A Method for Treatment of Crops

FIELD OF THE INVENTION

The present invention relates to a method of the treatment of crops and more particularly a method for preparing and applying a formulation, preferably in the form of a spray to the growing crop, for control of pathogen growth and to provide crop protection from pathogenic attack. The formulation may also be applied as a fruit and vegetable wash to remove harmful pathogens from surface of produce and extend shelf life and safety of the packed or stored produce as a post harvest application.

BACKGROUND OF THE INVENTION

Pathogen infections can result in significant losses to agricultural crops caused by pre-harvest damage, killing them outright or weakening them so as to decrease yields and render the plants, fruit or grains susceptible to primary and secondary infections. Post-harvest infections also results in significant loss of agricultural products during storage, processing and handling.

When fruit, vegetables and grains are to be eaten or processed it is essential that any treatment given to them does not lead to residues which exceed safe limits. Significant variation in allowable residues may exist between local and overseas markets.

Many pathogen treatments may produce residues, although very small, leave the treated product in breach of the law of the country to which it has been exported. Further, some current treatments also result in harm to select beneficial microorganisms present on the surface of the crop.

Further, some pathogen strains are found to have developed separate mechanisms of resistance to two or more unrelated fungicides and is termed ‘multiple resistance’. For example, strains of Botrytis cinerea are known to have become resistant to both benzimidazole and dicarboximide fungicides.

Despite a number of chemical agents having been developed for treating crops, there remains a need for the development of further methods of treatment, in particular in the development of bacteriacide and disinfectant control agents which are highly toxic to harmful pathogens yet safe for humans, crops and/or animals.

There exists a need to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

SUMMARY OF THE INVENTION

According to the invention there is provided a method of treating crops, including fruit, vegetables and grain, to provide protection against selected pathogens. There is further provided a method of treating crops, including fruit, vegetables and grain, to control pathogen growth. The pathogens may include plant pathogens, as well as bacterial, fungal and human pathogens.

In one aspect, the present invention provides a method for treating crops comprising the steps of:

- producing a dry composition comprising;
  - a metabisulphite,
  - a benzoate salt, and
  - a cellulose additive;
- preparing said dry composition as a formulation; and
- applying the prepared formulation to a crop,
  
  wherein said treatment is for prevention or reduction of crop damage by plant pathogens, or to reduce bacterial, fungal or human pathogens on said crop.

In a second aspect, the present invention provides a method for treating crops comprising the steps of:

- providing a dry composition comprising:
  - a metabisulphite,
  - a benzoate salt, and
  - a cellulose additive;
- preparing said dry composition as a formulation;
- applying the crop with a fungicide; and
- applying the formulation to the crop,
  
  wherein said treatment is for prevention or reduction of crop damage by plant pathogens or to reduce bacterial, fungal or human pathogens on said crop.

DETAILED DESCRIPTION

By a ‘dry composition’ as used herein is meant a mixture of components in a form substantially free of moisture. For example, the dry composition may be in powder or any other suitable physical form. A dry composition according to the invention may be presented in unit dosage form, for example in a sachet.

By ‘plant pathogen’ as used herein is meant an organism which is capable of causing harm or disease to a crop, wherein the plant pathogen may include pathogens which are also capable of causing harm or disease to a humans or animals.

In a preferred embodiment the metabisulphite is selected from any suitable metabisulphite salt. In a particularly preferred embodiment the metabisulphite salt is a sodium metabisulphite. In an alternatively preferred embodiment the metabisulphite salt is a potassium metabisulphite.

Preferably, the metabisulphite is in the physical form of a powder.

In a preferred embodiment, the benzoate salt is selected from any suitable benzoate salt. In a particularly preferred embodiment the benzoate salt is a sodium benzoate. In an alternative embodiment the benzoate is a potassium benzoate.

Preferably, the benzoate salt is in the physical form of a powder.

In a preferred embodiment, the dry composition comprises sodium metabisulphite blended with sodium benzoate at a ratio of approximately between 20:80 and 30:70 w/w, together with a cellulose additive. In a particularly preferred embodiment the dry composition comprises sodium metabisulphite blended with sodium benzoate at a ratio of approximately between 22:78 and 29:71 w/w, together with a cellulose additive.

In a preferred embodiment, the dry composition includes a cellulose additive at approximately between 0.5 to 3% by weight of the dry composition. In a further preferred embodiment the dry composition includes a cellulose additive at approximately between 0.8 to 2.0% by weight of the dry composition. In a further preferred embodiment the dry composition comprises a cellulose additive at approximately between 1.0 to 1.5% by weight of the dry composition.

By ‘formulation’ as used herein is meant a mixture comprising the ‘dry composition’ being further blended with a surfactant, additional additive or solution.

By ‘blended’ as used herein is meant any suitable form of mixing to form a substantially evenly distributed formulation. Preferably, the blending technique includes any method of mechanical or hand mixing, or any other suitable form of agitation to achieve a substantially evenly distributed formulation.

In a preferred embodiment the blending may be performed by a V blender, double blender, bin blender, drum blender, paddle blender, cement or concrete mixers, twin shaft mixers, or any other suitable blender or mixer.

By a ‘cellulose additive’ as used herein is meant any additional component containing cellulose. For example, the cellulose additive may be selected from alpha cellulose, cellulose, cellulose crystalline; cellulose gel, hydroxycellulose, microcrystalline cellulose, plastics, cellulosic, and sulfite cellulose.

In a preferred embodiment the cellulose additive is CAS #9000-34-6.

In a preferred embodiment the formulation comprises a dry composition being further blended with a surfactant, other suitable additive or solution. In a particularly preferred embodiment the formulation comprises a dry composition being further blended with a surfactant at a ratio of approximately between 0.5% to 10% w/w. of the final formulation. In a particularly preferred embodiment the formulation comprises a dry composition being further blended with a surfactant at a ratio of approximately between 0.8% to 8% w/w of the final formulation. In a particularly preferred embodiment the formulation comprises a dry composition being further blended with a surfactant at a ratio of approximately between 1.0% to 6% w/w of the final formulation.

The surfactant (otherwise referred to as wetting agents) optionally used in the present invention is selected from any suitable surfactant, said surfactant being suitable for human and/or animal consumption. Preferably the surfactant is selected from a non-ionic surfactant and an ionic surfactant.

By a ‘non-ionic surfactant’ as used herein is meant an organic compound containing covalently bonded oxygen-containing hydrophilic groups, bound to hydrophobic parent structures.

By an ‘ionic surfactant’ as used herein is meant a chemical compound containing a positively and/or negatively charged, polar functional ground bound to a hydrophobic parent structure. Ionic surfactants include anionic, cationic and zwitterionic molecules.

Preferably the surfactant is selected from polyethylene glycol, polyethylene oxide, dipropylene glycol and polysorbate 80.

By a ‘polyethylene glycol’ as used herein is meant a polyether organic compound preferably having a molecular weight less than 100,000 g/mol. By a ‘polyethylene oxide’ as used herein, is meant a polymer preferably having a molecular weight equal to or greater than 100,000 g/mol.

By an ‘organic compound’ is meant a chemical compound, the molecules of which contain the element carbon. In a preferred embodiment, the organic compound may be a hydrocarbon. By a ‘hydrocarbon’ is meant an organic compound containing, inter alia, the elements carbon and hydrogen.

In a preferred embodiment, the dry composition is capable of being stored for approximately up to 24 months prior to further blending/formulation or being administered to crops.

In a preferred embodiment the formulation may be diluted to produce a solution, prior to being administered to crops. In a further preferred embodiment the formulation may be diluted with an aqueous mixture to produce a solution. In a particularly preferred embodiment the formulation may be diluted with water to produce a solution used to wash crops.

The aqueous mixture may be of any suitable type. By “aqueous mixture” as used herein is meant a water based solvent or a solvent including at least approximately 50% water. In a preferred embodiment, the aqueous mixture is water.

Preferably the formulation is diluted with a solution no earlier than approximately 14 days prior to being administered the crops.

In a preferred embodiment, the solution has a pH of between approximately 2.0 to 7.5. In a further preferred embodiment, the solution has a pH of between approximately 3.0 to 6.5. In a particularly preferred embodiment, the solution has a pH of between approximately 4.0 and 6.0.

Preferably the solution is applied to a crop as either a pre-harvest spray or a post harvest wash. In a particularly preferred embodiment the solution is applied to the crop as a pre-harvest spray.

By ‘a crop’ as used herein is meant any food product suitable for human or animal consumption, or a tree, vine or other plant upon which the food product is grown. In a preferred embodiment the crop includes fruits, vegetables, grains, grasses and seeds.

In a particularly preferred embodiment the crop includes grapes and other fruit, vegetables or grains suitable for the production of wine or other beverages. In a further preferred embodiment the crop includes berries, stone fruits, citrus fruits, tropical fruits, melons, drupes, pomes or any other edible fruit. In a further preferred embodiment the crop includes tropical vegetables, bulb vegetables, brassica vegetables, fruiting vegetables, leafy vegetables, legumes, pulses, root and tuber vegetables, stalk and stem vegetables, cereal grains, tree nuts and herbs, including lettuce, garlic and pistachios. In a further preferred embodiment the crop includes seeds and seedlings of flowering crops, fruits and vegetables.

In a particularly preferred embodiment the crop to be treated is selected from apples, pears, cherries or grapes.

In an embodiment, the solution is applied to a crop upon expression of pathogens or at any combination of the following stages of crop maturation:

- Bud-swell;
- (20% to 30%) bloom and early petal-fall stages;
- One month to harvest;
- Two weeks to harvest.

In an alternative preferred embodiment, a fungicide is applied between approximately 2 to 12 hours prior to the solution. In a further preferred embodiment the fungicide contains an active ingredient which is applied at a rate of between approximately 5 to 25 ppm.

In a more preferred embodiment, the grape vine varieties may be selected from the group consisting of Vitis Vinifera, Vitis labrusca, Vitis riparia, Vitis rotundifolia, Vitis rupestris, Vitis aestivalis, Vitis mustangensis. Vitis coignetiae, Vitis californica, Vitis vulpina, Vitis amurensis, Muscadinia rotundifolia and Vitis romanetii. In a further preferred embodiment the grape vine varieties may be a cultivar or hybrid of any aforementioned species.

In a preferred embodiment, the crop may be a fruit that is susceptible to stem end rots, such as cherries. In this embodiment, the formulation of the present invention may be as a spray pre-harvest to help prevent or reduce stem end rots, and/or used after harvest to prevent or reduce stem end rots.

In a preferred embodiment, the solution is applied at no later than 3 days prior to harvest. In a further preferred embodiment, the solution is further applied upon expression of botrytis and at any combination of the following stages of grape maturation:

- approximately 10% flower crop;
- approximately 10% cap fall;
- approximately 30% cap fall;
- approximately end of flowering;
- approximately berry size approximately 4 mm;
- approximately bunch closure; and
- approximately veraison.

In a preferred embodiment the applied solution has a concentration of approximately between 1 g/L to 8 g/L. In a further preferred embodiment the applied solution has a concentration of approximately between 2 g/L to 6.5 g/L. In a further preferred embodiment the applied solution has a concentration of approximately between 3.5 g/L to 4.5 g/L. In a further preferred embodiment the applied solution has a concentration of approximately between 3.75 g/L to 4.25 g/L.

In a preferred embodiment the applied solution has a concentration of between approximately 2 g/L and approximately 8 g/L. In a particularly preferred embodiment, the applied solution has a concentration of 2 g/L, 4 g/L or 8 g/L.

In a preferred embodiment the applied solution results in a reduction of growth of crop pathogens. In a preferred embodiment, the applied solution results in a reduction of growth of crop pathogens selected from the group consisting of Botrytis cinerea, Xanthomonas spp E. coli, Monilina fructicola and Penicillium spp. In a further embodiment the applied solution results in a reduction of growth of the crop pathogen Xanthomonas campestris. In a further preferred embodiment the applied solution results in reducing growth of the crop pathogen Erwinia Carotovora.

Preferably, the applied solution is delivered at a rate between approximately 500-1600 L/Ha. Preferably the applied solution is delivered at a temperature of not more than approximately 30′C. Preferably the applied solution is applied at a humidity of less than approximately 75%.

In a preferred embodiment the applied solution may be applied at the above rates and delivery conditions for all growing crops described herein, from seedling through to harvest.

In a preferred embodiment use of the applied solution results in very low levels of residue of sulphites and the benzoates in the resulting crop and products thereof. These levels may be well below the limits for food safety standards.

For example, when the solution of the present invention is used on grape vines, as hereinbefore described, sulphite residue in the resulting wine, juice or pomace may be less than approximately 100 mg/L, more preferably less than 10 mg/L, more preferably between approximately 3 and 5 mg/L. In Australia, the maximum permitted levels of sulphites in wines varies from 200 to 300 mg/kg depending on the type of wine and residual sugar level.

For example, when the solution of the present invention is used on grape vines, as hereinbefore described, benzoate residue in the resulting wine, juice or pomace may be less than approximately 100 mg/L, more preferably less than 50 mg/L, more preferably between approximately 1 and 50 mg/L. In Australia, the maximum permitted level of benzoates in wines is 400 mg/kg.

In a preferred embodiment the applied solution may be used in a run to waste washing facility as a post harvest bacteriacide/disinfectant on produce such as fruit, vegetables and nuts. In this embodiment, capacity may be dosed through automatic control, preferably at rates of approximately 2 g/L or 4 g/L. Preferably the contact time is not less than approximately 2 minutes and not more than approximately 60 minutes.

In a preferred embodiment the applied solution may be used in a recirculating washing facility as a post harvest bacteriacide/disinfectant on produce as fruit, vegetables and nuts. In this embodiment, capacity may be dosed through automatic control, preferably at rates of approximately 2 g/L or 4 g/L. Preferably the contact time is not less than approximately 2 minutes and not more than approximately 60 minutes.

In a preferred embodiment, the applied solution may be used in conjunction with a filtration system.

In an alternative preferred embodiment the crop is treated with a solution of the composition, as described herein, post harvest. In a preferred embodiment the solution applied post harvest has a concentration of approximately between 1 g/L to 8 g/L. In a further preferred embodiment the solution applied post harvest has a concentration of approximately between 2 g/L to 6.5 g/L. In a further preferred embodiment the solution applied post harvest has a concentration of approximately between 3.5 g/L to 4.5 g/L. In a further preferred embodiment the solution applied post harvest has a concentration of approximately between 3.75 g/L to 4.25 g/L.

In a preferred embodiment the applied solution results in approximately between 10% to 30% reduction in Botrytis cinerea growth compared to an untreated crop. In a further preferred embodiment the applied solution results in approximately between 15 to 25% reduction in Botrytis cinerea growth compared to an untreated crop. In a particularly preferred embodiment the applied solution results in approximately between 17% to 23% reduction in Botrytis cinerea growth compared to an untreated crop.

In a preferred embodiment, the applied solution results in approximately greater than 50% reduction in Xanthomonas sp growth compared to an untreated crop. In a more preferred embodiment, the applied solution results in approximately greater than 75% reduction in Xanthomonas sp growth compared to an untreated crop. In a particularly preferred embodiment, the applied solution results in approximately greater than 90% reduction in Xanthomonas sp growth compared to an untreated crop.

