The anammox case - A new experimental manifesto for microbiological eco-physiology

The anaerobic ammonium oxidation process is a new process for ammonia removal from wastewater. It is also a new microbial physiology that was previously believed to be impossible. The identification of Candidatus Brocadia anammoxidans and its relatives as the responsible bacteria was only possible with the development of a new experimental approach. That approach is the focus of this paper. The approach is a modernisation of the Winogradsky/Beyerinck strategy of selective enrichment and is based on the introduction of the molecular toolbox and modern bioreactor engineering to microbial ecology. It consists of five steps: (1) postulation of an ecological niche based on thermodynamic considerations and macro-ecological field data; (2) engineering of this niche into a laboratory bioreactor for enrichment culture; (3) black-box physiological characterisation of the enrichment culture as a whole; (4) phylogenetic characterisation of the enriched community using molecular tools; (5) physical separation of the dominant members of the enrichment culture using gradient centrifugation and the identification of the species of interest in accordance with Koch's postulates; (6) verification of the in situ importance of these species in the actual ecosystems. The power of this approach is illustrated with a case study: the identification of the planctomycetes responsible for anaerobic ammonium oxidation. We argue that this was impossible using molecular ecology or conventional 'cultivation based techniques' alone. We suggest that the approach might also be used for the microbiological study of many interesting microbes such as anaerobic methane oxidisers.

Glycogen-accumulating organisms in laboratory-scale and full scale wastewater treatment processes

Laboratory-scale sequencing batch reactors (SBRs) as models for wastewater treatment processes were used to identify glycogen-accumulating organisms (GAOs), which are thought to be responsible for the deterioration of enhanced biological phosphorus removal (EBPR). The SBRs (called Q and T), operated under alternating anaerobic-aerobic conditions typical for EBPR, generated mixed microbial communities (sludges) demonstrating the GAO phenotype. Intracellular glycogen and poly-beta-hydroxyalkanoate (PHA) transformations typical of efficient EBPR occurred but polyphosphate was not bioaccumulated and the sludges contained 1.8% P (sludge Q) and 1.5% P (sludge T). 16S rDNA clone libraries were prepared from DNA extracted from the Q and T sludges. Clone inserts were grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism banding profiles. OTU representatives were sequenced and phylogenetically analysed. The Q sludge library comprised four OTUs and all six determined sequences were 99.7% identical, forming a cluster in the gamma-Proteobacteria radiation. The T sludge library comprised eight OTUs and the majority of clones were Acidobacteria subphylum 4 (49% of the library) and candidate phylum OPU (39% of the library). One OTU (two clones, of which one was sequenced) was in the gamma-Proteobacteria radiation with 95% sequence identity to the Q sludge clones. Oligonucleotide probes (called GAOQ431 and GAOQ989) were designed from the gamma-Proteobacteria clone sequences for use in fluorescence in situ hybridization (FISH); 92 % of the Q sludge bacteria and 28 % of the T sludge bacteria bound these probes in FISH. FISH and post-FISH chemical staining for PHA were used to determine that bacteria from a novel gamma-Proteobacteria cluster were phenotypically GAOs in one laboratory-scale SBR and two fullscale wastewater treatment plants. It is suggested that the GAOs from the novel cluster in the gamma-Proteobacteria radiation be named 'Candidatus Competibacter phosphatis'.

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria

Recently, two fresh water species, 'Candidatus Brocadia anammoxidans' and 'Candidatus Kuenenia stuttgartiensis', and one marine species, 'Candidatus Scalindua sorokinii', of planctomycete anammox bacteria have been identified. 'Candidatus Scalindua sorokinii' was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle-the anammoxosome-in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms

Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, Candidatus Accumulibacter phosphatis was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions. (C) 2003 Wiley Periodicals, Inc.

Avaliação de extrato de algas no progresso temporal da mancha de Mycosphaerella em cultivares de morangueiro

