RO127524A2 - Strain of bacillus subtilis antagonist to phytopathogenic fungi - Google Patents

Abstract

The invention relates to a strain of Bacillus subtilis B30 having the deposit number NCAIM (P) B001359 and GenBank EU334510, intended to be used as a bioinoculant for the treatment of seeds and soil, resulting from the stage selection of some natural soil isolates. The strain of B. Subtilis B30 exhibits broad spectrum of antifungal action against a series of phytopatogenic fungi and at the same time stimulates plant growth by auxins production. The strain also models the formation of microbial consensus consortiums with other bacteria employed as bioinoculants due to the lactonase activity influencing the AHL signals involved in achieving the group sensibility.

Description

The present invention relates to the strain of Bacillus subtilis B30, intended for use as a bioinoculant for the treatment of seed and / or soil, including in the form of complex biopreparations.

Several strains of Bacillus subtilis are known to have activity against phytopathogenic agents. WO 2007/043771 relates to compositions for the prevention of plant diseases based on KCCM 10639 or KCCM 10640 strains of B. subtilis and a process for applying these compositions. A number of US patents (6,060 051, 6 103 228, 6 291 426 and 6 417 163) protect the AQ / QST 713 strain of B. subtilis (with deposit number NRRL BO-21661) which is high in antibiotic and a broad spectrum of insecticidal, antifungal and antibacterial activity.

Also, several strains of Bacillus subtilis have been described so far that have concomitant activity of combating phytopathogenic agents and stimulating plant growth. US patent application 2008/0152684 describes a strain of B. subtilis WG6-14 (filing number NRRL B-30954), which exhibits both plant growth stimulation activity (due to the production of volatile metabolites, such as acetoin and 2 , 3 butandiol), as well as antagonistic activity against phytopathogenic fungi (Pythium aphanidermatum, Rhizoctonia solani, Colletotrichum gloesporoides, Sclerotium rolfsii) and against phytopathogenic bacteria (Xanthomonas axenopodis pv. Citri, X. axenoponasy or X. ). US Patent 7 211 428 discloses a B. subtilis strain (MTTC filing number 5130) with growth inhibiting features of pathogenic fungi (Rhizoctonia solani, Fusarium oxysporum, Fusarium semitectum, Pythium aphanidermatum, Curvularia andropogatusetrich, Collaria alternata, Collaria alternata, Collaria alternata capsicum, Colletotrichum gloeosporiodes, Corynespora cassiicola, Thielavia basicola) and which stimulates the development of crop plants (geranium and pyrethrum).

So far, no strains of Bacillus subtilis have been described that have antagonism with soil phytopathogenic agents, stimulate plant growth and modulate group sensitivity, with a role in the development of multi-spinal consortia and biopreparations.

The B. subtilis B30 strain has the following advantages:

rich growth on the usual media used to grow sporulated gram-positive bacilli;

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ability to modulate the formation of consensus microbial consortia with other bacteria used as bioinoculants due to the lactonase activity by which they influence the AHL signals involved in achieving group sensitivity;

wide range of action against the attack of numerous phytopathogenic fungi;

stimulation of plant growth due to the in situ production of phytohormones.

The present invention is illustrated by the example below

Example. The B30 strain of Bacillus subtilis was isolated at the Research and Development Institute for Plant Protection, Bucharest, from soil samples from Bărăgan. This strain was selected from a collection of over 250 isolates of Bacillus spp., Based on the beneficial action exercised on the different crop plants (tomatoes, cucumbers, sunflower), due to the protection of the plants against the following phytopathogenic fungi: Rhizoctonia solani, Pythium ultimum, Pythium aphanidermatum, Fusarium graminearum, Fusarium oxisporum f. Sp. radicis-lycopersici, Sclerotinia sclerotiorum, Sclerotium bataticola, Alternaria spp., Botrytis cinerea, Trichoderma viride), phytohormone production and lactonase activity by which they influence the AHL signals involved in group sensitivity.

For taxonomic classification, strain B30 was characterized morphologically (table 1), biochemically (table 2) and based on the 16S rDNA sequence.

Tab. 1. Morphology of Bacillus subtilis B30 colonies on different media after cultivation for 24 hours.

