Articles
PLUGS OF IMMATURE PEAR FRUIT TO ASSESS THE VIRULENCE OF ERWINIA AMYLOVORA AND TO STUDY IN THE LABORATORY THE INTERACTION BETWEEN BIOLOGICAL CONTROL AGENTS AND THE FIRE BLIGHT PATHOGEN
This assay has since been widely used for selecting potential biological control agents (Beer and Rundle, 1983; Beer et al., 1984a, 1984b; Kearns and Hale, 1995; McLaughlin et al., 1993; Nicholson et al., 1990; Psallidas et al., 1993; Wilson et al., 1990) as well as to determine the mode of action of some these biological control agents (lshimaru, et al., 1988; Kearns, 1993; Vanneste et al., 1990, 1992; Wright and Beer 1996). It consists of pipetting into a well bored in the cheek of a pear fruit cut longitudinally in half, a suspension of the biological control agent followed by a suspension of the pathogen.
The fruits are then incubated in a tray lined with wet paper (humid chamber) for up to 6 days, and are examined daily for appearance of exudate.
Because the time needed for the appearance of exudate is dependent on the humidity present in the experimental tray, only pears incubated in the same humid chamber can be compared.
This limits the number of biocontrol agents or of derivatives that can be compared per experiment, and makes the statistical analysis quite difficult since the number of half pears used per treatments is also limited.
We modified this assay by using plugs of immature pear fruit (ca. 2 mm thick and ca. 4 mm diameter) rather than half pears.
This change enabled us to routinely compare in one tray twelve different treatments (biological control agents, bacterial metabolites or chemicals), using 12 to 20 plugs per treatment, which allows statistical analysis of the results.
Pears were surface disinfected with ethanol 70%, sliced and cored using a sterile cork borer number 1. Cores, or plugs, were made only from the flesh of the fruit, vascular tissues were avoided.
The plugs were placed on a Petri dish itself placed on wet paper towel in a large tray.
Twenty μl of a biological control agent suspension or of MgSO4 10 mM, used as a negative control, were pipetted over the entire surface of the core and allowed to adsorb before the cores were inoculated with 10 μl of a suspension of the fire blight pathogen.
The tray was then bagged such as to act as a humid chamber, and incubated at 27°C. The flat surface of the core was examined daily for presence of exudate.
This bioassay proved quite sensitive especially when using the cultivars Beurre Bosc or Williams Bon Chretien.
It has been a useful test to confirm the identity of strains isolated from the orchard, since up to 20 isolates can easily be assayed using a single pear.
This pear core assay was also used to compare the virulence of different strains and derivatives of the fire blight pathogen (data not shown).
We used the bioassay to compare the ability of Eh252, a strain of Erwinia herbicola that reduces fire blight incidence in apple and Asian pear orchards (Beer et al. 1984; Vanneste and Yu 1990, 1993; Vanneste et al. 1995), to protect pear fruit from fire blight,