MD1072Z - Process for conservation of Pseudomonas aureofaciens strain with antifungal activity - Google Patents

Process for conservation of Pseudomonas aureofaciens strain with antifungal activity Download PDF

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MD1072Z
MD1072Z MDS20150151A MDS20150151A MD1072Z MD 1072 Z MD1072 Z MD 1072Z MD S20150151 A MDS20150151 A MD S20150151A MD S20150151 A MDS20150151 A MD S20150151A MD 1072 Z MD1072 Z MD 1072Z
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antifungal activity
strain
pseudomonas aureofaciens
cnmn
psb
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MDS20150151A
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Romanian (ro)
Russian (ru)
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Людмила БАТЫР
Валерина СЛАНИНА
Тамара СЫРБУ
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Институт Микробиологии И Биотехнологии Академии Наук Молдовы
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Priority to MDS20150151A priority Critical patent/MD1072Z/en
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Abstract

Изобретение относится к биотехнологии, а именно к способу консервирования штамма Pseudomonas aureofaciens с антифунгальной активностью.Способ, согласно изобретению, предусматривает культивирование Pseudomonas aureofaciens CNMN-PsB-07 на питательной среде в течение 48 часов при температуре 28…30°C, ресуспендирование в среде, состоящей из обезжиренного молока и 12% сахарозы с титром 10-8…10-9, быстрое замораживание при температуре -20°C, лиофилизацию и хранение при температуре + 4°C.Результат состоит в повышении антифунгальной активности штамма после хранения.The invention relates to biotechnology, and in particular to a method of preserving a strain of Pseudomonas aureofaciens with antifungal activity. The method according to the invention provides for the cultivation of Pseudomonas aureofaciens CNMN-PsB-07 on a nutrient medium for 48 hours at a temperature of 28 ... 30 ° C, resuspension in the medium, consisting of skim milk and 12% sucrose with a titer of 10-8 ... 10-9, quick freezing at -20 ° C, lyophilization and storage at + 4 ° C. The result is an increase in the antifungal activity of the strain after storage.

Description

Invenţia se referă la biotehnologie, şi anume la un procedeu de conservare a tulpinii Pseudomonas aureofaciens cu activitate antifungică, şi anume stimularea activităţii antifungice asupra diferitor tulpini de fungi fitopatogeni ca: Fusarium oxysporum, Fusarium solani, Alternaria alternata, Botrytis cinerea şi Aspergillus niger ce provoacă diferite boli la plantele de cultură. Datorită capacităţii de păstrare şi sporire a activităţii antifungice, procedeul dat poate fi utilizat la conservarea microorganismelor pe un termen îndelungat şi utilizarea acestora ca agenţi de prevenire şi protejare a plantelor de cultură de infectarea cu fitopatogeni. The invention relates to biotechnology, namely to a process for preserving the Pseudomonas aureofaciens strain with antifungal activity, namely stimulating the antifungal activity on different strains of phytopathogenic fungi such as: Fusarium oxysporum, Fusarium solani, Alternaria alternata, Botrytis cinerea and Aspergillus niger which cause various diseases in cultivated plants. Due to the ability to preserve and increase the antifungal activity, this process can be used for the long-term preservation of microorganisms and their use as agents for preventing and protecting cultivated plants from infection with phytopathogens.

Afectarea plantelor de cultură de către fitopatogeni duce la scăderea productivităţii lor, la micşorarea recoltei, precum şi la diminuarea calităţii produsului obţinut. Astfel, utilizarea microorganismelor în calitate de agenţi cu proprietăţi antimicrobiene pronunţate asupra fitopatogenilor ar permite atât majorarea productivităţii plantelor, cât şi îmbunătăţirea calităţii produsului. The damage of crop plants by phytopathogens leads to a decrease in their productivity, a decrease in the harvest, as well as a decrease in the quality of the product obtained. Thus, the use of microorganisms as agents with pronounced antimicrobial properties on phytopathogens would allow both an increase in plant productivity and an improvement in product quality.

