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).     

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     

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.     

Time-course study of the accumulation of hydroxyproline-rich glycoproteins in root cells of susceptible and resistant tomato plants infected byFusarium oxysporum f. sp.radicis-lycopersici

The accumulation of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants is thought to be involved in the defense response to pathogens. An antiserum raised against deglycosylated HRGPs from melon was used for studying the subcellular localization of these glycoproteins in susceptible and resistant tomato (Lycopersicon esculentum Mill.) root tissues infected by Fusarium oxysporum f.sp. radicis-lycopersici. A time-course of HRGP accumulation revealed that these glycoproteins increased earlier and to a higher extent in resistant than in susceptible cultivars. In the compatible interaction, increase in HRGPs was largely correlated with pathogen invasion and appeared to occur as a result of wall damage. In the incompatible interaction, HRGPs accumulated in the walls of uninvaded cells, thus indicating a possible role in the protection against fungal penetration. The occurrence of substantial amounts of HRGPs in papillae, known to be physical barriers formed in response to infection, and in intercellular spaces provides additional support to the concept that such glycoproteins play an important role in disease resistance.     

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.     

Interactive effects of selected pesticides on the two-spotted spider mite and its fungal pathogen Neozygites floridana

Benomyl affected populations of Tetranychus urticae by interfering with the pathogenic fungus, Neozygites floridana. Benomyl delayed but prolonged spider mite outbreaks. Few mites were infected with the pathogen when benomyl was used. Reductions in mite populations treated with fentin hydroxide were associated with a high incidence of N. floridana infection. Benomyl did not affect sporulation of N. floridana but appeared to inhibit conidial germination or growth of the fungus.

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Résumé Le bénomyl a modifié les populations de Tetranychus urticae Koch en interférant avec son champignon pathogène, Neozygites floridana (Weiser & Muma). Le bénomyl retardait mais prolongeait les pullulations de l'acarien. Peu d'acariens étaient infectés par le champignon quand on utilisait du bénomyl. Les réductions des populations d'acariens traitées avec l'hydroxyde de fentine étaient associées à un haut niveau d'infection par N. floridana. Le bénomyl ne modifiait pas la sporulation de N. floridana mais semblait inhiber la germination des conidies ou la croissance du champignon.

     

Colonisation patterns of root tissues byPhytophthora nicotianae var.parasitica related to reduced disease in mycorrhizal tomato

Tomato plants pre-colonised by the arbuscular mycorrhizal fungusGlomus mosseae showed decreased root damage by the pathogenPhytophthora nicotianae var.parasitica. In analyses of the cellular bases of their bioprotective effect, a prerequisite for cytological investigations of tissue interactions betweenG. mosseae andP. nicotianae v.parasitica was to discriminate between the hyphae of the two fungi within root tissues. We report the use of antibodies as useful tools, in the absence of an appropriate stain for distinguishing hyphae ofP. nicotianae v.parasitica from those ofG. mosseae inside roots, and present observations on the colonisation patterns by the pathogenic fungus alone or during interactions in mycorrhizal roots. Infection intensity of the pathogen, estimated using an immunoenzyme labelling technique on whole root fragments, was lower in mycorrhizal roots. Immunogold labelling ofP. nicotianae v.parasitica on cross-sections of infected tomato roots showed that inter or intracellular hyphae developed mainly in the cortex, and their presence induced necrosis of host cells, the wall and contents of which showed a strong autofluorescence in reaction to the pathogen. In dual fungal infections of tomato root systems, hyphae of the symbiont and the pathogen were in most cases in different root regions, but they could also be observed in the same root tissues. The number ofP. nicotianae v.parasitica hyphae growing in the root cortex was greatly reduced in mycorrhizal root systems, and in mycorrhizal tissues infected by the pathogen, arbuscule-containing cells surrounded by intercellularP. nicotianae v.parasitica hyphae did not necrose and only a weak autofluorescence was associated with the host cells. Results are discussed in relation to possible processes involved in the phenomenon of bioprotection in arbuscular mycorrhizal plants.     

