FRUIT FLY ATTRACTANTS AND BAITS

Abstract: A bait for fruit fly comprising live bacteria of at least one species of the family Enterobacteriacae on which fruit fly have been found to feed. The bait includes a substrate which is able to support the growth of the bacteria, and an agent toxic to the fruit fly, such as a pesticide. Preferred attractant bacteria species are Erwinia herbicola, Citrobacter freundii, Citrobacter diversus, Providencia rettgeri, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus vulgari, Serratia liquefaciens. Fruit fly attractants are also disclosed comprising the above bacteria and a substrate, without the toxic agent. These attractants are primarily used in fruit fly traps. Also disclosed are baits and attractant compositions including volatile compounds which are produced by the bacteria, and which serve as attractants to fruit fly. Preferred attractant compounds are 2-butanone, phenylacetate, butan-1-ol and isopentanol. Storage-stable pre-mixes are also described.

Description: "FRUIT FLY ATTRACTANTS AND BAITS" TECHNICAL FIELD The present invention concerns attractant traps, lures and baits for fruit flies belonging to the family Tephritidae. The invention also relates to methods for attracting and controlling these insects. BACKGROUND ART The terms "bait" and "lure" are generally used in the art to indicate similar things. These terms are often used interchangeably in the art, and do not appear to have a precisely defined meaning. In many cases, a "bait" comprises an attractant material, especially a food substance, and usually includes a pesticide. A "lure" may or may not include a pesticide, and often indicates that a sexual phero one is used as the insect attractant. However, as these terms are not used consistently in the literature, in this specification, the term "bait" is assumed to include the term "iure" , and the presence or absence of a pesticide is specifically indicated. The present invention is directed to attractant lures and baits for insects of the family Tephritidae, namely fruit flies. As used in this specification, the term "fruit flies" is used to indicate all flies belonging to the family Tephritidae (Diptera) , such as Daeus dorsalis (Oriental fruit fly) , Daeus tryoni (Queensland fruit fly) , Daeus cucurbitae (Melon Fly) , Ceratitis capitata (Mediterranean fruit fly) , and fruit flies of the genera Rhagoletis and Anastrepha, for example. Insects such as fruit fly cause considerable damage to agricultural crops throughout the world. Spraying and similar methods of applying the crops with pesticides are the most commonly practiced methods of controlling these insect pests. However, there are problems with such methods, as spraying crops makes it very difficult to select for specific insect pests. Also these indiscriminant application methods are wasteful of insecticide, and frequent application is generally required, against mobile insect pests. Also concern has been raised by insecticides causing damage to the environment. Therefore, other methods having fewer of these disadvantages have been developed for controlling insect pests in crops. One such method is the use of lures and baits which are able to attract particular types and species of insects. This method enables the selective control of insect pests. Troublesome pests will be controlled, while other harmless or beneficial insects in the vicinity are generally not affected. These lures and baits rely on the principle of providing an attractant for a particular insect pest. The attractant can be of food substance, or a sex pheromone, for example. The mechanism of the attraction is primarily chemical, although tactile and visual stimuli created by the attractant can also be important. The attractants are sometimes used to attract and catch insect pests in traps, or more generally cause the insect pest to come in contact with poisons thereby killing or damaging it, as well as controlling the insect population as a result. The use of traps also enables the measurement of population densities, which is important for timing spray applications of insecticide, for example. Tephritid fruit flies cause extensive damage to horticultural crops in almost all horticultural districts throughout the world. Damage to fruit and vegetables is caused by fruit fly larvae which tunnel through fruits and in some cases through stem tissue, spreading bacterial rot as they go. The bacteria, which are deliberately introduced into the plant host tissue by the adult female fruit fly along with her eggs, multiply at the expense of this plant tissue. The larvae then feed on the resultant bacterial "soup" inside the host tissue which is soon rendered unfit for human consumption. Adult fruit flies, on the other hand, and in particular the females, feed on naturally occurring leaf surface saprophytic bacteria. These latter bacteria may be, but need not always be, the same species which the female r e-gurgitates for inoculation into plant tissue with her eggs . The extent of damage caused by fruit flies is illustrated by the example of the American State of California, which spent $80 million in the course of eradicating the Mediterranean fruit fly, Ceratitis capitata ( ied.) following two outbreaks in 1980. It is noteworthy that the then available commercial baits were not particularly effective. Eradication was not achieved until aerial cover sprays were applied over residential areas, causing public outcry and concern at the environmental impact. In another example, the United States Congress is prepared to approve the spending of $500 million to eradicate pest fruit flies from the Hawaiian islands. Furthermore, the Australian Federal Government allocated about $25 million for eradication following a report in August 1975 of an outbreak of the Oriental fruit fly, Dacus dorsalis, in the Northern Territory of Australia. In each case the amount spent on eradication reflects the value of horticultural produce to be protected from loss, which can run to billions of dollars. Many fruit fly attractants have been used in the past, in association with both traps and poisoned foliar