几种水稻根际固氮菌与水稻联合共生固氮的研究

从水稻根际分离的两株固氮菌:肺炎克雷从伯氏菌(Klebsilla Pneumoniae)K—12,肠杆菌属(En—terobacter sp)E—25以及从国外引进的水稻根际固氮螺菌(Azospirill-um lipoferum FS),克雷伯氏菌(Klebsiella oxytoca NG13)菌株,分别接种于有水稻幼苗生长的无碳、无氮培养基中,于28℃暗培养条件下进行培养.以上各菌均能正常生长,并与水稻幼苗有联合固氮作用.其固氮活性分别如下:Azospirillum lipoferum FS菌株为10.5毫微克分子C_2H_4/株/小时,Klebsiella oxytoca NG13菌株为3.9毫微克分子,C_2H_4/株/小时,Klebsiella pneumoniae K—12菌株为4.9毫微克分子C_2H_4/株/小时,Enterobacter SP E—25菌株为2.9毫微克分子C_2H_4/株/小时.固氮菌与水稻联合共生于半固体培养基中时,菌在水稻胚乳周围生长旺盛、繁殖快、并形成菌环.用对DNA专一的荧光染料DAPI染色观察,以及扫描电镜观察均表明:这四种固氮菌均聚集并附着在水稻根表,大部分在根毛区.水稻根横切面的扫描电镜观察,以及对接种的水稻根内生菌的重新分离实验表明,部分固氮菌能够进入水稻根组织的皮层薄壁细胞内生长并繁殖.     

鼓槌石斛内生细菌分离、 鉴定及功能分析

为微生物农药和肥料生产提供菌种资源,用组织分离法从鼓槌石斛健康组织分离内生细菌,用对峙法和抑菌圈法筛选拮抗菌株;以平板法筛选解磷、解钾和固氮菌株;以田间试验检验固氮菌株的增产效果;用PCR扩增功能菌株的16S rDNA,结合菌株的菌体、菌落形态特征和部分生理生化特征,确定功能菌株分类地位。结果表明,从鼓槌石斛组织共分离到33株内生细菌,数量和种类为根〉茎〉叶;其中GB7属解淀粉芽孢杆菌（Bacillus amyloliquefaciens）,GB16属链霉菌（Streptomyces sp.）,GB8、GB9、GB21属芽孢杆菌（Bacillus spp.）,均有病害生防功能;GB2属不动杆菌（Acinetobacter calcoaceticus）,GB20属肠杆菌（Enterobacter sp.）,具解磷功能;菌株GB1属产酸克雷伯氏菌（Klebsiellaoxytoca）,具固氮功能。     

棉花多抗病性育种的抗性诱导研究

采用强致病力菌株,制备成棉子菌粉载菌体和孢子悬浮液,研究不同菌量、不同接菌方法和致病温度,诱导棉花苗期抗枯、黄萎病性的育种新技术。通过4种接种剂量梯度试验表明,棉花苗期诱导抗性的最佳接菌量是90 g.m-2棉子菌粉载菌体,播种前土壤接菌,棉黄萎病菌孢子悬浮液的最佳接菌浓度是2×107孢子.ml-1,2片真叶时伤根接菌10 ml,薄膜拱盖提温,20~25℃是诱导发病的最适温度,接菌15~20 d后,淘汰棉枯萎病发病8%以上和棉黄萎病12%以上的品种群体材料,并淘汰抗病群体材料中的感病个体,构建既抗棉枯萎病、又抗棉黄萎病性的多抗病性育种方法。     

传统法起泡红葡萄酒的关键酿造工艺研究

以宁夏贺兰山东麓产区黑比诺、西拉和马瑟兰葡萄为原料,对传统法起泡红葡萄酒酿造工艺的关键环节进行研究.确定最佳工艺条件为:黑比诺、西拉、马瑟兰的适时采摘期分别为9月10日、9月25日和10月1日;8℃冷浸渍时间为5 d;微氧处理溶氧量为15 mL/(L?月),西拉和马瑟兰的微氧处理时间为15 d和12 d;黑比诺、西拉和马瑟兰调配比例为50:25:25;瓶内陈酿时间为12-15个月.按此工艺可获酒液呈宝石红色,香气浓郁,口感舒顺,酒体饱满、起泡绵密的高品质起泡红葡萄酒.     

