两株自生固氮菌的鉴定及其应用基础研究

为鉴定从滨海盐土中分离的2株自生固氮菌,并探讨其环境适应性及小麦盆栽接种效应。采用形态特征、生理生化测定和16S r DNA基因序列分析的方法研究了2株菌的分类地位,采用乙炔还原法测定其固氮酶活性,用IAA分泌量表征环境适应性,利用小麦盆栽试验评价其对小麦幼苗的促生作用。初步鉴定菌株1为争论贪噬菌,菌株2为玉米固氮螺菌,固氮酶活性分别为4. 71,2. 84μmol/(h·m L)。菌株1、2对酸碱度具有较强的适应性。渗透压结果显示,菌1、2在5%Na Cl及以下浓度,IAA分泌能力较强。菌株1对甘露醇利用效果较好,菌株2对葡萄糖利用效果最好。外源低浓度NH_4~+提高菌株1分泌IAA能力,当NH_4~+浓度 0. 5 mmol/L,其分泌IAA能力受到不同程度抑制。菌株2对NH_4~+利用呈M型曲线。盆栽试验表明,菌株1、2显著提高小麦株高、地上部干质量,促进小麦氮、磷、钾吸收,提高了土壤速效养分含量。土壤脲酶活性增加了16. 00%,48. 15%,其中菌株2效果最佳。菌株1、2具有较高的固氮酶活性、较强的环境适应性,小麦盆栽接种效果良好,属于高效固氮耐盐菌株,可为新垦低产盐碱农田的培肥使用,并可为中高产盐碱农田优化施肥结构提供技术支撑。     

杜仲内生真菌DZJ07的GFP标记及在小麦植株中的定殖

杜仲内生真菌DZJ07对小麦纹枯病菌有较好的防治作用,为了阐明其在小麦植株中的生态适应性,通过农杆菌介导转化法将绿色荧光蛋白基因(gfp)表达载体质粒pDHt-bar-gfp转入DZJ07菌株中,获得了带绿色荧光且遗传稳定的转化子,比较了荧光转化子DZJ07-6和原始菌株在菌落形态、生长特性、拮抗特性以及对小麦纹枯病防治效果等方面的特性,结果发现,荧光转化子DZJ07-6和原始菌株在上述特性方面无明显差别。在此基础上,温室条件下研究了荧光转化子DZJ07-6在小麦植株的定殖动态,荧光显微观察发现荧光转化子DZJ07-6可侵染小麦根系,在小麦根和茎组织的细胞间隙和细胞内大量定殖,平板稀释结果表明,荧光转化子DZJ07-6在小麦根际、根和茎内均能定殖,截止生长的第34天,小麦根际、根内以及茎组织的DZJ07-6菌量分别为2.72×10~6,0.62×10~4,2.26×10~3cfu/g。结果为深入研究DZJ07菌株内生性及生防作用机理奠定了基础。     

粪产碱菌和阴沟肠杆菌与水稻联合共生的固~(15)N_2作用

应用同位素¹⁵N 示踪证实了粪产碱菌(Alcaligenes faecalis)A—15和阴沟肠杆菌(Enterobacter cloacae)E—26均有较高的固氮能力。用这两株菌接种水稻幼苗,在不添加碳源时,它们不论单独或混合接种均能在水稻根际正常生长和固氮,其部分固氮产物能迅速转移到水稻植株;若添加碳源,则固氮量剧增。     

