Proceedings of the Ecophyto Research & Innovation
2021 symposium

Umr 1347 Agroécologie, Xavier Reboud

► To cite this version:

Umr 1347 Agroécologie, Xavier Reboud. Proceedings of the Ecophyto Research & Innovation 2021 symposium: Synthesis report of the webinars presenting the results of the Pesticides 2014, JEVI 2016 and PSPE2 calls for projects. Ecophyto Research & Innovation 2021 symposium, Oct 2021, Visio-conference, 2022, 10.17180/8FC8-1102. hal-03762101

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Symposium proceedings

Synthesis report of the webinars presenting
the results of the Pesticides 2014, JEVI 2016
and PSPE2 calls for projects

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7 October – 16 December 2021

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Document translated from French to English by ABAQUE SAS
# Contents

**INTRODUCTION** ................................................................................................................ 4

## GENERAL SYNTHESIS OF THE WEBINARS

Thirty Ecophyto research projects shed light on the future of protecting plants.................. 7

### Webinar 1

In search of natural molecules to promote plant health.................................................... 10

### Webinar 2

Micro-organisms as the defenders of plant health................................................................ 14

### Webinar 3

The integration and optimisation of bio-control .................................................................. 18

### Webinar 4

A world of odours.................................................................................................................... 22

### Webinar 5

Macro-organisms: Sibling rivalry.......................................................................................... 26

### Webinar 6

Resistance as a way to protect crops .................................................................................. 30

### Webinar 7

Companion plants for healthy crops and soil ........................................................................ 34

### Webinar 8

Technical routes towards zero phyto .................................................................................... 38

### Webinar 9

Looking beyond the fields........................................................................................................ 42

## CONCLUSION

Three visions of the Ecophyto Research & Innovation Symposium.................................... 46

## Appendix

Project description sheets........................................................................................................ 49
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## INTRODUCTION

The public is crying out for a reduction in the use of phytopharmaceutical products, which in all cases is an essential part of preserving public health and nature's biodiversity. The successive Ecophyto plans are the embodiment of commitments to reducing the use of phytopharmaceutical products and achieving a pesticide usage policy compatible with sustainable development.

The 2021 Ecophyto Research & Innovation Symposium took the form of a series of 9 weekly webinars presenting and discussing the results of around thirty projects that responded to the 3 calls for projects launched since 2015, concerning:

* Contribution to the progress of biocontrol within the framework of the 'For and On the Ecophyto 2 plan' call for projects (PSPE2);

* The concepts of resistance in terms of pesticides: resisting pests, combating resistance to change in order to reduce risks (Pesticides 2014);

* Development of alternative solutions to the phytopharmaceutical products used in gardens, parks and infrastructures (JEVI 2016).

Research-innovation is an essential lever in achieving these objectives and its importance has been further

accented as the various plans have evolved. The "Research & Innovation" axis of the Ecophyto II+ (axis 2), guided by 4 French government ministries (in charge of Agriculture, Environment, Health and Research) with the support of the Research & Innovation Scientific Orientation Committee (CSO R&I), unites and organises the various research-innovation communities around the concept of developing and furthering knowledge and the tools that will be necessary to achieve the objectives set by the plan in terms of reducing the use of phytopharmaceutical products and the risks associated with them.

One major initiative of the Research & Innovation axis will be to launch calls for research-innovation projects and then make the best possible use of their results. The 2021 Ecophyto Research & Innovation Symposium is therefore aimed at a number of different sectors: research-innovation communities, agricultural and non-agricultural professionals, agricultural supplies manufacturers and professionals, teachers and students, decision-makers and local politicians.

For each webinar session, the results of 2 to 5 projects are presented by their project leaders, followed by participations from one or more expert guests:

* Webinar 1: In search of natural molecules to promote plant health - 7 October

* Webinar 2: Micro-organisms as the defenders of plant health - 14 October

* Webinar 3: The integration and optimisation of biocontrol - 21 October

* Webinar 4: A world of odours - 4 November

* Webinar 5: Macro-organisms: sibling rivalry... - 18 November

* Webinar 6: Resistance as a way to protect crops - 25 November

* Webinar 7: Companion plants for healthy crops and soil - 2 December

* Webinar 8: Technical routes towards zero phyto - 9 December

* Webinar 9: Looking beyond the fields - 16 December

The content of the symposium has been published or posted via a number of different media: on-line posts of the project presentation videos via the dedicated YouTube channel, publication of the webinar

brochures on the dedicated EcophytoPIC page, and the project results summaries alongside the main points raised during the webinars in this document.
ORGANISATION COMMITTEE

Research-Innovation axis specialists

Mariam Yalaoui (INRAE - UMR Agroécologie), Sibylle de Tarlé (INRAE - UMR Agroécologie), Damien Corazzi (INRAE - UMR Agroécologie),

Research & Innovation Scientific Orientation Committee (CSO R&I) members

Xavier Reboud (INRAE - UMR Agroécologie) ; Camille Dumat (Toulouse INP-ENSAT) ; Marc Gallien (DREETS Normandie) ; Philippe Nicot (INRAE - unité Pathologie végétale) ; Michel Duru (INRAE - UMR AGIR) ; Antoine Messéan (INRAE - unité Eco-Innov) ; Caroline Gilbert (Solagro) ; Thibaut Malausa (INRAE - ISA) ; Thierry Bordin (Chambre Régionale Agriculture Centre-Val de Loire) ; Carole Barthélémy (Université d'Aix Marseille)

Ministerial Research-Innovation axis co-organisers

Anne-Sophie Carpentier (MTE) ; Anna Grout (MTE) ; Antoine Le Gal (MAA) ; Enrique Barriuso Benito (MESRI) ; Jordan Barlemont (MSS)

OUTSIDE CONTRIBUTORS

Terre-écos ; AgroParisTech Services Etude ; Agreenium ; La boîte à verbe
The public is crying out for a reduction
in the use of phytopharmaceutical
products, which in all
cases is an essential part of preserving
public health and nature's biodiversity.

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Photo: © Nicolas Bertrand, INRAE
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# GENERAL SYNTHESIS OF THE WEBINARS

## Thirty Ecophyto research projects shed light on the future of protecting plants

The 2021 Ecophyto Research & Innovation Symposium took the form of a series of nine webinars, running from 7 October to 16 December 2021, presenting the results of thirty projects. Success, potential, deceptions, questions... A lot has been learnt from these ambitious projects that sought to discover new ways to regulate pest & disease pressure on plants and reduce the use of phytosanitary products and the risks associated with them. They have opened new positive prospects and evoked the clear need to continue consolidating knowledge on the subject and defining ways to apply it.

### Three tenders launched since 2015

The thirty projects presented during the 2021 Ecophyto Research & Innovation Symposium were all responses to the three tenders that have been launched since 2015. They concern contributions to progress in biocontrol approaches (For and On the Ecophyto II Plan, PSPE2), the concepts of resistance in terms of pesticides (Pesticides Programme) and the development of alternative solutions to phytosanitary products for gardens, parks and infrastructures (JEVI).

As part of the 2021 Ecophyto II+ Research-Innovation axis symposium, nine webinars were organised between 7 October and 16 December, involving almost 500 participants. Natural molecules, micro-organisms, pheromones, macro-organisms, agronomy practices or service plants... Thirty projects, outlining the future plant protection prospects responding to the three tenders that have been launched since 2015.

### Some projects have already met with success and resulting products are actually on the market!

Using living material, such as indigenous or exotic strains of terrestrial or marine fungus, to regulate pest & disease pressure is progressing well as a way to reduce the use of phytosanitary products. A number of the Écophyto projects have demonstrated significant reductions under experimental conditions, such as MilPomBio which has reduced fungicide dosages by 40 to 90% by exploring the use of phosphite based solutions to treat potato blight. The Lipocontrole project uses lipopeptides to achieve as much as 96% disease protection for wheat or green-house tomato crops. Some experiments have managed to develop pioneering organic alternatives for use on tomato crops, as can be seen with the Acarosol project to treat spider-mites affecting solanaceae. Others have even registered and marketed products, as with OptimPhero which has developed pheromones for the biological control of the pine processionary and box tree moth.

### Furthering solutions and knowledge for tomorrow

In addition to those products that have been marketed, identified or which are currently on trial, all of the projects presented are serving to consolidate knowledge for the agro-ecology of tomorrow. Living material has its own codes, and sometimes springs surprises that can cause solutions to fail completely: the Attract my fly project had to deal with this when working on melon fly; they found that using the males as vectors for getting spores of entomopathogenic fungus onto the females of the species in order to control egg production did not meet expectations.

Even when the effectiveness of biocontrol is proven, there is no magic wand. The DicaBio project stands as a clear example of this; natural molecules have shown their effectiveness in the laboratory, but the formulations generally require further work before they can achieve the desired objectives in the field. And the same can be said for the Rhizodia project, where releases of predator ladybirds were not sufficient to control scale insects on blackcurrent; this meant that further research was required to optimise the use of these biocontrol agents or better define the contexts in which they should be used.
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> The presented projects are consolidating knowledge for the agro-ecology of tomorrow.

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Pheromone balls.
Photo: © Nicolas Bertrand, INRAE

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> Finding an idea that works under laboratory conditions is not the end of the story, you need to take it out into the field.

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## Optimise and adjust agricultural practices

If the use of living material appears to be one viable alternative to phytosanitary products, instances of direct substitution, where one solution directly replaces another - like for like, remain very rare. The objective is therefore more about trying to create new solutions that can be integrated into developing general strategies, where the chemical ingredient is no longer the only response. Some products are clearly taking this approach, such as the **Biobot** project, which treats grey mould, caused by the Botrytis fungus, by combining a biocontrol approach with a preventive approach. Others highlight the benefits of service plants to both shelter and encourage the reproduction of auxiliaries, as in the **Pro-Bio-Taupin**, **Acarosol**, **Fleur** and **MacroPlus** projects, or to combat soil parasites, as with the **Serum** and **Biocou** projects.

This combined approach is gaining ground, but many cases still require experimental confirmation to prove their viability. This is another dimension which will need to be taken into consideration in research: that an idea that can be applied in the laboratory is not the end of the story. For many, the presented projects already have one foot in the real world, and often over a number of sites and a number of years to gain a broader view. For example, the **RegPuc** and **Ecoverger** projects propose a number of levers that can be integrated into technical routes to reduce fruit tree pest infestations, or the **Das-Revi** project which studies the possibility of using new disease resistant varieties of grape vines.

## Some important figures about these research projects

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## Collective thinking

Some projects presented during the symposium also question spatial dimensions by looking at the impacts of phytosanitary products on the landscape as a whole and the levers that will need to be implemented to manage such impacts (such as the **Rescape** project). They have even taken the approach further to involve everyone concerned, such as farmers and gardeners, in a participative approach to find suitable alternative real-world solutions to meet phytopharmaceutical product use and impact reduction objectives, as is the case for the **Resyst**, **Trajectoires** and **Althercol** projects.
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> The various
> Ecophyto plans
> consolidate the
> commitments
> undertaken to
> respond to this
> obligation and
> achieve a
> sustainable
> degree
> of plant
> protection
> product use.

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Agricultural landscape of the ZAPVS, cultivated field and bordering hedge, Rescape Project. Photo: © Céline Pelosi, INRAE

## Social issues, widespread distribution

The reduction of the use of phytosanitary products and the restriction of the risks associated with them is a subject that concerns the whole of society. Some projects have clearly raised the question of how acceptable biocontrol solutions really are for farmers... and also a general public that is developing an increasing curiosity of how their food is actually produced. The **2021 Ecophyto Research & Innovation Symposium** is not just aimed at the research-innovation communities but also at a wider public: agricultural and non-agricultural professionals, industrial and agro-supplies businesses, teachers and students, decision-makers and local politicians. The results will be interpreted on a number of different levels: from a purely scientific perspective, with articles published in peer reviewed academic journals, and on a broader scale through written summaries and videos on the projects published on a dedicated Youtube channel. All useful information can be found on the EcophytoPIC web-site.

## The Ecophyto II+ Research-Innovation axis

The reduction of the use of phytosanitary products is a major issue for society as a whole, as is the need to improve the health of the soil and all living things. The various Ecophyto plans consolidate the commitments undertaken to respond to this obligation and achieve a sustainable degree of plant protection product use, by reducing their use and restricting the associated risks **The Ecophyto II+ plan "Research & Innovation" axis** will involve different research communities working together to produce and complete the knowledge and tools necessary to achieve these objectives. It is led by the ministries of Agriculture and food, Ecology transition, Solidarity and healthcare as well as the Innovation and research in higher education; it also benefits from the expertise of the Research & Innovation Scientific orientation committee (CSO RI).

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Spanish fly, auxiliary insect, predator. Photo: © Nicolas Bertrand, INRAE
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In search of natural molecules
to promote plant health

<figure><img src="image_21.png" /><figcaption>*Arabidopsis Thaliana* plants in the greenhouses of the Jean-Pierre BOURGIN institute.
Versailles, 30 September 2021. Photo: © Nicolas Bertrand, INRAE</figcaption></figure>

Session programme

<table><tr><td>► DICABIO</td><td>Evaluation of Dicaffeoylquinic acid as a natural Biocontrol substance (PSPE2)</td><td>Myriam Siegwart (INRAE Avignon)</td></tr><tr><td>► LIPOCONTROLE</td><td>The search for new lipopeptides that could be used as biopesticides, by screening a collection of Pseudomonas (PSPE2)</td><td>Philippe Jacques (University of Liège)</td></tr><tr><td>► DESHERBAL</td><td>Development and study of the effectiveness of allelopathic substances to improve weed control for particularly resistant species (JEVI 2016)</td><td>Claire Richard (Clermont - Ferrand Chemistry Institute)</td></tr></table>

**EXPERT GUEST: Cédric Bertrand**, University of Perpignan, Head of the Chemistry Department, President of the PO²N Group, President of the Biocontrol and Integrated Biological Protection Academy, “Sharka” Chair of the UPVD Foundation, Scientific Director of AkiNaO

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You can see the complete recording of this session on [the symposium's YouTube channel](https://www.youtube.com/symposium)
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# Biocontrol, prospects and specifics for a number of natural substances

The DicaBio, Lipocontrole and Desherbal projects present the potentials and opportunities raised by natural molecules for the conception of biocontrol products... while also highlighting certain specific characteristics of these substances, which will need to be made clear well in advance before the full potential of these solutions can be realised.

What do peach-trees, the Bacillus and Pseudomonas bacteria and the Mediterranean Rock Rose (Cistus) have in common? They have all inspired projects that apply a single logic: the use of natural molecules to develop biocontrol solutions which could become viable alternatives to synthesised phytosanitary products as well as being respectful of the environment and human health. The results of these three projects, DicaBio, Lipocontrole and Desherbal respectively, have raised promising prospects for the Ecophyto 2+ Research-Innovation plan.

For all three projects, the rapidly biodegradable nature of the raw materials under consideration is worthy of some note. The active ingredients in question, diCT and diCQ acids for DicaBio, lipopeptides for Lipocontrole or terpenes for Desherbal, all bear this characteristic. Xavier Reboud, Ecophyto 2+ axis 2 coordinator, insists on this difference to a very large number of synthesised products.

## Combating resistance

Another projected, but as yet unproven, advantage of these biocontrol solutions is their potentially long action durations when compared to synthesised products which often seem to facilitate increased pest resistance instead. This is especially the case for the aphids targeted by DicaBio. According to the researchers, the two identified substances, diCT and diCQ, currently present little risk of resistance compared to synthesised pesticide products. Only one of the aphid clones tested has proven to be unaffected. "A better knowledge of the origin of this phenomenon will help us to identify how these substances work, and achieve a better understanding of the risk of aphids adapting to them", says Myriam Siegwart, researcher at INRAE Avignon.

In the case of Lipocontrole, this is beginning to be taken into account and University of Liège professor, Philippe Jacques explains that he has "high hopes" that the resistance mechanisms of the fungus pathogens targeted by his project will only be able to activate "very slowly", meaning that the solution will have a long-term action in protecting large-scale crops either in complement or as a substitute to chemical fungicides.

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> Synthesised chemical products are now forbidden for garden use.

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## Need to work on the formulation

For their part, Desherbal highlights the visual and highly educative nature of certain plant interactions. For this project, terpenes produced by a number of different plant species were tested for their capacity to restrict the germination of weeds at the bases of trees and in flower beds. This has been done in partnership with the town of Aubière (Puy de Dôme) in its town hall conservation gardens and the local school courtyard. "Synthesised chemical products are now forbidden for garden use" reminds Claire Richard, researcher at the Clermont-Ferrand Chemistry Institute. "The town council is very interested by our work, and plans to continue and broaden the scope of the trials."
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> We need to identify the pests, weeds or pathogens that the product could be used to control... but we also need to know about the "non-target" species.

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The three scientists' experiences have also led them towards work orientations specifically related to the nature of the substances they are working on. For example, researchers need to remain objective when encouraging results are produced under laboratory conditions and concentrate on finding the best way to use the new molecules in-situ. The DicaBio consortium is hoping that a "micro-emulsion" approach will resolve the very low penetration factor of diCT and diCQ in the plant tissues they are being used to protect, they will need to find an industrial partner to help them achieve this. Whereas for Desherbal, the actions of the terpenes present in Leylandii cypress needles has been tested as a concentrate or a mulch, to try to achieve a "very significant" reduction in the germination and growth rates of the clover species selected as control subject. For Lipocontrole, the finalisation of a formulation able to guarantee the conservation and potency of lipopeptides remains one element that still requires more work.

## A very broad field of research

This part of the research process highlights the advantage of collaborating with formulation specialists, especially phytosanitary product manufacturers. This also serves as a reminder that, in spite of the conclusions and knowledge gained, all three of these projects still require further research work. None of them are ready to be registered for commercialisation. "That's the way thing go when you are innovating from the ground up," says Xavier Reboud. "The relatively long period of time between the start of a project and its possible market launch means that one should never delay launching such a research program."

The selectivity of the substances being studied is one of the things that remains to be evaluated before considering any potential registration for commercialisation. In other words, their operational scope: what species will be affected? This is not a minor issue. We need to identify the pests, weeds or pathogens that the product could be used to control... but we also need to know about the "non-target" species, what you could call the potential collateral damage. DicaBio has made a start by establishing that diCQ is not toxic to bees. At the same time, the project leaders are planning a deployment on other targets beyond aphids, including certain fungal diseases affecting wheat, using diCT in particular. As for Lipocontrole, the performances measured under greenhouse conditions concern wheat and tomato crops, and have lead to disease protection levels of up to 96 %. However the planned applications are broader, the project hopes to extend to cover septoria leaf blotch on wheat, sclerotinia stem rot on rapeseed and grey mould on vines and tomatoes. The toxicological risks to other organisms are currently being evaluated by another project, Ceres (2020-23). And finally, Desherbal warns that it still remains to be proven as to whether terpenes from Leylandii Cypress needle mulch are totally harmless to soil-life. Other terpene rich plants are still to be identified and tested.

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Laboratory tests on the anti-fungal activity of P. syringae bacteria. Anti-fungal bacteria inhibiting fungal growth (inhibition zone). The LIPOCONTROLE project.

Photo: © Alexandre Bricout, University of Lille
► **The views of Cédric Bertrand, professor at the University of Perpignan**

**"Sourcing the raw materials is a question which must not be overlooked!"**

The guest expert for this webinar, Cédric Bertrand, confirms the trends raised by the various accounts. "Remember that it all comes from observing nature! The bio-active ingredients we are talking about here are chemical mediators which allow the plant to interact with the surrounding micro and macro-organisms. They use a number of operational methods to play a protecting role for the plant: fighting off bioagressors (insects, fungus, weeds, etc.) or stimulating its defences."

However, he used another project, *Folivarde*, to warn about the danger of just shifting the problem over to other crops The plant from which the project's main active ingredient is taken, *Dittrichia viscosa*, grows in waste grounds. "To be able to set up a large-scale production, you need to immediately ask the following question: are we able to harvest enough *Dittrichia viscosa* in the existing waste grounds, or will special farms need to be set up?" In this case, he mentions another important point: ensure that the production of this *Dittrichia viscosa* does not need synthetic phytosanitary products, in this case the problem would just be shifted from the protected crop to the one that is being grown to produce the natural molecules to protect it.

> It all comes from observing nature!