sprays, beginning with C.W. Mally's poisoned sugar and treacle baits in South Africa, 1904-1909. Very many different liquid lures were tested in glass traps in Queensland following the outbreak of Queensland fruit fly, Dacus tryoπi, in the Stanthorpe district in 1921. Orange juice and ammonium carbonate became the principal ingredients, and the fruit flies simply drowned in the liquid. In 1939 M. McPhail published an account of the attraction of fruit flies to protein lures, and in 1952 L.F. Steiner improved poisoned sweet foliar baits by adding hydrolysed plant protein. Steiner*s protein hydrolysate bait, with the modern insecticide Malathion, is still used in fruit fly control and eradication throughout the world today. Additionally, powerful male fruit fly attractants, ^-«» i.e. kerosene was discovered to attract males of C.capitata in Sydney. Currently used male lures can be divided conveniently into three basic groups: (a) the "cue-lure" group:- cue-lure, anisylacetone, and Willison's lure; (b) the "methyl eugenol" group:- citronella oil, huon pine oil, and methyl eugenol; (c) the "med-lure" group: - siglure, med-lure, and trimed-lure. Cue-lure is 4- (p-acetoxyphenyl) -2-butanone, anisylacetone is 4- (p-methoxyphenyl) -2-butanone, Willison's lure is 4- (p-hydroxyphenyl) -2-butanone, methyl eugenol is O-methyleugenol and trimedlure is t-butyl-4(or 5)- chloro-2-methyl cyclohexane carboxylate. These lures primarily attract male fruit flies, which is a substantial disadvantage in their use, as damage to fruit results from the female laying her eggs in the fruit. The cue-lure group of attractants were discovered many years ago by organic chemists working on cosmetics. Apparently, it was noticed that fruit flies were attracted to these compounds in a laboratory where the chemists were working with them at the time. However, it is not clearly understood why these particular chemicals attracted the fruit flies. One theory is that these substances are sex pheromones produced by female fruit flies. It is also known to use a fermenting bait to attract fruit flies. It is thought that fruit flies are attracted to this type of bait by it being a food source. A typical fermenting bait comprises a sugar solution to which yeast has been added, and which has been allowed to ferment. This bait has not been found to be particularly effective in attracting fruit flies, and is not commonly used these days. Another type of bait is the protein hydrolysate bait discovered by Steiner, and this bait is commonly used at present. In general, a protein hydrolysate is combined with an insecticide and, for example, sprayed on fruit trees. Fruit flies are attracted to the bait and will die after f " contact with the pesticide present in the bait. This type of bait will attract both male and female fruit flies. For example, protein-malathion hydrolysate bait sprays are suited to aerial application, and the protein solution is generally harmless to vegetation. Spraying is generally carried out using a high viscosity spray mixture, with large droplet size, in order to ensure the survival of non-target insect species, which will not be attracted to the mixture. (See M.A. Bateman Drew, Hooper and Bateman, Economic Fruit Flies of the South Pacific Region, Brisbane, 1982) Experiments involving the proteinaceous baits for controlling oriental fruit flies are described in an article by Paul L. Gow, entitled "Proteinaceous Bait for the Oriental Fruit Fly" in the Journal of Economic Entomology, 4_7 (1), 153-160, (1954). It is of interest that on page 156, Gow noted that "the attractants in the proteinaceous baits were produced by microbial action on the original protein material". In addition, Gow isolated .various bacterial strains obtained from the protein hydrolysate, and tentatively identified one bacterial colony as belonging to the genus Proteus. However, no further identification of this bacterial species was provided in the article or subsequently, and in the experiments the proteinaceous baits were colonised by random bacteria from the general environment. In fact, fourteen strains or species of microorganisms were noted by Gow and none of the organisms were discovered to predominate in the bait, which Gow believed indicated the presence of "a complex and unstable microbiological balance". However, the experiments by Gow did indicate that proteinaceous baits were more effective as baits than the fermenting type. Since that time interest has centred on the use of ammonia as an attractant for fruit flies (see Bateman and Morton, "The Importance of Ammonia in Proteinaceous Attractants for Fruit Flies", Aust J. Agric. Res. 1981, 32, 883-903) and in the volatile constituents of proteinaceous attractants (see Morton and Bateman "Chemical Studies on Proteinaceous Attractants for Fruit Flies Including the f f _. O Λ _ Identification of Volatile Constituents", Aust J. Agric. Res. 1981, Z 2_, 905-16). It has now been discovered that the natural food required by wild female fruit flies for egg production is leaf surface saprophytic micro-organisms. This discovery became the starting point for the present invention. It was not until this discovery that female fruit flies feed on leaf surface microorganisms, and that fruit fly are attracted by the microbial vapours from such microorganisms, that major progress has been made in the art of fruit fly baits and lures. DISCLOSURE OF INVENTION It has now been discovered that fruit flies, and especially female fruit flies, feed on bacterial colonies which grow on leaves of trees. It is now believed that these bacteria provide the necessary food for egg production. Furthermore, it has been discovered that vapours emitted by growing colonies of bacteria attract fruit fly. The present invention comprises a bait for fruit fly comprising one or more live bacteria species of the family Ente obacteriacae on which the fruit fly feed, and a substrate able to support the growth of said bacteria species, and an agent toxic to the fruit fly. The agent toxic to the fruit fly may be a pesticide for example. A suitable such pesticides is malathion. Other pesticides are those known