Different genetic groups of Frankia within the root nodules of Casuarina growing in Mexico

This study was carried out to examine the possible genetic diversity that may occur among Frankia strains that nodulate C. equisetifolia in Mexico growing in the site of introduction, the cost of the Golf of Mexico, as well in the highlands. DNA extracted from reference cultures of Casuarina infecting Frankia strains, from field collected nodules (two trees of each of 14 sites and 3–5 analyzed lobes of each nodule) or from young nodules obtained from plants growing at the growth chamber and inoculated with field nodules, were used as the template in PCR reaction with primers targeting two DNA regions, one of the ribosomal operon and the other in the nif operon. PCR products were analyzed by using a set of recommended restriction enzymes. Six PCR-RFLP groups were detected after digestion with combination of enzymes BstUI and CfoI of the nif region, and four PCR-RFLP groups with enzymes NciI and ScrFI in the ribosomal region. Reference strains showed similar patterns and were assigned in group 1 along with an uncultured strain. nifD-K PCR-RFLP groups allowed a better understanding of the diversity among nodular Frankia strains than those observed in the rrs PCR-RFLP groups.     

盐胁迫下木麻黄幼树营养元素的分配规律

利用盆栽试验,研究了盐胁迫下木麻黄幼树营养元素含量和分布规律。结果表明,不同施盐量处理的木麻黄幼树生长、营养元素含量及分布发生明显的变化。低浓度(5g/kg)NaCl处理促进了木麻黄的生长,而高浓度(20、25g/kg)则抑制木麻黄的生长。随着盐浓度的增加,木麻黄根中N、P、K,茎中N、P,小枝中N含量随之增加,并与盐浓度之间呈显著正相关,而茎和小枝中的K以及小枝中的Ca含量则与盐浓度之间呈显著负相关,说明盐胁迫下木麻黄营养元素的比例失调是导致盐害的主要原因之一。盐浓度对Mg元素影响较小。     

Growth response of Casuarina equisetifolia Forst. rooted stem cuttings to Frankia in nursery and field conditions

Casuarina equisetifolia Forst. is a tree crop that provides fuel wood, land reclamation, dune stabilization, and scaffolding for construction, shelter belts, and pulp and paper production. C. equisetifolia fixes atmospheric nitrogen through a symbiotic relationship with Frankia, a soil bacterium of the actinobacteria group. The roots of C. equisetifolia produce root nodules where the bacteria fix atmospheric nitrogen, which is an essential nutrient for all plant metabolic activities. However, rooted stem cuttings of elite clones of C. equisetifolia by vegetative propagation is being planted by the farmers of Pondicherry as costeffective method. As the vegetative propagation method uses inert material (vermiculite) for rooting there is no chance for Frankia association. Therefore after planting of these stocks the farmers are applying 150 kg of di-ammonium phosphate (DAP)/acre/year. To overcome this fertilizer usage, the Frankia-inoculated rooted stem cuttings were propagated under nursery conditions and transplanted in the nutrient-deficient soils of Karaikal, Pondicherry (India), in this study. Under nursery experiments the growth and biomass of C. equisetifolia rooted stem cuttings inoculated with Frankia showed 3 times higher growth and biomass than uninoculated control. These stocks were transplanted and monitored for their growth and survival for 1 year in the nutrient-deficient farm land. The results showed that the rooted stem cuttings of C. equisetifolia significantly improved growth in height (8.8 m), stem girth (9.6 cm) and tissue nitrogen content (3.3 mg g^?1) than uninoculated controls. The soil nutrient status was also improved due to inoculation of Frankia.     

Genetic variation of Casuarina equisetifolia subsp equisetifolia and C. equisetifolia subsp incana populations on the northern coast of Senegal

The genetic variation of 70 individual samples of Casuarina equisetifolia (L. Johnson) subsp equisetifolia and C. equisetifolia subsp incana growing along the northern coast of Senegal was analyzed with RAPD markers. Of the 160 primers tested, five were chosen; they generated 1396 reproducible bands and 61 polymorphic bands that were scored. This result showed a narrow genetic variation among (4.36%) and within (5.90%) C. equisetifolia subsp equisetifolia and C. equisetifolia subsp incana plantation sites. The genetic variation at each site revealed a high degree of polymorphism in Potou (5.90%) and low diversity in Retba (3.06%). In the dendrogram analyses, each sampling site was formed by two main groups. Similar results were found for the dendrograms based on the RAPD data gathered from the five different sites. These dendrograms revealed several polytomies in one of the subgroups, suggesting replication of the same specimens in different sites along the Senegalese coast. The RAPD data support the hypothesis that these populations are of the same provenance, subject to hybridization and inbreeding depression.     

