Synergist for improvement of effectiveness of biological agents against colorado potato beetle

FIELD: agriculture.

SUBSTANCE: improvement of effectiveness of insecticidal preparation is achieved through the use in sublethal doses of a mixture of fluorinated usnic acid (FUA) of the formula and II obtained by the reaction of (+)-usnic acid with perfluoropropene as a synergist of such insecticides. The decline of the latent period of the disease and increased mortality rate of larvae of Colorado potato beetle is marked using FUA and entomopathogenic microorganisms of Beauveria bassiana, Metarhizium anisopliae and Bacillus thuringiensis. The mixed composition of FUA at a concentration of 0.03% with a suspension of conidia of the fungus Bauveria bassiana with a titer of 3×10⁶ conidia/ml (in vitro) and 5×10⁶ conidia/ml (in the field) has proved to be the best in all respects.

EFFECT: increased biological efficiency of insecticides based on entomopathogenic fungi and bacteria.

1 cl, 4 tbl,5 ex

The invention relates to agriculture and can be used to improve the biological effectiveness of insecticide based on entomopathogenic microorganisms.

One of the most promising groups of microorganisms to control populations of insects are entomopathogenic fungi and bacteria. With a certain specificity with respect to the owners, they have a significantly lower compared with chemical insecticides effects on non-target objects [Pathogens insects: structural and functional aspects. VPO (as amended) M: all the year Round. 2001. 736 C.]. However, the use of drugs based on entomopathogenic microorganisms encounters a number of difficulties. In particular, mycosis insects are characterized by a long latency period. Stretched in time death occurs when the infection hyphomycetes insects various groups [Ogarkov B.N., Ogarkova BORN Entomopathogenic fungi of Eastern Siberia. Irkutsk: Publishing house of Irkutsk. Univ. 2000. 134 S.; palavinka G.P., Naumova, E.N. 1997. Entomopathogenic properties of the fungus Tolypocladium inflatum (Gams), isolated from meadow moth Loxostege sticticalis L. // Regulation of population invertebrates and phytopathogens. Novosibirsk: NSAU. Pp.42-46; Lednev BORN, hooks V.Y., Khodyrev VP, Levchenko M.V., Duisembekov B.A., Sagitov O.A., Glupov V.V. Dyn is ka death LMI simultaneous infection with entomopathogenic fungi (Metarhizium anisopliae, Beauveria bassiana) and the bacterium Pseudomonas sp. // Siberian ecological journal, 4, 2007, 527-531; Lomer C.J., Bateman R.P., Johnson D.L., Lagewald J., Thomas M. 2001. Biological Control of Locusts and Grasshoppers // Annu. Rev. Entomol. V.46. P.667-702]. During infection with larvae of the Colorado potato beetle, the hyphomycetes Beauveria bassiana (Bals.) Vuill or Metarhizium anisopliae (Metsch.) Sorokin in laboratory conditions, the period from infection to 95-100%mortality rate is between 10 and 17 days. In field conditions, this period is even more extended for 2 weeks dies only about 50% of the larvae, and the mortality observed in the care in the soil and metamorphosis [Secure A.I., Izhevsk S., Trofimova I. 1979. Microbiological means of control Colorado potato beetle. M. 52 S.; hooks V.Y., V. Serebrov, Malyarchuk A.A., Kopzhasarov B.K., pea-potato NS, Orynbayev A.K., Khodyrev VP prospects for the use of entomopathogenic hyphomycetes (Deuteromycota, Hyphomycetes) against the Colorado potato beetle in the conditions of South-Eastern Kazakhstan // Siberian Bulletin of agricultural science. 2007. No. 4. Pp.52-60]. Naturally, this fact limits the interest of practitioners to entomopathogenic fungi, because during latent development of mycosis insects can cause significant damage to plants. For bacterial infections, in contrast, is characterized by rather fast insects (2-7 days), but this is not always achieved 100% mortality. Often the younger ages are chuvstvitelnyy entomopathogenic bacteria, and the high is very resistant to him. If we take into account the temporal overlap of all stages of development in many insects, in particular in the Colorado potato beetle, it becomes apparent low efficiency of bacterial preparations.