in the art to kill or damage fruit fly. Alternatively the toxic agent may be a suitable fruit fly pathogen; for example a bacterium or virus which can kill the insect or otherwise be detrimental to it. As well as killing the fruit fly, the agent may damage, or destroy the reproductive abilities of the fly, or otherwise act to control the fly population. More than one pesticide or pathogen can be included. The chosen agent should not be harmful to the bacterial attractant. The invention also concerns a storage stable pre-mix for preparing the above described fruit fly bait. One form which these premixes can take is to combine a dried powder substrate with dried and inactive live bacteria. Such a dried bacteria/dried substrate composition can be stored for long periods. When it is desired to use the composition, it merely needs to be moistened and left for a period for the bacteria to begin growth again. Preferably the pre- ix bacterial culture is dried by freeze drying. Alternatively, the pre-mix may comprise a dried powdered substrate with inactive bacteria, without the toxic agent required for the bait. The toxic agent, for example a pesticide, is added when the bait is made up for use. Also, another pre-mix comprises the dried, inactive bacteria, preferably mixed with any desired additives such as dried CMC, glucose or urea. The substrate, and the pesticide are added when the bait or attractant is made up. The pre-mix may also be part of a kit, wherein the inactive bacteria is provided together with the substrate, either dried or ready for use. If the pre-mix is to be used as an attractant in a trap, for example, the pesticide or other toxic agent, need not be included in the attractant composition. When the pre-mix is made up, and moistened, the bacteria will become active, and begin growing on the substrate. The bacteria and substrate or storage stable pre-mix may include the pesticide, or the pesticide may be added at the time the bait is applied in the field. Any substrate conducive to the growth of the preferred bacterial inoculum may be used. Acid hydrolysates of proteinaceous material, such as soybean or wheat, or alternatively autolysates, may be used, but the preferred substrate is a yeast autolysate, extract, or powder. In dried form such a substrate may, for example, contain:- Moisture 4.1% Ash 11.5% Salt 1.3% Phosphate (As P 2 °5/ 3.2% Total Nitrogen 10.5% A ino Nitrogen 4.8% Alternatively non-hydrolysed material such as soy meal or pea flower may be used as a substrate, in which case tl preferred inoculum inself makes the initial breakdown of the substrate. In this case attraction to fruit flies may be delayed about a week, depending on temperature and other conditions, following inoculation. Such baits should be used in traps rather than as foliar applications. The substrate may contain one or more additives to promote, retard, or regulate bacterial growth in a manner consistent with maximum attractancy to fruit flies. Suitable additives to enrich the nutrient content of the substrate are, for example, glucose, other sugars, peptones, amino acids, urea, ammonium salts, potassium hydrogen phosphate salts, sodium thiosulphate, or B-vitamins. Other additives conducive to bacterial activity and the attractancy of the bait are those which retard dehydration or facilitate rehydration and the leakage of nutrients from within the applicable plant leaf or other surface to the attractant bait composition thereon. These water regulating materials may for example be ydrophilic gels or colloids, agars, extenders or food thickeners, or they may be organic films which present a barrier to evaporation. Sodium carboxy methyl cellulose and hydroxy propyl cellulose are examples of suitable extenders. Miller's "Nu-film 17" is an example of an anti-evaporative film. If required for optimal bacterial growth or optimal attractancy the pH of the attractant composition may be altered with suitable agents. In addition the substrate may be modified by additives or other means to selectively favour the growth of the preferred bacterial inoculum over and above the growth of volunteer species of microorganism already present on applicable plant surfaces, or in the general environment of those surfaces. For example sodium taurocholate (bile salt) inhibits many Gram-positive organisms which abound on leaf surfaces but which are not attractive to fruit flies. Without the use of such substrate modification to favour the chosen bacterial inoculum, the substrate will be more or less used up by random less attractant or non-attractant bacterial species or strains from the applicable surface general environment, and the attraction of the bait will be proportionately spoiled. As a further example of a means to select against undesirable volunteer micro-organisms, the substrate may be altered by the addition of what in medical terminology are called chemotherapeutic agents or antibiotics, some of which are bacteriocidal and others bacteriostatic, different examples having different spectra of activity against some but not other bacteria. For example Klebsiella aeruginosa, a very similar bacterium to K. neumoniae isolated below from the fruit fly Dacus dorsalis, is resistant to the Sulphonamides. Thus if K. aeruginosa were used as the inoculum, the addition of a sulphonamide to the bait would eliminate competition, not only from, for example, leaf surface Bacillus species, but also many other competing Enterobacteriaceae. In field trials against Dacus tryoni and D.neohumeralis in guava groves referred to in Example 1 below, it was frequently observed that uninoculated baits using the preferred yeast autolysate substrate were of low attractancy during the first day, and thereafter, or sooner, to varying degrees caught up with the inoculated baits in terms of the number of flies caught. It is believed that the degree to which they "caught up" reflects the attractancy of the volunteer species. In the table presented with Example 1 treatments No. 4 and No. 9 may be compared in this regard. Suitable additives of these nutritive, regulatory, selective and other kinds are well understood by