Fatty acid methyl ester (FAME) profiles as a tool to investigate community structure of two agricultural soils

Soil microbiological parameters may be the earliest predictors of soil quality changes. Recently, molecular techniques such as fatty acid methyl ester (FAME) profiles have been used to characterize soil microbial communities. Fatty acid methyl ester (FAME) from whole soil may be derived from live cells, dead cells, humic materials, as well as plant and root exudates. Our objective was to verify differences in FAME profiles from two agricultural soils with different plants. Soil samples were collected from Ritzville and Palouse silt loams for fatty acid analysis. Soil samples from wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), pea (Pisum sativum L.), jointed goatgrass ( Aegilops cylindrica L.) and downy brome (Bromus tectorum L.) rhizospheres were also collected for fatty acid analysis. Principal component analysis (PCA) of the two soils explained 42% of the variance on PC1, which accounted for Palouse soil. Ritzville soil accounted for 19% of the variance on PC2. Factor analysis showed that rhizosphere microbial communities from various plant species may differ depending on the plant species. Presence of Gram-positive bacteria as identified by a15:0, i15:0, a17:0 and i17:0 peaks were similar between rhizosphere and nonrhizosphere soils. Gram-negative bacteria characterized by short chain hydroxy acids (10:03OH and 12:03OH) as well as cyclopropane acids (cy17:0) were higher in rhizosphere soil than nonrhizosphere. This indicates a possible shift in the bacterial community to more Gram-negative bacteria and fewer Gram-positive bacteria in the rhizospheres of the plants species studied.     

Selection of bacteria able to control Fusarium oxysporum f. sp. radicis-lycopersici in stonewool substrate

AIMS: Tomato foot and root rot (TFRR), caused by Fusariumoxysporum f. sp. radicis-lycopersici (Forl), is an economically important disease of tomato. The aim of this study was to develop an efficient protocol for the isolation of bacteria, which controls TFRR based on selection of enhanced competitive root-colonizing bacteria from total rhizosphere soil samples. METHODS AND RESULTS: A total of 216 potentially enhanced bacterial strains were isolated from 17 rhizosphere soil samples after applying a procedure to enrich for enhanced root tip colonizers. Amplified ribosomal DNA restriction analysis, in combination with determination of phenotypic traits, was introduced to evaluate the presence of siblings. One hundred sixteen strains were discarded as siblings. Thirty-eight strains were discarded as potential pathogens based on the sequence of their 16S rDNA. Of the remaining strains, 24 performed equally well or better than the good root colonizer Pseudomonas fluorescens WCS365 in a competitive tomato root tip colonization assay. Finally, these enhanced colonizers were tested for their ability to control TFRR in stonewool, which resulted in seven new biocontrol strains. CONCLUSIONS: The new biocontrol strains, six Gram-negative and one Gram-positive bacteria, were identified as three Pseudomonas putida strains and one strain each of Delftia tsuruhatensis, Pseudomonas chlororaphis, Pseudomonas rhodesiae and Paenibacillus amylolyticus. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe a fast method for the isolation of bacteria able to suppress TFRR in stonewool, an industrial plant growth substrate. The procedure minimizes the laborious screens that are a common feature in the isolation of biocontrol strains.     

Interactions in the tomato rhizosphere of two Pseudomonas biocontrol strains with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici

The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite phenazine-1-carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.     

施磷对干旱胁迫下箭竹根际土壤养分及微生物群落的影响

以箭竹及其根际土壤作为研究对象,采用两因素随机区组实验,设置2种水分处理（正常浇水和干旱胁迫）和2种施磷量处理（施磷和不施磷）,探究施磷对干旱胁迫下箭竹根际土壤养分及微生物群落结构和多样性的影响。结果表明：（1）干旱胁迫显著降低了箭竹根际土壤中微生物量碳、可溶性有机氮和有效磷的含量,虽对箭竹根际土壤微生物群落的多样性无显著影响,但显著降低了箭竹根际土壤中总PLFA（phospholipid fatty acid contents）的含量和真菌、细菌、革兰氏阳性菌与革兰氏阴性菌的PLFA含量以及革兰氏阳性菌/革兰氏阴性菌的PLFA比值,显著改变了箭竹根际土壤微生物群落结构,结果显著降低了箭竹的生物量。（2）施磷显著增加了受旱箭竹根际土壤中微生物量碳和有效磷的含量,虽大体上对受旱箭竹根际土壤微生物群落的多样性无显著影响,但显著增加了受旱箭竹根际土壤中总PLFA和真菌PLFA的含量,并在一定程度上增加了细菌、革兰氏阳性菌、革兰氏阴性菌和放线菌的PLFA含量以及革兰氏阳性菌/革兰氏阴性菌和真菌/细菌的PLFA比值,也在一定程度上改善了受旱箭竹根际土壤微生物群落结构,从而改善受旱箭竹的生长。（3）主成分分析表明,干旱对箭竹根际土壤微生物群落结构的影响显著,而施磷的影响不明显。（4）相关分析发现,箭竹根际土壤微生物群落结构与箭竹根际土壤微生物量碳、可溶性有机氮及箭竹生物量呈显著正相关。综上,干旱降低了箭竹根际土壤养分含量和微生物生物量,改变了箭竹根际土壤微生物群落结构,抑制了箭竹的生长;施磷能增加受旱箭竹根际土壤养分含量和微生物生物量,改善受旱箭竹根际土壤微生物群落结构,进而改善受旱箭竹的生长。     

