Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents

Definitions

• This invention comprises several synergistic compositions, of the pesticide and antiparasitic kind, useful for the control of parasitic phytonematodes and zoonematodes, some diseases (fungal and bacterial), and the control of parasitic trematodes ( Fasciola hepatica ).
• Nematode control still falls short.
• the use of chemical nematicides is restricted each day more and more, because they have highly toxic and widespread action compounds.
• efforts have been made to identify the effective means to eliminate the damage caused by nematodes, in favor of reducing the use of chemical pesticides.
• One of the approaches is the use, of biological ones with specific mode of actions and relatively safer toxicological profiles, instead of chemical nematicides.
• Some of the alternative nematicides include ABG-9008, a Myrothecium verrucaria fungus metabolite and a combination of avermectines (or related compounds, like milbecines) with fatty acids (Abercrombie K. D. 1994.
• Another approach is to combine spores of Pasteuria penetrans a nematode bacterial parasite, with organophosphorated nematicides (Nordmeyer D. 1987. Synergistic nematocidal compositions of Pasteuria penetrans spores and an organophosphorus nematocide. 1987. CIBA-GEIGY AG Patent AU 06057386A1. 01/29/1987).
• Chitinolytic fungi and bacteria that share the nematode's habitat may have certain biological balance and somehow restrict nematode proliferation.
• Two strains of chitinolytic bacteria (Toda T. and Matsuda H. 1993. Antibacterial, anti-nematode and/or plant-cell activating composition, and chitinolytic microorganisms for producing the same. Toda Biosystem Laboratory, Japan. U.S. Pat. No. 5,208,159, May 4, 1993) have been claimed as antibacterial, antinematode and/or plant-cell activating composition.
• Sulfides are inhibitors in the electron transport breathing process of the aerobic organism, just like other metabolites produced by certain soil bacteria (Rodriguez-Kábana, R. 1991. Control biológico de nematodes parasitos de plantas. NEMATROPICA, 21(1), pp 111-22).
• PAECILTM also known as BIOACT or Nemachek
• BIOACT is a biological nematicide that contains a patented strain from Paecilomyces lilacinus , in a dry and stable spore concentration for soil and seed treatment. This fungal species is commonly found in all soils worldwide.
• the patented strain used as PAECILTM active ingredient has a particular effectiveness against plant parasitic nematodes. It was originally isolated at The Philippines University, and has been developed in Australia, Macquarie University. Furthermore, it has been broadly tested for the control of several kinds of nematodes that attack major crops in Australia, The Philippines, South Africa, and others.
• PAECILTM formulation is commercially available as a pesticide registered in The Philippines, under the name of BIOACT R ; in South Africa, under the name of PL PLUS; and Indonesia, under the name of PAECILTM.
• BIOACT R BIOACT R
• PL PLUS in South Africa
• Indonesia under the name of PAECILTM.
• the above-mentioned instances fail to solve all parasitic helminth problems. Therefore, the need to implement improved means for parasite control to substitute chemical pesticides and antiparasitic products still remains.
• Trematodes cause considerable economic damage to animal production and human health.
• the diversity of the species, relative benign pathogenicity and endemism in isolated regions seem to be essential factors that effect on the lack of knowledge on trematodes.
• intestinal trematodes are zoonotic and have a large number of reservoir hosts in each species.
• Fasciola hepatica the first known parasitic trematode; it affects man by inhabiting the bile conduits. Its egg is one of the largest, ovoid and operculated from helminthes, and causes digestive malfunction consisting in gastric disepsia, colon motility malfunction, liver and bile vesicle pain, fever and hepatic colic. Other signs may include cystic forms in lungs, eyes, brain, hepatic vein, and other tissues (Saleha A. 1991. Liver fluke disease (fasciolosis) epidemiology, economic impact and public health significance. Southeast Asian J. Trop. Med. Public health 22 supp 1dic. P 361-4)
• Zoohelminths have become significant pests to sheep and cattle. Antihelminthic resistance is wide, particularly in populations of small ruminant parasitic nematodes.
