A full factorial analysis of nine factors influencing in vitro antagonistic screens for potential biocontrol agents

Abstract

The effect of nine factors on the outcome of classic in vitro screens testing the antagonistic action of endophytic bacterial isolates from grape vines against virulent Agrobacterium vitis has been examined. These factors were (i) the strain of A. vitis, (ii) the strain of endophyte, (iii) the growth medium of the pathogen, (iv) the growth medium of the endophyte, (v) the temperature of growth of the pathogen, (vi) the temperature of growth of the endophyte, (vii) the pH of growth of the pathogen, (viii) the pH of growth of the endophyte, and (ix) the medium of the assay plate. Analyses of variance of the full factorial design incorporating main effects and two- and three-way interactions accounted for 66% of the variance. All nine factors had a significant effect on the diameter of inhibition zones (p < 0.001). An examination of the three-way interactions revealed that generalizations were difficult to draw; each target agrobacterium had a specific response to a given antagonistic isolate. It was possible to determine that the growth history of bacterial strains, before they were administered to an assay plate to test for antagonism (especially the composition of the growth medium and the temperature of growth), had a profound effect on the outcome of the test. Generally the more chemically defined media produced less inhibition whereas the lower growth temperature of 15 °C produced more inhibition. These findings could be relevant to in situ inhibitory activity. The method used to conduct the inhibitory screen (order of strain application and the medium of the assay plate) had a profound influence on the results. These influences add to the caution necessary in the use of in vitro antagonistic screens for finding successful biocontrol agents.Key words: in vitro antagonism, endophytic bacteria, grape crown gall disease, biocontrol, screening strategy.