Dahliae Inoculation Research Articles

Melatonin enhances cotton immunity to Verticillium wilt via manipulating lignin and gossypol biosynthesis.

SummaryPlants endure challenging environments in which they are constantly threatened by diverse pathogens. The soil‐borne fungus Verticillium dahliae is a devastating pathogen affecting many plant species including cotton, in which it significantly reduces crop yield and fiber quality. Melatonin involvement in plant immunity to pathogens has been reported, but the mechanisms of melatonin‐induced plant resistance are unclear. In this study, the role of melatonin in enhancing cotton resistance to V. dahliae was investigated. At the transcriptome level, exogenous melatonin increased the expression of genes in phenylpropanoid, mevalonate (MVA), and gossypol pathways after V. dahliae inoculation. As a result, lignin and gossypol, the products of these metabolic pathways, significantly increased. Silencing the serotonin N‐acetyltransferase 1 (GhSNAT1) and caffeic acid O‐methyltransferase (GhCOMT) melatonin biosynthesis genes compromised cotton resistance, with reduced lignin and gossypol levels after V. dahliae inoculation. Exogenous melatonin pre‐treatment prior to V. dahliae inoculation restored the level of cotton resistance reduced by the above gene silencing effects. Melatonin levels were higher in resistant cotton cultivars than in susceptible cultivars after V. dahliae inoculation. The findings indicate that melatonin affects lignin and gossypol synthesis genes in phenylpropanoid, MVA, and gossypol pathways, thereby enhancing cotton resistance to V. dahliae.

Disruption and Overexpression of the Gene Encoding ACC (1-Aminocyclopropane-1-Carboxylic Acid) Deaminase in Soil-Borne Fungal Pathogen Verticillium dahliae Revealed the Role of ACC as a Potential Regulator of Virulence and Plant Defense.

It has been suggested that some microorganisms, including plant growth-promoting rhizobacteria, manipulate the level of ethylene in plants by degrading 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, into α-ketobutyrate and ammonia, using ACC deaminase (ACCd). Here, we investigated whether ACCd of Verticillium dahliae, a soil-borne fungal pathogen of many important crops, is involved in causing vascular wilt disease. Overexpression of the V. dahliae gene encoding this enzyme, labeled as ACCd, significantly increased virulence in both tomato and eggplant, while disruption of ACCd reduced virulence. Both types of mutant produced more ethylene than a wild-type (70V-WT) strain, although they significantly differed in ACC content. Overexpression strains lowered ACC levels in the roots of infected plants, while the amount of ACC in the roots of plants infected with deletion mutants increased. To test the hypothesis that ACC acts as a signal for controlling defense, roots of WT and Never-ripe (Nr) tomato plants were treated with ACC before V. dahliae inoculation. Plants pretreated with ACC displayed less severe symptoms than untreated controls. Collectively, our results suggest a novel role of ACC as a regulator of both plant defense and pathogen virulence.

Early stage effects of Verticillium wilt of olive (WVO) on nutrient use in young olive trees grown in soils amended with compost and mineral fertilisation

Verticillium wilt, caused by the soil-borne pathogenic fungus Verticillium dahliae (Kleb), is one of the most severe diseases of olive trees in Mediterranean agriculture. At present, the use of organic amendments is considered an effective means to combat certain soil-borne plant diseases while in turn supplying plants with nutrients. The aim of this study was to determine the early stage effects of Verticillium wilt of olive (VWO) on macronutrient uptake and in planta mobility by analysing nutrients in young olive trees. Young olive trees were grown in unamended as well organically amended soils (employing two distinct olive mill waste composts, C1 and C2) in the presence or absence of mineral fertilisation. Half of the soils were inoculated with Verticillium dahliae (Vd) defoliating (D) pathotype, leaving the other half non-inoculated. At 22 and 100 days after inoculation, nutrient content (N, P, K, S, Ca, and Mg) was analysed in mature and young leaves. Olive mill compost enhanced N, P, K and S availability but did not prevent Verticillium infection in young olive trees. Pathogen inoculation affected nutrient content in mature and young leaves and reduced leaf nutrient input during the early stages of host colonization. Stronger effects were observed in pathogen inoculated plants grown in soil amended with C2, coinciding with high N and P and transiently low S availability. Leaf K input showed the highest sensitivity to V. dahliae inoculation. In high N conditions, deleterious effects caused by Vd on young olive trees may be overcome by promoting the growth of new shoots. We present evidence that Vd requires S during the initial stages of host colonization, in which plant-pathogen competition for this element is key, and that low levels of S in foliage during such early stages may enhance Verticillium wilt development.

