AU609073B2 - Method for treating cereal crop seed with chitosan to enhance yield, root growth, and stem strength - Google Patents

Description

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I'ELCULPOERYOG~ZTQ

AU-AI-81575/87

PCT

INTERNATIONAL APPLICA1 WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau rION E lELNE THTPATT COOPERATION TREATY (PCT) (51) International Patent Classification 4 l)Ynternational X lication Number: WO 89/ 01288 A01N 43/16 Al (43) International Publication Date: 23 February 1989 (23.02.89) (21) International Application Number: PCT/US87/01971 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (22) International Filing Date: 14 August 1987 (14.08.87) pean patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), SE (European patent), (71) Applicant (for all designated States except US): WASH- SU, US.

INGTON STATE UNIVERSITY RESEARCH FOUNDATION, INC. [US/US]; Washington State University Pullman, WA 99164-1045 Published With international search report.

(72) Inventor; and Inventor/Applicant (for US only): HADWIGER, Lee, A.

[US/US]; NW 330 Joe Street, Pullmann, WA 99163

(US),

(74) Agents: CANTOR, Herbert, I. et al.; Wegner Bretsch- 7 7 AR neider, 1233 20th Street, P.O. Box 18218, Wash.- 2 7 AP 1989 ington, DC 20036-8218 (US).

AUSTRALIAN

9 MAR 1989 This document contains the PATENT OFFICE amenme ments male under Section 49 and is dorrect fur printing, (54) Title: METHOD FOR TREATING CEREAL CROP SEED WITH CHITOSAN TO ENHANCE YIELD, ROOT GROWTH, AND STEM STRENGTH (57) Abstract Commercially produced chitosan applied to cereal crop seeds at rates of 60 pg to 1000 p.g per gram of seed enhances root development, crown diameter, mature straw strength and crop yield. Dry chitosan, when dissolved in dilute acid and neutralized, is applied directly to cereal crop seed with only minor modification to seed treating machinery and methods, In addition to a clear benefit in cereal crop yield, the chitosan treated seed can be planted early to reduce erosion and it can be planted in regions having soil infested with root rotting organims and not suffer extensive lodging that would prevent seed recovery by commercial harvesters.

WO 89/01288 PCr/US87!01971 -1- Method for Treating Cereal Crop Seed With Chitosan to Enhance Yield, Root Growth, and Stem Strength This appli-eat-ion is a continuation-in-part -ofmy- U.S. application Serial No. 795702, f ovember 1985, which was a cont' i n of my U.S. application Serial N ,084, filed October 5, 1984 and now a andoned.

Field of the Invention This invention pertains to methods for treating the seed of cereal crops, which are defined as members of the grass family (Graminae) that produce edible, starchy grains and are characterized by long, narrow blades, Wheat, barley, oats, rye, and rice are cereal crops.

Background of the Invention One of the major problems in growing cereal crops is the lodging (falling over) of plants prior to harvest, which prevents the mechanical recovery at harvest of high yielding plant heads. Researchers have tried to alleviate this problem by: 1. Breeding stiffer stemmed varieties; 2. Using chemical treatments such as benzamidazoletype fungicides (Benlate) to reduce root rot; 3. Recommending that planting dates for winter crops be delayed so that organisms have minimal time to initiate the root rotting process prior to the slow growth phase of winter; and 4. Recommending no-till or minimum tillage procedures that leave crop debris to reduce the erosion that is rampant when planting dates are delayed.

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WO 89/01288 PCT/US87/01971 -2- Each of these alternative procedures are partial solutions to yield losses. However, each has serious drawbacks.

It has been impossible to breed for straw strength and still retain all of the other desirable agronomic traits, winter hardiness, milling quality yield, disease resistance, etc., at the same time because the high yielding heads place unusually severe strain on the plant stem.

Lodging has been reduced by fungicides that are derivatives of methyl l-(butylcarbamoyl)-2-benzimidazole carbamate (Benlate), which reduce the incidence of root rotting and thus retain original straw strength.

Extended use of this chemical, however, has resulted in the selection of fungal pathogens that are resistant to its fungicidal effect and, thus, effectiveness is greatly diminished. Only emergency Food and Drug Administration clearance has been obtained in the United States of America for use of this chemical on wheat because of potential side effects. Finally, its cost of treatment is very high.

