Cyanobacterial diversity in the rhizosphere of rice and its ecological significance

This investigation was undertaken to characterize the abundance and genera-wise diversity of cyanobacteria in the rice rhizosphere and nitrogen-fixing ability of the isolated strains. The cyanobacterial strains belonging to the genera Nostoc and Anabaena comprised 80% of the rhizosphere isolates, which were also efficient in enhancing the germination and growth of wheat seeds and exhibited significantly high protein accumulation and IAA production. Distinct profiles for the cyanobacterial strains were obtained on amplification with extended Hip 1 primer — HipTG, indicative of the diversity among these strains. Our investigation helped in identifying promising cyanobacterial isolates from the rhizosphere of rice, which can be utilized in developing efficient plant growth promoting cyanobacterial inoculants.     

Characterization of Cyanobacteria by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene

Planktonic, filamentous cyanobacterial strains from different genera, both toxic and nontoxic strains, were characterized by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene. Total protein pattern analysis revealed the mutual relationships at the genus level. Restriction fragment length polymorphism (RFLP) of the 16S rRNA gene with reference strains proved to be a good method for the cyanobacterial taxonomy. The nonheterocystous strains outgrouped from the nitrogen-fixing ones. With both methods, Aphanizomenon clustered with Anabaena, and Nodularia with Nostoc. In the RFLP study of Anabaena, the neurotoxic strains were identical, but the hepatotoxic ones formed a heterogeneous group. Genetic distances found in the RFLP study were short, confirming that close genotypic relationships underlie considerable diversity among cyanobacterial genera. Received: 16 December 1996 / Accepted: 14 May 1997     

Phylogeny of symbiotic cyanobacteria within the genus <Emphasis Type="Italic">Nostoc</Emphasis> based on 16S rDNA sequence analyses

A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.The GenBank accession numbers for the cyanobacterial 16S rRNA gene sequences determined in this study are AY742447-AY742454.     

Determinative Value of a Portion of the nifH Sequence for the Genera Nostoc and Anabaena (Cyanobacteria)

The taxonomic positions of Nostoc and Anabaena strains are currently disputed. We selected three Nostoc and Anabaena strains, using the classic criteria of morphology and life cycle. DNA sequences of a part of the nifH gene were determined from these strains and aligned with homologous sequences from 10 other Nostoc/Anabaena strains in the public databases. Phylogenetic reconstructions were carried out to test the consistency of the taxonomic placement of these strains. The phylogenetic trees do not separate these strains into distinct groups. Our results are in agreement with other molecular-based phylogenies that also fail to differentiate the Nostoc-Anabaena groups. The data suggest that the currently recognized genera Nostoc and Anabaena may in fact belong within a single, broadly defined genus. Received: 14 February 2000 / Accepted: 21 March 2000     

Influence of diverse rice soil ecologies on cyanobacterial diversity and abundance

Cyanobacteria comprise a large group of structurally complex and ecologically significant gram-negative prokaryotes which flourish in rice paddies, and play a major role in sustaining the fertility of this ecosystem. This study aimed to characterize the abundance of cyanobacteria in various rice ecologies of India, identify the isolates and determine diversity indices in relation to the genera wise distribution. Average population counts (measured as MPN) of various locations clearly brought out the tremendous diversity among the locations sampled. Soil samples from Jeypore (Orissa state) recorded highest diversity and 20 cyanobacterial forms, spanning 9 genera were isolated. Nostoc and Anabaena were found to be the dominant genera in all the locations, in terms of their abundance and exhibited highest diversity indices. Our results suggest the need for practical utilization of these organisms towards developing region-specific inocula–which can establish better in their niche and provide maximum benefits to the crop.     

Molecular differentiation of the heterocystous cyanobacteria,Nostoc and Anabaena,based on complete NifD sequences

The segregation of Nostoc and Anabaena into separate genera has been debated for some time. The nitrogen fixation gene nifD was completely sequenced from representatives of these genera and analyzed phylogenetically, by using the representatives of other genera of the heterocystous cyanobacteria as outgroups. We were clearly able to differentiate between Nostoc and Anabaena in all analyses used. Our data suggest that Nostoc and Anabaena should remain as separate genera. Received: 16 November 2001 / Accepted: 14 December 2001     

Identification of a Diagnostic Marker To Detect Freshwater Cyanophages of Filamentous Cyanobacteria

