Genlisea hawkingii (Lentibulariaceae), a new species from Serra da Canastra, Minas Gerais, Brazil.

Genlisea hawkingii, which is a new species of Genlisea subgen. Tayloria (Lentibulariaceae) from cerrado in southwest Brazil, is described and illustrated. This species has been found in only one locality thus far, in the Serra da Canastra, which is located in the Delfinópolis municipality in Minas Gerais, Brazil. The new species is morphologically similar to Genlisea violacea and G. flexuosa, but differs from them in having a corolla with a conical and curved spur along with sepals with an acute apex and reproductive organs that only have glandular hairs. Moreover, it is similar to G. uncinata's curved spur. G. hawkingii is nested within the subgen. Tayloria clade as a sister group to all the other species of this subgenus. Therefore, both morphological and phylogenetic results strongly support G. hawkingii as a new species in the subgen. Tayloria.

Cultivated bacterial diversity associated with the carnivorous plant Utricularia breviscapa (Lentibulariaceae) from floodplains in Brazil

ABSTRACT Carnivorous plant species, such as Utricularia spp., capture and digest prey. This digestion can occur through the secretion of plant digestive enzymes and/or by bacterial digestive enzymes. To comprehend the physiological mechanisms of carnivorous plants, it is essential to understand the microbial diversity related to these plants. Therefore, in the present study, we isolated and classified bacteria from different organs of Utricularia breviscapa (stolons and utricles) and from different geographic locations (São Paulo and Mato Grosso). We were able to build the first bacterium collection for U. breviscapa and study the diversity of cultivable bacteria. The results show that U. breviscapa bacterial diversity varied according to the geographic isolation site (São Paulo and Mato Grosso) but not the analyzed organs (utricle and stolon). We reported that six genera were common to both sample sites (São Paulo and Mato Grosso). These genera have previously been reported to be beneficial to plants, as well as related to the bioremediation process, showing that these isolates present great biotechnological and agricultural potential. This is the first report of an Acidobacteria isolated from U. breviscapa. The role of these bacteria inside the plant must be further investigated in order to understand their population dynamics within the host.

Cultivated bacterial diversity associated with the carnivorous plant Utricularia breviscapa (Lentibulariaceae) from floodplains in Brazil.

Carnivorous plant species, such as Utricularia spp., capture and digest prey. This digestion can occur through the secretion of plant digestive enzymes and/or by bacterial digestive enzymes. To comprehend the physiological mechanisms of carnivorous plants, it is essential to understand the microbial diversity related to these plants. Therefore, in the present study, we isolated and classified bacteria from different organs of Utricularia breviscapa (stolons and utricles) and from different geographic locations (São Paulo and Mato Grosso). We were able to build the first bacterium collection for U. breviscapa and study the diversity of cultivable bacteria. The results show that U. breviscapa bacterial diversity varied according to the geographic isolation site (São Paulo and Mato Grosso) but not the analyzed organs (utricle and stolon). We reported that six genera were common to both sample sites (São Paulo and Mato Grosso). These genera have previously been reported to be beneficial to plants, as well as related to the bioremediation process, showing that these isolates present great biotechnological and agricultural potential. This is the first report of an Acidobacteria isolated from U. breviscapa. The role of these bacteria inside the plant must be further investigated in order to understand their population dynamics within the host.

Nectar trichome structure of aquatic bladderworts from the section Utricularia (Lentibulariaceae) with observation of flower visitors and pollinators.

In Utricularia, the flower spur is a nectary and in this organ, nectar is produced and stored. This study aimed to examine the structure of the nectary trichomes in four Utricularia species (Utricularia vulgaris L., U. australis R.Br., U. bremii Heer and U. foliosa L.) from the generic section Utricularia. We have investigated whether species with different spur morphology had similar spur anatomy and nectary trichome structure. In Utricularia flowers, nectar is produced by spur capitate trichomes (sessile or stalked). Our results showed that regardless of the various spur morphology, trichomes have similar architecture and ultrastructure. Head cells of these trichomes are transfer cells with an eccrine nectar secretion. Examined species differed in the micromorphology of papillae in spurs. The fly Eristalis tenax was found to be a pollinator of U. vulgaris. Small Halictidae bees seem to be pollinators of U. foliosa.

