Streptomyces lividans inhibits the proliferation of the fungus Verticillium dahliae on seeds and roots of Arabidopsis thaliana

Verticillium wilt, a vascular disease in more than 200 dicotyledonous plants, is due to the ascomycete fungus Verticillium dahliae. As documented by video-microscopy, the soil bacterium Streptomyces lividans strongly reduces the germination of V. dahliae conidia, and the subsequent growth of hyphae. Quantification by the use of DNA-intercalating dyes and Calcofluor-staining revealed that during prolonged co-cultivation, bacterial hyphae proliferate to a dense network, provoke a poor development of V. dahliae vegetative hyphae and lead to an enormous reduction of conidia and microsclerotia. Upon individual application to seeds of the model plant Arabidopsis thaliana, either the bacterial spores or the fungal conidia germinate at or within the mucilage, including its volcano-shaped structures. The extension of hyphae from each individual strain correlates with the reduction of the pectin-containing mucilage-layer. Proliferating hyphae then spread to roots of the emerging seedlings. Plants, which arise in the presence of V. dahliae within agar or soil, have damaged root cells, an atrophied stem and root, as well as poorly developed leaves with chlorosis symptoms. In contrast, S. lividans hyphae settle in bunches preferentially at the outer layer near tips and alongside roots. Resulting plants have a healthy appearance including an intact root system. Arabidopsis thaliana seeds, which are co-inoculated with V. dahliae and S. lividans, have preferentially proliferating bacterial hyphae within the mucilage, and at roots of the outgrowing seedlings. As a result, plants have considerably reduced disease symptoms. As spores of the beneficial S. lividans strain are obtainable in large quantity, its application is highly attractive.     

Vegetative compatibility and pathogenecity of Verticillium dahliae kleb. isolates from olive in Iran

Verticillium wilt caused by Verticillium dahliae is a serious problem of olive trees leading to significant reduction in yield. Verticillium wilt of olive trees was first recorded in Iran 1996 and confirm as due to Verticillium dahliae Kleb. 101 isolates of V. dahliae from olive trees at deferent locations in north provinces of Iran were assigned to vegetative compatibility groups (VCGS), using nitrate non-utilizing (Nit) mutants. A higher frequency of nit 1/nit 3 mutants (93%) was obtained compared with NitM (7%) with 10% of the isolates being assigned to VCG1 and 51% VCG4B and 19% VCG2A. 20% of isolates could not be classified in standard isolates. The pathogenecity of 15 randomly selected isolates (5 of each VCG) was tested on olive seedling (cv. Zard) and eggplant. The VCGs isolates were similarly aggressive on olive. However, VCG1 isolates were more aggressive on eggplant cv. Local than the VCG2A and VCG4B isolates as indicated by a higher colonization index. The pathogenecity tests of the pathogen on test plants (cotton cv. 'sahel', eggplant cv. 'local' and tomato cv. 'ps') show all isolates category in 2 pathogenecity groups defoliate and non-defoliate (with severe and mild subgroups). The morphology of V. dahliae isolates on C'zapeck's agar and water agar medium were different especially for microsclerotia appearance time in culture and their morphology.     

VdMsb regulates virulence and microsclerotia production in the fungal plant pathogen Verticillium dahliae

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.     

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.     

基因沉默技术在抗真菌病害中的应用和展望

真菌病害严重威胁作物的产量和品质,给国家和人民造成巨大的经济损失。尤其是引起维管束病害的土传真菌,化学农药的作用效果很不理想。利用抗性基因进行遗传育种是目前生物防治的重要手段之一,但对于缺乏抗性资源的物种,面对强大的土壤真菌病害,研究者也时常束手无策。近年来,利用RNA干扰技术发展而来的宿主诱导的基因沉默(Host induced gene silencing,HIGS)策略,在抗病虫害领域逐渐崭露头角,但由于真菌侵染的复杂多样性及土壤传播的特性,HIGS在土壤真菌病害中的应用充满神秘和挑战。本研究室近期揭示了棉花黄萎病(一种严重的土壤真菌病害)的"罪魁祸首"——大丽轮枝菌的侵染结构和侵染过程;并首次证明了宿主植物内源小RNA能够跨界进入病原菌细胞中降解致病基因表达的抗病作用;在此基础上,本研究室利用HIGS在棉花上获得了对黄萎病抗性较高的品系,成功地开辟了抗土壤黄萎真菌病害的新天地,研究结果显示出基因沉默技术在这一领域强大的应用潜力和前景。     

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.     

