Genotypic diversity among Brevibacillus laterosporus strains.

In comparison with other entomopathogenic Bacillus species, the genome of Brevibacillus laterosporus is poorly characterized. The aim of this study was to examine genetic variability in B. laterosporus by using a range of typing methodologies. Strains of B. laterosporus were examined for variation in 13 chromosomal genes encoding enzymes by multilocus enzyme electrophoresis. Optimal conditions of pulsed-field gel electrophoresis and randomly amplified polymorphic DNA were established that allowed analysis of the genome of B. laterosporus. None of these techniques allowed the identification of a convenient molecular marker for entomopathogenic strains, although one specific primer amplified only DNA from almost all mosquitocidal strains.     

Molecular Characterization of Brevibacillus laterosporus and Its Potential Use in Biological Control

Thirty-three strains of Brevibacillus laterosporus, including three novel strains isolated from Brazilian soil samples, were examined for genetic variability by the use of different PCR-based methods. Molecular markers that could characterize bacterial strains with regards to their pathogenic potential were investigated. In addition, toxicity was assessed by the use of insects belonging to the orders Lepidoptera and Coleoptera and the mollusk Biomphalaria glabrata. Among the targets tested, Biomphalaria glabrata demonstrated the highest degree of sensitivity to B. laterosporus, with some strains inducing 90 to 100% mortality in snails aged 3 and 12 days posteclosion. Larvae of the coleopteron Anthonomus grandis were also susceptible, presenting mortality levels of between 33 and 63%. Toxicity was also noted towards the lepidopteron Anticarsia gemmatalis. In contrast, no mortality was recorded among test populations of Tenebrio molitor or Spodoptera frugiperda. The application of intergenic transcribed spacer PCR and BOX-PCR generated 15 and 17 different genotypes, respectively. None of the molecular techniques allowed the identification of a convenient marker that was associated with any entomopathogenic phenotype. However, a 1,078-bp amplicon was detected for all strains of B. laterosporus when a primer for amplification of the BOXA1R region was used. Similarly, a 900-bp amplicon was generated from all isolates by use of the primer OPA-11 for randomly amplified polymorphic DNA analysis. These amplicons were not detected for other phenotypically related Brevibacillus species, indicating that they represent markers that are specific for B. laterosporus, which may prove useful for the isolation and identification of new strains of this species.     

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.     

Insecticidal Activity of Bacillus laterosporus

The Bacillus laterosporus strains 921 and 615 were shown to have toxicity for larvae of the mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens. The larvicidal activity of B. laterosporus was associated with spores and crystalline inclusions. Purified B. laterosporus 615 crystals were highly toxic for Aedes aegypti and Anopheles stephensi.     

Genotypic Diversity among Bacillus cereus and Bacillus thuringiensis Strains

Twenty-four strains of Bacillus cereus were analyzed by pulsed-field gel electrophoresis (PFGE) and compared with 12 Bacillus thuringiensis strains. In addition, the 36 strains were examined for variation in 15 chromosomal genes encoding enzymes (by multilocus enzyme electrophoresis [MEE]). The genome of each strain had a distinct NotI restriction enzyme digestion profile by PFGE, and the 36 strains could be assigned to 27 multilocus genotypes by MEE. However, neither PFGE nor MEE analysis could distinguish between the two species. Two of the B. cereus strains contained extrachromosomal DNA that hybridized to a cryIA insecticidal toxin probe, and seven strains contained DNA with homology to a Tn4430 transposon probe derived from B. thuringiensis. The results strongly indicate that B. cereus and B. thuringiensis should be regarded as one species.     

Electron microscopy of the surfaces of bacillus spores

The surface structures of the spores of Bacillus cereus, Bacillus thuringiensis, and Brevibacillus laterosporus were studied by transmission and scanning electron microscopy. Platinum deposition and negative staining with uranyl acetate revealed appendages and exosporium in B. thuringiensis and B. cereus. The exosporium structure was visualized by negative staining and ultrathin sectioning. For staining the exosporium polysaccharide, Alcian blue was used during fixation. The results obtained show the differences in structural organization of appendages and exosporium in different strains. Canoe-shaped inclusions were revealed in all Br. laterosporus strains, while strain IGM16-92 had a fibrillar capsule as well. Electron microscopy using a dual beam scanning electron microscope Quanta 200 3D provided the information of the spore surface relief without sample treatment (fixation and dehydration). The spores of Br. laterosporus strains had folded surface, unlike the smooth surface of B. cereus and B. thuringiensis spores. The diversity of external spore structures was shown within a species, which may be used for detection of bacteria at the strain level. Optimized procedures for visualization of spore surface by different electron microscopic techniques were discussed.     

