Induction of Defense-Related Ultrastructural Modifications in Pea Root Tissues Inoculated with Endophytic Bacteria

The stimulation exerted by the endophytic bacterium Bacillus pumilus strain SE34 in plant defense reactions was investigated at the ultrastructural level using an in vitro system in which root-inducing T-DNA pea (Pisum sativum L.) roots were infected with the pea root-rotting fungus Fusarium oxysporum f. sp. pisi. In nonbacterized roots, the pathogen multiplied abundantly through much of the tissue including the vascular stele, whereas in prebacterized roots, pathogen growth was restricted to the epidermis and the outer cortex In these prebacterized roots, typical host reactions included strengthening the epidermal and cortical cell walls and deposition of newly formed barriers beyond the infection sites. Wall appositions were found to contain large amounts of callose in addition to being infiltrated with phenolic compounds. The labeling pattern obtained with the gold-complexed laccase showed that phenolics were widely distributed in Fusarium-challenged, bacterized roots. Such compounds accumulated in the host cell walls and the intercellular spaces as well as at the surface or even inside of the invading hyphae of the pathogen. The wall-bound chitin component in Fusarium hyphae colonizing bacterized roots was preserved even when hyphae had undergone substantial degradation. These observations confirm that endophytic bacteria may function as potential inducers of plant disease resistance.     

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.     

Effect of time of inoculation on in vitro ectomycorrhizal colonization and nodule initiation in Acacia holosericea seedlings

The complex interactions that occur in systems with more than one type of symbiosis were studied using one isolate of Bradyrhizobium sp. and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch inoculated on to the roots of Acacia holosericea A. Cunn. ex G. Don in vitro. After a single inoculation with Bradyrhizobium sp., bacteria typically entered the roots by forming infection threads in the root hair cells via the curling point of the root hair and/ or after intercellular penetration. Sheath formation and intercellular penetration were observed on Acacia roots after a single inoculation with Pisolithus tinctorius but no radial elongation of epidermal cells. Simultaneous inoculation with both microorganisms resulted in nodules and ectomycorrhiza on the root system, occasionally on the same lateral root. On lateral roots bearing nodules and ectomycorrhiza, the nodulation site was characterized by the presence of a nodule meristem and the absence of an infection thread; sheath formation and Hartig net development occurred regularly in the region of the roots adjacent to nodules. Prior inoculation with Bradyrhizobium sp. did not inhibit ectomycorrhizal colonization in root segments adjacent to nodules in which nodule meristems and infection threads were clearly present. Conversely, in ectomycorrhizae inoculated by bacteria, the nodule meristem and the infection thread were typically absent. These results show that simultaneous inoculation with both microorganisms inhibits infection thread development, thus conferring an advantage on fungal hyphae in the competition for infection sites. This suggests that fungal hyphae can modify directly and/or indirectly the recognition factors leading to nodule meristem initiation and infection thread development.     

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.     

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.     

Effects of Fungal Root Pathogens on the Population Dynamics of Biocontrol Strains of Fluorescent Pseudomonads in the Wheat Rhizosphere

The influences of Gaeumannomyces graminis var. tritici (which causes take-all of wheat), Rhizoctonia solani AG-8 (which causes rhizoctonia root rot of wheat), Pythium irregulare, P. aristosporum, and P. ultimum var. sporangiiferum (which cause pythium root rot of wheat) on the population dynamics of Pseudomonas fluorescens 2-79 and Q72a-80 (bicontrol strains active against take-all and pythium root rot of wheat, respectively) in the wheat rhizosphere were examined. Root infection by either G. graminis var. tritici or R. solani resulted in populations of both bacterial strains that were equal to or significantly larger than their respective populations maintained on roots in the absence of these pathogens. In contrast, the population of strain 2-79 was significantly smaller on roots in the presence of any of the three Pythium species than on noninfected roots and was often below the limits of detection (50 CFU/cm of root) on Pythium-infected roots after 40 days of plant growth. In the presence of either P. aristosporum or P. ultimum var. sporangiiferum, the decline in the population of Q72a-80 was similar to that observed on noninfected roots; however, the population of this strain declined more rapidly on roots infected by P. irregulare than on noninfected roots. Application of metalaxyl (which is selectively inhibitory to Pythium spp.) to soil naturally infestated with Pythium spp. resulted in significantly larger rhizosphere populations of the introduced bacteria over time than on plants grown in the same soil without metalaxyl. It is apparent that root infections by fungal pathogens may either enhance or depress the population of fluorescent pseudomonads introduced for their control, with different strains of pseudomonads reacting differentially to different genera and species of the root pathogens.     

