Final Report Summary - BCA_GRAPE (New biocontrol agents for powdery mildew on grapevine)
The main goal of the BCA_GRAPE project was to develop prototypes of new biofungicide products based on new strains of fungal biological control agents (BCAs) of Erysiphe necator, the causal agent of grapevine powdery mildew.
The 'BCA_GRAPE' consortium involved six partners: four small and medium sized enterprises (SMEs) settled in four European countries: Agrifutur (Italy), Vellsam (Spain), Bioved (Hungary) and Biyotar (Turkey) and two research and technological development (RTD) performers: Università Cattolica del Sacro Cuore (Italy) and Plant Protection Institute of the Hungarian Academy of Sciences (Hungary).
The RTD performers selected 10 Ampelomyces strains from a worldwide collection of these mycoparasites containing over 1100 strains, owned by PPI HAS, based on their sporulation and growth patterns in culture and mycoparasitic activities against grapevine powdery mildew. Molecular markers were also developed by the RTD performers to distinguish the newly selected Ampelomyces strains from the already commercialised ones, as well as from other Ampelomyces mycoparasites that naturally occur in grapevine powdery mildew. To achieve this second goal, more than 110 Ampelomyces strains were isolated and genotyped from Hungarian, Italian and other European vineyards during the project. The new molecular markers have also been useful in monitoring the fate of the selected strain following their field applications.
During the project, a new field application strategy was developed based on the use of the newly selected strains. This was built on a comprehensive screen of the whole life cycle of the target plant pathogen, Erysiphe necator, during laboratory and field experiments. The results of these experiments showed that the new Ampelomyces-based BCAs can be successfully used in a sanitation strategy, i.e. to reduce the overwintering inoculum of E. necator and, as a consequence, the disease pressure in the next season.
The SME partners developed new mass production and formulation technologies of the new biofungicide prototype based on the selected Ampelomyces strains. The prototype and the application strategy developed during the project were tested in 15 demonstration fields organised across Europe (Italy, France, German, Spain, Greece, Turkey, Hungary and Slovakia) to confirm the results obtained at experimental level. The prototypes obtained during the project will be further developed by the SME partners to gain new commercial plant protection products according to the Regulation 1107/2009.
Project context and objectives:
Powdery mildew fungi (Erysiphales) are one of the most conspicuous groups of plant pathogens comprising more than 500 species that attack many plant species, including important crops, worldwide. Despite extensive research on their pathogenesis, epidemiology and control, powdery mildew infections remain among the most important plant pathological problems. In the European crop protection the largest amount of fungicides has been used to control powdery mildews in various crops.
The main goal of the BCA_GRAPE project was to develop prototypes of new biofungicide products based on new strains of fungal BCAs of Erysiphe necator, the causal agent of grapevine powdery mildew. The obtainable prototypes should be further developed by the SME partners to finally gain new commercial products. This motivation was based on the following considerations:
1. grapevine is an important and highly profitable crop in many countries of Europe;
2. powdery mildew is a key disease for both wine and table grape production all across Europe in traditional, integrated and organic viticulture;
3. this disease is currently controlled almost exclusively by chemical fungicides;
4. only one biofungicide product, commercialised as 'AQ10 Biofungicide®', initially developed in Israel and based on a strain of a common mycoparasite of powdery mildews, Ampelomyces quisqualis, is currently available on the European market to be used against grapevine powdery mildew;
5. this biofungicide is not considered by most of the growers as being effective enough against grapevine powdery mildew;
6. two other similar products, based on other strains of A. quisqualis, have already been developed in Asia but these are currently not available on the European market.
The interest of the participating SMEs in the development of these biofungicide prototypes raised from their strategic needs to increase their offers for important crops and diseases not targeted by their currently available products. Grapevine powdery mildew was one of the key plant diseases from this point of view.
The BCA_GRAPE consortium involves six partners: four SMEs settled in four European countries (Italy, Spain, Hungary and Turkey) and two RTD performers from Italy and Hungary. Other European countries (France, Germany, Slovakia and Greece) are involved in demonstration activities so that eight countries are involved in total in this project.
To meet the project objectives, the following four actions have been considered necessary which also represent the innovative elements of the planned project and advances in the knowledge on these mycoparasitic fungi known as BCAs of powdery mildews.
