Identification of the endophypte ofDryas andRubus (Rosaceae)

Summary Root nodules ofDryas drummondii are of the coralloid type (Alnus type). The endophyte is present in the middle cortical cells of the root-nodule tissue. Transmission electron micrographs revealed an actinorhizal endophyte with septate hyphae and non-septate spherical or ovoid vesicles. Vesicles always possess at the base a septum; septa formation in the endophyte is always associated with the presence of mesosomes. Branching of the endophyte is not necessarily correlated with septum formation. Hyphal structures are more prominent in the apical part of the root nodule and vesicles are more numerous in a broad zone below this. In the middle and towards the base of the root nodule the endophytic structures appear in a stage of disintegration. Vesicles appear in a broad region near the periphery of the host cell and regularly show no strict orientation towards the host-cell wall. In the center of the host cells only hyphae occur. In the intercellular spaces between the host cells theFrankia endophyte produces spore-like structures although the outline of the sporangia is often faint.The coralloid root ofRubus ellipticus shows characteristically a basal rootlet initiated below the dichotomous branching of the nodular lobes, but extending beyond the root nodule. The endophyte is only present in the outer cortex of the root nodule in a 1–2 cell wide layer. This endophytic layer is bounded, internally as well as externally, with a 4–5 cell wide layer of tannin-filled host cells. The implications of this situation are discussed. Tannin-filled cells occur regularly inRubus species and their arrangement has been used for taxonomic purposes within the genus. TheRubus endophyte is aFrankia species with septate hyphae and distinctly septate spherical vesicles. The ultrastructure of the vesicles of theRubus endophyte is very similar to that of theAlnus endophyte.     

Observations on the ultrastructure ofFrankia sp. in root nodules ofDatisca cannabina L.

Summary The fine structures of the microsymbiont inside the root nodules ofDatisca cannabina have been studied by light, by transmission- and by scanning-electron microscopy. The endophyte is prokaryotic and actinomycetal in nature. The hyphae are septate and branched, diameter 0.3–0.5 m. The tips of hyphae are swollen to form electron-dense, clubshaped to filamentous vesicles, ranging in diameter: 0.4–1.4 m. The endophyte penetrates through walls of the cortial cells. The infected zone is kidney shaped and confined to one side of the acentric stele. The orientation of infection is reversed from other actinorhizae exceptCoriaria. The hyphae are near the host cell wall and vesicles are directed towards the central vacuole. Vesicles are aseptate and no collapsing of the vesicle cell wall (void area) has been observed. Vesicle clusters structures are globular with an opening at one side of the cluster. The host cell is multinucleate or contains a lobed nucleus. Groups of mitochondria are located in between the hyphae, suggesting a strong association between the host and the endophyte for energy supply and amino acid production. The consequences of the inability to separate the mitochondria from the vesicle clusters in nodule homogenates in physiological studies have been discussed.Isolated vesicles clusters showed dehydrogenase activity, indicated by the presence of formazan crystals, after incubation with NADH and NBT. Strongest reducing activity was found within the vesicles. The possible role of filamentous vesicles in nitrogen fixation has been discussed.     