În prezent este cunoscut un procedeu de stimulare a activităţii antifungice a tulpinii Pseudomonas aureofaciens CNMN-PsB-07 asupra fungilor fitopatogeni în urma liofilizării, utilizând ca mediu protector succinat de Na + 12% zaharoză, în rezultatul căruia, după 6 ani de păstrare, zonele de inhibiţie a patogenilor au atins valori de 21,3…28,0 mm, în dependenţă de test-cultura studiată, însă dezavantajul acestuia constă în aceea că proprietăţile antifungice sunt slabe şi mai puţin pronunţate după păstrarea îndelungată [1]. Currently, a process is known for stimulating the antifungal activity of the Pseudomonas aureofaciens strain CNMN-PsB-07 on phytopathogenic fungi following lyophilization, using Na succinate + 12% sucrose as a protective medium, as a result of which, after 6 years of storage, the pathogen inhibition zones reached values of 21.3…28.0 mm, depending on the test culture studied, but its disadvantage is that the antifungal properties are weak and less pronounced after long-term storage [1].

Problema pe care o rezolvă prezenta invenţie constă în elaborarea unui procedeu de stimulare a activităţii antifungice a tulpinii Pseudomonas aureofaciens CNMN-PsB-07, în urma liofilizării, cu utilizarea unui mediu protector ce asigură stimularea proprietăţilor antifungice asupra fitopatogenilor, după 6 ani de păstrare. Procedeul revendicat constă în conservarea tulpinii Pseudomonas aureofaciens CNMN-PsB-07 cu capacitate antifungică prin liofilizarea acesteia, utilizând mediul de protecţie lapte degresat + 12% zaharoză. Zonele de inhibiţie a creşterii fitopatogenilor, după 6 ani de la conservare, ating valori de 28,0 mm pentru Fusarium oxysporum, 27,7 - Fusarium solani, 26,3 - Alternaria alternata, 28,0 - Botrytis cinerea şi 21,3 mm - Aspergillus niger. The problem solved by the present invention consists in developing a process for stimulating the antifungal activity of the Pseudomonas aureofaciens CNMN-PsB-07 strain, following lyophilization, with the use of a protective medium that ensures the stimulation of antifungal properties on phytopathogens, after 6 years of storage. The claimed process consists in preserving the Pseudomonas aureofaciens CNMN-PsB-07 strain with antifungal capacity by lyophilization thereof, using the protective medium skimmed milk + 12% sucrose. The zones of inhibition of the growth of phytopathogens, after 6 years of storage, reach values of 28.0 mm for Fusarium oxysporum, 27.7 - Fusarium solani, 26.3 - Alternaria alternata, 28.0 - Botrytis cinerea and 21.3 mm - Aspergillus niger.

Procedeul de conservare a tulpinii Pseudomonas aureofaciens cu activitate antifungică prevede cultivarea tulpinii Pseudomonas aureofaciens CNMN-PsB-07 pe un mediu nutritiv în decurs de 48 ore la temperatura de 28…30°C, resuspendarea într-un mediu constituit din lapte degresat şi 12% de zaharoză cu un titru de 10-8…10-9, congelarea rapidă la temperatura de -20°C, liofilizarea şi păstrarea la temperatura de + 4°C. The preservation process of the Pseudomonas aureofaciens strain with antifungal activity involves cultivating the Pseudomonas aureofaciens strain CNMN-PsB-07 on a nutrient medium for 48 hours at a temperature of 28…30°C, resuspension in a medium consisting of skimmed milk and 12% sucrose with a titer of 10-8…10-9, rapid freezing at a temperature of -20°C, lyophilization and storage at a temperature of + 4°C.

Rezultatul constă în majorarea activităţii antifungice a tulpinii după păstrare. The result is an increase in the antifungal activity of the strain after storage.

Procedeul de conservare a tulpinii Pseudomonas aureofaciens CNMN-PsB-07 permite stimularea evidentă a proprietăţilor antifungice asupra fitopatogenilor Fusarium oxysporum, Fusarium solani, Alternaria alternata, Botrytis cinerea şi Aspergillus niger după 6 ani de păstrare. The preservation process of the Pseudomonas aureofaciens strain CNMN-PsB-07 allows for the evident stimulation of antifungal properties on the phytopathogens Fusarium oxysporum, Fusarium solani, Alternaria alternata, Botrytis cinerea and Aspergillus niger after 6 years of storage.

