Rhamnose synthase activity is required for pathogenicity of the vascular wilt fungus Verticillium dahliae

The initial interaction of a pathogenic fungus with its host is complex and involves numerous metabolic pathways and regulatory proteins. Considerable attention has been devoted to proteins that play a crucial role in these interactions, with an emphasis on so‐called effector molecules that are secreted by the invading microbe to establish the symbiosis. However, the contribution of other types of molecules, such as glycans, is less well appreciated. Here, we present a random genetic screen that enabled us to identify 58 novel candidate genes that are involved in the pathogenic potential of the fungal pathogen Verticillium dahliae, which causes vascular wilt diseases in over 200 dicotyledonous plant species, including economically important crops. One of the candidate genes that was identified concerns a putative biosynthetic gene involved in nucleotide sugar precursor formation, as it encodes a putative nucleotide‐rhamnose synthase/epimerase‐reductase (NRS/ER). This enzyme has homology to bacterial enzymes involved in the biosynthesis of the nucleotide sugar deoxy‐thymidine diphosphate (dTDP)‐rhamnose, a precursor of L‐rhamnose, which has been shown to be required for virulence in several human pathogenic bacteria. Rhamnose is known to be a minor cell wall glycan in fungi and has therefore not been suspected as a crucial molecule in fungal–host interactions. Nevertheless, our study shows that deletion of the VdNRS/ER gene from the V. dahliae genome results in complete loss of pathogenicity on tomato and Nicotiana benthamiana plants, whereas vegetative growth and sporulation are not affected. We demonstrate that VdNRS/ER is a functional enzyme in the biosynthesis of uridine diphosphate (UDP)‐rhamnose, and further analysis has revealed that VdNRS/ER deletion strains are impaired in the colonization of tomato roots. Collectively, our results demonstrate that rhamnose, although only a minor cell wall component, is essential for the pathogenicity of V. dahliae.     

Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum

Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.     

Multiple forms of pectin trans-eliminase from Verticillium dahliae Klebahn -- cotton wilt agent

From the culture liquid filtrate of Verticillium dahliae--cotton wilt agent--pectin trans-eliminase (EC) was isolated. The enzyme was isolated and examined, using ultrafiltration, gel filtration, ion exchange chromatography, isoelectrofocusing, and electrophoresis. The fungus was found capable to produce several forms of pectin trans-eliminase that differed in their molecular weight, charge, synthesis and release regulation, substrate action (position of bonding breakdowns in the pectin polymer molecule). Pectin trans-eliminase activity was also detected in cell walls of the fungal mycelium. Possible origin of multiple forms of the enzyme is discussed.     

大丽轮枝菌黑色素合成相关基因与微菌核形成的关系

大丽轮枝菌是一种重要的土传植物病原真菌,以休眠结构微菌核作为初始接种体,可侵染660多种植物引致黄萎病。微菌核是致密的多细胞结构,表面附着大量的DHN黑色素。许多报道指出,在微菌核发育过程中,传统的DHN黑色素合成途径中有5种催化酶编码基因Vd PKS、Vd T4HR、Vd SCD、Vd T3HR和Vd LAC均被诱导表达,但这些基因与微菌核形成的关系目前尚无报道。本研究通过基因敲除技术,系统研究了传统DHN黑色素合成通路上这5种关键酶编码基因及一种缩链催化酶编码基因Vayg1在大丽轮枝菌黑色素合成及微菌核形成中的作用。结果表明,大丽轮枝菌DHN黑色素合成需要Vayg1基因的参与,且Vayg1和Vd T3HR基因还参与微菌核的形成过程。因此,Vayg1基因和Vd T3HR基因可作为黄萎病防治的新靶标。     

Ethylene perception via ETR1 is required in Arabidopsis infection by Verticillium dahliae