• Dictyocaulus viviparous a parasite that comes to sexual maturity and when adult, is lodged in the lung of cattle, particularly young animals.
• the diseased caused is known as verminose bronchitis, or bovine Dictyocaulosis, and infestation is produced after ingesting the 3 or infesting larvae, present in the pastures.
• the treatment requires antihelminthics (Borgsteede F. H. M, de Leeuw W. A. & Burg W. P. J. 1988.
• Trichoderma spp (Chet I, Inbar J. 1994 Biological control of fungal pathogens. Appl Biochem Biotechnol; 48(1): 37-43) a fungus whose action mechanism is largely discussed, where chitinases that degrade the cellular wall of the host fungus take part.
• chitinolytic action from fungi and bacteria used as fungal disease bioregulators (Herrera-Estrella A, Chet 1.1999. Chitinases in biological control. EXS; 87:171-84).
• Analyses of bacterium-bacterium interaction have shown there are three main types: antibiosis, substrate competition and parasitism.
• antibiosis some bacterial strains are known to release antibiotics in order to suppress the surrounding bacterial activity, which may be used for biological control of pathogenic species.
• substrate competition is a mechanism that may as well be used to achieve proper biological control, since the bioregulating organism is able to synthesize siderophores microelement quelant agents, which causes microelement deficiency, mainly iron, in the medium, thus inhibiting the respective pathogenic growth (Ongena M. 1998. Conference on biological controls. Training program in the area of biotechnology applied to agriculture and bioindustry. Gembloux, Belgium).
• the invention is related with a composition that contains, at least, one chitinolytic agent or a chitinolytic activity inducing agent, and sulfide or a sulfide producing agent from microorganisms or chemical compounds, where the chitinolytic agent or a chitinolytic activity inducing agent, and sulfide or sulfide producing agent from microorganisms or chemical compounds, are concurrently applied at a substantially minor degree than when each component is used independently to achieve effective control over helminths and causative agents of bacterial and fungal diseases.
• the invention is also related with the use of such compositions and/or the concurrent administration of the said compounds from different sources, such as, biologicals and chemicals for effective control over a wide spectrum of plant parasitic nematodes (Meloidogyne spp, Angina spp, Ditylenchus spp, Pratylenchus spp, Heterodera spp, Aphelenchus spp, Radopholus spp, Xiphinema spp, Rotylenchulus spp), animal parasitic nematodes and trematodes (Haemonchus spp, Trichostrongylus spp, Dictyocaulus spp.
• plant parasitic nematodes Malignant nematodes
• Angina spp Ditylenchus spp
• Pratylenchus spp Pratylenchus spp
• Heterodera spp Aphelenchus spp
• the chitinolytic agent or the chitinolytic activity inducing agent and sulfide, or sulfide producing agent can be appropriately mixed in the form of a solution, suspension, emulsion, powder or granulating mixture, and is applied to the plant or soil as a fertilizer, pre-packed soil, covert seed device, a powder, granulate, nebulizer, a suspension, liquid, or any of the indicated form in capsules for the control of parasitic helmiths, and bacterial and fungal diseases.
• a significant control over helminths, bacteria and fungi is achieved with a mixture of 1) a chitinase producing microorganism between 10 7 Colony Forming Units (CFU) and 10 12 CFU of a particular microorganism per composition gram or chitin between 1% and 50% of the composition; and 2) a sulfide producing microorganism between 10 7 CFU and 10 12 CFU of a particular microorganism per composition gram, or any sulfide producing chemical agent, where sulfide varies between 1.0 mg/minute per composition gram.
• CFU Colony Forming Units
• compositions with a microorganism between 10 7 CFU and 10 12 CFU per composition gram, that concurrently produces chitinolytic agents and sulfide, is appropriate for the control over helminths, bacteria and fungi.