1. Selection of a new Ampelomyces strain as a BCA of grapevine powdery mildew taking in consideration the genetic differences amongst the known strains of these mycoparasites.
2. Timing the application of the selected strain based on a comprehensive screen of the whole life cycle of the target plant pathogen during laboratory and field experiments.
3. Development of a new mass production and formulation technology for the delivery of the new biofungicide product.
4. Development of molecular tools to distinguish the selected strain from other Ampelomyces strains that commonly occur in powdery mildews in the field and/or have already been commercialised as BCAs and monitor the fate of the selected strain following its field application.
To achieve the prefixed objectives, the project was organised in nine work packages (WPs). WP1 to WP5 referred to research, technological development and innovation activities. RTD performers were mainly in charge of RTD activities but SME partners have also been involved in RTD works. The main objectives of the RTD activities were to:
1. find new Ampelomyces strains highly effective as BCAs of E. necator and well adapted to the European conditions (WP1 and WP2);
2. develop a technology for the industrial mass production and formulation of the new BCA (WP3);
3. develop protocols for the field application in order to maximise efficacy in both integrated and organic vineyards (WP4);
4. develop and validate molecular protocols for precise identification of the selected BCA strain(s) in order to distinguish them from other Ampelomyces strains, monitor their spread and survival in the vineyards and trace their presence along the grape production chain (WP5).
WP6 referred to demonstration activities that were aimed at validating the RTD results in commercial vineyards across Europe. WP7 to WP9 referred to other activities that facilitate the take-up of results by the SMEs, in particular to dissemination (WP7) and training (WP8). Dissemination activities included the organisation of an international meeting to present the results to the end-users, scientific and technical publications in national and international journals and a website. The RTD partners were in charge of training activities which included the training of the technical / management staff of the participating SMEs. These activities focused on the results and the technologies generated by the project. Knowledge management and IPR protection supporting the participating SMEs in using the research results to their best advantage and economic impact were included in WP9.
Project results:
In order to achieve the main objective of the BCA_GRAPE project, i.e. the development of prototypes of new biofungicide products based on new strains of fungal BCAs of Erysiphe necator, the first step was to develop a protocol for an efficient screening of Ampelomyces strains. Based on preliminary studies carried out by partner two (P02) and partner three (P03), a complex screening protocol was developed and used in the project.
The protocol was applied in WP1 to screen more than 700 Ampelomyces strains maintained at the fungal culture collection of Plant Protection Institute of the Hungarian Academy of Sciences (P03), which is the largest collection of Ampelomyces strains worldwide. To determine wether there was a considerable variation in the in vitro and the in planta characteristics, the strains were screened for growth and sporulation rate in culture and sporulation rate and spread in E. necator colonies on grapevine leaves. This second pattern of the strains characterised their mycoparasitic activity in grapevine powdery mildew. It was demonstrated that there is indeed a considerable diversity in many characteristics of different Ampelomyces strains including both culture patterns and mycoparasitic activities. Ten strains were selected as very promising potential BCAs of grapevine powdery mildew and these were given to all partners to be used for further assessments and for the development of adequate technologies for their mass production.
In particular, in WP2 it was investigated the effectiveness of the selected strains against E. necator in relation to its life cycle and the environmental conditions. To do this, leaves of potted grapevine plants were infected with conidia of E. necator and then inoculated with conidial suspensions of the selected Ampelomyces strains, at different developmental stages of the powdery mildew colonies and in different environmental conditions. Particular attention was paid to determine the ability of the different strains to parasitise the fruiting bodies (chasmothecia) of the powdery mildew fungus. It was demonstrated that these mycoparasites attack and destroy immature chasmothecia on grapevine leaves and the younger immature chasmothecia are more efficiently destroyed than those representing more advanced stages of chasmothecial development.
On the other hand, if applied against the asexually reproducing powdery mildew colonies, i.e. against the conidial stage of their life cycle, some strains were able to significantly reduce the conidial production of the pathogen. Whatever the target is, the mycoparasites need high relative humidity and moderate temperatures.
