Cultural techniques for improvement in biocontrol potential of fungal antagonists for biological control of Armillaria root rot of strawberry plants under glasshouse conditions

Several in vitro and in vivo experiments were conducted to develop an effective technique for culturing potential fungal antagonists (isolates of Trichoderma harzianum, Dactylium dendroides, Chaetomium olivaceum and one unidentified fungus) selected for activity against Armillaria mellea. The antagonists were inoculated onto (1) live spawn of the oyster mu shroom (Pleurotus ostreatus), (2) extra-moistened or sucrose-enriched mushroom composts containing living or autoclaved mycelia of P. ostreatus or Agaricus bisporus (button mushroom), (3) pasteurized compost with or without A. bisporus mycelium, wheat bran, wheat germ and (4) spent mushroom composts with living mycelia of A. bisporus, P. ostreatus or Lentinus edodes (the Shiitake mushroom). In one experiment, a representative antagonist (isolate Th2 of T. harzianum) was grown together with the A. bisporus mycelium, while in another one, the antagonist was first grown on wheat germ or wheat bran and then on mushroom compost with living mycelium of A. bisporus. Some of the carrier substrates were then added to the roots of potted strawberry plants in the glasshouse to evaluate their effectiveness against the disease. The antagonists failed to grow on the spawn of P. ostreatus even after reinoculations and prolonged incubation. Providing extra moisture or sucrose enrichment also did not improve the growth of Th2 on mushroom composts in the presence of living mycelia of A. bisporus or P. ostreatus. The antagonist, however, grew rapidly and extensively on mushroom compost with autoclaved mycelia, and also on wheat germ and wheat bran. Colonization of the substrates by the antagonist was positively correlated with its effectiveness in the glasshouse studies. Whereas only 33.3% of the inoculated control plants survived in one experiment monitored for 560 days, 100% survival was achieved when Th2 was applied on wheat germ or wheat bran. Growth of the antagonist alone on pasteurized or sterilized compost (without A. bisporus mycelia) and simultaneous growth of the antagonist and mushroom on pasteurized compost did not improve survival over the inoculated controls, but growth over mushroom compost with the living mycelium resulted in 50% survival rate. C. olivaceum isolate Co was the most effective, resulting in overall survival rate of 83.3% compared with only 8.3% for the inoculated and 100% for the uninoculated (healthy) controls. This antagonist gave the highest survival rate of 100% on spent mushroom compost with L. edodes. T harzianum isolate Th23, with 75% survival rate, was the most effective on spent mushroom compost with P. ostreatus, while D. dendroides isolate SP resulted in equal survival rates of 50% on all the three mushroom composts.

Fecal microbiota in patients receiving enteral feeding are highly variable and may be altered in those who develop diarrhea

Background: The pathogenesis of diarrhea in patients receiving enteral feeding includes colonic water secretion, antibiotic prescription, and enteropathogenic colonization, each of which involves an interaction with the gastrointestinal microbiota. Objective: The objective was to investigate temporal changes in the concentrations of fecal microbiota and short-chain fatty acids (SCFAs) in patients starting 14-d of enteral feeding and to compare these changes between patients who do and do not develop diarrhea. Design: Twenty patients starting exclusive nasogastric enteral feeding were monitored for 14 d. Fecal samples were collected at the start, middle, and end of this period and were analyzed for major bacterial groups by using culture independent fluorescence in situ hybridization and for SCFAs by using gas-liquid chromatography. Results: Although no significant changes in fecal microbiota or SCFAs were observed during enteral feeding, stark alterations occurred within individual patients. Ten patients (50%) developed diarrhea, and these patients had significantly higher concentrations of clostridia (P = 0.026) and lower concentrations (P = 0.069) and proportions (P = 0.029) of bifidobacteria. Patients with and without diarrhea had differences in the proportion of bifidobacteria (median: 0.4% and 3.7%; interquartile range: 0.8 compared with 4.3; P = 0.035) and clostridia (median: 10.4% and 3.7%; interquartile range: 14.7 compared with 7.0; P = 0.063), respectively, even at the start of enteral feeding. Patients who developed diarrhea had higher concentrations of total fecal SCFAs (P = 0.044), acetate (P = 0.029), and butyrate (P = 0.055). Conclusion: Intestinal dysbiosis occurs in patients who develop diarrhea during enteral feeding and may be involved in its pathogenesis. Am J Clin Nutr 2009; 89: 240-7.

