EP3229592A1 - Use of active culture of lactic acid bacteria for the preparation of a compound for capturing to control of drosophila suzukii and related compound - Google Patents

Use of active culture of lactic acid bacteria for the preparation of a compound for capturing to control of drosophila suzukii and related compound

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Publication number
EP3229592A1
EP3229592A1 EP15804575.7A EP15804575A EP3229592A1 EP 3229592 A1 EP3229592 A1 EP 3229592A1 EP 15804575 A EP15804575 A EP 15804575A EP 3229592 A1 EP3229592 A1 EP 3229592A1
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EP
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Prior art keywords
compound
lactic acid
acid bacteria
wine
use according
Prior art date
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Application number
EP15804575.7A
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German (de)
French (fr)
Inventor
Raffaele GUZZON
Gianfranco ANFORA
Alberto Grassi
Claudio IORIATTI
Giuseppe MADDALENA
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Fondazione Edmund Mach
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Fondazione Edmund Mach
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom

Definitions

  • a scope of the invention is to propose a new, effective, economical, and long lasting preparation alternative to the treatments based on chemical pesticides for controlling Drosophila suzukii.
  • This scope is achieved by using a culture of lactic acid bacteria, belonging to the Oenocccus oeni species, and put in a condition to warranty the production of metabolites strongly attractive for Drosophila suzukii for a long period of time, thanks to the original composition of the attractive blend contained in a trap.
  • Drosophila suzukii is an insect capable of causing substantial damage in different high-value arboreal, frutescent and vegetable cultures, including soft fruits, drupaceous fruits, and grape. Aggressiveness and adaptability to numerous contexts make it one of the major adversities in the agricultural field, capable of causing step-downs of production even greater than 90%. At present, the struggle against this insect is performed through the use of insecticides, massive capture traps, and other agronomic practices. None of these solutions led to a resolution of the problem.
  • the experimental data clearly shows that Drosophila melanogaster is more attracted by substrates being in fermentation, thanks to the presence of the Saccharomyces cerevisiae yeast, as compared to what observed in tests where the same, non-yeasting substrates were present.
  • Drosophila suzukii Little information is present in the literature on the chemical ecology of Drosophila suzukii. With respect to Drosophila melanogaster and to all remaining species of the same genus, Drosophila suzukii has a peculiarity in that it lays eggs on unwounded and healthy fruits, whereas it is attracted by marcescent and fermenting substrates for feeding purposes (Cini et al., 2012). Consequently, the knowledge acquired on the Drosophila melanogaster model insect and on other closely- related species cannot provide an exhaustive information for understanding the behavior of Drosophila suzukii.
  • the most effective traps for capturing Drosophila suzukii are those baited with different types of vinegar and wine (Landolt et al., 2012). Both these substrates result from fermentation, mediated both by yeast and by acetic bacteria, of carbonaceous compounds, mainly hexose sugars present in the raw material (McKenzie and Parsons, 1972; Becher et al., 2010, 2012; Barata et al., 2012). Other secondary compounds are also apparently relevant in attracting these insects, which justifies the greater effectiveness observed in the case of using food matrices like vinegar and wine to bait traps, with respect to that of pure compounds, for instance acetic acid and ethanol.
  • traps are marketed containing wine vinegar, apple vinegar, wine, fruit juices (Grassi et al., 2014).
  • such matrices are sterilized via pasteurization during the production step.
  • the traps known so far are not sufficiently active against Drosophila suzukii and feature a short duration in time. In order for the control strategies to provide acceptable results, it is therefore necessary to use a high number of traps per hectare, which can be estimated to amount to different thousands. Furthermore, the traps known so far require weekly service of the bait. The high number of traps and the complex operativity necessary for their maintenance make the current solution ineffective and unsustainable from the economical point of view.
  • the present invention refers to a bait aimed at monitoring and controlling Drosophila suzukii, which comprises the use of an active culture of lactic acid bacteria, preferably of the Oenococcus oeni species, preferably of the type used in oenology.
  • the concentration of the lactic acid bacteria equals at least 10 8 cells/ g and the mixture also comprises at least apple cider vinegar or wine vinegar or wine or fruit juices taken individually or added with at least one organic acid, sugar, preferably a fermentable one, and a fermentation activator, or with all of the three additives, namely organic acid, preferably fermentable sugar, and a fermentation activator.
