RO127467A2 - Strain of brevibacillus laterosporus antagonist against phytopathogenic fungi - Google Patents
Strain of brevibacillus laterosporus antagonist against phytopathogenic fungi Download PDFInfo
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- RO127467A2 RO127467A2 ROA201001159A RO201001159A RO127467A2 RO 127467 A2 RO127467 A2 RO 127467A2 RO A201001159 A ROA201001159 A RO A201001159A RO 201001159 A RO201001159 A RO 201001159A RO 127467 A2 RO127467 A2 RO 127467A2
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- brevibacillus laterosporus
- laterosporus
- phytopathogenic fungi
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Abstract
Description
TULPINA DE BREVIBACILUS LATEROSPORUS ANTAGONISTATULPINA DE BREVIBACILUS LATEROSPORUS ANTAGONIST
FATA DE CIUPERCI FITOPATOGENEGIRL OF PHYTOPATHOGEN MUSHROOMS
Prezenta invenție se referă la o tulpină nouă de Brevibacilus laterosporus (număr depozit DSM 23663), care prezintă, concomitent, antagonism față de ciupercile fitopatogene din sol și capacitate ridicată de colonizare a materialului vegetal și a țesuturilor plantelor, și care este destinată utilizării în exploatațiile agricole conservative, inclusiv în cele care utilizează muici format din culturi verzi de protecție.The present invention relates to a novel strain of Brevibacilus laterosporus (deposit number DSM 23663), which concurrently exhibits antagonism to phytopathogenic fungi in the soil and high ability to colonize plant material and plant tissues, and is intended for use in farms. agricultural conservatives, including those that use mosses made from green crops.
Sunt cunoscute o serie întreagă de tulpini de bacterii gram-pozitive sporulate, aparținând speciei Brevibacillus laterosporus și destinate utilizării în practică. Brevetul WO 2008/03887 descrie o tulpină de Brevibacillus laterosporus, UNISS18, nepatogenă pentru om, depozitată cu numărul 41419 la NCIMB, care este prezintă activitate insecticidă pentru dipterele care sunt vectori ai unor boli umane (Musca domestica, Culex quinquefasciatus, Anopheles maculipennis, Aedes aegypti). Tulpina este destinată combaterii biologice a acestor diptere prin aplicare în diferite tipuri de formulări. Brevetul SUA 5055293 se referă la tulpina P5 de Brevibacillus laterosporus (nr de depozit ATCC 53694), care este patogenă pentru de o serie întreagă de lepidoptere dăunătoare culturilor agricole, și în special față viermele vestic al porumbului (Diabrotica virgifera virgifera). Tulpina P5 se aplică ca tratament la sol sau al seminței pentru limitarea populațiilor lepidopterelor dăunătoare față de care este activă.A whole series of strains of sporulated gram-positive bacteria, belonging to the Brevibacillus laterosporus species, are intended for use in practice. Patent WO 2008/03887 describes a strain of Brevibacillus laterosporus, UNISS18, non-pathogenic to humans, filed with number 41419 at NCIMB, which is insecticidal in activity for dipterans that are vectors of human diseases (Musca domestica, Culex quinquefasciatus, Anopheles macul aegypti). The strain is intended for biological control of these diphtheres by application in different types of formulations. US Patent 5055293 refers to strain P5 of Brevibacillus laterosporus (deposit number ATCC 53694), which is pathogenic to a whole series of lepidopterans harmful to agricultural crops, and in particular to western corn worm (Diabrotica virgifera virgifera). P5 strain is applied as a soil or seed treatment for limiting populations of harmful lepidopterans to which it is active.
Nu s-au descris până în prezent tulpini de Brevibacillus laterosporus care să aibă, concomitent, activitate de antagonism față de agenții fitopatogeni din sol și capacitate de colonizare a materialului vegetal și a țesuturilor plantelor. Astfel de tulpini sunt necesare sistemelor de agricultură conservativă. In cadrul acestor sisteme lucrările mecanice ale solului sunt reduse, iar solul este acoperit cu cel puțin 30% resturi vegetale. Sistemele de agricultură conservativă reduc eroziunea solului, și poluarea apelor, de suprafață și din pânza freatică, dar au dezavantajul că mențin solul rece, reducând germinația și dezvoltarea plantelor cultivate, și favorizând dezvoltarea agenților de dăunare, în special ciuperci fitopatogene din sol cu spectru larg de acțiune.So far, no Brevibacillus laterosporus strains have been described that have, at the same time, antagonistic activity against soil phytopathogenic agents and ability to colonize plant material and plant tissues. Such strains are needed for conservative farming systems. In these systems, the mechanical works of the soil are reduced and the soil is covered with at least 30% plant debris. Conservative agriculture systems reduce soil erosion, and water pollution, surface and groundwater, but they have the disadvantage of keeping soil cold, reducing germination and development of cultivated plants, and favoring the development of harmful agents, especially phytopathogenic fungi from broad-spectrum soil. action.
In cadrul acestor sisteme de agricultură conservativă tulpinile de Brevibacillus laterosporus, care prezintă atât activitate de antagonism față de agenții fitopatogeni din sol cât și capacitate de colonizare a materialului vegetal și a țesuturilor plantelor, ar contribui la înlăturarea dezavantajului menționat mai sus, pentru că ar inhiba agenți fitopatogeni din sol, ar proteja plantele de cultură pe care le colonizează și ar asigura un management durabil al resturilor vegetale.Within these systems of conservative agriculture, the strains of Brevibacillus laterosporus, which exhibit both antagonistic activity against soil phytopathogenic agents and ability to colonize plant material and plant tissues, would help to remove the aforementioned disadvantage, because it would inhibit phytopathogenic agents from the soil, would protect the crop plants they colonize and ensure a sustainable management of plant debris.
