Gotowa bibliografia na temat „Serratia entomophila”

Strains of the bacteria Serratia entomophila and S proteamaculans cause amber disease in the grass grub Costelytra zealandica (Coleoptera Scarabaeidae) an important pasture pest in New Zealand Strains of both Serratia species occur naturally in New Zealand pasture soils Serratia entomophila has been developed as a commercial biological control agent bioshield The persistence of the applied strain in soil is currently measured by enumeration of bacterial colonies on Serratiaselective agar and subsequent biochemical tests are carried out to differentiate between the two Serratia species on the basis of their ability to utilise itaconate as a sole carbon source A speciesspecific DNA probe has been developed as an alternative to these laborious biochemical tests Tests against a range of Serratia species in colony dot blots showed the probe could be used to differentiate between S entomophila and S protemaculans recovered from treated soil when assessing persistence of bioshield inoculum and efficacy of the product in the field

Microbial control of soil dwelling pests and pathogens depends on the survival of microbial inocula in soil Three microbes Beauveria bassiana A6 Serratia entomophila 626 and Pseudomonas fluorescens CHA0Rif were inoculated into soil microcosms at three soil moistures and temperatures Survival was determined at regular intervals Beauveria bassiana survived well in soil; after 3 months the populations were maintained at levels close to those immediately following inoculation under most soil conditions Serratia entomophila and P fluorescens populations declined gradually Soil moisture impacted on survival of P fluorescens with populations declining most rapidly in the dry soil at all temperatures Pseudomonas fluorescens was not recovered after 54 days at 20C The rate of population decline of S entomophila increased with soil temperature but populations remained above the minimum level of detection after three months with soil moisture having little effect on survival Formulation of S entomophila into granules greatly improved the survival of this bacterium in soil

Eight bacteriophages specific to Serratia entomophila, a commercially available bacterial pathogen of the New Zealand grass grub (Costelytra zealandica), were characterized by host range determination, morphology and restriction endonuclease patterns of DNA. Phages were originally isolated from grass grub larvae and fermenter broth where phages had disrupted large-scale production of S. entomophila. Seven of the phages (CW1–CW5, BC, and BT) had heads similar in size (approximately 60 × 60 nm) and long noncontractile tails (185 × 10 nm). Phage AgRP8 (P8) had a smaller head and a short tail structure. Restriction endonuclease analysis divided the phages into four groups: CW2, CW4, CW5, BC, and BT gave identical patterns, while CW1, CW3, and P8 each gave different patterns. Six distinct phage groups were distinguished by host range determination, after screening phages against 70 bacterial isolates: CW1, CW2/CW4, CW3, CW5, BC/BT, and P8. While confirming the indicated groupings by DNA analysis, it was possible to distinguish between some of the phages in the largest group: CW2/4 could be distinguished from CW5 and BC/BT. Screening of soil bacterial isolates of S. entomophila against nondiluted phages will aid in monitoring the establishment and persistence of strains applied for biological control of the grass grub.Key words: Serratia entomophila, bacteriophage, morphology, phage typing, host range.

ABSTRACT Strains of Serratia spp. showed a high level of virulence when injected into the hemocoel of larvae Costelytra zealandica, with Serratia entomophila, S. plymuthica, and S. marcescens showing significantly higher virulence than S. proteamaculans. Toxicity was independent of the amber disease-causing plasmid pADAP, suggesting a generalized Serratia toxin.

A granular formulation of Serratia entomophila has been developed to improve shelflife and storage characteristics of this bacterium which is used as a microbial control agent of the New Zealand grass grub Bacterial establishment and survival of bacteria released from the granular and liquid formulations were assessed in a laboratory experiment Bacteria were enumerated by dilution plating onto Serratia selective agar Serratia entomophila populations in soil inoculated with granules remained stable in soil for up to five months at a range of soil moisture levels Bacterial numbers declined more rapidly when soil was inoculated with the liquid formulation High numbers of bacteria remained viable in the granules throughout the experiment demonstrating the potential for sustained release of inoculum after application of the biopesticide granules

