Nematicidal compositions and methods of using them

Abstract

There is disclosed method of killing nematodes comprising the step of applying an effective amount of a nematicidal composition comprising a terpene component and compositions suitable for use in the method. The terpene component is preferably in association with water, either as a solution or a suspension. An excipient may also be included, which is suitably hollow glucan particles which encapsulate the terpene component.

Claims

1. A method of killing plant parasitic nematodes, said method comprising applying an effective amount of a nematicidal composition comprising hollow glucan particles, wherein the hollow glucan particles encapsulate a terpene component comprising a combination of thymol and geraniol, wherein the hollow glucan particles have a lipid content greater than 5% w/w, and wherein the nematicidal composition kills plant parasitic nematodes and wherein the composition comprises a surfactant which holds the terpene component in solution.

2. The method according to claim 1 wherein the nematicidal composition further comprises an excipient.

3. The method according to claim 1 wherein the hollow glucan particles are yeast cell walls or hollow whole glucan particles.

4. The method according to claim 3 wherein the yeast cell walls are derived from Baker's yeast cells.

5. The method according to claim 3 wherein the hollow glucan particles are obtained from the insoluble waste stream of a yeast extract manufacturing process.

6. The method according to claim 3 wherein the glucan particles are alkali extracted.

7. The method according to claim 3 wherein the glucan particles are acid extracted.

8. The method according to claim 1 wherein the hollow glucan particles have a lipid content greater than 10% w/w.

9. The method according to claim 1 wherein the surfactant is selected from the group consisting of sodium lauryl sulphate, polysorbate 20, polysorbate 80, polysorbate 40, polysorbate 60, polyglyceryl ester, polyglyceryl monooleate, decaglyceryl monocaprylate, propylene glycol dicaprilate, triglycerol monostearate, polyoxyethylenesorbitan monooleate, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, polyoxyethylene (4) lauryl ether and mixtures thereof.

10. A method of preparing a composition for killing plant parasitic nematodes, the composition comprising hollow glucan particles encapsulating a terpene component and wherein the terpene component comprises a combination of thymol and geraniol, said method comprising; a) providing the terpene component and a surfactant which holds the terpene component in solution in water; b) providing hollow glucan particles; c) incubating the terpene component with the glucan particles under suitable conditions for terpene encapsulation; and d) recovering the glucan particles encapsulating the terpene component.

11. The method according to claim 10 further comprising the step of drying the glucan particles encapsulating the terpene component.

12. The method according to claim 11 wherein the drying is achieved by freeze drying, fluidized bed drying, drum drying or spray drying.

13. The method according to claim 10 wherein in step a) the terpene component is provided as a suspension in an aqueous solvent.

14. The method according to claim 10 wherein the solvent is water.

15. The method according to claim 10 wherein the surfactant is present at a concentration of 0.1 to 10% by volume of the nematicidal composition.

16. The method according to claim 10 wherein the terpene component is provided as a true solution in water.

17. The method according to claim 10 wherein in step b), the hollow glucan particles are provided as a suspension in a solvent.

18. The method according to claim 17 wherein the suspension comprises 1 to 1000 mg glucan particles per ml.

19. The method according to claim 18 wherein the suspension comprises 200 to 400 mg glucan particles per ml.

20. The method according to claim 10 wherein the hollow glucan particles are provided as a dry powder and added to the terpene-surfactant suspension.

21. The method according to claim 10 wherein the glucan particles are provided in between the hydrodynamic volume and 1.5 times the hydrodynamic volume of water.

22. The method according to claim 11 wherein the conditions of step c) are atmospheric pressure and a temperature of 20 to 37 C.

23. A method of killing plant parasitic nematodes, said method comprising applying an effective amount of a nematicidal composition comprising hollow glucan particles, wherein the hollow glucan particles encapsulate a terpene component and wherein the terpene component comprises a combination of thymol and geraniol, wherein the hollow glucan particles have a lipid content greater than 5% w/w, and wherein the nematicidal composition kills plant parasitic nematodes.

24. A method of killing plant parasitic nematodes, said method comprising: applying an effective amount of a nematicidal composition comprising hollow glucan particles, wherein the hollow glucan particles encapsulate a terpene component comprising a combination of thymol and geraniol, wherein the hollow glucan particles have a lipid content greater than 5% w/w, and wherein the nematicidal composition kills plant parasitic nematodes.

Description

(1) Embodiments of the present invention will now be described by way of example only, with reference to the figures in which:

(2) FIG. 1 represents a light micrograph of empty yeast cell walls;

(3) FIG. 2 represents a light micrograph of yeast cell walls encapsulating L-carvone;

(4) FIG. 3 represents a light micrograph of yeast cell walls encapsulating citral;

(5) FIG. 4 represents a light micrograph of terpene emulsion;

(6) FIG. 5 represents a light micrograph of yeast cell walls in hydrodynamic volume (HV) water;

(7) FIG. 6 represents a light micrograph of yeast cell walls encapsulating terpene in 5 times hydrodynamic volume (HV) of water;

(8) FIG. 7 represents a light micrograph of yeast cell walls encapsulating terpene in HV of water;

(9) FIG. 8 represents a light micrograph of yeast cell walls encapsulating terpene in HV plus 5% of water;

(10) FIG. 9 represents a light micrograph of yeast cell walls encapsulating terpene in HV plus 10% of water;

(11) FIG. 10 represents a light micrograph of yeast cell walls encapsulating terpene in HV plus 20% of water;

(12) FIG. 11 represents a light micrograph of yeast cell walls encapsulating terpene in HV plus 30% of water;

(13) FIG. 12 represents a light micrograph of yeast cell walls encapsulating terpene in HV plus 40% of water.

(14) FIG. 13 represents a light micrograph showing the dispersal of dried hollow glucan particles encapsulating a terpene component and no xanthan gum.

(15) FIG. 14 represents a light micrograph as in FIG. 13 where 0.07 g of 1% xanthan gum is included.

(16) FIG. 15 represents a light micrograph as in FIG. 13 where 0.14 g of 1% xanthan gum is included.

(17) FIG. 16 represents a light micrograph as in FIG. 13 where 0.28 g of 1% xanthan gum is included.

(18) FIG. 17 represents a light micrograph as in FIG. 13 where 0.55 g of 1% xanthan gum is included.

(19) FIG. 18 represents a light micrograph as in FIG. 13 where 1.1 g of 1% xanthan gum is included.

(20) FIG. 19 represents a light micrograph as in FIG. 13 where 2.2 g of 1% xanthan gum is included.

(21) FIG. 20 represents a light micrograph as in FIG. 13 where 4.4 g of 1% xanthan gum is included.

EXAMPLE 1

Preparation of a Terpene Emulsion or Suspension Using a Surfactant

(22) A terpene, terpene mixture, or liposome-terpene combination can be combined with a surfactant to form a suspension. The volumetric ratio of terpenes is generally about 1-99%, and the surfactant volumetric ratio is about 1-50% of the solution/mixture. The terpenes, comprised of natural or synthetic terpenes, are added to water. The surfactant, preferably polysorbate 80 or other suitable GRAS surfactant, is added to the water/terpene mixture and then blended to from a suspension. Citral is a suitable terpene.

EXAMPLE 2

Preparation of a Terpene Solution (without Surfactant)

(23) The solution can be prepared without a surfactant by placing the terpene, e. g. citral, in water and mixing under solution-forming shear conditions until the terpene is in solution.

(24) 0.5 ml citral was added to 1 liter water. The citral and water were blended in a household blender for 30 seconds.

(25) Alternatively, moderate agitation also prepared a solution of citral by shaking by hand for approximately 2-3 minutes.

(26) Greater than about zero ppm to about 1000 ppm of natural or synthetic terpenes such as citral, b-ionone, geraniol, carvone, terpeniol, carvacrol, anethole, or other terpenes with similar properties are added to water and subjected to a solution-forming shear blending action that forces the terpene(s) into a true solution. The maximum level of terpene(s) that can be solubilized varies with each terpene. Examples of these levels are shown in Table 1.

