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Vitamin C is a crucial antioxidant and cofactor for both plants and humans. Apple fruits generally contain low levels of vitamin C, making vitamin C content an interesting trait for apple crop improvement. With the aim of breeding high vitamin C apple cultivars it is important to get an insight in the natural biodiversity of vitamin C content in apple fruits. In this study, quantification of ascorbic acid (AsA), dehydroascorbic acid (DHA), and total AsA (AsA + DHA) in apple pulp of 79 apple accessions at harvest revealed significant variation, indicating a large genetic biodiversity. High density genotyping using an 8 K SNP array identified 21 elite and 58 local cultivars in this germplasm, with local accessions showing similar levels of total AsA but higher amounts of DHA compared to elite varieties. Out of the 79 apple cultivars screened, ten genotypes with either the highest or the lowest concentration of total AsA at harvest were used for monitoring vitamin C dynamics during fruit development and storage. For all these cultivars, the AsA/DHA ratio in both apple pulp and peel increased throughout fruit development, whereas the AsA/DHA balance always shifted towards the oxidized form during storage and shelf life, putatively reflecting an abiotic stress response. Importantly, at any point during apple fruit development and storage, the apple peel contained a higher level of vitamin C compared to the pulp, most likely because of its direct exposure to abiotic and biotic stresses.

We describe a semi in vivo pollination technique to determine the compatibility relation between different pear cultivars. This assay provides a valuable addition to existing tools in GSI research. The gametophytic self-incompatibility (GSI) system in Pyrus inhibits fertilization by pollen that shares one of the two S-alleles of the style. Depending on their S-locus genotype, two pear cultivars therefore either show a cross-compatible, semi-compatible or incompatible interaction. Because GSI greatly influences seed and fruit set, accurate knowledge of the compatibility type of a cultivar is key for both pear fruit production and breeding. Currently, compatibility relations between different pear cultivars are generally assessed via S-genotyping. However, this approach is restricted to the currently known S-alleles in pear, and does not provide functional assessment of the level of (self-)incompatibility. We here present an optimized semi in vivo pollination assay, that enables quantitative analysis of (self-)incompatibility in pear, and that can also serve useful for more fundamental studies on pollen tube development and pollen-style interactions. This assay involves in vitro incubation of cut pollinated styles followed by microscopic counting of emerging pollen tubes at a specific time interval. The validity and selectivity of this method to determine compatibility interactions in pear is demonstrated in the cultivars "Celina" and "Packham's Triumph." Overall, this technique constitutes a valuable tool for quantitatively determining in vivo pollen tube growth and (cross-)compatibility in pear.

Site-specific high throughput monitoring of airborne ascospores of Venturia inaequalis, the causal agent of apple scab, can improve existing warning systems. A new qPCR assay was developed to quantify ascospores collected by a simple rotating-arm spore sampler. The qPCR assay was highly specific and sensitive, with a limit of quantification of 20 ascospores per sample. The new detection system was compared to sampling with a traditional Burkard volumetric spore trap and to microscopic quantification. During controlled ascospore release experiments in a closed environment, strong correlations (ρ: 0.96 to 0.99) were observed between the two types of samplers and the two methods of quantification but significantly larger numbers of spores (log difference: 0.43 to 0.69) were obtained when using the rotating-arm sampler and when using molecular quantification. During comparisons under outdoor conditions over a three-year period, reasonable correlations between the techniques (average ρ = 0.61) were observed. When rotating-arm samplers operate continuously they can get saturated but their counts still correlated better with those from the Burkard sampler than when they only operate during rain and until two hours after. This suggests that ascospores were also captured outside of rain events. Based on these comparisons, molecular quantification of spores captured with the rotating-arm sampler appears to be a sensitive and reliable method to determine airborne ascospores of V. inaequalis and holds promise as a tool to guide targeted fungicide applications in commercial orchards as well as to increase our knowledge of the aerobiology of this pathogen.

Colletotrichum spp. infections are an increasing threat to the fruit production industry worldwide, as a postharvest disease. Gaining knowledge on the susceptibility of apple fruit cultivars to Colletotrichum species is essential for specific breeding programs aiming to decrease the disease susceptibility of commercial apple cultivars. We investigated fruit susceptibility of nine apple cultivars at harvest and after storage, and studied the relationship of the susceptibility with fruit ripening characteristics. An accurate artificial inoculation technique, by wounding and injecting the spore suspension, was used to consistently follow-up the relative susceptibility symptoms of fruits. The nine apple cultivars studied, cvs. Pinova, Nicoter, Nicogreen, Golden Delicious, Gala, Elstar, Jonathan, Idared and Topaz, showed clear differences in susceptibility to C. fioriniae, main species causing problems in Europe, based on lesion expansion growth rate data. Cultivar Nicoter and Nicogreen were evaluated as less susceptible cultivars, while cv. Golden Delicious and cv. Pinova were classified as high susceptible cultivars. After three months of storage all fruit cultivars showed a higher susceptibility than at harvest. We further explored the link between fruit ripening characteristics and postharvest pathogen developments. Results showed significant higher Brix, ethylene production and respiration and significantly lower firmness and titratable acids after storage, with the extent of these changes being cultivar dependent. The ripening parameters respiration and Brix had a positive and the firmness a negative correlation with the lesion expansion growth rate of C. fioriniae after artificial wound inoculations. These experiments help to give a better insight in the susceptibility of different cultivars. Moreover, our data support the hypothesis that the extent of the necrotrophic fungal development depends on the fruit ripening stage. ispartof: EUROPEAN JOURNAL OF PLANT PATHOLOGY vol:155 issue:3 pages:801-816 status: published

