MD1835Z - Nutrient medium for cultivation of Trichoderma longibrachiatum CNMN-FD-27 fungal strain - Google Patents
Nutrient medium for cultivation of Trichoderma longibrachiatum CNMN-FD-27 fungal strainInfo
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Abstract
Изобретение относится к микробиологии и биотехнологии, в частности к среде культивирования штамма гриба Trichoderma longibrachiatum CNMN-FD-27, которая обладает противомикробными и фитостимулирующими свойствами и может быть использована в сельском хозяйстве для биологической защиты, а также для стимуляции сельскохозяйственных растений. .Сущность изобретения состоит в том, что предложена оптимизированная питательная среда для глубинного культивирования штамма гриба Trichoderma longibraciatum CNMN FD 27 в течение 4 суток, которая содержит (г/л): кукурузный сироп - 20,0; глюкоза - 10,0; KH2РО4 - 1,0; MgSO4x7H2O - 1,0; NaNO3 - 1,0; СаСО3- 1,0; питьевая вода до 1 литра; рН 6,0-6,2.Технический результат изобретения заключается в сокращении продолжительности культивирования штамма с получением биопрепарата с более выраженными противогрибковыми и стимулирующими свойствами по сравнению с прототипом.The invention relates to microbiology and biotechnology, in particular to a medium for culturing the fungal strain Trichoderma longibrachiatum CNMN-FD-27, which has antimicrobial and phytostimulating properties and can be used in agriculture for biological protection, as well as for stimulating agricultural plants. The essence of the invention consists in that an optimized nutrient medium is proposed for submerged cultivation of the fungal strain Trichoderma longibraciatum CNMN FD 27 for 4 days, which contains (g / l): corn syrup - 20.0; glucose - 10.0; KH2PO4 - 1.0; MgSO4x7H2O - 1.0; NaNO3 - 1.0; CaCO3- 1.0; drinking water up to 1 liter; pH 6.0-6.2. The technical result of the invention consists in reducing the duration of culturing the strain with the production of a biopreparation with more pronounced antifungal and stimulating properties compared to the prototype.
Description
Invenţia se referă la microbiologie şi biotehnologie, în particular un mediu de cultivare a tulpinii de fungi Trichoderma longibrachiatum, ce posedă proprietăţi antimicrobiene şi fitostimulatoare şi poate fi utilizată în agricultură pentru protecţia biologică şi pentru stimularea plantelor de cultură. The invention relates to microbiology and biotechnology, in particular a culture medium for the fungal strain Trichoderma longibrachiatum, which possesses antimicrobial and phytostimulatory properties and can be used in agriculture for biological protection and for stimulating crop plants.
Este cunoscut mediul nutritiv de cultivare submersă a tulpinii de fungi Trichoderema sp. MG-97, producătoare de trihodermin, care conţine, g/L: zaharoză 25,4; extract de porumb 31,8; KH2PO4 3,14; MgSO4x7H2O 0,6; apă restul [1]. Cultivarea se efectuează pe mediul solid timp de 15 zile la temperatura de 28°C, apoi pelicula micelială obţinută se usucă şi se mărunţeşte până la forma de praf. The nutrient medium for submerged cultivation of the fungus strain Trichoderema sp. MG-97, producing trichodermin, is known, which contains, g/L: sucrose 25.4; corn extract 31.8; KH2PO4 3.14; MgSO4x7H2O 0.6; water the rest [1]. Cultivation is carried out on solid medium for 15 days at a temperature of 28°C, then the mycelial film obtained is dried and ground to a powder.
Dezavantajul acestui mediu constă în conţinutul de zaharoză, preţul căreia este înalt, perioada îndelungată de cultivare a tulpinii, numărul impunător de etape pentru obţinerea preparatului şi forma acestuia (praf), care este greu de utilizat. The disadvantage of this medium is the sucrose content, the price of which is high, the long period of cultivation of the strain, the impressive number of steps to obtain the preparation and its form (powder), which is difficult to use.
