BACKGROUND OF THE INVENTION

This invention relates to a means for biological control of certain soil borne plant pathogens. Biological control is defined as pathogen control by use of a second organism. The mechanisms of biologi­cal control are quite diverse. In some instances the control is caused by production of certain chemicals by the biological organism, with the chemicals being inhibitory. In other instances, there is competition for space with the control organism occupying space, and consuming nutrients which inhibit the organism to be controlled. In further instances it may be nothing more than competition for chemical nutrients. In any event, the concept of biological control when used herein is as defined in this paragraph.

The biological control mechanism of this invention is especially designed for control of seed corn rot and corn seedling blight. It is possible that the control method of the present invention may be used for certain other plants which suffer from similar diseases caused by high moisture content in the growing media at planting time. Thus, the inven­tion is not limited to control of corn seed rot and seedling blight, but instead is a method of control­ling any types of "damping off" or "root rot" that crops such as corn, alfalfa, soybeans, etc. are known to have.

In the typical progress of corn seed rot, and seedling blight the symptoms occur early in the spring soon after planting or seed germination in a disease conducive environment. Often the seedling will be destroyed, the leaves yellow and wilt, the roots rot and fail to properly develop, and the plant is significantly impaired. It may even die. This disease is especially common when there is not only high moisture during spring planting season, but also colder temperatures.

Currently, the best known methods for treatment are chemical treatment with fungicides such as Cap­tan. However, because of recent environmental con­cerns it is especially desirable to eliminate the use of potentially environmentally hazardous fungicides, such as Captan.

There is, therefore, a continuing high level of interest in developing other means for treating bacteria and fungi known to cause corn seed rot and seedling blight, especially those caused by Pythium Spp. and Fusarium graminearum. These are perhaps two of the most common species causing corn seed rot and seedling blight.

It is a primary objective of the present invention to provide a method of biological control of corn seed rot and seedling blight.

Another objective of the present invention is to provide a treating composition of inoculum for con­trol of corn seed rot and seedling blight, which comprises a protection effective amount of a bacteria which produces bioemulsifiers and/or biosurfactants. It is selected from the group consisting of strains 6519EO1, 6133DO2, 6109D01 and their respective genetic equivalents, combined with a satisfactory carrier.

Another objective of the present invention is to provide a method of biological control which compris­es placing the treating composition on the seed, or adjacent the seed in a growing medium, to protect said seed during its critical time periods just prior to and just after germination. Protection is provid­ed from hazards of blight caused by Pythium Spp. and Fusarium graminearum, etc.

The method and means of accomplishing the objectives of the present invention will become apparent from the detailed description which will follow hereinafter.

SUMMARY OF THE INVENTION

The cultures of the present invention are used for biological control by a bioemulsifier and biosur­factant producing bacteria. They are selected from the group consisting of strains 6519EO1, 6133DO2, 6109D01 and their respective genetic equivalents, as well as mixtures thereof. It is believed these strains are active in part because they produce bioemulsifiers and/or biosurfactants. These may interact with the aqueous media to potentially transport away from the seed, or the seedling after germination, aqueous media containing pathogens such as Pythium Spp. and Fusarium graminearum, which are known to induce corn seed rot and seedling blight.

DETAILED DESCRIPTION OF THE INVENTION

The bacterial strains selected for use in preparing the biological control systems of the present invention were selected after screening about 5000 strains from various growing media. Only the three strains of the present invention, and their genetic equivalents or mutants thereof, exhibit the unusual property of successful biological control of corn seed rots and seeding blights.

The bacterial strains of the present invention have been code designated as strains 6519EO1, 6133D02 and 6109DO1. These strains have been deposited at the American Type Culture Collection, Rockville, Maryland, and have been assigned ATTC Accession Nos. 53858 , 53859 and 53860 , respectively. The term genetic equivalents as used herein is to be under­stood to mean not only the precise strains of the present invention, but mutants thereof, or genetical­ly altered bacteria which nevertheless have the common identifying characteristic of the present invention, i.e. they produce biosurfactants and/or bioemulsifiers which behave in the same or similar or equivalent manner which may be important to providing effective biological control of pathogens causing corn seed rot and corn seed blight.