Currently, one of the most important challenges is to provide sufficient and affordable food and energy for a fast-growing world population, alongside preserving natural habitats and maintaining biodiversity. About 35% of the global food production depends on animals for pollination. In recent years, an alarming worldwide decline in pollinators has been reported, putting our food production under additional pressure. Therefore, there is an urgent need to find sustainable ways to ensure this crucial ecosystem service. Recent studies have shown that floral nectar is generally colonized by microorganisms, specifically yeasts and bacteria, which may alter nectar chemistry and enhance attraction of pollinators. In this study, we investigated changes in pollinator foraging behavior and pollination success in European pear (Pyrus communis L.) cultivars ‘Regal Red’ and ‘Sweet Sensation’ (red sports of ‘Doyenné de Comice’) after flower inoculation with the typical nectar-inhabiting microorganisms Metschnikowia reukaufii and Acinetobacter nectaris, and a combination of both. Pollination success was monitored by measuring the number of flower visits, fruit set and seed set in two consecutive years, 2019 and 2020. Results revealed that application of a mixture of M. reukaufii and A. nectaris resulted in significantly higher visitation rates of honeybees and hoverflies. By contrast, no effects on flower visits were found when yeasts and bacteria were applied separately. Fruit set and seed set were not significantly affected by any of the inoculation treatments. The only factors affecting fruit set were initial number of flower clusters on the trees and the year. The absence of treatment effects can most likely be attributed to the fact that pollination was not a limiting factor for fruit set in our experiments. Altogether, our results show that inoculation of flowers with nectar microbes can modify pollinator foraging patterns, but did not lead to increased pollination success under the conditions tested.

In the original publication, the positions of the SNP markers from the 50K SNP array are given according toGDDH13 v1.0 version of the apple reference genome and not according to GDDH13 v1.1 version as stated in the original publication.

‘Conference’ (Pyrus communis L.) is a self-incompatible cultivar, although it can also set fruit parthenocarpically. Stimulating parthenocarpy through gibberellin (GA) applications increases the fruit set, but it may also negatively affect the fruit size and shape. The aim of this study was to investigate the effects of a bumblebee (BB) amendment in combination with a GA treatment on the fruit set and fruit shape of ‘Conference’ pears. In the first experiment, we applied three treatments (GA, GA + BB & control) in a ‘Conference’ monoculture. In the second experiment, we applied two treatments (GA & GA + BB) in a ‘Conference’ orchard inter-planted with ‘Concorde’ as pollinizer trees. Both experiments showed that the GA application and bumblebee supplementation did not affect the fruit set. However, the BB treatment resulted in significantly higher amounts of normally shaped pears. Trees closer to the bumblebee hive had more normal shaped pears than trees further away. ispartof: Horticultural Science vol:47 issue:4 pages:211-216 status: published

There is an urgent need for novel, efficient and environmentally friendly strategies to control apple scab (Venturia inaequalis), for the purpose of reducing overall pesticide use. Fructans are recently emerging as promising &ldquo, priming&rdquo, compounds, standing out for their safety and low production costs. The objective of this work was to test a fructan-triggered defense in the leaves of apple seedlings. It was demonstrated that exogenous leaf spraying can reduce the development of apple scab disease symptoms. When evaluated macroscopically and by V. inaequalis-specific qPCR, levan-treated leaves showed a significant reduction of sporulation and V. inaequalis DNA in comparison to mock- and inulin-treated leaves, comparable to the levels in fosetyl-aluminum-treated leaves. Furthermore, we observed a significant reduction of in vitro mycelial growth of V. inaequalis on plates supplemented with levans when compared to controls, indicating a direct inhibition of fungal growth. Variations in endogenous sugar contents in the leaves were followed during priming and subsequent infection, revealing complex dynamics as a function of time and leaf ontogeny. Our data are discussed in view of the present theories on sugar signaling and fructan-based immunity, identifying areas for future research and highlighting the potential use of fructans in apple scab management in orchards.