  • Drosophila suzukii is an invasive species capable of causing major damages to cultivated fruits. For this reason it is presently managed by using chemical insecticides with big risks of damages to the environment, to the operator's health and to the population who stands on the cultivation areas of the cultures infected by Drosophila suzukii.
  • chemical insecticides with big risks of damages to the environment, to the operator's health and to the population who stands on the cultivation areas of the cultures infected by Drosophila suzukii.
  • mass trapping of the population of insects by means of specific traps.
  • so far traps did not demonstrate to be capable of solving the problem, as it appears from the major losses of production recorded even in the presence of these devices, because an adequately attractive and long-lasting bait has not been identified yet.
  • Studies carried out by the applicant demonstrate that Drosophila suzukii is particularly sensitive to some molecules like acetoin and diacetyl.
  • Oenococcus oeni a species of lactic acid bacteria exclusively isolated in oenological environments like wine in malolactic fermentation (Liu, 2002).
  • lactic acid bacteria belonging to the Oenococcus oeni species which is the scope of the present invention, in traps baited with properly modified mixes of vinegar and wine, makes it possible very effective massive captures, capable of representing an alternative to chemical treatments.
  • Oenococcus oeni is a species of lactic acid bacteria of a typically oenological origin (Versari, 1999). Such species is the only one belonging to the Oenocccus genus and its presence has been exclusively described in an oenological environment, in particular in wines during the malolactic fermentation. The very restricted environmental allocation of the species under consideration, as well as its poor intraspecific variability, is due to some of its physiological characteristics. In particular, Oenococcus oeni exhibits a poor vigor and a very low growth rate, thereby being rapidly overtaken in the colonization of the common fermentable substrate, including sugary juices of vegetal origins, by other species of bacteria or yeasts.
  • Oenococcus oeni exhibits an excellent resistance to low pH values and to the presence of ethanol in the environment, thereby resulting the dominant species in wine at the end of alcoholic fermentation, wherein it finds the organic acids, among which in particular the malic acid, as a primary feeding source (Liu, 2002).
  • Its characteristic metabolism consists of the decarboxylation of the malic acid into lactic acid, from which Oenococcus oeni is capable of taking energy thanks to the protonic gradient that is thus generated; it also produces secondary matabolites, including acetoin, diacetyl, and acetaldehyde (Liu, 2002).
  • Oenococcus oeni is also capable of warranting an increased attractive capacity of the conventional traps already in use, as demonstrated by experiments carried out by the applicant.
  • the applicant experimentally ascertained that adding Oenococcus oeni to the common attractants present in the traps for Drosophila suzukii induces a higher number of captures than that observed with traps baited with other microorganisms or other attractive formulations; such difference can be attributed, according to the present references and to experiments carried out by the applicant, to the production by Oenococcus oeni of volatile molecules that are attractive to Drosophila suzukii, for instance acetoin or diacetyl, significantly higher with respect to other species of microorganisms.
  • Droskidrink consisting of apple cider vinegar, wine vinegar, wine, fruit juices taken individually or mixed together
  • Oenococcus oeni do not enable Oenococcus oeni to express its metabolic activity at the best.
  • Droskidrink consisting of apple cider vinegar, wine vinegar, wine, fruit juices taken individually or mixed together
  • Oenococcus oeni do not enable Oenococcus oeni to express its metabolic activity at the best.
  • a better result is achieved with completely new modifications in the composition of the attractant, consisting of adding to a 3: 1 mix of apple cider vinegar and red wine of exogenous components like organic acids, for instance citric acid and L-malic acid, cane sugar, and nutritional factors including nitrogenous sources and vitamins.
  • the acidity of the mix has also been corrected, by adjusting its pH to a value ranging from 3.5 to 5 in order to warrant a maximum efficiency, in energy terms, of the decarboxylation reaction of the malic acid.
  • Such liquid bait is stabilized via a sterile filtration and is not thermally treated because it has been possible to experimentally demonstrate that the attractiveness of baits containing non thermally-treated substrates is greater, probably thanks to the preservation of the compounds with high biological activity, but thermolabile, secondary constituents, for instance vitamins.
  • the use of an immobilized bacterial culture makes it possible to recover the biomass in the periodical service and/ or replacement of the attractive substrate, making it possible its use in subsequent batches with surprising increases in the activity period of the trap. Note that the current time of use of the commercial traps is as low as one week.