Flower palate ultrastructure of the carnivorous plant Genlisea hispidula Stapf with remarks on the structure and function of the palate in the subgenus Genlisea (Lentibulariaceae).

In the genus Genlisea as well as in its sister genus Utricularia, the palate probably plays a key role in providing the colour, mechanical and olfactory stimuli to attract insect pollinators and to guide them to the generative structures and the nectary spur. However, information about the micro-morphology of the palate of Genlisea is scarce. This study aims to examine the structure of the palate in Genlisea hispidula in detail as well as the palate from other five species from the subgenus Genlisea. In particular, its aim is to ascertain whether these palates function as an area for the osmophores in the flower or whether they produce nectar. We showed that the palate in all of the species that were examined was the glandular type and that it had capitate, glandular trichomes, which had a similar general architecture across the species that were examined. No nectar secretion was observed on the palates. The ultrastructure of the palate trichomes showed that the palate glandular trichomes most probably function as scent glands that produce an olfactory stimulus for flower pollinators.

Hepatozoon caimani in Caiman crocodilus yacare (Crocodylia, Alligatoridae) from North Pantanal, Brazil.

Hepatozoon species are the most common intracellular hemoparasite found in reptiles. Hepatozoon caimani, whose vectors are Culex mosquitoes, has been detected in a high prevalence among caimans in Brazil by blood smears examinations. The present work aimed to detect and characterize the Hepatozoon spp. found in 33 caimans (24 free-ranging and 9 captive; 28 males and 5 females) (Caiman crocodilus yacare) sampled at Poconé, North Pantanal, state of Mato Grosso, Brazil, using blood smears examinations and molecular techniques. Hepatozoon spp.-gametocytes were found in 70.8% (17/24) and 88.8% (8/9) of blood smears from free-ranging and captive caimans, respectively. Hepatozoon spp. 18S rRNA DNA was found in 79.2% (19/24) and 88.8% (8/9) of free-ranging and captive caimans, respectively. Comparative analysis of parasitized and non-parasitized erythrocytes showed that all analyzed features were significantly different (P<0.05) for both linear and area dimensions. Phylogenetic analysis based on 18S rRNA sequences grouped the Hepatozoon spp. sequences detected in the present study together with H. caimani, recently detected in caimans in southern Pantanal.

Hepatozoon caimani in Caiman crocodilus yacare (Crocodylia, Alligatoridae) from North Pantanal, Brazil / Hepatozoon caimani em Caiman crocodilus yacare (Crocodylia, Alligatoridae) do Norte do Pantanal, Brasil

Abstract Hepatozoon species are the most common intracellular hemoparasite found in reptiles. Hepatozoon caimani, whose vectors are Culex mosquitoes, has been detected in a high prevalence among caimans in Brazil by blood smears examinations. The present work aimed to detect and characterize the Hepatozoon spp. found in 33 caimans (24 free-ranging and 9 captive; 28 males and 5 females) (Caiman crocodilus yacare) sampled at Poconé, North Pantanal, state of Mato Grosso, Brazil, using blood smears examinations and molecular techniques. Hepatozoon spp.-gametocytes were found in 70.8% (17/24) and 88.8% (8/9) of blood smears from free-ranging and captive caimans, respectively. Hepatozoon spp. 18S rRNA DNA was found in 79.2% (19/24) and 88.8% (8/9) of free-ranging and captive caimans, respectively. Comparative analysis of parasitized and non-parasitized erythrocytes showed that all analyzed features were significantly different (P<0.05) for both linear and area dimensions. Phylogenetic analysis based on 18S rRNA sequences grouped the Hepatozoon spp. sequences detected in the present study together with H. caimani, recently detected in caimans in southern Pantanal.

