Final Report Summary - QUALIREDFRUITS (New agricultural practices for quality production of red fruits enriched in healthy compounds)
Project context and objectives:
The QUALIREDFRUITS project was co-funded by the European Commission (EC) and involved seven small and medium enterprises (SMEs) and five research and technological development (RTD) performers altogether representing seven different European Member and Associate States. This project dealt with the improvement of quality and agricultural procedures of raspberries considered as a paradigm of red fruits because of their high value due to:
- a unique panel of flavours and sensory quality,
- a proven 'health promotion disease prevention' (HPDP) property, and
- a high but still increasing competition between production worldwide.
Despite its continuous increase in the world market during the last decade, the European market share has remained stable around 20 % and may decrease in the next decade. In this context, the major innovative aspects of the QUALIREDFRUITS project are to provide a consortium of SMEs with the necessary research activities aimed at combining - for the first time - varietal selection of raspberries for greater HPDP properties with new cultivation systems based on biotisation (inoculation of beneficial microorganisms) and elicitors (stimulators of natural defence), in order to optimize the nursing of healthy raspberry plantlets and ensure high-quality of the fruits without resorting to the use of polluting chemicals.
Moreover, the sustainable development of new production strategies requires that molecular tools be developed to:
- monitor beneficial microorganisms introduced into biotised raspberry culture systems,
- assess plant sanitary status,
- enable the tracing of raspberries on the market for producer protection against biopiracy and,
- guarantee long-term conservation and availability of germplasms of high quality raspberry varieties selected during the project.
Raspberry crop
In the global fruit production system, a dominant position belongs to the berry fruits and especially raspberry production. The major production regions are: Russia, Europe and the Pacific Coast of United States of America (USA) and Canada. Many other countries, such as Chile, New Zealand, Australia and China are entering into the market as they supply the fresh market during winter in the northern hemisphere.
In Europe, most of the raspberry production is in the northern and central countries and higher regions of Italy. Greece, Portugal and Spain are new comers. In many production areas, the fruit is grown for the high-value fresh market, but in Eastern Europe (Serbia, Poland, and Hungary) a high proportion of the crop is for further processing, i.e. frozen preparations including individual quickly frozen (IQF) fruits, juice, concentrate, and preserves. These products are then packaged and sold directly to the consumer or further processed into jam, jelly, dessert topping, pie filling, ice cream, yoghurt, etc. The popularity of raspberries continues to grow as shown by the increasing number of raspberry-containing products on supermarket shelves.
Key figures
Statistics about production are controversial at both world and national levels. Roughly, the worldwide production is about one million tonnes per year. A half of it enters into the regular market where an average 20 % comes from Europe. Since the market price of raspberries varies from EUR 1 to 7/kg paid to the producers and EUR 7 to 10 paid by the consumers, the economical significance of this crop on the global market is rather high.
Context of the project
The QUALIREDFRUITS project is undertaken at the crossroad between the evolution of the fruit market, especially red fruits, and the progress of plant physiology.
Market trends
In addition to the continuous request for increased productivity and decreased production costs, the new trends of the market are dominated by the increasing consumers' interest in the potential of red fruits in health promotion and disease prevention (HPDP) mainly resulting from their potentially high content in antioxidant molecules (AOM). Additionally, there is an increasing social awareness and requests for agricultural practices globally safer for the environment and free from noxious chemicals in the context of sustainable development and food safety. In the near future, these new features may significantly add to the conventional quality criteria which are: the fruit size and firmness, the colour, the ease of harvest and the taste (in priority order).
Scientific and technical progress in plant physiology
The mechanisms which regulate the interactions between the soil and the plant roots are not fully elucidated yet. Nevertheless, the role of fungi and bacteria in these interactions is better known and laboratories have developed a new knowhow to monitor the plant roots / bacteria / fungi symbiosis in order to protect plants from their major diseases such as fungi and viruses. Plant conditioning is known as 'biotisation'. Additionally, it has been established that the plant self-defences against several pathological agents can be enhanced by new treatments without pesticides or fungicides and known as 'elicitors'.
Major objectives
The current market trends and progresses in plant physiology and molecular biology have created a timely opportunity to identify raspberry varieties with higher quality in terms of AOM content and consistent with innovative cultural practices respectful of the environ-ment. The QUALIREDFRUITS key objectives of the project are to:
- screen raspberry varieties for their AOM content and compare with the varieties currently dominating the market;
- design and deliver on purpose cultural practices suitable for the selected varieties and making use of beneficial micro-organisms (biotisation) and natural elicitors of plant defence reactions in order to drastically reduce chemical input and increase AOM synthesis in the selected varieties.
Considering the improvement of the competitiveness of the SMEs involved in the project as the global and ultimate prospective, the newly design chain from plant biotisation, plant nursing and farming must be strengthened by an adequate quality assurance scheme for both plant production and further raspberry production. The project is thus meant to bring operational advantages to the SMEs participants while contributing, as a pioneer, to the evolution of fruit production in a broader prospective. This includes the development and adaptation of complementary technologies for:
- monitoring of the biotisation system towards large scale industrialisation,
- assessing plants sanitary status,
- tracing raspberries to combat biopiracy, and
- preserving germplasms of the processed varieties on a long term.
Ultimate challenges of the project
While the world market of raspberries has increased by 60 % in the last decade, the relative share of the products originating from Europe has increased in a lesser proportion although the production capacity per unit of cultivated area has doubled during the same period of time. Reasonable business anticipations suggest that the European Union (EU) dependence in volume and in value may unfortunately decrease by 10 % in the 5 years after the new varieties and innovative cultivation procedures resulting from the QUALIREDFRUITS project are actually demonstrated and consolidated at the field level. This reduction of imports to the benefit of European actors is expected to reach 10 % per 5-year time slot and should likely continue for 2 decades. This will require that several challenges be met and namely:
- broadening the offer to customers by introducing on the market new cultivars originating from European technologies;
- underpinning such new European varieties thanks to a standardised assessment of their content in bioactive compounds as the role of these compounds in HPDP is increasingly established by medical research worldwide;
- offering varieties that require significantly reduced chemical treatments as the result of innovative cultivation procedures;
- orienting the relevant regulatory framework towards the establishment of certification procedures based on easy genotyping of the new varieties and easy assessment of the sanitary status of the plants;
- forging arguments to promote the informative labelling of the marketed fruits and processed derivatives concerning their HPDP properties including measurement of AOM content.
Implementation strategy and expected outcomes
The main directions followed by the project are:
- selection of raspberry varieties with high fruit quality (including AOM) and tolerance / resistance to fungal diseases;
- development of new methods of plant biotisation and elicitor treatment for healthier plant production;
- conservation, genotyping and plant health assessment;
- production field trials.
Since the project approach from laboratories to fields had to deal with seasonal constraints, parts of the work was undertaken in parallel instead of sequential developments.
Objective indicators involved molecular markers for:
- the specific identification of varieties together with a cryopreservation protocol for germplasms' long-term conservation;
- the identification of beneficial and pathogenic micro-organisms in order to assess the sanitary status of plants;
- the biomarkers-based approach tested in real production trials, from in vitro culture to the field for validation.
Finally, appropriate controls and reproducibility tests enabled the partners to formulate recommendations for quality production of raspberries taking into due consideration both the fresh fruits market of higher quality and the processed fruit market.
Project results:
The results of the QUALIREDFRUITS project which had to be delivered to the SME participants by the RTD performers according to the transaction of the Grant Agreement and the description of work (DoW) are reminded.
Independently from the deliverables reporting the achievements in the project work packages (WPs), each eesult R01 to R09 has been formally delivered in a detailed internal report available to fix the joint exploitation agreement among the SME participants and to serve as foreground reference for any further work to be carried out after the end of the project.
The internal reports executive summaries are presented below.
Result ID: R01
Disclosing Institution: Agroscope Changins-Wädenswil Research Station ACW, Centre de recherche Conthey Route des vergers 18, 1964 Conthey, Switzerland
Authors: Zo-Norosoa Camps, Christoph Carlen P08-ACW, Kaloyan Kostov, Slavcho Slavov, Rossitza Batchvarova P10-ABI, Anton Ivancic, Andrej uek P09-UMFKVB, Tomasso Sozzi, Vivienne Gianinazzi-Pearson, Silvio Gianinazzi P06-INRA
Dissemination level: Confidential
Title: Identification of raspberry varieties with enhanced antioxidant molecule content and/or tolerant to pathogenic fungi that could be used as commercial varieties and/or in a breeding program of new commercial varieties
Date of disclosure: 30 November 2012 - Project month: 24
Summary:
- The aim of WP1 was to select cultivars riche in AOM and tolerant to diseases with a high exploitation potential with regard to specific cultivation procedures, to breeding and to intellectual property rights (IPR).
- A screening of about 100 varieties and genotypes of raspberries made it possible to define 37 available floricane and primocane fruiting raspberries varieties to be tested by entering them into the experimental approach of the project and finally support a future breeding programme.
- Concerning AOM synthesis of floricane fruiting cultivars the most interesting red cultivar is Elida. Elida showed high contents of Vitamin C, phenolics, ellagitannins and high antioxidant capacities. Elida may support a future breeding program in combination with a high yielding cultivars producing firm fruits such as Tulameen. Another approach is to breed a high yielding cultivar with big, light red fruits with Bristol or some wild genotypes (SL, F) to get progenies with high contents of health related compound. These progenies may have to be backcrossed with another high yielding cultivar to get a cultivar with big, red fruits and a high yield.
- Concerning AOM synthesis of primocane fruiting cultivars, the most interesting varieties were Joan J and Kweli. It would be an interesting approach to cross these two cultivars, because their positive attributes concerning AOM synthesis are very complementary. On the other hand, Kweli, a high yielding cultivar with big, firm fruits, has an interesting potential for healthy fruits and in combination with specific cultivation procedures (biotisation, elicitor treatments) to produce high value fruits.
- Concerning the tolerance to diseases (such as Botrytis cinerea and Leptosphaeria sp and Phytophthora sp.) of floricane fruiting cultivars, Elida showed good tolerance against Botrytis cinerea and Schopska Alena and Williamette against Phytophthora sp. The other cultivars were not different from each other concerning the tolerance to these diseases. Concerning the primocane fruiting cultivars Sugana, Autumn Bliss, Joan J (Dieffenbach) and Erika showed interesting tolerance to Botrytis cinerea and Leptosphaeria sp. Erika has a good tolerance to Leptosphaeria sp. and the two Bulgarian cultivars Iskra and Samodiva against Phytophthora sp.
- Data on technical aspects of hybridisation of new cultivars including pollination, seed harvesting, stratification of seeds and plantlets production were given related to experiences developed in this project. The biggest problems were to regenerate plantlets from seeds obtained from crossing of two genotypes.
- In order to capture the greatest value from plant-related intellectual property assets, a number of types of statutory intellectual properties rights for SME involved in breeding new varieties and/or SME involved in biotisation of plants are described and discussed such as plant variety protections (PVPs), plant patents, geographic indications, trademarks and trade secrets. SME participants should be aware of all options for further IP protection and use them to their greatest advantage. The various forms of intellectual property (IP) protection for plants can be used alone or in combination.
Result ID: R02
Disclosing institution: Institut National de la Recherche Agronomique, 17 Rue Sully, BP 86510, 21065 Dijon Cedex, France
Authors: Vivienne Gianinazzi-Pearson, Silvio Gianinazzi, Tommaso Sozzi
Nature: Report
Dissemination level: Confidential
Title: Identified beneficial microbes for plant biotisation
Date of disclosure: 30 November 2012
Summary:
- Three arbuscular mycorrhizal fungi are identified for efficient raspberry plant biotisation: Glomus intraradices isolates LPA8, LPA7 and LPA54.
- Two beneficial soil bacteria are identified for efficient raspberry plant biotisation: Pseudomonas fluorescens sp. I13 and Sinorhizobium meliloti.
- A composite inoculum has been defined based on the compatibility between the bacterium Sinorhizobium meliloti and the arbuscular mycorrhizal fungus Glomus intraradices isolate LPA8.
- A biotisation procedure is provided for the inoculation of Glomus intraradices iso-late LPA8 and Sinorhizobium meliloti during the acclimatisation of vitroplants or cuttings of different raspberry cultivars.
- The described biotisation procedure is highly reproducible and the methodology has the advantage of rapidity and applicability to a large number of plants.
