Discovery of 5-hydroxymethyl-2-furancarboxylic acid as a potential agent for controlling Verticillium dahliae and its action mechanism research

ABSTRACTThe infection of Macrophomina phaseolina often results in a grayish appearance with numerous survival structures, microsclerotia, on the plant surface. Past works have studied the development of fungal survival structures, sclerotia and microsclerotia, in the Leotiomycetes and Sordariomycetes. However, M. phaseolina belongs to the Dothideomycetes, and it remains unclear whether the mechanism of microsclerotia formation remains conserved among these phylogenetic clades. This study applied RNA-sequencing (RNA-Seq) to profile gene expressions at four stages of microsclerotia formation, and the results suggested that reactive oxygen species (ROS)-related functions were significantly different between the microsclerotia stages and the hyphal stage. Microsclerotia formation was reduced in the plates amended with antioxidants such as ascorbic acid, dithiothreitol (DTT), and glutathione. Surprisingly, DTT drastically scavenged H2O2, but the microsclerotia amount remained similar to the treatment of ascorbic acid and glutathione that both did not completely eliminate H2O2. This observation suggested the importance of over H2O2 in initiating microsclerotia formation. To further validate this hypothesis, the superoxide dismutase 1 (SOD1) inhibitor diethyldithiocarbamate trihydrate (DETC) and H2O2 were tested. The addition of DETC resulted in the accumulation of endogenous and more microsclerotia formation, but the treatment of H2O2 did not. The expression of SOD1 genes were also found to be upregulated in the hyphae to the microsclerotia stage, which suggested a higher endogenous stress presented in these stages. In summary, this study not only showed that the ROS stimulation remained conserved for initiating microsclerotia formation of M. phaseolina but also highlighted the importance of in initiating the hyphal differentiation to microsclerotia formation.IMPORTANCE Reactive oxygen species (ROS) have been proposed as the key stimulus for sclerotia development by studying fungal systems such as Sclerotinia sclerotiorum, and the theory has been adapted for microsclerotia development in Verticillium dahliae and Nomuraea rileyi. While many studies agreed on the association between (micro)sclerotia development and the ROS pathway, which ROS type, superoxide () or hydrogen peroxide (H2O2), plays a major role in initiating hyphal differentiation to the (micro)sclerotia formation remains controversial, and literature supporting either or H2O2 can be found. This study confirmed the association between ROS and microsclerotia formation for the charcoal rot fungus Macrophomina phaseolina. Moreover, the accumulation of but not H2O2 was found to induce higher density of microsclerotia. By integrating transcriptomic and phenotypic assays, this study presented the first conclusive case for M. phaseolina that is the main ROS stimulus in determining the amount of microsclerotia formation.

Volatile organic compounds (VOCs) produced by microorganisms are considered promising environmental-safety fumigants for controlling soil-borne diseases. Verticillium dahliae, a notorious fungal pathogen, causes economically important wilt diseases in agriculture and forestry industries. Here, we determined the antifungal activity of VOCs produced by Trichoderma koningiopsis T2. The VOCs from T. koningiopsis T2 were trapped by solid-phase microextraction (SPME) and tentatively identified through gas chromatography–mass spectrometry (GC/MS). The microsclerotia formation, cell wall-degrading enzymes and melanin synthesis of V. dahliae exposed to the VOC mixtures and selected single standards were examined. The results showed that the VOCs produced by strain T2 significantly inhibited the growth of V. dahliae mycelium and reduced the severity of Verticillium wilt in tobacco and cotton. Six individual compounds were identified in the volatilome of T. koningiopsis T2, and the dominant compounds were 3-octanone, 3-methyl-1-butanol, butanoic acid ethyl ester and 2-hexyl-furan. The VOCs of strain T2 exert a significant inhibitory effect on microsclerotia formation and decreased the activities of pectin lyase and endo-β-1,4-glucanase in V. dahliae. VOCs also downregulated the VdT3HR, VdT4HR, and VdSCD genes related to melanin synthesis by 29. 41-, 10. 49-, and 3.11-fold, respectively. Therefore, T. koningiopsis T2 has potential as a promising biofumigant for the biocontrol of Verticillium wilt disease.

