Clove oil as a plant fungicide

A clove oil formulation which inhibits soil-borne fungal diseases is disclosed. The formulation includes about 70-90% by weight rectified clove oil, and about 2-30% by weight surfactant. The surfactant includes about 15-95% by weight nonionic compounds such as ethoxylated monoglycerides, ethoxylated diglycerides, ethoxylated alcohols, silicone glycol copolymers, sorbitan fatty acid esters, ethoxylated alkyl phenols, ethylene oxide block copolymers, and propylene oxide block copolymers. The surfactant further includes about 5-85% by weight anionic compounds such as amine alkylaryl sulfonate, calcium alkylaryl sulfonate and phosphate esters. The formulation may also include up to about 30% by weight of a solvent such as alcohol, esters, glycol ethers, mineral oil, methyl esters and hydrocarbon solvents.

FIELD OF THE INVENTION 
The present invention relates to a clove oil formulation which inhibits 
soil-borne and foliar fungal diseases such as those caused, for example, 
by Pythium, Rhizoctonia, Botrytis, Alternaria, Penicillium, 
Colletotrichum, Xanthomonas, and Fusarium. 
BACKGROUND OF THE INVENTION 
Most plant pathogens, notably prevalent fungal plant pathogens, are 
commonly controlled by the application of synthetic organic chemicals to 
plants in the field, either in furrow or as foliar sprays. However, as 
general sensitivity to synthetic chemical pesticides spreads, there is a 
significant desire to find "natural" substitutes for these pesticides. In 
the absence of effective control, however, fungal plant pathogens can 
extract a significant toll in plant stand, vigor, survival and yield. Some 
researchers have examined the effects various natural compounds have 
against microorganisms, most commonly to find food preservatives. For 
example, 50 plant essential oils, including clove oil, were tested against 
25 bacteria (primarily aerobic), which might act to spoil food. The 
essential oils were applied both undiluted, and diluted in ethanol. The 
study also tested the activity of the oils based on the volatility of the 
oils. Among the findings of the study, both gram positive and gram 
negative bacterias were susceptible to clove oil. Deans et al., 
international Journal of Food Microbiology 5: 165-180, 1987. 
In another screening study, the anti-microbial effects of spice essential 
oils, including clove oil, were tested. The study was performed on spoiled 
fruit and vegetable products by the filtered paper disk diffusion method. 
The results indicated that several of the essential oils studied, 
including clove oil, had antimicrobial activity. Meena, et al., Journal of 
Food Service and Technology 31(1): 68-70, 1994. 
In another study of more interest to plant husbandry, an essential oil 
isolated from the weed Chenopodium ambrosioides was shown to exhibit 
fungicidal activity against the damping off fungus Rhizoctonia solani. The 
isolated essential oil was tested on soils, and in addition, seeds of 
Phaseolus aureus plants were soaked with the oil. The isolated essential 
oil was found to inhibit 44 fungi species. Kishore, et al., Trop. Sci. 29: 
171-176, 1989. 
The spice essential oil, clove oil, consisting primarily of the phenolic 
compound eugenol, has been discovered to be an effective insecticide 
against the cowpea weevil, a pest of stored legumes. Gunathilagaraj et 
al., Madras Agric. J. 54(7): 487-488, 1978. In this study, a solution of 
clove oil and water was either applied to weevils or poured over green 
gram seeds which were exposed to the insects. It was discovered that clove 
oil was toxic to the cowpea weevil, although the authors of the study 
indicated that clove oil is costly compared to other nontoxic 
insecticides. 
Clearly, these studies indicate that some natural compounds, such as clove 
oil, show promise in pest control for some specific agents. However, it is 
difficult to generalize since compounds which are anti-bacterial or 
anti-insecticidal may or may not be anti-fungal and may or may not control 
important plant pathogens. Moreover, because clove oil is hydrophobic, it 
is difficult to formulate into an emulsifiable concentrate in order to 
produce a stable emulsion upon dilution with water, a necessary 
preliminary step for standard applications of plant disease control 
agents. 