Vascular wilts caused by Verticillium spp. are very difficult to control and, as a result, are the cause of severe yield losses in a wide range of economically important crops. The responses of Arabidopsis thaliana mutant plants impaired in known pathogen response pathways were used to explore the components in defence against Verticillium dahliae . Analysis of the mutant responses revealed enhanced resistance in etr1-1 [ethylene (ET) receptor mutant] plants, but not in salicylic acid-, jasmonic acid- or other ET-deficient mutants, indicating a crucial role of ETR1 in defence against this pathogen. Quantitative polymerase chain reaction analysis revealed that the decrease in symptom severity shown in etr1-1 plants was associated with significant reductions in the growth of the pathogen in the vascular tissues of the plants, suggesting that impaired perception of ET via ETR1 results in increased disease resistance. Furthermore, the activation and increased accumulation of the PR-1 , PR-2 , PR-5 , GSTF12 , GSTU16 , CHI-1 , CHI-2 and Myb75 genes, observed in etr1-1 plants after V. dahliae inoculation, indicate that the outcome of the induced defence response of etr1-1 plants seems to be dependent on a set of defence genes activated on pathogen attack.     

Verticillium wilt of cacao in Uganda: the relationship between Verticillium dahliae and cacao roots

Isolates of Verticillium dahliae Kleb. from wilted cacao (Theobroma cacao L.), cotton (Gossypium hirsutum L.), and okra (Abelmoschus esculentus Medik.) penetrated all regions of living cacao tap and lateral roots and progressed intracellularly from the epidermis to the xylem in 4–6 days. The hypocotyl and tissues of the unerupted lateral roots beneath the epidermis resisted invasion. Host reactions included browning of extensively colonized cells, alteration (with apparent granulation) of the cytoplasm, and accumulation of materials in the lumina of endodermal cells. Resistance in the hypocotyl was associated with occasional thickening of inner tangential walls of colonized epidermal cells. The fungus formed conidia, microsclerotia, and narrow and wide hyphae within root tissues. The narrow hyphae predominated at the front of mycelial invasion of tissues while the broad hyphae developed behind this front. Limited studies under non-sterile conditions indicated that the apparent host-parasite interactions were similar to those observed with sterile roots and cultures of V. dahliae.     

Genetic relationships between virulence,vegetative compatibility and ISSR marker of Verticillium dahliae isolated from cotton

Verticillium dahliae is one of the most important pathogens causing Verticillium wilt. We studied the characterisation of the genetic relationship between virulence, vegetative compatibility groups (VCGs) and inter-simple sequence repeat (ISSR). A total of 48 V. dahliae isolates, in which 16 are collected from different cotton growing regions in China and 4 isolates belonged to all known VCGs, are used. Half of them were found highly virulent. Mutants (565) were obtained using the nitrate non-utilising mutant. These mutants were grouped into three VCGs: VCG1 (27 isolates), VCG 2 (14 isolates) and VCG 3 (7 isolates). Use of ISSR indicated two main clusters that were related to VCG and virulence. Genetic diversity lineages were obviously correlated to VCGs and ISSRs according to their geographical origin, virulence and ISSR genetic variation. This study could be useful to design and develop effective management strategies beside for quarantine purposes on Verticillium wilt control.     

VEA1 is required for cleistothecial formation and virulence in Histoplasma capsulatum

Histoplasma capsulatum is a pathogenic fungus dependent on dimorphism for virulence. Among the four described Velvet family genes, two of them, Ryp2 and Ryp3, have been shown to be required for dimorphism. It is known that Velvet A (VeA) is necessary for sexual development and toxin production in Aspergillus nidulans. However, the role of the VeA ortholog in H. capsulatum has not yet been explored. Vea1, H. capsulatum homolog of VeA, was studied to determine its role in cleistothecial formation, dimorphism, and virulence. H. capsulatum Vea1 restores cleistothecial formation and partially restores sterigmatocystin production in an A. nidulans veA deletion strain. Furthermore, silencing VEA1 in an H. capsulatum strain capable of forming cleistothecia abolishes cleistothecial formation. Silenced strains also switch to mycelial phase faster, and show impaired switching to the yeast phase once in mycelial phase. Virulence in mice and macrophages is attenuated in VEA1 silenced strains and silenced strains demonstrate increased sensitivity during growth under acidic conditions. These results indicate that H. capsulatum Vea1 shares a similar role in development as VeA. H. capsulatum is also more susceptible to growth in acidic conditions when VEA1 is silenced, which may contribute to the silenced strains' attenuated virulence in mice and macrophages.     

