Physiological and molecular diversity of feather moss associative N2-fixing cyanobacteria

Cyanobacteria colonizing the feather moss Pleurozium schreberi were isolated from moss samples collected in northern Sweden and subjected to physiological and molecular characterization. Morphological studies of isolated and moss-associated cyanobacteria were carried out by light microscopy. Molecular tools were used for cyanobacteria identification, and a reconstitution experiment of the association between non-associative mosses and cyanobacteria was conducted. The influence of temperature on N2 fixation in the different cyanobacterial isolates and the influence of light and temperature on N2-fixation rates in the moss were studied using the acetylene reduction assay. Two different cyanobacteria were effectively isolated from P. schreberi: Nostoc sp. and Calothrix sp. A third genus, Stigonema sp. was identified by microscopy, but could not be isolated. The Nostoc sp. was found to fix N2 at lower temperatures than Calothrix sp. Nostoc sp. and Stigonema sp. were the predominant cyanobacteria colonizing the moss. The attempt to reconstitute the association between the moss and cyanobacteria was successful. The two isolated genera of cyanobacteria in feather moss samples collected in northern Sweden differ in their temperature optima, which may have important ecological implications.     

Comparative analysis of cyanobacteria in the rhizosphere and as endosymbionts of cycads in drought-affected soils

Does the diversity of cyanobacteria in the cycad rhizosphere relate to the cyanobiont species found in the coralloid roots of these ancient plants? The aim of this study was to identify the diversity of soil cyanobacteria occurring in the immediate vicinity of 22 colonized coralloid roots belonging to members of the cycad genera: Macrozamia, Lepidozamia, Bowenia and Cycas. The majority of coralloid roots were sampled at depths >?10?cm below the soil surface. A total of 32 cyanobacterial isolates were cultured and their 16S rRNA gene partially sequenced. Phylogenetic analysis revealed nine operational taxonomic units of soil cyanobacteria comprising 30 Nostoc spp., a Tolypothrix sp. and a Leptolyngbya sp. Microscopy indicated that all isolates were unialgal and confirmed their genus identity. Rhizospheric diversity was compared to existing data on cyanobionts isolated at the same time from the cycad coralloid root. The same isolate was present in both the cycad coralloid root and rhizosphere at only six sites. Phylogenetic evidence indicates that most rhizosphere isolates were distinct from root cyanobionts. This weak relationship between the soil cyanobacteria and cycad cyanobionts might indicate that changes in the soil community composition are due to environmental factors.     

Nitrogen deprivation stimulates symbiotic gland development in Gunnera manicata

Gunnera is the only genus of angiosperms known to host cyanobacteria and the only group of land plants that hosts cyanobacteria intracellularly. Motile filaments of cyanobacteria, known as hormogonia, colonize Gunnera plants through cells in the plant's specialized stem glands. It is commonly held that Gunnera plants always possess functional glands for symbiosis. We found, however, that stem gland development did not occur when Gunnera manicata plants were grown on nitrogen (N)-replete medium but, rather, was initiated at predetermined positions when plants were deprived of combined N. While N status was the main determinant for gland development, an exogenous carbon source (sucrose) accelerated the process. Furthermore, a high level of sucrose stimulated the formation of callus-like tissue in place of the gland under N-replete conditions. Treatment of plants with the auxin transport inhibitor 1-naphthylphthalamic acid prevented gland development on N-limited medium, most likely by preventing resource reallocation from leaves to the stem. Optimized conditions were found for in vitro establishment of the Nostoc-Gunnera symbiosis by inoculating mature glands with hormogonia from Nostoc punctiforme, a cyanobacterium strain for which the full genome sequence is available. In contrast to uninoculated plants, G. manicata plants colonized by N. punctiforme were able to continue their growth on N-limited medium. Understanding the nature of the Gunnera plant's unusual adaptation to an N-limited environment may shed light on the evolution of plant-cyanobacterium symbioses and may suggest a route to establish productive associations between N-fixing cyanobacteria and crop plants.     

Physiological sources of reductant for nitrogen fixation activity in Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus.