Potential roles and clinical utility of prebiotics in newborns, infants, and children: proceedings from a global prebiotic summit meeting, New York City, June 27-28, 2008

Initial bacterial colonization, including colonization with health-positive bacteria, such as bifidobacteria and lactobacilli, is necessary for the normal development of intestinal innate and adaptive immune defenses. The predominance of beneficial bacteria in the gut microflora of breast-fed infants is thought to be, at least in part, supported by the metabolism of the complex mixture of oligosaccharides present in human breast milk, and a more adult-type intestinal microbiota is found in formula-fed infants. Inadequate gut colonization, dysbiosis, may lead to an increased risk of infectious, allergic, and autoimmune disorders later in life. The addition of appropriate amounts of selected prebiotics to infant formulas can enhance the growth of bifidobacteria or lactobacilli in the colonic microbiota and, thereby, might produce beneficial effects. Among the substrates considered as prebiotics are the oligosaccharides inulin, fructo-oligosaccharides, galacto-oligosaccharides, and lactulose. There are some reports that such prebiotics have beneficial effects on various markers of health. For example, primary prevention trials in infants have provided promising data on prevention of infections and atopic dermatitis. Additional well-designed prospective clinical trials and mechanistic studies are needed to advance knowledge further in this promising field. (J Pediatr 2009;155:S61-70).

Evaluation of the inclusion of a mixture of organic acids or lactulose into the feed of pigs experimentally challenged with Salmonella Typhimurium

A mixture of organic acids and lactulose for preventing or reducing colonization of the gut by Salmonella Typhimurium was evaluated in pigs. A total of 63 4-week-old commercial piglets were randomly distributed into three different experimental dietary groups: a plain diet without additives (PD) and the same diet supplemented with either 0.4% (w/v) formic acid and 0.4% lactic acid (w/v) (AC) or 1% (w/v) lactulose (LC). After 7 days of adaptation, two-thirds of the pigs (14 from each diet) were challenged with a 2-mL oral dose of 10(8) CFU/mL of Salmonella Typhimurium, leaving the remaining animals unchallenged (UC). After 4 and 10 days post-challenge, pigs were euthanized and the ileum and caecum content were aseptically sampled to (a) quantify lactic, formic, and short-chain fatty acids (SCFA), (b) quantify bacterial populations and Salmonella by fluorescence in situ hybridization and (c) qualitatively analyse bacterial populations through denaturing gradient gel electrophoresis (DGGE). Modification of fermentation products and counts of some of the bacterial groups analysed in the challenged pigs receiving the treatments AC and LC were minimal. Treatments only influenced the bacterial diversity after 10 days post-challenge, with AC generating a lower number of DGGE bands than UC(P < 0.05). Neither the inclusion of a mixture of 0.4% (w/v) formic and 0.4% (w/v) lactic acids nor of 1% (w/v) lactulose in the feed influenced numbers of Salmonella in the ileum and caecum of experimentally challenged pigs. (C) 2009 Elsevier B.V. All rights reserved.

Prebiotic capacity of inulin-type fructans

The human gut microbiota plays a significant role in human health through its ability to digest food ingredients and manufacture metabolites. This can be positive or negative for host welfare. Moreover, the microflora plays an active role in host defense whereby colonization resistance affords protection against pathogens. Prebiotics are nondigestible food ingredients that target beneficial components of the gut microflora (mainly colonic), particularly the bifidobacteria. In vitro and in vivo evidence has accumulated to confirm the prebiotic effects of inulin-derived fructans.

Confocal imaging of pseudomonas syringae pv. phaseolicola colony development in bean reveals reduced multiplication of strains containing the genomic island PPHGI-1.

Pseudomonas syringae pv. phaseolicola is the seed borne causative agent of halo blight in the common bean Phaseolus vulgaris. Pseudomonas syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene hopAR1 (located on a 106-kb genomic island, PPHGI-1, and earlier named avrPphB), which matches resistance gene R3 in P. vulgaris cultivar Tendergreen (TG) and causes a rapid hypersensitive reaction (HR). Here, we have fluorescently labeled selected Pseudomonas syringae pv. phaseolicola 1302A and 1448A strains (with and without PPHGI-1) to enable confocal imaging of in-planta colony formation within the apoplast of resistant (TG) and susceptible (Canadian Wonder [CW]) P. vulgaris leaves. Temporal quantification of fluorescent Pseudomonas syringae pv. phaseolicola colony development correlated with in-planta bacterial multiplication (measured as CFU/ml) and is, therefore, an effective means of monitoring Pseudomonas syringae pv. phaseolicola endophytic colonization and survival in P. vulgaris. We present advances in the application of confocal microscopy for in-planta visualization of Pseudomonas syringae pv. phaseolicola colony development in the leaf mesophyll to show how the HR defense response greatly affects colony morphology and bacterial survival. Unexpectedly, the presence of PPHGI-1 was found to cause a reduction of colony development in susceptible P. vulgaris CW leaf tissue. We discuss the evolutionary consequences that the acquisition and retention of PPHGI-1 brings to Pseudomonas syringae pv. phaseolicola in planta.