• the previous compositions involve combinations of the following agents in the above-mentioned proportions:
• the previous compositions are effective against a wide range of plant parasitic nematodes, including, not limiting Meloidogyne species, such as, M. incognita ; Angina species, such as A. tritici ; Ditylencus species, such as D. dipsaci ; Pratylenchus species, such as P. coffee; Heterodera species, such as H. glycines; Aphelenchus species, such as A. avenae; Radopholus species, such as R. similis; Xiphinema species, such as X. index; Rotylenchulus species, such as R.
• Meloidogyne species such as, M. incognita
• Angina species such as A. tritici
• Ditylencus species such as D. dipsaci
• Pratylenchus species such as P. coffee
• Heterodera species such as H. glycines
• Aphelenchus species such as A
• Zoonematodes such as: Haemonchus spp, Trichostrongylus spp, Ostertagia spp, Nematodirus spp, Cooperia spp, Ascaris spp, Bunostomum spp, Oesophagostomum spp, Chabertia spp, Trichuris spp, Strongylus spp, Trichonema spp., Dictyocaulus spp., Capillaria spp., Heterakis spp., Toxocara spp, Ascaridia spp, Oxyuris spp, Ancylostoma spp, Uncinaria spp, Toxascaris spp and Parascaris spp; trematodes, such as Fasciola hepatica; plant pathogenic bacteria, such as Erwinia chrysanthemi, Burkholderia glumae, and
• the Meloidogyne incognita larvae were removed from the sift net and carefully resuspended with a LB medium solution diluted 10 times in sterile distilled water. The final collecting and disinfecting results were checked by counting and registering the live larvae with an inverted Olympus microscope.
• the nematode's eggs and larvae were placed in a number of 100 individuals in approximately 2 ml of LB medium diluted 10 times. This volume was introduced into safety valves that allow the air to go through the liquid and, therefore, the gasses make contact with the eggs and larvae. Every valve was a replica for each treatment.
• the hydrogen sulfide was obtained by a reaction against the chloride acid of two sulfide salts (Na 2 S and FeS), and from an anaerobial fermentation of bacterium Desulfovibrio desulfuricans subs. desulfuricans ATCC 27774 (isolated from an ovine rumen).
• the chitinolytic enzyme used was chitinase SIGMA C 1650, from bacterium Serratia marcescens.
• Control treatment chitinase not applied, and air circulated through the valve.
• Chitinase treatment chitinase at a rate of 0.2 units per replica.
• Sulfide treatment hydrogen sulfide from Desulfovibrio desulfuricans with a 0.2 flux at 0.3 mg/minute.
• the expected effectiveness (EE) must be equal to the sum of the individual effects (EI), given by the effectiveness rendered to the chitinase action (Eq) and the effectiveness rendered to the hydrogen sulfide action (Esn, Esf and Esd), minus the intersection effect (ei) (Sigarroa, A. 1985. Biometr ⁇ a y dise ⁇ o experimental. 1ra. Parte. Minist. Educa Terms Sup. Ed. Pueblo y Educa Terms. Cap. 3. pag 69-107).
• Brown soil with neutral pH was selected: it was dried and sieved with a 0.5 cm net to remove the undesirable particles. It was sterilized in a vertical autoclave for 1 hour at 120° C. and 1 atmosphere (Sambrook J., Fritsch E. F. and Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. 2nd. Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA). It was dried at room temperature for 3-4 days to later make the foreseen mixtures in the treatments with river sand, soil worm humus and chitin (ICN catalogue number 101334).
• Control treatment soil 70%, river sand 25% and humus 5%.
• Microorganic treatment soil 70%, river sand 25%, humus 5% and D. desulfuricans , applied to a concentration of 1010 CFU-pot.
• Injection 1 ml or ⁇ l of head space.
• Carrier gas Nitrogen 1.5 ml/min.
• Purge gas Nitrogen 30 ml/min.
• Table 5 shows a summary of the sulfide gases analysis issued by the two strains at different times.
