Pyrazine derivatives as biocontrol and springtail attractant agents for controlling fungal diseases in cereals

Abstract

The present invention relates to novel pyrazine-derived compounds and to the use thereof as a biocontrol agent, in particular as a springtail attractant. These compounds are intended to be used in the biocontrol of phytopathogenic fungi affecting cereal crops, and in particular for the treatment and prevention of fusarium wilt and Septoria blotch in wheat. The application of these compounds to an area or a plant to be treated makes it possible to attract the springtails that are naturally present in an ecosystem, and these springtails will consume phytopathogenic fungi that cause these diseases, thus considerably and significantly reducing the surface area occupied by these pathogens, and consequently the diseases. The invention relates to a natural pheromone identified in H. nitidus, 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine and the synthetic analogues thereof.

Description

[0001] PYRAZINE DERIVATIVES AS BIOCONTROL AGENTS AND COLLEMBOLA ATTRACTANTS FOR CONTROLLING FUNGAL DISEASES OF CEREALS

[0002] Technical field of the invention:

[0003] The invention relates to the field of plant protection products and biocontrol. In particular, the invention relates to pyrazine derivatives and their use as biocontrol agents for fungal diseases in cereal crops, and in particular wheat. In particular, the compounds of the invention act as chemical mediators by attracting springtails naturally present in soils, arthropods acting as a natural biocidal agent by feeding on fungi pathogenic to crops, such as Zymoseptoria tritici and Fusarium graminearum.

[0004] State of the art:

[0005] With a global population of over 8 billion people, ensuring sufficient grain production is crucial. With a limited amount of arable land available, it's important to achieve high crop yields. In addition to the challenges of global warming, which inevitably impact crop yields due to water shortages or high temperatures, crops also suffer from numerous pathogens such as caterpillars and aphids, as well as bacteria, viruses, and fungi.

[0006] These pathogens have a significant impact on cereal production yield. Epidemics of Septoria, a fungal disease that attacks wheat leaves, cause yield losses of up to 50% on susceptible cultivars not treated with fungicides (Fones and Gurr, 2015). The fungicide market in Europe for this fungus is estimated by manufacturers at $1.2 billion (Torriani et al., 2015). However, their overuse has negative impacts on many organisms and leads to resistance against most active substances (Palumbi, 2001). Septoria due to Zymoseptoria tritici is the most observed disease on wheat crops. The spread of mutations leading to fungicide resistance in Zymoseptoria tritici populations is favored by its genetic variability (Linde et al., 2002). This highlights the need for control by alternative methods.

[0007] Fusarium head blight (FHB) of wheat, barley, and maize is a widespread disease worldwide (Goswami and Kistler, 2004). It causes yield losses of up to 50%, and the 1990s outbreak in the United States alone resulted in losses of $2.6 billion (McMullen et al., 1997). Fusarium graminearum, one of the major causative agents of FHB currently produces numerous toxic secondary metabolites such as deoxynivalenol (DON) (Miedaner, 1997), which reduces the quality of the remaining grains. Currently, FHB control relies on a combination of strategies including cultivar resistance and fungicides (Bai and Shaner, 2004), but crop protection products are still struggling to balance efficacy, food safety, and economic concerns (Jones, 2000).

[0008] For over fifty years, the massive use of pesticides or fungicidal agents has been a common solution to protect crops. Among the most effective and widely used fungicides worldwide against fungal diseases, including Septoria and Fusarium wilt, are succinate dehydrogenase (SDH) inhibitors (SDHIs). However, a study by Inserm and CNRS published in 2019 (Bénit et al 2019) shows the in vitro toxicity of the majority of SDHIs on human cells as well as on those of bees and earthworms. Anses (the French national food safety agency) has called for vigilance at the European and international levels, and emphasizes the need to strengthen research on the potential toxicological effects of SHDIs in humans.

[0009] There is therefore a great need for alternative methods to synthetic pesticides or insecticides, based on a complementary set of natural processes (Integrated Pest Management) of ecosystems for crop management. These alternatives must make it possible to preserve crops without affecting biodiversity and human health, or polluting or degrading the soil.

[0010] Biological control, also known as biocontrol, is another method of controlling pests and diseases using predators, parasites, or natural pathogens. It traditionally involves the deliberate introduction of one or more natural enemies, such as insects, mites, fungi, or bacteria, to suppress the population of a target pathogenic organism or promote resistance in cultivated plants. For example, patent application EP 3987068 A1 proposes the use of a strain of Trichoderma atroviride as a biological control agent against fungal diseases of wheat. Spraying this strain onto a seedbed would prevent contamination by Fusarium graminearum. In particular, Trichoderma atroviride is able to penetrate the roots and promote development, nutrition, and resistance to fungal diseases.

[0011] Biological control can thus be an alternative to chemical pesticides and is considered a sustainable and environmentally friendly approach to pest control. It is used in agricultural and horticultural settings, as well as in natural ecosystems, to maintain the balance between different species and reduce the negative impact of invasive species. Many insects can be used as biological control agents, one of the most widespread being the ladybug, a natural predator of aphids. By releasing ladybugs into an infested area, farmers and producers can reduce the need for chemical pesticides and promote a more sustainable approach to pest control. Alternatively, plant protection products such as chemical mediators are also considered biocontrol agents, including insect pheromones and kairomones (Article L.253-6 of the Rural and Maritime Fishing Code in France). For example, pheromones are used to mass trap a pest insect in a trap, or in sexual confusion techniques.

[0012] The present invention aims to implement a new biocontrol method using springtails. Springtails are a class of small arthropods, found in and on soil and litter (i.e. plant debris on the ground) worldwide. They are generally less than 6 mm long, have a head with antennae, and a soft body divided into several segments. They are harmless to humans and play an important role in soil health and nutrient cycling. Indeed, springtails are important decomposers in terrestrial ecosystems, helping to break down organic matter and release nutrients such as nitrogen, phosphorus, and potassium, thus facilitating their availability to crop plants. In general, springtails are omnivores that feed on various organic matter present in and on the soil (litter).They can consume a wide range of materials, including bacteria, fungi, terrestrial microalgae, and decaying plant matter. However, laboratory food choice experiments have shown that springtails preferentially select certain fungal taxa (Collembolan dietary specialization on soil-grown fungi, Jorgensen et al. 2003). Thus, when a springtail shows a marked palatability for a pathogenic fungus, it can represent an excellent method of biological control of the fungus.

[0013] Various scientific studies have highlighted the potential of springtails for the biocontrol of phytopathogenic fungi (Innocenti & Sabatini, 2018; Meyer-Wolfarth et al., 2017; Shiraishi et al., 2003; Lartey et al, 1994). However, implementing this biocontrol method, like any other insect biocontrol method, requires introducing these insects in sufficient quantities into an area to be treated. This introduction is difficult in practice, as sources of supply for biological control insects are not common, and moreover require much more restrictive introduction logistics than the application of a simple plant protection product. Regarding springtails, their introduction into crops is currently completely impossible because there is no method for mass production of these arthropods.

[0014] The general aim of the present invention is to propose a new method of biocontrol using springtails.

[0015] Description of the invention:

[0016] The inventors have demonstrated that with a particular class of chemical molecules, some of which are of natural origin and used in infinitesimal doses, it is possible to indirectly implement a method of biological control against phytopathogenic fungi using springtails. They achieved this result by using pyrazine derivatives not as pesticides, but by using them as attractants, in order to attract springtails naturally present in the soil and consuming fungi that are pathogenic for certain crops, particularly cereals. These pyrazine derivatives can therefore be used as biocontrol agents, in particular as chemical mediators acting as an aggregation pheromone and attractant.

[0017] Indeed, after numerous studies, the inventors identified and synthesized an aggregation pheromone naturally produced by the springtail species Heteromurus nitidus, which effectively attracts springtails, preferentially the species H. nitidus. Advantageously, this species is capable of consuming Zymoseptoria triti and Fusarium graminearum, the main phytopathogenic fungi attacking wheat, over the long term.

[0018] The natural pheromone identified in H. nitidus corresponds to the molecule 3-methoxy-2-(1 - methylpropyl)-5-(2-methylpropyl)pyrazine, illustrated below, and the invention relates to its use as a biocontrol agent.

