kinetics and strain speciﬁcity of rhizosphere and

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2003, p. 1783–1790 Vol. 69, No. 30099-2240/03/$08.00�0 DOI: 10.1128/AEM.69.3.1783–1790.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Kinetics and Strain Specificity of Rhizosphere and EndophyticColonization by Enteric Bacteria on Seedlings of Medicago

sativa and Medicago truncatulaYuemei Dong,1 A. Leonardo Iniguez,1 Brian M. M. Ahmer,2 and Eric W. Triplett1*Department of Agronomy, University of Wisconsin—Madison, Madison, Wisconsin 53706,1 and Department

of Microbiology, The Ohio State University, Columbus, Ohio 432102

Received 7 October 2002/Accepted 12 December 2002

The presence of human-pathogenic, enteric bacteria on the surface and in the interior of raw produce is asignificant 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 sproutcontaminations, along with Escherichia coli K-12, Salmonella enterica serotype Typhimurium strain ATCC14028, and an endophyte of maize, Klebsiella pneumoniae 342, were labeled with green fluorescent protein, andtheir abilities to colonize the rhizosphere and the interior of the plant were compared. These strains differedwidely in their endophytic colonization abilities, with K. pneumoniae 342 and E. coli K-12 being the best andworst 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 of102 CFU, although infection characteristics were different for each strain. For most strains, a strong correla-tion between endophytic colonization and rhizosphere colonization was observed. These results show signifi-cant strain specificity for plant entry by these strains. Significant colonization of lateral root cracks wasobserved, 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 inthe interior by a Salmonella endophyte than was the wild-type host. Endophytic entry of M. truncatula appearsto occur by a mechanism independent of the symbiotic infections by Sinorhizobium meliloti or mycorrhizal fungi.

Since 1995, there have been several reports of pathogenicSalmonella contaminating raw produce (1, 23). Salmonella out-breaks attributed to alfalfa sprout contamination have beenreported in Finland and in several states, including California,Kansas, Missouri, Nevada, Oregon, and Washington (18, 23,24). In each of these cases, the seeds were contaminated priorto germination. Various approaches to surface sterilize vege-tables do not eliminate the enteric pathogens (2, 24, 25, 26).This result led to the discovery that these enteric pathogensreside within the interiors of apples, lettuce, and alfalfa sprouts(4, 11, 16, 26). Gandhi et al. (11) showed that a SalmonellaStanley strain could reside within the interiors of alfalfasprouts to a depth of 18 �m from the surface. No cells weredetected on the surface.

Nothing is known about the genetics of the invasion of aplant’s intercellular spaces by bacteria. It is not known whetherthis process is regulated at the gene level by either the planthost or the bacterium. That is, do genetic determinants in thehost and/or the bacterium influence endophytic colonization?If strains of Salmonella differ in their abilities to colonize theplant host, it is reasonable to assume that genes within thebacteria can affect endophytic colonization. Similarly, if hostgenotypes of a given species differ in their abilities to be in-fected by a specific strain of Salmonella, it can be reasonably

assumed that there are genetic determinants in the hosts thatregulate infection by endophytes.

Similarly, nothing is known about the kinetics of endophyticinvasion by bacteria. For example, how many bacterial cellsmust be present in the inoculum for plant infection? Does theextent of endophytic colonization by the bacterium increasewith increasing inoculum size? Is the ability to colonize therhizosphere correlated with the invasion of the plant interior?How many salmonellae are found in the interiors of alfalfaseedlings, and how is this related to inoculum size? In thiswork, the kinetics of endophytic colonization by several Sal-monella strains was studied. Most of the Salmonella strainsused here were derived from salmonellosis outbreaks associ-ated with alfalfa sprouts. Rhizosphere colonization by thesebacteria was also assessed in this work.

