MD4304C1 - Strain of Bacillus thuringiensis subsp. kurstaki bacteria - bioinsecticide for the control of Lymantria genus lepidopterans - Google Patents
Strain of Bacillus thuringiensis subsp. kurstaki bacteria - bioinsecticide for the control of Lymantria genus lepidopterans Download PDFInfo
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- MD4304C1 MD4304C1 MDA20130057A MD20130057A MD4304C1 MD 4304 C1 MD4304 C1 MD 4304C1 MD A20130057 A MDA20130057 A MD A20130057A MD 20130057 A MD20130057 A MD 20130057A MD 4304 C1 MD4304 C1 MD 4304C1
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- Moldova
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- strain
- kurstaki
- bacillus thuringiensis
- thuringiensis subsp
- lymantria
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- 241001147758 Bacillus thuringiensis serovar kurstaki Species 0.000 title claims abstract description 14
- 241000721696 Lymantria Species 0.000 title claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 241000255777 Lepidoptera Species 0.000 abstract description 16
- 230000000749 insecticidal effect Effects 0.000 abstract description 10
- 230000001580 bacterial effect Effects 0.000 abstract description 6
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- 239000008272 agar Substances 0.000 description 6
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Изобретение относится к микробиологии и биотехнологии, и может быть использовано в сельском хозяйстве для борьбы с чешуекрылыми из рода Lymantria.Штамм бактерий Bacillus thuringiensis subsp. kurstaki, изолированный из природной среды, депонирован в Национальной Коллекции Непатогенных Микроорганизмов под номером CNMN-BB-04 и может быть использован для производства биоинсектицидов.Штамм бактерий Bacillus thuringiensis subsp. kurstaki CNMN-BB-04 проявляет выраженную инсектицидную активность против чешуекрылых рода Lymantria.The invention relates to microbiology and biotechnology, and can be used in agriculture to combat Lepidoptera of the genus Lymantria. The bacterial strain Bacillus thuringiensis subsp. kurstaki, isolated from the natural environment, has been deposited with the National Collection of Non-Pathogenic Microorganisms under the number CNMN-BB-04 and can be used for the production of bio-insecticides. Bacillus thuringiensis subsp. kurstaki CNMN-BB-04 exhibits marked insecticidal activity against Lepidoptera genus Lymantria.
Description
Invenţia se referă la microbiologie şi biotehnologie şi poate fi utilizată în agricultură pentru combaterea lepidopterelor din genul Lymantria. The invention relates to microbiology and biotechnology and can be used in agriculture to combat lepidoptera of the genus Lymantria.
Mai multe soiuri de fructe seminţoase, inclusiv de mere, pere şi gutui, sunt cultivate în zonele cu climă temperată. O diversitate bogată de insecte atacă aceste tipuri de fructe în toată lumea. Lepidopterele sunt considerate dăunători des întâlniţi în multe state din Europa, inclusiv în Republica Moldova (Жигальцева М.И. Комплекс листовёрток вредителей плодоводства в Молдавии с обоснованием методов борьбы//М.И. Жигальцева: Автореф. дис. канд. с.-х. наук. Ленинград, 1959, 20 p.). Several varieties of pome fruits, including apples, pears and quinces, are grown in temperate climates. A rich diversity of insects attack these types of fruit all over the world. Lepidoptera are considered common pests in many countries in Europe, including the Republic of Moldova (М.И. Жигалцева. Комплекс листоворток врадителей плодоводства в Молдавии с прозводнием методов больше//М.И. Жигалцева: Автореф. дис. канд. с.-х . наук. Leningrad, 1959, 20 p.).
Numeroase specii de lepidoptere pot provoca daune pomilor fructiferi, cele mai susceptibile specii depind atât de tipul de pomi fructiferi, cât şi de zona geografică (David J. Carter Pest Lepidoptera of Europe: with special reference to the British Isles. Springer Science&Business media, 1984, 431 p.). Numerous species of lepidoptera can cause damage to fruit trees, the most susceptible species depend both on the type of fruit trees and on the geographical area (David J. Carter Pest Lepidoptera of Europe: with special reference to the British Isles. Springer Science&Business media, 1984 , 431 p.).
