Control of bean rust with Bacillus subtilis
BACKGROUND OF THE INVENTION 
     1. Field of The Invention 
     This invention relates to the use of a particular isolate of Bacillus subtilis to control or to inhibit the development of rust on bean plants and other agricultural products. It also pertains to the use of a certain fraction obtained from purifying the isolate of B. subtilis for inhibiting the development of rust. 
     2. Description of The Art 
     The use of species of Bacillus as antagonists of plant pathogens in the phyllosphere is well known. Bacillus cereus subsp. mycoides has been found to control brown spot on tobacco leaves. Cereal rust has been controlled by spray treatment of wheat and oats with B. pumilus. The strain of B. subtilis used in this invention was found to suppress charcoal rot of potato, Plant Dis. Reptr. 61, 543-546, 1977. 
     SUMMARY OF THE INVENTION 
     An object of this invention is to provide a method for controlling or inhibiting the development of rust on beans and other agricultural products such as wheat that is safe for the growers and consumers of these agricultural products. 
     Another object is to provide a method for controlling rust which is effective on major varieties of dry and snap beans. 
     Still another object is to provide a method and an agent that is effective against major races of the highly variable pathogen, rust. 
     Another object is to provide a method and agent for controlling rust that is economical to use. 
     A further object is to reduce the need for breeding disease resistant lines of plants and for reducing the need to continually test these disease resistant lines. 
     A still further object is to increase the production of beans and other agricultural products by greatly reducing the losses that are caused by rust. 
     Another further object is to provide a means of improving the environment by reducing the need to use synthetic fungicides. 
     According to this invention the above objects are accomplished by treating bean plants and other agricultural plants with a particular strain of Bacillus subtilis designated as APPL.-1. The objects are also accomplished by treating the plants with an easily obtainable heat stable culture-extract obtained from the biocontrol agent APPL-1. Both APPL-1 and the culture-extract are applied as a spray or as an aerosol. 
     A viable culture of the strain of B. subtilis designated as Bacillus subtilis APPL-1 has been deposited with the Culture Collection at the Northern Regional Research Center, United States Department of Agriculture, Peoria, Illinois, 61604, and its accession number is NRRL B-15570. With reference to 886 O.G. 638, progeny of this strain will be available during pendency of the patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. All restrictions on the availability of progeny of the strain to the public will be irrevocably removed upon the granting of the patent of which the strain is the subject. 
     DESCRIPTION OF THE INVENTION 
     Rust is a major agricultural problem in the United States and bean rust, caused by Uromyces phaseoli, is a very major disease problem of dry and snap beans in the United States which causes very high yield and monetary losses. Synthetic fungicides, principally Manzate, a dithiocarbamate, aid in controlling bean rust, but are expensive, not completely effective, and undesirable because of possible toxic side effects to the environment and to man. Alternatives to present day pesticides are urgently needed in all phases of pest control and this is especially true for bean rust, nationwide as well as worldwide. Use of synthetic fugicides to control bear rust is widespread on fresh market and processing snap beans as well as on pinto, navy, and other dry beans. Breeding for resistance is a major effort because of the need to reduce dependence on synthetic fungicides. However, because of the highly variable nature of the pathogen causing the disease and the wide variety of geographical areas where beans are grown, this breeding program requires maximal and continual input of time, effort and resources worldwide. The present invention provides a new and different means for inhibiting the development of and therefore controlling all major races of the bean rust pathogen. 
     In order to develop the present invention we maintained a number of bacterial cultures on nutrient agar at 25° C. One of them was a cluture of Bacillus subtilis designated APPL-1. Another was a culture of Bacillus subtilis designated APPL-2, which was isolated from an entirely different soil than that from which APPL-1 was isolated. Other cultures were B. cereus subsp. mycoides isolate B 23, which controls lesion production on tobacco caused by Alternaria alternata, an isolate of B. thuringiensis HD-1, a culture of Erwinia ananas pv. uredovora, and an isolate of Pseudomonas syringae which is avirulent on beans. Each of the above bacterial species was transferred from nutrient agar to one liter of Eugon broth in a 2.8 liter fernbach flask. Eugon broth consists of the following ingredients: tryptose, 15 gm; Soytone, an enzymatic hydrolysate of soybean meal, 5 gm; dextrose, 5 gm; cystine, 0.2 gm; sodium chloride, 4 gm; sodium sulfite, 0.2  gm; sodium citrate, 1.0 gm. Other equivalent medium or broth may be used in place of Eugon broth. The cultures were incubated on a circulatory shaker at 25 rpm at 30° C. and in the dark until early stationary phase. Culture supernatant and other culture fractions tested for rust inhibition activity were applied to young plants of Phaseolus vulgaris cv. Lake Shasta with primary leaves one-fifth to one-third expanded. Five seeds were planted per four inch plastic pot, germinated in a greenhouse at 24° to 30° C., and thinned to three uniform plants per pot, five to seven days after seeding. The bacterial suspensions and test solutions were then sprayed onto the plants with a spray device from a distance of approximately 12 to 15 inches to give thorough coverage of all above ground plant surfaces. The plants were then allowed to dry. 
     Urediospores of Uromyces phaseoli were added to a solution of distilled water containing 0.01% Tween 20 and stirred on a magnetic stirrer for about five minutes. Any wetting agent similar to Tween 20 may be used in its place. Urediospore concentration was determined with a hemocytometer and adjusted to about 20,000 spores per ml. This suspension was lightly sprayed onto the upper surface of leaves of plants pretreated or nontreated with the bacterial suspensions. After the film dried, the plants were placed overnight in a dew chamber in which the relative humidity was 100% and the air temperature was 22° C. The plants were moved to a greenhouse the next morning. In eight to fourteen days uredial pustules had developed and were counted. All of these in vivo treatments were applied to at least ten replicate plants. 