Observations on the Responses of Lentil Root Cells to Hyphae of Fusarium oxysporum

The infection of lentil roots by Fusarium oxysporum Schlecht and the responses of the host cells to invading hyphae were examined by light microscopy. Hyphae from inoculum placed on the zone of cell elongation entered the roots at the juncture of epidermal cells within 8 h after inoculation. Although swollen hyphal apices were observed on the epidermal cells, root penetration occurred without formation of these structures or appressoria. The sheath of material found on the surface of uninoculated roots was absent from inoculated roots penetrated by hyphae. Prior to penetration, the epidermal cells became irregular in shape and their cytoplasm appeared to be plasmolysed or granular. Hyphae were observed in the cortex 10—12 h after inoculation and non–penetrated cortical cells were distinctly lobate. Often these lobed cells had a broad, peripheral band of diffuse cytoplasm. When hyphae were first observed in the cortical cells, the walls were ruptured and only slightly stained or unstained by toluidine blue. The inability of such walls to bind the stain may have been the result of the removal of wall components by fungal enzymes. Although extensive proliferation of hyphae was evident throughout the cortex after 24 h of incubation, the endodermis and vascular cylinder were free of hyphae for at least 72 h. Hyphae from inoculum placed on the root hairs or the root apex failed to penetrate the roots during the first 24 h of incubation. The cytological results herein are discussed in relation to the infection of field plantings by this pathogen.     

Structure and histochemistry of mycorrhizae synthesized between Arbutus menziesii (Ericaceae) and two basidiomycetes,Pisolithus tinctorius (Pisolithaceae) and Piloderma bicolor (Corticiaceae)

Arbutoid mycorrhizae were synthesized in growth pouches between Arbutus menziesii Pursch. (Pacific madrone) and two broad host range basidiomycete fungi, Pisolithus tinctorius (Pers.) Coker and Couch and Piloderma bicolor (Peck) Jülich. P. tinctorius induced the formation of dense, pinnate mycorrhizal root clusters enveloped by a thick fungal mantle. P. bicolor mycorrhizae were usually unbranched, and had a thin or non-existent mantle. Both associations had the well-developed para-epidermal Hartig nets and intracellular penetration of host epidermal cells by hyphae typical of arbutoid interactions. A. menziesii roots developed a suberized exodermis which acted as a barrier to cortical cell penetration by the fungi. Ultrastructurally, the suberin appeared non-lamellar, but this may have been due to the imbedding resin. Histochemical analyses indicated that phenolic substances present in epidermal cells may be an important factor in mycorrhiza establishment. Analyses with X-ray energy dispersive spectroscopy showed that some of the granular inclusions present in fungal hyphae of the mantle and Hartig net were polyphosphate. Other inclusions were either protein or polysaccharides.     

Ability of Nonpathogenic Fusarium oxysporum Strain Fo47 To Induce Resistance against Pythium ultimum Infection in Cucumber

The influence exerted by nonpathogenic Fusarium oxysporum strain Fo47 in triggering cucumber protection against infection by Pythium ultimum was investigated ultrastructurally. Macroscopic and microscopic observations of the pathogen colony in dual cultures revealed that reduction of Pythium growth was associated with marked disorders, including generalized disorganization of the host cytoplasm, retraction of the plasmalemma, and complete loss of the protoplasm. Cytochemical labeling of cellulose with an exoglucanase-gold complex showed that the cellulose component of the host cell walls was structurally preserved at a time when the host cytoplasm had undergone complete disorganization. A similar antagonistic process was observed at the root cell surface. Most striking and interesting was the finding that mycoparasitism, as evidenced by the frequent occurrence of Fo47 hyphae within nearly empty cells of the pathogen, occurred not only at the root surface but also within the invaded root tissues. The specific labeling pattern obtained with the exoglucanase-gold complex confirmed that Fo47 successfully penetrated cells of the pathogen, both in the rhizosphere and inside the root tissues. Pythium cells that could evade the first defensive line in the rhizosphere could penetrate the root epidermis, but their growth was restricted to the outermost tissues. Positive correlations between Fo47 treatment and induced resistance to infection by P. ultimum in cucumber were confirmed by (i) the reduction of pathogen viability; (ii) the elaboration of newly formed barriers, a phenomenon which was not seen in Fo47-free plants, where the pathogen proliferated in all root tissues within a few days; and (iii) the occlusion of intercellular spaces with a dense material likely enriched in phenolics. Taken together, our observations provide the first convincing evidence that Fo47 exerts a direct inhibitory effect on P. ultimum through a combination of antibiosis and mycoparasitism, in addition to being a strong inducer of plant defense reactions.     