鹰嘴豆锌指蛋白基因ZE1的克隆及表达分析

利用一段从PEG胁迫的鹰嘴豆幼苗叶片所构建的cDNA文库中得到的EST序列,通过3'RACE方法克隆到一个鹰嘴豆C_2H_2型锌指蛋白基因ZF1,该基因不含内含子,编码一条244个氨基酸残基的多肽,含有两个典型的Cys2/His2锌指结构.其氨基酸序列含有一个可能的核定位型号,农杆菌介导的洋葱表皮细胞GFP瞬时表达实验表明,ZF1蛋白位于细胞核内.半定量RT-PCR分析表明,ZF1在鹰嘴豆的根、茎、叶、花、幼荚和幼胚中均有表达,在茎和叶中表达较弱,为组成型转录因子.半定量RT-PCR和实时荧光定量PCR检测结果显示,ZF1不但受高温及干旱诱导,而且还受6-苄基腺嘌呤(6-BA)、脱落酸(ABA)、乙烯利(Et)、赤霉素(GA_3)、吲哚-3-乙酸(IAA)、茉莉酸甲酯(MeJA)、水杨酸(SA)和氧胁迫诱导.这些结果表明,ZF1基因可能作为一个核调控因子参与植物的生长代谢以及多种生物与非生物胁迫的应答.     

红壤旱地一株自生固氮菌的筛选鉴定及其固氮能力评估

为了鉴定从红壤中分离得到的一株自生固氮菌,并探讨其固氮能力。采用形态观察、生理生化测定和16S rDNA基因序列分析的方法研究了菌株的分类地位,采用乙炔还原法测定其固氮酶活性,并在室内培养条件下,通过花生、玉米的幼苗盆栽试验研究菌株对土壤MBN、矿化氮和植株氮素积累量的影响。结果表明：从种植于红壤的玉米根际,筛选出15株自生固氮菌,以菌株CM12固氮能力最强,初步鉴定CM12为伯霍尔德杆菌属(Burkholderia sp.),固氮酶活性达C₂H₄ 39.1 nmol/(h·mL)。室内培养试验结果表明,接种CM12菌株的处理,花生和玉米土壤MBN含量较CK处理分别提高了2.38和2.37倍,其中种植花生体系中,接菌处理与施用化学氮肥处理土壤MBN含量无显著差异。接种固氮菌影响了旱地红壤NO₃^--N和NH₄⁺-N比例,降低了土壤中NO₃^--N含量,且种植玉米体系中土壤NO₃^--N含量降低较明显。固氮菌短期接种增加了花生根系和玉米地上植株的氮素积累量。研究结果为该菌株在红壤旱地实际生产中的应用提供了理论基础。     

夏花生覆膜对根瘤中固氮酶和叶片硝酸还原酶活性影响的研究

覆膜夏花生有利于根瘤菌的侵染和根瘤的形成，使花生早结瘤、多结瘤、早进行共生固覆；覆膜降低结荚以前的固氮酶活性，但有利于中后期固氮酶活性的提高和维持，可延缓根瘤衰老、延长根瘤固氮时间。覆膜对花生叶片硝酸还原酶活性（ＮＲＡ）的影响与固氮酶活性正好相反，结荚以前较高、中后期较低。与露地相比，覆膜花生前期利用土壤和肥料氮较多，而中后期则可主要靠根瘤固氮来提供氮素营养。     

Effect of Water Regimes and Moisture Stress at Different Growth Stages on Nodule Dynamics,Nitrogenase Activity and Nitrogen Fixation in Soybean (Glycine max (L.) Merrill)*

The effect of water regimes and soil moisture stress on total nodule number, effective nodule number, nodule dry weight, nitrogenase activity and nitrogen fixation in soybean was studied. The effective nodule number, nodule weight, nitrogenase activity and nitrogen fixation increased due to increase in water regimes from 0.4 to 0.6 Cumulative Pan Evaporation (CPE). The stress during either flowering or pod filling reduced the effective nodule number, nodule weight, nitrogenase activity and nitrogen fixation. The amount of nitrogen fixed was maximum with 0.8 CPE (50.09 kg ha^-1 season^-1) followed by 0.6 CPE (42.08 kg ha^-1 season^-1) and 0.4 CPE (33.04 kg ha^-1 season^-1). The amount of nitrogen fixed was reduced by 17.71 % and 26.39 % due to stress during flowering and pod filling respectively compared to normal irrigation.     

