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Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola

Grapevine (Vitis vinifera L.) is susceptible to many pathogens, such as Botrytis cinerea, Plasmopara viticola, Uncinula necator, and Eutypa lata. Phytochemicals are used intensively in vineyards to limit pathogen infections, but the appearance of pesticide-resistant pathogen strains and a desire to protect the environment require that alternative strategies be found. In the present study, the beta-1,3-glucan laminarin derived from the brown algae Laminaria digitata was shown both to be an efficient elicitor of defense responses in grapevine cells and plants and to effectively reduce B. cinerea and P. viticola development on infected grapevine plants. Defense reactions elicited by laminarin in grapevine cells include calcium influx, alkalinization of the extracellular medium, an oxidative burst, activation of two mitogen-activated protein kinases, expression of 10 defense-related genes with different kinetics and intensities, increases in chitinase and beta-1,3-glucanase activities, and the production of two phytoalexins (resveratrol and epsilon-viniferin). Several of these effects were checked and confirmed in whole plants. Laminarin did not induce cell death. When applied to grapevine plants, laminarin reduced infection by B. cinerea and P. viticola by approximately 55 and 75%, respectively. Our data describing a large set of defense reactions in grapevine indicate that the activation of defense responses using elicitors could be a valuable strategy to protect plants against pathogens.

Oligogalacturonide signal transduction, induction of defense-related responses and protection of grapevine against Botrytis cinerea

Grapevine (Vitis vinifera L.) is vulnerable to a variety of pathogenic fungi, among them Botrytis cinerea, the causal agent of grey mould, is responsible for worldwide yield losses that would be even more important without a successful control that relies mainly on fungicides. In the present work we investigated an alternative way of using oligogalacturonides (OGA) to induce defense responses in grapevine and protection against B. cinerea. Kinetic experiments with grapevine cells showed that OGA induced a rapid and transient generation of H2O2, followed by differential expression of nine defense-related genes and stimulation of chitinase and β-1,3-glucanase activities. Inhibition of OGA-induced oxidative burst by diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, lowered induction levels of six genes and chitinase activity. Interestingly, the induction of three other genes and β-1,3-glucanase activity were inhibited by K252a, a protein kinase inhibitor, but not by DPI. Treatment of grapevine leaves with OGA also reduced infection by B. cinerea by about 55–65%. Accordingly, DPI or K252a with or without OGA increased the susceptibility of grapevine leaves to B. cinerea. We suggest that treatment of grapevine with OGA elicits different signalling pathways, which might act in tandem with the oxidative burst to increase grapevine defense responses required for protection against B. cinerea.

Osmotic stress-induced polyamine oxidation mediates defence responses and reduces stress-enhanced grapevine susceptibility to Botrytis cinerea

Abiotic factors inducing osmotic stress can influence the plant immune response and resistance to pathogen infections. In this study, the effect of polyethylene glycol (PEG)- and sucrose-induced osmotic stress on polyamine (PA) homeostasis and the basal immune response in grapevine plantlets before and after Botrytis cinerea infection was determined. Pharmacological approaches were also addressed to assess the contribution of osmotic stress-induced PA oxidation to the regulation of defence responses and the susceptibility of grapevine to B. cinerea. Following osmotic stress or pathogen infection, PA homeostasis was linked to enhanced activity of diamine oxidases (CuAO) and PA oxidases (PAO) and the production of 1,3-diaminopropane. These responses paralleled the accumulation of the main stilbenic phytoalexins, resveratrol and ε-viniferin and upregulation of gene transcripts including STS (a stilbene synthase), PR-2 (a β-1,3-glucanase), PR3-4c (acidic chitinase IV), and PR-5 (a thaumatin-like protein), as well as NCED2 involved in abscisic acid biosynthesis. It was also demonstrated that leaves pre-exposed to osmotic stress and later inoculated with B. cinerea showed enhanced PA accumulation and attenuation of CuAO and PAO activities. This was consistent with the impaired production of phytoalexins and transcript levels of defence- and stress-related genes following infection, and the enhanced susceptibility to B. cinerea. Pharmacological experiments revealed that, under osmotic stress conditions, CuAO and PAO were involved in PA homeostasis and in the regulation of defence responses. Specific inhibition of CuAO and PAO in osmotically stressed leaves strongly attenuated the induction of defence responses triggered by B. cinerea infection and enhanced susceptibility to the pathogen. Taken together, this study reveals a contribution of PA catabolism to the resistance state through modulation of immune response in grapevine following osmotic stress and/or after B. cinerea infection.

