A gene cluster that regulates both heterocyst differentiation and pattern formation in Anabaena sp strain PCC 7120

Wild-type Anabaena sp. strain PCC 7120, a filamentous nitrogen-fixing cyanobacterium, produces single heterocysts at semi-regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5' and 3' portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5' end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini-patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ-patU5-patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5-1087b were nitrogen-deprived, P-hetC-gfp was very weakly expressed, and in hetZ::Tn5-1087b, P-hetR-gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ-patU5-patU3 cluster plays an important role in co-ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.

Studies of polysaccharides from three edible species of Nostoc (cyanobacteria) with different colony morphologies: Structural characterization and effect on the complement system of polysaccharides from Nostoc commune

The cyanobacterium Nostoc commune Vaucher produces quite complex extracellular polysaccharides. The cyanobacterium is nitrogen fixing, and on growing the cyanobacterium in media with and without nitrogen, different types of extracellular polysaccharides were obtained. These were also different from the polysaccharides present in N. commune collected in the field. High pH anion exchange chromatography (HPAEC) of weak acid hydrolysates of the culture-grown material demonstrated that, in this case, HPAEC was useful for comparison of the different polymers. The main differences between the polymers from the field group and the culture-grown samples were the presence of substantial amounts of arabinose, 2-O-methylglucose, and glucuronic acid in the latter. Methylation studies also revealed a difference in the branching points on the glucose units between the field and cultured samples, being 1,4,6 for the first and 1,3,6 for the latter. The field acidic fraction gave, on weak acid hydrolysis and separation on BioGel P2 and HPAEC, 12 oligosaccharide fractions that were isolated and studied by different mass spectroscopy techniques. The structures of the oligosaccharides were determined, and two different series that can originate from two repeating pentamers were identified: GlcAl-4/6GlcM1-4Ga11-4Glc1-4Xyl and GlcAl-4/6Glc1-4Ga11-4Glc1-4Xyl. The difference between these oligosaccharides lies in the methyl substituent on carbon 2 of the glucose unit next to the nonreducing glucuronic acid unit. The polysaccharides from field material were shown to have a strong effect on the complement system.

固氮树木丛枝菌根真菌(AMF)遗传多样性的研究

丛枝菌根是自然生态系统中分布最广的内生菌根，在促进植物生存与生长、植被恢复以及生物多样性保护等方面有着非常重要的作用。 随着现代分子生物学技术的不断发展，丛枝菌根真菌研究得到空前发展。大量DNA分析新技术在丛枝菌根真菌的分子遗传、分类鉴定、种间及种内亲缘关系、菌株持久性等方面得到应用，与传统菌根研究方法相比，表现出巨大的优越性。 本项研究利用分子生物学技术和研究方法对中国吉林长白山地区非豆科固氮植物以及东北地区固氮树木的丛枝菌根真菌DNA分子多态性及其与宿主植物之间的相互关系等进行初步研究，旨在利用分子生态学理论和研究方法揭示丛枝菌根真菌多样性及其与宿主植物之间相互适应和协同进化的一般规律，为更好地保护和利用这一重要的微生物资源提供理论依据。 通过比较与筛选，建立起丛枝菌根真菌痕量DNA快速、简便、高效的提取纯化方法——改良CTAB法。经PCR检测，所得DNA满足进一步研究的要求。 根据丛枝菌根真菌18s rRNA 小亚基核基因片段的特点，利用“科”特异性引物进行半巢式标记PCR (Labelled Primers-PCR，LP-PCR) 及单链构象多态性（Single-Stranded Conformation Polymorphism，SSCP）分析技术研究了长白山赤杨在属水平上表现出的多样性。另外，利用巢式PCR-RFLP技术，分别对来源于长白山不同海拔的四种赤杨菌根样品的AMF侵染情况及其系统进化进行了研究。利用AMF特异性PCR技术对我国东北地区四种非豆科树木和5种豆科树木菌根侵染情况和系统发育规律进行了研究 研究结果显示：赤杨根内AMF存在丰富的基因多样性。AMF的侵染有从宿主混乱性向宿主专一性发展的趋势。 长白山地区赤杨属植物至少有东北赤杨、西伯利亚赤杨和色赤杨三个树种在其“属”的水平上与共生的球囊霉科（Glomaceae）至少一个“种” 的丛枝菌根真菌，即根内球囊霉（Glomus intraradix），在“种”的水平上表现出不相关于宿主海拔高度的某种相互选择性。 东北赤杨AMF菌的宿主专一性水平最强，球囊霉属已成为东北赤杨的优势侵染类群；对于其余三种赤杨，AMF则出现宿主混乱现象。宿主因素比海拔因素对AMF侵染特异性的影响更为重要。 豆科与非豆科样本的混乱性都比较强，在特定植物和AMF属之间无特异侵染规律，相对来说，非豆科树木比豆科树木对于AMF的选择性要更强一些，更倾向于和球囊霉属与无梗孢囊霉属的AMF构建共生体。

