NOVEL STRAINS OF AZOSPIRILLUM, METHODS OF GROWING THE STRAINS, COMPOSITIONS CONTAINING THEM AND USE THEREOF AS BIOFERTILIZER

Abstract: Bacteria of Azospirillum brasilense or lipoferum having enhanced pectinolytic activity relative to wild type Azospirillum are useful as biofertilizers for cereal crops. Presently preferred is A. brasilense strain ATCC No. 39199. A. brasilense strains having both enhanced pectinolytic activity and resistance to pesticides such as fungicides and herbicides are particularly useful. Presently preferred is ATCC No. 39200. Also, A. brasilense strains having resistance to pesticides are useful as biofertilizers including presently preferred strain ATCC No. 39201. Effective biofertilizing amounts of one or more of these strains may be incorporated into or applied onto soil in which cereals are grown directly, or in admixture with a suitable carrier, such as peat, or as a seed coating.

Description: NOVEL STRAINS OF AZOSPIRILLUM, METHODS OF GROWING THE STRAINS, COMPOSITIONS CONTAINING THEM AND USE THEREOF AS BIOFERTILIZER Background of the Invention Nitrogen is an essential plant nutrient. Unfortunately, it is usually not present in soil at concentrations sufficient for agricultural production of commercial crops. It therefore must be provided to the crops in the form of fertilizer. Of the commercially important crops, cereals such as corn, wheat, rice and barley require particularly large amounts of fertilizer. Leguminous crops, for example, soybeans, peas, beans and clover are able to obtain part of their nitrogen requirements from atmospheric nitrogen. This is accom¬ plished by virtue of a symbiotic relationship between legumes and soil bacteria of the genus Rήizobium. Such bacteria synthesize assimilable nitrogen compounds from atmospheric nitrogen and make them available to legum¬ inous plants. In recent years, naturally-occu ing bacteria of the genus Azospirillu (formerly known as Spirillum) have been found in association with cereals. Azospirillum bacteria are able to make atmospheric nitrogen avail¬ able to cereals, thus reducing fertilizer requirements. This association may also result in increased yields. For example, Azospirillum has been shown to increase total ear yield and average number of ears per plant in sweet corn; to increase panicle weight per hectare, percentage nitrogen present in seeds and panicle number per plant in Sorghum bicolor; and to increase leaf weight, ear yield, seed dry weight and total nitrogen OMH yield in Zea mays. [Kapulnik, Y. , et al., Experimental Agric. 17: 179-187 (1981).] Naturally occurring Azospirillum bacteria are free- living, aerobic, gram-negative, motile and nitrogen fixing. They generally prefer organic acids as carbon sources, e.g. malic or lactic acid, and fix nitrogen in the absence of a combined nitrogen source, under micro- aerophilic conditions (low oxygen tension) . They also have relatively short shelf lives and generally are not resistant to commonly used, commercial fungicides, herbicides and other pesticides or to antibiotics re¬ leased by different soil microorganisms. Finally, they have relatively low pectinolytic activity, that is, relatively little ability to break down pectin present in the cell walls of cereal plants in order to render their cell walls more permeable to minerals, hormones and the like and possibly to the bacteria themselves. In order to overcome certain disadvantages associated with naturally-occurring Azospirillum strains, the present invention proves novel strains having rela¬ tively improved survivability, or resistance to common¬ ly used pesticides or increased pectinolytic activity or a combination of these traits. Such strains are able to enhance crop yields or reduce nitrogen fer¬ tilizer requirements, or both. OMPI . VIrO Summary of the Invention Bacteria of the genus Azospirillum may be used as bio¬ fertilizers for cereal crops to provide enhanced crop yields or reduced fertilizer requirements or both. Azospirillum bacteria which may be so used include those having pectinolytic activity relative to the pectinolytic activity of wild type strains in the range from about 1.5:1 to 20:1, preferably in the range from about 5:1 to 15:1. Presently available bacteria in accordance with the invention are of the species brasilense and include the preferred strain deposited with the American Type Culture Collection, Rockville, Maryland 20852, United States of America, pursuant to the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure under accession number ATCC No. 39199. Additional bacteria within the scope of the present invention are of the species lipoferum. Such bacteria which additionally possess resistance to amounts of herbicides or fungicides which would kill or substantially retard the growth of wild type Azospirillum bacteria are particularly useful as bio- fertilzers. One such strain which is presently preferred has been deposited with the American Type Culture Collection pursuant to the provisions of the Budapest Treaty under accession number ATTC No. 39200. in addition, bacteria of the genus Azosperillum which possess pectinolytic activity in the aforementioned range and contain above about 1 percent dry weight poly-beta-hydroxybutyrate, preferably from about 15 percent to about 35 percent dry weight based upon the weight of the bacterium, are particularly useful. OMPI Desirably, the bacteria possess all three characteristics, i.e., pectinolytic activity in the stated range, resistance to amounts of herbicides, fungicides, or both, which would kill or substantially retard the growth of wild type Azospirillum bacteria and poly- beta-hydroxybutyrate content in the amounts set forth. In addition to the individual bacteria of the preceding types the invention also contemplates mixtures of the bacteria. Such mixtures may include two or more types of bacteria, e.g., bacteria having increased pectino¬ lytic activity and bacteria having resistance to herbicides or fungicides. Alternatively, the mixtures may be of bacteria of the same type but of different specific characteristics, e.g., bacteria having pectino¬ lytic activity in a given range admixed with bacteria having pectinolytic activity in a different range or bacteria having resistance to certain fungicides and herbicides admixed with bacteria having resistance to other fungicides and herbicides. Bacteria of the genus Azospirillum useful as biofertil¬ izers for cereal crops may be prepared by treating a naturally-occurring strain of an Azospirillum species to induce mutation therein, growing the treated bacteria under suitable conditions so as to select for bacteria having pectinolytic activity relative to that of wild type strains in the range from about 1.5:1 to 20:1 and recovering the resulting bacteria. By growing the bacteria so recovered in the presence of one or more herbicides, fungicides, or both, to which resistance is desired, the herbicides or fungi¬ cides being present in amounts sufficient to substan- tially retard the growth of or kill bacteria lacking _OMPI resistance, recovering