Diffusible Metabolites Research Articles

The Growth of <i>Steroidobacter agariperforans</i> sp. nov., a Novel Agar-Degrading Bacterium Isolated from Soil, is Enhanced by the Diffusible Metabolites Produced by Bacteria Belonging to <i>Rhizobiales</i>

An agar-degrading bacterium was isolated from soil collected in a vegetable cropping field. The growth of this isolate was enhanced by supplying culture supernatants of bacteria belonging to the order Rhizobiales. Phylogenetic analysis based on 16S rRNA gene sequences indicated the novel bacterium, strain KA5–BT, belonged to the genus Steroidobacter in Gammaproteobacteria, but differed from its closest relative, Steroidobacter denitrificans FST, at the species level with 96.5% similarity. Strain KA5–BT was strictly aerobic, Gram-negative, non-motile, non-spore forming, and had a straight to slightly curved rod shape. Cytochrome oxidase and catalase activities were positive. The strain grew on media containing culture supernatants in a temperature range of 15–37°C and between pH 4.5 and 9.0, with optimal growth occurring at 30°C and pH 6.0–8.0. No growth occurred at 10 or 42°C or at NaCl concentrations more than 3% (w/v). The main cellular fatty acids were iso–C15:0, C16:1ω7c, and iso–C17:1ω9c. The main quinone was ubiquinone-8 and DNA G+C content was 62.9 mol%. In contrast, strain FST was motile, did not grow on the agar plate, and its dominant cellular fatty acids were C15:0 and C17:1ω8c. Based on its phylogenetic and phenotypic properties, strain KA5–BT (JCM 18477T = KCTC 32107T) represents a novel species in genus Steroidobacter, for which the name Steroidobacter agariperforans sp. nov. is proposed.

A link between gut community metabolism and pathogenesis: molecular hydrogen-stimulated glucarate catabolism aids Salmonella virulence

Glucarate, an oxidized product of glucose, is a major serum organic acid in humans. Still, its role as a carbon source for a pathogen colonizing hosts has not been studied. We detected high-level expression of a potential glucarate permease encoding gene gudT when Salmonella enterica serovar Typhimurium are exposed to hydrogen gas (H2), a gaseous by-product of gut commensal metabolism. A gudT strain of Salmonella is deficient in glucarate-dependent growth, however, it can still use other monosaccharides, such as glucose or galactose. Complementation of the gudT mutant with a plasmid harbouring gudT restored glucarate-dependent growth to wild-type (WT) levels. The gudT mutant exhibits attenuated virulence: the mean time of death for mice inoculated with WT strain was 2 days earlier than for mice inoculated with the gudT strain. At 4 days postinoculation, liver and spleen homogenates from mice inoculated with a gudT strain contained significantly fewer viable Salmonella than homogenates from animals inoculated with the parent. The parent strain grew well H2-dependently in a minimal medium with amino acids and glucarate provided as the sole carbon sources, whereas the gudT strain achieved approximately 30% of the parent strain's yield. Glucarate-mediated growth of a mutant strain unable to produce H2 was stimulated by H2 addition, presumably owing to the positive transcriptional response to H2. Gut microbiota-produced molecular hydrogen apparently signals Salmonella to catabolize an alternative carbon source available in the host. Our results link a gut microbiome-produced diffusible metabolite to augmenting bacterial pathogenesis.

Characterization of antagonistic‐potential of two Bacillus strains and their biocontrol activity against Rhizoctonia solani in tomato

