secreted proteins remains enigmatic. On the other hand, the plant immune system can recognize some secreted proteins or their actions, resulting in disease resistance. We give an overview of proteins currently known to be secreted by vascular wilt fungi and discuss their role in pathogenicity and plant immunity.

Verticillium dahliae is a causative fungal pathogen and only a few genes have been identified that exhibit critical roles in disease resistance and few has shown positive effects on the resistance to Verticillium wilt in transgenic cotton. We cloned a receptor-like kinase gene (GbRLK) induced by Verticillium dahliae (VD) in the disease-resistant cotton Gossypium barbadense cv. Hai7124. Northern blotting revealed that the GbRLK was induced by VD at 96 h after inoculation. The functional GbRLK is from D subgenome since a single base deletion results in a frameshift or dysfunctional homologue in the A subgenome in tetraploid cotton. To verify the function of GbRLK, we developed the overexpression transgenic GbRLK cotton and Arabidopsis lines, and found that they all showed the higher resistance to Verticillium in the greenhouse and field trial. The results of the expression profile using transgenic and non-transgenic Arabidopsis thaliana revealed that the GbRLK regulated expressions of a series genes associated with biotic and abiotic stresses. Therefore, we propose that the increased resistance to Verticillium dahliae infection in transgnic plants could result from reduction in the damage of water loss and regulation of defense gene expression.

The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant.

Pseudomonas fluorescens PICF7 is an indigenous inhabitant of olive (Olea europaea L.) rhizosphere, able to display endophytic lifestyle in roots, to induce a wide range of defence responses upon colonization of this organ and to exert effective biological control against Verticillium wilt of olive (VWO) (Verticillium dahliae). We aimed to evaluate the involvement of specific PICF7 phenotypes in olive root colonization and VWO biocontrol effectiveness by generating mutants impaired in swimming motility (fliI) or siderophore pyoverdine production (pvdI). Besides, the performance of mutants with diminished in vitro growth in potato dextrose agar medium (gltA) and cysteine (Cys) auxotrophy was also assessed. Results showed that olive root colonization and VWO biocontrol ability of the fliI, pvdI and gltA mutants did not significantly differ from that displayed by the parental strain PICF7. Consequently, altered in vitro growth, swimming motility and pyoverdine production contribute neither to PICF7 VWO suppressive effect nor to its colonization ability. In contrast, the Cys auxotroph mutant showed reduced olive root colonization capacity and lost full biocontrol efficacy. Moreover, confocal laser scanning microscopy revealed that all mutants tested were able to endophytically colonize root tissue to the same extent as wild-type PICF7, discarding these traits as relevant for its endophytic lifestyle., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The soilborne fungus Verticillium dahliae is the major pathogen that causes the verticillium wilt disease of plants, which leads to huge economic loss worldwide. At the early stage of infection, growth of the pathogen is subject to the nutrition stress of limited nitrogen. To investigate the secreted pathogenic proteins that play indispensable roles during invasion at this stage, we compared the profiles of secreted proteins of V. dahliae under nitrogen starvation and normal conditions by using in-gel and in-solution digestion combined with liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS). In total, we identified 212 proteins from the supernatant of liquid medium, including 109 putative secreted proteins. Comparative analysis indicated that the expression of 76 proteins was induced, whereas that of 9 proteins was suppressed under nitrogen starvation. Notably, 24 proteins are constitutively expressed. Further bioinformatic exploration enabled us to classify the stress-induced proteins into seven functional groups: cell wall degradation (10.5%), reactive oxygen species (ROS) scavenging and stress response (11.8%), lipid effectors (5.3%), protein metabolism (21.1%), carbohydrate metabolism (15.8%), electron-proton transport and energy metabolism (14.5%), and other (21.0%). In addition, most stress-suppressed proteins are involved in the cell-wall remodeling. Taken together, our analyses provide insights into the pathogenesis of V. dahliae and might give hints for the development of novel strategy against the verticillium wilt disease., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Quantification of Verticillium dahliae microsclerotia is an important component of wilt management on a range of crops. Estimation of microsclerotia by dry or wet sieving and plating of soil samples on semiselective medium is a commonly used technique but this method is resource-intensive. We developed a new molecular quantification method based on Synergy Brands (SYBR) Green real-time quantitative polymerase chain reaction of wet-sieving samples (wet-sieving qPCR). This method can detect V. dahliae microsclerotia as low as 0.5 CFU g(-1) of soil. There was a high correlation (r=0.98) between the estimates of conventional plating analysis and the new wet-sieving qPCR method for 40 soil samples. To estimate the inoculum threshold for cotton wilt, >400 soil samples were taken from the rhizosphere of individual plants with or without visual wilt symptoms in experimental and commercial cotton fields at the boll-forming stage. Wilt inoculum was estimated using the wet-sieving qPCR method and related to wilt development. The estimated inoculum threshold varied with cultivar, ranging from 4.0 and 7.0 CFU g(-1) of soil for susceptible and resistant cultivars, respectively. In addition, there was an overall relationship of wilt incidence with inoculum density across 31 commercial fields where a single composite soil sample was taken at each field, with an estimated inoculum threshold of 11 CFU g(-1) of soil. These results suggest that wilt risk can be predicted from the estimated soil inoculum density using the new wet-sieving qPCR method. We recommend the use of 4.0 and 7.0 CFU g(-1) as an inoculum threshold on susceptible and resistant cultivars, respectively, in practical risk prediction schemes.

