Showing papers on "Phytoalexin published in 2013"

WRKY76 is a rice transcriptional repressor playing opposite roles in blast disease resistance and cold stress tolerance

Abstract: OsWRKY76 encodes a group IIa WRKY transcription factor of rice. The expression of OsWRKY76 was induced within 48h after inoculation with rice blast fungus (Magnaporthe oryzae), and by wounding, low temperature, benzothiadiazole, and abscisic acid. Green fluorescent protein-fused OsWRKY76 localized to the nuclei in rice epidermal cells. OsWRKY76 showed sequence-specific DNA binding to the W-box element in vitro and exhibited W-box-mediated transcriptional repressor activity in cultured rice cells. Overexpression of OsWRKY76 in rice plants resulted in drastically increased susceptibility to M. oryzae, but improved tolerance to cold stress. Microarray analysis revealed that overexpression of OsWRKY76 suppresses the induction of a specific set of PR genes and of genes involved in phytoalexin synthesis after inoculation with blast fungus, consistent with the observation that the levels of phytoalexins in the transgenic rice plants remained significantly lower than those in non-transformed control plants. Furthermore, overexpression of OsWRKY76 led to the increased expression of abiotic stress-associated genes such as peroxidase and lipid metabolism genes. These results strongly suggest that OsWRKY76 plays dual and opposing roles in blast disease resistance and cold tolerance.

Identification of rice Allene Oxide Cyclase mutants and the function of jasmonate for defence against Magnaporthe oryzae

Abstract: †‡ § ¶ SUMMARY Two photomorphogenic mutants of rice, coleoptile photomorphogenesis 2 (cpm2) and hebiba, were found to be defective in the gene encoding allene oxide cyclase (OsAOC) by map-based cloning and complementation assays Examination of the enzymatic activity of recombinant GST–OsAOC indicated that OsAOC is a functional enzyme that is involved in the biosynthesis of jasmonic acid and related compounds The level of jasmonate was extremely low in both mutants, in agreement with the fact that rice has only one gene encoding allene oxide cyclase Several flower-related mutant phenotypes were observed, including morphological abnormalities of the flower and early flowering We used these mutants to investigate the function of jasmonate in the defence response to the blast fungus Magnaporthe oryzae Inoculation assays with fungal spores revealed that both mutants are more susceptible than wild-type to an incompatible strain of M oryzae, in such a way that hyphal growth was enhanced in mutant tissues The level of jasmonate isoleucine, a bioactive form of jasmonate, increased in response to blast infection Furthermore, blastinduced accumulation of phytoalexins, especially that of the flavonoid sakuranetin, was found to be severely impaired in cpm2 and hebiba Together, the present study demonstrates that, in rice, jasmonate mediates the defence response against blast fungus

Modulation of Phytoalexin Biosynthesis in Engineered Plants for Disease Resistance

Abstract: Phytoalexins are antimicrobial substances of low molecular weight produced by plants in response to infection or stress, which form part of their active defense mechanisms. Starting in the 1950's, research on phytoalexins has begun with biochemistry and bio-organic chemistry, resulting in the determination of their structure, their biological activity as well as mechanisms of their synthesis and their catabolism by microorganisms. Elucidation of the biosynthesis of numerous phytoalexins has permitted the use of molecular biology tools for the exploration of the genes encoding enzymes of their synthesis pathways and their regulators. Genetic manipulation of phytoalexins has been investigated to increase the disease resistance of plants. The first example of a disease resistance resulting from foreign phytoalexin expression in a novel plant has concerned a phytoalexin from grapevine which was transferred to tobacco. Transformations were then operated to investigate the potential of other phytoalexin biosynthetic genes to confer resistance to pathogens. Unexpectedly, engineering phytoalexins for disease resistance in plants seem to have been limited to exploiting only a few phytoalexin biosynthetic genes, especially those encoding stilbenes and some isoflavonoids. Research has rather focused on indirect approaches which allow modulation of the accumulation of phytoalexin employing transcriptional regulators or components of upstream regulatory pathways. Genetic approaches using gain- or less-of functions in phytoalexin engineering together with modulation of phytoalexin accumulation through molecular engineering of plant hormones and defense-related marker and elicitor genes have been reviewed.