固氮树木丛枝菌根真菌（AMF）遗传多样性的研究

丛枝菌根是自然生态系统中分布最广的内生菌根，在促进植物生存与生长、植被恢复以及生物多样性保护等方面有着非常重要的作用。 随着现代分子生物学技术的不断发展，丛枝菌根真菌研究得到空前发展。大量DNA分析新技术在丛枝菌根真菌的分子遗传、分类鉴定、种间及种内亲缘关系、菌株持久性等方面得到应用，与传统菌根研究方法相比，表现出巨大的优越性。 本项研究利用分子生物学技术和研究方法对中国吉林长白山地区非豆科固氮植物以及东北地区固氮树木的丛枝菌根真菌DNA分子多态性及其与宿主植物之间的相互关系等进行初步研究，旨在利用分子生态学理论和研究方法揭示丛枝菌根真菌多样性及其与宿主植物之间相互适应和协同进化的一般规律，为更好地保护和利用这一重要的微生物资源提供理论依据。 通过比较与筛选，建立起丛枝菌根真菌痕量DNA快速、简便、高效的提取纯化方法——改良CTAB法。经PCR检测，所得DNA满足进一步研究的要求。 根据丛枝菌根真菌18s rRNA 小亚基核基因片段的特点，利用“科”特异性引物进行半巢式标记PCR (Labelled Primers-PCR，LP-PCR) 及单链构象多态性（Single-Stranded Conformation Polymorphism，SSCP）分析技术研究了长白山赤杨在属水平上表现出的多样性。另外，利用巢式PCR-RFLP技术，分别对来源于长白山不同海拔的四种赤杨菌根样品的AMF侵染情况及其系统进化进行了研究。利用AMF特异性PCR技术对我国东北地区四种非豆科树木和5种豆科树木菌根侵染情况和系统发育规律进行了研究 研究结果显示：赤杨根内AMF存在丰富的基因多样性。AMF的侵染有从宿主混乱性向宿主专一性发展的趋势。 长白山地区赤杨属植物至少有东北赤杨、西伯利亚赤杨和色赤杨三个树种在其“属”的水平上与共生的球囊霉科（Glomaceae）至少一个“种” 的丛枝菌根真菌，即根内球囊霉（Glomus intraradix），在“种”的水平上表现出不相关于宿主海拔高度的某种相互选择性。 东北赤杨AMF菌的宿主专一性水平最强，球囊霉属已成为东北赤杨的优势侵染类群；对于其余三种赤杨，AMF则出现宿主混乱现象。宿主因素比海拔因素对AMF侵染特异性的影响更为重要。 豆科与非豆科样本的混乱性都比较强，在特定植物和AMF属之间无特异侵染规律，相对来说，非豆科树木比豆科树木对于AMF的选择性要更强一些，更倾向于和球囊霉属与无梗孢囊霉属的AMF构建共生体.

非豆科固氮树木色赤杨联合共生体的研究

非豆科树木共生固氮体系抗逆性强，具有耐干旱、耐盐碱、耐低PH、耐水湿等特点，且分布广泛，资源丰富，因此开展Frankia-非豆科树木联合共生体系的理论与应用研究，不仅对丰富和发展联合共生理论具有重要意义，而且在加速树木生长、解决农村资源短缺、减少水土流失、改良土壤和改善生态环境等方面将产生重大的经济和生态环境效益.该文通过对非豆科固氮树木色赤杨的菌根、根瘤联合共生体人工构建技术以及赤杨联合共生增效作用的研究，选择出色赤杨的最适共生菌根菌、弗兰克氏菌，为联合共生体接种技术在造林生产实践尤其是在逆境条件下的应用提供理论依据.

