Characterization of Novel Virulent Broad-Host-Range Phages of Xylella fastidiosa and Xanthomonas

The xylem-limited bacterium Xylella fastidiosa is the causal agent of several plant diseases, most notably Pierce''s disease of grape and citrus variegated chlorosis. We report the isolation and characterization of the first virulent phages for X. fastidiosa, siphophages Sano and Salvo and podophages Prado and Paz, with a host range that includes Xanthomonas spp. Phages propagated on homologous hosts had observed adsorption rate constants of ∼4 × 10⁻¹² ml cell⁻¹ min⁻¹ for X. fastidiosa strain Temecula 1 and ∼5 × 10⁻¹⁰ to 7 × 10⁻¹⁰ ml cell⁻¹ min⁻¹ for Xanthomonas strain EC-12. Sano and Salvo exhibit >80% nucleotide identity to each other in aligned regions and are syntenic to phage BcepNazgul. We propose that phage BcepNazgul is the founding member of a novel phage type, to which Sano and Salvo belong. The lysis genes of the Nazgul-like phage type include a gene that encodes an outer membrane lipoprotein endolysin and also spanin gene families that provide insight into the evolution of the lysis pathway for phages of Gram-negative hosts. Prado and Paz, although exhibiting no significant DNA homology to each other, are new members of the phiKMV-like phage type, based on the position of the single-subunit RNA polymerase gene. The four phages are type IV pilus dependent for infection of both X. fastidiosa and Xanthomonas. The phages may be useful as agents for an effective and environmentally responsible strategy for the control of diseases caused by X. fastidiosa.     

Isolation and Characterization of Five Erwinia amylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages Ea1 and Ea7 and 3 novel phages named Ea100, Ea125, and Ea116C, were identified based on differences in genome size and restriction fragment pattern. Ea1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages Ea100, Ea7, and Ea125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. Ea116C contained an approximately 75-kb genome. Ea1, Ea7, Ea100, Ea125, and Ea116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. Ea116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10⁵ CFU.     

Discrimination and Detection of Erwinia amylovora and Erwinia pyrifoliae with a Single Primer Set

Erwinia amylovora and Erwinia pyrifoliae cause fire blight and black-shoot blight, respectively, in apples and pears. E. pyrifoliae is less pathogenic and has a narrower host range than that of E. amylovora. Fire blight and black-shoot blight exhibit similar symptoms, making it difficult to distinguish one bacterial disease from the other. Molecular tools that differentiate fire blight from black-shoot blight could guide in the implementation of appropriate management strategies to control both diseases. In this study, a primer set was developed to detect and distinguish E. amylovora from E. pyrifoliae by conventional polymerase chain reaction (PCR). The primers produced amplicons of different sizes that were specific to each bacterial species. PCR products from E. amylovora and E. pyrifoliae cells at concentrations of 10⁴ cfu/ml and 10⁷ cfu/ml, respectively, were amplified, which demonstrated sufficient primer detection sensitivity. This primer set provides a simple molecular tool to distinguish between two types of bacterial diseases with similar symptoms.     

Expression of lysozymes from Erwinia amylovora phages and Erwinia genomes and inhibition by a bacterial protein

Genes coding for lysozyme-inhibiting proteins (Ivy) were cloned from the chromosomes of the plant pathogens Erwinia amylovora and Erwinia pyrifoliae. The product interfered not only with activity of hen egg white lysozyme, but also with an enzyme from E. amylovora phage ΦEa1h. We have expressed lysozyme genes from the genomes of three Erwinia species in Escherichia coli. The lysozymes expressed from genes of the E. amylovora phages ΦEa104 and ΦEa116, Erwinia chromosomes and Arabidopsis thaliana were not affected by Ivy. The enzyme from bacteriophage ΦEa1h was fused at the N- or C-terminus to other peptides. Compared to the intact lysozyme, a His-tag reduced its lytic activity about 10-fold and larger fusion proteins abolished activity completely. Specific protease cleavage restored lysozyme activity of a GST-fusion. The bacteriophage-encoded lysozymes were more active than the enzymes from bacterial chromosomes. Viral lyz genes were inserted into a broad-host range vector, and transfer to E. amylovora inhibited cell growth. Inserted in the yeast Pichia pastoris, the ΦEa1h-lysozyme was secreted and also inhibited by Ivy. Here we describe expression of unrelated cloned 'silent' lyz genes from Erwinia chromosomes and a novel interference of bacterial Ivy proteins with a viral lysozyme.     