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。     

小麦抗白粉病SSH-cDNA文库中差异基因的表达模式

为了解白粉菌诱导下抗白粉病小麦的抗病机制,构建了白粉菌接种初期的抑制性消减杂交cDNA(SSH-cDNA)文库。从中随机挑取140个阳性克隆进行测序,去除冗余序列和重复序列后,得到94条EST,利用NCBI的BLAST在线序列比对工具对GenBank的蛋白质数据库进行同源性比对及功能分类。结果表明,49个EST与已知功能蛋白同源性较高,主要涉及初级代谢(2%)、能量代谢(24%)、细胞结构(2%)、转录(2%)、蛋白质合成加工与储藏(16%)、转运(4%)、信号转导(4%)和抗病与防御(30%)。经文库比较,筛选出与病程相关蛋白基因同源的EST(Z25-1)和与谷胱甘肽硫转移酶同源的EST(Z440-1),GenBank登录号为EX567369和EX567360。以其EST序列为依据设计引物,通过RT-PCR分析它们在白粉菌诱导下的表达模式,结果该2基因表达存在明显差异。其中,Z25-1在未接种白粉菌时具有一定的表达量,白粉菌侵染后表达量开始上升,侵染72 h时表达量最高,然后下降;Z440-1在未接种时也具有一定的表达量,但在接种初期表达微弱,甚至不表达,24 h后表达开始上升,到72 h时达最高,然后下降。本研究表明,病程相关蛋白和谷胱甘肽硫转移酶属于诱导型表达基因,且参与白粉病抗病反应,在白粉菌诱导72 h时表达量最高。     

皮肤癣菌微量液基稀释法抗真菌先导化合物敏感性试验方法的建立

为考察抗真菌先导化合物的抗皮肤癣菌活性.建立一种皮肤癣菌微量液基稀释法抗真菌先导化合物敏感性试验方法.以NCCLS(CLSI)M38-A产孢丝状真菌抗真菌药敏试验参考方案为基础,笔者对6种溶媒对红色毛癣菌生长系数的影响进行了考察.对红色毛癣菌的孢子接种量、所用培养基、孵育温度和时间以及终点判定等条件进行了修正.各种溶媒的工作浓度应该分别不高于1%(N,N-二甲基甲酰胺)、8%(聚乙二醇200)、2%(二甲基亚砜)、4%(丙酮)、8%(无水乙醇)、2%(1,2-丙二醇).红色毛癣菌孢子接种量在1×104～5.0×104CFU/ml、培养温度28℃、培养时间7d、80%的菌株生长受到抑制时作为判定的终点是理想的测定条件.改良后的M38-A方案,其试验结果可靠,具有很强的重现性,适合作为考察先导化合物的抗皮肤癣菌活性的药敏试验方法.     

小麦接种联合固氮菌增产效果研究

在田间条件下,小麦接种联合同氮菌W—43、W—432、W—91菌株,对小麦营养生长有明显促进作用,主要表现在单株次生根多0.8—1.0条,单株分蘖增加0.35—0.48个;旗叶面积增加1—3平方厘米,叶绿素含量提高12%,株高1—3厘米,每亩有效穗增加2.2—3.2万个,穗粒数增加1.4—1.5粒,千粒重增加1.4—1.5克,单产提高20.1—46.4公斤,增产率6.6—14.2%,同时改善小麦品质.     

Co胁迫下紫花苜蓿HPT基因的表达调控研究

钴(Co)胁迫会对植物产生多种毒害作用。为研究其植物毒性作用机理,需构建Co胁迫条件下紫花苜蓿的实时荧光定量PCR反应体系。研究选取了6个持家基因(actin2,GAPDH,UBI,18S,MSC27,EF1-α)作为备选内参基因,以紫花苜蓿植株为实验材料,检测Co胁迫条件下备选内参基因的表达稳定性。通过RefFinder和geNorm软件分析,确定Co胁迫下紫花苜蓿最佳内参基因组合为actin2和18S。将四种根际促生菌分别接种紫花苜蓿,并使用100 mg/L CoCl_2对接种后植株进行处理,使用筛选内参引物检测生育酚合成关键基因尿黑酸叶绿醇转移酶(HPT)的转录水平。研究结果显示Co胁迫可使HPT基因转录水平发生显著下调,根际促生菌铜绿假单胞菌Pseudomonas aeruginosa 1401可降低Co胁迫对苜蓿植株的毒害作用,接种该菌株植株的HPT基因相对表达水平为对照样本的6.63倍,植株株高和生物量分别达到对照样本的140.86%和193.62%。根据测试样本中HPT基因转录水平与植株Co耐受能力的变化趋势,推测紫花苜蓿的Co耐受能力增强与生育酚的合成途径调控或存在紧密联系。     

