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We combined traditional mRNA-seq and RNC-seq together to reveal post-transcriptional regulation events impacting gene expression and interactions between the serious fungal pathogen Verticillium dahliae and a susceptible host, Gossypium hirsutum TM-1. After screening the differentially expressed and translated genes, V. dahliae infection was observed to influence gene transcription and translation in its host. Interestingly, the asparagine synthase (ASN1) gene transcripts increased significantly with the increase of infection time, while the rate of ASN1 protein accumulation in host TM-1 was distinctly lower than that in resistant hosts. We knocked down the ASN1 gene in resistant plants (ZZM2), and found that Verticillium-resistance was significantly reduced upon knockdown of ASN1. Our study revealed both transcriptional and post-transcriptional regulation of gene expression in TM-1 cotton plants infected by V. dahliae, and showed that ASN1 functions in the V. dahliae resistance process. These insights support breeding of disease resistance in cotton.

Plants interact with a great variety of microorganisms that inhabit the rhizosphere playing critical roles in several aspects of plant growth and protection against abiotic and biotic diseases. In this study, we performed a screening of bacteria associated with the rhizosphere of Prunus domestica trees to identify bacterial strains with plant growth-promoting activity. Ten strains isolated from the rhizosphere of P. domestica showed multiple in vitro plant growth promoting rhizobacteria (PGPR) activity such as the production of indole acetic acid, hydrogen cyanide, ammonia, solubilization of phosphates and antifungal activity against Verticillium dalhiae and Fusarium oxysporum f.sp. melonis. In planta, they significantly increased the growth (stem length, number of leaflets, leaf area and root weight) and biochemical (nitrate reductase activity, proline and chlorophyll content) parameters of tomato, as well as the rate of seed germination. Two selected strains (Pr7 and Pr8) with higher antagonistic activity against V. dalhiae and F. oxysporum f.sp. melonis protected tomato plants against Verticillium wilt and salt stress. In addition, they enhanced acclimatization of Vitis vinifera cv. Pinot noir and the peach root stock GF305 from in vitro to the greenhouse. 16S rRNA sequencing identified strains Pr7 and Pr8 as Pseudomonas stutzeri and Bacillus toyonensis, respectively. Since these two PGPR inoculants exhibited multiple traits beneficial to the examined host plants, they may be applied in the development of safe, and effective seed treatments as an alternative to chemical fungicides and fertilization but also for successful acclimatization of micropropagated plants., Mohamed Faize was supported by funding from the ‘Ministère de l'Enseignement Supérieur, de la Recherche Scientifique de la Formation des Cadres’ (MERSFC, Morocco) within the framework of ARIMNet2 Project

As people desire contact with the natural environment and comfortable living, modern buildings are increasingly incorporating the design of courtyard spaces. Hence, it is of interest to investigate the fungal air quality of courtyard spaces. This study aimed to investigate seasonal patterns of airborne fungal concentration, particlesize distribution, and taxonomic composition by using air microbial samplers and combining culture and molecular techniques. The results indicated that fungal concentration varied between seasons, peaking in autumn (825 ± 260 CFU/m3) and being the lowest in winter (617 ± 197 CFU/m3). Spearman's correlation analysis showed that fungal concentrations were positively correlated with relative humidity and wind speed. The relative humidity and wind speed supported fungal dispersion, whereas temperature negatively affected fungal dispersion in a multiple linear regression model. The fungal concentrations corresponding to “not excellent” in the Air Quality Index (AQI: >50) were higher than those corresponding to “excellent” (AQI: 0–50). The airborne fungal particle sizes in the range 1.1–3.3 μm (>58%) were predominant in all four seasons. In all four seasons, the airborne fungi had count median diameters ranging from 1.60 μm to 1.80 μm, allowing the fungi to penetrate the terminal bronchi. Verticillium (75.9%) and Cladosporium (27.4%) were the most abundant fungal genera in spring and summer, respectively. Flammulina was predominant in autumn and winter, accounting for 35.2% and 31.3%, respectively. Moreover, 14 fungal genera that contained allergenic or pathogenic species were identified in all four seasons. The highest mean average daily dose rates of airborne fungi was found in autumn. These findings will provide information for creating a more reasonable, comfortable, and healthier courtyard space air environment.

Germin-like proteins (GLPs) play important roles in plant disease resistance but are rarely reported in cotton. We compared the expression of GLPs in Verticillium dahliae inoculate G. hirsutum (susceptible) and G. barbadense (resistant) and enriched 11 differentially expressed GLPs. 2741 GLP proteins identified from 53 species determined that GLP probably originated from algae and could be classified into 7 clades according to phylogenetic analysis, among which Clade I is likely the most ancient. Cotton GLP (two allopolyploids and two diploids) genes within a shared clade were highly conserved. Intriguingly, clade VII genes were mainly located in gene clusters that derived from the expansion of LTR transposons. Clade VII members expressed mainly in root which is the first battle against Verticillium dahlia and could be induced more intensely in G. barbadense than G. hirsutum. The GLP genes are resistant to Verticillium dahliae, which can be further investigated against Verticillium wilt.

Sunflower yellow wilt is a widespread and destructive disease caused by the soil-borne pathogen Verticillium dahliae (V. dahliae). To better understand the pathogenesis mechanism of V. dahliae in sunflower, T-DNA insertion library was generated via Agrobacterium tumefaciens mediated transformation system (ATMT). Eight hundred positive transformants were obtained. Transformants varied in colony morphology, growth rate, conidia production and pathogenicity in sunflower compared to the wild type strain. A mutant, named VdGn3-L2, was chosen for further analysis based on its deprivation on microsclerotia formation. The flanking sequence of T-DNA insertion site of VdGn3-L2 was identified via hiTAIL-PCR, and the interrupted gene encoded an initiation-specific α-1, 6-mannosyltransferase, named as VdOCH1. The deletion mutant ΔVdOCH1 was impaired in certain characteristics such as fungal growth, conidia production, and microsclerotia formation. Also, ΔVdOCH1 mutants were more sensitive to the cell wall perturbing reagents, such as SDS and Congo red, lost their penetration ability through cellophane membrane, and exhibited dramatically decreased pathogenicity to sunflower. The impaired phenotypes could be restored to the wild type level by complementation of the deletion mutant with full-length VdOCH1 gene. In conclusion, VdOCH1, encoded α-1,6-mannosyltransferase, manipulating the biological characteristics, microsclerotia formation and pathogenic ability of V. dahliae in sunflower.

Verticillium dahliae is a soil-borne, hemibiotrophic phytopathogenic fungus that causes Verticillium wilt in a broad range of economic crops. The microsclerotia (MS), which act as the main host inoculum, can survive long-term in soil resulting in uncontrollable disease. In order to clarify the mechanism of MS formation, we sequenced the whole genome-wide expression profile of V. dahliae strain V991. Compared with M1 (no MS formation), during the process of MS formation and maturation, 1354, 1571, and 1521 unique tags were significantly regulated in M2, M3, and M4 library, respectively. During MS formation, melanin synthesis-related genes were preferentially upregulated. The process is more likely to regulated by transcription factors (TFs) including C2H2, Zn2Cys6, bZIP, and fungal-specific TF domain-containing proteins; additionally, G-protein coupled receptors, Ca2+, small GTPases, and cAMP were involved in signalling transduction. Protein kinase-encoding (VDAG_06474) and synthase-encoding (VDAG_05314) genes were demonstrated to negatively and positively influence MS production, respectively. The gene expression dynamics revealed during MS formation provide comprehensive theoretical knowledge to further understanding of the metabolism and regulation of MS development in V. dahliae, potentially providing targets to control Verticillium wilt through interfering MS formation.

