Management of cherry leaf spot disease, caused by the fungus Blumeriella jaapii, with succinate d... more Management of cherry leaf spot disease, caused by the fungus Blumeriella jaapii, with succinate dehydrogenase inhibitor (SDHI) fungicides has been ongoing in Michigan tart cherry orchards for the past 17 years. After boscalid-resistant B. jaapii were first isolated from commercial orchards in 2010, premixes of SDHI fungicides fluopyram or fluxapyroxad with a quinone outside inhibitor were registered in 2012. Here, we report widespread resistance to fluopyram (FluoR), fluxapyroxad (FluxR), and boscalid (BoscR) in commercial orchard populations of B. jaapii in Michigan from surveys conducted between 2016 and 2019. A total of 26% of 1610 isolates from the 2016–2017 surveys exhibited the fully-resistant BoscR FluoR FluxR phenotype and only 7% were sensitive to all three SDHIs. Practical resistance to fluopyram and fluxapyroxad was detected in 29 of 35 and 14 of 35 commercial tart cherry orchards, respectively, in surveys conducted in 2018 and 2019. Sequencing of the SdhB, SdhC, and SdhD...
We assessed the effect of three antibiotics (streptomycin, oxytetracycline, and kasugamycin) on p... more We assessed the effect of three antibiotics (streptomycin, oxytetracycline, and kasugamycin) on populations of the fire blight pathogen Erwinia amylovora on apple flower stigmas during three field seasons. Application timing relative to E. amylovora presence on flower stigmas had little impact on population dynamics and subsequent disease incidence. Although E. amylovora populations on water-treated flowers increased to 106–7 cfu flower−1 after 4 to 5 days during each experiment, the antibiotics streptomycin and kasugamycin caused statistically significant reductions in stigma populations by as many as 4 to 5 logs over a 4- to 5-day period during two of the three experiments. In contrast, the effect of oxytetracycline on E. amylovora populations on stigmas was more variable, with reductions in E. amylovora populations only observed during one of the three experiments. In agreement with the population data, the disease incidence was significantly higher for oxytetracycline-treated fl...
Background Toxin-antitoxin (TA) systems, abundant in prokaryotes, are composed of a toxin gene an... more Background Toxin-antitoxin (TA) systems, abundant in prokaryotes, are composed of a toxin gene and its cognate antitoxin. Several toxins are implied to affect the physiological state and stress tolerance of bacteria in a population. We previously identified a chromosomally encoded hok-sok type I TA system in Erwinia amylovora, the causative agent of fire blight disease on pome fruit trees. A high-level induction of the hok gene was lethal to E. amylovora cells through unknown mechanisms. The molecular targets or regulatory roles of Hok were unknown. Results Here, we examined the physiological and transcriptomic changes of Erwinia amylovora cells expressing hok at subtoxic levels that were confirmed to confer no cell death, and at toxic levels that resulted in killing of cells. In both conditions, hok caused membrane rupture and collapse of the proton motive force in a subpopulation of E. amylovora cells. We demonstrated that induction of hok resulted in upregulation of ATP biosynthe...
Resistance to sterol demethylation inhibitor (DMI) fungicides in Monilinia fructicola, causal age... more Resistance to sterol demethylation inhibitor (DMI) fungicides in Monilinia fructicola, causal agent of brown rot of stone fruit, has been reported in the southeastern and eastern United States and in Brazil. DMI resistance of some M. fructicola isolates, in particular those recovered from the southeastern United States, is associated with a sequence element termed “Mona” that causes overexpression of the cytochrome demethylase target gene MfCYP51. In this study, we conducted statewide surveys of Michigan stone fruit orchards from 2009 to 2011 and in 2019, and we determined the sensitivity to propiconazole of a total of 813 isolates of M. fructicola. A total of 80.7% of Michigan isolates were characterized as resistant to propiconazole by relative growth assays, but the Mona insert was not uniformly detected and was present in some isolates that were not characterized as DMI resistant. Gene expression assays indicated that elevated expression of MfCYP51 was only weakly correlated wit...
Fungi in the genus Monilinia are known to cause devastating brown rot disease of stone and pome f... more Fungi in the genus Monilinia are known to cause devastating brown rot disease of stone and pome fruits. Here, we report the draft genome assemblies of four important phytopathogenic species: Monilinia fructicola, Monilinia fructigena, Monilinia polystroma, and Monilinia laxa. The draft genome assemblies were 39 Mb (M. fructigena), 42 Mb (M. laxa), 43 Mb (M. fructicola), and 45 Mb (M. polystroma) with as few as 550 contigs (M. laxa). These are the first draft genome resources publicly available for M. laxa, M. fructigena, and M. polystroma.
Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of app... more Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae-infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations ('Widely Prevalent' clade). Furthermore, we revealed that strains from the Widely Prevalent clade d...
