NZ587844A - Pre-harvest treatment of crops with C11-C20 mineral oils - Google Patents

Abstract

Disclosed is a pre-harvest prevention and/or treatment method for eradication of biological infestation in crops comprising the step of applying a mineral oil pre-harvest to the crop, wherein the oil is aromatic-free and is great than or equal to nC11 and less than nC18. In particular is is a nC13 and /or nC14 paraffinic oil applied to kiwifruit by spraying.

Description

RECEIVED at IPONZon 13 December 2010 Patent No. 5 - Complete Specification Pre-harvest treatment of crops We, Caltex Australia Petroleum Pty Ltd, of Level 11 MLC Centre, 19-29 Martin Place, Sydney, New South Wales 2000, hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement Freehills l\6986245 Page 1 6993881 RECEIVED at IPONZon 13 December 2010 1A Pre-Harvest Treatment of Crops Field of the invention The present invention relates to the pre-harvest treatment of crops. More particularly, the invention relates to the application of mineral oils and emulsions with specified 5 characteristics to crops such as fruit and vegetables for treating biological infestations including pests, parasites, funguses, moulds, etc. The invention will be primarily described with reference to its use on kiwifruit but it should be appreciated that the invention also has application with the treatment of infestation in other fruit, vegetables, ornamental plants and other crops.

Background of the invention Many crops are susceptible to biological infestation pre-harvest, which can be caused by certain pests. The pests may affect the crops in a number of ways, including using up the nutrients of the plants, eating away at the fruit or injecting toxins into the plant. This can lead to delays in the maturity of the plant and result in the production of 15 deformed fruit, subsequently affecting the yield of crops. Armoured scale pests are particularly significant pests of most perennial horticultural crops (e.g. apples, citrus, stonefruit and kiwifruit). Three key pest species of armoured scale insect include latania scale (Hemiberlesia tataniae), greedy scale (Hemiberlesia rapax) and oleander scale (Aspidiotus nerii). It is therefore desirable to treat fruit, vegetables and plants pre-20 harvest, in order to eradicate any such pests. A number of methods for control of biological infestation are currently employed, including biological pest control (the control of one species through the control and management of natural predators and parasites), field burning and use of pesticide sprays. However, not all methods are applicable to all crops, and some crops can be detrimentally affected by one type of 25 pest control method. For example, kiwifruit are affected in a detrimental manner by certain pesticides, the application of which can lead to premature fruit drop and phytotoxicity (as a result of the inhibitory effect of the pesticide on fruit respiration).

Kiwifruit growers have traditionally used nC20 to nC24 spray oils for the control of scale pests, as oils do not leave pesticide residues, which can affect the marketing of the fruit. 8249462 RECEIVED at IPONZ on 7 April 2011 2 However, the use of these "heavy" oils is restricted during certain periods when the vines are susceptible to damage from the oil.

Therefore, there remains a need for a method that is both effective against biological pests and that does not have a detrimental effect on crops.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in New Zealand or any other jurisdiction.

Summary of the invention The present invention provides a pre-harvest prevention and/or treatment method for 10 eradication of biological infestation in crops comprising the step of applying pre-harvest to the crop an oil.

In one embodiment, the oil is a mineral oil, which may be a very light mineral oil. Preferably, the mineral oil is greater than or equal to nC11 and less than nC20, and more preferably less than nC18. The most preferred oil is nC13 and/or /7C14. Preferably, the oil 15 is an aromatic-free oil. The oil may also be a paraffinic oil.

Preferably, the mineral oil has a median distillation point (ASTM D 86 or 2887) in the range of 200 to 300°C (at atmospheric pressure).

In one embodiment the oil is a volatile synthetic ester, which has a median distillation point (ASTM D 86 or 2887) in the range of 200 to 300°C (at atmospheric pressure).

In a further embodiment, the oil is applied by spraying. The oil may be applied as a low volume mist.

In another embodiment, the oil is applied as an oil-in-water formulation, most preferably an emulsion. Preferably, the oil is present in an amount of from about 0.5 to about 5% (v/v), more typically at about 2% (v/v). When the formulation is an emulsion it can include a 25 humectant and an emulsifier. The humectant may be glycerol mono-oleate. Typical emulsifiers include polyoxyethylene sorbitan trioleate, sorbitan mono-oleate, 8006990 3 cetyl-oleyl alcohol and mono-oleate polyoxyethylene sorbitan. Preferably, the emulsifier is a food grade aliphatic hydrocarbon.

In another embodiment, the crop is kiwifruit.

In another embodiment, the biological infestation is scale. The scale may be, for 5 example, oleander scale, greedy scale or latania scale.

Brief description of the drawings / figures Figure 1. The periods when there is a risk of phytotoxicity to Hayward and Hort.16A kiwifruit following sprays of mineral oil.

Figure .2., Oleander scale insect mortality 14 days after treatment with various 10 concentrations of nC13 mineral oil or D-C-Tron® Plus Spray Oil. The data have been corrected for control mortality and the vertical bars indicate 95% confidence intervals.

Figure 3. Hort16A fruit affected by an application of 1% ,nC20 oil at 105 days after fruit set: (a) rots on the side of fruit; (b) affected fruit ready to detach from the stalk.

Figure 4, Mean percentage of premature fruit drop from Hort16A vines following a single 15 spray of three new oil formulations and two commercial oil products at 76 days after fruit set. Error bars are SEMs.

