Effects of symbiosis with Rhizobium fredii on transport of fixed nitrogen in the xylem of soybean plants

An experiment was conducted to identify the main nitrogenous compound transported in the xylem sap of soybean plants nodulated with Rhizobium fredii. Soybean (Glycine max L. Merr.) cultivars, wild type Bragg (nod⁺, fix⁺) and its nitrate tolerant, hypernodulating mutant ntsll16 (nod⁺⁺, fix⁺) were used for this experiment. These soybean plants were inoculated with a slowgrowing rhizobium, Bradyrhizobium japonicum USDAllO or fast-growing rhizobia consisting of a mixture of R. fredii USDA191, USDA193, and USDA-194 and grown in a phytotron under natural light and controlled temperature conditions. Xylem sap was collected from Bragg and ntsll16 plants at the flowering and pod elongation stages. Acetylene reduction activity per plant or per nodule weight was not different between soybean lines and inoculums. The composition of the nitrogenous compounds in the xylem sap was compared between the symbionts, with B. japonicum and R. fredii. At the flowering stage, ureide-N and amide-N accounted for 53 to 70% and 20 to 27% respectively of the total N in the sap collected from the plants inoculated either with B. japonicum or R. fredii. At the pod elongation stage, ureide-N and amide-N accounted for 74 to 85%, and 7 to 19% of total sap N. With the growth of the soybean plants, the ratio of ureide-N in the xylem sap increased. These results suggest that in the case of wild soybean and the hypernodulating mutant line nodulated by R. fredii, ureide is transported as the main nitrogenous compound of fixed nitrogen in the xylem sap in the same way as in plants nodulated with B. japonicum.     

根瘤菌结瘤基因的表达调控研究概况

根瘤菌结瘤基因的表达调控在根瘤菌与植物的共生结瘤过程中起着十分重要的作用。随着研究的深入,发现根瘤菌的结瘤过程不仅与根瘤菌结瘤基因表达调控有关,而且与寄主植物的信号分子如黄酮类物质有关。根瘤菌结瘤基因的表达调控有一个复杂的过程,本文将简要地介绍这方面的研究成果。     

Effect of CO2-free air treatment on nitrogen fixation of soybeans inoculated with Bradyrhizobium japonicum and Rhizobium fredii

Soybean (Glycine max L. Merr.) cultivars Akisirome and Peking were inoculated with Bradyrhizobium japonicum Is-21 and Rhizobium fredii USDA 194, respectively, and were grown in cylindrical pots containing sterilized vermiculite which were aerated with CO₂-free air or ambient air to study the effects of CO₂ deficiency in the rhizosphere on plant growth, nodulation, and nitrogen fixation. The repressive effects of CO₂-free air treatment were more conspicuous in Peking than Akisirome, and nodule number, nodule mass, amount of biologically fixed N and plant growth of Peking were reduced remarkably by the CO₂-free air treatment.

Acetylene reduction activity (ARA) of Peking inoculated with USDA 194 and Akisirome inoculated Is-21 was assayed in the absence and presence of CO₂, ARA of Akisirome was not affected by the absence of CO₂, while that of Peking decreased drastically.

Based on these results, it was concluded that nitrogen fixation by Akisirome inoculated with B. japonicum Is-21 was not appreciably influenced by CO₂ deficiency in the rhizosphere, while that of Peking inoculated with R. fredii USDA 194 was severely repressed, and the decrease was estimated to be due to both the reduction of the nodule mass and specific nitrogen-fixing activity.     

抗春雷霉素大豆根瘤菌突变株的诱变及其生物固氮特性的分析

根瘤菌与豆科植物共生可以固定大量的氮。根瘤菌剂接种豆科作物是一项普遍推广应用、有效的农业技术。但由于大量土著根瘤菌的存在，产生竞争障碍，降低了接种菌剂的占瘤率。大多数的土著根瘤菌对春雷霉素敏感，因此接种抗春雷霉素的高效结瘤固氮根瘤菌，并用春雷霉素处理种子，可抑制土著根瘤菌，提高接种菌剂的占瘤率，从而达到高结瘤、高固氮和提高产量的目的。本文将探讨诱变对根瘤菌抗春雷霉素突变的作用，并对获得的抗性突变株的生物固氮特性进行分析。     

