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.     

Verticillium dahliae (Kleb.) Infecting Pumpkin Seed

This study investigated the natural occurrence of Verticillium dahliae (Kleb.) infection in pumpkin (Cucurbita pepo L.) seed. The mean incidence of infection was found to be 21.0%. Isolates recovered from seeds were pathogenic to pumpkin (cultivar ‘Jamaican squash’). Surface sterilization by immersion in 0.6% sodium hypochlorite for 20 min eradicated V. dahliae from infected pumpkin seeds without affecting germinability. Plating of seed components revealed that the fungus was present in the seed coat but not in the embryo or cotyledons. In a growing‐on test, 25% of 6‐week‐old plants grown from untreated seeds were infected. Germination and production of normal seedlings were unaffected by V. dahliae infection of seeds. Verticillium dahliae in pumpkin seed was found to be external and transmissible to plants. The findings of this study are important in devising disease control strategies.     

Effect of vesicular-arbuscular mycorrhizal fungi on verticillium wilt of cotton

The development of vesicular-arbuscular mycorrhizal fungi (VAMF): Glomus mosseae (Nicol and Gerd.) Gerdemann and Trappe, Glomus versiforme (Karsten) Berch, Sclerocystis sinuosa Gerdemann and Bakhi and Verticillium dahliae and the effects of the VAMF on the verticillium wilt of cotton (Gossypium hirsutum L. and Gossypium barbadense L.) were studied with paper pots, black plastic tubes and clay pots under natural growth conditions. All of the tested VAMF were able to infect all the cotton varieties used in the present experiment and typical vesicles and arbuscules were formed in the cortical cells of the cotton roots after inoculation. The cap cells, meristem, differentiating and elongating zones of the root tip were found to be colonized by the VAMF. In the case of most V. dahliae infection, the colonization occurred mostly from the root tip up to 2 cm. VAMF and V. dahliae mutually reduced their percentage of infection when inoculated simultaneously. VAMF inoculation reduced the numbers of germinable microsclerotia in the soil of the mycorrhizosphere, while the quantity of VAM fungal spores in the soil was not influenced by infection of with V. dahliae. The % of arbuscule colonization in roots was negatively correlated with the disease grades, while the numbers of vesicles in roots were not. These results suggest that certain vital competition and antagonistic reactions exist between VAMF and V. dahliae. VAMF reduced the incidence and disease indices of verticillium wilt of cotton during the whole growth phase. It is evident that cotton seedling growth was promoted, flowering was advanced, the numbers of flowers and bolls were increased, and this resulted in an increase in the yield of seed cotton. Among the VAMF species, Glomus versiforme was the most effective, and Sclerocystis sinuosa was inferior. So far as the author is aware, such an effect of VAMF on the increase of cotton wilt tolerance/resistance is reported here far the first time.     

Effect of Verticillium dahliae on Photosynthesis, Leaf Expansion and Senescence of Field-grown Sunflower

