一株弗兰克氏菌分类鉴定的研究

从中国沙棘(Nippophae rhamnoidel LI s叩．tinensis Roun)根瘤中分离出一株生孢囊的放线菌Fran&㈦P．HRl04。经回接试验证实,它可以侵染寄主植物,形成能够固氮的有效根瘤。通过形态和培养特征、生理生化特性、细胞壁组份及DNA中G+c含量等的研究,表明这株菌与已经研究和描述过的弗兰克氏菌有差别,特别是胞壁型不同,可能属于一个尚未研究过的新属。本文还对弗兰克氏菌的分类标准和鉴定方法进行了初步探讨。     

沙棘弗兰克氏菌的生物学特性研究

从辽宁省西部的沙棘(Hippophae rhamnoides L.)根瘤中分离到8株内生菌,它们都具有Frankia菌的典型形态特征。 回接鉴定表明,8株菌均可使寄主结瘤。各菌株的侵染能力不同;回接培养液的种类是影响侵染力的重要因素。分离于两个不同地区的菌株Frankia sp.Hr16和Hr32在GyA培养基上的培养特征有一定差异。实验菌株可利用多种碳源,其中丙酮酸盐是理想碳源。在以丙酮酸盐为唯一碳源,浓度为10mjlf的Bap培养基中,Hrl6菌株的对数期倍增时间可降至知小时,菌体在两周内达到最大生长量o Hrl6菌株的自生固氮活性亦受碳源的影响,在以5mM的丙酮酸盐的无氮Bap培养基中,其话性为176.5nmol. C2H4／mg蛋白/小时。     

沙棘共生固氮根瘤及其内生弗兰克氏菌

用透射电镜研究了中国沙棘（Ｈｉｐｐｏｐｈａｅ ｒｈａｍｎｏｙｄｅｓ Ｌ．）根瘤的超微结构。它的侵梁细胞位于皮层中部,非侵染细胞与之间排列,富含多酚和淀粉粒。在根瘤的发育过程中,具有以下特征：（１）早期侵染细胞中具有核仁联合体;（２）由内生菌丝趋核生长而形成的核膜内陷处,有许多核孔出现;（３）在中期侵染细胞的内生菌丝和泡囊的荚膜附近,有成束的微管存在;（４）在４、５月瘤样的维管束细胞、非侵染细胞及早     

弗兰克氏菌在构建新的共生固氮物种研究中的优越性

弗兰克氏菌在构建新的共生固氮物种研究中的优越性解放军石家庄医学高等专科学院０５００８１曲东明范浩南四川师范大学细胞研究室６１００６８韩善华弗兰克氏菌（Ｆｒａｎｋｉａ）能与多种木本被子植物建立共生固氮关系,它形成的根瘤具有较强的固氮能力,有些超过大豆根...     

弗兰克氏菌生物学特性的研究

对自欧美和亚洲不同地理环境、不同科属寄主植物根瘤分离的35株菌的研究结果表明,菌株均具有典型的弗兰克氏菌特征。其中由胡颓子属植物分离的4株菌具有串珠状特异菌丝体结构。全细胞氨基均含有meso-DAP,全细胞糖均为D型。均具有不同程度的固氮酶(乙炔还原)活性,并在乙炔注入后24—72小时活性倍增,同时在48—72小时活性达到最高。根据对糖和丙酮酸钠的利用,可将供试35株菌分为2群4亚群。     

弗兰克氏菌的分类研究进展

弗兰克氏菌是非豆科植物共生固氮菌。到目前为止,已发现它们可以分别与8个科、25个属的200多种非豆科植物共生[1],其中包括赤杨、杨梅、沙棘、胡颓子、马桑、木麻黄、悬钩子、仙女木等。这些植物对于荒地的开垦和防止水土流失具有重要意义。而弗兰克氏菌与这些植物共生形成可     

由四川桤木根瘤中分离和培养弗兰克氏菌

用蔗糖离心法从四川桤木的根癌中分离到内生菌——Frankia ACC 13,并获得纯培养。ACC 13是属慢生长型放线菌,形成的菌落紧密,无色素,菌丝分枝有隔,粗细不规整,一般为0．6一1．4μm。革兰氏阳性,菌丝居间或末端膨大,形成特征性的孢子囊。在孢子囊中形成孢子,圆形或椭圆形,直径1．3μm左右。在限制性培养基上诱导出分离菌的固氮泡囊。在根瘤悬液中不含抑制内生菌生长的“抑制物质”。该菌在pH 6．5—7．2范围内生长。以ACC13回接四川桤木幼苗,幼苗根系形成大量根瘤。根瘤的乙炔还原活性为15．7μmoIC2H4/g·鲜重．H。     

