NEMATOCIDALLY ACTIVE CHITIN-PROTEIN COMPLEX

Abstract: A chitin-protein complex is prepared from the chitin-containing biological waste material such as crustacean shells. The complex is different from both chitin and chitosan, and has useful nematostatic and nematocidal activity for agricultural and horticultural applications by admixing nematocidally effective amounts with a plant growth medium. The complex also provides a source of nitrogen in slow-release form, making it particularly suitable for combination with fertilizers, soil conditioners, etc.

Description: NEMATOCI DALLY ACTIVE CHITIN-PROTEIN COMPLEX Dp pp T PT T n i DF THF TNUPKITT r8_ Technical EJ.p1d.Q-f the lnm.nti.r-_ Thi invention relates to a process -for converting shell-fish wastes into use-ful products and for avoiding costly traditional methods for disposing of low economic value waste products of the shellfish processing industry. More particularly, the invention relates to methods for the isolation and recovery of a naturally occur ing ch i t i n-protei n complex from the tough polymer matrix of crustacean exoskeletons and to methods for using these polymeric composi t i ons for inhibiting the growth of plant-parasi ic and other nematodes of interest in horticulture and agriculture. Rrri-orniinfl Art Nematodes <nema — thread; oides — resembl ing), or unseg ented roundworms wi th elongated, fusiform, or sac! ike bodies covered with cuticle, which belong to the phylum Nemathel inthes, are virtually ubiquitous in nature, inhabitating soil , water and plants, and are importantly involved in a ~~wide range of animal and plant parasitic diseases. While there are some significant problems related to nematodiasis in animals, it seems likely that major interest will continue to focus on nematodes w ich parasitize the roots, stems, leaves and seeds of plants and are major contributing factors to crop losses and to serious economic losses to agricultural productivity on a worldwide basis. These include, by way of example, root-knot <M l,o,i,rinr|yn , -sjip.), root-lesion (Pratyl pnrhns spp . ) , spiral ( HPI inrntyl pnrhn spp.), and burrowing nematode <Rarinρhnlu e , imil ), which is highly destructive of citrus crops and more than 288 other species of plants. Damaging levels of stunt Tyl pnrhnrynrhn*- spp.) , reniform (__.tyl nr.hu In .-fifi.) and oliar (Am hgl onrhni HP=. g ,pp . ) nematodes are also found in foliage ornamentals. Ring g Marrn r-.thnn i a xp.nnpl as) and dagger (Xiph i np a __p.) nematodes infect peach orchards, and soybeans are often seriously infested with both the soybean cyst nematode (i-£.t___ripra glyrinp ) and root-knot (MPI oirioQynp spp..). In the United States alone, appro imatel two million acres of agricultural land are treated each year by prophylactic and quarantine measures, chemical control, soil fumigation, hot-water treatment, and resi tance-based selection methods in order to control nematodes. T i includes land used to r w nonfood crops (e.g., cotton and tobacco), field crops (e.g., corn and wheat), orchard crops <e.g., apples, citrus and nuts), vegetables (e.g., potatoes) and a wide range of ornamentals. Almonds, apples, asparagus, citrus (including oranges, grapefruit, lemons and limes), cotton, grapes, melons, peaches, peanuts, pineapples, soybeans and strawberries, among other edible plant foods, and essentially all vegetables are susceptible to nematode infestation, as are home gardens and lawns, commercial turf, ornamentals and most other plants. Furthe more, large areas of otherwise arable agricultural lands (i.e., croplands, pasturel ands, forest lands and lands in other agricultural use) may lie fallow or unused owing directly or indirectly . to overwhelming or uncontrolled nematode infestations. Plant-protection methods for nematode control, including crop rotation, soil-treatment and fertilization practices, and "green manuring 8 with sweet clover or mustard, as well as physical methods of soil treatment, such as steaming of soil and hot-water treatment of planting stocks, have generally met with only l imited success. Chemical methods, on the other hand, employing a range of systemic pesticides, have been reasonably successful , particularly in horticul ural practice, despite the fact that nematodes tend to be resistant to many of the pesticidal agents which have been marketed for application either in a gaseous form (fumigation ) or dispersed in soil in liquid or sol id forms; (see, for example, A.C. Tarjan and P.C, Cheo, "The Nematode Screening Program of the University of Rhode Island," Contribution 887, Agricultural Experiment Station, Kingston, R.I., March, 1956). Currently, only some 25 ne atoc idal chemicals are registered with the U.S. Environmental Protection Agency (EPA) for use on important food, feed and fiber crops. Most nematocides now available in commercial markets are, moreover, quite toxic to both man and animals, in large part being organic t icphosphate (phosphorot i oate and phosphorodi i oate) compounds and chol i nesterase inhibitors. Many of - them are also phytotoxic. Because of their adve-rse effects on the environment, several nematocides which are currently marketed are subject to review which may result in cancella ion of regi tration. Thus, issues of safety and efficacy as well as of agricultural economics are critical considerations in the control of plant-parasitic nematodes. A clear and present commerc i l need exists for ne atostat i or nemato idal materials, preferably biological control agents, which are non-toxic for plants, animals and man. Despite the widespread presence in almost all soils, especially those wi h a i h content of decomposing organic matter, protozoa, bacteria, predatory nematodes, and fungi which can in theory act as biological control agents against plant-parasitic nematodes, biological control programs have not been instituted on a large-scale anywhere in the world; see, for example, K. F. Baker and R. J. Cook, Rinlnnir l Cnntrnl .rιf..E.l,an ,-EathθQe.