persons skilled in the art of the present invention, and commonly known additives can be used in the invention. Additives may be incorporated into the attractant composition, or into any storage stable pre-mix preparation referred to above. It is slightly preferred that the protein autolysate and hydrolysate substrate has a low salt (NaCl) content. Protein autolysate and hydrolysate substrates which are readily available can contain added salt, and are known as "high salt" substrates, or contain small amounts of added salts, or no added salt, and are known as "low salt" or OMP "salt free" substrates. In general a high salt substrate will contain approximately more than about 5 % w/v salt and often between about 12 and about 16 % w/v salt. Low salt autolysates will typically contain about 1.5 % w/v salt, which is the natural amount salt of present in the protein autolysate without any additional salt being added. It is believed, that the major effect of the presence of a high salt level in a bait or attractant composition is that it gives the bait or attractant a repellent effect to fruit flies. As a salty bait dries on leaves or other applicable surfaces, the concentration of the salt increases. Fruit flies may still be attracted but are less likely to land on the composition or feed and hence come in contact with the pesticide, if there is a high salt level. This appears to be due to the acute sensitivity of the tactile receptors on the feet of the flies, reacting unfavourably to high salt or electrolyte levels. Alteration of the'pH of high salt acid hydrolysates such as Stayley's P.I.B.-7 has been found by various workers in different parts of the world to slightly increase the attractancy of these compositions. Acid hydrolysates however tend not to support strong bacterial growth and their comparatively low attractancy to fruit flies may possibly be due to de-naturing of the plant protein ingredient during acid hydrolysis rather than to their salt content. It is preferred that the bacteria species present in the attractant composition or bait be a single species. Two or more species can be included in a single composition or bait, but in this case the two or more strains will compete with one another, and one will predominate in the long run. Preferred bacteria species are of the genera: Erwinia, Citrobacter, Providencia, Klebsiella, Serratia or Salmonella. In addition, certain species of genus Proteus are also preferred. Earlier experiments by Gow isolated an unidentified bacteria of genus Proteus, and so no novelty is claimed for all bacteria of this genus. However, particular species of Proteus which have now been identified and found to be attractants for fruit fly are claimed. Preferred bacteria species are chosen from Erwinia herbicola, Citrobacter freundii, Citrobacter diversus, Providencia rettgeri, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus vulgaris. Salmonella cholerae-suis, or Serratia liquefaciens. Providencia rettgeri is synonymous with Proteus rettgeri. The bacteria of genus Salmonella are harmful to humans and animals, and are therefore not particularly preferred for use in the baits and traps of the invention. Also found is bacteria of species Enterobacter cloacae, which is a contaminant and which is not considered as one of the attractant bacteria of the invention. BEST MODES OF CARRYING OUT THE INVENTION The bacteria of the invention are leaf and fruit surface saprophytes belonging to family Enterobacteriaceae. Such bacteria grow strongly in a manner attractive to fruit flies where the plant surface substrate is rich in hydrolysed plant proteins, or in protein precursors such as amino acids and peptides. These bacteria are generally obtained by catching wild fruit flies of the species in question usually while they are feeding, and, using sterile dissection techniques, removing crop and stomach. The crop, in particular, and the stomach, contains, among other micro-organisms, the required inoculum. Enterobacter cloacae, a common inhabitant of the lower gut of fruit flies, presents an example of a non-required contaminant which may be found in the crop together with the ingested bacterial food. The crop and stomach contents may be spread on the surface of a suitable medium, such as a peptone yeast extract (PYE) agar (lOg peptone, 5g yeast extract, 5g NaCl, 15g agar, 1,000ml distilled water) and incubated at a suitable temperature. The most common colony types found in a series of plates from a number of crops and stomachs are selected and pure cultures obtained. Erwinia herbicola (synonymous with Enterobacter agglo erans) and Providencia rettgeri in family Enterobacteriaceae (Regn. Nos. UQM 2510 and 2514 respectively of the University of Queensland Microbiology Culture Collection) are examples of commonly isolated bacteria attractive to pest fruit flies Dacus tryoni (Queensland fruit fly) and Dacus neohumeralis and Dacus cacuminatus in South East Queensland. The attractant composition consists of a substrate with or without the hereinabove described additives, and a bacterial inoculum. The so called low salt yeast autolysate (LSA) produced by Mauri Foods of Toowoomba, Queensland, is a good example of a preferred substrate, although this Mauri Foods product does require refrigeration during storage. A powdered dried form of LSA could be stored for long periods at normal temperatures. Most preferably 50mls of LSA or other substrate, or 25g of protein solids, are added to each litre of water to make the bait. 20mls of a broth culture of the bacterial inoculum, at the log phase of growth, provides an example of an acceptable number of cells for inoculation of each litre of this bait. This quantity of inoculum is given to provide an indication of the number of cells required rather than the form in which the inoculum should be prepared. For a typical bait spray on green foliage, an insecticide can be added to the inoculated substrate. Malathion as the 103% emulsifiable concentrate, at the rate of approximately 0.1% is one example of a suitable pesticide. In addition, a food thickener or extender such as carboxy-methyl-cellulose (CMC) may