Seven new guanacastane-type diterpenoids, namely dahlianes E-K (1–7), and three known ones (8–10) were isolated from the cultures of the fungus Verticillium dahliae. Their structures were established by extensive spectroscopic data analysis along with Rh2(OCOCF3)4- and Mo2(OAc)4-induced electronic circular dichroism (ECD) experiment. Dahliane G showed an 80-fold potentiation effect on the sensitization of doxorubicin at the concentration of 15 μM when screening the reversal activity on doxorubicin-resistant human breast cancer cells (MCF-7/DOX).

The large-scale long-term plantation of cotton in the Xinjiang region has been accompanied by a regular and wide outbreak of soil-borne fungal diseases such as verticillium wilt, which significantly damaged the local cotton industry. High-throughput sequencing data showed that the cotton field cultivation management measures pose a significant influence upon the original ecological soil fungal community structure. During long-term continuous cropping of cotton, a new soil fungal community structure emerges after several repeated adjustments over five years. The number of verticillium wilt pathogens in the soil increased rapidly with prolonged continuous cropping time, reaching a maximum at around the 10th y; moreover, the abundance of the verticillium wilt pathogen only serves as one of numerous essential factors for disease occurrence. The fungal community structure and the abundance of verticillium wilt pathogens in local cotton fields are gradually formed under joint effects of year-long continuous cropping and supporting cultivation management measures.

Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. However, these functions have not been assessed in Verticillium dahliae, a soil-borne fungal pathogen that causes devastating wilt diseases in many crops. The discovery and identity of novel lncRNAs and their association with virulence may contribute to an increased understanding of the regulation of virulence in V. dahliae. Here, we identified a total of 352 lncRNAs in V. dahliae. The lncRNAs were transcribed from all V. dahliae chromosomes, typically with shorter open reading frames, lower GC content, and fewer exons than protein-coding genes. In addition, 308 protein-coding genes located within 10 kb upstream and 10 kb downstream of lncRNAs were identified as neighboring genes, and which were considered as potential targets of lncRNA. These neighboring genes encode products involved in development, stress responses, and pathogenicity of V. dahliae, such as transcription factors (TF), kinase, and members of the secretome. Furthermore, 47 lncRNAs were significantly differentially expressed in V. dahliae following inoculation of susceptible cotton (Gossyoiumhisutum) cultivar Junmian No.1, suggesting that lncRNAs may be involved in the regulation of virulence in V. dahliae. Moreover, correlations in expression patterns between lncRNA and their neighboring genes were detected. Expression of lncRNA012077 and its neighboring gene was up-regulated 6 h following inoculation of cotton, while the expression of lncRNA007722 was down-regulated at 6 h but up-regulated at 24 h, in a pattern opposite to that of its neighboring gene. Overexpression of lncRNA012077 in wild-type strain (Vd991) enhanced its virulence on cotton while overexpression of lncRNA009491 reduced virulence. Identification of novel lncRNAs and their association with virulence may provide new targets for disease control.

Verticillium wilt, caused by Verticillium dahliae, is a cancer of cotton which affects cotton quality and yield in China. In our previous study, a novel anti-Verticillium dahliae protein TUBP1 was obtained from Bacillus axarquiensis. Then, we have systematically studied the anti-V. dahliae activity and the pore formation action of TUBP1 protein on V. dahliae membrane. In present study, we provide detailed whether TUBP1 protein induced mitochondrial damaged and mitochondria-mediated apoptotic cell death in V. dahliae. In V. dahliae cells exposed to the TUBP1 protein, the mitochondrial dehydrogenases, F0F1-ATPase, malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) activities were reduced and reactive oxygen species (ROS), which is a major cause of apoptosis, were increased. The results demonstrated that mitochondria dysfunction and ROS-induced oxidative stress caused the release of apoptotic factors. The following cellular changes, which are characteristic of apoptosis, were measured including mitochondrial membrane potential (MMP), Cytochrome c (Cyt C) release, metacaspase activation, phosphatidylserine (PS) exposure, and DNA condensation and fragmentation. The results showed that an important feature of apoptosis, MMP, was caused by ROS. Significantly, cyt c was released, which is a factor in metacaspase activity after treatment with the TUBP1 protein. Number of stained cells with activated intracellular metacaspases exposed to TUBP1 protein was increased in a concentration-dependent manner. We also showed that in the early and late stages of apoptosis, the effects of the TUBP1 protein were mediated by PS and DNA fragmentation and condensation in the plasma membrane, respectively. There turned out that the TUBP1 protein led to mitochondria-mediated apoptotic cell death in V.dahliae. The results of this investigation indicated that TUBP1 stain or protein is a potent candidate against V.dahliae infections in crop species.

The objectives of this study were to screen chitinase producing microorganisms to inhibit Rigidoporus microporus (white root rot) and to analyze the bioactive compounds produced by the selected strain using gas chromatography mass spectrometry (GC–MS). A total of 148 microbial isolates were screened from different sources such as composts and soils for chitinolytic activity by colloidal chitin hydrolysis, and then tested for inhibition by dual culture technique. The result showed that 78 isolates exhibited inhibitory activity against R. microporus. Among them, the fungal strain F14 showed the most chitinase production. It was identified as Trichoderma asperellum based on 18S rDNA. T. asperellum F14 was then cultured in 1/5 potato dextrose broth for 28 days to obtain the bioactive compounds using various type of solvents including methanol, n-butanol, ethyl acetate and hexane. Crude ethyl acetate extract displayed the highest inhibitory activity against many plant pathogens such as Agrobacterium tumefaciens, Erwinia carotovora subsp. carotovora, Xanthomonas oryzae, Aspergillus parasiticus, Fusarium culmorum, F. merismoides, F. oxysporum f. sp. gladioli, R. microporus, Verticillium albo-atrum and V. dahliae. The GC–MS analysis of the bioactive compounds produced by T. asperellum F14 showed that they were composed of many known bioactivities. This study indicates that T. asperellum F14 can be used to inhibit white root rot disease and other plant diseases.

Long non-coding RNAs (lncRNAs) has significant role in of gene expression and silencing pathways for several biological processes in eukaryotes. lncRNAs has been reported as key player in remodeling chromatin and genome architecture, RNA stabilization and transcription regulation, including enhancer-associated activity. Host lncRNAs are reckoned as compulsory elements of plant defense. In response to pathogen attack, plants protect themselves with the help of lncRNAs -dependent immune systems in which lncRNAs regulate pathogen-associated molecular patterns (PAMPs) and other effectors. Role of lncRNAs in plant microbe interaction has been studied extensively but regulations of several lncRNAs still need extensive research. In this study we discussed and provide as overview the topical advancements and findings relevant to pathogen attack and plant defense mediated by lncRNAs. It is hoped that lncRNAs would be exploited as a mainstream player to achieve food security by tackling different plant diseases.

Verticillium wilt caused by soil-borne fungus of Verticillium dahliae Kleb. is one of the most devastating diseases of cotton. Since the hierarchically organized mitogen-activated protein kinase (MAPK) cascade plays pivotal roles in signaling plant defense against pathogen attack, and the key nodes of MAPKKs (MKKs) may serve as for the convergence and divergence of signals in MAPK cascades, the possible relations between MAPK signaling and cotton Verticillium resistance were examined in this study. A total of 24 MKK genes were identified in the Gossypium hirsutum L. genome and then classified based on phylogenetic analysis. Then the regulation roles of all types of cotton MKKs in activation of cotton disease resistance were tested with the virus-induced gene silencing (VIGS) method. The results showed that three types of MKKs (GhMKK4, GhMKK6 and GhMKK9) positively regulate, while GhMKK10 negatively regulate the cotton resistance to Verticillium wilt. Further, more subtle regulation of cotton resistance mediated by MKK genes were revealed. In GhMKK9, only Gh_A12G2448 and Gh_D12G2574 displayed positive regulation of cotton resistance; whereas only Gh_A12G1883 and Gh_D12G2062 displayed negative regulation of cotton resistance in GhMKK10. All these results show that MKK members in MAPK signal cascades play dual roles in subtly regulating of cotton resistance to Verticillium wilt.