And then there is the question of regulations: how can they be adjusted to allow this set of tools to be put on the market, whilst also allowing for the environmental impact that these molecules might have in relation to their scope of activity? The current commercial registration processes are long and complex, especially for complex extracts even if they can be used at lower doses than synthetic products. In the case of the *Folivarde* project "the first ecological screening processes, started with five plants back in 2010 but the 'race' from laboratory work to field trials may not finally result in commercial registration before 2024."

<figure><img src="image_29.png" /></figure>

A

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B

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C

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D

Effects of rock rose on the germination and growth of clover. A: control, 9 days after seeding; B: pot with cystus planted on right, 9 days after seeding; C: control, 5 months after seeding; D: pot with cystus plant on the right, 5 months after seeding. DESHERBAL project. Photo: © Claire Richard, ICF/CNRS
# Micro-organisms as the defenders of plant health

<figure><img src="image_33.png" /></figure>

Fungus strains grown in petri dish. Some of these micro-organisms are used to protect plants from disease or other bioaggressors. INRAE, Sophia-Antipolis. Photo: © Nicolas Bertrand, INRAE

## Session programme

<table><tr><td>► BIOTI-VIGNE</td><td>Biotisation of young nursery-grown grapevines to prevent trunk diseases (PSPE2)</td><td>Marc Fermaud (INRAE Bordeaux)</td></tr><tr><td>► Systemyc</td><td>Conception of crop-growing SYSTEmS based on the use of MYCorrhization for the biocontrol of earth-bound pests that damage tomato crops (PSPE2)</td><td>Marie Chave (INRAE Antilles-Guyane)</td></tr><tr><td>► NABUCO</td><td>New Bio-Marine Agents Used for Biological Control (Nouveaux Agents Bio-Marins Utilisables en Contrôle biologique) (PSPE2)</td><td>Thomas Guillemette (Université d'Angers)</td></tr><tr><td>► AttractMyFly</td><td>Development of attractors and entomopathogenic fungus entomovection processes to control melon fly Bactrocera cucurbitae (PSPE2)</td><td>Laurent Costet (CIRAD)</td></tr></table>

**EXPERT GUEST:** Bruno Le Cam Research Director at INRAE Angers, coordinator of the ENFIN! project (ANR EcoPhyto Maturation) - Development of a new plant protection concept applied to apple scab disease

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You can see the complete recording of this session on the symposium's YouTube channel
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# Micro-organisms and biocontrol, a research field full of promise and surprises

The use of micro-organisms as biocontrol agents raises many interesting prospects for the reduction of the use of phytosanitary products. The Ecophyto research projects that are working in this field must never lose sight off the fact that: living materials have their own codes, which are far from linear and sometimes full of surprises. This is a summary of some of the, sometimes unexpected, lessons learned by Ecophyto projects.

## 1+1 does not always equal two

The Bioti-vigne project has managed to reveal the potential of a strain of *Pythium oligandrum*, this fungus is likely to be able to protect vines from trunk diseases. It is effective in protecting against two major pathogens known to cause these 'diseases'. The project focuses mostly on the potential interest of bringing the vines into contact with this strain at a nursery level, long before the first signs of the diseases appear. The key factor is ensuring that the *Pythium oligandrum* remains effective over the long term. "The vine sees it as both an ally and a potential aggressor", says Marc Fermaud, INRAE researcher. It doesn't exactly roll out the red carpet, but nor does it try to eliminate it, so the *Pythium* can hang around for a while." And another piece of good news: the very good protection levels achieved on grafted plants with the two most common root-stock varieties used in France and other wine-growing countries. However *Pythium oligandrum* does not currently have a synergistic effect with other bacteria strains with demonstrated high biocontrol potential. "Intuitively speaking we are thinking of complementarity, but our tests have not revealed any additional affects: the advantages of each individual strain are not cumulated when different strains are combined", says Marc Fermaud.

> Can marine fungus save the apple?

<figure><img src="image_36.png" /></figure>

> The Mer Molécules Santé laboratory in Nantes has a library of 900 strains!

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

Trichoderma atroviride filament wrapping and parasiting filaments of the phytopathogen agent Botrytis cinerea. Scale: 10 µm. FBC: filament of *B. cinerea* ; FTA: filaments of *T. atroviride* ; SBC: spores of Botrytis cinerea. **Nabuco** project Photo © Franck Bastide, IRHS
<figure><img src="image_39.png" /></figure>

Vine with Esca, the initial symptoms of the disease are a discolouration (yellow and red) of the leaves. Over the more or less long-term, the disease will cause the grape vine to wither and die. Château Couhins, Villenave d'Ornon, Gironde, FRANCE, 21 June 2018.

Photo: © Nicolas Bertrand, INRAE

## Solutions from the sea

Can marine fungus save the apple? This rather surprising concept is producing encouraging results.

"New bio-marine active ingredients used for biological control"; this is what the project's acronym, **Nabuco**, stands for and that speaks for itself. The idea, not necessarily the first thing that would come to mind, involves looking for fungus in the marine ecosystem that can be used to protect crops on dry land. "Marine biodiversity is very rich, we are synthesising molecules many of whose properties have yet to be identified" says Thomas Guillemette, researcher at the University of Angers. "The Mer Molécules Santé laboratory in Nantes has a library of 900 strains! " **Nabuco** focuses its work on 50 marine strains of *Trichoderma*. According to the criteria analysed by the project, four of these strains have revealed a potential for cultivation and high degrees of antagonist activities far superior to the four strains currently on the market. **Nabuco** has also isolated twelve secondary metabolite extracts from these fungus. There is potential there too. One of these metabolites, and the strain it comes from, is being tested as a preventative treatment of stems and leaves without using any wetting agent, at this point, and already achieving protection levels of around 25% for septoria leaf blotch on wheat and apple scab disease. Could these results raise the interest of a commercial producer?

## Males, not always the best vectors for getting at the females

Melon fly does a great deal of damage to cucurbitaceae on the Island of Réunion. Most of the damage comes from the females when they lay their eggs. However the existing pheromone traps only work for males... The **Attract my fly** project originally aimed to use the males to transport *Beauveria hoplocheli* spores to the females, this being a proven pathogen affecting the female fly. The principle is as follows; capture the males, contaminate them, then release them so that they transmit the spores to the females during mating. Unfortunately "The results are below expectations", says Laurent Costet, researcher at Cirad. "The females are only affected if they mate within 24h of the male being contaminated." and the transmission of *Beauveria* using only this approach will doubtless never be enough. The most promising leads involve finding a way to trap the females. **Attract my fly** has field tested a number of kairomones that have been singled out as interesting under laboratory conditions. This combination will be applied as part of a known deployment strategy perspective under the name "attract and kill".
<figure><img src="image_40.png" /></figure>

The 50 farmers involved... noted that some of their existing habitual practices also encouraged mycorrhizal symbiosis

<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /></figure>

The acceptability of this kind of solution is an integral part of the project... Not just for farmers but for consumers and society as well.

<figure><img src="image_43.png" /></figure>

## Of micro-organisms... and men

Two projects, one of which has reached its finality and the other that has only just started, highlight one of the success criteria for micro-organism-based biocontrol solutions: the human factor!

Systemyc concentrates on interactions between plants and soil micro-organisms, such as mycorrhizal symbiosis. Whilst the technical aspects were evidently part of the research scope, the "social acceptance" dimension of any potential solutions would also have to be taken into account. A collective educational plan alternating workshops and surveys provided a means to approach the complexity and incertitude of the mycorrhizal processes. "The 50 farmers involved discovered some of the phenomena, which helped them to comprehend these invisible mechanisms... but they noted that some of their existing habitual practices also encouraged mycorrhizal symbiosis and this helped some of them to feel better about their working methods", says Marie Chave, INRAE researcher. A version of this educational plan will be made available for agricultural training programmes.

The Enfin! project (2022-2024) combines two patented INRAE inventions for the treatment of *Ventura inaequalis*, which causes apple scab rot. The first of these aims to force this fungus to reproduce with non-pathogenic strains, such that its offspring become non-virulent when they return the following spring. As well as reducing symptoms, this will significantly reduce the spread of this pathogen. The second patent involves applying the same non-pathogenic strains on the apple trees in spring in order to generate protective immunity in the trees. "The acceptability of this kind of solution is an integral part of the project," says Bruno Le Cam, scientific director (INRAE). "Not just for farmers but for consumers and society as well. We will try to identify the potential hurdles in order to outline ways to remove them where necessary. These limitations are doubtless not only related to technical, regulatory or economic issues."

<figure><img src="image_44.png" /><figcaption>Female melon fly affected by *Beauveria hoplocheii*.
Attract my fly project.
Photo: © Brice Derepas, CIRAD</figcaption></figure>
# The integration and optimisation of bio-control

<figure><img src="image_45.png" /><figcaption>a)</figcaption></figure>

<figure><img src="image_46.png" /><figcaption>b)</figcaption></figure>

<figure><img src="image_47.png" /><figcaption>c)</figcaption></figure>

a) Fields of various lettuce and chicory crops b) Field of new potatoes on the isle of Noirmoutier. Photo: © Jean-Marie Bossennec, INRAE

c) Monitoring ground beetle populations. Photo: © Christophe Maitre, INRAE

## Session programme

<table><tr><td>► MilPomBio</td><td>Potato blight: identification and development of biocontrol products for integrated crop protection (PSPE2)</td><td>Marie Turner (UMR Vegenov)</td></tr><tr><td>► BIOLIM</td><td>Innovative biocontrol techniques for the management of crop damaging land molluscs: Looking for an evaluation methodology suited to their ecophysiology (PSPE2)</td><td>André Chabert (ACTA)</td></tr><tr><td>► PRO-BIO-TAUPIN</td><td>Evaluation of biocontrol solutions for dealing with wireworm damage (PSPE2)</td><td>Jean-Baptiste Thibord (ARVALIS)</td></tr><tr><td>► BIOBOT</td><td>Optimisation, including the global and integrated management, of biocontrol solutions for the treatment of grey mould on grapevines (PSPE2)</td><td>Nicolas Aveline (INRAE Bordeaux)</td></tr></table>

**EXPERT GUESTS:** Anthony Ginez, Experiments officer at APREL

**Thomas Pressecq**, PhD student and researcher at the INRAE, working on a thesis entitled "Développer des outils d'aide à la décision pour optimiser l'usage du biocontrôle" (developing decision-making support tools for the optimisation of the use of biocontrol)

<figure><img src="image_48.png" /></figure>

You can see the complete recording of this session on [the symposium's YouTube channel](https://www.youtube.com/thesymposium)
<figure><img src="image_49.png" /></figure>

# Biocontrol, a lever to be integrated into a combination of solutions

There's no magic wand. If biocontrol appears to be viable alternative to synthetic phytosanitary products, instances of direct substitution, where one solution replaces the other, like for like, remain very rare. The objective is therefore more about trying to create new solutions that can be integrated when developing generalised strategies, where the chemical ingredient is no longer the only response. Here is a summary of some of the levers available to optimise the integration of biocontrol agents.

## Combining biocontrol with species selection to reduce dosages of synthetic phytosanitary products

The MilPomBio project aims to identify which solutions, out of a list of 42 already existing or preliminary stage biocontrol products, show the most potential for potato blight control. The most promising ones will be tested in the field after the completion of a laboratory experimentation phase. "We have identified some phosphite-based solutions as promising," says Antoine Menil, researcher for Vegenov. We have tested them in combination with different doses of a traditional fungicide, whilst also monitoring the genetic factor, each variety has its own degree of sensitivity to blight." This is one way of proposing more applied solutions. In this way, it has been seen that the dosage of traditional fungicide can be significantly reduced, but not uniformly across all varieties. For some, the use of fungicides can be reduced by up to 90% without affecting crop production. For others, this figure is closer to a 40% reduction in fungicide use.

<figure><img src="image_50.png" /></figure>

> For some varieties, the use of fungicides can be reduced by up to 90% without affecting production

<figure><img src="image_51.png" /></figure>

<figure><img src="image_52.png" /></figure>

Biotic and abiotic relations of slugs with their environment. The number of listed publications (between 1992 and 2018) for each biocontrol method is shown here in brackets.

Image: © André Chabert, ACTA
Integrating biocontrol into technical routes and associating “service plant” crops

Biolim has set its sights on slugs. Again, the objective was to establish a range of potential biocontrol leads to deal with this pest: natural substances, predators or “service plants”, such as using repulsive mustards as intermediate crops. Each biocontrol agent has its strengths and weaknesses but André Chabert, researcher at ACTA, tells us that one common denominator has been identified: “As far as slugs are concerned, it is quite difficult to separate agricultural practices from biocontrol implementation, whatever the selected solution!”. So performances need to be evaluated in terms of global coherency.

In the case of Pro-Bio-Taupin, the project's initial spectrum was also very broad. To deal with wireworm, a highly damaging beetle larva against which chemical solutions are becoming increasingly rare and ineffective, researchers have explored a number of possibilities, such as natural substances containing glucosinolate. “Large quantities are needed for it to work”, says Jean-Baptiste Thibord, researcher at Arvalis. One interesting approach would be to cultivate plants that produce these substances directly in the field, either before or during the cultivation of the crop to be protected.”
Today the intermediate crop concept is more advanced than that of companion plant associations. In all cases, these strategies require farmers to adjust their practices and crop-rotation methods.

“As far as slugs are concerned, it is quite difficult to separate agricultural practices from biocontrol implementation, whatever the selected solution!”

<figure><img src="image_53.png" /><figcaption>Grey slug on a corn leaf. BIOLIM project. Photo: © André Chabert, ACTA</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>Wire-worm, beetle larvae
Photo: © Serge CARRE, INRAE</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>Grey mould on grapes. BIOBOT project.
Photo: © Nicolas Aveline, IFV</figcaption></figure>
## Avoid damage through epidemic-surveillance and prevention

André Chabert, *Biolim* project leader, insists that the presence of pests in fields must be analysed in detail. “Detectors and traps allow us to survey slug populations and they are our first ally in crop protection, including biocontrol! "

In wine growing, grey mould, caused by the Botrytis fungus, reduces harvests and grape quality. For the *Biobot* project leaders, the 14 currently approved biocontrol solutions propose an interesting alternative. The project aimed to define the best ways to integrate biocontrol into grapevine cultivation practices. For Nicolas Aveline, what they have learnt is its complementarity with preventive measures. “One of them involves reducing the number of leaves on the vine to air the fruit and reduce the conditions that favour fungus proliferation. As well as a direct effect on the pathogen, this practice also has the advantage of making it easier to apply biocontrol products onto the grapes”, he explains.

<figure><img src="image_56.png" /></figure>

### ► The views of Anthony Ginez, experiments officer at Aprel

Anthony Ginez works at the Provençal vegetable research and experimentation association (APREL) on the protection of sheltered crops. For him, biocontrol must be used as part of a combined approach since its effectiveness is much less clear-cut than that of chemical solutions. “In my work, it is essential to activate more than one lever. Biocontrol is therefore combined with physical pest-control barriers such as nets, an active biodiversity that promotes the presence of auxiliaries and, as a last resort, phytosanitary products.” Sometimes he even combines biocontrol... with biocontrol! “For example, with the strawberry aphid we combine lacewing and ladybirds, as predators, with potassium salts of fatty acids, as a natural substance with an insecticide action that does not have a negative impact on the auxiliaries. "

> It is essential
to activate
more than one
lever

<figure><img src="image_57.png" /></figure>

## DST to support of biocontrol

Farmers often use Decision Support Tools (DST) to protect their crops. This technical term covers various ways to guide farmers in their practices. And biocontrol is no exception to this, the Ecophyto projects integrate them frequently in their work. In the case of *MilPomBio*, a DST is also used for the chemical treatments, Mileos in this case, it is used to define the real necessity and the right time to apply. *Biobot* has used the “Ciliberti index” to orient the best possible fungicide applications and has improved it for use with biocontrol. In other cases, specific DSTs have been developed, such as Deci Control which is currently at the prototype stage with the Provençal vegetable research and experimentation association (Aprel) for use in market gardens. “At the moment we are mostly working on a local scale, but the aim is to program the tool for nationwide use.” says Ginez, Experiments officer at APREL.
<figure><img src="image_58.png" /></figure>

Isomate®: pheromone diffuser in an integrated or organic fruit tree orchard. Photo: © Jean-Charles Bouvier, INRAE

## Session programme

<table><tr><td>► SEMIOTRAP</td><td>Development of a biocontrol trap to survey and monitor the palm borer moth, <i>Paysandisia archon</i> (JEVI 2016)</td><td rowspan="2">Johann Fournil (M2i)</td></tr><tr><td>► OPTIM'PHERO</td><td>Optimising Pheromones and Transposing the results obtained with the pine processionary to other insects, such as pest lepidoptera in Non-Agricultural Zones (<u>PSPE2</u>)</td></tr><tr><td>► AM&BAS</td><td>Agro-messages and treatment of insect pests in seedling production and large-scale crops (<u>PSPE2</u>)</td><td>Brigitte Frérot iEES Paris</td></tr></table>

EXPERT GUESTS: Emmanuelle Jacquin-Joly, Research Director at INRAE - iEES Paris

Philippe Lucas, Research Director at INRAE - iEES Paris

Denis Thiery, Research Director at INRAE - UMR SAVE

<figure><img src="image_59.png" /></figure>

You can see the complete recording of this session on the symposium's YouTube channel
<figure><img src="image_60.png" /></figure>

# Pheromones and kairomones, a chemical control solution from nature

We are all surrounded by odours which can attract us, disgust us or even guide us... And it's the same for pest insects! Sexual confusion has been a genuine success in French vineyards, research on using pheromones and kairomones to deal with crop-damaging pests is leading to a number of promising prospects. In this vast scientific and technical field, the Semiotrap, Optim'Phero and Am&Bas projects are shedding light on these intriguing prospects.

You can do anything with odours: trap, restrict mating or keep pests away from crops! This is what has been revealed by the Semiotrap, Optim'Phero and Am&Bas projects which are part of axis 2 of the Ecophyto plan. Whilst sexual confusion is now in use in 17% of French vineyards, chemical signals, especially pheromones and kairomones, are becoming increasingly promising solutions for biocontrol, with one particular major advantage: odours are environmentally friendly.

## Identify, formulate and observe the pheromone's capacity to attract

The Semiotrap project, originally set up to protect the heritage palm-trees of the Mediterranean coastlines from damage caused by *Paysandisia archon*, has made some major advances for biocontrol methods in general. "We had a large number of scientific obstacles to get around: would it be possible to define, synthesise and use a pheromone to trap this insect? If yes, how do we use it? How do we apply it? What kind of trap will we need to make for this insect?" says Johann Fournil of M2i. The results have been a genuine success, the teams have managed to identify, define and synthesise the pheromone to conclusively demonstrate its power of attraction over males and females. But one of the specific things about the insect, a large hovering moth, is that it has turned out to be highly resistant to traps. "We considered two types of trap: electrical and net", he continues. None of the solutions were effective since the moth resists electrical charge and does not fly into the net." Research will continue in 2022-2023 to find a suitable trap and market the system.

## Paint-ball and sprayers for sexual confusion, some solutions have already been put on the market

However, Optim'Phero is currently already on the market! "At the end of this Ecophyto project, we received two commercial registration approvals and have therefore been able to put two new products on the market", says Johann Fournil, M2i being also a partner for this project. This time pheromones are being used to hinder the reproduction of two major pests that cause both environmental and physical damage in France and Europe: the pine processionary and the box tree moth. The research work has allowed us to develop effective solutions. "For the pine processionary, we achieved a significant reduction of 78% in the population of the moths with a product action period running from June right through to September. For the Box tree moth, we have combined our product with a larvicide and this has resulted in dividing the population of moths by a factor of ten." The project team has taken things even further with the development of two new application method innovations. For the Box tree moth, the pheromones are applied with a hand sprayer, and for the Pine processionary, the researchers came up with the idea of... paint-ball! The solution is produced as a gel and packed into biodegradable balls, made exclusively from natural raw materials, which can be easily fired onto the tree using special launchers.