花椒主效化感物质分离鉴定及化感作用研究

近年来，随着对作物重茬障碍原因的深入研究，植物的化感作用越来越受到国内外众多学者的重视。花椒（Zanthoxy piperitum.）为芸香科植物，是一种收益早、用途广、价值高的经济树种，是川西干旱河谷地区的重要经济作物，其连作障碍也倍受关注，系统研究花椒化感作用将有助于理解和最终解决花椒连作障碍问题。本文首先通过萃取、层析等方法分离花椒主效化感成分；通过外加不同浓度的花椒叶水浸液研究了对土壤氮素养分循环的影响；研究了花椒叶水浸液对苜蓿生理生化、光合作用、氮素养分吸收的影响，并对外施氮肥对这种化感影响的缓解作用做了研究；研究了花椒化感潜力对全球变化——UV-B增强辐射的响应。主要研究结果如下： 1．用不同极性的有机溶剂对花椒叶水浸液浓缩浸膏萃取、柱层析，结合生物活性检测，分离得到主效化感作用组分的一种化感物质——对甲氧基苯酚。采用该物质纯品进行生物活性检测，证明其具有化感作用。 2．花椒叶水浸液处理土壤30天后，土壤硝态氮、铵态氮、无机氮（硝态氮＋铵态氮）与对照相比，随着花椒叶水浸液浓度的增加呈现降低的趋势，其中土壤铵态氮含量显著降低，而硝态氮含量的变化则不显著，无机氮含量也显著降低。土壤脲酶和蛋白酶的活性与无机氮含量的变化趋势相同。随着花椒叶水浸液浓度的增加，氨化细菌数量显著降低，固氮菌的数量变化不显著，硝化细菌和反硝化细菌数量有减少的趋势。60天后，硝态氮含量、铵态氮含量、无机氮随水浸液浓度增加的变化趋势与30天时相似；随着花椒叶水浸液浓度的增加，氨化细菌、固氮菌的数量显著减少，硝化细菌数量、反硝化细菌数量仍呈减少趋势；土壤脲酶、蛋白酶活性与第30天的变化趋势相同。第60天与第30天的结果相比，相同水浸液浓度处理的硝态氮、铵态氮、无机氮均有下降的趋势，但除了25g.L－1水浸液处理的外，其它相同浓度的处理间差异均不显著；除了12.5 g.L的处理外土壤脲酶活性均呈增强的趋势；蛋白酶活性都有不同程度的增加；花椒叶水浸液处理的土壤硝化细菌和反硝化细菌数量呈增加趋势。 3．随着花椒叶水浸液浓度的增加，显著抑制了苜蓿根长、地上地下生物量、叶绿素含量、叶片中可溶性蛋白的含量，净光合速率。苜蓿体内四种抗氧化酶（POD、SOD、CAT、APX） 活性随着水浸液浓度的增加而降低，而丙二醛含量则增加。苜蓿氮初级同化相关酶硝酸还原酶（NR）、谷氨酰合成酶(GS)、谷氨酸脱氢酶（GDH）的活性随着水浸液浓度的增加受到不同程度的影响。总的来说，苜蓿硝酸还原酶、谷氨酰合成酶的活性受到抑制，而谷氨酸脱氢酶活性的变化则比较复杂，根呈先降低后增加的趋势，叶片则无显著变化。外施两种不同浓度的硝酸铵氮肥后，对12.5、25 g.L－1花椒叶水浸液处理的苜蓿化感作用有显著的缓解作用，表现在株高、生物量、光合作用等方面，大多达到与对照（0 g.L－1）未施氮肥无显著差异的水平，而对50 g.L－1水浸液处理的苜蓿幼苗，虽有一定的缓解作用，但这种作用均未达到与对照（0 g.L－1）未施氮肥时无显著差异的水平。 4． UV-B增强辐射处理花椒后，花椒的化感潜力显著增强。花椒叶片内UV-B吸收物质的含量和总酚含量均显著增加。 In recent years, with profound research on the reasons of continuous cropping obstacles, allelopathy received increasing attention to many scholars at home and abroad. Zanthoxy bungeanum as a Rutaceae plant is a high economic value species which gains early and uses widely. Zanthoxylum is an important economic crop in the arid valley of western Sichuan region, and its not even has received much concern for the continuous cropping obstacles. The systematic study of allelopathy of Zanthoxylum will contribute to the understanding and final settlement of this issue. The major allelopathic composition was separated through the extraction, chromatography combined with other methods. The impact on soil nutrient cycling was also studied through the addition of different concentrations of water extracts of Zanthoxylum. Furthermore, the effects of water extracts of Zanthoxylum leaves on alfalfa leaf physiological and biochemical indexes, photosynthesis, soil enzymes and nutrient uptake of nitrogen and the mitigation of allelopathy through using external fertilizer were studied to put forward scientific resolvent for Zanthoxylum continuous cropping obstacles .The response of allelopathic potential of Zanthoxylum to global change - UV-B enhanced radiation was studied . The main findings are as follows: 1. Through extraction with different polar organic solvents on concentrated water extract of Zanthoxylum leaf and then using column chromatography combined with detection of biological activity, one of the main allelopathic components- methoxy-phenol was isolated. The biological activity testing of the pure material of methoxy-phenol proved that it does have allelopathic potential. 