Fungicide ingestion reduces net energy gain and microbiome diversity of the solitary mason bee.

Fungicides are frequently used during tree fruit bloom and can threaten insect pollinators. However, little is known about how non-honey bee pollinators such as the solitary bee, Osmia cornifrons, respond to contact and systemic fungicides commonly used in apple production during bloom. This knowledge gap limits regulatory decisions that determine safe concentrations and timing for fungicide spraying. We evaluated the effects of two contact fungicides (captan and mancozeb) and four translaminar/plant systemic fungicides (cyprodinil, myclobutanil, penthiopyrad, and trifloxystrobin) on larval weight gain, survival, sex ratio, and bacterial diversity. This assessment was carried out using chronic oral ingestion bioassays where pollen provisions were treated with three doses based on the currently recommended field use dose (1X), half dose (0.5X), and low dose (0.1X). Mancozeb and penthiopyrad significantly reduced larval weight and survival at all doses. We then sequenced the 16S gene to characterize the larvae bacteriome of mancozeb, the fungicide that caused the highest mortality. We found that larvae fed on mancozeb-treated pollen carried significantly lower bacterial diversity and abundance. Our laboratory results suggest that some of these fungicides can be particularly harmful to the health of O. cornifrons when sprayed during bloom. This information is relevant for future management decisions about the sustainable use of fruit tree crop protection products and informing regulatory processes that aim to protect pollinators.

Systemic pesticides in a solitary bee pollen food store affect larval development and increase pupal mortality.

Solitary bees are often exposed to various pesticides applied for pest control on farmland while providing pollination services to food crops. Increasing evidence suggests that sublethal toxicity of agricultural pesticides affects solitary bees differently than the social bees used to determine regulatory thresholds, such as honey bees and bumblebees. Studies on solitary bees are challenging because of the difficulties in obtaining large numbers of eggs or young larvae for bioassays. Here we show the toxic and sublethal developmental effects of four widely used plant systemic pesticides on the Japanese orchard bee (Osmia cornifrons). Pollen food stores of this solitary bee were treated with different concentrations of three insecticides (acetamiprid, flonicamid, and sulfoxaflor) and a fungicide (dodine). Eggs were transplanted to the treated pollen and larvae were allowed to feed on the pollen stores after egg hatch. The effects of chronic ingestion of contaminated pollen were measured until adult eclosion. This year-long study revealed that chronic exposure to all tested pesticides delayed larval development and lowered larval and adult body weights. Additionally, exposure to the systemic fungicide resulted in abnormal larval defecation and increased mortality at the pupal stage, indicating potential risk to bees from fungicide exposure. These findings demonstrate potential threats to solitary bees from systemic insecticides and fungicides and will help in making policy decisions to mitigate these effects.

The Rapid Apple Decline Phenomenon: Current Status and Expected Associated Factors in Korea.

Rapid apple decline (RAD) is a complex phenomenon affecting cultivated apple trees and particularly dwarf rootstocks on grafted young apple trees. Since its first appearance in the United States, RAD has been reported worldwide, for example in Canada, South America, Africa, and Asia. The phenomenon has also been observed in apple orchards in Korea, and it presented similar symptoms regardless of apple cultivar and cultivation period. Most previous reports have suggested that RAD may be associated with multiple factors, including plant pathogenic infections, abiotic stresses, environmental conditions, and the susceptibility of trees to cold injury during winter. However, RAD was observed to be more severe and affect more frequently apple trees on the Malling series dwarf rootstock. In this study, we reviewed the current status of RAD worldwide and surveyed biotic and abiotic factors that are potentially closely related to it in Korea.

Spore Dispersal Patterns of Colletotrichum fioriniae in Orchards and the Timing of Apple Bitter Rot Infection Periods.

