A Novel Approach Using H-Mutant Bacteriophages for the Biological Control of Bacterial Blight of Geranium. Final Report
A Novel Approach Using H-Mutant Bacteriophages For The Biological Control of Bacterial Blight of Geranium and Erwinia Soft Rot of Poinsettia
Final Report submitted by Dr. Brent Harbaugh, Professor of Floriculture, Dr. Jeffrey B. Jones, Professor of Bacteriology, Mr. Joseph E. Flaherty, Research Associate at the Univ. of Florida’s Gulf Coast Research and Education Center, Bradenton, FL., and Dr. Lee Jackson, Research Scientist, AgriPhi., Inc., Logan, UT.
Industry Needs and Project Objectives:
Bacterial diseases may well be the most difficult of all floricultural crop diseases to control. Current control measures are often directed at preventing disease and include using culture-indexed plants, destroying infected plants, and disinfecting equipment and growing structures. Only a few bactericides are labeled for ornamental use and these have achieved modest control at best; furthermore, bacteria are notorious for developing resistance to new products quickly. In addition, the public does not consider pesticides environmentally safe and the horticulture industry is constantly under pressure to find alternative control measures.
Our research, which was supported by the American Floral Endowment, is a novel approach utilizing bacteriophages (phages) as biological control agents for the prevention and control of bacterial diseases of ornamentals. Bacteriophages are viruses that kill bacteria. The life cycle of phages and manner in which phages kill bacteria is illustrated in Fig. 1.
Figure 1. Life cycle of bacteriophage.
These viruses are very specific for target bacteria and are nontoxic to workers and non-target bacteria. Thus, this research was initiated to develop an environmentally safe and effective alternative for prevention and control of bacterial diseases by utilizing bacterial viruses. We chose two model systems that would have application for control strategies for bacterial diseases of floricultural crops in greenhouse-field and propagation environments.
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Greenhouse-field studies:
Perhaps the most difficult problem to overcome in geranium production, and certainly the most serious disease of geraniums, is bacterial blight caused by Xanthomonas campestris pv pelargonii (X.c.p.). Bacterial blight, also referred to as bacterial stem rot, bacterial leaf spot or bacterial wilt, has destroyed entire geranium crops and caused millions of dollars in losses. Geranium growers fear this deadly disease because it is extremely difficult to control and can be spread through cuttings, soil, water, and insects. Experiments were completed on geraniums grown in plug trays and as 4-inch potted plants in the greenhouse.
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Propagation studies:
Bacterial soft rot of poinsettias, caused by Erwinia carotovora subsp. carotovora (E.c.c.), is a grave propagation disease of poinsettias. Propagation-associated diseases are often difficult to control because mist systems provide an ideal environment for bacterial survival. E.c.c. causes a soft rot on cuttings and can completely or partially prevent rooting of cuttings as well as lead to death of cuttings. In addition, infected cuttings have the potential to carry E.c.c. to production houses where the bacterium can spread and damage or kill potted crops.
Summary of Research
Development of phage mixtures:
- Phages for Xanthomonas campestris pv pelargonii (X.c.p.). We have successfully isolated phages for X.c.p. in soil samples from CA, FL, UT, and MN. After selection of these phages for increased virulence, 17 phage strains were screened against 21 X.c.p. strains. The most virulent strains were then improved by developing and selecting h-mutants as illustrated in Fig. 2. This procedure selects for bacteria resistant to the wild-type phages employed and then subjects these resistant bacteria to high concentrations of phages. This facilitates selection of mutant phages that can attack the formerly resistant bacteria. The following figure illustrates the developmental procedure for h-mutant bacteriophages.
Figure 2. Protocol for selecting for H-mutants from the wild-type phage (P1).
Unlike the past use of phage for prevention of disease in which a single wild-type phage was used, our approach utilized a mixture of four h-mutant phages. If the control application is a mixture of phages including h-mutants, any phage-resistant bacterial mutants arising in a natural bacterial-pathogen population will be destroyed by h-mutants in the multiphage composition. At the completion of each experiment, any surviving bacteria were tested against the phage mixture to determine if survival was due to resistance or application procedures. We did not detect any bacterial strains that were not killed by the h-mutant mixture indicating resistance was not a problem with this control strategy. Thus, we have been able to produce a mixture of phages that has been effective against over 30 X.c.p. strains collected from around the world, and we have not detected development of resistant strains.