Cyanophages are viruses that infect the cyanobacteria, globally important photosynthetic microorganisms. Cyanophages are considered significant components of microbial communities, playing major roles in influencing host community diversity and primary productivity, terminating cyanobacterial water blooms, and influencing biogeochemical cycles. Cyanophages are ubiquitous in both marine and freshwater systems; however, the majority of molecular research has been biased toward the study of marine cyanophages. In this study, a diagnostic probe was developed to detect freshwater cyanophages in natural waters. Oligonucleotide PCR-based primers were designed to specifically amplify the major capsid protein gene from previously characterized freshwater cyanomyoviruses that are infectious to the filamentous, nitrogen-fixing cyanobacterial genera Anabaena and Nostoc. The primers were also successful in yielding PCR products from mixed virus communities concentrated from water samples collected from freshwater lakes in the United Kingdom. The probes are thought to provide a useful tool for the investigation of cyanophage diversity in freshwater environments.     

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF PLANKTONIC CYANOBACTERIA FROM BELGIUM AND LUXEMBOURG1

For the first time in Belgium and Luxembourg, the diversity and taxonomy of 95 cyanobacterial strains isolated from freshwater blooms were assessed by the comparison of phenotypes and partial 16S rRNA gene sequences. The results showed the high diversity of nanoplanktonic, picoplanktonic, and benthic–periphytic cyanobacteria accompanying the main bloom‐forming taxa. Indeed, besides 15 morphotypes of bloom‐forming taxa, seven non‐bloom‐forming planktonic morphotypes and 11 morphotypes from benthic–periphytic taxa were isolated in culture from the plankton samples of 35 water bodies. The bloom‐forming strains belonged to the genera Microcystis, Woronichinia, Planktothrix, Anabaena, and Aphanizomenon, whereas the other strains isolated from the same samples were assigned to the nanoplanktonic Aphanocapsa, Aphanothece, Snowella, and Pseudanabaena; to the picoplanktonic Cyanobium; and to the benthic–periphytic Geitlerinema, Komvophoron, Leptolyngbya, Lyngbya, Phormidium, Calothrix, Nostoc, and Trichormus. The results supported both the polyphyletism of genera such as Aphanocapsa, Aphanothece, Leptolyngbya, Geitlerinema, Anabaena, and Aphanizomenon as well as the validity of genera such as Microcystis, Planktothrix, and Pseudanabaena with gas vesicles and cells constricted at the cross wall. The results obtained showed the close relationship between Snowella and Woronichinia for which very few sequences exist. The first sequence of Komvophoron appeared poorly related to other available cyanobacterial sequences. Although in a few cases a good agreement existed between phenotypic and genotypic features, there was generally a discrepancy. Strains with identical morphotypes show small differences in the 16S rRNA sequences, which might be related to the different chemical properties of their habitats. The results showed the importance of the polyphasic approach in order to improve the taxonomy of cyanobacteria.     

Nitrogen-fixing cyanobacteria as source of phycobiliprotein pigments. Composition and growth performance of ten filamentous heterocystous strains

Ten strains of filamentous, heterocystous nitrogen-fixing blue-green algae (cyanobacteria) were screened for growth performance and tolerance to temperature, pH, irradiance and salinity, together with their potential as producers of phycobiliprotein pigments. Phycobiliproteins typically accounted for about 50% total cell protein, the prevalent type being C-phycocyanin, followed by alloppycocyanin, with levels of 17 and 11% d.wt, respectively, in some strains of Anabaena and Nostoc. C-phycoerythrin was the major pigment in several Nostoc strains, reaching 10% d.wt. Some strains represent, therefore, excellent sources of one or more phycobiliproteins. All strains tolerated an irradiance of ca 2000 μmol photon m^-2 s^-1. Anabaena sp. ATCC 33047 and Nostoc sp. (Albufera) exhibited the widest optimum range of both temperature (30–45 and 25–40 °C) and pH (6.5–9.5 and 6.0–9.0) for growth, the former also showing significant salt tolerance. In an outdoor open system, productivity of cultures of two phycoerythrin-rich strains of Nostoc was over 20 g (d.wt) m^-2 d^-1 during summer. The growth performance of the allophycocyanin-rich Anabaena sp. ATCC 33047 in outdoor semi-continuous culture has been assessed throughout the year. Productivity values under optimized conditions ranged from 9 (winter) to 24 (summer) g (d.wt) m^-2 d^-1.     