ΪΟΚίαΰΓ5?3ΤΑΤ PE&TRU INVENȚII Șl MĂRCI î Cerere de brevet de invențieΪΟΚίαΰΓ5? 3ΤΑΤ PE & TRU INVENTIONS Ml MARK Î Patent application
C^-2 Ο 1 Ο - Ο 1 1 59 - 2 k -11- 2910C ^ -2 Ο 1 Ο - Ο 1 1 59 - 2 k -11- 2910
Tulpina Brevibacillus laterosporus 56.1s (număr de depozit DSM 23654, DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germania) prezintă acțiune antifungică față de ciupercile fitopatogene din sol și capacitate de colonizare a materialului vegetal și a țesuturilor plantelor.Brevibacillus laterosporus 56.1s strain (deposit number DSM 23654, DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) exhibits antifungal action against phytopathogenic fungi in soil and ability to colonize plant material and tissues.
Tulpina Brevibacillus laterosporus 56.1s prezintă următoarele avantaje:The Brevibacillus laterosporus 56.1s strain has the following advantages:
- creștere bogată pe mediile uzuale utilizate pentru creșterea bacililor gram pozitivi sporulați;- rich growth on the usual media used to grow sporulated gram positive bacilli;
- formarea de spori cu viabilitate îndelungată;- formation of spores with long-term viability;
- capacitate de a modula formarea consorțiilor microbiene de consens cu alte bacterii utilizate în biopreparate mixte datorită activității lactonazice prin care influențează semnalele AHL implicate în realizarea sensibilității de grup;- ability to modulate the formation of consensus microbial consortia with other bacteria used in mixed biopreparations due to the lactonase activity by which they influence the AHL signals involved in achieving group sensitivity;
- acțiune împotriva atacului principalelor ciupercilor fitopatogene de sol;- action against the attack of the main phytopathogenic soil fungi;
- mobilitate ridicată, capacitate de a coloniza material vegetal și de a forma biofilme;- high mobility, ability to colonize plant material and form biofilms;
- capacitate de colonizare a țesuturilor plantelor în primele faze de vegetație și de protejare împotriva atacului unor agenți fitopatogeni.- ability to colonize plant tissues in the early stages of vegetation and to protect against the attack of some phytopathogenic agents.
Prezenta invenție se ilustrează cu exemplul prezent mai jos.The present invention is illustrated by the example below.
Exemplu. Tulpina 56.1s de Brevibacillus laterosporus a fost obținută la Institutul de Cercetare-Dezvoltare pentru Protecția Plantelor, București, dintr-o proba de sol provenit din Bărăganul de sud. Pentru izolarea bacteriei s-a utilizat agar nutritiv (peptona - 5g/l, extract de carne - 3g/l, agar - 20g/l, la 1000 ml apa; pH 6.8-7.2), iar pentru cultivare s-a utilizat mediul Luria-Bertani agarizat (LBA: bactotriptona - 10g/I, extract de drojdie - 5g/l, NaCI - 10g/I, agar - 20g/l, la 1000 ml apa; pH 7.5.), la o temperatura optima de incubare de 28°C.Example. The strain 56.1s of Brevibacillus laterosporus was obtained at the Research and Development Institute for Plant Protection, Bucharest, from a soil sample from the southern Bărăganul. Nutrient agar (peptone - 5g / l, meat extract - 3g / l, agar - 20g / l, 1000 ml water; pH 6.8-7.2) was used for isolation of the bacterium, and agarized Luria-Bertani medium was used ( LBA: Bactotriptone - 10g / I, yeast extract - 5g / l, NaCl - 10g / I, agar - 20g / l, 1000 ml water; pH 7.5.), At an optimal incubation temperature of 28 ° C.
Această tulpină a fost selectată dintr-o colecție de peste 50 izolate de bacili sporulanți gram-pozitivi, pe baza acțiunii antagoniste față de ciupercile fitopatogene de sol (Fusarium graminearum, Alternaria spp, Sclerotinia sclerotiorum, Rhizoctonia solani, Fusarium oxisporum f. sp. radicis-lycopersici, Sclerotium bataticola), producerii de amilază și lactonază AHL, mobilității de migrare / swimming și agregare / swarming, formării de biofilme in vitro, capacității de colonizare a materialului vegetal, activității de protecție a plantulelor de porumb față de atacul de Fusarium graminearum.This strain was selected from a collection of over 50 isolates of gram-positive sporadic bacilli, based on antagonistic action against phytopathogenic soil fungi (Fusarium graminearum, Alternaria spp, Sclerotinia sclerotiorum, Rhizoctonia solani, Fusarium spisporic. -lycopersici, Sclerotium bataticola), AHL amylase and lactonase production, migration / swimming and aggregation / swarming mobility, in vitro biofilm formation, the ability to colonize plant material, the protection of corn seedlings against the attack of Fusarium graminearum .
In vederea încadrării taxonomice, tulpina 56.1 s a fost caracterizată pe baza unei taxonomii polifazice, respectiv a combinării caracterelor morfologice (tabel 1), cu cele fiziologice (tabel 2) și cu cele moleculare (secvența 16S rADN, tab.3, profilului de acizi grași din lipidele membranare, tab. 4). Coroborând toate acesteFor taxonomic classification, strain 56.1 was characterized on the basis of a multiphase taxonomy, respectively of the combination of morphological characters (table 1), with the physiological ones (table 2) and with the molecular ones (16S rDNA sequence, table 3, fatty acid profile). from the membrane lipids, tab. 4). Corroborating all these
CC 2 Π 1 Ο - Ο 1. ί 5 9 - 2 ί| -Ί· 2010 date, tulpina 56.1s a fost încadrată ca aparținând speciei Brevibacillus laterosporus.CC 2 Π 1 Ο - Ο 1. ί 5 9 - 2 ί | -Ί · 2010 data, strain 56.1s was classified as belonging to the species Brevibacillus laterosporus.
Tab. 1. Morfologia coloniilor de Brevibacillus laterosporus 56.1s pe diferite medii după cultivare timp de 24 ore.Tab. 1. Morphology of Brevibacillus laterosporus 56.1s colonies on different media after cultivation for 24 hours.
Tab. 2. Caracteristicile fiziologice ale tulpinii 56.1s.Tab. 2. Physiological characteristics of strain 56.1s.