The dynamic transcriptome and metabolomics profiling in Verticillium dahliae inoculated Arabidopsis thaliana

Verticillium wilt caused by the soil-borne fungus Verticillium dahliae is a common, devastating plant vascular disease notorious for causing economic losses. Despite considerable research on plant resistance genes, there has been little progress in modeling the effects of this fungus owing to its complicated pathogenesis. Here, we analyzed the transcriptional and metabolic responses of Arabidopsis thaliana to V. dahliae inoculation by Illumina-based RNA sequencing (RNA-seq) and nuclear magnetic resonance (NMR) spectroscopy. We identified 13,916 differentially expressed genes (DEGs) in infected compared with mock-treated plants. Gene ontology analysis yielded 11,055 annotated DEGs, including 2,308 for response to stress and 2,234 for response to abiotic or biotic stimulus. Pathway classification revealed involvement of the metabolic, biosynthesis of secondary metabolites, plant–pathogen interaction, and plant hormone signal transduction pathways. In addition, 401 transcription factors, mainly in the MYB, bHLH, AP2-EREBP, NAC, and WRKY families, were up- or downregulated. NMR analysis found decreased tyrosine, asparagine, glutamate, glutamine, and arginine and increased alanine and threonine levels following inoculation, along with a significant increase in the glucosinolate sinigrin and a decrease in the flavonoid quercetin glycoside. Our data reveal corresponding changes in the global transcriptomic and metabolic profiles that provide insights into the complex gene-regulatory networks mediating the plant’s response to V. dahliae infection.

English

The present study was carried out to evaluate the effect of reducing wilt disease through the medium of fresh chicken manure (FCM) mixed with soil before solarized and then artificial Verticillium dahliae (V.d) inoculation on yield of eggplant (Solanum melongena L.) under field conditions. According to the split-plot design, solarization of main plots, sub-plots with fresh chicken manure and V dahliae inoculation were established as a mini parcel in experiment with three replications. During solarization, the average temperature values were ensured by means of increasing the fresh chicken manure per unit area recorded 24 times per day (24 h) at intervals of one week. Among the 6 applications form, the highest average temperature was obtained at “12 kg FCM m-2 +solarization” application for soil surface, 10, 20 and 30 cm depth. There was increasing 18°C, a temperature according to control (no FCM), and this difference decreased with increasing soil depth, which was calculated as 8°C at 30 cm depth soil. During the day, the highest temperature values were ​​obtained in the middle of the day on the ground, and underground measurements were performed at night. Key words: Soil solarization, fresh chicken manure, Verticillium dahliae, eggplant, yield.

Host-induced gene silencing compromises Verticillium wilt in tomato and Arabidopsis.

Verticillium wilt, caused by soil-borne fungi of the genus Verticillium, is an economically important disease that affects a wide range of host plants. Unfortunately, host resistance against Verticillium wilts is not available for many plant species, and the disease is notoriously difficult to combat. Host-induced gene silencing (HIGS) is an RNA interference (RNAi)-based process in which small RNAs are produced by the host plant to target parasite transcripts. HIGS has emerged as a promising strategy for the improvement of plant resistance against pathogens by silencing genes that are essential for these pathogens. Here, we assessed whether HIGS can be utilized to suppress Verticillium wilt disease by silencing three previously identified virulence genes of V. dahliae (encoding Ave1, Sge1 and NLP1) through the host plants tomato and Arabidopsis. In transient assays, tomato plants were agroinfiltrated with Tobacco rattle virus (TRV) constructs to target V. dahliae transcripts. Subsequent V. dahliae inoculation revealed the suppression of Verticillium wilt disease on treatment with only one of the three TRV constructs. Next, expression of RNAi constructs targeting transcripts of the same three V. dahliae virulence genes was pursued in stable transgenic Arabidopsis thaliana plants. In this host, V. dahliae inoculation revealed reduced Verticillium wilt disease in two of the three targets. Thus, our study suggests that, depending on the target gene chosen, HIGS against V. dahliae is operational in tomato and A. thaliana plants and may be exploited to engineer resistance in Verticillium wilt-susceptible crops.