be added to thicken the inoculated substrate. A solution of CMC is preferred which will make the solution sufficiently thick without impeding the application onto foliage through a spray nozzle. "Spots" or "squirts" of this bait preparation may consist of about 100ml each, and is most preferably mainly applied to the undersides of green foliage. Aerial application to the upper surfaces of the foliage from light aircraft or helicopters, is also satisfactory but in this case the bait is preferably not dispersed with a fine spray nozzle. In orchard and urban baiting, "spots" may be applied on a regular grid spacing to combat sedentary fruit flies (M.A. Bateman ojo. cit. 1982) . where the target population of pest fruit flies is sexually immature, and therefore less sedentary, the distance between "spots" can be increased. This may apply in eradication programmes where teneral, or immature, flies are moving away from a locus of infestation. For estimating the size of fruit fly populations "spots" are applied and calico sheets may be spread beneath each spot to monitor the kill. In this instance, where the bait preparation is used to assess the size and age or sexual maturity or species composition of a fruit fly population, white sheets of apprximately 2 square metres in area below a "spot" of 1 square metre will provide the required result, of course provided that ants, frogs, skinks, and so forth are not permitted to remove the catch. This inoculated attractant preparation can be used in a trap, with or without insecticide depending on the mode of operation of the trap. Whereas fruit flies are not particularly attracted to protein hydrolysates made by acid hydrolysis when such are applied in wicks or traps, fruit flies are strongly attracted to inoculated substrates, in accordance with the invention, applied in such devices. Furthermore inoculated attractant preparations are attractive to fruit flies even when applied to unusual target surfaces, such as fence posts, brick walls, or telegraph poles for example. The non-inoculated preparations of the prior art are scarcely attractive at all on these non-living surfaces, and must be applied to green leaves and fruit which fruit flies normally frequent and where volunteer inocula may be present. A further aspect of the present invention relates to baits and attractants for fruit flies involving chemical compounds chosen from those naturally produced in bacterial volatile emissions. Several volatile compounds were identified by Lee, Smith and Freeman ("High-resolution chromatographic profiles of volatile organic compounds produced by microorganisms at refrigerated temperatures", Appl. Environ. Microbiol. 37, 85-90, 1979) from cultures of Pseudomonas putida. The compounds so identified are: 2-butanone, acetone, methyl butanal, methyl thiolacetate, methyl isothiocyanate, methyl isobutyrate, dimethyl disulphide, toluene, methyl-2-methyl butyrate, n-butyl acetate, methyl pent-2-enoate, dimethyl benzene, benzaldehyde, dimethyl trisulphide, methyl propionate, methyl benzoate, 2-nonanone, 1-undecene, 2-undecanone and/or methyl mercaptan. Of these compounds, 2-butanone was tested and found to act as an attractant for fruit fly. Other compounds, namely phenyl acetate, butan-1-ol and isopentanol were also found to be attractive' to fruit fly. It was found that the combination of these compounds functioned better than the individual compounds. However, 2-butanone was particularly effective as an attractant among the compounds tested under laboratory conditions . Further details are provided. in Example 1. If desired, these attractant compounds, (preferably stabilized chemically by adsorption on to inert powders such as talc, or by microencapsulation, for example) may be added to the bacterial lures discussed previously. It is believed the compounds identified by Lee, et al. will act as fruit fly attractants, as Dacus tryoni are attracted to Pseudomonas putida cultures on PYE agar under laboratory conditions. The family Pseudomonadaceae is fairly closely related to the family Enterobacteriaceae, and when grown on similar substrates or media, bacteria from the two families will produce many volatile compounds in common. Therefore attractant compositions including these compounds fall within the scope of the invention. As many of the attractant compounds are very volatile, it is preferred to use them in a controlled release formulation; for example, micr©encapsulated. Fruit fly can be caught in traps using the attractant compositions or baits of the invention. Any of the commonly known traps for catching insects can be used. Merely some examples of these which are used for catching fruit flies are the Steiner trap, the Israeli trap, the Bateman trap, the Jackson trap, or the McPhail trap. These traps are discussed in more detail in Drew, Hooper and Bateman, op.cit. (1982), on pages 129 to 133. Traps can catch fruit fly by having an opening which allows the flies to easily enter the trap but which restricts them from leaving it. Otherwise, the trap may incorporate a sticky surface to hold the fruit flies, and this restricts the fly from leaving the trap. The preferred attractant bacteria for use as the bacterial inoculum in the invention is Erwinia herbicola. A strain of Erwinia herbicola which is particularly effective has been deposited with the American Type Culture Collection (ATCC) at Rockville, Maryland, the United States of America, under the Applicant's reference number "BTA 751". The ATCC deposit number is lodged on . Example 1 - Synthetic bacterial volatiles In an experiment to test the attractancy of these compounds, a number of Steiner traps were set up at equal spaces around a field cage. Dacus tryoni were released into the room after thimbles, with a pin hole for release of the attractant, were placed in each trap. The thimbles contained the different chemicals with one empty as a control. No insecticide was used and each trap was examined at 60 minute intervals throughout the day. The contents of each trap were noted - the flies were counted and sexed - and released back into the room. Cue-lure was also included for