Este cunoscut mediul nutritiv de cultivare submersă a tulpinii de fungi Trichoderma virens CNMN-FD-13, ce posedă proprietăţi antifungice faţă de un spectru mai larg de fitopatogeni, care conţine, g/L: sirop de glucoză 20,0; zaharoză 10,0; KH2PO4 1,0; MgSO4x7H2O 1,0; NaNO3 1,0; extract de drojdii 10,0; CaCO3 2,0; apă restul; pH 5,5-6,0, cultivarea timp de 5 zile la temperatura de 28-30°C cu agitare continuă [2]. The nutrient medium for submerged cultivation of the fungal strain Trichoderma virens CNMN-FD-13 is known, which possesses antifungal properties against a wider spectrum of phytopathogens, which contains, g/L: glucose syrup 20.0; sucrose 10.0; KH2PO4 1.0; MgSO4x7H2O 1.0; NaNO3 1.0; yeast extract 10.0; CaCO3 2.0; water the rest; pH 5.5-6.0, cultivation for 5 days at a temperature of 28-30°C with continuous stirring [2].
Dezavantajul acestui mediu nutritiv constă în componenţa acestuia ce conţine sirop de glucoză, extract de drojdii şi zaharoză, care sunt costisitoare şi durata lungă de cultivare a tulpinii. The disadvantage of this nutrient medium is its composition, which contains glucose syrup, yeast extract and sucrose, which are expensive, and the long duration of strain cultivation.
În calitate de soluţie proximă s-a utilizat mediul nutritiv pentru cultivarea tulpinii de fungi Trichoderma lignorum CNMN-FD-14, ce posedă proprietăţi antifungice şi fitostimulatoare, care conţine, g/L: melasă de sfeclă 20,0; zaharoză 10,0; KH2PO4 2,0; MgSO4x7H2O 1,0; NaNO3 2,0; CaCO3 2,0; apă restul; pH 5,5 - 6,0 [3]. As a proximate solution, the nutrient medium for cultivating the fungal strain Trichoderma lignorum CNMN-FD-14 was used, which possesses antifungal and phytostimulating properties, containing, g/L: beet molasses 20.0; sucrose 10.0; KH2PO4 2.0; MgSO4x7H2O 1.0; NaNO3 2.0; CaCO3 2.0; water the rest; pH 5.5 - 6.0 [3].
Biopreparatul obţinut la cultivarea submersă a tulpinii Trichoderma longibrachiatum CNMN-FD-27 pe mediul menţionat, la temperatura de 28-30°C, în condiţii de agitare continuă, manifestă cea mai înaltă activitate antifungică după 4 zile de cultivare faţă de Fusarium sporotrichiella şi Botrytis cinerea, diametrul zonei de inhibiţie constituind respectiv 27,0 mm şi 40,3 mm, iar faţă de Sclerotinia sclerotiorum şi Alternaria alternata după 6 zile de cultivare. De asemenea, biopreparatul stimulează creşterea plantulelor de floarea soarelui. The biopreparation obtained from the submerged cultivation of the Trichoderma longibrachiatum strain CNMN-FD-27 on the mentioned medium, at a temperature of 28-30°C, under conditions of continuous agitation, exhibits the highest antifungal activity after 4 days of cultivation against Fusarium sporotrichiella and Botrytis cinerea, the diameter of the inhibition zone being 27.0 mm and 40.3 mm respectively, and against Sclerotinia sclerotiorum and Alternaria alternata after 6 days of cultivation. The biopreparation also stimulates the growth of sunflower seedlings.
Dezavantajul soluţiei proxime constă în preţul înalt al mediului, ce conţine zaharoză, melasă, o cantitate mare de săruri minerale, dar şi durata lungă de cultivare de 4-6 zile. The disadvantage of the proximal solution is the high price of the medium, which contains sucrose, molasses, a large amount of mineral salts, but also the long cultivation duration of 4-6 days.