Effects of Microbial Community Diversity on the Survival of Pseudomonas aeruginosa in the Wheat Rhizosphere

Ecological theory suggests that microbial communities with greater microbial diversity would be less susceptible to invasion by potential opportunistic pathogens. We investigated whether the survival of the opportunistic pathogen Pseudomonas aeruginosa in the wheat rhizosphere would be affected by the presence of natural and constructed microbial communities of various diversity levels. Three levels of microbial community diversity were derived from wheat roots by a dilution/extinction approach. These wheat rhizosphere inocula, as well as a gnotobiotic microbial community consisting of seven culturable wheat rhizobacterial isolates, were introduced into the nutrient solution of hydroponically grown wheat plants on the day of planting. Phenotypic characterization of the culturable microbial communities on R2A medium, Shannon microbial diversity index, community-level physiological profiles, and terminal restriction fragment length polymorphisms were used to assess the varying microbial diversity levels. At day 7 the roots were invaded with P. aeruginosa and the number of P. aeruginosa colony forming units per root were measured at day 14. The average number of surviving P. aeruginosa cells was 3.52, 4.90, 7.18, 6.65 log₁₀ cfu/root in the high, medium, low, and gnotobiotic microbial community diversity level treatments, respectively. The invasibility of the rhizosphere communities by P. aeruginosa was inversely related to the level of diversity from the dilution extinction gradient. The gnotobiotic community did not confer protection against P. aeruginosa invasion. Although these data indicate that invasibility is inversely related to diversity, further study is needed to both reproduce these findings and define the specific mechanisms of the diversity effect.     

Polymerase chain reaction amplification of naphthalene-catabolic and 16S rRNA gene sequences from indigenous sediment bacteria.

We report the amplification of bacterial genes from uninoculated surface and subsurface sediments by the polymerase chain reaction (PCR). PCR amplification of indigenous bacterial 16S ribosomal DNA genes was unsuccessful when subsurface sediment containing approximately 10(7) cells.g-1 was added directly to a PCR mixture. However, when 10 mg of sediment was inoculated with approximately 10(5) cells of Pseudomonas putida G7, the nahAc naphthalene dioxygenase gene characteristic of the P. putida G7 NAH7 plasmid was detected by PCR amplification. Southern blotting of the PCR amplification product improved sensitivity to 10(3) to 10(4) cells from samples inoculated with P. putida G7, but controls with no sediment added showed that the PCR was partially inhibited by the sediments. Lysozyme-sodium dodecyl sulfate-freeze-thaw DNA extraction was combined with gel electrophoretic partial purification in the presence of polyvinylpyrrolidone to render DNA from indigenous bacteria in surface or subsurface sediment samples amplifiable by PCR using eubacterial 16S ribosomal DNA primers. The nahAc gene could also be amplified from indigenous bacteria by using nahAc-specific primers when PCR conditions were modified by increasing Taq and primer concentrations. Restriction digests of the nahAc amplification products from surface and subsurface sediments revealed polymorphism relative to P. putida G7. The procedures for DNA extraction, purification, and PCR amplification described here demonstrate that the PCR is a potentially useful tool in studies of function- and taxon-specific DNA from indigenous microbial communities in sediment and groundwater environments.     

Direct cellular lysis/protein extraction protocol for soil metaproteomics

We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.     