The environment used Morphology of B30 strain colonies color exterior appearance dimensions (24h) Semi-synthetic gelled media potato decoction medium - glucose agar (CGA) brown rough, irregular edges in the form of filaments large colonies medium with beef extract cream-colored rough, center prominent, wrinkled, edges irregular large colonies bean decoction environment crembrun rough, irregular edges middle colonies medium with soil extract crembrun rough, irregular edges middle colonies Natural solid media slices of sterile potato brown rough, irregular edges, center with fine roughness large colonies Liquid media medium with potato decoction - glucose - thin film on the surface, rough, weak turbidity - medium with meat extract - surface thin film, rough, poor turbidity -

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Tab. 2. The physiological characteristics of the B. subtilis B30 strain.

The biochemical test B30 strain Gram reaction + The Voges-Proskauer reaction + Starch hydrolysis + Hydrolysis of gelatin + Reduction NO ₃ -> NO2 + Anaerobic growth - catalase + Carbon source: malonate + citrate + propionate - tartrate + trehalose + glucose + Acidifies: xylose + glucose + arabic wedding + mannitol + raffinose + cellobiose + NaCI tolerant 7% +

Identification based on 16S rDNA was achieved by PCR amplification using primers 27fm (5'-AGA GTT TGA TCM TGG CTC AG-3 ') and r1522 (5'-AAG GAG GTG ATC CAG CCG CA-3') (Weisberg et al. (1991). Enzyme amplification products were separated on 1% agarized gel, and the 16S rDNA fragment was extracted from the gel and purified with QIAGEN kit (QIAGEN Benelux BV, Venlo, The Netherlands). The sequencing was performed at ServiceXS (Leiden, The Netherlands), followed by processing with the Vector NTI 10 program (Invitrogen, USA). For the taxonomic identification based on the sequences, research was performed regarding the similarity of the sequences in Genbank, using the NCBI Taxonomy BLAST software. The resulting sequences were submitted to GenBank. The taxonomic classification of strain B30 was based on a similarity of at least 98% between the 16S rDNA sequence of this strain with those of other strains whose taxonomic classification is recognized. The 16S rDNA sequences for strain B30, filed with GenBank, have accession number EU334510.

In vitro antagonistic activity testing of strain B30 was performed on agar dextrose potato medium (CGA - PDA). The B30 strain (from a 24-hour culture) was seeded on the medium by bending a straight line with a 3 cm distance from a calibrated mycelium round (5mm) from the nine fungi studied. Petri dishes thus seeded were incubated at 28 ° C and analyzed in

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regarding the inhibition zone (mm) at 24, 48 and 72 hours. The experience was repeated three times.

Tab. 3. In vitro testing of antagonistic activity of Bacillus subtilis B30 strain on mycelial growth of phytopathogenic fungi (inhibition zone at 72 hours, mm).

Phytopathogenic fungus Inhibition zone induced by strain B30 (mm) Rhizoctonia solani 12 Pythium ultimum 9 Pythium aphanidermatum 6 Fusarium oxisporum f. Sp. graminearum 5 Fusarium oxisporum f. Sp. radicislycopersici 6 Sclerotinia sclerotiorum 5 Sclerotium bataticola 4 Trichoderma viride 8 Alternaria spp. 6 Botrytis cinerea 8

The results showed that B30 strain produces antifungal metabolites that inhibited the development of all fungi studied. The largest area of inhibition was recorded against R. solani fungus (12 mm), followed by P. ultimum (9 mm) and T. viride (8 mm).

B30 strain has been tested under greenhouse conditions in terms of its effectiveness in combating phytopathogenic soil fungi (Rhizoctonia solani, Pythium ultimum, Fusarium oxisporum, Sclerotinia sclerotiorum and Sclerotium bataticola), which commonly occur in the seedling / seedling stage and produce seed. losses of planting material (which can reach up to 30% seedlings) and implicitly reduce production.

