Emmanuelle Vigne

Corinne Schmitt-Keichinger

Véronique Komar

Lalaïna Rakotomalala

Olivier Lemaire

Christophe Ritzenthaler

Marc Fuchs

Symptom Determinants of Grapevine Fanleaf Virus in Nicotiana Species

INTRODUCTION

The mechanisms of symptom development following plant infection with viruses remain poorly understood although both, the crosstalk between the miRNA pathway and viral suppressors of RNA interference (VSR), and the induction of a hypersensitive response of the plant are involved. So far, no VRS encoded by a member of the genus Nepovirus, family Secoviridae, has been identified but RNAi and hypersensitive-like necrosis have been described for Tomato ringspot vius (ToRSV) in Nicotiana benthamiana [START_REF] Jovel | Salicylic acid-dependent restriction of Tomato ringspot virus spread in tobacco is accompanied by a hypersensitive response, local RNA silencing, and moderate systemic resistance[END_REF]. Also, little is known about viral sequences responsible for symptom development. Back in the 1970s, it was shown that RNA2 of Raspberry ringspot virus caused systemic yellowing in Petunia hybrida while RNA1 was responsible for the severity of systemic symptoms in Chenopodium quinoa and that both RNAs determined the lesion type in inoculated leaves of C. quinoa (2). More recently, the 5' untranslated region of Grapevine chrome mosaic virus was involved in the induction of necrotic symptoms in Nicotiana sp. when expressed from a viral vector, although typical symptoms were not reproduced, suggesting that this sequence may not act as a dominant determinant of symptomatology (1).

To get insight into nepoviral sequences involved in symptom development, we took advantage of two Grapevine fanleaf virus (GFLV) strains causing systemic infection in Nicotiana species but distinct symptomatology: strain GHu (4) induces a vein clearing on N. benthamiana and chlorotic spots on N. clevelandii while strain F13 (7) causes asymptomatic infections on both species. A reverse genetics approach was used to identify GFLV determinants of symptomatology by using infectious cDNA clones of strains F13 and GHu, which were available at the onset of this study [START_REF] Viry | Biologically active transcripts from cloned cDNA of genomic grapevine fanleaf nepovirus RNAs[END_REF] or developed over the course of this study, respectively. Here we describe how the use of assortants and chimeras of these clones were used in a gain-of-symptom approach to identify the determinants of symptomatology during systemic infection.

MATERIAL AND METHODS

GFLV strains F13 [START_REF] Vuittenez | Souches de virus à haute agressivités isolées de vignes atteintes de dégénérescence infectieuse[END_REF] and -GHu (4) were isolated from naturally infected grapevines. Full-length cDNA clones of GFLV-F13 RNA1 and RNA2 were used for in vitro synthesis of transcripts [START_REF] Viry | Biologically active transcripts from cloned cDNA of genomic grapevine fanleaf nepovirus RNAs[END_REF].

A full-length cDNA of RNA1 of GFLV-GHu was amplified by immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR) using primers providing a T7 RNA polymerase promoter and a restriction linearization site. The full-length cDNA of RNA2 of GFLV-GHu was obtained by three RT-PCR steps using total RNA extracted from infected plants. The restricted PCR fragments were successively assembled in a plasmid carrying a T7 promoter and a unique linearizing site.

Chimeric cDNAs of RNA1 clones were constructed by introducing unique restriction sites by site-directed mutagenesis with overlap extension (3) in the parental infectious clones. Targeted sequences were then swapped using PCR amplification and/or restriction enzymes.

Transcripts of cDNAs were obtained by in vitro transcription with the mMESSAGE mMACHINE T7 kit (Ambion) according to the manufacturer's instructions. Size and integrity of transcripts were verified by electrophoresis on denaturing agarose gels prior to inoculation.

Four leave-stage Chenopodium quinoa and N. benthamiana plants were mechanically inoculated with purified transcripts. Symptoms were monitored and systemic infection was assessed by DAS-ELISA in apical leaves. Crude sap of infected C. quinoa or N. benthamiana was then used for passages in C. quinoa, N. benthamiana or N. clevelandii. October 7-14, 2012 -39 -All the wild-type and chimeric cDNA clones were sequenced prior to transcription. The progeny viral RNAs were similarly checked for integrity by IC-RT-PCR followed by sequencing.

Coding cDNA sequences of interest were introduced in a binary vector for agroinfiltration assays in N. benthamiana. Electroporated Agrobacterium tumefaciens were grown at 28°C, centrifuged, resuspended in water and infiltrated using a needleless syringe.

RESULTS AND DISCUSSION

Infectious cDNA clones of GFLV-F13 RNA1 and RN2 (6) were re-sequenced for the purpose of this work. Although a few single nucleotide polymorphisms (SNPs) were detected compared to the published sequences, transcripts derived from the cDNA clones reproduced parental symptoms on C. quinoa plants for which a characteristic yellow mosaic developed on upper uninoculated leaves 6 to 8 days post-inoculation. On N. benthamiana and N. clevelandii the F13 cDNA derived transcripts also mimicked the parental virus, as these plants remained symptomless.

Infectious cDNA clones of GFLV-GHu RNA1 and RNA2 were developed and sequenced. A few SNPs were identified but, again, transcripts reproduced the parental symptoms on both C. quinoa and Nicotiana species.

We then assorted RNA1 from one strain with RNA2 from the other strain and could clearly conclude that the GFLV-GHu symptoms on N. benthamiana and N. clevelandii mapped to RNA1. Further experiments were undertaken to identify the coding region responsible for symptom development by swapping cDNA regions of RNA1 between the two strains. Eight chimeras were produced which allowed us to delineate a coding sequence responsible for typical GHu symptoms on Nicotiana species. This is the first identification of a nepoviral coding sequence eliciting symptoms in a plant host.

The RNA1 coding sequence that elicits symptoms on Nicotiana species was then tested in transient expression assays to determine whether it is able to suppress RNA silencing or induce a symptomatic response on its own. Neither silencing suppression nor symptoms were observed, suggesting that the mechanism underlying the induction of GHu symptoms on N. benthamiana and N. clevelandii is a complex phenomenon.

ACKNOWLEDGEMENTS

This work was supported by INRA and Hatch Act Formula Funds.


Gauthier Quesnel
email: gauthier.quesnel@toulouse.inra.fr

Ronan Trépos

Patrick Chabrier

Jennifer Baudet

Raphaël Duboz
email: raphael.duboz@cirad.fr

Éric Ramat
email: ramat@lisic.univ-littoral.fr

Observations in DEVS framework

Keywords: DEVS, observation, methodology and simulation

The observation of a model is a necessary process in the context of modeling and simulation as it offers to modelers the results of their simulations. In this paper, we focus our works on the observation mechanism which is generally not explicit nor clearly specified. This is generally not an issue unless we want to use our model in experimental frames or to avoid the observation mechanism to interfere with the simulation. In this paper, we introduce an extension to the Parallel Discrete Event System Specification (PDEVS) formalism, to observe models in various ways, by event (at each state transition of a model), at the end of the simulation or by a time step. Thus, we define a formal specification of this extension and its abstract simulators algorithms. Finally, we present an implementation in the DEVS framework VLE.

INTRODUCTION

In the Modeling and Simulation (M&S) activity, observation of the behavior of models is a major task. Observation is the process that catches the state of a model during or at the end of the simulation. It allows the modeler to test, prove, validate or generate data from simulations by connecting simulation software to output streams like files, databases, unit test or visualisation softwares for instance. Observations contribute to the modeller's representation of its studied system. Nevertheless, the observation processes are generally not explicit nor specified in formalism or in simulation software. Thus, they are often model dependant and they simply consist in source code extension. However, on the major M&S software, observations are integrated in the simulation model like in Ptolemy II [START_REF] Lee | Overview of the Ptolemy Project[END_REF] or in Modelica [START_REF] Michael | Introduction to Physical Modeling with Modelica[END_REF]. This is generally not useful to specify formally the observation process. However, it becomes necessary when we want the observation to be modeled as a part of an experimental frame [START_REF] Zeigler | Theory of modeling and simulation: Integrating Discrete Event and Continuous Complex Dynamic Systems[END_REF][START_REF] Traoré | Experimental Frames Methodology[END_REF][START_REF] Bonté | Recursive simulation and experimental frame for multiscale simulation[END_REF], where simulation results have to be captured following a precise experimental plan. For instance, in experimental frame, to calibrate a model or to make a sensitivity analysis, only a final observation of the system is necessary but in online optimisation by simulation or generally in dynamical experimental frames, we need the trajectories of variables of the models [START_REF] Gosavi | Simulation-Based Optimisation -Parametric Optimization Techniques and Reinforcement Learning[END_REF].

