Induction of systemic resistance to Pythium damping-off in cucumber plants by benzothiadiazole: ultrastructure and cytochemistry of the host response

Benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH, CGA 245704), a non-toxic, synthetic chemical, was applied as a foliar spray to cucumber plants and evaluated for its potential to induce defense mechanisms in root tissues infected by the soilborne pathogen, Pythium ultimum Trow. In non-treated cucumber plants, fungal colonization was intense and paralleled marked host tissue damage, whereas in BTH-treated plants, pathogen ingress towards the vascular stele was apparently halted by the massive deposition of a phenolic-enriched material which occluded a large number of cortical and vascular parenchyma cells. This considerable increase in the accumulation of phenolics was accompanied by cytological disorders of the invading pathogen at a time when the wall-bound cellulose component was preserved. In addition to phenolic compounds, the occluding material contained large amounts of beta-glucoside residues. These residues gradually decreased in the areas neighboring fungal cells whereas phenolic deposition appeared to be more uniformly distributed throughout the occluded host cells. Pathogen penetration in non-occluded cucumber root cells coincided with other changes, mainly characterized by both the deposition onto the inner surface of the cell walls of some heterogeneous wall appositions and the coating of some intercellular spaces with an electron-opaque material. Evidence is provided in this study that BTH has the ability to induce SAR in cucumber. Exogenous, foliar applications of the chemical sensitize susceptible cucumber plants to react more rapidly and more efficiently to P. ultimum attack, mainly through the massive accumulation of phenolic compounds at sites of attempted pathogen penetration.     

Ultrastructural Changes Caused by Fusarium oxysporum f. sp. lycopersici in Meloidogyne javanica Induced Giant Cells in Fusarium Resistant and Susceptible Tomato Cultivars

Tomato (Lycopersicon esculentum Mill.) seedlings, susceptible (cv. Pearson A-I Improved) and resistant (cv. Pearson Improved) to race 1 Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyd &Hans., were inoculated with Meloidogyne javanica (Trueb) Chitwood second-stage juveniles and 3 weeks later with race 1 F. oxysporum f. sp. lycopersici spores. One week after fungal inoculation, no fungus was visible in root tissue of the tomato cultivars and the giant cells were normal. Two weeks after fungal inoculation, abundant hyphae were visible in xylem tissues of Fusarium-susceptible but not of Fusarium-resistant plants. In susceptible plants, giant cell degeneration occurred, characterized by membrane and organelle disruption. In addition, where hyphae were in direct contact with the giant cell, dissolution of the giant cell wall occurred. Three weeks after fungal inoculation, fungal hyphae and spores were visible inside xylem tissues and giant cells in Fusarium-susceptible plants and in xylem tissue of the resistant plants. In susceptible and resistant plants, giant cell degeneration was apparent. Giant cell walls were completely broken down in Fusarium-susceptible tomato plants. In both cultivars infected by Fusarium, giant cell nuclei became spherical and dark inclusions occurred within the chromatin material which condensed adjacent to the fragmented nuclear membrane. No such ultrastructural changes were seen in the giant cells of control plants inoculated with nematode alone. Giant cell deterioration in both cultivars is probably caused by toxic fungal metabolites.     

Cell wall synthesis in cotton roots after infection with Fusarium oxysporum

Fusarium oxysporum f. sp. vasinfectum penetration hyphae infect living cells in the meristematic zone of cotton (Gossypium barbadense L.) roots. We characterized wall modifications induced by the fungus during infection of the protodermis using antibodies against callose, arabinogalactan-proteins, xyloglucan, pectin, polygalacturonic acid and rhamnogalacturonan I in high-pressure frozen, freeze-substituted root tissue. Using quantitative immunogold labelling we compared the cell walls before and after hyphal contact, cell plates with plasmodesmata during cytokinesis, and wall appositions induced by fungal contact. In the already-existing wall, fungal contact induced only minor modifications such as an increase of xyloglucan epitopes. Wall appositions mostly exhibited epitopes similar to the cell plate except that wall appositions had a much higher callose content. This study shows that wall appositions induced by Fusarium oxysporum hyphae are the result of normal cell wall synthesis and the addition of large amounts of callose. The appositions do not stop fungal growth.     

