Detection and identification of species-specific bacteria associated with synanthropic mites

Internal bacterial communities of synanthropic mites Acarus siro, Dermatophagoides farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae (Acari: Astigmata) were analyzed by culturing and culture-independent approaches from specimens obtained from laboratory colonies. Homogenates of surface-sterilized mites were used for cultivation on non-selective agar and DNA extraction. Isolated bacteria were identified by sequencing of the 16S rRNA gene. PCR amplified 16S rRNA genes were analyzed by terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning sequencing. Fluorescence in situ hybridization using universal bacterial probes was used for direct bacterial localization. T-RFLP analysis of 16S rRNA gene revealed distinct species-specific bacterial communities. The results were further confirmed by cloning and sequencing (284 clones). L. destructor and D. farinae showed more diverse communities then A. siro and T. putrescentiae. In the cultivated part of the community, the mean CFUs from four mite species ranged from 5.2?×?10(2) to 1.4?×?10(3) per mite. D. farinae had significantly higher CFUs than the other species. Bacteria were located in the digestive and reproductive tract, parenchymatical tissue, and in bacteriocytes. Among the clones, Bartonella-like bacteria occurring in A. siro and T. putresecentiae represented a distinct group related to Bartonellaceae and to Bartonella-like symbionts of ants. The clones of high similarity to Xenorhabdus cabanillasii were found in L. destructor and D. farinae, and one clone related to Photorhabdus temperata in A. siro. Members of Sphingobacteriales cloned from D. farinae and A. siro clustered with the sequences of "Candidatus Cardinium hertigii" and as a separate novel cluster.     

Dna profiling and strategies for in planta localisation of bacteria intimately associated with Billbergia magnifica ssp. acutisepalia

Plant Cell, Tissue and Organ Culture (PCTOC) -     

丛枝菌根真菌伴生细菌的研究进展

在丛枝菌根真菌（Arbuscular mycorrhizal fungi,AMF）的孢子、菌丝的表面或内部栖息着细菌,称之为AMF伴生细菌。AMF伴生细菌种类多样、分布广泛,生态位点包括孢子壁的表面或内部、细胞质、菌丝、孢子果等。其可能的生物学意义包括影响AMF孢子萌发、菌丝生长、菌根形成等过程。由于伴生细菌与AMF联系紧密,其对AMF和土壤微生物生态学具有重要的意义。国际上在该领域的研究已有30多年的历史,就其研究进展进行综述。     

Taxonomy and identification of bacteria associated with acute oak decline

Acute oak decline (AOD) is a relatively newly described disorder affecting native oak species in Britain. Symptomatic trees are characterised by stem bleeds from vertical fissures, necrotic lesions in the live tissue beneath and larval galleries of the two spotted oak buprestid (Agrilus biguttatus). Several abiotic and biotic factors can be responsible for tree death, however the tissue necrosis and stem weeping is thought to be caused by a combination of bacterial species. Following investigations of the current episode of AOD which began in 2008, numerous strains belonging to several different bacteria in the family Enterobacteriaceae have been consistently isolated from symptomatic tissue. The majority of these enterobacteria were found to be novel species, subspecies and even genera, which have now been formally classified. The most frequently isolated species from symptomatic oak are Gibbsiella quercinecans, Brenneria goodwinii and Rahnella victoriana. Identification of these bacteria is difficult due to similarities in colony morphology, phenotypic profile and 16S rRNA gene sequences. Current identification relies heavily on gyrB gene amplification and sequencing, which is time consuming and laborious. However, newer techniques based on detection of single nucleotide polymorphisms show greater promise for rapid and reliable identification of the bacteria associated with AOD.     

Detection and identification of cytokinins produced by mycorrhizal fungi

Several fungi including six species of the genus Rhizopogon, 22 species of Hebeloma and one of Agaricus have been screened for production of cytokinins. The screening was done by culturing cytokinin-requiring soybean callus tissue alongside the fungus on a medium lacking a cytokinin supply. Growth of the soybean callus indicated production of cytokinins by the fungus. Of the fungi tested, only R. ochraceorubens A. H. Smith gave off sufficient cytokinin to be detected. Although a number of mycorrhizal species are now known to make and give off cytokinins, an even larger number apparently do not do so under the conditions of screening employed. An unidentified ectendotrophic species definitely gave off trans-zeatin, which has been crystallized, and probably trans-ribosylzeatin. Suillus punctipes (Pk.) Sing. apparently produced the same two cytokinins.     