Bitter rot is a major disease of apple fruit in warm and humid regions. It is caused by various species in the Colletotrichum gloeosporioides and C. acutatum species complexes, of which C. fioriniae of the C. acutatum species complex is most common in the Mid-Atlantic region of the United States. While bitter rot management begins with good cultural practices, fungicides are generally used for consistent control. Fungicides should be applied before or during infection periods, but the timing of infection is unclear due to the hemibiotrophic lifestyle of the causal species. To determine when infection periods occur, we quantified C. fioriniae spore dispersal throughout three growing seasons and compared the temporal susceptibility of apples in two seasons of field trials. Spores were detected in rainwater from bud break to leaf drop, with the highest spore quantities in the summer and early fall correlating with optimal temperatures for C. fioriniae. Late-season-inoculated fruit had more bitter rot than early-season-inoculated fruit, but this was also positively correlated with periods of optimal temperatures and moisture for infection. In the context of previous experiments, these results suggest that infection periods are primarily determined by temperature and moisture rather than apple fruit phenology. Based on the relative numbers of spores and biotrophic and necrotrophic infections, only a tiny proportion of spores establish viable biotrophic infections, but a relatively high proportion of biotrophic infections switch to necrotrophy. We suggest bitter rot management should focus on preventing initial biotrophic infections by protecting apples during weather conditions that favor infection.

Would You Like Wood or Plastic? Bin Material, Sanitation Treatments, and Bin Inoculum Levels Impact Blue Mold Decay of Stored Apple Fruit.

Blue mold, caused primarily by Penicillium expansum, is a significant postharvest disease of apples. It not only causes economic losses but also produces mycotoxins that contaminate processed fruit products, which contributes to food waste and loss. Previous research has shown that packing and storage bins harbor Penicillium spores and that steam and hot water efficiently reduce spore inoculum levels. However, studies using wooden and plastic bins regarding their ability to harbor spores, the effect of chemical sanitation treatments on spore levels, and the impact of rinsate from treated bins on apple fruit decay have not been investigated for the Mid-Atlantic area (Okull et al. 2006; Rosenberger 2009). We evaluated different sanitation treatments (chemical and physical) to reduce P. expansum inoculum levels on wooden and plastic bins. We determined that wooden bins bound P. expansum spores four orders of magnitude higher than plastic. When both bin types were treated with steam (wooden) or sterile hot water (plastic), Thyme Guard, or Academy, all treatments resulted in significantly (P < 0.05) lower spore levels compared to untreated controls. Although, plastic bins retained lower numbers of spores after inoculation with contaminated spore rinsate and required much higher concentrations of P. expansum spores in rinsate to retain spores at levels that would lead to decay on apple fruit. Overall, we demonstrated that plastic bins retain fewer spores than wooden bins and that both can be sanitized by various physical or chemical treatments. We envision that our findings will be applicable in the future as the techniques implemented in this study were used to investigate industry-relevant questions. Our goal is that the research techniques and findings become feasible with advancements in technology and/or accompany other shifts in existing processes in commercial pome fruit packing and storage facilities.

Putative transcription antiterminator RfaH contributes to Erwinia amylovora virulence.

The gram-negative bacterium Erwinia amylovora causes fire blight disease of apple and pear trees. The exopolysaccharide amylovoran and lipopolysaccharides are essential E. amylovora virulence factors. Production of amylovoran and lipopolysaccharide is specified in part by genes that are members of long operons. Here, we show that full virulence of E. amylovora in apple fruitlets and tree shoots depends on the predicted transcription antiterminator RfaH. RfaH reduces pausing in the production of long transcripts having an operon polarity suppressor regulatory element within their promoter region. In E. amylovora, only the amylovoran operon and a lipopolysaccharide operon have such regulatory elements within their promoter regions and in the correct orientation. These operons showed dramatically increased polarity in the ΔrfaH mutant compared to the wild type as determined by RNA sequencing. Amylovoran and lipopolysaccharide production in vitro was reduced in rfaH mutants compared to the wild type, which probably contributes to the rfaH mutant virulence phenotype. Furthermore, type VI secretion cluster 1, which contributes to E. amylovora virulence, showed reduced expression in ΔrfaH compared to the wild type, although without an increase in polarity. The data suggest that E. amylovora RfaH directly, specifically, and exclusively suppresses operon polarity in the amylovoran operon and a lipopolysaccharide operon.