[0019] 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine

[0020] The invention also relates to novel functional analogues of said natural pheromone which can also be used as biocontrol agents, in particular as attractants for springtails, preferably H. nitidus.

[0021] In particular, the invention provides a pyrazine derivative compound of formula (I), or a salt thereof:

[0022] Formula (I) in which R 1 represents a group selected from C1-C10 alkyl, C1-C2 alkyl substituted by an aryl group, C1-C10 alkyl, and C1-C4 alkyl substituted aryl, and

[0023] R 2 represents a C1 to C4 alkyloxy group, and

[0024] R 3 represents a group selected from a C1 to C10 alkyl group.

[0025] More particularly, the invention relates to a pyrazine derivative compound of formula (I) or a salt thereof, wherein R1 represents a group selected from C1-C2 alkyl substituted by phenyl, butyl, isobutyl, butyl substituted by methyl, and benzyl substituted by methyl, and

[0026] R2 represents a methoxy group, and

[0027] R3 represents a group selected from isobutyl and secbutyl.

[0028] The following molecules are examples of pyrazine derivative compounds of formula (I) according to the invention: (hylbenzyl)-5-(2-methylpropyl) pyrazine,

[0029] -3-methoxy-2-(2-phenylethyl)-5-(2-methylpropyl) pyrazine,

[0030] - l)-5-(2-methylpropyl) pyrazine,

[0031] -3-methoxy-2-(3-methylbutyl)-5-(2-methylpropyl) pyrazine,

[0032] -3-methoxy-2-(1-methylbutan-4-ol)-5-(2-methylpropyl) pyrazine.

[0033] The present invention also relates to the use of at least one pyrazine-derived compound of formula (I) or a salt thereof as a biocontrol agent, and preferably as a biocontrol agent attracting springtails, and in which

[0034] -R 1 represents a group selected from C 1 -C 2 alkyl, C 1 -C 2 alkyl substituted by phenyl, butyl, isobutyl, sec-butyl, butyl substituted by methyl, and benzyl substituted by methyl, R 2 represents an oxo group or a C1 to C4 alkyl group, preferably a methoxy, and

[0035] R 3represents a hydrogen atom or a group selected from C1-C10 alkyl, C1-C2 alkyl substituted by phenyl, butyl, isobutyl, sec-butyl, butyl substituted by methyl, and benzyl substituted by methyl.

[0036] The following molecules are preferred examples of pyrazine derivative compound of formula I for said use:

[0037] -3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine,

[0038] -3-methoxy-2-(1-methylbutyl)-5-(2-methylpropyl)pyrazine,

[0039] -3-methoxy-2-(3-methylbutyl)-5-(2-methylpropyl)pyrazine,

[0040] -3-methoxy-2-(1-methylbenzyl)-5-(2-methylpropyl)pyrazine,

[0041] -3-methoxy-2-(2-phenylethyl)-5-(2-methylpropyl)pyrazine,

[0042] -3-methoxy-2-(butyl)-5-(2-methylpropyl)pyrazine, and

[0043] -3-methoxy-2-(1-methylbutan-4-ol)-5-(2-methylpropyl)pyrazine, and

[0044] -2-isopropyl-3-methoxypyrazine.

[0045] In particular, 2-isopropyl-3-methoxypyrazine is a molecule found in several species in nature and for which the inventors have discovered an attractiveness effect on springtails, and its potential as a biocontrol agent attracting springtails. Of course, in a preferred embodiment of the invention, said use relates to the pheromone naturally produced by H. nitidus, i.e. 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine.

[0046] 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine

[0047] The inventors have identified the existence of enantiomers and positional isomers of this molecule. The invention therefore also relates to the use described above of a compound of formula (I) in which R 1and R 2 are distinct and chosen from a secbutyl and isobutyl group, or a racemic mixture, or an R, or S enantiomer thereof.

[0048] In particular, said pyrazine-derived compound of formula I is used as a biocontrol agent attracting springtails, in particular the springtail species H. nitidus. Indeed, the invention proposes to attract fungivorous springtails already present in the soil by means of an aggregation pheromone and / or its analogues (and not to introduce springtails). Advantageously, said springtails feed on phytopathogenic fungi such as Zymoseptoria tritici and Fusarium graminearum. Therefore, said use is preferably intended for the treatment or prevention of septoria, and / or fusarium wilt of wheat. In practice, said use comprises the diffusion of said pyrazine-derived compound of formula (I) in a growing field, or the application of said pyrazine-derived compound of formula (I) to a crop or to the residues of a crop, such as a wheat crop or wheat debris.

[0049] According to one embodiment of the invention, the pyrazine derivative compound of formula I is used at a dose of less than 50 ng per application, preferably between 0.01 and 3 ng. According to another embodiment of the invention, the dose will be between 0.01 and 3 ng, and preferably between 0.05 and 1 ng in order to have the best attractant effect on springtails in the area of ​​application of said compound. The dose to be used will be adapted according to the conditions of application of these compounds.

[0050] Finally, the invention also relates to a composition comprising at least one compound of formula (I) among those described above or 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine, in powder form or in a gel or a hydrogel or a solution, as well as the use of said composition as an attractant for a biocontrol agent, such as springtails. In order to facilitate the use of said composition, the invention also provides a device containing said composition, said device comprising a reservoir in which said composition is placed, and being capable of diffusing said composition outwards. The composition will thus be protected from the external environment and its diffusion can be controlled by means of any prolonged release technique known to those skilled in the art.

[0051] The present invention is further illustrated, but not limited to, the following figures and examples.

[0052] List of figures

[0053] [Fig. 1] illustrates the principle of the biocontrol method proposed by the invention using a pyrazine derivative as an attractant for springtails in a wheat crop.

[0054] [Fig. 2] illustrates an agar culture of Zymoseptoria tritici and the ability of springtails (H. nitidus) to consume this phytopathogenic fungus over the long term.

[0055] [Fig. 3] illustrates an agar culture of Fusarium graminearum and the ability of springtails (H. nitidus) to consume this phytopathogenic fungus over the long term.

[0056] [Fig. 4] illustrates the methodology followed by the inventors to identify, synthesize and test the pyrazine-derived compounds of the invention.

[0057] [Fig. 5] illustrates the synthesis pathways of the so-called original pyrazine (3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine) identified in H. nitidus and its analogues.

[0058] [Fig. 6] shows the results of the attractiveness tests of the original pyrazine on springtails as a function of the pyrazine dose used, according to the Petri dish attractiveness test.

[0059] [Fig. 7] shows the results of attractiveness tests of one of the analogues of the original pyrazine, 3-methoxy-2-(3-methylbutyl)-5-(2-methylpropyl)pyrazine, on springtails as a function of the pyrazine dose used, according to the Petri dish attractiveness test. [Fig. 8] shows the results of attractiveness tests of the original pyrazine, (3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine) on springtails as a function of the pyrazine dose used, according to the olfactometry attractiveness test.

[0060] Detailed description of the invention

[0061] The present invention relates to novel pyrazine-derived compounds and their use as biocontrol agents. These compounds are intended for use in a biocontrol method using springtails as an agent for controlling phytopathogenic fungi. The invention is particular in that it does not propose to introduce the springtails into their site of action, but suggests attracting springtails already present in the soils by means of an aggregation pheromone and / or its synthetic functional analogues, in particular said pyrazine-derived compounds.

[0062] Figure 1 schematically illustrates the principle of the biocontrol method proposed by the invention. The latter aims to protect, in a non-limiting manner, cereal crops, and in particular wheat, against phytopathogenic fungi, such as the genera Fusarium and Zymoseptoria. To this end, the inventors propose using a springtail aggregation pheromone (or a synthetic analogue) so that its application in a target area induces the attraction and aggregation of springtails naturally present in the ecosystem. Once aggregated in the target area, the springtails will feed on the phytopathogenic fungi affecting this area. For example, phytopathogenic fungi can affect a cereal crop and the springtails will feed on them, thus allowing the crop to recover.Above all, springtails can act as a preventative measure when they are attracted to a post-harvest field containing crop residues contaminated by harmful fungi. Indeed, these contaminated residues are a common source of contamination for new crops. Springtails will help alleviate this problem because, attracted to the post-harvest field, they will consume these fungi.