Verticillium dahliae is a prominent generator of plant vascular wilting disease and sulfur (S)-enhanced defense (SED) mechanisms contribute to its in-planta elimination. The accumulation of S-containing defense compounds (SDCs) including elemental S (S(0) ) has been described based on the comparison of two near-isogenic tomato (Solanum lycopersicum) lines differing in fungal susceptibility. To better understand the effect of S nutrition on V. dahliae resistance both lines were supplied with low, optimal or supraoptimal sulfate-S. An absolute quantification demonstrated a most effective fungal elimination due to luxury plant S nutrition. High-pressure liquid chromatography (HPLC) showed a strong regulation of Cys levels and an S-responsive GSH pool rise in the bulk hypocotyl. High-frequency S peak accumulations were detected in vascular bundles of resistant tomato plants after fungal colonization by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Global transcriptomic analysis suggested that early steps of the primary S metabolism did not promote the SDCs synthesis in the whole hypocotyl as gene expression was downregulated after infection. Enhanced S fertilization mostly alleviated the repressive fungal effect but did not reverse it. Upregulation of glutathione (GSH)-associated genes in bulk hypocotyls but not in vascular bundles indicated a global antioxidative role of GSH. To finally assign the contribution of S metabolism-associated genes to high S(0) accumulations exclusively found in the resistant tomato line, a spatial gene expression approach was applied. Laser microdissection of infected vascular bundles revealed a switch toward transcription of genes connected with cysteine (Cys) synthesis. The upregulation of LeOASTLp1 suggests a role for Cys as key precursor for local S accumulations (possibly S(0) ) in the vascular bundles of the V. dahliae-resistant tomato line., (© 2014 Scandinavian Plant Physiology Society.)