αs, W. H. Freeman and Company, San Francisco, California, 1974 and H. Decker, Plant pmafprip and Th ir r.nrt r n1 ( Phy nnpm tnl Γ.Q> , publ i hed for the U.S. Department of Agriculture and the National Science Foundation, Washington, D.C. by Amerind Publ ishing Co., Ltd., New Delhi , India, 1981. Few efforts have been made to analyze in a systematic and orderly manner the biological effects of deliberate introduction into soils of relatively large amounts of the type of non-toxic organic wastes which may accumulate and cause serious environmental and economic consequences in specific geographic regions, such as the Chesapeake Bay (e.g., R. A. A. Muzzarelli and E. R. Pariser, Eds., Fi r<=t . Intarnat i nnal πnn-f p nr nn Hhi r in/Chi tncan , MIT Sea Grant Report, MITSG 78-7, Cambridge, Mass., 1978; B. L. Averbach, "Chi t in/Ch i tosan Production for Utilization of Shellfish Wastes," pp. 285-388, in W. S. Otwell , Ed. Rpaiporl _la__e Man pprnpnt in th 193H^. ; Cnn PΓPΠΓP Prnrppiiinn , Florida Sea Grant Program, Report No. 48, February, 1981; T. P. Cathcart et al . , "Composting Blue Crab Processing Plant Solid Waste", Annual Report, Department of Agriculture Engineering, University of Maryland, College Park, Maryland, December 31, 1981; and T. M. Cook, "Development of a Fermentation Process to Use Wastes from the Chesapeake Bay Industry", Report F-16-81-885, Department of Microbiology, University of Maryland, College Park, Maryl nd, March 19S2). Laboratory studies undertaken by Brown et al . have, however, shown that dried and powdered seafood wastes — including commercially available chitin and chitosan, alkal i -treated (3.5% NaOH for 24 hours) and non-treated shrimp shell wastes — when utilized as a soil amendment cause a stati tically significant decrease in root-knot infestation and a statisticall significant increase in the number of chitinolytic act inomycetes in both tomato and ornamental plants (see, for example, L. R. Brown et al . : "The Use of Chitinous Seafood Wastes for The Control of Parasitic Plant Nematodes", MMRC Project No. GR-7ά-884 and CO-76-828, Mississippi Marine Resources Chronical, Long Beach, Mississippi, October, 1977 and "The Use of Chitinous Seafood Wastes for the Control of Plant Parasitic Nematodes", BMR Project No. GR-ST-73-883 and GR-ST-73-884, Bureau of Marine Resources, Mississippi Department of * Wildlife Conservation, Long Beach, Mississippi, September, 1979). Problems associated with the accumulation of crabshell wastes in the Chesapeake Bay region (or, by way of further example, shrimp shell wastes around the Gulf of Mexico) and often conflicting observations on the role and effect of chitin and chitosan materials isolated from these wastes prompted the laboratory investigations which l d to the product and process of the present invention. Relevant observations include published reports that: (a) the addition to soils of crop residues and other carbonaceous materials appears to suppress both nematodes and certain fungi in soil populations ( e.g., M. B. Linford et al . , "Reduction of Soil Populations of the Root-Rot Nematode During Decomposi t i on of Organic Matter", Sύ.i.1 Sc.L._ i_5: 127, 1938, and C. B. Davey and G. C. Papavizas, "Effect of Organic Soil Ament±nents of the Rt nrtnπia Disease of Snap Beans", Aprnn, ,\■ , 51: 493, 1959 ) ; (b ) small additions of commercially available chitin, but not of chitosan or N-acetyl gl ucosami ne , seem able to stimulate ch i t inase-produc ing microorganisms in the soil and to reduce the severity of root-rot of beans caused by Eu_s__j__Lum (e.g., R. Mitchell and M. Alexander, "The Mycolytic Phenomenon and Biological Control of Fii e .ar.iιιm in Soil", Na.tm-a ISA: 1961, and R. Mitchell and M. Alexander, "Chitin and the Biological Control of E_.____iι__ Diseases", £1 ant Di=.pase Rppnr.tpr __-.: 487, July 15, 1961; (c) chitosan, but not chitin, inhibits the growth of many fungi , including plant and animal pathogens, in culture media (e.g., C. R. Allan and L. A. Hadwiger, "The Fungicidal Effect of Chitin on Fungi of Varying Cell Wall Composi ion", F_p. yrmingy 2; 285, (1979); and (d) commercial preparations of chitosan have l ittle or no effect in reducing either the chemical or biological oxygen demand of wastewater effluents from crab processing operations (e.g., F. W. Wheaton et al . , "Wastewater Characterization and Treatment System Development for a Blue Crab Processing Plant", WRRC Technical Report No. 65, University of Maryland, College Park, Maryland, April 1981.) Several attempts at developing useful methods for deal ing with chitin disposal have also been described in the patent l iterature, e.g., see Austin, U.S. Patents 3,879,377; 3,392,731 and 4,236,887; Balassa, U.S. Patents 3,983,268; 3,911,116 and 3,914,413; Dunn, U.S. Patent 3,847,397; Casey, U.S. Patent 4,859,897; Mural idhara, - U.S. Patent 4,293,893; and Muzzarell i , U.S. Patent 4,232,351, the contents of which are incorporated by reference herein. Various techniques are also known in the art for recovering chitosan from chitin e.g., Rigby, U . S. Paten t 2 ,848 , 879 , Penn i stcn , U . S . Paten ts 3 ,862, 122 , 4 , 195 , 175 and 4 , 199, 496, the contents of wh i ch are i ncorporated by reference here i n . P> i <=.r 1 n-.iirn r,-τ t h p T n u pn t i nn It is a general object of this invention to provide improved and economical ly advantageous methods for disposing of otherwise low economic value wastes remaining after commercial shellfish processing operati ons. Another object of this invention is to provide a process for converting chi t in-contain i ng biomass waste materials into industrially useful compositions, preferably into forms and compositions of matter which have use in agriculture, horticulture and animal husbandry. A further object of the invention is to provide an improved and inexpensive means for obtaining commercial quantities of materials from naturally occurring ch i t in-contain ing biomass which can be demonstrated to induce ne atostatic and nematoc idal activity in cul ure media and in soil samples. •A more particular object of the invention is to provide a newly isolated ch i t in-prote in complex which can be obtained from naturally occurring sources and has demonstrable nematoc idal activity for prototypical nematode species without evidence of a direct toxic effect on nematodes. Upon study of the specification and appended claims, further- objects, features and advantages of this invention will become more fully apparent to those skilled in the art to which this invention appl ies. The present invention involves the discovery that a nematoc idally active ch i t in-protei n complex can be easily and economically prepared by mild acid hydrolysis of crustacean shell wastes, with or without recovery of carbon dioxide and other volatile gases produced during demineral izat i on and partial protein degradation. The resulting c i t i n-prote in complex induces nematoc idal activity in nematode cultures la v i trn, characterized by microscopic evidence of premature senescence and gas vacuole formation accompanied by loss of mot i 1 i ty and death. Dead and dying nematodes, in sharp contrast to viable and highly motile forms, take up Brill iant Green and Brilliant Cresyl Blue stains. Because of the nematoc idal activity that is induced