Preliminary Screening on Antibacterial Crude Secondary Metabolites Extracted from Bacterial Symbionts and Identification of Functional Bioactive Compounds by FTIR, HPLC and Gas Chromatography-Mass Spectrometry.

Secondary metabolites, bioactive compounds produced by living organisms, can unveil symbiotic relationships in nature. In this study, soilborne entomopathogenic nematodes associated with symbiotic bacteria (Xenorhabdus stockiae and Photorhabdus luminescens) were extracted from solvent supernatant containing secondary metabolites, demonstrating significant inhibitory effects against E. coli, S. aureus, B. subtilus, P. mirabilis, E. faecalis, and P. stutzeri. The characterization of these secondary metabolites by Fourier transforms infrared spectroscopy revealed amine groups of proteins, hydroxyl and carboxyl groups of polyphenols, hydroxyl groups of polysaccharides, and carboxyl groups of organic acids. Furthermore, the obtained crude extracts were analyzed by high-performance liquid chromatography for the basic identification of potential bioactive peptides. Gas chromatography-mass spectrometry analysis of ethyl acetate extracts from Xenorhabdus stockiae identified major compounds including nonanoic acid derivatives, proline, paromycin, octodecanal derivatives, trioxa-5-aza-1-silabicyclo, 4-octadecenal, methyl ester, oleic acid, and 1,2-benzenedicarboxylicacid. Additional extraction from Photorhabdus luminescens yielded functional compounds such as indole-3-acetic acid, phthalic acid, 1-tetradecanol, nemorosonol, 1-eicosanol, and unsaturated fatty acids. These findings support the potential development of novel natural antimicrobial agents for future pathogen suppression.

Photorhabdus antumapuensis sp. nov., a novel symbiotic bacterial species associated with Heterorhabditis atacamensis entomopathogenic nematodes.

One motile, Gram-negative, non-spore-forming and rod-shaped symbiotic bacterium, strain UCH-936T, was isolated from Heterorhabditis atacamensis nematodes. Results of biochemical, physiological, molecular and genomic analyses suggest that it represents a new species, which we propose to name Photorhabdus antumapuensis sp. nov. Digital DNA-DNA hybridization shows that strain UCH-936T is more closely related to Photorhabdus kleinii DSM 23513T, but shares solely 50.5 % similarity, which is below the 70% cut-off value that delimits species boundaries in bacteria. Phylogenetic reconstructions using whole-genome sequences show that strain UCH-936T forms a unique clade, suggesting its novel and distinct taxonomic status again. Similarly, comparative genomic analyses shows that the virulence factor flagella-related gene fleR, the type IV pili-related gene pilL and the vibriobactin-related gene vibE are present in the genome of strain UCH-936T but absent in the genomes of its closest relatives. Biochemically and physiologically, UCH-936T differs also from all closely related Photorhabdus species. Therefore, Photorhabdus antumapuensis sp. nov. is proposed as a new species with the type strain UCH-936T (CCCT 21.06T=CCM 9188T=CCOS 1991T).

Morphological, molecular and ecological characterization of a native isolate of Steinernema feltiae (Rhabditida: Steinernematidae) from southern Chile.

BACKGROUND: Steinernema feltiae is an entomopathogenic nematode used in biological control programs with a global distribution. Populations of this species show phenotypic plasticity derived from local adaptation and vary in different traits, such as location and host penetration. The aim of this work was to describe a Chilean isolate of this nematode species, using integrative approaches. METHODS: Nematode morphological and morphometric studies were conducted along with molecular analysis of nuclear genes. The symbiotic bacterium was also identified by sequencing the 16S rRNA gene. Some ecological characteristics were described, including the temperature requirements for the nematode life cycle and the effect of soil water content for optimal reproduction. RESULTS: Morphometric characterization revealed a large intra-specific variability. The isolate identity was also corroborated with the analysis of nuclear genes. Based on the 16S gene, its symbiont bacteria, Xenorhabdus bovienii, was identified. The lowest, optimal and highest temperatures found to limit the infestation and reproduction on Galleria mellonella were 10, 20 and 30 °C, respectively; the emergence from the host larvae occurred approximately 10 days after inoculation. Differences were observed in offspring, and 120 infective juveniles (IJ)/larva was the most prolific dose at 20 °C. The soil water content did not affect the number of IJ invaders, penetration efficacy and IJ emergence time or offspring per larva, but it caused a delay in achieving full mortality at the permanent wilting point with respect to saturation and field capacity. CONCLUSIONS: For the first time, a Chilean isolate of S. feltiae is described in detail considering morphological, molecular and ecological aspects. The isolate was shown to be efficient in soil containing water, with optimal temperatures ranging from 15 to 25 °C for host infestation and production of an abundant offspring; these characteristics would allow its potential use as control agents in a wide geographical area of the country.