^2010-01159-2 4 -11- 2010^ 2010-01159-2 4-11-11
Identificarea pe baza secvenței 16S rADN s-a realizat prin aplicarea unui protocol de lucru caracterizat prin următoarele etape: obținerea de culturi pure colonii izolate, tehnica însămânțării prin epuizarea ansei; extracția ADN-ului bacterian; electroforeză în gel pentru detectarea ADN-ului; amplificarea secvenței 16S rADN prin tehnica PCR și electroforeză în gel; purificarea ADN-ului ribozomal; amplificare enzimatică a acizilor nucleici înainte de secvențializare; precipitarea și uscarea ADN-ului codificând pentru 16sRNA. Secvențierea nucleotidică a fost realizată cu metoda Dye Terminator Cycle Sequencing (Perkin Elmer, 1998), folosind un secvențiator automat de tip ABI PRISM 310 (Perkin Elmer). Secvențele au fost analizate folosind programul CHROMAS 2.33 (Technelysium Pty Ltd). Compararea secvențelor 16S rADN obținute cu secvențele existente în Banca de gene NCBI (Național Center for Biotechnology Information), s-a realizat cu ajutorul programului BLAST (Basic Local Alignment Search Tool). Rezultatele sunt prezentate în tab. 3, ele dovedind o similaritate de 99,8% cu alte tulpini de Brevibacillus laterosporus.Identification based on the 16S rDNA sequence was performed by applying a working protocol characterized by the following stages: obtaining pure cultures isolated colonies, the technique of seeding by exhausting the goose; extraction of bacterial DNA; gel electrophoresis for DNA detection; amplification of the 16S rDNA sequence by PCR and gel electrophoresis; purification of ribosomal DNA; enzymatic amplification of nucleic acids prior to sequencing; precipitation and drying of DNA encoding for 16sRNA. Nucleotide sequencing was performed using the Dye Terminator Cycle Sequencing method (Perkin Elmer, 1998), using an ABI PRISM 310 automatic sequencer (Perkin Elmer). The sequences were analyzed using the software CHROMAS 2.33 (Technelysium Pty Ltd). Comparison of the 16S rDNA sequences obtained with the existing sequences in the NCBI (National Center for Biotechnology Information) gene bank was performed using the BLAST (Basic Local Alignment Search Tool) program. The results are presented in the tab. 3, they show a similarity of 99.8% with other strains of Brevibacillus laterosporus.
Tab. 3. Identificarea tulpinii pe baza similarității secvenței 16 S rADN cu tulpinile din GenBank.Tab. 3. Identification of the strain based on the similarity of the 16 S rDNA sequence with GenBank strains.
1 - (perechi de baze - pb); 2 - pe baza similarității secvenței 16S rADN cu tulpinile din GenBank (nr. de referință - procentul de similaritate 1 - (base pairs - pb); 2 - based on the similarity of the 16S rDNA sequence with GenBank strains (reference no. - similarity percentage
Profilul acizilor grași membranari a fost determinat cu ajutorul unui sistem Sherlock® Microbial ID, prin folosirea procedurii Instant FAME™. Esterii acizilor grași din membrana bacteriană au fost separați pe un cromatograf de gaze GC 4The profile of membrane fatty acids was determined using a Sherlock® Microbial ID system, using the Instant FAME ™ procedure. Fatty acid esters from the bacterial membrane were separated on a GC 4 gas chromatograph
t\- 2 Ο 1 0 - Ο 1 1 5 9 - 2h -II- 2010t \ - 2 Ο 1 0 - Ο 1 1 5 9 - 2h -II- 2010
6890Ν (Agilent Technologies) echipat cu injector split/splitless clasic, coloana capilara tip Ultra 2 (cod Agilent 19091B-102, având lungimea de 25m, diametrul interior de 0,2mm, faza staționara 5% feriil metil siloxan, grosimea fazei staționare de 0,33 pm) și detector de ionizare în flacără (FID). Soiturile utilizate pentru achiziția si prelucrarea automata a datelor cromatografice au fost: GC ChemStation, versiunea B.01.03 [204] / 2005, și Sherlock Microbial Identification System, versiunea 6.1 / 2008. S-au folosit metode cromatografice dezvoltate si validate de producător (MIDI, Inc.), utilizând bibliotecile de profile de esteri metilici ai acizilor grași pentru microorganisme aerobe din mediu (RTSBA6, versiunea 6.0 / 2008). Ca material de referință s-a folosit un amestec etalon de esteri metilici ai acizilor grași cu catenă lineară cu număr de atomi de carbon in moleculă cuprins între 9 și 20 (etalonare cantitativă pentru metodele cromatografice Sherlock rapide și sensibile). Microorganismele de referință pentru verificarea protocoalelor microbiologice și chimice de procesare a probelor au fost Bacillus subtilis (tulpina ATCC 6633) și Stenotrophomonas maltophilia (tulpina ATCC 13637).6890Ν (Agilent Technologies) equipped with classic split / splitless injector, Ultra 2 capillary column (Agilent code 19091B-102, 25m long, 0.2mm inner diameter, stationary phase 5% ferriyl methyl siloxane, stationary phase thickness 0 , 33 pm) and flame ionization detector (FID). The varieties used for the acquisition and automatic processing of the chromatographic data were: GC ChemStation, version B.01.03 [204] / 2005, and Sherlock Microbial Identification System, version 6.1 / 2008. Chromatographic methods developed and validated by the manufacturer (MIDI) were used. , Inc.), using the fatty acid methyl ester profile libraries for aerobic microorganisms in the environment (RTSBA6, version 6.0 / 2008). As a reference material, a standard mixture of linear chain fatty acid methyl esters with a number of carbon atoms in the molecule between 9 and 20 was used (quantitative calibration for fast and sensitive Sherlock chromatographic methods). The reference microorganisms for the verification of microbiological and chemical protocols for sample processing were Bacillus subtilis (strain ATCC 6633) and Stenotrophomonas maltophilia (strain ATCC 13637).
Tab. 4. Profilul acizilor grași din membrana tulpinii 56.1s.Tab. 4. The profile of fatty acids in the membrane of strain 56.1s.