Fatty acid methyl ester (FAME) profiles as measures of soil microbial community structure

Analysis of fatty acid methyl ester (FAME) profiles extracted from soils is a rapid and inexpensive procedure that holds great promise in describing soil microbial community structure without traditional reliance on selective culturing, which seems to severely underestimate community diversity. Interpretation of FAME profiles from environmental samples can be difficult because many fatty acids are common to different microorganisms and many fatty acids are extracted from each soil sample. We used principal components (PCA) and cluster analyses to identify similarities and differences among soil microbial communities described using FAME profiles. We also used PCA to identify particular FAMEs that characterized soil sample clusters. Fatty acids that are found only or primarily in particular microbial taxa-marker fatty acids-were used in conjunction with these analyses. We found that the majority of 162 soil samples taken from a conventionally-tilled corn field had similar FAME profiles but that about 20% of samples seemed to have relatively low, and that about 10% had relatively high, bacterial:fungal ratios. Using semivariance analysis we identified 21:0 iso as a new marker fatty acid. Concurrent use of geostatistical and FAME analyses may be a powerful means of revealing other potential marker FAMEs. When microbial communities from the same samples were cultured on R2A agar and their FAME profiles analyzed, there were many differences between FAME profiles of soil and plated communities, indicating that profiles of FAMEs extracted from soil reveal portions of the microbial community not culturable on R2A. When subjected to PCA, however, a small number of plated communities were found to be distinct due to some of the same profile characteristics (high in 12:0 iso, 15:0 and 17:1 ante A) that identified soil community FAME profiles as distinct. Semivariance analysis indicated that spatial distributions of soil microbial populations are maintained in a portion of the microbial community that is selected on laboratory media. These similarities between whole soil and plated community FAME profiles suggest that plated communities are not solely the result of selection by the growth medium, but reflect the distribution, in situ, of the dominant, culturable soil microbial populations.     

Large Pseudomonas phages isolated from barley rhizosphere

Abstract: Five bacteriophages infecting common fluorescent pseudomonads ( Pseudomonas fluorescens and Pseudomonas putida ) were isolated from barley rhizosphere soil. Morphological and molecular characteristics of the phages are described together with selected phage-host interactions. All phages belonged to the Myoviridae family with isometrical heads on contractile tails; 4 of them were unusually large and had complex protein and DNA profiles. The large phages had estimated genome sizes of 200 kb or more. Restriction enzyme analyses and DNA-DNA hybridizations showed that all isolates represented different phage species. None of the isolates were observed to establish lysogeny with the main host strain, P. putida MM1. The large phages multiplied slowly on their hosts, producing very small plaques; one-step growth experiments with one of the large phages (Psp 4) hence demonstrated a long latent period (2.5 h) and a very small burst size (10 particles). One of the large phages (Psp 3) was abundant in the rhizosphere (approx. 10⁴ pfu g⁻¹ soil) and had a particularly broad host range which extended to both fluorescent ( Pseudomonas aeruginosa, P. fluorescens, P. putida and Pseudomonas chlororaphis ) and non-fluorescent (Pseudomonas stutzeri) Pseudomonas spp. occurring in soil. The ecological importance of the large Pseudomonas phages must be further studied, but their slow multiplication rates suggested a possible mechanism of balanced phage-host co-existence in the rhizosphere.     

Characterization of the rhizosphere microbial community from different Arabidopsis thaliana genotypes using phospholipid fatty acids (PLFA) analysis

Total and culturable rhizosphere microbial communities structure from three different genotypes of Arabidopsis thaliana growing on three different substrates was studied with phospholipid fatty acid analysis (PLFA) and multivariate statistical analyses: correspondence analysis (CA) and distance based redundancy analyses (db-RDA). In addition, microbial biomass from different groups (total bacteria, Gram+, Gram? and fungi) was calculated from biomarkers PLFA peak area, both from total and culturable microbial community. db-RDA analysis showed significant differences between soils but not between plant genotypes for culturable microbial community structure. Total microbial community was significantly different between soils, and also between plant lines in each soil. Biomass of different bacterial groups showed significant higher values in soil two rhizosphere irrespective of the plant line. In addition, significant differences between plant lines were also found for microbial biomass of different bacterial groups both in total and culturable microbial community. Throughout the work we have demonstrated that PLFA analysis has been able to show a different behaviour of total microbial community with regard to the culturable fraction analyzed in this work under the influence of plant roots. Microbial biomass of different microbial groups calculated with PLFA biomarkers was a suitable tool to detect differences between soils irrespective of the plant line, and differences in the same soil between plant lines. According to this data, a previous study should be carried out before GMPs are used in field conditions to evaluate the potential alterations that may take place on rhizosphere microbial communities structure which may further affect soil productivity. In conclusion, based on data presented in this work, GMPs alter rhizosphere microbial communities structure and this effect is different depending on the soil. Furthermore, total microbial community is affected to a greater extent than the culturable fraction analyzed.     

