1a. Objectives (from AD-416):
The long-term goal of this project is to develop an effective biologically-based and integrated program for controlling fire blight of apple and pear caused by Erwinia amylovora. Over the next 5 years we will focus on the following objectives: (1) Develop improved formulations of commercial biocontrol agent Pantoea agglomerans E325 that promote rapid colonization on blossom stigmas and effective preemptive exclusion of disease bacterium; (2) Increase biocontrol efficacy by using the bacterium E325 as a carrier of bacteriophages that preferentially attack the fire blight bacterium; (3) Develop yeast biocontrol agent that complements E325 and is highly tolerant of osmotic conditions in blossom nectaries where infection occurs; (4) Integrate optimized biocontrol mixtures with other fire blight management approaches, including agents that induce plant host resistance. Fire blight is generally initiated by populations of E. amylovora that become established on the surfaces of flower stigmas and later spread in surface moisture to the hypanthium where invasion occurs through nectary openings. Thus, the overall project strategy is to optimize suppression of the pathogen in two distinct microenvironments, i.e., stigmatic and hypanthial floral surfaces, which represent the first and second lines of defense against disease development, respectively. Work related to Objectives 1 through 3, representing bacterial, viral and fungal components of the biological control proposed, will be done separately and concurrently at first, but eventually merged as part of Objective 4.
1b. Approach (from AD-416):
Objective 1 will involve multiple approaches for improving the formulation of the bacterial biocontrol agent, Pantoea agglomerans strain E325, which is primarily adapted to the flower stigma where the disease organism (Erwinia amylovora) becomes established. To increase E325 survival in dry preparations and water-limited microenvironments, osmoadaptation involving the augmentation of growth media with sodium chloride and glycine betaine will be investigated. The delivery of E325 in alginate capsules the size of pollen grains will be examined both for protecting the biocontrol agent from environmental extremes and for increasing its dispersal by pollinating insects from dry non-secretory floral surfaces (including pollen-bearing anthers) to the stigmas and hypanthia of flowers emerging after or between spray applications. Strategies for enhancing biocontrol efficacy include amending the growth medium and product formulation with amino acids known to increase E325 production of a compound that specifically inhibits the disease organism. Further, efforts will be made to capture the active E325 compound from the growth medium and formulate it with the bacterial cells. To accomplish Objective 2, bacteriophage strains from a Canadian collection will be selected based on their compatibility with E325 as a phage carrier and their efficacy against strains of E. amylovora representative of the Pacific Northwest. Subsequently, a mixture of select phage strains will be tested in combination with E325 against E. amylovora on flowers in the laboratory and orchard. For Objective 3, a local collection of yeast strains originally isolated from apple flowers and shown to be adapted to flower stigmas, the tissue first contacted by treatment sprays, will be further screened for their adaptation and suppression of E. amylovora in the flower hypanthium, where infection usually occurs through nectary openings. Yeasts will also be evaluated for their tolerance to osmotic conditions typical of the nectar-rich hypanthium, compatibility with E325, and temperature growth range compared to the bacterium. Objective 4 will clear a path through the complexities of merging diverse biocontrol agents together with formulation strategies, including the use of a low-pH buffer to give advantage to biocontrol organisms over E. amylovora. It will also integrate biocontrol with other disease control approaches, such as the use of fast-acting “soft” chemicals that can be applied when predictive models indicate a high disease risk, or agents that enhance plant resistance to E. amylovora. In general, the above approaches will be tested in the laboratory and greenhouse prior to evaluation in research orchards.
3. Progress Report:
Fire blight is a potentially devastating disease of apple and pear trees that is generally initiated in flowers. Disease control historically depended on chemical control measures, but pathogen resistance and safety-related concerns prompted a search for alternative strategies. ARS researchers previously developed the biocontrol agent Pantoea agglomerans strain E325, now available commercially, and showed that strain E325 produces a peptidic inhibitor highly specific to the disease organism. Recent efforts to improve E325 through a process known as osmoadaptation led to modifications in its commercial production and formulation. As another attempt to improve product formulation, collaboration with nanotechnology scientists at the University of Illinois led to preliminary data indicating the potential of microencapsulation as a means of protecting biocontrol agents and controlling their release in orchard environments. Progress was also made toward the goal of enhancing biological control of fire blight through the use of microbial mixtures with complementary modes of action and ecological niches. In 2012 the yeast Cryptococcus infirmo-miniatus strain CIMyy6, which is already being developed commercially for controlling postharvest fruit diseases, was evaluated as a potential complement to strain E325 against fire blight. The above activities relate to Objective 1 (“Develop improved formulations…”) and Objective 3 (“Develop yeast biocontrol agent that complements E325…”).
Kim, I., Pusey, P.L., Zhao, Y., Korban, S.S., Choi, H., Kim, K. 2012. Controlled release of Pantoea agglomerans E325 for biocontrol of fire blight. Controlled Release Journal. 161:109-115.