Physiological response of two olive cultivars to secondary metabolites of Verticillium dahliae Kleb.

The effects of two purified fractions (formerly D-SXM and ND-SXM) produced in vitro by defoliating (Vd312D) and non-defoliating (Vd315ND) strains of Verticillium dahliae were studied on twigs of Olea europaea cvs Frantoio and Leccino. Symptoms, such as leaf curling, yellowing, vein clearing and defoliation, which are observed on the two cultivars naturally affected by Verticillium wilt, were produced by these fractions. Physiological changes were induced during the first seven days after the absorption of solutions containing ND-SXM or D-SXM. Both fractions increased the transpiration flow from abaxial leaf surfaces. Cell membrane and antioxidant activity were the most important action sites of ND-SXM and D-SXM. ND-SXM influenced malondialdehyde concentration in 'Leccino' leaves, while D-SXM increased the percentage of electrolyte leakage in 'Frantoio'. Both fractions reduced the total non-enzymatic antioxidant activity on the leaves of the treated twigs. The total phenol content increased in both cultivars, without differences to the control. Variations on electrolyte leakage and total antioxidant activity were effective in discriminating the two tested olive cultivars for V. dahliae tolerance or susceptibility. If V. dahliae strains Vd315ND and Vd312D produce ND-SXM and D-SXM in the infected plants, these metabolites may move via the xylem sap, accumulate in the leaves and induce changes that will lead symptoms on the leaf by compromising the cell membranes physiology.

In silico prediction and characterisation of secondary metabolite clusters in the plant pathogenic fungus Verticillium dahliae.

Fungi are renowned producers of natural compounds, also known as secondary metabolites (SMs) that display a wide array of biological activities. Typically, the genes that are involved in the biosynthesis of SMs are located in close proximity to each other in so-called secondary metabolite clusters. Many plant-pathogenic fungi secrete SMs during infection in order to promote disease establishment, for instance as cytocoxic compounds. Verticillium dahliae is a notorious plant pathogen that can infect over 200 host plants worldwide. However, the SM repertoire of this vascular pathogen remains mostly uncharted. To unravel the potential of V. dahliae to produce SMs, we performed in silico predictions and in-depth analyses of its secondary metabolite clusters. Using distinctive traits of gene clusters and the conserved signatures of core genes 25 potential SM gene clusters were identified. Subsequently, phylogenetic and comparative genomics analyses were performed, revealing that two putative siderophores, ferricrocin and TAFC, DHN-melanin and fujikurin may belong to the SM repertoire of V. dahliae.

Seven new guanacastane-type diterpenoids from the fungus Verticillium dahliae.

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).

Two new aromadendrane sesquiterpenes from Verticillium psalliotae.

The chemical constituents of the fungus Verticillium psalliotae were studied. Two new aromadendrane sesquiterpenes inonotin M (1) and inonotin N (2) were isolated from the EtOAc extract of the fungal culture broth. The structures of compounds were elucidated mainly by HRESIMS experiments, and 1D, 2D-NMR spectroscopy analysis.

Natural meroterpenoids isolated from the plant pathogenic fungus Verticillium albo-atrum with noteworthy modification action against voltage-gated sodium channels of central neurons of Helicoverpa armigera.

A new meroterpenoid, named acetoxydehydroaustin A (1) and the known meroterpenoid austin (2) were isolated from the plant pathogenic fungus Verticillium albo-atrum. Their structures were established based on general spectroscopic techniques and the relative configuration of compound 1 was determined by single-crystal X-ray diffraction analysis. We first investigated and identified their significant electrophysiological effects on the gating kinetics of voltage-gated sodium channels in central neurons acutely dissociated from Helicoverpa armigera using whole-cell patch clamp technique. Similar to the effects of pyrethroids on sodium late currents, both compounds produced concentration-dependent modification of sodium channels, prolonging the kinetics of channel inactivation to generate large persistent late currents during depolarization. However, different from the effects of tefluthrin and deltamethrin on sodium channels, two meroterpenoids did not induce tail currents during deactivation. Compounds 1 and 2 also caused depolarizing shifts in the voltage dependence of channel activation. The V0.5 shifted about 5.02mV and 6.32mV in the depolarizing direction by 50µM 1 and 50µM 2. The V0.5 of voltage-dependent inactivation shifted about 11.42mV and 11.62mV respectively in the hyperpolarizing direction by 50µM 1 and 100µM 2. In addition, they prolonged the time course of recovery from fast-inactivation for sodium channels. The effects of two compounds on the voltage-dependent gating substantially increased the size of sodium window currents. The overlapped area of window currents increased about 89.69% and 44.51% respectively by 10µM compound 1 and 10µM compound 2. These findings show that both compounds have effects on sodium channel activation, inactivation and window currents. The voltage-gated sodium channels in central neurons of H. armigera are the target sites of two meroterpenoid natural products.

