The influence of the pollination compatibility type on the pistil S-RNase expression in European pear (Pyrus communis).

The S-RNase gene plays an essential role in the gametophytic self-incompatibility (GSI) system of Pyrus. It codes for the stylar-expressed S-RNase protein which inhibits the growth of incompatible pollen tubes through cytotoxicity and the induction of programmed cell death in the pollen tube. While research on the Pyrus GSI system has primarily focused on the S-RNase gene, there is still a lack of insight into its spatiotemporal expression profile and the factors that regulate it. Previous studies have suggested that S-RNase expression in the style is influenced by pollination and is dependent on the compatibility type. We here continue on this basic hypothesis by analyzing the spatiotemporal expression of the S-RNase alleles in Pyrus communis "Conference" styles in response to different types of pollination; namely, upon full- and semi-compatible pollination and upon incompatible selfing. The results revealed that temporal dynamics of S-RNase expression are influenced by the pollen's compatibility type, indicating the presence of a signaling mechanism between pollen and style to control S-RNase production during pollen tube growth. In our experiment, S-RNase expression continuously decreased after cross-pollination and in the unpollinated control. However, after a fully incompatible pollination, S-RNase expression remained constant. Finally, semi-compatible pollination showed a initially constant S-RNase expression for both alleles followed by a strong decrease in expression. Based on these results and previous findings, we propose a regulatory mechanism to explain the effect of pollination and the associated compatibility type on S-RNase expression in the style. This proposed mechanism could be used as a starting point for future research.

Phenotyping, genetics, and "-omics" approaches to unravel and introgress enhanced resistance against apple scab (Venturia inaequalis) in apple cultivars (Malus × domestica).

Apple scab disease, caused by the fungus Venturia inaequalis, endangers commercial apple production globally. It is predominantly managed by frequent fungicide sprays that can harm the environment and promote the development of fungicide-resistant strains. Cultivation of scab-resistant cultivars harboring diverse qualitative Rvi resistance loci and quantitative trait loci associated with scab resistance could reduce the chemical footprint. A comprehensive understanding of the host-pathogen interaction is, however, needed to efficiently breed cultivars with enhanced resistance against a variety of pathogenic strains. Breeding efforts should not only encompass pyramiding of Rvi loci and their corresponding resistance alleles that directly or indirectly recognize pathogen effectors, but should also integrate genes that contribute to effective downstream defense mechanisms. This review provides an overview of the phenotypic and genetic aspects of apple scab resistance, and currently known corresponding defense mechanisms. Implementation of recent "-omics" approaches has provided insights into the complex network of physiological, molecular, and signaling processes that occur before and upon scab infection, thereby revealing the importance of both constitutive and induced defense mechanisms. Based on the current knowledge, we outline advances toward more efficient introgression of enhanced scab resistance into novel apple cultivars by conventional breeding or genetic modification techniques. However, additional studies integrating different "-omics" approaches combined with functional studies will be necessary to unravel effective defense mechanisms as well as key regulatory genes underpinning scab resistance in apple. This crucial information will set the stage for successful knowledge-based breeding for enhanced scab resistance.

Time Is of the Essence-Early Activation of the Mevalonate Pathway in Apple Challenged With Gray Mold Correlates With Reduced Susceptibility During Postharvest Storage.

Apple is typically stored under low temperature and controlled atmospheric conditions to ensure a year round supply of high quality fruit for the consumer. During storage, losses in quality and quantity occur due to spoilage by postharvest pathogens. One important postharvest pathogen of apple is Botrytis cinerea. The fungus is a broad host necrotroph with a large arsenal of infection strategies able to infect over 1,400 different plant species. We studied the apple-B. cinerea interaction to get a better understanding of the defense response in apple. We conducted an RNAseq experiment in which the transcriptome of inoculated and non-inoculated (control and mock) apples was analyzed at 0, 1, 12, and 28 h post inoculation. Our results show extensive reprogramming of the apple's transcriptome with about 28.9% of expressed genes exhibiting significant differential regulation in the inoculated samples. We demonstrate the transcriptional activation of pathogen-triggered immunity and a reprogramming of the fruit's metabolism. We demonstrate a clear transcriptional activation of secondary metabolism and a correlation between the early transcriptional activation of the mevalonate pathway and reduced susceptibility, expressed as a reduction in resulting lesion diameters. This pathway produces the building blocks for terpenoids, a large class of compounds with diverging functions including defense. 1-MCP and hot water dip treatment are used to further evidence the key role of terpenoids in the defense and demonstrate that ethylene modulates this response.

A semi in vivo pollination technique to assess the level of gametophytic self-incompatibility and pollen tube growth in pear (Pyrus communis L.).

KEY MESSAGE: 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.

Inoculation of pear flowers with Metschnikowia reukaufii and Acinetobacter nectaris enhances attraction of honeybees and hoverflies, but does not increase fruit and seed set.

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.

Sweet Immunity: The Effect of Exogenous Fructans on the Susceptibility of Apple (Malus × domestica Borkh.) to Venturia inaequalis.

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.

Dynamics of ascorbic acid content in apple (Malus x domestica) during fruit development and storage.

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.

Temporal and spatial variation in bacterial communities of "Jonagold" apple (Malus x domestica Borkh.) and "Conference" pear (Pyrus communis L.) floral nectar.

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.

Finding a Compatible Partner: Self-Incompatibility in European Pear (Pyrus communis); Molecular Control, Genetic Determination, and Impact on Fertilization and Fruit Set.

Pyrus species display a gametophytic self-incompatibility (GSI) system that actively prevents fertilization by self-pollen. The GSI mechanism in Pyrus is genetically controlled by a single locus, i.e., the S-locus, which includes at least two polymorphic and strongly linked S-determinant genes: a pistil-expressed S-RNase gene and a number of pollen-expressed SFBB genes (S-locus F-Box Brothers). Both the molecular basis of the SI mechanism and its functional expression have been widely studied in many Rosaceae fruit tree species with a particular focus on the characterization of the elusive SFBB genes and S-RNase alleles of economically important cultivars. Here, we discuss recent advances in the understanding of GSI in Pyrus and provide new insights into the mechanisms of GSI breakdown leading to self-fertilization and fruit set. Molecular analysis of S-genes in several self-compatible Pyrus cultivars has revealed mutations in both pistil- or pollen-specific parts that cause breakdown of self-incompatibility. This has significantly contributed to our understanding of the molecular and genetic mechanisms that underpin self-incompatibility. Moreover, the existence and development of self-compatible mutants open new perspectives for pear production and breeding. In this framework, possible consequences of self-fertilization on fruit set, development, and quality in pear are also reviewed.

The effect of polyploidization on tree hydraulic functioning.

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 ×domestica '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.

To disinfect or not to disinfect in postharvest research on the fungal decay of apple?

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.

A systematic evaluation of protocols for a proteomics analysis of (lyophilized) fruit tissues.

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.

Considerations to prevent the breakdown and loss of fruit carotenoids during extraction and analysis in Musa.

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.