• Sulfide gases analysis H 2 S flux mg/min (Sulfide flux detected) 18 24 Strains Samples 16 hours hours 20 hours 22 hours hours C-924 1 0.0673 0.2208 0.4779 0.3578 0.0672 2 0.0659 0.2160 0.4755 0.3552 0.0680
• DSM 1 0.0231 0.0416 0.1014 0.1863 0.0009 20162 2 0.0240 0.0422 0.1040 0.1887 0.0097
• the bacterial cultures of the studied strains were grown in LB medium at 28° C. and 100 rpm for 24 hours, followed by centrifugation at 3500 rpm; the supernatants were filtered through two 0.2 ⁇ m nets.
• the filtered product was assayed in plates prepared with a chitin colloidal suspension (0.5%), agarose was added too, up to 0.8%, to achieve the medium gelling and assure porosity to facilitate protein diffusion. After gelling, 5 mm diameter wells were opened, where 100 ⁇ l of the filtered supernatant from each bacterial strain was added. Three replicas were used for every plate, and were incubated at 28° C. in the dark.
• Eggs from parasite Fasciola hepatica were used. The egg collections were directly made from the infested liver bile of a bovine (cattle), previously sacrificed. The bile content was resuspended in a 3 times higher volume of distilled water and remained still for 2-3 hours at 28° C., to achieve egg precipitation. Then the greatest possible volume of supernatant liquid was removed. The precipitate was filtered through a sift net of 71 ⁇ m, where the eggs were trapped.
• This parasitic trematode's eggs hatch under the previously in vitro set conditions in about 15 days of incubation at 28° C.; a good preparation of the sample was considered when more than 60% of the eggs hatched at the end of the incubation period.
• the disinfected eggs were placed in a number of 100 individuals approximately, in 1 ml of LB medium diluted 10 times.
• the volume was uniformly introduced in 20 safety valves that allow the air passage through the liquid; hence, the gases make contact with the eggs.
• Each valve was a replica (4 per treatment) in all the five treatments.
• Control treatment Addition of 1 ml of LB medium diluted 10 times to every valve, with no chitinase, and air circulating through it.
• the expected effectiveness is given by the effectiveness rendered to the chitinase action (Eq) and the effectiveness rendered to the action of hydrogen sulfide (Esn, Esf and Esd), minus the intersection effect (ei) (Sigarroa, A. 1985. Biometria y dise ⁇ o experimental. 1ra. Parte. Minist. Educa Terms Sup. Ed. Pueblo y Educa Terms. Cap. 3. pag 69-107).
• fungus species were used: Pestalotia palmarum, Alternaria tabacina, Sarocladium orizae, Pitium debaryanum; and the following bacterial species: Erwinia chrysanthemi, Burkholderia glumae, Serratia marcescen ATCC 13880, Bacillus subtilis F 1695 and Escherichia coli ATCC 25922, were used as well.
• Table 9 shows the description of the results accomplished during the above mentioned interaction assays. TABLE 9 Results accomplished during interaction assays. Antagonistic effect of Species Description strain C-924. Pestalotia palmarum Fungus, Deuteromiceto, + + + phytopathogenic of foliage and fruits. Alternaria tabacina Fungus, Deuteromiceto, + + + phytopathogenic of tobacco leaves. Sarocladium orizae Fungus, Deuteromiceto, + + phytopathogenic of rice, it is involved in the acarus-fungus complex, affecting seeds, sheath and neck. Pytium debaryanum Fungus, Oomiceto, lives on the soil + and is part of the causative Damping-off complex.
• Erwinia Bacterium isolation of Dahlia + + + chrysanthemi stems with soft rottenness symptoms. Burkholderia glumae Bacterium, isolation of rice plants + + with apical and marginal necrosis. Bacillus subtilis Bacterium, isolation of potato ⁇ cepa F1695 rhyzosphere in rottenness-free on affected field. Biorregulator.
• antagonism was observed in the two pathogenic strains ( Erwinia crhysanthemi and Burkholderia glumae ), whereas antagonism was not observed in the case of Bacillus subtilis, as it is isolated from an antagonist soil with other microorganisms and; therefore, more resistant to adverse environmental factors.

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