Genetic diversity among and within cultured cyanobionts of diverse species of <Emphasis Type="Italic">Azolla</Emphasis>

The cyanobionts isolated from 10 Azolla accessions belonging to 6 species (Azolla mexicana, A. microphylla, A. rubra, A. caroliniana, A. filiculoides, A. pinnata) were cultured under laboratory conditions and analyzed on the basis of whole cell protein profiles and molecular marker dataset generated using repeat sequence primers (STRR(mod) and HipTG). The biochemical and molecular marker profiles of the cyanobionts were compared with those of the free-living cyanobacteria and symbiotic Nostoc strains from Anthoceros sp., Cycas sp. and Gunnera monoika. Cluster analysis revealed the genetic diversity among the selected strains, and identified 3 distinct clusters. Group 1 included cyanobionts from all the 10 accessions of Azolla, group 2 comprised all the symbiotic Nostoc strains, while group 3 included the free-living cyanobacteria belonging to the genera Nostoc and Anabaena. The interrelationships among the Azolla cyanobionts were further revealed by principal component analysis. Cyanobionts from A. caroliniana-A. microphylla grouped together while cyanobionts associated with A. mexicana-A. filiculoides along with A. pinnata formed another group. A. rubra cyanobionts had intermediate relationship with both the subgroups. This is the first study analyzing the diversity existing among the cultured cyanobionts of diverse Azolla species through the use of biochemical and molecular profiles and also the genetic distinctness of these free-living cyanobionts as compared to cyanobacterial strains of the genera Anabaena and Nostoc.     

满江红属系统学研究的新进展

本文从植物系统学角度对满江红属的研究进展作了全面综述。近年来从细胞研 究水平提出了将满江红属重新划分为〖WTBX〗Azolla和Tetrasporocarpia 2个亚 属,经典的三膘（Azolla）和九膘（Rhizosperma)〖WTBZ〗亚属作为新的Subgen Azolla中的2个组（Section),而原有九膘亚属中的A.nilotica 则成为Subgen.Tetrasporocarpia〖WTBZ〗的一个种。遗传育种和同工酶 的研究证据对A.mexicana,A.microphylla和A.caroliniana能否在Sect. Azolla中占据3个种的分类地位提出疑问。但要完全解决满江红属的系统 学问题,分子生物学技术将是重要手段。由于满江红为蕨 藻共生体,其样本的异质性影响了现有分子标记研究结果的可靠性。     

Influence of Azolla management on the growth,yield of rice and soil fertility

A field experiment conducted at Central Rice Research Institute, Cuttack, during three successive seasons showed that with the 120-day-duration variety Ratna two dual crops ofAzolla pinnata R. Brown (Bangkok isolate) could be achieved 25 and 50 days after transplanting (DAT) by inoculating 2.0 t ha⁻¹ of fresh Azolla 10 and 30 DAT respectively. One basal crop of Azolla could also be grown using the same inoculum 20 days before transplanting (DBT) in fallow rice fields. The three crops of Azolla grown—once before transplanting and twice after transplanting—gave an average total biomass of 38–63 and 43–64 t ha⁻¹ fresh Azolla containing 64–90 and 76–94 kg N ha⁻¹ respectively in the square and rectangular spacings. Two crops of Azolla grown only as a dual crop, on the other hand, gave 26–39 and 29–41 t ha⁻¹ fresh Azolla which contained 44–61 and 43–59 kg N ha⁻¹ respectively. Growth and yield of rice were significantly higher in Azolla basal plus Azolla dual twice incorporated treatments than in the Azolla dual twice incorporation, Azolla basal plus 30 kg N ha⁻¹ urea and 60 kg N ha⁻¹ urea treatments. Azolla basal plus 30 kg N ha⁻¹ urea and 60 kg N ha⁻¹ urea showed similar yields but Azolla dual twice incorporation was significantly lower than those. The different spacing with same plant populations did not affect growth and yield significantly, whereas Azolla growth during dual cropping was 8.3 and 64% more in the rectangular spacing than in the square spacing in Azolla basal plus Azolla dual twice incorporation and Azolla dual twice incorporation treatments.     

