Gnotobiotic cultures of rice plants up to ear stage in the absence of combined nitrogen source but in the presence of free living nitrogen fixing bacteriaAzotobacter vinelandii andRhodopseudomonas capsulata

Summary An all glass tight growth chamber, entirely sterilizable, has been constructed to carry out axenic and gnotobiotic cultures of rice plants (Oryza sativa L.). When grown in liquid medium and in the absence of combined nitrogen but in the presence of the diazotrophsAzotobacter vinelandii andRhodopseudomonas capsulata, rice plants exhibited a complete biological cycle from germination up to ear stage, during a period of time similar to the one encountered in french paddy soil of Camargue. In one experiment, mannitol was given to rice culture medium together withAzotobacter vinelandii andRhodopseudomonas capsulata. In another experiment, mannitol was not given together with Rhodopseudomonas, and still positive nitrogen gain was obtained, although it was less than culture with mannitol. When¹⁵N labeled cells of Rhodopseudomonas were added in rice culture medium,¹⁵N was partly transferred to rice plant. Among the nitrogen substances excreted from the bacteria in the rhizosphere medium, large organic molecules were shown to be the most abundant in our experimental conditions. Moreover, the concentration of free ammonia or aminoacids present in the rice rhizosphere were always compatible with a bacterial nitrogenase activity.     

Isolation of gas vacuole-less mutants of the blue-green alga Anabaenopsis raciborskii

From a bloom forming blue-green alga, Anabaenopsis raciborskii, spontaneous mutants, which had lost the ability to form gas vacuoles have been isolated; the mutant frequency was 4.8×10^-3. The filaments of gas vacuole-less mutants settled at the bottom of flasks in liquid culture media unlike the parent alga. The growth and nitrogen fixation were comparatively poor in the mutants.     

Pleiotropic mutants affecting the control of nitrogen metabolism in Aspergillus nidulans

Summary Mutants of Aspergillus nidulans with lesions in gene amdT are pleiotropically affected in their ability to utilize a wide variety of nitrogen sources in the presence of glucose. Ability to utilize a number of these compounds as sole sources of carbon and nitrogen is not altered. One of these mutants, amdT102, has properties consistent with it being derepressed for glucose repression of the utilization of most (but not all) nitrogen sources. The amdT102 mutant can grow strongly on histidine, lysine and cystine as sole nitrogen sources while the wild type strain grows extremely poorly on these amino acids. Similar but less extreme effects apply to many other nitrogen sources. The amdT19 mutant is unable to utilize most nitrogen sources in the presence of glucose, suggesting that it is subject to greatly increased repression of nitrogen source utilization. The amdT mutants are not affected in their ability to use many compounds as sole carbon sources. Carbon sources other than glucose also affect utilization of nitrogen sources in the amdT mutants.     

氮肥处理对氮素高效吸收水稻根系性状及氮肥利用率的影响

2011—2012年在土培条件下,以氮素吸收效率差异较大的15个常规籼稻为供试材料,研究氮肥运筹对不同氮效率品种根系性状、成熟期吸氮量及氮肥利用率的影响,分析影响氮高效水稻氮素吸收的主要根系性状。结果表明:(1)各氮肥处理下,成熟期吸氮量均表现为氮高效品种氮中效品种氮低效品种。适量增施氮肥及基肥+促花肥处理有利于氮高效品种吸氮量的增加,氮素吸收受品种、氮肥处理的显著影响。(2)在施氮量处理下,氮高效品种单株不定根数、单株根干重、单株不定根总长大或较大,单株根活力在常氮(N2)、高氮(N3)处理下有一定的优势;在施氮时期处理下,氮高效品种单株不定根数、单株不定根总长、单株根干重、单株根系总吸收面积、单株根系活跃吸收面积、抽穗期冠根比多数处理有优势;增施氮肥有利于促进氮高效品种单株不定根总长和单株根活力的提高,适量施氮有利于单株不定根数、单株根干重增加,前期施氮可促进不定根的发生和伸长,后期施氮有利于不定根的充实和根系生理性状的提高。此外,增施氮肥可提高各类品种冠根比;(3)在常氮、高氮处理下,氮高效品种氮肥利用率大于氮中效、氮低效品种。(4)提高单株不定根数、单株不定根总长、单株根活力及抽穗期冠根比有利于各类品种吸氮量的提高,增加根干重对氮高效品种吸氮量的提高也有显著的促进作用。结合相关分析与通径分析结果,抽穗期冠根比及单株不定根数、单株根活力、单株不定根总长、单株根干重是影响氮高效品种吸氮能力的主要根系性状。     

