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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
  • A01G18/00—Cultivation of mushrooms
  • A01G18/10—Mycorrhiza; Mycorrhizal associations
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
  • A01N63/22—Bacillus
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
  • A01N63/27—Pseudomonas
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus
  • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/38—Pseudomonas
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/38—Pseudomonas
  • C12R2001/39—Pseudomonas fluorescens

Definitions

• the invention relates to means for improving the growth of plants, more especially forest trees, by perfecting the techniques of controlled mycorrhization.
• the invention therefore aims to provide new bacteria capable of greatly accelerating the symbiosis between a mycorrhizal fungus and the host plant, and thereby allowing an increase in the growth of the plant.
• the invention also aims to provide a method of mycorrhization of high efficiency and .des compositions usable in the implementation of this method.
• the bacterial strains of the invention are characterized in that they are capable of specifically stimulating the formation of mycorrhizae by mycorrhizal fungi, these strains exerting an antagonistic effect with respect to fungi competing with the inoculated fungus.
• the specific stimulating effect on the inoculated fungus is such that the rate of mycorrhization, i.e. the ratio between the percentage of mycorrhized roots formed and the total number of roots, is significantly increased by at least about 10% compared to the system without the addition of bacteria.
• the antagonistic effect vis-à-vis a competing fungus is such that the percentage of mycorrhizal roots formed by it is significantly reduced to a value less than about 10%.
• the invention indeed provides means both for strengthening and accelerating the establishment of the symbiosis between the inoculated fungus and the host plant and for inhibiting the establishment of the competing fungus. It is thus possible to get rid of the soil disinfection operation which is currently essential before sowing and inoculation.
• Bacterial strains according 1'invention are constituted by Gram positive bacteria of the gender Bacillus.
• bacteria of this genus mention will be made, for example, of the species Bacillus subtil! S, in particular that hereinafter called A2 deposited at the CNCM (National Collection of Cultures of Microorganisms) on June 11, 1991 under No. 1 -1112 and Bacillus sp (hereinafter designated A15) deposited with the CNCM the same day, under the number 1-1113.
• Other bacteria of great interest for mycorrhization are Gram negative bacteria of the genus Pseudomonas.
• strain B5 and B8 The Pseudomonas sp and Pseudomonas fluorescens strains (hereinafter called B5 and B8 respectively), deposited at the C.N.C.M. June 11, 1991 under the respective numbers 1-1114 and 1-1115, are very effective in this regard. These strains were subjected to molecular characterization. As reported in Example 1, strain B8 is characterized by the presence in its 16S ribosomal DNA of the sequence:
• This strain is also characterized by its polypeptide profile, such as that shown in FIG. 7, obtained by one-dimensional electrophoresis under denaturing conditions on gel.
• the invention relates more specifically to the bacterial strains originating from lr. rhizosphere of forest trees.
• the bacterial strains of the invention are characterized in that they are isolated from the coats of mycorrhizae and from the carpophores of ectomycorrhizal fungi.
• the bacteria of the invention constitute auxiliaries of great interest with regard to ectomycorrhizal fungi.
• Ectomycorrhizae are found in many forest species. They are formed by many basidiomycete and higher ascomycete fungi.
• Fungi of this type having particularly high symbiotic efficacy include Laccaria s (e.g. Laccaria laccata, Laccaria __ ⁇ _____ L_____),
• Hebeioma sp for example: H. crustulinifQrme, H. cyl ndrosporus
• r Paxillus involutus r RhJZQPQgQn Sp for example R. vinicolor
• Suillus sp Scleroderma sp, Boletus edulis ..
• the efficiency of the strains of the invention with respect to fungi competing with the above ectomycorrhizal fungi is particularly high.
• T. terrestris one of the most common contaminants in the nursery, leads to an almost total inhibition of the development of this fungus.
• This effect is also important vis-à-vis undesirable strains compared to a given selected fungus, which can therefore include one of the fungi mentioned above, since it was not chosen for mycorrhization.
• Bacterial selectivity on the ectomycorrhizal fungus involves the emission of gaseous compounds by bacterial isolates.
• gas chromatographic analysis coupled with mass spectrometry has made it possible to identify, in cultures of Pseudomonas type bacteria, extracted using ethyl ether, various gaseous compounds, such as acid oleic, isoamyl alcohol and 2-phenyl ethanol. The issuance of such compounds therefore constitutes a characteristic of the strains of the invention.
• the bacterial strains of the invention are also characterized by their capacity to degrade the intercellular cement and to facilitate the penetration of the hyphae between the cells thanks to at least one of the following enzymatic activities: endoglucanase, cellobiose hydrolase, pectate lysase or xylanase.