There is an urgent need for novel, efficient and environmentally friendly strategies to control apple scab (Venturia inaequalis), for the purpose of reducing overall pesticide use. Fructans are recently emerging as promising “priming” compounds, standing out for their safety and low production costs. The objective of this work was to test a fructan-triggered defense in the leaves of apple seedlings. It was demonstrated that exogenous leaf spraying can reduce the development of apple scab disease symptoms. When evaluated macroscopically and by V. inaequalis-specific qPCR, levan-treated leaves showed a significant reduction of sporulation and V. inaequalis DNA in comparison to mock- and inulin-treated leaves, comparable to the levels in fosetyl-aluminum-treated leaves. Furthermore, we observed a significant reduction of in vitro mycelial growth of V. inaequalis on plates supplemented with levans when compared to controls, indicating a direct inhibition of fungal growth. Variations in endogenous sugar contents in the leaves were followed during priming and subsequent infection, revealing complex dynamics as a function of time and leaf ontogeny. Our data are discussed in view of the present theories on sugar signaling and fructan-based immunity, identifying areas for future research and highlighting the potential use of fructans in apple scab management in orchards.

Fruit production is of major importance in Flanders, with a production of 318,000 t of apples and 374,000 t of pears (2014), with ‘Jonagold’ and ‘Conference’ as main cultivars, respectively. In apple, it is believed that cross pollination can increase yield and improve yield stability and fruit quality, therefore, pollen donors are planted within the orchard, and pollinators such as honeybees (Apis mellifera L.) are commonly introduced. In contrast to apple, pear growers pay little attention to pollination. One problem is the limited attractiveness of pear nectar for pollinators, probably because of the relatively low sugar concentration and/or unattractive sugar composition. In this contribution we monitored the pollinator activity in an apple and pear orchard after introducing managed bumblebee hives, on the base of standardized transect walks. Furthermore, we compared the sugar content in the nectar between apple and pear flowers. Overall, flower visitation by bumblebees was very low, especially for pear where no bumblebees were observed. A first analysis confirms our hypothesis of higher sugar concentrations in the nectar of apple flowers (‘Jonagold’) compared to pear flowers (‘Conference’). Furthermore, the percentage of sucrose was noticeably lower in pear nectar (3%) compared to apple nectar (63%). This likely explains the low attractiveness of pear flowers compared to apple.

PREMISE OF THE STUDY: Recent research has highlighted the importance of living tissue in wood. Polyploidization can impact amounts and arrangements of living cells in wood, potentially leading to increased drought tolerance. Tetraploid variants were created from the apple cultivar Malus xdomestica 'Gala' (Gala-4x), and their vulnerability to drought-induced cavitation and their hydraulic capacitance were compared to those of their diploid predecessors (Gala-2x). Assuming a positive correlation between polyploidy and drought tolerance, we hypothesized lower vulnerability and higher capacitance for the tetraploid. METHODS: Vulnerability to drought-induced cavitation and the hydraulic capacitance were quantified through acoustic emission and continuous weighing of shoots during a bench-top dehydration experiment. To underpin the hydraulic trait results, anatomical variables such as vessel area, conduit diameter, cell wall reinforcement, and ray and vessel-associated parenchyma were measured. KEY RESULTS: Vulnerability to drought-induced cavitation was intrinsically equal for both ploidy variants, but Gala-4x proved to be more vulnerable than Gala-2x during the early phase of desiccation as was indicated by its significantly lower air entry value. Higher change in water content of the leafy shoot, higher amount of parenchyma, and larger vessel area and size resulted in a significantly higher hydraulic capacitance and efficiency for Gala-4x compared to Gala-2x. CONCLUSIONS: Both ploidy variants were typified as highly sensitive to drought-induced cavitation, with no significant difference in their overall drought vulnerability. But, when water deficit is short and moderate, Gala-4x may delay a drought-induced decrease in performance by trading hydraulic safety for increased release of capacitively stored water from living tissue.

Apple (Malus × domestica Borkh.) production is significantly affected by apple scab disease caused by the fungus Venturia inaequalis. Currently, management of the disease is largely based on fungicide applications and less on the inherent resistance in apple cultivars carrying resistance (Rvi) genes. Moreover, the durability of both approaches is questionable, since new virulent strains with resistance to fungicides and/or Rvi genes can develop. Hence, a combination of complementary management strategies is needed to tackle apple scab. To broaden the management options, a viable approach might be the breeding for polyploid cultivars and their implementation in commercial orchards. Apple germplasm is substantially characterized by polyploidy, which throughout the evolution of angiosperms often led to improved adaptation to adverse climatic conditions and abiotic stresses. However, the role of polyploidy in conferring resistance to biotic challenges is less clear. Here, we show that autopolyploidy in two apple cultivars, i.e. the susceptible ‘Gala’, and the resistant ‘Makali’ which carries the Rvi6 scab resistance gene, can reduce severity of apple scab symptoms upon inoculation with two different V. inaequalis isolates. Leaves of tetraploid plants show reduced sporulation symptoms in comparison with their diploid counterparts upon infection with both pathogenic strains, attributed to a reduced presence of V. inaequalis. These results suggest that polyploidy enhances resistance of apple cultivars against different V. inaequalis isolates. However, the degree of the enhanced resistance depends on a plant-pathogen-specific interaction. The knowledge provided here offers a relevant framework for improving our understanding of polyploidy-enhanced resistance to biotic stress in plants and may set the stage for implementing polyploidy breeding for further genetic improvement of apple.

