EP0214128A1 - Complexe de chitine-proteine a action nematocide - Google Patents

Complexe de chitine-proteine a action nematocide

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Publication number
EP0214128A1
EP0214128A1 EP85901194A EP85901194A EP0214128A1 EP 0214128 A1 EP0214128 A1 EP 0214128A1 EP 85901194 A EP85901194 A EP 85901194A EP 85901194 A EP85901194 A EP 85901194A EP 0214128 A1 EP0214128 A1 EP 0214128A1
Authority
EP
European Patent Office
Prior art keywords
chitin
growth medium
plant growth
protein
protein complex
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP85901194A
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German (de)
English (en)
Inventor
Robert Austin Milch
Russell J. Mccandliss
Barbara J. Eastwood
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Igi Biotechnology Inc
Original Assignee
Igi Biotechnology Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Igi Biotechnology Inc filed Critical Igi Biotechnology Inc
Publication of EP0214128A1 publication Critical patent/EP0214128A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C05G3/60—Biocides or preservatives, e.g. disinfectants, pesticides or herbicides; Pest repellants or attractants

Definitions

  • This invention relates to a process for converting shellfish wastes into useful 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 occuring chitin-protein complex from the tough polymer matrix of crustacean exoskeletons and to methods for using these polymeric compositions for inhibiting the growth of plant-parasitic and other nematodes of interest in horticulture and agriculture.
  • Nematodes nema -- thread; oides — resembling
  • unsegmented roundworms with elongated, fusiform, or saclike bodies covered with cuticle which belong to the phylum
  • Nemathelminthes 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 which 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.
  • Root-knot (Meloidogyne spp.), root-lesion (Pratylenchus spp.), spiral (Heliocotylenchus spp.), and burrowing nematode (Radopholus similis), which is highly destructive of citrus crops and more than 288 other species of plants.
  • Damaging levels of stunt (Tylenchorynchus spp.), reniform (Rotylenchulus spp.) and fol iar (Amphelenchoides spp .) nematodes are also found in foliage ornamentals.
  • Plant-protection methods for nematode control including crop rotation, soil-treatment and fertilization practices, and "green manuring” 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 limited success.
  • Chemical methods, on the other hand, employing a range of systemic pesticides have been reasonably successful, particularly in horticultural 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 solid forms; (see, for example, A.C. Tarjan and P.C, Cheo, "The Nemat ode Screening Program of the University of Rhode Island,” Contribution 887, Agricultural Experiment Station, Springfield, R.I., March, 1956).
  • nematocides now available in commercial markets are, moreover, quite toxic to both man and animals, in large part being organic thiophosphate (phosphorothioate and phosphorodithioate) compounds and cholinesterase inhibitors. Many of them are also phytotoxic. Because of their adverse effects on the environment, several n emat oc i de s which are currently marketed are subject to review which may result in cancellation of registration. 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 commercial need exists for nematostatic or nematocidal materials, preferably biological control agents, which are non-toxic for plants, animals and man.
  • GR-76-004 and CO-76-020 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-73-003 and GR-ST-73-004, Bureau of Marine Resources, Mississippi Department of Wildlife Conservation, Long Beach, Mississippi,
  • 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 Ch i t i n on Fungi of Varying Cell Wall Composition", Exp. Mycology 3: 285, (1979); and (d) commercial preparations of chitosan have little 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 Repor t No. 65, University of Maryland, College Park, Maryland, April 1981.)
  • Another object of this invention is to provide a process for converting chitin-containing 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 chitin-containing biomass which can be demonstrated to induce nematostatic and nematocidal activity in culture media and in soil samples.
  • a more particular object of the invention is to provide a newly isolated chitin-prote in complex which can be obtained from naturally occurring sources and has demonstrable nematocidal activity for prototypical nematode species without evidence of a direct toxic effect on nematodes.
  • the present invention involves the discovery that a nematocidally active chitin-protein 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 demineralization and partial protein degradation.
  • the resulting chitin-protein complex induces nematocidal activity in nematode cultures in vitro, characterized by microscopic evidence of premature senescence and gas vacuole formation accompanied by loss of motility and death. Dead and dying nematodes, in sharp contrast to viable and highly motile forms, take up Brilliant Green and Brilliant Cresyl Blue stains.
  • Figure 1 illustrates the process for producing the chitin- ⁇ rotein complex of this invention and identifies by-products which can be recovered.
  • Figure 2 illustrates the subunit c omp os ition of the protein component of crabshell waste treated according to the process described in Example 2.
