CALL: FP7-KBBE-2010-4
Proposal full title: Novel strategies for integrating land snail pests control of agricultural crops in Europe, with projection to Latin-America
Proposal acronym: Land snail’s life cycle as pest control core Type of funding scheme: Collaborative Project. CALL: FP7-KBBE-2010-4 Work programme topics
addressed:
Food, Agriculture and Fisheries, and Biotechnology Area: 2.1.2
Topic number: KBBE.2010.12-05
Name of the coordinating person: Dr José Castillejo Murillo
Departamento de Zoología y Antropología Física. Facultad de Biología.
Universidad de Santiago de Compostela. E-15782 Santiago de Compostela.
La Coruña. Galicia. España.
Mobile Phone: + 34 654 969 784 Tel: + 34 981563100
Fax: + 34 981 596904
E-mail: [email protected]
List of participants
Participant number Participant organitation name Country
1 (Coordinator) Universidad de Santiago de Compostela Spain 2
3 4 5 6 7 8 9 10 11 12 13 14 15
1
TITLE: Novel strategies for integrate land snail pests control at agricultural crops in Europe with projection to Latin-America.
1: Scientific and/or technical quality, relevant to the topics addressed by the call
1.1 Concept and objectives
To introduce in a series of crops, new strategies for the integrated land snail pest control. These strategies are based in the deep knowledge of the pest´s biology and ecology, so that the abundance and the activity periods can be predicted to elaborate an integrated pest control system and take decisions that can be applied in every develop phase (juvenile, adult, senile or eggs). Thanks to this the farmer will know when to apply the traditional molluscicides (to destroy the land snails), when to use the molluscicides ovicidal (to destroy the egg lays), when to apply the biological control through parasite nematodes or when to use the trap-plants. To develop this integrated control methods it will be necessary:
1. To know the biotic and a biotic factor that describe the land snails biological cycle in different areas of Europe and Latin-America, to elaborate the control method.
2. To know the specific land snails´ diet with the objective of finding the most attractive plant species to use them as trap-plants.
3. To understand the activity in function of the environment biotic and a biotic variables, with the aiming of developing an effective abundance and activity statistical prediction method.
4. To search plants with bio pesticide activity to be used as biomolluscicides and bio-ovicides against land snail and its eggs.
5. To know the ovicidal potential of the usual non residual agrochemicals to use them as molluscicides-ovicides in crops.
6. To know the ovicidal potential of cattle’s and swine’s slurries as molluscicides-ovicides in ecological farming.
7. To search in each study areas of Latin-America a parasite nematode (Phasmarhabdities alike) to use it as a biological control method against land snails.
8. To deliver to the horticultural industry an effective integrated crop management strategies, low chemical and biological protecting methods against land snails.
9. To deliver to the ecological farming effective integrated packages methods based on cattle and swine manure and plant traps, to protect the crops against land snail pests.
With these strategies we are settling the basis for an integrated control method, to achieve a more rentable crop due to; having less plant damages, less pesticides use, and a more respectful farming with the environment and the wild fauna. The land snail pest problem in Europe and Latin-America is increasing every day due the commercial globalization. The most dangerous land snail species in Europe are: Arion lusitancicus, Deroceras retiuclatum, Lehamnnia marginata, Milax gagates, Criptophalus aspersus and Theba pisana among others. The 90% of the land snail species that are pest in Latin-America are introduced species from Europe and other countries, this species proliferate indiscriminately because of the absence of natural predators, such as Acanthina fúlica, Deroceras reticulatum, Milax gagates, Lhemannia marginata, Arion intermedius, Criptophalus aspersus…. In some Caribbean regions native land snail species cause important damages, such is the case of Veronicella genus in Mexico and Cuba.
2 S&T objectives detailed description
This project requires the application of novel control strategies to control native or introduced land snail pests in crops that can be used in any agricultural pest. With the strategies proposed here we can anticipate to the damages caused by the land snail pests, due the application of a preventive method even before damages appear on the crops.
Our strategies are based on:
1. To understand the land snails biological cycle in the crops study areas.
2. To understand the land snails activity in function of the climatic variables and the crop type.
3. To destroy the land snail´s egg-lays thanks to the plant extracts and non residual standard agrochemicals collateral effect.
4. To rationalize standard molluscicides consumption in standard farming.
5. To introduce cattle and swine manure and trap-plants as control methods in organic farming.
6. To understand the collateral effects of the products used in this integrated pest control methods.
The understanding of the biological cycle is very important, because we attempt to use the molluscicides before damages appear. With the knowledge of the biological cycle we will know which time of the year is juvenile, adult or senile, we will know when the egg-lays are done, and in other words, we will know the sizes, structure and dynamics of their populations. The information provided by the biological cycle is important to implement this new strategy, as these molluscicides must be applied when the population density is lower and when there are fewer egg-lays in the soil. With this we obtain an optimal effectiveness destroying the egg-lays through ovicidal and also killing gastropods. By applying less molluscicides we save money and minimize the side effects on the environment.
Knowing the diet of land snails in the study areas will give us information on the possible use of trap-plants, to evaluate and estimate the damage that they actually produce on crops. By studying the stomach contents of a specified number of land snails we can know their preferences, in previous research we found that plants that had a low abundance in the environment, appeared with high frequency in the stomach of land snails, this means that they have positive selection for this type of plants. These plants can be used as trap-plants to protect vegetable crops deterring land snails to eat the trap-plants. It is a very useful strategy in organic farming.
Knowing the activity of terrestrial gastropods in terms of climatic variables and crop phenology is required to develop a statistical for activity prediction. With this model we can predict with 24-48 beforehand the activity, and provide the farmer information that land snails will be active, and thus may apply the traditional molluscicides at the right time, getting a greater effectiveness using smaller amount of molluscicides, leading to saving resources and reducing side effects on plants, soil and wildlife.
So far we have been talking about traditional molluscicides. The tradiotional molluscicides (metaldehyde, carbamate, iron sulphate, phasmarhadities...) are intended to kill the individuals, in other words, kill the terrestrial gastropods leaving intact the land snail´s egg-lays in the soil.
There is a growing body of evidence to suggest that in the past 4-5 decades there has been an excessive dumping of chemical toxins on the soil. As a result the soil in many places has become barren and the ground water toxic. Contrast with this the organic inputs that are safe, non toxic, and cost much less. 'Biopesticides' are certain types of pesticides derived from natural materials such as animals, plants, bacteria, and certain minerals.
Benefits of biopesticides include; effective control of insects pests, plant diseases and weeds, as well as human and environmental safety. Biopesticides also play an important role in providing pest management tools in areas where pesticide resistance, niche markets and environmental concerns, limit the use of chemical pesticide products. The idea of finding plant extracts with molluscicidal and ovicidal activity against land snails and their eggs is original and promising.