The bacterial biocontrol agent Serratia entomophila and the insecticide diazinon were applied as separate granular formulations with ryegrass seed and compared with a seedonly control treatment on three pastures of different ages and composition on the North Island volcanic plateau In the first 2 years diazinon and S entomophila significantly reduced healthy grass grub populations compared with the control However by the third year populations in the diazinon treatments had recovered and were significantly higher than in S entomophila or control plots Grass grub populations were reduced by disease outbreaks after S entomophila was applied which infected >40 of grass grub larvae in the treated plots in year two Bacterial extraction from soil a year after application confirmed establishment and persistence of S entomophila in treated plots Visual positive pasture growth responses were noted in both the S entomophila and diazinontreated plots

ABSTRACT Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grubCostelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 115-kb plasmid, pADAP, identified in S. entomophila is required for disease causation and, when introduced into Escherichia coli, enables that organism to cause amber disease. A 23-kb fragment of pADAP that conferred disease-causing ability on E. coli and a pADAP-cured strain of S. entomophila was isolated. Using insertion mutagenesis, the pathogenicity determinants were mapped to a 17-kb region of the clone. Sequence analysis of the 17-kb region showed that the predicted products of three of the open reading frames (sepA, sepB, and sepC) showed significant sequence similarity to components of the insecticidal toxin produced by the bacterium Photorhabdus luminescens. Transposon insertions in sepA, sepB, orsepC completely abolished both gut clearance and cessation of feeding on the 23-kb clone; when recombined back into pADAP, they abolished gut clearance but not cessation of feeding. These results suggest that SepA, SepB, and SepC together are sufficient for amber disease causation by S. entomophila and that another locus also able to exert a cessation-of-feeding effect is encoded elsewhere on pADAP.

Microbial control agents targeting soildwelling organisms need to be compatible with commonly used fertilisers The bacterium Serratia entomophila is used as a microbial control agent for control of the New Zealand grass grub Costelytra zealandica and Beauveria bassiana is an entomopathogenic fungus used to control a range of insect pests These biocontrol agents were formulated into granules and applied to pots together with five fertilisers commonly used on pastures throughout New Zealand Compatibility with S entomophila was also assessed in a field trial where treatments were applied by direct drilling and surface application There appeared to be no deleterious effect from the application of the fertiliser treatments on the establishment and survival of either S entomophila or B bassiana On the contrary there was a suggestion that some nitrogenous fertilisers may lead to an increase in numbers of the bacterial biocontrol agent

The bacterium Serratia entomophila is a naturally occurring pathogen causing amber disease of the New Zealand grass grub (Costelytra zealandica) A novel granular formulation of S entomophila Bioshieldtrade; was applied to 18 pasture sites in a largescale programme to demonstrate efficacy against grass grub No significant difficulties were encountered in application of the granules through conventional machinery There were high populations of the applied bacteria in soil within the first week of application and within 6 weeks of application there was an average of 3 x 104 viable S entomophila of the applied strain per gram of soil This resulted in a significant (Plt;005) 20 increase in the incidence of amber disease in the treated grass grub populations compared to untreated populations Successful establishment of the bacteria in the soil and target population following Bioshieldtrade; granule application was demonstrated on a wide range of sites under various farming conditions