Another important unanswered question is whether the pro-cess of infection by endophytic bacteria is related to the infec-tion of alfalfa roots by nitrogen-fixing root nodule bacteria ormycorrhizal fungi. This question encouraged us to examine theendophytic colonization of a mutant of Medicago truncatulathat is not infected by Sinorhizobium meliloti or Glomus sp. (5).This mutant is called dmi1. This experiment also addresses thequestion of whether host genotype matters in endophytic col-onization. Examination of the colonization patterns withinsuch host mutants may lead to effective strategies to preventthe entry of bacteria into many foods. An M. truncatula mutantwas chosen for this work since this plant is a model legume thatis closely related to alfalfa. In addition to its role as a modelplant, M. truncatula is used in agriculture as a forage legumecalled annual medic. In addition, there are several mutants of

* Corresponding author. Mailing address: University of Wisconsin—Madison, Department of Agronomy, 1575 Linden Dr., Madison, WI53706. Phone: (608) 262-9824. Fax: (608) 262-5217. E-mail:[email protected].

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M. truncatula available that are defective in plant-microbe in-teractions, particularly symbiotic associations.

The mode of entry of these human pathogens into plants isunknown. The nitrogen-fixing endophytes of sugarcane andother plants, Gluconacetobacter diazotrophicus and Herbaspiril-lum seropedicae, colonize lateral root junctions in high num-bers, making these junctions a likely site of plant entry (13, 14,20). These bacteria also enter the xylem and may infect thestem cortex through the xylem (13, 14). Here the ability ofenteric bacteria to colonize lateral root cracks and the plant’svascular tissue was examined.

MATERIALS AND METHODS

Bacterial strains and inoculum preparation. The bacterial strains used in thiswork are listed in Table 1. The organisms were cultured on solid Luria-Bertani(LB) medium (pH 7.3; Difco Laboratories, Detroit, Mich.) at 37°C for 24 h.Tetracycline (10 �g/ml) and ampicillin (50 �g/ml) were added for plasmid main-tenance following transformation with a stably maintained plasmid that expressesgreen fluorescent protein (GFP) in culture and in planta (see below). Bacteriawere suspended in phosphate-buffered saline (PBS; pH 7.2) to make concentra-tions of 108 CFU/ml for inoculation. The bacterial suspensions were then dilutedto provide inoculum levels of 107, 106, 105, 104, 103, 102, 101, and 100 CFU/seedling.

GFP expression and plasmid construction and stability. The plasmid gfpmut2(9) contains a mutated GFP (GFP-S65T, V68L, S72A) which under the controlof the promoter lac expresses a highly fluorescent GFP in Escherichia coli K-12MG1665. Since many of the Salmonella strains used in this work are ampicillinand kanamycin resistant but tetracycline sensitive, a tetracycline resistance cas-sette was inserted into gfpmut2 as follows. A 2.0-kb HindIII fragment containinga tetracycline cassette from pKRP12 (19) was inserted into the HindIII site ofgfpmut2, yielding pGFPTc2. Competent Klebsiella, Salmonella, and E. coli cellswere transformed with pGFPTc2 by the calcium chloride method described bySambrook et al. (22), which was slightly modified to use 50 mM rather than 100mM CaCl2. Transformed clones containing pGFPTc2 were directly selected asyellow-green colonies on solid LB medium containing ampicillin (50 �g/ml) andtetracycline (10 �g/ml). Only the GFP-labeled strains were used in this study.Thus, the GFP-tagged strains are referred to by their original names.

Plasmid stability in Salmonella enterica and E. coli strains was determined bygrowing those strains in LB liquid medium without selection pressure for over 24generations (doubling times) and then plating a dilution series on both LB agarand LB agar with ampicillin and tetracycline. Four replicates were included foreach treatment. The colonies on the plates were counted, and the ratio ofcolonies from plates with antibiotic selection pressure to those from plateswithout antibiotic selection pressure was determined.

Surface sterilization, germination, and inoculation of seeds. (i) Medicago

sativa cv. CUS101. Alfalfa seeds were obtained from J. W. Jung Seeds Co.(Randolph, Wis.). Alfalfa seeds (0.5 g per bacterial treatment) were surfacesterilized by immersing the seeds in 70% ethanol for 5 min followed by threewashes with sterile water. Seeds were then immersed in a 10% bleach solution for20 min followed by three washes with sterile water. Surface-sterilized seeds wereallowed to germinate on 0.5% agar LB plates overnight in the dark at roomtemperature. Seedlings with roots about 5 mm in length were transplanted intotest tubes (18 by 150 mm) containing 10 ml of Jensen’s N-free medium (15) with0.45% agar. The next day, sprouts were inoculated with the appropriate inocu-lum level of the GFP-labeled strain of interest by dropping the cell suspensionsdirectly to the seedling root area.