The operations involved consisted in the preparation of bacterial inoculum by refreshing in tubes (incubated at 27 ° C for 2-3 days) on cartofglucose-agar medium (CGA) and making bacterial suspensions in phosphate buffer titrated at 10 ⁸ cfu / ml. The fungal inoculum was obtained in Roux plates, on the natural environment consisting of double oat grains sterilized at 1 atm. for 20 minutes, by inoculation with mycelium and incubation at 27 ° C for 3-4 days.

The substrate used in the greenhouse consisted of 1/2 garden soil + 1/4 grain + 1/4 sand. It was mixed evenly with the fungal inoculum (~ 2 x 10 ⁶ spores / kg soil) and then distributed in plastic trays (32/24 cm) 48 hours before sowing.

The plant material, respectively sunflower seeds (Festive), tomatoes (Union) and cucumber (Cornichon) were treated before immersion for 20 minutes in bacterial suspensions whose titration was set at 10 ⁸ cfu / ml .

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In the first experiment, 5 strains of B. were tested together with B30.

subtilis namely: Bs23, Bs27, Bs36, Bs45, Bs48. The fungal inoculum applied consisted of the R. solani + F. oxysporum f. Sp. graminearum + P. de baryanum (RFP), S. sclerotiorum and S. bataticola. Sunflower seeds (Festive) and cucumbers (Cornichon) were used.

The experiment was analyzed after 3 weeks of sowing regarding the effectiveness of the strains in combating phytopathogenic fungi. Also, the effect of stimulating the growth of plants was analyzed by measuring the root length, the stem and the total length of the plants.

The analysis of the variant analysis was performed with the ANOVA program.

The analysis of the variant for the three parameters taken into account, the root length, the stem and the total length of the plants, for the two crops, the sunflower and cucumber highlighted the following:

In sunflower culture, root length was influenced by factor A (fungal inoculum) independent of factor B - bacterial inoculum (Tab. 4). The fungal complex R.F.P (Rhizoctonia-Fusarium-Pythlum) recorded the highest values, statistically assured compared to the other two fungi studied, which had an equal effect.

Tab. 4. Root length (mm) of sunflower plants (Festive cv.) Under the influence of biological treatments with different isolates of B. subtilis in seed and with fungal inoculants applied in soil.

Factor A (fungal inoculum) Tiradin 70 PU (4g / kg) Witness, inoculated Bs23 Bs27 Bs30 Bs36 Bs45 BS48 Medium factor A R-F-P 59,05bcd 27.77 68,68b 59,57bcd 94,82a 65,27bc 39,40ghi 55,87b-f 63.25 ss 49,03c-g 27.85 40,31f them 37,61ghi 60,83bc 30,96hi 59,76bcd 36,67ghi 45,78b sb 51,02c-g 27.33 40,35f them 32,44hi 41.79 I 26.51 i 56,78b-e 44,6d-h 41,19b Means factor B 53,04b 27.65 49,781 bc 43,208bc 65,817a 40,917cd 52,01b 45,70bc

The values followed by the same letter do not differ significantly for P <0.05 (DS A5% = 5,306 DS B5% = 10,202 DS AB5% = 15,919)

The study of the interaction of the biological treatments on the roots of the sunflower plants, revealed the following: (/) in case of the use of the phytopathogenic mushroom complex of soil R.F.P. strain B30 caused the largest growths of plant roots (statistically assured against untreated control) followed by isolates of B. subtilis Bs23 and Bs36; (//) in the variants of the soil inoculated with S. sclerotiorum fungus, the highest vegetative growths of the roots were noted in the seedlings resulting from the seeds bacterialized with the strain of B. subtilis B30.

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The fungal inoculum, independent of the bacterial one, has most significantly influenced the development of sunflower plants, respectively their total length, in the case of the Rhizoctonia-Fusarium-Pythium fungal complex.