<figure><img src="image_61.png" /></figure>

> The teams have managed to identify, define and synthesise the pheromone to conclusively demonstrate its power of attraction over males and females

<figure><img src="image_62.png" /></figure>

> We have already received two commercial registration approvals and have therefore been able to put two new products on the market

<figure><img src="image_63.png" /></figure>
<figure><img src="image_64.png" /><figcaption>Paysandia archon adult with wings spread.
Photo: © Jean Drescher, INRAE</figcaption></figure>

<figure><img src="image_65.png" /><figcaption>Pine processionary caterpillar.
Photo: © Olivier Bertel, INRAE</figcaption></figure>

## Kairomones are the new selection criteria for cultivated plant varieties

In the world of odours, the Am&Bas project is interested in another type of chemical message, the kairomone. Whilst pheromones are used to communicate within a single species, kairomones communicate between different species to cause the pest's behaviour to change. They are frequently emitted by plants to attract insects, they can also be used to trap them or alter their egg-laying behaviour. This is what the project team decided to base their work on, they have demonstrated the effectiveness of a kairomone in dealing with two pests affecting large-scale crops: the *Tychius aureolus* weevil on alfalfa and the European corn borer, *Ostrinia nubilalis*. Even if suitable trap designs remain elusive, the identification of this kairomone is opening up some very wide ranging genetic selection prospects, as Brigitte Frérot of the INRAE, explains: "The cultivated varieties are all very attractive! They produce the same volatile compounds that interest the pest insects. This data could orient selection towards resistant varieties that do not produce this kairomone." The Am&Bas project has therefore provided a solution that can concentrate egg-laying at a distance from the crops or trap large numbers of these two pests. It also opens up research prospects where kairomone production can become a plant variety selection criterion.

* **The views of Emmanuelle Jacquin-Joly and Philippe Lucas, INRAE research directors**

> **"Machine Learning and 3D modelling are accelerating chemical signal discoveries that could be useful for biocontrol"**

Attract, repel, block. These are the three main types of chemical signals that the INRAE staff working under Emmanuelle Jacquin-Joly are looking for. The researchers use a genuine innovation to identify them: the reverse chemical ecology approach: "we work on the insect's olfactory receptors, then we work our way back to the behaviour to study the effectiveness of the chemical signal for biocontrol." This approach is based on divergences between the olfactory receptors: each family of insects has its own receptors, which are also very different to those of vertebrates. In this way the treatment is targeted and there is no collateral damage to auxiliaries. "Once the desired receptor has been identified, we use it to screen the volatile molecules in vitro and in silico, and then we model its 3D structure in order to see which molecule will be able to correspond with its geometry and therefore produce a response," she explains. "It was quite

difficult to use this method before because we couldn't do 3D modelling: but this is now possible thanks to cryogenic electron microscopy!" The coupling of the chemical signal and receptor is then checked experimentally, and then the responses of the pest insect were tested. "With this method, between 60 and 80% of the tested molecules turn out to be active", says the research director. Philippe Lucas, who is also an INRAE research director, uses the PheroSensor project as an example to explain another application of olfactory receptors; the project was set up to develop bio-inspired sensors based on insect receptors which detect the presence of pest insects without capturing them since they can detect the pheromones that they emit into the environment. "To test out the concept, we are concentrating on 3 pests that cause considerable economic damage: the red palm weevil, the fall armyworm and the African cotton leaf worm" he explains.

> In this way the treatment is targeted and there is no collateral damage to auxiliaries
► **The views of Denis Thiery, INRAE Research Director**

**Sexual confusion in grapevines: avoid the white zones!**

When it comes to reducing the use of phytosanitary products, wine-growing is a major issue. “Grapevines represent 3% of French agricultural land for 20% of the national use of phytosanitary products”, explains Denis Thiery, INRAE Research Director. Using pheromones to provoke sexual confusion is becoming one of the leading organic farming techniques thanks to its 80 to 90% success rate, but it does have a few limitations: pest insects tend to mate at the end of the day when pheromone concentrations are quite low, the summer treatment period extended to the end of September, effectiveness depending on the pest population levels, difficulties in hot or windy climates, pheromones absorption by leaf cuticles. Denis Thiery proposes three possible ways of dealing with these limitations: “You have to avoid the white zones, where pheromone concentrations are too low, and use modern diffusion methods, such as the Puffer®, instead of passive distribution methods. We also need to gather a lot more data on pheromone concentrations using sensors placed directly in the fields.”

<figure><img src="image_66.png" /><figcaption>Tychius aureolus, the alfalfa seed weevil.
Photo: © Serge CARRE, INRAE</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>Larva of the European corn borer (Ostrinia nubilalis L.) inside a corn stem.
Boigneville (91720). Photo: © Pascal Thiebeau, INRAE</figcaption></figure>
# Macro-organisms: Sibling rivalry...

<figure><img src="image_68.png" /><figcaption>Adult *Perillus bioculatus* (two spotted stink bug) feeding on *Leptinotarsa decemlineata* (Colorado beetle) eggs. Photo: © Jeanne Daumal, INRAE</figcaption></figure>

## Session programme

<table><tr><td>► Rhizodia</td><td>Regulation of scale insect populations by releasing the scale-eating ladybird, Rhizobius lophantae (PSPE2)</td><td>Maria-Marta Fernandez (CTIFL)</td></tr><tr><td>► BIOCCYD</td><td>Biological control of the codling moth, Cydia pomonella (BIOControl of CYDia pomonella) (PSPE2)</td><td>Nicolas Borowiec (UMR ISA)</td></tr><tr><td>► ACAROSOL</td><td>Biological control of red spider mite on Solanaceae (PSPE2)</td><td>Marie-Stéphane Tixier (SupAgro)</td></tr></table>

EXPERT GUEST: **Cécilia Multeau**, Biocontrol Innovation and partnership officer – INRAE

**Marc Kenis**, Invasion ecology risk analysis manager – CABI

<figure><img src="image_69.png" /></figure>

You can see the complete recording of this session on the symposium's YouTube channel
<figure><img src="image_70.png" /></figure>

# Managing auxiliary populations to regulate pests naturally

Finding the right balance between pests and auxiliaries in order to naturally and sustainably regulate pest pressure on agricultural crops. Population dynamics are at the heart of organic pest control methods involving macro-organisms. The approaches of these three projects, Rhizodia, Acarosol and Bioccyd, range from introducing new species to increasing existing population levels; they are producing results that are going beyond just reducing the Treatment Frequency Index (TFI). They are defining the criteria for a highly effective agro-ecology.

Using auxiliary macro-organisms (invertebrates, insects, spider-mites or nematodes) highlights the potential for naturally regulated pest populations. The question of achieving a balance between auxiliary and pest populations is central to organic treatment, regardless of whether this involves increasing the population levels of an already existing species or introducing new species.

## Rhizodia investigated the overwintering of auxiliaries

The Rhizodia project sheds a lot of light on this issue as it concerns the biological control of scale insects on black-current crops. "Black-current farmers have been subjected to pest damage for the last 15 years, this has quite often forced them to tear up whole fields of plants", says Maria-Martha Fernandez of the Inter-professional Fruit and Vegetable Technical Centre (CTIFL). The project teams have identified the Rhizobius lophantae ladybird as a means to deal with this scale insect, this ladybird species is relatively easy to raise in large numbers, which is an essential criteria for biological control methods. There was a twofold objective: establish whether regulation can be effective and reduce operating costs. "The release programs represented between 20 and 45% of gross income, that's a big investment!" she insists. With two ladybird release operations, 6,000 individuals, at the stage where the scale insect larvae are colonising the plant, results where not conclusive. "We saw populations drop by 69% during the first year, but the pest would return by the third year, with a population explosion exactly in the same spot where we had originally released the ladybirds", explains Maria-Martha Fernandez. And most worrying of all, the ladybirds had completely disappeared from the field, which raised the question of how they overwintered... After a number of other attempts, modifying the locations and dates of the ladybird releases, researchers failed to achieve promising treatment effectiveness results. Research work continues and could now be redirected towards the identification and raising of new parasitoids for dealing with scale insects.

## The benefits of companion plants as homes to auxiliary populations

The Acarosol project seeks to deal with spider mite on solanaceae, especially *Aculops lycopersici* on tomato crops, it has successfully identified a promising predator from the Phytoseiidae family, *Typhlodromus (Anthoseius) recki*. "So far, all of the tests we have done with spider mite from this family have ended in failure", explains Marie-Stéphane Tixier, professor at Montpellier SupAgro. The structure of the tomato plant itself hinders the development and dispersion of these predator spider-mites and consequently their consumption of their prey." When identifying and collecting *Typhlodromus (Anthoseius) recki*, researchers noticed that this spider mite is naturally present in tomato crop fields. But they also discovered another particularity: the populations naturally occupy mint plants. So they decided to release the auxiliaries by placing branches of mint next to the tomato plants, in order to offer an environment that would facilitate the development and dispersion of populations. This was a good bet! Results confirmed the effectiveness of the auxiliary and that this was finally the

<figure><img src="image_71.png" /></figure>

> There was a twofold objective: establish whether regulation can be effective and reduce operating costs.

<figure><img src="image_72.png" /></figure>
first viable organic alternative for dealing with *Aculops lycopersici*. And maybe more, since as a general predator, *Typhlodromus (Anthoseius) recki* could turn out to be effective in dealing with other pests. This project also highlighted the benefits of developing companion plants in agriculture for use as reservoir habitats for auxiliaries.

## A new sustainable management eco-systemic service for dealing with the codling moth

Biocontrol methods for reducing the damage caused by the codling moth, *Cydia pomonella*, on apples do already exist but their performance is quite limited. The *Bioccyd* project managers therefore decided to add to these techniques by testing two new biological control methods, one using an already present parasitoid of the *Trichogramma* genus and the other involving the introduction and acclimatisation of a tropical parasitoid, *Mastrus ridens*. Whilst the first of these turned out to be of little interest, with low collection rates and no exclusivity for the codling moth as the target, the second seems to show more promise. "This parasitoid has already been introduced in other countries to control *Cydia pomonella*, explains Nicolas Borowiec of the INRAE. The researchers completed additional experiments to confirm the specificity of this parasitoid and received the necessary authorisations for introducing *Mastrus ridens* into the country and then to release it in agricultural fields around Avignon. It was quite an obstacle course! "The possibility of introducing Mastrus ridens into France opens up a good perspective for reducing treatment frequency indexes" he exclaims. Its permanent establishment and natural dispersion would lead to a sustainable regulation of codling moth populations. If this works, we would have a new eco-systemic service that would be free of charge for growers."

<figure><img src="image_73.png" /></figure>

> The possibility of introducing Mastrus ridens into France opens up a good perspective for reducing treatment frequency indexes

<figure><img src="image_74.png" /></figure>

### ► The views of Marc Kenis, researcher at the CABI research centre in Switzerland

> **"Organic pest control through acclimatisation involves major long-term economic and ecological benefits"**

Traditional or acclimatisation-based biological pest control methods involve the introduction of an exotic predator to re-establish the natural regulation of a pest insect. "Since 1889, there has been 6,164 introductions to combat 692 different pest insects", explains Marc Kenis, researcher at the CABI.

A well-established biological pest control programme provides constant control of the pest insects and very high economic benefits: during the 1990s the cost/benefit ratio of biological pest control methods for the cassava mealybug in Africa was 1 for 200 to 500. And this increases over time as the predator continues to control the pest!" The benefits are also ecological, they safeguard plant populations, often crops, from certain death: this was the case with the ladybirds that saved rubber trees on the Isle of Sainte-Hélène by naturally controlling mealybug populations. But there are also risks, notably negative effects on the surrounding biodiversity, but the strengthening of evaluation protocols and the biological study of auxiliary species have significantly reduced this. "Generally speaking, a parasite will not eliminate a non-target species because this will provoke an imbalance between prey and the biological control agents", he says. "Once the pest insect population begins to fall then that of the predator will do the same."

<figure><img src="image_75.png" /></figure>

> A well-established biological pest control programme provides constant control of the pest insects and very high economic benefits

<figure><img src="image_76.png" /></figure>
► **The views of Cécilia Multeau, Biocontrol Innovation and partnerships officer at INRAE**

**Macro-organisms are a dynamic sector!**

The biocontrol macro-organisms market sector is going through a phase of transformation. There are many issues involved: industrial scale production, legislation out of sync with EU legislation and the application of the Biodiversity Convention, the diversification of business models with the creation of insect farms as parts of cooperatives, and the monitoring of unintentional effects to evaluate the interference risks of auxiliaries, especially when exotic macro-organisms are introduced. Cécilia Multeau, Innovation and partnerships officer for the biocontrol sector at INRAE, gives some more information on a few major innovation orientations: "With genetic selection for example, a broad range of characteristics are now being screened such as longevity, or more specifically resistance to thermal stress. As far as mass production is concerned, the digital revolution and automation are having a major impact on processes. There have also been many introduction strategy and auxiliary deployment innovations. Finally, the sterile insect technique, is being deployed dynamically by the collectif TIS, with a transfer potential for new insect species such as *Drosophila suzukii*. "

<figure><img src="image_77.png" /><figcaption>The seven-spot ladybird (Coccinella septempunctata).
Photo: © Bastien Castagneyrol, INRAE</figcaption></figure>

<figure><img src="image_78.png" /><figcaption>Trichogramma brassicae parasiting the eggs of *Ostriania nubilalis* (european corn borer).
Photo: © Jeannine Pizzol, INRAE</figcaption></figure>

<figure><img src="image_79.png" /><figcaption>*Mastrus ridens* production room, a parasitoid of codling moth larvae (*Cydia pomonella*). INRAE Sophia Antipolis.
Photo: © Nicolas Betrand, INRAE</figcaption></figure>
<figure><img src="image_80.png" /></figure>

Resistance as a way to protect crops

<figure><img src="image_81.png" /></figure>

Sunset over the grapevines of the Château Couhins, Villenave d'Ornon, Gironde, FRANCE, 21 June 2018. Photo: © INRAE

Session programme

<table><tr><td>► DAS-REVI</td><td>Socio-technical development and appropriation of plant variety resistance in sustainable wine-growing (Pesticides 2014)</td><td>François Hochereau (UMR INRAE SAD-APT)</td></tr><tr><td>► MDRisque</td><td>Evaluation of the potential for multi-drug resistance in the wheat septoria leaf blotch agent, Mycosphaerella graminicola (Pesticides 2014)</td><td>Anne-Sophie Walker (INRAE BIOGER)</td></tr></table>

EXPERT GUEST: Marie-France Coriot-Costet, Research Director – INRAE UMR SAVE

Mourad Hannachi, Research Officer – INRAE UMR SADAPT

Marie Turner, R&D Quality Control and Plant Health Manager – Vegenov

<figure><img src="image_82.png" /></figure>

You can see the complete recording of this session on the symposium's YouTube channel
<figure><img src="image_83.png" /></figure>

# Crop protection: resistance development challenges

The protection of agricultural crops from the numerous, and often difficult to thwart, biotic threats present in their surrounding environment is becoming a vital challenge for the agricultural sector, often requiring a great deal of invention.

To cope with the frequent episodes of high past and disease pressure on crops, it will be necessary to find effective protective solutions. The intensive use of phytopharmaceutical produces is now being questioned in the light of their impact on human and environmental health. Their repeated application is actually increasing the resistance of certain pests to these products and putting crops under further danger. So there is clearly room for alternatives to synthetic products on this 'battleground'. This is a dynamic system, where natural selection competes with the resources applied by humanity to preserve plant health.

## Innovation through the hybridisation of varieties as a lever for combating grapevine diseases

The wine growing and making sector suffers heavily from the effects of pathogen fungi like mildew; this is why the sector is responsible for the consumption of two thirds of all of the fungicides sold in France. This huge quantity could even increase over the years to come according to INRAE researcher, Marie-France Corio-Costet: "The first instance of the resistance of grapevine powdery mildew to Qol type, mitochondrial respiration inhibitor, fungicides was detected in 2008; in just a few years it became almost total and generalised over all of the wine-growing regions of France". The acquisition of fungicide resistance by these pathogens has been caused by mutations that ensure a selective advantage for the survival and reproduction of the resistant strain. Which will then therefore multiply and spread as a result of natural selection. Generally, the first reflex would be to apply more phytosanitary products in the hope of greater effectiveness over these pests. But that just feeds a vicious circle, which means that the only way forwards is to find an alternative solution.

Using resistant grapevine varieties, such as Cabernet-Jura or Souvignier, is therefore a powerful lever in the fight against these diseases; and that merits attention. The DAS-REVI project is therefore committed to identifying the socio-economic benefits and pitfalls related to the cultivation of these hybrid varieties. "These varieties have allowed us to change our approach and reduce our use of phytosanitary products by as much as 90%", says Jérémy Ducourt, Bordeaux region wine grower and a pioneer in the use of these varieties since 2014. These varieties have many advantages: reduced overheads, protection of the environment, easy to grow... All of which are good reasons for taking a decision which might have appeared precarious at first: the wine-making and growing sector has been built on a very tradition-oriented image with a penchant for historic grape varieties, some of which have since become international brands, and a large number of consumer guarantee regulations (AOP and AOC). How to go about sustainably introducing these new varieties into a highly competitive market is still an open question that will be profoundly influenced by the reception of the product's consumers.

> These varieties have allowed us to change our approach and reduce our use of phytosanitary products by as much as 90%
## A crop protection arms race

A rather worrying phenomenon has been observed in farmland across France and Europe since 2017: some strains of *Zymoseptoria tritici*, the pathogenic fungus responsible for septoria leaf blotch in wheat, is developing a multi-drug resistance (MDR) to a number of phytosanitary products. Genome rearrangements cause an over-expressed efflux transport system that actively expels a wide variety of fungicides out of the pathogen cells. As a result the cells become less sensitive and continue to attack the crops in spite of the treatments. This recent phenomenon, studied at INRAE by Anne-Sophie Walker and Sabine Fillinger as part of the MDRisque project, presents a major threat to agricultural productions, since very few solutions currently exists for coming with the phenomena. "The implementation of a surveillance network to monitor and try to limit the large-scale selection of this mutation will be the first step", says Anne-Sophie Walker, "and the MDRisque project has allowed us to develop awareness of the dangers posed by the MDR mechanism".

On a wider scale, this resistance acquisition has become an integral part of phytosanitary product management policy, and this will need to be re-assessed according to Mourad Hannachi, Research Officer at INRAE. "What we are seeing now is that as soon as a new disease-resistant variety comes onto the market, a lot of growers are going to use it. And so the more the use is wide-scale, the higher the probability of it being rendered sensitive will increase in just a matter of years" he explains. Since these products have now been subjected to what could be referred to as the "Tragedy of the Commons"; and he suggests that they are treated and managed as such. "In this sense the Commons can be defined as a resource that struggles to remain exclusive and becomes a source of rivalry". To avoid draining this resource there needs to be a solution: implement the coordination of all of the parties involved in the industry and the farmlands, covering all of its aspects from plant breeding right up to end use. But this new form of management will involve fundamental changes and will certainly require the a genuine political willingness.

## Stimulate defences rather than fight the pests and diseases

After the encouraging results raised by RMT Elicitra demonstrating how plants can be partially protected by PDS (Plant Defence Stimulators), a new project, BESTIM (Stimulate plant health in agro-ecological systems), was launched at the start of 2021 with the aim of taking this field of research further, especially in terms of ecological immunity. Marie Turner, R&D, Quality Control and Plant Health Manager at Vegenov, defines this concept as "the external and internal environmental factors within which the plant develops and which contribute to stimulating its immunity system". For example we could consider that genetics, microbiota, climate conditions or interactions with other species are all factors that will influence plant health. When used alone or in association with other phytosanitary products to improve their effectiveness, PDS could now represent a new plant protection approach, and find numerous real-world applications allowing the agricultural industry to shift towards more sustainable production methods. However this vigorous stimulation of the immunity system must not turn out to have a negative effect on the development and growth of the plants themselves.

<figure><img src="image_84.png" /></figure>

> The more the use is wide-scale, the higher the probability of it being rendered sensitive will increase in just a matter of years

<figure><img src="image_85.png" /></figure>
<figure><img src="image_86.png" /></figure>

Vines under the autumn sun at Château Couhins. Villenave d'Ormon, 21 October 2021. Photo: © Nicolas Bertrand, INRAE

<figure><img src="image_87.png" /></figure>

Wheat septoria leaf blotch. Photo: © Frédéric Suffert, INRAE

<figure><img src="image_88.png" /></figure>

Defensive reactions in a sensitive plant treated with a natural defence stimulator (NDS): hypersensitive response of the damaged cells (brown-coloured cells) and deterioration of the mycelium of the parasitic fungus (in blue). Enlargement x 100.

