Conception, realization and experimentation of an auto-dissemination trap to control insect pest in perennial crops  (Ordonez & Goedert, 2003-pending patent)

 

Bio-control of Coleopters with micro-organisms is difficult as few biological compounds are really able to totally substitute chemical pesticides available at present on the market.

Entomopathogenic fungi such as the green muscardine, Metarhizium  anisopliae (Metschnikov), Sorokin and the white muscardine, Beauveria spp., have been studied as bio-control agents against the larvae and  adults of Coleopters (Kempkes et al, 2004). Nevertheless, the problemsinvolved with pathogenic disease dissemination in the larvae habitat have made their use arduous.

Experiments consisting in releasing the adults of Coleopters which transport pathogenic fungus were carried out at the end of the seventies (Zelazny et al, 1978). In the nineties, traps to disseminateentomo-pathogenic fungi against cabbage insect pests showed the
effectiveness of this method (Pell et al, 1993). Insect pathologists now know that it is possible to propagate a disease by using infected individuals which can disseminate fungi as well as bacteria (Payne J., 1991), thus controlling the level of a certain insect’s population. For instance, Klein et al (1999) developed an auto dissemination trap to contaminate Japanese beetles (Popillia japonica Newman) with green muscardine (Metarhizium anisopliae (Metschnikov), Sorokin).

Larvae of the pest insects (Coleopters) can be unreachable with the  usual treatments; therefore the auto-dissemination of entomopathogenic fungi techniques can be efficient.

Auto disseminatio trap Ordonez and  Goedert-2003
J.C. Gregoire from ULB, Belgium has invented a bottle-trap system for monitoring bark beetle forest pests (1999). Based on his trap system, we have designed and realized an auto-dissemination trap (prototype conceived and developed by Ordonez and Goedert, 2003).

 

Autodisemination trap: Ordonez and Goerdert 2003
Autodisemination trap, Ordonez & Goedert 2003

Description of our auto-dissemination trap:

To Gregoire’s trap system we have coupled an inoculation chamber. The composition of the trap is as follows:

1. A plastic funnel by which the insects penetrate into the trap. To the funnel we hang a sexual pheromone which attracts the insects.

2. A body with a plastic cylindrical tube is horizontally fixed. At the extremity of the tube, a pane lets the light pass through so that the insects attracted by the sexual pheromone (and eventually captured) will move towards an inoculation chamber fixed in the middle of the tube. The  other extremity of the pipe is closed by a grid in which an alcohol  scent is passed.

3. A bottle containing alcohol is fastened to the body of the trap. This feature is also a lure to attract the insects.

4. The inoculation chamber is a cylinder of 3 cm in height and diameter fixed on the body of the trap. The inoculation chamber is loaded with an entomo-pathogenic fungus powder formulation (60% of an active ingredient)plus an additive (30%) that helps to homogenize the active ingredient powder and a desiccant (10%).

For our first bio-essays we have placed in-between the inoculation chamber and the glazed end a bottle with holes which is used to collect some of the captured insects.

The principle of operation of our auto-dissemination trap is simple:

Insects are fattaly drawn initially by the odour emitted by the syntheticpheromone (distance pheromone) placed on the funnel and then by the alcohol vapours which act as attracters or alike resting pheromone (as itrecalls to insects the terpens compounds secreted by decaying trees).  Insects fatally drawn yet fall on a grid fixed onto the tube. The other role of the grid is to allow the rain water that is collected by the funnel to run-off.

Once the insects are caught into the trap body they will search for the light coming from the other end of the tube. In its walking progression,  they will mandatorily pass by the inoculation chamber and the only possibility they will have is to fall into the bottle with the holes.

In 2004 we captured Coleopters in a conifers forest. When the insects were brought to the laboratory conditions, we observed the presence of the pathogenic fungus -confirmation of Koch postulates-.

With this prototype trap we are searching to prove that dissemination is an efficient system to reach Coleopters larvae, particularly in the conifers plantation. Tests on perennial crops were carried out.