by the ch i t in-prote i n complex described herein and the ease and low cost of i s manufacture in commercial quantities, addition of this material to agricultural and horticultural soils for the purpose of control of plant-pathogeni nematodes provides an economically and environmentally attractive means for the use of otherwise low value shellfish wastes and a means for reducing food, fiber and economic losses due to nematode inf stations. Incorporation of these materials into animal feeds also offers a potential means for control of intestinal tract nematodiasis and a rich source of dietary protein. Bciaf. f)p=.rr ip.t inn r>f the Draui ΠQ=. The present invention will become more fully apparent to those skilled in the art from the following description, taken in conjunction with the annexed drawings, wherein: Figure 1 illustrates the process for producing the ch i t in ' -ρrote in complex of this invention and identifies by-products which can be recovered. Figure 2 illustrates the subunit co posi t i on of the protein component of crabshell waste treated according to the process described in Example 2. Figure 3 illustrates conventional processes used to treat shellfish wastes for the production of commercial chi in and chi osan products as they compare with the process disclosed herein. Also shown are the chemical structures of chitin and chitosan. Figure 4 illustrates the subunit composition of protein components of the ch i t i n-prote in complexes prepared in Examples 2, 3, and 4 and of commercial chitin and chitosan preparations. Figure 5 illustrates the subunit composi t ion of the protein component of the ch i t i n-prote in complex obtained by acid treatment of dried fermentor cake from a commercial gibberell in fermentation process as described in Example 5. Figure 6 is a plot of the alternating current (ac) conductivity of chitin, chitosan and the crabshell ch i i n-prote i n complex as a function of appl i ed vol tage . Figure 7 demonstrates the infrared spectra of (A) chitin, (B) the ch i t i n-prote in complex of this invention, and (C) chitosan. Figure 3 illustrates the infrared spectra of (A) untreated fungal fermentation cake and (B) an acid-treated fungal fermentation cake product of this invention. Figure 9 demonstrates the light microscopic appearance of Panaπrei lnc =_£ . nematodes in control culture media (A and B) at 16-28 days after innoculation and the appearance of nematodes at 16-28 days after innoculation in test media containing chitin the ch i t in-prote in complex (C and D) of this invention. Figure 18 shows living and dead nematodes stained with Brilliant Green . p pc.t Mnrlp 4nr C rrying Out thp Tnupntinn The c i i n-prote in complex of this invention can be prepared from any suitable chi in-containing biomass raw material. Such materials include but are not limited to invertebrate marine organisms having visible shells. Examples of such organisms are arthropods, including crustaceans, mollusks, marine benthic organisms and krill fish. Preferred shellfish waste is that obtained from crustaceans such as crabs, lobsters, crayfish, shrimp and prawns. Cell walls and f i 1 amentous masses of true fungi, including Phyco ycetes- and Asco ycetes species (which can be digested by one or more of the over 38 enzymes, including chitinase, glucanase and mannase , contained in the digestive juice of the snail H I i _ nm t i a or produced by certain bacteria, such as some soil scavenging miHmnna g species which have been isolated from soils) contain chitin but do not ordinarily provide a suitable raw material or feedstock for a commercial process because of the amounts of these materials presently available. Because it is the presently preferred embodiment, the preparation of the ch i t in-prot i n polymer complex obtained from the shells cf blue crabs (Hal 1 i nft.r tea =,ap i rius) harvested from the Chesapeake Bay will be described in detai 1. In conventional blue crab processing operations (see, for example, pages 286-287 in E. J. Mi ddl ebrooks, Industri l πll^tinn n trnl , Unliimp 1 : Anrn-Tndn r i P_, John Wiley and Sons, New York, 1979) crabs are dredged from the mud, caught in baited traps or l ines or scraped from grassy shores during the molt. Baited pots are used to trap Dungeness, Tanner and King crabs which are then stored in circulating seawater in shipboard and/or in landbased tanks prior to processing, usually by dry butchering. Blue crabs are transported live to the processing plant and are unloaded into trolleys for immediate steam cooking at 121°C for 18-28 minutes. The cooked crabs are then stored overnight in a cool ing locker after which the claws are removed and saved for later processing. After removal of the carapace and claws, the claws and sometimes the bodies of the crabs are either run through a mechanical picker or picked manually to separate residual meat from the shell . Crab processing waste is generally discharged to a waterway or a municipal sewer, hauled to a sanitary landfill or otherwise render-ed, frequently by drying and shredding for eventual use as a feed meal , especially for chickens (see, for example, -P. R. Austin et al . U.S. Patent 4,328,158; W. P. Ur i Yr ins, Jr. and T. M. Miller, "Prices Based on Nutritional Worth, Crab Meals and Crab Meal -Phosphor ic Acid Supplements in the Diets of Mo ogastric Animals", Final Report ( F15-31-885) to Department of Natural Resources, Maryl and Ti dewater Administra ion (March, 1932). The presently preferred starting or raw material for the herein disclosed invention (Figure 1) is such crab processing waste material which has been oven-dried and shredded to a small particle size. To reduce costs of raw materials, the drying step can be omitted. The exact particle size which is used affects the rate but not the nature of the process. Composition of the crabmeal raw material varies both with the season and with the thoroughness with which the meat is removed from the shells, but the raw material generally contains protein (48-58 ), calcium carbonate and small amounts of other mineral salts (about 58 ), and chitin (about 18%). Dried and shredded shell wastes are milled or ground to a desired particle size and either used directly or washed with hot or cold water to remove contaminants which may have developed during transportation, if required, to a s processing facil ity. Shell fragments are then 18 demineral ized in a stirred tank reactor using a dilute mineral acid, such as 1.8 N HC1 , for a period of 38-68 minutes, generally under ambient temperature and pressure. Acids such as sulfuric and phosphoric are not suitable since they result in insoluble calcium salts