Figure 5. Mean percentage of premature fruit drop from Hort16A vines following a single spray of three new oil formulations and 'two commercial oil products at 105 days after fruit set. Error bars are SEMs.

Figure 6. Marking to the skin of Hort16A fruit following a spray of 2% nC20 oil (a) or 2% DC-Tron® Plus-oil (b) at 76 days after fruit set.

Figure 7. The mean percentage of Hort16A fruit with skin damage (light and severe categories combined, severe only) following a single spray of five different oil formulations applied at 76 days after fruit set. Error bars are SEMs, 6006996 4 Figure 8. The mean percentage of Hort16A fruit with skin damage (light arid severe categories combined, severe only) following a single spray of five different oil formulations applied at 105 days after fruit set. Error bars are SEMs.

Figure 9. (a) Hayward cane (within white circle) infested with adult latania scale insects 5 suspended adjacent to the leader before spraying; (b) sprayed scale-infested cane suspended within canopy.

Figure 10. Mean mortality (%) of latania scale insects resulting from various oil treatments at concentrations of 1% and 2%, Vertical bars represent 95% confidence limits, Figure 11. Mean mortality (%) of oleander scale insects resulting from various oil treatments at concentrations of 1% and 2%. Vertical bars represent 95% confidence limits.

Figure 12. Sprayed potato infested with oleander scale insects, suspended adjacent to the kiwifruit leader.

Figure 13. Mean mortality (%) of latania-infested canes (left side) and oleander-infested potatoes (right side) resulting from various oil treatments at concentrations of 1 % and 2%. Vertical bars represent 95% confidence limits; numbers above bars denote sample size.

Detailed description of the embodiments As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features 25 mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. 8006996 In research leading to the invention, the inventors sought to provide new pesticide formulations, which eradicated pests and had no, or minimal, phytotoxic effect.

The inventors have surprisingly found that very light oils with certain characteristics can eradicate biological infestation when applied pre-harvest to crops and have no, or 5 minimal, phytotoxic effect.

Accordingly, the present invention relates to a pre-harvest prevention and/or treatment method for eradication of biological infestation in crops comprising the step of applying pre-harvest to the crop an oil.

Preferably the oil employed is a food grade aliphatic hydrocarbon, and is typically a very 10 light pa raff in ic oil of greater than or equal to nC11 and less than nC20, and more preferably less than nC18. The most preferred chain length of the oil is nC13 and/or nC14.

Within the context of the invention, the term "nCX", where X is a particular number, refers to an oil with a median normal paraffin equivalent carbon number of X. That is, 15 the temperature at which 50% of the oil's mass distils is that at which a normal paraffin, with X carbon atoms in the chain, distils.

Preferably, the mineral oil has a median distillation point (ASTM D 86 or 2887) in the range of 200 to 300°C (at atmospheric pressure). Mineral oils with a median distillation point (ASTM D 86 or 2887) in the range of 200 to 300°C are generally referred to as 20 hydrocarbon solvents or very light mineral oils. In accordance with the invention, such oils have been found to have a capacity to efficiently cover, spread and/or penetrate various crops (especially fruit) to which they are applied so as to penetrate and thus exterminate pests, parasites, funguses, mould, bacteria and the like.

The oil may also be a volatile synthetic ester, which has a median distillation point 25 (ASTM D 86 or 2887) in the range of 200 to 300°C (at atmospheric pressure).

As discussed previously, it has been found that certain crops, such as kiwifruit, are affected in a detrimental manner by certain pesticides, the application of which can lead 6008398 to skin damage and phytotoxicity (which usually manifests as premature fruit drop). The inventors have now found that very light paraffinic oils (e.g. ,oC13 and nC14 oils) do not cause skin damage to fruit or premature fruit drop. Without wishing to be bound by any theory or mode of action, the inventors propose that very light oils evaporate from the 5 fruit surface more rapidly than standard /iC20 to nC27 horticultural mineral oils, and have the advantage of allowing the fruit to respire, thus preventing skin damage and phytotoxicity due to oxygen depletion, which leads to fruit drop.

The inventors have also found that the method of the present invention can be used on the fruit at any time during the growing season, including periods when traditional spray 10 oils are known to damage the fruit. For example, in the case of kiwifruit, there are certain periods in the fruit growth cycle (e.g. a certain number of days after fruit set) when the fruit can be sprayed without causing the fruit to drop prematurely or causing oil spotting on the fruit. For example, in the case of the Hayward (Green) variety of kiwifruit, the safe oil spray period is from fruit set (the time at which the fruit forms) until 15 14 days later, The fruit then moves into an oil spotting susceptible phase until about 35 days after fruit set. Between 35 and about 100 days after fruit set the kiwifruit can be sprayed again but after this period the fruit is susceptible to fruit drop (Figure 1), In contrast, the oil susceptible period of the HortlSA (Gold) variety is from 25 to 65 days after fruit set for fruit speckling and then from 95 days after fruit set to harvest for the 20 fruit drop. In addition, some varieties are more susceptible to fruit drop than others (e.g. the Gold variety is generally more susceptible to fruit drop than the Green variety).

The terms "preventing" or "prevention" refer to applying to a crop an amount of an oil or an oil formulation, such that biological infestation is averted, delayed or reduced in frequency in the crop, relative to a crop which does not receive the composition, 25 Prevention does not require that the infestation is permanently avoided.