Modern methods of plant protection are based on integrated approaches, including the use of entomopathogenic microorganisms combination with chemical insecticides [Charnley A.K., Collins, S.A. 2007. Entomopathogenic fungi and their role in pest control // Environmental and microbial relationships. The Mycota: A comprehensive treatise on fungi as experimental systems for basic and applied research / Eds. Kubicek C.P., K. Esser, I.S. Druzhinina Berlin-Heidelberg: Springer-Verlag. V.4. P.159-187]. It is known that in some cases the combined use of insecticides and entomopathogenic microorganisms leads to the development of synergies that can significantly reduce the rates of consumption of insecticides and, as a consequence, contributes to the reduction of anthropogenic pressure on ecosystems [Benz, 1976. Synergism of microorganisms and chemical insecticides // Mashimaro (as amended) Microorganisms in the fight against harmful insects and mites. M.: Kolos. S-123; Serebrov CENTURIES, Gerber O.N., The Khodyrev V.P., V.P. Tsvetkov 2005. Prospects for joint use of entomopathogenic fungi and chemical insecticides // Mycology and Phytopathology. T. Issue 3. S.89-98]. Numerous studies have shown that the use of sublethal doses of insect is zidov of different nature together with the fungi Metarhizium and Beauveria leads to a synergistic effect on insects of different groups [Serebrov CENTURIES, Gerber O.N., The Khodyrev V.P., V.P. Tsvetkov 2005. Prospects for joint use of entomopathogenic fungi and chemical insecticides // Mycology and Phytopathology. T. Issue 3. S.89-98; D.G. Boucias, C.Stokes, G. Storey and J..Pendland. 1996. The effects of Imidacroprid on the termite Reticulitermes flavipes and its interaction with the mycopathogen Beauveria bassiana. Pflanzenschuts-Nachrichten Bayer V.49. P.103-144; Quintela E.D. and C.W.McCoy. 1997. Pathogenicity enhancement of Metarhizium anisopliae and Beauveria bassiana to first instars of Diaprepes abbreviates (Coleoptera: Curculionidae) with sublethal doses of imidacloprid Environ. Entomol. V.26. P.1173-1182].

At the same time, it is known that insects quickly develops resistance to insecticides. Therefore, it is necessary to expand the number of insecticides, showing a synergistic effect when combined with the use of entomopathogenic fungi.

Object of the invention is the creation of new effective tools used by sublethal doses together with entomopathogenic microorganisms would give a synergistic effect and it could be used as part of integrated plant protection, efficiency comparable with chemical insecticides.

The problem is solved by a composition comprising a mixture of products of interaction of usninovoy acid with performaing named authors trucks, as a synergist for insecticides based on entomopathogenic fungi and bacteria:

The specified synergist may be used as part of integrated plant protection, for example, the following composition:

Closest to the claimed invention of the patent in which the insecticides on the basis of pathogenic microorganisms as a synergist is usnic acid [Mpolweni, Nofilemenu, Oisin, VPO, Web, Immobility, Yea, Wourrynow; "Application of usninovoy acid as a synergist for insecticides based on entomopathogenic microorganisms, RF patent №2328493, priority from 09.01.2007]. In the prototype shows the increase in the percentage of destruction of caterpillars bee the liquidation Colorado potato beetle larvae and caterpillars Gypsy moth using the mixture of usninovoy acid and entomopathogen the micro-organisms, such as fungi Metarhizium anisopliae and Beauveha bassiana (conidia), the bacteria Bacillus turhingiensis (spore-crystal mass), nuclear polyhedrosis virus (SEM. Baculoviridae).

In the invention as an insecticide synergist based on entomopathogenic fungi and bacteria offers a mixture of products of interaction of usninovoy acid with performaing, which includes two main substances I and II.

Reaction of (+)-usninovoy acid with performaing described in the article [Fin, Oisin, Reaches, Mpolweni, Nofilemenu, Gaellic. Chemical modification of usninovoy acid. Message 1. The interaction of (+)-usninovoy acid with perfluoroolefins. The known. Acad. Sciences. Ser. chem. 2007. T. No. 6, str-2002]. The reaction of usninovoy acid with perftoran in the presence of K₂CO₃water and DMF (0.2% water) received a mixture of compounds I and II, which were isolated in individual form of column chromatography on silica gel.

For studies of the toxic properties of compounds in relation to the larvae of the Colorado potato beetle was tested as separate components, and are not separated after the reaction mixture - fours.

Synergistic properties FSD studied in larvae of the Colorado potato beetle of different ages collected on potato plants in Novosibirsk and in the environment is totah, Almaty. To infect the insects used for the culture of Beauveria bassiana (RAA-31) and Metarhizium anisopliae (R-72) from the collection of microorganisms of the Institute of animal systematics and ecology SB RAS. Conidial mass of fungi was developed on agar medium of čapek or sterile millet according to the recommendations Iaalay [Nicholas E. Methods of experimental Mycology (Ed. by V.I. Bilai). Kiev: Naukova Dumka. 1982. P.106-137]. In some experiments we used a strain 2495 B.thuringiensis tenebrionis (H8ab), isolated from dead larvae big meal hrusica (Tenebrio molitor) in laboratory populations in Isias SB RAS. Bacteria were grown on mesopatamia agar for 6 days, then washed with distilled water.

