<figure><img src="image_153.png" /></figure>

# Socio-technical development and appropriation of plant variety resistance in sustainable wine-growing

**Launch year:** 2015

**Completion year:** 2019

**Scientific manager**

François Hochereau, INRAE UMR

SADAPT francois.hochereau@inrae.fr

Laurent Delière, INRAE UMR

SAVE laurent.deliere@inrae.fr

**Partners**

INRAE UMR SADAPT ; INRAE UMR SAV ; IFV (French Grapevine Institute) ; Chambers of agriculture (Gironde, Hérault, Aude)

**Financing**

Total cost of the project: € 306,197

Ecophyto grant: € 134,868

**Key words:**

Wine growing; Improvement through variety selection; Disease resistant varieties; Environmental innovation; Observatory; Socio-economic evaluation

## Context and main objectives

The aim of the project was to study pioneering disease resistant grapevine variety introductions in France; in order to identify the main regulatory, technical and socio-economical levers and obstacles that come into play when deploying this kind of plant variety innovation (task 4).

German and Swiss context studies, where such varieties have been marketed since the early 2000s provide a rich source of feedback and a better understanding of the definitive methods which can be applied to a participatory experimental framework associating the different "involved parties" (task 2).

At the same time, we considered how the regulatory and professional bodies of the wine growing industry, notably the INAO, have influenced the development of the regulatory framework covering plant variety experimentation (task 1).

Finally, the project's objective was also to federate these pioneering disease resistant variety adoption practices by constituting a plant variety-related resistance observation network. This network is structured around the shared application of a protocol for the collection and sharing of data concerning the behaviour of disease resistant varieties (task 3).

## Principal results and interest in relation to the Ecophyto plan

In 2019, the disease resistant grape variety observation network covered 87 individual vineyards distributed over 43 different sites (wine growers, cooperatives, chambers of agriculture), most of which are in the Aquitaine and Occitanie regions. The observations were made between 2017 and 2019 and concerned 23 varieties of French, German and Swiss origin. It has shown a reduction of 96% in the TFI for fungicides compared to the agreste 2016 control reference with 80% of the treatment applications around the flowering season.

The study of restraints on plant variety innovations in France has shown:

* Plant variety innovation cycles are too long and above all obscure. French VATE (Environmental and Technical Agricultural Value) experimentation does not sufficiently integrate the wine-growers themselves in testing the wines produced or involve the nurseries in how they should prepare the plants for distribution.

* The very high degree of rigidity surrounding 'authentic regional origins' in the wine trade imposes endless experimentation constraints and 'origin' typicity criteria that are too strict.
German and Swiss wine makers are quite the opposite, they have a very flexible and much more participatory regulatory and experimental approach.

France has had to soften its experimental framework in the face of what is demanded in the field, even if this still remains too bureaucratic and tightly structured around the INRAE and IVF professional associations. In the same way the INAO has completely changed its own framework such that experimentation is now possible even if it is still restricted by a deep-seated hierarchical structure concerning the various 'regional origins'. A number of regional variety selection programs should lead to a major renewal of the grape variety landscape. To this end, the dynamic is now much more powerful in France than elsewhere, where the use of grape varieties remains secret even after 20 years of history.

As for those winegrowers who are prepared to adopt new varieties, the priority is placed on the quality of the wines produced rather than the variety's resistance to disease. Evidently all of this is central to the choices of new varieties, or there would be no reason to change the grape varieties. Professionals prefer a variety that produces greater quality even if it is less disease resistant (provided it isn't completely decimated by pests). They feel that they can better manage the risk of moderate levels of disease resistance with *ad hoc* control practices rather than face the commercial risk that the wine will not please consumers. The practice of wine blending in the South of France should favour the integration of a percentage of disease resistant varieties in the IGP regions, less rigid than the AOP 'regional origins' when it comes to homogeneous wine-typicity, and more interested in the marketability generated by the low environmental impact of their wines. Amongst the IGP of the North of France, where single grape variety wines are dominant, these varieties could find their place in the production of sparkling wines, since the wine-making qualities of the grape variety are less decisive. This is also the case (possibly even more so) for spirits such as Cognac and Armagnac.

More globally speaking the profession is waiting for the arrival, in about a dozen years, of varieties from regional retro-breeding programmes, where they hope to be able to combine wine typicity with variety related disease resistance. In the meantime, initiatives are concentrating on the zones of friction between wine-growers and residents of the new urban fringes, as a backdoor route to introducing varieties requiring little or no pesticide treatment. It would be probably best to support and federate experimental approaches for the IGP and cooperatives in order to build on the agricultural, pathological and quality capital of these collective organisations, since the observatory itself remains a simple research tool and sanitary watchdog before anything else.

## Prospects for transfer or research

### Transfer:

Originally the project was very optimistic considering that the distribution of disease resistant grape varieties remained quite confidential. Recent regulatory changes meant that planting did not really start until 2019. This is why regional exchange workshops have been planned in continuation of the project. Due to Covid, they will not be organised before the end of 2021.

The disease resistant grape variety monitoring observatory will ensure better education of wine-growers on the subject of how grape varieties behave in the face of disease. A doctorate on wine-making using disease resistant grape varieties is planned which should do a better job of analysing the relation between variety-related disease resistance and wine quality, and therefore provide a better response to the expectations of growers.

### Research:

The project has facilitated the launch of the follow-on project OSCAR, which aims to consolidate and extend the disease resistant grape variety observation structure. It will now extend into the PPR Curare on zero pesticides on grapevines, with a planned doctorate that aims to put together an experimental variety evaluation table, combining pathological, agricultural and oenological aspects.

## Publications and scientific symposia:

### SCIENTIFIC PUBLICATIONS:

* S. Tabouret, 2019, **Setting up a “good” experimentation: the case of vine varieties testing in Languedoc, Open Agriculture Journal**, Elsevier.

* S. Guimier, F. Delmotte, A.S. Miclot, F. Fabre, I. Mazet, C. Couture, C. Schneider, L. Delière, 2019. **OSCAR, a national observatory to support the durable deployment of disease-resistant grapevine varieties.** Acta Horticulturae

* S. Tabouret, 2020, **Résister ! Une aptitude commune aux vignes, aux agents pathogènes, aux professionnels et aux scientifiques**, L'immunité des plantes : Pour des cultures résistantes aux maladies (C. Lannou, D. Roby, V. Ravigné, M. Hannachi, B. Moury, eds.), Paris, Quae, p.235-46

* F. Hochereau, 2020, **La résistance variétale, un objet frontière à construire**, L'immunité des plantes : Pour des cultures résistantes aux maladies (C. Lannou, D. Roby, V. Ravigné, M. Hannachi, B. Moury, eds.), Paris, Quae, p. 257-58
## TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA:

* F. Hochereau, 2019, **Classement, déclassement et reclassement de l'innovation variétale : Une comparaison franco-suisse**, RT 29 Sciences et Techniques en Société, AFS congress, Aix en Provence

* F. Hochereau, 2019, **L'évolution des catégorisations du risque par l'adoption de cépages résistants en viticulture**, RT 38 sociologie de l'environnement et des risques, AFS congress, Aix-en Provence

* F. Delmotte 2018. **OSCAR — a national observatory for a sustainable deployment of disease-resistant grape varieties**. XII International Conference on Grapevine Breeding and Genetics

* L. Delière. 2017. **Déploiement des variétés résistantes en viticulture : observatoire Oscar. Rencontres régionales de la recherche, du développement et de la formation**. Montagne, 13 December 2017

* L. Delière. 2017. **Un Observatoire du déploiement des cépages résistants**. SITEVI conference, 29 November 2017

* F. Hochereau, 2017, **Sélectionneurs et pépiniéristes: Métiers ou Expertises au carrefour des mutations du monde de la vigne**, Métiers de la vigne et du vin, 20-21 November, Paris Saclay

* **PRESENTATION TO PROFESSIONAL OR DECISION-MAKING BODIES:**

* L. Delière 2018. **Table ronde Cépages résistants : quelles avancées ?** Cognac 2025

* L. Delière. 2018. **Comment accompagner le déploiement des nouvelles variétés de vigne résistantes au mildiou et à l'oïdium ?** Presented to: Séances de l'Académie d'Agriculture « Révolution variétale en viticulture ! De nouveaux cépages résistant aux maladies : création, déploiement et impact sur la filière viticole », Paris, France (2018-05-30 - 2018-05-30)

## PRACTICAL / EDUCATIONAL ARTICLES:

* **Un observatoire des cépages résistants pour lutter contre des maladies de la vigne.** Dépêche AFP, Bordeaux, 9 January 2017

* **Les cépages résistants en observation.** PHYTOMA n°718, November 2018
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# Development and study of the effectiveness of allelopathic substances to improve weed control for particularly resistant species

**Launch year:** 2017

**Completion year:** 2020

**Scientific manager**

Claire Richard, Clermont-
Ferrand Institute of Chemistry /
CNRS claire.richard@uca.fr

**Partners**

ICCF; Laboratoire de Physique et Physiologie
Intégratives de l'Arbre en environnement Fluctuant
(PIAF); INRAE; Mairie d'Aubière

**Financing**

Total cost of the project: € 287,550
Ecophyto grant: € 87,346

**Key words:**

JEVI; Weed control; Allelopathy; Phytotoxicity of secondary metabolites; Rock Rose; Hyssop; Mulch; Macerate; Cypress Leylandii

## Context and main objectives

The ban on chemical weedkillers has raised a number of practical difficulties for JEVI (Gardens, Parks and Infrastructures) maintenance managers. Manual weed control is time-consuming and expensive whilst weed control by heat is not very effective since the weeds re-grow very rapidly. Using plants to control weeds has become an attractive alternative. This involves making the best use of the plants' ability to produce and diffuse phytotoxic compounds.

We have selected plants that, on the basis of material published to date, are known to contain large quantities of terpenes and have set up experiments to evaluate their ability to control weeds when the terpenes are:

(i) Emitted by the plants;

(ii) Diffused via mulch or

(iii) Applied as a macerate.

These experiments should be able to reveal the allelopathic properties of terpene-rich plants and thus show how best they can be used. This project has a twofold objective: to propose new potential weed control solutions and increase specific knowledge in this field through fundamental research.

<figure><img src="image_155.png" /><figcaption>Figure 1: The phytotoxic compound emission paths of allelopathic plants. Image: © Claire Richard, ICCF/CNRS</figcaption></figure>

## Principal results and interest in relation to the Ecophyto plan

### Experiments on whole plants:

Lomelosia cretica; Centaurea bella; Hyssopus officinalis; Cistus creticus; Veronica polifolia were initially selected because they are strong-scented, hardy (-15°C), aesthetically pleasing, non-invasive, medium-sized (< 60 cm), evergreen and considered to be allelopathic by certain growers. Chemical analyses have shown that, apart from Veronica polifolia, all of these plants emit terpenes.

An initial series of tests in the Aubière municipal conservatory gardens have shown that rock rose and hyssop are the most promising of these plants (Fig. 2, following page); they were selected for a second series of tests under real conditions, where they were planted around the bases of trees in a school playground. This second series of experiments lasted barely more than a year, which did not provide the plants with enough time to develop and show their full capacities. However it was concluded that the results did confirm those obtained in the conservatory
gardens, rock-rose does appear to have interesting weed-control properties when planted alone or in combination with hyssop. And further tests in pots have also shown that rock-rose clearly reduces the growth of clover by reducing its access to water (fig. 3). This property could contribute to the effect it has previously been observed to have on weeds.

<figure><img src="image_156.png" /><figcaption>**Figure 2:** Tests on square plantations (x9) in the Aubière municipal conservatory gardens. A: *Lomelosia* May 2019; B: *Lomelosia* March 2020; C: Rock rose May 2019; D: Rock rose March 2020; E: Rock rose March 2020; showing ground underneath. Photo: © Claire Richard, ICCF/CNRS</figcaption></figure>

<figure><img src="image_157.png" /><figcaption>**Figure 3:** Effects of rock rose on the germination and growth of clover. A: Control, 9 days after seeding; B: Pot with rock-rose planted on right, 9 days after seeding; C: Control, 5 month after seeding; D: Pot with rock-rose planted on right, 5 months after seeding. Photo: © Claire Richard, ICF/CNRS</figcaption></figure>

## Experiments with Leylandii cypress extract:

Leylandii cypress, also terpene-rich, was selected for use as a mulch and a macerate.

This choice was based on information from published articles which presented encouraging results.

Germination tests under controlled laboratory conditions have shown that cypress needles, when applied as a concentrate or as a mulch, can very significantly reduce the germination or growth rates of clover and cress. The inhibition rate was > 90 % using an 8 day macerate. When applied as a 5cm layer of mulch, cypress needles reduce seedling growth rate by almost 50%. Experiments were also completed under real conditions around the bases of trees in a school playground. After a few months, the mulch turned out to be much more effective for weed control than a layer of the same thickness of chipped plant material (fig. 4, following page).

Chemical analyses of the macerate and cypress leaf ethanol extract were completed. In both cases a number of compounds were detected including terpenes that had oxidised to varying degrees. However, no clear relation has been established between the presence of any one particular compound and the observed property. The soil to which the mulch was applied was also tested after a year. This presented no unusual degree of acidification.

With these results, the Desherbal project has opened up prospects on the types of research to be undertaken in order to explore and understand the role played by terpenes in allelopathic phenomena. It also helped to implement operational solutions. Consequently, rock-rose, with or without the association of hyssop, and Leylandii cypress needle mulch can be used to help control weeds.
<figure><img src="image_158.png" /><figcaption>**Figure 4:** Test involving Leyland cypress mulch applied to the base of a tree. A: Chipped plant material, September 2019; B: Chipped plant material, June 2020; C: Cypress mulch, September 2019; D: Cypress mulch, June 2020. Photo Credit: © Claire Richard, ICCF/CNRS</figcaption></figure>

## Prospects for transfer or research

### Transfer:

This project has shown that rock-rose, either alone or in association with hyssop, can be used to control weeds in flower beds or around the bases of trees. Once in place, they require very little watering or maintenance. *Cistus creticus* produced better results than *cistus purpurus* but this could be a problem related to their growth-rates since the former was tested over two years whilst the latter was only tested over a year.

Additionally, Leylandii cypress clippings can be used as a mulch around the base of trees or in flower beds. Soil acidification remained very minimal.

### Research:

For the part of the project that concerned allelopathic plants, the project has been able to highlight the interesting properties shown by terpene-rich plants, but room for more precise or detailed research on the subject remains. Other terpene-rich plants could also be tested.

For the Leylandii cypress mulch part of the project, further soil analyses should be completed to establish whether compounds in Cypress leaves can be detected in the soil and to ensure that they are innocuous.

## Prospects for transfer or research (cont.)

### Publications and scientific symposia:

#### TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA

* **IUPAC on Crop plant protection. 19-24/05/2019. Gand, Belgium:** *Characterization of myrigalone photoproducts and evaluation of their anti-germinative properties*. Poster. A. Khaled, M. Sleiman, Y. Arbid, C. Sac, A. Corson, C. Bertrand, P. Goupil, C. Richard

* **European meeting on Environmental Chemistry (EMEC19). 03-07/12/2018. Clermont-Ferrand, France:** *Photodegradation of myrigalone, a natural herbicide, in water and on leaves*. Poster (Best poster prize). A. Khaled, M. Sleiman, C. Bertrand, C. Richard

#### Planned conference presentations

* **Congrès Biocontrole Perpignan (planned for September 2020, postponed due to Covid): Can cypress leaves be used as mulch to control weeds?** Amina Khaled, Etienne Darras, Guillaume Barrès, Mohamad Sleiman, Claire Richard

* **Congrès Biocontrole Perpignan (planned for September 2020, postponed due to Covid): Potential use of Cistus to control weed growth.** Amina Khaled, Etienne Darras, Guillaume Barrès, Mohamad Sleiman, Claire Richard

#### SCIENTIFIC PUBLICATIONS

* **Journal of Agricultural and Food Chemistry (n° 67, pp 7258-7265): Photodegradation of myrigalone A, an allelochemical from Myrica gale: photoproducts and effect of terpenes.** Amina Khaled, Mohamad Sleiman, Etienne Darras, Aurélien Trivella, Cédric Bertrand, Nicolas Inguimbert, Pascale Goupil, Claire Richard. 2019. https://pubs.acs.org/doi/10.1021/acs.jafc.9b01722

* **Forests (n°11-11, 1177, pp 1-12): Phytotoxic Effect of Macerates and Mulches from Cupressus leylandii Leaves on Clover and Cress: Role of Chemical Composition.** Amina Khaled, Mohamad Sleiman, Pascale Goupil and Claire Richard*. 2020. Open access https://link.springer.com/article/10.1007/s10311-020-01137-z; https://www.mdpi.com/1999-4907/11/11/1177

### Other practical works:

Article published in "La Montagne" http://ville-aubiere.fr/2018/07/26/espaces-verts-plantes-allelopathiques/-article published in "Métropole", December 2019 - January 2020
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DICABIO

# Evaluation of dicaffeoylquinic and dicaffeoyltartric acids as natural Biocontrol substances

**Launch year:** 2016

**Completion year:** 2020

**Scientific manager**

Jean-Luc Poëssel, INRAE

jean-luc-poessel@inrae.fr

**Partners**

INRAE PACA Avignon; SBM Développement

**Financing**

Total cost of the project: € 431,035

Ecophyto grant: € 110,658

## Key words:

Natural control methods; Plant extracts; Bio-aggressor resistance methods; Aphids; Fungal diseases; Bio-pesticides; Natural substances; Alternative elementary practices; Large-scale crops; Horticulture

## Context and main objectives

The aim of DicaBio is to develop natural substances for use as bio-pesticides. Whilst a large number of publications have shown that a number of botanical substances can have pesticide properties, this has only very rarely resulted in their use as biocontrol solutions due to the large number of obstacles to their development.

DicaBio aims to develop plant-based phenolic molecules, dicaffeoylquinic (diCQ) and dicaffeoyltartric (diCT) acids, as organic aphidicides and fungicides for use on a variety of crops. The lack of diversity in the range of aphid control methods currently on offer is in itself a factor for the increasing resistance of these pests and the resulting crop epidemics. For example, in 2020, viruses affecting beetroot transmitted by aphids led to major production losses and resulted in the return of neo-nicotinoid insecticides onto the market. The alternative to chemical products that we are proposing here aims to provide a very specific control solution for the target insects which can also be respectful of the environment and public health.

## Principal results and interest in relation to the Ecophyto plan

The results obtained by DicaBio cover every step of the bio-pesticide product development process.

* We have identified new natural production sources of diCT which could guarantee supplies, using dandelion or peanut for example.

* We have studied the chemical properties of diCT and diCQ. These two molecules are highly anti-oxidising. They breakdown rapidly through oxidation to neutral pH; diCT is slower than diCA, but both remain stable at low pH levels. These results made it possible for the partner company, SBM, to produce adapted formulations.

* Amongst the 5 geneartions of formulation developed, the most recent presents an environment in which the molecule remains stable. After developing a laboratory testing protocol for the aphidicide properties, the various formulation types (liquid, wettable powder, suspension, liquid concentrate) were studied for their aphidicide properties on the various species of aphid, with and without the addition of agents to facilitate the penetration of the active ingredient. In spite of the gradual adaptation of formulations to resolve issues as they arose, the laboratory tests did not show a clear aphidicide action. Our work on this action of the formulation has shown that the problem lies in the failure of the preparation to penetrate the foliar tissues in spite of the addition of wetting agents. Once these formulations actually penetrate into the leaves they show that they are truly effective. Our results have also indicated that the substance has a low persistence on leaves and in foliar tissue, indicating that it is probably metabolised very rapidly. This property demonstrates its very low environmental impact.
► We have also shown that diCT, like diCQ, has a potential fungicide effect under laboratory conditions against cereal seedling pathogens and septoria leaf blotch in wheat.

► Works on the risk of aphids adapting to treatment with diCT or diCQ have shown that, to the extent of existing knowledge, both of these active substances have a long action duration as an aphidicide on Myzus persicae, a species which is known to have developed resistance to a number of chemical insecticides. There is no reason for the observed resistance to chemical insecticides passes on to these two active substances. However we have observed that one Aphis craccivora clone was not affected at all by the two substances. Better knowledge of this insensibility phenomenon could help us to both understand how these substances function and better manage the risk of adaptation by aphid populations.

► The risks of unwanted side-effects were evaluated on domestic bees. Acute exposure to diCQ and diCT, either by contact or ingestion, or over a number of days does not cause mortality. However a reduction in feeding was noted after chronic exposure for diCT but not for diCQ. These results have raised potential unwanted effects which could be suffered by beneficial insects if these substances are not used correctly. However, the impact of these natural substances on bees appears to be much less serious than that of the chemical insecticides currently used in farming.

<figure><img src="image_160.png" /><figcaption>Dosage/response curves for different Myzus persicae populations.</figcaption></figure>

DiCT dosage/response curves for different Myzus persicae populations. Image: © Myriam Siegwart, INRAE

## Prospects for transfer or research

### Transfer:

We have clearly identified that the absence of product penetration is a serious obstacle to the expression of the full potential of these substances as aphidicides. Once the problems is resolved, new biocontrol products that respect both the environment and public health could be available for use in a number of agricultural domains such as large-scale crops, beetroot, rapeseed, or in fruit orchards, all of which are suffering from a serious lack of new solutions. The results obtained here are a major step along the road to using diCT and diCQ as bio-pesticides and, in a more general way, a significant contribution to the innovative development of other botanical biocontrol substances.

