# Patent

## Title
Control of insect pests

## Abstract

A composition for controlling insect pests comprising an extract from Clitoria ternatea comprising SPCs which have insecticidal activity and/or which repel the insect pest and/or deter the insect pest from laying eggs and/or influence the position of egg laying and/or deter the insect pest from feeding on a plant, wherein the composition optionally comprises a carrier.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a National Phase Application of International Application No. PCT/AU2012/000160, filed Feb. 21, 2012, which claims priority to Australian Patent Applications No. 2011900586, filed Feb. 21, 2011, which applications are incorporated herein fully by this reference.
TECHNICAL FIELD
The present invention relates to the use Clitoria ternatea for the control of insect pests, more particularly to the control of moths and their and larvae, and chewing or sap sucking insects generally, through insecticidal activity and/or repelling the insect pest and/or deterring the insect pest from laying eggs and/or influencing the position of egg laying and/or deterring the insect pest from feeding on an agricultural crop or other plant. In particular, the present invention relates to the use of extracts from Clitoria ternatea for the control of insect pests of agricultural crops and other plants, more particularly to the control of moths and their larvae and chewing or sap sucking insects on agricultural crops and other plants.
BACKGROUND ART
The Australian cotton industry still relies on repeated applications of synthetic pesticides to manage pests in crops. Accompanying problems associated with insecticide resistance, disruption of beneficial species, high cost of production, and environmental impact now require that alternative strategies be investigated for managing Helicoverpa spp. These include (but are not limited to) genetically engineered cotton crops containing insecticidal protein of Bacillus thuringiensis (Bt) and other host plant resistances, biopesticides, better management of beneficial species, trap crops, intercropping and companion planting, and manipulation of the behaviour of pests and beneficial insects. Genetically engineered (transgenic) crops are now grown in Australia and many countries to control lepidopteran pests and their introduction has reduced synthetic insecticide use against these pests. However, other pests are not affected by the toxin in the transgenic plants e.g. sucking pests. Also third to late stage Helicoverpa larvae that can tolerate the toxin have led to increased use of synthetic insecticides to control them on both transgenic and conventional cotton crops. Thus the need to develop new approaches to manage these pests is crucial.
One of the approaches with greater potential to revolutionalise the way insect pests are managed in broadacre crops such as cotton, is the use of natural plant chemical compounds or plant extracts. The natural plant extracts or secondary plant compounds (SPCs) in general can influence the behaviour of insects by functioning as cues stimulating an insect's “interest” or deter insects from infesting a particular host plant (Rhoades and Coates 1976). Many SPCs have evolved in plants to actually protect the plants against pest infestation (Rhoades and Coates 1976). This has led to several examples of SPCs being used as botanical insecticides to reduce pest damage when applied to crop plants. Some SPCs extracted from non-host plants and then sprayed on host plants can change the behaviour of a pest, particularly moths, which then avoid the host plant (Tingle and Mitchell 1984). Numerous studies into pest management have focussed on chemical compounds that kill the pest rather than behaviour modifying compounds (Tingle and Mitchell 1984, Mensah and Moore, 1999). Consequently, potentially useful compounds with more subtle modes of action that could lead to novel products have been overlooked (Mensah and Moore, 1999). Such compounds attract or repel pests over considerable distances; or stimulate or deter both feeding and egg-laying following contact. Deterrent compounds directly suppress oviposition and feeding by insects (Mensah, 1996, Mensah et al. 2000), they are considered more important than stimulants and in fact a deterrent effect is more commonly noted in SPCs (Bernays and Chapman 1994). It is plausible that the efficacy of a deterrent would be increased when used in combination with an attractant/stimulant applied to a non-valued resource (Miller and Cowles, 1990) in a push-pull strategy (Pyke et al. 1987). Therefore, a tool to modify egg laying and/or feeding behaviour of insect pests is a novel approach to pest management in agricultural crops and offers potentially very significant benefits.
SUMMARY OF THE INVENTION
In one aspect the present invention provides a composition for controlling insect pests comprising an extract from Clitoria ternatea comprising SPCs which have insecticidal activity and/or which repel the insect pest and/or deter the insect pest from laying eggs and/or influence the position of egg laying and/or deter the insect pest from feeding on a plant, wherein the composition optionally comprises a carrier.
In a further aspect the present invention provides a method of controlling one or more insect pests, the method comprising treating a locus with an SPC having insecticidal activity and/or which repels the insect pest and/or which deters the insect pest from laying eggs and/or influences the position of egg laying and/or which deters the insect pest from feeding on a plant, and which is derived from Clitoria ternatea. 



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows HPLC chromatograms of solid phase extraction (SPE) fractions from Clitoria ternatea together with UV spectra;
FIG. 2 graphically illustrates the feeding response of Helicoverpa armigera 3rd instar larvae on cotton leaves treated with Clitoria ternatea fractions (no-choice tests);
FIG. 3 graphically illustrates the antibiotic effects of Clitoria ternatea fractions on 2nd instar H. armigera resulting in mortalities after 48 hours;
FIG. 4 shows the no-choice feeding response of 2d instar H. armigera to the masking effects of Clitoria ternatea F2 on Lu Mein leaves (feeding attractant) on leaf is weight consumed;
FIG. 5 shows the no-choice masking effect of Clitoria ternatea fraction #2 on Lu Mein Leaves on larval weight of 2nd instar H. armigera (summary of 3 experiments);
FIG. 6 is a graph illustrating the efficacy of combined Clitoria ternatea formulated in hexane on oviposition of Helicoverpa spp. on cotton plants;
FIG. 7 shows the efficacy of different concentrations of Clitoria ternatea formulated in hexane on oviposition of Helicoverpa spp. on cotton plants;
FIG. 8 shows the efficacy of different concentrations of Clitoria ternatea formulated in hexane on the number of Helicoverpa spp. eggs and larvae per meter per sample date recorded on commercial conventional cotton fields;
FIG. 9 shows the efficacy of Clitoria ternatea formulated in (1) cotton seed oil (2) crude cotton seed oil and (3) canola oil on Helicoverpa spp. larvae from 2nd instar larvae until pupation on artificial diets; and
FIG. 10 is a HPLC profile at UV210 nm of a methanolic extraction of a composition comprising SPCs derived from Clitoria ternatea plant material at a pre-flower stage (comprising no flowers or pods) by contact with canola oil. The profile was obtained using an Agilent 1100 LCMSD with a Phenomex Luna C18 column at 40° C. using the following solvent gradient conditions:













Mobile Phase: A = 0.05% Trifluoroacetic acid (TFA)-Water


B = 0.05% TFA-Acetonitrile












Time (min)
% A
% B
Flow rate (mL/min)
