The terms "treating" or "treatment" refer to applying to a crop an amount of an oil or an oil formulation, such that undesired biological infestation is either completely eradicated, or eradicated to the desired extent. 6006996 7 The oil may be applied in any suitable form known to the skilled person, including as a low volume mist (100% oil v/v) or in the form of an oil-in-water formulation (e.g. an emulsion). In an emulsion, the oil may be present in an amount of from about 0.5 to about 5% (v/v), more typically at about 2% (v/v).

An emulsion is generally understood as a mixture of two or more immiscible liquids, e.g. oil and water, in which one liquid forms a discrete phase and another a continuous phase. Liquids are said to be immiscible if they are not able to mix, in any proportions, to form a homogenous solution. Generally, a solution is homogenous if the liquids that make up the solution are uniformly dispersed throughout the solution. An oil-in-water 10 emulsion is generally understood as meaning an emulsion wherein oil forms the discrete phase and water forms the continuous phase.

An emulsifier is generally understood as meaning a substance which stabilizes an emulsion by increasing its kinetic stability. In particular, once an emulsion has been established, the emulsifier minimises phase separation so that the relevant discrete and 15 continuous phases forming the emulsion are maintained. Phase separation may occur by the coalescence of the discrete oil droplets into larger droplets or creaming, where the oil migrates to the surface of the aqueous phase. Minimising can mean any of reducing, diminishing, lessening, curtailing or decreasing phase separation.

The person of ordinary skill in the art will understand that the dose of oil, as well as the 20 method and form of application will depend on factors such as plant type, planting density, plant age and stage of the season. In the case of kiwifruit, for example, more volume may be applied to the vines as the season progresses due to the increase in leaf mass as the vines grow, Therefore, the actual amount of oil applied to the crop according to the present invention will vary from case to case and should not be 25 restricted to the values provided herein. The present invention contemplates any dose of oil that is effective in treating and/or preventing biological infestation when applied to crops pre-harvest.

One particularly preferred application of the invention is to the pre-harvest treatment of kiwifruit. However, other "crops" to which the invention can be applied include citrus 6008996 8 fruit, peaches, apples, nut trees including macadamias, almonds and pecans, mangoes, table and wine grapes, ornamental plants including roses, gardenias and hibiscus, berry fruit, lycbees, avocadoes, star fruit and bananas, and vegetables including capsicum, tomatoes and pumpkin.

While the method of the present invention is particularly useful in the prevention and eradication of scale (e.g. oleander, greedy and latania scale), the method of the present invention can also be used to treat crops for other pests, such as larvae of light brown apple moth, mealy bugs and mites.

The oil may be applied to the crop using any suitable methods known to the skilled 10 person, including spraying.Typically when the formulation is an emulsion it can include a humectant and an emulsifier. The use of a humectant reduces water loss from treated fruit and vegetables (i.e. the water loss may sometimes be induced by certain mineral derived oils). A typical humectant employed is glycerol mono-oleate.

The emulsifier is selected to give desirable emulsifying characteristics to oil-in-water 15 formulations. For example, emulsifiers such as polyoxyethylene sorbitan trioleate, sorbitan mono-oleate, cetyl-oleyl alcohol and mono-oleate polyoxyethylene sorbitan impart desirable emulsifying characteristics. Emulsifiers such as polyoxyethylene sorbitan trioleate or sorbitan mono-oleate are physically and chemically compatible with the paraffinic base oil, being stable in the oil and imparting emulsibility to the oil.

Emulsifier content in the formulation can also be varied depending on the application (crop or application method) to optimise breaking characteristics so that desired oil coverage is achieved. The emulsifier is preferably of food grade standard (e.g. a food grade aliphatic hydrocarbon), particularly when used with crops for human consumption.

The examples that follow are intended to illustrate but in no way limit the present 25 invention. 6006998 9 Examples Example 1 This study was performed to assess the efficacy of an nC13 oil formulation against immature and mature oleander scale. Scale-infested potatoes were treated with the 5 nC13 mineral oil fonnulation at concentrations of 0.5%, 1% and 2%. Results were compared with those for scale-infested potatoes treated with the commercially available ,hC24 product D-C-Tron® Plus Spray Oil at the recommended rate of 1% and half the recommended rate (0.5%) as well as for untreated scale infested potatoes (control). The percentages of oil are v/v in water.

Method The bioassay was based on methods developed by Berry (Berry, J. A, (1983) "Aspects of the ecology and control of the greedy scale (Hemiberlesia rapax.y MSc Thesis, Auckland University, Auckland, p.. 112). This method is recognized as the standard laboratory based method for screening scalicides (Blank, R. H. et al. (1993) "Mineral oil 15 and diazinon to control armoured scale on kiwifruit", Proceedings of the Forty Sixth New Zealand Plant Protection Conference, p. 71-74). Forty-five 'Red Desiree' potatoes were washed and air dried. Each potato was seeded with approximately 300 crawlers obtained from oleander scale insect cultures maintained by Plant & Food Research at the Te Puke Research Centre, New Zealand. The infested potatoes were housed in 20 large 36-L ventilated plastic bins and maintained at 23°C ± 2°C, 55-75% RH. About four months later, 45 additional potatoes were set up and maintained as described above. Two weeks later, six potatoes containing mature scale insects (third instar adults), and six potatoes containing immature insects (second instar), which had been seeded in the second treatment, were randomly assigned to each treatment or the control.

