β－转换生长因子对牛体外受精卵体外发育的影响

本研究主要检测ＴＧＦ－β对牛早期胚胎体外发育和移植后的成活率的影响。在试验１和试验２，添加０．３～７．０ｎｇ／ｍＬ的ＴＧＦ－β至无论是复杂培养液还是各成分已知的简单培养液中，囊胚中内细胞团细胞都不同程度地增长，比对照组的囊胚多０．５～１．０倍；此外在含有３．０～５．０ｎｇ／ｍＬ的ＴＧＦ－β的培养液中的囊胚，其孵化率显著地高于对照组。在试验３中，添加３．０ｎｇ／ｍＬ的ＴＧＦ－β显著地促进与不同细胞共同培养的囊胚内细胞团的细胞增长，在颗粒细胞或输卵管细胞及其两者组合当中的单层细胞上的囊胚表现突出。在试验４中，用ＴＧＦ－β培养所获得的囊胚移植于受体母牛，约４５～９０ｄ将受体屠宰检查中发现，ＴＧＦ－β的囊胚成活率高于无ＴＧＦ－β的囊胚．试验结果表明，ＴＧＦ－β可促进体外发育的囊胚内细胞团的细胞增长以及提高囊胚移植后的成活率。     

牛促卵泡激素β亚基基因在哺乳动物细胞中的导入与表达

将牛ＦＳＨ－βｃＤＮＡ连接于ｐＭＭＴ中金属硫蛋白启动子下游，与带有二氢叶酸还原酶基因的质粒共转染中国仓鼠卵巢细胞ＣＨＯ－ｄｈｆｒ＾－，在ＤＨＦＲ＾＋转化细胞中所整合的ＭＴ－ｂＦＳＨ－β基因可重金属镉诱导。     

红壤中高岭类矿物的鉴定

用Ｆｏｍａｍｉｄｅ处理－ＸＲＤ，红外（ＩＲ）和透射电镜（ＴＥＭ）等方法对我国红壤的高岭类矿物进行了鉴定。结果表明：花岗岩发育的赤红壤中高岭类矿物为结晶良好的高岭石；玄武岩发育的砖红壤为无序高岭石；红壤中也以无序高岭石为主，部分样样含少量埃洛石，而发育度较低的黄壤，红砂土及泥页岩发育的黄红泥土则以埃洛石为主。     

在大肠杆蓖TG1中克隆和表达苏云金芽胞杆菌cryI基因

本研究分离并提纯苏云金芽胞杆菌８０１０菌株的质粒ＤＮＡ，经ＢａｍＨＩ／ＰｓｔＩ双酶解后与ＤＩＧ标记的ｃｒｙＩ基因ＥｃｏＲＩ－Ｆ片段的ＲＮＡ探针杂交，显示出分子量分别为６．５６ｋｂ和４．４ｋｂ的阳性片段。用Ｂｌａｓｓｍｉｌｋ回收４．４ｋｂ的阳性片段并与双功能载体ｐＲＩＴ５重组，转化感受态大肠杆菌ＴＧ１细胞，通过斑点杂交和快检获得含４．４ｋｂ片段的克隆株Ｔ２，ＳＤＳ－ＰＡＧＥ电泳表明它表达了１３０ｋＤ     

Frankia共生固氮基因的分离与克隆

利用ＤＮＡ－ＤＮＡ杂交方法，分离细枝木麻黄的Ｆｒａｎｋｉａ菌株Ｃｏ０１的ｎｉｆ克隆ｐＣｃ１ＧＸ，赤杨内生菌株Ａｔ４的ｎｉｆ克隆ｐＡｔ１ＧＸ及沙棘的ＦｒａｎｋｉａＨｒ１８的ｎｉｆ克隆ｐＨｒ１８ＧＸ、ｐＨｒ１１－③ＧＸ。用基因功能互补法，从Ｆｒａｎｋｉａ菌株Ａｔ４的基因文库中分离到二个可能可互补豌豆根瘤菌ｎｏｄ基因功能的克隆ｐＡｔ２ＧＸ、ｐＡｔ３ＧＸ，并制作了ｐＡｔ２ＧＸ、ｐＡｔ３ＧＸ的亚克隆，予进一步实验，获得充分的证据证明ｐＡｔ２ＧＸ、ｐＡｔ３ＧＸ是否带有Ｆｒａｎｋｉａ菌株Ａｔ４的ｎｏｄ基因．     