     As noted in Table 1, Lake Shasta bean plants pretreated with the cultures of B. subtilis, B. cereus subsp. mycoides, B. thuringiensis and E. ananas pv. uredovora and then sprayed with urediospores from U. phaseoli collection 73-16 had 80 to 99% fewer uredial pustules than those pretreated with avirulent P. syringae or sterile Eugon broth or given no treatment. The bacterial isolate most inhibitory to rust development was B. subtilis APPL-1, which consistently reduced the number of pustules by more than 90% as compared to the nontreated control. This is a significantly greater reduction than that obtained by treatment with APPL-2 or the other effective bacteria. There is no significant difference among the percent reduction in pustule numbers caused by B. subtilis APPL-2, B. cereus subsp. mycoides, B. thuringiensis or E. ananas pv. uredovora. 
     In order to determine if a culture of B. subtilis APPL-1 in early stationary phase was broadly effective, its effect on rust development on five bean cultivars was determined. The pathotype of U. phaseoli used in this experiment was rust collection 75-22. The cultivars tested included Lake Shasta, Mount Hood, Pinto 111, Mountaineer White Half-Runner, and BBL 47. All five cultivars are susceptible to rust collection 75-22. As noted in Table 2 a 96% to 98% reduction occurred in the number of pustules produced by rust collection 75-22 on all five cultivars. 
     The effect of a culture of B. subtilis APPL-1 in early stationary phase on symptom development on bean cv. Lake Shasta innoculated with five pathogenically unique collections of U. phaseoli was also determined. The bean cultivar was sprayed with the culture of APPL-1 and then innoculated with the five unique pathotypes of U. phaseoli. As seen in Table 3 there was no statistical difference among the five pathotypes of U. phaseoli. The reduction in number of necrotic spots caused by rust collection 73-23 on B. subtilis APPL-1 pretreated Lake Shasta did not significantly differ from the effects of pretreatment on the number of pustules that were produced by collections 79-4, 73-16, 75-22, and 73-32. Eleven additional collections of rust that contain unique pathotypes different from those listed in Table 3 were also tested and the B. subtilis APPL-1 pretreatment had an identical inhibitory effect on development of either the necrotic or pustule reaction, whichever was characteristic of the appropriate host-pathotype combination. The eleven addition collections of rust are 79-15F, 79-6-1, 79-6A-1, 79-6C-1, 79-15B-1, 79-15G-1, 79-15A-1, 79-4B-1, 81-10A-1, 79-15A-4, and 81-lOA-3-1. 
     We found that cultures which are in late stationary phase contain substantially more inhibitor than cultures in early stationary phase. We have also found that the activity of culture filtrates can be stabilized by autoclaving for 20 minutes. Autoclaving does not greatly effect the inhibitor, but appears to inactivate degradative enzymes which are also present in these filtrates. 
     A high molecular weight fraction or extract is obtained by removing with centrifugation the bacterial cells from a culture of B. subtilis APPL-1, in late stationary phase, processing the supernatant through molecular filtration and retaining only the high molecular weight portion, that is, the portion greater than 100,000 daltons. This purified and concentrated extract controls bean rust at levels of less than one part per million. 
     A more definitive description of the preparation of the high molecular weight fraction is as follows: 
     1. Prepare and sterilize one liter of Eugon broth in a 2.8 l. fernbach flask. 
     2. Inoculate medium with loopful of APPL-1 from a 24 hour-old culture grown on a nutrient agar plate at 30° C. 
     3. Incubate on a circulatory shaker at 125 rpm at 30° C. until the culture is in late stationary phase. 
     4. Separate bacterial cells from cultural broth by centrifugation at 8,000 rpm. 
     5. Autoclave the broth for 20 minutes. 
     6. Place the broth in an Amicon or equivalent ultrafiltration cell with a membrane of 100,000 molecular weight cut-off. Apply pressure of about 20 p.s.i. until the liquid is reduced to somewhat greater than 200 ml. 
     7. Wash the retained high molecular weight fraction by twice adding a volume of water to the cell and applying pressure until the volume is reduced to 200 ml. 
     For larger scale operations, the following procedure may be used: 
     1. Prepare and sterilize 15 liter of Eugon broth in a 20 l Nalgene or equivalent carboy fitted with an air inlet tubing and 0.2 micron air filter. 
     2. Inoculate medium with 500 ml of 24 hour-old culture of APPL-1 grown on Eugon broth on a shaker at 125 rpm and 30° C. 
     3. Set air flow to agitate broth to insure mixing and allow air to exit through carboy cap. Continue until culture is in late stationary phase. 
     4. Separate bacterial cells from broth by microporus filtration through a Millipore or equivalent pellicle chamber using 0.2 micron filters. 
     5. Ultrafilter the supernatant from 4 using a 100,000 molecular weight cut-off membrane. 
     6. Wash the material retained on filter in step 5 twice with distilled water and reduce the volume to 3 liters. 
     Field tests using three replicates of one meter rows of beans have shown that B. subtilis APPL-1 cultures and culture extracts are quite effective in controlling bean rust. The treatments include: (1) water, and (2) Eugon broth as control treatments; (3) early stationary phase and (4) late stationary phase cultures of B. subtilis APPL-1; and (5) high molecular weight fraction of a late stationary phase culture of B. subtilis APPL-1. As shown in Table 4, the B. subtilis APPL-1 cultures and the high molecular weight fraction reduced rust by about 70 to 84% on Pinto 111 and about 82% on Lake Shasta beans. The cultures and high molecular weight fraction were diluted with an equal volume of water and then spray or aerosol applied to the bean plants. When diluted, one volume of culture to five volumes of water, the cultures retained rust inhibiting activity. Approximately 500 ml of the diluted culture was used to treat about 75 bean plants. 
     Histological examination of the plant leaves was conducted by approved procedures. Leaves were removed from treated and control bean plants immediately after the plants were removed from the dew chamber. The leaves were sectioned with a scalpel into approximately one cm square pieces. The sections were immersed for 10 seconds in alcoholic lactophenol cotton blue modified to contain 0.1 g aniline blue per 40 ml. The sections were then rinsed briefly with 50% glycerin and mounted on a glass slide with cover slip for observation with an Olympus model BH-2 or equivalent light microscope. Leaf sections treated with B. subtilis had many nongerminated urediospores floating in the mounting solution. About 30% of the spores which remained on the leaf surface exhibited cytoplasm that had ballooned out but had remained attached to the spore cell wall. No normal germ tubes were observed on B. subtilis treated leaves. More than twice as many spores remained on the surface of the nontreated as on the treated leaves. Nearly 100% of the spores on the nontreated leaves had developed germ tubes 5 to 20 spore diameters in length during the overnight incubation. 
     