To investigate the biocontrol mechanism of two antagonistic Bacillus strains (Bacillus subtilis MB14 and Bacillus amyloliquefaciens MB101), three in vitro antagonism assays were screened and the results were concluded that both strains inhibited Rhizoctonia solani growth in a similar manner by dual culture assay, but the maximum percent of inhibition only resulted with MB101 by volatile and diffusible metabolite assays. Moreover, cell free supernatant (CFS) of MB101 also showed significant (p > 0.05) growth inhibition as compared to MB14, when 10 and 20% CFS mix with the growth medium of R. solani. After in vitro-validation, both strains were evaluated under greenhouse and the results concluded that strain MB101 had significant biocontrol potential as compared to MB14. Strain MB101 was enhanced the plant height, biomass and chlorophyll content of tomato plant through a higher degree of root colonization. In field trials, strain MB101 showed higher lessening in root rot symptoms with significant fruit yield as compare to strain MB14 and infected control. Next to the field study, the presence of four antibiotic genes (srfAA, fenD, ituC, and bmyB) also concluded the antifungal nature of both Bacillus strains. Phylogenetic analysis of protein sequences revealed a close relatedness of three genes (srfAA, fenD, and ituC) with earlier reported sequences of B. subtilis and B. amyloliquefaciens. However, bmyB showed heterogeneity in among both strains (MB14 and MB101) and it may be concluded that higher degree of antagonism, root colonization and different antibiotic producing genes may play an important role in biocontrol mechanism of strain MB101.

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Characterization of Novel Lipopeptides Produced by Bacillus tequilensis P15 Using Liquid Chromatography Coupled Electron Spray Ionization Tandem Mass Spectrometry (LC–ESI–MS/MS)

Microbes are known to interact and communicate with their neighbouring cells by releasing diverse types of low molecular weight diffusible metabolites. This paper describes the identification of iturins, fengycins and surfactins secreted by Bacillus tequilensis P15 isolated from sea coast of Jakhao, Kutch, India, using liquid chromatography coupled electron spray ionization tandem mass spectrometry. In methanol soluble fraction of acid precipitate harvested from cell free supernatant of B. tequilensis P15, 5 variants of iturins, 6 of fengycins and 39 surfactins could be identified. In particular, new surfactins with Ile/Leu at position 5 and Asp at position 6 in the peptide chain were discovered, which have not been previously reported. A novel class of novel surfactin consisting of Glu/methyl ester of Asp at position 5 in peptide chain was also identified. In addition, several linear forms of surfactins were also identified in the methanol soluble extracellular fraction of B. tequilensis P15. This is the first report on co-production of all the three classes of cyclic lipopeptides by a marine isolate B. tequilensis.

Acyl-homoserine lactone-based quorum sensing in the Roseobacter clade: complex cell-to-cell communication controls multiple physiologies.

Bacteria have been widely reported to use quorum sensing (QS) systems, which employ small diffusible metabolites to coordinate gene expression in a population density dependent manner. In Proteobacteria, the most commonly described QS signaling molecules are N-acyl-homoserine lactones (AHLs). Recent studies suggest that members of the abundant marine Roseobacter lineage possess AHL-based QS systems and are environmentally relevant models for relating QS to ecological success. As reviewed here, these studies suggest that the roles of QS in roseobacters are varied and complex. An analysis of the 43 publically available Roseobacter genomes shows conservation of QS protein sequences and overall gene topologies, providing support for the hypothesis that QS is a conserved and widespread trait in the clade.

In Vitro Antifungal Activity of Burkholderia gladioli pv. agaricicola against Some Phytopathogenic Fungi

The trend to search novel microbial natural biocides has recently been increasing in order to avoid the environmental pollution from use of synthetic pesticides. Among these novel natural biocides are the bioactive secondary metabolites of Burkholderia gladioli pv. agaricicola (Bga). The aim of this study is to determine antifungal activity of Bga strains against some phytopathogenic fungi. The fungicidal tests were carried out using cultures and cell-free culture filtrates against Botrytis cinerea, Aspergillus flavus, Aspergillus niger, Penicillium digitatum, Penicillium expansum, Sclerotinia sclerotiorum and Phytophthora cactorum. Results demonstrated that all tested strains exert antifungal activity against all studied fungi by producing diffusible metabolites which are correlated with their ability to produce extracellular hydrolytic enzymes. All strains significantly reduced the growth of studied fungi and the bacterial cells were more bioactive than bacterial filtrates. All tested Bulkholderia strains produced volatile organic compounds (VOCs), which inhibited the fungal growth and reduced the growth rate of Fusarium oxysporum and Rhizoctonia solani. GC/MS analysis of VOCs emitted by strain Bga 11096 indicated the presence of a compound that was identified as 1-methyl-4-(1-methylethenyl)-cyclohexene, a liquid hydrocarbon classified as cyclic terpene. This compound could be responsible for the antifungal activity, which is also in agreement with the work of other authors.