On the basis of known sunflower (Helianthus annuus L.) responsesto soil water deficit, it is proposed that the effect of thefungus Verticillium dahliae Klebahn on plant leaf area precedesand is greater than its effect on leaf photosynthesis and stomatalconductance. To test this hypothesis, we measured shoot andleaf area growth, leaf photosynthetic rate, stomatal conductanceand disease symptoms in a field experiment including hybridsof high (Sankol) and low (Dekasol 3900) susceptibility to V.dahliae. Plants inoculated with V. dahliae and controls werecompared. We also investigated the effect of V. dahliae on keycomponents of plant leaf area, leaf expansion and senescence,in inoculated and control plants of Sankol and Toba, a hybridof intermediate susceptibility to V. dahliae. Reduction in plantleaf area caused by V. dahliae was first detected 31 d afterinoculation (DAI), when visual symptoms of disease in inoculatedplants were slight (Sankol) or absent (Dekasol 3900). Reductionin leaf photosynthesis was first observed 66 DAI; stomatal conductanceand leaf dark respiration were both unaffected by V. dahliaeduring the whole experiment. In comparison with controls, V.dahliae reduced seasonal duration of plant leaf area by 25%in Dekalb 3900 and by 55% in Sankol, whereas the average reductionin leaf photosynthetic rate was 9%. In correspondence with thereduction in leaf area duration, inoculation reduced shoot drymatter of mature Sankol by 50%. In both experiments, less leafexpansion accounted for most of the early reduction in plantleaf area; as the disease progressed, increasing senescencealso contributed to reduced plant leaf area. It is concludedthat the response of sunflower to V. dahliae resembled the responseof the plant to soil water deficit: (1) plant leaf area, ratherthan leaf photosynthetic rate, accounted for the reduction ingrowth in mass; and (2) reduced leaf expansion early in theseason and faster leaf senescence in older plants accountedfor the decrease in plant leaf area. Copyright 2000 Annals ofBotany Company Helianthus annuus, Verticillium dahliae, allometry, apical dominance, drought, leaf expansion, leaf senescence, photosynthesis, stomatal conductance, growth     

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 Virulence Diversity of <i>Verticillium dahliae</i> from Okra (<i>Abelmoschus esculentus</i>) Plantations in Turkey and Evaluation of Okra Landraces for Resistance to <i>V. dahliae</i>

Forty-four V. dahliae isolates were collected from symptomatic vascular tissues of okra plants each from a different field in eight provinces located in the eastern Mediterranean and western Anatolia regions of Turkey during 2006- 2009. Nitrate-nonutilizing (nit) mutants of V. dahliae from okra were used to determine heterokaryosis and genetic relatedness among isolates. All isolates from okra plants were grouped into two vegetative compatibility groups (VCGs) (1 and 2) and three subgroups as 1A (13.6%, 6/44), 2A (20.5%, 9/44) and 2B (65.9%, 29/44) according to international criteria. Pathogenicity tests were performed on a susceptible local okra (A. esculentus) landrace in greenhouse conditions. All isolates from VCG1A and VCG2B induced defoliation (D) and partial defoliation (PD) symptoms, respectively. Other isolates from VCG2A gave rise to typical leaf chlorosis symptoms without defoliation. The obtained data showed that the virulence level of V. dahliae isolates from okra was related to their VCG belongings. Eighteen okra landraces from diverse geographical origins were screened for resistance to VCG2B and VCG1A of V. dahliae. The results indicated that all landraces were more susceptible to highly virulent VCG1A-D pathotype displaying D or PD symptoms depending on their susceptibility levels with a mean disease severity index of 3.52 than to less virulent VCG2B-PD pathotype of V. dahliae displaying PD and ND symptoms with a mean disease severity index of 2.52. Significant differences were observed among the landraces; however, none of them exhibited a level of resistance. Okra landraces; Çorum, Hatay Has and Şanlıurfa displayed the lowest level of susceptibility or little tolerance to both D and PD pathotypes. VCG2B of PD was prevailing in the surveyed areas and VCG1A of D was the most virulent of the VCGs identified. Introduction of resistant genotypes to Turkish okra germplasm from different sources and breeding new resistant okra cultivars are critical for the sustainability of okra production.     

Mowing during rainfall enhances the control of Cirsium arvense

Pastoral farmers in New Zealand have described dramatic demises in populations of the weed Cirsium arvense (L.) Scop., a perennial herb indigenous to Eurasia, following its mowing during rainfall. To test the hypotheses (1) that the mowing of C. arvense during rainfall increases the control of this weed and (2) that the causal organism in this ‘mowing-in-the-rain’ effect is the vascular wilt fungus Verticillium dahliae, two series of field experiments were carried out in C. arvense-infested pastures in New Zealand, one in autumn 2008 (Experiment series 1, 9 farms), and another in spring/summer 2008–2009 (Experiment series 2, 12 farms). The effect of mowing in the rain as compared to mowing in the dry was to reduce the % ground cover of the thistle in the spring following treatment by 21 and 32% in Experiment series 1 and 2, respectively. Correlations of this ‘rain versus dry’ effect with the incidence of V. dahliae in the subterranean parts of C. arvense shoots sampled in each of the two field experiment series provided no statistical evidence that the effect increased with V. dahliae incidence. Thus these experiments provide no support for the hypothesis that V. dahliae is the biological mechanism for the ‘mowing-in-the-rain’ effect. Nevertheless, they do support mowing during rainfall as a simple and effective management tactic for C. arvense.     