应用PCR-RFLP及PCR-SSCP技术研究我国水稻条纹病毒RNA4基因间隔区的变异

应用反转录－聚合酶链式反应（ＲＴ－ＰＣＲ）及限制性片段长度多态性（ＲＦＬＰ）和单链构象多态性（ＳＳＣＰ）分析技术,快速检测我国水稻条纹病毒（Ｒｉｃｅ ｓｔｒｉｐｅ ｖｉｒｕｓ,ＲＳＶ）ＲＮＡ４基因间隔区（ｉｎｔｅｒｇｅｎｉｃ ｒｅｇｉｏｎ,ＩＲ）的变异。ＩＲ经ＲＴ－ＰＣＲ扩增后得到一段长约６８０ｂｐ的片段,ＲＳＶ７个分离物之间片段大小相同,应用ＡＩｕＩ、ＮｄｅＩ在它们之间检测到ＲＦＬＰ,其中ＢＳ、     

生长因子对卵母细胞体外成熟及早期胚胎体外发育的影响

为了探讨生长因子（ＥＧＦ，ＰＤＧＦ）对牛卵母细胞体外成熟与早期胚胎体外发育的影响，本研究进行了两个实验。实验１：在卵母细胞体外成熟（ＩＶＭ）中，将卵母细胞平均随机的放入表皮生长因子浓度为０，２．５，２５，５０μｇ／Ｌ的成熟培养液和对照组中。实验２：在早期胚胎体外培养（ＩＶＣ）过程中，将受精卵平均随机分配入５个不同培养液的处理组：（１）ＴＣＭ－１９９＋１０％阉公牛血清（ＳＳ）；（２）ＴＣＭ－１９９＋ＥＧＦ＋ＰＤＧＦ；（３）ＴＣＭ－１９９＋ＥＧＦ；（４）ＴＣＭ－１９９＋ＰＤＧＦ；（５）ＴＣＭ－１９９＋无生长因子、其中ＥＧＦ：５０μｇ／Ｌ，ＰＤＧＦ：０．１μｇ／Ｌ。２，３，４，５组有０．１％ＰＶＡ和０．３％ＢＳＡ，并与颗粒细胞单层协同培养。卵母细胞的体外成熟（ＩＶＭ）、体外受精（ＩＶＦ）和早期胚胎体外培养（ＩＶＣ）的方法与Ｌｕ等（１９８７）报道的相同。结果表明：浓度为５０μｇ／Ｌ的处理组分裂率与对照组无差异（８９．０％，９２．４％，Ｐ＞０．０５），而浓度为０，２．５，２５μｇ／Ｌ的处理组分裂率均显著低于对照组（７８．６％，８２．３％，８４．３％，Ｐ＜０．０５），在囊胚率和第７ｄ一级胚胎率上各组均无差异。ＥＧＦ在体     

抗福美双假单胞菌株的NTG化学诱变及分子探针鉴定

利用微生物的抗药性筛选抗福美双的假单胞菌株Ｔ４６，然后通过亚硝基胍（ＮＴＧ）化学诱变获得了对福美双敏感的三株突变株Ｔ４６Ｎ１０、Ｔ４６Ｎ７和Ｔ４６Ｎ１７。利用这三个突变株作受体，通过基因克隆的方法筛选到了可使三个突变株恢复抗性的克隆，抗性基因分别被定位在７ｋｂ、２．５ｋｂ和２０ｋｂ的ＥｃｏＲⅠ片段上。将这三个克隆分别用（３２）Ｐ标记后制成分子探针，然后分别与三个突变菌株的总ＤＮＡ进行ＤＮＡ－ＤＮＡ分子杂交，结果表明，各突变株均存在与菌株Ｔ４６的抗福美双基因克隆的高度同源性，经结合形态、生理生化等分析后证实这三株突变株均源自于出发菌株Ｔ４６。     