一株灰潮土解磷菌的解磷特性及其对花生的促生作用

为研究解磷菌能使土壤中被铁、铝等吸附固定的难溶性的非有效态磷转化为易于被植物吸收利用的有效态磷,促进农作物的生长。从南京长江南岸的潮土中分离筛选获得了一株解磷效果好、性状稳定的解磷细菌Y1。观测了菌株Y1形态及生理生化性状,测定其16S rDNA的保守序列。通过菌株生长和发酵条件试验,研究了该菌株在不同培养条件下的解磷能力。通过设置盆栽实验,研究了Y1菌株对花生生长的作用。实验结果初步确定菌株Y1为荧光假单胞菌(Pseudomonas fluorescens),该菌株在碳源为葡萄糖、氮源为硝酸铵、初始pH 6~8、装液量为50/250 mL时,解磷效果最好。盆栽试验结果表明,接种菌株Y1的花生根长、根表面积、根直径分别比对照增加159.0%、133.0%、18.9%,植株对N、P、K的吸收量分别比对照高73.6%、26.6%、41.0%,差异均达显著水平。本实验从灰潮土中筛选出一株高效解磷菌Y1,不仅能够促进花生的生长、产量,而且能够增加花生植株对N、P、K的吸收。该研究结果为菌株Y1在农业生产中的应用提供了理论依据和实验基础。     

小麦黄花叶病毒人工侵染体系的研究

通过大量的接种实验,分析了影响病毒接种效率的各种因素,经ELISA和RT PCR方法进行检测,确立了较为稳定有效的小麦黄花叶病毒机械传毒体系。采用新鲜病叶或用氯化钙干燥保存的病叶作为接种毒源,在0 2mol·L-1磷酸缓冲液中研磨后,对低温下(11～13℃)培养的小麦幼苗(1～2叶龄)进行叶面接种,并将接种后的小麦低温培养可获得较高的接种效率(12 57%～13 43%)。对毒源类型、小麦苗龄和接种部位等其他可能影响接种效率的因素进行了测定。     

Biological Activity and Quantification of Suspected Allelochemicals from Alfalfa Plant Parts

Autotoxicity restricts reseeding of alfalfa (Medicago sativa L.) after alfalfa until autotoxic chemical(s) breaks down or is dispersed into external environments. A series of aqueous extracts from leaves, stems, roots and seeds of alfalfa ‘Vernal’ were bioassayed against alfalfa seedlings of the same cultivar to determine their autotoxicity. The highest inhibition was found in the extracts from the leaves. Extracts at 40 g dry tissue l^?1 from alfalfa leaves were 15.4, 17.5 and 28.7 times more toxic to alfalfa root growth than were those from roots, stems and seeds, respectively. A high‐performance liquid chromatography (HPLC) analysis with nine standard compounds showed that the concentrations and compositions of allelopathic compounds depended on the plant parts. In leaf extracts that showed the most inhibitory effect on root growth, the highest amounts of allelochemicals were detected. Among nine phenolic compounds assayed for their phytotoxicity on root growth of alfalfa, coumarin, trans‐cinnamic acid and o‐coumaric acid at 10^?3 m were most inhibitory. The type and amount of causative allelochemicals found in alfalfa plant parts were highly correlated with the results of the bioassay, indicating that the autotoxic effects of alfalfa plant parts significantly differed.     

Changes in Seed Yield and Quality with Maturity in Onion (Allium cepa L., cv. 'Early Cream Gold')

Development of onion (Allium cepa L., cv. ‘Early Cream Gold’) seed under cool climate conditions in Tasmania, Australia occurred over a longer duration than previously reported, but similar patterns of change in yield components were recorded. In contrast to previous studies, umbel moisture content declined from 85 to 67 % over 57 days while seed moisture content decreased from 85 to 31 %. Seed yield continued to increase over the duration of crop development, with increasing seed weight compensating for seed loss resulting from capsule dehiscence in the later stages of maturation. Germination percentage was high and did not vary significantly from 53 to 77 days after full bloom (DAF), but mean germination time declined and uniformity of germination increased significantly over the same time period. The percentage abnormal seedlings declined with later harvest date, resulting in highest seed quality at 77 DAF. The results of this study suggest that the decision to harvest cool climate onion seed crops before capsule dehiscence will result in a loss of potential seed yield and quality.     