2. Thirty days after treating soil with water extract of Zanthoxylum leaf, as compared with the control, the contents of soil nitrate, ammonium, nitrate plus ammonium nitrogen showed a trend of decrease with the increase of the concentration of water extract whereas the content of ammonium nitrogen showed a significant reduction, and the content of nitrate did not change significantly, the content of nitrate plus ammonium nitrogen also showed a significant (P <0.05) redction. The activity of soil urease and protease showed the same trend as the content of nitrate nitrogen plus ammonium nitrogen. With the increase in the concentration of water extract, the number of ammonification bacteria significantly reduced but nitrogen-fixing bacteria did not change significantly and there was a decreasing trend in the number of nitrifying bacteria and denitrifying bacteria. Sixty days after the treatment, with the increase in solution concentration of water extract of Zanthoxylum leaf, the content of nitrate、 ammonium nitrogen, nitrate plus ammonium nitrogen showed a similar change trend to 30 days’; the number of ammonification bacteria, nitrogen-fixing bacteria significantly reduced ; the number of nitrifying bacteria, denitrifying bacteria was still an downward trend; the activity of soil urease and protease showed the same trend as the 30th days’. Compared to the results of the 30th days’, the content of nitrate, ammonium, nitrate plus ammonium nitrogen showed a decrease trend between the treatment of same concentration, but there was no significant difference except the treatment of 25g.L-1 between the same concentration; the activity of soil urease showed enhanced trend except the treatment of 12.5 g.L-1; the activity of protease increased to varying degrees; the number of ammonification bacteria、 nitrifying bacteria and denitrifying bacteria were growing while nitrogen-fixing bacteria reduced.. 3. With the increase of the concentration of water extract of Zanthoxylum leaf, the water extract significantly inhibited the root length, aboveground biomass, content of chlorophyll and soluble protein in leaf and net photosynthetic rate. The activity of four antioxidant enzymes (POD, SOD, CAT, APX) reduced with the increase in concentration of the water extract but the content of MDA increased. The activity of enzymes related to primary nitrogen assimilation such nitrate reductase (NR), glutamyl synthetase (GS), glutamate dehydrogenase (GDH) were subject to different degrees with an increase in the concentration of water extracts. In general, the activity of nitrate reductase, glutamyl synthetase were inhibited, while change in the activity of glutamate dehydrogenase was more complex. The activity of glutamate dehydrogenase in leaf was first reduced and then increase,but did not change significantly in root. After using two external different concentrations of nitrogen fertilizer, there was a significant mitigation in inhibiton in plant height, biomass, photosynthesis, etc. in the treatment of 12.5,25 gL-1 of water extract of Zanthoxylum leaf, and most of these indexes showed no significant difference with the control (0 g.L-1, no external fertilizer was added) .Although there showed a certain degree of ease in the treatment of 50 g.L-1 , there was still a significant difference compared with the control (0 gL-1) in which no external fertilizer was used. 4.The allelopathic potential of Zanthoxylum positively responded to enhanced UV-B significantly. The content of UV-B absorbing compounds and the total phenol also significant increased.

Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.

Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.

