Performance ofin vitro propagatedAlnus glutinosa (L.) Gaertn. clones inoculated withFrankiae

Summary ThreeAlnus glutinosa (L.) Gaertn. clones, obtained byin vitro propagation techniques, were inoculated with four strains ofFrankia. The ability of these clones to nodulate and fix nitrogen was previously reported; this study deals with the performance of 12 different combinations of pairs of symbionts.Shoot fresh weight, shoot height and collar diameter were measured 60 and 82 days after inoculation. Shoot fresh weight seems to be more sensitive and reliable than the other parameters. Nitrogenase activity, measured by the acetylene reduction assay, was assayed 78 days after inoculation and was consistent with the biomass measurements.Better growth was observed when type N strains were used. Significant growth differences were observed between clones AG-2 and AG-8 on the one hand and clone AG-4 on the other. Thus, the use of genetically defined host plants and microsymbionts permitted the demonstration of significant performance variation even among cloned plants from the same provenance (AG-4 and AG-8).The duration of the experiment influenced the results with differences becoming less significant with time. This might be caused by an external limiting factor such as the pot size, competition for light,etc. But it could also be indicative of differences in nodulation speed among the treatments.     

Effect of phosphorus and zinc on nodulation and nitrogen fixation in chickpea (Cicer arietinum L.)

A green house study was conducted on the effect of P and Zn on nodulation and N fixation in chickpea (Cicer arietinum L.) in a loamy sand (Typic Torripsamments) using treatment combinations of five levels of P (0, 25, 50, 100 and 250 ppm), and six levels of Zn (0, 5, 10, 20, 40 and 100 ppm). The number, dry matter and leghaemoglobin content of nodules, and amount of N fixed generally increased with Zn alone upto 19 ppm and P alone upto 50 ppm, and decreased with their higher levels. Application of 25 to 50 ppm P and 5 to 10 ppm Zn counteracted to a greater extent the adverse effect of 40 and 100 ppm Zn, and 250 ppm P, resp. Maximum nodulation and N fixation (91 to 145% over zero P and Zn, at maturity) was recorded with 25 to 50 ppm P applied along with 5 to 10 ppm Zn. At 64 days, depletion in soil-N was noted, particularly when P was applied, whereas at maturity there was a gain in soil-N, ranging from 10.5 to 44.5 kg/2×10⁶ kg soil depending upon P and Zn treatments. The increase in nodulation and N fixation with balanced P and Zn nutrition might be attributed to an increase in leghaemoglobin, and K and Fe concentration in nodules, and increased plant growth, resulting into enhanced activity of N fixing organisms. The results showed that balanced P and Zn nutrition is essential not only for plant growth but also for maximum activity of Rhizobium for N fixation. Work done at Harvana Agricultural University, Hissar, India.     

锌对大叶相思-根瘤菌共生固氮体系影响研究

 研究报道了大叶相思(Acacia auriculaeformis)和美丽胡枝子(Lespedeza formosa)两种根瘤菌在Zn2+重金属环境中耐受性以及植物-根瘤菌共生固氮体系在这种环境中的结瘤、固氮和生长变化,讨论了大叶相思在Pb／Zn矿尾矿废弃地作为先锋植物克服定居初期氮素缺乏的可能性。结果表明：大叶相思根瘤菌对Zn2+的耐受性较强,可以忍受＜10.0 mM的Zn2+浓度,Zn2+对它的半致死浓度是7.91 mM。大叶相思在无重金属无菌砂培条件下固氮酶活性为2.71 C2H4 μg·g-1·h-     

甘露醇对渗透胁迫下的鱼腥藻(Anabaena)固氮活性的增效作用

外源甘露醇可在一定程度上增强鱼腥藻Anabaenasp．7120固氮的抗渗透胁迫能力．厌氧（Ar中）、能量供应受阻（暗处理、添加ATP形成的抑制剂）、合成固氮酶蛋白所需物质供应不足（单加N2不加CO2）以及分子氧下,甘露醇的有益作用减小或消失,反之（正常光照、通气环境、提高CO2浓度,同时供应H2和O2或CO2和N2）则这一作用增大。渗透胁迫下,外源蔗糖对甘露醇支持蓝藻固氮的作用不明显。     

Rock-degrading endophytic bacteria in cacti

A plant–bacterium association of the cardon cactus (Pachycereus pringlei) and endophytic bacteria promotes establishment of seedlings and growth on igneous rocks without soil. These bacteria weather several rock types and minerals, unbind significant amounts of useful minerals for plants from the rocks, fix in vitro N₂, produce volatile and non-volatile organic acids, and reduce rock particle size to form mineral soil. This study revealed the presence of large populations of culturable endophytic bacteria inside the seeds extracted from wild plants, from seeds extracted from the guano of bats feeding on cactus fruit, in seedlings growing from these seeds, in the pulp of fruit, and in small, mature wild plants, and are comparable in size to populations of endophytic populations in some agricultural crops. The dominant culturable endophytes were isolates of the genera Bacillus spp., Klebsiella spp., Staphylococcus spp., and Pseudomonas spp. Based on partial sequencing of the 16s rRNA gene, the isolated strains had low similarity to known strains in these genera. However, these strains have higher molecular similarity among endophytes obtained from seeds, endophytes from roots, and some bacterial strains from the rhizoplane. Seedlings developed from seeds with endophytes contain the similar species of endophytes in their shoots, possibly derived from the seeds. This study shows the involvement of endophytic bacteria in rock weathering by cacti in a hot, subtropical desert and their possible contribution to primary colonization of barren rock. This study proposes that cacti capable of acquiring diverse populations of endophytes may give them an evolutionary advantage to gain a foothold on highly uncompromising terrain.     

