CA2482090A1

CA2482090A1 - Method of protecting plants from bacterial and fungal diseases

Info

Publication number: CA2482090A1
Application number: CA002482090A
Authority: CA
Inventors: Joseph M. Dutcheshen
Original assignee: Northern Quinoa Corp
Current assignee: Northern Quinoa Corp
Priority date: 2003-09-22
Filing date: 2004-09-22
Publication date: 2005-03-22
Also published as: US20050261129A1

Abstract

Improved methods and compositions for protecting plants or seeds from plant diseases are provided, The compositions comprise (and preferably consist essentially of) saponin, such as triterpene type saponins extracted from quinoa or quillaja.
Compositions according to the invention may comprise (or consist essentially of) a quantity of a sapogenin compound, preferably a sapogenins selected from the group consisting of oleanolic acid, hederagenin, phytolaccinic acid, and quillaic acid. The methods comprise contacting the portion of the plant (e.g." foliage, shoot, etc.) to be treated with a disease-inhibiting or protective amount of the composition. The compositions can also be used to treat plant seeds or tubers prior to planting thereof, as well as soil adjacent a growing plant. The inventive compositions are particularly useful for the treatment, control and/or prevention of bacterial diseases. The saponins of the invention can be applied as liquids or dry particulates, and are especially suited for the treatment of tomato and potato plants and their respective seeds.

Description

_1_ METHOD OF PROTECTING PLANTS FROM
BACTERIAL AND FUNGAL DISEASES
RELATED APPLICATION
The present application claims the priority ofU.S. Provisional Application S/N
60/504,993, filed September22, 2003, which is incorporated by reference herein.
BACKGROUND OF THE INVENTION
Field of the Invention The present invention is broadly concerned with methods and compositions for protecting plants frombacterial diseases. More particularly, the inventive compositions comprise an aqueous solution of saponins or saponin derived compounds such as sapogenins. These compositions are directlyapplied to seeds, seedlings, shoots, foliage, etc. of the plant to be protected. In addition to bacterial diseases, the compositions are useful for protecting the plants against fungal and viral diseases.
Description of the Prior Art There are .numerous diseases which may harm or even kill plants. Fungal diseases are one such type ofdisease. For example, Rhizoetonia solani (Rhizoctonia Canker,, Black Scurf, or Helrninthospori~m solani (Silver Scurf)) and Phytophthora infestans (Late Blight) are both fungal diseases which are extremely dangerous to potato crops: In rhizoctonia infections, sclerotia or mycelium invade emerging sprouts, potato stems, roots, and stolons after germination occurring in early spring. On mature tubers or potatoes, the disease appears as black hard bodies known as Black Scurf, with the tuber skin underneath often remaining unharmed. The disease leads to a delay in the emergence of the sprouts and stems, and causes the sprouts and stems to have a reddish canker girdling them when they finally do emerge.
The symptoms of late blight first appear on older leaves soon after flowering of the plant. The leaves turn dark brown and brittle, while the tuber exhibits lesions which often appear around the eyes. Furthermore, the infected~portions of the tuber are granular in nature and penetrate as much as 2 cm into the tuber. All of these symptoms cooperate to reduce tuber yields and quality.
Both rhizoctohia and late blight readily infect potato plants and require extreme measures to avoid or minimize transmission thereof. For example, crop rotation is commonlypracticed in an attempt to avoid diseased crops: Additionally, growers often seek seeds that are certified as being disease-free: However, these and other currently available measures do not adequately protect against the diseases. There is a need for preventive treatments that will protect potatoes and other plants from these and other harmful diseases.
Bacterial diseases also pose significant problems to plants, especially tomatoes.
Such bacterial diseases include bacterial spot (caused byXanthomonas campestris pv.
vesicatoria) and target spot (caused by Corynesporia cassiicola). A number of bactericide compositions are presently available for combating these bacterial diseases.
KOCIDE, available from Griffin; L:L.C., Valdosta, Georgia, is a leading bactericide which utilizes copper as the primaryantibacterial agent. However, due to the prolonged use of copper antibacterial agents, bacteria are showing signs of increased resistance to copper, thereby reducing the effectiveness of the bactericide in controlling disease.
Furthermore, agricultural runoff from these agents is fording its way into coastal waters and potentially causing harm to various marine life; especially shrimp and other invertebrates.
Because ofthe problem ofbacteria developing resistance to various antibacterial compositions, attempts have been made to develop compositions which stimulate the plant's own defense genes to cause the plant to produce proteins which inhibit disease.
These products produce what is commonly known as a systemic activated resistance (SAR) response within the plant: ACTIGARD, available from Syngenta, Wilmington, Delaware, is one such product designed to stimulate a systemic response within the plant to combat the bacteria. ACTIGARD contains the active ingredient acibenzolar-s-methyl. While reasonably effective in controlling bacterial disease, it is a relatively expensive treatment option for farmers. Therefore, there is a need for an economical method for stimulating a plant's own immune system to combat bacterial diseases, preferably employing a naturally derived composition in order to lessen potential environmental concerns.
Quinoa is classified as a membex of the.Chenopodiaceae, a larg$ and varied familywhich includes cultivated spinach and sugarbeet. Quinoa is an extremely hardy and drought-resistant plant which can be grown under harsh ecological conditions - high altitudes, relatively poor soils, low rainfall, and cold temperatures - that other major cereal grains, such as corn and wheat, cannot tolerate.
Quinoa originated in the Andes region of South America where it was a staple grain in pre-Spanish Conquest times. Traditional uses of Quinoa declined after the Spanish Conquest. Cultivation and use of the grain was not widespread until a recent revival due to Western interest in this crop as a high lysine, high protein grain for human consumption. The principal obstacle to even wider human consumption of quinoa has been, and continues to be; the bitter taste of saponin glycosides present in the grain.
Saponins are a type of sterol glycoside widely distributed in quinoa as well as other plants. There are generally two types of saponins-triterpene saponins and steroidal saponins. Traditionallly, 5aponin has been removed by washing the grain in running water, although new methods have been developed recently (See, e.g., U.S.
Patent No. 6,355,249).
Saponins are compounds in which anoncarbohydrate moiety (the aglycone) is attached to a carbohydrate moiety through an acetal linkage. Sapogenins are created when the noncarbohydrate moiety is separated from the carbohydrate moiety. As with saponins, sapogenins may be either triterpenoidal or steroidal. The saponins from the quinoa seed are mainly of the triterpenoidal type.
Attempts have been made to utilize saponin as a synergist for other compounds which are useful for controlling the growtk of pathogens (e.g., fungi) on plants. For example, U.S. PatentNo. 5,639,794 to Emerson et al., is directed towards a method for treating agricultural crops comprising the step of applying a so-called "natural product"
in combination with at least one saponin to kill, retard growth of, or displace pathogenic organisms. The natural products combined with the saponin are the various aldehydes, and particularly aromatic aldehydes, and the saponins are used to enhance the activity -of the aldehyde (i.e., as a synergistj. However; the use of aldehydes increases the cost of treating the plants and, in many instances, may be undesirable to the grower due to environmental concerns as well as the extra effort involved in handling these aldehydes.
There is a need for a cost-effective, environmentally friendly composition for effectively treating andlor preventing diseases in plants.
SUMMARY OF THE INVINTION
The instant invention overcomes the problems of the prior art by broadly providing effective compositions and methods for treating and/or protecting plants from diseases, especially bacterial; fungal; and viral disease.
In more detail, the inventive compositions comprise (and preferably consist essentially of) saponins which act as a protectant for the plant independent of other compounds or agents (i.e., saponin is the principal and/or only active ingredient). As used herein, "plant" is intended to refer to anypart of a plant (e.g., roots, foliage, shoot) as well as trees, shrubbery, flowers, and grasses. "Seed" is intended to include seeds, tubers; tuber pieces, bulbs, etc., or parts thereof from which a plant is grown.
The saponin should be derived from a plant different than the plant that the final saponin composition is intended to protect. Suitable sources of saponins include Quinoa, Quillaja, Primula (Primulae sp.), Senega (Polygalasenega), Gypsophila, Horse chestnut (Aesculus sp.); Ginseng (Panax sp. and Eleutherr~cosus sp.), Licorice (Glycyrrhiza sp.), Ivy (Hedera sp.), Tea seed (Camellia cinensis), Alfalfa (Medicago sativa), Soya, Yucca (Yucca sp.), and Dioscorea. It is particularly preferred that the saponin be of the triterpene variety as found in Quinoa and Quillaj a versus the steroidal types found in yucca.
A preferred method of extracting saponins from Quinoa comprises placing a saponin-containing portion of a qu'tnoa plant in an aqueous alcohol (e.g., methanol, ethanol) solution to form a saponin-containing solution and an extracted, solid residue.
The alcohol is then removed from the solution followed by evaporatian of the water to yield the saponin-containing product. Those skilled in the art will appreciate that the saponins can also be extracted from quinoa by other methods for use in the instant invention.

