Title: Blend for Post-harvest Insects Feeding Deterrent Technical Field

This invention relates to post harvest pest management of food and agricultural commodities and particularly to a blend suitable for controlling the behaviour of insects, as well as methods of use of such compositions. BACKGROUND ART

The food 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 Prostephanus spp in dried food & agricultural products such as Maize {Zea mays) and Cassava (Manihot esculenta) among other crops.

One of the approaches with greater potential to change the way insect pests are managed in broad acre crops such as Maize, 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. Many SPCs have evolved in plants to actually protect the plants against pest infestation. 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. Numerous studies into pest management have focused on chemical compounds that kill the pest rather than behaviour modifying compounds.

Consequently, potentially useful compounds with more subtle modes of action that could lead to novel products have been overlooked. 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. They are considered more important than stimulants and in fact a deterrent effect is more commonly noted in SPCs. 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 in a push-pull strategy. Therefore, there is need for development of a novel tool that will modify behaviour of insect pests in pest management in agricultural crops.

Technical Problem

Maize (Zea mays) is one of the staple food crops in Kenya. Cassava {Manihot esculenta) forms another important food crop rich in carbohydrate, which is used as food and bio -fuel (fermentation) in ethanol production in Brazil and other parts of the world. The larger grain borer {Prostephanus truncatus Horn) is a serious primary pest of stored maize and dried cassava where it also breeds. LGB was first reported in East Africa in 1981 and in 1984 in West Africa. Since then, it has spread rapidly in the African continent. In Togo, soon after the discovery of P. truncatus mean losses of up to 30.2 % were reported on stored maize six months after storage. Stored dried cassava is also known to become heavily infested by P. truncatus, which may lead to cross infestation of maize. Hodges et al in 1985 reported 70 % loss in dried cassava roots after four months of storage due to this species. Prostephanus truncatus often attack maize cob in the field towards the end of the season, and then continue in the store. Adults bore into the cassava or maize husks, cobs or grain, making neat round holes and tunneling extensively producing large quantities of grain dust as they tunnel. The adults prefer grain on cobs to shelled grain, thus damage on unshelled maize is greater than on loose, shelled maize.

Various prior art concerning control of insects using plant-based pesticides have been disclosed in publications and patents. The closest prior art patent is PCT/AU2012/000160 by Innovate Ag Pty Limited dated Feb 21, 2012 concerning a composition for controlling insect pests comprising an extract from Clitoria ternatea comprising secondary plant compounds (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. However, this prior art is different from the present invention that is directed to the development of blended compositions suitable for controlling the behaviour of insects, as well as methods of use of such compositions. Objects of Invention

The first object of the present invention is to provide a solution to post-harvest pest management of food and agricultural commodities by using a feeding deterrent blend for post-harvest insect pests.

The second object of the present invention is to avail simple, efficacious and safe deterrent blend for post-harvest insect pests.

DISCLOSURE OF INVENTION The present invention describes a post-harvest feeding deterrent blend that contains 1,8-Cineole, fenchone, camphor, a-pinene, β-pinene, β-caryophyllene, γ-terpine and terpinen-4-ol present in Plectranthus marruboides and Tetradenia riparia, and related compounds are feeding deterrents of Prostephanus trancatus and other post-harvest insect pests of dry stored food and agricultural products.

Further, the invention disclose a 10% formulation of 1,8-cineole or fenchone with any combination of the other compounds listed here give 100% protection to maize against damage by the larger grain borer and weevils for up to 6 months in a single application. These chemicals, alone or in combination with one another and/or other feeding deterrents (natural/ synthetic), provide useful natural tools for protection of stored dried food products against damage by post-harvest insect pests. DETAILED DESCRIPTION OF INVENTION

Although it is known that LGB breeds only in maize and cassava and attack the crops from the farm before harvesting, potent phytochemical feeding deterrents of the LGB have not been identified.

In the first embodiment according to the present invention describes identification of natural feeding deterrents of LGB such as the ones described here represent ideal candidates for use in management of post-harvest insect pests that pose serious threat to food security through the damages that result in dried food products and other agricultural commodities.

Dried maize and cassava chips are severely damaged by the larger grain borer {Prostephanus truncatus Horn), which is a serious primary pest of these dry stored food products. The present invention is based on the characterization of key feeding deterrent constituents in the essential oils of Plectranthus marruboides and Tetradenia riparia, which play an important role in either killing the insect pest or deterring it from feeding thereby leading to death of the insect.

