The potential for breeding an improved lucerne-rhizobium symbiosis. 1. Assessment of genetic variation

Summary Six cultivars of lucerne (Medicago sativa L.) were grown in all possible combinations with eight strains of Rhizobium meliloti in order to assess genetic variation in symbiotic nitrogen fixation. Host genotype and Rhizobium genotype effects on nitrogen fixation were respectively 4.8% and 21.0% of total phenotypic variance. Genetic variation due to host cultivar × Rhizobium strain interactions accounted for a further 6.0% of phenotypic variance. The results indicated low heritability of general symbiotic effectiveness in the host with interaction effects being large enough to suggest that plant performance may be unpredictable with populations of R. meliloti in field soils. Joint regression analysis showed that about 50% of the interactive variation could be explained by generalized differences in the sensitivity of cultivars to alterations in the genotype of Rhizobium strains. One cultivar, Siriver, was relatively insentitive to changes in the Rhizobium genotype whilst still maintaining high average yield. The implications of the results for lucerne breeding are discussed.     

Variation in Nitrate Tolerance of Nitrogen Fixation in the Pea/Rhizobium Symbiosis

The short-term effects of nitrate on symbiotic nitrogen fixation were studied in six cultivars of pea (Pisum sativum L.) using two strains of Rhizobium leguminosarum. Plants grown symbiotically for three weeks, in order to eliminate the effect of NO₃ on nodule formation, were exposed to nil, 5 or 15 mM NO₃ for a period of seven days. Nodule mass, nitrogenase activity by acetylene reduction and the NO₂ and NO₃ concentrations in nodules were determined. Exposure to nitrate reduced the total acetylene reduction activity (ARA) in all cultivars, and a significant cultivar × nitrate treatment interaction was found. Nitrate influenced the components of N₂ fixation, nodule mass and specific ARA (ARA/mg nodule), differently within and among cultivars. Nodule mass was reduced in all cultivars, but a variation among cultivars in the degree of reduction was found. Nitrate also reduced the specific ARA to different extents in the cultivars- The specific ARA of nodules from NO₃-treated plants, expressed as per cent of the specific ARA of nodules from plants grown without NOT₃ was negatively correlated with the concentration of NO₃ in nodules. No relationship was found between the reduction of N₂ fixation and the nitrite concentration in nodules. The results indicate that it is possible to improve the nitrate tolerance of symbiotic N₂ fixation of the established pea/Rhizobium symbiosis by selection of suitable host plants.     

Investigation of the fenugreek (Trigonella foenum-graecum L.) genotypes for fresh weight, seed productivity, symbiotic activity, callus formation and aceumulation of steroids

Summary As a result of the analysis of 31 fenugreek (Trigonella foenum-graecum L.) genotypes we demonstrated that subsp. indica surpass significantly subsp. foenum-graecum in fresh weight, seed yield, nodulation ability, resistance to coot rot and the duration of the interstage and growing periods. There was no difference between the subspecies for leafiness and biochemical composition of seeds (concentrations of diosgenin and steroid glucosides, nitrogen, fat, inhibitors of tripsin and hemotripsin). The most promising genotypes (as compared with the standard cv. Nakhichevanskaya Shambala) yield 1650–1938 g/m² of fresh weight, 98.2–116.1 g/m² of seeds and accumulate 1.45–1.64% of diosgenin in seeds. High positive correlations (r=+0.53 ... +0.84; P_o<0.01) were found between fresh weight, seed yield, plant leafiness and height. Inoculation of the cv. Nakhichevanskaya Shambala with Rhizobium meliloti strains 851 and 852 increased its fresh weight by 94.5% and 93.6% and seed yield by 83.1% and 60.5%, respectively. Significant correlations of the nodule number with the fresh weight and seed yield (r=+0.70 and +0.77; P_o<0.01) allow us to assume that selection for increased symbiotic activity may be useful for improving the fenugreek productivity. The intersubspecies hybrids (four genotypes) surpass the subsp. indica (six genotypes) for the sizes of calli formed in presence of 2.4-D and kinetin. The sizes of calli formed by ten analysed genotypes in presence of BAP and kinetin (or 2.4-D) were negatively correlated with the nodule number (or fresh weight).     