The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype

Recessive mlo resistance alleles of the Mlo locus in barley control a non race-specific resistance response to infection by the obligate biotrophic fungus Erysiphe graminis f.sp. hordei. All the mlo alleles analysed stop fungal growth at the same developmental stage within a subcellularly restricted, highly localized cell wall apposition directly beneath the site of abortive fungal penetration. We report that near-isogenic lines carrying the alleles mlo ₁, mlo ₃ or mlo ₅ undergo dramatic spontaneous formation of cell wall appositions, not only in the absence of the fungal pathogen but also in sterile grown plants. A comparative study of spontaneous and infection-triggered cell wall appositions reveals a high degree of similarity with respect to structure, chemical composition and distinct localization within plant tissue. We show that the rate of spontaneous apposition formation is dependent on the genetic background of the plant and that its onset is under developmental control. Furthermore, spontaneous formation of wall appositions is specifically triggered by mlo alleles, since it is unaffected in the presence of the race-specific resistance allele Mlg. We propose a model for the function of the Mlo locus that suggests that both Mlo and mlo alleles control qualitatively the same apposition-based resistance mechanism, which, in the presence of the wild-type Mlo allele, is merely less efficient to provide protection against the currently common races of E. graminis f.sp. hordei.     

Fungus gnats vector Fusarium oxysporum f.sp. radicislycopersici1

Fungus gnat adults transported Fusarium oxysporum f.sp. radicis-lycopersici from Petri dish culture and infected host plants to the roots and hypocotyls of healthy tomato and bean plants. The source of the fungus did not affect the ability of fungus gnats to transport the fungus to healthy hosts. The presence of fungus gnat larvae in media in which young tomato plants were grown did not increase the incidence of plant infection by the pathogen. Fungus gnat adults appear to aid in the dissemination of F. oxysporum f.sp. radicis-lycopersici.     

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.     

Influence of substrate on the interaction ofGlomus intraradices andFusarium oxysporum f.sp.radicis-lycopersici on tomatoes

Summary The influence of five substrates on the interaction betweenGlomus intraradices andFusarium oxysporum f.sp.radicis-lycopersici and its effect on tomato plants development was investigated. The presence ofG. intraradices decreased root necrosis in all substrates and affected the Fusarium population with different intensity depending on the substrate used. Substrates were found to influence disease development, Fusarium population in the substrate, root colonization by the endomycorrhizal fungus and growth of the host plant. In addition to providing good experimental conditions, the use of calcined montmorillonite clay also facilitated washing, recuperation, necrosis evaluation and staining of roots. Its use is proposed as a standard medium for experimental work on the interactions between endomycorrhizal fungi, root pathogens and host plants.Contribution no J 981 of the Saint-Jean Research Station and no 268 of the Sainte-Roy Research Station     

Increased Growth and Yield of Hydroponically Grown Greenhouse Tomato Plants Inoculated with Arbuscular Mycorrhizal Fungi and Fusarium oxysporum f. sp. radicis-lycopersici

The arbuscular mycorrhizal fungi Glomus monosporum, G. vesiculiferum, G. deserticola, G. intraradices, G. mosseae, and two unidentified species were tested to determine their effect on plant growth and fruit production of tomato (Lycopersicon esculentum Mill.) cv. Trust inoculated with Fusarium oxysporum f. sp. radicis-lycopersici (FORL) under near-commercial greenhouse conditions. Inoculation with G. monosporum and G. mosseae significantly increased fruit yield and fruit number of tomato plants grown hydroponically in sawdust. Plant height and plant dry weight increased significantly when inoculated with G. monosporum and G. mosseae. Further, plants inoculated with G. monosporum and G. mosseae showed significantly lower FORL root infection than the untreated control plants.     

Chitinase-gold complex used to localize chitin ultrastructurally in tomato root cells infected by Fusarium oxysporum f. sp. radicis-lycopersici, compared with a chitin specific gold-conjugated lectin

Summary A cytochemical technique for the ultrastructural localization of chitin in tomato root cells infected byFusarium oxysporum f. sp.radicis-lycopersici is reported. Chitinase was complexed to colloidal gold and thin sections were incubated with the enzyme-gold complex. This technique yielded a more uniform distribution of gold particles over the fungus wall, compared to that obtained with the lectin-gold technique. Both techniques revealed no labelling of the fungus cytoplasm, except for organelles resembling Woronin bodies. No significant labelling of either healthy or infected root cells was seen except for the secondary walls of vessels and, occasionally, that of adjoining parenchyma cells. The importance of this technique in studying the development of the pathogen within host cells is discussed.     