the bacteria which grow under these conditions, growing them in the presence of in¬ creased amounts of the same herbicides or fungicides, and recovering the then-growing bacteria, one may prepare bacteria which also possess resistance to various amounts of herbicides, fungicides, or both. Additional bacteria also having increased poly-beta- hydroxybutyrate content may be prepared by fermenting bacteria of the genus Azosprillum which have both enhanced pectinolytic activity and resistance to herbicides, fungicides, or both, on a suitable carbon source in the absence of nitrogen and at about 0 percent dissolved nitrogen for a period of time sufficient to permit an increase to a desired level in poly-beta-hydroxybutyrate content. Further bacteria in accordance with this invention have resistance to amounts of herbicides, fungicides, or both, which substantially retard the growth of or kill wild type Azospirillum strains. Presently available bacteria are of the species brasilense and include the preferred strain deposited with the American Type Culture Collection pursuant to the provisions of the Budapest Treaty under accession number ATCC No. 39201. Additional strains within the scope of the invention are of the species lipoferum. Such bacteria which additionally contain above about 1 percent dry weight poly-beta-hydroxybutyrate, preferably from about 15 percent to about 35 percent dry weight poly-beta-hydroxybutyrate based upon the dry weight of the bacterium, are particularly useful as biofertilizers. Bacteria having resistance to amounts of a herbicide or _o..?ι_ fungicide which substantially retard the growth of or kill wild type Azospirillum strains may be prepared by growing bacteria of an Azosprillum species in the pres¬ ence of amounts of the herbicide or fungicide sufficient to substantially retard the growth of or kill the bac¬ teria lacking the desired resistance, recovering the bacteria which grow under these conditions, growing the bacteria in the presence of increased amounts of the same herbicide or fungicide and recovering the then- growing bacteria. Bacteria having resistance to amounts of herbicides or fungicides which substantially retard the growth of or kill wild type Azospirillum strains and also having poly-beta-hydroxybutyrate contents greater than about 1 percent, preferably in the range from about 15 percent to about 35 percent, dry weight based upon the dry weight of the bacteria may be prepared by first * preparing bacteria having resistance to the herbicides or fungi- cides using the method described hereinabove and ferment¬ ing the resulting bacteria on a suitable carbon source in the absence of nitrogen and at about 0 percent dis¬ solved oxygen for * a period of time sufficient to permit an increase in the amount of poly-beta-hydroxybutyrate present in the bacteria. The novel Azospirillum strains of this invention, singly or in combinations, may be combined or admixed in effec¬ tive biofertilizing amounts with a suitable agronomically acceptable carrier to produce biofertilizer compositions. They can also be used to coat cereal seeds prior to planting. By using the bacteria, biofertilizer composi¬ tions containing the bacteria or bacterial-coated cereal seeds, one may reduce fertilizer requirements or obtain increased yields, or both, in cereals such as maize, corn, sorghum, wheat, setaria and panicum. The invention also provides a method for growing a desired strain of Azospirillum comprising inoculating a single colony of the desired bacterial strain onto a suitable liquid culture medium, vigorously aerating the culture medium for an appropriate period of time, trans¬ ferring the culture medium containing the bacterial strain into a fer entor containing the liquid culture medium while the bacterial strain is in its logarithmic growth phase, growing the bacteria to a desired cell density and recovering the resulting bacteria from the fermentor. Detailed Description of the Invention Naturally-occurring strains of Azospirillum, particu¬ larly strains of Azospirillum brasilense and Azospirillum lipoferum, are useful as biofertilizers. When included in or applied onto soil in which cereal crops are grown, they can reduce the amount of externally provided nitro¬ gen fertilizer necessary or provide enhanced crop yields, or both. However, these naturally-occurring strains are characterized by certain disadvantages. They are generally not resistant to herbicides, fungicides and other pesticides. They are also generally not resis¬ tant to antibiotics produced by different soil micro¬ organisms and excreted into the soil. Furthermore, their relatively short shelf lives prevent them from obtaining the maximum commercial advantages which might otherwise be realized. Pectinolytic activity, that is, the ability to break down pectin present in the cell walls of cereal plants so as to render the cell walls more permeable to minerals, hormones and the like present in soil and possibly also to nitrogen-fixing bacteria such as those of the genus Azospirillum is relatively low in naturally-occurring Azospirillum strains. Since pectinolytic activity can be correlated with usefulness as biofertilizer, novel strains of Azospirillum having pectinolytic activity relative to the pectinolytic activity of naturally-occurring strains in the range from about 1.5:1 to 20:1 have been created. Presently, the pre¬ ferred strains are of the species brasilense. Option¬ ally, they are available as biologically pure, stable cultures. Particularly preferred are strains having pectinolytic activity relative to the pectinolytic activity of the wild type strain in the range from OMPI about 5:1 to 15:1 of which the strain ATCC No. 39199 is presently preferred. Such strains may be produced by treating bacteria of a naturally-occurring strain of an Azospirillum species, e.g., brasilense, to induce mutation in the bacteria and growing the treated bacteria under suitable conditions such that those having pectinolytic activity in the de¬ sired range can be identified or selected for. The novel bacteria having the desired level of pectinolytic activity are then recovered. Although the pectinolytic activity relative to that present in wild type strains may be in the range from about 1.5:1 to 20:1, it is preferred that it be in the range from about 5:1 to 15:1. Various methods of inducing genetic mutation are known and may be employed, including irradiation with ultraviolet light or contact with mutagenic chemicals such as nitrous acid. Preferred strain ATCC No. 39199 was prepared as a mutant of A_^ brasilense. It exhibits pectinolytic activity about 10 times that found in wild type Azosperillum strains. It also has 2 times the nitrogenase activity (i.e., ability to fix nitrogen) of wild-type strains. More particularly, this strain was produced by muta- genesis of colonies of naturally-occur ing A^ bra¬ silense strain cd [Nur, I. et al., J. Gen. Microbiol. 