Pure cultures of the symbiotic cyanobacterium-bryophyte association with Anthoceros punctatus were reconstituted by using Nostoc sp. strain UCD 7801 or its 3-(3,4-dichlorophenol)-1,1-dimethylurea (DCMU)-resistant mutant strain, UCD 218. The cultures were grown under high light intensity with CO2 as the sole carbon source and then incubated in the dark to deplete endogenous reductant pools before measurements of nitrogenase activities (acetylene reduction). High rates of light-dependent acetylene reduction were obtained both before starvation in the dark and after recovery from starvation, regardless of which of the two Nostoc strains was reconstituted in the association. Rates of acetylene reduction by symbiotic tissue with the wild-type Nostoc strain decreased 99 and 96% after 28 h of incubation in the dark and after reexposure to light in the presence of 5 microM DCMU, respectively. Supplementation of the medium with glucose restored nitrogenase activity in the dark to a rate that was 64% of the illuminated rate. In the light and in the presence of 5 microM DCMU, acetylene reduction could be restored to 91% of the uninhibited rate by the exogenous presence of various carbohydrates. The rate of acetylene reduction in the presence of DCMU was 34% of the uninhibited rate of tissue in association with the DCMU-resistant strain UCD 218. This result implies that photosynthates produced immediately by the cyanobacterium can supply at least one-third of the reductant required for nitrogenase activity on a short-term basis in the symbiotic association. However, high steady-state rates of nitrogenase activity by symbiotic Nostoc strains appear to depend on endogenous carbohydrate reserves, which are presumably supplied as photosynthate from both A. punctatus tissue and the Nostoc strain.     

Discovery of rare and highly toxic microcystins from lichen-associated cyanobacterium Nostoc sp. strain IO-102-I

The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda(5)]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda(5)]microcystin-LR and [d-Asp(3),ADMAdda(5)]microcystin-LR and a partial structure of three new [ADMAdda(5)]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.     

Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species.

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and (15)N(2) gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.     

Growth responses of larvae of the rice stem borer to rice plants treated with 2,4-D

Summary Growth response of rice stem borer larvae to rice plants treated with 2,4-D was investigated. In greenhouse rearing experiments, growth of the larvae was improved when they fed on 2,4-D treated plants. In aseptic rearing experiments, the larvae also grew better on stems of rice plants treated during growth with 2,4-D than on untreated stems. On the other hand, larval growth was not improved by addition of 2,4-D to a diet of sterilized rice stems. The nitrogen content of the 2,4-D treated plant is higher than that of the untreated plant. Because of the importance of nitrogenous compounds for larval growth, the improved growth on 2,4-D treated plants was apparently caused by the increased nitrogen content of the plants, and not as a direct effect of 2,4-D itself.Apparently the increased infestation of rice stem borer larvae on rice plants treated with 2,4-D arises because of the ability of such plants to support good larval growth.

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Zusammenfassung Es wurden die Wachstumsreaktionen vonChilo suppressalis-Larven auf Reispflanzen untersucht, die mit 2,4-D behandelt worden waren. In Gewächshausaufzuchten verbesserte sich das Wachstum der Raupen, wenn sie mit 2,4-D-behandelten Pflanzen ernährt wurden. Auch in aseptischen Zuchtversuchen wuchsen die Larven besser an Halmen von Reispflanzen, die während des Aufwuchses mit 2,4-D behandelt worden waren, als an unbehandelten Stengeln. Andererseits wurde das larvale Wachstum durch Hinzufügen von 2,4-D zu einer Diät sterilisierter Reishalme nicht verbessert. Der Stickstoffgehalt der mit 2,4-D behandelten Pflanzen ist höher als der unbehandelter. In Anbetracht der Bedeutung stickstoffhaltiger Verbindungen für die Raupenernährung wird die Wachstumssteigerung auf 2,4-D-behandelten Pflanzen offensichtlich durch den erhöhten Stickstoffgehalt der Pflanzen verursacht und nicht durch eine direkte Wirkung des 2,4-D selbst.Der erhöhte Befall 2,4-D-behandelter Pflanzen durchChilo suppressalis beruht offensichtlich auf der Fähigkeit solcher Pflanzen, gutes Raupenwachstum zu unterstützen.

     

2,4-D诱导固氮螺菌和慢生型大豆根瘤菌在小麦上的结瘤作用及其效果

分别在水培和砂培条件下进行了2,4-D诱导固氮螺菌和慢生型大豆根瘤菌在小麦根上的结瘤试验,结果表明2,4-D能诱发它们在小麦根系上形成“类根瘤”,扫描电镜结果证明只有个别细菌进入小麦根瘤细胞内,在细胞间隙有较多的细菌。用乙炔还原法仅检测到接种大豆根瘤菌的小麦根瘤有微量的固氮酶活性,但在盆栽植株的生长方面,看不到2,4-D,2,4-D+固氮螺菌或2,4-D+根瘤菌对小麦生长的促进作用。     

