Project description:European Bark Beetle Ips typographus is a secondary pest that affects dead and weakened spruce trees (Picea genus). Under certain environmental conditions, it has massive outbreaks, resulting in the attacks of healthy trees, becoming a forest pest. It has been proposed that the bark beetle's microbiome plays a key role in the insect's ecology, providing nutrients, inhibiting pathogens, and degrading tree defense compounds, among other probable traits yet to be discovered. During a study of bacterial associates from I. typographus, we isolated three strains identified as Pseudomonas from different beetle life stages. A polyphasic taxonomical approach showed that they belong to a new species for which the name Pseudomonas typographi sp nov. is proposed. Genome sequences show their potential to hydrolyze wood compounds and synthesize several vitamins; screening for enzymes production was verified using PNP substrates. Assays in Petri dishes confirmed cellulose and xylan hydrolysis. Moreover, the genomes harbor genes encoding chitinases and gene clusters involved in the synthesis of secondary metabolites with antimicrobial potential. In vitro tests confirmed the capability of the three P. typographi strains to inhibit several Ips beetles' pathogenic fungi. Altogether, these results suggest that P. typographi aids I. typographi nutrition and resistance to fungal pathogens.
Project description:During the exploration of microbial natural resources, two strains of Pseudomonas, PS14<sup>T</sup> and PS24<sup>T</sup>, were isolated from samples taken from Izu Oshima, a volcanic island located 120 km southwest of central Tokyo. Phylogenetic analysis based on 16S rRNA gene sequences showed that PS14<sup>T</sup> was most similar to Pseudomonas baetica a390<sup>T</sup> (99.6%) and Pseudomonas helmanticensis OHA11<sup>T</sup> (99.5%), and that PS24<sup>T</sup> was most similar to Pseudomonas qingdaonensis JJ3<sup>T</sup> (98.8%) and Pseudomonas lutea OK2<sup>T</sup> (98.7%). The major fatty acids of these two strains were C<sub>16:0</sub> and C<sub>17:0</sub> cyclo, summed feature 3 (C<sub>16:1</sub> ω6c and/or C<sub>16:1</sub> ω7c), and summed feature 8 (C<sub>18:1</sub> ω7c and/or <sub>18:1</sub> ω6c). The phylogenetic analyses, DNA-DNA hybridization results and phenotypic traits indicated that PS14<sup>T</sup> and PS24<sup>T</sup> constitute two novel species, Pseudomonas atagosis sp. nov. (type strain PS14<sup>T</sup> = CECT 9940<sup>T</sup>, = LMG 31496<sup>T</sup>) and Pseudomonas akappagea sp. nov. (type strain PS24<sup>T</sup> = CECT 9941<sup>T</sup>, = LMG 31497<sup>T</sup>), respectively. The sequence data of the draft genomes of PS14<sup>T</sup> and PS24<sup>T</sup> were deposited in the GenBank database under accession numbers VXCA00000000 and VXCP00000000, respectively, and the sequence data of their 16S rRNA genes were deposited in the GenBank database under accession numbers MN396717 and MN382268, respectively.
