Pesticide and antiparasitic compositions

FIELD: pest control.

SUBSTANCE: invention relates to controlling pests, in particular to compositions for controlling pests, diseases, and parasites damaging plants and animals. Composition comprises one chitinolytic agent or agent inducing chitinolytic activity, or sulfide or agent stimulating microorganisms to produce sulfides, or reacting with chemicals to release sulfides.

EFFECT: reduced amount of agent required to manifest high effect compared to effects of preparations formulated from any of above indicated compounds taken individually.

9 tbl, 5 ex

 

The present invention includes several synergistic compositions pesticide and antiparasitic type that can be used to combat parasitic phytonematode and themtoday, certain diseases (fungal and bacterial) and to fight parasitic trematodes (Fasciola hepatica).

The level of technology

Nematodes cause the greatest damage to agriculture in tropical, subtropical and temperate regions worldwide (Nickle W.R. (Editor). 1991. Manual of Agricultural Nematology, Marcel Dekker, Inc., New York, N.Y. Pub. 1035 pp). World production of bananas has about 20% of the losses associated only with nematodes, which is annually 178 million $ (J.N. Sasser and Freckman D.W. 1987. A world perspective on nematology: the role of the society. Vistas on nematology: a commemoration of the twenty-fifth anniversary of the Society of Nematologists/ edited by Joseph A. Veech and Donald W. Dickson. p.7-14). Bananas and banana plantations largely damaged Radopholus similis.

Meloidogyne spp is the most important parasitic nematode of the plant as loss because of its activity ranges from 11% to 25% of crop in almost all tropical regions (Sasser J.N. 1979. Root-knot nematodes. Ed. F. Lamberti & C.E.Taylor, Academic Press, London, p. 359). Therefore, there is a significant need in the fight against these parasites, they have struggled with chemical nematicides. Such compounds can be very effective, but many of them are the most harmful to the environment. In some cases, regulators have imposed restrictions on the introduced quantity or frequency of making (or both) of these compounds, thereby reducing their nematocidal efficiency.

Combating nematodes still does not bring success. The use of chemical nematicides every day more and more limited, as they are highly toxic and broad-spectrum compounds. In the result, attempts were made to find effective means to prevent damage caused by nematodes to reduce the use of chemical pesticides. One approach is to use instead of chemical nematicides biological objects with a specific type of action and relatively safe Toxicological profile. Some of the alternative nematicides include ABG-9008, a metabolite of the fungus Myrothecium verrucaria and combination of avermectins (or related compounds, such miletina) with fatty acids (Abercrombie K.D. 1994. Synergistic pesticidal compositions. Patent US 5346698. Mycogen Corporation. Sept.13). Similarly, the way that involves the simultaneous processing for elimination of plant damage caused by nematodes, land, soil or seeds that you want to process (a) a metabolite of the fungus Myrothecium verrucaria and (b) a chemical pesticide, and is also effective in this case blue is logical nematocidal composition, claimed in the patent (Warrior P., Heiman D.F. and Rehberger Linda A. 1996. Synergistic nematocidal compositions. Abbott laboratories. WO 9634529, 1996-11-07).

Another approach consists in the combination dispute Pasteuria penetrans, a bacterial parasite of nematodes, with organophosphorus 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).

However, when obtaining spores of P. penetrans on an industrial scale have to deal with the problem, consisting in the fact that this organism is an obligate parasite; therefore, it must be grown in situ, in nematodes isolated from Perevalov roots infected by nematodes.

Chitinolytic fungi and bacteria that live in the same environment that nematodes may have some biological equilibrium and some way to limit the spread of nematodes. Two strains of chitinolytic bacteria (Toda T. and Matsuda H. 1993. Antibacteriall, anti-nematode and/or plant-cell activating composition, and chitinolytic microorganisms for producing the same. Toda Biosystem Laboratory, Japan. Patent US 5208159, 05/04/1993) were reported as antibacterial, onlinemedia and/or activating the cells of the plant composition. There are several examples of chitinolytic effects on nematodes. Some of the most important are disclosed new strains of bacteria (Suslow T. and Jones, D.G. 1994. Novel chitinase-producing bacteria and plants. DNA Plant Technology Corporation, US 04940840, 07/10/1990), which are created as a result of the introduction the Oia DNA which encodes the production of chitinase, an enzyme that can break down the chitin in fungi and nematodes. These strains can be used to obtain chitinases for the inhibition of plant pathogens. Also described new resistant pathogens of plants, whose stability is determined by the introduction of the DNA that encodes the production of chitinases.

Also described other examples of microorganisms that reduce populations of nematodes that damage plants in natural conditions. Rodriguez-Kabana et al. (Rodriguez-Kabana R., J.W. Jordan, Hollis J.P. 1965. Nematodes: Biological control in rice fields-role of hydrogen sulfide. Science. 148: 524-26); Hollis and Rodriguez-Kabana (Hollis, J.P., y R. Rodriguez-Kabana. 1966. Rapid kill of nematodes in flooded soil. Phytopathology 56, pp. 1015-19) observed a correspondence between the bacterium Desulfovibrio desulfuricans, the production of hydrogen sulfide and parasitic nematodes of plants, their population was reduced by rice plantations of Louisiana. Sulfides are inhibitors of electron transport during respiration aerobic organisms, in the same way as other metabolites produced by some soil bacteria (Rodriguez-Kabana, R. 1991. Control biolgico de nematodosde plantas. NEMATROPICA, 21(1), pp. 111-22).

