RO127468A2 - Strain of bacillus amyloliquefaciens subsp. plantarum having various beneficial effects on crops - Google Patents

Abstract

The invention relates to a strain of Bacillus Amyloliquefaciens subsp. Plantarum B100 having the deposit number NCAIM (P) B001362, intended to be used as a bioinoculant in agricultural crops, for fortifying the grain cereal crops in regions of selenium deficit. The strain of B. Amyloliquefaciens B 100 produces lipopeptide and polyketide antibiotics and hydrolytic enzymes, protease and lactonase, exhibiting a broad spectrum of antifungal action against phytopatogenic fungi in the soil and the bacteria which affect fruit trees upon blossoming, having plant growth stimulation effects due to the endophytic production of growth factors and capacity of mineralizing the vegetable material due to the production of amylase, phytase and cellulase. The B 100 strain has the effects of solubilization of inorganic phosphorus, biodisponibilization of selenium and stimulation of selenium assimilation and accumulation by the wheat and maize plants.

Description

The present invention relates to a novel strain of Bacillus amyloliquefaciens subsp. plantarum B100, deposited NCAIM B001362, intended for use as a bioinoculant in agricultural crops, including for biofortification of cereal crop in areas with selenium deficiency.

Several strains of Bacillus amyloliquefaciens are known for use as agricultural bioinoculants. U.S. Patent No. 6,660,342 describes strain B190 (filed under CCRC 910182 at FIRDI, Hsin-Ciu, Taiwan) used to protect the Taiwanese lily (Tricyrtis formosana) against gray rot (Botrytis elliptica). U.S. Patent No. 76,156,66 discloses strain KTGB0202 of B. amyloliquefaciens (deposit number KCTC 10564BP, KCTC, Taejon, Republic of Korea) which is active against a wide range of phytopathogens. Patent application KR20060021162 protects the MJ-3 strain of B. amyloliquefaciens, which is concomitantly antagonistic to phytopathogens and with an activity promoting plant growth.

So far, no B. amyloliquefaciens strains have been described that show antagonism for phytopathogens, ability to stimulate growth of crop plants, phosphorus solubilization activity, mineralization of organic matter and bioavailability of selenium, although these bacteria are known. due to their high ability to produce exoenzymes, including exohydrolases active in lignocellulosic material degradation. US Patent 7452445 relates to the use of PMBP-m7 strain of B. amyloliquefaciens (filed under PTA-5819 at ATCC, Manassas, USA), along with other Bacillus strains, for the breakdown of plant material and the formation of fibrillar cellulose material. Although the resulting material is then used in agriculture (through organic moss formation), US Patent 7,452,445 does not describe features of antagonism for phytopathogens and / or plant growth stimulation for PMBP-m7 strain.

B. amyloliquefaciens strain B100 (NCAIM deposit number B001364, NCAIM, Budapest, Hungary) has a broad spectrum of antifungal action against phytopathogenic soil fungi, a significant ability to stimulate plant growth, increased activity by: involved in mineralization of plant material, solubilization of phosphorus from its organic and inorganic forms and bioavailability of selenium.

The B. amyloliquefaciens B100 strain has the following advantages:

THE ORCI STATE FOR INVENTIONS AND MARKETl

Patent application dc invention

No. | j _Ο ; · ;; · ;;: ·; “2ϊ -ΐ2 ^: 2οιιΐ '·' β 1.4 aoaaa aaea ea aaaa aaaaaa aj ^C V 201 0-01379- V

1 -12- 2010

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

- antagonism for a series of soil phytopathogenic fungi which allows the use as a bioinoculant for the protection of the first stages of the crop plants against these soil phytopathogenic agents;

- antagonism for bacteria that produce diseases during flowering (Erwinia amylovora, Pseudomonas ice ⁺ );

- stimulation of plant growth due to the in situ production of phytohormones and volatile compounds;

- ability to intensify mineralization of plant material;

- complementarity with the conservative agricultural technologies, in which, due to the fact that the soil works are reduced and more than 30% of the land is covered with vegetal debris, the seeds of the crop plants germinate harder and a favorable environment is created for the attack of the phytopathogenic agents;

- improvement of plant nutrition by solubilizing inorganic insoluble phosphorus through the production of organic acids, solubilizing organic insoluble phosphorus (phytic acid salts) by producing phytases, bioavailability of selenium and stimulating the take-up and accumulation by wheat and maize plants.

The present invention is illustrated by the example below

Example. The B100 strain of Bacillus amyloliquefaciens was obtained from the Research and Development Institute for Plant Protection, Bucharest, from the branches of Cydonia vulgaris. This strain was selected from a collection of over 150 isolates of gram-positive sporadic bacilli, based on: the action of protection of different crop plants against phytopathogenic soil fungi Fusarium graminearum, Alternaria spp, Botrytis tinerea, Sclerotinia sclerotiorum cepivorum, Gaeumannomyces graminis, Pythium debaryanum, Verticillium dahliae, Rhizoctonia solani, Fusarium oxisporum f. sp. radicislycopersici, Sclerotium bataticola), the in situ production of growth factors for plants, the ability of controlled degradation of plant material with polyamine formation, compounds that increase the resistance of plants to biotic and abiotic stresses, the solubilization of phosphorus from its inorganic and organic soluble forms. , bioavailability of selenium for wheat and maize plants.

For the taxonomic classification, strain B100 was characterized morphologically (table 1), biochemically - physiologically (table 2) and based on the 16S rDNA sequence (table 3).

2x9

Cv-2 O 1 O - O 1 3 7 9 - 2 1 -12- 2010

Tab. 1. Morphology of Bacillus amyloliquefaciens B1OO colonies on different media after cultivation for 24 hours.

The environment used Morphology of B100 strain colonies color exterior appearance dimensions (24h) Semi-synthetic gelled media potato glucose agar decoction medium (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 glucose decoction - thin film on the surface, rough, weak turbidity - medium with meat extract - surface thin film, rough, poor turbidity -

Tab. 2. The physiological characteristics of the B1OO strain.