Oligandrin. A proteinaceous molecule produced by the mycoparasite Pythium oligandrum induces resistance to Phytophthora parasitica infection in tomato plants

A low-molecular weight protein, termed oligandrin, was purified to homogeneity from the culture filtrate of the mycoparasitic fungus Pythium oligandrum. When applied to decapitated tomato (Lycopersicon esculentum Mill. var. Prisca) plants, this protein displayed the ability to induce plant defense reactions that contributed to restrict stem cell invasion by the pathogenic fungus Phytophthora parasitica. According to its N-terminal sequence, low-molecular weight, acidic isoelectric point, ultraviolet spectrum, and migration profile, the P. oligandrum-produced oligandrin was found to share some similarities with several elicitins from other Phytophthora spp. and Pythium spp. However, oligandrin did not induce hypersensitive reactions. A significant decrease in disease incidence was monitored in oligandrin-treated plants as compared with water-treated plants. Ultrastructural investigations of the infected tomato stem tissues from non-treated plants showed a rapid colonization of all tissues associated with a marked host cell disorganization. In stems from oligandrin-treated plants, restriction of fungal growth to the outermost tissues and decrease in pathogen viability were the main features of the host-pathogen interaction. Invading fungal cells were markedly damaged at a time when the cellulose component of their cell walls was quite well preserved. Host reactions included the plugging of intercellular spaces as well as the occasional formation of wall appositions at sites of potential pathogen entry. In addition, pathogen ingress in the epidermis was associated with the deposition of an electron-opaque material in most invaded intercellular spaces. This material, lining the primary walls, usually extended toward the inside to form deposits that frequently interacted with the wall of invading hyphae. In the absence of fungal challenge, host reactions were not detected.     

Irregular growth forms and cell wall modifications,polygalacturonase detection,and endocell formation in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">radicis-lycopersici</Emphasis> infecting tomato plants,as studied ultrastructurally and cytochemically

Pathogen cells of Fusarium oxysporum f.sp. radicis-lycopersici infecting container-grown tomato plants were characterized ultrastructurally, using gold-complexed probes, chitinase and wheat germ agglutinin to localize chitin, and polyclonal antibodies to a polygalacturonase to localize this enzyme. It was isolated and purified from the pathogen growing in culture. Many fungal cells were of irregular forms (microhyphal, frondose) with modified, thin or imperceptible lucent wall layers, in which were often included components seemingly of host origin. Gold particles of the polygalacturonase probe were concentrated on portions of penetration hyphae and in areas of associated altered host wall. Fine filamentous-like structures, often linked to fungal cells, reached into extracellular matter and into host walls. Examination of 0.2–0.25 μm-thick sections at 120 kV, and tilted at various angles, indicated that fungal cells frequently had a pronounced wavy contour. Labelling of thin walls for chitin was mostly nil, particularly in contact with host walls, as of also thicker walls in similar situations, or it was then associated with the outside opaque layer. Cells of diverse dimensions with thin or thicker walls and with altered or normal content, contained endocells. Walls of the encodcells and of the enclosing cells often labelled differently for chitin with both probes. Endocells mostly did not originate from proliferation of a living into a dead cell but often ensuing as an apparent fragmentation of the cell content or following its retraction. The bearing of these observations on the host-pathogen relationship, particularly concerning the role of thin-walled hyphae and irregular forms, is discussed.     

