Response of the microbial community associated with sweet potato (Ipomoea batatas) to Bacillus safensis and Bacillus velezensis strains.

Sweet potato is a subsistence crop cultivated worldwide. Although it is generally considered tolerant to different diseases, it is quite susceptible to the fungus Plenodomus destruens that causes foot-rot disease. Plant growth-promoting bacteria associated with sweet potato remain poorly studied, but some Bacillus strains may have potential as biological control agents. Here, we evaluate the persistence of two bacterial strains-Bacillus safensis T052-76 and Bacillus velezensis T149-19-in pot experiments and assess their impact on indigenous bacterial and fungal communities associated with sweet potato. Numbers of cells of both strains introduced into pots remained stable in the rhizosphere of sweet potato over the 180-day experiment. Denaturing gradient gel electrophoresis based on the rrs gene encoding bacterial 16S rRNA and the fungal ribosomal internal transcribed spacer region showed that bands corresponding to the introduced strains were not detected in plant endosphere. PERMANOVA and non-metric multidimensional scaling statistical analyses showed that: (1) strain T052-76 altered the structure of the indigenous bacterial community (rhizosphere and soil) more than strain T149-19; (2) T052-76 slightly altered the structure of the indigenous fungal community (rhizosphere and soil) and (3) strain T149-19 did not disturb the fungal community. Our results demonstrate the stability of both Bacillus strains in the sweet potato rhizosphere and, apart from the influence of B. safensis T052-76 on the bacterial community, their limited impact on the microbial community associated with this important crop plant.

The Endophytic Bacterial Microbiota Associated with Sweet Sorghum (Sorghum bicolor) Is Modulated by the Application of Chemical N Fertilizer to the Field.

Sweet sorghum (Sorghum bicolor) is a multipurpose crop used as a feedstock to produce bioethanol, sugar, energy, and animal feed. However, it requires high levels of N fertilizer application to achieve the optimal growth, which causes environmental degradation. Bacterial endophytes, which live inside plant tissues, play a key role in the health and productivity of their host. This particular community may be influenced by different agronomical practices. The aim of the work was to evaluate the effects of N fertilization on the structure, diversity, abundance, and composition of endophytic and diazotrophic bacterial community associated with field-grown sweet sorghum. PCR-DGGE, quantitative PCR, and high-throughput sequencing were performed based on the amplification of rrs and nifH genes. The level of N fertilization affected the structure and abundance but not the diversity of the endophytic bacterial communities associated with sweet sorghum plants. This effect was pronounced in the roots of both bacterial communities analyzed and may depend on the physiological state of the plants. Specific bacterial classes and genera increased or decreased when the fertilizer was applied. The data obtained here contribute to a better understanding on the effects of agronomical practices on the microbiota associated with this important crop, with the aim to improve its sustainability.

2,3-Butanediol production by the non-pathogenic bacterium Paenibacillus brasilensis.

2,3-Butanediol (2,3-BDO) is of considerable importance in the chemical, plastic, pharmaceutical, cosmetic, and food industries. The main bacterial species producing this compound are considered pathogenic, hindering large-scale productivity. The species Paenibacillus brasilensis is generally recognized as safe (GRAS) and is phylogenetically similar to P. polymyxa, a species widely used for 2,3-BDO production. Here, we demonstrate, for the first time, that P. brasilensis strains produce 2,3-BDO. Total 2,3-BDO concentrations for 15 P. brasilensis strains varied from 5.5 to 7.6 g/l after 8 h incubation at 32 °C in modified YEPD medium containing 20 g/l glucose. Strain PB24 produced 8.2 g/l of 2,3-BDO within a 12-h growth period, representing a yield of 0.43 g/g and a productivity of 0.68 g/l/h. An increase in 2,3-BDO production by strain PB24 was observed using higher concentrations of glucose, reaching 27 g/l of total 2,3-BDO in YEPD containing about 80 g/l glucose within a 72-h growth period. We sequenced the genome of P. brasilensis PB24 and uncovered at least six genes related to the 2,3-BDO pathway at four distinct loci. We also compared gene sequences related to the 2,3-BDO pathway in P. brasilensis PB24 with those of other spore-forming bacteria, and found strong similarity to P. polymyxa, P. terrae, and P. peoriae 2,3-BDO-related genes. Regulatory regions upstream of these genes indicated that they are probably co-regulated. Finally, we propose a production pathway from glucose to 2,3-BDO in P. brasilensis PB24. Although the gene encoding S-2,3-butanediol dehydrogenase (butA) was found in the genome of P. brasilensis PB24, only R,R-2,3- and meso-2,3-butanediol were detected by gas chromatography under the growth conditions tested here. Our findings can serve as a basis for further improvements to the metabolic capabilities of this little-studied Paenibacillus species in relation to production of the high-value chemical 2,3-butanediol.

Generally recognized as safe (GRAS) Lactococcus lactis strains associated with Lippia sidoides Cham. are able to solubilize/mineralize phosphate.