Field and laboratory studies conducted in 2006-2008 in the laboratory of the Novosibirsk Institute of animal systematics and ecology, SB RAS, Novosibirsk and in the surrounding area, Almaty on the basis of the laboratory of biotechnology, Institute of plant Protection of the Republic of Kazakhstan.

To assess the impact of the funds trucks on the development of infectious diseases conducted joint processing insects, entomopathogenic fungi, bacteria and tool trucks. This truck was dissolved in acetone, and then the acetone solution in varying amounts was added to a suspension of conidia of fungi, bacteria or a mixture of fungi and bacteria, recip is nnow mixture was used to infect insects. In the control options used processing insects, water, and handling insects individually entomopathogenic microorganisms and trucks. To show that the use of acetone as the solvent does not affect insects in control variants insects were treated water with the addition of equivalent amounts of acetone.

The efficiency of trucks as a synergist for biological products based on entomopathogenic fungi and bacteria can be demonstrated by the examples below.

Example 1. Receiving funds-synergist trucks from (+)-usninovoy acid.

Through a mixture of (+)-usninovoy acid (0.3 g) and K₂CO₃(0.3 g) in 10 ml aqueous DMF (0.2% water) under stirring and the temperature of 40-45°C for 3-4 hours missed performerin (~500-600 ml). The reaction mixture was poured into 300 ml of water, acidified with 5%hydrochloric acid, left for half an hour for the consolidation of loose fine sediment. The precipitate was filtered off, washed with 100 ml of water and dried in air for 24 hours. Obtained 0.26 g of the reaction mixture is then fours.

The selection I and II. The reaction mixture was divided on a column of silica gel (eluent - CH₂Cl₂). Highlighted components I (0.14 g) and II (0.06 g), crystalline substance yellow with the outputs 52 and 22%, respectively, the structure of which was established for the following range of the M.

2,6-Diacetyl-3,9-dihydroxy-8,9b-dimethyl-7-(1',1',2',3',3',3'-hexaphosphate)-9bH-dibenzofuran-1-he (I). NMR¹H (CDCl₃, δ ppm) 1.78 (3H, s, H-15), 2.12 (3H, s, H-10), 2.54 (3H, s, H-14), 2.64 (3H, s, H-12), 5.10 (1H, DM, H-17), 5.95 (1H, s, H-4), 11.1 (1H, s, OH-9), 18.8 (1H, s, OH-3). NMR¹³With (CDCl₃, δ ppm) 9.5 (C-10), 27.7 (C-12), 31.6 and 31.7 (C-14 and C-15), 58.9 (C-9b), 83.0-86.1 (DCV, C-17), 98.4 (C4), 99.4 (C18), 105.0 (C2), 111.0 (C9a), 113.2 (C8), 116-118 (C-16), 117.9 and 121.8 (DD, C-16), 119.2 (C-6), 144.9 (C-7), 152.3 (C-5A), 154.5 (C-9), 178.9 (C-4A), 191.5 (C-3), 194.7 (C-13), 197.4 (C-1), 201.7 (C-11).

(6bS)-8-acetyl-2,2,3-triflora-6,9-dihydroxy-5,6b-dimethyl-1-methylene-3-(trifluoromethyl)-2,3-dihydro-1H-benzofuro[2,3-f]chromen-7(6bH-he (II). NMR¹H (CDCl₃, δ ppm) 1.76 (3H, s, H-15), 2.16 (3H, s, H-10), 2.64 (3H, s, H-12), 5.99 (1H, s, H-4), 5.99, 6.45 and 6.50 (2H, m, H-14), 10.7 (1H, s, OH-9), 18.8 (1H, s, OH-3). NMR¹³With (CDCl₃, δ ppm) 8.0 (C-10), 27.7 (C-12), 31.7 (C-15), 58.8 (C-9b), 85.0-87.0 (C-17), 98.5 (C-4), 105.1 (C-2), 109.1 (C-9a), 111.2 (C-8), 116-118 (C-16), 119.3 (C-14), 120.0 (C-18), 126.1 (C-6), 147.8 (C-7), 151.2 (C-13), 152.5 (C-5), 152.8 (C-9), 179.2 (C-4A), 191.5 (C-3), 197.6 (C-1), 201.7 (C-11).

