Dying-arm disease in grapevines: diagnosis of infection with Eutypa lata by metabolite analysis

Dying-arm disease in grapevines, produced by infection with the ascomycete Eutypa lata, is responsible for major production losses in vineyards. Dieback of the shoots and cordon is believed to be due to acetylenic phenol metabolites produced by the fungus. To identify specific metabolites that could potentially be used for diagnosis of infection, eight E. lata isolates were grown in vitro on hot water extracts from grape varieties with various degrees of tolerance to the foliar symptoms of E. lata dieback. HPLC analysis showed that eutypinol was consistently produced in large amounts, together with smaller amounts of methyleutypinol and eulatachromene; eutypine, the putative toxin, was produced solely on Sauvignon Blanc extract and then in only barely detectable amounts. When E. lata isolates from Cabernet Sauvignon and Merlot were grown on identical media, the amounts of metabolites produced differed significantly between isolates but the pattern of metabolites was quite similar, with eutypinol again predominating. The consistent production of eutypinol indicated that this was the most suitable metabolite for which to analyze in order to diagnose the presence of E. lata. Extraction and analysis of grapevine tissues exhibiting symptoms of dieback failed to show the presence of any metabolites. However, when infected cordon sections were placed in water and cultured for 5 days, eutypinol was readily detected in the aqueous solution; metabolites were not produced from uninfected tissue. This provides a method for detection of infected tissue and indicates that the toxic metabolites react at the point of production, disrupting the vascular structure and inhibiting transport of nutrients, rather than being translocated to tissues that exhibit symptoms.     

Ethephon elicits protection against Erysiphe necator in grapevine

The grapevine (Vitis vinifera) is susceptible to many pathogens such as Botrytis cinerea, Plasmopara viticola, Erysiphe necator, and Eutypa lata. Phytochemicals are used extensively in vineyards to reduce pathogen infections, but the appearance of pesticide-resistant pathogen strains and the need for environmental protection require the use of alternative strategies. The phytohormone ethylene is assumed to play a role in the development of disease resistance. In the present study, we have treated grapevine foliar cuttings (Cabernet Sauvignon) with ethylene-releasing ethephon. This resulted in an increase in the number of pathogenesis-related protein (CHIT4c, PIN, PGIP, and GLU) gene copies and in an enhancement of phytoalexin biosynthesis by inducing the PAL and STS genes that correlated with the accumulation of stilbenes (antimicrobial compounds). Moreover, ethephon treatment triggered the protection of grapevine detached leaves and grapevine foliar cuttings against Erysiphe necator, the causal agent of powdery mildew (64% and 70%, respectively). These studies emphasize the major role of ethylene in grapevine defense.     

Assessment of quality characteristics of new Vitis vinifera L. cultivars for temperate climate vineyards

Since quality requirements for table grapes are mainly related to their physico-chemical and mechanical features, the aim of this work was to provide useful data for grape producers and consumers concerning the quality characteristics of table grapes of five new Vitis vinifera L. cultivars created in Romania and tending to spread within the temperate climate vineyards. During grape maturation, sugar content increased in a lesser extent than the reducing of total acidity, a balanced taste of grapes was given by the decrease in total acidity rather than sugar accumulation. Grapes of analysed cultivars showed lower concentrations of ascorbic acid and a higher content of organic nitrogenous compound. Berries with higher weight and volume (Transilvania cv.) incurred a higher mechanical deformation, while long berries (Gelu cv.) showed higher resistance to detachment from the pedicel, indicating higher resistance of grapes to packing, transport and storage. Antioxidant activity of mature grapes was high, but involvement of nonflavonoids in increasing the antioxidant capacity was statistically non-significant. Seeded table grapes of new Vitis vinifera L. cultivars showed superior physico-chemical and mechanical characteristics, indicating their high quality in the conditions of temperate continental climate, and promoting the studied cultivars as important grapevine germplasm resources.     

Induction of resistance to gray mold with benzothiadiazole modifies amino acid profile and increases proanthocyanidins in grape: primary versus secondary metabolism

Field treatments of grapevine (cv. Merlot) with the plant activator benzothiadiazole (BTH, 0.3 mM) induced resistance against gray mold caused by Botrytis cinerea. Both incidence and severity of the disease were reduced. The resistance was associated with an increase of total polyphenols in berry skins, in particular, the proanthocyanidin fraction, that increased up to 36%. The amino acid profile of leaves was also modified by treatments, particularly lysine, that augmented 4-fold. Other amino acids involved in resistance mechanisms to either biotic or abiotic stress increased as well. These results indicate that BTH treatments can be used to control gray mold, thereby limiting an excessive use of fungicides, and could be exploited to increase the content of micronutrients of high nutritional value, arising from both primary and secondary metabolisms.     