  • Figure 1 graphically represents a gaschromatography recording coupled with an electroantennography (GC-EAD) carried out by the applicant.
  • GC-EAD electroantennography
  • this preparation emits high quantities of diacetyl (the arrow indicates the peak of diacetyl at approximately lOmin of retention time) and of acetoin (the arrow indicates the peak of acetoin at approximately 13min of retention time). Both compounds induce significant electroantennographic responses (their corresponding arrows on the bottom side).
  • Droskidrink is normally formed of a mix of apple cider vinegar and red wine in a proportion 3: 1 with the addition of 4 gr of raw cane sugar.
  • the pH of the commercial Droskidrink is approximately 3.
  • Figure 2 shows the average number of captures of Drosophila suzukii by means of traps (no. 3/ theses 4/ weeks 5) by using an attractive mix inoculated with Oenococcus oeni, the result of the previously described test.
  • the values are represented in a logarithmic scale. Letters that are different for every week of checks indicate significant differences (ANOVA, Levene test; LSD test, PO.05).
  • the traps baited with a Droskidrink inoculated with a culture of O. oeni are always significantly more attractive than the remaining theses containing Droskidrink only in different conditions. Also, the presence of the bacteric culture in active growth made it possible to keep the trap effective for all the duration of the test without the need for completely replacing the liquid mix but just weekly refilling the contents lost by evaporation with new Droskidrink.
  • Figure 3 shows the results of a test carried out by the applicant similar to that previously described wherein different species of lactic acid bacteria, in particular of the Pediococcus and Lactobacillus genera, have been compared to each other.
  • the diagram shows the total number of captures during the 5 weeks of test of such species as compared to Oenococcus oeni, which results to always be significantly more attractive to Drosophila suzukii.

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  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Agronomy & Crop Science (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

Use of lactic acid bacteria belonging to the Oenococcus oeni species for preparing a attracting compound scilicet bait to be put in traps aimed at monitoring and controlling Drosophila suzukii.

Description

USE OF ACTIVE CULTURE OF LACTIC ACID BACTERIA FOR THE PREPARATION OF A COMPOUND FOR CAPTURING TO CONTROL OF DROSOPHILA SUZUKII AND RELATED COMPOUND
OBJECT OF THE INVENTION
The use of lactic acid bacteria, belonging to the Oenococcus oeni species, for preparing a capturing compound scilicet bait for monitoring and controlling Drosophila suzukii and its respective compound to be put in appropriate traps
SCOPE OF THE INVENTION
A scope of the invention is to propose a new, effective, economical, and long lasting preparation alternative to the treatments based on chemical pesticides for controlling Drosophila suzukii. This scope is achieved by using a culture of lactic acid bacteria, belonging to the Oenocccus oeni species, and put in a condition to warranty the production of metabolites strongly attractive for Drosophila suzukii for a long period of time, thanks to the original composition of the attractive blend contained in a trap.
BACKGROUND OF THE INVENTION
Drosophila suzukii is an insect capable of causing substantial damage in different high-value arboreal, frutescent and vegetable cultures, including soft fruits, drupaceous fruits, and grape. Aggressiveness and adaptability to numerous contexts make it one of the major adversities in the agricultural field, capable of causing step-downs of production even greater than 90%. At present, the struggle against this insect is performed through the use of insecticides, massive capture traps, and other agronomic practices. None of these solutions led to a resolution of the problem.
The researches published so far highlighted that the information concerning fundamental aspects for survival of insects, including the research for nutritional resources, escape from enemies, reproduction, and identification of the oviposition sites are mainly mediated by volatile chemical compounds perceived by olfaction (Schoonhoven et al, 1998; Dicke et van Loon, 2000; Bruce et al. 2005; Hilker et McNeil, 2007).
Numerous studies demonstrated the importance of the volatile compounds, produced by the host plant and by other organic substrates, in helping insects in identifying the foods and the oviposition site (Bruce et al, 2005. Bruce et Pickett, 201 1).
More recently for the Drosophila genus, some studies clarified the contribution given by the microbic component present in the substrates commonly frequented by the insects of this group, in producing chemical compounds capable of influencing their behavior. For instance, Becher et al. (2012) observed that such microbic component strongly influences Drosophila melanogaster in searching oviposition and food sites and that this insect features better performances, in terms of reproduction, whenever the substrate is contaminated by yeasts.