Liberation of amino acids by heterotrophic nitrogen fixing bacteria

Summary. Large amounts of amino acids are produced by nitrogen-fixing bacteria such as Azotobacter, Azospirillum, Rhizobium, Mesorhizobium and Sinorhizobium when growing in culture media amended with different carbon and nitrogen sources. This kind of bacteria live in close association with plant roots enhanced plant growth mainly as a result of their ability to fix nitrogen, improving shoot and root development suppression of pathogenic bacteria and fungi, and increase of available P concentration. Also, it has been strongly evidenced that production of biologically substances such as amino acids by these rhizobacteria are involved in many of the processes that explain plant-grown promotion. This paper reviews literature concerning amino acids production by nitrogen-fixing bacteria. The role of amino acids in microbial interactions in the rhizosphere and establishment of plant bacterial association is also discussed.     

Contribution to nitrogen fixation and soil fertility by photosynthetic bacteria

Summary The nitrogen fixation ability ofRhodopseudomonas capsulatus (a member of the photosynthetic bacteria) has been investigated. This organism can fix N₂ most effectively under illuminated anaerobic conditions. However, in mixed culture in symbiotic association with heterotrophic bacteria, this microorganism using pyruvic acid excreted by the heterotrophs is capable of fixing nitrogen even under an apparent aerobic environment. It has been demonstrated that some correlation exists between the growth of photosynthetic bacteria and the reproduction of the rice plant. Compared to the mineral fertilizer, application of photosynthetic bacteria at the reproductive stage of rice plants increased the yield of grain. This was confirmed by the fact that the root system of rice is capable of absorbing amino acids and nucleic acids excreted by photosynthetic bacteria. Uracil and proline have the most influence on rice reproduction. This is also true for tomato plants. Many toxic molecules such as hydrogen sulfide, amines, etc. are found in soil. They are metabolized by photosynthetic bacteria, which contributes to the detoxication of soil. Such findings were extended to the purification of polluted waste waters from industry and domestic sources,etc. It can be concluded that photosynthetic bacteria contribute very significantly to soil fertility and improvement of the plant growth condition.     

Relationship ofAzotobacter chrooccum siderophores with nitrogen fixation

In iron-limited medium, a siderophore producing soil isolate ofAzotobacter chroococcum showed a high level of hydroxamate with relatively low level of nitrogen fixation. Inclusion of iron in the medium resulted in increased nitrogen fixation with decreased hydroxamate production. Under shake culture conditions, the level of both hydroxamate and catechol type of siderophores decreased after 2 d of incubation in iron-deficient medium. However, under iron-sufficient conditions, both siderophore production and nitrogen fixation increased with time although the level of siderophore was quite low. A number of soil isolates and mutants ofA. chrococcum were tested for nitrogen fixation, hydroxamate and catechol type of siderophore production. Wide variation was observed in the siderophore level and nitrogen fixation in the cultures tested. Nitrogen fixation was higher in the iron-sufficient medium than in iron-limited one while hydroxamate yield was higher in iron-limited medium than in the iron-sufficient one in all the cultures. Inclusion of ammonium acetate in the medium induced catechol synthesis in more than 60% of the cultures.     