### Research:

It now seems necessary that innovative bio-pesticide product formulation approaches must be developed, such as nanoformulations; these are the subject of significant global research at the moment and could provide new solutions to resolve the low levels of active product penetration into foliar tissue, which has become the main product effectiveness obstacle raised by the DicaBio project. Additional research based on these innovative formulations would also be necessary in order to evaluate the possibility of developing a diCT-based fungicide to control wheat septoria leaf blotch and other fungal diseases.

## Publications and scientific symposia:

### Scientific symposia:

► Séminaire de restitution intermédiaire des appels à projets « PSPE 2 » et « Pesticides 2014 » (14-15/12/2017), Paris, France

► Natural Products and Biocontrol Conference (25-28/09/2018), Perpignan, France

► Resistance'19. (16-19/09/2019), Rothamsted, UK

### Technical workshop:

► Journée Métabolites Secondaires (16/09/2018), Avignon, France

### Scientific publications:

► Mode of action and sustainability of the use of caffeic acid derivatives in the control of aphids in agriculture (in preparation). Siegwart M., Sauge MH., Lecerf E., Mascle O., and Poëssel JL.
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ÉCOVERGER

# Designing low pesticide-use technical approaches for orchards that respect the current farming restrictions and objectives. An applied modelling approach for peach and mango trees

Launch year: 2015

Completion year: 2019

Scientific manager

Isabelle Grechi, CIRAD UPR HortSys

isabelle.grechi@cirad.fr

Partners

CIRAD UPR HortSys; INRAE UR PSH; University of Montpellier

Financing

Total cost of the project: € 348,935

Ecophyto grant: € 88,921

## Key words:

Fruit crops; Socio-economic evaluation; Technical route modification; Alternative treatment methods; Modelling; Growing practices; Production; Fruit quality; Farming strategy classification; Co-design

## Context and main objectives

Fruit tree farming is subject to strict social and governmental requirements in terms of reducing its impact on the environment and public health whilst, at the same time, increasing the production quality. Even if producers are increasingly turning to alternative methods, orchard pest and disease management still relies heavily on chemical products. Agricultural practices will need to be totally redefined to invent economically viable systems capable of producing quality fruit whilst using fewer pesticides.

The objective of the project was to develop specific technical route (or solution) design support tools and approaches for orchard productions; evaluated on the basis of their capacity to restrict the development and the damages caused by pests and diseases whilst also meeting agricultural performance and economic viability obligations. The project attempts to identify and develop viable alternatives to the use of pesticides in the form of integrated control and hands-on methods that will increase the resistance of orchards to pests and disease. The approach has been tested and proven on two different fruit crops: mangos in La Réunion and peach-nectarines in mainland France.

<figure><img src="image_162.png" /><figcaption>Peach-tree orchard
Photo: © Michel Génard, INRAE</figcaption></figure>

## Principal results and interest in relation to the Ecophyto plan

The Ecoverger project has used three case studies to evaluate the performance of a number of pest and disease control levers, which were selected for their action and their standards of innovation as pesticide alternatives for brown rot - peach, fruit fly - mango and gall midge - mango blossom.

Amongst the evaluated levers, those that aim to restrict crop disease and contamination are useful for limiting the development of pests and diseases along with the damage they cause, but they can have a potential negative impact on harvests. Moisture stress and moderate fruit loads will reduce the incidence of brown rot, increase fruit quality but reduce calibre and harvest potential. Early harvests of mango, when they are green-ripe and just turning yellow, will minimise the risk of fruit fly infestation on the fruit and will only have a minor effect on fruit quality and quantity.

The other evaluated levers to restrict pest and disease development are less consistent in their results but have no impact on harvests. Covering the ground with woven textiles or high weeds will reduce gall midge populations and the damage they cause to mango blossom. Blossom synchronisation using suitable practices would also reduce gall midge populations, but
only if the orchard is not exposed to a high degree of outside pest pressure. Winter sanitation harvests, involving the removal of any dessicated twigs or fruit from previous seasons, will only control brown rot incidence if the primary inoculum is almost entirely eliminated from the orchard in the first place. Removing infected peaches appears to be more effective when it is done during the growing season. Due to such partial effects, these levers should be preferred only in combination with other levers.

The substitution of phytosanitary treatments with certain levers may generate additional expenditure due to investment in equipment or increased labour resulting from the introduction of new technical operations (covering the ground with woven textile) or increase the technicality of a practice already in use (harvesting fruit at specifically targeted ripeness stages). Other levers are quite easy to implement since they are simply based on adapting practices already in place in the technical pathway (changes to weed-cutting dates, less irrigation and cleaning out).

The value, productivity and visual aspect of the fruit are the most important performance criteria for most producers, whereas indicators concerning the environmental pollution related to phytosanitary treatments are perhaps of lesser importance (at least in the mango trade). This implies that there is little motivation to move over to eco-responsible practices if there is no return on the investment, for example through a higher market value, or carefully adjusted subsidies to compensate for any additional expenditure or a diminished harvest.

To find out if these levers can be substituted, either totally or partially, for pesticides, it would be necessary to achieve a greater degree of objectivity on their performance; and establish the conditions for success and adoption when implemented in the field. The diversity of production profiles promotes the idea of seeking out solutions adapted for each specific production context allowing for the specific constraints and objectives of the users.

## Prospects for transfer or research

### Transfer:

The Ecoverger project has studied alternative control levers to pesticides for dealing with pests which attack the reproductive organs of fruit trees in temperate and sub-tropical climates. General and specific rules concerning the effectiveness and deployment methods for these levers have been established in terms of their action and the nature of the pests. Additionally, considering that certain potential obstacles to their adoption are essentially economic, measures to incite the adoption of eco-responsible practices along with better promotion and marketability for products with environmental and public health benefits could help to remove such obstacles.

### Research:

The Ecophyto II+ Plan ODACE<fnref n="1" /> project continues the work begun by the Ecoverger project with the operational and functional implementation of an orchard management approach which is less dependent on pesticides. To do this, it will use a larger range of tools and expertise as well as trying to do away with some of the Ecoverger project limitations. More specifically, it will develop an interactive evaluation tool to facilitate dialogue between researchers and growers, in which the combination of a number of alternative levers, multiple pest and disease vectors, and sustainability dimensions will be taken into account.

## Publications and scientific symposia:

### TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA:

* HortiModel 2016: Vth International Symposium on Models for Plant Growth, Environment Control and Farming

* Management in Protected Cultivation. 19-22 September 2016, Avignon, France: **A compartmental epidemiological model for brown rot spreading in fruit orchards.** Bevacqua D., Génard M., Turion-Quilot B., Oliveira Lino L., Mercier V., Lescourret F. (2016a).

* Plant Biology Europe EPSO/FESPB 2016 Congress. 26-30 June 2016, Prague, Czech Republic: **A compartmental epidemiological model for brown rot spreading in stone fruit orchards** [poster]. Bevacqua D., Génard M., Turion-Quilot B., Oliveira Lino L., Mercier V., Lescourret F., Bolzoni L. (2016b).

* XXVII Congresso Nazionale della Società Italiana di Ecologia, 12-15 September 2017, Napoli, Italy: **A model for temporal dynamics of brown rot spreading in fruit orchards.** Bevacqua D., Quilot-Turion B., Bolzoni L. (2017).

* XII International Mango Symposium, 10-16 July 2017, Baise, China: **How different pruning intensities and severities affect vegetative growth processes in "Cogshall" mango trees.** Persello S., Grechi I., Boudon F., Normand F. (2017).

<footnote n="1">¹ ODACE: Outil d'évaluation et de Dialogue entre acteurs et chercheurs, pour Accompagner la conCEption de solutions de protection des plantes dans le contexte arboricole (Evaluation and Dialogue Tool for professionals and researchers, to Support the design of plant protection solutions in an orchard context) (ECOPHYTO II+ OFB 2021-2023)</footnote>
► XII International Mango Symposium, 10-16 July 2017, Baise, China: **Effects of some cultural practices on**

**mango inflorescence and fruit pest infestation and damage in Reunion Island: recent progress, on-going**
**studies and future steps** [poster]. Ratnadass A., Grechi I., Caillat A., Préterre A.-L., Normand F., Graindorge
R. (2017).

► International conference: Models in Population Dynamics, Ecology and Evolution. 9-13 April, Leicester, UK: **A model for temporal dynamics of brown rot spreading in fruit orchards**. Bevacqua D., Quilot-Turion B., Bolzoni L. (2018a).

► XXVIII Congresso Nazionale della Società Italiana di Ecologia, 12-14 September, Cagliari, Italy: **Climate effects**

► **on the spread of brown rot disease: insights from an epidemiological model.** Bevacqua D., Vanalli C., Casagrandi R., Gatto M. (2018b).

► 30th International Horticultural Congress (IHC2018). 12-16 August 2018, Istanbul, Turkey: **Assessment of**

**mango tree - blossom gall midge management solutions from in-silico experiments: overview of an on-going modeling approach** [poster]. Grechi I., Saint Criq L., Soria C., Ratnadass A., Normand F., Amouroux P, Boudon F. (2018).

► UMT Si-Bio seminar, 21 June 2019, Avignon, France: **Évaluation d'itinéraires techniques pour la gestion de la moniliose en verger de pêcher-nectarine**. Borg J., Kerdraon M., Plénet D. (2019).

► The International Society for the Ecological Modelling - Global Conference 2019. 1-5 October 2019, Salzburg, Austria: **The mango tree - blossom gall midge system: in-silico assessment of its functioning and management**. Grechi I., Reyné B., Saint-Criq L., Memah M.M., Ratnadass A., Normand F., Boudon F. (2019).

► 2nd annual ESA International Branch Virtual Symposium. 8-10 April 2019, USA: **Plant organ hardness as a factor of crop resistance to insect pests** [poster]. Ratnadass A., Caillat A., Chantereau J., Chillet M., Fliedel G., Grechi I. (2019).

► EEF: Ecology across borders. Embedding ecology in sustainable development goals. 29 July-2 August 2019,

► Lisbon, Portugal: **Climate change and the spread of brown-rot disease in peach orchards: insights from an epidemiological model**. Vanalli C., Bevacqua B., Casagrandi R., Gatto M. (2019).

► 4th TEAM Meeting, 5-9 October 2020, La Grande Motte, France: **Potential of some cultural levers for fruit fly management on mango in Réunion** [poster]. Ratnadass A., Caillat A., Préterre A.L., Brunet-Lecomte C., Lardenois M., Grechi I. (2020)

**SCIENTIFIC ARTICLES:**

► Frontiers in Ecology and Evolution (n°5, p 170): **The crop load affects brown rot progression in fruit**

► **orchards: high fruit densities facilitate fruit exposure to spores but reduce the infection rate by decreasing fruit growth and cuticle cracking.** Bellingeri M., Quilot-Turion B., Oliveira Lino L., Bevacqua D. (2018). https://doi.org/10.3389/fevo.2017.00170

► Phytopathology (n° 108, pp 595-601): **A model for temporal dynamics of brown rot spreading in fruit orchards**. Bevacqua D., Quilo-Turion B., Bolzoni L. (2018). https://doi.org/10.1094/PHYTO-07-17-0250-R

► Scientific Reports (n°9, p 8519): **Coupling epidemiological and tree growth models to control fungal diseases spread in fruit orchards**. Bevacqua D., Génard M., Lescourret F., Martinetti D., Vercambre G., Valsesia P., Mirás-Avalos J.M. (2019). https://doi.org/10.1038/s41598-019-44898-6

► European Journal of Agronomy (n° 104, pp 85-96): **Nature abhors a vacuum: Deciphering the vegetative reaction of the mango tree to pruning**. Persello S., Grechi I., Boudon F., Normand F. (2019). https://doi.org/10.1016/j.eja.2019.01.007

► Acta Horticulturae (n° 1244, pp 159-166): **Effects of some cultural practices on mango inflorescence and**

**fruit pest infestation and damage in Reunion Island: recent progress, on-going studies and**
**future steps.** Ratnadass A., Grechi I., Graindorge R., Caillat A., Préterre A.L., Normand F. (2019). https://doi.org/10.17660/ActaHortic.2019.1244.24

► Annals of Botany (n° 126, pp 745-763): **V-Mango: A functional-structural model of mango tree growth, development and fruit production**. Boudon F., Persello S., Jestin A., Briand A-S., Grechi I., Fernique P., Guédon Y., Léchaudel M., Lauri P-E., Normand F. (2020). https://doi.org/10.1093/aob/mcaa089

► Acta Horticulturae (n° 1281, pp 633-641): **The mango tree - blossom gall midge system: toward in-silico assessment of management practices**. Grechi I., Saint Criq L., Ratnadass A., Normand F., Soria C., Brustel L., Amouroux P, Boudon F. (2020). https://doi.org/10.17660/ActaHortic.2020.1281.83

**Other promotional works:**

► Educational documents for schools and researchers: « L'agroécologie à la croisée des disciplines scientifiques » organised by the Cirad and Montpellier SupAgro (Saint-Pierre, Ile de la Réunion, 28 November – 2 December 2016) for which the Ecoverger project served as study material: Data-sheet: « Présentation du terrain mangues » - video: « La mangue, la culture de compromis »

► Tp dispensed in BTS GPN1 (Gestion & Protection de la Nature) at l'EPLEFPA in St-Paul, Ile de la Réunion

**ACTIVITY REPORTS FOR PRODUCERS:**

► **Analyse systémique des exploitations productrices de mangues à La Réunion : identification des déterminants influençant les choix techniques et les changements de pratiques des producteurs** pour la co-conception d'itinéraires techniques innovants apportant une alternative aux pesticides – Summary presentation of orchard types. 8p. Marchetti M. (2016).
► Compte rendu d'étude de l'effet de différentes modalités de couverture du sol sur la cécidomyie des fleurs du manguier, la phénologie de l'arbre, et sur l'infestation par les mouches des fruits des mangues précocement tombées au sol. 9p. Brustel L., Soria C., Wilt M., Ratnadass A. (2017).

► Evaluation des dynamiques des mouches des fruits, d'abondance/maturation des mangues et des niveaux d'infestation des fruits en vergers de manguiers. Préterre A.L., Grechi I., Ratnadass A. (2017). 2 x 8p.

► Compte rendu de l'analyse des résultats d'enquête et de classification : identification des profils de production. Kerdraon M. (2019). 4p+Appendix.

► Communication and educational posters/data-sheets:

► Quels leviers pour maîtriser les mouches des fruits et leurs dégâts sur mangue ? Grechi I., Caillat A., Préterre A-L., Brunet-Lecomte C., Ratnadass A. (2019).

► Un dispositif de recherche et de formation sur les options agroécologiques de régulation de la cécidomyie des fleurs du manguier. Ratnadass A., Amouroux P., Billot T., Briandy A., Brustel L., Grechi I., Normand F., Payet R-M., Reyné B., Saint Criq L., Soria C., Auré A., Wilt M. (2019a).

► Quels leviers pour maîtriser les cécidomyies des fleurs et leurs dégâts sur manguier ? Ratnadass A., Brustel L., Briandy A., Billot T., Grechi I. (2019b).

## SURVEY / PLACEMENT REPORTS:

► Ecoverger project – Déterminants des pratiques & indicateurs d'évaluation des systèmes techniques en vergers de manguiers. Cirad. 22p. Parrot L., Michels T., Brulard N. (2019).

► Analyse systémique des exploitations productrices de mangues à La Réunion : Identification des déterminants influençant les choix techniques et les changements de pratiques des producteurs pour la co-conception d'itinéraires techniques innovants apportant une alternative aux pesticides. Dissertation, ISTOM, 79p + Appendices Marchetti M. (2016).

► Comprendre les déterminants de la décision technique pour mieux accompagner l'innovation : le cas des producteurs de mangues réunionnais. Dissertation, UniLaSalle, 71p + Appendices Girard G. (2017).

► Evaluation des dynamiques des mouches des fruits, de l'abondance/ maturation des mangues et des niveaux d'infestation des fruits en vergers de manguiers et évaluation de la relation entre l'infestation des mangues et leur état de maturité. Placement year dissertation, AgroParisTech, FacForPro affiliation, 38p + Appendices Préterre A.-L. (2017).

► Effet de la taille sur la croissance végétative et la floraison du manguier Cogshall. Placement year dissertaion, Montpellier SupAgro, 29p. Stahl A. (2017).

► Evaluation de l'effet de pratiques culturales (paillage/ enherbement du sol/ récolte prophylactique précoce) en vergers de manguiers (Mangifera indica L.) sur la régulation de bio agresseurs de la floraison et de la fructification : les cas de la cécidomyie des fleurs (Procontarinia mangiferae) et des mouches des fruits (Diptera : Tephritidae). Dissertation, Ecole d'ingénieurs de Purpan, 95p + Appendices. Brustel L. (2018)

► Développement d'un outil pour co-concevoir des itinéraires techniques économies en pesticides en vergers. Master 2 dissertation, University of Grenoble Alpes, 29p + Appendices. Girard J. (2018).

► Modélisation du système manguier-cécidomyie des fleurs pour une évaluation de modes de gestion du ravageur et de ses dégâts. Master 2 dissertation, Université Paul Sabatier, Toulouse III, 60p + Appendices. Saint Criq L. (2018).

► Climate change and the spread of brown rot disease in peach orchards: insights from an epidemiological model. M2 dissertation, Politecnico di Milano. 109p. Vanalli C. (2018).

► Développement d'une démarche d'analyses statistiques de données d'un réseau pluriannuel d'expérimentations systèmes de culture en vergers de pêchers. Dissertation, ISPED - University of Bordeaux, 40p + Appendices. Bostal C. (2019).

► Evaluation du potentiel de leviers de gestion culturaux dans la lutte contre la mouche des fruits Bactrocera dorsalis (Hendel) sur manguier. Dissertation, ISTOM 80p + Appendices. Brunet-Lecomte C. (2019).

► Modélisation du système manguier-cécidomyie des fleurs pour une évaluation de modes de gestion du ravageur et de ses dégâts. Master 2 dissertation, Montpellier Science Faculty, 44p + Appendices. Reyné B. (2019).

► Growth and nutrient partitioning in deciduous trees: a modelling framework linking seasonal and inter-annual dynamics. M2 dissertation, Politecnico di Milano. 63p. Salvagno P. (2019).

► Evaluation de l'effet de pratiques culturales (type de couverture du sol / temps passé au sol par les fruits) et d'états physiologiques (couleur des fruits / état d'abscission / stade de maturité) sur l'infestation des mangues par les mouches des fruits (Diptera : Tephritidae) à la Réunion. Placement year dissertation, AgroParisTech, FacForPro affiliation, 36p + Appendices. Lardenois M. (2020). Project web-site link : https://cosaq.cirad.fr/projets/ecoverger.
<figure><img src="image_163.png" /></figure>

ENFIN !

# Development of a new plant protection concept for apple scab disease

**Launch year:** 2022

**Completion year:** 2024

**Scientific manager**

Bruno Le Cam, **INRAE, Centre Pays de Loire, UMR IRHS**, bruno.lecam@inrae.fr

**Partners**

**INRAE UMR IRHS**, CTIFL, IFPC, UEH d'Angers, UMR SADAPT de AgroParisTech

**Financing**

Total cost of the project: € 450,000
Ecophyto grant: 100 %

## Key words:

Technical approach modification; Plant stimulation; Arboriculture; Socio-economic evaluation, Management via macro-organisms; pesticide reduction

## Summary

Apple scab disease is caused by the *Venturia inaequalis* fungus and is the principal disease affecting apple-trees in temperate climates. Apple scab disease takes the form of blotches on the fruit making them unfit for consumption. Most of the leading commercial apple varieties are sensitive to this disease and phytosanitary products are used in orchards to cope with it. This represents an enormous financial and environmental cost (up to 30 fungicide applications per year to treat the disease effectively).

The ENFIN! project aims to test out a breakthrough technical approach which seeks to provoke a drastic fall in the population of this pathogen in orchards and thus considerably reduce the need to use fungicides. This approach uses a totally new biocontrol strategy which combines two INRAE patented inventions (FR 1915276 patent). The first invention renders the fungus non-virulent. It targets the reproductive phase of *V. inaequalis* and forces it to reproduce with non-pathogenic strains from the same species, thus generating non-virulent descendants for the following spring. The second innovation involves applying the same non-pathogenic strains during the spring time to provoke immunity in the apple trees, which will protect them from later apple scab disease infections. The project also aims to evaluate the durability of this approach in orchards (4 French experimental sites) and to evaluate the apple growing industry's acceptance and appropriation conditions for new inventions.

<figure><img src="image_164.png" /><figcaption>Apple scab disease</figcaption></figure>

Photo: © INRAE

## Ecophyto call for projects - Maturation (2021 edition) - Levers that can be implemented for a transition towards changing systems

In order to encourage research work that will contribute to reaching the Ecophyto II+ Plan objectives, the Ministries for Ecological transition and solidarity, Higher Education, Research and Innovation, Agriculture and Food, Solidarity and Health (Ministères de la Transition Ecologique et Solidaire, de l'Enseignement Supérieur de la Recherche et de l'Innovation, de l'Agriculture et de l'Alimentation, des Solidarités et de la Santé), have decided to launch a second edition of the "Ecophyto Maturation" call for projects.

This call for projects will promote the maturation of scientific works that have already succeeded in the context of a previously existing research program. The objective will be to take a breakthrough solution up to the stage where it can be applied using products, technologies or services. More precisely, the objective of this call for projects is to incite consortia of academic researchers and socio-economic entities (businesses, technical centres and institutes, professional bodies, consular chambers, NGOs, etc.) to work together to develop the research work necessary to be able to propose a solution which has been proven and rendered operational in the field whilst also responding fully to end-user requirements. On the TRL maturity scale, this would be the equivalent to level 5 or more.