w The saponin extract is preferably mixed with water to form the protective composition. If desired, the saponin extract can be mixed with water under mild heat (e.g., from about 10-35°C) in order to effect mixing. Alternately, the saponin extract can be applied as a dry composition alone, or blended with a suitable carrier.
Preferably, the composition comprises from about 25-300 g of saponin extract, and more preferably from about 50-200 g of saponin extract, per 100 liters of water, where the saponin extract has a triterpene saponin concentration of from about 10-70% by weight, and preferably at least about 50% by weight, based upon the total weight of the saponin extract taken as 100% by weight. Alternately, the saponins of the invention can be applied in a dry formulation using talc or some other particulate Garner.
In such -cases,. the: saponin_component should_be,present at_a;level of from about 8-46%_by weight more preferably from about 16-36% by weight.
The composition of the saponin within the scope of the present invention comprising one or more triterpene glycosides having the general formula (I) whereinRl R9 are independently H, OH, CH3, CH20H, COOCH3, or CHO, and where Rl°
is a .
saccharide comprising any combination of one or more glucose, arabinose, zylose, glucronic acid, galactose, rhamnose; fucose or any other naturally occuring monosaccharide.
Rg R too Formula I

a ~ _6_ Compounds according to the foregoing formula may be saturated or contain unsaturated carbon bonds at various locations in the molecule. The preferred saponin compositions are of the triterpene variety as found in quinoa or quillaja and contain glycosides of one or more of the following sapogenins (triterpenes): oleanolic acid, hederagenin, phytolaccinic acid (also referred to as phytolaccagenic acid), and quillaic acid, the chemical structures of which are given below.
Ha' Ho phytntaccixric aad q~u'tliac aad _7-It is also within the scope of the present invention'to provide a composition comprising or consisting essentially of a sapogenin that can be applied to plants or seeds to protect said plant or seed from disease. It is particularly preferred that the sapogenin be of the triterpene variety and having the structure of formula (I) wherein R,o is H (that is, the saccharide is replaced with H). Particularly preferred sapogenins include those whose structures are given above (oleanolic acid, hederagenin, phytolaccinic acid, and quillaic acid).
It is within the scope of the invention for the sapogenin compositionto be in the form of a concentrate wherein the composition comprises up to about 99% by weight of a compound according to formula (I) wherein R,° is H. The concentrates maybe diluted_(preferablywithwater) to form a use solution for_a~plication toplants and seeds.
Preferred sapogenin use solutions according to the present invention comprise from about 20-80% by weight of a compound according to formula (I), more preferably from about 30-70% by weight, and most preferably from about 40-fi0% by weight.
Given that sapogenins exhibit reduced solubility in water than saponins, a surfactant may be required in order to form more concentrated sapogenin use solutions.
In use; plants or seeds are treated with the inventive compositions by simply contacting one or more portions of a diseased plant or seed, or a plant or seed susceptible to attack by disease, with a disease-inhibiting or protective amount of the composition so as to elicit a protective response in the plant or seed. This can be accomplished by spraying the plant or seed as well as by submerging it in the aqueous composition. Those skilled in the art will appreciate that portions of a plant can be selectively treated (e.g., infected leaves can be treated individually or the roots. alone can be treated). Additionally, the seeds or tubers can be submerged in the aqueous composition and then planted and allowed to grow into a protected plant.
Furthermore, the soil around the plant or seed can be treated as well. When the plant to be treated is a tree; the composition is preferably introduced into the vascular system of the tree by conventional methods. In a similar way; the dry inventive compositions can be applied by dusting or coating a plant part or seed.
It has also been found that the compositions of the invention, and particularly the liquid versions thereof, are effective as foliar prays. Such sprays would normally contain concentrations of inventive composition of from about 1 g/10 L to 150 g/10 L, and more preferably from about 10 g/10 L to 100 g/10 L.
Preferably, the plant or seed is not pre-treated with some other type of pathogen-controlling agent. Furthermore, it is preferred hat the plant or seed is not treated with another pathogen-controlling agent simultaneous to treatment with the inventive composition. More specifically, it is preferred that the plants, seeds, or soils surrounding the plants have not been treated with some type of aldehyde composition.
Thus, the plant and plant surfaces or the seed and seed surfaces should be essentially aldehyde-free (e.g., less than about l mg of aldehyde groups, per square centimeter of plant surface area to be treated) prior to treatment according to the invention.
Virtually any~lant_can be_treated to prevent or lessen most plant diseases.
.For example, potato plants, tomato plants; sugar beets, canola, strawberries, chick peas, lentils, broccoli, cabbage, cauliflower; turf grass, tobacco, spinach, carrots, ginseng, radish, and field peas or seeds of any: of the foregoing can all be protected with the inventive compositions. Furthermore; the compositions can be used to treat, control and/or prevent fungal diseases (e.g., rhizoctonia, late Might), Aphanomyces;