Photo: © TROUVELOT Sophie - Université Bourgogne / INRAE
## Companion plants for healthy crops and soil

<figure><img src="image_89.png" /><figcaption>Marigold flower. Photo: © Corine Enard, INRAE</figcaption></figure>

<figure><img src="image_90.png" /><figcaption>Wild mustard in flower. Photo: © Corine Enard, INRAE</figcaption></figure>

<figure><img src="image_91.png" /><figcaption>Buckwheat flowers. Photo: © Jean Weber, INRAE</figcaption></figure>

## Session programme

<table><tr><td>► FLEUR</td><td>Manipulation of the wildflower biodiversity of cereal crop fields (PSPE2)</td><td>Joan van Baaren (UMR CNRS ECOBIO)</td></tr><tr><td>► MacroPlus</td><td>What techniques could be used to assist the installation of <i>Macrolophus pygmaeus</i>? (PSPE2)</td><td>Jérôme Lambion (GRAB)</td></tr><tr><td>► SERUM</td><td>Disinfecting soils in market gardens (PSPE2)</td><td>Ingrid Arnault (University of Tours)</td></tr></table>

EXPERT GUEST: Christophe Jarry, Director of Jardin Enchanté (Herblay)

<figure><img src="image_92.png" /></figure>

You can see the complete recording of this session on the symposium's YouTube channel
<figure><img src="image_93.png" /></figure>

# Using companion planting to improve crop and soil health

Phytopharmaceutical products are specifically designed to protect the health of cultivated plants. But they are not the only solution: one interesting alternative could be companion planting which can include hundreds of specific plant species cultivated either before or during cropping periods.

Ecosystems have their own interspecific biological pest and disease control mechanisms which could be exploited for their high crop and soil protection potential. The intelligent management of plant biodiversity would encourage the use of certain species for the beneficial role they can play within a crop ecosystem; this approach is relatively little used at the moment but it could be a potentially powerful lever for a more natural plant health protection policy. What follows is an analysis of these ways of using companion planting to support crop health.

## Using the capacity of certain flowers to attract auxiliaries and thus improve crop health

We are not the only ones to appreciate colourful scented flowers, insects love them too. The **FLEUR** project, under the leadership of Joan Van Baaren, CNRS researcher, is concerned with conservation based biological cereal crop pest and disease control techniques. "This involves organising the environment surrounding crop fields to encourage the presence of the natural enemies of crop pests", she explains. More precisely, this project is concentrated on a specific action lever: the installation of flower beds planted with specially chosen species, located such that they can be a suitable home and environment for auxiliaries, providing them with everything they need (such as pollen or nectar) as well as place to find refuge.

All we need to do now is identify the combinations of plant species which will provide the most effective protection. For example, to control the populations of aphids that pass on the Barley Yellow Dwarf Virus (BYDV) in autumn, a combination of buckwheat, cornflower, mustard and broad beans has been found to be particularly effective for cereal crops in Brittany. "Certain limitations must be taken into consideration", states Joan van Baaren. "The flowering seasons need to coincide with the growth and development phase of the crop, the predators you want to attract must not be dangerous to neighbouring crops. We also need to allow for the impact of climate change: some natural enemies are developing increasingly long periods of activity, this could lead to crops being under almost constant threat."

This solution can also be used in greenhouses. Jérôme Lambion, the **MacroPlus** project manager, has been concentrating on the *Macrolophus pygmaeus* stink bug which is a predator of certain pests that threaten tomato crops, such as spider mite, white fly or the tomato leaf miner, *Tuta absoluta... Macrolophus* was already known for its effectiveness and these stink bugs are indeed currently used on crops via one-shot release programs, although these can be quite costly. It would be better to attract them naturally, or ensure that there ate populations near to the crops or inside the greenhouses for long periods of time.

"Marigold plants are an ideal host for *Macrolophus*. So we set up beds of marigolds inside and outside the greenhouses". When that was not enough, the researchers also cut the stems of the marigolds so that they could be placed around the bases of the tomato plants and thus increase the active transfer of *Macrolophus* onto the crops that were to be protected. And it works! The spider mite, *Tuta absoluta* and white fly populations dropped off significantly on tomato plants grown near to marigolds. But

<figure><img src="image_94.png" /></figure>

> All we need to do now is identify the combination of plant species which will provide the most effective protection

<figure><img src="image_95.png" /></figure>
“I had to
commit the
whole of my
enterprise to
this dynamic”

Jérôme Lambion still maintains a certain relativism regarding these results: “Conservation based biological pest and disease control methods are indeed an effective lever for reducing the use of phytosanitary products, but they need to be integrated as part of a global approach, combined with other compatible techniques”. And there is the question of the companion plants own sensitivity to parasites: “Marigolds can be affected by powdery mildew (a fungus), which can make them less appetising for *Macrolophus*, but there is no risk of transmission to the crops since the different plants are not affected by the same species of mildew.

Some real-world applications of this plant protection method are already in place! Christophe Jarry is a horticulturist from the Paris region who has produced 15,000 chrysanthemums over the last 3 years without using any phytosanitary products. “It took a lot of testing to achieve this”, he tells us. “It wasn’t enough to just plant some flower beds in the unused parts of the greenhouse and between the rows of plants, I had to commit the whole of my enterprise to this dynamic. That was a lot of work, requiring training, maintenance and careful monitoring”. He has basically set up insect refuges, blue tit nests, owl nesting boxes, etc. “All of which also play a useful educational role, such that clients can learn about how we are doing all this”. Whilst the initial financial risks were quite high, Christophe Jarry is now deeply satisfied with the results, and intends to extend this plant protection approach to other flower species that he cultivates.

<figure><img src="image_96.png" /><figcaption>Row of marigold plants next to tomato crops. The MacroPlus project.
Photo: © Jérôme Lambion, GRAB</figcaption></figure>

“Biological pest and disease control methods... need to be integrated as part of a global approach, combined with other compatible techniques”
## Using plants to purify the soil

Protecting crops is not just about protecting the plants we are cultivating: soil health is also an essential aspect which must not be neglected. Many pest species, such as the agent that causes bacterial wilt in tropical regions or tomato nematodes, live in the soil and can cause serious damage to crops. These days soils are treated with a synthetic phytosanitary product: Dimethyl disulphide (DMDS). To reduce dependence on such phytosanitary products, the SERUM project has been studying possible alternatives on tomato crops in mainland France and the French Caribbean. "The choice of service plants evidently depends on their power to purify the soil as well as their geographical availability. Our tests have involved species of rattlepod and Allium (especially onions), under controlled laboratory conditions and in the field", explains Ingrid Arnault, manager of the Innophyt University Transfer and Expertise Centre (CETU) of the University of Tours. The idea: the service plant is cultivated on the field that needs to be treated, then cut down and left to break down on the surface of the soil as a "protective" layer (mulching). We can also grind up the service plant and plough it into the soil, or even cultivate the service plant and the crop plant in association. "Indian hemp has produced promising results, significantly reducing the presence of the bacterial wilt agent and tomato nematodes where previously the soil needed to be treated, and this without causing any unwanted side-effects to other insect species populations". However, using this method to protect crops can only be partial. But we could imagine combining it with an application of DMDS at significantly lower concentrations. But there still remains the fact that this method is quite difficult for producers to apply: for Indian hemp two seeding, growth, cutting and break-down cycles seem to be necessary to sufficiently purify the soil; and this means that the field would be out of productive action for around 200 days. During this period the growers would not be able to grow anything on the fields and this would involve an insupportable loss of earnings for many of them...

<figure><img src="image_97.png" /><figcaption>*Aphidius matricariae* feeding of buckwheat inflorescence. The **FLEUR** project. Photo: © Bernard Chaubert, INRAE</figcaption></figure>

<figure><img src="image_98.png" /><figcaption>Poppies and cornflower (Paris Region). Photo: © Jean Weber, INRAE</figcaption></figure>

<figure><img src="image_99.png" /><figcaption>C. Spectabilis mulch. The SERUM project. Photo: © Ingrid Arnault, CETU Innophyt</figcaption></figure>
<figure><img src="image_100.png" /><figcaption>INRAE experimental fields in Versailles. Flax in flower. Photo: © Nicolas Bertrand, INRAE</figcaption></figure>

<figure><img src="image_101.png" /><figcaption>Agricultural machinery working on a grapevines at the Château Couhins vineyard, Villenave d'Ornon, Gironde, FRANCE, 21 June 2018. Photo: © Nicolas Bertrand, INRAE</figcaption></figure>

Session programme

<table><tr><td>► Reg-Puc</td><td>Designing irrigation and fertilisation strategies for controlling green aphid populations in peach tree orchards? (Pesticides 2014)</td><td>Marie-Odile Jordan (INRAE PSH)</td></tr><tr><td>► ECOVERGER</td><td>Designing low pesticide-use technical routes for orchards that respect current farming restrictions and objectives. An applied modelling approach for peach and mango trees (Pesticides 2014)</td><td>Isabelle Grechi (CIRAD UPR HortSys)</td></tr><tr><td>► BIOCOU</td><td>Organic disease control for the for the grapevine fanleaf virus: the impact of integrating growing techniques involving intermediate crops and the use of a resistant root-stock on nematode populations; analysis of how nematode-killing ground-cover plants operate (Pesticides 2014)</td><td>Olivier Lemaire (University of Strasbourg)</td></tr></table>

EXPERT GUEST: Thierry Goujon, Director – Terre de Lin Cooperative

<figure><img src="image_102.png" /></figure>

You can see the complete recording of this session on the symposium's YouTube channel
# Technical routes towards zero phyto

The implementation of any innovations that involve the slightest dependence on phytosanitary products must integrate the specific restrictions that growers are now facing. We also asked ourselves about the possible contribution of various technical routes involving zero pesticide crop management. The development of alternative methods through scientific work on orchards, vineyards and flax crops must involve a detailed knowledge of each individual plot of land and a constant adaptation to the needs of the crops. If "zero phyto" still seems to be an ideal that we must strive towards; encouraging agricultural levers for the significant reduction of the use of phytosanitary products must involve essential concepts that will need to be mastered and combined for a successful agro-ecological transition.

## Dealing with pests in orchards by changing growing practices

Green aphid represents a serious threat to orchards, resulting in significant damage to fruit, foliage and stems as well as being a major vector for disease. Marie-Odile Jordan, INRAE researcher, has been working on strategies that seek to regulate green aphid populations through specific agricultural practices. "Preliminary studies have shown that water and nitrates applied to young trees result in increased aphid populations and more severe infestations", she tells us. Consequently, the RegPuc project aims to evaluate the possibility of effectively restricting aphid populations in commercial fruit-tree orchards by changing irrigation and fertilisation practices. This needed to cover the twin conditions of the fluctuating natural infestations of a number of aphid species and the need to guarantee production quality. "The severity of the infestation of a tree depends on the percentage of affected branches and the damage potential of the predominant species of aphid". Consequently, a twofold modulation of water and nitrate input managed to reduce aphid infestations in 2018 and 2019, because this altered the composition of the tree apex, with lower levels of soluble sugars and amino acids. "Weather and the effects of specific tree varieties must also be allowed for: such as the more vigorous and early-fruiting 'Ivory' peach tree variety which is more sensitive to aphids than the 'Conquise' variety". This had produced encouraging results, that should be handled with care: "The effects remain limited and these practices will not suffice in isolation. One idea would be to put together a "tool-box" of alternative approaches, that each grower can adapt to suit the needs of their plantations and the approaches they have already put in place".

The ECOVERGER project, under the leadership of Isabelle Grechi, CIRAD researcher, is concentrated on evaluating the feasibility of alternative technical solutions for dealing with the pests and diseases that threaten commercial orchards: brown rot, fruit fly or mango blossom gall midge. Once more, "we needed to allow for all of the technical and economic factors, not forgetting production quality", says Isabelle Grechi. A number of action levers have been identified: bringing the harvest forward to 20 days before full maturity for mangos and synchronising flowering seasons to restrict the sensitivity period for the fruit and the exposure of the blossom to pests; restrict irrigation to achieve slower development, making them less susceptible to infection from brown rot; systematically remove infected fruit to avoid the propagation of pathogen spores; or cover the ground with plastic sheets or ground-cover planting to disturb the ground-level development of the blossom gall midge. "These levers can be effective, and some are quite easy to put in place. Some restrictions could also be lifted if information can be shared better. For example, for early harvesting: contrary to what might be thought, if mango fruit are harvested 20 days before maturity that will only reduce their quality by 5% [estimated in terms of their fresh mass and sugar content compared to the fully ripe stage]", explains Isabelle

<figure><img src="image_103.png" /></figure>

> One idea would be to put together a "tool-box" of alternative approaches

<figure><img src="image_104.png" /></figure>

<figure><img src="image_105.png" /></figure>

> Some restrictions could also be lifted if information can be shared better

<figure><img src="image_106.png" /></figure>
Grechi. "Other levers would require some technical training and financial incentives to make it easier for growers to adopt them". The development of a tool to improve support for the conception of approaches that use alternative methods is being continued by the ODACE project which is carrying on where ECOVERGER left off.

Introducing nematode-killing ground cover plants to combat the grapevine fanleaf virus vector

The grapevine fanleaf virus, which is transmitted by the *Xiphinema index* nematode, causes major damage in the wine-growing industry. The infected plants wilt and the disease can remain in the soil as long as the grapevine remains there: growers are therefore obliged to completely remove any affected vines. The BIOCOU project concerns the use of Fabaceae such as sweet clover, red clover, trefoil and sanfoin, for their antagonist effects on this nematode. "Sanfoin and trefoil extracts are more effective under laboratory conditions, their root extracts can achieve 80% effectiveness on this nematode", explains Lise Negrel, researcher for Multifolia. "Whereas in the vineyards, the use of Fabaceae alone as an intercrop did not turn out to be effective. However, spreading Vitifolia granules [granulated sanfoin marketed by Multifolia] after replanting the vines has managed to reduce the return and spread of the disease". The high potential of these granules is starting to be thought of as being a potential long term solution. Olivier Lemaire, INRAE researcher, explains: "Since 2018, we have been monitoring the physical recontamination of plots where *Xiphinema index* was known to be present. These fields were left fallow with alfalfa for two years. Then at the start of the third year, we spread 10 tonnes per hectare of sainfoin granules before planting new vines during the spring of that year. The results were promising". There are still some aspects to be clarified, such as the maximum spreading dosages or the influence of soil-climate conditions on the effectiveness of the granules. "It is quite clear that there is not just one but a number levers that will need to be implemented to be able to deal with fanleaf virus without using phytosanitary products. Nematode killing granules and plants are proving to be a viable and easy to implement lever in this context.

<figure><img src="image_107.png" /><figcaption>Flax retting in Normandy. Photo: © Jean Weber, INRAE</figcaption></figure>

<figure><img src="image_108.png" /></figure>

> It is quite clear that there is not just one, but a number of levers to be implemented

<figure><img src="image_109.png" /></figure>
► **The views of Thierry Goujon, Director of the Terre de Lin Cooperative**

**“Combining different levers throughout the production cycle”**

Thierry Goujon, director of the Terre de Lin Cooperative which includes 700 Normandy flax producers, tells us about some progress made in the sector: “We are working on adapting our growing methods: we prioritise mechanical weed control or false-sowing techniques to deal with weeds. These methods are not perfect, especially since they are a highly dependent on climate conditions, but they do allow us to reduce our reliance on phytosanitary products”. Another lever: the use of resilient varieties developed through genetic innovations, has helped us to cope with fusarium which was rife around twenty years ago. The cooperative has also changed its seedling preparation methods. A steam disinfection technique has been developed to eliminate pathogens. “Its like a pasteurisation process for seedlings which complies perfectly with the applicable health standards. For 2021 we disinfected between 7,000 and 8,000 tonnes of seedlings in this way without having to use any phytosanitary products. The results are promising, which is helping us to strengthen the sector's reputation as the source of a quality natural product: a clear marketing advantage.

<figure><img src="image_110.png" /><figcaption>Colony of green aphids on a peach tree. Photo: © Yvon Robert, INRAE</figcaption></figure>

<figure><img src="image_111.png" /><figcaption>Fruit fly damage on mangos. The ECOVERGER project. Photo: © Isabelle Grechi, CIRAD</figcaption></figure>
<figure><img src="image_112.png" /></figure>

The Cantal landscape near to Salers. Photo: © Nicolas Bertrand, INRAE

Session programme

<table><tr><td>► <u>RESCAPE</u></td><td>REsistance of agricultural landSCAPEs to pesticide transfers in soils and living organisms (<u>Pesticides 2014</u>)</td><td>Céline Pelosi, Colette Bertrand (INRAE UMR EcoSys)<br/>Clémentine Fritsch (Chrono-Environnement CNRS/UBFC)</td></tr><tr><td>► <u>RESYST</u></td><td>The REsistance of Tropical agricultural SYStems to the reduction of pesticide pollution pressure on the watershed scale (<u>Pesticides 2014</u>)</td><td>Charles Mottes (CIRAD)</td></tr><tr><td>► <u>TRAJECTOIRES</u></td><td>Hereditary trajectories and the use of pesticides in major agricultural territories (<u>Pesticides 2014</u>)</td><td>Carole Barthélémy (University of Aix Marseille)<br/>Gilles Armani (University Lumière Lyon 2)</td></tr><tr><td>► <u>ALT'CIM</u></td><td>Heading towards zero phyto in Normandy cemeteries (<u>JEVI 2016</u>)</td><td>Flore Diradourian (ASTREDHOR)</td></tr><tr><td>► <u>ALTHERCOL</u></td><td>Weedkiller alternatives in urban allotment gardens. (<u>JEVI 2016</u>)</td><td>Francesca Di Pietro (University of Tours, UMR CITERES)</td></tr></table>

<figure><img src="image_113.png" /></figure>

You can see the complete recording of this session on the symposium's YouTube channel
# Looking beyond the fields

Reducing dependency on phytosanitary products will require the management of smaller crop volumes per field and some major changes to growing practices. However, the issues involved in the implementation of this dynamic in agricultural or non-agricultural regions are not going to be played out uniquely from an agricultural point of view. By "looking beyond the fields" we can see that they are an integral part of their surrounding landscape, with their own specific characteristics, restrictions and opportunities; and that the use of phytosanitary products also depends on the influences of the various parties involved (farmers, local government, citizens, etc.).

## Planning for a regionally coordinated phytosanitary product policy

A certain portion of the phytosanitary products used on a given field will affect the neighbouring ones via the atmosphere and/or the ground. The RESCAPE project is concerned with classifying these transfers on a countryside scale by measuring the contamination of farmland soil and the exposure of non-target wild-life to pesticides. "We have worked on 60 landscape slots representing different aspects of the farmland patchwork, with varying degrees of hedging or proportions of untreated zones [prairies and organic farming land]", explains Céline Pelosi, INRAE researcher. Fungicide, pesticide and herbicide residues were not only looked for in soil samples but also in the tissues of earthworms, ground beetles and small mammals. "We have been able to demonstrate the almost systematic contamination of all of the samples we studied: at least one pesticide residue was detected in each one of the soil or small mammal samples, and very frequently we found a cocktail of fungicides, herbicides and insecticides, such as epoxiconazole, diflufenicanil and imidacloprid". 40% of the soil samples contained concentrations higher than the chronic toxicity threshold for earthworms listed for at least one pesticide product. "We also completed a risk assessment for pesticide mixtures, concluding that there is a high risk factor for almost half of the soil samples studied, including the majority of conventional farmland samples but also a good number of the untreated zones. Fortunately these untreated zones are significantly less contaminated, which highlights their potential as "refuge zones" protecting living organisms from pesticides. The prairies and hedges which have not been treated with phytosanitary products will therefore retain a major importance in the landscape. Especially since a computer model of the atmospheric dispersion of pesticides has shown that the hedges act as a "barrier" restricting such products from reaching neighbouring non-target zones. Consequently, researchers now recommend that agro-ecosystems are managed on a countryside scale and not just in terms of individual fields, thus increasing the number of untreated zones and agro-ecological infrastructures such as hedges and shrub plantations. This management approach will require the effective coordination of the various parties involved in managing the countryside.

The importance of such coordination was also highlighted by the RESYST project under the leadership of Charles Mottes, researcher at CIRAD. The main interest of the project is to encourage change in the agricultural practices used in and around the Galion watershed in Martinique in the hope of restricting the pollution of the river. "Based on the observation that the island's three main industries [sugar cane, bananas for export and diverse productions for the local market] operate in isolation to each other, we needed to coordinate everyone involved so that they could work together to construct fifteen new region-wide innovations, notably including new grassing over techniques, that we then tested in real-world situations". The stated ambition: "to show that the countryside can provide resources and levers that the individual industries cannot come up with in isolation", by constructing new ties between farmers, by promoting the land and its resources, whilst always taking the specific nature of each local environment into consideration.