This call for projects was operated by the Agence Nationale de la Recherche (ANR) which also co-financed it with the Office Français pour la Biodiversité (OFB).
<figure><img src="image_165.png" /></figure>

# Manipulating the floral biodiversity of cereal crop fields

**Launch year:** 2015

**Completion year:** 2018

**Scientific manager**

Joan Van Baaren, CNRS-University of Rennes

joan.van-baaren@univ-rennes1.fr

**Partners**

University of Rennes-CNRS; INRAE / Agrocampus Ouest; Institut Sophia Agrobiotech; INRAE-AgroParisTech; SA PINAULT; Ter-Qualitechs; Dervenn; LTER Armorique

**Financing**

Total cost of the project: € 721,009

Ecophyto grant: € 141,748

**Key words:**

Floral plantings; Floral traits; Biological regulation; Trophic resources; Parasitoids; Predators; Winter season; Biological control by conservation; Global warming; Annual and perennial flower beds

## Context and main objectives

In conventional large-scale cereal crop farming phytopharmaceutical products, amongst others, are used to coat seedlings. This is to limit aphid populations which spread virus during the winter. However, such seedling treatments have been banned since September 2018. For rapeseed crops, very rare in organic farming, pest beetle species are resistant to insecticides. The FLEUR project aims to set up a biocontrol by conservation approach to increase the population and diversity of the natural enemies of these pest insects by increasing the plant biodiversity surrounding the fields through perennial and annual flowerbeds. The objectives of the FLEUR project were to (1) identify the plant species best suited to increasing the effectiveness of natural enemies, (2) define the most efficient spatial arrangements for these flowerbeds in relation to the crops to be protected, (3) define the risks to neighbouring crops associated with these flowerbeds.

<figure><img src="image_166.png" /></figure>

Aphidius avenae laying eggs inside an English grain aphid *Sitobion avenae*.

Photo: © Sonia Dourlot, University of Rennes 1

## Principal results and interest in relation to the Ecophyto plan

The main product of this project is a wild flower mix of 4 species of inexpensive and clean plants that do not attract other pests and are used by alternative hosts allowing parasitoid populations to develop; they flower in winter when there is no frost (therefore producing nectar that can be used by the parasitoids) and are attractive to parasitoids due to their dominant yellow colour (mustard); when located near to the crops they serve as a refuge for predator arthropods (ground and rove beetles, spiders) thanks to their attractive micro-climate. This mixture includes a number of varieties of buckwheat likely to flower right through the autumn, mustard which is very frost resistant and attractive, various varieties of broadbean which flower between autumn and winter, as well as cornflower.

In conventional agriculture in Brittany, a region where cereal crops (wheat and corn in rotation) are common, this annual mix can be sown amongst the winter cover crops that the farmers grow between September and March in areas where they plant corn in the spring. In organic farming, since farmers do not use winter cover crops, a part of the field surface area must be given over to the flowers in place of cereal (lost surface area). The results achieved are very similar for both conventional and organic farming, highlighting the lack of risk involved in growing these flowers near to crops.
The winter season has now become a favourable period for the implementation of biological pest control methods that use conservation, thus opening up the possibility of year-round biological pest control through plant diversity. With the effects of global warming, natural enemies are present in fields throughout the year and are dangerous in winter, the period when the aphids transmit virus such as the Barley Yellow Dwarf Virus.

Our results have highlighted the positive effects of these flowerbeds on parasitism rates and the predatory regulation of crop pests by their natural enemies and show how important these flower beds can be to encourage auxiliary populations throughout the year.

Demonstrating the benefits of planting annual flowerbeds near to crops during winter is a first for biological control by conservation which until then had been mostly concentrated on the growing season. Additionally, this project has also been able to highlight the benefits of perennial flowerbeds to maintain the population levels of natural enemies.

<figure><img src="image_167.png" /><figcaption>Winter manipulation (looking for aphid) in a wheat field.</figcaption></figure>

Photo: © Ouest-France 05 April 2018

## Prospects for transfer or research

### Transfer:

Our results show the benefits of planting wild flower mixes near to cereal crops during winter. In conventional agriculture in Brittany, which involves the rotation of wheat and corn crops, the transfer is relatively simple since farmers have to use winter cover crops and most of the farmers we contacted were not especially resistant to the idea of sowing a wildflower mix over the whole of the surface of their winter cover crops. If this method was adopted on a broad scale, it would probably have a synergistic effect over the whole of the landscape. Mass production of the wildflower mix is not in place yet.

### Research:

The benefits of perennial or annual cultivated plant diversity on the scale of farming landscapes would need to be evaluated. For the rapeseed and pulse crops that are attacked by pest beetles, these insects and their parasitoids only complete one reproductive cycle per year and disperse very widely in the surrounding landscape. This means that the effects of the perennial planting which facilitates natural regulation shifts over time and is diluted over the land and thus farmers find little reason to invest in this approach. Annual flowering winter cover crops that are widely distributed over the farming landscape would not only be beneficial to neighbouring fields but also to the whole of the landscape.

## Publications and scientific symposia:

### SCIENTIFIC PUBLICATIONS:

* Agriculture Ecosystems and Environment (n°247: pp 418-425): **Change in plant phenology during winter increases pest control but not trophic link diversity.** Damien M, Le Lann C, Desneux N, Alford L, Al-Hassan D, Georges R, Van Baaren J. 2017.

* Frontiers in Ecology and Evolution-Population and Evolutionary Dynamics: **Changes in host-parasitoid communities over the years in cereal crops of Western France: Does climate warming matters?** Tougeron K, Damien M, Le Lann C, Brodeur J & van Baaren J. 2018.

* Behavioral Ecology and Sociobiology (73:156): **Food or host : do physiological state and flower type affect foraging decisions of parasitoids?** Damien M, Barascou L, Ridel A, Van Baaren J, Le Lann C 2019. https://doi.org/10.1007/s00265-019-2758-9

* Entomologia generalis (40(2): 147 – 156): **How does floral nectar quality affect life history strategies in parasitic wasps?** Damien M, Llopis S, Desneux N, Van Baaren J and Le Lann C. 2020. Art No. ESP146004002003 DOI: 10.1127/entomologia/2020/0906S

### OPERATIONAL / EDUCATIONAL ARTICLES:

* Science ouest (n° 341, Avril 2016): **Des fleurs contre les pesticides.** http://www.espace-sciences.org/sciences-ouest/341/actualite/des-fleurs-contre-les-pesticides

* Horizon (n°130, April/May 2016): **Les parasitoïdes, des alliés actifs en hiver à favoriser.**
<figure><img src="image_168.png" /></figure>

# LIPOCONTROLE

## The search for new lipopeptides for use as biopesticides, by screening a collection of Pseudomonas

**Launch year:** 2015

**Completion year:** 2020

**Scientific manager**

Alice Rochex, University of Lille

alice.rochex@univ-lille.fr

**Partners**

University of Lille; INRAE; JUNIA; ULCO; Lipofabrik

**Financing**

Total cost of the project: € 254,261

Ecophyto grant: € 100,000

## Key words:

Biocontrol product; Large scale crops; Natural substances; Lipopeptides; Pseudomonas syringae; High flow-rate screening; Cryptogamic diseases

## Context and main objectives

Phytopathogenic fungi are one of the main pests and diseases affecting large-scale crops. Plant protection is currently largely based on the use of resistant varieties and chemical pesticides. There is a strong desire for the development of biocontrol plant protection products so that agriculture can become more sustainable and respectful of the environment and public health. Amongst these products, natural substances, mostly sulphur or plant based, occupy 63% of the market (in turnover, IBMA France 2019). Natural substances of microbial origin, such as lipopeptides produced by Bacillus and Pseudomonas bacteria, are little represented on the market but have been shown to have biocontrol potential against fungal diseases. The objective of the LIPOCONTROLE project is to identify new lipopeptides for use as biocontrol agents either in complement to or substitution of chemical pesticides in the treatment of the fungi that cause the cryptogamic diseases that cause the most economic damage.

## Principal results and interest in relation to the Ecophyto plan

The LIPOCONTROLE project has identified a number of new lipopeptides, extracted from cultures of *Pseudomonas* bacteria, which are effective against a wide range of fungal pathogens:

* Botrytis cinerea (responsible for grey mould on a number of plant species including grape vines and tomatoes)

* Sclerotinia sclerotiorum (responsible for white mould on a number of plant species including rapeseed and sunflower)

* Zymoseptoria tritici (responsible for wheat septoria leaf blotch)

* Phytophthora infestans (responsible for potato blight)

* Blumeria graminis f.sp. tritici (responsible powdery mildew on cereals)

* Oidium neolycopersici (responsible for oidium on tomatoes)

<figure><img src="image_169.png" /><figcaption>**Figure 1:** Lipopeptide structures produced by strains of the *P. syringae* complex. Linear or cyclic lipopeptides all have a hydrophobic fatty acid and a hydrophilous peptide chain.</figcaption></figure>

Image: © Alice Rochex, University of Lille
These new lipopeptides could become the active ingredients of future biocontrol products that would be effective in protecting large-scale crops, such as cereals, oil-seed and grapevines, from the principle fungal diseases. These biofungicide products would be classed as natural biocontrol substances and could be used in complement to or substitution for chemical fungicides. By proposing new natural biofungicide substances the results of the LIPOCONTROLE project will contribute to growth in biocontrol products and the reduction of the use of conventional phytosanitary products.

Using one molecule presents a number of advantages over the use of living bacteria. They avoid the storage and viability constraints during packing, formulation and field application as well as ensuring constant treatment effectiveness. These molecules are formulated as a liquid and can therefore be used and stored in the same way as a conventional phytosanitary product with no further constraints in comparison to current common practice.

The first limitation in the results is that the effectiveness of biomolecules has only been proven *in vitro* for *B. cinerea* and *S. sclerotiorum*, and *in vitro* and under glass for *Z. tritici*, *P. infestans*, *B. graminis f. sp. tritici* and *O. neolycopersci*. For *B. cinerea* and *S. sclerotiorum*, a protocol will have to be developed for *in planta* tests. For all of the pathogen-systems studied, the effectiveness of identified lipopeptides will have to be confirmed in the field.

<figure><img src="image_170.png" /><figcaption>**Figure 2:** Laboratory tests on the anti-fungal activity of *P. syringae* bacteria. The anti-fungal bacteria inhibit the growth of fungi (inhibition zone). Photo Credit: © Alexandre Bricout, University of Lille</figcaption></figure>

à The second limitation of these results is that the lipopeptides have only been produced on a laboratory scale (100 mL to 2L). A larger scale production and extraction process for these molecules will need to be designed and developed, this will need to produce enough of the molecule to fully evaluate their effectiveness in the field and show the feasibility of industrial scale production.

Finally, for the development of a new biocontrol product based on lipopeptides, there will need to be an evaluation of its toxicity, ecotoxicity and sustainability; and a formulation will have to be developed that will guarantee conservation and effective plant protection.

## Prospects for transfer or research

### Transfer:

*Psudomonas* lipopeptides are biocontrol products that show a high potential in spite of one major technological setback concerning its potential for mass industrial scale production: they are produced in very small quantities by the bacteria. Once the production and purification processes are available, the project results will be transferred to Lipofabrik a company specialised in the production of lipopeptides.
**Research:**

The works of LIPOCONTROLE will be continued by the ANR-19-ECOM-0007 CERES project (17 April 2020 - 27 October 2023), led by ULille and financed by APR ANR Ecophyto Maturation 2019. The CERES project aims to show that products based on Pseudomonas bacteria culture extracts containing lipopeptides are an operational biocontrol solution for managing wheat septoria leaf blotch.

**Publications and scientific symposia:**

**SCIENTIFIC PUBLICATIONS:**

► **The diversity of lipopeptides in the P. syringae complex parallels phylogeny and sheds light on structural diversification during evolutionary history.** Bricout A., Morris C.E., Chandeysson C., Boistel C., Chataigné G., Lecouturier D., Jacques P., Leclère V., Rochex A. Publication soumise.

**PHD THESIS:**

► **Mise en évidence d'une forte diversité structurale de lipopeptides chez P. syringae, un complexe bactérien aux activités antifongiques prometteuses.** Bricout A. (2020). Ademe - University of Lille

**SCIENTIFIC SYMPOSIA (ORAL PRESENTATIONS):**

► **Online conference, 16/06/2020, Akureyri, Iceland. Deciphering the structural diversity of lipopeptides produced by strains of the P. syringae complex. P. syringae.** Bricout A., Morris C.E., Bardin M., Nicot P., Chataigné G., Lecouturier D., Jacques P., Leclère V., Rochex A.

► **14th IUPAC International Congress of Crop Protection Chemistry. 19-24/05/2019, Gand, Belgium: Screening of Pseudomonas sp. strains for the biocontrol of septoria tritici blotch of wheat.** Bricout A., Morris C.E., Bardin M., Nicot P., Chataigné G., Siah A., Lecouturier D., Jacques P., Leclère V., Rochex A.

► **Biocontrol conferences. 04/04/2019, Reims, France: Stratégies de criblage pour la découverte et la caractérisation de lipopeptides antifongiques utilisables comme agents de biocontrôle.** Bricout A., Morris C.E., Bardin M., Nicot P., Chataigné G., Siah A., Lecouturier D., Jacques P., Leclère V., Rochex A.

► **Young Researchers Workshop Condorcet. 18-19/01/2018, Amiens, France: Développement de stratégies de criblage pour la recherche de lipopeptides antifongiques utilisables comme biopesticides.** Bricout A., Morris C.E., Bardin M., Nicot P., Chataigné G., Jacques P., Leclère V., Rochex A.
<figure><img src="image_171.png" /></figure>

MACROPLUS

Facilitating the installation of the whitefly, spider mite and aphid
predator *Macrolophus pygmaeus*

Launch year: 2015

Completion year: 2018

Partners

GRAB; SERAIL; APREL; Chambre d'Agriculture des
Bouches du Rhône; INRAE UE Alénya Roussillon

Scientific manager

Jérôme Lambion, GRAB
jerome.lambion@grab.fr

Financing

Total cost of the project: € 198,550
Ecophyto grant: € 99,233

Key words:

Management via macro-organisms; Redesigning crop systems; Functional biodiversity; Vegetable crops;
Host-plant; *Macrolophus*; Service plant; Co-design work with farmers

Context and main objectives

*Macrolophus pygmaeus* is a predatory plant bug which has been used to control white fly on tomatoes for the last twenty years. This plant bug is native to the South of France and can be used to regulate red spider mite, aphid, moths and tomato leaf miner populations provided that the crops are not intensively treated. Unfortunately this essential auxiliary species is costly and difficult to introduce sustainably.

The MacroPlus project aims to work on these last two
points by developing techniques to obtain larger numbers
of *M. pygmaeus* at a lower cost and earlier in the season.
The MacroPlus project has developed strong ties with
motivated producers that are curious about the planned
experiments. For the experiments, the techniques were
based on the use of *M. pygmaeus* host-plants such as the
common marigold. These plants were studied as winter
refuge zones or relay plants.

<figure><img src="image_172.png" /><figcaption>*Macrolophus* larvae and adults on marigolds.
Photo: © SERAIL</figcaption></figure>

The central issue for the MacroPlus project is that, beyond supplying technical references, it is the involvement of producers which will need to be strengthened such that they participate in the development of a biological control approach in which they play an active role rather than just being the clients of a supplier.

Principal results and interest in relation to the Ecophyto plan

Amongst the twenty species tested by the GRAB since 2007, it is the common marigold (*Calendula officinalus*) that has turned out to be the most interesting for encouraging the presence of *Macrolophus*. This hardy plant allows *Macrolophus* to feed, since they can feed off the plant juices via the stems and the pollen in the flowers. *Macrolophus* also finds prey on them. Observations during mid-winter have shown that *Macrolophus* were present and active on marigolds both inside and outside shelter areas and that they were able to reproduce during this, usually unfavourable, period.
The strategy throughout the project was to work on beds of marigolds as winter refuges for these predator plant bugs. Tests established in the fields of growers turned out to be conclusive. Marigolds were planted or sown under plastic sheets, inside unheated plastic greenhouses before the crop plants were ripped up in autumn. These marigolds were then able to serve as a refuge for *Macrolophus* during the winter where they could also reproduce; the marigold were then able to serve as a source of *Macrolophus* the following spring. Crops grown in tunnels bordered with marigold plantations had higher populations of *Macrolophus* and lower populations of pests than those without marigolds. Further assays have proven that ripping up or cutting the marigold plants served as a way to force the *Macrolophus* to transfer over to the crops at the best possible time and thus harmonise *Macrolophus* populations on the crops and strengthen their effectiveness as a biocontrol vector.

<figure><img src="image_173.png" /><figcaption>Cut Marigold stems bearing *Macrolophus* placed near to crops. Photo: © GRAB</figcaption></figure>

Transfer from the *Macrolophus* bearing marigold stems is very simple and can be just as easily done in the same tunnel as in another one. It would be useful to adjust the quantity of cut marigold stems in terms of the actual *Macrolophus* populations on the marigolds. The issue here is that there is no sudden interruption in the provision of a habitat and food to the auxiliaries, either on the marigolds or the crop plants.

When the marigolds cannot be planted or seeded in the ground, they could also be used as relay plants. They could then be sown or planted in pots or planters. These pots can be placed inside the tunnels with the tomato plants when they are ripped up at the end of the season. The *Macrolophus* then find refuge in the marigolds since the tomatoes have dried up and are no longer attractive. These pots are then placed in a tunnel (which does not need to be heated) for the winter where they should be watered sufficiently. The installation of insect-proof nets (or P17 in colder regions) over these pots will confine the *Macrolophus* populations and/or protect the plants and insects from the cold.

In springtime, after checking for the presence of *Macrolophus* and absence of pests (such as *Nesidiocoris*, white fly) on the marigolds, the pots can then be placed as early as possible near to the crops to be protected. The use of pots is also better suited where insecticide treatments are used on winter cultures or solarisation in summer. They can easily be moved from one tunnel to another to allow for crop rotation.

The results obtained in different production contexts (organic or conventional farming, different soil types) are consistent allowing for reliable conclusions. The success of strategies based on those worked on in the *Macrolophus* project in other regions of France further confirms these results. These references were acquired within a cold-frame growing context in the ground.

## Prospects for transfer or research

### Transfer:

Transfer has already begun through the growers' involvement in the project. This involvement was assisted by the close relations maintained by the experimentation stations with the professionals. The implementation of simplified monitoring on the sites of certain growers and the relay provided by the Dephy Fermes networks have amplified the application of the project's conclusions taking them beyond the experimental sites. Right from the start of the project, the care taken in listening to the expectations and constraints of growers ensured that the strategies would be realistic and that the transfer into the field would be rapid.

### Research:

In spite of the declared ambitions, budget limitations imposed certain choices. Winter cultures were not monitored for example, even if *Macrolophus*, still active in the flower beds during this period, could be used to regulate certain other pests (such as aphids on lettuce). The references were acquired within the project in a cold-frame, ground planting context. Hydroponic production constraints will be different. *Macrolophus* is an essential but costly auxiliary for these systems. It would therefore be very interesting to transpose the knowledge acquired for *Macrolophus* into a hydroponic production context. In the same way, working with other host-plant/auxiliary combinations such as *Geranium/Dicyphus* could also strengthen agro-ecosystem stability. The CASDAR ACOR project has taken on these objectives since it launched in 2020.
**Publications and scientific symposia:**

There have been a very large number of communication initiatives. These have been largely aimed at growers and technicians (professional journals, visits, conferences and participation in technical meetings). Some application operations have concentrated on scientific partners (Ecophyto, GTN functional biodiversity, GTN PBI symposia). The project results have also been integrated into functional biodiversity and Organic and Integrated P&D control training programs.

**EDUCATIONAL/FUNCTIONAL ARTICLES:**

* Culture Légumière (n°158 March April 2017): **Favoriser *Macrolophus pygmaeus*, un auxiliaire indigène**

* Biofil (n°111 May June 2017): **Lutte biologique sous abri : optimiser *Macrolophus pygmaeus***

* Serre et plein champ (n°378 June 2017): **Utilisation des soucis comme refuge hivernal pour les *Macrolophus***

* Phytoma n°724 May 2019: **M. pygmaeus, une punaise pleine de ressources**

**TECHNICAL DATA-SHEETS:**

* Technical resources data-sheet September 2018: **Le souci, plante-hôte de *Macrolophus*** 4p.
MDRisque

# Evaluation of the potential for multi-drug resistance in the wheat septoria leaf blotch agent, Zymoseptoria tritici

Launch year: 2016

Completion year: 2019

### Scientific manager

Anne-Sophie Walker, INRAE UMR BIOGER
anne-sophie.walker@inrae.fr

Sabine Fillinger, INRAE UMR BIOGER
sabine.fillinger@inrae.fr

### Partners

INRAE UMR BIOGER; Anses; Arvalis

### Financing

Total cost of the project: € 358,494

Ecophyto grant: € 103,120

## Key words:

Efflux; Adaptation; Fungicides; Resistance mechanisms; Genetics; Hybridisation; Field results; Pest and disease resistance management; Large-scale crops

## Context and main objectives

Wheat septoria leaf blotch caused by Zymoseptoria Tritici is the most common disease affecting wheat in Europe. This disease is largely controlled with fungicides which have the (unintentional) effect of provoking the natural selection of the more resistant individuals within populations. A new mechanism associated with a MultiDrug Resistance (MDR) phenotype was detected in 2007 although its complete genetic architecture remains to be fully elucidated. MDR is provoked by the intense efflux of fungicides with various action methods and which results in a non-specific resistance to a number of active ingredients. As a general mechanism, intense efflux can even have a potential impact on synthetic or natural active ingredients that will be marketed in the near future. The issue at the heart of this project is to promote wide ranging anti-fungal usage practices that would limit the selection of the MDR phenotype in order to maintain their sustainability and avoid unnecessary or useless applications.