Cercospora, Rhizopus, Sclerotium, ergot, Ascochyta, Fusarium, Anthracnose, Botrytis, and Ophiostoma (ceratocystis) ulmi (Dutch Elm disease). Plants or seeds treated according to the invention will remain essentially free of symptoms of the disease for at least about 20 days, preferably at least about 60 days, and more preferably at least about 100 days, after treatment: Thus, plants treated according to'the invention, or plants grown from seeds treated according to the invention, will at least exhibit reduced levels of the disease compared to nontreated .crops, and preferably will remain essentially free of symptoms of the disease throughout the natural growing season of the plant.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a graph illustrating average potato yield data for five test locations whereinpotato seed was coated with the saponin products of the invention versus a non-treated control, as. described in Example 2;
Fig. 2 is a graph illustrating Rhizoctonia level on tubers for two test locations, as described in Example 2;
Fig. 3 is a graph illustrating market yields obtained for various test compositions including the saponins of the invention in the late blight test described in Example 4;
Fig. 4 is a graph illustrating the percent of early blight plants recorded in Example 5;
Fig. 5 is a graph of;late.blightplants recorded in Example 5;_and _ _ _ Fig. 6 is a graph illustrating the percent late blight and number of stems per tuber recorded in Example 5.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The following examples set forth preferred methods in accordance with the invention. It is to be understood, however, that this example is provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.
Example 1 A quantity of the inventive composition was prepared by mixing 250 g of saponin extracted from quinoa with 200 liters of water. Fourth and fifth generation potato seeds were submerged in the solution after which the treated plants were planted along with untreated plants as the control. The quantity of treatment used was 125 g of saponin per acre of planted potatoes. The potato varieties tested were AC
Ptarmigan, Norland, Nordonna, Yukon Gold, and Frontier Russet.
Portions of the potatoes were harvested 14 days, 28 days, and 49 days after top kill. The potatoes harvested 14 and 28 days after top kill exhibited no black scurf while 2% of the tubers harvested 49 days after top kill had black scurf. The treated tubers showed an increased yield of 42% above that of the control tubers.
Furthermore, only ' CA 02482090 2004-09-22 -i0_ 11 % of the treated tubers weighed less than 4 ounces in size while 27% of the control tubers were less than 4 ounces in size. Finally, the control tubers exhibited visible signs of Rhizoctonia on one in three plants while the treated plants showed no signs of the disease until natural attrition or senescence of the plants. The treated plants exhibited vigorous, healthy growth.
Example 2 In this example, Norland potato seed pieces were treated with the preferred saponin material in accordance with the invention at five Canadian sites (Yorkton, Sk., Oakburn; Mb., Abernathy, Sk., and Quill Lake, Sk., and Lethbridge; Ab.) in order to determinethe efficacy__of the treatment for control of diseases during the growing season, and on crop produced: Stand establishment, disease development during the growing season, final yield, marketable yield and disease levels on harvested crop were measured.
In particular, at each location a total of five treatments were earned out, namely a no-treatment control and four coating reatments using different levels of a saponin product. This product was alcohol-extracted saponins from quinoa dried to a solid residue as described above, and then mixed with water under mild heat to give treatment dispersions of various concentration. This product is referred to as "94$15"
and contains more than three saponin active components (sapogenins); the three maj or active ingredients are oleanolic acid, hederagenin and phytolaccinic acid, or analogs thereof.
In particular, the treatments were: 0.02 g/L (treatment #1); 0.1 g/L
(treatment #2); 1 g/L (treatment #3); 10 g/L (treatment #4); and no-treatment control (treatment #5). Before coating, the trial seed pieces were suburized (the pieces were cut and allowed to heal) at Yorkton and Lethbridge, while at the remaining locations the treatment was carried out immediately after cutting (no suburization).
Thereupon, the seed pieces were simply dip-coated in the respective test dispersions.
In each case, there were eight;replications of the five treatments, using eight meter row lengths with common guard rows and two meter spacing between ends of blocks. The following randomized block design was used wlxere "g" refers to guard rows, the numbers 1-4 refer to the above, treatments and "C" refers to controls. The plot size in each case was 25 m x 25 m (1/6 acre).
Block Design Rep 1 Rep 2 Rep 3 Rep 4 g14C23g4123Cg43C21g1324Cg 8meterrows Rep 5 Rep 6 Rep 7 R.ep 8 2 meter space g4213CgC4132g124C3g3C421g 8meterrows The test seed pieces were planted 23 cm apart at a depth of I0" cm. At the time of planting, soil and air temperatures were recorded as well as soil type.
During the growing season, dates of emergence, rainfall amounts, irngation applications and the first visual symptoms of Rhizocto~cia in each row were recorded. The test plots were maintained weed free and were beetle ,controlled. Also, other observable diseases affecting plant growth were monitored, as well as Rhaaoctonia spores on stems (first appearance) in each row. Stem counts per row were made at 60 days after emergence.
Plants on replications 1, 3, 5 and 7 were pulledvat 60 days after emergence, and the following were recorded: Stem and cankor on plants, plant yield (tuber count, weight and range), and evidence of black scurf. At 70 days after emergence; a top kill was performed by shredding or spraying the plants with a commercial potato top dessicant, and harvesting took place at 90+ days after emergence. The following were recorded:
total row yield (weight), total marketable yield (weight), number of tubers of uniform shape and those greater than 45 mm; tuber count per row and size distribution (4 categories, less than 45 mm, 45-55 mm, 55-75 mm and over 7~ mrn) and the number of tubers showing black scurf and the percentage of surface covered.
The following Table 1 sets forth the averaged 90 day yield data from all sites, and Fig. 1 graphically illustrates this data.
Table 2 sets forth the Rhizoctonia data for two locations, and Fig. 2 graphically illustrates the data.

