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Enhancement of Biocontrol of Blue Mold with the Nutrient Analog 2-Deoxy-d-Glucose on Apples and Pears.

The glucose analog, 2-deoxy-d-glucose (2-DOG) enhanced biocontrol of blue mold (caused by Penicillium expansum), the most important postharvest disease of apples and pears. 2-DOG strongly inhibited P. expansum, and of the two antagonists investigated, Pseudomonas syringae was resistant to 2-DOG at 10 mg/ml whereas Sporobolomyces roseus was susceptible. A mutant of S. roseus resistant to 2-DOG was developed. Addition of 2-DOG at 4 mg/ml allowed more than a 10-fold reduction in the concentration of the antagonists applied to fruits without diminishing control. This effect was still apparent at an inoculum of 5 x 10 conidia of P. expansum per ml, which greatly exceeds concentrations found under normal commercial conditions. The effect of 2-DOG on biocontrol was stronger on apples than on pears. 2-DOG had no effect on populations of the antagonists, which increased greatly in wounds on apples and pears. The reduction in the concentration of the antagonists needed for control may improve the reliability and economy of the biocontrol system and may have a significant impact on commercialization of this new technology.

Journal Article↗

Inactivation of Escherichia coli O157:H7 in apple juice by irradiation.

Three strains (932, Ent-C9490, and SEA13B88) of Escherichia coli O157:H7 were used to determine the effectiveness of low-dose gamma irradiation for eliminating E. coli O157:H7 from apple juice or cider and to characterize the effect of inducing pH-dependent, stationary-phase acid resistance on radiation resistance. The strains were grown in tryptic soy broth with or without 1% dextrose for 18 h to produce cells that were or were not induced to pH-dependent stationary-phase acid resistance. The bacteria were then transferred to clarified apple juice and irradiated at 2 degrees C with a cesium-137 irradiator. Non-acid-adapted cells had radiation D values (radiation doses needed to decrease a microbial population by 90%) ranging from 0.12 to 0.21 kGy. D values increased to 0.22 to 0.31 kGy for acid-adapted cells. When acid-adapted SEA13B88 cells were tested in five apple juice brands having different levels of suspended solids (absorbances ranging from 0.04 to 2.01 at 550 nm), radiation resistance increased with increasing levels of suspended solids, with D values ranging from 0.26 to 0.35 kGy. Based on these results, a dose of 1.8 kGy should be sufficient to achieve the 5D inactivation of E. coli recommended by the National Advisory Committee for Microbiological Criteria for Foods.

Beverages↗

Combinations of intervention treatments resulting in 5-log10-unit reductions in numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 organisms in apple cider.

The U.S. Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log10 units. This study describes combinations of intervention treatments that reduced concentrations of mixtures of Escherichia coli O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or Salmonella typhimurium DT104 (DT104b, U302, and DT104) by 5 log10 units in apple cider with a pH of 3.3, 3.7, and 4.1. Treatments used were short-term storage at 4, 25, or 35 degrees C and/or freeze-thawing (48 h at -20 degrees C; 4 h at 4 degrees C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid [SA], or propionic acid). Treatments more severe than those for S. typhimurium DT104 were always required to destroy E. coli O157:H7. In pH 3.3 apple cider, a 5-log10-unit reduction in E. coli O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35 degrees C treatments. In pH 3.7 cider the 5-log10-unit reduction followed freeze-thawing combined with either 6 h at 4 degrees C, 2 h at 25 degrees C, or 1 h at 35 degrees C or 6 h at 35 degrees C alone. A 5-log10-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35 degrees C plus freeze-thawing, SA plus 12 h at 25 degrees C plus freeze-thawing, SA plus 6 h at 35 degrees C, and SA plus 4 h at 35 degrees C plus freeze-thawing. Yeast and mold counts did not increase significantly (P < 0.05) during the 6-h storage at 35 degrees C. Cider with no added organic acids treated with either 6 h at 35 degrees C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (P < 0.05). The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement.