The biochemical test B100 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 + arabinose + mannitol + raffinose + cellobiose + NaCI tolerant 7% +

ίλ-2 0 1 0 - 0 1 3 7 9 -2 1 -12- 2010

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; sequencing Reaction - PCR before sequencing; precipitation and drying of ribosomal DNA. 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 99.4% similarity with other strains of B. amyloliquefaciens.

Tab. 3. Identification of the strain based on the similarity of the 16 S rDNA sequence with GenBank strains.

Stem 16S rDNA sequence length (pb base pairs) The nucleotide composition of the sequence 16S rADN Identification of the strain based on the similarity of the 16S rDNA sequence with GenBank strains (reference no.); - the percentage of similarity B100 543 TGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAG ATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGT GAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGAT AACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTC TGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGC TACCACTTACAGATGGACCCGCGGCGCATTAGCTAGT TGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAG CCGACCTGAGAGGGTGATCGGCCACACTGGGACTGA GACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGG GAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAAC GCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCT CTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGG CGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCT AACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGC AAGCGTTGTCCGGAATTATTGGGCGTA Bacillus amyloliquefaciens FZB42 (CP000560.1) - similarity: 100%

Corroborating all the data obtained, strain B1OO was classified as belonging to the plantarum subspecies of B. amyloliquefaciens, recently described by Borriss et. al., 2010, doi: 10.1099 / ijs.0.023267-0 (the subspecies to which the FZB42 strain also belongs, described as a type strain, with which B100 has 100% similarity to the nucleotide sequence for 16S rDNA).

In vitro antagonistic activity testing of B100 strain was performed on potato agar dextrose (CGA) medium. B100 strain (from a 24-hour culture) a

ί \ - 2 Ο 1 Ο - Ο ι 3 7 9 - -

1 -12- 2010 was sown on the medium by a straight line bowing 3 cm from a calibrated mycelium round (5mm) of the seven fungi studied. Petri dishes thus seeded were incubated at 28 ° C and analyzed with respect to the inhibition zone (mm) at 24, 48 and 72 hours. The experience was repeated three times. The results (tab.4) showed that strain B100 produces antifungal metabolites that inhibited the development of all fungi studied. The largest zone of inhibition was in relation to the toxigenic fungi Aspergillus flavus (8.5 mm) and Fusarium graminearum (9.5 mm). The biological action is also significant for Gaeumannomyces graminis, R. solarii and F. oxisporum f. Sp. radicis lycopersici (8 mm), S. sclerotiorum, V. dahliae (7.5 mm), P. ultimum (7 mm), S. bataticola (6 mm).

Tab. 4. In vitro testing of antagonistic activity of Bacillus subtilis B1OO strain on mycelial growth of phytopathogenic fungi (inhibition zone at 72 h, mm).

Phytopathogenic fungus Inhibition zone (mm) induced by strain B100 Rhizoctonia solani 8 Pythium ultimum 7 Fusarium graminearum 9.5 Fusarium oxisporum f. Sp. radicislycopersici 8 Sclerotinia sclerotiorum 7 Sclerotium bataticola 6 Aspergillus flavus 8.5 Gaeumannomyces graminis 8 Verticilium dahliae 7.5

For the analysis of metabolic byproducts of policetidelor category and lipopeptides were extracted from the culture supernatant samples B100 Landy medium (Glucose 20 g MgSO _4. 7H ₂ 0 0.5 g; KCl 0.5 g; glutamic acid 5 g; MnSO ₄ H ₂ O 5 mg; Fe (SO ₄ ) 1% solution 0.1 ml; CuSO4 5H ₂ O solution 1% 0.02ml, water up to 1000 ml). Bacteria were grown 12 and 72 hours at 37 ° C. Samples were extracted on a Chromafix C18ec cartridge (Machery-Nagel, Duren, Germany). After binding and washing with MilliQ water (5 times the volume of the chromatographic bed in the cartridge), the metabolites were eluted with methanol (2 times the volume of the chromatographic bed in the cartridge), dried under vacuum and resuspended in 100 μΙ of methanol. Normalized samples were analyzed by high pressure chromatography in reverse phase, using a Zorbax C18 column, 4.6 mm in diameter and

ί \ - 2010-01379-2 1-12-2010

150 mm long, mounted on a 1290 Infinity LC system (Agilent, Palo Alto, USA, with diode array detector - "diode array") coupled with an Agilent 5230 Accurate TOF LC / MS mass spectrometer. Lipopetides were eluted in gradient, using 0.05% trifluoroacetic acid solutions in acetonitrile and milliQ water, at a flow rate of 1 ml / min. The wavelength for eluate detection was set at 280 nm, and the column temperature was maintained at 25 ° C.

The polycetides were eluted in a binary solvent system (solvent A: 0.1% formic acid in milliQ water; solvent B: 0.1% formic acid in acetonitrile) as follows: 30% B for 5 min, followed by 5 min gradient from 30% B to 45% B and a subsequent 25 min gradient from 45% B to 100% B. The flow rate was 0.5 ml / min at 40 ° C. The identity of each metabolite was obtained based on the molecular mass of the molecular ions detected by mass spectrometry, using the following ionization conditions (both positive and negative) source temperature: 150 ° C; desolvation temperature: 325 ° C; nitrogen flow: 550 l / min; cone voltage: 80 V. In the culture supernatant for 12 hours on Landy medium, peaks belonging to the following polycetide antibiotic classes were highlighted: macrolactin; bacillin, chlorotetane (table 8). In the case of the samples from the culture of 72 hours, three distinct groups of lipopeptide antibiotics were detected, which were identified as being of the surfactin, fengicin and iturine class (tab. 5).

