JPH01117810A - Method, composition and kit for increasing pharmachological effect of agricultural chemicals - Google Patents

Abstract

PURPOSE: To obtain an adjuvant composition capable of reinforcing the pharmacological effect of an agrochemical with a biodegradable adjuvant of enzyme by applying the enzyme capable of degrading at least a part of plant cell polymers to a plant before or simultaneously when the agrochemical is applied to the plant. CONSTITUTION: (a) An enzyme capable of degrading at least a part of the cell polymers of a plant on which an agrochemical has a pharmacological effect, preferably lipase, further preferably cutinase, especially Pseudomonas putida (ATCC 53352), or the mixture of cellulase, hemicellulase and pectinase, is selected, and (b) the enzyme as a biodegradable agrochemical adjuvant or a plant adjuvant is applied to the plant, before or simultaneously when the agrochemical is applied to the plant. The biodegradable adjuvant can reinforce the pharmacological effect of the agrochemical, and thereby enables to reduce the use amount of the agrochemical.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to the use of enzymes as agrochemical adjuvants. In particular, it relates to methods of increasing the effectiveness of pesticides with the use of the depolymerase enzymes of the present invention, and compositions of depolymerase enzymes and pesticides.

(Prior Art and Its Problems) An adjuvant is a drug added to a pesticide to increase its pharmacological effect. A wide range of adjuvants for plants are a variety of complex chemical types known to serve to enhance the action of pesticides. Classified by mode of action, oils, surfactants (e.g. wetting agents, spreading agents, penetrants),
Including stabilizers, solvents, moisture absorbers, deposit builders, blowing and antifoaming agents, buffers, activators, etc. These types of effects are not necessarily independent of each other. This great diversity of adjuvants is largely dependent on the varying conditions of the adjuvant used and the different results obtained with the particular pesticide used. However, many of the drawbacks of adjuvants are that they are not biodegradable.

Typical pesticides include herbicides, plant growth regulators, plant species specific toxins, fungicides, insecticides including chemically fungicidal pesticides, leaf colonizing microorganisms, and chemical fertilizers. With the availability of a variety of pesticides, there continues to be a need to provide plants with plant adjuvants with a wide range of applications.

It is an object of the present invention to provide a biodegradable agrochemical adjuvant and a method of increasing the pharmacological efficacy of pesticides using said botanical adjuvant.

It is also an object of the present invention to reduce the amount of pesticide used to achieve pharmacological effects.

Means for Solving the Problems The present invention therefore provides: a) selecting a devolimerase enzyme capable of degrading at least a portion of the plant cell polymers of the plant in which the pesticide is to have a pharmacological effect; b) applying the pesticide to the plant. Before or at the same time as applying
The present invention also relates to a method of increasing the pharmacological effect of a pesticide comprising the steps of applying a selected enzyme to a plant.The present invention also relates to a composition containing a plant devolimerase enzyme and a pesticide. The invention further relates to a kit of parts containing a plant polymerase enzyme and a pesticide.

In plants, the outer layer or membrane is a polymeric cellular structural component associated with water resistance and protection. The outermost layer is usually a biopolyester cutin with the air-clear part and the sperine, and the lower part and the scarred surface is a polymer containing polyester regions. Other membrane layers usually consist of cell walls containing polymers such as pectin, cellulases, hemicellulases, and proteins. Plant cell polymers then constitute the main protective barrier between the plant and its surroundings, acting as a biological barrier to control molecular scattering (Science, Vol. 208, May 30, 19BO). 990~
(See page 1000).

An advantage of the present invention is that the plant devolimerase selected for use in the present invention is biodegradable.

The choice of plant devolimerase is made in relation to the particular plant cell polymer to be degraded. Therefore, for example, when the cellular polymer is cutin, an appropriate lipase can be selected. Other devolimerases such as pectitases, hemicellulases, cellulases, and proteinases are combined with lipase alone.

Can be used together. Cutinase is a preferred lipase and is available from a variety of sources.

For a review of various cutinases useful in the practice of the present invention, see Fungi and Pol
len, P.E., Kolattukudy, 47
(pages 2 to 504) on cutinase. A preferred lipase is Pseudomonas O putida (Pseudomonas O putida).
It is a lipase that has been briefly isolated from A. onas Putida) AT CC53552, and has the following amino acid sequence.

