The diversity and beneficial characteristics of endophytic microorganisms have been studied in several host plants. However, information regarding naturally occurring seed-associated endophytes and vertical transmission among different life-history stages of hosts is limited. Endophytic bacteria were isolated from seeds and seedlings of 10 Eucalyptus species and two hybrids. The results showed that endophytic bacteria, such as Bacillus, Enterococcus, Paenibacillus and Methylobacterium, are vertically transferred from seeds to seedlings. In addition, the endophytic bacterium Pantoea agglomerans was tagged with the gfp gene, inoculated into seeds and further reisolated from seedlings. These results suggested a novel approach to change the profile of the plants, where the bacterium is a delivery vehicle for desired traits. This is the first report of an endophytic bacterial community residing in Eucalyptus seeds and the transmission of these bacteria from seeds to seedlings. The bacterial species reported in this work have been described as providing benefits to host plants. Therefore, we suggest that endophytic bacteria can be transmitted vertically from seeds to seedlings, assuring the support of the bacterial community in the host plant.

eThe promotion of sugarcane growth by the endophytic Pantoea agglomerans strain 33.1 was studied under gnotobiotic and greenhouse conditions. The green fluorescent protein (GFP)-tagged strain P. agglomerans 33.1::pNKGFP was monitored in vitro in sugarcane plants by microscopy, reisolation, and quantitative PCR (qPCR). Using qPCR and reisolation 4 and 15 days after inoculation, we observed that GFP-tagged strains reached similar density levels both in the rhizosphere and inside the roots and aerial plant tissues. Microscopic analysis was performed at 5, 10, and 18 days after inoculation. Under greenhouse conditions, P. agglomerans 33.1-inoculated sugarcane plants presented more dry mass 30 days after inoculation. Cross-colonization was confirmed by reisolation of the GFP-tagged strain. These data demonstrate that 33.1::pNKGFP is a superior colonizer of sugarcane due to its ability to colonize a number of different plant parts. The growth promotion observed in colonized plants may be related to the ability of P. agglomerans 33.1 to synthesize indoleacetic acid and solubilize phosphate. Additionally, this strain may trigger chitinase and cellulase production by plant roots, suggesting the induction of a plant defense system. However, levels of indigenous bacterial colonization did not vary between inoculated and noninoculated sugarcane plants under greenhouse conditions, suggesting that the presence of P. agglomerans 33.1 has no effect on these communities. In this study, different techniques were used to monitor 33.1::pNKGFP during sugarcane cross-colonization, and our results suggested that this plant growth promoter could be used with other crops. The interaction between sugarcane and P. agglomerans 33.1 has important benefits that promote the plant's growth and fitness.

The mangrove ecosystem is a coastal tropical biome located in the transition zone between land and sea that is characterized by periodic flooding, which confers unique and specific environmental conditions on this biome. In these ecosystems, the vegetation is dominated by a particular group of plant species that provide a unique environment harboring diverse groups of microorganisms, including the endophytic microorganisms that are the focus of this study. Because of their intimate association with plants, endophytic microorganisms could be explored for biotechnologically significant products, such as enzymes, proteins, antibiotics and others. Here, we isolated endophytic microorganisms from two mangrove species, Rhizophora mangle and Avicennia nitida, that are found in streams in two mangrove systems in Bertioga and Cananéia, Brazil. Bacillus was the most frequently isolated genus, comprising 42% of the species isolated from Cananéia and 28% of the species from Bertioga. However, other common endophytic genera such as Pantoea, Curtobacterium and Enterobacter were also found. After identifying the isolates, the bacterial communities were evaluated for enzyme production. Protease activity was observed in 75% of the isolates, while endoglucanase activity occurred in 62% of the isolates. Bacillus showed the highest activity rates for amylase and esterase and endoglucanase. To our knowledge, this is the first reported diversity analysis performed on endophytic bacteria obtained from the branches of mangrove trees and the first overview of the specific enzymes produced by different bacterial genera. This work contributes to our knowledge of the microorganisms and enzymes present in mangrove ecosystems.

Methylobacteriumstrains were isolated from mangrove samples collected in Bertioga, SP, Brazil, from locations either contaminated or uncontaminated by oil spills. The tolerances of the strains to different heavy metals were assessed by exposing them to different concentrations of cadmium, lead, and arsenic (0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM). Additionally, the genetic diversity ofMethylobacteriumspp. was determined by sequence analysis of the 16S rRNA genes. The isolates from the contaminated locations were grouped, suggesting that oil can select for microorganisms that tolerate oil components and can change the methylotrophic bacterial community. Cadmium is the most toxic heavy metal assessed in this work, followed by arsenic and lead, and two isolates ofMethylobacteriumwere found to be tolerant to all three metals. These isolates have the potential to bioremediate mangrove environments contaminated by oil spills by immobilizing the heavy metals present in the oil.