Characterizations of an Emerging Disease: Apple Blotch Caused by Diplocarpon coronariae (syn. Marssonina coronaria) in the Mid-Atlantic United States.

Apple orchards with minimal or reduced fungicide inputs in the Mid-Atlantic region of the United States have experienced outbreaks of severe premature defoliation with symptoms that matched those of apple blotch disease (ABD) caused by Diplocarpon coronariae. Fungal isolates obtained from symptomatic apple leaves and fruit produced uniform slow-growing, dark-gray colonies on peptone potato dextrose agar and had conidia. Internal transcribed spacer DNA sequences matched with D. coronariae and Koch's postulates were fulfilled when typical ABD symptoms occurred when reinoculated onto apple leaves and fruit. Spore dispersal in nonfungicide-treated orchards detected with quantitative PCR was low in early spring and dropped to undetectable levels in late May and early June before rising exponentially to highs in July and August, which coincided with symptom development. Only low spore numbers were detected in fungicide-treated orchards and nearby forests. In preliminary fungicide tests, fluxapyroxad, thiophanate methyl, and difenoconazole effectively inhibited mycelial growth of isolates in vitro. When apple cultivars Fuji and Honeycrisp were inoculated with D. coronariae, Honeycrisp showed delayed onset of symptoms and lower disease severity, and the transcription profile of seven host defense-related genes showed that PR-2, PR-8, LYK4, and CERK1 were highly induced in Honeycrisp at 2 and 5 days postinoculation. This is the first report of ABD in the Mid-Atlantic United States, which includes studies of seasonal D. coronariae spore dispersal patterns, preliminary fungicide efficacy, and host defense-related gene expression to assist development of best ABD management practices.

Fungicide Sensitivity of Colletotrichum Species Causing Bitter Rot of Apple in the Mid-Atlantic U.S.A.

Apple growers in the Mid-Atlantic region of the U.S.A. have reported increased losses to bitter rot of apple. We tested the hypothesis that this increase is because the Colletotrichum population has developed resistance to commonly used single-mode-of-action (single-MoA) fungicides. We screened 220 Colletotrichum isolates obtained from 38 apple orchards in the Mid-Atlantic region for resistance to 11 fungicides in Fungicide Resistance Action Committee (FRAC) groups 1, 7, 9, 11, 12, and 29. Eleven (5%) of these isolates were resistant to FRAC group 1 with confirmed ß-tubulin E198A mutations, and two (<1%) were also resistant to FRAC group 11 with confirmed cytochrome-b G143A mutations. Such low frequencies of resistant isolates indicate that fungicide resistance is unlikely to be the cause of any regional increase in bitter rot. A subsample of isolates was subsequently tested in vitro for sensitivity to every single-MoA fungicide registered for apple in the Mid-Atlantic U.S.A. (22 fungicides; FRAC groups 1, 3, 7, 9, 11, 12, and 29), and 13 fungicides were tested in field trials. These fungicides varied widely in efficacy both within and between FRAC groups. Comparisons of results from our in vitro tests with results from our field trials and other field trials conducted across the eastern U.S.A. suggested that EC25 values (concentrations that reduce growth by 25%) are better predictors of fungicide efficacy in normal field conditions than EC50 values. We present these results as a guideline for choosing single-MoA fungicides for bitter rot control in the Mid-Atlantic U.S.A.

Prevalence and Distribution of Listeria monocytogenes in Three Commercial Tree Fruit Packinghouses.