In this context the objectives of MDRisques were:

1. Formally demonstrate the involvement of the previously identified MDR mutations in the MDR phenotype

2. Detect and evaluate MDR mutations in populations

3. Evaluate the practical risk of MDR mechanism related resistance in Z. tritici.

## Principal results and interest in relation to the Ecophyto plan

Work completed within the MDRisque project provided a better description of the origin and characteristics of MultiDrug Resistance (MDR) in the major wheat pathogen Z. tritici. We have used a functional genetic approach to confirm that three types of MFS1 promoter inserts are responsible for the intense fungicide efflux mechanism in Z. trittici and, therefore, the MDR phenotype. We have developed a PCR tool that can detect these alleles on isolated strains. A quantification tool based on the amplicon sequencing technique using Nanopore® technology is being used routinely but still requires further work.

Through a Mendelian genetic approach and the phenotyping of descendants from two hybridisations, we were also able to show that the combination of efflux and target modification mechanisms, already common amongst populations, will significantly increase the risk of practical resistance to SDHI and IDM fungicides. This risk of association between specialist and generalist resistance mechanisms is high and realistic in natura due to the frequent and intense sexual reproduction of Z. tritici. Paradoxically, recombination would largely add to the risk associated with the alleles presenting low levels of resistance to SDHI (such as the *sdhC*<sub>T79N</sub> allele, which is constantly increasing its presence in French populations). In fact, if the risk of real-world resistance is low for strains with just this allele or the MFS1 alleles, their combination would lead to resistance levels compatible with a loss of effectiveness in the field. Additionally, no especially high costs associated with each of the five alleles studied were detected in the project, regardless of whether they were carried alone or in combination. This reduces the possibility of a natural decrease in the frequency of these alleles in the absence of natural selection.
Finally, we have shown that MDR was selected preferentially within a network of field assays by SDHI based programs. We have also demonstrated that the introduction of a multi-site such as chlorothalonil, not affected by these resistance mechanisms, into the programmes, especially at T1, tended to reduce the natural selection of MDR.

# Prospects for transfer or research

## Transfer:

This project has allowed professionals to develop their own awareness of the indirect risks associated with generalised resistance mechanisms such as intense efflux. This risk concerns active ingredients currently in use as well as those likely to be marketed in the near future including biocontrol fungicides The identification of practices which favour MDR has strengthened reflections on optimising treatment strategies, especially by limiting the use of SDHI in programmes, and will assist the transition towards other control methods, such as variety selections. MDR is now monitored annually in French populations using the diagnostic tools developed by this project.

## Research:

The MDRisque project has been associated with a 3 year training programme for an apprentice engineer.

This project has demonstrated the use of studying resistance mechanisms that are not related to the target in phytopathology, intense efflux being mainly studied by the medical community. The data obtained will also be used to explore the spatio-temporal adaptation dynamics observed in *Z. tritici*.

Finally, this project has led to the emergence of a thesis project (under way) to classify the distribution of MFS1 alleles in European populations and to elucidate the genetic mechanisms that regulate intense efflux in *Z. tritici*.

# Publications and scientific symposia:

## TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA:

* Walker A-S. (2019). **Research, monitoring and resistance management in France.** In: Annual meeting of the Phytopathological Society of Japan, Tokyo, Japan.

* Walker A-S. (2019). **Assessing the risk of resistance towards SDHIs in France.** In: Plant Health 2019, American Society of Phytopathology, Cleveland, USA.

* Couleaud G., Walker A-S., Maumené C. (2015). **Etude au champ de l'efficacité de la pression sélective exercée par des associations fongicides sur la sensibilité des populations de Zymoseptoria tritici ces dernières années en France.** In: *Proceedings of the "11ème Conférence Internationale sur les Maladies des Plantes"* (ed. AFPP), Tours (France).

* Omrane S., Audéon C., Ignace A., Duplaix C., Sghyer H., Aouini L., Kema G., Fillinger S., Walker A-S. (2015).

* **La résistance de type MDR chez l'agent de la septoriose du blé: dernières avancées sur un mode d'action**

* original. In: "11ème Conférence Internationale sur les Maladies des Plantes", Tours (France).

* Couleaud G., Maumené C., Maufras J-Y., Walker A-S. (2018). **Prévenir la sélection de la résistance MDR chez Zymoseptoria tritici.** In: *Proceedings of the « 12ème conférence internationale sur les maladies des plantes »* (ed. Végéphyl). Tours, 11 and 12 December 2018.

* Gazeau G. and Walker A-S. (2018). **Mécanisme de la résistance multidrogues (MDR) et prévention du risque de résistance aux anti-septoriose chez Zymoseptoria tritici.** In: *Proceedings of the « 12ème conférence internationale sur les maladies des plantes »* (ed. Végéphyl). Tours, 11 and 12 December 2018.

* Omrane S., Audéon C., Ignace A., Duplaix C., Sghyer H., Aouini L., Kema G., Walker A-S., Fillinger S. (2016).

* **Several mutations of Zymoseptoria tritici field strains lead to MFS1 overexpression and multi-drug resistance (MDR) (poster).** In: 18th International Reinhardsbrunn Symposium on Modern Fungicides and Antifungal Compounds, Friedrichroda (Germany).

* Walker A-S., Gazeau G. (2019). **Mechanism of multidrug resistance and risk assessment towards fungicides in Zymoseptoria tritici.** In: 19th International Reinhardsbrunn Symposium on Modern Fungicides and Antifungal Compounds, edited by H. B. Deising, B. Fraaije, A. Mehl, E. C. Oerke, H. Sierotzki and G. Stammler. Frierichroda, Germany: Deutsche Phytomedizinische Gesellschaft, Braunschweig.

* Omrane S., Audéon C., Ignace A., Duplaix C., Sghyer H., Aouini L., Kema G., Walker A-S., Fillinger S. (2015). **Genetic analysis of multi-drug resistance (MDR) in Mycosphaerella graminicola (Zymoseptoria tritici)** In: 28th Fungal Genetics Conference, Asilomar, USA.

* Omrane S., Audéon C., Ignace A., Duplaix C., Sghyer H., Aouini L., Kema G., Walker A-S., Fillinger S. (2015). **Multi-drug resistance (MDR) in septoria leaf blotch (oral pres.).** In: XVIII International Plant Protection Congress, Berlin (Germany).

* Walker A-S., Gazeau G. (2019). **Mechanism of multidrug resistance and risk assessment towards fungicides in Zymoseptoria tritici.** In: *International Symposium on Cereal Leaf Blights, 22-24 May 2019*, Dublin, Ireland.
► Gazeau G., Auclair C., Delestre G., Suffert F., Walker A-S. (2016). **Assessing the risk of recombination between MDR and specific resistance towards SDHIs in Zymoseptoria tritici.** In: *18th International Reinhardsbrunn symposium on modern fungicides and antifungal compounds*, Friedrichroda, Germany.

**SCIENTIFIC PUBLICATIONS:**

► Omrane S., Audéon C., Ignace A., Duplaix C., Aouini L., Kema G., Walker A-S., Fillinger S. (2017). **Plasticity of the MFS1 promoter leads to multidrug resistance in the wheat Pathogen Zymoseptoria tritici. mSphere 2 (5).**

► Garnault M., Duplaix C., Leroux P., Couleaud G., Carpentier F., David O., Walker A-S. (2019). **Spatiotemporal dynamics of fungicide resistance contrast quantitatively in the pathogenic fungus Zymoseptoria tritici. Pest Management Science 75 (7), 1794-1807.**

► Gazeau G., Duplaix C., Walker A-S. (*in prep*) **Assessing the risk of recombination between MDR and specific resistance towards SDHIs in Zymoseptoria tritici.**

**OTHER OPERATIONAL WORK:**

► Shared notes « Gestion de la résistance des maladies des céréales à paille ». https://www.r4p-inra.fr/fr/notes-communes/
<figure><img src="image_174.png" /></figure>

# Potato blight: identification and development of biocontrol products for integrated crop protection

**Launch year:** 2015

**Completion year:** 2018

**Scientific manager**

Marie Turner, VEGENOV

turner@vegenov.com

**Partners**

VEGENOV; INRAE UMR IGEPP; ARVALIS Institut du végétal; Bretagne Plants Innovation

**Financing**

Total cost of the project: € 235,561

Ecophyto grant: € 104,950

**Key words:**

Biocontrol; Blight; Potatoes; Combination of levers; Integrated protection; Variety resistance

## Context and main objectives

Potato blight (caused by phytophthora infestans) is the main disease affecting French potato crops. Conventional chemical treatments are widely used to cope with this pathogen resulting in a very high TFI for this crop (fungicide TFI > 10). At the time that this project was launched no biocontrol solution for this disease was available.

The objective of the MilPomBio project was to identify biocontrol products that could be effective against potato blight and then propose them to growers. These products have been integrated into technical approaches that associate them with the use of other phytosanitary products at lower doses, more resistant varieties and the Mileos DA tool to organise effective treatment campaigns. Under field production conditions, this has reached symptom levels compatible with satisfactory harvests as well as reducing TFI.

<figure><img src="image_175.png" /></figure>

Assays under controlled conditions.

Image: © MMedia

One of the difficulties faced was the transfer of results from controlled conditions to the field, where a significant loss in protection was noted.

## Principal results and interest in relation to the Ecophyto plan

The first phase of the MilPomBio project involved evaluating the effectiveness of around 40 biocontrol products (seaweed extracts, plant extracts, micro-organisms, minerals, organic compounds, phosphites) for the management of potato blight under controlled conditions after artificial inoculation. Under these conditions about 15 of the products were shown to be effective in reducing symptoms.

The most interesting of these products were then selected to:

1) Better understand how they work (natural plant defence stimulation and/or direct effects on the pathogen)

2) Study the effectiveness of these products in the field when they interact with the level of genetic resistance and the plant's structure

3) Evaluate the field protection effectiveness in combination with reduced fungicide doses for one variety.

Field screening was applied with the products whose effectiveness had been proven in the growing chambers. One plant extract based product also contained copper and did provide effective protection. This could not be included in a list of biocontrol products and was excluded from the study. A number of products containing phosphites were also shown to be effective in the field.
Some work was done on the importance of foliar structure in the disease's development as well as the performance of the biocontrol products. The conclusion was that a more open structure did not favour the disease. Over the 4 genotypes studied, the same phosphite-based product provided the same results. The effect of structure was visible but limited; no direct relation between biocontrol product performance and foliar structure was demonstrated.

The final stage of the project involved evaluating, under production conditions, a number of integrated protection strategies combining varieties with variable degrees of resistance, the Mileos DA, a biocontrol product and fungicides likely to maintain the disease at an acceptable level. This work was completed over two different sites with different soil types. A product was also evaluated on about a dozen different varieties to further investigate interactions with the plant's genetics.

In conclusion for the project, the use of a group of levers will reduce the need to use conventional phytosanitary products to treat potato blight. Notably, the results obtained with phosphite-based biocontrol products are encouraging and have lead to a reduction in the use of phytosanitary products of between 40% to more than 90% depending on the variety. These products are effective in protecting against potato blight for all of the varieties tested by the project, especially those with partial or high levels of resistance.

## Prospects for transfer or research

### Transfer:

Promising results will be communicated within the biocontrol consortium, notably the XP-BC potato blight/mildew network, such that assays can be completed on other crops.

Partnerships with businesses developing effective phosphites have been engaged which should lead to the registration of this solution for use on potato crops, or even for other crops.

Work on the evaluation of the active ingredient actually applied has been done. This has been broadened to cover other crops and protocols have been set up to assist producers to get a better understanding of the issue with the logic of encouraging them to reduce and optimise the quantity of products applied.

### Research:

These prospects concern different aspects of the project. A better understanding of how phosphites work would help to optimise their use in the field. Notably, research into the interaction between the plant's genetics and biocontrol products should be continued.

Work to identify other biocontrol products would appear essential such that a wider range can be made available to growers. A better understanding of why the results obtained under controlled conditions do not transfer into the field will also be crucial to the development of biocontrol solutions. Research into the combinations of integrated protection levers should be continued.

### Publications and scientific symposia:

* **Phytoma (n° 721 February 2019):** « Lutte contre le mildiou : allier le biocontrôle à d'autres méthodes (Projet MilPomBio) ». A. MENIL, J-E. CHAUVIN, R. PELLE, M. BOUSSEAU, D. GAUCHER, G. BEAUVALLET, C. MAUMENE, D. ANDRIVON, C. PASCO, F. VAL, A. BARBARY et M. TURNER

* **Potato Planet (n°39, July 2016, p. 82-83):** « Traitements alternatifs. Vegenov teste de nouveaux produits de biocontrôle » - M. TURNER, A. MENIL

### TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA:

* **AFPP Congress « Conférence internationale sur les maladies des plantes ». 10-12/12/2018, Tours: « Mildiou de la pomme de terre : recherche et maîtrise des produits de biocontrôle dans un schéma de protection intégrée des cultures (projet MilPomBio) ». Oral presentation.

* « Natural Product and Biocontrol » Congress. 25-28/09/2018, Perpignan: « Mildiou de la pomme de terre : recherche et maîtrise des produits de biocontrôle dans un schéma de protection intégrée des cultures (projet MilPomBio) ». Oral presentation.
<figure><img src="image_176.png" /></figure>

NABUCO

# New Bio-Marine Agents for Biological Control Use

**Launch year:** 2015

**Completion year:** 2018

**Scientific manager**

Thomas Guillemette, UMR 1345 IRHS University of Angers, thomas.guillemette@univ-angers.fr

**Partners**

UMR 1345 IRHS University of Angers; EA 2160 MMS

University of Nantes; Société AGRAUXINE by Lesaffre

**Financing**

Total cost of the project: € 534,412

Ecophyto grant: € 99,521

## Key words:

Biocontrol; Cryptogamic diseases; Marine fungi; *Trichoderma*; Secondary metabolites; Screening; Antagonism

## Context and main objectives

At the moment, biocontrol agents represent only a very small part of the global phytosanitary market and even that is dominated almost entirely by a narrow range of biological agents and substances. In this context, we are proposing to identify new biological control agents based on marine fungal micro-organisms. Marine environments are filled with an incredible microbial biodiversity which is able to synthesise molecules with diverse biological properties, in terms of plant protection agents we have barely scratched the surface of this biodiversity.

<figure><img src="image_177.png" /></figure>

A marine isolate of *Trichoderma atroviride* MMS1295 (TA) confronts the phytopathogenic agent *Botrytis cinerea* (BC) in a petri dish. The dotted white lines show the front lines of the MMS1295 colony. Photo: © Franck Bastide, IRHS

The identified biocontrol agents are intended for use in protecting against fungal pathogenic agents for which few effective biocontrol agents are currently available and which are responsible for high volumes of phytosanitary product consumptions. The objective is also to combine cultural and molecular tools and implement a method which could later be applied to other bio-resources in order to optimise and accelerate the sorting of strains and molecules for potential use in biological control agents.

## Principal results and interest in relation to the Ecophyto plan

This project has led to a number of advances in the search for new biological protection solutions to deal with fungal diseases affecting plants. Fungi of the genus *Trichoderma* are useful biological control agents since they have an action on phytopathogenic agents either through direct antagonism (parasitism, antibiosis) or indirect antagonism by stimulating the plant's natural defences.

First of all, a strain screening method was developed and applied to more than 50 marine *Trichoderma* and 11 secondary metabolites in order to evaluate the best candidates for use as biological control agents This covered both cultural characteristics (growth rate, spore production), environmental stress tolerance, mycotoxinogenic properties, compatibility with various pesticides, antagonist potential (antibiosis and parasitism) as well as the capacity to stimulate the natural defences of plants.
Various fungal growth monitoring tools (laser nephelometry micro-culture, time-lapse) or plant defence activation monitoring tools (qPFD) were used to optimise the screening process. qPFD remains difficult to apply to a large number of samples due to its relatively high cost. This screening process was useful for its capacity to highlight the major differences between the tested strains and then select the best ones. According to the analysed criteria, 4 marine strains of *Trichoderma* showed cultural potential and antagonistic activity superior to 4 other commercially available strains. This result validates the initial hypothesis that suggested that marine *Trichoderma* could be good candidates for use in plant protection.

At the same time, different secondary metabolites were extracted and purified from a variety of marine *Trichoderma* strains. This work was an opportunity to isolate and purify a series of secondary metabolites which had never before been described for this genus of fungi: peptaibols made up of 15 residues. *In vitro*, these secondary metabolites have demonstrated powerful inhibiting effects on pathogenic agents. These anti-fungal activities depend on the target pathogen and the nature of the secondary metabolites tested. The main difficulty with this action resides in the capacity to extract sufficient quantities of secondary metabolites compatible with the plant application thresholds.

<figure><img src="image_178.png" /><figcaption>*Trichoderma atroviride* filament wrapping and parasiting filaments of the phytopathogenic agent *Botrytis cinerea*. Scale: 10 µm. FBC: filament of *B. cinerea*; FTA: filaments of *T. atroviride*; SBC: spores of *Botrytis cinerea*.
Photo: © Franck Bastide, IRHS</figcaption></figure>

Finally, certain candidates were tested for their capacity to reduce apple scab disease and wheat septoria leaf blotch. When used as preventive treatment on the above ground parts of the plants without any wetting agent or additive, a secondary metabolite and the strain that produced it will achieve 24% level protection against wheat septoria leaf blotch. Under the same conditions, similar protection levels were achieved against apple scab disease after application of the same secondary metabolite with a slightly higher result (34%) after application of the strain itself.

The project's private sector partner is currently leading work to define the exact ecological/toxicological risks associated with the selected candidates (secondary metabolites and/or the *Trichoderma* that produce them), continue the research and development assays, evaluate the costs of applying for the necessary commercial registration and then finally to assess industrial and financial feasibility.

## Prospects for transfer or research

### Transfer:

This project must lead to the identification of new biological protection solutions to combat pathogenic agents that attack crops and lead to the intensive use of pesticides for which no alternative non-chemical solutions are currently available. Agrauxine by Lesaffre is continuing the work to define the exact ecological/toxicological risks associated with the selected candidates (secondary metabolites and/or the *Trichoderma* that produce them), continue the research and development assays, evaluate the costs of applying for the necessary commercial registration and then finally to assess industrial and financial feasibility.
**Research:**

Our results demonstrate that within the same species of *Trichoderma*, individual strains are far from presenting the same antagonistic performance and their parasitic capacities can vary significantly. In order to increase knowledge of these infectious processes and optimise the effectiveness of these biocontrol agents, it would appear essential to take a closer look at the fungal mechanisms that would explain variations in intra-species myco-parasitism. This type of approach should allow us to identify the molecular myco-parasitism aptitude markers that could be deployed for a more rapid screening of *Trichoderma* strains.

**Publications and scientific symposia:**

**TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA**

► **3<sup>rd</sup> International AFERP STOLON Symposium. 18-20/07/2018, Rennes, France:** *The NABUCO project: a search for new marine biocontrol agents against plant diseases*. Ruiz et al.

► **12<sup>ème</sup> Rencontres de Phytopathologie-Mycologie, Société Française de Phytopathologie. 15-19/01/2018, Aussois, France:** *Study of new fungal biocontrol isolates and their secondary metabolites against plant diseases*. Bastide et al.

► **1<sup>st</sup> International Symposium on plant bioprotection sciences and technologies. Reims, France 27-30/06/2017:** *Screening strategy to select new fungal biocontrol products against plant diseases*. Bastide et al.

**SCIENTIFIC PUBLICATIONS**

**Pentadecaibins I-V:** 15- Residue Peptaibols Produced by a Marine-Derived Trichoderma sp. of the Harzianum Clade. Van Bohemen Al, Ruiz N, Zalouk-Vergnoux A, Michaud A, Robiou du Pont T, Druzhinina I, Atanasova L, Prado S, Bodo B, Meslet-Cladière L, Cochereau B, Bastide F, Maslard C, Marchi M, Guillemette T, Pouchus YF. 2021. In press.
<figure><img src="image_179.png" /></figure>

OPTIM'PHERO

# Optimising Pheromones and Transposing results obtained with the pine processionary to other insects, pest lepidoptera in Non-Agricultural Zones
## Innovative biocontrol solutions for other lepidoptera.

**Launch year:** 2015

**Completion year:** 2018

**Scientific manager**

Jean-Claude MARTIN, INRAE UEFM

Jean-claude.martin@inrae.fr

**Partners**

INRAE UEFM; FREDON PACA; M2I Life Sciences

**Financing**

Total cost of the project: € 271,199

Ecophyto grant: € 98,393

**Key words:**

Biocontrol; Ecophyto; JEVI; Sexual confusion; PBI

## Context and main objectives

The OPTIM'PHERO project has its origins in the realisation that something was lacking in the alternative control strategies for pest lepidoptera in JEVI (Gardens, Parks and Infrastructures or non-agricultural zones). Four teams have combined forces and their multi-discipline skill base, covering fine chemicals, innovative patents and applied entomology. The objective of this project was to propose innovative biocontrol solutions for 3 pest insects: the pine and oak tree processionaries and box tree moth which all represent major threats. OPTIM'PHERO aimed to extend the range of available biocontrol products through new technology developed with their French partner M2i Life Sciences which proposes the controlled release of pheromone substances and innovative sexual confusion application methods suited to specific plant configurations. A biodegradable support for the pheromone (no diffuser, no system dismantling) would reduce costs whilst also optimising efficiency thanks to better suited application methods designed specifically for each insect and situation. A number of original application methods were tested over large surface areas including the development of paintball ammunition filled with micro-encapsulated pheromones or the application of a pheromonal gel directly onto the plants. These solutions represent interesting alternatives to the ground-level (aerial

treatment being forbidden) treatment methods currently used to control pine processionary, such as pick-up mounted turbines or lances. These latter are used to spray to great heights and are not at all ecologically friendly due to the vast quantities of product that falls back to the ground.