Microbes intimately associated with tissue and cell cultures of tropical Dioscorea yams

Plant Cell, Tissue and Organ Culture (PCTOC) -     

Detection and identification of ferricrocin produced by ectendomycorrhizal fungi in the genusWilcoxina

The ectendomycorrhizal fungiWilcoxina mikolae isolates CSY-14 and RMD-947 andW. rehmii isolate CSY-85 were grown in pure culture under iron-limiting conditions. All three isolates tested positive for siderophore formation using both the ferric perchlorate assay and a sensitive HPLC iron-binding assay. A peptide siderophore was isolated from the culture medium by HPLC and shown to contain the amino acids serine, glycine and ornithine in a 1:2:3 ratio. This siderophore was identified as ferricrocin on the basis of electrospray mass spectroscopy and its co-chromatography in two different HPLC systems with ferricrocin isolated fromAspergillus fumigatus. Ferricrocin was the only siderophore isolated from theseWilcoxina cultures. This is the first report of siderophore formation by ectendomycorrhizal fungi.     

Isolation and identification of soil bacteria growing at the expense of arbuscular mycorrhizal fungi

Soil-microorganism symbioses are of fundamental importance for plant adaptation to the environment. Research in microbial ecology has revealed that some soil bacteria are associated with arbuscular mycorrhizal fungi (AMF). However, these interactions may be much more complex than originally thought. To assess the type of bacteria associated with AMF, we initially isolated spores of Glomus irregulare from an Agrostis stolonifera rhizosphere. The spores were washed with sterile water and plated onto G. irregulare mycelium growing in vitro in a root-free compartment of bicompartmented Petri dishes. We hypothesized that this system should select for bacteria closely associated with the fungus because the only nutrients available to the bacteria were those derived from the hyphae. Twenty-nine bacterial colonies growing on the AMF hyphae were subcultured and identified using 16S rRNA gene sequences. All bacterial isolates showed high sequence identity to Bacillus cereus, Bacillus megaterium, Bacillus simplex, Kocuria rhizophila, Microbacterium ginsengisoli, Sphingomonas sp. and Variovorax paradoxus. We also assessed bacterial diversity on the surface of spores by PCR-denaturating gradient gel electrophoresis. Finally, we used live cellular imaging to show that the bacteria isolated can grow on the surface of hyphae with different growing patterns in contrast to Escherichia coli as a control.     

Detection of cellulolytic activity of bacteria and fungi growing on agar surfaces

Summary Cellulolytic activity of four fungal species growing on solid medium containing acid-swollen cellulose could be detected much more easily if fungal growth was partly inhibited by the detergent Triton X-100. The dye, aniline blue-black, did not affect growth but increased the sensitivity of detection of cellulolytic activity of both fungi and bacteria. Separating fungi from cellulose fibres by a layer of agar or by filters showed that cell-fibre contact is not necessary for cellulose degradation. Such degradation is clearer when contact is prevented.     

Ozone treatment inactivates common bacteria and fungi associated with selected crop seeds and ornamental bulbs

The use of disease-free seeds or bulbs is very crucial to ensure sustainable and profitable agricultural production. Seed-borne pathogens which are responsible for significant yield losses in various crops need to be successfully eliminated with appropriate seed treatments. In this study, we investigated the efficacy of gaseous ozone (O₃) and ozonated water treatments on the inactivation of seed-borne fungal and bacterial pathogens of widely cultivated vegetable and cereal seeds, and ornamental bulbs. We demonstrated that O₃ application to tomato and cucumber seeds inactivates Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum f. sp. radicis-lycopersici, Clavibacter michiganensis subsp. michiganensis, Pseuodomonas syringae pv. tomato, and Pseudomonas syringae pv. lachrymans, respectively, with no negative effect on seed germination rate. The sterilization capacity of O₃ has substantially increased when the seeds were soaked in water before the treatments. The saprophytic fungal load and the infection rate of Pectobacterium carotovorum subsp. carotovorum on several species and cultivars of ornamental bulbs were suppressed by O₃ treatment. A strong decrease in the infection rate of Tilletia caries was also shown in O₃-treated wheat seeds under field conditions. Overall, the current study indicated that O₃ treatment has great potential in ensuring the use of disease-free seeds or other propagation materials, which is indispensable at the beginning of crop production.     