722:27-32 (1981)] using nitrous acid (50mM sodium nitrite in 0.1M Na acetate buffer, pH 4.6, for 20 minutes at 35 C) , and screening resulting mutants for pectinolytic activity. Mutants having pectinolytic activity within the desired range were isolated. Pure cultures were grown on solid media. This procedure is described in greater detail in Example 1. The nitrogenase activity of this and other strains was measured by the acetylene reduction assay using conven¬ tional methods. Both nitrogenase and the acetylene reduction assay are described in detail in Hardy, R.W.F. and Holsten, R.D., (1977), "Methods for measurement of dinitrogen fixation," pp. 451-486 _in: Hardy, R.W.F. and A.H. Gibson (eds.) A Treatise on Dinitrogen Fixation IV: Agronomy and Ecology, J. Wiley and Sons, New York and London. 10 Although bacteria having pectinolytic activities higher than those of wild type Azospirillum strains would render cell walls permeable, it is undesirable for the pectinolytic activity to exceed about 20 times the J activity of wild type strains. Otherwise, the bacteria may completely destroy the cell walls of the cereal plants whose growth is desired. In general, pectinolytic activity about 10 times that of wild type Azospirillum strains is preferred. 20 Bacteria having pectinolytic activity relative to the activity of wild type strains in the range from about 1.5:1 to about 20:1 which additionally possess resistance to amounts of pesticides, e.g., herbicides or fungicides, £ -- J which substantially retard the growth of or kill wild type Azospirillum bacteria are generally preferred over those having only the desired levels of pectinolytic activity. Presently preferred is A_^ brasilense ATCC No. 39200, a strain which also has twice the nitrogenase 30 activity of wild type strains. It is desirable that the strains of Azospirillum be resistant to both fungicides and herbicides and desirably to antibiotics as well. Such strains may be isolated by genetic selection. A naturally-occurring or mutant OMPI strain of Azospirillum is cultured in the presence of an amount of a chemical antagonist such as a pesticide to which resistance is desired for a period of time sufficient to substantially retard the growth of or kill Azospirillum bacteria lacking resistance to the antagonist. Fast growing Azospirillum colonies emerging on the medium are isolated and recultured in the presence of an increased amount of the chemical antagonist. The amount of the chemical antagonist and the period of time for which the bacteria are cultured is again suf¬ ficient to substantially retard the growth of or kill the members of the bacterial population lacking resis¬ tance to the increased amount of the antagonist. This procedure may be repeated as needed, with increasing amounts of chemical antagonist, to provide an Azospirillum strain sufficiently resistant to the antagonist that it is capable of cell multiplication and of entering into a nitrogen-fixing symbiosis in the presence of agricul¬ turally effective amounts of one or more s ' uch chemical antagonists. To produce strains resistant to a plurality of such chemical antagonists, the same procedure is followed except that at each step the Azospi illum strain is cultured in the presence of differing amounts of each of the various chemical antagonists. Thus, bacteria possessing both higher pectinolytic activity and resistance to fungicides, herbicides, or both, may be prepared by treating bacteria of a naturally- occurring strain of an Azospirillum species to induce mutation in the bacteria, growing the treated bacteria under suitable conditions so as to select bacteria having pectinolytic activity relative to that of naturally-occurring strains in the range from about OMPI WΪPO 1.5:1 to 20:1, preferably from about 5:1 to 15:1, re¬ covering the resulting bacteria, growing the resulting bacteria in the presence of amounts of fungicides or herbicides sufficient to substantially retard the growth of or kill the bacteria lacking the desired resistance, recovering the resulting growing bacteria, growing these bacteria in the presence of increased amounts of the same herbicides or fungicides, recovering the then- growing bacteria and reculturing the bacteria so recovered, Additional bacterial strains useful in accordance with the teachings of this invention include novel Azospirillum strains which, in addition to pectinolytic activity within the desired range and resistance to specific pesticides, also contain more than about 1 percent dry weight poly-beta-hydroxybutyrate based upon the dry weight of the bacterium, preferably from about 15 to about 35 percent dry weight poly-beta-hydroxybutyrate. Such bacteria can be prepared by first producing bac- teria having the desired level of pectinolytic activity and optionally desired resistance to specific chemical antagonists and then fermenting these bacteria so chara- * cterized on a suitable carbon source, e.g. malic acid, lactic acid, succinic acid, fumaric acid, salts thereof, or glycerol, in the absence of nitrogen and at about 0 percent dissolved oxygen for a period of time suffi¬ cient to permit an increase in the amount of poly-beta- hydroxybutyrate -present in the bacteria. Other Azospirillum strains useful as biofertilizer are characterized by their resistance to amounts of herbi¬ cides, fungicides, or both, which substantially retard the growth of or kill wild type Azospirillum bacteria. Although resistance to fungicides or herbicides is desirable, resistance to both herbicides and fungicides and optionally to antibiotics as well is most desirable. Presently, the preferred strains are of the species brasilense. Desirably, they are in the form of bio¬ logically pure, stable cultures. An example of such a strain is A^ brasilense ATCC No. 39201, which is a nitrogen fixing strain. Such bacteria may be prepared by growing naturally- occurring bacteria of an Azospirillum strain in the presence of amounts of herbicides, fungicides, or both, sufficient to substantially retard the growth of or kill bacteria lacking resistance thereto, growing the bacteria in the presence of an increased amount of the same herbicides, fungicides, or both, recovering the then-growing bacteria and reculturing the bacteria so recovered. Using this approach, strains of Azospirillum have been isolated which are resistant to the following: (Details of the procedure are presented in Example II.) FUNGICIDES : Captan (1,2,3,6-tetryhydro-N- ( richloromethylthio) -Phthalimide) [Cp] ; TMTD (Tetramethyl thiuram disulphide) [TMTD] ; Caspan (ethyl- mercuro-chloride) [Cs] ; Benlate or Benomyl (1- (butylamino) carbonyl- IH-benzimidozo (-2-yl) Carbamic acid methyl ester) [B] . HERBICIDES : Atrazine (6-chloro-N-ethyl-N'-(1- methylethyl) -l,3,5-triazine-2,4- diamine) [At]; Trybonyl (1,3- dimeth l-3-(2-benzo-thiazolyl) - urea) [T] ; Illoxan (2-(4-(2'4'- dichlorophenozyl) Phenoxyl-methyl- propionate) [I] . OMPI CT/US83/01648 - 14 - ANTIBIOTICS Ampicillin [A] ; Kanamycin [K] ; Streptomycin [S] . BACTERICIDE/ BACTERIOSTAT Sodium Azide [Na] Illustrative strains including several having particu¬ larly desirable resistance characteristics, and their respective levels of resistance to specific