Acids↗

Assessment of the environmental fate of the biological control agent of fire blight, Pseudomonas fluorescens EPS62e, on apple by culture and real-time PCR methods.

The colonization of apple blossoms and leaves by Pseudomonas fluorescens EPS62e was monitored in greenhouse and field trials using cultivable cell counting and real-time PCR. The real-time PCR provided a specific quantitative method for the detection of strain EPS62e. The detection level was around 10(2) cells g (fresh weight)(-1) and the standard curve was linear within a 5-log range. EPS62e actively colonized flowers reaching values from 10(7) to 10(8) cells per blossom. In apple flowers, no significant differences were observed between population levels obtained by real-time PCR and plating, suggesting that viable but nonculturable (VBNC) cells and residual nondegraded DNA were not present. In contrast, on apple leaves, where cultivable populations of EPS62e decreased with time, significant differences were observed between real-time PCR and plating. These differences indicate the presence of VBNC cells or nondegraded DNA after cell death. Therefore, the EPS62e population was under optimal conditions during the colonization of flowers but it was stressed and poorly survived on leaves. It was concluded that for monitoring this biological control agent, the combined use of cultivable cell count and real-time PCR is necessary.

Colony Count, Microbial↗

Germination and adhesion of fungal conidia on polycarbonate membranes and on apple fruit exposed to mycoactive acetate esters.

The adhesion and germination of conidia of nine fungal species were assessed on polycarbonate membranes or on the skin of apple fruit in sealed glass bottles injected or not injected with acetate esters. Adhesion was determined after dislodging conidia from surfaces using a sonication probe. Adhesion and germination of conidia of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Penicillium citrinum, Penicillium claviforme, or Trichoderma sp. on membranes after 48 h were not increased in a 1.84 microg mL(-1) headspace of butyl acetate (BA), ethyl acetate, hexyl acetate, 2-methylbutyl acetate, pentyl acetate, or propyl acetate. Adhesion and germination of Botrytis cinerea, Penicillium expansum, and Penicillium roquefortii conidia were stimulated by all esters. Only conidia of B. cinerea and P. expansum exhibited increased adhesion and germination on the skin of apple fruit in bottles exposed to 0.92 microg mL(-1) of BA. Only conidia of B. cinerea and P. expansum produced decay in inoculated puncture wounds on fruit. Freshly made puncture wounds or 24-h-old puncture wounds in fruit were more adhesive than the unpunctured skin of fruit to conidia of B. cinerea or P. expansum. Fresh wounds were more adhesive to both fungi than 24-h-old puncture wounds. The skin and wounds of fruit were as adhesive to B. cinerea conidia as they were to P. expansum conidia. A 4-h exposure to 1.43 microg mL(-1) of BA increased adhesion of B. cinerea and P. expansum conidia in 24-h-old wounds. Results suggest that acetate-ester stimulation most likely is not a rare phenomenon in the fungi. For nutrient-dependent decay pathogens of apple fruit, acetate esters may be an alternative chemical cue used to maintain adhesion of conidia to wound surfaces.

Acetates↗

Biological control of apple blue mold with Pseudomonas fluorescens.