Rac1 is a small GTPase coordinating diverse cellular functions such as cell polarity, vesicular trafficking, the cell cycle and transcriptional dynamics in many organisms. In this study, we investigate the biological functions of VdRac1, a Rac1 homolog in the soil-borne, wilt-causing fungus Verticillium dahliae. The VdRac1 gene was deleted in a V. dahliae virulence strain Vd8 isolated from a local cotton cultivar. ΔVdrac1 mutants display drastic reduction in colony expansion and form compact, convoluted colonies, show hyper-branching, loss of polarity and ability to penetrate, leading to severely reduced virulence. The p21-activated kinase Cla4 (named as VdCla4 in V. dahliae) null mutants ΔVdcla4 share identical phenotypes with ΔVdrac1. Yeast two-hybrid studies prove that VdCla4 is an effector of VdRac1. Localizations of actin and reactive oxygen species (ROS) in ΔVdrac1 and ΔVdcla4 compared with the corresponding wild-type strain reveal that VdRac1 and VdCla4 play a primary role in polarized hyphal growth via organization of ROS and play only a minor role in the organization of actin. The Vdrac1 and Vdcla4 null mutants are defective in conidiation and trace elements can partially compensate for the defect. Our data demonstrate that VdRac1 regulates polarized growth and pathogenicity by interacting with its effector VdCla4 in V. dahliae., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Eleven essential oils isolated from higher plant species were assessed against the four isolates of Verticillium fungicola found on fruiting bodies of Agaricus bisporus. Eucalyptus citriodora and Lippia alba oils were more efficacious and completely inhibited the mycelial growth of fungal isolates. L. alba oil was fungistatic and fungicidal at 10- and 20-µL concentrations against all of the isolates, respectively, and was more potent than E. citriodora oil as well as some prevalent synthetic fungicides such as benomyl, ethylene dibromide, and phosphine. Eighty microliters of L. alba oil protected 500 g of fruiting bodies of A. bisporus for up to 7 d from infection of the fungus under in vivo conditions. The findings strengthen the possibility of L. alba oil as a plant-based protectant to enhance the shelf life of A. bisporus fruiting bodies.

The vascular wilt fungus Verticillium dahliae infects the roots of cotton plants and can seriously diminish the yield and quality of this and other dicotyledons. However, the key genes involved in V. dahliae infection and pathogenesis in cotton remain unclear. Msb encodes a transmembrane mucin that is highly conserved in the MAPK signal pathway. Msb has been implicated previously in pathogenicity in various aerial plant fungi. In this study, V. dahliae Msb (VdMsb) was found to be required for fungal virulence and microsclerotia production. Strains lacking VdMsb exhibited reduced conidiation and microsclerotia formation. Compared with wild-type and gene-complemented strains, the invasive growth and adhesive capacity of VdMsb deletion mutants were significantly decreased. These results suggest that VdMsb plays a role in development and virulence in V. dahliae., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A novel series of hybrid molecules 4a-i and 5a-i were prepared by condensation of 4-(trimethylsilylethynyl)benzaldehyde 1 with substituted o-phenylenediamines. These in turn were reacted with 2-(azidomethoxy)ethyl acetate in a Cu alkyne-azide cycloaddition (CuAAC) to generate the 1,2,3-triazole pharmacophore under microwave assistance. The newly synthesized compounds were examined for their in vitro antimicrobial activities against Gram-positive and Gram-negative bacteria and the phytopathogenic fungi Verticillium dahliae and Fusarium oxysporum f. sp. albedinis. 2-((4-(4-(5-Trifluoromethyl benzimidazol-2-yl)phenyl)-1,2,3-triazol-1-yl)methoxy)ethanol 5e showed a moderate inhibition of 30% in the Foa sporulation test., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Verticillium wilt, caused by Verticillium dahliae, is a destructive disease in lettuce, and the pathogen is seedborne. Even though maximum seed infestation rates of <5% have been detected in commercial lettuce seed lots, it is necessary to establish acceptable contamination thresholds to prevent introduction and establishment of the pathogen in lettuce production fields. However, introduction of inoculum into lettuce fields for experimental purposes to determine its long term effects is undesirable. Therefore, we constructed a simulation model to study the spread of Verticillium wilt following pathogen introduction from seed. The model consists of four components: the first for simulating infection of host plants, the second for simulating reproduction of microsclerotia on diseased plants, the third for simulating the survival of microsclerotia, and the fourth for simulating the dispersal of microsclerotia. The simulation results demonstrated that the inoculum density-disease incidence curve parameters and the dispersal gradients affect disease spread in the field. Although a steep dispersal gradient facilitated the establishment of the disease in a new field with a low inoculum density, a long-tail gradient allowed microsclerotia to be dispersed over greater distances, promoting the disease spread in fields with high inoculum density. The simulation results also revealed the importance of avoiding successive lettuce crops in the same field, reducing survival rate of microsclerotia between crops, and the need for breeding resistance against V. dahliae in lettuce cultivars to lower the number of microsclerotia formed on each diseased plant. The simulation results, however, suggested that, even with a low seed infestation rate, the pathogen would eventually become established if susceptible lettuce cultivars were grown consecutively in the same field for many years. A threshold for seed infestation can be established only when two of the three drivers of the disease-(i) low microsclerotia production per diseased plant, (ii) long-tail dispersal gradient, and (iii) low microsclerotia survival between lettuce crops-are present.

