Pienarūgščių bakterijų paieška ir jų identifikavimas migruojančių didžiųjų ančių (Anas platyrhynchos) žarnyne naudojant dalinių 16S rRNR geno sekų analizę ir kultivavimu paremtus metodus Klaidas VARNA Vilniaus Universiteto Ekologijos Institutas, Hidrobiontų Ekologijos ir Fiziologijos Laboratorija bei Populiacinės Genetikos Laboratorija, Akademijos-2, Vilnius-21, 08412, Lietuva. Šiame tyrime pavasarinių ir rudeninių didžiųjų ančių (Anas platyrhynchos) migrantų iš Nemuno deltos virškinamojo trakto pieno rūgšties bakterijų įvairovė buvo ištirta naudojant molekulinius metodus (polimerazės grandininės reakcijos amplifikacija ir dalinių 16S rRNR geno sekų sekvenavimas) ir kultivavimu paremtus metodus. Migruojančių didžiųjų ančių (Anas platyrhynchos) pieno rūgšties bakterijų paieška buvo atlikta pirmą kartą. Rudeniniai didžiųjų ančių migrantai plonojo žarnyno sienelėse (1.2×107 iki 2.1×107 k.f.v./g) ir jų turinyje (nuo 3.4×107 iki 1.1×108 k.f.v./g) turi didesnį pieno rūgšties bakterijų skaičių nei pavasariniai migrantai (atitinkamai nuo 3.2×106 iki 4.8×106 k.f.v./g ir nuo 1.0×107 iki 2.2×107 k.f.v./g). Tiek rudeninių tiek ir pavasarinių didžiųjų ančių migrantų plonojo žarnyno sienelėse ir jų turinyje dominavo kokinės pieno rūgšties bakterijų formos (atitinkamai 65% ir 83.5% bei 81.4% ir 91.6%), o lazdelių buvo mažiau (atitinkamai 35% ir 16.5% bei 18.6% ir 8.4%). Manoma, kad minėtus skirtumus įtakoja keli veiksniai: ilgai trunkanti migracija, perėjimo periodas, skirtingas maistas ir... [toliau žr. visą tekstą] / Identification of lactic acid bacteria in the migrant mallard ducks Anas platyrhynchos intestinal tract by partial 16S rRNA gene sequence analysis and using culture-based techniques Klaidas VARNA Institute of Ecology of Vilnius University, Laboratory of Hydrobionts Ecology and Physiology, Laboratory of Population Genetics, Akademijos-2, Vilnius-21, 08412, Lithuania. In this study the lactic acid bacteria diversity of the intestinal tract content of the vernal and autumnal migrant mallard ducks (Anas platyrhynchos) from Nemuno delta has been investigated by molecular methods: polymerase chain reaction amplification and sequencing of partial 16S rRNA genes and using culture-based techniques. The investigation of the lactic acid bacteria of the migrant mallard ducks has been performed the first time. Autumnal migrant mallard ducks in the small intestine walls (from 1.2×107 until 2.1×107 c.f.u./g) and in their content (from 3.4×107 until 1.1×108 c.f.u./g have the greatest number of the lactic acid bacteria then vernal migrants (respectively from 3.2×106 until 4.8×106 c.f.u./g and from 1.0×107 until 2.2×107 c.f.u./g). In the small intestine walls and in their content of the autumnal and vernal migrant mallard ducks, dominated cocci-shaped lactic acid bacteria (respectively 65% and 83.5%, 81.4% and 91.6%), whereas rod-shaped was under (respectively 35% and 16.5%, 18.6% and 8.4%). Supposedly, that these defferences determine some factors: a long migration, period of incubate... [to full text]

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Coral disease represents one of the many challenges facing coral reefs, and is a contributing factor to the overall decline in coral reef health worldwide. An increase in disease frequency, outbreaks and the emergence of new diseases has fuelled much concern over the impact of coral diseases and subsequently prompted research into their possible causes. Our understanding of putative coral pathogens has lagged behind the emergence of coral disease as a major threat to the health of coral reefs. The Great Barrier Reef (GBR) is the largest contiguous reef in the world, and is still regarded as one the healthiest and best managed coral reef ecosystems in existence today. Despite this, the frequency of coral disease has begun to increase sharply over the past decade, prompting researchers to focus on the aetiology, causal factors and ecological impact of coral disease within the Great Barrier Reef Marine Park (GBRMP). This PhD thesis