Interaction of Fusarium Oxysporum f.sp. Cubense with Pseudomonas Fluorescens Precolonized to Banana Roots

Effect of precolonization of banana cv Neeypovan roots with Pseudomonas fluorescens on infection with Fusarium oxysporum f.sp. cubense was studied. Under in vitro conditions Pseudomonas fluorescens clearly inhibited Fusarium oxysporum f.sp. cubense. Fluorescein isothiocyanate-tagged antibodies raised in a rabbit system for Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense separately were used to study the spread of both organisms in banana root. It was observed that precolonization with Pseudomonas fluorescens could reduce Fusarium oxysporum f.sp. cubense colonization by 72%, and also correlated with a number of structural changes in the cortical cells, mainly with densely stained amorphous material and polymorphic wall thickenings as revealed by light and electron microscopic studies. Massive depositions of unusual structures at sites of fungal entry was also noticed, which clearly indicated that bacterized root cells were signalled to mobilize a number of defence structures for preventing the spread of pathogen in the tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microconidia germination of the tomato pathogen Fusarium oxysporum in the presence of root exudates

Abstract

In this study we assessed microconidia germination of the tomato pathogens F. oxysporum f. sp. lycopersici (Fol) and F. oxysporum f. sp. radicis-lycopersici (Forl) in the presence of root exudates. Tomato root exudates stimulated microconidia germination and the level of stimulation was affected by plant age. Treatment of root exudates with insoluble polyvinylpolypyrrolidone, which binds phenolic compounds, indicated that tomato root exudates contain phenolic compounds inhibitory to F. oxysporum microconidia germination. Our study indicates that tomato root exudates similarly stimulate microconidia germination of both Fol and Forl. However, individual F. oxysporum strains differ in the degree of germination response to the root exudates. Furthermore, root exudates from non-host plants also contain compounds that stimulate microconidia germination of Fol. In general, the effects of root exudates from non-host plants did not differ considerably from those of tomato. The ability of phenolic compounds to inhibit germination of Fol seems not to be plant-specific.     

Developmental Regulation of Susceptibility and Tolerance to Fusarium Root Rot in Beans

The soil-borne fungus, Fusarium solani f. sp. phaseoli, attacks roots and hypocotyls of bean (Phaseolus vulgaris) plants causing a devastating disease called root and foot rot. In a study of the host-pathogen relationship it was found that young bean roots, with the radicle just emerging, were highly tolerant to the pathogen, whereas older bean seedlings, with a fully developed root system, were completely susceptible. Investigations by low-temperature scanning electron microscopy demonstrated that significantly fewer spores and hyphae were present on the root surface of young bean seedlings as compared to older ones. A similar pattern of attachment was found when bean roots were inoculated with spores of F. solani f. sp. pisi, a related pathogen causing disease on peas but not on beans. Light microscopic studies showed that F. solani f. sp. pisi did not penetrate the root but rapidly formed thick-walled resting spores on the root surface. F. solani f. sp. phaseoli on the other hand quickly penetrated the root and formed an extensive network of fungal hyphae. These results demonstrate that the ability of fungal propagules to adhere to and to penetrate host tissues are two distinct processes. Furthermore, the data indicate that young bean roots lack a surface component necessary for attachment of fungal spores which may help explain their tolerance to Fusarium root rot.     

Structural characterization of the O-chain polysaccharide from an environmentally beneficial bacterium Pseudomonas chlororaphis subsp. aureofaciens strain M71

Pseudomonas chlororaphis subsp. aureofaciens strain M71 was isolated from the root of a tomato plant and it was able to control in vivo Fusarium oxysporum f. sp. radicis-lycopersici responsible for the tomato crown and root rot. Recently, strain M71 was evaluated even for its efficacy in controlling Seiridium cardinale, the causal agent of bark canker of common cypress (Cupressus sempervirens L.). Strain M71 ability to persist on the tomato rhizosphere and on the aerial part of cypress plants could be related to the nature of the lipopolysaccharides (LPS) present on the outer membrane and in particular to the O-specific polysaccharide.A neutral O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide from P. chlororaphis subsp. aureofaciens strain M71. By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as the following linear trisaccharide.     

Modifications ultrastructurales provoquees par Puccinia graminis f. sp. tritici chez le ble

Susceptible varieties of wheat infected withPuccinia graminis (Pers.) f. sp.tritici Eriks. &E. Henn. were studied using chlorotic samples fixed with glutaraldehyde and postfixed in potassium permanganate or osmium tetroxide. Intercellular mycelium and haustoria are surrounded with a thick wall covered with dense material. Many kinds of organelles or structures were found in the electron-dense cytoplasm of fungi: nucleus, mitochondria, endoplasmic reticulum and lomasomes. Golgi apparatus was not recognized. Haustorium is characterized by pinocytosis figures lined with fungal plasmalemma and by numerous mitochondria. The haustorium is enclosed in an encapsulation of electron-dense, fibrillar or granular material. The thickness of the encapsulation increases with age. A membrane, which seems to be continuous with the host plasmalemma, forms the interface between the encapsulation and the host cytoplasm. This membrane is invaginated by lomasomes and small vesicles occur in his vicinity. The origin of the encapsulation and of the encapsulation membrane remains unknown.Many host organelles (mitochondria, endoplasmic reticulum, nucleus, chloroplasts) are closely appressed to the encapsulation membrane. The host nucleus appears to encase the haustorium; the chloroplasts develop excrescences and are devoid of starch, while photosynthesis is reduced. A large number of mitochondria encircles the haustorium and may be related with respiratory increase in the host. Rough-surfaced endoplasmic reticulum develops into long threads. Lipid droplets were observed in the host infected cytoplasm.

