Rapid Detection and Quantification of Patulin and Citrinin Contamination in Fruits.

Patulin (PAT) and citrinin (CTN) are the most common mycotoxins produced by Penicillium and Aspergillus species and are often associated with fruits and fruit by-products. Hence, simple and reliable methods for monitoring these toxins in foodstuffs are required for regular quality assessment. In this study, we aimed to establish a cost-effective method for detection and quantification of PAT and CTN in pome fruits, such as apples and pears, using high-performance liquid chromatography (HPLC) coupled with spectroscopic detectors without the need for any clean-up steps. The method showed good performance in the analysis of these mycotoxins in apple and pear fruit samples with recovery ranges of 55-97% for PAT and 84-101% for CTN, respectively. The limits of detection (LOD) of PAT and CTN in fruits were 0.006 µg/g and 0.001 µg/g, while their limits of quantification (LOQ) were 0.018 µg/g and 0.003 µg/g, respectively. The present findings indicate that the newly developed HPLC method provides rapid and accurate detection of PAT and CTN in fruits.

Evaluation of confirmatory data following Article 12 MRL review and modification of the existing MRLs in pome fruits for pyridaben.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Nissan Chemical Europe SAS submitted a request to the competent national authority in the Czech Republic to modify the existing maximum residue levels (MRLs) in pome fruits and to evaluate the confirmatory data identified in the framework of the MRL review under Article 12 of Regulation (EC) No 396/2005 as not available. To address the data gaps on residue trials, new trials according to the Good Agricultural Practices (GAPs) on apples, pears, medlars, quinces, loquats/Japanese medlars, apricots, peaches and beans with pods evaluated during the MRL review were not provided. These data gaps are not addressed. However, residue trials on apples and pears for an alternative GAP were provided and resulted by extrapolation in an MRL proposal for pome fruits lower than the current (tentative) MRL in EU legislation. The provided information may require a revision of the existing MRLs for pome fruits, apricots, peaches and beans with pods. Information on storage temperature of samples from the feeding study and a validated analytical method for animal commodities were submitted. The two data gaps on animal commodities were satisfactorily addressed. Adequate analytical methods for enforcement are available to control the residues of pyridaben in plant matrices under consideration and in all animal matrices, where currently an limit of quantification (LOQ) of 0.02 mg/kg is considered at the validated LOQ of 0.01 mg/kg. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the uses of pyridaben according to the reported agricultural practices is unlikely to present a risk to consumer health.

Changes in Reactive Oxygen Species, Antioxidants and Carbohydrate Metabolism in Relation to Dormancy Transition and Bud Break in Apple (Malus × domestica Borkh) Cultivars.

Understanding the biochemical mechanisms underlying bud dormancy and bloom time regulation in deciduous woody perennials is critical for devising effective strategies to protect these species from spring frost damage. This study investigated the accumulation profiles of carbohydrates, ROS and antioxidants during dormancy in 'Cripps Pink' and 'Honeycrisp', two apple cultivars representing the early and late bloom cultivars, respectively. Our data showed that starch levels generally declined during dormancy, whereas soluble sugars increased. However, the present study did not record significant alternations in the carbohydrate accumulation profiles between the two cultivars that could account for the differences in their bloom dates. On the other hand, H2O2 accumulation patterns revealed an apparent correlation with the dormancy stage and bloom dates in both cultivars; peaking early in the early-blooming cultivar, sustaining high levels for a longer time in the late-blooming cultivars, and fading by the time of bud burst in both cultivars. Also, the redox balance during dormancy appeared to be maintained mainly by catalase and, to a lesser extent, by glutathione (GSH). Overall, the present study concludes that differences in ROS and the bud redox balance could, at least partially, explain the differences in dormancy duration and bloom date among apple cultivars.

Molecular Characterization of the Coat Protein Gene of Greek Apple Stem Pitting Virus Isolates: Evolution through Deletions, Insertions, and Recombination Events.

A RT-PCR assay developed to amplify the full coat protein (CP) gene of apple stem pitting virus (ASPV) was evaluated using 180 Greek apple and pear samples and showed a broad detection range. This method was used to investigate the presence of ASPV in quince in Greece and showed a high incidence of 52%. The sequences of 14 isolates from various hosts with a distinct RFLP profile were determined. ASPV population genetics and the factors driving ASPV evolution were analyzed using the Greek ASPV sequences, novel sequences from Brazilian apple trees and Chinese botanical Pyrus species, and homologous sequences retrieved from GenBank. Fourteen variant types of Greek, Brazilian and botanical isolates, which differ in CP gene length and presence of indels, were identified. In addition, these analyses showed high intra- and inter-group variation among isolates from different countries and hosts, indicating the significant variability present in ASPV. Recombination events were detected in four isolates originating from Greek pear and quince and two from Brazilian apples. In a phylogenetic analysis, there was a tendency for isolates to cluster together based on CP gene length, the isolation host, and the detection method applied. Although there was no strict clustering based on geographical origin, most isolates from a given country tended to regroup in specific clusters. Interestingly, it was found that the phylogeny was correlated to the type, position, and pattern of indels, which represent hallmarks of specific lineages and indicate their possible role in virus diversification, rather than the CP size itself. Evidence of recombination between isolates from botanical and cultivated species and the clustering of isolates from botanical species and isolates from cultivated species suggest the existence of a possible undetermined transmission mechanism allowing the exchange of ASPV isolates between the cultivated and wild/ornamental hosts.