The treatments consisted of a standard mineral oil, D-C-Tron® Plus, at concentrations of 0.5% (half the recommended concentration for scale insects in kiwifruit) and 1.0% (the recommended concentration for scale insects in kiwifruit), and the test mineral oil, nC13, at concentrations of 0.5%, 1.0% and 2.0%. The treatments were made up to the desired concentrations in 2-L plastic containers with water. The top half of each potato 30 (marked with a 'T') was immersed into a well-agitated solution for 5 seconds, then 8008096 placed onto a plastic tray to dry within a laminar flow cabinet. The water control was administered in the same way.

The treated potatoes were held for 14 days at 20°C ± 2°C, 55-75% RH„ Assessments to determine the percentage of live and dead scale were conducted 14 days after 5 treatment. Assessments were performed using a dissecting microscope. Insects were identified as immature or mature, as previous use of this insecticide dipping technique has shown that mature scale are harder to kill than immature (Blank, R, H, & Olson, M. H. (1937) "Different toxicological responses of greedy scale stages to dazinon", Proceedings of the New Zealand Weed and Pest Control Conference, p, 161-164), If 10 the insect body was yellow and turgid (indicating a healthy insect), it was recorded as live' and if it was brown and flaccid or desiccated, it was recorded as 'dead'.

Data analysis Mortality was corrected using the standard (Abbott, W. S, (1925) "A method of computing the effectiveness of an insecticide", Journal of Economic Entymology, 18, p. 15 265-267) correction applied to proportions: Corrected % mortality = (% treatment mortality - % control mortality)/! 00 - % control mortality Differences between treatments were determined after calculating 95% confidence 20 intervals of corrected mortality (%). Confidence intervals (95%) were calculated using the CONFIDENCE function in Microsoft® Excel 2003, Results As shown in Figure 2, both concentrations of D-C-Tron® Plus resulted in 99-100% mortality, regardless of scale insect age, 14 days after treatment. nC13 Mineral oil also 25 resulted in 100% mortality of both mature and immature scale insects, but only when applied at a 2% concentration. The lower concentrations tested (0.5% and 1%) were not as effective, with lower mortality of the mature insects compared with the immature insects. However, the lowest mortality rate observed was still greater than 80%. This 6006998 11 difference in efficacy between the different life stages is believed to be related to the natural resistance of the insect. Physical and physiological differences, such as differences in the size of the insect and the thickness of the cap, are likely to contribute to differences in mortality observed. At 2%, nC13 mineral oil appears to have good 5 efficacy against oleander scale insects, as shown by the high levels of mortality of both immature and mature insects 14 days after treatment.

Example 2 This study was performed to assess the phytotoxicity risk to kiwifruit from sprays of three light oil formulations (nC14, r?C13 and nC20) from Caltex {Table 1). The potential 10 of the formulations to cause marking to the fruit skin was tested on Hayward kiwifruit at 14 days after fruit set {Study A), while the potential of the formulations to cause premature fruit drop was tested on Hort16A kiwifruit at 76 and 105 days after fruit set (Study B).

Study A - Method The trial was carried out in one-third of an 18-row Hayward block, at Plant & Food Research Orchard, No, 1 Road, Te Puke, New Zealand. The block contains 152 female vines with a plant spacing of 4.5 m x 5,5 m. Vines are trained on a pergola structure and received standard commercial kiwifruit husbandry including an application of the budburst enhancer, Hi-Cane®. A pre-blossom spray of thiacloprid {Calypso®) was applied to the vines 33 days later for control of armoured scale insects, as is routinely undertaken in normal commercial practice, and Bacillus thuringiensis (Bt) (Delfin® WG) was applied 69 and 85 days later for control of leafrollers. All pesticides were applied at the recommended rate for kiwifruit, that is, Calypso at 20 ml/100 L and Delfin WG at 50 g/100 L.

Two concentrations (1% and 2%) of each of the three new oil formulations (Table 1) were applied at 14 days after fruit set(Table 2). Each single vine plot was sprayed only once. Results were compared with those from the standard oil products D-CTron® Plus (DCT+) and D-C-Tron® Plus Organic (DCT+Org) applied at 1% and 2%, and an untreated control. 6006986 12 Table 1, The three new oil formulations applied to Hayward kiwifruit vines Description Formulation 1 (nC20) Emulsified nC20 polyalfaolefin Formulation 2 (nC14) Emulsified dearomatised hydrocarbon fluid (nC14 equivalent) Formulation 3 (nC13) Emulsified nC13 normal paraffin Table 2, Actual spray dates and the weather conditions during spraying of three new oil formulations and the commercial D-C-Tron® Pius oil products on Hayward kiwifruit Timing of spray Spray time (h) Weather conditions (14 days after fruit set) 1130-1315 21.7°C, 15% cloud cover, occasional very slight breeze 2.0 m/s direction 257°N, RH = 52%, approximate drying time = 30 minutes, crop surface dry at start of spraying. 1 Rainfall, following 24 h = nil, 48 h = nil, 1 week = 22.0 mm 1 Note rainfall data are cumulative Sprays were applied to run-off (c. 2000 L per ha equivalent) using a four-nozzle Sentra handgun (nozzle size D4) from a P48 Comet pump, with an operating pressure of 200 psi. All spraying was carried out when conditions gave fast drying (Table 1), Each replicate comprised a single-vine plot. There were four replicates per treatment in a randomised block design. Random numbers generated by the computer programme 10 Microsoft® Excel were used to assign treatments to each numbered vine within a block. Fruit and leaves were visually checked in situ for symptoms of phytotoxic damage 14 days after spray application. One hundred fruit per plot were picked without conscious bias 56 days after application and checked for damage to the fruit skin.