Apple is a seasonal product that is stored for long periods of time, during which fungal‐caused decay can occur. Previous infection experiments of intact ‘Jongaored’ apple with Botrytis cinerea displayed apparent differences in lesion expansion rate with respect to inoculation position on the fruit (on, above or below the equator). The goal of this study was to (i) investigate whether these differences are consistent or not, and, if so, to (ii) relate them to fruit characteristics. We measured the hue angle of the intended inoculation spots prior to inoculation, and firmness and total soluble solids content of fruit from the same batch. Results showed that firmness correlated somewhat (‐0.55 and ‐0.72 for shadow and sun side, respectively) with lesion diameter expansion rate. In a subsequent step, X‐ray imaging was carried out for samples from each position. Analysis of 3D reconstructions by micro computed tomography of these diffraction images showed that cell size was strongly correlated (0.996) to lesion diameter expansion rate. Finally, it was investigated if cell size could also be used to rank different apple cultivars for their susceptibility to B. cinerea. The result shows that there is a clear distinction between ‘Jonagold’ and Golden ‘Delicious’ (non‐blushing variety), which have a smaller cell size, and ‘Braeburn’ and ‘Kanzi’, which have a larger cell size (overall correlation of 0.87). This indicates that cell size may also be an important factor in determining susceptibility across cultivars. ispartof: PLANT PATHOLOGY vol:69 issue:1 pages:60-67 status: published

Acidity and sweetness are important qualities for apple breeders and understanding their genetic regulation can improve the breeding process. In previous QTL studies, fruit quality assessments were performed using instrumental measurements, leading to the identification of two major QTL Ma and Ma3. Here, we use sensorial data to investigate the role of known and unknown genetic factors in the perception of acidity and sweetness in three pedigreed full-sib families. For 2 years, these families were phenotyped by a trained panel, at harvest and after 2 months of cold storage, and genotyped with a new 50 K SNP array. FlexQTLTM analyses using both an additive and an additive + dominance model resulted in the identification of Ma and Ma3 for acidity as well as sweetness, whereas the use of the additive model yielded decisive evidence for the discovery of two additional QTL on LG1 and LG6 for acidity. QTL genotypes were qualified as the inverse of each other, which indicates that individuals with the less-acidity alleles of Ma and Ma3 are perceived sweeter. Due to the genetic configuration in the families studied, resulting from a link with the Pale Green Disorder locus, no incomplete dominance effect could be detected for the Ma locus, although previously reported in the literature. For the Ma3 locus, however, an incomplete dominance effect (58%) is reported here for the first time. The Ma3 locus was also further confined to a 2–4-cM region and a predictive marker for this locus was identified.

Production of many agricultural crops and fruits strongly depends on pollinators. For instance, pome fruits such as apple and pear are highly dependent on pollination for fruit set, fruit quality, and yield. Nectar is often inhabited by microbes, most often yeasts and bacteria, which may change nectar quality and therefore also affect plant–pollinator interactions. Here, we used high‐throughput 16S ribosomal RNA gene amplicon sequencing to investigate the temporal and spatial variation in bacterial communities in floral nectar of apple and pear. We sampled 15 apple (Malus x domestica Borkh.) and 15 pear (Pyrus communis L.) orchards distributed over the eastern part of Belgium over a timespan of seven days. Nectar bacterial community composition differed strongly among fruit species. Nectar of pear was dominated by Actinobacteria, followed by Proteobacteria and Firmicutes. Apple nectar was strongly enriched in Bacteroidetes, a phylum which until now has been found to be rarely associated with floral nectar. Nectar was dominated by only a few bacterial species, with Brevibacterium (Actinobacteria) and Undibacterium (Proteobacteria) as the most abundant bacteria in pear and apple nectar, respectively. Bacterial richness and diversity were found to fluctuate during flowering, likely due to changing environmental conditions. Additionally, spatial structure in nectar bacterial community composition was found in apple orchards, while this was not the case for pear. Differences in nectar bacterial communities between apple and pear nectar may differently affect the chemical and nutritional composition of the nectar, influencing pollinator attraction and visitation, and thus pollination efficacy in general., Nectar bacterial community composition was dominated by only a few bacterial species and differed strongly among fruit species. The nectar of pear (b) was dominated by Actinobacteria, Proteobacteria, and Firmicutes, while apple nectar (a) was strongly enriched by Bacteroidetes. Bacterial richness and diversity were found to fluctuate during flowering.