 

BEING A BEE…

Bees are marvellous creatures. They survived for millions of years without the assistance of human beings. The most powerful varieties are adapted to the environment in their struggle for life under the terms of natural selection (Darwinism).

Bees are hard workers and play an important role in nature, more so than one can think or imagine. All the products of the hive are natural elixirs for humans: wax, honey, royal jelly, propolis, venom…

Bees

In our world of fast and perpetual evolution, it is important to be able to keep the interest of rising generations in bee-keeping. Its ancestral techniques sometimes can appear exceeded, becoming a major risk factor for the perennial and the survival of agriculture. Indeed, if the number of bee-keepers is reduced more and more, how can we guarantee pollination up to now assured by bees?

At first glance, it is only one insect species which is likely to disappear, but it can become a major environmental catastrophe when it is announced. Wasn’t it Albert Einstein who had already stated that humanity would not survive more than four years with the disappearance of bees?

Our applied researches were unique, and were extremely useful and innovative for the Luxembourg environment. Knowing that in the Grand Duchy there were about 500 bee-keepers with more or less 12.000 hives, the hope in seeing increasing numbers was illusory. On the contrary, the future risk is being marked by a concentration of the production in the hands of a much reduced number of these workers.

Pol Goedert

To reduce the harmful input of chemical pesticides, we also investigate the effect of biological products on the environment. We are aware of the steps we have to follow for future homologation, necessary in any field trial. This is why it is of primary importance to test bio-products on non-target insects. Because of its importance on an ecological and economic level, bees are our non-target insect choice.

We used domestic bees for our experiments–those used in local bee-keeping—ergo, bees provide the experimental equipment for our tests.

Queen Mandibular Pheromone
Queen Mandibular Pheromone

But we must respect the social structure implied on a hive and place the bees under the closest conditions to their natural environment, hence to observe the swarm not as a whole of identical individuals independent from/to each other, but as a diffuse organization.

As a queen is a precious treasure for bee-keepers, we used the Queen Mandibular Pheromone (QMP) on our research with the experimental hives. The QMP– the only pheromone chemically identified in the honey bee (Apis mellifera) queen– makes it possible to keep the cohesion of the population, to block the ovarian development of workers and to preserve the capacities of altruism of these insects.

Desing of Experimental Hive: Ordonez and Goedert 2003
Desing of Experimental Hive: Ordonez and Goedert 2003

Pol and Lucas

Honey Extractor : Goedert 2003
Honey Extractor : Goedert 2003

For permission and information about utilization of our experimental hives please email us.


Use of the natural elicitors against fungi disease on wine crops

Wine crops, Vitis vinifera L., are a cultural inheritance in the Grand-duchy of Luxembourg. The wine and life-wine-related museums rooted in the Moselle region (Ehnen, Bech-Kleinmacher) are worth more than one visit. Luxembourg vineyards are very important in the economic and social plan but sensitive to the attack of diseases which are detrimental for the crop.

Powdery Mildew Oidium
Powdery Mildew Oidium

The principal diseases are the powdery mildew oidium, the downy mildew, the grey mould or Botrytis bunch rot/Botrytis rot. These three parasitic diseases are related to fungi which develop in wet conditions. The dry areas are thus, not very concerned.

The downy mildew is caused by a microscopic fungus (Plasmopara viticola) which attacks the leaves and the grapes. It produces a said yellowish tasks or “oil tasks” on the leaves, then the lower part of the leaves end up covered with white felting caused by mildew on the lower part of a wine-leaf. This fungus is also responsible for the tanning and drying of the grape-grains. The mildew contamination does not hibernate on the crop.

The powdery mildew oidium (Uncinula necator –sexual stage–, Oidium tuckeri –asexual stage) develops at the surface of the wine’s green bodies. Its presence is detected on the leaves by diffuse tasks of a white-grey dust. If the contamination takes place at inflorescent times, the grape-grains fall and if it takes place later, the grains are covered with grey powder, then they burst. The oidium hibernates on the crop. A first attack is a warning for the following year when for once it can be assured that the disease will be declared again.