Problema tehnică pe care o rezolvă prezenta invenţie constă în micşorarea duratei de cultivare a tulpinii Trichoderma longibraciatum CNMN-FD-27, obţinerea unui biopreparat cu proprietăţi antifungice şi fitostimulatoare sporite, ieftin, cu componente accesibile, săruri minerale în cantităţi mici. The technical problem solved by the present invention consists in reducing the cultivation time of the Trichoderma longibraciatum CNMN-FD-27 strain, obtaining a biopreparation with enhanced antifungal and phytostimulatory properties, cheap, with accessible components, mineral salts in small quantities.
Problema se rezolvă prin mediul nutritiv pentru cultivarea tulpinii de fungi Trichoderma longibrachiatum CNMN-FD-27, care conţine, g/L: sirop de porumb 20,0, glucoză 10,0, KH2PO4 1,0, MgSO4x7H2O 1,0, NaNO3 1,0, CaCO3 1,0, apă restul, cu pH 6,0-6,2. The problem is solved by the nutrient medium for cultivating the fungal strain Trichoderma longibrachiatum CNMN-FD-27, which contains, g/L: corn syrup 20.0, glucose 10.0, KH2PO4 1.0, MgSO4x7H2O 1.0, NaNO3 1.0, CaCO3 1.0, water the rest, with pH 6.0-6.2.
Pe mediul nutritiv optimizat propus tulpina de fungi Trichoderma longibraciatum CNMN- FD-27 manifestă cea mai înaltă activitate antifungică după 4 zile de cultivare. On the proposed optimized nutrient medium, the fungal strain Trichoderma longibraciatum CNMN-FD-27 exhibits the highest antifungal activity after 4 days of cultivation.
Rezultatul tehnic al invenţiei constă în reducerea duratei de cultivare a tulpinii, obţinerea unui biopreparat cu proprietăţi antifungice şi stimulatoare mai pronunţate faţă de soluţia proximă şi reducerea preţului mediului nutritiv de 2-3 ori. The technical result of the invention consists in reducing the duration of strain cultivation, obtaining a biopreparation with more pronounced antifungal and stimulating properties compared to the proximal solution and reducing the price of the nutrient medium by 2-3 times.
Exemple de realizare a invenţiei Examples of embodiments of the invention
Exemplul 1 Example 1
Tulpina Trichoderma longibrachiatum CNMN-FD-27 s-a cultivat în baloane Erlenmeyer de 0,5 L în care s-au introdus 100 mL de mediu nutritiv, g/L: melasă de sfeclă 20,0; zaharoză 10,0; KH2PO4 2,0; MgSO4x7H2O 1,0; NaNO3 2,0; CaCO3 2,0; apă restul; pH 5,5 - 6,0 şi paralel în baloane de 0,5 L cu 100 mL de mediu nutritiv, g/L: sirop de porumb 20,0; glucoză 10,0; KH2PO4 1,0; MgSO4x7H2O 1,0; NaNO3 1,0; CaCO3 1,0; apă până la 1 litru; pH 6,0-6,2 în condiţii de agitare continuă (180 rpm) la temperatura de 28-30°C, timp de 6 zile. Biopreparatul reprezintă lichidul cultural obţinut la separarea mediului de cultivare de biomasă prin filtrare. Culturile fitopatogene au fost însămânţate în cutii Petri pe mediul malţ-agar. La mijlocul cutiei s-au făcut godeuri de 0,8 mm în care s-au introdus câte 0,1 mL de biopreparat. Difuzia biopreparatului s-a produs la temperatura de 4°C timp de 24 de ore, după care cutiile au fost amplasate în termostat, la temperatura de 28-30°C, pentru incubare. Peste 4 zile de la incubare s-au efectuat măsurările zonei de inhibiţie a fungilor fitopatogeni. The Trichoderma longibrachiatum strain CNMN-FD-27 was cultivated in 0.5 L Erlenmeyer flasks in which 100 mL of nutrient medium were introduced, g/L: beet molasses 20.0; sucrose 10.0; KH2PO4 2.0; MgSO4x7H2O 1.0; NaNO3 2.0; CaCO3 2.0; water the rest; pH 5.5 - 6.0 and in parallel in 0.5 L flasks with 100 mL of nutrient medium, g/L: corn syrup 20.0; glucose 10.0; KH2PO4 1.0; MgSO4x7H2O 1.0; NaNO3 1.0; CaCO3 1.0; water up to 1 liter; pH 6.0-6.2 under conditions of continuous stirring (180 rpm) at a temperature of 28-30°C, for 6 days. The biopreparation represents the cultural liquid obtained when separating the cultivation medium from the biomass by filtration. The phytopathogenic cultures were seeded in Petri dishes on malt-agar medium. 0.8 mm wells were made in the middle of the box into which 0.1 mL of the biopreparation was introduced. The diffusion of the biopreparation occurred at a temperature of 4°C for 24 hours, after which the boxes were placed in a thermostat, at a temperature of 28-30°C, for incubation. After 4 days of incubation, measurements of the zone of inhibition of the phytopathogenic fungi were performed.