In each country, agricultural authorities allow a number of non residual agrochemicals for different uses and for different purposes, can be fertilizers, herbicides, acaricides, fungicides, etc... These products have gone through a series of tests to be authorized. In previous research we have tested non residual agrochemicals to see
3
if they had the potential to destroy the land snail´s egg-lays, and many of them had as side effect the egg-lays killing. Basing on these assumptions each participant must make a test series with non residual agrochemicals approved in their country, to discover which one or ones have a higher ovicidal power at the lowest concentration in the shortest time. In organic farming the use of synthetic chemicals such as fertilizers, pesticides, antibiotics, etc.., it´s forbidden, with the objective of preserving the environment, maintaining or enhancing soil fertility and provide food with all its natural properties. Fertilizers that can be used in these kinds of crops can be of two types, green fertilizers or livestock manure. In previous research we found that certain concentration of swine and cattle manure had ovicidal action on terrestrial gastropod egg-lays. Therefore each participant will have to do tests to discover the type and concentration of manure that has higher ovicidal power against the land snail´s egg-lays in their study areas.
Finding a new parasite-nematode with a biological cycle like Phasmorhadities hermaphrodita is crucial to have a new tool for biological land snail pests control in agriculture in Latin-America, as the European variety has problems at certain soil temperatures. The task to find European zoo parasitic nematode was made in previous 97- UE - Project.
As a consequence the of traditional and organic farmers are going to get a series of useful tools for land snail pests and, first they are going to have a new control strategy based on the application of the ovicidal- molluscicides at the correct time, determined by the life cycle of the terrestrial gastropods, and not by the crop phenology. It will be explained which chemicals they need to destroy land snail´s egg-lays. Through the predictive model, the farmers will have the information necessary to know which day and a time terrestrial gastropods will be active, thus apply the traditional molluscicides at the right, time, place and amount. This information must be transmitted through scientific meetings, counselling State Agricultural Agencies and through web pages of the State Servers.
4
COMPLIANCE WITH THE OBJETIVES OF THE WORK PROGRAMME AND ITS PRIORITIES
This project can definitely solve the problem of land snail pests in agriculture that has been globalized by the reform of the European Common Agricultural Policy and which it is forced to fulfil in the countries from which we import agricultural products.
This project is related to THEME 2 (Food, Agriculture and Fisheries, and Biotechnology), Area 2.1.2 KBBE.2010.1.2-05 "Integrated pest management in farming systems of major importance for Europe" out of the 7th Framework Program of Cooperation (FP7 Cooperation Work Program), and our project fits in perfectly in the topics (issues) of integrated pest control (management) (In the context of Integrated Pest Management - IPM-) in particular (specifically) can be said that:
1. It includes preventive measures such as, molluscicides application in the more labile phases of the life cycle of terrestrial gastropods and when there is less density of population and eggs in the soil, which generally coincides with (periods in which) (times when) there is nothing planted on farms .
2. The design of this strategy is based on the study of the biological cycle of pests and the study of their activity in terms of biotic and abiotic variables of the environment, representing perfect control and more accurate information to take preventive measures.
3. With this strategy control measures are applied at the right time, anticipating the emergence of the pest, which implies less molluscicide applied to achieve a better effect, besides the molluscicide is never next to plants, if the measures control are applied prior to planting. Our strategy is aimed at controlling or eradicating the pest to protect the crop, in other words, we use a preventive strategy.
4. It is an integrated control based on the decision-making through the statistical model prediction.
We only use low toxicity chemicals, we use the biological control of nematodes through zooparasites, not to mention the use of trap plants to deter terrestrial gastropods that attack crops or the use of swine and cattle manure as ovicidal all this applied at the time we enter the life cycle of the pest snail and the predictive model.
5. This strategy helped to reduce pesticide use on crops applying the necessary quantity at the right time, not introducing new chemicals in agricultural crops, but taking advantage of the farmers’ standard used non residual agrochemicals just giving it a different use or using the favourable side-effects. Thereby decreasing the amount of toxic agents that may be harmful to humans and to wildlife and soil.
6. This project combines "combine modelling and experimentation" because the entire strategy is based on the study of biological and ecological cycle of the pest, its dynamics, in order to achieve the greatest success of control with the least effort and with the least means.
7. The risks of not succeeding in this project are limited, first all the participants who are part of the research group have demonstrated ability to perform all the tasks outlined in the Work Packages, and also the USC team that coordinates this project has an extensive experience in developing predictive models of activity of the snails and slugs in agricultural crops of Galicia (Spain) and in controlling pests in vegetable crops and vineyards and many others European participants have similar experience in similar fields. Given the globalization of trade, over 90% of land snail species pests in Latin-America are of European origin, in other words, they are introduced species that we have been working with over 20 years.
8. The balance between costs and benefits will always be positive because we will control the final shape of land snail pests in agriculture, and we will bring to the traditional and organic farmers to have a number of tools to obtain a more profitable crop and seeding time management, very respectful with the environment.
9. Finally, all information obtained from this project and the control strategies are available for the farmer, either through meetings, workshops taught by the competent authorities or available through “on line” services were the farmers will resolve questions and provide information of the pest activity.
5 1.2 Progress beyond the state-of-the-art
At global scale, the economic damage caused by terrestrial gastropods has increased, thanks to the globalization of the trade market which leads us to talk of land snail species introduced from one continent to another, species that having no specific predators grow their population exponentially. While some land snails can reach pest status even in relatively arid regions, slugs are particularly problematic in temperate and rainy weathers, but even in this case, the extent of damage done to crops varies greatly at Regional and year to year scale (Port and Port, 1986). Many experts agree that the damage caused by gastropods has increased very significantly in the last 2 or 3 decades due to a combination of factors such as simplified cultivation techniques (reduced tillage, seeding direct), the reduction of predatory insects population caused by the insecticide products or by the use of new crop varieties more susceptible to be attacked by gastropods (Hommay, 1995, 2002, Godan, 1999; Speiser, 2002). Furthermore, the rising quality standards, makes the consumers tolerance to damaged products shrink, resulting in an intensification of the pest control measures.
Terrestrial gastropods Pest control is performed, almost exclusively, through the application of bait (pellets) containing between 2% and 8% of metaldehyde or carbamates (Godan, 1983, 1999, South 1992, Garthwaite and Thomas, 1996; Bailey, 2002; Speiser, 2002). The world's largest producer of metaldehyde is the Swiss company Lonza. The most widely used carbamate in land snail pest control is the metilocarbamate, which manufacture licensed is owned by the German company Bayer. Both compounds show similar efficacy in terms of reducing damage to plants (Bailey, 2002), and both have collateral effects on other animal groups populations (South, 1992; Bailey, 2002). Buchs, Heimbach and Czarnecki (1989) have noted the existence of the molluscicides metaldehyde baits have negative effects on some carabid populations; also Bieri, Schweizer, Christensen and Daniel (1989) have documented a reduction in the carabids and staphylinids populations, after the application of molluscicides metiocarbamate baits in meadows. Although currently all molluscicides baits incorporate pigments (usually blue) and other substances to reduce the mammals and birds ingestion risk, the cases of domestic animals poisoning because of molluscicides bait are frequent (Bailey, 2002). In the late 80s, molluscicides carbamate baits were banned in many U.S. states, due to the high frequency of poisoned bird cases were recorded (Sakovich, 1996). Tarrant and Westlake (1988) suggest that the use of molluscicides baits with
metiocarbamato is a serious threat to field mice (Apodemus sylvaticus) populations (Linnaeus, 1758). Gibson and Reynolds (1991) reported high acetaldehyde concentrations (resulting from the metaldehyde depolymerization in the gastrointestinal tract) in hedgehogs (Erinaceus europaeus) (Linnaeus, 1758) found dead on the field, and Gemmeke (1997) observed symptoms of poisoning cases and death in hedgehogs fed with slugs that had eaten metiocarbamate baits.