In tomato, Verticillium resistance is determined by the Ve gene locus encoding two leucine-rich repeat-receptor-like proteins (Ve1, Ve2). The resistance function usually is attributed to Ve1 alone, with two known alleles: Ve1, encoding a resistance protein, and ve1, with a premature stop codon encoding a truncated product. We have examined further Ve-gene expression in resistant and susceptible near-isolines of Verticillium-infected Craigella tomatoes, using both quantitative RT-PCR and an alternative RFLP assay. Ve1 is induced differentially in resistant and susceptible plants, while Ve2 is constitutively expressed throughout disease development. Contrary to their putative role in Verticillium resistance, these profiles were observed even with compatible Verticillium interactions, some bacterial pathogens, and transgenic tomato plants expressing the fungal Ave1 effector. This suggests broader roles in disease and/or stress. To determine the contribution of plant hormones, abscisic acid, methyl jasmonate, naphthaleneacetic acid or salicylic acid were infused independently via the tomato root and effects on Ve1 induction were confirmed using biosynthesis mutants. While all the hormones modulated Ve1-gene induction, abscisic acid and salicylic acid were not required while jasmonic acid appears to play a more direct role.

Avocado wilt complex (AWC) is an important disease and disorder of the avocado crop. It is associated with different causal agents that induce similar disease symptoms of expression, with Phytophthora cinnamomi, Verticillium sp., and abiotic hypoxia-anoxia being the most important ones under tropical conditions. The objectives of this work were to identify the edaphic variables associated with AWC foci and develop a GIS tool for the prevention of this disease. Microbiological, topographic, physical and chemical soil properties were performed, and AWC incidence and severity were estimated in plots planted with avocado. The effect of each of the soil properties on AWC was determined using a generalized additive model. Additionally, three categories of crop management were proposed and a GIS tool was designed to determine the risk of AWC presence. Diseased plants with AWC were more frequently found (P

Klebsiella pneumoniae (Kp) a common cause of pneumonia leads to intense lung injury and mortality that are correlated with infective exacerbations. A significant increase in the prevalence of imipenem resistant K. pneumoniae (IRKP) has been observed in common human pathogens. The rapid emergence of IRKP has limited the availability of anti-bacterial treatment options. Silver nano-particles (AgNPs) are one of the well-known antibacterial substances showing such multimode antibacterial action. Therefore, AgNPs are suitable candidates for use in combinations with imipenem in order to improve its antibacterial action. Fifteen fungal species were screened for mycosynthesis of silver nanoparticles (AgNPs), only eight fungal species were found to reduce the silver salt into silver nanoparticles which was characterized by UV–visible spectrophotometric analysis, Energy Dispersive Analysis of X-ray (EDX) and transmission electron microscopy. Consistent with characterized silver nanoparticles, mycosynthesized AgNPs by Verticillium albo-atrum (RCMB 039001) was found the higher in concentration (as detected by UV–visible spectrophotometric analysis) and the least in size (as detected by TEM analysis) so, it was chosen for further studies such as in vitro antibacterial activity against Imipenem resistant K. pneumonia (IRKP), MIC, FIC measurements and in vivo study. In this work a strong synergistic antibacterial effect between AgNPs and imipenem was detected in vitro (with FIC Index 0.07) and in vivo against IRKP strain. These results suggested that sliver nanoparticles have an effective antibacterial action on bacterial count, histopathology as well as protective immune response in an IRKP rat model.

A new species, Scopulariopsis gossypii, was found to be present in the vascular bundles of cotton plants (Gossypium hirsutum) infected by Verticillium dahliae which is an economically important pathogen in Hangzhou, China. The fungus was only present in the diseased plants, but it never became isolated from the healthy plants. Scanning electron microscopy showed that the fungus was present in parenchyma cells enclosing vessels in dark brown vascular tissues of stems, and produced asexual conidia within the tissues. Phylogenetic analysis of combined nuclear ribosomal D1/D2 region of the 28S rDNA as well as translation elongation factor 1-alpha (TEF1) and beta-tubulin (TUB) gene showed that S. gossypii represents an undescribed species of Scopulariopsis, Microascaceae. In this study, characteristics of sexual and asexual stages of the fungus were described, illustrated and compared with similar taxa. In addition, the molecular and morphological analyses indicated that S. gossypii was a distinct species of Scopulariopsis. The pathogenicity tests proved by inoculation of wounded roots confirmed that S. gossypii was an opportunistic pathogen causing leaf interveinal chlorosis and vascular browning of cotton plants. However S. gossypii did not infect host with undamaged roots. Moreover, coinoculation with S. gossypii and V. dahliae significantly increased disease severity.

Salicylic acid (SA) is a hormone connected with various cellular functions including the fight against invading pathogens. Priming of seeds pre-sowing is a very simple method to the farmers’ to produce better growth, yield and manage the pathogens. The present study was aimed to determine the growth and disease resistance ability in brinjal seeds primed with different concentrations (0.25, 0.5, 0.75 and 1.0 mM) of SA under greenhouse conditions. Priming of seeds with SA significantly increased seed germination and seedling vigor with a maximum of 84% and 859.18, respectively at 0.5 mM concentration. Seed priming with SA also reduced Verticillium wilt incidence to 39.25% (at 0.5 mM) under greenhouse conditions and also enhanced the vegetative growth parameters of the plant compared to control. The induced resistance obtained with SA was in line with higher expression of PR-protein (β-1,3-glucanase and chitinase) related defense enzymes. Further, an increase of 1.7, 2.9, 2.1, 2.5 and 2-fold increase in gene expression of IAA27, MPK1, GPX, chitinase and β-1,3-glucanase, respectively were observed in SA primed challenge inoculated seedlings than non-primed susceptible inoculated controls. The higher expression of IAA27, MPK1, GPX, chitinase and β-1,3-glucanase correlates with the plant growth promoting and disease protection studies as these genes are vital for increasing plant growth and inducing resistance during host-pathogen interaction. Enhanced activation of defense-related activities in plants upon priming with SA suggests that it alters plant physiology which in turn is useful for production and protection of brinjal.

The objective of the present study was to isolate and characterize effective lactic acid bacteria (LAB) with antifungal activities from some corn silages in Iran. The corn biomass was harvested from fields of three different regions, chopped, and ensiled in the lab silos. From 200 LABs isolates, 18 isolates with the highest acidic pH resistance (up to pH 3), salt resistance (up to 8%) and growth at 15–45 °C were selected for the next experiments. The highest (11.31 mg/ml) and the lowest (1.9 mg/ml) lactic acid production among the selected strains were belonged to the isolates E4 and G1, respectively. The antifungal properties of the selected strains were evaluated against some important plant pathogenic fungi, including Fusarium verticillioides , Penicillium sp. and Verticillium dahlia, and also against a parasitic oomycote “ Pythium aphani dematum ”. The in vitro experiments confirmed that the strains could inhibit from 0 to 40 mm of mycelial growth of different fungi. Based on amplification and sequencing of the16SrDNA gene, the strains were belonged to Lactobacillus fermentum (61.2%), L. plantarum (11.2%), L. paralimentaris (11.2%), L. pentosus (5.6%), L. buchneri (5.6%), and Sporolactobacillus (5.6%). The field experiments confirmed that different combinations of the selected strains could reduce disease index F. verticillioides up to 40%. This is the first report of antifungal activity of lactobacilli strains against F. verticillioides at lab and field levels. Finally, it could be concluded that the native LABs isolated form Iranian corn silages have high antifungal activities and via their biochemical activities may be used as silage additive to enhance quality and durability for ensiled corns.