chemicals follow. The figures in parentheses are in parts per million (ppm) of the compound, except for Na, which is expressed in millimoles (mM) . Strain Resistant To 1 A(100) T(50) ; 1(50) ; At(50) 2 S(250) T(50) ; 1(50) ; At(50) 3 K(100) T(50) ; 1(50) ; At(50) 4 A(100) S(250) K(100) ; T(50) K50) ; At(50) 5 Na(0.2) ; T(50) 1(50) ; At(50) 6 Na(0.2) S(200: B(20) ; T(50) 1(50) ; At(50) Na(0.2) : S(200) ; B(20) ; Cp(50-100) ; Cs(20) T(50) ; 1(50) ; At(50) 8* A(100) S(250) ; K(50) 9* At(50) B(20) ; Cp(50) 10* Cs(20) ATCC No. 39200* At(50) ; Cp(50) ; TMTD(50) ATCC NO. 39201 At(50); At(50) ; TMTD(50) *Derivative of ATCC No. 39199 possessing enhanced pectino¬ lytic activity as well as the chemical resistances indicated. OMP It will be appreciated by those skilled in the art that the foregoing resistance characteristics are exemplary of resistance to other chemicals within the same or similar chemical classes. Thus, resistance to Captan is representative of resistance to chlorinated hydro¬ carbons generally. Resistance to Caspan is repre¬ sentative of resistance to organic mercury compounds; to Benlate of carbamate compounds; to Atrazine of triazine-heterocyclic nitrogen derivatives; and to Tribonyl of urea derivatives, etc. The invention is therefore not limited to resistance to specific chemicals but embraces the classes of commonly used, agricultural chemicals including pesticides, e.g., herbicides and fungicides. In addition to resistance to herbicides, fungicides, or both, strains are useful which additionally contain more than about 1 percent dry weight poly-beta- hydroxybutyrate based upon the dry weight of the bac- teriu , preferably from about 15 percent to about 35 percent. This latter type strain may be prepared from strains having the desired levels of resistance to agricultural chemicals prepared using methods already described. The resistant strains are then fermented on a suitable carbon source in the absence of nitrogen at about 0 per¬ cent dissolved oxygen for a period of time sufficient to permit an increase . to the desired level in the amount of poly-beta-hydroxybutyrate present in the bacteria. Suitable carbon sources for use in this method include malic acid, lactic acid, succinic acid, fu aric acid, salts thereof, i.e., malate, lactate, succinate or fumarate, or glycerol. OMPI In addition to individual strains, mixtures of strains having different desirable properties may be employed. Alternatively, mixtures of strains having the same property but in differing degree may be used. For example, a mixture of two strains having different levels of pectinolytic activity and two strains each having resistance to different levels of different chemical antagonists may be preferred for some applica¬ tions. Effective biofertilizing amounts of the various novel Azospirillum strains of the present invention may be incorporated into biofertilizer compositions together with a suitable agronomically acceptable carrier. Gen- erally, effective biofertilizing amounts of microorganism will be in the range from about 10^ to lθlO bacteria per gram of the final composition or formulation, preferably from about 1 10^ to 5x10^ bacteria per gram of composition or formulation. Various conventional agronomically acceptable carriers may be employed. One such carrier is peat, e.g. finely ground peat. The carrier may contain additional compo¬ nents, including carbonate, a wetting agent, a suspending agent or an absorbing agent. In addition to such biofertilizer compositions, cereal seeds may be coated, with an effective biofertilizing amount of a bacterium in accordance with this invention. Effective biofertilizing amounts of microorganism typically range from about 10^ to lθlO bacteria per gram of coating formulation, preferably from about 1 x 10^ to 5 x 10^ bacteria per gram. Methods for seed coating are well known to those skilled in the art and are therefore not discussed in detail herein. Fertilizer requirements may be reduced or increased crop yields made possible, or both, by incorporating into or applying onto soil in which cereals such as maize, sorghum, corn, wheat, setaria or panicum are to be grown effective amounts of one or more Azospirillum bacterium according to the present invention or a biofertilizer composition containing one or more such bacterium or a cereal seed coated therewith. Cereals are then grown in a conventional manner. A microbial culture of Azospirillum produced or grown according to the above methods may be applied to crops by any of a number of known methods, including seed coating, wettable powder and granular formulations. One particularly successful formulation suitable for direct application to the soil as a spray is a wettable powder of finely ground peat. This formulation comprises: Finely ground or granular peat; CaCθ3 which alters the pH of the peat to about pH 7.0; A wetting agent: for example Nonyl phenol ethoxy- late - 10 Ethylene oxide (commercially available under the tradena es 9M10 [Dow Chemical Co.], NP-10 or Lanco) . Since it is a liquid, it is generally mixed with equal parts of milled silica before being mixed with the other dry ingredients; A suspending agent: e.g. Sodium ligno-sulphate; An absorbing agent: e.g. a milled silica such as that marketed under the tradename Wessalon S; [Degussa, W. Germany]; and A microbial culture of the desired Azospirillum strain or strains. Alternatively, a microbial culture of an Azospirillum strain may be applied to corn or other relatively large seeds as a coating with an adhesive such as Pelgel (the Nitragin Co., Clearwater, Florida, U.S.A.). Pelgel contains calcium carbonate, sugar and gum arabic. Example III provides a further example of a biofertilizer composition in accordance with the present invention. Most strains of Azospirillum are nitrogen-fixing. Some persons skilled in the art believe that the nitrogen- fixing capability is responsible for the beneficial ef¬ fects on cereal crop yield or fertilizer requirements. Others skilled in the art maintain that there is no causal relationship between nitrogen-fixing capability and the ability to increase crop yield and reduce fertil- izer requirements in cereal crops. The present invention contemplates both nitrogen-fixing and non-nitrogen- fixing strains useful as biofertilizers and is not to be construed as limited except in so far as the strains are limited by the claims set forth hereinafter. While Azospirillum strains have been previously grown in the laboratory, there has existed no suitable manufac¬ turing process for producing the bacteria in commercial¬ ly useful quantities while retaining the desired bacteri- al characteristics. Such a process has been developed. It is described in detail in Example IV. Desired strains of Azospirillum may be grown as follows. A single colony of the desired bacterial strain is inoculated onto a suitable liquid culture medium which OM Ir is then vigorously aerated for an appropriate period of time. The culture medium containing the bacterial strain is transferred into a fermentor containing the liquid