快,慢型大豆根瘤菌质粒接合重组的初步研究

通过含有Ｒ６８．４５质粒的大肠杆菌将快生型大豆根瘤ＡＣＣＣ１５０６７菌株的大质粒转到慢生型大豆根瘤菌６１Ａ７６菌株中去，共接合转移出２２个接合子菌株，对其中的１２个菌株进行了与８３－１１９大豆的共生接种试验，其效果是部分接合子菌株超过供体菌和受体菌对接合子ＢＦ２－Ａ１菌株进行了质粒检测。田间应用亦有增产趋势。     

水稻条纹病毒北京双桥(RSV-SQ)分离物RNA4片段序列分析

对我国水稻条纹病毒（Ｒｉｃｅ ｓｔｒｉｐｅ ｖｉｒｕｓ,ＲＳＶ）北京双桥（ＲＳＶ－ＳＱ）分离物ＲＮＡ４片段进行克隆及测序,结果表明ＲＮＡ４全长为２１５７ｂｐ。序列同源性比较发现,在核苷酸水平上,ＳＱ分离物与Ｔ、Ｍ及ＣＸ分离物的ｖＲＮＡ４ＯＲＦ同源性分别为９８．３％、９８．３％和９６．１％,ｖｃＲＮＡ４ＯＲＦ的同源性分别为９８．１％、９７．８％和９４．０％,ＲＮＡ４ＩＲ的同源性分别为９４．８％、８９     

Variations in infectivity of indigenous rhizobial isolates of some soils in the rainforest zone of Nigeria

Nodule formation in legumes is a process that starts with root infection by rhizobia. The present study assessed the population and infectivity of the indigenous rhizobial strains in rainforest soils of Nigeria. Soils were collected from three sites – Idi-Ayunre, Orile-Ilugun (OI) and the University of Ibadan Teaching and Research Farm (UITRF) – and analysed for physico-chemical properties and rhizobial population. Soybean varieties TGx1448-2E and TGx1456-2E and a cowpea variety IT89KD-288 were planted as trap crops on each of the soils, and rhizobia were isolated from their nodules. Infectivity assay was conducted using eight varieties of soybean and a cowpea variety. Most probable number estimate of the rhizobial population showed that the UITRF had significantly higher rhizobial population than the other two locations. OI and the UITRF soils planted with TGx1448-2E had significantly higher nodules and number of strains than other treatments. Among the 70 slow-grower strains isolated, only nine were infective. Three of the nine strains – IDC8, TRC2 and OISa-6e – nodulated at least seven of the eight soybean varieties used for infectivity test. Indigenous rhizobial infectivity of the studied locations was low, and cultivation of grain legume may require rhizobial inoculation for high productivity.     

Diversity of rhizobia isolated from an agricultural soil in Argentina based on carbon utilization and effects of herbicides on growth

Seventy-six rhizobial isolates belonging to four different genera were obtained from the root nodules of several legumes (Vicia sativa, Vicia faba, Medicago sativa, Melilotus sp., Glycine max and Lotus corniculatus). The action of five commonly used herbicides [2,4-dichlorophenoxyacetic acid (2,4-D), glyphosate (GF), dicamba, atrazine and metsulfuron-methyl] on the growth of rhizobial strains was assessed. Subsequently, GF and 2,4-D were tested in a minimum broth as C and energy sources for 20 tolerant strains. The ability of these strains to metabolize different carbon sources was studied in order to detect further differences among them. Tolerance of the bacteria to agrochemicals varied; 2,4-D and GF in solid medium inhibited and diminished growth, respectively, in slow-growing rhizobial strains. Among slow-growing strains we detected Bradyrhizobium sp. SJ140 that grew well in broth + GF as the sole C and energy source. No strain was found which could use 2,4-D as sole C source. The 20 strains studied exhibited different patterns of C sources utilization. Cluster analysis revealed three groups, corresponding to four genera of rhizobia: Rhizobium (group I), Sinorhizobium (group II) and Mesorhizobium–Bradyrhizobium (group III). On the basis of the results obtained on responses to herbicides and C sources utilization by the isolates investigated, it was possible to differentiate them at the level of strains. These results evidenced a considerable diversity in rhizobial populations that had not been previously described for Argentinean soils, and suggested a physiological potential to use natural and xenobiotic C sources.     