Identification of Pathogenicity-Related Genes in the Vascular Wilt Fungus <Emphasis Type="Italic">Verticillium dahliae</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated T-DNA Insertional Mutagenesis

Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.     

The symptoms and causal agents of early-dying disease (Verticillium wilt) of potatoes

The only characteristic symptom produced by Verticillium albo-atrum and V. dahliae in infected potato plants is unilateral chlorosis and necrosis: this was not shown until the approach of host maturity, and was distinguishable from symptoms of natural senescence only in its slightly earlier expression. Of six species of Verticillium tested against potato (King Edward), V. albo-atrum, V. dahliae, V. nigrescens and V. nubilum were pathogenic (all produced ‘wilt’ symptoms and relative virulence was in that decreasing order) but V. tricorpus and V. lateritium did not induce disease. Isolates of V. albo-atrum and V. dahliae, obtained from a number of other host plants, were also pathogenic to potato. Possible reasons are given for the fewness of records of ‘Early dying’ disease (Verticillium wilt) of potatoes in the field.     

Verticillium wilt of pea (Pisum sativum)

A wilt disease of garden pea (Pisum sativum) caused by Verticillium dahliae is described and the range of pathogenicity of the isolate investigated. It is pathogenic to potato, sweet pea, antirrhinum and broad bean and isolates of V. dahliae from potato, lucerne and sweet pea and V. albo-atrum from lucerne are pathogenic to pea. Since the most common disease symptoms, acropetal progression of chlorosis and necrosis of the leaves followed by premature defoliation are indistinguishable from natural senescence, it is probable that disease and senescence symptoms are confused in the field. The premature defoliation results in marked reduction in green leaf area, leaf dry weight and pod yield.     

Verticillium dahliae Asp1 regulates the transition from vegetative growth to asexual reproduction by modulating microtubule dynamic organization

Verticillium dahliae is a devastating pathogenic fungus that causes severe vascular wilts in more than 400 dicotyledonous plants. The conidiation of V. dahliae in plant vascular tissues is the key strategy for its adaptation to the nutrient-poor environment and is required for its pathogenicity. However, it remains unclear about the regulatory mechanism of conidium production of V. dahliae in vascular tissues. Here, we found that VdAsp1, encoding an inositol polyphosphate kinase, is indispensable for the pathogenicity of V. dahliae. Loss of VdAsp1 function does not affect the invasion of the host, but it impairs the colonization and proliferation in vascular tissues. The ΔVdAsp1 mutant shows defective initiation of conidiophore formation and reduced expression of genes associated with the central developmental pathway. By live-cell imaging, we observed that some of ΔVdAsp1 mutant hyphae are swollen, and microtubule arrangements at the apical region of these hyphae are disorganized. These results indicate that VdAsp1 regulates the transition from vegetative growth to asexual reproduction by modulating microtubule dynamic organization, which is essential for V. dahliae to colonize and proliferate in vascular tissues. These findings provided a potential new direction in the control of vascular wilt pathogen by targeting conidium production in vascular tissues.     

