FIELD OF THE INVENTION

This invention relates to methods and compounds for treatment of plants to obtain beneficial qualities, especially enhanced protein content, freeze protection, seed priming, seed toughening and increases in yield. SUMMARY OF THE INVENTION

The invention comprises novel chitosan salt treatments that may be used to enhance the protein nutrient content of the "fruits" of treated crops. The word "fruits" is used to refer to the desired harvest of the crop (grain, fruit, vegetable, root, flower, plant, timber, etc.). Plants treated according to the invention have also been found to increase pod set in legumes, give improved yields and enhance freeze protection. The novel chitosan treatments may also be used as seed priming agents. Methods by which crops can be treated with chitosan salts in accordance with this invention include seed coating, seed priming, pre-germination soaking, post-emergent or transplant drench irrigation, flood or overhead irrigation, root dip, and foliar spray applications. Any of the preceding may be accompanied or followed by the application of a fixing agent to extend the protein enhancing effects of chitosan salts. The invention supplies chitosan in appropriate molecular weights to the crop during its fruiting stage to enhance protein content of the fruits. The degree of enhancement of protein may vary depending upon the molecular weight of the chitosan salt applied, the timing and frequency of treatment, and the amount of chitosan salts utilized.

Accordingly, an object of this invention is to enhance protein in "fruits" of existing varieties of food or feed

crops over untreated crops grown under the same conditions. Another object of this invention is to provide treatments for enhancing freeze and disease protection of crops and for seed priming to speed emergence and increase germination in seeds, and to toughen delicate seeds.

These 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. DETAILED DESCRIPTION OF THE INVENTION Chitosan is a polymer made up of a hexosa ine sugar (glucosamine) whose molecules are linked (Bl 4) into chains that can easily exceed molecular weights of one million. Chitosan molecules in a range of up to and exceeding 1 x 10 (6) molecular weight (raw) are derived commercially from chitin. Chitin, an amino cellulose derivate, is the second most abundant polymer 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 deacetylation exceeds 70%, and it is most effective when exceeding 90% and even approaching 100% deacetylation, notably in foliar applications since polymer chains are usually shortened in production and the molecule is more readily absorbable. Deacetylation of 90% or greater is preferred for protein enhancement. Deacetylation of 80%-99%, where 85%-95% is preferred for enhancing yields by seed treatment. The degree of deacetylation and average molecular weights may easily be determined by methods disclosed in "Preparation and Characterization of Fully Deacetylated Chitosan", A. Domard and M. Rinaudo in International Journal of Macromoles r 1983, Vol. 5, February, and is hereby incorporated by reference.

Enhancement of protein content was first observed in hard red spring wheat crops harvested from chitosan-treated seed planted in conditions of high fertilization (the fertilizer is now believed to have acted as a "fixing agent" as described below). The treated crop's protein was slightly higher compared to untreated controls, although statistically insignificant. While the exact method of plant utilization of chitosan is still not fully understood, chitosan is known to stimulate production of enzymes to cleave chitosan in contact with the plant. This action has been shown to occur when the chitosan is a component in the cell wall of naturally occurring fungi. The direct application of chitosan to the plant, or seed, prior to planting, has been shown to elicit a similar response. Until recently, however, it was unknown that this reaction could cause a real increase at harvest of protein in the "fruits".

Protein in fruits of crops is measured in wheat with an infranalyzer after being milled on a cyclone mill with 0.5 mm screen. All other crop proteins have been measured by the Lowry Colorametric Method.

Also provided is an improved method of increasing germination, early growth, pod setting, the yield, and providing a healthy crop of soybeans by planting soybean seed, the improvement which comprises using as the soybean seed the chitosan coated soybean.

In a further aspect, there is provided a tree seed which is bonded to chitosan having a degree of deacetylation of at least 80%. Seed treated in accordance with the present invention results in essentially disease-free trees. Fir tree seed treated according to the present invention average about a 24% to 79% increased growth after only three weeks.

In other aspects there are provided similarly chitosan- ^■ coated seeds of flower, including chrysanthemum, gypsophila (Baby's Breath) , azalea, mum and dahlia. As an improved method there is provided the method of providing a healthy crop of flower from the seeds of flower, the improvement which comprises using as the seeds of flower the chitosan coated seed. When applied in a foliar spray, the chitosan solution described below appears to trigger a protective action in chrysanthemums inoculated with several fungal pathogens. Notably, all symptoms of fusarium oxysporum were eliminated in wounded inoculated plants. Thus, the solution can be applied as a foliar spray as well as "in coating seeds.

Also provided in further aspects of the invention are similarly chitosan-coated seeds of corn, lettuce, onions, parsley, radishes, rapeseed, sugar cane, milo, cotton, grass, sunflower, and potatoes.

By describing the various seeds as "bonded" to chitosan is meant seed having a film of chitosan thereon. The amount of chitosan solution used to treat the seed as described in the following examples is in the range of about 4 to about 32 oz. of 2.5% (w/w) aqueous chitosan solution per hundredweight (dwt) of seeds. The amount used can vary within the range, the optimum amount for each type of seed being easily determinable by one skilled in the art. The preferred treatment for soybeans and corn is about 16 oz./dwt. This results in about a 15-30% increase in soybean pod setting and ultimately in about a 7 bushel per acre increase in yield. In corn, it results in about 2.5 to 12 bushels per acre increase in yield.

It has been found that soybeans grown from untreated seeds have about 36 pods per plant (each pod containing 3 seeds) , while the plants grown from treated seeds have 44- 55 pods per plant. While increasing the yield, the

chitosan has no observable fungicidal or pesticidal effect. When used to treat other types of seed as mentioned above, the chitosan treatment results in enhanced growth, yields, and frequently improves (desirable agronomic characteristics, i.e., protein, milling, etc.) of the "fruit".

The molecular weight of chitosan salts used in practicing this invention will vary directly with the proximity of the treatment to harvest; the closer the treatment is to harvest of the crop the lower the molecular weight of the chitosan salt applied. Conversely, the farther the treatment is in time from harvest, higher chitosan molecular weights must be used for optimum effectiveness. Additionally, the presence of chitosan degrading enzymes, soil microbes or fauna in the growing plant's environment should be taken into account (except for foliar treatments) and the molecular weight of the chitosan salt increased accordingly to compensate for degradation in the chitosan salt's molecular weight. For below ground fruits, where seed treatment or treatment by drench irrigation may be preferable, higher molecular weights and dosages are required to obtain satisfactory results. The molecular weight of chitosan is determined from intrinsic viscosity measurements. Dry chitosan as either flakes, granules, or powder, can be obtained by deacetylating chitin. Preferably in finely dispersed form, the chitosan is first dissolved in a dilute aqueous acid, e.g., 0.5% to 5%, hydrochloric acid, sulfuric acid, formic acid, lactic acid, ascorbic acid, citric acid, malic acid, malonic acid, proprionic acid, pyruvic acid and succinic acid, or the like, with 1-2.5% preferred, acetic acid, to give a solution having a concentration of chitosan ranging from about 1% to about 10% by weight, based on the total weight of the solution. The solution is then

filtered to remove undissolved particles. The acidity of the solution is adjusted to a pH between a preferred range of about 5.7 to about 6.4 as desired using a non-phytotoxic alkali or base; sodium carbonate is preferred. Other bases suitable in such application would be NaOH, KOH, NH OH, sodium bicarbonate, and the like.