Associative cyanobacteria isolated from the roots of epiphytic orchids

Associative cyanobacteria were isolated from the rhizoplane and velamen of the aerial roots of the epiphytic orchids Acampe papillosa, Phalaenopsis amabilis, and Dendrobium moschatum and from the substrate roots of Acampe papillosa and Dendrobium moschatum. Cyanobacteria were isolated on complete and nitrogen-free variants of BG-11 medium. On all media and in all samples, cyanobacteria of the genus Nostoc predominated. Nostoc, Anabaena, and Calothrix were isolated from the surface of the A. papillosa aerial roots, whereas the isolates from the substrate roots were Nostoc, Oscillatoria, and representatives of the LPP-group (Lyngbia, Phormidium, and Plectonema, incapable of nitrogen fixation). On the D. moschatum substrate roots, Nostoc and LPP-group representatives were also found, as well as Fischerella. On the aerial roots of P. amabilis and D. phalaenopsis grown in a greenhouse simulating the climate of moist tropical forest, cyanobacteria were represented by Nostoc, LPP-group, and Scytonema in the D. phalaenopsis and by Nostoc, Scytonema, Calothrix, Spirulina, Oscillatoria, and the LPP-group in P. amabilis. For D. moschatum, the spectra of cyanobacteria populating the substrate root zhizophane and the substrate (pine bark) were compared. In the parenchyma of the aerial roots of P. amabilis, fungal hyphae and/or their half-degraded remains were detected, which testifies to the presence of mycorrhizal fungi this plant. This phenomenon is attributed to the presence of a sheath formed by cyanobacteria and serving as a substrate for fungi.     

Colonization of wheat by Azospirillum brasilense Cd is impaired by saline stress

The effect of saline stress on the colonization of wheat was analyzed by using Azospirillum brasilense Cd carrying the fusion of the reporter gene lacZ (β-galactosidase) with the N₂ fixation gene promoter nifA. Colonization was also studied by inducing para-nodules on wheat roots using 2,4-D, establishing that these structures acted as bacterium protected niches. Bacteria grown under standard conditions were distributed along the whole root system, except the elongation zone, and colonized the para-nodules. Bacteria experiencing saline stress were mainly localized at the root tips and the lateral roots. In 2,4-D treated plants, most of the bacteria were present around the basal surface of the modified lateral root structures. Using the MPN method, there were not statistical differences between the numbers of control and stressed bacteria. As this method estimates endophytic colonization in contrast with the one using X-gal, which emphasizes colonization on the root surface, both procedures demonstrated to be necessary, concluding that salt treatment reduced surface colonization (X-gal) but not colonization inside the root. The bacterial counts made on inoculated wheat roots indicated higher numbers of both control and stressed bacteria in roots treated with 2,4-D compared with untreated roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

The devR gene product is characteristic of receivers of two-component regulatory systems and is essential for heterocyst development in the filamentous cyanobacterium Nostoc sp. strain ATCC 29133.

Strain UCD 311 is a transposon-induced mutant of Nostoc sp. strain ATC C 29133 that is unable to fix nitrogen in air but does so under anoxic conditions and is able to establish a functional symbiotic association with the hornwort Anthoceros punctatus. These properties of strain UCD 311 are consistent with previous observations that protection against oxygen inactivation of nitrogenase is physiologically provided within A. punctatus tissue. Upon deprivation of combined nitrogen, strain UCD 311 clearly differentiates heterocysts and contains typical heterocyst-specific glycolipids; it also makes apparently normal akinetes upon phosphate starvation. Sequence analysis adjacent to the point of the transposon insertion revealed an open reading frame designated devR. Southern analysis established that similar sequences are present in other heterocyst-forming cyanobacteria. devR putatively encodes a protein of 135 amino acids with high similarity to the receiver domains of response regulator proteins characteristics of two-component regulatory systems. On the basis of its size and the absence of other functional domains, DevR is most similar to CheY and Spo0F. Reconstruction of the mutation with an interposon vector confirmed that the transposition event was responsible for the mutant phenotype. The presence of wild-type devR on a plasmid in strain UCD 311 restored the ability to fix nitrogen in air. While devR was not essential for differentiation of akinetes, its presence in trans in Nostoc sp. strain ATCC 29133 stimulated their formation to above normal levels in aging medium. On the basis of RNA analysis, devR is constitutively expressed with respect to the nitrogen source for growth. The devR gene product is essential to the development of mature heterocysts and may be involved in a sensory pathway that is not directly responsive to cellular nitrogen status.     