Tab. 5. Metabolites produced by Bacillus amyloliquefaciens B100 strain grown on Landy's environment.

metabolite The mass table observed Identification polyketide Macrolactină 425.4 [M + Na] + Macrolactin A 511.4 [M + Na] + 7-o-malonyl macrolactin A 525.4 [M + Na] + 7-o-succinyl macrolactin A 629.3 [M + H-H2OJ + Macrolactin D Bacilenă 583.5 [M + H] + Bacilene A 605.5 [M + Na] + Bacilene B Clorotetaină 289.2 [M + H] + Chlorothetaine ( ³⁵ CI) 291.1 [M + HJ + Chlorothetaine ( ³⁷ CI) lipopeptides surfactin 1044.8, 1060.8 [M + Na, K] + C14-surfactin 1058.8, 1074.8 [M + Na, K] + C15-surfactin Fengicină 1471.9, 1487.9 [M + Na, K] + Ala-6 C15-fengicin 1485.9, 1501.9 [M + Na, K] + Ala-6 C16-fengcine 1499.9, 1515.9 [M + Na, K] + Ala-6 C17-fengicin 1513.9, 1529.9 [M + Na, K] + Val-6 C16-fengicin 1527.8, 1543.8 [M + Na, K] + Val-6 C17-fengycin Iturine A 1066.1 [M + Na] + C14-iturine A 1079.7 [M + Na] + C15-iturine A

«-2010-01379-- | country

1 -12- 2010

Antibiotics produced by the B1OO strain exert its protective action against plant pathogens both directly and by inhibiting the development of phytopathogens and indirectly by activating the plant defense system (Ongena et al., 2007, Surfactin and fengycin lipopeptides of Bacillus subtilis as elicitors of induced systemic resistance in plants, Environ. Microb., 9, 1084-1090).

In another experiment, the biosurfactant activity of B. amyloliquefaciens B100 strain was tested. Biosurfactants produced by Bacillus antagonists are among the best known for their antagonistic properties, but they also play an important role in bacterial mobility. B100 strain biosurfactant activity was tested in undiluted bacterial cell free supernatant, diluted 5x and 10x. For this, the strain was inoculated in 10 ml medium Luria Bertani (LB) and in the optimized medium Jacques; The inoculated media were incubated for 18 hours at 150 rpm and 37 ° C. The supernatant from the two growth media was collected and dilutions were made. The surface tension was measured with a Du Nouy ring (K6 Kruss, GmbH, Harnburg, Germany) (Kuiper et al., 2004).

In both growth media similar curves of surface tension were recorded, but in the optimized environment the strain maintained a higher voltage at higher dilutions compared to the LB medium; this fact also demonstrates the production of antibiotics by the B100 strain, which expresses at a higher level the genes for the synthesis of biosurfactant lipopetide antibiotics in the Jacques optimized environment.

B100 bacteria have been shown to exhibit antagonism in vitro and in situ and to bacteria that affect flowering fruit trees (Erwinia amylovora, Pseudomonas ice ⁺ ). The in vitro assay was performed on agarized LB medium. The phytopathogenic strains used were E. amylovora NCAIM B 01108 and Pseudomonas syringae ATCC 53543. The stems, refreshed 24 hours before the test, were seeded simultaneously in stone plates with agarized LB medium, the phytopathogenic stems being streaked on the middle of the plate, and B100 of B. amyloliquefaciens perpendicular, at a distance of 2 mm. The plates were incubated at 28 ° C for 24-48 hours. The test was repeated five times. The positive result was appreciated by the appearance of the inhibition zones. The Pss 33 strain inhibited the growth of phytopathogens that affect flowering fruit trees in vitro (Erwinia amylovora, Pseudomonas syringae ice +).

The in situ test was performed by determining microbial interactions on detached flowers. The experimental model used was that of the isolated flowers (Pusey, 1997). Apple branches with buds were cut from trees 4-5 days before flowering

2.-2 0 1 0 - 0 1 3 7 9 -2 1 -12- 2010 and deposited at 4 ° C, with the base immersed in water. At the beginning of the experiments the branches were removed from the refrigerator and kept at room temperature (20 ... 23 ° C). The flower buds opened in two days. According to the protocol, the open flowers (petals and red anthers) were taken with the stalk intact (1.5 cm long) and placed in microcentrifuge tubes, with the base end of the stalk immersed in a sterile 20% sucrose solution. Suspensions of 10 ⁶ cfu / ml of the tested antagonistic strains were sprayed onto the flowers thus prepared. The suspensions were prepared in 10 mM phosphate buffer with 0.03% Tween 20. Eppendorf tubes were deposited in racks, and inoculated flower racks were transferred into 10 L plastic boxes, the bottom of which was stored a solution of glycerin 40% (to maintain a humidity of 93 ... 94%). At the same time as the treatment with antagonistic bacteria (depending on the tested variant) over the detached flowers, a suspension of 10 ⁶ cfu phytopathogenic bacteria (Erwinia amylovora NCAIM B 01108 and Pseudomonas syringae ATCC 53543) was added. It was incubated at 20 ° C (Erwinia amylovora) or at 0 ° C (bacteria that nucleate ice). After 24 hours, efficiency ratings were made, according to an efficiency scale with scores from 0 to 5, according to the following scale: 0 - total destruction of the flowers; 1 - 80 ... 90% of flowers destroyed; 2 - 60 ... 80% of flowers destroyed; 2 - 40 .. 60% of flowers destroyed; 3 - 20 ... 40% of flowers destroyed; 5 - 0 ... 20% of flowers destroyed. The B100 strain protected the flowers from destruction, under 20% of the flowers being destroyed in the variant in which it was applied, so it has antagonism to the bacteria that affect the fruit trees when flowering /

The B100 strain has been tested under controlled conditions in terms of its effectiveness in combating phytopathogenic fungi of soil Fusarium graminearum, which frequently attacks the seedling stage and causes damage.

Wheat karyops (Triticum aestivum), cv. Alex. Seeds previously disinfected with 4% hypochlorite solution were immersed in a mixture consisting of bacterial cell suspension titrated 1 x 10 ⁹ cfu / ml and 1% methylcellulose in phosphate buffer (w / v). The fungal inoculum (of Fusarium graminearum DSMZ 4527) 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 sterilized by gamma irradiation (2.5 KGy) and then mixed evenly with the fungal inoculum (~ 2 x 10 ⁶ spores / kg soil) and distributed in plastic trays (32/24 cm) 48 hours before sowing. Wheat grains were sown in the prepared substrate, whether or not inoculated with bacteria After 15 and 20 days

^ -2010-01379-2 1 -12- 2010 the number of sunflower and uniformly developed plants was determined. The results are presented in the tab. 6, compared with an untreated control and with a chemical treatment.