Bacterial etiolation and decline (BED), caused by Acidovorax avenae, is an emerging disease of cr... more Bacterial etiolation and decline (BED), caused by Acidovorax avenae, is an emerging disease of creeping bentgrass on golf courses in the United States. We performed the first comprehensive analysis of A. avenae on a nationwide collection of turfgrass- and maize-pathogenic A. avenae. Surprisingly, our results reveal that the turfgrass-pathogenic A. avenae in North America are not only highly divergent but also belong to two distinct phylogroups. Both phylogroups specifically infect turfgrass but are more closely related to maize pathogens than to each other. This suggests that, although the disease is only recently reported, it has likely been infecting turfgrass for a long time. To identify a genetic basis for the host specificity, we searched for genes closely related among turfgrass strains but distantly related to their homologs from maize strains. We found a cluster of 11 such genes generated by three ancient recombination events within the type III secretion system (T3SS) patho...
We report the utilization of site-directed and random mutagenesis procedures in the gene encoding... more We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined th...
Proceedings of the National Academy of Sciences, 2012
The successful growth of hypermutator strains of bacteria contradicts a clear preference for lowe... more The successful growth of hypermutator strains of bacteria contradicts a clear preference for lower mutation rates observed in the microbial world. Whether by general DNA repair deficiency or the inducible action of low-fidelity DNA polymerases, the evolutionary strategies of bacteria include methods of hypermutation. Although both raise mutation rate, general and inducible hypermutation operate through distinct molecular mechanisms and therefore likely impart unique adaptive consequences. Here we compare the influence of general and inducible hypermutation on adaptation in the model organism Pseudomonas aeruginosa PAO1 through experimental evolution. We observed divergent spectra of single base substitutions derived from general and inducible hypermutation by sequencing rpoB in spontaneous rifampicin-resistant (Rif R ) mutants. Likewise, the pattern of mutation in a draft genome sequence of a derived inducible hypermutator isolate differed from those of general hypermutators reporte...
Control strategies for Venturia inaequalis rely heavily on chemical fungicides. Single-site fungi... more Control strategies for Venturia inaequalis rely heavily on chemical fungicides. Single-site fungicides such as the quinone-outside inhibitors (QoI) have been used in Michigan apple orchards for more than 11 years. In 2008, we sampled eight commercial orchards in the Fruit Ridge growing region of Michigan in which apple scab control failures were observed on ‘McIntosh’ apple following applications of kresoxim-methyl or trifloxystrobin. QoI resistance was assessed in 210 total isolates (a total of 17 orchards) using a spore germination assay and in 319 isolates using a polymerase chain reaction (PCR) assay to detect the G143A mutation located within the V. inaequalis cytochrome b gene (CYTB). The G143A mutation is known to confer high-level QoI resistance in plant-pathogenic fungi. QoI resistance was confirmed in 50 and 64% of the isolates tested with the spore germination and PCR assays, respectively, and there was a 97% concordance observed between the assays. In 2009, we sampled an...
Eighteen apple cultivars were tested in the field and laboratory for their relative susceptibilit... more Eighteen apple cultivars were tested in the field and laboratory for their relative susceptibility to one of the bitter rot pathogens, Colletotrichum acutatum. Fruit were inoculated in the field at 3 to 4 weeks preharvest with cheesecloth strips soaked in a conidia suspension. In the laboratory, detached fruit were inoculated using a conidia suspension in capped, sterile microcentrifuge tubes attached to the fruit surface with modeling clay. The same fruit as above also were inoculated over a wound on the side opposite the nonwounded inoculation. Fruit were tested for relative susceptibility to the fungus with five criteria: disease incidence and severity of attached fruit in the field, disease incidence and severity of detached fruit in laboratory inoculations of nonwounded fruit, and disease severity in laboratory inoculations of wounded fruit. Relative cultivar ranks from field tests were not reproducible in the 2 years studied, whereas laboratory tests showed moderate reproducib...
This Focus Issue addresses the topic of food safety and the biology of human pathogens on plants,... more This Focus Issue addresses the topic of food safety and the biology of human pathogens on plants, a relatively new problem with a direct impact on public health. This critical aspect of produce safety is relevant to research in plant microbial ecology and intersects with numerous concepts that are explored in plant pathology. The emergence of outbreaks of human illness linked to the contamination of produce is likely one of the most important problems to face horticultural production at the beginning of this century. Epidemics of foodborne disease are not only a threat to public health but also erode consumer confidence in the causal food product and thus, impact the economic viability of the industry. Although researched extensively for nearly two decades, produce contamination with human pathogens continues to bring many important questions about the behavior of these pathogens on plants and the biotic and abiotic factors that contribute to their persistence in this habitat, there...