0
90
10
1.0



17.5
80
20
1.0



20
5
95
1.0



22.5
5
95
1.0



25
90
10
1.0



30
90
10
1.0










DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the control of one or more insect pests with SPCs derived from Clitoria ternatea. 
As used herein an SPC, or secondary plant compound, is chemical compound synthesised by a plant which is not essential to the survival of the plant. The SPCs of the present have insecticidal activity and/or repel the insect pest and/or deter the insect pest from laying eggs and/or influence the position of egg laying and/or deter the insect pest from feeding on the plant.
In an embodiment, the insect pest is a plant pest and the method comprises applying an extract from Clitoria ternatea to the plant or its surroundings.
Embodiments of the invention can be used to treat crops in order to limit or prevent insect infestation. The present invention is especially suitable for agronomically important plants, which refers to a plant that is harvested or cultivated on a commercial scale.
Examples of such agronomic plants (or crops) are cereals, such as wheat, barley, rye, oats, rice, maize or sorghum; beet, such as sugar or fodder beet; fruit, for example pome fruit, stone fruit and soft fruit, such as apples, pears, plums, prunes, peaches, almonds, cherries or berries, for example strawberries, raspberries or blackberries; legumes, such as beans, lentils, peas or soya beans; oil crops such as oil seed rape, mustard, poppies, olives, sunflowers, coconuts, castor, cacao or peanuts; the marrow family, such as pumpkins, cucumbers or melons; fibre plants such as cotton, flax, hemp or jute; citrus fruits such as oranges, lemons, grapefruits or tangerines; vegetables such as spinach, lettuce, asparagus, cabbage species, carrots, onions, chillies, tomatoes, potatoes, or capsicums; the laurel family such as avocado, Cinnamonium or camphor; and tobacco, nuts (such as walnut), coffee, egg plants, sugar cane, tea, pepper, grapevines, hops, the banana family, latex plants and ornamentals. Also important are forage crops such as grasses and legumes.
In an embodiment plants include fibre plants, grain crops, legume crops, pulse crops, vegetables and fruit, more particularly, cotton, maize, sorghum, sunflower, lucerne, various legumes especially soybean, pigeon pea, mung bean and chickpea, tomatoes, okra and like plants.
In an embodiment plants include ornamental plants. By way of example these ornamental plants may be orchids, roses, tulips, trees, shrubs, herbs, lawns and grasses, bulbs, vines, perennials, succulents, house plants.
In an embodiment, the insect pest is a pest of an animal and the method comprises applying an extract from Clitoria ternatea to the animal. In an embodiment the animal may be dogs, cats, cattle, sheep, horses, goats, pigs, chicken, guinea pig, donkey, duck, bird, water buffalo, camel, reindeer, goose, Llama, alpaca, elephant, deer, rabbit, mink, chinchilla, hamster, fox, emu, ostrich.
In an embodiment the method comprises treating a habitat.
The present invention encompasses applying a Clitoria ternatea extract in oil, water or any carrier product to a plant affected by the pest or its surroundings, or to an animal affected by the pest. Treatment can include use of an oil-based formulation, a water-based formulation, a residual formulation, wettable powder and the like. In some embodiments, combinations of formulations can be employed to achieve the benefits of different formulation types. The extract may added to the carrier or, in the case of a liquid formulation, the carrier may have been used to extract SPCs from Clitoria ternatea e.g. canola oil.
In an embodiment the formulation is an oil-based formulation which may further comprise a surfactant.
In an embodiment the formulation is an oil-based formulation and the oil is a C19-C27 hydrocarbon.
In an embodiment the formulation includes a extract from Clitoria ternatea in a polar solvent such as an alcohol, ketone, aldehyde or sulfoxide. In particular, the formulation may be an extract from Clitoria ternatea in alcohol.
In an embodiment the formulation includes a methanolic extract from Clitoria ternatea. In an embodiment the formulation includes an ethanolic extract from Clitoria ternatea. In an embodiment the formulation includes an extract from Clitoria ternatea in low molecular weight oil such as crude and refined cotton seed oil or canola oil. Other oils include white oils, DC Tron oil (nC 21 and nC 24 oils), Canopy oil (nC 27 oil), Biopest oil (nC 24 oils), dormant oil or summer oil, as known in the horticultural industry. Most of these oils are nC 19-nC 27 but other hydrocarbons having acceptable toxicity may be used. There are number of such products in the market which are suitable for use with the present invention. These are Sunspray oil, tea tree oil, Sunspray Ultra fine manufactured by the Sun Refining and Marketing Company.
The petroleum spray oil may be used in conjunction with suitable agronomically acceptable diluents and/or carriers and with other additives common in the art such as emulsifiers, wetting agents, surfactants, stabilizers, spreaders or the like.
In an embodiment the formulation includes an aqueous extract from Clitoria ternatea. 
In an embodiment the formulation includes an extract from Clitoria ternatea in a lower hydrocarbon solvent such as hexane.
In an embodiment the formulation includes a fraction of a crude extract from Clitoria ternatea 
In an embodiment the formulation includes a mixture of fractions of a crude extract from Clitoria ternatea. 
The term “carrier” as used herein means a liquid or solid material, which can be inorganic or organic and of synthetic or natural origin, with which the active compound is mixed or formulated to facilitate application a composition according to the invention or an SPC derived from Clitoria ternatea is applied to a locus to be treated, or to facilitate its storage, transport and/or handling. In general, any of the materials customarily employed in formulating insecticides are suitable.
The term “locus” as used herein refers to a place to which a composition according to the invention or an SPC derived from Clitoria ternatea is applied. It includes application to an individual plant, a group of plants such as a plant and/or its surrounds, an animal individually or in a group and the region in which plants may be planted or in which animals may congregate, as well application directly to an insect or insects and/or the vicinity in which they are located.
The compositions of the present invention can be employed alone or in the form of mixtures with such solid and/or liquid dispersible carrier vehicles and/or other known compatible active agents such as pesticides, or acaricides, nematicides, fungicides, bactericides, rodenticides, herbicides, fertilizers, growth-regulating agents, etc., if desired, or in the form of particular dosage preparations for specific application made therefrom, such as solutions, emulsions, suspensions, powders, pastes, and granules which are thus ready for use.
The compositions of the present invention can be formulated or mixed with, if desired, conventional inert insecticide diluents or extenders of the type usable in conventional pest control agents, e.g., conventional dispersible carrier vehicles in the form of solutions, emulsions, suspensions, emulsifiable concentrates, spray powders, pastes, soluble powders, dusting agents, granules or foams.
Typical emulsifiers that may be suitable for use in the compositions of the invention, include, but are not limited to, light molecular weight oils (e.g., canola, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), and non-ionic, anionic and cationic surfactants. Blends of any of the above emulsifiers may also be used in the compositions of the present invention.
Typical non-ionic surfactants include ethoxylated alkanols, in particular ethoxylated fatty alcohols and ethoxylated oxoalcohols, such as ethoxylated lauryl alcohol, ethoxylated isotridecanol, ethoxylated cetyl alcohol, ethoxylated stearyl alcohol, and esters thereof, such as acetates; ethoxylated alkylphenols, such as ethoxylated nonylphenyl, ethoxylated dodecylphenyl, ethoxylated isotridecylphenol and the esters thereof, e.g. the acetates alkylglucosides and alkyl polyglucosides, ethoxylated alkylglucosides; ethoxylated fatty amines, ethoxylated fatty acids, partial esters, such as mono-, di- and triesters of fatty acids with glycerine or sorbitan, such as glycerine monostearate, glycerine monooleate, sorbitanmonolaurate, sorbitanmonopalmitate, sorbitanmonostearate, sorbitan monooleate, sorbitantristearate, sorbitan trioleate; ethoxylated esters of fatty acids with glycerine or sorbitan, such as polyoxyethylene glycerine monostearate, polyoxyethylene sorbitanmonolaurate, sorbitanmonopalmitate, polyoxyethylene sorbitanmonostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitantristearate, polyoxyethylene sorbitan trioleate; ethoxylates of vegetable oils or animal fats, such as corn oil ethoxylate, castor oil ethoxylate, tallow oil ethoxylate; ethoxylates of fatty amines, fatty amides or of fatty acid diethanolamides.
Typical anionic surfactants include salts, in particular, sodium, potassium calcium or ammonium salts of alkylsulfonates, such as lauryl sulfonate, isotridecylsulfonate, alkylsulfates, in particular fatty alcohol sulfates, such as lauryl sulfate, isotridecylsulfate, cetylsulfate, stearylsulfate—aryl- and alkylarylsulfonates, such as napthylsulfonate, dibutylnaphtylsulfonate, alkyldiphenylether sulfonates such as dodecyldiphenylether sulfonate, alkylbenzene sulfonates such as cumylsulfonate, nonylbenzenesulfonate and dodecylbenzene sulfonate; sulfonates of fatty acids and fatty acid esters; —sulfates of fatty acids and fatty acid esters; sulfates of ethoxylated alkanols, such as sulfates of ethoxylated lauryl alcohol; sulfates of alkoxylated alkylphenols; alkylphosphates and dialkylphosphates; dialkylesters of sulfosuccinic acid, such as dioctylsulfosuccinate, acylsarcosinates, fatty acids, such as stearates, acylglutamates, ligninsulfonates, low molecular weight condensates of naphthalinesulfonic acid or phenolsulfonic acid with formaldehyde and optionally urea;
Typical cationic surfactants include quaternary ammonium compounds, in particular alkyltrimethylammonium salts and dialkyldimethylammonium salts, e.g. the halides, sulfates and alkylsulfates.
In some embodiments, the insect control compositions can be combined with one or more synthetic insecticides or pesticides. In one embodiment, the insecticide or pesticide is selected from one or more of endosulfan, dicofol, chlorpyrifos, dimethoate, disulfoton, omethoate, parathion, phorate, profenofos, sulprofos, thiometon, aldicarb, carbaryl, beta-cyfluthrin, deltamethrin, esfenvalerate, fenvalerate, fluvalinate, lamda-cyhalothrin, chlorfluazuron, piperonyl butoxide, and petroleum spray oils. In another embodiment, the pesticide is a biological pesticide selected from a nuclear polyhedrosis virus and/or a plant extract known to be anti-feedant of pests. In yet another embodiment, the insecticide or pesticide is used at a reduced label rate. For example, the insecticide or pesticide may be used at half or one-third of the label rate.
The compositions of the present invention can be used to control insects by either treating a host directly, or treating an area in which the host will be located. For example, the host can be treated directly by using a spray formulation, which can be applied to a plant individually or when grouped, such as an agricultural crop.
The formulation of the present invention may further comprise other formulation auxiliaries known in the art of agrochemical formulations in customary amounts. Such auxiliaries include, but are not limited to, antifreeze agents (such as but not limited to glycerine, ethylene glycol, propylene glycol, monopropylene glycol, hexylene glycol, 1-methoxy-2-propanol, cyclohexanol), buffering agents (such as but not limited to sodium hydroxide, phosphoric acid), preserving agents (such as but not limited to derivatives of 1,2-benzisothiazolin-3-one, benzoic acid, sorbic acid, formaldehyde, a combination of methyl parahydroxybenzoate and propyl parahydroxybenzoate), stabilizing agents (such as but not limited to acids, preferably organic acids, such as dodecylbenzene sulfonic acid, acetic acid, propionic acid or butyl hydroxyl toluene, butyl hydroxyl anisole), thickening agents (such as but not limited to heteropolysaccharide and starches), and antifoaming agents (such as but not limited to those based on silicone, particularly polydimethylsiloxane). Such auxiliaries are commercially available and known in the art.
In an embodiment the present invention uses fractions, active compounds and crude extracts of Clitoria ternatea formulated in oil and emulsifiers to control cotton pests.
Preferably, the Clitoria ternatea extracts and formulations are suitable for killing the insect. The extract or formulation in water when applied to the plant or insect penetrates the insect's cuticle layers or is ingested to kill the insects or the residue of the extract on the plant can repel insects or deter the insects from egg laying or feeding. The formulation can kill or deter insect egg laying or feeding within 3-4 days of application to the insect or the target crop.
In an embodiment the Clitoria ternatea extract, fractions, crude or active compounds in oil is dissolved in water and applied to the crops infested with the target insects. The rate of application of the composition of the invention is typically between 1-2 liters of oil formulated extracts or active compounds dissolved in 1-500 liters of water, preferably 60-100 liters of water per hectare. In an embodiment, the rate of application is about 2 liters of oil formulated extracts dissolved in about 100 liters of water per hectare of plants. In an alternative embodiment, the rate of application is about 2 liters of oil formulation. Typically the treatment may involve at least four sprays at 14-28 day intervals.
Alternatively, the method may comprise applying 1-3000 ml, preferably 1000-2000 ml, of oil formulated extracts or active compounds per hectare of plants in the absence of dissolution of the oil formulated extracts or active is compounds in water, particularly when smaller areas are to be treated. When, for example, plants in a greenhouse, are to be treated.
While not wishing to be bound by theory, it is believed that the present invention controls moths and their larvae and chewing or sap sucking pests by repelling the pests, suppression of egg laying, deterrence of feeding and direct contact activity of the plant extract which kills the insect. There may also be method of control of other non-target pests and conservation of natural enemies of the moths and their larvae and chewing or sap sucking pests through treatment of the habitat.
In an embodiment the Clitoria ternatea structure used is the leaves, stems, roots, pods, seeds and a combination of any of the plant parts. These may be used as fractions or crude extracts in formulations such as low and high molecular oil or water or any other carriers to control moths and their larvae and chewing or sap sucking pests through repellent action, suppression of egg laying, deterrence of feeding and direct contact activity.
Botanically, Clitoria ternatea belongs to the family Fabaceae and sub family Papilionaceae. The family Fabaceae is a large family of around 12,000 species that cover a wide range of life forms from annuals to rainforest trees. Widespread in tropical and temperate regions, and the source of many economically important food plants, fodder crops, ornamentals, timber species and weeds, Clitoria ternatea is a climbing perennial, with sparsely hairy leaves usually with five leaflets. The flowers are large, to 5 cm, solitary or in pairs, blue with a yellow blotch in the centre, produced between April and June. In Australia it is a garden escape pest which has naturalised on creek banks and around waterholes throughout the Kimberley; also around coastal settlements in the Pilbara and Gascoyne. It is also used as a forage crop in large part of north QLD. It is pantropical, and probably native to tropical America.
For purposes of simplicity, the term “insect” or its equivalents or derivatives such as “insecticidal” shall be used in this application; however, it should be understood that the term “insect” refers, not only to insects but to their immature forms and larvae.
Those skilled in the art will recognize that not all compounds are equally effective against all insects. In embodiments the compositions display activity against insect pests, which may include economically important agronomic, forest, greenhouse, nursery, ornamentals, food and fiber, public and animal health, domestic and commercial structure, household, and stored product pests. Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Coleoptera and Lepidoptera.
Larvae of the order Lepidoptera include, but are not limited to, armyworms, cutworms, loopers, and heliothines in the family Noctuidae Spodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia ni Hübner (cabbage looper); Pseudoplusia includens Walker (soybean looper); Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabra Fabricius (green cloverworm); Heliothis virescens Fabricius (tobacco budworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa messo{acute over (η)}a Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); borers, casebearers, webworms, coneworms, and skeleton izers from the family Pyralidae Ostrinia nubilalis Hübner (European corn borer); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo suppressalis Walker (rice stem borer); C. partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leaf roller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia g{acute over (η)}sella Fabricius (lesser wax moth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella HüObner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); and leafrollers, budworms, seed worms, and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller); and other Archips species, Adoxophyes orana Fischer von Rósslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (coding moth); Platynota flavedana Clemens (variegated leaf roller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermiiller (European grape vine moth); Spilonota ocellana Denis & Schiffermiiller (eyespotted bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hübner (vine moth); Bonagota salub{acute over (η)}cola Meyrick (Brazilian apple leaf roller); Grapholita molesta Busck (oriental fruit moth); Suleima helianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneura spp.
Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith (orange striped oakworm); Antheraea pernyi Guerin-Meneville (Chinese Oak Tussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculat{acute over (η)}x thurbeflella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut is caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hübner (elm spanworm); Erannis tiliaria Harris (linden looper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guerin-Meneville (grapeleaf skeletonizer); Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail, orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval & Leconte (Southern cabbageworm); Sabulodes aegrotata Guenee (omnivorous looper); Schizura concinna J. E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenee; Malacosoma spp. and Orgyia spp. Of interest are larvae and adults of the order Coleoptera including weevils from the families Anthribidae, Bruchidae, and Curculionidae (including, but not limited to: Anthonomus grandis Boheman (boll weevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (rice weevil); Hypera punctata Fabricius (clover leaf weevil); Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyx fulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (gray sunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug)); flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetles, and leafminers in the family Chrysomelidae (including, but not limited to: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabrotica virgifera virgifera LeConte (western corn rootworm); D. barberi Smith & Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber (southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn flea beetle); Phyllotreta cruciferae Goeze (corn flea beetle); Colaspis brunnea Fabricius (grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle); Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from the family Coccinellidae (including, but not limited to: Epilachna vatvestis Mulsant (Mexican bean beetle)); chafers and other beetles from the family Scarabaeidae (including, but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northern masked chafer, white grub); C. immaculata Olivier (southern masked chafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer); Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer (carrot beetle)); carpet beetles from the family Dermestidae; wireworms from the family Elatehdae, Eleodes spp., Melanotus spp.; Conoderus spp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; bark beetles from the family Scolytidae and beetles from the family Tenebhonidae.
Adults and immatures of the order Diptera are of interest, including leafminers Agromyza parvicornis Loew (corn blotch leafminer); midges (including, but not limited to: Contarinia sorghicola Coquillett (sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosis mosellana Gehin (wheat midge); Neolasioptera murtfeldtiana Felt, (sunflower seed midge)); fruit flies (Tephritidae), Oscinella frit Linnaeus (frit flies); maggots (including, but not limited to: Delia platura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly); and other Delia spp., Meromyza americana Fitch (wheat stem maggot); Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F. femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus (stable flies)); face flies, horn flies, blow flies, Chrysomya spp.; Phormia spp.; and other muscoid fly pests, horse flies Tabanus spp.; bot flies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deer flies Chrysops spp.; Melophagus ovinus Linnaeus (keds); and other Brachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; black flies Prosimulium spp.; Simulium spp.; biting midges, sand flies, sciands, and other Nematocera. Included as insects of interest are adults and nymphs of the orders Hemiptera and Homoptera such as, but not limited to, adelgids from the family Adelgidae, plant bugs from the family Miridae, cicadas from the family Cicadidae, leafhoppers, Empoasca spp.; from the family Cicadellidae, planthoppers from the families Cixiidae, Flatidae, Fulgoroidea, lssidae and Delphacidae, treehoppers from the family Membracidae, psyllids from the family Psyllidae, whiteflies from the family Aleyrodidae, aphids from the family Aphididae, phylloxera from the family Phylloxeridae, mealybugs from the family Pseudococcidae, scales from the families Asterolecanidae, Coccidae, Dactylopiidae, Diaspididae, Eriococcidae, Ortheziidae, Phoenicococcidae and Margarodidae, lace bugs from the family Tingidae, stink bugs from the family Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs from the family Lygaeidae, spittlebugs from the family Cercopidae squash bugs from the family Coreidae, and red bugs and cotton stainers from the family Pyrrhocoridae.
Agronomically important members from the order Homoptera further include, but are not limited to: Acyrthisiphon pisum Harris (pea aphid); Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black bean aphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicis Forbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecola Patch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid); Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxia Kurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantaginea Paaserini (rosy apple aphid); E{grave over (η)}osoma lanigerum Hausmann (woolly apple aphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopterus pruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnip aphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphum euphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potato aphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid); Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphis graminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcane aphid); Sitobion avenae Fabricius (English grain aphid); Therioaphis maculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer de Fonscolombe (black citrus aphid); and T. citricida Kirkaldy (brown citrus aphid); Adelges spp. (adelgids); Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaci Gennadius (tobacco whitefly, sweetpotato whitefly); B. argentifolii Bellows & Perring (silverleaf whitefly); Dialeurodes citri Ashmead (citrus whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) and T. vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris (potato leafhopper); Laodelphax striatellus Fallen (smaller brown planthopper); Macrolestes quadrilineatus Forbes (aster leafhopper); Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal (rice leafhopper); Nilaparvata lugens Stal (brown planthopper); Peregrinus maidis Ashmead (corn planthopper); Sogatella furcifera Horvath (white-backed planthopper); Sogatodes orizicola Muir (rice delphacid); Typhlocyba pomaria McAtee (white apple leafhopper); Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus (periodical cicada); lcerya purchasi Maskell (cottony cushion scale); Quadraspidiotus perniciosus Comstock (San Jose scale); Planococcus citri Risso (citrus mealybug); Pseudococcus spp. (other mealybug complex); Cacopsylla pyricola Foerster (pear psylla); Trioza diospy{acute over (η)} Ash mead (persimmon psylla).
Agronomically important species of interest from the order Hemiptera include, but are not limited to: Acrosternum hilare Say (green stink bug); Anasa tristis De is Geer (squash bug); Blissus leucopterus leucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lace bug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellus Herrich-Schaffer (cotton stainer); Euschistus servus Say (brown stink bug); E. va{acute over (η)}ol{acute over (η)}us Palisot de Beauvois (one-spotted stink bug); Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say (leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois (tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug); L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius (European tarnished plant bug); Lygocoris pabulinus Linnaeus (common green capsid); Nezara viridula Linnaeus (southern green stink bug); Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas (large milkweed bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper).
Furthermore, embodiments of the present invention may be effective against Hemiptera such, Calocoris norvegicus Gmelin (strawberry bug); Orthops campestris Linnaeus; Plesioco{acute over (η)}s rugicollis Fallen (apple capsid); Cyrtopeltis modestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly); Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocoris chlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onion plant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper); Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatus Fabricius (four-lined plant bug); Nysius ericae Schilling (false chinch bug); Nysius raphanus Howard (false chinch bug); Nezara viridula Linnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.; Pyrrhocoridae spp.; Timidae spp.; Blostomatidae spp.; Reduviidae spp.; and Cimicidae spp.
Also included are adults and larvae of the order Acari (mites) such as Aceria tosichella Keifer (wheat curl mite); Petrobia latens Müller (brown wheat mite); spider mites and red mites in the family Tetranychidae, Panonychus ulmi Koch (European red mite); Tetranychus urticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDaniel mite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestani Ugarov & Nikolski (strawberry spider mite); flat mites in the family Tenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust and bud mites in the family Ehophyidae and other foliar feeding mites and mites important in human and animal health, i.e. dust mites in the family Epidermoptidae, follicle mites in the family Demodicidae, grain mites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodes scapularis Say (deer tick); /. holocyclus Neumann (Australian paralysis tick); Dermacentor variabilis Say (American dog tick); Amblyomma americanum Linnaeus (lone star tick); and scab and itch mites in the families Psoroptidae, Pyemotidae, and Sarcoptidae. Insect pests of the order Thysanura are of interest, such as Lepisma saccharina Linnaeus (silverfish); Thermobia domestica Packard (firebrat).
In an embodiment the insects are selected from cotton bollworm, native budworm, green mirids, aphids, green vegetable bugs, apple dimpling bugs, thrips (plaque thrips, tobacco thrips, onion thrips, western flower thrips), white flies and two spotted mites.
In an embodiment the insect pests of animals include fleas, lice, mosquitoes, flies, tsetse flies, ants, ticks, mites, silverfish and chiggers.
Insect pests may be tested for insecticidal activity of compositions of the embodiments in early developmental stages, e.g., as larvae or other immature forms. The insects may be reared in total darkness at from about 20° C. to about 30° C. and from about 30% to about 70% relative humidity. Methods of rearing insect larvae and performing bioassays are well known to one of ordinary skill in the art.
In some embodiments, the insecticidal effect is an effect wherein treatment with a composition causes at least about 10% of the exposed insects to die. In some embodiments, the insecticidal effect is an effect wherein treatment with a composition causes at least about 25% of the insects to die. In some embodiments the insecticidal effect is an effect wherein treatment with a composition causes at least about 50% of the exposed insects to die. In some embodiments the insecticidal effect is an effect wherein treatment with a composition causes at least about 75% of the exposed insects to die. In some embodiments the insecticidal effect is an effect wherein treatment with a composition causes at least about 90% of the exposed insects to die.
Beneficial insects that can be conserved by the present invention include (1) predatory beetles (see table 11) Harmonia arcuata (Fabricius) adults, Diomus notescens (Blackburn) adults, Coccinella repanda (Thunberg) adults, Dicranolauis bellulus (Guerin); (2) predatory bugs such as Geocoris lubra (kirkaldy adults, Cermatulus nasalis (Westwood) adults, Nabis capsiformis (Germar), (3) Spiders especially salticidae, Araneus spp. Oxypes spp. and (Parasitoids) Pterocormus promissorius (Erichson), Heteropelma scaposum (Morley), Netelia producta (Brulle).
Embodiments of the invention are also directed to making an improved insect control agent by identifying one or more fractions in a complex agent, screening the one or more fractions using the methods disclosed herein, and characterizing the one or more fractions as having a positive or negative effect on potential activity against a target insect.
In some embodiments, one or more fractions in a complex agent (such as, for example, an essential oil) can be isolated using fractionation techniques including, for example, differential solvent extraction, fractional distillation, fractional crystallization, fractional freezing, dry fractionation, detergent fractionation, solvent extraction, supercritical CO2 fractionation, vacuum distillation, column chromatography, reverse-phase chromatography, high-pressure liquid chromatography, and the like. These methods are known to those of skill in the art.
Vacuum distillation is preferred, because it is relatively simple to employ and does not require the use of solvents.
In some embodiments, one or more fractions of a complex agent can be isolated by column chromatography using silica or alumina solid support. An organic solvent, including for example, alkanes such as hexanes and petroleum ether, toluene, methylene chloride (or other halogenated hydrocarbons), diethyl ether, ethyl acetate, acetone, alcohol, acetic acid, and the like, can be used alone or in combination as the column solvent, or mobile phase. In some embodiments, the complex agent is fractionated by column chromatography using an increasing concentration of a polar solvent as eluting solvent. Methods of column chromatography and solvents common for its use are well known in the art.
In some embodiments, SPCs can be isolated by solvent extraction. For example, an essential oil can be combined with an organic solvent, including, for example an organic solvent such as methanol, acetic acid, acetone, acetonitrile, benzene, 1-butanol, 2-butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-dichloroethane, diethyl ether, diethylene glycol, diglyme (diethylene glycol dimethyl ether), 1,2-dimethoxy-ethane (glyme, DME), dimethylether, dimethyl-formamide (DMF), dimethyl sulfoxide (DMSO), dioxane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide (HMPA), hexamethylphosphorous triamide (HMPT), hexane, methyl t-butyl ether (MTBE), methylene chloride, JV-methyl-2-pyrrolidinone (NMP), nitromethane, pentane, petroleum ether (ligroine), 1-propanol, 2-propanol, pyridine, tetrahydrofuran (THF), toluene, triethyl amine, water, heavy water, o-xylene, m-xylene, and/or -xylene. Other organic solvents known to those of skill in the art can also be employed. The mixture of the SPC and the organic solvent can then be combined with an extraction solvent that is not miscible in the organic solvent, including, for example, water, ethanol, and methanol. This combination is shaken vigorously in a glass container such as a separatory funnel for several minutes, then allowed to settle into separate phases for several minutes. The lower, denser phase is then allowed to drain from the separatory funnel. The organic phase can then be repeatedly re-extracted with the extraction solvent to further partition compounds that are soluble in the extraction solvent from the organic phase. The volume of the organic phase and the extracted phase can then be reduced in volume using rotary evaporation, yielding two separate fractions of the SPC.
In some embodiments, a method for identifying an improved agent against a target insect can include the identification of the compounds present in either a complex agent or individual isolated fractions of a complex agent and screening of the ingredient compounds for their activity. Identification of the compounds can be performed by analyzing the complex agent or an isolated fraction thereof by High-Performance Liquid Chromatography (HPLC) or gas chromatography (GC) coupled with Mass Spectrometry (MS). Ingredient compounds can also be identified by first enriching or purifying individual ingredients to homogeneity using techniques including, for example, differential solvent extraction, fractional distillation, vacuum distillation, fractional crystallization, fractional freezing, dry fractionation, detergent fractionation, solvent extraction, supercritical CO2 fractionation, column chromatography, reverse-phase chromatography, high-pressure liquid chromatography, and the like. Enriched or purified components can be identified using spectroscopy techniques, including, for example, infrared (IR) spectroscopy, Raman spectroscopy, nuclear magnetic resonance spectroscopy (NMR), and the like.
Some embodiments relate to the use of chemical derivatives or analogs of chemicals identified to generate an improved agent against a target insect. Chemical derivatives of the chemicals identified can include compounds derivatised with an inorganic or organic functional group. In some embodiments, the chemical derivative is a compound derivatised with an organic functional group. In some embodiments, the organic functional group can be an alkyl group. In some embodiments, the organic functional group can be a methyl, ethyl, propyl, butyl, ceryl, decyl, heptyl, hexyl, myricyl, myristyl, nonyl, octyl, palmityl, pentyl, stearyl, isopropyl, isobutyl, lignoceryl, pentacosyl, heptacosyl, montanyl, nonacosyl, pentan-2-yl, isopentyl, 3-methylbutan-2-yl, tert-pentyl, neopentyl, undecyl, tridecyl, pentadecyl, margaryl, nonadecyl, arachidyl, henicosyl, behenyl, tricosyl, cyclobutyl, cyclopropyl group, or the like. In some embodiments, the organic functional group can be an aryl group. In some embodiments, the organic functional group can be a phenyl or biphenyl-4-yl group.
In some embodiments, the improved agent against a target insect includes a chemical derivative that is a halogenated derivative of a compound identified. In some embodiments, the chemical derivative is a fluorinated, chlorinated, brominated, or iodinated derivative.
In some embodiments, the improved agent against a target insect includes a chemical derivative that is an alkenylated derivative of a compound. In some embodiments, the chemical derivative is an oleylated, allylated, isopropenylated, vinylated, prenylated, or phytylated derivative.
In some embodiments, the improved agent against a target insect includes a chemical derivative that is a hydroxylated derivative of a compound identified.
In some embodiments, the improved agent against a target insect includes a chemical derivative that is a thiolated derivative of a compound identified.
In some embodiments, the improved agent against a target insect includes a chemical derivative that is a carboxylated derivative of a compound identified.
In some embodiments, the improved agent against a target insect includes a chemical derivative that is an amidated derivative of a compound identified.
In some embodiments, the improved agent against a target insect includes a chemical derivative that is an esterified derivative of a compound identified.
In some embodiments, the improved agent against a target insect includes a chemical derivative that is acylated derivative of a compound identified.
In some embodiments, the improved agent against a target insect includes a chemical derivative that is a sulfonated derivative of a compound identified.
In some embodiments, the improved agent against a target insect includes a chemical derivative that is derivatised by introducing a homologue of a substituent group.
In some embodiments, the improved agent against a target insect includes a chemical derivative that is derivatised by moving a substituent around a ring to a different position.
In some embodiments of the invention, the efficacy of a test composition can be determined by conducting studies with insects. For example, the efficacy of a test composition for killing an insect, altering its propensity to feed or lay eggs, or the like, an insect can be studied using controlled experiments wherein insects are exposed to the test composition. In some embodiments, the toxicity of a test composition against an insect can be studied using controlled experiments wherein insects are exposed to the test composition.
In some embodiments, the formulations consist of an emulsifier of high solvency and the capacity to form stable emulsions of the total formulation in water and a “carrier” oil which may also have pesticidal properties. A preferred “carrier” oil is an esterified vegetable oil.
MODES OF CARRYING OUT THE INVENTION
A study was conducted to identify plants that could be used as a trap or refuge crop within a commercial cotton system to control cotton pests. In this study, a wide range of plants such as lucerne, pigeon pea, sorghum, sweet corn and Clitoria ternatea and cotton genotypes were planted in 12 meter row strips within commercial cotton fields. Pest succession on these crops especially Helicoverpa spp. eggs and larval counts were carried out fortnightly throughout the cotton season. The results of the trial revealed that Helicoverpa spp. and other cotton pest infestation on Clitoria ternatea was significantly lower than that on cotton and other refuge crops. Thus the plant was thought to be containing some SPCs that may either kill or modify the behaviour of insect pests. Since Clitoria ternatea had not received previous attention in terms of identification of SPCs and use of these SPCs to control pests, it was anticipated that a bioassay-directed fractionalisation of the plant may reveal compounds or fractions for biological pest control previously unknown.
Example 1
Clitoria ternatea: Toxicity Feeding Experiments
Based on the results of the field trial that revealed that Helicoverpa spp. and other cotton pest infestation of Clitoria ternatea was significantly lower than that on cotton and other refuge crops tested, an investigation of possible reasons for this and the SPCs involved was undertaken. The study also determined the location in the plant structures that may contain the toxic compounds and the use of Clitoria ternatea as a formulated product to manage pests on agricultural crops.
Toxicity of Plant Structures to Helicoverpa spp. 1st and 2nd Instar Larvae
Clitoria ternatea structures or parts (viz; new, middle, old leaves; whole and cut pods; green (immature), mature seeds; crushed immature seeds and crushed mature seeds) were prepared and placed individually on a filter paper moistened with 100 μL of distilled water in a petri dish 12 mm in diameter. One second instar larva of Helicoverpa spp. was placed on each plant structure in each petri dish and sealed. Each treatment was replicated four times. The dishes were then placed into a Labec incubator running at 25° C. (±2° C.) with 14 hours light/10 hours dark and checked daily for mortalities for up to 9 days. Percent mortalities for each treatment were calculated at days 1-2, 3-4 and 5-9 days.
Analysis of Data
All experimental data were analysed using the ANOVA procedures of Instat, version 2.03 (Graphpad Instat Software Inc., San Diego, Calif., USA). Tukey-Kramer multiple comparison tests were used to separate the means.
Results
Table 1 presents the mortality of Helicoverpa spp on different parts of Clitoria ternatea (Clitoria ternatea). The results show that the toxin(s) in Clitoria ternatea are located in all plant parts with the highest in the leaves. H. punctigera was found to be more susceptible to the toxin(s) in Clitoria ternatea than H. armigera larvae.