Stress Studies in Lentil (Lens esculenta Moench)

A pot experiment was conducted to see the effect of varying levels of soil sodicity on leaf growth, nitrate reductase activity, nodule development and nitrogenase activity in two lentil ( Lens esculenta Moench) genotypes. It was observed that in both the genotypes increasing level of soil sodicity decreased the plant height, leaf area, leaf dry weight, total biomass production and finally the grain yield. Nitrate reductase activity in leaf tissues and concentration of total nitrogen in different plant organs showed significant reduction with increase in soil sodicity. Increasing soil sodicity (15 and 20 ESP) was not only deleterious to nodulation and nitrogenase activity but also caused complete failure of nodulation process at 25 ESP of soil. Reduction in aforesaid characters due to increasing soil sodicity was less in variety PL-406 than that of local indicating thereby the relative tolerance of PL-406 to sodicity.
Prolonged and higher activities of nitrate reductase and nitrogenase at higher sodicity level in PL-406 might be the reasons for superiority of this variety. Significant and positive correlations were also observed between leaf characters and nitrogenase activity.     

The Effect of Nitrogen Fertilization and Population Density of the Field Bean (Vicia faba L. minor) of Indeterminate and Determinate Growth Habit on the Symbiosis with Root Nodule Bacteria and on the Seed Yield

Field beans of indeterminate (cv. Nadwiślański) and determinate (cv. Tibo) growth habits were grown on field plots with a density of 20, 40 and 80 plants m⁻² at two levels of nitrogen fertilization: low (20 kg N ha⁻¹) and very high (150 kg N ha⁻¹). At the phase of intense pod growth the number and the dry matter weight of root nodules as well as their nitrogenase activity and some features of the plants growth were determined, and in the period of ripeness the components of seed yield were established.
It was found that increased density of sowing as well as the high level of nitrogen fertilization inhibited the growth and development of root nodules and limited their nitrogenase activity in both cultivars. Plants of both varieties were characterized by a similar potential of forming the root nodules, however, in plants of the cv. Tibo the nitrogenase activity of nodules was much lower than in the cv. Nadwiślański.
Increased population density of the plants has in both varieties given increased seed yield when calculated per m² of the soil, compensating in excess the depression of the seed yield from one plant. Such compensation did not occur in the case of the depression of nitrogen fixation under intense nitrogen fertilization.     

减量施氮对带状套作大豆土壤通气环境及结瘤固氮的影响

为研究种植模式和施氮量对大豆土壤通气环境及结瘤固氮的影响,本研究采用二因素裂区试验设计,主区为种植模式:大豆单作(SS)、玉米/大豆带状套作(MS),副区为不同施氮量:不施氮(NN:0 kg hm^-2)、减量施氮(RN:45kghm^-2)和常量施氮(CN:60kghm^-2),监测了大豆生育期内土壤的O₂含量和土壤呼吸速率的动态变化规律,分析了各处理的土壤水稳性团聚体、容重和孔隙度差异,探讨了大豆生育期内结瘤量、固氮能力的变化特征。2年数据结果表明,与单作大豆相比,带状套作大豆可提高土壤O₂含量、>2 mm粒径团聚体的百分含量、显著增加土壤孔隙度、显著增强后期土壤呼吸的速率,显著降低<1mm粒径团聚体百分含量与土壤孔隙度;R5期根瘤干重差异不显著,但结瘤数量显著增加39.9%,固氮酶活性与固氮潜力也在此时显著高于单作大豆。各施氮量间,土壤O₂含量、0.25～1.00mm粒径的百分含量、带状套作大豆的土壤呼吸速率以RN处理最高,施氮能降低<0...     