                       TABLE 1                                                     
______________________________________                                    
                 Uredial pustules/leaf                                    
                   Number    Control                                      
Treatment          (mean)    (%).sup.y                                    
______________________________________                                    
Bacillus subtilis (APPL-1)                                                
                   0.5 ± .2                                            
                             99.sup.a                                     
B. subtilis (APPL-2)                                                      
                   6.9 ± 1.0                                           
                             84.sup.b                                     
B. cereus subsp. mycoides                                                 
                   9.2 ± 1.5                                           
                             80.sup.b                                     
B. thuringiensis   6.9 ± 1.6                                           
                             85.sup.b                                     
Erwinia ananas pv. uredovora                                              
                   6.4 ± 1.0                                           
                             86.sup.b                                     
Pseudomonas syringae                                                      
                   47.2 ± 7.1                                          
                              0.sup.c                                     
Untreated control  45.3 ± 6.5                                          
                              0.sup.c                                     
Culture medium control                                                    
                   45.7 ± 6.2                                          
                              0.sup.c                                     
______________________________________                                    
 .sup.y Percent control or reduction in number of pustules as compared to 
 the number on untreated plants. Any two figures followed by the same     
 letter are not significantly different by Duncan&#39;s Multiple Range test at
 p = 0.05.                                                                
 