Fireblight Erwinia amylovora and weather: a comparison of warning systems

Warning systems for fireblight Erwinia amylovora developed in New York, Illinois and California, USA, and in south-east England are compared. General principles which might be applicable in the different climates were sought. The consequences of applying threshold temperature values chosen for one area in a different climatic area were examined using Sacramento, California; Rochester, New York; Vlissingen, The Netherlands; Kent, England as examples. A graded system for assessing fireblight risks, derived from all the systems, is suggested. It takes into account both risks of infection and risks of high insect activity and it is best used in conjunction with Billing's incubation period assessment system.     

Comparative Genome Analysis of Listeria Bacteriophages Reveals Extensive Mosaicism,Programmed Translational Frameshifting,and a Novel Prophage Insertion Site

The genomes of six Listeria bacteriophages were sequenced and analyzed. Phages A006, A500, B025, P35, and P40 are members of the Siphoviridae and contain double-stranded DNA genomes of between 35.6 kb and 42.7 kb. Phage B054 is a unique myovirus and features a 48.2-kb genome. Phage B025 features 3′ overlapping single-stranded genome ends, whereas the other viruses contain collections of terminally redundant, circularly permuted DNA molecules. Phages P35 and P40 have a broad host range and lack lysogeny functions, correlating with their virulent lifestyle. Phages A500, A006, and B025 integrate into bacterial tRNA genes, whereas B054 targets the 3′ end of translation elongation factor gene tsf. This is the first reported case of phage integration into such an evolutionarily conserved genetic element. Peptide fingerprinting of viral proteins revealed that both A118 and A500 utilize +1 and −1 programmed translational frameshifting for generating major capsid and tail shaft proteins with C termini of different lengths. In both cases, the unusual +1 frameshift at the 3′ ends of the tsh coding sequences is induced by overlapping proline codons and cis-acting shifty stops. Although Listeria phage genomes feature a conserved organization, they also show extensive mosaicism within the genome building blocks. Of particular interest is B025, which harbors a collection of modules and sequences with relatedness not only to other Listeria phages but also to viruses infecting other members of the Firmicutes. In conclusion, our results yield insights into the composition and diversity of Listeria phages and provide new information on their function, genome adaptation, and evolution.The opportunistic pathogen Listeria monocytogenes is ubiquitous in nature and can become endemic in food processing environments, causing contamination of dairy products, meats, vegetables, and processed ready-to-eat food (14). L. monocytogenes is the causative agent of epidemic and sporadic listeriosis. The risk of infection is markedly increased among immunocompromised patients, newborns, pregnant women, and the elderly and is associated with a mortality rate of about 20 to 30% (37).Although all strains of L. monocytogenes are considered potentially pathogenic, epidemiological evidence has shown that certain serovars are more frequently associated with both sporadic cases and larger food-borne outbreaks. However, genetic variation within the virulence genes of wild-type strains appears to be limited and could not be directly linked to differences in pathogenicity (30) or environmental distribution.It is becoming increasingly clear that bacteriophages have an important role in bacterial biology, diversity, and evolution, as indicated by the advances in genome sequencing which revealed a high incidence of phage-related sequences in bacterial genomes. Many phages have been described for the genus Listeria, and lysogeny appears to be widespread (28). Availability of the genome sequences of different L. monocytogenes and L. innocua strains also revealed the presence of several different putative prophages in the genomes of these bacteria, constituting up to 7% of the coding capacities of the genomes (15, 32). Although the genomes of L. monocytogenes were found to be essentially syntenic, a significant portion of sequence variability is apparently based upon phage insertions and subsequent rearrangements. Investigations of prophage contributions to population dynamics in Salmonella suggest that prophages can improve the competitive fitness of the lysogenized host strains (5). This type of selective pressure also results in diversification and generation of new strains by lysogenic conversion. In the case of Listeria, however, the potential influence of prophages on their host strains, such as phenotypic variation or provision of selective benefits, has not been investigated. To gain more insight in bacteriophage-host interactions and the molecular biology and characteristics of Listeria phages, more information on the structure, information content, and variability of different Listeria phage genomes is required.Although a number of Listeria phages have been isolated and described (25, 27, 42), only limited information was available at the sequence level for phages PSA, A118, A511, and P100 (9, 19, 26, 41). As the result of a comprehensive study to determine the diversity of this group of bacterial viruses, we here report the complete nucleotide sequences of a representative set of six different Listeria phages from the Siphoviridae (A006, A500, B025, P35, and P40) and Myoviridae (B054) families in the order Caudovirales. In addition to molecular and in silico analyses, we also determined the physical genome structures and attachment loci of the temperate phages, and we describe integration of the B054 prophage into a highly conserved elongation factor gene. Another interesting finding is the unusual decoding used by some of the phages, which use programmed frameshifting to generate C-terminally modified structural proteins required for assembly of the capsid and tail.     