Nodulation of Medicago truncatula and Medicago polymorpha in two pastures of contrasting soil pH and rhizobial populations

The size of the background rhizobial population can often determine the success of field nodulation and persistence of inoculant rhizobia. Field experiments were conducted to determine the nodulation response of annual medics (Medicago spp.) in a pasture-wheat-pasture rotation when grown in soils of contrasting pH and rhizobial populations. Medicago truncatula Gaertn. and M. polymorpha L. were inoculated with one of three different strains of Sinorhizobium medicae (WSM540, WSM688) or S. meliloti (NA39) or left uninoculated and sown in two fields of pH (CaCl₂) 5.9 and 7.2 of differing soil rhizobial backgrounds (11 and 7.1 × 10⁴ cells/g soil, respectively). Nodulation was assessed in years 1 and 3 of the rotation. At the site with a small rhizobial background, M. polymorpha nodulated poorly when inoculated with the acid-sensitive strain NA39 but nodulated well when inoculated with acid-tolerant strains WSM688 and WSM540. M. truncatula had a similar extent of nodulation with each of the rhizobial inoculants. At the site with a large rhizobial background all treatments had greater than 85% of plants nodulated. Nodule occupancies, assessed by PCR, provided further insight: at the site with a small rhizobial background both medic species successfully nodulated with the acid-tolerant strains WSM540 and WSM688 and these strains persisted to year 3. However, at the site with large rhizobial background, only one strain, WSM688, was identified from M. truncatula nodules in year 3. This study highlights the importance of edaphic constraints and plant–rhizobia interactions to the successful development of nodulation in a field environment.     

Distribution and efficiency of Rhizobium leguminosarum strains nodulating Phaseolus vulgaris in Northern Spanish soils: Selection of native strains that replace conventional N fertilization

Phaseolus vulgaris is a legume extensively cultivated in Spain, León province being the most important producer. This province produces selected varieties of common bean highly appreciated by their quality that warrants a Protected Geographic Indication (PGI). In this work we analysed the rhizobia present in nodules of the variety “Riñón” in several soils from León province in order to select native rhizobial strains to be used as biofertilizers. The analysis of rrs and housekeeping genes of these strains showed that they belong to two phylogenetic groups within Rhizobium leguminosarum (I and II). Although the group II strains were most abundant in nodules, very effective strains were also found in group I. Strains LCS0306 from group I and LBM1123 from group II were the best nitrogen fixers among all strains isolated and were selected for field experiments. The field research showed that the biofertilization of common bean with native and selected rhizobial strains can completely replace the fertilization with chemical N fertilizers. The biofertiliser designed in such way, was valid for the whole agroecological area, regardless the specific properties of each soil and microclimatic conditions. This conclusion can be generalised as a strategy for the development of biofertilisers in different agroecological conditions worldwide.     