Delaying the planting date has reduced lodging; however, it prevents the time dependent development of large seedling plants needed for ground cover prior to the onset of winter rains and erratic snow melts that erode away large tonnages of soil each year. The latter problem can be reduced by minimal tillage practices that leave straw on top of the soil at planting time.

j However, minimal tillage, which produces generally lower yields, leaves weeds untilled as well and nust be accompanied by additional herbicide and pesticide treatments requiring expensive machinery for application.

Accordingly, a need exists for a method that will increase the straw strength and the root development of WO 89/01288 PCT/US87/01971 -3cereal crops at a commercially feasible cost while still maintaining or increasing the amount of yield.

Summary of the Invention This invention comprises a novel chitosan seed treatment that strengthens the stems of cereal crop plants, such as wheat, oats, barley, rye, and rice, helping to preserve their water-carrying capacity, greatly reducing lodging (plants falling over before harvest), and increasing yield. The lodging problem is most severe for winter crops when seed is planted early so that the plants can cover the ground prior to the soil eroding winter rains. Thus, by using this seed treatment, farmers will be able to plant crops early and reduce the erosion loss that exceeds 9071.8 kilograms per 0.4047 hectare in some parts of the United State of America.

While this invention is applicable to any of the cereal crops, primary work has been done with wheat, barley, and oats and this specification will discuss the invention using these cereal grains as an embodiment.

The problem of lodging of high yielding cereal plants can be rectified by seed treatment with the naturally occurring carbohydrate, chitosan. Commercially produced chitosan when applied in an aqueous form to cereal seeds is able, under field conditions, to greatly increase the development of the plant's root system, to substantially increase the diameter of the stem, and, in association with these specific and other intangible morphological and biochemical developments, to enhance yield. The chitosan treatment results in a plant that is beneficial to erosion control, resi ftant tq lodging, and superior in yield over non-treated plants. 4 the method of treatment comprises the direct application of chitosan derived from various shell sources, such as crab, lobster, shrimp and other marine life, in a nearly lllIll--NI -4neutral aqueous solution to wheat seeds prior to planting.

Accordingly, it is a primary object of the present invention to provide a method for increasing the straw strength and root development of cereal crops while increasing the amount of yield.

This and further objects and advantages will be apparent to those skilled in the art in connection with the detailed description of the preferred embodiments set forth below.

According to a first embodiment of the present invention there is provided a method of enhancing crop yield, straw strength, stem diameter, and root development of rice comprising applying chitosan in a neutral aqueous solution to rice seed at a rate of from 60uto 1000,ugof chitosan per gram of seed prior to planting.

According to a second embodiment of the invention there is provided S rice seed treated by the method of the first embodiment.

S~1 According to a third embodiment of this invention there is provided seed treated with chitosan in an amount effective to enhance yield, straw strength, stem diameter, and root development of plants ggerminating from said seed by applying chitosan in a neutral aqueous solution to seed at a rate of from 60p 3 to 10000gof chitosan per gram of seed prior to planting, said seed being selected from the group consisting of barley, rye, oats and S:rice.

According to a fourth embodiment of this invention there is provided a method of enhancing yield, straw strength, stem diameter, and root development in plants comprising the step of applying chitosan in a neutral aqueous solution to seed at a rate of from 60p to 1000gof chitosan per gram of seed prior to planting the chitosan being produced by deacetylating chitin to an extent exceeding 90% and wherein the seed is selected from the group consisting of barley, rye, oats and rice.

According to a fifth embodiment of the present invention there Is provided a method of enhancing yield and preventing lodging of plants comprising the steps of: dissolving in a dilute aqueous acid chitosan that has been produced by deacetylating chitin to an extent exceeding 90%, in order to form an aqueous acid solution of chitosan; neutralizing the aqueous acid solution of chitosan to a pH no greater than i~ -E Utl- 4A coating seed with the neutralized aqueous acid solution of chitosan at a rate of from 60pgto 0010E of chitosan per gram of seed prior to planting; and drying the seed to leave a cellophane-like surface chitosan on the seed; and wherein the seed is selected from the group consisting of barley, rye, oats and rice.