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.     

A fungal protein elicitor PevD1 induces Verticillium wilt resistance in cotton

Key message

We found that the elicitor PevD1 triggered innate immunity in cotton, which plays an important role in future cotton wilt disease control.

Abstract

Elicitors can induce defense responses in plants and improve pathogen resistance. PevD1 is a secreted protein from Verticillium dahliae and activates the hypersensitive response and systemic acquired resistance to tobacco mosaic virus in tobacco plants. To investigate the PevD1-induced disease resistance mechanisms in cotton (Gossypium hirsutum), we report that Escherichia coli expressing PevD1 enhanced cotton resistance and the defense response to the fungal pathogen V. dahliae. The results showed that recombinant PevD1 improved cotton resistance when infiltrated at a concentration as low as 4 μg ml^?1, and the highest disease reduction was 38.16 % on the 15th day post V. dahliae inoculation. This protein was able to systemically induce hydrogen peroxide production, nitric oxide generation, lignin deposition, vessel reinforcement and defense enzymes, including phenylalanine ammonia-lyase, peroxidase, and polyphenol oxidase. PevD1 also enhanced the expression of three pathogenesis-related genes, namely, β-1,3-glucanase, chitinase, and cadinene synthase, and three key genes, PAL, C4H1, and 4CL, from the cotton defense phenylpropanoid metabolism pathway. Our results demonstrated that PevD1 acted as an effector in cotton and V. dahliae interactions and triggered innate immunity in cotton, resulting in the upregulation of defense-related genes, metabolic substance deposition and cell wall modifications. PevD1 is a candidate plant defense activator for cotton wilt disease control.     

Systemic fungicides in the control of strawberry wilt (Verticillium dahliae)

In a series of experiments between 1970 and 1973 the application of benomyl or thiophanate methyl to field-grown strawberries, planted on Verticillium-mtested land, gave control of wilt for up to 5 months, the duration of control being related to the amount of fungicide applied in the spring. Treatment of inoculated plants grown on in chloropicrin-fumigated soil was effective for at least two seasons. An autumn-planted multi-factorial experiment in heavily-infested soil showed that, to achieve maximum wilt control, it was advantageous to grow cv. Gorella rather than cv. Cambridge Vigour; to drench the runners at planting rather than to dip them in the fungicide suspension; and to use a high concentration (0–075 % a-i-) and large volume (600 ml per plant) for a supplementary treatment in May rather than a lower concentration (0.025 %) ^or smaller volume (400 ml). There were small but significant advantages in applying benomyl rather than thiophanate methyl, and in using 0–2% a.i. suspension at planting rather than 0–05%. No advantage was gained by dividing the spring application into two equal doses applied 2 wk apart. Extrapolation from the logarithmic relation between wilt index and total dose of fungicide applied in the spring suggested that I.I g/plant would have given almost complete control until October; such control had been achieved in an earlier experiment in which 1–2 g/plant was applied. Crop yield in the second year was determined by the treatment applied in the first year, but although these treatments had given significantly better control of wilt in Gorella than in Cambridge Vigour, the greater growth and yield potentials of the latter cultivar had an over-riding effect on crop production; Cambridge Vigour yielded more than Gorella under all chemical treatments, but in the absence of treatment Gorella gave a larger crop than Cambridge Vigour. A proposed regime, entailing spring and autumn applications, is aimed at minimizing the colonization of the plant throughout the year, thus reducing the production of new inoculum and, by limiting the quantity of the pathogen in contact with the systemic chemicals, minimizing the probability of selecting fungicide-resistant variants of V. dahliae.     