The Response of Red Clover (Trifolium pratense L.) to Separate and Mixed Inoculations with Rhizobium leguminosarum and Azospirillum brasilense in Presence of Polycyclic Aromatic Hydrocarbons.

This study aimed to evaluate the impact of co-inoculation Rhizobium sp. and Azospirillum sp. on plant (Trifolium pratense L.) growth in the presence of polycyclic aromatic hydrocarbon (PAH) contamination (anthracene, phenanthrene, and pyrene). Eight strains from the genus Rhizobium leguminosarum bv. trifolii were selected for biotest analysis. Two methods of inoculation were used in the chamber experiment: (1) R. leguminosarum alone and (2) a combined inoculant (R. leguminosarum and Azospirillum brasilense). For comparison, non-contaminated controls were also used. The results demonstrated that co-inoculation of plants with Rhizobium and Azospirillum resulted in more root and shoot biomass than in plants inoculated with R. leguminosarum alone. The results indicated that application of a co-inoculation of bacteria from Rhizobium and Azospirillum species had a positive effect on clover nodulation and growth under the condition of PAH contamination.

Occurrence of diverse Bradyrhizobium spp. in roots and rhizospheres of two commercial Brazilian sugarcane cultivars.

The genus Bradyrhizobium harbors many endosymbionts of legumes, but recent research has shown their widespread presence in soils and in non-legumes, notably in roots of sugarcane. This study aimed to investigate the Bradyrhizobium sp. community density in the endosphere and the rhizosphere of two commercial sugarcane cultivars. Samples of the rhizosphere and root endosphere of two Brazilian sugarcane cultivars (RB867515 and IACSP95-5000) were collected, serially diluted, and inoculated on axenic cowpea (Vigna unguiculata) and the induction of nodules was evaluated. Based on the results, a density was estimated of at least 1.6 × 104 rhizobia g root-1 in rhizosphere samples and up to 105 rhizobia g root -1 in endosphere. BOX-PCR profiling of 93 Bradyrhizobium isolates revealed genetic variability, with some dominant (up to 18 representants) and less dominant genotypes. 16S rRNA and ITS sequence analyses confirmed nine phylotypes, six of which pertained to the B. elkanii clade and three to the B. japonicum clade. Five isolates were genetically similar to the recently described species B. sacchari. There was no effect of the factors "plant cultivar" and "root compartment" on Bradyrhizobium sp. community composition and the most abundant genotypes occurred both in rhizosphere and endosphere of both cultivars. Therefore, this study confirms the natural presence of diverse Bradyrhizobium spp. in sugarcane root systems (mainly the rhizosphere) and indicates that certain Bradyrhizobium phylotypes have a special affinity for sugarcane root colonization.

Native rhizobia from Zn mining soil promote the growth of Leucaena leucocephala on contaminated soil.

Plants on contaminated mining soils often show a reduced growth due to nutrient depletion as well as trace elements (TEs) toxicity. Since those conditions threat plant's survival, plant growth-promoting rhizobacteria (PGPRs), such as rhizobia, might be of crucial importance for plant colonization on TE-contaminated soils. Native rhizobia from mining soils are promising candidates for bioaugmented phytoremediation of those soils as they are adapted to the specific conditions. In this work, rhizobia from Zn- and Cd-contaminated mining soils were in vitro screened for their PGP features [organic acids, indole-3-acetic acid (IAA), and siderophore (SID) production; 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity; and Ca3(PO4)2 solubilization] and Zn and Cd tolerance. In addition, some type and reference rhizobia strains were included in the study as well. The in vitro screening indicated that rhizobia and other native genera have great potential for phytoremediation purposes, by exerting, besides biological N2 fixation, other plant growth-promoting traits. Leucaena leucocephala-Mesorhizobium sp. (UFLA 01-765) showed multielement tolerance and an efficient symbiosis on contaminated soil, decreasing the activities of antioxidative enzymes in shoots. This symbiosis is a promising combination for phytostabilization.