A quantity of 2.5% chitosan salt solution suitable for use in the invention may be mixed as follows: at ambient temperature, to obtain about 500 ml of chitosan acetate (or the like), add 12.5 g of chitosan of the desired molecular weight (as discussed above) to 450 ml of water in 600 ml beaker. This material is agitated with an overhead stirrer for several minutes. Then add 7 ml of 85% acetic acid (or other organic acid) (USP Grade) and allow to mix for one hour until the chitosan is dissolved. Continue agitation throughout this hour. The solution is then filtered and insoluble particles are removed.

Then, while continuing agitation, neutralize the solution with approximately 2 g sodium carbonate (or the like) mixed in about 30 ml of water. The neutralization solution should be slowly added to the chitosan salt solution until the pH reaches between about 6 to about 6.4. The total solution may then be diluted with water until the quantity of the total solution reaches about 500 ml. The aqueous chitosan salt solution should not have precipitated or otherwise undissolved particles of chitosan. The solution may be diluted to whatever concentration is called for by the method of treatment chosen.

The above-described process of preparing a chitosan salt solution suitable for use in the invention may be scaled up as desired for commercial or field use, as is well known to those in the art.

Some acids may be more appropriate in different applications of the invention; appropriateness of acids in

particular depends on the phyto toxicity of the resulting salts in various crops. Such salts may be those formed with chitosan on dissolution and/or those formed on neutralizing the aqueous chitosan-acid solution. Selection of acids and concentrations used may also effect shelf life due to ensuing chemical breakdown by hydrolysis of the chitosan polymer. In foliar applications, this may ensure relatively rapid yet sustained uptake of the polymer into plant cells. However, total degration of the chitosan polymer to simple amino sugar residues renders the invention relatively ineffective.

Chitosan acetate (the chitosan is dissolved in acetic acid) is generally preferred for the seed treatment, root dip, foliar spray, and irrigation methods of application of chitosan salts according to the invention. Chitosan lactate, if used shortly after production, is, however, the most desirable and perhaps effective, due to the superior non-phytotoxicity of chitosan lactate. Chitosan ascorbate is also particularly suitable for foliar spray methods of treatment in certain crops but is not particularly preferred over chitosan acetate or lactate.

The solution obtained as described above may be applied to crop seed at an appropriate concentration for the seed treatment embodiment of this invention. The method of application should preferably coat the entire seed. Cereal seed may be mixed with the chitosan salt solution by cement mixer-type devices or grain augers. More delicate seed, such as that of peas or beans, may be sprayed with the solution while in a rotating drum. However, the treating of such traditionally fragile seed with this solution while transferring such seed through an auger has been shown to "toughen" the seed adding resiliency and allowing equal or higher germination than untreated seed not subjected to mechanical stress. If a "fixing agent" is employed as

described below, the fixing agent may be applied to the seed either during or after the chitosan salt solution is applie .

Seed treatment offers significant cost effectiveness benefits due to its ideal placement at the root of the plant, immediate benefits to the germinating seedling, and extremely low dosage to obtain the desired response. Chitosan must be made available to the plant during the fruiting stage, however, for seed treatments to have a protein enhancing effect. The length of time between planting and fruition is important, therefore, because the chitosan polymer applied to the seed will be biodegraded with time and may not be available to the plant near maturity. In seed treatment the protein enhancing action of this invention is observed consistently only in short life-term crops unless supplemental solutions, metal salts, and/or polymer cross-linking agents which are not otherwise phytotoxic to the seed being treated are used as "fixing agents" at the time of treatment of the seed with the chitosan salt. Fixing agents are compounds designed to be applied concurrently or consecutively with chitosan seed treatment to immobilize and reduce the rate of degradation of the chitosan salt. Fixing agents may be solids, liquids or gases as long as they reprecipitate the chitosan salt film on the seed by the time the seed is planted. Examples of successful fixing agents are zinc oxide, sodium phosphate (dibasic) , potassium phosphate (monobasic or tribasic) , potato starch, corn starch and almost any fertilizer. The fixing agent greatly extends the benefits of the chitosan seed coat, although slight increases in chitosan salt dosage may be required to achieve beneficial results of this invention in some crops.

In intermediate life term crops which take about 45-90 days from planting to harvest, protein enhancement due to seed treatment with chitosan salts is observed inconsistently unless supplemented with a fixing agent or an additional treatment at about 45-60 days after planting. Such additional treatment may be via roots or by foliar application. Foliar applications proved more beneficial in above-ground crops. In row crops like spinach or lettuce, however, drench or flood treatments may be preferable to obtain a premium crop. Some leaf tissue irritation due to foliar treatments is observed in such crops with the use of chitosan ascorbate. This disadvantage may be overcome by utilizing a different salt of chitosan (such as chitosan acetate or lactate) or by treating further from harvest with increased molecular weights and/or lower dosages.

In longer life term crops (taking over 90 days from planting to harvest) desired protein increases from seed treatment with chitosan salts are rarely observed, unless supplemented by fixing agents or additional treatments with chitosan salts. This is believed to occur because the salts of chitosan have been totally degraded or provide only minimal influence to the fruit during the fruiting stage. Such crops require fixing agents or additional treatments with chitosan salts to consistently show the beneficial effects of the present invention.

The sustained release of chitosan to the seed results in consistent protein increases when used with seed treatments of any crop with an annual life cycle. Treatment of perennial crops (e.g., alfalfa) must be done at least annually to benefit from this invention.

Due to the relatively short life cycle of chitosan seed treatment in the soil (2-8 months estimated) , plants may be given a supplemental dose of chitosan salts as discussed above by irrigation or foliar spray. Such treatments may

be tailored to various molecular weights and dosages to provide the desired residual treatment effects. Alternatively, application of a fixing agent may be appropriate to keep the salts in the vicinity of the roots, particularly in the case of acid soils. Use of a fixing agent in acid soils can reduce mobility, leaching, and degradation of the chitosan salt polymer in irrigation applications of chitosan salts. Treatment by foliar methods may find a fixing agent beneficial if rain is expected a short time after treatment. Otherwise a fixing agent in foliar application may not be beneficial.