Comparative Proteomic Analysis of Gossypium thurberi in Response to Verticillium dahliae Inoculation.

Verticillium wilt is threatening cotton productivity globally. This disease is caused by soil-borne Verticillium dahliae which directly infects cotton roots, and exclusively colonizes and occludes xylem vessels, finally resulting in necrosis, defoliation, and most severely, plant death. For the first time, iTRAQ (isobaric tags for relative and absolute quantification) was applied to screen the differentially expressed proteins of Gossypium thurberi inoculated with V. dahliae. A total of 6533 proteins were identified from the roots of G. thurberi after inoculation with V. dahliae, and 396 showed up- and 279 down-regulated in comparison to a mock-inoculated roots. Of these identified proteins, the main functional groups were those involved in cell wall organization and reinforcement, disease-resistant chemicals of secondary metabolism, phytohormone signaling, pathogenesis-related proteins, and disease-resistant proteins. Physiological and biochemical analysis showed that peroxidase activity, which promotes the biosynthesis and accumulation of lignin, was induced early in the hypocotyl after inoculation with V. dahliae. Similarly, salicylic acid also accumulated significantly in hypocotyl of the seedlings after inoculation. These findings provide an important knowledge of the molecular events and regulatory networks occurring during G. thurberi-V. dahliae interaction, which may provide a foundation for breeding disease-resistance in cotton.

Genome-wide analysis of the gene families of resistance gene analogues in cotton and their response to Verticillium wilt.

BackgroundGossypium raimondii is a Verticillium wilt-resistant cotton species whose genome encodes numerous disease resistance genes that play important roles in the defence against pathogens. However, the characteristics of resistance gene analogues (RGAs) and Verticillium dahliae response loci (VdRLs) have not been investigated on a global scale. In this study, the characteristics of RGA genes were systematically analysed using bioinformatics-driven methods. Moreover, the potential VdRLs involved in the defence response to Verticillium wilt were identified by RNA-seq and correlations with known resistance QTLs.ResultsThe G. raimondii genome encodes 1004 RGA genes, and most of these genes cluster in homology groups based on high levels of similarity. Interestingly, nearly half of the RGA genes occurred in 26 RGA-gene-rich clusters (Rgrcs). The homology analysis showed that sequence exchanges and tandem duplications frequently occurred within Rgrcs, and segmental duplications took place among the different Rgrcs. An RNA-seq analysis showed that the RGA genes play roles in cotton defence responses, forming 26 VdRLs inside in the Rgrcs after being inoculated with V. dahliae. A correlation analysis found that 12 VdRLs were adjacent to the known Verticillium wilt resistance QTLs, and that 5 were rich in NB-ARC domain-containing disease resistance genes.ConclusionsThe cotton genome contains numerous RGA genes, and nearly half of them are located in clusters, which evolved by sequence exchanges, tandem duplications and segmental duplications. In the Rgrcs, 26 loci were induced by the V. dahliae inoculation, and 12 are in the vicinity of known Verticillium wilt resistance QTLs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0508-3) contains supplementary material, which is available to authorized users.

Determination of long-term effects of consecutive effective fresh chicken manure with solarization and verticillium wilt (Verticillium dahlia Klebb) on weed and its control in egg plant

The aim of the study was to determine the weed density and the most economical way of weed control in eggplant (Solanum melongena) fields contaminated with Verticillium dahliae (Kleb) after the application of fresh chicken manure and solarization in the second year as the same crop was grown. The effect of solarization on weed and the labor need in weed control continued in a diminishing way in the consecutive observations. With fresh chicken manure (FCM), number of weeds (number m-2) decreased but their green and dry biomass (weights g m-2) increased. The labor need (d ha-1) to control the weeds decreased. Similar results were also recorded for V. dahliae inoculation. As a result of the study, 50% of labor saving was achieved in the plots of solarization and either FCM rate combinations [sol x FCM (12 kg.m-2); sol x FCM (6 kg.m-2)] compared to the control plots. Achieved savings in labor can afford to cover the costs of solarization and FCM. Key words: Soil solarization, fresh chicken manure, Verticillium dahliae, eggplant, weed, weed control.

Analysis of upland cotton (Gossypium hirsutum) response to Verticillium dahliae inoculation by transcriptome sequencing.