Tab. 6. The effectiveness of the biopreparate in preventing the attack of Fusarium graminearum in wheat seedlings (cv. Alex)

Alternative T reatment % sunflower plants 15 days 20 days Substrate sterilized by irradiation, untreated - 95 96 Substrate sterilized by irradiation, the inoculum of F. graminearum, untreated - 25 22 Substrate sterilized by irradiation, F. graminearum inoculum, Propamocarb-HCI Splashing soil, 0.2% i.a., 12 hours before sowing 88 84 Substrate sterilized by irradiation, F. graminearum inoculum, inoculated with B100 Cariopse immersion 10 ⁹ cfu B100 / ml 94 87

Highlighting lactonase production was done through a stepwise experiment. Many pathogenic Gram-negative bacteria use acyl-homoserinlactone (AHL) for expression of group sensitivity (quorum sensing). An enzyme called AHL-lactonase produced by numerous strains of Bacillus sp. it is able to hydrolyze the ring of the homoserin-lactone molecule thus interfering with the communication system of these bacteria (quorum quencing). Chromobacterium violaceum biosensor strain CV026 (McCIean et al., 1997) was used as an indicator. The test itself consisted of inoculating strain B100 in 2 ml Luria Bertani liquid medium in which C6-hexanoyl-homoserin-lactone (C6-HHL) was added at a final concentration of 5 μΜ and incubated overnight at 28 ° C with stirring at 150 rpm. Simultaneously, the same medium only with C6-HHL was used as a negative control to see if the medium induces lactolysis. The test was performed on Petri dishes with LBA medium (Luria Bertani with agar) supplemented with 50 pg / ml kanamycin and inoculated in the beach with CV026 by the distribution in the form of a 250 μΙ of a 12-hour culture. On the surface of the plate thus inoculated were made wells with a diameter of 5 mm in which 100 μΙ of the supernatant of the bacterial culture to be tested were distributed. The petrioles were incubated overnight at 28 ° C and subsequently analyzed for the presence / absence of purple halos. The absence of the purple halos indicated that all C6-HHL in the growth medium was degraded.

B100 strain has very high lactonase production capacity, modulating the group-level communication of other bacteria. So it can be

χ 2019-01379-2 1 -12- 2010 used to combat phytopathogenic bacteria using C6-HHL or to achieve consensus microbial consortia in which quorum sensing modulation is required.

The following series of experiments aimed at highlighting the action to promote plant growth and development due to the production of phytohormones in situ by the microorganism in question. The aim was to highlight the activity of gibberelic phytohormones produced by sporulated gram-positive bacteria, useful for growing plants, in order to verify the biological quality of growth promoter.

The highlighting and estimation of the activity of the gibberelliric phytohormones was performed by the biotest of the α-amylase induction in the barley endosperm coupled with the gel diffusion technique. The stages of the procedure used were:

- induction of α-amylase biosynthesis in barley grain endosperm by phytohormones present in the test extracts;

- the radial diffusion in the agar-starch gel of the enzymes synthesized under the action of phytohormones and the consecutive hydrolysis of the starch;

- Highlighting the area of action of the enzyme by coloring the substrate.

Reagents used in this test were:

Agar gel - starch. In 100 ml substrate buffer pH 6.9 diluted with 100 ml water or hot dissolved 2.5 g agar and 1 g starch; boiled until it became clear. Substrate buffer solution pH 6.9; 590 mg sodium acetate + 1.47 g veronal sodium + 1.4 g NaCl + 780 mg CaCl ₂ were dissolved in distilled water and then 62 ml 0.1 N HCl was added and a final volume was added 250 ml with distilled water.

Development solution: 45 ml 0.1 N iodine with 55 ml absolute ethanol Biopreparate extract made by reverse osmosis.

Endosperm of barley. The barley beans were cut from the pallets and the part containing the embryo was removed by cutting.

The way of working involved the following steps:

- deposition of an agar - starch gel in thickness of 2-3 mm on a support represented by cellulose acetate sheets or microscopic slides;

- gel drilling of wells with a diameter of 3 mm;

- depositing in barley endosperm (half grain without embryo) and test extract (culture medium of microorganism);

- incubation 72 h at room temperature in a humid chamber;

- coloring with gel development solution;

- measurement of the diffusion area.

Giberelin phytohormones were extracted from a specific culture medium (LB) in which a suspension of 10 ⁶ cfu / ml bacteria was incubated overnight. extraction

CV-2 Ο 1 0 - 0 1 3 7 9 - -) 2 /

1 -12- 2010 was performed by reverse dialysis of the incubated mixture overnight. The bacterial suspension was passed into a dialysis bag, which was suspended for 24 hours in ethyl acetate (reverse dialysis). The biotest was performed against control reagents, resulting from the extraction, by reverse dialysis, of the LB environment. The B100 strain produced under the given conditions more than 28 pg gibberellic acid equivalents per ml, at over 2/3 of the level produced by a standard strain of Azosprillum brasiliense Sp001, recognized for its high production capacity of gibberell phytohormones.

In other experiments, the mobility test of strain B165 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 B165 was tested for mobility of agar surface migration (swimming) and aggregation due to chemotaxis (swarming), together with a control strain known as immobile, P. putida PCL1760.

The test consisted of the use of fresh cultures that were inoculated by staining in the center of Petri dishes with LB medium supplemented with 0.3% agar (for swimming) and 0.5% (for swarming). B165 strain showed that it possesses both types of mobility, this result also confirming the production by this protease and surfactin strain.