Partial Purification and Characterization of a Polysaccharide Depolymerase Associated with Phage-Infected Erwinia amylovora

Erwinia amylovora infected with bacteriophage ERA103 produced an enzyme which degraded the extracellular polysaccharide of noninfected cells. The depolymerase enzyme was purified 15-fold by a procedure which included ammonium sulfate precipitation, ultracentrifugation, CM-Sephadex batchwise separation, Sephadex G-50 column chromatography, and Sephacryl S-200 column chromatography. The enzyme had a molecular weight of approximately 21,000 and a pH optimum of 6.0. Activity was enhanced by supplements of 2-mercaptoethanol or dithiothreitol.     

Real-time PCR, a method fit for detection and quantification of Erwinia amylovora

Fire blight, a devastating disease of pome fruit trees continues to pose threat to agricultural production. Detection of its causative agent, bacterium Erwinia amylovora, is usually straightforward in symptomatic samples. Methods with increased sensitivity however, are sometimes needed for detection of E. amylovora and real-time PCR assays have been shown to have required sensitivity and reliability. Here we summarize our previous results on real-time PCR detection of fire blight and present new, fast and sensitive real-time PCR assay based on amsC gene performed on SmartCycler^? instrument. The setting is optimal for analysis of small number of samples in the laboratory or for on-site detection. Many advantages of real-time PCR assays warrant their use in detection and diagnosis of E. amylovora, particularly in detection of low concentrations of target bacteria e.g. in testing for latent infections. It is to be expected that the use of real-time PCR will increase in both diagnostics and in research, as a tool for target detection and quantification as well as for gene expression analysis.     

Bacteriophage Mu as a genetic tool to study Erwinia amylovora pathogenicity and hypersensitive reaction on tobacco

Erwinia amylovora 1430 was shown to be sensitive to Mu G(-) particles. Infection resulted either in lytic development or in lysogenic derivatives with insertion of the Mu genome at many sites in the bacterial chromosome. We used the Mu d1Bx::Tn9 (lac Apr Cmr) derivative, called Mu dX, to identify mutants affected in pathogenicity and in their ability to induce a hypersensitive reaction (HR) on tobacco plants. Inoculation of 1,400 lysogenic derivatives on apple root calli led to the identification of 12 mutants in three classes: (i) class 1 mutants were nonpathogenic and unable to induce an HR on tobacco plants; (ii) class 2 mutants were nonpathogenic but retained the ability to induce an HR; and (iii) class 3 mutants showed attenuated virulence. Of the 12 mutants, 8 had a single insertion of the Mu dX prophage. For class 1 and 2 mutants, reversion to pathogenicity was concomitant with the loss of the Mu dX prophage. Furthermore, revertants from the class 1 mutants also recovered the ability to induce an HR on tobacco plants. Five of the six class 3 mutants were impaired in exopolysaccharide production. No changes of the envelope structure (lipopolysaccharide and outer membrane proteins) were correlated with differences in pathogenicity. One class 3 mutant did not produce any functional siderophore, suggesting that iron uptake could be involved in pathogenicity.     