Study A - Results No damage was observed on the vine foliage or the fruit skin following applications of the test formulations nC14, r?C13 and nC20 or the commercial formulations, DCT+ and DCT+ Org. However, during the commercial harvest (approximately 5 months after 6006996 13 application) a picker observed some fruit had a dark, stained appearance. These 'darker' fruit were restricted to the four vines sprayed with 2% nC20. The amount of affected fruit was estimated at 5-15% per vine.

These results suggest that there is little or no risk of skin damage to Hayward kiwifruit 5 from sprays of the light oils nC14 or nC13, but nC20 equivalents may have potential to cause phytotoxic damage to the fruit. The oil formulations were applied during what is considered to be the period of safe oil use on Hayward kiwifruit (the high risk periods are generally considered to be between 15 and 35 days after fruit set and in the 4 to 6 weeks before harvest).

Study B - Method The trial was carried out in one third of an 18-row Hort16A block at the Plant & Food Research Orchard, No, 1 Road, Te Puke, New Zealand. The block contains 177 female vines, planted in every alternate row with a spacing of 3.5 m x 5,6. The remaining nine rows contain strip males. Vines are trained on a pergola structure and received 15 standard commercial kiwifruit husbandry including an application of the budburst enhancer, Hi-Cane®. A pre-blossom spray of spirotetramat (Movento®) plus the adjuvant Partner® were applied to the vines 66 days later for control of armoured scale insects, as would be routinely undertaken in commercial practice. D-C-Tron® Plus mineral oil and Bacillus thuringiensis (Bt) (Delfin® WG) were applied to all vines at petal 20 fall and 19 days later for control of armoured scale insects and leafrollers respectively. All pesticides were applied at the recommended rate for kiwifruit, that is, Movento at 20 ml/100L, D-C-Tron® Plus oil at 1% and Delfin WG at 50 g/100 L.

Two concentrations (1% and 2%) of each of the three new oil formulations (Table 1) were applied at 76 days after fruit set and at 105 days after fruit set (Table 3). These 25 dates were chosen based upon previous work, from which it is known that oil applied on the first date was expected to induce little or no premature fruit drop, while oil applied on the second date was expected to induce considerable premature fruit drop (McKenna, C. E. et at. (2007) "Mineral oil for control of armoured scale insects on 'Hort16A' kiwifruit", Acta Horticulturae, 753 (2), p. 703-710). Neither application date was 30 expected to result in skin damage to the fruit. Each single vine plot was sprayed only 6006996 14 once. Results were compared with those from the commercial oil products, DCT+ and DCT+Org applied at 1% and 2%, and an untreated control.

Table 3. Actual spray dates and the weather conditions during spraying of three new oil formulations and the commercial D-C-Tron® Plus on Hort16A kiwifruit Timing of spray Spray time (h) Weather conditions 76 days after fruit set 1100- 1430 °C, 30% cloud cover, still, RH = 47-49%, occasional very slight breeze, wind speed <2 m/s, crop surface dry at start, of spraying. Rainfall, following 24 h = nil, 48 h = 0.4 mm, 1 week = 14.0 mm 105 days after fruit set 1230-1530 23-25°C, 5% cloud cover, still, RH = 47-47%, occasional very slight breeze, wind speed 2.1 - 2.3 m/s, crop surface dry at start of spraying. Rainfall, following 24 h = nil, 48 h = nil 1 week = 11.6 mm 1 Note rainfall data are cumulative Sprays were applied to run-off (c. 2000 L per ha equivalent) using a four-nozzle Sentra handgun (nozzle size D4) from a P48 Comet pump, with an operating pressure of 200 psi. All spraying was carried out when conditions gave fast drying (Table 3).

A plot comprised the fruit on ten canes on a half vine. There were five replicates per 10 treatment in a randomised block design. Random numbers generated by the computer programme Microsoft® Excel were used to assign treatments to each numbered vine within a block. The crops were assessed as follows: 1 Premature fruit drop The numbers of fruit on each of ten tagged canes per vine were recorded just before 15 spray application. After oil application, the numbers of fruit remaining per plot were counted at regular intervals until 78 days after the second application, and the 6.006996 percentage fruit drop calculated. Results were compared with, those from an untreated control. 2 Skin marking During the first post-spray counts of fruit on the tagged canes, skin marking due to oil 5 damage was observed on some of the sprayed vines. To quantify this damage, 50 fruit per plot were picked without conscious bias 22 days after the second application and checked in the laboratory for damage to the fruit skin. Damage was recorded in two categories; Light = light marking spread over 1-3 cm2 at the stamen end of fruit. Acceptable for 10 export Severe = extensive marking >3 cm2 at the stamen end of fruit and/ or side of fruit. Not acceptable for export- Care was taken to avoid picking any of the fruit on the 10 tagged canes that were being assessed for premature fruit drop.

Study B - Data analysis 1 Premature fruit drop The effect of oil formulation, oil concentration and date of application (days after fruit set) on the percentage of premature fruit drop was compared using AN OVA. Data were logit transformed, LN((X+0.1)/(100-X+0.1)), before analysis. Tukey's HSD multiple 20 comparison procedure was used to find which means were significantly different from one another if the ANOVA resulted in p < 0.05. Raw data means are presented in the results. 2 Skin marking The effect of oil formulation, oil concentration and date of application (days after fruit 25 set) on the percentage of fruit with skin marking was compared using ANOVA, Treatments with zero damage were not included in the analysis. Tukey's HSD multiple 6006906 16 comparison procedure was used to find which means were significantly different from one another, if the ANOVA resulted in p < 0,05, Raw data means are presented in the results.

