Xanthomonas axonopodis pv. phaseoli var. fuscans is aggregated in stable biofilm population sizes in the phyllosphere of field-grown beans

Abstract

The occurrence of "Xanthomonas axonopodis pv. phaseoli var. fuscans" (proposed name) populations as biofilms on bean leaves was investigated during three field experiments on plots established with naturally contaminated bean seeds. Behavior of aggregated versus solitary populations was determined by quantification of culturable cells in different fractions of the epiphytic population separated by particle size. X. axonopodis pv. phaseoli var. fuscans population dynamic studies confirmed an asymptomatic and epiphytic colonization of the bean phyllosphere. For all years of experiment and cultivars tested, biofilms and solitary components of the populations were always detected. Biofilm population sizes remained stable throughout the growing season (around 10(5) CFU/g of fresh weight) while solitary population sizes were more abundant and varied with climate. According to enterobacterial repetitive intergenic consensus fingerprinting, aggregated bacterial isolates were not different from solitary isolates. In controlled conditions, application of a hydric stress resulted in a decrease of the solitary populations on the leaf surface while the biofilm fraction remained stable. Suppression of the hydric stress allowed solitary bacterial populations to increase again. Aggregation in biofilms on leaf surfaces provides protection to the bacterial cells against hydric stress.

FIG. 1.

Scanning electron microscopic micrographs of field-grown bean leaf surfaces colonized by seed-borne X. axonopodis pv. phaseoli. (A) Leaf surface showing mostly solitary bacterial populations (Sol. b.). Note the accumulation of bacterial cells in grooves (G) between epidermal cells. Bar, 10 μm. (B) Focus on a bacterial biofilm. Note the matrix (M) embedding bacterial cells constituting a typical biofilm. Bar, 1 μm.

FIG. 2.

X. axonopodis pv. phaseoli population dynamics on bean leaflets for two field experiments (1999 [A] and 2000 [B]) and two cultivars (Michelet [A] and Contender [B]). Epiphytic bacterial populations are divided into solitary and biofilm fractions. Error bars represent the standard error of the mean. For a given fraction of the population in each experiment mean population sizes followed by different letters are significantly (P < 0.05) different on the basis of the Newman-Keuls test. Water amounts of every rainfall higher than 10 mm are indicated by arrows. fr. wt., fresh weight.

FIG. 3.

Dynamics of different fractions of X. axonopodis pv. phaseoli populations on bean leaflets following application of a hydric stress. The figure shows total (A and D), epiphytic (B and E), solitary (C and F [rectangles]), and biofilm (C and F [triangles]) X. axonopodis pv. phaseoli var. fuscans CFBP4834-R population dynamics on plants submitted to hydric stress (dashed line) applied (↓) 4 days after spray inoculation compared to plants maintained in high-RH conditions (solid line) for two repetitions of the experiment (first trial, A to C; second trial, D to F). Stress was suppressed (↑) after sampling on day 6. Error bars represent the standard error of the mean. For a sampling date mean population sizes followed by different letters are significantly (P < 0.05) different on the basis of the Newman-Keuls test.