满江红鱼腥藻与其宿主的遗传多样性和协同性的RAPD分析

从16个代表不同种属或地域来源的满江红样本中分离出共生藻并通过处理获得无藻的满江红宿主,对二者同步进行了RAPD扩增,分别得到了大量DNA多态片段。通过建立满江红鱼腥藻及其宿主的UPGMA聚类关系图,看出二者在遗传分支上存在着一定程度的协同对应关系。但在种内的不同品系间,这种协同性有所减少,发现有的品系的共生藻发生了明显的变异。 Abstract:Symbiotic Anabeana azollae and its host plant Anabeana-free Azolla were isolated from 16 Azolla accessions representing different Azolla species or geographic origins.DNA polymorphic fragments were obtained by simultaneous RAPD amplification of both symbiont and host.The UPGMA clusters of Anabeana azollae and its host Azolla were established separately based on Dice coefficient caculation and a coordinated relationship was shown between Anabeana azollae and its Azolla host along both individual genetic divergence,but this genetic homology was reduced among different strains within Azolla species while the obvious mutants of Anabeana azollae were detected in some Azolla tested strains collected from different geographic area in the same host species.     

无藻萍的RAPD分析及其在三膘组满江红种间关系研究中的应用

从满江红Azolla Lam.萍-藻共生体中提取DNA进行的RAPD系统分析通常忽视了满江红样品的异质性。本研究通过获得无藻的满江红,比较有藻萍、无藻萍和离体藻之间的RAPD指纹图谱。发现从有藻萍中提取DNA的扩增反应来源于萍藻双方DNA的共同影响。依引物和植物样本的不同,共生双方对扩增产物的贡献结果不同,说明了用无藻萍进行RAPD检测的重要性。对满江红三膘组5个种的11个无藻萍样本进行了RAPD分析,由9个引物产生的127个DNA多态片段用于计算样本间的Jaccard相似系数和UPGMA树状聚类图。结果     

满江红属系统学研究的新进展

本文从植物系统学角度对满江红属的研究进展作了全面综述,近年来从细胞水平提出了将满江红属重新划分为Azolla和Tetrasporocarpia2个亚属,经典的三膘（Azolla）和九膘（Rhizosperma)亚属作为新的Shbgen,Azolla中的2个组（Section）,而原有九膘亚属中的A.nilotica则为Subgen,Tetrasporocarpia的一个种,遗传育种和同工酶的研究证据对A.mexicana,A.microphylla和A.caroliniana能否在Sect.A-zolla中占据3个种的分类地位提出疑问,但要完全解决满江红属的系统学问题,分子生物学技术是重要手段,由于满江红为蕨－藻共生体,其样本的异质性影响了现有分子标记研究结果的可靠性。     

Azolla and other plant-cyanobacteria symbioses: Aspects of form and function

Nostoc, a genus of filamentous, heterocystous, cyanobacteria, is widely distributed in the free-living state. It is also the most common phycobiont in N₂-fixing lichens and occurs as the N₂-fixing symbiont in a small and diverse group of green plants. These include several bryophyte genera (e.g. Anthoceros and Blasia), a pteridophyte genus (Azolla; while the symbiont is referred to asAnabaena azollae, it may be aNostoc spp.), a division of gymnosperms (the 10 cycad genera) and one angiosperm genus (Gunnera). In Gunnera the Nostoc apparently penetrates into the cells of the host. In the other associations Nostoc is extracellular but specific morphological modifications and/or structures of the host plant organs create an environment which fosters interaction and metabolite interchange.The individual group of Nostoc-green plant symbioses other than Azolla are summarized in regard to the current understanding of their establishment, perpetuation, and host-symbiont interaction. This includes available information on recognition and specificity, mode(s) of infection if applicable, and a synopsis of morphological modifications of the partners. The symbiosis withAzolla is then addressed separately with a more indepth account of the foregoing areas. In addition, the concept ofAzolla harboring a dominant, obiligately symbiotic Nostoc which has not been cultured as well as minor symbionts capable of free-living growth, the distinction between re-constituting and simply re-establishing the symbiosis, and current approaches to improving the symbiosis and to authenticating the establishment of new associations are considered.     

北方稻田生态系统研究Ⅰ．稻萍结合系统的结构研究

经过连续5年的实验,研究了北方稻区稻萍结合系统的不同结构对细绿萍产量和水稻产量的影响.结果表明.随着萍种用量的增加,水田中萍的产量也明显增加;尽管宽窄行交替的水稻栽培结构也有利于萍产量的增加,但这样的结构可导致水稻减产3～7%.在北方单季稻地区建议采用30cm等行距的水稻栽培方式和325kg·hm^-2(湿重)的萍接种量建立的稻萍结合系统.     

