Improved resistance against Botrytis cinerea by grapevine-associated bacteria that induce a prime oxidative burst and phytoalexin production

Bacteria such as Pantoea agglomerans (Pa-AF2), Bacillus subtilis (Bs-271), Acinetobacter lwoffii (Al-113), and Pseudomonas fluorescens (Pf-CT2), originating from the vineyard, can induce defense responses and enhance resistance of grapevine against the fungal pathogen Botrytis cinerea. The perception of these bacteria by plant cells or tissues in relation to their activities remains unknown. In this study, we examined the relationships between the activity of each bacterium to induce or prime some defense responses, and its effectiveness to induce resistance in grapevine against B. cinerea. We showed that all selected bacteria are capable of inducing early oxidative burst and phytoalexin (trans-resveratrol and trans-ε-viniferin) production in grapevine cells and leaves. Pf-CT2 and Al-113 induced higher H(2)O(2) and trans-resveratrol accumulations, and were able to further prime plants for accelerated phytoalexin production after B. cinerea challenge. These two bacteria were also the most effective in inducing local and systemic resistance. A similar level of induced resistance was observed with live Pa-AF2 which also induced but not primed a greater accumulation of trans-resveratrol. However, Bs-271, which was less effective in inducing resistance, induced a lower trans-resveratrol synthesis, without priming activity. Treatment of grapevine cells with growing medium or crude extract of the bacteria quickly and strongly enhanced oxidative burst compared with the live bacteria. However, both treatments resulted in comparable amounts of phytoalexins and induced local and systemic resistance to B. cinerea as compared with those induced by living bacteria, with extracts from Pf-CT2 and Al-113 being the most effective. Together, these results indicate that induced resistance can be improved by treatment with bacteria or derived compounds which induced or primed plants for enhanced phytoalexin accumulation.     

Stilbene oligomer phytoalexins in grape as a response to Aspergillus carbonarius infection

Stilbenes are grapevine phytoalexins elicited by biotic and abiotic agents; Aspergillus carbonarius is a widespread ochratoxin A producing fungus present in warm conditions, such as in Southern Italy. To increase the knowledge on biosynthesis of stilbene oligomers induced by A. carbonarius infection, grape berries of the Southern Italian grape cv. Negro Amaro were inoculated. Significant increase of trans-resveratrol and resveratrol dimers and oligomers, such as caraphenol, E-ε-viniferin, ω-viniferin, δ-viniferin, α-viniferin, E-miyabenol C, and two tetramers, was observed, and concomitant decrease of glycoside derivatives. These findings improve the knowledge on the phytoalexin production as response against this pathogen.     

Effect of Pythium spp. and glyphosate on phytoalexin production and exudation by bean (Phaseolus vulgaris L.) roots grown in different media

Kievitone, phaseollinisoflavan and phaseollin were detected in roots of bean seedlings (Phaseolus vulgaris L.) grown in natural soil. Comparison of phytoalexin production by roots grown in different media indicated that these phytoalexins were probably induced by microorganisms in soil. The influence of common root rot pathogens of bean, Pythium spp., on phytoalexin production was determined. Pythium ultimum elicited kievitone, phaseollinisoflavan and phaseollin in roots grown in sterilized silica sand. P. sylvaticum induced only kievitone and phaseollin in the same growth medium. Glyphosate did not significantly affect the accumulation of phytoalexins within 3 days. However, by day 5, significantly more phaseollin was detected in the roots of Pythium inoculated plants treated with glyphosate than in Pythium inoculated plants not treated with glyphosate. In a hydroponic system, both Pythium spp. elicited accumulation of kievitone and phaseollin in root tissue, and both phytoalexins were exuded into the bathing solution. Glyphosate application did not significantly affect accumulation or exudation of phytoalexins by bean roots in the hydroponic system. The results from this study illustrate the nature and extent of phytoalexin production by bean roots in the absence and presence of microbes.     

