Biological control of Erwinia amylovora by Erwinia herbicola
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
A naturally-occurring Erwinia herbicola strain, Eh1087, inhibitory to Erwinia amylovora (Burr.) Winslow et al. (1920) was isolated from apple blossom (Malus X. domestica Borkh.) in a Canterbury orchard. Total populations of E. herbicola on apple blossoms in Canterbury and Hawkes Bay orchards were low (less than 50 cfu.blossom-1) throughout flowering, increasing l00-fold at petal drop. E. Herbicola populations at petal drop were more dominant in Canterbury orchards (30-50% of total) than in Hawkes Bay orchards (1-2% of total) in the seasons surveyed. In vitro inhibition of E. amylovora was observed for 20% of all bacterial strains isolated from Canterbury orchards and from none of the strains isolated from Hawkes Bay orchards under the conditions used. Eh1087 established stable populations for at least 10 days post application when sprayed onto apple blossoms in the orchard. This strain produced a broad spectrum, non-peptide, β-lactam antibiotic in vitro that was bactericidal for E. amylovora. Eh1087 suppressed E. amylovora disease symptoms in immature pear fruit, excised apple shoots and in apple blossoms. The partially purified antibiotic of Eh1087 reduced the severity of symptom expression in immature pear fruit. TnphoA-insertion mutants of Eh1087 were created (Ant-) that failed to inhibit E. amylovora in vitro and in immature pear fruit. Ant- mutants no longer produced an antibiotic inhibitory to E. amylovora in vitro. TnphoA insertions in the Ant- mutants all mapped within a 1.5 kb region on a 200 kb indigenous plasmid of Ehl087. A genomic library of Eh1087 was constructed and cosmids which complemented Ant- mutants to the Ant+ phenotype in vitro and in immature pear fruit were selected. A HinIII 8 kb cosmid fragment subclone, pAH8, weakly complemented Ant- mutant EhA17g. This complementation was enhanced by the presence in trans of an EcoR1 5 kb cosmid fragment subclone, pBE5B, which mapped approximately 6 kb away from the region of TnphoA insertions. Analysis of the proteins produced by these cosmid fragment sub clones in Escherichia coli mini-cells revealed that pBE5B codes for three proteins, approximately 20, 34 and 41 kd in size and pAH8 codes for two proteins, approximately 28 and 39 kd in size. At least one of the proteins coded for by pAH8 must be essential to antibiotic synthesis in Eh1087. The possible relationship between the cosmid fragment-encoded proteins and b-lactam biosynthetic enzymes is discussed.