The experimental data clearly shows that Drosophila melanogaster is more attracted by substrates being in fermentation, thanks to the presence of the Saccharomyces cerevisiae yeast, as compared to what observed in tests where the same, non-yeasting substrates were present.
Little information is present in the literature on the chemical ecology of Drosophila suzukii. With respect to Drosophila melanogaster and to all remaining species of the same genus, Drosophila suzukii has a peculiarity in that it lays eggs on unwounded and healthy fruits, whereas it is attracted by marcescent and fermenting substrates for feeding purposes (Cini et al., 2012). Consequently, the knowledge acquired on the Drosophila melanogaster model insect and on other closely- related species cannot provide an exhaustive information for understanding the behavior of Drosophila suzukii. Preliminary experimental observations confirm that this insect too is attracted, in terms of nutritional sources, by wounded and presumably fermenting fruits, as suggested by the attraction exerted onto Drosophila suzukii by a variety of fermented liquids, including wine, vinegar, and liquid derivatives of fruits (Kanzawa, 1934; Landolt et al., 2012; Cha et al., 2012, 2013, 2014) and by the association, recently reported, of this insect with a community of yeast species basically formed of Hanseniaspora uvarum (Hamby et al., 2012).
A further confirmation of the interactions between Drosophila suzukii and microorganisms was found in capture tests, wherein baits inoculated with Saccharomyces cerevisiae gave improved results as referred to sterile baits (Knight et al., 2013). Conversely, no evidences have been published about the positive interactions, in terms of a greater attractiveness, of the insect with bacteria originating from the ambient or involved in the fermentation processes that is the base of the attractive mix commonly used in traps. However, tests carried out by the applicant demonstrate that the attractive capacity exerted by matrices activated with lactic acid bacteria belonging to species not commonly isolated in traps or in the agricultural context where these are located, is significantly higher than that observed by the mentioned species of yeast, the conditions being equal.
At present, the most effective traps for capturing Drosophila suzukii are those baited with different types of vinegar and wine (Landolt et al., 2012). Both these substrates result from fermentation, mediated both by yeast and by acetic bacteria, of carbonaceous compounds, mainly hexose sugars present in the raw material (McKenzie and Parsons, 1972; Becher et al., 2010, 2012; Barata et al., 2012). Other secondary compounds are also apparently relevant in attracting these insects, which justifies the greater effectiveness observed in the case of using food matrices like vinegar and wine to bait traps, with respect to that of pure compounds, for instance acetic acid and ethanol.
Among these secondary compounds it is worth mentioning, according to the present references, acetyl methyl carbinol, butyrate, 2-phenyletanol, present both in wine and in vinegar, methionol, isoamyl lactate, and diethyl succinate present in wine. All of these compounds are produced by the fermentation of yeasts and/ or bacteria (Cordente et al., 2012; Nielsen and Richelieu, 1999), which confirms the importance of the microflora in forming a set of volatile molecules attractive to Drosophila suzukii.
According to this knowledge, traps are marketed containing wine vinegar, apple vinegar, wine, fruit juices (Grassi et al., 2014). In order to warrant stability and marketability, such matrices are sterilized via pasteurization during the production step.
Tests carried out by the applicant demonstrated that the liquid matrices used so far in traps feature a limited activity, are not capable of warranting a sufficient effectiveness in the control strategies (mass trapping, attract and kill) and have a rather low duration in time, in the order of one week.
In summary, the traps known so far are not sufficiently active against Drosophila suzukii and feature a short duration in time. In order for the control strategies to provide acceptable results, it is therefore necessary to use a high number of traps per hectare, which can be estimated to amount to different thousands. Furthermore, the traps known so far require weekly service of the bait. The high number of traps and the complex operativity necessary for their maintenance make the current solution ineffective and unsustainable from the economical point of view. In summary, the present invention refers to a bait aimed at monitoring and controlling Drosophila suzukii, which comprises the use of an active culture of lactic acid bacteria, preferably of the Oenococcus oeni species, preferably of the type used in oenology. In a preferred preparation the concentration of the lactic acid bacteria equals at least 108 cells/ g and the mixture also comprises at least apple cider vinegar or wine vinegar or wine or fruit juices taken individually or added with at least one organic acid, sugar, preferably a fermentable one, and a fermentation activator, or with all of the three additives, namely organic acid, preferably fermentable sugar, and a fermentation activator.