Differentiation of mixed soil-borne fungi in the genus level using infrared spectroscopy and multivariate analysis.

Early detection of soil-borne pathogens, which have a negative effect on almost all agricultural crops, is crucial for effective targeting with the most suitable antifungal agents and thus preventing and/or reducing their severity. They are responsible for severe diseases in various plants, leading in many cases to substantial economic losses. In this study, infrared (IR) spectroscopic method, which is known as sensitive, accurate and rapid, was used to discriminate between different fungi in a mixture was evaluated. Mixed and pure samples of Colletotrichum, Verticillium, Rhizoctonia, and Fusarium genera were measured using IR microscopy. Our spectral results showed that the best differentiation between pure and mixed fungi was obtained in the 675-1800 cm-1 wavenumber region. Principal components analysis (PCA), followed by linear discriminant analysis (LDA) as a linear classifier, was performed on the spectra of the measured classes. Our results showed that it is possible to differentiate between mixed-calculated categories of phytopathogens with high success rates (~100%) when the mixing percentage range is narrow (40-60) in the genus level; when the mixing percentage range is wide (10-90), the success rate exceeded 85%. Also, in the measured mixed categories of phytopathogens it is possible to differentiate between the different categories with ~100% success rate.

Physiological and molecular characterization of compost bacteria antagonistic to soil-borne plant pathogens.

Disease suppressive composts have the potential to mitigate the risks associated with chemical pesticides. One of the main characteristics responsible for the suppressive nature of composts is their microbiological populations. To gain insight into the determinants responsible for their suppressive effects, we assayed composts to (i) isolate and identify beneficial antagonistic bacteria, (ii) quantify their antifungal and anti-oomycetal activities, (iii) extract inhibitory compounds produced by the bacteria, and (iv) identify antimicrobial lipopeptides produced by these bacteria. The antagonistic bacteria belonged to the genera Arthrobacter, Pseudomonas, Bacillus, Brevibacillus, Paenibacillus, and Rummeliibacillus and had the ability to antagonise the growth of Fusarium sambucinum, Verticillium dahliae, and (or) Pythium sulcatum. These bacteria produced antimicrobial compounds that affected the mycelial growth and (or) conidial germination of the pathogens. Mass spectrometry analyses showed the presence of various antimicrobial lipopeptides in Bacillus and Bacillus-related spp. extracts, demonstrating that they are responsible, at least in part, for the antagonistic activity of the bacteria. Results from this work provide greater insight into some of the biological, biochemical, and physiological determinants of suppressiveness in composts involved in the control of plant pathogens.

Enhanced production of microsclerotia in recalcitrant Verticillium dahliae isolates and its use for inoculation of olive plants.

AIMS: The optimization of a simple protocol for the mass production of viable microsclerotia (MS) of Verticillium spp., even for recalcitrant isolates, to the inoculation of olive cuttings. METHOD AND RESULTS: Four Verticillium spp. isolates were characterized by growth rate and morphology. Then, the production ability and the viability of MS over time were assessed in seven solid culture media and five aqueous media. The best culture medium, according to the quantity and the quality (size) of the MS produced, was the alkaline-modified sodium polipectate (AMSP) aqueous medium. The MS viability was higher in peat moss substrates. Finally, the MS obtained in this work were infective causing 100% incidence of Verticillium wilt (VW) disease in inoculated olive plants. CONCLUSION: This study demonstrates that the modified sodium polipectate medium amended with 0·1% agar is the most suitable for the production of MS of Verticillium dahliae isolates that have lost the ability to produce MS in standard culture media. SIGNIFICANCE AND IMPACT OF THE STUDY: Mass production of MS for artificial infestation of soil is critical to the study of epidemiological and control aspects of the VW. To overcome the failure in the production of MS in recalcitrant isolates, a culture media was optimized and a successful plant inoculation experiment was carried out with artificial MS.