Detailed Description of the Preferred Embodiments Chitosan is a polymer made up of a hexosamine sugar (glucosamine) whose molecules are linked 4) into chains that can exceed molecular *S 0 S weights of one million. Chitosan compounds in a range of up to and exceeding 1 x 106 molecular weight are derived commercially from chitin.

SChitin, an amino cellulose derlvite, is the second most abundant polymer S occurring in nature, existing, for example, in the cell walls of fungi, bovine cartilage, and the hard shells of insects and crustaceans. Wastes from the shrimp, lobster, and crab seafood industries contain 10-30% chitin. Chitosan is produced by deacetylating chitin. This invention is effective if the deacetylatlon exceeds about 90% and it is most effective when approaching 100% deacetylatioh.

i2 Dry chitosan as either flakes, granules, or powder is suitable as a starting material. The more finely dispersed the chitosan, the more quickly it can be dissolved in a dilute aqueous acid 1% acetic acid or dilute hydrochloric acid, sulfuric acid, or formic acid).

Typically, a quantity of chitosan suitable for planting a 64.75 hectare field (quarter section) of wheat would be mixed as follows: 1.089 kilograms of crab shell 0 i c i WO 89/01288 PCT/US87/01971 chitosan is dissolved in 30.28 liters of 1% acetic acid. 48 hours is allowed at room temperature for the chitosan to dissolve with stirring. H20 is then added to bring the volume to 98.42 liters or until the viscosity is reduced enough to feed through the dispenser of commercial seed treating machinery. (Most seed treatment machines utilized to handle water based seed treatments can be utilized with the aqueous solution of chitosan.) The solution is neutralized to pH 6.0 to 6.5 with 0.757 to 0.871 liters of 6.0 N NaOH. NaOH is added slowly with stirring, because localized changes to a pH higher than will cause the chitosan to precipitate. Once near neutrality, the viscosity of the opaque aqueous preparation of chitosan is reduced to the designed viscosity with H 2 0 to approximately 98.42 liters. From this, approximately 0.473 liters is added to each bushel (27.22 kg.) of wheat seed. This volume of aqueous chitosan increases the moisture percentage of 27.22 kilograms of wheat seed by 1.6%.

Chitosan seed treatments were found to be effective using rates ranging from 60 pg chitosan per gram of wheat seed to 1000 pg chitosan per gram of wheat seed. Optimal results were obtained at 250 ug chitosan per gram of wheat seed. This rate is 0.00635 kilogram chitosan per 27.22 kilograms (1 bushel) of wheat seed. 27.22 kilograms per 0.4047 hectare is the average seeding rate for wheat in most areas.

For barley, optional results are obtained at about a 60 ig per gram of barley seed. For oats, such results are obtained in the range 475 .g to 525 ug per gram of oats seed.

The native chitosan required to treat seed prior to planting is inexpensive. For winter wheat, chitosan treated wheat seed can be planted in late summer or as early as August as moisture is available. The chitosan treatment enhances stem diameter by approximately La j e~p~ i II I ICI IIIIC~-- WO 89/01288 PCT/US87/01971 -6- Although chitosan-treated plants grown in soils heavily infested with root rotting disease are susceptible to development of some rotting symptoms such as stem discoloration and white heads, the larger stem diameter and the extensive root system caused by the treatment maintain greater stem strength and an adequate water transporting capacity of stem vascular systems. As a result of seed treatment with this naturally occurring compound, 10-30% higher yields are obtained at a low cost, soil erosion is minimized, and the chitosan is readily degraded to simple amino sugar residues and/or metabolized by soil organisms.

Plants and micro-organisms contain chitosanase and other degradative enzymes with the potential to digest chitosan into smaller fragments and eventually into hexosamines that can be utilized as nutrients by soil microflora.

If send is treated in a humid environment, a posttreatment drying step must be added to reduce the moisture content of the treated grain to the 10-14% range in order to prevent premature germination of the seed; therefore, the more viscous the chitosan preparation, the less drying it will be required. Highly viscous chitosan preparations can be mixed with seed using any machinery marketed for cement mixing. Modifications of grain augering devices will also enable chitosan to be added to seed as it is being loaded aboard trucks just prior to transport to the field for planting. This eliminates the need for extensive drying to prevent seed 30 germination.