Genome Organization and Characterization of the Virulent Lactococcal Phage 1358 and Its Similarities to Listeria Phages

Virulent phage 1358 is the reference member of a rare group of phages infecting Lactococcus lactis. Electron microscopy revealed a typical icosahedral capsid connected to one of the smallest noncontractile tails found in a lactococcal phage of the Siphoviridae family. Microbiological characterization identified a burst size of 72 virions released per infected host cell and a latent period of 90 min. The host range of phage 1358 was limited to 3 out of the 60 lactococcal strains tested. Moreover, this phage was insensitive to four Abi systems (AbiK, AbiQ, AbiT, and AbiV). The genome of phage 1358 consisted of a linear, double-stranded, 36,892-bp DNA molecule containing 43 open reading frames (ORFs). At least 14 ORFs coded for structural proteins, as identified by SDS-PAGE coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The genomic organization was similar to those of other siphophages. All genes were on the same coding strand and in the same orientation. This lactococcal phage was unique, however, in its 51.4% GC content, much higher than those of other phages infecting this low-GC Gram-positive host. A bias for GC-rich codons was also observed. Comparative analyses showed that several phage 1358 structural proteins shared similarity with two Listeria monocytogenes phages, P35 and P40. The possible origin and evolution of lactococcal phage 1358 is discussed.The first sequenced genome of a phage infecting Lactococcus lactis (bIL67) was reported in 1994 (57). Its genomic characterization was performed with the prospect of a better understanding of lactococcal phage biology. L. lactis is a Gram-positive bacterium added to milk to produce an array of fermented dairy products. In this human-made environment, substantial amounts of lactococcal cells are cultivated on a daily basis in large fermentation vats, and these added cells randomly encounter virulent phages present in heat-treated but nonsterile milk. Moreover, it is widely acknowledged that the increased use of the same bacterial strains within existing dairy facilities inevitably leads to milk fermentation failures due to the multiplication of virulent phages. This biotechnological problem reduces yields and lowers the quality of fermented products (51).Over 700 lactococcal phage isolates have been reported in the literature (3). To date, more than 25 complete genome sequences of lactococcal phages are publicly available in the NCBI database, and the sequencing of others is under way. These numbers indicate that Lactococcus phages are among the most studied of the bacterial viruses. All lactococcal phages belong to the order Caudovirales and are included within two families according to their tail morphology: the Siphoviridae (long noncontractile tail [most lactococcal phages]) and the Podoviridae (short noncontractile tail [few lactococcal phages]) (14). Currently, phages infecting L. lactis strains have been divided into 10 genetically distinct groups (14). The complete genomic sequence is available for at least one representative of 8 of the groups.Early sequencing efforts concentrated on the genomes of lactococcal phages belonging to the 936, c2, and P335 groups (Siphoviridae), because members of these groups were regularly isolated in dairy plants (8, 36, 50). PCR-based methods were also devised to rapidly classify these phages (41). These Siphoviridae phages pose a significant risk to the dairy industry, and their characterization is important for developing adapted antiphage strategies to limit their propagation and evolution.In recent years, representatives of the less recognized lactococcal phage groups have been characterized, including phages Q54 (22), KSY1 (13), 1706 (23), asccφ28 of the P034 group (39), and P087 (63). Their molecular characterizations were aimed at understanding why some phage groups (936, c2, and P335) predominate while the others have remained marginal, at best. However, it was recently reported that P034-like phages may be emerging in certain regions (52). Genomic and microbiological analyses indicated that members of these rare phage groups were likely the result of recombination between different lactococcal phages and phages infecting other Gram-positive bacteria, and they may not be fit to multiply rapidly in milk. For example, lactococcal phage 1706 shares similarities with Ruminococcus and Clostridium prophages (23). Similarly, L. lactis phage P087 structural proteins share identity with gene products found in a prophage in the Enterococcus faecalis genome (63). It was also shown previously that lactococcal phage asccφ28 was related to Streptococcus pneumoniae phage Cp-1 and Bacillus subtilis φ29-like phages (39). It was suggested that phages 1706, asccφ28, and P087 acquired a receptor-binding protein complex from another lactococcal phage that enabled them to infect a L. lactis host.Here, we report the complete genome sequence and analysis of phage 1358, a virulent representative of the 9th lactococcal phage group.     

