BIOLOGICAL INOCULANT EFFECTIVE AGAINST APHANOMYCES

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.

Description: BIOLOGICAL INOCULANT EFFECTIVE AGAINST APHANO YCES Cross-Reference to Related Application This application is a continuation-in-part of application Serial No. 07/227,810 filed August 3, 1988. 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 Aphano yces 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 Aphano yces 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 piant-bacterium symbiosis. Reference is now made to U.S. Patent 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 "biocontrol agent", 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, CR 419, 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 "inoculum" 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 Figure 1 is a graph illustrating the results of the mass spectrometric analysis of the fatty acid profile of Pseudomonas cepacia AMMA? Figure 2 is a graph illustrating the results of the mass spectrometric analysis of the fatty acid profile of Pseudomonas cepacia AMMD; Figure 3 is a graph illustrating the results of the mass spectrometric analysis of the fatty acid profile of Pseudomonas fluorescens PRA25: and Figure 4 illustrates a schematic view of a plant bioassay useful for observing biocontrol activity. Figure 5 is a graph illustrating the results of the mass spectrometric analysis of the fatty acid profile of Corynebacterium flaccumfaciens 5A. Figure 6 is a graph illustrating the results of the mass spectrometric analysis of the fatty acid profile of bacterial strain AM. Figure 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, Wisconsin. 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 epiσotyl 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 β 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 "biologically pure culture" 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 "biologically non-interfering" 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 seed . 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 vermiσulite, 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: Bacterial ATCC Date of Strain Accession No, Deposit Pseudomonas cepacia AMMA 52796 7/22/88 Pseudomonas cepacia AMMD 53795 7/22/88 Pseudomonas fluorescens PRA25 53794 7/22/88 Corynebacterium flaccumfaciens 5A 53934 7/26/89 Bacillus/Corynebacterium AM 53933 7/26/89 Bacillus 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 "rhizosphere", as used herein, refers to the zone of soil subject to the influence of the plant roots. With reference to Figure 1, the results of the tests to determine the fatty acid profile for Pseudomonas cepacia AMMA are presented below in Table 1: TABLE 1 Feature 3 4 ,95 ECL deviates 0.003 14:0 30Iξ 16:l ISO 9.636 27640 0.042 0.941 15.819 16:1 CIS 9 15 ,97 ECL deviates 0.002 10 9.928 25281 0.044 0.940 15.999 16:0 14 ,58 ECL deviates -0.001 Ref -0.002 11.437 9844 0.047 0.936 16.890 17:0 CTCXO 5 ,66 ECL deviates 0.002 Ref 0.000 11.714 1646 0.047 0.936 17.052 16:1 20H 0 ,95 ECL deviates 0.005 12.034 1188 0.047 0.936 17.236 16:0 20H 0 68 ECL deviates 0.001 12.534 9443 0.048 0.937 17.524 16:0 30H 5 ,43 ECL deviates 0.004 15 12.937 733 0.047 17.757 13.053 73016 0.048 0.937 17.824 Sum In Feature 7 42 01 ECL deviates -0.001 18:1 TRANS 9/t6/cll 13.356 13070.0460.938 17.99818:0 075 ECL deviates -0.002 Ref -0.002 20 14.143 9040.066 18.452 14.922 53610.0500.943 18.90119:0 CΪCXO Cll-12 3 10 ECL deviates 0.001 Ref 0.001 15.251 39690.0550.945 19.09118:120H 2, 30 ECL deviates 0.003 ****** 8547 SUMMED 25 FEAS JKE 3 4 95 12:0 AUJE ? unkncwn 10.9 TftBIE 1 continued RT Area Ar Ht Respon ECL Name % Cαameπt 1 Cαaneπt 2 ****** 16:1 ISO 1/14:0 30H 14: 0 30H /16:l ISO I ****** 73016 SUMMED FEHURE 7 42.0118:1 CIS U t 9/t 618:1 TRANS 9/t6/cll ****** 18:1 IKME 6/t9/cll Solvent Ar Total Area Named Area % Named Total Amπt Nbr Ref ECL Deviation Ref ECL Shift 40785000 174842 173205 99.06 162924 6 0.002 0.001 TSBA [Rev 2.0] Eseudαrcπas 0.440 P. cepacia 0.440 P. c. cepacia GC subgroup B . . . 