The pH of the preparation ranges from 3.5 to 5 and is cold sterilized, preferably by filtration, before the first use.
According to an even more preferable preparation, the active culture of lactic acid bacteria is included in an organic matrix, preferably formed of an organic polymer of an alginate stabilized by bond to an element in a cationic form. Such containment form of microorganisms makes it possible to optimize their functionality, meant as a biosynthetic activity and a resistance to environmental stresses, and to warrant a use thereof extended in time as compared to the devices currently in use (Guzzon et al., 2012; Avnir et al., 2006).
The scientific works mentioned in the present document are listed below for the sake of completeness.
Avnir D., Coradin T., Lev O., Livage J., 2006. Recent bio- applications of sol-gel materials. Journal of Materials Chemistry. 16: 1013- 1030.
Barata A., Malfeito-Ferreira M., Loureiro V., 2012. The microbial ecology of wine grape berries. International Journal of Food Microbiology 153: 243-259.
Becher P.G., Flick G., Rozpedowska E., Schmidt A., Hagman A., Lebreton S., Larsson M.C., Hansson B.S., Piskur J., Witzgall
P., Bengtsson M., 2012. Yeast, not fruit volatiles mediate Drosophila melanogaster attraction, oviposition and development. Functional Ecology 26: 822-828.
Becher P.G., Bengtsson M., Hansson B.S., Witzgall P., 2010.
Flying the Fly: Long-range Flight Behavior of Drosophila melanogaster to Attractive Odors. Journal of Chemical Ecology 6: 599-607. Bruce T.J. A., Pickett J. A., 201 1. Perception of plant volatile blends by herbivorous insects — Finding the right mix. Phytochemistry 72: 1605-161 1.
Bruce T.J.A., Wadhams L.J., Woodcock CM., 2005. Insect host location: a volatile situation. Trends in Plant Science 10, 269-74.
Cha D., Adams T., Rogg H., Landolt P.J., 2012. Identification and field evaluation of fermentation volatiles from wine and vinegar that mediate attraction of spotted wing Drosophila, Drosophila suzukii. Journal of Chemical Ecology 38, 1419- 1431.
Cha D.H., Hesler S.P., Cowles R.S., Vogt H., Loeb G.M., Landolt P.J., 2013. Comparison of a synthetic chemical lure and standard fermented baits for trapping Drosophila suzukii (Diptera: Drosophilidae). Environmental Entomology 42: 1052-1060.
Cha D.H., Adams T., Werle C.T., Sampson B.J., Adamczyk Jr.
J.J., Rogg H., Landolt P.J., 2014. A four-component synthetic attractant for Drosophila suzukii (Diptera: Drosophilidae) isolated from fermented bait headspace. Pest Management Science 70: 324-331 Cini A., Ioriatti C, Anfora G., 2012. A review of the invasion of Drosophila suzukii in Europe and a draft research agenda for integrated pest management. Bulletin of Insectology 65: 149- 160. Cordente A., Curtin C, Varela C, Pretorius I., 2012. Flavour- active wine yeasts. Applied microbiology and biotechnology 96:3 601-618.
Dicke M., Van Loon J.J.A., 2000. Multitrophic effects of herbivore-induced plant volatiles in an evolutionary context. Entomologia Experimentalis et Applicata 97:237-249.
Grassi A., Anfora G., Maistri S., Maddalena G., De Cristofaro A., Savini G., Ioriatti C, 2014. Development and efficacy of Droskidrink, a food bait for trapping Drosophila suzukii. Book of Abstract VIII Workshop on Integrated Soft Fruit Production, IOBC WG "Integrated Protection of Fruit Crops",
Subgroup "Soft Fruits", Vigalzano di Pergine Valsugana (Trento), 26-28 May 2014, pages 105- 106.
Guzzon R., Carturan G., Krieger-Weber S., Cavazza A. 2012. Use of organo-silica immobilized bacteria produced in a pilot scale plant to induce malolactic fermentation in wines that contain lysozyme. Annals of Microbiology. 62: 381-390. Hamby K.A., Hernandez A., Boundy-Mills K., Zalom F.G., 2012. Associations of yeasts with spotted-wing drosophila (Drosophila suzukii; Diptera: Drosophilidae) in cherries and raspberries. Applied Environmental Microbiology 78: 4869- 4873.