For treatment by irrigation (as for seed treatment) the higher the molecular weight of chitosan salt used, the more marked the increase in protein content for "fruits" of longer life cycle crops. This sustained release of chitosan to the plant becomes increasingly important when chitosan salts are used as a seed coating on long life cycle plants. The probable lack of significant residues of chitosan to enhance protein in "fruits" of long life term plants (e.g., winter wheat) during their fruiting phase is very likely responsible for lack of protein enhancement in such crops when their seed is treated with chitosan prior to planting. Fixing agents or supplemental treatments are required for such crops. For foliar spraying prior to harvest very low molecular weights of chitosan salts are easily absorbed by the plant tissue. This stimulates the production of protein in the plant which is then transferred to the "fruit." Higher molecular weights of chitosan salts are excluded from immediate absorption and must be cleaved by plant enzymes prior to tissue absorption. Treatment times prior to harvest affect the recommended molecular weights to be used. Consequently such applications of the invention will be with increasingly lower molecular weight chitosan when

treated nearer to harvest. Applications to too high a dosage of chitosan can be detrimental to the crop and should be avoided.

Chitosan is commercially available in relatively low molecular weights (10 cps (centipoise) and above at about 230,000 mw) . However, for the purpose of this invention, extremely low molecular weights are desirable to rapidly induce the protein enhancement, particularly for foliar spray applications. The following process will produce extremely low molecular weight chitosan. Chitosan of 45 cps was ground to pass 60 mesh screen. Then up to 300 grams of chitosan is added to 3,000 ml of hot water (72 C) on a magnetic stirring hot plate, agitated with a stir bar at 100 rpm. To this is added 40 ml of hydrogen peroxide (50%) and then soda ash is slowly added to accelerate the oxidation and chemical reduction of the chitosan polymers. The material is allowed to mix for one hour until the reaction is complete and is then rinsed to neutrality of pH 7. The material is vacuum filtered and the resulting cake is pulverized and dried in an oven at low heat (80° C) and the viscosity is measured by Brookfield viscometer. The following measurements were utilized for obtaining the desired average molecular weights: mw 5,000 at about 1.1 cps; 10,000 at about 1.2 cps; 25,000 at about 2.0 cps; 125,000 at about 4.0 cps; 250,000 at about 12.0 cps; 1,000,000 at about 500 cps (1% chitosan in 1% acetic acid at ambient measured by Brookfield viscometer) . The procedure described above resulted in a drop from 45 cps to 6.0 cps and yielded 298 grams. 70 grams of the above (6 cps) chitosan sample can be degraded to 2.0 cps (25,000 mw) by mixing with 700 ml of water, adding 12.33 ml hydrogen peroxide (50%) at 75° C heat and then slowly adding 14 grams sodium carbonate while agitating. The mixture is rinsed and dried as above.

70 grams of the above (6 cps) chitosan sample may be degraded to 1.2 cps (10,000 mw) by being added to 700 ml of water, adding 23.8 ml hydrogen peroxide (50%) at 75° C heat and then slowly adding 14 grams of sodium carbonate while agitating. The mixture is rinsed and dried as above.

25 grams of the above (1.2 cps) chitosan sample may be degraded to l.l cps (5,000 mw) by adding it to 250 ml of water, adding 10 ml hydrogen peroxide (50%) at ambient temperature and slowing adding 5 grams of sodium carbonate while agitating for about four hours. The mixture is rinsed and dried as above.

The procedures for obtaining low molecular weight chitosan described above may be scaled up as desired. The mixture should not be allowed to brown during this procedure. Browning indicates that the chitosan in the mixture is turning into glucosamine.

Actual molecular weights may be obtained by multiple columnar separation checked by a spectrascope, reprecipitated, and lyophilized. These samples may be used to check viscometer reading of known molecular weights of chitosan in various acids. Average molecular weights represent a relatively inexpensive way to obtain polymers useful in this invention without extensive commercial processing. The table below gives effective ranges for molecular weights, concentrations, and dosages for chitosan salt applications and fixing agent applications useful in this invention.

Application mw's Concentrations Dosage

Chitosan salt, (5,000- (0.5-2.5% (50 μg/g- Seed Treatment 1,500,000) chitosan) i-ooo μg/g chitosan/seed

Fixing Agents various (.1 mM - .5M) (1 μg/g- 250 μg/g fixing agent/ seed)

Chitosan salt, (5,000- (10 ppm- (diluted in Irrigation 1,500,000)1,000 ppm) normal watering)

Chitosan salt, (1,000- 10 ppm- (0.25 g/

Acre

Foliar 250,000) 1,000 ppm) 38 g/Acre)

Seed Priming (250,000- (10 ppm- (10 μg/g- 1,500,000) 25,000 ppm) 25,000 μg/g chitosan/seed)

Tomato plants treated by spot irrigation on transplanting at rates from 1000 ppm to 250 ppm survived a "killer frost" at 108 days at rates of 40% to 60%; with supplemental treatment at 60 day intervals, survival rates were 20% to 80%, while control survival was only 5%. Yields at 1000 ppm were the least desirable, although freeze protection was the most enhanced. Yields after the freeze for 1000 ppm, however were over 300% of those from controls, but were still less than overall yields at 500 ppm and 250 ppm.

The improved freeze protection demonstrated by chitosan salt treatments is believed to be due to a slight increase of about 10% in cell wall thickness in the tissue of the plant. A small infusion of chitosan salts into the tissue is believed to be an additional cause of slightly increased freeze tolerance. Both factors are believed to be instrumental in keeping cell walls from rupturing to the extent of untreated plants. The most recently treated (18

days prior to freeze) plants and those treated at highest dosage were observed to be most protected.

Seed priming is a technique frequently used to revitalize old or damaged seed, improve seed germination, and improve stand in less than ideal temperatures or moisture conditions upon planting. Priming involves controlling the time seed is exposed to water, by keeping water temperature below optimum for germination, or by limiting water availability. Controlling the amount of water imbibed by seed and the rate of uptake is the most reliable and widely practiced method of seed priming. Osmotic agents are used to control rate of ^" water uptake and are frequently salts, polyethylene glycol (PEG), and mannitol. Seeds for some crops are adversely affected by the osmoticum of salt. For such crops, currently PEG is the most widely accepted osmoticum.

Aqueous chitosan salt solutions are a suitable alternative to PEG. Chitosan is not toxic to seed at adjusted pH and sufficiently high molecular weights which do not allow absorption of the chitosan polymer. In this application, aqueous chitosan salts having polymers of less than 250,000 mw are removed by column separation. Ideally, removal of short polymers should be practiced but lower molecular weight chitosan salts may be used, particularly in short dwell treatments.