Chitosan seed applications are not detrimentally influenced by fertilizer supplements, herbicide applications or irrigation programs. Other commercial seed treatments, insecticides and fungicides, should be applied, prior to chitosan. Components already on the seed will be attached to the seed by the chitosan,

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WO 89/01288 PCT/US87/01971 -7which leaves a "cellophane-like" surface on seed after drying. The chitosan-treated seed can be planted directly in any commercial planter. Special planters that automatically administer fertilizers, soil sterilants, herbicides, etc. can be utilized to treat seeds with chitosan as they are being planted. Chitosan labelled with tritium, [3H]-Chitosan, added to seeds was translocated to the developing plant indicating that a large portion of the chemical is distributed systemically.

Dry chitosan can be stored indefinitely at room temperature without loss of biological activity.

Chitosan can be mixed as described above at room temperature. Chitosan has no known toxicity and can be supplemental to the diet of animals without detrimental side effects, The physical irritation properties of chitosan have not been investigated in long term studies, however, and, therefore, the same basic precautions taken in the handling of other fibrous materials or powders, cotton fibers or flour, may apply to chitosan.

The root enhancing, stem diameter increasing, and strengthening effect of chitosan seed treatment is seen at both early and late seeding dates for winter crops; however, the major beneficial effects for erosion reduction are obtained with early seeding dates. This allows the development of the large seedling plants needed for ground cover prior to the onset of winter rains and snow melts while the chitosan minimizes the problem of root rotting.

The following data illustrate examples of enhanced properties obtained in wheat, oats, and barley through the use of this invention.

PCT/US87/01971 WO 89/01288 -8- EXAMPLE 1. Enhanced Seedling Development Seedlings from chitosan treated Daws wheat seed (200 u~g chitosan/g seed) 4 months after planting at Washtuchna, Washington under circle irrigation, October 15, 1983.

Seed Treatment Ave. diameter of crown (lower stem) Ave. length of stem from crown to first leaf Ave. wt.

root system per, 30 plants fresh wt dry wt g

H

2 0 control 2.3 3.7 1.002 2.690 .139 .330 Chitosan (200 pig/g seed) 3.7 3.8 EXAMPLE 2. Reduced Lodging Daws winter wheat 1983 outside row of 4' x 100' plot.

crop lodging reading on Treatment Stems lodged g chitosan/grau seed no.

Chitosan 62 Control Chitosan 125 Control ChItosan 250 Co~ntrol Chitosan 500 275 468 313 948 1.43 835 186 WO 89101288 PCT/US87/01971 -9- Control Chitosan 1000 Control EXAMPLE 3. Increased Stem Diameter 652 250 410 Effect of chitosan seed treatment on stem diameter of Daws wheat at maturity 1983 Chitosan applied per gram seed Stem diameter increase g mm Chitosan 62 3.872a Control 3.239 19 Chitosan 125 3.432 Control 3.231 6 Chitosan 250 3.606 Control 3.322 9 Chitosan 500 3.997 Control 3.651 9 aAverage diameter of 100 stems.

i i PCT/US87/01971 WO 89/01288 EXAMPLE 4. Enhanced Yield Effect of Chitosan Seed Treatment on Daws Winter Wheat Yield in 1983 Application kg/bushel Avg. Increase (27.2 kg) yield Over seed kh/hectare Control Chitosan 1000 pg/g .0268 6066 .0a 14% Control 5299.3 Chitosan 500 pg/g .0132 6032.3 13% Control 5232.1 Chitosan 250 pg/g .0064 6341.7 21% Control 5662.5 Chitosan 125 pg/g .0032 6153.4 8% a Yield was an average of four replications. Plot size was 1.22 m x 9.14 m. Lodging in control plots was up to 60%. Yield included lodged wheat recovered by hand at harvest.