In recent years has appeared on the market a new chemical molluscicide, under the Ferramola trade name manufactured by the German company Neudorff GMBH. This product is also presented in the bait format and contains iron phosphate as active ingredient. The tests conducted to corroborate its effectiveness (Iglesias and Speiser 2001, Speiser and Kistler, 2002) indicate that the iron phosphate it´s comparable to the classic chemical molluscicides such as metaldehyde and metiocarbamate. Unlike these which are totally synthetic, iron phosphate occurs naturally, as part of various minerals, especially strengite (Fe III PO 4 2 (H 2 O) orthorhombic) and metastrengita (Fe III PO 4 2 (H 2 O) monocyclic) (Roberts, Campbell and Rapp, 1990, Clark 1993) and it is a
compound with a very low toxicity (EPA, 1998).
The lack of gastropods pest control methods that are authorised in organic farming makes these animals to be considered as the most damaging ones by many professional associations in Britain and Switzerland in this kind of plantations (Peackock and Norton, 1990; Kesper and Imhof, 1998). The only currently marketed biological pest control agent for land snail, is the nematode Phasmarhabditis hermaphrodita (Schneider, 1859), released first in Britain in 1994. Numerous field tests in a wide variety of crops in European countries have shown that P.
hermaphrodita can reduce the damage done by land snails in plantations (Wilson, Glen and George, 1993; Wilson, Glen, George and Hughes, 1995, Wilson and Glen Hughes, 1995; Ester & Geelen, 1996; Iglesias, Castillejo &
Castro, 2001ab). Its effectiveness against the species D. reticulatum is beyond any doubt (Glen Wilson, Brain and Stroud, 2000), but there are signs that its effectiveness against other species might be low (Wilson et al. 1995a;
Coupland, 1995, Glen et al., 1996, Speiser and Andermatt, 1996; Speiser, Zaller and Neudecker, 2001, Iglesias and Speiser, 2001). The treatments with P. hermaphrodita effectiveness is very conditional to the soil moisture and
6
temperature, which greatly affects their survival, but has the advantage that the favourable moisture and temperature conditions for the land snails activity, are also the best suited for the nematode development, whereas chemical molluscicides effectiveness (as methaldeide baits) is greatly reduced due to this conditions, in which gastropods cause the most damages to plants (Glen et al., 1996). However, the high economic costs of the nematode pest control treatments makes their use restricted to high value crops such as ornamentals plants and some vegetables (Grunder, 2000).
The application of molluscicides is only a short terms control measure, it is a temporarily protection from the damage that might cause gastropods on plants. Also it doesn´t have a significant and lasting effect on gastropods populations residents in the plantation areas, so the risk of damage is permanent (Hommay, 2002, Port and Ester 2002). This is because applied molluscicides affect only the active portion of the population, as the gastropod egg-lays, which are found in soil, are not affected by conventional molluscicides treatments, resulting in a rapid population recovery (Glen, Wiltshire and Milson, 1988). It has been estimated that treatments based in molluscicides metaldehyde or carbamate baits kill less than 50% of the population of gastropods existing at the time of implementation (Glen & Wiltshire, 1986; Wiltshire and Glen 1989, Glen, Wiltshire and Butler, 1991). On the other hand, it is common that the amount of molluscicide bait ingested by gastropods has only a transient sublethal effects (Kemp & Newell, 1985; Wedgwood and Bailey 1986, Briggs and Henderson, 1987; Bourne, Jones
& Bowen 1988), and it has been shown that the fertility of individuals who experience such sublethal poisoning is not affected, so they continue laying eggs once they are recovered (Kemp & Newell, 1985).
In the 60 surges the Integrated Pest Management concept (IPM) (Stern, Smith, van der Bosch and Hagen, 1959), which currently is part of another broader concept, which is the sustainable development. The IPM involves the integration of many fields knowledge (biology, chemistry, agronomy, climatology, economics, etc). In order to develop the most appropriate control strategies, from the economic, environmental and Public Health viewpoint (Dent, 1991). While it is a system based on the combination of different methods in order to minimize the use of chemical pesticides is not excluded, a priori, the use of any control agent (Coombs and Hall, 1998).
Methodologically, the IPM can be described as a "decision making process", based on the analysis of all the available relevant data, deciding what action to take and when, to best control the plagues. Besides, the IPM searches the most effective, economic and less aggressive approach, from the economic and the the
environmental viewpoint (Bechinski, Mahler and Homan, 2002).
Currently, a lot of IPMs for many arthropods and fungi pest species are based on the use of prediction methods (forecasting systems) (Dent, 1991; Frahm, John and Volk, 1996). Predicting when a pest can cause significant damages to crops is essential, to know the exact moment to apply molluscicides (Buhler, 1996).
Furthermore, depending on the pesticide action mode, its effectiveness can be influenced by the organism life cycle or its activity level (Bailey, 2002). In short, criteria it´s needed to determine the need and desirability of applying pesticides.
7
The advances that the proposed project and patents will bring.
To this day, non residual agrochemicals with ovicidal action had not been used to control land snail pests in agriculture. The strategy proposed in this project is innovative, original and this pest control methods are very efficient, and makes it very easy to eradicate pest. It is original because it attempts to control the causative agent pest in the phase of their life cycle [biological cycle] where they are most fragile, in the egg stage. It is an original method that will develop statistical models to predict the abundance and activity of the land snail with which the farmer can act in a preventive manner so as not to damage because it will indicate the moment when which has to apply the ovicidal or the traditional molluscicides. It is original because it doesn´t introduce new pesticides in agriculture, it is based in non residual agrochemicals that the farmer habitually uses, it seeks the best profile to exploit its ovicidal activity. It is new because it puts into the hands of the organic farming a number of tools to control land snails pests using natural fertilizers or deterrent strategies implemented by trap plants deterring land snails from attacking crops.
It is a project that uses current technologies to see how the pests live, which are their weaknesses, and attack them manipulating it that way to minimize side effects on crops, and even on the man. With this project we are going to obtain results that will be patented. As a result we will be able to patent the pest control
strategies, establish a patent for the active use of non residual agrochemicals with ovicidal action against the land snail´s egg-lays, and finally be able to patent the Statistical Prediction Activity Model and abundance of land snail that are pests, and most likely be able to patent the use of a zooparasite nematodes for the land snail pests biological control.