Location of a high-lysine gene and the DDT-resistance gene on barley chromosome 7

Summary The high-lysine gene in Risø mutant 1508 conditions an increased lysine content in the endosperm via a changed protein composition, a decreased seed size, and several other characters of the seed. The designation lys3a, lys3b, and lys3c, is proposed for the allelic high-lysine genes in three Risø mutants, nos 1508, 18, and 19. Linkage studies with translocations locate the lys3 locus in the centromere region of chromosome 7. A linkage study involving the loci lys3 and ddt (resistance to DDT) together with the marker loci fs (fragile stem), s (short rachilla hairs), and r (smooth awn) show that the order of the five loci on chromosome 7 from the long to the short chromosome arm is r, s, fs, lys3, ddt. The distance from locus r to locus ddt is about 100 centimorgans.     

Simultaneous Determination of Four Phenolic Acids in Radix Salviae Miltiorrhizae Formula Granules by QAMS

[Objectives]This study aimed to establish a QAMS(quantitative analysis of multi-components by single-marker)method for simultaneous determination of four phenol...     

Pollen and pollination experiments. III. The viability of apple and pear pollen as affected by irradiation and storage

Summary Apple and pear pollen was irradiated with doses of 0, 50, 100, 250 and 500 krad (gamma rays) and stored at 4°C and 0–10% r.h. From the in-vitro germination percentages an average LD 50 dose of about 220 krad was estimated. For both irradiated and untreated pollen a close and corresponding lineair relationship existed between germination percentage and pollen tube growth.Irradiated pollen was much more sensitive to dry storage conditions than untreated pollen, resulting in less germination and more bursting. Apparently, irradiation caused the pollen cell membrane to lose its flexibility faster than normal. Rehydration of dry-stored, irradiated pollen in water-saturated air restored germination percentages up to their initial levels. The importance of this procedure in germination trials is stressed.     

Water Extraction Process of Chinese Herbal Compound Man Gan Ning

[Objectives]To optimize the water extraction process of Chinese Herbal Compound Man Gan Ning and establish a method for its extraction and content determination...     

Present status and future strategy in breeding faba beans (Vicia Faba L.) for resistance to biotic and abiotic stresses

Progress is being made, mainly by ICARDA but also elsewhere, in breeding for resistance to Botrytis, AScochyta, Uromyces, and Orobanche; and some lines have resistance to more than one pathogen. The strategy is to extend multiple resistance but also to seek new and durable forms of resistance. Internationally coordinated programs are needed to maintain the momentum of this work.Tolerance of abiotic stresses leads to types suited to dry or cold environments rather than broad adaptability, but in this cross-pollinated species, the more hybrid vigor expressed by a cultivar, the more it is likely to tolerate various stresses.     

Optimization of Extraction Technology and Determination of Content of Total Phenols of Leaves in Acanthopanax giraldii Harms

[Objectives] To determine the optimum extraction technology for total phenols of leaves in Acanthopanax giraldii Harms.[Methods]The single factor test and ortho...     