După 6 ani de cultivare tulpina Pseudomonas aureofaciens CNMN-PsB-07 atinge titrul de 10-4…10-5 log 10 UFC ml-1, astfel după fiecare pasaj care durează 24…48 ore putem creşte titrul cu 2…3 unităţi, ceea ce ne permite de a obţine o cantitate suficientă de cultură pentru a asigura securitatea acesteia. After 6 years of cultivation, the Pseudomonas aureofaciens strain CNMN-PsB-07 reaches a titer of 10-4…10-5 log 10 UFC ml-1, thus after each passage lasting 24…48 hours we can increase the titer by 2…3 units, which allows us to obtain a sufficient quantity of culture to ensure its safety.

Creşterea proprietăţilor antifungice, pot fi influenţate de diferenţa conţinutului mediului de protecţie utilizat, astfel în componenţa laptelui care este un produs proteic intră un şir de macro- şi microelemente, fermenţi şi vitamine care ulterior joacă un rol foarte important în sporirea sintezei fermenţilor şi metaboliţilor secundari cu activitate antifungică. The increase in antifungal properties can be influenced by the difference in the content of the protective medium used, thus the composition of milk, which is a protein product, includes a number of macro- and microelements, enzymes and vitamins which subsequently play a very important role in increasing the synthesis of enzymes and secondary metabolites with antifungal activity.

Rezultatul tehnic al invenţiei este condiţionat de faptul că, pentru prima dată în calitate de mediu protector pentru liofilizarea tulpinii Pseudomonas aureofaciens CNMN-PsB-07 se utilizează lapte degresat + 12% zaharoză. Proprietăţile antifungice evidente sunt asigurate datorită resuspendării tulpinii în mediul dat, care asigură un efect protector faţă de şocurile osmotice şi termice şi nu permite acestora de a influenţa negativ sinteza metaboliţilor cu activitate antifungică. The technical result of the invention is conditioned by the fact that, for the first time, skimmed milk + 12% sucrose is used as a protective medium for lyophilization of the Pseudomonas aureofaciens CNMN-PsB-07 strain. The evident antifungal properties are ensured due to the resuspension of the strain in the given medium, which provides a protective effect against osmotic and thermal shocks and does not allow them to negatively influence the synthesis of metabolites with antifungal activity.

Exemplu de realizare a invenţiei Example of embodiment of the invention

Se prepară mediul nutritiv agarizat King B cu următorul conţinut al ingredientelor: peptonă - 20,0 g; glicerină - 15,0 mL; K2HPO4 - 1,5 g; MgSO4·7H2O - 1,5 g; agar - 15,0 g; apă distilată până la 1,0 litru; pH-ul 7,0…7,2. Pe mediul preparat, în tuburi înclinate, se reînsămânţează tulpina Pseudomonas aureofaciens CNMN-PsB-07, după 48 ore de incubare la +28…30°C, cultura se resuspendă în mediul protector lapte degresat + 12% zaharoză până la titrul de 10-8…10-9, se repartizează câte 1,0…2,0 mL de suspensie în flacoane şi se congelează rapid la -20°C, după care probele se liofilizează. Probele liofilizate sunt sigilate şi plasate în frigider la temperatura de +4°C. După 6 ani de conservare în stare liofilizată tulpina se reînsămânţează pentru a determina activitatea antifungică. Prepare the King B agarized nutrient medium with the following content of ingredients: peptone - 20.0 g; glycerin - 15.0 mL; K2HPO4 - 1.5 g; MgSO4·7H2O - 1.5 g; agar - 15.0 g; distilled water up to 1.0 liter; pH 7.0…7.2. On the prepared medium, in inclined tubes, the Pseudomonas aureofaciens strain CNMN-PsB-07 is reseeded, after 48 hours of incubation at +28…30°C, the culture is resuspended in the protective medium skimmed milk + 12% sucrose up to a titer of 10-8…10-9, 1.0…2.0 mL of suspension is distributed into vials and quickly frozen at -20°C, after which the samples are lyophilized. The lyophilized samples are sealed and placed in a refrigerator at a temperature of +4°C. After 6 years of preservation in a lyophilized state, the strain is reseeded to determine antifungal activity.