2υ ala val ala asn phe
asp arg ser gly pro
tyr thrthr Ser Ser gi
n ser glu gly pro se
r Cys arg ileU gly pro ser thr tyr
ala gly leu ser
his trpala ser his gl
y phe vat vat ala al
a ala glue thrser asn
ala gly thr gly arg
glu met leu ala cys1
'6υ gly thr ser gly his
ser gln gay gly gly
gly ser11e met ala gl
y gln asp thr arg va
l arg thr thrala pro
ile gin pro tyr thr
Ieu gly leu gl)' h1s
'1 so υ pro tyr leu asn ala
gin pro vat tyr arg
arg alaasn vat pro va
l phe trp gly glue ar
g, arg tyr valser his
phe glu pro val gly
ser gly gly ala tyrs
er val gly arg arg g
In addition, selective substitutions of amino acids can be used to generate lipase or other devolimerase enzymes that are more selective for particular cutin substances or other polymers.

Pesticides for use on plants vary widely in both type and action, as discussed above. One thing that pesticides generally have is that their effectiveness increases with increased regular absorption.

The enzyme selected must be in a concentration large enough to degrade at least a portion of the corresponding polymer. This amount varies between enzymes and plants, and is also influenced by conditions such as weather, humidity, soil, etc. Those skilled in the art will be able to select such amounts without undue experimentation;
Generally, at least about 1 microgram per d of applied plant surface area is the desired amount to achieve an adjuvant effect. Enzymes can be used before or at the same time as the desired pesticide. The enzyme is mixed with a suitable diluent (eg water) to facilitate use, eg by spraying. So, for example, mix about 1 mg to 0.01 #Iff of enzyme to 1 - 1 of water and add the desired pesticide together or separately to 40 gallons (181,84 liters) per knee car (4046,8 Td). Apply.

A preferred concentration is 0.0.0% divolimerase per d of solvent. O5f
f1g or 0.5! It is rtg. A further advantage of the present invention is that pesticides (such as atrazine) normally required to be applied to roots or soil for ingestion can be applied to leaves or other similar tissues to provide efficacy or additional benefits.

The following examples are merely representative and are not limiting. A person skilled in the art will, based on what is disclosed herein, select suitable other depolymerase enzymes other than the lipases described herein when reading representative examples of giving a particular plant and the corresponding pesticide. be able to.

Examples Examples show that enzymes that break down plant surface polymers into monomers increase the effectiveness of herbicides.

Multiple herbicides from different chemical classes were compared in a multispecies screen for percent chlorosis, necrosis, and reduction in vigor with and without the enzyme from Pseudomonas putida AT CC53552 described above. . The herbicide was used at the manufacturer's recommended dosage and 10% and 40% of that dosage on the various plants.

Using two enzyme concentrations, 0.5111/ridl (
HE) and 18 at a concentration of 0.05 mg/d (LE).
1.849J (40 gallons) /404B, 8Td (1
(knee car) ratio was used. All spray solutions had a pH of 1O and each contained 100 m sodium phosphate. Evaluations were made 6 and 12 days after treatment. All experiments were conducted in a greenhouse.

Recommended amount 100% herbicide active W g/vt (lbs,/a
cre) Basagran (E-4) Made by BASF
0.1t2 (1) Atradi > (ATRZ)
(L-4) Made by Shell 0.224 (2) Calmesis (WO-80) Made by DuPont 0
．． 179 (1,8) Hyper XL (E-2) Manufactured by DuPont 0.448 (4) Rating Chlorosis - Number representing the percentage of plants exhibiting chlorosis.

Necrosis - A number representing the percentage of plants exhibiting necrosis.

Vitality - A number representing the percentage of plants whose vigor has decreased.

Herbicidal - A number representing the average percentage of chlorosis, necrosis and vigor for a given sample.

Example 1 Using the conditions and enzymes described above, one week and two weeks after administering Hyper.
The following results were obtained during the week. In the table, CH represents chlorosis, NEC represents necrosis, VIG represents vigor, and PHY represents the average herbicidal activity of these three readings.