<figure><img src="image_114.png" /></figure>

> The prairies and hedges will therefore retain a major importance in the landscape

<figure><img src="image_115.png" /></figure>
## Successful transition by allowing for socio-cultural factors

The implementation of new agricultural practices will also be determined by socio-cultural factors. The *Trajectoires* project led by Carole Barthélémy, professor-researcher in sociology, concerns the sociological causes that decide whether pesticide use reduction initiatives work or not. They have identified three trajectories that may or may not lead to change: the hereditary trajectory of changing phytopharmaceutical product usage from generation to generation, the material trajectory of the development of the phytosanitary products themselves and the trajectory of land-use transformations. "For the four agricultural regions that we studied, those who answered often spoke of the post-war period when phytosanitary products were being used systematically. Since then there has apparently been a rise in awareness, we were able to observe that individual or collective initiatives, both old and new, are seriously questioning this accepted norm". *Trajectoires* also raised the question of the effectiveness of the CERTIPHYTO training programme, the mandatory passport for all operatives who use phytosanitary products. "We wanted to identify the limits of this system, that was conceived with a philosophy of uniformity although addressed to social groups with very different cultural and economic capitals: some consider this training to be pointless whilst others can't afford to put its teachings into practice", says Carole Barthélémy. These works highlight the need to adapt to the real local limitations that are encountered by professionals.

The ALTHERCOL project is also interested in the links between the socio-demographical origins of allotment gardeners and their motivations to implement alternative weed control strategies. "The survey looked both at the organisations that manage these gardens (associations or local councils) which promote pesticide-free gardening without wanting to leave too much room for wild or spontaneous flora, and on the gardeners themselves, who could be divided up into 4 classifications depending on their pesticide use", explains Francesca Di Pietro, professor-researcher at the University of Tours. "Our surveys showed that the use of pesticides depended on the proximity between the gardeners' rurality, where they learned to garden during their childhoods, and their current residence. Additionally we discovered that young gardeners coming from built up areas had a higher tolerance for wild or spontaneous flora". And so, to help these gardeners on their way to reducing their use of phytosanitary products, the ALTHERCOL project proposes the implementation of training programmes, the diffusion of printed information and the organisation of debates between gardeners to encourage the exchange of knowledge and good practices.

<figure><img src="image_116.png" /></figure>

Upstream from one of the RESYST project's pilot zones: the Galion drainage basin
Photo: © Charles Mottes, CIRAD.

<figure><img src="image_117.png" /></figure>

> The work of the
> *Trajectoires*
> project
> highlights the
> need to adapt
> to the local
> restrictions
> encountered
> by
> professionals

<figure><img src="image_118.png" /></figure>
## Encouraging biodiversity in cemeteries

The ALT'CIM project aims to develop an alternative vegetation for cemeteries that will not require the use of phytosanitary products. A number of different plant species and introduction techniques were tested on eight cemeteries in Normandy. Grassing over the paths with trample-resistant Ray-Grass, herbaceous plants like camomile between and behind the tombs, wild flower prairies and pre-planted rolls are all examples of plantations that can be maintained easily without phytosanitary products. The extent to which these new planting schemes were accepted by cemetery users was surveyed, revealing that a large majority of users support the use of such vegetation since it encourages biodiversity and makes cemeteries more attractive, even if this did involve more work for the landscape managers.

<figure><img src="image_119.png" /><figcaption>Hommet d'Arthenay cemetery (50) - Hydroseeding. <u>Alt'Cim.</u> project Photo:
© David Philippart, FREDON Normandie</figcaption></figure>

<figure><img src="image_120.png" /><figcaption>Farmlands of the ZAPVS (Zone Atelier Plaine & Val de Sèvre), crop field and neighbouring hedges. <u>Rescape</u> project. Photo: © Céline Pelosi, INRAE</figcaption></figure>

> A large
> majority of
> users support
> the use of
> such
> vegetation
> since it
> encourages
> biodiversity
> and makes
> cemeterie
<figure><img src="image_121.png" /></figure>

CONCLUSION

Three visions of the Ecophyto Research &
Innovation Symposium

► **The views of Marion Bardy,**
deputy to the Food and Agriculture Ministry
vice-director of research, innovation
and international cooperation

This symposium was organised within the framework of the Ecophyto plan, which was launched in 2008 in response to high public expectations concerning the preservation of human health and the environment. The plan's ambitions have been regularly revised and we are now in the Ecophyto II+ plan. The projects presented during the symposium, which took the highly original form of a series of themed webinars, have been financed by the plan and are opening up new ways forward by proposing different phytosanitary product usage reduction strategies, either in an agricultural or JEVI (Jardins, Espaces Végétalisés et Infrastructures - Gardens, Parks and Infrastructures) context. "It was very important to take the time to share the results of these projects through this symposium and to debate the performances that might be achieved using the levers proposed by the projects as well as to identify the most suitable ways to implement them in the real world" explains Marion Bardy. "Additionally, the discussions that continued throughout these webinars will serve to feed reflections on the different orientations that the plan's Research & Innovation axis could take", she adds. These research projects contribute not only to knowledge but also to structuring research communities around these subjects. And what's more they are being expressed through projects from other programmes financed by the Ecophyto Plan and which are also supporting this agro-ecological transition such as the "Grow and protect differently" and "Maturation" calls for projects. And finally, it is important to spread knowledge of all that is produced, not just on a nationwide scale. "As for research, the INRAE has set up a European alliance around the question of constructing a pesticide-free agriculture. And as for the institutions, we are currently working with our European opposite numbers as part of the European Standing Committee on Agricultural Research SCAR. "

“It was very
important to take
the time to share
the results...and to
debate the
performances that
might be achieved
using the levers
proposed by these
projects”

► **The views of Xavier Reboud,**
president of the “Research & Innovation”
Scientific Orientation Committee (CSO R&I)

The subjects covered by this series of webinars fits perfectly with the scientific policy of the CSO R&I, notably the promotion of the acquisition of the knowledge that has been identified as essential for the achievement of the Ecophyto plan objectives. The session formats were set out in advance such that they present the projects financed by the Ecophyto plan in an informative way, and such that
useful exchanges between those presenting and those listening could occur. "We decided to attach an external viewpoint for each session, by inviting one or a number of guest experts. This allowed us to gain some perspective on the subjects" says Xavier Reboud. Some of the projects presented over the sessions managed to initiate partnerships to develop the marketed or almost-marketed alternative solutions, and we clearly noted that the promising content of some of the projects could also have taken things further. "Market repercussions will require the conjunction of two factors: the first being the expression of high expectations, the second being the presence of project supporters who are prepared to take on the risk associated with these opportunities. So it remains crucial to link these partnerships together as early as possible in order to ensure that both the scientific and the practising communities can work together", he says. Another positive aspect that was highlighted was the informational format of these sessions, the diffusion of which could reach a broader public and thus encourage wider partnerships. The symposium resources are available to the general public: the videos on the symposium's YouTube channel and the webinar brochures on the symposium's EcophytoPIC page.

“Some of the projects presented over the sessions managed to initiate partnerships to develop the marketed alternative solutions”

► **The views of Lucile Gauchet, Ecophyto plan project leader for the Ecology Transition Ministry's General Planning, Housing and Nature Directorate**

Amongst the projects presented during the symposium, the spotlight was placed on alternative techniques, such as biocontrol, to replace the use of synthetic phytosanitary products. "What really hit me was the diversity of the propositions, the new approaches for finding alternatives at different levels highlighting the various levers that will need to be incorporated into the technical routes and also promoting reflections beyond the management of individual fields" she says. Following this, it should be noted that the presented projects all come from the programmes launched a few years ago (2015 - 2016), which really demonstrates the anticipatory work of the Research & Innovation axis. "The work of the R&I CSO supported by the events hub, contributes to structuring the scientific community on the subject of reducing pesticide use". It should also be noted that the Ecophyto plan Research & Innovation axis also covers other crucial and current aspects such as health and environmental impact through other research proposition calls which were not presented at this symposium. Lucile Gauchet also points out that France has done pioneering work by setting the ambitious Ecophyto Plan objectives to reduce the use of phytopharmaceutical products, requiring further research efforts, and this has lead to an increase in the resources allocated to this axis via Ecophyto II+. The effects that are leading us to change in the here are now are becoming evident, as the European Union has also set ambitious goals via the Green Charter through the "from the Farm to the table" and "EU biodiversity" strategies." And finally there is the question of the scope of these research projects, especially in terms of passing on the necessary know-how to those concerned: farmers, consultants, public institutions etc. It would now seem essential to reconsider the resources necessary to make the best use of these leverage effects.

“What really hit me was the diversity of the propositions, the new approaches for finding alternatives at different levels”
<figure><img src="image_122.png" /></figure>

Appendix

Project description sheets

ACAROSOL

ALT'CIM

ALTHERCOL

AM&BAS

AttractMyFly

BIOBOT

BIOCCYD

BIOCOU

BIOLIM

BIOTI-VIGNE

DAS-REVI

DESHERBAL

DICABIO

ECOVERGER

ENFIN !

FLEUR

LIPOCONTROLE

MacroPlus

MDRisque

MilPomBio

NABUCO

OPTIM'PHERO

PRO-BIO-TAUPIN

Reg-Puc

Rescape

RESYST

Rhizodia

SEMIOTRAP

SERUM

Systemyc

TRAJECTOIRES
<figure><img src="image_123.png" /></figure>

# Biological control of spider mite on Solanaceae

**Launch year:** 2015

**Completion year:** 2018

**Scientific manager**

Marie-Stéphane Tixier,
Institut Agro-Montpellier SupAgro

marie-stephane.tixier@supagro.fr

**Partners**

Institut Agro-Montpellier SupAgro ; INRAE UMR
CBGP ; CTIFL-Ballandran ; SONITO ; Koppert

**Financing**

Total cost of the project: € 527,989

Ecophyto grant: € 90,347

**Key words:**

Spider-mite; Biological control; Tomatoes; Biodiversity; Agro-ecological transitions; Management via macro-organisms

## Context and main objectives

Spider mite can cause significant crop damage on tomatoes (as much as 65%), resulting in the need to use pesticides, which is highly unsatisfactory in environmental, financial and social terms. Predatory spider mites, Phytoseiidae, are the most effective natural enemy of the pest spider-mites. However, these auxiliaries have little effect on tomato crops because of the plant's physical and biochemical characteristics. This project is therefore concerned with identifying endemic European predators that are suited to Solanaceae and then testing their predatory characteristics in the laboratory and in experimental greenhouses. The effects of predators present in the non-cultivated areas of crop-fields were also studied. The main hypotheses tested were as follows:

1. As yet unidentified predators exist that are both endemic to Europe and suited to the tomato plant.

2. They have useful predatory characteristics.

3. They can be raised in large quantities for release within the crop fields.

4. They are naturally present on the plants in the non-cultivated areas bordering the fields.

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

Generally speaking, the project results met these expectations regarding the biological control of pest spider mites on tomato crops; notably the most widespread species, *Aculops lycopersici*, which causes tomato russet. The work was organised over three major phases.

### 1. Identification and classification of "useful" and original biodiversity

A number of field missions were completed; more than 100 plants (33 species of Solanaceae) were collected in France and Morocco. Four main species of Phytoseiidae were found.

Laboratory assays identified the predatory differences between the species and populations studies. One predator species (*T. (A.) recki*) was selected to continue the tests on a broader scale. Additionally, molecular and morphological analysis demonstrated the relations between these characteristics, the host plants and the predatory capacities of the identified (*T. (A.) recki*) populations.

### 2. Classification of the predator's dispersion capacities on tomato plants.

Laboratory tests have shown that females of the predators move around freely on the stems, and that they are attracted by food (pollen or *Aculops lycopersici*). However, the results of preliminary tests on whole plants were deceptive, showing low predator dispersion along the stems of the tomato plants. Consequently the perspective of large scale breeding by an industrial partner has been abandoned and an alternative solution had to be found.
### 3. Survey of biological effectiveness in experimental conditions under glass.

With large-scale breeding now considered to be impossible, two other predator "introduction methods" were studied: Companion planting: plants serving as a reservoir for natural enemies. During the predator diversity survey, we have shown that the selected species was present in very large numbers on plants of the mint family. Consequently, we have decided to study the impact of the presence of mint as a factor in the control of pests present on nearby tomato crops. Experiments showed that *T. (A.) recki* spreads from the mint to the infested tomato plants both on isolated leaves or whole plants. A planting density of 6 tomato plants to 3 mint plants seems sufficient to ensure this dispersion. *T. (A.) recki* seems to spread more effectively when the tomato plants are infested. This result opens up the perspective of pest control approaches involving relay plants.

Inoculation via predator introduction; considering the presence of large numbers of the predator on mint leaves, branches of mint were placed around the bases of the tomato plants under experimental greenhouse conditions. The branches of mint were introduced followed by the inoculated pests one week later (A. lycopersici). The greenhouse experiments demonstrated an unprecedented and satisfactory level of biological control over A. lycopersici.

It should be noted that the selected predator species has also been observed in the sample non-cultivated zones surrounding outdoor tomato crop fields which also opens up the perspective of an organic control approach via the preservation of biodiversity.

## Prospects for transfer or research

### Transfer:

This project has opened up a pathway to the development of two biological control approaches:

* **Use of companion planting.** Our results have opened up new market prospects for (i) nursery owners as the suppliers of companion plants and (ii) companies that supply auxiliaries and/or companion plants already colonised by populations of the desired auxiliary.

* **Biological control by increase.** Our results have provoked interest from biocontrol companies which would be able to develop additional testing programmes to prepare for marketing. The economical stakes are very high given the high global demand for biological control solutions for *A. lycopersici*.

### Research:

The project results are very positive because (i) this is the first time that a predator species has turned out to be so effective in controlling *Aculops lycopersici* and (ii) the fact that new biological control prospects using companion plants can now be envisaged. This work is being continued in an Ecophyto Maturation project, BioLycTom, with the more operational objective of testing (i) the ideal conditions for the introduction of Phytoseiidae, (ii) the impact of the predator on other tomato pests, (iii) the positive and potentially negative effects of the presence of companion plants inside crop-fields in biological, technical and socio-economical terms.

## Publications and scientific symposia:

### Technical workshops and scientific symposia:

* Gard B., Douin M., Tixier M.-S. 2021. A new hope for the biological control of *Aculops lycopersici* (Acari: Eriophyidae) with the predatory mite *Typhlodromus (Anthoseius) recki* Wainstein (Acari: Phytoseiidae). International Congress of Entomology, Helsinki. Planned for 2020 and postponed to 2021.

### Scientific publications:

* Tixier M.-S., Douin M., Kreiter S. 2020. Phytoseiidae (Acari: Mesostigmata) on plants of the family Solanaceae: results of a survey in the south of France and a review of world biodiversity. Experimental and Applied Acarology 81: 357-388

* Tixier M.-S., Perez Martinez S., Douin M. 2020. Markers for life traits: the example of variations in morphology, molecular and amino acid sequences within the species *Typhlodromus (Anthoseius) recki* Wainstein (Acari: Mesostigmata: Phytoseiidae). Journal of Linnean Society accepted. https://doi.org/10.1093/biolinnean/blaa103

* Tixier M.-S., Douin M., Oliva R., Gonzalez L., Pount B., Kreiter S. 2020. Distribution and biological features of the species *Typhlodromus (Anthoseius) recki* (Acari: Phytoseiidae) on *Tetranychus urticae, T. evansi* (Acari: Tetranychidae) and *Aculops lycopersici* (Acari: Eriophyidae). Acarologia Acarologia 60 (4): 684-697. DOI: 10.24349/ acarologia / 20204396
<figure><img src="image_124.png" /></figure>

# Heading towards zero phyto in Norman cemeteries

**Launch year:** 2017

**Completion year:** 2021

**Scientific manager**

Agnès Langlois, **ASTREDHOR**

agnes.langlois@astredhor.fr

**Partners**

**ASTREDHOR**; FREDON Normandie

**Financing**

Total cost of the project: € 193,845

Ecophyto grant: € 145,383

## Key words:

JEVI; Zero-phyto; Integrated landscape management for cemeteries; Planting solutions; Restricted spaces; Landscaping memorial sites

## Context and main objectives

The use of phytosanitary products in cemeteries will become illegal in 2022. Experience has shown that this kind of change can be very difficult for this type of site.

These environments involve a lot of hard surfaces, making it very difficult to tolerate even the smallest weed. This means that weedkillers are used massively to keep the sites "clean".

The project objectives are as follows:

1) Define and classify the different types of cemetery in Normandy and the specific issues that they face whilst also listing all of the currently available approaches and solutions.

2) Promote changes to cultural perceptions of cemeteries through specific communication and awareness initiatives.

3) Support local governments in their transition to a sustainable zero-phyto approach for their cemeteries by testing planting solutions that would reduce time spent on maintenance and encourage the acceptance of vegetation

4) Respect the purposes of the sites

5) Plantations must respect and improve both landscape and heritage.

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

The plantation tests and trials were completed between autumn 2017 and spring 2018. These tests took a number of different forms: grassing over, herbaceous planting, pre-planted solutions and mixed wildflower seed.

The grassing over techniques were generally tested on the cemeteries' main or secondary footpaths. Hydro-seeding was used for the sites where grassing over was more generalised. Whilst complicated to implement, this method has the benefit of assisting the initial growth stages. The results were rapid and encouraging, although the costs were higher than for more traditional grassing over techniques.

The grass was mostly seeded when it was to be used on the main, secondary and transversal paths.

Outside of any specific restrictions (localised shade) and within the scope of our trials, the best ground cover results were achieved with Top Green Euronature TPV1 and Routemaster 3 grasses. These varieties/mixes caught up with the performances of the hydro-seeded areas after only a few months.

Where the areas between the tombs were too narrow for grass, other solutions such as herbaceous planting were tested. They were planted as single species or in modules of a number of species. Varieties were tried on a number of sites. The study analysed the ground cover percentage, invasive impact, aesthetics... The following table provides a summary of the results.
<table><thead><tr><th></th><th>Establishment</th><th>Competition with weeds</th><th>Invasive impact</th><th>Aesthetic</th></tr></thead><tbody><tr><td>Centaurea simplicaulis</td><td>+++</td><td>++++</td><td>++</td><td>++++</td></tr><tr><td>Prunella vulgaris</td><td>++++</td><td>++++</td><td>+++</td><td>+++</td></tr><tr><td>Acaena microphylla</td><td>++++</td><td>++++</td><td>++++</td><td>+++</td></tr><tr><td>Frankenia laevis</td><td>+++</td><td>+++</td><td>++++</td><td>++</td></tr><tr><td>Muehlenbeckia complexa</td><td>++</td><td>+++</td><td>++</td><td>++</td></tr><tr><td>Erigeron karvinskianus</td><td>+++</td><td>++++</td><td>+</td><td>++++</td></tr><tr><td>Chamaemelum nobile</td><td>++++</td><td>+++</td><td>+</td><td>++++</td></tr><tr><td>Polygonum affine 'Dimity'</td><td>+++</td><td>+++</td><td>++++</td><td>++++</td></tr><tr><td>Sedum album 'Coral carpet'</td><td>++</td><td>+++</td><td>+</td><td>++++</td></tr><tr><td>Sedum spurium 'Woofoo'</td><td>++</td><td>+++</td><td>+</td><td>++++</td></tr><tr><td>Origanum vulgare 'Compactum'</td><td>+++</td><td>++</td><td>+</td><td>+++</td></tr></tbody></table>

Summary comparison of the most effective herbaceous varieties based on the criteria of adaptation and development, ALT'CIM test programme. Credits: Agnès Langlois, ASTREDHOR

Observations have identified some species that are interesting both for their adaptability and ground-cover capacity: *Centaurea simplicaulis*, *Prunella vulgaris* and *Chamaemelum nobile*.

<figure><img src="image_125.png" /><figcaption>Amières / Iton (27), *Chamaemelum nobile*.
Photo: © David Philippart, FREDON Normandie</figcaption></figure>

<figure><img src="image_126.png" /><figcaption>Régneville / mer (50): *Prunella vulgaris*. Photo: © David Philippart, FREDON Normandie</figcaption></figure>

<figure><img src="image_127.png" /><figcaption>Vaucelles, Caen (14): *Centaurea simplicaulis*.
Photo: © David Philippart, FREDON Normandie</figcaption></figure>

On the contrary, others have displayed a highly invasive nature: *Acaena microphylla*, *Frankenia laevis* and *Polygonum affine 'Dimity'*.