Strawberries are one of the most popular fruits in Germany. However, an expansion of the cultivation area, especially for organic strawberry production, is limited due to the high yield losses caused by diseases and arthropod pests. Therefore, the two soil strengthener RhizoVital ® 42 fl. and Trichostar ® were evaluated for their efficacy in stabilizing strawberry yields in the presence of soil-borne diseases. First, an integration of antagonists in an IPM strategy was proven by testing their compatibility with synthetic pesticides. When mixed for a maximum of four hours only few fungicides reduced germination of the antagonistic fungus contained in Trichostar ® . The germination of the bacterial antagonist (RhizoVital ® 42 fl.) was not influenced by any tested synthetic pesticide used in this study. Afterwards, the microbial based products were tested in two commercial strawberry fields in the Rheine-Main area, in which Verticillium -wilt was found. In one field, with just one treatment of Trichostar ® in 2013, the yield increase in 2013 and 2014 was 8 and 9%, respectively. On the second field, RhizoVital ® 42 fl. was applied both years and the increase was about 6% each year. Our results demonstrate that beneficial micro-organisms can promote health and can stabilize or increase yield of strawberries under field conditions. Integration of these organisms in IPM strategies seems to be possible.

Oxidation of α-euphorbol and 31-norlanostenol, two triterpenic compounds isolated from the latex of Euphorbia resinifera and Euphorbia officinarum respectively, yielded four products named 3β-tosyloxy-4α,14α-dimethyl-5α-cholesta-7,9-diene; 4α,14α-dimethyl-5α-cholesta-7,9-dien-3β-ol; 24-methylen-elemo-lanosta-8,24-dien-3-one and elemo-lanost-8-en-3,11,24-trione. They were evaluated for protection of tomato plants against Verticillium dahliae in a greenhouse. The four semisynthesized products were phytotoxic at higher concentrations as they completely inhibited tomato germination at 100 and 500 μg/ml. However at lower concentrations (10 and 50 μg/ml) germination and root length were not affected. Disease resistance against Verticillium wilt was assessed in tomato plants derived from seeds that germinated in the presence of 10 and 50 μg/ml of the four products. All of them were able to reduce significantly disease severity, with 10 μg/ml being more effective than 50 μg/ml. Reduction of leaf alteration index and of stunting index ranged from 52 to 68% and from 43 to 67%, respectively, while vessel discoloration was reduced by at least 95%. The compounds were also able to elicit H2O2 accumulation before and after fungal inoculation and to significantly enhance peroxidase and polyphenol oxidase activities. These results suggest that the hemisynthetized triterpenes can be used as elicitors of disease resistance.

The production of biocontrol factors by Pseudomonads is reported to be controlled at the post-transcriptional level by the GacS/GacA signal transduction pathway. This involves RNA-binding translational repressor proteins, RsmA and RsmE, and the small regulatory RNAs (sRNAs) RsmX, RsmY, and RsmZ. While the former represses genes involved in secondary metabolite production, the latter relieves this repression at the end of exponential growth. We have studied the fluorescent Pseudomonas strain Psd, possessing good biocontrol potential, and confirmed the presence of rsmY and rsmZ by PCR amplification. Gene constructs for all the three small RNAs (RsmX, RsmY and RsmZ) carried on broad host-range plasmid, pME6032 were mobilized into strain Psd. Expression analysis of gacA in the recombinant strains over-expressing rsmX (Psd-pME7320), rsmY (Psd-pME6359) and rsmZ (Psd-pME6918) revealed a significant upregulation of the response regulator. Besides, a remarkable down-regulation of rsmA was also reported in all the strains. The variant strains were found to produce comparatively higher levels of phenazines. Indole acetic acid levels were higher to some extent, and strain Psd-pME6918 also showed elevated production of HCN. The tomato seedlings infected with Fusarium oxysporum and Verticillium dahliae in the presence of culture filtrate of the recombinant strains showed better plant protection response in comparison to the wild-type strain Psd. These results suggest that small RNAs are important determinants in regulation of the biocontrol property of strain Psd.

To identify key genes expressed in Verticillium dahliae in early stages of infection of cotton roots, spore suspensions of eight V. dahliae isolates with different virulence levels were induced by cotton roots and genes expressed in these isolates during the early stages of infection were profiled. A gene that was differentially expressed between highly and less virulent strains was identified. Cloning and bioinformatics analysis of the gene suggested that it belongs to the putative Gβ-like/RACK1 protein family, and has seven WD40 domains. Targeted deletion of the gene revealed that it controls a number of growth-related phenotypes, including conidia and microsclerotia production, normal spore germination and hyphal development. RACK1 is a component of eukaryotic ribosomes, and here we found by qRT-PCR that disruption of RACK1 in V. dahliae (designated VdRACK1) significantly altered the transcriptional levels of other ribosomal proteins, suggesting possible global effects of VdRACK1 deletion on the protein translation of other genes. VdRACK1-null mutants lost the ability to penetrate intact cotton roots. However, the mutant strain was able to infect root-wounded cotton plants and, intriguingly, resulted in a hypervirulent phenotype, implicating a role for VdRACK1 in the restriction of rampant growth within the plant.

Tiamulin (TIA) is a widely used antibiotic in Thai swine farms and persists in swine manure storage. Significant amounts remain in swine manure after composting, posing risks related to the spread of the compound and the evolution of resistant genes when this compost is used as a fertilizer. Therefore, it is necessary to enhance TIA degradation. This study successfully purified TIA-degrading fungi, which have a high capacity for treating antibiotic-contaminated manure. Twelve strains were collected from swine farms and preliminarily examined for their ability to degrade recalcitrant compounds using brilliant green and crystal violet dyes at 0.05% (w/v). Three isolated fungi and three white-rot fungal strains–namely, Lasiodiplodia sp. F1, Fusarium sp. F5, Galactomyces sp. F8, Verticillium sp., Trametes versicolor , and Trametes hirsuta AK4–capable of developing considerable decolorized zones on both dyes exhibited their abilities to degrade 10 mg l −1 of TIA over a period of 12 days: 93.2 ± 0.0%, 82.4 ± 10.2%, 73.8 ± 5.6%, 89.3 ± 0.0%, 86.1 ± 0.0%, and 66.8 ± 4.8%, respectively. Lasiodiplodia sp. F1, Verticillium sp., and Trametes versicolor were able to remove TIA most efficiently (>85%) and have the potential to be applied to swine waste for the elimination of antibiotic substances. Manganese peroxidase (MnP) was predominantly produced; MnP expression was induced by tiamulin, suggesting its main role in TIA degradation.

Verticillium wilt, due to the soilborne fungus Verticillium dahliae , is a persistent disease affecting flax culture in Upper Normandy. This pathology has increased since the last decade, leading to yield losses for flax producers. In part due to the long survival of V. dahliae in soil and the difficulty of early diagnosis in affected plant, Verticillium flax wilt management remains problematic in the absence of efficient phytosanitary treatment. Pathogen avoidance and the reduction of soil inoculum through adapted cultural practices are the best alternatives to fight against Verticillium wilt. Therefore, the objective was here to optimize and validate a rapid and specific real-time PCR assay targeting the ribosomal DNA Internal Transcribed Spacers (ITS) to measure V. dahliae density in soil. This method was then used to assess and compare the pathogen density in fields from four diverse management systems: conventional, integrated with or without tillage, and organic. First, the real-time PCR assay provided sensitive and reliable quantification of V. dahliae in a range of artificially inoculated soils with known inoculum density. Then, this method was successfully applied in crop fields. Measured V. dahliae densities presented an intra-parcel heterogeneity, emphasizing the importance of an adapted sampling strategy to assess pathogen load in crop field. Furthermore, these densities appeared to be impacted by agricultural practices, particularly tillage. The influence of the previous crop on pathogen load had also to be considered with attention to manage efficiently this disease through crop rotation. Such knowledge of pathogen density in soils could provide critical information for stakeholders to identify infested fields and predict disease development. Molecular approach should be considered as a useful tool for Verticillium wilt management in flax culture.