Enzyme 1 week after treatment Hyper dog shrimp soybean
Giant Greentail CHLNECVIGP)TY C
HLNECVIGPHY CHLNECVIGPHY
Muaki 10% 30 25 30
28 55 75 75 88 4
0 30 50 40 low enzyme 10% 50
18 50 39 90 83 90 88 50
30 50 43 High enzyme 10% 25 10 5
0 28 25 50 50 42 Ill 1
8 30 22 Enzyme-free 40% 65 30 55
50 45 GO73595038554g low enzyme 40% 55 60 83 59 55 88
73 72 43 65 63 57 High enzyme 40
% 55 50 30 45 45 50 83 5
9 38 50 83 50 Enzyme-free 100% 5
0 80 75 62 100100100100
80 60 75 65 Enzyme Hyper Cub
, Alfalfa 5) IL NECWIG PHY
CHL NECVIG PRY enzyme-free 10%
70 91 90 84 10 5 10 8 low enzyme
10% 70 90 93 84 50 5 30
2B high enzyme 10% 50 85 83 71
18 0 10 10 Enzyme-free 40% 65 78
85 74 25 5 15 15 low enzyme 40%
93 95 95 94 30 30 50 37
High enzyme 40% 97 97 98 97 30
13 30 24 Enzyme-free 100% 100 95
90 95 50 10 50 37 These results are
Lower enzyme concentrations have been shown to increase the herbicidal activity of the herbicide on all tested plants. Very high concentrations reduce herbicidal activity in all cases. This indicates that optimal enzyme concentrations need to be used to obtain the best effect. The decrease in efficacy at high protein concentrations is due to an increase in the viscosity of the solution or to the incorporation of the drug into the leaves.

2 weeks after treatment Hyper dog shrimp Giant foxtail Alfalfa CHLNECVIGPHY CHLNECVI
GPHY CHLNEC VIGPHY enzyme-free 18
% 85 78 78 80 65 85 68 6
4 55 58 58 57 Low enzyme 18% 8
8 85 85 88 73 73 73 73 10
0100100100 High enzyme 18% 95 8
8 88 90 75 75 75 75 10010
0100100 High Hyper killed 100% of the plants after 2 weeks versus all other test conditions.

Evaluation two weeks after herbicide treatment also showed that the enzyme increased the herbicidal activity of Hyper.

Example 2 Next, the results of using the method of Example 1 using herbicides obtained from various chemicals are shown.

CI(L NECWIG Pi(Y C1(L)
NECVIG P)IY enzyme-free 10% 1
8 33 50 34 38 23 50
37 low enzyme 10% 38 58 48
48 50 38 50 4B high enzyme
10% 55 18 13 12 25
25 25 25 Enzyme-free 40% 55
83 55 58 70 70 83
74 low enzyme 40% 100 100 100
100 90 90 90 90 high enzyme
40% 80 88 88 85 75
88 88 84CHI, NECWIG P
HY CIIL NECVIG PHY enzyme-free
10% 5 5 13 8 25
25 38 29 Low enzyme 10% 18
4350 37 38 63 75 59 High enzyme 10% 18 38 73 43
38 50 83 50 With Basagran using 40% of the recommended amount, the plants completely died.

High enzyme 10% 0 0 0 0
18 15 0 11 Under these test conditions, the herbicidal activity of Calmetsis was low. At 40% of the recommended dose, Calmetsis increased herbicidal activity when sprayed with enzymes on turnips.

From these experiments it is clear that the use of depolymerase enzymes such as cutinase increases the herbicidal properties of herbicides obtained from the four chemical classes.

Example 3 The method of Example 1 was used, except that the enzyme was used 2 hours before administering the pesticide, and the results are shown below. Evaluation was made 2 weeks after treatment.

Basagran soybean mosquito
Buca NECVIG%PHY 5)L NE
CVIG%PHY This example shows that the enzyme can be sprayed before the herbicide and the herbicidal properties increase with the enzyme. This method is used to increase herbicidal properties when using chemicals that inhibit enzymes.

Example 4 Agricultural chemicals labeled with radioactive isotopes were ingested into leaf discoid tissues.