Plant-associated fungi are considered a vast source for biotechnological processes whose potential has been poorly explored. The interactions and diversity of sugarcane, one of the most important crops in Brazil, have been rarely studied, mainly concerning fungal communities and their interactions with transgenic plants. Taking this into consideration, the purpose of this study was, based on culture dependent strategy, to determine the structure and diversity of the fungal community (root endophytes and rhizosphere) associated with two varieties of sugarcane, a non-genetically modified (SP80-1842) variety and its genetically modified counterpart (IMI-1, expressing imazapyr herbicide resistance). For this, the sugarcane varieties were evaluated in three sampling times (3, 10 and 17 months after planting) under two crop management (weeding and herbicide treatments). In addition, a strain of Trichoderma virens, an endophyte isolated from sugarcane with great potential as a biological control, growth promotion and enzyme production agent, was selected for the fungal-plant interaction assays. The results of the isolation, characterization and evaluation of fungal community changes showed that the sugarcane fungal community is composed of at least 35 different genera, mostly in the phylum Ascomycota. Many genera are observed at very low frequencies among a few most abundant genera, some of which were isolated from specific plant sites (e.g., the roots or the rhizosphere). An assessment of the possible effects upon the fungal community showed that the plant growth stage was the only factor that significantly affected the community’s structure. Moreover, if transgenic effects are present, they may be minor compared to other natural sources of variation. The results of interaction studies using the Green fluorescent protein (GFP)-expressing T. virens strain T.v.223 revealed that this fungus did not promote any phenotypic changes in the host plant and was found mostly in the roots where it formed a dense mycelial cover and was able to penetrate the intercellular spaces of the root epidermis upper layers. The ability of T. virens to colonize plant roots suggests a potential for protecting plant health, inhibiting pathogens or inducing systemic resistance.

a b s t r a c tThe species of bacteria associated with the traps of the carnivorous plants Utricularia hydrocarpa Vahl and Genlisea filiformis A. St.-Hil. were identified by analysing 16S rRNA gene libraries. We observed larger bacterial diversity inside the traps of U. hydrocarpa than in G. filiformis. The Clostridium genus (Firmicutes) was the dominant group in G. filiformis, while Aeromonas (␥-Proteobacteria) and Acidobacterium (Acidobacteria) were the dominant genera in U. hydrocarpa. In general, the microbial community observed in these carnivorous plants was composed of Firmicutes (46.8%), Proteobacteria (33.9%), Acidobacteria (9.3%), Actinobacteria (4.4%), Bacteroidetes (0.8%), Chloroflexi (0.4%), Gemmatimonadetes (0.4%), Cyanobacteria (0.4%), Chlamydiae (0.4%) and Tenericutes (0.4%). Only 1.2% of the observed operational taxonomic units (OTU 0.03 ) were shared by U. hydrocarpa and G. filiformis. The present study describes the dominant bacterial species associated with the traps of the carnivorous plant G. filiformis and U. hydrocarpa and briefly discusses the possible role of bacteria in plant prey utilisation.

Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction.

-The work was carried out to test DNA extraction protocols and to characterize populations of Tibraca limbativentris Stål, an important rice insect-pest. Insects were collected in Joinville, Rio do Oeste and Turvo, in Santa Catarina State, and Agudo, Uruguaiana, Pelotas and Palmares do Sul, in Rio Grande do Sul State, and six literature-referenced protocols, besides a new one, were tested. DNA from ten individuals of each population was extracted using the best protocol and RAPD reactions were carried out with ten initiators. The new protocol showed the best results and was used in the PCR reactions, that generated 151 polymorphic bands, allowing to access genetic differences among all the populations; no individuals from one population were clustered with individuals from another. The largest intrapopulacional similarity was found in Uruguaiana (22%), and the smallest in Palmares do Sul (50%), which was also the most divergent population in relation to the others. The Gst was 0.5215, and the Nm was 0.4588; these values refl ect the low similarity between the populations. The smallest genic fl ow was obtained when Palmares do Sul and Pelotas were included in the comparisons, in accordance with the largest divergence of these two populations in relation to the others. There was no signifi cant relation between geographic distance and genetic similarity, which can refl ect unknown model of dispersion of T. limbativentris. New studies exploring the species dispersion strategies may help to understand the insect distribution and to unveil the main factors linked to the genetic variability within and between populations.KEY WORDS: Insecta, rice stem bug, irrigated rice, molecular marker RESUMO -O trabalho objetivou testar protocolos de extração de DNA e caracterizar populações de Tibraca limbativentris, Stål, importante inseto-praga do arroz. Os insetos foram coletados em Joinville, Rio do Oeste e Turvo, em Santa Catarina, e Agudo, Uruguaiana, Pelotas e Palmares do Sul, no Rio Grande do Sul. Testaram-se seis protocolos de extração de DNA citados na literatura, e um novo protocolo adequado à espécie em questão. DNA de dez indivíduos de cada população foi extraído usando o melhor protocolo e reações de RAPD foram realizadas com dez iniciadores. O novo protocolo mostrou os melhores resultados e foi utilizado nas reações de PCR, que geraram 151 bandas polimórfi cas, permitindo acessar diferenças genéticas entre todas as populações; não ocorreram indivíduos de uma população agrupados com os de outra. A maior similaridade intrapopulacional foi encontrada em Uruguaiana (22%), e a menor em Palmares do Sul (50%), também a população mais divergente das demais. O valor Gst foi 0,5215, e de Nm 0,4588; esses valores refl etem a pouca similaridade entre as populações. O menor Nm foi apresentado quando Palmares do Sul e Pelotas foram incluídos nas comparações, em consonância com a maior divergência apresentada por essas populações em relação às outras. Não se observou relação entre a distância geográfi ca e a similaridade ...