Responses of Lentil to Co-Inoculation with Phosphate-Solubilizing Rhizobial Strains and Arbuscular Mycorrhizal Fungi

The purpose of this study was to evaluate the responses of lentil (Lens culinariscv. ‘Ziba’) to co-inoculation with arbuscular mycorrhizal (AM) fungi and some indigenous rhizobial strains varying in phosphorus (P)-solubilizing ability in a calcareous soil with high pH and low amounts of available P and nitrogen (N). A factorial experiment with completely randomized block design was conducted under controlled greenhouse conditions. The treatments consisted of (1) three inoculants of Rhizobium leguminosarum bv. viciae strains and a mixed rhizobial inoculant with an effective P-solubilizer strain of Mesorhizobium ciceri, (2) two AM fungal species, Glomus mosseae and Glomus intraradices, (3) two P sources, superphosphate and phosphate rock. Four replications were prepared for each treatment and a related control. After the growth period of three months, the dry matter of shoots plus seeds, their P and N contents, and percent of root colonized by AM fungus were measured. The results showed that the effects of AM fungi, rhizobial strains, and P fertilizers were highly significant (p < 0.01) for all the characteristics studied. The rhizobial strain with P-solubilizing ability showed a more beneficial effect on plant growth and nutrient uptake than the strain without this ability, although both strains had similar effectiveness for N₂-fixation in symbiosis with lentil. Synergistic relationships were observed between AM fungi and some rhizobial strains that related to the compatible pairing of these two microsymbionts. The P-uptake efficiency was increased when P fertilizers were applied along with AM fungi and/or P-solubilizer rhizobial strains.     

Benefits of inoculation of the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) crop with efficient and competitive<Emphasis Type="Italic"> Rhizobium tropici</Emphasis> strains

Cropping in low fertility soils, especially those poor in N, contributes greatly to the low common bean (Phaseolus vulgaris L.) yield, and therefore the benefits of biological nitrogen fixation must be intensively explored to increase yields at a low cost. Six field experiments were performed in oxisols of Paraná State, southern Brazil, with a high population of indigenous common bean rhizobia, estimated at a minimum of 10³ cells g^–1 soil. Despite the high population, inoculation allowed an increase in rhizobial population and in nodule occupancy, and further increases were obtained with reinoculation in the following seasons. Thus, considering the treatments inoculated with the most effective strains (H 12, H 20, PRF 81 and CIAT 899), nodule occupancy increased from an average of 28% in the first experiment to 56% after four inoculation procedures. The establishment of the selected strains increased nodulation, N₂ fixation rates (evaluated by total N and N-ureide) and on average for the six experiments the strains H 12 and H 20 showed increases of 437 and 465 kg ha^–1, respectively,in relation to the indigenous rhizobial population. A synergistic effect between low levels of N fertilizer and inoculation with superior strains was also observed, resulting in yield increases in two other experiments. The soil rhizobial population decreased 1 year after the last cropping, but remained high in the plots that had been inoculated. DGGE analysis of soil extracts showed that the massive inoculation apparently did not affect the composition of the bacterial community.     

Simple method of genomic DNA extraction from Rhizobia in dried nodules of Phaseolus vulgaris for strain differentiation by PCR-based DNA fingerprinting

Repetitive DNA peR fingerprinting of bacterial genomic DNA is a useful tool for typing and differentiation of rhizobial strains. The method was reported to be suitable for strain differentiation of Rhizobia present in individual root nodules of some leguminous plants without the need for isolation and cultivation of the strains, in which rhizobial genomic DNA was extracted directly from each fresh or frozen nodule. We developed a new protocol of rhizobial genomic DNA extraction/purification from dried nodules of Phaseolus vulgaris for generating repetitive DNA peR fingerprints of Rhizobia present in the nodules. The simplified protocol consists of only three major steps, heat extraction of genomic DNA from rhizobial cells prepared from dried nodules, ethanol precipitation of the DNA and Sephadex G-50 column purification of the DNA, and generated fingerprints with good quality for differentiation of Rhizobia strains. The protocol will be useful to examine the nodule occupancy of inoculated rhizobial strains in field experiments.     

