George W. Sundin

Contribution of Erwinia amylovora Exopolysaccharides Amylovoran and Levan to Biofilm Formation: Implications in Pathogenicity

Erwinia amylovora is a highly virulent, necrogenic, vascular pathogen of rosaceous species that produces the exopolysaccharide amylovoran, a known pathogenicity factor, and levan, a virulence factor. An in vitro crystal violet staining and a bright-field microscopy method were used to demonstrate that E. amylovora is capable of forming a biofilm on solid surfaces. Amylovoran and levan production deletion mutants were used to determine that amylovoran was required for biofilm formation and that levan contributed to biofilm formation. In vitro flow cell and confocal microscopy were used to further reveal the architectural detail of a mature biofilm and differences in biofilm formation between E. amylovora wild-type (WT), Deltaams, and Deltalsc mutant cells labeled with green fluorescent protein or yellow fluorescent protein. Scanning electron microscopy analysis of E. amylovora WT cells following experimental inoculation in apple indicated that extensive biofilm formation occurs in xylem vessels. However, Deltaams mutant cells were nonpathogenic and died rapidly following inoculation, and Deltalsc mutant cells were not detected in xylem vessels and were reduced in movement into apple shoots. These results demonstrate that biofilm formation plays a critical role in the pathogenesis of E. amylovora.

Overexpression of the 14α-Demethylase Target Gene (CYP51) Mediates Fungicide Resistance in Blumeriella jaapii

ABSTRACT Sterol demethylation inhibitor (DMI) fungicides are widely used to control fungi pathogenic to humans and plants. Resistance to DMIs is mediated either through alterations in the structure of the target enzyme CYP51 (encoding 14α-demethylase), through increased expression of the CYP51 gene, or through increased expression of efflux pumps. We found that CYP51 expression in DMI-resistant (DMIR) isolates of the cherry leaf spot pathogen Blumeriella jaapii was increased 5- to 12-fold compared to that in DMI-sensitive (DMIS) isolates. Analysis of sequences upstream of CYP51 in 59 DMIR isolates revealed that various forms of a truncated non-long terminal direct repeat long interspersed nuclear element retrotransposon were present in all instances. Similar inserts upstream of CYP51 were not present in any of 22 DMIS isolates examined.

Identification of Erwinia amylovora Genes Induced during Infection of Immature Pear Tissue

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In this study, we used a modified in vivo expression technology system to identify E. amylovora genes that are activated during infection of immature pear tissue, a process that requires the major pathogenicity factors of this organism. We identified 394 unique pear fruit-induced (pfi) genes on the basis of sequence similarity to known genes and separated them into nine putative function groups including host-microbe interactions (3.8%), stress response (5.3%), regulation (11.9%), cell surface (8.9%), transport (13.5%), mobile elements (1.0%), metabolism (20.3%), nutrient acquisition and synthesis (15.5%), and unknown or hypothetical proteins (19.8%). Known virulence genes, including hrp/hrc components of the type III secretion system, the major effector gene dspE, type II secretion, levansucrase (lsc), and regulators of levansucrase and amylovoran biosynthesis, were upregulated during pear tissue infection. Known virulence factors previously identified in E. (Pectobacterium) carotovora and Pseudomonas syringae were identified for the first time in E. amylovora and included HecA hemagglutinin family adhesion, Peh polygalacturonase, new effector HopPtoC(EA), and membrane-bound lytic murein transglycosylase MltE(EA). An insertional mutation within hopPtoC(EA) did not result in reduced virulence; however, an mltE(EA) knockout mutant was reduced in virulence and growth in immature pears. This study suggests that E. amylovora utilizes a variety of strategies during plant infection and to overcome the stressful and poor nutritional environment of its plant hosts.

Effect of solar UV-B radiation on a phyllosphere bacterial community.

ABSTRACT The effect of solar UV-B radiation on the population dynamics and composition of the culturable bacterial community from peanut (Arachishypogeae L.) was examined in field studies using plants grown under UV-B−transmitting (UV-B+) or UV-B−excluding (UV-B−) plastic filters. Our data demonstrate that solar UV-B selection alters phyllosphere bacterial community composition and that UV tolerance is a prevalent phenotype late in the season. The total bacterial population size was not affected by either UV-B treatment. However, isolates from the UV-B+ plots (n = 368) were significantly more UV tolerant than those from the UV-B− (n = 363) plots. UV sensitivity was determined as the minimal inhibitory dose of UV that resulted in an inhibition of growth compared to the growth of a nonirradiated control. The difference in minimal inhibitory doses among bacterial isolates from UV-B+ and UV-B− treatments was mainly partitioned among nonpigmented isolates, with pigmented isolates as a group being characterized as UV tolerant. A large increase in UV tolerance was observed within isolate groups collected late (89 and 96 days after planting) in the season. Identification of 200 late-season isolates indicated that the predominant UV-tolerant members of this group were Bacilluscoagulans,Clavibactermichiganensis, andCurtobacteriumflaccumfaciens. We selected C. michiganensis as a model UV-tolerant epiphyte to study if cell survival on UV-irradiated peanut leaves was increased relative to UV survival in vitro. The results showed an enhancement in the survival of C.michiganensis G7.1, especially following high UV-C doses (300 and 375 J m−2), that was evident between 24 and 96 h after inoculation. A dramatic increase in the in planta/in vitro survival ratio was observed over the entire 96-h experiment period for C. michiganensis T5.1.