Activitatea antagonistă a tulpinii 56.1s față de diferite ciuperci fitopatogene, cum ar fi: Fusarium graminearum, Fusarium oxisporum radicis-lycopersici, Sclerotinia sclerotiorum, Sclerotium bataticola si Alternaria spp. s-a analizat in vitro prin utilizarea tehnicii culturilor duble sau prin înțeparea mediului cu biomasă bacteriană la maxim 2 cm de o rondea calibrată (5 mm) de miceliu. Culturile de ciuperci fitopatogene au fost împrospătate pe mediu CGA (cartof, dextroza, agar) și incubate la 28°C timp de 5 zile. Tulpina 56.1s a fost împrospătată pe mediu Luria-Bertani (LB) agarizat prin incubare la 28°C timp de 24 ore. Testarea activității antagoniste in vitro a fost efectuată pe mediul cu cartof glucoza agarThe antagonistic activity of strain 56.1s against different phytopathogenic fungi, such as: Fusarium graminearum, Fusarium oxisporum radicis-lycopersici, Sclerotinia sclerotiorum, Sclerotium bataticola and Alternaria spp. Was analyzed in vitro by using the double culture technique or by stinging the bacteria within 2 cm of a calibrated round (5 mm) of mycelium. Cultures of phytopathogenic fungi were refreshed on CGA medium (potato, dextrose, agar) and incubated at 28 ° C for 5 days. The 56.1s strain was refreshed on Luria-Bertani medium (LB) agarized by incubation at 28 ° C for 24 hours. In vitro antagonistic activity testing was performed on potato glucose agar medium
V ^-2 0 1 0 - 0 1 1 5 9 -2 4 -11- 20W (CGA). Plăcile Petri însămânțate cu microorganismele de testat au fost incubate la 28°C și analizate în ceea ce privește zona de inhibiție (mm) produsa de B.V ^ -2 0 1 0 - 0 1 1 5 9 -2 4 -11-20W (CGA). Petri dishes seeded with microorganisms to be tested were incubated at 28 ° C and analyzed for the inhibition zone (mm) produced by B.
laterosporus 56.1s, la 24, 48 și 72 ore. Experiența a fost repetată de trei ori.laterosporus 56.1s, at 24, 48 and 72 hours. The experience was repeated three times.
Tab. 5. Testarea in vitro a activității antagoniste a tulpinii de Brevibacillus laterosporus 56.1s asupra creșterii miceliene a unor ciuperci fitopatogene (zona de inhibiție la 72 h, mm).Tab. 5. In vitro testing of the antagonistic activity of Brevibacillus laterosporus 56.1s strain on mycelial growth of phytopathogenic fungi (inhibition zone at 72 h, mm).
Rezultatele au demonstrat ca tulpina 56.1s produce metaboliți antifungici care au inhibat dezvoltarea ciupercilor luate in studiu. Cea mai mare zonă de inhibiție s-a înregistrat față de ciuperca S. sclerotiorum (8 mm) aceasta fiind urmata de Fusarium graminearum, F. oxisporum f. sp. radicis-lycopersici si Alternaria spp. (5 mm).The results showed that strain 56.1s produced antifungal metabolites that inhibited the development of the fungi studied. The largest zone of inhibition was recorded against the fungus S. sclerotiorum (8 mm), followed by Fusarium graminearum, F. oxisporum f. Sp. radicis-lycopersici and Alternaria spp. (5 mm).
Tulpina 56.1s a fost analizată în ceea ce privește producerea de enzime care au un rol important în colonizarea materialului vegetal, în activitatea antagonistă și în modularea consorțiilor de consens formate de diferite tipuri de microorganisme.Stem 56.1s was analyzed in terms of the production of enzymes that play an important role in the colonization of plant material, in antagonistic activity and in the modulation of consensus consortia formed by different types of microorganisms.
Producerea de amilază s-a analizat prin însămânțarea sub forma de striu pe mediul nutrient agar (NA) + 0,4% amidon solubil. Plăcile au fost incubate la 28°C timp de 48 -72 ore, după care s-au tratat cu soluție de iod în iodură de potasiu prin inundare. Zonele clare din jurul creșterii bacteriene, după adăugarea soluției de iod, au indicat descompunerea amidonului din mediu și deci, producerea de amilază. Rezultatele au reflectat faptul ca tulpina 56.1s produce amilaza.The production of amylase was analyzed by sowing in the form of a strip on the nutrient medium agar (NA) + 0.4% soluble starch. The plates were incubated at 28 ° C for 48-72 hours, after which they were treated with iodine solution in potassium iodide by flood. Clear areas around bacterial growth, after the addition of iodine solution, indicated the breakdown of starch in the environment and thus the production of amylase. The results reflected that strain 56.1s produces amylase.
Producerea de lactonază (enzima implicată în quorum quencing, prin care se împiedică comunicarea dintre bacteriile dăunătoare care folosesc sistemul de quorum sensing de tip AHL) s-a analizat prin inocularea tulpinii 56.1s în 2 ml mediu lichid Luria Bertani în care s-a adăugat C6-hexanoil homoserin lactonă (C6-HHL) în concentrație finală de 5 μΜ urmata de incubarea peste noapte la 28°C și 150 rpm. Simultan, același mediu numai cu C6-HHL a fost utilizat ca martor negativ pentru a vedea daca mediul induce lactoliza. Testul a fost efectuat pe plăci Petri cu mediul LBA (Luria Bertani cu agar) suplimentat cu 50 pg/mlThe production of lactonase (the enzyme involved in quorum quencing, which prevents the communication between harmful bacteria using the AHL quorum sensing system) was analyzed by inoculating strain 56.1s in 2 ml Luria Bertani liquid medium to which C6-hexanoyl homoserin was added. lactone (C6-HHL) in final concentration of 5 μΜ followed by overnight incubation at 28 ° C and 150 rpm. Simultaneously, the same medium only with C6-HHL was used as a negative control to see if the environment induces lactolysis. The test was performed on Petri dishes with LBA medium (Luria Bertani with agar) supplemented with 50 pg / ml
Ofil O 1 o - o 115 9 - - ή -li- 2010 kanamicină și inoculat în plaja cu tulpina biosenzor de Chromobacterium violaceum CV026 prin distribuția sub formă de plaja unei cantități de 250 μΙ dintro cultură de 12 ore. Pe suprafața plăcii astfel inoculate au fost efectuate godeuri cu diametrul de 5 mm în care s-au distribuit 100 μΙ din supernatantul culturii 56.1s. Petriurile au fost incubate peste noapte la 28°C si apoi analizate pentru prezența halourilor violete. Absenta halourilor violete a indicat faptul ca tot C6-HHL din mediul de creștere a fost degradat. Rezultatele au evidențiat faptul că tulpina 56.1s a produs lactonază.Ofil O 1 o - o 115 9 - - ή -li- 2010 kanamycin and inoculated into the beach with the Chromobacterium violaceum CV026 biosensor strain through the distribution of 250 μΙ of a 12-hour culture within the beach. On the surface of the plate thus inoculated were made wells with a diameter of 5 mm in which 100 μΙ of the culture supernatant 56.1s were distributed. The petrioles were incubated overnight at 28 ° C and then analyzed for the presence of purple halos. The absence of the purple halos indicated that all C6-HHL in the growth medium was degraded. The results revealed that strain 56.1s produced lactonase.