Ultrastructural and cytochemical characterization of elicitor-induced structural responses in tomato root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici

Commercial chitosan and laminarin, as well as -glucans, isolated from either Phytophthora megasperma f.sp. glycinea or Saccharomyces cerevisiae, were applied to decapitated tomato (Lycopersicon esculentum Mill.) plants and evaluated for their potential to induce defense mechanisms in root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici. A significant decrease in disease incidence was monitored in elicitor-treated plants as compared to water-treated plants. No difference was detected in the capacity of the elicitors under study to confer enhanced protection against pathogen attack. Ultrastructural investigations of the infected root tissues from watertreated (control) plants showed a rapid colonization of all tissues including the vascular stele. Fungal ingress was lways associated with marked host cell disorganization and cell wall alteration. In root tissues from elicitortreated plants, restriction of fungal growth to the epidermis and the outer cortex, decrease in pathogen viability, and formation of numerous wall appositions at sites of attempted penetration were the main features of the hostpathogen interaction. The wall appositions were found to vary greatly in their appearance from multi-textured to multi-layered structures, from elongated deposits to hemispherical protuberances. Application of various goldcomplexed probes to root tissue sections revealed that callose, pectin and phenolic-like compounds (likely lignin) were the main components of the newly-formed barriers. By contrast, cellulose appeared confined to outer or intermediate layers resembling the host cell wall in terms of structure and architecture. In the absence of fungal challenge, the cytologically visible consequences of elicitation were restricted to a discrete deposition of electron-opaque substances in the vacuoles of some cells, and wall appositions were not detected. The key importance of fungal challenge in the elaboration of defense mechanisms is discussed in relation to the possibility that an alarm ignal provided by the pathogen itself is required for the expression of resistance in plants previously sensitized by an exogenous elicitor.Abbreviations AGL Aplysia gonad lectin - FORL Fusariumoxysporum f.sp. radicis-lycopersici The authors wish to thank Sylvain Noël for excellent technical assistance and Drs. J.P. Geiger and Michel Nicole (ORSTOM, Montpellier, France) for providing the purified laccase. This work was supported by a grant from the FCAR-CQVB (Fonds Québécois pour la Formation de Chercheurs et l'Aide à la Recherche and Centre Québécois de Valorisation de la Biomasse) and by a contract from the Company Tourbières Premier Ltée, Rivière-du-Loup, Québec.     

Ultrastructure at the Host-Parasite Interface of Phytophthora capsici in Roots and Stems of Capsicum annuum

The infecting hyphae of Phytophthora capsici grew intercellularly in infected tissues of roots and stems of pepper (Capsicum annuum). The vascular tissues were not markedly disorganized even when heavily infected. Intercellularly growing hyphae penetrated the host cells by forming haustorium-like bodies. The consistent features of ultrastructural changes in infected tissues of pepper roots and stems were degeneration of cell organelles and dissolution of host cell walls. The cytoplasm detached from the cell wall aggregated abundantly around some haustorium-like bodies or the penetration sites of fungal hyphae. The host cell walls were palely stained, thinned and swollen, possibly being biochemically altered by the action of fungal macerating enzymes. Electron-dense, wall-like material was apposed on the outer wall of xylem vessel contacted by fungal hyphae. The infecting hyphae were also surrounded by granular, dark-staining cytoplasm. Characteristics of host cell responses to the invading P. capsici were the deposition of papilla-like material on host cell walls next to hyphae and the encasement of haustorium-like bodies with wall appositions.     

Accumulation of beta-Fructosidase in the Cell Walls of Tomato Roots following Infection by a Fungal Wilt Pathogen