[0063] The inventors' studies (Figs. 2 and 3) confirm the ability of springtails to feed on Fusearium graminearum and Zymoseptoria tritici, the plant pathogens responsible for the Fusarium wilt and Septoria wilt outbreaks, respectively. The species H. nitidus is particularly advantageous for its ability to preferentially consume harmful fungi. As shown in Figures 2 and 3, and in the inventors' population assessment studies (results not shown), H. nitidus is capable of long-term feeding on Fusarium graminearum or Zymoseptoria tritici, as well as reproducing and increasing its population when feeding on these fungi. Similarly, the inventors experimentally confirmed that H. nitidus is capable of consuming Zymoseptoria tritici spores when present on a wheat leaf contaminated with Z. tritici, as well as long-term feeding and reproduction based on such a diet. H.H. nitidus is therefore able to preferentially feed on Zymoseptoria tritici when this fungus is found on a complex substrate such as wheat debris. In addition, H. nitidus is able to stop the spread of spores and the development of the fungus, as well as to eliminate almost all spores present on the residues.

[0064] Based on these results, the inventors set up an analysis of the volatile molecules produced by H. nitidus in order to identify at least one aggregation pheromone produced by this species. 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine, illustrated below (referred to as the original pyrazine in the figures), was identified as a molecule produced naturally in H. nitidus and capable of attracting and aggregating springtails.

[0065] 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine

[0066] The invention proposes to use this molecule as an aggregation pheromone or attractant for springtails, and preferably H. nitidus, in order to treat a crop or a crop area affected by a phytopathogenic fungus.

[0067] As schematically illustrated in Figure 4, following the identification of this pyrazine derivative, the inventors synthesized this pheromone, as well as functional analogs.

[0068] The invention thus provides new synthetic analogues of 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine, and in particular pyrazine derivative compounds of formula (I), formula (I) or a salt thereof, wherein R1 represents a group selected from C1-C2 alkyl substituted by phenyl, butyl, isobutyl, butyl substituted by methyl, and benzyl substituted by methyl, and

[0069] R2 represents an oxo or methoxy group, and

[0070] R3 represents a group selected from isobutyl and secbutyl.

[0071] All of these compounds have potential as biocontrol agents, in particular of the chemical mediator type acting as an aggregation pheromone. The present invention thus proposes their use as a biocontrol agent, in particular as a springtail attractant. In the present invention, the term "attractant" means a compound capable of attracting a biocontrol agent such as springtails. Depending on the dose applied, the pyrazine-derived compounds of the invention also have potential as an insect attractant or repellent.

[0072] De préférence, les analogues fonctionnels sont choisis parmi les dérivés de pyrazine suivants :la 3-methoxy-2-(1 -methylbenzyl)-5-(2-methylpropyl)pyrazine, la 3-methoxy-2-(2- phenylethyl)-5-(2-methylpropyl)pyrazine, la 3-methoxy-2-(butyl)-5-(2-methylpropyl)pyrazine, la 3-methoxy-2-(3-methylbutyl)-5-(2-methylpropyl)pyrazine, la 3-methoxy-2-(1 -methylbutyl)-5- (2-methylpropyl)pyrazine, la 3-methoxy-2-(1 -methylbutan-4-ol)-5-(2-methylpropyl)pyrazine et la 3-methoxy-2-(2-methylpropyl l)-5-(2-methylpropyl)pyrazine. Voir figure 5.

[0073] The inventors' work also demonstrated that 2-isopropyl-3-methoxypyrazine is also capable of attracting springtails, including H. nitidus. 2-isopropyl-3-methoxypyrazine is a natural molecule associated with the ladybug (Coccinella septempunctata), the monarch butterfly (Danaus plexippus), as well as Saccharomyces cerevisiae and certain plants (PubChem compound summary). However, no link between this molecule and springtails was previously known. The invention thus proposes the use of 2-isopropyl-3-methoxypyrazine as a biocontrol agent in a method for treating or preventing septoria and / or fusarium wilt of wheat using springtails, particularly as an attractant for H. nitidus.

[0074] Of course, 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine naturally present in H. nitidus is the preferred pyrazine derivative of the invention. The inventors have identified positional isomers of this molecule that can be used in the biocontrol method according to the invention, in the form of a racemic or non-racemic mixture, or of an R, or S, enantiomer thereof. According to one embodiment, the isomers are chosen from the following pyrazine derivative compounds:

[0075] In one embodiment, the R, S enantiomers are used in the form of a mixture having a molar ratio R:S equal to or between 1:100 or equal to or between 100:1. The present invention thus provides the use of the pyrazine-derived compounds of the invention as a plant protection product that can be easily applied, and allowing crops to be treated indirectly, by triggering a natural biocontrol method using springtails. The springtails, by consuming the phytopathogenic fungal species in the contaminated crop residues, prevent the contamination of subsequent crops by these fungi. Advantageously, the pyrazine-derived compounds of the invention make it possible to treat fusarium and septoria of wheat, and can be produced by a synthetic process in few steps, so that they are available in large quantities on the market.

[0076] The pyrazine derivatives of the invention are soluble in organic solvents. Advantageously, the inventors have demonstrated that the attraction of springtails is also effective when the molecules are dissolved in a very small quantity of organic solvent (a few milliliters of methanol) and the solution obtained is highly diluted in water (by a factor of approximately 1.10 e 6 ). It will therefore be possible to consider an aqueous solvent for the application of these compounds, and thus have a product with low toxicity for the environment. Alternatively, dilution in an organic solvent such as methanol can be used and achieve a result similar to that of pyrazine diluted in water and shown in Figure 6.

[0077] The pyrazine-derived compounds of the invention will, for example, be diffused into a growing area in order to attract springtails so that they participate in the elimination of phytopathogenic fungi present in this area. In addition, this method may be used in synergy with others (resistant wheat varieties, antagonistic fungi such as Trichoderma atroviride, etc.) in order to provide integrated pest management of these pests. The compounds of the invention may also be sprayed directly onto plants or crop residues. Finally, the pyrazine-derived compounds may be presented in the form of a composition in powder form, in solution, in gel or a hydrogel or may be integrated inside a device allowing its controlled release, such as a pheromone-diffusing device.

[0078] The methodology and experimental tests leading to this invention are described below.

[0079] EXPERIMENTAL PART:

[0080] 1. Biological material

[0081] The springtails (H. nitidus species) used for the experiments came from the MECADEV laboratory (National Museum of Natural History, Brunoy, France). They were kept in culture dishes (80 mm diameter, 52 mm height) in the dark on a moistened Fontainebleau sand substrate (Sordalab) and were fed with terrestrial microalgae (Pleurococcus spp.) and dried cow dung.

[0082] Fusearium graminearum was obtained from the fungal culture collection of the Muséum national d'Histoire naturelle (Paris, France). Zymoseptoria tritici was provided by INRAE ​​BIOGER (Palaiseau, France). All strains were cultured in Petri dishes on Mandels MM medium (Mandels and Reese, 1957) because this medium is very little consumed by springtails unlike others. Spore suspensions were spread on MM medium and the Petri dishes were incubated for two weeks in climate chambers at 25°C for complete colonization. Thereafter, all Petri dishes were kept in the dark at 4°C for a maximum of two weeks until further experiments.

[0083] 2. Ability of springtails to consume Fusarium graminearum and Zymoseptoria tritici in the long term

[0084] The long-term consumption experiment consisted of quantifying the consumption of two phytopathogenic fungi by springtails by evaluating the evolution of springtail population size and their grazing effects on fungal development up to about ten weeks. Three conditions were tested for each pathogenic fungus with ten replicates each. Thirty adult springtails were placed in the presence of either: (i) the fungus grown on MM medium, (ii) uncolonized MM medium, or (iii) sandy substrate alone (moistened Fontainebleau sand: H. nitidus culture medium without food resources). Conditions (ii) and (iii) were carried out to ensure that springtail growth and reproduction are the result of the diet and not of potential reserves made before the start of the experiment and that egg-laying is not the result of environmental stress.

