Appalachian Fruit Research Laboratory

Losses from postharvest fruit diseases range from 1 to 20 percent in the United States, depending on the commodity. The application of fungicides to fruits after harvest to reduce decay has been increasingly curtailed by the development of pathogen resistance to many key fungicides, the lack of replacement fungicides, negative public perception regarding the safety of pesticides and consequent restrictions on fungicide use. Biological control of postharvest diseases (BCPD) has emerged as an effective alternative. Because wound-invading necrotrophic pathogens are vulnerable to biocontrol, antagonists can be applied directly to the targeted area (fruit wounds), and a single application using existing delivery systems (drenches, line sprayers, on-line dips) can significantly reduce fruit decays. The pioneering biocontrol products BioSave and Aspire were registered by EPA in 1995 for control of postharvest rots of pome and citrus fruit, respectively, and are commercially available. The limitations of these biocontrol products can be addressed by enhancing biocontrol through manipulation of the environment, using mixtures of beneficial organisms, physiological and genetic enhancement of the biocontrol mechanisms, manipulation of formulations, and integration of biocontrol with other alternative methods that alone do not provide adequate protection but in combination with biocontrol provide additive or synergistic effects.

BACKGROUND: The necrogenic enterobacterium, Erwinia amylovora is the causal agent of the fire blight (FB) disease in many Rosaceae species, including apple and pear. During the infection process, the bacteria induce an oxidative stress response with kinetics similar to those induced in an incompatible bacteria-plant interaction. No resistance mechanism to E. amylovora in host plants has yet been characterized, recent work has identified some molecular events which occur in resistant and/or susceptible host interaction with E. amylovora: In order to understand the mechanisms that characterize responses to FB, differentially expressed genes were identified by cDNA-AFLP analysis in resistant and susceptible apple genotypes after inoculation with E. amylovora. RESULTS: cDNA were isolated from M.26 (susceptible) and G.41 (resistant) apple tissues collected 2 h and 48 h after challenge with a virulent E. amylovora strain or mock (buffer) inoculated. To identify differentially expressed transcripts, electrophoretic banding patterns were obtained from cDNAs. In the AFLP experiments, M.26 and G.41 showed different patterns of expression, including genes specifically induced, not induced, or repressed by E. amylovora. In total, 190 ESTs differentially expressed between M.26 and G.41 were identified using 42 pairs of AFLP primers. cDNA-AFLP analysis of global EST expression in a resistant and a susceptible apple genotype identified different major classes of genes. EST sequencing data showed that genes linked to resistance, encoding proteins involved in recognition, signaling, defense and apoptosis, were modulated by E. amylovora in its host plant. The expression time course of some of these ESTs selected via a bioinformatic analysis has been characterized. CONCLUSION: These data are being used to develop hypotheses of resistance or susceptibility mechanisms in Malus to E. amylovora and provide an initial categorization of genes possibly involved in recognition events, early signaling responses the subsequent development of resistance or susceptibility. These data also provided potential candidates for improving apple resistance to fire blight either by marker-assisted selection or genetic engineering.

A rapid assay to determine antifungal activity in plant extracts and essential oils is described. Wells in microtiter plates were loaded with Botrytis cinerea spores and plant extracts or essential oils. Subsequent changes in optical density following spore germination in the wells was measured after 24 h using an automatic microtiter plate reader driven by a software program developed for this purpose. Extracts from 345 plants and 49 essential oils were evaluated for their antifungal activity against B. cinerea. Among 345 plant extracts analyzed, 13 showed high levels of antifungal activity, with species of Allium and Capsicum predominating. Among the 49 essential oils tested, palmarosa (Cymbopogon martini), red thyme (Thymus zygis), cinnamon leaf (Cinnamomum zeylanicum), and clove buds (Eugenia caryophyllata) demonstrated the most antifungal activity against B. cinerea. The most frequently occurring constituents in essential oils showing high antifungal activity were: D-limonene, cineole; β-myrcene; α-pinene, β-pinene; and camphor.

The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10 degrees C but increased significantly on fresh-cut honeydew melons stored at 10 degrees C over 7 days. In addition, we examined the effect of lytic, L. monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L. monocytogenes populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced L. monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced L. monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of L. monocytogenes.

The brown marmorated stink bug (BMSB), Halyomorpha halys (Stål), is an invasive pentatomid introduced from Asia into the United States, Canada, multiple European countries, and Chile. In 2010, BMSB populations in the mid-Atlantic United States reached outbreak levels and subsequent feeding severely damaged tree fruit as well as other crops. Significant nuisance issues from adults overwintering inside homes were common. BMSB is a highly polyphagous species with a strong dispersal capacity and high reproductive output, potentially enabling its spread and success in invaded regions. A greater understanding of BMSB biology and ecology and its natural enemies, the identification of the male-produced aggregation pheromone, and the recognition that BMSB disperses into crops from adjacent wooded habitats have led to the development of behavior-based integrated pest management (IPM) tactics. Much is still unknown about BMSB, and continued long-term collaborative studies are necessary to refine crop-specific IPM programs and enhance biological control across invaded landscapes.