In a preferred embodiment, the applied solution results in approximately greater than 60% reduction in growth of E. coli compared to an untreated crop. In a more preferred embodiment the applied solution results in approximately greater than 70% reduction in growth of E. coli compared to an untreated crop. In a particular preferred embodiment the applied solution results in approximately greater than 80% reduction in growth of E. coli compared to an untreated crop.

Where this analysis is performed in a laboratory rather than in situ, the untreated crop may be a sample of an untreated crop.

In a further preferred embodiment, the applied solution results in no substantial effect on the growth rate of Saccharomyces cerevisae and Schizosaccharomyces pombe species.

In an embodiment the fungicide contains a halogen based active ingredient. In a preferred embodiment the halogen based fungicide contains an active ingredient selected from bromochlorodimethylhydantoin (BCDMH), Chlorine, Bromine, an active ingredient which releases a halogen, an active ingredient which releases hypobromous acid and/or hypochlorous acid, an active ingredient which releases chlorine and/or bromine, or a fungicide containing any suitable combination thereof.

By ‘bromochlorodimethylhydantoin (BCDMH)’ as used herein is meant 1-Bromo-3-chloro-5,5-dimethylhydantoin, 3-Bromo-1-chloro-3-chloro-5,5-dimethylhydantoin or any combination or mixture thereof.

In a preferred embodiment the fungicide is applied as a solution containing the halogen based active ingredient at a concentration of approximately between 1 to 100 ppm. In a further embodiment the fungicide is applied as a solution containing the halogen based active ingredient at a concentration of approximately between 2 to 50 ppm. In a preferred embodiment the fungicide is applied as a solution containing the halogen based active ingredient at a concentration of approximately between 5 to 10 ppm.

In an embodiment the crop is treated with both the formulation and fungicide pre harvest. In a further embodiment the crop is treated with both the formulation and fungicide pre harvest and the crop is further treated with the formulation post harvest. In a further embodiment the crop is treated with both the formulation and fungicide pre harvest and the crop is further treated with both the formulation and fungicide post harvest.

In an alternative embodiment the crop is treated with both the formulation and fungicide post harvest. In an alternative preferred embodiment the crop is treated with both the formulation and fungicide post harvest and the crop is treated with the formulation pre harvest.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows the necrosis of the untreated control at 15DAAB—Grapevine cv. Sauvignon Blanc, as described in Example 10.

FIG. 2 shows grapevine cv. Sauvignon Blanc following two applications of WOB NP1 at the lowest application rate of 35+119.6 g ai/100 L (15DAAB), as described in Example 10.

FIG. 3a shows necrosis of tissue on grapevine cv. Sauvignon Blanc following two applications of WOB NP1 at 70+239.2 g ai/100 L (15DAAB), as described in Example 10.

FIG. 3b shows necrosis (as indicated by circled regions) of tissue on grapevine cv. Sauvignon Blanc following two applications of WOB NP1 at 70+239.2 g ai/100 L (15DAAB), as described in Example 10.

FIG. 4a shows necrosis of tissue on grapevine cv. Sauvignon Blanc following two applications of WOB NP1 at 140+478.4 g ai/100 L (15DAAB), as described in Example 10.

FIG. 4b shows necrosis (as indicated by circled regions) of tissue on grapevine cv. Sauvignon Blanc following two applications of WOB NP1 at 140+478.4 g ai/100 L (15DAAB), as described in Example 10.

FIG. 5a shows necrosis of tissue on grapevine cv. Sauvignon Blanc following two applications of WOB NP1 at 280+956.8 g ai/100 L (15DAAB), as described in Example 10.

FIG. 5b shows necrosis (as indicated by circled regions) of tissue on grapevine cv. Sauvignon Blanc following two applications of WOB NP1 at 280+956.8 g ai/100 L (15DAAB), as described in Example 10.

FIG. 6a shows necrosis studies, leaf damage and bunch residue 114DAB as described in Example 11. (Clockwise from top left) Photograph 1: Untreated control bunches. Photograph 2: Untreated leaves. Photograph 3: WOB NP1 (35.0+119.6 g ai/100 L) bunches. Photograph 4: WOB NP1 (35.0+119.6 g ai/100 L) leaves.

FIG. 6b shows necrosis studies, leaf damage (as indicated by circled regions) and bunch residue 114DAB as described in Example 11. (Clockwise from top left) Photograph 1: Untreated control bunches. Photograph 2: Untreated leaves. Photograph 3: WOB NP1 (35.0+119.6 g ai/100 L) bunches. Photograph 4: WOB NP1 (35.0+119.6 g ai/100 L) leaves.

FIG. 7a shows necrosis studies, as described in Example 11. (Clockwise from top left) Photograph 5: WOB NP1 (70.0+239.2 g ai/100 L) bunches. Photograph 6: WOB NP1 (70.0+239.2 g ai/100 L) leaves. Photograph 7: WOB NP1 (140.0+478.4 g ai/100 L) bunches. Photograph 8: WOB NP1 (140.0+478.4 g ai/100 L) leaves.

FIG. 7b shows necrosis studies, as described in Example 11. (Clockwise from top left) Photograph 5: WOB NP1 (70.0+239.2 g ai/100 L) bunches. Photograph 6: WOB NP1 (70.0+239.2 g ai/100 L) leaves with leaf damage as indicated by circled regions. Photograph 7: WOB NP1 (140.0+478.4 g ai/100 L) bunches. Photograph 8: WOB NP1 (140.0+478.4 g ai/100 L) leaves with leaf damage as indicated by circled regions.

FIG. 8a shows necrosis studies, as described in Example 11. (Clockwise from top left) Photograph 9: WOB NP1 (280.0+956.8 g ai/100 L) bunches. Photograph 10: WOB NP1 (280.0+956.8 g ai/100 L) leaves. Photograph 11: Standard control program bunches. Photograph 12: Standard control program leaves.

FIG. 8b shows necrosis studies, as described in Example 11. (Clockwise from top left) Photograph 9: WOB NP1 (280.0+956.8 g ai/100 L) bunches. Photograph 10: WOB NP1 (280.0+956.8 g ai/100 L) leaves with leaf damage as indicated by circled regions. Photograph 11: Standard control program bunches. Photograph 12: Standard control program leaves.

FIG. 9 shows Log 10 of cfu/g+1 of fungi on pears washed with either water, WOB NP1, BCDMH or BCDMH+WOB NP1. LSD=1.166.

FIG. 10 shows Log 10 of cfu/g+1 of E. coli on pears washed with either water, WOB NP1, BCDMH or BCDMH+WOB NP1 LSD=0.593.

FIG. 11 shows Log 10 of cfu/g+1 of fungi on apples washed with either water, WOB NP1, BCDMH or BCDMH+WOB NP1 LSD=0.869.

FIG. 12 shows Log 10 of cfu/g+1 of E. coli on apples washed with either water, WOB NP1, BCDMH or BCDMH+WOB NP1. One obvious outlier was removed from the unwashed data prior to analysis. LSD=1.352.

FIG. 13 shows Incidence of rots after storage on pears washed with either water, WOB NP1, BCDMH or BCDMH+WOB NP1.

FIG. 14 shows Incidence of rots after storage on apples washed with either water, WOB NP1, BCDMH or BCDMH+WOB NP1.

EXAMPLE 1—PREPARATION OF THE DRY FORMULATION

25 kg of sodium metabisulphite is combined with 67 kg of a sodium benzoate powder and then 1 kg of Diacel 150 (CAS #9000-34-6) is further added. The resulting mixture is then blended by addition to a cement mixer. The resulting mixture is then blended by addition to a cement mixer (100 L capacity revolving drum mixer with a 880 W 1440 RPM electric motor). The mixture is blended for 10 minutes, allowed to stand for 10 minutes and further blended for an additional 10 minutes. The described process provides 93 kg of the dry composition.

EXAMPLE 2—PREPARATION OF A FORMULATION COMPRISING A SURFACTANT

To 93 kg of the dry composition is added 5 kg of polyethylene glycol and the resulting composition is blended by addition to a cement mixer (100 L capacity revolving drum mixer with a 880 W 1440 RPM electric motor). The mixture is blended for 10 minutes, allowed to stand for 10 minutes and further blended for an additional 10 minutes. The described process provides of 98 kg of the desired formulation.

40 g of the pre-prepared formulation is added to 10 L of water and mixed with agitation and the resulting dispersion is allowed to stand for 10 minutes to ensure the powder formulation is dissolved.

EXAMPLE 3—PH STUDY FOR DILUTED ‘DRY COMPOSITIONS’

Preparation of Products

WOB NP 1 and WOB PH1 were prepared according to the general method of Example 1, wherein sodium sulphite is substituted for sodium metabisulphite in the case of WOB PH 1. The method of Example 1 was further modified whereby the sodium benzoate added was in the form of a prill bead rather than a powder.

The water used throughout the projects is rainwater held in the dark in a plastic tank with stable pH value of 6.25. Controls were set up by replacing actives with tank water only.

Products were dissolved in tank water before application to the agar plants. Tank water (pH 6.25) was adjusted to the respective pH levels prior to adding the actives to determine the change in pH caused by the actives.

Tank water was adjusted to pH 4.0, 5.5, and 7.0 before adding sodium benzoate, sodium metabisulphite and WOB NP1, each at 0.8%.

Tank water was adjusted to pH 7.0, 7.5 and 8.4 before adding sodium benzoate, sodium sulphite and WOB PH1, each at 0.8%.

TABLE 1

Recorded pH of Sodium metabisulphite, sodium benzoate and

WOB NP1 in tank water (pH range 4.0-7.0).

pH of water
pH of water
pH after active added

before
after
in unamended

product added
product added
tank water

Tank water
6.25

Na metabisulphite
4.0
3.75
4.74

5.5
5.52

7.0
6.14

Na Benzoate
4.0
6.15
4.78

5.5
6.34

7.0
6.60

WOB NP1
4.0
4.8
5.14

5.5
5.77

7.0
6.39

TABLE 2

Recorded pH of Sodium sulphite, sodium benzoate and

WOB NP1 in tank water (pH range 7.0-8.4).

pH of water before
pH of water after
pH after active added in

product added
product added
unamended tank water

Tank water
6.25

Na sulphite
7.0
6.78
5.24

7.5
6.78

8.4
6.99

Na
7.0
6.74
4.78

Benzoate
7.5
6.80

8.4
6.85

WOB PH1
7.0
6.68
5.24

7.5
6.66

8.4
6.67

EXAMPLE 4—IN VITRO STUDIES FOR INHIBITION OF CROP PATHOGENS (STUDY 1—DILUTED DRY FORMULATION)

Preparation of Test Media

The fungal and bacterial pathogens Erwinia carotovora (bacterial) and Botrytis cinerea (fungal) were cultured on to Nutrient Agar (NA) and potato dextrose agar (PDA), respectively and incubated at ambient temperature until sporulating or well grown.

Multiple plates of PDA were inoculated with B. cinerea and allowed to sporulate. Multiple plates of NA were inoculated with E. carotovora and allowed to grow into a thick lawn.

Curative Activity:

Plates of PDA and NA were inoculated with fungal spores and bacterial cells, respectively, and allowed to grow into a lawn covering the agar surfaces. Three replicates were used for each product and each pH. Following the results from the preliminary tests, pH 4.0 and 7.0 were selected for all further product pH tests.

When the lawns were well grown and sporulating in the case of the fungal pathogen, five discs soaked with 200 uL of each product (sodium metabisulphite, sodium benzoate, WOB NP1, sodium sulphite, and WOB PH1) at appropriate pH levels were laid onto the sporulating surface or cell lawn surface for the fungal pathogen and the bacterial pathogen, respectively.

The plates were incubated at ambient temperature (14-25° C.). Inhibition zones were measured at 24 hours, 48 hours and 7 days.

Preventative Activity:

Plates of agar containing each product (Na metabisulphite, Na Benzoate, WOB NP1 of Example 3) and (Na sulphite, Na Benzoate, WOB PH1) at concentrations equivalent to 0.8% concentration were made up and poured into sterile disposal Petri dishes. Three replicates for each product and pH (4.0 and 7.0) were used.

Sterile agar discs covered with bacterial cells or fungal hyphae and spores were cut from respective plates of B. cinerea and E. carotovora. Three discs were each laid culture surface down onto the amended agar surface, incubated at ambient temperatures (14-25° C.) and observed for inhibition zones at 24 hours, 48 hours and 7 days.

TABLE 3

Sodium metabisulphite activity on the growth of E. carotovora.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

Na

E.

24 hrs
1
4.0
No effect
No growth away from core onto

metabisulphite

carotovora

2

agar surface. Growth 2-3 mm

3

onto agar from core. Cells not

freely spreading

48 hrs
1

Clear 2-3 mm
Limited growth

2

back from
onto agar surface

3

active disc
2-3 mm

7 days
1

Clearing
Limited growth

2

around disc
onto agar surface

3

still apparent.
2-3 mm

Active is still

affecting

pathogen

TABLE 4

Sodium metabisulphite activity on the growth of B. cinerea.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

Na

B. cinerea

24 hrs
1
4.0
No effect
Sporulation

metabisulphite

2

heavy on core.

3

Some hyphae

growing on

agar.

48 hrs
1

Hyphae unhealthy
Some hyphae

2

around discs.
on agar

3

surface.

7 days
1

No sporulation
Restricted

2

immediately around
hyphal growth.

3

active discs. Hyphae
Unhealthy—

appeared unhealthy
little sporing on

with loss of turgor.
agar.

Collapsing hyphae.

Sporulation reduced.

TABLE 5

Sodium benzoate activity on the growth of E. carotovora.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

Na

E. carotovora

24 hrs
1
4.0
No obvious
Strong growth around plugs on all

benzoate

2

effect
reps. Cells compacted & not spreading

3

freely.

48 hrs
1

No obvious
Strong growth around plugs on all

2

effect
reps. Cells compacted & not spreading

3

freely.

7 days
1

Growth
Growth out from

2

restricted around
plug but clumping

3

disc. No growth
and restricted in

onto discs.
spread.

TABLE 6

Sodium benzoate activity on the growth of B. cinerea.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

Na

B. cinerea

24 hrs
1
4.0
No obvious effect
Heavy sporulation on plug. Hyphae

benzoate

2

grown onto agar surface

3

but not into agar containing active.

48 hrs
1

Sporulation up to
Heavy sporulation on plug

2

discs. Some
Hyphae grown onto agar surface

3

collapsing of

hyphae and

conidiophores.

7 days
1

Reduced
Unhealthy hyphae & restricted

2

sporulation
sporing on plugs. Restricted growth

3

around discs.
on agar. Hyphae very unhealthy—

Hyphae
loss of turgor.

collapsing.

TABLE 7

WOB NP1 activity on the growth of E. carotovora.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

WOB NP1

E. carotovora

24 hrs
1
4.0
No obvious
Strong growth out from plugs

2

effect

3

48 hrs
1

No obvious
Strong but restricted growth

2

effect
out from plugs

3

7 days
1

1-2 mm of
Growth rings less

2

restricted growth
than on pH 7.0 plates.

3

around discs.