PAECILTMalso known as BIOACT or Nemachek, is the biological nematocidal, which contains a patented strain of Paecilomyces lilacinus in dry form and with a stable concentration of spores, printing handling the key soil and seeds. These types of fungi are usually found in all soils all over the world. Patented strain used as the active ingredient PAECILTMhas very good efficacy against parasitic nematodes of plants. Originally, the project was highlighted in the University of the Philippines and was developed in Australia, Macquarie University. In addition, it was tested on a wide program to combat with some types of nematodes that damage agricultural crops in Australia, Philippines, South Africa, etc. Composition PAECILTMavailable as a pesticide registered in the Philippines, under the trademark BIOACTR; in South Africa called PL PLUS; in Indonesia called PAECILTM. Currently, the Australian National Registration Agency considers this product as a pesticide (Holland, R. PAECILTM. 1998. http://www.ticorp.com.au/article1.htm).

However, the above cases cannot solve all of the problems associated with parasitic helminths. So still the urgent need to create improved means which would ensure the fight against parasites and would replace chemical pesticides and protivodiabeticheskie products. Trematodes cause significant economic losses in the area of productive livestock and harm to human health. A variety of the species, relative malignant pathogenicity and endemism in some regions, it seems, are the significant factors that influence the lack of knowledge about the trematodes. Generally speaking, intestinal trematodes are zoonotic and have a large number of backup hosts in each of the types.

From the point of view of the economy one of the most important trematodes is Fasciola hepatica, the first known parasitic trematode; it affects the human body in the bile ducts. Her egg is one of the largest egg coated worms and causes disorders of the digestive system consisting of gastric dyspepsia, disorders of motility in the colon, pain in the liver and gall bladder, fever and intestinal colic. Other signs may include cystic forms in the lungs, eyes, brain, liver Vienna and in 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).

Tagelement have become significant pests for sheep and cattle. Anthelminthic resistance is widespread, especially in populations of parasitic nematodes of small ruminants.

Developed new advanced techniques and others are in the research phase. The fungus Duddingtonia flagrans is a predator, notariorum grid creates a broad wall, motionless disputes: clamidospores able to survive passage through the gastrointestinal tract of cattle, horses, sheep and swine (Larsen M. 1999. Biological control of helminths. Int. J. Parasitol. Jan.; 29(1): 139-46, and Larsen, M. 2000. Prospects for controlling animal parasitic nematodes by predacious micro fungi. Parasitology, 120, S120-S121).

Studies of D. flagrans in Denmark, France, Australia, USA and Mexico confirmed the high potential of biological control of this fungus.

Like many other important countries producing sheep in South Africa is undergoing a great crisis from the point of view of anthelminthic resistance, especially against gastrointestinal nematodes in sheep and goats. Important parasitic helminths are involved in this phenomenon; however, this causes a particular problem with rennet hematophagous parasite At contortus. Research has revealed that more than 90% of strains of this parasite of the most important sheep regions of South Africa demonstrate some degree of drug resistance in three of the four anthelminthic groups available on the market in South Africa. Even on the areas of conventional pastures in the Northern Province, it was found in five herds surveyed in 1993 (van Wyk J.A., G.F. Bath and F.S. Malan 2000. The need for alternative methods to control nematode parasites of ruminant livestock in South Africa. World Animal Review. http://www.fao.org/ag/AGA/AGAP/FRG/FEEDback/War/contents.htm).

The problem of increasing ustoichivosti serious, as it was also detected in other areas. Recently held a series of anthelminthic research in four Latin American countries: Argentina (Eddi, C., Caracostantogolo, J., Peya, M., Schapiro, J., Marangunich, L., Waller, P.J. & Hansen, J.W. 1996. The prevalence of anthelmintic resistance in nematode parasites of sheep in southern Latin America: Argentina. Vet. Parasitol., 62: 189-197); in Brazil (F. Echevarria, Borba M.F.S., A.C. Pinheiro, Waller P.J. & Hansen J.W. 1996. The prevalence of anthelmintic resistance in nematode parasites of sheep in southern Latin America: Brazil. Vet. Parasitol., 62: 199-206); Paraguay (S. Maciel, Giminez A.M., Gaona, C., Waller, P.J. & Hansen J.W. 1996. The prevalence of anthelmintic resistance in nematode parasites of sheep in southern Latin America: Paraguay. Vet. Parasitol., 62: 207-212); and Uruguay (Nari A., Salles J., Gil, A., Waller, P.J. & Hansen J.W. 1996. The prevalence of anthelmintic resistance in nematode parasites of sheep in southern Latin America: Uruguay. Vet. Parasitol., 62: 213-222).

One of nematodes, which most strongly affects cattle, is Dictyocaulus viviparous, the parasite reaches sexual maturity, and as an adult enters the lungs of cattle, especially in light of young animals. Called when this disease is known as helminthic bronchitis, or cow Dictyocaulosis, and infection occurs by eating food contaminated with the larvae. Treatment requires anthelminthic drugs (Borgsteede F.H.M, W.A. de Leeuw & Burg W.P.J. 1988. A comparison of the efficacy of four different long-acting boluses to prevent infections with Dictyocaulus viviparus in calves. The Veterinary Quarterly, Vol. 10, No. 3), but success is determined by the high prices because of new strains resistant to the to learning about your medicine, what makes the further treatment of the infected animal more difficult. The high cost of these products is a limiting factor for countries with a large number of resources, and the use of these drugs harm the environment. International problem anthelminthic resistance is complemented by the fact that, although chemotherapy remains the cornerstone in the fight against parasites, apparently, there is little hope that any new chemically unrelated anthelminthic drug appears for at least the next decade (Soll, M.D. 1997. The future of anthelmintic therapy from an industry perspective. In J.A. van Wyk & P.C. van Schalkwyk, eds. Managing anthelmintic resistance in endoparasites, p.1-5. Proceedings of the 16th International Conference of the World Association for the Advancement of Veterinary Parasitology, Sun City, South Africa, 10-15 August 1997).