Our works take place in the M&S theory defined by B. P. Zeigler [START_REF] Zeigler | Theory of modeling and simulation: Integrating Discrete Event and Continuous Complex Dynamic Systems[END_REF]. M&S theory tends to be as general as possible. It addresses major issues of computer sciences from artificial intelligence to model design and distributed simulations. M&S theory aims to develop a common framework, formal and operational, for the specification of dynamical systems. In this paper, we consider the Discrete Event Specification System (DEVS) formalism [START_REF] Zeigler | Theory of modeling and simulation: Integrating Discrete Event and Continuous Complex Dynamic Systems[END_REF] from B. P. Zeigler works to develop a formal observation for the M&S theory. The DEVS formalism is well situated to clearly specify both system dynamics and experimental frames [START_REF] Zeigler | Theory of modeling and simulation: Integrating Discrete Event and Continuous Complex Dynamic Systems[END_REF][START_REF] Traoré | Experimental Frames Methodology[END_REF]. Nevertheless, even if it is possible to specify observations using DEVS without any modification, it appears that the addition of a specific function dedicated to observation process can be very useful and computationally more efficient than a classic DEVS implementation (cf. figure 1). In this paper, we propose to extend the DEVS formalism with an observation process. These changes allow the modeler to observe its models in different ways.

METHOD

Before proposing the formalisation of the extension observation, we briefly describe, in the following section, the PDEVS formalism.

PDEVS

DEVS [START_REF] Zeigler | Theory of modeling and simulation: Integrating Discrete Event and Continuous Complex Dynamic Systems[END_REF] is a well-known and accepted formalism for the specification of complex discrete or continuous systems abstracted as a network of concurrent, timed and interacting atomic and coupled models. PDEVS [START_REF] Chow | Parallel DEVS: a parallel, hierarchical, modular, modeling formalism[END_REF] extends the Classic DEVS [START_REF] Zeigler | Theory of modeling and simulation: Integrating Discrete Event and Continuous Complex Dynamic Systems[END_REF] essentially by allowing bags of inputs to the external transition function. Bags can collect inputs which are built at the same date, and process their effects in future bags. This formalism offers a solution to manage simultaneous events that could not be easily managed with Classic DEVS. For a detailed description, we encourage the reader to read the chapter three in [START_REF] Zeigler | Theory of modeling and simulation: Integrating Discrete Event and Continuous Complex Dynamic Systems[END_REF].

PDEVS defines an atomic model as a set of input and output ports and a set of state transition functions:

M = X,Y, S, δ int , δ ext , δ con , λ, ta
Where:

X is the set of input values Y is the set of output values S is the set of sequential states ta : S → R + 0 is the time advance function Q = {(s, e)|s ∈ S, 0 ≤ e ≤ ta(s)}
Q is the set of total states, e is the time elapsed since last transition δ int : S → S is the internal transition function

δ ext : Q × X b → S is the external transition function X b is a set of bags over elements in X δ con : S × X b → S is the confluent transition function, subject to δ con (s, / 0) = δ int (s) λ : S → Y is the output function
If no external event occurs, the system will stay in state s for ta(s) time. When e = ta(s), the system changes to the state δ int . If an external event, of value x, occurs when the system is in the state (s, e), the system changes its state by calling δ ext (s, e, x). If it occurs when e = ta(s), the system changes its state by calling δ con (s, x). The default confluent function δ con definition is:

δ con (s, x) = δ ext (δ int (s), 0, x)
The modeler can prefer the opposite order:

δ con (s, x) = δ int (δ ext (s,ta(s), x))
Of course, this function can be completely defined by the modeler.

Every atomic model can be coupled with one or several other atomic models to build a coupled model. This operation can be repeated to form a hierarchy of coupled models. The set of atomic and coupled models and their connections is named the structure of the model. A coupled model is defined by:

N = X,Y, D, {M d }, {I d }, {Z i,d }
Where X and Y are input and output ports, D the set of models and:

∀d ∈ D, M d is a PDEVS model ∀d ∈ D ∪ {N}, I d is the influencer set of d : I d ⊆ D ∪ {N}, d / ∈ I d ∀d ∈ D ∪ {N}, ∀i ∈ I d , Z i,d is a function, the i-to-d output translation: Z i,d : X → X d , if i = N Z i,d : Y i → Y, if d = N Z i,d : Y i → X d , if i = N and d = N
The influencer set of d is the set of models that interact with d and Z i,d specifies the types of relations between models i and d.

In previous section, we present succinctly the PDEVS formalism. In the following paragraphs and sections, we propose the formalization and abstract simulators of our observation extension.

Formal specification

The construction of a DEVS platform for modeling and simulation (like presented in section 3.) implies to observe models and their evolution during the simulation. Observation of DEVS models involves watching or capturing their states. These captures can be done when a change of state occurs (in DEVS terminology, in δ int , δ ext or δ con transition functions) or at specific dates.

Observation of models is a necessary process in the context of the modeling and simulation. To develop a useful simulator software based on DEVS or which accepts DEVS models and theirs computation like OSA [START_REF] Ribault | OSIF: a framework to instrument, validate, and analyze simulations[END_REF] or James II [START_REF] Himmelspach | The JAMES II Framework for Modeling and Simulation[END_REF], for the modellers, the observation can be separated from the models behaviour. OSA or James II use classical software engineering paradigm to develop observation. Respectively "aspect oriented" techniques for OSA or the "design pattern observer" in James II. In this paper, we consider the development of the observation mechanism into the DEVS formalism itself. This work allows to clearly and formally defines when and how to observe models in a "all DEVS" vision of the simulation.

In DEVS, the commonly used solution (as presented in figure 1), is to connect models, both inputs and outputs, to an observation model. This model has in charge to send observation messages and to process the responses of the models. For instance, to build a discrete observation, the observation model relies on a constant ta function in order to send, at each time step, an event to observed models. Then, observed models output observation values that are sent to the observer.

This solution forces the modelers to merge the state graphs between observation and behavior of their models (See figure 2 for an explanation). In addition, by merging the state graphs, the modeler may make the results of its models dependent to the observation he makes. For example, if a model is observed asynchronously with its behaviour, the computation of the e or internal state variables, can give different results with or without observations. Precisely, this mismatch can be explained by the difficulty in representing reals in computer engineering [START_REF] Goldberg | What every computer scientist should know about floating-point arithmetic[END_REF] (see the IEEE Standard for Floating-Point Arithmetic, IEEE 754) .

Finally, in DEVS, a model can have several states at the same date (when at least one call to the ta(s) function returns 0). These states are known as transitory states. It seems obvious that the observation of the transitory states makes no sense. However, this solution does not guarantee to provide only observations of real model states. For example, if the observation is required at a specific time of simulation, the state observed can be one of these transitory states.

In this section, we propose a formal and operational representation of observations in the PDEVS formalism. This extension is based on a function of observation in atomic models and a second graph of connections in coupled models. This extension to the PDEVS formalism integrates necessary characteristics:

• first is to ensure that this extension does not disturb the simulation i.e. we need to conserve the same result with or without the observation.

• second is to provide several types of observation. We propose, in this paper, two modes: time step and finish which define respectively: observations when a model reaches a time and observations at the end of the simulation. (See figure 3).

• finally, in DEVS, a model may change several times statements to the same date (when its ta returns 0 or an external events disturb it). In this case, we observe the last state of the model.

Thus, to develop this extension we need to add function to the atomic models, to add new set in coupled model and add specific observation models. We develop these changes in the next section.

PDEVS Atomic model

To develop this extension in the PDEVS formalism, we extend the PDEVS atomic model with new sets of input and output ports (X obs? and Y obs ). The first one is used for observation request, the second one is used for routing values returned by modeler. We attach to this port a new output function called λ obs . The new PDEVS atomic model is defined such as:

M = X,Y, S, X obs? ,Y obs , δ int , δ ext , δ con , λ, λ obs , ta
Where:

X obs? are the ports used to catch observation requests Y obs are the ports used to send observation values λ obs : X obs? × s × t obs → Y obs s ∈ S, t obs current time of the simulation.

This definition allows us to represent both observed models and classical PDEVS models as defined in section 2.1.. Indeed the model X,Y, S, X obs? ,Y obs , δ int , δ ext , δ con , λ, λ obs , ta with no observations1 is equivalent to the classical PDEVS In the next charts, we use an arrow to show when an observation occur. Thus, in the second chart, we show a finish observation at the end of the simulation, in the third chart, we show a time step observation with a duration of ∆ t = 0.5 between two observations and finally, we show an event view, when the value of the observed variable crosses thresholds. atomic model X,Y, S, δ int , δ ext , δ con , λ, ta . In fact, we should distinguish observed models from observer models.

Observer models

In this paper, we suggest two types of observers. O timed , and O finish to respectively, send observation following a given time-step or at the end of the simulation.

• The discrete time observer model, O timed , needs to send output events on its output port Y obs every ∆ t unit time.

O timed reads observation events from its port X obs and stores data transported by event. This model is : where d is the state of the output stream once X obs has been stored,

O timed = X
X obs? = / 0 Y obs = / 0 ∀d ∈ Data : ta((WAIT, d)) = end
where end is the duration of simulation ∀d ∈ Data : ta((END, d)) = +∞ ∀s ∈ S : λ(s) = build request(s) λ obs is a null function The function build request is a user defined function that identifies in Y obs? the observation request that should be sent to the observed models.

Coupled model

As seen earlier in the introduction and in the section 2.1., the I d variable and the function Z i,d define the graph of connections of the models by calculating the influencees models. Our observation extension of the PDEVS formalism uses the same principle. It proposes two subsets I o and I r which, respectively identify the influencees of atomic models (responses of the observations) and the influencees of observer models (models sending the request of observation).