Chitosan salt seed treatments are most effective in wet and/or cold soils. This effect may be due to the seed priming characteristics of the chitosan salt seed coat. Treatment with chitosan salts provides an osmotic membrane shown to be beneficial to seed imbibition. This osmoticum is particularly beneficial to seed planting in wet and cold environments. It is also beneficial on roots. Shrubs being planted into marsh—like conditions that had their roots dipped into a chitosan salt solution dip all

survived, whereas only 30% of the untreated plants survived. Whether the chitosan salts merely metered the uptake of water or provided some source of oxygen to the roots, or both, is unknown. The dip included an acid known to oxidize chitosan and rapidly degrade the polymer. The osmotic coating certainly reduces excess water uptake and consequently may reduce oxygen requirements of the plant to keep it from drowning.

A chitosan salt membrane certainly provides seed priming benefits to rejuvenate old or damaged seed lots. In field trials very visible stand differences were observed in germination of seed only one year old. In laboratory trials very old seed with a chitosan salt coating germinated at a rate of 50-300% higher than untreated seed. The following data illustrate examples of enhancing protein in "fruits" of crops through the use of this invention. The crops examined to demonstrate the applicability of this invention were selected entirely at random. For the sake of brevity only a representative selection of such crops and their treatment is given in the working examples below. Replicative results should be obtainable in nearly all crops, since the mode of action for chitosan salt applied in accordance with this invention appears to be similar in all plants. Each crop tested demonstrated a slight to significant positive response to treatment carried out according to this invention. EXAMPLE I - Protein Enhancement Resulting From Seed Treatment

Seeds of radishes were treated with 62 μg to 1000 μg chitosan salt (average molecular weight: 5,000 mw) per gram of seed; 62 μg to 1000 μg chitosan salt (average molecular weight: 25,000 mw) per gram seed; 62 μg to 1000 μg chitosan salt (average molecular weight: one million mw) per gram seed. The chitosan salt seed treatments were

tested against untreated controls. Untreated seed was also planted for drench control irrigation-only treatments, and for subsequent foliar-only treatments.

Avg. Protein

Initial Supplemental Harvested Avg. Yield % Control Treatment Treatment Plants % % Control 12 day; 24 da ^y

a. Very low molecular weight (avg. 5,000 mw) chitosan lactate seed treatment in radishes and supplemented with a fixing agent (FA) where shown of KH ₂ P0 (0.25 molar) at about 10 μg/g seed.

Control None 100 100 62 μg/g None 100 105 62 μg/g FA 100 103 125 μg/g None 100 107 125 μg/g FA 100 105 250 μg/g None 100 109 250 μg/g FA 100 110 500 μg/g None 94 98 500 μg/g FA 100 104 looo μg/g None 88 92 looo μg/g FA 96 102

b. Moderately low molecular weight (avg. chitosan lactate seed treatment in radishes.

c. Low molecular weight (avg. 125,000 mw) chitosan lactate seed treatment in radishes.

d-e. Treatment at higher tested molecular weights (500 K mw and 1 M mw) were not quite as beneficial to protein as in the examples noted above although yields were about the same. Phytotoxicity was reduced at higher treatment levels as chitosan salt molecular weights increased or when treated with a fixing agent. In chitosan salts of lower molecular weights use of a fixing agent reduced the phytotoxic effects of chitosan salts.

EXAMPLE II - Protein enhancement due to one time pot emergent irrigation at 8 days with varying strengths (ppm) and molecular weights (mw) on radishes not given any seed treatment.

Avg. Protein

Initial Supplemental .ield % Control Treatment Treatment ntrol 12 day; 24 day

Control None

None lOppm 5k mw

None 50ppm 5k mw

None 250ppm 5k mw

None 500ppm 5k mw

None lOpp 125k mw

None 50ppm 125k mw

None 250ppm 125k mw

None 500ppm 125k mw

EXAMPLE III - Protein enhancement due to foliar spray application at 8 or 16 days (d) with chitosan ascorbate at varying strengths (ppm) and molecular weights (mw) in radishes; no seed treatment or irrigation treatment.

Avg. Protein

Initial Supplemental Harvested Avg. Yield % Control Treatment Treatment Plants % % Control 12 day; 24 day

None 8 d lOppm 5k mw 105 101 None 8 d 50ppm 5k mw 103 103 None 8 d 250ppm 5k mw 92 90* None 8 d 500ppm 5k mw 60 80*

None 8 d lOppm 25k mw 102 104 None 8 d 50ppm 25k mw 105 107 None 8 d 250ppm 25k mw 95 96* None 8 d 500ppm 25k mw 65 70*

None 8 d lOppm 125k mw 100 102 None 8 d 50ppm 125k mw 102 102 None 8 d 250ppm 125k mw 98 100 None 8 d 500ppm 125k mw 72 76*

None 16 d lOppm 5k mw 105 102 None 16 d 50ppm 5k mw 102 105 None 16 d 250ppm 5k mw 100 97* None 16 d 500ppm 5k mw 86 94*

None 16 k lOppm 25k mw 100 106 None 16 d 50ppm 25k mw 104 106 None 16 d 250ppm 25k mw 101 102 None 16 d 500ppm 25k mw 90 92*

* Phytotoxic effects observed in foliar application at higher chitosan concentrations at lower molecular weights.

EXAMPLE IV - Chitosan with Fixing Agent

Chitosan salts were applied with a fixing agent to prevent initial over stressing of seed and to extend the chitosan salt's ability to function as a protein enhancer. Rice seed was treated with an aqueous solution of chitosan salt prepared as described in the specification above. After coating the seed with the chitosan salt solution, a fixing agent was applied consisting of zinc oxide in a water slurry, at the rate of 4 oz. zinc oxide to 100 lbs. seed. This fixing agent reprecipitated the chitosan directly upon the seed. Reprecipitation of the chitosan is required since the direct seed soaking frequently utilized in rice treatment was phytotoxic to the seedling and caused reduced germination. The reprecipitated chitosan-salt treated seed was allowed to dehydrate for six hours and then soaked in the usual manner with water prior to seeding. Seed treatments are given in terms of ug of chitosan per g of seed.

Seed Subsequent Yield Protein

Treatment Treatment % Control % Control

a. Treated seed with and without a fixing agent were planted against a control.

100%

1005

1015

99%

103? 1015

101% 103% 108% 104% 106%

b. Chitosan lactate (molecular weight: 10K mw) foliar spray on treated rice 15 days prior to harvest at 10 ppm strength

102% 104% 106% 103% 105%

c. Chitosan lactate (molecular weight: 10K mw) foliar spray on treated rice 30 days prior to harvest at 50 ppm strength.