With regard to bacteria and pathogenic fungi, there are extensive reports of biological drugs, which are mainly based on antagonism, and that many of these drugs are commercially available. Here are some of them: Conquer (Pseudomonas fluorescens, which is an antagonist Pseudomonas tolassii), Galltrol-A (Agrobacterium radiobacter, which controls Agrobacterium tumefaciens), Bio-Fungus (Trichoderma spp, which controls the following fungi: Phytophthora, Rhizoctonia solani, Pythium spp, Fusarium, Verticillium), Aspire (Candida oleophila I-182, which controls Botrytis spp. and Pencillium spp), etc.

One biofungicides with the widest spectrum of activity, is Trichoderma spp (Chet I., J. Inbar 1994 Biological control of fungal pathogens. Appl Biochem Biotechnol; 48(1):37-43), fungus, mechanism of action which has been widely discussed, and this is the mechanism involved chitinases that degrade the cell wall of the host fungus. Moreover, there is experimental evidence chitinolytic action of fungi and bacteria, which are used as bioregulators of fungal diseases (Herrera-Estrella A, Chet I.1999. Chitinases in biological control. EXS; 87:171-84). However, this is not the only way bacteria against plant pathogenic fungi; there are other ways of struggle, based on the production of secondary metabolites, such hydrocyanic acid, which can inhibit root pathogenic fungi (C. Blumer and Haas D. 2000. Mechanism, regulation, and ecological role of bacterial cyanide biosynthesis. Arch Environ Mar; 173(3):170-7), in the specific case of fungi strain P. fluorescens CHAO.

Analysis of interactions of type bacterium-bacterium has shown that there are three basic types of interaction: antibiosis, substrate competition and parasitism. In the case of antibiosis some strains of bacteria known to produce antibiotics to inhibit the activity surrounding bacteria that can be used for biological method to control pathogenic species. nelogicno substrate competition is a mechanism which can also be used to achieve the proper biological control, as bioregulatory the body is able to synthesize agents, chelating siderophore trace elements that cause deficiency of trace elements, mainly iron, environment, thus is the inhibition of growth of the relevant pathogens (Ongena M. 1998. Conference on biological controls. Training program in the area of biotechnology applied to agriculture and bioindustry. Gembloux, Belgium).

The invention

The present invention relates to a composition, which contains at least one chitinolytic agent or agent, inducing chitinolytic activity, and sulfide or agent producing sulfide from microorganisms or chemical compounds, where chitinolytic agent or agent, inducing chitinolytic activity, and sulfide or agent producing sulfide from microorganisms or chemical compounds put together in a substantially smaller amount than if each component is used independently, to achieve effective control of helminths and the causative agents of bacterial and fungal diseases.

The present invention relates also to the use of such compositions and/or to the simultaneous introduction of these compounds from various sources, such as biological and chemical, to effect the main struggle with a wide range of parasitic nematodes of plants (Meloidogyne spp, Angina spp, Ditylenchus spp, Pratylenchus spp, Heterodera spp, Aphelenchus spp, Radopholus spp, Xiphinema spp, Rotylenchulus spp), parasitic nematodes of animals and trematodes (At spp, Trichostrongylus spp, Dictyocaulus spp. y Fasciola hepatica), bacterial agents causing disease (Erwinia chrysanthemi, Burkholderia glumae) and fungal agents causing disease (Pestalotia palmarum, Alternaria tabacina, Sarocladium orizae).

The chitinolytic activity of the agent or agent, inducing chitinolytic activity, and sulfide or producing sulfide agent helminths, bacteria and fungi was previously demonstrated or reported. However, in the present study first demonstrated a synergistic effect with the simultaneous use of both components.

If chitinolytic agent or agent, inducing chitinolytic activity, and sulfide or agent producing sulfide, used separately, the effect is always less than if you simultaneously use both the agent.

When used in the compositions of the present invention, chitinolytic agent or agent, inducing chitinolytic activity, and sulfide or agent producing sulfide, can be appropriately mixed in the form of a solution, suspension, emulsion, powder or granulated mixture and apply to the plant or the soil in the form of fertilizers, pre-Packed soil, shell, seed, then, is the Cabinet, granules, aerosols, suspensions, liquids, or any of the above forms in capsules to combat parasitic helminth and bacterial and fungal diseases.

The optimal dose application of chitinolytic agent or agent, inducing chitinolytic activity, and sulfide or agent, producing a sulfide, for the specific case of nematodes, trematodes, bacteria or fungi, and for the case of specific conditions, the intervals of doses is determined experimentally in vitro, in the greenhouse or in the field.

In accordance with the results disclosed in the present invention, a significant success in the fight against helminths, bacteria and fungi can be achieved using a mixture of 1) producing a chitinase microorganism from 107craniopathy units (CFU) of up to 1012CFU specific microorganism per gram of the composition or of chitin from 1% to 50% of the composition; and 2) producing sulfide microorganism from 107CFU up to 1012CFU specific microorganism per gram of the composition and any producing sulfide chemical agent, where the output of the sulfide in the range from 0.1 mg/min to 1.0 mg/min per gram of composition.