In parallel, the efficacy against powdery mildew on grapevine of the selected Ampelomyces strains was evaluated in natural conditions, i.e. commercial vineyards (WP4). These strains were applied in experimental plots (in Italy and Hungary) twice during fall 2008 and three times in spring 2009, to compare their effectiveness in reducing powdery mildew in comparison with untreated control plots, plots treated with commercial Ampelomyces products and with a normal fungicide schedule. It was observed that the fall applications reduced significantly the number of the first ascosporic infections in spring and delayed the appearance of secondary infections as well as their severity. On the contrary, early season (i.e. spring) applications of Ampelomyces did not lead to any significant reduction of the grapevine powdery mildew epidemic development.
Applications were repeated in fall 2009 and observations performed in spring 2010 confirmed the results obtained in the precedent year regarding the reduction and delay of ascosporic infections. Through these experiments it was possible to define protocols (WP8) for the optimal application methodology of the mycoparasites, i.e. in late afternoon when the temperature decreases and the relative humidity increases.
Mycoparasitic activity of Ampelomyces strains applied in autumn, i.e. parasitism of immature chasmothecia on grapevine leaves sprayed with different strains and Ampelomyces-based products, was regularly observed 5 to 10 days after treatments.
In WP5, as a first step towards developing molecular markers for distinguishing the selected strains of Ampelomyces from the already commercialised strains and also from the strains that naturally occur in grapevine powdery mildew, nuclear ribosomal deoxyribonucleic acid (nrDNA) internal transcribed spacer (ITS) sequences were newly determined in more than 100 strains in this project. As expected based on data in the literature, many strains exhibited identical ITS sequences, thus other, more polymorphic markers were also tested. An approximately 850 bp-long portion of the actin gene was amplified and sequenced in a total of 77 Ampelomyces strains including the 10 strains selected in this project. These sequences distinguished more strains from each other compared to ITS sequences but, as expected, were not useful for strain-specific identifications in all cases. For this purpose, nine polymorphic microsatellite simple sequence repeat (SSR) markers were newly developed in this project, in addition to testing six SSR markers already known from the literature and nine inter-simple sequence repeat (ISSR) markers were also newly selected to readily distinguish Ampelomyces strains from each other. A large culture collection of more than 110 strains was established by isolating Ampelomyces strains from E. necator in different parts of Europe and these were used to test the usefulness of the newly developed molecular markers in distinguishing the strains selected as promising BCAs in this project from Ampelomyces strains that naturally occurr in grapevine powdery mildew. Also, Ampelomyces mycoparasites were re-isolated many times from the experimental vineyards treated in this project with selected strains of Ampelomyces. The re-isolated strains were genotyped using the markers developed in this project. The results showed that:
1. the strains used in an inundative way quickly colonised the treated powdery mildew mycelia because these were re-isolated two to three weeks after treatments; and
2. these strains did not persist for a year in the treated experimental plots because a year later only other, naturally occurring Ampelomyces strains were isolated from those plots.
In conclusion, the molecular markers developed in WP5 can be used for strain-specific identifications of any Ampelomyces mycoparasites isolated from grapevine (or other) powdery mildew species. In this way the environmental fate of Ampelomyces strains used as BCAs can be monitored in the field and the genetic variability of naturally occurring strains can also be studied.
The Ampelomyces strains selected by RTD performers were delivered to the SMEs partners AGF (P01) and Bioved (P04) that studied in WP3 a number of various inexpensive nutritive media and the best conditions to grow all strains. Fermentation in submerged and solid state were tested at a pilot level performing mass production in monoaxenic systems montoring and controlling the growth paramenters. It was obtained with the best two strains a high viable concentration of Ampelomyces conidia. Formulation of the active ingredients with drying and freeze drying techniques was performed. Final formulations were done considering the shelf life, dispersion in water, UV sunscreen and other co-formulants. In formulation were involved also Vellsam (P05) and Biyotar (P06). The SMEs partners have been trained continuously during the project by the RTD performers: how to grow Ampelomyces strains, to induce sporulatation and to check the viability and the infectivity. Two product prototypes were obtained with a good shelf life. An estimation of cost production was done and a first feasibility study gave good result.
The prototype developed in WP3 was tested in 15 demonstration fields (WP6) across Europe (France, Greece, Germany, Hungary, Italy, Slovakia, Spain and Turkey) in the most important grape-growing European regions. Each field was divided in two plots, one plot was treated as usual while the other plot was treated as usual plus two applications of Ampelomyces product in two fall sprays.