- Phage for Erwinia carotovora subsp. carotovora. We have isolated six phages that will kill E.c.c. isolated from poinsettia. We have also tested these phages against E.c.c. from other plant material, such as caladium and Kalenchoe. While we are concentrating on poinsettia, we hope to gain additional strains that will be effective against E.c.c. from other vegetatively propagated ornamentals. We are currently developing h-mutants from the six-phage/poinsettia strains.
Greenhouse/propagation house experiments:
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Efficacy of h-mutants phage mixtures on plug and pot geraniums.
We have shown that daily applications of h-mutant phage mixtures can provide similar or better control of bacterial blight of geranium when compared to plants treated with copper sulfate pentahydrate (Phyton-27) applied at 10 or 14 day intervals. Disease incidence and severity were less for plants treated daily with phage than for those treated less frequently with phages or Phyton-27. The high disease pressure present in our tests may have overcome the bactericidal effects of Phyton27, and further signifies the potential of bacteriophages for controlling outbreaks of bacterial spot in the greenhouse. Given the reduction of bacterial blight observed by daily mist applications of phages under conditions of high disease pressure, less frequent applications may serve to adequately control the disease under conditions encountered by growers.
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Use of phages specific for E.c.c. on poinsettia in a mist system.
Poinsettia cuttings were dipped in a solution of bacteriophages found to be specific to E. carotovora subsp. carotovora to determine if a single application could reduce the incidence of Erwinia soft-rot. Results gained from an initial test have shown that cuttings dipped in water alone and placed in oasis cubes soaked in a suspension of pathogenic Erwinia can result in over 50% of cuttings getting soft-rot. If dipped in a concentrated solution of phages prior to placement in the Erwinia containing cubes, the incidence of soft-rot was reduced to less than 10%. While still in the preliminary stage, results from this and other tests indicate control strategies utilizing phages in the propagation environment (mist-propagation) will significantly reduce the loss of cuttings due to Erwinia soft rot.
Application methods:
- Xanthomonas campestris pv pelargonii (X.c.p.). It was reported that phages had the potential to move systemically within tomato plants and that phages applied in the soil were recovered in tomato leaves and flowers. We have not been able to demonstrate either the uptake of phages or that phages move systemically within geranium plants. Thus, application of phages will be as foliar sprays. We have shown that phage longevity on leaf surfaces decreases significantly after 24 hours. In conditions of a severe outbreak of bacterial blight (similar to the disease pressure used in our tests), daily applications gave better control than applications once or twice per week. Recognizing that daily applications may not be economically feasible, we are currently evaluating compounds that may extend the longevity of phages in the cropping environment.
- Erwinia carotovora subsp. carotovora. We have initiated experiments to determine if dipping cuttings in a phage solution, drenching oasis rooting cubes in phage solutions, or using phages injected into the mist system would provide the best strategy for control of Erwinia soft rot in a mist system. While we are still evaluating these strategies, it appears that a combination of treatments will provide the highest degree of control. However, all methods are relatively easy to adapt to cutting production and would not appear to be an economical or practical deterrent to their use.
Future Plans and Anticipated Benefits to the Industry
We are continuing to seek and analyze plant and soil samples from growers from around the U.S. that have bacterial blight of geraniums and Erwinia soft rot of poinsettias. It is important to find many strains of bacteriophages to insure a broad range of effectiveness against all strains of X.c.p. or Erwinia. We are continuing to develop a phage mixture that can attack a broad range of Erwinia species that infect various ornamental hosts. Additional research needs to be conducted on application strategies for use of bacteriophages in harsh environments. We have identified an inexpensive compound that extends the life of phages in the cropping environment, and it may serve to reduce the application frequencies required for use of bacteriophages in greenhouse or field environments.
This research has been a pioneering effort and the need for further research is great if products are to be developed and control strategies precisely defined. However, these research results are far reaching and indicate this new technology can significantly benefit the floriculture industry. We believe the American Floral Endowment has made an important contribution to the future success of our industry by supporting this work on biological control of bacterial diseases. Our research has demonstrated that h-mutant bacteriophage mixtures can be developed for control of bacterial diseases affecting floricultural crops. We have shown that h-mutant bacteriophage mixtures have the potential to be integrated into control strategies for crops grown in greenhouse and propagation house environments. The level of control in our tests utilizing h-mutant bacteriophages for bacterial blight of geraniums was similar or better than the control achieved using a standard copper-based bactericide. In addition to being environmentally safe, we have not seen the development of resistance to h-mutant bacteriophages. The utilization of h-mutant bacteriophages and the application procedures tested in this research have the potential to be developed for control of other bacterial diseases of floricultural crops including cut flowers, flowering potted plants, and bedding plants.