Împotriva lepidopterelor se utilizează un spectru larg de insecticide, însă aplicarea acestor preparate manifestă acţiuni negative asupra organismelor nevizate (Гулий В.В., Памужак Н.Г. Справочник по защите растений для фермеров. Кишинев, Universitas, Москва: Росагросервис, 1992, 464 p.). A wide spectrum of insecticides is used against lepidoptera, but the application of these preparations shows negative effects on non-target organisms (Гулий В.В., Памужак Н.Г. Справочник по защите растений для фермеров. Chişinău, Universitas, Moscow: Росагросервис, 1992, 464 pp.).
Elaborarea unor metode inofensive şi totodată eficiente de combatere a lepidopterelor este una dintre problemele stringente şi actuale. The development of harmless and at the same time effective methods to combat lepidoptera is one of the urgent and current problems.
Controlul microbiologic al insectelor dăunătoare a fost adesea considerat ca o alternativă favorabilă folosirii insecticidelor chimice, care posedă o acţiune nespecifică, poluează mediul ambiant şi se acumulează în aer, apă şi sol. Microbiological control of harmful insects has often been considered as a favorable alternative to the use of chemical insecticides, which have a non-specific action, pollute the environment and accumulate in air, water and soil.
Bacillus thuringiensis (Bt) este o bacterie cunoscută ca un agent important în controlul biologic. Aceasta este utilizată pe scară largă ca bioinsecticid pentru combaterea multor insecte dăunătoare şi constituie baza a peste 90% din biopesticidele disponibile în comerţ, datorită absenţei efectelor toxice asupra omului şi altor organisme homeoterme, precum şi datorită nepoluării mediului ambiant. Bacillus thuringiensis (Bt) is a bacterium known as an important agent in biological control. It is widely used as a bioinsecticide to combat many harmful insects and is the basis of more than 90% of commercially available biopesticides, due to the absence of toxic effects on humans and other homeothermic organisms, as well as due to the non-pollution of the environment.
Este cunoscută tulpina Bacillus thuringiensis subsp. kurstaki Z-52, utilizată pentru obţinerea preparatului ”Lepidocid”- insecticid biologic pentru combaterea lepidopterelor [1]. The strain Bacillus thuringiensis subsp. kurstaki Z-52, used to obtain the preparation "Lepidocide" - biological insecticide for combating lepidoptera [1].
Cea mai apropiată după esenţă şi rezultatul tehnic este tulpina de bacterii Bacillus thuringiensis subsp. kurstaki B-159/252Bt, activă împotriva dăunătorilor din ordinul Lepidoptera [2]. The closest in terms of essence and technical result is the Bacillus thuringiensis subsp. kurstaki B-159/252Bt, active against pests of the order Lepidoptera [2].
Dezavantajul acestei tulpini constă în faptul că activitatea ei insecticidă este mai puţin eficientă. Respectiv, pentru obţinerea tulpinii în cantităţi industriale se vor consuma mai mulţi reactivi pentru mediul de cultivare, implicând cheltuieli suplimentare, ca urmare sporind costul produsului. Un alt dezavantaj este cantitatea mare de spori per mililitru introdusă în mediul ambiant, pentru asigurarea efectului insecticid. The disadvantage of this strain is that its insecticidal activity is less effective. Respectively, to obtain the strain in industrial quantities, more reagents for the cultivation environment will be consumed, involving additional expenses, as a result increasing the cost of the product. Another disadvantage is the large amount of spores per milliliter introduced into the environment, to ensure the insecticidal effect.
Problema pe care o rezolvă prezenta invenţie constă în obţinerea din mediul natural a unei tulpini autohtone noi de bacterii Bacillus thuringiensis, care ar asigura producerea de proteine cu efecte insecticide şi cu o activitate mai înaltă contra lepidopterelor. The problem that the present invention solves consists in obtaining from the natural environment a new autochthonous strain of Bacillus thuringiensis bacteria, which would ensure the production of proteins with insecticidal effects and with a higher activity against lepidoptera.
