Skip to main content

Predictive diagnostics based on gene expression for rapid assessment of crop load programs and bitter pit disorder in apple fruit

Final Report Summary - APPLEGENIE (Predictive diagnostics based on gene expression for rapid assessment of crop load programs and bitter pit disorder in apple fruit)

Executive Summary:
Ineffective thinning programs for crop load management and fruit losses caused by the occurrence of bitter pit remain two major burdens for apple growers in Europe.

Fruit thinning is the single most important, yet difficult to manage, strategy that determines the annual profitability of apple orchards. Despite over 40 years’ experience with chemical thinning, it remains an unpredictable part of apple production with large variations from year to year.

At present, the apple sector lacks tools to get relevant information supporting decisions regarding the choice for a specific thinning program (heavy or light) or a second treatment.

Comparable difficulties arise around the occurrence of bitter pit. In certain years this post-harvest disorder is a serious threat while in other years the disorder is hardly observed. Insight in the risk at a certain moment is needed to prevent superfluous calcium sprayings in the orchard or unforeseen losses upon long term storage.

The AppleGenie project aimed at developing tools to support decisions regarding these issues, directed to prevent losses. Tools will be based on molecular diagnostics. Information from relevant changes in gene activity of the apples forms the basis of the tests. The idea behind this approach is that each change in the physiology and eventually in the quality of an apple starts with a change in the activity of related genes. Also the way in which a plant will react to certain treatments can most probably be read from the gene-activity fingerprint. For each situation, specific so-called indicator genes need to be identified.


The AppleGenie project was driven by SME’s, all four of them deeply involved in agricultural practice. The combination with prestigious research institutes from Southern Europe and presumed end users secured the value of the study performed. Furthermore, NSure, a company realizing diagnostics based on gene activity measurements, coordinated this project.

First and most important step in the process was to identify the actual needs regarding the molecular tests to be developed. This led to the conclusion that the focus should be on tools that support the following decisions: which thinning program to choose/repeat thinning or not/apply Ca sprayings for curation of bitter pit/store batches of apples for long or not.

Trials were realized aiming at generating variability. Variability is needed for proper molecular indicator selection. Already after the first trials it appeared that encountered variability exceeded by far the expectations. This immediately made clear that the issues under study are complicated and directed by a multitude of factors. The findings were supported by the molecular analysis. The high degree of variability hampers specific indicator selection to a great extent. Anyhow putative indicators have been selected and validated for thinning response and bitter pit risk prediction. However the outcome of this study made clear that before a test could be released to the market, additional research is needed.

Next to that effort has been put in an inventory focused on the technology for measuring gene activity to be applied on location, once the indicators have been fully validated. This effort led to a detailed description of techniques that can be used nowadays.

Although no commercial tests are available at the end of the AppleGenie project, valuable and deep insight is gained in the complexity of the issues surrounding thinning practice and bitter pit prevention.

The knowledge in this sense contributes definitely to the search for decision support tools that will make agriculture more sustainable. Moreover, information obtained from the sector during publicity actions, consistently confirmed the need for this kind of research.

Project Context and Objectives:
Ineffective thinning programs for crop load management and fruit losses caused by the occurrence of bitter pit remain two major burdens for apple growers in Europe.
Fruit thinning is the single most important, yet difficult to manage, strategy that determines the annual profitability of apple orchards. Despite over 40 years’ experience with chemical thinning, it remains an unpredictable part of apple production with large variations from year to year.
In certain years, chemical thinning applied according to a standard protocol, is hardly effective while in other years over-thinning occurs, both resulting in severe losses. Once the first spraying has been performed it is difficult to judge whether a second spraying is needed. At present, the apple sector lacks tools to get relevant information supporting decisions regarding the choice for a specific thinning program (heavy or light) or a second treatment.
Comparable difficulties arise around the occurrence of bitter pit. In certain years this post-harvest disorder is a serious threat while in other years the disorder is hardly observed. Insight in the risk at a certain moment is needed to prevent superfluous calcium sprayings in the orchard or unforeseen losses upon long term storage.
The AppleGenie project aims at developing tools to support decisions regarding these issues, directed to prevent losses. The project is SME driven focused on the development of practical tools. Prestigious research institutes like IRTA (Spain), University of Bologna (Italy) and CTIFL (France) combine their efforts with stakeholders; end users and suppliers of high-end consultancy well involved in horticultural practice.
Tools will be developed based on molecular diagnostics. Information on relevant changes in gene activity of the apples forms the basis of the tests. The idea behind this approach is that each change in the physiology and eventually in the quality of an apple starts with a change in the activity of related genes. Also the way in which a plant will react to certain treatment can most probably be read from the gene-activity fingerprint.

The main objectives of the project AppleGenie have therefore been defined as follows.

First an inventory will be realized in order to specify the user needs regarding the tools to be developed.
Starting from the information obtained from both End Users that are part of the consortium, additional potential users should be questioned regarding their needs. The main question is, at what moments do they need additional information supporting their decisions? The inventory supposed to be realized in three different Southern European countries thereby securing the relevance of the choices to be made.
Also the expert knowledge of the SME’s and not the least of the scientists forming the consortium secures relevant choices. The exercise and the results, that supposed to drive the trial design, needs to result in a specific deliverable, D1.1 – Report on user requirements and specifications of the diagnostic services.

Once the specifications have been clarified, trials should be realized in France, Italy, Spain and even New Zealand thereby obtaining a high diversity of samples. Because the consortium decided to take full advantage of all available seasons within the three years of the project, trials should start already before all information regarding the End Users needs became available and immediately after kicking of the project. Expert knowledge present within the consortium will support proper choices to be made regarding the trial design. At least two subsequent years, covering four seasons, supposed to be needed for sample selection. Next to trials aiming at sampling from fields that show natural variation, which is sometimes risky, trials supposed to be organized aiming at introducing variability. Because of the expected complexity of the trials as a whole, results should be summarized in a specific deliverable, D2.1 – Protocols for sample generation.