TABLE 8

WOB NP1 activity on the growth of B. cinerea.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

WOB

B. cinerea

24 hrs
1
4.0
No obvious
Little sporulation

NP1

2

effect
but some hyphal

3

growth on & in

agar.

48 hrs
1

Hyphal growth
Little sporulation

2

unhealthy-
but some hyphal

3

reduced
growth on & in

sporing.
agar.

7 days
1

Hyphal growth
Sporulation

2

unhealthy-
restricted to plug-

3

reduced
little on agar.

sporing.
Hyphae unhealthy.

TABLE 9

Sodium sulphite activity on the growth of E. carotovora.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

Na

E. carotovora

24 hrs
1
7.0
Growth out from
Growth out

sulphite

2

plug.
from plug

3

onto agar.

48 hrs
1

More growth
More growth

2

but restricted &
but limited

3

clumping

7 days
1

Growth onto
Growth onto

2

agar. More than
agar greater

3

at pH 4.0
than pH 4.0.

TABLE 10

Sodium sulphite activity on the growth of B. cinerea.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

Na

B. cinerea

24 hrs
1
7.0
No effect
Less sporulation

sulphite

2

than on pH 4.0

3

plates

48 hrs
1

Hyphae
Greater spread of

2

unhealthy
hyphae than on pH

3

around active
4.0 plates

discs.

7 days
1

No sporulation
Heavy sporulation

2

immediately
on plugs.

3

around discs
Restricted hyphal

containing
growth with some

active. Hyphae
sporulation onto

appeared
agar.

unhealthy with

loss of turgor.

TABLE 11

Sodium benzoate activity on the growth of E. carotovora.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

Na

E. carotovora

24 hrs
1
7.0
No effect
Strong growth

benzoate

2

around plugs.

3

Greater than on

pH 4.0 plates

48 hrs
1

Reduced
No increase in

2

growth
spread but cells

3

back from disc
piling onto top

of each other-ie

restricted outward

growth

7 days
1

Clearing around
Growth on

2

discs still
agar greater

3

apparent. Active
than pH 4.0

is still affecting

pathogen

TABLE 12

Sodium benzoate activity on the growth of B. cinerea.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

Na

B. cinerea

24 hrs
1
7.0
No effect
Greater hyphal

benzoate

2

growth on & in

3

agar but no

sporing.

48 hrs
1

Hyphae
Greater hyphal

2

unhealthy around
growth on & in

3

discs
agar but no

sporing.

7 days
1

No sporulation
Similar to pH 4.0

2

immediately
plates but more

3

around discs
sporulation on

containing active.
the agar hyphae.

Hyphae appeared

unhealthy with

loss of turgor.

Growth greater

than on pH 4.0

plates

TABLE 13

WOB PH1 activity on the growth of E. carotovora.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

WOB

E. carotovora

24 hrs
1
7.0
No obvious
Clumped growth

PH1

2

effect
around plugs.

3

48 hrs
1

No obvious
Restricted growth

2

effect
around plugs.

3

7 days
1

No growth onto
Restricted growth

2

the discs.
around plugs but

3

rings of growth.

TABLE 14

WOB PH1 activity on the growth of B. cinerea.

Results

Active
Pathogen
Time
Reps
PH
Curative
Preventative

WOB

B. cinerea

24 hrs
1
7.0
No obvious
Little sporulation.

PH1

2

effect
Hyphal growth on

3

& in agar

48 hrs
1

Minimal
Little sporulation.

2

sporulation onto
Hyphal growth on

3

discs
& in agar

7 days
1

Damaged
Little sporulation in

2

hyphae around
hyphae on agar

3

discs. Effect of
but sclerotia

active
forming on hyphae

persisting.
on agar. Sclerotia

sign of unhealthy

colony.

The observed results for the two products as (WOB NP1 and WOB PH1) were not as expected. Both WOB products were observed to have little or no effect on curative or preventative inhibition of E. carotovora and B. cinerea pathogen growth.

EXAMPLE 5—IN VITRO STUDIES FOR INHIBITION OF CROP PATHOGENS (STUDY 2—LIQUID FORMULATION, UNADJUSTED WATER PH)

Further WOB NP 1 and WOB PH 1 products were prepared, according to the general method of Example 1, wherein the sodium benzoate added was is the form of a powder rather than a prill bead of Example 4. These products were subsequently prepared as a liquid formulation according to the method of Example 2.

Water was used unmodified and agars were made up of the 6 products using them at the pH resulting after dissolving to 0.8% concertation. Curative and preventative plates were prepared as described for Example 4 except that pHs were as dissolved (tank water not adjusted prior to dissolving/diluting product).

TABLE 15

Sodium metabisulphite activity on the growth of

E. carotovora (unadjusted water pH)

Results

Active
Path
Time
Reps
Curative
Preventative

Na

E.

24 hrs
1
No obvious effect
No obvious effect

metabisulphite

carotovora

2

3

48 hrs
1
1 mm av. reduced
Restricted growth onto

2
growth of cells
agar containing active.

3
away from active.
Clumping effect

just off plug.

7 days
1
3-4 mm av. reduced
Restricted growth onto

2
growth of cells
agar containing active.

3
away from active.
Clumping effect

just off plug.

3-4 mm clumped growth

around plug on agar

containing active.

TABLE 16

Sodium metabisulphite activity on the growth of B. cinerea

(unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

Na

B. cinerea

24 hrs
1
No obvious effect
No obvious effect

metabisulphite

2

3

48 hrs
1
Hyphae around
No growth off plug into

2
active looking
agar containing active.

3
unhealthy-losing

turgor-conidiophores

collapsing around

active.

7 days
1
No growth onto
Kill. No growth off plug

2
active discs.
into agar or away from

3
Hyphae and
agar on plug. Hyphae

conidiophores
collapsed and no

carrying sporing
sporulation on any

heads at apex all
replicate.

collapsing out from

active.

TABLE 17

Sodium benzoate activity on the growth of

E. carotovora (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

Na

E. carotovora

24 hrs
1
No obvious
No obvious effect

benzoate

effect

2

3

48 hrs
1
No obvious
Cells clumped around

2
effect
plug. Piling suggesting

3

move away from active

in agar. Vertical rather

than linear growth.

7 days
1
Reduction in
Cells clumped around

2
cells numbers
plug. Piling suggesting

3
around active
move away from active

disc. 3-4 mm
in agar. Vertical rather

reduction zone.
than linear growth.

Growth very restricted.

TABLE 18

Sodium benzoate activity on the growth of

B. cinerea (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

Na

B.

24 hrs
1
No obvious effect
No obvious effect

benzoate

cinerea

2

3

48 hrs
1
Growth up to but
No growth into agar

2
not on disc
but a little on surface.

3
containing active.
No sporulation.

7 days
1
Growth up to but
No growth into agar

2
not on disc
containing active.

3
containing active.
Effect less than for

Hyphae unhealthy.
Na metabisulphite.

Effect less than
No sporulation.

with Na

metabisulphite.

TABLE 19

WOB NP1 (liquid formulation) activity on the growth of E. carotovora

(unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

WOB NP1

E. carotovora

24 hrs
1
No obvious effect
No obvious effect

2

3

48 hrs
1
No growth onto
Colonies clumping

2
active discs.
around plug.

3
Reduced density of

cells around active

discs.

7 days
1
No growth onto
Colonies clumping

2
active discs.
around plug. Vertical

3
Reduced density of
rather than lateral

cells around active
growth. Growth

discs.
restricted to 3-4 mm

from plug.

TABLE 20

WOB NP1 (liquid formulation) activity on the growth of

E. carotovora(unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

WOB NP1

B.

24 hrs
1
No obvious effect
No obvious

cinerea

2

effect

3

48 hrs
1
No growth onto active
No growth onto

2
discs. Hyphae around disc
or in agar

3
collapsing but not as much
containing

as with Na metabisulphite.
active.

Sporulation reduced.

7 days
1
No growth onto active
Kill. No growth

2
discs. Hyphae around disc
onto or in agar

3
collapsing but not as much
containing

as with Na metabisulphite.
active.

Sporulation reduced.

EXAMPLE 6—IN VITRO STUDIES FOR INHIBITION OF CROP PATHOGENS (STUDY 3—STORAGE EFFECTS)

The curative and preventative experiments were repeated according to the method of Example 5 using the liquid WOB NP1 and WOB PH 1 formulations and the solid actives sodium metabisulphite, sodium benzoate and sodium sulphite.

The liquid WOB formulations were divided into 3 aliquots; one was used immediately—time zero; one stored at ambient temperate (15-27° C.) for one week and experiments repeated; one kept refrigerated (5° C.) for one week and experiments repeated. The bottles used for storage of the aliquots did not allow light penetration into the product.

TABLE 21

WOB NP1 dissolved in sterile water and applied at t = 0, activity

on the growth of E. carotovora(unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

pre WOB

E. carotovora

24 hrs
1
No obvious effect
No obvious effect

NP1

2

3

48 hrs
1
1 mm av. reduced
No growth off plug.

2
growth of cells away
Cells not

3
from active.
multiplying.

7 days
1
3-4 mm av. reduced
Kill. Cells under

2
growth of cells away
plug in contact

3
from active.
with active in agar

not multiplying.

TABLE 22

WOB NP1 dissolved in sterile water and applied at t = 0, activity on the

growth of B. cinerea (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

pre WOB

B.

24 hrs
1
No obvious effect
No obvious effect

NP1

cinerea

2

3

48 hrs
1
Hyphae around
No growth off plug into

2
active looking
agar containing active.

3
unhealthy-losing

turgor-conidiophores

collapsing around

active.

7 days
1
No growth onto active
Kill. No growth off plug

2
discs. Hyphae and
into agar or away from

3
conidiophores carrying
agar on plug. Hyphae

sporing heads at apex
collapsed and no

all collapsing out from
sporulation on any

active.
replicate.

TABLE 23

WOB PH1 dissolved in sterile water and applied at t = 0, activity on the

growth of E. carotovora(unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

post WOB

E.

24 hrs
1
No obvious effect
No obvious effect

PH1

carotovora

2

3

48 hrs
1
Reduced growth of
3-5 mm bacterial

2
cells away from
growth around plug.

3
active. Less effect
Cells clumping.

than WOB pre.

7 days
1
Reduced growth of
3-5 mm bacterial

2
cells away from
growth around plug.

3
active. Less effect
Cells clumping.

than WOB pre.

TABLE 24

WOB PH1 dissolved in sterile water and applied at t = 0, activity on the growth of

B. cinerea (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

post WOB PH1

B. cinerea
24 hrs
1
No obvious effect
No obvious effect

2

3

48 hrs
1
No growth onto discs.
Restricted growth

2
More sporulation
onto and into agar

3
around active discs
containing active.

than for WOB pre.
Some sporulation

but restricted

around plug.

7 days
1
No growth onto discs.
Some hyphal

2
More sporulation
growth out from

3
around active discs
plug. Restricted

than for WOB pre.
growth but some

Hyphae collapsing.
sporulation around

Conidiophores
plug.

collapsing.

TABLE 25

WOB NP1 dissolved in sterile water and applied at t = 7 days with storage at 5° C., activity on

the growth of E. carotovora (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

WOB NP1

E. carotovora
24 hrs
1
No obvious effect
Some growth

2

onto agar

3

containing

active.

48 hrs
1
Reduced growth of cells
Restricted

2
away from active.
growth to

3

around plug.

7 days
1
Reduced growth of cells
Restricted

2
away from active. No
growth to

3
obvious difference in
around plug.

growth when compared

with Time 0.

TABLE 26

WOB NP1 dissolved in sterile water and applied at t = 7 days with storage at 5° C., activity on

the growth of B. cinerea (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

WOB NP1

B. cinerea
24 hrs
1
No obvious effect
No obvious

2

effect

3

48 hrs
1
Hyphae around active
No growth off

2
looking unhealthy-losing
plug into agar

3
turgor-conidiophores
containing

collapsing around active.
active.

7 days
1
No growth onto active
Kill. No growth

2
discs. Hyphae and
off plug into

3
conidiophores carrying
agar or away

sporing heads at apex all
from agar on

collapsing out from
plug.

active.

TABLE 27

WOB PH1 dissolved in sterile water and applied at t = 7 days with storage at 5° C., activity on

the growth of E. carotovora (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

WOB PH1

E. carotovora
24 hrs
1
No obvious effect
No obvious effect

2

3

48 hrs
1
Reduced growth of
Bacterial growth

2
cells away from
around plug. Cells

3
active. Less effect
clumping.

than pre.

7 days
1
Reduced growth of
Bacterial growth

2
cells away from
around plug. Cells

3
active. Less effect
clumping.

than pre.

TABLE 28

WOB PH1 dissolved in sterile water and applied at t = 7 days with storage at 5° C., activity on

the growth of B. cinerea (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

WOB NP1

B. cinerea
24 hrs
1
No obvious effect.
No obvious effect.

2

3

48 hrs
1
No growth onto discs.
Restricted growth

2
More sporulation
onto and into agar

3
around active discs
containing active.

than for pre.
Some sporulation

but restricted

around plug.

7 days
1
No growth onto discs.
Some hyphal

2
More sporulation
growth out from

3
around active discs
plug. Restricted

than for pre. Hyphae
growth but some

collapsing.
sporulation around

Conidiophores
plug. More hyphae

collapsing.
onto agar.

TABLE 29

WOB NP1 dissolved in sterile water and applied at t = 7 days with storage at ambient

temperature (15-27° C.), activity on the growth of E. carotovora (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

WOB NP1

E. carotovora
24 hrs
1
No obvious effect
Some growth onto

2

agar containing active.

3

48 hrs
1
Reduced growth
Growth onto agar

2
of cells away from
containing active.

3
active. Less effect
Little restriction in cell

than refrigerated.
colony formation.

7 days
1
Reduced growth
Growth onto agar

2
of cells away from
containing active.

3
active. Less effect
Little restriction in cell

than refrigerated.
colony formation.

TABLE 30

WOB NP1 dissolved in sterile water and applied at t = 7 days with storage at ambient

temperature (15-27° C.), activity on the growth of B. cinerea (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

WOB NP1

B. cinerea
24 hrs
1
No obvious effect.
No obvious effect.

2

3

48 hrs
1
Hyphae around active
Growth and

2
looking unhealthy-
sporulation out

3
losing turgor-
from plug. More

conidiophores
hyphae in agar.

collapsing around

active.

7 days
1
No growth onto active
Growth and

2
discs. Hyphae and
sporulation out

3
conidiophores
from plug. More

carrying sporing
hyphae in

heads at apex all
agar. More growth

collapsing out from
than 7 days

active.
refrigerated.

TABLE 31

WOB PH1 dissolved in sterile water and applied at t = 7 days with storage at ambient

temperature (15-27° C.), activity on the growth of E. carotovora (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

WOB NP1

E. carotovora
24 hrs
1
No obvious effect
Growth on agar

2

containing active

3

but restricted to

around plugs.

48 hrs
1
Reduced growth of
Growth on agar

2
cells away from active.
containing active

3
Less effect than pre.
but restricted to

Less effect than
around plugs.

refrigerated.