8 1.3 S/T methodology and associated work plan
1.3.1 Overall strategy of the work plan (1 page). Detailed description of the proposed work
The whole strategy is designed to perform integrated land snail pests control in conventional and organic farming, and it is here to introduce the use of new molluscicides ovicidal action, capable of destroying the egg- lays, the use of trap-plants as a deterrent, and the rational use of traditional molluscicides.
WP. 1 .-To understand the biology of land snail that are pests in a range of vegetable crops, it is necessary to have information of the size, structure and dynamics of their populations.
WP. 2 .-To study food and qualitative and quantitative composition of the diet of terrestrial gastropods that are pests in order to find plants that can be used as trap plants in organic farming and traditional.
WP. 3 .-To develop a statistical model that could explain and predict the abundance and activity of the land snail as a function of environmental variables such biotic and abiotic.
WP. 4 .-To investigate the feasibility of using plant extracts as biomolluscicides and bio-ovicides. To carry this out it will be necessary:
4.1. Laboratory tests on filter paper (direct contact) and artificial soil (standard soil) to select the plant extracts with molluscicidal and /or ovicidal activity.
4.2. Mini plots experiments on horticultural soil to evaluate the efficacy of the selected plant stract against lands snails and its eggs.
4.3. Mini plots analysis to know the collateral effect of plant extract selects on invertebrate soil.
4.4. Chemical analysis to find the plant extracts active principle by analytic steps.
WP. 5 .-To investigate the feasibility of using commercial agrochemical activity as ovicidals with control land snail egg-lays for key Conventionally grown horticultural crops. To carry this out it will also be necessary:
5.1. Laboratory tests on filter paper (direct contact) and artificial soil (standard soil) to select the agrochemical which best suits the egg types of the pest species and soil type in the study area.
5.2. Field experiments on horticultural crops to evaluate the efficacy of the selected agrochemical as Ovicidal-molluscicides for the pest control of key conventional grown horticultural crops.
5.3. Field analysis to know the collateral effect of agrochemical selects on invertebrate soil fauna and border effect on wild land snails in conventional horticultural crops.
WP. 6 .- To investigate the feasibility of using swine and cattle manure of killing land snail eggs and plant- tramp strategy of land snail pest control for key organic horticultural crops grown. To carry this out it will also be necessary:
6.1. Laboratory testing to determine concentrations of swine and cattle manure that are effective against the egg-lays of terrestrial gastropods pest. Discriminant trials will be made on filter paper (direct contact) and artificial soil.
6.2. Field experiments to evaluate the efficacy of swine and cattle manure land snail egg-lays as control for key organic horticultural crops.
6.3. Field analysis to investigate the collateral effect of swine and cattle manure on soil invertebrate fauna and border effect on wild land snails in organic horticultural crops.
6.4. Field experiments to use plants as tramp-deterrent method to protect organic horticultural crops WP. 7 .- Make field trials in traditional crops to compare the effectiveness of the control strategy of pest land snail proposed by us versus conventional approaches of applying chemical molluscicides when observed damage to the crops. Field experiments in Conventional key horticultural crops to evaluate the efficacy of selected agrochemical ovicidal with activity against land snail egg-lays in relation to other standard commercial low-chemical methods of killing animals land snail. Final trial.
WP. 8 .- Field experiments organics in key horticultural crops to evaluate the efficacy of organic Molluscicides-ovicides and the use of plant-traps as land snails method to control pests.
WP. 9 .- To identify improved strains of nematodes Phasmarhabditis Which are more effective biocontrol agents of larger land snail species in Hispano-America.
9
METHODOLOGY AND RESEARCH WORK PACKAGES Work Package 1
Field research to investigate life cycle of land snail past in horticultural crops Participants 1 (……. Man-month)
Participants 2 (……. Man-month) Participants 3 (……. Man-month) Participants 4 (……. Man-month) Participants 5 (……. Man-month) Participants 6 (……. Man-month) Participants 7 (……. Man-month) Participants 8 (……. Man-month) Participants 9 (……. Man-month) OBJETIVES
To investigate size, structure and dynamic of land snail for a key horticultural crops Estudiar el tamaño, estructura y dinámica de sus poblaciones.
BACKGROUND
Methodological review. Many methods have been used to make quantitative studies of land snail populations. According to South (1992) these methods can be qualified in three categories:
A. Absolute methods, expresses the number of individuals per unit area.
B. Relative methods, expresses the number of individuals per unit effort or the relation to non-standardized traps.
C. Indirect methods, expresses sizes of population in terms of traces left or the effects produced by land snails (for example, depending on the damage extension done to the crop, or according to bait consumption).
Relative methods are faster and much more comfortable but they have the disadvantage that the estimates are highly dependent of the land snail activity levels, this can lead to incorrect population sizes because of
weather conditions that have an important effect on the land snail activity (Getz, 1959; Hunter, 1968a; South, 1992).
Absolute Methods involve the absolute quantification of individuals per surface area. This can be done on site by applying an irritant substance to a determined area. For years formaldehyde was used for this purpose, but South (1964) rejected this method when he realized that most of the land snails died before they could reach the surface. Högger (1993) proposed a new method. First he determines an area, limiting it with a metal ring of 15 cm of height; then he introduces it in to the ground to a depth of 5 cm. Once done this, he applies mustard oil to the ground and captures the land snails that come out to the surface. Another method, proposed by Ferguson, Barratt & Jones (1989), it is based on the placement of shelter traps located inside of the area determined by the metal ring, which in this occasion is covered with a top that prevents the escape of land snails and helps to keep the humidity (moisture) inside. The shelter traps and the area inside the ring are inspected each day removing the land snails caught, until no new individuals appear.
Another way of obtaining absolute estimates is to take soil samples of known surfaces and transfer them to the laboratory for further land snail extraction and quantification. The extraction can be done by; the progressive flooding of the soil samples to make land snails surface, or by washing the soil sample on sieves with water. South (1964) compared the last sieving method with absolute methods (flooding with cold or hot water, extraction with chemicals, dry sieving) and relative methods (trapping, Direct observation during night). South concluded that the water sieving is the most reliable method, since it allows the recovery of almost the 100% of the land snails contained in one soil samples. Hunter (1968a) proved the efficiency of this method when he recovered almost all individuals of a known population; South (1964) concludes that the water sieving is the most accurate method;
It´s also is the only reliable method to obtain and quantify land snail lays.
10
Almost all the authors agree that land snails and it´s lays are located in the uppermost stratum of the soil.