Em morangueiros, a mancha das folhas, causada por Mycosphaerella fragariae, é uma das doenças mais comuns na cultura, o que torna importantes os estudos de controle alternativo e de cultivares, sobre o progresso da epidemia. Em busca de práticas alternativas para seu controle, avaliou-se o efeito de cultivares de morangueiro e do extrato da alga Ascophyllum nodosum, na expressão dos sintomas de mycosphaerella, além de avaliar o ajuste de modelos de crescimento para o progresso temporal da incidência e severidade da doença. Utilizou-se o extrato de algas a 29%, na dose de 2 L ha-1, aplicado no solo, na folha e em ambos. As cultivares Dover, Toyonoka, Albion, Camarosa, Ventana, Campinas, Tudla e Camino Real foram avaliadas, quinzenalmente, quanto a incidência e severidade da doença. Utilizou-se o delineamento inteiramente casualizado, com parcelas subdivididas e três repetições. Com os dados de incidência e severidade, calculou-se a área abaixo da curva de progresso da doença e utilizou-se a análise de variância para avaliar o efeito de cultivares e do extrato de algas. Aos dados do progresso temporal da incidência e da severidade, em cada cultivar, foram ajustados os modelos monomolecular, logístico e de Gompertz. O extrato de algas não apresentou efeito ou interação com cultivares, no controle da mycosphaerella. Houve efeito de cultivar com menor intensidade da doença, em Albion e Ventana, devido, principalmente, ao atraso na epidemia, pela redução do inóculo inicial efetivo. Aos dados de progresso temporal da incidência e da severidade ajustaram-se, respectivamente, os modelos logístico e monomolecular, para todas as cultivares.

MOLECULAR INVESTIGATION OF HEMOTROPIC MYCOPLASMAS IN HUMAN BEINGS, DOGS AND HORSES IN A RURAL SETTLEMENT IN SOUTHERN BRAZIL

SUMMARY The aims of this study were to determine the prevalence of hemoplasmas in a rural Brazilian settlement's population of human beings, their dogs and horses, highly exposed to tick bites; to identify the tick species parasitizing dogs and horses, and analyze factors associated with their infection. Blood samples from 132 dogs, 16 horses and 100 humans were screened using a pan-hemoplasma SYBR green real-time PCR assay followed by a species-specific TaqMan real-time PCR. A total of 59/132 (44.7%) dog samples were positive for hemoplasmas (21 Mycoplasma haemocanisalone, 12 ' Candidatus Mycoplasma haematoparvum' alone and 21 both). Only 1/100 (1.0%) human sample was positive by qPCR SYBR green, with no successful amplification of 16S rRNA or 23 rRNA genes despite multiple attempts. All horse samples were negative. Dogs >1 year of age were more likely to be positive for hemoplasmas ( p= 0.0014). In conclusion, although canine hemoplasma infection was highly prevalent, cross-species hemoplasma transmission was not observed, and therefore may not frequently occur despite overexposure of agents and vectors.

Understanding the microbial ecology and ecophysiology of enhanced biological phosphorus removal processes through metabolic modelling and experimental studies

Dissertation to obtain the degree of Master in Chemical and Biochemical Engineering

Ocorrência de endotoquia matricida em Meloidogyne javanica (Nematoda, Heteroderidae)

In the past years, several writers described eudotokia matricida in nematodes belonging to the family Rbabditidae. In 1958, LOOF dealt with two cases he observed in Apbeltncboides fragariae and Pratylencbus coffeae , showing that such phenomenon eventually occurs in the Tylencbida, families Apbelencboididae and Tylencbidae respectively. A third case referring to another tylenchid nematode is described in this paper, the species envolved having been identified as Meloidogyne javanica, of the family Heteroderidae.

Multilocus sequence analysis reveals the genetic diversity of European fruit tree phytoplasmas and supports the existence of inter-species recombination

The genetic diversity of three temperate fruit tree phytoplasmas ‘Candidatus Phytoplasma prunorum’, ‘Ca. P. mali’ and ‘Ca. P. pyri’ has been established by multilocus sequence analysis. Among the four genetic loci used, the genes imp and aceF distinguished 30 and 24 genotypes, respectively, and showed the highest variability. Percentage of substitution for imp ranged from 50 to 68% according to species. Percentage of substitution varied between 9 and 12% for aceF, whereas it was between 5 and 6% for pnp and secY. In the case of ‘Ca P. prunorum’ the three most prevalent aceF genotypes were detected in both plants and insect vectors, confirming that the prevalent isolates are propagated by insects. The four isolates known to be hypo-virulent had the same aceF sequence, indicating a possible monophyletic origin. Haplotype network reconstructed by eBURST revealed that among the 34 haplotypes of ‘Ca. P. prunorum’, the four hypo-virulent isolates also grouped together in the same clade. Genotyping of some Spanish and Azerbaijanese ‘Ca. P. pyri’ isolates showed that they shared some alleles with ‘Ca. P. prunorum’, supporting for the first time to our knowledge, the existence of inter-species recombination between these two species.