Native to China, Japan, Korea, and Taiwan, the brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) was first detected in the United States in the mid-1990s. Since establishing in the United States, this invasive species has caused significant economic losses in agriculture and created major nuisance problems for home and business owners, especially in the mid-Atlantic region. Basic and applied questions on H. halys have been addressed in its native range in Asia since the mid-1900s and the research outcomes have been published in at least 216 articles from China, Japan, and the Republic of Korea. In Asia, H. halys is described as an occasional or outbreak pest of a number of crops such as apple, pear, persimmon, and soybeans. This species is considered a nuisance pest as well, particularly in Japan. This review summarizes 100 articles primarily translated from Chinese, Japanese, and Korean to English. The content of this review focuses on the biology, ecology, and management of H. halys in Asia, with specific emphasis on nomenclature, life history, host range, damage, economic importance, sampling and monitoring tools, and management strategies. This information from the native range of H. halys provides greater context and understanding of its biology, ecology, and management in North America.

BACKGROUND: MicroRNAs (miRNAs) and their regulatory functions have been extensively characterized in model species but whether apple has evolved similar or unique regulatory features remains unknown. RESULTS: We performed deep small RNA-seq and identified 23 conserved, 10 less-conserved and 42 apple-specific miRNAs or families with distinct expression patterns. The identified miRNAs target 118 genes representing a wide range of enzymatic and regulatory activities. Apple also conserves two TAS gene families with similar but unique trans-acting small interfering RNA (tasiRNA) biogenesis profiles and target specificities. Importantly, we found that miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. These miRNA target sites are differentially conserved among MYBs, which is largely influenced by the location and conservation of the encoded amino acid residues in MYB factors. Finally, we found that 10 of the 19 miR828-targeted MYBs undergo small interfering RNA (siRNA) biogenesis at the 3' cleaved, highly divergent transcript regions, generating over 100 sequence-distinct siRNAs that potentially target over 70 diverse genes as confirmed by degradome analysis. CONCLUSIONS: Our work identified and characterized apple miRNAs, their expression patterns, targets and regulatory functions. We also discovered that three miRNAs and the ensuing siRNAs exploit both conserved and divergent sequence features of MYB genes to initiate distinct regulatory networks targeting a multitude of genes inside and outside the MYB family.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

Laboratory and greenhouse experiments were conducted to determine the herbicidal effect of plant-derived oils and to identify the active ingredient in an oil with herbicide activity. Twenty-five different oils were applied to detached leaves of dandelion in the laboratory. Essential oils (1%, v/v) from red thyme, summer savory, cinnamon, and clove were the most phytotoxic and caused electrolyte leakage resulting in cell death. Each of these essential oils in aqueous concentrations from 5 to 10% (v/v) plus two adjuvants (nonionic surfactant and paraffinic oil blend at 0.2% [v/v]) were applied to shoots of common lambsquarters, common ragweed, and johnsongrass in the greenhouse; shoot death occurred within 1 h to 1 d after application. Essential oil of cinnamon had high herbicidal activity, and eugenol (2-methoxy-4-[2-propenyl]phenol) was determined to be this oil's major component (84%, v/v). Dandelion leaf disk and whole-plant assays verified that eugenol was the active ingredient in the essential oil of cinnamon. Essential oils are extracted from plants and thus may be useful as “natural product herbicides” for organic farming systems.

Although it is difficult to determine the full extent of postharvest losses due to diseases, conservative estimates place losses to fruits and vegetables from spoilage at 24% of the harvested crop in the United States (U.S. Dept. of Agriculture, 1965) and 50% in underdeveloped, tropical countries

Plants and animals mediate early steps of the innate immune response through pathogen recognition receptors (PRRs). PRRs commonly associate with or contain members of a monophyletic group of kinases called the interleukin-1 receptor-associated kinase (IRAK) family that include Drosophila Pelle, human IRAKs, rice XA21 and Arabidopsis FLS2. In mammals, PRRs can also associate with members of the receptor-interacting protein (RIP) kinase family, distant relatives to the IRAK family. Some IRAK and RIP family kinases fall into a small functional class of kinases termed non-RD, many of which do not autophosphorylate the activation loop. We surveyed the yeast, fly, worm, human, Arabidopsis, and rice kinomes (3,723 kinases) and found that despite the small number of non-RD kinases in these genomes (9%-29%), 12 of 15 kinases known or predicted to function in PRR signaling fall into the non-RD class. These data indicate that kinases associated with PRRs can largely be predicted by the lack of a single conserved residue and reveal new potential plant PRR subfamilies.