Result ID: R03
Disclosing Institution: Scottish Agricultural College, West Mains Road, Edinburgh EH9 3JG, UK
Authors: Dale Walters
Nature: Report
Dissemination level: Confidential
Title: Identified elicitors of natural plant defence mechanisms for improving raspberry resistance against pathogenic fungi
Date of disclosure: 26 June 2012
Summary:
- Cultivar differences exist in raspberry responses to foliar or root drench application of the plant defense elicitors Bion (acibenzolar-S-methyl), BABA (beta-aminobutyric acid), Yea Foliar (chitosan) and methyl jasmonate.
- In Tulameen cultivar plants, only Bion reduces root rot damage by P. fragariae var rubi and increases root development.
- In Meeker cultivar plants, all four elicitors significantly decrease root rot damage and increase resistance to P. fragariae var rubi, with best control achieved using methyl jasmonate.
- The elicitor Bion is identified as effectively increasing resistance to the root rot pathogen in both Tulameen and Meeker raspberry cultivars.
- Increased resistance elicited by Bion in Tulameen plants is associated with enhanced activity of the defence-related enzymes peroxidase, beta-1,3-glucanase and cinnamoyl alcohol dehydrogenase (CAD) in leaves.
- BABA appears to increase 'tolerance' to raspberry root rot in Tulameen and Meeker plants, and enhance CAD activity in Tulameen leaves.
Result ID: R04
Disclosing institution: Institut National de Recherche Agronomique (INRA), France
Authors: Vivienne Gianinazzi-Pearson, Silvio Gianinazzi,Tommaso SozziI at INRA and Dale Walters at Scottish Agricultural College (SAC), Scotland, Christoph Carlen, Zo-Norosoa Andrianjaka-Camps at ACW - Eidgenoessisches Volkswirtschaftsdepartement, Switzerland
Nature: Report
Dissemination level: Confidential
Title: Defined protocol for the combined use of biotisation and elicitor treatment
Date of disclosure: 20 November 2012
Summary:
- Indicators have been defined for monitoring compatibility in raspberry plants between biotisation and plant defence elicitor application: mycorrhizal development, plant growth, defence-related enzyme activities, fruit production and AOM content / antioxidant activities in leaves and fruits.
- Protocols are described for the combined use of biotisation and elicitor application in greenhouse and real production systems of raspberry plants and fruits.
- Treatment of raspberry plants with the plant defence elicitors BION, BABA, methyl jasmonate or chitosan is compatible with biotisation with a composite microbial inoculum (glomus intraradices isolate LPA8 / sinorhizobium meliloti) under greenhouse production conditions.
- The compatibility of BION application with plant biotisation has been confirmed in a real production system of organic farming for raspberries.
Result ID: R05 - R06 - R07
Disclosing institution: University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 10, 2311 Hoce, Slovenia
Authors: A. uek, M. iko, A. Ivancic at University of Maribor, Faculty of Agriculture and Life Sciences, V. Gianinazzi-Pearson, T. Sozzi at INRA, R. Batchvarova at Agrobioinstitute (ABI), A. Gollotte at INOPLANT, France
Nature: Report
Dissemination level: Confidential
Title: Conservation, genotyping and plant sanitary status assessment
Date of disclosure: 14 November 2012
Summary:
- The cultivars Meeker, Glen Ample, Rose de Core d'Or, JJR, Sicoly S1 and Polka were successfully identified with microsatellites markers.
- Specific molecular primers for five plant pathogenic fungi (Phytophthora fragariae, Thielaviopsis basicola, Leptospheria coniothyrium, Aphanomyces euteiches, Pythium ulitmum) were designed.
- Specific molecular primers for three beneficial bacteria (Sinorhizobium meliloti, Pseudo-monas fluorescens sp. I13, Paenibacillus sp. B2) were designed.
- Specific molecular primers for two beneficial fungi (Glomeromycota, Glomus intra-radices) were designed.
- Cryopreservation protocol based on encapsulation / vitrification was developed for raspberry varieties Tulameen and Meeker.
Result ID: R08
Disclosing institution: SRUC (formerly SAC), West Mains Road, Edinburgh EH9 3JG, UK
Authors: Dale Walters
Nature: Report
Dissemination level: Confidential
Title: Innovative scheme for raspberry plant production from in vitro to field, requiring low amounts of chemical input and more compatible than conventional with respect to the environment
Date of disclosure: 14 December 2012
Summary:
- The aim of WP4 was to test raspberry varieties and methods developed in WPs 1, 2 and 3 under real production conditions. In order to achieve this aim, field trials were conducted in Switzerland, France, Slovenia, Bulgaria and Scotland, using the same basic design and layout of the trials and joint protocols for treatment. The overall aim was to test the effect of biotisation and elicitor (Bion) treatments, alone and in combination, on growth and yield of raspberry varieties, and on control of diseases, particularly raspberry root rot. The sanitary status of the plants was examined to ensure the presence of mycorrhiza and the absence of pathogens. The content of antioxidant molecules (AOM) and the antioxidant capacity of the plants receiving the different treatments were also determined.
- The results highlight the importance of geography / location, and genotype, in determining the response of a raspberry crop to agronomic treatments.
- Biotisation and elicitor treatments had no significant effect on factors affecting fruit yield in Switzerland.
- There were significant effects of the treatments on growth of raspberry plants and fruit yield in Bulgaria, although genotype differences were observed in fruit yield.
- Genotype differences were also found in Slovenia, where elicitor, and biotisation plus elicitor treatments, provided increased fruit yields in Polka, but not in Tulameen.
- In France, the combination of biotisation and elicitor provided good fruit quality and yield, while the elicitor treatment provided good vegetative growth and root development. Here, the elicitor treatment (Bion) was associated with some phytotoxicity.
- Under Scottish conditions, poor weather in 2011 and extremely poor weather in 2012 affected the trial, again highlighting the role of geography, with its accompanying differences in weather, on crop performance. Nevertheless, the field trial in Scotland confirmed the genotype differences observed in the other field trials, and also showed that in Polka, the elicitor can provide some protection against Phytophthora root rot, while in Tulameen, the combination of biotisation and elicitor provided best protection against root rot, under very heavy inoculum potential.
- Analysis of AOM contents and antioxidant activities in the leaves and fruits from plants in the different field trials also highlighted the importance of genotype, followed by geographic location, while agronomic treatments had little effect.
- In most of the field trials, elicitors and biotisation were compatible, suggesting that in most production systems for raspberries, the elicitor Bion and biotisation can be used together.
Result ID: R09
Disclosing Institution: SRUC, West Mains Road, Edinburgh EH9 3JG, UK
Authors: Dale Walters
Nature: Report
Dissemination level: Confidential
Title: Formal set of recommendations to SMEs for producing and using biotised plants, and for elicitor treatment procedures
Date of disclosure: 14 December 2012
Summary:
- Based on the experiments carried out in the four work packages comprising this project, recommendations can be offered to SMEs.
- A protocol has been defined for the combined use of biotisation and elicitor treatment, based on:
(i) isolates of mycorrhizal fungi and beneficial bacteria selected by P06 INRA for their efficiency and compatibility,
(ii) a procedure optimised by P03 INOPLANT for biotisation of raspberry plants before outplanting, and
(iii) an effective elicitor treatment developed by P07 SAC for reducing development of Phytophthora root rot symptoms in plant production systems.
- Specifically, the following recommendations can be given to SMEs:
i. Since the plant defence elicitor BION is compatible with biotisation when applied under greenhouse conditions, its application can be recommended during the standardised procedure for large scale production of biotised raspberry plants to provide effective control of Phytophthora root rot.
ii. In some environments (e.g. Bulgaria, France and Slovenia), and in certain cultivars (e.g. Polka and perhaps Tulameen), biotisation and the elicitor Bion can be recommended to protect raspberry plants against Phytophthora root rot and to increase plant growth under field conditions.
iii. In Scotland, depending on the raspberry variety, biotisation and / or the elicitor Bion can be recommended to reduce the damaging effects of root rot under field conditions.
Detailed achievements of the project
Selection of raspberry varieties with high fruit quality and tolerance to fungal diseases
Current interest by consumers, media and medical actors in the health benefits of several components of fruit and vegetable aliments having pharmaceutical properties (often referred to as 'nutraceutical' in English and 'Alicament' in French) has created an increasing interest in the berry industry. There is a consensus that berries are a nutritional and nutraceutical powerhouse, and that raspberries rank near the top of all fruits for antioxidant strength. Past and current medical research shows likely benefit of regularly consuming raspberries against cardiovascular diseases, diabetes, inflammation, some cancers and age-related degenerative diseases. Such benefit is proven to result from antioxidant molecules (AOM) (Kresty et al., 2001; Puupponen-Pimiä et al., 2005; Coates et al., 2007). The concept of AOM refers mainly to non-nutrient compounds in foods and covers a broad range of molecules including flavonoid or non-flavonoid polyphenolics, phenolic acids, vitamins and other potential organic antioxidants (Beekwilder et al., 2005). Anthocyanins and ellagitannins are the major contributors to the total antioxidant activities of raspberry (Mullen et al., 2002; Beekwilder et al., 2005; Battino et al., 2009): ellagitannins contribute around 50 % and anthocyanins about 25 %. AOM synthesis in raspberries varies between plant varieties; most red raspberry varieties presently on the market contain relatively low levels of AOM compared to black raspberry, blackberry and wild raspberry.
Raspberry plants are prone to parasites, fungal diseases and virus infections, which make the use of pesticides a conventional cultivation practice. Chemical treatments generate potentially noxious food residues which counterbalance the positive effect of berry diet upon human health (?ozowicka et al., 2012), but little success has been achieved to find alternatives to fungicides to control fungal diseases (Ançay et al., 2005; Xu et al., 2012). Three fungal pathogens have a high potential to cause serious raspberry yield loss. Root rot due to Phytophthora fragariae var. rubi, the main cause of raspberry plant death, cannot be effectively controlled by chemicals and only prophylactic measures can be adopted by planting healthy raspberry plants in non-contaminated soils (Ançay et al., 2005). Leptosphaeria coniothyrium, which attacks raspberry canes, causes variable disease se-verity depending on the year, situation, weather and varieties (Williamson et al., 1986). Finally, Botrytis cinerea causes the fruit grey mould responsible for significant economic loss (Xu et al., 2012; O'Neill et al., 2012).
Technical data on raspberry varieties / cultivars with a high exploitation potential related to enriched AOM content of fruits and tolerance to disease are therefore an important pre-requisite to an integrated approach including the selection of plant varieties more resistant to main pathogens and breeding-based creation of new genotypes with combined producer and consumer defined traits.
Pre-selection and evaluation of raspberry varieties
About 100 commercially and collection available varieties and genotypes of raspberry were screened on the basis of literature and project partner experiences for their high economical importance and contrasting traits concerning consumer criteria (fruit sensory quality, firmness), farming criteria (yield, fruit size), potentially higher quantities of antioxidant molecules (HPDP properties) and/or tolerance to major fungal diseases in raspberry production systems. A total of 33 available floricane and primocane fruiting raspberries cultivars as well as 4 wild or semi-wild floricane genotypes were defined and grown to fruit for further analyses.
- AOM content and antioxidant capacities
- Appropriate methodologies for extraction and analysis of health-related compounds in raspberry fruits and leaves were defined and applied to 23 floricane and 11 primocane cultivars or genotypes for the characterisation of AOM compound contents (vitamin C, total phenolics, anthocyanins, ellagitannin) and antioxidant capacities (DPPH radical inhibition, ferric reducing antioxidant power, oxygen radical absorption capacity).
- Overall, the variation in antioxidant capacities of floricane and primocane cultivars followed that in terms of AOM content, with cultivars rich in the major phytochemicals showing the higher antioxidant activities. Results clearly indicated that leaf antioxidant analyses cannot be used to predict fruit antioxidant capacities. No correlation was found between leaf and fruit antioxidant capacities, or between leaf antioxidant capacities and fruit AOM content.
- In general, floricane fruiting cultivars with interesting agronomical, physical and sensorial quality parameters had relatively poor antioxidant activity and health related compounds. From factorial discriminant analyses, floricane raspberries could be classified in four principal groups depending on their anthocyanin constituents and in four main clusters according to ellagitannin patterns, whilst primocane genotypes were distinguished into three categories for their anthocyanin patterns and two main classes for ellagitannin composition.
- Three primocane cultivars gave a significantly higher yield and fruit size than all other cultivars whilst four other cultivars (two floricane, two primocane) and some of the wild genotypes showed high potential in terms of AOM content and antioxidant capacities. These could be promising for future breeding in combination with high yielding cultivars producing large, firm fruits.