According to South (1964), 100% of the lays and land snails of D. reticulatum species is located in the first 2 or 3cm of soil in field areas. In crop areas, Hunter (1966) found out that 83% of individuals of D. reticulatum were located in the soil first 7´5cm, and a 6% were located above 15cm. Also in crop areas, Runham & Hunter (1970) observed that in the first 10cm of soil contained the 97% of D. reticulatum. Rollo & Ellis (1974) pointed out that the 90% of snail lays are also located in the same soil layers. According to Marquet (1985), in standard conditions any land snail appears above the first 5cm of soil, However in exceptionally severe winters up to a 15% of
individuals may appear at depths between 10 and 20cm. South (1992) suggests that sampling the soil´s first 10cm it´s enough to quantify land snail populations.
IMPORTANCE: The WP.1 is important because it will provide us with information of the size, structure and dynamics of the land snail pest populations. This information will be used in the development of a predictive model.
PROGRESS ASSESSMENT AND RESULTS: Through semi-annual and annual reports, and by regular personal controls carried out by the coordinator to each one of the participants in the concerned country.
--- Task 1.1
Objetives
Studying the land snail population size in crops where the study will be conducted.
Participants: all participants
Materials and Methods
The methodology used is based on absolute estimation methods; land snails quantification of known surface soil samples.
Soil sampling.
The choice of the sampling location is randomly selected. To carry this out the plot is divided in a grid composed at least of 50 frames of 4 x 4 m; each one subdivided in four quadrants. The quadrants are determined using the last two digits of the randomly generated value of the “random” (ran) calculator function. Once selected 20 frames, we proceed to determine which quadrant of each frame, by using again the calculator random number generator following the next code: 0,000-0,249 for the upper left quadrant; 0,250-0,499 for the upper right quadrant; 0,500-0,749 for the lower left quadrant; 0,750-0,999 for the lower right quadrant.
The sample extraction is performed with a rectangular spade mark with the depth to be achieved (10 centimeters). First is selected the area to take the soil sample, then we place on the ground an aluminum frame of 25 x 25 cm, then the spade is stuck in to the ground along its entire contour until the marked depth and extract the sample. Each sample is introduced in a properly labeled opaque plastic bag; these bags are conserved in a cold storage at 4ºC in the dark, for its further laboratory analysis in the next 3 days.
To determine the number of soil samples needed its used an statistical software; The means and variances of the average land snails and eggs are calculated, to find out all the possible combinations of 2 samples,
3samples, 4samples till the total of 20 samples. For each number of samples the degree of error is calculated dividing the standard deviation by the arithmetic mean. In previous investigations we observed that to obtain a degree of error less or equal to 10%, 18 samples are enough in the case of land snails and 16 in the case of eggs.
The abundance of land snails and eggs is estimated by washing the soil samples precedent from the study plots. On a monthly basis 20 soil samples of 25x25 cm square and 10 cm deep.
Soil samples treatment. Each sample is placed individually on a white plastic tray; in the first place the sample is thoroughly inspected to capture any snail that might be in the soil surface, once done this the vegetal cover is removed, cutting it with scissors; then the soil sample is washed in sieves, with a decreasing mesh sizes from 4mm to 1mm. The soil samples are crumbled to smaller pieces with the help of the water jet. The thicker roots are cut to smaller pieces. The sieves content is carefully inspected under a 10X magnifying glass and a powerful white light source.
11
Larger land snails are retained in the upper sieve, and in the lower sieve the smaller land snails and the eggs. The collected land snails are kept in a tray and the eggs in a Petri dish, both with a humid filter paper. Once finished separating eggs and land snails from the soil samples we proceed to identify them.
--- Task 1.2
Objetives
Field experiments to investigate the land snails pests populations dynamics and structure.
Participant: All participants
Materials and Methods
To study the population structures and variation over time it´s necessary to determine the maturity state of the land snails constituting the population along time. Because of this, Bett (1960), Hunter (1968a), and Hunter &
Symonds (1971), based their studies exclusively on sperms presence or absence along the genital tract. South (1989a) used the same methodology, but he also obtained the gonad and albumin gland mass, (Hermaphrodite Gland Index, H.G.I. and Albumin Gland Index A.G.I. respectively, of each individual. This index express the % of corporal mass represented by each gland, that have a characteristically variation along the maturation cycle of D.
reticulatum.
Duval & Banville (1989) and Barker (1991), in adition to calculating the H.G.I. and A.G.I., incorporated in their work the gonad cytological analysis of individuals, and determined their maturity level using as reference the previous studies of Runham & Laryea (1968), based on the presence and relative abundance of each cellular type in gonad gametogenisis in D. reticulatum gonad. Haynes et al. (1996) classified the land snails in five categories based on the body mass index H.G.I. A.G.I. instead of using the cytological gonad analysis to describe the population structure. The population dynamic and structure study are only done on the species that really are a pest; in our previous studies (Barrada, 2003) we focused exclusively on Deroceras reticulatum that really constitute a pest in the European crops.
Individual management. Individuals belonging to the pest species are weight on a laboratory balance to the hundredth of milligram, and then they are sacrificed by a brief immersion in water at 50ºC. This method is a modification from the Haynes, Rushton y Port (1996) method, which is to dip them in boiling water. The sacrificed individuals are introduced in properly labeled glass tubes, with preservation 70% alcohol. Then the individuals are dissected and they have their hermaphrodite gland and albumin gland removed, which is used to determine the maturity state of individuals.
Following Barker (1991) approach, individuals with BMI(body mass index) exceeding 20 mg are not dissected, assuming that they don´t have a differentiated gonad. The hermaphrodite and albumin gland, extracted from individuals with BMI greater or equal to 20 mg, the glands are weighed up to the hundredth of milligram immediately after its extraction. The hermaphrodite gland is fixed with Carnoy for 24 hours and preserved in alcohol 70%.
Determining the maturity degree. It will follow the methodology used by Duval & Banville (1989) and Barker (1991), the sexual maturity status of individuals at each monthly sampling is determined by gonad cytological analysis, using as a reference the states defined by Runham & Laryea (1968) . To this end, each individual gonad was dehydrated by a series of ethanol baths of progressively higher degree (70%, 96% and 100%), ending with 2 baths of toluene. Next the sample is included in a paraffin block; which is sectioned at 8 μ thick. Obtained sections are rehydrated by reversing the previous dehydration, replacing toluene by xylene and dipping them in distilled water. Then the sections are stained using hematoxylin-eosin staining, and finally dehydratated again.
Each cellular type that appear in the selected land snails gonad and the maturity states are available in Pelluet & Watts (1951), Watts (1952), Bridgeford & Pelluet
12
(1952), Henderson & Pelluet (1960), Smith (1966), Runham & Laryea, (1968), Bailey (1973), Hill & Bowen (1976), Parivar (1978, 1980, 1981), Nicholas (1984), South (1992), Fawcett (1987) and Lutchel et al. (1997) works.
Each captured individual was identified as belonging to one of the following sexual maturity stages: i) undifferenciated spermatogonia, ii) spermatocyte, iii) spermatid, iv) espermatozoa, v) oocyte and vi) senescent.
The first three states correspond to immature land snails, with no reproduction ability; sexually mature land snails are those that are in spermatozoon and oocyte state (Runham y Laryea, 1968; South 1989a).