• the bacterial strains of the invention are characterized in that they are as obtained by a process comprising:
• the invention also relates to a process for obtaining bacterial strains as defined above.
• the invention therefore relates to the use of the above bacterial strains for the preparation of compositions which can be used as adjuvants for the controlled mycorrhization of plants.
• compositions are characterized in that they comprise a bacterial strain as defined above, in association with an inert solid or liquid vehicle.
• a bacterial suspension advantageously containing from 10 7 to 1012, preferably from 108 to 1010 bacteria / 1.
• These suspensions contain adjuvants necessary for preservation such as magnesium sulfate, polyethylene glycol or glycerol. These doses will be easily adjusted by those skilled in the art taking into account the effectiveness of a given strain.
• the suspension medium is an inert liquid vehicle. Conveniently, sterile water can be used.
• the suspension is diluted at the time of use.
• This suspension can be inoculated as a mixture with a solid vehicle, such as a gel, or a porous particulate material, inert towards bacteria.
• a solid vehicle such as a gel, or a porous particulate material, inert towards bacteria.
• Suitable gelling materials include polymers of plant or animal origin, hydrogels, alginates being particularly preferred.
• the particulate materials mention will be made of vermiculite, peat, or mixtures thereof, and expanded clay.
• the bacterial strain is " in the form of a liquid or solid inoculum mixed with the fungus.
• the bacterium is incorporated into the mycorrhizal inoculum, advantageously at doses of 106 to 109 bacteria per gram of dry weight of mycelium.
• a vehicle is used to handle and apply the inoculum easily. It is more especially a solid vehicle, in particular a gel or a porous particulate material as mentioned above.
• An advantageous mixed inoculum is thus in the form of calcium alginate beads.
• the inoculum can be directly associated with the seed.
• Mixed inocula particularly preferred for the culture of conifers such as Douglas, or oak comprise at least one of the bacteria of the invention in association with L.accata.
• the invention relates in particular to mixed inocula of L. laccata with one of the bacteria deposited at the C.N.C.M., mentioned above.
• mixed inocula include H. crustuliniforme or P.involutus associated with the bacteria of the invention.
• the mycelial inoculum used together or not with the bacteria is advantageously prepared as described in "Mycorrhizae of trees and cultivated plants", by DG Strullu, ed. Lavoisier, Paris, 1991. This preparation includes solid fermentation of the substrate.
• a liquid fermentation is carried out followed by inclusion in an inert vehicle such as a gel.
• the invention also relates to a method of controlled mycorrhization of plants characterized in that it comprises the use of an effective amount of bacterial strain as defined above.
• the invention relates in particular to the mycorrhization of forest species.
• conifers such as Douglas fir, spruces, pines, larches and tsugas or leafy species such as oaks, beeches and eucalyptus trees.
• FIGS. 1 to 7 respectively represent:
• FIG. 6 the DNA restriction fragments of strains of the invention after agarose gel electrophoresis, compared with known strains, and
• Example 1 selection of bacteria that aid mycorrhization.
• Douglas plants are obtained from seeds of Douglas pseudotsuga menziesii (Mirb).
• the bacterial strains are isolated from the carpophores, and surface sterilized mycorrhizae, of Laccaria laccata associated with young Douglas plants in pot, greenhouse and nursery and plantation experiments.
• the sporocarps are brush cleaned and broken to open them. Pieces of tissue from inside the hat are homogenized in sterile water using an UltraturaxR homogenizer. The mycorrhizae are washed with running water, surface sterilized in 1.5% NaCIO, 2 min, rinsed 20 times in sterile water and homogenized under the same conditions as the sporocarp tissues.
• the sterilization of the surface is checked by spreading the water from the last rinse on nutritious agar.
• the separate colonies are isolated and returned to culture on the same medium.
• Mycorrhizal isolates are designated by Ax and those of carpophores by Bx, x corresponding to the number of the test.
• Morphological characteristics of the bacterial strains used in the screening are Morphological characteristics of the bacterial strains used in the screening.
• NIT nitrate reductase
• TRP tryptophanase
• ADH arginine dihydrolase
• URE action of ⁇ -glucosidase
• GEL proteolysis of gelatin
• OX cytochrome oxidase
• the bacteria are cultivated for 48 h in a liquid TSB medium (Tryptic Soy Broth) at 25 ⁇ C.
• Amplification Four ⁇ l of the bacterial culture are placed in a microcentrifuge tube containing 96 ⁇ l of the following amplification solution: 500 pmol of each of the two oligonucleotide primers fDl (AGAGTTTGATCCTGGCTCAG) and rDl (AAGGAGGTGATCCAGCC) in dNTP (400 ⁇ M) , Tris-HCl buffer pH 8.8 (10 M), MgC 2 (1.5 mM), KC1 (50 M), Tri on-X (0.1).