Tab. 5. The total length of sunflower plants (mm) under the influence of biological treatments with bacterial inoculants in seed and fungus applied in the soil

Factor A (fungal inoculum) Tiradin 70 PU (4g / kg) Witness nebact. inoculated Bs23 Bs27 Bs30 Bs36 Bs45 BS48 Medium factor A R-F-P 136,11d-f 73,11lm 150,39cd 134,68def 186.53 129,35efg 104,15ij 117,23g-j 133,57a ss 115,66g-j 65,23m 124,66fgh 123,36fgh 127,74efg 113,71g-j 133,86def 128,29efg 119,60b sb 127,23e-h 78,22lm 118,80f-j 100,24jk 144,51cde 105,88ij 121,38f them 113,83g-j 118,43b Means factor B 126,34bc 72,19d 131,28b 119,42bc 152,92a 116,32c 119,80bc 119,78bc

The values followed by the same letter do not differ significantly for P <0.05 (DS A5% = 6,263 DS B5% = 12,041 DS AB5% = 18,789)

B. subtilis isolates, independent of the soil fungi tested, caused the largest increases in total plant length on soil infected with R.F.P. and the one infected with the S.bataticola fungus, statistically insured against the untreated control, in the bacterial variant with B. subtilis B30. These had distinctly significant values compared to strains Bs23, Bs27, Bs45, Bs48 and Bs36.

The interaction of the two types of biological inoculum showed that the B30 strain significantly favored the total growth of the flowering plants compared to the untreated control and the chemically treated variant.

The calculation of the efficacy of the B. subtilis treatments in the flowering seed (Festive cv.), In preventing the attack of the soil fungi studied, reflected that the B30 strain had an efficacy in combating the phytopathogenic soil fungi complex R.F.P. higher (77%) compared to the standard chemical control (70%).

The total length of the cucumber plants (tab.6) was influenced by the biological treatments with the fungal inoculants, distinctly significant values, statistically assured compared to the untreated control, resulting in the variant inoculated with S. bataticola. Treatment with B. subtilis B30 strain, independent of treatments with fungal pathogens, positively influenced the overall length of the plants, the values resulting from the analysis of the variance being distinctly significant compared to the untreated control. The interaction of the two types of inoculants, bacterial and fungal, revealed that strain B30 caused distinctly significant increases in the total length of cucumber plants compared to the untreated control and the chemically treated variant.

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Tab. 6. Total length of cucumber plants (Cornichon) under the influence of bacterial inoculum applied to seeds (factor B) and fungal (factor A) applied in soil.

Factor A (fungal inoculum) Tiradin 70 PU (4g / kg) Witness nebact. inoculated Bs23 Bs27 Bs30 Bs36 Bs45 BS48 Medium factor A R-F-P 100,34d-f 43,33lm 78.11 hk 77,77h-k 123,59ab 95,74e-g 88.55 I 89,44e them 89,921b ss 110,20bc 35,82m 97,06eg 87,33f-j 119,00abc 81,26g them 104,11cde 73,83ijk 91,56ab sb 88,626e-j 38,92m 65,08j it 93,030eh 125,85ab 101,10df 104,167cde 114,627bcd 95,97a Means factor B 99,725b 39.35 80,08d 86,04cd 122.81 92,70bc 98,94b 92,63bc

The values followed by the same letter do not differ significantly for P <0.05 (DS A5% = 5.413; DS B5% = 10.408; DS AB5% = 16.241).

Tab. 7. Effectiveness of some B. subtilis strains in combating telluric mushrooms in cucumber culture (Cornichon) under greenhouse conditions (21-24 ° C and 70% relative humidity).

Fungal inoculum * R.F.P. S. s. S.b. Variant (isolated code) % healthy sunflower plants effectiveness (%) % healthy sunflower plants effectiveness (%) % healthy sunflower plants Effectiveness (%) Bs 23 60 27 87 74 80 57 27s 58 24 70 40 87 72 Bs 30 98 96 100 100 90 79 Bs 36 72 49 78 56 73 43 Bs 45 74 53 79 58 79 55 Bs 48 79 62 82 64 85 68 Tiradin 70 PU (4g / kg) 93 87 98 96 90 79 Mt. untreated 45 - 50 - 53

* RFP = R. solani + F. oxysporum + P. de baryanum; Ss = S. sclerotiorum; Ss = S. bataticola.

Calculation of effectiveness (%) of B. subtilis treatments in cucumber seed (Cornichon), tab.7, reflected the following:

- In the variants infected with the R.F.P. and with S. sclerotiorum the highest efficacy (96 and 100% respectively) was recorded in the variants treated with strain B30.

- In the variant infected with S. bataticola, the highest efficacy resulted in the use of strain Bs30 (79%).