Pipecolic acid enhances resistance to bacterial infection and primes salicylic acid and nicotine accumulation in tobacco

Abstract: Distinct amino acid metabolic pathways constitute integral parts of the plant immune system. We have recently identified pipecolic acid (Pip), a lysine-derived non-protein amino acid, as a critical regulator of systemic acquired resistance (SAR) and basal immunity to bacterial infection in Arabidopsis thaliana. In Arabidopsis, Pip acts as an endogenous mediator of defense amplification and priming. For instance, Pip conditions plants for effective biosynthesis of the phenolic defense signal salicylic acid (SA), accumulation of the phytoalexin camalexin, and expression of defense-related genes. Here, we show that tobacco plants respond to leaf infection by the compatible bacterial pathogen Pseudomonas syringae pv tabaci (Pstb) with a significant accumulation of several amino acids, including Lys, branched-chain, aromatic, and amide group amino acids. Moreover, Pstb strongly triggers, alongside the biosynthesis of SA and increases in the defensive alkaloid nicotine, the production of the Lys catabolites Pip and α-aminoadipic acid. Exogenous application of Pip to tobacco plants provides significant protection to infection by adapted Pstb or by non-adapted, hypersensitive cell death-inducing P. syringae pv maculicola. Pip thereby primes tobacco for rapid and strong accumulation of SA and nicotine following bacterial infection. Thus, our study indicates that the role of Pip as an amplifier of immune responses is conserved between members of the rosid and asterid groups of eudicot plants and suggests a broad practical applicability for Pip as a natural enhancer of plant disease resistance.

Biosynthesis of phytoalexins and regulatory mechanisms of it in rice.

Abstract: We performed extensive functional characterization of diterpenoid phytoalexin biosynthetic genes in rice, and found that the genes for the biosynthesis of the major diterpenoid phytoalexins, phytocassanes and momilactones, are clustered on chromosomes 2 and 4, and that their expression is coordinately induced in rice cells after elicitation. Isopentenyl diphosphate, an early precursor of diterpenoid phytoalexins, was found to be synthesized through the plastidic methylerythritol phosphate pathway. We also found that chitin elicitor receptor kinase OsCERK1 and a mitogen-activated protein kinase cascade, the OsMKK4-OsMPK6 cascade, play essential roles in the elicitor-induced production of diterpenoid phytoalexins. In addition, a basic leucine zipper transcription factor, OsTGAP1, was identified as a key regulator of the coordinated expression of the clustered genes and the methylerythritol phosphate pathway genes. Naringenin 7-O-methyltransferase (OsNOMT) was also identified as a key enzyme in the biosynthe...

OsJAR1 Contributes Mainly to Biosynthesis of the Stress-Induced Jasmonoyl-Isoleucine Involved in Defense Responses in Rice

Abstract: Jasmonate plays key roles in plant growth and stress responses, as in defense against pathogen attack. Jasmonoyl-isoleucine (JA-Ile), a major active form of jasmonates, is thought to play a pivotal role in plant defense responses, but the involvement of JA-Ile in rice defense responses, including phytoalexin production, remains largely unknown. Here we found that OsJAR1 contributes mainly to stress-induced JA-Ile production by the use of an osjar1 Tos17 mutant. The osjar1 mutant was impaired in JA-induced expression of JA-responsive genes and phytoalexin production, and these defects were restored genetically. Endogenous JA-Ile was indispensable to the production of a flavonoid phytoalexin, sakuranetin, but not to that of diterpenoid phytoalexins in response to heavy metal stress and the rice blast fungus. The osjar1 mutant was also found to be more susceptible to the blast fungus than the parental wild type. These results suggest that JA-Ile production makes a contribution to rice defense responses with ...