In other words, each captured individual is characterized by their body mass (mg), by their maturity state and by the mass (mg) of the hermaphrodite and albumin gland. From these values it´s calculated for each individual, the hermaphrodite gland index (H.G.I.) and the albumin gland index (A.G.I.), as follows,
H.G.I. = Hermaphrodite gland mass 100 / individual mass A.G.I. = Albumin gland mass 100 / individual mass
For each sample occasion, individuals who have similar characteristics referring to its sexual maturity state and body mass index values, H.G.I. and A.G.I., are considered as belonging to the same land snail generation.
In this research project we follow the methodology used by Duval & Banville (1989) and Barker (1991) for the population structure study. As these authors did, we assume that individuals with body mass exceeding 20mg are land snails with completely undifferentiated gonads (from the cytological viewpoint) are not analyzed. In this regard, it should be mentioned that South (1989a) indicates a value of 40 mg body mass as a limit from which, the maturity state of D. reticulatum can be defined by studying the cytology of the testis. Previous obtained results agree with the values set by South (1989a).
13
Work Package 2
Field research to investigate the feeding habits and the qualitative and quantitative diet of land snails in horticultural crops.
Objetives
To Study land snail feeding and qualitative and quantitative composition of their diet in order to find plant species that can be used as trap plants in organic farming.
Participants 1 (……. Man-month) Participants 2 (……. Man-month) Participants 3 (……. Man-month) Participants 4 (……. Man-month) Participants 5 (……. Man-month) Participants 6 (……. Man-month) Participants 7 (……. Man-month) Participants 8 (……. Man-month) Participants 9 (……. Man-month) Background
The study of the material ingested by land snails is carried out through microscopic analysis of the small plant fragments found in their feces or inside the animal’s digestive tract. This method has been used by Grime, Blythe and Thornton (1970), Mason (1970), Wolda et al. (1971), Chatfield (1975), Richardson (1975), Williamson and Cameron (1976), Szlavecz (1986), Speiser and Rowell-Rahier (1991), Hatziioannou et al. (1994), to study the land snails diet as Cepaea nemoralis, Oxychilus cellarius (Müller, 1774), Oxychilus alliarius (Miller, 1822), Discus rotundatus (Müller 1774), Arianta arbustorum (Linnaeus, 1758), Monadenia hillebrandi (Smith, 1957), Monacha cantiana (Montagu, 1803), Monacha cartusiana (Müller, 1774), Braybaena fructicum (Müller, 1774), Helix lucorum (Linnaeus, 1758), Sandy Xeropicta (Ziegler 1827) and Cepaea vindobonensis (Férussac 1821). Hunter (1968b), Pallant (1969, 1972), and Jennings and Barkham (1975) (it) has been used to study the slugs diet as Deroceras reticulatum, Tandonia budapestensis (Hazay, 1881), Arion hortensis (Férussac, 1819), and Arion ater.
The study of feces is a faster method than the analysis of the stomach contents, as it does not require the slaughter or dissection of the animals. However, the substances present in the feces have passed through all the animal´s digestive tract and are more degraded, making them harder to identify than those extracted from the stomach (Cook and Radford, 1988; Hatziioannou et al., 1994).
The feces study is a suitable method to verify the presence or absence of certain elements in the diet of the animals, but the proportion of unidentified substances in the feces is often so high that it is not an effective method to perform a quantitative characterization of the diet (Williamson and Cameron, 1976; Szlavecz, 1986;
Speiser and Rowell-Rahier 1991). Vadas (1977) points out that the most numerous and more easily recognizable substances in the animals feces are metabolically the least used and perhaps, are also eaten in smaller amounts, so the feces analysis leads to an overestimation of the little nutrients consumed; on the other hand, the most consumed nutrients are underestimated because they are ingested in great quantities and therefore become more scarce and difficult to identify in the feces.
The analysis of the animals digestive tract contents is a tougher method, but it provides a more realistic picture of the diet of the animals (Norbury and Sanson, 1992). Pallant (1969, 1972) studied the diet of D.
reticulatum in natural populations (forests and grasslands) through the analysis of stomach contents. To minimize gastrointestinal degradation of the food eaten by the captured land snails and slugs and to facilitate their
identification, Pallant (1969, 1972) introducing the captured animals directly in 70% alcohol and made their dissection to extract the contents of the digestive tube tract during the following 2 hours after they were caught.
Triebskorn and Florschutz (1993) conducted a study on the transit of the food through the digestive tract of D.
reticulatum, using a special food preparation (lettuce, corn and milk powder) radioactively marked. Subsequently x-rays were done to the animals at regular intervals; according to their results, the ingested food penetrates immediately in the maw, stomach and anterior intestine, where it remains for a minimum period of at least two hours and a half before starting to move through the intestine; the maw and the stomach are completely emptied in the following thirteen hours after ingestion.
14
Importance: the information obtained in this study will be useful to designing the strategy of the plant- traps use as deterrent agents, and also to understand and assess the real damages on crops.
Progress assessment and results. Through semi-annual and annual reports, and through regular personal controls, that the Coordinator will carry out at each participants concerned country.
Work and methodology Description
Sampling. The diet study is done by the analysis of the digestive tract content of 20 land snails captured in monthly samplings. The captures of these animals are randomly done in the course of a run across the whole plot.
Only the collection of small land snails is avoided, because their processing is very difficult. The capture of the land snails is done during the next 3 or 4 hours after sunset, which is when it takes place the main feeding activity of land snails and D. reticulatum in particular (Dobson & Bailey, 1982, Rollo, 1988ab; South, 1992; Hommay, Lorvelec & Jacky, 1998). Every plant species, where each land snail is captured, is recorded. The land snails are individually introduced in plastic containers with a piece of damp cotton for its further transport and storage in the laboratory.
The day after the capture of the land snails, the plot´s vegetation composition is determined. The different plant species coverage percentage is estimated using a measuring tape extended along the plot´s principal axis, at intervals of 0.5 meters. The tape´s intersection points with the different plant species are noted, and with this data the coverage percentage for each species is calculated. Graminaceous are considered as a whole.
Individual Processing. The land snails caught for the diet analysis are transported and processed within 2 hours, in order to minimize digestive degradation of their stomach contents (Pallant, 1969, 1972).
Land snails are weighed on a scale up to the hundredth of milligram, before being sacrificed by immersion in hot water (50 º C). Then the individuals are dissected and have their maw removed, which is placed onto a slide. Under a binocular microscope the maw is open along the longitudinal axis and all its contents are collected, using a fine brush. Each land snail´s stomach contents is weighed to the hundredth of milligram and immediately introduced into a small plastic tube, were 2 milliliters of 1N hydrochloric acid is added to eliminate the mucus and epithelial debris from the maw sample (Hatziioannou et al., 1994). Stomach contents are always analyzed in the following two days after its capture.
Qualitative diet determination. The qualitative diet study is based on the food fragments identification, found in the snails´ digestive tract. The plant fragments identification is possible through epithelial formations such as stomata and trachoma. The appearance and distribution of these formations are characteristic for each plant species.