  • Figure 3 illustrates conventional processes used to tr eat sh e l l f i sh wastes for the production of commercial chitin and chitosan produc t s 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 chitin-protein complexes prepared in Examples 2, 3, and 4 and of commercial chitin and chitosan preparations.
  • Figure 5 illustrates the subunit composition of the protein component of the chitin-protein complex obtained by acid treatment of dried fermentor cake from a commercial g i bbere l l i n fermentation process as described in Example 5.
  • Figure 6 is a plot of the alternating curr en t (ac) conductivity of chitin, ch i tosan and the crabshell chitin-protein complex as a function of applied voltage.
  • Figure 7 demonstrates the infrared spectra of (A) chitin, (B) the chitin-protein 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 Panagrellus spp. 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 chitin-protein complex (C and D) of this invention.
  • Figure 18 shows living and dead nematodes stained with Brilliant Green.
  • the chitin-protein complex of this invention can be prepared from any suitable chitin-containing biomass raw material.
  • suitable chitin-containing biomass raw material 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.
  • Ascomycetes species (which can be digested by one or more of the over 30 enzymes, including chitinase, glucanase and mannase, contained in the digestive juice of the snail Helix pomatia or produced by certain bacteria, such as some soil scavenging Pseudomonas 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 chitin-protein polymer complex obtained from the shells cf blue crabs (Callinectes sapidus) harvested from the Chesapeake Bay will be described in detail .
  • Crab processing waste is generally discharged to a waterway or a municipal sewer, hauled to a sanitary l an d f i l l or otherwise rendered, frequently by drying and shredding for eventual use as a f eed meal, especially for chickens (see, for example, P. R. Austin et al. U.S. Patent 4,328,158; W. P. Uri Yrains, Jr. and T. M.
  • the presently preferred starting or raw material for the here i n disclosed invention 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 (40-50%), calcium carbonate and small amounts of other mineral salts (about 50%), and chitin (about 10%).
  • 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 processing facility.
  • Shell fragments are then demineralized in a stirred tank reactor using a dilute mineral acid, such as 1.8 N HCl , for a period of 30-60 minutes, generally u nder 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 demineralization reaction which is accompanied 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 chitin-protein complex which has distinctly different gel electrophoretic properties from the product resulting from demineralization of crabshells by chelating agents such as ethylenediaminetetracetic acid (EDTA) ( Figure 4) and from chitin-protein complexes isolated from fungal residues ( Figure 5).
  • chelating agents such as ethylenediaminetetracetic acid (EDTA) (Figure 4)
  • chitin-protein complexes isolated from fungal residues Figure 5
  • the solid-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).
  • DM/PM demineralization and protein modification
  • mild acid hydrolysis usually at about 68 minutes after the start of the hydrolysis reaction
  • the resulting chitin-protein material is washed until neutral (pH 7.8) with water or weak soda ash (Na 2 CO 3 ) 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 facility.
  • the resulting chitin-protein complex is then dried in a suitable drier and ground, if desired, to a particle size of less then 0.5mm. No further treatment, as is required in conventional chitin and chitosan processing operations ( Figure 3), is needed.
  • the resultant chitin-protein complex ( Figures 4, 6 and 7) is: (i) insoluble in neutral and in dilute acid solutions but solubilized with significant decomposition of the protein component in concentrated mineral acids; (ii) low in ash content; (iii) high in bound nitrogen content owing to the presence of the protein moiety; and (iv) a naturally occurring, biodegradable material which, when added to nematode cultures in vitro, results in a significant reduction in the number of living organisms ( Figures 9-18).
  • the product can be produced commercially in better yield and at substantially less cost than can either ch i t i n or chitosan derived from crab, lobster, shrimp or other shellfish processing wastes or from the walls of chitin-containing fungi, molds and yeasts. Morphological changes induced in nematode cultures in in vitro 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 application of the chitin-protein 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 requisite nutrients. More preferred rates are in the range of 2 to 20 weight percent; the presently most preferred rates are in the range of 5 to 10 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 limited to disease to be controlled, the type of crop, stage of development of the crop and the interval between applications. Applications within the range given may need to be repeated one or more times at intervals of 1 to 6 months.
  • the chitin-protein 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 solids such as talcs, natural clays, pyrophyllite, di atomaceous earth; flours such as wheat, redwood, and soya bean; or inorganic substances such as magnesium carbonate, calcium carbonate, calcium phosphate, sodium siliocoaluminate, sulfur and the like.
  • the choice of a particular diluent is based on consideration of the physical and ch em i c a l 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 compositions are made by thoroughly blending the active ingredient with the diluent and other additives.