Table 1 Yield Summar of ble Locations Five Gom ara Lethbridge must its own tonia is be reviewed on because grouped differenti . the % count for Rhizoc S 1: 1 m/50L YIELDS:
ALL SITES
k TREAT PLANTS TUBERS WEIGHT

Oakburn 90da 125 447 ' 61 Yorkton 90da 119 789 111 Abernath 90da 94, 293 61 Lethbrid a 90da 120 1340 165 uill 90da 94 791 108 Average lgm/SOL 110 732 101 2: 1 m/lOL YIELDS:
ALL STTES

Oakburn 90da 131 619 11'2 _. ~a~~~$ _-____.__._.__________~~- _:_____..~_:___-.._-___~_____..___:.
_ ..__.__._ uw Abernath 90da 95 311 ' 65 Lethbrid a 90da 119 1346 169 2~ uill90da 114 876 120 Average 1 gmllOL L17 843 121 3: 1 mlL YIELDS:
ALL SITES

TREAT PLANTS TUBERS WEIGHT

2S Oakburn 90da 130 612 116 Yorkton 90da 122 897 127 Abernath 90da 100 322 81 Lethbrid a 90da 120 1296 ' 185 uill 90da 98 836 117 3~ Average lgmlL 114 793 121 3: 10 m/L YIELDS:
ALL SITES

TREAT PLANTS TUBERS WEIGHT

Oakburn 90da 132 607 122 3S Yorkton 90da 132 932 122 Abernath 90da 99 362 77 _ Lethbrid a 90da 118 1204 144 uili 90da 10i 863 110 Average lOgmfL 116 794 115 ' -13-Control YIELDS: ALL
SITES

TREAT PLANTS TUBERS: WEIGHT

Oakburn 90da 126 384 54 Yorkton 90da 117 986 134 $ Abernath 90da 92 196- 31 Lethbrid a 90da 120 1276 170 uill 90da 111 804 99 Average Control 113 729 98 lO SUMMARY YIELDS: ALL
SITES

TREAT PLANTS TUBERS WEIGHT

Avers a 1 m/50L 110 732 101 Avers a 1 mllOL 117 843 121 Avers a 1 mIL 114 793 121 1$ Avers a 10 m/L 116 794 115 Avers a Control 113 729 98 Avers a all 94815115 790 114 ~orkton-tnbers froze-in-thewsoir~rence-rhizoctoxia-~aCav at ~iseit~ -- --... _ _.
_. ._..

uill Lake: no rhizoctonia data su lied.

Table 2 Oakburn a nd Abernath~ umma 90 dad Tuber Rhizoctonia S

1 m/50L Number of tubers with Rhizoctonia.;
2 locations 0% <1% 1-5% 5-10% >10%

Oakburn 90da 248 128 67 14 0 Abernath 90da212 54 27 0 0 Average 1gm150L230 91 47 7 0 1 mllOL Number of tubers with Rhizoctonia:, 2 locations 0% <1% 1-5% 5-i0% >10%

Oakburn 90da 403 154 61 0 0 Abernath 90da244 43 24 0 0 Average lgmIlOL324 99 43 0 0 1 m!L Number rs with of tube Rhizoctonia, 2 locations 0% <1% I-5% 5-10% >10%

Oakburn 90da 514 89 9 0 0 Abernath 90da253 68 1 0 0 Average lgm/L384 ?9 5 0 0 -14=
m/1L Number of tubers'hizoctonia 2 locations 0~ <1% 1-S% 5-10% >10%

Oakbura 90da 477 113 14 0 O

Abernath 90da301' 53 8 0 0 $ Average lOgmllL389 83 11 0 O

Control Numbet of tubers W th Rhizoctonia 2 locations 0% <l% 1-5%a 5-10% >10%

Oakburn 90da 129 118 100 37 0 10 Abernath 90da96 49 34 17 0 Average control113 84 67 27 0 Summar Number exs with of tub Rhizoctonia 2 locations p% ' G1/b 1-S% S-10% >lO%
-Average lgm/50L23Q 91 47 7 0 Average lgm/lOL324 99 43 0 O

-. average 1gm/~__ .-~.~_..__ _._-.,~__._ _ _ _._ ._ . _-._____ _._.. . _ ~._ __ _ -_.~ _ _ _ _ ..._ _..~
_ .