comparison purposes. The totals for each chemical were: Chemicals NO. Of No. of % Female Total Males F Feemm.aalleess (a) Cue-lure 413 3 399 9 9 452 (b) 2-Butanone 658 2 26633 2 299 921 (c) Phenyl acetate 219 9 955 3 300 314 (d) Butan-1-ol 171 7 700 3 300 241 (e) Isopentanol 135 4 400 2 233 175 (f) Control 50 3 311 3 388 81 f OMPI A s ignif icant feature is the number of females caught by 2-butanone as the lure in comparison with cue-lure . By catching a s ignificant number of females , 2-butanone f unctioned much better than cue-lure, which hardly ever catches females at all. In a further experiment to test these compounds, 2-butanone was placed in a trap in bushland removed from the presence of the other chemicals of the previous experiment which pervaded the air in the experimental room. In this experiment, the 2-butanone only attracted males, and at a rate equal to, or less than, the known cue-lure. From these experiments, it appears that the female fruit fly may respond to 2-butanone only in the presence of other chemicals, for example phenyl acetate and/or butan-1-ol. Thus she may respond to a complex array of bacterial odours and not to any one constituent on its own. By varying the concentrations of the various constituents of the lures to re-produce the chemical emmisions of particular leaf surface bacterial colonies and by the use of controlled release methods, such as microencapsulation, to regulate volatility of the constituents, a high proportion of the females, can be trapped. In one embodiment, the attractant or bait mixture may be used to attract fruit flies into a plastic or glass or other trap containing the attractant mixture. In another embodiment the bait may be applied to green foliage in orchards, as, for example, a controlled release formulation of the preferred compounds. In each case, the mixture may be used in association with a suitable insecticide such as malathion, dichlorvos, or pyrethroids. The insecticide may also be given controlled release formulation either separately or after mixing with the attractants. The constituents of the attractant mixture, their relative concentrations, and the methods of distributing the attractant mixture, can be varied to suit the particular applicable conditions. The attractant mixture can provide an effective lure for the damage causing female fruit flies which are not, if barely, attracted to the known synthetic lures . Example 2 - Field attractancy of baits Five experimental trials were conducted against Dacus tryoni and D.neohumeralis in abandoned Guava groves in South East Queensland during autumn 1984. 100ml spots of various preparations, using different recipes and different bacteria were tested under these field conditions. In each trial, 2-square-metre calico sheets were suspended beneath each spot to monitor the catch. In a trial conducted at Palmwoods in April, there were 11 treatments and each treatment was assigned four spots, each in a guava tree about ten metres distant from the next spot. The spots were applied at 9 a.m. on the first day, and catches recorded for four days. Fly numbers from each treatment are here presented as the sum of four spots. "2-day brew", for example, refers to the time, at 26°C, between inoculation and application of the bait to the guava foliage. The substrate used was generally Mauri Foods low salt yeast autolysate (LSA) , and the inoculum was prepared as a broth and added at the rate of 50ml/litre unless otherwise indicated. The bacterial species in this case was Providencia rettgeri. The pesticide used was Malathion at a concentration of 0.1%. The results are presented in Table 1, following. Table of experimental results from a trial in a guava grove at Palmwoods, Queensland, in April 1984. JϋRE OMPI TABLE Cumulative total male + female D. tryoni + D. Treatments neohumeralis after 1 and 4 days 1. P.rettgeri (2-day brew) in PYE broth P.rettgeri (2-day brew) 37 49 in autoclaved LSA [LSA = Mauri Foods Low Salt Autolysate] P. ettgeri (2-day brew) in 42 68 non-autoclaved LSA. P.rettgeri (not brewed) 93 137 not autoclaved LSA 5. P.rettgeri (250ml/l of 52 81 inoculum broth in LSA substrate) 6. P . retgerr i (250ml/l of 31 40 inoculum and 2 -day brew) LSA substrate 7 * P. ettgeri (with 2 1/2% CMC) 118 210 LSA substrate 8. Control (Malathion only) 8 10 9. LSA without inoculation 62 94 10. LSA stored at Alice Springs 43 63 without refrigeration 11. Lanes acid hydrolysate 49 87 without inoculation As a res ult of all f ive tr ials , the following were noted. (a) An LSA substrate should preferably not be autoclaved or otherwise heated before use. (b) A liquid LSA substrate should preferably not be stored without refrigeration. (c) An inoculated bait using autolysate or hydrolysate substrates should preferably be held for as short a time as possible before application. (d) Where an inoculum is added as a broth, a small volume (20ml/litre) is preferred. (e) Food thickeners or extenders will enhance the efficiency of inoculated baits. (f) In most cases the resident population of fruit flies in the vicinity of each spot appeared to have been killed on the first day. Further kills on subsequent days were attribited to immigrant flies responding to the baits. (g) Female flies out-numbered males 2:1. The same pattern of sex ratios, and teneral/mature ratios, prevailed over all five experiments. (h) Erwinia herbicola proved a superior species of inoculum to Providencia rettgeri under these field conditions. Escherichia coli proved less attractive, and Pseudomonas putida of family Pseudomonadaceae much less attractive under the same field conditions. (i) After 8 days, inoculated LSA with 2 1/2% CMC and 0.1% malathion were still catching large numbers of flies although acid hydrolysates were no longer effective. It was subsequently noted that bacterial growth in the latter was 1/10th that in the LSA baits. (j) In the table of results presented. Treatment No. 7 gave the best results against D.tryoni and D.neohumeralis in South East Queensland. The high knock-down rate on the first day with preferred treatments indicated their value in eradication campaigns where frequent