A technique To quantify the population size and composition of the biofilm component in communities of bacteria in the phyllosphere

Abstract

The presence of microbial biofilms in the phyllosphere of terrestrial plants has recently been demonstrated, but few techniques to study biofilms associated with living plant tissues are available. Here we report a technique to estimate the proportion of the bacterial population on leaves that is assembled in biofilms and to quantitatively isolate bacteria from the biofilm and nonbiofilm (solitary) components of phyllosphere microbial communities. This technique is based on removal of bacteria from leaves by gentle washing, separation of biofilm and solitary bacteria by filtration, and disintegration of biofilms by ultrasonication. The filters used for this technique were evaluated for their nonspecific retention rates of solitary bacteria and for the efficiency of filtration for different concentrations of solitary bacteria in the presence of biofilms and other particles. The lethality and efficiency of disintegration of the sonication conditions used here were also evaluated. Isolation and quantification of bacteria by this technique is based on use of culture media. However, oligonucleotide probes, sera, or epifluorescent stains could also be used for direct characterization of the biofilm and solitary bacteria in the suspensions generated by this technique. Preliminary results from estimates of biofilm abundance in phyllosphere communities show that bacteria in biofilms constitute between about 10 and 40% of the total bacterial population on broad-leaf endive and parsley leaves.

FIG. 1

Steps in the method for quantifying the population sizes of biofilm and solitary bacteria in the phyllosphere.

FIG. 2

Effect of ultrasonication on the culturability of total (A and B) and fluorescent (C and D) epiphytic bacteria. Bars represent the mean population densities in three aliquots sonicated for each combination of time and amplitude. Error bars indicate the standard error. Panels A and C and panels B and D represent the results of two independent trials.

FIG. 3

Effect of ultrasonication on the number of total (A and B) and fluorescent (C and D) bacterial CFU retained on Isopore polycarbonate filters with 5-μm-diameter pores. Bars represent the mean population densities retained on filters after sonication of three aliquots for each combination of time and amplitude. Error bars indicate the standard error. Panels A and C and panels B and D represent the results of two independent trials.