Electron microscopy of the endophyte ofAlnus glutinosa

Earlier light microscopic investigations have revealed that the endophyte ofAlnus glutinosa presents itself in three different forms. In the present study this is confirmed by electron microscopy; also, new data on the cytology of the endophyte have been obtained.The host cells are primarily infected by the hyphal form of the endophyte. A plant cell nucleus and mitochondria can be found in the infected host cells.In the majority of the infected cells, so-called vesicles develop at the tips of the hyphae. Electron micrographs show that these vesicles, as well as the hyphae, are surrounded by the host-cell cytoplasmic membrane. The endophyte cytoplasm inside the vesicles is divided in all directions by cross walls, many of which are incomplete. Plasmalemmosomes are conspicuous. Some vesicles look vigorous but others shrunken or nearly devoid of cytoplasm as if being digested.A minority of host cells situated between the vesicle-containing ones are completely filled by bacteria-like cells. These host cells, in contrast to the other ones, do not contain a nucleus nor mitochondria, nor are the endophyte cells in them enveloped by a host cell cytoplasmic membrane: these host cells are dead. Vesicles are not found in these cells.It is inferred that a living host cell exerts a stimulus on the endophyte to which the latter responds by forming vesicles at the tips of the hyphae. At a later stage the host cells digest the vesicles and the hyphae. On the other hand, if a host cell does not survive the infection, the hyphae divide into bacteria-like cells, which are not digested owing to the absence of host cytoplasm.According to the cytology of the hyphae, the endophyte is an actinomycete.The cytology of the endophyte needs further elucidation. Its plasmalemmosomes, or membranous bodies connected with the cytoplasmic membrane, are beautifully developed. The striated bodies described on p. 359 under 4) may be a new feature, which may turn up in other actinomycetes or bacteria.     

Studies of an effective strain ofFrankia fromAllocasuarina lehmanniana of the Casuarinaceae

Summary Seedlings ofCasuarina spp. andAllocasuarina spp. were grown from seed in the greenhouse and inoculated with a nodule suspension fromC. equisetifolia. Plants ofCasuarina spp. nodulated regularly and were effective in nitrogen-fixation. Only one species ofAllocasuariona, A. lehmanniana formed root nodules. Using these plants as source of inoculum, the isolation of a newFrankia sp. HFPA11I1 (HFP022 801) was made and the strain was grown in pure culture.Frankia sp. HFPA11I1 grows well in a defined medium and shows typical morphological characteristics. In media lacking combined nitrogen, the filamentours bacterium forms terminal vesicles in abundance and differentiaties large intrahyphal or terminal sporangia containing numerous spores. This strain, used as inoculum, nodulates effectively seedlings ofC. equisietifolia andC. cunninghamiana, forming nodules with verically-growing nodule roots. Although effective in acetylene reduction, the endophyte within the nodules is filamentous and lacks veiscles. When used to inoculated seedlings ofA llocasuarina lehmanniana, Frankia sp. HFPA11I1 induces root nodules which are coralloid and lacking nodule roots. The nodules are effective in acetylene reduction and the filamentous hyphae ofFrankia within the nodule lobes lack vesicles. Effective nodulation inA. Lehmanniana depends upon environmental conditions of the seedlings and proceeds much more slowly than in Casuariana.     

Fine structure of actinorhiza of theRhamnaceae growing in Chile I.Talguenea quinquenervia (Gill et Hook)

Summary Root nodulesTalguenea quinquenervia Gill et Hook (Rhamnaceae) are restricted to the middle region of the root cortex. The root endophyte possesses hyphae which are septate and vesicles. The vesicles are spherical and are continuous with that of the hyphae. The endophyte fine structure is similar to otherFrankia-induced root nodules.     

Nodulation of actinorhizal plants byFrankia strains capable of both root hair infection and intercellular penetration

Summary A morphological analysis of the initiation and development of root nodules ofElaeagnus angustifolia andMyrica cerifera inoculated with pure-culturedFrankia strains DDB 011610 or DDB 020110 was undertaken. From ultrastructural observations it was determined that both of theseFrankia strains can infectElaeagnus by an intercellular penetration mechanism andMyrica by the root hair infection mechanism. This indicates that both of these strains have the ability to infect host plant roots by either of two mechanisms. The reverse, thatElaeagnus orMyrica could be infected by both mechanisms, was not observed. The infection and nodule development processes of these two plants in combination with these strains were similar to observations made in previous studies (Miller andBaker 1985,Torrey andCallaham 1979). However, one exception was identified in the development of the prenodule ofMyrica when infected with strain 011610, in that endophytic hyphae developed vesicles within the cells of the prenodule. This event has not been described before for any of the actinorhizal genera and may be an indication of less than optimal compatibility between the host plant and the symbiont.Contribution no. 876 of the Battelle-Kettering Laboratory.     