Any composition containing a microorganism from 107CFU up to 1012CFU per gram of composition, which simultaneously produces chitinolytic agents and sulfide, the year is raised to combat worms, bacteria and fungi. The above compositions include combinations of the following agents in the above proportions:

1. The chitinase and Na2S.

2. The chitinase and FeS.

3. The chitinase and the bacterium Desulfovibrio desulfuricans.

4. Chitin and Na2S.

5. Chitin and FeS.

6. Chitin and bacterium Desulfovibrio desulfuricans.

7. The microorganism, which simultaneously produces chitinolytic activity and H2S.

Previously known compositions are effective against a wide variety of parasitic nematodes of plants, including (but not limited to) the Meloidogyne species, such as M. incognita; Anginaspecies, such as A. tritici; Ditylencus species such as 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. reniformis; zoonematodes, such as: At spp, Trichostrongylus spp, Ostertagia spp, Nematodirus spp, Cooperia spp, Ascaris spp. and Haemonchus spp, the recommended dose rate of 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; pathogenic bacteria of plants, such as Erwinia chrysanthemi, Burkholderia glumae, and pathogenic fungi of plants, such as Pestalotia palmarum, Alternaria tabacina and Sarocladium orizae.

EXAMPLES

Example 1: In vitro evaluation nematocidal effect of hydrogen sulfide from chemical sources and chitinolytic enzyme.

Use eggs AOR the nematodes At the spp and Trichostrongylus colubriformis and Dictyocaulus viviparous, and larvae of parasitic nematodes (juvenile 2) Melodoigyne incognita.

Gather At the spp and Trichostrongylus colubriformis nematodes of sheep (sheep) and bovine (cattle) abomasum, respectively. Adult female nematodes are washed in physiological solution and process “Hibitane” (Chlorhexidine) in a concentration of 0.5%, within 1 minute, and the process is carried out at 37°C. to About 100 pre-disinfected females placed in the Erlenmeyer flask containing 50 ml LB medium, diluted tenfold with distilled sterile water, and leave to lay their eggs at night (8-10 hours).

Collection of nematodes D. viviparous exercise of infected lung pre userswindows cows (cattle). The same procedure is used for At spp. and T. colubriformis; however, females leave to lay eggs for 2-3 hours.

Since then, the manipulation is carried out in aseptic conditions in a vertical laminar flow, using the 24-hole plates to tissue culture. The full volume of medium containing females and eggs, filtered using a mesh sieve of 60 μm. Eggs of nematodes remain on the grid of the second sieve 30 μm. They are placed in the Hibitane solution concentration of 0.5% for 3 minutes, then thrice washed with LB medium, diluted 10 times with sterile distilled water.

After the procedure, disinfection of eggs recovered SITA and actor is tenderly again suspended in a solution of LB medium, diluted 10 times with sterile distilled water. The end result of the distribution check, counting and registering eggs in each well using a microscope with inverted Olympus, in this phase shall also monitor the homogeneity of evolutionary status.

At spp and T. colubriformis hatched from eggs in the time interval from 24 to 48 hours incubation at 28°C, whereas D. vivparus hatched from eggs up to 24 hours. Good sample preparation is, if all untreated controls more than 60% of vilaplana occurs in the above time intervals for each of the types.

Collect piles of eggs of Meloidogyne incognita carried out from the roots of pumpkin (Cucurbita pepo), previously infected and grown in greenhouses. For this operation use a stereoscopic microscope and needles with appropriately modified tips. Piles are placed in sterile distilled water in Petri dishes at 28°C in the amount of 50 heaps in the Cup. Conduct daily surveillance to monitor vilaplana from eggs. After about 72 hours enough larvae to start their collection and disinfection.

The entire volume of water containing a handful of eggs and larvae, filtered through the sieve mesh 60 ám. From this point on, all manipulations are carried out under aseptic conditions in a vertical Lamin is Pnom thread using the 24-hole plates to tissue culture. Eggs, separated from piles, not able to vilaplana and remain on the sieve mesh 30 μm; larvae collected on the following grid 5 μm. They are placed in the Hibitane solution with a concentration of 0.5% for 3 minutes, and then thrice washed with LB medium, diluted 10 times with sterile distilled water.

After disinfecting the larvae of Meloidogyne incognita removed from mesh sieve, and gently again suspended in a solution of LB medium, diluted 10 times with sterile distilled water. The final concentration and the results of the disinfection test, counting and recording of live larvae using a microscope with inverted Olympus.

Eggs of nematodes and larvae are placed in quantities of 100 pieces approximately 2 ml of LB medium, diluted 10 times. This volume is placed in a bubble cell, which ensure the passage of air through the liquid, and therefore, is provided with a contact of the gases with eggs and larvae. Each cell is duplicated for each treatment.

The sulphide get in the reaction of two sulfide salts (Na2S and FeS) with hydrochloric acid and the resulting anaerobic fermentation bacteria Desulfovibrio desulfuricans subs. desulfuricans ATCC 27774 (isolated from sheep rumen). As a chitinolytic enzyme using chitinase SIGMA C 1650 from the bacterium Serratia marcescens.

The studied eggs and larvae of nematodes subjected to edusim treatments within 24 hours:

1. Control treatment: the chitinase is not added, and the air is circulated through the cell.

2. Treatment with chitinase: chitinase in the amount of 0.2 units on the duplicate.

3. Processing sulfide: hydrogen sulfide from Na2S at a flow of 0.2 at 0.3 mg/min

4. Processing sulfide: hydrogen sulfide from FeS at a flow of 0.2 at 0.3 mg/min

5. Processing sulfide: hydrogen sulfide from Desulfovibrio desulfuricans at a flow of 0.2 at 0.3 mg/min

6. Combined treatment: simultaneous implementation of treatments 2 and 3.

7. Combined treatment: simultaneous implementation of treatments 2 and 4.

8. Combined treatment: simultaneous implementation of treatments 2 and 5.

All the above processing is repeated 4 times.

Twenty-four hours after the start of the experiment, calculate hatched larvae (At sp., T. colubriformis and D. viviparous) and the number of live larvae in all treatments. Results efficiency (E) are presented in table 1. This value represents the average of 4 repetitions of each treatment. For the obtained results for each of the species studied nematodes using analysis of variance (ANOVA) separately; use test Duncan (Lerch G. 1977. Laen las cienciasy agrcolas. 1rap.p.203-308, Editorial Cientifico-Tecnica, La Habana), the results of which are presented in t the blitz 1. The same letters indicate that among the dimensions, there were no significant differences (p<0,05).