Burkholderia cepacia XXVI siderophore with biocontrol capacity against Colletotrichum gloeosporioides

Colletotrichum gloeosporioides is the causal agent of anthracnose in mango. Burkholderia cepacia XXVI, isolated from mango rhizosphere and identified by 16S rDNA sequencing as a member of B. cepacia complex, was more effective than 6 other mango rhizosphere bacteria in inhibiting the model mango pathogen, C. gloeosporioides ATCC MYA 456. Biocontrol of this pathogen was demonstrated on Petri-dishes containing PDA by > 90 % reduction of surface colonization. The nature of the biocontrol metabolite(s) was characterized via a variety of tests. The inhibition was almost exclusively due to production of agar-diffusible, not volatile, metabolite(s). The diffusible metabolite(s) underwent thermal degradation at 70 and 121 °C (1 atm). Tests for indole acetic acid production and lytic enzyme activities (cellulase, glucanase and chitinase) by B. cepacia XXVI were negative, indicating that these metabolites were not involved in the biocontrol effect. Based on halo formation and growth inhibition of the pathogen on the diagnostic medium, CAS-agar, as well as colorimetric tests we surmised that strain XXVI produced a hydroxamate siderophore involved in the biocontrol effect observed. The minimal inhibitory concentration test showed that 0.64 μg ml(-1) of siderophore (Deferoxamine mesylate salt-equivalent) was sufficient to achieve 91.1 % inhibition of the pathogen growth on Petri-dishes containing PDA. The biocontrol capacity against C. gloeosporioides ATCC MYA 456 correlated directly with the siderophore production by B. cepacia XXVI: the highest concentration of siderophore production in PDB on day 7, 1.7 μg ml(-1) (Deferoxamine mesylate salt-equivalent), promoted a pathogen growth inhibition of 94.9 %. The growth of 5 additional strains of C. gloeosporioides (isolated from mango "Ataulfo" orchards located in the municipality of Chahuites, State of Oaxaca in Mexico) was also inhibited when confronted with B. cepacia XXVI. Results indicate that B. cepacia XXVI or its siderophore have the potential to be used as a biological control agent against C. gloeosporioides; thus diminishing environmental problems caused by the current practices to control this disease.

Truffle brûlé: an efficient fungal life strategy

The terms 'brûlé' and 'burnt' are used to describe vegetation-devoid areas of the ground around a range of woody plants interacting with certain truffle species. Increasing interest is currently focused on a systematic search for and study of volatile organic compounds (VOCs) emitted by truffles in the course of their life cycle. These metabolites are now recognized as biochemicals with an important impact on burnt formation. Based on current molecular approaches, Tuber melanosporum is emerging as an aggressive colonizer of the brûlé, dominant in competition with indigenous brûlé-associated organisms, suppressing their richness and biodiversity. There is compelling evidence that mycelia, mycorrhizae, and fruiting bodies of brûlé-forming truffles have evolved diffusible metabolites for their survival, typically characterized as having harmful effects on weeds, impairing seed germination, altering root morphogenesis and plant hormonal balance, or inhibiting the native rhizospheric microflora regularly associated with the brûlé. These effects can be widely interpreted as allelopathic phenomena, and the brûlé may thus be regarded as a promising opportunity to study truffle allelopathy. Considering the outstanding success of the genome analysis in T.melanosporum, we are facing a very difficult task to proceed from the molecular to the ecological level.

