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PubMed2004

Cloning and linkage mapping of resistance gene homologues in apple.

Apple ( Malus x domestica Borkh.) sequences sharing homology with known resistance genes were cloned using a PCR-based approach with degenerate oligonucleotide primers designed on conserved regions of the nucleotide-binding site (NBS). Sequence analysis of the amplified fragments indicated the presence of at least 27 families of NBS-containing genes in apple, each composed of several very similar or nearly identical sequences. The NBS-leucine-rich repeat homologues appeared to include members of the two major groups that have been described in dicot plants: one possessing a toll-interleukin receptor element and one lacking such a domain. Genetic mapping of the cloned sequences was achieved through the development of CAPS and SSCP markers using a segregating population of a cross between the two apple cultivars Fiesta and Discovery. Several of the apple resistance gene homologues mapped in the vicinity, or at least on the same linkage group, of known loci controlling resistance to various pathogens. The utility of resistance gene-homologue sequences as molecular markers for breeding purposes and for gene cloning is discussed.

Amino Acid Sequence↗
PubMed2005

Linkage map positions and allelic diversity of two Mal d 3 (non-specific lipid transfer protein) genes in the cultivated apple (Malus domestica).

Non-specific lipid transfer proteins (nsLTPs) of Rosaceae fruits, such as peach, apricot, cherry, plum and apple, represent major allergens for Mediterranean atopic populations. As a first step in elucidating the genetics of nsLTPs, we directed the research reported here towards identifying the number and location of nsLTP (Mal d 3) genes in the apple genome and determining their allelic diversity. PCR cloning was initially performed on two cultivars, Prima and Fiesta, parents of a core apple mapping progeny in Europe, based on two Mal d 3 sequences (AF221502 and AJ277164) in the GenBank. This resulted in the identification of two distinct sequences (representing two genes) encoding the mature nsLTP proteins. One is identical to accession AF221502 and has been named Mal d 3.01, and the other is new and has been named Mal d 3.02. Subsequent genome walking in the upstream direction and DNA polymorphism analysis revealed that these two genes are intronless and that they could be mapped on two homoeologous segments of linkage groups 12 and 4, respectively. Further cloning and sequencing of the coding and upstream region of both Mal d 3 genes in eight cultivars was performed to identify allelic variation. Assessment of the deduced nsLTP amino acid sequences gave a total of two variants at the protein level for Mal d 3.01 and three for Mal d 3.02. The consequences of our results for allergen nomenclature and the breeding of low allergenic apple cultivars are discussed.

Alleles↗
PubMed2006

The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants.

A selection system based on the phosphomannose-isomerase gene (pmi) as a selectable marker and mannose as the selective agent was evaluated for the transformation of apple (Malus domestica Borkh.). Mannose is an unusable carbon source for many plant species. After uptake, mannose is phosphorylated by endogenous hexokinases to mannose-6-phosphate. The accumulation of mannose-6-phosphate leads to a block in glycolysis by inhibition of phosphoglucose-isomerase, resulting in severe growth inhibition. The phosphomannose-isomerase is encoded by the manA gene from Escherichia coli and catalyzes the conversion of mannose-6-phosphate to fructose-6-phosphate, an intermediate of glycolysis. Transformed cells expressing the manA gene can therefore utilize mannose as a carbon and survive on media containing mannose. The manA gene along with a beta-glucuronidase (GUS) gene was transferred into apple cv. 'Holsteiner Cox' via Agrobacterium tumefaciens-mediated transformation. Leaf explants were selected on medium supplemented with different concentrations and combinations of mannose and sorbitol to establish an optimized mannose selection protocol. Transgenic lines were regenerated after an initial selection pressure of 1-2 g l(-1) mannose in combination with 30 g l(-1) sorbitol followed by a stepwise increase in the mannose concentration up to 10 g l(-1) and simultaneous decrease in the sorbitol concentration. Integration of transgenes in the apple genome of selected plants was confirmed by PCR and southern blot analysis. GUS histochemical and chlorophenol red (CPR) assays confirmed activity of both transgenes in regenerated plants. The pmi/mannose selection system is shown to be highly efficient for producing transgenic apple plants without using antibiotics or herbicides.