SUMMARY OF THE INVENTION 
The present invention discloses a clove oil formulation which inhibits 
soil-borne and foliar fungal diseases. The formulation includes about 
70-90% by weight rectified clove oil, and about 2-30% by weight 
surfactant. The surfactant includes about 15-95% by weight nonionic 
compounds such as ethoxylated monoglycerides, ethoxylated diglycerides, 
ethoxylated alcohols, silicone glycol copolymers, sorbitan fatty acid 
esters, ethoxylated alkyl phenols, ethylene oxide block copolymers, and 
propylene oxide block copolymers. The surfactant further includes about 
5-85% by weight anionic compounds such as amine alkylaryl sulfonate, 
calcium alkylaryl sulfonate and phosphate esters. In another embodiment of 
the present invention, up to about 30% by weight of a solvent is included 
in the formulation. Examples of suitable solvents include alcohol, esters, 
glycol ethers, mineral oil, methyl esters and hydrocarbon solvents. 
It is an object of the present invention to provide a clove oil formulation 
for inhibiting soil-borne fungal diseases. 
It is another object of the present invention to provide a method to 
inhibit soil-borne fungal diseases, which method minimizes adverse side 
affects. 
Other objects, features and advantages of the present invention will become 
apparent after examination of the specification and claims. 
DETAILED DESCRIPTION OF THE INVENTION 
The spice derivative clove oil is a natural compound which according to the 
literature has shown promise as an antimicrobial agent. The present 
invention is directed toward the use of clove oil on plants to combat 
plant pathogens. Clove oil formulations may be used in standard methods of 
application of disease control compounds, that is, soil drench and 
incorporation into soil prior to planting or foliar sprays, and fungal 
disease will be inhibited. The hydrophobic nature of clove oil, however, 
has made it difficult to formulate into a stable emulsion upon dilution 
with water. The present specification discloses that such stable emulsions 
can be made and used to control plant pathogenic fungi. The pathogenic 
fungi that can be controlled include, but are not limited to Pythium, 
Rhizoctonia, Botrytis, Alternaria, Penicillium, Colletotrichum, 
Xanthomonas, and Fusarium strains. 
In the present invention, it was discovered that a special formulation of 
rectified clove oil and surfactant composed of ionic and anionic compounds 
was highly effective in inhibiting fungal diseases. The formulation, which 
forms a stable emulsion when diluted with water, is applied as an aqueous 
solution. Upon dilution, the clove water formulation is highly effective 
in application as a soil drench, or when incorporated into the soil prior 
to planting for soil-borne diseases or as a foliar spray for foliar 
diseases. 
Rectified clove oil, the main active ingredient, consists of 84-88% v/v 
eugenol, 10-15% of .beta.-caryophellene, 1-4% of .alpha.-caryophellene, 
together with less than 5% traces of eugenol acetate. Clove oil is denser 
than water, having a density in the range of 1.036 grams g/cc to 1.04 
g/cc. To create the formulation of the present invention, approximately 
70-90% by weight rectified clove oil is mixed with about 2-30% by weight 
of surfactant (preferably 10-30%) composed of about 15-95% by weight 
nonionic compounds, and about 5-85% by weight anionic compounds. Suitable 
surfactant nonionic compounds include ethoxylated monoglycerides, 
ethoxylated diglycerides, ethoxylated alcohols, silicone glycol 
copolymers, sorbitan fatty acid esters, ethoxylated alkyl phenols, 
ethylene oxide block copolymers, and propylene oxide block copolymers. 
Suitable surfactant anionic compounds include amine alkylaryl sulfonate, 
calcium alkylaryl sulfonate and phosphate esters. The surfactant should 
have a neutral pH value, and an HLB (Hydrophilic-Lipophilic Balance) value 
of 10 to 15. HLB value is a number between 1 and 20 assigned to 
emulsifiers based on the percent weight of hydrophobe to lipophobe in a 
molecule. The clove oil formulation is diluted with water prior to 
application. A preferred dilution of the formulation for application 
brings the rectified clove oil into the range of 0.05 to 1% by weight, and 
the surfactant into the range of 0.001 to 0.01% by weight. 