which interfere with recovery of the product. The demineral i zation reaction, which is accompan ied by significant modification of the protein component of the crab shells (Figure 2 ) and by the release of carbon dioxide gas containing detectable amounts of the "fishy" odors characteristic of alkyl amines, can be followed by titration or by observation of gas release. The insoluble end-product of the reaction which is of specific interest to this disclosure is a chi t i n-prote in complex which has distinctly different gel electrophoretic properties from the product resulting from demineral izat ion of crabshells by chelating agents such as ethyl enedi ami netetrace i c acid (EDTA) (Figure 4) and from ch i t in-prote i n complexes isolated from fungal residues (Figure 5). The sol id-state electrical properties of such ch i t in-prote i n material are also distinctly different from those of commercial preparations of chitin and chitosan which are produced by substantially more vigorous subsequent treatments (Figures 3 and 6). After comp1 et i on of demineral i zat i on and protein modification (DM/PM ) by mild a id hydrolysis, usually at about 68 minutes after the start of the hydrolysis reaction, the resulting c i t in-prote in material is washed until neutral (pH 7.8) with water or weak soda ash ( N 2Cθ3 ) solutions. Effluents from the DM/PM and wash water tanks can be recycled for recovery of low molecular weight peptides, amino acids and calcium chloride, brine, or can be simply discharged to an approved waterway or wastewater treatment facil ity. The resulting ch i ti -prote in complex is then dried in a suitable drier and ground, if desired, to a particle size of less then 8.5mm. No further treatment, as is required in conventional chitin and chitosan processing operations ( Figure 3), is needed. The resultant c i t in-prote i n complex (Figures 4, 6 and 7) is: (i) insoluble in neutral and in dilute acid solutions but solubil ized with significant decomposition of the protein component in concentrated mineral acids; (i i) low in ash content; (iii) high in 1 1 bound nitrogen content owing to the presence of the protein moiety; and (iv) a naturally occurring, biodegradable material which, when added to nematode cul tures la _ i trrt, results in a significant reduction in the number of l iving organisms (Figures 9-18). The product can be produced commercially in better yield and at substantially less cost than can either chitin or chitosan derived from crab, lobster, shrimp or other shellfish processing wastes or from the walls of c i i n-contai n i ng fungi , molds and yeasts. Morphological changes induced in nematode cultures in la v i tr culture media are also distinctly different from those which are seen following exposure of prototypical nematode species to chitin or chitosan (Figure 9). Preferred rates for appl ication of the ch i t i -prote i n complex of this invention to plant growth media range from 1 to 58 weight percent, generally, in admixture with a plant growth medium containing the requisi e nutrients. More preferred rates are in the range of 2 to 28 weight percent; the presently most preferred rates are in the range of 5 to 18 weight percent. The optimum -amount within this.range depends upon a number of variables which are well known to those skilled in the art of plant protection. These variables include but are not l imited to disease to be controlled, the type of crop, stage of development of the crop and the interval between ap li a ions. Appl ications within the range given may need to be repeated one or more times at intervals of 1 to 6 months. The chi ti n-prote i n complex of this invention can be applied in a variety of formulations, preferably as granules, pellets, etc. Powder and dust preparations can be made by blending the active ingredient, with or without surfactant, with finely divided sol ids such as talcs, natural clays, pyrophyll ite, di ato aceous earth; flours such as wheat, redwood, and soya bean; or inorganic substances such as magnesium carbonate, calcium carbonate, calcium phosphate, sodium si 1 i ocoal uminate , sulfur and the l ike. The choice of a particular diluent is based on consideration of the physical and chemical properties required of the product, the chemical and physical properties and concentration of the active ingredient, and the use for which the formulation is intended. The composi t i ons are made by thoroughly blending the active ingredient with the diluent and other addi t i ves. Powdered compositions can be converted to granules by adding a l iquid, treating mechanically, and usually drying. Mechanical devices such as granulating pans, mixers and extruders can be used. Compaction devices can be used even without a liquid in the mixture. Water soluble binders, e.g. inorganic salts, urea, l ignin sulfonates, methyl cellulose, other water soluble polymers and the l ike, can be included in these part ϊcu1 ate formulations in amounts up to about 25% by weight of the finished granule or pellet. Such materials also aid in disintegration of the pellet and release of the active ingredient under- field conditions. Alternatively, a suspension of the active ingredient can be sprayed on the surface of preformed granules of clay, vermicul ite, corn cob and the like. Surfactants may al o be included in formulations of the latter type. The composi i ons of the invention can contain, in addition, to the active ingredient of this invention, conventional insecticides, iticides, b cter ic i dεs, other nematocides, fungicides or other agricultural chemicals such as fruit set agents, fruit thinning compounds, fertil izer ingredients- and the l ike, so that the compositions can serve useful purposes in addition to its nematoc i da! act i v i ty. Because of its protein, the ch i t in-prote i n complex of this invention contains about 18% nitrogen in a si cw-rel ease form; it can advantageously be mixed with sources of metabol izabl e phosphorous and/or potassium to provide a balanced fertilizer. The nitrogen content can be enhanced by the further addition of other nitrogen fertil izer sources which are well known in the art. The presently preferred embodiment of this invention is for use in a potting mixture with soil or a particulate inorganic material such as vermicul ite, e.g. for growing greenhouse plants or nursery stock. In one embodiment, Harpn c ,pnr iiim fungus is added to enhance the nematoc ida! ac ivity of the ch i t in-prote in corri lex. Pathogenic plant nematodes which may be controlled in accordance with the present invention include but are not l imited to those set forth in the following table. 