Physiological and symbiotic characteristics of Rhizobium fredii isolated from subtropical-tropical soils

Summary Physiological and symbiotic characteristics were identified in Rhizobium fredii isolated from subtropical-tropical soils. The generation times of R. fredii Taiwan isolated-SB 357 and -SB 682 were 1.7 and 2.5 h, respectively. These strains were associated with acid production in yeast-extract mannitol medium. They were able to use hexoses, pentose, sucrose, trehalose and raffinose. Strain SB 357 can resist a high concentration of kanamycin (100 g ml^–1 and penicillin (400 g ml^–1). It can tolerate up to 2.34% NaCl and 1031.3 mosmol kg^–1 (23.4 bars). The growth rate of R. fredii SB 357 under the concentration of approximately 450 mosmol kg^–1 (10.2 bars) was not affected by salinity, but responded to osmotic pressure. Both strains (SB 357 and SB 682) isolated from subtropical-tropical soils were able to form an effective N₂-fixing symbiosis with the US soybean cv Clark lanceolate leaflet.     

Nodulation competitiveness of Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici strains measured by glucuronidase (gus) gene fusion

Summary The nodulation competitiveness of 17 Rhizobium leguminosarum bv. phaseoli and 3 R. tropici strains was analysed in growth pouches, at pH 5.2 and 6.4. All 20 strains were coinoculated with a gus ⁺ strain of R. leguminosarum bv. phaseoli strain KIM5s. The gus⁺ phenotype, carrying the glucuronidase gene, was used to type nodules directly in the growth pouches. Nodule occupancy ranged from 4% for the least competitive to 96% for the most competitive R. leguminosarum bv. phaseoli strain. The R. tropici strains showed low rates of nodule occupancy at pH 6.4 but their competitiveness improved significantly under acid conditions. CIAT 895 was the only R. leguminosarum bv. phaseoli strain that was less competitive (P<0.05) at the lower pH. The competitiveness of all the other R. leguminosarum bv. phaseoli strains was unaffected by pH. Various physiological and genetic properties of the strains were analysed in search of correlations with nodulation competitiveness. Hybridisation patterns with three different DNA probes (nif KDH, common nod genes, and hup genes) and the metabolism of 53 different C sources were compared. No general correlations were found between hybridisation or growth pattern and competitiveness. The less competitive R. tropici strains had a unique DNA hybridisation pattern and were not able to use shikimate, ferulate, coumarate, or asparagine as C sources. Most of the less competitive R. leguminosarum bv. phaseoli strains could not metabolize either ferulate or coumarate. This might indicate a relationship between nodulation competitiveness and the ability to degrade aromatic compounds.     

Efficiency of N2 fixation in Vicia faba L. in combination with different Rhizobium leguminosarum strains

The efficiency of symbiotic dinitrogen (N₂) fixation in Vicia faba L. in combination with 3 different Rhizobium leguminosarum strains was studied in a pot experiment during vegetative and reproductive growth. The objective of the experiments was to assess variability among Rhizobium strains inoculated on single legume species and determine possible reasons for observed variations. Dry matter formation, N₂ fixation and the carbon (C) costs of N₂ fixation were determined in comparison with nodule free plants grown with urea. Nodule number and the capacity of different respiratory chains in the nodules were also measured. The plants inoculated with the Rhizobium strain A 37 formed less dry matter and fixed less N compared to the other two Rhizobium strains (Vic 1 and A 150). This coincided with a lower number of nodules and higher C costs of N₂ fixation. The C costs for N₂ fixation were in all cases significantly lower during reproductive growth compared to vegetative growth. Neither the latter nor the differences in C expenditure for N₂ fixation between the Rhizobium strains could be explained in terms of differences or shifts in the capacity of different respiratory chains in the nodules.     

Effect of nodulation with Bradyrhizobium japonicum and Shinorhizobium fredii on xylem sap composition of Peking (Glycine max L. Merr )

Five barley cultivars were grown together in complete, low-P·low-pH and high-Al medium containing only NO₃, only NH₄ or both NO₃ and NH₄ as N sources, respectively using an automatic control system of pH for water culture, and the relationship between the differential Al tolerance and the plant-induced pH change of medium among the barley cultivars was investigated.