Immunogold localisation of hemoglobin inCasuarina root nodules

Summary Immunocytochemical localisation of hemoglobin on sections ofFrankia-infecledCasuarina glauca nodule tissue confirmed its presence in nitrogen-fixing infected cells. Using colloidal gold as the marker, hemoglobin was shown to be restricted to the cytoplasm and nucleus of infected cells. None was associated with endophyte hyphae or uninfected cells. As infection developed, with its associated thickening and modification of host cell walls, the level of label, and by implication, the level of hemoglobin increased.Abbreviations SPL semi-permeable layer - GA glutaraldehyde - PFA paraformaldehyde     

Scanning electron microscopy of the Alnus crispa var. mollis Fern. root nodule endophyte

Nitrogen-fixing root nodules of the Alnus crispa var. mollis Fern. were studied by scanning electron microscopy (SEM). The critical point drying of glutaraldehyde-osmium fixed nodular tissue permitted an excellent morphological preservation of the three-dimensional structures of the host and endophyte cells. The nodule endophyte was observed as two forms: the hypha which can be branched, and the vesicle which developed at the parental hypha tip. The actinomycetal endophyte penetrated through the host cortical cell wall and became enveloped by a membrane. This enclosing membrane is suggested to be the invaginated host plasmalemma. Perforations of the cell wall of the host infected cell were observed. These perforations are suggested to be the result of an enzymatic degradation process, probably regulated by the penetrating endophyte hyphae. In addition to the polymorphic endophyte, endogenous bacterial contaminants were observed in the nodular tissue. The present SEM study confirms previous light microscopy and transmission electron microscopy studies of the same species of root nodule symbiosis.     

DEVELOPMENT OF ENDOPHYTIC FRANKIA SPORANGIA IN FIELD- AND LABORATORY-GROWN NODULES OF COMPTONIA PEREGRINA AND MYRICA GALE

Field-collected nodules of Comptonia peregrina (L.) Coult. and Myrica gale L. (Myricaceae), infected by the nitrogen-fixing actinomycete Frankia sp., were of two types: those that lacked sporangia entirely, designated spore(-), and those that showed extensive sporangial development, designated spore(+). In spore(+) nodules of C. peregrina, sporangia began to develop after the differentiation of endophytic vesicles and the concomitant onset of nitrogenase activity. At the onset of sporangial differentiation, infected host cells appeared healthy. However, endophytic vesicles and host cell cytoplasm and nuclei began to senesce rapidly as sporangia developed. Staining of sectioned material with the fluorescent stain Calcofluor White suggested that vesicles, hyphae and young sporangia were enclosed within a host-derived encapsulation layer, but mature sporangia were no longer encapsulated. In both C. peregrina and M. gale, vesicles were more short-lived in spore(+) than in spore(-) nodules. Field-collected spore(+) M. gale nodules exhibited a pronounced seasonality of sporangial formation. Sporangia began to differentiate in June, after the formation of vesicles and became more prominent in late summer. Inter- and intraspecific cross-inoculations suggest that the ability to form sporangia in the symbiotic state is controlled by endophytic strain type rather than host genotype or host/endophyte combination. The host may, however, influence the number and seasonal appearance of sporangia formed.     

A comparison of cultural characteristics and infectivity ofFrankia isolates from root nodules ofCasuarina species

Summary The isolations of three new strains ofFrankia were made from root nodules ofCasuarina cunninghamiana growing aeroponically. Two strains, HFPCCI1 and HFPCcI2 isolated by Lopez are typicalFrankia strains, producing sporangia among filamentous mats in culture and, in the absence of combined nitrogen, forming vesicles and showing acetylene reduction. They are red-pigmented and, although failing to nodulateCasuarina hosts, effectively nodulatedElaeagnus andHippophae. A third strain HFPCcI3 isolated by Zhang from the same source, also a typicalFrankia, can form sporangia and vesicles in culture and reduce acetylene, is unpigmented, fails to nodulateElaeagnus but effectively nodulatesC. cunninghamiana andC. equisetifolia. Comparisons are made among all of theCasuarina isolates in our collection from around the world (twelve in all) with regard to their cultural characteristics and capacity to infect host plant species. Questions are raised about the specificity of the various isolates and their possible affinities. Opportunities are suggested for inoculation of seedlings for forestry and field application using the infective, effective strains now available.     