(27) TABLE-US-00001 TABLE 1 Solution levels for various terpenes. Terpene Solution Level Citral 1000 ppm Terpeniol 500 ppm b-ionone 500 ppm Geraniol 500 ppm Carvone 500 ppm

EXAMPLE 3

Potency of Solution

(28) Terpenes will break down in the presence of oxygen. The rate at which they decay varies for each particular terpene.

(29) Citral is a terpene aldehyde and will decay over a period of days. Two protocols are described below which quantify the rate of decay of citral.

(30) The following protocol was used to determine the rate of decay of citral in a sealed container:

(31) Test Material

(32) A solution prepared as described in Example 2 containing citral at 1000 ppm was prepared in distilled water. This solution was stored in a capped glass vial for the duration of the test.

(33) Procedure

(34) A standard curve was prepared with citral and B-ionone as internal standard.

(35) At the beginning of the study and weekly for four weeks the 1000 ppm suspension was analyzed using a gas chromatography procedure. The concentration of citral was determined by plotting it on the standard curve.

(36) The results are shown below in Table 2.

(37) TABLE-US-00002 TABLE 2 Stability of citral Percentage of citral remaining Day 1 Week 1 Week 2 Week 4 Citral 100 32 27 22 (1000 ppm)

(38) The following protocol was used to determine the rate of decay of citral in a container with a porous lid.

(39) To determine the concentration of citral in water the following protocol was used.

(40) Test Material

(41) A solution containing citral at 1000 ppm was prepared in distilled water. This solution was stored in a beaker covered with porous paper for the duration of the test.

(42) Procedure

(43) A standard curve was prepared with citral and B-ionone as internal standard.

(44) At the beginning of the study and after a week the 1000 ppm suspension was analyzed using a gas chromatography procedure. The concentration of citral was determined by plotting it on the standard curve.

(45) The results are shown below in Table 3.

(46) TABLE-US-00003 TABLE 3 Stability of citral Percentage of citral remaining Day 1 Week 1 Citral 100 21.5% (1000 ppm)

EXAMPLE 4

Extraction of Nematode Eggs from Soil and Counting Nematode Numbers

(47) Extraction of Eggs and Quantification of Soil Populations

(48) The following is an outline of a suitable technique to determine the population densities of soybean cyst nematodes SCN in soil samples, although it would be applicable to other soil nematodes. The procedure has three stages: extracting the cysts from the soil; crushing the cysts to extract the eggs; and, microscopic observation of the suspension of eggs for counting.

(49) Extraction of Cysts from Soil

(50) Cysts of soybean cyst nematode are recovered from soil through a combination of wet-sieving and decanting. The technique is a modification of the Cobb (Cobb, N. A. 1918. Estimating the nema population of soil. U.S. Dept. Agr. Bur. Plant Ind. Agr. Tech. Cir., 1:1-48) sifting and gravity technique.

(51) The procedure is as follows: 1. Combine a well mixed 100 cm.sup.3 soil sample (approx. cup) in a bucket with two (2) quarts (2.27 liters) of water. 2. Break any clumps with your fingers and mix the soil suspension well for 15 seconds. 3. Pour the soil suspension through an 8-inch-diameter #20 (850 mm pore) sieve into another bucket. Briefly rinse the debris caught on the 20 mesh sieve. 4. Pour the soil suspension in the second bucket through a #60 (250 mm pore) sieve. 5. Backwash the debris caught on the 60 mesh screen into a pan. 6. Repour the suspension through the 60 mesh screenhold the screen at an angle to concentrate the cysts and debris. 7. Backwash into a pan using a minimal (<250 ml) amount of water. 8. Pour the cysts and debris into a 250 ml beaker. NOTE: Discard the heavier material that quickly settles to the bottom of the buckets/pans during the above sieving process.

(52) Extraction of Eggs from the Cysts

(53) The above technique will result in a suspension of SCN cysts, along with organic debris and sediments similar in size to the cysts. The cysts in this suspension could be counted using a simple dissecting microscope. Some laboratories that analyze soil for soybean cyst nematode report results in the form of cysts per 100 cm.sup.3 of soil. Egg content of cysts is highly variable, and will not yield reliable counts of the SCN population in the sample. Therefore, it is preferable if eggs are extracted from the cysts and results are reported back as eggs and second stage juveniles (J-2) per 100 cm.sup.3 of soil.

(54) The procedure used to extract eggs from cysts is as follows: 1. Allow cysts/debris to settle for ca 30 minutes in the 250 ml beakers. Pour off excess water, resuspend sediments and transfer to 50 ml beakers. 2. Allow cysts to settle in the 50 ml beakers. 3. Pour off excess water (30 ml) and transfer the cyst/debris suspension to a 55 ml Wheaton Potter-Elvehjen tissue grinder. 4. Grind at 7500 RPM for 10 seconds. Rinse pestle into grinding tube. 5. After grinding, pour the suspension in the tube through an 8-inch-diameter #200 (75 mm pore) sieve over a stainless steel #500 (25 mm pore) sieve. 6. Rinse the tube several times with tap water, each time pouring the contents through the sieves. Discard sediments caught on the #200 sieve. 7. Carefully wash sediments and eggs caught on the #500 sieve into a clean beaker with as little water as possible.

(55) Counting Eggs with the Nematode Counting Slide:

(56) The volume of the egg suspension should be brought up to exactly 50 ml with tap water. Fill the chamber of the nematode counting slide with a well-mixed suspension using a pipette. The specially made nematode counting slides are constructed so that the volume of egg suspension observed over the grid is exactly 1 ml. Consequently, simply count the number of eggs that appear within the grid of the slide to determine the number of eggs per ml of suspension. The total number of eggs in the sample can then be calculated by multiplying the number of eggs per ml by 50.

(57) Sources of Materials and Equipment

(58) Sieves: Fisher Scientific, 1600 W. Glenlake Avenue, Itasca, Ill. 60143-(800) 223-9114 VWR Scientific, P.O. Box 66929, O'Hare AMF, Chicago, Ill. 60666-(800) 932-5000

(59) Tissue Grinder: Fisher Scientific, 1600 W. Glenlake Avenue, Itasca, Ill. 60143-(800) 223-9114

(60) Motorized Stirrer:

(61) The motorized laboratory stirrer is a Talboys Model 101 stirrer. This stirrer can be purchased through VWR Scientific or directly through Talboys Engineering Corporation, South Montrose, Pa. 18843.

(62) Nematode Counting Slides:

(63) The specially made nematode counting slides can be purchased from Advanced Equine Products, 5004 228th Avenue S.E., Issaquah, Wash. 98029, (425) 391-1169, FAX (425) 391-6669.

EXAMPLE 5

Effect of Terpenes on Nematode Egg Hatching and Juvenile Survival

(64) The effect of various terpene containing compositions was assessed in relation to nematode eggs and juvenile nematodes.

(65) The protocol used was as follows:

(66) The live eggs were treated in the various samples for one hour, rinsed, put back into distilled water and counted 24 hours later. The samples were made up as shown in Table 4a:

(67) TABLE-US-00004 TABLE 4a Sample Components NM1 10% Tween 80 45% d-limonene 45% b-Ionone NM3 10% Tween 80 45% citral 45% b-Ionone NM5 10% Tween 80 45% citral 45% a-terpineol NM6 10% Brig 30 45% a-terpineol 45% b-Ionone NM7 10% Tween 80 45% a-terpineol 45% b-Ionone

(68) The results of the protocol are shown below in Table 4b.

(69) TABLE-US-00005 TABLE 4b Results Sample Egg batch Juveniles Designation Conc. (%) (%) alive (%) Control 19 86 NM1 0.5 3 0 0.1 10 19 0.05 17 67 NM3 0.5 2 1 0.1 5 3 0.05 10 31 NM5 0.5 4 0 0.1 9 16 0.05 16 37 NM6 0.5 11 13 0.1 17 36 0.05 16 48 N6 0.5 26 53 0.1 26 58 0.05 15 60 NM7 0.5 13 74 0.1 13 58 0.05 17 75

(70) Observations: The combinations containing citral (NM3 and NM5) were more effective. The Brig surfactant was not as effective as Tween 80. The aldehyde worked better than the alcohols.