TABLE 1







Mortality of second instar larvae of Helicoverpa


spp. on different structures of Clitoria ternatea










Plant
Percent Mortality













Species
Structure
Part
Day 1-2
Day 3-4
Day 5-9
Total
















H. armigera
Leaf
New
20.0
10.0
20.0
50.0




Middle
10.0
30.0
0
40.0




Old
20.0
30.0
31.1
81.1



Whole
Green
31.1
0
20.0
51.1



seed
Mature
63.3
—
—
63.3



Crushed
Green
20.0
11.1
40.0
71.1



seed
Mature
0
41.1
20.0
61.1


H. punctigera
Leaf
New
88.9
—
—
88.9




Middle
77.8
—
—
77.8




Old
88.9
—
—
88.9



Whole
Green
12.5
0
10.0
22.5



seed
Mature
50.0
—
—
50.0



Crushed
Green
44.4
0
10.0
54.4



seed
Mature
11.1
62.5
—
73.6





1 Control treatment: Newly opened cotton leaf as compared to Clitoria ternatea's leaf; Cotton square and boll as compared to Clitoria ternatea's pod and cotton seed as compared to Clitoria ternatea's seed. Larval mortality on cotton (control mortality) was used for calculation of corrected mortality.






Example 2
Crude Extraction of Clitoria ternatea 
Choice of Solvent and Extraction Procedure
Three solvents with different polarity used for extraction were water (high polarity), methanol (intermediate polarity) and hexane (apolar). Both surface rinsing and homogenisation were employed in the early stages of the study. The washing process involved shaking the leaves in solvent for 30 seconds and evaporating the solvent to a volume that achieved a concentration equivalent to 1 g fresh leaf weight per mL. Homogenates were prepared by submerging leaves in solvent overnight before blending in an industrial Waring blender for 10 minutes. The blended leaf material was then filtered and evaporated to the same adjusted volume as for the surface wash. In the case of hexane extraction, any aqueous phase was eliminated using a separating funnel and the hexane evaporated to the appropriate volume.
Methanol and hexane extracts were concentrated using a rotary evaporator and water extracts with a freeze drier.
Methanol Extract
For the preparation of methanolic extracts for SPE fractionation and later supply of fractions for bioassay, freeze-dried plant material was used. Up to twelve containers, each containing up to ca. 45 g plant material, were dried to constant weight (24-30 hours). Freeze-dried weight was generally 25%-30% of the wet weight.
Freeze-dried material (weight) was chopped into 2-3 mm portions and weighed into a 150 mL beaker. HPLC-grade methanol, 50 mL, was added and the sample ultrasonicated for 20 minutes. The solvent was filtered and the plant mass treated a further three times in the same way, with filtrates being combined, then evaporated to dryness using a rotary-evaporator. Water-bath temperature was maintained at 40° C.
Ethanol Extract
An exhaustive extraction was made on 500 g of air-dried, ground plant material using hot ethanol. The Soxhlet extractor was run for 24 hours and on evaporation of the solvent yielded 80 g of extract (16%). For comparison another extraction was performed on another sample using ethanol at ambient temperature and steeping over 24 hr. This process yielded 25 g of extract (5%) on the first steeping. No repeated extracts were made.
Fractionation of Extracts
Solid phase extraction (SPE) was used to fractionate extracts and to provide fractions for biological assays against insects using C18 hydrophobic silica-based solid phase SPE cartridges comprising a 40 μm/120 μm irregularly-shaped acid-washed silica, (60 Å mean porosity) with a trifunctional octadecyl bonded functional group (Varian Bond Elute (Part No. 12256001) or Phenomenex Strata C18-E (Part No. 8B-S001-JCH)). All cartridges were processed simultaneously on an Alltech Vacuum manifold (Part No. 210351)). The SPE cartridges were used to accommodate 1.5 g wet-weight of freeze-dried plant material and gave a better band definition when dried extracts were dispersed on Celite filter aid (Merck Celite 545, 0.02-0.1 mm) before being added to a conditioned cartridge as a solid. To this end, the bulb end of a Pasteur pipette was used to deliver a volume, measured to the constriction in the pipette neck (ca. 0.2 g), of Celite, 6 times to the remaining 9 mL of sample, which had been transferred, with rinsing, to a small pre-weighed vial which was attached to the rotary evaporator. After thorough mixing of the Celite, the vial contents were dried to a paste under N2, then to completeness using the rotary evaporator. The SPE cartridges were conditioned by passing through 5 mL of methanol followed by almost all of 5 mL of water. One-sixth of the dried sample/Celite residue was transferred by spatula to each of six conditioned SPE cartridges. The general procedure then involved passing 5 mL water through the column followed by aliquots of methanol/water mixtures, pure methanol, then acetone, with all eluates being collected.
HPLC Analysis
An Agilent 1100 instrument with diode array detector was used for the analytical-scale chromatography. The column was a silica based Phenomenex Luna 5 μm C18 (2), 150 mm×4.6 mm, maintained at 30° C. Varying volumes, but normally 20 μL, of filtered sample were analysed via an acidified methanol/water gradient elution program from 5% MeOH to 100% MeOH in 20 minutes, held to 28 minutes and returned to 5% MeOH by 30 minutes and held to 37 minutes to equilibrate. Both components of the mobile phase contain 0.5% acetic acid. Flow rate was usually 1 mL/min. The photo diode-array detector collected data at 254 nm, 280 nm, 360 nm, 430 nm and 450 nm. All chromatograms illustrated below were those acquired at 280 nm.
Results
The chemical and structural analyses of SPE fractions from a methanolic extract of Clitoria ternatea are given in FIG. 1. The uppermost trace is that of the crude methanol extract of the freeze-dried plant material. Those below represent the various fractions (1-6) sequentially eluted from an SPE cartridge as solvent polarity is increased. As expected, the reversed-phase separation process common to both the SPE fractionation and the HPLC analysis results in chemical components with greater affinity for the stationary phase appearing at longer retention times in later fractions.
Example 3
Efficacy of Clitoria ternatea Fractions on Oviposition of Helicoverpa spp. on Cotton Plants in the Mesh House (No-Choice Test)
The experiment was conducted to determine oviposition deterrent activities of fractions isolated from Clitoria ternatea. The Clitoria ternatea fractions tested were (1) Fraction 1, (2) Fraction 2, (3) Fraction 3, (4) Fraction 4, (5) Fraction 5 and (6) Fraction 6, and (7) Untreated (control). Each treatment was replicated 4 times in a complete randomised design. Plants used in the experiment were grown in 8 cm diameter pots in black soil (from the field) and watered three times a week. The plants were fertilised once. Potted plants were kept and maintained in the mesh house which allowed a greater degree of exposure to the natural environment but protected them from potential pest infestation. Once the plants had reached the 4-true-leaf stage, 0.25 mL of extract of each treatment was placed on each leaf (1 mL in one pot) and spread evenly over the surface. The plants were then covered and three mated (5 day post emergence) female moths were released into the cages. Eggs were counted three days after treatment. The Oviposition Deterrent Index (ODI) was calculated as follows: ODI=100×(C−T)/(C+T) where C=total eggs laid in control; T=total eggs in treated filter paper.
Analysis of Data
All experimental data were analysed using the ANOVA procedures of Instat, version 2.03 (Graphpad Instat Software Inc., San Diego, Calif., USA). Tukey-Kramer multiple comparison tests were used to separate the means
Results
No choice tests conducted under laboratory conditions using H. armigera adult females confirmed that Clitoria ternatea fraction 3 and fraction 4 (Table 2) had significantly lower (P<0.001) number of eggs per plant laid on them than all the other fractions tested (Table 2). Thus the two fractions may contain oviposition deterring compounds.







TABLE 2







No-choice oviposition test of Helicoverpa


armigera females on filter papers treated with Clitoria


ternatea fractions 1-3












No. eggs/plant ±
1Oviposition



Treatments
SE
Deterrent Index (ODI)







Fraction 1
32.75 ± 25.39 a
 20.1 a



Fraction 2
41.00 ± 14.41 a
 9.1 a



Fraction 3
17.50 ± 7.84 b
 47.6 b



Fraction 4
 6.50 ± 1.19 a
 42.2 a



Fraction 5
58.25 ± 22.96 b
−56.9 b



Fraction 6
35.00 ± 10.40 b
−37.3 b



Control (water)
49.25 ± 17.21 a
 0.0 a







Means within columns followed by the same letter are not significantly different (P > 0.05) (Tukey-Kramer Multiple comparison test).



1Oviposition Deterrent Index (ODI) was calculated as follows: ODI = 100 × (C − T)/(C + T); where C = Total eggs laid in control; T = total eggs in treated filter paper.






