Draft Genomes, Phylogenetic Reconstruction, and Comparative Genomics of Two Novel Cohabiting Bacterial Symbionts Isolated from Frankliniella occidentalis

Obligate bacterial symbionts are widespread in many invertebrates, where they are often confined to specialized host cells and are transmitted directly from mother to progeny. Increasing numbers of these bacteria are being characterized but questions remain about their population structure and evolution. Here we take a comparative genomics approach to investigate two prominent bacterial symbionts (BFo1 and BFo2) isolated from geographically separated populations of western flower thrips, Frankliniella occidentalis. Our multifaceted approach to classifying these symbionts includes concatenated multilocus sequence analysis (MLSA) phylogenies, ribosomal multilocus sequence typing (rMLST), construction of whole-genome phylogenies, and in-depth genomic comparisons. We showed that the BFo1 genome clusters more closely to species in the genus Erwinia, and is a putative close relative to Erwinia aphidicola. BFo1 is also likely to have shared a common ancestor with Erwinia pyrifoliae/Erwinia amylovora and the nonpathogenic Erwinia tasmaniensis and genetic traits similar to Erwinia billingiae. The BFo1 genome contained virulence factors found in the genus Erwinia but represented a divergent lineage. In contrast, we showed that BFo2 belongs within the Enterobacteriales but does not group closely with any currently known bacterial species. Concatenated MLSA phylogenies indicate that it may have shared a common ancestor to the Erwinia and Pantoea genera, and based on the clustering of rMLST genes, it was most closely related to Pantoea ananatis but represented a divergent lineage. We reconstructed a core genome of a putative common ancestor of Erwinia and Pantoea and compared this with the genomes of BFo bacteria. BFo2 possessed none of the virulence determinants that were omnipresent in the Erwinia and Pantoea genera. Taken together, these data are consistent with BFo2 representing a highly novel species that maybe related to known Pantoea

Beneficial Bacteria Isolated from Grapevine Inner Tissues Shape Arabidopsis thaliana Roots

We investigated the potential plant growth-promoting traits of 377 culturable endophytic bacteria, isolated from Vitis vinifera cv. Glera, as good biofertilizer candidates in vineyard management. Endophyte ability in promoting plant growth was assessed in vitro by testing ammonia production, phosphate solubilization, indole-3-acetic acid (IAA) and IAA-like molecule biosynthesis, siderophore and lytic enzyme secretion. Many of the isolates were able to mobilize phosphate (33%), release ammonium (39%), secrete siderophores (38%) and a limited part of them synthetized IAA and IAA-like molecules (5%). Effects of each of the 377 grapevine beneficial bacteria on Arabidopsis thaliana root development were also analyzed to discern plant growth-promoting abilities (PGP) of the different strains, that often exhibit more than one PGP trait. A supervised model-based clustering analysis highlighted six different classes of PGP effects on root architecture. A. thaliana DR5::GUS plantlets, inoculated with IAA-producing endophytes, resulted in altered root growth and enhanced auxin response. Overall, the results indicate that the Glera PGP endospheric culturable microbiome could contribute, by structural root changes, to obtain water and nutrients increasing plant adaptation and survival. From the complete cultivable collection, twelve promising endophytes mainly belonging to the Bacillus but also to Micrococcus and Pantoea genera, were selected for further investigations in the grapevine host plants towards future application in sustainable management of vineyards

Detection and identification of wheat and barley phytopathogens in the Russian Federation