EXAMPLE 6

Effect of Terpenes on Mature Root-Knot, Ring and Citrus Nematodes

(71) The effect of various terpene containing compositions was assessed in relation to Root-Knot nematodes (Meloidogyne), Ring nematodes (Criconemella xenoplax) and Citrus nematodes (Tylenchulus semipenetrans).

(72) The protocol used was as follows:

(73) Nematodes: A single 5 ml volume with pre-counted nematode numbers was used as the initial inoculum. Nematodes were collected, identified and maintained from commercial agricultural crops soils. The nematodes were counted and evaluated for good health for the duration of the study.

(74) Nematicidal compositions: In this protocol the terpene used in the nematicidal composition was citral. The relevant details of the citral used are as follows: Chemical Name: CITRAL Common Name: Lemongrass Oil Formulation: CITRAL FCC Product Trade Name: CITRAL FCC Product code: 03-29200 Source: Penta Manufacturing Lot Numbers: 77887 Type: Liquid Carrier: Distilled Water

(75) Storage Conditions: Ambient indoor room temperature 65 F. (28.3 C.).

(76) Stability: Insoluble in water above 1,000 ppm.

(77) 3 different concentrations of citral were used to assess the efficacy of terpenes in killing the nematodes. These were untreated control (UTC), 500 ppm and maximum soluble terpene concentration (900 ppm). The terpenes were combined with water as a solution by mixing at a solution forming shear. The 900 ppm concentration value was not be measured, but estimated at the maximum soluble concentration that can be obtained with distilled water at 65 F. (28.3 C.). 3 replicates of the 900 ppm concentration were used (R1, R2 and R3) and one replicate of the 500 ppm concentration and UTC.

(78) Test mixtures of nematodes and the nematicidal compositions were made up according to Table 5.

(79) TABLE-US-00006 TABLE 5 Test Mixtures Terpene Added Nematode + Nematode Conc. Terpene Terpene Treatment Label Vol. ml ppm Vol. Ml Vol. Conc. ppm UTC 5.0 0.0 5.0 10.0 0.0 1.0 5.0 500.0 15.0 20.0 375.0 R1 5.0 900.0 15.0 20.0 675.0 R2 5.0 900.0 15.0 20.0 675.0 R3 5.0 900.0 15.0 20.0 675.0

(80) The terpene and nematode containing water was combined to form a final dilution volume and maintained in vials between evaluations. The nematodes were exposed to the terpenes for between 48 to 72 hours depending on their survival.

(81) Evaluations: Nematodes were be counted and their appearance assessed by microscope. The microscope used for assay provided for only 5 ml to be viewed at one time. Therefore, the 20 ml of total terpene nematode sample water was divided into 4 parts for each assay and recombined afterwards. The rating of degree of efficacy of the test samples was determined by observing nematode mobility, mortality, and internal disruption or vacuolation over time.

(82) The results are shown below in Table 6.

(83) TABLE-US-00007 TABLE 6 Results Root-Knot Ring Citrus Sample I.D. Meloidogyne CX TS Day Treatment Time Alive Dead alive Dead alive dead (pretreatment reading) 1 UTC 11:00 am 351 0 357 0 148 0 1 1.0 11:00 am 359 0 325 0 119 0 1 20 ml-R1 11:00 am 326 0 264 0 132 0 1 20 ml-R2 11:00 am 347 0 260 0 141 0 1 20 ml-R3 11:00 am 328 0 442 0 137 0 (postreatment readings) 1 UTC 6:00 pm 348 0 350 0 144 0 1 1.0 6:00 pm 355 0 319 0 114 0 1 20 ml-R1 6:00 pm 320 0 258 0 128 0 1 20 ml-R2 6:00 pm 341 0 255 0 139 0 1 20 ml-R3 6:00 pm 325 0 436 0 134 0 2 UTC 6:00 am 344 0 348 0 140 0 2 1.0 6:00 am 350 0 312 0 112 0 2 20 ml-R1 6:00 am 140 176 91 0 160 0 2 20 ml-R2 6:00 am 168 169 110 141 46 84 2 20 ml-R3 6:00 am 137 184 181 248 70 59 2 UTC 6:00 am 340 0 342 0 135 0 2 1.0 6:00 am 340 6 304 4 101 8 2 20 ml-R1 6:00 am 0 302 0 239 0 109 2 20 ml-R2 6:00 am 0 322 0 236 0 116 2 20 ml-R3 6:00 am 0 305 0 402 0 117 3 UTC 6:00 am 330 3 336 1 126 5 3 1.0 6:00 am 189 149 190 108 47 51

(84) There was a small nematode loss from one reading to another due to nematodes hanging up on the sides of dishes and vials. These populations are usually under 5 nematodes per reading.

(85) Observations: Day 1pretreatment readings showed no dead nematodes and the nematodes were all moving and had no internal disruption or vacuolation. Day 16 pm (20 mlR1+R2+R3) treatments all appeared to have slowed movement but they had no internal disruption or vacuolation. Day 16 pm (1.0 and UTC) treatments showed no slowing of movement or internal disruption or vacuolation. Day 26 am (UTC and 1.0) treatments all appeared normal with no loss of movement and no internal disruption or vacuolation. Day 26 am (20 mlR1+R2+R3) treatments had some dead (dead had no movement and their internal body structures were highly vacuolated). The living nematodes were still moving, although slowly, but no internal disruption or vacuolation. Day 26 pm (UTC) treatment all appeared normal with no loss of movement and not internal disruption or vacuolation. Day 26 pm (1.0) treatment had some dead. Dead had no movement with internal disruption and vacuolation. Some of the living had slowed movement and some did not, but none had any internal disruption or vacuolation. Day 26 pm (20 mlR1+R2+R3) treatments were all dead with no movement and internal disruption with vacuolation. Day 36 am (UTC) treatments showed a few dead or dyeing nematodes. They had no movement but showed no internal disruption or vacuolation. The rest of the nematodes, listed as alive, still had good movement. Day 36 am (1.0) treatments showed about 50% dead and both internal disruption and vacuolation. The alive nematodes showed some slowing of movement but no internal disruption or vacuolation.

(86) As can be clearly seen from the results, on day two by 6 pm, compositions R1, R2 and R3 had killed all nematodes. This demonstrates the highly nematicidal properties of compositions R1, R2 and R3 and consequently the nematicidal properties of citral.

EXAMPLE 7

Effect of Citral Alone and Citral and Thymol on Root-Knot Nematode Juveniles

(87) Treatment samples were prepared as follows: Cital1 ml citral was added to 400 ml of sterile distilled water and mixed using a household blender for 40 seconds. This was labelled 2500 ppm and was diluted to provide test solutions at 500, 250, 125 and 62.5 ppm. Citral and Thymol1.0 g of thymol was dissolved in 1 ml of citral and blended in 400 ml of water as for citral alone. This was marked 2500 ppm and diluted to provide test solutions at 500, 250, 125 and 62.5 ppm. ControlWater was used as the control.

(88) Nematode juveniles were collected in water and 0.1 to 0.15 ml added to each well of a plastic assay plate. 1.0 ml of the test solutions was added to each well. Observations were made microscopically after 24 and 48 hours as described in Example 4. Dead nematodes adopt a straight position and do not move when probed with a fine needle. Living nematodes move in an undulating, wave-like motion.

(89) The results of two experiments are provided below in Tables 7 and 8. The figures given are for the percentage of nematodes found to be dead upon microscopic examination and are the average of 2 replicates.

(90) TABLE-US-00008 TABLE 7 Effect of test solutions of root-knot juveniles after 24 and 48 hours Citral and Thymol Cital (ppm) Control Test (ppm) 500 250 125 500 250 125 Water 24 h 100 100 100 98 100 100 10 48 h 100 91 50 97 91 24 31

(91) TABLE-US-00009 TABLE 8 Effect of test solutions of root-knot juveniles after 24 hours. Citral and Thymol Cital (ppm) Test (ppm) Control 250 125 62.5 250 125 62.5 Water 24 h 97 96 94 94 94 98 6

(92) The results demonstrate the ability of citral alone and a citral and thymol mixture to kill nematodes at low concentrations. Kill rates in table 7 after 48 hours were over 90% for both mixtures at 250 ppm and 500 ppm concentrations. The 125 ppm concentration showed a lower kill rate. The kill rates in Table 8 show high kill rates after 24 hours for concentration as low as 62.5 ppm.