In another embodiment of the present invention, up to 30% by weight of a 
solvent is included in the formulation. Examples of suitable solvents 
include alcohol, esters, glycol ethers, mineral oil, methyl esters, and 
hydrocarbon solvents. 
In yet another embodiment, a formulation can contain up to 90% solvent, 
7-10% clove oil and up to 3% surfactant. 
The preferred embodiments of this formulation comprise 5-30% of the solvent 
along with 50-85% rectified clove oil and 10-30% of a surfactant blend. 
It has been found that clove oil formulations are effective as inhibitors 
of at least some fungal plant disease organisms regardless of method of 
application. As described below, several important plant pathogens are 
effectively inhibited by clove oil formulations. Such clove oil 
formulations can be applied in-furrow, as soil drenches or as foliar 
sprays. The clove oil application results in less fungal disease as 
reflected by better stands of plants and more vigorous plant growth.

EXAMPLE 1 
The activity of the clove oil formulation of the present invention was 
tested against Rhizoctonia solani, a soil pathogen causing damping-off of 
many greenhouse seedlings, including zinnia. Activity was tested by using 
a soilless mix infested with pathogen prior to planting of the zinnia 
seeds. The soilless medium was placed in a plastic bag, moistened, and 
infested with R. solani, and incubated for 2 days at 25.degree. C. in the 
dark. 
Zinnia seed were planted in 12.times.16 cm market packs with 4 rows each 
containing 10 seed constituting a replication with 4 
replications/treatment. After planting, market packs were placed in a 
growth chamber at 26.5.degree. C. with alternating 10 hours light, 14 
hours dark, periods. Market packs were arranged in a randomized complete 
block design. Disease was assessed by recording the number of healthy 
seedlings in each treatment replicate 2 weeks after planting. 
The clove oil formulation used in this Example had the following 
composition: 15 parts Witco Sponto AK 32-03, 15 parts Silwet L-77, and 70 
parts rectified clove oil. The formulation was applied as an aqueous 
solution, at various v/v dilutions, as described below. 
Treatments used were 0.25% (v/v) clove oil formulation incorporated into 
the soilless medium immediately prior to incubation, 0.125% (v/v) and 
0.25% (v/v) clove oil applied as a drench immediately after planting, and 
Banrot (0.3 g product/L) applied as a drench immediately after planting. 
All treatments were applied as solutions at the equivalent of 120 ml/pack. 
These treatments were compared against a pathogen and healthy check. After 
the seeded packs were watered and treatments drenched onto the surface 
equal amount of water were applied to move the treatment into the medium. 
The results are presented in Table 1. 
TABLE 1 
______________________________________ 
AVR % of 
STAND HEALTHY 
EXPLANATION OF TREATMENT 
COUNT PLANTS 
______________________________________ 
PATHOGEN ALONE 21.3 58.0 
0.25% CLOVE OIL INCORP INTO SOIL 
33.8 92.6 
0.125% CLOVE OIL DRENCH 
32.7 89.6 
0.25% CLOVE OIL DRENCH 
31.3 85.8 
BANROT DRENCH (0.3 g/L) 
34.5 94.5 
HEALTH CHECK 36.5 100.0 
______________________________________ 
These results demonstrate that the clove oil formulation of the present 
invention, applied as a drench at rates of 0.125% or 0.25% or incorporated 
at 0.25%, was efficacious in suppressing damping-off caused by R. solani. 
Seeding counts are not markedly different between the fungicide standard, 
Banrot, and clove oil treatments. It appears that the clove oil 
formulation of the present invention, used in the greenhouse, could 
provide a less toxic alternative to commercially available fungicides, and 
an alternative that may be considered more widely acceptable. 
The method of this example is an effective bioassay for the efficacy of 
other clove oil formulations. This zinnia-Rhizoctonia bioassay can be used 
to determine what other clove oil formulations are effective against plant 
fungal pathogens. 