(ii) M. truncatula Gaertn. cv. A17 and dmi1. Both wild-type M. truncatula andthe dmi1 mutant, which is deficient in root nodulation by Sinorhizobium melilotiand/or mycorrhizal infection by Glomus, were kindly provided by D. Cook (Uni-versity of California, Davis). Scarified seeds were surface sterilized by immersionin 70% ethanol for 2 min followed by three washes with sterile water, 5 min ofimmersion in 10% bleach, and another three washes with sterile water. Seedgermination and bacterial inoculation were done as described above for alfalfa.

Plant culture conditions and harvesting. All plants were placed in a growthchamber under a day and night cycle of 15 and 9 h, respectively, at 22°C. Alfalfaand M. truncatula plants were harvested 5 and 7 days after inoculation, respec-tively. Plants were removed from the growth medium, and the leaves werediscarded. The fresh weights of the roots and hypocotyls were measured shortlyafter the removal of the leaves. There were four replicates for each treatment,with each replicate containing root and hypocotyl systems from four plants.

Determination of microbial populations in rhizosphere and inside surface-sterilized plant roots. Plant tissues were suspended in a PBS solution containing20% glycerol and subjected to a vortex at full speed for 1 min followed bysonication in a water bath for 1 min. The PBS solution was decanted, seriallydiluted in 10-fold increments, and cultured on solid LB medium with ampicillin(50 �g/ml) and tetracycline (10 �g/ml) to determine the microbial populations inthe rhizosphere as described previously (21).

Roots and hypocotyls were gently dried, weighed, and immersed in 25 ml of asurface sterilization solution (1� PBS, 1% bleach, 0.1% sodium dodecyl sulfate,0.2% Tween 20). Plant tissues were subjected to a vortex for 1 min followed byfour washes with sterilized water. The samples were then crushed manually witha mortar and pestle for about 40 s. The homogenates were resuspended in 1 mlof PBS containing 20% glycerol. This suspension was serially diluted in 10-foldincrements and cultured on LB plates supplemented with ampicillin (50 �g/ml)and tetracycline (10 �g/ml) in order to determine the microbial populationsinside surfaced-sterilized roots and hypocotyls. To determine the efficacy of thesurface sterilization, sterilized roots and hypocotyls were placed on LB platescontaining ampicillin and tetracycline and incubated for 15 min prior to crushing.The roots were then removed, and the plate was further incubated at 37°C for24 h as described by Gandhi et al. (11). In addition, the wash from the last rootrinse was cultured to determine the efficiency of sterilization. In the very fewcases in which the last wash yielded colonies with GFP, the number of those

TABLE 1. Bacterial strains used in this study

Strain Comment Reference or source

K. pneumoniae 342 Endophyte isolated from maize 7S. enterica serovar Cubana strain H7976 Isolated from patient infected with S.

enterica during alfalfa sprout-associatedoutbreak

Larry Beuchat, Center for Food Safety,University of Georgia

S. enterica serovar Infantis strain H3517 Isolated from patient infected with S.enterica during alfalfa sprout-associatedoutbreak

Larry Beuchat, Center for Food Safety,University of Georgia

S. enterica strain 8137 Isolated from patient with salmonellosisfrom alfalfa sprout contamination inWisconsin

Wisconsin State Hygiene Laboratory

S. enterica serovar Typhimurium strainATCC 14028

Clinical isolate used in many labs to examineSalmonella pathogenesis

American Type Culture Collection

E. coli K-12 strain MG1665 E. coli type strain 3E. coli O157:H7 strain F4546 Isolated from patient infected with E. coli

O157:H7 during outbreak associated withalfalfa sprouts

Robert Mandrell, Western RegionalResearch Center, USDAAgricultural Research Service,Albany, Calif.

1784 DONG ET AL. APPL. ENVIRON. MICROBIOL.

colonies was subtracted from the inside population, as described by Gyaneshwaret al. (12).