Esenţa invenţiei constă în faptul că se propune o tulpină nouă de bacterii Bacillus thuringiensis subsp. kurstaki, depozitată în Colecţia Naţională de Microorganisme Nepatogene a Institutului de Microbiologie şi Biotehnologie al AŞM sub nr. CNMN BB-04 în calitate de bioinsecticid pentru combaterea lepidopterelor din genul Lymantria. The essence of the invention consists in the fact that a new strain of bacteria Bacillus thuringiensis subsp. kurstaki, stored in the National Collection of Nonpathogenic Microorganisms of the Institute of Microbiology and Biotechnology of the ASM under no. CNMN BB-04 as a bioinsecticide for the control of lepidoptera of the genus Lymantria.
Rezultatul tehnic al invenţiei constă în faptul că tulpina propusă: The technical result of the invention consists in the fact that the proposed strain:
- posedă o activitate insecticidă mai înaltă asupra lepidopterelor faţă de cea mai apropiată soluţie; - has a higher insecticidal activity on lepidoptera compared to the closest solution;
- este o tulpină autohtonă, ceea ce reduce semnificativ costurile pentru obţinerea bioinsecticidelor şi asigură micşorarea gradului de poluare a mediului ambiant. - it is an autochthonous strain, which significantly reduces the costs for obtaining bioinsecticides and ensures the reduction of environmental pollution.
Rezultatul tehnic, în special efectul insecticid obţinut, este determinat de particularităţile biochimice ale tulpinii, şi anume de capacitatea de a sintetiza δ-endotoxine. The technical result, especially the insecticidal effect obtained, is determined by the biochemical particularities of the strain, namely by the ability to synthesize δ-endotoxins.
Cultura propusă nu este patogenă pentru organismele homeoterme şi se păstrează în tuburi pe medii agarizate T3 (Travers R., Martin P., Reichelderfer C. Selective process for efficient isolation of soils Bacillus spp. Appl. Environ. Microbiol., 1987, 53 (6), p. 1263-1266), la temperatura de 4°C în Colecţia Naţională de Microorganisme Nepatogene a Institutului de Microbiologie şi Biotehnologie al AŞM. The proposed culture is not pathogenic for homeothermic organisms and is kept in tubes on T3 agar media (Travers R., Martin P., Reichelderfer C. Selective process for efficient isolation of soils Bacillus spp. Appl. Environ. Microbiol., 1987, 53 ( 6), p. 1263-1266), at a temperature of 4°C in the National Collection of Nonpathogenic Microorganisms of the Institute of Microbiology and Biotechnology of the ASM.
Tulpina a fost izolată în cultură pură din corpul insectei Phyllobius oblongus L. (Thiery I., Frachon E. Identification, isolation, culture and preservation of enthomopathogenic bacteria, 1997, p. 55-73. In: Lacey A.L. (eds) Manual of Techniques in Insect Pathology, Academic Press, London), colectate pe teritoriul Republicii Moldova. The strain was isolated in pure culture from the body of the insect Phyllobius oblongus L. (Thiery I., Frachon E. Identification, isolation, culture and preservation of enthomopathogenic bacteria, 1997, p. 55-73. In: Lacey A.L. (eds) Manual of Techniques in Insect Pathology, Academic Press, London), collected on the territory of the Republic of Moldova.
Pentru cultivarea tulpinii poate fi utilizat mediul nutritiv cu următoarea componenţă (g/l): tripton (sau Casein Trypsic Peptone) - 3; triptoză - 2; extract de drojdii - 1,5; NaH2PO4 × H2O - 6,9; MnCl2 × 4H2O - 0,008; agar - 15; pH - 6,8. For the cultivation of the strain, the nutrient medium with the following composition (g/l) can be used: tryptone (or Casein Trypsic Peptone) - 3; tryptose - 2; yeast extract - 1.5; NaH2PO4 × H2O - 6.9; MnCl2 × 4H2O - 0.008; agar - 15; pH - 6.8.