    
     
                       TABLE 2                                                     
______________________________________                                    
            Number of uredial pustules/leaf.sup.a                         
              B. subtilis                                                 
              treated    Nontreated                                       
                                   Control.sup.b                          
Bean CV       (mean)     (mean)    %                                      
______________________________________                                    
Lake Shasta   9 ± 2.9 231 ± 24.3                                    
                                   96                                     
Mount Hood    12 ± 4.4                                                 
                         317 ± 29.4                                    
                                   96                                     
Pinto 111     8 ± 2.9 346 ± 19.9                                    
                                   98                                     
Mountaineer White                                                         
              8 ± 2.1 322 ± 27.6                                    
                                   98                                     
Half Runner                                                               
BBL 47        3 ± .6  159 ± 27.9                                    
                                   98                                     
______________________________________                                    
 .sup.a The primary leaves of nine plants were counted and averaged for   
 each treatment and control.                                              
 .sup.b Percent control or reduction in number of pustules as compared to 
 the number on untreated plants.                                          
 
    
     
                       TABLE 3                                                     
______________________________________                                    
        Average No. Uredia/leaf                                           
          Treated     Control    Control.sup.b                            
Collection                                                                
          (mean)      (mean)     %                                        
______________________________________                                    
79-4      1.6 ± .5  39.1 ± 14.5                                     
                                 96                                       
.sup. 73-23.sup.a                                                         
          0.7 ± .37                                                    
                      14.5 ± 1.1                                       
                                 95                                       
73-16     0.5 ± .2 14.7 ± 4.0                                       
                                 97                                       
75-22     0.6 ± .19                                                    
                      26.8 ± 5.5                                       
                                 98                                       
73-32     0.4 ± .21                                                    
                      12.0 ± 2.7                                       
                                 97                                       
______________________________________                                    
 .sup.a Collection 7323 causes necrotic spots without sporulation on cv   
 Lake Shasta, therefore, necrotic spots were counted rather than uredia.  
 .sup.b Percent control or reduction in the number of pustules as compared
 to the number on untreated plants. There is no significant difference    
 between these figures at either the 95 or 99% level of significance.     
 
    
     
                       TABLE 4                                                     
______________________________________                                    
                           Average                                        
Treatment       Cultivar   percent rust                                   
______________________________________                                    
1.    Water Control Pinto III  95                                         
                    Lake Shasta                                           
                               40                                         
2.    Eugonbroth    Pinto III  85                                         
      Control       Lake Shasta                                           
                               15                                         
3.    B. Subtilis   Pinto III  22                                         
      APPL-1 Culture                                                      
                    Lake Shasta                                           
                                7                                         
      in early                                                            
      stationary phase                                                    
4.    B. subtilis   Pinto III  15                                         
      APPL-1 Culture                                                      
                    Lake Shasta                                           
                                7                                         
      in late                                                             
      stationary phase                                                    
5.    B. subtilis APPL-1                                                  
                    Pinto III  28                                         
      High MW Fraction                                                    
                    Lake Shasta                                           
                                5                                         
______________________________________                                    
 .sup.a Percent leaf area was estimated using the modified Cobb scale and 
 the figures given are the averages from three replicates.                
 .sup.b Each treatment was applied on every Monday, Wednesday, and Friday 
 for five weeks from time of seedling emergence.