A 2-year longitudinal study of three tree fruit packinghouses was conducted to determine the prevalence and distribution of Listeria monocytogenes. Samples were collected from 40 standardized non-food-contact surface locations six different times over two 11-month production seasons. Of the 1,437 samples collected, the overall prevalence of L. monocytogenes over the course of the study was 17.5%. Overall prevalence did not differ significantly (p > 0.05) between each year. However, values varied significantly (p ≤ 0.05) within each production season following packing activity levels; increasing in the fall, peaking in early winter, and then decreasing through spring. L. monocytogenes was most often found in the packing line areas, where moisture and fruit debris were commonly observed and less often in dry cold storage and packaging areas. Persistent contamination was attributed to the inability of water drainage systems to prevent moisture accumulation on floors and equipment during peak production times and uncontrolled employee and equipment traffic throughout the facility. This is the first multiyear longitudinal surveillance study to compare L. monocytogenes prevalence at standardized sample sites common to multiple tree fruit packinghouses. Recommendations based on our results will help packinghouse operators to identify critical areas for inclusion in their L. monocytogenes environmental monitoring programs.

Bitter Rot of Apple in the Mid-Atlantic United States: Causal Species and Evaluation of the Impacts of Regional Weather Patterns and Cultivar Susceptibility.

Apple growers in the Mid-Atlantic region of the United States have been reporting an increase in losses to bitter rot of apple and are requesting up-to-date management recommendations. Management is complicated by variations in apple cultivar susceptibility, temperature, rainfall, and biology of the Colletotrichum spp. that cause bitter rot. Over 500 apple fruit with bitter rot were obtained from 38 orchards across the Mid-Atlantic and the causal species were identified as Colletotrichum fioriniae and C. nymphaeae of the C. acutatum species complex and C. chrysophilum, C. noveboracense, C. siamense, C. fructicola, C. henanense, and C. gloeosporioides sensu stricto of the C. gloeosporioides species complex, the latter two being first reports. Species with faster in vitro growth rates at higher temperatures were more abundant in warmer regions of the Mid-Atlantic, while those with slower growth rates at higher temperatures were more abundant in cooler regions. Regional bloom dates are earlier and weather data show a gradual warming trend that likely influenced but was not necessarily the main cause of the recent increase in bitter rot in the region. A grower survey of apple cultivar susceptibility showed high variation, with the increase in acres planted to the highly susceptible cultivar Honeycrisp broadly corresponding to the increase in reports of bitter rot. These results form a basis for future studies on the biology and ecology of the Colletotrichum spp. responsible, and suggest that integrated bitter rot management must begin with selection of less-susceptible apple cultivars.

Genetic Diversity of Listeria monocytogenes Isolated From Three Commercial Tree Fruit Packinghouses and Evidence of Persistent and Transient Contamination.

Whole genome analysis was performed on 501 isolates obtained from a previous survey which recovered 139 positive environmental sponge samples (i.e., up to 4 isolates per sample) from a total of 719 samples collected at 40 standardized sites in 3 commercial apple packinghouse facilities (i.e., P1, P2, and P3) over 3 successive seasons in a single production year. After excluding duplicated isolates, the data from 156 isolates revealed the clonal diversity of L. monocytogenes and allowed the detection of transient contamination, persistent contamination, and cross-area transmission events. Facility P2 with the poorest sanitary conditions had the least diversity (Shannon's index of 0.38). P2 contained a Clonal Complex (CC) 554, serogroup IVb-v1 strain that persisted throughout the year and spread across the entire facility, a singleton Sequence Type (ST) 1003, lineage III strain that persisted through two seasons and spread across two areas of the facility, and 3 other clones from transient contaminations. P1 and P3, facilities with better sanitary conditions, had much higher diversity (i.e., 15 clones with a Shannon's index of 2.49 and 10 clones with a Shannon's index of 2.10, respectively) that were the result of transient contamination. Facilities P1 and P3 had the highest incidence (43.1%) of lineage III isolates, followed by lineage I (31.3%) and lineage II (25.5%) isolates. Only 1 isolate in the three facilities contained a premature stop codon in virulence gene inlA. Fourteen samples yielded 2-3 clones per sample, demonstrating the importance of choosing appropriate methodologies and selecting a sufficient number of isolates per sample for studying L. monocytogenes diversity. Only 1 isolate, belonging to CC5 and from facility P3, contained a known plasmid, and this was also the only isolate containing benzalkonium chloride tolerance genes. The persistent CC554 strain did not exhibit stronger sanitizer resistance than other isolates and did not contain any confirmed molecular determinants of L. monocytogenes stress resistance that were differentially present in other isolates, such as genes involved in sanitizer tolerance, heavy metal resistance, biofilm-forming, stress survival islet 1 (SSI-1), stress survival islet 2 (SSI-2) or Listeria genomic island (LGI2).