Verticillium wilt caused by Verticillium dahliae Kleb. is a major disease in cotton. We found that pectin lyase can enhance cotton resistance to Verticillium wilt and induce cell apoptosis of V. dahliae strain Vd080. The biocontrol effect of pectin lyase on Vd080 reached 61.9%. Pectin lyase increased ERG4 (Delta (24 (24 (1)))-sterol reductase) expression, the ergosterol content of the cell membrane, the collapse of mitochondrial membrane potential, hydrogen peroxide content, metacaspase activity, and Ca2+ content in the cytoplasm in the Vd080 strain and induced endoplasmic reticulum (ER) stress. Pectin lyase also increased the expression levels of the ER molecular chaperone glucose regulating protein Grp78 (BiP), protein disulfide isomerase (PDI) and calnexin (CNX), reduced the expression levels of the protein Hsp40. When the PDI and BiP genes of Vd080 were knocked out, the mutants △BiP and △PDI had reduced sensitivity to pectin lyase. In the absence of external stress, ER stress appeared in mutant △BiP cells. Pectin lyase affects the ergosterol content of the Vd080 cell membrane, which causes ER stress and increases the level of BiP to induce Vd080 cell apoptosis. These results demonstrate that pectin lyase can be used to control Verticillium wilt in cotton.

Guizhou Suantang (GZST), a type of sour soup, is a traditional fermented food that can be classified into Hong Suantang (HST) and Bai Suantang (BST). GZST possesses unique flavors arising from various microbiota in fermentation ecosystems. However, the association between these microbiota and flavors remains poorly understood. Accordingly, this study analyzed the volatile components and microbial communities of GZST via headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry and high-throughput 16S rRNA and internal transcribed spacer sequencing techniques. Results showed that 133 compounds, including alcohols, esters, phenols, hydrocarbons, ketones, aldehydes, nitriles, acids, and sulfides, were identified from GZST. Moreover, principal component analysis found significant variances in the composition of volatile compounds among different samples. The bacterial genus level indicated that all GZST samples were dominated by Lactobacillus. At the fungal genus level, BST was dominated by Pichia, Debaryomyces, Mortierella, unclassified, Meyerozyma, and Dipodascus. Meanwhile, HST was dominated by Pichia, Candida, Kazachstania, Debaryomyces, Archaeorhizomyces, and Verticillium. The potential correlations between microbiota and volatile components were also explored through bidirectional orthogonal partial least squares-based correlation analysis. Nine bacterial genera and eight fungal taxa were identified as functional core microbiota for flavor production on the basis of their dominance and functionality in the microbial community. In addition, excessive Lactobacillus inhibited the formation of certain flavor substances. These findings provided basic data for the isolation, screening, and fermentation regulation of functional microorganisms in GZST. The information provided in this study is valuable for the development of effective strategies for selecting beneficial bacterial and fungal strains to improve the quality of GZST.

Studies were conducted to identify candidate soil microbes responsible for observed differences in strawberry vigour at a small spatial scale, which was not associated with visual disease symptoms. Samples were obtained from the soils close to the rhizosphere of ‘big’ and ‘small’ plants from small plots which exhibited large local heterogeneity in plant vigour. A metabarcoding approach was used to profile bacterial and fungal compositions, using two primer pairs for 16S ribosomal RNA genes (16S rDNA) and one for the fungal internal transcribed spacer (ITS) region. Of the two 16S rDNA primer sets, the 341F/805R resulted in sequences of better quality. A total 28 operational taxonomic units (OTUs) had differential relative abundance between samples from ‘big’ and ‘small’ plants. However, plausible biological explanation was only possible for three fungal OTUs. Two were possible phytopathogens: Verticillium spp. and Alternaria alternata although the latter has never been considered as a main pathogen of strawberry in the UK. For samples from ‘small’ plants, the abundance of these OTUs was much greater than from ‘big’ plants. The opposite was true for a mycorrhizal OTU. These results suggest that soil microbes related to crop production can be identified using metabarcoding technique. Further research is needed to assess whether A. alternata and Verticillium spp. could affect strawberry growth in the field.

Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, poses a major threat to a broad host range of more than 400 plant species, including economically important cotton (Bell, 1992). V. dahliae is especially difficult to control because it persists in soil as resting structures, called microsclerotia, for several years in the absence of a host plant. The dormant microsclerotia are the primary infectious propagules and germinate when they are stimulated by root exudates. Infection of cotton roots by V. dahliae in soil naturally leads to the colonization of vascular tissues, from the parasitic to saprophytic phase, when mycelia and melanized dormancy microsclerotia are produced in the infected cotton, resulting in vessel blockage and cotton wilt disease (Gerik and Huisman, 1988).

The fungus Verticillium dahliae has gained worldwide notoriety as a destructive plant pathogen, causing vascular wilt diseases on diverse plant species. V. dahliae produces melanized resting bodies, known as microsclerotia, which can survive for 15 years in the soil, and are thus critically important in its disease cycle. However, the molecular mechanisms that underpin microsclerotia formation, survival, and germination remain poorly understood. In this study, we observed that deletion of VdHog1 (ΔVdHog1), encoding a homolog of a high-osmolarity glycerol (HOG) response mitogen-activated protein kinase, displayed decreased numbers of melanized microsclerotia in culture, heightened sensitivity to hyperosmotic stress, and increased resistance to the fungicide fludioxonil. Through RNA-Seq analysis, we identified 221 genes differentially expressed in the ΔVdHog1 strain. Interestingly, the expression levels of genes involved in melanin biosynthesis, as well as the hydrophobin gene VDH1, involved in the early stage of microsclerotia formation, were significantly decreased in the ΔVdHog1 strains relative to the wild-type expression levels. The ΔVdHog1 strains exhibited decreased virulence relative to the wild type strain on smoke tree seedlings. These results indicate that VdHog1 regulates hyperosmotic stress responses in V. dahliae, and establishes the Hog1-mediated pathway as a target to further probe the up- and downstream processes that regulate asexual development in this fungus.

Six new compounds, including three acremonidins F–H ( 1 , 3 , and 5 ), two acremoxanthones F–G ( 7 and 8 ), and acremonoxide ( 10 ), along with eight known compounds ( 2 , 4 , 6 , 9 , 11 – 14 ) were isolated from Verticillium sp. BCC33181. Their structures were determined based on extensive 1D and 2D NMR spectral analyses, mass spectrometric data and by comparison with the known compounds reported previously. The relative configurations were identified based upon NOESY spectroscopic information as well as comparison of CD spectrum with the known analogs. Both acremonidins and acremoxanthones exhibited anti- Bacillus cereus activity (MIC 0.39–25 μg/mL), while acremonidins F ( 1 ), A ( 2 ), G ( 3 ), and C ( 4 ) also displayed anti- Enterococcus faecium activity (MIC 1.56–25 μg/mL). In addition, acremoxanthones F ( 7 ) and G ( 8 ) showed antimalarial activity against Plasmodium falciparum K1 strain, multidrug–resistant strain, with respective IC 50 values of 2.38 and 1.71 μg/mL, while others were inactive.