Tulpina 56.1s a fost testata în ceea ce privește mobilitatea de migrare la suprafața agarului (swimming) și de agregare (swarming). Ca martor a fost utilizată o tulpina martor, modificată genetic pentru a fi imobilă, P. putida PCL1760 (Validov, 2007). Pentru aceasta, tulpinile au fost împrospătate pe mediul LB suplimentat cu 1,8% agar si crescute peste noapte la 28°C. Petriuri cu 25 ml mediu LB suplimentat cu 0,3% agar, pentru swimming si cu 0,5% agar pentru swarming au fost preparate și lăsate să se usuce pentru 20-30 minute în hota cu flux laminar înainte de utilizare. Tulpinile s-au inoculat prin înțepare în centrul mediului cu betișoare sterile din lemn. După 18 ore incubare la 28°C plăcile s-au analizat în ceea ce privește mobilitatea. Rezultatele au evidențiat faptul că tulpina 56.1s a prezentat ambele tipuri de mobilitate.Stem 56.1s was tested in terms of mobility of migration on agar surface (swimming) and aggregation (swarming). A control strain, genetically modified to be immobile, P. putida PCL1760 was used as a control (Validov, 2007). For this, the strains were refreshed on LB medium supplemented with 1.8% agar and grown overnight at 28 ° C. Petri dishes with 25 ml LB medium supplemented with 0.3% agar for swimming and 0.5% agar for swarming were prepared and allowed to dry for 20-30 minutes in the laminar flow hood before use. The stems were inoculated by stinging in the middle of the environment with sterile wooden seedlings. After 18 hours incubation at 28 ° C the plates were analyzed for mobility. The results showed that strain 56.1s exhibited both types of mobility.
Tulpina 56.1s a fost testate in vitro in ceea ce privește formarea biofilmului prin cultivarea in mediul CM (Fall et al., 2004) si mediul M63 (O'Toole et al., 1998). împreuna cu tulpina 56.1s au fost testate si doua tulpini martor, Pseudomonas fluorescens WCS 365 (Simons et al. 1996) considerata martor pozitiv si Bacillus subtilis B168 (Burkholder et al., 1947) cunoscuta ca fiind slaba producătoare de biofilm.Stem 56.1s was tested in vitro for biofilm formation by cultivation in CM (Fall et al., 2004) and M63 (O'Toole et al., 1998). Along with strain 56.1s, two control strains, Pseudomonas fluorescens WCS 365 (Simons et al. 1996) considered positive control and Bacillus subtilis B168 (Burkholder et al., 1947) known as biofilm producers, were tested.
Testul a constat în împrospătarea celulelor pe mediul LB prin incubare la 28°C timp de 18 ore, după care 10 μΙ au fost utilizați pentru inocularea a 0,5 ml mediu CM si respectiv M63, distribuit in tuburi Eppendorf din polipropilenă. Tuburile astfel inoculate au fost incubate peste noapte la 37°C fără agitare. In scopul analizei fenotipice a biofilmului, după eliminarea mediului din tuburi si clătirea cu apa distilata sterila, celulele aderente au fost colorate cu 1% w/v cristal violet timp de 10-15 minute.The assay consisted of refreshing the cells on the LB medium by incubation at 28 ° C for 18 hours, after which 10 μΙ were used to inoculate 0.5 ml of CM medium and M63 respectively, distributed in Eppendorf polypropylene tubes. The tubes thus inoculated were incubated overnight at 37 ° C without stirring. For the purpose of phenotypic analysis of the biofilm, after removing the medium from the tubes and rinsing with sterile distilled water, the adherent cells were stained with 1% w / v violet crystal for 10-15 minutes.
Formarea biofilmului, a fost cuantificată prin adiționarea a 2x 200 μΙ etanol 95% și spălarea celulelor aderente colorate cu cristal violet. Etanolul a fost apoi transferat intr-un tub Eppendorf de 1,5 ml si s-a ajustat volumul la 1 ml cu apa distilata sterila. Absorbanta s-a analizat la 540 nm cu ajutorul spectrofotometrului.Biofilm formation was quantified by adding 2x 200 μΙ 95% ethanol and washing the adherent cells stained with purple crystal. The ethanol was then transferred to a 1.5 ml Eppendorf tube and the volume adjusted to 1 ml with sterile distilled water. The absorbance was analyzed at 540 nm using the spectrophotometer.