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Résumé L'étude de l'ultrastructure de variétés de Blé sensibles parasitées parPuccinia graminis (Pers.) f. sp.tritici Eriks. &E. Henn a été conduite sur des échantillons chlorotiques fixés à la glutaraldéhyde et postfixés par le permanganate de potassium ou l'acide osmique. Une paroi peu dense et recouverte d'exsudat entoure le mycélium intercellulaire et les haustorias. Le cytoplasme, dense aux électrons, renferme diverses structures ou organites: lomasomes, mitochondries, noyau, réticulum endoplasmique. Aucun dictyosome n'a été relevé. L'haustoria se caractérise par des figures de pinocytose qui tapissent la membrane plasmique et par la présence de nombreuses mitochondries. Une encapsulation de densité variable selon la fixation, encapsulation qui s'épaissit en vieillissant, sépare l'haustoria du protoplaste de l'hôte. Elle contient des produits fibrillaires ou granuleux, plus opaques aux électrons. La membrane qui recouvre l'encapsulation au contact du cytoplasme de l'hôte semble en continuité avec le plasmalemme de ce dernier. Elle est interrompue par des lomasomes et de nombreuses vésicules la cotoient. L'origine de l'encapsulation et de cette membrane demeure hypothétique.De nombreux organites de la plante (mitochondries, réticulum endoplasmique, noyau, chloroplastes) établissent des liens étroits avec le parasite. Le noyau devient lobé et les chloroplastes présentent parfois des excroissances. L'absence d'amidon indique un ralentissement de la photosynthèse. L'élévation du rythme respiratoire se traduit par la présence de nombreuses mitochondries qui encerclent l'haustoria. Le réticulum endoplasmique, souvent long et flexueux est riche en ribosomes. Le cytoplasme des cellules infectées contient des globules lipidiques.

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Contribution No. 105 de la Faculté d'Agriculture, Université Laval, Québec 10e.     

Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata

Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 × 10⁵ CFU/g fresh weight. Scanning electron micrographs showed that C. rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and β-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C. rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen.     

Induction of defense-related proteins in tomato roots treated with Pseudomonas fluorescens Pf1 and Fusarium oxysporum f. sp. lycopersici

Pseudomonas fluorescens isolate Pf1 was found to protect tomato plants from wilt disease caused by Fusarium oxysporum f. sp. lycopersici. Induction of defense proteins and chemicals by P. fluorescens isolate Pf1 against challenge inoculation with F. oxysporum f. sp. lycopersici in tomato was studied. Phenolics were found to accumulate in bacterized tomato root tissues challenged with F. oxysporum f. sp. lycopersici at one day after pathogen challenge. The accumulation of phenolics reached maximum at the 5^th day after pathogen challenge. In pathogen-inoculated plants, the accumulation started at the 2^nd day and drastically decreased 4 days after the pathogen inoculation. Activities of phenylalanine ammonia-lyase (PAL), peroxidase (PO) and polyphenol oxidase (PPO) increased in bacterized tomato root tissues challenged with the pathogen at one day after pathogen challenge and activities of PAL and PO reached maximum at the 4^th day while activity of PPO reached maximum at the 5^th day after challenge inoculation. Isoform analysis revealed that a unique PPO1 isoform was induced and PO1 and PPO2 isoforms were expressed at higher levels in bacterized tomato root tissues challenge inoculated with the pathogen. Similarly, -1,3 glucanase, chitinase and thaumatin-like proteins (TLP) were induced to accumulate at higher levels at 3-5 days of challenge inoculation in bacterized plants. Western blot analysis showed that chitinase isoform Chi2 with a molecular weight of 46 kDa was newly induced due to P. fluorescens isolate Pf1 treatment challenged with the pathogen. TLP isoform with molecular weight of 33 kDa was induced not only in P. fluorescens isolate Pf1-treated root tissues challenged with the pathogen but also in roots treated with P. fluorescens isolate Pf1 alone and roots inoculated with the pathogen. These results suggest that induction of defense enzymes involved in phenylpropanoid pathway and accumulation of phenolics and PR-proteins might have contributed to restriction of invasion of F. oxysporum f. sp. lycopersici in tomato roots.