Verticillium wilt is one of the main diseases in cotton (Gossypium hirsutum), severely reduces yield and fiber quality, and is difficult to be con-trolled effectively. At present, the molecular mechanism that confers resistance to this disease is unclear. Transcriptome sequencing is an important method to detect resistance genes, explore metabolic pathways, and study resistance mechanisms. In this study, the transcriptome of a disease-resistant inbred cot-ton line inoculated with Verticillium dahliae was sequenced. A total of 126,402 unigenes were obtained using de novo assembly and data analysis, 99,712 (78.88%) of which were annotated into the Nr, Nt, Swiss-Prot, KEGG, COG, and GO databases. The expression patterns of 16 candidate disease-resis-tance genes showed that some genes were upregulated soon after V. dahliae inoculation and others were upregulated later, which may indicate instanta-neous basal defense and lagged specific defense, respectively. We conducted a preliminary analysis of the transcriptome database, which will contribute to further research regarding the cloning of disease-resistance genes.

A fungal protein elicitor PevD1 induces Verticillium wilt resistance in cotton

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

Island cotton Gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae.

Verticillium wilt caused by soilborne fungus Verticillium dahliae could significantly reduce cotton yield. Here, we cloned a tomato Ve homologous gene, Gbve1, from an island cotton cultivar that is resistant to Verticillium wilt. We found that the Gbve1 gene was induced by V. dahliae and by phytohormones salicylic acid, jasmonic acid, and ethylene, but not by abscisic acid. The induction of Gbve1 in resistant cotton was quicker and stronger than in Verticillium-susceptible upland cotton following V. dahliae inoculation. Gbve1 promoter-driving GUS activity was found exclusively in the vascular bundles of roots and stems of transgenic Arabidopsis. Virus-induced silencing of endogenous genes in resistant cotton via targeting a fragment of the Gbve1 gene compromised cotton resistance to V. dahliae. Furthermore, we transformed the Gbve1 gene into Arabidopsis and upland cotton through Agrobacterium-mediated transformation. Overexpression of the Gbve1 gene endowed transgenic Arabidopsis and upland cotton with resistance to high aggressive defoliating and non-defoliating isolates of V. dahliae. And HR-mimic cell death was observed in the transgenic Arabidopsis. Our results demonstrate that the Gbve1 gene is responsible for resistance to V. dahliae in island cotton and can be used for breeding cotton varieties that are resistant to Verticillium wilt.

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.

Grafting Eggplant onto Tomato Rootstock to Suppress Verticillium dahliae Infection: The Effect of Root Exudates

Verticillium wilt (caused by Verticillium dahliae), a soilborne disease, often causes significant reductions of yield in eggplant (Solanum melongena L.) production where crop rotation is limited. Rootstock replacement through grafting is considered an effective method to control this disease. This 2-year study investigated the eggplant yield, resistance to verticillium wilt, and allelochemicals in root exudates of eggplant grafted onto a tomato rootstock. Both disease incidence and disease severity on grafted eggplant were markedly lower than those of nongrafted eggplants. Fifteen days after V. dahliae inoculation, grafted eggplants did not exhibit any infection, whereas the disease incidence and disease severity index of the nongrafted eggplants were 68.3% and 37.8% in 2006 and 66.7% and 36.3% in 2007, respectively. Twenty-five days after inoculation, disease incidences on grafted eggplants were only 8.1% and 9.5% in 2006 and 2007, respectively, but those of the nongrafted eggplants increased to 100%. As a result, early yield, total yield, and average fruit weight were significantly increased by grafting when inoculated with V. dahliae in 2006 and 2007. Mycelium growth of V. dahliae was inhibited by the root exudates of grafted eggplants. In contrast, the root exudates of nongrafted eggplants enhanced the mycelium growth. The gas chromatography–mass spectrometry analysis revealed that the composition in the root exudates released by grafted eggplants differed not only from the nongrafted eggplants, but also from the tomato rootstock plants. Ten chemical classes were isolated and identified in root exudates of grafted eggplants. Carbazoles, amines, azulene, and fluorene were only detected in the grafted eggplants. The relative contents of ester compounds were the highest in the root exudates from the grafted eggplant followed by derivatives of benzene, whereas the relative contents of benzene derivatives were much higher than that of the ester compounds in the root exudates from the nongrafted eggplant and tomato rootstock.