- Tolerance to fungal diseases and fruit malformation
-Eleven floricane and 9 primocane fruiting cultivars were examined for tolerance to three pathogenic fungi causing major diseases in raspberry production systems. Detached leaf and stem bioassays against infection by Phytophthora fragariae var. rubi, Leptosphaeria coniothyrum, Botrytis cinerea) showed that none of the studied floricane fruiting cultivars possess good tolerance to all three fungal diseases. However, one floricane cultivar demonstrated good tolerance towards B. cinerea, and two others against P. fragariae.
- For primocane fruiting cultivars, one showed significant tolerance to all three fungal diseases. An interesting level of tolerance against B. cinerea and L. coniothyrum was observed for another three cultivars, and against P. fragariae for two.
- None of the floricane or primocane fruiting cultivars grown in a project field site showed more than 2 % of fruit malformation. No virus or MLO was detected and canes deformed fruits in one year did not necessarily have them in the following year. Conditions during flowering, especially extreme temperatures, may impact on fruit malformation
Pilot hybridisations
- In order to set the pace for breeding innovative raspberry genotypes with high exploitation potential related to increase the AOM content of raspberry fruits tolerance to diseases, pilot hybridisations were performed and successfully achieved between some interesting genotypes and varieties. Technical procedures (emasculation, pollination, seed harvesting, seed stratification, plant regeneration) were optimised and guidelines were provided to SMEs. Intellectual property rights relative to novel plant-related assets have also been communicated.
- Higher leaf antioxidant activities and total phenolics content were identified in some hybrids between the cultivated and wild genotypes. However, plants were too young to be transferred to the field for fruit evaluation and this will be performed in the 2013 season.
In conclusion, the expectancies of the project SMEs have been met with regards to obtaining reliable information and defining several raspberry cultivars or genotypes which are enriched with healthy compounds, and which meet farmer and consumer criteria requirements. Also in line with the SME expectancies, new analytical tools have been introduced for the identification of raspberries having a high AOM content and thus enabling new marketing approaches to promote raspberry consumption based on objective quality criteria. In this context, knowledge about the qualitative and quantitative contents in health-promoting compounds of raspberry plants or fruits, as well as of their tolerance to diseases, can provide competitive arguments for SMEs in order to increase their market share of fresh fruits or derivative products from fruits and leaves. Moreover, the information can support a future breeding program by suggesting cultivars with a potential to increase AOM content of the fruits or leaves of newly developed cultivars, or their tolerance to three major fungal diseases in raspberry production systems. In the context of a future breeding programme, various types of statutory intellectual properties rights (IPR) for plants such as plant variety protections (PVPs), plant patents, geographic indications, trademarks or trade secrets have been communicated to the project consortium.
Raspberry plant genotyping, conservation and health assessment
Variety / cultivar registration is an important issue of plant genetic resource characterisation and utilisation. Traditional methods to identify raspberry cultivars for the granting of plant breeder's rights are based on phenotypic observations. The incorporation of new methodologies into plant material certification schemes will accelerate and optimise the identification process of genotypes and offer guarantee of plant sanitary status. Genetic fingerprinting (genotyping) can provide exact data on the genetic diversity existing in raspberry collections of available genetic resources for breeding programmes, and independently of developmental stage or environmental factors that may influence the phenotype. Genotyping techniques based on microsatellites, also known as simple sequence repeats (SSRs) or short tandem repeats (STRs), are highly informative (show high polymorphism), technically simple (simpler than other techniques), robust and suitable for automated allele detection and sizing (Rafalski and Tingey 1993). In addition, the utilisation of fluorescence-based automated DNA detection and fragment sizing offers a potential improvement of the efficiency and affordability of variety testing (Hayden et al., 2008). Since raspberry species are vegetatively propagated (not by seed), the identification of genotypes will offer a standardised reference for certifica-tion of any cultivar and for controlling its propagation. This approach may prove to be crucial in the context of variety ownership rights in the event of the creation of new genotypes.
Novel techniques for ensuring long term conservation of raspberry materials, like cryopreservation (Reed et al., 2001; Wang et al., 2005; Keller et al., 2006; Reed, 2008), can provide producers with easy access to stable genetic resources and a back-up to recover varieties when genetic divergence or sanitary problems occur. In this context, the development of molecular tests to detect beneficial or pathogenic micro-organisms in plant materials, soil and substrates will provide rapid and reliable information that is crucial for raspberry cultivation systems. The basic methods for detection and identification of plant-inhabiting micro-organisms applied in practice include symptom observation, isolation from attacked tissue or soil, and morphological characterization of the isolated organism. Molecular techniques based on PCR provide additional information and certainty of the microbial detection and identification process (Bonants et al., 1997, Vandemark et al., 2000, Gollotte et al., 2004, Farmer et al., 2007, Nilsson et al., 2008). The combination of both approaches in the routine observation of plant sanitary status would facilitate the accuracy of the diagnostics and lead to an optimised regime of the plant protection activities.
Plant genotyping
- A molecular technique to genotype raspberry material was developed based on the discriminating power of SSR sequences in 19 microsatellite loci. SSR primer sets designed from the microsatellite loci were used in PCR of DNA extracted from fresh, young leaf tissue to evaluate the genetic diversity in a germplasm collection of 49 raspberry genotypes (semi-wild, wild, floricane, primocane). Capillary electrophoresis of PCR products generated SSR sequences which revealed 167 polymorphic alleles at the 19 microsatellite loci.
- Dendrogram analysis of genetic relationships based on SSR data arranged the 49 investigated raspberry genotypes in five main clusters, with a larger genetic distance between genotypes than between clusters. One cluster included wild and semi-wild raspberry germplasm, whilst one local variety and genotypes resembling cultivated floricane or primocane cultivars were grouped in the other four clusters. Analyses of genetic diversity in the cultivars identified 8 duplicate varieties.
- An SSR-based PCR protocol was optimized for the identification of raspberry varieties considered to be commercially important. A specific fingerprint (electrotrace), which can be used for identification, was obtained for each variety using combinations of SSR primer sets for selected microsatellite loci. The most specific microsatellite loci were selected for each variety from PCR reactions conducted with the 19 optimised SSR primers.
- The optimised protocol was successfully applied to six raspberry varieties. Seven of the most specific microsatellite loci were used for one floricane variety, and combinations of six loci were used for two other floricane varieties and three primocane varieties. To reduce labour and costs for PCR product detection, a multiplex of three primer sets labelled with two different fluorescent dyes were combined. Applicability of the developed protocol for variety identification was verified in a 'blind' test on anonymous DNA samples from five raspberry genotypes.
Conservation of raspberry genotypes
- In the context of long-term conservation and stability of high quality raspberry varieties / genotypes, a cryopreservation technique adapted from published literature was first successfully applied to Medicago sativa seeds in pre-experimentation trials. The same protocol, however, gave toxic effects when applied to shoot tips of three raspberry varieties. An alternative procedure was developed for raspberry tissues, in which treatment with low sugar concentrations, together with adapted osmotic loading and plant vitrification solutions, improved tissue survival and enabled some plant regeneration from raspberry shoot tips or root fragments after storage in liquid nitrogen.
Plant sanitary status assessment
- A molecular method, based on PCR detection, was developed to track the presence or absence of beneficial or pathogenic micro-organisms in raspberry cultivation systems. Specific PCR primers were designed on sequences in the DNA region coding for rRNA and used on soil or plant material from raspberry cultures.
- Molecular marker sets were obtained to detect three beneficial bacteria and one mycorrhizal fungus used in biotisation technologies, and for six fungal pathogens, of which three are important pests in raspberry production systems (Phytophthora fragariae, Leptospheria coniothyrium, Botrytis cinerea).
- Specificity of the PCR detection method was verified under different laboratory conditions and on soil or raspberry plant tissues colonised by the targeted organisms. The approach was successfully applied to check for the absence of pathogenic fungi from substrates or composite inoculum used for biotisation.
- Applicability of the molecular protocols for routine monitoring of the targeted fungi under real production conditions was demonstrated directly on fungal-colonised raspberry plant tissues using DNA from leaves infected by P. fragariae var. rubi or L. coniothyrium, and of roots or soil colonized by P. fragariae var. rubi or AM fungi, sampled from field trials.
In conclusion, the methodology developed for genotyping of raspberry material can be exploited in furthering certification procedures for registration of established or new variety / cultivar cultivars and for their control during propagation or across the market. The protocols defined for detection of beneficial or pathogenic micro-organisms provide rapid and reliable procedures for plant sanitary status assessment, and so promote the production of healthy biotised raspberry plants not contaminated by pathogens. Both these achievements meet the SME expectancies of proven methods for ensuring plant quality in future certification procedures. In addition, optimization of the cryopreservation technique opens the possibility of long-term, genetically stable conservation of pathogen-free raspberry material which represents an added value resource for producers.
Development of new methods of plant biotisation and elicitor treatment for healthier plant production
Biotisation is a biotechnology consisting in the inoculation of young plants with beneficial micro-organisms (bacteria and/or fungi) in order to increase plant vigour (Gianinazzi et al., 2003). In the case of raspberry, biotisation of young plants during the acclimatisation phase has proved useful for improving plant survival and growth, and for increasing tolerance to P. fragariae var rubi (Gollotte et al., 2009; Lemoine et al., 2000). Arbuscular mycorrhizal (AM) fungi are the most significant of beneficial soil micro-organisms as they establish a symbiosis at the interface between soil and the roots of most high value agricultural and horticultural plants. Although AM fungi are present in most ecosystems, their populations are generally limited in the field under conventional agriculture because of their sensitivity to practices using large quantities of fertilisers and pesticides. Moreover, micro-propagated plants and substrates used in raspberry production are usually devoid of AM fungi so that biotisation can be very useful in such cases to ensure plant vigour and health. Biotisation can also be beneficial to field-grown plants, for example under organic farming conditions or when reduced amounts of fertilisers and pesticides are used.
Fighting against cryptogamic diseases is still a problem in raspberry cultivation, although a few raspberry varieties can be identified which show good tolerance to Phytophthora fragariae var. rubi (see above), and producers have to resort to the use of chemical fungicides which can be pejorative to the environment and human health. Elicitors of natural plant defence mechanisms have been shown to provide useful disease control in many crops against a range of economically important pathogens (Walters, 2011), and various natural agents (e.g. cell wall fragments, plant extracts) can induce plant resistance to subsequent pathogen attacks, both locally and systemically (Walters et al., 2005; Walters and Heil, 2007). This induced resistance does not lead to complete disease control, but rather to a reduction in pathogen development and disease symptoms. The defence mechanisms involved are those used in other forms of plant resistance to pathogens and include enhanced defence-related enzyme activities as well as synthesis and accumulation of a range of phenolic compounds, many of which possess antioxidant properties. Agents which mimic natural elicitors of resistance have been developed and commercialised for broad-spectrum disease control using the plant's own resistance mechanisms, but these have not been tested on raspberry (Walters and Heil 2007).
AOM synthesis in plants depends on agricultural practices as well as plant varieties and climatic conditions (Anttonen et al., 2005). Mycorrhization has been reported to increase polyphenol content in grapevine and sweet basil, carotenoid level in sweet potato, and antioxidant activity in raspberry (Krishna et al., 2005; Farmer et al., 2007; Toussaint et al., 2007; Gollotte et al., 2009). Also, treatment with plant defence elicitors has been reported to increase total phenolic content and antioxidant activity in sweet basil (Kim et al., 2005), and to increase total phenolic, anthocyanin and antioxidant content of raspberries and blackberries when sprayed onto fruits during the ripening phase (Wang and Lin, 2000; Wang and Zheng, 2005; Wang et al., 2008). However, the impact of combining these innovative agricultural practices on plant content in AOM has yet to be characterised.
Whilst the identification of plant defence elicitors which enhance resistance to major fungal diseases in raspberry represents a new alternative to chemical control, knowledge about their compatibility, or synergy, with biotisation in raspberry plant production systems is so far inexistent, although positive or no effects have been reported in other plants (Murphy et al., 2000; Sonnemann et al., 2000; Tosi and Zazzerini, 2000; Hause et al., 2007). The enhancement and the consequent standardisation of raspberry plant biotisation coupled with elicitor treatment are expected to be of competitive advantage for the SMEs in terms of healthier plant production and fruit quality.
Raspberry biotisation
- Suitable mycorrhizal fungi and beneficial bacteria were selected for their compatibility in the bioti-sation of raspberry microplants, and a multiplication procedure was established for the production of appropriate mycorrhiza and bacteria inocula. A first composite inoculum for biotisation in raspberry production was defined based on the compatibility of one beneficial bacterium with a mycorrhizal fungus, together with its persistence after inoculation and its synergistic effect on mycorrhizal plant growth.