Example 4
Efficacy of Clitoria ternatea Fractions on the Feeding of Helicoverpa spp. 2nd Instar Larvae on Leaf Discs of Cotton Plants in the Laboratory (No-Choice Test)
The experiment was conducted using cotton leaves in a no-choice test. H. armigera 2nd instar larvae were used for the feeding bioassays. The Clitoria ternatea fractions tested were (1) Fraction 1, (2) Fraction 2, (3) Fraction 3, (4) Fraction 4, (5) Fraction 5, (6) Fraction 6, and (7) Water-treated (control). Each treatment was replicated 4 times in a complete randomised design. Three day-old cotton leaves taken from plants grown in the glasshouse were used for the study. The leaves were treated with 1 mL of extract of each treatment. To prevent preference for or avoidance of the extract, 0.5 mL was applied to each side of the leaf surface. The leaves were allowed to air dry in the fume hood for 1 hour.
Once air dried, a 25 mm disc of each treated leaf was cut out, weighed and placed in a 55 mm petri dish. The filter paper was moistened with 100 μL of distilled water to prevent the disc from drying out. One larva of the desired size was weighed and placed into each dish. The dishes were then placed into a Labec incubator running at 25° C. (±2° C.) with 14 hours light/10 hours dark for 48 hours.
Forty-eight hours after treatment, both the leaf discs and the larvae were then weighed. The differences in weights of both the leaf discs and larvae before and after the experiments were calculated for each treatment and control to determine any antifeedant effect of the treatments.
Analysis of Data
All experimental data were analysed using the ANOVA procedures of Instat, version 2.03 (Graphpad Instat Software Inc., San Diego, Calif., USA). Tukey-Kramer multiple comparison tests were used to separate the means
Results
In no-choice bioassays conducted using the fractions of Clitoria ternatea and H. armigera 2nd and 3rd instar larvae, leaves treated with fractions 2, 4 and 6 were consumed at lower levels and resulted in lower weight gains by the larvae compared to the other fractions and the control tested (FIG. 2a). Fraction 2 appears to have a stronger deterrent effect than fraction 4 and 6 so much so that the 2nd instar resulted in a weight loss (FIG. 2b).
Example 5
Efficacy of Clitoria ternatea Fractions on Toxicity of Helicoverpa spp. 2nd Instar Larvae in the Laboratory (No-Choice Test)
The experiment was using cotton leaves in a no-choice test. H. armigera 2nd instar larvae were used for the feeding bioassays. The Clitoria ternatea fractions tested were (1) Fraction 1, (2) Fraction 2, (3) Fraction 3, (4) Fraction 4, (5) Fraction 5, (6) Fraction 6, and (7) Water-treated (control). Each treatment was replicated 4 times in a complete randomised design. Three day-old cotton leaves taken from plants grown in the glasshouse were used for the study. The leaves were treated with 1 mL of extract of each treatment. To prevent preference for or avoidance of the extract, 0.5 mL was applied to each side of the leaf surface. The leaves were allowed to air dry in the fume hood for 1 hour.
Once air dried, one 2nd instar larva was placed in a petri dish with a leaf disc 25 mm in diameter, on a filter paper moistened with 100 μl of distilled water and sealed. The dishes were then placed into a Labec incubator running © 25° C. (±2° C.) with 14 hours light/10 hours dark and checked daily for larval mortalities for 3 days.
Analysis of Data
All experimental data were analysed using the ANOVA procedures of Instat, version 2.03 (Graphpad Instat Software Inc., San Diego, Calif., USA). Tukey-Kramer multiple comparison tests were used to separate the means.
Results
The results of the study also showed that the Clitoria ternatea fractions had antibiotic effects on 2nd instar H. armigera larvae. The experiments showed that fractions 2, 3 and 4 contain compounds toxic to the larvae due to the higher mortality rates in these treatments after 48 hours than the control (FIG. 3). This figure indicates that after 48 hours 7 of the 10 larvae tested were dead within 48 hour period.
Example 6
Feeding Deterrent Activity of Clitoria ternatea Fraction 2 Applied to Leaves of Susceptible Cotton Genotype (Lumein)
Masking experiments were conducted to observe and quantify the feeding response effects or antifeedant effects of Helicoverpa spp. 2nd instar larvae towards a cotton genotype known to stimulate Helicoverpa spp. larval feeding. Tests were conducted in the laboratory at ACRI using H. armigera 2nd instar larvae to determine the effects of the interactions of using a combination of an identified feeding stimulant (Lu Mein fraction 3) and a feeding deterrent (Clitoria ternatea fraction 2).
In this experiment, Clitoria ternatea fraction 2 was applied to leaf discs of a cotton genotype called Lu Mein. A second leaf disc was treated with water. Once air dried, one 2nd instar larva was placed in a petri dish with a leaf disc 25 mm in diameter, on a filter paper moistened with 100 μL of distilled water and sealed. The experiment was replicated 10 times in a randomised complete block design. The dishes were then placed into a Labec incubator running at 25° C. (±2° C.) with 14 hours light/10 hours dark. The larvae were left to feed for 48 hours and the weight of leaf consumed and larval weight were calculated and analysed for differences in leaf consumed and weight loss or gain between the Clitoria ternatea fraction 2-treated and water-treated leaf discs.
Analysis of Data
All experimental data were analysed using the ANOVA procedures of Instat, version 2.03 (Graphpad Instat Software Inc., San Diego, Calif., USA). Tukey-Kramer multiple comparison tests were used to separate the means.
Results
The results of the study showed that Clitoria ternatea fraction 2 masked the feeding stimulant effect of the Lu Mein leaves reducing the quantity of leaf consumed per larvae compared to the water-treated (FIG. 4). This resulted in a lower larval weight gain compared to the control (FIG. 5).
Example 7
Effect of Clitoria ternatea Fractions Formulated in Hexane on Oviposition of Helicoverpa spp. on Cotton Plants
Clitoria ternatea fractions 2, 3 and 4 were combined and formulated in hexane for studies to determine the oviposition of Helicoverpa spp. in the mesh house. Three experiments were conducted in a mesh cage (100 cm×50 cm×70 cm) when the plants were at a 6 true leaf stage. Four treatments representing concentrations of 0 (control), 20% v/v, 15% v/v and 10% v/v of the Clitoria ternatea formulations were used. Ten cotton plants were randomly allocated to each treatment and enclosed in the mesh cage. Each treatment was applied to run-off using a small hand-held sprayer. The control plants were sprayed with water. Each treatment was replicated 4 times in different cages in a complete randomised design. Twenty mated female moths were released into each cage to lay on the treated plants. At three days after treatment, the number of eggs per plant in each treatment was counted and the number of eggs per plant calculated. Data was expressed as number of eggs per plant per treatment.
Analysis of Data
All experimental data were analysed using repeated measures ANOVA (Graphpad Instat Software, Inc., v2.03, San Diego, Calif., USA). Treatment and sample dates were the independent variables. Tukey-Kramer Multiple Comparisons test was used to separate the means.
Results
The results of the study showed that the number of eggs per meter recorded on cotton plants treated with combined Clitoria ternatea fractions at 10%, 15% and 20% v/v were significantly lower (P<0.001) than the untreated (control) plants in all 3 experiments conducted (FIGS. 6a,b and 7). No significant difference (P>0.05) was detected among the Clitoria ternatea concentrations indicating 10% v/v concentration was as efficacious as the 15 and 20% v/v concentrations (FIGS. 6A,B and 7). It was also observed that the Hexane formulation was toxic to the cotton plants causing burns on the leaves.
Example 8
Field Experiments with Clitoria ternatea fractions—Effect of Combined Clitoria ternatea Fractions Formulated in Hexane on Oviposition of Helicoverpa spp. on Cotton Plants
Clitoria ternatea formulations 2, 3 and 4 were formulated in hexane and used for field trials on conventional cotton crops against Helicoverpa spp. The trial was conducted on commercial conventional cotton crops. The Clitoria ternatea formulation was evaluated at 3 different concentrations (1) 20 v/v (2) 15 v/v (3) 10% v/v and (4) Control (Unsprayed) for efficacy against is Helicoverpa spp. eggs and larvae. Each treatment was replicated 4 times in a randomised complete block design. Each replicate or treated plot measured 100 meters long and 6 meters or rows wide. Foliar application of the different rates of the formulated Clitoria ternatea fractions was applied in 15 liters of water twice during the trial 24 days apart. Helicoverpa spp. eggs and larvae were assessed visually on one meter row of randomly selected cotton plants. Pre-treatment counts were taken one day before treatment application, and post-treatment counts 7, 14, 21 and 28 days after treatment. Data was expressed as number of Helicoverpa spp. eggs and larvae per meter per sample date.
Analysis of Data
All experimental data were analysed using repeated measures ANOVA (Graphpad Instat Software, Inc., v2.03, San Diego, Calif., USA). Treatment and sample dates were the independent variables. Tukey-Kramer Multiple Comparisons test was used to separate the means.
Results
A significant difference (P<0.001) was found among cotton plants treated with Clitoria ternatea fractions and the untreated (control) plants (FIG. 8). However, no significant difference (P>0.05) was detected in oviposition deterrent activity between the Clitoria ternatea concentrations (FIG. 8).
The number of Helicoverpa spp. very small and small (1st-3rd instar) larvae per meter per sample date recorded on plots treated with different concentrations of Clitoria ternatea were significantly lower (P<0.001) than the untreated (control) plots (FIG. 7). However, no significant difference (P>0.05) was found among the different concentrations of the Clitoria ternatea products tested (FIG. 8).
Example 9
Efficacy of Clitoria ternatea Formulations on Oviposition of Helicoverpa armigera Females on Cotton Plants in the Mesh House
The oviposition response of mated Helicoverpa spp. to potted cotton plants treated with different concentrations of Clitoria ternatea in Canola oil and Crude cotton seed oil was evaluated. The plants used in the study were potted squaring cotton plants of the same age. The experiment was conducted under no-choice conditions. The treatments evaluated were (1) 1% Clitoria ternatea in Canola oil, (2) 2% Clitoria ternatea in Canola, (3) 1% Clitoria ternatea in crude Cotton seed oil, (4) 2% Clitoria ternatea in crude cotton seed oil and (5) Control (water). Each treatment was replicated 8 times with each treatment containing 10 plants. Each treatment was applied to the respective cotton plants until run-off. Each treatment replicate of 10 plants were enclosed in a plastic cage containing 4 mated H. armigera female to oviposition the plants. Four days after treatment, the eggs on each plant was counted and recorded. Data was expressed as number of eggs per plant.
Analysis of Data
All experimental data were analysed using the ANOVA procedures of Instat, version 2.03 (Graphpad Instat Software Inc., San Diego, Calif., USA). Tukey-Kramer multiple comparison tests were used to separate the means.
Results
No significant difference in the number of eggs per plant was detected among plants treated with the different Clitoria ternatea formulations (Table 3). However significant differences in the number of eggs per plant were detected between the treated and the control plants (Table 3). Significantly higher (P<0.0001) eggs per plant was recorded on the control plants compared to the Clitoria ternatea-Oil formulations (Table 3).







TABLE 3







Effect of Clitoria ternatea in oil formulation on


oviposition of H. armigera. (replicates per treatment)


(n = 100 mated pairs of H. armigera females).










Treatments
No. eggs per plant







1% Clitoria ternatea +
18.25 ± 3.16 a



Canola oil



2% Clitoria ternatea +
21.13 ± 1.84 a



Canola oil



1% Clitoria ternatea +
21.38 ± 3.06 a



Cotton seed oil



2% Clitoria ternatea + crude
18.63 ± 2.87 a



cotton seed oil



Control (Unsprayed)
37.88 ± 3.62 b







Means between treatments followed by the same letters are not significantly different (P > 0.05) by Tukey-Kramer Multiple comparison test






Example 10
Effect of Clitoria ternatea Formulations on Mortalities of H. armigera Larvae in the Laboratory
The study was conducted on Helicoverpa spp. artificial diet in the laboratory at ACRI. During this experiment the laboratory was maintained at a temperature of 25° C. and a relative humidity of 55-60%. The treatments evaluated were (1) 1% v/v Clitoria ternatea in Cotton seed oil, (2) 1% v/v Clitoria ternatea in crude cotton seed oil, (3) 1% v/v Clitoria ternatea in Canola oil and (4) Control (water). For each concentration, I sprayed a total of 48 2nd instar larvae (12 larvae/replicate) until run off. After spray application, larvae from each treatment were transferred and kept separately in 35 mL clear plastic containers (P10M; Solo, Urbana, Illionis, USA) containing a soybean-based artificial diet mixed individually with each treatment. Each treatment was replicated 4 times. The number of dead larvae were counted and recorded in each treatment at 14 days after treatment when all larvae in the control had pupated.
Analysis of Data
All experimental data were analysed using the ANOVA procedures of Instat, version 2.03 (Graphpad Instat Software Inc., San Diego, Calif., USA). Tukey-Kramer multiple comparison tests were used to separate the means.
Results
At 14 DAT, the number of dead larvae was highest in the 1 v/v Clitoria ternatea in crude cotton seed oil, followed by 1 v/v Clitoria ternatea in canola and cotton seed oils (FIG. 9). No deaths were recorded in the larvae treated with water (control) (FIG. 9). Additionally, most of the larvae that were not dead in the Clitoria ternatea treated plots were sick and sluggish (FIG. 9). There were no sick larvae in the control and all larvae had pupated. Overall, the development of the Clitoria ternatea treated larvae was delayed by an average of 4 days relative to the control.
Example 11
Efficacy of Clitoria ternatea Oil Formulations on Pests and Beneficial Insects in Conventional Cotton
The study was conducted on late season commercial conventional cotton crop. The trial did not target any specific pest but treatments were applied to the cotton crops and evaluated for efficacy against any pest and predatory insects that were on the crop during that period of the cotton season in the lower Namoi region. The treatment evaluated were (1) 1 v/v Clitoria ternatea in Canola oil, (2) 2% v/v Clitoria ternatea in Canola oil, (3) 1% v/v Clitoria ternatea in Crude cotton seed oil, (4) 2% v/v Clitoria ternatea in Crude Cotton seed oil, (5) Unsprayed (control). Each treatment was replicated 3 times in a randomised complete block design. Each replicate measured 100 meters long and 6 rows or meters wide.
Foliar application of each treatment was made on day 1. Visual counts of the following pests green mirids (Creontiades dilutus), Cotton loopers (Anomis flava), Green vegetable bug (Nezara viridula) and Apple dimpling bug (Campylomma liebknechti), and predatory insects (predatory beetles, bugs, lacewings and spiders) which is were abundant in the study site at the period of the trials in each treatment were made 24 hours before treatment and 3, 5 and 7 days after treatment. Counts were made in a two randomly selected 1 meter lengths of row of cotton in each treatment replicate, i.e. a total of 6 meters per treatment. Counts were separated into the various pests and predators. The beneficial insects were grouped into predatory beetles, bugs, lacewings and spiders. Data were expressed in numbers per meter.
Analysis of Data
All experimental data were analysed using repeated measures ANOVA (Graphpad Instat Software, Inc, v2.03, San Diego, Calif., USA). Treatment and sample dates were the independent variables. Tukey-Kramer Multiple comparisons tests were used to separate means.
Results
Predominant pests in the trial site during the trial period were green mirids, cotton loopers, green vegetable bugs and apple dimpling bugs. Helicoverpa spp. were absent at the trial site. Therefore, the data reported here are on those pests that were abundant during the test period.
Effect of Clitoria ternatea on Green Mirids
The number of green mirids per meter recorded in the study plots ranged from 0.17 to 0.33 per meter (Table 4). At 3 DAT, all the Clitoria ternatea treated plots had no mirids recorded in them whereas the unsprayed plot recorded 0.33 per meter (Table 4). At 5 DAT, the number of mirids per meter recorded in plots treated with 2% v/v Clitoria ternatea were significantly different (P<0.01) than unsprayed plots but were not significantly different (P>0.05) from plots treated with 1% v/v Clitoria ternatea (Table 4). The number of green mirids recorded on plots treated with 1% v/v Clitoria ternatea was not is significantly different from the unsprayed (control) plots at 5 DAT (Table 4). At 7 DAT, number of green mirids per meter recorded on treated and control; plots were not significantly different (Table 4). This may be due to hatching of mirid eggs that were laid on the plants pre-treatment and also lack of good coverage of the products as many of the plants were dislodged as a result of the heavy boll load.
Effect of Clitoria ternatea on Cotton Loopers They were the most abundant insect species on the cotton crop at the study site during the trials. The study showed that significantly lower (P<0.05) cotton loopers per meter were recorded on Clitoria ternatea treated plots compared to the control (Table 5). At 3 DAT, Cotton looper mortalities was highest on plots treated with 2% Clitoria ternatea in Crude cotton seed oil (58.9%), followed by 11 Clitoria ternatea in crude cotton seed oil (47.2%) (Table 5). The 1 and 2% Clitoria ternatea in Canola seed oils caused 26.6 and 42.9% mortalities respectively (Table 5). The unsprayed plot recorded a 10% increase in Cotton looper population at the same time (Table 5).