  • Powdered compositions can be converted to granules by adding a liquid, 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, lignin sulfonates, methyl cellulose, other water soluble polymers and the like, can be included in these particulate 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.
  • a suspension of the active ingredient can be sprayed on the surface of preformed granules of clay, vermiculite, corn cob and the like.
  • Surfactants may also be included in formulations of the latter type.
  • compositions of the invention can contain, in addition, to the active ingredient of this invention, conventional insecticides, miticides, bactericides, other nematocides, fungicides or other agricultural chemicals such as fruit set agents, fruit thinning compounds, fertilizer ingredients and the like, so that the compositions can serve useful purposes in addition to its nematocidal activity.
  • the chitin-protein complex of this invention contains about 10% nitrogen in a slow-release form; it can advantageously be mixed with sources of metabolizable phosphorous and/or potassium to provide a balanced fertilizer.
  • the nitrogen content can be enhanced by the further addition of other nitrogen fertilizer 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 vermiculite, e.g. for growing greenhouse plants or nursery stock.
  • Harpospocium fungus is added to enhance the nematocidal activity of the chitin-protein complex.
  • Pathogenic plant nematodes which may be controlled in accordance with the present invention include but are not limited to those set forth in the following table.
  • Crabmeal processing waste obtained from a commercial supplier was water washed, dried in a hot-air oven at 100°C for approximately 16 hours and then shredded mechanically to a particle size such that all of the material passed through a No. 25 USA Standard Testing Sieve.
  • the resulting particles had a moisture content of 5-10% and contained approximately 40-50% protein and 30-50% CaCO 3 on a dry weight basis.
  • Elemental composition 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! 240B Elemental Analyzer.
  • the subunit composition was determined by electrophoresis on a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS). Shown in Figure 2 are sc an s of the gels after staining of the proteins with Coomassie Brilliant Blue R.
  • the positions indicated by the arrows are the positions of standard molecular weight (given in daltons in parentheses) marker proteins: (a) myosin (200,000), (b) beta-galactosidase (116,500), (c) phosphorylase B (97,400), (d) bovine serum albumin (66,200), (e) ovalbumin (45,000), (f) carbonic anhydrase (31,000), (g) soybean trypsin inhibitor (21,500), and (h) lysozyme (14,400).
  • the samples run were 5 mg of (A) starting material, (B) starting material after 18 minutes in acid, (C) after 38 minutes, (D) 60 minutes, and (E) the final product after acid treatment, washing, and drying.
  • Example 2 Four hundred grams of the raw material described in Example 1 were slowly added over a 30 minute period to 2 liters of 1.0 N HCl with continuous stirring. The reaction caused rapid demineralization of the CaCO 3 phase of the raw material feedstock as evidenced by foaming of the reaction mixture and the release of CO 2 gas containing readily detectable amine odors. Approximately 40 mls. concentrated HCl were then added in small aliquots to the reaction mixture (to maintain acidity at approximately pH 1.5) over a period of about 60 minutes, after which no further foaming was observed. The insoluble residue remaining after the demineralization and partial hydrolysis procedure was collected on a No. 270 U.S.A.
  • the neutral (pH 7.0) product was oven-dried at 100°C overnight and then ground to a particle size of less than 0.5 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 Table III.
  • Figure 4 illustrates the subunit composition of protein components of the chitin-protein complexes prepared in Examples 2, 3, and 4 and of commercial chitin and chitosan preparations as determined by electrophoresis on a 10% pol yacrylamide ge! containing 0.1% SDS. Shown are scans of Coomassie Brilliant Blue R - stained gels.
  • Samples are 5mg of (A) untreated crabshell wastes described in Example 1, (B) chitin-protein complex obtained by mild acid hydrolysis described in Example 2, (C) chitin-protein complex prepared by demineralization with ethylenediaminetetraacetic acid (EDTA) described in Example 3, (D) chitin-protein complex prepared as in Example 4, (E) chitin obtained commercially, and (F) chitosan obtained commercially. Arrows indicate positions of migration of molecular weight markers as in Figure 2.
  • Dried fermentor cake obtained from a commercial gibberellin 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 1,000 ml. of 1.0 N HCl and the mixture stirred continuously for a period of one hour. There was no appreciable release of gas nor any significant neutralization of the HCl solution during the course of the reaction. Residual insoluble material was collected by centrifugation for 10 minutes at 10,000 rpm in a Sorvall GSA rotor at 4°C. The pellet was resuspended in water and centrifuged again as described above.
  • Figure 5 illustrates the subunit composition of the protein component of the chitin-protein complex obtained by acid treatment of dried fermentor cake from a commercial gibberellin fermentation 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.
  • Example 6