• the primers were synthesized and supplied by Bioprobe Systems (Montreuil-sous-Bois, France).
• the tube is then heated to 95 ° C for 10-20 min in order to burst the cells and release the DNA, and 2.5 units of Taq DNA polymerase (Bioprobe Systems, Montreuil-sous-Bois, France) are added .
• a drop of mineral oil (Sigma Chemistry) deposited on the surface of the solution prevents evaporation.
• the tube is placed in a GeneATAQ Controller thermoblock (Pharmacia-LKB) and the reaction mixture is subjected to 25-30 amplification cycles. Each cycle consists of three phases: 2 min at 95 ⁇ C (DNA denaturation), 25 s at 50 ° C (hybridization) and 2 min at 72 ° C (polymerization). All 25-30 cycles are preceded by an initial denaturation period (3 min at 95 ⁇ C) and followed by a final polymerization period (10 min at 72 ° C) _.
• a bacteria-free control is carried out to verify the absence of any contaminating DNA in the reagents.
• the degree and specificity of the amplification are evaluated by electrophoresis on 1% agarose gel (Sambrook et al., 1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York).
• the strain B8 was also the subject of the characterization of its polypeptide profile by one-dimensional electrophoresis under denaturing conditions on discontinuous polyacrylamide gel according to the Laemmli technique in Nature, 227, 680-685, 1970.
• the bacterium was cultivated 48 h, homogenized, and the proteins precipitated with acetone at -20 "C. After centrifugation, the protein pellet was dissolved in the Laemmli migration buffer.
• the polyacrylamide concentration of the concentration gel was 5% and that separation gel of 15%, and the intensity of the current of 15 then 30 mA. After revealing the polypeptides by silver staining, the gel was dried and the optical densities measured with a spectrophotometer at 540 nm.
• FIG. 7 gives the polypeptide profile obtained, with the measurements absorbance on the ordinate and position of the peaks on the gel in mm, on the abscissa. The indication of molecular weights is reported in the upper part of the figure.
• Example 2 Characterization of the strains according to their agonist and antagonist effects defined above.
• the three components of the system (bacteria, fungus and plant) are compared in 95 ml polyethylene cups filled with a mixture not disinfected with vermiculite and peat (1/1. vol / vol) and fungal inoculum (1/10; vol / vol).
• a syringe 5 ml are injected into each container of a concentrated bacterial suspension (more than 108 cells / ml) in 0.1 M MgSO4, 7.H20.
• a control " treatment the fungus and the buffer solution are inoculated, without bacteria. 3 seeds are sown per cup.
• each treatment is represented by a tray containing 40 cells.
• the trays are moved each month on the shelves of -serre to compensate gradients microclimatiq 'ues.
• the photoperiod (16 hours) was the same in the two experiments (daylight supplemented with artificial light).
• the rate of mycorrhization was measured by determining the number of short mycorrhized roots in relation to the total number of short roots, and transformed by arc sin (square root). All results are treated statistically, the differences are considered significant at a risk of 5%.
• the mean value of the mycorrhization rate of each treatment was compared with that of the control.
• the empty part corresponds to short non-mycorrhized roots.
• FIG. 3 The results obtained at the 18th week are reported in FIG. 3.
• the hatched part corresponds to the control without addition of bacteria of the invention, and the black part, to the deficit of mycorrhizae resulting from the effect of the bacteria. .
• isolates tested are those of the previous example, namely A1 to A15 and B1 to B10.
• the three components of the system (bacteria, fungus, plant) are compared under aseptic conditions in glass test tubes (3 x 15 cm) filled with a treated vermiculite-peat mixture (1/1; vol / vol) in an autoclave (120 ⁇ C, 20 min), moistened with the nutritive solution used in the greenhouse and mixed with the fungal inoculum 1:10 (vol: vol).
• the roots are then maintained in axenic conditions, while the aerial part of the plant develops freely outside the tube.
• the plants are left to grow for 4 weeks in a room with a controlled climate (23 ° C during the day, 17 ° C at night, 16 hours of photoperiod with 400 ⁇ E.m-2s-l, 80% humidity).
• the determination of the rate of mycorrhization carried out as in the previous experiments, shows in each case a strong increase compared to the control.
• Laccaria bicolor D-101 (positive control)
• Laccaria bicolor D-101 (positive control)
• the fungal inoculum was prepared using the technique indicated above in Example 1.