Enhanced salt-induced antioxidative responses involve a contribution of polyamine biosynthesis in grapevine plants

The possible involvement of polyamines in the salt stress adaptation was investigated in grapevine (Vitis vinifera L.) plantlets focusing on photosynthesis and oxidative metabolism. Salt stress resulted in the deterioration of plant growth and photosynthesis, and treatment of plantlets with methylglyoxal-bis(guanylhydrazone) (MGBG), a S-adenosylmethionine decarboxylase (SAMDC) inhibitor, enhanced the salt stress effect. A decrease in PSII quantum yield (Fv/Fm), effective PSII quantum yield (Y(II)) and coefficient of photochemical quenching (qP) as well as increases in non-photochemical quenching (NPQ) and its coefficient (qN) was observed by these treatments. Salt and/or MGBG treatments also triggered an increase in lipid peroxidation and reactive oxygen species (ROS) accumulation as well as an increase of superoxide dismutase (SOD) and peroxidase (POX) activities, but not ascorbate peroxidase (APX) activity. Salt stress also resulted in an accumulation of oxidized ascorbate (DHA) and a decrease in reduced glutathione. MGBG alone or in combination with salt stress increased monodehydroascorbate reductase (MDHAR), SOD and POX activities and surprisingly no accumulation of DHA was noticed following treatment with MGBG. These salt-induced responses correlated with the maintaining of high level of free and conjugated spermidine and spermine, whereas a reduction of agmatine and putrescine levels was observed, which seemed to be amplified by the MGBG treatment. These results suggest that maintaining polyamine biosynthesis through the enhanced SAMDC activity in grapevine leaf tissues under salt stress conditions could contribute to the enhanced ROS scavenging activity and a protection of photosynthetic apparatus from oxidative damages.

Enzymatic basis for fungicide removal by Elodea canadensis

PurposePlants can absorb a diversity of natural and man-made toxic compounds for which they have developed diverse detoxification mechanisms. Plants are able to metabolize and detoxify a wide array of xenobiotics by oxidation, sugar conjugation, glutathione conjugation, and more complex reactions. In this study, detoxification mechanisms of dimethomorph, a fungicide currently found in aquatic media were investigated in Elodea canadensis.MethodsCytochrome P450 (P450) activity was measured by an oxygen biosensor system, glucosyltransferases (GTs) by HPLC, glutathione S-transferases (GSTs), and ascorbate peroxidase (APOX) were assayed spectrophotometrically.ResultsIncubation of Elodea with dimethomorph induced an increase of the P450 activity. GST activity was not stimulated by dimethomorph suggesting that GST does not participate in dimethomorph detoxification. In plants exposed to dimethomorph, comparable responses were observed for GST and APOX activities showing that the GST was more likely to play a role in response to oxidative stress. Preincubation with dimethomorph induced a high activity of O- and N-GT, it is therefore likely that both enzymes participate in the phase II (conjugation) of dimethomorph detoxification process.ConclusionsFor the first time in aquatic plants, P450 activity was shown to be induced by a fungicide suggesting a role in the metabolization of dimethomorph. Moreover, our finding is the first evidence of dimethomorph and isoproturon activation of cytochrome P450 multienzyme family in an aquatic plant, i.e., Elodea (isoproturon was taken here as a reference molecule). The detoxification of dimetomorph seems to proceed via hydroxylation, and subsequent glucosylation, and might yield soluble as well as cell wall bound residues.

How Streptomyces anulatus Primes Grapevine Defenses to Cope with Gray Mold: A Study of the Early Responses of Cell Suspensions

Gray mold, caused by Botrytis cinerea, is one of the most destructive diseases of grapevine and is controlled with an intense application of fungicides. As alternatives to chemicals, beneficial microbes may promote plant health by stimulating the plant’s immune system. An actinomycete, Streptomyces anulatus S37, has been screened from the rhizosphere microbiome of healthy Vitis vinifera on the basis of its ability to promote grapevine growth and to induce resistance against various phytopathogens, including B. cinerea. However, molecular mechanisms involved locally after direct perception of these bacteria by plant cells still remain unknown. This study focuses on local defense events induced in grapevine cells during interactions with S. anulatus S37 before and after pathogen challenge. We demonstrated that S. anulatus S37 induced early responses including oxidative burst, extracellular alkalinization, activation of protein kinases, induction of defense gene expression and phytoalexin accumulation, but not the programmed cell death. Interestingly, upon challenge with the B. cinerea, the S. anulatus S37 primed grapevine cells for enhanced defense reactions with a decline in cell death. In the presence of the EGTA, a calcium channel inhibitor, the induced oxidative burst, and the protein kinase activity were inhibited, but not the extracellular alkalinization, suggesting that Ca2+ may also contribute upstream to the induced defenses. Moreover, desensitization assays using extracellular pH showed that once increased by S. anulatus S37, cells became refractory to further stimulation by B. cinerea, suggesting that grapevine cells perceive distinctly beneficial and pathogenic microbes.