Project description:Antimicrobial resistance is a worldwide problem that threatens the effectiveness of treatments for microbial infection. Consequently, it is essential to study unexplored niches that can serve for the isolation of new microbial strains able to produce antimicrobial compounds to develop new drugs. Bark beetles live in phloem of host trees and establish symbioses with microorganisms that provide them with nutrients. In addition, some of their associated bacteria play a role in the beetle protection by producing substances that inhibit antagonists. In this study the capacity of several bacterial strains, isolated from the bark beetles Ips acuminatus, Pityophthorus pityographus Cryphalus piceae, and Pityogenes bidentatus, to produce antimicrobial compounds was analyzed. Several isolates exhibited the capacity to inhibit Gram-positive and Gram-negative bacteria, as well as fungi. The genome sequence analysis of three Pseudomonas isolates predicted the presence of several gene clusters implicated in the production of already described antimicrobials and moreover, the low similarity of some of these clusters with those previously described, suggests that they encode new undescribed substances, which may be useful for developing new antimicrobial agents. Moreover, these bacteria appear to have genetic machinery for producing antitumoral and antiviral substances. Finally, the strain IA19T showed to represent a new species of the genus Pseudomonas. The 16S rRNA gene sequence analysis showed that its most closely related species include Pseudomonas lutea, Pseudomonas graminis, Pseudomonas abietaniphila and Pseudomonas alkylphenolica, with 98.6, 98.5 98.4, and 98.4% identity, respectively. MLSA of the housekeeping genes gyrB, rpoB, and rpoD confirmed that strain IA19T clearly separates from its closest related species. Average nucleotide identity between strains IA19T and P. abietaniphila ATCC 700689T, P. graminis DSM 11363T, P. alkylphenolica KL28T and P. lutea DSM 17257T were 85.3, 80.2, 79.0, and 72.1%, respectively. Growth occurs at 4-37°C and pH 6.5-8. Optimal growth occurs at 28°C, pH 7-8 and up to 2.5% NaCl. Respiratory ubiquinones are Q9 (97%) and Q8 (3%). C16:0 and in summed feature 3 are the main fatty acids. Based on genotypic, phenotypic and chemotaxonomic characteristics, the description of Pseudomonas bohemica sp. nov. has been proposed. The type strain is IA19T (=CECT 9403T = LMG 30182T).
Project description:Ips subelongatus is a major pest that infects larch plantations over large areas of northern and northeastern China. Ips species are closely associated with ophiostomatoid fungi that are morphologically well-adapted for dispersal by beetles. These associations result in important threat for coniferous forests worldwide. The aim of this study was to characterize the ophiostomatoid communities associated with I. subelongatus infesting Larix species and sympatric Pinus sylvestris var. mongolica in northeastern China forests. Morphological and multilocus phylogenetic approaches (based on six markers: ITS, LSU, 60S, β-tubulin, EF-1α, and CAL gene regions) allowed identifying 14 species of four genera (Ceratocystiopsis, Endoconidiophora, Leptographium and Ophiostoma). Eight species are showed to be new to science. Most strains resided in two Ophiostoma species complexes, viz. the O. clavatum and the O. ips complexes, all together accounting for 76.8% of all isolates. Ophiostoma hongxingense sp. nov., O. peniculi sp. nov., and O. subelongati sp. nov. (O. clavatum complex) and O. pseudobicolor sp. nov. (O. ips complex) were the four dominant species. The ophiostomatoid communities associated with larch bark beetles, I. cembrae and I. subelongatus, in Europe and Asia, China and Japan, also were compared. These comparisons showed distinct, specific assemblage patterns.
Project description:The timber and pulp industries of Finland rely heavily on importations from Russia as source of raw timber. These imports raise the risk of accidentally importing forest pests and pathogens, especially bark beetles and their associated fungi, into Finland. Although ophiostomatoid fungi have previously been reported from Finland and Russia, the risks of accidentally moving these fungi has prompted a first survey to compare the diversity of conifer-infesting bark beetles and associated fungi from boreal forests on both sides of the Finnish-Russian border. The aim of the present study was to identify and characterise Ophiostoma species isolated in association with 11 bark beetle species infesting Pinus sylvestris and Picea abies during this survey in the eastern parts of Finland and neighbouring Russia. Fungal isolates were grouped based on morphology and representatives of each morphological group were subjected to DNA sequence comparisons of the internal transcribed spaced region (ITS1, 5.8S, ITS2) and ?-tubulin gene region. A total of 15 species of Ophiostoma were identified, including seven known species, five new species, and three species for which the identity remains uncertain. In the O. piceae-complex we identified O. canum, O. floccosum, O. karelicum and O. rachisporum sp. nov., and related to these, some isolates belonging to the European clade of O. minus in the O. minus-complex. Ophiostoma bicolor and O. fuscum sp. nov. were identified in the O. ips-complex, while O. ainoae, O. brunneo-ciliatum, O. tapionis sp. nov. and O. pallidulum sp. nov. were shown to group close to, but not in a strict monophyletic lineage with species of the O. ips-complex. Together with a single O. abietinum-like isolate, the only species that grouped close to the Sporothrix schenckii- O. stenoceras complex, was O. saponiodorum sp. nov.