根瘤菌在大麦和水稻根上形成拟瘤的细胞结构

用3种方法使紫云英根瘤菌(Rhizobium astragali Huikui)、田菁根瘤菌(R.sesbania sp.)分别入侵大麦(hordeum vulgare L.)和水稻(Oryza sativa L.),形成拟瘤状组织。一是用一定磁场强度处理根瘤菌和植物,并接种培养。二是用含有水稻幼苗根提取物的培养基培养根瘤菌,接种水稻。三是重复别人用2,4-D外源激素处理植物,接种根瘤菌。镜检观察,用紫云英根瘤菌接种形成的大麦根拟瘤细胞结构非常精细,保持各种细胞器。有侵入线结构和根瘤菌从侵入线释放。根瘤菌被宿主细胞来源的膜包围,成为拟菌体。这些形态结构与豆科根瘤细胞相似,有共生状态特征,但拟菌体有泡状化现象。田菁根瘤菌入侵水稻根形成的拟瘤,在细胞间隙和细胞内都有细菌分布。受侵染的细胞结构粗糙,根瘤菌裸露,无胞膜包围。用2,4-D处理接种根瘤菌的拟瘤细胞结构也如此,但在维管系统内有大量密集的细菌存在。这种结构完全不同于豆科根瘤细胞结构,细菌与植物细胞的形态学相互关系是一种非共生联合作用。     

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.     

Possible contribution of Bradyrhizobium on nitrogen fixation in sweet potatoes

Aims

Sweet potato (Ipomoea batatas) is known for its ability to grow under nitrogen-limited conditions. To clarify the possible contribution of biological nitrogen fixation, we tried to isolate and identify diazotrophic bacteria from sweet potatoes.

Methods

By using cultivation technique, we isolated putative endophytes, which possess nifH genes, from surface-sterilized sweet potatoes. Their nitrogen-fixing abilities were demonstrated by the acetylene reduction assay in a semi-solid malate medium and sweet potato extracts. We also examined the colonization of an isolated strain (AT1) in sweet potatoes and their influence on growth and nitrogen fixation in plants as assessed by an acetylene reduction assay and ¹⁵N-isotope dilution technique.

Results

The isolates were identified as strains of Bradyrhizobium sp. AT1, Paenibacillus sp. AS2 and Pseudomonas sp. T16 based on their 16S rRNA gene sequences. They showed acetylene reduction activity (ARA) in the semi-solid malate medium. Among them, B. sp. AT1 showed ARA in sweet potato extracts under micro-aerobic conditions whereas both P. sp. AS2 and P. sp. T16 showed no ARA. The inoculation of B. sp. AT1 to the sweet potatoes resulted in increases in the fresh weights and detection of ARA in the inoculated plants. Moreover, the reduction of ¹⁵N atom % was observed in the inoculated plants compared to uninoculated controls.

Conclusions

B. sp. AT1 actively expresses nitrogenase activity in sweet potatoes and may contribute to the nitrogen nutrition of host plants.     

Effects of inoculation ofPoa pratensis andTriticum aestivum with root-associated,N2-fixing Klebsiella,Enterobacter and Azospirillum

Strains ofKlebsiella pneumoniae, Klebsiella terrigena, Enterobacter agglomerans andAzospirillum lipoferum were compared as diazotrophic inoculants in association withPoa pratensis andTriticum aestivum. Each strain colonized both plants in numbers ranging from 10⁴ to 10⁷ bacteria per root, and electron microscopy and immunofluorescence staining of inoculated roots revealed bacteria mainly on root hairs. Indirect immunofluorescence with specific antifimbriae antibodies showed that the enteric bacteria expressed their fimbria in both associations. All associations were positive in an acetylene reduction test but only in half of them was atmospheric nitrogen transferred to the plant. In the inoculated plants, variable effects in the dry matter and N yields in both hosts were observed and no correlation was found between dry matter, nitrogen content or the amount of fixed nitrogen. In infected plants, the number of root hairs and lateral roots increased and the length of the zone of elongation decreased. The changes in root morphology were more evident in associations with enteric bacteria than with Azospirillum. The results give further evidence on the importance of bacterial adhesion in associative N₂ fixation and suggest that bacteria-induced physiological changes in plant roots may be more important than the amount of nitrogen transferred to the plant.     