Table 1

Processing efficiency (E*)
Processing efficiency (E*)
1.Ec

control
2.Eq

3.

Esn
4.

Esf
5.Esd6.Eqsn

(2+3)
7.Eqsf

(2+4)
8.Eqsd

(2+5)
aemonchus0,00

(a)
0,32

(b)
0,41

(c)
0,40

(c)
0,37

(b,c)
0,86

(d)
0,85

(d)
0,82

(d)
Trichostrongilus0,00

(a1)
0,37

(b1)
0,40

(b1c1)
0,39

(b1c1)
0,38

(b1c1)
0,88

(d1)
0,88

(d1)
0,83

(d1)
Dictyocaulus0,00

(a2)
0,35

(b2)
0,44

(c2)
0,42

(c2)
0,40

(b2c2)
0,91

(d2)
0,90

(d2)
0,86br>
(d2)
Meloidogyne0,00

(a3)
0,39

(b3)
0,51

(c3)
0,52

(c3)
0,47

(c3)
0,95

(d3)
0,93

(d3)
0,90

(d3)
*Efficiency (E) is the result of subtracting the value of the active frequency (Fr) for vilaplana or live larvae of 1, regardless of case. Fr is the ratio between the number vyluplivaetsya or live larvae in each treatment (Ntto) and the number vyluplivaetsya or live larvae in treatment 1 (Nc):

E=1-Fr, where Fr=Ntto-Nc; therefore, E=1-Ntto/Nc

To identify synergies for treatments 6, 7 and 8 took that acts that take place in them, are not excluded.

For this type of analysis, the expected efficiency (EE) should be equal to the sum of the individual effects (EI), defined by the efficiencies associated with the action of chitinases (Eq) and the efficiencies associated with the action of hydrogen sulphide (Esn, Esf and Esd), minus the effect of intersection (ei) (Sigarroa, A. 1985. Biometra y diseo experimental. 1ra. Parte. Minist.Sup. Ed. Pueblo yCap. 3. pag 69-107).

EE=Eq+Es-ei, where ei=Eq×Es

If the experimental efficiency (E) in those treatments where the combined DV is nematocide agent (processing 6, 7, 8), more than the expected efficiency (EE) for these treatments, you can be sure that there are synergies in terms nematocides chitinolytic activity of the agent (chitinases) and hydrogen sulfide, if both agent simultaneously used in the same processing. The values obtained are presented in table 2.

Table 2.

Experimental (E) and expected (IT) efficiency
Experimental (E) and expected (IT) efficiency
6Processing 7Processing 8
EITEITEIT
aemonchus0,860,600,850,590,820,57
Trichostrongilus0,880,620,880,620,830,61
Dictyocaulus0,910,640,900,620,860,61
Meloidogyne0,950,700,930,710,90 0,68

For these three treatments, in which simultaneously the United chitinase and hydrogen sulfide, the experimental efficiency (E) was higher than expected efficiency (EE) for the four studied nematodes that statistically demonstrates the existence of synergism between the two compounds (if they act simultaneously in nematocidal activity.

No significant differences were observed as regards the sources of sulfides and their nematocidal effect (table 1).

Example 2: Evaluation in the greenhouse nematocidal effect of the agent inducing chitinolytic activity (chitin), and agent, producing hydrogen sulfide (Desulfovibrio desulfuricans subps. desulfuricans ATCC 29577 extracted from soil).

Choose brown soil with a neutral pH value: it is dried and sieved on a 0.5 cm mesh to remove unwanted particles. It is sterilized in a vertical autoclave for 1 hour at 120°C and a pressure of 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 is dried at room temperature for 3-4 days in order later to cook the mixture for treatments with river sand, humus earthworms and chitin (ICN catalog number 101334).

Twenty pots (15 cm diameter, 13 cm depth and capacity 1 liter) fill in these soothes the deposits for the following treatments:

1. Control treatment: soil 70%, river sand 25% and humus 5%.

2. Treatment with chitin: soil 70%, river sand 25%, humus 4% and chitin 1%.

3. Micro-organic treatment: soil 70%, river sand 25%, humus 5% and D. desulfuricans, added to a concentration of 1010CFU/pot.

4. Combined treatment: soil 70%, river sand 25%, humus 4% and D. desulfuricans, added to a concentration of 1010CFU/pot.

Each treatment is carried out 5 times (5 pots).

For treatments 2 and 4 of the provisional mixture of humus and chitin are produced in the ratio of 4:1, and then prepare the final mixture of soil and sand. For treatments 3 and 4 D. desulfuricans contribute with 100 ml of deionized water in each pot. These volumes contribute evenly during the first irrigation.

For all treatments in pots inoculant 500 nematodes species Radopholus similes, pre-assembled from infected naturally roots of bananas. Use the centrifugation-flotation (Jenkins, W. R. 1964. A rapid centrifugal-flotation tecnique for separation nematodes from soil. Plant Disease Reporter, 48: 692); samples were diluted in 5 ml of distilled water and contribute evenly to a depth of 5 cm below the soil surface.

The pots are placed in the greenhouse and leave there at 3 days after treatment and insulinopenia nematodes. At this stage, carry out daily watering to keep conditions sufficient wet the STI. Before the fourth day after treatments in pots planting plants of banana var. Cavendish, obtained by the method of tissue culture in vitro. From this point on, perform a hard mode of irrigation, which provides continuous soil moisture to the field capacity.

