PCR-amplified 16S rRNA sequence analysis to confirm nodulation of Datisca cannabina L. by the endophyte of Coriaria nepalensis Wall

Different Frankia strains and crushed nodule suspensions were tested for their ability to nodulate Coriaria nepalensis and Datisca cannabina. Datisca cannabina seedlings were nodulated effectively by both crushed nodule suspension from Coriaria nepalensis and Datisca cannabina. The origin of the endophyte in Datisca nodules induced by crushed nodules of Coriaria was confirmed by comparing partial PCR-amplified 16S rRNA sequences with those of the endophytes of both plants. Coriaria seedlings could only be nodulated by crushed nodule suspensions of Coriaria nepalensis. All pure cultures of Frankia used as a single inoculum source or in combinations with a nodule filtrate, failed to induce nodulation on Coriaria. Two atypical Frankia strains Cn3 and Cn7 isolated from Coriaria nodules showed no acetylene reduction activity and did not induce nodulation on the host seedlings.     

Nitrogen-fixing root nodules inDatisca Cannabina L.

Summary The occurrence of root nodules inDatisca cannabina is reported. The nodules are typically of the Alnus type, forming dichotomously-branched coralloid clusters. The enlarged cortical cells contain vesicle clusters of the endophyte. The nodules reduced acetylene to ethylene at a rate of 5.5 moles ethylene per g fresh wt of nodules per hour.     

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.     

Enhanced nodule initiation on alfalfa by wild-typeRhizobium meliloti co-inoculated withnod gene mutants and other bacteria

Nodule formation on alfalfa (Medicago sativa L.) roots was determined at different inoculum dosages for wild-typeRhizobium meliloti strain RCR2011 and for various mutant derivatives with altered nodulation behavior. The number of nodules formed on the whole length of the primary roots was essentially constant regardless of initial inoculum dosage or subsequent bacterial multiplication, indicative of homeostatic regulation of total nodule number. In contrast, the number of nodules formed in just the initially susceptible region of these roots was sigmoidally dependent on the number of wild-type bacteria added, increasing rapidly at dosages above 5·10³ bacteria/plant. This behavior indicates the possible existence of a threshold barrier to nodule initiation in the host which the bacteria must overcome. When low dosages of the parent (10³ cells/plant) were co-inoculated with 10⁶ cells/plant of mutants lacking functionalnodA, nodC, nodE, nodF ornodH genes, nodule initiation was increased 10- to 30-fold. Analysis of nodule occupancy indicated that these mutants were able to help the parent (wild-type) strain initiate nodules without themselves occupying the nodules. Co-inoculation withR. trifolii orAgrobacterium tumefaciens cured of its Ti plasmid also markedly stimulated nodule initiation by theR. meliloti parent strain. Introduction of a segment of the symbiotic megaplasmid fromR. meliloti intoA. tumefaciens abolished this stimulation.Bradyrhizobium japonicum and a chromosomal Tn5 nod^- mutant ofR. meliloti did not significantly stimulate nodule initiation when co-inoculated with wild-typeR. meliloti. These results indicate that certainnod gene mutants and members of theRhizobiaceae may produce extracellular signals that supplement the ability of wild-typeR. meliloti cells to induce crucial responses in the host.Abbreviations EH emergent root hairs - kb kilobase - RDU relative distance unit - RT root tip This is journal article No. 188-87 of the Ohio Agricultural Research and Development Center     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

The electrochemical proton gradient and its influence on citrate uptake in tonoplast vesicles of Hevea brasiliensis

The relationship between the electrochemical proton gradient, _H+ ^– , and citrate transport has been studied in tonoplast vesicles from Hevea brasiliensis (the rubber tree). Vesicles were generated from lyophilized samples of fresh vacuoles obtained from the latex sap. Methylamine was used to measure intravesicular pH and lipophilic ions to determine the electrical potential difference () across the tonoplast. When incubated at pH 7.5 in the absence of ATP, the tonoplast vesicles showed a pH of 0.6 units (interior acid) and a of about-100 mV (interior negative). This potential is thought to be made up of contributions from an H⁺ diffusion potential, diffusion potentials from other cations and a Donnan potential arising from the presence of internal citrate. In the presence of 5 mol m^-3 MgATP the pH was increased to about 1.0 unit and the to about-10 mV. Under these conditions the proton-motive force ( _{p H+} ^– /F) became positive and reached +50 mV. These effects were specific to MgATP (ADP and Mg²⁺ having no significant effect) and were prevented by the protonophore p-trifluoromethoxycarbonylcyanidephenylhydrazone (FCCP). Citrate uptake by the vesicles was markedly stimulated by MgATP; ADP and Mg²⁺ again had no effect. Nigericin greatly increased pH and this was associated with a large increase in citrate accumulation. The results indicate that the vesicle membrane possesses a functional H⁺-translocating ATPase. The _H+ ^– generated by this ATPase can be used to drive citrate uptake into the vesicles. The properties of the tonoplast vesicles are compared with those of the fresh latex vacuoles.Abbreviations _H+ ^– electrochemical proton gradient - electrical potential difference across membrane - p proton-motive force ( _H+ ^– /F) - FCCP p-trifluoromethoxycarbonylcyanidephenylhydrazone - TPMP⁺ triphenylmethylphosphonium ion     