7 days
1
Reduced growth of
Growth on agar

2
cells away from active.
containing active

3
Less effect than pre.
but restricted to

around plugs.

TABLE 32

WOB PH1 dissolved in sterile water and applied at t = 7 days with storage at ambient

temperature (15-27 °C.), activity on the growth of B. cinerea (unadjusted water pH).

Results

Active
Path
Time
Reps
Curative
Preventative

WOB PH1

B. cinerea
24 hrs
1
No obvious effect
No obvious

2

effect.

3

48 hrs
1
No growth onto discs.
Growth

2
More sporulation around

3
active discs than for pre.

7 days
1
No growth onto discs.
—

2
More sporulation around

3
active discs than for pre.

Hyphae collapsing.

Conidiophores collapsing.

EXAMPLE 9—IN VITRO STUDIES FOR INHIBITION OF CROP PATHOGENS (STUDY 3—VARIED FUNGAL PATHOGENS)

This trial was set up to determine the efficacy of a formulation comprising WOB-NP1 as a curative against the fungal pathogen, Botrytis cinerea, and two bacteria strains, E. coli and Xanthomonas sp.

The effect of WOB NP1 on two wild type yeasts, Saccharomyces cerevisae and Schizosaccharomyces pombe were also further investigated.

The organisms were transferred from culture collection mother cultures to fresh media and checked for purity.

Preparation of Test Medium

20 mL of Potato Dextrose Agar (PDA) agar was poured into Petri plates to give the thickness of agar necessary to take 600 μLs of product in each well. Botrytis cinerea, Saccharomyces cerevisae and Schizosaccharomyces pombe were cultured on PDA and grown until sporulating or growing freely across the medium.

Preparation of Products

A formulation was prepared according to the method described in Example 1 (referred to as WOB NP1). Prior to adding formulation to plates, pH readings of the WOB NP1 solutions were taken over a 30 min period to determine stability of the product in solution.

Two identical solutions of WOB-NP1, originating from separate yet identical dry composition batches (WOB-NP1 A and WOB-NP1 B), were produced at 4 g/L (4% v/v) in boiled water.

TABLE 33

pH recordings prior to inoculation.

Product
Unadjusted pH

WOB NP1 A
5.58

WOB NP1 B
5.55

Preparation of Cell/Spores for Trials:

Sterile boiled water was added to the surface of the Botrytis cinerea lawn plates and rubbed gently with sterile hockey sticks to loosen cells (conidia). A known volume—1 mL—of Botrytis cinerea conidia or yeast cells was lifted aseptically from the culture plates and dispersed by shaking gently into 9 mL of 1% peptone water. Serial dilutions were carried out until haemocytometer counts showed between 103 and 104 colony forming units (cfus) per mL. Two×300 μLs were added to each of the wells in each plate for the respective organisms. The plates were incubated at 22° C. and observed for reactions between the product and organism at 24 and 10 days. The reaction would be zones of inhibition for the yeast cells or fungal hyphae dying or growing away from the product.

Trials were carried out using direct immersion in product as a curative, using the WOB NP1 formulation A and B, with sterile boiled water as a control tested against Botrytis cinerea conidia (spores), E. coli and Xanthomonas species in triplicate on potato dextrose agar (PDA) and nutrient agar (NA). WOB NP1 A prepared in 2015, just prior to testing in November 2015 and WOB NP1 B prepared two years prior in November 2013, being stored at room temperature in dry conditions until testing.

Exposure time to the products was 5 mins after which 50 μL was applied to each of the replicate plates and spread evenly across the agar surface using sterile disposable hockey sticks.

The plates incubated inverted at 22° C. and counts were read at 48 hours. The above method was followed to make another set of plates where the spores/cells were exposed to the products for 48 hours.

TABLE 34

Qualitative assessment of response of organisms to products WOB NP1 A and WOB NP1 B.

Exposure

WOB NP1 A
WOB NP1 B

time
Organism
pH 5.55
pH 5.58

5 mins

Xanthomonas sp
50% reduction
50-60% reduction when

compared with control
compared with control

48 hours

Xanthomonas sp
100% reduction when
100% reduction when

compared with control
compared with control

5 mins

E. coli
No effect
No effect

48 hours

E. coli
75-80% reduction
90% reduction compared

compared with control
with control

5 mins

Botrytis
cinerea

No effect
No effect

48 hours

Botrytis
cinerea

100% reduction when
100% reduction when

compared with control
compared with control

EXAMPLE 10—GROWTH STUDIES FOR CONTROL OF BOTRYTIS CINEREA IN GRAPEVINES CV. SAUVIGNON BLANC

A trial was conducted within a commercial vineyard to evaluate WOB NP1 for the control of botrytis (Botrytis cinerea) and for crop safety in grapevines cv. Sauvignon Blanc. A WOB NP1 formulation was prepared according to the method described in Examples 1 and 2. WOB NP1 (comprising active ingredients sodium metabisulphite+sodium benzoate) was applied at 35+119.6, 70+239.2, 140+478.4 and 280+956.8 g ai/100 L and compared with Teldor 500 SC at 50 g ai/100 L and an untreated control.

Materials and Methods

TABLE 35

Products used in the study for control of Botrytiscinerea.

Concentration

Product

of active

name
Active ingredient(ai)
ingredient
Formulation

WOB NP1
sodium metabisulphite
175 g/kg +
Powder

as sulphur dioxide +
598 g/kg

sodium benzoate as

benzoic acid

Teldor 500 SC
fenhexamid
500 g/L
Suspension

concentrate

TABLE 36

Treatment levels and application schedule summary.

Rate

Product
Active

(g or mL/
ingredient

No.
Product
100 L)
(g ai/100 L)*
Application schedule

1
Untreated
Nil
Nil
N/A

control

2
WOB NP1
200 g
35 + 119.6
A total of six foliar

applications to grapevines

at 7-26 day intervals

3
WOB NP1
400 g
70 + 239.2
commencing at BBCH 61

4
WOB NP1
800 g
140 + 478.4
(10% flowering).

5
WOB NP1
1600 g
280 + 956.8
Treatments applied as a

6
Teldor 500
100 mL
50
dilute spray prior to the

SC

point of run-off when

temperature was below

20° C. and humidity

below 70%.

*WOB NP1 773 WG formulation containing sodium metabisulphite + sodium benzoate.

Treatments were applied as six dilute foliar sprays just prior to the point of run-off in spray volumes from 700-900 L/ha, commencing at the BBCH 61 (10% flowering) crop stage.

At an assessment conducted three days after application F (3DAAF), although all WOB NP1 treatments appeared to reduce the incidence of botrytis in grapevine bunches, only WOB NP1 at 280+956.8 g ai/100 L had significantly less botrytis than the untreated control. The incidence of botrytis was less in bunches sprayed with Teldor when compared with each of the WOB NP1 treatments (Table 40).

At 3DAAF, the severity of botrytis was significantly less in all WOB NP1 treatments when compared with an untreated control. Disease severity in bunches sprayed with WOB NP1 at 70+239.2 and 280+956.8 g ai/100 L was also statistically comparable with Teldor (Table 40).

At 15DAAB, WOB NP1 at 70+239.2, 140+478.4 and 280+956.8 g ai/100 L caused some phototoxicity to grapevine leaves but phytotoxicity was absent in grape bunches. Necrotic spotting was observed on leaves sprayed with WOB NP1 at 70+239.2, 140+478.4 and 280+956.8 g ai/100 L with the most severe damage at the highest rate of WOB NP1 (Table 41, FIGS. 1-5b).

TABLE 37

Outlining the chronology of events stages of application of the

WOB NP-1 formulation on the grape vine test subjects.

Days after

application
Crop stage

timing
BBCH

(DAA#)
scale
Description
Event

0DAAA
61
10% flowering
Application A

7DAAA
68
80% flowering
Application B

15DAAB
75
Berries pea size
Crop safety photographs taken

Crop safety assessment

24DAAB
77
Berries beginning
Application C

to touch
Crop safety assessment

26DAAC
81
Veraison
Application D

Crop safety assessment

21DAAD
83
Berries softening
Application E

Crop safety assessment

12DAAE
83
Berries softening
Application F

3DAAF
83
Berries softening

Botrytis assessment

Crop safety assessment

Application Details—Spray

Table 38 and 39 describe details of the application spray and conditions at each time point throughout the application schedule.

TABLE 38

Outlining specifics of the application spray and conditions at application time

points A, B and C.

Application equipment

Method
Dilute foliar application just prior to the point of run-off

Equipment
Motorised backpack sprayer with hand-held lance

Nozzle type
Spraying Systems TG-3 full cone

Nozzle number and spacing
1

Spray quality
Medium

Spray volume (L/ha)
700-900

Pressure (kPa)
500

Treatment applications

Application timing
A
B
C

Days after application timing
0DAAA
7DAAA
24DAAB

Times
08:30-09:45
10:45-12:00
08:45-10:00

Treatments applied
2-6
2-6
2-6

Spray volume (L/ha)
700
700
900

Temperature (° C.)
15
18
17

Relative humidity (%)
67
59
63

Cloud cover (%)
100
10
80

Wind direction
NE
Variable
NW

Wind speed (kph)
5-10
0-3
0-5

Leaf wetness
Nil
Nil
Nil

Disease level
Nil
Nil
Nil

Crop stage description
10% flowering
80% flowering
Berries beginning

to touch

Crop stage (BBCH)
61
68
77

TABLE 39

Outlining specifics of the application spray and conditions at application time

points D, E and F.

Application equipment

Method
Dilute foliar application just prior to the point of run-off

Equipment
Motorised backpack sprayer with hand-held lance

Nozzle type
Spraying Systems TG-3 full cone

Nozzle number and
1

spacing

Spray quality
Medium

Spray volume (L/ha)
900

Pressure (kPa)
500

Treatment applications

Application timing
D
E
F

Days after application
26DAAC
21DAAD
12DAAE

timing

Times
11:30-13:00
08:30-09:30
10:00-11:15

Treatments applied
2-6
2-6
2-6

Spray volume (L/ha)
900
900
900

Temperature (° C.)
14
19
21

Relative humidity (%)
52
55
47

Cloud cover (%)
40
100
20

Wind direction
W
NW
W

Wind speed (kph)
5-10
0-5
10-12

Leaf wetness
Nil
Nil
Nil

Disease level
Nil
Nil

Botrytis present

Crop stage description
Veraison
Berries softening
Berries softening

Crop stage (BBCH)
81
83
83

Results

TABLE 40

Botrytis incidence and severity at three days after application F (3DAAF)

Botrytis control on grapevine bunches

3DAAF

Rate
Incidence
Severity (% bunch

No.
Treatment
(g ai/100 L)*
(% bunches affected)
area affected)

1
Untreated control
Nil
41
a
6.9
a

2
WOB NP1
35 + 119.6
34
ab
4.0
b

3
WOB NP1
70 + 239.2
29
ab
2.3
bc

4
WOB NP1
140 + 478.4
33
ab
3.1
b

5
WOB NP1
280 + 956.8
24
b
2.8
bc

P-value
0.0034
0.0009

LSD (P ≤ 0.05)
13.1
2.23

*WOB NP1 formulation containing sodium metabisulphite + sodium benzoate. Means followed by the same letter are not significantly different (P = 0.05, LSD)

DAA# = Days after application timing

TABLE 41

Grapevine bunch crop safety

Rate
Grapevine bunch crop safety (% bunch area affected by phytotoxic symptoms)

(g ai/

No.
Treatment
100 L)*
15DAAB
24-DAAB
26DAAC
21DAAD
3DAAF

1
Untreated
Nil
0
0
0
0
0

control

2
WOB
35 +
0
0
0
0
0

NP1
119.6

3
WOB
70 +
0
0
0
0
0

NP1
239.2

4
WOB
140 +
0
0
0
0
0

NP1
478.4

5
WOB
280 +
0
0
0
0
0

NP1
956.8

6
Teldor
50
0
0
0
0
0

500 SC

P-value
1.0000
1.0000
1.0000
1.0000
1.0000

LSD (P ≤ 0.05)
NSD
NSD
NSD
NSD
NSD

*WOB NP1 formulation containing sodium metabisulphite + sodium benzoate.

DAA# = Days after application timing

NSD = No significant difference due to a P-value > 0.05

TABLE 42

Describes the methods used to assess the crops including methods of statistical

analysis for results observed. Botrytis assessment

Days after
3DAAF

application timing

Sample size
40 bunches per plot

Method
Percent area affected by botrytis (Botrytiscinerea) from 40 grape

bunches per plot was visually estimated with results presented as

mean percent bunch area affected. Incidence was calculated in

ARM2018 from severity data collected.

Crop safety assessment-grape bunches

Daysafter
15DAAB
24DAAB
26DAAC
21DAAD
3DAAF

application timing

Sample size
Whole plot (4 vines)

Method
All grape bunches were visually assessed for symptoms of

phytotoxicity including, but not limited to discolouration, necrosis

or developmental effects.

Statistical
Analysis of variance (ANOVA) test and Fisher’s least significant

analysis
difference (LSD) test were conducted using ARM2018. When

data violated the assumptions of ANOVA (homogeneity of

variance and normality) data correction transformations were

conducted. Original plot means are presented in Results tables

with analysis of variance and letters of separation from

transformed data. Note, treatment data with the same number

but different letters of separation can result from statistics relying

on transformed data.

TABLE 43

Botrytis incidence and severity at three days after

application F (3DAAF)

Pest Name
Botrytis
Botrytis

Part Rated
BUNCH P
BUNCH P

Rating Type
PESINC
PESSEV

Rating Unit
%
% AREA

Sample Size, Unit
40 BUNCH
40 BUNCH

Reporting Basis, Unit
1 PLOT
1 BUNCH

Trt-Eval Interval
3DAAF
3DAAF

Trt
Trt.

Rate

No.
Name
Rate*
Unit
1
2

1
Untreated

41
a
6.9
a

control

2
WOB NP1
35 +
g ai/100 L
34
ab
4.0
b

119.6

3
WOB NP1
70 +
g ai/100 L
29
ab
2.3
bc

239.2

4
WOB NP1
140 +
g ai/100 L
33
ab
3.1
b

478.4

5
WOB NP1
280 +
g ai/100 L
24
b
2.8
bc

956.8

6
Teldor 500 SC
50
g ai/100 L
11
c
0.8
c

LSD (P = .05)
13.1
2.23

Standard Deviation
8.7
1.48

CV
30.26
44.44

Bartlett's X2
2.213
1.187

P(Bartlett's X2)
0.819
0.946

Skewness
−0.6714
0.6929

Kurtosis
0.0788
0.0161

Replicate F
0.752
1.018

Replicate Prob(F)
0.5379
0.4122

Treatment F
5.862
7.662

Treatment Prob(F)
0.0034
0.0009

*WOB NP1 formulation containing sodium meta bisulphite + sodium benzoate. Means followed by same letter do not significant ly differ (P=.05, LSC >) Mean comparisons performed only when AOV Treatment P(F) is significant at mean comparison OSL

Part Rated

BUNCH=bunch

P=Pest is Part Rated

Rating Type

PESINC=pest incidence

PESSEV=pest severity

Rating Unit

%=percent

% AREA=percent of area

BUNCH=bunch

PLOT=total plot

TABLE 44

Grapevine bunch crop safety profile

Pest Name

Part Rated
BUNCH C

Rating Type
PHYGEN

Rating Unit
%AREA

Sample Size, Unit
4 VINE

Reporting Basis, Unit
1 PLOT

Trt-Eval Interval
15DAAB
24DAAB
26DAAC
21DAAD
3DAAF

Other

Trt
Trt.