ANTAGONISTIC ACTIVITIES OF Bacillus amyloliquefaciens FROM PHYLLOSPHERE OF SUGAR BEET AGAINST Cercospora beticola sacc.

Sixteen isolates of Bacillus amyloliquefaciens isolatedfrom phyllosphere of sugar beet leaves were tested for its antagonistic activity against Cercospora beticola the causal agent of sugar beet leaf spot disease. The tested isolates showed a significant antagonistic potential against C. beticola in vitro. The inhibition of the radial growth of the pathogen ranged from 77.8% to 90%. The diffusible metabolites and volatile gases produced by B. amyloliquefaciens dramatically inhibited the pathogen growth. The antifungal proteins (AFP) extracted from culture filtrates showed an antagonistic effect on the pathogen and inhibition zones ranged from 8.17 mm to 16.45 mm. The antifungal metabolites (AFM) produced by the tested B. amyloliquefaciens isolates showed a high thermal stability. The activity of the AFM at pH 3.0 upto 5.0 reduced by 20% and started to increase at pH 6.0 then reached its maximum at pH 7.0. All the tested isolates produced Sidrophores and β1,3,glucanase while only three isolates were able to produce chitinase. In the greenhouse experiments, the AFM and bacterial cells treatments decreased the severity of cercospora leaf spot. It was found that the treatments with AFM or bacterial cells before inoculation with the pathogen was more efficient than the treatments after inoculation with the pathogen. This may be the first report on using B. amyloliquefaciens as a biocontrol agent against C. beticola

Assessment of root-associated paenibacillus polymyxa groups on growth promotion and induced systemic resistance in pepper.

Twenty-nine P. polymyxa strains isolated from rhizospheres of various crops were clustered into five genotypic groups on the basis of BOX-PCR analysis. The characteristics of several plant growth-promoting factors among the isolates revealed the distinct attributes in each allocated group. Under gnotobiotic conditions, inoculation of pepper roots with P. polymyxa isolates significantly increased the biomass in 17 of total 29 treated plants with untreated plants. Experiments on induced systemic resistance (ISR) against bacterial spot pathogen Xanthomonas axonopodis pv. vesicatoria in pepper by P. polymyxa strains were conducted and only one isolate (KNUC265) was selected. Further studies into ISR mediation by the KNUC265 strain against the soft-rot pathogen Erwinia carotovora subsp. carotovora in tobacco demonstrated that the tobacco seedlings exposed to either bacterial volatiles or diffusible metabolites exhibited a reduction in disease severity. In conclusion, ISR and plant growth promotion triggered by P. polymyxa isolates were systemically investigated on pepper for the first time. The P. polymyxa KNUC265 strain, which elicited both ISR and plant growth promotion, could be potentially used in improving the yield of pepper and possibly of other crops.

Identification and Biological Characterization of Isolates with Activity Inhibitive against Macrophomina phaseolina (Tassi) Goid

The Macrophomina phaseolina (Tassi) Goid fungus, causal agent of charcoal root rot, is considered one of the most important pathogens in forest nurseries. Recent studies in Chile have verified the antagonistic capacity of diverse bacterial isolates against M. phaseolina in vitro as well as in field testing. The objectives of this study were to identify Bacillus isolates and establish the mechanisms of antagonism against M. phaseolina. Isolates were identified with the bioMerieux biochemical kit. Tests were done in vitro and consisted in determining the production of volatile metabolites, siderophores, and the effect of different culture temperatures (20, 25, 30, and 35 °C) on the production of diffusible metabolites and inhibition of mycelial growth of the pathogen. The isolates were identified as Bacillus subtilis IX 007, B. amyloliquefaciens VII 015 and VIII 016, B. pumilus IX 030, and B. stearothermophilus TM 008. It was not possible to identify the (-) Gram VI 009 isolate. At all the evaluated temperatures, isolates inhibited M. phaseolina, reaching 75% reduction of mycelial growth (B. subtilis IX 007 at 25 °C). Some isolates showed volatile metabolites and siderophore production as possible mechanisms of antagonism.

