A Method for Treatment of Crops
20220361504 · 2022-11-17
Assignee
Inventors
Cpc classification
A01N59/00
HUMAN NECESSITIES
A01N37/10
HUMAN NECESSITIES
A01P1/00
HUMAN NECESSITIES
A01N59/00
HUMAN NECESSITIES
A01N37/10
HUMAN NECESSITIES
International classification
A01N37/10
HUMAN NECESSITIES
A01P1/00
HUMAN NECESSITIES
Abstract
The present invention provides a method for treating crops in field or in a processing facility 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 formulation to a crop, wherein said treatment is for prevention or reduction of crop damage by plant pathogens, or reduction of bacterial, fungal or human pathogens.
Claims
1-66. (canceled)
67. 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 formulation to a crop, wherein said treatment is for prevention of crops damage by plant pathogens or to reduce bacterial, fungal or human pathogens on said crop.
68. A method according to claim 67, wherein the dry composition comprises a metabisulphite and benzoate salt blended at a ratio of approximately between 20:80 and 30:70 w/w.
69. A method according to claim 67, wherein the metabisulphite is selected from sodium metabisulphite and potassium metabisulphite and wherein the benzoate salt is selected from sodium benzoate and potassium benzoate; and wherein the benzoate salt and/or metabisulphite is optionally in the form of a powder.
70. A method according to claim 67, wherein the cellulose additive is present at approximately between 0.5% to 3% by weight of the dry composition, preferably at approximately between 0.8% to 2% by weight of the dry composition, more preferably at approximately between 1.0% to 1.5% by weight of the dry composition; and wherein the cellulose additive optionally has a particle size between approximately 20 μm to 2500 μm.
71. A method according to claim 67, wherein the formulation comprises a dry composition being further blended with a surfactant.
72. A method according to claim 71, wherein the formulation comprises a dry composition and a surfactant blended such that the surfactant is present at approximately between 0.5% to 10% w/w of the final formulation, preferably at approximately between 0.8% to 8% w/w of the final formulation, more preferably at approximately between 1.0 to 6% w/w of the final formulation.
73. A method according to claim 71, wherein the surfactant is a non-ionic surfactant selected from the group consisting of polyethylene glycol, polyethylene oxide, dipropylene glycol and polysorbate 80.
74. A method according to claim 67, wherein the formulation is diluted to produce a solution, preferably wherein the formulation is diluted with an aqueous mixture to produce the solution, more preferably wherein the formulation is diluted with water to produce the solution; and wherein the applied solution preferably has a concentration of approximately between 1 g/L to 8 g/L, more preferably a concentration of approximately between 2 g/L to 6.5 g/L, more preferably a concentration of approximately between 3.5 g/L to 4.5 g/L, more preferably a concentration of approximately between 3.75 g/L to 4.25 g/L.
75. A method according to claim 74, wherein the solution has a pH of between approximately 2.0 and 7.5, preferably between approximately 3.0 and 6.5, more preferably between approximately 4.0 and 6.0.
76. A method according to claim 74, wherein the solution is applied to the crop as either a pre-harvest spray or a post-harvest wash.
77. A method according to claim 67, wherein the crop treated is selected from fruits, vegetables, grains, grasses and seeds, preferably wherein the crop to be treated is selected from berries, stone fruits, citrus fruits, tropical fruits, melons, drupes, pomes or any other edible fruit, more preferably wherein the crop to be treated is selected from apples, pears, cherries or grapes, more preferably wherein the crop treated is a grape selected from the species 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, more preferably wherein the crop treated includes a cultivar or hybrid species.
78. A method according to claim 77, wherein the formulation is further applied upon expression of botrytis and at any combination of the following stages of grape maturation: approximately 10% flower crop; approximately 30% cap fall; approximately end of flowering; approximately berry size approximately 4 mm; approximately bunch closure; and approximately veraison; or wherein the formulation is applied to the 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; and Two weeks to harvest.
79. A method according to claim 67, wherein the formulation is applied at no later than 3 days prior to harvest, preferably wherein the crop is further treated with a solution of the composition post harvest, preferably wherein the post harvest treatment solution has a concentration approximately between 1 g/L and 8 g/L.
80. A method according to claim 67, wherein the crop is treated with a solution of the composition post harvest, preferably wherein the post harvest treatment solution has a concentration approximately between 1 g/L and 8 g/L.
81. A method according to claim 67, wherein the applied formulation results in reducing growth of crop pathogens selected from the group consisting of Botrytis cinerea, Xanthomonas spp, E. coli, Monilina fructicola, Penicillium spp. and Erwinia carotovora.