Cotton is an important economic crop. Cotton Verticillium wilt caused by Verticillium dahliae seriously damages production. Phytosterols play roles in plant-pathogen interaction. To explore the function and related mechanism of phytosterols in the interaction between Verticillium dahliae and cotton plants, and the resistance to Verticillium wilt, in this study, we analyzed the changes of sterol composition and content in cotton roots infected by Verticillium dahliae, and identified the sterol C22-desaturase gene GhCYP710A1 from upland cotton. Through overexpressing and silencing the gene in cotton plant, and ectopically expressing the gene in Arabidopsis, we characterized the changes of sterol composition and the resistance to Verticillium wilt in transgenic plants. The infection of Verticillium dahliae resulted in the content of total sterol and each sterol category decreasing in cotton root. The ratio of stigmasterol to sitosterol (St/Si) increased, indicating that the conversion of sitosterol to stigmasterol was activated. Consistently, the expression level of GhCYP710A1 was upregulated after infection. The GhCYP710A1 has the conservative domain that is essential for sterol C22-desaturase in plant and is highly expressed in root and stem, and its subcellular location is in the endoplasmic reticulum. The ectopic expression of GhCYP710A1 gene promoted the synthesis of stigmasterol in Arabidopsis. The St/Si value is dose-dependent with the expression level of GhCYP710A1 gene. Meanwhile, the resistance to Verticillium wilt of transgenic Arabidopsis increased and the permeability of cell membrane decreased, and the content of ROS decreased after V991 (a strain of Verticillium dahliae) infection. Consistently, the resistance to Verticillium wilt significantly increased in the transgenic cotton plants overexpressing GhCYP710A1. The membrane permeability and the colonization of V991 strain in transgenic roots were decreased. On the contrary, silencing GhCYP710A1 resulted in the resistance to Verticillium wilt being decreased. The membrane permeability and the colonization of V991 were increased in cotton roots. The expression change of GhCYP710A1 and the content alteration of stigmasterol lead to changes in JA signal transduction, hypersensitivity and ROS metabolism in cotton, which might be a cause for regulating the Verticillium wilt resistance of cotton plant. These results indicated that GhCYP710A1 might be a target gene in cotton resistance breeding.

BackgroundVerticillium wilt is a serious soil-borne vascular disease that causes major losses to upland cotton (Gossypium hirutum L.) worldwidely every year. The protein VIP1 (VirE2 interaction protein 1), a bZIP transcription factor, is involved in plant response to many stress conditions, especially pathogenic bacteria. However, its roles in cotton response to Verticillium wilt are poorly understood.ResultsThe GbVIP1 gene was cloned from resistant sea-island cotton (G. barbadense) cv. Hai 7124. Expression of GbVIP1 was up-regulated by inoculation with Verticillium dahliae and exogenous treatment with ethylene. Results of virus-induced gene silencing suggested that silencing of GbVIP1 weakened cotton resistance to Verticillium wilt. The heterologous expression of GbVIP1 in tobacco showed enhanced resistance to Verticillium wilt. The PR1, PR1-like and HSP70 genes were up-regulated in GbVIP1 transgenic tobacco after Verticillium wilt infection.ConclusionOur results suggested that GbVIP1 increased plant resistance to Verticillium wilt through up-regulating expressions of PR1, PR1-like, and HSP70. These results provide new approaches to improving resistance to Verticillium wilt in upland cotton and also have great potential for disease-resistance breeding of cotton.