The pH of the medium containing only NO₃ as N source tended to increase, whereas the pH of the other media containing only NH₄ or both NO₃ and NH₄ as N sources tended to decrease, but the fluctuations of the medium pH could be maintained within the value of 0.2 pH in the complete medium and within the value of 0.1 pH in the high-Al medium.

Barley cultivars still differed in their Al tolerance in the medium which was continuously stirred and circulated at a constant pH. The pattern of Al tolerance was not affected by the N sources in the medium. The plant-induced pH change of medium for each cultivar was influenced by the N sources in the medium, and was not correlated positively with Al tolerance. The contents of Al and Ca or other nutrient cations in roots were positively correlated with Al tolerance and positive correlations were recognized also between the contents of Al and Ca or some other nutrient cations in the roots.

In conclusion, the following mechanisms are proposed. Al tolerant barley cultivars exclude Al actively outside the plasmalemma of the root cells, and the excluded Al may polymerize and or react with P to form Al precipitates. Consequently, in the Al tolerant barley cultivars the Al content may be low in the root protoplasts, high in the whole root tissues and the contents of Ca or other nutrients may be high in the roots. The plant-induced pH change of medium is not considered to be the cause of the differential Al tolerance among barley cultivars.     

Study on the Effects of Organic and Inorganic Nitrogen Fertilizer on Yield,Yield Components,and Nodulation State of Chickpea (Cicer arietinum L.)

To study the effects of organic and inorganic nitrogen (N) on yield and nodulation of chickpea (Cicer arietinum L.) cv. ILC 482, a spilt-plot experiment based on randomized complete block design with four replications was conducted in 2008 at the experimental farm of the Agriculture Faculty, University of Mohaghegh, Ardabili. Experimental factors were inorganic N fertilizer at four levels (0, 50, 75, and 100 kg ha^?1) in the main plots that applied in the urea form, and two levels of inoculation with Rhizobium bacteria (with and without inoculation) as subplots. Nitrogen application and Rh. inoculation continued to have positive effects on yield and its attributes. The greatest plant height, number of primary and secondary branches, number of pods per plant, number of filled and unfilled pods per plant, number of grains per plant, grain yield, and biological yield were obtained from the greatest level of N fertilizer (100 kg urea ha^?1) and Rh. inoculation. Application of 75 and 100 kg ha^?1 urea showed no significant difference in these traits. Furthermore, the greatest rate of N usage (100 kg urea ha^?1) adversely inhibited nodulation of chickpea. Number and dry weight of nodules per plant decreased significantly with increasing N application rate. The lowest values of these traits recorded in application of 100 kg ha^?1 urea. Results indicated that application of suitable amounts of N fertilizer (i.e., between 50 and 75 kg urea ha^?1) as starter can be beneficial to improve nodulation, growth, and final yield of inoculated chickpea plants.     

Productivity,profitability and sustainability of rain-fed chickpea under inorganic and biofertilization in foothills of north-west Himalayas

A 4-year (2008–2009 to 2011–2012) study was conducted on the effect of mineral phosphorus (P) + sulphur (S) and biofertilizers on rain-fed chickpea (Cicer arietinum L.) at the Punjab Agricultural University’s Research Station, Ballowal Saunkhri, India. The experiment comprised of five combinations of P and S, viz. control (P₀S₀), no P + 10 kg S ha^?1 (P₀S₁₀), 15 kg P + 10 kg S ha^?1 (P₁₅S₁₀), no P + 20 kg S ha^?1 (P₀S₂₀) and 30 kg P + 20 kg S ha^?1 (P₃₀S₂₀); and three seed inoculation levels, viz. control, Rhizobium and phosphate-solubilizing bacteria (PSB), were laid out in randomized complete block design. Combined application of P + S resulted in improved growth, nodulation, yield attributes and yield. The increase in seed yield over control due to P + S ranged from 11.8% to 17.7%. Seed inoculation with Rhizobium recorded the highest growth, nodulation, yield attributes and yield of chickpea and was statistically at par with PSB and significantly better than no inoculation. Highest benefit/cost ratio (B:C, 2.19) was obtained in P₃₀S₂₀. In view of environmental pollution and high costs of chemical fertilizers, biofertilizers alone or in combination may help to achieve sustainable and ecological agricultural production.     