13 TABLE I PΔTHΠRFNTΓ PI __MT MFMΔτnr>FS NFMATΠΠF Pi NT HflSI Aphpl f Strawberry Pi tyl pnrhn rii spar i Root crops Hetprodera , r.ostnrh i sn e , i e , Potato Hi tyl nrhim dp<-.trnr tnr Potato Ej.atylpr.chus pp.nptran Tobacco, apple, cherry Xiphinpma a pr ; r mim Grasses, Citrus, Tomato MPIΠJ ngynp hapla Potato Xylpnchi.il s ■■semij-pnetran Ci trus Pi tyl r.rhπc myr P 1 i nph anna. Mushroom Tyli-nchorhyrt£.hιιs rlaytnni Tobacco Hpmi c i noe oi p?. , rb. ' t ttnnd i, CarrieIlia Hpmi ryr 1 i nphnra ar-pnaria Ci trus Cel sry Paratyl pnrhu prQj,ectu„ Grass Dr tyl n hn ze.as. Corn Soybean -ix&i Corn, strawber ies, etc. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, util ize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. All temperatures are set forth uncorrected in degrees Celsius; unless otherwise indicated, all pressures are ambient and all parts and percentages are by weight. The values obtained by elemental analysis are within the usual l imits of experimental error; all new products gave the expected parent peaks in 1R. 14 F-. amp l ρ 1 Pr pp an a t i r.n r.-f Paι_ Ma t p i a l Crabmeal processing waste obtained from a commerc i al suppl ier was water washed, dried in a hot-air oven at 188°C for approximately 16 hours and then shredded mechanically to a particle size such that al 1 of the material passed through a No. 25 USA Standard Testing Sieve. The resulting particles had a moisture content of 5-18% and contained appro imately 48-58% protein and 38-58% CaCQs on a dry weight basis. Elemental composi t i on of three representative batches of otherwise untreated raw material feedstock is illustrated in Table II. Protein content was determined by the method of Lowry ( which measures tyrosine and peptide bond content). Nitrogen content was determined using combustion analysis based on the Pregl-Dumas method and a Perkin-Elmer mode! 248B Elemental Analyzer. 1! Table II Cnmpnsi tinp-o ,.,ShPl If ish Starting Matprial FIFMFNT 1INFRACT!nNATFI) (1) ST7FD <: RRΠIM (3) Calcium (Hg/g) 92988.8 . 94999.8 95988.8 Sodium (Hg/g) 9478.8 12888.8 18588.8 Magnesium ( g/g) 4338.8 5178.8 5838.8 Potassium ( g/g) 4168.8 3398.8 4428.3 Strontium (Hg/g) 1878.8 1888.8 1178.8 Iron (Hg g) 361.8 418.8 716.8 Aluminum (Hg/g) 188.8 183.8 286.8 Barium (Hg/g) 25.5 23.5 34.6 Boron (Hg/g) 4.5 4.5 6.6 Cadmium (Pg/g) <8.5 <8.5 <8.5 Chromium ( g/g) 1.8 1.5 1.6 Cobalt (Pg/g) <i.B <l.β <3 °t Copper ( g/g) 27.8 25.8 36.3 Lead (Hg g) <2.5 <2.5 Manganese (Hg/g) 158.8 145.8 223.8 Mo1ybdenurn ( "g/g) <1.8 <l.β <3.3 Zinc <μg/g) 69.8 57.5 74.2 Carbon (%) 29.84 34.76 34.81 Hydrogen (%) 3.96 4.74 5.59 Ni trogen (%) 5.46 6.29 7.19 Protein (Lowry) (%) 32.4 43.8 41.3 Ash (%) 45.76 36.65 37.22 Moisture (%) 7.41 18.47 8.11 (1) Shredded shellwastes, as received (Batch 118) (2) Shredded shellwastes, sized through ASTM 48 mesh screen (Batch 118) (3) Shredded shellwastes, ground to <8.5mm size (Batch 524) F-_ m le, ? I.ahnratnry-Rral I e .nl at i ππ ,ρf Crabshpll C it i n'-Prntp in πnmplp* b Arid pminpral i 7a inn The subunit composition was determined by el ectrophoresi on a 18% polyacryl amide gel containing 8.1% sodium dodecy! sulfate ( SDS). Shewn in Figure 2 are scans of the gels after staining of the proteins with Coo assie Brill iant Blue R. The positions indicated by the arrows are the positions of standard molecular weight (given in da! tons in parentheses) marker proteins: (a) myosin (288,888 ) , ( b) beta-gal actosidase (116,588), (c) phosphory! ase B (97,488), (d) bovine serum albumin (66,288 ) , (e) ovalbumin (45,888), (f) carbonic anhydrase ( 31 ,888 ) , ( g ) soybean trypsϊn inhibitor (21 ,588), and (h) lysozyme (14,488). The samples run were 5 mg of (A) start ing mater i al , (B) starting material after 18 minutes in acid, (C) after 38 minutes, (D) 68 minutes, and (E) the final product after acid treatment, washing, and drying. Four hundred grams of the raw material described in Example 1 were slowly added over a 38 minute period to 2 l iters of 1.8 N HC1 with continuous stirring. The reaction caused rapid demineral izat ion of the CaCθ3 phase of the raw material feedstock as evidenced by foaming of the reaction mixture and the release of CO2 gas containing readily detectable amine odors. Approximatel 48 mis. concentrated HC! were then added in small al iquots to the reaction mixture (to maintain acidity at appro imately pH 1.5 ) over a period of about 68 minutes, after which no furthe foaming was observed. The insoluble residue remaining after the deminera! izati on and partial hydrolysis procedure was collected on a No. 278 U.S.A. Standard Testing Sieve and washed with water until both the product and the washings were neutral (pH 7.8 ) . The insoluble product was oven-dried at 188^0 overnight yielding 128 grams of product (32% yield ) . The dried product was ground in a Wiley Laboratory Mil! to a particle size of less than 8.5 mm. for use in al! subsequent studies. E'- am l p 3 Treatmpnt .ui th . a„„Cl.β .at i ng. Agpnt Ten grams of the raw material feedstock described in Example 1 were added to 1 l iter of 8.1M ethylenediaminetetraacet ic acid (EDTA), pH 7.5, and the mixture stirred continuously at 25°C for 72 hours. The residual insoluble product was collected on a No. 278 U.S.A. Standard Testing Sieve and washed exhaustively with water. The resulting product was oven-dried at 188°C overnight with recovery of 2.75g of dry product (27"/. yield). F--ampl P ,4 P M nt -Sr a . P Pr ep ar a t i on n-f Pr a h=.hp 1 1 Ph i t i n -Pr nt P i n Pnn.p l P Y Fifteen kilograms of the raw material feedstock described in Example 1 were slowly added to 188 l i ers of 1.25 N HC1 in a 288 l iter stainless steel stirred tank reactor. The rate of addition of feedstock was regulated so as to minimize foaming over the course of a 68-minute deminer l i za i on and acid hydrolysis reaction. Insoluble product remaining after demi era! i zat i on and acid hydrolysis was collected on a S e o^ Vi bro-Energy'^-* separator equipped with a 158-mesh sel -cl aning stainless steel screen, washed with water and then 1% Na2C03 and, finally, washed with water again to remove all soluble carbonates. The neutral (pH 7.8) product was oven-dried at 188°C overnight and then ground to a particle size of less than 8.5 mm. prior to use. The elemental compositions of the preparations from Examples 2, 3, and 4 as compared to chitin and chitosan are shown in Tabl e 111. Figure 4 illustrates the subunit composition of protein components of the ch i t i n-prote i n complexes prepared in Examples 2, 3, and 4 and of commercial chitin and chitosan preparations as determined by electrophoresis on a 18% pol yacry! ami de ge! containing 8.1% SDS. Shown are