[0085] A specific experimental setup was designed for each of the two fungi because Z. tritici, unlike F. graminearum, has a yeast-like development. As a result, Z. tritici colonies do not homogeneously cover the entire MM culture medium. Thus, cubes were taken from the parts of the culture where the coverage of the MM medium by Z. tritici was the densest. This made it possible to offer approximately the same amount of fungi to the springtails in all replicates. The cubes covered with fungal culture were replaced every three weeks (immediately after the measurements on springtail populations at weeks 3, 6, and 9) to ensure that excess fungal mycelium was always available for all H. nitidus individuals. These boxes were then left in the dark in climate chambers at 15°C for ten weeks.Adult and juvenile springtails and eggs were then counted three times each week by the same observer using a binocular microscope. Dead individuals and exuviae (growth proxies) were also counted and removed each week. Finally, a photograph of the mycelial cover on the agar medium was taken weekly for 3 and 9 weeks. Mycelial cover, i.e., the areas of agar covered by white aerial mycelium, was measured using Image J (Schneider et al., 2012). This software allows counting pixels to distinguish these areas from those grazed by springtails using their color difference. Generalized linear models were performed with a Poisson distribution and a logarithmic link function using the Ime4 package (Bates et al., 2007) in R software because the population indicators were count data.

[0086] Mycelium cover was analyzed as continuous proportion data (thus bounded to the interval [0,1]) using beta regressions with a link logit function (Douma and Weedon, 2019) with the betareg (Zeileis et al., 2016) and glmmTMB (Magnusson et al., 2017) packages in R software.

[0087] The results show that H. nitidus is able to feed and reproduce in the long term using Zymoseptoria tritici (Fig. 2) or Fusarium graminearum (Fig. 3) as a food source. Springtails significantly reduced (p = 4.24e -12 ) by more than 55% on average, the surface occupied by the mycelium of Z. tritici, compared to the control boxes without springtails. The results are very spectacular concerning Fusarium graminearum, for which we observe after ten weeks the almost total elimination of this fungus in certain culture boxes, with a highly significant average reduction (p = 7.1 e-4 ) of the occupied surface. In addition, the number of adult and total springtails (adults + juveniles) is significantly higher in the presence of phytopathogenic fungi than in their absence. These results indicate significant reproduction of the springtail population initially introduced into the box and very low, if not non-existent, mortality compared to boxes without fungi, which demonstrates the sustainable nature of this method.

[0088] 3. Analysis of volatile molecules produced by H. nitidus and identification of pyrazine (GC-MS protocol)

[0089] Molecules produced by springtails (H. nitidus) in their environment were collected using SPME (Solid-Phase MicroExtraction) fibers capable of capturing a wide spectrum of molecules (divinylbenzene / Carboxen / Polydimethylsiloxane DVB / CAR / PDMS fibers, Merck). These molecules were then analyzed and identified by gas chromatography (GC) directly coupled with Shimadzu QP2010-Plus mass spectrometry (MS) analysis at the CRC laboratory (National Museum of Natural History).

[0090] Forty-eight hours before GC-MS analysis, 100 H. nitidus (adults) were placed in a culture dish (97g Fontainebleau sand; 21 mL water) with 60mg of Pleurococcus spp. algae as food. The dishes were then placed in the dark at ~20°C. Abundant food, darkness, and humidity are favorable conditions that encourage springtails to produce aggregation pheromones. In parallel with these dishes, springtail-free dishes with sand alone or sand + food were also prepared to identify molecules not originating from springtails and which would come from the crystal polystyrene of the dishes, the sand, or the food. Finally, boxes containing springtails on sand but without food are prepared in order to differentiate the molecules produced by fasting springtails and feeding springtails, the potential aggregation pheromones being produced only under favorable conditions.

[0091] Twenty-four hours after the preparation of the boxes, the SPME fiber is conditioned for 30 min at 270°C in the GC injectors. After conditioning, the SPME fiber is introduced into the boxes so that it captures the molecules (pheromones) emitted in these boxes prepared the day before. The capture of the molecules by the fiber lasts 24 hours in the dark at ~20°C. The fiber is then removed from the boxes, placed on a fiber holder and then inserted into the injector of the GC-MS device. The analysis of the molecules desorbed from the fiber, by GC-MS lasts 34.5 minutes. The different parameters of the analysis are: temperature of the GC injector: 250°C, values ​​of the flow of the Helium carrier gas: Flow rate 1.0 mL / min; pressure: 49.7kPa, splitless injection mode (for 0.4 min), temperature program applied: 1 min at 40°C then 8°C / min up to 300°C with a final plateau of 1 minute, characteristics of the stationary phase GC column: 5%phenyl-95%dimethylpolysiloxanes DB5 Agilent, GC-MS interface temperature 300°C). The mass spectrometer scans at a speed of 5000 u s. -1, over a mass range of 45 to 500 u, under electron ionization conditions at 70 eV. Data control is performed using GCMSsolution software (Shimadzu). The gain value of the mass spectrometer detector is reduced by a value of 0.9 at the beginning of the chromatogram (in order to reduce the risks of saturation of the detector and its automatic shutdown). The peaks corresponding to the molecules produced only in the presence of springtails are deduced by superimposing the spectra of the “Food” and “Springtails + Food” boxes. An analysis of the results is carried out with the GCMS Postrun Analysis reprocessing software (Shimadzu) and the support of the NIST 2011 mass spectra library.

[0092] 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine was thus identified as a volatile molecule produced by H. nitidus only in the presence of food. The ability of this molecule to act as an aggregation pheromone or attractant for H. nitidus was then demonstrated.

[0093] 4. Attractiveness tests of pyrazine and analogues on H. nitidus

[0094] The attractive effect of the pyrazine-derived compounds of the invention is demonstrated by choice tests in Petri dishes or by means of an olfactometer. Whatever the test used, the previously fasted springtails are exposed to the presence of a solution of the pyrazine to be tested and a control solution, then the springtails must make a choice in their direction of movement, either to move towards the pyrazine solution or to move towards the control solution. The attractive effect is confirmed when the springtails show a significant preference for moving towards the source of pyrazine.For reference, the article "Olfactometric study of the behavior of Oryctes monoceros olive tree (coleoptera, dynastidae) in the presence of the pheromone 4-methyl-octanoate ethyl (4-moe) and synergistic plant material in Ivory Coast" DIBY et al 2010 "Afrique Science 06(1)(2010)74-85 gives a detailed example of an olfactometric study capable of studying the attractiveness of a pheromone.

[0095] 4. 1 The attractiveness of 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine

[0096] The attractiveness of 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine was tested at doses ranging from 0.01 ng to 50 ng, after dilution in two solvents, demineralized water and methanol.

[0097] Five doses of pyrazine were tested with the solvent “deionized water”: 50 ng, 10 ng, 1 ng, 0.1 ng and 0.01 ng.

[0098] For the experiment with methanol as solvent, 3 microliters of solvent (methanol without pyrazine) were placed on a glass fiber filter and 3 microliters of the pyrazine solution were placed on a second glass fiber filter and four doses were tested: 10 ng, 1 ng, 0.1 ng and 0.01 ng. The two filters were placed inside a Petri dish 1.5 cm apart and equidistant from the edges. Then, the movement of 10 springtails inside the Petri dish was observed, and their position was recorded for 3 hours. The final choice of springtails for pyrazine was analyzed using generalized linear mixed models (GLMM) following Bolker et al. (2009) with the Ime4 package (Bates et al., 2007).

[0099] The results are shown in Figure 6, and indicate an attractive effect of 3-methoxy-2-(1 - methylpropyl)-5-(2-methylpropyl)pyrazine depending on the dose used in each test. Very high or very low doses (50 ng and 0.01 ng) do not appear effective in comparison with the control, while an attractive effect is clearly visible between 10 ng and 0.1 ng. Under the conditions studied, doses from 1 ng to 0.1 ng are the most advantageous (significant effect with p-values ​​of 0.00136 and 0.000176 respectively). The results with methanol are similar and the same effect of attractiveness and dose could be observed (significant attractiveness with p-values ​​of 0.0185 and 6.35.10 -7respectively). Figure 8 shows the results of this same test but carried out with one of the analogues, 3-methoxy-2-(3-methylbutyl)-5-(2-methylpropyl)pyrazine. A dose-dependent attractiveness effect is also observed, the most significant effect (highly significant with p = 1.22.10- 6 ) being obtained with a dose of 0.1 ng.