Effects of Rhizobium inoculation and magnesium application on growth and nodulation of soybean (Glycine max L.)

Abstract

Magnesium (Mg) deficiency is one of the major nutritional problems in tropic and subtropic areas, where the most soils are acidic. In this study, the effects of Mg application and Bradyrhizobium inoculation on growth, nodulation, symbiotic nitrogen (N) fixation as well as N nutrition status in soybean (Glycine max L.) were investigated in hydroponics under greenhouse conditions. With the increase of Mg up to 0.75?mM at low N and up to 0.5?mM at high N solutions, the dry weights of shoots, roots, and pod grain yield in soybean were increased, while further increase in Mg supply inhibited soybean growth. The availability of Mg was found to entail an improved uptake of N by plants and nodulation process in the root by Bradyrhizobium. Inoculation with rhizobial inoculants not only formed many nodules, but also increased soybean shoot, root biomass and yield, as well as plant N nutrient status.     

Isolation,selection and evaluation of Rhizobium under controlled conditions

Abstract

Rhizobia were screened in growth chamber and greenhouse tests with the host plants: bean (Phaseolus vulgaris L.), lentil (Lens esculenta Moench.), cowpea (Vigna unguiculata (L.) Walp) and peanut (Arachis hypogaea L.). Rhizobial isolates varied in effectiveness, time to nodulation, and host plant specifities. Initial screening procedures in plastic growth pouches allowed selection of infective strains of Rhizobium. These tests enabled the selection of a small number of highly effective isolates for more critical evaluation. Highly significant correlations (p=0.01) were obtained between shoot dry weight and total nitrogen, suggesting that the technique was applying sufficient selection pressure to differentiate variation in N₂ fixation among strains of Rhizobium. Results indicate that highly effective rhizobia can be efficiently selected under controlled conditions following a step‐wise procedure.     

Nodulation Productivity and Nutrient Uptake of Cowpea (Vigna unguiculata L. Walp) with Phosphorus and Potassium under Rainfed Conditions

Low availability of phosphorus (P) and potassium (K) in acidic soil is a major constraint for crop production. Therefore, a field study was conducted to determine the effects of K and P on nodulation, productivity, and nutrient uptake of cowpea (Vigna unguiculata L. Walp) under rainfed conditions. The K and P were subjected to main and subplots, respectively with 100, 75, and 50% of fertilizer application. The growth and yield attributes were better using 100% K with 100% P. However, 100% K resulted in 20.9 and 16.9% greater green pod and stover yield than 50% K. Similarly, 100% P recorded 20.2 and 15.6% greater green pod and stover yield than 50% P. Uptake of nutrients such as nitrogen (N), P, and K followed the trend of greater to lower, 100% > 75% > 50%, in order for K and P. Similarly, nutrient-use efficiencies and production efficiency followed the trend of nutrient uptake.     

Low root zone temperature effects on bean (Phaseolus vulgaris L.) plants inoculated with Rhizobium leguminosarum bv. phaseoli pre-incubated with methyl jasmonate and/or genistein

Abstract

Methyl jasmonate (MeJA) has recently been shown to act as a plant-to-bacteria signal. We tested the hypothesis that pre-induction of Rhizobium leguminosarum bv. phaseoli cells with genistein and/or MeJA would at least partially overcome the negative effects of low root zone temperature (RZT) on bean nodulation, nitrogen fixation and plant growth. Otebo bean plants were grown at constant air temperature (25^oC) and two RZT regimes (25 and 17^oC) and inoculated with R. leguminosarum bv. phaseoli pre-induced with MeJA and/or genistein. Our results indicate that low RZT inhibited nodulation, nitrogen fixation and plant growth. The plants growing at low RZT began fixing nitrogen seven days later compared to those at higher RZT. The low RZT plants had fewer nodules, lower nodule weight, less N fixation, slower plant growth, fewer leaves, smaller leaf area, and less dry matter accumulation comared to plants at a higher RZT. Rhzobium leguminosarum bv. phaseoli cells induced with genistein and/or MeJA enhanced bean nodulation, nitrogen fixation and growth at both optimum and suboptimum RZTs. The results of this study indicate that MeJA improves bean nitrogen fixation and growth at both optimum and suboptimum RZTs, and can be used alone or in combination with genistein to partially overcome the low RZT induced inhibitory effects on nodulation and nitrogen fixation.     