Grey mould or Botrytis bunch rot/Botrytis rot (Botryotinia fuckeliana -sexual stage rare!-, Botrytis cinerea–asexual stage–) attack mainly the grape-grains. These get brown then rot while being covered with a grey felting. In fact, the types of vines with thin skins are the most sensitive.

The input of harmful phyto-pesticides is heavily seen in wine crops. Moreover, the far-flung use of chemicals employed to control these diseases bring about serious economic consequences and exert influence on  wine quality as well as damage to the environment and human health. The intensive consummation of these products has lead to the development of resistances of micro-organisms to several fungicides. Due to their high toxicity, a myriad of these merchandise are on the way to prohibition. Indeed, an EU decree (Official Journal September 24th, 2002) has banned the utilization of more than one hundred active molecules in agriculture, including fungicides which are represented by many marketed products. A fixed deadline enlightened the need to reduce the application of fungicides and develop products harmless to the environment.

Mating Disruptor

Vineyards Jean Marie Vesque

Our pioneer strategy in the Grand-duchy began in 2003. After that, a national collection of pathogens was successfully established at the Musée National d’Histoire Naturelle (MNHN) in Luxembourg. Alternative of research to improve protection of wine-crops were settled.

The visionary open-mind of the wine growers from the Domain Cep D’Or, Hëttermillen (Luxembourg/Moselle) cued this innovative research.
Hettermillen

The controlled stimulation of the plant defence mechanisms is an alternative to the use of pesticide products. The activity (applied research) on the vineyards consisted in the activation of wine tree defences by elicitors in order to test the efficacy of more effective formulations developed by our French partner INRA, Dijon and private partners of fundamental research.

Our French partners  brought their scientific knowledge and provided us with products (elicitors) which we tested. Elicitors are currently defined as a physical, chemical or biological stimulus that triggers defence responses in plants. In addition, two French private firms supplied us with their formulations. It is important to state that these goods are available in the French market and thus already underwent the process of homologation. They currently passed tests in France (Champagne, Bourgogne and Bordeaux) and also in the Grand-duchy of Luxembourg (Hëttermillen).

The management of the patent rights remains confidential between partners.


 

BOOKS SCIENCE

Bioinventaire
Bioinventaire

Ana Isabel Ordonez: écologie et contrôle intégré des ravageurs au Luxembourg

Zanter 1999 ass d’Dr. Ana Isabel Ordonez als Wëssenschaftlerin fir de Naturmusée an zanter kuerzem fir de Centre de Recherche Publique Gabriel Lippmann aktiv. Déi gebiirteg Kolumbianerin huet an hirer Heemecht Agronomie studéiert, 1993 op der „Université de Bourgogne“ hir Dokterthèse iwwert sexuell Reizstoffer (d’Pheromonen) bei den Matten ofgeschloss, huet e.a. an der Schwaiz an an England Expérienzen a Saachen Parasiten gesammelt. An hirem naie Buch „écologie et contrôle intégré des ravageurs au Luxembourg“ faasst sie Resultater aus deene leschten 5 Joer zesummen, e.a. beschreiwt se hir Zesummenarbecht mam Wënzer Jean-Marie Vesque.

De “Borkenkäfer“, op lëtzebuergesch léif „den Décken“ genannt, zerstéiert an der Iwwerzuel an eise Bëscher -zesumme mat aanere Schiedléngen, wéi Paiperlékslarven- Eechen a Bichen. D’Gedéiesch, daat ënnert der Schuel vum Baam sëtzt a Gäng griewt, bekämpft een am beschte nët mat Pestiziden (vun 2007 uns wäerten iwwert 200 Produi’en wéinst Gesondheetsrisken verbueden sin), ma mat Schëmmelpilzen, déi nët d’Planz ugraifen, ma nëmmen d’Parasiten éliminéieren.