0.440 TABIE 1 continued Cαtπparison with TSBA [Rev 2.0]: P___uώtnonas-cQ * acia-cεpacia GC subgroup B Distance: 3.80 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 12:0 -*- 11:0 ISO.30H. *- 13.1 AT 12-13 *- 14:0 . • " ■ +__ ■ 16:1 CCS 9 16:1 C 16:0 17:0 CYCIO OM- 17:0 *- 16:120H.. -*- 16:020H.. 16:030H.. - X- 18:0 19:0 CYCXD Cll-12.. -X4- 18:120H — *— SUMMED FEAΪURE 3 -*- SUMMED FEATURE 7 With reference to Figure 2, the results of the fatty acid profile for Pseudomonas cepacia AMMD are presented below in Table 2: -13- TABLE 2 Feature 3 4.59 ECL deviates 0.003 14:0 30_yi6:l ISO 9.634 30599 0.043 0.941 15.818 16:1 CIS 9 14.02 ECL deviates 0.001 1° 9.928 36362 0.044 0.940 16.000 16: 0 16.63 ECL deviates 0.000 Ref -0.002 10.083 1560 0.070 16.092 11.436 16563 0.048 0.936 16.890 17:0 CΪCLO 7.55 ECL deviates 0.002 Ref 0.000 11.714 1747 0.046 0.936 17.052 16:1 20H 0.80 ECL deviates 0.005 12.034 1387 0.049 0.936 17.237 16:0 20H 0.63 ECL deviates 0.002 15 12.532 11342 0.048 0.937 17.524 16: 0 30H 5.17 ECL deviates 0.004 12.934 1066 0.044 17.756 13.051 86323 0.047 0.937 17.823 Sum In Feature 7 39.38 ECL deviates 0.001 18:1 CIS 11/t 9/t 6 20 13.354 1722 0.047 0.938 17.998 18:0 0.79 ECL deviates -0.002 Ref -0.003 14.143 1445 0.067 18.453 14.489 1051 0.057 18.652 15.921 9636 0 048 0.943 18.901 19:0 CΪCL0 CU-12 4.42 ECL deviates 0.001 Ref 0.000 25 15.249 5288 0.057 0.945 19.091 18:1 20H 2.43 ECL deviates 0.003 17.137 1003 0.043 20.191 > max rt -14- TABIE 2 continued RT Area Ar/Ηt ResDon EC Name % _ Cαπmeπt 1 Comment 2 9988 SUMMED FEATURE 3 4.59 12: 0 AIDE ? unknown 10.9 16:1 ISO 1/14:0 30H 14:0 30H /16:1 ISO I 86323 SUMMED FEATURE 7 39.3818:1 CIS 11/t 9/t 618:1 TRANS 9/t6/cll ****** 18:1 TRANS 6/t9/Cll Solvent Ar Total Area N * *t * re Area % Named Total Amπt Nbr Ref ECL Deviation Ref ECL Shift 40748000 223561 218439 97.71 205479 0.002 0.002 TSBA [Rev 2.0] PsBudαrcnas 0.591 P. cepacia 0.591 P. c. cepacia GC subgroup B . . . 0.591 TABLE 2 continued comparison with TSBA [Rev 2.0]: P_βudcs__røs-cepac__ϊ-cepacia GC subgroup B-Distancε: 3.04 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 12:0 -*— 11:0 ISO 30H. *- 13.1 AT 12-13 *- 14:0 -+x- 16:1 CIS 9 *-+- 16:1 C * 16:0 17:0 CYCIO... . we 17:0 *- 16:120H -*- • • 16:020H -*- 16:030H -.X- 18:0 " .... -*- . 19:0 CΪCLO Cll-12.. * 18:120H —*— SUMMED FEATURE 3 -*- SUMMED FEAIURE 7 -X- With reference to Figure 3, the results of the tests to determine the fatty acid profile for Pseudomonas fluorescens PRA25 are presented below in Table 3: TABLE 3 RT Area Ar Ht Respon ECL Name % Consent 1 Q * __ ι__πt 2 1.613 40050000 0.080 7.047 SOLVENT PEAK ..... <min rt 3.958 4169 0.030 1.069 11.429 10:0 30H 5.14 ECL deviates 0.006 4.444 1045 0.039 1.041 12.000 12:0 1.26 ECL deviates 0.000 Ref -0.002 0 5.759 5852 0.036 0.996 13.181 12:0 20H 6.72 ECL deviates 0.003 6.122 4845 0.037 0.988 13.460 12:0 30H 5.52 ECL deviates 0.005 9.632 30810 0.043 0.942 16.818 16:1 CIS 9 33.48 ECL deviates 0.001 9.925 29005 0.043 0.941 15.999 16:0 31.46 ECL deviates -0.001 Ref -0.002 