Active defense in plants is associated with marked metabolic alterations, but little is known about the exact role of the reported changes in specific activity of several enzymes in infected plant tissues. β-Fructosidase (invertase), the enzyme that converts sucrose into glucose and fructose, increases upon infection by fungi and bacteria. To understand the relationship between fungal growth and β-fructosidase accumulation, we used an antiserum raised against a purified deglycosylated carrot cell wall β-fructosidase to study by immunogold labeling the spatial and temporal distribution of the enzyme in susceptible and resistant tomato (Lycopersicon esculentum) root tissues infected with the necrotrophic fungus, Fusarium oxysporum f. sp. racidis-lycopersici. In susceptible plants, the enzyme started to accumulate in host cell walls about 72 hours after inoculation. Accumulation occurred only in colonized cells and was mainly restricted to areas where the walls of both partners contacted each other. In resistant plants, accumulation of β-fructosidase was noticeable as soon as 48 hours after inoculation and appeared to reach an optimum by 72 hours after inoculation. Increase in wall-bound β-fructosidase was not restricted to infected cells but occurred also, to a large extent, in tissues that remained uncolonized during the infection process. The enzyme also accumulated in wall appositions (papillae) and intercellular spaces. This pattern of enzyme distribution suggests that induction of β-fructosidase upon fungal infection is part of the plant's defense response. The possible physiological role(s) of this enzyme in infected tomato plants is discussed in relation to the high demand in energy and carbon sources during pathogenesis.     

Antagonistic bacteria of composted agro-industrial residues exhibit antibiosis against soil-borne fungal plant pathogens and protection of tomato plants from Fusarium oxysporum f.sp. radicis-lycopersici

Rhizospheric and root-associated/endophytic (RAE) bacteria were isolated from tomato plants grown in three suppressive compost-based plant growth media derived from the olive mill, winery and Agaricus bisporus production agro-industries. Forty-four (35 rhizospheric and 9 RAE) out of 329 bacterial strains showed in vitro antagonistic activity against at least one of the soil-borne fungal pathogens, Fusarium oxysporum f.sp. radicis-lycopersici (FORL), F. oxysporum f.sp. raphani, Phytophthora cinnamomi, P. nicotianae and Rhizoctonia solani. The high percentage of total isolates showing antagonistic properties (13%) and their common chitinase and β-glucanase activities indicate that the cell wall constituents of yeasts and macrofungi that proliferate in these compost media may have become a substrate that favours the establishment of antagonistic bacteria to soil-borne fungal pathogens. The selected bacterial strains were further evaluated for their suppressiveness to tomato crown and root rot disease caused by FORL. A total of six rhizospheric isolates, related to known members of the genera Bacillus, Lysinibacillus, Enterobacter and Serratia and one RAE associated with Alcaligenes faecalis subsp. were selected, showing statistically significant decrease of plant disease incidence. Inhibitory effects of extracellular products of the most effective rhizospheric biocontrol agent, Enterobacter sp. AR1.22, but not of the RAE Alcaligenes sp. AE1.16 were observed on the growth pattern of FORL. Furthermore, application of cell-free culture extracts, produced by Enterobacter sp. AR1.22, to tomato roots led to plant protection against FORL, indicating a mode of biological control action through antibiosis.     

Anatomical study on the interaction between the root endophytic fungus <Emphasis Type="Italic">Heteroconium chaetospira</Emphasis> and Chinese cabbage

Chinese cabbage roots colonized by the dematiaceous fungal taxon Heteroconium chaetospira were previously found to become highly resistant to clubroot and Verticillium yellows. The dematiaceous fungus possesses an endophytic nature, but no detailed anatomical studies on endophyte–host plant interactions have so far been provided. Light and electron microscopy revealed that hyphae of H. chaetospira were abundant on and inside the root epidermal cells by 3 weeks following inoculation. The penetration pegs easily breached into epidermal cells, and the infection hyphae penetrated into cortical cells. Some appressorium-like swollen structures formed from intracellular hyphae, but no visible degradation of the host cell walls was evident where the hyphae contacted. No visible signs of host reactions and no invagination of the host plasma membrane around the hyphae were seen in the host cells. By 8 weeks following inoculation, masses of closely packed fungal cells had been formed in some cells of the epidermis and cortical layers, but further hyphal ingress was halted, mostly in the inner cortical cell layer. Thus, root vascular cylinders remained intact.     