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log(10) 5 colony forming units g(-1) fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.     

Genes and their organization in the replication origin region of the bacterial chromosome

Genes and their organization are conserved in the replication origin region of the bacterial chromosome. To determine the extent of the conserved region in Gram-positive and Gram-negative bacteria, which diverged 1.2 billion years ago, we have further sequenced the region upstream from the dnaA genes in Bacillus subtilis and Pseudomonas putida. Fifteen open reading frames (ORFs) and 11 ORFs were identified in the 13.6 kb and the 9.8 kb fragments in B. subtilis and P. putida, respectively. Eight consecutive P. putida genes, except for one small ORF (homologous to gene 9K of Escherichia coli) in between, are homologous in sequence and relative locations to genes in B. subtilis. Altogether, 12 genes and their organization are conserved in B. subtilis and P. putida in the origin region. We found that the conserved region terminated on one side after the orf290 in P. putida (orf282 in B. subtilis). In the B. subtilis chromosome, five additional ORFs were found in between the conserved genes, suggesting that they are added after Gram-positive bacteria were diverged from the Gram-negative bacteria. One of the ORFs is a duplicate of the conserved gene. The third non-translatable region containing multiple repeats of DnaA-box (second in the case of P. putida) was found flanking gidA in both organisms. This result shows clearly that E. coli oriC and flanking genes gidA and gidB have been translocated by the inversion of some 40 kb fragment.     

Endophytic and ectophytic potato-associated bacterial communities differ in structure and antagonistic function against plant pathogenic fungi

Differences between endophytic and ectophytic bacterial communities with stress on antagonistic bacteria, were studied by comparing the composition of communities isolated from the rhizosphere, phyllosphere, endorhiza and endosphere of field-grown potato plants using a multiphasic approach. Terminal restriction fragment length polymorphism analysis of 16S rDNA of the bacterial communities revealed discrete microenvironment-specific patterns. To measure the antagonistic potential of potato-associated bacteria, a total of 2648 bacteria were screened by dual testing of antagonism to the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. Composition and diversity of bacterial antagonists were mainly specific for each microenvironment. The rhizosphere and endorhiza were the main reservoirs for antagonistic bacteria and showed the highest similarity in their colonisation by antagonists. The most prominent species of all microenvironments was Pseudomonas putida, and rep-PCR with BOX primers showed that these isolates showed microenvironment-specific DNA fingerprints. P. putida isolates from the rhizosphere and endorhiza gave nearly identical fingerprints confirming the high similarity of bacterial populations. The phlD gene, involved in the production of the antibiotic 2,4-diacetyl-phloroglucinol, was found only among Pseudomonas isolates from the rhizosphere and endorhiza. Evaluation of the bacterial isolates for biocontrol potential based on fungal antagonism and physiological characteristics resulted in the selection of five promising isolates from each microenvironment. The most effective isolate was Serratia plymuthica 3Re4-18 isolated from the endorhiza.     

Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting mRNA and rRNA

A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences was labeled with Cy3 fluor, and the probe specific to the tom genes was labeled with Alexa647 fluor. Probe specificity was first determined, and hybridization temperature was optimized using three rhizosphere bacteria pure cultures as controls, along with the P. putida TOM20 strain. The probes were highly specific to the respective targets, with minimal non-specific binding. The recombinant strain was inoculated into wheat seedling rhizosphere. Colonization of P. putida TOM20 was confirmed by extraction of root biofilm and growth of colonies on selective agar medium. Confocal microscopy of hybridized root biofilm detected P. putida TOM20 cells emitting both Cy3 and Alexa647 fluorescence signals.     

Changes in Microbial Communities,Including both Uncultured and Culturable Bacteria,with Mid-Ocean Ballast-Water Exchange during a Voyage from Japan to Australia

We assessed changes in the microbial communities in ballast water during a trans-Pacific voyage from Japan to Australia that included a mid-ocean ballast-water exchange. Uncultured (i.e., total) and culturable bacteria were counted and were characterized by using denaturing gradient gel electrophoresis (DGGE). There was a clear decrease over time in numbers of uncultured microorganisms, except for heterotrophic nanoflagellates, whereas the abundance of culturable bacteria initially decreased after the ballast-water exchange but then increased. The increase, however, was only up to 5.34% of the total number of uncultured bacteria. Cluster analysis showed that the DGGE profiles of uncultured bacteria clearly changed after the exchange. In contrast, there was no clear change in the DGGE profiles of culturable bacteria after the exchange. Multidimensional scaling analysis showed changes in microbial communities over the course of the voyage. Although indicator microbes as defined by the International Convention for the Control and Management of Ships'' Ballast Water and Sediments were occasionally detected, no coliform bacteria were detected after the exchange.     