Wild nodules can be broken: proteomics of Frankia in field-collected root nodules

With the genomes of three Frankia strains available, high-throughput proteomics methods can be used to reveal the set of proteins expressed by these bacteria in symbiosis with plants. A question we address is the degree to which the known genomes can be used to study proteomes of uncharacterized frankiae growing in field-collected root nodules. To this end, we have characterized the symbiotic proteomes of Frankia from three plant species, Alnus incana subsp. rugosa, Ceanothus americanus, and Elaeagnus angustifolia. Root nodule proteins were identified using two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC MS/MS) of trypsin-digested protein samples. We identified 1300 Frankia proteins in A. incana nodules using the Frankia alni ACN14a genome and 1100 proteins from E. angustifolia nodules using the EAN1pec genome. In addition, over 100 proteins were identified from C. americanus nodules using a more limited one dimensional LC MS/MS analysis. Many of the most abundant proteins identified are involved in energy and nitrogen metabolism. The enzyme nitrogenase and the nitrogenase iron protein were among the most abundant proteins, reflecting the major process occurring in symbiosis. Several hundred plant proteins were also identified. We highlight the power of proteomics to uncover the physiology of symbiotic Frankia in the environment using heterologous genome information.     

施肥对短枝木麻黄幼苗总酚和可溶性缩合单宁含量的影响

研究了施用氮肥和磷肥对短枝木麻黄幼苗总酚(total phenolics,TP)和可溶性缩合单宁(extractable condensed tannin,ECT)含量的影响,探讨短枝木麻黄单宁形成的养分效应.结果表明:施加氮肥使短枝木麻黄幼苗小枝的TP和ECT含量显著降低,支持碳氮平衡假说和生长分化平衡假说,但对氮含量没有显著影响,从而导致TP/N和ECT/N降低;施加磷肥对TP和ECT含量没有显著影响;随着处理时间的延长,短枝木麻黄幼苗小枝TP含量升高了9.91%～14.32%,而ECT含量降低了14.32%～298.88%;TP或ECT与有机物质含量的关系则相反,表明不同类型单宁的合成途径不同,但由于TP和ECT均与氮含量无显著相关性,故不支持蛋白质竞争模型;在贫瘠土壤条件下,TP/N和ECT/N的水平较高,有利于提高短枝木麻黄的防御水平,降低凋落物的分解率,减少养分损失,从而保持较高的生产力.     

Purity of Frankia preparations from root nodules of Alnus incana

Frankia vesicle clusters were prepared from Alnus incana (L.) Moench root nodules by a homogenization-filtration procedure. The preparation was examined by transmission electron microscopy and computerized picture analysis to quantify contamination from the host plant. Special attention was paid to plant mitochondria. Mitochondria were only found in 30% of the 50 sections of clusters examined. In sections containing mitochondria the mean number of mitochondria per cluster section was 1.5. The relative volume of all objects found in the vesicle clusters was calculated. More than 98% of the volume of a preparation consisted of Frankia vesicles and hyphae, while only 0.4% of the volume was host plant mitochondria. The frequency of mitochondria in a preparation could be further decreased by osmotic shock. It is concluded that Frankia vesicle clusters, prepared from Alnus incana by the homogenization-filtration technique used here, are sufficiently pure to be used for studies of Frankia metabolism.     

Symbiosis between Frankia and actinorhizal plants: root nodules of non-legumes

In actinorhizal symbioses, filamentous nitrogen-fixing soil bacteria of the genus Frankia induce the formation of nodules on the roots of a diverse group of dicotyledonous plants representing trees or woody shrubs, with one exception, Datisca glomerata. In the nodules, Frankia fixes nitrogen and exports the products to the plant cytoplasm, while being supplied with carbon sources by the host. Possibly due to the diversity of the host plants, actinorhizal nodules show considerable variability with regard to structure, oxygen protection mechanisms and physiology. Actinorhizal and legume-rhizobia symbioses are evolutionary related and share several features.     

