Showing papers on "Phytoalexin published in 2008"

The glutathione-deficient mutant pad2-1 accumulates lower amounts of glucosinolates and is more susceptible to the insect herbivore Spodoptera littoralis.

Abstract: Summary Plants often respond to pathogen or insect attack by inducing the synthesis of toxic compounds such as phytoalexins and glucosinolates (GS). The Arabidopsis mutant pad2-1 has reduced levels of the phytoalexin camalexin and is known for its increased susceptibility to fungal and bacterial pathogens. We found that pad2-1 is also more susceptible to the generalist insect Spodoptera littoralis but not to the specialist Pieris brassicae. The PAD2 gene encodes a gamma-glutamylcysteine synthetase that is involved in glutathione (GSH) synthesis, and consequently the pad2-1 mutant contains about 20% of the GSH found in wild-type plants. Lower GSH levels of pad2-1 were correlated with reduced accumulation of the two major indole and aliphatic GSs of Arabidopsis, indolyl-3-methyl-GS and 4-methylsulfinylbutyl-GS, in response to insect feeding. This effect was specific to GSH, was not complemented by treatment of pad2-1 with the strong reducing agent dithiothreitol, and was not observed with the ascorbate-deficient mutant vtc1-1. In contrast to the jasmonate-insensitive mutant coi1-1, expression of insect-regulated and GS biosynthesis genes was not affected in pad2-1. Our data suggest a crucial role for GSH in GS biosynthesis and insect resistance.

A stress-inducible resveratrol O-methyltransferase involved in the biosynthesis of pterostilbene in grapevine.

Abstract: Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl3 treatment.

A Stress-Inducible Resveratrol O-Methyltransferase Involved in the Biosynthesis of Pterostilbene

Abstract: Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl3 treatment.

Localized production of phytoalexins by peanut (Arachis hypogaea) kernels in response to invasion by Aspergillus species.

Abstract: Peanuts respond to fungal infection by synthesizing phytoalexins, most of which are antibiotic stilbenes. The mechanism and dynamics of phytoalexin formation in the peanut have not been studied. One of the most popular peanut cultivars in the southeastern United States, Georgia Green, was investigated for its ability to produce phytoalexins in response to infection by soil fungal strains. The experimental design allowed for study of phytoalexin production in peanut kernels layer-by-layer. The layers were dissected from different depths of the kernel starting from the infected area down to healthier tissues. Six peanut phytoalexins, trans-resveratrol, trans-arachidin-1, trans-arachidin-2, trans-arachidin-3, trans-3-isopentadienyl-4,3',5'-trihydroxystilbene, and SB-1, were detected in the kernel slices and quantitated. All of the fungal strains triggered phytoalexin production; however, the composition of phytoalexins varied significantly by layer. After incubation for 24 h, tissues remote from the infected area produced almost exclusively trans-resveratrol, whereas closer to the infected area tissues synthesized all six phytoalexins. In all of the experiments, after 48 h of fungal growth, deeper layers produced all tested phytoalexins. There was a significant difference in phytoalexin production elicited by some fungal isolates. No association was observed between phytoalexin production and toxigenic potential of fungal strains that elicited the production in mature peanut kernels.

Systemic resistance and lipoxygenase-related defence response induced in tomato by Pseudomonas putida strain BTP1.

Abstract: Previous studies showed the ability of Pseudomonas putida strain BTP1 to promote induced systemic resistance (ISR) in different host plants. Since ISR is long-lasting and not conducive for development of resistance of the targeted pathogen, this phenomenon can take part of disease control strategies. However, in spite of the numerous examples of ISR induced by PGPR in plants, only a few biochemical studies have associated the protective effect with specific host metabolic changes. In this study, we showed the protective effect of this bacterium in tomato against Botrytis cinerea. Following treatment by P. putida BTP1, analyses of acid-hydrolyzed leaf extracts showed an accumulation of antifungal material after pathogen infection. The fungitoxic compounds thus mainly accumulate as conjugates from which active aglycones may be liberated through the activity of hydrolytic enzymes. These results suggest that strain BTP1 can elicit systemic phytoalexin accumulation in tomato as one defence mechanism. On another hand, we have shown that key enzymes of the lipoxygenase pathway are stimulated in plants treated with the bacteria as compared with control plants. Interestingly, this stimulation is observed only after pathogen challenge in agreement with the priming concept almost invariably associated with the ISR phenomenon. Through the demonstration of phytoalexin accumulation and LOX pathway stimulation in tomato, this work provides new insights into the diversity of defence mechanisms that are inducible by non-pathogenic bacteria in the context of ISR.