11.434 1943 0.045 0.935 16.889 17:0 CΪCTO 2.10 ECL deviates 0.001 Ref 0.000 15 13.046 13300 0.045 0.934 17.821 Sum In Feature 7 14.32 ECL deviates -0.00118:1 CIS 11/t 9/t 6 17.779 5280.026 20.569 >ma__ rt ****** 13300 SUMMED 20 FEATURE 7 14.32 18:1 CIS 11 t 9/t 6 18:1 TRANS 9/t6/cll ****** 18:1 TRANS 6/t9/σll -17- TAHTE 3 continued Solvent Ar Total Area Named Area % Named Total Amπt Nbr Ref ECL Deviation Ref ECL Shift 40050000 90969 90969 100.00 86707 3 0.003 0.002 TSBA [Rev 2.0] Pseu≤αnαnas 0.661(P. fluorescens D) 5 P. chlororaphis 0.661(P. fluoirescens D) P. aureofaciens 0.515(P. fluorescens E) P. fluorescens 0.422 P. f. A 0.422 P. f. G 0.320 10 P. f. C 0.265 -18- TABTE 3 continued Comparison with TSBA [Rev 2.0]: I--seudα_cπas- ιlororaphis (P. fluorescens D)-Distance: 2. 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 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 bacteria are aerobic, catalase positive, oxidase negative and grow on TTC agar. To further firmly fix the species classification, it would be -19- necessary to perform a thin layer σhromatographic 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 % C rrmeπt 1 Cαπmeπt 2 1.613 407170000.081 7.051 SOLVENT PEAK < min rt 6.328 43540.0360.979 13.61814:0 ISO 1.23 ECL deviates -0.000 Ref -0.003 7.747 342920.0400.956 14.62115:0 ISO 9.51 ECL deviates -O.000 Ref -0.002 7.884 1986500.0400.955 14.71315:0 ANTET.SQ 54.98 ECL deviates 0.002 Ref 0.000 8.310 10690.0430.950 15.00015:0 0.29 ECL deviates -0.000 Ref -0.002 9.321 686830.0420.943 15.62516:0 ISO 18.77 ECL deviates -0.001 Ref -0.003 9.927 110800.0430.940 16.00016:0 3.02 ECL deviates -0.000 Ref -0.002 10.993 55600.0450.937 16.62917:0 ISO 1.51 ECL deviates 0.000 Ref -0.002 11.150 393660.0450.937 16.72217:0 ANIEISO 10.69 ECL deviates 0.000 Ref -0.003 16.434 11610.365 19.784 * >max ar/ht 17.908 27710.261 20.644 >max rt TABIE 4 continued Solvent Ar Total Area Nf-rcred Area % Named Total Amπt Nbr Ref ECL Deviation Ref ECL Shift 40717000 364215 363054 99.68 345020 0.001 0.002 TSBA [Rev 2.0] Bacillus 0.161 B. poly yxa 0.161 Oarparisαn with TSBA [Rev 2.0] : Bacillus-polymyxa Distance: 5.670 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 11:0 ISO 30H. Λ - . ι 4:0 ISO -X + -. 14:0 X-+-. 15:0 ISO. 1 — X- 15:0 ANIEISO. . 15:0 *- . 16:0 ISO 1 16:1 A x-l — . 16:0 x 1 17:0 ISO -x. — 17:0 ANIEISO. . 1 — 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 ArHt Respon ECL Name % Cαπmeπt l Comment 2 1.613 406080000.081 7.052 SOLVENT PEAK <min rt 10 1.811 8150.020 7.485 .* <min rt 6.021 6200.0370.985 13.38114:0 ISO E 0.15 ECL deviates -0.007 6.329 35740.0360.979 13.61814:0 ISO 0.84 ECL deviates -0.000 Ref -0.002 7.749 413150.0400.956 14.62115:0 ISO 9.53 ECL deviates -0.000 Ref -0.001 7.886 2172900.0410.955 14.71315:0 ANIEISO 50.05 ECL deviates 0.002 Ref 0.001 15 8.311 9680.0420.950 14.99915:0 0.22 ECL deviates -0.001 Ref -0.001 9.325 825430.0420.943 15.62616:0 ISO 18.78 ECL deviates 0.000 Ref -0.001 9.928 113510.0440.940 15.99916:0 2.57 ECL deviates -0.001 Ref -0.002 10.995 124260.0440.937 16.62917:0 ISO 2.81 ECL deviates 0.000 Ref -0.001 11.153 665660.0440.937 16.72217:0 ANIEISO 15.04 ECL deviates 0.000 Ref -0.001 20 14.004 8050.050 18.372 14.494 8080.053 18.655 TAHLE 5 continued Solvent Ar Total Area Nc-rrnpd Area % Named Total Aiππt Nbr Ref ECL Deviation Ref ECL Shift 40603000 438266 436653 99.63 414520 0.002 0.001 TSBA [Rev 2.0] Bacillus 0.021 B. circulans 0.021 B. polymyxa . 0.014 Cαπparison with TSBA [Rev 2.0] : Eacillus-ci_*culans Distance: 8.257 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 10 13:0 ISO il — 14:1 ISO *- 14: 0 ISO -X- 14.0 X 1 . 