In another series of experiments, the biotestation of the stimulatory effect of the B100 strain was followed, along with other strains on the growth of sunflower seedlings, and it had a total of 12 variants in 4 repetitions each. Each rehearsal had 5 plants, and each variant had 20 plants. Bacterial strains were grown on liquid LB medium at 28 ° C, aerated and stirred at 150 rpm for 16 hours. Sunflower seeds (Favorite cv.) Were disinfected in two stages. The first disinfection was performed in 70% ethanol for 30 seconds with stirring at 60 rpm. After the ethanol was removed, the seeds were rinsed three times with sterile distilled water. The second disinfection was done with 4% sodium hypochlorite solution for 15 minutes. Subsequently, rinsing with distilled water was carried out, from 25 to 25 minutes for two hours. The seeds were inoculated by immersion in 3 ml bacterial suspension in a concentration of 10 ⁷ cfu / ml in phosphate buffer and 2% carboxy-methyl cellulose, then sown in sterile "Cyg" growth bags (Mega International). Plants 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 ). After sowing and throughout the experiment, the bags were moistened with 0.25% Hoagland nutrient solution. The root growth of sunflower plants was analyzed 15 days after sowing (tab. 7).

λ-2310-0 1379-2 1-12-2010

Tab. 7. The total length (mm) of the roots of the sunflower plants (Favorite cv.) At 15 days from sowing.

Alternative Total root length (mm) Uninoculated witness 184.29 b WCS 365 219.05 of B49 b 187 5 b FL400 174.54 b Ps 33 173 5 b 56. 1s 184.75 b Hm s1 200.86 ab Cn s2 214.38 of Sp s2 181.93 b Tm s2 187 b B100 225.79 of 77.3 292 6 b

B100 strain showed the highest capacity to stimulate the development of sunflower seedlings, statistically assured against the uninoculated control. The activity of stimulating plant growth of strain B100 was also tested against wheat, Triticum aestivum, cv. Alex. In order to inoculate, the wheat karyops were disinfected in two stages. The first disinfection was performed in 70% ethanol for 30 seconds with stirring at 60 rpm. After the ethanol was removed, the seeds were rinsed three times with sterile distilled water. The second disinfection was done with 4% sodium hypochlorite solution for 15 minutes. Subsequently, rinsing with distilled water was carried out, from 25 to 25 minutes, for two hours. The disinfected karyops were kept at 4 ° C, in the dark, on moistened filter paper. The B100 strain was grown on liquid LB medium at 28 ° C and 150 rpm for 16 hours. Seed inoculation was performed at two concentrations of 10 ⁶ or 10 ⁸ cfu / ml. Bacterial karyopsis was performed by immersion in bacterial suspension for 15 minutes, at room temperature, and stirring at 20 rpm. As a control, wheat karyops were used, sterilized as above and immersed in sterile distilled water.

Wheat cariopses inoculated according to the method described above were germinated in Petri dishes (9.4 cm in diameter), on 0.8% agarized water, in number of 20 karyops / plate, in 5 repetitions. Each variant consisted of 100 karyops. The incubation temperature was 28 ° C. Comments were made regarding the number of seeds germinated at 24, 48 and 72 hours.

To determine the influence of B100 bacteria on the development of wheat seedlings, wheat karyops inoculated with bacterial suspension at a concentration of 10® or 10® ufc / ml were distributed in Petri dishes (with agarized water, 9.4 cm plate diameter), each 10 seeds / plate, in 3 repetitions (30 karyops per variant). 5 ml of the medium were added to the agarized water

C-2 010-01379-2 1-12-2010

1 O nutrient mineral medium for plants of the formula: 0.4 g NH4H2PO4; 2.4 g KNO ₃ ;

1.6 g Ca (NO ₃ ) 2; 0.8 g MgSC> 4, agarized with 2 g per liter. The plates were incubated in the dark at 28 ° C. Observations were made regarding the stimulating effect of root growth and wheat seedlings after 72 hours, determining root length and seedling height.

Determinations regarding the influence of the strain on the germination and development of the seedlings were made compared to a control represented by sterile distilled water, and against strains of bacilli with proven phytostimulatory activity. The results, presented in the tab. 8 and 9, support the existence of a phytostimulatory effect exerted by B100 bacteria on wheat seedlings, and especially on the root system.

Tab. 8. Influence of bacterial inoculum on germination capacity of wheat karyops. (cv. Alex).

Bacterial strain taxonomic classification Inoculum concentration (ufc / ml) % of cariopse germinated after 24 h 48 h 72 h Mt sterile distilled water - 75 88 92 B49b Bacillus subtilis 10 ⁶ 79 89 98 FL400 Paenibacillus graminis 10 ⁶ 71 88 88 B100 Bacillus amyliquefaciens var. plantarum 10 ⁶ 88 82 99

Tab. 9. Testing the stimulating effect of wheat plant growth (cv. Alex) by the tested bacteria strains (72 hours post-inoculation).

Bacterial strain Inoculum concentration (ufc / ml) Germination Nr. Rad. Red length. (Cm) seedling height (cm) From 30 weeks % B49b 10 ⁶ 24 80 4 3.79 2.02 B49b 10 ⁸ 29 96.7 4 3.05 1.89 FL400 10 ⁸ 26 86 3 3.93 2.27 B100 10 ⁶ 29 96.7 4 4.05 2.52 B100 10 ⁸ 30 100 4 4.27 2.62 Mt - 25 83.3 4 3.68 2.54

DL 5% 0.36 0.29

The ability of strain B100 to produce enzymes with a role in mineralization of plant material was also tested, determining cellulase, protease and amylase activity. Cellulase activity was determined by

Ο Μ - β ί 3 7 9 - 2 I -12- 2010 seeding on minimal medium, without carbon source, distributed in petri dishes, in which 1% carboxy-methyl cellulose was added. They were incubated at 28 ° C for 5 days and then stained for 30 minutes with 0.3% Congo red, followed by rinsing with tap water and fixing the dye by incubation for 15 minutes with 10% acid. acetic. The decomposition of the carboxy-methylcellulose substrate and, therefore, cellulase production was indicated by the appearance of a clear area around the bacterial growth. B100 strain produces cellulase, which is a feature of the plantarum subspecies of Bacillus amyloliquefaciens.

The production of amylase was evidenced by sowing in the form of a strip on agar nutrient (NA) supplemented with 0.4% soluble starch. The plates were incubated at 28 ° C for 48-72 hours, then flooded with iodine solution in potassium iodide. The clear areas around the bacterial growth, after adding the iodine solution indicated the breakdown of the starch in the environment, therefore, the production of amylase. Tests have indicated that B100 strain produces amylase.