EXAMPLE 2 
This Example studied inhibition of Botryosphaeria dothidea mycelial growth 
and conidial germination by botanical extracts, insecticidal, soap and the 
clove oil formulation of the present invention. Mycelial inhibition and 
inhibition of conidial germination was evaluated in vitro for several 
aqueous botanical extracts (turmeric, yarrow, ginger, black pepper, 
pomegranate, lemongrass, basil), insecticidal soap, and the clove oil 
formulation. Preliminary tests indicate that the clove oil formulation of 
the present invention was the only one to markedly inhibit mycelial 
growth. The clove oil formulation used in this Example had the following 
composition: 15 parts Witco Sponto AK 32-03, 15 parts Silwet L-77, and 70 
parts rectified clove oil. The clove oil formulation was applied as an 
aqueous solution, and the formulation's inhibitory activity on mycelial 
growth was compared at 7 concentrations of the aqueous solution between 1% 
and 25% (v/v dilutions). 
Mycelial inhibition was measured in Petri dishes filled with potato 
dextrose agar by placing a 5 mm mycelial agar plug of the fungus along one 
edge of a dish, opposite to a 5 mm well containing sterile, distilled 
water (control) or 3 drops of one of the clove oil formulations. Dishes 
were incubated at 25.degree. C. and colony radius was measured in all 
treatments when mycelial growth in the control had covered the diameter of 
the dish (-3 days). 
The zone of mycelial inhibition was calculated as the difference between 
colony radius of the control and colony radius in the treatment dishes. To 
assess inhibition of conidial germination, test solutions were sprayed 
over a glass slide containing two dried water agar droplets onto which 
conidial extrusions from pure cultures of the fungus had been topically 
applied before the agar had completely solidified. Each slide was enclosed 
in a glass Petri dish containing moistened filter paper to maintain high 
humidity conditions, and dishes were incubated at 25.degree., 30.degree., 
or 40.degree. C. for 24 hr. The droplets were stained with 0.5% cotton 
blue in lactic acid following incubation, and germination of conidia and 
the condition of the germ tubes were evaluated for 60 conidia in each 
treatment combination. 
As shown by the results presented in Table 2, the clove oil formulation of 
the present invention inhibited mycelial growth at 25.degree. C. when 
concentrations of 6% (v/v) or higher were used. A lower concentration of 
clove oil (1% v/v) inhibited conidial germination completely at all 
temperatures tested, in contrast to the control in which germination 
approached 100%. Despite the high germination rate of conidia in the 
control treatments at all temperatures, germ tubes had ceased growing in 
most conidia from 30.degree. and 40.degree. C., suggesting that high 
temperature alone could inhibit germ tube extension growth. The percentage 
of germination at 25.degree. and 30.degree. C. was high for most of the 
botanical extracts and soap (73-97%), but germ tubes in all but the basil 
treatment appeared unhealthy and malformed. Germination was completely 
inhibited by most treatments at 40.degree. C. As shown by the results 
presented in Table 3, the clove oil formulation was most active in 
inhibiting growth of the pathogen. 
TABLE 2 
______________________________________ 
MYCEL MYCEL 
TREATMENT.sup.1 
INHIBITION.sup.2 
TREATMENT INHIBITION 
______________________________________ 
CLOVE OIL 1% 
0.00 CLOVE OIL 12% 
2.70 
CLOVE OIL 3% 
0.00 CLOVE OIL 15% 
5.00 
CLOVE OIL 6% 
3.30 CLOVE OIL 25% 
4.00 
CLOVE OIL 9% 
2.00 
______________________________________ 
.sup.1 Means of mycelial inhibition, measured in mm from 3 Petri dishes. 
TABLE 3 
______________________________________ 
% GERMINA- % GERMINA- % GERMINA- 
TREATMENT.sup.1 
TION 25.degree. C. 
TION 30.degree. C. 
TION 40.degree. C. 
______________________________________ 
WATER 84 87 98 
CLOVE OIL 0 0 0 
GINGER 73 30 67 
BASIL 97 93 93 
BLACK PEPPER 
75 93 0 
POMEGRANATE 
90 85 0 
YARROW 70 87 0 
LEMONGRASS 77 80 0 
INSECTICIDAL 
75 90 0 
SOAP.sup.2 
______________________________________ 
.sup.1 Aqueous extracts were prepared using 30 g leaf tissue per 150 ml o 
water. 