We extend the PDEVS model coupled in order to introduce a second connection network (see the figure 4). This second network is dedicated to the observer models and to the classical PDEVS atomic models to observe through their ports X obs? and Y obs . The new PDEVS coupled model is defined as:

N = X,Y, D, O, R, X obs? , X obs ,Y obs? ,Y obs , {M d }, {I d }, {Z i,d }, {M o }, {O d }, {Z o,d }, {M r }, {R d }, {Z r,d }
Where X obs , Y obs and X obs? , Y obs? are input and output ports to route observation and request events. O is the set of names of observed models O ⊆ D, {M o } is the set of observed models {M o } ⊆ {M d } , R is the set of names of observer models R ⊆ D and {M r } is the set of observer models {M r } ⊆ {M d }.

∀r ∈ R ∪ {N}, I r is the influencer set of observed models of r :

I r ⊆ O ∪ {N}, r / ∈ I r ∀o ∈ O ∪ {N},
I o is the influencer set of observed models of o :

I o ⊆ R ∪ {N}, o / ∈ I o
The observation network is however very constrained. Thus, the functions Z o,d and Z r,d are very different and provide additional constraints from the classical Z i,d function. These additional constraints ensure that a DEVS atomic model cannot send an observation event to another DEVS atomic model, and an observation model cannot send request observation to another observation model. The figure 4 shows an example of connections between observed and observer models.

∀r ∈ R ∪ {N}, ∀d ∈ I r , ∃ an output translation function Z r,d : Z r,d : X obs? → X obs? d , if r = N Z r,d : Y obs? r → Y obs? , if d = N Z r,d : Y obs? r → X obs? d , if r = N and d = N ∀o ∈ O ∪ {N}, ∀d ∈ I o , ∃ an output translation function Z o,d : Z o,d : X obs → X obs d , if o = N Z o,d : Y obs o → Y obs , if d = N Z o,d : Y obs o → X obs d , if o = N and d = N
Closure under coupling As we do not change the internal S of coupled or atomic models nor all sets of the PDEVS definition (IMM, INF(s), Figure 4. In this picture, we illustrate the second network in the coupled model to deal with observations graph. Plain connections are observation requests from output port Y obs? of observation models, to atomic model input port X obs? or to coupled model's output port Y obs? , or from coupled model input port X obs? to atomic model X obs? . Dashed connections constitute response of observation request from extended PDEVS atomic model (on output port Y obs ) to observation model X obs or coupled model output port Y obs . The atomic model B is observed by an external observation model on its port X obs2 . It sends observations to its output port Y obs2 . For this figure and for a better understanding, we distinguish observation ports for the model B. CONF(s), EXT(s) and UN(s) of the coupled model), the PDEVS functions have the same content (See chapter 7 in [START_REF] Zeigler | Theory of modeling and simulation: Integrating Discrete Event and Continuous Complex Dynamic Systems[END_REF] to the complete formalization), and the closure under coupling property of the extension observation for parallel DEVS is still verified.

Algorithms

Root coordinator

The root-coordinator implements the overall simulation loop. It sends messages to its direct subordinate (simulator or coordinator). The root-coordinator first sends an initialize message (i-message), and loop on internal transition (*message) from its child to perform the simulation cycles until some termination conditions. As describe in previous algorithm, the root coordinator is not modified in this extension.

Coordinator

In the coordinator algorithm, we add an additional variable tb which indicates the last date of the simulation. This variable tb detects the last PDEVS bags to ensure to observe the latest state of a model. We add a new bag, called mailobs to store all observation events to atomic models at a specific date. When tb detect a change, the mailobs is flushed into the observation network using the functions Z r,d and Z o,d . • l. 4-7: the classic PDEVS functions: ta, δ int , δ ext , λ and δ con .

• l. 8: the observation method is called when an observation event arrived on a input observation port X obs . λ obs is also a constant function to prevent user to modify the state of its model. This function returns a Value (simple type as integer, real, boolean, string, and complex type as set, dictionary, matrix etc.).

Experimental frames in VLE

The study of the models is very important in the modeling and simulation cycle. It is generally carried out using experimental frames. Experimental frames as sensitivity analysis, replicas generation, etc. are used to study the possibilities of models. One important motivation in formalising the observation process in PDEVS is to develop generic experimental frames, which ones rely on intensive observation of models. In the VLE environment, we propose tools for managing the experimental frames. These tools provide methods to:

• Assign parameters or experimental conditions to atomic models.

• Define observation of atomic models, frequency, type, output.

• Build instances of the experimental design by performing combinations between different experimental conditions and applying a number of replicas with particular seed.

• Execute these instances of the experimental design on the grid computation composed of workstations and/or clusters (distributed and parallelized).

The VLE environment is based on a set of libraries called VFL (VLE Foundation Libraries). The development of new programs, based on these libraries, is easily achievable. Thus, in order to collaborate with users of statistical tools, we provide an interface to the program R [START_REF] Development | R: A Language and Environment for Statistical Computing[END_REF]: a tool and language for statistical computing. This package, called RVLE, has the same capabilities as the VFL and provided an easy-to-use tool to exploit and to explore model output based on VLE simulations. In this context, the observation extension is fundamental. The link between the simulator and the scripting language used a piece of software to transform data of observations to R data frames. Following the same idea, we propose, pyvle and jvle respectively for Python and Java interpreters.

CONCLUSION AND PERSPECTIVES

In this paper, in section 2., we extend the PDEVS formalism to introduce the observation of models in the formalism. This development was motivated by the separation between the dynamics of the system and its observation as a fundamental issue. This addition to the formalism is closed under coupling and allows to build observations in a hierarchical and modular manner. The abstract simulators necessary to run were also formally described. And a implementation respectful to the proposed add-on has been provided. This DEVS extension is crucial for us to develop experimental design and to abstract observation from models' dynamic. However, a type of observation is missing in these works. It concerns the event observation of atomic models. In PDEVS terminology, after each change in δ int , δ ext or δ con transition functions. We work to formalize this new type of observation. It will be done in a future paper. . These pictures show the GVLE modeling tool. In the left picture, GVLE is used to build hierarchy of models and connections, to attach behaviours, experimental conditions and observations to atomic models. In the right picture is used to parametrize the observer models.

Finally, from the user point of view of the M&S framework the delivery of a reliable service of observation that can be used either in simple case or in complex experimental frame is a true advantage when the need is to study simulations of heterogeneous coupled models.

Figure 2 .

 2 Figure 2. These pictures show state graphs of an atomic model. In left picture, we show the model without observation and in the right part, the same model with the capacity to be observed at any time (dotted lines represent external transitions). In the computation of the observation states Obs init , Obs A or Obs B (in δ ext , δ int , δ conf or ta functions), simulators can introduce floating-point errors when computing the time elapsed since last transition (e in DEVS terminology) for example.

Figure 1 .

 1 Figure1. This picture shows the classic observation mechanism to observe models in the DEVS formalism. Three atomic models A, B and C are observed by an observation model O which sends event for request observation on output Y obs and waits the results on input port X obs in order to store it in output files, databases etc.

Figure 3 .

 3 Figure3. This figure shows three modes for observing the state of a system (in the top chart). In the next charts, we use an arrow to show when an observation occur. Thus, in the second chart, we show a finish observation at the end of the simulation, in the third chart, we show a time step observation with a duration of ∆ t = 0.5 between two observations and finally, we show an event view, when the value of the observed variable crosses thresholds.

1 6 t = t 0 7 send 8 t

 678 Parallel-Devs-Root-Coordinator 2 variables: 3 t // current simulation time 4 child // direct subordinate devs-simulator or devs-coordinator 5 initialization message (i,t) to child = tn of its child 9 loop 10 send ( * ,t) message to child 11 t = tn of its child 12 until end of simulation 13 end Parallel-Devs-Root-Coordinator

1

  Parallel-Devs-Coordinator 2 variables: 3 DEVN = (X, Y , D, {M d }, {I d }, {Z i,d }) 4 parent // parent coordinator 5 tl // time of last event 6 tn // time of next event 7 eventlist // list of element (d, tn d ) sorted by tn d 8 mail // output mail bag 9 mailobs // observation mail bag 10 O event // event-list of event observation model 11 y parent // output message bag to parent 12 y d // set of output message bags for each child d 13 14 when receive xobs-message (x obs ,t) 15 if not (tl ≤ t ≤ tn) then 16 error: bad synchronisation 17 receivers = {o|o ∈ children, o ∈ I r } 18 for each o ∈ receivers 19 send x-message (Z r,d (x) with input value Z r,d to o 20 21 when receive xobs?-message (x obs? ,t) 22 if not (tl ≤ t ≤ tn) then 23 error: bad synchronisation 24 receivers = {r|r ∈ children, r ∈ I o } 25 for each r ∈ receivers 26 add r, x obs? to mailobs 27 28 when receive yobs-message (y obs ,t) from o 29 for each child d ∈ Z o,d 30 send xobs-message to d 31 for each d ∈ Z o,d and d = N 32 send yobs-message to parent 33 34 when receive yobs?-message (y obs? ,t) from r 35 for each child d ∈ Z r,d 36 send yobs-message to d 37 for each d ∈ Z r,d and d = N 38 send yobs?-message to parent 39 40 // finally, we append these algorithms in the following message 41 This simplified class defines:

Figure 5

 5 Figure 5. These pictures show the GVLE modeling tool. In the left picture, GVLE is used to build hierarchy of models and connections, to attach behaviours, experimental conditions and observations to atomic models. In the right picture is used to parametrize the observer models.