Glomus intraradices and Gigaspora margarita arbuscular mycorrhizal associations differentially affect nitrogen and potassium nutrition of Plantago lanceolata in a low fertility dune soil

Two controlled microcosm experiments aimed at a critical re-assessment of the contributions of divergent arbuscular mycorrhizal (AM) fungi to plant mineral nutrition were established that specifically targeted Plantago lanceolata–Glomus intraradices (B.B/E) and –Gigaspora margarita (BEG 34) symbioses developed in a native, nutrient limited, coastal dune soil. Plant tissue nitrogen (N), phosphorus (P) and potassium (K) status as well as plant growth parameters and levels of mycorrhizal colonization were assessed at harvest. In addition to the general well-established mycorrhizal facilitation of P uptake, the study was able to demonstrate a G. intraradices-specific contribution to improved plant nitrogen and potassium nutrition. In the two respective experiments, G. intraradices-inoculated plants had 27.8% and 40.8% more total N and 55.8% and 23.3% more total K when compared to Gi. margarita inoculated counterparts. Dissimilar overall contribution of the two isolates to plant nutrition was identified in AM-genus specific differences in plant tissue N:P:K ratios. G. intraradices inoculated and non-mycorrhizal plants generally exhibited N:P:K ratios indicative of P limitation whereas for Gi.margarita mycorrhizal plants, corresponding ratios strongly implied either N or K limitation. The study provides further evidence highlighting AM functional biodiversity in respect to plant nutrient limitation experienced by mycorrhizal P. lanceolata in an ecologically relevant soil system.

IsdA protects Staphylococcus aureus against the bactericidal protease activity of apolactoferrin

An important facet of the Staphylococcus aureus host-pathogen interaction is the ability of the invading bacterium to evade host innate defenses, particularly the cocktail of host antimicrobial peptides. In this work, we showed that IsdA, a surface protein of S. aureus which is required for nasal colonization, binds to lactoferrin, the most abundant antistaphylococcal polypeptide in human nasal secretions. The presence of IsdA on the surface of S. aureus confers resistance to killing by lactoferrin. In addition, the bactericidal activity of lactoferrin was inhibited by addition of phenylmethylsulfonyl fluoride, implicating the serine protease activity of lactoferrin in the killing of S. aureus. Recombinant IsdA was a competitive inhibitor of lactoferrin protease activity. Reciprocally, antibody reactive to IsdA enhanced killing of S. aureus. Thus, IsdA can protect S. aureus against lactoferrin and acts as a protease inhibitor.

The restoration of phytophagous beetles in species-rich chalk grasslands

This study focuses on the restoration of chalk grasslands over a 6-year period and tests the efficacy of two management practices, hay spreading and soil disturbance, in promoting this process for phytophagous beetles. Restoration success for the beetles, measured as similarity to target species-rich chalk grassland, was not found to be influenced by either management practice. In contrast, restoration success for the plants did increase in response to hay spreading management. Although the presence of suitable host plants was considered to dictate the earliest point at which phytophagous beetles could successfully colonized, few beetle species colonized as soon as their host plants became established. Morphological characteristics and feeding habits of 27 phytophagous beetle species were therefore tested to identify factors that limited their colonization and persistence. The lag time between host plant establishment and colonization was greatest for flightless beetles. Beetles with foliage-feeding larvae both colonized at slower rates than seed-, stem-, or root-feeding species and persisted within the swards for shorter periods. Although the use of hay spreading may benefit plant communities during chalk grassland restoration, it did not directly benefit phytophagous beetles. Without techniques for overcoming colonization limitation for invertebrate taxa, short-term success of restoration may be limited to the plants only.