Combined Effect of Phosphate-Solubilizing Microorganisms,Rhizobium and Enterobacter on Root Nodulation and Physiology of Soybean (Glycine max L.)

Although phosphate-solubilizing microorganisms play a positive role in plant growth, their role in plant growth and root nodulation in combination with Rhizobium and Enterobacter has not been fully elucidated. Furthermore, only information exists regarding the effect of inoculation at successive stages of nodulation. The present study aimed to monitor the changes in the production of Indole acetic acid (IAA) and Gibberellin (GA₃) in the roots of soybean during and after nodulation (25 DAS and 40 DAS) and in the rhizosphere soil following inoculation with two different strains of phosphate-solubilizing microorganisms (PSM I, strain CA 18 and PSM II, strain 54RB), Enterobacter strain A and BradyRhizobium Japonicum strain Tal 377. The effects of inoculation on the available NO^?3, K, and P content of soil were studied at harvest. Beneficial effects of inoculation with Rhizobium, Enterobacter, and PSM I and II were obtained in all growth parameters of soybean. Co-inoculation resulted in maximum increase in IAA and GA content, plant biomass, root nodulation, number, weight and length of pods as compared with control (non-inoculated) and single-inoculation plants. The soil of the inoculated plants also showed higher IAA and GA content over.     

Synergistic effect of bio-inoculants on yield,nodulation and nutrient uptake of chickpea (Cicer arietinum L) under rainfed conditions

The use of efficient bio-inoculants in chickpea is the best way to increase crop productivity under rainfed conditions. To assess the combined effect of bio-inoculants on crop yield, field experiments were conducted during Rabi seasons at Research Station, Punjab Agricultural University, Ballowal Saunkhri, Punjab, India. The application of different bio-inoculants significantly improved number of pods, grain and straw yield of chickpea over the un-inoculated treatment. The combined application of Rhizobium + PSB?+?AM fungi?+?azotobactor inoculums as seed treatment with 75% of recommended phosphorus produced highest grain yield. The nodule count, nodule weight, per cent root colonization of AM fungi and different enzymes activities in soil were also highest in combined bio-inoculants treatment. The present study concluded that combined application of bio-inoculants (Rhizobium, PSB, AM fungi and azotobactor) can save 25% of recommended phosphorus by sustaining the crop yield and improving the soil health.     

Intragenomic variation in the internal transcribed spacer regions between 16S–23S rRNA genes among the three copies of Sinorhizobium fredii strains

The soybean-nodulating Sinorhizobium fredii strain has been reported to possess three copies of rRNA gene operons. In the present study, we investigated the diversity of the 16S–23S rDNA internal transcribed spacer (ITS) regions of S. fredii strains. Based on the sequences of the ITS regions, we divided the sequences of the S.   fredii strains into two groups, type A and type B. A dot-matrix analysis indicated that the region flanked by tRNA-Ile and tRNA-Ala is longer in type A than in type B, whereas type B sequences possess longer regions upstream of tRNA-Ile and downstream of tRNA-Ala than those of the type A sequence. Restriction fragment length polymorphism of polymerase chain reaction product (PCR-RFLP) of the ITS region in the cloned plasmids as templates could reconstruct the PCR-RFLP pattern from the total DNA as a template. The results of Southern hybridization using the insert sequence between tRNA-Ile and tRNA-Ala in type A as a probe indicated differences in the copy numbers of the type A ITS regions among the strains tested. These results indicated that S. fredii strains possess the type A and type B sequences of the ITS regions at ratios of 3:0, 2:1, 1:2 or 0:3. These S. fredii strains may be useful biological materials for the study of intraspecific variations.     