Slide preparation for microscopy and SCLM. At least two seedlings from eachof three independent inoculations were collected at 5, 7, and 9 days after inoc-ulation. Samples of plant roots and hypocotyls were first surface sterilized andstored at 4°C for 2 h for the formation of the GFP chromophore (8). Root andhypocotyl pieces and hand-cut transverse sections of the hypocotyls and thematuration zones of the primary roots were mounted with an antifade kit (Pro-long; Molecular Probes) and 0.25% agarose under a coverslip for microscopicobservation. The coverslip was sealed with nail polish. Scanning confocal lasermicroscopy (SCLM) was done on a Bio-Rad MRC-1000 SCLM system attachedto a Nikon Diaphot 200 inverted microscope. GFP-labeled cells were excitedwith the 488-nm laser line, and the autofluorescence from the plant tissues wasexcited with the 568-nm laser line. Images from both channels were collectedsequentially in a z-series from 30 to 60 optical sections ranging from 0.5 to 2 �min thickness. All microscopy experiments were repeated at least four times, andmultiple samples were examined each time.

Data analysis. Microsoft Excel 2000 was used to plot the endophytic andrhizosphere colonization data. Each plotted data point represents a mean and issurrounded by a representation of the standard error of the mean of the resultsfrom four replicates. Minitab 12 for Windows was used to perform Student’s ttests to determine the significance of the differences between the means. Cor-relations between endophytic colonization and rhizosphere colonization weredetermined by regression analysis with Minitab 12. Where significant differencesare described, the 95% confidence interval was used. Confocal photomicro-graphs were analyzed with the aid of Confocal Assistant 4.02 and Adobe Pho-toshop 5.0.

RESULTS

Assurance of endophytic colonization. To ensure that theendophytic colonization numbers presented reflect only thenumber of cells within the interior of the plant tissues, a spe-cific method of surface sterilization was developed. This ster-ilization method is sufficient to kill and/or wash away the sur-face bacteria while maintaining the survival of the interiorbacteria. Three control procedures were performed to ensurethat proper surface sterilization had been used in our experi-ments. First, root samples were examined by confocal micros-copy for GFP-containing cells remaining on the plant surfaceafter surface sterilization. No cells were ever observed by thisapproach. Second, no bacterial growth was observed on LBsolid medium 2 days after the mounting of surface-sterilizedroots. And third, bacterial growth was rarely observed whenthe last set of washes used to rinse the tissues was placed on LBmedium. Colonies were observed in less than 1% of thesewashes. In those very few cases in which colonies were ob-served, the number of those colonies was subtracted from thefinal interior count of that particular sample. Based on theresults of these three controls, the endophytic colonizationnumbers presented reflect only the number of cells within theinterior of the tissues. Also, there were replicate inoculationsfor each treatment in each experiment. The standard error ofthe mean represented by each data point is presented in eachfigure.

Gandhi et al. (11) inserted the gfp gene into the chromosomeof the Salmonella strain of interest to ensure the stability of gfpexpression. Such stability is important to ensure accuratecounting of cells on and within plant tissues. In this work, gfpwas expressed on a plasmid that was stably maintained in thebacterial host for at least 24 generations. Plasmid-borne gfpwas used in this work so that several strains could be labeledeasily. As a result of this plasmid stability and the carefulsurface sterilization of plant tissues when needed, these results

accurately reflect the numbers of enteric bacteria in the rhizo-spheres and the plant interiors.

Endophytic colonization of alfalfa by enteric bacteria. At thefour lowest inoculum levels, Klebsiella pneumoniae 342 colo-nized the interior of alfalfa in higher numbers than any otherstrain tested (Fig. 1A and Fig. 2A). At the four highest inoc-ulum levels, only S. enterica serovar Cubana strain H7976 wasable to match the level of endophytic colonization by K. pneu-moniae 342 (Fig. 1A and Fig. 2A). Also, at the four highestinoculum levels, serovar Cubana strain H7976 colonized the

FIG. 1. Numbers of GFP-labeled bacterial CFU recovered frominteriors (A) and rhizospheres (B) of alfalfa seedlings 5 days afterinoculation of 1.5-day-old seedlings with different inoculum levels. Thedata points represent the means and the bars represent the standarderrors of the means of results from four replicate treatments. Fourseedling samples (roots and hypocotyls) were taken from each repli-cate. SCH7976, S. enterica serovar Cubana strain H7976; Kp342, maizeendophyte K. pneumoniae 342; SIH3517, S. enterica serovar Infantisstrain H3517; S8137, S. enterica strain 8137; 14028, S. enterica serovarTyphimurium strain ATCC 14028; gfw, gram (fresh weight).