focused on two distinct disease elements: brown band (BrB) and white syndrome (WS). These two diseases affect corals within the GBRMP, yet their microbiology and ecology is largely unknown. The research project investigated the microbiology and ecology of WS and BrB affecting acroporids, using culture-dependent and independent methods to characterise the microbial community associated with healthy and diseased corals and identify putative coral pathogens. The lifecycle and diurnal cycles of BrB ciliates were also explored to gain a greater understanding of the effect of these ciliates on coral health. Ecological surveys were carried out at Heron Island sites commonly used for the collection of corals to ascertain the prevalence and significance of these diseases in the context of laboratory results. Surveys at five sites revealed a mean prevalence of 8.11% of tabular acroporids affected by WS, which is consistent with previous studies. BrB revealed a much lower prevalence of less than 0.04%. Bacterial 16S rRNA gene clone libraries were constructed from Acropora hyacinthus samples derived from a healthy control colony, and a healthy section and lesion border of a WS affected colony. Distinct shifts in the microbial community and partitioning between the lesion border and healthy section of the diseased colony were observed. In addition, the healthy section of the diseased colony displayed a different microbial community to the control colony, supporting previous data that a microbial shift occurs preceding visible signs of infection. A number of bacteria from the healthy section of diseased coral shared close sequence affiliations to a number of Vibrio spp., including potentially pathogenic Vibrio species. Sequences retrieved from the lesion border of WS affected Acropora hyacinthus were dominated by Pseudoalteromonas spp., although these species have not been previously implicated in coral disease. The coral disease BrB is characterised by the presence of a brown ciliate band and these ciliates have been identified as a new species belonging to the class Oligohymenophorea, subclass Scuticociliatia. Within BrB-affected Acropora muricata, numerous filamentous, coccoid and rod bacteria were observed to be closely associated with the ciliate band, but absent in coral tissue adjacent to the typical brown band. It is unknown whether the bacteria associated with the mass of ciliates are the primary pathogens, a food source for the ciliates or simply opportunistic pathogens. Several isolates retrieved from BrB corals were tested for their pathogenicity in controlled infection trials using Acropora muricata. The preliminary results identified at least two isolates of interest (CC1 and HB-8). However, the results of a replicated infection trial failed to conclusively identify the bacteria as the causative agents of this disease. The findings from the cross-infection trials and ecological surveys suggest that BrB is an infectious but not highly contagious coral disease. This study revealed important aspects of both WS and BrB that were previously unknown. The research carried out has built a greater understanding, and a platform for future research directed at understanding key processes involved in these coral diseases. This research has highlighted the need for ongoing infection trials in diseases, even when a pathogen has been identified. The discovery of possible key bacterial species involved in WS and BrB warrants further research aimed at understanding the mechanisms in which bacteria may affect the coral holobiont. In conclusion, this research has further supported the notion that corals are a complex community with bacterial, animal and protistan partners, which when disturbed may see one or several of the previous benign partners becoming pathogenic. In a rapidly changing climate, this conclusion is consistent with the idea that coral diseases are on the rise due to changing environmental circumstances disturbing the balance between these interdependent partners.