Ácaros predadores e o fungo Neozygites floridana associados a tetraniquídeos em soja no Rio Grande do Sul

O objetivo deste trabalho foi verificar a ocorrência de ácaros predadores e do fungo Neozygites floridana, associados a ácaros tetraniquídeos, em soja (Glycine max L.), no Estado do Rio Grande do Sul. Entre 2003 e 2005, foram avaliadas amostras de folhas de soja das principais regiões produtoras do Estado. Essas amostras foram coletadas de diferentes posições da planta e aleatoriamente na lavoura. As espécies de ácaros predadores encontradas foram: Neoseiulus anonymus, N. californicus, Phytoseiulus fragariae, P. macropilis, Proprioseiopsis cannaensis e Galendromus annectens. O fungo N. floridana apresentou ampla distribuição e ocorreu em todas as espécies de tetraniquídeos encontradas: Mononychellus planki, Tetranychus desertorum, T. gigas, T. ludeni e T. urticae.

Reação de cultivares de morangueiro ao oídio sob condições de casa de vegetação

O objetivo deste trabalho foi avaliar a reação de dez cultivares de morangueiro ao oídio (Sphaerotheca macularis f.sp. fragariae). Plantas-matrizes foram transplantadas em bandejas de 72 células, contendo substrato autoclavado. Em seguida, bandejas com plantas de morangueiro severamente infestadas com oídio foram colocadas na casa de vegetação. Utilizou-se do delineamento experimental de blocos casualizados, com parcelas subdivididas e quatro repetições, contendo dez plantas. As parcelas foram constituídas de dez cultivares de morangueiro (Aromas, Bürkley, Camarosa, Campinas, Dover, Milsei-Tudla, Oso Grande, Santa Clara, Sweet Charlie e Vila Nova) e as subparcelas de três épocas de avaliação da severidade da doença nas três folhas mais novas totalmente expandidas (8ª, 10ª e 12ª semana após o transplantio das plantas-matrizes). Utilizou-se de uma escala de notas, onde 0; 1; 2 e 3 representaram, respectivamente, 0%, 1-25%, 26-50% e > 51% de área foliar coberta com estruturas do fungo. A severidade da doença diminuiu com a proximidade do verão, devido às maiores temperaturas médias diárias. Verificou-se alta variabilidade das cultivares quanto à reação ao oídio, sendo: 'Milsei-Tudla' altamente resistente; 'Camarosa', 'Aromas', 'Oso Grande', 'Sweet Charlie' e 'Campinas' medianamente resistentes; 'Dover' pouco resistente; e 'Bürkley', 'Vila Nova' e 'Santa Clara' altamente suscetíveis.

Estimativa dos impactos econômicos decorrentes de eventual introdução do huanglongbing (HLB) no estado da Bahia

A Bahia é o segundo mais importante Estado produtor de citros do Brasil, responsável por 5,5% da produção do País. 80% da produção vem de propriedades de base familiar, as quais dependem desse cultivo para seu sustento econômico. O Huanglongbing (HLB) nunca foi registrado na Bahia, mas está disseminando-se em outros três Estados do Brasil (São Paulo, Paraná e Minas Gerais), um dos quais faz fronteira com a Bahia. Assim, este estudo teve como objetivo estimar o impacto econômico potencial de uma eventual introdução do HLB na Bahia. Foram utilizados os modelos de Gompertz e logístico associados a diferentes estratos de idade das plantas para estimar, respectivamente, a evolução da incidência e da severidade da doença no parque citrícola baiano, num horizonte de 20 anos. O progresso da doença foi usado para estimar os danos à produção em três cenários. No cenário-base (A), os esforços da agência estadual de defesa agropecuária previniria a entrada do HLB. No cenário B, a doença seria introduzida e não haveria esforços para seu controle, levando a uma disseminação nos anos seguintes. No cenário C, após a detecção da doença, os produtores adotariam medidas de controle: eliminação de plantas sintomáticas e supressão das populações do inseto vetor. Os custos do controle foram calculados considerando-se a necessidade de pulverizações, inspeções periódicas e eliminação das plantas doentes. O valor presente líquido (VPL) foi usado para comparar os três cenários. Os resultados indicam que as perdas seriam muito significativas caso o HLB fosse introduzido na Bahia. Assim sendo, se as medidas de controle não fossem aplicadas, os prejuízos poderiam superar R$1,8 bilhão.