Explaining the mechanisms underlying patterns of species diversity and composition in riverine networks is challenging. Historically, community ecologists have conceived of communities as largely isolated entities and have focused on local environmental factors and interspecific interactions as the major forces determining species composition. However, stream ecologists have long embraced a multiscale approach to studying riverine ecosystems and have studied both local factors and larger-scale regional factors, such as dispersal and disturbance. River networks exhibit a dendritic spatial structure that can constrain aquatic organisms when their dispersal is influenced by or confined to the river network. We contend that the principles of metacommunity theory would help stream ecologists to understand how the complex spatial structure of river networks mediates the relative influences of local and regional control on species composition. From a basic ecological perspective, the concept is attractive because new evidence suggests that the importance of regional processes (dispersal) depends on spatial structure of habitat and on connection to the regional species pool. The role of local factors relative to regional factors will vary with spatial position in a river network. From an applied perspective, the long-standing view in ecology that local community composition is an indicator of habitat quality may not be uniformly applicable across a river network, but the strength of such bioassessment approaches probably will depend on spatial position in the network. The principles of metacommunity theory are broadly applicable across taxa and systems but seem of particular consequence to stream ecology given the unique spatial structure of riverine systems. By explicitly embracing processes at multiple spatial scales, metacommunity theory provides a foundation on which to build a richer understanding of stream communities.

Developing peach (Prunus persica L. Batsch ;Redskin') fruit were used to characterize the role of peroxidases in lignification. During development, the endocarp of these drupes becomes lignified while the mesocarp remains parenchymatous. Acidic peroxidase from lignifying endocarp were similar to those of the fleshy mesocarp. The endocarp had a larger amount and number of basic peroxidases than the mesocarp. Cultured peach leaf cells are thought to be lignified because their walls give a positive reaction with phloroglucinol-HCI. These cells also secreted a basic peroxidase. Peroxidases were difficult to extract from endocarp tissue as they lignified. This was also demonstrated by tissue printing on nitrocellulose. Flesh, but not endocarp peroxidase was evident in tissue prints. This suggests that tissue printing may fail to reveal the presence of enzymes which are firmly attached to the cell.

Objective: Interest in protective factors for adversity has burgeoned, but the set of examined protective factors remains limited and most studies have focused on a single or narrow set of adversities. Using the resilience portfolio model as a conceptual framework, this study seeks to identify promising protective factors for individuals exposed to violence and other adversities. We include strengths drawn from the positive psychology literature in addition to established protective factors. We also explore the utility of the concept of poly-strengths, or the number of different types of protective factors an individual has. Method: Participants were 2,565 adolescents and adults from a rural, low-income community in southern Appalachia (64% female). Three kinds of adversity were assessed (victimization, stressful life events, financial strain) along with 23 protective factors representing 3 broader domains that are the focus of the resilience portfolio model: self-regulation, interpersonal strengths, and meaning-making. Results: The combination of strengths and adversities accounted for 42% of the variance in trauma symptoms, 50% of the variance in posttraumatic growth, and 58% of the variance in subjective well-being. Strengths associated with thriving included purpose, optimism, religious involvement, emotional regulation, emotional awareness, psychological endurance, compassion, generativity, and community support. Poly-strengths was uniquely associated with well-being after controlling for other protective factors. Conclusions: Expanding the range of studied protective factors and considering poly-strengths hold considerable promise to better understand resilience. A more strengths-based approach to prevention and intervention could improve outcomes in individuals who have experienced adversity.

Four commercial orchards in the mid-Atlantic region of the United States were surveyed weekly in 2010 and 2011 for the presence of brown marmorated stink bug and the injury caused to both apple and peaches. Among tested sampling techniques, pyramid traps baited with the aggregation pheromone of Plautia stali Scott, methyl-(2 E ,4 E ,6 Z )-decatrienoate, yielded the most brown marmorated stink bug adults and nymphs, followed by visual observations. Brown marmorated stink bugs began to feed on apples and peaches soon after fruit set and continued to feed on fruit throughout the growing season. Injury to apple was relatively inconsequential until after mid-June, whereas feeding on peaches resulted in immediate economic injury as the surface became distorted, dented, discolored, and the flesh beneath turned brown. Significantly more apples were injured and with greater severity in 2010 than in 2011. Likewise, percent injury on the exterior portion of each apple plot was significantly greater than injury reported from the interior in both years. Growers increased the number of insecticide applications nearly 4-fold from 2010 to 2011. In addition to the increased number of targeted insecticide applications, growers also reduced the interval between treatments in 2011. A metric was created to compare the relative intensity of each grower's commercial management program between seasons and amongst each other.