Glucuronidase↗
PubMed2006

Evaluation of resistance pattern to fenpyroximate and pyridaben in Tetranychus urticae collected from greenhouses and apple orchards using lethal concentration-slope relationship.

This study aimed to monitor the present and future developments of the resistance of Tetranychus urticae Koch to fenpyroximate and pyridaben, using the relationship of the LC(50) and slope of the concentration-mortality line in a probit model, for the provision of reliable resistance management tactics. Tetranychus urticae populations were collected from 16 commercial greenhouses, where various crops were cultivated, as well as from 10 apple orchards throughout Korea. The resistance to fenpyroximate and pyridaben of each population was estimated by calculating the median lethal concentration (LC(50)), resistance ratio (RR) and slope of the concentration-mortality regression. Most of the greenhouse populations exhibited moderate levels of resistance, whereas the apple orchard populations showed only low levels, indicating that T. urticae populations in greenhouses were more strongly selected than those in apple orchards. Four population groups were established based on either the habitats (greenhouse and apple orchard) or acaricides (fenpyroximate and pyridaben). To test the hypothesis, "the slope is greatest at low and high levels of resistance," the slopes were regressed as a function of the LC(50), and fitted to a polynomial regression. The polynomial regression model explained this relationship well for the four population groups (p < 0.05), indicating that the development of resistance toward fenpyroximate or pyridaben was consistent with the gradient. A laboratory selection study agreed with the results from both acaricide field populations. These results suggest that the gradient was a good indicator of the susceptibility of T. urticae to genetic variations, which was related to the LC(50). The application of these findings is also discussed in relation to the resistance management of T. urticae.

Animals↗
PubMed1985

Olfactory, gas chromatographic and mass-spectral analyses of fecal volatiles traced to ingested licorice and apple.

Volatile components of two foodstuffs with characteristic aromas, apple and licorice, and fecal samples obtained from subjects on high-apple and licorice diets, were analyzed by head-space gas chromatography and gas chromatography-mass spectrometer. The volatile compounds characteristic of the odors of apple and licorice were detected by "sniffing" the GC effluent. The aromatic component of licorice was identified by GC-MS as anethole [1-methoxy-4-(2-propenyl) benzene]. The aromatic component of apple could not be chemically characterized by our GC-MS system even though readily detected by the nose. Head-space, GC-MS analysis of fecal volatiles offers a means of tracing odorous and non-odorous components to ingested foodstuffs with potential applications to clinical and forensic medicine and anthropology.

Allylbenzene Derivatives↗
PubMed1994

Binding geometry, stoichiometry, and thermodynamics of cyclomalto-oligosaccharide (cyclodextrin) inclusion complex formation with chlorogenic acid, the major substrate of apple polyphenol oxidase.

The inclusion complexes of cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD), cyclomaltooctaose (gamma-CD), and polymerized beta-CD (beta-CDn) with chlorogenic acid (CA), the major substrate of apple fruit polyphenol oxidase (PPO), were studied with regard to pH, ionic strength, and temperature in model buffer systems and apple juice. The thermodynamics of CD.CA inclusion complex formation, which were studied in solution using UV spectrophotometry, displayed enthalpy-entropy compensation typical of processes driven by solvation phenomena. We also found that the apparent association constants (K) of the CD.CA equilibrium were relatively insensitive to pH for beta-CD, compared to alpha- and gamma-CDs, but were subject to substantial enhancement at low ionic strengths. The beta-CD.CA inclusion complex was also characterized for binding geometry and stoichiometry at 9.4 T and 25 degrees C in 0.05 M Na phosphate buffer by 1H NMR spectroscopy. A 1:1 stoichiometric ratio for the complex was found using the method of continuous variations. 1H Spin-lattice relaxation and chemical-shift data indicate that the phenolic ring of CA docks within the cavity of beta-CD. The Ks for beta-, alpha-, and gamma-CD determined in apple juice, which contains a mixture of PPO substrates, were found to correlate with PPO activity-related data. Apple juice, treated with beta-CDn, did not brown until CA was added back. These latter findings strongly argue that the mechanism for inhibition of juice browning with cyclodextrins was mainly due to the binding of PPO substrates and not some other means such as enzyme inactivation via sequestration of Cu2+ by CDs.