Management of cherry leaf spot disease, caused by the fungus Blumeriella jaapii, with succinate d... more Management of cherry leaf spot disease, caused by the fungus Blumeriella jaapii, with succinate dehydrogenase inhibitor (SDHI) fungicides has been ongoing in Michigan tart cherry orchards for the past 17 years. After boscalid-resistant B. jaapii were first isolated from commercial orchards in 2010, premixes of SDHI fungicides fluopyram or fluxapyroxad with a quinone outside inhibitor were registered in 2012. Here, we report widespread resistance to fluopyram (FluoR), fluxapyroxad (FluxR), and boscalid (BoscR) in commercial orchard populations of B. jaapii in Michigan from surveys conducted between 2016 and 2019. A total of 26% of 1610 isolates from the 2016–2017 surveys exhibited the fully-resistant BoscR FluoR FluxR phenotype and only 7% were sensitive to all three SDHIs. Practical resistance to fluopyram and fluxapyroxad was detected in 29 of 35 and 14 of 35 commercial tart cherry orchards, respectively, in surveys conducted in 2018 and 2019. Sequencing of the SdhB, SdhC, and SdhD...
We assessed the effect of three antibiotics (streptomycin, oxytetracycline, and kasugamycin) on p... more We assessed the effect of three antibiotics (streptomycin, oxytetracycline, and kasugamycin) on populations of the fire blight pathogen Erwinia amylovora on apple flower stigmas during three field seasons. Application timing relative to E. amylovora presence on flower stigmas had little impact on population dynamics and subsequent disease incidence. Although E. amylovora populations on water-treated flowers increased to 106–7 cfu flower−1 after 4 to 5 days during each experiment, the antibiotics streptomycin and kasugamycin caused statistically significant reductions in stigma populations by as many as 4 to 5 logs over a 4- to 5-day period during two of the three experiments. In contrast, the effect of oxytetracycline on E. amylovora populations on stigmas was more variable, with reductions in E. amylovora populations only observed during one of the three experiments. In agreement with the population data, the disease incidence was significantly higher for oxytetracycline-treated fl...
Background Toxin-antitoxin (TA) systems, abundant in prokaryotes, are composed of a toxin gene an... more Background Toxin-antitoxin (TA) systems, abundant in prokaryotes, are composed of a toxin gene and its cognate antitoxin. Several toxins are implied to affect the physiological state and stress tolerance of bacteria in a population. We previously identified a chromosomally encoded hok-sok type I TA system in Erwinia amylovora, the causative agent of fire blight disease on pome fruit trees. A high-level induction of the hok gene was lethal to E. amylovora cells through unknown mechanisms. The molecular targets or regulatory roles of Hok were unknown. Results Here, we examined the physiological and transcriptomic changes of Erwinia amylovora cells expressing hok at subtoxic levels that were confirmed to confer no cell death, and at toxic levels that resulted in killing of cells. In both conditions, hok caused membrane rupture and collapse of the proton motive force in a subpopulation of E. amylovora cells. We demonstrated that induction of hok resulted in upregulation of ATP biosynthe...
Resistance to sterol demethylation inhibitor (DMI) fungicides in Monilinia fructicola, causal age... more Resistance to sterol demethylation inhibitor (DMI) fungicides in Monilinia fructicola, causal agent of brown rot of stone fruit, has been reported in the southeastern and eastern United States and in Brazil. DMI resistance of some M. fructicola isolates, in particular those recovered from the southeastern United States, is associated with a sequence element termed “Mona” that causes overexpression of the cytochrome demethylase target gene MfCYP51. In this study, we conducted statewide surveys of Michigan stone fruit orchards from 2009 to 2011 and in 2019, and we determined the sensitivity to propiconazole of a total of 813 isolates of M. fructicola. A total of 80.7% of Michigan isolates were characterized as resistant to propiconazole by relative growth assays, but the Mona insert was not uniformly detected and was present in some isolates that were not characterized as DMI resistant. Gene expression assays indicated that elevated expression of MfCYP51 was only weakly correlated wit...
Fungi in the genus Monilinia are known to cause devastating brown rot disease of stone and pome f... more Fungi in the genus Monilinia are known to cause devastating brown rot disease of stone and pome fruits. Here, we report the draft genome assemblies of four important phytopathogenic species: Monilinia fructicola, Monilinia fructigena, Monilinia polystroma, and Monilinia laxa. The draft genome assemblies were 39 Mb (M. fructigena), 42 Mb (M. laxa), 43 Mb (M. fructicola), and 45 Mb (M. polystroma) with as few as 550 contigs (M. laxa). These are the first draft genome resources publicly available for M. laxa, M. fructigena, and M. polystroma.
Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of app... more Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae-infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations ('Widely Prevalent' clade). Furthermore, we revealed that strains from the Widely Prevalent clade d...