The Influence Of Salinity On Verticillium dahliae In Stem Cuttings Of Five Olive Cultivars

Verticillium dahliae represents one of the main limiting factors in olive production in the Mediterranean countries. Increasing shortage of fresh water and land, increase the pressure on using alternative sources of marginal or saline water, and land previously cropped with V. dahliae host plants. The objective of the present study was to evaluate the influence of salinity on V. dahliae expression in olive stem cuttings. V. dahliae‐inoculated cuttings of cvs. Picual, Frantoio, Mansanillo and Barnea, showed higher senescence symptoms than their non‐inoculated controls. Colonization levels obtained in cv. Picual were significantly higher than in cv. Frantoio. Manzanillo was the most sensitive cultivar to salinity alone, with significant senescence symptoms in 4 and 6 dS/m NaCl treatments. When cv. Manzanillo was exposed to both salinity and V. dahliae, significantly higher senescence symptoms were obtained as compared with each of them separately. Senescence symptoms of cv. Picual exposed to V. dahliae, whether or not in combination with saline solutions, were significantly higher than those when cuttings were exposed to a saline solution alone. In cv. Frantoio, which is more resistant to salinity than the other cultivars, significantly high senescence symptoms were observed only in combination of V. dahliae and high saline concentration (8 dS/m). The fungal colonization index in cv. Manzanillo in high salinity (8 dS/m) was significantly higher than in the treatment without salt. In cv. Barnea, colonization index in 8 dS/m salinity was significantly higher than in the 4 dS/m concentration or control (fresh water). In conclusion, our findings demonstrate the interaction between V. dahliae and saline irrigation in various cultivars. Thus, stem cuttings could serve as an effective screening method in breeding olive clones for V. dahliae resistance, salt tolerance and their interaction.     

Live-cell imaging elaborating epidermal invasion and vascular proliferation/colonization strategy of Verticillium dahliae in host plants

The soilborne ascomycete fungus Verticillium dahliae causes destructive vascular wilt disease in hundreds of dicotyledonous plant species. However, our understanding of the early invasion from the epidermis to the vasculature and the prompt proliferation and colonization in the xylem tissues remains poor. To elaborate the detailed infection strategy of V. dahliae in host plants, we traced the whole infection process of V. dahliae by live-cell imaging combined with high-resolution scanning electron microscopy. The 4D image series demonstrated that the apex of invading hyphae becomes tapered and directly invades the intercellular space of root epidermal cells at the initial infection. Following successful epidermal invasion, the invading hyphae extend in the intercellular space of the root cortex toward the vascular tissues. Importantly, the high-resolution microscopic and live-cell images demonstrated (a) that conidia are formed via budding at the apex of the hyphae in the xylem vessels to promote systemic propagation vertically, and (b) that the hyphae freely cross adjacent xylem vessels through the intertracheary pits to achieve horizontal colonization. Our findings provide a solid cellular basis for future studies on both intracellular invasion and vascular colonization/proliferation during V. dahliae infection and pathogenesis in host plants.     

Seedling vaccination by stem injecting a conidial suspension of F2, a non-pathogenic Fusarium oxysporum strain, suppresses Verticillium wilt of eggplant

Several bacterial and fungal strains have been evaluated as biocontrol agents (BCAs) against Verticillium dahliae. In these studies, the BCAs were applied as a root drenching inoculum; however, this application method may have an adverse effect on the native, beneficial for the plants, microbial community. In the present study, it was evaluated whether endophytic application by stem injecting a conidial suspension of the non pathogenic Fusarium oxysporum F2 strain, isolated from a V. dahliae suppressive compost amendment, could reduce significantly Verticillium wilt symptom development in eggplants. It was revealed that stem injection of F2 seven days before transplanting the seedlings to soil infested by V. dahliae microsclerotia resulted in reduced disease severity compared to the control treatment. To visualise F2 ramification into the plant vascular system eggplant stems were injected with an EGFP transformed F2 strain. It was shown that F2 colonises effectively the plant vascular tissues over a long period of time as it was assessed by F2 DNA levels. In parallel, qPCR analysis showed that the application of F2 reduced significantly the amount of V. dahliae DNA in the stem tissues compared to the control treatment.     