The production and utilisation of monoclonal antibodies for identification of a Frankia strain utilised as inoculum for Casuarina equisetifolia

Monoclonal antibodies were raised against Frankia 0RS020607, a strain isolated originally by H.G. Diem from nodules of Casuarina equisetifolia from Senegal. One of these antibodies, mAb8C5, was shown by ELISA to have high, but not absolute specificity for 0RS020607. This antibody was employed to investigate the mobility and persistence of 0RS020607 in plantations of C. equisetifolia. Seedlings were inoculated in pots of sand in a forest nursery with 0RS020607, with local crushed nodule suspensions or were left uninoculated. They were planted out after 5 months in experimental plots on a moderately fertile black soil site and on a low organic, oxidised red soil site. Compared with crushed nodule inoculated seedlings or uninoculated controls, growth of seedlings at transplant was improved by inoculation with Frankia 0RS020607. However, 4 years after transplant to experimental plots, the growth of trees receiving different treatments was similar. The possibility that movement of ORS 020607 between treatment plots contributed to new nodulation and enhanced growth of uninoculated trees was tested using mAb8C5 in ELISA of Frankia mycelium, extracted from the nodules of trees of the three treatments. No significant differences in reactivity were detected between nodules from uninoculated and 0RS020607 inoculated trees at either the black or the red soil sites, showing that 0RS020607 moved between treatment plots at both sites. However, at both sites, nodules from plots of trees that were inoculated originally with local crushed nodules gave reactions in ELISA that were significantly lower than values for 0RS020607 inoculated trees, possibly due to the competitive effects for new nodulation of enhancement of the indigenous population of Frankia. Serological techniques using antibodies of high specificity against Frankia strains have value for rapid screening of field samples as a preliminary for further analysis by more discriminatory techniques based on assays of genetic polymorphisms.     

福建东山短枝木麻黄小枝氮磷含量及其再吸收率季节动态

对福建东山赤山林场短枝木麻黄纯林小枝中的N和P含量、N ∶ P比、养分再吸收率的季节动态进行研究.结果表明,随着小枝的成熟和衰老,各季节N和P的含量依次降低,其中衰老小枝中N和P含量分别为(3.97±0.21)～(8.64±0.58) mg · g~(-1)和(0.04±0.03)～(0.10±0.03) mg · g~(-1),基本上表现为N和P的完全再吸收.成熟小枝N ∶ P比的季节动态介于(18.67±3.24)～(37.98±1.32)之间,均高于16,表明木麻黄的生长受到不同程度的P限制.N、P再吸收率的季节变化分别为(51.02±4.66)%～(63.00±8.61)%和(81.24 ±5.08)%～(91.78±5.84)%,P再吸收率显著高于N.N和P的再吸收率之间以及成熟叶中N、P含量与二者再吸收率之间没有显著相关性,但在衰老叶中存在显著负相关,表明养分再吸收程度越高,其再吸收率就越高.因此,木麻黄通过小枝衰老过程中的养分再吸收,减少养分的损失,从而适应贫瘠的立地条件.     

滨海沙地木麻黄(Casuarina equisetifolia)人工林细根养分与能量动态

2005年1月到2005年11月对福建省惠安县赤湖林场不同林龄木麻黄人工林细根养分和能量的季节动态进行了观测,结果表明:(1)6种元素的含量在不同林龄木麻黄细根中都具有明显的季节变化。各林龄细根的N含量一般在冬夏季节较高,且死细根的N浓度高于相同林龄的活细根,除12林龄活细根P浓度在7月份有最大值外,其他各林龄活、死细根在一年中呈波动性下降,K含量在冬季较高,而在其他季节变化幅度不大,除5林龄活细根和18林龄死细根在3月份和7月份有两个峰值外,其他林龄细根Ca含量随季节变化较小,Mg含量随季节变化总体呈下降趋势,而在11月份上升;各林龄C则呈波浪形变化;(2)随着林龄的增大,细根N、P、Mg含量的变化模式相似,都呈先增加,后降低,再增加的趋势,K和Ca含量变化趋势相似,但变动幅度存在差别,C则呈波浪形变化;(3)随着季节的变化,灰分、干重热值和去灰分热值呈"V"形变动,一年中都存在两个峰值,分别在3月份和7月份或9月份;(4)灰分、干重热值和去灰分热值随林龄的增大表现为波浪形增加。由此可见,不同森林类型的细根养分和能量动态具有季节和林龄的特殊性,在进行整个地区森林生态系统物质循环和能量流动研究时,应考虑不同森林类型的特性。     

Characterization of four defense-related genes up-regulated in root nodules of Casuarina glauca