82. A method according to claim 81, wherein the applied formulation results in approximately between 10% to 30% reduction in Botrytis cinerea growth compared to an untreated crop, preferably wherein the applied formulation results in approximately between 15 to 25% reduction in Botrytis cinerea growth compared to an untreated crop; or wherein the applied formulation results in approximately greater than 50% reduction in Xanthomonas spp growth compared to an untreated crop, preferably wherein the applied formulation results in approximately greater than 75% reduction in Xanthomonas spp growth compared to an untreated crop, more preferably wherein the applied formulation results in approximately greater than 90% reduction in Xanthomonas spp growth compared to an untreated crop; or wherein the applied formulation results in approximately greater than 60% reduction in growth of E. coli compared to an untreated crop, preferably wherein the applied formulation results in approximately greater than 70% reduction in growth of E. coli compared to an untreated crop, more preferably wherein the applied formulation results in approximately greater than 80% reduction in growth of E. coli compared to an untreated crop.
83. A method according to claim 67, wherein the applied formulation results in substantially no effect on the growth rate of Saccharomyces cerevisae and/or Schizosaccharomyces pombe species.
84. 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 formulation to the crop, and applying a fungicide 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.
85. A method according to claim 84, wherein the fungicide contains a halogen based active ingredient, preferably wherein the halogen based fungicide includes an active ingredient selected from 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 any suitable combination thereof.
86. A method according to claim 84, wherein the formulation is diluted to produce a solution, preferably wherein the formulation is diluted with an aqueous mixture to produce the solution, more preferably wherein the formulation is diluted with water to produce the solution; and wherein the solution preferably has a concentration of approximately between 1 g/L to 8 g/L.
87. A method according to claim 84, wherein the method results in reducing growth of crop pathogens selected from the group consisting of Botrytis cinerea, Xanthomonas spp, E. coli, Monilina fructicola and Penicillium spp.
88. A method according to claim 84, wherein the crop is treated with both the formulation and fungicide pre harvest, preferably wherein the crop is further treated with the formulation post harvest; alternatively wherein the crop is treated with both the formulation and fungicide post harvest, preferably wherein the crop is further treated with the formulation pre harvest.
89. A method according to claim 84, wherein the applied fungicide contains a halogen based active ingredient at a concentration of between 1 to 100 ppm.
90. A method according to claim 84, wherein the fungicide is applied sequentially before the formulation.
Description
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
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EXAMPLE 1—PREPARATION OF THE DRY FORMULATION
[0113] 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
[0114] 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.
[0115] 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’
[0116] Preparation of Products
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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%.
[0121] 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-US-00001 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-US-00002 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)
[0122] Preparation of Test Media
[0123] 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.
[0124] 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.
[0125] Curative Activity:
[0126] 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.
[0127] 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.
[0128] The plates were incubated at ambient temperature (14-25° C.). Inhibition zones were measured at 24 hours, 48 hours and 7 days.
[0129] Preventative Activity:
[0130] 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.
[0131] 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-US-00003 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-US-00004 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-US-00005 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-US-00006 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-US-00007 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-US-00008 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-US-00009 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-US-00010 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-US-00011 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-US-00012 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-US-00013 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-US-00014 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.
[0132] 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)
[0133] 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.
[0134] 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-US-00015 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-US-00016 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-US-00017 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-US-00018 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-US-00019 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-US-00020 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)
[0135] 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.
[0136] 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-US-00021 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-US-00022 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-US-00023 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-US-00024 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-US-00025 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-US-00026 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-US-00027 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-US-00028 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-US-00029 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-US-00030 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-US-00031 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-US-00032 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)
[0137] 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.
[0138] The effect of WOB NP1 on two wild type yeasts, Saccharomyces cerevisae and Schizosaccharomyces pombe were also further investigated.
[0139] The organisms were transferred from culture collection mother cultures to fresh media and checked for purity.
[0140] Preparation of Test Medium
[0141] 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.
[0142] Preparation of Products
[0143] 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.
[0144] 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-US-00033 TABLE 33 pH recordings prior to inoculation. Product Unadjusted pH WOB NP1 A 5.58 WOB NP1 B 5.55
[0145] Preparation of Cell/Spores for Trials:
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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-US-00034 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
[0150] 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.
[0151] Materials and Methods
TABLE-US-00035 TABLE 35 Products used in the study for control of Botrytis cinerea. 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-US-00036 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.
[0152] 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.
[0153] 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).
[0154] 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).
[0155] 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,
TABLE-US-00037 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
[0156] Application Details—Spray
[0157] Table 38 and 39 describe details of the application spray and conditions at each time point throughout the application schedule.