culture medium while the bacterial strain is in its logarithmic growth phase. The bacteria are grown to the desired cell density and the resulting bacteria are recovered from the fermentor. One suitable liquid culture medium includes appropriate amounts of K-HP0 4 , KH 2 P0 4 , MgS0 4 *7H 2 0, ferric ammonium citrate, NH.C1 and a carbon source. Another suitable medium contains appropriate amounts of F^HPO., KH-PO., MgS0 4 '7H 2 0, FeCl.,, a nitrogen source and a carbon source. Carbon sources useful in the invention include malate, lactate, succinate, fumarate salts or the acids from which they are derived, or glycerol. Nitrogen sources include ammonia or an ammonium salt. One method of growing desired strains of Azospirillum comprises growing cells of a chosen or desired strain on a solid medium. After dilution plating, a single colony is inoculated onto a liquid culture medium in baffled erlenmeyer flasks with glycerol as the carbon source. A particularly successful liquid culture medium comprises: K 2 HP0 4 , 6.0g/l; KH 2 P0 4 , 4.0g/l; MgS0 4 -7H 2 0, 0.2g/l; Ferric ammonium citrate, O.Olg/l; glycerol in a concentration from 5.0 to 10.0g/l with NH 4 C1 preferably in the concentration * 1.5 to 3.0g/l. The inoculated flasks are vigorously aerated, as on a rotary shaker, until a desired concentration of organisms is reached. It is essential that the contents of these flasks, which are to be used as inoculum for the follow¬ ing fermentation stage be kept in the ' logarithmic growth phase and not reach the stationary phase. The inoculum is inoculated into a fermentor having the same liquid culture medium to which a carbon source and a nitrogen source is continuously added. The carbon source preferably comprises glycerol or may comprise malate, lactate, succinate or fumarate. The nitrogen source preferably comprises ammonia or an ammonium salt. The pH and temperature are controlled during this growth phase. The first stage of this growth takes about 20- 24 hours, and terminates when the dissolved oxygen (D.O.) begins to rise after all the glycerol has been consumed. Additional carbon source is then added to the culture in the fermentor. This carbon source may be of any suitable organic acid, such as a concentrated solution of malic acid, succinic acid or fumaric acid, or additional glycerol. The addition of this carbon source results in decreased D.O. content and increased pH. Growth is continued until the desired concentration of cells is reached. This generally coincides with the point at which the D.O. begins to increase again, indi- eating that all the NH4 + in the fermentor has been exhausted. The absence of H4 + may be confirmed by the Nessler test. [Dawson, R.M.C., et al., Data for Biochemical Research, Oxford Press, page 619.] It is a particular feature of the present invention that it yields extremely high cell densities of Azospirillum, while utilizing an optimal carbon/nitrogen ratio for organism growth. About 0.3% of the dry weight of a naturally-occurring Azospirillum strain comprises a preservative material, poly-beta-hydroxybutyrate. It has been shown in labora¬ tory tests that the presence of increased amounts of poly-beta-hydroxybutyrate enhances long term bacterial survival. [Daws, E.A. and Senior, P.J., Advances in OMPI Microbial Physiology K 135-266 (1973)] In other words, both shelf life of the stored product prior to use and survival of the bacterium in the soil during the initial period of plant growth are enhanced by the presence of poly-beta-hydroxybutyrate in the organism. It was therefore desirable to obtain a method for in¬ creasing the amount of poly-beta-hydroxybutyrate within a desired Azospirillum bacterium in order to enhance its long term survival. It has recently been found that poly-beta-hydroxybutyrate is also useful as a biopolymer, and has piezoelectric properties useful in various electrical apparatus. It will be appreciated that the following method of enrich- ing Azospirilla with poly-beta-hydroxybutyrate is also useful for the production of such material itself, to be followed by its removal from the bacterium. A method of increasing the percentage of poly-beta- hydroxybutyrate in an Azospirillum strain has been developed. It is described in detail in Example V. It involves continuing the growth of a desired culture of an Azospirillum strain in the absence of nitrogen at about 0 percent dissolved oxygen and with a continuously fed carbon source, such as malic acid. Alternatively, a ripe culture of Azospirillum previously grown may be returned to the fermentor and cultured under these conditions. Suitable organic acids useful as carbon sources include succinic acid, lactic acid, fumaric acid and malic acid. Alternatively, the salts of these acids may be used, i.e., succinate, lactate, fumarate and malate. In the absence of nitrogen, the culture does not continue to grow. Instead the carbon source is converted into poly-beta-hydroxybutyrate within the individual Azospirillum bacterium. At the end of this stage, the mature micro-organisms are ready for incor¬ poration into a soil inoculant mixture. Desirably, more than 0.3% of the dry weight of the resulting Azospirillum or bacterium will be poly-beta-hydroxy- butyrate. Preferably, from about 15-35% of the organisms' dry weight will be poly-beta-hydroxybutyrate. It is a particular feature of the present invention that growth of a desired strain of Azospirillum and enrichment thereof according to the above-described methods not only yields extremely high cell densities of Azospirillum, but also allows the harvesting of cells containing a high poly-beta-hydroxybutyrate con¬ centration which is important for survival of the bac- teria and may be important to their ability to overcome competing soil organisms. EXAMPLE I Isolation of Azospirillum having higher pectinolytic activity. Colonies of naturally-occurring A_. brasilense strains were treated with 50 mM sodium nitrite in 0.1 M Na acetate buffer, pH 4.6, for 20 minutes at 35 C to cause mutagenesis. The resulting mutants were screened for pectinolytic activity. A culture of mutant A^ brasilense strains was plated on a solid medium containing 0.5% pectin as the sole carbon source. Of the few colonies that developed, a particu¬ larly large colony was selected and restreaked on fresh solid medium. Of the colonies that then grew, the largest was picked. This is strain ATCC No. 39199. Its pectinolytic enzyme activity was confirmed to be higher than the wild type's by a standard cup-plate assay. [Dingle, J. et al., Sci. Food Agric. zl : l 49 -155 (1953)]. This involved growing A_ ; _ brasilense ATCC No. 39199 on pectin (0.5%) in liquid culture, centrifuging the culture broth, and applying an aliquot of supernatant to a well in an agar plate containing pectin. The pectinases, when present, attack the pectin and leave a clear zone on the plate surrounding the well. Although this is a semiquantitive assay, the results clearly indicated that ATCC No.39199 had activity about 1 ' 0-fold greater than the activity associated with naturally-occurring