Auxin-independent growth of maize tissue culture cells

Wall-regenerating protoplasts and suspension culture cells of Zea mays L. ‘Black Mexican Sweet’ were co-cultivated] with Ti plasmid-containing Agrobacterium tumefaciens strain ACH5. After elimination of the bacteria, putative transformants were selected for their ability to grow in the absence of added auxin. Althoug some of the treated cells grew, untreated controls and cells treated with the Ti plsmidless strain ACH5C3 also proliferated. The frequency of auxin-independent growth was similar in all treatments. Because none of 75 candidate transforms contained detectable T-DNA, the hormone-independent phenotype appears to be a consequence of habituation. Although some of the habituated colonies grew as undifferentiated callus, others produced rootlike structures. Miaze cells subcultured in liquid media containing progressively reduced concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) also became habituated.     

Inoculation of forage grasses with N2-fixing Enterobacteriaceae

Summary Previous investigations indicated some forage grass roots in Texas are heavily colonized with N₂-fixing bacteria. The most numerous N₂-fixing bacteria were in the genera Klebsiella and Enterobacter. In the present investigation inoculation experiments were conducted using 18 isolates of these bacteria to determine if a N₂-fixing association could be established between the bacteria and the grassesCynodon dactylon andPanicum coloratum. Plants were grown in soil for approximately 5 months in a greenhouse and were measured periodically for dry matter, nitrogen accumulation, and acetylene reduction activity. Results of the investigation indicated that 25% of the plant-soil systems were active in acetylene reduction and the activity was high enough to indicate agronomically significant quantities of N₂ were being fixed (>8kg N ha⁻¹). However, plant systems extrapolated to fix>8 kg N ha⁻¹ contained less nitrogen and accumulated less dry matter than plants less active in acetylene reduction. Inocula could not be re-isolated from healthy grass roots indicating that the N₂-fixing activity may have not have been closely assiciated with plant roots. Future research is needed to determine factors limiting colonization of grass roots.     

Regulation of expression of glutamine synthetase in a symbiotic Nostoc strain associated with Anthoceros punctatus.

A characteristic of N2-fixing cyanobacteria in symbiotic associations appears to be release of N2-derived NH4+. The specific activity of the primary ammonium-assimilating enzyme, glutamine synthetase (GS), was found to be three- to fourfold lower in Nostoc sp. strain 7801 grown in symbiotic association with the bryophyte Anthoceros punctatus than in free-living Nostoc sp. strain 7801. Quantitative immunological assays with antisera against GS purified from Nostoc sp. strain 7801 and from Escherichia coli indicated that similar amounts of the GS protein were present in symbiotic (50 micrograms mg-1) and free-living (68 micrograms mg-1) cultures. The conclusion from these experiments is that GS is regulated by a posttranslational mechanism in Anthoceros-associated Nostoc sp. strain 7801. However, the results of comparative catalytic and immunological experiments between N2- and NH4+-grown free-living Nostoc sp. strain 7801 implied control of GS synthesis. A correlation was not observed between the level of GS expression and the extent of symbiotic heterocyst differentiation in Nostoc sp. strain 7801 associated with A. punctatus.     

Formation of artificial nitrogen-fixing symbiosis with rape (Brassica napus var. napus) plants in nonsterile soil]

The treatment of rape plants grown in nonsterile soil with 2,4-dichlorophenoxyacetic acid (auxin-like growth-promoting substance) or their inoculation with the bacterial association Micrococcus sp. + Rhodococcus sp. and/or with the mixed nitrogen-fixing culture Azotobacter nigricans + Bacillus sp. led to the formation of paranodules on the rape roots. The introduced bacteria were detected both in the intercellular space and inside the cells of the paranodules and the rape roots. The nitrogen-fixing activity of the paranodulated plants was two times higher than that of the inoculated plants lacking paranodules and five times higher than that of the control (i.e., not inoculated) plants. The paranodulation led to a 40% increase in the crop yield of rape plants and provided for a statistically significant increase in the total nitrogen as well as protein nitrogen contents of the plants.     

Reassociation of a modified mycorrhiza with the host plant roots (Pinus radiata) and the transfer of acetylene reduction activity

Summary Experiments were carried out to assess the potential for reassociation of modified strains of the mycorrhizal fungus Rhizopogon sp., capable of acetylene reduction activity in vitro, with the roots of its host plant (Pinus radiata). Reassociation was effected and acetylene reduction assays indicated that nitrogenase activity was present in the reassociated whole plants. Those host plants symbiotic with the modified strains had higher levels of nitrogen than those associated with the wild type fungus under nitrogen deprived conditions. Uptake of phosphate was unimpaired in the modified mycorrhiza. Electron microscopy showed that hyphae of the modified strains as well as lying in the intercellular spaces were often found within the cells of the root cortex. This was in contrast with the wild type strains where no such intracellular growth was found. One strain was sound to be pathogenic to seedlings of Pinus radiata. re]19760603