Long-distance dispersal and its influence on adaptation to host resistance in a heterogeneous landscape

Small propagules like pollen or fungal spores may be dispersed by the wind over distances of hundreds or thousands of kilometres,even though the median dispersal may be only a few metres. Such long-distance dispersal is a stochastic event which may be exceptionally important in shaping a population. It has been found repeatedly in field studies that subpopulations of wind-dispersed fungal pathogens virulent on cultivars with newly introduced, effective resistance genes are dominated by one or very few genotypes. The role of propagule dispersal distributions with distinct behaviour at long distances in generating this characteristic population structure was studied by computer simulation of dispersal of clonal organisms in a heterogeneous environment with fields of unselective and selective hosts. Power-law distributions generated founder events in which new, virulent genotypes rapidly colonized fields of resistant crop varieties and subsequently dominated the pathogen population on both selective and unselective varieties, in agreement with data on rust and powdery mildew fungi. An exponential dispersal function, with extremely rare dispersal over long distances, resulted in slower colonization of resistant varieties by virulent pathogens or even no colonization if the distance between susceptible source and resistant target fields was sufficiently large. The founder events resulting from long-distance dispersal were highly stochastic and exact quantitative prediction of genotype frequencies will therefore always be difficult.

Transcriptional regulators of the GAD acid stress island are carried by effector protein-encoding prophages and indirectly control type III secretion in enterohemorrhagic Escherichia coli O157:H7

P>Type III secretion (T3S) plays a pivotal role in the colonization of ruminant hosts by Enterohemorrhagic Escherichia coli (EHEC). The T3S system translocates effector proteins into host cells to promote bacterial attachment and persistence. The repertoire and variation in prophage regions underpins differences in the pathogenesis and epidemiology of EHEC strains. In this study, we have used a collection of deletions in cryptic prophages and EHEC O157 O-islands to screen for novel regulators of T3S. Using this approach we have identified a family of homologous AraC-like regulators that indirectly repress T3S. These prophage-encoded secretion regulator genes (psr) are found exclusively on prophages and are associated with effector loci and the T3S activating Pch family of regulators. Transcriptional profiling, mutagenesis and DNA binding studies were used to show that these regulators usurp the conserved GAD acid stress resistance system to regulate T3S by increasing the expression of GadE (YhiE) and YhiF and that this regulation follows attachment to bovine epithelial cells. We further demonstrate that PsrA and effectors encoded within cryptic prophage CP933-N are required for persistence in a ruminant model of colonization.

Lactobacilli antagonize the growth, motility, and adherence of brachyspira pilosicoli: a potential intervention against avian intestinal spirochetosis

Avian intestinal spirochetosis (AIS) results from the colonization of the ceca and colorectum of poultry by pathogenic Brachyspira species. The number of cases of AIS has increased since the 2006 European Union ban on the use of antibiotic growth promoters, which, together with emerging antimicrobial resistance in Brachyspira, has driven renewed interest in alternative intervention strategies. Probiotics have been reported as protecting livestock against infection with common enteric pathogens, and here we investigate which aspects of the biology of Brachyspira they antagonize in order to identify possible interventions against AIS. The cell-free supernatants (CFS) of two Lactobacillus strains, Lactobacillus reuteri LM1 and Lactobacillus salivarius LM2, suppressed the growth of Brachyspira pilosicoli B2904 in a pH-dependent manner. In in vitro adherence and invasion assays with HT29-16E three-dimensional (3D) cells and in a novel avian cecal in vitro organ culture (IVOC) model, the adherence and invasion of B. pilosicoli in epithelial cells were reduced significantly by the presence of lactobacilli (P < 0.001). In addition, live and heat-inactivated lactobacilli inhibited the motility of B. pilosicoli, and electron microscopic observations indicated that contact between the lactobacilli and Brachyspira was crucial in inhibiting both adherence and motility. These data suggest that motility is essential for B. pilosicoli to adhere to and invade the gut epithelium and that any interference of motility may be a useful tool for the development of control strategies.