Photosynthetic efficiency and nitrogen distribution under different nitrogen management and relationship with physiological N-use efficiency in three rice genotypes

Nitrogen fertilization strategies were widely adopted to enhance grain production and improve nitrogen utilization in rice all over the world. For fertilization timing strategy, ear fertilization was usually employed in recent years. For fertilization amount strategy, nitrogen fertilization would continually increase to meet the demands of increasing people for food. However, under heavy ear fertilization as well as great nitrogen amount (NA), physiological N-use efficiency (PE, defined as grain production per unit nitrogen uptake by plants) decreased. Under three NA and two ratios of fertilization given during ear development period to total NA (ear fertilization distribution ratio, EFDR), net photosynthetic rate (Pn), Pn to nitrogen content per unit area (photosynthetic N-use efficiency, Pn/N), nitrogen accumulation in plant tissues and PE of three rice (Oryza sativaL.) genotypes, Jinyou 253, Liangyoupeijiu and Baguixiang were screened in the first and second seasons in 2002 so as to understand the fluctuation patterns of Pn/N and nitrogen distribution in leaf blades under great NA & EFDR and relationship with PE in rice. Results showed that under greater NA & EFDR, Pn in flag leaves at heading and plant nitrogen accumulation at maturity always increased and PE & Pn/N always decreased in spite of increased grain production. Rice distributed more nitrogen in leaf blade under greater NA and EFDR. PE indicated significantly (P<0.05) positive relationship with Pn/N and negative relationship with nitrogen distribution ratio in leaf blades at heading and maturity, and no association with Pn in two growing seasons. Results suggested that low PE in rice under great NA and heavy ear fertilization is associated to more nitrogen distribution in leaf blades and decreases in photosynthetic efficiency.     

Azide-resistant mutants ofAzorhizobium caulinodans with enhanced symbiotic effectiveness

Azide-resistant mutants ofAzorhizobium caulinodans strains Sb3, S78, SrR13 and SrS8 were isolated and screened for nitrate reductase activity. Selected nitrate reductase negative mutants were inoculated onSesbania bispinosa andS. rostrata under sterile conditions in chillum jars to study their symbiotic behavior. Azide-resistant mutants exhibited either similar or higher symbiotic effectiveness than the parent strain after 30 d of plant growth. Nodule mass, nitrogenase activity and uptake hydrogenase activity of the mutants varied depending on the host as well as on the plant growth stage. In comparison to wild-type parent strains, four azide-resistant mutants, Sb3Az18, S78Az21, SrR13Az17 and SrS8Az6 showed significant increase in nodulation and nitrogen fixation as well as shoot dry mass of the inoculated plants.     

Isolation and characterisation of nitrate reductase mutants and regulation of nitrate reductase and nitrogenase in the cyanobacterium Nostoc muscorum

Summary Chlorate resistant mutants of the cyanobacterium Nostoc muscorum isolated after N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis were found to be defective/blocked in nitrate reductase (NR).The parent strain possessed active NR in the presence of nitrogen as nitrate and only basal levels of activity in ammonia and N-free grown cultures. Addition of ammonia suppressed the NR activity in the parent strain whereas addition of L-methionine DL-sulphoximine (MSX) restored NR activity. A similar repression by ammonia, glutamine and derepression with MSX were also observed for nitrogenase synthesis.One class of mutants lacked NR activity (nar ^-) whereas the specific activity of NR was low in another class of mutants (nar ^def). Unlike the parent, the mutants synthesized nitrogenase and differentiated heterocysts in the presence of nitrate nitrogen. Uptake studies of nitrite and ammonia in mutants revealed that they possessed both nitrite reductase and glutamine synthetases (GS) at low levels, and the same level respectively in comparison with the parent.     

Improved Nutrient Use Efficiency Increases Plant Growth of Rice with the Use of IAA-Overproducing Strains of Endophytic Burkholderia cepacia Strain RRE25