Verticillium dahliae is a soil-borne phytopathogenic fungus that causes vascular wilt diseases in a wide variety of crop plants, resulting in extensive economic losses. In the past 5 years, progress has been made in elaborating the interaction between this hemibiotrophic fungus and its host plants. Some genes responsible for the vegetative growth and/or pathogenicity in V. dahliae have been identified. Plants have accrued a series of defense mechanisms, including inducible defense signaling pathways and some resistant genes to combat V. dahliae infection. Here, we have reviewed the progress in V. dahliae-plant interaction research.

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

Verticillium longisporum is a soil-borne vascular pathogen causing economic loss in rape. Using the model plant Arabidopsis this study analyzed metabolic changes upon fungal infection in order to identify possible defense strategies of Brassicaceae against this fungus. Metabolite fingerprinting identified infection-induced metabolites derived from the phenylpropanoid pathway. Targeted analysis confirmed the accumulation of sinapoyl glucosides, coniferin, syringin and lignans in leaves from early stages of infection on. At later stages, the amounts of amino acids increased. To test the contribution of the phenylpropanoid pathway, mutants in the pathway were analyzed. The sinapate-deficient mutant fah1-2 showed stronger infection symptoms than wild-type plants, which is most likely due to the lack of sinapoyl esters. Moreover, the coniferin accumulating transgenic plant UGT72E2-OE was less susceptible. Consistently, sinapoyl glucose, coniferyl alcohol and coniferin inhibited fungal growth and melanization in vitro, whereas sinapyl alcohol and syringin did not. The amount of lignin was not significantly altered supporting the notion that soluble derivatives of the phenylpropanoid pathway contribute to defense. These data show that soluble phenylpropanoids are important for the defense response of Arabidopsis against V. longisporum and that metabolite fingerprinting is a valuable tool to identify infection-relevant metabolic markers., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Six transcription regulatory genes of the Verticillium plant pathogen, which reprogrammed nonadherent budding yeasts for adhesion, were isolated by a genetic screen to identify control elements for early plant infection. Verticillium transcription activator of adhesion Vta2 is highly conserved in filamentous fungi but not present in yeasts. The Magnaporthe grisea ortholog conidiation regulator Con7 controls the formation of appressoria which are absent in Verticillium species. Vta2 was analyzed by using genetics, cell biology, transcriptomics, secretome proteomics and plant pathogenicity assays. Nuclear Vta2 activates the expression of the adhesin-encoding yeast flocculin genes FLO1 and FLO11. Vta2 is required for fungal growth of Verticillium where it is a positive regulator of conidiation. Vta2 is mandatory for accurate timing and suppression of microsclerotia as resting structures. Vta2 controls expression of 270 transcripts, including 10 putative genes for adhesins and 57 for secreted proteins. Vta2 controls the level of 125 secreted proteins, including putative adhesins or effector molecules and a secreted catalase-peroxidase. Vta2 is a major regulator of fungal pathogenesis, and controls host-plant root infection and H2 O2 detoxification. Verticillium impaired in Vta2 is unable to colonize plants and induce disease symptoms. Vta2 represents an interesting target for controlling the growth and development of these vascular pathogens., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