Part of respiratory nitrate reductase of Klebsiella aerogenes is intimately associated with the peptidoglycan.

Lysozyme digestion and sonication of sodium dodecyl sulfate (SDS)-purified Klebsiella aerogenes murein sacculi resulted in the quantitative release of both subunits of nitrate reductase, as well as a number of other cytoplasmic membrane polypeptides (5.2%, by weight, of the total membrane proteins). Similar results were obtained after lysozyme digestion of SDS-prepared peptidoglycan fragments, which excluded the phenomenon of simple trapping of the polypeptides by the surrounding peptidoglycan matrix. About 28% of membrane-bound nitrate reductase appears to be tightly associated with the peptidoglycan. Additional evidence for this association was demonstrated by positive immunogold labeling of SDS-murein sacculi and thin sections of plasmolyzed bacteria. Qualitative amino acid analysis of trypsin-treated sacculi, a tryptic product of holo-nitrate reductase, and amino- and carboxypeptidase digests of both nitrate reductase subunits indicated the possible existence of a terminal anchoring peptide containing the following amino acids: (Gly)n, Trp, Ser, Pro, Ile, Leu, Phe, Cys, Tyr, Asp, and Lys.     

Denitrification by the mix-culturing of fungi and bacteria with shell

Denitrification by pure and mixed culture of Fusarium oxysporum and Pseudomonas stutzeri in different mineral medium and in synthetic wastewater were examined. The results obtained revealed that a rapid N2 evolution by F. oxysporum and P. stutzeri in mineral medium and synthetic wastewater was observed. In co-cultures of F. oxysporum and P. stutzeri, N2O produced by F. oxysporum was rapidly consumed by P. stutzeri. This indicated that mixed culture of F. oxysporum and P. stutzeri could be used for efficient nitrate and nitrite removal. Using synthetic wastewater, about 87% of nitrate was reduced by co-denitrification of F. oxysporum and P. stutzeri after incubation for 6 days. In the further denitrification tests, the interaction of shell and mixed culture of F. oxysporum and P. stutzeri was investigated. The dinitrogen was rapidly evolved (442.48 micromol N2 produced from 1.0 mmol of NO3(-) in 36 h). These results clearly showed that shell provide a suitable microenvironment for P. stutzeri, which is beneficial to the denitrification.     

空气环境中几种细菌检测与16S rRNA测序鉴定

采集空气环境微生物,根据菌落和革兰氏染色特征,分离5株细菌。提取分离株DNA,采用设计的16SrRNA引物扩增DNA片段;纯化PCR产物,进行测序反应并再纯化,上机读序。拼接与校对DNA序列,在NCBI网站进行Blast,鉴定细菌型别。5株菌株分别是表皮葡萄球菌、头状葡萄球菌、施氏假单胞菌、溶血葡萄球菌和大肠杆菌;其中溶血葡萄球菌是致病菌,其他4株是条件致病菌。这提示,应警惕空气环境中致病菌的感染。     

Small RNAs from plants,bacteria and fungi within the order Hypocreales are ubiquitous in human plasma

Background

The human microbiome plays a significant role in maintaining normal physiology. Changes in its composition have been associated with bowel disease, metabolic disorders and atherosclerosis. Sequences of microbial origin have been observed within small RNA sequencing data obtained from blood samples. The aim of this study was to characterise the microbiome from which these sequences are derived.

Results

Abundant non-human small RNA sequences were identified in plasma and plasma exosomal samples. Assembly of these short sequences into longer contigs was the pivotal novel step in ascertaining their origin by BLAST searches. Most reads mapped to rRNA sequences. The taxonomic profiles of the microbes detected were very consistent between individuals but distinct from microbiomes reported at other sites. The majority of bacterial reads were from the phylum Proteobacteria, whilst for 5 of 6 individuals over 90% of the more abundant fungal reads were from the phylum Ascomycota; of these over 90% were from the order Hypocreales. Many contigs were from plants, presumably of dietary origin. In addition, extremely abundant small RNAs derived from human Y RNAs were detected.

Conclusions

A characteristic profile of a subset of the human microbiome can be obtained by sequencing small RNAs present in the blood. The source and functions of these molecules remain to be determined, but the specific profiles are likely to reflect health status. The potential to provide biomarkers of diet and for the diagnosis and prognosis of human disease is immense.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-933) contains supplementary material, which is available to authorized users.     

