Development of a Bacterial Cell Enrichment Method and its Application to the Community Analysis in Soybean Stems

A method was developed for enriching bacterial cells from soybean stems which was recalcitrant for a culture-independent analysis of bacterial community due to the interference with plant DNA. Stem homogenates were fractionated by a series of differential centrifugations followed by a Nycodenz density gradient centrifugation. The efficiency of bacterial cell enrichment was assessed by ribosomal intergenic spacer analysis (RISA). The intensity and the number of bacterial amplicons of RISA were markedly increased in the DNA extracted from the enriched bacterial cells compared to that in the DNA directly extracted from soybean stems. The phylogenetic diversity of the enriched bacterial cells was evaluated by analyzing a clone library of 16S rRNA gene in comparison with those of the culturable fractions of the enriched and non-enriched stem-associated bacteria, endophytic bacteria, and epiphytic bacteria. The results indicated that the method was able to enrich both endophytic and epiphytic bacteria from soybean stems, and was useful to assess the bacterial diversity based on a 16S rRNA gene clone library. When the sequence data from all clones (1,332 sequences) were combined, 72 operational taxonomic units were affiliated with Proteobacteria (Alpha-, Beta-, and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes, which also provided the most comprehensive set of data on the bacterial diversity in the aerial parts of soybeans.     

Optimizing Phytoplasma DNA purification for genome analysis.

Genome analysis of uncultivable plant pathogenic phytoplasmas is hindered by the difficulty in obtaining sufficient quantities of phytoplasma enriched DNA. We investigated a combination of conventional enrichment techniques such as cesium chloride (CsCl) buoyant gradient centrifugation, and new methods such as rolling circle amplification (RCA), suppression subtractive hybridization (SSH), and mirror orientation selection (MOS) to obtain DNA with a high phytoplasma:host ratio as the major first step in genome analysis of Candidatus Phytoplasma australiense. The phytoplasma:host ratio was calculated for five different plasmid libraries. Based on sequence data, 90% of clones from CsCl DNA enrichment contained chromosomal phytoplasma DNA, compared to 60% from RCA CsCl DNA and 20% from SSH subtracted libraries. Based on an analysis of representative libraries, none contained plant DNA. A high percentage of clones (80-100%) from SSH libraries contained extrachromosomal DNA (eDNA), and we speculate that eDNA in the original DNA preparation was amplified in subsequent SSH manipulations. Despite the availability of new techniques for nucleic acid amplification, we found that conventional CsCl gradient centrifugation was the best enrichment method for obtaining chromosomal phytoplasma DNA with low host DNA content.     

New insights on molecular regulation of biofilm formation in plant-associated bacteria

Biofilms are complex bacterial assemblages with a defined three-dimensional architecture, attached to solid surfaces, and surrounded by a self-produced matrix generally composed of exopolysaccharides, proteins, lipids and extracellular DNA. Biofilm formation has evolved as an adaptive strategy of bacteria to cope with harsh environmental conditions as well as to establish antagonistic or beneficial interactions with their host. Plant-associated bacteria attach and form biofilms on different tissues including leaves, stems,vasculature, seeds and roots. In this review, we examine the formation of biofilms from the plant-associated bacterial perspective and detail the recently-described mechanisms of genetic regulation used by these organisms to orchestrate biofilm formation on plant surfaces. In addition, we describe plant host signals that bacterial pathogens recognize to activate the transition from a planktonic lifestyle to multicellular behavior.     

Perspectives of plant-associated microbes in heavy metal phytoremediation

"Phytoremediation" know-how to do-how is rapidly expanding and is being commercialized by harnessing the phyto-microbial diversity. This technology employs biodiversity to remove/contain pollutants from the air, soil and water. In recent years, there has been a considerable knowledge explosion in understanding plant-microbes-heavy metals interactions. Novel applications of plant-associated microbes have opened up promising areas of research in the field of phytoremediation technology. Various metabolites (e.g., 1-aminocyclopropane-1-carboxylic acid deaminase, indole-3-acetic acid, siderophores, organic acids, etc.) produced by plant-associated microbes (e.g., plant growth promoting bacteria, mycorrhizae) have been proposed to be involved in many biogeochemical processes operating in the rhizosphere. The salient functions include nutrient acquisition, cell elongation, metal detoxification and alleviation of biotic/abiotic stress in plants. Rhizosphere microbes accelerate metal mobility, or immobilization. Plants and associated microbes release inorganic and organic compounds possessing acidifying, chelating and/or reductive power. These functions are implicated to play an essential role in plant metal uptake. Overall the plant-associated beneficial microbes enhance the efficiency of phytoremediation process directly by altering the metal accumulation in plant tissues and indirectly by promoting the shoot and root biomass production. The present work aims to provide a comprehensive review of some of the promising processes mediated by plant-associated microbes and to illustrate how such processes influence heavy metal uptake through various biogeochemical processes including translocation, transformation, chelation, immobilization, solubilization, precipitation, volatilization and complexation of heavy metals ultimately facilitating phytoremediation.     