Submitted by Luciana de Cenço Correa Lacerda (lulacerda2@yahoo.com.br) on 2018-06-21T19:31:05Z No. of bitstreams: 1 TESE Luciana 21.06.18 PRONTA.pdf: 1525467 bytes, checksum: a6d6df3286d60500baf769d3035234e1 (MD5) / Approved for entry into archive by Neli Silvia Pereira null (nelisps@fcav.unesp.br) on 2018-06-25T18:40:29Z (GMT) No. of bitstreams: 1 lacerda_lcc_dr_jabo.pdf: 1525467 bytes, checksum: a6d6df3286d60500baf769d3035234e1 (MD5) / Made available in DSpace on 2018-06-25T18:40:29Z (GMT). No. of bitstreams: 1 lacerda_lcc_dr_jabo.pdf: 1525467 bytes, checksum: a6d6df3286d60500baf769d3035234e1 (MD5) Previous issue date: 2018-05-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Lesões na pele podem resultar em feridas que, dependendo do tempo de reparação tissular podem ser classificadas como agudas ou crônicas, sendo crônicas aquelas que não apresentaram cicatrização dentro do período de quatro semanas. A ferida é contaminada por diferentes espécies bacterianas, sendo o sistema imunológico da pele o responsável por impedir que tais contaminações evoluam para infecções. No entanto, muitas vezes o quadro infeccioso é instalado, havendo necessidade de tratamento com antimicrobianos. Tendo em vista que o mau uso de antimicrobianos provoca resistência a multidrogas em estirpes bacterianas potencialmente patogênicas, este trabalho teve como objetivo identificar as bactérias prevalentes em feridas agudas e crônicas de cães por meio de sequenciamento da região 16S rRNA e testar a sensibilidade dos isolados a diferentes antimicrobianos. Para tanto, foram amostradas 20 feridas, sendo cada uma de um cão atendido no Hospital Veterinário da UNESP, Câmpus de Jaboticabal. De cada ferida foram obtidos dez isolados, os quais foram selecionados para o sequenciamento de DNA por meio de comparação entre os perfis genéticos obtidos pelo emprego de marcador molecular randômico. Foram sequenciados 74 isolados identificados como pertencentes a oito gêneros de bactérias gram-negativas, Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, Enterobacter spp., Acinetobacter baumannii, Klebsiella spp., Kluyvera georgiana e Providencia stuartii, e três de gram-positivas, Enterococcus sp., Staphylococcus spp. e Bacillus spp. Casos de cães com feridas agudas ou crônicas, associadas a mais de um gênero bacteriano, foram de 85,7% e 30,7%, respectivamente. Isolados da espécie S. aureus apresentaram amplificação para quatro genes codificadores das enterotoxinas sea, seh, see e hlg, enquanto um isolado de B. cereus foi positivo para a presença dos genes hblA, hblC, hblD, nheA, nheB, nheC e entFM. Dois dos isolados de E. coli (6%) apresentaram o gene blaCTX-M-2 e provaram ser resistentes à cefotaxima, um antibiótico do grupo dos β-lactâmicos. Todos os isolados avaliados apresentaram resistência a pelo menos um dos antimicrobianos testados, sendo metronidazol, cefalexina e cefazolina, aqueles pelos quais os isolados mostraram maior resistência. Cinco pacientes que estavam sob antibioticoterapia no momento da coleta possuíam estirpes resistentes aos antimicrobianos pelos quais estavam sendo tratados. Tendo em vista que há uma grande diversidade bacteriana resistente à multidrogas colonizando feridas cutâneas agudas e crônicas de cães, a implantação de antibiogramas previamente à recomendação de antimicrobianos é prática imprescindível e deve ser implantada para preservação da saúde animal e, consequentemente, pública. / Skin lesions can result in cutaneous wounds that may be classified as acute or chronic depending on the period of time spent in tissue repairment. Wounds that have not healed within four weeks are generally classified as chronic. The wound is contaminated by different bacterial species and the immune system of the skin is responsible for preventing infections. Nonetheless, the infectious process is often developed and antimicrobial treatment become necessary. Considering that the misuse of antimicrobials provokes multidrug resistance in potentially pathogenic bacterial strains, this work aimed to identify prevalent bacteria in acute and chronic wounds of dogs by 16S rRNA sequencing and to test the sensitivity of the isolates to different antimicrobials. For that, 20 wounds were sampled, each one from a dog admitted at the Veterinary Hospital of UNESP, Jaboticabal, São Paulo, Brazil. From each wound ten isolates were obtained, which were selected for DNA sequencing through genetic profiles comparison by applying a random molecular marker. Seventy-five isolates were identified as belonging to eight Gram-negative bacteria genera, Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, Enterobacter