- A highly reproducible procedure for biotisation of raspberry microplants or cuttings with the composite inoculum was optimised during plant acclimatisation using an appropriate substrate and fertilisation régime. This was subsequently up-scaled to provide material on a large scale for tunnel and field production trials of four raspberry varieties.
- The feasibility of using biotisation was tested under the conditions of field soils or inert substrates used by SMEs to grow raspberry plants. When biotised and acclimatized vitroplants of two floricane raspberry varieties were outplanted into an organic-based field production system, mycorrhizal levels were maintained and non-biotised plants quickly developed mycorrhiza after out-planting. Plant-derived liquid manure proved to be compatible with mycorrhiza development and had a slight positive effect on raspberry fruit size of biotised plants.
- Four commercial potting substrates, currently used by SME partners for raspberry plant or fruit production, proved to be poorly compatible with mycorrhiza development in a biotised floricane variety under greenhouse conditions. Nevertheless, residual beneficial effects of initial biotisation on plant behaviour were observed (absence of mildew, better growth, no lodging). Also, higher fruit yields were obtained in plants that had previously been biotised in two of the commercial substrates and then outplanted into a small field trial.
- Introduction of better adapted mycorrhizal fungi, modications in the P fertilisation regime and/or amendment of the substrate composition overcame the problems of incompatibility in a commercial substrate and greatly improved mycorrhiza development in biotised raspberry plants.
Elicitor treatments
- Four plant defence elicitors (reported to control fungal diseases in other plants) were tested under greenhouse conditions in order to define the most effective product for control of P. fragariae var. rubi in raspberry production systems. Plantlets of two floricane raspberry varieties were treated with the elicitors by foliar spraying or root drench and challenged with P. fragariae two days later. None of the elicitors significantly affected growth of either raspberry variety in the absence of Phytophthora.
- Effectiveness of the tested elicitors on root rot development was genotype-dependent: all four elicitors (E1-E4) clearly reduced development of disease symptoms, indicative of resistance, in one raspberry variety whilst only one of the elicitors (E1) was effective in the other variety. In the case of another elicitor (E2), the growth of the treated plants in both raspberry varieties was less affected by pathogen attack in spite of the development of root rot symptoms, which suggests an increased tolerance to the disease induced by the elicitor.
- Analyses of raspberry root and leaf samples for the response of three enzymes known to be related to defence activation in other plants showed that elicitor treatments differentially affected the early response of enzyme activities to P. fragariae var. rubi depending on the elicitor, the raspberry variety and the treated plant tissue. The elicitor E1 showed most effect, stimulating all three enzymes in the leaves of one raspberry variety, and one enzyme in the roots of both varieties. The effect of the other three elicitors was limited to one enzyme in roots (E3, E4) or in leaves (E2).
Compatibility and synergy between elicitor treatments and biotisation
Compatibility between biotisation and elicitor treatment was evaluated under greenhouse and organic farming conditions using as indicators: mycorrhizal development, plant growth, defence-related enzyme activities, fruit production and AOM content / antioxidant activities in leaves and fruits.
Under greenhouse conditions
- The four elicitors which can reduce Phytophthora root rot in raspberry plants were compared for effects on mycorrhizal development and plant growth during the large scale greenhouse production of biotised raspberry plants. Elicitors were applied three times as an aerial spray at two week intervals to non-biotised or biotised raspberry plants after acclimatization, and effects monitored up to eight weeks. All four elicitors were compatible with biotisation. Although mycorrhizal development decreased slightly following a second elicitor treatment, levels subsequently increased to reach those comparable to plants not treated with the elicitors. None of the elicitors altered the beneficial effect of biotisation on raspberry plant growth as compared to non-biotised plants.
- Root and leaf samples of the raspberry plants were analysed after the last elicitor application for the response of the three defence-related enzymes. Effects were not conclusive and varied between the enzymes, plant parts and biotisation. Whilst two enzyme activities were decreased by the elicitor in the leaves of all plants, two showed increased activities in the leaves or roots of elicitor-treated, biotised plants.
- The elicitor (E1)-treated raspberry plants were transferred into fresh substrate and overwintered in an unheated, unlighted greenhouse; the elicitor was foliar applied again at early and late flowering stages. Mycorrhizal colonization in biotised plants was not significantly affected by the elicitor treatment and was maintained through the overwintering period. Biotised plants developed a higher number of buds and flowers than non-biotised ones in spring. During the first five weeks of harvest, total fruit yield and number were greater in biotised compared to non-biotised plants with values for the yield of biotised plants ranging from +37 % to +60 % in presence or absence of the elicitor. At the last harvest after nine weeks, no differences were observed between treatments.
- The influence of biotisation and/or elicitor treatment on AOM content and antioxydant activities in the overwintered raspberry plants was not consistent between leaves and fruits. Leaflets of non-biotised plants treated with the elicitor showed an increase in some AOM and antioxidant activities whilst in biotised plants, the levels of AOM and antioxidant activities decreased as compared to non-biotised plants and this trend was accentuated by elicitor treatment. Different observations were made for fruits where elicitor application tended to decrease AOM and antioxidant activities in non-biotised plants, whereas in biotised plants treated with the elicitor, on the contrary, the levels of AOM, but not antioxidant activities, were higher in fruits as compared to biotised or elicitor-treated, non-biotised plants
Under organic farming field conditions
- Organic farming conditions enhanced compatibility in elicitor-treated, biotised plants in a pilot field trial. Biotised and non-biotised plants of two floricane raspberry cultivars transplanted into an organic production system were foliar treated or not with a plant defence elicitor (E1) at 4, 8 and 12 weeks after plantation and at bud break, early and full flowering the following year. Treatment with the elicitor had no negative effect on mycorrhizal development, whatever the raspberry cultivar. At 12 weeks after transplantation, there was a synergistic effect of two plant-based liquid manures on mycorrhizal development for the two raspberry cultivars and mycorrhizal levels were higher in the elicitor-treated plants compared to untreated plants.
- No significant variation was observed in the mean fresh weight of fruits from either raspberry cultivar between plants treated or not with the elicitor, whether they were biotised or not before out-planting, probably due to mycorrhization of the non-biotised plants by AM fungi indigenous to the field soil. However, application of both liquid manures increased fruit size in previously biotised plants of one cultivar compared, and this synergistic effect was accentuated in elicitor-treated plants.
- Clear differences in AOM content and antioxidant activities existed between the two raspberry cultivars grown under organic farming field conditions. The impact of biotisation and/or elicitor treatment on AOM contents and antioxidant capacities varied between the two raspberry cultivars, having no effect in one cultivar, across treatments, and decreasing slightly with elicitor treatment alone or combined with biotisation at outplanting.
In conclusion, the objectives of associating biotisation and elicitor treatment in the production of raspberry microplants or cuttings have been attained through the combined use of:
(i) growth-promoting and compatible AMF isolates and beneficial bacteria,
(ii) an optimised procedure for large scale biotisation of raspberry plantlets before outplanting, and
(iii) effective elicitor treatment for reducing development of Phytophthora root rot in raspberry plants.
The successful utilisation of both biotisation and plant defence elicitors resulted in improved plant growth / fruit yield, even though treatments did not consistently influence AOM content or antioxidant activities of raspberry fruits under the experimental conditions. The defined methodology has the advantages of being highly reproducible, rapid and applicable to a large number of plants, without excessive cost, destined to greenhouse or field production systems. Also, the adaptation of commercial substrates to biotisation is important in order to give a competitive advantage to SMEs in soil-less as well as soil-based production systems. Altogether, these results represent a promising step towards the development of innovative plant production procedures respectful of the environment.
Biotisation and elicitor application in real production systems
In order to evaluate the response of raspberry varieties to biotisation and plant defence elicitor treatment under real production conditions, protocols based on a common experimental design and layout were defined for use either in the field or under polythene tunnels, in soil or in soil-less substrates, depending on the trial location. Recommendations were formulated for the application of one elicitor (E1) with a clear potential to reduce development of root rot symptoms in different raspberry varieties whilst being compatible with biotisation. Four raspberry varieties (three floricane, one primocane) were subjected to four treatments (untreated, biotised before outplanting, elicitor, elicitor and biotised) at seven locations in five countries (Switzerland, Slovenia, Bulgaria, Scotland, France). Elicitor treatment was programmed to protect raspberry plants against four major diseases: root rot, cane blight, spur blight and grey mould. Fertilisation practices were applied according to the producer.
Productivity in soil-based cultivation systems
- No drastic changes were observed in the composition of microorganisms in soils as a result of biotisation and/or elicitor application. Mycorrhizal levels remained high in all biotised plants out-planted to soil, and roots of non-biotised plants became colonised by indigenous mycorrhizal fungi to comparable levels after two to three months. Elicitor applications had no consistent effect on mycorrhiza development, whatever the trial location. Low phosphate fertigation favoured mycor-rhiza development.
- Biotisation and elicitor treatment had significant positive effects on raspberry fruit yield at a Bulgarian field site, although genotype differences were observed. A primocane raspberry variety responded best to each treatment separately whilst biotisation, with or without elicitor, was most effective for a floricane variety. Genotype differences were also found in soil-based production system under tunnel in Slovenia, where elicitor or biotisation plus elicitor treatments provided increased fruit yields in the primocane variety, but not in a floricane variety, although in this case, the genotype variation may have been related rather to differences in cold tolerance during overwintering.
- Genotype differences were again observed in responses to biotisation and/or elicitor treatments between primocane and floricane varieties outplanted into a P. fragariae var. rubi-heavily infested field site in Scotland, in this case in sensitivity to root rot. Elicitor treatment of primocane plants provided some protection against Phytophthora, while the combination of biotisation and elicitor provided best protection against the fungal pathogen in the floricane variety. In contrast, biotisation and/or elicitor treatments had no significant effect on factors affecting fruit yield of a floricane variety outplanted into another Phytophthora-infested field site in Switzerland.
Productivity in soil-less cultivation systems
- Mycorrhization of biotised plants tended to be low after outplanting and then increased to reach good levels in the second year of raspberry plant production. Mycorrhiza development was not adversely affected by elicitor treatment and was enhanced by suppression of P fertilization in a P-rich substrate.
- Biotisation and/or elicitor treatments had no significant effect on factors affecting fruit yield of a floricane variety produced in a soilless greenhouse production system in Switzerland, whilst the combination of biotisation and elicitor provided best fruit yield of primocane plants in a soilless production system under plastic tunnel in France. Here, elicitor treatment provided good vegetative growth and root development, although elicitor treatment induced some early phytotoxicity symptoms.
AOM content and antioxidant capacities
- Growing (geographical) location, including climate and culture conditions, significantly influenced AOM content and antioxidant capacities of raspberry fruits with, in general, fruits from the field site in Bulgaria showing the highest contents of health related compounds. Fruits from production on soilless substrate had higher contents of anthocyanins but lower contents of total phenols and antioxidant capacities compared to fruits from plants grown in soil.
- Plant genotype was also an important factor influencing AOM content and antioxidant activities in raspberry fruits. Taking all the growing locations and cultural treatments together, one floricane variety was significantly richer in total phenolic content, the primocane variety in total anthocyanin content and another floricane variety was intermediate for both AOM.
- Neither biotisation nor elicitor treatment had any clear impact on AOM production and antioxidant activities in fruits or leaves at each location. Nevertheless, leaves or fruits of some raspberry varieties from production systems with higher mycorrhizal colonisation showed overall higher total phenols and antioxidant capacities than elsewhere.
In conclusion, the compatibility observed between plant defence elicitor treatment and biotisation in the production trials, conducted over two years in the field, under polythene tunnels, in soil or using soil-less substrates, indicate that these agronomic practices can be used together in most production systems for raspberries. Results highlight the importance of geography / location and plant genotype in determining the response of a raspberry crop to biotisation and/or elicitor application. There is a clear potential to use these agronomic innovations in some environments and in certain raspberry cultivars, to increase fruit yield or to improve plant protection from the damaging effects of root rot by P. fragariae var rubi., although treatments did not consistently influence AOM content or antioxidant activities of raspberry fruits. Recommendations have been made to the project SMEs for the successful utilisation in raspberry production of both biotisation to improve plant growth / fruit yield and plant defence elicitors which effectively reduce Phytophthora root rot, even though treatments did not consistently influence AOM content or antioxidant activities of raspberry fruits under greenhouse or organic farming conditions.