Study B - Results Fruit damage in the form of premature fruit drop was first observed one week after the application of oil, and continued for at least eight weeks. The affected fruit softened with ripe rots developing at the petiole end and on the side of the fruit (Figure 3). These fruit detached from the stalk soon afterwards.

Fruit losses following applications of 1% or 2% oil at 76 days after fruit set were £1.1% 10 (Table 4, Figure 4). These losses were a result of factors such as beak end shrivel or accidental removal, and cannot be attributed to the oil spray.

In contrast to the results achieved at 76 days after fruit set, applying nC20 or the DCT+ formulations at 105 days after fruit set resulted in significant fruit drop (Table 4, figure 5} (p < 0.001). At a 1% concentration, fruit losses ranged from 7.5% to 19.2%, but more 15 than doubled to 26.3 to 40.7% with the 2% oil concentration. The greatest loss at both the 1 % and 2% concentration was caused by the test formulation nC20. No significant fruit loss was recorded following a spray of nC14 and nC13 at 105 days after fruit set. 6006998 17 Table 4, The mean percentage of premature fruit drop from Hort16A vines following a single spray of three new oil formulations and two commercial oil formulations at 76 or 105 days after fruit set Oil formulation Mean percent fruit drop 76 days after fruit set 105 days after fruit set 1% 2% 1% 2% Formulation 1 (nC20) 0.8 0.8 19,2 40.7 Formulation 2 (nC14) 0.3 0.6 0.8 1.3 Formulation 3 (nC13) 0.8 1.1 1,4 1.3 D-C-Tron® Plus 0.2 0.7 11.1 26.3 D-C-Tron® Plus Organic 0.7 0.2 7.5 32.3 Untreated control 0.9 One of the experimental formulations and both commercial formulations resulted in 5 marking to the fruit skin: Formulation 1 /iC20, DCT+, and DCT+Org. Marking caused by the r?C20 formulation presented as a raised brown area on the fruit skin, while the marking caused by the commercial DCT+ formulations presented as a dark speckling on the fruit skin (Figure 6).

Sprays of nC14 or oC13 oil did not cause damage to the fruit skin, but a significant 10 amount of fruit had skin marking following a spray of nC20 oil at 76 or 105 days after fruit set (Table 5, Figures 7 and 8} (p < 0.001). The majority of this marking was in the severe category and the affected fruit would not be acceptable for export. Increasing the concentration of r?C20 oil applied from 1% to 2% significantly increased both the amount and severity of damage at 76 days after fruit set (p < 0.001). A. spray of DCT+ or 15 DCT+Org also resulted in significant amounts of fruit with skin marking, but only when applied at a 2% concentration. In each case where damage was recorded, significantly more damage resulted from the spray at 76 days after fruit set, than 105 days after fruit set. 8006998 18 Table 5, The mean percentage of Hort16A fruit with skin damage (light and severe categories combined, severe only) following a single spray of five different oil formulations, applied at 76 or 105 days after fruit set Oil formulation Mean percent fruit with damage (light and severe damage categories combined} 76 days after fruit set 105 days after fruit set 1% oil 2% oil 1% oil 2% oil Formulation 1 (nC20) 47.6 I 85.2 26.0 18.8 Formulation 2 (r?C14) 0 0 0 0 Formulation 3 (nC13) 0 0 0 0 D-C-Tron® Plus 0 44.8 0 8.0 D-C-Tron® Plus Organic 0 36.4 0 9.6 Oil formulation Mean percent fruit wit h severe damage only 76 days after fruit set 105 days after fruit set 1% oil 2% oil 1%oil 2% oil Formulation 1 (nC20) 34.8 85.2 18.4 .8 Formulation 2 (nC14) 0 0 0 0 Formulation 3 (nC13) 0 0 0 0 D-C-Tron® Plus 0 18.4 0 0 7.2 D-C-Tron® Plus Organic 0 13.6 4.8 The r>C13-14 equivalent oil formulations have potential for use on Hort16A vines without 5 causing phytotoxic damage to the fruit. Whereas the nC20 equivalent oil formulation resulted in unacceptable amounts of fruit with skin marking or premature fruit drop, the nC13 formulations caused no damage, even when applied at a 2% concentration during the period when oil sprays can induce considerable fruit drop.

The nC13-14 oils also compared favourably with the commercial DCT+ products. As 10 expected, both DCT+ and DCT+Org caused significant amounts of fruit drop when applied at 105 days after fruit set. However, the occurrence of skin marking following the applications of the DCT+ products at both 76 and 105 days after fruit set was not expected, and suggests that Hort16A may have a second period of susceptibility to skin 6006996 19 marking. Further evidence of this comes from other research studies completed in 2010 (McKenna, C. et al. (2010) "Mineral oil use in Hort16A kiwifruit crops in summer for Project MA1033", Report to Zespri Group Ltd. Plant and Food Research Client Report No, 35871), In these studies, skin damage to Hort16A fruit was observed following 5 sprays of oil applied between 70 and 98 days after fruit set. The fact that no damage was recorded from sprays of nC13-14 oils during this period further supports the conclusion that there is a very low risk of phytotoxicity to kiwifruit from sprays of nC13-14 oils.