The isolation,culture and infectivity of aFrankia strain fromGymnostoma papuanum (Casuarinaceae)

A strain ofFrankia was isolated fromGymnostoma papuanum(Casuarinaceae) nodules harvested from rooted cuttings which had been inoculated with a suspension of crushedCasuarina equisetifolia nodules. Designated HFPGpI1 (catalogue #HFP021801), this strain is pigmented and similar to other pigmentedFrankia strains in cultural characteristics. A previously unknown spiraled hyphal morphology was observed at very low frequency in some cultures of this strain. HFPGpI1 is infective and effective onG. papuanum but not on anyCasuarina species tested. It also infects members of the family Elaeagnaceae andMyrica gale. The host plantG. papuanum can be infected with a wide range ofFrankia isolates and thus can be considered a promiscuous host, unlike its close relatives in the genera Casuarina and Allocasuarina which are very restrictive as to which strains may nodulate them.     

Root-nodules formation byPenicillium sp. onAlnus glutinosa andAlnus incana

A non identified species ofPenicillium induced the formation of nodules on the root system of two species of alder (Alnus glutinosa and A.incana). These so-called myconodules looked like young actinorhizae. Namely only some cortical cells of the young transformed root were invaded by the mycelium. Plasmalemma of the host-cell surrounded the hyphae when they penetrated in the cell, but then the fungus colonized all the cell, the contents of which degenerated. In spite of this necrophytic relationships the plant showed no evident damage after the infection.     

Interaction between an isolate of dark-septate fungi and its host plant <Emphasis Type="Italic">Saussurea involucrata</Emphasis>

A dark-septate endophytic (DSE) fungus EF-M was isolated from the roots of an alpine plant Saussurea involucrata Kar. et Kir. ex Maxim. The fungus was identified by sequencing the PCR-amplified rDNA 5.8S gene and ITS regions. The sequence was compared with similar sequences in the GenBank, and results showed that EF-M was congeneric to Leptodontidium. Resynthesis study was conducted to clarify the relationship between the root endophyte EF-M and the host plant S. involucrata using the material grown in sterile culture bottle. In roots recovered 6 weeks after inoculation, epidermal cells were colonized by intercellular and intracellular hyphae and “microsclerotia” formed within individual cells in the epidermis layers. However, hyphae did not invade the cortex and the stele. There were no profound effects of endophyte EF-M on plant root development, but significant differences were detected in plant height and shoot dry weight between the treatments. The present study is the first report hitherto on DSE fungi in S. involucrata.     

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     

The initiation,development and structure of root nodules inElaeagnus angustifolia L. (Elaeagnaceae)