(93) The mixture of thymol and citral did not show a significant increase in kill rate over citral alone.

(94) The results show that citral is an effective nematicide even at low concentrations.

EXAMPLE 8

Effects of Citral on Root-Knot Nematodes Vs Sarprophagous Nematodes

(95) The purpose of this experiment was to demonstrate that citral selectively kills the harmful root-knot nematodes over saprophagus nematodes, which are not harmful, and indeed may be beneficial to the plant and soil. Such selective killing is a surprising effect that means treatment with terpenes may kill parasitic nematodes, but not eliminate the beneficial micro-fauna in the soil.

(96) Aqueous text mixtures comprising 250 ppm citral alone and 250 ppm citral and 10% tween were produced according to the techniques described in Example 7 above. These compositions were then incubated with root-knot and saprophagus nematodes and the kill rate assessed microscopically. Living saprophagus nematodes move rapidly in water. The control used was the nematodes in water alone.

(97) The results are provided in Tables 9 and 10 below. The figures given are for the percentage of nematodes found to be dead upon microscopic examination and are the average of 2 replicates.

(98) TABLE-US-00010 TABLE 9 Nematicidal activity of citral on root-knot nematodes (% dead) Citral + Citral + Tween 80 Citral Tween 80 Citral (250 ppm) (250 ppm) (250 ppm) (250 ppm) Control 24 h 87 87 89 88 17 48 h 100 100 100 100 22

(99) TABLE-US-00011 TABLE 10 Nematicidal activity of citral on Saprophagous nematodes (% dead) Citral + Citral + Tween 80 Citral Tween 80 Citral (250 ppm) (250 ppm) (250 ppm) (250 ppm) Control 24 h 45 43 51 50 15 48 h 50 50 53 52 19

(100) The results clearly show that citral kills the pathogenic root-knot nematodes at a much higher kill rate than the beneficial saprophagus nematodes. After 48 hrs the kill rate for root-knot nematodes was 100% for all test mixtures, whereas for Saprophagus nematodes it was only 50-53%. The results were not significantly effected by the inclusion of Tween 80.

(101) The results demonstrate that terpenes have the ability to selectively kill pathogenic nematodes whilst allowing beneficial nematodes to survive in the soil. This would result in a more healthy soil environment post treatment than a treatment which kills the entire nematode population in the soil. Firstly this is because beneficial nematodes would be present in the soil post treatment, and secondly there would not be a nematode vacuum in the soil which could be filled with pathogenic nematodes or other pathogens.

(102) It could be expected that at a very high concentration of terpene may result in a higher kill rate of saprophagus nematodes, thus reducing the selectivity of the treatment. Therefore in use in the field the minimum concentration that achieves the desired kill rate in root-knot or other parasitic nematodes may be selected, thus maximising the selectivity.

EXAMPLE 9

Effect of pH on the Nematicidal Activity of Citral Containing Compositions

(103) The following protocol was performed to assess the affect of pH on test solutions containing citral.

(104) Solutions were made up of citral at 250, 125 and 62.5 ppm concentrations. Test solutions of these three concentrations were prepared at different pHs by adjusting the pH with HCl or NaOH to pH 4, 7 and 10.

(105) One batch of test solutions was used immediately and another was left for 24 hours before use. The method of administration to the nematodes and counting the kill rate is the same as for previous protocols.

(106) The results are shown below in Tables 11 and 12. The figures given are for the percentage of nematodes found to be dead upon microscopic examination and are the average of 2 replicates.

(107) TABLE-US-00012 TABLE 11 Effect of fresh citral at three pH levels on root-knot nematodes (% nematodes dead) 250 ppm 125 ppm 62.5 ppm PH 4 7 10 4 7 10 4 7 10 Water 24 h 75 73 83 31 44 39 48 39 32 21 48 h 73 72 87 50 47 39 50 44 45 30

(108) TABLE-US-00013 TABLE 12 Effect of one-day old citral at three pH levels on root-knot nematodes (% nematodes dead) 250 ppm 125 ppm 62.5 ppm PH 4 7 10 4 7 10 4 7 10 water 24 h 90 40 47 27 25 25 40 30 16 10 48 h 90 33 52 31 33 32 27 27 21 14

(109) The results demonstrate that, in general, the test solutions lose efficacy if left for one day before use. However, it was observed that the citral solutions at the low pH (i.e. 4) did not lose efficacy to such an extent and, in fact the 250 ppm sample actually increased in efficacy after being left for a day. At all concentrations tested, the low pH samples did not demonstrate nearly such a significant a drop of efficacy after being left when compared to the neutral and high pH counterparts.

(110) This demonstrates that low pH of citral is beneficial in terms of retaining the efficacy of citral as a nematocide over time. The reasons for this are unclear, but may be the result of stabilising the citral and preventing degradation.

(111) It is therefore clear that adjusting the pH of a citral containing nematicidal composition to be acid (i.e. a pH below 7) would be beneficial in terms of prolonging its action.

EXAMPLE 10

Comparison of Nematicidal Activity High Purity Citral (98% Pure) with Low Purity Citral (80% Pure)

(112) Citral is commercially available in 2 formsregular (98% pure) and technical (80% pure). The following protocol was carried out to determine if technical citral is a viable-alternative to pure citral.

(113) Compositions of regular and technical ciral at 250 and 125 ppm were produced in 1% Tween 80 and incubated with root-knot nematodes a in the same way as previously described. Observations of the kill rate (percentage dead) were made at 21 and 42 hours.

(114) The results are shown below in Table 13 and are the average of four replicates.

(115) TABLE-US-00014 TABLE 13 average percentage dead Citral Citral 1% Tween (98% pure) (80% pure) 80 Water Ppm 250 125 250 125 21 h 87 23 89 29 14 7 42 h 87 22 96 27 17 18

(116) The results indicate that both regular and technical citral kill nematodes effectively at concentrations of 250 ppm. Thus technical citral may be used as a cheaper alternative to regular citral.

EXAMPLE 11

Nematicidal Effects of Citral in Soil

(117) The following protocol was carried out to assess the nematicidal properties of nematodes in soil.

(118) Methodology: Nematodes used for the analysis originated from commercial agricultural crop soils. Species of nematode included root-knot and citrus. Prior to commencement of each study the nematodes were counted and evaluated for viability. In each experiment soil samples were infected with only one species of nematode. Three measured quantities of soil (250 g) were placed into large PVC plastic containers.

(119) Soil moisture was assessed by weighing a soil sample and then drying the sample in a drying oven. Soil moisture content was confirmed using a Hydroscout instrument. In all cases the moisture content measured by both methods was within the resolution of the instruments. By determining the water content of the soil it was possible to calculate the volume of terpene solution which would be diluted when mixed with the soil.

(120) A series of citral dilutions in water were prepared (500 ppm to 62.5 ppm) such that when they were added to the soil samples, they would yield the required ratios. These dilutions were by volume not the more commonly used mass ratios. The reason for using volume dilutions was simply one of convenience enabling the use of a micropipette or cylinder to measure the terpene. The mass ratio of the in soil and in water solution could be simply calculated by multiplying the ppm of terpene by it's density (0.92 g/ml).

(121) The terpene solution was added to each test tube containing a weighed sample of nematode infected soil. The terpene solution and soil were mixed by inverting the test tube several times. The test tubes containing the soil and terpene solution were left to stand in racks in the laboratory for 48 hours-72 hours depending on the survival of the untreated nematodes. In each experiment a control group was treated with distilled water. The % mortality (kill) rates in the treatment groups was compared with the control population.

(122) The nematodes were extracted by Sieving & mist extraction (Ayoub, S. M. 1977) prior to being counted.

(123) Criteria for Evaluation: Nematode counts were performed to determine the proportion of nematodes which survived and were killed in each treatment group.