东北旱田土壤中Anabaena伴生细菌的分离与鉴定

[背景]鱼腥藻(Anabaena)在农田土壤中广泛分布,具有固碳和固氮功能。明确伴生细菌与蓝细菌的关系,对提高农田土壤中Anabaena的功能具有重要意义。[目的]从东北不同旱田土壤中分离Anabaena sp.PCC7120的伴生细菌,初步鉴定伴生细菌的分类归属,推测伴生细菌的功能,为明确旱田土壤蓝细菌与伴生细菌的关系提供数据支撑。[方法]采用平板分离、PCR-DGGE、克隆测序技术测定并分析不同旱田土壤中伴生细菌的16S rRNA基因序列,确定伴生细菌的分类地位。[结果]PCR-DGGE图谱显示东北旱田14个土样中分离获得Anabaena sp.PCC7120伴生细菌数量和种类不同;PCR-克隆测序获得伴生细菌的16S rRNA基因序列37条,可鉴定到种水平的菌株36条,主要归为鞘氨醇盒菌属(Sphingopyxis)、贪噬菌属(Variovorax)、黄杆菌属(Flavobacterium)和红球菌属(Rhodococcus)等,推测这些伴生细菌具有适应寡营养、富集微量元素、清除毒素等功效。[结论]东北旱田不同土壤中Anabaena sp.PCC7120伴生细菌种类和数量各异,这...     

Rapid identification and detection of pine pathogenic fungi associated with mountain pine beetles by padlock probes

Fifteen million hectares of pine forests in western Canada have been attacked by the mountain pine beetle (Dendroctonus ponderosae; MPB), leading to devastating economic losses. Grosmannia clavigera and Leptographium longiclavatum, are two fungi intimately associated with the beetles, and are crucial components of the epidemic. To detect and discriminate these two closely related pathogens, we utilized a method based on ligase-mediated nucleotide discrimination with padlock probe technology, and signal amplification by hyperbranched rolling circle amplification (HRCA). Two padlock probes were designed to target species-specific single nucleotide polymorphisms (SNPs) located at the inter-generic spacer 2 region and large subunit of the rRNA respectively, which allows discrimination between the two species. Thirty-four strains of G. clavigera and twenty-five strains of L. longiclavatum representing a broad geographic origin were tested with this assay. The HRCA results were largely in agreement with the conventional identification based on morphology or DNA-based methods. Both probes can also efficiently distinguish the two MPB-associated fungi from other fungi in the MPB, as well as other related fungi in the order Ophiostomatales. We also tested this diagnostic method for the direct detection of these fungi from the DNA of MPB. A nested PCR approach was used to enrich amplicons for signal detection. The results confirmed the presence of these two fungi in MPB. Thus, the padlock probe assay coupled with HRCA is a rapid, sensitive and reproducible method for the identification and detection of these ophiostomatoid fungi.     

Detection and identification of bacterial endosymbionts in arbuscular mycorrhizal fungi belonging to the family Gigasporaceae

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.     

A protocol for the simultaneous identification of chitin-containing particles and their associated bacteria

Chitin is the second most abundant polymer on Earth, playing a crucial role in the biogeochemical cycles. A core issue for studying its processing in aquatic systems is the identification and enumeration of chitin-containing particles and organisms, ideally in a manner that can be directly linked to bulk chitin quantification. The aim of this study was the development of such a technique. We successfully combined the methodology of bulk chitin determination using wheat germ agglutinin (FITC-WGA) for staining chitin-containing particles and organisms along with CARD-FISH staining of either chitin-containing eukaryotic cells or bacteria associated with them. Environmental chitin staining was successfully applied to natural water samples. Fungal hyphae, diatoms, and dinoflagellates, sestonic aggregates and chitin-containing structures derived from metazoa were observed. Also, hybridized bacteria attached to chitinaceous debris were clearly visualized. Finally, as proof of principle, cultured yeast cells were simultaneously-targeted by FITC-WGA and the EUK516 probe without exhibiting any interference between both stains. The presented approach appears as a powerful tool to evaluate the contribution of different size classes and organisms to chitin production and consumption, opening the possibility for application of single-cell approaches targeting the ecophysiology of chitin transformations in aquatic systems.