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.     

Infection of clover by plant growth promoting Pseudomonas fluorescens strain 267 and Rhizobium leguminosarum bv. trifolii studied by mTn5-gusA

Plant growth promoting Pseudomonas fluorescens strain 267, isolated from soil, produced pseudobactin A, 7-sulfonic acid derivatives of pseudobactin A and several B group vitamins. In coinoculation with Rhizobium leguminosarum bv. trifolii strain 24.1, strain 267 promoted clover growth and enhanced symbiotic nitrogen fixation under controlled conditions. To better understand the beneficial effect of P. fluorescens 267 on clover inoculated with rhizobia, the colonization of clover roots by mTn5-gusA marked bacteria was studied in single and mixed infections under controlled conditions. Histochemical assays combined with light and electron microscopy showed that P. fluorescens 267.4 (i) efficiently colonized clover root surface; (ii) was heterogeneously distributed along the roots without the preference to defined root zone; (iii) formed microcolonies on the surface of clover root epidermis; (iv) penetrated the first layer of the primary root cortex parenchyma and (v) colonized endophytically the inner root tissues of clover.     

Penetration of Phytophthora cinnamomi into disease tolerant and susceptible eucalypts

The mechanisms of penetration of Phytophthora cinnamomi Rands into seedling eucalypt roots were studied by light and electron microscopy. Culture grown seedlings of root-rot tolerant Eucalyptus st johnii and root-rot susceptible Eucalyptus obliqua were inoculated with both zoospores and mycelium. Zoospores encysted on roots of both species and the germ tubes penetrated without the formation of appressoria. Swellings, previously described as appressoria, were formed when the germ tube was slow to enter the host by intracellular penetration. Vegetative hyphae penetrated both inter- and intracellularly into the zones of root elongation and differentiation, often through root hairs. Evidence of hydrolysis of the host cell-wall at the point of penetration was observed in electron micrographs. Several hours after the germ tube penetrated the epidermis, a thick plug of amorphous material formed in the germ tube slightly below the level of the outer walls of the epidermal cells, sealing off the hypha within the root. Behaviour of zoospores and germ tubes and the mechanism of penetration were similar on both hosts. Micrographs do not suggest any kind of a hypersensitive reaction by the host cells during the early stages of infection.     

Endophytic Pseudomonas fluorescens Endo2 and Endo35 induce resistance in black gram (Vigna mungo L. Hepper) to the pathogen Macrophomina phaseolina

Abstract

The effect of endophytic Pseudomonas fluorescens isolates Endo2 and Endo35 on induced systemic disease protection against dry root rot of black gram (Vigna mungo L. Hepper) caused by Macrophomina phaseolina was investigated under glasshouse conditions. When the bacterized black gram plants were inoculated with dry root rot pathogen, the activities of peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL) were stimulated in addition to accumulation of phenolics and lignin. Activity of phenylalanine ammonia-lyase (PAL) reached the maximum 24 h after pathogen challenge inoculation, whereas the activities of PO and PPO reached the maximum at 72 h and 48 h, respectively. Isoform analysis revealed that a unique PPO3 isozyme was induced in bacterized black gram tissues inoculated with the pathogen. Phenolics were found to accumulate in bacterized black gram tissues challenged with M. phaseolina one day after pathogen challenge. The accumulation of phenolics reached maximum at the third day after pathogen inoculation. Similar observation was found in the lignin content of black gram plants. In untreated control plants, the accumulation of defence enzymes and chemicals started at the first day and drastically decreased 3 days after pathogen inoculation. These results suggest that induction of defense enzymes involved in phenylpropanoid pathway and accumulation of phenolics and PR-proteins might have contributed to restricting invasion of Macrophomina phaseolina in black gram roots.     