E26菌剂对葡萄悬浮细胞防卫反应基因pal和sts表达的影响

摘要：以苯丙氨酸解氨酶（phenylalanine ammonia-lyase, PAL）和1,2-二苯乙烯合成酶（stilbene synthase, STS）作为抗性反应指标，对秋黑（Vitis romanetii cv.Qiuhei）和赤霞珠（V. vinifera cv. Cabernet Sauvignon）葡萄悬浮细胞系在根癌病生防制剂—E26(Agrobacterium vitis )菌剂的不同组分（E26菌体细胞、发酵液和代谢液）处理后的PAL酶活性及防卫反应基因pal和sts的转录进行了研究。在E26菌体细胞处理后12~48 h、E26发酵液处理后的4~48 h和E26代谢液处理后的12~48 h，秋黑和赤霞珠的PAL酶活性均明显高于对照。Dot blotting显示，在E26菌体细胞处理后的12~48 h，秋黑和赤霞珠的pal和sts的转录表达量（mRNA）均明显高于对照; E26发酵液处理后，秋黑在4~48 h的pal转录表达量明显高于对照，赤霞珠在4~12 h的pal转录表达量明显高于对照；秋黑和赤霞珠在4~48 h的sts转录表达量均明显高于对照; E26代谢液处理后，秋黑在4~48 h的pal转录表达量明显高于对照，赤霞珠在4~24 h的pal转录表达量明显高于对照；秋黑和赤霞珠在6~48 h的sts转录表达量均明显高于对照。     

Trichoderma brevicompactum complex: Rich source of novel and recurrent plant-protective polypeptide antibiotics (peptaibiotics)

Three strains of Trichoderma brevicompactum and another four that are closely related to that species (Trichoderma cf. brevicompactum) were analyzed for the formation of polypeptide antibiotics (peptaibiotics) by LC/ESI-MS(n). These isolates were selected because of an antagonistic potential against Eutypa dieback and Esca disease of grapevine and have not yet been investigated for the production of peptide antibiotics. Fully grown cultures on potato dextrose agar were extracted with CH2Cl2/MeOH, and this extract was subjected to SPE using C18 cartridges. The methanolic eluates were analyzed by LC/ESI-MS(n). All strains were found to produce membrane-active alamethicins F30. In addition to that, novel peptaibiotics were detected, namely, 14 12-residue trichocryptins B, 12 11-residue trichocryptins A, 19 11-residue trichobrevins A and B, 6 10-residue trichoferins, and 17 8-residue trichocompactins. These compounds may partially be responsible for the plant-protective action of the producers. Chemotaxonomic considerations also indicated the necessity to introduce another new species that is closely related to T. brevicompactum.     

Conversion of the mycotoxin patulin to the less toxic desoxypatulinic acid by the biocontrol yeast Rhodosporidium kratochvilovae strain LS11

The infection of stored apples by the fungus Penicillium expansum causes the contamination of fruits and fruit-derived products with the mycotoxin patulin, which is a major issue in food safety. Fungal attack can be prevented by beneficial microorganisms, so-called biocontrol agents. Previous time-course thin layer chromatography analyses showed that the aerobic incubation of patulin with the biocontrol yeast Rhodosporidium kratochvilovae strain LS11 leads to the disappearance of the mycotoxin spot and the parallel emergence of two new spots, one of which disappears over time. In this work, we analyzed the biodegradation of patulin effected by LS11 through HPLC. The more stable of the two compounds was purified and characterized by nuclear magnetic resonance as desoxypatulinic acid, whose formation was also quantitated in patulin degradation experiments. After R. kratochvilovae LS11 had been incubated in the presence of (13)C-labeled patulin, label was traced to desoxypatulinic acid, thus proving that this compound derives from the metabolization of patulin by the yeast. Desoxypatulinic acid was much less toxic than patulin to human lymphocytes and, in contrast to patulin, did not react in vitro with the thiol-bearing tripeptide glutathione. The lower toxicity of desoxypatulinic acid is proposed to be a consequence of the hydrolysis of the lactone ring and the loss of functional groups that react with thiol groups. The formation of desoxypatulinic acid from patulin represents a novel biodegradation pathway that is also a detoxification process.     