Implication of Pectic Components in Cell Surface Interactions between Tomato Root Cells and Fusarium oxysporum f. sp. radicis-lycopersici: A Cytochemical Study by Means of a Lectin with Polygalacturonic Acid-Binding Specificity

Aplysia gonad lectin, a polygalacturonic acid-binding lectin isolated from the sea mollusc Aplysia depilans, was complexed to colloidal gold and used for localizing polygalacturonic-acid-containing molecules in tomato root tissues infected with Fusarium oxysporum f. sp. radicis-lycopersici (FORL). Colonization of host tissues by FORL was associated with striking wall modifications including disruption and even loss of middle lamellae. According to the labeling pattern observed in host wall areas adjacent to fungal penetration channels, it is likely that FORL pectolytic enzymes act through localized wall degradation. The release of polygalacturonic acid-rich wall fragments and the accumulation of polygalacturonic acid-containing molecules in some altered phloem cells were frequently observed and considered to be specific host reactions to fungal attack. The heavy deposition of such molecules at strategic sites such as wall oppositions and intercellular spaces provides support to their implication in the plant defense system. The possible interrelation between polygalacturonic acid-containing molecules and other polymers such as lignin and phenolic compounds remains to be investigated further. The role of these molecules in host-pathogen interactions is discussed in relation to plant defense.     

Induction of resistance against Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial strain: ultrastructure and cytochemistry of the host response

The potential of Bacillus pumilus (PGPR strain SE 34), either alone or in combination with chitosan, for inducing defense reactions in tomato (Lycopersicon esculentum Mill.) plants inoculated with the vascular fungus, Fusarium oxysporum f. sp. radicis-lycopersici, was studied by light and transmission electron microscopy and further investigated by gold cytochemistry. The key importance of fungal challenge in the elaboration of defense mechanisms is discussed in relation to the possibility that an alarm signal provided by the pathogen itself is required for the expression of resistance in plants previously sensitized by biotic agents. Ultrastructural investigations of the infected root tissues from water-treated (control) plants showed a rapid colonization of all tissues including the vascular stele. In root tissues from bacterized tomato plants grown in the absence of chitosan, the limited fungal development coincided with marked changes in the host physiology. The main facets of the altered host metabolism concerned the induction of a structural response at sites of fungal entry and the abnormal accumulation of electron-dense substances in the colonized areas. A substantial increase in the extent and magnitude of the cellular changes induced by B. pumilus was observed when chitosan was supplied to bacterized tomato plants. These changes were characterized by a considerable enlargement of the callose-enriched wall appositions deposited onto the inner cell wall surface in the epidermis and the outer cortex. The use of the wheat germ agglutinin-ovomucoid-gold complex provided evidence that the wall-bound chitin component in Fusarium cells colonizing bacterized tomato roots was not substantially altered. One of the most-typical fungal cell reactions, observed only when bacterized tomato plants were grown in the presence of chitosan, was the formation of abnormal chitin-enriched deposits between the retracted plasma membrane and the cell wall. Results of the present study provide the first evidence that combination of biocontrol approaches is a promising step towards elaborating integrated pest management programmes. Received: 6 June 1997 / Accepted: 8 July 1997     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

Immunolocalization of 1,3‐β‐Glucanases Secreted by Gaeumannomyces graminis var. tritici in Infected Wheat Roots

The distribution of extracellular 1,3‐β‐glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3‐β‐glucanase secreted by the pathogenic fungus. A specific antibody of 1,3‐β‐glucanase, anti‐GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3‐β‐glucanase of Ggt, but not for 1,3‐β‐glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3‐β‐glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen‐infected wheat roots with anti‐1,3‐β‐glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non‐inoculated wheat roots. The results indicate that 1,3‐β‐glucanase secreted by Ggt may be involved in pathogenesis of the take‐all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers.     