Analysing diversity among Indian isolates of Anabaena (Nostocales, Cyanophyta) using morphological, physiological and biochemical characters

A set of 24 strains belonging to the genus Anabaena (Phylum Cyanobacteria), isolated from diverse geographic locations in India, were evaluated along with three International type strains of Anabaena (ATCC 29414, ATCC 29208 and ATCC 27899) for their morphological, physiological and biochemical diversity. The morphological dataset, consisting of 58 variants for 15 characters, and SDS-PAGE protein profiles comprising 17 polymorphic bands were utilized to differentiate the selected Anabaena strains and explore the patterns of diversity through cluster analysis. Physiological and biochemical characterization with respect to nitrogen fixation and accumulation of chlorophyll and phycobiliproteins led to the identification of some highly promising Anabaena strains for use as biofertilizers and source of pigments. The study highlighted the tremendous inter and intraspecific diversity within the Anabaena isolates and indicated the potential as well as constraints of the morphological and protein profiling datasets for unambiguous differentiation and analyses of diversity among the Anabaena strains.     

The role of extracellular polysaccharides produced by the terrestrial cyanobacterium Nostoc sp. strain HK-01 in NaCl tolerance

The terrestrial cyanobacterium Nostoc sp. HK-01 was more tolerant to NaCl stress than the aquatic cyanobacterium Anabaena sp. PCC 7120 (also called Nostoc sp. PCC 7120) which is similar to Nostoc sp. HK-01 in phylogeny. We determined the amount of extracellular polysaccharides (capsular and released polysaccharides) from the cells of both strains cultured with or without 200 mM NaCl. The amount of capsular polysaccharides from Nostoc HK-01 reached approximately 65% of the dry weight whereas that from Anabaena PCC 7120 only occupied approximately 18% of the dry weight under NaCl stress. Anabaena PCC 7120 grew well under NaCl stress when both polysaccharides from Nostoc HK-01 were added to the culture. However, Anabaena PCC 7120 barely grew under NaCl stress when both of its polysaccharides were added. Extracellular polysaccharides from Nostoc HK-01 contained abundant fucose and glucuronic acid in comparison with those from Anabaena PCC 7120. Under NaCl stress, the composition ratios of sugars in the extracellular polysaccharides from Anabaena PCC 7120 hardly changed in comparison with those in ordinary culture conditions. By contrast, the composition ratios of sugars in the extracellular polysaccharides from Nostoc HK-01 changed under NaCl stress. These results suggest that the effect of extracellular polysaccharides from Nostoc HK-01 on NaCl tolerance comes from the increased amount of capsular polysaccharides, the sugar composition, and the change of the sugar composition ratio under NaCl stress.     

Antioxidant activity and phenolic profile in filamentous cyanobacteria: the impact of nitrogen

In the present study, ethanolic extracts of ten cyanobacterial strains cultivated under different nitrogen conditions were assessed for the phenolic content and antioxidant activity. The amount of detected phenolic compounds ranged from 14.86 to 701.69 μg g^?1 dry weight (dw) and HPLC-MS/MS analysis revealed gallic acid, chlorogenic acid, quinic acid, catechin, epicatechin, kaempferol, rutin and apiin. Only catechin, among the detected phenolics, was present in all the tested strains, while quinic acid was the most dominant compound in all the tested Nostoc strains. The results also indicated the possibility of increasing the phenolic content in cyanobacterial biomass by manipulating nitrogen conditions, such as in the case of quinic acid in Nostoc 2S7B from 70.83 to 594.43 μg g^?1 dw. The highest radical scavenging activity in DPPH assay expressed Nostoc LC1B with IC₅₀ value of 0.04?±?0.01 mg mL^?1, while Nostoc 2S3B with IC₅₀ =?9.47?±?3.61 mg mL^?1 was the least potent. Furthermore, the reducing power determined by FRAP assay ranged from 8.36?±?0.08 to 21.01?±?1.66 mg AAE g^?1, and it was significantly different among the tested genera. The Arthrospira strains exhibited the highest activity, which in the case of Arthrospira S1 was approximately twofold higher in comparison to those in nitrogen-fixing strains. In addition to this, statistical analysis has indicated that detected phenolics were not major contributor to antioxidant capacities of tested cyanobacteria. However, this study highlights cyanobacteria of the genera Nostoc, Anabaena, and Arthrospira as producers of antioxidants and phenolics with pharmacological and health-beneficial effects, i.e., quinic acid and catechin in particular.     

