A hydrophobin gene, VDH1, is involved in microsclerotial development and spore viability in the plant pathogen Verticillium dahliae

The wilt fungus Verticillium dahliae Kleb. produces desiccation- and cold-tolerant resting structures, known as microsclerotia, which are the primary source of disease inoculum in the field. In an exploration of the molecular mechanisms involved in the development of these important structures, we have identified in V. dahliae a differentially expressed, class II hydrophobin gene (VDH1). vdh1 mutants generated through targeted gene disruption show a severe reduction in microsclerotia production, indicating that the gene is important for this type of development. Although vdh1 mutants do produce normal conidiophores and spores, desiccation-tolerance of the spores is reduced. The VDH1 gene is not, however, needed for normal disease development in tomato. VDH1's functions are multi-faceted, and seem generally relevant to long-term survival in V. dahliae.     

A glutamic acid-rich protein identified in Verticillium dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes wilt disease in a wide range of crops, including cotton. The life cycle of V. dahliae includes three vegetative phases: parasitic, saprophytic and dormant. The dormant microsclerotia are the primary infectious propagules, which germinate when they are stimulated by root exudates. In this study, we report the first application of Agrobacterium tumefaciens-mediated transformation (ATMT) for construction of insertional mutants from a virulent defoliating isolate of V. dahliae (V592). Changes in morphology, especially a lack of melanized microsclerotia or pigmentation traits, were observed in mutants. Together with the established laboratory unimpaired root dip-inoculation approach, we found insertional mutants to be affected in their pathogenicities in cotton. One of the genes tagged in a pathogenicity mutant encoded a glutamic acid-rich protein (VdGARP1), which shared no significant similarity to any known annotated gene. The vdgarp1 mutant showed vigorous mycelium growth with a significant delay in melanized microsclerotial formation. The expression of VdGARP1 in the wild type V529 was organ-specific and differentially regulated by different stress agencies and conditions, in addition to being stimulated by cotton root extract in liquid culture medium. Under extreme infertile nutrient conditions, VdGARP1 was not necessary for melanized microsclerotial formation. Taken together, our data suggest that VdGARP1 plays an important role in sensing infertile nutrient conditions in infected cells to promote a transfer from saprophytic to dormant microsclerotia for long-term survival. Overall, our findings indicate that insertional mutagenesis by ATMT is a valuable tool for the genome-wide analysis of gene function and identification of pathogenicity genes in this important cotton pathogen.     

Microsclerotial germination of Verticillium dahliae as affected by rape rhizosphere

Abstract The germination of nylon net-trapped microsclerotia of Verticillium dahliae pathogenic to rape ( Brassica napus ) was assessed in different systems by fluorescence microscopy using fluorescein diacetate. The influence of the culture's age and the size of the microsclerotia on germination percentages was assessed in water, mineral salts solution and mineral salts solution plus sucrose for 3 V. dahliae isolates. Large microsclerotia germinated better than smaller ones. The microsclerotia of 2 isolates showed decreased germination percentages with culture age over a 4–11-week period. Microsclerotial germination percentages were always higher in mineral salts solution plus sucrose than in mineral salts solution alone or water. In a sand culture system with the intact rape plant, microsclerotial germination percentages were high close to the root and decreased in a steep gradient to background levels within 5 mm from the root.     

In vitro analysis of the role of glucose oxidase from Talaromyces flavus in biocontrol of the plant pathogen Verticillium dahliae.

Culture filtrates from Talaromyces flavus grown on glucose contained high levels of glucose oxidase activity, while culture filtrates from T. flavus grown on xylan contained negligible glucose oxidase activity. Culture filtrates from T-flavus grown on both media contained complex protein profiles. However, only culture filtrates from T. flavus grown on glucose inhibited germination of microsclerotia of Verticillium dahliae in in vitro inhibition assays. A polyclonal antiserum preparation, pABGO-1, raised against purified glucose oxidase from T. flavus was highly specific for glucose oxidase. Only one protein band in culture filtrates (from glucose medium), migrating at 71 kDa, was detected in Western blots (immunoblots) with this antiserum. This band comigrated with purified glucose oxidase. No bands were detected in culture filtrates from the xylan medium. Glucose oxidase was removed via immunoprecipitation from culture filtrates of T. flavus grown in glucose medium, resulting in filtrates which no longer inhibited in vitro microsclerotial germination. When glucose oxidase-depleted filtrates were amended with purified glucose oxidase from T. flavus, the ability to kill microsclerotia in vitro was restored to original levels. We conclude that glucose oxidase is the only protein in culture filtrates of T. flavus responsible for inhibition of germination of microsclerotia of V. dahliae.     