A specific number of species were tested in association, planted between high tombs, which potentially invasive plants would not be able to cover. Independent to the plant combinations,

<figure><img src="image_128.png" /><figcaption>Vaucelles, Caen (14), *Polygonum affine 'Dimity'*, Photo: © David Philippart, FREDON Normandie</figcaption></figure>

<figure><img src="image_129.png" /><figcaption>Vaucelles, Caen (14), *Frankenia laevis*. Photo: © David Philippart, FREDON Normandie</figcaption></figure>

Hernaria glabra, *Polygonum affine*, *Thymus serpyllum*, *Saponaria ocymioides* and *Stachys byzantina* provided the best results.

Other planting solutions such as pre-grown rolls were used on three sites. These were installed around the tomb stones. The pre-planted rolls or mats includes both the plants (sedum or herbaceous) and the planting media necessary for their growth with or without coco matting to hold it all together. These systems are very quick to install and provide immediate aesthetic results. These were compared to individually planted sedum.

The pre-grown rolls are a very effective planting solution, since they cover the ground directly thus avoiding the emergence of spontaneous plants and making establishment easier. The sedums have the advantage of being closer to the ground than the herbaceous plants which makes them more acceptable near to monuments. Whilst the planted rolls have shown quicker and more effective results, they will be kept for restricted and strategic areas because of their high cost.
<figure><img src="image_130.png" /><figcaption>Regnéville / mer (50): herbaceous
mat. Photo: © David
Philippart, FREDON Normandie</figcaption></figure>

<figure><img src="image_131.png" /><figcaption>Regnéville / mer (50): sedum
mat. Photo: © David
Philippart, FREDON Normandie</figcaption></figure>

Wildflower mixes were tested in different locations over three sites. Generally speaking, wildflower mixes are recommended for 2-3 years. Beyond this the mix loses its diversity, and a new sowing operation should be done.

## Prospects for transfer or research

### Transfer:

The results obtained by the ALT'CIM project will provide a source of guidance for local councils who want to go zero-phyto in their cemeteries. The diversity of the solutions tested means that one or more solutions can be proposed for each of the complex spaces of these memorial sites: main and secondary footpaths, inter-tombs, inter-rows, waiting areas, decoration of some monuments...

The project has shown that grassing over is one of the easiest solutions to implement. However, given that configurations are different for each cemetery, planting with herbaceous plants or sedums has also been shown to be of interest.

### Publications and scientific symposia:

#### Technical workshops and scientific symposia:

* The “Que vont devenir les cimetières en Normandie, et ailleurs ?” symposium, 30-31/08 and 01/09/2017. CCIC Cerisy-la-Salle (FLORYSAGE Intervention), by the 'Fédération Normande pour la sauvegarde des cimetières et du patrimoine funéraire'.

* The FLORYSAGE 2017 "Paysage et entretien des cimetières" symposium, 29/11/17. Eure Departmental Council.

* "Vers le zéro-phyto dans les cimetières de la Manche" half-day conference. Manche Departmental Council, February 2018.

* "Végétalisation des espaces contraints : massifs de voiries et cimetières" technical workshop, 06/04/18. Jean Rostand Professional College (Offranville, 76).

* CAUE14/FLORYSAGE "Quand le zéro-phyto nourrit le projet de paysage" technical workshop, May 2018.

* "Zéro-phyto dans l'espace public : pour aller plus loin que la réglementation" Technical workshop, Caen la Mer and Plante & Cité, in partnership with FLORYSAGE, Hortis, Unep, September 2018.

* ASTREDHOR Open Days, Seine-Manche and Pays de la Loire, September 2018.

* Second "Champs d'Innovation" forum organised by the Normandy Chamber of Agriculture, Caen, 18/10/18.

* "Le zéro-phyto au quotidien dans ma commune : je végétalise le cimetière" Technical half-day workshop, partners: FREDON/Le Havre Seine Métropole / FLORYSAGE, April 2019.

* Intervention at the Salon Vert: "Sensibiliser et accompagner les collectivités dans la végétalisation de leur cimetière, afin de faciliter la transition zéro-phyto", Saint-Chéron, 23/09/2020.

#### Prtactical / educational articles:

* Paris Normandie, October 2017: "Le « zéro pesticides » arrive au cimetière de La Bonneville-sur-lton".

* Ouest France, October 2018: "Quand on dit zéro phytos, c'est zéro".

* Le Lien Horticole, January 2018: "Cimetières normands : Accompagner les collectivités dans la transition zéro-phyto de leur cimetière".

#### Other practical works:

* "(Ré)aménager et entretenir les cimetières de Normandie". Brochure published in 2018.

* "Diagnostic du paysage funéraire normand". Brochure published in 2019.

* "Passer au zéro-phyto dans mon cimetière : la solution de la végétalisation". Unpublished brochure.

* "Info'Charte" n°13 and n°15. FREDON Haute-Normandie (April and October 2018).

* "Comment (Ré)aménager et entretenir les cimetières en Normandie?". Unpublished handbook.

* "Végétaliser son cimetière pour le gérer sans produit phytosanitaires", FLORYSAGE Technical data-sheet, (For members, 2020).

* ALT'CIM "Information kit" (Poster, panels, flyers).
<figure><img src="image_132.png" /></figure>

ALTHERCOL

# Weedkiller alternatives in urban allotment gardens

**Launch year:** 2017

**Completion year:** 2020

**Scientific manager**

Francesca Di Pietro, University of Tours UMR
CITERES

dipietro@univ-tours.fr

**Partners**

University of Tours UMR CITERES; France
Nature Environnement Centre Val de Loire (FNE-CVdL)

**Financing**

Total cost of the project: € 174,760

Ecophyto grant: € 119,760

## Key words:

Allotment gardens; Horticultural practices; Pesticides; Socio-demography; Spontaneous flora; Gardeners; Motivations; Representations; Urban biodiversity; Biocontrol

## Context and main objectives

Two thirds of pesticides on the non-agricultural market are aimed at private gardens; pesticides for amateur gardeners have an impact on a vast majority of the population. In allotment gardens, as with private gardens, people use the whole range of horticultural practices, from the consumption of vast quantities of pesticides to the most environmentally friendly techniques. But due to the nature of collective gardens, they can serve as an ideal place for gardeners to experiment and pick up or learn about biocontrol methods. We have postulated that **allotment gardens are an opportunity to encourage the appropriation of biocontrol methods and the abandon of pesticide use in amateur gardening.** It would seem like an important action to assist gardeners in making this transition towards more environmentally respectful practices and towards the adoption of biocontrol methods, especially given that pesticide stocks could still last for a few more years and that inappropriate alternative practices are still sometimes implemented.

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

We have mobilised specific methodologies for:

1) Identifying all of the allotment gardens in two urban centres: Tours and Orléans (see photo and maps)

2) Interviewing allotment garden managers (17 interviews)

3) Interviewing gardeners (30 initial interviews and 150 questionnaires)

4) Learning about the structure and spontaneous flora of allotment gardens (150 allotment structure surveys, 150 botanic allotment surveys, 150 botanic footpath surveys)

Our survey showed that, according to the questionnaires completed by gardeners in allotment gardens, ecological gardening practices are increasingly wide spread, and most of the garden managers have committed to these new practices through regulations, training and discussions with

<figure><img src="image_133.png" /><figcaption>Allotments and footpaths, two very distinct spaces within family gardens. Photo: © F. Di Pietro, University of Tours</figcaption></figure>
the gardeners. However, for many of them, this change can be reduced to simply replacing one set of products with other, less toxic, ones (example: 'bordeaux mixture'). Additionally allotment gardens are considered to be vegetable production areas by their managers. Even if auxiliary life is accepted or even encouraged there, "weeds" are the subjects of hostility since they are considered to be completely contrary to the production function.

Initial interviews with allotment gardeners allowed us to classify the diversity of the gardening practices in use as well as the motivations and attitudes of the gardeners in relation to this activity. More specifically we have also defined the spectrum of synthetic pesticide usage and the implementation of alternative methods. We have highlighted a large number of links between the positions of individual gardeners on this spectrum, their type and their socio-demographic profile. This classification allowed us to target the problems raised by each type of gardener and imagine ways to resolve them. The use of synthetic pesticides is not influenced by socio-demographic factors, but related to where the gardener grew up (countryside) and their identification with the countryside. However, age and social class, as well as where they grew up influence not only how they accept the wild flora and fauna, but also how they manage the soil, fertilisation and choice of vegetable varieties as well as their openness to changing habits. These elements were confirmed by a larger sample of 150 allotment gardeners that were interviewed via questionnaires.

Structural analysis of the gardens and the wild plants in the allotments and footpaths have shown that **the internal structure of the allotments** varies significantly between the different sites and gardens, and the associations have a significant influence on the number of trees planted by the gardeners. Women tend to cultivate less surface area than men. The wild plants found in the footpaths correlate strongly with gardening practices and the structure of the gardens: the share of perennial species increases significantly with the proportion of hedges and, rather surprisingly, diminishes with the proportion of non-cultivated areas and the proportion of vine planting. **The wild plants found in the allotments** vary in terms of the number of ornamental trees (related to perennial species). Additionally, the gardening practices, especially hand tilling of the soil and use of 'bordeaux mixture', have a major influence on the specific variety and proportion of perennial species present in the allotments and footpaths.

<figure><img src="image_134.png" /><figcaption>Family garden allotments can be intensively
cultivated spaces.</figcaption></figure>

Photo: © F. Di Pietro

Prospects for transfer or research

Transfer:

4 orientations were found to be useful for supporting the transition of gardeners towards more environmentally
friendly practices:

1) Organise “debates” involving gardeners and a mediator: a priority for those gardeners who use the most synthetic pesticides.

2) Distribute printed information (even if this might exclude gardeners who are illiterate or do not speak much French) and on-line content (even if this is largely the domain of gardeners who do not use a lot of pesticides).

3) Train gardeners on the basis of the techniques they already use.

4) Train the garden managers on biological gardening and inform them about other aesthetic garden forms.

Research:

Research perspectives point towards the conditions surrounding the place where food and ecological objectives meet (conservation of urban biodiversity). The results are currently being analysed in detail and published in scientific journals. Within the framework of the Ecophyto programme events and the ALTHERCOL project, exchanges have been able to form ties with the Orléans INRAE (Jérôme Rousselet). These exchanges have resulted in a partnership for the OSTils project (direction: Jérôme Rousselet), one of the major chapters will cover private gardeners' reasons for their planting choices and tree species preferences. The financing for this project has been approved and it has now been launched.

<figure><img src="image_135.png" /><figcaption>Organise “debates” involving gardeners and a
mediator: a priority for those gardeners who use the
most synthetic pesticides.</figcaption></figure>

Photo: © F. Di Pietro
## Publications and scientific symposia:

### TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA:

* Di Pietro F. (2020), hosting of Workshop 3 (Pourquoi jardinez-vous ?), Biocontrol for amateur gardeners symposium, Paris - La Défense (France), 11 February 2020.

* Di Pietro F. & Poiré M. (2018), **Évolution des fonctions et des pratiques horticoles dans les jardins familiaux**, oral presentation, Biocontrol symposium. Les nouvelles pratiques pour un jardinage au naturel, Fondettes (France), 13 December 2018

### SCIENTIFIC PUBLICATIONS:

#### Published

* **Faire nature en ville. Les jardins familiaux entre production alimentaire et loisirs : quelle place pour la biodiversité ? Attitude des gestionnaires vis-à-vis des pratiques de jardinage, de la faune et de la flore spontanées.** Di Pietro F. & Poiré M., Carrière JP. et al. (Eds.), 2021. Published by Harmattan.

#### Pending publishing

* Dynamiques Environnementales n°47: **Nature spontanée, nature maitrisée : quelle place pour la biodiversité dans les jardins familiaux ?** Poiré M. & Di Pietro F. (submission)

* Espaces et sociétés : **Motivations, pratiques de jardinage et rapport à la nature dans les jardins familiaux.** Di Pietro F., Gardair E. & Poiré M. (to be published in early 2023)

* Nouvelles Perspectives en Sciences Sociales n°17(2): **Rapport à la nature et contrôle de l'alimentation dans les jardins familiaux.** Di Pietro F., Gardair E., Poiré M. & Gosset S.

Two other science papers are currently being prepared, one discussing the questionnaire results, the other on the botanical analyses.

### Practical / educational articles:

* Microscoop, Hors-série n° 18 (October 2018, pp14-15) : **Jardins collectifs en ville : nature spontanée, nature maitrisée.** Di Pietro F. & Poiré M. Link to document: http://www.dr8.cnrs.fr/CNRS-Hebdo/Documents/542/Document.aspx
<figure><img src="image_136.png" /></figure>

AM&BAS

# Agro-messages and treatment of insect pests in seedling production and large-scale crops

**Launch year:** 2015

**Completion year:** 2017

**Scientific manager**

Brigitte Frérot, INRAE IEES Paris

brigitte.frerot@inrae.fr

**Partners**

INRAE IEES Paris; ARVALIS; FNAMS; M2i

**Financing**

Total cost of the project: € 277,432

Ecophyto grant: € 100,000

**Key words:**

Tychius aureolus; Ostrinia nubilalis; Alfalfa; Sweetcorn; Chemical landscape; Attraction; Biocontrol; Chemical mediators; Kairomone; Mass trapping; Improving varieties

## Context and main objectives

Following the ban on the majority of conventional insecticides due to their negative impact on public health and the environment, a number of industry sectors now find themselves in a technical no-man's land, especially seedling specialists and large-scale crop producers. These producers will now have to evolve in keeping with the Ecophyto plan, but with their priorities being to maintain the performance, quality and sustainability of their operations.

Chemical mediators are one of the biocontrol solutions which can be used as a viable alternative to conventional insecticides. The objective of the AM&BAS project is to use them to control European corn borer and alfalfa weevil populations. The project could be seen as a detailed investigation of the process of taking a proposal from the laboratory to the field; it aims to validate the potentials of kairomone-type chemical mediators for the control of two pest species. A priority was placed on developing biodegradable diffusers to add a further technical benefit. The project involves an industrial producer, an inter-professional, technical institutes and an INRAE research laboratory working together to explore the attractive potentials raised by previous research programs.

<figure><img src="image_137.png" /><figcaption>Trials with INRAE diffusers</figcaption></figure>

Photo: © Brigitte Frérot, INRAE

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

The AM&BAS project continues existing laboratory research work which has shown that insect host-plant location and recognition processes are very closely related to the olfactory detection of specific chemical signals emitted by the plant.

There is no doubt that host-plants attract fertilised female corn borers and that fruiting-stage alfalfa plants attract *Tychius*. The chemical signals emitted by these plants have been identified. The perception of certain compounds by the olfactory organs of the two insects has been validated.

Behavioural laboratory tests on European corn borers have validated the attraction of an identified mixture and have provided evidence of an impact on egg-laying. This action has also been confirmed in the field. The attraction of the mixture has not yet been confirmed in the field, but none of the traps tested were suited to capturing the female of the European corn borer. Studies on the perception of the identified molecules by the insects' antennae has shown that there are key molecules that block the insect's localisation of the plant if they are not emitted. The attraction of fertilised females raises a new approach to corn crop protection but this does involves a complex system in which the insect's detection capacities have been demonstrated to be very precise.

A specific mixture appears to attract *Tychius aureolus*, it seems to be able to concentrate insects within the zone where the mixture was diffused. It could be used to establish trap areas. However, for the moment we have not identified a trap suited to the behaviour of this insect. Certain compounds essential to the relation have also been identified, especially for the seedpod-development stage.
The hypothesis of the attraction of pests outside of the field or along its edges has been validated for *Tychius aureolus*. The positive first year results for the European corn borer were not validated for the second year, perhaps due to the influence of excessive population levels.

The diffusion parameters of the INRAE and M2i diffusers have been calibrated. Experiments have allowed us to improve and advance the M2i diffusion matrices. These matrices are biodegradable and easy to use. The proposed attractor for the project was evaluated using a trap that has already proven its usefulness for capturing male moths attracted by a sexual pheromone. In the context of our study, the results were not able to validate the effectiveness of the attractor for capturing females. Further work would be merited.

<figure><img src="image_138.png" /></figure>

Concentration of European corn borer eggs - Average number of eggs counted on 10 corn plants within areas close to the B6 attractor at different doses

Image: © Brigitte Frérot, INRAE

# Prospects for transfer or research

## Transfer:

The INRAE has submitted an Invention and Usable Results Declaration (DIRV) for the Corn plant - Corn borer pairing. Competition in the presence of the host plant must be reinforced but we have all the information necessary to improve the assemblies of synthetic molecules.

A scientific article is currently being drafted for the Alfalfa-*Tychius* pairing.

For both of the pairings, we have identified the key compounds of the process whereby the host-plant is colonised by the insect. This knowledge could be used to select resistant varieties. Knowledge built up on the olfactory relation between the insect and the host-plant will enable the selection of resistant plants that do not emit the key chemical signals.

## Research:

Research work on the olfactory relations between pest insects and their host-plants must continue since it could produce new pest insect control solutions or new selection criteria for crop varieties resistant to pest insects. This latter point raised a new research path concerning key chemical signal genes. They could be selected to produce plants that specialist pest insects would be unable to identify. This selection could be advanced as a compatible clean and biological pest control solution. Since the gene maps of most cultivated plants are already known, it would be easy to remove a light alcohol and a terpene.

# Publications and scientific symposia:

## SCIENTIFIC SYMPOSIA:

* **Perspective de lutte contre les insectes monophages. Du paysage chimique à la protection des plantes.** AFPP - Sixth alternative protection methods for integrated production conference. Ene LEPPIK, Centina PINIER, Céline ROBERT, Véronique BIARNES, Pierre TAUPIN, Jean-Baptiste THIBORD, Brigitte FREROT - Lille, on 21, 22 and 23 March 2018.

* **L'attraction des femelles fécondées, une nouvelle voie de recherche pour la protection des plantes cultivées:** AFPP - New contributions to crop protection from pests. Ene LEPPIK, Centina PINIER, Magali GRANGER, Jean-Baptiste THIBORD and Brigitte FREROT - Montpellier on 24 October 2017.

## PRACTICAL / EDUCATIONAL ARTICLES:

* **Peut-on leurrer la pyrale avec le parfum du maïs ?** Phytoma, n° 707, 26-29 (2017). Ene LEPPIK, Centina PINIER, Magali GRANGER, Jean-Baptiste THIBORD and Brigitte FREROT

* **L'attraction des femelles fécondées de lépidoptères ; une nouvelle voie de recherche pour la protection des plantes cultivées.** Phloem (2018). Brigitte FREROT.

* **Ravageurs du maïs - Le « parfum » du maïs synthétisé.** Perspectives-agricoles (2018). See with Paloma Cabeza-Orcel - p.cabeza@perspectives-agricoles.com

* **« Tromper les insectes ravageurs des cultures grâce à l'écologie chimique »** (21/06/2018) [Article available here](https://www.sciencedirect.com/science/article/pii/S1477095X18300006)

## OTHER PRACTICAL WORK:

* Confirmation of previous results of work on the identification of signals emitted by the host-plant: DIRV (Invention and Usable Results Declaration) for an attractor for European corn borer

* Optimisation of biodegradable distributors (M2i)

* **2018 and 2019 agricultural shows** with Arvalis and Passion céréales 2018 "using odours in Agriculture"

* **Un parfum pour protéger les plantes** / Passion céréales: [Video available online here](https://www.youtube.com/watch?v=dQw4w9WgXcQ)
<figure><img src="image_139.png" /></figure>

ATTRACTMYFLY

# Development of attraction and entomopathogenic fungus auto-dissemination techniques to control melon fly, *Zeugodacus cucurbitae*

**Launch year:** 2015

**Completion year:** 2018

**Scientific manager**

Laurent Costet, UMR PVBMT CIRAD

laurent costet@cirad.fr

**Partners**

CIRAD UMR PVBMT; ARMEFLHOR; AB7

Innovation Natural Plant Protection SA and Betel

Réunion SAS (Arysta LifeScience Group)

**Financing**

Total cost of the project: € 262,426

Ecophyto grant: € 100,000

## Key words:

Olfactory pest control and traps; Elementary alternative practices; Vegetable crops; Specific attractors for fruit fly; Kairomones; Entomopathogens; *Beauveria* spp; Auto-dissemination; "Attract and disseminate"

## Context and main objectives

Fruit fly (*Tephritidae*) are pests that damage fruit and vegetable crops in tropical and temperate zones, including mainland France and its overseas territories. Synthetic insecticides are used in vast quantities to control them. Alternative control methods, such as large-scale trapping, have produced encouraging results, but their development remains limited for certain species where no specific attractor for the female insects that cause the damage has yet been identified. *Zeugodacus curcurbitae*, the melon fly causes a lot of damage to cucurbitaceous crops. Kairomones, substances emitted by the host-plant that attract the female, have been identified. One of the objectives of the AttractMyFly project was to develop a mass female fly trapping system including a polymer kairomone diffuser. Another objective was to evaluate an auto-contamination-dissemination strategy involving entomopathogenic fungus.