Synthesis of C-glycosyl flavonoid phytoalexins as a site-specific response to fungal penetration in cucumber

Leaf tissue harvested from cucumber plants (Cucumis sativus L.) expressing induced resistance against the powdery mildew fungus Podosphaera xanthii (syn. Sphaerotheca fuliginea, Castagne; Braun and Shishkoff) was extracted and analyzed for phytoalexin compounds. Fluorescence microscopy was then used to observe the production of these compounds in planta, and laser scanning confocal microscopy observations were made to locate the subcellular sites of phytoalexin accumulation. Phytochemical analyses and fluorescence microscopy observations revealed the production of autofluorescent C-glycosyl flavonoid phytoalexins within the epidermal tissues of disease-resistant plants undergoing fungal ingress. Phytoalexin production was triggered by the combination of an eliciting/inoculation treatment, and tissue autofluorescence of color characteristic of the phytoalexins reached a maximum 48 h after elicitation prior to subsiding following the collapse of the pathogen. After a second eliciting treatment, disease-resistant plants produced phytoalexins more rapidly in response to fungal challenge. At the cellular level, autofluorescent C-glycosyl flavonoid phytoalexins were observed associated with the plasma membrane of infected epidermal cells immediately following elicitation. In the hours that preceded the collapse of conidial chains, phytoalexins accumulated inside the haustorial complexes of the pathogen within the epidermal cells of disease-resistant plants. Taken together, the results of this study show the timely synthesis of C-glycosyl flavonoid phytoalexins at precise subcellular locations as a key defense reaction used by cucumber to create incompatible interactions with powdery mildew.     

Formation of phytoalexins within and outside lesions of Botrytis cinerea in French bean leaves

Rapidly spreading lesions and lesions restricted in size developed in primary leaves of French bean (Phaseolus vulgaris) in response to infection byBotrytis cinera isolates BC-1 and BC-5, respectively. These isolates caused similar differential lesions in leaves of cucumber, flax, lettuce and tomato. To determine whether phytoalexin accumulation was correlated with the resistant reaction in bean leaves, accumulation of phytoalexins was examined in necrotic areas of both types of lesions and in their surrounding green tissues. Phaseollin was the predominant phytoalexin, both inside and outside lesions, whereas phaseollidin and sometimes also phaseollinisoflavan were always present in lower concentrations. Phaseollin accumulated earlier and to higher levels within and around lesions of isolate BC-5 than of isolate BC-1. Relatively low concentrations of phaseollin were detected in the more remote green areas, including the petiole, of leaves bearing a spreading lesion. The phaseollin metabolite, 6a-hydroxyphaseollin, was found only inside lesions and in a narrow zone around lesions of both types. The authors consider the possibility that the differing concentrations of phytoalexins in the infected tissues are not a determining factor for the differential interactions betweenB. cinerea and bean leaves, but are rather the result of it.     

Silicon-mediated accumulation of flavonoid phytoalexins in cucumber

ABSTRACT The controversial role of silicon in plant disease resistance, described mostly as a passive mechanical protection, has been addressed. Conclusive evidence is presented that silicon is involved in the increased resistance of cucumber to powdery mildew by enhancing the antifungal activity of infected leaves. This antifungal activity was attributable to the presence of low-molecular-weight metabolites. One of these metabolites, described here as a phytoalexin, was identified as a flavonol aglycone rhamnetin (3,5,3',4'-tetrahydroxy-7-O-methoxyflavone). This is the first report of a phytoalexin for this chemical group in the plant kingdom and of a flavonol phytoalexin in cucumber, a chemical defense long believed to be nonexistent in the family Cucurbitaceae. The antifungal activity of leaf extracts was better expressed after acid hydrolysis, extending to another plant species the concept that some phytoalexins are synthesized as glycosylated phytoalexins or their precursors.     

Characterization of a Pterostilbene Dehydrodimer Produced by Laccase of Botrytis cinerea

ABSTRACT In the interaction between grapevines and Botrytis cinerea, one of the main aspects of pathogenicity is fungal ability to degrade phytoalexins synthesized by the plant in response to infection. Laccase-like stilbene oxidase activity in liquid cultures of B. cinerea has been shown to be related to the decrease of phytoalexin concentrations. Recent research and results presented in this paper determined the chemical structure of a pterostilbene metabolite produced by B. cinerea. Study of degradation of pterostilbene that has just one free hydroxy phenyl group function allowed us to determine the oxidative dimerization process undergone by grapevine phytoalexins after B. cinerea infection. The phytopathological significance of this degradation process in the B. cinerea interaction has also been discussed.     