(124) TABLE-US-00015 TABLE 14 Pretreatment nematode counts Sample ID Root-Knot Citrus Mean nematode 659.25 12,711.75 counts (N = 8)

(125) The results are shown below in Tables 15 and 16.

(126) TABLE-US-00016 TABLE 15 Treatment of Root Knot nematodes with terpene solution. Terpene concentration No of Replicates Mean % killed 500 ppm 8 67.10 250 ppm 8 23.66 125 ppm 8 4.34 62.5 ppm 8 18.87 untreated 8 5.71

(127) TABLE-US-00017 TABLE 16 Treatment of Citrus nematodes with terpene solution Terpene concentration No of Replicates Mean % killed 500 ppm 8 95.53 250 ppm 8 91.66 125 ppm 8 46.29 62.5 ppm 8 2.84 untreated 8 13.7

(128) The protocol was repeated, this time using only citral at 500 ppm concentration. The results are shown below on Table 17 to 19.

(129) TABLE-US-00018 TABLE 17 Pretreatment nematode counts Sample ID Root-Knot Citrus Mean nematode 1225.25 10755.5 counts (N = 8)

(130) TABLE-US-00019 TABLE 18 Treatment of Root-Knot nematodes with terpene solution Terpene concentration N Mean % killed 500 ppm 10 99.6

(131) TABLE-US-00020 TABLE 19 Treatment of Citrus nematodes with terpene solution Terpene concentration N Mean % killed 500 ppm 10 99.9

(132) The experiment was performed once again, this time with the following changes: Dose range of 125 ppm-750 ppm was used. Glass tubes containing 150 g of soil were used as opposed to PVC tubes in previous experiments.

(133) The results are shown below in Table 20.

(134) TABLE-US-00021 TABLE 20 Treatment of Root Knot nematodes with terpene solution Terpene Mean % concentration N killed 750 ppm 8 99.42 500 ppm 8 100 250 ppm 8 97.37 125 ppm 8 74.51

(135) The results all show that terpenes are effective nematicides in soil. This supports the data already provided showing that terpenes are effective nematicides in vitro. Concentrations of terpene as low as 125 ppm demonstate strong nematicidal activity in soil, though concentrations of 250 ppm and above showed more consistent high kill rates.

EXAMPLE 12

Demonstration of Terpene Loading into Bakers Yeast Particles and Purified Yeast Glucan Particles

(136) The following protocol was performed to demonstrate that terpenes would load into yeast cell walls and other yeast glucan particles.

(137) Emulsions of citral and L-carvone were prepared by mixing 150 l of the terpene with 100 l of 10% Tween 80 in water and 250 l of water.

(138) Baker's yeast particles (YP) or Levacan yeast glucan particles (YGP), available from Savory Systems International, Inc., Branchburg, N.J., were mixed with water to form a 250 mg/ml suspension.

(139) 500 l of the YP or YGP suspension and 250 l of the terpene emulsion were mixed together and incubated overnight under constant agitation. 500 l YP or YGP suspension and 500 l of water were used as a control. The particles were then washed with water until free from external emulsion. The particle preparations were then frozen and lyophilised until dry.

(140) The particles were then rehydrated and examined under light microscope. The results are shown in FIGS. 1 to 4.

(141) FIG. 1 shows spherical structures with a dark area at their centre, these are empty hollow glucan particles. FIGS. 2 and 3 shows spherical structures with a swollen appearance with a light coloured interior, these are particles with terpene encapsulated in the central cavitycitral in FIG. 2 and L-carvone in FIG. 3. In FIGS. 2 and 3 small blobs of free terpene can also be seen, e.g. at the top of FIG. 2, just left of centre. FIG. 4 shows the terpene emulsion as small blebs of terpene suspended in water.

EXAMPLE 13

Determination of Maximal Citral and L-Carvone Loading Levels in Baker's Yeast Particles (YP)

(142) The following protocol was performed to determine the maximal amounts of terpenes that would load into YP. L-carvone and citral emulsions were prepared by sonicating 4.5 g of the terpene with 0.3 ml water. 10% Tween-80 solution was prepared by sonicating 4.5 g Tween-80 in 40.5 mls water. YP suspension was prepared by mixing YP with water to form 20 mg/ml suspension. Encapsulation reactions were set up as described in Table 21.

(143) Citral or L-carvone-water emulsion was mixed with YP and Tween 80 surfactant overnight at room temperature. Samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer was scored. The results are shown in the right hand column labelled free terpene of Table 21.

(144) The expression free terpene refers to the visible presence of terpene in the centrifuged reaction mixture. The absence of free terpene indicates complete absorption of the terpene by the particles. The highest volume of terpene absorbed by the particles, as evidenced by the absence of free terpene, was recorded as the maximal volume of absorbed terpene emulsion.

(145) TABLE-US-00022 TABLE 21 20 mg/ml 10% Tween- YP Terpene Vol 80 Free Tube l Emulsion l l Terpene 1 500 500 2 500 L-carvone 0.5 500 3 500 L-carvone 1.65 500 4 500 L-carvone 5 495 5 500 L-carvone 16.5 483.5 6 500 L-carvone 50 450 + 7 500 L-carvone 165 335 + 8 500 L-carvone 500 + 9 500 Citral 0.5 500 10 500 Citral 1.65 500 11 500 Citral 5 495 12 500 Citral 16.5 483.5 +/ 13 500 Citral 50 450 + 14 500 Citral 165 335 + 15 500 Citral 500 +

(146) As can be seen from the results, YP is capable of absorbing and encapsulating at least 16.5 l of L-carvone terpene emulsion or at least 5 l of citral emulsion per 10 mg of YP.

EXAMPLE 14

Demonstration of Improved Terpene Loading with Surfactant and Determination of Optimal Tween-80:Terpene Ratio

(147) The following protocol was performed to demonstrate that the presence of surfactant improves terpene loading and to determine the minimum level of Tween-80 surfactant required for the YP terpene loading reaction. L-carvone and citral emulsions were prepared by sonicating 4.5 g of the terpene with 0.3 ml water. 10% Tween-80 solution was prepared by sonicating 4.5 g Tween-80 in 40.5 ml water. Baker's YP suspension was prepared by mixing YP with water to form 250 mg/ml suspension.

(148) Loading reactions were set up as shown in Table 22 below.

(149) Citral or L-carvone-water emulsion was mixed with YP with 0-10% v/v Tween 80 surfactant overnight at room temperature. Samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer was scored. The results are shown in the right hand column labelled free terpene of Table 22.

(150) The expression free terpene refers to the visible presence of terpene in the centrifuged reaction mixture. The absence of free terpene indicates complete absorption and encapsulation of the terpene by the YP. The highest volume of terpene absorbed by the YP, as evidenced by the absence of free terpene, was recorded as the maximal volume of absorbed terpene emulsion.

(151) TABLE-US-00023 TABLE 22 250 mg/ml 10% Tween- YP Terpene Vol 80 Water Free Tube ml Emulsion l l l Terpene 1 500 500 2 500 L-carvone 150 0 350 Sl 3 500 L-carvone 150 5 345 Sl 4 500 L-carvone 150 10 340 Sl 5 500 L-carvone 150 33 317 Sl 6 500 L-carvone 150 100 250 7 500 L-carvone 150 200 150 8 500 L-carvone 150 350 9 500 L-carvone 400 0 100 ++ 10 500 L-carvone 400 5 95 ++ 11 500 L-carvone 400 10 90 ++ 12 500 L-carvone 400 33 77 ++ 13 500 L-carvone 400 100 + 14 500 L-carvone 400 20 l 100% 30 + 15 500 Citral 113 0 387 + 16 500 Citral 113 5 382 + 17 500 Citral 113 10 377 + 18 500 Citral 113 33 354 Sl 19 500 Citral 113 100 287 Sl 20 500 Citral 113 200 187 21 500 Citral 113 350 37 22 500 Citral 250 0 250 ++ 23 500 Citral 250 5 245 ++ 24 500 Citral 250 10 240 ++ 25 500 Citral 250 33 217 + 26 500 Citral 250 100 150 + 27 500 Citral 250 20 l 100% 230 + Sl = slight

(152) As can be seen from the results a Tween-80 concentration of 1% (i.e. 100 l of 10% Tween-80 in 1000 l of reaction mixture) is sufficient to allow complete uptake of the terpene in the above reaction. A 2% Tween-80 causes no improvement in results, whereas with a 0.33% concentration free terpene was observed. This indicates that: a) Terpenes are absorbed into YP particles in the absence of a surfactant, but the presence of surfactant significantly increases terpene absorption. b) A Tween-80 concentration of around 1% is optimum for YP loading as it ensures proper loading whilst maximising the terpene payload of the YP particles.