According to the invention, there is provided the use of compounds represented by formulae (I), (II), (III), (IV), (V), (VI) and (VII) in protecting dried food products and agricultural commodities from damage by post-harvest insect pests, which include but not limited to the larger grain borer, Prostephanus truncatus (Horn), which is serious primary post-harvest insect pest of dried maize and cassava chips:

9 10

Where X=0 or OR of which R is either a hydrogen or an alkyl group (CH ₃ , CH ₃ CH ₂ etc ), or a Carbonyl (COCH ₃ , COCH ₂ CH ₃ etc) and at either CI & C8, CI & C4, CI & C3, CI & C2, C3 & C8

(Π) (III) (IV) (V)

Where in (II) and (III) Y= O, or OR of which R is a Hydrogen or Alkyl group (CH ₃ , CH ₃ CH ₂ etc) or Carbonyl (COCH ₃ , COCH2CH3 etc); R', R" and R'" are Alky groups (CH ₃ , CH2CH3 etc);

while in (IV) C=C bond is either at CI & C2, or C8 & C9, Z = OR where R is a hydrogen or alkyl group (CH ₃ , CH3CH2, etc) or a carbonyl (COCH3, COCH ₂ CH ₃ etc) and can be at either CI or C2, or C3 or C4 or C8. In (V) W = CH ₂ when V = CH ₂ ; or W = CH ₃ when V = CH.

Where (VI) represents the caryophyllenes in which case Q = CH ₂ when C8-C9 bond is saturated for β-caryophyllene; or Q = CH ₃ when C8-C9 bond is a double bond as the C9-C1 bond of the four (4) member ring opens up to form a double bond at C1-C2 for a-Caryophyllene (also known as a-humulene); or Q = CH ₂ when C8-C9 bond is saturated (single bond) and an epoxidation reaction occurs between C4-C5 carbon double bond as in caryophyllene oxide.

Wherein (VII) the ring is saturated or unsaturated in which case they are terpinenes whereby there are two pairs of C=C bonds at CI & C2 and C3 & C4; or CI & C2 and C4 & C5; or CI & C2 and C4 & C8; or C4 & C5 and CI & C7.

As an important feature of the invention, there is provided the use at least two of any of the above compounds represented by formulae (I), (II), (III), (IV), (V), (VI) and (VII) in

combinations, with other compounds, synthetic or naturally occurring, in protection of stored dry food products and agricultural commodities against damage by post-harvest insect pests.

It is to be understood that the invention is meant to include the use of all compositions containing the compounds represented by formulae (I), (II), (III), (IV), (V), (VI) and (VII), alone or in blends, in varying proportions, with additional constituents, in killing or deterring post-harvest insect pests from feeding on stored dry food products and agricultural commodities or masking the natural attractive host odor. These may include formulations or concentrates containing diluents, solvents, or dispersing agents, surface-active agents, additives such as antioxidants and UV-screens etc., added for convenience of use or protection of the active agent(s) or its (their) increased effectiveness against damages caused by post-harvest insect pests in dry maize and cassava among other stored dry food products. Likewise, it is also to be understood that the invention includes all controlled-release formulations or devices prepared for convenience and/or extending the duration of performance of the individual compounds or blends deployed directly on dry stored food products or in space protection. Additionally, it is to be understood that the invention is meant to include the use of the said compounds, as formulations or in appropriate devices, with additional deterrent or repellent or insecticidal constituents, in post-harvest control management systems that involve protection of dry food products. Best Modes of Carrying out the Invention

The best way of practicing the present invention is illustrated but not limited by the following examples.

Example 1

Feeding Deterrence of synthetic blend from Plectranthus marruboides

Deterrence activity of the formulation of the plant oil and synthetic blends was evaluated in a dark room. Maize grain seeds (100g) were weighed and put into transparent plastic jars (50 ml) with perforations of small holes all over the container to allow air circulation between the container and the surrounding environment. The maize in the containers got separately treated with formulation of synthetic blends of the components identified in the P. marruboides oil. This blend comprised of 1,8-cineole, γ-terpinene, camphor, terpinen-4-ol and β-caryophyllene in the ratio 37:7:26:3:27, respectively, which represent the proportions in which the components exist in the plant leaf oil. The following 5 component blends were prepared:

Blend (i) was prepared using 5 components in relative proportion present in the typical plant leaf oil that also exhibited deterrent activity against the larger grain borer. These compounds included 1,8-cineole (37 wt%); γ-terpinene (7 wt%); camphor (26 wt%); terpinen-4-ol (3 wt%) and β- caryophyllene (27 wt%).