Irrigation water disinfestation is an unexplored option for reducing Verticillium dahliae inoculum in water and consequently for more efficiently managing Verticillium wilts in Andalusia. We assessed Suppressive Efficacy (SE; water was infested and subsequently treated) and Preventive Efficacy (PE; previously treated water was subsequently inoculated) of OX-VIRIN®, OX-AGUA AL 25® and Deccoklor® in reducing water infestations by V. dahliae conidia. Five concentrations of each disinfestant, the lowest three being recommended by the manufacturer, were tested in vitro against six V. dahliae isolates. Validation assays were carried out in experiments under natural environmental conditions in spring. The four highest concentrations of OX-VIRIN® (0.8–51.2 mL L−1), the three highest of OX-AGUA AL 25® (46.4–417.5 μL L−1) and the two highest of Deccoklor® (0.375 and 3.75 mL L−1), showed an in vitro-efficacy (SE and PE) of 96.2, 80.0 and 100.0% after 30, 5 to 30 and 15 days respectively. Therefore, recommended concentrations for OX-VIRIN® and OX-AGUA AL 25® showed a greater in vitro-effectiveness than those recommended for Deccoklor®. Assays in natural environmental conditions proved that OX-VIRIN® at the recommended concentration of 3.2-mL L−1, applied every 28 days to water, was the most effective treatment (SE and PE), with a 100% reduction of the average relative viability after 56 days. Other chemical treatments showing high in vitro-efficacy, such as OX-VIRIN® at 0.8 mL L−1 and OX-AGUA AL 25® at 46.4 μL L−1 showed an SE of 99.9% after 14 and 28 days when applied every 28 and 14 days, respectively. However, PE of OX-AGUA AL 25® at 46.4 μL L−1 was only 59 and 38% after 28 and 14 days respectively, depending on the experiment.

The tomato Ve1 gene and several Ve1 homologues are involved in the resistance to Verticillium dahliae. Here, we report on another Ve homologous gene, Gbvdr3, from a Verticillium wilt-resistant cotton cultivar, Gossypium barbadense Hai7124, which has a 3207-bp region that encodes a predicted receptor-like protein. Transient expression analyses indicated that Gbvdr3 is localized in the plasma membrane, and virus-induced gene silencing of Gbvdr3 compromised the resistance of Hai7124 cotton to a defoliating strain of V. dahliae, V991, but not to a non-defoliating strain, BP2. This resistance pattern was further confirmed by over-expression of Gbvdr3 in transgenic Arabidopsis, which significantly elevated the expression of the ethylene-regulated gene GST2, the ethylene- and jasmonic acid-regulated defense-related genes PR3 and PDF1.2, and the salicylic acid-regulated genes PR1 and PR5, but not the PR2 gene. It also triggered the accumulation of hydrogen peroxide and callose at early time points during infection by the V991 defoliating strain. In contrast, elevated accumulation of hydrogen peroxide or callose in Gbvdr3-expressed Arabidopsis leaves was not apparent under infection by the non-defoliating strain, BP2. These results suggested that Gbvdr3 is involved in the resistance to a unique spectrum of defoliating V. dahliae strains.

MicroRNAs (miRNAs) participate in plant development and defence through post-transcriptional regulation of the target genes. However, few miRNAs were reported to regulate cotton plant disease resistance. Here, we characterized the cotton miR164-NAC100 module in the later induction stage response of the plant to Verticillium dahliae infection. The results of GUS fusing reporter and transcript identity showed that ghr-miR164 can directly cleave the mRNA of GhNAC100 in the post-transcriptional process. The ghr-miR164 positively regulated the cotton plant resistance to V. dahliae according to analyses of its over-expression and knockdown. In link with results, the knockdown of GhNAC100 increased the plant resistance to V. dahliae. Based on LUC reporter, expression analyses and yeast one-hybrid (Y1H) assays, GhNAC100 bound to the CGTA-box of GhPR3 promoter and repressed its expression, negatively regulating plant disease resistance. These results showed that the ghr-miR164 and GhNAC100 module fine-tunes plant defence through the post-transcriptional regulation, which documented that miRNAs play important roles in plant resistance to vascular disease.

The soil-born vascular disease Verticillium wilt, which is caused by fungal pathogen Verticillium dahliae, is a devastating disease of cotton worldwide. In the last decade, a large number of genes have been found to participate in cotton-V. dahliae interactions, but the detailed mechanisms of cotton resistance to V. dahliae remain unclear. Here, we functionally characterized MPK3, a MAPK gene from cotton. MPK3 was induced in the roots of both resistant and susceptible cotton cultivars by V. dahliae inoculation. Transgenic cotton and tobacco with constitutively higher GbMPK3 expression conferred higher V. dahliae susceptibility, while MPK3 knockdown in cotton has limited effect on cotton resistance to V. dahliae. Expression profiling revealed that SA-mediated defense pathway genes (WRKY70, PR1, and PR5) accumulated after V. dahliae inoculation in roots of both wild-type and transgenic cotton, and the expression levels of these genes were higher in GbMPK3-overexpressing plants than in wild-type plants, indicating that GbMPK3 upregulation may reduce plant resistance to V. dahliae through regulating salicylic acid signaling transduction.

Verticillium wilt of cotton, caused by the fungal phytopathogen Verticillium dahliae (V. dahliae), is one of the most devastating diseases of upland cotton. While Verticillium dahliae-resistant phenotypes have been identified in several plant species, the underlying mechanisms of resistance need further clarification. Here, the induction of resistance to V. dahliae through over-expression of the GhARPL18A-6 (Ribosomal protein L18A) in Gossypium hirsutum (G. hirsutum) was demonstrated. GhARPL18A-6 was highly induced in G. hirsutum acc. Zhongzhimian2 (Zz2) after inoculation with Verticillium dahliae. Knock-down of GhARPL18A-6 in Zz2 led to disease-sensitive phenotypes to V. dahliae. Further studies showed that increased lignin deposition, an enhanced reactive oxygen species (ROS) burst, and phenylpropanoid biosynthesis defense response pathways all contributed to a decrease in colonization by V. dahliae. RNA-seq analysis of GhARPL18A-6 over-expression and wild-type cotton revealed differentiate-expression of cell wall biosynthesis, sucrose signaling and metabolism, lignin biosynthesis, and ROS response genes in resistant plants inoculated with V. dahliae. Given the susceptibility of most commercially valuable cotton lines, this research provides insight into the mechanisms of resistance to Verticillium wilt as well as novel germplasm for cotton breeding.

Naphthoflavones (benzoflavones) are synthetic flavonoids commonly used in drug metabolism studies as selective activators or inhibitors of cytochrome P-450 enzymes. Nowadays they are also used as a component of food supplements for body builders. There is no data regarding naphthoflavone microbial metabolism. In the present studies sixty-three fungal strains have been screened for their ability to transform α-naphthoflavone (7,8-benzoflavone) or β-naphthoflavone (5,6-benzoflavone). Five strains belonging to the genera Penicillium, Cladosporium, Aspergillus and Verticillium transformed α-naphthoflavone and β-naphthoflavone to the corresponding 4′-hydroxy derivatives. These selected fungi have been used in a further study on biotransformation of naphthoflavones with a differently substituted B-ring. Only 4′-methoxy derivatives have been transformed to the related 4′-hydroxy products. Selected strains are good biocatalysts to obtain 4′-hydroxy naphthoflavones in the one step reaction.