  • Samples of crabshell raw material and each of the test materials were analyzed for carbon, hydrogen, nitrogen, ash and metal contents; for total protein and amino acid content (Tables II, III, and IV); for solid-state electric properties (Figure 6); and by infrared spectroscopy ( Figures 7-8). Elemental composition was determined using Inductively Coupled Plasma Emission Spectroscopy (ICP) for metal analysis and a Perkin-Elmer 240B Elemental Analyzer for carbon, hydrogen and nitrogen analysis. A Perkin-Elmer Model 1320 Infrared Spectrophotometer was used to measure infrared spectra of all materials.
  • ICP Inductively Coupled Plasma Emission Spectroscopy
  • Perkin-Elmer 240B Elemental Analyzer for carbon, hydrogen and nitrogen analysis.
  • a Perkin-Elmer Model 1320 Infrared Spectrophotometer was used to measure infrared spectra of all materials.
  • Total protein content was determined by extraction of each of the materials with 1.0 N NaOH for 48 hours at 25°C, followed by determination of the protein content in the solution by the Lowry method.
  • samples were hydrolyzed in vacuo in 6 N HCl for 24 hours at 110°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. Chrom. 1, 222-228 (1983). Solid-state electrical properties were measured by an electrical testing laboratory using standard techniques.
  • the amino acid compositions are shown in Table V.
  • the chitin-protein complex was analyzed by infrared spectroscopy and compared with commercially available chitin and chitosan (Figure 7).
  • the chitin-protein complex gives a spectrum very similar to that of chitin with the exception of an extra absorption band at 1738 cm -1 , possibly due to the protein component.
  • the relative intensities of this band in chitin and the chitin-protein complex indicate that there is very little deacetylation of chitin during preparation of the chitin-prote in complex.
  • the chitin-protein complex has solubility 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 with 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.
  • PanagrplIus a saprophytic nematode obtained from Dr. Julius Felcfenesser at the U.S. Department of Agriculture Plant Protection Institute, Beltsville, Maryland, was cultured in a commerc ially available oatmeal cereal (Gerber Products Co., Fremont, Michigan).
  • the nematodes were cultured in 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 inoculated with approximately 2000 nematodes suspended in 2 ml of sterile distilled water. The cultures were then incubated at 30°C for 21 days.
  • Control cultures contained only oatmeal cereal, distilled water, and nematodes in the above proportions. Materials to be tested for nematocidal activity were autoclaved and added to the individual culture dishes at the level of 0.2 grams per dish. Both control and test cultures were set up in series of five samples,
  • Nematoc idal activity in nematode test populations included a variety of events evident in all stages of development. Loss of motility, a standard determination of death in nematodes, was reinforced by avital staining with Brilliant Green, (CI. 42040) and Brilliant Cresyl Blue (CI. 51010). Staining was performed by adding a drop of a 0.057. aqueous solution to a .preparation of the organisms on a microscope slide. Within three minutes, there was a clear distinction between living and dead organisms (Figure 10). The living organisms were not stained, while the dye was taken up by the dead organisms. Cuticle disruption was evident as shown in Figure 9.
  • Soil chosen randomly from agricultural fields was mixed with the chitin-protein complex at ratios of .05, .025 and .01 complex/soil (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, predominantly Panagrellus sp. and Phabditis sp. The numbers of living and dead organisms in the cultures were counted. As shown in Table VII, the maximum killing was obtained with 5% chitin-protein complex. Control experiments with chitin and chitosan showed substantially less efficient killing of only 33% and 49%, respectively, at day 33.
  • the present invention is industrially useful in converting chitin-containing biological waste material into product having nematostatic and nematocidal properties useful in horticultural and agricultural applications.

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Abstract

Un complexe de chitine-protéine est préparé à partir de déchets biologiques contenant de la chitine, tels que les carapaces de crustacés. Le complexe diffère aussi bien de la chitine que du chitosan, et a une action nématostatique et nématocide utile pour des applications agricoles et horticoles lorsqu'il est ajouté en quantités efficaces pour tuer des nématodes à un milieu de croissance végétale. Le complexe fournit également une source d'azote à libération lente, ce qui fait qu'il peut être combiné de façon particulièrement appropriée avec des fertilisants, des produits d'amélioration du sol, etc.
EP85901194A 1985-02-08 1985-02-08 Complexe de chitine-proteine a action nematocide Withdrawn EP0214128A1 (fr)

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PCT/US1985/000192 WO1986004586A1 (fr) 1985-02-08 1985-02-08 Complexe de chitine-proteine a action nematocide

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Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2040879A (en) * 1934-06-21 1936-05-19 Du Pont Substantially undegraded deacetylated chitin and process for producing the same
US3862122A (en) * 1972-02-16 1975-01-21 Quintin P Peniston Method of recovering chitosan and other by-products from shellfish waste and the like
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