Summary A correlated light and electron microscopic study was undertaken of the initiation and development of root nodules of the actinorhizal tree species,Elaeagnus angustifolia L. (Elaeagnaceae).Two pure culturedFrankia strains were used for inoculation of plants in either standing water culture or axenic tube cultures. Unlike the well known root hair infection of other actinorhizal genera such asAlnus orMyrica the mode of infection ofElaeagnus in all cases was by direct intercellular penetration of the epidermis and apoplastic colonization of the root cortex. Root hairs were not involved in this process and were not observed to be deformed or curled in the presence of the actinomyceteFrankia. In response to the invasion of the root, host cells secreted a darkly staining material into the intercellular spaces. The colonizingFrankia grew through this material probably by enzymatic digestion as suggested by clear dissolution zones around the hyphal strands. A nodule primordium was initiated from the root pericycle, well in advance of the colonizingFrankia. No random division of root cortical cells, indicative of prenodule formation was observed inElaeagnus. As the nodule primordium grew in size it was surrounded by tanninised cells of a protoperiderm. The endophyte easily traversed this protoperiderm, and once inside the nodule primordium cortex ramified within the intercellular spaces at multiple cell junctions. Invasion of the nodule cortical cells occurred when a hyphal branch of the endophyte was initiated and grew through the plant cell wall, again by apparent enzymatic digestion. The plant cell plasmalemma of invaded cells always remained intact and numerous secretory vesicles fused with it to encapsulate the advancingFrankia within a fibrous cell wall-like material. Once within the host cell some endophyte cells began to differentiate into characteristic vesicles which are the presumed site of nitrogen fixation. This study clearly demonstrates that alternative developmental pathways exist for the development of actinorhizal nitrogen-fixing root symbioses.     

Preliminary characterization of an ineffective Frankia derived from a spontaneously neomycin-resistant strain

Five strains of Frankia isolated from actinorhizae of 3 alder species were cultivated with various concentrations of neomycin in the medium. For one strain, resistance to neomycin was associated with lost of effectiveness. The ineffective strain was as infective as the parent strain. Protein and isoenzymes patterns showed that the ineffective and the parent strains were quite similar. The ineffective strain differentiated vesicles inside the infected host-cells.     

Variable compatibility of clonedAlnus glutinosa ecotypes against ineffectiveFrankia strains

Nodulation tests onin-vitro propagated clones ofAlnus glutinosa ecotypes (forest ecotype, pioneer ecotype) withFrankia strains originating from both ecotypes indicated differences in host-plant compatibility. Inoculated plants of the pioneer ecotype clone were not infected by strains, that were unable to fix nitrogen in pure culture. Nodulation could only be induced on the clone of the forest ecotype, but no nitrogen-fixing activity could be detected. Ultra-structural observations of the nodules by SEM and TEM indicated that ineffectivity of these strains was correlated with the lack of vesicles in the infected cells. Cells were only filled with hyphae: neither sporangia nor vesicles could be detected. In contrast, effective nodules could be obtained on both alder clones after inoculation with an effective strain, showing normal development of vesicle clusters in infected cells. In pure culture the ineffective strains produced no vesicles; sporangia were found only during early stage of growth. The results demonstrate the existence ofFrankia strains which were either non-infective or ineffective on different clones ofAlnus glutinosa.     

同一生境下白及与黄花白及内生菌群差异的初步研究

为揭示白及与黄花白及内生菌群落特征异同的内在机制,该文运用末端限制性酶切片段长度多态性分析技术对白及和黄花白及叶、茎、块茎、根等组织中内生细菌16S rDNA、内生真菌ITS区进行检测,分析内生菌丰度及多样性指数,运用主成分分析、聚类分析和相关性分析对比白及与黄花白及内生菌差异.结果表明:(1)白及和黄花白及各组织内生...     

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.     

Mycotrophy of Annona cherimola and the morphology of its mycorrhizae

The mycotrophic character of Annona cherimola (Magnoliales), a tropical/subtropical plantation crop of interest, is described for the first time. This crop seems to depend on mycorrhizae (arbuscular) for optimal growth, with Glomus deserticola being the most effective endophyte tested. Study of the morphology of the arbuscular mycorrhizae in Annona roots showed exclusively intracellular hyphal development, with cell-to-cell fungal passage and an abundance of arbuscules and coiled hyphae within cells. Intercellular distributive hyphae were not observed. The morphology and the pattern of spread of the mycorrhizal colonization were similar for the different endophytes involved and appeared to be dependent on the host root. Such features of mycorrhizal colonization are characteristic of host species lacking intercellular air channels and have been described for some species of ecological interest, but they are not commonly noted in the mycorrhizal literature, especially that dealing with crop species. Some ecophysiological consequences of this pattern of colonization are discussed.