Symbiotic variability in Vicia faba. 3. Genetic effects of host plant,rhizobium strain and of host × strain interaction

Summary Six Vicia faba populations were grown in all possible combinations with six Rhizobium leguminosarum strains in order to estimate the relative importance of the three genetic components of symbiotic variability. Additive genetic effects of host genotype and rhizobium genotype accounted for only 8.9% and 11.8% of the total phenotypic variation. Non-additive variation attriabutable to specific host genotype × rhizobium genotype interactions was by far the largest component of variation and accounted for 73.8% of phenotypic differences. Therefore the greatest improvement in symbiotic nitrogen fixation is likely to arise from simultaneous selection of both symbionts.     

根瘤菌的分类、鉴定及应用技术研究现状

豆科植物(legumines)作为重要的粮食及经济作物,一直以来被广受重视。根瘤菌(Rhizbium)是一类普遍存在于土壤中的革兰氏阴性杆菌,可与豆科植物形成共生固氮体系,不仅可以提高植物的产量而且对周边生态环境无影响。根瘤菌与豆科植物的共生体系是生物固氮效率最高的,占生物固氮总量的65%以上。深入研究这种共生固氮体系对中国农业的可持续发展具有重要意义。近年来,越来越多的学者参与到根瘤菌的分类、鉴定以及应用技术的研究。本文主要从根瘤菌的分类、形态水平、生理生化水平、细胞组分、核酸分子水平的鉴定和根瘤菌的应用等方面进行了综述,为根瘤菌的实际应用提供参考依据。     

Interactive Effects of Salinity and Biological Nitrogen Fixation on Chickpea (Cicer arietinum L.) Growth

The effect of salinity on the nodulation, N-fixation and plant growth of selected chickpea- Rhizobium symbionts was studied- Eighteen chickpea rhizobial strains were evaluated for their growth in a broth culture at salinity levels of 0 to 20 dS m⁻¹ of NaCl + Na₂SO₄. Variability in response was high. Salinity generally reduced the lag phase and/or slowed the log phase of multiplication of Rhizobium. Nine chickpea genotypes were also evaluated for salt tolerance during germination and early seedling growth in Petri dishes at five salinity levels (0–32 dS m⁻¹). Chickpea genotypes ILC-205 and ILC-1919 were the most salt-tolerant genotypes. The selected rhizobial strains and chickpea cultivars were combined in a pot experiment aimed at investigating the interactive effect of salinity (3, 6 and 9 dS m⁻¹) and N source (symbiosis vs. inorganic N) on plant growth. Symbiotic plants were more sensitive to salinity than plants fed mineral N. Significant reductions in nodule dry weight (59.8 %) and N fixation (63.5 %) were evident even at the lowest salinity level of 3 dS m-¹. Although nodules were observed in inoculated plants grown at 6 dS m-¹, N-fixation was completely inhibited. The findings indicate that symbiosis is more salt-sensitive than both Rhizobium and the host plant, probably due to a breakdown in one of the processes involved in symbiotic-N fixation. Improvement of salinity tolerance in field grown chickpea may be achieved by application of sufficient amounts of mineral nitrogen.     

Genotypic lipoxygenase variation in soybean seeds and response to nitrogen nutrition

Decreasing the lipoxygenase content of soybean (Glycine max (L.) Merr.) seed is a prerequisite for its increased use in human foods. These investigations aim to analyse the variation of lipoxygenase contents in relation to the genotype or the nitrogen nutrition of the plant. Four genotypes from a divergent selection for N₂ fixation and the recurrent parent, Weber, were grown in pots, and two Bradyrhizobium japonicum strains (G.49 and SMGS1) were used. Dinitrogen fixation was estimated in situ using the acetylene–reduction assay. The lipoxygenase activity was determined polarographically on seeds taken from plants grown under controlled conditions in the presence or absence of symbiotic N₂ fixation. The large genotypic variation observed for the lipoxygenase contents was enhanced by high N₂ fixation. The variation range of lipoxygenase contents, which was obtained through the combination of the genotypic variation and the N₂ fixation activity, was relatively large with a ratio of 2 on a seed dry–weight basis and a ratio of 2.6 when the total proteins were considered. Decreased lipoxygenase content and increased total protein content can thus be considered in parallel in a genetic context, together with more efficient N₂ fixation.     