Binding Sites↗
PubMed1983

Cold-stage scanning electron microscope measurement of ice morphology in apple tissue as a function of freezing rate.

A cold-stage SEM was used to document the morphology of ice in apple tissue and a sucrose solution frozen at rates ranging from 450 to 0.03 degrees K/min. Freezing rates of 3-mm-thick apple discs were measured with a differential thermocouple technique, which gave measurement of the growth velocity and the temperature gradient through the solidified specimen as well as the cooling rate during solidification. Cold-stage SEM micrographs were used to measure the dendritic spacing of the ice structures, and these data were found to correlate linearly with the square root of the cooling rate during solidification as would be predicted by a theoretical analysis of mass transfer in the formation of dendrites. Comparison of freeze-substituted and freeze-dried apple-tissue micrographs with those from a cold-stage SEM showed that the cold-stage SEM technique was the only one which correctly represented ice morphology in apple tissue.

Food Preservation↗
PubMed1995

Estimating the risks and benefits of pesticides: considering the agroecosystem and integrated pest management in the use of EBDC fungicides on apples.

Calculating the risks and benefits of agricultural pesticides determines whether a pesticide will be registered for use on commodities. Historically, the US Environmental Protection Agency (EPA) has used very conservative estimates of health risks, particularly in the absence of data regarding actual use patterns of the pesticide and actual pesticide residue at the point of food purchase. However, when grower groups and manufacturers were faced with the problem of losing the ethylene bisdithiocarbamate (EBDC) fungicides, they approached EPA offering to provide hard data in place of estimates. Plant pathologists familiar with apple agroecosystems pointed out that different fungicides have highly specific uses in integrated pest management (IPM) programs, and that banning EBDC use on apples would probably raise total pesticide use in apples by compromising the pesticide reductions obtained under existing IPM programs. In addition, an early study showed that use-patterns could significantly reduce the amount of EBDC residue on apples. This approach led to a more realistic appraisal of both risks and benefits of the fungicides, lowering risks and raising benefits. The process ended with a registration prescribing use-patterns which result in minimal residue at harvest, thus providing valuable tools for use in IPM programs.

Journal Article↗
PubMed2006

Annurca apple-rich diet restores long-term potentiation and induces behavioral modifications in aged rats.

The impairment of brain functions during aging could be prevented by an increase in antioxidative defenses. In fact, dietary manipulation with antioxidants or with antioxidant-rich extracts from vegetables and fruit can ameliorate age-associated behavioral alterations. Moreover, dietary supplements of antioxidants can reverse the age-dependent impairment to sustain long-term potentiation (LTP). There is evidence that fresh fruit could be more effective than antioxidant supplements or fruit and vegetable extracts but the effect of a diet enriched in fresh fruit on brain aging process has not been investigated until now. The aim of the present study was to verify whether dietary manipulation with fresh apples could influence age-induced changes in motor and emotional behavior and in LTP in rats. Groups of aged and young rats were fed with a standard diet or a diet supplemented with fresh apples of annurca variety for 10 weeks and assessed for behavioral performance in the open field and elevated plus maze tests and for the ability to sustain LTP. The brains were then removed, histochemically stained for superoxide dismutase (SOD) and SOD activity was measured by computer-assisted image analysis. The aged rats fed with the enriched diet showed a significant decrease in the anxiety level. Moreover, they improved in the ability to sustain LTP, reaching the level of the young rats. SOD activity was increased in the aged rats fed with the standard diet whereas SOD activity in the hippocampus of the aged rats treated with annurca apple was at the level of the young animals. These results suggest that a diet rich in annurca apple could have an important role in health-care during aging.

Aging↗
PubMed2006

Fourier transform infrared spectroscopy, detection and identification of Escherichia coli O157:H7 and Alicyclobacillus strains in apple juice.