In spring 2010, periodic observations were performed to assess the presence and severity of the disease in the demonstration fields, in order to demonstrate the efficacy of the late season Ampelomyces-based prototype sprays.
The results achieved by the Ampelomyces prototype product in demonstration fields confirmed that late season treatments can reduce the incidence and the severity of the disease in the following season as already showed by experimental plots in WP2 and WP4.
SMEs partner organised visits in the demonstration fields to show the results of the application of Ampelomyces based product to local growers and technicians.
Potential impact:
The project led to important scientific results, some of them were new for the whole scientific community and were disseminated also outside of the consortium by RTD partners during the activity of dissemination WP7 (peer review publications, publications in technical journals and congress). The most important result was the design and validation of a new strategy of application of Ampelomyces in the field that led to a much higher rate of mycoparasitism of grapevine powdery mildew in commercial vineyards than the already existing and commercially recommended, application procedure. The results achieved by the RTD performers were transferred to the SMEs partners during the activities of training: management and technical personnel belonging to the SMEs visited the two RTD institutions and vice versa (WP8). In particular, PPI (P03) transferred to SMEs partners' protocols for molecular methods to identify and detect the new Ampelomyces strains, methods and techniques to manage them in the laboratory and UCSC (P02) transferred to all the partners involved in WP6 methods and schedules to apply the new Ampelomyces products in the demonstration vineyards.
The results achieved in the mass production and in the field using the new Ampelomyces-based prototype product bring to the conclusion that it is feasible economically and technologically to have in the near future, after the registration process in European Union (EU) a new European commercial product on the market. This is fully in accordance with the new European policy on the 'Sustainable use of pesticide' (Directive 128/2009). It is a new effective and low environmental impact tool to control the powdery mildew in grapevine and in many other cultivated plants like tomato, cucumber, strawberry and roses. The new product based on newly selected Ampelomyces strains in integrated pest management strategy reduces the losses caused by powdery mildew and in the same time the use of chemical fungicide products and, as a consequence, the amount of fungicide residues in the food chain.
The results obtained during the project have been published by RTD performers in technical journals of national and international relevance targeted to technicians, advisors and growers, in agreement with the SMEs. The RTD performers have also prepared posters and presentations to disseminate the results of the project during scientific international congresses.
The SMEs partners prepared leaflets targeted to technicians, advisors and growers to present the new BCA product prototypes. These are available in nine languages and are freely downloadable from the project website.
The results of the project are disseminated in the website http://www.bca-grape.eu ; the project with its main goals and highlights is described in the home page. There are other pages that show project activities and a table with the contacts of all partners.
The international conference, with a scientific content mainly addressed to the end-users along the entire 'grapevine-chain', was organised on 21 March 2011 in Piacenza (Italy) and the congress proceedings are freely downloadable from the website.
Project website:
http://www.bca-grape.eu
Beneficiaries:
P01 (coordinator):
Agrifutur srl
AGF Agrifutur srl, Parco Tecnologico Padano, via Einstein 1, 26900 Lodi (Italy)
http://www.Agrifutur.com
Dr Roberto Kron Morelli
P02:
Università Cattolica del Sacro Cuore (UCSC), Institute of Entomology and Plant Pathology
via E. Parmense 84, Piacenza (Italy)
http://www.unicatt.it
Dr Vittorio Rossi
P03:
Plant Protection Institute (PPI) of the Hungarian Academy of Sciences
H-1022 Budapest, Herman O. ut 15, (Hungary)
http://www.nki.hu
Dr Levente Kiss
P04:
Bioved Ltd (BIOV)
BIOVÉD Biological Plant Protection Product Producing Ltd
2005 KFT Kemesmáil út 23 9923 Kamestaródfa (Hungary)
http://www.bioved.hu
Dr Gyula Bohar
P05:
Vellsam Materias Bioactivas S.L. (VELL)
Vellsam Materias Bioactivas S.L.
Ctra Nacional 340 Km 477 04200 Tabernas Almeria (Spain)
http://www.ing.vellsam.com
Dr Francisco Castillo Ferreira
P06:
Biyotar (BIYO)
BIYOTAR, Biyolojik Tarim Danismanlik ve Mühendislik Hizmetleri Ltd Sti (Biological Agriculture Consulting and Engineering Co.)