.sup.2 MPede .TM. (Mycogen Corp., San Diego, CA) at a concentration of 
19.6 .mu.l/ml water. 
EXAMPLE 3 
In this Example, Rhizoctonia solani was the pathogen studied on the host 
Zinnia "State Fair", using a general drench procedure. After planting 
seeds into infested soil, market packs were moistened lightly, and 
drenched, followed by 125 ml of water to move the treatments into the 
soilless medium. 15 parts Witco Sponto AK 32-03, 15 parts Silwet L-77, and 
70 parts rectified clove oil. Drenches (125ml) were applied to each market 
pack containing 250 ml of soil. Banrot (8 oz/100 gal/800 sq ft)=0.3 g/L 
applied at 1 pt/sq ft=125 ml/market pack. Market packs were placed in a 
growth chamber at 25.degree. C. for two weeks. A series of experiments 
were performed, with the results presented in Tables 4, 5 and 6. 
1. Damping-off at 28% plant loss in pathogen treated: 
TABLE 4 
______________________________________ 
AVG. HEALTHY COUNT 
% 
TREATMENTS 2-WEEKS CONTROL 
______________________________________ 
Healthy 37 100 
Rhizoctonia 26 0 
Banrot 34 72 
0.18% form. Clove oil 
32 55 
0.36% form. Clove oil 
33 67 
______________________________________ 
2. Damping off at 42% plant loss in pathogen treated: 
TABLE 5 
______________________________________ 
AVG. HEALTHY COUNT 
% 
TREATMENTS 2-WEEKS CONTROL 
______________________________________ 
Healthy 37 100 
Rhizoctonia 21 0 
Banrot 35 88 
0.18% form. Clove oil 
33 80 
0.36% form. Clove oil 
31 67 
______________________________________ 
3. Damping-off at one week=65%, at 2 wk=88%: 
TABLE 6 
______________________________________ 
AVERAGE AVERAGE 
HEALTHY HEALTHY 
TREAT- COUNT % COUNT % 
MENTS 1 WEEK CONTROL 2 WKS CONTROL 
______________________________________ 
Healthy 37 100 36 100 
Rhizoctonia 
14 0 4 0 
Banrot (1) 
30 69 21 53 
0.18% form. 
18 18 9 15 
Clove oil 
0.36% form. 
29 65 20 50 
Clove oil 
______________________________________ 
(1) Two flats were not treated properly with Banrot and the stand count i 
low due to applicator's error. 
EXAMPLE 4 
In this Example, Pythium ultimum was the pathogen studied on the host 
Zinnia "State Fair", using a general drench procedure. Analogous to the 
procedure described in Example 3, after planting seeds and then infesting 
soil, market packs were moistened lightly, and drenched, followed by 125 
ml of water to move the treatments into the soilless medium. The 
formulation used was 15 parts Witco Sponto 32-03, 15 parts Silwet L-77, 
and 70 parts rectified clove oil. Drenches (125 ml) were applied to each 
market pack containing 250 ml of soil. Banrot (8 oz/100 gal/800 sq ft)=0.3 
g/L applied at 1 pt/sq ft+125 ml/market pack. Market packs were placed in 
a growth chamber at 18.degree. C. for one week. Table 7 presents the 
results of damping off at 55.5% knockdown. 
TABLE 7 
______________________________________ 
AVG. HEALTHY COUNT 
TREATMENTS 2-WEEKS CONTROL 
______________________________________ 
Healthy 36 100 
Rhizoctonia 16 0 
Banrot 35 95 
0.18% form. Clove oil 
33 85 
0.36% form. Clove oil 
31 75 
______________________________________ 
The results of Examples 3 and 4, as presented in Tables 6 and 7, clearly 
indicate that the clove oil formulation of the present invention is active 
against two soil pathogens (Pythium and Rhizoctonia) in plant bioassays. 
Clove oil formulation treatments increased zinnia stands over the pathogen 
check and work near to or as well as the commercial fungicide, Banrot. 
EXAMPLE 5 
In this Example, Uromyces appendiculatus was the pathogen studied on the 
host, pinto bean (Phaseolus vulgaris cv. Pinto). 