Azospirillum. EXAMPLE II Isolation of Azospirillum strains resistant to various agricultural chemicals. A naturally-occurring (wild type) strain of A^ brasilense (cd) and A_^ brasilense ATCC No.39199 were each grown on liquid minimal media. Single colonies of each strain were then plated on solid minimal media of the following composition (g/1) : K2HPO4, 6.0; KH2PO4, 4.0; NH4CI, 1.5; MgSθ4*7H2θ, 0.2; ferric ammonium citrate, 0.01; malic acid, 5.0; and Agar, 20.0, containing a low concen¬ tration of one or more agricultural chemicals or anti¬ biotics, on the order of about 5 ppm. The chemicals were each dissolved in 1 ml 95% ethanol at a concentra¬ tion sufficient to produce the desired final concentra- tion in the medium when the 1 ml was added to 1 liter of medium. The ethanol solution was added to the medium after the medium was autoclaved and allowed to cool. Fast growing colonies emerging on the plates were transferred onto the solid minimal media containing an QMPI_ . increased amount of the agricultural chemical, on the order of about 15 ppm, depending upon the amount of resistance exhibited by the Azospirillum to the low concentration of chemical. Again, fast emerging colonies were isolated, purified and replated on media containing an increased amount of the chemical, for example, 30 ppm. These steps were repeated at increasing concentrations of the chemical until the desired resistance was achieved. EXAMPLE III Formulation for application of Azospirillum to crops. An inoculant formulation was prepared in the following manner. The figures in parenthesis are in percent w/w calculated on the final product. The following ingredients were mixed: Peat which, after drying at 105°C, was milled so that at least 80% passed a 325 mesh [particle size below 45mm] (57) ; CaC03 (3) ; [In the event this amount does not correct the pH of the peat to 7.0, the percentages may be altered to obtain a total of 60% for peat and CaC03 combined] Wetting agent 9M10 (1) ; Sodium ligno-sulphate (3) ; and Wessalon S (11) , a portion of which was mixed with the NP10 before addition to the other dry ingredients, The mixture of the above ingredients was sterilized by Gamma rays at a dose of 5 Megarad. To the sterile mixture, the culture of Azospirillum, as obtained at the end of the growth process, was added without centri- fugation or other manipulation at a concentration of 25%. As the culture had a cell concentration of about 1 to 3 x lθlO/ml, the final formulated inoculant had a concentration of about 5 x 10 $ cells per gram. The inoculant was stored in aseptic sealed packages and maintained at room temperature until use. The sprayed inoculant was obtained by adding lOOOg of inoculant to 100 1 of water. This quantity is sufficient to spray one hectare of soil. In applications of Azospirillum strains to crops in a granular formulation, between 5 and 60 kilograms are required per hectare depending upon the particular crop and the plant stand. Cereal crops on which formulations comprising a microbial culture of Azospirillum according to the present inven¬ tion are particularly useful include maize, sorghum corn, setaria, panicum and wheat. EXAMPLE IV Growth of agriculturally useful quantities of Azospirillum. The cells of a chosen strain of A^ brasilense were grown in Petri dishes on a solid medium of the following OMPI composition (g/1) : K2HPO4, 6.0; KH2PO4, 4.0; NH4CI, 1.5; gSθ4*7H2θ, 0.2; Ferric ammonium citrate, 0.01; Malic acid, 5.0; Agar, 20.0. After dilution plating, a single colony was inoculated onto liquid culture medium in baffled erlenmeyer flasks filled to 20% of nominal capacity. The medium had the following composition (g/1): K2HPO4, 6.0; KH2PO4, 4.0; gSθ4*7H2θ, 0.2; Ferric ammonium citrate, 0.01. The carbon source was glycerol rather than malic acid in a concentration from 5.0 to 10.0, while the NH4CI added was in the range from 1.5 to 3.0. The medium was adjusted to pH 7.2 and the flasks were aerated on a rotary shaker at 200 ' rpm. The temperature was maintained at 35°C. After about 20-24 hours, the Optical Density (O.D.) measured at 660 n was 5.0 to 9.0, corresponding to an organism concentration of 1 to 5 x loVml. A quantity of inoculum from the culture flasks was inoculated into liquid culture medium in a fermentor, nominal volume 16 liters. The inoculum was 5 to 10% of the final volume of the fermentor, so that the initial Optical Density was 0.1. The composition of the culture medium was: K2HPO4, 2.0; KH2PO4, 1.3; MgSθ4*7H2θ, 0.2; H2O, 0.2; Ferric ammonium citrate, 0.01; Glycerol, 10.0; NH4CI, 3.0. The pH of the medium was adjusted, after sterilization, to 7.0 by addition of 5N NaOH. A Silicone antifoam agent, Sigma Antifoam C Emulsion, was added by means of a peristaltic pump controlled by a foam sensing probe. The growth temperature was maintained at 35°C. Dissolved oxygen (D.O.) was maintained at between 30 and 60% CM * « ?Λ « saturation (although D.O. up to 100% saturation at this stage will produce the desired results) by adjustment of both agitation and sparging. This adjustment may be made automatically by a D.O. controller or manually when only a D.O. analyzer is present. The pH is regulated at 7.0 by a pH regulator which adds 5N NaOH when necessary, The first stage of fermentor growth required about 20 to 24 hours and terminated when the D.O. began to rise, because all the glycerol had been consumed. Malic acid as a carbon source was added in a 500g/l solution. The initial addition was made equivalent to 7.5g/l of malic acid. This solution was adjusted to pH 5.5 with NaOH. As a result of the malic acid addition, the D.O. de- creased and the pH rose to 6.0-6.2. The concentrated malic acid was continually fed into the fermentor through the acid peristaltic pump of the pH regulator. The utilization of the malic acid by the bacteria raised the pH, which the regulator maintained at 7.0 by adding malic acid. Growth continued until all the NH4 " in the fermentor was exhausted, at which point the D.O. again began to rise. The absence of NH4 + was determined by the Nessler test on a sample of the culture from which the cells had been removed. At this stage the Optical Density was between 22 and 28 and the organism concentration about 1 to 3 x lθl°/ml. EXAMPLE V Enrichment for poly-beta-hydroxybutyrate. Fermentation of the culture produced according to the method of Example IV was continued and the dissolved oxygen reduced to about 0 percent, but avoiding anaerobic conditions. The pH controller maintained the flow of malic acid for 3 to 4 more hours, during which there was no further growth and the malic acid was converted to poly-beta-hydroxybutyrate in the absence of nitrogen, all the nitrogen in the culture medium having been previously consumed. The culture broth so produced was utilized in formulations for application to plants. The amount of poly-beta-hydroxybutyrate accumulated in the bacteria was determined to be in the range 15-35% using known methods [Williamson and Wilkinson, J. Gen. Microbiol. 3^:198-209 (1958); Law and Slepcky, J. Bacteriol. _82:33-36 (1961)].