自辽东赤杨根瘤分离的弗兰克氏菌的分类研究

自辽东赤杨(41nus sibirica)的根瘸中分离得到AS,菌株。该菌株形成圆形或棒状孢囊和圆形泡囊。细胞壁化学组分lII型,糖型为D。不利用阿拉伯糖、葡萄糖、甘油、麦芽糖、蔗糖、山梨醇和木糖。在S培养基中,菌的生长受Tweea一80和葡萄糖协同作用的影响,属于生理型B。DNA的G+c含量为77．34 mol(Tin)o 因此,该菌株定为Frankia gp-AS zo     

弗兰克氏菌Hr138菌株的生理学研究

用根瘤切片液体培养方法,从沙棘(Hippophae themnoides L.)根瘤中分离得到弗兰克氏菌Hr138菌株,对其生理特性和培养条件进行了研究。菌株能在多种培养液中生长,最适宜生长的培养液为Tween／Cas。它能利用吐温-80、琥珀酸、丙酸、醋酸钠、葡萄糖、麦芽糖或半乳糖作为碳源：利用酪蛋白水解物、蛋白胨或氯化铵作为氮源。在吐温-80为碳源的培养液中,加入葡萄糖对生长有抑制作用。此外,就温度和pH对生长的影响也进行了研究。     

Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina

Five free-living Frankia strains isolated from Casuarina were investigated for occurrence of hydrogenase activity. Nitrogenase activity (acetylene reduction) and hydrogen evolution were also evaluated. Acetylene reduction was recorded in all Frankia strains. None of the Frankia strains had any hydrogenase activity when grown on nickel-depleted medium and they released hydrogen in atmospheric air. After addition of nickel to the medium, the Frankia strains were shown to possess an active hydrogenase, which resulted in hydrogen uptake but no hydrogen evolution. The hydrogenase activity in Frankia strain KB5 increased from zero to 3.86 μ mol H₂ (mg protein)⁻¹ h⁻¹ after addition of up to 1.0 μ M Ni. It is likely that the hydrogenase activity could be enhanced even more as a response on further addition of Ni. It is indicated in this study that absence of hydrogenase activity in free-living Frankia isolated from Casuarina spp. is due to nickel deficiency. Frankia living in symbiosis with Casuarina spp. show hydrogenase activity. Therefore, the results also indicate that the hydrogenase to some extent is regulated by the host plant and/or that the host plant supplies the symbiotic microorganism with nickel. Moreover, the result shows that this Frankia is somewhat different from Frankia isolated from Alnus incana and Comptonia peregrina ., i.e., Frankia isolated from A. incana and C. peregrina showed a small hydrogen uptake activity even without addition of nickel.     

Total and CO-reactive heme content of actinorhizal nodules and the roots of some non-nodulated plants

Summary The concentration of total and CO-reactive heme was measured in actinorhizal nodules from six different genera. This gave the upper limit to hemoglobin concentration in these nodules. Quantitative extraction of CO-reactive heme was achieved under anaerobic conditions in a buffer equilibrated with CO and containing Triton X-100. The concentration of CO-reactive heme in nodules of Casuarina and Myrica was approximately half of that found in legume nodules, whereas in Comptonia, Alnus and Ceanothus the concentrations of heme were about 10 times lower than in legume nodules. There was no detectable CO-reactive heme in Datisca nodules, but low concentrations were detected in roots of all non-nodulating plants examined, includingZea mays. Difference spectra of CO treated minus dithionite-reduced extracts displayed similar wavelengths of maximal and minimal light absorption for all extracts, and were consistent with those of a hemoglobin. The concentration of CO-reactive heme was not correlated to the degree to which CO inhibited nitrogenase activity nor was it affected by reducing the oxygen concentration in the rooting zone. However, there was a positive correlation between heme concentration and suberization or lignification of the walls of infected host cells. These observations demonstrate that, unlike legume nodules, high concentrations of heme or hemoglobin are not needed for active nitrogen fixation in most actinorhizal nodules. Nonetheless, a significant amount of CO-reactive heme is found in the nodules of Alnus, Comptonia, and Ceanothus, and in the roots ofZea mays. The identity and function of this heme is unknown.     

木麻黄共生固氮菌Frankia的分离鉴定及固氮效应

从浙江省短枝木麻黄(Casuarina equisetifolia)和细枝木麻黄(C.curninghamiana的根瘤中共分离获得14株共生菌株.形态观察表明,菌株具有分枝状菌丝、多腔孢囊、泡囊等典型的Frankia结构、16S rDNA测序结果表明,供试菌株均为Frankia,其中4株属于生理类群A,7株属于生理类群B,3株属于生理类群AB.固氮效应研究表明,菌株均具有固氨酶生物学活性,但菌株之间存在显著差异,其中菌株ZCN192固氨酶活性最强,可达2.897 μmol·mg-1·h-1,菌株ZCN199最低,固氮酶活性为0.056 μmol·mg-1·h-1.活体固氮试验显示,与阴性对照相比,供试菌株能显著提高苗高、地径和干重,且一般情况下,离体固氮酶活性强的菌株在活体接种时能获得更明显的固氮效应.     