The three bacterial strains selected involve two members of the genus Pseudomonas, and a third bacte­ria from the genus Serratia. These may be described in the following manner.

Organisms (6519E01 and 6133D02) are gram negative, produce white colonies on yeast-dextrose calcium carbonate agar, and are fluorescent on King's medium B placing them in the genus Pseudomonas. They were positive in oxidase and arginine dihydrolase tests, positive in gelatinase tests, were able to grow at 5°C, and unable to grow at 41°C. These results place them in the Pseudomonas fluorescens group.

Pseudomonas fluorescens (6519E01) is an isolate which most closely resembles P. fluorescens biovar V, although it differs from most biovar V strains in the utilization of D-alanine. It is variable in the use of this carbon source and strains of biovar V are by definition unable to utilize this carbon source.

Pseudomonas fluorescens biovar I (6133D02) is an isolate able to utilize sucrose and D-alanine as carbon sources and produces levan. It is unable to reduce nitrate and cannot utilize L-tartrate as a sole carbon source. By these criteria it fits the biovar one classification.

The third organism (6109DO1) is of the genus Serratia and is a member of the family Enterobacteri­aceae. Essential characteristics of the genus Serra­tia include the production of extracellular gelatin­ase, lecithinase, and DNase. Extensive carbon source utilization tests were used to identify strains of the genus serratia to species. Some differential tests might include: inability to grow on adonitol or erythritol as sole carbon sources, positive for growth at 5°C, and negative for growth at 40°C. Identification to species was carried out in accor­dance with the work of Grimont and Grimont. Strain 6109D01 is Serratia plymuthica.

From time to time it is referred to herein as a biologically pure or substantially pure culture of these above referred-to strains. This is intended to refer to a culture which contains no other bacterial species in quantities sufficient that they interfere with the pathogen-treating effectiveness of the strains above referred to. The term "seed treating effective amount" or "protecting amount" as used herein refers to an amount of the biological pathogen control composition which is sufficient to produce effective field treatment to provide a noticeable field decrease of corn seed rot and corn seed blight.

The common denominator for each of the referred to strains herein are that each produce biosurfac­tants. The term "biosurfactants" as used herein is intended to include both emulsifiers and surfactants. Generally speaking, each of the three strains, their genetic equivalents, or their mutants are known to reduce the surface tension of an aqueous medium from its normal level of about 72.5 dynes/cm downwardly as much as 40%, preferably within the range of at least 40% to 80% and especially from about 50% to about 75% surface tension reduction. Generally, all of the organisms useful for biological control in this invention will successfully reduce the surface ten­sion of water from its normal 72.5 dynes/cm down to 40 dynes/cm and at least in one instance with respect to strain 6519EO1, will reduce the surface tension down to at least 20 dynes/cm.

It is not known precisely why the biological controls of the present invention work. While appli­cants do not wish to be bound by a theory of opera­bility, it is believed that they work in part because of the characteristic nature of each of the organisms of producing biosurfactants. The biosurfactants may result in pathogen control by a number of different mechanisms. One of those mechanisms may well be to aid in the redistribution of soil borne plant pathogens on a natural basis. Put another way, the biological control agents of the invention when either on the seed or adjacent thereto, produce the biosurfactants which then interact with the aqueous media, aiding in protecting from attacks by soil borne plant pathogens, such as Pythium Spp. and Fusarium graminearum. It may also be possible that the biosurfactants are simply potentiating the activity of other protective agents by facilitating dispersal around the seed.

The biological controls may either be applied to the seed or directly applied to the growing medium, but are preferably applied to the seed. They may be applied to the seed by mixing with a suitable carri­er, and applying a thin coating on the seed. This can be done by soaking followed by planting of the seed. It can also be done by low water volume spray application. Neither the carrier nor the precise manner of either dry application or soaking application in a liquid medium appears to be critical. Suitable carriers are those which do not inhibit the growth of the bacteria, nor are they harmful to the seed. Generally speaking, suitable carriers may be water and for example aqueous methyl cellulose.