Claims: - 29 - WHAT IS CLAIMED IS: 1. A bacterium of the genus Azosperillum useful as a bio¬ fertilizer having pectinolytic activity relative to the pectinolytic activity of the wild type strain in the range from about 1.5:1 to about 20:1. 2. A bacterium in accordance with claim 1 having pec- tynolytic activity in the range from about 5:1 to 15:1. 3. A bacterium in accordance with claim 1 of the species brasilense. 4. A bacterium in accordance with * claim 1 of the species lipoferum. 5. A biologically pure, stable culture of a bacterium in accordance with claim 1. 6. A bacterium in accordance with claim 3 comprising Azobrasilense ATCC No. 39199. 7. A bacterium in accordance with claim 1 additionally having resistance to an amount of a chemical antagonist capable of substantially retarding the growth of or of killing wild type Azosperillum strains. 8. A bacterium in accordance with claim 7, wherein the chemical antagonist is a pesticide. 9. A bacterium in accordance with claim 8, wherein the pesticide is a herbicide or a fungicide. 10. A bacterium in accordance with claim 9, wherein the herbicide is atrazine, trybonyl or illoxan. 11. A bacterium in accordance with claim 9, wherein the fungicide is captan, TMTD, capsan, or benlate or benomyl. 12. A bacterium in accordance with claim 7 comprising ATCC No. 39200. 13. A bacterium in accordance with claim 1 or claim 7 containing more than about 1 percent by weight poly-beta- hydroxybutyrate based upon the dry weight of the bacter- ium. 14. A bacterium in accordance with claim 13 containing from about 15 percent to about 35 percent by weight poly- beta-hydroxybutyrate based upon the dry weight of the bacterium. 15. A mixture of bacteria, each being in accordance with claim 1. 16. A method of producing a bacterium in accordance with claim 1 comprising treating bacteria of a naturally- occurring strain of an Azospirillum species to induce mutation in the bacteria, growing the treated bacteria under suitable conditions so as to select for bacteria having pectinolytic activity relative to that of the wild type strain in the range from about 1.5:1 to 20:1 and recovering the resulting bacteria. 17. A method in accordance with claim 16, wherein said mutation-inducing treatment comprises contacting the Azospirillum with sodium nitrite. 18. A method in accordance with claim 16, wherein the suitable conditions comprise growing the mutant bacteria on a solid medium containing about 0.5 to 2.0 percent pectin. OMPI 19. A method in accordance with claim 16, wherein the Azospirillum species is brasilense. 20. A method in accordance with claim 16, wherein the Azospirillum species is lipoferum. 21. A bacterium produced by the method of claim 16. 22. A method of producing a bacterium in accordance with claim 7 comprising treating bacteria of a naturally- occurring strain of an Azospirillum species so as to induce mutation in the bacteria, growing the treated bacteria under suitable conditions so as to select bac¬ teria having pectinolytic activity relative to that of the wild type strain in the range from about 1.5:1 to 20:1, recovering the resulting bacteria, growing the resulting bacteria in the presence of amounts of a chemi¬ cal antagonist or antagonists sufficient to substantially retard the growth of or kill bacteria lacki'ng resistance, growing the surviving bacteria in the presence of an increased amount of the chemical antagonist or antago¬ nists, recovering the then-growing bacteria and growing the bacteria so recovered. 23. A bacterium produced by the method of claim 22. 24. A biofertilizer comprising an effective biofertiliz¬ ing amount of a bacterium in accordance with any of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21 or 23 or a mixture thereof, and a suitable agronomically ac¬ ceptable carrier. 25. A biofertilizer composition in accordance with claim 24, wherein the effective biofertilizing amount is in the OMPI range from about 10> to lOl bacteria per gram of com¬ position. 26. A biofertilizer composition in accordance with claim 25, wherein the effective biofertilizing amount in the range from about 1 x 10^ to 5 x 10^ bacteria per gram of composition. 27. A biofertilizer composition in accordance with claim 24, wherein the suitable agronomically acceptable carrier is peat. 28. A biofertilizer composition in accordance with claim 24, wherein the carrier additionally includes carbonate. 29. A biofertilizer composition in accordance with claim 24, wherein the carrier additionally includes a wetting agent, a suspending agent and an absorbing agent. 30. A cereal seed coated with an effective biofertilizing amount of a bacterium in accordance with any of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21 or 23, or a mixture thereof. 31. A method of reducing fertilizer requirements or of providing increased crop yields in cereals or both com¬ prising incorporating in or applying to soil in which the cereals are grown an effective amount of a bacterium in accordance with any of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21 or 23, a biofertilizer composi¬ tion in accordance with any of claims 24, 25, 26, 27, 28 or 29, or a coated cereal seed in accordance with claim 30. OlSΛ_ " 32. A method in accordance with claim 31, wherein the cereal is maize, sorghum, corn, wheat, setaria or panicum. 33. A bacterium of the genus Azosprillum useful as a biofertilizer having resistance to an amount of a chemical antagonist capable of substantially retarding the growth of or of killing wild type Azospirillum strains. 34. A bacterium in accordance with claim 33, wherein the chemical antagonist is a pesticide. 35. A bacterium in accordance with claim 33, wherein the pesticide is a herbicide or a fungicide. 36. A bacterium in accordance with claim 35, wherein the herbicide is atrazine, trybonyl or illoxan. 37. A bacterium in accordance with claim 35, wherein the fungicide is captan, TMTD, capsan, or benlate or benomyl. 38. A bacterium in accordance with claim 33 comprising ' ATCC No. 39201. 39. A bacterium in accordance with claim 33 of the species brasilense. 40. A bacterium in accordance with claim 33 of the species lipoferum. 