Targeted application of micro-encapsulated pheromone responds to a high demand for solutions to deal with pests that depend on trees (Pheroball) as well as direct manual methods applied to the shrub layer (micro-encapsulated pheromone gel).

## Principal results and interest in relation to the Ecophyto plan

### Pine Pheroball

Three years of experimental and optimising work on the Pine Pheroball, a product that results directly from the project, has demonstrated an average reduction, through sexual confusion, of processionary populations of between 29% (300 balls/ha, over a 4 ha field) and 58% (30 balls per pine tree, isolated trees in urban areas), depending on the tested dose. The study was completed in wooded and urban zones.

<figure><img src="image_180.png" /></figure>

Photo: © M2i LIFE SCIENCES
PHEROBALL PIN - Ball filled with micro-encapsulated pheromone for application by Paintball gun
This study raised a number of limits associated with this control strategy:

1) **The effectiveness** of the doses tested in 2017, between 300 and 400 balls per hectare over large wooded areas (4 ha), corresponds to a population reduction between 29 and 36% whilst the tests were conducted on low population levels of pine processionary, a situation which is more favourable for a sexual confusion control strategy.

2) In terms of the **ball application time** (6 to 8 hectares per day for 2 operatives) and frequent gun cleaning. In forests, the precise ball application route following a 10m x 10m grid to apply is very time consuming which increases the overall cost of this control strategy. However the cost and ease of use remains attractive when compared to traps or BT.

3) In terms of **cost**: Pine Pheroball requires further experimental work to evaluate the effectiveness of higher doses.

## Pheromone gel

Tests of targeted applications of biodegradable pheromone gel were completed on pine processionary and box tree moth as a control strategy using sexual confusion. Results were encouraging for pine processionary with a more than 87% descent reduction; the pheromone gel was applied onto the crowns of the trees. This was done with a single testing dose; the experiment needs to be repeated to validate the result. For box tree moth, a population reduction of 25%, compared to the control, may well be significant but does not reduce foliar damage. Further work is required to establish the correct dose.

On oak processionary, the complexity involved in synthesising the pheromone was a genuine technical obstacle throughout the 3 test seasons.

<figure><img src="image_181.png" /><figcaption>Firing PINE PHEROBALL In urban zones
(Avignon 2017)</figcaption></figure>

Photo: © INRAE; M2i LIFE SCIENCES

## Prospects for transfer or research

### Transfer:

By the end of the project, M2i obtained a Biocide AMM for pine tree confusion solutions in France, by derogation for 2017 and then permanent as of 2018 followed by the same for Spain, Portugal and Algeria. This sexual confusion pheromone application method has been available in agriculture since a paintball product for walnut trees has just been authorised for the French market. OPTIM'PHERO has also contributed to the development of a sexual confusion product for box tree moth, this is now authorised for sale in France with a biocontrol AMM since 2019 and the same for Benelux, Italy, Spain, Portugal and the United Kingdom.

UEFM will continue to develop the AGIIR (Alerter et Gérer les Insectes Invasifs et/ou Ravageurs - Alert and Manage Invasive Insects and/or Pests) smartphone application as a control support tool for the project's 3 target pests. This application can be downloaded free for Android and IOS. The publication of the technical guide and real-time updates will continue.

### Research:

**Pine Pheroball in forests:** the effectiveness of Pine Pheroball has been demonstrated by the programme but its use in forests (large densely wooded areas) remains to be proven. M2i will continue forest application protocol optimisation experiments after the end of the project until authorisation for sale is awarded.

**Pheromone gel and effectiveness:** M2i has developed a new application protocol for box tree moth based on the project work which increases dosage and the number of treatments and includes the combination of a larvicide to develop a fully effective solution.

The solution's conclusive results on pine processionary open up prospects to extend application to other pest lepidoptera that affect JEVI or even agricultural zones thanks to its innovative pheromone application method.

### Sexual confusion with oak processionary

M2i will be continuing its research since sexual confusion assays will be launched in cooperation with Cardiff Univeristy, Great Britain for 2022. The INRAE has continued its research work on this pest through other projects and has yet to identify a pheromone that is suitably effective for monitoring, or other methods involving sexual pheromones (low or non-existent capture rates for pheromone comparisons).
**Publications and scientific symposia:**

**TECHNICAL DOCUMENTS:**

► Brinquin A.S. and Martin J-C., 2017: **Les clés pour lutter contre la processionnaire du pin.**

https://www6.paca.inra.fr/entomologie_foret_med/Insectes-ravageurs-et-protection-durable/Aide-a-la-decision

**TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA:**

► Technical workshops organised by the UEFM and its institutional partners to train managers on new alternative pest control techniques.

► Box tree bioaggressor symposium Tours 16 & 17 October 2018 organised by Végéphyl

https://www.academie-agriculture.fr/sites/default/files/agenda/programmebuisenfrancaisv17-09.pdf

**PRACTICAL / EDUCATIONAL ARTICLES:**

► E. Pal, M. Corréard, F. Rei, J. Thévenet, S. Lantus, E. Pezzini, M. Buradino, A-S Brinquin and J-C. MARTIN (2018). **Processionnaire du pin : un traitement bille en tête.** Phytoma 715, 28-32.

► E. Pal, M. Correard, M. Buradino, E.Morel, S. Touzeau, J.Thévenet, D. Vauthier, O.Gilg and J-C. Martin (2017).

**Processionnaire du pin : le piégeage se perfectionne.** Phytoma 709, 27-29.

► J-C. Martin, M. Buradino, A-S. Brinquin, M. Correard, J. Thévenet, D. Vauthier, E. Morel, A. Gilli, M. Vénard et E. Tabone (2018). **Réguler la pyrale du buis Cydalima perspectalis : limites d'utilisation de la phéromone sexuelle de synthèse.**
<figure><img src="image_182.png" /></figure>

PRO-BIO-TAUPIN

# Evaluation of biocontrol solutions for protecting crops from the damage caused by wireworms

**Launch year:** 2016

**Completion year:** 2019

**Scientific manager**

Jean-Baptiste Thibord, ARVALIS

jb.thibord@arvalis.fr

**Partners**

**ARVALIS:** UMR DGIMI INRAE - University of Montpellier; ACTA; CTIFL; Invenio; ACPEL; APREL; SudExpé; Sonito

**Financing**

Total cost of the project: € 210,807

Ecophyto grant: € 113,891

**Key words:**

Wireworm; Biocontrol; Entomopathogen nematodes; Entomopathogen fungi; natural substances

## Context and main objectives

Protection against wireworms is often essential to maintaining large-scale and vegetable crop harvests and/or quality standards. For a number of years growers have seen the number of phytopharmaceutical solutions become increasingly rare, with those that remain on the market often limited or insufficient in terms of effectiveness. It has therefore become necessary, even urgent, to develop protection strategies integrating biocontrol solutions either as a substitution to phytopharmaceutical solutions for low wireworm population situations, or in addition to conventional solutions to limit the damage caused by the pest in high population situations.

The objective of the PRO-BIO-TAUPIN project was to explore the technical interest of various potential biocontrol solutions;

* Entomopathogen nematodes;

* An entomopathogen fungus, more specifically a product based on *Metarhizium anisopliae*.

* Natural substances, essentially glucosinolate based products.

The assays were completed under controlled conditions (entomopathogen nematodes) and field conditions (entomopathogen fungus, natural substances) to protect various crops: potato, carrot, melon, asparagus, tomato and corn.

## Principal results and interest in relation to the Ecophyto plan

Implementation work on entomopathogen nematodes (EPNs) has highlighted the fact that the insect's physical barriers (particularly its highly resistant cuticle) clearly restrict the entrance of nematodes into the larvae thus explaining the weak performance of these biocontrol agents on wireworm (see image on next page). This key stage of the nematode's parasitic cycle is the main obstacle to be dealt with before considering any field application of EPNs to control wireworm. The direct injection of EPN symbiotic partner bacteria into the insects' haemolymphes has been able to show that it could be interesting to use a high performance bacteria strain to kill wireworm larvae: X. *kozodoii* FR48. To our knowledge, this is the first time that an EPN symbiotic bacterium is pathogenic in isolation to wireworm larvae. Bio-assays involving injections have also shown that other species of the X. *kozodoii* species were conversely quite ineffective on wireworm larvae. This result means that it would be interesting to launch genome studies comparing the virulent strain, X. *kozodoii* FR48 and the other non-virulent strains of X. *kozodoii* to identify any genes or genome regions involved in the pathogenic property of X. *kozodoii* FR48 on wireworms. Complete genomes of the pathogenic X. *kozodoii* FR48 strain and the non-virulent FR71 and FR74 strains have been sequenced.
<figure><img src="image_183.png" /></figure>

Entomopathogen nematode mortality tests associating different bacteria on wireworm larvae. The histograms show the average percentage of wireworm larvae mortality (3 replicas) 21 days after infection with strains of *S. boemarii* and *S. carpocapsae*. N = 30 wireworm larvae; infestation dose = 500 EPNs per larva; negative control = Ringer liquid; positive control = SK27 T2. Image: © INRAE

<figure><img src="image_184.png" /></figure>

The study of entomopathogen fungi (axis 2) was concentrated on the MET 52 granule commercial speciality, based on Metarizhium anisopliae F52. Plant protection assays involving wireworm larvae produced very encouraging results with, in some situations, a degree of effectiveness comparable to those achieved with conventional products (50% effectiveness).

However protection level variations, the high cost of the currently available commercial product and supply chain problems mean that this solution cannot be used as a year round crop protection solution. Initial works were implemented as part of a strategy to lower wireworm populations through repeated introductions into the fields. Whilst the initial results were encouraging, further work needs to be done to confirm the interest of this control method.

Evaluation of MET 52 GR applied and incorporated into the ground before sowing corn. 10 assay summary (2012-2017) - Image: © ARVALIS

A number of liquid formulations involving glucosinolates are available to growers. Having been unable to test them all, those that were the subject of experiments organised by the project's partners (axis 3) did not produce particularly interesting results in spite of the diversity of application methods tested. However a formulation of Abyssinia mustard seed, *Brassica carinata*, press cakes as sold by Biofence, could be a solution to reduce wireworm attacks on corn and potato crops. This seems to be less effective on other crops but might be useful against other under-soil pests - such as symphyman (on asparagus) and cyst nematodes (on potatoes). However the required doses could imply a lack of selectivity under certain circumstances. With the Biofence product being very expensive, doses were reduced and locally targeted. The results on corn crops were interesting but such experiments would need to be taken further to prove the effectiveness of the techniques in the event of severe attacks. At the same time, a number of assays were completed to evaluate the effectiveness of mustard bran which is authorised as a base substance and can be used as a substitution product. Whilst the results were not very encouraging, improvements could be made by improving the chemical composition of the bran used (see image on next page).
<figure><img src="image_185.png" /></figure>

Wireworm attack intensity on asparagus leading to rejected harvests.

Image: © INVENIO

## Prospects for transfer or research

### Transfer:

Even if none of the solutions evaluated by the PRO-BIO-TAUPIN project turned out to be satisfactory for crop protection under production conditions (especially for financial reasons), the results have been presented to growers in the various crop industries. Additionally, these results have enabled the launch of further research work programmes. The work on wireworm pathogen bacteria will be continued by UMR DGIMI. New work on entomopathogen fungi has been launched as part of a collaboration with the University of 'Pau et des Pays de l'Adour'. Finally, further works are planned on the production of glucosinolates in the field via service plants.

### Research:

The results obtained via the first axis of the PRO-BIO-TAUPIN project are very promising thanks to the identification of a pathogen bacterium that is effective against wireworm larvae. However it will be difficult to implement this strategy in the field for the moment considering that the *Xenorhabdus* entomopathogen bacteria cannot survive without its host nematode. One possibility would be to identify the pertinent toxins present in these bacteria.

The technical interest of *Metarizhizium anisopliae* and certain press cakes containing glucosinolates has been confirmed. However, it will be necessary to analyse the active ingredients that affect wireworm larvae in order to develop knowledge on the most effective strains and substances and define the conditions which would favour their presence in the field. This would allow for an optimisation of doses and render these solutions compatible with the budgetary constraints involved in producing these crops.

## Publications and scientific symposia:

### SCIENTIFIC SYMPOSIA:

* **International Congress Natural Products and Biocontrol (Perpignan, France. 25-28/09/2018): Évaluation des nématodes entomopathogènes et leurs bactéries symbiotiques pour lutter contre les taupins (poster présenté).** Frayssinet Marie, Pagès Sylvie, Lawac Floriane, Duvic Bernard, Givaudan Alain and Ogier Jean-Claude

* **11ème Conférence Internationale sur les Ravageurs et Auxiliaires en Agriculture, Montpellier 25-26/10/2017: Intérêt de l'utilisation de Metarhizium brunneum pour lutter contre les taupins (Agriotes sp.) en grandes cultures.** Larroudé Ph. and Thibord J.B. – 2017

### SCIENTIFIC PUBLICATIONS:

* **M1 placement report (IMHE master, University of Montpellier): Étude des facteurs de virulence de la bactérie entomopathogène Xenorhabdus kozodoii vis-à-vis du ravageur taupin (Agriotes sordidus).** Océane Bueno, 2018.

* **M1 placement report (Master in Agronomics and Food industry, SEPMET, SupAgro Montpellier): Évaluation de la virulence des complexes némato-bactériens entomopathogènes contre les taupins.** Floriane Lawac, 2017.
# Designing irrigation and fertilisation strategies for controlling green aphid populations in peach tree orchards?

Launch year: 2015

Completion year: 2019

## Scientific manager

Marie-Odile Jordan, INRAE PSH

marie-odile.jordan@inrae.fr

## Financing

Total cost of the project: € 248,235

Ecophyto grant: € 90,000

## Key words:

Alternative elementary practices; Technical approach changes; Irrigation; Fertilisation; Orchards; *Prunus persica*; *Myzus percicae*; Branch composition; Branch development

## Context and main objectives

"Bottom-up" methods, involving controlling pests and disease by modifying the host-plant's vulnerability, are not very well known. This vulnerability is related to the dynamic equilibrium between plant development and structure; it can be optimised through various techniques, such as irrigation and fertilisation. However the effects of these techniques will vary depending on the plant's initial condition (size, shape and structure). The implementation of these techniques will therefore have to involve an assessment of the variability of their effects in terms of the trophic and hydric states of the plants. This is the main subject of this project which concentrates on the possibility of adjusting irrigation and nitrate fertilisation practices to control green aphid infestations on peach tree orchards.

This objective involves:

1) Identifying the specific operational characteristics of the plant to which aphids are attracted

2) Identify the effects of irrigation and fertilisation practices on these key plant-pest/disease interactions and on fruit production

3) Select the most adequate seeming practices from these combinations

This is the approach we have adopted, and which we will combine with experimental and modelling techniques.

<figure><img src="image_186.png" /></figure>

Damage to fruit in production
orchards.

Photo: © Alice Diot, INRAE

## Principal results and interest in relation to the Ecophyto plan

The project was divided into three phases:

1) Experiments under controlled conditions to identify the main determining factors for plant vulnerability

2) Multi-criteria and multi-year (2 year) validation of results in commercial orchards that are subject to severe natural infestations.

3) Modelling work: development and simulation of the long-term effects of the tested practices.

For container planted trees, infestation severity is positively related to branch development (leaves, secondary structures) and the non-structural amino acid and carbon content in the apical buds, and negatively related to polyphenol concentrations. These relations depend on the variety's hydric state.

In orchards, the trees that have been subjected to a Nitrate limitation were able to maintain their development and production potential during the first year by drawing on their own reserves. However they were infested later on, but less severely. During the second year, infestations were less severe on the least vigorous branches and those trees which had been limited in their Nitrate uptake. Again, these practices had no effect on production.
Reducing Nitrate fertilisation therefore reduces the prevalence of infestations, but the frequency of periods where the tree is fertilised correctly such that it can reconstitute its reserves has yet to be defined. The impact of irrigation depends on the environment surrounding the orchard: the apparition of hydric stress will be later in relation to winter precipitation quantities. However this is only effective if it is applied (even temporarily) at the start of the infestation and not around the peak.

Simulations using "simple models" show that growth is more sensitive to fertilisation than to the presence of aphids. Production is only affected during the second year: the decrease in the growth of shoots related to severe infestation will reduce the number of fruit for year (n+1) since these are produced on year old wood. This drop in production stabilised after 4 years.

RegPuc has facilitated the integration of QualiTree (a function structure model), a module which formalises the absorption of nitrates from the soil, its distribution amongst organs and the effects on development and growth. This was a preparative for the inclusion of the "aphid model" which is currently being developed.

<figure><img src="image_187.png" /><figcaption>Figure 1: Tree-aphid interaction structure. Impact of development variables (in pale grey) and those relating to structure (in pale green) on aphid populations, shown in orange. Positive if marked with + and negative is marked with O. The interdependence of "plant" variables" is indicated by arrows, black where the result comes from the work presented here and pale grey if it is drawn from previous work (Jordan et al. 2011). Details of the effects of irrigation and fertilisation on plant variables are shown on the left side of the graphic.
Image: © Marie-Odile Jordan, INRAE</figcaption></figure>

Our results confirm that fertilisation and irrigation practices can be adapted to meet a twofold objective: maximised production and aphid control. However further studies will have to establish any long-term consequences (> 2 years) on the durability and productivity of orchards. Additionally, other practices such as hedge planting, ground cover plant selection could also be applied at the same time to increase performance, notably by encouraging predators. Consequently growers will have to have access to a set of methods to be applied depending on the specifics of each orchard. These can be more or less vulnerable to aphids depending on their surroundings which will determine the number of fundatrices, and the condition of the tree on which the future of the first colonies will depend. However, no existing alternative control approach targets all of these aspects. They should therefore be combined to reach the same degree of effectiveness as pesticides. The effects of these method combinations have not been studied to any great extent so far.

## Prospects for transfer or research

### Transfer:

Since one of the partners is an association of tree farmers, the transfer has occurred in "real-time": project progress presentation to growers from the network during themed workshops, (assay) site visits and publications in the network's news-sheet. Finally, once sanitary conditions allow, the presentation of the final results will be organised in the form of a summary presentation to growers in the network and the technical coordination units of the PACA and Languedoc-Roussillon Regions. Finally a shorter presentation will be published in "La France Agricole" as part of a collaboration between the magazine and the GRCETA.
## Research:

The results of RegPuc have enabled the INRAE partner (PSH unit) to participate in APITree, a European C-IPM project investigating the aphid's choices in terms of host-tree when returning in autumn. This project was an opportunity to initiate collaboration with the G. Alins (IRTA Lleida ESP) and L. Sigsgaard (University of Copenhagen, Sweden) teams.

Additionally, INRAE PSH is currently coordinating a PPR project: CAP Zéro Phyto, for which it is implementing a long-term experimentation programme (4 years) in peach-tree orchards concerning the combined use of different levers (fertilisation, irrigation, ground-cover and service plants, mechanical stress, etc.) on aphid-plant interactions.

## Publications and scientific symposia:

### Scientific publications:

* **Ecological Modelling (n° 338, pp 11-16): The consequences of aphid infestation on fruit production become evident in a multi-year perspective: Insights from a virtual experiment.** Bevacqua D. Grechi I. Génard M. Lescourret F. (2016)

* **Agronomy for Sustainable Development (n° 38, pp 1-17): Harnessing the aphid lifecycle to reduce insecticide reliance in apple and peach orchards: A review.** Rousselin A. Bevacqua D. Sauge M-H. Lescourret F. Moody K. Jordan M-O. (2017)

* **Pest Management Science (n° 76, pp 797-806): Chemical and growth traits of the peach tree may induce higher infestation rates of the green peach aphid, Myzus persicae (Sultzer).** Jordan M-O., Sauge M-H., Vercambre G. (2020)

* **Journal of the royal society Interface (n°17: 20200356): An eco-physiological model of plant-pest interactions: the role of nutrient and water availability.** Zaffaroni M., Cunniffe N. J., Bevacqua D (2020)

### Scientific symposia:

* **International Crop Modelling Symposium: Crop Modelling for Agriculture and Food Security under Global Change (15-17/03/2016. Berlin, Germany): Consequences of aphid infestation over fruit tree production: the peach tree-green aphid case.** Bevacqua D., Grechi I., Genard M., Lescourret F.

* **9th international conference on integrated protection of fruit crops, 4-8/09/2016, Tessaloniki (Greece). IOBC WPRS Bulletin, (n° 123, pp 79-85): Combining irrigation, fertilization and pruning techniques helps control aphid populations in apple and peach orchards.** Jordan M-O. Rousselin A. Vercambre G. Sauge M-H.

* **IIIrd International Symposium on Horticulture in Europe – SHE2016 (17-20/10/2016, Chaina, Greece): Modelling fruit plant-pest interactions, their control by cultivation practices and their link to fruit quality.** Lescourret F. Gibert C. Grechi I. Génard M. Bevacqua D.

* **BAPOA (Biologie Adaptative des Pucerons et Organismes Associés) network workshops, (19-20/10/2017, Colmar, France): Variables écophysiologiques (croissance, architecture, composition biochimique) affectant l'attractivité du pêcher pour le puceron vert.** Jordan M-O. Rousselin A. Sauge M-H.