(1-2 Ο 1 Ο - Ο 1 1 59 - 2 h -11- 2010(1-2 Ο 1 Ο - Ο 1 1 59 - 2 h -11- 2010
Rezultatele, prezentate in tabelul 6, reflecta faptul ca tulpina 56.1s a format biofilm pe cele doua tipuri de medii testate. Pe ambele medii de creștere, analiza cantitativa a biofilmului a evidențiat diferența nesemnificativa dintre tulpina WCS365 si tulpina 56.1s. Pe mediul de creștere CM s-a observat o mai buna dezvoltare a biofilmului, pentru toate tulpinile studiate, comparative cu mediul M63.The results, presented in Table 6, reflect that strain 56.1s formed biofilm on the two types of media tested. On both growth media, the quantitative analysis of the biofilm revealed the insignificant difference between strain WCS365 and strain 56.1s. On the CM growth medium, a better biofilm development was observed, for all the strains studied, compared to the M63 environment.
Tab. 6. Cuantificarea biofilmului după 24 ore de creștere in condiții statice la 37°C a tulpinii 56.1s, pe mediile CM si M63.Tab. 6. Quantification of biofilm after 24 hours of growth under static conditions at 37 ° C of strain 56.1s, on CM and M63 media.
Formarea biofilmului este o caracteristica importanta a microorganismelor benefice, acest mecanism fiind direct implicat în protecția plantelor prin colonizarea substratului de creștere și competiția pentru nișa.Biofilm formation is an important feature of beneficial microorganisms, this mechanism being directly involved in plant protection by colonizing the growth substrate and competing for the niche.
Tulpina 56.1s a fost analizata in ceea ce privește inocuitatea pe larve de Galleria mellonella in conformitate cu protocolul descris de Seed et al. in 2008. Pentru aceasta, tulpina 56.1s de Brevibacillus laterosporus a fost cultivata pe mediul LB la 28°C cu agitare la 150 rpm timp de 48 ore, după care a fost centrifugata la 4000 rpm iar sedimentul a fost resuspendat in 10mM MgSO4 suplimentat cu 1,2 mg/ml ampicilina. Concentrația inocului utilizat pentru tratarea larvelor a fost masurata cu spectrofotometrul la OD 600 (cunoscându-se ca OD600=1 reprezintă 108 ufc/ml) si este indicata in tabelul 7.Stem 56.1 was analyzed for the safety of larvae of Galleria mellonella in accordance with the protocol described by Seed et al. In 2008. For this, strain 56.1s of Brevibacillus laterosporus was grown on LB medium at 28 ° C with stirring at 150 rpm for 48 hours, then centrifuged at 4000 rpm and the sediment was resuspended in 10mM MgSO 4 supplemented. with 1.2 mg / ml ampicillin. The concentration of the inoculum used to treat the larvae was measured with the spectrophotometer at OD 600 (knowing that OD600 = 1 represents 10 8 cfu / ml) and is shown in Table 7.
Tab.7. Valoarea densității optice la OD600nm a inoculului de 56.1s utilizat in testul de inocuitate.Tab.7. The optical density value at OD600nm of the 56.1s inoculum used in the safety test.
Larvele de G. mellonella au fost crescute pe mediul Haydak la 30°C. Pentru testul de inocuitate, larvele in ultimul stadiu au fost tinute la 4°C timp de 5 minute, după care au fost injectate cu 5μΙ suspensie bacteriana, utilizând o seringa Hamilton. Fiecare varianta de dilutie a cuprins 30 larve, iar zece larve auLarvae of G. mellonella were grown on Haydak medium at 30 ° C. For the safety test, the larvae in the last stage were kept at 4 ° C for 5 minutes, after which they were injected with 5μΙ bacterial suspension, using a Hamilton syringe. Each dilution variant comprised 30 larvae and ten larvae
^‘2 0 1 0 - 0 1 1 5 9 -2 4 -11- 2010^ '2 0 1 0 - 0 1 1 5 9 -2 4 -11- 2010
fost utilizate pentru fiecare repetitive. Varianta martor a constat in larve injectate cu 5μΙ 10mM MgSO4 si 1,2 mg/ml ampicillin in scopul analizării efectelor fizice letale determinate de injecții. Larvele inoculate au fost menținute apoi pe mediul Haydak la 30°C în întuneric. La 24, 48 si 72 ore după infecție larvele au fost analizate, iar cele moarte au fost numărate. Larvele care nu au prezentat semne vitale la atingerea cu vârful pipetei au fost considerate moarte. Rezultatele au evidențiat faptul că tulpina de Brevibacillus laterosporus 56.1s, nici atunci când a fost aplicată nediluată, nu a determinat o mortalitate mai mare de 50%, iar la diluția de 103 ufc/mL'1 nu s-a determinat mortalitatea larvelor de G. mellonella (tabel 8). Deci tulpina de Brevibacillus laterosporus 56.1s nu este patogenă pentru organisme nețintă, fapt demonstrat prin mortalitatea redusă a larvelor de G. mellonella tratate.were used for each repetitive. The control variant consisted of larvae injected with 5µΙ 10mM MgSO 4 and 1.2 mg / ml ampicillin in order to analyze the lethal physical effects caused by the injections. Inoculated larvae were then maintained on Haydak medium at 30 ° C in the dark. At 24, 48 and 72 hours after infection the larvae were analyzed, and the dead ones were counted. Larvae that did not show vital signs upon reaching the tip of the pipette were considered dead. The results revealed that the strain of Brevibacillus laterosporus 56.1s, even when applied undiluted, did not cause a mortality greater than 50%, and at the dilution of 10 3 cfu / mL ' 1 the mortality of G. larvae was not determined. mellonella (Table 8). Thus, the strain of Brevibacillus laterosporus 56.1s is not pathogenic to target organisms, as evidenced by the reduced mortality of the treated G. mellonella larvae.
Tab. 8. Procentul larvelor de G. mellonella moarte la 24, 48 si 72 ore după inoculare cu tulpina B. laterosporus 56.1s.Tab. 8. Percentage of G. mellonella larvae die at 24, 48 and 72 hours after inoculation with B. laterosporus 56.1s strain.