Before starting the samplings, land snails are captured around the study area, they are fed with a
monospecifical plant diet, and their feces are used to make an image collection of the fragments found in the land snails´ stomachs. These land snails are kept inside a climate chamber with a photoperiod of 12 hours, 18 º C and 90% relative humidity. All land snails are housed inside transparent plastic boxes 20 × 20 × 10 cm, with the
bottom covered with a damp filter paper, and all the plastic boxes are perforated to allow the air renewal. In each case four individuals are kept. After a period of 72 hours without food all land snails will be fed with a diet
consisting on a single plant species collected in the study plot. Then after six hours, the land snails are processed as described in the previous section, and the fragments found in their stomachs are photographed under a microscope (Barrada, 2003).
For each plant we have a large reference image collection of each one after being eaten by land snails. The captured land snail´s digestive tract is studied using the same microscope equipment. Fragments found in the land snails´ digestive tract are compared with the reference images collection for their further identification.
Quantitative diet determination. To determine the land snails´ quantitative diet composition, we follow the methodology used by Hatziioannou et al. (1994). A stomach sample content of each land snail is taken; the surface area of each fragment is measured with the help of a software image analysis (SPSS ® Sigma Scan Pro Image Analysis Version 5.0.0.). The surface areas of all the same type fragments are added together and the percentage is calculated and represented in relation with the sum of all the fragments contained in the same sample surface areas. According to this, each plant contribution to the land snail diet is estimated.
Using 0.24 ml sample taken from the stomach contents of each land snail, diluted with 2 ml of hydrochloric acid and then homogenized with the aid of an agitator pressure SBS ® AT-1 or similar. All fragments of food contained in the sample are photographed, identified and measured.
The sample volume used for each digestive tract is previously determined from the land snails´ stomach contents study, whose diet is known. To this purpose four land snails are individually housed in each plastic box.
15
After a fasting period of 72 hours, the land boxed land snails are provide with a small piece of 3 different plant species, and 6 hours after they are sacrificed and their stomach contents are removed, as previously described.
Their stomach contents are diluted in 2 ml of HCl for their analysis in 6 successive 0.08 ml samples. In previous researches (Barrada, 2003), it was determined that a 0.16 ml sample (8% of total volume) is an accurate
representation of the land snails stomach contents composition, equivalent to a 0.48 ml sample (24% of the total volume). To minimize the error degree it is always used a 0.24 ml sample (12% of total volume) for the digestive tract contents representation.
Diversity and selection Indexes. The diversity land snails´ diet and the vegetation variety in the study plots, is calculated through the Shannon-Weaver index, H '= Σ (pi) (log2pi), where pi is the frequency for each component of the diet or vegetation (Margalef, 1982).
The index C is used as a selection index, Pearre (1982), this index reflects the outcome of predator-prey interaction taking into account the abundance in the environment for each prey type. This index has a value ranging from +1 and -1, where C = 0 indicates no selection. It shows if a plant is consumed above (positive values) or below (negative values) it´s expected consumption according to their availability in the nature. This index is based on the χ2 test that allows establishing the significance of the selection degree for any sample size.
16
Work Package 3
Objetives
To develop a statistical method to explain and predict the land snails´ activity according to atmospheric conditions.
Participants 1 (……. Man-month) Participants 2 (……. Man-month) Participants 3 (……. Man-month) Participants 4 (……. Man-month) Participants 5 (……. Man-month) Participants 6 (……. Man-month) Participants 7 (……. Man-month) Participants 8 (……. Man-month) Participants 9 (……. Man-month) Background
The terrestrial gastropods activity is regulated by complex mechanisms that involve both, external (environmental) and internal (endogenous rhythms) factors (Bailey and Lazaridou-Dimitriadou, 1986; Aupinel 1987, Young & Port, 1989, Cook, 2001).
As a general rule, it is recognized that these animals spend their days idle at their shelters, their activity is mostly nocturne, and the weather conditions largely determine their state (Hommay et al., 1998). However, it recognizes the existence of interspecific differences in regard to their activity patterns and the influence of environmental factors on them (Cook, 2001). It has also hinted at the existence of intraspecific differences in the relative importance of different environmental factors that control the activity, depending on the microclimate or the type of living environment in each population (Lorvelec and Daguzan 1990, Iglesias and Castillejo, 1996 ). The close dependence of terrestrial gastropods have regarding environmental conditions qualities make them ideal models for studying the relationship between animal behavior and climate (Rollo, 1982). His character of agricultural pests is another aspect that helps explain the interest shown by many researchers in the study of their activity in relation to climatic conditions (Dainton, 1954ab; Getz, 1963; Webley, 1964; Newell, 1968, Cook and Ford 1989, Young & Port 1991, Young, Port, Emmet and Green, 1991; Hommay, Lorvelec and Jack, 1998, Grimm and Kaiser, 2000, Barred, 2003).
One way to deal with this study, which is receiving much attention in recent years due to its applicability in the pest control field, is the development of predictive models or expert models (Bohan et al., 1997, Cook, 2001).
Predictive models of activity as a matter of pest animals represent a useful tool from the applied point of view, since they allow to predict periods when crops can suffer further damage and to optimize use of the pesticides used to control reducing economic and environmental costs resulting from its application at times when there is no risk of damage to crops (Frahm, John, and Volk, T. 1996; Hommay et al., 1998; Hommay, 2002; Port and Ester, 2002).
Importance: data obtained after this investigation will be used to develop a statistical model to predict abundance and activity.
Progress assessment and results. Through semi-annual and annual reports, and through regular personal controls, that the Coordinator will carry out at each participants concerned country.
Methodology
The Barrada(2003)methodology is followed to develop the activity models, this methodology is based on the ones designed by Young & Port (1989) and Young et al. (1991). These authors identified, for the diverse environmental conditions, the limits that define the high land snail activity nights, without attempting to mathematically describe the limits that define those lines, but only giving the extreme values to define the
17
optimal range values for each variable. These models predict that high activity nights will be those with all the variables comprehend between their optimal ranges.
Sampling. The land snail´s activity analysis is done in the same study area used to analyze its feeding habits.
The samplings are done during three consecutive nights per month; this means a total of 72 nights during the sampling period. Every night two investigators examine during three hours the study area, searching for active land snails. The tours are done thoroughly, searching the soil and vegetation, but no effort is done to locate those out of the observer´s sight.
At the beginning of each sampling and subsequently, at 1 hour intervals, the soil temperature and the relative humidity are registered at 5 cm above ground. The soil temperature can be registered with an electronic thermometer fixed in the ground at 10 cm deep, and the temperature and the relative humidity are registered with an electronic thermo-higrometer or a similar instrument.
