3.5.4
BIOLOGICAL CONTROL OF FIRE BLIGHT USING MICROCIN AND HONEY BEES


JL VANNESTE, J YU, DC CORNISH, and MD VOYLE

HortResearch, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand.

Background and objectives
Few treatments are available for the control of fire blight, a bacterial disease which affects apples, pears, and some ornamentals [1]. We are developing a strategy of biological control using beneficial bacteria. This strategy also includes a delivery mechanism. We are using honey bees to distribute beneficial bacteria to apple and pear flowers which are the main port of entry for Erwinia amylovora the causal agent of fire blight [1]. This system takes full advantage of the fact that we are using organisms which can multiply and colonise easily apple and pear flowers. Most of the work presented here concerns Erwinia herbicola Eh252, an epiphytic bacterium which reduces fire blight incidence when sprayed on apple or pear blossoms [2].

Results and conclusions
We showed that Eh252 produces an antibiotic type compound which inhibits the fire blight pathogen on plates. By comparing the ability in reducing fire blight of the wild type strain (Eh252) with that of transposon induced isogenic mutants which do not produce this antibiotic (Ant- mutants), and by comparing the ability of Eh252 to protect immature pear fruits inoculated with a wild type strain of E. amylovora or with a derivative resistant to the antibiotic produced by Eh252, we showed that this antibiotic was one of the major mechanism leading to reduction of fire blight on immature pear fruits. Clones of Escherichia coli carrying the genes necessary for antibiotic production were identified in a gene library of Eh252 by their ability to inhibit on plate the growth of E. amylovora. Analysis of nested deletions and transposon induced mutants revealed that a 2.2 Kb fragment was necessary to complement all Ant- mutants of E. herbicola and to confer antibiotic production to E. coli. Analysis of the DNA sequence of this fragment and biochemical characteristics of the compound indicate that this antibiotic belongs to a family of low molecular weight peptides called microcins. Production of this compound, now called mccEh252 is not induced by SOS mechanism. It is influenced by temperature of growth, but not by carbon sources or osmolarity. MccEh252 has a molecular weight of less than 3,000 , it retains activity after boiling, and after exposure to extreme pH, but looses its activity in presence of proteinases and histidine.

We showed that bees do carry freeze dried bacteria if a powder of such bacteria is placed in a pollen insert fixed at the exit of the hive. We found that after using a powder of freeze dried bacteria for 3 days, up to 84 % of the flowers were colonised by the beneficial bacteria. After 8 days this percentage peaked to 98 %. Beneficial bacteria were still detected on 95% of the flowers 11 days after the last loading of lyophilised cells. This indicates that bees and other insects picked up beneficial bacteria from the already colonised flowers and brought them to newly opened flowers. The population of bacteria found on flowers varied between 8 x 103 and 7 x 105 bacteria per flower. These figures are quite high considering that in the laboratory the carrying capacity of apple flowers for Eh252 is about 5 x 106 bacteria per flower.

References
1. Vanneste JL, 1995. Pathogenesis and Host-Parasite Specificity in Plant Diseases, eds. K Khomoto, RP Singh and US Singh, Pergamon Press, Oxford, U.K, pp.21-46.
2. Vanneste JL, 1996. Biocontrol News and Information 17, 67N-78N.