spp., Acinetobacter baumannii, Klebsiella spp., Kluyvera georgiana and Providencia stuartii, and to three gram-positive bacteria genera, Enterococcus sp., Staphylococcus spp. and Bacillus spp. Cases of dogs with acute or chronic wounds associated to more than one bacterial genus were 85.7% and 30.7%, respectively. Staphylococcus aureus isolates presented amplification to four enterotoxin encoding the genes sea, seh, see and hlg, whereas a B. cereus isolate was positive for hblA, hblC, hblD, nheA, nheB, nheC and entFM genes. Two of the E. coli isolates (6%) presented the blaCTX-M-2 gene and proved to be resistant to cefotaxime, a β-lactam antibiotic. All isolates evaluated were resistant to at least one of the antimicrobials tested, being metronidazole, cephalexin and cefazolin the ones for those the isolates showed to be more resistant. Five patients undergoing antibiotic therapy at the same period of sampling presented resistants bacterial strains to the antibiotics by which the patients were being treated. Considering that there is great multidrug resistant bacterial diversity colonizing acute and chronic cutaneous wounds of dogs, the implantation of antibiograms prior to the antimicrobials recommendation is a practice to be implemented in order to preserve animal and, consequently, public health.

With an adoption of modern methods of polyphasic approach to the study of cyanobacteria, an increased demand for the revision of the traditional taxonomy has emerged. This thesis is devoted to the systematic revisions of selected terrestrial cyanobacteria at several taxonomic levels. The methodology included thorough morphological characterization of cultured cyanobacterial strains using light and electron microscopy complemented with analyses of the molecular data: DNA sequencing, phylogenetic analyses based on 16S rRNA gene and the adjacent 16S-23S ITS region, and comparison of the predicted secondary structures of this region. Descriptions of new species, genera, families and an in-depth characterization of a previously poorly known family were achieved.

The study of the gut microbiota of fish began in the 1930’s and since that time a considerable amount of information has been collated on its composition and diversity. These studies have revealed that the microbial communities of the fish gastrointestinal tract are generally difficult to culture on bacteriological media and mainly consist of bacteria, archaea, viruses, yeasts and protists. The bacteria appear to be the most abundant of these microbial groups and their activity may have major implications for host health, development, immunity and nutrition. Therefore, much of the most recent published research has focused on developing improved methods of identifying the extent of the bacterial diversity within the fish gut and unravelling the potential influence of these microorganisms on the health of farmed fish species. However, whilst such studies have improved our knowledge of the dominant bacterial groups present in the rainbow trout gastrointestinal tract, the limited resolution capacity of many of the methods used has meant that our understanding of their baseline composition in healthy fish remains poorly understood. In this study, the bacterial communities that inhabit the intestine, now commonly referred to as the ‘microbiome’, of farmed Rainbow trout (Oncorhynchus mykiss) were characterized using a culture independent high-throughput molecular sequencing method. The microbiome of the intestinal lumen and mucosa was investigated to ascertain the true extent of the bacterial diversity present in this fish species prior to further experiments. It was found that the diversity of the intestinal microbiome was greater than previous studies had reported with a total of 90 and 159 bacterial genera being identified in both the lumen and mucosal regions respectively. The dominant bacterial phyla identified in both of the regions investigated were Proteobacteria, Firmicutes, Fusobacteria, Bacteroidetes and Actinobacteria. Furthermore, the data collected suggested that the intestinal microbiome may be similar in structure between individual fish, and illustrate the utility of next generation molecular methods in the investigation of the fish gut microbiome. A study was conducted to examine the effect of diet on the composition of the intestinal microbiome of rainbow trout. Two diets, one control and one treatment, were prepared which were identical apart from that the treatment diet contained a microalgal component at 5% of the total formulation. These diets were fed to rainbow trout