Pentru determinarea capacității de colonizare a materialului vegetal s-au utilizat paie de grâu sterilizate prin iradiere gamma. Paie sterile de circa 6... 7 cm au fost depuse aseptic pe plăci petri cu diametrul de 9 cm, care conțineau hârtei de filtru umectată cu tampon fosfat salin steril. Pe un capăt al paiului de grâu s-au depus 10 pl suspensie bacteriană conținând 106 ufc/ml 56.1s. După 48 ore s-a prelevat aseptic capătul celălalt al paiului (cca 2 cm), care s-au trecut într-o epubetă cu 10 ml tampon fosfat salin steril. S-a fiert timp de 10 min, iar din izolatul răcit s-au prelevat 0,5 ml care s-au diluat serial de trei ori și s-au inoculat pe nutrient agar. Coloniile formate au fot re-identificate ca fiind Brevibacillus laterosporus, pe baza unui test de microetalare fenotipică de tip Biolog și a testelor API 20E. Concluzia experimentului a fost că tulpina bacteriană 56.1s prezintă capacitate de colonizare a materialului vegetal.To determine the colonization capacity of the plant material, wheat straw sterilized by gamma irradiation was used. Sterile straws of about 6-7 cm were aseptic deposited on 9 cm diameter petri dishes containing filter paper moistened with sterile saline phosphate buffer. On one end of the wheat straw were deposited 10 µl bacterial suspension containing 10 6 cfu / ml 56.1s. After 48 hours, the other end of the straw (approx. 2 cm) was aseptically taken, which were passed through a 10 ml sterile saline phosphate buffer. It was boiled for 10 minutes, and from the chilled isolate 0.5 ml were taken which were diluted serial three times and inoculated on agar nutrient. The colonies formed were re-identified as Brevibacillus laterosporus, based on a Biolog phenotypic microalgae test and API 20E tests. The conclusion of the experiment was that the bacterial strain 56.1s has the ability to colonize the plant material.
Pentru a verifica activitatea de protecție a plantelor de porumb împotriva atacului de Fusarium graminearum s-a realizat o experiență în condiții controlate. Semințele de porumb sterilizate chimic prin spălări repetate cu hipoclorit au fost bacterizate cu o suspensie bacteriană de 10® ufc/ml. S-a lucrat față de un martorIn order to verify the protection activity of maize plants against the attack of Fusarium graminearum, an experiment was conducted under controlled conditions. Corn seeds sterilized chemically by repeated washing with hypochlorite were bacterialized with a bacterial suspension of 10® cfu / ml. It worked against a witness
<C-2 010-01159-- \<C-2 010-01159-- \
4 -11- 2010 neinoculat și cu un martor etalon, tratat cu un produs chimic (2g/kg, metiltiofanat 70%). Infecția s-a realizat prin tratatarea soluției nutritive cu o suspensie concentrată de spori, astfel încât numărul de spori per ml de soluție nutritivă a fost de 104 ufc/ml. Variantele experimentale au fost: V! - 56.1s; V2 - 56.1s + F. graminearum', V3 - metil tiofanat 70% 2 g/kg + F. graminearum-, V4 - Martor neinoculat cu bacterii sau ciuperci. Fiecare variantă a fost realizată în 5 repetiții. S-au utilizat pungi de creștere cyg (Mega Internațional), iar in fiecare punga s-au plasat 3 semințe pregerminate. Pungile au fost amplasate randomizat într-o cameră de creștere. Plantulele au fost crescute în condiții controlate (temperatură de 22°C± 0,2°C, iluminare 12 ore pe zi cu 250 pmol fotoni m'2s‘1) timp de 10 zile. Rezultatele finale au arătat că tulpina 56.1s are o eficacitate de peste 90%, cu 2% mai mult decât etalonul chimic tiofanat-metil. Deci tulpina 56.1s prezintă un antagonism semnificativ pentru F. graminearum și in vivo, în condiții controlate.4 -11- 2010 not inoculated and with a standard control, treated with a chemical (2g / kg, 70% methylthiophanate). The infection was achieved by treating the nutrient solution with a concentrated spore suspension, so that the number of spores per ml of the nutrient solution was 10 4 cfu / ml. The experimental variants were: V! - 56.1s; V 2 - 56.1s + F. graminearum ', V 3 - methyl thiophanate 70% 2 g / kg + F. graminearum-, V 4 - Uninoculated control with bacteria or fungi. Each variant was made in 5 repetitions. Cyg growth bags (Mega International) were used, and 3 pre-seeded seeds were placed in each bag. The bags were randomly placed in a growth chamber. Seedlings were grown under controlled conditions (temperature of 22 ° C ± 0.2 ° C, illumination 12 hours a day with 250 pmol photons m ' 2 s' 1 ) for 10 days. The final results showed that strain 56.1s has an efficiency of over 90%, with 2% more than the thiophanate-methyl chemical standard. Thus strain 56.1s exhibits significant antagonism for F. graminearum and in vivo under controlled conditions.
S-au prelevat aseptic părți din tulpinița plantulelor provenite din semințe pre-germinate tratate cu tulpina 561.s. Tulpinițele s-au trecut într-o eprubetă cu 10 ml tampon fosfat salin steril. S-a fiert timp de 10 min, iar din izolatul răcit s-au prelevat 0,5 ml care s-au diluat serial de trei ori și s-au inoculat pe nutrient agar. Coloniile formate au fot re-identificate ca fiind Brevibacillus laterosporus, pe baza unui test de microetalare fenotipică de tip Biolog și a testelor API 20E. S-a concluzionat că tulpina 56.1s are capacitatea de a coloniza endofit plantele de porumb pe care le protejează împotriva atacului unor patogeni majori.Aseptic parts of the seedling stems from pre-germinated seeds treated with strain 561. were taken aseptically. The stems were passed into a 10 ml sterile saline phosphate buffer tube. It was boiled for 10 min, and from the cooled isolate 0.5 ml were taken which were diluted three times serial and inoculated on agar nutrient. The colonies formed were re-identified as Brevibacillus laterosporus, based on a Biolog phenotypic microalgae test and API 20E tests. It was concluded that strain 56.1s has the ability to colonize endophyte maize plants that it protects against the attack of major pathogens.