The QUALIREDFRUITS project was co-funded by the European Commission (EC) and involved seven small and medium enterprises (SMEs) and five research and technological development (RTD) performers altogether representing seven different European Member and Associate States. This project dealt with the improvement of quality and agricultural procedures of raspberries considered as a paradigm of red fruits because of their high value due to:
- a unique panel of flavours and sensory quality,
- a proven 'health promotion disease prevention' (HPDP) property, and
- a high but still increasing competition between production worldwide.
Despite its continuous increase in the world market during the last decade, the European market share has remained stable around 20 % and may decrease in the next decade. In this context, the major innovative aspects of the QUALIREDFRUITS project are to provide a consortium of SMEs with the necessary research activities aimed at combining - for the first time - varietal selection of raspberries for greater HPDP properties with new cultivation systems based on biotisation (inoculation of beneficial microorganisms) and elicitors (stimulators of natural defence), in order to optimize the nursing of healthy raspberry plantlets and ensure high-quality of the fruits without resorting to the use of polluting chemicals.
Moreover, the sustainable development of new production strategies requires that molecular tools be developed to:
- monitor beneficial microorganisms introduced into biotised raspberry culture systems,
- assess plant sanitary status,
- enable the tracing of raspberries on the market for producer protection against biopiracy and,
- guarantee long-term conservation and availability of germplasms of high quality raspberry varieties selected during the project.
Raspberry crop
In the global fruit production system, a dominant position belongs to the berry fruits and especially raspberry production. The major production regions are: Russia, Europe and the Pacific Coast of United States of America (USA) and Canada. Many other countries, such as Chile, New Zealand, Australia and China are entering into the market as they supply the fresh market during winter in the northern hemisphere.
In Europe, most of the raspberry production is in the northern and central countries and higher regions of Italy. Greece, Portugal and Spain are new comers. In many production areas, the fruit is grown for the high-value fresh market, but in Eastern Europe (Serbia, Poland, and Hungary) a high proportion of the crop is for further processing, i.e. frozen preparations including individual quickly frozen (IQF) fruits, juice, concentrate, and preserves. These products are then packaged and sold directly to the consumer or further processed into jam, jelly, dessert topping, pie filling, ice cream, yoghurt, etc. The popularity of raspberries continues to grow as shown by the increasing number of raspberry-containing products on supermarket shelves.
Key figures
Statistics about production are controversial at both world and national levels. Roughly, the worldwide production is about one million tonnes per year. A half of it enters into the regular market where an average 20 % comes from Europe. Since the market price of raspberries varies from EUR 1 to 7/kg paid to the producers and EUR 7 to 10 paid by the consumers, the economical significance of this crop on the global market is rather high.
Context of the project
The QUALIREDFRUITS project is undertaken at the crossroad between the evolution of the fruit market, especially red fruits, and the progress of plant physiology.
Market trends
In addition to the continuous request for increased productivity and decreased production costs, the new trends of the market are dominated by the increasing consumers' interest in the potential of red fruits in health promotion and disease prevention (HPDP) mainly resulting from their potentially high content in antioxidant molecules (AOM). Additionally, there is an increasing social awareness and requests for agricultural practices globally safer for the environment and free from noxious chemicals in the context of sustainable development and food safety. In the near future, these new features may significantly add to the conventional quality criteria which are: the fruit size and firmness, the colour, the ease of harvest and the taste (in priority order).
Scientific and technical progress in plant physiology
The mechanisms which regulate the interactions between the soil and the plant roots are not fully elucidated yet. Nevertheless, the role of fungi and bacteria in these interactions is better known and laboratories have developed a new knowhow to monitor the plant roots / bacteria / fungi symbiosis in order to protect plants from their major diseases such as fungi and viruses. Plant conditioning is known as 'biotisation'. Additionally, it has been established that the plant self-defences against several pathological agents can be enhanced by new treatments without pesticides or fungicides and known as 'elicitors'.
Major objectives
The current market trends and progresses in plant physiology and molecular biology have created a timely opportunity to identify raspberry varieties with higher quality in terms of AOM content and consistent with innovative cultural practices respectful of the environ-ment. The QUALIREDFRUITS key objectives of the project are to:
- screen raspberry varieties for their AOM content and compare with the varieties currently dominating the market;
- design and deliver on purpose cultural practices suitable for the selected varieties and making use of beneficial micro-organisms (biotisation) and natural elicitors of plant defence reactions in order to drastically reduce chemical input and increase AOM synthesis in the selected varieties.
Considering the improvement of the competitiveness of the SMEs involved in the project as the global and ultimate prospective, the newly design chain from plant biotisation, plant nursing and farming must be strengthened by an adequate quality assurance scheme for both plant production and further raspberry production. The project is thus meant to bring operational advantages to the SMEs participants while contributing, as a pioneer, to the evolution of fruit production in a broader prospective. This includes the development and adaptation of complementary technologies for:
- monitoring of the biotisation system towards large scale industrialisation,
- assessing plants sanitary status,
- tracing raspberries to combat biopiracy, and
- preserving germplasms of the processed varieties on a long term.
Ultimate challenges of the project
While the world market of raspberries has increased by 60 % in the last decade, the relative share of the products originating from Europe has increased in a lesser proportion although the production capacity per unit of cultivated area has doubled during the same period of time. Reasonable business anticipations suggest that the European Union (EU) dependence in volume and in value may unfortunately decrease by 10 % in the 5 years after the new varieties and innovative cultivation procedures resulting from the QUALIREDFRUITS project are actually demonstrated and consolidated at the field level. This reduction of imports to the benefit of European actors is expected to reach 10 % per 5-year time slot and should likely continue for 2 decades. This will require that several challenges be met and namely:
- broadening the offer to customers by introducing on the market new cultivars originating from European technologies;
- underpinning such new European varieties thanks to a standardised assessment of their content in bioactive compounds as the role of these compounds in HPDP is increasingly established by medical research worldwide;
- offering varieties that require significantly reduced chemical treatments as the result of innovative cultivation procedures;
- orienting the relevant regulatory framework towards the establishment of certification procedures based on easy genotyping of the new varieties and easy assessment of the sanitary status of the plants;
- forging arguments to promote the informative labelling of the marketed fruits and processed derivatives concerning their HPDP properties including measurement of AOM content.
Implementation strategy and expected outcomes
The main directions followed by the project are:
- selection of raspberry varieties with high fruit quality (including AOM) and tolerance / resistance to fungal diseases;
- development of new methods of plant biotisation and elicitor treatment for healthier plant production;
- conservation, genotyping and plant health assessment;
- production field trials.
Since the project approach from laboratories to fields had to deal with seasonal constraints, parts of the work was undertaken in parallel instead of sequential developments.
Objective indicators involved molecular markers for:
- the specific identification of varieties together with a cryopreservation protocol for germplasms' long-term conservation;
- the identification of beneficial and pathogenic micro-organisms in order to assess the sanitary status of plants;
- the biomarkers-based approach tested in real production trials, from in vitro culture to the field for validation.
Finally, appropriate controls and reproducibility tests enabled the partners to formulate recommendations for quality production of raspberries taking into due consideration both the fresh fruits market of higher quality and the processed fruit market.
Project results:
The results of the QUALIREDFRUITS project which had to be delivered to the SME participants by the RTD performers according to the transaction of the Grant Agreement and the description of work (DoW) are reminded.
Independently from the deliverables reporting the achievements in the project work packages (WPs), each eesult R01 to R09 has been formally delivered in a detailed internal report available to fix the joint exploitation agreement among the SME participants and to serve as foreground reference for any further work to be carried out after the end of the project.
The internal reports executive summaries are presented below.
Result ID: R01
Disclosing Institution: Agroscope Changins-Wädenswil Research Station ACW, Centre de recherche Conthey Route des vergers 18, 1964 Conthey, Switzerland
Authors: Zo-Norosoa Camps, Christoph Carlen P08-ACW, Kaloyan Kostov, Slavcho Slavov, Rossitza Batchvarova P10-ABI, Anton Ivancic, Andrej uek P09-UMFKVB, Tomasso Sozzi, Vivienne Gianinazzi-Pearson, Silvio Gianinazzi P06-INRA
Dissemination level: Confidential
Title: Identification of raspberry varieties with enhanced antioxidant molecule content and/or tolerant to pathogenic fungi that could be used as commercial varieties and/or in a breeding program of new commercial varieties
Date of disclosure: 30 November 2012 - Project month: 24
Summary:
- The aim of WP1 was to select cultivars riche in AOM and tolerant to diseases with a high exploitation potential with regard to specific cultivation procedures, to breeding and to intellectual property rights (IPR).
- A screening of about 100 varieties and genotypes of raspberries made it possible to define 37 available floricane and primocane fruiting raspberries varieties to be tested by entering them into the experimental approach of the project and finally support a future breeding programme.
- Concerning AOM synthesis of floricane fruiting cultivars the most interesting red cultivar is Elida. Elida showed high contents of Vitamin C, phenolics, ellagitannins and high antioxidant capacities. Elida may support a future breeding program in combination with a high yielding cultivars producing firm fruits such as Tulameen. Another approach is to breed a high yielding cultivar with big, light red fruits with Bristol or some wild genotypes (SL, F) to get progenies with high contents of health related compound. These progenies may have to be backcrossed with another high yielding cultivar to get a cultivar with big, red fruits and a high yield.
- Concerning AOM synthesis of primocane fruiting cultivars, the most interesting varieties were Joan J and Kweli. It would be an interesting approach to cross these two cultivars, because their positive attributes concerning AOM synthesis are very complementary. On the other hand, Kweli, a high yielding cultivar with big, firm fruits, has an interesting potential for healthy fruits and in combination with specific cultivation procedures (biotisation, elicitor treatments) to produce high value fruits.
- Concerning the tolerance to diseases (such as Botrytis cinerea and Leptosphaeria sp and Phytophthora sp.) of floricane fruiting cultivars, Elida showed good tolerance against Botrytis cinerea and Schopska Alena and Williamette against Phytophthora sp. The other cultivars were not different from each other concerning the tolerance to these diseases. Concerning the primocane fruiting cultivars Sugana, Autumn Bliss, Joan J (Dieffenbach) and Erika showed interesting tolerance to Botrytis cinerea and Leptosphaeria sp. Erika has a good tolerance to Leptosphaeria sp. and the two Bulgarian cultivars Iskra and Samodiva against Phytophthora sp.
- Data on technical aspects of hybridisation of new cultivars including pollination, seed harvesting, stratification of seeds and plantlets production were given related to experiences developed in this project. The biggest problems were to regenerate plantlets from seeds obtained from crossing of two genotypes.
- In order to capture the greatest value from plant-related intellectual property assets, a number of types of statutory intellectual properties rights for SME involved in breeding new varieties and/or SME involved in biotisation of plants are described and discussed such as plant variety protections (PVPs), plant patents, geographic indications, trademarks and trade secrets. SME participants should be aware of all options for further IP protection and use them to their greatest advantage. The various forms of intellectual property (IP) protection for plants can be used alone or in combination.
Result ID: R02
Disclosing institution: Institut National de la Recherche Agronomique, 17 Rue Sully, BP 86510, 21065 Dijon Cedex, France
Authors: Vivienne Gianinazzi-Pearson, Silvio Gianinazzi, Tommaso Sozzi
Nature: Report
Dissemination level: Confidential
Title: Identified beneficial microbes for plant biotisation
Date of disclosure: 30 November 2012
Summary:
- Three arbuscular mycorrhizal fungi are identified for efficient raspberry plant biotisation: Glomus intraradices isolates LPA8, LPA7 and LPA54.
- Two beneficial soil bacteria are identified for efficient raspberry plant biotisation: Pseudomonas fluorescens sp. I13 and Sinorhizobium meliloti.
- A composite inoculum has been defined based on the compatibility between the bacterium Sinorhizobium meliloti and the arbuscular mycorrhizal fungus Glomus intraradices isolate LPA8.
- A biotisation procedure is provided for the inoculation of Glomus intraradices iso-late LPA8 and Sinorhizobium meliloti during the acclimatisation of vitroplants or cuttings of different raspberry cultivars.
- The described biotisation procedure is highly reproducible and the methodology has the advantage of rapidity and applicability to a large number of plants.