  SENT } : δ ext ((s, d), e, X obs ) = (s, d ) where d is the state of the output stream once X obs has been stored, ∀d ∈ Data : ta((IDLE, d)) = ∆ t (the time step) ∀d ∈ Data : ta((SENT, d)) = 0 ∀s ∈ S : λ(s) = build request(s) λ obs is a null function• The finish observer model, O finish , produces observation events at the end of the simulation on it Y obs port and wait observation events from its port X obs . O finish is the same model as O timed but, its ta function returns the end of the simulation to build observation event, or +∞ after.

	Where:
	X obs is the set of observation values,
	Y obs? is the set of observation requests,
	S : {IDLE, SENT} × Data, with:
	IDLE, SENT: automata finite states,
	Data: output stream states (files, database, etc.).
	X obs? = / 0
	Y obs = / 0
	∀d ∈ Data : δ int ((IDLE, d)) = (SENT, d)
	∀d ∈ Data : δ int ((SENT, d)) = (IDLE, d)
	∀s ∈ {IDLE,

obs ,Y obs? , S, X obs? ,Y obs , δ int , δ ext , δ con , λ, λ obs , ta O timed = X obs ,Y obs? , S, X obs? ,Y obs , δ int , δ ext , δ con , λ, λ obs , ta Where: X obs is the set of observation values, Y obs? is the set of observation requests, S : {WAIT, END} × Data, with: WAIT, END: automata finite states, Data: output stream states (files, database, etc.). ∀d ∈ Data : δ int ((WAIT, d)) = (END, d) ∀d ∈ Data : δ int ((END, d)) = (END, d) ∀s ∈ {WAIT, END} : δ ext ((s, d), e, X obs ) = (s, d )

X obs? = / 0, Y obs = / 0 and λ obs is a null function.

when receive i-message (i,t) at time t • from l. 1 to l. 38: routes the message or stores in mailobs.

• from l. 40 to the end: to update the tb variable and to send xobs?-message to the atomic models.

Simulator

Lastly, this last section on the abstract simulators develops algorithm for the simulator of the atomic model. As presented previously, the management of the observations events is very simple for the modeler since only one function is called λ obs . This function is called only for classical atomic models when they receive an X obs? event. When observation models receives X obs event, they use the classical way when receiving input message (See x-message in PDEVS abstract simulators). • l. 12: when receives an xobs?-message from the parent, simulator computes the observation in the λ obs function and sends the data (wrapped into an yobs-message) to the parent coordinator.

To validate this work, these abstract simulators are implemented in VLE, a platform of modeling and simulation VLE.

RESULT

Our works have started with multi-disciplinary issues emerging from the interaction between computer scientists and biologists. Considering these works, we think that the integration of heterogeneous models and the respect of the M&S cycle are the key issues to provide a complete and reliable software environment for natural complex systems modelling. So, VLE (Virtual Laboratory Environment) has evolved toward a complete multi-modelling and simulation environment [START_REF] Quesnel | The Virtual Laboratory Environment -An operational framework for multi-modelling, simulation and analysis of complex dynamical systems[END_REF] and is now a generic environment for M&S, in Environmental Sciences, in Industry or Medicine. It is used in many projects from two major French research institutes INRA and Cirad like the RECORD project [START_REF] Chabrier | Toward a simulation modeling platform for studying cropping systems management: the record project[END_REF]. RECORD is a platform designed for developing models of cropping systems, including crops, soils, pests, pathogens, and farmers, at different spatial and temporal scales. Scientists will use the RECORD platform to develop new models as modular components, to re-use and combine them in order to represent cropping systems and to share them with the community.

Technically, VLE is a set of softwares and libraries which supports multi-modeling and simulation by implementing the PDEVS abstract simulators and a DEVS bus. VLE is oriented toward the integration of heterogeneous formalisms like integration of ordinary differential equations (DESS, QSS 1 and 2), petri nets, finite state automata (moore, mealy, UML statechart), cellular automata (Cell-DEVS and Cell-QSS) and agent. Furthermore, VLE is able to integrate specific models developed in most popular programming languages into one single multi-model. Finally, VLE uses an open-source license and all the source code are available on its website including this observation extension (see the figure 5 for screenshots of VLE).

Implementation

In our implementation of the PDEVS abstract simulator, we define an atomic model as a classical C++ class. This class must be inherited to override the observation function and to benefit to the observation extension developed in the previous section.


Francis Fleurat-Lessard

Jean-Michel Savoie

Florence Richard-Forget

Christian Barreau

Nadia Ponts

Vessela Atanasova-Penichon

Laetitia Pinson-Gadais

Véronique Pronier

Thierry Gibard

Marie-France Neveux

Corine Grimaldi

Christos Athanassiou

Maria Otilia Carvalho

Shlomo Navarro

George Opit

Catherine Regnault-Roger

Matthias Schöller

Vaclav Stejskal

Phyllis Weintraub

S Savoldelli

Ali A Isikber

H K Tofel

R Guruprasad

Cornel Adler

Matthias Schoeller

Subramanyam

A A Hameed

Canadas Pedraza

De Paredes

Weichel L Birner
email: erich.birner@basf.com

and by-products

Keywords: 20 Suppression of Plodia interpunctella moth population by mating disruption Cephalonomia tarsalis control Integrated pest management, food industry, monitoring, insecticides. Session 1: Recent advances in IPSP and current trends in post-harvest Metal bin, grain, insects, molds, remote monitoring, phosphine recycling, fumigation, liquid insecticides 3 -Carifend™: an alternative method remote monitoring, phosphine recycling, fumigation, liquid insecticides Stored tobacco, insect, IPM, Carifend™, tobacco quality, tobacco

and Food Safety" of the INRA Research Centre of Bordeaux-Aquitaine.

This scientific meeting, held every two years, under the auspices of the International Organization for Biological and integrated Control (IOBC), regularly brings together scientists, engineers, industry players, stakeholders and professionals in post-harvest agrofood chain sanitation quality and safety to report the state of progress in applied research in stored product integrated protection. The socio-economic reality of increasing safety and quality traceability concern by foodstuffs purchasers, processed food manufacturers, food supply chains, consumer demand, and the application of recent European Regulations of "Hygiene package" and "Pesticide package" in all food chains accelerate the need for greater production of applied research results. The real challenge of developing new approaches and the application of necessary changes in current programs of integrated pest management in food processing plants and in stored products is impeded by a global reduction of human and material resources available for research and development on pests and pathogens contamination of durable agricultural products and derived food products.

Regardless of this critical situation, the 9th IPSP 2013 Conference is meeting the challenge for the transition towards an efficient control of post-harvest pests and pathogens with both the limitation chemical use and the improvement of risk control of infestation or contamination of durable foods by insect pests, molds or their impurities and toxins. At IPSP 2013 Conference, the optimal solutions for tackling this difficult challenge will be presented through 90 communications comprising the scientific program. Their authors come from more than 25 countries, confirming the interest of the international scientific community for this IPSP Conference.

The Local Organizing Committee of IPSP 2013 wishes to thank all the contributors making this Conference intriguing and relevant. We are grateful to IOBC and INRA for their advice and support for the organisation. We really appreciate the financial support of our sponsors and organizations and wish to thank the Scientific Advisory Committee for their advice in building the scientific program of this Conference. Thank you also to all the authors of communications for making the work of the Organizing Committee possible.

Avant-propos

La 9 ème Conference OILB-SROP sur la Protection Intégrée des Produits stockés (PIPS) est organisée pour la 1 ère fois par l'U.R. INRA "Mycologie et Sécurité des Aliments" (MycSA) du Centre INRA de Bordeaux-Aquitaine.

Cette Conférence biennale, placée sous les auspices de l'Organisation Internationale de Lutte Biologique et Intégrée (OILB), regroupe des scientifiques, ingénieurs, acteurs économiques et professionnels du monde entier intéressés par la maîtrise de qualité et la sécurité sanitaire dans les chaînes alimentaires post-récolte et pour faire l'état d'avancement de la recherche appliquée en Protection Intégrée des Produits stockés. Dans le contexte socio-économique actuel, la sécurité sanitaire et la traçabilité sont prioritairement exigées par les traders de produits alimentaires, les entreprises de transformations, les distributeurs ou les consommateurs. L'application des récents règlements européens du "paquet hygiène" et du "paquet pesticide" dans toutes les chaînes alimentaires accélère les besoins en résultats de recherche. Le challenge à relever consiste à développer de nouvelles approches et applications pratiques de solutions innovantes en protection intégrée contre les ravageurs et les contaminations des chaînes alimentaires après récolte. Cet objectif est contrecarré par la diminution constante du nombre de chercheurs qui se consacrent à ce domaine de recherche dans le monde. Toutefois, la 9 ème Conférence PIPS 2013 contribue à relever ce challenge de la transition vers une maitrise des ravageurs, agents pathogènes ou contaminants des denrées aprèsrécolte en limitant l'utilisation des pesticides tout en améliorant la prévision et la maîtrise des risques d'infestation ou de contamination des produits alimentaires durables par leurs impuretés ou leurs toxines. Pour la Conférence PIPS 2013, 90 communications ont été présentées pour contribuer à relever ce challenge. Leurs auteurs sont issus de plus de 25 pays différents, ce qui confirme l'intérêt de la communauté scientifique internationale pour cette thématique de la protection intégrée.