VOL. 69, 2003 BACTERIAL ENDOPHYTIC COLONIZATION OF MEDICAGO 1785

interior of alfalfa in much higher numbers than all other strainsexcept K. pneumoniae 342 (Fig. 1A and Fig. 2A). Among theother salmonellae, S. enterica serovar Typhimurium strainATCC 14028, a clinical isolate used in many labs to examineSalmonella pathogenesis, colonized the interior in higher num-bers than did S. enterica serovar Infantis strain H3517 and S.enterica strain 8137 over a range of inoculum levels from 102 to105 CFU per plant (Fig. 1A). At low inoculum levels, S. en-terica strain 8137 was the poorest colonizer, but at high inoc-ulum doses, this strain colonized the plant in numbers similarto those for all other Salmonella strains tested except serovar

Cubana strain H7976 (Fig. 1A). At low inoculum levels, thelevels of colonization by the two E. coli strains were betweenthose of K. pneumoniae 342 and serovar Cubana strain H7976(Fig. 2A). At the highest inoculum levels, the E. coli O157:H7strain derived from an outbreak of diseases associated withalfalfa sprouts colonized the interior in higher numbers thandid E. coli K-12 (Fig. 2A).

Rhizosphere colonization by enteric bacteria. K. pneu-moniae 342 colonized the alfalfa rhizosphere at significantlyhigher levels than the other enteric bacteria tested at the fourlowest levels of inoculum (Fig. 1B and Fig. 2B). At higherinoculum levels, K. pneumoniae 342 and serovar Cubana strainH7976 were very similar in their levels of colonization of thealfalfa rhizosphere (Fig. 1B and Fig. 2B). Over most inoculumlevels, the Salmonella strains differed little in the levels ofrhizosphere colonization (Fig. 1B). At levels of inoculum be-tween 102 and 105 CFU/plant, serovar Infantis strain H3517was a relatively poor rhizosphere colonizer compared to mostsalmonellae tested (Fig. 1B). At the two lowest inoculum lev-els, E. coli K-12 was a better rhizosphere colonizer than E. coliO157:H7 (Fig. 2B). At the two highest inoculum levels, E. coliO157:H7 was a better rhizosphere colonizer than E. coli K-12(Fig. 2B). No difference was observed between these twostrains at the middle levels of inoculum (Fig. 2B). Across allinoculum levels, K. pneumoniae 342 was a better rhizospherecolonizer than either strain of E. coli (Fig. 2B).

Correlation between rhizosphere colonization and interiorcolonization of alfalfa. There was a strong correlation betweenthe abilities of all strains tested to colonize the rhizosphere andto colonize the interior of alfalfa. The correlation coefficients(r2) of alfalfa interior colonization and rhizosphere coloniza-tion by K. pneumoniae 342, serovar Cubana strain H7976, se-rovar Typhimurium strain ATCC 14028, serovar Infantis strainH3517, S. enterica strain 8137, E. coli O157:H7, and E. coliK-12 were 0.729, 0.891, 0.951, 0.949, 0.825, 0.803, and 0.017,respectively. Although this suggests a strong relationship be-tween the two habitats, the strain most capable of entering thealfalfa plant, K. pneumoniae 342, has the second lowest corre-lation between rhizosphere colonization and endophytic colo-nization of the strains tested. In stark contrast to the otherstrains tested, E. coli K-12 showed no correlation betweenrhizosphere colonization and endophytic colonization. E. coliK-12 was a poor colonizer of the interior across all inoculumlevels but a better-than-average colonizer of the rhizosphere atlow inoculum doses.

