Patent Abstract:
A bacterial inoculant is disclosed for controlling root rot in peas caused by Aphanomyces fungus. The inoculum is obtained from general bacterial strains including strains of Pseudomonas cepacia, Pseudomonas fluorescens, Corynebacterium flaccumfaciens, and two other Bacillus strains of uncertain taxonomy.

Full Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of Ser. No. 861,991 filed Feb. 18, 1992, abandoned, which was a continuation of Ser. No. 387,919 filed Jul. 31, 1989 abandoned, which was a continuation in part of Ser. No. 227,810 filed Aug. 3, 1988, abandoned. 
    
    
     FIELD OF THE INVENTION 
     The present invention is generally directed to inoculants for plants, and particularly directed to a biological inoculant effective in controlling root rot of plants, such as peas, caused by the fungus Aphanomyces euteiches. 
     BACKGROUND OF THE INVENTION 
     Farm crops are continually plagued by a variety of pests which can stunt or damage crop growth or even completely destroy the crop. Some of the pests are in the form of weeds which grow similarly to the desired plant and compete for the nutrients provided by soil and water. Other pests are in the form of pathogens such as fungi and bacteria which are found in association with many plants. 
     One of the more serious problems associated with fungal pathogens in plants is root rot. For example, pea root rot caused by the fungus Aphanomyces euteiches is a serious problem in pea-growing areas, particularly in Wisconsin and other Great Lake states. The Aphanomyces fungus infects not only peas, but also snap beans and alfalfa, accounting for 10 to 15% losses in yield. In extreme cases, some fields, where the fungus population has been built up over the period of several years, have become essentially useless for these crops. 
     Despite efforts to develop fungicides and commercially acceptable pea cultivars with resistance to this pathogen, there is presently no commercially available product capable of controlling Aphanomyces. Currently, the best way to avoid the disease loss is to avoid planting susceptible crops in soils with a high population of the Aphanomyces fungus. Unfortunately, the fungus can survive for many years in field soil and a long rotational time to other crops is not practical. As a result, there is a need to find an alternative disease control strategy to eliminate root rot caused by Aphanomyces and possibly other fungi. 
     There is increasing interest in the use of living organisms to control such diseases. Microscopic organisms are present in soil in populations of approximately 1 billion per cubic inch of soil. Some of the microorganisms cause disease and some are beneficial. The beneficial microorganisms are of major interest. It has long been known in agriculture that certain of these microbial inoculants can be used to facilitate the growth of certain plant species or to assist the plants in suppressing particular pathogenic organisms. For example, it has been a common practice to inoculate soybeans and other legumes at planting with bacterial cultures of the genus Rhizobium so that nitrogen-fixing nodules will form as a result of the plant-bacterium symbiosis. 
     Reference is now made to U.S. Pat. No. 4,588,584 to Lumsden, et al. which discloses a particular species of Pseudomonas cepacia which is effective in controlling Pythium diseases of cucumber and peas. There is also much literature on the use of Pseudomonas fluorescens as a biocontrol agent against various plant diseases, but not against the fungus Aphanomyces. The term &#34;biocontrol agent&#34;, as used herein, refers to a living organism which controls diseases. 
     SUMMARY OF THE INVENTION 
     It is therefore an object of the invention to provide a biocontrol agent which is effective in biologically controlling pea root rot in the field. 
     It is also an object of the present invention to provide a biocontrol agent which is effective in reducing plant mortality in peas and other vegetable and field crops. 
     It is further an object of the present invention to provide a process for increasing the crop yield in Aphanomyces-infested soils. 
     These and other objects are met by the present invention which is directed to a process for controlling Aphanomyces fungal diseases of plants by inoculating the plants with an effective amount of an essentially biologically pure culture of a bacterial strain selected from the group consisting of strains AMMA, AMMD, PRA25, 5A, AM, CRK419, and mixtures thereof to control Aphanomyces. 
     The present invention is also directed to a process for increasing seed germination, decreasing plant mortality and increasing yield of a pea plant by inoculating the pea plant with a growth promotional effective amount of an essentially biologically pure culture of a bacterial strain selected from the group consisting of Pseudomonas cepacia and Pseudomonas fluorescens. 
     The present invention is also directed to a biological inoculant for controlling Aphanomyces fungal diseases on plants comprising an essentially biologically pure culture of a bacteria selected from the group consisting of strains AMMA, AMMD, PRAZ5, 5A AM, CRK419, and mixtures thereof. 
     The present invention is also directed to an agriculturally useful composition comprising a pea seed inoculated with an inoculant of either Pseudomonas cepacia or Pseudomonas fluorescens. 
     The inoculum which controls Aphanomyces on field crops, such as peas, is also disclosed in this invention. As used herein, the term &#34;inoculum&#34; means a biological control agent which is introduced onto a host substance or into soil. The inoculum comprises an essentially biologically pure culture of the bacteria mentioned in the previous paragraphs. 
     The bacterial strains and their process of use, disclosed in the present invention, represent a significant advance in controlling Aphanomyces. Because the bacterial strains are a biologically pure culture of a natural biological organism, massive quantities of the inoculum can be applied to the Aphanomyces infested area with little danger of environmental contamination. In view of public concern for ground water contamination and aerial pollution from pesticides, the form of control disclosed in the present invention is an attractive and economic alternative to chemical pesticides and other methods of control. 