Microbiota from Rhabditis regina may alter nematode entomopathogenicity.

Here we report the presence of the entomopathogenic nematode Rhabditis (Rhabditoides) regina affecting white grubs (Phyllophaga sp. and Anomala sp.) in Mexico and R. regina-associated bacteria. Bioassays were performed to test the entomopathogenic capacity of dauer and L2 and L3 (combined) larval stages. Furthermore, we determined the diversity of bacteria from laboratory nematodes cultivated for 2 years (dauer and L2-L3 larvae) and from field nematodes (dauer and L2-L3 larvae) in addition to the virulence in Galleria mellonella larvae of some bacterial species from both laboratory and field nematodes. Dauer and non-dauer larvae of R. regina killed G. mellonella. Bacteria such as Serratia sp. (isolated from field nematodes) and Klebsiella sp. (isolated from larvae of laboratory and field nematodes) may explain R. regina entomopathogenic capabilities. Different bacteria were found in nematodes after subculturing in the laboratory suggesting that R. regina may acquire bacteria in different environments. However, there were some consistently found bacteria from laboratory and field nematodes such as Pseudochrobactrum sp., Comamonas sp., Alcaligenes sp., Klebsiella sp., Acinetobacter sp., and Leucobacter sp. that may constitute the nematode microbiome. Results showed that some bacteria contributing to entomopathogenicity may be lost in the laboratory representing a disadvantage when nematodes are cultivated to be used for biological control.

Oil spill dispersants induce formation of marine snow by phytoplankton-associated bacteria.

Unusually large amounts of marine snow, including Extracellular Polymeric Substances (EPS), were formed during the 2010 Deepwater Horizon oil spill. The marine snow settled with oil and clay minerals as an oily sludge layer on the deep sea floor. This study tested the hypothesis that the unprecedented amount of chemical dispersants applied during high phytoplankton densities in the Gulf of Mexico induced high EPS formation. Two marine phytoplankton species (Dunaliella tertiolecta and Phaeodactylum tricornutum) produced EPS within days when exposed to the dispersant Corexit 9500. Phytoplankton-associated bacteria were shown to be responsible for the formation. The EPS consisted of proteins and to lesser extent polysaccharides. This study reveals an unexpected consequence of the presence of phytoplankton. This emphasizes the need to test the action of dispersants under realistic field conditions, which may seriously alter the fate of oil in the environment via increased marine snow formation.

Rhizobia and other legume nodule bacteria richness in brazilian Araucaria angustifolia forest

The Araucaria Forest is a sub-type of the Atlantic Forest, dominated by Araucaria angustifolia, which is considered an endangered species. The understory has a high diversity of plant species, including several legumes. Many leguminous plants nodulate with rhizobia and fix atmospheric nitrogen, contributing to forest sustainability. This work aimed at bacteria isolation and phenotypic characterization from the root nodules of legumes occurring in Araucaria Forests, at Campos do Jordão State Park, Brazil. Nodule bacteria were isolated in YMA growth media and the obtained colonies were classified according to their growth characteristics (growth rate, color, extra cellular polysaccharide production and pH change of the medium). Data were analyzed by cluster and principal components analysis (PCA). From a total of eleven collected legume species, nine presented nodules, and this is the first report on nodulation of five of these legume species. Two hundred and twelve bacterial strains were isolated from the nodules, whose nodule shapes varied widely and there was a great phenotypic richness among isolates. This richness was found among legume species, individuals of the same species, different nodule shapes and even among isolates of the same nodule. These isolates could be classified into several groups, two up to six according to each legume, most of them different from the used growth standards Rhizobium tropici, Bradyrhizobium elkanii and Burkholderia sp. There is some evidence that these distinct groups may be related to the presence of Burkholderia spp. in the nodules of these legumes.