Hilker M., McNeil J., 2007. Chemical and behavioral ecology in insect parasitoids: How to behave optimally in a complex odorous environment? Chapter 5 in Wajnberg E, Bernstein C, van Alphen J, eds. Behavioral Ecology of Insect Parasitoids: From Theoretical Approaches to Field Applications. Oxford (United Kingdom): Blackwell.
Landolt, P.J., Adams, T. and Rogg, H., 2012. Trapping spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), with combinations of vinegar and wine, and acetic acid and ethanol. Journal of Applied Entomology 136: 148- 154.
Liu S.Q., 2002. Malolactic fermentation in wine - beyond deacidification. Journal of Applied Microbiology. 92: 589- 601.
Nielsen J.C. and Richelieu M., 1999. Control of flavor development in wine during and after malolactic fermentation by Oenococcus oeni. Applied and Environmental Microbiology. Vol. 65, 740-745. Kanzawa T., 1934. Research into the fruit fly Drosophila suzukii Matsumura.- Yamanashi Prefecture Agricultural Experiment Station Report, October 1934, 48.
Knight A., Yee W., Hilton R., 2013. Developing a new bait for spotted-wing drosophila in organic cherry production.
Proceedings of Ilnd International Organic Fruit Symposium. Acta Horticulturae 1001: 147- 152.
McKenzie J.A., Parsons P.A., 1972. Alcohol tolerance: an ecological parameter in the relative success of Drosophila melanogaster and Drosophila simulans. Oecologia 10 : 373-
388.
Schoonhoven L.M., Jermy T., Van Loon J.J.A, 1998. Insect-plant biology, from physiology to evolution. Chapman & Hall, London, UK. Versari A., Parpinello G.P., Cattaneo M., 1999. Leuconostoc oenos and malolactic fermentation in wine: a review. Journal of Industrial Microbiology & Biotechnology 23, 447-455.
DISCLOSURE OF THE INVENTION
Drosophila suzukii is an invasive species capable of causing major damages to cultivated fruits. For this reason it is presently managed by using chemical insecticides with big risks of damages to the environment, to the operator's health and to the population who stands on the cultivation areas of the cultures infected by Drosophila suzukii. Among the few existing alternatives there is the mass trapping of the population of insects by means of specific traps. However, so far traps did not demonstrate to be capable of solving the problem, as it appears from the major losses of production recorded even in the presence of these devices, because an adequately attractive and long-lasting bait has not been identified yet. Studies carried out by the applicant demonstrate that Drosophila suzukii is particularly sensitive to some molecules like acetoin and diacetyl. Such molecules are not naturally present in significant quantities in the attractive mixes presently used, however they can be produced in high quantities by Oenococcus oeni, a species of lactic acid bacteria exclusively isolated in oenological environments like wine in malolactic fermentation (Liu, 2002). The addition of lactic acid bacteria belonging to the Oenococcus oeni species, which is the scope of the present invention, in traps baited with properly modified mixes of vinegar and wine, makes it possible very effective massive captures, capable of representing an alternative to chemical treatments.
Oenococcus oeni is a species of lactic acid bacteria of a typically oenological origin (Versari, 1999). Such species is the only one belonging to the Oenocccus genus and its presence has been exclusively described in an oenological environment, in particular in wines during the malolactic fermentation. The very restricted environmental allocation of the species under consideration, as well as its poor intraspecific variability, is due to some of its physiological characteristics. In particular, Oenococcus oeni exhibits a poor vigor and a very low growth rate, thereby being rapidly overtaken in the colonization of the common fermentable substrate, including sugary juices of vegetal origins, by other species of bacteria or yeasts. Contrary to other species Oenococcus oeni exhibits an excellent resistance to low pH values and to the presence of ethanol in the environment, thereby resulting the dominant species in wine at the end of alcoholic fermentation, wherein it finds the organic acids, among which in particular the malic acid, as a primary feeding source (Liu, 2002). Its characteristic metabolism consists of the decarboxylation of the malic acid into lactic acid, from which Oenococcus oeni is capable of taking energy thanks to the protonic gradient that is thus generated; it also produces secondary matabolites, including acetoin, diacetyl, and acetaldehyde (Liu, 2002). Such peculiar deacidification activity has been considered so far of an exclusively oenological interest, in that the action usually exerted by lactic acid bacteria in food matrices entails an acidification of the means through a consumption of sugars (e.g. dairy productions) and production of acid. The disadvantages of the known traps are therefore solved by the present invention. It is based on the original use of Oenococcus oeni in capturing Drosophila suzukii thanks to the surprising increase in attractiveness of the traps that this microorganism, added to bait, causes. Such application is totally surprising with respect to the current and one context of use of this microbial species, i.e. malolactic fermentation into wine (Liu, 2002).