Investigations on influencing plant-associated bacteria in tissue cultures of black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.)

During tissue culture of black locust (Robinia pseudoacacia L.), serious problems with plant-associated bacteria led to a reduction of propagation potential in several clones. Four dominant strains of plant-associated bacteria could be isolated and were assigned to the genera Acidovorax, Dyella, Microbacterium and Sphingomonas. Out of five essential oils tested, thyme and lemongrass oil at a concentration of 0.03% each and 0.015% of both oils in combination clearly inhibited the growth of these bacteria strains on bacteriologic medium. There were no significant differences in total bacterial population density when penicillin, thyme and lemongrass oil or thyme plus lemongrass oil were added to the plant propagation media. The use of lemongrass oil changed the proportion of dominant bacterial strains.     

Plant-by-Plant Variations of Bacterial Communities Associated with Leaves of the Nickel Hyperaccumulator Alyssum bertolonii Desv.

Bacteria associated with tissues of metal-hyperaccumulating plants are of great interest due to the multiple roles they may play with respect to plant growth and resistance to heavy metals. The variability of bacterial communities associated with plant tissues of three populations of Alyssum bertolonii, a Ni hyperaccumulator endemic of serpentine outcrops of Central Italy, was investigated. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes was applied to DNA extracted from leaf tissues of 30 individual plants from three geographically separated serpentine outcrops. Moreover, T-RFLP fingerprinting was also performed on DNA extracted from the same soils from which the plants were collected. Fifty-nine unique terminal-restriction fragments (TRFs) were identified, with more than half of the taxonomically interpreted TRFs assigned to Alpha- and Gamma-Proteobacteria and Clostridia. Data were then used to define the extent of variation of bacterial communities due to single plants or to plant populations. Results indicated a very high plant-by-plant variation of leaf-associated community (more than 93% of total variance observed). However, a core (numerically small) of plant-specific TRFs was found. This work demonstrates that plant-associated bacterial communities represent a large reservoir of biodiversity and that the high variability existing between plants, even from the same population, should be taken into account in future studies on association between bacteria and metal-hyperaccumulating plants.     

The host species affects the microbial community in the goat rumen

AIMS: This study was carried out to determine whether bacterial and ciliate populations in goat rumen vary significantly between different goat species living in the same environment. METHODS AND RESULTS: Bacterial and ciliate communities in the rumen of three goat species were analysed at the molecular level using denaturing gradient gel electrophoresis. The microbial community varied considerably among goats living in the same environment. Interspecies variation in the bacterial population was noticeably greater than intraspecies variation. In contrast, there was considerable variation in the ciliate population among goats within the same species, and intraspecies similarities were no greater than those observed across species. CONCLUSIONS: Because environmental factors and diets were identical for all goats, differences in bacterial populations reflect species-specific differences in rumen microbes. SIGNIFICANCE AND IMPACT OF THE STUDY: Factors related to the host species have an important effect on determining the bacterial composition in the goat rumen.     

Predominance of Gram-positive bacteria in house dust in the low-allergy risk Russian Karelia

Simple living conditions and farming environment have been associated with reduced risk for allergic diseases such as atopy and asthma but the factors responsible for this effect remain unresolved. We examined the bacterial composition of house dusts obtained from Finnish and Russian Karelia, two adjacent areas with high and low occurrence of atopic diseases respectively. Two dust mixes, both composed of 10 randomly selected dust samples from 349 Finnish and 417 Russian Karelian households were studied for bacterial biomarkers (DNA, Limulus-active endotoxin, 3-OH fatty acids, muramic acid) and for 16S rRNA gene sequences. Overall, the DNA cloning revealed more taxons (94 different genera) of dustborne bacteria than seen in any previous study on residential environments. Majority (67%) of the bacterial DNA clones in house dust from the low-allergy Russian Kareliarepresented Gram-positive bacteria (Firmicutes and Actinobacteria), predominantly Staphylococcaceae and Corynebacteriaceae. Russian Karelian dust showed up to 20-fold higher contents of muramic acid (marker of Gram-positive bacteria) and a sevenfold higher number of clones of animal-associated species, whereas in Finnish Karelian dust Gram-negatives (mainly Proteobacteria) predominated. Clones of plant-associated bacterial species and of chloroplast, indicating plant biomass, were more numerous in Finnish than in Russian Karelian dust. In conclusion, this study revealed major disparities between Finnish and Russian house dusts. The higher bacterial content and the predominance of Gram-positive bacteria in Russian dust may have implications for occurrence of atopy.     