AI.O.: Les champignons ont été isolés du sol luxembourgeois, ils existent partout. On les a mis en collection, en dépôt officiel à la mycothèque de l’Université Catholique de Louvain, car le Musée d’Histoire Naturelle ne dispose pas d’infrastuctures pour garder des microorganismes. Et puis il fallait aller aux congrès scientifiques, expliquer ce que j’étais en train de faire, puis avoir l’acronyme, pour s’assurer que les gens soient conscients qu’on ne fait pas de bio-terrorisme, mais qu’on travaille avec des substances qui viennent de l’environnement, de la nature, et qui servent aussi à l’environnement.

Zemols d’Baienziichter hun sech iwwert d’Chimie, déi an de Wéngerten agesaat gouf, beklot, vu dat d’ Baienpopulatiounen duerch déi toxesch Substanzen staark ofgeholl hun.

AI.O.: C’est sous cette vue-là que je me suis orientée vers la vigne. Et j’ai trouvé un partenaire, le professeur Bernard Paul de l’Université de Dijon, qui prépare des cocktails de bactéries de levure et de champignons, qui empêchent la poussée de botrytis. Ces microorganismes sont antagonistes. Les Cocktails réveillent en plus les défenses de la vigne, donc la production de substances qui sont intéressantes pour la plante, mais aussi pour la santé de l’homme.

J.-M. V.: D’Fiichtegkeet ass eppes, waat d’Pilzen als Liewensraum gären hun, da kommen heiansdo am Summer och nach héich Temperaturen dobai. Fir eis, fir déi nördlech Waibaurégiounen, ass ët bestëmmt eng ganz interessant Saach.

AI.O.: Nous pensons monter un projet européen, Europe plus Nouvelle-Zélande. Je tiens à souligner qu’il est très rare de trouver des viticulteurs qui veulent vraiment se mettre sur cette voie. A côté des cocktails de champignons il y a aussi les phéromones qui sont très importants.

J.-M. V.: Ech erklären : mir hänken Ampoulen an de Wéngerten op. Do ass Parfum dran. Daat lackelt d’Männecher un, well sie mengen, d’Weibecher géifen do sëtzen. Déi befruchten déi dann do, léen do hir Eer of, an doduerch entstin kéng nai Parasiten. Ech mengen, daat ass e ganz flotte System, deen ass an Daitschland scho wait verbreed, wou en och mat groussem Erfolleg agesaat gët. T’ass allerdéngs méi opwänneg. Déi Ampoulen mussen all Joers frësch opgehaangen gin, daat maachen mer zu Briedemës mat dem Club des Jeunes, dee mer dofir engagéieren, dat mer eng 30-35 Lait hun. Do si mer 2 Deeg am gaangen. T’ass och méi daier wéi d’Pestiziden.

Ët geet der Ana Isabel Ordonez nët drëms Insektenzorten z’exterminéieren, ma hir Zuel ze kontrolléieren. Sie huet vlaicht nët d’Ecologie nai erfonnt, ma mat hirer Energie an Ausdauer huet d’Wëssenschaftlerin Lait aus ganz ënnerscheedlechen domainen aus verschiddene Länner zesummenbruecht. Fuerscher sollen an hiren Aen nët am Labo hannert de Bicher oder dem Mikroskop verstëpsen, ma eng Responsabilitéit iwwerhuelen, agéieren.

AI.O.: Depuis le temps de Pasteur les microorganismes ont aidé. Depuis les vieux temps on se soigne qu’avec des produits naturels. La chimie est venue, parce qu’il y a eu l’essor des boîtes chimiques. Dans les sciences on n’est que des êtres humains, donc il faut philosopher. Je trouve que dans la recherche il ne faut pas seulement faire le fondamental, mais quelque chose qui donne un impact économique. Et quel est l’impact économique plus grand pour le pays que la vigne.

Der Ana Isabel Ordonez hiren Bréifdréier war vun «écologie et contrôle intégré des ravageurs au Luxembourg » begeeschtert ; d’Buch riicht sech also nët nëmmen u Wëssenschaftler oder Wënzer. 