Accumulation of isoflavonoid phytoalexins in leaves of Arachis hypogaea differing in reaction to rust (Puccinia arachidis) and early leafspot (Cercospora arachidicola)

Groundnut leaves, naturally infected in the field with Cercospora arachidicola or artificially infected in the greenhouse with C. arachidicola or Puccinia arachidis , accumulated isoflavonoid phytoalexins. Major compounds included medicarpin, formononetin, demethylmedicarpin, three isoflavanones and the isoflavone, daidzein. In both greenhouse and field experiments cv. Egret was less susceptible than cv. P84/5/244 to both pathogens, and accumulated phytoalexin (medicarpin) concentrations which were up to three times greater. Higher concentrations and greater variation of phytoalexins were detected when both cultivars were infected with C. arachidicola than when they were infected with P. arachidis.     

The possibility of factors other than phytoalexin accumulation preventing fungal growth in the incompatible interactions of soybean with Phytophthora megasperma f. sp. glycinea

Soybean ( Glycine max ) cv. Harosoy 63 is resistant to race 1 and susceptible to race 9 of Phytophthora megasperma f. sp. glycinea (Pmg). In detached primary leaves inoculated with zoospores, growth of race 1 was completely suppressed 16 h after inoculation, while race 9 was unaffected. The amount of the phytoalexin glyceollin that accumulated, however, was not significantly different in either the incompatible or compatible interaction 16 h after inoculation. At the circumference of the inoculated area, a slight accumulation of phytoalexin was observed only in the incompatible interaction 20 h or more after inoculation. Tolerance of race 9 to the phytoalexin was significantly higher than that of race 1 when the phytoalexin was added to agar. Moreover, race 9 degraded glyceollin faster than race 1. On leaves inoculated at separate points with either race, the lesion associated with race 9 never colonized areas inoculated with race 1. These results suggest that factor(s) other than the accumulation of phytoalexin in soybean tissue might cause cessation of growth of Pmg.     

Accumulation of sorghum phytoalexins induced by Colletotrichum graminicola at the infection site

Microspectrophotometry was performed on intact, pigmented vesicle-like inclusions within living sorghum cells that were accumulating phytoalexins as a response to attempted fungal infection. The results indicate that the deoxyanthocyanidin phytoalexins are present in inclusions. Moreover, the phytoalexin concentration within a single inclusion, based on luteolinidin, was calculated to be 0·15 m. The amounts of luteolinidin and apigeninidin in cells involved in the phytoalexin response at individual infection sites were also determined. The data showed that luteolinidin accumulated to levels of 0·48–1·20 ng/cell whereas apigeninidin accumulated to levels of 0·24–0·91 ng/cell. The results of both analyses confirmed that at the infection site the deoxyanthocyanidins accumulate to levels in substantial excess of those required for inhibition of the fungus Colletotrichum graminicola.     

Effect of glucans from different races of Phytophthora infestans on defense reactions in potato tuber

It has been proposed that susceptibility of potato to Phytophthora infestans would be a consequence of suppression and /or delaying of defense reactions by a soluble glucan which is released by compatible races of the fungus. In this report, the reaction of potato tuber slices (Solanum tuberosum cv. Huinkul) infected with either race I (1,4,7,8,10,11) or C (1,4,10,11) of Phytophthora infestans was studied. Race C grew better on slices than race I. Glucans from both races were isolated and their effect on the accumulation of phytoalexins and glucanases in tuber slices was studied. The glucans from the less virulent race (I) did not affect the accumulation of phytoalexins and glucanases in tuber slices infected or elicited with eicosapentaenoic acid, whereas the glucans from race C produced 70% inhibition of phytoalexin accumulation and reduced by 50% the induction of glucanase activities. Purified glucanases from potato degraded the glucans from race C but not from race I. The results reported here show that, at least on this cultivar, glucans from both races affected defense responses in a different manner, which could reflect structural differences between these glucans.     