EXAMPLE 5. Enhanced Yield Fielder spring wheat treatment yield of control 1982 Chitosan (320 g/g) seed treatment only Control 100 I WO 89/01288 PCT/US87/01971 -11- EXAMPLE 6. Enhanced Yield Daws winter wheat treatment 1982yield of control Chitosan (500 pg/g seed treatment 107 Control 100 EXAMPLE 7. Enhanced Disease Resistance Effects of Chitosan Seed Treatment Daws Wheat Pseudocercosporella herpotrichoides Disease symptoms on Wheat Straw at Harvest No. of Straws/Sympton Value Category (Chitosan treatment and control) Symptom Value 0 1 2 3 4 62 Ug/g 4 22 35 22 17 Control 8 28 40 24 8 125 pg/g 5 13 24 12 46 Control 1 8 21 49 21 250 pg/g 2 15 19 48 18 Control 0 8 39 48 500 Pg/g 7 19 37 21 16 Control 0 4 35 59 7 1000 vg/g 0 8 32 Control 1 9 38 48 4 Mature Stem Symptom Values Clean Straw 0 Slight Discoloration 1 Scattered Lesions 2 Coalescing Lesions a 3 Diseased Broekn 4 PCT/US87/01971 WO 89/012,88 -12- EXAMPLE 8. Enhanced Yield KAMIAK BARLEY WINTER GROWN Whitlow Farm, Pullman, Washington TREATMENT kg/hectare OF CONTROL Controla 8135.20 1000 pg/S seedb 7568.77 93 500 pg/g seedb 8301.23 102 250 pig/g seedb 8276.79 101 125 pJg/g seedb 8P~60.43 110 62 ig/g seedb 10010.31 123 a Average of 10 plots (generally 1.22 m x 9.14 in); ch~itos--i was applied with water an equivalent amount of water without the chitosan was applied to the control plots.

b Average of 2 plots (generally 1.22 mn x. 9.14 mn).

EXAMPLE 9. Enhanced Yield CORRET OATS SPRING PLANTED Plant Pathology Farm, Pullman, Washington TREATMENT kg/hectare OF CONTROL controla 2485.47--- 1000 pig/g seedb 2734.57 110 500 pg/~g seedb 3344.90 135 Ipg/9 seedb 2758.95 ill 125 pig/g seedb 2392.72 96 62 jjg/g seeab 2148.56 86 j WO 89/01288 PCT/US87/01971 wo 89/01288 -13a -Average of 10 plots (generally 1.22 m x 9.14 m); chitosan was applied with water an equivalent amount of water without the chitosan was applied to the control plots.

b Average of 2 plots (generally 1.22 m x. 9.14 m).

EXAMPLE 10. Wheat Seedling Weights Variety: VONA Date harvested: 18 December 1985 Location: Alva, Oklahoma mean wt mean wt per stem per root (g) fresh dry fresh dry Group I Control 2.01 0.38 1.98 0.04 Chitosan solution 0.473 liter 3.13 0.47 2.87 0.01 of control 156 124 145 Group 11 Control 2.3 0.23 0.43 0.04 Chitosan solution 0.473 liter 2.4 0.24 0.33 0.05 of control 104 104 77 125 Application rate o£ chitosan solution is 0.473 liter per 45.36 kilograms of seed, Chitosan solution is 2% chitosan and 98% inert ingredients.

i mnnr -e PCT/US87/01971 WO 89/01288 -14- EXAMPLE 11 Wheat Seedling Test Data Seedling Crown Diameter Variety: VONA Planted: 1 November 1984 Harvested: 5 January 1985 Location: Alva, Oklahoma No. of samples Treatment mean crown diameter (cm) Statistical Deviation of control Control Chitosan 250 pg/g 5.347 8.893 1.147 5.154 100 166 EXAMPLE 12.

1984-85 Vona Wheat Yield Date of harvest: 5 June 1985 Location: Alva, Oklahoma No. of kg Statistical cf Treatment plots hectare Deviation control control Chitosan 250 Pg/g 2858.1 3355.8 5.6 5.9 100 117 PCI'/US87/01971 WO 89/01288 EXAMPLE 13.

1983-84 TAM W-101 Wheat Yield Date of harvest: 12 June 1984 Location: Alva, Oklahoma No. of kq Statistical of Treatment plots hectare Deviation control control 6 3349.1 5.9 100 Chitosan 250 pg/g 6 3685.3 6.2 110 EXAMPLE 14.