The impact of temperature on the production and fitness of microsclerotia of the fungal bioherbicide Mycoleptodiscus terrestris

The impact of growth temperature was evaluated for the fungal plant pathogen Mycoleptodiscus terrestris over a range of temperatures (20–36°C). The effect of temperature on biomass accumulation, colony forming units (cfu), and microsclerotia production was determined. Culture temperatures of 24–30°C produced significantly higher biomass accumulations and 20–24°C resulted in a significantly higher cfu. The growth of M. terrestris was greatly reduced at temperatures above 30°C and was absent at 36°C. The highest microsclerotia concentrations were produced over a wide range of temperatures (20–30°C). These data suggest that a growth temperature of 24°C would optimize the parameters evaluated in this study. In addition to growth parameters, we also evaluated the desiccation tolerance and storage stability of air-dried microsclerotial preparations from these cultures during storage at 4°C. During 5 months storage, there was no significant difference in viability for air-dried microsclerotial preparations from cultures grown at 20–30°C (>72% hyphal germination) or in conidia production (sporogenic germination) for air-dried preparations from cultures grown at 20–32°C. When the effect of temperature on germination by air-dried microsclerotial preparations was evaluated, data showed that temperatures of 22–30°C were optimal for hyphal and sporogenic germination. Air-dried microsclerotial preparations did not germinate hyphally at 36°C or sporogenically at 20, 32, 34, or 36°C. These data show that temperature does impact the growth and germination of M. terrestris and suggest that water temperature may be a critical environmental consideration for the application of air-dried M. terrestris preparations for use in controlling hydrilla.     

Liquid culturing of microsclerotia of Mycoleptodiscus terrestris,a potential biological control agent for the management of hydrilla

Mycoleptodiscus terrestris has potential as an inundative biological control agent for the management of hydrilla, one of the world’s worst aquatic weeds. Essential to producing a marketable bioherbicidal product was the development of liquid culture procedures that would yield propagules that maintained biocontrol efficacy. Since M. terrestris did not produce conidia in liquid culture, various nutritional conditions were evaluated as a means to produce high concentrations of stable fungal propagules such as microsclerotia. Evaluations of propagule formation and biomass yield were carried out in liquid culture media containing a basal salts solution amended with corn steep liquor powder or cottonseed meal combined with 4% or 6% glucose. Hyphal aggregation was observed by day 2, and by day 8 abundant melanized microsclerotia were present in the broth cultures. When applied as a liquid inoculum to hydrilla at rates of 0.1 and 0.2 ml/l, the microsclerotial matrix was capable of significantly reducing hydrilla shoot biomass by as much as 99%. Air-dried microsclerotia were capable of hyphal germination in 24 h and sporogenic germination in 72 h. These capabilities have significance for the use of microsclerotia of M. terrestris as the preferred inoculum for biocontrol purposes. Hyphae germinating from microsclerotia on hydrilla plant surfaces can establish initial infection sites followed several days later by secondary infections resulting from the development and release of spores from the surface of the microsclerotia. The capability of microsclerotia of M. terrestris to remain stable as a dry preparation and to germinate both hyphally and sporogenically upon rehydration enhances the potential of this fungus for use as a nonchemical, biological control agent for hydrilla.     