Variables and Statistical analysis. In the model, the activity is divided in three categories (low, average and high) according to the number of active land snails registered during 72 nights in each study area. This activity levels made up the dependent models or response variables, in other words, (the variable whose value was expressed in terms of the values of the independent or predicted variables). Within these environmental variables were considered measures on site during the samplings are considered as independent variables measures on site during the samplings is considered as independent variables: i) the atmospheric conditions registered on site during the samplings; ii) atmospheric conditions corresponding to the sampling day and the days before it, according to the data registered in a near thermo-pluviometric station; iii) time variables (month, season); and iv) related variables with the land snails population dynamics in the sample area. All considered variables are compiled in table 5.1.
Because of the data nature, qualitative response variables with more than two categories (three
categories: low, average and high) and a serial of qualitative (factors) and quantitative (co variables) variables as independent variables, the statistical procedure to relate the land snails activity level with the independent variables was the ordinal regression (McCullagh, 1980; McCullagh & Nelder, 1989). To carry out the data analysis it can be used the SPSS, using the PLUM method for the ordinal regression (SPSS documents), or any related statistic programs (Barrada, 2003).
18
Work Package 4
To search for a Biopesticide elaborated of plant extracts with molluscicide and ovicide action.
Participants 1 (……. Man-month) Participants 2 (……. Man-month) Participants 3 (……. Man-month) Participants 4 (……. Man-month) Participants 5 (……. Man-month) Participants 6 (……. Man-month) Participants 7 (……. Man-month) Participants 8 (……. Man-month) Participants 9 (……. Man-month)
Objetives
To investigate the feasibility of using plant extracts as biomolluscicides and bio-ovicides.
Background
Farmers in their traditional knowledge have identified and used a variety of plant products and extracts as pest control, especially in storage. 2121 plant species are reported to have pest management properties, 1005 species showed insecticide properties, 384 with antifeed properties, 297 with repellant properties, 27 with attractant properties and 31 with growth inhibition properties. The most commonly used plants are neem (Azadirachta indica), pongamia (Pongamia glabra) and mahua (madhuca indica). 2-5 % neem or mahua seed kernel extract has been found effective against rice cutworm, tobacco caterpillar, rice green leafhopper, and several species of aphids and mites. The efficacy of vegetable oils in preventing infestation of stored product pests such as bruchids, rice and maize weevils has been well documented. Root extracts of Tagetes or Asparagus as nematicide and Chenopodium and Bougainvillea as antivirus have also been reported 'Biopesticide' can reduce pesticide risks, because- (a) Biopesticides are the best choice instead of the conventional pesticides, and usually inherently less toxic. (b) Biopesticides generally affect only the target pest and a wide spectrum ofclosely related organisms, conventional pesticides that may affect organisms as rent as birds, insects and mammals. (c)
Biopesticides are often effective in very small quantities and decompose quickly, thereby resulting in lower exposures and widely avoiding the pollution problems caused by conventional pesticides. (d) When used as a fundamental component of Integrated Pest Management (IPM) programs, biopesticides can greatly decrease the use of conventional pesticides, while crop yields remain high. (e) Amenable to small-scale, local production in developing countries and products available in small, niche markets that are typically unaddressed by large agrochemical companies.
The European Commission has published a proposed European Regulation concerning the placing on the market and use of biocidal products. The new European Regulation would replace the current regulatory regime for biocides, which is laid out in the Biocidal Products Directive 98/8/EC and transposed into UK law by the Biocidal Products Regulations 2001. Once in force (scheduled for January 2013(2019?)), the European Regulation would be directly acting on all Member States, including the UK.
Methodology
1.-MATERIAL AND METHODS
Extracts preparation for the tests:
The plants selection will be made using two parameters: abundance, medicinal and therapeutic
properties. Previously it is necessary to consult references on bibliography. The plants will be collected in the field and identified.
19
The extracts will be made with different fresh plant parts, after drying in an oven at 50ºC (Medina and Woodbury, 1979 & Makkar et al 1991) or at temperature room?(climaticed room). The different plant parts (leaves, steams, fruits, flowers, roots and seeds) will be separated and crushed to powder. The powder of the plant will be put in glass flasks, where they will be spilled to the solvent, leaving it macerate during 24 hours at a temperature room. Passed these 24 hours the samples with acetone/water will be extracted with the help of a Rotary Evaporator machine (Mendes et al, 1993).
The concentrations used will be: 80,000 ppm, 40,000 ppm and 8,000 ppm to the water extracts; 100,000 ppm and 50,000 ppm to the extracts made with acetone/water; and: 100,000 ppm, 50,000 ppm, 10,000 ppm, 1,000 ppm and 100 ppm for both extracts.
Molluscicide activity:
The tests will be made using 10 cm diameter glass Petri dishes with a 9 cm diameter filter paper on the bottom (Albet 400, of 90g/m2 of weight and a thickness of 0.21mm). 1 ml extract will be put in each dish, using a pipette that had a milk filter (Alfa Laval Agri) in the tip to avoid that the pipette gets plugged with the plants´
powder. The dish with the filter will be dried off at a temperature room; once it´s completely dry it will be moistened with 1 ml of distilled water, then the eggs-lays will be deposited on the filter (5 eggs in each dish, ater the presvious eggs selection).- They will stay in the incubation chamber until the death or hatching. To check the effect of the extracts on the embryo the eggs will be observed each 24 hours, with a binocular magnifying glass using an oviscope (glass tube).
The water extract controls will be made with distilled water, while controls for the acetone/water extracts will be made with an acetone/water mixture (in the proportion: 7:3), then the acetone will be removed with the Rotary Evaporator Machine and the water will be used as controls.
To do the tests, 1 ml of water will be put on the filter papers, when the paper dries off at the temperature room, another millilitre will be put on to it and then the eggs-lays will be placed on the moist paper. The Petri Dishes will be closed and put in the incubation chamber.
The same process will be made with the standard soil and with the commercial substratum. At this time plastic Petri dishes of 9 cm of diameter will be used to these purposes. 1 ml of extract will be put on 25g of standard soil with a humidity of 35%, and the same process will be made with the substratum.
Task 4.1.
To carry out laboratory experiments to find plant extracts that can be used as biopesticides to control agricultural land snail pests.
Objectives.
Laboratory tests on filter paper (direct contact) and standard soil to select plant extracts with molluscicidal and /or ovicidal action.
Materials and Methods
Egg-lays. The land snails are collected in the crops to obtain the egg-lays to make the experiments once in the laboratory. The individuals are kept in plastic boxes (25 x 25 x 15 cm) with perforated walls and lids and the floor covered with a moist filter paper. Black small polyethylene tube pieces are used as shelters for land snails, using as food, lettuce, carrots, cabbages, runner beans, potatoes, and mushrooms, supplemented with powdered CO3Ca. The cages are placed in a climatic chamber at 17ºC day/15ºC night with a 12D:12L photoperiod and 85%
relative humidity. Cleaning and food replacement will be done twice weekly. Since land snail breeding takes place whenever environmental conditions are suitable (Carrick, 1938; South, 1989), the cages are inspected looking for egg-lays every day. The land snails laid their eggs directly on the filter paper, mainly in those places covered by