E- 5 Karayolu Üzeri, Kisik Mevki PK 23 Erzin / Hatay (Turkey)
http://www.biyotar.com
Dr Lerzan Bakircioglu Erkilic.
The 'BCA_GRAPE' consortium involved six partners: four small and medium sized enterprises (SMEs) settled in four European countries: Agrifutur (Italy), Vellsam (Spain), Bioved (Hungary) and Biyotar (Turkey) and two research and technological development (RTD) performers: Università Cattolica del Sacro Cuore (Italy) and Plant Protection Institute of the Hungarian Academy of Sciences (Hungary).
The RTD performers selected 10 Ampelomyces strains from a worldwide collection of these mycoparasites containing over 1100 strains, owned by PPI HAS, based on their sporulation and growth patterns in culture and mycoparasitic activities against grapevine powdery mildew. Molecular markers were also developed by the RTD performers to distinguish the newly selected Ampelomyces strains from the already commercialised ones, as well as from other Ampelomyces mycoparasites that naturally occur in grapevine powdery mildew. To achieve this second goal, more than 110 Ampelomyces strains were isolated and genotyped from Hungarian, Italian and other European vineyards during the project. The new molecular markers have also been useful in monitoring the fate of the selected strain following their field applications.
During the project, a new field application strategy was developed based on the use of the newly selected strains. This was built on a comprehensive screen of the whole life cycle of the target plant pathogen, Erysiphe necator, during laboratory and field experiments. The results of these experiments showed that the new Ampelomyces-based BCAs can be successfully used in a sanitation strategy, i.e. to reduce the overwintering inoculum of E. necator and, as a consequence, the disease pressure in the next season.
The SME partners developed new mass production and formulation technologies of the new biofungicide prototype based on the selected Ampelomyces strains. The prototype and the application strategy developed during the project were tested in 15 demonstration fields organised across Europe (Italy, France, German, Spain, Greece, Turkey, Hungary and Slovakia) to confirm the results obtained at experimental level. The prototypes obtained during the project will be further developed by the SME partners to gain new commercial plant protection products according to the Regulation 1107/2009.
Project context and objectives:
Powdery mildew fungi (Erysiphales) are one of the most conspicuous groups of plant pathogens comprising more than 500 species that attack many plant species, including important crops, worldwide. Despite extensive research on their pathogenesis, epidemiology and control, powdery mildew infections remain among the most important plant pathological problems. In the European crop protection the largest amount of fungicides has been used to control powdery mildews in various crops.
The main goal of the BCA_GRAPE project was to develop prototypes of new biofungicide products based on new strains of fungal BCAs of Erysiphe necator, the causal agent of grapevine powdery mildew. The obtainable prototypes should be further developed by the SME partners to finally gain new commercial products. This motivation was based on the following considerations:
1. grapevine is an important and highly profitable crop in many countries of Europe;
2. powdery mildew is a key disease for both wine and table grape production all across Europe in traditional, integrated and organic viticulture;
3. this disease is currently controlled almost exclusively by chemical fungicides;
4. only one biofungicide product, commercialised as 'AQ10 Biofungicide®', initially developed in Israel and based on a strain of a common mycoparasite of powdery mildews, Ampelomyces quisqualis, is currently available on the European market to be used against grapevine powdery mildew;
5. this biofungicide is not considered by most of the growers as being effective enough against grapevine powdery mildew;
6. two other similar products, based on other strains of A. quisqualis, have already been developed in Asia but these are currently not available on the European market.
The interest of the participating SMEs in the development of these biofungicide prototypes raised from their strategic needs to increase their offers for important crops and diseases not targeted by their currently available products. Grapevine powdery mildew was one of the key plant diseases from this point of view.
The BCA_GRAPE consortium involves six partners: four SMEs settled in four European countries (Italy, Spain, Hungary and Turkey) and two RTD performers from Italy and Hungary. Other European countries (France, Germany, Slovakia and Greece) are involved in demonstration activities so that eight countries are involved in total in this project.
To meet the project objectives, the following four actions have been considered necessary which also represent the innovative elements of the planned project and advances in the knowledge on these mycoparasitic fungi known as BCAs of powdery mildews.
1. Selection of a new Ampelomyces strain as a BCA of grapevine powdery mildew taking in consideration the genetic differences amongst the known strains of these mycoparasites.