GhHb1: a nonsymbiotic hemoglobin gene of cotton responsive to infection by Verticillium dahliae

Verticillium wilt of cotton is a widespread and destructive disease that is caused by the fungus pathogen Verticillium dahliae. Although no cotton cultivar is immune to the disease, some genotypes exhibit superior wilt tolerance. To gain an insight into the molecular mechanisms responsible for wilt tolerance, we employed the method of suppression subtractive hybridization (SSH) to isolate genes whose expression is up-regulated after inoculation of the pathogen in a wilt-tolerant cotton cultivar (Gossypium hirsutum cv. BD18). Among the identified candidate ESTs, a cDNA representing a nonsymbiotic hemoglobin gene (designated GhHb1) was further characterized in this study. Northern blot hybridization demonstrated that GhHb1 shares similar characteristics to some other nonsymbiotic hemoglobin genes including the hypoxic stress-induced expression. Sub-cellular localization analysis indicated that GhHb1 proteins were predominantly present in the nucleus with a minor amount appearing in the cytoplasm. Two novel features of GhHb1 were also identified, indicating that GhHb1 expression is activated in the cotton roots after inoculation with V. dahliae and that exogenous hydrogen peroxide induces GhHb1 expression. These results suggest that the GhHb1 may play a role in the defense response of G. hirsutum against V. dahliae invasion.     

Quantitative assessment of the interaction between the antagonistic fungus Talaromyces flavus and the wilt pathogen Verticillium dahliae on eggplant roots

Quantitative aspects of the interaction between the antagonist Talaromyces flavus, the pathogen Verticillium dahliae and eggplant roots, were studied. When eggplant roots were inoculated with T. flavus, prior to the infection with the pathogen, the population density of T. flavus on V. dahliae-infected roots was at least 3 times higher than on healthy uninfected roots, and the proliferation of T. flavus on diseased eggplant roots was related to the severity of wilt symptoms, in the two levels of application of T. flavus studied. However, in all classes of disease severity tested (disease index, 0–3), the population density of T. Flavus on eggplant roots treated with 10⁶ ascospores g^–1 rooting mixture was significantly (p=0.05) higher than with 10⁵ ascospores g^–1. In roots treated with 10⁵ and 10⁶T. flavus ascospores g^–1 rooting mixture, the population density of V. dahliae was reduced by 51% and 69%, respectively. When testing the relationships between the population density of V. dahliae in the roots and disease severity, no significant (p=0.05) difference was found between disease indexes 2 and 3. However, the density of V. dahliae on roots of plants with disease index 1 was significantly (p=0.05) lower than disease indexes 2 and 3. The positive relationship between the inoculum concentration of V. dahliae and the population density of T. flavus developed on eggplant roots was significant (p=0.001), linear, and highly correlated (r=0.945) on a logarithmic scale. In addition, the analysis of these data revealed a significant (p=0.05), high, negative and linear correlation (r=–0.985) between the log concentration of V. dahliae inoculum and the disease reduction achieved by T. flavus.     

Influence of inoculum density of Verticillium dahliae, temperature and relative humidity on epidemics of verticillium wilt of cotton in northern Iran

During 1992--2003, frequency of Verticillium dahliae propagules, disease incidence and severity of verticillium wilt of cotton were determined in several cotton growing fields in Golestan province, northeastern Iran. Inoculum density varied among fields and different years ranging between 2-47 propagules/g of air-dried soil with an average of 18.96+/-0.73. In addition, the pattern of diseased plants varied with type of field and year. Simple regression analysis showed a linear relationship between inoculum density of V. dahliae at planting time on one hand, disease incidence and severity for all years on the other. The straight line model described the increase in disease intensity index over the accumulated physiological time from sowing. The number of days above 28 degrees C (T) and the area under relative humidity (RH) had significant effects on inoculum density in soil (MS) and final disease development (Y) and fitted the Y = 65.840 - 0.0034 RH + 0.57 MS - 1.7T model with R2 = 0.859 and significant F-function (p<0.0001).