Effect of indole acetic acid (IAA)-overproducing mutants of Burkholderia cepacia (RRE25), a member of β-subclass of Proteobacteria and naturally occurring rice endophyte, was observed on the growth of rice (Oryza sativa L.) plants grown under greenhouse conditions. Nine mutants were characterized for altered biosynthesis of IAA after nitrous acid mutagenesis. These mutants were grouped into two classes: class I mutants have reduced production of IAA as compared to the wild type, while class II mutants showed overproduction of IAA. Mutants of both classes and RRE25, the parent (wild type), were inoculated on rice seedlings of two cultivars (Sarjoo-52 and NDR-97). Uptake of nitrogen, phosphorous, and potassium was estimated in these plants. Significant increase in the amount of uptake of all three elements was observed when inoculated with the IAA-overproducing mutants over control as well as in the plants inoculated with the wild type (RRE25). Effect of inoculation of IAA-overproducing mutants was more pronounced on the uptake of phosphorous in cultivar NDR-97 than Sarjoo-52, while it was opposite with respect to potassium uptake. Any significant difference was not observed in nitrogen uptake among the two cultivars. It shows that the host also plays an important role in the beneficial endophytic association. It was concluded from these results that one of the possible mechanisms of growth promotion of rice plants inoculated with bacterial endophytes is their effects on an increase in the capability of nutritional uptake possible through the effect of IAA production which results in proliferation of root system that could mine more nutrients from the soil.     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N₂ at 39° but grew normally at 30° on N₂ and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation.     

Control of synbiotic nitrogen fixation in Rhizobia. Regulation of NH4+ assimilation.

This communication is concerned with physiological, biochemical, and genetic studies of the regulation of ammonium (NH4+) assimilation by Rhizobia (root nodule bacteria) that infect leguminous plants. The major conclutions are (i) physiological studies show that Rhizobia are able to assimilate NH4+ for growth only when supplemented with certain organic nitrogen sources (e.g., L-aspartate, L-leucine, L-serine). Addition of as little as 2 mug/ml of L-aspartate supported growth on NH4+ as nitrogen source. In contrast, addition of glutamate in combination with NH4+-blocked NH4+ utilization; (ii) biochemical analysis show that glutamate synthase activity (NADP- and NAD-linked) is always present in cells capable of assimilating NH4+; also cells without glutamate synthase activity were found to be incapable of NH4+ utilization. Glutamate synthase levels were observed to fluctuate markedly depending on the available nitrogen source and on the growth stage of the culture; (iii) mutants were selected in which assimilation of NH4+ is no longer subject to inhibition (repression?) by glutamate. The levels of glutamate synthase activity (NADP-linked) (in the presence of glutamate) show approximately a two-fold increase over the level in the parent strain. The mutants no longer require supplementation with small amounts of organic nitrogen for growth in medium containing inorganic nitrogen (e.g., NH4+ or NO3-); (iv) these findings are discussed in relation to the working model of symbiotic nitrogen fixation recently proposed (O'Gara and Shanmugam (1976), Biochim. Biophys. Acta 437, 313--321).     

Associative effect of cellulolytic fungi andAzospirillum lipoferum on yield and nitrogen uptake by wheat

The associative effect of cellulolytic fungi, such asAspergillus awamori andA. niger, with the nitrogen fixer,Azospirillum lipoferum was studied in a soil amended with rice straw. All the inoculants gave significantly higher grain and straw yield and nitrogen uptake by wheat crop than did the uninoculated treatment. The doubling of chemical nitrogen dose significantly increased the yield and nitrogen uptake. It was observed thatA awamori performed significantly better followed byA. niger andA. lipoferum. The maximum benefit was obtained with combined inoculation ofA. awamori andA. lipoferum. Another experiment was conducted in the subsequent year in soil amended with and without rice straw using cellulolytic culture eitherA. awamori orSclerotium rolfsii, andA. lipoferum. Application of straw in soil significantly reduced the yield and N-uptake by wheat crop as compared to the controls. All the inoculants exceptS. rolfsii gave significantly higher grain yield. However, N-uptake by grain was significantly increased only by combined inoculation ofA. lipoferum and either one of the cellulolytic fungi. Similar trends on yield and N-uptake of straw due to inoculants were observed. The maximum benefit was obtained with combined inoculation ofA. awamori andA. lipoferum followed byA. awamori alone on grain yield and only combined inoculants on N-uptake by the crop.     