Oenococcus oeni is also capable of warranting an increased attractive capacity of the conventional traps already in use, as demonstrated by experiments carried out by the applicant. The applicant experimentally ascertained that adding Oenococcus oeni to the common attractants present in the traps for Drosophila suzukii induces a higher number of captures than that observed with traps baited with other microorganisms or other attractive formulations; such difference can be attributed, according to the present references and to experiments carried out by the applicant, to the production by Oenococcus oeni of volatile molecules that are attractive to Drosophila suzukii, for instance acetoin or diacetyl, significantly higher with respect to other species of microorganisms. On the contrary, other experiments performed by the applicant demonstrated a non statistically significant intraspecific variability, i.e. internally to the Oenococcus oeni species, with respect to the attractive activity toward Drosophila suzukii. Such poor variability is reasonably to be attributed to the fact that the main differences highlighted between strains belonging to this species are in charge to genes encoding resistances to environmental stress factors and do not concern the energetic metabolisms essential to life of cells, which the production of the mentioned attractive molecules is related to.
An increased capability of traps in terms of attractiveness, thanks to the addition of Oenococcus oeni, is already evident by using traps already available on the market.
The applicant observed that the common attractants, especially that most commonly used in the Province of Trento, called Droskidrink, consisting of apple cider vinegar, wine vinegar, wine, fruit juices taken individually or mixed together, do not enable Oenococcus oeni to express its metabolic activity at the best. Conversely, a better result is achieved with completely new modifications in the composition of the attractant, consisting of adding to a 3: 1 mix of apple cider vinegar and red wine of exogenous components like organic acids, for instance citric acid and L-malic acid, cane sugar, and nutritional factors including nitrogenous sources and vitamins. The acidity of the mix has also been corrected, by adjusting its pH to a value ranging from 3.5 to 5 in order to warrant a maximum efficiency, in energy terms, of the decarboxylation reaction of the malic acid. Such liquid bait is stabilized via a sterile filtration and is not thermally treated because it has been possible to experimentally demonstrate that the attractiveness of baits containing non thermally-treated substrates is greater, probably thanks to the preservation of the compounds with high biological activity, but thermolabile, secondary constituents, for instance vitamins. Also, the use of an immobilized bacterial culture makes it possible to recover the biomass in the periodical service and/ or replacement of the attractive substrate, making it possible its use in subsequent batches with surprising increases in the activity period of the trap. Note that the current time of use of the commercial traps is as low as one week.
EXPERIMENTAL SECTION
Figure 1 graphically represents a gaschromatography recording coupled with an electroantennography (GC-EAD) carried out by the applicant. By this technique, the individual compounds eluted by the gaschromatograph reach the antenna of an insect specifically prepared in an electroantennograph. The substances perceived by the antenna of an insect induce an electrical response which is displayed as a depolarization. In this recording a solution of volatile compounds collected from a sample of Droskidrink added with a culture of Oenococcus oeni (red line on the upper part) has been injected into a gaschromatograph and coupled with an antenna of a female Drosophila suzukii (black line on the bottom side). As compared to the tests carried out in the absence of lactic acid bacteria, this preparation emits high quantities of diacetyl (the arrow indicates the peak of diacetyl at approximately lOmin of retention time) and of acetoin (the arrow indicates the peak of acetoin at approximately 13min of retention time). Both compounds induce significant electroantennographic responses (their corresponding arrows on the bottom side).
The applicant performed a field test in an applicant's experimental vineyard of the Teroldego variety, simple pergola trailing system and located in the municipality of San Michele all'Adige (Trento) to evaluate the attractive effectiveness to Drosophila suzukii of a bacterial culture of Oenococcus oeni combined with the Droskidrink commercial product, i.e. the commercial attractant commonly used in the province of Trento and in other fruit growing regions. Droskidrink is normally formed of a mix of apple cider vinegar and red wine in a proportion 3: 1 with the addition of 4 gr of raw cane sugar. The pH of the commercial Droskidrink is approximately 3.