佳西热带雨林土壤芽胞杆菌分离与多样性分析

采用热处理法从海南省佳西热带雨林土壤中分离到147株芽胞杆菌,并利用16S rDNA PCR-RFLP与序列分析技术对其遗传多样性进行了研究。16S rDNA PCR-RFLP酶切图谱UPGMA聚类分析结果表明,在100%的相似性水平上,这些芽胞杆菌分属13个遗传类群。不同遗传类型代表菌株的16S rRNA基因序列分析结果显示,它们分布在Bacillaceae、Planococcaceae和Paenibacillaceae科的Bacillus、Lysinibacillus、Paucisalibacillus、Bhargavaea和Paenibacillus五个属,其中Bacillus为优势属(占50%);有3株芽胞杆菌的16S rRNA基因序列与数据库中相应模式菌株的最大相似性在98.3%~98.9%之间。结果表明,佳西热带雨林土壤中芽胞杆菌有着较为丰富的遗传多样性。     

Sucrose synthase and RuBisCo expression is similarly regulated by the nitrogen source in the nitrogen-fixing cyanobacterium Anabaena sp.

In higher plants and cyanobacteria, sucrose (Suc) metabolism is carried out by a similar set of enzymes. The function and regulation of Suc metabolism in cyanobacteria has begun to be elucidated. In strains of Anabaena sp., filamentous nitrogen-fixing cyanobacteria, Suc synthase (SuS, EC 2.4.1.13) controls Suc cell level through the cleavage of the disaccharide. The present work shows that there are two sus genes in Anabaena (Nostoc) sp. that are co-regulated regarding the nitrogen source; however, only susA accounts for the extractable SuS activity and for the control of the Suc level. Primer extension analysis has uncovered the sequence of the Anabaena susA and susB ammonium-activated putative promoters, which share a high sequence similarity with that of rbcLS encoding ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) and other ammonium up-regulated genes. Moreover, susA and rbcLS expression is developmentally co-localized to the vegetative cells of the nitrogen-fixing cyanobacterial filaments. Our results strongly suggest the existence of a regulatory network that would coordinate the expression of key genes for Suc and nitrogen metabolism, carbon fixation, and development in Anabaena sp.     

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.Non-standard abbreviations bp base pairs - kbp kilobase pairs - RFLP's restriction fragment length polymorphisms     

入侵植物欧洲千里光内生固氮菌和溶磷菌多样性

【背景】菊科(Asteraceae)外来入侵植物欧洲千里光(Senecio vulgaris L.)来源于欧洲,广泛分布于我国西南和东北地区,在湖北高海拔山区也有分布。在入侵过程中,内生细菌可能在其获取氮磷营养方面起到了一些关键性作用。【目的】探究欧洲千里光内生固氮菌和溶磷菌的多样性和功能,为理解其入侵机制及防治提供参考。【方法】选择来自6个不同种群的种子,萌发后转移到花盆生长6-8周,并从每个种群中各挑选9株生长情况良好的植株,对其叶片和根组织表面进行消毒处理。使用基于nifH基因(固氮功能基因)的高通量测序方法对植物的固氮微生物群落结构和多样性进行研究。通过涂布平板法和平板划线法,在固体无氮培养基(Ashby)和无机磷培养基(inorganic phosphate, NBRIP)上对植物内生菌进行分离、纯化,对纯化的固氮菌株和溶磷菌株进行16S rRNA基因测序。采用钼锑抗比色法分析纯化溶磷菌株的溶磷能力。【结果】基于nifH基因的内生菌高通量测序结果表明,欧洲千里光叶样本中固氮菌多样性显著高于根样本;固氮菌群落中丰度最高的属是慢生根瘤菌属(Bradyrhizobium,30.9%...     

Hydrogen uptake in Nostoc sp. strain PCC 73102. Cloning and characterization of a hupSL homologue

Structural genes encoding an uptake hydrogenase of Nostoc sp. strain PCC 73102 were isolated. From partial libraries of genomic DNA, two clones (pNfo01 and pNfo02) were selected and sequenced, revealing the complete sequence of both a hupS (960 bases) and a hupL (1,593 bases) homologue in Nostoc sp. strain PCC 73102. A comparison between the deduced amino acid sequences of HupS and HupL of Nostoc sp. strain PCC 73102 and Anabaena sp. strain PCC 7120 showed that the HupS proteins are 89% identical and the HupL proteins are 91% identical. However, the noncoding region between the genes in Nostoc sp. strain PCC 73102 (192 bases) is longer than that of Anabaena sp. strain PCC 7120 and of many other microorganisms. Southern hybridizations using DNA from both N₂-fixing and non-N₂-fixing cells of Nostoc sp. strain PCC 73102 and different probes from within hupL clearly demonstrated that, in contrast to Anabaena sp. strain PCC 7120, there is no rearrangement within hupL of Nostoc sp. strain PCC 73102. Indeed, 6 nucleotides out of 16 within the potential recombination site are different from those of Anabaena sp. strain PCC 7120. Furthermore, we have recently published evidence demonstrating the absence of the bidirectional/reversible hydrogenase in Nostoc sp. strain PCC 73102. The present knowledge, in combination with the unique characteristics, makes Nostoc sp. strain PCC 73102 an interesting candidate for the study of deletion mutants lacking the uptake-type enzyme. Received: 20 August 1997 / Accepted: 24 November 1997     