Once results from the trials haven been described and interpreted accurately, choices should be made regarding the samples to be analyzed. Next Generation Sequencing, a technology that is being used nowadays to study the full transcriptome of a plant(part) at a certain moment supposed to be applied. This technique is however costly, so care should be taken which sample collection needs to be used. The choice has implications for the possibilities to select genes for which the activity correlates well enough with the phenomena under study. Data piles up quickly using this kind of techniques therefore results should be described in a dedicated deliverable, D3.1 – Marker gene libraries.
This information forms the basis for making the choice for specific selections of genes that can be used as indicators taking part in the different tests to be developed (based on the information summarized in Deliverable 1.1). Once the putative indicators have been selected, validation can be performed in additional samples, collected from the same trials or additional trials.
After a first round of validation, indicator selections need to be refined towards numbers that can be envisioned taking part of (routinely applied) diagnostics. These selection (for each one of the diagnostic tests to be developed) should be unequivocally defined. Therefore, these selection should be described in a specific deliverable, D4.1 – First prototype of kits.

These putative indicators will be validated in subsequent years. The duration of the project is three years. By including Plant and Food Research from New Zealand in the consortium, the benefits of two growing seasons every year are utilized, thus reinforcing validation related activities.

An additional requirement regarding the tests to be developed is that results are obtained quickly and in close vicinity of the companies that incorporate the result in decision making. Decision making in order to take measures based on the test results is sometimes a matter of hours. That is why part of the project budget is being used to design a so-called Mobile Lab. The idea is to develop a platform that measures the test specific gene activities on site. On site means at a central site in the neighborhood of the orchards. This speeds up the delivery of the test results and lowers the barriers to use the tests.
The effort to reach the goal: a real platform that can be used for analysis, depends strongly on the progress made regarding the selection of useful indicators. It is foreseen that final indicators (and thus diagnostic tests) did not undergo validation enough within three years. However, the platform to be used, once the tests will be validated enough, should be realizable. Starting point for a first inventory of techniques should be the PCR based measurement of the indicators. PCR based analysis is a worldwide accepted method and applied in a diversity of diagnostic fields.
A first inventory should aim at various easy and alternative techniques that can be applied by personal with low level skills. This exercise should be summarized in deliverable D4.2 – Operational Prototype of a Compact Lab. Next to that, and after further exploration until the end of the AppleGenie project, final conclusions should be drawn and described in a separate deliverable, D4.3 – Final version of the compact lab.
The budget allocated for this task doesn’t allow for the development of completely new devices so the project partners involved should focus on clever combinations of existing tools. In this respect, the combination of Turoni and NSure, supposed to be very effective. Turoni as a supplier of a high diversity of tools for quality measurements in agriculture, NSure as a developer of molecular diagnostics.

Once choices regarding the analysis platform has been made, first attempts to check perception by potential customers should be made in order to be sure that proposed end users could indeed work with the solutions provided. Results of the findings supposed to be summarized in a separate deliverable D5.1 – Report on efficacy of kits.

The different steps and predefined workpackages have been summarized in the accompanying figure (Figure 1). The project as a whole is managed by NSure, one of the SME’s, thereby securing practical relevance of the findings. Every opportunity, using every available tool, regarding publicity supposed to be used. Not only to inform but also to verify the need of what the consortiums aimed at.
An overview of the most relevant activities supposed to be present in a specific deliverable D6.3 – Final plan for dissemination of Foreground. This report should also include the IP rights connected to the foreground developed during the project.

Project Results:
This part is presented as an attachment because it contains many figures that are closely connected to the text.
Potential Impact:
Throughout the AppleGenie project, interaction with the national and international community has been realized. Not only to inform stakeholders about the effort but also to evaluate continuously the need for this form of diagnostics to be developed within the project.
Molecular diagnostics as a basis for tools that support difficult decisions was and still is new for the sector. So at each contact moment in depth information about the background of such diagnostics had to be given. This starts with informing the participants of the project as such. Project meetings with the consortium members have been used for that. This enables individual participants to spread the word, once they are acting on their own. Besides, regular skype meetings and the provision of material for publishing, assisted the individual participants.
The composition of the consortium was valuable. Possible end users and SME’s involved, secured the practical orientation of the project. This also implicates that the requirements regarding the diagnostics were, and still are, strict. This means that from a scientific point of view a lot of valuable, although sometimes difficult to recapitulate, information have been collected whereas in practice the results of the project appeared to be limited. This is outlined in the various reports that have been delivered about the work. A simple, nevertheless important, conclusion could be that reality is too complex in relation to the aims of the project. Anyhow, this insight is already valuable and will steer future research.
The fact that not any ready-to-use test has been developed yet has had implications for dissemination and further exploitation of the results obtained.
The major dissemination efforts have been described in a separate report (Deliverable 6.3). This report also contains rules for further exploitation of the results, specified for each kind of participant.
Research partners supposed to have different objectives in regard to use of data than commercial companies than end users.
Throughout the project and supported by each one of the contact moments with potential stakeholders the wish for diagnostics like the one being developed within AppleGenie is alive. Deeper insight in complex processes like fruit thinning and the occurrence of a disorder like bitter pit will sooner or later result in tools for optimization of horticultural measures and prevention of loss upon storage of fruits. It is widely recognized that the AppleGenie project definitely contributed to that.

List of Websites:
Public website: www.applegenie.eu

Contact: Peter Balk, NSure bv, Wageningen
peter.balk@nsure.nl