EXAMPLE 15

Determination of Maximal Terpene Loading and Encapsulation at High Baker's Yeast Particles (YP) Levels

(153) The following protocol was performed to determine the maximal amounts of terpenes that would load into YP at high YP levels. L-carvone and citral emulsions were prepared by sonicating 4.5 g of the terpene with 3 ml 1% Tween. 5% Tween-80 solution was prepared by sonicating 0.5 g Tween-80 in 9.5 ml water. YP suspension was prepared by mixing YP with water to form 250 mg/ml suspension. Encapsulation reactions were set up as shown in Table 23.

(154) Citral or L-carvone-water emulsion was mixed with YP and Tween 80 surfactant overnight at room temperature. Samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer was scored. The results are shown in the right hand column labelled free terpene of Table 23.

(155) The expression free terpene refers to the visible presence of terpene in the centrifuged reaction mixture. The absence of free terpene indicates complete absorption of the terpene by the YP. The highest volume of terpene absorbed by the YP, as evidenced by the absence of free terpene, was recorded as the maximal volume of absorbed terpene emulsion.

(156) TABLE-US-00024 TABLE 23 250 mg/ml 1% Tween- YP Terpene Vol 80 Free Tube l Emulsion l l Terpene 1 500 500 2 500 L-carvone 15 485 3 500 L-carvone 37.5 462.5 4 500 L-carvone 75 425 5 500 L-carvone 112.5 387.5 6 500 L-carvone 150 350 Sl+ 7 500 L-carvone 225 275 + 8 500 L-carvone 450 50 + 9 500 Citral 15 485 10 500 Citral 37.5 462.5 11 500 Citral 75 425 12 500 Citral 112.5 387.5 Sl+ 13 500 Citral 150 350 + 14 500 Citral 225 275 + 15 500 Citral 450 50 +

(157) As can be seen from the results in Table 9, YP is capable of absorbing and encapsulating terpenes at high YP concentration. YP absorbed and encapsulated at least 112.5 l of L-carvone terpene emulsion or at least 75 l of citral emulsion per 125 mg of YP. This demonstrates that the terpene encapsulation reaction is independent of YP concentration within the ranges tested.

EXAMPLE 16

Screen Commercially Available Particles for Terpene Absorption

(158) The following protocol was performed to analyse the loading properties of different types of particles. The particles studied were Baker's Yeast Particles (Sigma Chemical Corp., St. Louis, Mo.), Nutrex Walls (Sensient Technologies, Milwaukee, Wis.), SAF-Mannan (SAF Agri, Minneapolis, Minn.), Nutricept Walls (Nutricepts Inc., Burnsville, Minn.), Levacan (Savory Systems International, Inc., Branchburg, N.J.) and WGP (Alpha-beta Technology, Inc. Worcester, Mass.).

(159) L-carvone and citral emulsions were prepared by sonicating 7 g terpene+3 ml 3.3% Tween-80.

(160) Table 24 below compares the purity with the number of yeast particles per mg and the packed solids weight/volume ratio.

(161) TABLE-US-00025 TABLE 24 Purity % Beta 1,3- Yeast Particle glucan No. particles/mg Mg particles/ml Bakers 11.2 4 10.sup.7 250 Nutrex 24.5 1.7 10.sup.8 58.8 SAF Mannan 33.4 2.4 10.sup.8 41.7 2.7 10.sup.8 Nutricepts 55.7 5.2 10.sup.8 37 Levacan 74.6 1 10.sup.8 19.2 WGP 82.1 3.5 10.sup.8 10

(162) From Table 24 it can be concluded that the number of particles per mg is inversely proportional to purity. Thus the number of particles per mg of WGP is almost 10-fold higher than Baker's YP.

(163) The YP suspensions were prepared as follows: Baker's yeast particle suspension (YP) was prepared by mixing 250 mg YP/ml 1% Tween 80. Nutrex suspension was prepared by mixing 163 mg Nutrex YGP/ml 1% Tween 80. SAF Mannan suspension was prepared by mixing 234 mg Biospringer YGP/ml 1% Tween 80. Nutricepts suspension was prepared by mixing 99 mg Nutricepts YGP/ml 1% Tween 80. Levacan suspension was prepared by mixing 217 mg Lev YGP/ml 1% Tween 80. WGP suspension was prepared by mixing 121 mg WGP YGP/ml 1% Tween 80.

(164) The packed volume of the above particles is identical which means that equal numbers of particles were assayed.

(165) Loading reactions were set up as shown in Table 25 and left to incubate overnight. Samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer and the color of the encapsulated terpenes in the pellet was scored. The results are shown in the two right hand columns of Table 25. The highest volume of terpene absorbed by particles as evidenced by the absence of free terpene was recorded as the volume of absorbed terpene emulsion.

(166) TABLE-US-00026 TABLE 25 conc Terpene Vol Tube Particle mg/ml l Emulsion l 1% Tween 80 l Free Terpene Colour 1 Baker's 250 500 L-carvone 125 375 W 2 Nutrex 163 500 L-carvone 125 375 W 3 SAF Mannan 234 500 L-carvone 125 375 W 4 Nutricepts 99 500 L-carvone 125 375 + W 5 Levacan 217 500 L-carvone 125 375 + W 6 WGP 121 500 L-carvone 125 375 + W 7 Baker's 250 500 Citral 100 375 Y 8 Nutrex 163 500 Citral 100 375 Y 9 SAF Mannan 234 500 Citral 100 375 W 10 Nutricepts 99 500 Citral 100 375 + Y 11 Levacan 217 500 Citral 100 375 + int 12 WGP 121 500 Citral 100 375 + int 13 L-carvone 125 875 + 14 Citral 100 900 + Y W = white; Y = yellow; sl = sligh int = intermediate

(167) From the results the following conclusions were reached: Purified particles with a low lipid content were less effective at absorbing terpenes. Less pure particles were more effective at absorbing terpenes. Yellow degradation product of citral was not formed when encapsulated in SAF-Mannan. Based on qualitative loading at the single terpene level tested, SAF Mannan appears to be best, Nutrex second and Baker's third.

EXAMPLE 17

Kinetics of Terpene Loading into Various Types of Particles and Different Incubation Temperatures

(168) The following protocol was adopted to compare the loading kinetics of various types of yeast particles.

(169) L-carvone and citral emulsions were prepared by sonicating 7 g terpene with 3 ml 3.3% Tween-80.

(170) 1% Tween-80 solution was prepared by sonicating 1 ml 10% Tween-80 in 10 ml water. Baker's YP was prepared by mixing 5 g of bakers YP in 20 ml 1% Tween-80. Nutrex YGP suspension was prepared by mixing 2 g Nutrexm YGP in 20 ml 1% Tween-80. SAF Manna suspension was prepared by mixing 2 g SAF Mannan in 20 ml 1% Tween-80.

(171) Loading reactions were set up as shown in Table 26.

(172) The reactions were incubated for 1, 3, 6, 9 and 24 hours at room temperature or 37 C. After incubation samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer was scored. The results are shown in the two right hand columns of Table 26. The highest volume of terpene absorbed by the particles as evidenced by the absence of free terpene was recorded as the volume of absorbed terpene emulsion. Colour of the encapsulated pellet was scored at 24 hours.