TABLE 4







Efficacy of different rates of Clitoris ternatea


on the number of green mirids per metre recorded on


commercial cotton crops











Treatments
Pre-treatment
3 DAT
5 DAT
7 DAT





1% Clitoria
0.17 ± 0.17 a
0.00 ± 0.00 a
0.33 ± 0.21ab
0.33 ± 0.21a


ternatea + Canola oil


2% Clitoria
0.17 ± 0.17 a
0.00 ± 0.00 a
0.17 ± 0.17 a
0.17 ± 0.17a


ternatea + Canola oil


1% Clitoria
0.33 ± 0.21 a
0.00 ± 0.00 a
0.33 ± 0.21ab
0.33 ± 0.21a


ternatea +


Crude Cotton oil


2% Clitoria
0.17 ± 0.17 a
0.00 ± 0.00a
0.17 ± 0.17 a
0.00 ± 0.00a


ternatea + Crude


Cotton oil


Control (Unsprayed)
0.17 ± 0.17 a
0.33 ± 0.21 b
0.50 ± 0.22 b
0.33 ± 0.21a





Means within columns followed by same letters are not significantly different (P > 0.05) (Tukey-Kramer Multiple Comparison Test).













TABLE 5







Efficacy of different rates of Clitoria ternatea


on the number of Cotton Looper (Anomis flava) per metre


recorded on commercial cotton crops











Treatments
Pre-treatment
3 DAT
5 DAT
7 DAT





1% Clitoria ternatea +
5.00 ± 1.16 a
3.67 ± 0.76 a
4.33 ± 0.96 a
5.83 ± 0.95a


Canola oil


2% Clitoria ternatea +
5.83 ± 0.91 a
3.33 ± 1.02 a
3.00 ± 0.68 a
3.33 ± 0.62a


Canola oil


1% Clitoria ternatea +
6.00 ± 1.48 a
3.17 ± 0.87 a
3.67 ± 0.88 a
3.83 ± 1.33a


Crude Cotton oil


2% Clitoria ternatea +
5.67 ± 1.09 a
2.33 ± 0.67a
3.83 ± 0.75 a
2.67 ± 0.80a


Crude Cotton oil


Control (Unsprayed)
8.33 ± 1.41a
9.17 ± 1.72 b
6.83 ± 1.92 a
7.17 ± 0.83a





Means within columns followed by same letters are not significantly different (P > 0.05) (Tukey-Kramer Multiple Comparison Test).






Effect of Clitoria ternatea on Apple Dimpling Bug

The Clitoria ternatea formulation at different concentrations had significant effect on ADB population in the study site at 3 DAT (Table 6). The highest ADB kill at 3 DAT was recorded in plots treated with 2% Clitoria ternatea in Crude cotton seed oil (65.9%), followed by 2% Clitoria ternatea in Canola oil (64.0%) (Table 6). The 1% Clitoria ternatea in Canola and crude cotton seed oils recorded 56.8 and 52.1% mortalities respectively at 3 DAT (Table 6).







TABLE 6







Efficacy of different rates of Clitoria ternatea


on the number of Apple Dimpling bugs (Campylomma


liebknechti) per metre recorded on commercial cotton crops











Treatments
Pre-treatment
3 DAT
5 DAT
7 DAT





1% Clitoria
4.67 ± 0.84 a
0.67 ± 0.33 a
0.50 ± 0.22ab
0.33 ± 0.21a


ternatea + Canola oil


2% Clitoria
2.67 ± 0.42 a
0.50 ± 0.22 a
0.00 ± 0.00 a
0.00 ± 0.00a


ternatea + Canola oil


1% Clitoria
3.33 ± 0.84 a
0.83 ± 0.31 a
0.17 ± 0.17 a
0.33 ± 0.21a


ternatea +


Crude Cotton oil


2% Clitoria
4.67 ± 0.65 a
0.83 ± 0.31a
0.17 ± 0.17 a
0.00 ± 0.00a


ternatea + Crude


Cotton oil


Control (Unsprayed)
4.17 ± 0.70 a
2.17 ± 0.31 b
1.00 ± 0.26 b
0.83 ± 0.17a





Means within columns followed by same letters are not significantly different (P > 0.05) (Tukey-Kramer Multiple Comparison Test)






Beneficial Insects

Beneficial insects identified from the treated plots were predominantly predators and these include predatory beetles, bugs, lacewings and spiders (Table 7).







TABLE 7







Predators of cotton pests sampled and identified


from study plots.










Order
Family
Species
Group





Coleoptera
Coccinellidae
Coccinella transversalis
Predatory




(Fabricius)
beetles




Diomus notescens




(Blackburn)



Melyridae
Dicranolauis bellulus




(Guerin-Meneville)


Hemiptera
Nabidae
Nabis capsiformis
Predatory




(Germar)
bugs



Lygaeidae
Geocoris lubra




(Kirkaldy)



Pentatomidae
Cermatulus nasalis




(Westwood)




Ochelia schellenbergii




(Guerin-Meneville)




Coranus triabeatus




(Horvath)


Neuroptera
Chrysopidae
Chrysopa spp.
Predatory





lacewings



Hemerobiidae
Micromus tasmaniae




(walker)


Araneida
Lycosidae
Lycosa spp.
spiders



Oxyopidae
Oxyopes spp.



Salticidae
Salticidae spp.



Araneidae
Araneus spp.









No significant difference was detected among the treatments and control in the number of predatory beetles (Table 8), bugs (Table 9), lacewings (Table 10) and spiders (Table 11). This indicates that Clitoria ternatea products have no effect on beneficial insects in cotton.







TABLE 8







Efficacy of different rates of BC639 fungal


insecticides on the number of predatory beetles per metre


on commercial conventional cotton crops.












Pre-





Treatments
treatment
3 DAT
5 DAT
7 DAT





1% Clitoria
0.50 ± 0.22 a
1.17 ± 0.48 a
1.00 ± 0.37 a
0.67 ± 0.21 a


ternatea + Canola


oil


2% Clitoria
1.33 ± 0.42 a
1.33 ± 0.49 a
0.83 ± 0.31 a
1.33 ± 0.21 a


ternatea + Canola


oil


1% Clitoria
1.00 ± 0.26 a
1.17 ± 0.31 a
1.83 ± 0.70 a
1.00 ± 0.21 a


ternatea +


Crude Cotton oil


2% Clitoria
0.67 ± 0.21 a
1.33 ± 0.33 a
0.87 ± 0.21 a
0.67 ± 0.00 a


ternatea + Crude


Cotton oil


Control (Unsprayed)
0.67 ± 0.21 a
2.17 ± 0.17 a
2.83 ± 0.87 a
1.67 ± 0.22a





Means within columns followed by same letters are not significantly different (P > 0.05) (Tukey-Kramer Multiple Comparison Test)













TABLE 9







Efficacy of different rates of BC639 fungal


insecticides on the number of predatory bugs per metre on


commercial conventional cotton crops.












Pre-





Treatments
treatment
3 DAT
5 DAT
7 DAT





1% Clitoria
1.00 ± 0.37 a
2.17 ± 0.40 a
2.33 ± 0.33 a
2.17 ± 0.48 a


ternatea + Canola


oil


2% Clitoria
0.83 ± 0.31 a
2.50 ± 0.43 a
2.83 ± 0.48 a
2.17 ± 0.31 a


ternatea + Canola


oil


1% Clitoria
1.00 ± 0.37 a
2.33 ± 0.33 a
2.67 ± 0.62 a
2.00 ± 0.31 a


ternatea +


Crude Cotton oil


2% Clitoria
0.83 ± 0.31 a
1.33 ± 0.21 a
1.83 ± 0.48 a
2.33 ± 0.33 a


ternatea + Crude


Cotton oil


Control (Unsprayed)
1.50 ± 0.43 a
2.50 ± 0.34 a
2.67 ± 0.42 a
4.17 ± 0.26 a





Means within columns followed by same letters are not significantly different (P > 0.05) (Tukey-Kramer Multiple Comparison Test)













TABLE 10







Efficacy of different rates of BC639 fungal


insecticides on the number of predatory lacewings per


metre on commercial conventional cotton crops.












Pre-





Treatments
treatment
3 DAT
5 DAT
7 DAT





1% Clitoria
0.50 ± 0.22 a
1.00 ± 0.26 a
0.83 ± 0.17 a
0.50 ± 0.22 a


ternatea + Canola


oil


2% Clitoria
0.33 ± 0.21 a
1.50 ± 0.43 a
1.00 ± 0.26 a
0.33 ± 0.21 a


ternatea + Canola


oil


1% Clitoria
0.33 ± 0.21 a
1.67 ± 0.42 a
0.83 ± 0.31 a
0.50 ± 0.22 a


ternatea +


Crude Cotton oil


2% Clitoria
0.50 ± 0.22 a
0.67 ± 0.33 a
1.00 ± 0.26 a
0.33 ± 0.21 a


ternatea + Crude


Cotton oil


Control (Unsprayed)
0.67 ± 0.21 a
0.83 ± 0.31 a
0.83 ± 0.33 a
0.50 ± 0.34 a





Means within columns followed by same letters are not significantly different (P > 0.05) (Tukey-Kramer Multiple Comparison Test)













TABLE 11







Efficacy of different rates of BC639 fungal


insecticides on the number of spiders per metre on


commercial conventional cotton crops












Pre-





Treatments
treatment
3 DAT
5 DAT
7 DAT





1% Clitoria
6.33 ± 1.54 a
3.50 ± 1.15 a
3.00 ± 0.78 a
4.17 ± 0.60 a


ternatea + Canola


oil


2% Clitoria
3.00 ± 0.68 a
3.33 ± 0.56 a
3.33 ± 0.67 a
2.33 ± 0.72 a


ternatea + Canola


oil


1% Clitoria
5.17 ± 0.79 a
2.50 ± 0.43 a
2.83 ± 0.83 a
3.17 ± 0.40 a


ternatea +


Crude Cotton oil


2% Clitoria
3.67 ± 0.84 a
3.00 ± 1.00 a
2.00 ± 0.68 a
2.83 ± 0.60 a


ternatea + Crude


Cotton oil


Control (Unsprayed)
5.67 ± 0.95 a
4.17 ± 0.87 a
3.17 ± 1.07 a
3.17 ± 0.91 a





Means within columns followed by same letters are not significantly different (P > 0.05) (Tukey-Kramer Multiple Comparison Test)






Example 12
Formulation of Clitoria ternatea for Laboratory Trials Against Cotton Pests
Clitoria ternatea material was harvested at different growth stages at three different locations. The extracts were made by heating dry, macerated plant (30 g for each solvent) in an 80° C. water bath for 1 hour. Filtration and evaporation were carried out after 24 hours. The solutions were evaporated under vacuum at 400° C., while the evaporation of the water solution was performed using the water bath. The solutions were evaporated until fractionalized concentrates were obtained. The fractionalized concentrates were formulated in surfactants (C12-C15 Ethoxylate and Teric® (Huntsman), an emulsifier (Termul® 3000) and a light molecular weight oil such as crude cotton seed oil, refined cotton seed oil and canola oil. Vacuum and pressure was applied to ensure efficient filtration.
Four formulations were developed for use in laboratory and field bioassays against cotton pests and beneficial insects. The formulations were:
(1) Clitoria ternatea formulation in oil without emulsifiers (Formulation A),
(2) Formulation with emulsifiers but without Clitoria ternatea (Formulation B),
(3) Clitoria ternatea formulations with emulsifiers (Formulation C) and
(4) Clitoria ternatea formulations with emulsifiers (Formulation D).
Effect of Different Rates of Clitoria ternatea on Mortalities of Green Mirids in the Laboratory
The four formulations were evaluated at 0.5%, 1.0%, 1.5% and 2.0% (v/v) in the laboratory at a temperature of 25° C. and relative humidity of 55-60%. Water was used as control. For each formulation, a total of 6 (3 pairs) adult female and male mirids per treatment (1 pair/replicate) on beans were sprayed until run off. In addition the beans were also dipped for 60 seconds in each treatment. After treatment applications, a pair of green mirids (male and female) were transferred and released on dipped beans kept separately in 35 mL clear plastic containers (P101M; Solo, Urbana, Illionis, USA). Each treatment was replicated 3 times. The number of dead mirid adults were counted and recorded daily until all insects in the most efficacious treatment die. The percent mortality was calculated relative to the control.
Analysis of the Data
All experimental data were analysed using repeated measures ANOVA (Graphpad Instat and Prism Software, Inc. v. 2.03, San Diego, Calif., USA). Treatment and sample dates were the independent variables. Tukey-Kramer Multiple comparisons tests were used to separate means.
Results
Significant differences (P<0.01) were detected among treatments (Table 12). Fewer mirids were killed when Clitoria ternatea was applied at 0.5% v/v. As the rate of application increased the number of green mirids killed increased significantly (Table 12). Formulation A caused the highest mortalities to green mirid adults at all the rates tested (Table 12). At 1% v/v rate Formulation A caused 55.7% mortality compared to 44.3% caused by Formulation C and Formulation D respectively (Table 12). The highest mortality was caused by Formulation A (100%), Formulation C (89%) and Formulation D (100%) when the products were applied at 2% v/v rate (Table 12). The mortality to green mirids caused by Formulation B which did not contain Clitoria ternatea was significantly lower (P<0.01) than the other formulations containing Clitoria ternatea, and was not significantly different from the insects treated with water (control) (Table 12).







TABLE 12







Effect of direct spray application and residues


of canola-oil based Clitoria ternatea on mortalities of


green mirid adults in the laboratory.












0.5% v/v
1.0% v/v
1.5% v/v
2.0% v/v


Treatments
(% mortality)
(% mortality)
(% mortality)
(% mortality)





Formulation A
2.00 ± 0.58 a
1.33 ± 0.33 a
0.33 ± 0.33 a
0.00 ± 0.00 a



(33.3%)
(55.7%)
(89.0%)
(100.0%)


Formulation B
2.67 ± 0.33 b
2.67 ± 0.33 b
2.67 ± 0.33 b
2.67 ± 0.33 b



(11.0%)
(11.0%)
(11.0%)
 (11.0%)


Formulation C
2.00 ± 0.58 a
1.67 ± 0.67 a
1.67 ± 0.33 a
0.33 ± 0.33 a



(33.3%)
(44.3%)
(44.3%)
 (89.0%)


Formulation D
2.33 ± 0.33 a
1.67 ± 0.33 a
1.33 ± 0.33 a
0.00 ± 0.00 a



(22.3%)
(44.3%)
(55.7%)
(100.0%)


Water
3.00 ± 0.33 b
3.00 ± 0.33 b
3.00 ± 0.33 b
3.00 ± 0.33 b


(control)


Level of
P < 0.01
P < 0.001
P < 0.01
P < 0.01


significance





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.






Example 13
Efficacy of Combination of Direct Application and Residues of Clitoria ternatea on Survival of Green Mirid Adults
The Clitoria ternatea formulations Formulation A, Formulation C and Formulation D were evaluated at 1 and 2 L/ha in the laboratory with temperature at 25° C. and 60-70% RH. Water was used as a control. For each formulation, a total of 6 (3 pairs) adult female and male mirids per treatment (1 pair/replicate) were sprayed on beans until run off. The beans were also dipped for 60 seconds in each formulation and transferred and kept separately in 35 mL clear plastic containers (P101M; Solo, Urbana, Illionis, USA). Thereafter, one male and female green mirids (a pair) were released onto the beans in the plastic containers. Each treatment was replicated 3 times. The number of dead mirid adults (males and females) were counted and recorded at 3, 5, 7, 9 and 11 days after treatment and the percent mortality was calculated relative to the control.
Analysis of the Data
All experimental data were analysed using repeated measures ANOVA (Graphpad Instat and Prism Software, Inc. v. 2.03, San Diego, Calif., USA). Treatment and sample dates were the independent variables. Tukey-Kramer Multiple comparisons tests were used to separate means.
Results
Efficacy on Mortality of Green Mirid Adult Males
At 3 DAT, none of the Clitoria ternatea treatments caused mortalities to green mirid adult males exception of 2 L/ha Formulation A that caused 33.3% mortality (Table 13). At 5 DAT, Formulation D and Formulation A applied at 2 L/ha caused 33.3% mortality whereas the lower rates of Clitoria ternatea products did not kill any mirid. The 1 L/ha rate of the Clitoria ternatea products caused 33.3% mortality of green mirids at 7 DAT whereas the 2 L/ha rate caused 66.7% (Table 13). At 9 and 11 DAT, 2 L/ha Formulation A and Formulation D caused 100% kill of the mirids whereas the 1 L/ha rates and 2 L/ha Formulation C caused 66.7% kill (Table 13).