Tabelul 1 Table 1
Activitatea antifungică a biopreparatului în dependenţă de durata de cultivare Antifungal activity of the biopreparation depending on the duration of cultivation
a tulpinii Trichoderma longibrachiatum CNMN-FD-27 of the strain Trichoderma longibrachiatum CNMN-FD-27
Test culturile Durata de cultivare, zile Diametrul zonei de inhibiţie, mm Mediul proxim Mediul optimizat 3 4 5 6 3 4 5 6 Alternaria alternata 27,3 ± 2,8 26,7 ± 1,7 25, 3 ± 0,7 28,0 ±2,3 28,3 ±1,7 36, 0 ± 2,0 25, 0 ± 3,4 18,0 ± 2,3 Sclerotinia sclerotiorum 30,7 ± 1,3 28,0 ± 2,0 30,7 ± 3,5 34,7 ± 3,5 31,0 ± 4,1 32,3 ± 2,8 31,7 ± 2,3 30,7 ± 1,3 Fusarium sporotrichiella 26,0 ± 2,0 27,0 ± 2,0 24,4 ± 2,4 22,0 ± 2,3 26,7 ± 3,3 27,7 ± 2,8 21,3 ± 1,3 18,7 ± 1,7 Botrytis cinerea 38,7 ± 1,3 40,3 ± 0,7 34,3 ± 1,3 38,3 ± 3,3 40,0 ± 1,1 41,7 ± 0,7 27,7± 2,8 25,3 ± 0,7Test cultures Cultivation duration, days Inhibition zone diameter, mm Proximal medium Optimized medium 3 4 5 6 3 4 5 6 Alternaria alternata 27.3 ± 2.8 26.7 ± 1.7 25.3 ± 0.7 28.0 ±2.3 28.3 ±1.7 36.0 ± 2.0 25.0 ± 3.4 18.0 ± 2.3 Sclerotinia sclerotiorum 30.7 ± 1.3 28.0 ± 2.0 30.7 ± 3.5 34.7 ± 3.5 31.0 ± 4.1 32.3 ± 2.8 31.7 ± 2.3 30.7 ± 1.3 Fusarium sporotrichiella 26.0 ± 2.0 27.0 ± 2.0 24.4 ± 2.4 22.0 ± 2.3 26.7 ± 3.3 27.7 ± 2.8 21.3 ± 1.3 18.7 ± 1.7 Botrytis cinerea 38.7 ± 1.3 40.3 ± 0.7 34.3 ± 1.3 38.3 ± 3.3 40.0 ± 1.1 41.7 ± 0.7 27.7 ± 2.8 25.3 ± 0.7
Maximul activităţii antifungice a biopreparatului faţă de Fusarium sporotrichiella şi Botrytis cinerea a fost obţinut pe mediul proxim după 4 zile de cultivare (diametrul zonei de inhibiţie - 27,0 mm, respectiv 40,3 mm), iar faţă de Alternaria alternata şi Sclerotinia sclerotiorum după 6 zile de cultivare (diametrul zonei - 28 mm, respectiv 34,7 mm). The maximum antifungal activity of the biopreparation against Fusarium sporotrichiella and Botrytis cinerea was obtained on the proximal medium after 4 days of cultivation (diameter of the inhibition zone - 27.0 mm, respectively 40.3 mm), and against Alternaria alternata and Sclerotinia sclerotiorum after 6 days of cultivation (diameter of the zone - 28 mm, respectively 34.7 mm).
