Articles
DESIGNING A BIOLOGICAL CONTROL OF FIRE BLIGHT: EXPRESSION OF A NEW PEPTIDE ANTIBIOTIC GENE IN ERWINIA AMYLOVORA AND OF THE HARPIN GENE IN ERWINIA HERBICOLA
Several laboratories have, over the last decades, isolated numerous bacteria which can reduce the incidence of fire blight, when sprayed prior to the pathogen, on apple or pear flowers (see for a review Vanneste 1996). However, only one of these bacteria, Pseudomonas fluorescens A506, is today commercially available (BlightBan A506 Plant Health Technologies, Boise, ID). Although a few other strains might be commercialised in the future, the majority of the strains isolated so far will never be on the market, either because of a lack of consistency or a lack of efficiency.
The probability of finding a wild type strain that consistently prevents or reduces significantly the incidence of fire blight might be low.
Since the modes of action of several of these biological control agents have been identified, we now have the opportunity to construct biological control agents that will combine characteristics from different strains and therefore perform better and/or more consistently.
Some of the characteristics we are trying to combine are ability to colonise the same ecological niche as the pathogen; ability to produce a compound which inhibits the pathogen and ability to induce plant defence mechanisms.
To be effective, a biocontrol agent has to be able to multiply in the same ecological niche as the pathogen.
This results in competition for space and nutrients which in itself, in some cases, is enough to confer protection (Pusey 1997, Wilson and Lindow 1993; Johnson and Stockwell 1998). Non-pathogenic derivatives of E. amylovora are well adapted to compete with the fire blight pathogen for space and nutrients.
Such derivatives would be more effective if they also produced antibacterial compounds which inhibit E. amylovora. Therefore we tried to express in E. amylovora genes which code for the immunity and production of the peptide antibiotic produced by Erwinia herbicola Eh 252. E. herbicola Eh252 produces a small peptide antibiotic we identified as a new microcin (Vanneste and Yu 1996). This microcin has been shown to be one of the mechanisms which allows Eh252 to reduce incidence of fire blight on immature pear fruit (Vanneste et al., 1992). The genes necessary for the production of this compound and immunity were cloned on a multicopy plasmid and introduced in E. amylovora by electroporation. E. amylovora derivatives which contain this plasmid, express these genes and produce large amounts of this antibacterial compound.
They also inhibit the growth of all other E. amylovora strains.
We then tried to determine whether a biological control agent, either a non virulent strain of E. amylovora which still expresses harpin or a strain of E. herbicola which would be given the ability to express harpin, would be able to induce a plant defence reaction, which would then result in an increase of protection against fire blight.
The plasmid pCPP430 that contains and expresses all the genes necessary for production and export of harpin (Wei et al., 1992) was introduced by electroporation in a E. herbicola Eh252 derivative and in a strain of Escherichia coli. A culture supernatant of E. coli pCPP430 induced hypersensitivy on tobacco leaves, indicating it contains harpin and also protected immature pear fruit from fire blight.
However, when we used a strain of E. herbicola which carried pCPP430 or cell extracts from that same strain, the fruits were damaged even in the absence of any pathogens.
Immature pear fruits may not be