Guanacastane-type diterpenoids from the insect-associated fungus Verticillium dahliae.

Four new guanacastane-type diterpenoids, namely dahlianes A (1), B (2), C (3), and D (4), were isolated from cultures of Verticillium dahliae. Their structures were elucidated on the basis of extensive spectroscopic data analysis. Their absolute configurations were determined by a combination of Mo2(OAc)4-induced electronic circular dichroism experiment and Mosher ester method. In cytotoxicity evaluation against human tumor cell lines, compounds 2 and 3 exhibited significant cytotoxicities against MCF-7 cell lines with IC50 values of 3.35 and 4.72 µM, respectively.

Bioinformatics Analysis Reveals Abundant Short Alpha-Helices as a Common Structural Feature of Oomycete RxLR Effector Proteins.

RxLR effectors represent one of the largest and most diverse effector families in oomycete plant pathogens. These effectors have attracted enormous attention since they can be delivered inside the plant cell and manipulates host immunity. With the exceptions of a signal peptide and the following RxLR-dEER and C-terminal W/Y/L motifs identified from the sequences themselves, nearly no functional domains have been found. Recently, protein structures of several RxLRs were revealed to comprise alpha-helical bundle repeats. However, approximately half of all RxLRs lack obvious W/Y/L motifs, which are associated with helical structures. In this study, secondary structure prediction of the putative RxLR proteins was performed. We found that the C-terminus of the majority of these RxLR proteins, irrespective of the presence of W/Y/L motifs, contains abundant short alpha-helices. Since a large-scale experimental determination of protein structures has been difficult to date, results of the current study extend our understanding on the oomycete RxLR effectors in protein secondary structures from individual members to the entire family. Moreover, we identified less alpha-helix-rich proteins from secretomes of several oomycete and fungal organisms in which RxLRs have not been identified, providing additional evidence that these organisms are unlikely to harbor RxLR-like proteins. Therefore, these results provide additional information that will aid further studies on the evolution and functional mechanisms of RxLR effectors.

The secretome of vascular wilt pathogen Verticillium albo-atrum in simulated xylem fluid.

Verticillium albo-atrum is a vascular wilt pathogen capable of infecting many important dicotyledonous plant species. Fungal isolates from hop differ in aggressiveness, causing either mild or lethal symptoms in infected plants. As in other plant pathogenic fungi, extracellular proteins, such as cell wall-degrading enzymes and effectors, are thought to be crucial in the pathogenesis process. In this study, mild and lethal isolates from three countries were grown in simulated xylem medium and secretome analysis by 2D-DIGE showed low qualitative and high quantitative variability among the isolates. Functional classification of 194 identified proteins representing 100 unique protein accessions revealed an arsenal of cell wall-degrading enzymes and potential effectors. The set of proteins that were more abundant in at least two lethal isolates included enzymes acetylcholinesterases, lipases, polygalacturonases, pectate lyase, rhamnogalacturonan acetylesterases, acetylxylan esterase, endoglucanase, xylanases, mannosidases, and a protein similar to alginate lyase and also potential effectors necrosis- and ethylene-inducing protein, small basic 14 kDa hypothetical protein and 79 kDa hypothetical proteins. Other proteins associated with virulence showed different expression profiles between mild and lethal isolates. The results suggest that the increased virulence of lethal isolates has little background shared by all three lethal isolates and that upregulation of isolate specific sets of proteins may be most important.

Rapid evaluation technique to differentiate mushroom disease-related moulds by detecting microbial volatile organic compounds using HS-SPME-GC-MS.

Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used to analyse microbial volatile organic compounds (MVOCs) of mushroom disease-related microorganisms. Mycogone perniciosa, Lecanicillum fungicola var. fungicola, and Trichoderma aggressivum f. europaeum species, which are typically harmful in mushroom cultivation, were examined, and Agaricus bisporus (bisporic button mushroom) was also examined as a control. For internal standard, a mixture of alkanes was used; these were introduced as the memory effect of primed septa in the vial seal. Several different marker compounds were found in each sample, which enabled us to distinguish the different moulds and the mushroom mycelium from each other. Monitoring of marker compounds enabled us to investigate the behaviour of moulds. The records of the temporal pattern changes were used to produce partial least squares regression (PLS-R) models that enabled determination of the exact time of contamination (the infection time of the media). Using these evaluation techniques, the presence of mushroom disease-related fungi can be easily detected and monitored via their emitted MVOCs.

