中国沙棘(Hippophae rhamnoides ssp. Sinensis)根瘤内Frankia菌的遗传多样性

为了研究中国沙棘亚种共生菌Frankia的遗传多样性,利用PCR-RFLP分子标记方法,对从青海西宁到内蒙古库伦17个地点采集的106个中国沙棘根瘤样品进行遗传多样性分析.供试样品nifD-nifK基因间隔区(IGS)扩增产物分别用3种内切酶(HinfⅠ、HaeⅢ和MboⅠ)酶切,共产生21条酶切谱带,其中17条为多态性条带,多态位点百分比(PPL)为80.99%,所有样品可被划分为9个基因型.结果表明中国沙棘根瘤内的Frankia菌有丰富的遗传多样性,土壤质量较好地点的丰富度高于土壤质量较差地点,海拔较高地区的丰富度高于海拔较低地区,多数地点至少有2种不同基因型的Frankia 菌.聚类分析显示Frankia 菌不同基因型间的遗传距离在4.88%～55.96%之间,它们在不同地点的分布是不均匀的,没有发现不同基因型菌间的亲缘关系与地点有相关性.中国沙棘根瘤中Frankia菌可分为两个基因型组,组内基因型分布比较一致,而组间有明显差异.     

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.     

福建省放线菌结瘤植物共生菌Frankia遗传多样性研究

[目的]了解福建省放线菌结瘤植物共生固氮菌Frankia的遗传多样性.[方法]利用16S-23SrDNA间隔区(rrn)和nifD-K基因间隔区的PCR扩增和RFLP技术,分析了福建省木麻黄、杨梅、桤木、胡颓子等共生Frankia纯培养菌株的遗传差异.[结果]17个菌株获得rrn扩增片段,2个杨梅菌株和1个胡颓子菌株扩增未成功,酶切图谱经聚类分析表明6个地点的细枝木麻黄、短枝木麻黄、粗枝木麻黄12个共生Frankia菌株同源性高,属于一个类群,2个地点的4个杨梅菌株和1个四川桤木菌株亲缘关系近,为另一类群.25个Frankia菌株的,nifD-K基因间隔区PCR-RFLP分析结果显示,7个地点的3种木麻黄14个菌株聚类为一个类群,4个地点的7个杨梅菌株、2个地点的2个四川桤木菌株以及1个台湾桤木菌株聚类为另一个类群,胡颓子菌株则为独立的类群.[结论]研究结果表明福建省共生Frankia遗传多样性丰富.     

Immunogold localization of hydrogenase in free-living Frankia CpI1

Abstract The free-living Frankia strain CpI1 cultured under nitrogen-fixing and non-nitrogen-fixing conditions was investigated for occurrence of hydrogenase protein by Western blots. Transmission electron microscopy and immunocytological labelling were used to study the distribution of hydrogenase in the Frankia strain.
Western immunoblots revealed that a 72-kDa protein in the Frankia strain CpI1 was immunologically related to the large subunit of a dimeric hydrogenase purified from Alcaligenes latus . Immunolocalization showed that the hydrogenase protein is located both in vesicles and hyphae in Frankia strain CpI1 grown in a nitrogen-free medium. Earlier reports that nitrogenase is localized in the vesicles [1,2], together with this finding, point out a possible role for hydrogenase in increasing relative efficiency of nitrogen fixation. In CpI1 grown in media containing nitrogen (lacking vesicles), the enzyme was evenly distributed in the hyphae. The impact of this result has to be further analysed.     

自然环境胁迫对旱冬瓜Frankia菌基因多样性的影响

利用rep-PCR方法,研究云南鸡足山及无量山不同生境下旱冬瓜根瘤内Frankia菌基因多样性及其变化,以了解不同自然环境胁迫对Frankia菌基因多样性的影响。结果表明,多样性随地域、海拔和坡向不同而变化,鸡足山Frankia菌基因类型比无量山丰富。鸡足山旱冬瓜根瘤内的Frankia菌在山底2300m处,Shannon指数平均为0．90;山顶海拔2650m以上,Shannon指数随之上升到1．33。南坡Frankia菌多样性高于北坡,表明多样性指数与环境胁迫大小成正相关,自然环境胁迫是产生和保持Frankia菌基因多样性的重要因子之一。     

Frankia菌在非豆科放线结瘤植物根际内的存活与分布

提取Ｆｒａｎｋｉａ菌１６ＳｒＲＮＡ,制备具专一性的寡核苷酸探针,通过同源杂交,在人工接种的木麻黄根际内检测出痕量Ｆｒａｎｋｉｓ菌结果表明,Ｆｒａｎｋｉａ可在环境因子的作用下被动迁移不论接种点的位置如何,经６个月的时间稳定后,其分布状态基本相同,主要集中于土层下８～３０ｃｍ处     

Laser-based micromanipulation for separation and identification of individual Frankia vesicles

In studies of symbiotic efficiency it is of great importance to identify and separate individual Frankia strains from a nodule. Therefore, a new laser-based micromanipulation technique has been developed in which individual vesicles from root nodules of two Frankia-Alnus symbioses have been successfully cut loose and separated from clusters of vesicles in sterile conditions under light microscopy using a laser scalpel and optical tweezers. Vesicles from the Alnus incana-Frankia AvCI1 symbiosis were successfully isolated and grown in culture using this technique. The DNA from both Frankia sources was amplified by polymerase chain reaction (PCR). The work shows that a combination of laser-based manipulation techniques and PCR can be used for the separation and study of individual vesicles. This novel laser-based micromanipulation technique opens up various new possibilities, for instance, to study whether several Frankia strains can grow simultaneously in the same root nodule.     