Microbe-induced gene silencing targeting VdEno of Verticillium dahliae for the control of cotton Verticillium wilt

Verticillium wilt (VW) of cotton poses a serious threat to the quality and yield of cotton. Verticillium dahliae is the primary causal agent of cotton VW. Moreover, V. dahliae can infect more than 200 species of dicotyledonous plants. The fungal cell wall plays a crucial role in its growth, development and pathogenicity. However, the mechanism of cell wall synthesis in V. dahliae and its role in pathogenesis remains unclear. In this study, we identified two chitin synthase (CHS) genes VdChs5 and VdChs7 containing myosin motor-like domain (MMD) and characterized their role in virulence of V. dahliae. The results showed that the functions of VdChs5 and VdChs7 were largely redundant, and target deletion of both VdChs5 and VdChs7 in V. dahliae did not affect vegetative growth, but reduced conidial production. ΔVdChs5Chs7 deletion mutant failed to colonize and proliferate in cotton vascular tissue, and exhibited significantly reduced virulence on cotton, suggesting that VdChs5 and VdChs7 are necessary for pathogenesis. In addition, the thickness of the cell wall in ΔVdChs5Chs7 showed significantly decreased, and ΔVdChs5Chs7 mutant exhibited hypersensitivity to cell wall perturbing agents and reactive oxygen species (ROS), indicating that VdChs5 and VdChs7 play key roles in cell wall integrity. Further, host-induced gene silencing (HIGS) silenced transcripts of VdChs5 and VdChs7 in susceptible cotton (Gossypium hirsutum L. acc. TM-1) enhanced resistance to cotton VW. Taken together, our data demonstrated that VdChs5 and VdChs7 play pivotal roles in proliferation, cell wall integrity, and pathogenicity, and provided a novel strategy to improve Verticillium wilt resistance in cotton and other susceptible host plants.

Verticillium wilt of cotton, caused by Verticillium dahliae, is one of the most devastating soilborne fungal diseases in cotton production, urgently demanding the development of effective control measures. Myxobacteria, a group of higher prokaryotes exhibiting multicellular social behaviors, possess predatory activity against plant pathogenic fungi and bacteria, giving them unique potential for application in plant disease biocontrol. In this study, based on a previously myxobacterial strain collection, a myxobacterial strain, HM-E, exhibiting broad-spectrum antifungal activity was screened. Through morphological observation, physiological and biochemical characterization, and multi-locus sequence analysis, this strain was identified as Cystobacter fuscus HM-E. C. fuscus HM-E not only significantly lysed V. dahliae hyphae but also inhibited its spore germination. Both its cell-free fermentation filtrate and volatile metabolites exhibited certain antifungal activity. Greenhouse pot assays showed that the fermentation broth of C. fuscus HM-E had a control efficacy of only 23.01% against cotton Verticillium wilt, whereas the solid agent formulated with white star flower chafer (Protaetia brevitarsis) frass achieved a significantly higher control efficacy of 70.90%, and the myxobacterial solid agent also significantly promoted cotton seedling growth. Furthermore, the crude extracts concentrated using macroporous resin and acid precipitation showed no antifungal activity against V. dahliae, whereas the crude protein obtained by ammonium sulfate precipitation disrupted not only the cell wall and cell membrane of V. dahliae hyphae, induced intracellular reactive oxygen species (ROS) burst but also lysed spores and inhibited spore germ tube elongation. Enzyme substrate profile assays indicated that several peptidases, lipases, and glycoside hydrolases secreted by C. fuscus HM-E might play important roles in its antifungal process and are potential biocontrol factors. This study suggests C. fuscus HM-E, as a novel biocontrol agent, has great potential for application in the combating of cotton Verticillium wilt.

Mechanismal analysis of resistance to Verticillium dahliae in upland cotton conferred by overexpression of RPL18A-6 (Ribosomal Protein L18A-6)

Verticillium dahliae was one of the most important diseases caused Verticilliumwiltofcotton. In our previous study, Bacillus axarquiensis TUBP1 was screened and found to be an antagonistic strain against V. dahliae with 43% biocontrol effect in the cotton field. In order to uncover the functional mechanism of B. axarquiensis against Verticillium wilt in cotton, the colonization of B. axarquiensis labeled with a green fluorescent protein (GFP) was investigated in cotton plants and the rhizosphere soil. Firstly, a plasmid (pHT-315) containing gfp gene was successfully transformed into wild B. axarquiensis TUBP1 and labeled a green fluorescence by electroporation, which didn't change the bioactivity in vitro. In gnotobiotic conditions, cotton seeds were then inoculated with the gfp-labeled strain and grown in green house. Observation with a confocal laser scanning microscope and a scanning electron microscope showed that GFP-labeled B. axarquiensis TUBP1 infected cotton roots and widely distributed in epidermis, cortical parenchyma, intercellular spaces, the xylem vessels, and pith cells as well as root hair cells through cracks formed at the lateral root junctions, followed by a slow migration from roots to stems and leaves. Quantitative fluorescence and flow cytometry (FACS) approaches showed a gradual decrease in the number of TUBP1-315gfp with increasing inoculation time. However, TUBP1-315gfp levels were detectable till 45days after planting. In contrast, no fluorescence signal was detected in the non-inoculated groups. Therefore, GFP-labeled B. axarquiensis TUBP1 exhibited colonization in different parts of cotton plants from the rhizosphere soil.