* **10th International Conference on Dynamical Systems Applied to Biology and Natural Sciences (DSABNS) (03-06/02/2019, Napoli, Italy): A general eco-physiological framework to model the interactions between phloem feeder pests and plants.** Zaffaroni M., Bevacqua D.

* **XXIX Congresso SttE. (10-12/09/2019, Ferrara Italy): An eco-physiological model coupling plant growth and aphid population dynamics.** Bevacqua D., Cunniffe N., Zaffaroni M.

* **14th International Symposium « Ecology of Aphidophaga » (16-20/09/2019, Montreal, Canada): Limiting irrigation and fertilisation enables to limit the aphid populations in commercial apple (Malus domestica) and peach (Prunus persica) orchards.** Jordan M-O., Drevet Giraud A., Vercambre G., Hucbourg B.

* **The International Society for Ecological Modelling Global Conference (ISEM) (01-05/10/2019, Salzburg, Austria): An agro-ecological model coupling plant growth and pest population: highlights on the role of fertilization and irrigation.** Zaffaroni M., Bevacqua D.

### Operational / educational articles:

* **Info CETA (n°5, May 2017): Pêcher : quelles stratégies d'irrigation et de fertilisation pour réguler les populations de Myzus persicae.** Drevet Giraud A.

### Technical workshops - GR-CETA Growers network:

* **Participation in 11 technical workshops (2015-2019): presentation of assay and results.**

### Communication during technical coordination operations:

* **16-18/10/2019: "Rencontre CETA des techniciens" (involving CETA South-East region Technicians association members): results presentation**
<figure><img src="image_188.png" /></figure>

# RESistance of agricultural landSCAPES to pesticide transfers into soil and living organisms

**Launch year:** 2015

**Completion year:** 2019

**Scientific manager**

Céline Pelosi, UMR EMMAH INRAE - Avignon
University

celine.pelosi@inrae.fr

Clémentine Fritsch, UMR Chrono-environnement
CNRS - University of Bourgogne Franche-Comté -
Usc INRAE

clementine.fritsch@univ-fcomte.fr

Colette Bertrand, UMR EcoSys INRAE -
AgroParisTech

colette.bertrand@inrae.fr

**Partners**

UMR EMMAH INRAE - Avignon University; UMR Chrono-environnement CNRS - University of Bourgogne Franche-Comté - Usc INRAE ; UMR EcoSys INRAE - AgroParisTech; Luxembourg Institute of Health - Dpt of Population Health; INSA Centre Val de Loire; UMR 7372 CEBC - CNRS - University of La Rochelle; Laboratoire d'Analyse des Sols d'Arras; UMR 5280 Institut des Sciences analytiques INRAE - CNRS - University of Lyon

**Financing**

Total cost of the project: € 721,208

Ecophyto grant: € 111,540

**Key words:**

Pesticides; Transfers; Contamination of agricultural environments; Exposure; Functional biodiversity;
Earthworms; Ground beetles; Micro-mammals; Undesired effects; Landscape approach

**Context and main objectives**

Agricultural landscapes are made up of a patchwork of cultivated fields and semi-natural features (hedges, woods, prairies, etc.), which can be reservoirs of beneficial organism and pests but alsos sources and wells of pesticides (used on the crops and potentially transferred outside of the fields). The proportion and arrangement of these various agricultural and non-agricultural features can influence how landscapes resist pesticide transfer into the soil and living organisms.

The RESCAPE project fits into the landscape ecotoxicology framework, and aims to increase knowledge of the relations between phytosanitary practices, environmental contamination and biodiversity. The objective of the project was to identify the effects of land use (landscape composition and configuration) and agricultural management (cultural practices) on the spatial distribution of pesticides, the exposure of non-target organisms and impacts on them.

**Principal results and interest in relation to the Ecophyto plan**

The RESCAPE project has enabled the development of multi-residue analysis methods to measure soil and non-target organism exposure to pesticides, these revealed three major results:

1) The exposure of soils and living organisms is ubiquitous. We have found pesticide residues in all types of habitats (conventional and organic [AB] agriculture cereal fields, prairies and hedges). Pesticide concentrations measured in 180 soil samples demonstrated a high risk of negative effects on earthworm reproduction in 46% of cases, this applied equally to both cereal crop treated with insecticides and the untreated zones which are very important to biodiversity.

2) Exposure to cocktails of molecules involving at least one insecticide (imidaclopride), one fungicide (epoxiconazole) and one herbicide (most frequently diflufenican).

3) For both soils and non-target organisms, the pesticide treated zones were the most contaminated. AB fields and semi-natural habitats (such as prairies and hedges) often present lower concentrations and numbers of molecules, and could be used as refuges for organisms within the agricultural landscapes.
Additionally, a modelling approach has revealed that the predominant factors controlling the atmospheric diffusion of pesticides and their later entry into non-target ecosystems were (i) the positions of hedges in relation to the treated field, (ii) the width of the hedges, (iii) the physical-chemical properties of the pesticides and the application season. In this way, the presence of certain habitats (hedges, prairies) as well as an increase in the surface areas of untreated habitats (AB fields, wooded features) play a positive role in limiting soil and non-target living organism exposure to pesticides.

This project raised a number of questions on the following points:

- The pertinence and effectiveness of current assessment methods, currently in use for the environment and biodiversity, concerning the risks associated with pesticides (PEC and toxic risk measurements) and the innocuousness of authorised pesticides.

- The need to proceed with pesticide residue analyses for soils and non-target living organisms within the framework of post molecule registration monitoring.

Regarding the role of landscape context in limiting the unwanted effects of pesticides, the project results indicate:

- That local and agricultural landscape scale pesticide use should be limited (increase untreated zones via the installation of agro-ecological infrastructures).

- Consider the surface area and size of features (such as hedges) and their position within the landscape to ensure the effectiveness of agro-ecological infrastructures.

However, in order to be able to propose pertinent landscape management recommendations that will result in landscape feature arrangement optimisation propositions able to limit the unwanted negative effects of pesticides and favour the kind of agricultural biodiversity that would be a benefit to agro-ecosystems, it would seem necessary to consolidate these initial results by broadening the current data set (e.g.: over more years, more geographical zones and agro-soil-climate contexts). Additionally, it will be essential to work on a better evaluation of the eco-toxicological effects caused by the exposure of living organisms to the pesticides cocktails found in natura.

## Prospects for transfer or research

### Transfer:

The produced data could be useful to organisations in charge of pharmacovigilence (ANSES) as well as public decision-makers; it would contribute to national expertise on the molecules currently "in play". The project results could also be used to contribute to establishing recommendations for limiting the unwanted effects of pesticides and encourage a beneficial and useful biodiversity within agro-ecosystems (e.g.: Increase the presence of agro-ecological infrastructures in the landscape; reduce the field and landscape scale use of pesticides by encouraging the presence of untreated zones within the landscape patchwork).

### Research:

New research programmes will be necessary in order to propose management policies that would be pertinent and applicable for the whole of the country:

- Evaluate the temporal and geographical variability of pesticide contamination.

- Study other production systems (polyculture/livestock, vineyards) to evaluate the possibility of transposing recommendations.

- Consider other types of landscape configurations that could restrict unwanted side-effects.

- Improve the understanding of ecological and eco-toxicological mechanisms at work (e.g.: impacts on trophic chains and public health).

## Publications and scientific symposia:

### Technical workshops:

- Scientific workshops for the Sèvre Valley and plain workshop zone, 17/06/2016. RESCAPE. C. Pelosi & C. Fritsch.

- MP ECOSERV BIOSERV, MP SMACH ACE, MP SMACH ESPACE & MP SMACH PING (PSPE RESCAPE) projects presentation day 2018:

- Approches spatiales en écotoxicologie du paysage sur la distribution spatiale de polluants dans les sols et les transferts à la faune. C. Fritsch.

- C. Bertrand. Réponse des carabes aux facteurs locaux et paysagers.

- INRAE SMaCH / ACTA ITA day, 20 December 2018. PING and RESCAPE projects. C. Fritsch. & C. Bertrand.

### Scientific symposia:

- French Pesticide Research Group Congress, Nancy, 2017. Gaëlle D., Lafay F., Pelosi C., Fritsch C., Bretagnolle V., Vulliet E. **Développement d'une méthode multi-résidus par LC-MS/MS pour la quantification de traces de pesticides dans les vers de terre.**
► SFECOLOGIE symposium, Rennes, 2018 :

- Colette Bertrand, Pierre Zagatti, Sébastien Bonthoux, Gaëlle Daniele, Florent Lafay, Emmanuelle Vulliet, Vincent Bretagnolle, Clémentine Fritsch, Céline Pelosi. **Assessing the impact of farming practices and landscape heterogeneity on ground beetles' exposure to pesticides.**

- S. Bonthoux, C. Fritsch, C. Bertrand, P. Zagatti, V. Bretagnolle, M. Coeurdassier, G. Danielef F. Lafay, F. Raoul, R. Scheifler, E. Vulliet & C. Pelosi. **Disentangling the effects of pesticides, soil characteristics and landscape features on earthworms, carabids and small mammals.**

- French Pesticide Research Group Congress, Montpelier, 2019:

- Bertrand C., Daniele G., Lafay F., Vulliet E., Bretagnolle V., Zagatti P., Fritsch C., Pelosi C. **Exposition des sols agricoles et d'organismes non-cibles aux pesticides : quelle influence de la mosaïque paysagère ?** (Poster)

- Fritsch C., Coeurdassier M., Raoul F., Scheifler R., Burkart L., Hardy E., Palazzi P., Schaeffer C., Bretagnolle V., Bertrand C., Appenzeller B., Pelosi C. **Exposition des micromammifères aux pesticides actuellement utilisés : différences entre espèces, rôle de l'habitat et du paysage.** (Poster)

- Djouhri M., Loubet B., Benoit P., Mamy L., Bedos C. **Modélisation de l'effet de la configuration paysagère et des pratiques culturales sur la dispersion et le dépôt des pesticides utilisés en agriculture.** (Oral presentation).

- XVI<sup>th</sup> Symposium in Pesticide Chemistry Advances in risk assessment and management, Piacenza, 3-5 September 2019. Djouhri M., Loubet B., Benoit P., Mamy L., Bedos C. **Modélisation de l'effet de la configuration paysagère et des pratiques culturales sur la dispersion et le dépôt des pesticides utilisés en agriculture.**

- SETAC Europe 14<sup>th</sup> Special Science Symposium, Brussels, 19-29 November 2019:

- Bertrand C., Zagatti P., Bonthoux S., Daniele G., Lafay F., Vulliet E., Bretagnolle V., Fritsch C., Pelosi C. **Ground-dwelling beetles' exposure to pesticides at large scales.**

- Pelosi C., Bertrand C., Bonthoux S., Daniele G., Lafay F., Vulliet E., Bretagnolle V., Fritsch C. **Earthworm exposure to pesticides in agricultural landscapes.**

**Scientific publications:**

- Pelosi C., Bertrand C., Daniele G., Coeurdassier M., Benoit P., Nélieu S., Lafay F., Bretagnolle V., Gaba S., Vulliet E. Fritsch C., 2021. **Residues of currently used pesticides in soils and earthworms: a silent threat?** Agriculture, Ecosystems & Environment. 305, 107167, DOI: 10.1016/j.agee.2020.107167

- Daniele G., Lafay F., Pelosi C., Fritsch C., Vulliet E. 2018. **Development of a method for the simultaneous determination of multi-class pesticides in earthworms by liquid chromatography coupled to tandem electrospray mass spectrometry.** Analytical and Bioanalytical Chemistry 410(20):5009-5018. DOI: 10.1007/s00216-018-1151-2

**Other practical work:**

- La France agricole. **Produit phytosanitaire, Des résidus de pesticides dans les sols et dans les vers.** February 2021,

- INRAE website. **Des résidus de pesticides dans les sols et les vers de terre : une réalité omniprésente et insidieuse.** 14 January 2021. https://www.inrae.fr/actualites/residus-pesticides-sols-terre-realite-omnipresente-insidieuse

- Charlie Hebdo. **Lettre des animaux aux humains reconfinés : le lombric.** Allain Bougrain-Dubourg published on-line, 1 December 2020. https://charliehebdo.fr/2020/12/ecologie/lettre-des-animaux-aux-humains-reconfines-le-lombric/

- Journal La Libre Belgique. **Les pesticides intoxiquent aussi les vers de terre... et donc leurs prédateurs.** S. Devillers, 25 November 2020.

https://www.lalibre.be/planete/environnement/les-pesticides-intoxiquent-aussi-les-vers-de-terre-et-donc-leurs-predateurs-5fbd5ad3d8ad586f5119c890

- Solagro, Osaé. **Des niveaux alarmants de pesticides mesurés dans les sols et les vers de terre.**

17 November 2020. https://osez-agroecologie.org/des-niveaux-alarmants-de-pesticides-mesures-dans-les-sols-et-les-vers-de-terre-168-actu-169

- Forskare (Swedish agricultural publication). **Omfattande spridning av växtskyddsmedel,**

12 November 2020. https://www.atl.nu/lantbruk/forskare-omfattande-spridning-av-vaxtskyddsmedel/

- Le Monde. **Des niveaux alarmants de pesticides dans les sols - Une étude montre une large contamination de l'environnement, où plusieurs produits s'accumulent.** 30 October 2020.

- Sciences et Avenir. **Des pesticides retrouvés partout dans l'environnement, même dans les vers de terre,** 2 November 2020. https://www.sciencesetavenir.fr/nature-environnement/pollution/les-pesticides-migrent-partout-dans-l-environnement_148889

- Leblob.fr. **Vers de terre : une étude alerte sur l'impact des pesticides,** 6 November 2020. https://leblob.fr/videos/vers-de-terre-une-etude-alerte-sur-impact-des-pesticides

- Life Sciences UPSaclay. **La menace silencieuse de la contamination des sols et des vers de terre par les pesticides,** 5 November 2020. http://sco.lt/5slHLk

Project web-site link: https://rescape.univ-fcomte.fr
<figure><img src="image_189.png" /></figure>

# The RESistance of Tropical agricultural SYStems to the reduction of pesticide pollution pressure at the watershed scale

**Launch year:** 2015

**Completion year:** 2019

**Partners**

UPR HortSys CIRAD; UAG Ceregmia-INRAE; UMR Tetis CIRAD; UMR SCBPA CIRAD; UMR SADAPT AgroParisTech

**Scientific manager**

Charles Mottes, UPR HortSys CIRAD
charles.mottes@cirad.fr

**Financing**

Total cost of the project: € 246,623
Ecophyto grant: € 106,276

**Key words:**

Environment contamination; Territorial approach; Tropical crops; Co-design work with farmers and other local professionals

## Context and main objectives

The French Caribbean (Antilles) is facing generalised pollution by chlordecone. And despite this situation, large quantities of pesticides continue to be applied across the archipelago. The Caribbean environment is extremely vulnerable to pesticide pressure. For the moment there is relatively little awareness of the factors which maintain such pesticide usage and the processes that lead to the pollution of waterways. It should also be noted that no tools are in place to support changes or agro-ecological transition over to agricultural practices better suited to managing pollution situations under tropical conditions.

In this context, the objective of the project is to construct a support structure that will accompany changes in socio-ecosystem practices in the watersheds affected by agricultural pollution. This will involve the participative construction of a shared representation of the risks related to pollution from pesticide use, including the agronomic, economic and social representations. The practical objective will be to limit the pollution of rivers by changing practices across whole regions.

<figure><img src="image_190.png" /><figcaption>Upstream from one of the RESYST project's pilot zones: the Galion watershed. Photo: © Charles Mottes, Cirad</figcaption></figure>

## Principal results and interest in relation to the Ecophyto plan

The first results from the correlated analysis of pollution pressures and the pollution observed in the waterways of the watersheds have shown a **high herbicide usage pressure** on the watersheds since such herbicide use is common to the various agricultural systems in place. This intense pressure (applied quantities and application frequency considering the scale of the watersheds) is **generating chronic pollution in waterways**.

Pesticide use is a two-edged sword for the community. The concept of utility and danger co-exist without necessarily generating a shared representation by the different members of the community. In order to clear up this ambivalence, debates on perceptions of pesticide usage in the areas which influence local waterways have generated the need to create a space where points of view and knowledge can be exchanged on the subject. The second result concerns this space, which has taken the form of **action schools**.
Our analysis and participation in debates have revealed a **severe disconnect between the different professionals of the regions.** In this way changes in agricultural practices as well as the different agricultural sector support strategies for these changes are only mobilising the networks within the sectors without any interaction between them. Each sector favours its own production interests, highlighting its own constraint frameworks to the detriment of any regional development which would mobilise the diverse services and resources of the region. Our results have also shown that there is no "miracle" solution in terms of technical innovations that can be applied directly to the fields. Alternative techniques to the use of herbicides generate significant extra labour, operational and training costs.

However these costs could be offset by creating value on a region-wide scale through the available proximity resources (such as processing units, livestock, tourism, etc.) and by recognising the quality and diversity of local productions. In this way agriculture could have a leverage effect on tourism and promote the region through recognised "green" practices, such as excluding herbicide use, ending in a form of quality label for the region and its products. Agriculture would then be including in the strengthened economic development dynamic

with the creation of an eco-tourism circuit in the watershed area combined with short supply chain sales outlets frequented by tourists that would also allow farmers to diversify their revenues sources. Products exported from the area (such as bananas) could also carry this image outside of the local region. This type of scenario shows the ambitions and powerful interconnections that are necessary for regional innovations when their objective is to create a virtuous circle of economic development associated with a reduction in pesticide use.

These innovations and how they will be presented have been co-constructed and debated with the local professionals thanks to the project's tools and models (such as DPSIR, region level C-K methodology, role playing).

<figure><img src="image_191.png" /></figure>

Schematic representation of the professional network and influences concerning over-grassing management practices in the Galion watershed. From Deffontaines et al. (2020)

## Prospects for transfer or research

### Transfer:

Regional innovation seems to be a promising way to support change in agricultural practices. It would appear essential that such exchange and co-construction dynamics concerning the available options on a regional scale must be maintained between all professionals and all sectors. Environmental contracts must play a very specific role. The available tools (action schools, serious games) must facilitate these organisation and co-design systems. However, special attention must be paid to the long-term participation and commitment of the professionals involved. Sufficient and permanent resources must therefore be dedicated to this regional organisation.

### Research:

The RESYST project has shown that it will be necessary:

1. to construct and maintain perennial meeting places where the future of agriculture can be debated in full, to propose localised objectives for new public policies and lead reflections and to negotiate and allocate the resources that will be necessary for the transition.

2. to consider the regional scaling in order to elaborate phytosanitary product usage reduction innovations and use more localised public policies to support their implementation and/or regional transitions. The "Sustainable Territories" project should integrate the analysis of government transition support policies.

## Publications and scientific symposia:

### TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA:

* 14th ESA Congress. 2016, Edinburgh, Écosse: **Assessment of the pesticide pressure at the watershed scale: the challenge of the Water Framework Directive (INRAE, 2 p).** M. Lesueur Jannoyer, C. Mottes, M. Le Bail, P. Cattan.

* 14th ESA Congress. 2016, Edinburgh, Écosse: **Locating the contribution of cropping systems within a watershed to water pollution by pesticides (INRAE, 2p).** Mottes C., Lesueur Jannoyer M., Della Rossa P., Le Bail M., Cattan P.
► Tropentag 2017. 20-22/09/2017, Bonn, Allemagne: **Future Agriculture: Social-ecological transitions and bio-cultural shifts. Sociotechnical System Analysis of Weeding, A Key Step for Designing Agro-ecological Systems at the Watershed Scale.** University of Bonn, 1 poster. Della Rossa P., Mottes C., Cattan P., Le Bail M., Jannoyer M.

► Doctorate registered as a case study for the Idiss platform (INDISS spotlight project: INnovation and Design In Sociotechnical Systems) by Labex BASC (Biodiversité, Agroécosystèmes, Sociétés, Climat), seminar, 2017.

► OPDE symposium **“Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation”**. Montpellier, France, OPDE network: Ecole-acteurs aux Antilles, un espace de représentation partagée de la question des pollutions agricoles. 13 p. Tonneau, J.-P., et al. (2016).

**PRESENTATION TO PROFESSIONAL OR DECISION-MAKING BODIES:**

► **Agriculture et qualité de l'eau, comprendre leurs relations pour mieux les gérer.** Presented to the "Assises de l'agricultures de Martinique", Agriculture, environnement et santé, Schœlcher, 5 December 2017.

► **Action schools** including professionals, institution representatives and researchers:

* Action school 1: November 2016, action school dedicated to the presentation of the action school concept

* Action school 2: March 2017, action school dedicated to innovations and innovation systems

* Action school 3: June 2017, action school dedicated to the theme: "awareness without guilt".

* Action school 4: November 2017, action school dedicated to the theme: "how to get by without glyphosate"

* Action school 5: February 2019, action school dedicated to population expectations regarding agriculture

* Action school 6: September 2019, action school dedicated to the theme: "What future for agriculture in Guadeloupe".

* Action school 7: Finally, an action school in Martinique, April 2019. The objective was to work on constructing a diffused pollution model for Martinique's drainage basins.

* Action school 8: November 2019, on the food industry.

* K Workshop: March 2018, knowledge exchange workshop on innovations between Martinique's agricultural professionals.