1985 Comparison of Yield for Chitosan vs. Benlate Location: Whitlow Farm, Pullman, Washington Kg/hectare of control I. Hill 81

H

2 0 Controla Benlate Treatedb 1000 j.jg/g Benlatec 500 i'g/g Benlatec 250 pig/g Benlatec 125 pig/g BenlateC 62 pig/g Benlatec 1000 Uig/g c 500 pig/g C 250 iig/g C 125 ug/g

C

62 uig/g c 4794.9 3651.7 3396 .3.

3961.0 4041.7 4176.2 3254.9 4088.8 4673.9 5756.6 5427.1 4922. 7 76 71 83 84 87 68 97 3 113 103 I W; V -I

I

PCTIUS87/01971 WO 89/01288 -16- II. Daws

H

2 0 C ontrol 0 BenlateC 1000 lpg/g BenlateC 500 11g/g Benlate 0 250 14g/g Benlate 0 1000 Pg/g C 500 pig/g C 250 iig/g C 2757.3 1667 .8 1943 .5 1049.1 1499.7 2710.2 3961.0 3799.6 38 54 98 144 138 a Averaged over 6 reps 3.048 b Averaged over 8 reps 3.048 c Averaged over 2 reps 3.048 0.356I.

0.356 u 0.356 EXAMPLE 1982-83 Stephens Wheat Harvest Yield Treatment kg/hectare of control

H

2 0 Controla 6536.7-- Chitosan 500 pig/g b 7081.4 108 Chibosan 250 p.g/g b 6691.4 102 Chitosan 125 pig/g b 6395.5 98 Chitosan 62 i'g/g b 6247.5 96 a Average b Average of 7.62 of 7.62 x 1.22 m x 1.22 m replications.

replications.

L- C WO 89/01288 PCr/US87/01971 -17- EXAMPLE 16.

1984-85 Daws Wheat Harvest Yield Location: Whitlow Farm, Pullman, Washington Treatment kg/hectare of Control Control 3214.6 1000 pg/g 3712.2 115 500 pg/g 4008.1 125 250 pg/g 3880.3 121 All values averaged over four 3.048 m x 0.356 m replications.

EXAMPLE 17.

1984-85 Hill '81 Wheat Yield Location: Whitlow Farm, Pullman, Washington Treatment kg/hectarq of Control

H

2 0 Controla 4129.2 1000 pg/g b 3920.7 500 pg/g b 4337.6 105 250 pg/g b 4963.1 120 125 pg/g b 4922.7 119 62 ug/g b 4297.3 104 a Average of 50 3.048 m x 0.356 m replications.

b Average of 10 3.048 m x 0.356 m replications.

*1~ PCr/US87/01971 WO 89/01288 EXAMPLE 18.

1985 Corret Spring Oat Yield Location: Whitlow Farm, Pullman, Washington Treatment kg/hectare of Control

H

2 0 Controla 1001.6 500 iig/g b 876.4 87 250 p~g/g b 793.0 79 125 14g/g b 1544.2 154 62 iig/g b 667.8 67 a Average of 35 3.048 m x 0.356 m replications.

b Average of 7 3.048 m x 0.356 m replications.

EXAMPLE 19.

1985-86 Boyer Barley Yield Location: Soil Conservation Service Field Station Pullman, Washington No. of kq% of Treatment plots hectare control H0Control Chitosan solution 0.1183 liters/ 45.36 kg seed

H

2 0 control Chitosan solution 0.1479 liters/ 45.36 kg seed 4718.6 5884.2 4920.3 125 100 13 5839.4 119

I

WO 89/01288 PCT/US8701971 -19-

H

2 0 Control 4965.1 100 Chitosan solution 0.1775 liters/ 45.36 kg seed

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2 0 Control Chitosan solution 0.2366 liters/ 45.36 kg seed

H

2 0 Control Chitosan solution 0.2958 liters/ 45.36 kg seed 7677.5 6881.7 8013.7 6298.9 155 100 116 100 113 7139.5 Plot size 3.048 m x 0.357 m Chitosan solution is 2% ingredients.

1.087 m 2 chitosan and 98% inert Having fully described the present invention, it will be apparent to those skilled in the art that modifications to the method described herein may be made without departing from the scope of the present invention. While the embodiments described involve wheat, oats, and barley the process is generally applicable to cereal crops. Only the wheat, oats, and barley embodiments have been included for the sake of brevity.