     Other objects, advantages and features of the present invention will become apparent from the following specification when taken in conjunction with the accompanying drawing. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a graph illustrating the results of the mass spectrometric analysis of the fatty acid profile of Pseudomonas cepacia AMMA; 
     FIG. 2 is a graph illustrating the results of the mass spectrometric analysis of the fatty acid profile of Pseudomonas cepacia AMMD; 
     FIG. 3 is a graph illustrating the results of the mass spectrometric analysis of the fatty acid profile of Pseudomonas fluorescens PRA25: and 
     FIG. 4 illustrates a schematic view of a plant bioassay useful for observing biocontrol activity. 
     FIG. 5 is a graph illustrating the results of the mass spectrometric analysis of the fatty acid profile of Corynebacterium flaccumfaciens 5A. 
     FIG. 6 is a graph illustrating the results of the mass spectrometric analysis of the fatty acid profile of bacterial strain AM. 
     FIG. 7 is a graph illustrating the results of the mass spectrometric analysis of the fatty acid profile of bacterial strain CRK419. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is directed to improving the growth and survival rate of field crops infested with the fungus Aphanomyces, and particularly the strain Aphanomyces euteiches, by inoculating the field crop with a biologically pure bacterial inoculant of the selected strains of the species Pseudomonas cepaci, Pseudomonas fluorescens, Corynebacterium flaccumfaciens, and a strain of Bacillus. The particular strains of the bacteria involved in the present invention were discovered by the inventor and are identified by the following nomenclature, as presently known: 
     Pseudomonas cepacia AMMA 
     Pseudomonas cepacia AMMD 
     Pseudomonas fluorescens PRA25 
     Corynebacterium flaccumfaciens 5A 
     Bacillus/Corynebacterium sp AM 
     Bacillus CRK419 
     The above-referenced bacterial strains were initially isolated from over 200 strains of bacteria associated with pea plants in the field. The bacterial strains Pseudomonas fluorescens PRA25 and strains 5A and AM were initially isolated from the rhizosphere of healthy appearing pea plants grown in soil at the University of Wisconsin - Arlington Experimental Farms Pea Root Rot Nursery, a naturally infested pea root rot area. The bacterial strains Pseudomonas cepacia AMMA and Pseudomonas cepacia AMMD were initially isolated from the rhizosphere of healthy appearing pea plants grown in containment in soil known to be infested with the Aphanomyces fungus from the University of Wisconsin-Arlington Experimental Farm. The strain of uncertain taxonomy (probably Bacillus) designated CRK419 was isolated from a corn field in which peas were previously cultivated near Fredonia, Ozaukee County, Wis. 
     The targeted bacterial strains were collected in the following manner. The root system from the pea plants was removed and agitated to shake off the excess soil. The hypocotyl and epicotyl segments of the roots were placed in distilled water, sonicated, and thereafter isolated in a plate dilution process, known to the art, in a TSAC (tryptic soy agar cyclohexamide) medium. Cyclohexamide is an anti-fungal agent. Colonies were thereafter selected and screened according to methods known to the art. The bacterial strains were thereafter stored in a DMSO solution at approximately -80° C. until required for use. 
     It has been found that the bacterial strains may be mass produced in culture with relative ease. The strains are cultured in a suitable culture medium such as a commercially available nutrient broth yeast (NBY) extract. As the bacterial strains grow and multiply, essentially biologically pure cultures of the strains are formed which may be collected. The term &#34;biologically pure culture&#34; is used herein to refer to cultures of bacteria that have essentially no concentration of other strains of bacteria. 
     The bacteria strains were then screened for biocontrol activity in soil which has been naturally infested or artificially infested with the Aphanomyces fungus in order to determine which bacterial strains are effective biocontrol agents. 
     The selected biocontrol agents were coated onto the plant seeds, e.g., the pea seeds, prior to planting. A preferred method for coating the seeds is to combine the bacterial strain with a biologically non-interfering liquid carrier for application onto the seeds. A carrier shall be deemed &#34;biologically non-interfering&#34; if it does not prevent the bacterial strains from growing, and if it does not affect Aphanomyces in the absence of the bacteria. The nutrient medium in which the bacteria were cultured has been found to be a satisfactory medium. A suitable fungicide, i.e., captan, may also be coated on the seeds. The cultures survive air-drying after seed coating. The preferred carrier is a water-based liquid, preferably sterile distilled water. 
     Although coating the seed with the bacterial strain is preferred, other processes which provide a convenient means for distributing the bacterial strain to the Aphanomyces fungi fall within the scope and spirit of the invention. For example, the bacterial strain may be directly applied to the soil prior to planting the seeds. 
     Whether the inoculant is coated actually on the plant seed or inserted into the furrows into which the seeds are planted, the inoculant is preferably diluted with a suitable carrier or extender so as to make the inoculant easier to handle and to provide a sufficient quantity of material so as to be capable of easy human handling. Examples of suitable carriers include water, granular minerals, such as vermiculite, soils or peat. 
     To enable others to obtain a culture of these strains, samples have been deposited with the American Type Culture Collection (ATCC), being identified by the accession number and date of deposit as follows: 
     