A Floresta de Araucária é um sub-tipo da Mata Atlântica, cujo dossel é dominado por Araucaria angustifolia, uma espécie ameaçada de extinção. O sub-bosque dessa floresta tem alta diversidade, incluindo muitas espécies de leguminosas. Estas plantas podem formar nódulos e fixar nitrogênio atmosférico, contribuindo para a sustentabilidade da floresta. Efetuou-se o levantamento de leguminosas no Parque Estadual de Campos do Jordão e o isolamento de bactérias dos nódulos radiculares destas plantas, seguido da caracterização fenotípica dos isolados. As bactérias dos nódulos foram isoladas em meio de cultura YMA, sendo classificadas de acordo com suas características de crescimento (velocidade de crescimento, cor, produção de polissacarídeo extracelular e mudança de pH do meio). Os resultados foram analisados por análise de agrupamento e análise de componentes principais (PCA). De um total de onze espécies de leguminosas, nove apresentaram nódulos, sendo seis espécies descritas como nodulantes pela primeira vez. Duzentas e doze estirpes de bactérias foram isoladas, havendo variação no formato de nódulos e alta riqueza fenotípica das bactérias isoladas. Essa riqueza ocorreu entre as espécies de leguminosas, entre indivíduos da mesma espécie, entre diferentes formatos de nódulos e, inclusive, entre bactérias isoladas de um mesmo nódulo. As bactérias puderam ser classificadas em vários grupos, de dois a seis de acordo com cada leguminosa, a maioria deles diferentes de Rhizobium tropici, Bradyrhizobium elkanii e Burkholderia sp., que foram utilizados como padrões de crescimento. Existem algumas evidências de que estes grupos distintos podem estar relacionados à presença de Burkholderia spp. nos nódulos destas leguminosas.

Rhizobia and other legume nodule bacteria richness in brazilian Araucaria angustifolia forest

The Araucaria Forest is a sub-type of the Atlantic Forest, dominated by Araucaria angustifolia, which is considered an endangered species. The understory has a high diversity of plant species, including several legumes. Many leguminous plants nodulate with rhizobia and fix atmospheric nitrogen, contributing to forest sustainability. This work aimed at bacteria isolation and phenotypic characterization from the root nodules of legumes occurring in Araucaria Forests, at Campos do Jordão State Park, Brazil. Nodule bacteria were isolated in YMA growth media and the obtained colonies were classified according to their growth characteristics (growth rate, color, extra cellular polysaccharide production and pH change of the medium). Data were analyzed by cluster and principal components analysis (PCA). From a total of eleven collected legume species, nine presented nodules, and this is the first report on nodulation of five of these legume species. Two hundred and twelve bacterial strains were isolated from the nodules, whose nodule shapes varied widely and there was a great phenotypic richness among isolates. This richness was found among legume species, individuals of the same species, different nodule shapes and even among isolates of the same nodule. These isolates could be classified into several groups, two up to six according to each legume, most of them different from the used growth standards Rhizobium tropici, Bradyrhizobium elkanii and Burkholderia sp. There is some evidence that these distinct groups may be related to the presence of Burkholderia spp. in the nodules of these legumes.

A Floresta de Araucária é um sub-tipo da Mata Atlântica, cujo dossel é dominado por Araucaria angustifolia, uma espécie ameaçada de extinção. O sub-bosque dessa floresta tem alta diversidade, incluindo muitas espécies de leguminosas. Estas plantas podem formar nódulos e fixar nitrogênio atmosférico, contribuindo para a sustentabilidade da floresta. Efetuou-se o levantamento de leguminosas no Parque Estadual de Campos do Jordão e o isolamento de bactérias dos nódulos radiculares destas plantas, seguido da caracterização fenotípica dos isolados. As bactérias dos nódulos foram isoladas em meio de cultura YMA, sendo classificadas de acordo com suas características de crescimento (velocidade de crescimento, cor, produção de polissacarídeo extracelular e mudança de pH do meio). Os resultados foram analisados por análise de agrupamento e análise de componentes principais (PCA). De um total de onze espécies de leguminosas, nove apresentaram nódulos, sendo seis espécies descritas como nodulantes pela primeira vez. Duzentas e doze estirpes de bactérias foram isoladas, havendo variação no formato de nódulos e alta riqueza fenotípica das bactérias isoladas. Essa riqueza ocorreu entre as espécies de leguminosas, entre indivíduos da mesma espécie, entre diferentes formatos de nódulos e, inclusive, entre bactérias isoladas de um mesmo nódulo. As bactérias puderam ser classificadas em vários grupos, de dois a seis de acordo com cada leguminosa, a maioria deles diferentes de Rhizobium tropici, Bradyrhizobium elkanii e Burkholderia sp., que foram utilizados como padrões de crescimento. Existem algumas evidências de que estes grupos distintos podem estar relacionados à presença de Burkholderia spp. nos nódulos destas leguminosas.