Rate

No.
Name
Other Rate*
Unit
3
4
5
6
7

1
Untreated control

0a
0a
0a
0a
0a

2
WOB NP1
35 +
g ai/
0a
0a
0a
0a
0a

119.6
100 L

WOB NP1
70 +
g ai/
0a
0a
0a
0a
0a

239.2
100 L

4
WOB NP1
140 +
g ai/
0a
0a
0a
0a
0a

478.4
100 L

5
WOB NP1
280 +
g ai/
0a
0a
0a
0a
0a

956.8
100 L

6
Teldor 500 SC
50
g ai/
0a
0a
0a
0a
0a

100 L

LSD P = .05

Standard Deviation
0.0
0.0
0.0
0.0
0.0

CV
0.0
0.0
0.0
0.0
0.0

Bartlett's X2
0.00
0.00
0.00
0.00
0.00

P(Bartlett's X2)

Skewness

Kurtosis

Replicate F
0.000
0.000
0.000
0.000
0.000

Replicate Prob(F)
1.0000
1.0000
1.0000
1.0000
1.0000

Treatment F
0.000
0.000
0.000
0.000
0.000

Treatment Prob(F)
1.0000
1.0000
1.0000
1.0000
1.0000

*WOB NP1 formulation containing sodium metabisulphite + sodium benzoate formulation Means followed by same letter or symbol do not significantly differ (P = .05, LSD) Mean comparisons performed only when AOV Treatment P(F) is significant at mean comparison OSL Could not calculate LSD (% mean diff) for columns 3,4,5,6,7 because error mean square = 0

Part Assessed

BUNCH=bunch

C=Crop is Part Rated

Assessment Type

PHYGEN=phytotoxicity—general/injury

Assessment Unit

% AREA=percent of area

VINE=vine PLOT=total plot

TABLE 45

Botrytis incidence and severity at three days after application F (3DAAF)

Pest Name
Botrytis
Botrytis

Part Rated
BUNCH P
BUNCH P

Rating Type
PESINC
PESSEV

Rating Unit
%
% AREA

Sample Size, Unit
40 BUNCH
40 BUNCH

Reporting Basis, Unit
1 PLOT
1 BUNCH

Trt-Eval Interval
3DAAF
3DAAF

Trt
Treatment

No.
Name
Rate*
Rate Unit
Plot
1
2

1
Untreated

102
45
8.1

control

204
50
8.2

301
35
4.0

405
35
7.4

Mean =
41
6.9

2
WOB NP1
35 +
g ai/100 L
101
28
3.0

119.6

206
35
2.9

304
38
6.0

402
38
4.3

Mean =
34
4.0

3
WOB NP1
70 +
g ai/100 L
104
38
2.3

239.2

202
35
4.5

306
23
1.4

401
20
1.3

Mean =
29
2.3

4
WOB NP1
140 +
g ai/100 L
103
25
2.9

478.4

205
40
4.7

302
28
1.7

406
40
3.3

Mean =
33
3.1

5
WOB NP1
280 +
g ai/100 L
106
8
0.6

956.8

201
30
3.4

303
25
3.1

404
35
4.0

Mean =
24
2.8

6
Teldor 500
50
g ai/100 L
105
8
0.2

SC

203
0
0.0

305
18
0.7

403
18
2.4

Mean =
11
0.8

**WOB NP1 formulation containing sodium metabisulphite + sodium benzoate.

Part Rated

BUNCH=bunch

P=Pest is Part Rated

Rating Type

PESINC=pest incidence

PESSEV=pest severity

Rating Unit

%=percent

% AREA=percent of area

BUNCH=bunch

PLOT=total plot

TABLE 46

Meteorological details (part 1 of 2) throughout study period.

Location: Low Head, Tasmania, Australia

Day
Event
Min ° C.
Max ° C.
mm*
Event
Min ° C.
Max ° C.
mm*

1

19.0
19.7
0

15.8
20.6
0

2

13.8
17.3
13.0

14.8
19.9
0

3

8.8
15.9
28.0

12.4
20.0
0

4

10.0
18.3
0.2

14.6
19.8
0

5
Treat
10.7
17.3
0
Treat
12.5
21.8
0

Assess

6

11.5
20.8
0

14.4
20.6
0

7

12.9
19.3
0

15.6
22.3
0

8

11.9
18.7
0

15.2
20.6
0

9

14.1
20.1
2.4

15.8
20.5
0

10

15.2
19.7
0

12.9
20.3
2.2

11

14.6
21.0
0

14.0
21.4
0

12
Treat
13.1
21.1
0

18.8
20.8
0

13

14.8
21.2
0

13.7
20.3
5.2

14

16.9
20.8
0

10.4
21.6
0.2

15

13.4
19.5
0

13.8
27.3
0

16

15.4
20.4
0

12.5
20.7
0

17

11.0
19.4
0

15.1
21.6
0

18

15.2
22.4
0

14.3
21.6
0

19

16.9
21.8
0

15.9
24.6
0

20

16.7
19.5
4.8

16.9
21.7
0

21

14.0
19.4
0

16.3
21.8
0.4

22

15.4
20.6
0

18.9
22.8
0

23

15.7
19.8
0.8

15.8
22.1
0

24

13.4
18.9
0

16.2

25

11.2
20.1
0

16.1
23.7
0

26

12.4
20.6
0

15.3
23.8
0

27
Photos
15.5
21.2
0

18.3
24.1
0

Assess

28

18.3
23.0
4.8

21.2
25.2
0

29

16.7
19.3
0

21.4
23.5
0

30

15.3
18.7
1.8

15.1
22.4
11.4

31

14.2
19.2
0
Treat
11.2
20.8
0

Assess

Total

55.8

19.4

*mm = recorded rainfall at the corresponding time point.

TABLE 47

Meteorological details (part 2 of 2) throughout study period.

Location: Low Head, Tasmania, Australia

Day
Event
Min
° C.
Max ° C.
mm*
Event
Min ° C.
Max ° C.
mm*

1

10.9
20.2
0

14.8
23.7
0

2

13.4
21.0
0

14.9
21.0
0

3

16.2
22.6
0

14.3
21.2
0

4

17.8
22.8
0

14.5
20.4
0

5

15.8
21.5
0
Treat
12.0
21.0
0

6

17.3
21.9
0

12.5
21.1
0

7

15.8
23.2
0

12.6
20.6
0

8

19.4
25.9
0
Assess
13.2
21.6
0

9

18.9
23.0
0

14.2
22.6
0

10

20.2
22.1
0

14.4
22.9
0

11

16.8
21.7
3.4

16.2
24.1
0

12

13.2
21.8
0

14.8
20.2
0

13

11.3
21.3
0

15.5
21.7
0

14

15.6
17.9
6.0

14.6
21.1
0

15

13.8
19.0
3.2

15.0
19.6
0

16

13.9
19.3
1.4

12.9
19.2
1.0

17

14.7
19.6
0

14.0
23.4
2.2

18

16.3
20.4
0

17.3
18.2
11.8

19

13.6
22.7
0

12.9
19.1
10.0

20

10.6
19.4
0

12.1
18.4
0.6

21
Treat
13.0
19.6
0

9.2
18.5
0

Assess

22

13.4
21.3
0

11.1
18.9
0

23

16.5
19.7
0

15.0
19.2
0

24

18.2
22.7
22.0

16.5
19.0
0

25

11.3
20.7
0.4

14.4
18.5
23.6

26

11.9
19.3
0

12.1
18.9
13.6

27

12.2
21.1
0

12.5
18.1
0.2

28

15.3
21.0
0

13.0
21.0
0

29

15.1
19.3
0.2

30

15.7
18.2
4.6

31

11.3
17.6
0

Total

36.4

67.8

*mm = recorded rainfal at the corresponding time point

EXAMPLE 11—GROWTH CONTROL OF BOTRYTIS CINEREA IN GRAPEVINES CV. CABERNET SAUVIGNON

Formulations comprising sodium metabisulphite and sodium benzoate (WOB NP1 773 WG) were applied as dilute canopy sprays to grapevines cv. Cabernet Sauvignon for the control of grey mould (Botrytis cinerea). WOB NP1 773 WG was applied at 30% capfall, the end of flowering, when berries were 4 mm, during bunch closure and at veraison. The standard grey mould control program of Teldor 500 SC applied at end of flowering followed by Switch 625 WG when berries were 4 mm diameter was used for comparison.

Crop safety was assessed during flowering, at fruit set, just prior to bunch closure, at early and late veraison and just prior to harvest. WOB NP1 caused necrosis and browning of the leaf margins, with the area damaged increasing significantly with rate and with subsequent applications. The lower rate of WOB NP1 showed up to 28% of leaves damaged with a severity of 0.3% LAD (leaf area damaged), whilst the high rate showed 100% of the leaves damaged with up to 10.9% LAD. No visible damage was seen on bunches, however higher rates of WOB NP1 left residues on bunches.

The test site was chosen as all fruit from the previous season was rejected due to high levels of grey mould. Grey mould was first seen in the untreated control ten days after commercial harvest, when 8.7% of bunches were damaged by grey mould at a severity index of 2.2%. No grey mould was observed in any treatment, providing no dose response to WOB NP1 rates. All rates of WOB NP1 were equivalent to the standard spray program for the control of grey mould.

TABLE 48

Products employed in the study for growth control of Botrytiscinerea in

grapevines cv. Cabernet Sauvignon

Concentration

Active ingredient
of active

Product name
(ai)
ingredient
Formulation

WOB NP1 773
sodium metabisulphite as
175 g/kg +
Water dispersible

WG
sulphur dioxide + sodium
598 g/kg
granule

benzoate as benzoic acid

Teldor 500 SC
fenhexamid
500 g/L
Suspension

concentrate

Switch 625 WG
fludioxonil + cyprodinil
250 g/kg +
Water dispersible

375 g/kg +
granule

TABLE 49

Treatment schedule employed in the growth control of Botrytiscinerea study.

Rate

Active

Product
ingredient*
Application

No.
Product
(mL or g/100 L)
(g ai/100 L)
schedule

1
Untreated control
Nil
Nil
N/A

2
WOB NP1 773 WG
200 g
35.0 + 119.6
Applied at 30%

3
WOB NP1 773 WG
400 g
70.0 + 239.2
capfall (A), end of

4
WOB NP1 773 WG
800 g
140.0 + 478.4
flowering (B), 4 mm

5
WOB NP1 773 WG
1600 g
280.0 + 956.8
berries (C), bunch

closure (D) and

veraison (E)

6
Teldor 500 SC
100 mL
50.0
End of flowering (B)

Switch 625 WG
80 g
20.0 + 30.0
4 mm berries (C)

*WOB NP1 773 WG formulation containing sodium metabisulphite + sodium benzoate.

TABLE 50

Chronology of events throughout the growth control of Botrytiscinerea study.

Days after
Spray
Crop stage

budburst
interval
Modified

(DAB)
(days)
E-L scale
Description
Event

0
—
04
Budburst
Budburst

51
—
17-18
Pre-flowering
Prosper 500 EC + Avatar 300 WG

(Powdery mildew + garden weevil control)

73
—
21
30% capfall
Vivando 500 SC

(Powdery mildew control)

74
—
21
30% capfall
Application A

80
—
24
60% capfall
Crop phytotoxicity assessment

85
—
26
End of flowering
Vivando 500 SC + Revus 250 SC

(Powdery mildew + downy mildew control)

86
12
26
End of flowering
Application B

93
—
27
Beginning of fruit
Applaud 440 SC

set
(Mealy bug control)

Crop phytotoxicity assessment

99
13
29
4 mm berries
Application C

100
—
29
4 mm berries
Talendo 200 EC

(Powdery mildew control)

114
—
31
7 mm berries
Crop phytotoxicity assessment

129
30
33
Bunch closure
Application D

156
27
36
Veraison-colour
Crop phytotoxicity assessment

change 90%
Application E

190
—
37
Berries not quite
Crop phytotoxicity assessment

ripe
Grey mould bunch assessment

204
—
38
Berries harvest
Crop phytotoxicity assessment

ripe
Grey mould bunch assessment

214
—
39
Berries over ripe
Grey mould bunch assessment

Results

TABLE 51

Crop safety-bunch damage

Rate

100 L)*
Application
Mean bunch area damaged (%)

No.
Treatment
(g ai/
schedule
80DAB
93 DAB
114DAB
156DAB
190DAB
204DAB

1
Untreated
Nil
Nil
0.0
0.0
0.0
0.0
0.0
0.0

control

2
WOB NP1 773
35.0 +
ABCDE
0.0
0.0
0.0
0.0
0.0
0.0

WG
119.6

3
WOB NP1 773
70.0 +
ABCDE
0.0
0.0
0.0
0.0
0.0
0.0

WG
239.2

4
WOB NP1 773
140.0 +
ABCDE
0.0
0.0
0.0
0.0
0.0
0.0

WG
478.4

5
WOB NP1 773
280.0 +
ABCDE
0.0
0.0
0.0
0.0
0.0
0.0

WG
956.8

6
Teldor 500 SC
50
B
0.0
0.0
0.0
0.0
0.0
0.0

Switch 625 WG
20 + 30
C

P-value
1.0000
1.0000
1.0000
1.0000
1.0000
1.0000

LSD (P<0.05)
NSD
NSD
NSD
NSD
NSD
NSD

**WOB NP1 773 WG formulation containing sodium metabisulphit e + sodium benzoate.

DAB = Days after budburst

NSD = No significant difference due to a p-value > 0.05

TABLE 52

Crop safety-leaf necrosis incidence

Rate

Mean leaf necrosis incidence

(g ai/
App.
(% of leaves damaged)

No.
Treatment
100 L)*
schedule
80DAB
93DAB
114DAB
156DAB
190DAB
204DAB

1
Untreated
Nil
Nil
0.0
c
0.0
e
0.0
d
0.0
d
0.0
d
0.0
c

control

2
WOB NP1
35.0 +
ABCDE
0.0
c
25.0
d
28.0
c
19.2
c
0.0
d
0.0
c

773 WG
119.6

3
WOB NP1
70.0 +
ABCDE
11.0
c
59.0
c
65.0
b
55.0
b
36.0
c
0.0
c

773 WG
239.2

4
WOB NP1
140.0 +
ABCDE
54.0
b
86.0
b
99.0
a
93.0
a
79.0
b
70.0
b

773 WG
478.4

5
WOB NP1
280.0 +
ABCDE
95.0
a
99.0
a
100
a
93.0
a
95.0
a
87.0
a

773 WG
956.8

6
Teldor 500
50
B
0.0
c
0.0
e
3.0
d
9.0
c
0.0
d
0.0
c

SC Switch
20 + 30
C

625 WG

P-value
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001

LSD (P ≤ 0.05)
11.45
tA
tA
tA
tA
tA

*WOB NP1 773 WG formulation containing sodium metabisulphite + sodium benzoate.