Experimental method Leaf discs (1 cm in diameter) from 9 to 13 day old soybeans.
) and placed nitrocellulose paper pre-soaked with 100 μg/rttl stocbutomycin in a vetri dish. The radioactive drug was added to 0.05% Triton x-i
Dissolved in 100o+M sodium phosphate containing oo. Triton, uracil solution containing 0.4% ethanol ("5), atrazine containing 10% ethanol ("5), indoleacetic acid containing 2% ethanol ("5) and 0.2% ethanol (,145 ) Urea was added.

5 μl of radioactive drug solution (9/μcl/IR1) was applied to the leaf discs and incubated for 5-7 hours at room temperature. After incubation, the leaves were rinsed three times with 0.025% Triton X-100 solution and transferred to another scintillation bottle. The radioactivity absorbed by each leaf is 1 in each layer.
After adding 0- Ecolite scintillation liquid, measurement was performed using a scintillation counter. Ten leaf tissues were used for each data point for all experiments.

Radioactive chemicals were obtained from Amersham (U.S.A.). Specific drug activities are listed below.

Indolyl (2-”5) acetic acid, 319 μcl/#(ethyl-1-”5) atrazine, 118 pallIQ (ethyl 145) diuron, 243 μcl/+19(”5)
Urea, 1 Ilci/η (2-"5) Uracil, 474 .mu.c!/Q Enzyme The same cutinase enzyme dissociation enzyme as described above, GC219 is a commercially available enzyme mixture consisting of cellulase, pectinase and hemicellulase.

The protein control inactive for cutinase was a cutinase mutant that substituted alanine at position 12B for serine. This produced an inactive enzyme. For GC219, an equivalent amount of bovine serum albumin was used as an inactive protein control.

Example of drug ingestion into plant leaves: increased radioactivity (cpH) of uracil (5) ingested by cutinase and dissociation enzyme (GC219) Cutinase GC219 inactive protein p)I
u'J/l1dl m9/rIdl Control Active enzyme 7.0 200
818±214 4070±11607.0
100 2180±724
8075±11827.0 50
1340±696 3470±68410
200 481±124
869±364to 100
234± 37 477±2307.0
15.7 1941±315 51
91±1745* Intake without added protein at pH 7.0 is 2090±549, and at pH 1o it is 507±16
It was 2.

Results: ■Active curinase and dissociating enzymes increase uracil uptake.

2. pH 7.0 is better for uracil intake than PHIO.

Plant Growth Regulator Conclusion 3: A combination of enzymes is better for IAA intake than each enzyme alone.

Herbicide example Increased uptake of atrazine and diuron by cutinase 7.0 200 31097±2959
54133±2874 Atrazine 7.0 100 24832±2834
42684±2333 Diuron 10 200 8878±2128
Diuron intake without addition of 8592±803m protein was 5739±1376.

conclusion Active cutinase increased herbicide uptake.

Fertilizer example: Increase in urea uptake by cutinase (5) 7.0
200 934±168 116
7±10110 200 488±
36 911±13910 100
224±80 471±48398±4
It was 3.

conclusion ■ Cutinase increases urea uptake into leaf tissue.

2. Urea intake at PI(10 is better than pt(7,0).

Example 5 Experimental method for ingestion of fungicides in soybean plants Futaba leaflets of 9 to 13 days old soybean were treated with cutinase (1
00μ9/d), benomyl (IIftg/IIdri,
and GC219C12, 8m9/1rtl) alone, and a solution of benomyl and enzyme. The solution was dissolved in 1100ff1 of sodium phosphate and 0.05
% of Triton X-100 to bring the pH to 7. In addition to Triton X-100, Benomyl contained 5% dimethylformamide. For each experiment, 100μ of the solution was added in a 5μ drop to each Futaba leaflet. Plants were treated repeatedly in this way. The treated plants were incubated for 2 hours under normal greenhouse conditions. The same solution used for these plants was incubated for 2 hours under greenhouse conditions to ensure chemical stability. After incubation, treated leaves were cut at the petiole and rinsed with 0.05% Triton X-100 solution. Both leaves were then ground in 1.5 m of 100 mM sodium phosphate pH 7 buffer using a mortar and pestle. The extract was centrifuged and the supernatant was sterile filtered through a 0.45 μm disk. The supernatant was then added to CMA medium containing 2% agar to a final volume of 10 m m. 5d was then injected into one well of a six-well culture dish. 1=1 plant extract in agar
．． Each solution was serially diluted at 1:2 and 1:4 dilutions.