Mining the metagenome of activated biomass of an industrial wastewater treatment plant by a novel method

Metagenomic libraries herald the era of magnifying the microbial world, tapping into the vast metabolic potential of uncultivated microbes, and enhancing the rate of discovery of novel genes and pathways. In this paper, we describe a method that facilitates the extraction of metagenomic DNA from activated sludge of an industrial wastewater treatment plant and its use in mining the metagenome via library construction. The efficiency of this method was demonstrated by the large representation of the bacterial genome in the constructed metagenomic libraries and by the functional clones obtained. The BAC library represented 95.6 times the bacterial genome, while, the pUC library represented 41.7 times the bacterial genome. Twelve clones in the BAC library demonstrated lipolytic activity, while four clones demonstrated dioxygenase activity. Four clones in pUC library tested positive for cellulase activity. This method, using FTA cards, not only can be used for library construction, but can also store the metagenome at room temperature.     

胜利油田单12高温油藏非培养和富集培养样品的细菌组成特性

[目的]通过比较分析油藏样品的微生物群落结构特点,认识油藏微生物的生态功能.[方法]利用3种油藏微生物研究中常用的富集培养方法,对胜利油田单12区块S12-4油井产出水样品进行了选择性富集培养,运用构建16S rRNA基因文库的方法分析了富集样品和非培养样品的细菌多样性.[结果]通过16S rRNA基因序列比对发现,非培养样品、异养菌富集样品、烃降解菌富集样品和硫酸盐还原菌富集样品中的优势菌分别为Pseudomonas属,Thermotoga属,Thermaerobacter属和Thermotoga属的成员.多样性分析结果表明,非培养样品的微生物多样性最丰富,同时非培养样品和富集样品的微生物群落结构存在很大的差异,富集样品中的微生物包括优势菌在油藏原位环境中含量很低.[结论]细菌组成差异的比较结果,对油藏微生物的生态功能研究和微生物驱油潜力评估具有重要意义.     

Plant associated protists—Untapped promising candidates for agrifood tools

The importance of host-associated microorganisms and their biotic interactions for plant health and performance has been increasingly acknowledged. Protists, main predators and regulators of bacteria and fungi, are abundant and ubiquitous eukaryotes in terrestrial ecosystems. Protists are considered to benefit plant health and performance, but the community structure and functions of plant-associated protists remain surprisingly underexplored. Harnessing plant-associated protists and other microbes can potentially enhance plant health and productivity and sustain healthy food and agriculture systems. In this review, we summarize the knowledge of multifunctionality of protists and their interactions with other microbes in plant hosts, and propose a future framework to study plant-associated protists and utilize protists as agrifood tools for benefiting agricultural production.     

Integration of environmental and host-derived signals with quorum sensing during plant-microbe interactions

Many plant-associated microbes use secreted autoinducer molecules, including N-acylhomoserine lactones (AHLs), to regulate diverse behaviours in association with their population density (quorum sensing). Often, these responses are affected by environmental conditions, including the presence of other AHL-producing bacterial species. In addition, plant-derived metabolites, including products that arise as a direct result of the bacterial infection, may profoundly influence AHL-regulated behaviours. These plant products can interact directly and indirectly with the quorum-sensing network and can profoundly affect the quorum-sensing behaviour. Local conditions on a microscopic scale may affect signal molecule longevity, stability and accumulation, and this could be used to give information in addition to cell density. Furthermore, in many Gram-negative bacteria, AHL signalling is subservient to an additional two-component signalling system dependent upon homologues of GacS and GacA. The signal(s) to which GacS responds are not known, but recent research suggests that a self-produced ligand may be being detected. This review will focus on two well-studied examples of AHL-regulated plant-associated behaviour, Erwinia carotovora and Agrobacterium tumefaciens, to illustrate the complexity of such signalling networks.     

Construction of a bacterial artificial chromosome library, determination of genome size, and characterization of an Hsp70 gene family in Phytophthora nicotianae

The oomycete plant pathogen Phytophthora nicotianae causes diseases on a wide range of plant species. To facilitate isolation and functional characterization of pathogenicity genes, we have constructed a large-insert bacterial artificial chromosome (BAC) library using nuclear DNA from P. nicotianae H1111. The library contains 10,752 clones with an average insert size of 90 kb and is free of mitochondrial DNA. The quality of the library was verified by hybridization with 37 genes, all of which resulted in the identification of multiple positive clones. The library is estimated to be 10.6 haploid genome equivalents based on hybridization of 23 single-copy genes and the genome size of P. nicotianae was estimated to be 95.5 Mb. Hybridization with a nuclear repetitive DNA probe revealed that 4.4% of clones in the library contained 28S rDNA. Hybridization of total genomic DNA to the library indicated that at least 39% of the BAC library contains repetitive DNA sequences. A BAC pooling strategy was developed for efficient library screening. The library was used to identify and characterize BAC clones containing an Hsp70 gene family whose four members were identified to be clustered within approximately 18 kb in the P. nicotianae genome based on the physical mapping of eight BACs spanning a genomic region of approximately 186 kb. The BAC library created provides an invaluable resource for the isolation of P. nicotianae genes and for comparative genomics studies.     