Pseudomonas fluorescens isolate 1100-6 was evaluated as a potential biological control agent for apple blue mold caused by Penicillium expansum or Penicillium solitum. Both the wild-type isolate 1100-6 and a genetically modified derivative labeled with the gene encoding the green fluorescent protein (GFP) were compared. The P. fluorescens isolates with or without GFP equally reduced the growth of Penicillium spp. and produced large zones of inhibition in dual culture plate assays. Cell-free metabolites produced by the bacterial antagonists reduced the colony area of Penicillium isolates by 17.3% to 78.5%. The effect of iron chelate on the antagonistic potential of P. fluorescens was also studied. The use of iron chelate did not have a major effect on the antagonistic activity of P. fluorescens. With or without GFP, P. fluorescens significantly reduced the severity and incidence of apple decay by 2 P. expansum isolates after 11 d at 20 degrees C and by P. expansum and P. solitum after 25 d at 5 degrees C when the biocontrol agents were applied in wounds 24 or 48 h before challenging with Penicillium spp. Populations of P. fluorescens labeled with the GFP were determined 1, 9, 14, and 20 d after inoculation at 5 degrees C. The log CFU/mL per wound increased from 6.95 at the time of inoculation to 9.12 CFU/mL (P < 0.05) 25 d after inoculation at 5 degrees C. The GFP strain did not appear to penetrate deeply into wounds based on digital photographs taken with an inverted fluorescence microscope. These results indicate that P. fluorescens isolate 1100-6 could be an important new biological control for apple blue mold.

Green Fluorescent Proteins↗

Purification and characterization of the major allergen from apple and its allergenic cross-reactivity with Bet v 1.

The major allergen from apple extract was concentrated by anion exchange chromatography and further purified by reverse-phase HPLC. A distinct peak with a high degree of homogeneity was obtained. The isolated protein has a MW of 18 kD and specific IgE-binding capacity (immunoblotting, IgE-binding inhibition). N-terminal amino acid analyses of the allergen allowed 37 cleavages and showed 67.6% identity to Bet v 1, the major allergen of birch pollen. Enzyme immunoassay inhibition studies with serum of birch/apple-allergic patients showed that besides cross-reacting structures to Bet v 1, apple-specific IgE antibodies could exist. Monoclonal antibodies (mAbs) were raised against the 18-kD allergen from apple and characterized by means of immunoblotting and ELISA. Only three of the eight studied mAbs reacted with Bet v 1.

Allergens↗

A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential.

Polyphenol oxidase (PPO) is responsible for enzymatic browning of apples. Apples lacking PPO activity might be useful not only for the food industry but also for studies of the metabolism of polyphenols and the function of PPO. Transgenic apple calli were prepared by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistant gene and antisense PPO gene. Four KM-resistant callus lines were obtained from 356 leaf explants. Among these transgenic calli, three calli grew on the medium containing KM at the same rate as non-transgenic callus on the medium without KM. One callus line had an antisense PPO gene, in which the amount and activity of PPO were reduced to half the amount and activity in non-transgenic callus. The browning potential of this line, which was estimated by adding chlorogenic acid, was also half the browning potential of non-transgenic callus.

Agrobacterium tumefaciens↗

Ergonomic analysis of New York apple harvest work using a Posture-Activities-Tools-Handling (PATH) work sampling approach.

Although back, neck, and shoulder strains are common among migrant and seasonal orchard workers, little data currently exist regarding the ergonomic factors contributing to this problem. We adapted Posture-Activities-Tools-Handling (PATH) instruments and methods for ergonomic job analysis of apple harvest work in three New York orchards, and used the resulting protocol to quantify hazardous activities, loads, and postures. Using a prototype developed previously, we trained twelve contract orchard observers with classroom training and supervised orchard practice. The PATH data were then collected on 14 orchard workers over four days (2,900 observations). Mean coefficients of variation ranged from a low of 0.212 (standing leg neutral) to a high of 0.603 (trunk moderate flexion). Most frequently observed activities were: picking (62.9%), placing and moving apples in the bag (8. 7%), and walking (8.1%). Weight bearing (>10 lb, >4.54 kg) was observed 78.5% of the time throughout a range of activities. Apple harvest work is comparable with other ergonomically high-risk occupations. Future research should focus on low-cost interventions that reduce load and awkward postures.

Agricultural Workers' Diseases↗

Effects of simulated acid rain on the pollen germination and pollen tube growth of apple (Malus sylvestris Miller cv. Golden).