Purified galactooligosaccharide, derived from a mixture produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium adhesion and invasion in vitro and in vivo

The prebiotic Bimuno (R) is a mixture containing galactooligosaccharides (GOSs), produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 411 71 using lactose as the substrate Previous in vivo and in vitro studies demonstrating the efficacy of Bimuno (R) in reducing Salmonella enterica serovar Typhimurium (S Typhimurium) colonization did not ascertain whether or not the protective effects could be attributed to the prebiotic component GOS Here we wished to test the hypothesis that GOS, derived from Bimuno (R) may confer the direct anti-invasive and protective effects of Bimuno (R) In this study the efficacy of Bimuno (R), a basal solution of Bimuno (R) without GOS [which contained glucose, galactose, lactose, maltodextrin and gum arabic in the same relative proportions (w/w) as they are found in Bimuno (R)] and purified GOS to reduce S Typhimurium adhesion and invasion was assessed using a series of in vitro and in vivo models The novel use of three dimensionally cultured HT-29-16E cells to study prebiotics in vitro demonstrated that the presence of similar to 5 mg Bimuno (R) ml(-1) or similar to 2 5 mg GOS ml(-1) significantly reduced the invasion of S Typhimurium (SL1344nal(r)) (P<0 0001) Furthermore, similar to 2 5 mg GOS ml(-1) significantly reduced the adherence of S Typhimurium (SU 344nal(r)) (P<0 0001) It was demonstrated that cells produced using this system formed multi-layered aggregates of cells that displayed excellent formation of brush borders and tight junctions In the murine ligated deal gut loops, the presence of Bimuno (R) or GOS prevented the adherence or invasion of S Typhimurium to enterocytes, and thus reduced its associated pathology This protection appeared to correlate with significant reductions in the neutral and acidic mucins detected in goblet cells, possibly as a consequence of stimulating the cells to secrete the mucin into the lumen In all assays, Bimuno (R) without GOS conferred no such protection, indicating that the basal solution confers no protective effects against S Typhimurium Collectively, the studies presented here clearly indicate that the protective effects conferred by Bimuno (R) can be attributed to GOS

A mixture containing galactoollgosaccharide, produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium infection in mice

The prebiotic Bimuno (R) is a mixture containing galactooligosaccharide, produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41 .vertical bar 71 in the presence of lactose. Previous studies have implicated prebiotics in reducing infections by enteric pathogens, thus it was hypothesized that Bimuno (R) may confer some protection in the murine host from Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. In this study, infection caused by S. Typhimurium SL1344nal(r) in the presence or absence of Bimuno (R) was assessed using tissue culture assays, a murine ligated ileal gut loop model and a murine oral challenge model. In tissue culture adherence and invasion assays with HT-29-1 6E cells, the presence of similar to 2 mM Bimuno) significantly reduced the invasion of S. Typhimuriurn SL1 344nal(r) (p < 0.0001). In the murine ligated ileal gut loops, the presence of Bimuno (R) prevented colonization and the associated pathology of S. Typhimurium. In the BALB/c mouse mocel, the oral delivery of Bimuno prior to challenge with S. Typhimurium resulted in significant reductions in colonization in the five organs sampled, with highly significant reductions being observed in the spleen at 72 and 96 h post-challenge (P=0.0002, < 0.0001, respectively). Collectively, the results indicate that Bimuno (R) significantly reduced the colonization and pathology associated with S. Typhimurium infection in a murine model system, possibly by reducing the invasion of the pathogen into host cells.

An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157: H7 with bovine intestinal epithelium

Enterohaemorrhagic Escherichia coli O157 : H7 is a bacterial pathogen that can cause haemorrhagic colitis and haemolytic uremic syndrome. In the primary reservoir host, cattle, the terminal rectum is the principal site of E. coli O157 colonization. In this study, bovine terminal rectal primary epithelial cells were used to examine the role of H7 flagella in epithelial adherence. Binding of a fliC(H7) mutant O157 strain to rectal epithelium was significantly reduced as was binding of the flagellated wild-type strain following incubation with H7-specific antibodies. Complementation of fliC(H7) mutant O157 strain with fliC(H7) restored the adherence to wild-type levels; however, complementation with fliC(H6) did not restore it. High-resolution ultrastructural and imunofluorescence studies demonstrated the presence of abundant flagella forming physical contact points with the rectal epithelium. Binding to terminal rectal epithelium was specific to H7 by comparison with other flagellin types tested. In-cell Western assays confirmed temporal expression of flagella during O157 interaction with epithelium, early expression was suppressed during the later stages of microcolony and attaching and effacing lesion formation. H7 flagella are expressed in vivo by individual bacteria in contact with rectal mucosa. Our data demonstrate that the H7 flagellum acts as an adhesin to bovine intestinal epithelium and its involvement in this crucial initiating step for colonization indicates that H7 flagella could be an important target in intervention strategies.