Average lOgm/1L389 83 11 0 0 Average control113 84 67 27 O

This example demonstrates the usefulness of the saponins of the invention in preventing and/or minimizing the effects of Rhizoctonia, and further shows that the saponin treatment increases potato yields. Other data conf'ums that. better results are obtained when the sapoliin treatment is carried out on freshly cut (non-suburized) seed pieces, or on whole, uncut seed; versus suburized seed pieces.
Example 3 In this example, various potato seed piece treatments were compared for their effectiveness in the control of Rhizoctonia, Fusarium and Helrrainthosporium solani.
The materials tested are set forth in Table 3; where "Trigger" is a saponin dispersion in accordance with the invention. All products save for the saponin dispersions are commercially available or- experimental products designed for control of potato diseases.
METHODS: The test site was located in New Glasgow, Prince Edward Island.
Soil type was a sandy loam with a pH of 6.0 and an organic matter content of 2.8%.
The potato seed (Atlantic variety) used in the tudy had high levels of Rhizoctonia and was also infected with Fusarium by dipping the cutting knives in a slurry of diseased tissue before each cut. Seed was cut by hand on May 17, 2000 and seed treatments were applied on May 18, 2000 by shaking the seed pieces and the appropriate treatment in a plastic tote for two minutes: The saponin dispersion and in-furrow treatments were applied at planting on May 22, 2000. In-furrow treatments were dripped over uncovered seed .pieces prior to row closure using a 1 row, hand-held, COZ
sprayer equipped with a drop tube. The saponin dispersion was applied by placing cut seed in the saponin dispersion (10 g dry saponin dispersed in 10 L water) for 30 seconds. After 30 seconds seed was removed from solution and allowed to dry prior to planting.
Control treatments received no treatment. Treatments and rates of all products are outlined in Table 3. Each treatment was replicated 4 times in a randomized complete block_design. Seed was planted into-rows spaced 0.9.rn_apart_w th_a seed spacing of 35 cm and a depth of 15 cm. Plots were 6 ~ long and 4 rows wide. 15-15-15 fertilizer was banded during planting at a rate of 775 kg/ha. Insects were controlled with an in-furrow application of Admire (imidacloprid) at 850 ml produet/ha at planting. Weeds were controlled with Sencor (metribuzin) at 2:0 kg productlha on June 4, 2000, and Fusilade II (fluazifop-p-butyl) at 2.O L productJha on July 13, 2000. A regular preventative foliar, fizngicide program was maintained throughout the growing season. In-season data collected included plant emergence, stem counts, vigor, final stand count, incidence of Fusarium in non-emerged seed pieces, and Rhizoctonia canker on stolons.
Plant emergence, stem counts, vigor and final stand count was based on all plants in the two center rows of the plots. Incidence of Fusarium was completed by digging up non-emerged seed pieces in the two centeir rows. The non-emerged seed piece was identified as being infected by Fusarium, infected with some other pathogen or as a miss (blind seed piece). The percent Rhizoctonia canker rating was taken based on 5 plants per plot. Plots were top-killed on September 10; 2000, using Reglone (diquat) at 2.0 L product/ha and harvested on October, 4, 200Q. Total and marketable yield was determined at harvest. From the harvested tubers, 10 random tubers per plot were collected for determining the incidence of Silver Scurf (F.~elminthosporium) and Rhizoctonia after a period in storage. Each 10 tuber sample was placed in a plastic bag with holes punched in it and a wet paper towel was added to each bag to increase the humidity. All samples were stored in the dark until ratings were conducted.

-rs-Rhizoctonia and Silver Scurf ratings were completed on November 14, 2000.
Silver Scurf tuber ratings involved looking at each uber with a microscope and identifying the fungus as present or not present. Rhizoctonia tuber ratings involved washing the tubers and estimating the percent area covered by the-fungus. Data was subjected to analysis of variance and mean separation was conducted where analysis indicated significant differences at the O.OS level.
RESULTS: No significant differences were seen in emergence or final stand between any of the treatments. Two vigor assessments indicated all the treatments had higher vigor than Trt.#2 (infected check), however, not significantly different. A stem count indicated significant differences between some of the treatments. Txt.#7 (Maxim MZ) had the. highest number of stems, while. Trt_#9_ and Trt.#
10,,produced_the. least number of stems. Assessment for incidence ofFusarium on non-emerged seed pieces indicated that no Fusarium was seen in any of the treatments. No significant differences were seen in Rhizoctonia stolon canker, however, Trt.#4 (TM-10%) produced slightly better control. Crop harvest revealed no differences in tuber yield or number of tubers in any size category between anyof the treatments. Disease incidence at harvest yielded no significant amounts of late blight or Fusarium.
Following approximately 6 weeks in storage Silver Scurf was easily visible on the tuber surface. -Results indicated Maxim PSP provided 100% control of Silver Scurf infection, while Maxim MZ provided similar results. Both checks had nearly 50% infection, much higher than most treatments, indicating all treatments provided some control of the fungus. Maxim and Maxim MZ also provided the best control of Rhizoctonia on the tuber surface. All other treatments were not significantly different in their control of Rhizactonia as compared to the checks. Treatments 3 and 10 did not provide any control of Rhizoctonia, while all other treatments provided some control when compared to the checks, although not significantly different.
CONCLUSION: With the exception of slight differences in stem numbers, no differences between treatments were observed during the growing season or at harvest.
Significant differences between the treatm~ts became evident when storage ratings of Rhizoctonia andHelminthosporium were conducted. MaximPSP and Maxim MZ gave much better control ofHelminthosporium and Rhizoctonia on harvested tubers than any _I7_ other treatment. All other treatments gave some control.ofHelmihthospo~ium and all treatments except Nos. 9 and lU provided some control ofRhizoctania.
This example demonstrates that'the saponin product of the invention is at least equivalent to commercially available products. However; the cost of the saponin dispersions is much less than the commercial products. Therefore, effective control can be obtained at less expense and using a preferable natural source product.
Table 3 Treatments Used in Study Treatment Pioduct Application Rate 1 1 Healthy Check -----2 2 Infected Check -----3 3 PST - Mancozeb 10g/kg seed 4 4 TM - I O% 5gfkg seed 5 5 TM 2.5% and MancozeblOgllcg seed 6%

6 6 Maxim PSP Skg/kg seed 7 7 Maxim MZ Sg/kg seed 8 8 Trigger liquid seed dipped in lg saponin/L
treatment H20 9 9 Treatment No: 9 SOmI/I OOm2 IO 10 Treatment No. 10 50m1/IOOm2 IOg/kg seed Treatment Nos. 9 and 10 are experimental potato treatment agents.
Example 4 In this example, potato seed pieces were treated with various compositions to :determine the effect thereof upon the control of late blight. The following Tables 4 and 5 set forth the treatment protocol and the coating compositions, respectively.