for a total of 15 weeks. At the end of the trial period a total of 12 fish, three from each of four tanks, were sacrificed from each of the control and treatment groups and their intestinal tissue was sampled in order to compare the composition of the microbiome of both groups. The results revealed that both groups of fish shared similar microbiome compositions, with the Tenericutes being by far the most dominant phylum observed. The structure of the intestinal microbiome was not significantly different between both populations of trout tested. An increased level of bacterial diversity was noted in the treatment fish, however, this was not found to be statistically significant. A limited number of bacterial taxa were discriminatory between diets and were significantly elevated in the treatment group. These taxa were predominantly lactic acid bacteria of the genera Streptococcus, Leuconstoc, Lactobacillus, Lactococcus and Weissella. The results of this study suggested that the minor difference in the diets fed resulted in a correspondingly minor alteration in the intestinal microbiome of the tested rainbow trout. This may indicate that diet composition can modify the composition of the intestinal microbiome of these fish. A further study was conducted to investigate the structure of the intestinal microbiome from groups of fish reared in both freshwater cages and aquarium systems, in order to assess whether or not fish raised in different environments share similar microbiomes. This study also employed a novel computational tool, PICRUSt, to analyse the predicted functional capacity of the microbial communities of individual fish sampled from both environments. The data collected suggested that the structure of the intestinal microbiome was similar regardless of where the fish were raised, with the Tenericutes, Firmicutes, Proteobacteria, Spirochaetae and Bacteroidetes representing the dominant bacterial phyla recorded in the rainbow trout intestine. This suggests that the host may regulate the formation of the intestinal microbiome. A significant difference was however noted in community membership between the fish populations tested, which may point to an environmental influence on the intestinal microbiome. These data suggest that both deterministic host factors and stochastic environmental influences play important roles in shaping the composition of the bacterial communities in the intestine of these fish. The PICRUSt analysis revealed that gene pathways relating to metabolism, transport and cellular processes were enhanced in all of the fish studied, which may signal an involvement of these communities in the digestive processes of rainbow trout. In conclusion, this study used high-throughput sequencing methods in order to improve our understanding of the intestinal microbiome of farmed rainbow trout, and the effect of dietary and environmental factors on its composition. This research has generated scientific information relating to baseline bacterial community compositions in healthy fish, which may be used in future experiments including screening these baselines against the effects of novel aquafeed formulations, environmental perturbations or pathogenic challenges.

abstract: Obesity is a worldwide epidemic accompanied by multiple comorbidities. Bariatric surgery is currently the most efficient treatment for morbid obesity and its comorbidities. The etiology of obesity is unknown, although genetic, environmental, and most recently, microbiome elements have been recognized as contributors to this rising epidemic. The role of the gut microbiome in weight-loss or weight-gain warrants investigation, and bariatric surgery provides a good model to study influences of the microbiome on host metabolism. The underlying goals of my research were to analyze (i) the factors that change the microbiome after bariatric surgery, (ii) the effects of different types of bariatric surgeries on the gut microbiome and metabolism, (iii) the role of the microbiome on the success of bariatric surgery, and (iv) temporal and spatial changes of the microbiome after bariatric surgery. Roux-en-Y gastric bypass (RYGB) rearranges the gastrointestinal tract and reduces gastric acid secretions. Therefore, pH could be one of the factors that change microbiome after RYGB. Using mixed-cultures and co-cultures of species enriched after RYGB, I showed that as small as 0.5 units higher gut pH can aid in the survival of acid-sensitive microorganisms after RYGB and alter gut microbiome function towards the production of weight loss-associated metabolites. By comparing microbiome after