Result ID: R03
Disclosing Institution: Scottish Agricultural College, West Mains Road, Edinburgh EH9 3JG, UK
Authors: Dale Walters
Nature: Report
Dissemination level: Confidential
Title: Identified elicitors of natural plant defence mechanisms for improving raspberry resistance against pathogenic fungi
Date of disclosure: 26 June 2012
Summary:
- Cultivar differences exist in raspberry responses to foliar or root drench application of the plant defense elicitors Bion (acibenzolar-S-methyl), BABA (beta-aminobutyric acid), Yea Foliar (chitosan) and methyl jasmonate.
- In Tulameen cultivar plants, only Bion reduces root rot damage by P. fragariae var rubi and increases root development.
- In Meeker cultivar plants, all four elicitors significantly decrease root rot damage and increase resistance to P. fragariae var rubi, with best control achieved using methyl jasmonate.
- The elicitor Bion is identified as effectively increasing resistance to the root rot pathogen in both Tulameen and Meeker raspberry cultivars.
- Increased resistance elicited by Bion in Tulameen plants is associated with enhanced activity of the defence-related enzymes peroxidase, beta-1,3-glucanase and cinnamoyl alcohol dehydrogenase (CAD) in leaves.
- BABA appears to increase 'tolerance' to raspberry root rot in Tulameen and Meeker plants, and enhance CAD activity in Tulameen leaves.
Result ID: R04
Disclosing institution: Institut National de Recherche Agronomique (INRA), France
Authors: Vivienne Gianinazzi-Pearson, Silvio Gianinazzi,Tommaso SozziI at INRA and Dale Walters at Scottish Agricultural College (SAC), Scotland, Christoph Carlen, Zo-Norosoa Andrianjaka-Camps at ACW - Eidgenoessisches Volkswirtschaftsdepartement, Switzerland
Nature: Report
Dissemination level: Confidential
Title: Defined protocol for the combined use of biotisation and elicitor treatment
Date of disclosure: 20 November 2012
Summary:
- Indicators have been defined for monitoring compatibility in raspberry plants between biotisation and plant defence elicitor application: mycorrhizal development, plant growth, defence-related enzyme activities, fruit production and AOM content / antioxidant activities in leaves and fruits.
- Protocols are described for the combined use of biotisation and elicitor application in greenhouse and real production systems of raspberry plants and fruits.
- Treatment of raspberry plants with the plant defence elicitors BION, BABA, methyl jasmonate or chitosan is compatible with biotisation with a composite microbial inoculum (glomus intraradices isolate LPA8 / sinorhizobium meliloti) under greenhouse production conditions.
- The compatibility of BION application with plant biotisation has been confirmed in a real production system of organic farming for raspberries.
Result ID: R05 - R06 - R07
Disclosing institution: University of Maribor, Faculty of Agriculture and Life Sciences, Pivola 10, 2311 Hoce, Slovenia
Authors: A. uek, M. iko, A. Ivancic at University of Maribor, Faculty of Agriculture and Life Sciences, V. Gianinazzi-Pearson, T. Sozzi at INRA, R. Batchvarova at Agrobioinstitute (ABI), A. Gollotte at INOPLANT, France
Nature: Report
Dissemination level: Confidential
Title: Conservation, genotyping and plant sanitary status assessment
Date of disclosure: 14 November 2012
Summary:
- The cultivars Meeker, Glen Ample, Rose de Core d'Or, JJR, Sicoly S1 and Polka were successfully identified with microsatellites markers.
- Specific molecular primers for five plant pathogenic fungi (Phytophthora fragariae, Thielaviopsis basicola, Leptospheria coniothyrium, Aphanomyces euteiches, Pythium ulitmum) were designed.
- Specific molecular primers for three beneficial bacteria (Sinorhizobium meliloti, Pseudo-monas fluorescens sp. I13, Paenibacillus sp. B2) were designed.
- Specific molecular primers for two beneficial fungi (Glomeromycota, Glomus intra-radices) were designed.
- Cryopreservation protocol based on encapsulation / vitrification was developed for raspberry varieties Tulameen and Meeker.
Result ID: R08
Disclosing institution: SRUC (formerly SAC), West Mains Road, Edinburgh EH9 3JG, UK
Authors: Dale Walters
Nature: Report
Dissemination level: Confidential
Title: Innovative scheme for raspberry plant production from in vitro to field, requiring low amounts of chemical input and more compatible than conventional with respect to the environment
Date of disclosure: 14 December 2012
Summary:
- The aim of WP4 was to test raspberry varieties and methods developed in WPs 1, 2 and 3 under real production conditions. In order to achieve this aim, field trials were conducted in Switzerland, France, Slovenia, Bulgaria and Scotland, using the same basic design and layout of the trials and joint protocols for treatment. The overall aim was to test the effect of biotisation and elicitor (Bion) treatments, alone and in combination, on growth and yield of raspberry varieties, and on control of diseases, particularly raspberry root rot. The sanitary status of the plants was examined to ensure the presence of mycorrhiza and the absence of pathogens. The content of antioxidant molecules (AOM) and the antioxidant capacity of the plants receiving the different treatments were also determined.
- The results highlight the importance of geography / location, and genotype, in determining the response of a raspberry crop to agronomic treatments.
- Biotisation and elicitor treatments had no significant effect on factors affecting fruit yield in Switzerland.
- There were significant effects of the treatments on growth of raspberry plants and fruit yield in Bulgaria, although genotype differences were observed in fruit yield.
- Genotype differences were also found in Slovenia, where elicitor, and biotisation plus elicitor treatments, provided increased fruit yields in Polka, but not in Tulameen.
- In France, the combination of biotisation and elicitor provided good fruit quality and yield, while the elicitor treatment provided good vegetative growth and root development. Here, the elicitor treatment (Bion) was associated with some phytotoxicity.
- Under Scottish conditions, poor weather in 2011 and extremely poor weather in 2012 affected the trial, again highlighting the role of geography, with its accompanying differences in weather, on crop performance. Nevertheless, the field trial in Scotland confirmed the genotype differences observed in the other field trials, and also showed that in Polka, the elicitor can provide some protection against Phytophthora root rot, while in Tulameen, the combination of biotisation and elicitor provided best protection against root rot, under very heavy inoculum potential.
- Analysis of AOM contents and antioxidant activities in the leaves and fruits from plants in the different field trials also highlighted the importance of genotype, followed by geographic location, while agronomic treatments had little effect.
- In most of the field trials, elicitors and biotisation were compatible, suggesting that in most production systems for raspberries, the elicitor Bion and biotisation can be used together.
Result ID: R09
Disclosing Institution: SRUC, West Mains Road, Edinburgh EH9 3JG, UK
Authors: Dale Walters
Nature: Report
Dissemination level: Confidential
Title: Formal set of recommendations to SMEs for producing and using biotised plants, and for elicitor treatment procedures
Date of disclosure: 14 December 2012
Summary:
- Based on the experiments carried out in the four work packages comprising this project, recommendations can be offered to SMEs.
- A protocol has been defined for the combined use of biotisation and elicitor treatment, based on:
(i) isolates of mycorrhizal fungi and beneficial bacteria selected by P06 INRA for their efficiency and compatibility,
(ii) a procedure optimised by P03 INOPLANT for biotisation of raspberry plants before outplanting, and
(iii) an effective elicitor treatment developed by P07 SAC for reducing development of Phytophthora root rot symptoms in plant production systems.
- Specifically, the following recommendations can be given to SMEs:
i. Since the plant defence elicitor BION is compatible with biotisation when applied under greenhouse conditions, its application can be recommended during the standardised procedure for large scale production of biotised raspberry plants to provide effective control of Phytophthora root rot.
ii. In some environments (e.g. Bulgaria, France and Slovenia), and in certain cultivars (e.g. Polka and perhaps Tulameen), biotisation and the elicitor Bion can be recommended to protect raspberry plants against Phytophthora root rot and to increase plant growth under field conditions.
iii. In Scotland, depending on the raspberry variety, biotisation and / or the elicitor Bion can be recommended to reduce the damaging effects of root rot under field conditions.
Detailed achievements of the project
Selection of raspberry varieties with high fruit quality and tolerance to fungal diseases
Current interest by consumers, media and medical actors in the health benefits of several components of fruit and vegetable aliments having pharmaceutical properties (often referred to as 'nutraceutical' in English and 'Alicament' in French) has created an increasing interest in the berry industry. There is a consensus that berries are a nutritional and nutraceutical powerhouse, and that raspberries rank near the top of all fruits for antioxidant strength. Past and current medical research shows likely benefit of regularly consuming raspberries against cardiovascular diseases, diabetes, inflammation, some cancers and age-related degenerative diseases. Such benefit is proven to result from antioxidant molecules (AOM) (Kresty et al., 2001; Puupponen-Pimiä et al., 2005; Coates et al., 2007). The concept of AOM refers mainly to non-nutrient compounds in foods and covers a broad range of molecules including flavonoid or non-flavonoid polyphenolics, phenolic acids, vitamins and other potential organic antioxidants (Beekwilder et al., 2005). Anthocyanins and ellagitannins are the major contributors to the total antioxidant activities of raspberry (Mullen et al., 2002; Beekwilder et al., 2005; Battino et al., 2009): ellagitannins contribute around 50 % and anthocyanins about 25 %. AOM synthesis in raspberries varies between plant varieties; most red raspberry varieties presently on the market contain relatively low levels of AOM compared to black raspberry, blackberry and wild raspberry.
Raspberry plants are prone to parasites, fungal diseases and virus infections, which make the use of pesticides a conventional cultivation practice. Chemical treatments generate potentially noxious food residues which counterbalance the positive effect of berry diet upon human health (?ozowicka et al., 2012), but little success has been achieved to find alternatives to fungicides to control fungal diseases (Ançay et al., 2005; Xu et al., 2012). Three fungal pathogens have a high potential to cause serious raspberry yield loss. Root rot due to Phytophthora fragariae var. rubi, the main cause of raspberry plant death, cannot be effectively controlled by chemicals and only prophylactic measures can be adopted by planting healthy raspberry plants in non-contaminated soils (Ançay et al., 2005). Leptosphaeria coniothyrium, which attacks raspberry canes, causes variable disease se-verity depending on the year, situation, weather and varieties (Williamson et al., 1986). Finally, Botrytis cinerea causes the fruit grey mould responsible for significant economic loss (Xu et al., 2012; O'Neill et al., 2012).
Technical data on raspberry varieties / cultivars with a high exploitation potential related to enriched AOM content of fruits and tolerance to disease are therefore an important pre-requisite to an integrated approach including the selection of plant varieties more resistant to main pathogens and breeding-based creation of new genotypes with combined producer and consumer defined traits.
Pre-selection and evaluation of raspberry varieties
About 100 commercially and collection available varieties and genotypes of raspberry were screened on the basis of literature and project partner experiences for their high economical importance and contrasting traits concerning consumer criteria (fruit sensory quality, firmness), farming criteria (yield, fruit size), potentially higher quantities of antioxidant molecules (HPDP properties) and/or tolerance to major fungal diseases in raspberry production systems. A total of 33 available floricane and primocane fruiting raspberries cultivars as well as 4 wild or semi-wild floricane genotypes were defined and grown to fruit for further analyses.
- AOM content and antioxidant capacities
- Appropriate methodologies for extraction and analysis of health-related compounds in raspberry fruits and leaves were defined and applied to 23 floricane and 11 primocane cultivars or genotypes for the characterisation of AOM compound contents (vitamin C, total phenolics, anthocyanins, ellagitannin) and antioxidant capacities (DPPH radical inhibition, ferric reducing antioxidant power, oxygen radical absorption capacity).
- Overall, the variation in antioxidant capacities of floricane and primocane cultivars followed that in terms of AOM content, with cultivars rich in the major phytochemicals showing the higher antioxidant activities. Results clearly indicated that leaf antioxidant analyses cannot be used to predict fruit antioxidant capacities. No correlation was found between leaf and fruit antioxidant capacities, or between leaf antioxidant capacities and fruit AOM content.
- In general, floricane fruiting cultivars with interesting agronomical, physical and sensorial quality parameters had relatively poor antioxidant activity and health related compounds. From factorial discriminant analyses, floricane raspberries could be classified in four principal groups depending on their anthocyanin constituents and in four main clusters according to ellagitannin patterns, whilst primocane genotypes were distinguished into three categories for their anthocyanin patterns and two main classes for ellagitannin composition.
- Three primocane cultivars gave a significantly higher yield and fruit size than all other cultivars whilst four other cultivars (two floricane, two primocane) and some of the wild genotypes showed high potential in terms of AOM content and antioxidant capacities. These could be promising for future breeding in combination with high yielding cultivars producing large, firm fruits.
