Kinetics and strain specificity of rhizosphere and endophytic colonization by enteric bacteria on seedlings of Medicago sativa and Medicago truncatula

Abstract

The presence of human-pathogenic, enteric bacteria on the surface and in the interior of raw produce is a significant health concern. Several aspects of the biology of the interaction between these bacteria and alfalfa (Medicago sativa) seedlings are addressed here. A collection of enteric bacteria associated with alfalfa sprout contaminations, along with Escherichia coli K-12, Salmonella enterica serotype Typhimurium strain ATCC 14028, and an endophyte of maize, Klebsiella pneumoniae 342, were labeled with green fluorescent protein, and their abilities to colonize the rhizosphere and the interior of the plant were compared. These strains differed widely in their endophytic colonization abilities, with K. pneumoniae 342 and E. coli K-12 being the best and worst colonizers, respectively. The abilities of the pathogens were between those of K. pneumoniae 342 and E. coli K-12. All Salmonella bacteria colonized the interiors of the seedlings in high numbers with an inoculum of 10(2) CFU, although infection characteristics were different for each strain. For most strains, a strong correlation between endophytic colonization and rhizosphere colonization was observed. These results show significant strain specificity for plant entry by these strains. Significant colonization of lateral root cracks was observed, suggesting that this may be the site of entry into the plant for these bacteria. At low inoculum levels, a symbiosis mutant of Medicago truncatula, dmi1, was colonized in higher numbers on the rhizosphere and in the interior by a Salmonella endophyte than was the wild-type host. Endophytic entry of M. truncatula appears to occur by a mechanism independent of the symbiotic infections by Sinorhizobium meliloti or mycorrhizal fungi.

FIG. 1.

Numbers of GFP-labeled bacterial CFU recovered from interiors (A) and rhizospheres (B) of alfalfa seedlings 5 days after inoculation of 1.5-day-old seedlings with different inoculum levels. The data points represent the means and the bars represent the standard errors of the means of results from four replicate treatments. Four seedling samples (roots and hypocotyls) were taken from each replicate. SCH7976, S. enterica serovar Cubana strain H7976; Kp342, maize endophyte K. pneumoniae 342; SIH3517, S. enterica serovar Infantis strain H3517; S8137, S. enterica strain 8137; 14028, S. enterica serovar Typhimurium strain ATCC 14028; gfw, gram (fresh weight).

FIG. 2.

Numbers of bacterial CFU recovered from interiors (A) and rhizospheres (B) of alfalfa seedlings 5 days after inoculation of 1.5-day-old seedlings with different inoculum levels. The data points represent the means and the bars represent the standard errors of the means of results from four replicate treatments. Four seedling samples (roots and hypocotyls) were taken from each replicate. SCH7976, S. enterica serovar Cubana strain H7976; Kp342, maize endophyte K. pneumoniae 342; K12, E. coli K-12 strain MG1665; O157, E. coli O157:H7 strain F4546; gfw, gram (fresh weight).

FIG. 3.

Longitudinal section of alfalfa hypocotyls (A to H) and M. truncatula hypocotyls (J to L) and transverse section of alfalfa hypocotyls (I) showing colonization by the following GFP-labeled bacteria: E. coli K-12 (B), E. coli O157:H7 (C), K. pneumoniae 342 (D), S. enterica strain 8137 (E), S. enterica serovar Infantis strain H3517 (F), S. enterica serovar Typhimurium strain ATCC 14028 (G), and S. enterica serovar Cubana strain H7976 (H and I). (A) Uninoculated control. (J, K, and L) M. truncatula wild-type plant (J) and dmi1 mutant (K and L) inoculated with S. enterica serovar Cubana strain H7976. (B to L) Sections were visualized 9 days after inoculation. The inoculum level was 10⁴ CFU/plant. Arrows point to GFP-tagged bacterial cells. Bars, 50 μm.

FIG. 4.

Longitudinal section of alfalfa roots (A to I) and M. truncatula roots (J to L) showing colonization by the following GFP-labeled bacteria: E. coli K-12 (B), E. coli O157:H7 (C), K. pneumoniae 342 (D), S. enterica strain 8137 (E), S. enterica serovar Infantis strain H3517 (F), S. enterica serovar Typhimurium strain ATCC 14028 (G), and S. enterica serovar Cubana strain H7976 (H and I). (A) Uninoculated control. (J) M. truncatula without inoculation. (K and L) M. truncatula wild-type plant (K) and dmi1 mutant (L) inoculated with S. enterica serovar Cubana strain H7976. (B to L) Sections were visualized 9 days after inoculation. The inoculum level was 10⁴ CFU/plant. Arrows point to the lateral roots. Bars, 50 μm.

FIG. 5.

Numbers of CFU recovered from interiors (A) and rhizospheres (B) of M. truncatula plant tissues 7 days after inoculation with different inoculum levels. SCH7976-WT, M. truncatula wild type inoculated with S. enterica serovar Cubana strain H7976; SCH7976-“Dmi1,” M. truncatula dmi1 mutant inoculated with S. enterica serovar Cubana strain H7976; gfw, gram (fresh weight).