Grain export is an important branch of the food business in the Russian Federation. The countries of Europe, Asia, Africa, and South America are importers of Russian grain. Each importing country has its own requirements for the phytosanitary condition of imported products. One important requirement for importers is the absence of pathogens that can cause bacterial diseases of grain crops, such as Pectobacterium rhapontici, Rathayibacter tritici, Pseudomonas fuscovaginae, Pseudomonas syringae pvs., Acidovorax avenae subsp. avenae, Xanthomonas translucens pvs., Rathayibacter rathayi, and Pseudomonas cichorii. Reliable information on the distribution of these bacterial strains in the Russian Federation is limited. Methods for the isolation and identification of these bacterial pathogens have not been developed to date, which increases the risk of the spread of phytopathogens that could cause significant economic harm to agriculture.The purpose of this study was to isolate and identify the causative agents of bacterial diseases of wheat and barley. In order to do this, we collected samples of plant material of wheat and barley in the Rodionovo-Nesvetaysky, Myasnikovsky, Zernogradsky, Azovsky, and Martynovsky districts of the Rostov Oblast. Various bacterial strains were isolated from the obtained samples using the appropriate cultural media. The strains were tested by polymerase chain reaction (PCR) using primers designed for the 16S ribosomal RNA (PSF/PSR and 8UA/519B) and SyD1/SyD2 primers selected for the Pseudomonas syringae genome (GenBank CP047267.1) with subsequent sequencing according to the Sanger method. As a result, the following bacterial strains were isolated and identified from wheat and barley samples: Curtobacterium sp., Paenibacillus sp., Enterobacteriaceae, Pseudomonas azotoformans, P. poae, P. azotoformans, P. hibiscicola, P. fluorescens, Stenotrophomonas sp., P. syringae pv. syringae, P. syringae pv. atrofaciens, Bacillus sp., Erwinia sp., Pantoea sp., and Pantoea agglomerans.Grain export is an important branch of the food business in the Russian Federation. The countries of Europe, Asia, Africa, and South America are importers of Russian grain. Each importing country has its own requirements for the phytosanitary condition of imported products. One important requirement for importers is the absence of pathogens that can cause bacterial diseases of grain crops, such as Pectobacterium rhapontici, Rathayibacter tritici, Pseudomonas fuscovaginae, Pseudomonas syringae pvs., Acidovorax avenae subsp. avenae, Xanthomonas translucens pvs., Rathayibacter rathayi, and Pseudomonas cichorii. Reliable information on the distribution of these bacterial strains in the Russian Federation is limited. Methods for the isolation and identification of these bacterial pathogens have not been developed to date, which increases the risk of the spread of phytopathogens that could cause significant economic harm to agriculture.The purpose of this study was to isolate and identify the causative agents of bacterial diseases of wheat and barley. In order to do this, we collected samples of plant material of wheat and barley in the Rodionovo-Nesvetaysky, Myasnikovsky, Zernogradsky, Azovsky, and Martynovsky districts of the Rostov Oblast. Various bacterial strains were isolated from the obtained samples using the appropriate cultural media. The strains were tested by polymerase chain reaction (PCR) using primers designed for the 16S ribosomal RNA (PSF/PSR and 8UA/519B) and SyD1/SyD2 primers selected for the Pseudomonas syringae genome (GenBank CP047267.1) with subsequent sequencing according to the Sanger method. As a result, the following bacterial strains were isolated and identified from wheat and barley samples: Curtobacterium sp., Paenibacillus sp., Enterobacteriaceae, Pseudomonas azotoformans, P. poae, P. azotoformans, P. hibiscicola, P. fluorescens, Stenotrophomonas sp., P. syringae pv. syringae, P. syringae pv. atrofaciens, Bacillus sp., Erwinia sp., Pantoea sp., and Pantoea agglomerans

The complete genome sequence of Pantoea ananatis AJ13355, an organism with great biotechnological potential

Pantoea ananatis AJ13355 is a newly identified member of the Enterobacteriaceae family with promising biotechnological applications. This bacterium is able to grow at an acidic pH and is resistant to saturating concentrations of L-glutamic acid, making this organism a suitable host for the production of L-glutamate. In the current study, the complete genomic sequence of P. ananatis AJ13355 was determined. The genome was found to consist of a single circular chromosome consisting of 4,555,536 bp [DDBJ: AP012032] and a circular plasmid, pEA320, of 321,744 bp [DDBJ: AP012033]. After automated annotation, 4,071 protein-coding sequences were identified in the P. ananatis AJ13355 genome. For 4,025 of these genes, functions were assigned based on homologies to known proteins. A high level of nucleotide sequence identity (99%) was revealed between the genome of P. ananatis AJ13355 and the previously published genome of P. ananatis LMG 20103. Short colinear regions, which are identical to DNA sequences in the Escherichia coli MG1655 chromosome, were found to be widely dispersed along the P. ananatis AJ13355 genome. Conjugal gene transfer from E. coli to P. ananatis, mediated by homologous recombination between short identical sequences, was also experimentally demonstrated. The determination of the genome sequence has paved the way for the directed metabolic engineering of P. ananatis to produce biotechnologically relevant compounds