Resumo Espécies do gênero Hepatozoon são os hemoparasitas intracelulares mais comumente encontrados em répteis. Hepatozoon caimani, cujos vetores são mosquitos do gênero Culex sp., têm sido detectados em uma alta prevalência entre jacarés no Brasil, por meio da análise de esfregaços sanguíneos. O presente estudo objetivou detectar e caracterizar parasitas do gênero Hepatozoon spp. em 33 jacarés (24 de vida-livre e 9 de cativeiro; 28 machos e 5 fêmeas) (Caiman crocodilus yacare) amostrados em Poconé, região norte do Pantanal, estado do Mato Grosso, Brasil, por meio da análise de esfregaços sanguíneos e técnicas moleculares. Gametócitos de Hepatozoon spp. foram encontrados em 70,8% (17/24) e em 88,8% (8/9) dos esfregaços sanguíneos de jacarés de via-livre e cativeiro, respectivamente. 18S rRNA DNA de Hepatozoon spp. foi detectado em 79,2% (19/24) e 88,8% (8/9) das amostras de sangue de jacarés de vida-livre e cativeiro, respectivamente. A análise comparativa de eritrócitos parasitados e não parasitados mostrou diferença significativa (P<0,05) em todas as variáveis lineares e de área analisadas. A análise filogenética baseada em sequências de DNA do 18S rRNA agrupou as sequências de Hepatozoon spp. detectadas no presente estudo juntamente com aquelas de H. caimani, recentemente detectadas em jacarés do Pantanal do Mato Grosso do Sul.

Flower palate structure of the aquatic bladderworts Utricularia bremii Heer and U. minor L. from section Utricularia (Lentibulariaceae).

There is an enormous diversity in the structure of the flower palate of the carnivorous rootless genus Utricularia. This study aims to examine the structure of the palates in Utricularia bremii Heer and U. minor L of the Utricularia sect. Utricularia, which have a glandular palate type. In both species, the palate has only one type of glandular trichomes. Because of the occurrence of cell wall ingrowths in its glandular cells, any exudation may be transported via eccrinous secretion. It was proposed that the palate trichomes of the examined species act as scent glands and that the palate may play a role as an unguentarium. Both U. bremii and U. minor are of an open flower type. Thus, U. bremii and U. minor flowers can be penetrated by small, weak insects, which then easily have access to their generative structure. Small Hymenoptera (member of families Mymaridae and Braconidae) were observed as flower visitors of the male-sterile species Utricularia bremii.

Floral ultrastructure of two Brazilian aquatic-epiphytic bladderworts: Utricularia cornigera Studnicka and U. nelumbifolia Gardner (Lentibulariaceae).

Utricularia cornigera and Utricularia nelumbifolia are giant, aquatic-epiphytic species of carnivorous bladderwort from southeastern Brazil that grow in the central 'urns' of bromeliads. Both species have large, colourful flowers. The main aim of our study is to ascertain whether the prominent floral palate of U. cornigera and U. nelumbifolia functions as an unguentarius-i.e. an organ that bears osmophores. Floral tissues of both species were investigated using light microscopy, scanning electron microscopy, transmission electron microscopy and histochemistry. Floral palates of U. cornigera and U. nelumbifolia provide clear visual signals for pollinating insects. In both species, the palate possesses diverse micro-morphology, comprising unicellular, conical to villiform papillae and multicellular, uniseriate, glandular trichomes that frequently display terminal branching. The most characteristic ultrastructural feature of these papillae was the presence of relatively large, polymorphic plastids (chromoplasts) containing many plastoglobuli. Similar plastids are known to occur in the fragrance-producing (osmophores) and oil-producing (elaiophores) tissues of several orchid species. Thus, these palate papillae may play a key role in providing the olfactory stimulus for the attraction of insect pollinators. Nectariferous trichomes were observed in the floral spurs of both species, and in U. nelumbifolia, free nectar was also recorded. The location, micro-morphology, anatomy and ultrastructure of the floral palate of the two species investigated may thus indicate that the palate functions as an unguentarius. Furthermore, the flowers of these taxa, like those of U. reniformis, have features consistent with bee pollination.

Plant or fungal sequences? An alternative optimized PCR protocol to avoid ITS (nrDNA) misamplification

The nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) from leaves of Drosera (Droseraceae) were amplified using "universal" primers. The analysis of the products demonstrated most samples were a molecular mixture as a result of unsuccessful and non-specific amplifications. Among the obtained sequences, two were from Basidiomycota fungi. Homologous sequences of Basidiomycota were obtained from GenBank database and added to a data set with sequences from Drosera leaves. Parsimony analysis demonstrated that one sequence was amplified from an Ustilaginomycetes fungus, and another from a Heterobasidiomycetes. Possibly these fungi were associated to leaves of Drosera, and not because of samples contamination. In order to provide optimization and a better specificity of PCR (polymerase chain reaction), a very successful method was demonstrated using dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) in reactions.