The availability of a specific attractor for males raised the possibility of attracting them into the traps in order to use them as vectors for spreading spores to contaminate the females during mating.

<figure><img src="image_140.png" /></figure>

Experimental in-field trap evaluation system: the traps are attached to an iron post 50cm above the ground. They are located along the edge of the field with a distance of 10 metres between each trap.

Image and photo: © Anne-Sophie Zoogones, CIRAD

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

Starting from 10 compounds potentially involved in attracting females of the melon fly, *Zeugodacus cucurbitae*, a combination of two volatile synthetic compounds, C1 and C2, were identified in the laboratory and considered to be attractive in the same way as the odour of fresh fruit. One of the objectives was to incorporate kairomones into a polymer diffuser and develop a mass trapping system for the females.

These polymer diffusers were developed and then assessed in the laboratory using a one-way olfactometer. Polymers with C1 and C2 were found to be just as attractive as the odour of fresh fruit. The trapping capacity of the various systems was then assessed under semi-controlled conditions inside large outdoor cages. We tested the different traps using different combinations of the kairomones inside different polymers. Kairomones C1 and C2 were confirmed as attractive but were ten times less effective than they had been under laboratory conditions. A TephriTrap system filled with detergent and water was selected. This system's trapping capacity was then assessed in the field between June 2017 and January 2018 on thirteen different *cucurbitaceae* crop fields on Réunion Isle using different quantities of the C1 and C2 kairomones in the polymers. The systems with quantities higher than or equal to 27 mg of C1 and C2 trapped significantly larger quantities of females than the control. During the project we also developed an "Attract and Kill" system based on the kairomone attractors that target females.
It was also possible to attract the males into a system containing spores, using a specific attractor: cue-lure. These were then used as vectors for the spores in order to contaminate the females during mating. Another objective was to develop an auto-contamination-dissemination system for entomopathogenic fungus.

<figure><img src="image_141.png" /><figcaption>*Beauveria hoplocheli* strain.
Photo: © Isabelle Merle, CIRAD</figcaption></figure>

The first step was to identify a biocontrol agent that could be approved for marketing. We evaluated the pathogenic capacity of three commercially available strains of *Beauveria* on *Z. cucurbitae*. The I-2961 strain was the most pathogenic for the melon fly and was selected for the rest of the study.

The auto-contamination-dissemination concept specifically targeting the females is based on the possibility of transferring spores of *Beauveria* from the male onto the female during mating. The second step was to test this transfer process on *Z. cucurbitae*.

The transmission of spores from the males onto the females during mating will significantly increase the mortality of the female if the males are able to mate less than an hour after contamination. However, 24 hours after contamination, there was no mortality amongst females.

Finally, we have designed an attraction contamination system. This is based on attracting males with a polymer emitting cue-lure, a formulation of spores of the I-2961 strain and a modified Tephitrap. This system works in the laboratory. We were therefore able to prove the effectiveness of this auto-contamination-dissemination method. However: on one hand, the window of opportunity during which the spores can be transferred successfully seems too short for this strategy to have an effect on populations in the field, and on the other hand, we have shown that a part of the inoculate transported by the males was deposited into the surroundings thus creating a potential for spreading this disease to other individuals.

<figure><img src="image_142.png" /><figcaption>From left to right. Schematic representation of a female of the melon fly. Image: Toulassi Nurbel, CIRAD. *Beauveria hoplocheli* and auto-contamination system. Photo: Isabelle Merle, CIRAD. Female melon fly affected by *Beauveria hoplocheli*. Photo: © Brice Derepas, CIRAD</figcaption></figure>
# Prospects for transfer or research

## Transfer:

The mass trapping strategy for the melon fly, based on the use of kairomones, has a short-term transfer and usage potential. It could be possible to use the traps developed during the project after a validation phase. The pathogenicity tests on the commercially available strains of *Beauveria* have shown that the concept could be immediately extended for other pest species. The *Beauveria* spore auto-dissemination strategy targeting females shows promise but requires further research work.

## Research:

The continuation of research work on the mass-trapping strategy using kairomones should focus on the optimisation of the traps by considering the diffusers, attractor quantities and numbers as well as the trapping system. The use of kairomone compounds would also require further studies on the specificity of the traps and toxicological issues. As for the *Beauveria* spore auto-dissemination strategy targeting females, further work should be done to optimise the auto-contamination system especially concerning the spore formulation and to evaluate its genuine field potential.

## Publications and scientific symposia:

### TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA

* **International Congress on Invertebrate Pathology and Microbial Control (24-28/07/2016): Abstract of the 49th Annual Meeting of the Society for Invertebrate Pathology:** Characterization of the pathogenicity of commercial or precommercial Beauveria sp. strains against the melon fly *Bactrocera cucurbitae*. Rohrlich C., Merle I., Payet-Hoarau M., Télismart H., Besse S., Nibouche S., & Costet L. (2016). Tours, France [Poster FU 5].

### SCIENTIFIC PUBLICATIONS

* **PLOS one**, 13(7), e0199199: Variation in physiological host range in three strains of two species of the entomopathogenic fungus *Beauveria*. Rohrlich C., Merle I., Hassani I. M., Verger M., Zuin M., Besse S., Robene I., Nibouche S. & Costet L. (2018). https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199199.

### PRACTICAL / EDUCATIONAL ARTICLES:

* **Fertile: Bulletin de l'Association Réunionnaise pour la Modernisation de l'Economie Fruitière Légumière et HORticole (Armefhlor).** (n°41, p 14) : Lutte biologique contre la mouche du melon : des résultats prometteurs. Graindorge R. et Costet L. (2018).

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

* AttractMyFly steerage committee, Launch meeting, 10/04/2015

* AttractMyFly steerage committee, 22/09/2016

### OTHER PRACTICAL WORK:

#### Educational

* **PhD thesis in Population Biology and Ecology - Université de la Réunion:** Lutte biologique à base de champignons entomopathogènes du genre *Beauveria* en zone tropicale. Presented on 3 October 2018. 149 p. Rohrlich C. (2018)

* **Dissertation Master 2 Healthcare Sciences and Technology - Plant Biology and Technology, Plant Production and Technology (Protev), Université d'Angers:** Caractérisation de la capacité de transfert de spores de champignon entomopathogène *Beauveria* bassiana des mâles aux femelles de la mouche du melon *Zeugodacus cucurbitae* lors de l'accouplement. 33 p. Verger D. (2017)

* **Dissertation, Agronomics Engineer studies, ESA d'Angers:** Caractérisation de la capacité de transfert de spores de *Beauveria* bassiana des mâles aux femelles de la mouche du melon *Zeugodacus cucurbitae* lors de l'accouplement. 84 p. Venard J. (2016)

* **Dissertation Agronomics Engineer Studies, ENSAIA, specialisation: crop protection and Université de Lorraine:** Caractérisation du pouvoir pathogène de souches de *Beauveria* sp. sur la mouche du melon *Bactrocera cucurbitae* et de la capacité de transfert horizontal des spores du champignon lors de l'accouplement. 39 p. Merle I. (2015)
<figure><img src="image_143.png" /></figure>

BIOBOT

# Optimisation, including the global and integrated management, of biocontrol solutions for the treatment of grey mould on grapevines

**Launch year:** 2015

**Completion year:** 2019

**Scientific manager**

Nicolas Aveline,
IFV (French Vine and Wine Institute)

nicolas.aveline@vignevin.com

**Partners**

IFV; INRAE UMR SAVE; Gironde Chamber of Agriculture

**Financing**

Total cost of the project: € 141,394

Ecophyto grant: € 96,845

## Key words:

Wine-making; Botrytis; Grey mould; Biocontrol; Micro-organisms; Biological control agents; Decision-making tools; Changes to technical approaches

## Context and main objectives

Grey mould is a disease that affects grapes towards the end of the growing season (powdery greyish mould), once it affects one grape it rapidly spreads over the whole of the bunch. In addition to its effect on harvests (quantity), the fungus affects the taste of the wine produced if more than 5% of the harvest is affected. Generally speaking, the phytosanitary treatment for this disease can reach between 1 and 2 TFI, which regularly results in the presence of product residues in the wine.

Biocontrol alternatives exists, they were even amongst the first biocontrol products for wine-growing to be officially registered. Unfortunately the use of these products remains restricted and difficult to evaluate: especially the very diverse operational methods which raise a number of questions on their ideal role and application strategies. In addition, Botrytis is difficult to treat preventively and its sudden development at the end of the year can be explosive.

<figure><img src="image_144.png" /></figure>

Grapes affected by Botrytis.
Photo: © Nicolas Aveline, IFV

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

A lot of work has been done to assess the biocontrol products available for treating Botrytis on vines; over 3 years of assays on two different locations in the Gironde region: 14 products or substances were tested as part of a series of specially designed comparative experiments: 6 applications during the season and, for some of the products, application of the "Ciliberti index" decision-making tool to organise the post-ripening applications as efficiently as possible. The results generally showed a low level of protection effectiveness. One of the tested products, Armicarb® (potassium bicarbonate) seems to be the only solution that makes a consistent difference when compared to an untreated control. Some of the products are micro-organisms from the *Bacillus* family: Amylo-X®, S38 (INRA model bacteria) and Rhapsody® have regularly shown themselves to be effective. The other products produce results that are too variable from year to year for any conclusions to be reached on their usefulness.

The validity and above all consistency of the results obtained by the project remain complex: the 3 harvests were contrastingly different in terms of climate (heavy frosts in 2017) and the botrytis effect was mostly middling and light. Additional measurements (vigour, tightness of bunches, grape worm effect, etc.) were taken and still have not shown any clear intra-vineyard correlations. *B. cinerea* contamination rate tests on flower caps have turned out to be interesting in spite of inconsistencies from one year to the next and identification limitations. It is useful for the knowledge gained about the disease and as an indicator of parasite impact at the start of the season. It is a tool with potential for the evaluation of the early effects of biocontrol products on the pathogen, but it still requires some methodological improvements.
The post ripening "Ciliberti Index" decision making tool developed by INRAE can be used to simulate a daily botrytis development climate index (temperature and hygrometry) covering the 4 major risk categories. It allows for effective and judicious end-of-season biocontrol applications in relation to pre-identified risk periods.

It was tested on a number of candidate products on a large vineyard in 2017; one year later it was transferred to the partner experiment specialists. Interpretation and the decision making rules have changed during the project to finish with the 2019 on-line calculation version.

## Links with the Ecophyto plan:

* **Biocontrol development:** the strategies proposed and studied by the project are 100% biocontrol, no traditional products used in alternation or association were assessed. With the strict theme of lowering intrants, this project aims to define the biocontrol strategies that would cope with the residue issues generally raised by more traditional anti-botrytis treatments.

* **Optimising the use of biocontrol products:** the use and organisation of biocontrol products has been studied, especially at the end of the season and right up to just a few days before harvesting, the crucial period when the use of chemical products is restricted. We now have new intervention possibilities for dealing with botrytis.

* **Development of a decision-making tool:** the Ciliberti index was used and improved (definition of decision making rules and thresholds) to judiciously program the use of certain biocontrol products either after ripening or during maturity.

## Prospects for transfer or research

### Transfer:

The candidate biocontrol solutions which produced interesting performance results are currently in use (2019, 2020, etc.) in other regional applied research programs (Alt'Fongi Biocontrol project, BEE project). The aim is to include them in a global protection programme that uses as many biocontrol products and preventive approaches as possible along with an absolute minimum of conventional pesticides.

The use of the Ciliberti index opens up research potential on the position of anti-Botrytis products and the crucial phases of the disease's development. Its transfer through global tools distributed by the IFV (French Wine and Vine Institute), such as the on-line solution DECITRAIT®, should provide the means to integrate it into global protection management and provide optimised biocontrol application recommendations for grey mould.

The experimental "flower cap contamination rate" early warning indicator is very useful for evaluating the botrytis pressure on the inflorescence as well as measuring the effect of some of the biocontrol products applied during this period. Improving identification techniques will require more work (the molecular tool?) to identify and evaluate botrytis more effectively. This could then be integrated into the Botrytis Receptivity Potential (PRB) indicator published annually by the INRAE and allow for the publication of an early-use effectiveness rating for the tested solutions.

Finally, our trials have shown that the *B. ginsengihumi* S38 bacteria, isolated by the INRAE, has a very interesting anti-Botrytis potential for grape vines.

### Research:

The work completed during the project has shown the importance of advance knowledge, both concerning Botrytis and its development and on the operation of biocontrol products. Questions remain on the pathogen's epidemiology and the prediction of its development, but some clear progress has been achieved with certain risk indicators used by BIOBOT. Research programmes and projects have been launched on this subject by the UMT SEVEN, including a project for the identification and evaluation of aerial spore dissemination on grapevines, or dynamic studies of the agro-climate factors that influence the spread of the disease. All of this research work will help any control indicators that can be developed to improve protection management and potential biocontrol applications.

<figure><img src="image_145.png" /><figcaption>Petri dish culture of flower caps.
Photo: © Nicolas Aveline, IFV</figcaption></figure>
It is worth noting that there is a need for a biocontrol micro-organism traceability tool (for use with all strains) to know if the strain remains present on the plants and fruit, to have a better knowledge of its installation and survival conditions and organise suitable scheduling decisions.

The work by INRAE UMR SAVE on the *B. ginsengihum* S38 bacteria (Calvo et al., 2019) confirms how the micro-organisms naturally present in vineyards can be enormously useful. Research work could be continued to improve the use of this type of bacteria; especially in terms of mass production and formulation.

Finally, and more generally speaking, research on biocontrol and biostimulant products considered potentially effective in the treatment of grey mould should be continued: action methods such as defence stimulation, physical barrier strengthening etc., are not that common in the biocontrol list. The European "basic substances" status could also turn out to be a good source of alternatives to chemical fungicides which could have a regular and significant effect on grey mould.

## Publications and scientific symposia:

### TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA:

* **Rencontres Viticoles d'Aquitaine, Lycée Viticole de Blanquefort 2018: Le biocontrôle pour lutter contre la Pourriture Grise : the BIOBOT and ALB's projects (RVA 2018) [PPT]**. Aveline N.

### SCIENTIFIC PUBLICATIONS:

* **Frontiers in Plant Science (n° 10, 2019, p 105): Microbial Antagonism Toward Botrytis Bunch Rot of Grapes in Multiple Field Tests Using One Bacillus ginsengihumi Strain and Formulated Biological Control Products.** Calvo Garrido C., Dupin S., Aveline N., Roudet J., Davidou L., Fermaud M.

### PRACTICAL / EDUCATIONAL ARTICLES:

* **Guide ACTA: Guide ACTA Biocontrôle 2018: Lutte contre la Pourriture Grise sur Vigne.** Aveline N., Fermaud M., Chantelot E.

* **Guide ACTA: Guide ACTA Biocontrôle 2019: Lutte contre la Pourriture Grise sur Vigne.** Aveline N, Fermaud M, Chantelot E.

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

* **Union Girondine des Vins de Bordeaux (2017, pp 53-54): Utiliser les produits de biocontrôle pour protéger la vigne contre la pourriture grise en viticulture biologique : résultats des essais menés dans le RESAQ Vitibio.**
  Aveline N., Davidou L.

### OTHER PRACTICAL WORK:

* **Bordeaux ISVV: Master 2 course: Grape vine sciences, oenology, EU wine-growing pathology (2017): Epidémiologie et lutte contre Botrytis cinerea au vignoble.** Aveline N., Fermaud M.

* **Poster presentation, Perpignan (2018): BIOBOT: Evaluation de produits de biocontrôle contre la pourriture grise (B. cinerea) au vignoble.** Aveline N., Dupin S., Davidou L., Calvo Garrido C., Roudet J., Fermaud M.
<figure><img src="image_146.png" /></figure>

# Biological control of the codling moth, *Cydia pomonella* (BIOControl of *CYDia pomonella*)

**Launch year:** 2015

**Completion year:** 2018

**Scientific manager**

Nicolas Ris, **INRAE ISA**
nicolas.ris@inrae.fr

**Partners**

Institut Sophia Agrobiotech; **BIOLINE Agrosciences**

**Financing**

Total cost of the project: € 303,142

Ecophyto grant: € 103,918

**Key words:**

Acclimatisation; Augmentation; *Cydia pomonella*; Biological control; Parasitoid

## Context and main objectives

Codling moth, *Cydia Pomonella* (Lepidoptra: Torticidae) is one of the major macro-organism pests affecting apple orchards in France, and across the globe; due to (i) its extensive geographical distribution, (ii) the economic impact of the damage it causes, (iii) the excessive use of pesticides it provokes and (iv) the limits of the currently available control solutions. Amongst the available biocontrol solutions, a lot has been invested in a sexual confusion approach and the use of carpovirus; but both of these methods have major limitations. Elimination methods based on releasing sterile insects have also been tried locally but have yet to prove effective. Finally, the possibility of effectively controlling codling moth by encouraging the local biodiversity (organic control through conservation) also seems to have its limits. It would therefore appear that biocontrol methods need to be diversified to complement the already existing approaches and, potentially, limit the possibility of this pest developing resistance to them. In this context, the BIOCCYID project aims to evaluate two biocontrol methods based on two types of entomophagic macro-organisms: egg-eating parasitoids of the *Trichogramma* genus (*Hymenoptera*: Trichogrammatideae) and a pre-pupal parasitoid *Mastrus ridens* (*Hymenoptera*: Ichneumonidae).

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

Regarding the "Organic pest control by increase using trichogramma" part of the project: trichogramma sample collections were taken between 2014 and 2016 over a large part of the South of France in a coordinated program involving the "BIOCCYD", "INDREGARB" PSPE1 2013-2015 and "TRIPTIC" ANR 2014-2019 projects. For the potential host plants for *Cydia Pomonella*, the trichogramma collection rate was established as mid to low. The diversity of the species collected was estimated using molecular approaches (COI region "barcoding") and compared to those observed on other plant categories: (i) Rosaceae not host to *C. Pomonella* and sympatric with *C. pomonella* hosts; (ii) other non-host plants for *C. Pomonella* and sympatric to *C. pomonella* hosts; (iii) Rosaceae not host to *C. pomonella* and allopatric to *C. Pomonella* hosts. Except for the molecular taxon known as "misG" (only to be found in cultivated apple trees), the main species found on *C. pomonella* hosts turned out to be common and not very specialised. Regarding "misG", studies are currently under way to define its precise taxonomical status. Considering both these trichogramma diversity results and the strategy of BIOLINE Agrosciences, investigations on these subjects was ceased by mutual agreement.

Regarding the "Organic pest control by acclimatisation using *Mastrus ridens*" a number of *M. ridens* strains have been successfully introduced into our facilities (from New Zealand: LOA n°FR15PA00001 dated 22/05/2015; from Chilli: LOA n°FR16PA00001 dated 19/01/2016 and LOA n°FR16PA00002 dated 01/03/2016) after authorisation had been received (19/05/2015). *M. ridens* is relatively easy to reproduce for population level maintenance purposes, but much more difficult to reproduce in large numbers. Based on (i) the introduction applications made in other countries, (ii) articles on the subject and (iii) experiments we have completed on non-target hosts, an application for its introduction into the environment was submitted to the DGAL and the ANSES on 04/03/2016. This 37 page report summarises knowledge acquired on the biology of *M. ridens*, describing the communities of natural enemies to *C. Pomonella* present in France and across the world, and sets out the potential advantages and risks that could be associated with such an introduction into the environment. On this basis and with the recommendations of independent experts, the ANSES issued its approval on 04/01/2017 and a decree cosigned by the Ministry of Agriculture and the Ministry of the Environment was officially issued on 09/05/2017. As with all first-time introductions of exotic auxiliaries, the aim is now to use these rare opportunities to set up multi-site post-release monitoring programmes over a number of years, in order to evaluate the performance of *M. ridens* as precisely as possible and answer population biology issues.
Considering the available logistical and financial resources, only one site in the Vaucluse was used for releasing *M. ridens* during the second half of 2018. Attempts to raise further funding to complete a more precise evaluation of the establishment capacities of *M. ridens* were unsuccessful in 2017 and 2018. However in 2019, financial support was obtained from FranceAgriMer ("BIOCCYD-Mastrus" project; Manager: Nicolas BOROWIEC, INRAE).