Identification of acid-stress-tolerant proteins from promising cyanobacterial isolates

Cyanobacterial cultures were isolated from acidic (pH 4.9–6.2) rice grown soils in Tamil Nadu, India. The predominant genera were Anabaena (50%), Westiellopsis (17.5%), Nostoc (15%), Oscillatoria (5%) and others that were unicellulars (12.5%) viz., Microcystis, Calothrix and Phormidium. The levels of tolerance to acidity varied among these strains, which were tested and authenticated for their acid tolerance capacity under both in vitro and pot culture conditions. Westiellopsis sp. was found to predominate from pH 4.9 to pH 6.2, indicating its adaptability. Cultures tolerant to acidic conditions were characterized for growth, biomass production and biochemical constituents. Under acidic conditions, Westiellopsis sp. showed pronounced chlorophyll a content, phycobilin pigment content, ammonia excretion and nitrogenase activity compared to normal conditions. Molecular characterization, particularly isozyme and random amplified polymorphic DNA (RAPD) analysis, were also carried out. Three strains of Westielliopsis sp. strains were selected, of which two were able to grow at an acidity level of pH 4.0, while one strain was able to sustain growth at an acidity level of 5.0. These three cultures, along with acid susceptible strains of Westielliopsis sp. and Anabaena sp. PCC 7120 (standard check) were subjected to acid shock for different time intervals. Protein profiling of both the acid-tolerant and acid-susceptible strains was carried out with samples collected at different time intervals. Based on the presence/absence of protein bands in the tolerant/susceptible strains, some low- and medium-molecular weight proteins can be linked to conferring acid tolerance. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

A NUMERICAL TAXONOMIC STUDY OF NOSTOC AND ANABAENA1

Thirty characteristics of 14 Nostoc and 10 Anabaena species were analyzed from previously published data. Using standard numerical taxonomic methods, simple matching coefficients were calculated and a phenogram drawn. The analysis revealed that some of the central characteristics of Nostoc are: a punctiforme stage; motile reproductive stage; plant mass with a dull to shiny luster, non-veined surface, and nonfimbriate margin; some spherical vegetative cells; no cylindrical heterocysts; and some spherical, but no cylindrical akinetes. Some of the central characteristics of Anabaena that were revealed are: no punctiforme stage; a motile vegetative stage; plant mass with a shiny luster, veined surface, and fimbriate margin; no spherical vegetative cells; some cylindrical heterocysts; and some cylindrical, but no spherical, akinetes. In general, Anabaena has larger akinetes and vegetative cells than Nostoc. Based on 30 morphological characteristics and the clustering data of the phenogram, keys were constructed for the Nostoc and Anabaena species studied. The data clearly support two separate and distinct, though similar genera and, less sharply, the separation of the 24 species. The more useful characteristics for separation of the species are size and shape of akinetes, vegetative cells, and heterocysts; color and luster of plant mass; veined plant mass surface; margin fimbriate; and shape of plant mass in nature.     

Genomic diversity and CRISPR-Cas systems in the cyanobacterium Nostoc in the High Arctic

Nostoc (Nostocales, Cyanobacteria) has a global distribution in the Polar Regions. However, the genomic diversity of Nostoc is little known and there are no genomes available for polar Nostoc. Here we carried out the first genomic analysis of the Nostoc commune morphotype with a recent sample from the High Arctic and a herbarium specimen collected during the British Arctic Expedition (1875–76). Comparisons of the polar genomes with 26 present-day non-polar members of the Nostocales family highlighted that there are pronounced genetic variations among Nostoc strains and species. Osmoprotection and other stress genes were found in all Nostoc strains, but the two Arctic strains had markedly higher numbers of biosynthetic gene clusters for uncharacterised non-ribosomal peptide synthetases, suggesting a high diversity of secondary metabolites. Since viral–host interactions contribute to microbial diversity, we analysed the CRISPR-Cas systems in the Arctic and two temperate Nostoc species. There were a large number of unique repeat-spacer arrays in each genome, indicating diverse histories of viral attack. All Nostoc strains had a subtype I-D system, but the polar specimens also showed evidence of a subtype I-B system that has not been previously reported in cyanobacteria, suggesting diverse cyanobacteria–virus interactions in the Arctic.