Response of Vigna Unguiculata Grown Under Different Soil Moisture Regimes to the Dual Inoculation with Nitrogen-Fixing Bacteria and Arbuscular Mycorrhizal Fungi

The interaction between legumes, rhizobial and arbuscular mycorrhizal (AM) partners benefits plant nutrition and improves plant tolerance to water stress. The present research evaluated the effectiveness of symbioses between cowpea plants (Vigna unguiculata (L.) Walp.), AM fungi (Glomus intraradices) and two strains of Bradyrhizobium japonicum on the mycorrhization, acid phosphatase activity (APase), enzymes related to nitrogen fixation and assimilation, and biomass accumulation at three soil moisture levels. The results revealed that the soil moisture optimal for the formation of active symbiotrophic associations in cowpea cultivation was about 60% water-holding capacity (WHC), where both Bradyrhizobium strains and AM fungi function well with respect to mycorrhization, nitrogen and phosphorus uptake, nitrogen fixation and plant biomass production. Under conditions of reduced water supply, the symbiotic association between Br. japonicum-273 and Gl. intraradices was better for cowpea cultivation, while in elevated soil moisture association between Br. japonicum-269 and Gl. intraradices was more appropriate.     

The potential for rhizobial inoculation to increase soybean grain yields on acid soils in Ethiopia

In Ethiopia, inoculation of soybean with rhizobial inoculants is not common practice, but could provide an option to increase grain yields in low nitrogen (N) acidic soils. In these acid soils, the selection of acid tolerant rhizobia is one strategy that may increase the performance of soybean. In this study, rhizobial strains isolated from Ethiopian soils were evaluated for their acid tolerance and symbiotic N fixation efficiency with soybean, in controlled environments. Following this, four isolated rhizobial strains were evaluated in six field experiments in major soybean growing areas of Ethiopia. Inoculation with the commercial strain or with one of two locally sourced isolates, that were developed as inoculants, improved soybean yield. The yield increase due to inoculation with the commercial strain was consistent and greater than other treatments, while the increase due to the two locally sourced strains was comparable to, or greater than, application of 46 kg N/ha in soils, where the resident rhizobial population was ≤1.4 × 10³ cfu/g soil. For soils with high background rhizobial populations, there was no response to inoculation. In one of the experimental sites (Bako), the percentage of N fixed (%Ndfa) was 55 for the commercial strain and 35 for the local strain, ES3. This study demonstrated that field validation is a necessary step in the selection of acid-tolerant strains of rhizobia to increase soybean production for Ethiopia.     

Differences in common bean rhizobial populations associated with soil tillage management in southern Brazil

Progressive adoption of no-tillage (NT) agriculture in the tropics is finally reversing physical, chemical, and biological erosion of soil and in Brazil, an estimated 19 Mha are now devoted to NT. Common bean (Phaseolus vulgaris L.) is a main component of Brazilian agriculture, and enhancement of yields has been achieved under NT as a result of mitigation of environmental stresses, resulting in higher N₂ fixation. However, the effects of NT on rhizobial diversity are poorly understood. This study evaluated rhizobial diversity in soils planted to common bean under NT or conventional tillage (CT) systems that were compared with natural grassland used for grazing, in the State of Santa Catarina, southern Brazil. Genetic diversity was assessed by the amplification of the DNA by PCR with specific primers (BOX-PCR) and by RFLP-PCR analyses of the 16S rDNA region. A high level of diversity was observed among strains from all three systems, such that the similarity in the clustering analysis of BOX-PCR products ranged from 36% under natural grassland to only 23% for CT strains. High polymorphism was confirmed in the RFLP-PCR analysis; forty-seven different profiles were obtained, none sharing high similarity with the profiles of reference species of common bean rhizobia. These results indicate that other tropical rhizobial species remain to be described. Genetic diversity was higher among the NT than the CT rhizobial strains, especially when the RFLP-PCR profiles were considered. Genetic diversity in the natural grassland was lower than in the cropped systems, possibly due to absence of the host plant and stubble burning in winter. Average yields in the area under NT (e.g. common bean, approximately 1500 kg ha⁻¹) have been about 30% higher than under CT, therefore high rhizobial diversity may be a parameter indicative of superior soil quality.