## Link with the Ecophyto plan:

The initial objective of the BIOCCYD project was evidently to reduce the use of phytosanitary products to control the codling moth, *C. pomonella*, in combination with other biocontrol methods (sexual confusion, carpovirus, biological pest control through conservation). Considering the results achieved, one particularly interesting perspective raised by our work is the possibility of introducing *Mastrus ridens* into France and its potential establishment. If this is successful a new eco-systemic service will be created that regulates coddling moth populations at no cost to the agriculturalists involved.

## Prospects for transfer or research

### Transfer:

Organic pest control by acclimatisation is generally seen as a public service mission: (i) "anticipatory" evaluation of candidate auxiliaries, (ii) introduction into all or part of the territory and (iii) post release evaluations are all generally completed by research institutes and technical-agronomic bodies (Technical institutes, Experimental stations, Farming networks, etc.). The involvement of the private sector (bio-factories) may be possible (see the "chestnut gall wasp" case) but is generally limited over time.

### Research:

The most evident research perspective is clearly the **multi-year and multi-site evaluation of Mastrus ridens** in terms of its local dynamic (local establishment and populations), geographical dispersion and the ecological niches generated (effective use against *C. pomonella* on the various host-plants; any impact on non-target species). In many ways, this post-release evaluation will respond to scientific expectations (biology of introduced populations) and farming expectations (evaluation of the service provided by *M. ridens* and its impact on other codling moth control methods).

Through the introduction of *Mastrus ridens* in the Vaucluse (2018) and perhaps some other similar operations, **the influence of environmental heterogeneity** around the release site (< 1km) on local dynamics and dispersions is currently being evaluated (Thesis of David MURU; Institut Sophia Agrobiotech).

Another particularly interesting question is **the evaluation of the gender determination method (sl-CSD) of M. ridens on its population dynamics.** The lack of genetic variability at the sl-CSD locus leads to the production of diploid males that are reproductively non-functional which then compete with the normal (haploid) males to mate with females. Monitoring the frequency of diploid males in the wild and restoring genetic diversity at the sl-CSD locus, are both issues that deserve further specific investigation.

## Publications and scientific symposia:

### TECHNICAL DOCUMENTS:

* Borowiec N., Malausa T., Ris N. 2015. **Document technique relatif à l'introduction en milieu confiné de l'hyménoptère parasitoïde exotique, Mastrus ridens.** 18pp.

* Borowiec N., Muru D., Malausa T., Ris N. 2016. **Document technique relatif à l'introduction dans l'environnement de l'hyménoptère parasitoïde exotique, Mastrus ridens.** 37pp.

* Marchand A., Sellier N., Warot S., Ion-Scotta M., Ris N., Groussier-Bout G. 2017. **Formalisation d'un Centre de ressources biologiques dédié aux parastoïdes oophages : CRB EP-Coll.** Cahier des Techniques de l'INRA

### PRACTICAL / EDUCATIONAL ARTICLES:

* Muru D., Auguste A., Fauvergue X., Malausa T., Ris N., Thaon M., Vercken E., Borowiec N. 2018. **Un parasitoïde exotique pour lutter contre le carpocapse.** Phytoma, la Défense des Végétaux. 710: 37-41.

### SCIENTIFIC PUBLICATIONS:

* Muru D., Marchand A., Calcagno V., Cruaud A., Rasplus J-Y., Ris N., Vercken E., Warot S., Groussier G. in prep. **Survey of the diversity of Trichogramma species in France and neighbouring areas with information related to their host plants and habitats**

* Warot S., Cruaud A., Groussier G., Malausa T., Martinez-Rodriguez P., Pintureau B., Seguret J., Ris N. **Insights into the molecular diversity and species delineation in the genus Trichogramma with a focus on West Palearctic**
# Biological control of the grapevine fanleaf virus: the impact of integrating growing techniques involving intermediate crops and the use of a resistant root-stock on nematode populations

Unfortunately this summary data-sheet is not available.
<figure><img src="image_147.png" /></figure>

BIOLIM

# Innovative biocontrol techniques for the management of crop damaging land molluscs: Looking for an evaluation methodology suited to their ecophysiology

**Launch year:** 2015

**Completion year:** 2018

**Scientific manager**

André Chabert, ACTA

andre.chabert@acta.asso.fr

**Partners**

ACTA; Université de Rennes; ARVALIS Institut du végétal; Phyteurop; Bayer-CropScience; De Sangosse

**Financing**

Total cost of the project: € 194,696

Ecophyto grant: € 100,000

**Key words:**

Slugs; Biocontrol; Large-scale crops; Ecophysiology; Elementary alternative practices; Changing techniques; Redesigning crop systems; Management via macro-organisms

## Context and main objectives

For most annual plants that are subject to slug damage, chemical treatment becomes practically systemic during high rainfall years. This project aims to contribute to the development of new biocontrol approaches and therefore contribute to reducing the use of non-biocontrol phytosanitary products. The main contributions are as follows:

* Evaluate the performance of new biocontrol approaches and products for dealing with slugs;

* Use chemical ecology to explore new evaluation approaches to study the semiochemical relations between slugs and plants as well as ground beetles;

* Evaluate the effectiveness of biological control methods in terms of the agricultural levers that can be mobilised to deal with this pest;

* Define the conditions that apply to their use in reducing populations of this pest within crop systems through the combination of identified and evaluated levers to re-design integrated protection strategies;

* Create an innovation research dynamic by assembling a public/private sector partnership.

The methodologies were first communicated to the partner experimentation networks of the BIOLIM project which has facilitated the exploration of new research approaches by private companies; they were then communicated to the principal activity sectors concerned by slug damage.

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

This BIOLIM PSPE2 project includes a public partner (University of Rennes 1) for its expertise in mollusc eco-physiology along with technical institute and private business partners for their contribution to experimentation procedures and their knowledge of crop systems. Consequently this slug biocontrol product dedicated project has facilitated the compilation of a panorama of all current knowledge on the subject and the launch of new experimental programmes. It has also established an inventory of preventive agricultural levers and their effectiveness on slug populations.

The first phase was dedicated to completing a meta-analysis type approach based on:

i) Data from the CasDAR RESOLIM project

ii) Textual sources covering slug control techniques

iii) The various available biocontrol approaches.

The specific quality of this approach was to allow for interactions between slug biology, crop systems and all of the currently available biocontrol techniques. These resources are diverse and reflect the relations existing between slugs and their biotic and abiotic environments (see Fig 1 on the next page).
<figure><img src="image_148.png" /><figcaption>**Figure 1:** Biotic and abiotic relations of slugs with their environment. The number of listed publications (between 1992 and 2018) for each biocontrol method is shown here in brackets. Image: © André Chabert, ACTA</figcaption></figure>

Following this, these organic pest control methods have been studied using specially designed experimental methodologies to evaluate their performance, allowing for the specific aspects of slug life-cycles, and their potential for integration into crop systems. These studies could be resumed as follows:

**1) The use of service plants as an intermediary crop**

In order to clarify the usage potential for service plants, the University of Rennes studies were designed to evaluate the biochemical interactions between the slugs and the plants when the former are eating the latter. A protocol was set up to establish the feeding reaction of the slugs to the rapeseed metabolites emitted at the seed leaf stage. It has been shown that the phenylalanine present in the seedlings produces a phagostimulant effect, but this property is lost when glucosinolate emitted when the plant is attacked is added to the mix. Slugs are therefore always capable of eating rapeseed seedlings even if their natural defences are active, but their consumption is reduced.

The ARVALIS laboratory studies concentrated on the use of repellent mustard varieties as intermediate crops. They were able to evaluate the feeding preferences of slugs in the presence of 4 different mustard varieties. These methodologies clearly showed the differences between the plant species that need to be known in order to anticipate their real effects on slug populations and thus provide suitable recommendations.

**2) Natural (mineral or plant) substances with toxic or repellent properties**

ACTA lab-tested a caffeine based formulation on grey slugs, this provoked mortalities on contact but did not function as bait. The companies that own these formulations decided not to continue with the development of this method. Other studies have shown that decoctions of garlic for plant protection did not have a sufficiently repellent effect to restrict attacks from slugs.

**3) Macro-organisms: predators such as ground and rove beetles, spiders or parasites such as nematodes**

Monitoring campaigns using traps were completed in Boigneville to try to find out if ground beetle populations could regulate those of slugs. These trapping campaigns showed that slug populations were much higher in fields where the preceding crop was barley compared to those where the preceding crop had been rapeseed. However the total number of ground beetles (*Poecilus cupreus* and *Pterostichus melanarius*) trapped was almost exactly the same for both fields. In this case, no relation has been detected between the two populations. For this study it would appear that variations in slug population levels are more closely related to the preceding crop.

**► Results summary**

The performance results for the biocontrol techniques tested were summarised collectively by all of the partners. We have calculated ratios to compare the different works taken from the bibliography with those completed within the context of the project. Table 2 in the appendix, shows ratios that evaluate the impacts of different techniques on slugs, and the damage they incur calculated from the analysis of the bibliography and the works completed by BIOLIM.
# Prospects for transfer or research

## Transfer:

Regarding slugs present in large-scale crops, and probably any other crop-fields, it is difficult to isolate the agricultural practices from the implementation of biocontrol techniques. This might seem obvious, however the knowledge required, the practical issues raised by the implementation of biocontrol phytosanitary products are numerous, complex and therefore often avoided when there is no available expertise on the subject. This then leads to over-simplified recommendations that are possibly unsuited to the myriad of different localised and climate-related situations. This is where working as part of a network is important. However, a large number of scientific and technical questions concerning the evaluation of biocontrol projects could only be resolved by analytical tests which require significant resources if they are to be done independently.

## Research:

One research approach could be to collate the large sets of data obtained at different levels (laboratory, fields, systems, farms, etc.), which would require the collective work done here to continue, doubtless along with further collaborations around other projects. The continuation of this work will need to concentrate on these two points:

1) Maintain an experimental network concerning slugs in order to evaluate biocontrol phytosanitary products independently and maintain a network of expertise in place. Current chemical ecology knowledge would need to be shared and applied to find new biocontrol approaches for slugs.

2) This network should also contribute to the enlargement of the current meta-analysis approach as well as the development of slug risk evaluation tools. These two approaches could be extended further to cover other pests affecting large-scale or other crops.

# Publications and scientific symposia:

## TECHNICAL WORKSHOPS AND SCIENTIFIC SYMPOSIA:

* **REOLIM project results presentation symposium. 18/03/2016, Paris:** Presentation of the BIOLIM project and its current works.

* **Terres Inovia Workshop 26/01/17, Évreux:** presentation of the RESOLIM and BIOLIM project results.

## SCIENTIFIC PUBLICATIONS:

* **Phytoma (n°694, pp 34-37):** État des connaissances sur le biocontrôle vis-à-vis des limaces. Mottin E., Tamine M., Chabert A. and Charrier M. (2016)

* **Discrimination of oilseed rape seedlings by the field slug Deroceras reticulatum and storage of salicylic acid in its digestive gland.** Tamine M., Jonard C., Van Der Linde M. & Charrier M. Article in preparation

## PRESENTATION TO PROFESSIONAL OR DECISION-MAKING BODIES:

Contacts established with various Ecophyto plan participants during the results presentations:

* At the PSPE seminar, 2 December 2017

* At a workshop with farming professionals in Bourges, January 2017

* To people involved in one of the Ecophyto projects (action 30,000) on the reduction of the use of anti-slug products, 10 September 2018

## PRACTICAL / EDUCATIONAL ARTICLES:

* **Creation of an instruction sheet for a biocontrol anti-slug product as part of a solution contract**

* **Creation of part of the ACTA biocontrol index**

* **Master 2 Natural heritage and Biodiversity, University of Rennes (2015, 21 p): Quels moyens de biocontrôle pour les ravageurs de culture ? Le cas des limaces.** Tamine M.

* **Master 2 Natural heritage and Biodiversity, University of Rennes (2016, 25 p): Étude expérimentale de l'interaction trophique entre le colza et son ravageur, la limace grise Deroceras reticulatum : facteurs stimulant la nutrition et réponses métaboliques interspécifiques.** Tamine M.

* **Master 1 Natural heritage and Biodiversity, University of Rennes (2017, 15 p): Sélection gustative de métabolites du colza par la limace grise Deroceras reticulatum et devenir des phytohormones dans les tissus de la limace.** Van Der Linde M.

## MATERIALS AVAILABLE ON DEMAND:

* Mendeley data-base deliverables including the 310 articles

* Excel spreadsheets with the analysis of around one hundred articles

* Various seminar presentations

* Final project report

* Project web-site link: https://ecophytopic.fr/recherche-innovation/proteger/projet-biolim
<figure><img src="image_149.png" /></figure>

BIOTI-VIGNE

# Biotisation of young nursery-grown grapevines to prevent trunk diseases

**Launch year:** 2015

**Completion year:** 2017

**Scientific manager**

Dr Marc Fermaud, UMR SAVE INRAE

marc.fermaud@inrae.fr

**Partners**

UMR SAVE INRAE ; Mercier Frères SARL and
Biovitis

**Financing**

Total cost of the project: € 253,938

Ecophyto grant: € 88,848

**Key words:**

Wine growing; elementary alternative practised; Biotisation, Biological pest and disease management; Biocontrol

## Context and main objectives

The French grape and wine industry is a major chemical pesticide user: around 20% of all pesticides applied to only 3% of French agricultural land. The industry is also suffering from a state of vegetative decline, largely due to grapevine trunk diseases (GTD). Biocontrol for GTD is rapidly becoming a major subject considering the dire need to avoid any increase in the use of chemical fungicides to deal with these diseases. The biotisation of plants that have been grafted in nurseries, which is at the heart of this project, involves inoculating the plants with beneficial micro-organisms which will improve their tolerance to biotic stress (GTD pathogen fungus infections).

The project's originality is based on:

1) Various biotisation agents, micro-organism strains, all taken from French vineyards

2) Isolation and/or combination tests

3) Application to plants at a very early age

The 2 major objectives are:

1) Improving and limiting the possibility of varying protection levels using the candidate biotisation agents identified by our research work, such as bacterial strains and *Pythium oligandrum*

2) Evaluating the effect of root-stock choice on the persistence and effectiveness of biotisation agents in nurseries.

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

Two extensive experimental campaigns were completed in 2015 and 2016. The main objectives, methodology and results of these were presented in a scientific article (L. Daraignes et al., 2018 Biological Control 119:59-67, doi: 10.1016/j.biocontrol.2018.01.008 https://hal.inrae.fr/hal-02626154) and on a dedicated Web-page(http://www.maladie-du-bois-vigne.fr/Programmes-de-recherche/Les-projets-en-cours/Les-projets-de-biocontrole/BIOTIVIGNE/Les-resultats-du-projet).

► The first conclusion confirmed that the selected strain of *Pythium oligandrum* "Po" (oomycete) is a biocontrol or biotisation agent with a very high potential for the protection of grapevines from GTD. Biotisation assays have demonstrated effectiveness, as early as during nursery production, of the tested biotisation agents against 2 major GTD pathogen fungi: Phaeomoniella chlamydospora (Pch) and Neofusicoccum parvum (Np).

<figure><img src="image_150.png" /></figure>

Vine wood with brown bands under the bark,
a common sign of Esca.

Photo: © Pascal Lecomte, INRAE

<figure><img src="image_151.png" /></figure>

Microscope image of *Pythium oligandrum*,
successful biotisation agent for controlling
the fungi known to be the causes of
grapevine trunk diseases.

Photo: © Jonathan Gerbore, BIOVITIS
For both assay campaigns, the extent of dieback was significantly reduced by as much as 39-40% for Pch and 52-64% for Np in plants that had been biotised with *P. oligandrum* (Po). The absence of contact between the pathogen, present in the graft, and the Po biocontrol agent which colonises the root stock, indicates that the action occurs remotely through the stimulation of the vine's defence system. The mechanism is therefore an induction by Po in the roots with a vegetative elicitation which limits the infection and colonisation of the trunk by the two studied pathogenic fungi. In addition to this the Biotisation agent bacteria, Pantoea agglomerans (Pa) also significantly reduced dieback associated with the Np pathogen by 22-38%.

<figure><img src="image_152.png" /><figcaption>Cross-section view of mother plant root-stock trunks showing the extent of, central and/or sector type, Esca associated dieback.</figcaption></figure>

Photo: © Jean-Michel Liminana, INRAE

* The second major conclusion came from the combined implementation of Po in association with bacteria strains pre-identified as having a high biocontrol potential (UMR SAVE, INRAE results). These biotisation agent bacteria are confirmed as effective but less so than Po. In addition to this, the accumulation of different biotisation agents in the same plant does not generate a synergic effect (greater effectiveness compared to Po alone). This may not be general to other disease systems and/or other microbe combinations. Consequently the combination of two Biotisation agents (Po and Pa) has reduced the extent of Np associated dieback by 32-52% depending on the year.

* The third major conclusion shows that the presence of Po in the rhizosphere and/or roots of the grafted vine cuttings can be maintained for long periods of time, 2 to 3 months. Po's high capacity for colonising the rhizosphere of grafted vine cuttings has therefore been shown for the first time and in an innovative and applicable way for the wine growing industry (previous studies had only shown this for cuttings that had not been grafted).

* The fourth conclusion, of major interest for the development of future innovations, concerns the very promising levels of grafted plant protection using the two tested root stocks (101-14 and SO4), which are widely used in France and other wine-growing countries. The extrapolation of our results to cover wine-growing techniques has therefore been significantly increased, even if this project was based on assays carried out in nursery greenhouses (MERCIER), further experiments will be necessary under different wine-growing conditions. These have been launched and an essential applied perspective, as a direct follow-up to the BiotiVigne project, will be the process set up to achieve authorisation for the *Pythium oligandrum*-strain biocontrol agent that was used for these tests.

## Prospects for transfer or research

### Transfer:

A certain degree of consistency can be noted in this study, the tested micro-organisms also show antagonistic capacities under other conditions, as shown in other biocontrol studies on pathogen fungi affecting grapevines (such as the research works by UMR SAVE, INRAE Bordeaux). For the partner company Biovitos, this project has confirmed the interest and effectiveness of the ground surface application of the *P. oligandrum* (Po) micro-organism. The company is therefore planning to follow the Biotivigne project with field trials in vineyards, in order to get authorisation to market this biocontrol agent. Thanks to this project, Mercier has confirmed its decision to explore the development of one or a number of commercial products ranges involving biotised graft cuttings.

### Research:

In addition to opening up new potential biocontrol approaches, this project has raised a number of essential research issues. The biotisation procedure needs to be taken further in order to evaluate the duration of its beneficial effects, which are notable on young plants; this research would need to cover the whole productive life of a long-lived plant like the grapevine. One other question which is raising debate is that of the consistency of our major results: the association of a number of antagonist biocontrol micro-organisms is not always more effective than introducing a single one of the targeted candidate micro-organisms (this will almost certainly depend on the patho-system and/or the association of micro-organisms in question).

## Publications and scientific symposia:

* Biological Control (n°119, pp 59–67, 2018): Efficacy of *P. oligandrum* affected by its association with bacterial BCAs and rootstock effect in controlling grapevine trunk diseases. Leslie Daraignes, Jonathan Gerbore, Amira Yacoub, Laure Dubois, C. Romand, O. Zekri, Jean Roudet, P. Chambon, and Marc Fermaud. Doi: 10.1016/j.biocontrol.2018.01.008. URL: https://hal.inrae.fr/hal-02626154
**PRACTICAL / EDUCATIONAL ARTICLES:**

► INRA biocontrol brochure "les conquêtes de l'INRA pour le biocontrôle" to which Marc Fermaud and Jonathan Gerbore have made contributions (including the BIOTIVIGNE project), INRA or INRAE, July 2018 (https://www.inrae.fr/sites/default/files/pdf/180529_presse_BIOCONTROLE_BD_.pdf)

**OTHER PRACTICAL WORK:**

► **The project's dedicated web-site:** https://www.maladie-du-bois-vigne.fr/Programmes-de-recherche/Les-projets-en-cours/Les-projets-de-biocontrole/BIOTIVIGNE

► **BiotiVigne RESULTS WEB-page:** http://www.maladie-du-bois-vigne.fr/Programmes-de-recherche/Les-projets-en-cours/Les-projets-de-biocontrole/BIOTIVIGNE/Les-resultats-du-projet