Production of a major stilbene phytoalexin, resveratrol in peanut (Arachis hypogaea) and peanut products: a mini review

Abstract: As a major stilbene phytoalexin, resveratrol is produced or elicited in several plant species as a part of defense systems protecting plants against diseases. Resveratrol can be present in both the trans- and cis-isomeric forms, and only the trans-form increases the life expectancy and lowers the risk of cardiovascular diseases as the most bioactive form. In addition to the usages for diet and industry, peanut plant (Arachis hypogaea) and peanuts are getting higher attention due to their containment of resveratrol in the kernels and other parts of peanut plant, such as leaves, roots, and peanut shell. Recently, natural resveratrol derived from peanuts has also become a promising nutraceutical agent, promoting human health. Resveratrol has also been detected in peanut products including peanut butters, roasted peanuts, and boiled peanuts. Although, smaller and immature peanuts contain higher levels of resveratrol than mature peanuts, resveratrol in peanuts can also be preserved by cooking or manufacturing processes. Moreover, the amount of resveratrol in peanut plants and peanuts has been found to increase by external stimuli including microbial infection, wounding, UV light irradiation, ultrasonication, yeast extract treatment and by plant stress hormones. In addition, molecular level analysis has confirmed that four resveratrol synthase (RS) genes (RS1, RS2, RS3 and RS4) which catalyze synthesis of resveratrol have been identified in peanuts, and up-regulation of the genes is positively correlated to the increased contents of resveratrol. In this review, we summarize the natural biosynthesis of resveratrol in peanuts and peanut plants, as well as the occurrence of this natural phytoalexin in various peanut products. A brief knowledge on the biosynthetic pathway of resveratrol synthesis has been described. This review also deals on highlighting the effect of various external stimuli (biotic and abiotic stresses) in order to achieve the maximum induction and/or elicitation of resveratrol in peanuts and peanut plants.

Effect of salicylic acid and structurally related compounds in the accumulation of phytoalexins in cotyledons of common bean (Phaseolus vulgaris L.) cultivars.

Abstract: In the present work, isoflavonoid phytoalexin production in response to the application of salicylic acid in cotyledons of four common bean (Phaseolus vulgaris) cultivars (SA) was evaluated. The time-course and dose-response profiles of the induction process were established by quantifying the isoflavonoids by HPLC. Cotyledons of anthracnose-resistant cultivars induced by SA produced substantially higher phytoalexin contents as compared to the susceptible ones. In addition, maximum levels of phytoalexins (50–100 fold increases) were reached between 96 and 144 h, and when a concentration of SA from 3.62 to 14.50 mM was used. The observations also indicate that there was a relatively good correlation between the phytoalexin contents and the inhibitory effect against C. lindemuthianum; the higher antifungal activity was observed during the first 48 hours for extracts from cotyledons treated with SA at 1.45 and 3.62 mM, and between 96 and 144 h after induction. Finally, compounds structurally related to SA (dihydro-quinazolinones and some imines) showed a strong elicitor effect. Moreover, induced extracts from cotyledons treated with these potential elicitors, besides the properly elicitors, displayed a weak to moderated antifungal activity. These compounds may be considered good candidates for developing of new phytoprotectants. Furthermore, phytoalexin-eliciting substances may contribute for selecting disease resistant cultivars.

Production of Phytoalexins in Peanut (Arachis hypogaea) Seed Elicited by Selected Microorganisms

Abstract: Under favorable conditions, the peanut plant demonstrates appreciable resistance to fungal invasion by producing and accumulating phytoalexins, antimicrobial stilbenoids. This mechanism for resistance is little understood, yet it is crucial for breeding and genetically modifying peanut plants to develop new cultivars with fungal resistance. The dynamics of phytoalexin production in peanut seeds and embryos challenged by selected important fungi and bacteria was investigated. Different biotic agents selectively elicited production of major peanut stilbenoids, resveratrol, arachidin-1, arachidin-3, and SB-1. Aspergillis species, compared to other biotic agents, were more potent elicitors of stilbenoids. Embryos demonstrated significantly higher production of stilbenoids compared to cotyledons and may serve as a convenient source of genetic material in isolating genes for peanut plant defense enhancement.