Homogeneity of superoxide dismutase patterns in Frankia strains from Casuarinaceae

Abstract Seven Frankia strains from Casuarina and Allocasuarina were analyzed by polyacrylamide gel electrophoresis to determine the patterns of several enzymes. No relatedness could be established between the strains as far as polyphenol oxidase, esterase and diaphorase were concerned. A first attempt at grouping the Frankia isolates could be achieved with catalase. It was confirmed by the study of superoxide dismutase: only one activity band, with the same mobility, was found in all cases. We propose to use this enzyme as a marker for identification of Frankia strains issued from Casuarinaceae.     

Saprophytic growth of inoculated Frankia sp. in soil microcosms

The potential of two Frankia strains to grow saprophytically was studied in nonsterile soil microcosms with ground leaf litter of Alnus glutinosa as the sole carbon and nitrogen sources. Strains Ag45/Mut15 and ArI3, which represent two taxonomic subgroups within the Alnus host infection group were inoculated alone, or together to investigate potential competition. Their growth was analyzed by in situ and dot-blot hybridization. A significant increase in cell numbers and filament length was observed during the first 6 weeks after inoculation for strain Ag45/Mut15, both alone and in mixed culture with strain ArI3, followed by a decrease until the end of the study after 12 weeks. The number of filaments remained unchanged. In contrast, the cell numbers and filament length of strain ArI3 were reduced significantly during the first 2 weeks and were undetectable for the remainder of the study. These results were comparable with those obtained in sterile mineral medium amended with leaf litter of A. glutinosa, although reductions in cell numbers and filament length were less pronounced than in soil microcosms. In concomitant control studies without leaf litter amendments for both experimental setups, filaments of both strains could only be detected immediately after inoculation. These results were matched in all experimental setups by concomitant shifts in the rRNA content of both strains, i.e., an immediate decline in the rRNA content for strain ArI3 after inoculation, and an increase in the rRNA content, followed by a late decline during incubation for strain Ag45/Mut15. These results demonstrated that Frankia strain Ag45/Mut15 could grow saprophytically in soil with complex carbon and nitrogen sources such as leaf litter, while the growth of strain ArI3 was not supported.     

Isolation of Elaeagnus-compatible Frankia from soils collected in Tunisia

The occurrence and diversity of Frankia nodulating Elaeagnus angustifolia in Tunisia were evaluated in 30 soils from different regions by a Frankia-capturing assay. Despite the absence of actinorhizal plants in 24 of the 30 soils, nodules were captured from all the samples. Eight pure strains were isolated from single colonies grown in agar medium. On the basis of 16S rRNA and GlnII sequences, seven strains were clustered with Frankia, colonizing Elaeagnaceae and Rhamnaceae in two different phylogenetic groups while one strain described a new lineage in the Frankia assemblage, indicating that Frankia strains genetically diverse from previously known Elaeagnus-infective strains are present in tunisian soils. Genomic fingerprinting determined by rep-PCR, and tDNA-PCR-SSCP, confirmed the wide genetic diversity of the strains.     

Frankia in acid soils of forests devoid of actinorhizal plants

The capacity of some acid forest soils to induce nodulation on a hybrid between Alnus incana (L.) Moench and A. glutinosa (L.) Gaertn. was investigated. Soil was sampled from tree stands devoid for decades of actinorhizal hosts. Seven-week-old Alnus seedlings growing m liquid culture were inoculated with soil dilutions. The nodules were counted after 6 weeks and classified as Sp⁻, if they lacked spores, or as Sp⁺. if spores were present, according to microscopy of microtome sections. Frankia was found in all the forest soils studied, apart from a soil from a Betula swamp. The highest nodulation capacities on Alnus , caused predominantly by Frankia of the Sp⁻ type. were observed in mineral soil sites with Betula stands — even higher than in soil from an A. incana stand. A positive correlation was found between the pH and the noduiation capacity of the soil.     