Chromosome sizes and phylogenetic relationships between serotypes of Actinobacillus pleuropneumoniae

The genome size of Actinobacillus pleuropneumoniae was determined by pulsed field gel electrophoresis of AscI and ApaI digested chromosomal DNA. The genome size of the type strain 4074^T (serotype 1) was determined to be 2404±40 kb. The chromosome sizes for the reference strains of the other serotypes range between 2.3 and 2.4 Mb. The restriction pattern profiles of AscI, ApaI and NheI digested chromosomes showed a high degree of polymorphism among the different serotype reference strains and allowed their discrimination. The analysis of the macrorestriction pattern polymorphism revealed phylogenetic relationships between the different serotype reference strains which reflect to some extent groups of serotypes known to cross-react serologically. In addition, different pulsed fields gel electrophoresis patterns also revealed heterogeneity in the chromosomal structure among different field strains of serotypes 1, 5a, and 5b, while strains of serotype 9 originating from most distant geographical places showed homogeneous ApaI patterns in pulsed field gel electrophoresis.     

Detection and Identification of Bursaphelenchus Species with DNA Fingerprinting and Polymerase Chain Reaction

We have evaluated the potential of DNA-based methods to identify and differentiate Bursaphelenchus spp. and isolates. The isolation of a DNA probe, designated X14, and development of a DNA fingerprinting method for the identification and differentiation of Bursaphelenchus species and strains is described. Polymerase chain reaction (PCR) amplification of DNA isolated from Bursaphelenchus species using two primers derived from the sequence of the cloned repetitive DNA fragment X14 resulted in multiple band profiles. A 4-kb fragment thus amplified from B. xylophilus DNA was not amplified from B. mucronatus or B. fraudulentus DNA. In addition to this fragment, several other fragments are amplified from the three species. The banding patterns obtained allowed species identification and may have value in determining taxonomic affinities.     

Characterizing the mode of action of Brevibacillus laterosporus B4 for control of bacterial brown strip of rice caused by A. avenae subsp. avenae RS-1

Biological control efficacy of Brevibacillus laterosporus B4 associated with rice rhizosphere was assessed against bacterial brown stripe of rice caused by Acidovorex avenae subsp. avenae. A biochemical bactericide (chitosan) was used as positive control in this experiment. Result of in vitro analysis indicated that B. laterosporus B4 and its culture filtrates (70 %; v/v) exhibited low inhibitory effects than chitosan (5 mg/ml). However, culture suspension of B. laterosporus B4 prepared in 1 % saline solution presented significant ability to control bacterial brown stripe in vivo. Bacterization of rice seeds for 24 h yielded a greater response (71.9 %) for controlling brown stripe in vivo than chitosan (56 %). Studies on mechanisms revealed that B. laterosporus B4 suppressed the biofilm formation and severely disrupted cell membrane integrity of A. avenae subsp. avenae, causing the leakage of intracellular substances. In addition, the expression level of virulence-related genes in pathogen recovered from biocontrol-agent-treated plants showed that the genes responsible for biofilm formation, motility, niche adaptation, membrane functionality and virulence of A. avenae subsp. avenae were down-regulated by B. laterosporus B4 treatment. The biocontrol activity of B. laterosporus B4 was attributed to a substance with protein nature. This protein nature was shown by using ammonium sulfate precipitation and subsequent treatment with protease. The results obtained from this study showed the potential effectiveness of B. laterosporus B4 as biocontrol agent in control of bacterial brown stripe of rice.     

Physical and genetic chromosomal maps of Streptococcus agalactiae, serotypes II and III; rRNA operon organization

A detailed analysis of two Streptococcus agalactiae (group B streptococcus, GBS) strains was performed by pulsed field gel electrophoresis (PFGE). Digestion of the chromosomal DNA with SmaI and SgrAI endonucleases, followed by separation and analysis of fragments by PFGE was carried out. Physical chromosomal maps of serotype II/(α+β) and III/α strains of S. agalactiae were constructed. The GBS genome size was estimated to be 2200 kb. Sixteen GBS genes were used as probes and were located on the restriction maps of both strains by DNA-DNA hybridization. Six copies of ribosomal operons were found in the genome of the analyzed strains. Significant differences in the restriction patterns of chromosomal DNA and DNA-DNA hybridization between the two analyzed strains were detected so that DNA restriction patterns may be used to trace outbreaks of disease. The overall GBS chromosomal organization as determined is fairly conserved.     