_lg_ Table 4 Series 2000, Late Blight Seed Treatment Prosper, ND Non-Irrigated Trials; 2000 Plot Design: 2 X 25 ft. rows; 4 replications/treatment;
RCBD.

Planting Date:May 25, 2000 Row Width: 38 inches.

Plant Spacing:I2 inches:

Cultivar: Norvalley Fertilizer: 250# 24-12-5 banded at planting., Herbicide: Treflan (previous fall) Poast.@ 1.5_pt/a on July 10, 2_000 Matrix @ 1.S oz:/a on July 10, 2tJ00 Insecticide: Admire @ 18 oz./a banded at planting Asana @ 6 oz./a broadcast on July 10, 2000 InoculationMethod:
LateBlight:Fortrials inclusive, 200 fresh cut seed pieces were tumbled with 7 infected seed pieces, infected pieces were then removed and seed was immediately planted; for trials 2001 and 2009-2011, inclusive; induced airborne infection by planting infected plants adjacent the trials.

Early Blight:Natural infection Fungicide Application ates: July 9; July 13, July 20, July 27, August D 3, August 10, August 18; August 24 - -Vine-kill: August 30, 2000, rotobeat.

Harvest: September 16, 2000.

Grade: September 18, 2000:

NOTE: Due to heavy rain on June 19, 2000 replications I and II were destroyed.

Table 5 Late Blight Seed Treatment Prosper (2000 Series) Acc. # Treatment Rate NOTES

2001 Uninoculated, UNTREATED

......... _ _ .... ..
~

2002 Inoculated, __ UNTREATED
' .:
i . .. 12 oz/cwt 2003 Inoculated, Fungicide #1 _ ': ~:.: '" ' ' !
~~

2004 Inoculated, Fungicide 8 oz/cwt #1 ~~ ~ ' ,~ ~ ~ t ;
.

2005 Inoculated, Fungicide 8 oz/cwt #2 ' ~ ll' ' ! : , :,:: . 8 oz/cwt .. : . ' a 2006 Inoculated, Fungicide #3 ~ ~ _ _ :.
._.._._..,~_ ...:._~_ _ ~_ _,.:. _.

2007 Inoculated, Funycide 8 oz/cwt #4 "~ " _ ~ ~
' "
'~

' 16 oz/cwt .
.
I. . .
2008 Inoculated, Fungicide#5 . ~ ~ ! ,:
~ ;~ x 2009 Uninoculated, Trigger I g/L no foliar fungicide (60 sec dip) ... ..... ,.,._. _..:. _v:.:e..,~. ... ' ... ... .._ _.""F, :c. . ..
_ _. . . _ .':

2010 Uninoculated, Tngger 1 g/L Bravo or MZ foliar, (60 sec dip) ' full season ': .
[

:. . . .;;' :. ., a :'. . . ~ ':': no foliar fungicide V ., ' .
2011 Uninocuiated, Trigger 2 glL
(60 sec dip) Variety: Norvalley Planted: 5/25/2000 The "Trigger" products' were Saponin dispersions in accordance with the invention; all other products were experimental fungicides.
Test results demonstrated that use of the "Trigger" products gave a beneficial effect in controlling late blight, and it is believed that the product will have substantial utility in this context; particularly when used in conjunction with foliar applications through the growth period:
Fig. 3 is a graph depicting the marketable yields for the various treatments.
It should be noted that the yield using Treatment #2009 was slightly greater than that using Treatment #2010. However, given the fact that Treatment #2009 involved no application of foliar fungicide, it will be seen that the Trigger product itself protected the plants. This is a substantial advantage inasmuch as the cost of repeated foliar fungicide applications was saved.

Example 5 In this example, the saponins of tile invention were tested for potato late blight control at Outlook, Sk. The test was conducted exactly as set forth in Example 2.
However, late blight infection set in during the growing season: Data was then recorded on the top growth, and mechanical top killing on August l U, 2000 destroyed the top growth. The tubers were left in the 'ground and harvested on September I, 2000, and fmrther data recorded. The following Table 6 sets forth the important data.
Figs. 4-6 illustrate further the important data derived from this test.
Table 6 Data Averages Average-of Reps_for.Each Tr~tment -_ _OutLatnk,_ Sk. ._ _ . _ TreatmentMainstemLate EariyblightBlack Canker LeafrollBlack # (# / blight(/u) leg (# of ( # scurf Plot) (%) (# Plaats)of (%) of Plants) Plants) i:Igm/SOL104 18.75 3.75 1 0 0 0.00 2:Igm110L101 17.50 2.50: 0 0, 0 0.00 3:lgm/1L104 15.00 1.25 0 0 0 0.00 4: IOgm/IL106 18.75 2.50 0 ' 0 0 0.00 ' CONTROL98 23.13 2.501 1 0 0 0.00 The data from this test also indicated that.the treatment of the invention had an effect in controlling leaf roll virus.
Example 6 In this example, the saponin products of the invention were used to treat elm trees infected with Dutch Elm disease. Six elm trees.in Winnipeg; Mn. were discovered showing signs of Dutch Elm disease, ranging from 20-45 % infected. The diseased trees were injected with a sapon?n dispersion (1 g saponinlL H20) prepared as described in Example 1. The application rate was 1 g of dry saponin/cm of trunk diameter at breast height. The dispersions were conventionally injected at the root flare, with the trees absorbing the dispersion within about 48 hours. The treated trees were monitored every day for approximately six weeks. The trees remained stable and exhibited no further wilt, flagging or leaf loss.

_,, CA 02482090 2004-09-22 .~
' -2I-Example 7 In this example, the efficacy of several commercially available compositions was compared with that of the inventive composition (an aqueous solution consisting essentially of saponin extracted from quinoa) in controlling bacterial spot in tomatoes, more specifically transplanted greenhouse tomato plants (tomato cultivar BHN-555).
The experiment was conducted at a farm located in Quincy, Florida. The tomato plants were transplanted from the greenhouse into plots of 20 plants arranged in a single row with 50 x 180 cm plant spacing in randomized complete block design.
Five-week-old seedlings were transplanted on to raised beds previously fumigated with methyl bromide (67%) and chloropczin (33%) and covered with white palyethyl~ mulch. The plants.were drig_irxiga ed, staked, and fertili~est vyith19~_-6.0 195 lb/acre N-P205-KZO. Foliar spray applications initially employed a spray volume of 26 gpa and were increased to a maximum of 65 gpa. Disease severity was assessed three times for bacterial spot (caused byXanthomonus campestris pv, vesicatoria) and IS target spot (caused by Cory~cesporia cassiicola) over approximately a three month period. Fruit was,subsequently harvested from 12 plants per plot.
The specific treatment methods are described in detail in Table 7. Each treatment method described below was replicated four times during the experiment.