Cytometric determination of genome size and base composition of tree species of three genera of Casuarinaceae

The genome size and base composition of diploid plant species from three genera of the Casuarinaceae family were determined by flow cytometry. Casuarina glauca Sieb. ex Spring. and Gymnostoma deplancheana (Miq.) L. Johnson showed a small genome with 2C = 0.70 pg, 58.6% AT, 40.5% GC for the first species and 2C = 0.75 pg, 58.7% AT, 40.5% GC for the second. Allocasuarina verticillata (Lam.) L. Johnson had a larger genome: 2C = 1.90 pg, 59.3% AT, 41.1% GC. One haploid genome of C. glauca is therefore about 340×10⁶ base pairs. In leaves, roots or bark of these three species, polysomaty was virtually absent: a maximum frequency of 4C nuclei of only 0.08 was found in bark of C. glauca. The genome sizes of C. glauca and G. deplancheana are among the smallest described for higher plants. Small genome size, diploidy and the absence of polysomaty are advantageous traits for facilitating molecular approaches to improvement of these actinorhizal plants and developing the study of their symbiotic interactions with Frankia. Received: 20 December 1997 / Revision received: 13 March 1998 / Accepted: 30 March 1998     

Enzymes of the tricarboxylic acid cycle and the malate-aspartate shuttle in the N2-fixing endophyte of Alnus glutinosa

The occurrence and localization of enzymes involved in energy supply and biosynthesis was studied in root nodules of Alnus glutinosa (L.) Vill. Vesicle clusters of the endophyte, Frankia sp., contain NADP-dependent isocitrate dehydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase. The data indicate that both the endophyte and the host are capable of metabolizing carbon compounds via the tricarboxylic acid cycle. Both vesicle clusters of the endophyte and root nodule cells contain glutamate-oxaloacetate transaminase which can function in a malate-aspartate shuttle. This might enable transport of reducing equivalents from the host cell cytoplasm to the endophyte.     

Rapid,exponential growth and increased biomass yield of someFrankia strains in buffered and stirred mineral medium (BAP) with phosphatidyl choline

Rapid, exponential growth ofFrankia sp. BR (ORS 020608) isolated fromCasuarina equisetifolia was found to require stirring (magnetic bar) and adequate buffering (MES or MOPS buffers). We used a mineral medium (BAP) at 28°C with propionate and NH₄ ⁺ as the sole carbon and nitrogen source respectively. Sporulation was enhanced by stirring, but the addition of phosphatidyl choline was found to impair sporulation and slightly improve growth. In these conditions a generation time of 14–16 hours was observed with a maximal biomass yield of 108 g of protein × mL^-1 (Bicinchoninic acid) at day 4 when growth ceased. In stirred conditionFrankia growth as microcolonies (30–200 m diameter) showing an open mesh type of mycelium and also a random distribution of growing hyphae inside and in the periphery of the microcolonies (acridine orange vital staining). In static conditions macrocolonies and only peripheric growth can be observed. Optimal growth in buffered and stirred conditions in mineral medium has been also observed with Cj and CeFFrankia isolates. The reasons for rapid and balanced hyphal growth in stirred conditions are discussed.     

几株不同种属来源的木麻黄植物根瘤共生放线菌-Frankia的研究

用根瘤切片法,从长骨木麻黄(C. glauca Sieb.)、鸡冠木麻黄(C. cristata)和海滨木麻黄(A. littoralis)野生根瘤中分离出具有侵染能力的Frankia sp、NNCG01、NNCCR03和NNACL05菌株,它们都具有Frankia的典型形态特征,在无氮培养条件下,形成大量的顶囊和孢子囊。各菌株对简单碳源和氮源的利用比对复杂碳源和氮源的利用更好,而且都具有较高的耐盐(NaC1)能力,在0.2M盐浓度中,生长受到的影响很小,在盐浓度为0.5M时,也有一定生长。这三株Frankia菌株都能交叉感染木麻黄属(Casuarina)和异木麻黄属(Allocasuarina)中的一些放线菌结瘤植物,因此,从木麻黄属植物根瘤分离的Frankia菌株和从异木麻黄属植物根瘤分离的Frankia菌株属于同一个互接种簇。这三株Frankia的自生固氮活性与它们的共生固氮活性呈正比,而且同一菌株与不同种木麻黄植物共生固氮的效率没有明显的差异,为初步筛选高效共生固氮的Frankia菌株提供了一个快速简便的方法,具有重要的实际意义。