15:0 ISO Hx- 15 15JO ANIEISO. . 15:0 *- . 16:0 ISO E... *— . 16:0 ISO 1 — -x- 16:1 A — I 20 16:0 — 17:0 ISO -+-X-. 17:0 ANIEISO. H The strain CR 419 is a Bacillus strain, perhaps of Bacillus firπius. 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 % Cc_u____ιfc l π HiiWHiiL 2 1.613 40859000 0.081 7.054 SOLVENT PEAK <.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 14.00214:0 1.28 ECL deviates 0.002 Ref 0.000 7.749 33670 0.039 0.956 14.62115:0 ISO 8.47 ECL deviates -0.000 Ref -0.001 7.884 47762 0.040 0.955 14.71115:0 ANIEISO 12.00 ECL deviates 0.000 Ref 0.000 8.314 920 0.047 0.950 15.00115:0 0.23 ECL deviates 0.001 Ref 0.001 9.326 1334 0.037 0.943 15.62616:0 ISO 0.33 ECL deviates -0.000 Ref 0.000 0 9.381 6042 0.045 0.943 15.659 unknownl5.6651.50 ECL deviates -0.006 9.539 4827 0.0460.942 15.757 16:1 A 1.20 ECL deviates 0.000 9.636 43960.044 0.941 15.817 16:1 CIS 9 1.09 ECL deviates 0.000 9.695 4484 0.042 0.941 15.853 Sum in feature 4 1.11 ECL deviates -0.003 16:1 TRANS 5 9/15i20H 9.783 958 0.042 0.941 15.90816:1 C 0.24 ECL deviates -0.000 9.933 108370 0.043 0.094 16.00116:0 26.80 ECL deviates 0.001 Ref 0.001 10.484 2818 0.0460.938 16.38517:1 ISO E 0.70 ECL deviates -0.002 10.745 1397 0.054 0.937 16.480 Sum in 0 feature 5 0.34 ECL deviates 0.00417:1 ISO I/ANTEI B 10.996 115970.046 0.937 16.628 17:0 ISO 2.86 ECL deviates -0.001 Ref -0.001 11.154 10474 0.044 0.937 16.72117:0 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 25 11.438 833 0.053 0.936 16.889 17:0 CΪCIO 0.21 ECL deviates 0.001 Ref 0.001 TABTE 6 continued RT Area Ar/Ht Respon ECL Name % Cαπtneπt 1 Cαπmeπt 2 12.866 2291 0.054 0.937 17.716 Sum in feature 6 0.56 ECL deviates -0.00418:2 CIS 9,12/18:0a 12.961 26913 0.0460.937 17.77118:1 CIS 9 6.64 ECL deviates 0002 13.055 988670.0480.937 17.825 Sum in feature 7 24.39 ECL deviates 0 00018:1 TRANS 0.0490.938 18.00018:0 1.26 ECL deviates 0.000 Ref -0.001 0.053 18.591 0.045 18.701 0.0470.943 18.81419:1 TRANS 7 1.16 ECL deviates -0. 009 0.0480.943 18.871 Sum in feature 9 4.40 ECL deviates 0. 004 19: 0 C¥C3_0 C9-10/un 0.047 19.689 0.049 20.569 > max rt 0.047 20.665 . max rt 0.052 20.784 > max rt 0.069 21.499 max rt 0.049 21.619 > max rt SUMMED FEATURE 4 1.11 15:0 ISO 20H 16: lt9 16: 1 TRANS 25 9/15i20H TABLE 6 continued RT Area Ar/Ht Resoαn ECL Name % Cαmneπt 1 Cαπmeπt 2 ****** 397 SUMMED FEATURE 5 0.3417:1 ISO I/ANTEI B 17:1 ANTEISO E i ****** 2291 SUMMED FEATURE 6 0.56 18:2 CIS 9,12/18:0a 18:0 ANTET.SQ/18:2 ****** 98867 SUMMED FEATURE 7 24.39 18:1 CIS U/t 9/t 6 18:1 TRANS 9/t6/cll ****** 18:1 TRANS 6/t9/cll ****** 17717 SUMMED FEATURE 9 4.40 un 18.846/18.858 un 18.858 ****** 19:0 CΪCIO C9-10/un TABIE 6 continued Solvent Ar Total Area Named Area % NamBd Total Amπt 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-firrπus Distance: 53.724 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 14:0 ISO X- 14.0 -x- 10 5.i ANIEISO AxH— 15:0 ISO. 15: 0 ANIEISO. 15:0 __. 16:1 ISO E... x H 15 16:1 ISO.H.. . *- 16: 0 ISO x— unknown 15.665 +x 16:1 A -X- 0 16:1 CIS 9. . . +X . 16:1 C X-i — . 16:0 H TAELE 6 continued 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 17:1 ISO.E... -x-i 17:1 ANIEISO.A*- . 17:0 ISO —H - 17:0 ANIEISO. x+- 17:1 B *- . 17:0 CΪCLO... *- . 18:1 CIS 9... +- 18:0 -*- 19:1 TRANS 7. +x SUMMED FEATURE 4.. -*- SUMMED EEA3TJRE 5.. X- SUMMED FEATURE 6.. +X SUMMΞD FEATURE 7.. -+- . . . . X. SUMMED FEATURE 9.. -t— X 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 "captan" refers to a fungicide having the chemical name N-(Trichloromethylthio) tetrahydrophthali ide. 