Protease production was tested on minimal mineral medium, supplemented with 5% casein. The strains of test bacteria were inoculated and the plates were incubated at 28 ° C for 48-72 hours. After incubation, agar plates were treated with 50% methanol, 10% glacial acetic acid, 40% distilled water, and haloes around bacterial colonies were stained with a solution of 60 mg / l Coomasie Blue R-250 in 10% acetic acid. The results established that the B100 strain is a protease producer.

Because the results revealed that the B100 strain of B. amyloliquefaciens produces enzymes involved in the degradation of plant material, it was also tested for the mineralization ability of the organic substrate (by determining the respiration, using as plant material substrate and the release of soluble carbohydrates and phosphorus. from plant material).

In order to highlight the degradation capacity of the plant material, an experiment was carried out in which the oxygen consumption was tracked and the release of different compounds from plant material treated with strains of microorganisms. The vegetable material (hairs of hairy moss in the case of gram-positive sporulated bacteria) was ground in the blender and then passed on the 0.250 mm sieve. 10 g of powder were packed in polyethylene bags, which were sterilized by gamma irradiation (at IRASM, IFIN, Bucharest). 0.1 g of the sterile powder was aseptically taken aseptic and brought aseptically into a sterile 50 ml Erlenmayer. To the finely ground powder, 19 ml sterile phosphate buffer was aseptically added. The contents were homogenized by shaking and then inoculated with 1 ml of microbial suspension, normalized to 10 ⁸ cfu / ml. It was kept in the shaker for 24 hours, at a temperature of 28 ° C, after which it was aseptic in a breathing vessel.

c \ -201 0 - 01379-2 '-12- 2010 ί

Strathox (Strathkelvin Instruments Limited, Glasgow, United Kingdom). Respiration / carbon dioxide determination measurements were performed for 12 hours. After performing the respiration determinations, the supernatant was filtered off, in which the Total Organic Carbon (TOC) was determined with a Formacs HT apparatus (Skalar Analytical BV, Breda Netherlands), reducing carbohydrates (with DNS reagent) and total soluble phosphorus (with molybdate). of ammonium and chlorostan reagent).

In order to determine the mineralization capacity of the plant substrate, 7 bacterial strains were tested: 56.1 s; 77.3; 58.2, 77.1s and 82.1s from soil samples from southern Bărăganul; B1OO isolated from garlic rhizosphere (South Oltenia) and Mz.sl isolated from pea soil (South Dobrogea) and 3 Trichoderma strains: Td67 and Td85 isolated from South Bărăgan and Tm, isolated from South Oltenia. The samples were analyzed in comparison with two controls, one control without bacterial inoculum and one control in which the bacterium was grown in the usual growth medium (Luria Bertani liquid).

The results are presented in Table 10. These results demonstrate the existence of high activity of hydrolysis of plant material by most strains of microorganisms tested, including by B100.

Tab. 10. The activity of degradation of the plant material by the strains of microorganisms tested *.

Stem Breathing (mg / l O ₂ consumed, hourly average) Total organic carbon content in supernatant (mcg / l) Soluble carbohydrates (mcg / l) Soluble phosphorus (Mcg / l) 56.1s 0.12 + 0,03c 0.24 + 0,02c 1.67 ± 0,06c 0.02 + 0,01c 77.3 1.56 ± 0,32b 3.87 ± 1.17b 12.24 + 0,28b 17.87 + 2,17b 58.2 1.51 + 0,05b 4.54 ± 1,05b 12.64 ± 0,41b 16.54 ± 1,25b 77.1s 1.54 ± 0,24b 4.54 ± 1,25b 12.64 ± 0,41b 18.42 ± 1,54b 82.1s 1.85 ± 0,18ab 5.87 ± 3.17ab 22.24 ± 0,28ab 27.87 ± 4,17ab B100 2.15 ± 0.04 7.24 + 2.52 29.87 ± 0.36 35.24 ± 3.52 Mz.sl 1.62 ± 0,12b 3.24 ± 1,12b 11.87 ± 0,63b 15.24 ± 2,27b Td67 1.58 ± 0,28b 3.87 ± 1.72b 12.24 ± 0.28 17.87+ 3.23b Td85 2.24 ± 0.14 7.54 ± 1.25 27.64 ± 0.81 36.44 ± 4.07 Tm 1.87 ± 0,16ab 5.24 ± 2,85ab 16.72 ± 0,74b 15.42 ± 2,23b

'The values followed by the same letter do not differ significantly for P> 0.05.

The negative control confers real-time information on the behavior of ideal samples, without biological activity of degradation of the plant substrate, under the conditions of the test. in this variant,

¢ ^ 2010-01379-2 1 -12- 2010 The plant substrate was not inoculated with microorganisms and was maintained under identical incubation conditions with those of the test samples. It provides information on the maximum amount of oxygen that could be found in samples without biodegradation activity. The positive control provides information on the development capacity of the analyzed strain, under the conditions of cultivation on the usual growth medium. This environment allows the microorganism to multiply due to proper feeding and under the incubation conditions offered during the test.

The B100 strain has a very high degradation capacity of the plant material, at the level of fungi of the genus Trichoderma, mushrooms widely used to accelerate the development of the plant material.