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 10⁶ T. 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.     

iTRAQ-based proteomics analysis of autophagy-mediated immune responses against the vascular fungal pathogen Verticillium dahliae in Arabidopsis

The mechanisms underlying the functional link between autophagy and plant innate immunity remain largely unknown. In this study, we investigated the autophagy-mediated plant defense responses against Verticillium dahliae (V. dahliae) infection by comparative proteomics and cellular analyses. An assessment of the autophagy activity and disease development showed that autophagic processes were tightly related to the tolerance of Arabidopsis plant to Verticillium wilt. An isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics analysis was performed, and we identified a total of 780 differentially accumulated proteins (DAPs) between wild-type and mutant atg10-1 Arabidopsis plants upon V. dahliae infection, of which, 193 ATG8-family-interacting proteins were identified in silico and their associations with autophagy were verified for several selected proteins. Three important aspects of autophagy-mediated defense against V. dahliae infection were revealed: 1) autophagy is required for the activation of upstream defense responses; 2) autophagy-mediated mitochondrial degradation (mitophagy) occurs and is an important player in the defense process; and 3) autophagy promotes the transdifferentiation of perivascular cells and the formation of xylem hyperplasia, which are crucial for protection against this vascular disease. Together, our results provide several novel insights for understanding the functional association between autophagy and plant immune responses.     

Verticillium dahliae VdBre1 is required for cotton infection by modulating lipid metabolism and secondary metabolites

The soil-borne ascomycete Verticillium dahliae causes wilt disease in more than two hundred dicotyledonous plants including the economically important crop cotton, and results in a severe reduction in cotton fiber yield and quality. During infection, V. dahliae secretes numerous secondary metabolites, which act as toxic factors to promote the infection process. However, the mechanism underlying how V. dahliae secondary metabolites regulate cotton infection remains largely unexplored. In this study, we report that VdBre1, an ubiquitin ligase (E3) enzyme to modify H2B, regulates radial growth and conidia production of V. dahliae. The VdBre1 deletion strains show nonpathogenic symptoms on cotton, and microscopic inspection and penetration assay indicated that penetration ability of the ∆VdBre1 strain was dramatically reduced. RNA-seq revealed that a total of 1643 differentially expressed genes between the ∆VdBre1 strain and the wild type strain V592, among which genes related to lipid metabolism were significantly overrepresented. Remarkably, the volume of lipid droplets in the ∆VdBre1 conidia was shown to be smaller than that of wild-type strains. Further metabolomics analysis revealed that the pathways of lipid metabolism and secondary metabolites, such as steroid biosynthesis and metabolism of terpenoids and polyketides, have dramatically changed in the ∆VdBre1 metabolome. Taken together, these results indicate that VdBre1 plays crucial roles in cotton infection and pathogenecity, by globally regulating lipid metabolism and secondary metabolism of V. dahliae.     

MiR395 Overexpression Increases Eggplant Sensibility to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> Infection

Verticillium wilt (V. wilt), a notorious wilt disease caused by Verticillium dahliae, often leads to the reduction of eggplant (Solanum melongena L.) production. MiRNAs, as a class of small RNAs, can regulate gene expression and then affect growth and development in plants. MiR395 has been proven to respond to sulfate-deficient stress in Arabidopsis thaliana and sulfate is well known to have a close relationship with plant disease resistance. To explore the function of eggplant miR395, we examined its expression in V. dahliae-infected eggplant by qRT-PCR and found miR395 exhibited a gradual reduction trend with time after infection. We then expressed pre-miR395 from Arabidopsis thaliana in Suqi eggplant and resistance analysis showed that miR395 overexpressed plants were hypersensitive to V. dahliae infection. We further measured the content of GSH and activities of POD and SOD and the results indicated that the index of GSH/POD/SOD in the overexpressed plants was lower than that of the wild-type control under V. dahliae infection. These results suggest that miR395 plays a negative role in eggplant response to V. dahliae infection.