壳聚糖对NaCl胁迫下菜用大豆结瘤固氮的影响

【目的】研究壳聚糖对盐胁迫抑制菜用大豆结瘤固氮的缓解效应,为进一步探讨壳聚糖抗逆机理提供新的线索。【方法】以蛭石为基质,以菜用大豆‘特早王’–根瘤菌共生体系为研究对象,采用人工气候箱培养,研究NaCl胁迫下壳聚糖对菜用大豆根瘤形成、生物固氮的影响。菌种为与‘特早王’共生匹配性较好的快生根瘤菌N18。接种后的植株进行如下4个处理:1)叶面喷施清水,根部浇灌无氮营养液(CK);2)叶面喷施壳聚糖水溶液,根部浇灌无氮营养液(CTS);3)叶面喷施清水,根部浇灌溶有NaCl的无氮营养液(Cl);4)叶面喷施壳聚糖水溶液,根部浇灌溶有NaCl的无氮营养液(CTS+Cl)。上述各处理施用的水或水溶液均为无菌水配制,NaCl处理的浓度为50 mol/L,CTS处理的适宜浓度为200 mg/L。接种30天后,将大豆植株取出,用清水将根部蛭石冲洗干净后,立即测定根瘤固氮酶活性、根瘤数及根瘤鲜重,然后测定根瘤豆血红蛋白含量和根系活力,最后测植株干重和全氮量。【结果】氯化钠胁迫下,植株干重显著下降,与CK相比降幅达49%,喷施壳聚糖后(CTS+Cl),降低幅度显著减小,但依然显著低于CK (P <0.05)。无盐条件下,与CK相比,壳聚糖处理(CTS)增加植株干重的效果不明显。喷施壳聚糖显著增加了菜用大豆的根瘤数、根瘤鲜重、植株含氮量、根系活力、豆血红蛋白含量及固氮酶活性(P <0.05)。NaCl胁迫显著抑制了菜用大豆的结瘤固氮作用,其中根瘤数、根瘤鲜重分别较CK下降了79%、90%,而壳聚糖处理(CTS+Cl)使菜用大豆在盐逆境下的结瘤数、根瘤鲜重、植株全氮含量、根系活力、豆血红蛋白含量及固氮酶活性等均显著回升,增幅分别达对照的29%、20%、17%、48%、19%、21%,但均显著低于CK。【结论】非NaCl胁迫下,喷施壳聚糖可以显著促进菜用大豆结瘤,提高豆血红蛋白含量及固氮酶活性,最终增加植株含氮量。在NaCl胁迫下,外源壳聚糖可以显著缓解氯化钠胁迫导致的对根系活力和结瘤固氮的影响。因此,叶面喷施壳聚糖是促进菜用大豆结瘤固氮和生长的有效措施。     

Interaction of the fungicide mancozeb and Rhizobium sp. in pure culture and under field conditions

Information on the compatibility of Rhizobium sp. with seed-protectant chemicals is controversial because of variations in the methods used and the lack of quantitative data. The present study was conducted to determine the influence of the fungicide mancozeb (ethylenebis-dithiocarbamate), at recommended doses, on the growth, survival and symbiotic properties of Rhizobium sp. infecting peanut plants (Arachis hypogaea) under laboratory and field conditions. The results indicated that mancozeb decreased growth in pure culture by 50% of both Rhizobium sp. USDA 3187 and a strain isolated from peanut nodules. However, no differences were found in peanut seed yields under field conditions. These results suggest that the soil environment could reduce the probability of the direct, harmful effects of mancozeb on bacterial growth. Received: 21 June 1996     

Interaction of Rhizobium and Pseudomonas with Wheat (Triticum Aestivum L.) in Potted Soil with or Without P2O5

The effect of inoculation of Rhizobium or Pseudomonas, or both, in the presence and absence of phosphorus pentoxide (P₂O₅) fertilizer on wheat yield was tested. The experiment was conducted outdoors in potted soil during two consecutive years under natural conditions. Rhizobial strain (Thal 8-chickpea nodulating bacteria) and Pseudomonas strain (54RB), both indigenous P solubilizers were applied in broth culture at seedling stage. Fertilizer in the form of P₂O₅ was an additional treatment applied at sowing time. Results revealed that inoculation of Pseudomonas strain in presence of phosphorus (P) fertilizer increased yield up to 41.8% when it was compared with only P fertilizer applied. Co-inoculation of Rhizobium and Pseudomonas strains with the P₂O₅ treatment however resulted in increased grains yield by 10% over the P₂O₅ treatment alone. It is inferred that inoculation with Rhizobium and Pseudomonas combined with P₂O₅ is not only environmentally beneficial but also economically sound and productive, with wheat yield increases of 10% to 42%.