Caracterele morfo-culturale ale tulpinii: bacterii gram-pozitive cu spori. Celulele vegetative sunt mari şi mobile, dispuse câte una, în perechi sau în lanţuri. Mărimea celulelor variază de la 1,5…1,7 până la 3,6…6,2 µm. Formează şi produc incluziuni proteice cristalice de formă bipiramidală. Pe mediul NA peste 24 ore tulpina formează colonii de culoare albă-gri cu marginea filiformă şi suprafaţa mată. Morpho-cultural characters of the strain: gram-positive bacteria with spores. Vegetative cells are large and mobile, arranged one by one, in pairs or in chains. Cell size ranges from 1.5…1.7 to 3.6…6.2 µm. They form and produce bipyramidal crystalline protein inclusions. On the NA medium, after 24 hours, the strain forms white-grey colonies with a filiform edge and a matte surface.
Caracteristica fiziologo-biochimică a tulpinii Physiological-biochemical characteristic of the strain
Microorganism facultativ anaerob. Tulpina creşte în limitele de temperatură de +5...+40ºC. Temperatura optimă de creştere este de +28...+32ºC. Valoarea optimă a pH-ului pentru dezvoltarea tulpinii este de 5,2. Tulpina produce catalază, hidrolizează amidonul şi cazeina, lichefiază gelatina. Formează acetilmetilcarbinol, lecitinază şi urează. Nu descompune esculina şi salicina. Nu formează acizi şi gaze din maltoză, lactoză şi zaharoze. Asimilează glucoza, manoza cu formarea de acizi. Nu formează sulfat de hidrogen şi indol. Tulpina produce δ-endotoxine cu proprietăţi insecticide. Facultative anaerobic microorganism. The strain grows within the temperature limits of +5...+40ºC. The optimal growth temperature is +28...+32ºC. The optimum pH value for the development of the strain is 5.2. The strain produces catalase, hydrolyzes starch and casein, liquefies gelatin. It forms acetylmethylcarbinol, lecithinase and urea. It does not break down esculin and salicin. It does not form acids and gases from maltose, lactose and sucrose. It assimilates glucose, mannose with the formation of acids. It does not form hydrogen sulfate and indole. The strain produces δ-endotoxins with insecticidal properties.
Gradul de puritate al tulpinii The degree of purity of the strain
Tulpina a fost izolată în cultură pură din corpul insectei Phyllobius oblongus L. (Thiery I., Frachon E. Identification, isolation, culture and preservation of enthomopathogenic bacteria, 1997, p. 55-73. In: Lacey A.L. (eds) Manual of Techniques in Insect Pathology, Academic Press, London), colectate pe teritoriul Republicii Moldova. Pentru cultivare necesită utilizarea mediului agarizat T3. Gradul de puritate al tulpinii a fost verificat prin Reacţia de Polimerizare în Lanţ cu utilizarea primerilor specifici ITS (Internal Transcribed Spacer) 16S-23S şi amplificarea porţiunii de genă Cry1. The strain was isolated in pure culture from the body of the insect Phyllobius oblongus L. (Thiery I., Frachon E. Identification, isolation, culture and preservation of enthomopathogenic bacteria, 1997, p. 55-73. In: Lacey A.L. (eds) Manual of Techniques in Insect Pathology, Academic Press, London), collected on the territory of the Republic of Moldova. Cultivation requires the use of T3 agar medium. The degree of purity of the strain was verified by the Polymerization Chain Reaction with the use of ITS (Internal Transcribed Spacer) 16S-23S specific primers and the amplification of the Cry1 gene portion.