TABLE 13







Effect of Clitoria ternatea residues on mortalities of green mirid adult in the laboratory














Pre-
3 DAT
5 DAT
7 DAT
9 DAT
11 DAT


Treatments
treatment
% mortality
% mortality
% mortality
% mortality
% mortality
















1 L/ha
0
0a
0a
33.33a
66.67a
 66.67a


Formulation C


1 L/ha
0
0a
0a
33.33a
66.67a
100.00b


Formulation D


1 L/ha
0
0a
0a
33.33a
66.67a
100.00b


Formulation A


2 L/ha
0
0a
0a
33.33a
66.67a
100.00b


Formulation C


2 L/ha
0
0a
  33.33a
66.67b
100.00b 
100.00b


Formulation D


2 L/ha
0
  33.33a
  33.33a
66.67b
100.00b 
100.00b


Formulation A


Control
0
0a
0a
0c  
0c  
0c 


(water)





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.






Example 14
Efficacy on Mortality of Green Mirid Adult Females
The mortality of green mirid adult females at 3 DAT was zero but was 33.3% at 5 DAT (Table 14). The mortality when the female adults and beans were treated with 2 L/ha Formulation A and Formulation D was 66.7% at 7 DAT compared to 33.3% when they were treated with 1 L/ha Formulation D and Formulation A (Table 14). None of the insects treated with 1 L/ha Formulation C were dead at 7 DAT and only 33.3% died at 9 and 11 DAT. In contrast, application of 2 L/ha Formulation A and Formulation D killed 100 percent of the female adults at 9 DAT, whereas the 2 L/ha Formulation C killed 77.7% and the 1 L/ha Formulation D and Formulation A killed 66.7% of the insects. At 11 DAT, all the treated insects have died with the exception of insects treated with 1 L/ha Formulation C and 1 L/ha Formulation D where only 33.3 and 66.7% respectively died (Table 14).







TABLE 14







Effect of Clitoria ternatea residues on mortalities


of green mirid adult females in the laboratory














Pre-
3 DAT
5 DAT
7 DAT
9 DAT
11 DAT


Treatments
treatment
% mortality
% mortality
% mortality
% mortality
% mortality





1 L/ha
0
0
0b  
0a  
33.33a
 33.33a


Formulation C


1 L/ha
0
0
33.33a
33.33b
66.67b
 66.67b


Formulation D


1 L/ha
0
0
33.33a
33.33b
66.67b
100.00c


Formulation A


2 L/ha
0
0
33.33a
66.67c
77.70b
100.00c


Formulation C


2 L/ha
0
0
33.33a
66.67c
100.00c 
100.00c


Formulation D


2 L/ha
0
0
33.33a
66.67c
100.00c 
100.00c


Formulation A


Control
0
0
0b  
0a  
0d  
0d 


(water)





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.






Example 15
Oviposition Deterrent Activity of Clitoria ternatea on Green Mirid Adults on Beans in the Laboratory: Effect on Total Egg Lay
The Clitoria ternatea formulations Formulation A, Formulation B, Formulation C and Formulation D were evaluated at 0.5%, 1.0%, 1.5% and 2.0% (v/v) in the laboratory with 25° C. temperature and 40% RH. Water was used as a control. For each formulation, a total of 6 (3 pairs) green mirid adult females and males mirids per treatment (1 pair/replicate) were sprayed on filter papers until run off. Beans were dipped for 60 seconds in each formulation and then transferred and kept separately in 35-mL clear plastic containers (P101M; Solo, Urbana, Illionis, USA). Three pairs of treated green mirid adults were released onto the treated beans in each plastic container. Each treatment was replicated 4 times. The number of eggs were counted under a binocular microscope and recorded daily until 9 days after treatment when all the insects in one of the treatments died. Data was expressed as number of eggs laid per female and compared among the treatments.
Analysis of the Data
All experimental data were analysed using repeated measures ANOVA (Graphpad Instat and Prism Software, Inc. v. 2.03, San Diego, Calif., USA). Treatment and sample dates were the independent variables. Tukey-Kramer Multiple comparisons tests were used to separate means.
Results
Oviposition of green mirids on beans were significantly lower (P<0.001) at all concentrations in insects treated with Formulation A than the other treatments and control (Table 15). At 0.5% v/v Clitoria ternatea extract, 1.17 eggs/female was laid on beans treated with Formulation A compared to 8.58 and 7.25 per female respectively in Formulation C and Formulation D treated plants (Table 15). The Formulation B and the unsprayed beans had 11 and 12.42 eggs/female respectively (Table 15). At 1.0% v/v, the number of eggs laid per female on the insects and beans treated with Formulation D were the same as Formulation A, Formulation B and Formulation C but Formulation A was different from Formulation B and Formulation C. The number of eggs recorded per female on Formulation A and Formulation D treated plants were significantly lower (P<0.0009) than the control (Table 15). At 1.5 and 2 L/ha rates, no significant differences was detected in the number of eggs per female in Formulation A, Formulation C and Formulation D treated insects and beans. However, the number of eggs per female on Formulation A treated insects and beans were significantly lower (P<0.005 and P<0.01) than Formulation B and the unsprayed (control) (Table 15).







TABLE 15







Effect of direct spray application and residues


of Clitoria ternatea formulations on oviposition of green


mirid adults on beans in the laboratory.











Treatments
0.5% v/v
1.0% v/v
1.5% v/v
2.0% v/v





Formulation A
 1.17 ± 0.44 a
 1.35 ± 0.65 a
 1.25 ± 1.33 a
 2.00 ± 1.00 a


Formulation B
11.00 ± 0.76 b
10.35 ± 1.52 bc
11.00 ± 1.20 b
13.00 ± 2.08 b


Formulation C
 8.58 ± 2.36 b
12.65 ± 1.15 bc
 4.00 ± 2.65 ab
 7.33 ± 2.18 ab


Formulation D
 7.25 ± 0.95 b
 5.00 ± 0.35 ab
 5.65 ± 0.55 ab
 6.65 ± 1.20 ab


Water
12.42 ± 3.33 b
20.65 ± 3.38 c
11.65 ± 3.35 b
12.65 ± 3.35 b


(control)


Level of
P < 0.0001
P < 0.0009
P < 0.005
P < 0.01


significance





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.






Example 16
Oviposition Deterrent Activity of Clitoria ternatea on Green Mirid Adult Females on Beans in the Laboratory: Days after Treatment and Adult Female Egg Lay
The Clitoria ternatea formulations Formulation C, Formulation D and Formulation A were evaluated at 1 and 2 L/ha in the laboratory with 25° C. temperature and 40-55% RH. Water was used as control. For each formulation, a total of 6 adult mirid females and males (3 pairs) per treatment (1 pair/replicate) were sprayed on a filter paper until run off. Three beans were dipped for 60 seconds in each treatment or formulation. Each mirid pair and a treated bean from each treatment were transferred and kept separately in 35 mL clear plastic containers (P101M; Solo, Urbana, Illionis, USA). Each treatment was replicated 3 times. The numbers of green mirid eggs laid per female in each treatment were counted under a binocular microscope on 3, 5, 7, 9 and 11 days after treatment. The number of eggs per female per treatment were calculated and compared among treatments and control.
Analysis of the Data
All experimental data were analysed using repeated measures ANOVA (Graphpad Instat and Prism Software, Inc. v. 2.03, San Diego, Calif., USA). Treatment and sample dates were the independent variables. Tukey-Kramer Multiple comparisons tests were used to separate means.
Results
Green mirid adult females did not lay any eggs on beans treated with 2 L/ha Formulation A at 3 to 11 DAT (Table 16). Beans treated with 1 L/ha Formulation A had no eggs recorded on them at 3 DAT but at 5 to 11 DAT the number of eggs recorded on the 1 L/ha Formulation A treated beans ranged from 0.67 to 2.00 per female (Table 16). The number of eggs laid on the on beans treated with 1 L/ha Formulation C, Formulation D and water at 3 DAT were 0.33, 0.67 and 2.00 per female respectively (Table 16). At the same period no eggs were recorded on beans treated with 2 L/ha Formulation C and Formulation D respectively (Table 16). At 5 DAT the beans treated with 1 L/ha Formulation C and Formulation D had 10 and 8.67 eggs/female respectively and the control had 5.33 per female. When the application rate of Formulation C and Formulation D were doubled to 2 L/ha, the number of eggs laid per female reduced to 4.00 (Formulation C) and 6.00 is (Formulation D) and this was significantly different (P<0.01) from the 1 L/ha rate (Table 16). At 7 to 11 DAT, the number of eggs laid on beans treated with 2 L/ha Formulation C and Formulation D were not significantly different (P>0.05) from the 1 L/ha rate and the control (Table 16).







TABLE 16







Effect of Clitoria ternatea on the number of eggs laid by green mirid adult females in the laboratory















3 DAT
5 DAT
7 DAT
9 DAT
11 DAT



Pre-
No. eggs
No. eggs
No. eggs
No. eggs
No. eggs


Treatments
treatment
laid/female
laid/female
laid/female
laid/female
laid/female





1 L/ha
0
0.33 ± 0.33a
10.00 ± 4.91a 
7.33 ± 0.67a
8.67 ± 2.03a
7.00 ± 1.37a 


Formulation C


1 L/ha
0
0.67 ± 0.33a
8.67 ± 0.88a
10.33 ± 0.8a 
10.67 ± 1.21a 
8.87 ± 1.21a 


Formulation D


1 L/ha
0
0a
0.67 ± 0.33c
2.00 ± 1.00b
2.00 ± 1.00b
1.07 ± 1.00bc


Formulation A


2 L/ha
0
0a
4.00 ± 2.00b
7.00 ± 1.85a
6.33 ± 1.67a
4.53 ± 2.03ab


Formulation C


2 L/ha
0
0a
6.00 ± 3.79b
 5.67 ± 0.57ab
 4.67 ± 1.45ab
4.27 ± 0.97ab


Formulation D


2 L/ha
0
0a
0c
0b
0b
0c


Formulation A


Control
0
2.00 ± 1.16a
 5.33 ± 2.67ab
9.67 ± 2.85a
8.00 ± 0.58a
7.13 ± 0.58a 


(water)


Level of

P < 0.0001
P < 0.01
P < 0.01
P < 0.01
P < 0.05


significance





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.






Example 17
Efficacy of Clitoria ternatea Formulations Against Cotton Pests
The trial was conducted in an irrigated commercial conventional cotton crops at the Australian Cotton Research Institute farm in Narrabri. A sunflower strip was planted on one side of the field to generate high densities of green mirids and other cotton pests and beneficial insects.
The following treatments were evaluated against cotton pests especially green mirids: (1) 1 L/ha Formulation C, (2) 1 L/ha Formulation D, (3) 1 L/ha Formulation A, (4) 1 L/ha Formulation B, (5) Conventional insecticides (62.5 ml/ha Fipronil and 0.80 L/ha Steward) and (6) Unsprayed (untreated) control. The treatment plots were arranged in a randomised complete block design with 4 replicates per treatment. Each replicated plot measured 8 m wide and 100 meters long.
Foliar applications of each treatment were made over a period of 3 months. The decision to apply the treatment was made based on the IPM Guidelines and CottonLogic recommended economic threshold of 0.5 green mirids per meter. In all, 3 applications of each treatment were applied throughout the season.
Pre-treatment counts were made visually of green mirid adults and nymphs, green vegetable bugs, non target arthropods such as apple dimpling bugs, thrips, jassids and aphids on cotton plants. Post-treatment counts were made on 3, 7 and 14 days after treatment application. In each counts, two randomly selected 2 meter lengths of row of each treatment replicate, i.e. a total of 8 meters were examined per treatment. Data were expressed as numbers per meter for each treatment for each spray application and number per meter per sample date at the end of the season.
Analysis of the Data
All experimental data were analysed using repeated measures ANOVA (Graphpad Instat and Prism Software, Inc. v. 2.03, San Diego, Calif., USA). Treatment and sample dates were the independent variables. Tukey-Kramer Multiple comparisons tests were used to separate means.
Results
Efficacy of Clitoria ternatea Oil Based Formulations on Helicoverpa spp. in Commercial Cotton
Approximately equal number of Helicoverpa spp. eggs per meter per sample date were found on plots treated with conventional insecticide (2.22±0.26) compared with 1.64, 1.71 and 1.57 on 1 L/ha Formulation C (1.64), 1 L/ha Formulation D (1.71) and 1 L/ha Formulation A (Table 17). The number of Helicoverpa eggs per meter per sample date found on the plots treated with 1 L/ha Formulation B (formulation without Clitoria ternatea i.e. Clitoria ternatea carrier) was significantly higher (P<0.0001) than the other Clitoria ternatea formulations but was the same as plots treated with conventional insecticides (Table 17). The unsprayed plot (3.90 per meter) recorded the highest number of Helicoverpa eggs per meter per sample date than all the other treatments (Table 17). The mortalities of eggs on the conventional insecticide treated plots (43.1%) was significantly lower (P<0.001) than the Formulation C (58.0%), Formulation D (56.2%) and Formulation A (59.7%)-treated plots (Table 1). The Formulation B treated plots had 26.2% mortalities of Helicoverpa spp. eggs (Table 17).
The number of VS+S larvae was the same in conventional insecticide (1.19) compared with 1.70, 1.65 and 1.46 On the Formulation C (1.70), Formulation D (1.65) and Formulation A (1.46) treated plots (Table 17). Plots treated with Formulation B had significantly higher (P<0.0001) number of Helicoverpa spp. VS+S larvae than the other plots treated with the other Clitoria ternatea formulations and conventional insecticide (Table 17). The unsprayed plot had the highest number of VS+S larvae among the treatments but this was not significantly different (P>0.05) from the Formulation B treated plots (Table 17). The mortalities of VS+S larvae on the conventional insecticide treated plots (53.9%) was higher than the Formulation C (34.1%), Formulation D (36.1%) and Formulation A (43.4 W) (Table 17). The mortality of VS+S larvae on the Formulation B (10.1%) treated plots was the lowest among the Clitoria ternatea formulations.
The number of Helicoverpa spp. M+L larvae found on the conventional insecticide plots (0.30) was the same as the Formulation C (0.45), Formulation D (0.46) and Formulation A (0.26) treated plots (Table 17). The number of M+L larvae per meter per sample date found on the Formulation B treated plots (0.70) was the same as the unsprayed plots but significantly higher than the other Clitoria ternatea formulations (Table 17). The mortalities of M+L larvae on the conventional insecticide plots (68.1%) were lower than the Formulation A treated plots (73.3%). The mortalities of M+L larvae on plots treated with Formulation C (52.1%) and Formulation D (51.1%) was lower than Formulation A plots (73.3%) but higher than Formulation B treated plots (25.5%) (Table 17).







TABLE 17







Overall effect of Clitoria ternatea extracts on


Helicoverpa spp. eggs, very small and small larvae and


medium and large larvae on commercial conventional cotton


crops











Helicoverpa eggs/
Helicoverpa VS + S
Helicoverpa



metre/sample
larvae/metre/
M + L/metre/



date
sample date (%
sample date


Treatments
(% mortality)
mortality)
(% mortality)





1 L/ha
1.64 ± 0.26 a
1.70 ± 0.22 a
0.45 ± 0.11 a


Formulation C
(58.0%)
(34.1%)
(52.1%)


1 L/ha
1.71 ± 0.26 a
1.65 ± 0.25 a
0.46 ± 0.10 a


Formulation D
(56.2%)
(36.1%)
(51.1%)


1 L/ha
1.57 ± 0.24 a
1.46 ± 0.21 a
0.26 ± 0.06 a


Formulation A
(59.7%)
(43.4%)
(73.3%)


1 L/ha
2.88 ± 0.23 b
2.32 ± 0.25 bc
0.70 ± 0.13 bc


Formulation B
(26.2%)
(10.1%)
(25.5%)


Conventional
2.22 ± 0.26 ab
1.19 ± 0.20 a
0.30 ± 0.06 a



(43.1%)
(53.9%)
(68.1%)


Unsprayed
3.90 ± 0.33 c
2.58 ± 0.29 c
0.94 ± 0.19 c


(Control)
(0)
(0)
(0)


Level of
p < 0.0001
P < 0.0001
P < 0.0001


significance





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.