scans of Coomassie Brill iant Blue R - stained gels. Samples are 5mg of (A) untreated crabshell wastes described in Example 1, (B) c i i -prote i n complex obtained by mild acid hydrolysis described in Example 2, (C) ch i t in-prote i n complex prepared by demineral i zat i on with ethylenediaminetetraacetic acid (EDTA) described in Example 3, (D) I S chi t in-prote i n complex prepared as in Example 4, (E) chitin obtained commercially, and (F) chitosan obtained commerc ial'ly. Arrows indicate positions of migration of molecular weight markers as in Figure 2. Table III £αφfl--lι_Q--i-£xl _^_£____i_D^__-.d,r.h_l__φιjιt__a_£_-p-g_ EDTA - Chitin ill _____ _ __ _ £_ IΪXI -p£J3__lB_£ )OHkp___ Deπiineral 12. ELE_E_I __ι__h_H_ iSi£j___ __ι_≤__li _B_i___li_l ___i_____4_ i_ ____S£_flil Uactr- ζ Calcium g g ) ό?88.6 237.8 16388.8 288.8 166.8 151.6 7676.8 2156.6 Sodium (fg-'g) 155.8 182.8 456.8 1538.6 23.3 42.8 996.6 135.6 Magnesium (C .'g^ 316.8 48.6 661.6 45. v 73.5 24.3 318.8 419.8 Potassium d'g/gi <23.» 27.8 <36.4 i _ _ 13.4 <24.2 <26.8 <49.2 Strontiuir. (fg.'g) 118.8 1.4 254.6 5.8 2.8 1.2 92.3 4.9 Iron (Pg/g) 2688.8 86.7 337.8 137.8 689.8 257.8 188.6 246.8 Aluminum (Hg g> 40.8 28.3 45.6 16.6 184.6 42.5 51.6 52.2 Bar turn (fg.'g) 2.3 1.4 1.5 8.8 1.3 3.6 4.8 86.2 Boron (P 2.3 1.4 1.5 8.3 1.3 1.2 <2.8 2.5 Cadmium ^P -'g) α.i <1.4 <1.5 8.3 <8,6 (1.2 <l.θ 2.5 Chromium g/ i 5.7 1.4 6.8 18.3 1.3 1.2 1.6 2.5 Cobalt (I'g.'g ) <2.3 62.7 <3.8 <l.ό <1.3 <2.4 <2.θ 2.4 Copper (Pg/gi 4.6 2.7 3.8 1.6 24.8 26.7 26.6 (4.9 Lead <Pg g) <5.7 <-.7 <7.6 <4.1 <3.3 ' <6.8 <5.8 24.6 Manganese (P .'g ) 35.6 <1.4 12.2 3.3 4.6 1.2 22.6 <12.3 Molybdenum ( g/g) <2.3 <2.7 <3.8 <1.6 <1.3, 2.4 <2.θ 2.5 Zinc ( /g 188.8 1 1 35.8 2.5 3.3 2.4 16.6 <4.9 Carbon C 45.36 46.98 42.16 45.13 53.27 51.54 48.18 7.4 Hydrogen (" 6.41 6.86 6.74 7.89 7.45 7.56 7.68 7.15 Nitrogen '" 6.61 6.92 7.75 3.18 11.12 18.92 9.99 11.11 Protein (Lowp iC- .8 <l.β <l.β <1.B 74.4 76.9 54.4 51.8 A.ll {V. ) 2.32 6.88 1.61 r.d. B 3.36 3.88 3.88 n.d. Moisture i'. ) 7.86 6.33 9.49 n.d. 3.74 4.78 3.58 n.d. a n.d. - not determined F'_ amp 1 P 5 I ahnr a t nry- r a l P T c.nl a t i nn n-f Ph i <• i n-Prnt p i π Hrjrt l p-. Frnro Dr i er! Fungal Binmass Dried fermentor cake obtained from a commercial gibberell in fermentation process was used as a raw material feedstock in place of the crabshell raw material feedstock described in Examples 1 through 4, Two hundred grams of dried fungal biomass were added to I ,8Θ_ ml . of 1.8 N HC1 and the mixture stirred continuously for a period of one hour. There was no appreciable release of gas nor any significant neutral ization of the HC1 solution during the course of the reaction. Residual insoluble material was collected by centrifuga ion for 13 minutes at 18,888 rp in a Sorval 1 GSA rotor at 4°C. The pellet was resuspended in water and centrifuged again as described above. Thi procedure was repeated four times, by which point the residual insoluble biomass material and the washings were neutral (pH 7.8). Insoluble material remaining after the fifth centrifugation procedure was oven-dried overnight at 18Θ°C with recovery of 'S3 grams of sol id material ^44% yield). T i material was ground in a Wiley Laboratory Mill to a particle size of less than 8.5 mm. prior to use. Its composition is shown in Table IV. Figure 5 illustrates the subunit composi ion cf the protein component of the ch i in-prot in complex obtained by acid treatment of dried fermentor cake from a co merc i l gibberell in f rmentation process as described in Example 5. Scan <A) represents the acid-treated material and scan ( B) represents untreated fungal fermentor cake. Arrows indicate the positions of migration of molecular weight markers as in Figure 2. 21 Table IV r.nm n^i t inn n-f Fungal Prppara t i nnς Ac i d-Treated Fl FMFNT Cal ium (Pg/g) 766.8 174.8 Sodium <Hg/g) 182.8 168.3 Magnesium (Hg/g) 1888.8 23.5 Potassium <l*g g) 8278.8 132.8 Strontium <Hg/g) 3.2 1.? Iron (Hg/g) 186.3 237.8 Aluminum <Hg/g> 26.9 23.1 Barium <μg/g) 1.1 <8.9 Boron <Hg/g) 2.1 3.7 Cadmium <H /g) <ι.e <8.9 Chromium (^g/g) 1.3 <1.9 Cobal <μg/g) <2.1 1.9 Copper <Hg/g) 3.6 1 ■ ? Lead (Hg/g) <5.4 <4.7 Manganese (Hg/ ) 3.6 2.3 Molybdenum (Hg/g) <2.i <1.9 Zinc (Hg/g) 22.7 1.9 Carbon C 52.74 54.41 Hydrogen < * 0 7.87 7.61 Nitrogen OO 6.73 6.28 Protein <Lowry) C 31.46 29.32 Ash OO 4.96 38.75 Moisture < ) 4.84 5.33 22 F.amplp 6 r.hapartcr i?at inn n- tha rh i t i n Pr-nte i n r. m 1 -PC Samples of crabshell raw material and each of the test materials (particle size less than 8.5 mm.) were analyzed for carbon, hydrogen, nitrogen, ash and metal contents; for total protein and amino acid content (Tables II, III, and IM) ; for solid-state electric properties (Figure 6); and by infrared spectroscopy (Figures 7-3). Elemental composition was determined using Inductively Coupled Plasma Emission Spectroscopy (ICP) for metal analysis and a Perkin-Elmer 248B Elemental Analyzer for carbon, hydrogen and nitrogen analysis. A Perkin-Elmer Model 1328 Infrared Spe rcφhotometer was used to measure infrared spectra of all materials. Total protein content was determined by extraction of each of the materials with 1.8 N NaOH for 43 hours at 25°C, followed by determina ion of the protein content in the solution by the Lowry method. For amino acid analysis, samples were hydrolyzed in yariu. in 6 N HC1 for 24 hours at 118°C and the. amino acids were measured after separation by high performance liquid chromatography (HPLC) according to standard methods such as those recently reviewed by M.W. Dong and J.C. DeCesare in Liq. Chro . i, 222-223 (1?33). Solid-state electrical properties were measured by an electrical testing laboratory using standard techniques. The amino acid composi i ons are shown in Table V. F-. amp 1 p 7 Hharartpp i 7ai i nn nf th<- Prntpin πnm npnt n4 th Ch i t in-g_o_ρ in Cnmplp-. During the ac i d-demineral ϊza i on of crab shell wastes, the amount of high molecular weight protein extractable by sodium dodecyl sulfate decreases (Figure 2). The total amount of protein, however, remains nearly constant (Table III) and the protein can only be removed by extended alkaline hydrolysis, indicating that in the acid treatment the protein is either made more detergent insoluble, is partially degraded, is covalently linked to the chitin matrix, or a combination of these possibil ities. The amino acid analysis of the starti ng mater i l and of the c i t in-prote in complex ( Table V) demonstrates no significant change in amino a id composition during mild acid hydrolysis, indicating that the ' relative amounts of the proteins present in the starting materials and in the. final product are not very different. As seen in Figure 4, there is no significant amount of protein in commercial preparations of chi in or chitosan. The ch i t in-prote in complex prepared using EDTA to demineral ize the material, on the other hand, contains a significant amount of protein which has not been modified. 