Therefore, the scope of this invention is not intended to be limited except as may be required by the lawful scope of the following claims.

Claims (13)

1. A method of enhancing crop yield, straw strength, stem diameter, and root development of rice comprising applying chitosan in a neutral aqueous solution to rice seed at a rate of from 60p9to 1000 3 of chitosan per gram of seed prior to planting.

2. The method of claim 1 wherein said chitosan is dissolved in a dilute aqueous acid to form a solution prior to application.

3. The method of claim 2 wherein said solution of chitosan dissolved in a dilute aqueous acid is subsequently neutralized with a base to a pH no greater than

4. The method of claim 2 or claim 3, wherein the dilute aqueous S acid for the solution of chitosan is selected from the group consisting of acetic acid, hydrochloric acid, sulfuric acid and formic acid. Rice seed treated by the method of any one of claims 1 to 4.

6. Seed treated with chitosan In an amount effective to enhance yield, straw strength, stem diameter, and root development of plants ggerminating from said seed by applying chitosan in a neutral aqueous solution to seed at a rate of from 60 jto 1000oQof chitosan per gram of S seed prior to planting, said seed being selected from the group consisting of barley, rye, oats and rice.

7. The product of claim 6, wherein the seed Is treated with chitosan In an amount between 225pg per gram of seed and 275pg per gram Sof seed. o: 8. The product of claim 6 or 7, wherein the seed is treated with chitosan In an amount of about 250p.g per gram of seed. "0 9. A method of enhancing yield, straw strength, stem diameter, and root development in plants comprising the step of applying chitosan In a neutral aqueous solution to seed at a rate of from 60Qpto 1000pyof chitosan per gram of seed prior to planting, the chitosan being produced by deacetylating chltin to an extent exceeding 90% and wherein the seed Is selected from the group consisting of barley, rye, oats and rice. The method of claim 9 in which chitosan is applied to the seed as a dilute neutralized aqueous acid solution of chitosan.

11. The method according to claim 10 in which the dilute aqueous acid for the solution of chitosan is selected from the group consisting of acetic acid, hydrochloric acid, sulfuric acid and formic acid. 1849y 21

12. The method according to claim 10 or claim 11 in which the solution of chitosan is neutralized to a pH no greater than

13. The method according to any one of claims 10 to 12 in which the dissolved chitosan solution is neutralized to a pH of 6.0 to

14. The method according to any one of claims 9 to 13 in which the application of chitosan to the seed leaves a cellophane-like surface on the seed after drying. The method according to claim 14 in which the rate of application of the chitosan is between 225Rg per gram of seed and 275pg V:49 per gram of seed. •16. The method according to claim 14 or claim 15 in which the rate of application of the chitosan is about 250 g per gram of seed. eg17. A method of enhancing yield and preventing lodging of plants comprising the steps of: dissolving in a dilute aqueous acid chitosan that has been produced by deacetylating chitin to an extent exceeding 90%, in order to form an aqueous acid solution of chitosan; neutralizing the aqueous acid solution of chitosan to a pH no greater than coating seed with the neutralized aqueous acid solution of chitosan at a rate of from 60pto l000pof chitosan per gram of seed prior to planting; and a drying the seed to leave a cellophane-like surface chitosan on the seed; and wherein the seed is selected from the group consisting of barley, rye, oats and rice.