Microsclerotia development in Verticillium dahliae: Regulation and differential expression of the hydrophobin gene VDH1

The vascular wilt fungus Verticillium dahliae produces persistent resting structures, known as microsclerotia, which are important for this plant pathogen's long-term survival. Previously, we identified a hydrophobin gene (VDH1) that is necessary for microsclerotial production. The current study of VDH1's expression, and its regulation, was undertaken to provide insight into the largely uncharacterized molecular mechanisms relevant to microsclerotial development. Reporter gene analysis showed that VDH1 is specifically expressed in developing microsclerotia, as well as in hyphal fusions and conidiophores, suggesting that VDH1 mediates the development of microsclerotia from conidiophores and other hyphal structures. We report also on the effects of nutrient availability on the regulation of microsclerotial development in V. dahliae; the gene's activity appears to be regulated in response to carbon availability. Lastly, constitutive expression of VDH1 results in delayed disease symptom development, but has no noticeable effect on in vitro microsclerotial development.     

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.     

SOME SOIL FACTORS AFFECTING VERTICILLIUM WILT OF ANTIRRHINUM

Variations in the application rates of chalk and superphosphate and the omission of all the fertilisers had no visible effect on the incidence of wilt caused by any of the five Verticillium species.
he literature dealing with the influence of soil conditions upon the Verticillium wilt of a wide range of host plants is reviewed. It is shown that the species V. alboatrum, V. dahliae, V. nigrescens, V. nubilum and V. tricorpus vary inter se in their pathogenicity to Antirrhinum majus, and that their infectivity may be influenced by soil treatments. Thus, in pot cultures, the incidence of antirrhinum wilt induced by Verticillium dahliae and V. nigrescens was reduced by increasing sulphate of potash or ammonium sulphate; or by decreasing soil moisture. Very wet soil and heavy dressings of hoof-and-horn were the only conditions under which V. nubilunt and V. tricorpus induced wilt symptoms. Z7. albo-atrunt was the most virulent species tested; none of the soil treatments decreased its pathogenicity.     

The Arabidopsis thaliana DNA-binding protein AHL19 mediates verticillium wilt resistance

Verticillium spp. are destructive soilborne fungal pathogens that cause vascular wilt diseases in a wide range of plant species. Verticillium wilts are particularly notorious, and genetic resistance in crop plants is the most favorable means of disease control. In a gain-of-function screen using an activation-tagged Arabidopsis mutant collection, we identified four mutants, A1 to A4, which displayed enhanced resistance toward the vascular wilt species Verticillium dahliae, V. albo-atrum and V. longisporum but not to Fusarium oxysporum f. sp. raphani. Further testing revealed that mutant A2 displayed enhanced Ralstonia solanacearum resistance, while mutants A1 and A3 were more susceptible toward Pseudomonas syringae pv. tomato. Identification of the activation tag insertion site in the A1 mutant revealed an insertion in close proximity to the gene encoding AHL19, which was constitutively expressed in the mutant. AHL19 knock-out alleles were found to display enhanced Verticillium susceptibility whereas overexpression of AHL19 resulted in enhanced Verticillium resistance, showing that AHL19 acts as a positive regulator of plant defense.     

大丽轮枝菌微菌核形成能力的遗传*

带有硝酸盐利用缺陷型遗传标记的大丽轮技菌Verticilliumdahliae黑色菌核型和白色菌丝型菌株在25℃下配对培养,形成野生型融合菌落带,对融合带的分生孢子后代进行遗传分析的结果表明,融合带中的异核体表现不稳定,分布不均匀。微菌核遗传因子可随亲本细胞质在异核体中的运动和交换而发生迁移。     

大丽轮枝菌微菌核形成能力的遗传

带有硝酸盐利用缺陷型遗传标记的大丽轮技菌Verticilliumdahliae黑色菌核型和白色菌丝型菌株在25℃下配对培养,形成野生型融合菌落带,对融合带的分生孢子后代进行遗传分析的结果表明,融合带中的异核体表现不稳定,分布不均匀。微菌核遗传因子可随亲本细胞质在异核体中的运动和交换而发生迁移。     

高效大丽轮枝菌(Verticillium dahliae) 基因敲除体系的构建

[目的]为了深入研究大丽轮枝菌(Verticillium dahliae)致病基因的功能,构建高效大丽轮枝菌基因敲除体系.[方法]融合PCR构建基因敲除载体;利用农杆菌介导法转化大丽轮枝菌;使用在T-DNA之间加入致死基因的双元载体,使T-DNA随机插入转化子在添加5-氟脱氧尿苷的培养基上不能存活,实现对随机插入转化子的"反向筛选".[结果]对大丽轮枝菌腺嘌呤合成酶基因和几丁质合成酶基因进行基因敲除验证,基因敲除转化子在总转化子中的比例分别达到87%和44%.[结论]成功构建大丽轮枝菌高效基因敲除体系,为大丽轮枝菌致病基因的功能验证提供了技术平台.     