Colonization of a symbiosis mutant of M. truncatula by se-rovar Cubana strain H7976. Significantly higher numbers ofserovar Cubana strain H7976 bacteria were able to colonizethe interiors of the roots and hypocotyls of M. truncatula dmi1than were able to colonize those of wild-type M. truncatula(Fig. 3J, K, and L; see also Fig. 5A). Furthermore, an evengreater difference between the two host genotypes was foundby examining rhizosphere colonization by serovar Cubanastrain H7976. The numbers of serovar Cubana strain H7976cells in the rhizospheres were approximately 100-fold higheron the roots of dmi1 than on those of the wild-type plant at allbut the highest inoculum level (Fig. 4J, K, and L and Fig. 5B).The correlation between rhizosphere colonization and endo-phytic colonization was high for both genotypes, dmi1 (r2 �0.655) and the wild type (r2 � 0.741). However, the correlation

FIG. 2. Numbers of bacterial CFU recovered from interiors(A) and rhizospheres (B) of alfalfa seedlings 5 days after inoculation of1.5-day-old seedlings with different inoculum levels. The data pointsrepresent the means and the bars represent the standard errors of themeans 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. coliO157: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 strain8137 (E), S. enterica serovar Infantis strain H3517 (F), S. enterica serovar Typhimurium strain ATCC 14028 (G), and S. enterica serovar Cubanastrain 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. Arrowspoint to GFP-tagged bacterial cells. Bars, 50 �m.


FIG. 4. Longitudinal section of alfalfa roots (A to I) and M. truncatula roots (J to L) showing colonization by the following GFP-labeledbacteria: 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 strainH7976. (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 �m.


between these modes of colonization was much higher whenserovar Cubana H7976 was inoculated on alfalfa (r2 � 0.891).

Lateral root crack colonization. Cells of K. pneumoniae 342,S. enterica strain 8137, serovar Infantis strain H3517, serovarTyphimurium strain ATCC 14028, and serovar Cubana strainH7976 congregate at the lateral root emergence sites of alfalfa(Fig. 4). SCLM shows that these cells also colonize the interi-ors of alfalfa roots and hypocotyls. While S. enterica and E. colistrains showed the same patterns of colonization of lateral rootemergence sites as K. pneumoniae 342, the numbers of S.enterica and E. coli cells at these sites appeared to be lower(Fig. 4). Serovar Cubana strain H7976 and K. pneumoniae 342cells were observed in high numbers in hypocotyls by SCLM.

DISCUSSION

Several new aspects of the interaction between alfalfa andenteric bacteria are presented here. First, the number of cellsthat must be present in the inoculum for plant entry has beendetermined. With two of the four Salmonella strains used inthis work, a single CFU in the inoculum was sufficient to causeinvasion of the plant interior as determined 5 days after inoc-

ulation. This is a troubling result from a food protection per-spective. This suggests that no contamination can be toleratedif the infection of alfalfa sprouts by certain salmonellae underthe conditions used here is to be prevented. However, it is notknown whether low contamination levels can cause significantinvasion of the alfalfa apoplast under conditions in whichsprouts are grown commercially. The presence of nonpatho-genic bacteria on alfalfa seeds and in the nutrient solution andmedium used to culture these plants commercially may excludeplant invasion by low numbers of salmonellae and/or may pre-vent the multiplication of these pathogens in planta.

Second, the rigorous surface sterilization procedure usedhere allowed the quantification of salmonellae within the plant.The numbers of salmonellae in planta increased with inoculumdose and reached levels as high as 4.5 log10 per gram of tissue.These levels were nearly as high as those achieved by theKlebsiella endophyte. Thus, surface sterilization of tissue aloneis not sufficient to eliminate the pathogen. The numbers ofcells found in these plants are likely to be more than sufficientto cause disease if just one gram of tissue was available forhuman consumption, according to models by Latimer et al.(17). Gandhi et al. (11) and Charkowski et al. (6) determinedthe number of enteric pathogens on alfalfa seedlings. However,no surface sterilization was done in either study, so the num-bers of surface and endophytic bacteria cannot be distin-guished in these experiments. In this work, very thorough sur-face sterilization allows us to distinguish between rhizospherecolonization and endophytic colonization.

Third, four Salmonella strains were examined in this workwhile only one strain was studied by Gandhi et al. (11). Thefour strains differed greatly in their abilities to enter alfalfaroots and hypocotyls. Since strains can differ in their abilities toenter the same host genotypes cultured under the same envi-ronmental conditions, there must be genetic determinants inthe bacteria than can affect endophytic colonization. Thesestrain differences in endophytic colonization may occurthrough the presence of genes involved in this process orthrough a change in the expression of genes present in allstrains. In either case, these data suggest that endophytic col-onization by salmonellae is an active process and is not simplythe passive diffusion of bacteria into the intercellular spaces ofplants.