Metabolic Changes in Roots of the Oilseed Canola Infected with the Biotroph Plasmodiophora brassicae: Phytoalexins and Phytoanticipins

Abstract: Analyses of metabolite production and accumulation in roots of canola ( Brassica napus L. spp. oleifera) infected with the phytopathogen Plasmodiophora brassicae (clubroot) allowed the identification of 45 metabolites. HPLC analysis corroborated by metabolite isolation and NMR spectroscopic data demonstrated for the first time that phytoalexins and phytoanticipins were produced in roots of canola infected with a soilborne biotroph. In addition, six new indolyl metabolites were identified, synthesized, and tested against three fungal pathogens of canola. Multivariate data analysis using principal component analysis (PCA) revealed distinct metabolic responses of canola to P. brassicae infection during a six-week period. At late harvest days (five and six weeks), a clear clustering was observed among samples of infected roots because of the higher concentration of phytoalexins, while higher concentration of phytoanticipins contributed to the differentiation between three and four weeks samples of infected and control roots. Altogether, the data shows that canola roots under biotrophic attack are able to produce a complex blend of phytoalexins and other antimicrobial metabolites as a defensive response and that the metabolic regulation of phytoanticipins and phytoalexins appeared to correlate with the infection period.

Sub‐lethal Levels of Electric Current Elicit the Biosynthesis of Plant Secondary Metabolites

Abstract: Many secondary metabolites that are normally undetectable or in low amounts in healthy plant tissue are synthesized in high amounts in response to microbial infection. Various abiotic and biotic agents have been shown to mimic microorganisms and act as elicitors of the synthesis of these plant compounds. In the present study, sub-lethal levels of electric current are shown to elicit the biosynthesis of secondary metabolites in transgenic and non-transgenic plant tissue. The production of the phytoalexin (+)-pisatin by pea was used as the main model system. Non-transgenic pea hairy roots treated with 30-100 mA of electric current produced 13 times higher amounts of (+)-pisatin than did the non-elicited controls. Electrically elicited transgenic pea hairy root cultures blocked at various enzymatic steps in the (+)-pisatin biosynthetic pathway also accumulated intermediates preceding the blocked enzymatic step. Secondary metabolites not usually produced by pea accumulated in some of the transgenic root cultures after electric elicitation due to the diversion of the intermediates into new pathways. The amount of pisatin in the medium bathing the roots of electro-elicited roots of hydroponically cultivated pea plants was 10 times higher 24 h after elicitation than in the medium surrounding the roots of non-elicited control plants, showing not only that the electric current elicited (+)-pisatin biosynthesis but also that the (+)-pisatin was released from the roots. Seedlings, intact roots or cell suspension cultures of fenugreek (Trigonella foenum-graecum), barrel medic, (Medicago truncatula), Arabidopsis thaliana, red clover (Trifolium pratense) and chickpea (Cicer arietinum) also produced increased levels of secondary metabolites in response to electro-elicitation. On the basis of our results, electric current would appear to be a general elicitor of plant secondary metabolites and to have potential for application in both basic and commercial research.

Effect of Methyl Substitution on the Antioxidative Property and Genotoxicity of Resveratrol

Abstract: Resveratrol (trans-3,4′,5-trihydroxystilbene) is a natural phytoalexin with various biological activities including inhibition of lipid peroxidation and free radical scavenging properties. In addition to its beneficial effects, resveratrol also has significant genotoxicity that leads to a high frequency of chromosome aberration together with micronucleus and sister chromatid exchanges. To enhance the radical scavenging activities and to reduce the genotoxicity of resveratrol, we designed 4′-methyl resveratrol analogues where a methyl group was introduced at the ortho position relative to the 4′-hydroxy group, which is responsible for both antioxidative activities and genotoxicity of resveratrol. These synthesized methyl analogues of resveratrol showed increased antioxidative activities against galvinoxyl radical as an oxyl radical species. Furthermore, the methyl analogues also surprisingly showed reduced in vitro genotoxicities, suggesting that methyl substitution may improve resveratrol efficacy.