Microbiome-mediated signal transduction within the plant holobiont

Microorganisms colonizing the plant rhizosphere and phyllosphere play crucial roles in plant growth and health. Recent studies provide new insights into long-distance communication from plant roots to shoots in association with their commensal microbiome. In brief, these recent advances suggest that specific plant-associated microbial taxa can contribute to systemic plant responses associated with the enhancement of plant health and performance in face of a variety of biotic and abiotic stresses. However, most of the mechanisms associated with microbiome-mediated signal transduction in plants remain poorly understood. In this review, we provide an overview of long-distance signaling mechanisms within plants mediated by the commensal plant-associated microbiomes. We advocate the view of plants and microbes as a holobiont and explore key molecules and mechanisms associated with plant–microbe interactions and changes in plant physiology activated by signal transduction.     

A Genome-Wide Analysis of Antibiotic Producing Genes in Streptomyces globisporus SP6C4

Soil is the major source of plant-associated microbes. Several fungal and bacterial species live within plant tissues. Actinomycetes are well known for producing a variety of antibiotics, and they contribute to improving plant health. In our previous report, Streptomyces globisporus SP6C4 colonized plant tissues and was able to move to other tissues from the initially colonized ones. This strain has excellent antifungal and antibacterial activities and provides a suppressive effect upon various plant diseases. Here, we report the genome-wide analysis of antibiotic producing genes in S. globisporus SP6C4. A total of 15 secondary metabolite biosynthetic gene clusters were predicted using antiSMASH. We used the CRISPR/Cas9 mutagenesis system, and each biosynthetic gene was predicted via protein basic local alignment search tool (BLAST) and rapid annotation using subsystems technology (RAST) server. Three gene clusters were shown to exhibit antifungal or antibacterial activity, viz. cluster 16 (lasso peptide), cluster 17 (thiopeptide-lantipeptide), and cluster 20 (lantipeptide). The results of the current study showed that SP6C4 has a variety of antimicrobial activities, and this strain is beneficial in agriculture.     

A feeling for the micro-organism: structure on a small scale. Biofilms on plant roots

Biofilms are structured communities of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface; they have clinical, industrial and environmental impacts. Biofilms that are established by bacteria on plants are found on the surfaces of roots, leaves, seeds and internal vascular tissues where the microbes live in commensal, mutualistic or parasitic/pathogenic associations with their host. The study of the structure of plant-associated biofilms has been considerably helped by the development of techniques using fluorescent markers coupled with confocal scanning laser microscopy as well as scanning electron microscopy. We review several of these techniques as well as some of the research that has dealt with plant-associated biofilms. Our investigations focus on biofilm formation in the early stages of the Rhizobium –legume symbiosis, in which Gram-negative rhizobia provide fixed nitrogen to a host legume, and in return, the legume provides carbon-containing molecules. Because root colonization is an important early step in the establishment of the nitrogen-fixing symbiosis, we looked at Sinorhizobium meliloti attachment and biofilm establishment on the roots of its legume hosts, Medicago sativa L. and Melilotus alba Desr. We also examined biofilm formation by Rhizobium leguminosarum bv. viciae on the roots of Arabidopsis thaliana (L.) Heynh., a non-legume and non-host. Our ultimate goal is to characterize the rhizobial genes involved in aggregation and attachment to roots because several of these appear to be shared in biofilm formation and rhizobial entry of legume root cells.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 79–88.     

Plant symbionts: keys to the phytosphere

The exterior and interior of plants, aboveground and belowground, comprise a complex plant micro-ecosystem, known in recent years as the phytosphere. There are three components: the phyllosphere, endosphere, and rhizosphere. Although in comparison with other ecosystems the phytosphere is small, it similarly includes a great variety of functional microbes. Among these are certain microbes that live in symbiotic relationships with plants; these microbes are known as plant symbionts. Recent research has shown that these symbionts have tremendous effects on plant growth, confer resistance to abiotic stresses and pathogens, aid in the accumulation of metabolites, and have crucial relationships with other plant-associated microbes in the phytosphere. We review the ecological effects of plant symbionts on other microbes, and interactions between plant symbionts in the phytosphere. In addition, we discuss internal mechanisms and suggest future hot spots for research.