Free amino nitrogen concentration correlates to total yeast assimilable nitrogen concentration in apple juice.

Yeast assimilable nitrogen (YAN) is essential for yeast growth and metabolism during apple (Malus x domestica Borkh.) cider fermentation. YAN concentration and composition can impact cider fermentation kinetics and the formation of volatile aroma compounds by yeast. The YAN concentration and composition of apples grown in Virginia, USA over the course of two seasons was determined through analysis of both free amino nitrogen (FAN) and ammonium ion concentration. FAN was the largest fraction of YAN, with a mean value of 51 mg N L-1 FAN compared to 9 mg N L-1 ammonium. Observed YAN values ranged from nine to 249 mg N L-1, with a mean value of 59 mg N L-1. Ninety-four percent of all samples analyzed in this study contained <140 mg N L-1 YAN, a concentration generally considered the minimum level needed in grape-based wines for yeast to fully utilize all of the fermentable sugars. FAN concentration was correlated with total YAN concentration, but ammonium concentration was not. Likewise, there was no correlation between FAN and ammonium concentration.

Rapid and sensitive detection of pyrimethanil residues on pome fruits by Surface Enhanced Raman Scattering.

Surface Enhanced Raman Scattering (SERS) supported by gold nanoparticles (AuNPs) was applied to detect and quantify residues of pyrimethanil on pome fruits, a widely used fungicide in horticultural species. Spheroidal AuNPs with different size were fabricated and compared in this study. The analytical procedure was set up on a silicon dioxide flat substrate to standardize SERS methodology. A Raman mapping strategy was exploited to increase signal reproducibility and to minimize bias due to different local surface morphologies. Univariate and multivariate regressions were compared for calibration. Multivariate PLS approach demonstrated acceptable repeatability and method stability (RMSECV = 4.79 ppm; RMSEP = 4.31 ppm) in the range 0-40 mg kg-1, providing higher accuracy and intra-day repeatability with a mean percentage error of 18.7% and 32.8% for PLS and univariate calibration, respectively. The method here proposed can be reliably applied for PMT detection on pome fruits within the European law limits.

Experimental Bin Drenching System for Testing Biocontrol Agents to Control Postharvest Decay of Apples.

A portable drencher capable of drenching a single bin of fruit was built to simulate the commercial application of chemicals to harvested apples in small orchard operations in the central and eastern United States. The drencher required as little as 125 liters of the treatment solution and permitted various bin travel speeds. Wounded apples were placed midway between the bottom and top of the bin, in the center, and near the four corners of the bin (20 fruit per location) and covered with enough unwounded apples to fill the bin. The bins were drenched with a suspension containing Penicillium expansum at 2 × 104 conidia per ml in 2000, 5 × 103 conidia per ml in 2001, and 3 × 103 conidia per ml in 2002 and 2003. In 2000 and 2003, the additional treatments included a combination of P. expansum with the yeast Metschnikowia pulcherrima at ~;1.2 × 107 CFU/ml, and in 2003 a combination with 2% sodium bicarbonate (SB) or a mixture of the yeast and SB. After 3 months of storage at ~;2°C, at all P. expansum conidial concentrations, more than 90% of wounded fruit developed decay on 'Golden Delicious', 'Delicious', and 'Rome' apples in the 2000-02 experiments. In 2003, 66 and 33.1% of the wounded fruit developed decay on 'Delicious' and 'Golden Delicious', respectively. The application of the antagonist reduced decay to 39 and 3.3% on 'Golden Delicious' in 2000 and 2003, respectively, and to 26% on 'Delicious' in 2003. The addition of SB reduced decay on both cultivars and, in combination with the yeast, was the most effective treatment on 'Golden Delicious'. This portable drencher can be very useful for evaluating different treatments applied to apples after harvest at the commercial level.

Susceptibility of the Stem Pull Area of Mechanically Harvested Apples to Blue Mold Decay and Its Control with a Biocontrol Agent.

A new harvester, which uses a rapid displacement actuator on the main scaffolds to remove apples from trees with narrow-inclined trellises, has shown good potential. With this technique, stem loss (stempulls) during harvest ranges from 20 to 57%, depending on the cultivar. This can create a potential point of entry for pathogens. We evaluated the susceptibility of the stem cavity area, with and without stems, to blue mold decay (Penicillium expansum) on three cultivars of mechanically harvested apples, and tested the effectiveness of the antagonist Pseudomonas syringae (used in BioSave 110) in controlling decay. Fruit with stempulls were more susceptible to blue mold decay than fruit with stems. On fruit with stempulls inoculated with P. expansum and stored for 2 months at 1°C, decay incidence was 0% on 'Pink Lady', 8.3% on 'Ace Spur Delicious', and 41% on 'Empire'. On fruit with stems, there was no decay on all three cultivars. P. syringae reduced decay on 'Empire' with stempulls to 3.3%, and no decay occurred on the other two cultivars. Similar trends were observed on fruit stored at 22°C for 14 days, but the incidence of decay was higher, and only 'Pink Lady' had no decay on the antagonist-treated fruit. Although mechanical harvesting can predispose the stem cavity to decay in some cultivars, this problem can be alleviated using biological control without resorting to the use of synthetic pesticides.