Brassinin oxidase, a fungal detoxifying enzyme to overcome a plant defense – purification, characterization and inhibition

Abstract: Blackleg fungi [Leptosphaeria maculans (asexual stage Phoma lingam) and Leptosphaeria biglobosa] are devastating plant pathogens with well-established stratagems to invade crucifers, including the production of enzymes that detoxify plant defenses such as phytoalexins. The significant roles of brassinin, both as a potent crucifer phytoalexin and a biosynthetic precursor of several other plant defenses, make it critical to plant fitness. Brassinin oxidase, a detoxifying enzyme produced by L. maculans both in vitro and in planta, catalyzes the detoxification of brassinin by the unusual oxidative transformation of a dithiocarbamate to an aldehyde. Purified brassinin oxidase has an apparent molecular mass of 57 kDa, is approximately 20% glycosylated, and accepts a wide range of cofactors, including quinones and flavins. Purified brassinin oxidase was used to screen a library of brassinin analogues and crucifer phytoalexins for potential inhibitory activity. Unexpectedly, it was determined that the crucifer phytoalexins camalexin and cyclobrassinin are competitive inhibitors of brassinin oxidase. This discovery suggests that camalexin could protect crucifers from attacks by L. maculans because camalexin is not metabolized by this pathogen and is a strong mycelial growth inhibitor.

Local and systemic gene expression of sesquiterpene phytoalexin biosynthetic enzymes in plant leaves

Abstract: Production of antimicrobial metabolites known as phytoalexins is considered as one of the initial and main barriers to inhibit pathogen development in local infected aerial tissues. Capsidiol is the main bicyclic sesquiterpene phytoalexin in tobacco (Nicotiana tabacum) and chili pepper (Capsicum annuum). Production of 5-epi-aristolochene by the corresponding sesquiterpene cyclase enzymes is considered the critical step in capsidiol biosynthesis. To analyze the transcriptional activation of chili pepper 5-epi-aristolochene synthase gene expression in response to several pathogen-associated molecular patterns, a 1,455 bp promoter fragment upstream start codon was fully sequenced and fused to β-glucuronidase reporter gene. Analyses of spatial and temporal patterns of hybrid gene expression were carried out in transgenic tobacco plants. Surprisingly β-glucuronidase was detected in both, the locally treated and the phylotactically adjacent leaves. A particular systemic gene expression was localized in the immediate vascular tissue. The activation patterns of 5-epi-aristolochene synthase transcripts and detection of capsidiol in corresponding tobacco and pepper systemic leaves confirmed these results. This expression pattern might be mediated by reactive oxygen species. This is the first report of a highly localized systemic gene expression of enzymes directly involved in sesquiterpene phytoalexin biosynthesis in leaves, elicited by pathogen-associated molecular patterns.

Caracterização parcial de frações obtidas de extratos de Cymbopogon nardus com atividade elicitora de fitoalexinas em sorgo e soja e efeito sobre Colletotrichum lagenarium

Abstract: Medicinal plants present potential to control phytopathogens due to the direct effect or by the activation of defense mechanisms like phytoalexins. Then, the present work had the objective to verify the antifungal and elicitor effect of citronella (Cymbopogon nardus) obtained from methanolic and ethanolic extracts. It was studied the accumulation of phytoalexins in the mesocotyls of sorghum and in cotyledon of soybean, in response to application of fractions from crude extracts methanolic (EME) and ethanolic extracts (EET) of citronella. The EME and EET extracts were fractioned by gel filtration chromatography (CFG) and three fractions were obtained from the methanolic extract (FMI, FMII and FMIII), and two fractions from the ethanolic extract (FEI and FEII). The molecular weight for the FMI, FMII, FMIII, FEI and FEII were 69.29, 40.51, 18.72, 65.89 and 24.11 kDa, respectively. The fractions obtained from the CFG were used in a bioassay of germination of C. lagenarium spores and of induction of phytoalexins in the mesocotyls of sorghum and cotyledon of soybean. There was no significative effect of the fractions EME and EET on germination and appressorium formation by the pathogen. In the accumulation of phytoalexins in soybean cotyledons there was no significant effect of the fractions FMI, FMII, FMIII, FEI and FEII. However, it was detected a significative effect of fractions obtained by CFG on the production of phytoalexins in mesocotyls of sorghum. The highest accumulation of phytoalexin in mesocotyls of sorghum was obtained in the FMI and FMIII fractions, which differed significantly to the control. Based on the obtained results, it is possible to conclude that fractions partially purified obtained from EME of C. nardus present potential to induce phytoalexins in sorghum mesocotyls.