Bacterial etiolation and decline (BED), caused by Acidovorax avenae, is an emerging disease of cr... more Bacterial etiolation and decline (BED), caused by Acidovorax avenae, is an emerging disease of creeping bentgrass on golf courses in the United States. We performed the first comprehensive analysis of A. avenae on a nationwide collection of turfgrass- and maize-pathogenic A. avenae. Surprisingly, our results reveal that the turfgrass-pathogenic A. avenae in North America are not only highly divergent but also belong to two distinct phylogroups. Both phylogroups specifically infect turfgrass but are more closely related to maize pathogens than to each other. This suggests that, although the disease is only recently reported, it has likely been infecting turfgrass for a long time. To identify a genetic basis for the host specificity, we searched for genes closely related among turfgrass strains but distantly related to their homologs from maize strains. We found a cluster of 11 such genes generated by three ancient recombination events within the type III secretion system (T3SS) patho...
We report the utilization of site-directed and random mutagenesis procedures in the gene encoding... more We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined th...
Proceedings of the National Academy of Sciences, 2012
The successful growth of hypermutator strains of bacteria contradicts a clear preference for lowe... more The successful growth of hypermutator strains of bacteria contradicts a clear preference for lower mutation rates observed in the microbial world. Whether by general DNA repair deficiency or the inducible action of low-fidelity DNA polymerases, the evolutionary strategies of bacteria include methods of hypermutation. Although both raise mutation rate, general and inducible hypermutation operate through distinct molecular mechanisms and therefore likely impart unique adaptive consequences. Here we compare the influence of general and inducible hypermutation on adaptation in the model organism Pseudomonas aeruginosa PAO1 through experimental evolution. We observed divergent spectra of single base substitutions derived from general and inducible hypermutation by sequencing rpoB in spontaneous rifampicin-resistant (Rif R ) mutants. Likewise, the pattern of mutation in a draft genome sequence of a derived inducible hypermutator isolate differed from those of general hypermutators reporte...
Control strategies for Venturia inaequalis rely heavily on chemical fungicides. Single-site fungi... more Control strategies for Venturia inaequalis rely heavily on chemical fungicides. Single-site fungicides such as the quinone-outside inhibitors (QoI) have been used in Michigan apple orchards for more than 11 years. In 2008, we sampled eight commercial orchards in the Fruit Ridge growing region of Michigan in which apple scab control failures were observed on ‘McIntosh’ apple following applications of kresoxim-methyl or trifloxystrobin. QoI resistance was assessed in 210 total isolates (a total of 17 orchards) using a spore germination assay and in 319 isolates using a polymerase chain reaction (PCR) assay to detect the G143A mutation located within the V. inaequalis cytochrome b gene (CYTB). The G143A mutation is known to confer high-level QoI resistance in plant-pathogenic fungi. QoI resistance was confirmed in 50 and 64% of the isolates tested with the spore germination and PCR assays, respectively, and there was a 97% concordance observed between the assays. In 2009, we sampled an...
Eighteen apple cultivars were tested in the field and laboratory for their relative susceptibilit... more Eighteen apple cultivars were tested in the field and laboratory for their relative susceptibility to one of the bitter rot pathogens, Colletotrichum acutatum. Fruit were inoculated in the field at 3 to 4 weeks preharvest with cheesecloth strips soaked in a conidia suspension. In the laboratory, detached fruit were inoculated using a conidia suspension in capped, sterile microcentrifuge tubes attached to the fruit surface with modeling clay. The same fruit as above also were inoculated over a wound on the side opposite the nonwounded inoculation. Fruit were tested for relative susceptibility to the fungus with five criteria: disease incidence and severity of attached fruit in the field, disease incidence and severity of detached fruit in laboratory inoculations of nonwounded fruit, and disease severity in laboratory inoculations of wounded fruit. Relative cultivar ranks from field tests were not reproducible in the 2 years studied, whereas laboratory tests showed moderate reproducib...
This Focus Issue addresses the topic of food safety and the biology of human pathogens on plants,... more This Focus Issue addresses the topic of food safety and the biology of human pathogens on plants, a relatively new problem with a direct impact on public health. This critical aspect of produce safety is relevant to research in plant microbial ecology and intersects with numerous concepts that are explored in plant pathology. The emergence of outbreaks of human illness linked to the contamination of produce is likely one of the most important problems to face horticultural production at the beginning of this century. Epidemics of foodborne disease are not only a threat to public health but also erode consumer confidence in the causal food product and thus, impact the economic viability of the industry. Although researched extensively for nearly two decades, produce contamination with human pathogens continues to bring many important questions about the behavior of these pathogens on plants and the biotic and abiotic factors that contribute to their persistence in this habitat, there...
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