The pollens of apple flowers have been treated with simulated acid rain solutions in range of pHs 2.9 to 5.0 in order to determine the threshold proportion values that lead the observed symptoms of detriments of acid rain. Compared to controls (pH 6.5), pollen germination decreased by 41.75% at pH 3.3 and pollen tube elongation decreased by 24.3% at pH 3.4. Acid rain threshold proportion value was around pH 3.3 and 3.4 for apple pollen germination and pollen tube elongation, respectively. Furthermore, pollen tube elongation was determined to be more sensitive to acid rain than pollen germination. The pH values below 3.1 resulted in complete destruction of pollen tubes. Pollen germination entirely stopped at around pH 3.0. Finally, it has been shown that the acid rain has a blocking effect on pollen germination and pollen tube elongation in apple. The conclusion is that not only pH value but also the quantity of acid rain is important factor in germination. The results were found statistically significant through the LSD test at levels of p < 0.05 and p < 0.01.

Acid Rain↗

Comparison of pheromone-mediated mating disruption and conventional insecticides for management of tufted apple bud moth (Lepidoptera: Tortricidae).

Large-plot studies were used to compare pheromone-mediated mating disruption and conventional insecticide applications for management of tufted apple bud moth, Platynota idaeusalis (Walker), in North Carolina in 1993 and 1994. Pheromone trap catches were reduced in mating disruption blocks, and traps placed in the lower stratum of the canopy had a higher level of trap capture reduction compared with traps placed in the upper stratum. First-generation tufted apple bud moth exposure to either pheromones for mating disruption or insecticides affected second generation pheromone trap catches in the lower and upper canopy. More second generation male moths were caught in pheromone traps placed in the upper compared with the lower canopy in blocks treated with pheromones for mating disruption during the first generation, whereas the opposite was true in blocks treated with insecticides during the first generation. Despite reduced trap catches in pheromone-treated blocks, egg mass densities were not reduced in these blocks compared with insecticide-treated blocks. Furthermore, fruit damage was not significantly different between mating disruption blocks and conventionally treated blocks in orchards with relatively low populations of tufted apple bud moth, but damage was greater in mating disruption blocks in orchards with higher moth densities.

Animals↗

Monitoring plum curculio, Conotrachelus nenuphar (Coleoptera: Curculionidae), populations in apple and peach orchards in the mid-Atlantic.

We evaluated responses of plum curculio, Conotrachelus nenuphar (Herbst), to four trap types in commercial and unsprayed apple and peach orchards. Trap types included black pyramid and clear Plexiglas panel traps deployed outside the orchard 2 m from the border row, and branch-mimicking cylinder and trunk-mounted screen traps attached to trees in the border row. Bait treatments evaluated in conjunction with each trap type included the synthetic fruit volatile benzaldehyde, the aggregation pheromone grandisoic acid (GA), benzaldehyde in combination with GA, and an unbaited control treatment. In commercial apple orchards, significantly more plum curculio were captured in traps baited with benzaldehyde + GA compared with traps baited with other treatments. Furthermore, significantly more plum curculio were captured by screen traps baited with benzaldehyde + GA compared with unbaited control traps. Significantly more plum curculio were captured by screen traps compared with other trap types in an unsprayed apple orchard. Very few captures were recorded in commercial peach orchards. Dissections of trapped females indicate that bivoltine populations are present in the mid-Atlantic. In general, correlations between timing and amount of trap captures and timing and amount of fruit injury inflicted concurrently or 1 wk after trap captures were very weak for all trap types and bait combinations. Our results agree with previous studies in the northeastern United States in which trap captures are increased by presence of semiochemical baits but fail to serve as reliable tools to determine need for and timing of insecticide application against plum curculio.

Animals↗

Incidence and control of dogwood borer (Lepidoptera: Sesiidae) and American plum borer (Lepidoptera: Pyralidae) infesting burrknots on clonal apple rootstocks in New York.