Benzothiadiazole enhances resveratrol and anthocyanin biosynthesis in grapevine, meanwhile improving resistance to Botrytis cinerea

Pre-harvest multiple treatments of grapevine (cv. Merlot) with the plant activator benzothiadiazole (BTH, 0.3 mM) enhanced trans-resveratrol content in berries by about 40%. An even more striking effect was observed on anthocyanin synthesis, particularly on malvidine 3-glucoside, malvidine 3-(6-O-acetyl)glucoside and malvidine 3-(6-O-p-coumaroyl)glucoside, whose amounts were more than doubled. These data were obtained with a novel and time-saving HPLC method, set up for the simultaneous detection of stilbenes and anthocyanins, using an RF-10Axl fluorimetric detector instrument, with excitation at 330 nm and emission at 374 nm, and a SPD-Avp UV detector with absorption at 520 nm. Furthermore, BTH treatments induced systemic acquired resistance in grapevine, as assessed by inoculating clusters from treated and untreated plants with Botrytis cinerea. Disease severity, estimated according to the percentage of infected berries per cluster, was significantly reduced in grapes from BTH-treated plants. These results indicate that BTH treatments, besides improving the content of two important classes of nutraceuticals, with their well-known antioxidant, antitumoral, and phytoestrogenic activities, could be exploited in vineyard to protect grape against gray mould infection, thereby limiting an excessive use of fungicides     

Influence of vine vigor on grape (Vitis vinifera L. Cv. Pinot Noir) and wine proanthocyanidins

The relationships between variations in grapevine (Vitis vinifera L. cv. Pinot noir) growth and resulting fruit and wine phenolic composition were investigated. The study was conducted in a commercial vineyard consisting of the same clone, rootstock, age, and vineyard management practices. The experimental design involved monitoring soil, vine growth, yield components, and fruit composition (soluble solids, flavan-3-ol monomers, proanthocyanidins, and pigmented polymers) on a georeferenced grid pattern to assess patterns in growth and development. Vine vigor parameters (trunk cross-sectional area, average shoot length, and leaf chlorophyll) were used to delineate zones within both blocks to produce research wines to investigate the vine-fruit-wine continuum. There was no significant influence of vine vigor on the amount of proanthocyanidin per seed and only minimal differences in seed proanthocyanidin composition. However, significant increases were found in skin proanthocyanidin (mg/berry), proportion of (-)-epigallocatechin, average molecular mass of proanthocyanidins, and pigmented polymer content in fruit from zones with a reduction in vine vigor. In the wines produced from low-vigor zones, there was a large increase in the proportion of skin tannin extracted into the wine, whereas little change occurred in seed proanthocyanidin extraction. The level of pigmented polymers and proanthocyanidin molecular mass were higher in wines made from low-vigor fruit compared to wines made from high-vigor fruit, whereas the flavan-3-ol monomer concentration was lower.     

Occurrence of pyranoanthocyanins in sparkling wines manufactured with red grape varieties

The anthocyanin pigments in rosé (Vitis vinifera cv. Garnacha) and blanc de noir (V. vinifera cv. Monastrell) base and sparkling wines were studied by LC-DAD/ESI-MS. Anthocyanins of grape origin and pyranoanthocyanins resulting from C-4/C-5 cycloaddition of the former ones with pyruvic acid, acetaldehyde, 4-vinylphenol, 4-vinylguaiacol, and 4-vinylcatechol were identified in the different wines. Rosé wines presented a higher total pigment content than blanc de noir wines. Pyranoanthocyanins represented 68.9-76.0% of total pigment content in rosé wines and 49.4-60.7% in blanc de noir wines. Malvidin 3-glucoside-pyruvate was the most abundant pigment in both rosé and blanc de noir base wines. Important qualitative and quantitative changes were observed in terms of the anthocyanin and pyranoanthocyanin pigments after the second (bottle) fermentation and 9 months of aging on yeast lees, but not after a further time (3-9 additional months) of aging on lees. Evaluation of the wine color characteristics was consistent with a greater color stability for the rosé sparkling wines that could be associated with the high content, structural diversity, and spectroscopic features of the pyranoanthocyanins present in these wines.     