Biocontrol activities of Bacillus amyloliquefaciens DGA14 isolated from banana fruit surface against banana crown rot-causing pathogens

Two bacteria, isolated from the surface of banana fruits, one forming a creamy white colony and the other, dry yeast like colony were screened for in vitro antagonism toward Lasiodiplodia theobromae. Both isolates, identified as Bacillus amyloliquefaciens, were further tested for antibiosis against other crown rot-causing pathogens ( Thielaviopsis paradoxa, Colletotrichum musae, and Fusarium verticillioides). The creamy white colony strain, coded as B. amyloliquefaciens DGA14, was subjected to laboratory and field studies. B. amyloliquefaciens DGA14 produced a diffusible metabolite that inhibited all test pathogens in culture. In addition, bacteria moved and attached to pathogens significantly affecting mycelial growth and conidial germination in liquid medium. Following inoculation, B. amyloliquefaciens DGA14 survived and colonized banana fruits after 2 d. Interparasitic relationships were observed between the antagonist and pathogens on artificial media and the natural substrate. Postharvest application of B. amyloliquefaciens DGA14 in the packing house reduced the incidence of crown rot to a level significantly lower than in fungicide treated or control fruits.

Mesenchymal Stem Cells as Suicide Gene Therapy Vehicles for Organ-Confined and Metastatic Prostate Cancer (PCa).

The efficacy of mesenchymal stem cells (MSC) is currently being examined as a clinical regenerative medicine for multiple sclerosis, cirrhosis, liver disease, tibial fractures, heart failure and graft versus host disease. MSC display an inherent tumor-tropic property that has been exploited for the targeted delivery of therapeutic genes to metastatic melanoma, glioma, breast and colon carcinoma in animal models. Advantages of using MSC include their ability for: self-renewal, ease of propagation and gene modification ex vivo; secretion of high levels of therapeutic protein; evasion of immune rejection; differentiation into connective tissue and tumor stroma; and long-term engraftment in vivo. This study explores the utility of MSC to deliver reporter or suicide genes to advanced Pca which is currently incurable using the syngeneic RM1 mouse model. Sca-1bright CD45− cells sorted from adherent bone marrow cells were shown to be true MSC by their ability to undergo tri-lineage differentiation in adipogeneic, osteogenic and chondrogenic media and their characteristic CD44+, CD90+, CD73+ and CD106+ phenotype. Lentiviral vectors showed sustained green fluorescent protein (GFP) reporter gene expression in MSC (MSC-GFP) for 50 passages by flow cytometry. When MSC-GFP (10e6 cells) were implanted into the mouse prostate with or without RM1 tumor cells on day 0, examination by full body fluorescence imaging (IVIS 100; Xenogen/Caliper) showed MSC persisted only within the tumor-bearing prostate (p<0.05; day 18). No MSC were detected in any other organ examined. To test their systemic homing ability, MSC-GFP (10e6 cells) were infused every second day (2–14) via the tail vein of mice in the presence or absence of RM1 lung pseudometastases. MSC persisted within the lungs of RM1 tumor-bearing mice alone (p<0.01) with no detectable MSC in any other organ examined (day 18). These results suggest MSC engraft organ-confined Pca and home to metastatic Pca. Gene directed enzyme prodrug therapy (GDEPT) describes the transfer of a suicide gene, not expressed in mammalian cells, into tumors using viral vectors. This renders tumors sensitive to prodrugs that are non-toxic to normal tissues. In our pre-clinical study, prostate tumors established from RM1 tumor cells stably transfected with fusion suicide gene cytosine deaminase/uracil phosphoribosyltransferase (CDUPRT) followed by systemic treatment with prodrug 5-fluorocytosine, showed significant reductions in prostate tumor growth and pseudometastases in the lungs (1). More recently, we stably transfected MSC with CDUPRT prior to implantation into established RM1 prostate tumors. In the presence of prodrug MSC-CDUPRT showed similar levels of Pca killing observed in the published experiment. In both experiments CDUPRT in the presence of prodrug significantly reduced (∼75%; p<0.05) the weight of RM1 prostate tumors compared to the control gene or no prodrug control mice. These results demonstrate that MSC can deliver suicide genes to developing Pca and efficiently convert prodrug to a toxic diffusible metabolite to suppress tumor growth. MSC implantation was well tolerated without observed toxicity and therefore show promise as a novel form of cell-directed suicide gene therapy.