Cytoplasmic male sterility,resistance to gall mite and mildew,and season of leafing out in black currants

Summary Cytoplasmic male sterility (cms) is described in the F₁ hybrids Ribes × carrierei (R. glutinosum albidum × R. nigrum) and R. sanguineum × R. nigrum. In backcrosses to R. nigrum, progenies with R. glutinosum cytoplasm were either all male sterile, or segregated for full male fertility (F) and complete (S) and partial (I) male sterility. Ratios of F:I+S suggested that two linked genes controlled cms, F plants being dominant for one (Rf ₁) and recessive for the other (Rf ₂).Segregation for cms in relation to three linded genes, Ce (resistance to the gall mite, Cecidophyopsis ribes), Sph ₃(resistance to American gooseberry mildew, Sphaerotheca mors-uvae) and Lf ₁(one of two dominant additive genes controlling early season leafing out) indicated that Rf ₁and Rf ₂were in this linkage group. The gene order and approximate crossover values appeared to be: % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqef0uAJj3BZ9Mz0bYu% H52CGmvzYLMzaerbd9wDYLwzYbItLDharqqr1ngBPrgifHhDYfgasa% acOqpw0xe9v8qqaqFD0xXdHaVhbbf9v8qqaqFr0xc9pk0xbba9q8Wq% Ffea0-yr0RYxir-Jbba9q8aq0-yq-He9q8qqQ8frFve9Fve9Ff0dme% aabaqaciGacaGaamqadaabaeaafaaakeaacaWGdbGaamyzamaamaaa% baGaaiiiaiaacccacaGGWaGaaiOlaiaacgdacaGG0aGaaiiiaiaacc% caaaGaaiiiaiaacccacaGGGaGaamOuaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaaccdacaGGUaGaaiOmaiaacs% dacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaaaacaWGsbGaamOzaSGa% aGOmaOWaaWaaaeaacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccaaaGaamitaiaadAgaliaaigdakmaamaaa% baGaaiiiaiaacccacaGGGaGaaiiiaiaacccacaGGGaGaaiiiaiaacc% cacaGGGaGaaiiiaiaacccacaGGGaaaaiaadofacaWGWbGaamiAaSGa% aG4maaaa!6E4D!\[Ce\underline { 0.14 } Rf1\underline { 0.24 } Rf2\underline { } Lf1\underline { } Sph3\]. Crossover values of 0.36 for Ce-Lf ₁, and 0.15 for Lf ₁-Sph ₃were estimated from the relative mean differences in season of leafing out between seedlings dominant and recessive for Ce and Sph ₃.It is suggested that competitive disadvantage of lf ₁-carrying gametes and/or zygotes at low temperatures may be implicated in the almost invariable deficit of plants dominant for the closely linked mildew resistance allele Sph ₃. Poor performance of lf ₁- (and possibly lf ₂-) carrying gametes and young zygotes during periods of low temperature at flowering might also account for the liability of some late season cultivars and selections to premature fruit drop (running off).     

Screening techniques and sources of resistance to parasitic angiosperms

Parasitic angiosperms cause great losses in many important crops under different climatic conditions and soil types. The most widespread and important parasitic angiosperms belong to the genera Orobanche, Striga, and Cuscuta. The most important economical hosts belong to the Poaceae, Asteraceae, Solanaceae, Cucurbitaceae, and Fabaceae. Although some resistant cultivars have been identified in several crops, great gaps exist in our knowledge of the parasites and the genetic basis of the resistance, as well as the availability of in vitro screening techniques. Screening techniques are based on reactions of the host root or foliage. In vitro or greenhouse screening methods based on the reaction of root and/or foliar tissues are usually superior to field screenings and can be used with many species. To utilize them in plant breeding, it is necessary to demonstrate a strong correlation between in vitro and field data. The correlation should be calculated for every environment in which selection is practiced. Using biochemical analysis as a screening technique has had limited success. The reason seems to be the complex host-parasite interactions which lead to germination, rhizotropism, infection, and growth of the parasite. Germination results from chemicals produced by the host. Resistance is only available in a small group of crops. Resistance has been found in cultivated, primitive and wild forms, depending on the specific host-parasite system. An additional problem is the existence of pathotypes in the parasites. Inheritance of host resistance is usually polygenic and its transfer is slow and tedious. Molecular techniques have yet to be used to locate resistance to parasitic angiosperms. While intensifying the search for genes that control resistance to specific parasitic angiosperms, the best strategy to screen for resistance is to improve the already existing in vitro or greenhouse screening techniques.