Identification of Growth-promoting Bacteria from Rhizosphere of Pastures and Their Effects on Growth of Lotus corniculatus L.

[Objectives] This study was conducted to explore the interaction between nitrogen-fixing and phosphate-solubilizing strains and the optimal combination of different functional strains,in order to provide a theoretical basis for the development of PGPR compound fertilizers suitable for local environment.[Methods] In this study,16S rDNA gene sequence analysis was used to identify fast-growing and competitive strains from pasture nodules and rhizosphere soils in Guizhou Province,and three representative Rhizobia and phosphorus-solubilizing bacteria were chosen for the test of bacterial combination when reducing 50% of nitrogen and 30% of phosphorus.The effects of different strain combinations on the plant height,root length,aboveground and underground biomass of Lotus corniculatus L.were investigated,and the total nitrogen and total phosphorus contents of the plants were determined.[Results] The mixed bacterial agents could promote the increase of root biomass,and the effects of A1,A3,B3 and C3 were the most obvious.The fresh weight and dry weight of the roots of L.corniculatus increased by 30.35%-168.45% and 26.43%-180.00%,respectively,and A3,B3,B2 and C3 had the best effects.The total phosphorus content of the plants increased by 12.79%-55.25% compared with the CK2;and most of the bacterial agents with significant growth-promoting effects showed decreased total nitrogen contents,while those with non-significant growth-promoting effects showed significantly-increased total nitrogen contents,which were not as much as the CK1.Comprehensively,the most productive combination was C3,namely R27-2 Rhinohizobium fredii and P33-3 Stenotrophomonas rhizophila.[Conclusions] This study can provide a theoretical basis for the production and promotion of bacterial fertilizers.

Effects of Condensed Tannins from Quebracho Extract on the Kinetic of in vitro Gas Production on Trifofium repens, Lotus corniculatus and Lofium perenne

The aim of this work was to study the effect of condensed tannins （CT） on the kinetic of gas production in vitro, on the three main species of Azorean pasture： Trifolium repens, Lotus corniculatus and Lolium perenne, using Quebracho extract as the source of CT, and also to calculate total tannins and condensed tannins on the species studied. Lotus corniculatus, Lolium perenne and Trifolium repens were tested in vitro with the inclusion of Quebracho extract in doses of 0%, 2.5% and 5% DM. The total content in tannins was determined in the 3 species. We have found, in comparison with standard tannic acid, that tannins exist in the flower of T. repens （0.81 equivalents of tannic acid） and in L .corniculatus （1.07 equivalents of tannic acid） but not in L .perenne （0 equivalents of tannic acid）. The concentration of condensed tannins in the 3 species was： 0.34 mg/mL for T. repens; 0.83 mg/mL for L. corniculatus and 0 mg/ml for L. perenne. We verified that a significant reduction （P 〈 0.05） of gas production happened for doses of 5% of CT in comparison with the other two concentrations： 0% and 2.5% of CT. The results of this study express a reduction of the fermentation rate, which implies a reduction of gas production, so, a reduction of methane emission to the atmosphere and an increase of exploitation of the protein by ruminants.

TRANSFORMATION OF Lotus corniculatus L.MEDIATED BY Agrobacterium tumefaciens

A simple and effective system for the transformation and regeneration of a leguminous plant has been developed. The cotyledon explants of Lotus corniculatus (var. Leo) were infected by Agrobacterium tumefaciens that contained a non-oncogenic Ti plasmid vector. The vector carried a chimaeric npt-Ⅱ gene and a nopaline synthase gene (nos). On the selective medium that contained kanamycin, 40% of the explants formed buds within 3 weeks. Developed shoots were cut off and transferred to rooting medium. Normal-looking plants were recovered and grew well after being transplanted into soil, bloomed and set seeds. Foreign genes were integrated onto the L. corniculatus genome, expressed, and inherited through sexual reproduction, which was proven by nopaline detection, NPT-Ⅱ enzyme activity detection and DNA hybridization test of the transformed plants and the nopaline detection of the progeny.

干旱胁迫及复水处理对百脉根叶片丙二醛含量及抗氧化酶活性的影响

以栽培型和野生型百脉根(Lotus corniculatus L.)为试验材料,对干旱胁迫4,8,12,16 d和复水处理下4,8 d百脉根的部分生理生化指标的变化进行了研究。结果表明:随着干旱胁迫时间的延长,叶片相对含水量逐渐下降,叶绿素含量、MDA含量、SOD活性和POD活性呈先升高后降低的变化趋势。复水后,各指标得到了不同程度的恢复,但POD的活性明显仍低于对照(P<0.05);从MDA含量变化来看,复水后细胞膜受到的伤害仍然没有修复。干旱胁迫处理下,栽培型和野生型材料的生理生化变化具有一定的差异性,说明来自湿润环境中的野生百脉根的耐旱性相对较差。

百脉根高频再生体系建立和猪瘟病毒E2基因的遗传转化

在建立百脉根(Lotus corniculatus L.)高频再生体系的基础上,通过农杆菌介导,将猪瘟病毒(CSFV)表面抗原E2基因转化进入百脉根,获得转基因阳性株系5个。并对遗传转化体系中关键性因素进行优化,结果表明,最优化体系为子叶预培养3 d后,用OD600nm为0.5±0.05的菌液侵染30 min,共培养3 d并添加AS(乙酰丁香酮),选择培养基中卡那霉素浓度为50 mg/L,可获得理想的转化效率。为抗猪瘟可食性疫苗的研制奠定基础。