Medicago truncatula Vapyrin is a novel protein required for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal (AM) symbiosis is a widespread mutualism formed between vascular plants and fungi of the Glomeromycota. In this endosymbiosis, fungal hyphae enter the roots, growing through epidermal cells to the cortex where they establish differentiated hyphae called arbuscules in the cortical cells. Reprogramming of the plant epidermal and cortical cells occurs to enable intracellular growth of the fungal symbiont; however, the plant genes underlying this process are largely unknown. Here, through the use of RNAi, we demonstrate that the expression of a Medicago truncatula gene named Vapyrin is essential for arbuscule formation, and also for efficient epidermal penetration by AM fungi. Vapyrin is induced transiently in the epidermis coincident with hyphal penetration, and then in the cortex during arbuscule formation. The Vapyrin protein is cytoplasmic, and in cells containing AM fungal hyphae, the protein accumulates in small puncta that move through the cytoplasm. Vapyrin is a novel protein composed of two domains that mediate protein–protein interactions: an N‐terminal VAMP‐associated protein (VAP)/major sperm protein (MSP) domain and a C‐terminal ankyrin‐repeat domain. Putative Vapyrin orthologs exist widely in the plant kingdom, but not in Arabidopsis, or in non‐plant species. The data suggest a role for Vapyrin in cellular remodeling to support the intracellular development of fungal hyphae during AM symbiosis.     

Biological control ofFusarium oxysporum f.sp.cubense in banana by inoculation withPseudomonas fluorescens

Native strains ofPseudomonas fluorescens exhibitedin vitro antibiosis towards isolates of races 1 and 4 ofFusarium oxysporum f.sp.cubense, the Panama wilt pathogen of banana. The seedlings ofMusa balbisiana seedlings treated withP. fluorescens showed less severe wilting and internal discolouration due toF. oxysporum f.sp.cubense infection in greenhouse experiments. In addition to suppressing Panama wilt, bacterized seedlings ofM. balbisiana also showed better root growth and enhanced plant height.     

Bacterial effects on arbuscular mycorrhizal fungi and mycorrhiza development as influenced by the bacteria, fungi, and host plant

Bacterial strains from mycorrhizal roots (three belonging to Comamonadaceae and one to Oxalobacteraceae) and from non-mycorrhizal roots (two belonging to Comamonadaceae) of Medicago truncatula and two reference strains (Collimonas fungivorans Ter331 and Pseudomonas fluorescens C7R12) were tested for their effect on the in vitro saprophytic growth of Glomus mosseae BEG12 and on its colonization of M. truncatula roots. Only the Oxalobacteraceae strain, isolated from barrel medic mycorrhizal roots, and the reference strain P. fluorescens C7R12 promoted both the saprophytic growth and root colonization of G. mosseae BEG12, indicating that they acted as mycorrhiza helper bacteria. Greatest effects were achieved by P. fluorescens C7R12 and its influence on the saprophytic growth of G. mosseae was compared to that on Gigaspora rosea BEG9 to determine if the bacterial stimulation was fungal specific. This fungal specificity, together with plant specificity, was finally evaluated by comparing bacterial effects on arbuscular mycorrhizal symbiosis when each of the fungal species was inoculated to two different plant species (M. truncatula and Lycopersicon esculentum). The results obtained showed that promotion of saprophytic growth by P. fluorescens C7R12 was expressed in vitro towards G. mosseae but not towards G. rosea. Bacterial promotion of mycorhization was also expressed towards G. mosseae, but not G. rosea, in roots of M. truncatula and L. esculentum. Taken together, results indicated that enhancement of arbuscular mycorrhiza development was only induced by a limited number of bacteria, promotion by the most efficient bacterial strain being fungal and not plant specific.     