Paralel se pregătesc culturile de fungi fitopatogeni care sunt inoculate pe mediul Czapek cu următoarea componenţă (g/L): zaharoză - 30,0; NaNO3 - 3,0; KH2PO4 - 1,0; MgSO4·7H2O - 0,5; KCl - 0,5; FeSO4 - 0,01; agar - 20,0; apă distilată până la 1,0 litru; pH-ul = 7,0…7,2. Suspensia de spori a fungilor fitopatogeni cu titrul 10-7…10-8 se reînsămânţează submers în plăcile Petri. In parallel, phytopathogenic fungi cultures are prepared and inoculated on Czapek medium with the following composition (g/L): sucrose - 30.0; NaNO3 - 3.0; KH2PO4 - 1.0; MgSO4·7H2O - 0.5; KCl - 0.5; FeSO4 - 0.01; agar - 20.0; distilled water up to 1.0 liter; pH = 7.0…7.2. The spore suspension of phytopathogenic fungi with a titer of 10-7…10-8 is reseeded submerged in Petri dishes.

Determinarea activităţii antifungice a tulpinii Pseudomonas aureofaciens CNMN-PsB-07 după 6 ani de la liofilizare pe mediul protector lapte degresat + 12% zaharoză a fost efectuată conform metodei de difuziune a metaboliţilor prin utilizarea discurilor de geloză. În calitate de culturi de referinţă în experimentul in vitro au fost folosite tulpinile fitopatogene de micromicete: Fusarium oxysporum, Fusarium solani, Alternaria alternata, Botrytis cinerea şi Aspergillus niger. După 4…5 zile de incubare se măsoară zonele de inhibiţie aparente în jurul discurilor de geloză. Determination of the antifungal activity of the Pseudomonas aureofaciens strain CNMN-PsB-07 after 6 years of lyophilization on the protective medium skimmed milk + 12% sucrose was performed according to the metabolite diffusion method using agarose discs. As reference cultures in the in vitro experiment, the phytopathogenic strains of micromycetes were used: Fusarium oxysporum, Fusarium solani, Alternaria alternata, Botrytis cinerea and Aspergillus niger. After 4…5 days of incubation, the apparent inhibition zones around the agar discs were measured.

Proprietăţile antifungice pronunţate depistate în urma elaborării procedeului de conservare şi stimulare a activităţii antifungice a tulpinii Pseudomonas aureofaciens CNMN-PsB-07 asupra fitopatogenilor prezintă interes din punct de vedere al extinderii arsenalului de microorganisme cu capacitate antifungică pronunţată, cu utilizarea acestora ca agenţi de protejare a plantelor de cultură. The pronounced antifungal properties detected following the development of the process for preserving and stimulating the antifungal activity of the Pseudomonas aureofaciens strain CNMN-PsB-07 on phytopathogens are of interest from the point of view of expanding the arsenal of microorganisms with pronounced antifungal capacity, with their use as crop plant protection agents.

Rezultatele studiului activităţii antifungice după 6 ani de conservare, în urma liofilizării tulpinii Pseudomonas aureofaciens CNMN-PsB-07, utilizând ca mediu protector laptele degresat + 12% zaharoză, sunt prezentate în tabel, care relevă o activitate mai sporită faţă de soluţia cea mai apropiată. Astfel, datele experimentale obţinute demonstrează că procedeul revendicat manifestă activitate antifungică mai sporită faţă de fitopatogenii Fusarium oxysporum, Fusarium solani, Alternaria alternata, Botrytis cinerea şi Aspergillus niger, care depăşeşte de până la 2,1 ori caracteristicile analoage ale procedeului descris în cea mai apropiată soluţie. The results of the study of antifungal activity after 6 years of preservation, following lyophilization of the Pseudomonas aureofaciens strain CNMN-PsB-07, using skimmed milk + 12% sucrose as a protective medium, are presented in the table, which reveals a higher activity compared to the closest solution. Thus, the experimental data obtained demonstrate that the claimed process exhibits a higher antifungal activity against the phytopathogens Fusarium oxysporum, Fusarium solani, Alternaria alternata, Botrytis cinerea and Aspergillus niger, which exceeds by up to 2.1 times the analogous characteristics of the process described in the closest solution.

