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Development of sequence-characterized amplified regions (SCARs) from amplified fragment length polymorphism (AFLP) markers tightly linked to the Vf gene in apple.

Amplified fragment length polymorphism (AFLP) markers have become widely used in saturating the region of a gene of interest for the ultimate goal of map-based cloning of the gene or for marker-assisted selection. However, conversion of AFLP markers into restriction fragment length polymorphism (RFLP) or polymerase chain reaction (PCR)-based markers will greatly expand their usefulness in genetic applications. Previously, we have identified 15 AFLP markers tightly linked to the Vf gene conferring scab resistance in apple. In this study, we have successfully converted 11 of these AFLPs into sequence-characterized amplified region (SCAR) markers. Of the remaining four nonconverted AFLP markers, one, ET2MC8-1, has been found to be very short (83 base pairs) and is an A/T rich (90%) marker; a second, EA2MG11-1, has shown identical sequences between Malus floribunda 821 (the original source of the Vf gene) and scab-susceptible apple cultivars; while the other two, EA12MG16-1 and ET8MG1-1, have not been cloned. Using the 11 converted SCAR markers along with 5 previously identified SCAR markers, a high-resolution linkage map around the Vf gene has been constructed, and found to be consistent with its corresponding AFLP map. Three converted SCAR markers (ACS-3, -7, and -9) are inseparable from the Vf gene; whereas one (ACS-6) is located left of, and the remaining seven (ACS-1, -2, -4, -5, -8, -10, and -11) are located right of the Vf gene at genetic distances of 0.4 and 0.2 cM, respectively. A reliable and robust procedure for development of SCAR markers from AFLP markers is presented.

Base Sequence↗

The effects of a pesticide program on non-target epiphytic microbial populations of apple leaves.

The epiphytic microbial community was quantitatively and qualitatively altered when a standard pesticide schedule that comprised applications of an insecticide, a bactericide, and a fungicide was applied to McIntosh apple trees. Effects on non-target organisms were observed for two seasons by three indirect methods and three direct methods: plating of leaf washings, imprinting of leaves onto five different media, spore fall patterns, light microscopy, scanning electron microscopy, and isolation of propagules from leaves incubated in humidity chambers. Magnitude of reduction of bacteria, filamentous fungi, yeasts, and actinomycetes varied annually and between categories of microflora. Populations from treated leaves were reduced 10- to 1000-fold in 1976 and up to 50-fold in 1977. Qualitatively, fluorescent pseudomonads and lactic acid-type bacteria were among those depressed by pesticide. Fungal populations on treated leaves were less diverse than on control leaves. Aureobasidium was only slightly affected and incidence of Sporobolomyces was substantially higher on treated leaves than on controls. The results suggest that numbers of antagonists to foliar pathogens of apple which may occur as part of the natural epiphytic microbial community may be reduced by current pesticide programs and hence have possible implications for the development of biological approaches to integrated control strategies.

Bacteria↗

A procedure for quantifying adhesion of conidia of Botrytis cinerea to the skin of apple fruit.

Ultrasonication was evaluated as a nonchemical means to quantitatively remove conidia of Botrytis cinerea from the skin of Golden Delicious apple (Malus domestica Borkh.) fruit. A probe immersed in a suspension of conidia and generating 20 kHz at 150 W for 30- or 60-s pulses destroyed 13.3% or 29% of conidia, respectively. Destruction at 150 W for 10 s or at 30-120 W for up to 60 s was <2%. The procedure for quantifying adhesion of conidia to the skin of fruit consisted of pipetting a 50-microL water droplet containing 5 x 10(4) conidia onto the skinside of a slice of fruit, incubating the slices inside sealed 500 cm3 glass jars, excising a 1 cm diameter piece of skin bearing the droplet, and sonicating the skin in 8 mL of ice-cold water at 150 W for 10 s. The skin was removed, the suspension was centrifuged at 1250 x g for 15 min, and the supernatant was reduced to 1 mL by vacuum suction using a pipet. Conidia were stained with crystal violet and counted in a hemacytometer. Adhesion of conidia to skin was 3.0%, 14.6%, 20.8%, 39.4%, 57.6%, and 73.1% after 0, 2, 4, 8, 12, and 24 h incubation, respectively. Sonication was more effective than two other procedures for recovery of conidia. Conidia on the skin of fruit exposed to 4 microL of butyl acetate in the headspace of glass jars for 4 h at 23 degrees C increased the adhesion of conidia 107% above that for unexposed conidia. Sonication with a programmable power- and time-controlled probe was a simple, rapid, safe, and effective method for quantifying adhesion of B. cinerea conidia to the skin of apple fruit.