Characterization of Verticillium dahliae and V. tricorpus Isolates from Lettuce and Artichoke.

Verticillium isolates collected from lettuce and artichoke were characterized for morphology, growth and pathogenicity. Several isolates were identified as Verticillium tricorpus based on morphological and cultural characteristics, including the production of dark resting mycelia, chlamydospores, microsclerotia, and yellow to orange pigmentation in culture. Compared with isolates of V. dahliae, these isolates also produced microsclerotia and conidia that were significantly larger and exhibited a distinct growth pattern at varying temperatures. Using database sequence information, primers were developed from the internal transcribed spacer region to produce a diagnostic 337-bp product specific to V. tricorpus and used to confirm the identification of isolates. Pathogenicity tests indicated that isolates of V. tricorpus were weak pathogens, causing a median disease severity (DS) of <1 (0-to-5 scale) on lettuce and artichoke. In contrast, isolates of V. dahliae consistently caused severe wilt with a median DS of >3.5 on lettuce and 5.0 on artichoke. Although lettuce and artichoke inoculated with isolates of V. tricorpus exhibited reduced height and fresh foliar and root weight, the reductions were not statistically significant, unlike in plants inoculated with isolates of V. dahliae. Lettuce co-inoculated with isolates of V. tricorpus and V. dahliae exhibited reduced symptoms of Verticillium wilt and improved growth relative to those inoculated with V. dahliae alone. The early introduction of V. tricorpus in soil-drench inoculations appeared to provide better relief from subsequent V. dahliae inoculation than when the two species were co-inoculated simultaneously using the root-dip method, suggesting competitive exclusion as a plausible mechanism. A spore-polymerase chain reaction assay developed using cultured spores directly as template and primers specific to V. tricorpus confirmed the presence of V. tricorpus on inoculated roots. This work demonstrates the potential use of V. tricorpus to directly reduce the effect of V. dahliae on lettuce and artichoke and, to our knowledge, is the first reported characterization of V. tricorpus isolates collected from lettuce and artichoke.

Evidences for involvement of endogenous cAMP in Arabidopsis defense responses to Verticillium toxins

Although there were reports suggesting the involvement of endogenous cAMP in plant defense signaling cascades, there is no direct evidence supporting this notion yet and the detailed mechanism is unclear. In the present study, we have used pathogenic fungi Verticillium dahliae and Arabidopsis plants as a model system of plant-microb interaction to demonstrate the function of endogenous cAMP in Arabidopsis defense responses. Both V. dahliae inoculation and Verticillium toxins injection induced typical "wilt" symptoms in Arabidopsis seedlings. When either 8-Br-AMP (a membrane permeable cAMP analogue) or salicylic acid (SA) was applied to Arabidopsis, the plants became resistant to V. dahliae toxins. However, addition of 8-Br-AMP did not increase the resistance of Arabidopsis transgenic plants deficient in SA to the toxins, suggesting that cAMP might act upstream of SA in plant defense signaling pathway. Indeed, 8-Br-cAMP and forskolin, an activator of adenylyl cyclase, significantly stimulated the endogenous SA level in plants, whereas DDA, an inhibitor of adenylyl cyclase dramatically reduced toxin-induced SA increase. Both the endogenous cAMP and SA increased significantly in Arabidopsis seedlings treated with toxins. Furthermore, transcription level of pathogenesis-related protein 1 gene (PR1) was strongly induced by both 8-Br-cAMP and the toxin treatment. Taken together, our data demonstrate that endogenous cAMP is involved in plant defense responses against Verticillium-secreted toxins by regulating the production of the known signal SA in plant defense pathway.

Modification of fatty acids in eggplant affects its resistance to Verticilliumdahliae

Expression of the yeast Δ-9 desaturase gene in eggplant increased its levels of 16:1, 18:1, and 16:3 fatty acids and enhanced its resistance to Verticillium dahliae. Eggplants responded to V. dahliae inoculation with transitory upsurges in phospholipase A2and lipoxygenase activities, as well as levels of free fatty acids and lipid peroxides. The upsurges of certain fatty acids, in particular the 16:1 and 16:3 fatty acids, were more pronounced in the transgenic plants. cis -Δ9 16:1 was found to directly inhibit the growth of V. dahliae. The results suggest that increasing the production of 16:1 in plants could be an approach to enhance their resistance to fungal diseases