Identificação e caracterização de espécies de Colletotrichum associadas à antracnose de anonáceas no estado de Alagoas

No Brasil, Colletotrichum gloeosporioides é a única espécie associada à antracnose de anonáceas. Contudo, apenas características morfológicas têm sido utilizadas na identificação. Assim, o objetivo do trabalho foi identificar e caracterizar espécies de Colletotrichum que causam a antracnose nas culturas da pinha e da graviola no Estado de Alagoas. Cinquenta e um isolados, obtidos de folhas de pinheira e gravioleira com sintomas típicos da doença, foram coletados em Maceió, Palmeira dos Índios e União dos Palmares. Os isolados foram inoculados em folhas destacadas de ambas as culturas e caracterizados morfologicamente através da morfometria de conídios e apressórios, e molecularmente por meio do sequenciamento da região ITS. Verificou-se que todos os isolados foram patogênicos a ambas as espécies. Na caracterização morfológica, os isolados foram agrupados em três grupos: M1, M2 e M3. O grupo M1 foi formado por 32 isolados com características relacionadas a C. gloeosporioides. No grupo M2, constituído de 15 isolados, predominaram características de C. boninense, enquanto, no grupo M3, composto por quatro isolados, as características foram típicas de C. fragariae. A análise filogenética, da mesma forma, também resultou em três grupos (F1, F2 e F3), os quais, de modo geral, estiveram em concordância com os dados da morfologia. O grupo filogenético F1 concentrou os isolados do grupo morfológico M1 e sequências de referências de C. gloeosporioides e C. fragariae. O grupo F2, que agrupou as sequências de C. boninense, concentrou os isolados do grupo morfológico M2. Finalmente, o grupo F3 incluiu sequências de C. magna e outros quatro isolados desse estudo. Assim, foi possível comprovar que quatro espécies de Colletotrichum são responsáveis pela antracnose em pinheira e gravioleira em Alagoas: C. gloeosporioides, C. boninense, C. fragariae e C. magna.

Cyanobacterial diversity and a new Acaryochloris-like symbiont from Bahamian sea-squirts

Symbiotic interactions between ascidians (sea-squirts) and microbes are poorly understood. Here we characterized the cyanobacteria in the tissues of 8 distinct didemnid taxa from shallow-water marine habitats in the Bahamas Islands by sequencing a fragment of the cyanobacterial 16S rRNA gene and the entire 16S-23S rRNA internal transcribed spacer region (ITS) and by examining symbiont morphology with transmission electron (TEM) and confocal microscopy (CM). As described previously for other species, Trididemnum spp. mostly contained symbionts associated with the Prochloron-Synechocystis group. However, sequence analysis of the symbionts in Lissoclinum revealed two unique clades. The first contained a novel cyanobacterial clade, while the second clade was closely associated with Acaryochloris marina. CM revealed the presence of chlorophyll d (chl d) and phycobiliproteins (PBPs) within these symbiont cells, as is characteristic of Acaryochloris species. The presence of symbionts was also observed by TEM inside the tunic of both the adult and larvae of L. fragile, indicating vertical transmission to progeny. Based on molecular phylogenetic and microscopic analyses, Candidatus Acaryochloris bahamiensis nov. sp. is proposed for this symbiotic cyanobacterium. Our results support the hypothesis that photosymbiont communities in ascidians are structured by host phylogeny, but in some cases, also by sampling location.

Permissivity of fish cell lines to three Chlamydia-related bacteria: Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae.

Epitheliocystis is an infectious disease affecting gills and skin of various freshwater and marine fishes, associated with high mortality and reduced growth of survivors. Candidatus Piscichlamydia salmonis and Clavochlamydia salmonicola have recently been identified as aetiological agents of epitheliocystis in Atlantic Salmon. In addition, several other members of the Chlamydiales order have been identified in other fish species. To clarify the pathogenicity of Chlamydia-like organisms towards fishes, we investigated the permissivity of two fish cell lines, EPC-175 (Fathead Minnow) and RTG-2 (rainbow trout) to three Chlamydia-related bacteria: Waddlia chondrophila, Parachlamydia acanthamoebae and Estrella lausannensis. Quantitative PCR and immunofluorescence demonstrated that W. chondrophila and, to a lesser extent, E. lausannensis were able to replicate in the two cell lines tested. Waddlia chondrophila multiplied rapidly in its host cell and a strong cytopathic effect was observed. During E. lausannensis infection, we observed a limited replication of the bacteria not followed by host cell lysis. Very limited replication of P. acanthamoebae was observed in both cell lines tested. Given its high infectivity and cytopathic effect towards fish cell lines, W. chondrophila represents the most interesting Chlamydia-related bacteria to be used to develop an in vivo model of epitheliocystis disease in fishes.