DAB = Days after budburst

Means followed by the same letter are not significantly different (p = 0.05, LSD).

tA = Original plot means are presented with analysis of variance and letters of separation from data transformed using y = Arcsine square root percent (x)

TABLE 53

Crop safety-leaf necrosis severity

Rate

Mean leaf necrosis severity

(g ai/
App.
(% leaf area damaged)

No.
Treatment
100 L)*
schedule
80DAB
93DAB
114DAB
156DAB
190DAB
204DAB

1
Untreated
Nil
Nil
0.0
c
0.0
e
0.0
e
0.0
d
0.0
c
0.0
c

control

2
WOB NP1
35.0 +
ABCDE
0.0
c
0.3
d
0.3
d
0.2
cd
0.0
c
0.0
c

773 WG
119.6

3
WOB NP1
70.0 +
ABCDE
0.1
c
0.8
c
0.8
c
0.7
c
0.7
c
0.0
c

773 WG
239.2

4
WOB NP1
140.0 +
ABCDE
0.6
b
1.4
b
2.0
b
2.6
b
5.3
b
1.6
b

773 WG
478.4

5
WOB NP1
280.0 +
ABCDE
1.6
a
3.3
a
4.4
a
6.8
a
10.9
a
4.4
a

773 WG
956.8

6
Teldor 500
50
B
0.0
c
0.0
e
0.0
e
0.1
d
0.0
c
0.0
c

SC Switch
20 + 30
C

625 WG

P-value
0.0001
0.0001
0.0001
0.001
0.0001
0.0001

LSD (P ≤ 0.05)
tA
tA
tL
tL
tL
tS

*WOB NP1 773 WG formulation containing sodium metabisulphite + sodium benzoate.

DAB = Days after budburst

Means followed by the same letter are not significantly different (p = 0.05, LSD).

tL = Original plot means are presented with analysis of variance and letters of separation from data transformed using y = Log (x + 1)

tS = Original plot means are presented with analysis of variance and letters of separation from data transformed using y = SQRT (x + 0.5)

tA = Original plot means are presented with analysis of variance and letters of separation from data transformed using y = Arcsine square root percent (x)

TABLE 54

Grey mould incidence and severity-Berries not quite ripe

Mean grey mould bunch

damage-Berries not quite

ripe 190DAB

Rate
Application
Incidence
Severity index

No.
Treatment
(g ai/100 L)*
schedule
(%)
(%)

1
Untreated control
Nil
Nil
0.0
0.0

2
WOB NP1773 WG
35.0 + 119.6
ABCDE
0.0
0.0

3
WOB NP1773 WG
70.0 + 239.2
ABCDE
0.0
0.0

4
WOB NP1773 WG
140.0 + 478.4
ABCDE
0.0
0.0

5
WOB NP1773 WG
280.0 + 956.8
ABCDE
0.0
0.0

6
Teldor 500 SC
50
B
0.0
0.0

Switch 625 WG
20 + 30
C

P-value
1.0000
1.0000

LSD (P ≤ 0.05)
NSD
NSD

*WOB NP1 773 WG formulation containing sodium metabisulphite + sodium benzoate.

DAB = Days after budburst

Damage severity index (%) = Σ (Frequency × damage rating) × 100/[total # (eg. 100) × max. rating (i.e. 10)]

NSD = No significant difference due to a p-value > 0.05

TABLE 55

Grey mould incidence and severity-Harvest ripe

Mean grey mould bunch

damage-Harvest ripe

204DAB

Rate
Application
Incidence
Severity index

No.
Treatment
(g ai/100 L)*
schedule
(%)
(%)

1
Untreated control
Nil
Nil
0.0
0.0

2
WOB NP1 773 WG
35.0 + 119.6
ABCDE
0.0
0.0

3
WOB NP1 773 WG
70.0 + 239.2
ABCDE
0.0
0.0

4
WOB NP1 773 WG
140.0 + 478.4
ABCDE
0.0
0.0

5
WOB NP1 773 WG
280.0 + 956.8
ABCDE
0.0
0.0

6
Teldor 500 SC
50
B
0.0
0.0

Switch 625 WG
20 + 30
C

P-value
1.0000
1.0000

LSD (P ≤ 0.05)
NSD
NSD

*WOB NP1 773 WG formulation containing sodium metabisulphite + sodium benzoate.

DAB = Days after budburst

Means followed by the same letter are not significantly different (p = 0.05, LSD)

Damage severity index (%) = Σ (Frequency × damage rating) × 100/[total # (eg. 100) × max .rating (i.e. 10)]

TABLE 56

Grey mould incidence and severity-Berries overripe

Mean grey mould bunch damage-

Berries overripe

Rate
Application
214DAB

No.
Treatment
(g ai/100 L)*
schedule
Incidence (%)
Severity index (%)

1
Untreated control
Nil
Nil
8.7
a
2.2
a

2
WOB NP1 773 WG
35.0 + 119.6
ABCDE
0.0
b
0.0
b

3
WOB NP1 773 WG
70.0 + 239.2
ABCDE
0.0
b
0.0
b

4
WOB NP1 773 WG
140.0 + 478.4
ABCDE
0.0
b
0.0
b

5
WOB NP1 773 WG
280.0 + 956.8
ABCDE
0.0
b
0.0
b

6
Teldor 500 SC
50
B
0.0
b
0.0
b

Switch 625 WG
20 + 30
C

P-value
0.0107
0.0205

LSD (P ≤ 0.05)
5.21
1.40

*WOB NP1 773 WG formulation containing sodium metabisulphite + sodium benzoate.

DAB = Days after budburst

EXAMPLE 12—GROWTH CONTROL OF PATHOGENS ON CHERRIES, CV. REGINA

WOB NP1 at 200, 400 and 800 g/100 L was applied in a five spray program commencing at early flowering for the control of bacterial spot (Xanthomonas campestris) and brown rot (Monilinia fructicola) and penicillin mould (Penicillium spp.) in cherries cv. Regina. These treatments were compared with an industry standard program including Bavistin 500 SC at 50 ml/100 L, Polyram 700 OF and Tilt 250 SC applied on three occasions during flowering only, an industry standard program followed by two applications of WOB NP1 at rates of 200, 400 or 800 g/100 L prior to harvest and an untreated control. All sprayed treatments were applied as dilute sprays to the point of run-off.

TABLE 57

Treatment protocol

No.
Treatment
Product
Application Timing

1
Untreated control
Nil
Nil

2
WOB NP1 (Full program)
200
g
10% flowering: −WOB NP1

3
WOB NP1 (Full program)
400
g
50% flowering: −WOB NP1

4
WOB NP1 (Full program)
800
g
Petal fall: −WOB NP1

1 & 5 days prior to harvest: −WOB NP1

5
Standard program:

Standard program + WOB NP1

Bavistin 500 SC 50 ml
50
mL
10% Flowering: −Tilt 250 EC + Polyram 700

Polyram 700 DF
150
g
OF

Tilt 250 EC
25
mL
50% Flowering: −Tilt 250 EC + Polyram 700

+WOB NP1
+200
g
DF

6
Bavistin 500 SC 50 ml
50
mL
Petal fail: −Bavistin 500 SC

Polyram 700 DF
150
g
+5 days and 1 day prior to harvest: −WOB NP1

Tilt 250 EC
25
mL

+WOB NP1
+400
g

7
Bavistin 500 SC 50 ml
50
mL

Polyram 700 DF
150
g

Tilt 250 EC
25
mL

+WOB NP1
+800
g

8
Bavistin 500 SC 50 ml
50
mL
Standard program

Polyram 700 DF
150
g
10% Flowering: −Tilt 250 EC + Polyram 700

Tilt 250 EC
25
mL
OF

50% Flowering: −Tilt 250 EC + Polyram 700

DF

Petal fall: −Bavistin 500 SC

TABLE 58

Chronology of Events

Days after application number

(DAA#) Days after harvest (DAH)
Crop Stage
Event

ODAA1
20% flowering
Treatment 1

3DAA1
50% flowering
Treatment 2

14DAA1 | 11 DAA2
Petal fall
Treatment 3

94DAA 1, 91 DAA2, 80DAA3
Colouring
Treatment 4

advanced

99DAA 1, 96DAA2 85DAA3,
Fruit mature
Treatment 5

5DAA4

1OODAA1, 97DAA2, 86DAA3,
Harvest
Harvest

6DAA4, 1 DAA5

Assessment

22DAH
Post harvest
Post harvest

assessment

TABLE 59

Mean percentage of healthy green fruit stalk and post harvest

penicillin mould infections twenty two days after harvest

(22DAH).

Mean %

cherries

infected

Mean %
with

Product
healthy

Penicillium

(mL or
green stalk
spp.

No.
Treatment
g/100 L)
(22DAH*)
(22DAH*)

1
Untreated control
Nil
21
4

2
WOB NP1
200
g
46
6

(Full program)

3
WOB NP1
400
g
39
6

(Full program)

4
WOB NP1
800
g
41
6

(Full program)

5
Standard program:

36
2

Bavistin 500 SC 50 ml
50
mL

Polyram 700 DF
150
g

Tilt 250 EC
25
mL

+WOB NP1
+200
g

6
Bavistin 500 SC 50 ml
50
mL
52
0

Polyram 700 DF
150
g

Tilt 250 EC
25
mL

+WOB NP1
+400
g

7
Bavistin 500 SC 50 ml
50
mL
43
2

Polyram 700 DF
150
g

Tilt 250 EC
25
mL

+WOB NP1
+800
g

8
Bavistin 500 SC 50 ml
50
mL
45
0

Polyram 700 DF
150
g

Tilt 250 EC
25
mL

*DAH-Days after harvest.

EXAMPLE 13—POST HARVEST TREATMENT FOR GROWTH CONTROL OF PATHOGENS ON CHERRIES, CV. REGINA

Fruit obtained from the studies discussed in Example 6 were also used to evaluate WOB NP1 at 400, 240 and 160 g/100 L when used as a post harvest treatment. The use of WOB NP1 as a post harvest wash was investigated using both WOB NP1 and the industry standard program as a pre-harvest wash, as discussed in Example 6.

TABLE 60

Post harvest treatment product information

Active
Concentration

Product
ingredient
of active

name
(ai)
ingredient
Formulation

WOB NP1
WOB NP1
500 g/kg
Wettable Powder

WOB NP2
WOB NP2
700 g/kg
Wettable Powder

WOB NP3
WOB NP3
250 g/kg
Wettable Powder

TABLE 61

Treatment protocol

No.
Treatment
Product
Application Timing

1
Untreated control
Nil
Nil

2
Untreated control + WOB
Nil
Untreated control

NP1 (Post Harvest Dip)
400
g
+Post Harvest Dip: −WOB NP1

3
WOB NP1
400
g
Full WOB NP1 program:

10% flowering: −WOB NP1

50% flowering: −WOB NP1

Petal fall: −WOB NP1

+1 & 5 days prior to harvest: −WOB NP1

4
WOB NP1
400
g
Full WOB NP1 program

+WOB NP1
+400
g
+1 & 5 days prior to harvest: −WOB NP1

(Post Harvest Dip)

+Post harvest dip: −WOB NP1

5
Standard program:

Grower Standard Program:

Bavistin 500 SC
50
mL
10% flowering: −Polyram 700 DF + Tilt 250

Polyram 700 DF
150
g
EC

Tilt 250 EC
25
mL
50% flowering: −Polyram 700 DF + Tilt 250

EC

Petal fall: −Bavistin 500 SC

6
Standard program:

Grower Standard program

Bavistin 500 SC 50 ml
50
mL
+5 days and 1 day prior to harvest: −WOB NP1

Polyram 700 DF
150
g

Tilt 250 EC
25
mL

+WOB NP1
+400
g

7
Standard program:

Grower Standard program

Bavistin 500 SC 50 ml
50
mL
+1 & 5 days prior to harvest: −WOB NP1

Polyram 700 DF
150
g
+Post harvest dip: −WOB NP1

Tilt 250 EC
25
mL

+WOB NP1
+400
g

+WOB NP1
+400
g

(Post Harvest Dip)

8
Standard program:

Grower Standard program

Bavistin 500 SC 50 ml
50
mL
+1 & 5 days prior to harvest: −WOB NP1

Polyram 700 DF
150
g
+Post harvest dip: −WOB NP1

Tilt 250 EC
25
mL

+WOB NP2
+120
g

+WOB NP1
+400
g

(Post Harvest Dip)

9
Untreated control + WOB
Nil
Untreated control

NP3 (Post Harvest Dip)
+160
g
+Post Harvest Dip: −WOB NP3

10
WOB NP1
400
g
Full WOB NP1 program:

+WOB NP3
+160
g
+1 & 5 days prior to harvest: −WOB NP1

(Post Harvest Dip)

+Post harvest dip: −WOB NP3

11
Standard program:

Grower Standard program

Bavistin 500 SC 50 ml
50
mL
+1 & 5 days prior to harvest: −WOB NP2

Polyram 700 DF
150
g
+Post harvest dip: −WOB NP3

Tilt 250 EC
25
mL

+WOB NP2
+120
g

+WOB NP3
+160
g

(Post Harvest Dip)

12
WOB NP1
+400
g
Full WOB NP1 program

+WOB NP2
+240
g
+1 & 5 days prior to harvest: −WOB NP1

(Post Harvest Dip)

+Post harvest dip: −WOB NP2

TABLE 62

Chronology of Events

Days after application number

(DAA#) Days after harvest (DAH)
Crop Stage
Event

ODAA1
20% flowering
Treatment 1

3DAA1
50% flowering
T reatment 2

14DAA1 | 11 DAA2
Petal fall
Treatment 3

94DAA 1, 91 DAA2_,80DAA3
Colouring advanced
Treatment 4

99DAA 1, 96DAA2 85DAA3, 5DAA4
Fruit mature
Treatment 5

1OODAA1, 97DAA2, 86DAA3,
Harvest
Harvest Assessment

6DAA4, 1 DAA5

7DAH
Post harvest
Photographs

16DAH
Post harvest
Photographs

22DAH
Post harvest
Post harvest assessment

TABLE 63

Mean percentage of healthy green fruit stalk and post harvest penicillin mould

infections twenty two days after harvest (22DAH)

Post Harvest dip

Mean %

Sprayed applications
application
Mean %
cherries

Product

Product
healthy
infected with

rate (g or

rate (g or
green stalk

Penicillium

No.
Treatments
mL/100 L
Treatments
mL/100 L
(22DAH*)
(22DAH*)

1
Untreated control
Nil
Nil
Nil
21
4

2
Untreated control
Nil
WOB NP1
400 g
52
2

3
WOB NP1
400 g
Nil
Nil
38.8
4

(full program)

4
WOB NP1
400 g
WOB NP1
400 g
60
2

(full program)

5
Grower Program:
50 mL
Nil
Nil
45
0

Bavistin 500 SC
150 g

Polyram 700 DF
25 mL

Tilt 250 EC

6
Grower Program:
50 mL
Nil
Nil
52
0

Bavistin 500 SC
150 g

Polyram 700 DF
25 mL +

Tilt 250 EC +
400 g

WOB NP1

7
Grower Program:
50 mL
WOB NP1
400 g
42
6

Bavistin 500 SC
150 g

Polyram 700 DF
25 mL +

Tilt 250 EC +
400 g

WOB NP1

8
Grower Program:
50 mL
WOB NP1
400 g
42
6

Bavistin 500 SC
150 g

Polyram 700 DF
25 mL +

Tilt 250 EC +
120 g

WOB NP2

9
Untreated control
Nil
WOB NP3
160
56
0

10
WOB NP1
400 g
WOB NP3
160
59
2

(full program)

11
Grower Program:
50 mL
WOB NP3
160
51
2

Bavistin 500 SC
150 g

Polyram 700 DF
25 mL +

Tilt 250 EC +
120 g

WOB NP2

12
WOB NP1
400 g
WOB NP2
240
48.4
2

(full program)

*DAH-Days after harvest.