Eight strains isolated from the stems of Lippia sidoides were identified as belonging to Lactococcus lactis, a bacterial species considered as "generally recognized as safe". Their capacity to solubilize/mineralize phosphate was tested in vitro with different inorganic and organic phosphorus (P) sources. All strains were able to solubilize calcium phosphate as an inorganic P source, and the best result was observed with strain 003.41 which solubilized 31 % of this P source. Rock phosphate, a mined rock containing high amounts of phosphate bearing minerals, was solubilized by five strains. When calcium phytate was the organic P source used, the majority of the strains tested showed phosphate mineralization activity. Moreover, all strains were able to solubilize/mineralize phosphate from poultry litter, a complex P source containing inorganic and predominantly organic P. The presence of genes coding for phytase and alkaline phosphatase was searched within the strains studied. However, only gene sequences related to alkaline phosphatase (phoA and phoD) could be detected in the majority of the strains (excepting strain 006.29) with identities varying from 67 to 88 %. These results demonstrate for the first time the potential of L. lactis strains for phosphate solubilization/mineralization activity using a broad spectrum of P sources; therefore, they are of great importance for the future development of more safe bioinoculants with possible beneficial effects for agriculture.

Whole-Genome Sequence of Rummeliibacillus stabekisii Strain PP9 Isolated from Antarctic Soil.

The whole genome of Rummeliibacillus stabekisii PP9, isolated from a soil sample from Antarctica, consists of a circular chromosome of 3,412,092 bp and a circular plasmid of 8,647 bp, with 3,244 protein-coding genes, 12 copies of the 16S-23S-5S rRNA operon, 101 tRNA genes, and 6 noncoding RNAs (ncRNAs).

Monitoring of the Parasite Load in the Digestive Tract of Rhodnius prolixus by Combined qPCR Analysis and Imaging Techniques Provides New Insights into the Trypanosome Life Cycle.

BACKGROUND: Here we report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI). These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection. METHODOLOGY/PRINCIPAL FINDINGS: qPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the T. cruzi repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H). Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3-4 post-infection (p.i.), the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes. CONCLUSIONS/SIGNIFICANCE: These data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence.

Endophytic microbial community in two transgenic maize genotypes and in their near-isogenic non-transgenic maize genotype.

BACKGROUND: Despite all the benefits assigned to the genetically modified plants, there are still no sufficient data available in literature concerning the possible effects on the microbial communities associated with these plants. Therefore, this study was aimed at examining the effects of the genetic modifications of two transgenic maize genotypes (MON810--expressing the insecticidal Bt-toxin and TC1507--expressing the insecticidal Bt-toxin and the herbicide resistance PAT [phosphinothricin-N-acetyltransferase]) on their endophytic microbial communities, in comparison to the microbial community found in the near-isogenic non-transgenic maize (control). RESULTS: The structure of the endophytic communities (Bacteria, Archaea and fungi) and their composition (Bacteria) were evaluated by denaturing gradient gel electrophoresis (DGGE) and the construction of clone libraries, respectively. DGGE analysis and the clone libraries of the bacterial community showed that genotype TC1507 slightly differed from the other two genotypes. Genotype TC1507 showed a higher diversity within its endophytic bacterial community when compared to the other genotypes. Although some bacterial genera were found in all genotypes, such as the genera Burkholderia, Achromobacer and Stenotrophomonas, some were unique to genotype TC1507. Moreover, OTUs associated with Enterobacter predominated only in TC1507 clone libraries. CONCLUSION: The endophytic bacterial community of the maize genotype TC1507 differed from the communities of the maize genotype MON810 and of their near-isogenic parental genotypes (non-Bt or control). The differences observed among the maize genotypes studied may be associated with insertion of the gene coding for the protein PAT present only in the transgenic genotype TC1507.

Does the essential oil of Lippia sidoides Cham. (pepper-rosmarin) affect its endophytic microbial community?

BACKGROUND: Lippia sidoides Cham., also known as pepper-rosmarin, produces an essential oil in its leaves that is currently used by the pharmaceutical, perfumery and cosmetic industries for its antimicrobial and aromatic properties. Because of the antimicrobial compounds (mainly thymol and carvacrol) found in the essential oil, we believe that the endophytic microorganisms found in L. sidoides are selected to live in different parts of the plant. RESULTS: In this study, the endophytic microbial communities from the stems and leaves of four L. sidoides genotypes were determined using cultivation-dependent and cultivation-independent approaches. In total, 145 endophytic bacterial strains were isolated and further grouped using either ERIC-PCR or BOX-PCR, resulting in 76 groups composed of different genera predominantly belonging to the Gammaproteobacteria. The endophytic microbial diversity was also analyzed by PCR-DGGE using 16S rRNA-based universal and group-specific primers for total bacteria, Alphaproteobacteria, Betaproteobacteria and Actinobacteria and 18S rRNA-based primers for fungi. PCR-DGGE profile analysis and principal component analysis showed that the total bacteria, Alphaproteobacteria, Betaproteobacteria and fungi were influenced not only by the location within the plant (leaf vs. stem) but also by the presence of the main components of the L. sidoides essential oil (thymol and/or carvacrol) in the leaves. However, the same could not be observed within the Actinobacteria. CONCLUSION: The data presented here are the first step to begin shedding light on the impact of the essential oil in the endophytic microorganisms in pepper-rosmarin.