The ability to produce polyamines by the selected bacteria was also tested. The mechanism of action by which the vegetable mulch of legumes, and in particular of the hairy mosses, exerts the action of stimulating the growth of the plants is the one of modulation of the level of polyamines in plants (compounds with action of regulating the metabolic processes of plants recognized in the last decade) . The modulation is achieved by the contribution of endogenous polyamines and precursors, compounds formed due to the decomposition of the vegetable material of legumes, with a high protein content. The gram-positive sporulated bacteria that are most suitable for the formation of the bioactive mulch are therefore those microorganisms that have the highest capacity to produce polyamines from the plant material of hairy bean. It was worked with hairy pea powder, obtained according to the method already described. 0.1 g of plant material powder was aseptically taken from the sterilized powder, which was aseptic in a sterile 50 ml Erlenmayer. A further 19 ml sterile phosphate buffer was aseptically added to the finely ground powder. It was homogenized by stirring and then inoculated with 1 ml bacterial suspension, from culture for 12 hours on LB medium, normalized to 10 ⁸ cfu / ml. It was kept in the shaker for 72 hours, at 28 ° C, after which the bacterial culture was separated by centrifugation at 5000xg for 20 min at 4 ° C. The supernatant was acidified with perchloric acid until a final concentration of 5% was obtained. The acidified supernatant was derivatized by dance (Smith and Meeuse, 1996, Garcia-Moruno et al., 2005, Cassan et al., 2009). The polyamines were aspirated and identified by thin layer chromatography, using mixtures chloroform-triethanolamine (9: 1) and n-hexane- ethyl acetate (2: 1) and comparing them with the values of standard danylated compounds. The silicon plates were observed in UV light, the selected spots were quantitatively passed from the plate into a tube. Silica gel was eluted from polyethylene danylated with methanol-toluene solution (9: 1). Fluorescence was measured by

<2010-013792 1 -12- 2010 spectrofluorimetry, using excitation light of 415 nm and emission light of

510 nm.

The results showed that the B100 strain has the highest production capacity of polyamines (compounds mainly involved in plant response to different forms of stress) from the plant material of hairy bean. Its capacity is higher than the standard strain Az39 of Azospirillum brasiliense, cited in the literature as producing polyamines. (Cassana et al., 2009, Cadaverine production by Azospirillum brasilense and its role in plant growth promotion and osmotic stress mitigation. Eur J. Soil Biology 45: 12-19).

In another series of experiments the ability of B100de bacteria to solubilize phosphorus from its insoluble compounds was tested. To determine the solubility of mineral phosphorus, the Pikovskaya agar medium was used, which contains (per liter): 0.5 g yeast extract, 10 g glucose, 5 g Ca ₃ (PO ₄ ) ₂ , 0.5 g (NH ₄ ) ₂ SO ₄ , 0.2 g KCl, 0.1 g MgSO ₄ .7H ₂ O, 0.0001 g MnSO ₄ .H ₂ O, 0.0001 g FeSO ₄ .7H ₂ O and 15 g agar. In this environment, B100 strain bacteria produced halo around the colonies, so they solubilized mineral phosphorus.

The ability to solubilize organic phosphorus was tested on PSM (phytate screening medium), which contains (per liter): 10 g glucose, 4 g sodium phytate, 2 g CaCl ₂ , 5 g NH ₄ NO ₃ , 0.5 g KCI, 0.5 g MgSO ₄ -7H ₂ O, 0.01 g FeSO ₄ -7H ₂ O, 0.01 g MnSO ₄ H ₂ O, 15 g agar. And on this medium the bacteria of strain B100 produced halo around the colonies, so they solubilized the organic phosphorus from phytates.

In order to determine the influence of strain B100 on the bioavailability of selenium for wheat and maize plants, greenhouse and field experiments were performed. In the greenhouse experiments we worked with growth substrate (red preluvosol: meal: sand - 2: 1: 1, distributed in vegetation vessels with an area of 0.050 m ² and with a volume of 15 l), to which treatments were applied of soil at the seeding site (in the furrow) at a ratio of 1 ml per vegetation vessel (corresponding to 200 l / ha).

Corn kernels (PR36D79 Pioneer) and wheat (cv. Boema) were disinfected by maintaining 5 minutes in 5% hypochlorite and repeated washing with distilled water. The disinfected grains were placed in the vegetation vessels (2 corn kernels in one nest, 20 uniformly distributed wheat grains). At the sowing place of each grain were applied treatments with the solution of application, 1 ml for the two corn kernels, 50 pl for each grain of wheat. The application solution contained 10 ⁷ or 10 ⁸ cfu strain B100, 3,0g / l sucrose; 0.5g / l K ₂ HPO ₄ ; 0.2g / l MgSO ₄ x7H ₂ O; 0.1 g / l NaCl; 7 g / l yeast plasma

^ 2010-01379-2 1-12-2010

supplemented with 100 ± 5 mg Se / 1OO plasmolysed 0.5g / l sodium glutamate; 0.2g / l K2M0O4. Bacteria introduced as a concentrated suspension in the solution obtained by dissolving each ingredient in standard hard water in a row (considered in the literature as a standard for well water).

Standard hard water was obtained by mixing 68.5ml solution A with 17ml solution B in a 1000ml berzelius. It was then diluted with about 800ml of distilled water and the pH was brought to 6-7 with the addition of O, 1N NaOH. It was transferred to a 1000 ml graduated flask and made with distilled water.

Solution A was prepared as follows: 4g of calcium carbonate were weighed and transferred into a 50ml conical flask, with a minimum amount of distilled water. Or slowly added 82 ml of 1N HCl, stirring the contents. When all the calcium carbonate was dissolved, the solution was diluted to about 4OOml with distilled water and boiled to remove CO ₂ . The solution was cooled and then two drops of methyl red solution were added and neutralized with 1N ammonia solution to an intermediate orange color. It was transferred quantitatively into a 1000ml graduated flask.

Solution B was prepared as follows: exactly 1,613g of magnesium oxide was weighed and transferred into a 500ml vial with a minimum amount of distilled water. 82 ml of 1N HCl was added. It was slowly reheated until dissolved, diluted to about 400 ml with distilled water and boiled to remove CO ₂ . The solution was cooled and then two drops of methyl red solution were added and neutralized with a 1N ammonia solution to an intermediate orange color. It was transferred quantitatively into a 1000 ml graduated flask, made up to the mark with distilled water and transferred to storage in a polyethylene bottle.

The experimental variants that have been realized are:

V-ι - untreated control with (seeds / soil);

V ₂ - control treated in the furrow with seleniated application solution, not bacterial;

V ₃ - soil treated with B. amyloliquefaciens B100 10 ⁷ in the application solution;

V ₄ - soil treated with B. amyloliquefaciens B1OO 10 ⁸ in the application solution

V ₅ - control foliar treatment on the 14th day after sunrise (5 ml solution 0.1 mg% Se as sodium selenate, equivalent to 10 g / ha selenium).