MORPHOGENESIS IN VERTICILLIUM: A SELF-PRODUCED,DIFFUSIBLE MORPHOGENETIC FACTOR

A microsclerotial isolate of Verticillium albo-atrum produces a diffusible morphogenetic factor (DMF). At certain levels, DMF stimulates production of microsclerotia and melanin and inhibits hyphal elongation and sporulation. At higher concentrations it can inhibit production of microsclerotia and melanin as well as germination of Verticillium spores. Near-ultraviolet radiation (mostly 3200–4000 A, emission peak 3650 A), which inhibits production of microsclerotia and melanin, appears to act by suppressing synthesis of DMF. Once formed, DMF withstood 5 days’ exposure to near-UV. Dark-reared liquid cultures (shake or still) produced DMF while those reared under near-UV did not. DMF is active from pH 4 to pH 10, seems to be non-volatile, is dialyzable and water-soluble. In preliminary tests, it was insoluble in ether, ethanol and methylene dichloride. Acetone inactivated it. It may be 1 substance or a group of substances. We have developed assays for DMF in agar and liquid media employing the germination of Verticillium spores.     

Characterization and localization of prodiginines from Streptomyces lividans suppressing Verticillium dahliae in the absence or presence of Arabidopsis thaliana

The ascomycete Verticillium dahliae causes worldwide vascular wilt of many field and horticultural plants. The melanized resting structures of this fungus, so-called microsclerotia, survive for many years in soils and continuously re-infect plants. Due to the absence of known fungicides, Verticillium wilt causes immense crop losses. We discovered that the Gram-positive, spore-forming soil bacterium Streptomyces lividans expresses members of the prodiginine family during co-cultivation with V. dahliae. Using HPLC and LC-MS analysis of cultures containing S. lividans alone or grown together with V. dahliae, we found that undecylprodigiosin [394.4 M+H](+) is highly abundant, and streptorubin B [392.4 M+H](+) is present in smaller amounts. Within co-cultures, the quantity of undecylprodigiosin increased considerably and pigment concentrated at and within fungal hyphae. The addition of purified undecylprodigiosin to growing V. dahliae hyphae strongly reduced microsclerotia formation. Undecylprodigiosin was also produced when S. lividans grew on the roots of developing Arabidopsis thaliana plants. Furthermore, the presence of the undecylprodigiosin producer led to an efficient reduction of V. dahliae hyphae and microsclerotia on plant-roots. Based on these novel findings and previous knowledge, we deduce that the prodiginine investigated leads to multiple cellular effects, which ultimately impair specific pathways for signal transduction and apoptosis of the fungal plant pathogen.     

从功能基因到生物学性状:大丽轮枝菌致病性形成的分子基础

大丽轮枝菌为典型的土传维管束病原真菌,针对其致病相关基因的挖掘与功能解析一直是植物病理学研究的热点.大丽轮枝菌具有定殖维管束、"毒素"致萎、形成微菌核、种群分化多元化等特征,这些性状最终支撑或决定了病原对寄主的致病性基础.从进化角度来说,功能基因决定生物学性状.截至目前,在大丽轮枝菌中已鉴定出上百个功能基因;但针对其与...     

Effect of Temperature on the Formation of Microsclerotia of Verticillium dahliae

Microsclerotium formation by six isolates of Verticillium dahliae was studied at different temperatures both in vitro and in Arabidopsis thaliana . In vitro mycelial growth was optimal at 25°C, but microsclerotium formation was greatest at 20°C (two isolates) or 15–20°C (one isolate). Seedlings of A. thaliana were root-dipped in a conidial suspension, planted, and either placed at 5, 10, 15, or 25°C, or left at 20°C until the onset of senescence, after which some of the plants were placed at 5, 10, 15, or 25°C. The amount of microsclerotia per unit of shoot weight was assessed in relation to isolate and temperature. The optimal temperature for production of microsclerotia was 15–25°C. Two isolates each produced about 10 times more microsclerotia than each of the other four isolates. For these isolates, high R ²_adj.-values of 0.77 and 0.66 were obtained, with temperature and its square as highly significant (P   < 0.001) independent variables. R ²_adj.-values for the other isolates varied between 0.28 and 0.39. Moving plants to different temperatures at the onset of senescence led to microsclerotial densities that were intermediate between densities on plants that had grown at constantly 20°C and plants grown at other temperatures. This suggests that vascular colonization rate and rate of microsclerotium formation are similarly affected by temperature. The senescence rate of plants appeared unimportant except for plants grown at 25°C, which showed the highest amounts of microsclerotia per unit of plant weight in the most rapidly senescing plants.     