Le Comité d'Organisation de la Conférence PIPS 2013 remercie tous ceux qui ont contribué au succès et à l'attractivité de cette Conférence et en particulier l'OILB et l'INRA. Le soutien des entreprises et institutions qui ont participé financièrement à cette entreprise a été très apprécié. Le travail du comité scientifique pour effectuer la sélection difficile des communications orales ainsi que les efforts de l'ensemble des auteurs des communications (orales ou affichées) sont également à remercier pour la qualité et la rigueur scientifique de leurs prestations.

Francis Fleurat-Lessard Abstract: Acoustic detection of stored grain insects is based on their ability to generate sound (by feeding, flying, moving, probing for oviposition etc). In recent decades, acoustic devices of various kinds have been marketed for commodity inspection. The efficacy of acoustic devices in detecting the insects hidden inside the grain mass depends on many factors such as the sensor type, substrate structure, the interface between sensor and substrate, the assessment duration, the size and behavior of the insect, the density of the infestation, and the distance between the insects and the sensors. During the present study, adults and larvae of the most common pests of stored wheat have been detected in varying population density (0.1, 0.5, 1, 2, 10 & 50 per kg of wheat). Acoustic emissions of adults of Sitophilus oryzae, Rhyzopertha dominica, Tribolium confusum, Oryzaephilus surinamensis, Trogoderma granarium, Cryptolestes ferruginneus and Lasioderma serricorne and larvae of T. confusum, T. granarium, Ephestia kuehniella and Plodia interpunctella were recorded. The acoustic detector system consisted of a piezoelectric sensor mounted on the end of a probe that was pushed into the grain and a portable acoustic emission amplifier (AED-2010L, Acoustic Emission Consulting, Inc.) connected with a computer. All sound recordings took place in the lab using a sound insulated box in order to dampen background sound. Each recording lasted 3 minutes and was replicated 10 times. The system was 100% precise in negative predictions (predicting the absence of a pest when none is present) and considerably successful in positive predictions (predicting the presence of the pest when at least one individual is present in the grain mass). More analytically, prediction for infestation was rated falsely negative in 50-70%, 30-60%, 0-20% and 0-10% when actual population density was 0.1, 0.5, 1 and 2 adults / kg, respectively, irrespective of pest species. Respective values for larvae were 60-70%, 30-60, 0-40% and 0-30%. The system was always successful in detecting infestation when population density was > 2 individuals / kg irrespective of pest species or developmental stage. Our results are discussed on the basis of enhancing the use of acoustic devices as detection tools in storage facilities.

Keywords: Acoustic detection, stored wheat, piezoelectric sensor, sound analysis.

-The use of bio-test in the disinfestation of food industries

Abstract type: Poster

Süss L. 1 , Cassani S. 2 & Gallizia F. 1
Abstract: The proper use of bio-test in food industries, in the case of treatments with use of sulphuryl fluoride or hot air, is essential for assessing the results obtained.

In 2012 were given to several Pest Control Companies n. 490 bio-tests. Two special containers were prepared: one sized cm 7 x 5 x 4 h for placement on floors or in the crevices and the other, of sized cm 6 x 6 x 7 h to be hanged at different heights in empty silos. The treatments were carried out in mills and food industries. The species employed in the bio-tests were Tribolium confusum (stage egg, larvae, pupa, adult or mix population), Ephestia kuehniella (eggs and larvae), Plodia interpunctella (eggs and larvae), Sitophilus oryzae (adults and a mix population of eggs, larvae and pupae in rice) and Lasioderma serricorne (adults and a mix population of eggs, larvae and pupae in biscuits). Detailed operational guidelines were provided. Three TNT bio-test for controlling the pre-treatment mortality were also prepared for each group of bio-test at any treatment; one of the three to be kept in the laboratory and two others to be transported together with the Bio-test submitted to treatments. All the insects of any stage placed in the TNT-Bio-test resulted fully alive. Overall complete mortality occurred in 368 of 387 total, while in 19 cases survival was recorded. Obviously, at some points of the work processes have not been reached the desired saturation of sulphuryl fluoride at the temperature of hot air required to achieve a complete mortality. In these few cases and few sites localized treatments with pesticides became indispensable. These mechanisms for survival under conditions of water stress have been studied in several species. Despite its importance in stored grains there has been no study on the water balance in S. zeamais. Thus, this work aims to assess the water balance and aggregation behavior in pyrethroid resistant (with and without associated fitness cost) and susceptible strains, seeking to understand their potential. Our objective was to compare female and male adults of susceptible and resistant strains observing: (1) the moisture necessary for the survival;

Keywords

(2) differences in longevity (this response may be due to a balance of water in particular) and if these results may be related to insecticide resistance (and associated fitness costs); and also (4) if the aggregation behavior of this species could regulate water loss through the formation of groups of insects. The results indicated that S. zeamais was extremely vulnerable to low relative humidities in the absence of food. The three strains showed loss of water of approximately 30% which is a relatively low value, but enough to significantly compromise their survival. It was still possible to observe differences between the individual and the group in the conservation of water, but there are no differences between group size. The group retains more water than the insect isolated.

Keywords

:

  Sitophilus zeamais, water balance, insecticide resistance

14:00 -15:50 Session 2: Biology and ecology of S-P pests and pathogens in IPSP Wednesday 03 July Room "Badiane" 08:40 -10:20 Session 5: Chemical control in stored product IPM programs 15:10 -15:40 Coffee break (Poster removal) 15:40 -16:50 Session 6: IPSP decision support tools and integrated approach Chairpersons: Bhadriraju Subramanyam & Francis Fleurat-Lessard Page

	15:40 -16:10	Aspects related to decision support tools and IPM in food chain	P. Trematerra	119
		Technical and economic analysis of
	16:10 -16:30	pest management practice in 14 grain	K. Crepon	120
		elevators
		Influence of storage practices and kind	M.-P. Leblanc &
	16:30 -16:50	of structure on insect presence in grain	F. Fleurat-
		bulks	Lessard

121 16:50 -17:15 Closing Ceremony

	C. Athanassiou & F.
	Fleurat-Lessard

List of posters Session 1: Recent advances in IPSP and Post-Harvest IPM current trends Chairpersons: Phyllis Weintraub & M. Otilia Carvalho No. Page

	Integration of Isaria fumosorosea with DE for R. dominica control	Waqas Wakil	7
	Combination of B. hebetor and γ-radiation on Ephestia control in dates	A.A. AL-Taweel	8
	Using the parasitoid T. evanescens with
	pheromone traps in date palm orchards and in	A.A. AL-Taweel	9
	date warehouses
	Session 2:

Biology and ecology of S-P pests and pathogens useful in IPSP Chairpersons: George Opit & Christos Athanassiou

	Occurrence and identification of insect pests in sunflower seeds and rape	S. Dauguet	18
	Population density estimation of stored grain pests based on their acoustic emissions	P.A. Eliopoulos	19
	Detection of insect infestation in stored wheat based on their acoustic emissions	P.A. Eliopoulos	19 2
	The influence of plant characteristics on the field infestation	M. A. Amro	20
	Susceptibility of different pulse grain to infestation of cowpea beetle	M.A. Amro	21
	Effect of different temperatures and host density of Ephestia on B. hebetor efficacy	A.A. AL-Taweel	22
	Mycotoxicological Analysis of stored groundnuts in Southwest of Algeria	H. Meliani & A. Moussaoui	23
	The use of bio-test in the disinfestation of food industries	L. Süss & al.	24
	Sitophilus zeamais: water balance and behavior	N.M. Pinho Guedes	25
	Session 3:

Phytochemicals and semio-chemicals use in stored-product IPM Chairpersons: Catherine Regnault-Roger & Ali A. Isikber

	Study and analysis of Insecticidal & antimicrobial activity of essential oils Effectiveness of Laurus Nobilis and Mentha pullegium on S. zeamais Identification of pure compounds in apricot and apple electrophysiologically A. F. Ndomo Insecticidal effect of ozone against Plodia, F. Mouhouche M.G. Barros Tribolium, Cryptolestes & Oryzaephilus spp. C. Athanassiou et al. Investigations on microsporidian pathogens of P. interpunctella in Turkey K.F. Acar Insecticidal properties of peas homologous proteins (Pisum sativum L.) A. Mebarkia Stored-Product insects natural enemies in Sicilian wheat P. Suma Testing insecticidal efficacy of pure and combined natural substances on S. granarius S. Trdan Effect of fluctuating temperatures on the development of V.canescens S. Andreadis First application in France of heat disinfestation of a large wheat mill B. Vacquer Potential of Anisopterus calandrae and Lariophagus distinguendus as biocontrol agents of Callosobruchus maculatus O. Benkhellat Biological and molecular analysis of an isolated granulovirus Tecia solanivora against potato tuber moths A. Arroyo Natural occurrence of entomopathogenic fungi isolated from stored grain insect species in Punjab W. Wakil Session 5:	38 39 60 61 62 63 64 65 66 67 68

Chemical control in Stored-Product IPM programs Chairpersons: Shlomo Navarro & Yann Ciesla No. Page

	A comparison of efficacy of current insecticides with bio-based and mineral active substances	F. Fleurat-Lessard et al.	82
	Feeding deterrent activity of new alpha-asarone congeners	B. Lozowicka	83
	Weevil x insecticide does 'personality' matter?	R.N.C. Guedes	84
	Phosphine resistance in Cryptolestes ferrugineus from stored wheat in Oklahoma	C. Konemann	85
	Insecticidal effect of chlorantraniliprole against stored cereal product insect pests	N. Kavallieratos	86
	Insecticidal efficacy of thiamethoxam against T. confusum J. du Val	V. Goundy	87
	Determination of hexamethylentetramine in foods by GC-MS	Meehye Kim	88

25 -Sitophilus zeamais: water balance and behavior

  : bio-test, hot air, sulphuryl fluoride, food industries Water availability and temperature are the two most important abiotic variables that influence the distribution and abundance of insects. Insects have physiological and behavioral mechanisms that enable their survival in the absence of water for a long period.