Worldwide Colletotrichum spp. have been identified as a problem in the apple production. This is the first study executed and confirming the presence of Colletotrichum spp. causing the postharvest disease bitter rot on apple fruits in Belgium. The identification, genetic diversity of Colletotrichum isolates (present in Belgian apple orchards) their morphological traits and pathogenicity on two apple cultivars (cvs. Pinova and Nicoter) with a different level of susceptibility were studied. Based on sequence analysis of six different gene regions beta-tubuline (TUB2), histone H3 (HIS3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 gene (CHS-1), actin (ACT) and the Internal Transcriber Spacer (ITS) gene region, six different Colletotrichum spp., belonging to either the C. acutatum or C. gloeosporioides complexes, were isolated from twenty-one apple cultivars in three Belgian orchards: C. fioriniae, probably C. kahawae, C. salicis, C. rhombiforme, C. acutatum and C. godetiae. Colletotrichum godetiae was found to be the most present and pathogenic species in Belgian orchards. The species C. rhombiforme was found and identified on apple fruit for the first time. Reliable morphological discrimination between species, based on features such as in vitro growth rate, colony colour and spore measurements, is not possible. As such, molecular identification appears to outperform morphological analysis and was in this study the most ideal tool for identifying unknown isolates of Colletotrichum species. Inoculation assays on two apple cultivars revealed a significant difference in pathogenicity among isolates and among Colletotrichum species. The pathogenicity tests also showed that isolates coming from another host species, e.g. strawberry, are also pathogenic on apple fruits. Cultivar Pinova appeared to be more susceptible to bitter rot than cv. Nicoter. Given the difficulties with managing Colletotrichum infections, additional knowledge on the pathogen and the plant-pathogen interaction is essential for effective disease control. ispartof: EUROPEAN JOURNAL OF PLANT PATHOLOGY vol:153 issue:1 pages:47-63 status: published

Postharvest losses of fruit and vegetables can reach up to 30%, the main cause being microbial decay. For apple fruit, mostly fungal pathogens, such as Penicillium expansum, Colletotrichum spp., Neofabraea spp. and Botrytis cinerea, are important. As such losses are unsustainable in many ways, it is necessary that research is conducted to prevent them. Generally, for plants and fruit grown under non-sterile field conditions, disinfection is carried out prior to the start of a phytopathological experiment. The motivation for this practice is the removal of background contamination so that it will not affect the experimental outcome and its interpretation. In literature, a plethora of disinfection methods exists, differing in disinfectant, strength and duration. The following two disinfectants are commonly used: sodium hypochlorite (NaOCl) and ethanol. This article presents a targeted investigation into the effects of these two disinfectants on apple fruit surface and physiology. The results clearly demonstrate that both were affected by both disinfectants. NaOCl caused oxidative damage to the apple's wax layer, causing it to crack. Ethanol affected a redistribution of the wax on the fruit surface and altered the wax composition and/or metabolism. Both NaOCl and ethanol treatment resulted in an increased respiration rate. Therefore, apple and possibly other fruit should not be disinfected in phytopathological studies. A negative control, as is typically used, is not solving this issue, as we clearly demonstrate that the living tissue shows metabolic effects following disinfection, and hence the study objects are changed, hampering a clear interpretation of the experimental outcomes. Moreover, fungal inoculation during experiments is typically taking place at rather large levels in wounded tissue (as infection success is the exception), outnumbering the variable levels of background population, if present.

© 2017 Elsevier Ltd Several papers highlight methodological challenges related to the lifecycle assessment (LCA) of fruit production systems. These concern both the limited number of impact categories assessed in current LCA literature and the narrow view on the full production phase of an orchard cycle. This article addresses these challenges and contributes to improving the scientific knowledge on impacts associated with less productive years within an orchard cycle, and how these affect the impact associated with an entire fruit growing cycle. Using apple as a case study, an LCA is performed to obtain the impacts associated with young and old low productive trees, alongside those associated with trees in full production. Using the ILCD impact assessment method, the LCA is based on a large dataset of apple orchards in Flanders (Belgium), covering three production systems (conventional, integrated and organic production) and accounting for input and yield variability. The annual median impact values are used to describe a “typical” orchard for each orchard phase and production system. In conventional farming, lowest annual median impacts are mostly found in the full production phase while highest impacts are observed in the old low productive orchards. In integrated and organic production on the other hand, the lowest annual median impacts mostly occur in the old low productive orchards while highest impacts are found for the young low productive trees. Results for organic farming must however be interpreted with care, following the small sample size of organic producers. To calculate the impacts associated with a full orchard cycle, an orchard model was built based on the annual median impact within each apple bearing orchard phase and production system and using weighting factors based on the yields obtained within each of the orchard phases. Across the three production systems, the two low production phases are responsible for 27 to 38% of the calculated orchard cycle impact. Calculated impacts for the entire orchard cycle are, on average across the impact categories, higher than full production impacts in conventional and integrated farming, while lower in the case of organic farming. A mere focus on high productive trees leads to an underestimation of, on average, 18% in conventional farming and 11% integrated farming, versus an overestimation of 11% for organic farming. Inclusion of non-productive phases such as nursery and planting and destruction of the orchard, would further alter the orchard cycle impacts. publisher: Elsevier articletitle: Life cycle assessment (LCA) for apple orchard production systems including low and high productive years in conventional, integrated and organic farms journaltitle: Agricultural Systems articlelink: http://dx.doi.org/10.1016/j.agsy.2017.01.007 content_type: article copyright: © 2017 Elsevier Ltd. All rights reserved. ispartof: Agricultural Systems vol:153 pages:81-93 status: published