**SCIENTIFIC PUBLICATIONS:**

► **Agricultural Systems (n° 179, 102769): How farmers learn to change their weed management practices: Simple changes lead to system redesign in the French West Indies.** Deffontaines, L., Mottes, C., Della Rossa, P., Lesueur-Jannoyer, M., Cattan, P., Le Bail, M., 2020. https://doi.org/10.1016/j.agsy.2019.102769

► **Agronomical Sustainable Development (n° 40, 10): Innovations developed within supply chains hinder territorial ecological transition: the case of a watershed in Martinique.** Della Rossa, P., Le Bail, M., Mottes, C., Jannoyer, M., Cattan, P., 2020. https://doi.org/10.1007/s13593-020-0613-z

► **Conception collective d'organisations territoriales innovantes pour une évolution coordonnée de systèmes de production agricoles (p328): Cas d'une réduction de la pollution herbicide d'une rivière en Martinique.** Université Paris-Saclay, Saint Aubin, France. Della Rossa, P., 2020.

**Other practical works:**

* Presentation and use of WATPPASS-Game tools with a LEGTA class from Robert (December 2018).

* Presentation and training of technical staff from the Martinique Chamber of Agriculture on the use of the WATPPASS-Game solution (Novelber 2018).

* Réseau Canope - Gestion des pollutions à l'échelle des bassins versants (2017).

* France Antilles Martinique - Chlordécone : où en est la dépollution des sols ? - 9/10 June 2018.

* Bernard Crutzen, interview for the documentary: "Pour quelques bananes de plus" (June 2019).
<figure><img src="image_192.png" /></figure>

# Regulating scale insect populations on black-current crops by releasing the scale-eating ladybird *Rhizobius lophantae* - Transposition to other fruit crops

**Launch year:** 2016

**Completion year:** 2019

**Scientific manager**

Maria-Martha FERNANDEZ, CTIFL

maria-martha.fernandez@ctifl.fr

**Partners**

CTIFL; Station d'Expérimentation La Morinière;
Chambre Départementale d'Agriculture de Côte d'Or;
INRAE PACA; KOPPERT

**Financing**

Total cost of the project: € 149,576

Ecophyto grant: € 87,128

**Key words:**

Scale insect; Biological pest control by augmentation; Scale-eating lady-bird; *Rhizobius lophantae*; *Pseudaulacaspis pentagona*; Black-current; Auxiliary releases; Perennial culture; Fruit crops; Multi-sites experimentation

## Context and main objectives

Even if its is considered to be only a minor crop, black-current remains a major economic sector on a high value market where French production is much sought after (liquors, perfumes). For the last fifteen years, this crop has been severely affected by the white black-berry scale insect (*Pseudaulacaspis pentagona*), which was accidentally

introduced into Italy in the 19th century via the silk road, since then it has spread further North to affect French black-current crops. This pest regularly causes crop losses or even the entire destruction of plantations; it is not very well controlled by chemical solutions and has become a major problem for the black-current industry. The RhizoDia project proposes a multi-site experiment programme, involving releases of the specific lady bird predator for scale insect *Rhizobius lophantae*, to demonstrate the effectiveness of a biological pest control by augmentation approach which could be applied by black-current growers. Beyond the model studied, it also aims to transfer this same biocontrol solution and its experimental methodology to other fruit crops that are affected by scale insects.

<figure><img src="image_193.png" /><figcaption>Figure 1: Initial condition of test sites and evolutions in the pest pressure for the release zones.</figcaption></figure>

Image: © CTIFL/IGN (for map details)
## Principal results and interest in relation to the Ecophyto plan

Experiments were completed in production orchards using conventional agriculture; it involved 5 sites in the Valdu-Loire and one in the Côte d'Or presenting a variety of different pest pressure situations. The experimental method used was proven during the project and can now be proposed for this type of study: 2 plantations of the same variety and of the same age were chosen, located in the same geographical zone but at least 500m apart, one was used for the introduction of auxiliaries (0.5 ha) and the other as a control site.

<figure><img src="image_194.png" /><figcaption>Figure 2: Winter shelters used by the project.
Left: cardboard strips. Right: bamboo bundles
Photo: © Station La Morinière</figcaption></figure>

The control site was managed in keeping with a full phytosanitary treatment schedule including at least one specific scale insect treatment during the second swarming (REF(+)), except for a block of about 50 ares which was left without scale treatment, used as an "untreated" (REF(-)) control. The *Rhizobius* releases were annual, during each swarming period following a protocol which could also be used elsewhere for its ease of use: the auxiliaries were received ready for release, in bags with a ring which could be fitted to the base of the plant; each bag contained 200 adults, these were distributed across 15 release sites per 0.5 ha, so an installation time of 15 mins.

3 indicators were used to evaluate the effectiveness of this protection by release approach:

* The density of live scale insects per surface area unit, counted on samples of infested branches taken from the affected plants;

* The degree of plant infestation measured using a visual scale that covers the presence and severity of the symptoms;

* The number of *Rhizobius* captures, obtained by tapping the plants on a Japanese parasol.

The aptitude of the introduced auxiliaries to install themselves permanently was evaluated by analysing their ability to overwinter, measured by a hatchery experiment in which the viability of winter hatching was observed and by installing winter shelters where the occupation rates were observed at the end of winter to verify adult survival rates. A COI gene sequencing genetic classification protocol was applied to over 135 *Rhizobius* ladybirds captured before and after the releases as well as a sample taken directly from the producers, to attempt to confirm the contributions of the introduced auxiliaries over those of any established populations.

Under moderate pressure, the releases were effective during the first year of the project, with a scale insect regression rate of 48% in the introduction zones as opposed to 14% in the reference zones, a clear peak in *Rhizobius* re-captures after the releases and plants were visually in better health. But this was not confirmed later, since at the end of year 3 the situation degraded by 54% compared to the initial state of the introduction sites, whilst it remained stagnant for the reference sites. This failure could not be explained by phytosanitary pressure or global climate change, even if we can conclude that while the releases are effective, all of the factors for the method's success were not under control by the end of the project. Methodological improvements were also proposed, notably a more detailed classification of the fields, their surroundings and events occurring around the release operations in order to broaden the statistical analysis of the multi-sites experimentation in order to find explanatory factors.

<figure><img src="image_195.png" /><figcaption>Figure 3: Bags of 200 *Rhizobius* at the base of infested black-current plants
Photo: © Station La Morinière</figcaption></figure>

## Link with the Ecophyto plan:

The completion of the project did not result in the desired deployment of this biocontrol method. However it did confirm the highly variable effectiveness of phytosanitary treatments for the targeted pest, which further implies that work needs to continue to find alternative methods.
# Prospects for transfer or research

## Transfer:

The objectives laid down at the start of the project were not achieved; no transfer to application under production conditions can be planned at this time. Some methodological aspects, especially a number of points raised by the project report could be used for researchers working on similar projects.

## Research:

In order to terminate exploration of the use of Rhizobius lophante to control scale insect on black-current, a new study program will test the method on young plants, targeting the zones where the first colonies appear and limiting release operations to the second summer swarming to allow for climate restrictions; the objective being to break up the pest's installation cycle as early as possible as well as lower the costs of this protection approach, under the project conditions costs were more than 40% of net product. Two research subjects will also be proposed: 1) the use of other gene portions or other genetic classification techniques on the auxiliary, the method used was not sufficiently discriminatory, and (2) research for non-destructive active scale insect detection tools.

# Publications and scientific symposia:

## PLACEMENT REPORTS:

* DAUFFOUIS, S. 2018. **Projet RhizoDia : essais en protection du cassis par Rhizobius lophantae contre la cochenille blanche du mûrier.** GIS-Fruits placement report 2018. https://www.gis-fruits.org/content/download/3876/38621/version/1/file/Simon_Dauffouis_RhizoDia2018.pdf

* GAY, A. 2017. **Etude de la régulation de populations de cochenilles diaspines en culture de cassis par des lâchers de coccinelles coccidiphages.** End of studies dissertation (unpublished)

* GOUVRION, J. 2016. **La coccinelle Rhyzobius lophanthae, agent de bio-contrôle pertinent dans la lutte contre la cochenille Pseudaulacaspis pentagona ?** GIS-Fruits placement report 2016. https://www.gis-fruits.org/content/download/3697/36570/file/RhizoDia-Gouvrion.pdf

## PRACTICAL / EDUCATIONAL ARTICLES:

* FERNANDEZ, MM. 2021. **Cassis - Des coccinelles contre la cochenille.** Réussir Fruits et Légumes, n° 416: 36-37.

* FERNANDEZ, MM. 2020. **Une piste biocontrôle en culture de cassis ? Les lâchers de coccinelles contre la cochenille blanche du mûrier.** Infos CTIFL, n° 365: 18-32.

## SCIENTIFIC SYMPOSIA:

* GUIGNEBAULT, P. 2018. **Biological control of the White Peach Scale on blackcurrant: the RhizoDia project.** Comments from the 6th World blackcurrant congress, Angers 6-8 June 2018. https://www.blackcurrant-iba.com/event/wp-content/uploads/2018/07/5-Rhizodia-program.pdf
<figure><img src="image_196.png" /></figure>

SEMIOTRAP

# Development of a biocontrol trap to monitor and control the palm borer moth, *Paysandisia archon*

**Launch year:** 2018

**Completion year:** 2021

**Partners**

INRAE - iEES Paris; Sauvons nos palmiers (SNP); M2i Group

**Scientific manager**

Brigitte Frérot, INRAE - iEES Paris
brigitte-frerot@inrae.fr

**Financing**

Total cost of the project: € 226,902
Ecophyto grant: €120,000

**Key words:**

Attraction; Pheromone; VOC; Trap; Biocontrol; Chemical mediators; Palm trees; *Paysandisia archon*

## Context and main objectives

*Paysandisia archon* is a moth of Uruguayan origin. It is not considered as a pest in its native habitat. It was unintentionally introduced into Europe at the end of the 1990s, where its larvae now cause significant damage to palm tree species. A pheromone has been identified which is emitted by the male to attract females. The insect is large with a wing span of 11cm. Conventional or biological control solutions are ineffective since it develops inside the trunk. The use of pheromones can help to adapt treatments or direct control techniques such as the mass trapping of fertilised females.

The project seeks to develop an understanding of how the pheromone produced by the male works and should conclude with its use for mass trapping or pest monitoring programmes. In order to achieve this, the project includes behavioural and physical-chemical studies to optimise the composition and dosage of the pheromone. The project is committed to designing a trap suitable for this moth; commercially available traps are not suited to the size and behaviour of this pest. After this the project concentrated on the Volatile Organic Compounds (VOC) emitted by the palm tree crowns where the eggs are laid.

This project combines the work of three partners: The Versailles INRAE UMR iEES 1392 chemical ecology laboratory, M2i and th.e Sauvons Nos Palmiers (SNP - Save our Palm-trees) association, which is a collective of palm-tree growers. This collaboration serves to combine scientific expertise on the identification and formulation of chemical mediators with an industrial company specialised in synthesising Lepidoptera pheromones and formulating them on innovative biodegradable matrices. The SNP partner is deeply involved in communication and information concerning palm-tree pests. This partner played an essential role in the project by providing host-sites for the necessary field installations.

*Paysandisia archon*
Photo: © Brigitte Frérot - INRAE iEES Paris

The hoped for result was a commercial biocontrol product including a trap and one or two types of lure designed to control the moth. This product will improve the epidemic-surveillance of the pest and its rapid management as well as its final control through mass-trapping operations. This type of solution will improve data collection and limit the need to use phytosanitary products. As part of an integrated control program in association with the currently available biocontrol solutions, the arrival of this solution should also help to achieve the "zero pesticide" objective for the control of this pest.

<figure><img src="image_197.png" /></figure>
# Principal results and interest in relation to the Ecophyto plan

## I) Identification, formulation and observation of the effect of the pheromones

The SEMIOTRAP project continued work on:

1. the identification of the pheromone produced by the males and the demonstration that it attracts females independently to their visual senses.

2. the demonstration of the attraction of fertilised females by the odour of the palm-tree trunk.

During the synthesised pheromone attraction study; we demonstrated that the identified compound is never as attractive as the natural pheromone, and that the attraction rate remains much lower than that expected for a trapping system. The pheromone has therefore been re-investigated and two new minor compounds have been detected, only one of which has been identified: Z9-18: AC.

Various combinations were tested in flight tunnels, but none of them were as effective as the natural extract, except for E2Z13ODOL+ Z9-ODAC

<table><thead><tr><th>Retention time (min)</th><th>Retention index (RI)</th><th>Identified compound</th><th>Washingtonia</th><th>Trachycarpus</th><th>Phoenix</th></tr></thead><tbody><tr><td>2.36</td><td>854.3</td><td>Hexanal</td><td>7.84%</td><td>6.69%</td><td>2.14%</td></tr><tr><td>2.95</td><td>890.5</td><td>Ethylbenzene</td><td>2.50%</td><td>2.39%</td><td>1.55%</td></tr><tr><td>3.05</td><td>896.4</td><td>p-Xylene</td><td>4.55%</td><td>3.63%</td><td>2.43%</td></tr><tr><td>3.2</td><td>905.5</td><td>3-Heptanone</td><td>2.20%</td><td>1.56%</td><td>2.50%</td></tr><tr><td>3.39</td><td>916.8</td><td>heptanal</td><td>5.40%</td><td>5.59%</td><td>2.86%</td></tr><tr><td>3.84</td><td>944.1</td><td>α-Pinene</td><td>1.04%</td><td>0.90%</td><td>3.79%</td></tr><tr><td>4.09</td><td>959.4</td><td>2-Ethylhexanal</td><td>3.89%</td><td>2.28%</td><td>3.03%</td></tr><tr><td>4.24</td><td>968.6</td><td>Benzaldehyde</td><td>5.56%</td><td>7.19%</td><td>9.79%</td></tr><tr><td>4.83</td><td>1004.1</td><td>Octanal</td><td>3.89%</td><td>3.10%</td><td>2.74%</td></tr><tr><td>5.26</td><td>1030.1</td><td>Limonene</td><td>20.34%</td><td>32.19%</td><td>22.47%</td></tr><tr><td>5.34</td><td>1035.6</td><td>Benzyl alcohol</td><td>14.50%</td><td>13.82%</td><td>11.92%</td></tr><tr><td>6.5</td><td>1105.5</td><td>Nonanal</td><td>7.10%</td><td>4.99%</td><td>5.48%</td></tr><tr><td>7.22</td><td>1147.2</td><td>3-Methylheptyl acetate</td><td>0.45%</td><td>0.48%</td><td>0.84%</td></tr><tr><td>7.4</td><td>1156.3</td><td>Isomenthone</td><td>0.37%</td><td>0.19%</td><td>0.00%</td></tr><tr><td>7.63</td><td>1171</td><td>2-Decenol</td><td>0.93%</td><td>0.69%</td><td>0.91%</td></tr><tr><td>7.85</td><td>1183.3</td><td>p-Ethylbenzaldehyde</td><td>0.00%</td><td>0.33%</td><td>0.67%</td></tr><tr><td>8.03</td><td>1194.6</td><td>Methyl salicylate</td><td>0.00%</td><td>0.08%</td><td>0.00%</td></tr><tr><td>8.25</td><td>1207</td><td>Decanal</td><td>5.43%</td><td>5.19%</td><td>4.66%</td></tr><tr><td>8.49</td><td>1220.8</td><td>2-phenoxyethanol</td><td>11.37%</td><td>8.38%</td><td>21.59%</td></tr><tr><td>9.15</td><td>1259.3</td><td>p-Anisaldehyde</td><td>0.89%</td><td>0.34%</td><td>0.00%</td></tr><tr><td>9.58</td><td>1285.5</td><td>Anethole</td><td>0.96%</td><td>0.00%</td><td>0.00%</td></tr><tr><td>12.24</td><td>1448.9</td><td>cis-Geranylacetate</td><td>0.78%</td><td>0.00%</td><td>0.64%</td></tr></tbody></table>

Identifications of the compounds emitted by various palm-tree species. (tested compounds in blue) Image: INRAE

(10µg+0,1µg), which provokes 20% of landings, but was only tested on a small number of individuals.

The second major result concerns the demonstration of the attraction of males to the pheromone that they or their peers emit. There can be no doubt that the males return to the spots marked by their peers of the same sex or those they marked themselves. We have also demonstrated that this was not the result of any significant pheromonal polymorphism in the males.

## II) Trap assays:

None of the traps tested were effective. Electric harp or dome traps compatible with safety regulations do not produce a strong enough discharge to kill the insects. Net and cage traps are not compatible with the behaviour of this insect.

## III) Palm-tree VOC classification:

In 2019 we broadened the range of molecules and tested most of the molecules produced by palm-tree crowns, along with some other molecules often emitted by the plants such as DMNT, caryophyllene, etc. Some of these products were very well perceived by the antennae of the unfertilised females with reactions close to 2 mV. This was the case with Methyl salicylate, 1-phenoxy-2-propanol, phenol, butyrolactone, anisaldehyde and, above all, caryophyllene. Alpha-pinene, present in the odours emitted by palm-tree crowns, described as an attraction inhibitor for RPW (Soroker et al.), is not very well perceived by *Paysandisia archon*.

# Prospects for transfer or research

## Transfer:

The *Paysandisia* is a complex insect in terms of its biology and dimension. The host plant is disseminated across the urban landscape.

Studies of the attraction of males and females by the synthetic pheromone and to identify the third compound need to be continued. This will be a long process but it could lead to reducing populations by trapping both sexes.

Current results have not lead to a solution but have contributed to furthering knowledge of the chemical mediators which affect the reproduction and egg-laying of this insect. We know that the plant attracts fertilised females and the VOC have been identified. Potential egg-laying site location inhibitors have been proposed and will need to be tested.
**Research:**

The marketing of a new technique based on chemical mediators will not be easy and will have to involve a long-term behavioural approach in cases where insects react outside of the known model.

An optimal pheromone mix will need to be identified; the roles of the VOC produced by the plants will need to be investigated in order to produce more than one lure: a sexual lure and a pre-egg-laying lure.

M2i will continue its research work, notably on optimising the design of a net trap for capturing attracted insects.

**Publications and scientific symposia:**

**Operational / educational articles:**

► RACHID HAMIDI AND BRIGITTE FRÉROT, INRAE, UMR 1392, IEES, Route de St-Cyr, Versailles F-78000. **Les médiateurs chimiques, clés de voûte du cycle de Paysandisia archon.** Study of chemical mediators that are involved in the palm-borer moth's development cycle must lead to new control solutions. Phytoma 740 May 2021

**Technical workshops and scientific symposia:**

► The presence of SNP members amongst the consortium members guarantees, *via* their annual "Les rencontres ravageurs de palmiers de Monaco" symposium and regular public interventions, the awareness of those involved regarding biological control and highlighted progress made by the programme with the local PACA region councils and the general public: https://www.sauvonsnospalmiers.fr/les-quatriemes-rencontres-ravageurs-de-palmiers-de-monaco.html?id_document=3402.
<figure><img src="image_198.png" /></figure>

SERUM

# Disinfecting soils in market gardens

**Launch year:** 2015

**Completion year:** 2019

**Scientific manager**

Ingrid Arnault, CETU Innophyt, University of Tours ingrid.arnault@univ-tours.fr

**Partners**

CETU Innophyt, University of Tours; CIRAD; CIRAD/IRD/ SupAgro / UM2 UMR LSTM; INRAE; IRBI UMR CNRS 7261 University of Tours; EBI UMR CNRS 7267 Université Poitiers; GRAB-Avignon; CERTIS Europe; Delbon SARL

**Financing**

Total cost of the project: € 349,070

Ecophyto grant: € 140,930

**Key words:**

Tomato; *Allium*; *Crotalaria*; Nematodes; Bacterial wilt; DMDS; Mulch; Preceding crop; Ground-sheeting; Unwanted side-effects; Biostimulation; Allelopathy.

## Context and main objectives

In Martinique, *Ralstonia solanacearum*, the bacteria behind bacterial wilt (quarantine organism), is the leading pest/disease affecting solanaceae and cucurbitaceae, resulting in losses of up to 100%. No authorised product currently exists to treat this. The root-knot nematode, *Meloïdogyne*, causes 40% losses in green-house grown tomatoes in the South of France. Only dichloropropene is currently authorised for use. There is now a very significant need for agro-ecological solutions to protect harvests for market gardeners. The biocontrol strategies studied by SERUM are based on the bio-disinfection of soil using service plants such as rattlepods and Alliaceae as precedent crops or mulch (figs 1 and 2) and/or incorporated dry or fresh chippings, or as companion planting. Dimethyl disulfide (DMDS), a volatile molecule emitted by Alliaceae, is a viable soil disinfection solution (nematodes, fungi, weeds), it has already received authorisation for sale as a soil fumigation agent for specific crops in a number of countries. Its antibacterial properties against bacterial wilt have been explored for use in an integrated agriculture context.

<figure><img src="image_199.png" /><figcaption>Figure 1: *C. Spectabilis* mulch.
Photo: © Ingrid ARNAULT, CETU Innophyt</figcaption></figure>
## Principal results and interest in relation to the Ecophyto plan

The project has demonstrated the interest of service plants in soil disinfection for tomato crops from three different production and issue angles.

1) Against the **agent that causes bacterial wilt** in tropical agriculture, *C. juncea* as a **two cycle precedent crop followed by mulch** (see diagram) has been found to seriously reduce incidences of the disease.

<figure><img src="image_200.png" /><figcaption>Bio-disinfection design to deal with the agent that causes bacterial wilt in tomato crops in Martinique.</figcaption></figure>

Image: © Ingrid ARNAULT, CETU Innophyt

However this usage does present some technical disadvantages for professionals. **The use of *C. juncea* as a powder or chipping mixed into the soil would be a more realistic solution.** A production industry for dry forms of *C. juncea* should be set up for better integration into crop systems.