These results indicate that /1C13-14 formulations could be used safely on kiwifruit during 10 periods when the use of oil is currently not recommended, in particular, during the later part of the season against the second generation of scale insects.

Example 3 The objective of this study was to assess the efficacy of four experimental light oil formulations in the orchard against two armoured scale insect pests of kiwifruit, latania 15 {Hemiberlesia lataniae) and oleander scale (Aspidiotus nerii).

Population establishment Six-month-old kiwifruit canes were harvested from Hayward vines, wrapped in polythene and stored at 0°C Two months later, the canes were removed from cool-storage and 255 40-cm lengths were cut. Buds were cut from the cane sections, and 20 grafting wax applied to all exposed tissue, with the exception of the bottom end of the cane, which was placed into a plastic cup containing water (Hill, M. G. et al. (2010) "Measuring resistance to armoured scale insects in kiwifruit (Actinidia) germplasm", New Zealand Journal of Crop and Horticultural Science, iFirst, p. 1-17).

Wool was wound loosely around each cane to provide a settling site for the crawlers. 25 One hundred latania scale crawlers were seeded onto each of 200 canes, and 100 oleander scale crawlers were seeded onto each of the remaining 55 canes. Two months later, 40 additional canes and 10 potatoes were seeded with latania and oleander scale crawlers, respectively. All canes and potatoes were seeded using crawlers from scale 6006998 insect cultures maintained by staff at Plant & Food Research, and were held within controlled environment rooms maintained at 20°C ± 2°C, 55 - 65% RH.

Data analysis Differences among treatments were determined using confidence intervals and one-way 5 ANOVAs. All data were analysed and summarised using Minitab®15.1.0.0. Means were separated using Tukey's (HSD) test. All analysis were performed at the p=0.05 level of significance.

Study C - Method This experiment was an addition to the early season experiment to screen oils for 10 phytotoxicity on Hayward vines. The same vines, sprays and experimental setup were used (see Example 2, Study A for details).

Before spraying, which occurred approximately three months after the first seeding, canes infested with third instar (adult) scale insects (85 days old at 20°C) were secured into the canopy, adjacent to the leader, using electrical tape (Figure 9). Four canes were 15 randomly assigned to each of the 55 treatment vines and suspended within each vine (three infested with latania scale, one infested with oleander scale) (Figure 9), exposing 100-200 scale insects per vine to the oil applications.

Immediately after each treatment was applied, adequacy of spray coverage of the canes was checked by visually inspecting the wetting of each cane. Once dry (within 20 one hour), the canes were removed from the vines, put into cups containing water, and maintained in a controlled environment room at 20°C ± 2°C and 55 - 65% RH. After two weeks, all scale insects were examined under a dissecting microscope and assessed as live or dead; a yellow and turgid insect body was recorded as "live", a brown and flaccid or desiccated insect body was recorded as "dead".

Study C - Results Spray application resulted in patchy coverage on some of the suspended kiwifruit canes. This occurred when a portion of the cane was hard up against the leader. Scale 6006996 21 insects on this portion of the cane were not exposed to the spray. Mortality results reported herein were corrected for these coverage inconsistencies by removing canes from the analysis that had areas of live scale insects surrounded by large quantities of dead insects.

Armoured scale insects construct an outer cover or cap that functions as protection from the external environment (Foldi, I. (1990} "The scale cover" in Rosen, D. (ed) The Armoured Scale Insects, Their Biology, Natural Enemies and Control, Vol B, Elsevier Science Publishers B.V., Amsterdam, The Netherlands, p. 43-54). The cap varies in thickness and permeability between scale insect species (Beardsley, J. W. & Gonzalez, 10 R, H. (1975) "The biology and ecology of armoured scales", Annu. Re v. Entomol. 20, p. 47-73). Scale cap properties affect the susceptibility of a scale species to insecticides. Oleander scale insects were more susceptible than latania scale to the oil treatments applied (Table 6). This is probably because of the permeability of the scale cap to the insecticide, with the oleander cap being thin and soft compared with the latania cap, 15 There were very few differences in mortality of oleander scale among the treatments applied, indicating that oleander scale is more susceptible to a wider range of oils.

Mortality of latania and oleander scale insects was greater following treatment with the commercial oil products (DCT+ and DCT+Org) than following treatment using an experimental oil formulation (Table 6, Figures 10 and 11). The greatest mortality within 20 the experimental formulations was observed with nC20 (1%, 2%) and nC13 (2%). Inferior rates of mortality were achieved using nC14 (1%, 2%) and nC13 (1%). Oil applied at 2% tended to result in higher scale insect mortality than 1%; however, the difference between 1% and 2% was marginal in some cases. 6008996 22 Table 6. Mortality {%) of latania and oleander scale insects following treatment with various oil formulations at concentrates of 1% and 2%, Values within a column followed by the same letter are not significantly different (p>0.05).