	Abstract type: Poster
	Guedes N.M.P. 1 , Braga L.S. 1 , Silva L.B. 2 , Guedes R.N.C. 1 1 Dep. of Entomology, Federal University of Vicosa (UFV), 36570-000, Vicosa, MG, Brasil 2 Federal University of Piaui, Campus Bom Jesus, PI, Brasil
	E-mail: nmpguedes@gmail.com
	Abstract:

Università degli Studi di Milano-DEFENS, Via Celoria

2, 20133 Milano, Italy 2 Entomologia applicata, Laboratorio di RicercaAgroblu srl, via San Bernardo 35, 20017 Rozzano (Milano), Italy E-mail: guglielmo.cassani@agroblu.com

This year, the 9th

 Bourlaux, CS 20032, F-33882Villenave d'Ornon, France E-mail: francis.fleurat-lessard@bordeaux.inra.fr Abstract: A laboratory test-bench was built up to simulate high-temperature-short-time (HTST) exposure of infested grain samples by hidden stages of Sitophilus granarius or S. oryzae in a fluidized bed in order to model temperature-exposure time relationship. The reduction of emergence of the two weevil species (S. granarius in bread making wheat and S. oryzae in durum wheat) was determined after different exposure times in the fluidized bed exposure chamber crossed by 2 m.s -1 airflow at inlet air temperatures of 60, 90, 120 and 150°C. The influence of a grain moisture content from 14 to 19% w.b. on lethal time 50% (LT 50 ) and lethal time 99% (probit 8 or LT 100 ) was also observed. The main microbiological and technological characteristics checked on grain samples after heat treatment were measured. Higher the moisture content longer the exposure time required for the same mortality rate to be achieved. The "temperature-exposure time products" assessed at LT 50 and LT 100 were modeled as asymptotic negative regression curves. LT 100 was observed in less than 10 s exposure time for temperature at air inlet of 120 and 150°C, whatever the moisture content level. The reduction of fungal colonies was more important with long exposure time at moderately high temperature (90°C) than with short exposure time at very high temperature (150°C) (giving similar levels of insect mortality rate). For bread-making wheat, it was not observed significant changes in alveogramme rheological test parameters processed with white flour extracted from heattreated wheat samples, provided that air temperature and initial grain moisture content are at moderately high level (less than 150°C and 15% m.c.). The HTST treatment of cereal grain is an efficient and rapid method of control of weevil internal feeder stages and may be developed at practical scale for organic cereals or high-added-value use for special healthy cereal food disinfestation.

Keywords: Sitophilus granrius, Sitophilus oryzae, high-temperature-short-time treatment, wheat grain sample, fluidized-bed, quality characteristics changes. Abstract: As a result of high infestation of Dates by Ephestia spp. during its storage period which was for about 3-6 months before manufacturing and marketing it , Iraq used to apply Methyl bromide as fumigant agent to stop its infestation with Ephestia spp ,but because this fumigant agent proved to be carcinogen and ozone depleting agent , a decision was taken to stop using it internationally and nationally. This decision made Iraqi scientist to search for another methods to control Ephestia spp. or to decrease infestation to the lowest percentage. Therefore, Iraqi scientist recently concentrated on using IPM specifically biological control agents (Egg parasitoid, Trichogramma evanescens and larval parasitoid, Bracon hebetor in addition to the pheromone traps for this purpose. The research carried out into two date warehouses, one in Karbala province, while the other in the Babylon province which belong to the Iraqi company for manufacturing and marketing Dates. In these two date warehouses, the biological agents mentioned above were released periodically in addition to hanging pheromone traps. The results revealed that the percentage of preserving date in these two date warehouses were 97.5 and 98.1 % in comparison with 90.5 and 92.5 % into the control dates warehouses in which biological agents and pheromone traps did not used. Furthermore, to prove that this method for controlling Ephestia spp. in date warehouse was succeeded, we examined date from another date warehouse containing about 3250 tons of date packed in polyethylene bags and treated with methyl bromide to protect it from Ephestia spp. infestation. Unfortunately the percentage of preserving dates was only 91.7%. Finally our results showed that the mean number of Ephestia spp. captured by pheromone traps in the control warehouse which treated with methyl bromide was 18 times more than that captured into warehouses treated with biological agents mentioned above and pheromone traps.

-Application of Biological Control Agents and Pheromone Traps For Controlling Ephestia spp. in Date Warehouses of Two Provinces in Iraq

Keywords: Ephestia spp. date warehouses, pheromones traps, capture limitation, biocontrol species, Bracon hebetor 87

-Stored-product insect natural enemies in wheat industry in Sicily

Abstract type: Poster Suma P., Bella S., La Pergola A. & Russo A.

Dept of Agri-food and Environmental Systems Management -Applied Entomology sec. University of Catania, via S. Sofia, 100, 95123 Catania, Italy E-mail: suma@unict.it Abstract: The cultivation of wheat in Sicily (Italy) has its origins in ancient times and today it turns out to be the one that invests the largest areas in the island. Connected to this cultivation, a flourishing industry of primary and secondary processing has been developed and is particularly competitive in the global market thanks to the qualitative features of sicilian durum wheat. In the last decade research activities were undertaken in order to implement quality by studying infesting arthropods and by developing integrated control methods. In this context, during the monitoring activities of the stored grain pests carried out over the past decade in sevral grain industries and warehouses in Sicily, the following natural enemies were collected: Withius piger (Simon) (Pseudoscorpionida; Whiitidae), Xylocoris flavipes (Reuter) (Hemiptera; Anthocoridae), Anisopteromalus calandrae (Howard) (Hymenoptera; Pteromalidae), Habrobracon hebetor Say (Hymenoptera; Braconidae), Theocolax elegans (Westwood) (Hymenoptera; Pteromalidae), Venturia canescens (Gravenhorst) (Hymenoptera; Ichneumonidae), Cephalonomia sp. (Hymenoptera; Bethylidae). More extensive researches are currently in progress for some of these species. Here we discuss their potential use in the framework of the IPM programs.

Keywords: stored grain, predators, parasitoids, Italy.

-Testing the insecticidal efficacy of individual and combined use of four different natural substances against granary weevil (Sitophilus granarius) under laboratory conditions

Abstract type: Poster

Trdan S.

University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, Ljubljana, Slovenia. E-mail: stanislav.trdan@bf.uni-lj.si Abstract: Laboratory experiment was carried out to evaluate the impact of different natural substances on the mortality of the granary weevil (Sitophilus granarius) adults. We tested the insecticidal properties of diatomaceous earth (commercial formulation SilicoSec®), quartz sand of local origin, Neem leaf powder (offered in a local market as fertilizer and plant strengthener), common beech wood ash, combination of SilicoSec® and wood ash, combination of Neem leaf powder and wood ash, combination of quartz sand and wood ash, combination of all 4 substances tested. Natural substances in a form of powder were mixed with grain in different dose rates. Wheat grains were treated with 450 and 900 ppm of SilicoSec® and quartz sand as self-based formulation, wood ash (5 weight (w)% and 2.5 w%) as self-based formulation, Neem leaf dust (1.25 w% and 2.5 w%). Individual treatments were evaluated as mixtures of SilicoSec (450 ppm) and wood ash (2.5 w%), Neem leaf powder (2.5 w%) and wood ash (2.5 w%); quartz sand (450 ppm) and wood ash (2.5 w%); SilicoSec® (225 ppm), wood ash (2.5 w%), Neem leaf powder (0.625 w%) and quartz sand (225 ppm). Control treatment was presented as untreated grain. Specific treatment was composed from 500±0.01 g of wheat grain and concentration of natural substance and it was replicated three times. Mortality of adults was evaluated seventh, fourteenth and twenty-first day after exposure. Insecticidal efficacy was tested at three different temperatures (20, 25, and 30°C) and two different relative humidity levels (55 and 75%). Significantly the highest mortality of adults was evaluated in treatment treated with formulation consisting of wood ash and SilicoSec® (66.38±2.64%), meanwhile wood ash (2.5 w%) provoked 63.35±2.74 % mortality. In general, mortality after 21 days reached 48.52±0.01% and it was also higher at lower humidity level (46.02±1.66%). Interactions between different factors of experiment will be presented, and suggestions for the practical use of the most effective substance will be given.