103% 106% 114% 110% 103%

d. Chitosan lactate (molecular weight: 10k mw) foliar spray on untreated rice 15 days prior to harvest.

None Foliar 10 ppm 101% 102*

None Foliar 50 ppm 102% 104%

None Foliar lOOppra 99% 105%

e. Chitosan lactate (molecular weight: 50k mw) foliar spray on untreated rice 15 days prior to harvest.

None Foliar 10 ppm 99% 101% None Foliar 50 ppm 100% 101%

None Foliar 100 ppm 95% 102%

f. Chitosan lactate (molecular weight: 10k mw) foliar spray on untreated rice 30 days prior to harvest.

None Foliar 10 ppm 104% 103

None Foliar 50 ppm 101% 107 3.

None Foliar 100 ppm 103% 108o.

g. Chitosan lactate (molecular weight: 50k mw) foliar spray on untreated rice 30 days prior to harvest.

None Foliar 10 ppm 102% 104%

None Foliar 50 ppm 101% 110% None Foliar 100 ppm 98% 109% EXAMPLE V - As a Protein and Yield Enhancer

a. Potatoes

Seed starts of potatoes were treated prior to planting with chitosan acetate at 125-250 μg/g (based on the dehydrated weight of the seed starts (10-12% moisture)). No fixing agent was applied since it was known that the starch in the potatoes would serve as a fixing agent to bind the chitosan salt and give residual effects of protein

enhancement. Prior trials based on same treatment levels using fresh weight basis of potato starts resulted in significantly depressed yields.

Treatment Protein %

100 110 115

b. Peas

Seeds of peas, were treated with chitosan lactate at the rate of 250 μg/g and then treated with a fixing agent of zinc oxide (10%) slurry with water at the rate of one ounce zinc oxide per hundred pounds seed.

Treatment Yield % Protein %

Control 100 100

250 115 108

c. Soybeans

Seeds of soybeans were treated with chitosan acetate at the rate of 250 μg/g and then treated with a fixing agent of potato starch (20%) in water at the rate of one ounce of potato starch per hundred pounds seed.

Treatment Yield % Protein % Control 100 100

250 112 117

d. Corn

Seeds of corn were treated with chitosan lactate at the rate of 500 μg/g and then fixed with a weal solution of corn starch at the rate of one ounce of corn starch per hundred pounds seed.

Treatment Yield % Protein % Control 100 100

500 105 113

e. Lettuce

Seeds of lettuce were treated at the rate of 125 μg/g and fixed with a weak salt of potassium phosphate K3PO4 (0.05 molar) .

Treatment Yield % Protein %

Control 100 100 125 107 111

EXAMPLE VI - Fertilizer as a fixing agent

Most fertilizers in high concentrations or very alkaline soils have the ability to act as a fixing agent for chitosan salts. Fertilizers, however, may be ineffective in reprecipitating the chitosan salts directly on the seed at other than high fertilization levels. Sufficiently high rates allow the residual effects of protein enhancement in crops. Fertilization requirements are frequently estimated based on expected moisture for dryland crops to preclude "burning" of the crop or to reduce potential for lodging. However, when high rates of fertilizer are used, it may serve as an adequate fixing agent to cause both increased yields and enhanced protein content.

Seeds of wheat were treated with aqueous chitosan acetate at the rate of 250 μg/g. A very weak salt of disodium phosphate Na ₂ HP0 ₄ (ImM) was used as a fixing agent (FA) where shown. Untreated controls were planted at corresponding fertilizer rates.

EXAMPLE VII - As a Seed Priming Agent

Seeds of cabbage were treated with aqueous chitosan lactate (average molecular weight 1M+ mw) lactate (2.5% solution), pH neutralized to 6.3, diluted with water and soaked at the concentration of 250 to 1000 g/kg. Priming took place at 15°C for 1 to 14 days under normal priming conditions with oxygen being injected. Seedlings were tested for emergence times and percent germination. Seeds were planted in both warm, moist and cold, wet soils with the following results.

Treatment Emergence & Germination % Solution Temp Duration Control Treated a. Seeded in normal warm, moist soil

Control 10 days 100%

Aqueous Chitosan Lactate 250g/kg 15° C

500g/kg 15° C lOOOg/kg 15° C

b. Seeded in wet, cold soil

Control 14 days 82 ^:

Aqueous Chitosan Lactate

250g/kg 15° C 500g/kg 15° C lOOOg/kg 15° C

c. At 30 days plant fresh weights were taken for the 250g/kg treatment and showed a 7% increase over the controls. At harvest protein showed a 14% increase over controls and yielded 3% more by weight of heads. d. One year old radish seed was treated with chitosan lactate at an application rate of 250 μg/g. 100% of the treated seed germinated as against 92% for control (untreated) seed. e. One year old Daws winter wheat seed was treated with chitosan acetate at an application rate of 250 μg/g. 100% of the treated seed germinated whereas 75% of the untreated control seed germinated. f. Three year old barley seed was treated with chitosan acetate at a 62 μg/g application rate. 67% of the treated

seed germinated whereas only 12= of the untreated seed germinated. g. Four year old tomato seed was treated with chitosan acetate at a 250 μg/g application rate. 16% of the treated seed germinated. 0% of the untreated control seed germinated.

EXAMPLE VIII - Spot Irrigation & Improved Freeze Protection Tomato plants were spot irrigated upon transplant with 16 oz. of dilute aqueous salts of chitosan ascorbate (CA) . Plants were about 6 inches tall at time of transplanting. Plants were spaced at 24" X 36" and alternated with rows of controls. The following tables illustrate overall results:

ENHANCED YIELDS

Supplemental treatments were applied to immediate root areas. Highest treatment level (1000 ppm) caused significant yellowing in plant tissue. Water soluble fertilizer treatment, which acted as a fixing agent, subsequently brought back healthy green colors to plants, but there was some stunting of growth during the 15 day period immediately following initial treatment. Fertilizer treatments were subsequently applied immediately after

chitosan treatment. Some indications of phytotoxicity persisted at 1000 pm treatment levels.

IMPROVED FREEZE PROTECTION

At 108 days from transplanting, a severe, killing frost was encountered and general yield data collection halted. Almost all control plants, 57 of 60, were killed. Treatments at successively higher doses showed increasing immunity to effects of freeze, particularly when administered just prior to the freeze.