Fourier Transform Infrared (FT-IR) spectroscopy (4000-400 cm(-1)) combined with multivariate statistical methods were used to identify and detect Escherichia coli O157:H7 from Alicyclobacillus spp. recovered from apple juice. Four treatments and a control in triplicate experiments (N=3) were studied; the first three treatments of pasteurized apple juice were inoculated with E. coli O157:H7 ATCC 35150, Alicyclobacillus acidoterrestris 1016 and Alicyclobacillus spp. C-Fugi-6 respectively. The fourth treatment was a 1:1 (v:v) mixed culture of both A. acidoterrestris 1016 and Alicyclobacillus spp. C-Fugi-6. The control was uninoculated pasteurized apple juice. The second derivative transformation and loadings plot over the range of 1800-900 cm(-1) highlighted the most distinctive variations among bacterial spectra. Loadings 1 and 2 were distinctively representative of the bacterial spectral data and accounted for 73% of the total variability. Treatments were noticeably segregated with distinct clustering by principal component analysis (PCA). Using soft independent modeling of class analogy (SIMCA) analysis, 88.3% of (E. coli O157:H7 ATCC 35150) spectra, 75.0% of (A. acidoterrestris 1016) spectra, 88.3% of (Alicyclobacillus spp. C-Fuji-6) spectra, and 80.0% of the mixed culture of both Alicyclobacillus strains spectra were correctly classified. Using the spectral features of bacterial cellular constituents such as nucleic acids, proteins, phospholipids, peptidoglycan, and lipopolysaccharides from examined bacterial cells, pure and mixed cultures of Alicyclobacillus spp. cells, and the pathogenic E. coli cells could be detected in apple juice.

Beverages↗
PubMed2006

Fibres as carriers for Lactobacillus rhamnosus during freeze-drying and storage in apple juice and chocolate-coated breakfast cereals.

The capability of different fibre preparations to protect the viability and stability of Lactobacillus rhamnosus during freeze-drying, storage in freeze-dried form and after formulation into apple juice and chocolate-coated breakfast cereals was studied. In freeze-drying trials wheat dextrin and polydextrose proved to be promising carriers for the L. rhamnosus strains: both freeze-drying survival and storage stability at 37 degrees C were comparable to the control carrier (sucrose). Using apple fibre and inulin carriers resulted in powders with fairly good initial freeze-drying survival but with poor storage stability at 37 degrees C. When fresh L. rhamnosus cells were added into apple juice (pH 3.5) together with oat flour with 20% beta-glucan the survival of the cells was much better at 4 degrees C and at 20 degrees C than with sucrose, wheat dextrin and polydextrose, whereas with freeze-dried cells no protective effect of oat flour could be seen. The stability of freeze-dried L. rhamnosus cells at 20 degrees C was higher in chocolate-coated breakfast cereals compared to low pH apple juice. Similar to freeze-drying stability, wheat dextrin and polydextrose proved to be better carriers than oat flour in chocolate-coated breakfast cereals. Regardless of their differing capability to adhere to fibre preparations the two L. rhamnosus strains studied gave parallel results in the stability studies with different carriers.

Beverages↗
PubMed2006

Ontogenetic variation in chemical and physical characteristics of adaxial apple leaf surfaces.

The reaction of plants to environmental factors often varies with developmental stage. It was hypothesized, that also the cuticle, the outer surface layer of plants is modified during ontogenesis. Apple plantlets, cv. Golden Delicious, were grown under controlled conditions avoiding biotic and abiotic stress factors. The cuticular wax surface of adaxial apple leaves was analyzed for its chemical composition as well as for its micromorphology and hydrophobicity just after unfolding of leaves ending in the seventh leaf insertion. The outer surface of apple leaves was formed by a thin amorphous layer of epicuticular waxes. Epidermal cells of young leaves exhibited a distinctive curvature of the periclinal cell walls resulting in an undulated surface of the cuticle including pronounced lamellae, with the highest density at the centre of cells. As epidermal cells expanded during ontogenesis, the upper surface showed only minor surface sculpturing and a decrease in lamellae. With increasing leaf age the hydrophobicity of adaxial leaf side decreased significantly indicated by a decrease in contact angle. Extracted from plants, the amount of apolar cuticular wax per area unit ranged from only 0.9 microgcm(-2) for the oldest studied leaf to 1.5 microgcm(-2) for the youngest studied leaf. Differences in the total amount of cuticular waxes per leaf were not significant for older leaves. For young leaves, triterpenes (ursolic acid and oleanolic acid), esters and alcohols were the main wax components. During ontogenesis, the proportion of triterpenes in total mass of apolar waxes decreased from 32% (leaf 1) to 13% (leaf 7); absolute amounts decreased by more than 50%. The proportion of wax alcohols and esters, and alkanes to a lesser degree, increased with leaf age, whereas the proportion of acids decreased. The epicuticular wax layer also contained alpha-tocopherol described for the first time to be present also in the epicuticular wax. The modifications in the chemical composition of cuticular waxes are discussed in relation to the varying physical characteristics of the cuticle during ontogenesis of apple leaves.