[0100] These results are in no way limiting of the useful dose of the pyrazine derivative compounds of the invention. Indeed, the efficacy of doses according to the present tests is dependent on the conditions of the test carried out, and are not comparable to field application conditions. In practice, the effective doses will naturally be higher because the molecule will be highly diluted when released into the ambient air.

[0101] The attractiveness effect of 2-isopropyl-3-methoxypyrazine was also evaluated using this test at a dose of 1 ng. The results show that 2-isopropyl-3-methoxypyrazine is also capable of attracting springtails, including H. nitidus (average of 5.5 springtails vs. 3.3 on the "water" control with a p-value of 0.000596. 2-isopropyl-3-methoxypyrazine therefore has a significant attractive effect on springtails.

[0102] 4.2 Attractiveness test by olfactometry

[0103] The effectiveness of 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine was also studied using an olfactometer. Using this device, springtails must choose between two routes that are distinguished only by the presence of a solution of the pyrazine to be tested or a control solution without pyrazine. The springtails are placed at the branch of a Y-shaped duct and must choose between one or the other route of the duct.

[0104] The pyrazine solutions used are solutions in deionized water at 5 concentrations: S0=1 |jg.L 1 , S1 = 100ng.L -1 , S2=10ng.L -1 , S3=1 ng.L -1 , S4=0.1 ng.L -1 The olfactometry experiments were carried out with two series of ten individuals, giving a total of 100 individuals subjected to olfactometry: 20 individuals per dose of 0.01 ng, 0.1 ng, 1 ng, 10 ng and 100 ng.

[0105] All statistical analyses were performed using R v 4.1.1 software (R Core Team, 2021). The choices of individuals who chose a side were analyzed using binomial tests with the null hypothesis that individuals chose the control as much as the molecule (p=0.05).

[0106] The results of this test are shown in Figure 8 where we observe once again an attractiveness effect of the original pyrazine derivative (3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine) on springtails. This effect is strongly dependent on the dose used. In this experiment, a slight attractiveness effect is observed for the doses of 100ng, 10ng and 0.01ng, while a strong attractiveness (significant with a p value of 0.0009766) is observed at the doses of 1ng and 0.1ng. Once again, the doses to be used of the compounds derived from the invention will be adapted on a case-by-case basis according to the conditions of application of these compounds in the field.

[0107] 5. Synthesis of the original pyrazine and analogues

[0108] The original pyrazine, 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine, and its analogues were synthesized according to the synthetic routes illustrated in Figure 5, and detailed below.

[0109] In a flask were added 1 equivalent (eq) of 3-methoxy-5-bromo-2-pyrazinamine, toluene (12 mL / mmol), H2O (2.5 mL / mmol), K2CO3 (6 eq), (PPh3)4 (0.1 eq) and isobutyl-boronic acid (2eq). The mixture was stirred at 100°C for 3 h then the second equivalent of boronic acid was added and the mixture was stirred overnight at 100°C. After cooling to room temperature, the aqueous layer was extracted twice with AcOEt (ethyl acetate). The combined organic layers were then washed with saturated NaCl solution, dried with anhydrous MgSO4 and concentrated in vacuo. The crude material was purified on silica gel pad (85 / 15 Dichloromethane / ethyl acetate “DCM / AcOEt”) to give the desired 3-methoxy-5-isobutyl-2-pyrazinamine in 64% yield.

[0110] 1H RMN (300 MHz, CDCh) 5 ppm : 7.32 (s, 1 H) ; 4.69 (brs, 2H) ; 3.94 (s,3H) ; 2.39 (d, J=7.2 Hz, 2H) ; 2.0 (m,1 H) ; 0.90 (d, J=6.Q Hz, 6H)

[0111] 13 C RMN (75 MHz, CDCh) 5 ppm : 148,0, 143,2, 141 ,2, 131 ,7, 53,1 , 43,0, 28,4, 22,3 IR cm-1 : 3317, 3194, 2867, 1618, 1475, 1405, 1227, 1199, 1028, 588, 474 HRMS : calculé pour [C9HI5N3O+H]+ m / z=182.1293 trouvé 182.1289 m.p. : 31.8-32.6°C.

[0112] In a sealed flask, 3-methoxy-5-isobutyl-2-pyrazinamine (1 eq), acetonitrile (4 mL / mmol), H2O (6 mL / mmol), and 48% HI (3 mL / mmol) were added. The solution was cooled to 0°C before a solution of NaNO2 (15 eq) in water (4 mL / mmol) was added dropwise. Caution: The reaction is highly exothermic. After the addition was complete, the flask was sealed, and the mixture was stirred at 50°C overnight. After cooling to room temperature, the aqueous layer was extracted twice with diethyl ether. The combined organic layers were then washed with saturated NaCl and NaCl. The organic layer was dried with anhydrous MgSO4 and concentrated in vacuo. The precipitate was purified on silica gel in DCM to give 2-iodo-3-methoxy-5-isobutyl-pyrazine in 93% yield.

[0113] 1H RMN (300 MHz, CDCI3) 5 ppm : 7.75 (s, 1 H), 3.99 (s,3H), 2.50 (d, J=7.1 Hz, 2H), 2.07 (m,1 H),0.92 (d, J=6.Q Hz, 6H)

[0114] 13 C RMN (75 MHz, CDCI3) 5 ppm : 158,4, 152,8, 137,0, 103,3, 54,6, 42,9, 28,5, 22,3

[0115] IR cm’ 1 : 2954.78, 2868.15, 1358.78, 1047.55

[0116] HRMS : calculé pour [CgHi3N20l+H]+ m / z=293.0151 trouvé 293.0151

[0117] 5.1 General Procedure A for Suzuki Coupling of Thiophenyl Boronic Acid 1 eq of 2-iodo-3-methoxy-5-isobutylpyrazine, toluene (12 mL / mmol), H2O (2.5 mL / mmol), K2CO3 (6 eq), (PPh3)4 (0.1 eq), and boronic acid (2 eq) were added to a flask. The mixture was stirred at 100°C for 3 h, then the second equivalent of boronic acid was added, and the mixture was stirred overnight at 100°C. After cooling to room temperature, the aqueous layer was extracted twice with AcOEt. The combined organic layers were then washed with saturated NaCl solution, dried with anhydrous MgSO4, and concentrated in vacuo. The crude material was purified on a pad of silica gel.

[0118] 5.2 General procedure B for the Suzuki coupling of thiophenyl-boronic acid:

[0119] In a flask were added 2-iodo-3-methoxy-5-isobutyl-pyrazine (1 eq), 4 mL of dioxane, 1 mL of H2O, K2CO3 (2 eq), and boronic acid (2 eq). Argon was blown into the mixture before adding XPhos Pd G2 catalyst (0.1 eq). The mixture was stirred at 100 °C overnight. After cooling to room temperature, the aqueous layer was extracted twice with AcOEt. The combined organic layers were then washed with saturated NaCl solution and dried with anhydrous MgSC and concentrated in vacuo. The crude material was purified on silica gel.

[0120] Prepared according to procedure A with a yield of 88%.

[0121] 1 H NMR (300 MHz, CDCh) 5 ppm: 8.19 (dd, J=3.0; 1.2 Hz, 1 H), 7.99 (s, 1 H), 7.91 (dd, J= 5.1 Hz; 1.2 Hz, 1 H), 7.35 (dd, J= 5.1 Hz; 3.0 Hz, 1 H), 4.08 (s, 3H), 2.58 (d, J=7.1 Hz, 2H),2.15 (m,1 H) 0.97 (d, J=6.6 Hz, 6H)

[0122] 13C NMR (75 MHz, CDCI3) 5 ppm: 156.5, 151.2, 137.6, 136.0, 135.3, 129.0, 128.3, 128.2, 126.2,

[0123] 124.7, 53.4, 43.7, 28.6, 22.4

[0124] IR cm-1: 2953, 2867, 1534, 1523, 1451, 1340, 1300, 1220, 1167, 1077, 1037, 1024, 979, 895, 863, 808, 775, 692 628, 588

[0125] HRMS: calculated for [Ci3Hi6N2OS+H]+ m / z=249.1062 found 249.1053.

