Colonization of Pinus radiata D. Don Seedling Roots by Biocontrol Bacteria Erwinia billingiae and Bacillus simplex

Abstract

Erwinia billingiae S31R1 and Bacillus simplex S11R41, isolated from the rhizosphere of a healthy tree located in a Pinus radiata D. Don plantation with high presence of fungal pathogens, are antagonists of pine root rot fungi Heterobasidion annosum and Armillaria mellea in vitro and in young trees. For effective biocontrol of these pathogens, the bacteria must stably colonize P. radiata roots following their application. To determine root colonization patterns, the bacteria were transformed with stable plasmids encoding green fluorescent protein (GFP). Transformed E. billingiae was visualized on roots 24 days after soil inoculation by confocal and epifluorescence microscopy, and GFP was detected by ELISA 31 days after inoculation. The presence of E. billingiae microcolonies, in some cases in root intercellular spaces, suggests that bacterial growth was active and localized. Fluorescence of B. simplex S11R41 was visualized on P. radiata roots 31 days after inoculation and its colonization pattern changed from scattered cells to localized microcolonies. Although the populations decreased over time, microcolony formation and localization in specific regions of roots indicated that E. billingiae, normally considered to be an epiphyte, and B. simplex can stably colonize roots of P. radiata.