Influence of Diet, Sex, and Viral Infections on the Gut Microbiota Composition of Spodoptera exigua Caterpillars

The gut microbiota plays essential roles in processes related with metabolism, physiology, and immunity in all organisms, including insects. In the present work, we performed a broad analysis of the Spodoptera exigua gut microbiota, a major agricultural pest. We analyzed the influence of multiple parameters such as diet, geographic location, sex, or viral infections on S. exigua caterpillar gut microbiota composition. Our study revealed a high variability in bacterial composition among individuals, and a major influence of environmental bacteria (including those acquired through diet) on the gut microbiota composition, supporting previous studies that claim resident microbiota are lacking in caterpillars. Previous studies with laboratory-reared insects showed that changes in caterpillar gut bacterial composition affect the insecticidal properties of entomopathogenic viruses and bacteria. Our study revealed different microbiota composition in field insects carrying a natural viral infection with Spodoptera exigua nucleopolyhedrovirus (SeMNPV) and/or Spodoptera exigua iflavirus 1 (SeIV1). Few taxa can be specifically associated with the infection, suggesting microbiota influence the infective process of these natural pathogens, and providing new strategies for insect pest management.This work has been supported by projects from the Spanish Ministry of Science, Innovation and Universities (No. AGL2014-57752-C2-2R and RTI2018-094350-B-C32) and the VIROPLANT project which has received funding from the European Union’s Horizon 2020 Research and Innovation Program under grant agreement no. 774208Peer reviewe

Broad spectrum microarray for fingerprint-based bacterial species identification

BACKGROUND: Microarrays are powerful tools for DNA-based molecular diagnostics and identification of pathogens. Most target a limited range of organisms and are based on only one or a very few genes for specific identification. Such microarrays are limited to organisms for which specific probes are available, and often have difficulty discriminating closely related taxa. We have developed an alternative broad-spectrum microarray that employs hybridisation fingerprints generated by high-density anonymous markers distributed over the entire genome for identification based on comparison to a reference database. RESULTS: A high-density microarray carrying 95,000 unique 13-mer probes was designed. Optimized methods were developed to deliver reproducible hybridisation patterns that enabled confident discrimination of bacteria at the species, subspecies, and strain levels. High correlation coefficients were achieved between replicates. A sub-selection of 12,071 probes, determined by ANOVA and class prediction analysis, enabled the discrimination of all samples in our panel. Mismatch probe hybridisation was observed but was found to have no effect on the discriminatory capacity of our system. CONCLUSIONS: These results indicate the potential of our genome chip for reliable identification of a wide range of bacterial taxa at the subspecies level without laborious prior sequencing and probe design. With its high resolution capacity, our proof-of-principle chip demonstrates great potential as a tool for molecular diagnostics of broad taxonomic groups

Oligo-DNA Custom Macroarray for Monitoring Major Pathogenic and Non-Pathogenic Fungi and Bacteria in the Phyllosphere of Apple Trees

BACKGROUND: To monitor the richness in microbial inhabitants in the phyllosphere of apple trees cultivated under various cultural and environmental conditions, we developed an oligo-DNA macroarray for major pathogenic and non-pathogenic fungi and bacteria inhabiting the phyllosphere of apple trees. METHODS AND FINDINGS: First, we isolated culturable fungi and bacteria from apple orchards by an agar-plate culture method, and detected 32 fungal and 34 bacterial species. Alternaria, Aureobasidium, Cladosporium, Rhodotorula, Cystofilobasidium, and Epicoccum genera were predominant among the fungi, and Bacillus, Pseudomonas, Sphingomonas, Methylobacterium, and Pantoea genera were predominant among the bacteria. Based on the data, we selected 29 major non-pathogenic and 12 phytopathogenic fungi and bacteria as the targets of macroarray. Forty-one species-specific 40-base pair long oligo-DNA sequences were selected from the nucleotide sequences of rDNA-internal transcribed spacer region for fungi and 16S rDNA for bacteria. The oligo-DNAs were fixed on nylon membrane and hybridized with digoxigenin-labeled cRNA probes prepared for each species. All arrays except those for Alternaria, Bacillus, and their related species, were specifically hybridized. The array was sensitive enough to detect 10(3) CFU for Aureobasidium pullulans and Bacillus cereus. Nucleotide sequencing of 100 each of independent fungal rDNA-ITS and bacterial 16S-rDNA sequences from apple tree was in agreement with the macroarray data obtained using the same sample. Finally, we analyzed the richness in the microbial inhabitants in the samples collected from apple trees in four orchards. Major apple pathogens that cause scab, Alternaria blotch, and Marssonina blotch were detected along with several non-phytopathogenic fungal and bacterial inhabitants. CONCLUSIONS: The macroarray technique presented here is a strong tool to monitor the major microbial species and the community structures in the phyllosphere of apple trees and identify key species antagonistic, supportive or co-operative to specific pathogens in the orchard managed under different environmental conditions