         ______________________________________              ATCC        Date ofBacterial Strain   Accession No.                          Deposit______________________________________Pseudomonas cepacia AMMA              52796       7/22/88Pseudomonas cepacia AMMD              53795       7/22/88Pseudomonas fluorescens PRA25              53794       7/22/88Corynebacterium flaccumfaciens 5A              53934       7/26/89Bacillus/Corynebacterium AM              53933       7/26/89Bacillus CRK419    53935       7/26/89______________________________________ 
    
     To further identify the bacterial strains, a fatty acid profile for each rhizosphere culture was determined by mass spectrometric analysis. The term &#34;rhizosphere&#34;, as used herein, refers to the zone of soil subject to the influence of the plant roots. 
     With reference to FIG. 1, the results of the tests to determine the fatty acid profile for Pseudomonas cepacia AMMA are presented below in Table 1: 
     
                                           TABLE 1__________________________________________________________________________RT   Area  Ar/Ht           Respon                ECL  Name       %    Comment 1    Comment__________________________________________________________________________                                                  2 1.61340785000      0.081           --    7.051                     SOLVENT PEAK    &lt;min rt 4.4471548  0.036           1.036                12.000                     12:0       0.98 ECL deviates Ref  0.000 6.8284415  0.038           0.969                14.000                     14:0       2.63 ECL deviates Ref -0.001 9.1088547  0.042           0.944                15.493                     Sum In                     Feature 3  4.95 ECL deviates 14:003                                                  30H/16:1 ISC 9.63627640 0.042           0.941                15.819                     16:1 CIS 9 15.97                                     ECL deviates  0.002 9.92825281 0.044           0.940                15.999                     16:0       14.58                                     ECL deviates -0.001                                                  Ref -0.00211.4379844  0.047           0.936                16.890                     17:0 CYCLO 5.66 ECL deviates Ref  0.00011.7141646  0.047           0.936                17.052                     16:1 20H   0.95 ECL deviates  0.00512.0341188  0.047           0.936                17.236                     16:0 20H   0.68 ECL deviates  0.00112.5349443  0.048           0.937                17.524                     16:0 30H   5.43 ECL deviates  0.00412.937733   0.047           --   17.757          --13.05373016 0.048           0.937                17.824                     Sum In                     Feature 7  42.01                                     ECL deviates -0.001                                                  18:1 TRANS                                                  9/t6/c1113.3561307  0.046           0.938                17.998                     18:0       0.75 ECL deviates -0.002                                                  Ref -0.00214.143904   0.066           --   18.452                     --         --14.9225361  0.050           0.943                18.901                     19:0 CYCLO                     C11-12     3.10 ECL deviates Ref 0.00115.2513969  0.055           0.945                19.091                     18:1 20H   2.30 ECL deviates  0.003******8547  --   --   --   SUMMED                     FEATURE 3  4.95 12:0 ALDE ?  unknown 10.9******--    --   --   --              --   16:1 ISO I/14:0                                                  14:0 30H/                                                  16:1 ISO I******73016 --   --   --   SUMMED                     FEATURE 7  42.01                                     18:1 CIS 11/t 9/t                                                  18:1 TRANS                                                  9/t6/c11******--    --   --   --              --   18:1 TRANS 6/t9/c11__________________________________________________________________________Solvent Ar  Total Area          Named Area                  % NAmed Total Amnt                                 Nbr Ref                                        ECL Deviation                                                 Ref ECL__________________________________________________________________________                                                 Shift40785000  174842  173205  99.06   162924 6      0.002    0.001__________________________________________________________________________TSBA [Rev 2.0]      Pseudomonas               0.440               P. cepacia                0.440               P. c. cepacia GC subgroup B                                         0.440__________________________________________________________________________Comparison with TSBA [Rev 2.0 ]: Pseudomonas-cepacia-cepacia GC subgroupB Distance: 3.8051015202530354045505560657075................12:0-*--...............11:0 ISO.30H.*-...............13:1 AT 12-13*-...............14:0-+X---..............16:1 CIS 9..----------*------ ---..........16:1 C*...............16.0...-X----*-----..........17:0 CYCLO.----X+-----.............17:0*-...............16:1 20H-*-...............16:0 20H-*-...............16:0 30H.-+X-..............18:0-*-...............19:0 CYCLO-X+-------... ..........C11-1218:1 20H--*--..............SUMMED.-*-..............FEATURE 3SUMMED......-----*---------X.......FEATURE 7__________________________________________________________________________ 
    
     With reference to FIG. 2, the results of the fatty acid profile for Pseudomonas cepacia AMMD are presented below in Table 2: 
     
                                           TABLE 2__________________________________________________________________________RT   Area  Ar/Ht           Respon                ECL  Name        %   Comment 1    Comment__________________________________________________________________________ 1.61340748000      0.081           --    7.052                     SOLVENT PEAK                                 --  &lt;min rt 4.4461993  0.031           1.036                12.000                     12:0        1.00                                     ECL deviates Ref -0.001 6.8265489  0.038           0.969                14.000                     14:0        2.59                                     ECL deviates -0.000                                                  Ref -0.002 9.1079988  0.042           0.944                15.493                     Sum In                     Feature 3   4.59                                     ECL deviates 14:003                                                  30H/16:1 ISO 9.63430599 0.043           0.941                15.818                     16:1 CIS 9  14.02                                     ECL deviates  0.001 9.92836362 0.044           0.940                16.000                     16:0        16.63                                     ECL deviates Ref -0.00210.0831560  0.070           --   16.092           --11.43616563 0.048           0.936                16.890                     17:0 CYCLO  7.55                                     ECL deviates Ref  0.00011.7141747  0.046           0.936                17.052                     16:1 20H    0.80                                     ECL deviates  0.00512.0341387  0.049           0.936                17.237                     16:0 20H    0.63                                     ECL deviates  0.00212.53211342 0.048           0.937                17.524                     16:0 30H    5.17                                     ECL deviates  0.00412.9341066  0.044           --   17.756           --13.05186323 0.047           0.937                17.823                     Sum In                     Feature 7   39.38                                     ECL deviates 18:1 CIS                                                  11/t 9/t613.3541722  0.047           0.938                17.998                     18:0        0.79                                     ECL deviates -0.002                                                  Ref -0.00314.1431445  0.067           --   18.453           --14.4891051  0.057           --   18.652           --15.9219636  0.048           0.943                18.901                     19:0 CYCLO                     C11-12      4.42                                     ECL deviates Ref 0.00015.2495288  0.057           0.945                19.091                     18:1 20H    2.43                                     ECL deviates  0.00317.1371003  0.043           --   20.191           --  &gt;max rt******9988  --   --   --   SUMMED                     FEATURE 3   4.59                                     12:0 ALDE ?  unknown 10.9******--    --   --   --               --  16:1 ISO I/14:0                                                  14:0 30H/                                                  16:1 ISO I******86323 --   --   --   SUMMED                     FEATURE 7   39.38                                     18:1 CIS 11/t 9/t                                                  18:1 TRANS                                                  9/t6/c11******--    --   --   --               --  18:1 TRANS 6/t9/c11__________________________________________________________________________Solvent Ar  Total Area          Named Area                  % Named Total Amnt                                 Nbr Ref                                        ECL Deviation                                                 Ref ECL__________________________________________________________________________                                                 Shift40748000  223561  218439  97.71   205479 6      0.002    0.002__________________________________________________________________________TSBA [Rev 2.0]      Pseudomonas               0.591               P. cepacia                0.591               P. c. cepacia GC subgroup B                                         0.591__________________________________________________________________________Comparison with TSBA [Rev 2.0]: Pseudomonas-cepacia-cepacia GC subgroupB-Distance: 3.0051015202530354045505560657075................12:0-*--...............11:0 ISO 30H.*-...............13:1 AT 12-13*-...............14:0-+X---...... ........16:1 CIS 9..--------*-+---------..........16:1 C*...............16.0...-------X-+-----..........17:0 CYCLO.------+X----.............17:0*-...............16:1 20H-*-...............16:0 20H-*-...............16:0 30H.-+X-..............18:0- *-...............19:0 CYCLO---*------.............C11-1218:1 20H--*--..............SUMMED.-*-..............FEATURE 3SUMMED......-----+-----X-.......FEATURE 7__________________________________________________________________________ 
    