Thirteen Virulent and Temperate Bacteriophages of Lactobacillus bulgaricus and Lactobacillus lactis Belong to a Single DNA Homology Group

Thirteen virulent phages and two temperate phages of two closely related bacterial species (Lactobacillus lactis and L. bulgaricus) were compared for their protein composition, their antigenic properties, their restriction endonuclease patterns, and their DNA homology. The immunoblotting studies and the DNA-DNA hybridizations showed that the phages could be differentiated into two groups. One group contained 2 temperate phages of L. bulgaricus and 11 virulent phages of L. lactis. Inside each group, at least two common proteins of identical sizes could be detected for each phage. These proteins were able to cross-react in immunoblotting experiments with an antiserum raised against one phage of the same group. Temperate phage DNAs showed partial homology with DNAs from some virulent phages. These homologies seem to be located on the region coding for the structural proteins since recombinant plasmids coding for one of the major phage proteins of one phage were able to hybridize with the DNAs from phages of the same group. These results suggest that temperate and virulent phages may be related to one another.     

Complete genome sequences of three Erwinia amylovora phages isolated in north america and a bacteriophage induced from an Erwinia tasmaniensis strain

Fire blight, a plant disease of economic importance caused by Erwinia amylovora, may be controlled by the application of bacteriophages. Here, we provide the complete genome sequences and the annotation of three E. amylovora-specific phages isolated in North America and genomic information about a bacteriophage induced by mitomycin C treatment of an Erwinia tasmaniensis strain that is antagonistic for E. amylovora. The American phages resemble two already-described viral genomes, whereas the E. tasmaniensis phage displays a singular genomic sequence in BLAST searches.     

Survival and Possible Spread of Erwinia amylovora and Related Plant-Pathogenic Bacteria Exposed to Environmental Stress Conditions

The fire blight pathogen Erwinia amylovora was assayed for survival under unfavourable conditions such as on nitrocellulose filters, in non‐host plants as well as in inoculated mature apples and in infested apple stem sections. In a sterile dry environment, an E. amylovora EPS (exopolysaccharide) mutant, and to a lesser extent its parental wild‐type strain decreased within 3 weeks to a low titre. However, under moist conditions the decrease of viable cells occurred only partially for both strains. Very low cell titres were recovered after application of E. amylovora onto the surface of tobacco leaves, whereas infiltration into the leaves produced lesions (hypersensitive response, HR), in which the bacteria survived in significant amounts. A similar effect was found for the necrotic zones of HR in tobacco leaves caused by E. pyrifoliae, by Pseudomonas syringae pathovars and HR‐deficient E. amylovora mutants or mutants deficient in EPS synthesis and disease‐specific genes. During 7 years of storage, the viability of E. amylovora in wood sections from fire blight‐infested apple trees declined to a low titre. In tissue of mature apples, E. amylovora cells slowly dispersed and could still be recovered after several weeks of storage at room temperature. A minimal risk of accidental dissemination of E. amylovora apart from infested host plants can experimentally not be excluded, but other data confirm a very low incidence of any long distance distribution.     

HrpW of Erwinia amylovora,a New Harpin That Contains a Domain Homologous to Pectate Lyases of a Distinct Class