Apple is an economically important product worldwide. Because it is stored for long periods under low temperature and controlled atmosphere (CA) conditions fungal-caused decay during storage can occur. This complex multi-pathogen problem, caused by fungi with different infection mechanisms and conditions, requires various control strategies. However, very little information is available on which pathogens primarily cause this decay. Therefore, the goal of this study was to collect decayed samples from CA stored apples and isolate and identify the pathogens. Additionally, we aimed at investigating whether there is a link between rot incidence and fruit history and characteristics. In this experiment ‘Nicoter’ apple showed the highest abundance for the genus Penicillium, followed by Fusarium, Botrytis, Neonectria and Monilinia. We detected a significant correlation between fruit mass and rot incidence, most likely due to a relation between fruit mass and pre/postharvest factors. Larger fruit may have been in a more advanced ripening stage, making them more susceptible.

Malus × domestica Borkh."Golden Delicious" is one of the most important apple cultivars worldwide. However, if damaged, the fruit becomes susceptible to opportunistic infection by postharvest phytopathogens such as Botrytis cinerea (gray mould) and Penicillium expansum (blue mould) which annually lead to large economic losses worldwide. Therefore, a proteomic study of the fruit response to wounding would contribute to better understanding of the physiological mechanisms underlying fruit stress response. In this study, we provide the first systematic description of time-dependent protein changes in wounded"Golden Delicious" apples, using 2D-PAGE and MS for protein identification. Publicly available protein and EST databases were used for protein identification and annotation. Proteins that did not change in abundance after wounding were mainly categorized in the biological process of"metabolism","response to stress", and"oxidation-reduction processes", whereas proteins with changes in abundance after wounding were mainly categorized in"response to stress". We suggest that fruits react to mechanical damage by synthesizing a broad range of PR proteins. Increasing our knowledge of apple fruit proteins in wounded fruit tissues will be useful in developing new strategies to minimize postharvest losses.

This study represents a systematic evaluation of protocols for protein extraction and cleanup for fruit proteomic analysis. Procedures were optimized using pooled lyophilized banana fruit pulp, which is known to be particularly tricky due to high concentrations of soluble polysaccharides, phenolics, and other substances that interfere with protein extraction and purification. A total of 18 combinations of three protein extraction procedures (SDS-based, Triton X-100-based, and phenol-based), three protein precipitating agents (ammonium acetate/methanol, TCA/acetone, and acetone), and two resolubilization buffers (classical Rabilloud and the so-called R2D2) were compared for total protein yields and efficiency of recovery. The results demonstrate that while losses in total recovered protein are unavoidable, the degree of these losses depends on the method combinations used. Combinations based on buffer-saturated phenol always gave the highest yields, and overall recovery and purity was highest when acetone was combined with the R2D2 buffer for protein purification and concentration. Comparative 2D-PAGE analysis confirmed that this method combination produced high-quality and reproducible gels and the largest numbers of spots per gel. The usefulness of this methodology was demonstrated on ripe fruits from several other species and shown to give excellent results.

Novel and existing hyperspectral vegetation indices were evaluated in this study, with the aim of assessing their utility for accurate tracking of leaf spectral changes due to differences in biophysical indicators caused by apple scab. Novel indices were extracted from spectral profiles by means of narrow-waveband ratioing of all possible two-band combinations between 350 nm and 2500 nm at nanometer intervals (2 311 250 combinations) and all possible two-band derivative combinations. Narrow-waveband ratios consisting of wavelengths of approximately 1500 nm and 2250 nm, associated with water content, have proven to be the most appropriate for detecting apple scab at early developmental stages. Logistic regression c-values ranged from 0.80 to 0.88. At a more developed infection stage, vegetation indices such as R440/R690 and R695/R760 exhibited superior distinction between non-infected and infected leaves. Identified derivative indices were located in similar regions. It therefore was concluded that the most appropriate indices at early stages of infection are ratios of wavelengths situated at the water band slopes. The choice of appropriate indices and their discriminatory performances, however, depended on the phenological stage of the leaves. Hence, an undisturbed 20-day growth profile was examined to assess the effect of physiological changes on spectral variations at consecutive growth stages of leaves. Results suggested that an accurate distinction could be made between different leaf developmental stages using the 570 nm, 1460 nm, 1940 nm and 2400 nm wavelengths, and the red-edge inflection point. These results are useful to crop managers interested in an early warning system to aid proactive system management and steering.