Induction of systemic acquired resistance in tomato plants against early blight disease

Abstract: The effect of five antioxidants (citric acid, salicylic acid, benzoic acid, ascorbic acid, and sodium citrate) on the resistance of tomato plants (Lycopersicon esculentum Mill) to early blight disease incited by Alternaria solani was investigated in vitro and in vivo. The results indicated that all of the antioxidants tested markedly inhibited the mycelial growth of A. solani in vitro. The degree of inhibition was directly proportional with the antioxidant concentrations. The mycelial growth was greatly inhibited at the highest concentration (10.0 mM) of all antioxidants tested. Salicylic acid, ascorbic acid, and citric acid were more effective throughout the concentration ranges in inhibiting the mycelial growth than sodium citrate and benzoic acid respectively. Soaking tomato seeds in the antioxidants prior to sowing markedly increased tomato resistance to early blight disease by A. solani. All antioxidantse employed at different concentrations, caused a marked reduction in the appearance of early blight disease on tomato plants and the disease incidence was completely inhibited at the highest concentrations of every antioxidants tested. Salicylic acid, ascorbic acid, and citric acid were the most effective in controlling the disease; followed by sodium citrate, and benzoic acid. Also data indicated that the increasing in stem average length and leaves number per plant was corresponded with the reduction of disease severity. Phytoalexin (tomatine) production was greatly increased in antioxidant-treated inoculated tomato plants. In all treatments, the level of tomatine was higher in the leaf than stem of inoculated tomato plants compared with untreated inoculated control and treated uninoculated plants. Maximum production of tomatine was achieved in tomato plants treated with salicylic acid, ascorbic acid, or citric acid, and inoculated with fungal spores where it was not detected in leaf and stem of uninoculated control plants. These results suggested that antioxidants may play an important role in controlling the early blight disease through inducing production of phytoalexin from cells of tomato plants. Some antioxidants are used as food grade so they are safe for use as antifungal without any risk to human, animal, and environment.

Assessment of potential for biological control of botrytis cinerea by an indigenous trichoderma harzianum isolate with a novel detached leaf-droplet inoculation bioassay and correlated increase in phytoalexin production

Abstract: A simple detached leaf-droplet inoculation bioassay was devised in order to investigate the potential for biological control of Botrytis cinerea by indigenous Trichoderma harzianum isolates. Following preliminary in vitro screening of the antagonistic capacity of four local isolates of Trichoderma harzianum (T1-T4) against B. cinerea, T3 spore suspensions were then tested for their ability to control infection development of B. cinerea on T3 pre-inoculated lettuce and broad bean leaves, using different nutrient concentrations in B. cinerea infection inocula. Again, T3 proved to significantly control infection development in all but one of the treatments, as long as T3 spores were introduced in a nutrient solution. This bioassay provides a rapid and simple method of assessing biocontrol efficacy under in vivo conditions. T3 was also tested for its ability to induce plant defense mechanisms in the form of phytoalexin production after inoculation of broad bean cotyledons with T3 spore suspension. Thin layer chromatography indicated that T3 lead to the induction of relatively high phytoalexin yields, suggesting that this could be another potential ‘indirect’ mechanism of protection from pathogens by T. harzianum strain T3.

Selective Elicitation of the Phytoalexin Rutalexin in Rutabaga and Turnip Roots by a Biotrophic Plant Pathogen

Abstract: Stress responses of roots of rutabaga (Brassica napus ssp. napobrassica) and turnip (B. rapa ssp. rapa) were analyzed. Phytoalexin production due to biotic elicitation by the biotroph Albugo candid...