Temporal dynamics of microbial communities in the rhizosphere of two genetically modified (GM) maize hybrids in tropical agrosystems

The use of genetically modified (GM) plants still raises concerns about their environmental impact. The present study aimed to evaluate the possible effects of GM maize, in comparison to the parental line, on the structure and abundance of microbial communities in the rhizosphere. Moreover, the effect of soil type was addressed. For this purpose, the bacterial and fungal communities associated with the rhizosphere of GM plants were compared by culture-independent methodologies to the near-isogenic parental line. Two different soils and three stages of plant development in two different periods of the year were included. As evidenced by principal components analysis (PCA) of the PCR-DGGE profiles of evaluated community, clear differences occurred in these rhizosphere communities between soils and the periods of the year that maize was cultivated. However, there were no discernible effects of the GM lines as compared to the parental line. For all microbial communities evaluated, soil type and the period of the year that the maize was cultivated were the main factors that influenced their structures. No differences were observed in the abundances of total bacteria between the rhizospheres of GM and parental plant lines.     

Maintenance and Impacts of an Inoculated mer/luc-Tagged Pseudomonas fluorescens on Microbial Communities in Birch Rhizospheres Developed on Humus and Peat

Antagonistic bacteria represent promising biocontrol agents for improving forest production in seedling nurseries or forest soils. The fate of an introduced mer/luc-tagged antagonistic Pseudomonas fluorescens 31K3 was monitored in the rhizosphere of silver birch (Betula pendula) seedlings grown in microcosms containing forest humus or nursery peat. The inoculated strain (10(8) cfu g(-1) soil) was unable to establish in significant numbers in either soil type and turned nonculturable in humus. Detection in both soils was possible only via luminescence of enrichment cultures 80 days post-inoculation. Despite low P. fluorescens survival, inoculation had a positive effect on seedling growth. Limited impact of inoculation on the indigenous microbial communities was identified following analyses of respiration and denitrification potential, community-level physiological profiles and molecular fingerprinting of fungi and eubacteria, and Pseudomonas community structures. The minor changes observed in the indigenous microbial communities, including mycorrhiza development, were not consistent between humus and peat growth substrates. It was concluded that the rhizosphere-related microbial communities developed in both of these highly organic soil systems are highly buffered against introduction of foreign bacteria.     

Detection of Pseudomonas putida B in the rhizosphere by RAPD

A method was developed for the detection of Pseudomonas putida B MM12 released into the rhizosphere of non-sterile barley, using a Random Amplified Polymorphic DNA (RAPD)-generated probe for hybridization with RAPD products generated from DNA extracted from the rhizosphere. The detection procedure involves extraction of rhizosphere bacteria by sonication, extraction of DNA by boiling, RAPD and Southern hybridization with RAPD products and the selected probe. The level of detection of MM12 was at least 1·9×10⁴ cells g⁻¹ barley root. MM12 was detected in rhizosphere when it constituted as little as 0·5% of the culturable population.     

Bacterial endophyte-mediated naphthalene phytoprotection and phytoremediation

Polyaromatic hydrocarbons (PAHs) are major and recalcitrant pollutants of the environment and their removal presents a significant problem. Phytoremediation has shown much promise in PAH removal from contaminated soil, but may be inhibited because the plant experiences phytotoxic effects from low-molecular-weight PAHs such as naphthalene. This paper describes the construction of a naphthalene-degrading endophytic strain designated Pseudomonas putida VM1441(pNAH7). This strain was found to be an efficient colonizer of plants, colonizing both the rhizosphere and interior root tissues. The inoculation of plants with P. putida VM1441(pNAH7) resulted in the protection of the host plant from the phytotoxic effects of naphthalene. When inoculated plants were exposed to naphthalene, both seed germination and plant transpiration rates were higher than those of the uninoculated controls. The inoculation of plants with this strain also facilitated higher (40%) naphthalene degradation rates compared with uninoculated plants in artificially contaminated soil.