An Autoradiographic Study of the Azolla-Anabaena azollae Relationship

The symbiotic relationships between Azolla and Anabaena azollae were studied by means of autoradiography after the Azolla was administered by 3H-thymidine, 3H-uridine, 3H-leucine and 3H-glucose. The experimental results showed that the four labeled compounds mentioned above were transfered from Azolla to Anabaena azollae through cavity hairs. This indicated that there was a transfer way of substances from fern to algae. It is suggested that the symbiotic relationship between Azolla and Anabaena azollae is more complicated than we have known up to now. The fern not only get the NH3 which was formed by symbiont-blue alga, but also supplied some nitrogen-containing substances, such as amino acids (or proteins), ribonucleotides for symbiotic algae. Although the symbiont still retained photosynthetic ability, the ability of nitrogen fixation might be developed and the photosynthetic autotrophic ability might be dropped gradually in the long symbiotic life and the Anabaena azolla needed take a portion of substances from the Azolla as replenishment.     

Effect of phosphorus on Azolla and its utilization in rice culture in Niger

The trials to use Azolla as a green manure for rice culture were made in the Niger basin.Azolla pinnata (Niger isolate) was used for the experiments. The effect of phosphorus on the growth and N₂-fixation was examined in the field and in the laboratory. The growth rate and N content were maximum with P 3.1 ppm culture solution under laboratory conditions. The threshold P content for the growth was 0.5–0.6% in the dry matter. Maximum N content was 4.1% in the laboratory culture. In the field culture, the effect of P fertilizer on the growth and N yield of Azolla was tested. The split application of 6.5 kg P ha⁻¹ per 13 days was most effective in stimulating the growth of Azolla. One kg of P as triple superphosphate produced 3.66 kg N in the Azolla. Maximum growth rate and N content in the field trials was 4.3 days (doubling time) and 2.3%, respectively. The lower productivity in the field in comparison with the laboratory culture was considered to be due to higher temperature and light intensity. the growth of Azolla was suppressed in the hot season in the Niger basin. The growth rate and N content were reduced during the high temperature period over 30°C on an average. The effect of inoculation of Azolla on rice yield was tested in the field experiment. The grain yield was increased 27% by Azolla incolation over the treatment without Azolla inoculation in — N fertilizer treatments. While the growth of Azolla with rice plants did not attain saturated density (1.8 kg fresh weight m⁻²), the effect on the grain yield was comparable to 40 kg N ha⁻¹ as urea.     

Production potential of aquatic plants in systems mixing floating and submerged macrophytes

1. To investigate whether the areal production of aquatic plant systems can be increased by growing floating and submerged plants together, the floating fern Azolla filiculoides was grown at six densities and two nutrient availabilities in the presence and absence of the submerged macrophyte Elodea canadensis .
2. High nutrient availability did not affect the total areal biomass production, but raised the internal N concentration of Azolla and Elodea by 34% and 152%, respectively, and the internal P concentration by 50% and 378%.
3. High Azolla density reduced Elodea production, whereas Elodea did not affect the production of Azolla . A maximal total production of 4.0 g DW m⁻² day⁻¹ was obtained at an Azolla density at and above full surface cover, when Elodea contribute with less than 10% to the total. This contribution did not raise the combined production of Azolla and Elodea significantly above the production of Azolla grown alone. Thus, maximum production was not enhanced by growing floating and submerged macrophytes together.     

The Eocene Arctic Azolla bloom: environmental conditions, productivity and carbon drawdown

Enormous quantities of the free-floating freshwater fern Azolla grew and reproduced in situ in the Arctic Ocean during the middle Eocene, as was demonstrated by microscopic analysis of microlaminated sediments recovered from the Lomonosov Ridge during Integrated Ocean Drilling Program (IODP) Expedition 302. The timing of the Azolla phase (~48.5 Ma) coincides with the earliest signs of onset of the transition from a greenhouse towards the modern icehouse Earth. The sustained growth of Azolla , currently ranking among the fastest growing plants on Earth, in a major anoxic oceanic basin may have contributed to decreasing atmospheric p CO₂ levels via burial of Azolla -derived organic matter. The consequences of these enormous Azolla blooms for regional and global nutrient and carbon cycles are still largely unknown. Cultivation experiments have been set up to investigate the influence of elevated p CO₂ on Azolla growth, showing a marked increase in Azolla productivity under elevated (760 and 1910 ppm) p CO₂ conditions. The combined results of organic carbon, sulphur, nitrogen content and ¹⁵N and ¹³C measurements of sediments from the Azolla interval illustrate the potential contribution of nitrogen fixation in a euxinic stratified Eocene Arctic. Flux calculations were used to quantitatively reconstruct the potential storage of carbon (0.9–3.5 10¹⁸ gC) in the Arctic during the Azolla interval. It is estimated that storing 0.9 10¹⁸ to 3.5 10¹⁸ g carbon would result in a 55 to 470 ppm drawdown of p CO₂ under Eocene conditions, indicating that the Arctic Azolla blooms may have had a significant effect on global atmospheric p CO₂ levels through enhanced burial of organic matter.     