Systems level analysis of two-component signal transduction systems in Erwinia amylovora: Role in virulence, regulation of amylovoran biosynthesis and swarming motility

BackgroundTwo-component signal transduction systems (TCSTs), consisting of a histidine kinase (HK) and a response regulator (RR), represent a major paradigm for signal transduction in prokaryotes. TCSTs play critical roles in sensing and responding to environmental conditions, and in bacterial pathogenesis. Most TCSTs in Erwinia amylovora have either not been identified or have not yet been studied.ResultsWe used a systems approach to identify TCST and related signal transduction genes in the genome of E. amylovora. Comparative genomic analysis of TCSTs indicated that E. amylovora TCSTs were closely related to those of Erwinia tasmaniensis, a saprophytic enterobacterium isolated from apple flowers, and to other enterobacteria. Forty-six TCST genes in E. amylovora including 17 sensor kinases, three hybrid kinases, 20 DNA- or ligand-binding RRs, four RRs with enzymatic output domain (EAL-GGDEF proteins), and two kinases were characterized in this study. A systematic TCST gene-knockout experiment was conducted, generating a total of 59 single-, double-, and triple-mutants. Virulence assays revealed that five of these mutants were non-pathogenic on immature pear fruits. Results from phenotypic characterization and gene expression experiments indicated that several groups of TCST systems in E. amylovora control amylovoran biosynthesis, one of two major virulence factors in E. amylovora. Both negative and positive regulators of amylovoran biosynthesis were identified, indicating a complex network may control this important feature of pathogenesis. Positive (non-motile, EnvZ/OmpR), negative (hypermotile, GrrS/GrrA), and intermediate regulators for swarming motility in E. amylovora were also identified.ConclusionOur results demonstrated that TCSTs in E. amylovora played major roles in virulence on immature pear fruit and in regulating amylovoran biosynthesis and swarming motility. This suggested presence of regulatory networks governing expression of critical virulence genes in E. amylovora.

Comparative Analysis of Differentially Expressed Genes in Shewanella oneidensis MR-1 following Exposure to UVC, UVB, and UVA Radiation

We previously reported that Shewanella oneidensis MR-1 is highly sensitive to UVC (254 nm), UVB (290 to 320 nm), and UVA (320 to 400 nm). Here we delineated the cellular response of MR-1 to UV radiation damage by analyzing the transcriptional profile during a 1-h recovering period after UVC, UVB, and UVA exposure at a dose that yields about a 20% survival rate. Although the SOS response was observed with all three treatments, the induction was more robust in response to short-wavelength UV radiation (UVB and UVC). Similarly, more prophage-related genes were induced by short-wavelength UV radiation. MR-1 showed an active detoxification mechanism in response to UVA, which included the induction of antioxidant enzymes and iron-sequestering proteins to scavenge reactive oxygen species. In addition, a great number of genes encoding multidrug and heavy metal efflux pumps were induced following UVA irradiation. Our data suggested that activation of prophages appears the major lethal factor in MR-1 following UVC or UVB irradiation, whereas oxidative damage contributes greatly to the high UVA sensitivity in MR-1.

Fire Blight: Applied Genomic Insights of the Pathogen and Host

The enterobacterial phytopathogen Erwinia amylovora causes fire blight, an invasive disease that threatens a wide range of commercial and ornamental Rosaceae host plants. The response elicited by E. amylovora in its host during disease development is similar to the hypersensitive reaction that typically leads to resistance in an incompatible host-pathogen interaction, yet no gene-for-gene resistance has been described for this host-pathogen system. Comparative genomic analysis has found an unprecedented degree of genetic uniformity among strains of E. amylovora, suggesting that the pathogen has undergone a recent genetic bottleneck. The genome of apple, an important host of E. amylovora, has been sequenced, creating new opportunities for the study of interactions between host and pathogen during fire blight development and for the identification of resistance genes. This review includes recent advances in the genomics of both host and pathogen.

Field Evaluation of Biological Control of Fire Blight in the Eastern United States

The bacterial antagonists Pseudomonas fluorescens A506, Pantoea agglomerans C9-1, and Pantoea agglomerans E325 and preparations of Bacillus subtilis QST 713 containing bacterial endospores and lipopeptide metabolites were evaluated for efficacy in controlling fire blight in Michigan, New York, and Virginia. When examined individually, the biological control materials were not consistently effective in reducing blossom infection. The average reduction in blossom infection observed in experiments conducted between 2001 and 2007 was variable and ranged from 9.1 to 36.1%, while control with streptomycin was consistent and ranged from 59.0 to 67.3%. Incidence of blossom colonization by the bacterial antagonists was inconsistent, and <60% of stigmata had the antagonists present in 12 of 25 experiments. Consistent control of blossom infection was observed when the biological control materials were integrated into programs with streptomycin, resulting in a reduction of the number of streptomycin applications needed to yield similar levels of control. Our results indicate that the prospects for biological control of fire blight in the eastern United States are currently not high due to the variability in efficacy of existing biological control options.