Botrytis↗

Mutational analysis of amino acid positions crucial for IgE-binding epitopes of the major apple (Malus domestica) allergen, Mal d 1.

BACKGROUND: Individual amino acid residues of the major birch pollen allergen, Bet v 1, have been identified to be crucial for IgE recognition. The objective of the present study was to evaluate whether this concept was applicable for the Bet v 1-homologous apple allergen, Mal d 1. METHODS: A Mal d 1 five-point mutant was produced by PCR techniques, cloned into pMW 172 and expressed in Escherichia coli BL21(DE3) cells. To evaluate the allergenic properties of the engineered protein compared to Mal d 1 wild-type IgE immunoblotting, ELISA, peripheral blood monocytes proliferation assays, and skin prick tests were performed. RESULTS: The Mal d 1 mutant showed reduced capacity to bind specific IgE as compared to wild-ype Mal d 1 in in vitro assays in the majority of the sera tested. In ELISA, 10 out of 14 serum samples displayed an 88-30% decrease in IgE binding to Mal d 1 mutant compared to wild-type Mal d 1. Skin prick tests in apple-allergic patients (n = 2) confirmed the markedly decreased ability of the Mal d 1 mutant to induce allergic reactions in vivo. However, the relevant T cell epitopes were present in the mutated molecule according to peripheral blood mononuclear cell proliferation assays. CONCLUSIONS: Our findings suggest that it is possible to modulate the IgE-binding properties of allergens by single amino acid substitutions at crucial positions which might be useful for future immunotherapy of birch-pollen-associated food allergies which are not ameliorated by birch pollen immunotherapy.

Allergens↗

Allergenicity assessment of apple cultivars: hurdles in quantifying labile fruit allergens.

BACKGROUND: Assessment of allergenicity of foods is important for allergic consumers and regulators. Immunoassays to measure major food allergens are widely applied, often giving variable results. Using the major apple allergen Mal d 1 as a model, we aimed to establish at the molecular level why different immunoassays for assessing allergenicity of apple cultivars produce conflicting outcomes. METHODS: Mal d 1 was measured in 53 cultivars from Italy and 35 from The Netherlands, using four different immunoassays. Purified Mal d 1 standards were molecularly characterized by size-exclusion chromatography (SEC) and mass spectrometry (MS). RESULTS: Three immunoassays using an identical standard gave similar results. Minor differences in sample preparation already resulted in significant loss of allergenicity. The fourth assay, using a different Mal d 1 standard, gave 10- to 100-fold lower outcomes. By SEC, this standard was shown to be almost fully aggregated. This aggregation was accompanied by a decrease of the mass of the Mal d 1 molecule by approximately 1 kDa as analyzed by MS. The deviating immunoassay was shown to selectively recognize this aggregated form of Mal d 1, whereas the other three assays, including the one based on IgE antibody recognition, preferentially bound non-aggregated allergen. CONCLUSIONS: Variable and poorly controllable major allergen modification in both extracts and standards hamper accurate allergenicity assessments of fruits.

Allergens↗

Reduction of cholesterol transported in apo B-rich lipoproteins in spontaneously hypercholesterolemic hamsters fed an apple-supplemented diet.

The influence of an apple-supplemented diet on the distribution of cholesterol among the lipoproteins in plasma was studied using a special animal model: FEC hamsters which spontaneously develop hypercholesterolemia with aging when fed a commercial balanced diet. This metabolic disorder did not occur in animals freely consuming apples in addition to the standard diet. This combined diet essentially decreased the cholesterol content in the VLDL and LDL, the apo B-rich lipoproteins in plasma.

Animals↗

[Life styles and blood pressure: the protective effect of apple-eating habits on high blood pressure in a high-salt population].

Prospective epidemiological studies of blood pressure in a high-salt population in northeastern Japan were investigated along with dietary habits such as miso soup, rice, apple, fish, milk and sake consumption as well as smoking habits. Blood pressures of the populations in 3 villages were determined once or twice a year by mass surveys from 1954, 1957 or 1958 through 1975. The means and transitions of the personal blood pressure were calculated by regression analysis of the data obtained during each entire period. The number of persons was 1127 males and 1369 females and the response rate was 98.7 percent. The average number of times of determination of blood pressure for a person was 12.9. Stepwise multiple regression analyses were run with the means and transitions of systolic and diastolic blood pressure as the dependent variables and the life styles of the population in 1958 as an independent variable based on data of persons whose blood pressures were determined 5 or more times during the entire period. According to the backward stepwise method this study confirmed the positive relationship of age and sake drinking and the negative relationship of apple eating habits to blood pressure.