applications are made over a short period of time. Example 3 - Isolation of bacteria (a) A number of female fruit flies were obtained in order to isolate and identify the bacteria on which they had been feeding, and hence by which they had been attracted. Wild female fruit flies were caught while feeding. They were obtained from various locations, and belonged to various species. Sterile dissection techniques were employed to remove the crop and isolate bacteria as hereinabove described. Table 2 (following) describes the results of standard identification procedures for determining the species of bacteria so obtained. The standard tests are listed down the left hand side of the table, and the isolated bacterial colonies are listed by internal reference number along the top. Originals of the bacterial isolates by fruit fly species and location are also listed. TABLE 2 CODE B II 12 Jl J2 J3 Kl K2 K3 K4 KS 3A ISOLATEO D.CACUM D.TRYONI 0.TRYONI 0.TRYONI D.TRYONI D.TRYONI D.TRYONI 0.TRYONI D.TRYONI 0.TRYONI D.TRYONI 0.TRYONI LOCATION QUEENSLAND QUEENSLAND QUEENSLAND QUEENSLAND QUEENSLAND QUEENSLAND NSW NSW NSW NSW NSW QUEENSLAND GENUS* ERWINIA CITROBACTER KLEBSIELLA PROTEUS KLEBSIELLA ENTEROBACT CITROBACTER SERRATIA SERRATIA PROVIDENCIA KLEBISELLA CITROBACTE SPECIES" HERBICOLA FREUNDII OXYTOCA VULGARIS OXYTOCA CLOACAE FREUNDII LIQUEFAC LIQUEFAC RETTGERI PNEUMONIAE FREUNDII ROD . + + GRAH STAIN - CATALASE t + + OXIDASE MOTILITY t NITRATE i- t LYSINE t ORNITHONE - H2S t GLUCOSE t t + t + t t t t MANITOL . t + t t t t * t XYLOSE . t + t + ♦ t t t ONPG + t t + t t t INDOLE t t UREASE t t V.P. t t t t CITRATE t t + + TOA GELATIN HALONATE t INOSITOL t + SORBITOL t t t + RHAHNOSE t -t- t t + SUCROSE t + t LACTOSE +/- t ARBINOSE + + t t AOONITOL + + t t RAFFINOSE + + t SALICIN + t t t ARGININE AAC/136e GENUS AND SPECIES NAMES USED HERE ARE BASED ON LITERATURE FOR MICROBACT 2.E IDENTIFICATION KITS (DISPOSABLE PRODUCTS ADELAIDE SOUTH AUST.) TABLE 2 continued. CODE 22 23A 23B 2SA 26 I1A 31B 44A 44B ISOLATED 0.TRYONI 0.TRYONI 0.TRYO I 0.DOR A IS D.DORSA IS D.DORSALIS D.DORSALIS O.DORSALIS O.DORSALIS LOCATION QUEENSLAND QUEENSLAND QUEENSLAND HAWAII HAWAII HAWAII HAWAII HAWAII HAWAII GENUS" ENTEROBACT PROTEUS PROVIDENCIA KLEBSELLA SALMONELLA KLEBSELLA SPECIES" CLOACAE VULGARIS RETTGERI OXYTOCA CHOL. SUIS OXYTOCA ROD GRAM STAIN CATALASE OXIOASE HOTILITY t t NITRATE t + t t LYSINE t t t ORNITHONE t t H2S t GLUCOSE t t HANITOL + t XYLOSE t/- t t ONPG + t INOOLE t UREASE + t t .P. t t CITRATE + t t TOA GELATIN MALONATE t + t INOSITOL + + t SORBITOL + t RHAHNOSE t t SUCROSE . t t LACTOSE + ARBINOSE t ADONITOL t + RAFFINOSE * + SALICIN t + t ARGININE AAC/136C GENUS AND SPECIES NAMES USED HERE ARE BASED ON LITERATURE FOR MICROBACT 24E IDENTIFICATION KITS (DISPOSABLE PRODUCTS ADELAIDE SOUTH AUST. ) TABLE 2 continued. CODE 3B 5A SB 7 9A 9B 12A 12B 18A 20 21 ISOLATED D.TRYONI D.TRYONI D.TRYONI 0.TRYONI D.TRYONI D.TRYONI D.TRYONI 0.TRYONI 0.TRYONI 0.TRYONI D.TRYONI LOCATION QUEENSLAND QUEENSLAND QUEENSLAND QUEENSLAND QUEENSLAND QUEENSLAND QUEENSLAND QUEENSLAND QUEENSLAND QUEENSLAND QUEENSLAND GENUS- CITROBACTER CITROBACTER PROTEUS ERWINIA CITROBACTER KLEBSELLA SPECIES* FREUNDII FREUNDII VULGARIS HERBICOLA FREUNDII OXYTOCA ROD GRAM STAIN CATALASE OXIOASE +/- MOTILITY + t NITRATE + t LYSINE + ORNITHONE t +/- H2S +/- + * GLUCOSE + ♦ t t t MANITQL t + + t t t * XYLOSE + + + + t t ONPG + t t t INOOLE t t t UREASE + t t t .P. t t t t CITRATE + + t t t TDA + t t GELATIN t MALONATE t INOSITOL t t t t SORBITOL t t RHAMNOSE t t t t SUCROSE t t LACTOSE t/- ARBINOSE t t ADONITOL t RAFFINOSE t SALICIN * ARGININE AΛC/.36e GENUS AND SPECIES NAMES USED HERE ARE BASED ON LITERATURE FOR MICROBACT 24E IDENTIFICATION KITS (DISPOSABLE PRODUCTS ADELAIDE SOUTH AUST.) Example 4 - Isolation of bacteria (b) Using the same selection and identification techniques as discussed previously, fruit flies were collected from the Hawaiian Island of Oahu and dissected and the bacterial isolates identified. The results are shown in the following table Fruit Fly Species Bacteria 1. Ceratitis capitata Klebsiella oxytoca 2. Dacus cucurbitae Citrobacter freundii 3. Citrobacter diversus 4. Klebsiella oxytoca 5. Dacus dorsalis Erwinia herbicola 6. Citrobacter freundii 7. Klebsiella pneumoniae Note: (1) The bacteria- in sample 1 were isolated from a laboratory culture of fruit flies that had been in a field cage at sea level, outside this species normal range, for several weeks. Example 5 - Comparative attractiveness of bacteria An experiment was conducted to determine the comparative attractancy of three bacterial isloates obtained from fruit flies. In a 14 cubic metre field cage containing 600 sexually mature D.tryoni, 12 Golden Delcious apples were coated with inoculated baits and suspended at head height. The baits contained 6% Mauri Foods' low salt yeast autolysate, 1.5% glucose, 2% sodium CMC, and one of the three inocula. Four apples were assigned at random to each bait. The number of flies feeding on each apple were then counted at 30 minute intervals. At each count the flies were removed and each apple rotated two stations clockwise in the cage. The following results were obtained. OMPI Count Inoculum Apple 1 2 3 4 5 1 36 30 32 26 49 Erwinia 2 40 15 6 18 9 herbicola 3 13 32 27 48 36 4 18 14 11 21 14 Total 107 91 76 113 108 1 9 16 19 3 8 Citrobacter 2 8 18 19 11 13 freundii 3 22 19 17 11 9 4 5 16 17 14 9 Total 44 69 72 39 39 1 31 9 9 9 13 Providencia 2 13 30 27 30 37 rettgeri 3 19 13 21 19 10 4 15 20 21 13 16 Total 78 72 78 71 76 The results clearly indicate that bacterial isolates differ in their attractancy, and that, under the conditions of the experiment, Erwinia herbicola was superior. For D.tryoni the results was confirmed by a further experiment with teneral (immature) adult flies. During seven counts over a four hour period E.herbicola attracted a total of 298 flies while C.freundii attracted 144 and P.rettgeri 125. Furthermore, among female Dacus dorsalis collected on the Hawaiian Islands, several had full crops containing only E.herbicola. These results demonstrate the importance of choosing the right inoculum and also the advantage of using a selective bait medium to favour the preferred inoculum over volunteer plant surface bacteria. Example 6 - Nutrient additives An experiment was designed to measure the effect of added sugar on the attractancy of a bait using Mauri Foods low salt yeast autolysate (LSA) as the substrate. The inoculum was Providencia rettgeri. The two inoculated LSA treatments included 2 1/2% CMC. In one inoculated LSA treatment, 3% glucose was added, and this treatment was referred to as "Queenslure II" or "Q-II". For comparison LSA without additives or innoculum and also the two commonly known acid hydrolysate fruit fly baits, "Lanes Protein Hydrolysate" and "Staley's P.I.B.