Exemplu de utilizare a invenţiei Example of use of the invention
Tulpina Bacillus thuringiensis subsp. kurstaki este cultivată timp de o zi pe un agitator la temperatura de 30°C pe mediul nutritiv agarizat T3 cu următoarea componenţă (g/l): tripton - 3; triptoză - 2; extract de drojdii - 1,5; NaH2PO4 × H2O - 6,9; MnCl2 × 4H2O - 0,008; agar - 15; pH - 6,8. Celulele sunt precipitate, resuspensionate în apă distilată şi incubate pe un agitator circular pentru o noapte la 30°C, timp în care celulele lizează eliberând spori şi cristale. Ulterior este determinată concentraţia de spori şi cristale de δ-endotoxine din lichidul cultural rezultat. Concentraţia de spori este determinată după numărul de colonii în creştere pe mediul agarizat T3 după însămânţarea diluţiei. Prezenţa cristalelor este monitorizată cu ajutorul microscopiei prin contrast de fază. Prezenţa δ-endotoxinelor este determinată prin reacţia de precipitare, iar conţinutul lor cantitativ - folosind ELISA (Enzyme Linked Immunosorbent Assay). The strain Bacillus thuringiensis subsp. kurstaki is cultivated for one day on a shaker at a temperature of 30°C on a T3 agar nutrient medium with the following composition (g/l): tryptone - 3; tryptose - 2; yeast extract - 1.5; NaH2PO4 × H2O - 6.9; MnCl2 × 4H2O - 0.008; agar - 15; pH - 6.8. The cells are precipitated, resuspended in distilled water and incubated on a circular shaker overnight at 30°C, during which the cells lyse releasing spores and crystals. Afterwards, the concentration of δ-endotoxin spores and crystals in the resulting culture liquid is determined. Spore concentration is determined by the number of colonies growing on T3 agar medium after dilution seeding. The presence of crystals is monitored using phase contrast microscopy. The presence of δ-endotoxins is determined by the precipitation reaction, and their quantitative content - using ELISA (Enzyme Linked Immunosorbent Assay).
Se pregăteşte suspensia de contaminare în apă distilată cu diferite concentraţii: 0,9 × 109 spori/ml; 0,9 × 108 spori/ml; 0,9 × 107 spori/ml; 0,9 × 106 spori/ml; 0,9 × 105 spori/ml. Cea mai efectivă dintre concentraţiile testate, pentru Lymantria dispar L. 1758, s-a dovedit a fi 0,9 × 108 spori/ml. The contamination suspension is prepared in distilled water with different concentrations: 0.9 × 109 spores/ml; 0.9 × 108 spores/ml; 0.9 × 107 spores/ml; 0.9 × 106 spores/ml; 0.9 × 105 spores/ml. The most effective of the concentrations tested, for Lymantria dispar L. 1758, proved to be 0.9 × 108 spores/ml.
În experienţă se utilizează dăunătorul pomilor fructiferi Lymantria dispar Linnaeus, 1758 (Lepidoptera). In the experiment, the fruit tree pest Lymantria dispar Linnaeus, 1758 (Lepidoptera) is used.
Eficacitatea tulpinii de bacterii propuse a fost evaluată prin monitorizarea efectivului numeric al dăunătorilor colectaţi după prelucrare şi cantitatea exemplarelor de insecte pierite după infestare. The effectiveness of the proposed bacteria strain was evaluated by monitoring the number of pests collected after processing and the amount of insects killed after infestation.
Exemplul 1 Example 1
Evaluarea activităţii insecticide a tulpinii Bacillus thuringiensis subsp. kurstaki a fost testată pe larvele provenite din populaţia naturală a speciei Lymantria dispar Linnaeus, 1758 (Lepidoptera, Erebidae). Se prepară o serie din cinci diluţii ale culturii bacteriene în soluţie de 0,9% NaCl cu pasul 10 (concentraţiile indicate mai sus). În calitate de martor a fost utilizată soluţia de 0,9% NaCl. În suspensiile pregătite sunt introduse frunze de măr de aproximativ aceeaşi mărime, care sunt uscate la temperatura de 20°C şi plasate în vase Petri. Pentru fiecare diluţie sunt utilizate câte 10 insecte şi 2…5 frunze de măr tratate. Mortalitatea insectelor a fost cuantificată după 3 zile de incubaţie în incubator la 21°C. Evaluation of the insecticidal activity of the strain Bacillus thuringiensis subsp. kurstaki was tested on larvae from the natural population of the species Lymantria dispar Linnaeus, 1758 (Lepidoptera, Erebidae). Prepare a series of five dilutions of the bacterial culture in 0.9% NaCl solution with step 10 (concentrations indicated above). The 0.9% NaCl solution was used as a control. Apple leaves of approximately the same size are introduced into the prepared suspensions, which are dried at a temperature of 20°C and placed in Petri dishes. For each dilution, 10 insects and 2...5 treated apple leaves are used. Insect mortality was quantified after 3 days of incubation in the incubator at 21°C.