大豆PIN-Like(PILS)基因家族的鉴定、表达分析及在根瘤共生固氮过程中的功能

植物激素生长素在植物的生长发育过程中发挥了至关重要的作用,它的稳态和浓度梯度建立控制了几乎所有器官的极性建成。生长素在特定细胞中合成、运输、感知以及代谢降解建立了符合器官发育的生长素浓度梯度。在豆科植物中,根与土壤微生物互作形成了根瘤这一特殊的器官,进行生物固氮。然而,生长素稳态控制生物固氮的功能还未知。拟南芥中的研究表明,PIN-Like (PILS)蛋白协助调节的细胞内生长素稳态,并介导下游细胞核内的生长素信号传递。本研究以大豆作为研究模型,在大豆基因组中鉴定获得19个PILS家族基因(GmPILS),不均匀分布于大豆10条染色体上。GmPILS在大豆9种组织部位中表现出多种表达模式,且具有明显的组织表达特异性。GmPILS1e和GmPILS1f在根瘤菌体区域富集表达,使用人工微RNA沉默(artificialmicro RNA interference,amiRNAi)下调GmPILS1e和GmPILS1f在根瘤的表达,导致根瘤的固氮酶活性上升,而过量表达GmPILS1f导致根瘤的固氮酶活性下降,因此GmPILS1e和GmPILS1f可能参与大豆固氮酶活性的调节。这些结果为进一步解析大豆GmPILS家族基因的功能和作用机制奠定了基础,同时也为结瘤固氮在农业育种中的应用提供了有价值的基因资源。     

Nitrogen Fixation and Nitrate Assimilation of Determinate, Semi-determinate and Undeterminate Soybeans (Glycine max L.)

Soybean ( Glycine max L.) nitrogen nutrition is ensured by both symbiotic nitrogen fixation and mineral nitrogen assimilation. The relationship between these two modes of N nutrition was analysed in 3 growth types (determinate, semi-determinate and undeterminate) of soybean. The nitrate reductase activity and nitrogenase activity (acetylene reduction) of plants grown in the field and greenhouse showed that these enzymatic activity acted simultaneously or successively during the growth cycle, depending on the availability of inorganic nitrogen in the growing medium. Undeterminate soybean types had a higher potential nitrate reductase activities than determinate types.
The proportion of N₂ fixed as measured by ¹⁵N labelling or stem ureide content indicated that determinate soybeans derived a higher proportion of their N from N₂ fixation than the undeterminates.     

Einfluß des Lichtangebotes auf das Wachstum und die biologische Stickstoff-Fixierung von Weißklee (Trifolium repens L.)

Influence of light quantity on growth and biological nitrogen fixation of white clover ( Trifolium repens L.)
The influence of photon irradiance (E_p; 100 to 500 μmol m⁻² s⁻¹) and of the photoperiod (16 or 11 h) on growth and nitrogenase activity of nodulated white clover plants was studied in growth chambers at two nitrate levels (1.0 and 7.5 mM NO₃⁻).
Total dry mass production, the root proportion and nitrogenase activity increased with increasing E_p and photoperiod. Nitrogenase activity generally increased proportionally to root mass. Only at low E_p (100 μmol m⁻² s⁻¹) and under a short photoperiod (11 h) was the specific nitrogenase activity per unit root mass reduced. An abrupt change in E_p led to a rapid and parallel change in nitrogenase activity and relative growth rate.
A higher NO₃⁻ concentration in the nutrient solution (7.5 mM) led to a marked decrease in specific nitrogenase activity, but increased growth between 200 and 500 μmol m⁻² s⁻¹ during early development only. At 100 μmol m⁻² s⁻¹, there was no growth response to nitrate, although its effect on nitrogenase activity was more marked than at a higher E_p.
The results show that with changing light quantity, biological nitrogen fixation of white clover adapts to the existing demand for nitrogen and does not limit growth except during early development, even when light supply is low.