Effect of Azolla on flooded rice production

O experimento foi conduzido com arroz irrigado (Oryza sativa L.), em campo, na Estação Experimental Palmital, do Centro Nacional de Pesquisa de Arroz e Feijão - CNPAF, em Goiânia, GO. Os tratamentos testados foram: testemunha, Azolla, 60 kg/ha de N, e 60 kg/ha de N + Azolla. Com a consorciação de Azolla, houve um aumento de, aproximadamente, 15% na produção em relação à testemunha. Os tratamentos de 60 kg/ha de N e 60 kg/ha de N + Azolla apresentaram aumentos de produção de 46 e 56%, respectivamente, em comparação com a testemunha. Estes resultados preliminares mostram que existe possibilidade de uso de Azolla como fonte de nitrogênio para a cultura de arroz irrigado.A field experiment with Azolla and flooded rice (Oryza sativa L.) was conducted at the Estação Experimental  Palmital of the Centro Nacional de Pesquisa de Arroz e Feijão, in Goiânia - GO, Brazil. The treatments included: control (no added nitrogen), Azolla, 60 kg of N/ha, and Azolla + 60 kg of N/ha. An increase of 15% in the grain yield of rice was observed with the Azolla treatment. Additions of 60 kg N/ha and Azolla + 60 kg N/ha resulted in rice grain yield increase of 46 and 56%, respectively, in relation to the control. The results indicate that it is possible to use Azolla as a source of N for flooded rice culture

The effect of Azolla species on growth response of flooded rice.

Realizou-se um experimento em casa de vegetação com o objetivo de verificar o efeito de sete diferentes espécies deAzolla no desenvolvimento de arroz inundado quando empregada como fonte alternativa de nitrogênio (N), incorporada ao solo ou cultivada simultaneamente com o arroz. Os tratamentos utilizados foram: sem adição de fonte de N (testemunha), 30kg/ha de N, 60kg/ha de N, incorporação e cultivo simultâneo da Azolla. O melhor desenvolvimento do arroz foi alcançado com a aplicação de 30 e 60kg/ha de N e incorporação de Azolla no solo. As maiores contribuições foram dadas pela A. caroliniana, A.filiculoides, A. mexicana e A. microphylla. Os resultados indicam a necessidade de selecionar espécies de Azolla que ofereçam maior potencial de utilização do N-Azolla pelo arroz, bem como confirmam os benefícios desse adubo verde no desenvolvimento dessa cultura.An experiment was carried out under greenhouse conditions to assess the effect of seven different Azolla species on the growth of flooded rice using Azolla as an alternative source of nitrogen, incorporated into the soil of grown in dual culture. The treatments used were: no added nitrogen (control), 30 and 60 kg/ha of N, Azolla incorporated into the soil and grown in dual culture. Rice development was better using 30 and 60 kg/ha of N and when Azolla was incorporated into the soil. The A. caroliniana, A. filiciloides, A. mexicana and A. microphyllashowed good response. The results suggest the necessity of selecting Azolla species with a high potential as nitrogen source to flooded rice, as well as confirm their value as green manure for this culture

Hepatotoxin microcystin-LR extraction optimization

Several cyanobacterial genera produce toxic secondary metabolites, the most well-known of which are the hepatotoxic microcystins (MCYSTs). Microcystin analyses in drinking water are a requirement of the Health Ministry (Regulation 518/2004) in Brazil, but this regulation does not establish which extraction and analytical method should be used; toxin quantification is usually carried out by ELISA (enzyme-linked immunosorbent assay) or HPLC (high performance liquid chromatography), the efficiency of which depends on the extraction method used. In this work a simple, fast and cheap method of extraction was developed for the isolation and identification of MCYSTs. For this, the strain Microcystis aeruginosa NPLJ-4, reported to be a MCYST-LR producer, was selected. Eight different treatments were tested to determine the best MCYST extraction. Samples were applied in LC-MS (liquid chromatography-mass spectrometry), ELISA and Q-TOF (quadrupole time-of-flight). The most efficient extraction was achieved by sonicating samples diluted in water. The proposed method permits rapid sample processing, and establishes an extraction method for both the analysis and identification of MCYST-LR and other variants.Vários gêneros de cianobactérias produzem metabólitos secundários tóxicos, entre eles as hepatotoxinas microcistinas. A análise de microcistinas em águas para abastecimento humano é uma exigência do Ministério da Saúde (Portaria 518/2004), mas essa portaria ainda não estabelece o método de extração e análise a serem usados e a quantificação da toxina é comumente realizada por ELISA ("enzyme-linked immunosorbent assay") ou HPLC (cromatografia líquida de alta eficiência), cuja eficiência depende do método de extração utilizado. Neste trabalho foi desenvolvido um método simples, rápido e barato de extração para o isolamento e identificação de microcistinas. Para isso, selecionou-se a linhagem Microcystis aeruginosa NPLJ-4 descrita como produtora de microcistina-LR. Oito diferentes tratamentos foram testados para determinar a melhor extração da toxina. As amostras foram analisadas por LC-MS (cromatografia líquida acoplada a espectrometria de massas), ELISA e Q-TOF ("quadrupole time-of-flight"). Os resultados mostraram que a melhor extração foi a que usou sonicação das amostras diluídas em água. O método proposto permite o processamento rápido das amostras e estabelece um método de extração para análise e identificação de microcistina-LR e outras variantes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Convergent evolution of [D-Leucine(1)] microcystin-LR in taxonomically disparate cyanobacteria