Example 18
Efficacy of Clitoris ternatea Oil Based Formulations on Sucking Pests on Commercial Cotton Green Mirids
The number of green mirids per meter per sample date was the same in conventional insecticide (0.33) compared with 1.07, 1.09 and 0.77 on the Formulation C (1.07), Formulation D (1.09) and Formulation A (0.77) treated plots (Table 18). Plots treated with Formulation B had significantly higher (P<0.0001) number of green mirids per meter per sample date than the other plots treated with the Clitoria ternatea formulations and conventional insecticide (Table 18). The unsprayed plot had the highest number of green mirids per meter per sample date among all treatments but was not significantly different (P>0.05) from the Formulation B treated plots (Table 18). The mortalities of green mirids on the conventional insecticide treated plots (81.5%) was higher than the Formulation C (39.9%), Formulation D (38.8%) and Formulation A (56.7%) (Table 18). The mortality of green mirids on the Formulation B-treated plots (32.6%) was the lowest among the Clitoria ternatea formulations (Table 18).
Green Vegetable Bugs
The number of green vegetable bugs per meter per sample date was the same in conventional insecticide (0.19) compared with 0.44, 0.35 and 0.17 on the Formulation C (0.44), Formulation D (0.35), Formulation A (0.17) and Formulation B (0.54) treated plots (Table 18). Plots treated with Formulation B (0.54) had significantly higher (P<0.0001) number of green vegetable bugs per meter per sample date than the other plots treated with the Clitoria ternatea formulations and conventional insecticide (Table 18). The unsprayed plot had the highest number of green vegetable bugs per meter per sample date among all treatments but was not significantly different (P>0.05) from the Formulation B treated plots (Table 18). The mortalities of green vegetable bugs on the conventional insecticide treated plots (81.5%) was higher than the Formulation C (39.9%), Formulation D (38.8%) and Formulation A (56.7%) (Table 18). The mortality of green vegetable bugs on the Formulation B-treated plots (32.6%) was the lowest among the Clitoria ternatea formulations (Table 18).
Aphids
Approximately equal number of cotton aphids per meter per sample date were found on plots treated with conventional insecticide (1.20) compared with 2.09, 2.06, 0.17 and 2.62 on the Formulation C (2.09), Formulation D (2.06), Formulation A (1.36) and Formulation B (2.62) treated plots (Table 18). Plots treated with Formulation B (2.62) had significantly higher (P<0.0001) number of cotton aphids per meter per sample date than the other plots treated with the Clitoria ternatea formulations and conventional insecticide (Table 18). The unsprayed plot had the highest number of cotton aphids per meter per sample date among all treatments but was not significantly different (P>0.05) from the Formulation B treated plots (Table 18). The mortalities of cotton aphids on the conventional insecticide treated plots (63.4%) was higher than the Formulation C (36.3%), Formulation D (37.2%) and Formulation A (58.5%) (Table 18). The mortality of aphids on the Formulation B-treated plots (20.1%) was the lowest among the Clitoria ternatea formulations (Table 18).







TABLE 18







Efficacy of Clitoria ternatea extracts on green


mirids, green vegetable bugs and cotton aphids in


commercial cotton crops











No. Green
No. Green vege
No. Aphids/



mirids/metre/
bug/metre/
metre/sample



sample date
sample date
date


Treatments
(% mortality)
(% mortality)
(% mortality)





1 L/ha
1.07 ± 0.19 a
0.44 ± 0.09 ab
2.09 ± 0.57 a


Formulation C
(39.9%)
(47.6%)
(36.3%)


1 L/ha
1.09 ± 0.18 a
0.35 ± 0.11 ab
2.06 ± 0.40 a


Formulation D
(38.8%)
(58.3%)
(37.2%)


1 L/ha
0.77 ± 0.14 ab
0.17 ± 0.07 a
1.36 ± 0.29 a


Formulation A
(56.7%)
(79.8%)
(58.5%)


1 L/ha
1.20 ± 0.21 ac
0.54 ± 0.14 bc
2.62 ± 1.07 a


Formulation B
(32.6%)
(35.7%)
(20.1%)


Conventional
0.33 ± 0.11 b
0.19 ± 0.07 a
1.20 ± 0.27 a



(81.5%)
(77.4%)
(63.4%)


Unsprayed
1.78 ± 0.25 c
0.84 ± 0.18 c
3.28 ± 0.53 a


(Control)
(0)
(0)
(0)


Level of
P < 0.0001
P < 0.0001
P > 0.05


significance





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.






Apple Dimpling Bugs

The number of apple dimpling bugs per meter per sample date found on plots treated with conventional insecticide (2.03) were lower than those on plots treated with Formulation C (3.59), Formulation D (3.88), Formulation A (2.70) and Formulation B (4.32) (Table 19). Plots treated with Formulation B (4.32) had significantly higher (P<0.0001) number of Apple dimpling bugs per meter per sample date than the other plots treated with the Clitoria ternatea formulations and conventional insecticide (Table 19). The unsprayed plot (4.57) had the highest number of Apple dimpling bugs per meter per sample date among all treatments but was not significantly different (P>0.05) from the Formulation B treated plots (Table 19). The mortalities of Apple dimpling bugs on the conventional insecticide treated plots (55.6%) was higher than the Formulation C (21.4%), Formulation D (15.1%) and Formulation A (40.9%) (Table 19). The mortality of Apple dimpling bugs on the Formulation B-treated plots (5.5%) was the lowest among the Clitoria ternatea formulations (Table 19).
Green Jassids
The number of green jassids per meter per sample date found on plots treated with conventional insecticide (2.03) was the same as those found on plots treated with Formulation C (2.93), Formulation D (2.88), Formulation A (2.16) and Formulation B (3.13) treated plots (Table 19). Plots treated with Formulation A (2.16) had significantly lower (P<0.0002) number of green jassids per meter per sample date than the plots treated with Formulation B (3.13). Plots treated with Formulation B had the same number of green jassids per meter per sample date than plots treated with Formulation C, Formulation D and the unsprayed plot (3.78) (Table 19). The survival rates of green jassids on the conventional insecticide treated plots (28.6%) were lower than plots treated with Formulation A (42.9%). The mortalities of green jassids on the Formulation C and Formulation D treated were approximately the same as plots treated with Formulation B (17.2) (Table 19).
Thrips
Approximately equal number of thrips per meter per sample date were found on plots treated with conventional insecticide (11.06) compared with Formulation C (10.00), Formulation D (10.19), Formulation A (10.62), Formulation B (11.04) treated plots and the control plots (12.22) (Table 19). Plots treated with Formulation A (10.62) had the same number of thrips per meter per sample date as plots treated with Formulation B (11.04). Plots treated with both Formulation A and Formulation B had the same number of thrips per meter per sample date as plots treated with Formulation C, Formulation D, conventional insecticides and unsprayed plot (12.22) (Table 19). The mortalities of thrips on the conventional insecticide treated plots (9.5%) were the same as plots treated with the Clitoria ternatea formulations Formulation A (Table 19).







TABLE 19







Efficacy of Clitoria ternatea extracts on apple


dimpling bugs, green jassids and thrips in commercial


cotton crops











No. Apple Dimpling
No. Green




bug/metre/sample
jassids/metre/
No. Thrips/metre/



date
sample date
sample date


Treatments
(% mortality)
(% mortality)
(% mortality)





1 L/ha
3.59 ± 0.53 ab
2.93 ± 0.32 abc
10.00 ± 1.13 a


Formulation C
(21.4%)
(22.5%)
(18.2%)


1 L/ha
3.88 ± 0.52 a
2.88 ± 0.33 ac
10.19 ± 1.21 a


Formulation D
(15.1%)
(23.8%)
(16.6%)


1 L/ha
2.70 ± 0.46 bc
2.16 ± 0.22 b
10.62 ± 1.12 a


Formulation A
(40.9%)
(42.9%)
(13.1%)


1 L/ha
4.32 ± 0.62 a
3.13 ± 0.29 ac
11.04 ± 1.08 a


Formulation B
 (5.5%)
(17.2%)
 (9.7%)


Conventional
2.03 ± 0.33 c
2.70 ± 0.31 ab
11.06 ± 1.25 a



(55.6%)
(28.6%)
 (9.5%)


Unsprayed
4.57 ± 0.56 a
3.78 ± 0.36 c
12.22 ± 1.44 a


(Control)
(0)
(0)
(0)


Level of
P < 0.0001
P < 0.0002
P > 0.33


significance





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.






Example 19
Efficacy of Clitoria ternatea Formulations Against Key Beneficial Insects
The trial was conducted in an irrigated commercial conventional cotton crops at the Australian Cotton Research Institute farm in Narrabri. A sunflower strip was planted on one side of the field to generate high densities of green mirids and other cotton pests and beneficial insects.
The following Clitoria ternatea formulations were evaluated against beneficial insects on cotton crops (1) 1 L/ha Formulation C, (2) 1 L/ha Formulation D, (3) 1 L/ha Formulation A, (4) 1 L/ha Formulation B, (5) Conventional insecticides (62.5 ml/ha Fipronil and 0.80 L/ha Steward) and (6) Unsprayed (untreated) control. The treatment plots were arranged in a randomised complete block design with 4 replicates per treatment. Each replicated plot measured 8 m wide and 100 meters long.
Foliar applications of each treatment were made over 3 months. The decision to apply the treatment was made based on the IPM Guidelines and CottonLogic recommended predator to pre ratio of 0.5. In all, 3 applications of each treatment were applied throughout the season.
Pre-treatment counts were made visually of beneficial insects such as predatory beetles, predatory bugs, predatory lacewings and spiders on cotton plants. Post-treatment counts were made on 3, 7 and 14 days after treatment application. In each counts, two randomly selected 2 meter lengths of row of each treatment replicate, i.e. a total of 8 meters were examined per treatment. Data were expressed as numbers per meter for each treatment for each spray application and number per meter per sample date for each treatment at the end of the season.
Analysis of the Data
All experimental data were analysed using repeated measures ANOVA (Graphpad Instat and Prism Software, Inc. v. 2.03, San Diego, Calif., USA). Treatment and sample dates were the independent variables. Tukey-Kramer Multiple comparisons tests were used to separate means.
Results
Predatory insects identified from the treated plots were predatory beetles, bugs, lacewings and spiders (see Table 7).
Predatory Beetles
No significant difference (P>0.05) in the number of predatory beetles per meter per sample date were recorded in plots treated with the Clitoria ternatea formulations and the control (unsprayed) plots (Table 20). However, significant differences were found between plots treated with conventional insecticides and plots treated with Clitoria ternatea formulations (Table 20). Plots treated with conventional insecticides had 1.51 predatory beetles per meter per sample date compared with 2.25, 2.19, 2.00, 2.20 and 2.51 for Formulation C (2.25), Formulation D (2.19), Formulation A (2.00), Formulation B (2.20) and control (2.51) plots (Table 20).
Predatory Bugs
The highest number of predatory bugs per meter per sampling date was recorded on the Control plots (1.78) but this was not significantly different (P>0.05) from plots treated with Formulation B (1.65), Formulation A (1.25), Formulation C (1.16) and Formulation D (1.25) (Table 20). The conventional insecticide-treated cotton crops had the least number of predatory bugs (P<0.0001) (Table 20).
Predatory Lacewings
No significant difference (P>0.05) in the number of predatory lacewings per meter per sampling were found on plots treated with Clitoria ternatea formulations and the conventional insecticide treated plots (Table 20). The highest number of lacewings per meter was recorded in the unsprayed plot (1.01) but was not significantly different (P>0.05) from the plots treated with the Clitoria ternatea formulations (Table 20). In contrast the least number of lacewings per meter was recorded on the conventional insecticide treated plots and this was significantly different (P<0.03) from the control plots (Table 20).
Spiders
The number of spiders per meter per sampling date were recorded on the unsprayed (control) plots (3.28) compared with 2.52, 3.10, 2.78, 2.73 and 2.13 on the Formulation C (2.52), Formulation D (3.10), Formulation A (2.78), Formulation B (2.73) and the conventional insecticide (2.13) plots (Table 20). The difference between the spider numbers per meter per sample date found on the control plots was not significantly different (P>0.05) from plots treated with Clitoria ternatea formulations but was different (P<0.0003) from the conventional insecticide treated plots (Table 20). The number of spiders recorded on the conventional insecticide treated plots was not significantly different (P>0.05) from plots treated with Clitoria ternatea formulations exception is Formulation D treated plots (Table 20).







TABLE 20







Effect of Clitoria ternatea extracts on


predatory insects on commercial conventional cotton crops












No. Predatory






beetles/
No. Predatory
No. Predatory
No.



metre/sample
bugs/
lacewings/metre/
Spiders/metre/


Treatments
date
metre/sample date
sample date
sampling date





1 L/ha
2.25 ± 0.19 a
1.16 ± 0.19 ab
0.73 ± 0.10 ab
2.52 ± 0.21 ab


Formulation C


1 L/ha
2.19 ± 0.17 a
1.38 ± 0.22 ab
0.86 ± 0.13 ab
3.10 ± 0.22 a


Formulation D


1 L/ha
2.00 ± 0.19 a
1.25 ± 0.18 ab
0.99 ± 0.14 ab
2.78 ± 0.25 ab


Formulation A


1 L/ha
2.20 ± 0.21 a
1.65 ± 0.21 b
0.74 ± 0.11 ab
2.73 ± 0.24 ab


Formulation B


Conventional
1.51 ± 0.17 b
0.97 ± 0.17 a
0.58 ± 0.98 a
2.13 ± 0.19 b


Unsprayed
2.51 ± 0.18 a
1.78 ± 0.26 b
1.01 ± 0.14 b
3.28 ± 0.30 a


(Control)


Level of
P < 0.001
P < 0.001
P < 0.03
P < 0.0003


significance





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.






Example 20
Efficacy of Clitoria ternatea Formulations on Silverleaf Whiteflies
The following Clitoria ternatea formulations were used in the trials to determine the efficacy of the Clitoria ternatea product against Bemisia tabaci—b-biotype (silverleaf whitefly) adults and nymphs: They are

    (1) Formulation A (Clitoria ternatea formulation in oil without emulsifiers),
    (2) Formulation D (Clitoria ternatea formulations with emulsifiers)

Effect of Clitoria ternatea Extracts on Bemisia tabaci on Commercial Cotton Crops at Merah North Near Wee Waa



The trial was conducted in an irrigated conventional cotton crops in a commercial cotton farm at Merah north near Wee Waa. Two trials were conducted for a two week period. The treatments evaluated were (1) 2 L/ha Formulation A (2) 2 L/ha Formulation D (3) Unsprayed (untreated) control. The treatment plots were arranged in a randomised complete block design with 3 replicates per treatment. Each replicated plot measured 24 m wide 100 m long.
Foliar applications of each treatment were made on day 1. Pre-treatment counts were made visually of B. tabaci adults and nymphs, on the under-surface of leaves of cotton plants in each treatment. Post-treatment counts were made on 3, 7 and 14 days after treatment application. In each sampling date, twenty (20) plants from each treatment replicate were randomly selected and B. tabaci adults on a leaf from the 5th node below the terminal of each plant (during the early morning hours (9-10 am) were counted visually by carefully turning the leaf over and counting the number of individual adults present.
In the case of nymphs, one leaf from the 5th node below the terminal of each of the 20 plants was cut, removed and carefully placed individually in a plastic bag. The plastic bags containing individual leaves were brought to the laboratory and the B. tabaci nymphs were counted under a binocular microscope. Data of both adults and nymphs were expressed as numbers per leaf for each treatment.
Analysis of the Data
All experimental data were analysed using repeated measures ANOVA (Graphpad Instat and Prism Software, Inc. v. 2.03, San Diego, Calif., USA). Treatment and sample dates were the independent variables. Tukey-Kramer Multiple comparisons tests were used to separate means.
Results
The B. tabaci strains present in the study site were predominantly the b-biotype. The number of B. tabaci adults per leaf recorded in plots treated with Formulation A and Formulation D were significantly lower (P<0.01 on 3 DAT, P<0.05 on 7 DAT and P<0.0001 on 14 DAT) than the unsprayed plots (Table 21). The mortality of B tabaci adults recorded on plots treated with 2 L/ha Formulation D on 3 DAT was 74.5% compared with 54.8 on 2 L/ha Formulation A treated plots (Table 21). However, on 7 and 14 DAT mortalities on Formulation A treated plots increased to 77.3 and 73.4% respectively whereas those on the Formulation D plots were 60.4 and 73.2% respectively relative to the control (Table 21).
In the case of B. tabaci nymphs per leaf, no significant difference (P>0.05) were found between plots treated with Formulation A and Formulation D but were significantly different (P<0.0001) from the unsprayed control on 3, 7 and 14 DAT (Table 22). However, the mortalities of B. tabaci nymphs on the Formulation D treated plots (74.9%) on 3 DAT were significantly higher than the Formulation A (28.2%) treated plots relative to the control (Table 22). On 7 and 14 DAT, mortalities recorded on the Formulation A treated plots increased to 73.6 and 55.6% whereas the mortality on the Formulation D treated plots were 75.3 and 67.0% respectively relative to the control (Table 22).







