Comprehensive genomic analysis of Bacillus velezensis AL7 reveals its biocontrol potential against Verticillium wilt of cotton.

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.

Genome-Wide Study of NOT2_3_5 Protein Subfamily in Cotton and Their Necessity in Resistance to Verticillium wilt.

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 VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity.

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.

RNA interference core components identified and characterised in Verticillium nonalfalfae, a vascular wilt pathogenic plant fungi of hops.

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.

The α-1,6-mannosyltransferase VdOCH1 plays a major role in microsclerotium formation and virulence in the soil-borne pathogen Verticillium dahliae.

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.

Identification, Characterization, Pathogenicity, and Distribution of Verticillium alfalfae in Alfalfa Plants in China.

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.

Verticillium dahliae transcription factors Som1 and Vta3 control microsclerotia formation and sequential steps of plant root penetration and colonisation to induce disease.

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.

Characterization and mechanism of lead and zinc biosorption by growing Verticillium insectorum J3.

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.

Verticillium Wilt on Fiber Flax: Symptoms and Pathogen Development In Planta.

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 plant-specific transcription factors CBP60g and SARD1 are targeted by a Verticillium secretory protein VdSCP41 to modulate immunity.

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.

The APSES transcription factor Vst1 is a key regulator of development in microsclerotium- and resting mycelium-producing Verticillium species.

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.

Biological control of wilt disease complex on tomato crop caused by Meloidogyne javanica and Fusarium oxysporum f.sp. lycopersici by Verticillium leptobactrum.

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.

Soil Microbiomes Associated with Verticillium Wilt-Suppressive Broccoli and Chitin Amendments are Enriched with Potential Biocontrol Agents.

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.

FreB is involved in the ferric metabolism and multiple pathogenicity-related traits of Verticillium dahliae.

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 FeSO4, FeCl3 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 H2O2 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.

Fluorescent pseudomonads pursue media-dependent strategies to inhibit growth of pathogenic Verticillium fungi.

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.

Copper complexes of the 1,3,4-thiadiazole derivatives modulate antioxidant defense responses and resistance in tomato plants against fungal and bacterial diseases.

The metallic complexes µ-chloro-µ-[2,5-bis (2-pyridyl)-1,3,4-thiadiazole] aqua chlorocopper (II) dichlorocopper (II) (abbreviated 2PTH-Cu2-Cl4); aquabis [2,5-bis (2-pyridyl)-1,3,4-thiadiazole-κ2N2,N3] (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-Cu2-Cl4 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 H2O2 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 H2O2 accumulation.

The C2H2 transcription factor VdMsn2 controls hyphal growth, microsclerotia formation, and virulence of Verticillium dahliae.

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 C2H2 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.

Evaluation of the Biocontrol Potential of Purpureocillium lilacinum QLP12 against Verticillium dahliae in Eggplant.

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.

A RACK1-like protein regulates hyphal morphogenesis, root entry and in vivo virulence in Verticillium dahliae.

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 Gß-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.

The mononuclear nickel(II) complex bis(azido-κN)bis[2,5-bis(pyridin-2-yl)-1,3,4-thiadiazole-κ2 N2 ,N3 ]nickel(II) protects tomato from Verticillium dahliae by inhibiting fungal growth and activating plant defences.

BACKGROUND: The antifungal properties of the nickel(II) complex bis(azido-κN)bis[2,5-bis(pyridin-2-yl)-1,3,4-thiadiazole-κ2 N2 ,N3 ]nickel(II) [NiL2 (N3 )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: NiL2 (N3 )2 exhibited in vitro an elevated inhibition of radial growth of three strains of the pathogen. According to the strain, the EC50 values ranged from 10 to 29 µg mL-1 for NiL2 (N3 )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 H2 O2 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.