24 TAR1 F EDTA - demineral ized Chi in Prr its in. Complex Crab ώminn Λr i ή& Start i no Matpr i al Ratrh rr?4 ύrh 11 ft Asp b 18.3 3.3 18.5 13.3 Thr 3.6 3.6 4.5 13.6 C Ser 4.9 zi .5 4.3 Glu b 9.9 13.4 12.9 12.2 Gly 5.3 3.9 3.9 4.2 Ala 5.6 5.3 5.4 5.5 Val 3.6 5.2 5.9 6.8 M t 1.3 3.3 3. ? 3.1 lie 4.4 5.4 5.5 5.1 Leu 18.5 3.5 9.8 7.4 Tyr 4.9 5.9 5.7 5.9 Phe 13.3 9.3 9.3 5.9 Lys 12.5 7.3 O -5 7.5 His 3.8 2.9 3.2 3.8 Arg 3. '3 7.1 7.8 7.2 a Cysteine, prol ine, and tryptophan were not determined. " Asparagine and glutamine are included with Asp and Glu, respec ively. c Ser + Thr 25 E_ampls,..S r.h arar tpp i 7 t i nn n-t t hp Hh i τ i n Cn pnnpn t nf thp P. i tin-Prnt in πnmnlp* The ch i t in-prot in complex was analyzed by infrared spectroscopy and compared with commercially available chitin and chitosan (Figure 7). The ch i t i n-prote in complex gives a spectrum very similar to that of chitin with the exception of an extra absorption band at 1733 cm-*-, possibly due to the protein component. The absorption band at 1558 m-* in chitin and in the ch i t in-prote i n complex, which is shifted in chitosan, appears related to acetylation of the amino groups in chitin. The relative intensities of this band in chitin and the ch i i n-prote in complex indicate that there is very l ittle deacetyl at ion of chitin during preparation of the ch i t i n-prote i n complex. Infrared spectra of the fungal preparations were measured (Figure 3 ) . The spectra are 'j sry simil r to that of chitin, and the spectra of both preparations (untreated, acid) are similar, indicating 'jsry l ittle change i the form cf the chitin in the materials. The ch i t i n-prote i n complex has solubil ity properties similar to those of chitin, i .e. it is insoluble in most ordinary solvents. The protein portion can be partially solubil ized by detergents and other protein solvents such as urea or guanidinium salts, or by treatment wi h alkal i . Chitosan, on the other hand, is soluble in dilute organic acids (1% acetic, lactic, propionic, and formic acids). All of the materials are soluble in concentrated mineral acids, but significant degradation occurs. F.flm lp 9 Pr- ppar a t i nn n4 th p Mpττ.a t nr i ΠP TP C Ϊ Syc t pro Panagrpl In , a saprophy ic nematode obtained from Dr. Jul ius Felcfenesser at the U.S. Department of Agriculture Plant Protection Institute, Beltsville, Maryland, was cultured in a commer ial ly available oatmeal cereal (Gerber Products Co., Fremont, Michigan). The nematodes were cul ured i 68 x 22 mm sterile plastic petri dishes containing 6 grams of autoclaved oatmeal cereal and 23 ml of sterile distilled water. The dishes were i nocul ted wi h approximatel 2888 nematodes suspended in 2 ml of sterile distilled water. The cultures 26 were then incubated at 38°C for 21 days. Control cultures contained only oatmeal cereal , distilled water, and nematodes in the above proportions. Materials to be tested for nemato idal activity were autoclaved and added to the individual culture dishes at the level of 8.2 grams per dish. Both control and test cultures were set up in series of five samples, Fxamp,1 P 13 M <tιιr m t n4 NJpma tnr i Ha 1 ήr f i u i ty Cultures of anagr p l 1 ne were prepared and observed according to the test system presented in Example 9. Observa ions were made beginning day 6 and continued through day 21, or until the cultures died. Microscopic observations were made using the wet mount sl ide technique on 8.81 ml samples withdrawn from the active surface zone of the cul ures where Banagrpl l s existed. An average of the numbers obtained by counting the samples withdrawn from each -of the culture dishes in the series was used to determine the relative population. Counts were made beginning on the sixth and continuing through to the 21st day. Averaged results are shown in Table VI, where a significant reduction in numbers of nematodes in cultures treated with ch i t i n-prot i n complex of this invention can be seen. I___l____. Fffp.ct.of Variou Prpparat i nn=, nn thp Nnmhpr of i i u i nn Mpma tnπpc i n P.n l tnr p e. Nnmhpr n-f I i u i n fVnan i gjnc * Control 1,388 1,588 Chi in b 688 738 Chi osan 988 1 ,338 Chi tin-Protein Complex b 488 a - Motile organisms counted in a 8.31 ml sample taken from the surface of the test plates b - Additions were present at 3% (w/w) The most significant and reproducible reduction in the total number of organisms was seen at day 17 and later. While there was a reduction in the population in the cultures treated with the ch i t in-protein complex, chitin, and chitosan, the most significant reduction was observed with the ch i i n-prote i n complex of this invention which also offers significant economic advantages over the use of the more highly purified preparations of chitin and chitosan. The fungal fermentation cake is not presently available in quantities sufficient to consider it as a practical raw material for this process. Nematoc idal activity in nematode test popula ions, as documented by photos shown in Figure 9 (A - D) , included a variety of events evident in all stages of development. Loss of motil ity, a standard determination of death in nematodes, was reinforced by avital staining with Brill iant Green, (CI. 42848) and Brill iant Cresyl Blue (CI. 51818). Staining was performed by adding a drop of a 8.857. aqueous solution to a .preparation of the organisms on a microscope sl ide. Within three minutes, there was a clear distinction between living and dead organ i ms (Figure 18). The l iving organisms were not stained, while the dye was taken up by the dead organisms. Cuticle disruption was evident as shown in Figure 9. Unique to the c i t i n-prot i n complex treated cul ures was the appearance of large vacuoles, indicating premature senescence, in nematodes of all stages as early as day 13. This appearance of vacuoles was not observed in cultures treated wi h any of the other materials. Npma t n=.t a t i r a n d N m t nr i Ha 1 Ar t i u i t v nf Hh i t i n -Pr nt P i n Cnrn ftϋ i n Cii l ii pp c i i t h fir, ; 1 Soil chosen randomly from agricultural fields was mixed with the ch i t i n-prote i n complex at ratios of .35, .825 and .81 compl ex/soi 1 (wt wt). Identical portions of these mixtures were then spread on water agar plates and incubated at room temperature. During this time the endogenous population of saprophytic