Differences in nitrogen fixation among field-grown red clover strains at different levels of 15N fertilization

Summary Genetic variation in fixed nitrogen (N) yield of red clover (Trifolium pratense L.) strains and cultivars was investigated using the ¹⁵N isotope dilution method under three regimes of N fertilization: 0.5, 30, and 60 N (kg N ha^–1 per cut). The yield of fixed N per cut (the mean of eight cuts over 2 production years) varied among the strains (progenies of crosses between inbred parents) from 148 to 443 mg per plant at 0.5 N, from 76 to 324 mg at 30 N, and from 69 to 300 mg at 60 N. There were significant and consistent strain differences in the percentage of clover N derived from the atmosphere (% N_dfa). However, %N_dfa was positively correlated with dry mass yield. Consequently, ranking of the strains according to fixed N yield reflected that of dry mass yield. There were only minor strain × N fertilizer interactions, suggesting that selection for enhanced N fixation can be carried out at a single rate of fertilizer N. For a selected pair of strains, the difference in yield of fixed N was confirmed in an Italian ryegrass-red clover mixture, both without and with the addition of N fertilizer (50 kg N ha^–1 per cut). Results with 7-week-old seedling plants in a growth chamber, although obtained in the presence of mineral N and with the isotope dilution method, did not adequately predict field performance. It is concluded that selection for dry matter or total N yield is likely to result in an enhanced yield of fixed N at any level of mineral N availability.Abbreviations % N_dfa percentage of clover nitrogen derived from the atmosphere by symbiotic nitrogen fixation - S₂-F₁-n progeny of pair cross between inbred parents obtained after two generations of selfing     

Difference in Allelopathic Potential as Influenced by Root Periderm Colour of Sweet Potato (Ipomoea batatas)

Laboratory bioassay and high‐performance liquid chromatography (HPLC) analysis were conducted to determine the allelopathic potentials of aqueous or methanol extracts from three different coloured sweet potato [Ipomoea batatas L. (Lam)] cultivars by plant part. The aqueous extracts applied on filter paper significantly inhibited root growth of alfalfa (Medicago sativa L.). Aqueous leaf leachates at 40 g dry tissue l^?1 (g l^?1) from white sweet potato cultivar ‘Sinyulmi’ showed the highest inhibition against alfalfa, followed by yellow ‘Sinhwangmi’ and purple ‘Jami’. Alfalfa root growth was significantly inhibited by methanol extracts of the same plants as the concentration increased. Aqueous and methanol extracts from leaves showed the most inhibitory effect on alfalfa root growth followed by stems and roots. By means of HPLC analysis, leaf samples of sweet potato had the highest amount of phenolic compounds followed by stems and roots. Total content of these compounds was highest for leaf extracts (37.7 mg 100 g^?1), detected in EtOAc fraction, especially trans‐cinnamic acid (20.9 mg 100 g^?1). These results suggest that sweet potato plants are allelopathic and that their activities differ depending on plant part as well as root periderm colour.     

Residual Effects of Rhizobium and Preplant N Fertilizer on Newly Established Alfalfa1

In areas where ineffective nodulation may be a problem, ‘starter’ nitrogen fertilization is often suggested as a useful management tool to sustain ineffectively nodulated seedling alfalfa (Medicago sativa L.) until an effective population of Rhizobium can develop. Although ineffective nodulation has been reported from several locations around the world, there have been no studies examing this possibility. A field experiment was conducted during 1982 and 1983 on a Woodburn silt loam soil (Aquultic Argixeroll) which contained a symbiotically ineffective native population of Rhizobium meliloti Dang. This study examined the residual and interactive effects of preplant N fertilizer (0, 45, 90 kg N ha^?1) and inoculation on the N nutrition and yield of seedling alfalfa during the first year of growth. The sustaining effects of the fertilizer N were transient and resulted in decreased N₂ fixation until 35 days following harvest. It was concluded that during the establishment year, managerial emphasis should be placed on inoculation and not on N fertilization to obtain a consistent, economical source of N for herbage protein and dry matter production.     