BACKGROUND: Many important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly. RESULTS: Here, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu(1)] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA(2) adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA(2) adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu(1)] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met(1)] microcystin-LR. CONCLUSIONS: Phylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu(1)] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations.Peer reviewe

Composto antifúngico produzido pelo endófito de mandioca Bacillus pumilus MAIIIM4a

Na busca de novos organismos e novos metabólitos secundários, um estudo foi conduzido visando avaliar a diversidade de bactérias endofíticas de etnovariedades de mandioca cultivadas por tribos indígenas da Amazônia brasileira e também para estudar metabólitos secundários produzidos por Bacillus pumilus. Sessenta e sete bactérias endofíticas de mandioca foram identificadas através do seqüenciamento do gene 16S rRNA e por meio da análise de ácidos graxos (FAME). Essas análises revelaram que 25% de todos os endofíticos pertenciam ao gênero Bacillus. O isolado Bacillus pumilus MAIIIM4a apresentou forte ação inibitória contra os fitopatógenos Rhizoctonia solani, Pythium aphanidermatum e Sclerotium rolfsii. Os metabólitos secundários deste isolado foram extraídos do sobrenadante usando-se hexano, diclorometano e acetato de etila. Esses extratos foram utilizados nas análises de bioautografia e LC-MS, as quais permitiram a identificação do composto pumilacidina, um antifúngico produzido por B. pumilus MAIIIM4a. A localização das bactérias endofíticas foi confirmada examinando-se o tecido celular da mandioca através de microscopia eletrônica.In the search for new organisms and new secondary metabolites, a study was conducted to evaluate the diversity of endophytic bacteria from ethnovarieties of cassava cultivated by Brazilian Amazon Indian tribes and also to study the secondary metabolites produced by a Bacillus pumilus strain. Sixty seven cassava endophytic bacteria were subjected to 16S rRNA sequencing and FAME analysis. The bacterial profile revealed that 25% of all endophytic isolates belonged to the genus Bacillus. The isolate B. pumilus MAIIIM4a showed a strong inhibitory activity against the fungi Rhizoctonia solani, Pythium aphanidermatum and Sclerotium rolfsii. Secondary metabolites of this strain were extracted using hexane, dichloromethane and ethyl acetate. Extracts were subjected to bioautography and LC/MS analysis, which allowed the identification of pumilacidin, an antifungal compound produced by B. pumilus MAIIIM4a. The bacterial endophytic localization was confirmed by cassava cell tissue examination using scanning electron microscopy