Identification of a novel casbane-type diterpene phytoalexin, ent-10-oxodepressin, from rice leaves.

Abstract: A 70% methanol extract of UV-irradiated rice leaves (400 g) was separated by chromatographic methods to give UV-induced compound 1 (2.1 mg) which showed a possible molecular ion at m/z 300 in the GC/MS analysis. Its structure was determined by NMR and MS methods. The 1H- and 13C-NMR spectra of 1 were identical to those of 10-oxodepressin (2), a casbane-type diterpene derived from the soft coral, Sinularia depressa. The specific rotation of 1 was positive, whereas that of 2 was negative. We therefore established 1 as ent-10-oxodepressin. The accumulation of 1 was also induced by an inoculation of the rice blast fungus. Compound 1 inhibited spore germination (IC50 30 ppm) and germ tube growth (IC50 10 ppm) of the rice blast fungus. We thus concluded that 1 was a novel rice phytoalexin.

The oxygenase CAO-1 of Neurospora crassa is a resveratrol cleavage enzyme.

Abstract: The genome of the ascomycete Neurospora crassa encodes CAO-1 and CAO-2, two members of the carotenoid cleavage oxygenase family that target double bonds in different substrates. Previous studies demonstrated the role of CAO-2 in cleaving the C40 carotene torulene, a key step in the synthesis of the C35 apocarotenoid pigment neurosporaxanthin. In this work, we investigated the activity of CAO-1, assuming that it may provide retinal, the chromophore of the NOP-1 rhodopsin, by cleaving β-carotene. For this purpose, we tested CAO-1 activity with carotenoid substrates that were, however, not converted. In contrast and consistent with its sequence similarity to family members that act on stilbenes, CAO-1 cleaved the interphenyl Cα-Cβ double bond of resveratrol and its derivative piceatannol. CAO-1 did not convert five other similar stilbenes, indicating a requirement for a minimal number of unmodified hydroxyl groups in the stilbene background. Confirming its biological function in converting stilbenes, adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light, a key regulator of carotenoid metabolism, did not alter them. Targeted Δcao-1 mutants were not impaired by the presence of resveratrol, a phytoalexin active against different fungi, which did not significantly affect the growth and development of wild-type Neurospora. However, under partial sorbose toxicity, the Δcao-1 colonies exhibited faster radial growth than control strains in the presence of resveratrol, suggesting a moderate toxic effect of resveratrol cleavage products.

Phytoalexin formation in fire blight-infected apple

Abstract: Biosynthesis of phytoalexins is a plant defence strategy against pathogens. Shoots of the apple (Malus × domestica) cultivar ‘Holsteiner Cox’ formed biphenyls and dibenzofurans when inoculated with the fire blight bacterium, Erwinia amylovora. The phytoalexins were only present in the transition zone of stems, whereas the leaves were devoid of the defence compounds. The scaffold of the phytoalexins is formed by biphenyl synthase (BIS), a type III polyketide synthase. In apple, BIS is encoded by a gene family, members of which fall into four subfamilies. Representative BIS cDNAs were cloned from fire blight-infected shoots of ‘Holsteiner Cox’ and functionally expressed. The preferred starter substrates were benzoyl-CoA and salicoyl-CoA, leading to the formation of 3,5-dihydroxybiphenyl and 4-hydroxycoumarin, respectively, in the presence of malonyl-CoA as extender molecule. The four subfamilies were differentially regulated after inoculation of shoots with E. amylovora. The BIS3 gene was expressed in stems, with maximum transcript levels in the transition zone. The BIS3 protein was immunochemically localized to the parenchyma of the bark. Dot-shaped immunofluorescence was restricted to the junctions between neighbouring cortical parenchyma cells. Leaves contained transcripts for BIS2 which, however, were not translated into immunodetectable BIS protein. The understanding of phytoalexin metabolism may aid in improving apple resistance to fire blight.

Involvement of mitogen activated protein kinase kinase 6 in UV induced transcripts accumulation of genes in phytoalexin biosynthesis in rice.