Diversity of bacteriocinogenic lactic acid bacteria isolated from Mediterranean fish viscera

Nine lactic acid bacteria strains showing bacteriocin-like activity were isolated from various fresh fish viscera. The following species were identified based on 16S rDNA sequences: Enterococcus durans (7 isolates), Lactococcus lactis (1) and Enterococcus faecium (1). These strains were active against Listeria innocua and other LAB. Random amplified polymorphic DNA analyses showed four major patterns for the E. durans species. PCR analyses revealed a nisin gene in the genome of the Lc. lactis strain. Genes coding enterocins A, B and P were found in the genome of the E. faecium isolate. Enterocins A and B genes were also present in the genome of E. durans GM19. Hence, this is the first report describing E. durans strains producing enterocins A and B. Electrospray ionization mass spectrometry revealed that the purified bacteriocin produced by the E. durans GMT18 strain had an exact molecular mass of 6,316.89 Da. This bacteriocin was designated as durancin GMT18. Edman sequencing failed to proceed; suggesting that durancin GTM18 may contain terminal lanthionine residues. Overall, the results obtained revealed the presence of a variety of enterococci in Mediterranean fish viscera, as evidenced by their genetic profiles and abilities to produce different bacteriocins. These strains could be useful for food biopreservation or as probiotics.     

Genetic diversity and vegetative compatibility among Trichoderma harzianum isolates

Trichoderma harzianum is the collective name of a set of asexual fungal strains which exhibit heterogeneity in genome structure, DNA sequence and behavior. Contour-clamped homogeneous field (CHEF) electrophoresis of the chromosomes of ten isolates of T. harzianum revealed six clearly distinct electrophoretic karyotypes. Of the ten isolates analyzed, four (GH12, G109, Y and YF) could be classified in a single group with identical karyotypes, while the strains T35 and 315 formed a second group. The genome size characteristic of the different isolates fell into a broad range varying from 29.6 to 56.1?Mb. Gene assignments to the resolved chromosomes showed that all genes analyzed were localized on equivalent chromosomes in the isolates belonging to the same group. Analysis of randomly amplified polymorphic DNAs from the ten isolates confirmed the classification into groups and allowed us to distinguish between isolates T35 and 315, as well as between isolates GH12, G109, Y and YF. Direct confrontation assays using isolates of the same group showed compatible interactions, whereas the same experiment carried out with isolates of different groups showed an incompatible interaction characterized by an area of cell damage. Microscopic observation of the compatible interactions showed hyphal fusions between the isolates, similar to those described for vegetative compatible groups in other fungi. The molecular karyotypes correlated well with the compatibility of the isolates. In addition, we have evaluated both electrophoretic karyotype and randomly amplified polymorphic DNAs analysis as criteria for grouping isolates within the genus according to their capacity for biocontrol of plant pathogens.     

Variability in the distribution and composition of insertion sequence-like elements in strains of Mycoplasma fermentans

Mycoplasma fermentans has been reported to be pathogenic for man. All fourteen strains tested contain an insertion sequence-like element (ISLE) which may be present in multiple copies. To determine whether ISLE copies are similarly distributed in different strains of M. fermentans, restriction enzyme digest fragments of genomic DNA from 14 isolates, from a variety of sources, were separated by electrophoresis, blotted and hybridized to a biotin labelled polymerase chain reaction (PCR) amplified fragment of ISLE. A range of patterns was observed suggesting that the element has a tendency to undergo rearrangement within the genome. Analysis of ISLE sequences revealed inter- and intra-strain polymorphisms.     

Naturally occurring antibiotic resistance inBacillus sphaericus andBacillus licheniformis

Bacillus sphaericus is an aerobic, spore-forming, gram-variable bacillus. Certain strains of this organism are extremely pathogenic for mosquito larvae. Strains from four different serotypes ofB. sphaericus were found to be naturally resistant to streptomycin and to chloramphenicol. Both entomocidal and nonentomocidal strains of this organism exhibited similar patterns of resistance to these antimicrobial agents. In contrast, other members of the genusBacillus, includingB. subtilis, B. thuringiensis, B. laterosporus, andB. amyloliquefaciens, proved to be quite sensitive to these antibiotics. Four strains ofB. licheniformis were found to be resistant to chloramphenicol but sensitive to streptomycin.     