Keywords: Granary weevil, physical control, diatomaceous earth, quartz sand, neem leaf powder, common beech ash, mortality, efficacy. 90 Abstract: Thermal disinfestations of flour mill structures and equipment were traditionally used before the 1960's, but they have been replaced by methyl bromide fumigation until the international ban of methyl bromide in 2005. During the last decade, heat disinfestation of flour mills was developed in Northern America and in some European countries from the 2000's workshops of heat disinfestation of wheat mills were carried out. In France, the first industrial application of this technology was attempted in 2010 for the disinfestation of a large wheat mill and was carried out by Agronet Company in collaboration with INRA Research Laboratory in Bordeaux. The heating of the flour mill was performed from electric heat generators of Thermonox® technology (Germany). The electric heaters are forced heat air ventilators enabling to reach the target temperature of 50 to 55°C in about 10-12 h in all parts of the mill. Then, temperature is maintained at this level during 24 to 36 h and optimised from the indications of temperature sensors in order to obtain complete mortality of insects, whatever their development stage. Encaged insects (Tribolium castaneum and Rhyzopertha dominica) placed in the mill during mill heating were killed at more than 97%. Very few survivals were only observed on the floor at the ground level of the mill. The reproductive capacity of individuals surviving thermal exposure was severely reduced. This first industrial application of heat disinfestation process to a large wheat mill confirmed the results of laboratory studies performed earlier at INRA Laboratory. The economic evaluation of the heat treatment in the conditions described above revealed that it is competitive compared to fumigation with similar requirements in the preparation of the building before treatment. This technology seems very promising for periodic eradication of insect colonies in cereal processing industries without use of pesticide.

-First application in France of heat disinfestation of a large wheat mill

Keywords: Wheat mill, electric heat generator, Thermonox® technology, heat disinfestation, industrial scale, stored-product insect, complete mortality.

Abstract: Technical audits were achieved in 14 grain elevators in order to record pest management practices and to evaluate the cost of insect control management practices. Stored grain elevators that were selected for this study were representative of the different ways of insect pest prevention and control system in France. The selection of storage sites was based on the results of a previous study led on 95 elevators that identified 6 keyfactors correlated with pest infestation: type of storage bin (vertical silo or 'flat' store), preventive sanitation of empty storage structures, grain cleaning, grain aeration, insecticide treatment on grain, grain temperature monitoring equipment. By combining these 6 factors, 7 modalities of pest management have been described. For each modality, two grain elevators that theoretically apply each modality have been sampled among the 95 elevators surveyed in Leblanc et al. surveys in 2010 and2011. The evaluation carried out after the visit of the selected storage facilities revealed that pest management was mainly based on chemical treatment whatever the kind of insect management system was applied. Although all the visited elevators were equipped with ambient-air aeration systems, the airflow rate and the cooling time were often found inadequate to efficiently cool all stored grain bins. Grain cleaning cannot be made systematically at reception because harvested grain lots needing cleaning are delivered at a much higher rate than the grain cleaner can receive. Pest management costs were assessed in the range 0.15 €/t to 1.40 €/t, according to the 7 modalities. The major variation factor was the type of pesticide used. The economic loss related to price penalties in the case of quality defect at the supply of domestic or export grain markets for cereals are not considered as a driving factor for insect pest management policy adaptation by grain-elevator managers. Bourlaux, CS 20032, 33882 Villenave d'Ornon, France E-mail: marie-pierre.leblanc@franceagrimer.fr Abstract: A survey of stored-wheat bulks in France was carried out during two years in spring season 2010 and 2011 to improve the knowledge about the relationships between grain storage practices, preventive sanitation, grain insecticidal treatment, stored-grain temperature management, and presence of insect species in grain samples. 95 wheat storage sites were surveyed each year (after 8-9 month storage). Besides the quantitative analysis of grain samples for insect presence and residues content, a questionnaire was submitted to grain-store managers to record their storage practices, the characteristics of their equipment for grain quality preservation, and the preventive and corrective means they use for insect control. The data from these two groups of variables were processed in a multidimensional statistical analysis to reveal significant correlations between grain management practices for sanitary quality maintenance and the level of presence of insects in wheat samples. It was observed a strong relation between the kind of storage structure and the frequency of insect presence in wheat samples. Flat storage of large grain bulks was showed the most risky storage structure vs. vertical storage in metal or concrete bins. A relation was observed between insect presence frequency in samples and stored grain temperature level and also, with the lack of temperature measurement equipment installed inside grain bins linked to cooling aeration systems. Preventive sanitation before grain harvest period, i.e. systematic cleaning and insecticide spraying of unfilled bins, was related to 50% reduction of insect presence frequency in corresponding grain samples. The preventive chemical treatment of harvested grain before long-term storage was not related to a limitation of insect presence frequency in samples taken after a 8-9-month storage period. The results of this representative survey give objective arguments for the implementation of good IPM practices and risk prevention for grain sanitary quality preservation during long-term storage periods.

Keywords

Keywords: Grain bulk sampling, grain storage site, national survey, insect presence frequency, insecticide residues content, storage practices, multidimensional data processing.

Authors of oral presentations (chronological order of presentation)

127 NOTES NOTES


come

Temperature effect on respiration and ethylene production of two apricot varieties

  Barbara Gouble, Patrice Reling, Jean Marc Audergon, Catherine M.G.C. Renard To cite this version: Barbara Gouble, Patrice Reling, Jean Marc Audergon, Catherine M.G.C. Renard. Temperature effect on respiration and ethylene production of two apricot varieties. 15. International Symposium, Jun 2011, Yerevan, Armenia. hal-02745887


Jean Le Maguet

Gérard Hommay

Monique Beuve

Olivier Lemaire

Etienne Herrbach
email: etienne.herrbach@colmar.inra.fr

New Progress in the Study of Grapevine Leafroll Disease in France

INTRODUCTION

Grapevine leafroll disease is one of the most severe viral diseases of grapevine worldwide. It is associated to several virus species classified in the family Closteroviridae: Grapevine leafroll-associated virus (GLRaV) -1, -3 and some other species (genus Ampelovirus), GLRaV-2 (Closterovirus) and GLRaV-7 ('Velarivirus'). Leafroll ampeloviruses are disseminated by exchange of infected material, as well as by natural vectors, the scale insects (Hemiptera Coccoidea). Moreover, 'rugose wood'-associated Grapevine virus A (GVA) and B (GVB) (genus Vitivirus), often found in leafroll-affected vines, are also coccoid-transmissible. This raises questions about possible interactions between ampelo-and vitiviruses during transmission (Herrbach et al., in press). Improving the protection of vineyards against leafroll requires further research in virology, entomology and epidemiology. The present communication aims at updating the results and prospects of the studies run in our institute.

BIOLOGY OF TRANSMISSION

Ampeloviruses are known to be transmitted by mealybugs (Pseudococcidae) and soft scales (Coccidae). In order to enrich our knowledge on virus-vector specificity, transmission experiments were performed with the mealybug species Phenacoccus aceris (Signoret) and the soft scale Neopulvinaria innumerabilis (Rathvon), both present in Burgundy and Beaujolais vineyards. Experiments were done using aviruliferous insects, given a 48 h-acquisition access period (AAP) on detached leaves of virus-infected grapevine followed by a 48 h-inoculation access period (IAP) on healthy vine cuttings.

The mealybug P. aceris was found to vector efficiently the ampeloviruses GLRaV-1, -3, -4, -5, -6, and -9, as well as GVA and GVB (Le Maguet et al., 2012a). It is the first vector species identified so far for GLRaV-6. Thus, P. aceris is shown to vector many grapevine viruses in both Ampelovirus and Vitivirus genera, which emphasizes the attention to be paid to this Holarctic widespread species. Moreover, the first instar larvae (L1) of P. aceris were shown to transmit more efficiently GLRaV-1 and -3 than do second instars (L2).

The soft scale N. innumerabilis was also shown to transmit GLRaV-1, -3 and GVA (Le Maguet, 2012). This species is increasingly present in France, causing direct damage where its population level is high. In regions where this species is present, its vector ability is also to be considered carefully in protection of grapevine against leafroll and rugose wood.

Moreover, the retention of particles in the viruliferous vector has been assessed. The RNA of GLRaV-1, -3 and GVA remained detectable up to 14 days in starving L2 of the mealybug Heliococcus bohemicus. Conversely, starving L2 of the coccid Parthenolecanium corni lost rapidly the detectability of GLRaV-1 and GVA by RT-PCR; however, this species retained the viral RNA up to 18 days when placed on a plant non-host of the viruses.