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 Figure 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 x 10 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: -30- TABLE 7 Bacterial % Shoot Wt. Increase Strain Com ared to Control CR 4 9 5A CRK424 AMMD PRA44 CRK419 PRA25 PRA42 PRA48 CRK 68 CRK478 PRA15 AMMA 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 2 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. (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. 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 bacteria 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 * 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 otility 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 * 3.0 NBY * 3.0 Bacillus cereus 1.6 AMMD 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: -35- TABLE 12 Bacterial Strain % Germlings NBY * 58.6 AMMD 22.6 lake water * 17.4 Bacillus 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 dffects 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 A hanom ces treatment. The results are * Values are means of 5 replicates. Example 7 Example 7 was conducted to compare the effects of different bacterial strains on mycelial growth, zoospore otility 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 * 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 58.6 AMMD 22.6 Example 9 Example 9 was designed to test the in vitro effects of Pseudomonas 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 Figure 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 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. ( g) Control 5330 BC 4804 5A 5716 AMMD 9199 * AM 11533 * AMMA 11742 * Values are means of 15 replicates per treatment. Values marked with an asterisk are significantly different than the controls (Dunnett'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. % Emergence Greenhouse Experiment Bacterial Treatment: 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 Pythiu . 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 soils Seed Treatment Rochelle Arlin ton Hancock Muck This example was a test of the effectiveness of these bacterial inoculants derived from pea fields on Pythium ultimu disease in cucumber. Cucumber seeds variety "Straight Edge" 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 "Apron" or with a standard root clonizing bacteria, designated "standard" 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. 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:

Claims: CLAIMS What is claimed is: 1. A process for controlling Aphanomyces fungal diseases of plants comprising inoculating the plants with an Aphanomyces disease-controlling effective amount of an essentially biologically pure culture of a bacterial strain selected from the group consisting of Pseudomonas cepacia AMMA (ATCC Accession No. 52796) , Pseudomonas cepacia AMMD (ATCC Accession No. 53795) and mixtures thereof. 2. The process of Claim 1 wherein the plants are pea plants. 3. The process of Claim 1 wherein the Pseudomonas cepacia is diluted in a carrier to no less than effective concentration. 4. A process for controlling Aphanomyces fungal diseases of plants comprising inoculating the plants with an Aphanomyces disease-controlling effective amount of an essentially biologically pure culture of Pseudomonas fluorescens PRA25 of the strain ATCC Accession No. 53794. 5. The process of Claim 4 wherein the plants are pea plants. 