[0126] Prepared according to procedure A with a yield of 74%.

[0127] 1 H NMR (300 MHz, CDCI3) 5 ppm: 8.22 (s, 1 H), 8.02 (s, 1 H), 7.82 (m, 2H), 7.35 (m, 2H), 4.17 (s, 3H), 2.61 (d, J=7.1 Hz, 2H), 2.16 (m, 1 H) 0.98 (d, J= 6.6 Hz, 6H)

[0128] 13C NMR (75 MHz, CDCI3) 5 ppm: 156.1, 152.2, 141.2, 140.9, 140.3, 135.5, 135.1, 125.1,

[0129] 125.0,

[0130] 124.4, 124.3, 122.2, 53.7, 43.8, 28.6, 22.4

[0131] IR cm-1: 2946, 1525, 1451, 1431, 1370, 1305, 1275, 1174, 1156, 1087, 1026, 982, 916, 854,

[0132] 831, 738, 722, 564, 535

[0133] HRMS: calculated for [Ci7Hi8N2OS+H]+ m / z=299.1218 found 299.1230.

[0134] Prepared according to procedure A with a yield of 69%.

[0135] 1 H NMR (300 MHz, CDCI3) 5 ppm: 8.60 (dd, J=5.3; 1.8 Hz, 1 H); 8.23 (s, 1 H); 8.13 (s,1 H); 7.90 (dd, J=5.8; 1.6 Hz, 1 H); 7.41 (m, 2H); 4.06 (s, 3H); 2.64 (d, J=7.1 Hz, 2H); 2.2 (m, 1 H); 1.01 (d, J = 6.6 Hz, 6H).

[0136] 13C NMR (75 MHz, CDCI3) 5 ppm: 157.1, 151.1, 139.8, 138.1, 136.9, 135.0, 130.5, 129.1, 124.9, 124.5, 124.4, 122.4, 53.5, 43.7, 28.6, 22.5.

[0137] IR cm-1: 3051, 2953, 2867, 1528, 1456, 1443, 1375, 1327, 1176, 1024, 982, 950, 853, 810, 759, 733, 712,

[0138] HRMS: calculated for [Ci7Hi8N2OS+H]+ m / z=299.1218 found 299.1230.

[0139] Prepared according to procedure A with a yield of 84%.

[0140] 1 H NMR (300 MHz, CDCh) 5 ppm: 9.99 (s, 1 H); 8.58 (d, 1.4 Hz, 1 H); 8.57 (t, J=1.2 Hz);

[0141] 8.01 (s, 1 H), 4.10 (s,3H), 2.60 (d, J=7.1 Hz, 2H), 2.15 (m,1 H) 0.97 (d, J= 6.6 Hz, 6H)

[0142] 13C NMR (75 MHz, CDCI3) 5 ppm: 183.3, 156.5, 152.6, 143.3, 138.9, 137.4, 135.6, 134.8,

[0143] 134.3, 53.6, 43.7, 28.6, 22.4

[0144] IR cm-1: 3123, 2949, 2922, 2870, 1669, 1526, 1457, 1381, 1327, 1194, 1174, 1160, 1040, 1024, 975, 905, 868, 845, 807, 779, 666, 622, 589

[0145] HRMS: calculated for [CuHieNgOgS+H]-!- m / z=277.1011 found 277.1016

[0146] In a flask was added the aldehyde (1 eq), 10 mL of ethanol. The mixture was cooled to 0 °C before adding NaBH4 (1 eq). After 1 h, the solution was hydrolyzed with 15 mL of water. The aqueous layer was extracted twice with AcOEt. The combined organic layers were then washed with saturated NaCl solution and were dried with anhydrous MgSO4 and concentrated in vacuo. The residue was purified on silica gel (80 / 20 cyclohexane / AcOEt) to give the alcohol in 85% yield.

[0147] 1 H NMR (300 MHz, CDCI3) 5 ppm: 8.1 1 (d, J= 1.4 Hz, 1 H); 7.94 (s, 1 H); 7.77 (d, J= 1.2 Hz, 1 H); 4.84 (s, 2H); 4.06 (s,3H); 2.57 (d, J=7.1 Hz, 2H); 2.15 (m,1 H); 0.95 (d, J= 6.6 Hz, 6H).

[0148] 13 C NMR (75 MHz, CDCI3) 5 ppm: 156.5, 151.3, 143.6, 137.1, 135.7, 135.3, 128.8, 126.6, 126.2, 60.2, 53.5, 43.7, 28.6, 22.4.

[0149] IR cm 1: 3391, 2962, 1527, 1441, 1334, 1180, 1168, 1146, 1039, 1011, 979, 856, 848, 764, 628,589.

[0150] HRMS: calculated for [Ci4Hi8N2O2S+H]+ m / z=279.1167 found 279.1 163.

[0151] Prepared according to procedure B with a yield of 58%.

[0152] 1 H NMR (300 MHz, CDCh) 5 ppm: 7.97 (dd, J = 3.7; 0.9 Hz, 1 H); 7.94 (s, 1 H); 7.41 (dd, J = 5.1; 0.9 Hz, 1 H); 7.12 (dd, J= 5.1 Hz; 3.7 Hz, 1 H); 4.01 (s,3H); 2.57 (d, J= 7.1 Hz, 2H); 2.13 (m, 1 H); 0.95 (d, J=6.6 Hz, 6H).

[0153] 13 C NMR (75 MHz, CDCh) 5 ppm: 155.3, 151.4, 140.7, 135.4, 135.3, 128.2, 128.0, 127.8,

[0154] 53.5, 43.8, 28.8, 22.5

[0155] IR cm-1: 2954, 2868, 1526, 1451, 1370, 1356, 1341, 1274, 1200, 1180, 1028, 983, 851, 819, 703

[0156] HRMS: calculated for [Ci3Hi6N2S+H]+ m / z=249.1062 found 249.1049

[0157] Prepared according to procedure B with a yield of 64%.

[0158] 1 H NMR (300 MHz, CDCh) 5 ppm: 7.93 (s, 1 H); 7.78 (d, J=1.2 Hz, 1 H); 7.00 (d, J=1.2 Hz 1 H); 4.10 (s, 3H); 2.57 (d, J= 7.1 Hz, 2H); 2.31 (s, 3H) 2.15 (m, 1H); 0.95 (d, J=,6.6 Hz, 6H 13 C NMR (75 MHz, CDCh) 5 ppm: 155.3, 151.3, 140.4, 138.7, 135.5, 135.4, 130.6, 123.6, 53.6, 43.8, 28.8, 22.6, 16.1

[0159] IR cm 1 : 2954, 2927, 2867, 1526, 1475, 1452, 1371, 1361, 1349, 1193, 1180, 1028, 989, 945, 856, 741, 590

[0160] HRMS: calculated for [Ci4HisN2OS+H]+ m / z=263.1218 found 263.1224.

[0161] 5.3 General procedure C for the desulfurization of Ranev nickel:

[0162] In a flask were added the thiophene derivative (100 mg), 10 mL of ethanol and 1 g of Raney Nickel and put under atmospheric pressure of hydrogen (H2 flask). The reaction was stirred at 60 °C overnight. After cooling to room temperature, the reaction was filtered through a pad of silica gel (9 / 1 cyclohexane / AcOEt). The filtrate was concentrated to give the product with generally sufficient purity. If necessary, it can be purified by flash chromatography.