-7" also without additives or innoculum were included in the trial. The experiment was conducted with Dacus tryoni (about 1000 flies) . An old cream room on a dairy in South East Queensland served as the field cage. The room was 3m x 3m and the ceiling 2m above the floor. The walls were of fine insect gauze to permit free flow of air, ' and the roof was insulated. Pieces of old hardwood floor board 28cm x 8cm were hung from ceiling hooks equidistant around the perimeter of the room. 15ml of each attractant composition were spread on one face of each board remote from the room and the excess allowed to drain off before the boards, wet with the five treatments, and one dry control board, were hung at each of the six stations around the room at 9.30 a.m. ' At approximately half hour intervals the number of flies on each board were counted and recorded. After each count the flies were shaken off each board, and the boards all rotated one station clockwise around the room to counteract strong bias due to stations being favoured by the position of the sun or other factors. The results are given in the following table and indicate that the preferred attractant composition should contain some sugar. The results were entirely consistent with hereinabove described guava field trials with respect to comparisons among various substrates. Table- the effect of added sugar Time umber of Flies Control No Glucose Glucose Staley's Lanes L.S.A. 10.15am 0 5 6 18 7 9 10.45 0 3 14 6 0 7 11.25 0 15 49 4 0 1 11.55 0 20 11 3 0 2 12.25pm 0 9 15 2 0 3 12.45 0 10 25 1 1 3 1.15 2 8 25 5 0 0 2.15 0 14 25 0 3 0 2.45 1 15 13 0 1 2 3.15 2 4 25 0 2 0 3.35 0 3 14 0 4 2 4.15 0 3 16 0 2 0 Total 5 109 238 39 20 29

Claims: CLAIMS: 1. A bait for fruit fly comprising live bacteria of at least one species of the family Enterobacteriacae on which fruit fly feed, a substrate able to support the growth of the bacteria, and an agent toxic to fruit fly. 2. The bait of claim 1 wherein the bacteria are of the genus; Erwinia, Citrobacter, Providencia, Klebsiella, Serratia or Salmonella. 3. The bait of claim 1 wherein the bacteria are of the species; Erwinia herbicola, Citrobacter freundii, Citrobacter diversus, Providencia rettgeri, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus vulgari, Serratia liquefaciens or Salmonella cholerae-suis. 4. The bait of claim 3 wherein a single species is employed. 5. The bait of claim 1 wherein the substrate is a protein autolysate. 6. The bait of claim 5 wherein the substrate also contains at least one additive to facilitate or regulate growth of the bacteria. 7. The bait of claim 6 wherein the additive is; carboxymethylcellulose, glucose, or urea, or a mixture of two or more thereof. 8. The bait of claim 7 wherein the substrate has a low NaCl content. 9. The bait of claim 1 wherein the agent is malathion. 10. A bait for fruit fly comprising at least one of volatile compound produced by live bacteria of at least one species of the family Enterobacteriacea on which fruit fly feed, and an agent toxic to said fruit fly. 11. The bait of claim 10 wherein the volatile compound is 2-butanone, phenyl acetate, butan-1-ol or isopentanol, or a mixture of two or more thereof. 12. The bait of claim 11 which also comprises; 2-aminoacetophenone, methyl thiolacetate, dimethyldisulfide, methyl-2-methyl butyrate, methyl pent-2-enoate, dimethyltrisulfide, methyl butanol, methyl isothiocyanate, benzaldehyde, methyl propionate, methyl benzoate, 2-nonanone, 1-undecene, 2-undecanone, acetone, methyl isobutyrate, toluene, n-butylacetate, dimethyl benzene or methyl mercaptan, or a mixture of two or more thereof. 13. A storage-stable pre-mix for preparing a bait for fruit fly, comprising inactive, live bacteria of at least one species of the family Enterobacteriacae on which fruit fly feed, and a substantially dry substrate, which when moist is able to support the growth of the bacteria, and an agent toxic to fruit fly. 14. The storage-stable pre-mix of claim 13, wherein the substantially dry substrate contains one or more substantially dry additives, which facilitate or regulate growth of bacteria, when the substrate is moistened. 15. A trap for catching fruit fly comprising a container having an opening to allow fruit fly to enter and being adapted to substantially restrict fruit fly from leaving, and containing a fruit fly attractant comprising ■ live bacteria of at least one species of the family Enterobacteriacae on which fruit fly feed, and a substrate able to support the growth of the bacteria, and optionally an agent toxic to fruit fly. 16. A trap for catching fruit fly comprising a container having an opening to allow fruit fly to enter and being adapted to substantially restrict fruit fly from leaving, and containing a fruit fly attractant comprising at least one volatile compound produced by live bacteria species of the family Enterobacteriacae on which fruit fly feed, and optionally an agent toxic to fruit fly. 17. A method of controlling fruit fly comprising exposing a fruit fly bait as defined in any one of claims 1 to 12 in or near a fruit fly habitat. 18. A method of capturing fruit fly comprising exposing a trap for fruit fly as defined in claim 15 or 16 in or near a fruit fly habitat. OMP

Inventors: COURTICE ANTONY COLIN, COURTICE, ANTONY, COLIN
Applicants: COURTICE ANTONY COLIN, COURTICE, ANTONY, COLIN
Application year: 1984
Main classification: A01N63
IPC codes: A01N25, A01N31, A01N35, A01N37, A01N63