6. The process of Claim 4 wherein the Pseudomonas fluorescens is diluted in a carrier to no less than effective concentration. 7. A process for increasing germination, decreasing mortality and increasing yield of a pea plant comprising 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 AMMA (ATCC Accession No. 53796), Pseudomonas cepacia AMMD (ATCC Accession No. 53795) , Pseudomonas fluorescens PRA25 (ATCC Accession No. 53794), and combinations thereof. 8. The process of Claim 7 wherein the culture is diluted in a carrier to no less than effective concentration. 9. A biological inoculant for plants comprising a biologically pure culture of Pseudomonas cepacia AMMA (ATCC Accession No. 53796) . 10. A biological inoculant for plants comprising a biologically pure culture of Pseudomonas cepacia AMMD (ATCC Accession No. 53795) . 11. The biological inoculant of Claim 10 wherein the Pseudomonas cepacia is diluted in a carrier to no less than effective concentration. 12. A biological inoculant for plants comprising a biologically pure culture of Pseudomonas fluorescens PRA25 (ATCC Accession No. 53794) . 13. The biological inoculant of Claim 12 wherein the Pseudomonas fluorescens is diluted in a carrier to no less than effective concentration. 14. A biological inoculant for plants comprising a biologically pure culture of Corynebacterium flaccufaciens 5A (ATCC Accession No. 53934) . 15. A biological inoculant for plants comprising a biologically pure culture of Bacillus strain AM (ATCC Accession No.53933) . 16. A biological inoculant for plants comprising a biologically pure culture of Bacillus strain CRK419 (ATCC Accession No.53935 ) . 17. An agriculturally useful composition comprising a pea seed inoculated with an inoculant of a culture of a Pseudomonas cepacia strain selected from the group consisting of AMMA (ATCC Accession No. 53796) and AMMD (ATCC Accession No. 53795) . 18. The biological inoculant of Claim 18 wherein the Pseudomonas cepacia is diluted in a carrier to no less than effective concentration. 19. An agriculturally useful composition comprising a pea seed inoculated with an inoculant of Pseudomonas fluorescens PRA25 (ATCC Accession No. 53794) . 20. The biological inoculant of Claim 19 wherein the Pseudomonas cepacia is diluted in a carrier to no less than effective concentration. 21. A biologically pure culture of Pseudomonas cepacia AMMA (ATCC Accession No. 53796) . 22. A biologically pure culture of Pseudomonas cepacia AMMD (ATCC Accession No. 53795) . 23. A biologically pure culture of Pseudomonas fluorescens PRA25 (ATCC Accession No. 53794) . 24. A biologically pure culture of Corynebacterium flaccufaciens 5A (ATCC Accession No.53934 ) . 25. A biologically pure culture of Bacillus strain AM (ATCC Accession No. 53933 ) . 26. A biologically pure culture of Bacillus strain CRK419 (ATCC Accession No. 53935) . 27. A biological inoculant for plants comprising an essentially biologically pure culture of bacteria selected from the group consisting of Pseudomonas cepacia AMMA (ATCC Accession No. 53796) , Pseudomonas cepacia AMMD (ATCC Accession No. 53795) , Pseudomonas fluorescens PRA25 (ATCC Accession No. 53794) , Corynebacterium flaccumfaciens 5A (ATCC Accession No. 53934) _ Bacillus strain AM (ATCC Accession No. 53933) , Bacillus strain CRK419 (ATCC Accession No. 53935) , and combinations thereof.

Inventors: PARKE JENNIFER L, PARKE, JENNIFER, L.
Applicants: WISCONSIN ALUMNI RES FOUND, WISCONSIN ALUMNI RESEARCH FOUNDATION
Application year: 1989
Main classification: A61K35
IPC codes: A01N63