     With reference to FIG. 3, the results of the tests to determine the fatty acid profile for Psuedomonas fluorescens PRA25 are presented below in Table 3: 
     
                                           TABLE 3__________________________________________________________________________RT   Area  Ar/Ht           Respon                ECL  Name       %    Comment 1    Comment__________________________________________________________________________                                                  21.61340050000      0.080           --    7.047                     SOLVENT PEAK                                --   &lt;min rt3.9584169  0.030           1.069                11.429                     10:0 30H    5.14                                     ECL deviates  0.0064.4441045  0.039           1.041                12.000                     12:0        1.26                                     ECL deviates Ref -0.0025.7595852  0.036           0.996                13.181                     12:0 20H    6.72                                     ECL deviates  0.0036.1224845  0.037           0.988                13.460                     12:0 30H    5.52                                     ECL deviates  0.0059.63230810 0.043           0.942                16.818                     16:1 CIS 9 33.48                                     ECL deviates  0.0019.92529005 0.043           0.941                15.999                     16:0       31.46                                     ECL deviates -0.001                                                  Ref -0.00211.4341943  0.045           0.935                16.889                     17:0 CYCLO  2.10                                     ECL deviates Ref  0.00113.04613300 0.045           0.934                17.821                     Sum In                     Feature 7  14.32                                     ECL deviates -0.001                                                  18:1 CIS                                                  11/t 9/t617.779528   0.026           --   20.569          --   &gt;max rt******13300 --   --   --   SUMMED                     FEATURE 7  14.32                                     18:1 CIS 11/t 9/t                                                  18:1 TRANS                                                  9/t6/c11******--    --   --   --              --   18:1 TRANS 6/t9/c11__________________________________________________________________________Solvent Ar  Total Area          Named Area                  % Named Total Amnt                                 Nbr Ref                                        ECL Deviation                                                 Ref ECL__________________________________________________________________________                                                 Shift40050000  90969   90969   100.00  86707  3      0.003    0.002__________________________________________________________________________TSBA [Rev 2.0]  Pseudomonas      0.661 (P. fluorescens D)           P. chlororaphis  0.661 (P. fluorescens D)           P. aureofaciens  0.515 (P. fluorescens E)           P. fluorescens   0.422           P. f. A.         0.422           P. f. G.         0.320           P. f. C.         0.265__________________________________________________________________________Comparison with TSBA [Rev 2.0]: Pseudomonas-chlororaphis (P. fluorescensD)-Distance: 2051015202530354045505560657075................10.0 30H.-+X-..............12:0X+ -...............12:0 20H.-+-X..............12.1 30H*-...............12:0.30H.+-X..............14:0*-...............16:1 CIS 9.......-X-+----........16:0......---+X---.........17:0.CYCLO--*--..............18:0*-...............SUMMED...---X+----...... ......FEATURE 7__________________________________________________________________________ 
    
     FIG. 5 illustrates the fatty acid profile, determined by mass spectrometer for strain 5A. The exact taxonomical classification of strain 5A is not certain, although it is in the Corynebacterium or Bacillus groups, and it is currently believed that the organism is properly classified as Corynebacterium flaccumfaciens. It is a gram positive, non-motile rod and on NBY forms smooth bright yellow colonies, with margins entire. The bacterial are aerobic, catalese positive, oxidase negative and grow on TTC agar. To further firmly fix the species classification, it would be necessary to perform a thin layer chromatographic analysis of the whole organism methanolysates. The results of the fatty acid analysis are recapitulated in the following Table 4: 
     
                                           TABLE 4__________________________________________________________________________RT   Area  Ar/Ht           Respon                ECL  Name       %    Comment 1    Comment__________________________________________________________________________                                                  21.61340717000      0.081           --    7.051                     SOLVENT PEAK    &lt;min rt6.3284354  0.036           0.979                13.618                     14:0 ISO   1.23 ECL deviates -0.000                                                  Ref -0.0037.74734292 0.040           0.956                14.621                     15:0 ISO   9.51 ECL deviates -0.000                                                  Ref -0.0027.884198650      0.040           0.955                14.713                     15:0 ANIEISO                                54.98                                     ECL deviates Ref  0.0008.3101069  0.043           0.950                15.000                     15:0       0.29 ECL deviates -0.000                                                  Ref -0.0029.32168683 0.042           0.943                15.625                     16:0 ISO   18.77                                     ECL deviates -0.001                                                  Ref -0.0039.92711080 0.043           0.940                16.000                     16:0       3.02 ECL deviates -0.000                                                  Ref -0.00210.9935560  0.045           0.937                16.629                     17:0 ISO   1.51 ECL deviates Ref -0.00211.15039366 0.045           0.937                16.722                     17:0 ANIEISO                                10.69                                     ECL deviates Ref -0.00316.4341161  0.365           --   19.784                     --         --   &gt;max ar/ht17.9082771  0.261           --   20.644                     --         --   &gt;max rtSolvent Ar  Total Area          Named Area                  % Named Total Amnt                                 Nbr Ref                                        ECL Deviation                                                 Ref ECL__________________________________________________________________________                                                 Shift40717000  364215  363054  99.68   345020 8      0.001    0.002TSBA [Rev 2.0]        Bacillus              0.161                 B. polymyxa           0.161__________________________________________________________________________Comparison with TSBA [Rev 2.0]: Bacillus-polymyxa  Distance: 5.670051015202530354045505560657075................11:0 ISO30H.*-...............14:0 ISO- X+-...............14:0X-+ -...............15:0 ISO.-----+--X-.............15:0 ANIEISO..........-------X----+------------.15:0*-...............16:0 ISO.-------+------X......... ...16:1 AX+--...............16:0. X - - - +- - - -...............17:0 ISO.-X+--..............17:0 ANIEISO.---+--X.............__________________________________________________________________________ 
    