The final assessment is conducted three months after the beginning of the experiment; the roots of the plants gently released from the soil. Then record the number of instances (larvae and adult nematodes) and living nematodes collected from plants using the method of centrifugation-flotation (Jenkins, W. R. 1964. A rapid centrifugal-flotation tecnique for separation nematodes from soil. Plant Disease Reporter, 48: 692), and inverted microscope for counting. The results achieved efficiency for different treatments are presented in table 3. They represent the average of 5 repetitions for each of the treatments. To process the results using analysis of variance (ANOVA)followed by Duncan test (Lerch G. 1977. La Experimentacin en las cienciasy agrcolas. 1rap.p.203-308, Editorial Cientifico-Tecnica, La Habana), the obtained results are presented in table 3. The same letters indicate no significant differences (p<0,05) between treatments.

Table 3.

Processing efficiency (E)*
Processing efficiency (E)*
1.Ec2. Eq3. Esd4. Eqsd
Radopholus similis0,00(a)0,21(b)0,18 (b)0,48(c)
*Efficiency (E) is the result of subtracting the magnitude of the relative frequency of live specimens (Fr) of 1. Fr is the ratio of the number of live instances in each of the treatments (Ntto) and the number of live instances to process 1 (Nc):

E=1-Fr, where Fr=Ntto/Nc, so E=1-Ntto/Nc.

To determine the possible synergistic effect when processing 4, it was assumed that the current acts (nematocidal effect) cannot be excluded.

Analogously to example 1, the expected efficiency (EE) should be equal to the sum of the individual effects (EI), presents efficiency due to the action of chitin (Eq) as an inductor of the chitinolytic activity of microorganisms present in a mixture of soil and humus, and efficiency, due to the action of hydrogen sulphide (Esd), secreted by the bacteria D. desulfuricans; minus the cross-effect (ei) between the two treatments (Sigarroa, A. 1985.experimental. 1ra. Parte. Minist.Sup. Ed. Pueblo y Educacin. Cap.3. pag 69-107)

EE=Eq+Es-ei, where ei=Eq×Es

If the experimental efficiency (E) for processing 4, g is e combined two nematocide agent, it turns out higher than expected efficiency (EE), you can be sure that there is a synergism between the agent inducing chitinolytic activity (chitin) and hydrogen sulphide (from D. desulfuricans), when they are used simultaneously for the same treatment. The values obtained are given in table 4.

Table 4

Experimental (E) and expected (IT) efficiency
Experimental (E) and expected (IT) efficiency
EIT
Treatment 4
Radopholus similis0,480,35

When processing 4 initiator chitinolytic activity (chitin), and the biological source of hydrogen sulfide (D. sulfuricans) combined. In this case, the experimental efficiency (E) was higher than expected efficiency (EE), which proves the existence of synergism (in relation to their nematocidal activity) for these two compounds, if they are applied to the soil at the same time.

Example 3: Demonstration of chitinolytic activity and production of sulfide by bacteria Corynebacterium paurometabolum C-924 and Tsukamurella paurometabola DSM 20162.

Determination of the production of sulfide:

In capsules the La gas collection volume 100 ml draw samples of gas, released during the fermentation of strains C-924 and DSM 20162 5 liter bioreactors. Full time cultivation is 24 hours. The formation of hydrogen sulphide is first detected after 16 hours.

The samples are treated the same way as the processing when receiving the H2S. the Analysis is carried out using a gas chromatograph Varian in the following conditions:

- Flame photometric detector with a filter sensitive to serosoderjaschei connections.

A sample of hydrogen sulfide: 43,2 ng/ml (in duplicate).

Samples: two samples for each time of sampling.

- Input: 1 ml or ál in the head part.

- Column: DB-5 (15 m×0.53 mm).

- Temperature: 35°C.

- Carrier gas: nitrogen 1.5 ml/min

Detector: FPD-S

- The purge gas: nitrogen, 30 ml/min

Table 5 presents the results of analyses of sulfide gases emitted by the two strains at different points in time.

Table 5.

Analysis of sulfide gases
The flow of H2S mg/min (Detected sulfide stream)
StrainSample16 hour18 h20pm22 h24 hour
C-9241 0,06730,22080,47790,35780,0672
20,06590,21600,47550,35520,0680
DSM 2016210,02310,04160,10140,18630,0009
20,02400,04220,10400,18870,0097

Both strains produce sulfides, but the flow in strain C-924 stronger than in strain DSM 20162.

Determination of chitinolytic activity:

Use strains of Corynebacterium paurometabolum C-924, Tsukamurella paurometabola DSM 20162, Serratia marcescen ATCC 13880 and E. coli ATCC 25922

Bacteria cultures of the studied strains grown in LB medium at 28°C and a rotation speed of 100 rpm for 24 hours, then centrifuged at a speed of 3500 rpm; the supernatant liquid is filtered through two 0.2 μm mesh. The filtered product analyzed in tablets prepared with colloidal chitin suspension (0.5%), and also add the agarose up to 0.8%for secondary jelly and provide porosity to facilitate diffusion of the protein. After the formation of the jelly wells with diameter of 5 mm open and add 100 ál of filtered supernatant from each bacterial strain. Each tablet to repeat the accelerate in triplicate and incubated at 28° C in the dark.

Since 48 hours, and a decrease in turbidity of the medium, resembling a halo, which demonstrates the hydrolysis of chitin. The following table (table 6) presents the quantitative results from hydrolysis halos at different times of incubation with the supernatant fluids of different cultures of the studied strains.

The two strains (C. paurometabolum and T. paurometabola) demonstrate the chitin-hydrolysis halo, as used positive control (S. marcescen), whereas when using a strain of E. coli (negative control) hydrolysis halo is not formed.