41. A biologically pure, stable culture of a bacterium in accordance with claim 33. 42. A bacterium in accordance with claim 33 containing more than about 1 percent by weight poly-beta-hydroxy- butyrate based upon the dry weight of the bacterium. 43. A bacterium in accordance with claim 42 containing from about 15 percent to about 35 percent by weight poly- beta-hydroxybutyrate based upon the dry weight of the bacterium. 44. A mixture of bacteria each being in accordance with claim 33. 45. A method of producing a bacterium in accordance with claim 33 comprising growing bacteria of an Azosprillum species in the presence of an amount of a chemical antago¬ nist or antagonists sufficient to substantially retard the growth of or to kill bacteria lacking the desired resistance, recovering the bacteria which grow under these conditions, growing the bacteria in the presence of an increased amount of the chemical antagonist or antag¬ onists and recovering the then-growing bacteria. 46. A method in accordance with claim 45, wherein the Azosperillum species is brasilense. 47. A method in accordance with claim 45, wherein the Azosperillum species is lipoferum. 48. A bacterium produced by the method of claim 45. 49. A method of producing a bacterium in accordance with claim 42 comprising growing bacteria of an Azosperillum species in the presence of an amount of a chemical antag- onist or antagonists sufficient to substantially retard the growth of or to kill bacteria lacking the desired resistance, recovering the bacteria which grow under these conditions, growing the bacteria in the presence of an increased amount of the chemical antagonist or antag- onists, recovering the resulting bacteria and fermenting OMPI the bacteria so recovered on a suitable carbon source in the absence of nitrogen and at about 0 percent dissolved oxygen for a period of time sufficient to permit an in¬ crease in the amount of poly-beta-hydroxybutyrate present in the bacteria. 50. A method in accordance with claim 49, wherein the Azosperillum species is brasilense. 51. A method in accordance with claim 49, wherein the suitable carbon source is malic, lactic, succinic or fumaric acid, or a salt thereof, or glycerol. 52. A bacterium produced in accordance with the method of claim 49. 53. A biofertilizer composition comprising an effective biofertilizing amount of a bacterium in accordance with any of claims 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 48 or 52, or a mixture thereof, and a suitable agro¬ nomically acceptable carrier. 54. A biofertilizer composition in accordance with claim 53, wherein the effective biofertilizing amount is an amount in the range from about 10^ to 10^ bacteria per gram of composition. 55. A biofertilizer ' composition in accordance with claim 54, wherein the effective biofertilizing amount is an amount in the range from about 1 x 10^ to 5 x 10^ bacteria per gram of composition. 56. A biofertilizer composition in accordance with claim 53, wherein the suitable agronomically acceptable carrier is peat. 57. A biofertilizer composition in accordance with claim 53, wherein the carrier additionally includes carbonate. 58. A biofertilizer composition in accordance with claim 53, wherein the carrier additionally includes a wetting agent, a suspending agent and an absorbing agent. 59. A cereal seed coated with an effective biofertilizing amount of bacterium in accordance with any of claims 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 48 or * 52, or a mixture thereof. 60. A method of reducing fertilizer requirements or of providing increased crop yields in cereals, or both, comprising incorporating in or applying to soil in which the cereals are grown an effective amount of a bacterium in accordance with any of claims 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 48 or 52, or a mixture therof, bio¬ fertilizer composition in accordance with any of claims 53, 54, 55, 56, 57 or 58 or a coated cereal seed in accordance with claim 59. 61. A method in accordance with claim 60, wherein the cereal is maize, sorghum, corn, wheat, setaria or panicum. 62. A method of growing a desired strain of Azosperillum comprising inoculating a single colony of the desired bacterial strain onto a suitable liquid culture medium for an appropriate period of time, transferring the culture medium containing the bacterial strain into a fermentor containing the liquid culture medium while the bacterial strain is in its logarithmic growth phase, growing the bacterial to a desired cell density and recovering the resulting bacteria from the fermentor. OMPI Www 63. A method in accordance with claim 62, wherein the liquid culture medium contains suitable amounts of •K2HPO4, KH2 O4, M Sθ4*7H2θ, ferric ammonium citrate, NH4CI and a carbon source. 64. A method in accordance with claim 62, wherein the liquid culture medium contains suitable amounts of 2HPO4, KH2PO4, MgSθ4'7H2θ, FeCl3, a nitrogen source and a carbon source. 65. A method in accordance with claim 63 or 64, wherein the carbon source is malic, lactic, succinic or fumaric acid or a salt thereof, or glycerol.. 66.- A bacterium of the genus Azospirillum containing more than about 1 percent by weight poly-beta-hydroxy¬ butyrate based upon the dry weight of the bacterium. 67. A bacterium in accordance with claim 66 containing from about 15 percent to about 35 percent by weight poly- beta-hydroxybutyrate based upon the dry weight of the bacterium. 68. A method of preparing a bacterium in accordance with claim 66 which comprises fermenting a bacterium of the genus Azospe illum on a suitable carbon source in the absence of nitrogen and at about 0 percent dissolved nitrogen for a period of time sufficient to permit an increase in the amount of poly-beta-hydroxybutyrate present to above about 1 percent by weight.

Inventors: AVIV HAIM, FISCHER MEIR, HENIS YIGAL, KANNER DOV, OKON YAACOV, AVIV, HAIM, FISCHER, MEIR, HENIS, YIGAL, KANNER, DOV, OKON, YAACOV
Applicants: BIOTECH GENERAL CORP, BIOTECHNOLOGY GENERAL CORP.
Application year: 1983
Main classification: A01C1
IPC codes: A01C1, C05F11, C12N1, C12P7