two different bariatric surgeries, RYGB and laparoscopic adjustable gastric banding (LAGB), I revealed that gut microbiome structure and metabolism after RYGB are remarkably different than LAGB, and LAGB change microbiome minimally. Given the distinct RYGB alterations to the microbiome, I examined the contribution of the microbiome to weight loss. Analyses revealed that Fusobacterium might lessen the success of RYGB by producing putrescine, which may enhance weight-gain and could serve as biomarker for unsuccessful RYGB. Finally, I showed that RYGB alters the luminal and the mucosal microbiome. Changes in gut microbial metabolic products occur in the short-term and persist over the long-term. Overall, the work in this dissertation provides insight into how the gut microbiome structure and function is altered after bariatric surgery, and how these changes potentially affect the host metabolism. These findings will be helpful in subsequent development of microbiome-based therapeutics to treat obesity. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016

Salmonellosis or “food poisoning” is a foodborne infection brought on by the pathogen Salmonella from the ingestion of the bacterium on contaminated foods such as vegetables. Infection from Salmonella leads to the highest incidence of hospitalizations and deaths each year, compared to any other bacterial foodborne illness. South Florida is the second largest agricultural winter vegetable producer in the United States, and contamination of vegetables is often observed in preharvest practices. A hardy bacterium, Salmonella, has been shown to live up to 6 weeks in soil and water up to 42°C without a host. The Florida Everglades is a tropical wetland that plays a large role in South Florida’s watershed. It can be divided into agricultural, conservation, and urban areas that connect Lake Okeechobee to Florida Bay by canals, swamps, and rivers. Inland canals tightly regulate water levels in South Florida as a means of flood control for residential and agricultural land. With the influences of anthropomorphic run off from agricultural and urban use, we hypothesized that microbial communities would significantly differ between three select sites in western (Collier county) versus three sites in more urban eastern Florida (Broward county): natural standing water, manmade drainage canal in agricultural areas, and manmade drainage canals in urban areas. We also hypothesized that pathogenic like Salmonella would be present in these habitats. Deep sequencing and ecological genetics analyses of the 16s rRNA V4 region yielded a total of 163,320 unique bacterial OTUs from a total of 139 samples collected monthly for one year in 2015 and part of 2016. Salmonella is not considered an abundant taxon within the microbial population. With the knowledge that Salmonella resides within the microbial population isolates were cultured from soil and water samples that were taken monthly from each site using a modified version of the Food and Drug Administration Bacterial Analytical Methods manual (FDA-BAM). The culturing resulted in 234 isolates obtained and 31 different serovars of Salmonella. Culturing showed that Salmonella favored months with high standing water and high-water temperatures that would lead to the ideal environment for survival. The most commonly occurring isolates within the sample set are those associated with agricultural animals. Though Salmonella may be a rare taxon within the microbial population given the correct environmental conditions such as warm temperatures it is possible to observe Salmonella year round within the South Florida environment.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A castanheira-do-Brasil (Bertholletia excelsa H.B.K.), Lecythidaceae, é uma espécie nativa de potencial interesse para sistemas agroflorestais (SAFs) na Amazônia. Uma das dificuldades da propagação da castanheira é seu processo germinativo desuniforme, que constitui um problema no estabelecimento de mudas para cultivos. Pouco se sabe sobre os micro-organismos benéficos associados às raízes de castanheira-do-Brasil, sendo que o uso de micro-organismos promotores do crescimento vegetal pode ser uma biotecnologia positiva para a produção de mudas de qualidade. O objetivo desse trabalho foi caracterizar fenotípica e genotipicamente bactérias isoladas de raízes de castanheira-do-Brasil cultivadas e nativas e avaliar a presença de características para promoção do crescimento vegetal. Foram isoladas 303 bactérias de raízes de castanheiras em Roraima, sendo 81 oriundas da área de monocultivo de castanheira, 154 de SAF e 