Actinorhizal plants are capable of high rates of nitrogen fixation, due to their capacity to establish a root-nodule symbiosis with N₂-fixing actinomycetes of the genus Frankia. Nodulation is an ontogenic process which requires a sequence of highly coordinated events. One of these mechanisms is the induction of defense-related events, whose precise role during nodulation is largely unknown. In order to contribute to the clarification of the involvement of defense-related genes during actinorhizal root-nodule symbiosis, we have analysed the differential expression of several genes with putative defense-related functions in Casuarina glauca nodules versus non-inoculated roots. Four genes encoding a chitinase (CgChi1), a glutathione S-transferase (CgGst), a hairpin-inducible protein (CgHin1) and a peroxidase (CgPox4) were found to be up-regulated in mature nodules compared to roots. In order to find out to which extend were the encoded proteins involved in nodule protection, development or both, gene regulation studies in response to SA and wounding as well as phylogenetic analysis of the protein sequences were performed. These were further characterized through expression studies after SA-treatment and wounding, and by phylogenetic analysis. We suggest that CgChi1 and CgGst are involved in defense or microsymbiont control and CgPox4 is involved in nodule development. For CgHin1 the question “defense, development or both” remains open.     

The Cuticle, Epidermis and Stomatal Ontogeny of Casuarina equisetifolia Forst

The cuticle, epidermis and stomatal ontogeny of Casuarina equisetifoliaForst. is described. The cuticle shows well marked impressionsof the epidermal cells and stomata. The epidermis of leaf andstem shows transversely oriented, tetracytic, mesoperigenousstomata with two lateral mesogene subsidiaries and two polarperigene neighbouring cells. Although the epidermal structureof Casuarina shows a good deal of resemblance with that of theBennettitales, it may not indicate any phylogenetic relationshipssince there are important differences in the structure and reproductionof the plants of these two groups.     

Casuarina root exudates alter the physiology, surface properties, and plant infectivity of Frankia sp. strain CcI3

The actinomycete genus Frankia forms nitrogen-fixing symbioses with 8 different families of actinorhizal plants, representing more than 200 different species. Very little is known about the initial molecular interactions between Frankia and host plants in the rhizosphere. Root exudates are important in Rhizobium-legume symbiosis, especially for initiating Nod factor synthesis. We measured differences in Frankia physiology after exposure to host aqueous root exudates to assess their effects on actinorhizal symbioses. Casuarina cunninghamiana root exudates were collected from plants under nitrogen-sufficient and -deficient conditions and tested on Frankia sp. strain CcI3. Root exudates increased the growth yield of Frankia in the presence of a carbon source, but Frankia was unable to use the root exudates as a sole carbon or energy source. Exposure to root exudates caused hyphal "curling" in Frankia cells, suggesting a chemotrophic response or surface property change. Exposure to root exudates altered Congo red dye binding, which indicated changes in the bacterial surface properties at the fatty acid level. Fourier transform infrared spectroscopy (FTIR) confirmed fatty acid changes and revealed further carbohydrate changes. Frankia cells preexposed to C. cunninghamiana root exudates for 6 days formed nodules on the host plant significantly earlier than control cells. These data support the hypothesis of early chemical signaling between actinorhizal host plants and Frankia in the rhizosphere.     

Genetic heterogeneity among Frankia isolates from root nodules of individual actinorhizal plants

Abstract Genetic variations among selected Frankia isolates from nitrogen-fixing root nodules harvested from an individual actinorhizal plant ( Elaeagnus angustifolia L. or Shepherdia argentea Nutt.) were estimated by restriction fragment analysis of their total genomic DNA. The presence of plasmids and their restriction enzyme patterns were used as additional criteria. Certain isolates from separate nodules on the same plant were found indistinguishable, being probably clones of the same strain. An endophytic passage of a strain isolated from S. argentea on another host plant, Hippophaë rhamnoides L., did not modify the structural characteristics of the genome in the reisolates obtained. However, in some cases, especially when restriction endonucleases cleaving Frankia DNA into relatively small fragments were used, multiple infection of the actinorhizal plants with different Frankia strains and the presence of more than one strain in a nodule were demonstrated. Some aspects of variability in natural populations of Frankia are discussed.     

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.     

Morphological and molecular characterization of Frankia sp. isolates from nodules of Alnus nepalensis Don

Nodules collected from Alnus nepalensis growing in mixed forest stands at three different sites around Shillong, were crushed in various culture media to obtain isolates of Frankia. The isolates were found to have typical Frankia morphology as revealed by the scanning electron microscope. Seedlings inoculated with isolates or crushed nodules formed nitrogen fixing nodules. Frankia specific DNA probes amplified the DNA of the tested isolate AnpUS4. Partial nucleotide sequence of the 16S rRNA gene indicated that AnpUS4 was phylogenetically distinct from all other Frankia strains characterized so far.