EXAMPLE 14—EFFICACY OF WOB NP1 AND BCDMH ON APPLES AND PEARS

Studies performed to determine pathogen growth inhibition by WOB NP1, a formulation comprising the active ingredients sodium metabisulphite and sodium benzoate, and BCDMH a formulation comprising the active ingredient Bromochloro dimethyl hydantoin and a process where fruit where dipped with WOBNP1, BCDMH+WOBNP1+BCDMH.

Eight replicates of apples cv Jonagold and pears cv Beurre Bosc were used for each treatment. The fruit were contained in 36 litre plastic produce crates stacked on pallets in groups of 8.

The fruit had previously been washed and stored at 0° C. in air for approximately 4 months. Before the trial the fruit were wounded slightly by tipping once from one crate into another. Any fruit with rots or other disorders were removed at this time.

The fruit were inoculated with Penicillium expansum and a mixture of 4 strains of E. coli. Inoculation was achieved by dipping each crate of fruit in a 1001 tank of inoculum suspension. Separate tanks were used for apples and pears and the concentration of inoculum determined before and after dipping. The apple inoculum contained an average of 5.7×103 cfu/ml of P. expansum and 1.81×106 cfu/ml of E. coli. The Pear inoculum contained an average of 4.8×103 cfu/ml P. expansum and 2.09×106 cfu/ml of E. coli.

Fruit were then allowed to dry overnight at 0° C. Prior to treatment a sample of fruit was taken (unwashed control). Four apples or pears were selected from 4 different crates on each pallet and stored at 0° C. in sealed plastic bags.

Each batch of fruit was drenched for a contact time of 2 minutes then allowed to drain at room temperature for 2 hours before returning to storage at 0° C.

After drying overnight a sub-sample of 4 fruit was removed from each of 4 replicates of each treatment. These were stored in sealed plastic bags at 0° C. Microbiological testing was carried out the same day.

Microbiological testing was done on a bulked 25 g sample taken from 4 fruit for each replicate. Each 25 g sample was added to 250 ml of sterile 0.1% neutralized bacteriological peptone (pH 7.0-7.4) and stomached for 2 minutes. One ml of stomached samples was plated onto E. coli/coliform and Yeast and Mould Petrifilm plates (3M Microbiology Products) and incubated at 37° C. and 20° C. respectively before assessing, according to the manufacturer's instructions.

Following the drenching treatment and 24 hours drying pallets were stacked in groups of 2 and wrapped in plastic film to maintain high humidity. They were stored at 0° C. for approximately 3 months. Including the previous storage there was a total storage time of 7 months. Fruit were removed from cold storage on 9/10 (pears) and 12/10 (apples) and placed in a 21° C. room for 3 days (pears) or 3.5 days (apples) to allow rots to develop before assessing. Fruit were assessed visually and scored for the occurrence of Penicillium rots and “other” rots.

TABLE 64

SUMMARY MICROBIOLOGICAL PRODUCE TESTS

Yeast and
Faecal

Mould
Conforms

E.coli

Sample
(CFU/g)
(CFU/g) *
(CFU/g) *

Apples-Unwashed
9872
0
784

Apples-Water
8297
0
176

Apples-WOB NP1
5819
0
145

Apples-BCDMH
4593
0
162

Apples-BCDMH + WOB
3363
0
23

NP1

Pears-Unwashed
1880
0
31

Pears-Water
1626
0
19

Pears-WOB NP1
113
0
0

Pears-BCDMH
302
0
0

Pears-BCDMH WOB
21
0
0

NP1

* Average of 4 replicates

TABLE 65

SUMMARY OF POST-STORAGE ROT ASSESSMENTS

Penicillium

Other rots
Total rots

(Average %
(Average %
(Average %

Sample
Incidence) *
Incidence) *
Incidence) *

Apples-Water
30.8
2.3
33.1

Apples-WOB NP1
18.6
2.4
21.0

Apples-BCDMH
24.3
1.6
25.8

Apples-BCDMH +
18.2
2.2
20.4

WOB NP1

Pears-Water
25.8
15.8
41.6

Pears-WOB NP1
14.9
10.1
25.0

Pears-BCDMH
19.0
16.2
35.2

Pears-BCDMH +
15.7
12.4
28.1

WOB NP1

* Average of 8 replicates

Results

Results were analyzed by Analysis of Variance using GenStat for Windows 11th Edition (Lawes Agricultural Trust, IACR-Rothamsted) and significance determined using LSDs at the 5% level.

Microbiological Tests

Pears

For pears WOB NP1 (formulation comprising sodium metabisulphite and sodium benzoate, WOB NP1) and BCDMH (formulation comprising the active ingredient BromoChloroDimethylHydantoin)+WOB NP1 significantly reduced the level of contamination by fungi compared to the unwashed sample while BCDMH and water did not (FIG. 9). There were no significant differences in the levels of fungi between WOB NP1 and BCDMH+WOB NP1, or between BCDMH and water (FIG. 9).

Three treatments (WOB NP1, BCDMH and BCDMH+WOB NP1) reduced the levels of E. coli on pears to zero. There was no significant difference between water and unwashed (FIG. 10).

Apples

For apples only the BCDMH+WOB NP1 treatment significantly reduced the level of contamination by fungi compared to the unwashed sample (FIG. 11). There were no significant differences in the levels of fungi or E. coli between any of the treatments (FIGS. 11 and 12). Bozul, BCDMH+WOB NP1 or water significantly reduced the level of contamination with E. coli compared to the unwashed treatment (FIG. 12).

Post Storage Rot Assessments

Pears

For pears, all sanitizer treatments were significantly better than water in reducing Penicillium rots. For “other” rots only WOB NP1 was significantly better than water, while for “total” rots only WOB NP1 or WOB NP1 plus BCDMH were better (FIG. 13).

Apples

WOB NP1 and WOB NP1+BCDMH were significantly better at reducing Penicillium rots and “total” rots on apples than washing with just water, while BCDMH was not significantly different to water. Other rots were at very low incidences in all treatments (FIG. 14).

EXAMPLE 15—RESIDUE STUDY

This study was conducted to determine the presence and persistence of sulfur dioxide and benzoic acid residues in wine grapes and processed commodities (wine, juice and pomace) following six applications of WOB NP1 (prepared according to the method of Example 1 and 2).

The wine grapes to be treated as treatment 2 received six applications of WOB NP1 at a nominal rate of 212.8 g a.i./100 L sodium metabisulphite (equivalent to 140 g a.i./100 L sulfur dioxide) and 478.4 g a.i./100 L sodium benzoate; the actual application rates were 230.4 g a.i./100 L sodium metabisulphite (equivalent to 155.3 g a.i./100 L sulfur dioxide) and 513.6 g a.i./100 L sodium benzoate.

The wine grapes to be treated as treatment 3 received six applications of WOB NP1 at a nominal rate of 425.6 g a.i./100 L sodium metabisulphite (equivalent to 280 g a.i./100 L sulfur dioxide) and 956.8 g a.i./100 L sodium benzoate; the actual application rates were 460.8 g a.i./100 L sodium metabisulphite (equivalent to 310.6 g a.i./100 L sulfur dioxide) and 1027.2 g a.i./100 L sodium benzoate.

TABLE 66

Treatment table.

Rate of Test

Treatment

Item
Rate of Active

Number
Test item
(g/100 L)
(g a.i./100 L)
Application Timing

T1
Untreated
Nil
Nil
N/A

Control

T2
WOB NP1
800
212.8 (140) Sodium
A
B
C
D
E
F

Metabisulphite¹+

478.4 Sodium Benzoate

T3
WOB NP1
1600
425.6 (280) Sodium
A
B
C
D
E
F

Metabisulphite¹+

956.8 Sodium Benzoate

N/A = Not applicable

Note

¹Nominal and actual rates of active are sodium metabisulphite with results in brackets indicating the equivalent of sulfur dioxide.

Application A: 5% capfall;

Application B: 80% capfall

Application C: pre bunch closure

Application D: pre bunch closure to veraison

Application E: Veraison

Application F: 2 days before commercial harvest

TABLE 67

Test site 1 (Tasmania)

Formulated

Rates of Test
Nominal
Actual Rates of Active

Test
Active
Substance
Rates of Active
(g a.i./100 L)

Trt.
Substance
Ingredient
(g/100 L)
(g a.i./100 L)
A
B
C
D
E
F

T1
Untreated
Nil
Nil
Nil
—
—
—
—
—
—

Control

T2
WOB NP1
Sodium
800
212.8
230.4
230.4
230.4
230.4
230.4
230.4

Metabisulphite

(140)²
(155.3)
(155.3)
(155.3)
(155.3)
(155.3)
(155.3)

Sodium Benzoate

478.4
513.6
513.6
513.6
513.6
513.6
513.6

T3
WOB NP1
Sodium
1600
425.6
460.8
460.8
460.8
460.8
460.8
460.8

Metabisulphite

(280)³
(310.6)
(310.6)
(310.6)
(310.6)
(310.6)
(310.6)

Sodium Benzoale

956.8
1027.2
1027.2
1027.2
1027.2
1027.2
1027.2

Note

¹Rates are corrected for the concentration show on the Certificate of Analysis.

Note

²Nominal and actual rates of active are sodium metabisulphite with results in brackets indicating the equivalent of sulfur dioxide.

Comment-Actual rates applied were within 10.9% of the nominated rates.

TABLE 68

Test site 2 (Western Australia)

Formulated

Rates of Test
Nominal
Actual Rates of Active

Test
Active
Substance
Rates of Active
(g a.i./100 L)

Trt.
Substance
Ingredient
(g/100 L)
(g a.i./100 L)
A
B
C
D
E
F

T1
Untreated
Nil
Nil
Nil
—
—
—
—
—
—

Control

T2
WOB NP1
Sodium
800
212.8
230.4
230.4
230.4
230.4
230.4
230.4

Metabisulphite

(140)²
(155.3)
(155.3)
(155.2)
(155.3)
(155.3)
(155.3)

Sodium Benzoate

478.4
513.6
513.6
513.6
513.6
513.6
513.6

T3
WOB NP1
Sodium
1600
425.6
460.8
460.8
460.8
460.8
460.8
460.8

Metabisulphite

(280)²
(310.6)
(310.6)
(310.6)
(310.6)
(310.6)
(310 6)

Sodium Benzoate

956.8
1027.2
1027.2
1027.2
1027.2
1027.2
1027.2

Note

¹Rates are corrected for the concentration shown on the Certificate of Analysis.

Note

²Nominal and actual rates of active are sodium metabisulphite with results in brackets indicating the equivalent of sulfur dioxide.

Comment-Actual rates applied were within 10.9% of the nominated rates.

A minimum of 1 kg of grape bunches were sampled for residue samples from the treated plots at 0, 1, 2 and 3 days after last application (DALA). 2 DALA coincided with normal commercial harvest (NCH). Samples from the untreated control were collected at 2 DALA (NCH) to coincide with sampling from the treated plots.

A minimum of 5 kg of grape bunches were sampled for processing samples from the treated plots at 0, 1, 2 and 3 days after last application (DALA). 2 DALA coincided with normal commercial harvest (NCH). Samples from the untreated control were collected at 2 DALA (NCH) to coincide with sampling from the treated plots. These were for processing into wine, juice and pomace.

The analytical phase of the study was conducted by The Australian Wine Research Institute (AWRI) at their Urrbrae, South Australia facilities. Frozen samples of grapes were processed in accordance with AWRI SOP6—Preparation of fresh, frozen and dried fruit and vegetables and plant materials, and Vinification of fresh and frozen grapes. Samples of juice, wine and pomace were stored frozen prior to analysis or analysed within 14 days of generation. Samples were prepared and analysed as outlined below.

Grape study samples were analysed as whole commodity without caps and stems. Samples were partially defrosted and prepared as per AWRI SOP6—Preparation of fresh, frozen and dried fruit and vegetables and plant material. Approximately 500 g of berries were subsampled from all bunches in the sample and added to a Retsch Grindmix and homogenised for twenty seconds. Processing study samples were subsampled to generate an approximately 1 kg and 800 g subsamples of grapes for juicing and/or vinification respectively.

Vinification subsamples were thawed overnight then manually crushed and the must added to a 1 L glass fermentation vessel to which approximately 50 mg/L sulfur dioxide, as potassium metabisulphite, and 200 mg/L diammonium phosphate solution was added. The must was then inoculated with rehydrated active dried wine yeast, AWRI 796, and fermented on skins at 25° C., with daily mixing of the skin and liquid. After 7 days, the ferment was pressed twice, each time at approx. 19 Nm for 2 minutes, with mixing of the marc between pressings.

The wine was returned to the original vessel and allowed to ferment to dryness (<1 g/L residual sugar) at 25° C. Once fermentation was established as complete using CInitest strips and the wine were racked from the gross lees and a 200 mL subsample taken and stored at approx. 4° C. prior to analysis. The wine study samples were centrifuged prior to analysis to improve clarification.

Juice and pomace samples were generated by thawing the samples overnight then pressing the grapes at 19 Nm for two minutes, missing and repeating the processing. Juicing samples were taken. The pomace samples were taken for analysis and moisture content determination.

Pomace was subsampled and added to a Retsch Grindomix and homogenised for twenty (20) seconds or until the sample was considered homogenous. A subsample of homogenate was taken for analysis and a further 250 g taken as a backup.

Juice and wine study sample were analysed with no further preparation.

Analytical Method—Benzoic Acid

The analytical procedure used for determination of benzoic acid in the wine, juice and pomace study samples was performed using liquid chromatography with tandem mass spectrometry (LC/MS/MS). For grape and juice samples, a 15 g subsample of a sample homogenate was weighed into a 50 mL centrifuge and 0.05 mL of surrogate standard solution (12.5 μg/mL d5-atrazine) added. 15 mL of acetonitrile (1% acetic acid) was added and the tube shaken for approx. 2 minutes then cooled in a laboratory freezer for 15 minutes. Magnesium sulphate (6 g) and sodium acetate (1.5 g) was added with 2 glass beads and the sample shaken for a further 1 minute.

The extract was centrifuged and a 6 mL aliquot of supernatant was taken and added to a 15 mL dispersive solid-phase extraction (dSPE) tube containing 400 mg primary-secondary amine and 1200 mg magnesium sulphate. The sample tube was shaken for 1 minute then centrifuged.