68 de área de floresta nativa. As bactérias foram isoladas utilizando meios semissólidos NFB, LGI, JMV e DYGS. A caracterização fenotípica dos isolados foi avaliada através das análises de proteína totais por SDS-PAGE, solubilização de fosfato de cálcio em meio sólido, solubilização de fosfato de alumínio em meio liquido e produção de ácido indol acético. Baseado nos perfis de proteínas (SDS-PAGE) foi estimado o índice de diversidade de Shannon e análise de rarefação. A caracterização genotípica foi realizada através da avaliação da presença ou ausencia do gene nifH e pelo sequenciamento parcial do 16S rRNA. Das 303 bactérias foram obtidas o perfil proteico de 290. Através da análise dos perfis proteicos foram gerados quatro dendrogramas referentes a cada meio de isolamento, JMV, LGI, DYGS e NFB. A partir desses dendrogramas, foi realizada uma seleção de bactérias a partir de grupos formados a 80% de similaridade em cada meio de isolamento, sendo selecionadas 100 bactérias. Na avaliação da diversidade em cada ambiente, os índices de diversidade foram similares para as três localidades de onde foram isoladas as bactérias. Das 100 bactérias avaliadas quanto a capacidade de solubilização de fosfato de cálcio em meio sólido, observou-se que 69% apresentaram esta característica e 90% apresentaram capacidade de solubilizar fosfato de alumínio em meio liquido. Setenta bactérias sintetizaram acido indol acético, onde 44 sintetizaram em meio sem triptofano, 61 em meio com triptofano e 35 apresentaram nos dois meios. Das 100 bactérias submetidas à amplificação da região do gene nifH apenas 18 apresentaram bandas específica de amplificação correspondente do gene nifH, sendo oito isoladas em meio semisseletivo para Azospirilum sp. (NFB e LGI), e 10 isoladas de meio não seletivo (DYGS). Entre as bactérias avaliadas, 24 se destacaram para futuros testes de inoculação na produção de mudas, por apresentarem no mínimo duas ou mais caracaterísticas de promoção do crescimento vegetal. Através da análise do gene 16S rRNA, as sequências de nucleotídeos obtidas resultaram na distribuição das bactérias em 18 gêneros distintos. Foram detectadas sequências de gêneros pertencentes às classes α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Bacilli e Actinobacteria. Estes resultados indicam uma elevada diversidade de gêneros de bactérias endofíticas promotoras do crescimento presentes em raízes de castanheira-do-Brasil. Trata-se do primeiro trabalho de caracterização de bactérias endofíticas promotoras de crescimento em raízes de castanheira-do-Brasil.

A superfície das folhas de árvores (filosfera) oferece uma grande área de habitat para os micro-organismos, mas constitui um ambiente extremo. O desenvolvimento de comunidades microbianas é dependente de fonte de carbono e de certos nutrientes essenciais inorgânicos comumente liberados pela planta para a sua superfície. No entanto, um grupo especial de bactérias, Cyanobacteria, é menos dependente da planta para sua nutrição, pois vários destes organismos são autotróficos para carbono e nitrogênio. Portanto, Cyanobacteria é particularmente interessante para se avaliar neste ambiente. Neste estudo, linhagens de cianobactérias presentes na superfície das folhas secretoras de sal da planta de manguezal Avicennia schaueriana foram isoladas e caracterizadas morfológica, molecular e ultraestruturalmente. O potencial destes isolados para sintetizar moléculas bioativas também foi avaliado. Para isso, folhas de A. schaueriana foram coletadas em um manguezal com histórico de contaminação por petróleo, localizado próximo ao Rio Iriri, em Bertioga-SP. O isolamento das cianobactérias foi realizado usando quatro meios de cultura (BG-11, SWBG-11, BG-11o e SWBG-11o) e dois métodos: a) esfregaço das folhas nos meios sólidos em placas de Petri; e b) submersão das folhas em frascos Erlenmeyer contendo meios líquidos. Após a obtenção de culturas puras, os isolados foram crescidos em meios líquidos, as células foram concentradas e usadas para extração de DNA genômico. O gene de RNAr 16S de cada isolado foi amplificado por PCR usando iniciadores específicos (27F/1494Rc), clonado e sequenciado. As sequências de RNAr 16S foram usadas na construção de árvore filogenética. O potencial dos isolados para sintetizar moléculas bioativas foi acessado pela amplificação de PCR usando iniciadores específicos para sequências gênicas codificadoras de peptídeo sintetase (NRPS), policetídeo sintase (PKS), cianopeptolina, aeruginosina, saxitoxina, anatoxina-a/homoanatoxina-a e microcistina. Como resultado, trinta morfotipos foram isolados em meio líquido e quatro em