A new diphenyl ether from the endophytic fungus Verticillium sp. isolated from Rehmannia glutinosa.

AIM: To investigate the chemical constituents of the endophytic fungus Verticillium sp. isolated from Rehmannia glutinosa. METHODS: The compounds were isolated and purified by repeated column chromatography, and their structures were determined on the basis of physicochemical properties and spectral analysis. Their cytotoxic and antifungal activities were evaluated. RESULTS: Ten compounds were obtained and their structures were identified as 2, 4-dihydroxy-2', 6-diacetoxy-3'-methoxy-5'-methyl-diphenyl ether (1), paecilospirone (2), α-acetylorcinol (3), 2-methoxy-1,8-dimethyl-xanthen-9-one (4), 4-hydroxy-α-lapachone (5), enalin A (6), 2,3,4-trimethyl-5,7-dihydroxy-2,3-dihydrobenzofuran (7), 4-hydroxyethyl-phenol (8), 2,4-dihydroxy-3,5,6-trimethyl- methylbenzoate (9), and 3-isopropenyl-(Z)-monomethyl maleate (10). CONCLUSIONS: Compound 1 is a new diphenyl ether, and showed cytotoxic activity against HL-60 cells (IC50 2.24 µg · mL(-1)), and antifungal activities against Candida albicans (MIC 8 µg · mL(-1)) and Aspergillus fumigatus (MIC 16 µg · mL(-1)).

Glucogalactan: a polysaccharide isolated from the cell-wall of Verticillium lecanii.

A water-soluble polysaccharide was extracted with alkali from the cell wall of Verticillium lecanii (also called Lecanicillium lecanii). After freezing and thawing, the water-soluble fraction was purified by gel filtration chromatography on Sepharose CL-6B and eluted as one peak by HPSEC/RID. Monosaccharide analysis showed galactose and glucose (1.1:1), with traces of mannose (<1%). The structural characteristics were determined by spectroscopic analysis, FT-IR and 1D and 2D (1)H and (13)C NMR, and methylation results. On the basis of the data obtained, the following structure of the polysaccharide (E3SIV fraction) was established: [Formula: see text] where n≈22 and m≈22.

Oxidation of aromatic N-heterocyclic compounds to N-oxides by Verticillium sp. GF39 cells.

Verticillium sp. GF39, catalyzing the oxidation of 1-methylisoquinoline to 1-methylisoquinoline N-oxide, was found to be the highest N-oxide producer. Under the optimized reaction conditions, the whole cells of Verticillium sp. GF39 formed 5 mM 1-methylisoquinoline N-oxide from 1-methylisoquinoline with a molar conversion yield of 100% after a 10-h incubation at 20°C. The whole cells also acted on pyridine, 2-methylpyridine, quinoline and isoquinoline and formed the corresponding N-oxides.

Purification, crystallization and preliminary X-ray diffraction analysis of the effector protein PevD1 from Verticillium dahliae.

The effector protein PevD1 from the pathogenic fungus Verticillium dahliae was purified and crystallized using the hanging-drop vapour-diffusion method. Native crystals appeared in a solution consisting of 4.0 M sodium formate. A native data set was collected at 1.9 Šresolution at 100 K using an in-house X-ray source. Because of the absence of useful methinione in the protein sequence, derivative crystals that contained iodine were obtained by soaking in 1.25 M potassium iodide, and a data set that contained anomalous signal was collected using the same X-ray facility at a wavelength of 1.54 Å. The single-wavelength anomalous dispersion method was used to successfully solve the structure based on the anomalous signal generated from iodine.

Identification of fungal phytopathogens using Fourier transform infrared-attenuated total reflection spectroscopy and advanced statistical methods.