Evaluation of Rhizobium mdiloti Strains Isolated from Indigenous Populations in Northern Greece

Eight different Rhizobium meliloti strains (designated as strains 3, 6, 8, 15, 17, 18, 19 and 22) were isolated from fields cultivated with alfalfa. The growth characteristics of the isolated strains under different NaCl concentrations were investigated. Strains 15 and 22 showed marked tolerance to high NaCl concentration.
Symbiosis of one alfalfa variety ( Medicago sativa cv. Ipati ) inoculated with these strains showed marked differences in nitrogen fixing characteristics (nodule number, dry weight and total nitrogen content). Strains 8, 15 and 22 demonstrated the best performance with respect to all characteristics examined.
The supply of low levels of nitrate to inoculated alfalfa plants affected all the nitrogen fixing characteristics. Nodule number per plant decreased and the ranking of the different strains with respect to nodule number was altered. Dry weight was significantly increased by the supply of the nitrate, even in the case of symbioses with the highly effective strains 8, 15 and 22. Total nitrogen content was increased by the supply of nitrate. This increase was higher in plants inoculated with the less effective strains.     

Regenerative trait and cold hardiness in highly productive cultivars of alfalfa and red clover

Summary In earlier work on improvement of persistance in forage legumes, we selected genotypes from highly productive cultivars of alfalfa, Algonquin and Apica (Euphytica 45: 105–112, 1990) and cv. Florex red clover (Plant Cell Reports 8: 395–398, 1989) capable of in vitro regeneration from callus and cell culture. The alfalfa germplasm and its F₁ progeny as well as an F₂ red clover population were tested for cold stress tolerance. Plantlets were hardened in culture tubes at 2 or 5°C, 8h photoperiod, for at least four weeks and then subjected to freezing temperatures, –16 or –10°C for alfalfa and red clover, respectively. Survival of regenerative genotypes was significantly higher than of the non-regenerative ones in both species. A strong oositive correlation (r=0.78) between the regenerative trait and plant survival was found in alfalfa. The experiments indicate that in vitro selection for regenerative trait may improve cold stress tolerance of alfalfa and red clover.     

Genetic Effects on Host × Strain Interaction in the Symbiosis of Vicia faba and Rhizobium leguminosarum

50 Vicia faba lines were inoculated with 4 strains of Rhizobium leguminosarum in all possible combinations under greenhouse conditions in 5 replications. A root-sterile method was used for the cultures. Characters such as plant height, leaf colour, number and colour of nodules, the dry weights of shoot, root and nodules, and the nitrogen content were recorded, after 10 weeks. It was demonstrated by analysis of variance that, for example, for the dry matter of the shoot only 1.9 % of the total phenotypic variance was caused by interaction between Vicia and Rhizobium genotypes, but 24.7 % Was conditioned by the faba bean genotypes and 73.4 % by the Rhizobium strains. Differences of Vicia faba genotypes in their reaction to different Rhizobium strains were detected by joint regression analysis.     

Effect of Agronomic Factors on Annual Leguminous Forage Crop in Mediterranean Environments

Dry matter and seed production of five annual leguminous species hairy vetch ( Vicia villosa Roth cv. Orsara) , crimson clover ( Trifolium incarnatum L. ecotype Campano) , squarrosum clover ( T. squarrosum L. ecotype Calabro) , persian clover ( T. resupinatum cv. Accadia) and fenugreek ( Trigonella foenum-graecum L. ecotype Maltese) was estimated in 1989–90 under two density and irrigation rates and with two cutting treatments. The experiments were done at the Forage Crops Institute of Foggia, which has a typical Mediterranean environment. The forage cut severely affected the regrowth of the plants in all species. The year and irrigation factors affect dry matter, seed production and seed yield components more than the plant density rates. The averages over the years, for dry matter and seed yield were 21.0 % and 20.5 % higher in the irrigated than the not irrigated ones. The lower plant density rate, for seed yield trait, gave better result in hairy vetch in irrigated and non-irrigated treatment. Whereas in the other species, the lower rate of planting gave greater yield under non-irrigated and the higher rate under irrigated growing condition. The seed yield components most influenced by irrigation were: thousand seed weight and seeds/pod in hairy vetch and heads/tiller and seeds/head in clovers species. Fenugreek was the earliest forage crop (cut at the beginning of March), followed by crimson and persian clovers (cut at the middle of April) and squarrosum clover and hairy vetch (cut in the middle-late of May). All the cultivars and ecotypes considered not regrowth after cut and could represent an important fresh forage resource for the period March–May for the Mediterranean regions.     