TABLE-US-00038 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-US-00039 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
[0158] Results
TABLE-US-00040 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-US-00041 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-US-00042 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 (Botrytis cinerea) 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-US-00043 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
[0159] Part Rated
[0160] BUNCH=bunch
[0161] P=Pest is Part Rated
[0162] Rating Type
[0163] PESINC=pest incidence
[0164] PESSEV=pest severity
[0165] Rating Unit
[0166] %=percent
[0167] % AREA=percent of area
[0168] BUNCH=bunch
[0169] PLOT=total plot
TABLE-US-00044 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
[0170] Part Assessed
[0171] BUNCH=bunch
[0172] C=Crop is Part Rated
[0173] Assessment Type
[0174] PHYGEN=phytotoxicity—general/injury
[0175] Assessment Unit
[0176] % AREA=percent of area
[0177] VINE=vine PLOT=total plot
TABLE-US-00045 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.
[0178] Part Rated
[0179] BUNCH=bunch
[0180] P=Pest is Part Rated
[0181] Rating Type
[0182] PESINC=pest incidence
[0183] PESSEV=pest severity
[0184] Rating Unit
[0185] %=percent
[0186] % AREA=percent of area
[0187] BUNCH=bunch
[0188] PLOT=total plot
TABLE-US-00046 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-US-00047 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
[0189] 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.
[0190] 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.
[0191] 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-US-00048 TABLE 48 Products employed in the study for growth control of Botrytis cinerea 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-US-00049 TABLE 49 Treatment schedule employed in the growth control of Botrytis cinerea 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-US-00050 TABLE 50 Chronology of events throughout the growth control of Botrytis cinerea 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
[0192] Results
TABLE-US-00051 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-US-00052 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-US-00053 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-US-00054 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-US-00055 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-US-00056 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
[0193] 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-US-00057 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-US-00058 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-US-00059 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
[0194] 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-US-00060 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-US-00061 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-US-00062 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.sub., 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-US-00063 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
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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-US-00064 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-US-00065 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
[0204] Results
[0205] 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.
[0206] Microbiological Tests
[0207] Pears
[0208] 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 (
[0209] 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 (
[0210] Apples
[0211] For apples only the BCDMH+WOB NP1 treatment significantly reduced the level of contamination by fungi compared to the unwashed sample (
[0212] Post Storage Rot Assessments
[0213] Pears
[0214] 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 (
[0215] Apples
[0216] 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 (
EXAMPLE 15—RESIDUE STUDY
[0217] 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).
[0218] 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.
[0219] 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-US-00066 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 Metabisulphite.sup.1 + 478.4 Sodium Benzoate T3 WOB NP1 1600 425.6 (280) Sodium A B C D E F Metabisulphite.sup.1 + 956.8 Sodium Benzoate N/A = Not applicable Note .sup.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-US-00067 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).sup.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).sup.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 .sup.1Rates are corrected for the concentration show on the Certificate of Analysis. Note .sup.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-US-00068 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).sup.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).sup.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 .sup.1Rates are corrected for the concentration shown on the Certificate of Analysis. Note .sup.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.
[0220] 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.
[0221] 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.
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 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.
[0227] 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.
[0228] Juice and wine study sample were analysed with no further preparation.
[0229] Analytical Method—Benzoic Acid
[0230] 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.
[0231] 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.
[0232] 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).
[0233] 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.
[0234] 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).
[0235] Analytical Methods—Sulfur Dioxide
[0236] 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
[0237] 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.
[0238] 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.
[0239] 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.
[0240] 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-US-00069 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 SO.sub.2 Acid Site Type Specimen sample code number Test Item (g/100 L) (DALA.sup.1) 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 .sup.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-US-00070 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 SO.sub.2 Acid Site Type Specimen sample code number Test Item (g/100 L) (DALA.sup.1) 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 .sup.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-US-00071 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 SO.sub.2 Acid Site Type Specimen sample code number Test Item (g/100 L) (DALA.sup.1) 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 .sup.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-US-00072 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 SO.sub.2 Acid Site Specimen sample code number Test Item (g/100 L) (DALA.sup.1) 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 .sup.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-US-00073 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 SO.sub.2 Acid Site Specimen sample code number Test Item (g/100 L) (DALA.sup.1) 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 .sup.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-US-00074 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 SO.sub.2 Acid Site Specimen sample code number Test Item (g/100 L) (DALA.sup.1) 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 .sup.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-US-00075 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 SO.sub.2 content weight’ weight’ Site sample code number Test Item (g/100 L) (DALA.sup.1) 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 .sup.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-US-00076 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 SO.sub.2 content weight’ weight’ Site code number Test Item (g/100 L) (DALA.sup.1) 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 .sup.1DALA days after last application *D denotes duplicate LoD: limit of detection (0.100 mg/kg) LoQ: limit of quantitation (0.200 mg/kg)
[0241] 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.