T reatment Latania Latania N Mortality (%) N Mortality (%) Control 774 8 d 140 d nC20 (1%) 708 c 156 41 b nC20 (2%) 844 18 be 137 50 ab nC14 (1%) 795 9 d 99 be nC14 (2%) 713 d 123 26 be r?C13 (1%) 828 d 93 c nC13 (2%) 771 17 c 115 be f**v #•*%. '"*f™ jCS r"% i / 4 rt / v D-C-Tron® Plus (1%) 615 29 b 140 52 ab D-C-Tron® Plus (2%) 516 51 a 68 63 ab D-C-Tron® Plus Organic (1%) 590 b 114 I 55 ab D-C-Tron® Plus Organic (2%) 350 48 a 129 I 58 ab Study D - Method This experiment was initiated after the first experiment (Study C), where canes were used as a substrate, produced unexpectedly low mortality of scale insects compared with prior laboratory experiments, which used potatoes as a substrate (Mauchline, N. & McKenna, C, (2009) "The efficacy of nC13 mineral oil against oleander scale, 10 Aspidiotus neriC, Report to Caftex Australia Petroleum PTY LTD. Report No. 2463). This experiment was designed to test the effect of substrate (potatoes versus canes) on scale mortality. The canes and potatoes were exposed to the sprays applied to Hort16A vines(see Example 2, Study B for details). Treatments nC20 (1%, 2%) and DCT+ (1%, 2%) were used to compare armoured scale mortality on canes and potatoes suspended 15 within Hort16A vines. A total of 30 latania infested canes (two canes per vine on each of three vines per treatment) and 10 oleander infested potatoes (two potatoes per vine on each of two vines per treatment) were randomly assigned and suspended within the 6006996 23 canopy (Figures 9 and 12), Mortality assessments were completed two weeks after application as described in Study C, Study D - Results Significant differences in insect mortality were apparent between scale infested canes 5 and potatoes when treated with DCT+ (1%, 2%) (p < 0,001), Insect mortality was greater on excised kiwifruit canes than on potatoes. This is probably because each scale insect is more exposed on a cane, whereas the scale insects tend to cluster and sometimes overlap on a potato. Although oleander would generally be considered more susceptible than latania scale insect to oil treatments (Figures 11 and 13), this was not 10 the case in this trial, which lends some weight to the hypothesis that substrate can significantly influence mortality rate.

The differences observed in rates of mortality between the first application and the second application might have been a result of differing canopy densities, Lighter canopies facilitate more rapid evaporation and degradation of the oil from the vines. As 15 the season progresses, the canopy gets considerably denser, enabling greater stability and longevity of the oils.

Study E - Method This experiment augmented the screening of Formulation 4 (F4, Table 7) for phytotoxicity to Hort16A (see Example 2, Study B for details). A total of eight canes 20 infested with latania scale insects (two canes per vine on each of two vines per oil concentration) were randomly assigned and suspended within the canopy as described in Study C. Mortality assessments were completed two weeks later.

Table 7. The fourth new oil formulation applied to Hayward kiwifruit vines Description Formulation 4 (nC13) Emulsified nC13 equivalent isoparaffin 6008998 24 Study E - Results F4 resulted in 26% and 38% mortality of latania insects on excised kiwifruit caries at a 1% and 2% concentration, respectively. F4 achieved greater mortality of latania scale than all experimental oil formulations applied in Experiment one, and resulted in a 5 mortality rate comparable to that with DCT+ (1%) (p = 0.51). The efficacy of F4 was, however, much lower compared with the efficacy of all oils applied in Study D. This suggests that F4 has potential to contribute to scale control in kiwifruit crops at certain times of the year. Residues of the standard mineral oil products can act as a repellent against scale crawler settlement on kiwifruit fruit for several weeks, depending on the 10 time of year. This repellency is likely to contribute to scale insect control on kiwifruit vines.

Claims (19)

RECEIVED at IPONZ on 18 July 2011 25 The claims defining the invention are as follows:

1. A pre-harvest prevention and/or treatment method for eradication of biological infestation in crops comprising the step of applying a mineral oil pre-harvest to the crop, wherein the oil is aromatic-free and is greater than or equal to nC11 and less than 5 nC18.

2. A method according to claim 1, wherein the oil is nC13 and/or nC14.

3. A method according to claim 1 or 2, wherein the oil is a paraffinic oil.

4. A method according to any one of the preceding claims, wherein the oil has a median distillation point (ASTM D 86 or 2887) in the range of 200 to 300°C (at 10 atmospheric pressure).

5. A method according to claim 1, wherein the oil is a volatile synthetic ester, which has a median distillation point (ASTM D 86 or 2887) in the range of 200 to 300°C (at atmospheric pressure).

6. A method according to any one of the preceding claims, wherein the oil is applied 15 by spraying.

7. A method according to claim 6, wherein the oil is applied as a low volume mist.

8. A method according to claim 6, wherein the oil is applied as an oil-in-water formulation.

9. A method according to claim 8, wherein the formulation is an emulsion. 20

10. A method according to claim 8 or 9, wherein the oil is present in an amount of from about 0.5 to about 5% (v/v).

11. A method according to claim 10, wherein the oil is present in an amount of about 2% (v/v).

12. A method according to any one of claims 9 to 11, wherein the emulsion further 25 includes a humectant and an emulsifier.

13. A method according to claim 12, wherein the humectant is glycerol mono-oleate. 8249462 RECEIVED at IPONZ on 7 April 2011 26

14. A method according to claim 12 or 13, wherein the emulsifier is polyoxyethylene sorbitan trioleate, sorbitan mono-oleate, cetyl-oleyl alcohol, mono-oleate polyoxyethylene sorbitan.

15. A method according to any one of claims 12 to 14, wherein the emulsifier is a 5 food grade aliphatic hydrocarbon.

16. A method according to any one of the preceding claims, wherein the crop is kiwifruit.

17. A method according to any one of the preceding claims, wherein the biological infestation is scale. 10

18. A method according to claim 17, wherein the scale is oleander scale, greedy scale or latania scale.

19. A method according to claim 1, substantially as hereinbefore described. 15