New strains obtained after UV treatment and protoplast fusion of native Trichoderma harzianum: their biocontrol activity on Pyrenochaeta lycopersici

The obtainment of 30 new strains from native Trichoderma harzianum after UV light irradiation (UV-A and UV-C), and of 82 strains resulted from protoplast fusion were accomplished. The new strains, initially selected for their growing rate under low temperature and high pH conditions, as well as for their innocuousness on tomato plants, were tested for in vitro inhibition of Pyrenochaeta lycopersici in dual cultures and due to secretion of volatile and diffusible metabolites. All the UV-A and UV-C selected candidate mutants were innocuous to tomato plants, but none of them showed improvement in their biocontrol activity on P. lycopersici . Th12A20.1 increased 1.3 and 1.9 fold the total fresh weight of Fortaleza tomato plants when compared to its parental strains Th12 and Th11, respectively. The selected candidate mutants obtained through protoplast fusion were also innocuous to tomato plants, but only ThF1-2 and ThF4-4 inhibited 1.3 fold (in dual cultures) and 5 fold (due to secretion of volatile metabolites) the growth of P. lycopersici , respectively, in relation to the mean inhibitory effect of both parents. Therefore, these candidate mutants could be included in experiments under field conditions.

The role of antibiosis in the antagonism of different bacteria towards Helminthosporium solani, the causal agent of potato silver scurf

Des antagonistes bactériens envers Helminthosporium solani ont été soumis à différents essais en vue de déterminer le rôle de l'antibiose dans l'antagonisme observé. Parmi les souches bactériennes évaluées, sept (Alcaligenes piechaudii, Aquaspirillum autotrophicum, Cellulomonas fimi, Pseudomonas chlororaphis, Pseudomonas putida (souches 94-19 et E-30) et Streptomyces griseus) ont produit dans le milieu gélosé des métabolites inhibant la croissance mycélienne et/ou la germination des conidies de H. solani. Les métabolites produits en culture pure et en culture mixte ont présenté une activité différente sur la croissance mycélienne de H. solani. Les résultats présentés indiquent que la méthodologie employée peut être un facteur déterminant dans l'identification de l'antibiose comme mode d'action d'un agent antagoniste.

Desferrioxamine E produced by Streptomyces griseus stimulates growth and development of Streptomyces tanashiensis

The authors previously reported that interspecific stimulatory events between Streptomyces species for antibiotic production and/or morphological differentiation mediated by putative diffusible metabolites take place at a high frequency. This paper reports the isolation and characterization of a substance produced by Streptomyces griseus that stimulates the growth and development of Streptomyces tanashiensis. The substance was purified from the culture supernatant of S. griseus by using anion-exchange chromatography, gel filtration chromatography and reverse-phase HPLC. FAB-MS and NMR analyses of the purified preparation indicated the substance to be desferrioxamine E (synonym: nocardamine), a siderophore that is widely produced by Streptomyces species and related organisms. Similar stimulatory effects on the growth and development of S. tanashiensis were exerted by desferrioxamine E produced by another actinomycete strain, but not by other siderophores tested, including ferrichrome and nocobactin and free ferric ion. An exogenous supply of desferrioxamine E stimulated secondary metabolite formation and/or morphological differentiation in various actinomycete strains. Disruption of the desferrioxamine biosynthesis gene cluster in Streptomyces coelicolor A3(2) abolished the production of desferrioxamine E and the activity to stimulate the growth and differentiation of S. tanashiensis. The S. coelicolor mutant showed impaired growth and development on Bennett's/glucose agar medium, but it was rescued by the exogenous supply of desferrioxamine E. These results indicate that desferrioxamines play an important role in streptomycete physiology. Similar to several pathogenic bacteria and fungi, S. tanashiensis may be defective in the production of siderophores; however, it can utilize the siderophores excreted by other organisms.