Serratia entomophila, the causative agent of amber disease, is an endemic bacterium used for the biocontrol of New Zealand grass grub larvae. Although the available biopesticide is effective, its use is limited to areas where sub-surface application is feasible, and is also impacted by soil conditions such as moisture levels and osmolarity. The aim of this study was to elucidate the responses of S. entomophila to osmotic and desiccation stresses in relation to challenges encountered during production, storage and soil application, with the goal of developing a more robust and versatile biocontrol agent. RpoS is a key factor in the stress response of many enteric bacteria. In order to dissociate the effect of RpoS from subsequent cellular stress studies, an rpoS mutant was constructed by site-directed mutagenesis. Assessment of the rpoS mutant showed that RpoS was not implicated in NaC1 or desiccation tolerance of S. entomophila. The rpoS mutant was instead found to have enhanced salt tolerance and could be distinguished from the wild-type by the ability to ferment arabinose, a phenotype that was confirmed through complementation. Complete abolition of the amber disease process was observed using an rpoS strain also missing the Sep virulence genes, suggesting that RpoS is a regulator of the S. entomophila anti-feeding prophage (Afp). These findings indicate a subtle interplay between NaC1 tolerance, virulence and RpoS-mediated regulation of amber disease in S. entomophila. A transposon mutagenesis screen was carried out to identify genes associated with NaC1 tolerance in S. entomophila. Fourteen mutants displaying NaC1 sensitivity were identified, two of which had mutations in genes with potential implications for the formulation of the bacterium as a biocontrol agent. The gene leuO that encodes a LysR-family transcriptional regulator was found to be essential for S. entomophila NaC1 tolerance. The toxicity of increased cellular LeuO from an over-expression vector led to the investigation of the effects of leuO mutation on the proteome. Multiple protein changes observed by two-dimensional gel analysis suggested that LeuO may be a global regulator in S. entomophila, as has been hypothesised for Salmonella species. A second NaC1-sensitive mutant contained an insertion in afp15, the product of which is thought to be involved in assembly of the Afp. As well as being sensitive to NaC1, the afp15 mutant was unable to induce the anti-feeding component of amber disease, again highlighting the link between stress tolerance and virulence in S. entomophila. This study also determined that pre-exposure to NaC1 in conjunction with the provision of exogenous glycine betaine significantly enhanced the survival of S. entomophila either in a desiccated state or after application to soil, regardless of the soil moisture content. The implication of this finding on the future formulation of S. entomophila led to investigation of the underlying genetic mechanisms involved in glycine betaine synthesis and NaC1 tolerance. The genes involved in glycine betaine biosynthesis from choline were identified through genomic comparison, degenerate PCR and primer walking. A 6.5 kb region was sequenced and found to contain four genes with homology and similar chromosomal arrangement to the E. coli bet genes (betTIBA). The S. entomophila betIBA genes comprised an operon, flanked by the divergently-transcribed betT gene whose product is responsible for choline transport. To ascertain the relative transcription levels of components of the bet operon, quantitative RT-PCR was performed. Results of qRT-PCR showed that choline in conjunction with NaC1 induced the greatest levels of bet gene transcription, and that levels of the betA transcript were significantly lower than those of the other bet genes. Examination of the betA 5� non-coding region identified a previously undetected hairpin region, possibly accounting for the observed decrease in betA transcript levels. The findings of this study have significantly advanced our understanding of how S. entomophiia responds to stress, and will contribute to the development of formulation strategies for the production of a robust product capable of application to pasture by a range of teclmiques. In addition, there is significant potential to utilise these findings in the development of other bacterial inocula for a range of biotechnological applications.

Amber disease caused by Serratia entomophila to larvae of Costelytra zealandica (Coleoptera: Scarabaeidae), is characterized by the production of two symptoms: anti-feeding effect (AFE) and amber coloration (AC). This study was aimed to identify and characterize the virulence factors involved in the disease. Three factors were identified: i) MRE-HA fimbriae; ii) an extracellular protease and, iii) an anti-feeding toxin. i) Fimbriae type 1, 3 and MRE-HA were identified and characterized in S. entomophila by haemagglutination tests and electron microscopy. Analysis of nonpathogenic mutants suggested that the MRE-HA fimbriae were associated with pathogenicity. ii) The locus coding for the extracellular protease of S. entomophila was identified and cloned. Examination and complementation assays of pathogenic and nonpathogenic strains showed that the protease is not directly involved, but it might potentiate the disease. It was suggested that the protease might be linked with pathogenicity by a common regulator factor. iii) A locus named amb2 was identified, isolated and cloned. Genetic evidence and complementation assays with nonpathogenic mutants demonstrated that amb2 is responsible for the AFE. SDS-PAGE analysis of the amb2 gene products expressed in minicells showed the synthesis of two proteins of 21 and 25 kDa, named AnfA and AnfB. The genes encoding these proteins were mapped by deletion analysis and lacZ-gene fusions. DNA sequencing of the anfA gene revealed that another protein of ~12 kDa (AnfA2) was also encoded by amb2. Consensus sequences with homology to the binding sites of the bacterial regulators CAP, Fur and ToxR were identified in the promoter regions. Homology of 50% was found between a hydrophobic motif of the δ-endotoxin of Bacillus thuringiensis and AnfA1, The results suggest that AnfA1 , AnfA2 and AnfB might be subunits of a toxin causing the AFE. It was concluded that virulence determinants in S. entomophila including the MRE-HA fimbriae, the extracellular protease and the anti-feeding toxin act in collaboration to produce amber disease.