- In the variant infected with S. Sclerotiorum the efficacy of the treatment of the seeds with B30 was equal to that of the variant of the chemical standard control (79%).

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In another series of experiments cucumber seeds (Cornishon variety) were inoculated by immersion with the following strains of Bacillus subtilis; Bs 1.98a; Bs salt; Bs lobes; Bs 98 and B30 while analyzing the ability of cucumber crop protection against infection with 5 phytopathogenic fungi: Fusarium oxysporum, Sclerotium bataticola, Pythium de baryanum, Rhizoctonia solani and Sclerotinia sclerotiorum.

Table 9. Effectiveness of B. subtilis strains in combating phytopathogenic soil fungi in cucumber culture (Cornichon) under greenhouse conditions (2124 ° C and 70% relative humidity).

inoculum Fusarium Sclerotium Sclerotinia Pythium Rhizoctonia fungic-> oxysporum bataticola sclerotiorum ultimum solani Variant % effective % effective % effective % Effective % effective of plants Dad plants Dad plants Dad plant -tate plants Dad (code healthy condition (%) healthy condition (%) healthy condition (%) healthy (%) healthy condition (%) isolated) -it -it -it -it -it sprung sprung sprung sprung sprung Bs 70 50 70 40 90 82 70 50 45 15 1.98 Good morning 76 60 80 60 93 87 77 62 63 43 Bs lobes 87 78 83 66 98 96 90 88 87 80 Bs 98 70 50 70 40 90 82 65 42 83 74 Bs 30 93 88 90 80 100 100 100 100 93 89 Tiradin 77 65 90 80 87 76 93 88 77 65 70 PU (4g / kg) Mt. 40 - 50 - 45 - 40 35 - untreated

Calculation of the effectiveness of B. subtilis treatments applied to cucumber seeds led to the following observations (tab. 9):

- in the variants treated with B. subtilis B30 the highest efficacy values were recorded for all 5 fungi studied, and in those where the soil was infected with S. sclerotiorum and P. ultimum, the efficacy was 100% , absent value in the chemical standard control variant (which recorded values of 76 and 88% respectively).

- In the variants infected with F. oxysporum, the highest efficacy was obtained following the use of B. subtilis B30 strain (88%) followed by B. subtilis Bs lob 1 (78%).

- On soil infected with S. bataticola, the variant inoculated with B. subtilis B30 had the highest efficacy (80%). The lowest efficacy was registered in the variants treated with strains Bs 1.98a and Bs 98 (they had an equal efficiency of 40%).

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- On the soil infected with P. ultimum, the maximum efficacy was registered in the variant treated with the strain of B. subtilis B30 (100%), this being followed by the treatment treated with B. subtilis Bs lob 1 (88%).

- The efficacy of the biological treatments on the soil infected with R. solani, revealed the variant B. subtilis strain B30 (89%), followed by the strains of B. subtilis Bs lobes and Bs 98. The lowest efficacy had the variant in which the seeds were treated with B. subtilis Bs 1.98 (15%).

- On the soil infected with S. sclerotiorum, the highest efficacy was obtained in the variant treated with B. subtilis B30 (100%), followed by the variant B. subtilis Bs lobes (96%).

For the extraction of antifungal metabolites, the most favorable environment for their production was selected, namely the Landy environment (Akpa et al., 2001) and the optimized environment for the production of antibiotics (Jacques, 1999). Also extraction solvents were selected, namely ethylacetate + 1% formic acid, which allowed to obtain a crude extract with the highest antagonistic action against phytopathogenic soil fungi.

Erlenmayer flasks (250 ml) with 25 ml Landy medium were inoculated with strain B30 and incubated at 28 ° C with shaking at 150 rpm for 72 hours. The supernatant was mixed in 1: 1 ratio with the solvent and incubated at 28 ° C and stirred at 150 rpm / min for 8 hours, followed by extraction itself by centrifugation at 4000 rpm / min and 4 ° C. 20 minutes.

The organic phase was collected and concentrated on a rotary evaporator in vacuum until drying.