Bottles provided with side holes filled with 200 ml of liquid attractant have been used in the test.
The following theses have been evaluated:
1) a Droskidrink whose pH had been increased up to a value of approximately 4 through the addition of KOH (such pH value makes the growth of lactic acid bacteria easier) added with approximately 3.5 ml of a culture medium with O. oeni.
2) a Droskidrink featuring a pH of approximately 4 3) an unmodified Droskidrink featuring a pH of approximately 3 (commercial Droskidrink)
4) a commercial Droskidrink added with 1 gr of tetracycline (an antibiotic) .
Every thesis has been repeated 3 times on different rows in a randomized block. Service of traps has been made weekly and the number of D. suzukii captures are expressed as an average of the 3 traps, account being taken of the summation of males and females of the insect.
The traps baited with O. oeni at the moment of the weekly control have been filtered from the captured insects and refilled with a quantity of Droskidrink up to reaching the quantity of 200 ml again. Conversely, the traps of the theses containing Droskidrink only, according to the commercial protocol, have been completely emptied and baited again with a fresh attractant.
The results have been analyzed via a Levene test to check the standard distribution of the values. Analysis of variance (ANOVA) and LSD test have been performed and plotted in the following figures.
Figure 2 shows the average number of captures of Drosophila suzukii by means of traps (no. 3/ theses 4/ weeks 5) by using an attractive mix inoculated with Oenococcus oeni, the result of the previously described test. The values are represented in a logarithmic scale. Letters that are different for every week of checks indicate significant differences (ANOVA, Levene test; LSD test, PO.05).
The traps baited with a Droskidrink inoculated with a culture of O. oeni are always significantly more attractive than the remaining theses containing Droskidrink only in different conditions. Also, the presence of the bacteric culture in active growth made it possible to keep the trap effective for all the duration of the test without the need for completely replacing the liquid mix but just weekly refilling the contents lost by evaporation with new Droskidrink.
Figure 3 shows the results of a test carried out by the applicant similar to that previously described wherein different species of lactic acid bacteria, in particular of the Pediococcus and Lactobacillus genera, have been compared to each other. The diagram shows the total number of captures during the 5 weeks of test of such species as compared to Oenococcus oeni, which results to always be significantly more attractive to Drosophila suzukii.

Claims

1 A use of an active culture of lactic acid bacteria (6) for preparing a capturing compound scilicet bait aimed at monitoring and controlling Drosophila suzukii.
2 A use according to claim 1, wherein the active culture of the lactic acid bacteria is of the type used in oenology. 3 A use according to claim 1, wherein the lactic acid bacteria
(6) of the compound belong to the Oenococcus oeni species.
4 A use according to claim 1, wherein the concentration of the lactic acid bacteria in the compound is of at least 108 ufc/g.
5 A use according to claim 1, wherein the active culture of lactic acid bacteria is included in an organic matrix.
6 A use according to claim 5, wherein the organic matrix is formed of an organic polymer.
7 A use according to claim 1, wherein said compound comprises said lactic acid bacteria in association with at least one of the following liquids: vinegar, wine, fruit juices. 8 A use according to claim 1, wherein said compound comprises said lactic acid bacteria in association with at least one of the following liquids: fruit vinegar, wine vinegar, wine, fruit juices, and added with at least one of the following elements: an organic acid, a fermentable sugar, a fermentation activator.
9 A use according to claim 5, wherein said compound is cold stabilized. 10 A use according to claim 5, wherein said compound is cold stabilized by filtration.
11 An attracting compound scilicet bait aimed at monitoring and controlling Drosophila suzukii, characterized in that it comprises an active culture of lactic acid bacteria belonging to the Oenococcus oeni species associated with at least one of the following liquids: fruit vinegar, wine vinegar, wine, fruit juices.
12 A compound according to claim 11, characterized in that the compound is added with at least one of the following elements: organic acid, sugar, fermentation activator.
13 A compound according to claim 11, characterized in that the compound is added with an organic acid and a sugar, and a fermentation activator. 14 A compound according to claim 10, characterized compound features a pH ranging from 3.5 to 5.
EP15804575.7A 2014-11-06 2015-11-05 Use of active culture of lactic acid bacteria for the preparation of a compound for capturing to control of drosophila suzukii and related compound Withdrawn EP3229592A1 (en)

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