Controlling Bacterial Infection by Inhibiting Intercellular Signalling

The opportunistic human pathogen Pseudomonas aeruginosa, which causes fatal lung infections in cystic fibrosis patients, regulates the expression of virulence factors by N-acyl-Lhomoserine lactone (AHL) mediated gene expression. AHL mediated gene expression, also known as quorum sensing, involves the production of and response to a small diffusible metabolite. This gene expression mechanism allows populations of P. aeruginosa to coordinate expression of virulence factors to maximise the success of infection. AHL mediated gene expression offers a new target for the control of bacterial infection that is not dependent on the use of compounds with antibiotic activity. This review summarises investigations that have demonstrated the efficacy of marine natural products in inhibiting AHL mediated gene expression and controlling P. aeruginosa infection. Keywords: quorum sensing, delisea, homoserine lactone, infection, pseudomonas aeruginosa

Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus trichoderma harzianum rifai 1295-22

We investigated the capability of the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22 (T-22) to solubilize in vitro some insoluble or sparingly soluble minerals via three possible mechanisms: acidification of the medium, production of chelating metabolites, and redox activity. T-22 was able to solubilize MnO2, metallic zinc, and rock phosphate (mostly calcium phosphate) in a liquid sucrose-yeast extract medium, as determined by inductively coupled plasma emission spectroscopy. Acidification was not the major mechanism of solubilization since the pH of cultures never fell below 5.0 and in cultures containing MnO2 the pH rose from 6.8 to 7.4. Organic acids were not detected by high-performance thin-layer chromatography in the culture filtrates. Fe2O3, MnO2, Zn, and rock phosphate were also solubilized by cell-free culture filtrates. The chelating activity of T-22 culture filtrates was determined by a method based on measurement of the equilibrium concentration of the chrome azurol S complex in the presence of other chelating substances. A size exclusion chromatographic separation of the components of the culture filtrates indicated the presence of a complexed form of Fe but no chelation of Mn. In liquid culture, T. harzianum T-22 also produced diffusible metabolites capable of reducing Fe(III) and Cu(II), as determined by the formation of Fe(II)-Na2-bathophenanthrolinedisulfonic acid and Cu(I)-Na2-2, 9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonic acid complexes. This is the first report of the ability of a Trichoderma strain to solubilize insoluble or sparingly soluble minerals. This activity may explain, at least partially, the ability of T-22 to increase plant growth. Solubilization of metal oxides by Trichoderma involves both chelation and reduction. Both of these mechanisms also play a role in biocontrol of plant pathogens, and they may be part of a multiple-component action exerted by T-22 to achieve effective biocontrol under a variety of environmental conditions.

The effect of diffusible metabolites of Trichoderma harzianum on in vitro interactions between basidiomycete isolates at two different temperature regimes

As part of a study aimed at developing a biological control of decay in freshly felled pine the action of Trichoderma harzianum on the interactions between target decay basidiomycetes was investigated. An in vitro bilayer system allowed the action of the mould's diffusible metabolites, as distinct from the other modes of antagonism, to be studied. Seven basidiomycete isolates, representing commonly found decay fungi in pine, were challenged against each other in the presence of Trichoderma metabolites and also on control (uninoculated) bilayers. Challenges were carried out at two temperatures, 10° and 22 °C. Both temperature and the presence of Trichoderma influenced the outcome of the interactions seen on the bilayers. Temperature had a marked effect on some of the challenges for instance switching a deadlock interaction to overgrowth of one individual by the other. Similarly, the same challenge could have contrasting outcomes depending on whether the interaction occurred on Trichoderma or control bilayers. The relevance of the results to biological control of wood decay is discussed.