In order to separate and identify the active fractions from the crude extract, silica gel chromatographic aluminum plates (HPTLC 20x20 cm silica gel60 F ₂₅ 4 _S , Merck) were revealed in the chloroform - methanol - water solvent system (65: 25: 4 v / v / v). From the crude extract were applied 10 μΙ per spot, and the migration front was limited to 7 cm. The plates were viewed in UV light at 254 nm and retention factors were noted.

To identify the active spots against Fusarium oxysporum f. Sp. radicis-lycopersici (Forl) and Rhizoctonia solani (Rs), the bioautography test was performed, which consisted of covering with 1% potato agar dextrose medium (PDA), inoculated with mushroom spores at a concentration of 2x10 ⁶ spores / ml. Chromatographic plates were incubated at 28 ° C for 72-96 hours and then visualized.

The results are shown in table 10. The results showed that B30 produces 2 antifungal metabolites, namely Bacilysin and Iturin A, a fact confirmed by the clear areas present on the auto (bio) graphic plates ^ -2010-01381-2 1 "12-2010

Table 10. The Rf values of the crude extract from strain B30 in the chloroform-methanol-water system (65: 25: 4 v / v / v) viewed at 254 nm and on the (bio) graphic plates.

Stem Rf values in the methanol-water chloroform system (65: 25: 4 v / v / v) viewed at 254 nm Antifungal antifungal (Loeffer, 1986) Rf values of active spots compared to: Fort Rs B30 0.09 0.20 0.35 0.40 Bacilizină Iturine A ++ +++ ++ ++++

Caption: inhibition zone ++> 5 mm; +++> 7 mm; ++++> 10 mm; Rf = retention factor

B30 strain has been characterized in terms of the production of compounds that can contribute to its biological protection activity. Chitinase and proteases play a role in antagonism by degrading the fungal cell wall, cellulose is important for plant root colonization, and lactonase acts on gram-negative phytopathogens that use acylhomoserine-lactone (AHL) as a signal involved in group sensitivity (quorum sensing).

The test for the production of proteases was performed on Bazai medium (BM; Meyer and Abdallah, 1978) agarized supplemented with 5% sour milk. The B30 strain was inoculated by staining the medium, after which the plates were incubated at 28 ° C. After 5 days, these were analyzed for the presence of clear areas around the inoculation site, indicating the production of proteases.

The cellulase activity was determined by decomposition of the carboxy-methyl-cellulose substrate. The B30 strain was seeded on plates with agarized LC medium which were coated on top with an agar layer + 1% carboxy-methyl cellulose. After 5 days incubation at 28 ° C, the plates were stained for 30 minutes with 0.3% Congo red. They were then rinsed with tap water and the dye was fixed by incubation for 15 min with 10% acetic acid. The presence of a clear area indicated cellulase production.

Chitinase production was analyzed by decomposition of CM-chitin-RBV substrate (Carboxy-methyl-chitin coupled with Remazol Brilliant Violet, Loewe Biochemica GmbH, Germany). B30 was seeded on plates with agarized BM medium, which at the top were covered with a + 50% (v / v) CM-Chitin-RBV agar layer as the carbon source. After 5 days, these were analyzed for the presence of clear areas around the inoculation site, indicating the production of chitinases.

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B30 lactonase production was analyzed by inoculating 2 ml of LB supplemented with 5 μΜ C6-hexanoyl-homoserin-lactone (C6-HHL) and raised overnight at 28 ° C and stirring 150 rpm / min. A negative control from a non-inoculated medium was used that was incubated under the same conditions to check for lactolysis. Petri dishes with medium LB + 50 pg / ml Km were seeded with the Cv026 biosensor strain of Chromobacterium violaceum (its transformation does not produce A / -acyl homoserin lactone). Cut wells (5 mm 0) in the inoculated environment in the beach with Cv06 were filled with 100 μΙ of B30 culture. The plates were incubated overnight at 28 ° C and analyzed for the presence / absence of purple halos around wells inoculated with bacteria. The absence of color indicated that all C6-HHL was degraded and therefore lactonase production.

The results showed that strain B30 produces proteases, cellulase and lactonase, but does not produce chitinase.