Quantification of Colletotrichum fioriniae in Orchards and Deciduous Forests Indicates It Is Primarily a Leaf Endophyte.

Colletotrichum fioriniae of the C. acutatum species complex is an important hemibiotrophic pathogen of vegetables and fruits in temperate regions worldwide. In apple, it is one of the primary species responsible for bitter rot disease. Understanding the disease cycle is complicated because many broadleaf plants can be hosts of C. fioriniae. By detecting and quantifying rain-splashed C. acutatum species complex conidia in more than 500 samples from heavily bitter-rot-infected apple orchards and nearby forested woodlots over two summers, we show that conidial quantities were higher in the woodlots than in the orchards. Testing of more than 1,000 surface-disinfected leaves of apple and 24 different forest plant species showed that overall C. fioriniae was an abundant leaf endophyte, with high variation in leaf colonization area. Endophytic isolates from leaves were pathogenic on apples, and multilocus sequence analysis showed 100% identity between most isolates from leaves and diseased fruits. Apple leaves endophytically infected with C. fioriniae were present in a conventionally managed orchard and abundant in an untreated orchard. These lines of evidence, in the context of previously published research, lead us to hypothesize that the main ecological role of C. fioriniae is that of a leaf endophyte, which we present as a generalized C. fioriniae infection cycle that provides an updated framework for its integrated management in agricultural systems.

Author Correction: Identification and characterization of Colletotrichum species causing apple bitter rot in New York and description of C. noveboracense sp. nov.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Virulence Genetics of an Erwinia amylovora Putative Polysaccharide Transporter Family Member.

The Gram-negative enterobacterium Erwinia amylovora causes fire blight disease in apple and pear trees. Lipopolysaccharides and the exopolysaccharide amylovoran are essential E. amylovora virulence factors. We found that mutations in rfbX disrupted amylovoran production and virulence in apple fruits and tree shoots and that the deletion of yibD suppressed the rfbX mutant phenotype. The level of expression of yibD was about 10-fold higher in the ΔrfbX mutant than the wild type. A forward genetic suppressor screen in the ΔrfbX mutant uncovered multiple mutations in yibD and supported the conclusion that the virulence defect of rfbX mutants is due to reduced amylovoran production. The yibD and rfbX genes are expressed as a two-gene operon, yibD rfbX The rfbX gene encodes a previously uncharacterized putative polysaccharide subunit transporter, while yibD encodes a predicted glycosyltransferase. Mutation of rfbX did not have a detectable effect on lipopolysaccharide patterns; however, the overexpression of yibD in both the wild-type and ΔyibD ΔrfbX genetic backgrounds disrupted both amylovoran and lipopolysaccharide production. Additionally, the overexpression of yibD in the ΔyibD ΔrfbX mutant inhibited bacterial growth in amylovoran-inducing medium. This growth inhibition phenotype was used in a forward genetic suppressor screen and reverse-genetics tests to identify several genes involved in lipopolysaccharide production, which, when mutated, restored the ability of the ΔyibD ΔrfbX mutant overexpressing yibD to grow in amylovoran-inducing medium. Remarkably, all the lipopolysaccharide gene mutants tested were defective in lipopolysaccharide and amylovoran production. These results reveal a genetic connection between amylovoran and lipopolysaccharide production in E. amylovoraIMPORTANCE This study discovered previously unknown genetic connections between exopolysaccharide and lipopolysaccharide production in the fire blight pathogen Erwinia amylovora This represents a step forward in our understanding of the biology underlying the production of these two macromolecules. Fire blight is an economically important disease that impacts the production of apples and pears worldwide. Few fire blight control measures are available, and growers rely heavily on antibiotic applications at bloom time. Both exopolysaccharide and lipopolysaccharide are E. amylovora virulence factors. Our results indicate that the overexpression of the yibD gene in E. amylovora disrupts both lipopolysaccharide production and exopolysaccharide production. This effect could potentially be used as the basis for the development of an antivirulence treatment for the prevention of fire blight disease.