Aged↗

Isolation of a cDNA encoding a 31-kDa, pathogenesis-related 5/thaumatin-like (PR5/TL) protein abundantly expressed in apple fruit (Nalus domestica cv. Fuji).

A fruit-specific and pathogenesis-related 5/thaumatin-like (PR5/TL), 31-kDa protein was isolated by 2D-PAGE from fully-grown apples (Malus domestica cv. Fuji) and named Mdtl1 (Malus domestica thaumatin-like protein 1). Using the N-terminal sequence of the protein, the full-length cDNA encoding Mdtll was isolated. The cDNA clone (Mdtl1) consists of 944 bp with an open reading frame (ORF) of 744 bp encoding a protein of 247 amino acids. The deduced amino acid sequence of Mdtl1 shows high similarity to the sequences of PR5/TL proteins. Mdtl1 is a slightly acidic protein with a putative signal peptide and a putative N-glycosylation site, and lacks a C-terminal extension. This suggests that Mdtl1 is an apoplastic glycoprotein. Results of northern blotting indicated that expressions of Mdtl1 are developmentally regulated. Southern blot analysis showed that Mdtl1 may be present as a single copy, and there exist other genes closely related to Mdtl1 in the apple genome.

Amino Acid Sequence↗

S-RNases from self-incompatible and -compatible apple cultivars: purification, cloning, enzymic properties, and pollen tube growth inhibitory activity.

Four S-RNases (RNase associated with self-incompatibility) were purified from the styles of two apple cultivars (Malus domestica), a self-incompatible cv., Starking Delicious (SD), and a self-compatible cv., Megumi (MG). Each cultivar produced two S-RNases and their enzymatic properties such as specific activity, pH optimum, thermal stability, and molecular mass, were characterized. The four S-RNases inhibited the tube growth of apple pollen in an in vitro bioassay at 25 microg/ml (1.0 microM), but did not distinguish self from non-self pollen. The cDNAs of four S-RNases were cloned, and the nucleotide and deduced amino acid sequences were analyzed. The nucleotide sequence of SD-Se RNase was a new one and the other was identical to that of Sc-RNase of cv. Fuji. In MG one was identical to the sequence of SD-Sc RNase and the other to that of Sa-RNase of cv. Golden Delicious except for one base. From results of the isolation amounts and the Western blot analysis for stylar crude extracts the amount of S-RNases in MG was apparently less than that in SD.

Amino Acid Sequence↗

Ribonuclease inhibitors in Malus x domestica (common apple): isolation and partial characterization.

A ribonuclease inhibitory activity was detected in the fruits of common apple, Malus x domestica, cv. Fuji, and purified by affinity chromatography on ribonuclease A-Sepharose. It inhibited hydrolysis of cyclic-2':3'-CMP by bovine pancreatic ribonuclease A with an apparent inhibition constant of about 5 x 10(-8) M. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the purified protein gave two peaks corresponding to the mass numbers of 55,658 and 62,839, while three bands of 43-, 34-, and 21-kDa were detected by SDS-PAGE. These results suggested that the inhibitor preparation was a mixture of two proteins comprised of 43- and 21-kDa subunits or of 34- and 21-kDa subunits. Attempts to separate these two proteins were unsuccessful. Amino acid composition and N-terminal amino acid sequence of these subunits were also identified and N-terminal sequences showed some similarity to that of cottonseed storage globulin. The significance of the presence of ribonuclease inhibitors in apple fruits is not clear, but it might allow some speculation about their possible involvement in the control of the self-incompatibility ribonuclease of Rosaceae plants.

Amino Acid Sequence↗

Nondestructive quantification of the soluble-solids content and the available acidity of apples by fourier-transform near-infrared spectroscopy.

This research evaluated the feasibility of using Fourier-transform near-infrared (FT-NIR) spectroscopy to quantify the soluble-solids content (SSC) and the available acidity (VA) in intact apples. Partial least-squares calibration models, obtained from several preprocessing techniques (smoothing, derivative, etc.) in several wave-number ranges were compared. The best models were obtained with the high coefficient determination (r2) 0.940 for the SSC and a moderate r2 of 0.801 for the VA, root-mean-square errors of prediction of 0.272% and 0.053%, and root-mean-square errors of calibration of 0.261% and 0.046%, respectively. The results indicate that the FT-NIR spectroscopy yields good predictions of the SSC and also showed the feasibility of using it to predict the VA of apples.