Project description:The ophiostomatoid fungi (Microascales and Ophiostomatales, Ascomycota) are common associates of Ips typographus, and include tree pathogens and species responsible for blue-stain of timber. Fungal assemblages associated with I. typographus have varied considerably between studies but few investigations have attempted to explain this variation. For this reason, we assessed the overall cultivable fungal diversity associated with I. typographus in a storm-felled spruce forest in south-eastern Finland. Fungi were isolated from the individually collected beetles as well as their phoretic mites in spring, summer and autumn, including different life stages of the beetle (hibernation, dispersal flight and first generation). The internal transcribed spacer (ITS) gene region was used to identify the fungi. A total of 32 operational taxonomic units (OTUs) were found and these resided in four fungal phyla/subphyla (24 Ascomycota, 2 Basidiomycota, 5 Mucoromycotina, 1 Mortierellomycotina) in association with adult bark beetles. Ophiostomatoid species were the most commonly detected fungal associates. A generalized linear model analysis showed a clear association between fungal communities and season, indicating seasonal succession among I. typographus-associated fungi. The season of sampling appears to be an important factor that has resulted in inconsistencies between results in previous studies. Many of these fungi were also found on phoretic mites and their presence or absence could have influenced variation in patterns of association.
Project description:<i>Xanthomonas translucens</i> is the etiological agent of the wheat bacterial leaf streak (BLS) disease. The isolation of this pathogen is usually based on the Wilbrink's-boric acid-cephalexin semi-selective medium which eliminates 90% of other bacteria, some of which might be novel species. In our study, a general purpose nutrient agar was used to isolate 49 bacterial strains including <i>X. translucens</i> from necrotic wheat leaf tissues. Maximum likelihood cluster analysis of 16S rRNA sequences grouped the strains into 10 distinct genera. <i>Pseudomonas</i> (32.7%) and <i>Pantoea</i> (28.6%) were the dominant genera while <i>Xanthomonas, Clavibacter</i> and <i>Curtobacterium</i> had 8.2%, each. <i>Erwinia</i> and <i>Sphingomonas</i> had two strains, each. BLAST and phylogenetic analyses of multilocus sequence analysis (MLSA) of specific housekeeping genes taxonomically assigned all the strains to validly described bacterial species, except three strains (10L4B, 12L4D and 32L3A) of <i>Pseudomonas</i> and two (23L3C and 15L3B) of <i>Sphingomonas</i>. Strains 10L4B and12L4D had <i>Pseudomonas caspiana</i> as their closest known type strain while strain 32L3A was closest to <i>Pseudomonas asturiensis</i>. <i>Sphingomonas</i> sp. strains 23L3C and 15L3B were closest to <i>S. faeni</i> based on MLSA analysis. Our data on MLSA, whole genome-based cluster analysis, DNA-DNA hybridization and average nucleotide identity, matrix-assisted laser desorption/ionization-time-of-flight, chemotaxonomy and phenotype affirmed that these 5 strains constitute three novel lineages and are taxonomically described in this study. We propose the names, <i>Sphingomonas albertensis</i> sp. nov. (type strain 23L3C<sup>T</sup> = DOAB 1063<sup>T</sup> = CECT 30248<sup>T</sup> = LMG 32139<sup>T</sup>), <i>Pseudomonas triticumensis</i> sp. nov. (type strain 32L3A<sup>T</sup> = DOAB 1067<sup>T</sup> = CECT 30249<sup>T</sup> = LMG 32140<sup>T</sup>) and <i>Pseudomonas foliumensis</i> sp. <i>nov.</i> (type strain 10L4B<sup>T</sup> = DOAB 1069<sup>T</sup> = CECT 30250<sup>T</sup> = LMG 32142<sup>T</sup>). Comparative genomics of these novel species, relative to their closest type strains, revealed unique repertoires of core secretion systems and secondary metabolites/antibiotics. Also, the detection of CRISPR-Cas systems in the genomes of these novel species suggests an acquired mechanism for resistance against foreign mobile genetic elements. The results presented here revealed a cohabitation, within the BLS lesions, of diverse bacterial species, including novel lineages.