Example 4: In vitro assessment of the actions provided by sulfides and chitinases produced by the bacteria Corynebacterium paurometabolum C-924 and Tsukamurella paurometabola DSM 20162, the parasite Fasciola hepatica (trematode).

Use the eggs of the parasite Fasciola hepatica. Egg collection is carried out directly from the bile of infected liver pre userswindows cows (cattle). The contents of the bile again suspended in three times the volume of distilled water and leave to stand for 2-3 hours at 28°C, to settle eggs. Then remove as much as possible of the supernatant liquid. The precipitate is filtered through a mesh sieve 71 μm, which delayed eggs.

From this point forward, all manipulations carried out in acept the economic conditions in a vertical laminar flow using the 24-hole plates to tissue culture. A sieve with eggs F.hepatica placed in the Hibitane solution concentration of 0.5% for 3 minutes, then thrice washed with LB medium, diluted 10 times with sterile distilled water. After the procedure, disinfection of eggs removed from the sieve and gently again suspended in a solution of LB medium, diluted 10 times with sterile distilled water. The end result of the collection and disinfection test, counting and registering eggs in each well using a microscope with inverted Olympus.

This phase shall also monitor the homogeneity of evolutionary status.

These eggs of parasitic trematodes in the above for the in vitro conditions are hatched in about 15 days incubation at 28°C; a good sample preparation is considered that more than 60% of the eggs hatch by the end of the incubation period.

For the development of the experiment disinfected eggs are placed in an amount of about 100 pieces in 1 ml of LB medium, diluted 10 times. This volume is evenly placed in 20 bubble cells that allow passage of air through the liquid; and so the gases can come into contact with eggs. Each cell used in multiple instances (4 per treatment) for all five treatments.

Eggs of F. hepatica is subjected to the following treatments during the last 4 days of incubation:

1. Control treatment: in each cuvette was added 1 ml of LB medium, diluted 10 times, without chitinases, and through it circulates the air.

2. To each cuvette was added 1 ml of chitinolytic supernatant without bacterial cells from the culture 1010colony forming units per milliliter (CFU/ml) Corynebacterium paurometabolum C-924.

3. To each cuvette was added 1 ml of chitinolytic supernatant without bacterial cells from 1010CFU/ml of Tsukamurella paurometabola DSM 20162.

4. Pass through the sample cell, the flow of gases from continuous culture of Corynebacterium paurometabolum C-924 at 1010CFU/ml

5. Pass through the sample cell, the flow of gases from continuous culture Tsukamurella paurometabola DSM 20162 at 1010CFU/ml

6. Combined treatment: simultaneous treatments 2 and 4.

7. Concurrent processing: Simultaneous treatments 3 and 5.

On the fourth day after the beginning of the experiment, calculate the hatched eggs. In the case of F. hepatica was not possible to calculate hatched larvae (miracides) because of their great mobility; therefore, observation through a microscope focused on the eggs. The results of efficiencies under different treatments are presented in table 7. This averages to 4 repetitions for each of the experiments. The same letters indicate no significant differences (p<0.05) between treatments is kami.

Table 7

Processing efficiency (E*)
Processing efficiency (E*)
1.E

control
2.Eq

C-924
3.Eq

DSM

20162
4.Es

C-924
5.Es

DSM

20162
6.E

(2+4)
7.E

(3+5)
Facsiola hepatica0,00

(a)
0,18

(b)
0,11

(c)
0,29

(d)
0,16

(b,c)
0,52

(e)
0,28

(d)
Efficiency* represents the result of subtracting the relative frequency vilaplana (Fr) of 1. Fr is the ratio between the number of hatched eggs in each of the treatments (Ntto) and the number of hatched eggs when processing 1 (Nc):

E=1-Fr, where Fr=Ntto/Nc; therefore, E=1-Ntto/Nc

To determine possible synergies for treatments 6 and 7, it was assumed that the observed in these cases, the action (antiparasitically effect) cannot be excluded.

For this type of analysis expected efficiency (EE) is defined by the efficiency associated with the action of chitosan (Eq) and efficiency associated with the action of hydrogen sulphide (Esn, Esf and Esd), minus p is crossed effect (ei) (Sigarroa, A. 1985.experimental. 1ra. Parte. Minist.Sup. Ed. Pueblo y Educacin. Cap.3. pag 69-107).

EE=Eq+Es-ei, where ei=Eq×Es

If the experimental efficiency (E) for treatments, which combine two protivoaritmicheskih agent (treatment 6 and 7), more than expected efficiency for these treatments, you can be sure that there is a synergy with protivorahiticescoe chitinolytic activity of the agent (chitinase) and hydrogen sulfide at their simultaneous use in the same process. The values obtained are summarized in table 8.

Table 8.

Experimental (E) and expected (EE) efficiency.
Experimental (E) and expected (EE) efficiency.
6Processing 7
EEEEEE
Fasciola hepatica0,520,310,280,25

When the treatments were combined chitinase and hydrogen sulfide, the experimental efficiency (E) was higher than expected efficiency (EE), which demonstrates the presence of synergism is La two compounds in relation to their nematocidal activity when they act simultaneously.

Example 5: In vitro evaluation of bacterial strain (Corynebacterium paurometabolum C-924), which produces hydrogen sulfide and has chitinolytic activity of some bacteria and fungi.

We used the following types of fungi: Pestalotia palmarum, Alternaria tabacina, Sarocladium orizae, Pitium debaryanum; and the following types of bacteria: Erwinia chrysanthemi, Burkholderia glumae, Serratia marcescen ATCC 13880, Bacillus subtilis F 1695 and were also used Escherichia coli ATCC 25922.

A) Analysis of fungi.