After the agar hardens, each depression is filled with Trichoderma reesei (
Trichoderflla reesei) Q M
6 A strain was inoculated. Plants were incubated at 30° C. for 24 hours to assess Trichoderma reesei growth.

material Enzyme: a) Cutinase enzyme as described above.

b) GC219 (Dissociation Enzyme Blend) is an enzyme mixture sold by Genencor.

Strain: Trichoderma Ezei 0M6A-obtained from ATCC (ATCC 13831) Fungicide: Benomyl and purchased from Rod McClain Media:
Streptomycin CMA medium at 0.05Q/rtdl: 2% agar 1.0 g Bacto-beptone 20 g
Malt extract 20g glucose evaluation for 1 DIR, O No inhibition 10 Weak inhibition ++ Good inhibition +++ Strong inhibition Description of results of fungicide ingestion on plant leaves Inhibition of fungal growth Untreated control - Buffer
-Benomyl (1:1 dilution) -〃
(1:2 dilution) −〃 (1:4 dilution) − Cutinase (1:1 dilution) −〃 (1:2
- (1:4 dilution) - Cutinase and Benomyl (1:1 dilution) +++ Cutinase and Benomyl (1:2 dilution) ++ (1:4 dilution) + GC219 (1:1 dilution) - (1 :
2 dilution) −” (1:4 dilution) − G C219 and Benomyl (1:1 dilution) ++〃(1:
2 dilution) + 〃 (1:4 dilution) - Conclusion Cutinase and dissociation enzymes increased the uptake of benomyl to soybean plants.

Example 5 Hyper uptake by whole plants; Effect of dissociation enzyme (GC219) Experimental method Beefsteak tomato seedlings 13 to 10 days old were treated with Hyper and GC219 solutions. Each solution of Hyper and GC219 was made with DIH20 and 1% ethanol, and Saihaku p84 was prepared using 0.1 NHC9J.
．． (adjust to 0). Apply 10μ of that solution to each cotyledon,
A 5 μl solution was applied to each secondary five-leaf leaflet. Ten tomato seedlings were used for each test solution. After 4 days of treatment, observations were made daily.

Evaluation - No herbicidal + Some herbicidal in the treated area ++ Herbicidal outside the treated area Materials Enzyme - C219 is a mixture of enzymes sold by Genencor.

Hyper XL-compound herbicide (DuPont) Hyper was diluted 1:200 or 1:400 for each experiment.

Herbicidal data Evaluation 1st time 2nd time 1, untreated -2, hyper only (1:200 dilution) -13, hyper +6.28#/
InIGc219 − ++1st time 2nd time 1, control −−2, hyper(
1:40G dilution)-13, hyper+8.28IRg/dGc219+
++4, hyper +12.6ay/dGc219
++ ++ Conclusion Dissociation enzyme (GC219) enhances the uptake of Hyper into tomato plants.

Claims (1)

[Scope of Claims] 1) a) selecting an enzyme capable of degrading at least a portion of the plant cell polymers of the plant on which the pesticide is to have a pharmacological effect; b) before or simultaneously with applying the pesticide to the plant;
A method of increasing the pharmacological effect of pesticides, consisting of steps of applying selected enzymes to plants. 2) The method according to claim 1, wherein the selected enzyme is lipase. 3) The method according to claim 2, wherein the lipase is cutinase. 4) Lipase is Pseudomonas putida ATCC5
3552. The method of claim 2. 5) A method according to claim 1, wherein the selected pesticide is normally absorbed essentially through the roots of the plant, and which comprises applying the enzyme directly to the plant leaves as part of step b). 6) A composition containing a pesticide and a cell membrane degrading enzyme. 7) A kit of parts containing a cell membrane degrading enzyme and a pesticide. 8) A method according to claim 1, wherein the selected enzyme is a mixture of cellulases, hemicellulases and pectinases.