From the experiments carried out, approx. 2..3 g of vegetable material, when the wheat has reached the twinning phase, respectively when the corn has reached the phase of 8 complete leaves. The plant material was weighed to an accuracy of 1 mg and brought into a 250 cm ³ stoppered balloon. 20 cm ³ of concentrated nitric acid were added and left at room temperature for 2 days. Thereafter, 2 cm ³ concentrated perchloric acid was added; and a refrigerant with

Μ-2 Ο 1 ο - ο 1 3 7 9 - 2 1 -12- 2010 spiral. It was placed on a sand bath with a temperature of 180 ° C, at the chemical niche and boiled for 16 hours. The refrigerant was removed, 1 cm ³ of concentrated sulfuric acid was added, followed by evaporation under the niche for approx. 3 hours, up to a volume of 1-2 cm ³ . The solution was cooled, 1 cm ³ of concentrated nitric acid was added and kept on the water bath for 10 minutes. The mineralized thus obtained was transferred quantitatively into a Berzelius glass of 100 cm ³ . To the sample thus disaggregated were added 5 cm ³ masking solution after which the pH of the solution was brought to 2 with ammonium hydroxide solution. 5 cm ³ of 0.1% 2,3-diamino-naphthalene solution was added and left in the dark for two hours. After the formation of the complex, the solution was transferred quantitatively into a separating funnel. The piazselenolic complex formed was repeatedly extracted with cyclohexane 2x5 cm ^3, stirring intensely for 2 minutes. The organic phases were combined and the fluorescence was determined at an excitation light of 380 nm and an emission of 519 nm. The obtained results are presented in the tab. 11. They demonstrate that strain B100 is a strain that has high ability to bioavailable selenium for wheat and maize plants.

Tab.11. Levels of total selenium (gg / kg) in plant tissues from seeds treated in the furrow with a selenium solution and strain B100.

Experimental variant Wheat Corn Vi - untreated control (seeds / soil); 44.3 ± 5.2 ^C 46.3 ± 4.5 ^C V ₂ - control treated in the furrow with the seleniated application solution, not bacterialized 54.6 ± 4.6 ^b 56.7 ± 3.7 ^b V ₃ - Soil treated with B. amyloliquefaciens B100 10 ⁷ in the application solution 66.2 ± 5.5 ^ab 64.6 ± 6.3 ^ab V ₄ - Soil treated with B. amyloliquefaciens B100 10 ⁸ in the application solution 72.4 ± 4.8 ^a 72.8 ± 5.3 ^a V ₅ - control foliar treatment day 14 68.8 ± 7.1 ^a 71.2 ± 5.6 ^a

The B100 strain was also tested in a large field experiment in Dobrogea. The bacteria were applied in the furrow at the same time as the sowing by the use of specific agricultural equipment for applying solutions in the furrow, at working pressures between 0.5 ... 3 bar of the application equipment by the spray in the furrow and at an average speed of movement of the sowing unit of 7 km / h. The application solution included the same components, which were dissolved in well water. The experiment was performed in Latin square, with 4 variants in 4 repetitions.

The 4 variants of the field experiment were:

V ₁ - untreated control (seeds / soil);

ce 2 3 1 Ο - Ο 1 3 7 9 - 2 ί -12- 2010

V ₂ - control treated in the furrow with seleniated application solution, not bacterial;

V ₃ - soil treated with B. amyloliquefaciens B1OO, 10 ⁸ in the application solution;

V ₄ - day foliar treatment control during spike phase in bellows (500 liters 2 mg% Se solution, in the form of sodium selenate equivalent to 10 g / ha selenium).

From the experiment were taken samples in the phenophase of grain formation and at harvest, grain samples. In these samples the total selenium level was determined by wet digestion of soil samples with a mixture of nitric, sulfuric, perchloric acids for the conversion of all molecular species Se into selenate (SeO ₄ ² ), its reduction with NaBH ₄ to H ₂ Se and its dosing by HG-AAS (hydride generation with selenium dosing by flame atomic absorption spectroscopy). The results are presented in the tab. 12.

Tab.12. Influence of inoculation treatment in the furrow with B1OO strain of B. amyloliquefaciens on selenium uptake and accumulation (pg / kg).

Experimental variant Plant material Wheat grains Vi - untreated control (seeds / soil); 39.5 ± 5.2 ^C 106.3 ± 4.5 ^c V ₂ - control treated in the furrow with the seleniated application solution, not bacterialized 53.6 ± 4.6 ^bc 116.7 ± 5.7 ^b V ₃ - soil treated with B. amyloliquefaciensBIOO 10 ⁸ in the application solution 79.8 ± 8.2 ^a 142.7 ± 12.3 ^a V ₄ - control foliar treatment day 14 62.8 ± 7.1 ^b 121, ± 7.9 ^b

B100 strain of B. amyloliquefaciens stimulates the accumulation and uptake of selenium by wheat plants under field conditions and can be used for selenium biofortification of wheat harvest.

Claims (1)

1. Stem of Bacillus amyloliquefaciens subsp. plantarum B100, with deposit number NCAIM (P) B001362, characterized in that it is antagonistic to phytopathogenic fungi of the soil Fusarium graminearum, Alternaria spp, Botrytis cinerea, Sclerotinia sclerotiorum, Sclerotium cepivorum, Gaeumannomythia rhyme, Pyramidium, Pyramidium, Pyramids solani, Fusarium oxisporum f. sp. radicis-lycopersici, Sclerotium bataticola and against the bacteria that affect the fruit trees during the flowering, Erwinia amylovora, Pseudomonas ice ⁺ , due to the production of lipopeptide and polycetidic antibiotics and of hydrolytic enzymes, proteases and lactonase, and which also stimulates activity plants, due to the endophyte production of growth factors, and mineralization capacity of the plant material, due to the production of amylase, phytase and cellulase, and which solubilizes inorganic phosphorus, bioavailability to selenium and stimulates the taking and accumulation of selenium by wheat and maize plants.