NATURAL SPREAD OF LEAFROLL VIRUSES

The spatio-temporal dispersal of leafroll in two Pinot noir vineyards in Burgundy has been surveyed over 8 years (Le Maguet, 2012;Le Maguet et al., submitted). On the first vineyard, planted with certified plant material, leafroll symptoms (mainly due to GLRaV-1) spread rapidly from the edges to virtually the entire plot. Biostatistical and molecular analyses showed that the ampelovirus and the vectors originated from the adjacent plots, planted several years earlier, heavily infested by P. aceris and highly infected by GLRaV-1. This highlights the risk generated by diseased neighboring plots to newly planted plots. The second vineyard displayed no or very little spread of leafroll. This vineyard was not surrounded by mealybug-infested and leafroll-infected plots.

Proceedings of the 17 th Congress of ICVG, Davis, California, USA October 7-14, 2012 -47 -

NATURAL SPREAD OF SOFT SCALE CRAWLERS

Field experiments were set up to estimate the possible natural spread by the wind of P. corni L1 and L2 larvae from infested vineyards to a newly planted plot [START_REF] Hommay | Poster at the present meeting[END_REF]. Wind-borne nymphs were caught using glue-covered cylindrical traps, set up in the young plot during the crawler phase (L1) in late spring and during autumn migration of L2 nymphs down to the stock. Results reveal that crawlers were commonly trapped and that part of them were viruliferous. Very few L2 were caught in autumn.

PROSPECTS

Our studies shed new light on the vector biology of P. aceris and other species, and pave the way towards deciphering the virus-vector interactions. Pending questions are the relationships between vector activity and virus transmission, the possible interaction between ampelo-and vitiviruses during transmission [START_REF] Hommay | Grapevine virus A transmission by larvae of Parthenolecanium corni[END_REF], the localization of virus particles within the alimentary system of vectors, as well as the identification of viral determinants of the specific retention in the vector. A French transmissible isolate of GLRaV-1 is being fully sequenced for this purpose.

Finally, a more precise understanding of vector dispersal and virus spread in vineyard plots requires further experiments on vector biology, particularly with species such as H. bohemicus and P. corni, also able to vector viruses and abundant in many French vineyards. Data from such trials, together with in-depth knowledge of virus-vector interactions and improvement of virus detection methods, will provide new insights into leafroll epidemiology and help to improve the protection of grapevine against this major viral disease.

ACKNOWLEDGEMENTS

We are most grateful to three French regional viticulture committees (Comité Interprofessionnel des Vins d'Alsace, Bureau Interprofessionnel des Vins de Bourgogne, Comité Interprofessionnel du Vin de Champagne) and to the FranceAgriMer agency for financial support.


Gérard Hommay
email: gerard.hommay@colmar.inra.fr

Louis Wiss

Jean Le Maguet

Monique Beuve

Etienne Herrbach

First Results on Wind Dispersal of Parthenolecanium corni Larvae in a Newly Planted Vineyard

INTRODUCTION

Many species of mealybugs (Pseudococcidae) and soft scales (Coccidae) living on grapevine have been shown to vector grapevine leafroll ampeloviruses (e.g. GLRaV-1 and -3) and 'rugose wood'-associated vitiviruses (e.g. GVA) in throughout grape-growing regions in the world (Herrbach et al., in press) and they represent a non negligible way of virus dispersal over short distances. It has been demonstrated that some mealybug species are able to spread rapidly leafroll from infected plots to new plantations [START_REF] Beuve | Viral sanitary status of declining grapevine Syrah clones and genetic diversity of Grapevine Rupestris stem pitting-associated virus[END_REF][START_REF] Maguet | Epidémiologie de l'enroulement viral de la vigne dans les vignobles français septentrionaux et transmission par cochenilles vectrices[END_REF]Le Maguet et al., submitted), but the dispersal and virus spreading by soft scales in the vineyard is not documented.

Several ways of natural dispersal can be observed in the vineyard. Larvae can easily crawl from one leaf to the other, and then to adjacent plants. They can also be transported by winegrowers and their engines during the different winegrowing works. Scale-attending ants, that carry crawlers from vine to vine, may also contribute to the spread of viruses [START_REF] Daane | Impacts of Argentine ants on mealybugs and their natural enemies in California's coastal vineyards[END_REF][START_REF] Mgocheki | Spatial distribution of ants (Hymenoptera: Formicidae), vine mealybugs and mealybug parasitoids in vineyards[END_REF]. However, the main factor of passive dispersal is probably the wind, as previously observed for several scale species (Greathead, 1997, Grasswitz and[START_REF] Grasswitz | Movement of grape mealybug, Pseudococcus maritimus, on and between host plants[END_REF]. In addition, wind dispersal of fallen leaves bearing larvae is also possible (Lo et al., 2005(Lo et al., -2006)).

Parthenolecanium corni (Bouché) is a soft scale that thrives in northern European vineyards and is able to vector GLRaV-1 as well as GVA [START_REF] Hommay | Grapevine virus A transmission by larvae of Parthenolecanium corni[END_REF]. The establishment of a new plantation between plots being both virus-infected and P. corni-infested offered the opportunity to evaluate whether nymphs may be detached from their support by the wind, during their active dispersal after hatching ('crawler' phase) and during their migration as second instars (L2) down the stocks to hibernate. The aim of this study was to assess whether nymphs can be transported by the wind and carry leafroll viruses or GVA, susceptible to contaminate new stocks.

MATERIALS AND METHODS

The young vine plot was planted in Nothalten (Alsace, north-eastern France) at spring 2008 with certified rootstocks (34 EM), on a strip of 94 m x 11 m. The plot was arranged in four rows, south-north oriented along the slope. The plantation was surrounded by plots infested by P. corni and infected by GLRaV-1, -2, -3 and GVA in various combinations, the plot to the west being the most heavily infested.

Sticky traps consisted of transparent PVC cylinders (height 30 cm, diameter 14 cm), wrapped up with a 30 cm x 45 cm transparent polythene sheet sprayed with glue (Soveurode™) and staked at a height of ca. 1.2 m (i.e. approximately at the level where larval density was the highest on the neighboring plots). A grid of such traps was set up in the young plot: five (2009) or six (2010 and 2011) traps placed at 20 m intervals within each of the four rows of plantation. From July 2009, a control trap was placed on a neighboring plot at ca. 50 cm height close to a highly infested vine infected by GLRaV-1, -3 and GVA. Traps were checked every week during the crawler phase and during autumnal migration of L2. The egg-laying period of females was controlled in order to settle traps just before the first hatchings. Sheets were examined under binocular microscope and nymphs were marked and counted on a grid divided into eight sectors corresponding to wind directions.

Trapped crawlers were then collected from the glue, if possible in samples of min. 20 individals, and tested in a quadriplex RT-PCR for the presence of GLRaV-1, -2, -3 and GVA (Beuve et al., submitted). Populations of L2 larvae were counted at spring on each vine of the first two first rows of the neighboring plots. Infection of the most infested vines was checked by ELISA.

In spring 2011 and 2012, distribution of L2 was controlled on the young plantation. In 2011, larvae and winter canes of the most infested vines were tested by the same quadriplex RT-PCR procedure. Mean temperatures, rainfall, maximal wind speed and wind direction were obtained from a meteorological station situated at ca. 12 km from the experimental plot.

RESULTS AND DISCUSSION

The total number of P. corni crawlers caught in spring 2009, 2010 and 2011 was respectively 145, 251 and 611 in all traps set up in the young plot (7, 10 and 25,5 in average per trap). The distribution of catches in the young plot seemed to be related to the main prevailing winds and to population density in the immediate vicinity. However, the settling of P. corni on the young plot displayed no significant structure. In Autumn, very few L2 nymphs were caught, probably because their heavier weight. About 30% of crawlers batches and 50% of L2 batches carried by the wind contained one virus or more. There is therefore a possibility that wind-borne larvae could contaminate young vines, provided they are able to attain a plant and feed on it. However, the relative part of such a way to disperse a virus is unknown. Moreover, our detection tests could not reveal the presence of either virus transmitted by P. corni (GLRaV-1 and GVA) in the colonized vines in the young plot. It is possible that the number of larvae is too low (less than 20 L2 on the most infested young vines) or that the virus, if transmitted, was not yet detectable. New monitorings are underway in 2012, as well as virus detection tests in young vines.

ACKNOWLEDGEMENTS

We are most grateful to the FranceAgriMer agency for financial support and to M. Patrick Meyer for allowing us to experiment in his vineyard.


est destinée au dépôt

Genome sequencing of two exoelectrogenic bacteria isolated from soil and assessment of their metabolic profile by means of Phenotype Microarrays Stefano Mocali, Elena Perrin, Ilaria Palchetti, Melania Migliore, Marco Fondi, Serena Laschi, Carlo Galeffi, Alessandro Florio, Francesco Canganella, Maria Teresa Dell'Abate, et al. To cite this version: Stefano Mocali, Elena Perrin, Ilaria Palchetti, Melania Migliore, Marco Fondi, et al.. Genome sequencing of two exoelectrogenic bacteria isolated from soil and assessment of their metabolic profile by means of Phenotype Microarrays. 4.International Congress Eurosoil 2012 -Soil Science for the Benefit of Mankind and Environment, Jul 2012, Bari, Italy. , 2675 p., 2012, EUROSOIL 2012, 4th International Congress of the European Confederation of Soil Science Societies (ECSSS), 2-6 July 2012, Bari, Italy. hal-01823229