Project description:Conifer-feeding bark beetles are important herbivores and decomposers in forest ecosystems. These species complete their life cycle in nutritionally poor substrates and some can kill enormous numbers of trees during population outbreaks. The Eurasian spruce bark beetle (Ips typographus) can destroy >100 million m<sup>3</sup> of spruce in a single year. We report a 236.8 Mb I. typographus genome assembly using PacBio long-read sequencing. The final phased assembly has a contig N<sub>50</sub> of 6.65 Mb in 272 contigs and is predicted to contain 23,923 protein-coding genes. We reveal expanded gene families associated with plant cell wall degradation, including pectinases, aspartyl proteases, and glycosyl hydrolases. This genome sequence from the genus Ips provides timely resources to address questions about the evolutionary biology of the true weevils (Curculionidae), one of the most species-rich animal families. In forests of today, increasingly stressed by global warming, this draft genome may assist in developing pest control strategies to mitigate outbreaks.
Project description:Pseudomonas isolates have frequently been isolated from the rhizosphere of plants, and several of them have been reported as plant growth-promoting rhizobacteria. In the present work, tomato (Solanum lycopersicum) seeds were germinated in greenhouse conditions, and the seedling height, length of plants, collar diameter and number of leaves were measured from plants grown in soil inoculated by bacterial isolates. Pseudomonas isolates were isolated from the rhizosphere. We used the Newman-Keuls test to ascertain pairwise differences. Isolates were identified as a new Pseudomonas species by rpoD gene sequencing. The results showed that isolates of Pseudomonas sp. (Q6B) increased seed germination (P?=?0.01); Pseudomonas sp. (Q6B, Q14B, Q7B, Q1B and Q13B) also promoted seedling height (P?=?0.01). All five isolates promoted plant length and enlarged the collar diameter (P?=?0.01). Pseudomonas sp. (Q1B) also increased leaf number (P?=?0.01). The investigation found that Pseudomonas isolates were able to solubilize phosphate, produce siderophores, ammonia, and indole-3-acetic acid and colonize the roots of tomato plants. This study shows that these five novel Pseudomonas sp. isolates can be effective new plant growth-promoting rhizobacteria.
Project description:The bacterial strain 2-92T, isolated from a field plot under long-term (>40?years) mineral fertilization, exhibited in vitro antagonistic properties against fungal pathogens. A polyphasic approach was undertaken to verify its taxonomic status. Strain 2-92T was Gram-reaction-negative, aerobic, non-spore-forming, motile by one or more flagella, and oxidase-, catalase- and urease-positive. The optimal growth temperature of strain 2-92T was 30?°C. 16S rRNA gene sequence analysis demonstrated that the strain is related to species of the genus Pseudomonas. Phylogenetic analysis of six housekeeping genes (dnaA, gyrB, recA, recF, rpoB and rpoD) revealed that strain 2-92T clustered as a distinct and well separated lineage with Pseudomonassimiae as the most closely related species. Polar lipid and fatty acid compositions corroborated the taxonomic position of strain 2-92T in the genus Pseudomonas. Phenotypic characteristics from carbon utilization tests could be used to differentiate strain 2-92T from closely related species of the genus Pseudomonas. DNA-DNA hybridization values (wet laboratory and genome-based) and average nucleotide identity data confirmed that this strain represents a novel species. On the basis of phenotypic and genotypic characteristics, it is concluded that this strain represents a separate novel species for which the name Pseudomonas canadensis sp. nov. is proposed, with type strain 2-92T (=LMG 28499T=DOAB 798T). The DNA G+C content is 60.30?mol%.