Richard R. Bélanger

Cytological Evidence of an Active Role of Silicon in Wheat Resistance to Powdery Mildew (Blumeria graminis f. sp. tritici).

ABSTRACT Silicon (Si) amendments in the form of exogenously supplied nutrient solution or calcium silicate slag protect wheat plants from powdery mildew disease caused by the fungus Blumeria graminis f. sp. tritici. The most striking difference between Si- and Si+ plants challenged with B. graminis f. sp. tritici was the extent of epidermal cell infection and colonization by B. graminis f. sp. tritici. Histological and ultrastructural analyses revealed that epidermal cells of Si+ plants reacted to B. graminis f. sp. tritici attack with specific defense reactions including papilla formation, production of callose, and release of electron-dense osmiophilic material identified by cytochemical labeling as glycosilated phenolics. Phenolic material not only accumulated along the cell wall but also was associated with altered integrity of haustoria in a manner similar to localized phytoalexins as reported from other pathosystems. These results strongly suggest that Si mediates active localized cell defenses against B. graminis f. sp. tritici attack.

Silicon Enhances the Accumulation of Diterpenoid Phytoalexins in Rice: A Potential Mechanism for Blast Resistance

ABSTRACT Although several reports underscore the importance of silicon (Si) in controlling Magnaporthe grisea on rice, no study has associated this beneficial effect with specific mechanisms of host defense responses against this fungal attack. In this study, however, we provide evidence that higher levels of momilactone phytoalexins were found in leaf extracts from plants inoculated with M. grisea and amended with silicon (Si(+)) than in leaf extracts from inoculated plants not amended with silicon (Si(-) ) or noninoculated Si(+) and Si(-) plants. On this basis, the more efficient stimulation of the terpenoid pathway in Si(+) plants and, consequently, the increase in the levels of momilactones appears to be a factor contributing to enhanced rice resistance to blast. This may explain the lower level of blast severity observed on leaves of Si(+) plants at 96 h after inoculation with M. grisea. The results of this study strongly suggest that Si plays an active role in the resistance of rice to blast rather than the formation of a physical barrier to penetration by M. grisea.

The protective role of silicon in the Arabidopsis–powdery mildew pathosystem

The role and essentiality of silicon (Si) in plant biology have been debated for >150 years despite numerous reports describing its beneficial properties. To obtain unique insights regarding the effect of Si on plants, we performed a complete transcriptome analysis of both control and powdery mildew-stressed Arabidopsis plants, with or without Si application, using a 44K microarray. Surprisingly, the expression of all but two genes was unaffected by Si in control plants, a result contradicting reports of a possible direct effect of Si as a fertilizer. In contrast, inoculation of plants, treated or not with Si, altered the expression of a set of nearly 4,000 genes. After functional categorization, many of the up-regulated genes were defense-related, whereas a large proportion of down-regulated genes were involved in primary metabolism. Regulated defense genes included R genes, stress-related transcription factors, genes involved in signal transduction, the biosynthesis of stress hormones (SA, JA, ethylene), and the metabolism of reactive oxygen species. In inoculated plants treated with Si, the magnitude of down-regulation was attenuated by >25%, an indication of stress alleviation. Our results demonstrate that Si treatment had no effect on the metabolism of unstressed plants, suggesting a nonessential role for the element but that it has beneficial properties attributable to modulation of a more efficient response to pathogen stress.

Silicon induced resistance in cucumber plants against Pythium ultimum

Abstract The effect of the amendment of nutrient solutions with soluble potassium silicate on the response of cucumber (cv. Corona) root and hypocotyl tissues infected by Pythium ultimum was examined by light and electron microscopy, and by energy dispersive X-ray analysis (EDX). Plants were grown in 0 or 1·7 m m Si-amended nutrient solutions, and root and hypocotyl samples were collected at different times after inoculation with P. ultimum . By 48 h after infection, striking differences in the expression of defence reactions were observed between Si-amended and Si-free cucumber plants. Treatment of plants with Si markedly stimulated the accumulation of an electron-dense, phenolic-like material in infected host tissues, and significantly increased the percentage of cells filled with this material. Fungal hyphae colonizing occluded host cells were seriously damaged, and were often reduced to empty hyphal shells. Additionally, Si-treated cucumber plants responded to P. ultimum infection by forming electron-dense layers along primary and secondary cell walls, as well as over pit membranes of xylem vessels. EDX analysis failed to reveal the presence of silica deposits in P. ultimum -infected plants grown in Si-supplemented media. Our results suggest that a relationship exists between Si treatment, resistance to P. ultimum attack, and expression of plant defence mechanisms.

Evidence of Phytoalexins in Cucumber Leaves Infected with Powdery Mildew following Treatment with Leaf Extracts of Reynoutria sachalinensis

Phenolic compounds extracted from cucumber (Cucumis sativus L.) leaves were separated and analyzed for their differential presence and fungitoxicity in relation to a prophylactic treatment with Milsana (Compo, Munster, Germany) against powdery mildew (Sphaerotheca fuliginea). Based on our extraction and purification procedures, at least eight separate phenolic compounds with antifungal activity were identified as intrinsic components of cucumber plants. Of these compounds, six displayed a significant increase in concentration as a result of elicitation with Milsana, this being particularly evident when the plant was stressed by the pathogen. The combined amounts of these antifungal compounds in treated plants was nearly five times the level found in control plants. One week after Milsana application, some of the antifungal compounds obtained through hydrolysis of their glycosidic links were also detected in their free form, indicating that they are likely liberated from conjugated phenolics by enzymatic hydrolysis in planta. To our knowledge, these results provide the first direct evidence that cucumber plants produce elevated levels of phytoalexins in response to an eliciting treatment after infection.