Activitatea biologică a tulpinii, exprimată în LC50, a fost calculată după formula lui Kerber conform valorilor concentraţiilor de spori în diluţiile culturii bacteriene: lg LC50 = lgCM-Σ (ΣL - 0,5), unde CM este concentraţia maximă testată; Σ - logaritmul multiplicării diluţiei; ΣL - suma valorilor L pentru toate concentraţiile. Astfel o reducere cu 50% a efectivului dăunătorului Lymantria dispar L. poate fi obţinută la aplicarea culturii bacteriene Bacillus thuringiensis subsp. kurstaki în concentraţie de 1,392 × 105 spori/ml, în soluţie de 0,9% NaCl. Rezultatele sunt prezentate în tabel. The biological activity of the strain, expressed in LC50, was calculated according to Kerber's formula according to the values of spore concentrations in bacterial culture dilutions: lg LC50 = lgCM-Σ (ΣL - 0.5), where CM is the maximum concentration tested; Σ - logarithm of dilution multiplication; ΣL - sum of L values for all concentrations. Thus a 50% reduction in the number of Lymantria dispar L. pests can be obtained when applying the bacterial culture Bacillus thuringiensis subsp. kurstaki in a concentration of 1.392 × 105 spores/ml, in a 0.9% NaCl solution. The results are presented in the table.
Tabel Table
Nivelul pieirii dăunătorului Lymantria dispar L. după prelucrare cu suspensie The level of destruction of the pest Lymantria dispar L. after processing with suspension
din cultura bacteriană a tulpinilor de Bacillus thuringiensis subsp. kurstaki from the bacterial culture of strains of Bacillus thuringiensis subsp. kurstaki
Nr. Tulpina Titrul sporilor, mlrd spori/ml Activitatea insecticidă LC50, spori/ml 1 Bacillus thuringiensis subsp. kurstaki 0,9 1,392 × 105 2 Bacillus thuringiensis subsp. kurstaki B159/252Bt 4,03 2,377 × 105 No. Strain Spore titer, billion spores/ml Insecticidal activity LC50, spores/ml 1 Bacillus thuringiensis subsp. kurstaki 0.9 1.392 × 105 2 Bacillus thuringiensis subsp. kurstaki B159/252Bt 4.03 2.377 × 105
Datele din tabel denotă că tulpina bacteriană propusă Bacillus thuringiensis subsp. kurstaki posedă o activitate insecticidă pronunţată asupra lepidopterelor şi poate fi utilizată ca agent biologic în controlul efectivului lor numeric. Tulpina este ecologic inofensivă, deoarece a fost extrasă din mediu natural, nu este patogenă pentru plante şi organismele homeoterme. În natură nu sunt introduse organisme noi, ceea ce exclude deteriorarea ecosistemelor, cum se observă în cazul utilizării altor patogeni şi preparate. The data in the table show that the proposed bacterial strain Bacillus thuringiensis subsp. kurstaki has a pronounced insecticidal activity on lepidoptera and can be used as a biological agent to control their numbers. The strain is ecologically harmless, because it was extracted from the natural environment, it is not pathogenic for plants and homeothermic organisms. No new organisms are introduced into nature, which excludes damage to ecosystems, as is observed in the case of the use of other pathogens and preparations.
1. SU 769787 A1 1984.01.23 1. SU 769787 A1 1984.01.23
2. RU 2179392 C1 2002.02.20 2. RU 2179392 C1 2002.02.20
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Owner name: INSTITUTIA PUBLICA UNIVERSITATEA DE STAT DIN MOLDOVA, MD STR. A. MATEEVICI NR. 60, MD-2009, CHISINAU, REPUBLICA MOLDOVA Free format text: PREVIOUS NAME OF PROPRIETOR: INSTITUTUL DE ZOOLOGIE AL ACADEMIEI DE STIINTE A MOLDOVEI, MD STR. ACADEMIEI NR. 1, MD-2028, CHISINAU, REPUBLICA MOLDOVA |