The plant-pathogenic fungus Verticillium longisporum is a causal agent of early senescence and ripening in cruciferous crops like Brassica napus. Verticillium wilts have become serious agricultural threats in recent decades. Verticillium species infect host plants through the roots and colonize xylem vessels of the host plant. The xylem fluid provides an environment with limited carbon sources and unbalanced amino acid supply, which requires V. longisporum to induce the cross-pathway control of amino acid biosynthesis. RNA-mediated gene silencing reduced the expression of the two CPC1 isogenes (VlCPC1-1 and VlCPC1-2) of the allodiploid V. longisporum up to 85%. VlCPC1 encodes the conserved transcription factor of the cross-pathway control. The silenced mutants were highly sensitive to amino-acid starvation, and the infected plants showed significantly fewer symptoms such as stunting or early senescence in oilseed rape plant infection assays. Consistently, deletion of single CPC1 of the haploid V. dahliae resulted in strains that are sensitive to amino-acid starvation and cause strongly reduced symptoms in the plant-host tomato (Solanum lycopersicum). The allodiploid V. longisporum and the haploid V. dahliae are the first phytopathogenic fungi that were shown to require CPC1 for infection and colonization of their respective host plants, oilseed rape and tomato.

Background: The soilborne fungus, Verticillium dahliae, causes Verticillium wilt disease in plants. Verticillium wilt is difficult to control since V. dahliae is capable of persisting in the soil for 10 to 15 years as melanized microsclerotia, rendering crop rotation strategies for disease control ineffective. Microsclerotia of V. dahliae overwinter and germinate to produce infectious hyphae that give rise to primary infections. Consequently, microsclerotia formation, maintenance, and germination are critically important processes in the disease cycle of V. dahliae., Results: To shed additional light on the molecular processes that contribute to microsclerotia biogenesis and melanin synthesis in V. dahliae, three replicate RNA-seq libraries were prepared from 10 day-old microsclerotia (MS)-producing cultures of V. dahliae, strain VdLs.17 (average = 52.23 million reads), and those not producing microsclerotia (NoMS, average = 50.58 million reads). Analyses of these libraries for differential gene expression revealed over 200 differentially expressed genes, including up-regulation of melanogenesis-associated genes tetrahydroxynaphthalene reductase (344-fold increase) and scytalone dehydratase (231-fold increase), and additional genes located in a 48.8 kilobase melanin biosynthetic gene cluster of strain VdLs.17. Nearly 50% of the genes identified as differentially expressed in the MS library encode hypothetical proteins. Additional comparative analyses of gene expression in V. dahliae, under growth conditions that promote or preclude microsclerotial development, were conducted using a microarray approach with RNA derived from V. dahliae strain Dvd-T5, and from the amicrosclerotial vdh1 strain. Differential expression of selected genes observed by RNA-seq or microarray analysis was confirmed using RT-qPCR or Northern hybridizations., Conclusion: Collectively, the data acquired from these investigations provide additional insight into gene expression and molecular processes that occur during MS biogenesis and maturation in V. dahliae. The identified gene products could therefore potentially represent new targets for disease control through prevention of survival structure development.

A long-term experiment on the effect of chitin addition to soil on the suppression of soilborne pathogens was set up and monitored for 8 years in an experimental field, Vredepeel, The Netherlands. Chitinous matter obtained from shrimps was added to soil top layers on two different occasions, and the suppressiveness of soil toward Verticillium dahliae, as well as plant-pathogenic nematodes, was assessed, in addition to analyses of the abundances and community structures of members of the soil microbiota. The data revealed that chitin amendment had raised the suppressiveness of soil, in particular toward Verticillium dahliae, 9 months after the (second) treatment, extending to 2 years following treatment. Moreover, major effects of the added chitin on the soil microbial communities were detected. First, shifts in both the abundances and structures of the chitin-treated soil microbial communities, both of total soil bacteria and fungi, were found. In addition, the abundances and structures of soil actinobacteria and the Oxalobacteraceae were affected by chitin. At the functional gene level, the abundance of specific (family-18 glycoside hydrolase) chitinase genes carried by the soil bacteria also revealed upshifts as a result of the added chitin. The effects of chitin noted for the Oxalobacteraceae were specifically related to significant upshifts in the abundances of the species Duganella violaceinigra and Massilia plicata. These effects of chitin persisted over the time of the experiment.