Classificacao taxonomica, baseada na caracterizacao molecular, das estirpes de rizobio recomendadas para a culturas da soja e do feijoeiro.

As culturas da soja [Glycine max (L.) Merril] e do feijoeiro (Phaseolus vulgaris) sao de grande importancia economica e social para o Brasil e ambas podem ser capazes de suprir suas necessidades do nutriente nitrogenio pela simbiose com bacterias da familia Rhizobiaceae. Para garantir a maximizacao do processo biologico, porem, deve-se proceder a inoculacao das sementes com estirpes de rizobio eficientes e competitivas, recomendadas pela pesquisa. No Brasil, sao comercializadas, anualmente, cerca de 13 milhoes de doses de inoculantes, sendo 99% destinadas a cultura da soja. Neste trabalho, determinou-se a posicao taxonomica das estirpes de rizobio recomendadas comercialmente para as duas culturas por duas tecnicas, o RFLP-PCR da regiao correspondente ao 16S rRNA (regiao conservada entre bacterias mas suficientemente variavel e carregando informacoes que permitem a determinacao das relacoes filogeneticas entre bacterias) e o sequenciamento parcial dos genes desta regiao. O sequenciamento parcial permitiu definir que duas das estirpes recomendadas para a cultura da soja, SEMIA 587 e SEMIA 5019, pertencem a especie Bradyrhizobium elkanii e as duas outras, SEMIA 5079 e SEMIA 5080, a especie B. japonicum. As duas estirpes recomendadas para a cultura do feijoeiro, SEMIA 4077 e SEMIA 4080, pertencem a especie Rhizobium tropici. As sequencias obtidas para essas estirpes foram depositadas no banco mundial de genes.

Un medio de cultivo exento de nitrógeno combinado para el desarrollo de diferentes especies bacterianas fijadoras de N2

p.15-18

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.

Efeitos da toxicidade por metais pesados em Rhizobium leguminosarum bv. trifolii: diversidade e resposta de população isoladas de uma mina de chumbo