nematodes developed. Several species were seen, predomi antly Panapr l lu_. sp . and PhahHi t i g. sp . The numbers of l iving and dead organisms in the cul ures were counted. As shown in Table VII, the maximum kill ing was obtained with 23 5 * /. ch i t in-prote in complex. Control experiments with chi i and chitosan showed substantially less efficient killing of only 33% and 49%, respectively, at day 33. Microscopic examination showed the nematophagous fungus H rpn^pnr inm to be present in the cultures where maximum killing occurred. This may reflect the mode of action of the chi t in-prote in complex, which may stimulate nematophagous fungi . The following table shows the nemato idal activity observed in tripl icate experiments, three plates per sample, of the ch i t in-prote i n complex on the growth of nematodes cultured in the presence of soil: TARI F _'TT Tn Uitrn fi i 1 Artiuitv Percentages of dead organisms on test plates; 33.days __LJ±_J_- 68.days Con trol 1 % 16% 19% 1% c i t i n-pr ote i n 52% 53% 91% co l x 2.5% " " 49.7"/. 52% 77% 5"% " ■ .93.6% 96V. 38 .7-% The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or cφerating conditions of this invention for those specifically used in the examples. From the foregoing description, one skilled in the art to which this invention pertains can easily ascertain the essential characteristics thereof and, without departing from the spirit and scope of the present invention, can make various changes and modifications to adapt it to various usages and condi ions. 29 Industr i al, Appl.trahi 1 i ty As can be seen from the preceding disclosure, the present invention is industrially useful in converting chi in-containing biological waste material into product having nematostatic and nematocidal properties useful in horticultural and agricultural appl ications.

Claims: 38 WHAT IS CLAIMED IS Claim 1. A composition of matter comprising a nematoc idally active chi tin-protein complex derived from ch i t in-contain ing waste material and consisting essentially of a water-insolubl e demineral ized chitin component complexed with a water-insoluble protein compnent, said complex being essentially free of low molecular weight peptides, amino acids, and calcium chloride brine formed by acid hydrolysis of such waste material , said complex characte ized by: a) a Lowry protein content of at least about 58% by weight, an ash content of not more than about 15% by weight, and a moisture content of less than about 18% by weight, based on the total composition; b) having solubility properties similar to those of chitin in being insoluble in neutral dilute acid solutions but solubil ized with significant decomposition of the protein component i concentrated mineral acids; c) having an IR spectrum similar to that of chitin but with a ' characteristic extra absorption band at 1733 cm-* ; d) the water-i soluble, demi neral ized chitin component having an acetyl content substantially identical to that of chitin as shown by a characte istic infrared spectrum absorption band at 1558 cm-*, but which is substantially free of carbonates and contains not more than about 15% of the ash content of chitin; e) the watei—insoluble protein component having an amino acid composition substantially identical to that of the untreated chi tin- containing waste material , a molecular weight primarily in the range of 18 to 53 kdal as determined by sodium dodecyl sulfate gel el e trophoresi s, and being essentially insoluble in common protein solvents; and f) said complex being in the form of dry particles having a diameter of less than about 8.5 mm. Claim 2. A composition according to claim 1, having a Lowry protein content of at least about 78% by weight, an ash content of not more than about 5% by weight, and a moisture content of less than about 5% by weight, based on the total composition. Claim 3. A composition according to claim 1, in the form of a pulverul ent sol i d. Claim 4. A composi t ion of matter comprising a plant growth medium in admixture with a nemato idal 1y effective amount of the ch i t in-prote in complex according to claim 1. Claim 5. A composition according to claim 4, wherein the plant growth medium is soil . Claim 6. A composition according to claim 5, wherein the plant growth medium is a plant potting soil suitable for growing nursery stoc . Claim 7. A composition according to claim 6, wherein the plant growth medium is planted with a l iving plant susceptible to nematode infection. Claim 3. A composition according to claim 4, wherein the plant growth medium is a particulate inorganic material . Claim 9. A composition according to claim 3, wherein the plant growth medium is an expanded mica. Claim 18. A composition according to claim 3, wherein the plant growth medium is planted with a l iving plant susceptible to nematode inf ct ion . Claim 11. A composi t i on according to claim 3, further comprising Harpnspnr i urn fungus in an amount effective to enhance the nematocidal activity of said ch i t in-prote i n complex. Claim 12, A method for inhibiting the growth of saprophytic nematodes in a plant growth medium capable of supporting such growth, which comprises admixing at least a nematosta i cal ly effective amount of the ch i in-prote i n complex according to claim 1 with said plant growth medium to inhibit the growth of said nematodes. Claim 13. A method according to claim 12, wherein the plant growth medi u i soi 1. Claim 14. A method according to claim 12, wherein the plant growth medium is a particulate inorganic material . Claim 15. A method according to claim 12, wherein the plant growth medium is suitable for growing nursery stock. Claim 16. A method according to claim 12, wherein a nematoc i dal ly effective amount of the c i t in-prote i n complex is admixed with the pl nt growth medium. Claim * 17. A method according to claim 16, wherein the nematoc i dal! y effective amount is at least about 5% by weight of the plant growth medium. Claim 13. A ne atocidal composition comprising a nematoc i dal ly effective amount of the ch i t in-proteiπ complex according to claim 1 in admixture with a hor icul turall y acceptable carrier material . Claim 19. A compos-ition according to claim 13 in the form of a pulverulent sol id composi t ion . Claim 28. A composition according to claim 13 wherein the carrier material includes a soil conditioning agent.

Inventors: EASTWOOD BARBARA J, MCCANDLISS RUSSELL J, MILCH ROBERT AUSTIN, EASTWOOD, BARBARA, J., MCCANDLISS, RUSSELL, J., MILCH, ROBERT, AUSTIN
Applicants: IGI BIOTECHNOLOGY INC, IGI BIOTECHNOLOGY, INC.
Application year: 1985
Main classification: C07G7
IPC codes: A01N63, C05G3, C08B37