18. Barley, rye, oats or rice seed treated with chitosan by the method of any one of claims 9 to 17.

19. Seed treated with chitosan as claimed in claim 6, substantially as herein described with reference to Examples 8, 9, 18 or 19 but excluding any control. A method of enhancing yield, straw strength, stem diameter, and root development in plants as claimed in claim 17, substantially as herein described with reference to any one of Examples 8, 9, 18 or 19 but excluding any control, 22 DATED this FOURTEENTH day of NOVEMBER 1990 Washington State University Research Foundation, Inc. Patent Attorneys for the Applicant SPRUSON FERGUSON 0 0 09 00 so 0 B 6O 0 0O 0 INTERNATIONAL SEARCH REPORT International Arplicnlion NrpCT U S 8 7 019 97 1 I- CLASSIFICATION OF SUBJECT MATTER oif Several classilic~tion Symbol$ apply, indicate all)I Accoroing to international Patent Classification IIPC) or to both National Claaaifl-at~on and IPC IPC(4): AOIN 43/16 US CL: 71177 -1t, FIELDS SEARCHED Minimum Documentation Searchect 4 Classification System IClassification Symbols U.S. 71/77,88 47/57. 6 Documentation Searched other than Minimum Documentaiizn to the Extent trial such Documents are Included In the Fields Searched CHEMICAL ABSTRACTS VOL. 56 VOL. 105 CHIITO SAN Ill. DOCUMENTS CONSIDERED TO Bn RELEVANT I& Category Citation of Document, 14 with indication, where appropriate, of the relevant passages I Iti Relevant to Claim No, 14 Wheat Research Review For Washington 1-18 Wheat Commission, Washington Associat- ion of Wheat Growers And State Depart- I ment Of Agriculture, 1984, (Pullman, Washington), pp.64-65. Research Grant Proposal, L.A. Hadwiger, "Mode of Actton of Chitosan In Reducing Disease-Caused Lodging of Winter Wheat". X Washington, Sea Grant Program, 1985. 1-18 Pullman, Washington). See Entire Document. Research Grant Proposal, L.A. EHadwiger, "Chitosan And Enhanced Wheat Yield". x L.A. Hadwiger, 'Chitosan, A Natural 11-18 Regulator in Plant-Fungal Pathogen Interactions, Increases Crop Yields". Chitino Chitosan And Related Enzymes, Acedemic Press Inc., 1984, (New York, New York), pp. 291-302. ScOial Categories of cited documental IS ITI later document oublished alter the internationat tiling dais *AK 4 document defining the general Stats of the art evhith. is not or tiriority date and not in conflict vvith the atitilication but Coflsidotsd to be at particular relevance Cited t0 untderstand the principle Or thory Underlying the ealier document but published on oat it the in1toonalline 11' document of oar'Iiculat relevancet the Ctaimed invvention filing date Canot be considered nlovel or Cannot be considered to

111.1 document which may throw doubte on priority claim(s) or involve an inventive step whtaiohnI att o ebihther ppblialtiensdat(as s&hother "Yf" document oft earticular relevancel the Claimed invention dI~ton Oherspeial easn a spcifed)Cannot be tCosdeted to Involve an inventive &ltp when the 110" document reltring to an Oral disclosure, use, exitibiton or documen11toI combinod with one or more othef such docu- ohthe means 1 ent3111 Such Combination being Obvious to a personi skilled 1116" document Oubtished prior to the tnteriatidnalt filin date but )fl the Ott. later than the jriority date claimed document member *t the same Patent tamill IV, CIRTIFICATION Oate, of the Actual Comletion of t~e International $earch I bilte of Matting of this Intrnational search Reorott 12 JANUARY,. 198FEB I9aw Ifltrnaitionat 446erhlol Authority I Signature of Authotized Offcer ISA/US :Catherine L. Mills fohrm 11C',01SA)211O leatoeW esetI (May 1111111) 1 International Application No. PCT/US87/01971 111, DOC~UMENTS CONSIDERED TO Ut RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation of Docunment, 14with indication, where appropriate, of the relevant passage$ It Relevant to Claim No is X R.L. Rawls, "Prospects Brighten 1-18 Converting Chitin Wastes To Valuable Products", Chemical And Engi.neering New, 1984, (Washington, D.C. Vol. 62, pp. 42-45. x Wheat Research Review For Washington 1-18 Wheat Commission, Washington Associat- ion Of Wheat Growers And State De- partment Of Agriculture, 1983, (Pullman, Washington), pp. 27- 28. Research Grant Proposal, L.A. Hadwiger, "Evaluation of Chitosan As A Seed Treatment"'. X Wheat Research Review For Washincjton 1-18 Wheat Commission, Washington Associat- ion of Wheat Growers And State Depart- ment Of Agriculture, 1981, (Pullman, Washington), pp. 63-64. Research Grant Proposal, L.A. H-adwiger, "Evaluation of Chitosan As A Systemic Fungicide". P&Vni PCSA12101lettrttie aiiet) IMay I1111111