Table 7 Treatment Description Control Untreated control KOCIDE DF KOCIDE at a concentration of 4:8 g/L
+ and MANZATE also at MANZATE' 4.8 g!L were applied weekly, as a foliar spray.

ACTIGARD ACTIGARD at a concentration of 60 mg/L
was applied every 14 days as a foliar spray; for a total of six applications.

Q.S.1 Quinoa saponin at a concentration of 1 g/L applied one time as a root dip immediately before to transplanting.

Q.S. 2 Quinoa saponin at a concentration of 1 g/L applied one time as . . . . . . a,-~Li~ applicati,or~ 5:.days be~oreara~asplanting:
. . ... ..

Q.S. 3 Quinoa saponin at a concentration of 1 g/L applied twice, once as a foliar application 5 days before transplanting and once as a root dip immediately before transplanting.

Q.S. 4 Quinoa saponin applied one time as a foliar spray three days after transplanting:

' A fungicide (manganese ethylene bisdithiocarbamate) available from Griffin, L.L.C.
The results of tomato plant trials are shown in Table 8 below. The effectiveness of the treatment method is indicated by the combined severity of bacterial and target spot diseases.
Table 8 Disease Severity Treatment 7 weeks after9 weeksafter 11 weeks after tans lant: traps lant traps lant Control 2.6: 13.4 69.9 KOCIDE DF 1.4 4.4 42.1 +MANZATE 75DF

ACTIGARD 1.7 7.3 61:7 .S. 1 2: 3 8:7 60:9 Q.S.2 2:0 8:1 64.0 .S. 3 4:0 9:9 53:9 Q.S. 4 2:0 7.3 59.3 -~ CA 02482090 2004-09-22 As expected, the untreated control plants exhibited the greatest disease severity, with almost 70% of the plants affected, and the KOCIDE and MANZATE treated plants exhibited the lowest disease severity. However, most notable is the comparisonbetween the ACTIGARD and quinoa saponin treatments, both designed to elicit a protective response from the tomato plant's own immune system. In most cases, the quinoa saponin treatment method performed ; comparably with or even outperformed the ACTIGARD treatment method with the best results obtained by the Q. S, 3 method. The Q.S. 3 method also presents the advantage that all treatment was performed in the greenhouse thereby eliminating the need for application in the field.
_ _ Fxam~ple 8 . _ : . _ _. . _ . .
In this example, the efficacy of several commercially available biocontrol compounds (ELEXA; MYCONATE and MESSENGER) for controlling common scab (Streptomyces scabies) in potatoes was compared with a preferred inventive saponin solution extracted from quinoa (Q:S.). MYCONATE; available from VAMTech LLC, Lansing, Michigan, is a water-soluble formulation of the potassium salt of 4'-methoxy, 7-hydroxy isoflavone, which is isolatedfrom theroots of clover plants that were stressed by phosphorous deficiency. ELEXA; available from Glyco Genesysis, Boston, Massachusetts, is a complex carbohydrate formulation containing no toxic active ingredients which inhibits fungal infections in a variety of plants.
MESSENGER, available from Eden Bioscience Corp., Bothell, Washington, is a harpin protein containing composition.
The experiments were performed in Bath, Michigan. Potatoes with minimal surface scab were selected for use in this example, and potato seeds were prepared by cutting two days prior to planting. The seeds were planted two-row by 20 ft.
plots with approximately 10 in. between plants so as to give a target population of 50 plants at 34 in. row spacing. The planting pattern was replicated four times in a randomized block design: The two-row beds were separated by a 5 ft. unplanted row.
In half of the trials, application of the biocontrol compound occurred in furrow over the seed at planting. Tn the in furrow trials, tfe compound was applied with a R&D
spray boom delivering 5 gaI/acre (80 psi) and one spray nozzle per row. In the remaining trials, the biocontrol compound was applied as a foliar spray. The first foliar application occurred approximately 5 weeks after planting with a second occurring a week later. Both applications were performed with an ATV rear-mounted R&D
spray boom delivering 25 gal/acre (80 psi) with three nozzles per row.
Weeds were controlled byhilling and with the following herbicide applications:
DUAL 8E at 2 pt/acre 10 days after planting (DAP), Basagran at 2 pt/acre 20 and 40 DAP, and POAST at 1.5 pt/acre 58DAP. Insects were controlled by application of the following insecticides: Admire 2F at 1.25 ptlacre at planting, SEVIN 80S at 1.25 lb./acre 3l and 55 DAP, THIODAN 3 EC at 2.33 pt/acre 65 and 87 DAP, and POUNCE 3.2EC
at 8 oz/acre 48 DAP.
Fertilizer vvas .drilled into the phts hefore planting. end formulated based on_.the results of soil testing. Additional nitrogen was applied o the growing crop with irngation 45 DAP. Once the plant canopy was about 50% closed, Bravo WS 6SC
fungicide was applied at a rate of 1.5 pt/acre on a seven-day interval for a total of 8 applications. A permanent irrigation systemwas established prior to the commencement of fungicide sprays and the fields were maintained at soil moisture capacity throughout the season by frequent (minimum S day) irngations: The vines were killed with REGLONE 2EC at 1 pt/acre approximately 14 weeks after planting. The plots were harvested one week after the vines were killed.
Of the biocontrol materials tested, each was applied :as an in furrow treatment and as a foliar treatment: In furrow applications were made over the seed at planting using a single nozzle R&D sprayboom delivering S gal/acre (80 psi) and using one spray nozzle per row. Foliar applications were applied' were performed approximately 5 and 6 weeks after planting using an ATV rear-mounted R&D spray boom delivering 25 gal/acre (80 psi) and using three spraynozzles per row. The application rates for each agent is given in Table 9 _25_ Table 9 Treatment ApplicationApplication rate Method ELEXA in furrow Manufacturer's suggested application rate.