- Tolerance to fungal diseases and fruit malformation
-Eleven floricane and 9 primocane fruiting cultivars were examined for tolerance to three pathogenic fungi causing major diseases in raspberry production systems. Detached leaf and stem bioassays against infection by Phytophthora fragariae var. rubi, Leptosphaeria coniothyrum, Botrytis cinerea) showed that none of the studied floricane fruiting cultivars possess good tolerance to all three fungal diseases. However, one floricane cultivar demonstrated good tolerance towards B. cinerea, and two others against P. fragariae.
- For primocane fruiting cultivars, one showed significant tolerance to all three fungal diseases. An interesting level of tolerance against B. cinerea and L. coniothyrum was observed for another three cultivars, and against P. fragariae for two.
- None of the floricane or primocane fruiting cultivars grown in a project field site showed more than 2 % of fruit malformation. No virus or MLO was detected and canes deformed fruits in one year did not necessarily have them in the following year. Conditions during flowering, especially extreme temperatures, may impact on fruit malformation
Pilot hybridisations
- In order to set the pace for breeding innovative raspberry genotypes with high exploitation potential related to increase the AOM content of raspberry fruits tolerance to diseases, pilot hybridisations were performed and successfully achieved between some interesting genotypes and varieties. Technical procedures (emasculation, pollination, seed harvesting, seed stratification, plant regeneration) were optimised and guidelines were provided to SMEs. Intellectual property rights relative to novel plant-related assets have also been communicated.
- Higher leaf antioxidant activities and total phenolics content were identified in some hybrids between the cultivated and wild genotypes. However, plants were too young to be transferred to the field for fruit evaluation and this will be performed in the 2013 season.
In conclusion, the expectancies of the project SMEs have been met with regards to obtaining reliable information and defining several raspberry cultivars or genotypes which are enriched with healthy compounds, and which meet farmer and consumer criteria requirements. Also in line with the SME expectancies, new analytical tools have been introduced for the identification of raspberries having a high AOM content and thus enabling new marketing approaches to promote raspberry consumption based on objective quality criteria. In this context, knowledge about the qualitative and quantitative contents in health-promoting compounds of raspberry plants or fruits, as well as of their tolerance to diseases, can provide competitive arguments for SMEs in order to increase their market share of fresh fruits or derivative products from fruits and leaves. Moreover, the information can support a future breeding program by suggesting cultivars with a potential to increase AOM content of the fruits or leaves of newly developed cultivars, or their tolerance to three major fungal diseases in raspberry production systems. In the context of a future breeding programme, various types of statutory intellectual properties rights (IPR) for plants such as plant variety protections (PVPs), plant patents, geographic indications, trademarks or trade secrets have been communicated to the project consortium.
Raspberry plant genotyping, conservation and health assessment
Variety / cultivar registration is an important issue of plant genetic resource characterisation and utilisation. Traditional methods to identify raspberry cultivars for the granting of plant breeder's rights are based on phenotypic observations. The incorporation of new methodologies into plant material certification schemes will accelerate and optimise the identification process of genotypes and offer guarantee of plant sanitary status. Genetic fingerprinting (genotyping) can provide exact data on the genetic diversity existing in raspberry collections of available genetic resources for breeding programmes, and independently of developmental stage or environmental factors that may influence the phenotype. Genotyping techniques based on microsatellites, also known as simple sequence repeats (SSRs) or short tandem repeats (STRs), are highly informative (show high polymorphism), technically simple (simpler than other techniques), robust and suitable for automated allele detection and sizing (Rafalski and Tingey 1993). In addition, the utilisation of fluorescence-based automated DNA detection and fragment sizing offers a potential improvement of the efficiency and affordability of variety testing (Hayden et al., 2008). Since raspberry species are vegetatively propagated (not by seed), the identification of genotypes will offer a standardised reference for certifica-tion of any cultivar and for controlling its propagation. This approach may prove to be crucial in the context of variety ownership rights in the event of the creation of new genotypes.
Novel techniques for ensuring long term conservation of raspberry materials, like cryopreservation (Reed et al., 2001; Wang et al., 2005; Keller et al., 2006; Reed, 2008), can provide producers with easy access to stable genetic resources and a back-up to recover varieties when genetic divergence or sanitary problems occur. In this context, the development of molecular tests to detect beneficial or pathogenic micro-organisms in plant materials, soil and substrates will provide rapid and reliable information that is crucial for raspberry cultivation systems. The basic methods for detection and identification of plant-inhabiting micro-organisms applied in practice include symptom observation, isolation from attacked tissue or soil, and morphological characterization of the isolated organism. Molecular techniques based on PCR provide additional information and certainty of the microbial detection and identification process (Bonants et al., 1997, Vandemark et al., 2000, Gollotte et al., 2004, Farmer et al., 2007, Nilsson et al., 2008). The combination of both approaches in the routine observation of plant sanitary status would facilitate the accuracy of the diagnostics and lead to an optimised regime of the plant protection activities.
Plant genotyping
- A molecular technique to genotype raspberry material was developed based on the discriminating power of SSR sequences in 19 microsatellite loci. SSR primer sets designed from the microsatellite loci were used in PCR of DNA extracted from fresh, young leaf tissue to evaluate the genetic diversity in a germplasm collection of 49 raspberry genotypes (semi-wild, wild, floricane, primocane). Capillary electrophoresis of PCR products generated SSR sequences which revealed 167 polymorphic alleles at the 19 microsatellite loci.
- Dendrogram analysis of genetic relationships based on SSR data arranged the 49 investigated raspberry genotypes in five main clusters, with a larger genetic distance between genotypes than between clusters. One cluster included wild and semi-wild raspberry germplasm, whilst one local variety and genotypes resembling cultivated floricane or primocane cultivars were grouped in the other four clusters. Analyses of genetic diversity in the cultivars identified 8 duplicate varieties.
- An SSR-based PCR protocol was optimized for the identification of raspberry varieties considered to be commercially important. A specific fingerprint (electrotrace), which can be used for identification, was obtained for each variety using combinations of SSR primer sets for selected microsatellite loci. The most specific microsatellite loci were selected for each variety from PCR reactions conducted with the 19 optimised SSR primers.
- The optimised protocol was successfully applied to six raspberry varieties. Seven of the most specific microsatellite loci were used for one floricane variety, and combinations of six loci were used for two other floricane varieties and three primocane varieties. To reduce labour and costs for PCR product detection, a multiplex of three primer sets labelled with two different fluorescent dyes were combined. Applicability of the developed protocol for variety identification was verified in a 'blind' test on anonymous DNA samples from five raspberry genotypes.
Conservation of raspberry genotypes
- In the context of long-term conservation and stability of high quality raspberry varieties / genotypes, a cryopreservation technique adapted from published literature was first successfully applied to Medicago sativa seeds in pre-experimentation trials. The same protocol, however, gave toxic effects when applied to shoot tips of three raspberry varieties. An alternative procedure was developed for raspberry tissues, in which treatment with low sugar concentrations, together with adapted osmotic loading and plant vitrification solutions, improved tissue survival and enabled some plant regeneration from raspberry shoot tips or root fragments after storage in liquid nitrogen.
Plant sanitary status assessment
- A molecular method, based on PCR detection, was developed to track the presence or absence of beneficial or pathogenic micro-organisms in raspberry cultivation systems. Specific PCR primers were designed on sequences in the DNA region coding for rRNA and used on soil or plant material from raspberry cultures.
- Molecular marker sets were obtained to detect three beneficial bacteria and one mycorrhizal fungus used in biotisation technologies, and for six fungal pathogens, of which three are important pests in raspberry production systems (Phytophthora fragariae, Leptospheria coniothyrium, Botrytis cinerea).
- Specificity of the PCR detection method was verified under different laboratory conditions and on soil or raspberry plant tissues colonised by the targeted organisms. The approach was successfully applied to check for the absence of pathogenic fungi from substrates or composite inoculum used for biotisation.
- Applicability of the molecular protocols for routine monitoring of the targeted fungi under real production conditions was demonstrated directly on fungal-colonised raspberry plant tissues using DNA from leaves infected by P. fragariae var. rubi or L. coniothyrium, and of roots or soil colonized by P. fragariae var. rubi or AM fungi, sampled from field trials.
In conclusion, the methodology developed for genotyping of raspberry material can be exploited in furthering certification procedures for registration of established or new variety / cultivar cultivars and for their control during propagation or across the market. The protocols defined for detection of beneficial or pathogenic micro-organisms provide rapid and reliable procedures for plant sanitary status assessment, and so promote the production of healthy biotised raspberry plants not contaminated by pathogens. Both these achievements meet the SME expectancies of proven methods for ensuring plant quality in future certification procedures. In addition, optimization of the cryopreservation technique opens the possibility of long-term, genetically stable conservation of pathogen-free raspberry material which represents an added value resource for producers.
Development of new methods of plant biotisation and elicitor treatment for healthier plant production
Biotisation is a biotechnology consisting in the inoculation of young plants with beneficial micro-organisms (bacteria and/or fungi) in order to increase plant vigour (Gianinazzi et al., 2003). In the case of raspberry, biotisation of young plants during the acclimatisation phase has proved useful for improving plant survival and growth, and for increasing tolerance to P. fragariae var rubi (Gollotte et al., 2009; Lemoine et al., 2000). Arbuscular mycorrhizal (AM) fungi are the most significant of beneficial soil micro-organisms as they establish a symbiosis at the interface between soil and the roots of most high value agricultural and horticultural plants. Although AM fungi are present in most ecosystems, their populations are generally limited in the field under conventional agriculture because of their sensitivity to practices using large quantities of fertilisers and pesticides. Moreover, micro-propagated plants and substrates used in raspberry production are usually devoid of AM fungi so that biotisation can be very useful in such cases to ensure plant vigour and health. Biotisation can also be beneficial to field-grown plants, for example under organic farming conditions or when reduced amounts of fertilisers and pesticides are used.
Fighting against cryptogamic diseases is still a problem in raspberry cultivation, although a few raspberry varieties can be identified which show good tolerance to Phytophthora fragariae var. rubi (see above), and producers have to resort to the use of chemical fungicides which can be pejorative to the environment and human health. Elicitors of natural plant defence mechanisms have been shown to provide useful disease control in many crops against a range of economically important pathogens (Walters, 2011), and various natural agents (e.g. cell wall fragments, plant extracts) can induce plant resistance to subsequent pathogen attacks, both locally and systemically (Walters et al., 2005; Walters and Heil, 2007). This induced resistance does not lead to complete disease control, but rather to a reduction in pathogen development and disease symptoms. The defence mechanisms involved are those used in other forms of plant resistance to pathogens and include enhanced defence-related enzyme activities as well as synthesis and accumulation of a range of phenolic compounds, many of which possess antioxidant properties. Agents which mimic natural elicitors of resistance have been developed and commercialised for broad-spectrum disease control using the plant's own resistance mechanisms, but these have not been tested on raspberry (Walters and Heil 2007).
AOM synthesis in plants depends on agricultural practices as well as plant varieties and climatic conditions (Anttonen et al., 2005). Mycorrhization has been reported to increase polyphenol content in grapevine and sweet basil, carotenoid level in sweet potato, and antioxidant activity in raspberry (Krishna et al., 2005; Farmer et al., 2007; Toussaint et al., 2007; Gollotte et al., 2009). Also, treatment with plant defence elicitors has been reported to increase total phenolic content and antioxidant activity in sweet basil (Kim et al., 2005), and to increase total phenolic, anthocyanin and antioxidant content of raspberries and blackberries when sprayed onto fruits during the ripening phase (Wang and Lin, 2000; Wang and Zheng, 2005; Wang et al., 2008). However, the impact of combining these innovative agricultural practices on plant content in AOM has yet to be characterised.
Whilst the identification of plant defence elicitors which enhance resistance to major fungal diseases in raspberry represents a new alternative to chemical control, knowledge about their compatibility, or synergy, with biotisation in raspberry plant production systems is so far inexistent, although positive or no effects have been reported in other plants (Murphy et al., 2000; Sonnemann et al., 2000; Tosi and Zazzerini, 2000; Hause et al., 2007). The enhancement and the consequent standardisation of raspberry plant biotisation coupled with elicitor treatment are expected to be of competitive advantage for the SMEs in terms of healthier plant production and fruit quality.
Raspberry biotisation
- Suitable mycorrhizal fungi and beneficial bacteria were selected for their compatibility in the bioti-sation of raspberry microplants, and a multiplication procedure was established for the production of appropriate mycorrhiza and bacteria inocula. A first composite inoculum for biotisation in raspberry production was defined based on the compatibility of one beneficial bacterium with a mycorrhizal fungus, together with its persistence after inoculation and its synergistic effect on mycorrhizal plant growth.