[0163] Prepared according to procedure C with a yield of 77%. 1 H NMR (300 MHz, CDCh) 5 ppm: 7.86 (s, 1 H) 3.92 (s, 3H), 3.10 (m, 1 H) 2.50 (d, J= 7.1 Hz, 2H), 2.10 (m, 1 H) 1.77 (m, 1 H) 1.56 (m, 1 H) 1.21 (d, J= 6.90 Hz, 2H); 0.93 (d, J=6.6 Hz, 6H) 0.83 (t, J= 7.4 Hz, 3H)

[0164] 13 C NMR (75 MHz, CDCh) 5 ppm: 157.5, 150.1, 148.2, 134.8, 53.1, 43.6, 36.0, 28.5, 28.2, 22.7, 22.5, 18.4.

[0165] IR cm-1: 2956, 2925, 2856, 1720, 1454, 1368, 1264, 1172, 1101, 1030, 911, 729 HRMS: calculated for [Ci3H22N2O]+ m / z=222.1732, found 222.1721.

[0166] Prepared according to procedure C with a yield of 94%.

[0167] 1 H NMR (300 MHz, CDCh) 5 ppm: 7.88 (s, 1 H); 7.25, (m, 5H); 3.95 (s,3H); 3.08 (m, 4H);

[0168] 2.55 (d, J= 7.2 Hz, 2H); 2.14 (m,1 H); 0.97 (d, J=6.6 Hz, 6H)

[0169] 13 C NMR (75 MHz, CDCh) 5 ppm: 157.8, 150.9, 143.5, 141.9, 134.8, 128.5, 128.3, 125.9, 53.2,

[0170] 43.6, 34.0, 33.6, 28.5, 22.5

[0171] IR cm 1 : 2956, 2925, 2856, 1720, 1454, 1368, 1264, 1172, 1 101 , 1030, 91 1 , 729 HRMS: calculated for [CI?H22N2O]+ m / z=270.1732, found 270.1727.

[0172] Prepared according to procedure C with a yield of 33%.

[0173] 1 H RMN (300 MHz, CDCh) 5 ppm : 7,84 (s, 1 H) ; 3,92 (s, 3H) ; 3,58 (m, 2H) ; 3,20 (m, 1 H) ; 2,49 (d, J= 7.1 Hz, 2H) ; 2.09 (m,1 H) ; 1.85 (m, 1 H) ; 1.44 (m, 1 H) ; 1.60 (m, 2H) ; 1.21 (d, J=6.8 Hz,3H) ; 0.92 (d, J=6.6 Hz, 6H)

[0174] 13C RMN (75 MHz, CDCh) 5 ppm : 157.3, 150.3, 147.6, 134.7, 62.8, 53.1 , 43.5, 34.0, 30.9, 30.6,28.4, 22.4, 19.0

[0175] IR cm-1 : 3364, 2955, 2869, 1540, 1447, 1373, 1351 , 1172, 1065, 1057, 1033, 586

[0176] HRMS : calculé pour [Ci4H24N2C>2+H]+ m / z=253.1916 trouvé 253.1911 .

[0177] Préparé selon la procédure C avec un rendement de 47%.

[0178] 1 H RMN (300 MHz, CDCh) 5 ppm :7.83 (s, 1 H) ; 7.20 (m, 6H) ; 4.44 (q, J= 7.1 Hz, 1 H) ; 3.92 (s,3H) ; 2.42 (d, J= 7.1 Hz, 2H) ; 2.01 (m,1 H) ; 1 .56 (d, J=7.1 Hz, 3H) ; 0.84 (d, J=6.6 Hz, 6H). 13C NMR (75 MHz, CDCh) 5 ppm: 157.2, 150.9, 146.2, 144.6, 134.9, 128.3, 127.9, 126.2, 53.3, 43.7, 40.1, 28.5, 22.5, 19.9

[0179] IR cm 1 : 2955, 2928, 2869, 1540, 1446, 1367, 1349, 1172, 1029, 698

[0180] HRMS: calculated for [CI?H22N2O+H]+ m / z=271 .1810, found 271 .1800.

[0181] Prepared according to procedure C with a yield of 63%.

[0182] 1 H NMR (300 MHz, CDCI3) 5 ppm: 7.81 (s, 1 H); 3.93 (s, 3H); 2.76 (d, J=7.6 Hz, 1 H); 2.73 (d, J=7.6 Hz, 1 H); 2.49 (d, J= 7.1 Hz, 2H); 2.09 (m, 1 H); 1.65 (m, 2H); 1.38 (m, 2H) 0.92 (d, J=6.6 Hz, 6H)

[0183] 13 C NMR (75 MHz, CDCI3) 5 ppm: 157.9, 150.5, 144.7, 134.8, 53.2, 43.6, 31.9, 29.8, 28.6, 22.7, 22.5, 14.0

[0184] IR cm 1 : 2956, 2928, 2870, 1542, 1450, 1370, 1171, 1065, 1032

[0185] Prepared according to procedure C with a yield of 91%.

[0186] 1 H NMR (300 MHz, CDCI3) 5 ppm: 7.79 (s, 1 H); 3.91 (s, 3H); 2.74 (d, J=7.6 Hz, 1 H); 2.70 (d, J=7.6 Hz, 1 H); 2.47 (d, J= 7.1 Hz, 2H); 2.06 (m,1 H); 1.56 (m, 3H); 0.90 (d, J=6.6 Hz, 12H)

[0187] 13 C NMR (75 MHz, CDCI3) 5 ppm: 157.8, 150.4, 144.9, 134.7, 53.1, 43.6, 36.6, 30.1, 28.5,

[0188] 28.2, 22.6, 22.4

[0189] IR cnr 1 : 2955, 2929, 2869, 1542, 1450, 1370, 1171, 1032

[0190] HRMS: calculated for [Ci4H24N2O+H]+ m / z=237.1976 found 237.1963.

[0191] Prepared according to procedure C with a yield of 86%.

[0192] 1 H NMR (300 MHz, CDCI3) 5 ppm: 7.85 (s, 1 H); 3.92 (s, 3H); 3.2 (m,1 H); 2.49 (d, J = 7.1 Hz, 2H); 2.10 (m,1 H); 1.70 (m, 1 H); 1.50 (m, 1 H); 1.20 (d, J=6.9 Hz, 3H); 0.92 (d, J=6.6 Hz, 6H); 0.87 (t, J=7.3 Hz, 3H)

[0193] 13C NMR (75 MHz, CDCI3) 5 ppm: 157.5, 150.2, 148.6, 135.0, 53.3, 43.8, 37.8, 34.2, 28.6, 22.6, 20.9, 19.0, 14.4

[0194] IR crrr 1 : 2956, 2929, 2870, 1540, 1447, 1372, 1350, 1172, 1077, 1032

[0195] HRMS: calculated for [Ci4H24N2O+H]+ m / z=237.1967 found 237.1960

[0196] 5.4 Peptide coupling synthesis procedure

[0197] The chiral molecules (S) of 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine and 3-methoxy-2-(2-methylpropyl)-5-(1-methylpropyl l)pyrazine, as well as 3-methoxy-2-(2-methylpropyl)-5-(2-methylpropyl)pyrazine were synthesized according to the present procedure based on the methodology for the synthesis of chiral pyrazynones according to Remya Ramesh, Michael T. Bovino, Yibin Zeng, Jeffrey Aubé, J. Org. Chem. 2019, 84, 3647-3651. This procedure is described below. In a round-bottom flask, a solution of DCC (dicyclohexylcarbodiimide) in DCM (dichloromethane) is added dropwise to a 0°C solution of amino acid (1 eq) and NHS (1 eq) in DCM. The reaction is stirred for 4 hours at room temperature. A solution of amino alcohol (1.2 eq) is added to the reaction mixture. The mixture is stirred overnight, then filtered and washed with water. The aqueous phase is extracted twice with DCM.The combined organic phases are washed with water, brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the dipeptide as a white solid. The crude product is purified on a silica gel column.

[0198] 1 H-NMR (300 MHz, CDCI3) 5 ppm: 0.86 (2H, d, J=7.4 Hz), 0.92 (10H, m), 1.43 (10H, s), 1.66 (3H, m), 2.98 (1 H, s), 3.66 (2H, q, J=4.0 Hz), 3.76 (1 H, m), 4.05 (1 H, q, J=7.2 Hz), 5.00 (1 H, s), 6.51 (1 H, d, J=7.9 Hz).