Blend (ii) contained all the compounds of blend (i) except 1,8-cineole.

Blend (iii) contained all the compounds of blend (i) except β-caryophyllene

Blend (iv) contained all the compounds of blend (i) except camphor Blend (v) contained all the compounds of blend (i) except oxygenated ones (1,8-cineole, camphor and terpinen-4-ol)

Blend (vi) contained oxygenated hydrocarbons, i.e. all the compounds of blend (i) except γ- terpinene and β-caryophyllene

Each blend was evaluated at five levels of dilutions (0.625, 1.25, 2.5, 5 and 10% w/w) and kept at room temperature (25°C) and 65-70% R.H. Ten unsexed adult P. truncates were introduced into the treated grains and allowed to feed. Four replicates of each concentration of the synthetic blends were tested and control experiments were performed.

The control experiments were two; the first involved treatment of the maize with hexane solvent which was used in formulation of the synthetic blends and the second one was just maize not treated by the hexane solvent and test compounds. After 30 days, dead and surviving LGBs were counted and the consumption of the maize was evaluated by counting: i. Total number of maize destroyed, ii. Total number of holes per maize.

Results

The results of the above experiment (Table 1) show that even though all the five components play a role in deterring the insect pest from feeding; 1,8-cineole and β-caryophyllene are crucial components of the blend because their removal registered some level of damage in maize (7) in the second month after application of the blend formulation. Non-oxygenated hydrocarbons, blend (v) was as active as the oxygenated blend (vi).

Table 1: Mean number of maize damaged by the Prostephanus trancatus after exposure to various treatments of chemical standards, blends of the standards and P. marruboides oil

Example 2

Feeding Deterrence of synthetic blend from Tetradenia riparia

Deterrence activity of the formulation of the plant oil and synthetic blends was evaluated in a dark room. Maize grain seeds (lOOg) were weighed and put into transparent plastic jars (50 ml) with perforations of small holes all over the container to allow air circulation between the container and the surrounding environment. The maize in the containers got separately treated with formulation of synthetic blends of the components identified in the T. riparia oil. This blend comprised of -pinene, β-pinene, 1,8-cineole, γ-terpinene, fenchone and caryophyllene O.lmg/ml) in the ratio of 6:11:24:41:53:1 respectively, which represent the proportions in which the components exist in the plant leaf oil. The following 6 components blend were prepared: Blend (vii) was prepared using 6 components in relative proportion present in the typical plant leaf oil that also exhibited deterrent activity against the larger grain borer. These compounds included:

a-pinene (4.4 wt%);

β-pinene (8 wt%); iii. 1,8-cineole (17.6 wt%);

iv. γ-terpinene (30 wt%);

v. fenchone (39 wt%) and

vi. β-caryophyllene (0.7 wt%).

5 Blend (viii) contained all the compounds of blend (vii) except fenchone.

Blend (ix) contained all the compounds of blend (vii) except 1,8-cineole.

Blend (x) contained 1,8-cineole and fenchone.

Each of the mentioned blends and some two single individual constituents (1,8-cineole and fenchone) were evaluated at five levels of dilutions (0.625, 1.25, 2.5, 5 and 10% w/w) and kept

10 at room temperature (25°C) and 65-70% R.H. Ten unsexed adult P. truncates were introduced into the treated grains and allowed to feed. Four replicates of each concentration of the synthetic blends were tested and control experiments on were performed. The control experiments were two; the first involved treatment of the maize with hexane solvent which was used in formulation of the synthetic blends and the second one was just maize not treated by the hexane solvent and

15 test compounds. After 30 days, dead and surviving LGBs were counted and the consumption of the maize was evaluated by counting: i. Total number of maize destroyed, ii. Total number of holes per maize.

Results

20 The results of the above experiment are summarized in table2. From these results 1,8-cineole and fenchone are key components of the deterrent blend because removal of 1,8-cineole and fenchone in blends (viii) and (ix), respectively led to some damage in maize by the larger grain borer. The deterrent activity of 1,8-cineole and fenchone were further confirmed when each of these compounds individually and as a blend (x) gave 100% protection of maize against damage

25 by the larger grain borer. Table 2: Mean number of maize damaged by the Prostephanus trancatus after exposure to various treatments of chemical standards, blends of the standards and T. riparia oil

INDUSTRIAL APPLICATION

The present invention has developed a feeding deterrent blend for post-harvest insect pests that is useful in post-harvest pest management of food and agricultural commodities.