2. Timing the application of the selected strain based on a comprehensive screen of the whole life cycle of the target plant pathogen during laboratory and field experiments.
3. Development of a new mass production and formulation technology for the delivery of the new biofungicide product.
4. Development of molecular tools to distinguish the selected strain from other Ampelomyces strains that commonly occur in powdery mildews in the field and/or have already been commercialised as BCAs and monitor the fate of the selected strain following its field application.
To achieve the prefixed objectives, the project was organised in nine work packages (WPs). WP1 to WP5 referred to research, technological development and innovation activities. RTD performers were mainly in charge of RTD activities but SME partners have also been involved in RTD works. The main objectives of the RTD activities were to:
1. find new Ampelomyces strains highly effective as BCAs of E. necator and well adapted to the European conditions (WP1 and WP2);
2. develop a technology for the industrial mass production and formulation of the new BCA (WP3);
3. develop protocols for the field application in order to maximise efficacy in both integrated and organic vineyards (WP4);
4. develop and validate molecular protocols for precise identification of the selected BCA strain(s) in order to distinguish them from other Ampelomyces strains, monitor their spread and survival in the vineyards and trace their presence along the grape production chain (WP5).
WP6 referred to demonstration activities that were aimed at validating the RTD results in commercial vineyards across Europe. WP7 to WP9 referred to other activities that facilitate the take-up of results by the SMEs, in particular to dissemination (WP7) and training (WP8). Dissemination activities included the organisation of an international meeting to present the results to the end-users, scientific and technical publications in national and international journals and a website. The RTD partners were in charge of training activities which included the training of the technical / management staff of the participating SMEs. These activities focused on the results and the technologies generated by the project. Knowledge management and IPR protection supporting the participating SMEs in using the research results to their best advantage and economic impact were included in WP9.
Project results:
In order to achieve the main objective of the BCA_GRAPE project, i.e. the development of prototypes of new biofungicide products based on new strains of fungal BCAs of Erysiphe necator, the first step was to develop a protocol for an efficient screening of Ampelomyces strains. Based on preliminary studies carried out by partner two (P02) and partner three (P03), a complex screening protocol was developed and used in the project.
The protocol was applied in WP1 to screen more than 700 Ampelomyces strains maintained at the fungal culture collection of Plant Protection Institute of the Hungarian Academy of Sciences (P03), which is the largest collection of Ampelomyces strains worldwide. To determine wether there was a considerable variation in the in vitro and the in planta characteristics, the strains were screened for growth and sporulation rate in culture and sporulation rate and spread in E. necator colonies on grapevine leaves. This second pattern of the strains characterised their mycoparasitic activity in grapevine powdery mildew. It was demonstrated that there is indeed a considerable diversity in many characteristics of different Ampelomyces strains including both culture patterns and mycoparasitic activities. Ten strains were selected as very promising potential BCAs of grapevine powdery mildew and these were given to all partners to be used for further assessments and for the development of adequate technologies for their mass production.
In particular, in WP2 it was investigated the effectiveness of the selected strains against E. necator in relation to its life cycle and the environmental conditions. To do this, leaves of potted grapevine plants were infected with conidia of E. necator and then inoculated with conidial suspensions of the selected Ampelomyces strains, at different developmental stages of the powdery mildew colonies and in different environmental conditions. Particular attention was paid to determine the ability of the different strains to parasitise the fruiting bodies (chasmothecia) of the powdery mildew fungus. It was demonstrated that these mycoparasites attack and destroy immature chasmothecia on grapevine leaves and the younger immature chasmothecia are more efficiently destroyed than those representing more advanced stages of chasmothecial development.
On the other hand, if applied against the asexually reproducing powdery mildew colonies, i.e. against the conidial stage of their life cycle, some strains were able to significantly reduce the conidial production of the pathogen. Whatever the target is, the mycoparasites need high relative humidity and moderate temperatures.
In parallel, the efficacy against powdery mildew on grapevine of the selected Ampelomyces strains was evaluated in natural conditions, i.e. commercial vineyards (WP4). These strains were applied in experimental plots (in Italy and Hungary) twice during fall 2008 and three times in spring 2009, to compare their effectiveness in reducing powdery mildew in comparison with untreated control plots, plots treated with commercial Ampelomyces products and with a normal fungicide schedule. It was observed that the fall applications reduced significantly the number of the first ascosporic infections in spring and delayed the appearance of secondary infections as well as their severity. On the contrary, early season (i.e. spring) applications of Ampelomyces did not lead to any significant reduction of the grapevine powdery mildew epidemic development.