Pathway leading to the formation of anthocyanin-vinylphenol adducts and related pigments in red wines

On the basis of observations from Vitis vinifera cv. Pinotage wines and experiments performed in model wine medium, a new chemical pathway responsible for the formation of anthocyanin-vinylphenol adducts in red wines is described. Until now, these pigments have been considered to be reaction products of anthocyanins and vinylphenols, the latter being generated during fermentation by enzymatic decarboxylation of the respective cinnamic acids. The mechanism of the novel pathway, involving intact hydroxycinnamic acid and anthocyanin, is explained. Only cinnamic acids with electron-donating substituents on the aromatic ring, such as coumaric acid, ferulic acid, caffeic acid, and sinapic acid, undergo this conversion, as they stabilize an intermediately formed carbenium ion. Decarboxylation and oxidation of the pyran moieties are the final steps in the generation of the corresponding 4-vinylphenol, 4-vinylguaiacol, 4-vinylcatechol, and 4-vinylsyringol adducts of anthocyanins in red wine.     

酵母菌对果蔬采后病害防治的研究进展

该文对酵母菌对果蔬采后病害防治的研究现状进行了综述，其中包括防治效果、防治机理、防治的优势及最新研究成果。许多酵母菌对果蔬采后病害具有明显的抑制作用，酵母菌抑制霉菌的机理主要在于营养与空间的竞争、对病原菌的直接寄生作用及诱导寄主产生抗病性。酵母菌与其它生防微生物相比，具有拮抗效果好、不产生毒素、可以和化学杀菌剂共同使用等优点。使用生物工程技术改造拮抗酵母菌从而提高其生防效果，是酵母菌对果蔬采后生物防治的最新研究领域。该文还指出了酵母菌对果蔬采后生物防治存在的问题，并对未来的研究提出了新的思路。     

Complement of integrated fertilizer management and integrated pest management concepts to ameliorate faba bean growth and yield

Twenty-one microbial preparations recommended for controlling pathogenic fungal strains causing root rot and wilt diseases of faba bean were investigated for antibiosis against several symbiotic and associative diazotrophs adopting a modified agar-plate-inhibition-zone assay. Rhizobium and Bradyrhizobium exhibited a somewhat similar susceptibility to biocontrol agents while associative diazotrophs showed variable responses. Azotobacter, compared to others, was severely inhibited by such bio-candidates. The members of the biofertilizer formulation ‘Biofertan’ did bear mixed cultivation with the majority of biocontrol agents. Among those, Bacillus subtilis was deemed the pioneer. In pot experiments, almost all the antagonists significantly restricted the severity of root rot and wilt diseases besides modifying faba bean seedling stand and improving plant development. This was very obvious with shoot biomass increases of >?50%. Moreover, the bioagents successfully recovered the legume establishment, seriously injured due to pathogenic fungal infection. Simultaneous inoculation with Rhizobium and biocontrol agents provided more growth stimulation compared to either when introduced individually. Field-grown faba beans were inoculated with the diazotroph and representatives of biocontrol strains by two different methods, seed coating and over-head soil. Growth parameters determined were the highest when the legume plant was seed-coated by Rhizobium simultaneously over-head soil inoculated with the bacterial bioagents; this was reported with Bacillus subtilis and Pseudomonas aerugenosa. The significance of combined application of biofertilizer and bioagent to ensure cheap, clean and safe farm products is discussed.     

Major flavonoids in grape seeds and skins: antioxidant capacity of catechin, epicatechin, and gallic acid

Grape seeds and skins are good sources of phytochemicals such as gallic acid, catechin, and epicatechin and are suitable raw materials for the production of antioxidative dietary supplements. The differences in levels of the major monomeric flavanols and phenolic acids in seeds and skins from grapes of Vitis vinifera varieties Merlot and Chardonnay and in seeds from grapes of Vitis rotundifolia variety Muscadine were determined, and the antioxidant activities of these components were assessed. The contribution of the major monomeric flavonols and phenolic acid to the total antioxidant capacity of grape seeds and skins was also determined. Gallic acid, monomeric catechin, and epicatechin concentrations were 99, 12, and 96 mg/100 g of dry matter (dm) in Muscadine seeds, 15, 358, and 421 mg/100 g of dm in Chardonnay seeds, and 10, 127, and 115 mg/100 g of dm in Merlot seeds, respectively. Concentrations of these three compounds were lower in winery byproduct grape skins than in seeds. These three major phenolic constituents of grape seeds contributed <26% to the antioxidant capacity measured as ORAC on the basis of the corrected concentrations of gallic acid, catechin, and epicatechin in grape byproducts. Peroxyl radical scavenging activities of phenolics present in grape seeds or skins in decreasing order were resveratrol > catechin > epicatechin = gallocatechin > gallic acid = ellagic acid. The results indicated that dimeric, trimeric, oligomeric, or polymeric procyanidins account for most of the superior antioxidant capacity of grape seeds.     