In other experiments, the mobility test of strain B30 was performed. Mobility plays a major role in colonizing plant roots and is the direct factor involved in the biological control mechanism through competition for nutrients and space. Bacillus subtilis B30 was tested for mobility of agar surface migration (swimming) and aggregation due to chemotaxis (swarming), along with a control strain known as immobile, P. putida PCL1760.

The test consisted of using fresh cultures that were inoculated by staining in the center of the Petri dishes with LB medium supplemented with 0.3% agar (for swimming) and 0.5% (for swarming).

B30 strain has been shown to possess both types of mobility, this result also confirming the production of this protease and surfactin strain.

The presence of auxin in the B. supertilis B30 strain culture supernatant was detected by a colorimetric method specific for indole compounds. Briefly, four strains of B. subtilis were inoculated into broth nutrient medium (NB: pepton 5 g; yeast extract 1.5 g, meat extract 1.5 g, NaCl 5 g) with and without tryptophan (auxin precursor; 100 mg / ml) and incubated at 30 ° C and 150 rpm. After one day, 4 and 8 days after incubation, 5 ml was centrifuged at 13,000 rpm for 10 minutes. Two milliliters of the supernatant were transferred to a new tube, to which were added 100 μΙ 10 mM orthophosphoric acid and 4 ml of reagent Salkowski (2% FeCl ₃ solution in 35% 0.5 M perchloric acid). The absorbance was read at 530 nm, after maintaining the tubes for 30 minutes at room temperature.

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When auxyric compounds are produced in the environment, the addition of Salkowski reagent causes the color to turn pink. The concentration of indolyl acetic acid 3 (IAA) in the studied cultures was determined by using a standard curve made with different concentrations of auxin. All the analyzed strains reached the stationary growth phase in 24 hours, but at that time no auxin was detected in the culture media. After 8 days of cultivation in the NB environment, with and without tryptophan, all the strains investigated produced auxin.

All tested Bacillus subtilis strains tested positive for tryptophan in the environment. B30 strain produced 6.64 µg / ml tryptophan-free auxin and 8.4 µg / ml tryptophan. The amount of auxin produced by all tested isolates ranged from 6 to 6.64 pg / ml (NB without tryptophan) and 7.64 - 8.4 pg / ml with tryptophan (NB supplemented with tryptophan, 100 pg / ml on average). .

In conclusion, strain B30 has shown that it has the qualities of biological control agent against a large number of phytopathogenic soil fungi, also having an activity of stimulating the growth of plants due to auxin production.

B30 strain produces bacilizine and iturine A, lipopolipeptide antibiotics, synthesized nonribosomal, with action to inhibit the development of phytopathogenic fungi. Also strain B30 produces cellulase, protease and lactonase, enzymes that play an important role in stopping / inhibiting mechanisms that underlie the phytopathogenicity of microorganisms. Lactonase interferes with the group sensitivity of bacteria that use AHL signals, so it can modulate the formation of bacterial consortia and can be used to produce polypulpine biopreparations.

B. subtilis B30 has been shown to be mobile on (semi) agarized media, a property that can be correlated with protease production.

B30 strain has the ability to convert amino acid precursors (tryptophan) into auxin phytohormones.

The use of B. subtilis strain B30 (NCAIM deposit number (P) B001359 and GenBank number EU 334510) as a bioinoculant represents an alternative solution to the use of pesticides in the fight against phytopathogenic fungi.

Claims (1)

Claim 1. Bacillus subtilis strain B30, with deposit number NCAIM (P) B001359, characterized in that: it is antagonistic to phytopathogenic fungi: Rhizoctonia solani, Pythium ultimum, Pythium aphanidermatum, Fusarium graminearum, Fusarium oxisporum f. radicis-lycopersici, Sclerotinia sclerotiorum, Sclerotium bataticola, Alternaria spp., Botrytis cinerea, Trichoderma viride, due to the production of lipopetide antibiotics, baciizine and iturine A, as well as hydrolytic enzymes: cellulase and proteases, has the activity of stimulating the growth of plants. , cucumbers and sunflower due to the production of auxin hormones, modulate the formation of consensus microbial consortia with other bacteria used as bioinoculants due to the lactonase activity by which they influence the AHL signals involved in achieving group sensitivity.