(173) TABLE-US-00027 TABLE 26 T conc Terpene Vol 1% Free Terpene (hr) Tube C. Particle mg/ml l Emulsion l Tween-80 1 3 6 9 24 Color 1 Rt Bakers 250 3500 L-carvone 788 2712 + W 2 37 Bakers 250 3500 L-carvone 788 2712 + W 3 Rt Nutrex 100 3500 L-carvone 1050 2450 + W 4 37 Nutrex 100 3500 L-carvone 1050 2450 + W 5 Rt SAF 100 3500 L-carvone 1050 2450 <+ W 6 37 SAF 100 3500 L-carvone 1050 2450 <+ W 7 Rt Bakers 250 3500 Citral 525 2975 + Y 8 37 Bakers 250 3500 Citral 525 2975 + VY 9 Rt Nutrex 100 3500 Citral 788 2712 + Y 10 37 Nutrex 100 3500 Citral 788 2712 + VY 11 Rt SAF 100 3500 Citral 788 2712 + W 12 37 SAF 100 3500 Citral 788 2712 + W White, W; Yellow, Y; Very Yellow, VY; Room Temperature, Rt

(174) From the results shown in Table 26 and other observations the following conclusions can be made: Terpene loading reaction takes between 1 and 3 hours. Terpene loading occurs faster at 37 C. than at room temperature. SAF Mannan appears to be preferable particles for two reasons: Faster and more complete uptake of both terpenes. Citral remains stable when loaded as evidenced by the absence of yellow colour, characteristic of citral degradation, after 24 hours at 37 C.

EXAMPLE 18

Screen a Range of Single Terpenes and Terpene Combinations for Particle Loading

(175) The following protocol was adopted to compare the loading efficiency of Baker's YP versus SAF Mannan.

(176) Terpene emulsions were prepared as follows: L-carvone4.5 g L-carvone in 1.5 ml 3.3% Tween-80. Citral4.5 g citral in 1.5 ml 3.3% Tween-80. Thymol/L-carvone mixture (T/L)-2.25 g thymol and 2.25 g L-carvone in 1.5 ml 3.3% Tween-80. Eugenol4.5 g eugenol in 1.5 ml 3.3% Tween-80. Geraniol4.5 g geraniol in 1.5 ml 3.3% Tween-80. Citral/L-carvone/Eugenol mixture (C/L/E)1.5 g citral, 1.5 g L-carvone, 1.5 g eugenol in in 1.5 ml 3.3% Tween-80.

(177) Emulsions composed of terpene:water:surfactant ratio of 0.75:0.3:0.05 were used for these experiments.

(178) Increasing volumes of terpene emulsion were mixed with 250 mg/ml Baker's YP or 250 mg/ml SAF Mannan overnight at room temperature as shown in Tables 27 and 28. Samples were centrifuged at 14,000g for 10 minutes and the appearance of free terpene floating on the aqueous layer was scored. The highest volume of terpene emulsion absorbed by Baker's YP or SAF Mannan as evidenced by the absence of free terpene was recorded as the volume of absorbed terpene emulsion. Colour of encapsulated terpenes in the pellet was recorded. The results in Tables 27 and 28 show that all single and terpene combinations were efficiently loaded into both Baker's YP or SAF Mannan particles.

(179) TABLE-US-00028 TABLE 27 Evaluation of Baker's YP Loading of Different Terpenes and Terpene Mixtures. Baker Terpene Vol 1% Tween- Free Tube (l) Emulsion (l) 80 (l) Terpene Colour 1 500 500 W 2 500 L-carvone 15 485 W 3 500 L-carvone 37.5 462.5 W 4 500 L-carvone 7 425 +/ W 5 500 L-carvone 112.5 387.5 +/ W 6 500 L-carvone 150 350 + W 7 500 L-carvone 225 275 + W 8 500 L-carvone 450 50 ++ W 9 500 Citral 15 485 Y 10 500 Citral 37.5 462.5 Y 11 500 Citral 75 425 Y 12 500 Citral 112.5 387.5 +/ Y 13 500 Citral 150 350 + Y 14 500 Citral 225 275 + Y 15 500 Citral 450 50 + Y 16 500 T/L 15 485 W 17 500 T/L 37.5 462.5 W 18 500 T/L 75 425 W 19 500 T/L 112.5 387.5 +/ W 20 500 T/L 150 350 + W 21 500 T/L 225 275 + W 22 500 T/L 450 50 + W 23 500 Eugenol 15 485 W 24 500 Eugenol 37.5 462.5 W 25 500 Eugenol 75 425 W 26 500 Eugenol 112.5 387.5 +/ W 27 500 Eugenol 150 350 + W 28 500 Eugenol 225 275 + W 29 500 Eugenol 450 50 + W 30 500 Geraniol 15 485 W 31 500 Geraniol 37.5 462.5 W 32 500 Geraniol 75 425 W 33 500 Geraniol 112.5 387.5 + W 34 500 Geraniol 150 350 + W 35 500 Geraniol 225 275 + W 36 500 Geraniol 450 50 + W 37 500 C/L/E 15 485 Y 38 500 C/L/E 37.5 462.5 Y 39 500 C/L/E 75 425 Y 40 500 C/L/E 112.5 387.5 +/ Y 41 500 C/L/E 150 350 + Y 42 500 C/L/E 225 275 + Y 43 500 C/L/E 450 50 + Y

(180) TABLE-US-00029 TABLE 28 Evaluation of SAF Mannan Loading of Different Terpenes and Terpene Mixtures. SAF Terpene 1% Tween- Free Tube (l) Emulsion Vol 80 (l) Terpene Colour 1 500 500 W 2 500 L-carvone 15 485 W 3 500 L-carvone 37.5 462.5 W 4 500 L-carvone 75 425 W 5 500 L-carvone 112.5 387.5 W 6 500 L-carvone 150 350 +/ W 7 500 L-carvone 225 275 +/ W 8 500 L-carvone 450 50 + W 9 500 Citral 15 485 W 10 500 Citral 37.5 462.5 W 11 500 Citral 75 ul 425 W 12 500 Citral 112.5 387.5 W 13 500 Citral 150 350 +/ W Inverted 14 500 Citral 225 275 + W Inverted 15 500 Citral 450 50 + W Inverted 16 500 T/L 15 485 W 17 500 T/L 37.5 462.5 W 18 500 T/L 75 425 W 19 500 T/L 112.5 387.5 W 20 500 T/L 150 350 +/ W 21 500 T/L 225 275 + W 22 500 T/L 450 50 + W 23 500 Eugenol 15 485 W 24 500 Eugenol 37.5 462.5 W 25 500 Eugenol 75 425 W 26 500 Eugenol 112.5 387.5 +/ W 27 500 Eugenol 150 350 + W 28 500 Eugenol 225 275 + W 29 500 Eugenol 450 50 + W 30 500 Geraniol 15 485 W 31 500 Geraniol 37.5 462.5 W 32 500 Geraniol 75 425 W 33 500 Geraniol 112.5 387.5 W 34 500 Geraniol 150 350 W 35 500 Geraniol 225 275 W Inverted 36 500 Geraniol 450 50 + W Inverted 37 500 C/L/E 15 485 W 38 500 C/L/E 37.5 462.5 W 39 500 C/L/E 75 425 W 40 500 C/L/E 112.5 387.5 W 41 500 C/L/E 150 350 W 42 500 C/L/E 225 275 +/ W 43 500 C/L/E 450 50 + W Inverted = Phase Inverted solids floating on top no free oil; W = white; Y = yellow.

(181) From the results the following observations were made: All terpenes appeared to load into Baker's YP and SAF Mannan. SAF Mannan has a higher terpene loading capacity than bakers YP. The two and three way mixtures of terpenes also appear to efficiently load. The terpene Eugenol appears to have a higher density than the particles and water as it was found associated with the pellet. For the SAF Mannan, the higher load levels and lighter particles resulted in loaded particles floating on the surface of the aqueous layer for citral and geraniol. Citral was protected from oxidation by the SAF Mannan but not by the Baker's YP.

(182) The approximate maximal loading for each particle type was determined and is shown in tables 29 and 30 below. Percentage loaded represents a ratio of the amount of terpene loaded to the amount of particle present (weight for weight).