Benzothiadiazole-Mediated Induced Resistance to Fusarium oxysporum f. sp. radicis-lycopersici in Tomato

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic chemical, was applied as a foliar spray to tomato (Lycopersicon esculentum) plants and evaluated for its potential to confer increased resistance against the soil-borne pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In nontreated tomato plants all root tissues were massively colonized by FORL hyphae. Pathogen ingress toward the vascular stele was accompanied by severe host cell alterations, including cell wall breakdown. In BTH-treated plants striking differences in the rate and extent of fungal colonization were observed. Pathogen growth was restricted to the epidermis and the outer cortex, and fungal ingress was apparently halted by the formation of callose-enriched wall appositions at sites of fungal penetration. In addition, aggregated deposits, which frequently established close contact with the invading hyphae, accumulated in densely colonized epidermal cells and filled most intercellular spaces. Upon incubation of sections with gold-complexed laccase for localization of phenolic-like compounds, a slight deposition of gold particles was observed over both the host cell walls and the wall appositions. Labeling was also detected over the walls of fungal cells showing signs of obvious alteration ranging from cytoplasm disorganization to protoplasm retraction. We provide evidence that foliar applications of BTH sensitize susceptible tomato plants to react more rapidly and more efficiently to FORL attack through the formation of protective layers at sites of potential fungal entry.

Ultrastructural and Cytochemical Aspects of Silicon-Mediated Rice Blast Resistance

ABSTRACT Although exogenous application of silicon (Si) confers efficient control of rice blast, the probable hypothesis underlying this phenomenon has been confined to that of a mechanical barrier resulting from Si polymerization in planta. However, in this study, we provide the first cytological evidence that Si-mediated resistance to Magnaporthe grisea in rice correlates with specific leaf cell reaction that interfered with the development of the fungus. Accumulation of an amorphous material that stained densely with toluidine blue and reacted positively to osmium tetroxide was a typical feature of cell reaction to infection by M. grisea in samples from Si+ plants. As a result, the extent of fungal colonization was markedly reduced in samples from Si+ plants. In samples from Si- plants, M. grisea grew actively and colonized all leaf tissues. Cytochemi-cal labeling of chitin revealed no difference in the pattern of chitin localization over fungal cell walls of either Si+ or Si- plants at 96 h after inoculation, indicating limited production of chitinases by the rice plant as a mechanism of defense response. On the other hand, the occurrence of empty fungal hyphae, surrounded or trapped in amorphous material, in samples from Si+ plants suggests that phenolic-like compounds or phytoalexins played a primary role in rice defense response against infection by M. grisea. This finding brings new insights into the complex role played by Si in the nature of rice blast resistance.

The effects of plant extracts of Reynoutria sachalinensis on powdery mildew development and leaf physiology of long English cucumber

An aqueous formulation of concentrated extracts (Milsana flussig) from leaves of the giant knotweed, Reynoutria sachalinensis, applied weekly at a concentration of 2%, provided control of powdery mildew (Sphaerotheca fuliginea) on long English cucumber that was as effective as benomyl. In two separate experiments, this treatment significantly reduced the severity of powdery mildew compared to control plants. Fruit yield was not affected by the treatment, even though repeated applications of Milsana induced a greener and glossier coloration of the leaves, which became brittle to the touch. A rapid and distinct accumulation of fungitoxic phenolic compounds occurred in leaves treated with Milsana, especially in infected leaves. A slight inhibition of conidial germination was the only direct effect of Milsana on S. fuliginea. These results support the hypothesis that Milsana may act indirectly by inducing plant defense reactions and that it may be useful in the integrated management of cucumber powdery mildew

Specificity and Mode of Action of the Antifungal Fatty Acid cis-9-Heptadecenoic Acid Produced by Pseudozyma flocculosa

ABSTRACT cis-9-Heptadecenoic acid (CHDA), an antifungal fatty acid produced by the biocontrol agent Pseudozyma flocculosa, was studied for its effects on growth and/or spore germination in fungi. Inhibition of growth and/or germination varied considerably and revealed CHDA sensitivity groups within tested fungi. Analysis of lipid composition in these fungi demonstrated that sensitivity was related primarily to a low intrinsic sterol content and that a high level of unsaturation of phospholipid fatty acids was not as involved as hypothesized previously. Our data indicate that CHDA does not act directly with membrane sterols, nor is it utilized or otherwise modified in fungi. A structural mechanism of CHDA, consistent with the other related antifungal fatty acids produced by P. flocculosa, is proposed in light of its activity and specificity. The probable molecular events implicated in the sensitivity of fungi to CHDA are (i) partitioning of CHDA into fungal membranes; (ii) a variable elevation in fluidity dependent on the buffering capability (sterol content) in fungi; and (iii) higher membrane disorder causing conformational changes in membrane proteins, increased membrane permeability and, eventually, cytoplasmic disintegration.

Methyl Ester ofp-Coumaric Acid: A Phytoalexin-Like Compound from Long English Cucumber Leaves

Methanolic extracts from long English cucumber leaves were fractionated and tested for their fungitoxicity by direct bioassay againstCladosporium cucumerinum on TLC plates. After separation by several preparative TLCs and flash chromatography and purification through reverse-phase HPLC, a crystalline compound possessing antifungal properties was isolated and identified asp-coumaric acid methyl ester. This antifungal compound was found to increase following infection and treatment with Milsana®, which could explain in part the prophylactic properties of the natural inducer. As such,p-coumaric acid methyl ester is the first identified compound to be associated with phytoalexin-like behavior in cucumber.