Surveys were conducted in the major apple growing regions of New York state to determine the incidence of borers infesting burrknots on clonal apple rootstocks. Dogwood borer, Synanthedon scitula (Harris), was generally prevalent throughout the state, but American plum borer, Euzophera semifuneralis (Walker), was limited to western New York apple orchards near infested stone fruit trees. Insecticides evaluated in the field for efficacy against both borers were chlorpyrifos, endosulfan, indoxacarb plus oil, methoxyfenozide, fenpropathrin, and kaolin clay. Also, white latex paint was tested alone and mixed with chlorpyrifos. One application of chlorpyrifos applied at the petal fall developmental stage was equivalent to chlorpyrifos applied at petal fall and again in mid-July, and it provided season-long control of dogwood borer and American plum borer. One application of chlorpyrifos applied any time between the half-inch green developmental stage and petal fall, or after harvest the previous season, controlled both overwintered and summer brood larvae of dogwood borer. Multiple applications of fenpropathrin, indoxacarb plus oil, and endosulfan applied during the dogwood borer flight period controlled the summer brood.

Animals↗

Monitoring stink bugs (Hemiptera: Pentatomidae) in mid-Atlantic apple and peach orchards.

Pyramid traps coated with "industrial safety yellow" exterior latex gloss enamel paint and baited with Euschistus spp. aggregation pheromone, methyl (2E,4Z)-decadienoate captured more stink bugs than all other baited and unbaited trap types in both apple and peach orchards in 2002 and 2003. Commercial sources of dispensers of methyl (2E,4Z)-decadienoate deployed in association with pyramid traps had a significant impact on trap captures. Captures in pyramid traps were four-fold greater when baited with lures from IPM Technologies, Inc. (Portland, OR) than with lures from Suterra (Bend, OR). Variation in yellow pyramid trap color ("industrial safety yellow" and "standard coroplast yellow") and material (plywood, plastic, and masonite) did not affect trap captures. Brown stink bug was the predominant species captured (58%), followed by dusky stink bug, Euschistus tristigmus (Say) (20%); green stink bug, Acrosternum hilare (Say) (14%); and other stink bugs (Brochymena spp. and unidentified nymphs) (8%). Captures in baited pyramid traps were significantly correlated with tree beating samples in both managed and unmanaged apple orchards and with sweep netting samples in the unmanaged apple orchard. However, problems associated with trapping mechanisms of pyramid trap jar tops and jar traps likely resulted in reduced captures in baited traps. Improved trapping mechanisms must be established to develop an effective monitoring tool for stink bugs in mid-Atlantic orchards.

Animals↗

Apple juice prevents oxidative stress induced by amyloid-beta in culture.

Increased oxidative stress contributes to the decline in cognitive performance during normal aging and in neurodegenerative conditions such as Alzheimer's disease. Dietary supplementation with fruits and vegetables that are high in antioxidant potential have in some cases compensated for oxidative stress. Herein, we examined whether apple juice could alleviate the neurotoxic consequences of exposure of cultured neuronal cells to amyloid-beta (Abeta), since at least a portion of the neurotoxicity of Abeta is due to oxidative stress. Apple juice concentrate (AJC; 70 degree brix) was diluted into culture medium of SH-SY-5Y human neuroblastoma cells that had been differentiated for 7 days with 5 microM retinoic acid concurrent with the addition of 20 microM Abeta. AJC prevented the increased generation of reactive oxygen species (ROS) normally induced by Abeta treatment under these conditions. AJC also prevented Abeta-induced calcium influx and apoptosis, each of which results in part due to increased ROS. These findings suggest that the antioxidant potential of apple products can prevent Abeta-induced oxidative damage.

Amyloid beta-Peptides↗

Supplementation with apple juice attenuates presenilin-1 overexpression during dietary and genetically-induced oxidative stress.