Additional yield after 108 days (22 days of extended growing season) :

Treatment % Survival Yield % Impact of -1.5°C freeze Control 5 (3/60) 100 Nearly total plant kill; some new growth seen in 5 days

1000 ppm 80 (12/15) 310 Moderate damage to all; heavy new growth seen in 2 days

500 ppm 60 (9/15) 185 Extensive damage to most 250 ppm 33 (5/15) 145 Severe damage to all

125 ppm 7 (1/15) 115 Extreme damage to all

The above table shows the effects on tomatoes. Similar although less dramatic effects were observed on watermelons, cantaloupe, cucumbers, summer squash and green peppers at the same location.

EXAMPLE IX Improved Soybean Yield 2.4 lbs. of crab shell chitosan is dissolved in 8 gallons of 1% acetic acid. 48 hours is allowed at room temperature for the chitosan to dissolve with stirring. Water is then added to bring the volume to 26 gallons 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 about pH 6.0 to about 6.5 with 0.2 to 0.23 gallons of 6.0 N NaOH. NaOH is added slowly with stirring, because localized changes to a pH higher than 7.0 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 O to approximately 26 gallons. From this, approximately 16 ounces is added per hundredweight of soybeans. This treatment increases the yield by up to 20- 30%.

The sodium hydroxide solution can be from about 5.0 to about 7.5 N, but 6.0 N is preferred. Any alkali, such as potassium hydroxide, ammonium hydroxide, and soda ash can be used in place of the sodium hydroxide. The preferred alkali is soda ash. In that case, a solution of from about 63 to about 77 pounds of soda ash, preferably 75 pounds, in about 750 gallons of water is first made up. Enough of the soda ash solution is used to raise the pH to about 6.0 to about 6.5.

Chitosan seed treatments are applied using rates ranging from at least about 12, and preferably at about 16 oz. (of 2.5% concentration w/w) chitosan solution per dwt. of soybeans.

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 seed is treated in a humid environment, a post- treatment 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

that will be required. Highly viscous chitosan preparations can be mixed with seed using any machinery marketed for cement mixing. Modifications of grain angering 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 germination.

Chitosan seed applications are not detrimentally influenced by fertilizer supplements, herbicide applications or irrigation programs. Other commercial seed treatments, e.g., insecticides and fungicides, can be applied prior to chitosan. Components already on the seed will be attached to the seed by the chitosan, which 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, [ ³ H]- 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, e.g., cotton fibers or flour, may apply to chitosan.

EXAMPLE X

Soybeans treated with chitosan generally along the lines of Example IX have been tested in field trials. In one season where there was "normal" (about the same as the past 5 year average) weather, the trials met the objectives; yield was increased 18%.

EXAMPLE XI The following season, there were field tests conducted in a total of over 10 trials. Much of the trials were damaged by drought conditions. Average yields were increased in the order of 1-2 bushels/acre.

EXAMPLE XII Tree seeds treated with chitosan generally along the lines of Example IX are being tested with favorable results. The applicability of this invention is widespread, including conifer, pine, fir, other evergreen trees and deciduous trees including fruit trees. Data has been positive as to treated seed, which also tested disease-free, while every control developed disease symptoms. Fir tree seeds averaged a 25-79 percent increased growth after only 3 weeks.

The tree seeds are treated with chitosan generally along the lines of Example IX. Alternatively, the aqueous chitosan solution may be obtained using acetic acid, hydrochloric acid, formic acid, dilute sulfuric acid, or any other dilute acid. The applicability of this aspect of the invention is to tree seed. Chitosan seed treatments are applied using rates ranging from about 12 to about 64 oz. (2.5% concentration)/dwt. EXAMPLE XIII

When applied in a foliar spray, the chitosan solution appears to trigger a protective action in chrysanthemums inoculated with several fungal pathogens. It is also being tested on gypsophila (Baby's Breath) , azaleas, mums and

dahlias. Accordingly, in a further aspect of the invention, there are provided chrysanthemums seeds treated with chitosan. Alternatively, the chitosan may be obtained using acetic acid, hydrochloric acid, formic acid, dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 12 to about 32 oz. (2.5% concentration)/dwt.

EXAMPLE XIV In a further aspect of the invention, there are provided corn seeds treated with chitosan generally along the lines of Example IX. Alternatively, the chitosan may be obtained using acetic acid, hydrochloric acid, formic acid, dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 12 to about 64 oz. (2.5% concentration)/dwt. , preferably about 16 oz./dwt.

EXAMPLE XV In a further aspect of the invention, there are provided lettuce seeds treated with chitosan generally along the lines of Example IX. Alternatively, the chitosan may be obtained using acetic acid, hydrochloric acid, formic acid, dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 4 to about 16 oz. (2.5% concentration)/dwt. EXAMPLE XVI

In a further aspect of the invention, there are provided onion seeds treated with chitosan generally along the lines of Example IX. Alternatively, the chitosan may be obtained using acetic acid, hydrochloric acid, formic acid, dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 12 to about 32 oz./dwt.

EXAMPLE XVII In a further aspect of the invention, there are provided parsley seeds treated with chitosan generally along the lines of Example IX. Alternatively, the chitosan may be obtained using acetic acid, hydrochloric acid, formic acid, dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 12 to about 32 oz./dwt.

EXAMPLE XIX In a further aspect of the invention, there are provide radish seeds treated with chitosan generally along the lines of Example IX. Alternatively, the chitosan may be obtained using acetic acid, hydrochloric acid, formic acid, or dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 12 to about 32 oz./dwt.

EXAMPLE XX In a further aspect of the invention, there are provided rapeseed seeds treated with chitosan generally along the lines of Example IX. Alternatively, the chitosan may be obtained using acetic acid, hydrochloric acid, formic acid, dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 12 to about 32 oz./dwt. EXAMPLE XXI

In a further aspect of the invention, there are provided sugar cane seeds treated with chitosan generally along the lines of Example IX. Alternatively, the chitosan may be obtained using acetic acid, hydrochloric acid, formic acid, dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 12 to about 32 oz./dwt.

EXAMPLE XXII In a further aspect of the invention, there are provided milo seeds treated with chitosan generally along the lines of Example IX. Alternatively, the chitosan may be obtained using acetic acid, hydrochloric acid, formic acid, dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 12 to about 32 oz./dwt.

EXAMPLE XXIII In a further aspect of the invention, there are provided cotton seeds treated with chitosan generally along the lines of Example IX. Alternatively, the chitosan may be obtained using acetic acid, hydrochloric acid, formic acid, dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 12 to about 32 oz./dwt.

EXAMPLE XXIV In a further aspect of the invention, there are provided grass seeds treated with chitosan generally along the lines of Example IX. Alternatively, the chitosan may be obtained acetic acid, hydrochloric acid, formic acid, or dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 12 to about 32 oz./dwt. EXAMPLE XXV

In a further aspect of the invention, there are provided potato seeds treated with chitosan generally along the lines of Example IX. Alternatively, the chitosan may be obtained using acetic acid, hydrochloric acid, formic acid, or dilute sulfuric acid, or any other dilute acid. Chitosan seed treatments are applied using rates ranging from about 12 to about 32 oz./dwt.