     The strain AM is also not unequivocally classified. It appears to belong to the Bacillus polymyxa/circulans/macerans group. It may also, however, be Corynebacterium as well. With reference to FIG. 6, the results of the fatty acid profile for strain AM are presented below in Table 5: 
     
         TABLE 5  RT Area Ar/Ht Respon ECL Name % Comment 1 Comment   1.613 40608000 0.081 --  7.052 SOLVENT PEAK  &lt;min rt 1.811 815 0.020 --  7.485 -- -- &lt;min rt 6.021 620 0.037 0.985 13.381 14:0 ISO E 0.15 ECL deviates -0.007 6.329 3574 0.036 0.979 13.618 14:0 ISO 0.84 ECL deviates -0.000 Ref -0.002 7.749 41315 0.040 0.956 14.621 15:0 ISO 9.53 ECL deviates -0.000 Ref -0.001 7.886 217290 0.041 0.955 14.713 15:0 ANIEISO 50.05  ECL deviates  0.002 Ref  0.001 8.311 968 0.042 0.950 14.999 15:0 0.22 ECL deviates -0.001 Ref -0.001 9.325 82543 0.042 0.943 15.626 16:0 ISO 18.78  ECL deviates  0.000 Ref -0.001 9.928 11351 0.044 0.940 15.999 16:0 2.57 ECL deviates -0.001 Ref -0.002 10.995  12426 0.044 0.937 16.629 17:0 ISO 2.81 ECL deviates  0.000 Ref -0.001 11.153  66566 0.044 0.937 16.722 17:0 ANIEISO 15.04  ECL deviates  0.000 Ref -0.001 14.004 805 0.050 -- 18.372 -- -- 14.494   808 0.053 -- 18.655 -- --                Solvent Ar Total Area Named Area % Named Total Amnt Nbr Ref ECL Deviation Ref ECL Shift   40608000 438266 436653 99.63 414520 8 0.002 0.001   TSBA [Rev 2.0] Bacillus 0.021  B. circulans 0.021  B. polymyxa 0.014     Comparison with TSBA [Rev 2.0]: Bacillus-circulans  Distance: 8.257 051015202530354045505560657075 ................ 13:0 ISO*--..........  14:1 ISO*-.... ........... 14:0 ISO-X---+------------------........... 14.0X---+----.............. 15:0 ISO-------+-X-------------........... 15:0 ANIEISO......-------------------+- -X---------------.. 15:0*-.........  16:0 ISO E*--............... 16:0 ISO--------+---------X-........... 16:1 AX--+-----.............. 16:0---X--------+--- -----------------.......  17:0 ISO-+-X-............... 17:0 ANIEISO.-----+--------X............ 
    
     The strain CRK419 is a Bacillus strain, perhaps of Bacillus firmus. Referring now to FIG. 7, the results of the fatty acid profile of the Bacillus strain CRK419 is presented referring also to the following Table 6: 
     
         TABLE 6  RT Area Ar/Ht Respon ECL Name % Comment 1 Comment   1.613 40859000 0.081 --  7.054 SOLVENT PEAK  &lt;min rt 6.326 1464 0.037 0.979 13.616 14:0 ISO 0.38 ECL deviates -0.002 Ref -0.005 6.829 5020 0.038 0.969 14.002 14:0 1.28 ECL deviates  0.002 Ref  0.000 7.749 33670 0.039 0.956 14.621 15:0 ISO 8.47 ECL deviates -0.000 Ref -0.001 7.884 47762 0.040 0.955 14.711 15:0 ANIEISO 12.00  ECL deviates  0.000 Ref 0.000 8.314 920 0.047 0.950 15.001 15:0 0.23 ECL deviates  0.001 Ref 0.001 9.326 1334 0.037 0.943 15.626 16:0 ISO 0.33 ECL deviates -0.000 Ref  0.000 9.381 6042 0.045 0.943 15.659 unknown 15.665 1.50 ECL deviates -0.006 9.539 4827 0.046 0.942 15.757 16:1 A 1.20 ECL deviates 0.000 9.636 4396 0.044 0.941 15.817 16:1 CIS 9 1.09 ECL deviates  0.000 9.695 4484 0.042 0.941 156.853 Sum in      feature 4 1.11 ECL deviates -0.003 16:1 TRANS         9/15i20H 9.783 958 0.042 0.941 15.908 16:1 C 0.24 ECL deviates -0.000 9.933 108370 0.043 0.094 16.001 16:0 26.80 ECL deviates  0.001 Ref  0.001 10.484  2818 0.046 0.938 16.385 17:1 ISO E 0.70 ECL deviates -0.002 10.745  1397 0.054 0.937 16.480 Sum in feature 5 0.34 ECL deviates  0.004 17:1 ISO         I/ANTEI B 10.996 11597 0.046 0.937 16.628 17:0 ISO 2.86 ECL deviates -0.001 Ref -0.001 11.154  10474 0.044 0.937 16.721 17:0 ANIEISO 2.58 ECL deviates -0.001 Ref -0.000 11.274  1078 0.058 0.936 16.792 17:1 B 0.27 ECL deviates 0.000 11.438  833 0.053 0.936 16.889 17:0 CYCLO 0.21 ECL deviates  0.001 Ref 0.001 12.866  2291 0.054 0.937 17.716 Sum in      feature 6 0.56 ECL deviates -0.004 18:2 CIS         9,12/18:0a 12.961  26913 0.046 0.937 17.771 18:1 CIS 9 6.64 ECL deviates  0.002 13.055  98867 0.048 0.937 17.825 Sum in      feature 7 24.39  ECL deviates  0.000 18:1 TRANS   9/t6/c11 13.358  5115 0.049 0.938 18.000 18:0 1.26 ECL deviates  0.000 Ref -0.001 14.379  3110 0.053 -- 18.591 -- -- 14.570  3110 0.045 -- 18.701 -- -- 14.765  4691 0.047 0.943 18.814 19:1 TRANS 7 1.16 ECL deviates -0.009 14.863  17717 0.048 0.943 18.871 Sum in      feature 9 4.40 ECL deviates  0.004 19:0 CYCLO         C9-10/un 16.278  6987 0.047 -- 19.689 -- -- 17.787  4189 0.049 -- 20.569 -- -- &gt;max rt 17.953  10626 0.047 -- 20.665 -- -- &gt;max rt 18.156 2298 0.052 -- 20.784 -- -- &gt;max rt 19.384 1248 0.069 -- 21.499 -- -- &gt;max rt 19.590 12895 0.049 -- 21.619 -- -- &gt;max rt ****** 4484 -- -- -- SUMMED      FEATURE 4 1.11 15:0 ISO 20H/16:1t9 16:1 TRANS         9/15i20H ****** 1397 -- -- --  SUMMED FEATURE 5 0.34 17:1 ISO I/ANTEI B 17:1         ANTEISO B/i ****** 2291 -- -- -- SUMMED      FEATURE 6 0.56 18:2 CIS 9,12/18:0a 18:0 ANTEISO/18:2 ****** 98867 -- -- -- SUMMED      FEATURE 7 24.39  18:1 CIS 11/t 9/t 6 18:1 TRANS         9/t6/c11 ****** -- -- -- -- -- -- 18:1 TRANS 6/t9/c11 ****** 17717 -- -- -- SUMMED      FEATURE 9 4.40 un 18.846/18.858 un 18.858/         .846/19c ****** -- -- -- -- -- -- 19:0 CYCLO C9-10/un   Solvent Ar Total Area Named Area % Named Total Amnt Nbr Ref ECL Deviation Ref ECL Shift   40859000 416245 403038 96.83 379910 11 0.003 0.002   TSBA [Rev 2.0] *NO MATCH* Comparison with TSBA [Rev 2.0]: Bacillus-firm us  Distance: 73.724 051015202530354045505560657075 ................ 14:0 ISOX--+------............. 14.0-X-+-----.............. 15.1 ANTEISOX+----............. . A 15:0 ISO-------X-----------------+----------  .. 15:0 ANIEISO----------X------+-------------------------.......       -- 15:0X+ 16:1 ISO EX----+----------- ----........... 16:1 ISO.H*-............  16:0 ISOX--------+------------------------......... unknown 15.665+X......  16:1 A-X--+----------............ 16:1 CIS 9+X............... 16:1      .. CX+-- 16:0--------+----------------X---------........ 17:1 ISO.E-X+---.....  17:1 ANIEISO.*-............ ... A 17:0 ISO--+X-.............. 17:0 ANIEISO---X+-----............. 17:1 B*-............... 17:0 CYCLO*-........  18:1 CIS 9+-.X.............. 18:0-*- -............... 19:1 TRANS        .. 7+X.... SUMMED-*----.............. FEATURE 4 SUMMEDX--+-------.............  FEATURE 5 SUMMED+X............... FEATURE 6 SUMMED+-... .X........... FEATURE 7 SUMMED+-X............... FEATURE 9 
    