Assessing Nuclear and Mitochondrial DNA Sequence Variation Within Steinernema (Rhabditida: Steinernematidae)

DNA sequence analysis was used to characterize the nuclear ribosomal DNA ITS1 region and a portion of the COII and 16S rDNA genes of the mitochondrial genome from Steinernema entomopathogenic nematodes. Nuclear ITS1 nucleotide divergence among seven Steinernema spp. ranged from 6 to 22%, and mtDNA divergence among five species ranged from 12 to 20%. No intraspecific variation was observed among three S. feltiae strains. Phylogenetic analysis of both nuclear and mitochondrial DNA sequences confirms the existing morphological relationships of several Steinernema species. Both the rDNA ITS1 and mtDNA sequences were useful for resolving relationships among Steinernema taxa.     

Efficacy of entomopathogenic bacteria for control of Musca domestica

The aim of this study was to evaluate the larvicidal activity, and sub lethal effects of entomopathogenic bacteria Brevibacillus laterosporus, Bacillus thuringiensis var. israelensis, B. thuringiensis var. kurstaki, and a commercial formulation of Bacillus sphaericus on Musca domestica. Bacterial suspensions were prepared in different concentrations and added to the diet of newly-hatched larvae which were monitored until the adult stage. The larvae were susceptible to the B. laterosporus, B. thuringiensis var. israelensis, and B. thuringiensis var. kurstaki bacteria in varied concentration levels. These bacteria have larvicidal and sub lethal effects on the development of flies, reducing both adult size, and impairing the reproductive performance of the species.     

Differentiation of the Gene Clusters Encoding Botulinum Neurotoxin Type A Complexes in Clostridium botulinum Type A, Ab, and A(B) Strains

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.     

Characterization of Six Leuconostoc fallax Bacteriophages Isolated from an Industrial Sauerkraut Fermentation

Six bacteriophages active against Leuconostoc fallax strains were isolated from industrial sauerkraut fermentation brines. These phages were characterized as to host range, morphology, structural proteins, and genome fingerprint. They were exclusively lytic against the species L. fallax and had different host ranges among the strains of this species tested. Morphologically, three of the phages were assigned to the family Siphoviridae, and the three others were assigned to the family Myoviridae. Major capsid proteins detected by electrophoresis were distinct for each of the two morphotypes. Restriction fragment length polymorphism analysis and randomly amplified polymorphic DNA fingerprinting showed that all six phages were genetically distinct. These results revealed for the first time the existence of bacteriophages that are active against L. fallax and confirmed the presence and diversity of bacteriophages in a sauerkraut fermentation. Since a variety of L. fallax strains have been shown to be present in sauerkraut fermentation, bacteriophages active against L. fallax are likely to contribute to the microbial ecology of sauerkraut fermentation and could be responsible for some of the variability observed in this type of fermentation.     

Microsatellite markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in different environments: Technical validation and first applications

Here, we report on the application of five previously developed microsatellite markers (simple sequence repeats, SSRs) to monitor an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. in different environments. Discriminatory power of these SSR markers was assessed in two commercialized B. bassiana isolates as well as in 16 B. bassiana isolates from a world-wide collection, and three of the five SSR markers were estimated to allow a confident discrimination among the given isolates. Sensitivity thresholds of 0.1 pg DNA were subsequently determined for all SSR markers in case pure genomic fungal B. bassiana DNA was used as a template for PCR assays, but threshold levels varied depending on the environment (soil, plant) of the PCR assay. Furthermore, presence of a commercialized B. bassiana isolate was monitored via these SSR markers in three different types of potting substrates over a period of 14 weeks. With two SSR markers, strain-specific products were detected up to 14 weeks after application of B. bassiana to the substrate. Infectivity of B. bassiana conidia in the respective soil samples was confirmed by the Galleria baiting technique. Together these results indicate that molecular markers like SSRs specific for commercialized strains of entomopathogenic fungi are important tools to monitor a particular fungal strain in complex environmental samples such as bulk soil or plant DNA.