TABLE 21







Effect of Clitoria ternatea extracts on Bemisia


tabaci adults per leaf on commercial Bollgard cotton crops












Pre-treatment
3 DAT
7 DAT
14 DAT


Treatments
counts
(% mortality)
(% mortality)
(% mortality)





2.0 L/ha
74.33 ± 6.15 a
32.33 ± 3.90 a
15.56 ± 1.70 a
15.56 ± 1.70 a


Formulation A

(54.8%)
(77.3%)
(73.4%)


2.0 L/ha
70.89 ± 7.99 a
18.22 ± 3.62 a
27.11 ± 5.19 a
15.67 ± 2.08 a


Formulation D

(74.5%)
(60.4%)
(73.2%)


Unsprayed
53.56 ± 7.51 a
71.56 ± 8.85 b
68.45 ± 5.70 b
58.44 ± 5.82 b


(Control)

(0)
(0)
(0)


Level of
P > 0.05
P < 0.001
P < 0.05
P < 0.0001


significance





Means between treatments within columns followed by the same letter are not significantly different (p > 0.05); Tukey-Kramer multiple comparison test.













TABLE 22







Effect of Clitoria ternatea extracts and


conventional insecticides on Bemisia tabaci nymphs per


leaf on commercial Bollgard cotton crops












Pre-treatment





Treatments
counts
3 DAT
7 DAT
14 DAT





2.0 L/ha
75.56 ± 11.35 a
51.11 ± 8.08 a
 44.78 ± 7.15 a
121.33 ± 15.91 a


Formulation A

(28.2%)
(73.6%)
(55.6%)


2.0 L/ha
75.56 ± 11.18 a
17.89 ± 2.90 a
 41.78 ± 4.85 a
 90.11 ± 5.58 a


Formulation D

(74.9%)
(75.3%)
(67.0%)


Unsprayed
77.22 ± 9.06 a
71.22 ± 8.35 b
169.33 ± 25.10 b
273.22 ± 24.21 b


(Control)

(0)
(0)
(0)


Level of
P > 0.05
P < 0.0001
P < 0.0001
P < 0.0001


significance





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.






Example 21
Effect of Clitoria ternatea Extracts and Conventional Insecticides on Bemisia tabaci on Commercial Cotton Crops
The trial was conducted in an irrigated conventional cotton crops in a cotton farm. The following treatments were evaluated against B. tabaci—b-biotype adults and nymphs: (1) 2 L/ha Formulation A (2) 2 L/ha Formulation D (3) 0.80 L/ha Diafenthiuron at Merah north (conventional insecticides) and (4) Unsprayed (control). The treatment plots were arranged in a randomised complete block design with 3 replicates per treatment. Each replicated plot measured 24 m wide 100 m long.
Foliar applications of each treatment were made over two weeks. Pre-treatment counts were made visually of B. tabaci adults and nymphs, on the under-surface of leaves of cotton plants in each treatment. Post-treatment counts were made on 3, 7 and 14 days after treatment application. Data of both adults and nymphs were expressed as numbers per leaf for each treatment.
Analysis of the Data
All experimental data were analysed using repeated measures ANOVA (Graphpad Instat and Prism Software, Inc. v. 2.03, San Diego, Calif., USA). Treatment and sample dates were the independent variables. Tukey-Kramer Multiple comparisons tests were used to separate means.
Results
The B. tabaci strains present in the study site were predominantly the b-biotype. Approximately equal number of B. tabaci adults per leaf were found on plots treated with conventional insecticides (15.33) on 3 DAT compared with 18.00, 11.33 and 25.00 on Formulation D, Formulation A and unsprayed plots (Table 23). On 7 DAT, the number of adults per leaf recorded on Formulation A treated plots were the same as Formulation D and conventional insecticide plots but was significantly different (P<0.0001) from the control plots (Table 23). In contrast, the number of B. tabaci adults per leaf found on Formulation D and conventional insecticide plots were not significantly different (P>0.05) from the control plots (Table 23). On 14 DAT, no significant differences (P>0.05) were recorded among the treatments and control plots (Table 23). However, the mortalities of B. tabaci adults on the Formulation D treated plots (54.7 t) on 3 DAT was higher than the Formulation A plots (28.0%) and conventional insecticide plots (38.7%) (Table 23). On 7 DAT, mortalities of B. tabaci adults (66.7%) was higher than is the Formulation D (38.9 t) and conventional insecticide (32.2%) relative to the control (Table 23). In contrast, the difference in mortality among treatments was approximately the same relative to the control on 14 DAT (Table 23).
The number of B. tabaci nymphs per leaf found on plots treated with Formulation A and Formulation D were not significantly different (P>0.05) but were significantly different (P<0.06) from the conventional insecticide and the unsprayed plots on 3 DAT (Table 24). No significance difference (P>0.05) was detected on 3 DAT between conventional insecticide treated and unsprayed plots (Table 24). No significant differences (P>0.05) were found among treatments and control on 7 DAT but a significant difference was found between Clitoria ternatea treated plots and the conventional and unsprayed plots (Table 24). In terms of mortalities, Formulation A and Formulation D treated plots recorded 53.3 and 36.4% mortalities of B. tabaci nymphs compared with conventional insecticide treated plots which recorded a 12 percent increase of nymphs over the control plot (Table 24). On 7 DAT, the Formulation D treated plots had 47.5 percent mortality compared with Formulation A (28.8%) and conventional insecticide (22.1%). On 14 DAT, the mortality caused individual treatments was increased to 77.2 percent (Formulation A), 79.5% (Formulation D) and 60.6% (conventional insecticides) (Table 24).







TABLE 23







Effect of Clitoria ternatea extracts and


conventional insecticides on Bemisia tabaci adults per


leaf on commercial Bollgard cotton crops












Pre-treatment
3 DAT
7 DAT
14 DAT


Treatments
counts
(% mortality)
(% mortality)
(% mortality)





2.0 L/ha
24.33 ± 2.96 a
18.00 ± 2.89 a
10.00 ± 1.73 a
14.67 ± 2.90 a


Formulation A

(28.0%)
(66.7%)
(20.0%)


2.0 L/ha
28.00 ± 3.51 a
11.33 ± 0.88 a
18.33 ± 5.04 ab
12.33 ± 2.33 a


Formulation D


(38.9%)
(32.7%)


0.80 L/ha
25.67 ± 1.76 a
15.33 ± 1.45 a
20.33 ± 2.40 ab
12.00 ± 0.88 a


Diafenthiuron

(54.7%)
(32.2%)
(34.5%)


Unsprayed
23.00 ± 4.73 a
25.00 ± 2.65 b
30.00 ± 5.77 b
18.33 ± 2.33 a


(Control)

(38.7%)
(0)
(0)


Level of
P > 0.05
P < 0.0001
P < 0.0001
P > 0.05


significance





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.













TABLE 24







Effect of Clitoria ternatea extracts and


conventional insecticides on Bemisia tabaci nymphs per


leaf on commercial Bollgard cotton crops












Pre-treatment
3 DAT
7 DAT
14 DAT


Treatments
counts
(% mortality)
(% mortality)
(% mortality)





2.0 L/ha
29.67 ± 5.78 a
12.00 ± 1.00 a
14.00 ± 3.06 a
 9.67 ± 2.19 a


Formulation A

 (53.3%)
(28.8%)
(77.2%)


2.0 L/ha
38.00 ± 2.52 a
16.33 ± 2.33 a
10.33 ± 0.33 a
 8.67 ± 1.33 a


Formulation D

 (36.4%)
(47.5%)
(79.5%)


0.80 L/ha
40.00 ± 9.87 a
28.67 ± 7.22 b
15.33 ± 2.40 a
17.00 ± 1.73 b


Diafenthiuron

(−12.0%)
(22.1%)
(60.6%)


Unsprayed
17.67 ± 3.33 a
25.67 ± 7.22 b
19.67 ± 0.33 a
42.33 ± 3.93 b


(control)

(0)
(0)
(0)


Level of
P > 0.05
P < 0.06
P > 0.05
P < 0.001


significance





Means between treatments within columns followed by the same letter are not significantly different (P > 0.05); Tukey-Kramer multiple comparison test.






REFERENCES
The disclosure of the following documents is incorporated herein by reference:

    1 Bernays, E. A. and Chapman, R. F. (1994). Host Plant Selection by Phytophagous Insects. Chapman and Hall. London.
    2 Mensah, R. K., Verneau, S. and Frerot, B. (2000).


Deterrence of oviposition of adult Ostrinia nubilalis (Hubner) by a natural enemy food supplement Envirofeast® on maize in France. International Journal of Pest Management 46 (1), 49-53

    3 Mensah, R. K. (1996). Suppression of Helicoverpa spp. oviposition by use of natural enemy food supplement “Envirofeast”. Australian Journal of Entomology, 35, 323-329.
    4 Mensah, R. and Moore, C. J. (1999). A Review of Behaviour Modifying Chemicals in Relation to Pest Host Selection and Management on Australian Cottons. CRDC.
    5 Miller, J. R. and Cowles, R. S. (1990). Stimulo-deterrent diversion: a concept and its possible application to onion maggot control. J. Chem. Ecol. 16: 3197-3212.
    6 Pyke, B., Rice, M., Sabine, G. and Zalucki, M. (1987). The push-pull strategy-behavioural control of Heliothis. Australian Cotton Grower 9: 7-9.
    7 Rhoades, D. F. and Coates, R. H. (1976). Towards a general theory of plant anti-herbivore chemistry. In: J. W. Wallace and L. Mansell, eds. Biochemical Interactions Between Insects and Plants. Pp. 168-213. Plenum, New York.
    8 Tingle, F. C. and Mitchell, E. R. (1984). Aqueous extracts from indigenous plants as oviposition deterrents for Heliothis virescens. J. Chem. Ecology 10: 101-113.

## Claims

1. A composition for controlling insect pests of plants comprising:
an extract from leaves of Clitoria ternatea comprising SPCs which have insecticidal activity and/or which repel the insect pest and/or deter the insect pest from laying eggs and/or influence the position of egg laying and/or deter the insect pest from feeding on a plant; and
one or more emulsifiers, wherein at least one of the one or more emulsifiers is non-natural, wherein a HPLC analysis of a methanolic extract of said composition produces a chromatogram comprising the major peaks present in the chromatogram of FIG. 1; and
wherein the Clitoria ternatea is at a pre-flower stage.

2. The composition of claim 1, further comprising a carrier.

3. A composition comprising:
a solvent selected from the group consisting of polar organic solvents, lower hydrocarbon solvents and low molecular weight oils;
an extract from leaves of Clitoria ternatea disposed in the solvent, the extract comprising secondary plant compounds (SPCs) which have insecticidal activity and/or which repel an insect pest and/or deter an insect pest from laying eggs and/or influence the position of egg laying and/or deter an insect pest from feeding on a plant; and
one or more emulsifiers, wherein at least one of the one or more emulsifiers is non-natural; and
wherein the Clitoria ternatea is at a pre-flower stage.

4. The composition as claimed in claim 3, further comprising one or more agriculturally acceptable excipients.

5. The composition as claimed in claim 3, wherein the composition comprises more than one emulsifier.

6. The composition as claimed in claim 3, wherein the plant is selected from the group consisting of cotton, a grain crop, a legume crop, a pulse crop, a vegetable crop and a fruit crop.

7. The composition as claimed in claim 3, wherein the plant is an ornamental plant.

8. The composition of claim 3, wherein the SPCs deter the insect pest from feeding on a plant.

9. The composition of claim 3, wherein the SPCs deter the insect pest from laying eggs.

10. The composition of claim 3, wherein the SPCs deter the insect pest from laying eggs and deter the insect pest from feeding on a plant.

11. The composition as claimed in claim 3, wherein the insect pest is selected from the group consisting of cotton bollworm, native budworm, green mirids, aphids, green vegetable bugs, apple dimpling bugs, plaque thrips, tobacco thrips, onion thrips, cotton thrips, western flower thrips, whiteflies, two spotted mites and leaf hoppers.

12. The composition as claimed in claim 3, wherein non-target insect species numbers are not significantly affected thereby conserving natural enemies of the insect pests to supplement control of insect infestation.

13. The composition as claimed in claim 3, wherein a HPLC analysis of a methanolic extract of said composition produces a chromatogram comprising the major peaks present in the chromatogram of FIG. 1.

14. The composition as claimed in claim 13, wherein the major peaks are present in the HPLC chromatogram of Fractions 2, 4 and/or 6.

15. The composition as claimed in claim 14, wherein the major peaks have retentions times at about 4 minutes corresponding to the peak with the same retention time in the HPLC chromatogram of Fraction 2, at about 12 minutes corresponding to the two peaks with the same retention times in the HPLC chromatogram of Fraction 4, and at about 17 to about 19 minutes corresponding to the peaks with the same retention times in the HPLC chromatogram of Fraction 6.

16. The composition as claimed in claim 3, wherein the one or more emulsifiers is selected from the group consisting of a non-ionic, an anionic, a cationic surfactant, and a blend thereof.

17. The composition as claimed in claim 16, wherein the one or more emulsifiers is a non-ionic surfactant.

18. The composition as claimed in claim 3, wherein the one or more emulsifiers is selected from the group consisting of an ethoxylated ester of a fatty acid, an ethoxylated alkanol, a fatty acid, an ethoxylate of a vegetable oil or animal fat, an alkyl polysaccharide, and a combination thereof.

19. The composition as claimed in claim 3, further comprising a carrier.

20. The composition as claimed in claim 19, wherein the carrier is Canola oil.

21. The composition as claimed in claim 19, wherein the carrier is polyethylene glycol.

22. The composition as claimed in claim 19, wherein the carrier is a non-polar solvent or an oil.

23. The composition as claimed in claim 22, wherein the oil is canola oil or cotton seed oil.

24. The composition as claimed in claim 3, wherein the extract is from a whole plant of Clitoria ternatea.

25. The composition of claim 3, wherein the solvent is a polar organic solvent selected from the group consisting of alcohols, ketones, aldehydes and sulfoxides.

26. The composition of claim 25, wherein the polar organic solvent is methanol.

27. The composition of claim 25, wherein the polar organic solvent is ethanol.

28. A method for controlling insect pests of plants comprising treating a plant suffering from an infestation of insect pests or a plant prone to such infestations with the composition of claim 2.

29. A method as claimed in claim 28, further comprising one or more agriculturally acceptable excipients.

30. A method as claimed in claim 29, wherein the composition comprises more than one emulsifier.

31. A method as claimed in claim 28, wherein the composition further comprises a carrier; and wherein the carrier is a non-polar solvent or an oil.

32. A method as claimed in claim 31, wherein the oil is canola oil or cotton seed oil.

33. A method as claimed in claim 28, wherein said extract from Clitoria ternatea is an extract in a polar organic solvent.

34. A method as claimed in claim 33, wherein the polar organic solvent is selected from the group consisting of alcohols, ketones, aldehydes and sulfoxides.

35. A method as claimed in claim 34, wherein the polar organic solvent is an alcohol.

36. A method as claimed in claim 35, wherein the polar organic solvent is methanol.

37. A method as claimed in claim 35, wherein the polar organic solvent is ethanol.

38. A method as claimed in claim 28, wherein the plant is selected from the group consisting of cotton, a grain crop, a legume crop, a pulse crop, a vegetable crop and a fruit crop.

39. A method as claimed in claim 38, wherein the plant is an ornamental plant.

40. A method as claimed in claim 28, wherein the control of insect infestation is by reducing egg laying.

41. A method as claimed in claim 28, wherein the control of insect infestation is by deterrence of insect feeding.

42. A method as claimed in claim 28, wherein the control of insect infestation is by direct kill.

43. A method as claimed in claim 28, wherein said one or more insect pests is selected from the group consisting of cotton bollworm, native budworm, green mirids, aphids, green vegetable bugs, apple dimpling bugs, plaque thrips, tobacco thrips, onion thrips, cotton thrips, western flower thrips, whiteflies, two spotted mites and leaf hoppers.

44. A method as claimed in claim 28, wherein non-target insect species numbers are not significantly affected thereby conserving natural enemies of the insect pests to supplement control of insect infestation.