Fourth, the abilities of each of these strains to colonize therhizosphere were assessed. This allows a comparison of rhizo-sphere colonization and interior colonization for each of thestrains tested. A strong correlation was found between rhizo-sphere colonization and interior colonization for all strainsexcept E. coli K-12, for which no correlation existed. The othersix strains did exhibit a strong correlation between these twomodes of colonization. However, it remains unclear whetherthe ability to colonize the rhizosphere enhances endophyticcolonization. For example, the strain with the best ability tocolonize the plant’s interior, K. pneumoniae 342, showed thelowest correlation between rhizosphere colonization and en-dophytic colonization among the six strains with a strong pos-itive correlation. In addition, the strain with the highest corre-lation, serovar Typhimurium strain ATCC 14028, was anaverage colonizer of both sites and is the only strain among thesix strains that was not derived directly from plants or isolatedfrom patients believed to have been infected through plant

FIG. 5. Numbers of CFU recovered from interiors (A) and rhizo-spheres (B) of M. truncatula plant tissues 7 days after inoculation withdifferent inoculum levels. SCH7976-WT, M. truncatula wild type inoc-ulated with S. enterica serovar Cubana strain H7976; SCH7976-“Dmi1,” M. truncatula dmi1 mutant inoculated with S. enterica serovarCubana strain H7976; gfw, gram (fresh weight).


consumption. Nevertheless, it seems reasonable that a highernumber of cells on the roots gives the strain a higher likelihoodof multiple infection events, which would result in a higherinterior colonization.

Fifth, a search was done with SCLM for bacterial coloniza-tion of lateral root cracks. Nitrogen-fixing endophytes colonizesuch cracks in high numbers (13, 14, 20). Micrographs pre-sented here are representative of the extensive colonization ofthese regions on alfalfa roots by enteric bacteria. This findingsuggests that these bacteria may enter the plant through thesecracks. Alternatively, these root cracks may be a higher sourceof plant nutrients for the enteric bacteria than the rhizosphere.In addition, root hair deformation was not observed followinginoculation with any of these bacteria. Thus, infection throughroot hairs, as in the infection of legume root hairs by rhizobia(10), is unlikely.

This work also expands on previous work in that a cleareffect of the host genotype on endophytic colonization wasobserved. The dmi1 mutant of M. truncatula is defective informing a symbiosis with either Sinorhizobium meliloti or my-corrhizae (5). This mutant is defective in a common and veryearly infection event in both symbioses. Prior to inoculationwith serovar Cubana strain H7976, the expectation was that thedmi1 mutation would have no effect on endophytic coloniza-tion. A result that was thought to be less likely was that endo-phytic colonization would be impaired in the dmi1 mutantcompared to that in the wild type. In contrast to both of thesepossibilities, both rhizosphere colonization and endophytic col-onization were enhanced in the dmi1 mutant compared tothose in the wild type, particularly at low inoculum doses. Theeffect on rhizosphere colonization was more pronounced. Asthe biochemical nature of the dmi1 mutation is unknown, thebiological basis for these observations is not known. It appearsthat a blockage of infection by symbionts somehow reduces thebarriers to endophytic infection at low inoculum doses. Thehigher level of endophytic colonization may be the effect ofincreased rhizosphere colonization. This alone may give morebacteria an opportunity to enter the plant root.

In summary, several important features of endophytic colo-nization by enteric bacteria have been characterized in thiswork. Very few cells must be present in the inoculum forcolonization of the interior of the plant. Rhizosphere coloni-zation and endophytic colonization are usually highly corre-lated. Strains of enteric bacteria can differ greatly from eachother in their abilities to invade the plant interior and tocolonize roots. As both bacterial and host genotypes werefound to influence endophytic colonization, this infection islikely mediated by genetic determinants in both partners.

ACKNOWLEDGMENTS

We acknowledge support for this work from the College of Agricul-tural and Life Sciences of the University of Wisconsin—Madison andthe Consortium for Plant Biotechnology Research.

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kinetics and strain speciﬁcity of rhizosphere and

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