The following examples are offered to further illustrate but not limit the process and composition of the present invention.

EXAMPLE 1

Seeds were coated with bacterial cells grown in specific media as medium composition has been found to influence efficacy. All Serratia plymuthica strains were grown in half-strength tryptic soy broth. P. fluorescens strain 6133DO2 was grown in nutrient broth. P. fluorescens strain 6519E01 was grown in potato dextrose broth. Stationary phase cultures were used in all cases. Seed was coated by one of two methods, either high volume coating or low volume coating.

In high volume coating, the Pseudomonad treat­ments were coated onto seeds by centrifugation and resuspending the cells in approximately 15mls of culture supernatant for every 125 seeds to be coated. The resulting cell suspension was approximately 5 x 10⁸ cfu per ml. Seeds were allowed to soak in this suspension for at least 30 minutes. They were then removed from the cell suspension and dryed in moving air.

In low volume coating, the Serratia treatments were coated onto seed by a similar method except that cells were resuspended into only 1ml of culture supernatant and applied to 200 seed in a small rotat­ing drum before air drying. This treatment has since been found to be less desirable than the first, at least for some organisms.

Field trials were conducted in the following manner. A randomized complete block design was used at all stations. Four or five replications per entry were used depending on the location.

The field trials were conducted at six sites in the spring of 1988. Algona, Iowa; 1 row plot, 50 seeds per row; planted April 8th. Johnston, Iowa; 1 or 2 row plots, 50 or 100 seed, April 4, 8, 11. Princeton, Illinois; 2 row plot; 25 seed per row; April 12th. Willmar, Minnesota; 1 row plot; 50 seed per row; April 19th. Windfall, Indiana; 2 row plot; 50 seed per row; April 12th. York, Nebraska; 2 row plot; 50 seed per row; March 29th.

Non-treated seed and seed treated with one of the following were planted using standard agronomic practices: Apron (fungicide), Captan (fungicide), 6109D01, 6109D06, 6171B01, 6120A04, 6133D02, 6519E01 + 6109D01, or 6509E01. Those organisms not designat­ed as part of the present invention were selected for field test, but were not successful biological con­trols and are presented here for data comparison only.

All stations measured the standard trait generally referred to as early stand count or seed­ling emergence. This is simply a numerical count of the number of emerging seedlings. At Johnston station data on biomass production was also collected.

Standard analysis of variance and mean separation tests were carried out on the data from locations showing significant reduction in stand due to fungal disease.

Data was grouped from plots where a significant difference in emergence was found between raw seed and seed treated with any of the chemical fungicides. The raw data from a location were expressed as a fraction of the mean emergence of all treatnents within that one location. This variable was called standardized emergence and was used to generate all statistical analyses presented here. Analysis of the non-transformed data set resulted in identical con­clusions except that Station and Location were addi­tional sources of significant variability. The normalization by location mean was instituted to reduce variability in the data set due to pooling the data from several stations and locations within a station. The distribution of the new variable was found to approximate a normal distribution.

Analysis of variance showed two significant sources of variability in this data set. They were treatment and the interaction of treatment with the trial site (station). The interaction of trial site and treatment was expected as edaphic, climatic, and environmental factors were all known to influence strain efficacy.

Table 1 presents mean separation for standard emergence at the six locations where fungal disease was present.

(Multiple Range Test Results)

Treatment

Standardized Mean Emergence

Duncan Grouping

Plots/Treatment Across All Locations

Apron

1.09

A

25

Captan

1.08

A

25

6133D02

1.04

AB

25

6519E01+6109D01

1.03

AB

25

6519E01

1.01

BC

25

6109E01

0.98

BCD

25

6120A04

0.97

BCD

25

6109D06

0.96

CD

25

6171B01

0.93

D

25

RAW

0.93

D

25

In table 1 the term "Duncan Grouping" refers to a statistical method of data analysis. This is the generally accepted method of analysis for this type of data. Treatment means followed by the same Duncan grouping letter do not differ significantly at the 95% confidence level.