Gain-of-function mutations in the presenilin-1 (PS-1) promote Alzheimer's disease (AD) by increasing reactive oxygen species, at least part of which is derived by an accompanying increase in generation of amyloid-beta (Abeta). Additional studies indicate that impaired Apolipoprotein E function, which also increases oxidative stress and is also associated with AD, potentiates the deleterious activity of PS-1. Folate deficiency is also associated with AD and potentiates the impact of both ApoE deficiency and beta exposure. More recently, folate deficiency has been shown to increase PS-1 expression. Since dietary supplementation with apple juice provides neuroprotection against ApoE deficiency, Abeta exposure and folate deficiency, we examined the impact of apple juice on PS-1 overexpression. Herein, we demonstrate that dietary deficiency in folate and vitamin E increased PS-1 expression in juvenile and adult normal C57B1/6J and ApoE-/- mice and in aged normal mice. Supplementation with apple juice concentrate (AJC) attenuated or prevent these increases. Prior studies demonstrate that impaired DNA methylation resulting from a deficiency in S-adenosylmethionine (SAM, which is rapidly depleted following folate deprivation) leads to PS-1 overexpression, and that direct supplementation with SAM attenuates PS-1 overexpression. We determined that AJC contained levels of SAM comparable to those capable of suppressing PS-1 overexpression, suggesting that the SAM content of AJC represents a potential mechanism for preventing PS-1 overexpression, and further highlighting the possibility that AJC provides neuroprotection by mechanisms in addition to its antioxidant potential.

Acetylcholine↗

Expansion of the sooty blotch and flyspeck complex on apples based on analysis of ribosomal DNA gene sequences and morphology.

Sooty blotch and flyspeck (SBFS) is a late-season disease of apple and pear fruit that cosmetically damages the cuticle, resulting in produce that is unacceptable to consumers. Previous studies reported that four species of fungi comprise the SBFS complex. We examined fungal morphology and the internal transcriber spacer (ITS) and large subunit (LSU) regions of rDNA of 422 fungal isolates within the SBFS complex from nine orchards in four Midwestern states (USA) and compared them to previously identified species. We used LSU sequences to phylogenetically place the isolates at the order or genus level and then used ITS sequences to identify lineages that could be species. We used mycelial and conidial morphology on apple and in culture to delimit putative species. Thirty putative species found among the Midwest samples were shown to cause SBFS lesions on apple fruit in inoculation field trials. Among them Peltaster fructicola and Zygophiala jamaicensis have been associated previously with SBFS in North Carolina. The LSU analyses inferred that all 30 SBFS fungi from Midwestern orchards were Dothideomycetes; one putative species was within the Pleosporales, 27 were within Dothideales, and two putative species could not be placed at the ordinal level. The LSU sequences of 17 Dothideales species clustered with LSU sequences of known species of Mycosphaerella.

Ascomycota↗

Biological control of Botrytis cinerea growth on apples stored under modified atmospheres.

The combined effect of modified-atmosphere packaging and the application of a bacterial antagonist (Erwinia sp.) on Botrytis cinerea growth on apples (cv. 'Golden Delicious') was investigated. Inoculated apples were stored in polyethylene bags at 5 degrees C. The initial gas composition in each bag was set according to a central composite experimental design involving five levels of O2 (1 to 15%) and CO2 (0 to 15%). Control samples under ambient conditions were also included. Without the antagonist, measurements of mold colony diameter over time showed that O2 had no effect on the growth of B. cinerea, while increased CO2 levels delayed its growth by about 4 days. Application of the antagonist resulted in a significant interaction between O2 and CO2. At low O2 levels, CO2 had no effect on mold growth, but at high O2, CO2 enhanced mold growth. O2 and the antagonist worked synergistically to reduce mold growth by about 6 days at low levels of CO2. However, at high CO2 levels, O2 had no effect. The strongest antagonistic effect was observed under ambient conditions. Overall, results showed that high CO2 atmospheres can slow the growth of B. cinerea and that Erwinia sp. was an effective antagonist against B. cinerea growth on apples, particularly under ambient conditions.

Analysis of Variance↗