百脉根组织培养的研究

以UM和MS为基本培养基,通过调整激素种类与浓度等培养条件,按正交实验设计的原则,摸索出建立百脉根组织培养体系的最佳条件:愈伤诱导最佳培养基为UM+2,4-D 2 mg.L-1+KT3 mg.L-1;最佳分化培养基为MS+NAA0.1 mg.L-1+6-BA 1 mg.L-1;最佳生根培养基为MS+NAA0.05 mg.L-1。

Introduction, Collection and Characterization of Lotus spp. Germplasm in Chile

The information related with results of a project on germplasm characterization of three Lotus species, important as forage plants, curried out in five environments of the central-southern zone of Chile, was reviewed. An evaluation was made of 12 L. corniculatus cultivars introduced from different parts of the world, as well as germplasm naturalized in Chile, being 11 accessions of L. tenuis and 21 accessions ofL. uliginosus collected from 33°S to 38°S and 38°S to 45°S, respectively. Phenological, morphological, and agronomical characters were evaluated. Nitrogen fixation in field conditions and concentrations of condensed tannins in herbage were also included. It is concluded that there are cultivars of high value in L. corniculatus introduced germplasm which can be recommended for different environments of the central zone of Chile, as well as for local species breeding programmes. Information obtained from L. tenuis and L. uliginosus accessions shows genetic variability within both species. L. tenuis germplasm is adapted to clay, medium-textured, or sandy soils with water restrictions and phosphorous deficiency. Therefore, characterized accessions could be used to breed cultivars for low input production systems in constrained environments. L. uliginosus accessions include genotypes collected in sites with acid soils and variable tannin content in plant tissues that may be promissory genetic materials for breeding programmes with the objective of producing cultivars for animal production systems for the wet and acid soils in the southern zone of the country.

Root Exudation of Organic Acids of Herbaceous Pioneer Plants and Their Growth in Sterile and Non-Sterile Nutrient-Poor,Sandy Soils from Post-Mining Sites

Nutrient-poor, sandy soils form the prevailing substrate at post-mining sites of the Lusatian region(Brandenburg, Germany) and present a challenge for vegetation development. We studied the organic acid quantity and composition of three commonly occurring pioneer plant species, the legumes Lotus corniculatus L. and Trifolium arvense L. and the grass Calamagrostis epigeios(L.) Roth, to determine if plant growth and exudation differed with(non-sterilized soil) and without(sterilized soil) an indigenous soil microbial community. We investigated whether organic acids were found in the rhizosphere and surrounding soil and whether this influenced nutrient mobilization. This study consists of linked field investigations and a greenhouse experiment. Plants were grown in the greenhouse in either sterilized or non-sterilized sandy soil from a reclamation site in the Lusatian mining landscape(Welzow Su¨d, East Germany). After seven months, the plant biomass, root morphology, organic acids, and water-soluble nutrients and root colonization with arbuscular mycorrhizal fungi(AMF) and dark septate endophytes(DSE) were analyzed. Roots of all three plants in the field and greenhouse experiments were highly colonized with AMF. Calamagrostis epigeios and T. arvense had a significantly higher colonization frequency with DSE than L. corniculatus. The quantity and composition of organic acids strongly differed among plant species, with the highest number of organic acids found for L. corniculatus and lowest for C. epigeios. The quantity of organic acids was greatly reduced in all plants under sterilized soil conditions. However, the composition of organic acids and plant growth in sterilized soil were reduced for both legumes, but not for C. epigeios, which had a higher biomass under sterilized conditions. Changes in nutrient concentrations in the field rhizosphere soil relative to those in the control were measurable after seven months. While the spectrum of organic acids and the growth of legumes seemed to be dependent on a highly diverse soil microbial community and a symbiotic partner, the grass C. epigeios appeared capable of mobilizing enough nutrients without an indigenous microbial community, and might be more competitive on sites where soil microbial diversity and activity are low.

Selection of rhizobial strains differing in their nodulation kinetics under low temperature in four temperate legume species

Background:Winter climate change including frequent freeze-thaw episodes and shallow snow cover will have major impacts on the spring regrowth of perennial crops.Non-bloating perennial forage legume species including sainfoin,birdsfoot trefoil,red clover,and alsike clover have been bred for their adaptation to harsh winter conditions.In parallel,the selection of cold-tolerant rhizobial strains could allow earlier symbiotic nitrogen(N)fixation to hasten spring regrowth of legumes.Methods:To identify strains forming nodules rapidly and showing high N-fixing potential,60 rhizobial strains in association with four temperate legume species were evaluated over 11 weeks under spring soil temperatures for kinetics of nodule formation,nitrogenase activity,and host yield.Results:Strains differed in their capacity to form efficient nodules on legume hosts over time.Strains showing higher nitrogenase activity were arctic strain N10 with sainfoin and strain L2 with birdsfoot trefoil.For clovers,nitrogenase activity was similar for control and inoculated plants,likely due to formation of effective nodules in controls by endophyte rhizobia present in seeds.Conclusions:Selection based on nodulation kinetics at low temperature,nitrogenase activity,and yield was effective to identify performant rhizobial strains for legume crops.The use of cold-tolerant strains could help mitigate winter climatic changes.