• B5 and B8 bacterial isolates were cultured for 8 days with shaking in 300 ml Erlenmeyer flasks containing 100 ml of 0.3% TSB medium in a culture chamber (25 ° C., darkness). These suspensions were then centrifuged (2400g, 10 min), the supernatant was removed and the bacterial cells were resuspended in 100 ml of 0.1 M magnesium sulfate. This bacterial suspension was diluted in 7.5 liters of reverse osmosis water. The final suspension contained approximately 1012 bacterial cells. For the control treatment (without bacteria), 100 ml of 0.1 M MgSO 4 were diluted in 7.5 liters of water.
• the fungal inoculation was carried out at the rate of 2 liters of inoculum per m. . Uninoculated treatment was performed.
• the bacteria diluted in water were inoculated using a watering can at the rate of 2.5 liters per tray (101 cfu.m-2). These two applications having been carried out, the 2 inocula were mixed with the soil to a depth of approximately 10 cm.
• results obtained show the stimulating effect of mycorrhization by L-laccata and L-biCQlor exerted by B5 and B8 in the nursery. These results are confirmed under axemic conditions.
• Example 4 Effects of Bacteria of the Invention on Mycorrhizae of L.accata with the Pedunculate Oak (Sequential Inoculation).
• Seedlings of peduncle oaks are obtained from acorns collected in the autumn, soaked in water at 41 ° C for two hours (to prevent necrosis of cotydelons caused by Ciboria batschiana) r powdered with one gram per kilo of iprodione (against Penicillium spp. and other storage molds) and kept at 4 "C.
• L.laccata is mixed in peat beds from the nursery at a dose of 1.1 l of inoculum / m2, then the acorns are sown.
• the L.laccata strain is that used in the other examples relating to Douglas-fir.
• the seedlings are grown under a large polyethylene tunnel (6 x 42 m) in a layer of peat 20 cm thick, watered with a mist system.
• the same peat in the same tunnel has previously been used for 7 years for -Growth oak or 'beech alternately to avoid 1'apparition of root diseases such as Pythium ultimum.
• the bacteria are inoculated using a suspension in 0.1 M MgSO 4.
• the bacteria are grown for one week in 500 ml of TSB (Trysic Soy Broth) medium on a stirring table at 25 ° C.
• TSB Transsic Soy Broth
• the cultures which are then in stationary phase are centrifuged in 300 ml tubes at 2400 g for 15 minutes and the pellet is resuspended in 600 ml of 0.1 M MgSO 4.
• Bacteria five bacterial strains obtained according to Example 1 are used, namely:
• strains are cultivated for 8 days at 25 ° C. in glass flasks containing TSB liquid medium at a concentration of 3 g / 1.
• the concentrated bacterial suspensions (more than 10 11 cells per ml) are centrifuged (2400 g, 10 min), the supernatant is eliminated and the centrifugation residue is resuspended in 200 ml of 0.1 M MgSO 4 or in 500 ml sterile water (in the case of an alginate inoculum).
• the mixed Laccar-ia laccata mycelium and the bacteria are resuspended in a solution of sodium alginate (10 g / liter) containing crushed peat (50 g / liter). Small 4 mm beads of mycelium and bacteria are obtained by dripping the suspension in 100 g per liter of calcium chloride.
• the nursery floor is disinfected with cold methyl bromide.
• Laccaria laccata is inoculated at the rate of 2 liters of peat-vermiculite inoculum per m.2. Five bacterial treatments are carried out with each of the above bacteria, inoculated at three different doses: 105, 107 and 109 cfu per m for B6 and 10 * 5, 108 and 1010 cfu per m.2 for the other 4.
• mycorrhization in the control of 60% It is noted that the bacterial treatments with A2, A15, and B8 significantly increase the establishment of ectomycorrhizal symbiosis. The rates of mycorrhization with these bacteria are indeed respectively 88%, 85% and 83%.
• the soil has been disinfected as described above.
• the plate was divided into plots, separated from each other by uninoculated and unseeded areas.
• the plots were inoculated with L.laccata (1 liter of 1 alginate beads per m, containing the fungus and the bacteria).
• the alginate beads in the controls were bacteria-free. The crop was kept well watered throughout the growing season. After 3 and 5 months of culture, the mycorrhization rates in the controls are 26% and 83% respectively.
• the biomass also appears increased when the bacteria of the invention encapsulated with L.laccata are applied.
• These bacteria are particularly useful in controlled mycorrhization techniques in the forest nursery on disinfected soils as well as advantageously on non-disinfected soils, which avoids the use of disinfectants such as methyl bromide.

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• 1991
  • 1991-06-26 FR FR9107882A patent/FR2678281A1/fr active Granted
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