Concentration-dependent influence of quercetin on nodulation process and the main characteristics of soybean inoculated with Bradyrhizobium japonicum

Soybean plants cv. Corsoy were grown in greenhouse conditions on sterilized quartz sand. They were inoculated with Bradyrhizobium japonicum, strain 542. The plants were treated with different concentrations of quercetin (ranging from 10 nM to 1M) at regular intervals during the experiment. The experiment was terminated at flower development. The following parameters, important for symbiosis efficiency were determined: shoot, root and nodule weights, nodule number, total leghemoglobin in the nodules,total nitrogen and soluble protein concentrations in shoots and roots, as well as chlorophyll concentration in the leaves.The results obtained partly confirmed the earlier findings that quercetin inhibits nodulation since increasing quercetin concentration decreased the number of nodules. However, at very low concentrations, quercetin stimulated the number of nodules. Quercetin also exerted a stimulating influence on other characteristics of the plant and nodules which did not correlate with nodule number and quantity of N fixed. These are: nodule weight, leghemoglobin concentration, total soluble protein content in shoots and roots as well as shoot and root weight.     

The effect of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis

A study was conducted to investigate the effects of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis. The effects of P on growth and survival of Frankia in the rhizosphere was assessed by examing Frankia growth and survival in flasks of basal nutrient solution. There was no growth in the nutrient solution during the experimental period. However, the viability of Frankia in the nutrient solution without P supply was half that of the initial level, whereas, with P supply, there was only a minor decline during the first week. In a growth pouch experiment, supplying P increased plant and nodule growth, irrespective of P status of the inoculant Frankia culture. There were no effects of P status on any growth or nodulation parameters measured when the inoculants had been standardized on the basis of viability. In a split root experiment, Frankia inoculation and application of P together or separately did not cause any significant difference. This suggests that growth and nodulation respond only to total P supply. Increasing P from 0.1 to 10 M significantly increased plant growth but not N concentrations. Both nitrogen-fixation and nitrate supported growth were strongly increased as P increased from 0.1 to 1.0 M. This study indicates that P deficiency limits the growth of host plants more severely than nitrogen fixation processes and P deficiency on nodulation and symbiotic nitrogen fixation in Casuarina cunninghamiana operated indirectly via reducing host plant growth.     

Effects of Brassmosteroids on Growth, Nodulation, Phytohormone Content and Nitrogenase Activity in French Bean Under Water Stress

Effect of pre-treatments of 1 and 5 M epibrassinolide or homobrassinolide prior to water stress induction on changes in root nodulation and contents of endogenous abscisic acid (ABA) and cytokinin trans-zeatin riboside (ZR), and nitrogenase activity was investigated in the nodulated roots of Phaseolus vulgaris L. cv. Arka Suvidha. Brassinosteroids in the unstressed plants increased root nodulation, ZR content and nitrogenase activity, and also ameliorated their stress-induced decline in the nodulated roots. The ABA contents in the nodules of control or stressed plants were not altered by brassinosteroids treatment. There was an increase in pod yield by brassinosteroids treatment (5 M) in the irrigated control as well as stressed plants without influencing pod number or pod length. Among the brassinosteroids, epibrassinolide was relatively more effective.     

Polyamines and proline are affected by cadmium stress in nodules and roots of soybean plants

The effect of moderate (50 M) and high (200 M) doses of Cd were studied in relation to polyamine (Pas) metabolism, proline level and the glutamine synthetase/glutamate synthase system (GS/GOGAT) activity in nodules and roots of soybean plants during 6 days of treatment. The lower Cd concentration increased putrescine (Put) in both nodules and roots, while 200 M Cd increased Spm only in nodules and Put in roots. Spermidine (Spd) decreased in roots under both Cd concentrations. Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were both involved in Put biosynthesis in roots. In nodules, Put formation could mainly be attributed to ODC activity. Diamine oxidase (DAO) activity was severely reduced by 50 and 200 M Cd either in nodules or roots. The GS/GOGAT system activity was depressed either with 50 or 200 M Cd, but most significantly with the highest metal concentration. Under 200 M Cd, GS activity decayed to 25% or 60% of the control in nodules and roots, respectively, while GOGAT decreased 85% in nodules and 79% in roots by day 4 of treatment. Ammonium increased greatly in nodules (200% over the controls) and roots (100%) under 200 M Cd. Proline concentration increased significantly in nodules and roots under both Cd treatments, more markedly under 200 M Cd. The relationship between Pas and proline accumulation and nitrogen assimilation is discussed.     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Structure of root nodules formed by Rhizobium on the non-legume Trema cannabina var. scabra