Biological nitrogen fixation associated with flooded rice varieties

A fixação biológica de N2 (FBN) associada com arroz cultivado em vasos contendo solo alagado marcado com 15N foi determinada em condições in situ durante dois anos subsequentes. A técnica da diluição isotópica, o balanço de nitrogênio e a atividade da redução de acetileno (ARA) foram usados para avaliar a contribuição da fixação de nitrogênio em 15 cultivares de arroz. Isolamentos de algumas bactérias heterotróficas fixadoras de nitrogênio foram incluídos para correlacionar resultados positivos (15N, balanço de N, ARA) com esses organismos - da rizosfera, rizoplano e hitosfera de arroz. A diluição de 15N e nitrogênio total nas plantas indicaram diferenças significativas entre os cultivares. A contribuição da FBN em plantas foi calculada a partir de um cultivar com baixo potencial de fixação de N2 (CNA-3314), que apresentou o maior conteúdo de 15N na planta. Estimativas da FBN pela diluição isotópica de 15N mostraram valores entre 1,5 a 16,5% do total de N na planta no primeiro ano e 9,2 a 23,9% no segundo ano. IAC-4440, HUA-CHOU, BR-IRGA-409, IAC-1278 e BR-IRGA-410 mostraram superioridade em FBN, contribuindo com quantidades de 193, 148, 133, 130 e 123 mg N.vaso-1, respectivamente, no primeiro ano e 101, 213, 168, 108 e 140 mg N.vaso-1 no segundo ano. A ARA no período da emergência da panícula foi alta, variando de 0,6 a 1,6 µmol C2H4.plant-1.8h-1. Bactérias heterotróficas foram encontradas em associação com todos os cultivares, incluindo Azospirillum, Pseudomonas e Enterobacter. A menor contagem através do método de NMP foi obtida no cv. CNA-3314, que também mostrou ser mais restrito para as bactérias estudadas.Biological N2 Fixation (BNF) associated with rice grown in pots containing 15N-labelled soil in shallow water was determined under in situ conditions, during two subsequent cropping. 15N dilution technique, total nitrogen balance as well as acetylene reduction activity (ARA) were used to assess the contribution of nitrogen fixation to 15 rice varieties. Isolation of some heterotrophic N2-fixing bacteria was also included to correlate positive data (15N, N-balance, ARA) with these organisms - from rhizosphere, rhizoplane and hitosphere of rice. 15N dilution and total nitrogen indicated significant differences in N2-fixing ability among the varieties. Contribution of BNF to nitrogen in plants was calculated from a poor N-fixing rice variety (CNA-3314), which showed the highest 15N content in the plant. Estimates of BNF by N isotope dilution gave values from 1.5 to 16.5% of total N in the first year and 9.2 to 23.9% in the second year. IAC-4440, HUA-CHOU, BR-IRGA-409, IAC-1278, and BR-IRGA-410 varieties were superior in BNF contributing with total amounts of 193, 148, 133, 130 and 123 mg N.pot-1, respectively, in the first year and 101, 213, 168, 108 and 140 mg N.pot-1 in the second year. ARA at heading stage was high ranging from 0.6 to 1.6 µmol C2H4.plant-1.8h-1. Heterotrophic bacteria were found in association with all varieties, including Azospirillum, Pseudomonas and Enterobacter. The least MPN count was obtained with var. CNA-3314 which also showed more restriction to bacterial growth

Quorum Sensing Detected By Atomic Force Microscopy Imaging Of Corrals Surrounding Multicellular Arrangement Of Bacteria.

Connectivity of the glycocalyx covering of small communities of Acidithiobacillus ferrooxidans bacteria deposited on hydrophilic mica plates was imaged by atomic force microscopy. When part of the coverage was removed by water rinsing, an insoluble structure formed by corrals surrounding each individual bacterium was observed. A collective ring structure with clustered bacteria (>or=3) was observed, which indicates that the bacteria perceived the neighborhood in order to grow a protective structure that results in smaller production of exopolysaccharides material. The most surprising aspect of these collective corral structures was that they occur at a low bacterial cell density. The deposited layers were also analyzed by confocal Raman microscopy and shown to contain polysaccharides, protein, and glucoronic acid.71112-