Insecticidal activity of components I and II, each individually tested on larvae of the Colorado potato beetle in laboratory conditions. When handling insects component I or II was first dissolved in 0.5 or 1 ml of acetone was then added respectively in 9.5 or 19 ml of water and abruptly shook. In the control options (process water) was added an appropriate amount of acetone. The tested larvae and food was immersed in an aqueous suspension with a certain concentration is the situation of components I and II. Regression analysis showed no significant differences in mortality of larvae from compounds I and II. However, there was a trend of increasing mortality from component II. Thus, at concentrations of 0.01%, 0.02% and 0.05% in this case the marked 35, 75 and 100% mortality respectively, while from component I - 20, 40 and 95%, respectively. In all cases, delayed growth and molting of larvae treated with the investigated components. Given data on the components I and II were isolated and obtained in the reaction of usninovoy acid with performaing mixture of compounds used as a means of trucks.

Example 2. The best way in a laboratory experiment on the larvae of the Colorado potato beetle proved to the tool trucks at a concentration of 0.03% with a suspension of conidia of the fungus Beauveria bassiana with a titer of 3×10⁶conidia/ml In the experience used larvae of the Colorado potato beetle II, III and IV instars. When handling insects trucks were first dissolved in 0.5 or 1 ml of acetone was then added respectively in 9.5 or 19 ml of a suspension of conidia of the fungus or water and abruptly shook. In the control options (processing water suspension of conidia of the fungus) was added an appropriate amount of acetone. The tested larvae and food was immersed in water suspension for 3-5 seconds. Replace the feed was carried out on the second day after treatment and then every day. Larvae were kept in a PLA is teak cups with a volume of 700 ml, covered by the grid. To prevent drying of the leaves and their petioles were placed in Eppendorf tubes (1.5 ml), was swabbed with a damp cotton wool. Each option was put in 3 replicates of 15 birds each. Accounting for mortality was performed every day for 12 days. The results of laboratory tests are shown in table 1. The effects obtained by biotesting agents in the indicated concentrations, can be classified as additive.

Found that FSD has the property to substantially increase the mortality of Colorado potato beetle larvae as younger and older ages when their infection with entomopathogenic fungi (table 1). It is important to note that in addition to increasing the total mortality of larvae (12 day experience) versions of C. bassiana+FSD observed earlier insects. The average half-lethal time (LT50) for III and IV instars when processing the combined mixture of B.bassiana+FT decreased by 2-3 days in contrast to variants with processing only by the fungus. In addition, variants of trucks and B.bassiana+FT larvae have registered strong growth retardation and considerably less feed consumption.

This blended composition - fours at a concentration of 0.03% with a suspension of conidia of the fungus Beauveria bassiana with a titer of 5×10⁶conidia/ml was further tested in field trials.

Example 3. In the field malcode anachem the experiment was carried out to study the efficacy of drugs based on entomopathogenic fungi and the effect on their efficiency trucks. Experience laid on private planting potatoes. Processing plants held in the evening in calm weather with the help of manual knapsack sprayer. Infectious burden was made at the rate of 1×10⁹conidia/m²according to the adopted abroad standards of consumption: 5×10¹²up to 5×10¹³conidia/ha [S.P.Wraight, M.Sporleder, T.J.Poprawski, L.A. Lacey. 2007. Application and evaluation of entomopathogens in potato // Field manual of techniques in invertebrate pathology. Application and evaluation of pathogens for control of insects and other invertebrate pests / ed. L.A.Lacey, H.K.Kaya. Springer, P.329-359]. The fluid flow was about 40 ml/m²the concentration of conidia of fungi in the working solution of 5×10⁶conidia/ml (increase of the titer of the fungus in the working solution in this experiment is due to the fact that under the influence of solar radiation is the loss of a certain percentage of conidia, resulting in lower mortality insects from mycosis) and 0.03% FSD, respectively. In the control processing plants did not. The experiment also laid the experiences in the use of trucks separately from entomopathogenic fungi to assess its insecticidal properties. For accurate accounting of mortality on the branches of potatoes wore charge from the grid. Experiment was repeated 4 times. The number of individuals per one repetition of 15-20. The results are shown in table 2.