Applications were repeated in fall 2009 and observations performed in spring 2010 confirmed the results obtained in the precedent year regarding the reduction and delay of ascosporic infections. Through these experiments it was possible to define protocols (WP8) for the optimal application methodology of the mycoparasites, i.e. in late afternoon when the temperature decreases and the relative humidity increases.
Mycoparasitic activity of Ampelomyces strains applied in autumn, i.e. parasitism of immature chasmothecia on grapevine leaves sprayed with different strains and Ampelomyces-based products, was regularly observed 5 to 10 days after treatments.
In WP5, as a first step towards developing molecular markers for distinguishing the selected strains of Ampelomyces from the already commercialised strains and also from the strains that naturally occur in grapevine powdery mildew, nuclear ribosomal deoxyribonucleic acid (nrDNA) internal transcribed spacer (ITS) sequences were newly determined in more than 100 strains in this project. As expected based on data in the literature, many strains exhibited identical ITS sequences, thus other, more polymorphic markers were also tested. An approximately 850 bp-long portion of the actin gene was amplified and sequenced in a total of 77 Ampelomyces strains including the 10 strains selected in this project. These sequences distinguished more strains from each other compared to ITS sequences but, as expected, were not useful for strain-specific identifications in all cases. For this purpose, nine polymorphic microsatellite simple sequence repeat (SSR) markers were newly developed in this project, in addition to testing six SSR markers already known from the literature and nine inter-simple sequence repeat (ISSR) markers were also newly selected to readily distinguish Ampelomyces strains from each other. A large culture collection of more than 110 strains was established by isolating Ampelomyces strains from E. necator in different parts of Europe and these were used to test the usefulness of the newly developed molecular markers in distinguishing the strains selected as promising BCAs in this project from Ampelomyces strains that naturally occurr in grapevine powdery mildew. Also, Ampelomyces mycoparasites were re-isolated many times from the experimental vineyards treated in this project with selected strains of Ampelomyces. The re-isolated strains were genotyped using the markers developed in this project. The results showed that:
1. the strains used in an inundative way quickly colonised the treated powdery mildew mycelia because these were re-isolated two to three weeks after treatments; and
2. these strains did not persist for a year in the treated experimental plots because a year later only other, naturally occurring Ampelomyces strains were isolated from those plots.
In conclusion, the molecular markers developed in WP5 can be used for strain-specific identifications of any Ampelomyces mycoparasites isolated from grapevine (or other) powdery mildew species. In this way the environmental fate of Ampelomyces strains used as BCAs can be monitored in the field and the genetic variability of naturally occurring strains can also be studied.
The Ampelomyces strains selected by RTD performers were delivered to the SMEs partners AGF (P01) and Bioved (P04) that studied in WP3 a number of various inexpensive nutritive media and the best conditions to grow all strains. Fermentation in submerged and solid state were tested at a pilot level performing mass production in monoaxenic systems montoring and controlling the growth paramenters. It was obtained with the best two strains a high viable concentration of Ampelomyces conidia. Formulation of the active ingredients with drying and freeze drying techniques was performed. Final formulations were done considering the shelf life, dispersion in water, UV sunscreen and other co-formulants. In formulation were involved also Vellsam (P05) and Biyotar (P06). The SMEs partners have been trained continuously during the project by the RTD performers: how to grow Ampelomyces strains, to induce sporulatation and to check the viability and the infectivity. Two product prototypes were obtained with a good shelf life. An estimation of cost production was done and a first feasibility study gave good result.
The prototype developed in WP3 was tested in 15 demonstration fields (WP6) across Europe (France, Greece, Germany, Hungary, Italy, Slovakia, Spain and Turkey) in the most important grape-growing European regions. Each field was divided in two plots, one plot was treated as usual while the other plot was treated as usual plus two applications of Ampelomyces product in two fall sprays.
In spring 2010, periodic observations were performed to assess the presence and severity of the disease in the demonstration fields, in order to demonstrate the efficacy of the late season Ampelomyces-based prototype sprays.