Accumulation of phytoalexins in susceptible and resistant near-isogenic lines of tomato infected with Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici

The time course of accumulation of two phytoalexins, the terpenoid rishitin and the polyacetylene cis-tetradeca-6-ene-1,3-diyne-5,8-diol, was determined in near-isogenic susceptible and resistant tomato lines inoculated with either Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici.Cultivars containing the Ve gene for verticillium wilt resistance accumulated phytoalexins at a rate similar to that in susceptible plants following stem inoculation with V. albo-atrum. Higher amounts of phytoalexins were isolated from susceptible than from resistant plants at 11 days after inoculation. Inoculum concentrations of 10⁵, 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ had no differential effect on phytoalexin accumulation at 3 days after inoculation. Also, no differences were observed between fungal growth in susceptible and resistant cultivars during that period.A cultivar containing the I-1 gene for fusarium wilt resistance contained more rishitin than did susceptible plants at 2 and 3 days after inoculation with 10⁷ conidia of F. oxysporum f.sp. lycopersici ml⁻¹, but at 7 and 11 days after inoculation more rishitin had accumulated in the susceptible plants.No difference was observed between the rate of accumulation of phytoalexin in stem segments from resistant and susceptible plants inoculated by vacuum-infiltration.To estimate the concentration of phytoalexins in the xylem fluid, sap was expressed from vascular tissue and amounts of phytoalexins were determined in the sap and in the expressed tissue. Less than 5% of the phytoalexins present in stem segments was recovered from the sap, indicating that their concentration in the xylem fluid may be relatively low.The role of phytoalexins in resistance to verticillium and fusarium wilt is discussed.     

<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylsphingosine,an inhibitor of sphingosine kinase,induces phytoalexin production and hypersensitive cell death of Solanaceae plants without generation of reactive oxygen species

Plant recognition of elicitors derived from pathogens induces various resistant reactions, including production of reactive oxygen species, hypersensitive cell death and accumulation of phytoalexins. Previously, we isolated a ceramide elicitor from Phytophthora infestans, which activates O₂ ⁻ production of potato suspension-cultured cells. In this study, we employed nine ceramide-related chemicals to test their elicitor activity. Although, none of the tested chemicals induced O₂ ⁻ production, N,N-dimethylsphingosine (DMS) induced accumulation of phytoalexin in potato tubers. In potato, tobacco and Nicotiana benthamiana, DMS also induced rapid cell death. DMS-treated potato cells stained with 4′,6-diamidino-2-phenylindole (DAPI) showed chromatin condensation, and isolated DNA from DMS-treated cells had ladder pattern, confirming that DMS-induced plant cell death is a hypersensitive reaction-like programmed cell death. Involvement of ceramide signaling in induction of plant defense reactions is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Induction of systemic resistance to Agrobacterium tumefaciens by endophytic bacteria in grapevine

Crown gall disease of grapevine, caused by Agrobacterium tumefaciens, often results in severe economic loss to grape production worldwide. This study demonstrated the ability of the endophytic bacteria Pseudomonas sp. Sn48 and Pantoea sp. Sa14 isolated from domesticated and wild grapevines to induce resistance in both above- and belowground tissues of grapevines infected with A. tumefaciens. Our results provide evidence that both strains can colonize roots and/or shoots. We showed that the strains Pseudomonas sp. Sn48 and Pantoea sp. Sa14 are capable of inducing stilbenic phytoalexin production in grapevine tissues and to further prime plantlets for enhanced phytoalexin production after A. tumefaciens inoculation. We also showed that in the majority of treatments, polyamine accumulation remained unchanged or slightly increased in plantlets treated with Pseudomonas sp. Sn48 and Pantoea sp. Sa14 compared with the control. Our findings indicated that the levels of polyamines remain unchanged or significantly decrease in plantlets treated with endophytic bacteria after A. tumefaciens challenge compared to the control and plantlets treated with individual endophytic bacterial strains. PR1, PR2, and PR4 gene expression levels of plantlets treated with Pseudomonas sp. Sn48 and Pantoea sp. Sa14 significantly increased after A. tumefaciens inoculation. The findings revealed the efficacy of the selected endophytic bacteria in triggering grapevine resistance against A. tumefaciens and the possible use of these strains as an alternative to chemical control methods in grapevine crown gall disease management.     