Application of cyanobacteria in radioactive waste

The extremely toxic radioactive wastes whose radioactivity persists for thousands of years are accumulated through nuclear power plants, mining companies, industries, and research centers, among others. However, the lack of technology for the treatment and removal of radioactivity from radioactive waste becomes a bottleneck for research. Currently, there are temporary solutions, and the radioactive waste continues to increase. Given this scenario it is necessary to seek alternatives for the treatment of radioactive waste with efficiency and low cost, without polluting the environment, and that is not harmful to human health. One way to reduce this type of pollution is to use microorganisms, especially cyanobacteria, which comprise one of the largest, most ecologically diverse, successful, and important group of bacteria on Earth. These bacteria have a great ability to remove pollutants, such as heavy metals, textile dyes, pesticides, etc., but their role in the degradation of recalcitrant and radioactive compounds is still scarce. The present work aimed to investigate the potential of cyanobacteria isolated from different environments to remove radiolabeled molecules from radioactive waste (containing 14C radioisotopes). Seven cyanobacteria, Synechococcus sp. CENA136 (isolated from the Mangrove of Cardoso Island), Phormidium autumnale UTEX 1580 (collection of UTEX cultures - fish aquarium), Nostoc sp CENA420 (Antarctica), Limnothrix sp. CENA458 (isolated from the reservoir), Oscillatoria acuminata CENA525 (isolated from the Pantanal), Nodularia sp CENA215 (isolated from Caatinga), and Trichormus SP UFV-56 (isolated from the Federal University of Viçosa-MG) were used in the present study. Cyanobacteria were maintained in Z8, BG-11, and SWBG-11 liquid culture medium under constant fluorescent lighting of 40 µmol photons·m-2·s-1 and a controlled temperature of 23 ± 1°C. Radioactive waste containing several 14C organic molecules, solubilized in organic solvents was used. The inoculums were obtained from 50 mL of culture medium. The radioactive waste was added at concentrations of 0, 5, 10 and 15%. To investigate the removal of the radioactivity present in the waste, analysis was performed by Liquid Scintillation Counting (LSC) in the beginning and final cultivation and HPLC (coupled to a flow scintillation analyzer) in order to evaluate the profile of radioactive molecules present in the waste. Intracellularly accumulated radioactivity was also assessed by LSC after cell disruption. The ability to accumulate radioactive molecules intracellularly was observed in all cyanobacteria, but Nostoc sp CENA420 accumulated 43% and Trichormus sp UFV-56 accumulated 68%, both cultivated with 15% of radioactive waste. These results showed that the cyanobacteria Nostoc sp CENA420 and Trichormus sp UFV-56 consumed 87 and 86% of radioactive molecules, respectively, being of great potential for the removal of radiolabeled radioactive waste molecules. Research using cyanobacteria to remove radiolabeled molecules from radioactive waste is still at an early stage but is a promising alternative to biological treatment

Estimating genetic structure and diversity of cyanobacterial communities in Atlantic forest phyllosphere

Cyanobacterial communities on the phyllosphere of four plant species inhabiting the endangered Brazilian Atlantic Forest biome was evaluated using cultivation-independent molecular approaches. Total genomic DNA was extracted from cells detached from leaves surface of Euterpe edulis, Guapira opposita, Garcinia gardneriana and Merostachys neesii sampled in two Brazilian Atlantic Forest locations along an elevational gradient, i.e., lowland and montane forest. The DNA fingerprinting method PCR-DGGE revealed that the cyanobacterial phyllosphere community structures were mainly influenced by the plant species, with a low effect of the geographical location of the plant. The 16S rRNA gene sequences obtained by clone libraries showed a predominance of nitrogen-fixing cyanobacteria of the order Nostocales, even though the majority of retrieved OTUs (~60% of the sequences) showed similarity only to uncultured cyanobacteria phylotypes. The leaf surfaces of Guapira opposita had the highest richness and diversity of cyanobacteria, whereas the M. neesii (bamboo) had the largest number of copies of cyanobacterial 16S rRNA gene per cm2 of leaf. This study investigated cyanobacteria diversity and its distribution pattern in Atlantic forest phyllosphere. The results indicated that plant species is the main driver of cyanobacteria community assemblage in the phyllosphere, and that these communities are comprised by a high diversity of cyanobacterial taxa to be discovered.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Heterocyte-forming cyanobacteria from Brazilian saline-alkaline lakes

Studies investigating the diversity of cyanobacteria from tropical environments are scarce, especially those devoted to the isolation and molecular characterization of the isolated strains. Among the Brazilian biomes, Pantanal has mainly been examined through microscopic observation of environmental samples, resulting in lists of morphotypes without any genetic information. Recently, two studies were conducted evaluating the morphologic and genetic diversity of cultured non-heterocytous cyanobacteria in this biome, which resulted in the separation and description of two novel genera. In order to complement the diversity of cultured cyanobacteria from saline-alkaline lakes in Pantanal, the present study is dedicated to the examination of cultured nitrogen-fixing heterocytous cyanobacteria from this extreme and underexplored environment. A total of fourteen cyanobacterial strains were isolated. According to morphological examination they belong to the order Nostocales and to the subsections IV.I and IV.II, according to the International Code of Nomenclature for Algae, Fungi and Plants and the Bergey’s Manual of Systematic Bacteriology, respectively. Phylogenetic evaluation of their 16S rRNA gene sequences resulted in the formation of five clusters. Among them, one is clearly related to the genus Anabaenopsis whilst the remaining clusters may represent new genetic lineages. These novel sequences aid in the delimitation of problematic groups, especially those containing sequences belonging to mixed genera. The application of both morphologic and phylogenetic studies has proven to be an important tool in resolving problematic groups in cyanobacteria systematics. This strategy is essential in order to detect novel cyanobacteria genera from other tropical environments