An innovative index for LAI estimation over orchard canopies based on the combination of two near-infrared narrow wavebands (1050 nm and 1250 nm) is presented. The index is insensitive to physiological changes on the leaf level even under changing pheneological developmental stage. In the meantime, the index is highly sensitive for variations at the canopy level. Values of LAI estimated by means of this newly developed index, called sLAIDI (standardized LAI Determining Index), resulted in excellent agreements with in-situ measured LAI values of citrus trees, with determination coefficients (R2) of 0.83. Based on in-situ measured data and simulated data (PROSPECT and ACRM), the newly developed sLAIDI was also found to perform outstanding at high LAI values in contrary to well-known LAI related indices such as the Normalized Difference Vegetation Index (NDVI) and the Modified Chlorophyll Ratio Index 2 (MCARI2) that saturate at LAI values of 5 and 8 respectively. Moreover, the sLAIDI index was independent of changing pigment contents. Consequently, this index was regarded a valuable tool to more accurately extract biochemical parameters after normalizing the considered spectral data for LAI. By implementing sLAIDI in existing chlorophyll-related indices (e.g., GM index), the performance to estimate the leaf chlorophyll content from spectral citrus and peach canopy level data is increased (R2 increases from 0.68 to 0.77). Moreover, a correction factor was proposed to enable the application of sLAIDI on fields with strong variations of water content.

Hyperspectral canopy reflectance measurements are usually obtained under ambient solar illumination. In some specific cases, however, the use of artificial light is preferred. This raises issues, since artificial light is usually non-parallel and non-homogeneous over the area and height profile of the target. This study therefore attempts to address the characteristics of artificial illumination and the consequences for remote sensing. A low-cost experimental setup is proposed to counter the main issues of using artificial light in hyperspectral research.

The use of hyperspectral approaches for early detection of plant stress caused by Venturia inaequalis (apple scab) was investigated to move towards more efficient and reduced application of pesticides, fertilizers or other crop management treatments in apple orchards. Apple leaves of the resistant cultivar, Rewena and the susceptible cultivar, Braeburn, were artificially inoculated with conidia of V. inaequalis in a controlled greenhouse environment. The research focused on (i) determining if leaves infected with V. inaequalis could be differentiated from non-infected leaves, (ii) investigating at which developmental stage V. inaequalis infection could be detected, and (iii) selecting wavelengths that best differentiated between infected and non-infected leaves. Hyperspectral data were used because of their contiguous nature and the abundance of narrow wavelength bands in the electromagnetic reflectance spectrum, thereby providing the spectral sensitivity needed to detect subtle variations in reflectance. Processing the data, however, presented challenges, given the need to avoid data redundancy, identification of data extraction techniques, and maintaining modeling accuracy. Statistical techniques therefore had to be robust. Logistic regression, partial least squares logistic discriminant analysis, and tree-based modeling were used to select the hyperspectral bands that best defined differences among infected and non-infected leaves. Results suggested that good predictability (c-values > 0.8) could be achieved when classifying infected plants based on these supervised classification techniques. It was concluded that the spectral domains between 1350–1750 nm and 2200–2500 nm were the most important regions for separating stressed from healthy leaves immediately after infection. The visible wavelengths, especially around 650–700 nm, increased in importance three weeks after infection at a well-developed infection stage. Identification of such critical spectral regions constitutes the logical first step towards development of robust stress indicators based on hyperspectral imagery. © 2007 Elsevier B.V. All rights reserved.

One-year-old, ‘Granny Smith’ apple ( Malus × domestica Borkh.) shoots were selected randomly from commercial orchards in 1999 and 2003, cold stored at temperatures between 1 and 13 °C for varied periods, following a 12/12 h freezing temperature pre-treatment of −1/13 °C (supposedly non-chilling temperatures) for 1 or 2 weeks. The rate of budburst, determined after forcing at 25 °C, was used to follow the progression of bud dormancy. The freezing pre-treatment clearly enhanced (deepened) bud dormancy in all experiments. Clearly definable influences due to the presence of leaves during the pre-treatment were not observed on shoots cold stored at chilling temperatures. Trees were defoliated before leaf drop in late summer/autumn under field conditions by hand (2001) and using a spray containing 3% urea and 1.8% zinc sulphate in 2003. In 2001 the first two hand defoliation dates significantly enhanced dormancy, while in 2003 chemical defoliation had no effect. These data indicate that the leaves are not clearly involved in the perception of factors that are responsible for dormancy induction. As with dormancy release it is possible that the perception of induction factors, i.e. low temperatures, and as shown in these data, freezing temperatures, occurs within the buds themselves.