Evaluation of Kasugamycin for Fire Blight Management, Effect on Nontarget Bacteria, and Assessment of Kasugamycin Resistance Potential in Erwinia amylovora

The emergence and spread of streptomycin-resistant strains of Erwinia amylovora in Michigan has necessitated the evaluation of new compounds effective for fire blight control. The aminoglycoside antibiotic kasugamycin (Ks) targets the bacterial ribosome and is particularly active against E. amylovora. The efficacy of Ks formulated as Kasumin 2L for control of fire blight was evaluated in six experiments conducted over four field seasons in our experimental orchards in East Lansing, MI. Blossom blight control was statistically equivalent to the industry standard streptomycin in all experiments. E. amylovora populations remained constant on apple flower stigmas pretreated with Kasumin and were ≈100-fold lower than on stigmas treated with water. Kasumin applied to apple trees in the field also resulted in a 100-fold reduced total culturable bacterial population compared with trees treated with water. We performed a prospective analysis of the potential for kasugamycin resistance (Ks(R)) development in E. amylovora which focused on spontaneous resistance development and acquisition of a transferrable Ks(R) gene. In replicated lab experiments, the development of spontaneous resistance in E. amylovora to Ks at 250 or 500 ppm was not observed when cells were directly plated on medium containing high concentrations of the antibiotic. However, exposure to increasing concentrations of Ks in media (initial concentration 25 μg ml(-1)) resulted in the selection of Ks resistance (at 150 μg ml(-1)) in the E. amylovora strains Ea110, Ea273, and Ea1189. Analysis of mutants indicated that they harbored mutations in the kasugamycin target ksgA gene and that all mutants were impacted in relative fitness observable through a reduced growth rate in vitro and decreased virulence in immature pear fruit. The possible occurrence of a reservoir of Ks(R) genes in orchard environments was also examined. Culturable gram-negative bacteria were surveyed from six experimental apple orchards that had received at least one Kasumin application. In total, 401 Ks(R) isolates (42 different species) were recovered from apple flowers and leaves and orchard soil samples. Although we have not established the presence of a transferrable Ks(R) gene in orchard bacteria, the frequency, number of species, and presence of Ks(R) enterobacterial species in orchard samples suggests the possible role of nontarget bacteria in the future transfer of a Ks(R) gene to E. amylovora. Our data confirm the importance of kasugamycin as an alternate antibiotic for fire blight management and lay the groundwork for the development and incorporation of resistance management strategies.

Diversity and Biogeography of Sooty Blotch and Flyspeck Fungi on Apple in the Eastern and Midwestern United States

Sooty blotch and flyspeck (SBFS) fungi on apple fruit were sampled from nine orchards in four midwestern U.S. states during 2000 and 30 orchards in 10 eastern U.S. states during 2005 in order to estimate taxonomic diversity and discern patterns of geographic distribution. Forty apple fruit per orchard were arbitrarily sampled and colonies of each mycelial phenotype were counted on each apple. Representative colonies were isolated, cultures were purified, and DNA was extracted. For representative isolates, the internal transcribed spacer (ITS) and large subunit (LSU) regions of ribosomal DNA were amplified and sequenced. In total, 60 SBFS putative species were identified based on ITS sequences and morphological characteristics; 30 of these were discovered in the 2005 survey. Modified Kochs postulates were fulfilled for all 60 species in an Iowa orchard; colonies resulting from inoculation of apple fruit were matched to the original isolates on the basis of mycelial type and ITS sequence. Parsimony analysis for LSU sequences from both surveys revealed that 58 putative SBFS species were members of the Dothideomycetes, 52 were members of the Capnodiales, and 36 were members of the Mycosphaerellaceae. The number of SBFS species per orchard varied from 2 to 15. Number of SBFS species and values of the Margalef and Shannon indexes were significantly (P < 0.05) lower in 21 orchards that had received conventional fungicide sprays during the fruit maturation period than in 14 unsprayed orchards. Several SBFS species, including Schizothyrium pomi, Peltaster fructicola, and Pseudocercosporella sp. RH1, were nearly ubiquitous, whereas other species, such as Stomiopeltis sp. RS5.2, Phialophora sessilis, and Geastrumia polystigmatis, were found only within restricted geographic regions. The results document that the SBFS complex is far more taxonomically diverse than previously recognized and provide strong evidence that SBFS species differ in geographic distribution. To achieve more efficient management of SBFS, it may be necessary to understand the environmental biology of key SBFS species in each geographic region.