Abstract: Ultra violet radiation leads to accumulation of phytoalexins (PA) in rice (Oryza sativa) which are typically accumulated when the plants are infected with rice blast pathogen Magnaporthe oryzae. Although extensive works have been done in elucidating phytoalexin biosynthesis, UV stress signal transduction leading to accumulations of rice phytoalexin is largely unknown. In the present study, the involvement of mitogen activated protein kinase (MAPK) cascade has been shown in UV induced regulation of genes in phytoalexin biosynthesis in rice. UV induced activation of MAPK and expression of PA biosynthesis genes were shown to be inhibited with staurosporin and MAPK inhibitors. Transcript regulation studies and kinase assays indicated involvement of OsMKK6 in the process. Transgenic rice overexpressing constitutive active OsMKK6 EE exhibited higher expression of genes of PA biosynthesis pathway upon UV stress and also upon infection with M. oryzae. These results suggest a key role of OsMKK6 in regulation of UV responsive expression of genes of PA biosynthesis in rice. This study will help to elucidate the intricate signalling components of UV leading to phytoalexins biosynthesis in rice.

The Medicago truncatula-Mycosphaerella pinodes interaction: a new pathosystem for dissecting fungal-suppressor-mediated disease susceptibility in plants

Abstract: A model pathosystem involving Mycosphaerella pinodes, causal agent of Mycosphaerella blight on pea and barrel medic(k) Medicago truncatula has been developed. Nineteen M. truncatula ecotypes were evaluated for disease susceptibility to M. pinodes strain OMP-1, using detached leaves inoculated with a pycnospore suspension. Inoculation of ecotype R108-1 with the pycnospores allowed direct penetration into the host’s epidermal cells, eventually causing leaf spots. Since pycnidia formed abundantly in infected tissues by 3 days post inoculation, M. pinodes is considered to have completed its infection cycle as on a natural host plant. Expression of phenylalanine ammonia-lyase (PAL)-, chalcone synthase (CHS)- and isoflavone reductase (IFR) mRNAs as well as subsequent accumulation of a major phytoalexin, medicarpin, was induced in ecotype R108-1 with an elicitor preparation from M. pinodes, whereas this phytoalexin response was markedly attenuated by supprescins from the same fungus. A transcriptional study of a salicylic acid (SA)-regulated gene encoding pathogenesis-related protein 10-1 (PR10-1) indicated that the suppressor likely interferes with the signal transduction process leading to inducible defenses in M. truncatula. Indeed, the suppressor rendered the host tissues (ecotype R108-1) susceptible to unrelated nonpathogenic fungi, probably through targeting ATPase activity of the host cells. Accordingly, the resistant and susceptible response of pea can be recapitulated in M. truncatula. This model pathosystem thus will allow us to verify the molecular basis underlying the fungal suppressor-mediated plant susceptibility.

Resveratrol analog 4-[2-(3,5-dimethoxyphenyl)vinyl]pyridine reduces differentiation of the 3T3-L1 adipocyte.

Abstract: Context: Resveratrol (3,5,4′-trihydroxystilbene) is a phytoalexin synthesized by plants, most notably grapes, against microbial invasion or ultraviolet stimulation, and is known to exert antioxidant, anticancer, and antiobesity effects.Objective: This study was conducted to find resveratrol derivatives with higher anti-obesity activity compared to resveratrol and to verify their mechanism of action.Materials and methods: The inhibitory effect of resveratrol and its derivatives on adipocyte differentiation in 3T3-L1 cells was studied using Oil Red O staining, and the effects on the intracellular expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were measured via Western blot analysis.Results: A derivative of resveratrol, 4-[2-(3,5-dimethoxyphenyl)vinyl]pyridine (DPVP), exerted inhibitory effects against 3T3-L1 adipocyte differentiation (IC50 = 13.5 µM) and FAS expression. Notably, it displayed higher activity at concentrations lower than 25 µM compared to resveratrol.Discussion and c...