Mosquitoes have been becoming serious vectors worldwide thus effective and safe control strategies should be established. Entomopathogenic fungi can be alternative controlling agents by substituting chemical insecticides. Herein we assayed 12 soil-borne entomopathogenic fungi against Asian tiger mosquito (Aedes albopictus) larvae in laboratory conditions and tried to establish an effective application method using millet granular formulation (GR). Twelve fungal isolates which belong to 6 genera (Beauveria, Cordyceps, Metarhizium, Paecilomyces, Purpureocillium and Verticillium) were assayed; M. anisopliae JEF-003 showed the fastest mosquitocidal activity, approximately 73% mortality rate at 2 days post-inoculation (dpi.) and > 90% mortality rate at 5 dpi. Conidia of M. anisopliae JEF-003 also showed a dosage dependent activity at 1 × 105, 1 × 106 and 1 × 107 conidia ml− 1. Hyphal growth of M. anisopliae JEF-003 in the Ae. albopictus larvae was observed by infection of an M. anisopliae JEF-003 EGFP-transformant, which was generated by restriction enzyme-mediated integration (REMI) method. GR of M. anisopliae JEF-003 showed high virulence to Ae. albopictus larvae (> 90% mortality) after 5 days of application. These results suggest that M. anisopliae JEF-003 has a potential to control A. albopictus larvae and GR can be practically used for management of the serious vector in water environment.

C2H2 zinc finger (CZF) proteins are a major class of transcription factors that play crucial roles in fungal growth, development, various stress responses, and virulence. Little genome-wide data is available regarding the roles of CZF proteins in Verticillium dahliae, a destructive pathogen that causes vascular wilt disease in more than 200 plant species. We identified a total of 79 typical CZF genes in V. dahliae. Comparative analysis revealed that four plant pathogenic fungi, V. dahliae, Fusarium oxysporum, Magnaporthe oryzae, and Botrytis cinerea, have comparable numbers of predicted CZF genes with similar characteristics. Phylogenetic analysis identified a C2H2-homeobox subfamily in V. dahliae containing seven genes with similar gene structures. V. dahliae and F. oxysporum (Hypocreomycetidae) have more genes of this subfamily than M. oryzae (Sordariomycetidae) and B. cinerea (Leotiomycetes). Furthermore, gene-expression analysis of the smoke tree wilt fungus V. dahliae strain XS11 using digital gene-expression profiling and RT-qPCR revealed that a number of CZF genes were differentially expressed during microsclerotia formation, nutritional starvation, and simulated in planta conditions. Furthermore, the expression profiles revealed that some CZF genes were overrepresented during multiple stages, indicating that they might play diverse roles. Our results provide useful information concerning the functions of CZF genes in microsclerotia formation, nutritional stress responses, and pathogenicity in V. dahliae, and form a basis for future functional studies of these genes.

The soilborne fungus Verticillium dahliae is the major pathogen that causes the verticillium wilt disease of plants, which leads to huge economic loss worldwide. At the early stage of infection, growth of the pathogen is subject to the nutrition stress of limited nitrogen. To investigate the secreted pathogenic proteins that play indispensable roles during invasion at this stage, we compared the profiles of secreted proteins of V. dahliae under nitrogen starvation and normal conditions by using in-gel and in-solution digestion combined with liquid chromatography–nano-electrospray ionization tandem mass spectrometry (LC–nanoESI-MS). In total, we identified 212 proteins from the supernatant of liquid medium, including 109 putative secreted proteins. Comparative analysis indicated that the expression of 76 proteins was induced, whereas that of 9 proteins was suppressed under nitrogen starvation. Notably, 24 proteins are constitutively expressed. Further bioinformatic exploration enabled us to classify the stress-induced proteins into seven functional groups: cell wall degradation (10.5%), reactive oxygen species (ROS) scavenging and stress response (11.8%), lipid effectors (5.3%), protein metabolism (21.1%), carbohydrate metabolism (15.8%), electron–proton transport and energy metabolism (14.5%), and other (21.0%). In addition, most stress-suppressed proteins are involved in the cell-wall remodeling. Taken together, our analyses provide insights into the pathogenesis of V. dahliae and might give hints for the development of novel strategy against the verticillium wilt disease.

In this study, we identified a defense-related major latex protein (MLP) from upland cotton (designated GhMLP28) and investigated its functional mechanism. GhMLP28 transcripts were ubiquitously present in cotton plants, with higher accumulation in the root. Expression of the GhMLP28 gene was induced by Verticillium dahliae inoculation and was responsive to defense signaling molecules, including ethylene, jasmonic acid, and salicylic acid. Knockdown of GhMLP28 expression by virus-induced gene silencing resulted in increased susceptibility of cotton plants to V. dahliae infection, while ectopic overexpression of GhMLP28 in tobacco improved the disease tolerance of the transgenic plants. Further analysis revealed that GhMLP28 interacted with cotton ethylene response factor 6 (GhERF6) and facilitated the binding of GhERF6 to GCC-box element. Transient expression assay demonstrated that GhMLP28 enhanced the transcription factor activity of GhERF6, which led to the augmented expression of some GCC-box genes. GhMLP28 proteins were located in both the nucleus and cytoplasm and their nuclear distribution was dependent on the presence of GhERF6. Collectively, these results demonstrate that GhMLP28 acts as a positive regulator of GhERF6, and synergetic actions of the two proteins may contribute substantially to protection against V. dahliae infection in cotton plants.

The vascular plant pathogen Verticillium dahliae colonizes the xylem fluid where only low nutrient concentrations are provided. Biosynthesis of the vitamin thiamine is connected to oxidative stress. The highly conserved VdThi4 protein is localized in fungal mitochondria and is required under vitamin B1 limiting conditions. Deletion of the corresponding VdTHI4 gene by Agrobacterium-mediated transformation resulted in strains which were impaired in growth on thiamine-free medium and could be rescued by additional vitamin supply or by complementation with the original gene after protoplastation. Furthermore, we show that VdThi4 increases fungal stress tolerance such as UV-damage or oxidative stress. The orthologous sti35 gene of Fusarium oxysporum, another vascular wilt fungus, was shown to be involved in stress response, however to be dispensable for pathogenicity on tomato. In contrast, VdTHI4 is required for fungal-induced tomato disease demonstrated by infection assays with a V. dahliae ΔVdTHI4 deletion strain which is still able to invade plants through the roots but is asymptomatic. Our results suggest remarkable differences between two vascular tomato pathogens where VdThi4 is required for pathogenicity of V. dahliae, whereas F. oxysporum still causes disease when the corresponding Sti35 protein is absent.

A semifield assay was conducted from 2009 to 2011 to distinguish between different preventive methods of reducing Verticillium spp. in spinach seed production. The seed treatments for controlling seed infection levels included Thiram, Signum, Trichoderma harzianum , Gliocladium roseum and Natural II, and these were tested in naturally infested and uninfested soil. Even though seed treatment by Thiram and Signum in all cases reduced the incidence of Verticillium spp. on the harvested seed, the soil type had a large influence on the subsequent disease pressure as a significant effect of soil was seen in 2010 and 2011 and a non-significant effect of seed treatment on Verticillium spp. was observed in all experimental years. As no interaction between soil and seed treatment was observed, the seed treatment had no effect on the disease pressure on the seeds subsequently harvested from the infested soil.

Verticillium longisporum and Verticillium dahliae are soil borne vascular pathogens. Under standard laboratory growth conditions both fungi infect Arabidopsis thaliana ecotype Col-0 plants via primary and lateral (secondary) root tips and colonize the vascular cylinder including proto- and metaxylem. After entry into the hypocotyl both Verticillium species spread into petioles and leaves, but with different efficiency and distinct disease symptom patterns. V. longisporum typically induces early senescence whereas V. dahliae infections cause wilting. In both cases, these symptoms coincide with the switch from a biotrophic to a necrotrophic life style. Fungal proliferation analyses monitored by quantitative real-time PCR and anatomical studies show that V. dahliae is able to colonize aerial plant parts more efficiently compared to V. longisporum. Moreover, V. longisporum-infection triggers hyperplastic xylem formation and cellular transdifferentiation in Arabidopsis, whereas V. dahliae-infection induces enhanced xylem lignification. We have recently shown that V. longisporum induced de novo xylem formation results in enhanced drought stress tolerance, suggesting a conditionally mutualistic interaction. Here we show that on the contrary V. dahliae infections do not provide protection of the plant against drought. Together, our results demonstrate that V. dahliae and V. longisporum induce distinct and species-specific developmental and structural alterations in Arabidopsis, which differently affect plant fitness under concomitant abiotic drought stress conditions.