Verticillium wilt of cotton is a severe soil-borne disease caused by Verticillium dahliae worldwide. Phytotoxin of V. dahliae (VD-toxin) plays a crucial role in inducing the wilt syndrome in cotton. In this study, spores of four antagonistic isolates were used in the form of powdered biocontrol agent (BCA) to protect cotton from the toxic and wilting effect of VD-toxin. The application of powdered Streptomyces BCA to soil at the time of sowing considerably enhanced defense-related enzyme activities and increased ortho-dihydric phenol and lignin contents in cotton plants after 6 week cultivation in a greenhouse. Compared to the control treatment, Streptomyces BCA treatment severely enhanced defense-related responses in cotton plants within 24 h post-VD-toxin treatment, which further decreased the accumulation of malondialdehyde and mitigated the losses of root activity, leaf total chlorophyll content, and leaf water content. Ultimately, the Streptomyces BCA treatment weakened the pathogenic effect of VD-toxin on cotton and reduced Verticillium wilt by 20.5% to 68.2% at 72 h. These results indicate that the induction of systemic disease resistance and the reduction of VD-toxin’s pathogenic effect are two important mechanisms of antagonistic Streptomyces BCA for biocontrol of Verticillium wilt of cotton.

Investigating the genetic regulatory mechanisms underlying complex traits forms the foundation for crop improvement. Verticillium wilt (VW), caused by Verticillium dahliae (V. dahliae), is one of the most devastating diseases affecting crop production worldwide. However, the genetic basis underlying crop resistance to V. dahliae remains largely obscure, hindering progress in the genomic selection for VW resistance breeding. Here, we unraveled the genetic architectures and regulatory landscape of VW resistance in cotton by combining genome‐wide association studies (GWAS) and transcriptome‐wide association studies (TWAS) using 1152 transcriptomes derived from 290 cotton accessions. We identified 10 reliable quantitative trait loci (QTLs) associated with VW resistance across multiple environments. These QTLs showed a pyramiding resistance effect and exhibited promising efficacy in the genomic prediction of cotton's VW resistance supported by an F2:3 population. Moreover, trace analysis of these elite alleles revealed a notably increased utilization of Lsnp1, Lsnp4, Lsnp5, Lsnp8, and Lsnp9, which potentially contribute to the improvement of VW resistance in Chinese cotton breeding since the 1990s. We also identified remarkable gene modules and expression QTL (eQTL) hotspots related to the regulation of reactive oxygen species (ROS) homeostasis and immune response. Furthermore, 15 candidate causal genes were prioritized by TWAS. Knocking down eight genes with a negative effect significantly enhanced cotton resistance to V. dahliae. Among them, GhARM, encoding an armadillo (ARM)‐repeat protein, was verified to modulate cotton resistance to V. dahliae by regulating ROS homeostasis. Overall, this study updates the understanding of the genetic basis and regulatory mechanisms of cotton's VW resistance, providing valuable strategies for VW management through genomic selection in cotton breeding.