Nas últimas décadas verificou-se um aumento da contaminação dos solos com metais pesados resultante de processos antropogénicos. As descargas de efluentes industriais, a actividade mineira e a aplicação de lamas residuais e de fertilizantes são as principais fontes de metais pesados. Em certas regiões, a acumulação destes elementos nos solos tem atingido níveis preocupantes para o equilíbrio dos ecossistemas. Vários estudos têm demonstrado que os metais influenciam os microrganismos afectando adversamente o seu crescimento, morfologia e actividades bioquímicas resultando num decréscimo da biomassa e diversidade. Entre os microrganismos do solo, as bactérias pertencentes ao género Rhizobium têm um elevado interesse científico, económico e ecológico devido à sua capacidade para fixar azoto. Deste modo, o trabalho desenvolvido ao longo desta tese incidiu sobre o efeito da toxicidade imposta pelos metais nas bactérias fixadoras de azoto, em particular em Rhizobium leguminosarum bv. trifolii e teve como principais objectivos: determinar o efeito dos metais pesados na sobrevivência e na capacidade de fixar azoto dos isolados de rizóbio; avaliar a influência dos metais na diversidade das populações de rizóbio isoladas de solos contaminados; determinar os níveis de tolerância do rizóbio a diferentes metais e analisar a resposta ao stresse oxidativo imposto pelo cádmio. A Mina do Braçal foi o local de estudo escolhido uma vez que os seus solos estão muito contaminados com metais em resultado da extracção de minério durante mais de 100 anos. Foram escolhidos 3 solos com diferentes graus de contaminação, o solo BC com concentrações reduzidas de metais, escolhido por estar numa zona já fora da mina e designado por solo controlo e os solos BD e BA considerados medianamente e muito contaminados, respectivamente. O Pb e o Cd foram os metais predominantes nestes solos, assim como o metalóide As, cujas concentrações ultrapassaram largamente os limites previstos na lei. Sendo as enzimas do solo boas indicadoras da qualidade do mesmo, foi determinada a actividade de algumas como a desidrogenase (DHA) e a catalase (CAT). Ambas as enzimas correlacionaramse negativamente com as concentrações de metais nos solos. A dimensão das populações indígenas de rizóbio nos solos contaminados (BD e BA) foi bastante baixa, 9,1 bactérias g-1 de solo e 7,3 bactérias g-1 de solo, respectivamente, quando em comparação com a população do solo BC (4,24x104 bactérias g-1 de solo). Estes resultados parecem estar relacionados com o elevado conteúdo em metais e com o pH ácido dos solos. A capacidade simbiótica também foi afectada pela presença de metais, uma vez que os isolados originários do solo BD mostraram menor capacidade em fixar azoto do que os isolados do solo controlo. A diversidade das populações de rizóbio foi determinada com recurso à análise dos perfis de plasmídeos, perfis de REP e ERIC-PCR de DNA genómico e perfis de proteínas e lipopolissacarídeos. No conjunto dos 35 isolados analisados foram identificados 11 plasmídeos com pesos moleculares entre 669 kb e 56 kb. Embora a incidência de plasmídeos tenha sido superior nos isolados do solo BC verificou-se maior diversidade plasmídica na população isolada do solo BD. Resultados similares foram obtidos com os perfis de REP e ERIC-PCR e perfis de proteínas, que indicaram maior diversidade nas populações dos solos contaminados (BD e BA), contrariamente ao verificado por outros autores. O grau de tolerância aos metais pesados e ao arsénio dos vários isolados testados dependeu do metal e do local de origem. No geral, os isolados do solo BD mostraram maior tolerância aos metais do que os isolados do solo controlo, o que está de acordo com o esperado uma vez que geralmente as populações dos locais contaminados são mais tolerantes. Contudo, os isolados do local mais contaminado (BA) foram muito tolerantes apenas ao chumbo mostrando-se sensíveis aos restantes metais. A inoculação dos solos BC, BD e BA após irradiação com estirpes seleccionadas de rizóbio permitiu avaliar a sua sobrevivência ao longo de 12 meses em condições mais realistas. Verificou-se que após um decréscimo inicial, os isolados inoculados no solo BC conseguiram recuperar a dimensão das suas populações para números similares aos inicialmente introduzidos, contrariamente ao verificado no solo BD onde o número de rizóbios decresceu ao longo dos 12 meses. As condições adversas do solo BA apenas permitiram a sobrevivência de 4 isolados até aos 3 meses e apenas dois deles conseguiram sobreviver após 12 meses, designadamente C 3-1 e A 17-3. Estes isolados possuem um plasmídeo de 669 kb que poderá estar na base da sobrevivência destas estirpes. Por outro lado, o último isolado é originário do solo contaminado e por isso estará também mais adaptado a sobreviver às elevadas concentrações de Pb existentes no solo BA. Por fim, constatou-se que o cádmio, um dos metais presente em concentrações mais elevadas nos solos em estudo, é um indutor de stresse oxidativo nos isolados de rizóbio menos tolerantes o que foi confirmado pelo aumento de ROS e danos celulares ao nível dos lípidos.

Contribution des légumineuses, des champignons endophytes et mycorhiziens dans la nutrition azotée des prairies indigènes semi-arides