The results achieved by the Ampelomyces prototype product in demonstration fields confirmed that late season treatments can reduce the incidence and the severity of the disease in the following season as already showed by experimental plots in WP2 and WP4.
SMEs partner organised visits in the demonstration fields to show the results of the application of Ampelomyces based product to local growers and technicians.
Potential impact:
The project led to important scientific results, some of them were new for the whole scientific community and were disseminated also outside of the consortium by RTD partners during the activity of dissemination WP7 (peer review publications, publications in technical journals and congress). The most important result was the design and validation of a new strategy of application of Ampelomyces in the field that led to a much higher rate of mycoparasitism of grapevine powdery mildew in commercial vineyards than the already existing and commercially recommended, application procedure. The results achieved by the RTD performers were transferred to the SMEs partners during the activities of training: management and technical personnel belonging to the SMEs visited the two RTD institutions and vice versa (WP8). In particular, PPI (P03) transferred to SMEs partners' protocols for molecular methods to identify and detect the new Ampelomyces strains, methods and techniques to manage them in the laboratory and UCSC (P02) transferred to all the partners involved in WP6 methods and schedules to apply the new Ampelomyces products in the demonstration vineyards.
The results achieved in the mass production and in the field using the new Ampelomyces-based prototype product bring to the conclusion that it is feasible economically and technologically to have in the near future, after the registration process in European Union (EU) a new European commercial product on the market. This is fully in accordance with the new European policy on the 'Sustainable use of pesticide' (Directive 128/2009). It is a new effective and low environmental impact tool to control the powdery mildew in grapevine and in many other cultivated plants like tomato, cucumber, strawberry and roses. The new product based on newly selected Ampelomyces strains in integrated pest management strategy reduces the losses caused by powdery mildew and in the same time the use of chemical fungicide products and, as a consequence, the amount of fungicide residues in the food chain.
The results obtained during the project have been published by RTD performers in technical journals of national and international relevance targeted to technicians, advisors and growers, in agreement with the SMEs. The RTD performers have also prepared posters and presentations to disseminate the results of the project during scientific international congresses.
The SMEs partners prepared leaflets targeted to technicians, advisors and growers to present the new BCA product prototypes. These are available in nine languages and are freely downloadable from the project website.
The results of the project are disseminated in the website http://www.bca-grape.eu ; the project with its main goals and highlights is described in the home page. There are other pages that show project activities and a table with the contacts of all partners.
The international conference, with a scientific content mainly addressed to the end-users along the entire 'grapevine-chain', was organised on 21 March 2011 in Piacenza (Italy) and the congress proceedings are freely downloadable from the website.
Project website:
http://www.bca-grape.eu
Beneficiaries:
P01 (coordinator):
Agrifutur srl
AGF Agrifutur srl, Parco Tecnologico Padano, via Einstein 1, 26900 Lodi (Italy)
http://www.Agrifutur.com
Dr Roberto Kron Morelli
P02:
Università Cattolica del Sacro Cuore (UCSC), Institute of Entomology and Plant Pathology
via E. Parmense 84, Piacenza (Italy)
http://www.unicatt.it
Dr Vittorio Rossi
P03:
Plant Protection Institute (PPI) of the Hungarian Academy of Sciences
H-1022 Budapest, Herman O. ut 15, (Hungary)
http://www.nki.hu
Dr Levente Kiss
P04:
Bioved Ltd (BIOV)
BIOVÉD Biological Plant Protection Product Producing Ltd
2005 KFT Kemesmáil út 23 9923 Kamestaródfa (Hungary)
http://www.bioved.hu
Dr Gyula Bohar
P05:
Vellsam Materias Bioactivas S.L. (VELL)
Vellsam Materias Bioactivas S.L.
Ctra Nacional 340 Km 477 04200 Tabernas Almeria (Spain)
http://www.ing.vellsam.com
Dr Francisco Castillo Ferreira
P06:
Biyotar (BIYO)
BIYOTAR, Biyolojik Tarim Danismanlik ve Mühendislik Hizmetleri Ltd Sti (Biological Agriculture Consulting and Engineering Co.)
E- 5 Karayolu Üzeri, Kisik Mevki PK 23 Erzin / Hatay (Turkey)
http://www.biyotar.com
Dr Lerzan Bakircioglu Erkilic.