Ultrastructure of infected cells in the actinorhizal root nodules ofGymnostoma papuanum (Casuarinaceae) prepared by high pressure freezing and chemical fixation

Summary Actinorhizal root nodules ofGymnostoma papuanuum (Casuarinaceae) were examined with transmission electron microscopy after being either fixed with glutalaldehyde and osmium tetroxide or frozen with liquid nitrogen at high pressure and freeze-substituted. Much better preservation was obtained by the cryopreservation method. Mitochondria, plastids, membranes und ribosomes were much better preserved in the frozen specimens than in the chemically fixed tissues. No nucleoids were observed in the microsymbiont in frozen specimens. In contrast nucleoid regions were present in chemically fixed specimens. The actinomycete microsymbiont differentiates small spherical-shaped symbiotic vesicles after the hyphae have grown and penetrated into most regions of the cytoplasm.Dedicated to the memory of Professor Oswald Kiermayer     

Effect of oxygen on substrate utilization for nitrogen fixation and growth in Frankia spp.

Frankia, the actinomycete partner in the nitrogenfixing symbiosis of certain woody non-legumes, has been shown to fix nitrogen in pure culture under aerobic conditions. The sensitivity of in vivo nitrogen-fixation (acetylene reduction) to oxygen tension in the gas phase was measured in short-term assays with two Frankia isolates designated ARI3 and CcI3. The carbon source utilized had an effect on the optimum O₂ concentration for acetylene reduction. Cells utilizing an organic acid, e.g., propionate or pyruvate had maximum nitrogenase activity at an oxygen concentration of 15 to 20%. In contrast, cells respiring a sugar, e.g., trehalose or glucose, or endogenous reserves (glycogen or trehalose) had maximum acetylene reduction activity at 5 to 10% in the gas phase. Oxygen uptake kinetics showed that respiration in vesicle-containing cells utilizing trehalose had a biphasic response to oxygen concentration with a diffusion limited component at oxygen concentrations of 20 M to more than 300 M. These results suggested that trehalose was oxidized in the vesicles as well as in the vegetative hyphae. Oxygen concentration also had an effect on the trehalose-supported growth of cells (non nitrogenfixing, [⁺NH₄Cl]). Cells grown with 5–10% O₂ in the gas phase had a doubling time approximately half those grown with 20% O₂ (atmospheric). Propionate-grown cells showed similar growth rates at the two oxygen tensions, and grew faster (almost 2x) than the trehalose cells at 5–10% O₂. Trehalose also supported approximately 40% lower rates of oxygen uptake than propionate in vesicle-containing cells.     

Purification and properties of trehalase in Frankia ArI3

Trehalase was purified from cultures of Frankia strain ArI3 grown on media with or without NH₄Cl. The purified enzyme was specific for trehalose, exhibited a broad pH optimum of pH 4.5 to 5.3 and had a K _m for trehalose of 4.2 mM. The trehalase was inhibited in vitro completely by sucrose, glucose and mannose and partially by mannitol and sorbitol. In addition to the specific trehalase, a mixture of non-specific - and -glucosidases which exhibited some activity with ,-trehalose as a substrate were also partially purified in Frankia extracts made from nitrogen-fixing cells. These enzymes were not detected in the purifications of crude extracts made from non-nitrogen-fixing cells (grown on media supplemented with NH₄Cl). Trehalase activity in crude extracts increased over time when cells were induced to fix nitrogen, and the maximum specific activity of trehalase from nitrogen-fixing cultures was 4 times the maximum activity from non-fixing cultures. Trehalase activity was also examined in crude extracts made from Frankia vesicle clusters isolated from Alnus rubra nitrogen-fixing nodules infected with ArI3. The maximum activity of trehalase in these clusters was 6–7 times greater than in the nitrogenfixing pure cultures of ArI3 and 26–33 times greater than the non-fixing pure cultures.Abbreviations pcv packed cell volume - DTE dithioerythritol - PMSF phenylmethylsulphonylfluoride - EDTA sodium ethylenediaminetetraacetate     

Cloning of pEA3, a large plasmid ofEnterobacter agglomerans containing nitrogenase structural genes

Summary A clone bank of an indigenous plasmid ofEnterobacter agglomerans containing structural nitrogen-fixation (nif) genes was established in a non-mobilisable, multicopy derivative of the cosmid vector pHC79. The restriction enzyme Bam HI was used to establish the clone bank and it was found that 96% of the clones contained inserts. The clones containingnif-genes were identified by Southern hybridisation usingKlebsiella pneumoniae nif DNA (KpnifHDKY) as the radioactive probe. Thenif-genes ofE. agglomerans showed extensive homology to those ofK. pneumoniae but the restriction enzyme fragment patterns of thenif-genes ofE. agglomerans were different. The plasmid bornenif-genes ofE. agglomerans are clustered as inK. pneumoniae.