 


 

Projet de recherche:
Bioinventaire, caractérisation, protection et exploitation des champignons entomopathogènes présents dans les sols forestières du Grand-Duché du Luxembourg; Développement des agents biologiques de contrôle d’insectes ravageurs.

Chef du projet :

Dr. A.I. Ordonez
Musée National d’Histoire Naturelle

Dr. Dave Moore. CABI Biosciences
UK Centre Bakeham Lane, Egham, Surrey TW20 9TY UK.

Dr. Roy Bateman, CABI Biosciences
UK Centre, Silwood Park, Ascot, Berks. SL5 7TA UK.

J.P. Arend
Administration des Eaux et Forêts, Service de l’Aménagement des Bois et de l’Économie Forestière. BP. 2513. L-1025 Luxembourg.
La plus grande partie des pertes biotiques en bois résineux sur pied sont dues à des attaques du bostryche, qui peuvent engendrer une perte monétaire des bois infectés de 20% et plus.

En ce qui concerne les essences feuillues ce sont surtout le chêne et le hêtre qui souffrent des surpopulations périodiques de certains lépidoptères. Même si les arbres surmontent ces attaques, leur accroissement annuel est considérablement réduit. A ces pertes il faut ajouter également les coûts du contrôle du ravageur (pest-management).

L’opinion publique s’oppose de plus en plus, et à juste raison, à une utilisation de pesticides en forêt, ce qui a fait naître une demande en agents de contrôle biologiques spécifiques aux hôtes, et inoffensifs pour l’environnement. Les champignons entomopathogènes infectant leurs hôtes par pénétration à travers la cuticule semblent être une alternative intéressante. Ils produisent un large spectre de composés insecticides actifs, mais également d’autres composés pharmaceutiques. La grande quantité des souches naturelles présentes au Luxembourg représente un vaste réservoir de matériel pour la recherche et le développement.

Comme l’écosystème forestier est stable, la forêt est un endroit idéal pour utiliser des agents de contrôle biologiques qui ont une tendance à agir plus lentement, comparés aux insecticides synthétiques et chimiques.

L’objectif de cette recherche est de quantifier les champignons deutéromycètes, ubiquistes et hétérogènes, en utilisant des techniques d’extraction, afin de décrire l’occurrence naturelle et la densité de ces entomopathogènes dans les forêts feuillues et résineuses. La palette d’hôtes qui peuvent être combattus par ces entomopathogènes sera étudiée, afin de détecter la virulence et la viabilité de ces agents de contrôle.

Les aspects concernant la sécurité d’utilisation de ces genres de microorganismes seront abordés, et plus particulièrement les effets sur les organismes non-ciblés, qu’il s’agisse de mammifères ou d’invertébrés. Les techniques d’isolation, les répertoires, la protection et l’utilisation de strategies de mycocontrôle seront développés en collaboration avec d’autres centres de recherche.

Le projet a été défini en collaboration avec une biologiste (Dr. Ana-Isabel Ordonez) ayant obtenu une bourse recherche auprès du Ministère de la Culture, de l’Enseignement Supérieur et de la Recherche. Madame Ordonez, qui aupart-avant a géré des projets de recherche dans le domaine des ravageurs agricoles, notamment en Angleterre et en Suisse, possède beaucoup d’expérience dans le secteur des microorganismes.

Le choix des points d’échantillonnage, qui devraient se trouver de préférence dans des peuplements qui ont récemment subit une attaque d’insectes, ainsi que les travaux de terrain proprement dits, sont exécutés avec l’aide du Service de l’Aménagement des Bois et de l’Économie Forestière. Faute de pouvoir mettre à disposition des laboratoires appropriés, les analyses et les travaux d’isolation des différentes souches des champignons sont faits au sein du Centre de Recherche Scientifique du Musée National d’Histoire Naturelle. Le projet s’est terminé en 2004.

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