水杨酸诱导水稻幼苗抗瘟性的生化机制

 以0.01mM水杨酸(SA)喷雾处理或稻瘟菌孢子悬浮液接种稻苗后,叶片中苯丙氨酸解氨酶(PAL)和定位于细胞不同部位的过氧化物酶(POD),特别是与细胞壁结合的POD和细胞间隙POD的活性迅速升高,但没有明显的新POD同功酶带出现。同时叶片中木质素含量迅速增加。在SA处理后的稻叶中提取到能抑制稻瘟菌分生孢子萌发、含有Momilactone A的抑菌物质,并检测到分子量分别为48、52、59、106.5KDa的碱性病程相关蛋白(PRs)。这些生化指标的时序变化与SA诱导抗瘟性的表现相吻合。对SA诱导水稻幼苗抗瘟性的可能生化机理作了归纳。     

Fungal polygalacturonase activity reflects susceptibility of carnation cultivars to fusarium wilt

Carnation cultivars with different levels of partial resistance were inoculated with race 2 of Fusarium oxysporum f.sp. dianthi and monitored for accumulation of host phytoalexins, fungal escape from compartmentalization, production of fungal pectin-degrading enzymes and development of external disease symptoms. Accumulation of phytoalexins, assessed after 10 days in the first 5 cm above the inoculation site, was weakly (methoxydianthramide S) or not (hydroxydianthalexin B) correlated with resistance levels after 12 weeks. Fungal escape from compartmentalization, assessed after 3 weeks as percentages colonized plants at 8 cm above the inoculation site, was highly correlated with expression of susceptibility after 12 weeks. Polygalacturonase (PG) activity, assessed after 4 weeks in the first 5 cm above the inoculation site, was highly correlated to final disease development. Linear increases in disease severity were accompanied by quadratic increases in PG activity. In contrast to water-treated plants, that lacked any PG activity, inoculated plants contained two main groups of fungal PGs, the dominant forms of which had estimated pI values of 7.0 and minimally 9.5, respectively. Compared to those of the first group, enzymes of the second group were produced only in trace amounts in liquid media containing pectin or polygalacturonate as sole source of carbon. On these media, the fungus also produced a pectin methyl esterase (PME) with an estimated pI of 9.3. Besides PMEs of host origin, inoculated plants of susceptible cultivars contained the fungal PME while no more than traces were found in resistant ones.Assessment of phytoalexin production by the host during defense responses cannot replace monitoring of external symptoms as a resistance test. Assessment of fungal growth, whether by reisolations above the compartmentalization area or by measurement of PG activity, provides a both rapid and reliable prediction of disease development.     

Phytoalexin accumulation during infection of bean and soybean by ascospores and mycelium of Sclerotinia sclerotiorum

Infection by ascospores of Sclerotinia selerotiorum caused hypersensitivity in epidermal cells in leaves and etiolated hypocotyls of bean and soybean. In bean, phaseollin and phaseollidin accumulated in leaves but kievitone alone in hypocotyls. In soybean, no phytoalexins were detected in leaves but glyceollin accumulated in hypocotyls.
Mycelial infection caused water-soaked spreading lesions in leaves and etiolated hypocotyls of both hosts. In bean, no phytoalexins were detected in leaves but kievitone alone accumulated in hypocotyls. In soybean, glyceollin accumulated in leaves but was not sought in hypocotyls.
Transfer of bean hypocotyls infected with mycelium from 18 to 28°C caused lesion limitation and marked accumulation of phaseollin and kievitone.
Phaseollin, kievitone and glyceollin inhibited ascospore germination and growth of hyphae from preformed germ-tubes and established mycelia, phaseollin being most active and glyceollin least active. Hyphal growth from mycelia was least affected by the phytoalexins.     

Signal Mediators for Phytoalexin Production in Defense Response of Oats Elicited by Victorin as a Specific Elicitor

Signals mediating phytoalexin (PA) production were analyzed in primary leaves of oats cv. Iowa X469 treated with an elicitor victorin. Production of the PA avenanthramide A was inhibited by DPI, an inhibitor of NADPH oxidase/nitric oxide synthase (NOS) and the NOS inhibitors l-NMMA and 1,3-PBIT. However, catalase and superoxide dismutase (SOD) hardly suppressed it. From the data, NO functions as a major reactive oxygen species in signal transduction leading to PA production in the defense response of oats. EGTA, verapamil and ruthenium red inhibited PA production, suggesting that Ca²⁺ influx into the cytoplasm and intracellular Ca²⁺ movement are involved in the defense response. Trifluoperazine, a calmodulin function inhibitor, and K-252a, a serine/threonine kinase inhibitor, also suppressed the accumulation, whereas okadaic acid, a serine/threonine phosphatase inhibitor, did not suppress it, suggesting the involvement of calmodulin and protein kinase, but not of phosphatase in PA production. Received 24 December 1999/ Accepted in revised form 4 February 2000