(183) TABLE-US-00030 TABLE 29 Maximal terpene loading in Baker's YP. Terpene Vol. Loaded l % Loaded w/w L-carvone 37.5 33.3 Citral 75 67% Thymol/L-carvone 1:1 75 67% Eugenol 75 67% Geraniol 75 67% Citral/L-carvone/ 75 67% Eugenol (1:1:1)

(184) TABLE-US-00031 TABLE 30 Maximal terpene loading in SAF Mannan. Terpene Vol. loaded l % Loaded w/w L-carvone 112.5 100% Citral 150 133% Thymol/L-carvone 1:1 112.5 100% Eugenol 112.5 100% Geraniol 150 133% Citral/L-carvone/ 150 133% Eugenol (1:1:1)

EXAMPLE 19

Evaluation of Terpene Stability in Aqueous Emulsions and Encapsulated Terpene Formulations

(185) Terpene stability was assessed by the observation of citral formulations for the formation of a yellow colored oxidation product. As noted in the right hand column in Tables 25-28 citral emulsions and citral encapsulated Bakers YP turned a progressively increasing yellow color over time. However, citral encapsulation in SAF Mannan increased citral stability as evidenced by a reduction or absence of yellow color over time.

EXAMPLE 20

Loading of Terpenes in Minimal Water

(186) The following protocol was carried out to evaluate the possibility that terpene loading and encapsulation into YP could be carried out at a very high Yeast Particles (YP) solids level to allow for direct extrusion of the loaded formulation into a fluidised bed drier. The minimal amount of water to completely hydrate the SAF Mannan particles was determined to be 3.53 g water per g solids. This defines the hydrodynamic volume (HV) or water absorptive capacity of the particles. At this level of water the hydrated particles have a consistency of a stiff dough which is thixotropic, i.e. shear thinning like mayonnaise. Addition of water up to 40% above the HV results in a thick flowable paste. The standard reaction that has been used in the above examples was carried out at 3HV water.

(187) A series of terpene (L-carvone) loading reactions were carried out keeping the ratio of particle:terpene:Tween (1:0.44:0.04) constant and varying the amount of water in the system from the HV (3.53 g) to HV+40% water (4.92 g). Controls were the standard loading system which uses 3HV water, particles only and terpene only reactions. Following overnight incubation samples of the mixtures were evaluated microscopically for free terpene and evidence of terpene uptake into the particles and for material flow characteristics by assessing flow in inverted tubes over 15 minutes. In addition, the presence of free oil was assessed by hydrating the reaction mixture with 5HV, vortexing to obtain a complete dispersion of particles and centrifugation to sediment the particle encapsulated terpene. The results are shown in Table 31 and FIGS. 7 to 12. FIGS. 7 to 12 show the loading results of the following tubes: FIG. 7Tube 3 FIG. 8Tube 5 FIG. 9Tube 6 FIG. 10Tube 8 FIG. 11Tube 10 FIG. 12Tube 11

(188) TABLE-US-00032 TABLE 31 Terpene Weight Water Free Tube SAF g Emulsion (g) (g) Terpene Flow 1 L-carvone 4.64 4.5 + + 2 1 8.0 + 3 1 L-carvone 4.64 4.5 + 4 1 L-carvone 4.64 5 1 L-carvone 4.64 0.17 6 1 L-carvone 4.64 0.35 7 1 L-carvone 4.64 0.52 Sl 8 1 L-carvone 4.64 0.7 Mod 9 1 L-carvone 4.64 0.87 High 10 1 L-carvone 4.64 1.05 High 11 1 L-carvone 4.64 1.39 High

(189) The results shown in Table 31 and FIGS. 7 to 12 demonstrate that terpene loading and encapsulation into the particles occurred at all water ratios evaluated. Surprisingly, equivalent loading occurred even when the loading reaction was taking place in a reaction with the consistency of a stiff dough using the minimal amount of water to hydrate the particles. The absence of free terpene was observed microscopically (FIGS. 7 to 12) and in the low level of terpene in the supernatants, as evidenced by a marked reduction in the turbidity of the supernatant compared to the terpene only control.

(190) These results extend our understanding of the conditions to load terpenes into hollow glucan particles. The flexibility to use a minimal volume of water to hydrate the particles during the loading process will allow loading of the terpenes under conditions where the reaction mixture is a malleable dough-like consistency using standard food-grade swept surface dough mixers. The consistency of the final high solids terpene loaded mixture is suitable for direct extrusion to form noodles and pellets for fluidised bed drying.

(191) Suitable facilities to scale up production in this manner would require: Gaulin homogeniser, or equivalent to produce stable terpene emulsion. Swept surface dough mixing tank. Extruder. Fluidised bed drier.

EXAMPLE 21

Evaluation of an Interstitial Hydrocolloid Agent to Aid Dispersion in Dried Hollow Glucan Particles Encapsulating a Terpene Component Dispersion when Re-Hydrated

(192) The following protocol was adopted to evaluate the effect of an interstitial hydrocolloid to increase dried hollow glucan particle encapsulated terpene formulations to disperse when hydrated. SAF Mannan particles 0.1% Tween 80 L-carvone Xanthan Gum1% w/v in 0.1% Tween 80

(193) The effect of increasing xanthan gum levels on dry hollow glucan particle encapsulated L-carvone dispersion in water was assessed by loading L-carvone into SAF Mannan by incubating 1.1 g of an L-carvone emulsion (L-carvone:water:surfactant ratio of 0.75:0.3:0.05) with 1 g SAF Mannan and 4.4 g 0.1% Tween 80 containing 0-1% xanthan gum as shown in Table 32.

(194) TABLE-US-00033 TABLE 32 L-carvone 0.1% 1% Emulsion Tween-80 Xanthan Visual Tube SAF g (g) (g) (g) Observations 1 1 1.1 4.4 0 Large non- uniform clumps 2 1 1.1 4.33 0.07 Uniform suspension 3 1 1.1 4.26 0.14 Uniform suspension 4 1 1.1 4.12 0.28 Uniform suspension 5 1 1.1 3.85 0.55 Uniform suspension 6 1 1.1 3.3 1.1 Finer Uniform suspension 7 1 1.1 2.2 2.2 Finer Uniform suspension 8 1 1.1 0 4.4 Finer Uniform suspension

(195) The results in Table 32 and FIGS. 13 to 20 demonstrate that the inclusion of a high molecular weight hydrocolloid during the drying of the particle encapsulated terpene aids in the hydration and dispersion of the microparticles into a uniform suspension. Other examples of such hydrocolloid agents are maltodextrin, alginates, or the like.

(196) It may also be worthwhile to include a pellet coating to increase the stability of the loaded terpenes, and to provide a sustained release of terpene.

EXAMPLE 22

Nematocidal Activity of Encapsulated Terpenes

(197) Preparations of yeast cell walls encapsulating citral were prepared according to the procedures described above. The hollow glucan particles contained 17.5% citral, and the particles were present at in the test preparations at a concentration of 1000 ppm. This means that terpenes were effectively present at a concentration of 175 ppm.

(198) 1.0 ml of the test preparations was added to 0.1 to 0.15 ml of water containing root-knot nematodes. 1.0 water was added to the nematodes as the control.

(199) Observations were made as [revopis; u described and the kill rate assessed (i.e. percentage dead) after 24 and 48 hrs. The results shown below in Table 13 are an average of 2 sets of results.

(200) TABLE-US-00034 TABLE 33 Nematicidal activity of encapsulated terpene solution (17.5% citral @ 1000 ppm) Kill Rate Time Test Control 24 h 45 17 48 h 56 21

(201) The results demonstrate that hollow glucan particles encapsulating terpenes are effective at killing root-knot nematodes at a particle concentration of 1000 ppm, which corresponds to a citral concentration of only 175 ppm.

(202) Thus hollow glucan particles encapsulating terpenes appear to be as effective as terpenes in solution or with surfactant as nematicides. The nematicidal activity is retained despite the terpene being encapsulated within the particle. It can be expected that higher concentrations of terpenes within the hollow glucan particles, or higher concentrations of the particles would result in an even higher kill rate, as is the case for terpenes in solution or with surfactant.