Discriminative Phytoalexin Accumulation in Lotus japonicus against Symbiotic and Non-Symbiotic Microorganisms and Related Chemical Signals

Abstract: The phytoalexin response of Lotus japonicus seedlings to selected microbes and chemical signals was analyzed. The symbiotic rhizobium induced vestitol production weakly, while non-symbiotic rhizobia and potential pathogens led to increases in its accumulation. Whereas chitin-related molecules were ineffective, a flagellin-derived peptide not of symbiont origin induced phytoalexin production, indicating discriminative antibiotic production by the plant host.

Method for producing phytoalexin and phytoalexin produced by the same method

Abstract: PURPOSE: A method for preparing phytoalexin is provided to enhance the content of phytoalexin in seeds, to obtain a large amount of natural phytoalexin, and to apply to pharmaceutical, food, or cosmetic products CONSTITUTION: A method for preparing phytoalexin comprises: a step of germinating seeds; and a step of fermenting the seeds The seeds do not include Glycine max Phytoalexin does not contain coumestrol The germination is performed by contacting a part of the seeds with oxygen or air and microorganism The microorganism is fungi, yeast, or lactic acid bacteria

Effect of metabolites produced by beneficial fungi on the plant metabolome, physiology and agronomic performance

Abstract: The production of different metabolites has been proposed, in some cases, to be related to the activity of various beneficial fungi used world-wide for crop protection and fertilization. Metabolomic analysis of the interactions between plants, fungal phytopathogens and beneficial fungi has aided in the identification of several bioactive fungal metabolites that positively affect plant metabolism. Some of these compounds have direct activity against phytopathogens, but also increase disease resistance by triggering the plant defence system, and/or enhance root, stem, shoot and leaf vegetative growth, which may produce significant yield increases that are detectable in the field. Beneficial fungi belonging to the genus Trichoderma are well known producers of bioactive metabolites, and are presently marketed as biopesticides and biofertilizers, due to their ability to protect plants by containing pathogen populations, as well as increase plant growth and development under different soil conditions. In this study, we have investigated the possibility of improving the effectiveness and the usefulness of fungal biocontrol agents by using selected metabolites [6-pentyl--pyrone (6PP), harzianic acid (HA) and hydrophobin 1 (HYTRA1)] able to: i) inhibit phytopathogens; ii) promote Trichoderma antagonistic activity; iii) induce resistance mechanisms in the plant; iv) stimulate growth and development of different cultures. To achieve this goal, we combined biochemical characterization of SMs and fungal strains with tests for agronomic performance of the biocontrol agents, and used agriculturally important plants (Solanum lycopersicum, Brassica rapa, Vitis vinifera) as well as Arabidopsis thaliana. Application of metabolites isolated and characterized from Trichoderma strains improved plant growth in terms of seed germination (tomato plant), shoot length and dry weight (tomato, broccoli, grapevine and A. thaliana). Moreover, HA and 6PP stimulated systemic resistance mechanisms, including the production of glucosinolates in broccoli plant, while 6PP increased also the antioxidant activity and the polyphenol content in the grapes. The effect on the antioxidant activity and the polyphenol content was generally comparable with, or better that one obtained by using the commercial formulation based on the highly-effective strain T22 of T. harzianum. We have examined, by using LC-ESI-QToF MS, the metabolic changes occurring in A. thaliana treated with three Trichoderma metabolites found to be involved in the induction of disease resistance and growth promotion,. The effects of treatments on A. thaliana growth were observed. Applications of 6PP, HA and HYTRA1 increased plant root-length by 48%, 51% and 40%, respectively, as compared to the untreated control. The Trichoderma metabolite treatments were found to produce significant changes in the plant metabolome, particularly in the production of major hormones, by acting on specific pathways involved in plant defence (i.e phytoalexin production). The results obtained allow a better understanding of the role of some metabolites in the important beneficial interaction of Trichoderma fungi with various crop and model plants.