Harpins, such as HrpN of Erwinia amylovora, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified hrpW of E. amylovora, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream of hrpW, and Western blot analysis of hrpL mutants indicated that the production of HrpW is regulated by hrpL. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and hrp secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of E. amylovora consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that hrpW is conserved among several Erwinia species, and hrpW, provided in trans, enhanced the HR-inducing ability of a hrpN mutant. However, HrpW did not increase the virulence of a hrpN mutant in host tissue, and hrpW mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts.Most gram-negative plant-pathogenic bacteria contain clusters of genes termed hrp that are required for elicitation of a rapid localized defense response called the hypersensitive reaction (HR) in incompatible plants and that are required for pathogenicity in susceptible plants (1). Proteins encoded by hrp genes are involved in the regulation of the expression of other hrp genes and in a specialized secretion process called the Hrp or type III pathway (9). Harpins, a major class of proteins that travel the pathway (including HrpN of Erwinia species, HrpZ of Pseudomonas syringae, and PopA of Ralstonia solanacearum), elicit the HR when infiltrated into the apoplast of leaf tissue (reference 1 and references therein). They are heat stable, rich in Gly and/or Ser, lack Cys, and differ in their primary sequences. In Erwinia amylovora, mutation of hrpN results in substantially reduced Hrp phenotype (4, 6, 45).E. amylovora causes the devastating fire-blight disease on many rosaceous plants, such as apple, pear, and cotoneaster. Cosmids pCPP430 and pCPP450, which harbor the hrp gene cluster of E. amylovora Ea321, enable Escherichia coli to elicit the HR in tobacco (7). The region of pCPP430 essential for the Hrp phenotype encodes two-component regulatory proteins, a ς⁵⁴ enhancer-binding protein, a sigma factor, secretory proteins, and the HrpN harpin (11, 27, 42–45). In contrast, the locus next to hrp genes, designated dsp, contains pathogenicity genes, and P. syringae pv. glycinea containing the E. amylovora dsp locus causes the HR rather than disease in soybean plants (10). This locus encodes a Hrp-secreted protein and a probable chaperone of the secreted protein (8, 10, 17).Additional HR elicitors in E. amylovora have been suspected based on the HR-variable phenotype of E. amylovora hrpN mutants (references 4 and 6; see also Table ​Table1).1). We report here the identification and characterization of a novel harpin of E. amylovora, HrpW, the C-terminal domain of which surprisingly is homologous to fungal pectate lyases (PLs). We show that HrpW, the production of which is controlled by hrpL, is delivered by the E. amylovora Hrp pathway. HrpW elicits the HR in plants, and the HR necrosis is not due to the potential PL activity of HrpW. Finally, we provide evidence that HrpW is not required for the HR and pathogenicity, although when overexpressed it enhances the HR-eliciting activity of a hrpN mutant. Preliminary reports on E. amylovora HrpW have been made (28, 29), and, while this article was under revision, a paper describing HrpW from E. amylovora CFBP1430 (16) appeared.

TABLE 1

HR induction and virulence of E. amylovora Ea321 and mutant derivatives^a

The use of luciferase as a reporter for response of plant cells to the fireblight pathogen Erwinia amylovora

Summary We have introduced the gene encoding luciferase from Photinus pyralis into pear and tobacco cells in order to judge the reaction of plant tissue to damaging conditions such as incubation at high temperature or inoculation with a pathogen. The constitutive expression of the luciferase gene via a strong promoter slowly decreased during propagation of the transformed pear cell line. After various stress treatments the resulting luciferase activity and the ATP content of the plant cells were determined by bioluminescence and found to correspond to each other. Inoculation of transformed pear cells with Erwinia amylovora resulted in a continuous decrease of luciferase activity in contrast to tobacco cells, where the enzyme activity was significantly higher in the first period after inoculation with bacteria compared to the untreated control cells. The pattern of the luciferase activity reflected the slow damage of the host-plant cells by E. amylovora and the elevated metabolism of the non-host cells after inoculation with the pathogen.Abbreviations 2,4-D 2,4-dichloro-phenoxyacetic acid - CaMV Cauliflower mosaic virus - DTT dithiothreitol - EDTA ethylenediaminetetraacetate - FDA fluorescein diacetate - HEPES (hydroxyethyl)piperazine(ethanesulfonic acid) - HR hypersensitive reaction - Tris tris (hydroxymethyl)amino-methane     

Characterization of Bacillus strains from apple and pear trees in South Africa antagonistic to Erwinia amylovora

In order to find reasons for the absence of fire blight in most countries of the Southern hemisphere, bark samples from apple and pear trees in orchards of the Western Cape region in South Africa were extracted for bacteria which could be antagonistic to Erwinia amylovora. Screening was done in the late growth season and mainly Gram-positive bacteria were isolated. Approximately half of them produced growth inhibition zones on a lawn of E. amylovora. Most isolates were classified as Bacillus megaterium by microbiological assays and in API 50 test systems. They were visualized in the light microscope as non-motile large rods. These strains may not be responsible for the absence of fire blight in orchards, but they may indicate unfavourable climatic conditions for Gram-negative bacteria including E. amylovora. They may reduce the ability of E. amylovora to establish fire blight and could also be useful for application in biological disease control.