MESSENGER in furrow 1.4 oz per 1000 ft. of row Q.S. in furrow 0.9 oz per 1000 ft. of row MYCONATE in furrow Manufacturer's suggested application rate.

ELEXA foliar Manufactarer's suggested application rate.

MESSENGER foliar 0.42 lbs. per acre per application Q.S. foliar 0.25 lbs. per acre per application MYCONATE foliar Manufacturer's uggested application rate.

The results of the potato scab treatments are shown in Table 10. Effectiveness of the treatment was determined by measuring the percentage of the surface area of the tuber which the scab affected. Tubers exhibiting less than 5% affected surface area were considered to be marketable:

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00pp ~ N 00 cn , ~ "_, r,.,~"-, N

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:
.-i M d' - d' V1 d' ~ d' V1 d' _~_.M - ~- V7 -O M- M..p _ _ _ ~

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v', o ~n _27_ The results of this trial indicate that the quinoa saponin solution was extremely effective in controlling common scab when applied as a foliar spray. About 72.6% of all tubers treated in this manner showed 5% or less affected urface area. This method also showed the smallest average affected surface area of all methods tested.
S
Example 9 In this example, the efficacy of several experimental systemic acquired resistance (SAR) compositions in controlling bacterial spot (Xanthomonas campestris pv.
vesicatoria) and bacterial speck (Pseudomonas syringae pv. tomato) in tomato plants was determined. The SAR compositions tested are noted in Table 11.
Table 11 SAR Composition Active Ingredient Sapogenin l 85 mg of Phytolaccagenic acid Sapogenin 2 85 mg of I-Iederagenin Sapogenin 3 85 mg of Oleanolic acid Sapogenin 4 170 mg of Phytolaccagenic acid Sapogenin S 170 mg of Hederagenin Sapogenin 6 170 mg of ~leanolic acid ACTIGUARD SOWG Acibenzolar Tomatoes were seeded in a green house and the SAR compositions applied three separate times, first as a drench nine days after seeding, the second at the 2-leaf stage 15 days after seeding, and finally as a foliar application in the filed approximately two months from seeding. Each treatment was followed up by a 5 ppm application of a plant growth regulator comprising paclobutrazol available under the name BONZI from Syngenta, Greensboro, NC. The tomatoes were transplanted in single twin-row plots, seven meters in length with rows spaced 1:65 m apart. This procedure was replicated four times in a randomized complete block design. The seedlings were transplanted approximately 1.5 months after seeding using a commercial transplanter. The foliar applications were applied using a specialized small plot research COZ sprayer with a CA 02482090 2004-09-22 ~'~

three nozzled hand-held boom applying 200 L/ha of spray mixture. Foliar bacterial disease assessments were made prior to transplanting and in the field approximately 3: 5 months after seeding. Phytotoxicity ratings were also taken in greenhouse prior to transplanting. The results of this trial are noted in Table I2.
Table 12 GreenhouseEvaluationsField Evaluations Application Treatments Rate Bacterial Foliar VigourFoliar Disease Cluster Ratings Ratings counts (0-10) (0-10) Sapogenin lg 12.3 57 6.8 + product/L

BONZI4 5ppm I5 Sapogenin 1 g 19.0 5.3 6:8 + product/L

BONZI4 5ppm Sapogenin l g 2.7 4.7 6.5 + productlL

BONZI4 5ppm Sapogenin lg 19:7 4.3 7.8 + product/L

BONZI4 Sppm Sapogenin lg 23.3 4.3 8.0 + product/L

BONZI4 5pprn Sapogenin lg 31.3 4.3 8.0 + product/L

BONZI4 5ppm ACTIGUARD 30 ppm 7:7 2.7 7.8 BONZI 4 5 ppm Control - ' 32.3 6.0 5.8 (no treatment) Bacterial Cluster Counts: number of bacterial lesions observed an a total of 100 tomato seedlings. The lower the number the more effective the material.
Foliar Vigour Ratings (0-10) - 0, severe foliar injury; 10, foliage green in excellent health.
Foliar Damage Ratings (0-10) - 0, no control, foliage severely damaged; 10, complete control.
The sapogenin compositions all proved effective in disease control versus the control. Sapogenin compositions 4-6 exhibited particularly good foliar disease ratings performing as well or better than the ACTIGUARD treated plants.

Claims

1. A method of protecting a plant or seed from disease comprising the step of applying a composition to said plant or seed comprising from about 20-80 %
by weight of a saturated or unsaturated compound having the formula where R1-R2 are independently H; OH; CH3, CH2OH, or CHO, so as to elicit a protective response in the plant or seed.

2. The method of claim 1, said composition being applied to said plant as a foliar spray.

3. The method of claim 1, said composition being applied to said plant by dipping at least a portion of the roots of the plant in said composition.

4. The method of claim 1, said composition being applied to said seed by at least partially coating the seed with said composition.

5. The method of claim 1, said plant being a tomato or potato plant.

6. The method of claim 1, said seed being a tomato seed.

7. The method of claim 1, said compound being a member selected from the group consisting of oleanolic acid; hederagenin, phytolaccinic acid, and quillaic acid.

8. A method of protecting a plant or seed from disease comprising the step of applying a composition to said plant or seed consisting essentially of a saturated or unsaturated compound having the formula wherein R1-R9 are independently H, OH, CH3, CH2OH, or CHO, so as to elicit a protective response in the plant or seed.

9. The method of claim 8, said composition being applied to said plant as a foliar spray.

10. The method of claim 8, said composition being applied to said plant by dipping at least a portion of the roots of the plant in said composition.

11. The method of claim 8, said composition being applied to said seed by at least partially coating the seed with said composition.

12. The method of claim 8, said plant being a tomato or potato plant.

13. The method of claim 8, said seed being a tomato seed.

14. The method of claim 8, said compound being a member selected from the group consisting of oleanolic acid, hederagenin, phytolaccinic acid, and quillaic acid.