The early diagnosis of phytopathogens is of a great importance; it could save large economical losses due to crops damaged by fungal diseases, and prevent unnecessary soil fumigation or the use of fungicides and bactericides and thus prevent considerable environmental pollution. In this study, 18 isolates of three different fungi genera were investigated; six isolates of Colletotrichum coccodes, six isolates of Verticillium dahliae and six isolates of Fusarium oxysporum. Our main goal was to differentiate these fungi samples on the level of isolates, based on their infrared absorption spectra obtained using the Fourier transform infrared-attenuated total reflection (FTIR-ATR) sampling technique. Advanced statistical and mathematical methods: principal component analysis (PCA), linear discriminant analysis (LDA), and k-means were applied to the spectra after manipulation. Our results showed significant spectral differences between the various fungi genera examined. The use of k-means enabled classification between the genera with a 94.5% accuracy, whereas the use of PCA [3 principal components (PCs)] and LDA has achieved a 99.7% success rate. However, on the level of isolates, the best differentiation results were obtained using PCA (9 PCs) and LDA for the lower wavenumber region (800-1775 cm(-1)), with identification success rates of 87%, 85.5%, and 94.5% for Colletotrichum, Fusarium, and Verticillium strains, respectively.

The purification and characterization of a novel hypersensitive-like response-inducing elicitor from Verticillium dahliae that induces resistance responses in tobacco.

PevD1, a novel protein elicitor from the pathogenic cotton verticillium wilt fungus, Verticillium dahliae, induced a hypersensitive response in tobacco plants. In this paper, the elicitor was purified and analyzed using de novo sequencing. The protein-encoding pevD1 gene consists of a 468-bp open reading frame that produces a polypeptide of 155 amino acids, with a theoretical molecular weight of 16.23 kDa. The sequence of elicitor protein PevD1 was matched to the genomic sequence (GenBank accession no. ABJE 01000445.1) of a putative protein from V. dahliae strain vdls.17, but a function had not yet been reported. The pevD1 gene was expressed in Escherichia coli, and the recombinant protein was characterized for its ability to confer systemic acquired resistance to tobacco mosaic virus (TMV). Recombinant PevD1-treated plants exhibited enhanced systemic resistance compared to control, including a significant reduction in the number and size of TMV lesions on tobacco leaves. The elicitor protein-induced hydrogen peroxide production, extracellular-medium alkalization, callose deposition, phenolics metabolism, and lignin synthesis in tobacco. Our results demonstrate that elicitor-PevD1 triggers defense responses in intact tobacco plants.

Studies on calcium release and H2O2 level produced by the elicitor induced plant cell by fluorescence probing.

Using fluorescence probing technology, we studied the mechanism and interrelations of calcium release and H(2)O(2) production in situ in living tissues of tobacco and cotton plants which were induced by pathogen elicitor, salicylic acid (SA) and pectinase respectively. Results showed that (1) pathogen elicitors could induced H(2)O(2) response in epidermis cells regardless of environmental calcium, but in mesophyll protoplast, H(2)O(2) response could only be induced at calcium condition. Similarly, SA and pectinase induced H(2)O(2) response could only be observed at calcium condition; (2) pathogen elicitors could induce calcium response in both epidermis cells and protoplasts regardless of environmental calcium, while calcium response couldn't be induced at non-calcium condition by SA and pectinase; (3) H(2)O(2) response and calcium response in protoplast were faster than that in the whole cell. These results indicated that pathogen elicitors can induce the release of cell wall calcium and the cell wall calcium release is independent to pectinase. And it is concluded that free calcium influx is necessary for the oxidative burst and cell wall calcium has an irreplaceable role in defense signal transduction.

Hydrogen peroxide modulates the dynamic microtubule cytoskeleton during the defence responses to Verticillium dahliae toxins in Arabidopsis.

The molecular mechanisms of signal transduction of plants in response to infection by Verticillium dahliae (VD) are not well understood. We previously showed that NO may act as an upstream signalling molecule to trigger the depolymerization of cortical microtubules in Arabidopsis. In the present study, we used the wild-type, and atrbohD and atrbohF mutants of Arabidopsis to explore the mechanisms of action of H(2)O(2) signals and the dynamic microtubule cytoskeleton in defence responses. We demonstrated that H(2)O(2) may also act as an upstream signalling molecule to regulate cortical microtubule depolymerization. The depolymerization of the cortical microtubules played a functional role in the signalling pathway to mediate the expression of defence genes. The results indicate that H(2)O(2) modulates the dynamic microtubule cytoskeleton to trigger the expression of defence genes against V. dahliae toxins (VD-toxins) in Arabidopsis.