In the field experiments established that the FSD has the property to increase significantly bend the ü insects infected their entomopathogenic fungi B.bassiana. Table 2 presents the dynamics of the death of the insects recorded in field experiments. These effects can be classified as reinforcing synergies and temporal synergism [Benz, 1976. Synergism of microorganisms and chemical insecticides // Mashimaro (as amended). Microorganisms in the fight against harmful insects and mites. M.: Kolos. S-123]. In a field experiment in variants of trucks and B. bassiana+FSD noted weak damaged leaves. Defoliation of potato 10-12 day experience was in these embodiments, only 5-15%, whereas in the variant B.bassiana - 60-80%, and in control - 60-100%).

Thus, the application of FSD as an insecticide synergist based on entomopathogenic fungi B.bassiana increases the level of mortality of larvae of the Colorado potato beetle and significantly reduced their consumption of biomass in comparison with the usual infection with entomopathogenic fungi.

Example 4. Along with the above examples have conducted a series of laboratory experiments according to the method described above (Example 2), which used different concentrations of conidia of the fungus Beauveria bassiana and trucks. The results are shown in table 3.

Material of table 3 shows that the most rapid time of the death of the larvae was observed when combining mushroom with FSD in concentrations of 0.05%, however, a need to reduce this concentration within the range of 0.0 to 0.05%, and the title of the conidia of the fungus in 2-3 times to get more pronounced additive or synergistic effect.

Example 5. In laboratory experience on the larvae of the 3rd age of the Colorado potato beetle tested the combined effect of FSD and combined experimental drug on the basis of the fungus Metarhizium anisopliae and the bacterium Bacillus thuringiensis tenebrionis [Kryukov V.Yu., Khodyrev V.P., Yaroslavtseva O.N., Kamenova A.S., Duisembekov B.A., Glupov V.V. 2009. Synergistic Action of Entomopathogenic Hyphomycetes and the Bacteria Bacillus thuringiensis ssp. morrisoni in the Infection of Colorado Potato Beetle Leptinotarsa decemlineata // Applied Biochemistry and Microbiology. Vol.45. No. 5. P.511-516]. To identify all possible effects tool trucks tested as a combined mixture of fungus and bacteria, and both microorganisms separately. Concentration trucks amounted to 0.03%, titer B.thuringiensis - 5×10⁶crystals/ml titer .anisopliae - 2×10⁶conidia/ml Method of preparation of the mixtures, process and content of insects is similar to described above (Example 2). The experimental data presented in table 4.

Analysis of data on mortality of Colorado potato beetle showed that adding trucks reduces the latent period of mycosis, bacteriosis, as well as mixed infection. Adding trucks to the suspension of bacteria there are significant differences not only in the time of the death of the larvae, but in their final death. The latter effect can be classified as additional synergies [B who, NC 1976. Synergism of microorganisms and chemical insecticides // Mashimaro (as amended). Microorganisms in the fight against harmful insects and mites. M.: Kolos. S-123].

However, all compositions presented in example 5 showed far less effective compared to the composition of the conidia of the fungus Beauveria bassiana and trucks described in examples 2 and 3.

Thus, it was found that FSD has the property to substantially increase the mortality of Colorado potato beetle larvae as younger and older ages when their infection with entomopathogenic fungi (tables 1, 2). In a variant of the trucks at a concentration of 0.03% with a suspension of conidia of the fungus Beauveria bassiana with a titer of 3×10⁶conidia/ml (laboratory experiments) and 5×10⁶conidia/ml (field experiment) was observed not only an increase in total mortality, but the earlier death of the insect. The average half-lethal time (LT50) when processing such a combined mixture of unlike options for processing only by the fungus decreased by 2-3 days in the laboratory and on 9 days in the natural. This shortening of the death of insects has led to a decrease in the level of defoliation of potato by 50% or more.

Agents of biological control of Colorado potato beetle on the basis of entomopathogenic fungi cause specific mortality of insects, but they can't prevent defoliation of potato due to the active power and the normal growth of larvae during the course of fungal infections. Bacterial agents control act mostly on younger larvae. The older age of the larvae survive, actively feed and give the next generation beetle. As for the Colorado potato beetle is characterized by the temporal overlap of all stages of development (the presence at one time of all instars), bacterial preparations may not be highly effective against the pest. Therefore, the combination of fungal pathogens and the means of trucks offered as a synergist, leads to a synergistic effect in mortality at all ages Colorado potato beetle larvae, reducing the time of their death, to reduce the number of feed consumed, which ultimately contributes to the conservation of potato harvest.

The application of a mixture of porpoising usninovoy acids of formulas I and II

obtained by the reaction of (+)-usninovoy acid with performaing, as a synergist for insecticides based on entomopathogenic microorganisms against Colorado potato beetle larvae.