Malus↗
PubMed2006

Biosynthesis of flavan 3-ols by leucoanthocyanidin 4-reductases and anthocyanidin reductases in leaves of grape (Vitis vinifera L.), apple (Malus x domestica Borkh.) and other crops.

Catechin and epicatechin biosyntheses were studied of grape (Vitis vinifera L.), apple (Malus x domestica Borkh.) and other crop leaves, since these monomers and the derived proanthocyanidins are important disease resistance factors. Grape and apple leucoanthocyanidin 4-reductase (LAR; EC 1.17.1.3) enzymes were characterized on basis of plant and recombinant enzymes. In case of grape, two LAR cDNAs were cloned by assembling available EST sequences. Grape and apple leaf anthocyanidin reductase (ANR; EC 1.3.1.77) cDNAs were also obtained and the respective plant and recombinant enzymes were characterized. Despite general low substrate specificity, within the respective flavonoid biosyntheses of grape and apple leaves, both enzyme types deliver differently hydroxylated catechins and epicatechins, due to substrate availability in vivo. Furthermore, for LAR enzymes conversion of 3-deoxyleucocyanidin was shown. Beside relevance for plant protection, this restricts the number of possible reaction mechanisms of LAR. ANR enzyme activity was demonstrated for a number of other crop plants and its correlation with (-)-epicatechin and obvious competition with UDP-glycosyl:flavonoid-3-O-glycosyltransferases was considered.

Catechin↗
PubMed2000

Determination of fenpyroximate in apples by supercritical fluid extraction and packed capillary liquid chromatography with UV detection.

A method using off-line supercritical fluid extraction (SFE) and micro liquid chromatography (microLC) with UV detection at 260 nm, was developed for selective determination of fenpyroximate in apple samples. The packed capillary liquid chromatography method utilises 20 microl injection volumes with on-column focusing. A 350x0.32 mm capillary column packed with Kromasil 100-C18 of 5 microm particle size was used with a mobile phase of acetonitrile-10 mM ammonium acetate (85:15, v/v) at a flow of 5 microl/min. A two-step SFE procedure was used to extract fenpyroximate selectively in 2 g apple samples, with Hydromatrix (HMX) added as a water absorbent at a 1:1 (w:w) ratio. Fenpyroximate was extracted at 200 bar and 90 degrees C for 15 min using carbon dioxide at a flow of 2 ml/min, and solvent trapping collection in 10 ml acetonitrile. The volume of the acetonitrile extract was reduced by evaporation and water was added to a final composition of acetonitrile-water (40:60, v/v). The resulting 2.0 ml solution was filtered using a 0.45 microm poly(vinylidene difluoride) syringe filter before microLC analysis. Validation of the method was accomplished with apple samples spiked with fenpyroximate, covering the range of 0.1 to 1.0 microg/kg. The within-day and between-day repeatabilities were in the range 4-18% relative standard deviation. Accuracy, measured as recovery, was found to be approximately 60%. Apple samples from a field treated with fenpyroximate were analysed. None of the samples contained fenpyroximate above the quantification level.

Benzoates↗
PubMed2001

Determination of phenolic acids and flavonoids of apple and pear by high-performance liquid chromatography.