新疆棉花黄萎病菌拮抗细菌的分离、筛选与鉴定

【背景】棉花黄萎病是由大丽轮枝菌(Verticillium dahliae Kleb.)引起的一种世界性病害,近年来对该病害的生物防治因具有环境友好和人畜安全的特性而倍受关注。【目的】筛选棉花黄萎病高效拮抗细菌并对其进行鉴定,为棉花黄萎病的生物防治扩充菌种资源。【方法】采用稀释涂布平板法分离细菌,并进行拮抗细菌的初筛和复筛,通过形态特征、生理生化特征和16S rRNA基因序列分析对筛选到的细菌进行鉴定,确定其分类地位。【结果】初筛分离到535株对病原菌具有拮抗作用的细菌,并选取了108株拮抗细菌进行复筛,最终筛选到了4株优势拮抗细菌。通过形态观察、生理生化特征和16SrRNA基因序列分析,将菌株BHZ-29、SHT-15、SHZ-24和SMT-24分别鉴定为贝莱斯芽孢杆菌(Bacillusvelezensis)、枯草芽孢杆菌斯皮兹仁亚种(Bacillus subtilis subsp. spizizenii)、萎缩芽孢杆菌(Bacillus atrophaeus)和香草芽孢杆菌(Bacillus vanillea)。【结论】获得了4株高效拮抗细菌,并且首次报道了香草芽孢杆菌对棉花黄萎病菌具有抑制作用。     

Root-colonizing Fungi from Oilseed Rape and their Inhibition of Verticillium dahliae

Root-colonizing fungi obtained from thoroughly washed root segments of symptomless young oilseed rape plants growing in field soil were examined for their inhibitory effect on the wilt fungus, Verticillium dahliae Kleb. The isolated fungi that were possible to identify belonged to the different genera Trichoderma , Gliocladium , Mortierella , Fusarium and Alternaria . Oilseed rape seedlings from seeds exposed to both sporulating and nonsporulating fungal isolates were shown to be colonized by their hyphae in gnotobiotic conditions and the emergence/root growth was significantly affected in their presence. Inoculation with a few isolates not only covered the root surface but also seemed to extensively colonize the inner root cortical tissues partly by producing characteristic structures in the roots. All of these suppressed the pathogen in in vitro assays but the mode of suppression in most cases did not seem to be due to production of antifungal substances. In nonsterile field soil, several of the isolates conferred protection against V. dahliae by delaying symptom development. None of the isolates was shown to increase wilt symptoms. In addition to protection, two isolates, yet to be identified, also showed increased shoot dry weight compared with that of healthy controls. A majority of the isolates showed proteolytic activity whereas only three produced chitinolytic enzymes.
Results for the isolates obtained by the method selected for this study could be considered as a starting point for evaluating antagonists capable of both competing for root colonization and preventing root infection, with the final aim being to achieve successful biological control of the wilt fungus.     

Ultrastructure and germinability of Verticillium dahliae microsclerotia parasitized by Talaromyces flavus on agar medium and in treated soil

The interactions between microsclerotia (ms) of the fungal plant pathogen Verticillium dahliae and the mycoparasite Talaromyces flavus were followed in soil and on agar medium. Germinability of ms, which had been incubated for 14 days in soil treated with 0.5% of a T. flavus ‐ wheat bran preparation, decreased from 84% to 17%, as compared with 81% and 74% in untreated soil and in soil treated with a sterilized biocontrol preparation respectively. Germinability of ms which had been buried in treated soil for 4 days decreased to 70%, all ms being parasitized by T. flavus. Upon transfer of the ms to untreated soil for 10 more days, germinability decreased further to 20%, indicating that T. flavus continued to parasitize sclerotia in the untreated soil. Scanning electron micrographs showed heavy fungal colonization and typical T. flavus conidia on the surface of the ms buried in the treated soil, but not in control soils. Transmission electron micrographs of ms incubated with T. flavus on agar revealed parsitism involving invasion of some host cells by means of small penetration pegs; the host cell walls were mainly lysed at their site of contact with the parasite hyphal tips. Further colonization of the ms cells occurred simultaneously with the degradation of the invaded host cell contents, rather than the cell walls. Mycoparasitism of V. dahliae ms by T. flavus hyphae may be involved in the biological control of verticillium wilt disease.