Biopreparatul obţinut la cultivarea tulpinii Trichoderma longibrachiatum CNMN-FD-27 pe mediul propus a demonstrat activitatea maximă faţă de toţi fitopatogenii testaţi după 4 zile de cultivare (diametrul zonelor de inhibiţie variază în limitele 27,7- 41,7 mm). The biopreparation obtained from the cultivation of the Trichoderma longibrachiatum strain CNMN-FD-27 on the proposed medium demonstrated maximum activity against all phytopathogens tested after 4 days of cultivation (the diameter of the inhibition zones varies within the limits of 27.7-41.7 mm).
Astfel, durata de cultivare a tulpinii T. longibrachiatum CNMN-FD-27 pe mediul propus pentru obţinerea biopreparatului cu activitate antifungică înaltă faţă de toţi fitopatogenii testaţi este cu 2 zile mai mică faţă de activitatea biopreparatului obţinut pe mediul proxim. Thus, the cultivation duration of the T. longibrachiatum CNMN-FD-27 strain on the medium proposed for obtaining the biopreparation with high antifungal activity against all tested phytopathogens is 2 days shorter than the activity of the biopreparation obtained on the proximate medium.
Exemplul 2 Example 2
Seminţele de floarea soarelui au fost tratate cu biopreparatele obţinute (pe mediul proxim şi pe mediul propus) timp de 1 oră, apoi introduse în sol şi cultivate. După 14 zile de cultivare la temperatura camerei de 20-22°C au fost determinaţi indicii: lungimea plantulei, masa a 100 de plantule, masa rădăcinilor la 100 de plantule. Sunflower seeds were treated with the obtained biopreparations (on the proximate medium and on the proposed medium) for 1 hour, then introduced into the soil and cultivated. After 14 days of cultivation at room temperature of 20-22°C, the following indices were determined: seedling length, mass of 100 seedlings, mass of roots per 100 seedlings.
Tabelul 2 Table 2
Activitarea stimulatoare a biopreparatului la floarea soarelui în dependenţă de mediul de cultivare a tulpinii Trichoderma longibrachiatum CNMN-FD-27 Stimulating activity of the biopreparation in sunflower depending on the cultivation environment of the Trichoderma longibrachiatum strain CNMN-FD-27
Indicii corporali Tratarea seminţelor cu biopreparat proxim Tratarea seminţelor cu biopreparat optimizat Lungimea plantulei, cm 18,0± 0,3 19,3 ± 0,28 Masa a 100 de plantule, gr 124,2±4,5 148,6±3,2 Masa rădăcinilor la 100 de plantule, gr 22,4±0,4 24,8±0,45Body indices Seed treatment with proximate biopreparation Seed treatment with optimized biopreparation Seedling length, cm 18.0± 0.3 19.3 ± 0.28 Mass of 100 seedlings, gr 124.2±4.5 148.6±3.2 Root mass per 100 seedlings, gr 22.4±0.4 24.8±0.45
Masa a 100 de plantule şi masa rădăcinilor la 100 de plantule de floarea soarelui sub influenţa biopreparatului obţinut pe mediul optimizat a sporit cu 20,0% şi respectiv 10,7%, iar lungimea plantulelor cu 7,2% faţă de preparatul obţinut pe mediul proxim. The mass of 100 seedlings and the mass of roots per 100 sunflower seedlings under the influence of the biopreparation obtained on the optimized medium increased by 20.0% and 10.7%, respectively, and the length of the seedlings by 7.2% compared to the preparation obtained on the proximal medium.
1. RU 2171580 C1 2001.08.10 1. RU 2171580 C1 2001.08.10
2. MD 4249 B1 2013.08.31 2. MD 4249 B1 2013.08.31
3. MD 4251 B1 2013.09.30 3. MD 4251 B1 2013.09.30
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