EXAMPLE XXVI In a further aspect of the invention, there are provided soybean seeds treated with chitosan through an auger generally along the lines of Example IX. The chitosan coating "toughened" the seeds, providing greatly improved resiliency and protection from mechanical damage not found in seeds run through the same mechanical device without the application of chitosan. Seeds which were treated with a similar amount of distilled water only showed slightly less damage than untreated seeds. Treated seeds showed only 2% decreased germination in 100% germinable seed. In seed lots rated at 80-90%, the treated seed consistently exceeded 5-15% by that of the totally untreated controls and also exceeded the seed run through the auger with water only by 5-10%.

EXAMPLE XXVII In a further aspect of the invention are provided some heretofore unknown combinations of chitosan with certain substituted phenoxy trialkylamines and with linear dextrin which, at very low levels of use, significantly enhance the performance of emergence of chitosan and influence on timing of treatments. These compounds also apparently work by gene derepression and are not single enzyme or single gene activators. They do appear to allow plants to maximize the yield hydrocarbons and are recommended for use during period of dormancy or stress. These compounds work best in conjunction with certain thiols or alcohols. These hydrocarbon inducers are the subject of Henry Yokoyama et al., U.S. Patent No. 4,204,859 of May 27, 1980 and J. Szejtli et al., U.S. Patent No. 4,272,276 of June 9, 1981 which was examined as a fixing agent for chitosan salts, are hereby incorporated by reference. In greenhouse trials, treatment of seeds with chitosan acetate (ChA) on the same day of planting unless otherwise shown of soybeans

(R. Sherman) and wheat (Reardon) were conducted with the results shown below:

with 1 m solution DCPTA 30 da s

EXAMPLE XXVIII In a further aspect of the invention are provided results of early growth in the utilization of the seed generally treated with the compounds of Example XXVII where the

following results expressed as % control from 12 plants per treatment on 4 harvest from 14 days to 23 days were obtained:

o ean R he n'

The above table shows synergistic effects on early growth of the combination of chitosan with the compound DCPTA cited in Example XXVII and U.S. Patent No. 4,204,859 by H. Yokoyama et al. of May 27, 1980. Similar synergistic results were observed with the Compound identified therein as ITA.

EXAMPLE XXIX In a further aspect of the invention are provided field test yield results in wheat utilizing the compound DCPTA cited in Example XXVII. This table shows the benefit of somewhat reduced planting rates in conjunction with the proposed dosages and also the inability of either compound to exceed the untreated controls in these essentially ideal

growing con _d itions, but the excellent synergism of the two together on ultimate yields.

Wheat (Hill 81 Variety; SWWW)

PLANTING RATES: a = 40 pounds of Wheat seed per acre, b = 84 pounds of Wheat seed per acre.

All weights are recorded in grams

EXAMPLE XXX In a further aspect of the invention, is the compound chitosan in conjunction with that of its insoluble parent chitin. It is proposed that the effect of chitin powder (60 mesh or finer) in the immediate presence of the developing roots provides a host for beneficial soil micro¬ organisms which provide some benefit to developing seedlings from various pathogens and parasites (i.e., nematodes) . Performance of this particular aspect wherein select micro-organisms could be incorporated with or without chitin into the treatment or could produce highly active and selective biological controls due to the immobilization aspects of solubilized chitosan salts, their systemic properties making endophyte inoculations ideal, and the carbohydrate structure of the cited polysaccharides making an ideal food source for certain friendly biologies. Accepted biologies have been used as a top dressing over chitosan seed dressings with no adverse effects providing excellent and uniform root inoculations in soybeans. The table below is designed to show the synergistic effects of chitin additions directly to the seed in yields and the increased immune defense against a black mold which formed in controls after rain was received during maturity shortly before harvest of winter wheat where Disease Index (D.I.) 0 indicates free thereof and 10 indicates total infection.

39

Chitin is believed to play an important role in reduction of symptoms from cephalosporium stripe where benefits were also observed and may have been responsible for increasing yields in the above trials.

EXAMPLE XXXI In further aspect of the invention is the compound of chitosan salts which were prepared as previously stated but immediately after solubilization and neutralization spray dried utilizing commercially available equipment to form a non-clumping high viscosity water soluble product for field testing. Such a product is deemed to a high commercial value due to ease of handling, lack of preservatives since chitosan salts dehydrated to low moisture levels is relatively inert and not subject to biodegradation or significant chemical degradation which enhances shelf life. Also, transport of the finished product is greatly reduced since only 2.5% of chitosan is able to be solubilized without encountering virtually unworkable viscosities (too high) and further dilution would increase shipping costs and require much higher levels of moisture to be applied to the seed. Also, viscosities higher than those presently used due to the above limitations are frequently desirable

to maximize yield increase, obtain protein increases, and in general sustain the release of the chitosan salt. Further, the ease of application of such a product is obvious in that it would lend itself well to "drill box" applications or allow more normal seed treating equipment to apply it in its powdered form using a carrier (i.e., acetones, thiols, alcohols, etc. in which the powder being water soluble is insoluble. And finally the product would also obviously allow the current aqueous solution application with solubilization being available on site. In support of the above, the following example table using spray dried chitosan"acetate (ChAP) is submitted:

(ChA powder (ChAP)

Treatment: solubilized on site w/16 oz. H2θ/dwt)

ChAP@ 440μg/g ChAP@330μg/g ChA@ 250μg/g(notpowder) ChA@ 500μg/g(notpowder)

Although the exact treatment rates were not duplicated in the above table to get direct correlation, the trends of the data all at the same location in adjacent plots, shows the same relative performance versus untreated controls and represents a significant commercial advancement in working with this natural biochemical. It also makes other organic acids potentially usable which on solubilization had caused relatively short shelf life problems of only 1-2 months to be available for product formulation.