Os espaçadores internos transcritos do DNA nuclear ribossomal (ITS1 e ITS2) de folhas de Drosera (Droseraceae) foram amplificados com o emprego de iniciadores "universais". A análise demonstrou que a maior parte das amostras continha uma mistura resultante de amplificações não-específicas. Dentre as sequências de DNA obtidas, duas delas foram de fungos basidiomicetos. Sequências homólogas foram obtidas do GenBank e analisadas junto às sequências obtidas de folhas de Drosera. Através das análises filogenéticas de máxima parcimônia foi possível identificar uma seqüência como sendo de um Ustilaginomycetes e outra de Heterobasidiomycetes (Basidiomycota). Possivelmente esses organismos estavam associados às folhas de Drosera e assim não sejam resultantes de contaminação. Com o objetivo de otimizar e buscar uma melhor especificidade das reações de PCR, um protocolo bem sucedido foi demonstrado com o uso de dimetilsulfóxido (DMSO) e soroalbumina bovina (BSA).

16s-23S rDNA: polymorphisms and their use for detection and identification of Xylella fastidiosa strains

Strains of Xylella fastidiosa from several hosts (coffee, citrus, grape, almond, oleander, peach, plum, etc.) were characterized by analyzing the content of the nucleotide sequences of 16S-23S rDNA (coding for a small subunit ribosomal RNA) spacer region (ITS). Current methods for sequencing the ITS region yields partial sequences that do not contribute with significant information. According to this fact, new primers have been designed in order to obtain a complete sequence and facilitate the sequencing. The complete 16S-23S sequences from 08 strains were amplified through PCR using the primers designed in our lab. The 16S-23S sequences obtained were compared with 52 others sequences entries in GenBank database. The results revealed a higher level of variation than that found in 16S gene sequences, with similarity values ranging from 0.79-1.00. The dendogram based on similarity data revealed 5 main groups. This spacer sequence contains two genes for tRNA (tRNAala and tRNAile). The sequence analysis of the tRNA content showed a conserved region with a few differences in the nucleotide composition.

Linhagens de Xylella fastidiosa de diferentes hospedeiros (café, uva, amêndoa, ameixa, etc.) foram caracterizados analisando as sequências de nucleotídeos do espaço intergênico 16S-23S (ITS). Os métodos atuais para o sequenciamento da região ITS produzem fragmentos parciais das sequências que não contribuem com informações significantes. Em vista disso, novos primers foram desenhados a fim de obter uma sequência completa e facilitar o sequenciamento. A sequencia completa da região ITS de 08 linhagens de X. Fastidiosa foram amplificadas via PCR, sequenciadas e comparadas com outras 52 sequencias depositadas no GenBank. Os resultados revelaram um alto nível de variação sendo maior que os níveis encontrados quando se utiliza o gene 16S para este tipo de análise, com valores variando entre 0.79 a 1.00. O dendograma baseado em dados de similaridade agrupou as linhagens de X. fastidiosa em 5 grupos principais. Duas sequencias codificadoras de tRNA's foram encontradas (tRNAala e tRNAile) e mostram-se conservadas entre as linhagens analisadas com pouca diferença entre a composição nucleotídica.

Phylogenetic relationships of Xylella fastidiosa strains based on 16S-23S rDNA sequences.

In spite of the lack of resolution of Xylella fastidiosa phylogenetic relationships, parsimony analysis of the 16S-23S rDNA sequence from a wide range of hosts has been evaluated in this research. In order to establish an easier method for sequencing the spacer region completely, a new primer pair was designed. The sequences obtained revealed a higher level of variation than that found in 16S gene sequences, with similarity values ranging from 0.80 to 1.00. The cladogram constructed allowed the clustering of two major clades. From these results it has been possible to recognize the monophyletic grouping of some strains belonging to the same host, possibly representing only one infection process. However, for other hosts there is paraphyletic and polyphyletic grouping. This methodology followed from promising results regarding strain-host clustering. With the parsimony approach, hypothetical genealogical relationship among Xylella strains may be inferred.