Table 1 shows that across all locations where fungicide treatment significantly increased emergence, treatment of seed with strain 6133D02 or the mix of strains 6519E01 and 6109D01 provided emergence equal to commercial fungicides. Seed treatment with strain 6519E01 alone provided protection significantly better than no treatment.

At the Johnston station above ground biomass production was also measured for treatments 6519E01, 6120A04, 6133D02, and the controls on Pioneer Seed Corn® hybrid 3475. Total biomass production by plants grown from seed treated with 6519E01 (27.0g) was significantly greater than the total biomass of the non-treated seed (17.9g) and was equal to that of Captan (22.0g) and Apron (22.9g). Strain 6133D02 did not significantly increase total biomass (23.2g) above the non-treated seed. However, it equaled biomass production by either Captan or Apron treated seed. In addition, the biomass per plant of plants from seed treated with 6519E01 (0.61g) was signifi­cantly greater than the biomass per plant of plants from non-treated seed (0.46g) or from seed treated with Captan (0.50g) and was not significantly differ­ent from the biomass per plant of plants from seed treated with Apron (0.52g).

Strain 6519E01 may be able to promote seedling growth as well as prevent attack by fungal pathogens. At the one location where data was collected on above ground biomass production seed treatment with 6519E01 resulted in significantly larger plants as well as significantly higher emergence which equaled the protection provided by chemical fungicides.

EXAMPLE 2

Each of the three strains of the present invention were further analyzed for their character­istic development of biosurfactant and bioemulsifier properties. The data for the surfactant and bioemul­sifier characterization studies are presented below.

Emulsification was determined by a modification of the method of Gerson and Zajic: 6mL of kerosene or vegetable oil and 4 mL of aqueous surfactant, con­sisting of X mL (0.1 mL:1.0 mL) of whole broth and 4­X mL of distilled water, were vigorously vortexed in a test tube for two minutes. If an emulsion formed, the percentage of the total volume occupied by the emulsion was measured with time as a variable. From a plot of log percent emulsion versus time the slope of the straight line was determined and divided into 0.693 to give the half-life of the emulsion in hours.

Each of the bacterial strains of the present invention exhibited either trivial or poor emulsifying properties after fermentation growth times of about 7 hours. However, at times varying from 24 hours upwardly, the bioemulsifier capabilities significantly improved.

The following examples illustrate the biosur­factant emulsion activity of the protective composi­tions produced by the bacterial strains of the present invention. The tables illustrate the kinet­ics of decay of emulsion under gravity using the protocol earlier described.

In Table 3 below the organism was Pseudomonas fluorescens strain 6519EO1 and the growth medium was potatoe dextrose broth (PDB). The fermentation growth time was 48 hours.

% Emulsion

Stability Time (hours)

71

0

71

1

69

4

69

6

64

24

63

48

62

72

In Table 3 below the organism was Pseudomonas fluorescens strain 6133D02 and the growth medium was nutrient broth (NB). The fermentation growth time was 40 hours.

Stability Time

% Emulsion

(hours)

Kerosene

Oil

a. Oxygen Level 1

0

80

70

1

65

65

6

63

60

16

62

57

24

62

55

b. Oxygen Level 2

0

84

64

1

73

64

6

70

54

16

69

53

24

68

53

In Table 4 below the organism was Serratia plymuthica strain 6109D01. The fermentation growth time was 40 hours.

% Emulsion

Stability time (hours)

a. Oxygen Level 1

78

0

76

1

77

3

78

24

78

48

78

72

b. Oxygen Level 2

78

0

78

3

78

5

76

21

76

24

76

48

76

72

It can be seen that P. fluorescens 6519EO1, P. fluorescens 6133DO2, and S. plymuthica 6109D01 all were effective bioemulsifiers. The best surface tension measurements or each of these respective strains from numerous fermentation runs is the fol­lowing. For P. fluorescens 6519E01, 26.8 dynes/cm; for P. fluorescens 6133DO2, 58 dynes/cm; and for S. plymuthica 6109D01, 43 dynes/cm.

It can be seen from the field test data that each of these strains are noticeably effective as successful biological controls for seed corn rot and blight. The biosurfactant data shows they also produce efficient biosurfactants.