[0199] 13 C-NMR (75 MHz, CDCI3) 5 ppm: 11.3, 15.5, 22.1, 22.9, 24.8, 25.4, 28.3, 35.5, 40.6, 53.5, 56.1, 63.6, 80.4, 156.0, 173.2

[0200] HRMS: calculated for [Ci7H34N2O4+H]+ m / z= 331 .2597, found 331 .2589. 1H-NMR (300 MHz, CDCh) 5 ppm : 0.85 (11 H, m), 1.37 (9H, s), 1.57 (3H, m), 3.07 (1 H, s), 3.46 (1 H, q, J=5.4 Hz), 3.59 (1 H, d, J=5.5 Hz), 3.97 (2H, m), 4.99 (1 H, s), 6.38 (1 H, d, J=7.7 Hz).

[0201] 13 C-NMR (75 MHz, CDCI3) 5 ppm : 22.1 , 22.9, 23.1 , 24.8, 28.29, 40.0, 40.7, 50.1 , 53.4, 66.0, 80.3, 156.0, 173.1.

[0202] HRMS : calculé pour [Ci7H34N2O4+H]+ m / z= 331 .2597, trouvé 331 ,2586.

[0203] 5.4. 1 Procédure Générale de cyclisation :

[0204] In a round-bottom flask, dipeptide (1 eq), periodinane Dess Martin (1.5 eq), and DCM are added. The mixture is stirred at room temperature for 1.5 hours. Saturated Na2S2O3 and saturated NaHCO3 solutions are added and stirred vigorously to give two clear phases. The organic phase is separated, and the aqueous layer is extracted twice with DCM. The combined organic phases are dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give the crude material, which is directly used in the next step. Trifluoroacetic acid TFA (20 eq, based on the dipeptide) is added to the crude material in the DCM solution, and the mixture is stirred in air for 16 hours at room temperature.The mixture is diluted with DCM, washed with saturated NaCl, dried over magnesium sulfate, and concentrated under reduced pressure to give the crude pyrazinone, which is purified on a silica gel column (dichloromethane / ethyl acetate 9:1 v / v). The following molecules are obtained using this procedure. d, J=7.0 Hz), 1.70 (2H, m), 2.19 (1 H, m), 2.56 (1 H, q, =7.1 Hz), 2.65 (2H, d, J=7.1 Hz), 7.23 (1 H, s).

[0205] 13 C-NMR (75 MHz, CDCl3) 5 ppm: 11.9, 18.7, 22.7, 27.1, 28.5, 37.4, 41.4, 121.3, 143.2, 156.9, 158.6. HRMS: calculated for [Ci2H2iN2O+H]+ m / z=209.1654, found 209.1654 ppm: 0.97 (12H, m), 2.05 (1 H, m), 2.22 (1 H, m), 2.39 (2H, d, J=7.3 Hz), 2.65 (2H, d, J=7.1 Hz), 7.20 (1 H, s).

[0206] 13 C-NMR (75 MHz, CDCI3) 5 ppm: 22.2, 22.7, 27.0, 28.2, 39.5, 41.6, 123.2, 137.9, 156.8, 158.4.

[0207] HRMS: calculated for [CI2H2I N2O+H]+ m / z=209.1654, found 209.1657.

[0208] 5.4.3 General methylation procedure:

[0209] In a round-bottom flask, pyrazinone was dissolved in DCM and silica was added (0.04-0.063 mm 10 eq by mass). The mixture was cooled in an ice bath and TMSCHN2 was added (2 M 5 eq). The solution was left stirring for 30 minutes. Glacial acetic acid was added until the yellow color disappeared. The mixture was filtered through a silica pad, eluting with AcOEt. The organic phase was washed with water, dried over MgSC, filtered, and concentrated under reduced pressure to give the pyrazine. The crude product was purified on a silica column (Cyclohexane / dichloromethane 3 / 7). The following molecules are obtained according to this procedure. 155.2, 158.2. HRMS: calculated for [Ci3H 22 N2O+H]+ m / z=223.1810, found 223.1813. ppm : 0.92 (12H, m), 2.12 (2H, m), 2.51 (2H, d, J=7.1 Hz), 2.64 (2H, d, J=7.2 Hz), 3.93 (3H, s), 7.84 (1 H, s).

[0210] 13 C-NMR (75 MHz, CDCI3) 5 ppm : 21.4, 21.5, 26.5, 27.5, 39.9, 42.6, 52.1 , 133.7, 142.9,

[0211] 149.5, 157.1.

[0212] HRMS : calculé pour [Ci3H22N2O+H]+ m / z=223.1810, trouvé 223.1801 .

Claims

CLAIMS 1. Pyrazine derivative compound of formula (I), or a salt thereof, wherein R 1 represents a group selected from C1-C10 alkyl, C1-C2 alkyl substituted by phenyl, butyl, isobutyl, butyl substituted by methyl, and benzyl substituted by methyl, and R 2 represents an oxo or methoxy group, and R 3 represents a group selected from isobutyl and secbutyl.

2. Pyrazine-derived compound of formula (I) according to claim 1, chosen from one of the following molecules: -3-methoxy-2-(1-methylbenzyl)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(2-phenylethyl)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(butyl)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(3-methylbutyl)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(1-methylbutyl)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(1-methylbutan-4-ol)-5-(2-methylpropyl)pyrazine and -3-methoxy-2-(2-methylpropyl)-5-(2-methylpropyl)pyrazine.

3. Use of a pyrazine derivative compound of formula (I) or a salt thereof as a biocontrol agent, and preferably as a springtail attractant biocontrol agent, and for which: -R 1 represents a group selected from C1-C10 alkyl, C1-C2 alkyl optionally substituted by phenyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, butyl substituted by methyl, and benzyl substituted by methyl, R 2 represents an oxo group or a C1 to C4 alkyl group, preferably a methoxy, and R 3represents a hydrogen atom or a group selected from C1 to C10 alkyl, C1 to C2 alkyl optionally substituted by phenyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, butyl substituted by methyl, and benzyl substituted by methyl.

4. Use according to claim 3, in which the pyrazine-derived compound is chosen from the following molecules: -3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(1-methylbutyl)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(3-methylbutyl)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(1-methylbenzyl)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(2-phenylethyl)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(butyl)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(1-methylbutan-4-ol)-5-(2-methylpropyl)pyrazine, -3-methoxy-2-(2-methylpropyl)-5-(2-methylpropyl)pyrazine and -2-isopropyl-3-methoxypyrazine.

5. Use according to claim 3 of a pyrazine derivative compound of formula (I) in which R 1 and R 2 are distinct and chosen from a secbutyl and isobutyl group, or a racemic mixture, or an R, or S, enantiomer thereof.

6. Use according to one of claims 3 to 5, wherein said pyrazine-derived compound of formula I is used as a biocontrol agent attracting springtails, in particular the springtail species H.nitidus.

7. Use according to claim 6, wherein said springtails consume phytopathogenic fungi such as Zymoseptoria tritici and Fusarium graminearum.

8. Use according to one of claims 3 to 7, wherein said use is intended for the treatment or prevention of wheat septoria, and / or wheat fusarium wilt.

9. Use according to one of claims 3 to 8, comprising the diffusion of said pyrazine-derived compound of formula (I) in a growing ground, or the application of said pyrazine-derived compound of formula (I) to a crop or crop residues.

10. Composition comprising at least one compound according to formula (I) according to one of claims 1 to 2 or 3-methoxy-2-(1-methylpropyl)-5-(2-methylpropyl)pyrazine or mixtures thereof in the form of powder, in solution, in gel or in hydrogel.

11. Use of a composition according to claim 10 as an attractant for a biocontrol agent, such as springtails.

12. Device containing a composition according to claim 10, said device comprising a reservoir in which said composition is placed, and being capable of diffusing said composition outwards.

3. Use of 2-isopropyl-3-methoxypyrazine as a biocontrol agent in a method of treating or preventing septoria and / or fusarium wilt of wheat using springtails, in particular as an attractant for H. nitidus.

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