Identification of a common cyanobacterial symbiont associated with Azolla spp. through molecular and morphological characterization of free-living and symbiotic cyanobacteria.

Symbiotically associated cyanobacteria from Azolla mexicana and Azolla pinnata were isolated and cultured in a free-living state. Morphological analyses revealed differences between the free-living isolates and their symbiotic counterparts, as did restriction fragment length polymorphism (RFLP) analyses with both single-copy glnA and rbcS gene probes and a multicopy psbA gene probe. RFLP analyses with Anabaena sp. strain PCC 7120 nifD excision element probes, including an xisA gene probe, detected homologous sequences in DNA extracted from the free-living isolates. Sequences homologous to these probes were not detected in DNA from the symbiotically associated cyanobacteria. These analyses indicated that the isolates were not identical to the major cyanobacterial symbiont species residing in leaf cavities of Azolla spp. Nevertheless, striking similarities between several free-living isolates were observed. In every instance, the isolate from A. pinnata displayed banding patterns virtually identical to those of free-living cultures previously isolated from Azolla caroliniana and Azolla filiculoides. These results suggest the ubiquitous presence of a culturable minor cyanobacterial symbiont in at least three species of Azolla.     

The pore of the leaf cavity of Azolla species: teat cell differentiation and cell wall projections

The differentiation of the specialized secretory teat cells of the leaf cavity pore of Azolla species was investigated at the ultrastructural level with emphasis on their peculiar cell wall projections. The results indicated that the projections are formed as soon as the teat cells complete their differentiation and that their production is principally associated with changes in endoplasmic reticulum profiles. The number of projections increases with the teat cell age and is stimulated under salt and P deficiency stresses. Salt stress also promotes their emergence on Azolla species that under normal conditions do not produce projections. Cytochemical tests on different Azolla species showed that the projection composition is almost identical: proteins, acidic polysaccharides, and pectin are always detected. This study revealed that Azolla teat cell projections differ fundamentally from other types of hitherto described cell wall projections that are considered as remnant structures from cell separation. In contrast, in Azolla teat cells projections are actively produced and compounds are excreted by an exocytotic mechanism. The possible role of the projections in the symbiosis of Azolla spp. with Anabaena azollae is discussed.     

The recovery of polyphenols from olive mill waste using two adsorbing vegetable matrices

Olive mill wastewater (OMW) is considered one of the most pollutive waste materials in the Mediterranean basin. However, its phenolic fraction should be recovered, since it has been shown to have incredible benefits for health. In the present study, the adsorbent and desorbent capacities of Azolla and granular activated carbon (GAC) were investigated. The GAC was found to be more efficient than Azolla in both the adsorption and the desorption of phenols. The total characterization of two powder products obtained from Azolla and GAC desorption is reported, together with their antioxidant and antiradical activities. In the Azolla powder product, total polyphenols were more than twice as numerous as those found in the GAC powder product. The GAC powder contained hydroxytyrosol in concentrations that were 3.5 times higher than those of Azolla. On the other hand, both powder products showed great antiradical activities: the IC?? was found to be 102 mg ml?1 for the Azolla and 199 mg ml?1 for the GAC powders respectively. The oxygen radical absorbance capacity was very high: 4097 μmol TE g?1 Azolla powder product and 1277 μmol TE g?1 of GAC powder products.     

Identification of a common cyanobacterial symbiont associated with Azolla spp. through molecular and morphological characterization of free-living and symbiotic cyanobacteria.

Symbiotically associated cyanobacteria from Azolla mexicana and Azolla pinnata were isolated and cultured in a free-living state. Morphological analyses revealed differences between the free-living isolates and their symbiotic counterparts, as did restriction fragment length polymorphism (RFLP) analyses with both single-copy glnA and rbcS gene probes and a multicopy psbA gene probe. RFLP analyses with Anabaena sp. strain PCC 7120 nifD excision element probes, including an xisA gene probe, detected homologous sequences in DNA extracted from the free-living isolates. Sequences homologous to these probes were not detected in DNA from the symbiotically associated cyanobacteria. These analyses indicated that the isolates were not identical to the major cyanobacterial symbiont species residing in leaf cavities of Azolla spp. Nevertheless, striking similarities between several free-living isolates were observed. In every instance, the isolate from A. pinnata displayed banding patterns virtually identical to those of free-living cultures previously isolated from Azolla caroliniana and Azolla filiculoides. These results suggest the ubiquitous presence of a culturable minor cyanobacterial symbiont in at least three species of Azolla.     