Journal Article↗

Pulmonary lipogranulomatosis due to excessive consumption of apples.

A 55-year-old man was admitted for treatment of unstable angina. Chest x-ray films showed a micronodular pattern, but there were no respiratory symptoms. Autopsy, carried out following sudden death, revealed pulmonary granulomas surrounding lipid crystals. The same crystals were found elsewhere: lymph nodes, spleen, liver, adrenal glands, and were composed of aliphatic hydrocarbons of vegetal origin, mainly C29H60. Investigation of the case history revealed excessive consumption of apples, to which the hydrocarbon deposition could be attributed, since C29H60 is a natural constituent of the cuticular wax in apple peel. This is a unique example of pulmonary granulomatosis due to storage of vegetal hydrocarbons of dietary origin.

Dietary Fats↗

Structure-activity relationship study of host-specific phytotoxins (AM-toxin analogs) using a new assay method with leaves from apple meristem culture.

AM-toxins are host-specific phytotoxins of the Alternaria alternata apple pathotype, which induce necrosis on apple leaves. In this study, we developed a new assay to measure the necrotic activity of AM-toxin analogs using cultured leaves from meristem cells. This method was not only more sensitive to AM-toxin I, but also more reliable than the previous one that used tree leaves due to the homogeneous nature of cultured leaves and to the method of application of toxins. Using this assay method we investigated a structure-activity relationship of AM-toxin analogs synthesized in this study. Most residues and the macrocyclic ring structure were strictly recognized by AM-toxin putative receptor, whereas the L-Ala binding subsite of the receptor allowed for side chain structures with various stereoelectronic properties. These findings are important for designing ligands for further experimental probing of the nature of the receptor.

Alternaria↗

Volatiles from apple (Malus domestica) eliciting antennal responses in female codling moth Cydia pomonella (L.) (Lepidoptera: Tortricidae): effect of plant injury and sampling technique.

The antennal responses of codling moth females, Cydia pomonella, to volatiles from apple branches with green fruits were recorded by electroantennography coupled to gas chromatography. The antennae strongly responded to 4,8-dimethyl-1,3(E),7-nonatriene, linalool, beta-caryophyllene, (E)-beta-farnesene, germacrene D, (Z,E)-alpha-farnesene, (E,E)-alpha-farnesene and methyl salicylate. These compounds were all present in volatile collections on Porapak Q from both living and cut branches. Analysis by the solid phase microextraction technique (SPME) showed that the emission of some electrophysiologically active compounds increased after branches had been cut, especially 4,8-dimethyl-1,3(E),7-nonatriene, linalool and (E,E)-alpha-farnesene. The identification of apple volatiles eliciting antennal responses is the first step towards the identification of compounds mediating host-finding and oviposition in codling moth females.

Acyclic Monoterpenes↗

Simulating the impact of cross resistance between Bt toxins in transformed clover and apples in New Zealand.

Simulation were conducted to guide development of resistance management strategies aimed at prolonging the usable life of B. thuringiensis (Bt) endotoxins in multiple cropping situations, where different crops expressing Bt endotoxins are host plants for a common pest. We used the New Zealand apple and clover model ecosystem to explore the relative impact on the rate of resistance development of varying levels of cross-resistance between different toxins expressed in these 2 potentially Bt-transformed crops. These 2 crops are hosts for a complex of leaf-rollers in New Zealand, including the light-brown apple moth, used here as the model pest. Cross-resistance was varied between 0.0 and 0.5 (zero to partial cross-resistance) to allow for the case in which selection by one plant has a potential effect on resistance to the toxin in another plant. The largest factor affecting the evolution of resistance was the total habitat area occupied by transgenic orchards. The proportion of the clover habitat that was transformed was also an important factor, even in the absence of cross-resistance. The effect of increasing the proportion of the second transformed crop (clover) acted on resistance evolution mainly by reducing the external refuge of susceptibility for the transgenic orchards. Hence, the ecological implications of reducing the available source of susceptible insects from clover, which can help to slow resistance development in the orchard ecosystem, had a more significant impact than the presence of cross-resistance. Partial cross-resistance between different toxins in the separate crops was overall of relatively minor importance. These simulations have implications for deployment decisions for individual transformed crops in multiple cropping systems, where there is the potential for the crops to serve as refugees for each other. These decisions may need to focus less on cross-resistance between toxins, than on economic trade-offs between the relative roles of individual crops as refugia maintaining susceptibility in the system as a whole.