A new HPLC stationary phase has been applied to the analysis of phenolic acids and flavonoids with diode array and mass spectrometric detection. The separation of 26 standard compounds was achieved within 1 h. The stationary phase displayed excellent resolution especially of flavonol glycosides. The analytical system has been used for the determination of phenolic compounds in apple pomace and apple juice, and in extracts of pear fruits of different cultivars. Apple pomace was found to be a promising source of phenolics. However, yields are affected by the drying conditions applied. Furthermore, the applicability of the analytical system for the authenticity control of apple and pear juice was demonstrated by determination of characteristic quercetin and isorhamnetin glycosides, and dihydrochalcones, respectively. Since isorhamnetin-3-glucoside was present in all pear cultivars investigated, the usefulness of arbutin as a specific marker of pear products appears to be doubtful.

Chromatography, High Pressure Liquid↗
PubMed2001

Induced emissions of apple fruit volatiles by the codling moth: changing patterns with different time periods after infestation and different larval instars.

The changes in the emission of volatiles from mature apple fruits in response to larval feeding by the codling moth (Cydia pomonella) under laboratory conditions are reported. A time course experiment investigated the emission of volatiles throughout the period of larval development following infestation. The volatiles consisted mainly of esters, a few aldehydes, and the terpene alpha-farnesene. Infested apples emitted the same compounds as healthy apples. The quantities of volatiles released were much higher for infested as compared to healthy fruits for an initial three day period. Following this period there was a decrease in volatile emissions (days 6-9), eventually declining back to the levels emitted from healthy apples or below by 9-21 days after infestation. In a separate experiment, the volatile emissions from healthy and artificially damaged fruits were compared to those from herbivore damaged fruits for each of the five larval instars of C. pommonella. The results from the discriminant analysis indicate that the most effective induction of volatiles occurred when fruits were infested with first instar larvae. Induction by first instar larvae was generally higher than after infestation by later instars, and for most compounds it also exceeded the emission from artificially damaged fruits.

Animals↗
PubMed2002

Removal of dyes from a synthetic textile dye effluent by biosorption on apple pomace and wheat straw.

This paper deals with two low-cost, locally available, renewable biosorbents; apple pomace and wheat straw for textile dye removal. Experiments at total dye concentrations of 10, 20, 30, 40, 50, 100, 150, and 200 mg/l were carried out with a synthetic effluent consisting of an equal mixture of five textile dyes. The effect of initial dye concentration, biosorbent particle size, quantity of biosorbent, effective adsorbance, dye removal and the applicability of the Langmuir and Freundlich isotherms were examined. One gram apple pomace was found to be a better biosorbent, removing 81% of dyes from the synthetic effluent at a particle size of 2 mm x 4 mm and 91% at 600 microm. Adsorption of dyes by apple pomace occurred at a faster rate in comparison to wheat straw. Both the isotherms were found to be applicable in the case of dye adsorption using apple pomace.

Adsorption↗
PubMed2000

Biological control of blue mould on apple by a strain of Candida sake under several controlled atmosphere conditions.

The biocontrol potential of the yeast Candida sake (CPA-1) against Penicillium expansum decay of apples under several controlled atmosphere conditions was investigated. In a laboratory trial under different commercial cold storage conditions, increasing concentrations of C. sake improved decay control. A maximum reduction of decay was achieved at 3% O2-3% CO2 atmosphere. It amounted to a 97% lesion reduction after treatment with a suspension containing 2.4 x 10(6) CFU/ml of C. sake (CPA-1). In a semi-commercial trial at 1 degree C with wounded fruits, the reduction in decay diameter caused by C. sake exceeded 80% after 60 days at 21% O2 and 60% after 120 days of storage under controlled atmosphere conditions. For seven controlled atmosphere conditions studied, a significant influence by C. sake on the P. expansum decay was observed, and the lesion size was reduced more than 70% by C. sake at 10(7) CFU/ml. The populations of C. sake (CPA-1) on the apple surface followed the same pattern under all controlled atmosphere conditions studied. They decreased 4-10-fold during the first 2 weeks, followed by an increase to the initial level after 45 days, and thereafter the count remained constant for the period of 90 days examined. This indicated the capacity of C. sake (CPA-1) to colonize the surface of apples under various storage conditions. The ability to colonize was even higher in apple wounds.

Atmosphere↗