A 0.2 mL aliquot of the supernatant was added to a 2 mL amber vial and diluted with 0.8 mL 25% methanol/0.005% formic acid/0.01% EDTA solution and mixed. The final extract was then analysed using an Agilent 1290 liquid chromatography (LC) with a 6460A tandem mass spectrometer (MS/MS).

For pomace samples, 3 g sample was taken and rehydrated with 12 mL of MilliQ water prior to extraction as above, except the dSPE tube contained 400 mg primary-secondary amine, 400 mg C18 and 1200 mg magnesium sulphate.

For wine samples a 15 mL aliquot of wine was taken and the procedure as outlined for grape study samples followed with the exception that a 1 mL aliquot was taken from the centrifuged dSPE tube and evaporated to dryness in a TurboVap then reconstituted using 0.1 mL methanol, vortexed and 0.1 mL 25% methanol/0.005% formic acid/0.01% EDTA solution. The final extract was added to a 2 mL amber vial containing a 0.3 mL insert then analysed using an Agilent 1290 liquid chromatograph (LC) with a 6460A tandem mass spectrometer (MS/MS).

Analytical Methods—Sulfur Dioxide

The free sulfur determination is based on the reaction between free sulfur in an acidic medium with a mixture of pararosanline and formaldehyde to give a pink colour which is measured at 575 nm. The method requires two tests to be analysed concurrently, one with pyruvic acid (FSO2A) and one without (FSO2B). A third method (FSO2C) is sued to determine the solpe (m). The free SO2 is calculated by the following formula:

FSO2=m(FSO2A−FSO2B)−Blank

The total sulfur determination is performed by diluting with pH 8 buffer, stabilizing, then taking a zero measurement. DTNB reagent is then added, which reacts with a free sulfhydryl group to yield a mixed disulphide and 2-nitro-5-thiobenzoic acid product. This yellow product is measured at 412 nm.

All samples, both wine and juice (including grape and pomace as juice), were centrifuged at 3500 rpm for 5 minutes prior to analysis, and were analysed as close to room temperature as possible. Samples volume of 7 mL of each sample was sued for analysis.

Tabulated below is a summary of residue results applicable for the harvest interval range for wine grapes treated with the formulation under test. Results are reported in mg/kg, or less than the limit of quantification (<LoQ) or limit of detection (<LoD) as appropriate.

Benzoic acid results for ‘dry weight’ are based on a calculation using residue results from the ‘wet weight’ then adjusted for the moisture content of the sample. Benzoic acid results reported as <LoD and <LoQ for ‘dry weight’ are based entirely on the calculated ‘wet weight’ result.

TABLE 69

The residual benzoic acid and sulfur dioxide remaining in grapes at study site 1

Test

Rate of
sample

Benzoic

Sample

Treatment

Test Item
timing
AWRI
Total SO₂
Acid

Site
Type
Specimen sample code
number
Test Item
(g/100 L)
(DALA¹)
Sample ID
(mg/L)
(mg/kg)

1
Grapes
WOB17483-FB001-FB
T1
Untreated
Nil
2
AE51697
<3
<LoD

control

WOB17483-FB002-FB
T2
WOB NP1
800
0
AE51698
<3
3.507

WOB17483-FB003-FB
T2
WOB NP1
800
1
AE51699
<3
4.471

WOB17483-FB004-FB
T2
WOB NP1
800
2
AE51700
<3
1.090

WOB17483-FB005-FB
T2
WOB NP1
800
3
AE51701
<3
1.351

WOB17483-FB006-FB
T3
WOB NP1
1600
0
AE51702
<3
9.670

WOB17483-FB006-FB
T3
WOB NP1
1600
0
AE51702D
<3
10.476

WOB17483-FB007-FB
T3
WOB NP1
1600
1
AE51703
<3
9.992

WOB17483-FB008-FB
T3
WOB NP1
1600
2
AE51704
<3
5.289

WOB17483-FB009-FB
T3
WOB NP1
1600
3
AE51705
<3
4.456

¹DALA days after last application

*D denotes duplicate

LoD: limit of detection (0.100 mg/kg)

LoQ: limit of quantitation (0.200 mg/kg)

TABLE 70

The residual benzoic acid and sulfur dioxide remaining in grapes at study site 2

Test

Rate of
sample

Benzoic

Sample

Treatment

Test Item
timing
AWRI
Total SO₂
Acid

Site
Type
Specimen sample code
number
Test Item
(g/100 L)
(DALA¹)
Sample ID
(mg/L)
(mg/kg)

2
Grapes
WOB17483-FB010-FB
T1
Untreated
Nil
2
AE51715
<3
<LoD

control

WOB17483-FB011-FB
T2
WOB NP1
800
0
AE51716
4
3.078

WOB17483-FB012-FB
T2
WOB NP1
800
1
AE51717
4
2.150

WOB17483-FB013-FB
T2
WOB NP1
800
2
AE51718
3
1.265

WOB17483-FB014-FB
T2
WOB NP1
800
3
AE51719
3
1.000

WOB17483-FB015-FB
T3
WOB NP1
1600
0
AE51720
3
10.908

WOB17483-FB015-FB
T3
WOB NP1
1600
0
AE51720D
3
10.911

WOB17483-FB016-FB
T3
WOB NP1
1600
1
AE51721
<3
7.303

WOB17483-FB017-FB
T3
WOB NP1
1600
2
AE51722
<3
5.088

WOB17483-FB018-FB
T3
WOB NP1
1600
3
AE51723
<3
4.493

¹DALA days after last application

*D denotes duplicate

LoD: limit of detection (0.100 mg/kg)

LoQ: limit of quantitation (0.200 mg/kg)

TABLE 71

The residual benzoic acid and sulfur dioxide remaining in grapes at study site 3

Test

Rate of
sample

Benzoic

Sample

Treatment

Test Item
timing
AWRI
Total SO₂
Acid

Site
Type
Specimen sample code
number
Test Item
(g/100 L)
(DALA¹)
Sample ID
(mg/L)
(mg/kg)

3
Grapes
WOB17483-FB001-JF
T1
Untreated
Nil
2
AE51735
<3
<LoD

control

WOB17483-FB002-JF
T2
WOB NP1
800
0
AE51738
<3
4.383

WOB17483-FB003-JF
T2
WOB NP1
800
1
AE51741
<3
6.330

WOB17483-FB004-JF
T2
WOB NP1
800
2
AE51744
<3
3.668

WOB17483-FB005-JF
T2
WOB NP1
800
3
AE51747
<3
1.110

WOB17483-FB006-JF
T3
WOB NP1
1600
0
AE51750
<3
14.332

WOB17483-FB006-JF
T3
WOB NP1
1600
0
AE51750D
<3
14.569

WOB17483-FB007-JF
T3
WOB NP1
1600
1
AE51753
<3
11.346

WOB17483-FB008-JF
T3
WOB NP1
1600
2
AE51756
<3
7.609

WOB17483-FB009-JF
T3
WOB NP1
1600
3
AE51759
<3
5.556

¹DALA days after last application

*D denotes duplicate

LoD: limit of detection (0.100 mg/kg)

LoQ: limit of quantitation (0.200 mg/kg)

TABLE 72

The residual benzoic acid and sulfur dioxide remaining in wine at study site 2

Test
Wine

Rate of
sample

Benzoic

Treatment

Test Item
timing
AWRI
Total SO₂
Acid

Site
Specimen sample code
number
Test Item
(g/100 L)
(DALA¹)
Sample ID
(mg/L)
(mg/L)

2
WOB17483-FB010-JF
T1
Untreated
Nil
2
AE51762
<3
<LoD

control

WOB17483-FB011-JF
T2
WOB NP1
800
0
AE51765
4
4.965

WOB17483-FB012-JF
T2
WOB NP1
800
1
AE51768
7
7.191

WOB17483-FB013-JF
T2
WOB NP1
800
2
AE51771
5
4.187

WOB17483-FB014-JF
T2
WOB NP1
800
3
AE51774
4
2.921

WOB17483-FB015-JF
T3
WOB NP1
1600
0
AE51777
4
13.797

WOB17483-FB015-JF
T3
WOB NP1
1600
0
AE51777D
4
13.946

WOB17483-FB016-JF
T3
WOB NP1
1600
1
AE51780
<3
12.629

WOB17483-FB017-JF
T3
WOB NP1
1600
2
AE51783
4
11.357

WOB17483-FB018-JF
T3
WOB NP1
1600
3
AE51786
3
8.498

¹DALA days after last application

*D denotes duplicate

LoD: limit of detection (0.100 mg/kg)

LoQ: limit of quantitation (0.200 mg/kg)

TABLE 73

The residual benzoic acid and sulfur dioxide remaining in juice at study site 1

Test
Juice

Rate of
sample

Benzoic

Treatment

Test Item
timing
AWRI
Total SO₂
Acid

Site
Specimen sample code
number
Test Item
(g/100 L)
(DALA¹)
Sample ID
(mg/L)
(mg/L)

1
WOB17483-FB001-JF
T1
Untreated
Nil
2
AE51733
<3
<LOD

control

WOB17483-FB002-JF
T2
WOB NP1
800
0
AE51736
<3
2.879

WOB17483-FB003-JF
T2
WOB NP1
800
1
AE51739
<3
2.617

WOB17483-FB004-JF
T2
WOB NP1
800
2
AE51742
<3
1.235

WOB17483-FB005-JF
T2
WOB NP1
800
3
AE51745
<3
0.881

WOB17483-FB006-JF
T3
WOB NP1
1600
0
AE51748
<3
11.109

WOB17483-FB006-JF
T3
WOB NP1
1600
0
AE51748D
<3
11.065

WOB17483-FB007-JF
T3
WOB NP1
1600
1
AE51751
<3
9.196

WOB17483-FB008-JF
T3
WOB NP1
1600
2
AE51754
<3
4.949

WOB17483-FB009-JF
T3
WOB NP1
1600
3
AE51757
<3
4.972

¹DALA days after last application

*D denotes duplicate

LoD: limit of detection (0.100 mg/kg)

LoQ: limit of quantitation (0.200 mg/kg)

TABLE 74

The residual benzoic acid and sulfur dioxide remaining in juice at study site 2

Test
Juice

Rate of
sample

Benzoic

Treatment

Test Item
timing
AWRI
Total SO₂
Acid

Site
Specimen sample code
number
Test Item
(g/100 L)
(DALA¹)
Sample ID
(mg/L)
(mg/L)

2
WOB17483-FB010-JF
T1
Untreated
Nil
2
AE51760
<3
<LOD

control

WOB17483-FB011-JF
T2
WOB NP1
800
0
AE51763
<3
8.083

WOB17483-FB012-JF
T2
WOB NP1
800
1
AE51766
<3
9.928

WOB17483-FB013-JF
T2
WOB NP1
800
2
AE51769
<3
4.938

WOB17483-FB014-JF
T2
WOB NP1
800
3
AE51772
<3
32.42

WOB17483-FB015-JF
T3
WOB NP1
1600
0
AE51775
<3
21.944

WOB17483-FB015-JF
T3
WOB NP1
1600
0
AE51775D
<3
21.922

WOB17483-FB016-JF
T3
WOB NP1
1600
1
AE51778
<3
15.432

WOB17483-FB017-JF
T3
WOB NP1
1600
2
AE51781
<3
15.621

WOB17483-FB018-JF
T3
WOB NP1
1600
3
AE51764
<3
12.499

¹DALA days after last application

*D denotes duplicate

LoD: limit of detection (0.100 mg/kg)

LoQ: limit of quantitation (0.200 mg/kg)

TABLE 75

The residual benzoic acid and sulfur dioxide remaining in pomace at study site 1

Pomace

Benzoic

Rate of
Test

acid
Benzoic

Test
sample

Total
Moisture
‘wet
Acid ‘dry

Specimen
Treatment

Item
timing
AWRI
SO₂
content
weight’
weight’

Site
sample code
number
Test Item
(g/100 L)
(DALA¹)
Sample ID
(mg/L)
(%)
(mg/kg)
(mg/kg)

Site 1
WOB17483-FB001-
T1
Untreated
Nil
2
AE51734
<3
68.33
<LoD
<LoD

JF

control

WOB17483-FB002-
T2
WOB NP1
800
0
AE51737
<3
67.79
2.553
7.924

JF

WOB17483-FB003-
T2
WOB NP1
800
1
AE51740
<3
68.59
1.891
8.019

JF

WOB17483-FB004-
T2
WOB NP1
800
2
AE51743
<3
67.7
1.368
4.234

JF

WOB17483-FB005-
T2
WOB NP1
800
3
AE51746
<3
68.13
0.788
2.474

JF

WOB17483-FB006-
T3
WOB NP1
1600
0
AE51749
<3
69.80
13.821
45.770

JF

WOB17483-FB006-
T3
WOB NP1
1600
0
AE51749D
<3
69.80
13.502
44.713

JF

WOB17483-FB007-
T3
WOB NP1
1600
1
AE51752
<3
69.68
10.304
33.981

JF

WOB17483-FB008-
T3
WOB NP1
1600
2
AE51755
<3
67.54
5.056
15.579

JF

WOB17483-FB009-
T3
WOB NP1
1600
3
AE51758
<3
67.79
4.693
14.588

JF

¹DALA days after last application

*D denotes duplicate

LoD: limit of detection (0.100 mg/kg)

LoQ: limit of quantitation (0.200 mg/kg)

TABLE 76

The residual benzoic acid and sulfur dioxide remaining in pomace at study site 1

Pomace

Benzoic

Rate of
Test

acid
Benzoic

Test
sample

Total
Moisture
‘wet
Acid ‘dry

Specimen sample
Treatment

Item
timing
AWRI
SO₂
content
weight’
weight’

Site
code
number
Test Item
(g/100 L)
(DALA¹)
Sample ID
(mg/L)
(%)
(mg/kg)
(mg/kg)

Site 2
WOB17483-FB010-
T1
Untreated
Nil
2
AE51761
4
65.43
<LoD
<LoD

JF

control

WOB17483-FB011-
T2
WOB NP1
800
0
AE51764
5
62.98
7.186
19.410

JF

WOB17483-FB012-
T2
WOB NP1
800
1
AE51767
5
61.96
9.992
26.269

JF

WOB17483-FB013-
T2
WOB NP1
800
2
AE51770
4
60.22
4.795
12.052

JF

WOB17483-FB014-
T2
WOB NP1
800
3
AE51773
4
60.72
2.932
7.466

JF

WOB17483-FB015-
T3
WOB NP1
1600
0
AE51776
4
62.91
24.133
65.074

JF

WOB17483-FB015-
T3
WOB NP1
1600
0
AE51776D
4
62.91
24.437
65.892

JF

WOB17483-FB016-
T3
WOB NP1
1600
1
AE51779
4
63.94
15.31
42.461

JF

WOB17483-FB017-
T3
WOB NP1
1600
2
AE51782
3
62.29
17.683
46.894

JF

WOB17483-FB018-
T3
WOB NP1
1600
3
AE51785
<3
65.59
4.887
14.021

JF

¹DALA days after last application

*D denotes duplicate

LoD: limit of detection (0.100 mg/kg)

LoQ: limit of quantitation (0.200 mg/kg)

Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

A Method for Treatment of Crops

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