Identification and characterization of Colletotrichum species causing apple bitter rot in New York and description of C. noveboracense sp. nov.

Apple bitter rot caused by Colletotrichum species is a growing problem worldwide. Colletotrichum spp. are economically important but taxonomically un-resolved. Identification of Colletotrichum spp. is critical due to potential species-level differences in pathogenicity-related characteristics. A 400-isolate collection from New York apple orchards were morphologically assorted to two groups, C. acutatum species complex (CASC) and C. gloeosporioides species complex (CGSC). A sub-sample of 44 representative isolates, spanning the geographical distribution and apple varieties, were assigned to species based on multi-locus phylogenetic analyses of nrITS, GAPDH and TUB2 for CASC, and ITS, GAPDH, CAL, ACT, TUB2, APN2, ApMat and GS genes for CGSC. The dominant species was C. fioriniae, followed by C. chrysophilum and a novel species, C. noveboracense, described in this study. This study represents the first report of C. chrysophilum and C. noveboracense as pathogens of apple. We assessed the enzyme activity and fungicide sensitivity for isolates identified in New York. All isolates showed amylolytic, cellulolytic and lipolytic, but not proteolytic activity. C. chrysophilum showed the highest cellulase and the lowest lipase activity, while C. noveboracense had the highest amylase activity. Fungicide assays showed that C. fioriniae was sensitive to benzovindiflupyr and thiabendazole, while C. chrysophilum and C. noveboracense were sensitive to fludioxonil, pyraclostrobin and difenoconazole. All species were pathogenic on apple fruit with varying lesion sizes. Our findings of differing pathogenicity-related characteristics among the three species demonstrate the importance of accurate species identification for any downstream investigations of Colletotrichum spp. in major apple growing regions.

Blistering1 Modulates Penicillium expansum Virulence Via Vesicle-mediated Protein Secretion.

The blue mold fungus, Penicillium expansum, is a postharvest apple pathogen that contributes to food waste by rotting fruit and by producing harmful mycotoxins (e.g. patulin). To identify genes controlling pathogen virulence, a random T-DNA insertional library was created from wild-type P. expansum strain R19. One transformant, T625, had reduced virulence in apples, blistered mycelial hyphae, and a T-DNA insertion that abolished transcription of the single copy locus in which it was inserted. The gene, Blistering1, encodes a protein with a DnaJ domain, but otherwise has little homology outside the Aspergillaceae, a family of fungi known for producing antibiotics, mycotoxins, and cheese. Because protein secretion is critical for these processes and for host infection, mass spectrometry was used to monitor proteins secreted into liquid media during fungal growth. T625 failed to secrete a set of enzymes that degrade plant cell walls, along with ones that synthesize the three final biosynthetic steps of patulin. Consequently, the culture broth of T625 had significantly reduced capacity to degrade apple tissue and contained 30 times less patulin. Quantitative mass spectrometry of 3,282 mycelial proteins revealed that T625 had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. T625 also had reduced proteins controlling mRNA surveillance and RNA processing. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles. These data reveal that Blistering1 affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance.