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     

The infection process ofFusarium oxysporum in cotton root tips

Summary Conidia ofFusarium oxysporum f. sp.vasinfectum started to germinate on the roots of cotton (Gossypium barbadense L.) 6 h after inoculation and formed a compact mycelium covering the root surface. 18 h later, penetration hyphae branched off and infected the root. The number of penetration hyphae increased with the number of conidia used for inoculation. The optimal temperature for penetration was between 28 and 30 °C. The highest numbers of penetration hyphae were found in the meristematic zone, 40 percent less in the elongation and root hair zones, and none in the lateral root zone. The fine structure of the infection process was studied in protodermal cells of the meristematic zone and in rhizodermal cells of the elongation zone. The penetration hyphae were well preserved after freeze substitution and showed a Golgi equivalent consisting of three populations of smooth cisternae. Plant reactions were found already during fungal growth on the root surface. In the meristematic zone, a thickening of the plant cell wall due to an apposition of dark and lightly staining material below the hyphae occurred. This wall apposition increased in size around the hypha invading the plant cell and led to the formation of a prominent wall apposition with finger-like projections into the host cytoplasm. In the elongation zone, the deposits around the penetration hypha appeared less thick and the dark inclusions were less pronounced. High pressure freezing of infected cells revealed, thatF. oxysporum penetrates and grows within the host cells without inducing damages such as plasmolysis, cell degeneration or even host necrosis. We suggest thatF. oxysporum has an endophytic or biotrophic phase during colonization of the root tips.Abbreviation Ph penetration hyphae     

Flavonoids in roots of white clover: interaction of arbuscular mycorrhizal fungi and a pathogenic fungus

The effects of two arbuscular mycorrhizal fungi (AMF) (Glomus mosseae and G. claroideum) and a pathogenic fungus (Pythium ultimum) on the production of eight flavonoids in roots of two white clover (Trifolium repens L.) cultivars were evaluated. Quantification of AM and pathogenic fungi in the roots showed that the AM symbiosis significantly reduced P. ultimum biomass and in some cases prevented infection. The flavonoid productions in clover roots varied depending on the presence of beneficial and/or pathogenic fungi, fungal isolate or plant cultivar. Only plants colonized with G. claroideum showed detectable concentrations of either coumestrol or kaempferol (cultivar-dependant). In addition, inoculation with G. claroideum resulted in significantly higher concentrations of coumestrol in cv. Sonja and medicarpin in cv. Milo. A low production of coumestrol and kaempferol in mycorrhizal plants may be G. mosseae-specific. Only the concentrations of formononetin and daidzein increased in clover roots in response to infection with P. ultimum. These flavonoids are supposedly stress metabolites, synthesized or produced from glycosides in response to pathogen infection. However, the presence of one or both AMF significantly lowered the formononetin and daidzein concentrations, and overruled the inductive effect of P. ultimum. Therefore the antagonistic action of AM against the pathogen must take place through another mechanism.     

Immunological detection of the fungal parasite,Pythium sp.; the causative organism of red rot disease in Porphyra yezoensis

The red rot disease of Porphyra yezoensis Ueda (Rhodophyta) is caused by a parasitic fungus, Pythium sp. To facilitate the detection of this pathogen in infected thalli of P. yezoensis, polyclonal and monoclonal antibodies were prepared. Antibodies were raised against antigen prepared from an isolate of fungal hyphae obtained from red-rot infected thallus of P. yezoensis from Aichi Prefecture. Polyclonal antibody was obtained from the antisera of immunized rabbits. Monoclonal antibody was obtained from the culture supernatant of a hybridoma which had been established by cell fusion between a myeloma cell line and spleen cells of immunized mice. Hyphae were detected by means of indirect fluorescent antibody technique. Titers of polyclonal antibodies obtained were too low to recognize fungal hyphae that had penetrated the thalli of P. yezoensis; however, monoclonal antibody was useful for the detection of fungi that had penetrated algal thalli. The monoclonal antibody was specific for the Pythium sp. from red-rot infected thalli of P. yezoensis from Saga (western Japan) and from Aichi Prefectures (central Japan), but was ineffective for infections from Miyagi Prefecture (northern Japan). It is evident, therefore, that Pythium sp. can give rise to immunologically distinct groups of red rot disease. Based on chemical and enzymatic treatments, the antigenic determinant appeared to localize on the sugar chains of glycoconjugates or the polysaccharides of the hyphal cell wall.