The structure of the nodules formed by Rhizobium on the non-legume Trema cannabina var. scabra was studied using the light microscope. The overall features of the nodules showed greater resemblance to the non-legume rather than the legume nodule. Nodule squashes yielded bundles of infection threads and bacteroids with morphological differences from rhizobial cells grown on yeast-mannitol-glucose agar. Two types of cell infection occurred within the bacterial zone; plant cells were either, like legumes, filled with rhizobia released from the infection threads (less than one third of infected cells) or were filled with the extensive growth and development of the infection thread. The rate of nitrogen fixation in the Trema nodule was high. It seemed that host cells filled with threads were active in N fixation.     

Callus induction and plant regeneration from excised zygotic embryos of the seed-propagated yams Dioscorea composita Hemsl. and D. cayenensis Lam.

A tissue culture method for regeneration of plantlets from calluses of Dioscorea composita Hemsl. and Dioscorea cayenensis L. is described. Zygotic embryos were used as initial explants. Calluses were obtained on Murashige & Skoog basal medium supplemented with 18 M 2,4-D and plantlets were regenerated on media containing 0.1 M zeatin and 3.3 mM glutamine according to previously described protocols [3]. Inclusion of 0.3% (w/v) activated charcoal in media did not increase callusing. Regeneration of plantlets from D. cayenensis calluses occurred only at low levels of 2,4-D (2.25 M) contained in the media tested. The results indicated that there were genotype-dependent differences between the yam species in their ability to regenerate plantlets in vitro.     

Nitrogen fixation byRubus ellipticus J. E. Smith

Summary Nitrogenase activity as assayed by acetylene reduction was observed in detachedRubus ellipticus J. E. Smith root nodules collected in the field and tested under ambient conditions. The nitrogenase activity was 8.4 moles C₂H₄.gfr. wt nodule^–1.h^–1 or 24.0 moles C₂H₄.g dry wt nodule^–1.h^–1 being at a rate comparable with that measured in some other non-legumes assayed in Java at the same time under similar conditions. Nodule morphology bore little resemblance to the root nodules of other non-leguminous plants and nodule structure was different from the other rosaceous examples.The endophyte inhabiting the root nodules was actinomycetal.     

Aerial root nodules in the tropical legume,Pentaclethra macroloba

Summary Symbiotic nitrogen fixation in angiosperms normally occurs in buried root nodules and is severely inhibited in flooded soils. A few plant species, however, respond to flooding by forming nodules on stems, or, in one case, submerged roots with aerenchyma. We report here the novel occurrence of aerial rhizobial nodules attached to adventitious roots of the legume,Pentaclethra macroloba, in a lowland tropical rainforest swamp in Costa Rica. Swamp sapdings (1–10 cm diameter) support an average 12 g nodules dry weight per plant on roots 2–300 cm above water, and nodules remain in aerial positions at least 6 months. Collections from four swamp plants maintained linear activity rates (3–14 moles C₂H₄/g nodule dry weight/hr) throughout incubations for 6 and 13 hrs; excised nodule activity in most legumes declines after 1–2 hrs. Preliminary study of the anatomy and physiology suggest aerial nodules possess unusual features associated with tolerance to swamp conditions. High host tree abundance and nodulation in the swamp compared to upland sites indicate the aerial root symbiosis may contribute more fixed nitrogen to the local ecosystem than the more typical buried root symbiosis.     

Intracellular pH inSchizosaccharomyces pombe — Comparison withSaccharomyces cerevisiae

We examined cytoplasmic pH regulation inSchizosaccharomyces pombe andSaccharomyces cerevisiae using pH-sensitive fluorescent dyes. Of several different fluorescent compounds tested, carboxy-seminaphthorhodafluor-1 (C.SNARF-1) was the most effective. Leakage of C.SNARF-1 fromS. pombe was much slower than leakage fromC. cerevisiae. Using the pH-dependent fluorescence of C.SNARF-1 we showed that at an external pH of 7, mean resting internal pH was 7.0 forS. pombe and 6.6 forS. cerevisiae. We found that internal pH inS. pombe was maintained over a much narrower range in response to changes in external pH, especially at acidic pH. The addition of external glucose caused an intracellular alkalinization in both species, although the effect was much greater inS. cerevisiae than inS. pombe. The plasma membrane H⁺-ATPase inhibitor diethylstilbestrol reduced both the rate and extent of alkalinisation, with an IC₅₀ of approximately 35 M in both species. Amiloride also inhibited internal alkalinisation with IC₅₀'s of 745 M forS. cerevisiae and 490 M forS. pombe.Abbreviations C.SNARF-1 carboxy-seminaphthorhodafluor-1 (-AM-acetoxy-methylester) - DES diethylstilbestrol - IC₅₀ apparent inhibitory constant - BCECF 2,7-bis-(carboxyethyl)-5(6)-carboxyfluorescein (-AM--pentaacetoxymethyl ester) - FDA fluorescein diacetate     