Isolation of nitrogen deregulated mutants of erythromycin biosynthesis

Summary Ammonium represses erythromycin synthesis bySaccharopolyspora erythraea and insensitive mutants to this effect were isolated. Six mutants were selected and one of them produces 50% more antibiotic than the wild type in 100 mM NH₄Cl as nitrogen source. Glutamine synthetase and alanine dehydrogenase levels in the mutants were determined and no differences with wild type strain were observed.     

Algal nitrogen fixation in the tropics

Summary a)Nitrogen fixation in rice fields. Nitrogen-fixing blue-green algae grow abundantly in tropical regions and are particularly common in paddy fields. Their possible role in the nitrogen accumulation of soil has been studied. The most vigorous nitrogen-fixing blue-green algae have been assessed for use as green manure in rice fields and favorable effects have been reported in India and other countries. b)Nitrogen fixation by algae in water. The planktonic blue-green algae occur abundantly at certain time of the year in sea water and lake water, and some of them are known to be nitrogen fixers. Certain Japanese species of blue-green algae can withstand high temperatures including ten nitrogen-fixing species from hot-spring waters. c)Nitrogen fixation by symbiotic blue-green algae. Certain species of blue-green algae form associations with other organisms such as fungi, liverworts, ferns and seed plants. The relationship between these two organisms is on one occasion commensal and on others symbiotic. Certain symbiotic blue-green algae are provided with the ability to fix the atmospheric nitrogen.     

Assay of nitrogen supplying capacity of tropical rice soils

Summary The nitrogen supplying capacity of 39 wetland rice soils evaluated by two anaerobic incubation methods and six chemical methods was compared with N uptake of IR 26 rice grown on these soils under flooded conditions in a greenhouse pot study. The uptake of N by rice correlated highly with the N supplying capacity determined by anaerobic incubation methods involving incubation of soils at 30°C for 2 weeks (r=0.84^**) or at 40°C for 1 week (r=0.82^**) as well as with the organic carbon (r=0.82^**) and total N (r=0.84^**) contents of soils. Among the chemical indexes, available N determined by the oxidative release of soil N by alkaline permanganate, acid permanganate, acid dichromate and hydrogen peroxide also provided good index of soil N availability to rice. According to these results soil organic carbon and total N contents seem to be good indexes of available nitrogen in tropical wetland rice soils.     

Enhanced OsNLP4‐OsNiR cascade confers nitrogen use efficiency by promoting tiller number in rice

Increased use of nitrogen fertilizers has deleterious impact on the environment. Increase in yield potential at low nitrogen supply is regarded as a cereal breeding goal for future agricultural sustainability. Although natural variations of nitrogen transporters have been investigated, key genes associated with assimilation remain largely unexplored for nitrogen use efficiency (NUE) enhancement. Here, we identified a NIN‐like protein NLP4 associated with NUE through a GWAS in rice. We found that OsNLP4 transactivated OsNiR encoding nitrite reductase that was critical in nitrogen assimilation in rice. We further constructed quadrupling NREs (Nitrate‐responsive cis‐elements) in the promoter of OsNiR (p4xNRE:OsNiR) and enhanced nitrogen assimilation significantly. We demonstrated that OsNLP4‐OsNiR increased tiller number and yield through enhancing nitrogen assimilation and NUE. Our discovery highlights the genetic modulation of OsNLP4‐OsNiR signalling cascade as a strategy for high NUE and yield breeding in rice.     

Effect of rates of nitrogen and relative efficiency of sulphur-coated urea and nitrapyrin-treated urea in dry matter production and nitrogen uptake by rice

Summary A field experiment conducted for two rainy seasons (1974 and 1975) on a sandy clay loam soil at the Indian Agricultural Research Institute, New Delhi showed that at 100kg N/ha the apparent recovery of urea nitrogen by the rice crop was only 28%, which was raised to 41.7% by treating urea with Nitrapyrin and to 47.4% by coating urea withneem (Azadirachta indica Juss) cake. The recovery with sulphur-coated urea was 37.7%. Dry matter production nitrogen concentration in plant and uptake by rice were increased as the rate of nitrogen was increased from 0 to 150kg N/ha. Advantage of treating urea with Nitrapyrin or coating withneem cake was seen more in grain than straw yield.