Erwinia amylovora Auxotrophic Mutant Exometabolomics and Virulence on Apples.

The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic E. amylovora mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type E. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents.IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic E. amylovora auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

Extragenic Suppression of Elongation Factor P Gene Mutant Phenotypes in Erwinia amylovora.

Elongation factor P (EF-P) facilitates the translation of certain peptide motifs, including those with multiple proline residues. EF-P must be posttranslationally modified for full functionality; in enterobacteria, this is accomplished by two enzymes, namely, EpmA and EpmB, which catalyze the ß-lysylation of EF-P at a conserved lysine position. Mutations to efp or its modifying enzymes produce pleiotropic phenotypes, including decreases in virulence, swimming motility, and extracellular polysaccharide production, as well as proteomic perturbations. Here, we generated targeted deletion mutants of the efp, epmA, and epmB genes in the Gram-negative bacterium Erwinia amylovora, which causes fire blight, an economically important disease of apples and pears. As expected, the Δefp, ΔepmA, and ΔepmB mutants were all defective in virulence on apples, and all three mutants were complemented in trans with plasmids bearing wild-type copies of the corresponding genes. By analyzing spontaneous suppressor mutants, we found that mutations in the hrpA3 gene partially or completely suppressed the colony size, extracellular polysaccharide production, and virulence phenotypes in apple fruits and apple tree shoots but not the swimming motility phenotypes of the Δefp, ΔepmA, and ΔepmB mutants. The deletion of hrpA3 alone did not produce any alterations in any characteristics measured, indicating that the HrpA3 protein is not essential for any of the processes examined. The hrpA3 gene encodes a putative DEAH-box ATP-dependent RNA helicase. These results suggest that the loss of the HrpA3 protein at least partially compensates for the lack of the EF-P protein or ß-lysylated EF-P.IMPORTANCE Fire blight disease has relatively few management options, with antibiotic application at bloom time being chief among them. As modification to elongation factor P (EF-P) is vital to virulence in several species, both EF-P and its modifying enzymes make attractive targets for novel antibiotics. However, it will be useful to understand how bacteria might overcome the hindrance of EF-P function so that we may be better prepared to anticipate bacterial adaptation to such antibiotics. The present study indicates that the mutation of hrpA3 could provide a partial offset for the loss of EF-P activity. In addition, little is known about EF-P functional interactions or the HrpA3 predicted RNA helicase, and our genetic approach allowed us to discern a novel gene associated with EF-P function.

Whole-genome comparisons of Penicillium spp. reveals secondary metabolic gene clusters and candidate genes associated with fungal aggressiveness during apple fruit decay.

Blue mold is a postharvest rot of pomaceous fruits caused by Penicillium expansum and a number of other Penicillium species. The genome of the highly aggressive P. expansum strain R19 was re-sequenced and analyzed together with the genome of the less aggressive P. solitum strain RS1. Whole genome scale similarities and differences were examined. A phylogenetic analysis of P. expansum, P. solitum, and several closely related Penicillium species revealed that the two pathogens isolated from decayed apple with blue mold symptoms are not each other's closest relatives. Among a total of 10,560 and 10,672 protein coding sequences respectively, a comparative genomics analysis revealed 41 genes in P. expansum R19 and 43 genes in P. solitum RS1 that are unique to these two species. These genes may be associated with pome fruit-fungal interactions, subsequent decay processes, and mycotoxin accumulation. An intact patulin gene cluster consisting of 15 biosynthetic genes was identified in the patulin producing P. expansum strain R19, while only a remnant, seven-gene cluster was identified in the patulin-deficient P. solitum strain. However, P. solitum contained a large number of additional secondary metabolite gene clusters, indicating that this species has the potential capacity to produce an array of known as well as not-yet-identified products of possible toxicological or biotechnological interest.