Seasonal patterns of proteins and of cold hardiness were characterized in bark and xylem tissues of genetically related (sibling) deciduous and evergreen peach (Prunus persica [L.] Batsch). In contrast with deciduous trees, which entered endodormancy and abscised leaves in the fall, evergreen trees retained their leaves and exhibited shoot elongation under favorable environmental conditions. A successive increase in the cold hardiness of bark and xylem was observed during the fall in both genotypes. This was followed by a subsequent decrease from midwinter to spring. Xylem tissue in both genotypes exhibited deep supercooling and a significant correlation (r = 0.99) between the midpoint of the low-temperature exotherm and the subzero temperature at which 50% injury occurred (assessed by electrolyte leakage) was noted. The maximum hardiness level attained in deciduous trees was more than twofold that of evergreens. Seasonal pattern of proteins from bark and xylem of the sibling genotypes was characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Among other qualitative and quantitative changes, accumulation of a 19-kilodalton polypeptide in the bark of both genotypes was observed during fall followed by a decrease in spring. This polypeptide accumulated to higher levels in the deciduous peach compared with the evergreen. Additionally, a 16-kilodalton protein exhibited the same pattern in deciduous trees but not in the evergreen trees. Both the 19- and a 16-kilodalton bark proteins conform to the criteria of a bark storage protein. The relationship of seasonal changes in protein to cold hardiness and dormancy in these genetically related peach genotypes is discussed.

Perception of extracellular signals by cell surface receptors is of central importance to eukaryotic development and immunity. Kinases that are associated with the receptors or are part of the receptors themselves modulate signaling through phosphorylation events. The rice (Oryza sativa L.) XA21 receptor kinase is a key recognition and signaling determinant in the innate immune response. A yeast two-hybrid screen using the intracellular portion of XA21, including the juxtamembrane (JM) and kinase domain as bait, identified a protein phosphatase 2C (PP2C), called XA21 binding protein 15 (XB15). The interaction of XA21 and XB15 was confirmed in vitro and in vivo by glutathione-S-transferase (GST) pull-down and co-immunoprecipitation assays, respectively. XB15 fusion proteins purified from Escherichia coli and from transgenic rice carry PP2C activity. Autophosphorylated XA21 can be dephosphorylated by XB15 in a temporal- and dosage-dependent manner. A serine residue in the XA21 JM domain is required for XB15 binding. Xb15 mutants display a severe cell death phenotype, induction of pathogenesis-related genes, and enhanced XA21-mediated resistance. Overexpression of Xb15 in an XA21 rice line compromises resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae. These results demonstrate that Xb15 encodes a PP2C that negatively regulates the XA21-mediated innate immune response.

The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples. Li et al. ( MPMI-12-14-0398-FI ) reported similar results and conclusions in their recently accepted paper.

Pathogenic Escherichia coli O157:H7, as well as nonpathogenic strains ATCC 11775 and ATCC 23716, grew exponentially in wounds on Golden Delicious apple fruit. The exponential growth occurred over a longer time period on fruit inoculated with a lower concentration of the bacterium than on fruit inoculated with a higher concentration. The bacterium reached the maximum population supported in the wounds regardless of the initial inoculum concentrations. Populations of E. coli O157:H7 in various concentrations of sterilized apple juice and unsterilized cider declined over time and declined more quickly in diluted juice and cider. The decline was greater in the unsterilized cider than in juice, which may have resulted from the interaction of E. coli O157:H7 with natural populations of yeasts that increased with time. Experiments on the transmission of E. coli by fruit flies, collected from a compost pile of decaying apples and peaches, were conducted with strain F-11775, a fluorescent transformant of nonpathogenic E. coli ATCC 11775. Fruit flies were easily contaminated externally and internally with E. coli F-11775 after contact with the bacterium source. The flies transmitted this bacterium to uncontaminated apple wounds, resulting in a high incidence of contaminated wounds. Populations of the bacterium in apple wounds increased significantly during the first 48 h after transmission. Further studies under commercial conditions are necessary to confirm these findings.

Particle film technology is a new tool for tree fruit production systems. Trials were performed in Santiago, Chile, and Washington and West Virginia to evaluate the effect of particle film treatments on apple [ Malus sylvestris (L.) Mill var domestica (Borkh.) Mansf.] fruit temperature and the incidence of solar injury. Fruit surface temperature was reduced by the application of reflective particles and the amount of temperature reduction was proportional to the amount of particle residue on the fruit surface. Effective solar injury suppression was achieved with spray applications of 45 to 56 kg·ha -1 of a reflective, processed-kaolin particle film material in concentrations ranging from 3% to 12% in some of the locations. The timing of application to suppress solar injury was not clearly defined. The processed-kaolin particle film material was highly reflective to the ultraviolet wavelengths and this characteristic may be important in reducing solar injury to both fruit and leaves.