Performance of Timothy-based Grass/Legume Mixtures in Cold Winter Region

This study sought to identify grass/legume mixtures that increase the yield and persistence of forage stands with improved nutritive quality in cold‐winter regions, compared with the standard mixture of timothy (Phleum pratense L.)/red clover (Trifolium pratense L.)/alsike clover (Trifolium hybridum L.). Timothy was mixed with either perennial ryegrass (Lolium perenne L.), meadow fescue (Festuca pratensis L.) or Kentucky bluegrass (Poa pratensis L.). The legumes in mixtures were red clover, alfalfa (Medicago sativa L.) or white clover (Trifolium repens L.). Averaged over three production years, the majority of mixtures had greater dry matter (DM) yields than the standard (8.35 t ha^?1). Timothy, grown alone and in three mixtures, outyielded the standard by 19–30 %. Yield reductions in mixtures over the 3‐year period were greatest with red clover, and least with bluegrass. Mixtures with alfalfa were highest in nitrogen (28.4 g kg^?1), while grasses grown alone (24.6 g kg^?1) and the standard mixture (25.1 g kg^?1) were the lowest in N. Mixtures with red clover or alfalfa had the least neutral detergent fibre (NDF), averaging 418 and 429 g kg^?1 respectively. Mixtures including white clover were initially low in NDF at 347 g kg^?1 in year 1 but increased to 550 g kg^?1 in year 3 as white clover composition declined in the sward.     

野生大豆GsGST19基因的克隆及其转基因苜蓿的耐盐碱性分析

谷胱甘肽S-转移酶对植物抵御逆境胁迫和解除细胞毒素起着重要作用。本研究从野大豆盐碱胁迫基因表达谱中筛选并克隆得到GsGST19基因,将其转化苜蓿,获得超量表达的转基因苜蓿,并对转基因苜蓿进行耐盐碱性分析。结果显示在正常培养条件下,转基因苜蓿株系19-4和19-9的GST酶活性分别是非转基因株系的1.52倍和1.49倍。在100 mmol L^–1 NaHCO₃处理14 d后转基因株系生长状态良好,而非转基因对照株系明显萎蔫、失绿、甚至死亡;转基因株系的丙二醛含量和相对质膜透性显著低于非转基因株系(P<0.05),而叶绿素含量和根系活力显著高于非转基因对照(P<0.05),说明超量表达GsGST19基因增强了苜蓿的耐盐碱能力。     

Effects of Soil Moisture Deficit on Forage Quality, Digestibility, and Protein Fractionation of Kura Clover

Kura clover (Trifolium ambiguum M.B.) is a persistent rhizomatous forage legume with great potential for permanent pastures. The effects of a soil moisture deficit on forage quality, digestibility and protein fractionation of two cultivars of Kura clover (Endura and Rhizo) were investigated in this study for 1 year. The responses of alfalfa (Medicago sativa L.) and red clover (Trifolium pratense L.) were also characterized. Stands of each species were field‐grown and submitted to two soil water regimes promoting soil moisture deficits and well‐watered (i.e. control) conditions. There were no interactions between species and soil water regimes. Soil moisture deficit increased acid detergent fibre (ADF) but reduced acid detergent lignin (ADL) content and consequently increased forage digestibility. It had only minor effects on protein content and fractionation. Species varied for most parameters measured. Kura clover generally had the lowest neutral detergent fibre (NDF), ADF and ADL contents, and consequently the greatest digestibility (83.9 %). Species also differed in their crude protein fractionation. Kura clover and red clover had a lower proportion of non‐protein nitrogen (NPN; A fraction) and a greater proportion of true protein (B fraction) (30.9 and 64.5 %, respectively) than alfalfa (36.4 and 57.4 %, respectively). Kura clover also had the lowest proportion (i.e. 4.7 %) of acid detergent insoluble protein (ADIP; C fraction) of all species tested. Endura Kura clover often had a higher forage quality than Rhizo. This study confirms that Kura clover produces high‐quality forage and provides the first estimates of protein fractionation in this species.     