Role of ethylene in the protection of tomato plants against soil-borne fungal pathogens conferred by an endophytic Fusarium solani strain

An endophytic fungal isolate (Fs-K), identified as a Fusarium solani strain, was obtained from root tissues of tomato plants grown on a compost which suppressed soil and foliar pathogens. Strain Fs-K was able to colonize root tissues and subsequently protect plants against the root pathogen Fusarium oxysporum f.sp. radicis-lycopersici (FORL), and elicit induced systemic resistance against the tomato foliar pathogen Septoria lycopersici. Interestingly, attenuated expression of certain pathogenesis-related genes, i.e. PR5 and PR7, was detected in tomato roots inoculated with strain Fs-K compared with non-inoculated plants. The expression pattern of PR genes was either not affected or aberrant in leaves. A genetic approach, using mutant tomato plant lines, was used to determine the role of ethylene and jasmonic acid in the plant's response to infection by the soil-borne pathogen F. oxysporum f.sp. radicis-lycopersici (FORL), in the presence or absence of isolate Fs-K. Mutant tomato lines Never ripe (Nr) and epinastic (epi1), both impaired in ethylene-mediated plant responses, inoculated with FORL are not protected by isolate Fs-K, indicating that the ethylene signalling pathway is required for the mode of action used by the endophyte to confer resistance. On the contrary, def1 mutants, affected in jasmonate biosynthesis, show reduced susceptibility to FORL, in the presence Fs-K, which suggests that jasmonic acid is not essential for the mediation of biocontrol activity of isolate Fs-K.     

Chitosan Treatment: An Emerging Strategy for Enhancing Resistance of Greenhouse Tomato Plants to Infection by Fusarium oxysporum f.sp. radicis-lycopersici

The potential of chitosan, a non-toxic and biodegradable polymer of beta -1,4-glucosamine, for controlling fusarium crown and root rot of greenhouse-grown tomato caused by Fusarium oxysporum f.sp. radicis-lycopersici (FORL) was investigated. The amendment of plant growth substratum with chitosan at concentrations of 12.5 or 37.5 mg l-1 significantly reduced plant mortality, root rot symptoms and yield loss attributed to FORL. Maximum disease control was achieved with chitosan at 37.5 mg l-1, when plant mortality was reduced by more than 90% and fruit yield was comparable with that of non-infected plants. In the absence of FORL, chitosan did not adversely affect plant growth and fruit yield. Cytological observations on root samples from FORL-inoculated plants revealed that the beneficial effect of chitosan in reducing disease was associated with increased plant resistance to fungal colonization. In chitosan-treated plants, fungal growth was restricted to the epidermis and the cortex. Invading hyphae showed marked cellular disorganization, characterized by increased vacuolation and even complete loss of the protoplast. The main host reactions included the formation of structural barriers at sites of attempted fungal penetration, the deposition of an opaque material (probably enriched with phenolics according to its electron density) in intercellular spaces and the occlusion of xylem vessels with tyloses, polymorphic bubbles and osmiophilic substances. Although chitosan may also have antifungal properties, the ultrastructural observations provide evidence that chitosan sensitizes tomato plants to respond more rapidly and efficiently to FORL attack. Chitosan has the potential to become a useful agent for controlling greenhouse diseases caused by soil-borne pathogens.     

Pre-inoculation of Ri T-DNA-transformed pea roots with Pseudomonas fluorescens inhibits colonization by Pythium ultimum Trow: an ultrastructural and cytochemical study