As for the local *allium fistulosum* which has interesting anti-bacterial properties, its use as a mulch will not be possible since it is highly sought after for cooking.

2) Against **root-knot nematode**, *Meloidogyne*, in (French) mainland **market garden green-house productions**, *C. spectabilis* as a **preceding crop/mulch followed by incorporation into the soil** seems to reduce root-knot on lettuce. The incorporation of powdered rattlepod into the soil has also provided interesting results in the reduction of root-knot on tomatoes.

3) Against **root-knot nematode**, *Meloidogyne*, in (French) mainland **tunnel productions**, assays have demonstrated a slight interest in using **ground up chives and onions incorporated into the soil**. However, experimental design research needs to continue: the optimal dose of dry or fresh ground material incorporated into the soil combined with suitable ground-sheeting, use as a precedent crop/mulch, association with rattlepods and *Allium* sp., etc.

When parasite pressures are very high, as was the case for the nematode and *R. solanacearum* assays, service plants as crop precedents/mulch or as ground material incorporated into the soil did not always fully protect productions. A strategy involving pesticides during the growing phase could be envisaged to definitively reduce pathogens.

The project has highlighted the anti-bacterial properties of DMDS. This product is a promising alternative for use against this bacteria which is a pathogen in tropical region and potentially invasive in temperate climates. The prospects for the use of DMDS, currently unauthorised for use in this context, depend on the currently pending application for European commercial registration.

As for unwanted side-effects, observations have been mostly positive for the Service Plants. These Service Plants stimulate microbial populations in the soil and growth in tomatoes. The only toxic effect of *C. juncea* extracts and DMDS occur in very high doses, much higher than those used in the fields during the project, but should be considered for any potential effect on woodlouse.

<figure><img src="image_201.png" /><figcaption>Figure 2: Left: cross-section of *C. Spectabilis*; Photo : © Ingrid ARNAULT, CETU Innophyt</figcaption></figure>
# Prospects for transfer or research

## Transfer:

The question of the generic nature of the method is very important for biocontrol. As regards the project results, any transposition to other market garden crops would be possible but delicate, provided that the exact same production conditions apply: tunnel, green-house or field. For example, we have been able to demonstrate that *C. Spectabilis* has an anti-nematode effect as a precedent crop followed by application as a mulch for lettuce crops.

## Research:

Strategies combining preventive approaches using Service Plants as precedent crops, and curative approaches using DMDS during the growth season for tomatoes, will permanently reduce pathogens.

Considering the prospects, and where possible, it would be useful to test this strategy with a crop rotation to reduce the infectious potential of the soils. Actions will be different and probably complementary.

Bio-stimulation of tomato plant roots and soil microbe populations via Service Plants suggests furthering studies of the rhizosphere and symbiotic interactions: mycorrhiza, antagonist bacteria, irregular bacteria, etc.

## Publications and scientific symposia:

* Poster: Biocontrol and Natural Products, 25-28 September 2018, Perpignan. I. Arnault, P. Deberdt, G. Dubreuil, Y. Prin, P. Fernandes, H. Védie, P. Sunder, T. Fouillet, G. Dufretay, C. Souty-Grosset, N. Pourtau, M. Zimmermann, D. Giron. **Biodésinfection des sols en culture de tomate.**

* Poster: Future IPM 3.0: towards a sustainable agriculture. 16-20 October 2017, Italy. **Allelopathic effects of *Crotalaria juncea* and dimethyldisulfide (DMDS) on tomato plants in the future development of a biocontrol method against root-knot nematodes.** G. Dubreuil, N. Pourtau, N. Moreau, C. Leboissetier, M. Piot, D. Giron, and I. Arnault.

* Poster: 13e Rencontres Plantes-Bactéries. 29 January-2 February 2019, Aussois. **Beneficial effect of the use of the tropical legume *Crotalaria* spp for organic greenhouse vegetable production in nematodes-infested soil of South of France.** Y. Prin, F. Cussonneau, C. Le Roux, E. Tournier, B. Vincent, R. Duponnois, F. Deleuze, A. Galiana.

* Manuscript submitted to the European Journal of Agronomy (6 March 2019). **Beneficial effect of the use of the tropical legume *Crotalaria* spp for organic greenhouse vegetable production in nematodes-infested soil of South of France.** Y. Prin, F. Cussonneau, C. Le Roux, E. Tournier, B. Vincent, R. Duponnois, F. Deleuze, A. Galiana.

* Proceedings of IX International Symposium on Soil and Substrate Disinfestation (SD 2018) 9-13 September 2018, Heraklion, Crète. **Soil disinfestation with dimethyl disulfide (DMDS) to manage the bacterial wilt of tomato in the tropics.** P. Deberdt, R. Coranson-Beaudu, C. Thibaut, N. Le Roch, T. Fouillet, P. Sunder and I. Arnault.
<figure><img src="image_202.png" /></figure>

SYSTEMYC

# Conception of crop-growing SYSTEmS based on the use of MYCorrhization for the biocontrol of earth-bound pests on tomato crops

Launch year: 2015

Completion year: 2018

Scientific manager

Marie Chave, UR 1321 ASTRO INRAE

marie.chave@inrae.fr

Partners

UR 1321 ASTRO INRAE; University of the Antilles; INRA UR Ecodéveloppement; INRA UE Alénya-Roussillon; Institut Sophia Agrobiotech

Financing

Total cost of the project: € 528,074

Ecophyto grant: € 99,297

## Key words:

Soil biodiversity; Mycorrhiza; Biocontrol; Redesigning crop systems; Vegetable crops; Co-design work with agriculturalists; Socio-technical blocking; Learning; Educational tool

## Context and main objectives

French mainland and Caribbean market garden crops are currently affected by severe soil parasites resulting in serious damage to their harvests. With the recent withdrawals of authorisations to use certain phytopharmaceutical products (nematicides, methyl bromide, etc.), the development of biocontrol strategies has become a necessity. Interactions between plants and micro-organisms in the soil, such as mycorrhizal symbioses, could provide new solutions for the agro-ecological management of crop health. In this context; the SYSTEMYC project objectives were:

i. Co-design crop-growing systems based on the use of mycorrhiza for the biocontrol of under soil pests

ii. Construct systems able to identify the obstacles and levers involved in the use of mycorrhiza by farmers and the training that will be necessary to use them for biocontrol.

iii. Evaluate innovative strategies for using mycorrhiza in temperate and tropical zone market garden crop systems.

## Principal results and interest in relation to the Ecophyto plan

If the "one pest/disease, one solution" approach remains valid for some biocontrol products, it does not apply to mycorrhiza. In fact, mycorrhiza 1) have a multi-pest/disease effect, 2) have different direct and indirect actions, and 3) are impacted by most agricultural practices.

Using mycorrhiza for biocontrol will therefore require an overhaul of crop protection approaches. The main results of the SYSTEMYC project are:

1. A collective MYMYX1 training system<fnref n="1" /> ("Mimic Mycorrhizal networks"), which alternates participative workshops (figures 1 and 2) and farm surveys and has enabled the achievement of an understanding of the complexities and uncertainties associated with mycorrhiza processes and the identification of the obstacles and levers that will be involved in their use. From the 50 agriculturalists involved, we were able to distinguish two groups with different characteristics in terms of their proposition dynamics and the obstacles they identify regarding the implementation of mycorrhization: a "proactive group" and a "reserved group".

<figure><img src="image_203.png" /><figcaption>Figure 1: The MYMYX game board. Photo: © Marie Chave, INRAE</figcaption></figure>

<footnote n="1">¹ MYMYX: ("Mimic Mycorrhizal networks") is a support which can be used to get an understanding of under-soil interactions by creating and maintaining a network through the development of mycorrhizal filament segments (in white). For the players this involves constructing a strategy, a set of practices (crop/inter-crop, tilling, fertilisation, phyto-protection, introduction of commercial or other mycorrhiza, etc.) to design a crop system that will allow the roots (shown in the four corners of the board) to access the nutritive resources they need as rapidly as possible (e.g.: phosphates in yellow, water in blue, etc.) whilst maintaining protection from pestattacks (in red).</footnote>
The "proactive group" agriculturalists display a capacity to adopt a re-designed approach whilst those of the "reserved group" show a lesser propensity to integrate different or new concepts. We have observed some diversity in regional responses to the propositions raised.

<figure><img src="image_204.png" /><figcaption>**Figure 2:** Organisation of 5 sequences of the participative "Sharing knowledge" workshop implemented in the 4 studied regions (Martinique, Guyana, Guadeloupe and Provence).</figcaption></figure>

2. An innovative design approach based on the MYMYX system was able to open up mycorrhiza usage prospects for biocontrol purposes. When applied in a diverse range of contexts, it leads to the production of a hierarchical tree of the farmers' propositions organised in three levels: concepts, sub-concepts and practices. The explored concepts are (C1) the introduction of standard commercial or native (natural) strain propagules, (C2) the connection of mycorrhiza fungi with plants through associations and crop rotations, (C3) the intensification of the mycorrhiza networks by reducing tilling operations and additives (fertilisers, phytopharmaceutical products) (figure 3). This large, yet still incomplete, range of solutions which can be adapted to each individual context as part of a systemic logic, will allow farmers to understand that they are all potential mycorrhiza producers.

<figure><img src="image_205.png" /><figcaption>**Figure 3:** Extract from the mycorrhiza mobilisation levers propositions library (from the 4 study regions: Martinique, Guyana, Provence and Guadeloupe), CMA: Arbuscule Mycorrhizal fungi. Image: © Marie Chave, INRAE</figcaption></figure>

3. The development of innovative strategies and systems has enabled the application of native mycorrhiza networks for the biocontrol of under-soil pests/diseases in market garden crops. The experimental evaluation of this strategy has been undertaken by local professionals in Martinique and Guyana. The evaluation of an innovative mycorrhiza strategy, completed at the same time by the experimental units (INRAE Alénya and PEYI), based on multiplying native mycorrhiza to 'pre-mycorrhize' tomato plants has shown: 1. in nurseries: the feasibility and effectiveness of this strategy in mycorrhization container prototypes, 2. in the field: the interest of early mycorrhization for crop health, 3. under controlled conditions: the effectiveness of the mycorrhization of a tomato plant using a mycorrhiza network in comparison to mycorrhization by propagule inoculation and the bio-protector effect of mycorrhization regarding root-knot nematodes.
# Prospects for transfer or research

## Transfer:

The results and tools developed by the SYSTEMYC project have been used by the CasDAR projects (Mycoagra and Mycado). The MYMYX educational tool has been redesigned with agricultural training and advice. This type of usage redesign approach is necessary to help professionals through the transition process, as they change their positions in order to produce/educate/advise/research in new ways.

Whilst the 'approach of applying biodiversity in the interests of crop health' developed by SYSTEMYC was concentrated on mycorrhiza, it remains generic and applicable to other natural regulation processes (mobilising auxiliaries for examples).

## Research:

Continuing the analysis work on plant interactions via mycorrhiza networks will open up numerous crop health management possibilities combining the bioprotector and disinfection effects of mycorrhizotrophic plants (see the MultiServ, INRAE, SuMCrop projects).

On the crop systems scale, it will be necessary to continue field evaluations of the impacts of numerous mycorrhiza usage strategies for biocontrol (see the Dephy Expé Cabiosol project supported by FREDON Martinique).

On the territorial scale, analysis of the obstacles and levers involved in the implementation of biocontrol should be continued (see the Ecophyto Interlude project).

## Publications and scientific symposia:

### TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA:

* **International Conference On Mycorrhizae, Mexico (30/06/2019 - 05/07/2019)**: *A participatory approach to harness native mycorrhizae*. Chave M, Angeon V, Harter AC, Goasduff M, Paul G, Quinquenel S, Ster H.

* **International Conference on Plant Protection, Boston, USA (29/07/2018 - 03/08/2018)**: *Agroecological engineering for biocontrol of soil pests- examples from the French Caribbean*. Chave M, Angeon V.

* **Scientific Advisory Board of the SMaCH meta-programme, Versailles, FRA (29/01/2018)**: *Harnessing mycorrhiza for crop health: an agroecological approach*. Chave M, Angeon V.

* **54th Caribbean Food Crops Society symposium. Belize (07-15/07/2018)**: *Agroecological production and consumption models in the context of transition: the approach of the economies of worth*. Angeon V, Crevoisier O.

* **Salon Tech & Bio. 20-21/09/2017. Valence:** *Poster and workshop: "Cultivez vos mycorhizes !"*. Chave M., Paut R., Angeon V., Lefèvre A., Dufils A., Tchamitchian M.

* **TANDEM network presentation seminar, Avignon, FRA (16/10/2017)**: *Les systèmes de culture maraîchers : des laboratoires pour la co-conception d'innovations agroécologiques ? Focus sur les réseaux mycorhiziens*. Chave M, Angeon V, Paut R, Tchamitchian M.

* **54th ASRDLF symposium, ERSA-GR conference, Athens, GRC (05-07/07/2017)**: *Mettre en œuvre la transition agréocologique : un défi d'apprentissage et de partage de connaissances par et pour l'action*. Angeon V, Chia E, Chave M, Auricoste C.

* **52nd Caribbean Food Crops Society symposium. Gosier, Guadeloupe (07-15/07/2016)**: *How to foster mycorrhiza ? From brakes to levers*. Chave M, Paut R, Angeon V, Dufils A, Lefèvre A, Tchamitchian M 2016.

### PRACTICAL / EDUCATIONAL ARTICLES:

* **INRA technical notebooks:** *Des tomates mycorhizées dès la pépinière pour favoriser la nutrition et la protection des plantes : développement d'un dispositif-pilote*. Julianus P, Perrin B, Chave M. 2019.

* **Data-sheet available online via geco.ecophytopic.fr**: « Les mycorhizes : des réseaux vivants au service de la protection des cultures ». Chave M., Paut R., Perrin B., Dufils A. 2017.
http://geco.ecophytopic.fr/documents/20182/21720/pdf_Cultiver_des_esp_ces_mycorhizes_2.pdf

* **Data-sheet available online via geco.ecophytopic.fr**: « Multiplier des champignons mycorhiziens sur son exploitation ». Chave M., Paut R., Perrin B., Dufils A. 2017
http://geco.ecophytopic.fr/documents/20182/21720/pdf_Multiplier_des_champignons_mycorhiziens sur son_ex_ploitation_1.pdf

* **INRA EA-SAD key-note 2016** : MYMYX, un dispositif participatif de conception d'innovations agroéco-logiques pour valoriser les réseaux mycorhiziens. Chave M., Angeon V. 2016.
PRESENTATION TO PROFESSIONAL OR DECISION-MAKING BODIES:

► **Annual APCA-RESOLIA training (2019)**: Développer les symbioses mycorhiziennes au profit des cultures.
Formation à destination des conseillers agricoles. Hirissou F, Chave M, Auricoste C, Lendure A.

► **Presentation to the DEPHY Ferme Bouches du Rhône network (28/01/2016)**: Comment valoriser les mycorhizes en agriculture ? Paut R., Chave M., Angeon V.

► **INRA EA-SAD key-note 2016**: MYMYX, un dispositif participatif de conception d'innovations agroécologiques pour valoriser les réseaux mycorhiziens. Chave M., Angeon V. 2016.

► **Présentation dans le cadre du Forum territorial Culture Science PACA (22/092016)**: Accompagner la transition écologique en agriculture par le jeu. Paut R, Chave M, Angeon V., Tchamitchian M.

SCIENTIFIC PUBLICATIONS:

► **Agronomy for Sustainable Development** (n°39, p 48) : Codesigning biodiversity-based agrosystems promotes alternatives to mycorrhizal inoculants. Chave M, Angeon V, Paut R, Collombet R, Tchamitchian M. 2019.

► **Phytopathologia Mediterranea** (n°59[2], pp 377-384) : Protective effects of mycorrhizal association in tomato and pepper against Meloidogyne incognita infection, and mycorrhizal networks for early mycorrhization of low mycrotrophic plants. Rodriguez-Heredia M, Djian-Caporalino C, Ponchet M, Lapeyre L, Canaguier R, Fazari A, Marteau N, Industri B, Chave M.

► **Agroecological transitions: from theory to practice in local participatory design** (pp 99-120) : A plurality of viewpoints regarding the uncertainties of the agroecological transition. Magda D., Girard N., Angeon V., Cholez C., Raulet-Croset N., Sabbadin R., Salliou N., Barnaud C., Monteil C., Dubois Peyrard N. 2019. In: E. Audoin, JE. Bergez, O. Therond, dir.

► **Innovations Agronomiques** (n°64, pp 97-111) : Du partage de connaissances à la co-conception d'innovations agroécologiques : Exemple de la mobilisation des mycorhizes en Guyane. Chave M, Angeon V. 2018.

► **Mycorrhiza** (n° 27, pp 719-723) : Rhizophagus irregularis MUCL 41833 transitorily reduces tomato bacterial wilt severity caused by Ralstonia solanacearum under in vitro conditions. Chave M, Crozilhac P, Deberdt P, Plouznikoff K, Declerck S. 2017.

OTHER PRACTICAL WORKS:

► **MYMYX, board-game that aims to encourage knowledge sharing on mycorrhiza and the design of crop systems which will facilitate their success.** (DI-RV-15-15-0020. Enveloppes Soleau n°582327 et n°582992. Marque n°174341832 déposée au nom de l'INRA en classes 9, 28 et 41.) Chave M, Angeon V, Giraud N. 2017.

► **Redesign of the MYMYX tool and production of mobile elements.** N Giraud. 2016.

► **Development of a limited series of 4 MYMYX board games, UE INRA Alénya-Roussillon.** Girard G. 2016.

► **Les Mycorhizes. Kamannyok radio show.** Guadeloupe Première. Chave M., Angeon V. 2016. 22 October 2016
<figure><img src="image_206.png" /></figure>

# Trajectoires

## Hereditary trajectories and the use of pesticides in major agricultural lands

**Launch year:** 2015

**Completion year:** 2019

**Scientific manager**

Carole Barthélémy, LPED Aix-Marseille University

carole.barthelemy@univ-amu.fr

**Partners**

LPED Aix-Marseille University; INRAE Lyon et

Montpellier; Bureaux d'études Ressources

**Financing**

Total cost of the project: € 177,916

Ecophyto grant: € 89,991

## Key words:

Sociological analysis; Hereditary trajectories; Pesticide use; Interdisciplinary approach; Wine growing; Rice growing; Oilseed culture; Tree culture

## Context and main objectives

Within the framework of the national Ecophyto (2018, 2 and 2+) policy, and if we refer to the indicator used to evaluate the effects of this policy - the Number of Unit Doses (Nombre de Doses Unité - NODU), the overall results are disappointing in terms of the stated objective of reducing pesticide use by 50% over a ten year period<fnref n="1" />. Guichard et al.<fnref n="2" /> have highlighted two major obstacles behind this result. The first is related to the lack of a systemic vision of the Ecophyto plan, which is exclusively concentrated on farmers and their consultants, thus failing to pay sufficient attention to "socio-technical blocks"<fnref n="3" />, this aligns neatly with criticism laid out in the Potier report which mentioned a "vision that was too narrowly technical" and "not systemic enough"<fnref n="4" />. The second obstacle concerns the choice and use of monitoring indicators which remain essentially quantitative, thus blocking any chance of noting more intimate changes, or emerging changes.

It is in this context that the "Trajectoires" project seeks to contribute to reflections on changes in phytosanitary usage practices. As regards the limitations of an approach based on an evaluation using only quantitative indicators and figures, this research is mostly built on qualitative data from sociological interviews, taking into account the viewpoints and experiences of farmers and agricultural professionals.

## Principal results and interest in relation to the Ecophyto plan

The sociological research was established on the basis of a comparison between four agricultural regions with different crops and crop systems and looked at how they could reduce their use of phytosanitary products: rice farming in the Camargue, oilseed culture in Alpilles-Sud; wine-growing in the Beaujolais and in the heart of the Hérault; and tree-farming in the Comtat (Nord-Alpilles). 146 semi-guided interviews were completed with 120 agriculturalists and 26 agriculture professionals, all of these were subjected to a comparative content analysis.

In order to encourage inter-disciplinary dialogue, four seminars were organised in each of the survey regions. Each seminar was organised around a visit to the region involving the project team and one or more agriculturalists and agriculture professionals. Group interviews were also organised, which encouraged the environmental science researchers to take interest in what the farmers had to say. An analysis exercise of certain statements was completed by these researchers. Finally, further inter-disciplinary dialogue was opened up between the ergotoxicology and sociology fields in order to confront these two disciplinary visions on the subject of the Certiphyto system.

The first element involved what was a largely stable point across all of the interviews, concerning the farmers' opinions of a systematic application period without any major protection from phytosanitary products. The structuring element was built on the fact that, for each of the regions, we have become aware of the individual and collective initiatives of various age-groups which suggest a desire to take a step back from the previously

<footnote n="1">¹ 2017 supervisory note, Ministère de la Transition écologique et solidaire (French Ministry for Ecological transition and solidarity), Ministère de l'Agriculture et de l'Alimentation (French Ministry of Agriculture and Food), July 2018.</footnote>

<footnote n="2">² Laurence Guichard et al., « Le plan Ecophyto de réduction d'usage des pesticides en France : décryptage d'un échec et raisons d'espérer », *Cahiers Agricultures* 26, no 1, 2017.</footnote>

<footnote n="3">³ Guichard et al., p.1.</footnote>

<footnote n="4">⁴ Report by Dominique Potier, Pesticides et agro-écologie, les champs du possible, November 2014, p. 61.</footnote>