Signals and metabolic consequences during the interaction of Brassicaceae and Verticillium longisporum

Abstract: The soil-borne, hemibiotrophic pathogen Verticillium longisporum is a severe threat for oilseed rape (Brassica napus) cultivation. In order to develop effective, defensive treatments for infected plants a thorough understanding of the plant-pathogen interaction is necessary. Therefore this work was conducted to find and identify metabolic changes and involved metabolite pathways in the pathogenic fungus or in the invaded host plant. For that a metabolite fingerprinting approach was optimized by adjusting the analytical parameters and adapting extraction methods to the analysed types of samples. In order to identify extracellular metabolites that are produced by V. longisporum in the xylem vessel, an in vitro assay was established resulting in the identification of seven metabolites from the tryptophan metabolism that accumulates in a provided xylem like environment. Nevertheless none of the identified substances produced by the fungus in vitro was detectable in planta after V. longisporum infection. Hence all accumulating metabolites in planta were analysed. A comprehensive analysis of apoplastic fluids and different plant organs in a time course from 5 to 35 days past infection in B. napus revealed a strong change in the metabolite pattern caused by V. longisporum infection. The identification of ubiquitously accumulating infection markers resulted in the detection of the phytoalexin cyclobrassinin and 22 related newly described substances. Structure elucidation was performed by MS/MS and pseudo-MS/MS/MS fragmentation analysis. The distribution of the cyclobrassinin related compounds within the plant correlated with the amount of fungal DNA detected in the plant organs pointing to a high contamination and a strong metabolic response in the hypocotyl tissue. By combining the obtained structural information and a proposed pathway for the phytoalexin camalexin in Arabidopsis thaliana an analogue model for the biosynthetic pathway of cyclobrassinin is proposed. A subgroup of the identified infection markers hints to a degradation of cyclobrassinin resulting in 2-mercapto-indole-3-carboxylic acid (MICA) derivatives. When samples from Camelina sativa – whose infection process with V. longisporum was accessed for the first time in this work - and A. thaliana were included in the comprehensive analysis of Brassicaceae it was found, that other groups of infection markers are more general than the species specific cyclobrassinin related markers. Raphanusamic acid (RA), a substance putatively related to phytoalexin or glucosinolate metabolism, pipecolic acid and three glycosylated salicylic acid (SA) derivatives proved to accumulate in the three Brassicaceae. Two trihydroxy fatty acids were identified infection markers from apoplastic fluids of B. napus and A. thaliana. Furthermore, five groups of infection markers were identified exclusively in the apoplastic wash fluid (AWF). The saturated dicarboxylic acids and their monoamide derivatives are infection markers in B. napus as well as in A. thaliana. Furthermore, diamides, aldehydes and alcohol derivatives were found to accumulate in B. napus plants. The mono- and diamide derivatives were unequivocally identified by chemically synthesized authentic standards. Three polyamine infection markers were additionally measured in a targeted analysis whereas the glucosinolates often involved in plant-pathogen interactions were not found to be reliable infection markers in B. napus. Preliminary results from a priming experiment of B. napus with the C9 dicarboxylic acid, azelaic acid, indicated only a shift in cyclobrassinin biosynthesis but no enhanced resistance to V. longisporum infection. An infection resembling metabolite pattern could not be achieved by flotation on several identified infection markers like SA or RA. The elicitation of B. napus leaves with CuCl2 demonstrated that the cyclobrassinin related substances were plant derived substances. They as well as markers like RA, SAG and pipecolic acid accumulate upon biotic and abiotic stress in B. napus. In summary more than 70 metabolites were unequivocally identified either as fungal substances that accumulate in in vitro assays or as plant-derived metabolites in Brassicaceae upon V. longisporum infection.

The Relation-Ship between Phytoalexin Production in Plant and Plant Resistance to Fungal Infection.

Abstract: The phytoalexin accumulation in the leaves and whole plants of two cultivators of Alfalfa plants, resistant and susceptible to the fungus Verticillium albo-atrum isolates (V1) Virulent and (V2) AVirulent and many species were investigated, the results showed that different isolates and species of Verticillium fungus induce the resistant and susceptible cultivar of Alfalfa to produce different amount of phytoalexin. But the two cultivars produce similar amount of phytoalexin in response to either pathogenic isolates (V1) or the non pathogenic isolates (V2).