Les prairies indigènes présentent une source importante d'alimentation pour le pâturage du bétail dans les prairies Canadiennes semi-arides. L'addition de légumineuses fixatrices d'azote et de phosphore dans les prairies indigènes peut améliorer la productivité et la valeur nutritive de fourrage. Ces pratiques peuvent induire des modifications de la structure et de la diversité des communautés fongiques du sol, ce qui peut en retour avoir un impact sur la production et le contenu nutritionnel du fourrage. L’objectif de cette étude était de développer un système de pâturage à bas niveau d’intrants, productif, autonome et durable. À court terme, nous voulions 1) déterminer l'effet des légumineuses (Medicago sativa, une légumineuse cultivée ou Dalea purpurea, une légumineuse indigène) et la fertilité en phosphore du sol sur la productivité et la valeur nutritive des graminées indigènes, comparées avec celles de la graminée introduite Bromus biebersteinii en mélange avec le M. sativa, 2) identifier l'effet de ces pratiques sur la diversité et la structure des communautés des champignons mycorhiziens à arbuscules (CMA) et des champignons totaux, 3) identifier l'effet des légumineuses et des CMA sur les interactions compétitives entre les graminées de saison fraîche et les graminées de saison chaude. Les expériences menées au champ ont montré que M. sativa améliorait les teneurs en azote et en phosphore des graminées indigènes au début de l'été, ainsi que la teneur en azote de la graminée de saison chaude Bouteloua gracilis à la fin de l'été de l'année sèche 2009. Par contre, la fertilité en phosphore du sol n'ait pas affecté la productivité des plantes. D'autre part, l'inclusion des légumineuses augmentait la diversité des CMA dans le mélange de graminées indigènes. Cette modification présentait des corrélations positives avec la productivité et la quantité totale d'azote chez le M. sativa et avec la teneur en phosphore des graminées indigènes, au début de l'été. La structure des communautés de champignons totaux était influencée par l'interaction entre le mélange des espèces et la fertilité en phosphore du sol seulement en 2008 (année humide). Cet effet pourrait être lié en partie avec la productivité des plantes et l'humidité du sol. Les expériences menées en chambre de culture ont montré que les CMA peuvent favoriser la productivité des graminées de saison chaude au détriment des graminées de saison fraîche. En effet, Glomus cubense augmentait la productivité de la graminée de saison chaude B. gracilis, en présence de M. sativa. Cet effet pourrait être associé à l’effet négatif du G. cubense sur la fixation de l’azote par le M. sativa et à la diminution de l’efficacité d’utilisation de l'azote de certaines graminées de saison fraîche résultant en une augmentation de la disponibilité de l'azote pour B. gracilis. Par contre, le Glomus sp. augmentait la biomasse de Schizachyrium scoparium, autre graminée de saison chaude, en absence de légumineuse. Ce phénomène pourrait être attribuable à une amélioration de l’efficacité d’utilisation du P de cette graminée. En conclusion, mes travaux de recherche ont montré que la légumineuse cultivée M. sativa peut améliorer la valeur nutritive des graminées indigènes au début de l'été ainsi que celle de la graminée de saison chaude B. gracilis, dans des conditions de sécheresse sévère de la fin de l'été. De plus, l'addition de M. sativa dans le mélange de graminées indigènes peut contribuer à augmenter le nombre des espèces bénéfiques des CMA pour la production et la nutrition du fourrage au début de l'été.

Occurrence and Distribution of a Diatom-Diazotrophic Cyanobacteria Association during a Trichodesmium Bloom in the Southeastern Arabian Sea

Symbiotic diatom-diazotrophic cyanobacteria association (DDA) of Rhizosolenia hebetata and Rhizosolenia formosa with endosymbiotic cyanobacteria Richelia intracellularis was noticed and documented for the first time during a bloom of the cyanobacterium Trichodesmium erythraeum in the oligotrophic shelf waters along Kochi and Mangalore transects, southeastern Arabian Sea (SEAS), during spring intermonsoon (April 2012). Although the host is frequently seen, the symbiont is rarely reported in the Indian EEZ. The presence of nitrogen-fixing symbiotic association of Rhizosolenia-Richelia along with Trichodesmium erythraeum highlights the significance of DDAs on the nutrient and energy budgets of phytoplankton in the oligotrophic environments of the Arabian Sea during spring intermonsoon

Calcium carbonate solubilization through H-proton release from some legumes grown in calcareous saline-sodic soils

Increasing levels of CO2 and H+ proton in the rhizosphere from some legumes may play an important role in calcite dissolution of calcareous salt affected soils. Soils planted with white and brown varieties of cowpea (Vigna unguiculata L.) and hyacinth bean (Dolichos lablab L.) relying on either fertilizer N (KNO3) or N-fixation were compared against soils to which gypsum was applied and a control without plants and gypsum application to study the possibility of Ca2+ release from calcite and Na+ leaching. As compared to plants relying on inorganic N, leachates from all pore volumes (0·5, 1·0, 1·5, 2·0 pore volume) in lysimeters planted with N-fixing hyacinth bean contained significantly higher concentrations of HCO with lower concentrations from lysimeters planted with white cowpea relying on N-fixation. However, the lowest concentrations of HCO were recorded in the gypsum and control treatments. In initial leaching, lysimeters planted with N-fixing plants maintained similar leachate Ca2+ and Na+ concentrations compared to gypsum amended soils. However, gypsum amended soils were found to have a prolonged positive effect on Na+ removal. It might be concluded that some legumes that are known to fix N in calcareous salt affected soils may be an alternative ameliorant to the extremely expensive gypsum through calcite solubilization and a consequent release of Ca2+.