A fost realizat și un experiment de testare în condiții de câmp a eficacității tulpinii 56.1s. Experimentele de cultivare au fost realizate pe un cernoziom cambie, la Amzacea (Dobrogea). Cultura de mazăre de toamnă, Pisum sativus subsp. arvense, cv. Enduro a fost semănată direct în miriștea de grâu, la sfârșitul lunii septembrie. S-a folosit o mașină de semănat direct în miriște și s-a semănat la o densitate de 70...75 semințe germinabile de mazăre/m2, corespunzând unei cantități de 65...75 kg/ha. Adâncimea de semănat a fost de 7...8 cm. In primăvară s-a transformat cultura de mazăre în muici bioactiv prin tăvălugire, și tratare cu 900 litri de suspensie care include un erbicid total pe bază de glifosat, aplicat în doză de 1,25 s.a./ha, și suspensie de 105 ufc/ml spori Brevibacillus laterosporus 56.1s. S-a însămânțat o cultură de porumb, hibrid mediu, la o densitate de 5... 7 boabe germinabile/m2, după 14 zile de la mulcire. Cultura de porumb a fost întreținută până la jumătatea lunii mai, când s-a aplicat o fertirigare cu 250 m3/ha, cu o doză de 60 îngrășăminte complexe care conțin 15% azot, 5% fosfor, 20% potasiu și 2% microelemente. S-a întreținut în continuare cultura de porumb și s-a recoltat la sfârșitul lunii septembrie. S-a lucrat față de o variantă martor intensiv, la care s-a realizat arătură de toamnă adâncă, tratarea cu erbicide pre-mergente șiAn experiment of field test of the effectiveness of strain 56.1s was also performed. Cultivation experiments were performed on a cambium cernozoma, at Amzacea (Dobrogea). Autumn pea culture, Pisum sativus subsp. arvense, cv. Enduro was sown directly in the wheat stubble at the end of September. A seed drill was used directly in the stubble and was sown at a density of 70 ... 75 germinated pea seeds / m 2 , corresponding to an amount of 65 ... 75 kg / ha. The sowing depth was 7 ... 8 cm. In the spring, the pea crop was transformed into bioactive moss by rolling, and treatment with 900 liters of suspension including a total herbicide based on glyphosate, applied in a dose of 1.25 s / ha, and a suspension of 10 5 cfu / ml spores. Brevibacillus laterosporus 56.1s. A corn crop, medium hybrid, was seeded at a density of 5 ... 7 germinated seeds / m 2 , after 14 days after mulching. Corn cultivation was maintained until mid-May, when a fertilization with 250 m 3 / ha was applied, with a dose of 60 complex fertilizers containing 15% nitrogen, 5% phosphorus, 20% potassium and 2% microelements. The maize crop continued to be maintained and harvested at the end of September. Work was carried out on an intensive control variant, which included deep autumn plowing, treatment with pre-submerged herbicides and
io a-2 O 1 O - O 1 159 - v 2 h -11- 2010 post-emergente, fertirigarea în aceeași perioadă. De asemenea s-a folosit și o variantă la care nu s-a realizat bioactivarea mulciului vegetal cu bacterii 56.1s.io a-2 O 1 O - O 1 159 - v 2 h -11- 2010 post-emergence, fertilization during the same period. Also a variant was used in which the bioactivation of the vegetal mulch with bacteria 56.1s was not performed.
Fiecare variantă a fost realizată în 4 repetiții.Each variant was made in 4 repetitions.
In stadiul V6 al dezvoltării porumbului s-a determinat vigoarea plantelor, înălțimea și biomasa aeriană. Vigoarea plantelor s-a diferențiat semnificativ în stadiul V6. Pe o scară de la 1 la 9, la sistemul intensiv vigoarea plantelor a fost de doar 7, în pofida condițiilor climatice foarte favorabile pentru porumb în anul de experimentare. La sistemul în care s-a folosit muici vegetal vigoarea a crescut la 7,5. La sistemul cu muici bioactivat, tratament al mulciului cu suspensie de bacterii 56.1s. Plantele de porumb au prezentat o vigoare sporită de 8,5. înălțimea plantelor nu a fost influențată semnificativ, la mulciul bioactivat plantele au acumulat o semnificativ mai multă biomasa. In final vigoarea suplimentară a plantelor de porumb protejate și în condiții de câmp de atacul ciupercilor fitopatogene din sol s-a concretizat într-un spor de producție de aprox.10%. Datele sunt prezentate în tab. 9.At stage V6 of maize development, the vigor of the plants, the height and the aerial biomass were determined. The vigor of the plants differed significantly in stage V6. On a scale from 1 to 9, in the intensive system the vigor of the plants was only 7, despite the very favorable climatic conditions for maize in the experimentation year. In the system in which vegetable moss was used, the force increased to 7.5. In the bioactivated moss system, mulch treatment with bacterial suspension 56.1s. The maize plants had an increased force of 8.5. The height of the plants was not significantly influenced, in the bioactivated mulch the plants accumulated significantly more biomass. Finally, the additional vigor of the maize plants protected and under field conditions by the attack of the phytopathogenic fungi in the soil resulted in a production increase of approx. 10%. The data are presented in the tab. 9.
Tab. 9. Influența diferitelor sisteme tehnologice asupra creșterii și producției porumbului1.Tab. 9. The influence of different technological systems on the growth and production of maize 1 .
H — - 1 — — — . . i - - 1 - — — — 1—H - - 1 - - -. . i - - 1 - - - - 1 -
- valorile urmate de aceeași literă nu diferă semnificativ pentru P>0,05.- the values followed by the same letter do not differ significantly for P> 0.05.
2- stadiul V6, vigoarea 1 - plante mici cu frunze mici; 9 - plante mari cu frunze robuste 2 - stage V6, force 1 - small plants with small leaves; 9 - large plants with robust leaves
Creșterile de producție s-au realizat în condițiile unui sistem conservativ, în care sunt reduse costurile de producție aferente folosirii erbicidelor și arăturii, iar folosirea unei culturi de protecție este încurajată prin plățile compensatorii asociate măsurilor de agro-mediu.The production increases were realized under the conditions of a conservative system, in which the production costs related to the use of herbicides and plows are reduced, and the use of a protection crop is encouraged by the compensatory payments associated with the agri-environment measures.
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