Bacterial community associated with the trunk latex of Hancornia speciosa Gomes (Apocynaceae) grown in the northeast of Brazil.

Prevention or cure of different illnesses through the use of plant latex is a worldwide known concept. The antifungal activity of Hancornia speciosa latex has been observed against Candida albicans. However, H. speciosa latex is not a sterile plant exudate and secondary metabolites produced by bacteria could be involved in fungal inhibition. In the present study, the bacterial communities of the latex from three H. speciosa trees were characterized using traditional plating and molecular methods. Twelve strains isolated from the latex samples were clustered into four groups by amplified ribosomal DNA restriction analysis (ARDRA). One representative of each group was sequenced and they were identified as belonging to the genera Bacillus, Klebsiella, Enterobacter and Escherichia. None of the 12 isolates showed antifungal activity against C. albicans. A lack of a microbial origin for the antifungal properties of latex was noted. DGGE profiles generated from each of the three latex samples showed unique patterns. Sequencing of the DGGE bands demonstrated the affiliation with the genera Klebsiella, Pantoea, Enterobacter and Burkholderia. In addition, clone libraries were generated and the phylogenetic distribution of the 50 analyzed clones was similar to that obtained using DGGE. The presence of some potential pathogens should be considered before using H. speciosa latex in folk medicine.

Comparison of the bacterial community and characterization of plant growth-promoting rhizobacteria from different genotypes of Chrysopogon zizanioides (L.) Roberty (vetiver) rhizospheres.

Molecular approaches [PCR-denaturing gradient gel electrophoresis (DGGE)] were used to determine whether three different vetiver (Chrysopogon zizanioides) genotypes, commercially used in Brazil and considered economically important over the world, select specific bacterial populations to coexist in their rhizospheres. DGGE profiles revealed that the predominant rhizospheric bacterial community hardly varies regarding the vetiver genotype. Moreover, using traditional cultivation methods, bacterial strains were isolated from the different rhizospheres. Colonies presenting different morphologies (83) were selected for determining their potential for plant growth promotion. More than half of the strains tested (57.8%) were amplified by PCR using nifH-based primers, specific for the enzyme nitrogenase reductase. The production of siderophores was observed in 88% of the strains, while the production of antimicrobial substances was detected in only 14.5% of the isolates when Micrococcus sp. was used as the indicator strain. Production of indole-3-acetic acid and the solubilization of phosphate were observed in 55.4% and 59% of the isolates, respectively. In total, 44 strains (53%) presented at least three characteristics of plant growth promotion and were submitted to amplified ribosomal DNA restriction analysis. Twenty-four genetic groups were formed at 100% similarity and one representative of each group was selected for their identification by partial 16S rRNA gene sequencing. They were affiliated with the genera Acinetobacter, Comamonas, Chryseobacterium, Klebsiella, Enterobacter, Pantoea, Dyella, Burkholderia, or Pseudomonas. These strains can be considered of great importance as possible biofertilizers in vetiver.

Cyclodextrin production and genetic characterization of cyclodextrin glucanotranferase of Paenibacillus graminis.

Paenibacillus graminis strains were described recently as cyclodextrin (CD) producers. Cyclodextrins are produced by cyclodextrin glucanotransferase (CGTase) which has not been characterized in P. graminis. Similar amounts of alpha- and beta-CDs were produced by P. graminis (MC22.13) and P. macerans (LMD24.10(T)). Primers were designed to sequence the gene encoding CGTase from P. graminis. A phylogenetic tree was constructed and P. graminis CGTase protein showed to be closer (79.4% protein identity) to P. macerans |P31835|. Hybridization studies suggested that the gene encoding CGTase is located in different positions in the genomes of P. macerans and P. graminis.

Evaluation of the diversity of cyclodextrin-producing Paenibacillus graminis strains isolated from roots and rhizospheres of different plants by molecular methods.

To address the diversity of cyclodextrin-producing P. graminis strains isolated from wheat roots and rhizospheres of maize and sorghum sown in Australia, Brazil, and France, restriction fragment length polymorphism analysis of part of genes encoding RNA polymerase (rpoB-RFLP) and DNA gyrase subunit B (gyrB-RFLP) was used to produce genetic fingerprints. A phylogenetic tree based on rpoB gene sequences was also constructed. The isolates originated from Brazil could be separated from those from Australia and France, when data from the rpoB-based phylogenetic tree or gyrB-RFLP were considered. These analyses also allowed the separation of all P. graminis strains studied here into four clusters; one group formed by the strains GJK201 and RSA19T, second group formed by the strains MC22.02 and MC04.21, third group formed by the strains TOD61, TOD 221, TOD302, and TOD111, and forth group formed by all strains isolated from plants sown in Cerrado soil, Brazil. As this last group was formed by strains isolated from sorghum and maize sown in the same soil (Cerrado) in Brazil, our results suggest that the diversity of these P. graminis strains is more affected by the soil type than the plant from where they have been isolated.