     The following non-limitative examples are designed to illustrate the present invention: 
     EXAMPLES 
     Example 1 
     Example 1 was conducted to isolate and determine particular bacterial strains which are effective biocontrol agents for the Aphanomyces fungus. Approximately 200 bacterial strains were isolated from pea roots grown in Wisconsin soils infested with Aphanomyces. Each isolate was grown in a nutrient broth (NBY) and coated onto a captan-treated pea seed (Perfection 8221). The term &#34;captan&#34; refers to a fungicide having the chemical name N-(Trichloromethylthio) tetrahydrophthalimide. The coated seeds were air-dried prior to planting. 
     The coated seeds and the control seeds were then planted in 60 cc. cone-shaped containers, as illustrated in FIG. 4, containing either pasteurized soil or naturally infested (with Aphanomyces) field soil. Unless otherwise defined, the control in each of the experiments was a captan-treated pea seed. The pasteurized soil was inoculated with 2×10 4  Aphanomyces zoospores six days after planting. The plants were then grown under greenhouse conditions for approximately three weeks, after which the disease symptoms and shoot dry weights were measured. 
     The following bacterial strains, listed in Table 7, were identified as the best strains in terms of improvement in shoot dry weights and decreased disease symptoms over control conditions: 
     
                       TABLE 7______________________________________Bacterial   % Shoot Wt. IncreaseStrain      Compared to Control______________________________________CRK449      19.55A          19.9CRK424      20.0AMMD        20.2PRA44       20.2CRK419      20.6PRA25       21.2PRA42       22.6PRA48       23.0CRK468      25.1CRK478      27.7PRA15       45.2AMMA        52.7______________________________________ 
    
     The bacterial strains which showed the greatest promise in reducing pea root rot and disease severity, as well as increasing shoot dry weight, were then tested under field conditions (Examples 2 and 3). 
     EXAMPLE 36 
     Example 2 was designed to test the twelve bacterial strains, which showed the greatest promise from Example 1, for biocontrol activity. The bacterial strains were cultured and coated onto pea seeds according to the methods described in Example 1. The seeds were then planted in a plot of 17 foot rows of 100 seeds each, each replicated 5 times in a randomized block design. The plants were allowed to grow for one season (8 weeks). Plant mortality was evaluated weekly and the plant yield was determined using the dry weight of the pea plants measured. It is to be noted that the disease was so prevalent in this experiment that no pea pods formed. The results of Example 2 are presented below in Table 8. 
     
                       TABLE 8______________________________________Bacterial    Mean Shoot %Strain       Dry Wt., g Difference**______________________________________control      61         --PRA48        36         -41PRA44        44         -28CRK424       47         -23CRK168       61          +1CRK468       66          +8PRA42        68         +12PRA15        69         +22CRK419       79         +31PRA25        85         +415A            92*       +52AMMD          94*       +55AMMA         103*       +70______________________________________ *P less than .05 Dunnett Test **Between the treatments (Bacterial Strain) and the control. 
    
     EXAMPLE 3 
     This example, which is similar to Example 2, comprised field trials conducted in locations representing a range of Aphanomyces densities. Example 3 was designed to test five bacterial strains plus a control. The methods and materials were conducted in a manner similar to Example 2. The plant mortality due to Aphanomyces was evaluated weekly. Plant yield was determined using the dry weight of the peas at dry seed stage. The results of this experiment are presented below in Table 9. 
     