meio sólido. Estes morfotipos foram identificados como pertencentes a quatro ordens diferentes (12 Nostocales, 9 Pseudanabaenales, 8 Chroococcales e 5 Oscillatoriales). Entre os isolados, alta abundância de linhagens potencialmente fixadoras de N2 foi encontrada, indicando que elas possivelmente são uma importante fonte de nitrogênio neste habitat. As sequências do gene de RNAr 16S de vinte e quatro isolados ficaram distribuídas em onze clados distintos na árvore filogenética e mostraram baixas similaridades com gêneros já descritos. Na análise da ultraestrutura destas linhagens, destacou-se a presença de grânulos de elevado volume em uma cianobactéria unicelular e de um arranjo de tilacoides incomum em uma cianobactéria filamentosa homocitada com morfologia aparentemente simples. Sequências gênicas codificadoras de PKS foram detectadas em dezessete linhagens, de aeruginosina em sete linhagens e de cianopeptolina em dez linhagens. Entretanto, sequências gênicas codificadoras de NRPS e das cianotoxinas microcistina, saxitoxina e anatoxina-a/homoanatoxina-a não foram detectadas. A superfície das folhas de A. schaueriana apresenta elevado número de cianobactérias não descritas, provavelmente um resultado das condições peculiares tanto da filosfera quanto do manguezal estudado. Este é o primeiro relato de isolamento de cianobactérias da superfície de folhas de A. schaueriana / The tree leaf surface (phyllosphere) offer a large habitat area for microorganisms but constitute an extreme environment. The development of microbial communities is dependent of carbon source and certain essential inorganic nutrients commonly released from the plant to its surface. However, a special group of bacteria, Cyanobacteria, is less dependent of the plant for their nutrition since several of these organisms are autotrophic for carbon and nitrogen. Therefore, cyanobacteria are particularly interesting to be evaluated in this environment. In this study, cyanobacterial strains present in the salt-excreting leaf surface of the mangrove Avicennia schaueriana were isolated and morphologically, molecularly and ultrastructurally characterized. The potential of these isolates to synthesize bioactive molecules was also evaluated. To this purpose, A. schaueriana leaves were collected in a mangrove with history of oil contamination located near to the Iriri river in Bertioga-SP. The isolation of cyanobacteria was achieved using four culture media (BG-11, SWBG-11, BG-11o and SWBG-11o) and two methods: a) smearing of leaves into solid media in Petri dishes; and b) submersion of leaves in Erlenmeyer flasks containing liquid media. After obtaining pure cultures, the isolates were grown into liquid media, and the cells were concentrated and used for genomic DNA extraction. The gene of rRNA 16S of each isolate was amplified by PCR using specific primers (27F/1494Rc), cloned and sequenced. The 16S rRNA sequences were used for the construction of a phylogenetic tree. The potential of the isolates to synthesize bioactive molecules was assessed by PCR amplification using primers specific for gene sequences encoding non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS), cyanopeptolin, aeruginosin, saxitoxin, anatoxin-a/homoanatoxin-a and microcystin. As results, thirty morphotypes were isolated in liquid media and four in solid media. These morphotypes were identified as belonging to four different orders (12 Nostocales, 9 Pseudanabaenales, 8 Chroococcales and 5 Oscillatoriales). Among the isolates, it was found a high abundance of potentially N2-fixing strains, what indicates that they possibly are an important source of nitrogen in this habitat. The 16S rRNA gene sequences of twenty-four isolates were distributed into eleven distinct clades in the phylogenetic tree and showed low similarities with described genera. In the ultrastructural analyses of these strains, the highlight was the presence of granules of high volume in a unicellular cyanobacterium and an unusual thylakoid arrangement in a homocytous filamentous cyanobacterium with apparently simple morphology. Gene sequences encoding for PKS were detected in seventeen strains, for aeruginosine in seven strains and cyanopeptolin in ten strains. Gene sequences encoding for NRPS and for the cyanotoxins microcystin, saxitoxin, and anatoxin-a/homoanatoxin-a were not found. The leaf surface of A. schaueriana presents a high number of undescribed cyanobacteria, probably as a result of the peculiar conditions of the phyllosphere and the studied mangrove. This is the first report of isolation of cyanobacteria from the leaf surface of A. schaueriana