Endophyte transmission and activity in the Anabaena-Azolla association

Summary The survival of Azolla was studied in an artificial system which simulated the soil/water interface and the desiccation of soil during a fallow period in lowland rice culture. Tests with non-sporulating and sporulating Azolla fronds showed that Azolla only survives with sporulated fronds. At their reappearance the Azolla fronds already harboured the Anabaena endophyte. A detailed light microscopic and transmission electron microscopic study of macro- and micros-porocarp formation and development revealed that the endophyte is transmitted by the macrosporocarps and not by the microsporocarps. The Anabaena cells within the macrosporocarps are found just below the indusium cap. These cells are not nitrogen-fixing akinetes. The free-living Anabaena cells at the stem apex and below the overarching developing leaves do not bear heterocysts and accordingly are non nitrogen-fixing. During the development of the leaf the Anabaena enters the leaf cavity, but later the pore of this, cavity closes and the imprisoned cyanobacteria are lysed before the leaf decays. As the Azolla leaves age a nitrogen-fixing capability is successively built up concomittantly with the production of heterocysts. Heterocyst frequencies of 40–50% can be found inAnabaena azollae. Usually a gradient of nitrogen-fixing capacity occurs along the Azolla rhizome with two distinct peaks at leaf number 7/8 and at leaf number 13/14 from the apex.     

Azolla: Botany,physiology, and use as a green manure

This is a comprehensive review of literature pertaining to the aquatic fern Azolla and its nitrogen-fixing algal symbiont, Anabaena azollae. The preceding decade has witnessed an explosive growth in research on Azolla, and hopefully this paper will facilitate those efforts. The paper is broken into three major categories: botany, physiology and biochemistry, and agriculture. The botany section includes a world distribution map and reference tables for the 6 Azolla species, and includes the first review of literature on Anabaena azollae. The physiology and biochemistry section covers the range of topics from environmental factors to life processes and nitrogen fixation. Tables on the effect of growth regulators and on the rate of nitrogen fixation measured by acetylene reduction are presented. The agriculture section draws extensively from literature published in the People’s Republic of China and in the Democratic Republic of Vietnam. The major focus of this section is on the history and management practices for Azolla cultivation as a green manure for rice. The effect of weed suppression, use as a fish food and animal fodder, and the insects and diseases of Azolla are also discussed.     

细叶满江红大孢子果的扫描电镜观察

细叶满江红(Azolla filicufoides Lam.)是优良的水生绿肥和饲料作物,又是用孢子果越夏和过冬的好材料,植物学者对其进行广泛研究已有悠久的历史。本文记述了电镜扫描观察到的大孢子果及小孢子囊泡胶块的图象;认为大孢子果表面同小孢子囊泡胶块的附着力,与大孢子果的萌发力有相关性;指出孢子果带菌,将是导致高温季节大田满江红发生毁灭性病害“自然倒萍”的主要因素之一。     

Effect of varying periods of inoculation ofAzolla pinnata R. Brown on its growth,nitrogen fixation and response to rice crop

Summary Inoculation of water fernAzolla pinnata R. Brown (Bangkok isolate) at the rate of 500kg fresh weight ha⁻¹ in rice fields at weekly intervals after planting in addition to 30 kg N ha⁻¹ as urea showed a decrease in its growth and N₂-fixation with delay in application. Use of Azolla up to 3 weeks after planting (WAP) during wet and 4 WAP during dry season produced significantly more grain yield than 30 kg N ha⁻¹, whereas its application upto one WAP produced more grain yield than 60 kg N ha⁻¹. Grain yield with Azolla applied at the time of planting was similar to that of 60 kg N treatment during the wet season. Higher grain yields in zero and one WAP Azolla treatments resulted due to increase in both number of panicles m⁻² and number of grains/panicle while the subsequent Azolla inoculations increased grain yield mainly by producing more number of grains/panicle. Dry matter and total N yields at maturity of rice crop were more with Azolla application upto 3 WAP during wet and 2 WAP during dry season while the reduction in sterility (%) was observed upto one WAP over 30 kg N ha⁻¹ during both seasons. Number of tillers m⁻² and dry matter production at maximum tillering and flowering were more than 30 kg N ha⁻¹ with the use of Azolla upto one WAP. Increased grain N yield was observed with the use of Azolla upto 4 WAP during two seasons whereas straw N yield increased upto one WAP during wet and 2 WAP during dry season.