Identification of VdASP F2-interacting protein as a regulator of microsclerotial formation in Verticillium dahliae

Verticillium dahliae, a notorious phytopathogenic fungus, causes vascular wilt diseases in many plant species. The melanized microsclerotia enable V. dahliae to survive for years in soil and are crucial for its disease cycle. In a previous study, we characterized the secretory protein VdASP F2 from V. dahliae and found that VdASP F2 deletion significantly affected the formation of microsclerotia under adverse environmental conditions. In this study, we clarified that VdASP F2 is localized to the cell wall. However, the underlying mechanism of VdASP F2 in microsclerotial formation remains unclear. Transmembrane ion channel protein VdTRP was identified as a candidate protein that interacts with VdASP F2 using pull-down assays followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and interaction of VdASP F2 and VdTRP was confirmed by bimolecular fluorescence complementary and coimmunoprecipitation assays. The deletion mutant was analysed to reveal that VdTRP is required for microsclerotial production, but it is not essential for stress resistance, carbon utilization and pathogenicity of V. dahliae. RNA-seq revealed some differentially expressed genes related to melanin synthesis and microsclerotial formation were significantly downregulated in the VdTRP deletion mutants. Taken together, these results indicate that VdASP F2 regulates the formation of melanized microsclerotia by interacting with VdTRP.     

Sequence tag analysis of gene expression during pathogenic growth and microsclerotia development in the vascular wilt pathogen Verticillium dahliae

Two cDNA libraries were constructed from cultures of the vascular wilt fungus Verticillium dahliae, grown either in simulated xylem fluid medium (SXM) or under conditions that induce near-synchronous development of microsclerotia. Expressed sequence tags (ESTs) were obtained for over 1000 clones from each library. Most sequences in the two EST collections were unique; nearly 55% of the translated ESTs had strong similarity to protein sequences in the NCBI nonredundant database. ESTs corresponding to melanin biosynthetic enzymes were exclusive to the developing microsclerotia (DMS) collection, and sequences corresponding to extracellular hydrolases (plant cell wall degrading enzymes) were more abundant in that collection. ESTs corresponding to proteins involved in transport and cell growth were more abundant in the SXM collection. The results of this preliminary analysis suggest that the in vitro growth conditions used here provide useful model systems that will facilitate studies of pathogenesis and microsclerotia development in V. dahliae.     

HWP1 Functions in the Morphological Development of Candida albicans Downstream of EFG1, TUP1, and RBF1

The morphological plasticity of Candida albicans is an important determinant of pathogenicity, and nonfilamentous mutants are avirulent. HWP1, a hypha-specific gene, was identified in a genetic screen for developmentally regulated genes and encodes a cell surface protein of unknown function. Heterozygous and homozygous deletions of HWP1 resulted in a medium-conditional defect in hyphal development. HWP1 expression was blocked in a Deltaefg1 mutant, reduced in an Deltarbf1 mutant, and derepressed in a Deltatup1 mutant. Therefore, HWP1 functions downstream of the developmental regulators EFG1, TUP1, and RBF1. Mutation of CPH1 had no effect on HWP1 expression, suggesting that the positive regulators of hyphal development, CPH1 and EFG1, are components of separate pathways with different target genes. The expression of a second developmentally regulated gene, ECE1, was similarly regulated by EFG1. Since ECE1 is not required for hyphal development, the regulatory role of EFG1 apparently extends beyond the control of cell shape determinants. However, expression of ECE1 was not influenced by TUP1, suggesting that there may be some specificity in the regulation of morphogenic elements during hyphal development.     

大丽轮枝孢微菌核的形成条件

以棉花黄萎病菌Verticillium dahliae XJ2008菌株为试材,研究了培养基类型、pH值、温度等因素对棉花黄萎病菌微菌核形成的影响,确立了微菌核形成的最佳培养条件,并对采自新疆、江苏、河南、陕西、山东等地的15个棉花黄萎病菌菌株进行了测试。结果表明,适合于棉花黄萎病菌微菌核大量产生的培养基为基础改良培养基（BMM）、pH值为9.5－11.5、温度为20℃。在该条件下,15个大丽轮枝孢菌株在接种后第12天即可产生大量的微菌核,不同菌株产生微菌核的数量及大小间存在着显著差异,但均与病菌的致病性