Assessment of Azotobacter chroococcum’s Ability to Produce Hydrolytic Enzymes and Enhance Growth of Vicia faba

A. chroococcum isolate was examined for its ability to produce the hydrolytic enzymes chitinase, pectinase, protease, and lipase, in an effort to enhance the growth of fava bean (Vicia faba). Biological experiment was conducted in pots with complete random design (CRD). The experiment includes three treatments: control (plant without treatment) (P), NPK fertilizer (plant + fertilizer) (PF), and A. chroococcum inoculum (plant + A. chroococcum) (PA). These treatments were performed with sterile and non-sterile soil, which were planted with fava beans. At the end of experiment (seven weeks from planting), length and weight of plant shoot and plant root were calculated. The results show that the isolate was able to produce chitinase, pectinase, protease, and lipase enzymes. Plant growth increased with fertilizer and A. chroococcum inoculum addition. Fertilizer treatments (PF) were the best growth indicators, with the averages of shoot length and weight, and root length and weight of 19.52 cm, 15.55 gm, 19.93 cm, and 10.52 gm respectively.  The averages of these indicators in A. chroococcum treatment (PA) were 16.4 cm, 8.85 gm, 17.52 cm, and 6.88 gm respectively. The percentages of increase caused by A. chroococcum inoculum addition (PA) in contrast with control treatment (P) were 12.83% and 34.4% in plant shoot length and weight respectively, and 60.98% and 38.99% in plant root length and weight. In general, growth indicators were higher in non-sterile than sterile soil, but these increases were not significan

Participation of type VI secretion system in plant colonization of phosphate solubilizing bacteria

In mutualistic endophytic bacteria, the type VI secretion system (T6SS) is related to important functions, such as interbacterial competition, stress response, quorum sensing, biofilm formation, and symbiosis. The presence of T6SS in beneficial endophytic bacterial population associated with different plants suggests that it plays an important role in its interaction with the eucaryotic partner. Within plant promoting bacteria, those with phosphate solubilizing activity constitute a group of great relevance to the rhizosphere as they provide phosphorus to plants. Among them, those with endophytic colonization capacity have survival advantages. The aim of this study was to determine whether the T6SS of a native peanut phosphate solubilizing bacterium is involved in its colonization in this legume. Initially, an in silico analysis looking for genes related to T6SS in the genome of the Enterobacter sp. J49 strain enabled us to identify almost all the tss genes, except for the tssE gene. A T6SS mutant of the Enterobacter sp. J49 strain was obtained by interrupting one of the essential tss genes. Then, the Enterobacter sp. J49-hcp strain was inoculated on peanut plants to analyze its colonization capacity. In addition, properties associated with endophytic colonization were analyzed, such as the formation of biofilms and the production of pectinase and cellulase enzymes. The results obtained indicated a significant decrease in the epiphytic and endophytic colonization of the mutant with respect to the wild strain. It is possible to conclude that T6SS, although not essential, may participate in bacterial colonization, either by accelerating the infection or by promoting other mechanisms involved in it.Fil: Lucero, Cinthia Tamara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Departamento de Química; ArgentinaFil: Lorda, Graciela Susana. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Departamento de Química; ArgentinaFil: Ludueña, Liliana Mercedes. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Nievas, Fiorela Lujan. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Bogino, Pablo Cesar. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Angelini, Jorge Guillermo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones Agrobiotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Agrobiotecnológicas; ArgentinaFil: Ambrosino, Mariela Lis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Pampa. Facultad de Ciencias Exactas y Naturales. Departamento de Química; ArgentinaFil: Taurian, Tania. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; Argentin