Tabel Table

Activitatea antifungică a tulpinii Pseudomonas aureofaciens CNMN-PsB-07, după 6 ani de conservare în stare liofilizată conform procedeului propus în invenţie şi celei mai apropiate soluţii Antifungal activity of the Pseudomonas aureofaciens strain CNMN-PsB-07, after 6 years of preservation in a lyophilized state according to the process proposed in the invention and the closest solution

Metoda utilizată Zona de inhibiţie a fungilor fitopatogeni (mm) Fusarium oxysporum Fusarium solani Alternaria alternata Botrytis cinerea Aspergillus niger Conform celei mai apropiate soluţii Liofilizarea pe mediul succinat de Na + 12% zaharoză 13,3±0,7 15,0±1,1 24,7±0,7 23,0±0,7 19,0±1,1 Conform soluţiei propuse în invenţie Liofilizarea pe mediul lapte degresat + 12% zaharoză 28,0±2,3 27,7±1,3 26,3±1,3 28,0±1,1 21,3±1,7Method used Inhibition zone of phytopathogenic fungi (mm) Fusarium oxysporum Fusarium solani Alternaria alternata Botrytis cinerea Aspergillus niger According to the closest solution Lyophilization on Na succinate medium + 12% sucrose 13.3±0.7 15.0±1.1 24.7±0.7 23.0±0.7 19.0±1.1 According to the solution proposed in the invention Lyophilization on skim milk medium + 12% sucrose 28.0±2.3 27.7±1.3 26.3±1.3 28.0±1.1 21.3±1.7

Datele din tabel demonstrează că zonele de inhibiţie determinate conform procedeului propus în invenţie sunt mai mari şi le depăşesc de până la 2,1 ori pe cele descrise în cea mai apropiată soluţie. The data in the table demonstrate that the inhibition zones determined according to the method proposed in the invention are larger and exceed by up to 2.1 times those described in the closest solution.

Proprietăţile depistate ale tulpinii Pseudomonas aureofaciens CNMN-PsB-07, liofilizată conform procedeului revendicat, prezintă interes din punct de vedere al extinderii arsenalului de preparate cu acţiune antifungică pronunţată asupra fungilor fitopatogeni - dăunători ai plantelor de cultură. The discovered properties of the Pseudomonas aureofaciens strain CNMN-PsB-07, lyophilized according to the claimed process, are of interest from the point of view of expanding the arsenal of preparations with pronounced antifungal action on phytopathogenic fungi - pests of cultivated plants.

1. Batîr L., Slanina V., Sârbu T., Chiseliţa O. Activitatea antifungică a unor tulpini autohtone de microorganisme după 3 şi 6 ani de conservare în stare liofilizată. Revista de Proprietate Intelectuală Intelectus. nr.3, 2015, p. 96-104 1. Batîr L., Slanina V., Sârbu T., Chiseliţa O. Antifungal activity of some indigenous strains of microorganisms after 3 and 6 years of preservation in lyophilized state. Intellectual Property Journal Intelectus. no. 3, 2015, p. 96-104

Claims (1)

Procedeu de conservare a tulpinii Pseudomonas aureofaciens cu activitate antifungică, care prevede cultivarea tulpinii Pseudomonas aureofaciens CNMN-PsB-07 pe un mediu nutritiv în decurs de 48 ore la temperatura de 28…30°C, resuspendarea într-un mediu constituit din lapte degresat şi 12% de zaharoză cu un titru de 10-8…10-9, congelarea rapidă la temperatura de -20°C, liofilizarea şi păstrarea la temperatura de + 4°C.Process for preserving the Pseudomonas aureofaciens strain with antifungal activity, which provides for the cultivation of the Pseudomonas aureofaciens strain CNMN-PsB-07 on a nutrient medium within 48 hours at a temperature of 28…30°C, resuspension in a medium consisting of skimmed milk and 12% sucrose with a titer of 10-8…10-9, rapid freezing at a temperature of -20°C, lyophilization and storage at a temperature of + 4°C.
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