Effect of lime-induced leaf chlorosis on ochratoxin A, trans-resveratrol, and epsilon-viniferin production in grapevine (Vitis vinifera L.) berries infected by Aspergillus carbonarius

Berries of Vitis vinifera L. cv. Merlot, grown on a neutral or calcareous soil, were infected, at phenological phases of veraison and ripening, by a conidial suspension of Aspergillus carbonarius to control ochratoxin A production and trans-resveratrol- and epsilon-viniferin-induced synthesis as affected by the soil lime content. Chlorosis occurrence was evaluated by a visual rating scale at veraison, and the leaves from vines growing on the calcareous soil showed the typical yellowing, whereas those grown on the neutral soil were dark green. Berry mineral element yield was recorded at veraison and ripening. Infection symptoms on berries were more severe at ripening in bunches collected from vines grown in calcareous soil. Ochratoxin A concentration increased at phenological phase of veraison in berries harvested from vines cultivated in calcareous soil. A. carbonarius enhanced trans-resveratrol and epsilon-viniferin production in infected berries more than in the control samples. Moreover, at veraison their concentration in the berries collected from vines grown in calcareous soil was greater than that recorded from berries collected from vines grown in the neutral soil. The lowest symptom severity was observed on berries containing the highest copper concentration.     

Oxidative transformation of natural and synthetic phenolic mixtures by Trametes versicolor laccase

The efficiency of Trametes versicolor laccase in the transformation of phenols (caffeic acid, catechol, hydroxytyrosol, methylcatechol, protocatechuic acid, syringic acid, m-tyrosol, 3-hydroxybenzoic acid, 3-hydroxyphenylacetic acid, 2,6-dihydroxybenzoic acid, 4-hydroxybenzaldehyde) usually present in waste water, such as that derived from an olive oil factory, was investigated. According to their response to 24 h laccase action the 11 phenolic compounds were classified in three groups: reactive (88-100% transformation), intermediate reactive (transformation lower than 50%), and recalcitrant (not transformed at all). The enzyme was able to transform the 11 substrates even when they were present in a mixture and also toward a phenolic extract from a Moroccan olive oil mill waste water (OMW) sample. The disappearance of protocatechuic, 3-hydroxyphenylacetic, and 2,6-dihydroxybenzoic acids, and 4-hydroxybenzaldehyde was enhanced whereas that of caffeic acid and m-tyrosol was depressed when the phenols were present in the mixture. A reduction of enzyme activity occurred in single and/or complex phenolic mixtures after enzymatic oxidation. No correspondence between phenol transformation and disappearance of enzymatic activity was, however, observed. The overall results suggest that laccases are effective in the transformation of simple and complex phenolic mixtures.     

Stilbene content of mature Vitis vinifera berries in response to UV-C elicitation

A method using HPLC analysis has been used to compare the level of resveratrol and its derivatives, piceid, pterostilbene and epsilon-viniferin, in grapevine berries of three Vitis vinifera varieties. The concentration of these compounds has been evaluated in healthy and Botrytis cinerea infected grape clusters, both in natural vineyard conditions and in response to UV elicitation.     

Biological control: a sustainable and practical approach for plant disease management

ABSTRACT

This review deals with the mechanism of antagonistic action of bacterial and fungal biocontrol agents such as the production of antibiotics, siderophores, enzyme secretion, competition for nutrition, plant growth promotion by rhizosphere microorganism. The utilisation of synthetic pesticides has been the predominant control processor for diseases brought about by phytopathogenic microorganisms. Notwithstanding, their open and improper application in intensive agriculture has realised issues that have prompted ecological contamination, considerable residues in agricultural products and phytopathogen resistance. They are likewise disrupting the quantity of beneficial microorganism which is available in the soil and capable of expanding soil fertility. Along these lines, there is a need to look through the option of synthetic pesticides that are safe, environmental and monetarily feasible to confront this problem. Biocontrol agent’s utilisation is the best alternative method to control the different kinds of diseases, such as nematode infestation, fungal pathogen and bacterial pathogen. Nowadays, biocontrol agents assume a significant role in the field of agriculture. It is a financially savvy, environment-friendly and inhibits the advancement of pathogenic microorganism sustainably. This review emphasises the role of biocontrol agents against different pathogenic microorganisms and their significance potentiality to improve plant growth and enhance defence system of plants.