Wilting and damping off disease caused by Verticillium sp. and Phytophthora sp. are some of the common diseases that occur in Solanum melongena L. ( S. melongena L.) plants. Piriformospora indica has the ability to colonize broad host spectrum and offers resistance to various plant pathogens. Hence the present study was undertaken to investigate the effectiveness of Piriformospora indica as a biocontrol agent against Verticillium sp . and Phytophthora sp. infected Solanum melongena L. grown under greenhouse conditions. Solanum melongena L. plants colonized by Piriformospora indica significantly showed lesser disease incidence/ severity caused by Verticillium and Phytophthora . Accumulation of proline and defense enzymes such as peroxidase and polyphenol oxidase was found to be high with Piriformospora indica colonized Solanum melongena L. plants. Unlike arbuscular mycorrhiza fungi, Piriformospora indica can be easily cultivated on synthetic media and thus can be exploited to reduce disease resistance with increase crop productivity.

The vascular wilt fungus Verticillium dahliae infects the roots of cotton plants and can seriously diminish the yield and quality of this and other dicotyledons. However, the key genes involved in V. dahliae infection and pathogenesis in cotton remain unclear. Msb encodes a transmembrane mucin that is highly conserved in the MAPK signal pathway. Msb has been implicated previously in pathogenicity in various aerial plant fungi. In this study, V. dahliae Msb (VdMsb) was found to be required for fungal virulence and microsclerotia production. Strains lacking VdMsb exhibited reduced conidiation and microsclerotia formation. Compared with wild-type and gene-complemented strains, the invasive growth and adhesive capacity of VdMsb deletion mutants were significantly decreased. These results suggest that VdMsb plays a role in development and virulence in V. dahliae.

In our previous study, PevD1 was characterized as a novel protein elicitor produced by Verticillium dahliae inducing hypersensitive response (HR) and systemic acquired resistance (SAR) in tobacco plants; however, the detailed mechanisms of PevD1's elicitor activity remain unclear. In this study, five mutant fragments of PevD1 were generated by polymerase chain reaction-based mutagenesis and the truncated proteins expressed in Escherichia coli were used to test their elicitor activities. Biological activity analysis showed that the N-terminal and C-terminal of PevD1 had distinct influence on HR and SAR elicitation. Fragment PevD1ΔN98, which spans the C-terminal 57 amino acids of PevD1, was critical for the induction of HR in tobacco plants. In contrast, fragment PevD1ΔC57, the N-terminal of 98 amino acids of PevD1, retained the ability to induce SAR against tobacco mosaic virus (TMV) but not induction of HR, suggesting that the induction of HR is not essential for SAR mediated by PevD1. Our results indicated that fragment PevD1ΔC57 could be a candidate peptide for plant protection against pathogens without causing negative effects.

Hop (Humulus lupulus L.) is an important industrial plant providing ingredients for brewing and pharmaceutical industry worldwide. Its intensive production is challenged by numerous diseases. One of the most lethal and difficult to control is verticillium wilt, a vascular disease caused by the fungal pathogen Verticillium nonalfalfae. The disease can be successfully controlled by the host resistance. Despite various studies that already researched resistance mechanisms of hops, only limited number of resistance genes and markers that could be utilized for efficient resistance breeding has been identified. In this study we aimed to follow fungus colonization pattern and the differential expression of selected genes during pre-symptomatic period of susceptible (Celeia) and resistant (Wye Target) hop cultivars. Results of gene expressions and fungal colonisation of compatible and incompatible interactions with V. nonalfalfae suggest that the hop plant is challenged already at the very early fungal colonisation stages. In total, nine out of 17 gene targets investigated in our study resulted in differential expression between inoculated and control plants of susceptible and resistant cultivars. The difference was the most evident in stems at an early stage of colonisation (6 dpi), showing relatively stronger changes in targeted gene expression to infection in the resistant cultivar than in the susceptible one. Analysed gene targets are involved in the overall defence response processes of nucleic acid binding, signalling, protein ubiquitination, cell oxidative burst, hydroxylation, peroxidation, alternative splicing, and metabolite biosynthesis. The up-regulation of some genes (e.g. glycine-rich RNA-binding family protein, protein phosphatase, cysteine-rich receptor-like protein kinase, zinc finger CCCH domain-containing protein 40, cinnamic acid 4-hydroxylase, class III peroxidase, putative MAPK2, peroxiredoxin-2F) upon infection in incompatible interactions might reflect defence activation, restriction of disease spreading throughout the plant and successful response of resistant genotype.

Verticillium wilt causes massive annual losses of cotton yield, but the mechanism of cotton resistance to Verticillium dahliae is complex and poorly understood. In this study, a comparative proteomic analysis was performed in resistant cotton (Gossypium barbadense cv7124) on infection with V. dahliae. A total of 188 differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) analysis and could be classified into 17 biological processes based on Gene Ontology annotation. Most of these proteins were implicated in stimulus response, cellular processes and metabolic processes. Based on the proteomic analysis, several genes involved in secondary metabolism, reactive oxygen burst and phytohormone signaling pathways were identified for further physiological and molecular analysis. The roles of the corresponding genes were further characterized by employing virus-induced gene silencing (VIGS). Based on the results, we suggest that the production of gossypol is sufficient to affect the cotton resistance to V. dahliae. Silencing of GbCAD1, a key enzyme involving in gossypol biosynthesis, compromised cotton resistance to V. dahliae. Reactive oxygen species and salicylic acid signaling may be also implicated as regulators in cotton responsive to V. dahliae according to the analysis of GbSSI2, an important regulator in the crosstalk between salicylic acid and jasmonic acid signal pathways. Moreover, brassinosteroids and jasmonic acid signaling may play essential roles in the cotton disease resistance to V. dahliae. The brassinosteroids signaling was activated in cotton on inoculation with V. dahliae and the disease resistance of cotton was enhanced after exogenous application of brassinolide. Meanwhile, jasmonic acid signaling was also activated in cotton after inoculation with V. dahliae and brassinolide application. These data provide highlights in the molecular basis of cotton resistance to V. dahliae.

During the course of our investigation on the selective isolation and in vitro pathogenicity tests of the nematode egg parasite and endoparasitic Catenaria anguillulae and the nematode-trapping fungus Dactylaria brochopaga against Anguina tritici , and Meloidogyne graminicola, the two biocontrol agents were also examined for their capabilities to colonize wheat seed gall and also to reduce the M. graminicola , and A. tritici in wheat ( Triticum aestivum L.). Using seed galls as bait was found effective for isolating the soilborne natural colonizers of seed galls of wheat caused by A. tritici . Seed galls were found severely infested with cellulose decomposers as well as nematode trapping fungi i.e., Fusarium spp ., Verticillium spp., Aspergillus spp., Penicillium spp. Arthrobotrys oligospora , Arthrobotrys superba , Arthrobotrys dactyloides , Arthrobotrys musiformis , D. brochopaga , Monacrosporium eudermatum, Stylopaga hadra, Paecilomyces spp., and C. anguillulae . Co-inoculation of D. brochopaga DBS-105, and C. anguillulae CAS101 significantly reduced the root knot and seed gall in wheat and increased the plant growth parameters including length, and dry weight of root and shoot as well as yield attributing characters like spike length; number of seed per spike, test weight etc. under greenhouse conditions as compared to pathogen challenged plants without any bioagents/chemical nematicide. These bioagents have the potential to enhance production of quality seeds of wheat being free from hazardous pesticides and thus, can further be developed into marketable formulations in order to promote eco-friendly and sustainable means of crop production.