                       TABLE 9______________________________________          Mean Dry  % YieldBacterial Strain          Wt. Peas, g                    Difference______________________________________control        175       --AM             158       -105A             189        8PRA25          210       12CRK419         215       23AMMD           282       61______________________________________ 
    
     From Table 3, it can be seen that the bacterial strain AMMD increased the average seed yield by 61%, compared to the non-coated controls. 
     EXAMPLE 4 
     Like Example 3, Example 4 was designed to test strains of bacterial in the field. Six bacterial strains plus a control were tested under conditions similar to Example 2. Unlike Example 2, the yield here was determined using the fresh weight of peas. The results of Example 4 can be found below in Table 10: 
     
                       TABLE 10______________________________________          Mean Fresh % YieldBacterial Strain          Wt. Peas, g                     Difference______________________________________control        105        --UW85           119        13CRK419         155        41PRA25          166        585A             177        69AMMD           188        79AM              209*      99______________________________________ *P less than .05 Dunnett Test. 
    
     Several bacterial strains increased pea yield by 13-99%. Pseudomonas cepacia strain AMMD increased yield by 79%, and Pseudomonas fluorescens strain PRA 25 increased yield by 58% compared to the control treatment. It is to be noted that none of the bacterial strains increased the pea yield in fields with less than 1 Aphanomyces propagule per gram of soil. 
     The next experiments, Examples 5-13, were designed to provide information as to how the bacterial strains work. Although the mechanism of biocontrol by the bacteria is unknown, it has been suggested from tests conducted in petri dishes that the biocontrol bacteria produce a substance which limits the growth of the fungus. This substance may act as an antibiotic in reducing the growth of the fungus in the soil. 
     EXAMPLE 5 
     Example 5 was conducted to test the effects of the bacterial cultures on the Aphanomyces zoospores. Prior to conducting the test, it was determined that the growth medium, a 1% solution of NBY broth, does not affect the motility of Aphanomyces zoospores. The bacterial strains Pseudomonas cepacia AMMD and Bacillus cereus (UW85) were grown in the NBY growth medium under conditions explained previously with respect to culturing the bacterial strains. The bacterial strains were then diluted to 1% of their original solution and added to a petri dish containing zoospores of the Aphanomyces fungus. The Aphanomyces zoospores were tested for motility after 30 minutes exposure to the bacterial strains, and cyst germination was quantified after 6 hour exposure to the bacterial strains. Cyst germination is a test of the viability of the fungus. 
     The motility rating scale is as follows: 
     0=no motility 
     1=a few motile cells 
     2=roughly half 
     3=most cells motile 
     4=full motility as seen at initial release in check treatment. 
     After 30 minutes exposure to the bacteria and the controls (lake water and 1% NBY broth), the effects on zoospore motility are presented on Table 11: 
     
                       TABLE 11______________________________________Bacterial Strain           Motility______________________________________lake water*     3.0NBY*            3.0Bacillus cereus 1.6AMMD            0.2______________________________________ *Control 
    
     Table 12 below illustrates the effect of exposure of the bacterial strains and controls to cyst development in the Aphanomyces fungus after 6 hours: 
     
                       TABLE 12______________________________________Bacterial Strain          % Germlings______________________________________NBY*           58.6AMMD           22.6lake water*    17.4Bacillus cereus          13.6______________________________________ *Control 
    
     Replicates of these procedures also demonstrated that AMMA also eliminates zoospore motility in 10 minutes and delays cyst germination. 
     EXAMPLE 6 
     Example 6 was conducted to compare the effects of certain bacterial strains with a control treatment in zoospore motility of Aphanomyces fungus. The experimental procedure described in Example 5 was followed. The effects on zoospore motility was observed 10 minutes after the bacterial strains (or control) was added to the Aphanomyces treatment. The results are illustrated below in Table 13. 
     
                       TABLE 13______________________________________Treatment*    Motility______________________________________broth alone   2.0AM            2.0PRA25         1.9CRK419        1.9BC            1.85A            1.4AMMA          0.0AMMD          0.0______________________________________ *Values are means of 5 replicates. 
    
     EXAMPLE 7 
     Example 7 was conducted to compare the effects of different bacterial strains on mycelial growth, zoospore motility and cyst germination of Aphanomyces. The experimental procedure of Example 5 was followed with respect to Example 7. The results of Example 7 are illustrated below in Table 14. 
     
                       TABLE 14______________________________________Bacterial   Mycelial     Zoospore CystStrain  Growth       Motility Germination______________________________________AMMA     +++*        +++      +++AMMD    +++          +++      +++PRA25   +++          -        -AM      +++          -        -CRK419   -**         -        -5A      -            +        -UW85      +***       -        -______________________________________ *cessation of activity **no change in activity ***slight decrease in activity 
    
     EXAMPLE 8 
     Example 8 was conducted to compare the effects of the bacterial strain Pseudomonas cepacia AMMD with a control (NBY broth) on cyst germination. The procedure of Example 5 was used with respect to Example 8. The results of this experiment are illustrated below in Table 15. 
     