河南省优良草种区划的探索

通过对河南省主要草种的生物学特点、生态学特点以及经济价值的分析,结合当前农牧业发展对饲草的需要特点的分析,采用系统学优化理论,确定了河南省当家草种应具备的条件,制定了全省草种区划方案。结果表明：适合河南省的当家草种有10种,分别是：紫花苜蓿、沙打旺、苇状羊茅、多年生黑麦草、小冠花、红三叶、杂三叶、鸡脚草、无芒雀麦;适合河南省草业优质发展的草种区域有6个,分别是：豫北平原牧草种植区、豫西北黄土丘陵和太行山地种植区、豫中东低洼平原和南阳盆地种植区、豫西山地种植区、淮南与桐柏山区种植区、豫东平原种植区,并且明确了各区域内以当家草种为核心的草种优化组合。河南省当家草种的制定和草种区划的确定有利于为河南省各地草种的正确选择和草业未来大发展提供理论依据。     

Genotypic Differences in Root Hair Deformation and Subsequent Nodulation for Red Clover under Different Additions of Starter N Fertilization

Red clover cultivars, including diploid and tetraploid, are commonly used in legume‐based pasture mixtures. However, information on nodulation under different starter N regimens is limited. We hypothesized that there is genetic variability among different red clover cultivars for nodulation. A root hair deformation assay was conducted using three diploid (AC Christie, Tapani and CRS15) and three tetraploid (Tempus, CRS18 and CRS39) red clover cultivars by inoculating them with Rhizobium leguminosarum biovar trifolii. Nodulation and morphological characteristics of two selected red clover cultivars, AC Christie and Tempus, were determined under five starter N concentrations (0, 0.2, 0.4, 0.8 and 1.6 mg per plant). Inoculation with rhizobia increased root hair deformation with significant interaction across cultivars. Nodulation was delayed under high starter N concentrations, and genotypic differences were evident for days‐to‐nodule initiation. There was a positive quadratic response to starter N for AC Christie and a negative quadratic response for Tempus for nodulation. Tempus had more active nodules (92 %) than AC Christie (73 %). The genetic variability of red clover cultivars should be considered in N fixation studies and their response to availability of initial N.     

Intercropping fenugreek (Trigonella foenum‐graecum L.) and barley (Hordeum vulgare L.) with and without biochar: Tests along a competition gradient

Integrating soil amendment biochar to legume‐based intercropping systems may amplify the intercropping benefits and lead to more sustainable production due to its positive effects on the soil physicochemical and biological environment. Fenugreek (Trigonella foenum‐graecum) is a legume used in an intercropping system in several countries in Southern Europe, the Middle East, and Asia. A barley cultivar and an Iranian fenugreek ecotype were studied in two replacement series experiments to find the best combination of the two species as well as the effect of adding biochar to the soil. Fenugreek and barley were grown in 4‐L pots in five density combinations (20:0, 15:5, 10:10, 5:15, 0:20) with and without application of biochar in sandy loam soil. The biomass, nitrogen (N) and carbon (C) amount of the two crops were measured fifty days after sowing, and the Relative Yield Total (RYT) was estimated. Our results showed that biochar application resulted in a higher total biomass, N and C amount in all combinations of fenugreek and barley. Total biomass was increased by biochar 19.2% when fenugreek was grown alone and 8.1 and 12.9% in series with 25 and 50% barley when biochar was added, respectively. Biochar increased dry matter, N and C accumulation in both crop species in the mixtures. An exception was the C amount of barley which was not influenced by biochar in intercropping. RYT values were largest for biomass, C and N amount in mixtures with 15 fenugreek plants per pot and 5 barley plants per pot corresponding to approximately 400 fenugreek plants per m mixed with 130 barley plants per m². Biochar application increased RYT of the total dry weight by 2.7 and 5.5% in mixtures of 25 and 50% barley plants, RYT of the total nitrogen by 0.8% in mixtures with 25% barley plants, and RYT of the total carbon by 2.7 and 6.6% in mixtures with 25 and 50% barley plants. The RYT values declined with increasing number of barley plants as a result of a less competitive ability of fenugreek when growing in high barley densities. The yield was highest with intercropping but monoculture of barley also resulted in high yields. The total highest yield was found when approximately 130 fenugreek plants per m² were mixed with 400 barley plants per m². Mixtures with 75% fenugreek and 25% barley obtained RYT values larger than 1 for biomass, carbon and nitrogen percentages.