内蒙古沙棘共生菌Frankia的遗传多样性

利用RFLP分子标记方法,在自然条件下对内蒙古东、西部8个地点采集的24个沙棘根瘤样品进行沙棘共生菌Frankia的遗传多样性分析。结果表明,供试样品nifD-nifK基因间隔区(IGS)扩增片段的大小约1 100bp;不同样品的酶切图谱有明显差异;有些样品产生复合RFLP型,揭示在自然状态下不同基因型的Frankia菌株可共同侵染同一沙棘寄主。聚类分析显示,来源于相同地点和不同地点的根瘤样品内的Frankia菌株间均有遗传多样性;没有发现Frankia菌株遗传多样性的分布与采样地点有相关性。     

Studies of Seedling Inoculation of Frankia in Elaeagnus mollis Diels

The research on the preparation of Frankia solution, seedling culture and inoculation was carried out. Results indicated that the collective amount of cells may be increased when the logarithm phase cells were used as the inoculum and regularly homogenizing and transplanting inoculum by the method of magnetic stirring were adopted during the cultural process of Frankia. The inoculative tests verified that the strain with the highest ability to infect Elaeagnus mollis Diels is SIB 1301118 isolated from Elaeagnus angustifolia L. other than SIB 1332281 isolated from Elaeagnus mollis Diels. The effectivities of inoculation for germinating seeds and the seedlings to be transplanted were seed soaking and root soaking in Frankia solution preparel respectively. Early inoculation was beneficial to the growth and nodulation of seedings. The fefectivity of late inoculation was bad. Sand-soil mixture (2:1) was properly suitable to the seed germination and seedling growth. The seedling growth and nodulative rate could be promoted when 1/4 Sileris-Yong nutrient solution was suppplimented regularly.     

Successful nodulation of Casuarina by Frankia in axenic conditions

AIMS: In order to depict the fine interactions that lead to nodulation, absolute microbiological control of the symbiotic partners is required, i.e. the ability to obtain in vitro axenic nodulation, a condition that has never been fulfilled with the Casuarina-Frankia symbiosis. The effects of culture conditions on plant growth and nodule formation by Casuarina cunninghamiana were investigated. METHODS AND RESULTS: Axenic (capped tubes with different substrates), and nonaxenic cultures (Gibson tubes, pot cultures) were tested. In axenic conditions, C. cunninghamiana, inoculated with Frankia, had poor growth and did not form nodules at 6 weeks. Plants cultivated in Gibson tubes reached the four axillary shoots stage within 6 weeks and formed nodules 4 weeks after inoculation. Sand-pot cultures allowed us to relate the plant development stage at inoculation with nodulation. CONCLUSIONS: The sterile replacement of the cap by a plastic bag increased plant growth and enabled nodule formation 6 weeks after inoculation. The new system of plant culture allows the axenic nodule formation 6 weeks after inoculation. Nodulation behaviour is related to plant development and confinement. SIGNIFICANCE AND IMPACT OF THE STUDY: This axenic plant nodulation system is of major interest in analysing the roles of Frankia genes in nodulation pathways.     

ARDRA对植物根瘤内共生放线菌Frankia多样性的研究

应用原核生物16SrDNA特异性引物rD1和fD1,通过ARDRA（amplified ribosomal DNA restriction analysis）法直接扩增自中国云南、东北地区赤杨属3种植物和沙棘属1种植物根瘤内FrankiaDNA,得到一长约1500bp的扩增产物,选用两种内切酶HaeⅢ、AfaⅠ联合对扩增产物进行酶切,得到稳定的酶切图谱,将所测48个感染赤杨的Frankia样本区分为3个不同的组,所测43个感染沙棘的Frankia样本区分为3个不同的组,显示根瘤内Frankia存在丰富的遗传多样性。     

Effects of phosphorus supply on in vitro growth and phosphatase activity of Frankia isolates from Casuarina

An in vitro experiment was conducted to investigate the effects of different sources and levels of P supply on growth, viability and phosphatase activity of three tropical Frankia strains isolated from Casuarina. P concentration for optimum growth was between 0.1 and 10.0 M in the absence of external combined nitrogen. Specific viability was not influenced by P supply. Morphological features of Frankia, such as hyphal length and vesicle numbers, were found to largely mirror growth. Phosphatase activity was detected in all three Frankia strains and was highest when P was omitted from the culture solution. There were more than 10-fold differences between the Frankia strains in the level of phosphatase activities shown. This study suggested that soils low in P are unlikely to restrict micro-symbiont growth activity.     

高黎贡山旱冬瓜Frankia的IGS PCR-RFLP分析

在云南省高黎贡山自然保护区海拔1310～2400m的范围内,采集30个旱冬瓜根瘤样品,直接从根瘤中提取Frankia DNA,对其,nifD-nifK基因间隔区(intergenic spacer,IGS)和16S-23S rDNA IGS进行PCR—RFLP分析．结果表明,nifD-nifK IGS的PCR产物长度差异很大,经HaeⅢ和Afa I双酶切后,得到15种酶切带型,检测到多种基因型的菌株同时与同一株宿主植物共生;16S-23S rDNA IGS的PCR产物长度相似,酶切后亦区分出15种酶切带型．通过对两个基因间隔区的PCR-RFLP联合分析,发现高黎贡山旱冬瓜Frankia存在20种基因型．