Animals↗

Particle films for suppression of the codling moth (Lepidoptera: Tortricidae) in apple and pear orchards.

Studies were conducted in 1997 and 1998 to evaluate the effects of three particle film formulations consisting of kaolin and adjuvants on neonate larvae, ovipositing adult females, and eggs of the codling moth, Cydia pomonella (L.). Neonate larval walking speed, fruit discovery rate, and fruit penetration rate on apple host plants coated with particle films were significantly lower than on host plants without particle films in laboratory assays. Females oviposited less on host plants covered with a particle film residue than on untreated plants in laboratory choice and no-choice tests. Hatch rate of codling moth neonate larvae was unaffected by particle films sprayed on host plants either before or after oviposition. Fruit infestation rates were significantly reduced on particle film-treated trees compared with untreated trees for both first- and second-generation codling moth in field trials in both apple and pear orchards. Particle films appear to be a promising supplemental control approach for codling moth in orchards where moth density is high, and may represent a stand-alone method where moth densities are lower.

Animals↗

Comparison of pheromone application rates, point source densities, and dispensing methods for mating disruption of tufted apple bud moth (Lepidoptera: Tortricidae).

Small-plot (approximately 0.1 ha) studies were used to evaluate different pheromone dispensing systems, application rates, and point-source densities for mating disruption of the tufted apple bud moth, Platynota idaeusalis (Walker). Using polyvinyl chloride spirals impregnated with tufted apple bud moth pheromone (1:1 ratio of E11-tetradecenyl alcohol/E11-tetradecenyl acetate), pheromone rates of > or = 1,482 spirals per hectare (74.1 g pheromone per hectare) were superior to a rate of 988 spirals per hectare (49.4 g pheromone per hectare) in decreasing male response to pheromone traps in 1995, whereas no differences were detected among rates of 988, 1,482 and 1,975 spirals per hectare in 1996. Within a range of 370-988 pheromone dispensers per hectare, point source densities were equally effective in suppressing male response to pheromone traps. Pheromone-impregnated paraffin disks were equally effective at inhibiting male response to pheromone traps compared with polyvinyl chloride spirals. However, a paraffin emulsion formulation of pheromone applied with a hand-held grease gun provided longer residual communication disruption effects than polyvinyl chloride spirals. Dilution of paraffin emulsion pheromone formulations in water for application with a backpack sprayer and airblast sprayer rendered them ineffective in reducing male response to pheromone traps. The releases of pheromone from polyvinyl chloride spirals and paraffin disks aged in the field were described by a linear and negative logarithmic curve, respectively, indicating that dispenser life time should be longer for spirals. The ratio of acetate to alcohol components of pheromone released from spirals increased over time, whereas the release ratio remained more constant for paraffin disks. This suggests that the disruption efficacy of spirals may be prematurely reduced because of imbalance of the released components.

Animals↗

Influence of timing and prey availability on fruit damage to apple by Campylomma verbasci (Hemiptera: Miridae).

Campylomma verbasci Meyer is a zoophytophagous mirid that feeds on small arthropods as well as apple (Malus domestica Borkhausen) fruits, causing economic damage to some cultivars. The influence of timing and prey availability on the amount of fruit damage was studied to determine whether either factor could be used to refine a management program. C. verbasci nymphs were caged on branches of fruiting 'Golden Delicious' apple trees during the period from bloom through early fruit set. The greatest amount of fruit damage occurred during the bloom period; little or no damage occurred after fruit reached approximately equals 13 mm in diameter. The availability of prey did not reduce the incidence of fruit damage by C. verbasci, nor did it influence the survival of nymphs. Nymphal survival was higher, however, in cages where a blossom or fruitlet was present versus a vegetative spur. These data support the hypothesis that post petal fall insecticide applications (those made after the fruit is greater than approximately equals 10-13 mm in diameter) are not useful in preventing economic levels of fruit damage in Washington State, and that petal fall applications would only prevent a fraction of the total amount of damage by this pest. The data from this study do not support the hypothesis that manipulating arthropod prey species of C. verbasci will prevent fruit damage. There was evidence to support the hypothesis that nymphs can survive a relatively short period (7 d) without arthropod prey.

Animals↗