EXAMPLE XXXII In further aspect of the invention is the insecticidal benefit observed in treating roses with the compound to obtain at least a dietary preference away from treated plants. Although this has certainly not been observed in all plants in roses with every other plant in a row receiving a dose of 32 oz. of 1000 ppm chitosan acetate solution, the aphids failed to choose those plants about 90% of the time. The full observed benefits lasted from 30 to 75 days depending upon watering and rate of fertilization. The entire benefit was not lost for the entire growing season. The mode of action while unclear presents the possibility of chitinase or other elicited enzymes or proteins causing digestive difficulties in the aphids or the possibility of transferring or stimulating endophytes within such plants. Similar observations in certain varieties of wheat with aphids have been observed. With the altering of certain genes taken from Bacillus Thuringiensis, a single cell soil bacterium, scientists have been able to develop genes which produce proteins which are deadly to corn borers. The corn can be colorized with such endophytes which occur naturally in bermuda grass, but not in corn. Transference capabilities of such natural predators or defenses is highly probable with chitosan salts. Further genetic stimulation of certain natural proteins for the plant's bioinsecticidal defense with chitosan salts alone cannot be dismissed. Certainly, the potential for biologic inoculation with chitosan has been demonstrated in trials on soybeans with Guzobium organisms.

EXAMPLE XXXIII A further apsect of this invention is the ability of chitosan salts to bind previously applied agrichemicals notably slurries which present personal health hazards,

environmental safety, and waste disposal problems for treaters, handlers, and users. Such slurry applied chemicals are only loosely adhered to seeds at the time of application and unless planted quickly, are easily dislodged becoming airborne or accumulating at the bottom of bags, transport containers, or invading the air breathed by anyone in the vicinity. It has been shown that minute applications of atomized vapor carrying salts of chitosan preferably acetic in a non-neutralized solution prepared as previously discussed in a 2.5% (w/w) concentration of chitosan in a 1% concentration of acetic acid and applied to seed immediately after treatment at the rate of about 1 μg/g which is far too low to otherwise effectively accomplish the purposes of this invention can effectively reduce "dust off" or slurry chemical/seed separation by over 80% and at 5 μg/g it is reduced by about 98%.

Application of this solution by high pressure spray immediately upon discharge from a bin by an auger or most gravity bins when nozzles are properly placed virtually eliminates "dust off" when a 5 μg/g rate is used and it is diluted in water and applied at the rate of 4 oz./dwt. Injection into the base of an auger as the grain is being outloaded is extremely effective in eliminating "dust off" and its associated potential health problems. It is expected that such protection would cost about one penny per 100 lbs. of treated seed.

Such a binder coat would ensure treated seed was sowed with most of the chemical applied to it.

Further, the "cellophane like" wrapper thereby obtained tends to immobilize the slurried chemicals in the immediate vicinity of the seed (decreasing leaching) , and enhancing effectiveness of the chemical for its intended purpose. It appears that dosages of such chemicals may be open to

reduction at higher levels of treatment approaching 5-10 μg/g which would easily offset the cost of such treatments.

Commercialization of this aspect of chitosan salts would reduce personal risks, environmental pollution, and improve efficiency in chemical seed treatments.

EXAMPLE XXXIV A further aspect of this invention is the application of chitosan salts to serve as an encapsulation coat for various agri-chemicals to provide sustained release. The ability to reduce leaching of such chemicals as herbicides, insecticides, or fertilizers and to provide sustained release thereof is a further way to improve agrichemical efficiency, reduce health risks to the public at large, and minimize adverse toxicologic impacts on the environment. As such, a novel method of treating dry fertilizers with salts of chitosan has been developed wherein an atomized spray of aqueous solution is applied directly to such granules at concentrations of about 5-50 μg/g which allows a very sustained release of the fertilizer. Further and perhaps the biggest advantage is its tendency not to release such fertilizing agents without sufficient moisture. In very dry environments it will tightly bind such fertilizer decreasing the probability of crop burning even with high dosages of fertilizer in marginal moisture years. Such treatments can be effectively applied in large fertilizer batch blenders by injection of the atomized aqueous chitosan salts.

EXAMPLE XXXV As a further aspect of this invention is the ability of the compound chitosan to improve the yield in perennial alfalfa when sprayed on the crop at the rate of 1 pint of 2.5% chitosan acetate solution diluted into 10 gallons and applied uniformly to one acre. The height of the alfalfa was visually checked and measured showing an additional six

inches height on the treated field and resulted in a 12% increase in tonnage from the treated field.

EXAMPLE XXXVI In a further aspect of the invention, a quantity of 2% chitosan solution suitable for use in the invention may be mixed as follows: at ambient temperature, to obtain about 500 ml of aqueous chitosan acetate, add 10 g of chitosan of high molecular weight (over 500,000 mw preferred) to 450 ml of water in a 600 ml beaker. This material is agitated with an overhead stirrer for several minutes. Then add 5 ml of glacial acetic acid (USP Grade) and allow to mix for one hour until the chitosan is dissolved. Continue agitation throughout this hour. The concentrated solution is then filtered and insoluble particles are removed. Other ingredients may be added to this base formulation including but not limited to the following:

1. A wetting agent may be added for oily seeds (i.e., canola) but is not required.

2. The solution pH may be raised but when diluted adequately no ill effects to germination were seen.

3. The aqueous chitosan may be combined with a small amount of hydrating agent (such as polyethylene glycol) which improves film flexibility and reduces its tendency to fracture during post dried handling. For this purpose up to 10% by volume of a hydrating agent may be added to the concentrated solution prior to packaging during production or before dilution and application.

This concentrate is then diluted immediately prior to use to provide a rate of 1/2 fl. oz. to 1 fl. oz. of concentrate per 100 lbs. For large or dense seeds with relatively small surface areas (i.e., peas, beans, cereal grains, corn, canola) the lower rate provides adequate coverage. For small, light seeds (i.e., grasses, lettuce,

etc.) with larger surface areas, the higher rate provides a more uniform treatment film.

Additional benefits observed by such treatment was enhanced germination rate for seeds planted into cold wet soils and in old seed lots.

The binder coat treatment was effective in virtually eliminating dust in later handling of seed. Even after it had been dried for a prolonged time.

Seed may be treated by first diluting from 4 up to 10 parts or more water to 1 part aqueous concentrate chitosan solution (prepared as described above) and by injecting a fine spray or fog mist into the seed transfer device. This may be pneumatic tubes, an auger, or a similar device for seed transfer. The application point is after all other suspension treatments are applied (preferably immediately thereafter) and the final seed coating. Injection of the fine spray or mist causes small droplets of dilute aqueous chitosan to be deposited on the prior seed coats and seed causing any powdery material to be adhered to the polymer which is in turn bound to itself and the seed. The additional travel through the seed transfer device causes further dispersion of the coating until it thoroughly covers the seed.

Thus, methods have been provided for enhancing protein content, providing improved freeze protection, and obtaining seed priming for crops with the use of chitosan salts. Those skilled in the art will appreciate that the conception upon which this disclosure is based may be used as a basis for the perfection of other methods, for carrying out the several purposes of the invention. The claims, therefore, should be regarded as including such equivalent methods as do not depart from the spirit and scope of the invention, which is intended to be defined by the appended claims.