The influence exerted by Pseudomonas fluorescens, strain 63-28R, in stimulating plant defense reactions was investigated using an in-vitro system in which Ri T-DNA-transformed pea (Pisum sativum L.) roots were subsequently infected with Pythium ultimum. Cytological investigations of samples from P. fluorescens-inoculated roots revealed that the bacteria multiplied abundantly at the root surface and colonized a small number of epidermal and cortical cells. Penetration of the epidermis occurred through the openings made by the disruption of the fibrillar network at the junction of adjacent epidermal cell walls. Direct cell wall penetration was never observed and bacterial ingress into the root tissues proceeded via an intercellular route. Striking differences in the extent of fungal colonization were observed between bacterized and non-bacterized pea roots following inoculation with P. ultimum. In non-bacterized roots, the pathogen multiplied abundantly through most of the tissues while in bacterized roots, pathogen growth was restricted to the epidermis and the outer cortex. At the root surface, the bacteria interacted with the pathogen, in a way similar to that observed in dual culture tests. Most Pythium cells were severely damaged but fungal penetration by the bacteria was never observed. Droplets of the amorphous material formed upon interaction between the bacteria and the host root were frequently found at the fungal cell surface. Incubation of sections with a -1,4-exoglucanase-gold complex revealed that the cell wall of markedly altered Pythium hyphae was structurally preserved. Successful penetration of the root epidermis was achieved by the few hyphae of P. ultimum that could escape the first defensive line in the rhizosphere. Most hyphae of the pathogen that penetrated the epidermis exhibited considerable changes. The unusual occurrence of polymorphic wall appositions along the host epidermal cells was an indication that the host plant was signalled to defend itself through the elaboration of physical barriers.Abbreviations AGL Aplysia gonad lectin - PGPR plant growth-promoting rhizobacteria The authors wish to thank Sylvain Noël for excellent technical assistance. This study was supported by grants from the Fonds Québécois pour la formation de chercheurs et l'Aide à la Recherche (FCAR), the Natural Sciences and Engineering Council of Canada (NSERC) and the Ministère de l'Industrie, du Commerce, de la Science et de la Technologie (SYNERGIE).     

Agrobacterium-Mediated Transformation of Tomato with rolB Gene Results in Enhancement of Fruit Quality and Foliar Resistance against Fungal Pathogens

Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.     

Cytology of infection of 35S-bean chitinase transgenic canola plants by Rhizoctonia solani: cytochemical aspects of chitin breakdown in vivo

Transgenic canola plants containing high, constitutive levels of bean endochitinase have been shown to be more resistant to infection by the soil-borne pathogen, Rhizoctonia solani, than are wild-type plants that lack the chimeric chitinase gene. To determine whether the resistance of the 35S-chitinase plants to Rhizoctonia infection results from an antimicrobial activity of the bean chitinase in planta, an ultrastructural and cyto-chemical study was performed on infected control and transgenic canola plants. Analysis of root tissues of infected wild-type canola plants revealed that R. solani was capable of extensive tissue colonization including the xylem vessels. Pathogen ingress towards the vascular system was associated with marked host cell wall alterations such as disruption of middle lamella matrices that occurred in advance of fungal penetration. Fungal hyphae colonizing these tissues appeared metabolically active as judged by their typical morphological features and their extensive multiplication. In infected transgenic plants, however, the pattern of fungal colonization was different to that observed in wild-type plants. Penetration of the host cuticle and epidermis was frequently observed, but fungal colonization was usually restricted to the cortex although, in a few cases, some fungal cells could be seen in xylem vessels. In all samples examined, severe hyphal alterations ranging from increased vacuolization to cell lysis were seen. Hyphae occasionally seen in xylem vessels were markedly damaged and often reduced to convoluted wall fragments. Cytochemical labeling of chitin using the WGA/ovomucoid-gold complex showed that hyphal alterations correlated with extensive chitin degradation. Thus, reduction in fungal biomass, increase in hyphal alterations leading to fungal lysis and chitin breakdown appear to be typical features observed in transgenic canola plants. Because these features were not seen in infected wild-type plants, it is likely that constitutive expression of the bean endochitinase gene is, at least in part, responsible for the enhanced protection against fungal attack observed in these plants. It is not known, however, if other components of the host defense response contribute to the resistance phenotype.