- A highly reproducible procedure for biotisation of raspberry microplants or cuttings with the composite inoculum was optimised during plant acclimatisation using an appropriate substrate and fertilisation régime. This was subsequently up-scaled to provide material on a large scale for tunnel and field production trials of four raspberry varieties.
- The feasibility of using biotisation was tested under the conditions of field soils or inert substrates used by SMEs to grow raspberry plants. When biotised and acclimatized vitroplants of two floricane raspberry varieties were outplanted into an organic-based field production system, mycorrhizal levels were maintained and non-biotised plants quickly developed mycorrhiza after out-planting. Plant-derived liquid manure proved to be compatible with mycorrhiza development and had a slight positive effect on raspberry fruit size of biotised plants.
- Four commercial potting substrates, currently used by SME partners for raspberry plant or fruit production, proved to be poorly compatible with mycorrhiza development in a biotised floricane variety under greenhouse conditions. Nevertheless, residual beneficial effects of initial biotisation on plant behaviour were observed (absence of mildew, better growth, no lodging). Also, higher fruit yields were obtained in plants that had previously been biotised in two of the commercial substrates and then outplanted into a small field trial.
- Introduction of better adapted mycorrhizal fungi, modications in the P fertilisation regime and/or amendment of the substrate composition overcame the problems of incompatibility in a commercial substrate and greatly improved mycorrhiza development in biotised raspberry plants.
Elicitor treatments
- Four plant defence elicitors (reported to control fungal diseases in other plants) were tested under greenhouse conditions in order to define the most effective product for control of P. fragariae var. rubi in raspberry production systems. Plantlets of two floricane raspberry varieties were treated with the elicitors by foliar spraying or root drench and challenged with P. fragariae two days later. None of the elicitors significantly affected growth of either raspberry variety in the absence of Phytophthora.
- Effectiveness of the tested elicitors on root rot development was genotype-dependent: all four elicitors (E1-E4) clearly reduced development of disease symptoms, indicative of resistance, in one raspberry variety whilst only one of the elicitors (E1) was effective in the other variety. In the case of another elicitor (E2), the growth of the treated plants in both raspberry varieties was less affected by pathogen attack in spite of the development of root rot symptoms, which suggests an increased tolerance to the disease induced by the elicitor.
- Analyses of raspberry root and leaf samples for the response of three enzymes known to be related to defence activation in other plants showed that elicitor treatments differentially affected the early response of enzyme activities to P. fragariae var. rubi depending on the elicitor, the raspberry variety and the treated plant tissue. The elicitor E1 showed most effect, stimulating all three enzymes in the leaves of one raspberry variety, and one enzyme in the roots of both varieties. The effect of the other three elicitors was limited to one enzyme in roots (E3, E4) or in leaves (E2).
Compatibility and synergy between elicitor treatments and biotisation
Compatibility between biotisation and elicitor treatment was evaluated under greenhouse and organic farming conditions using as indicators: mycorrhizal development, plant growth, defence-related enzyme activities, fruit production and AOM content / antioxidant activities in leaves and fruits.
Under greenhouse conditions
- The four elicitors which can reduce Phytophthora root rot in raspberry plants were compared for effects on mycorrhizal development and plant growth during the large scale greenhouse production of biotised raspberry plants. Elicitors were applied three times as an aerial spray at two week intervals to non-biotised or biotised raspberry plants after acclimatization, and effects monitored up to eight weeks. All four elicitors were compatible with biotisation. Although mycorrhizal development decreased slightly following a second elicitor treatment, levels subsequently increased to reach those comparable to plants not treated with the elicitors. None of the elicitors altered the beneficial effect of biotisation on raspberry plant growth as compared to non-biotised plants.
- Root and leaf samples of the raspberry plants were analysed after the last elicitor application for the response of the three defence-related enzymes. Effects were not conclusive and varied between the enzymes, plant parts and biotisation. Whilst two enzyme activities were decreased by the elicitor in the leaves of all plants, two showed increased activities in the leaves or roots of elicitor-treated, biotised plants.
- The elicitor (E1)-treated raspberry plants were transferred into fresh substrate and overwintered in an unheated, unlighted greenhouse; the elicitor was foliar applied again at early and late flowering stages. Mycorrhizal colonization in biotised plants was not significantly affected by the elicitor treatment and was maintained through the overwintering period. Biotised plants developed a higher number of buds and flowers than non-biotised ones in spring. During the first five weeks of harvest, total fruit yield and number were greater in biotised compared to non-biotised plants with values for the yield of biotised plants ranging from +37 % to +60 % in presence or absence of the elicitor. At the last harvest after nine weeks, no differences were observed between treatments.
- The influence of biotisation and/or elicitor treatment on AOM content and antioxydant activities in the overwintered raspberry plants was not consistent between leaves and fruits. Leaflets of non-biotised plants treated with the elicitor showed an increase in some AOM and antioxidant activities whilst in biotised plants, the levels of AOM and antioxidant activities decreased as compared to non-biotised plants and this trend was accentuated by elicitor treatment. Different observations were made for fruits where elicitor application tended to decrease AOM and antioxidant activities in non-biotised plants, whereas in biotised plants treated with the elicitor, on the contrary, the levels of AOM, but not antioxidant activities, were higher in fruits as compared to biotised or elicitor-treated, non-biotised plants
Under organic farming field conditions
- Organic farming conditions enhanced compatibility in elicitor-treated, biotised plants in a pilot field trial. Biotised and non-biotised plants of two floricane raspberry cultivars transplanted into an organic production system were foliar treated or not with a plant defence elicitor (E1) at 4, 8 and 12 weeks after plantation and at bud break, early and full flowering the following year. Treatment with the elicitor had no negative effect on mycorrhizal development, whatever the raspberry cultivar. At 12 weeks after transplantation, there was a synergistic effect of two plant-based liquid manures on mycorrhizal development for the two raspberry cultivars and mycorrhizal levels were higher in the elicitor-treated plants compared to untreated plants.
- No significant variation was observed in the mean fresh weight of fruits from either raspberry cultivar between plants treated or not with the elicitor, whether they were biotised or not before out-planting, probably due to mycorrhization of the non-biotised plants by AM fungi indigenous to the field soil. However, application of both liquid manures increased fruit size in previously biotised plants of one cultivar compared, and this synergistic effect was accentuated in elicitor-treated plants.
- Clear differences in AOM content and antioxidant activities existed between the two raspberry cultivars grown under organic farming field conditions. The impact of biotisation and/or elicitor treatment on AOM contents and antioxidant capacities varied between the two raspberry cultivars, having no effect in one cultivar, across treatments, and decreasing slightly with elicitor treatment alone or combined with biotisation at outplanting.
In conclusion, the objectives of associating biotisation and elicitor treatment in the production of raspberry microplants or cuttings have been attained through the combined use of:
(i) growth-promoting and compatible AMF isolates and beneficial bacteria,
(ii) an optimised procedure for large scale biotisation of raspberry plantlets before outplanting, and
(iii) effective elicitor treatment for reducing development of Phytophthora root rot in raspberry plants.
The successful utilisation of both biotisation and plant defence elicitors resulted in improved plant growth / fruit yield, even though treatments did not consistently influence AOM content or antioxidant activities of raspberry fruits under the experimental conditions. The defined methodology has the advantages of being highly reproducible, rapid and applicable to a large number of plants, without excessive cost, destined to greenhouse or field production systems. Also, the adaptation of commercial substrates to biotisation is important in order to give a competitive advantage to SMEs in soil-less as well as soil-based production systems. Altogether, these results represent a promising step towards the development of innovative plant production procedures respectful of the environment.
Biotisation and elicitor application in real production systems
In order to evaluate the response of raspberry varieties to biotisation and plant defence elicitor treatment under real production conditions, protocols based on a common experimental design and layout were defined for use either in the field or under polythene tunnels, in soil or in soil-less substrates, depending on the trial location. Recommendations were formulated for the application of one elicitor (E1) with a clear potential to reduce development of root rot symptoms in different raspberry varieties whilst being compatible with biotisation. Four raspberry varieties (three floricane, one primocane) were subjected to four treatments (untreated, biotised before outplanting, elicitor, elicitor and biotised) at seven locations in five countries (Switzerland, Slovenia, Bulgaria, Scotland, France). Elicitor treatment was programmed to protect raspberry plants against four major diseases: root rot, cane blight, spur blight and grey mould. Fertilisation practices were applied according to the producer.
Productivity in soil-based cultivation systems
- No drastic changes were observed in the composition of microorganisms in soils as a result of biotisation and/or elicitor application. Mycorrhizal levels remained high in all biotised plants out-planted to soil, and roots of non-biotised plants became colonised by indigenous mycorrhizal fungi to comparable levels after two to three months. Elicitor applications had no consistent effect on mycorrhiza development, whatever the trial location. Low phosphate fertigation favoured mycor-rhiza development.
- Biotisation and elicitor treatment had significant positive effects on raspberry fruit yield at a Bulgarian field site, although genotype differences were observed. A primocane raspberry variety responded best to each treatment separately whilst biotisation, with or without elicitor, was most effective for a floricane variety. Genotype differences were also found in soil-based production system under tunnel in Slovenia, where elicitor or biotisation plus elicitor treatments provided increased fruit yields in the primocane variety, but not in a floricane variety, although in this case, the genotype variation may have been related rather to differences in cold tolerance during overwintering.
- Genotype differences were again observed in responses to biotisation and/or elicitor treatments between primocane and floricane varieties outplanted into a P. fragariae var. rubi-heavily infested field site in Scotland, in this case in sensitivity to root rot. Elicitor treatment of primocane plants provided some protection against Phytophthora, while the combination of biotisation and elicitor provided best protection against the fungal pathogen in the floricane variety. In contrast, biotisation and/or elicitor treatments had no significant effect on factors affecting fruit yield of a floricane variety outplanted into another Phytophthora-infested field site in Switzerland.
Productivity in soil-less cultivation systems
- Mycorrhization of biotised plants tended to be low after outplanting and then increased to reach good levels in the second year of raspberry plant production. Mycorrhiza development was not adversely affected by elicitor treatment and was enhanced by suppression of P fertilization in a P-rich substrate.
- Biotisation and/or elicitor treatments had no significant effect on factors affecting fruit yield of a floricane variety produced in a soilless greenhouse production system in Switzerland, whilst the combination of biotisation and elicitor provided best fruit yield of primocane plants in a soilless production system under plastic tunnel in France. Here, elicitor treatment provided good vegetative growth and root development, although elicitor treatment induced some early phytotoxicity symptoms.
AOM content and antioxidant capacities
- Growing (geographical) location, including climate and culture conditions, significantly influenced AOM content and antioxidant capacities of raspberry fruits with, in general, fruits from the field site in Bulgaria showing the highest contents of health related compounds. Fruits from production on soilless substrate had higher contents of anthocyanins but lower contents of total phenols and antioxidant capacities compared to fruits from plants grown in soil.
- Plant genotype was also an important factor influencing AOM content and antioxidant activities in raspberry fruits. Taking all the growing locations and cultural treatments together, one floricane variety was significantly richer in total phenolic content, the primocane variety in total anthocyanin content and another floricane variety was intermediate for both AOM.
- Neither biotisation nor elicitor treatment had any clear impact on AOM production and antioxidant activities in fruits or leaves at each location. Nevertheless, leaves or fruits of some raspberry varieties from production systems with higher mycorrhizal colonisation showed overall higher total phenols and antioxidant capacities than elsewhere.
In conclusion, the compatibility observed between plant defence elicitor treatment and biotisation in the production trials, conducted over two years in the field, under polythene tunnels, in soil or using soil-less substrates, indicate that these agronomic practices can be used together in most production systems for raspberries. Results highlight the importance of geography / location and plant genotype in determining the response of a raspberry crop to biotisation and/or elicitor application. There is a clear potential to use these agronomic innovations in some environments and in certain raspberry cultivars, to increase fruit yield or to improve plant protection from the damaging effects of root rot by P. fragariae var rubi., although treatments did not consistently influence AOM content or antioxidant activities of raspberry fruits. Recommendations have been made to the project SMEs for the successful utilisation in raspberry production of both biotisation to improve plant growth / fruit yield and plant defence elicitors which effectively reduce Phytophthora root rot, even though treatments did not consistently influence AOM content or antioxidant activities of raspberry fruits under greenhouse or organic farming conditions.