The impact of Corynebacterium paurometabolum C-924 on fungi was investigated for the following fungi: Pestalotia palmarum, Alternaria tabacina, Sarocladium orizae and Pytium debayianum. Strain Serratia marcescen ATCC 13880 used as a positive control and E. coli strain ATCC 25922 was used as negative control in determining fungicidal activity. Bacterial cultures were grown, through the usual shaking and ambient temperature for all species within 24 hours. Conducted the necessary dilution to when measuring the absorption at a wavelength of λ 530 nm to provide a cell concentration of 109CFU/ml, the Samples were placed in Petri dishes containing medium (PDA agar potato-dextrose), insulinopenia carried out on the Central line using a microbiological loop. The cups were incubated for 48 hours at 28°C, then inoculable discs with diameter of 8 mm from a variety W is ammo fungi, grown earlier (tablets containing PDA medium were placed on the surface of the tablets on each side of the midline inoculated bacteria. Each subject of the study of fungi was investigated in triplicate, and incubated for 10 days at 28°C. the Results are read, starting from the fifth day from the beginning of the experiment.

b) Analysis of bacteria.

The presence of the interaction of Corynebacterium paurometabolum C-924, E. coli ATCC 25922 and Bacillus subtilis F 1695 investigated the following bacteria: Erwinia chrysanthanem and Burkholderia glumae. Bacillus subtilis strain F 1695 used as a positive control to determine antagonism with other bacteria, for the negative control used E. coli strain ATCC 25922. The bacterial strains were grown in LB medium at normal shaking and in a normal temperature conditions within 24 hours. These crops have performed the necessary dilution to the previous value of absorption at a wavelength of λ 530 nm provided a cell concentration of 109CFU/ml In the case-924 added 5 µl drops on three different tablets plot with LB medium, two different plot for positive control, and two other different plot for negative control, respectively. The plates were incubated at 28°C for 48 hours. After this time they were processed by the flow of chloroform for 3 minutes (for inactivation to avoid dispersion in the subsequent stages), then the tablets were kept in laminar flow, semi-open, to eliminate the excess gas. Exercise insulinopenia stimulating strains of Erwinia chrysanthemi and Burkholderia glumae, which start with pure cultures of each of the microorganisms from which are taken the necessary amount to bring the cell concentration to 109CFU/ml, after you add up to three ounces semi-solid LB medium (with 0.1% technical agar No. 3). The mixture is spread on tablets containing stimulating strains, incubated at 28°C for 48 hours prior to evaluation.

Table 9 describes the results obtained during the above analysis of the interaction.

As can be seen from the results of table 9, there is a noticeable antagonistically the effect of the strain Corynebacterium paurometabolum C-924 against fungi Pestlotioa palmarum, Alternaria tabacina and Sarocladium orizae, which are characterized by a high content of chitin in their structures. There is only weak antagonism caused by the action of hydrogen sulfide. In case of interaction with the investigated bacteria antagonism is observed for the two pathogenic strains (Erwinia crhysanthemi and Burkholderia glumae), while antagonism was not observed in the case of Bacillus subtilis, as this organism is isolated from antagonistic with other soil microorganisms and therefore b is more resistant to harmful environmental factors.

1. Composition for use in agriculture and veterinary medicine, which includes at least one chitinolytic agent or agent, inducing chitinolytic activity, and sulfide or agent producing sulfide, which is independently used for effective control of helminths and the causative agents of bacterial and fungal diseases, to deal effectively with a wide range of parasitic nematodes plant parasitic nematodes of animals and trematodes, bacterial agents that cause disease, and fungal agents causing disease.

2. The composition according to claim 1, where the specified chitinolytic agent is a chitinase.

3. The composition according to claim 1 where the specified chitinolytic agent is a microorganism producing the chitinase.

4. The composition according to claim 3, which contains from 107craniopathy units (CFU) of up to 1012CFU specified microorganism per gram of composition.

5. The composition according to claim 1, where the specified agent, inducing chitinolytic activity is chitin.

6. The composition according to claim 5, where the chitin content is from 1 to 50 %.

7. The composition according to claim 1, where producing the specified sulfide agent is a microorganism.

8. The composition according to claim 7, which contains from 107CFU up to 1012CFU specified microorganism per gram of the composition./p>

9. The composition according to claim 1, where indicated, producing sulfide agents are chemical agents.

10. The composition according to claim 1, where producing the sulfide is from 0.1 to 1.0 mg/min per gram of composition.

11. The composition according to claim 1, where chitinolytic agent and producing sulfide agent is a microorganism.

12. The composition according to claims 1 to 11 for combating parasitic themtoday.

13. The composition according to item 12, where parasitic themtoday are At spp., Trichostrongylus spp and Dictiocalus spp.

14. The composition according to claims 1 to 11 for combating parasitic nematodes of plants.

15. The composition according to 14, where parasitic nematodes of plants are Meloidogyne spp. and Radopholus similis.

16. The composition according to claims 1 to 11 to control pathogenic bacteria of plants and animals.

17. The composition according to item 16, where pathogenic bacteria are Erwinia and Burkholderia spp.

18. The composition according to claims 1 to 11 to control pathogenic fungi of plants and animals.

19. The composition according to p, where pathogenic fungi are Pestalotia palmarum, Alternaria tabacina and Sarocladium orizae.

20. The composition according to claims 1 to 11 for combating parasitic trematodes.

21. The composition according to claim 20, where the parasitic trematode is Fasciola hepatica.

22. Composition according to any one of the preceding paragraphs for use in agriculture and/or in veterinary medicine, where specified suitable media, such as fertilizer, pre is varicella Packed soil, the shell of the seed, powder, granules, aerosols, suspension, liquid, or any of the above forms, presented in the form of encapsulated compositions for combating parasitic helminths, bacteria and pathogenic fungi.



 

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