Inadequate iron supply and high bicarbonate impair the symbiosis of peanuts (Arachis hypogaea L.) with different Bradyrhizobium strains

In the present study, we examined the effects of iron deficiency in an acid solution and in an alkaline solution containing bicarbonate on the growth and nodulation of peanuts inoculated with different bradyrhizobial strains or supplied with fertilizer nitrogen.Inadequate iron supply in acid solution decreased the number of nodule initials, nodule number and nodule mass. Alleviating the iron deficiency increased acetylene reduction but not bacteroid numbers in nodules. Nitrogen concentrations in shoots of inoculated plants increased as iron concentrations in solution increased when determined at day 30 but not at day 50. Higher iron concentrations in solution were required for maximum growth of plants reliant on symbiotic nitrogen fixation than for those receiving fertilizer nitrogen.Adding bicarbonate to the solution with 7.5 M Fe markedly depressed nodule formation. This effect was much more severe than that of inadequate iron supply alone. Bicarbonate also decreased nitrogenase activity but did not decrease bacteroid concentrations in nodules.Both NC92 and TAL1000 nodulated peanuts poorly when bicarbonate was present. However, an interaction between iron concentrations in acid solutions and Bradyrhizobium strains on nodulation of peanuts was observed. Alleviating iron deficiency increased the number of nodule initials and nodules to a much greater extent for plants inoculated with TAL1000 than for plants inoculated with NC92.     

Two new species of Isospora (Apicomplexa: Eimeriidae) from passeriform birds of South America

Summary Two new species of Isospora (Apicomplexa: Eimeriidae) are described from the faeces of passeriform birds of South America. I. cyanocoracis n. sp. is described from Cyanocorax chrysops (Passeriformes: Corvidae) and I. paroariae n. sp. from Paroaria coronata (Passeriformes: Emberizidae). I. cyanocoracis oocysts are spherical or subspherical, 28.7×26.8 m (25.0–30.5×24.5–29.0), with bi-layered wall about 2.0 m thick. Micropyle and oocyst residuum are absent; large polar granule present. Sporocysts are ovoid, 19.3×11.4 m (17.0–21.0×10.5–12.2), with smooth, single-layered wall about 0.8 m thick. Stieda and substiedal bodies and sporocyst residuum are present. Sporozoites 12.2×4.2 m (10.5–15.0×3.5–4.5), possess spherical anterior and posterior refractile bodies. I. paroariae oocysts are spherical or subspherical, 22.3×21.4 m (19.5–25.5×18.5–24.0), and have bi-layered wall about 1.8 m thick. Micropyle, polar granule, and oocyst residuum are absent. Sporocysts ovoid, 15.2×10.0 m (14.0–16.5×8.0–11.5), possess smooth, single-layered wall about 0.7 m thick. Stieda and substiedal bodies and sporocyst residuum are present. Sporozoites elongate, 11.3×3.4 m (10.0–13.5×3.2–4.0), have single, large, posterior refractile body. ac]19840712     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

In vitro clonal propagation of Capsicum spp.

The effect of various concentrations of benzyladenine (BA 4.4–177.5 M) or kinetin (4.7–185.9 M) on shoot proliferation from shoot-tip explants was investigated in C. praetermissum Heiser & Smith and C. annuum L. Maximum number of shoots were obtained on Murashige & Skoog's medium with 66.6 M BA or 92.9 M kinetin in C. praetermissum, and 88.8 M BA or 116.2 M kinetin in C. annuum after 4 weeks of culture. Combining 1 M 2, 3, 5-triiodobenzoic acid (TIBA) with low levels of BA or kinetin significantly increased shoot number as compared to using either cytokinin alone. Rooting of regenerated shoots was achieved on MS medium containing 5.7 M indoleacetic acid. Best rooting (80–100%) was observed in shoots from TIBA plus BA or kinetin media while only 40–50% of shoots from the BA or kinetin treatments were rootable. Plantlets obtained from TIBA plus BA or kinetin were normal diploids while those from BA or kinetin alone revealed distinct chromosomal aberrations in their root tip squashes. Regenerants from TIBA plus BA or kinetin media were successfully established in the soil (86% survival rate), where they flowered and showed normal meiotic behaviour with 100% pollen viability.Abbreviations BA benzyladenine - IAA indole-3-acetic acid - MS Murashige & Skoog's medium - TIBA 2, 3, 5-triiodobenzoic acid