                       TABLE 15______________________________________Treatment   % Cyst Germination______________________________________broth alone 58.6AMMD        22.6______________________________________ 
    
     EXAMPLE 9 
     Example 9 was designed to test the in vitro effects of Psuedomonas cepacia AMMD on Aphanomyces zoospores. The Pseudomonas cepacia AMMD bacterial strain was compared to three controls: (1) lake water; (2) cell-free filtrate from NBY-AMMD culture: and (3) NBY growth broth alone. All solutions were diluted to 1% of original in milli-Q water. The motility of the zoospores was rated according to the table illustrated in FIG. 5. Table 16 below illustrates the mean of 5 repetitions at 10 and 30 minutes from the following treatments: 
     Treatment 1--lake water control 
     Treatment 2--AMMD in NBY broth 
     Treatment 3--Cell-free filtrate from NBY-AMMD culture 
     Treatment 4--NBY broth alone 
     
                       TABLE 16______________________________________Time      Treatment: 1     2       3   4______________________________________10 min.              3.4   0       3.6 2.630 min.              3.8   0       3.4 2.8______________________________________ 
    
     As illustrated in Table 16, Pseudomonas cepacia AMMD in the NBY broth eliminates zoospore motility after 10 minutes. However, the NBY broth alone and the cell-free cultures of AMMD do not effect zoospore motility. 
     EXAMPLE 10 
     Example 10 was conducted to test the effects of sugar beet seeds coated with certain bacterial strains on Aphanomyces cochlioides zoospores. Sugar beet seeds were coated with the bacterial strains and planted in a growth chamber under conditions similar to Example 1. The soils were inoculated 6 weeks later with 20 ml. of 103 zoospores per milliliter of Aphanomyces cochlioides. Approximately 6 weeks later, the plants were harvested and the shoots dried and weighed. The results of the shoot dry weight are illustrated below in Table 17. 
     
                       TABLE 17______________________________________Treatment   Shoot Dry Wt. (mg).sup.1______________________________________Control     5330BC          48045A          5716AMMD         9199*AM          11533*AMMA        11742*______________________________________ .sup.1 Values are means of 15 replicates per treatment. Values marked wit an asterisk are significantly different than the controls (Dunnett&#39;s test p = 0.05). 
    
     The bacterial strains Pseudomonas cepacia AMMA, AM, and AMMD significantly increased sugar beet shoot dry weight compared to the control treatment without bacteria. 
     EXAMPLE 11 
     Example 11 was conducted to compare the effects of various bacterial strains with or without the addition of captan on the emergence of pea shoots. The tests were conducted in soil naturally infested with Aphanomyces euteiches. Pea seeds were coated with the bacteria using the same procedure as for Example 1. In one experiment, treated seeds were planted into flats of soil in the greenhouse. The next experiment was conducted in the field using only three of the bacteria. In both cases, seeds without bacteria served as the check treatments. The results of Example 11 are shown below in Table 18. 
     
                                           TABLE 18__________________________________________________________________________Effects of Bacteria On Pea Emergence.       % EmergenceBacterial Treatment:       None           5A PRA25                  AMMA AM CRK419                               AMMD__________________________________________________________________________Greenhouse Experimentwith captan 84 e           79 e              88 de                  92 e 85 e                          81 e 62 cwithout captan       13 a           29 ab              63 cd                  61 c 31 b                          25 ab                               --Field Experimentwith captan 88 d   92 d     88 d    89 dwithout captan       40 a   56 b     56 b    72 c__________________________________________________________________________ 
    
     Treatments within each experiment that are not followed by the same letter are significantly different at the P=0.05 level using the Least Significant Difference test. 
     As illustrated in Table 18, the bacteria strain Pseudomonas cepacia AMMA and Pseudomonas fluorescens PRA25 significantly improved pea emergence from seeds not treated with captan in the greenhouse experiment. Comparing seeds without captan in the field experiment, Pseudomonas cepacia AMMD and Pseudomonas fluorescens PRA25 significantly increased the emergence of peas compared to those without bacteria. None of the bacteria tested improved the emergence of peas treated with captan. 
     EXAMPLE 12 
     This example was conducted to examine the effects of strains PRA25 and AMMD on pre-emergence damping off caused by the fungal pathogen Pythium. Four soils naturally invested with Pythium species were used for this greenhouse experiment. Each replicate consisted of 25 pea plants planted in each of four sorts. Pea seeds without captan were coated with PRA25 or AMMD as previously described and planted in flats containing infested soil. Untreated seeds without bacteria or captan, and seeds treated with captan were used as controls. There were three replicates per treatment, and the protocol was repeated three times. Percentage of pea seedling emergence was determined eight days after planting. The results of these experiments are summarized in the following Table 19. 
     
                       TABLE 19______________________________________Pea Seedling Emergence in Pythium-infested soilsSeed Treatment     Rochelle  Arlington Hancock Muck______________________________________Untreated 46.7      32.0      45.7    49.7PRA25     72.0      51.5      81.3    42.7AMMD      91.5      89.8      92.9    63.5Captan    95.5      95.1      97.3    77.8______________________________________ 
    
     EXAMPLE 13 
     This example was a test of the effectiveness of these bacterial inoculants derived from pea fields on Pythium ultimum disease in cucumber. Cucumber seeds variety &#34;Straight Edge&#34; were planted into potted soils invested with the pathogenic Pythium and were inoculated with an overnight liquid culture of the strains tested. Cucumber seeds were treated with the commercial standard fungicide &#34;Apron&#34; or with a standard root clonizing bacteria, designated &#34;standard&#34; for controls. In addiiton untreated seeds were planted both in infected and uninfected soils as controls. Emergence and post-emergence damping off are expressed as percentages in the following Tables 20 and 21. Stand represents a percentage of total plants surviving of those planted and vigor was calculated on the mean distance to first leaf compared to the control. 
     
                       TABLE 20______________________________________Treatment    % Emergence  % Damping-Off                              % Stand______________________________________Test 1Uninoculated    80            0           80Inoculated    68           35           44Apron    96            0           96Standard 90           16           785A       90           13           80AM       72           21           58AMMA     92           18           78AMMD     94           28           70CRK419   64           43           40PRA25    88           16           80Test 2Uninoculated    92            0           92Inoculated    70           38           46Apron    98            0           98Standard 98            0           985A       89           13           76AM       82            2           80AMMA     96            9           88AMMD     78            8           72CRK419   72           12           62PRA25    98            0           98______________________________________ 
    
     
                       TABLE 21______________________________________    %          %          %      %Treatment    Emergence  Damping-Off                          Stand  Vigor______________________________________Uninoculated    100        0          100    100Inoculated    74         15         66     72Apron    100        0          100    104Standard 100        0          100    83AMMA     98         3          95     92AMMD     96         0          96     95PRA25    96         2          94     106______________________________________ 
    
     It is understood that the invention is not confined to the particular construction and arrangement herein illustrated and described, but embraces such modified forms thereof as come within the scope of the following claims:

Technology Classification (CPC): 2