This study represents a systematic evaluation of protocols for protein extraction and cleanup for fruit proteomic analysis. Procedures were optimized using pooled lyophilized banana fruit pulp, which is known to be particularly tricky due to high concentrations of soluble polysaccharides, phenolics, and other substances that interfere with protein extraction and purification. A total of 18 combinations of three protein extraction procedures (SDS-based, Triton X-100-based, and phenol-based), three protein precipitating agents (ammonium acetate/methanol, TCA/acetone, and acetone), and two resolubilization buffers (classical Rabilloud and the so-called R2D2) were compared for total protein yields and efficiency of recovery. The results demonstrate that while losses in total recovered protein are unavoidable, the degree of these losses depends on the method combinations used. Combinations based on buffer-saturated phenol always gave the highest yields, and overall recovery and purity was highest when acetone was combined with the R2D2 buffer for protein purification and concentration. Comparative 2D-PAGE analysis confirmed that this method combination produced high-quality and reproducible gels and the largest numbers of spots per gel. The usefulness of this methodology was demonstrated on ripe fruits from several other species and shown to give excellent results.

Scab resistance is one of the most important goals of apple breeding, typically achieved by time-consuming and expensive conventional breeding techniques. Cisgenesis, which is the genetic modification of a recipient organism with genes from a crossable—sexually compatible—organism, is a promising tool for plant breeding to develop disease resistance in a rapid way. A cisgenic, scab-resistant line of the apple variety ‘Gala’ expressing the native apple scab resistance gene Rvi6 (formerly HcrVf2) under control of its own regulatory sequences has been recently developed. In this paper, we present the results from a phenotypic, molecular and biochemical evaluation of clonal replicates of this line (C11.1.53). The phenotype (shoot length, shoot diameter, internode length, number of leaves, leaf length and leaf width) of C11.1.53 was compared to that of the Gala parental background over a period of 108 days. Only a few statistically significant differences were detected, which are probably due to small differences in the quality of the budwood used for grafting rather than effects related to the presence of the cisgene. As the expression of a resistance gene can affect the downstream cascade of plant defence responses, a selection of apple defence-related genes was analyzed by quantitative real-time PCR analysis. These genes are also known as major allergen genes in apple. Even if three out of ten apple allergen genes tested in the leaves differed in the cisgenic line compared to both Gala (background) and ‘Florina’ (the variety from which the Rvi6 gene was cloned), using 2D-PAGE, we were unable to find any significant difference in the expressed proteomes of the leaves of C11.1.53 compared to Gala. Results are discussed in the context of a possible use of cisgenic lines for fruit crop improvement.

The impact of treatments aimed at improving the robustness of protocols for the analysis of carotenoids in fruit of banana and plantain were examined. Neither the inclusion of polyvinylpolypyrrolidine in the extraction buffer, nor vigorous homogenisation with glass beads influenced recoveries or chromatographic profiles. By contrast, heating lead to losses of up to 53% and to the formation of degradation products that are no longer detectable on our RP-HPLC system. Carotenoid extracts are unstable and most sensitive to exposure to light. However, even in the dark at -20 degrees C and in the presence of antioxidants breakdown rates of around 5% per day were observed. ispartof: Journal of Chromatography A vol:1216 issue:30 pages:5759-5762 ispartof: location:Netherlands status: published

Early detection of biotic and abiotic stresses and subsequent steering of agricultural systems using hyperspectral sensors potentially could contribute to the pro-active treatment of production-limiting factors. Venturia inaequalis (apple scab) is an important biotic factor that can reduce yield in apple orchards. Previous hyperspectral research focused on (i) determining if Venturia inaequalis leaf infections could be differentiated from healthy leaves and (ii) investigating at which developmental stage Venturia inaequalis infection could be detected. Logistical regression and partial least squares discriminant analysis were used to select the hyperspectral bands that best define differences among treatments. It was clear that hyperspectral data provide the contiguous, high spectral resolution data that are needed to detect subtle changes in reflectance values between healthy and stressed vegetation. The research was extended to include tree-based modeling as an alternative classification method. Results suggested that good predictability could be achieved when classifying infected plants based on this supervised classification technique. It was concluded that the spectral domain around 1600 nm was best suited to discriminate between infected and non-infected leaves immediately after infection, while the visible spectral region became more important at a well-developed infection stage. Research was focused on young leaves, because of the decreased incidence of infection in older leaves, the so-called 'ontogenic resistance'. Additional research was performed to gain a better understanding of the processes occurring during the first days after leaf unfolding and to evaluate the natural spectral variability among leaves. An undisturbed 20-day growth profile was examined to assess variations in the reflectance spectra due to physiological changes at the different growth stages of the leaves. Results suggested that an accurate distinction could be made between different leaf developmental stages using the 570 nm, 1940 nm, and 1460 nm wavelengths, and the red edge inflection point. Based on these results and the outcome of some existing chlorophyll indices, it was concluded that the chlorophyll content in leaves increased remarkably during the first 20 days after unfolding.