Verticillium wilt in cotton caused by Verticillium dahliae is one of the most serious plant diseases worldwide. Because no known fungicides or cotton cultivars provide sufficient protection against this pathogen, V. dahliae causes major crop yield losses. Here, an isolated cotton endophytic bacterium, designated Bacillus amyloliquefaciens 41B-1, exhibited greater than 50% biocontrol efficacy against V. dahliae in cotton plants under greenhouse conditions. Through high-performance liquid chromatography and mass analysis of the filtrate, we found that the antifungal compounds present in the strain 41B-1 culture filtrate were a series of isoforms of iturins. The purified iturins suppressed V. dahliae microsclerotial germination in the absence or presence of cotton. Treatment with the iturins induced reactive oxygen species bursts, Hog1 mitogen-activated protein kinase (MAPK) activation and defects in cell wall integrity. The oxidative stress response and high-osmolarity glycerol pathway contribute to iturins resistance in V. dahliae. In contrast, the Slt2 MAPK pathway may be involved in iturins sensitivity in this fungus. In addition to antagonism, iturins could induce plant defence responses as activators and mediate pathogen-associated molecular pattern-triggered immunity. These findings suggest that iturins may affect fungal signalling pathways and mediate plant defence responses against V. dahliae.

Verticillium wilt in cotton (Gossypium hirsutum) is primarily caused by Verticillium dahliae. Previous data suggest that prenylated RAB acceptors (PRAs) play essential roles in environmental plant adaptation, although the potential roles of PRA1 in cotton are unclear. Therefore, in this study, PRA1 family members were identified in G. hirsutum, and their roles in biotic and abiotic stresses were analyzed. Thirty-seven GhPRA1 family members were identified in upland cotton, which were divided into eight groups. Gene structure and domain analyses revealed that the sequences of GhPRA1 members in each group were highly conserved. Many environmental stress-related and hormone-response cis-acting elements were identified in the GhPRA1 promoter regions, indicating that they may respond to biotic and abiotic stresses. Expression analysis revealed that GhPRA1 members were widely expressed in upland cotton. The GhPRA1 genes responded to abiotic stress: drought, cold, salt, and heat stress. GhPRA1.B1-1A expression increased after V. dahliae infection. Furthermore, the functional role of GhPRA1.B1-1A was confirmed by overexpression in Arabidopsis thaliana, which enhanced the resistance to V. dahliae. In contrast, V. dahliae resistance was significantly weakened via virus-induced gene silencing of GhPRA1.B1-1A in upland cotton. Simultaneously, reactive oxygen species accumulation; the H2O2, salicylic acid, and jasmonic acid contents; and callose deposition were significantly decreased in cotton plants with GhPRA1.B1-1A silencing. These findings contribute to a better understanding of the biological roles of GhPRA1 proteins and provide candidate genes for cotton breeders for breeding V. dahliae-resistant cultivars.

SummaryVerticillium wilt (VW), caused by soil‐borne fungi of the genus Verticillium, is a serious disease affecting a wide range of plants and leading to a constant and major challenge to agriculture worldwide. Cotton (Gossypium hirsutum) is the world's most important natural textile fibre and oil crop. VW of cotton is a highly devastating vascular disease; however, few resistant germplasms have been reported in cotton. An increasing number of studies have shown that RNA interference (RNAi)‐based host‐induced gene silencing (HIGS) is an effective strategy for improving plant resistance to pathogens by silencing genes essential for the pathogenicity of these pathogens. Here, we have identified and characterized multifunctional regulators of G protein signalling (RGS) in the Verticillium dahliae virulence strain, Vd8. Of eight VdRGS genes, VdRGS1 showed the most significant increase in expression in V. dahliae after treating with the roots of cotton seedlings. Based on the phenotype detection of VdRGS1 deletion and complementation mutants, we found that VdRGS1 played crucial roles in spore production, hyphal development, microsclerotia formation and pathogenicity. Tobacco rattle virus‐mediated HIGS in cotton plants silenced VdRGS1 transcripts in invaded V. dahliae strains and enhanced broad‐spectrum resistance to cotton VW. Our data demonstrate that VdRGS1 is a conserved and essential gene for V. dahliae virulence. HIGS of VdRGS1 provides effective control against V. dahliae infection and could obtain the durable disease resistance in cotton and in other VW‐susceptible host crops by developing the stable transformants.

Surfactin and fengycin contribute differentially to the biological activity of Bacillus subtilis NCD-2 against cotton verticillium wilt