Propagation: Bean seed plants approximately 1.3 cm deep in moist potting 
soil at 4 seed/10cm pots were covered with black plastic on a greenhouse 
bench at 24.degree. C. for 2-3 days to germinate the bean seedlings. Pots 
were thinned to the three most uniform plants/pot when the first 
trifoliate was approximately 1/2 expanded. 
Treatment: Treatments of the clove oil formulation, in aqueous solution, 10 
parts Witco Sponto AK 32-03, 5 parts Ethylene Glycol n-Butyl Ether, and 85 
parts rectified clove oil (all w/w), were applied to the lower and upper 
primary leaf surfaces using a handheld, pump-type sprayer. The treatment 
was allowed to dry before inoculation with the pathogen. 
Inoculation: An urediniospore suspension (20,000 spores/ml) of the U. 
appendiculatus was prepared in water containing one drop of Tween 
20/liter. The suspension was agitated constantly on a stir plate using a 
spin bar until the spores were wetted and evenly dispersed. The suspension 
was then atomized onto the lower leaf surfaces only using a Crown 
Spra-Tool aerosol sprayer. The inoculum was allowed to dry before placing 
the plants into a dew chamber for a 16 hr. Infection period at 
18.degree.-21.degree. C. The plants were removed to a warm greenhouse 
(min. 21.degree. C. night temp.) to allow rust pustule development. 
Disease assessment: The plants were rated for disease 7-8 days after 
inoculation by assessing the number of rust pustules/10cm.sup.2. The 
results, presented in Table 8, clearly indicate that the clove oil 
formulation of the present invention reduces the number of bean rust 
pustules per leaf. 
TABLE 8 
______________________________________ 
PRE- POST- 
TREATMENTS TREATMENTS TREATMENTS 
______________________________________ 
Pathogen alone 2.6(a) 2.9 
0.36% Clove oil 0.7 3.5 
(Treatment was not phytotoxic) 
0.18% Clove Oil 1.2 3.1 
0.09% Clove Oil 1.2 3.1 
0.04% Clove Oil 1.0 3.1 
0.02% Clove Oil 1.7 3.4 
______________________________________ 
(a) These numbers denoting the number of rust pustules/cm.sup.2 were 
obtained using a visual rating scale from (0-7), where 0 = no 
pustules/cm.sup.2, 1 = less than 2 pustules/cm.sup.2, 2 = approximately 4 
pustules/cm.sup.2, 3 = approximately 7 pustules/cm.sup.2 and 4 = 
approximately 18 pustules/cm.sup.2, and so on. 
The activity of the clove oil formulation of the present invention against 
bean rust appears to be prior to inoculation only. Formulated clove oil 
applied at 0.36% exhibits good control of bean rust. Formulated clove oil 
at 0.36% was not phytotoxic in the test above. 
EXAMPLE 6 
The volatile activity of the clove oil formulation of the present invention 
was tested by dipping filter paper disks into an aqueous solution of 25% 
(v/v) clove oil formulation. 
The filter paper disk was then placed on the underside of the lid of the 
petri plate and a 3 mm plug of each test fungus was placed on potato 
dextrose agar at the edge of the plate. Plates were inverted and incubated 
at 25.degree. C. in the dark as many as 10 days depending on the growth 
rate of the fungus. Test fungi were Pythium ultimum, Rhizoctonia solani, 
Botrytis cinerea, and Fusarium roseum. 
Measurements were obtained over time by measuring mycelial growth. 
Measurements began 2 days after the mycelial plugs were placed onto the 
agar surface. The zone of inhibition was calculated by taking the 
difference between the mycelial growth of the control plate and the 
treated plate. Data indicated that the rate and inhibition of fungal 
growth was affected by the volatile activity of the clove oil formulation 
applied at a rate of 25%. The rate of fungal growth in the treated plate 
was greatly delayed as compared to the control plate for all fungi tested. 
Zones of inhibition for each fungus is as follows: P. ultimum, 34 mm; R. 
solani, 40 mm; B. cinerea, 39 mm; and F. roseum, 34 mm.