JOURNAL OF BACTERIOLOGY, Mar. 1971, p. 728-732Copyright i 1971 American Society for Microbiology

Vol. 105, No. 3Printed in U.S.A.

Formation of Tumor-Like Structures on LegumeRoots by Rhizobium

A. N. MACGREGOR' AND M. ALEXANDER

Laboratory ofSoil Microbiology, Department ofAgronomy, Cornell University, Ithaca, New York 14850

Received for publication 28 September 1970

Tumor-like structures appeared on the roots of Medicago sativa, Alysicarpusvaginalis, and Trifolium pratense inoculated with a non-nodulating strain of Rhizo-bium trifolii or with irradiated cultures of either of two nodulating Rhizobiumstrains. The structures were composed of disorganized plant tissues which, on thebasis of microscopic examination, were devoid of bacterial cells. Rhizobia whichcould nodulate legumes of one cross-inoculation group and which were able to in-duce formation of such tumor-like structures on plants of a second cross-inocula-tion group were isolated from extracts of these root growths. The apparent tumoro-genic activity of some of the rhizobia, but not their nodulating capacity, was lostwhen the bacteria were transferred in laboratory media.

The infection of leguminous plants by membersof the genus Rhizobium does not always lead toan association capable of nitrogen fixation. Intheir study of root nodules of certain Trifoliumspecies, Bergersen and Nutman (3) found ineffec-tive nodules may be made up of disorganized ortumorized plant tissue, by contrast with the dis-tinct organization of the effective nodules. Theformation of ineffective nodules on legumes ofthis genus was ascribed to the presence of a reces-sive plant gene. Atypical structures, termed"pseudonodules," were also noted on the roots oflegumes grown in the field (2; 0. N. Allen and E.K. Allen, Hawaiian Acad. Sci. Proc., BishopMuseum Spec. Pub. no. 35, p. 15-16, 1940).Analogous atypical structures made up of swollenroot tips and showing radial enlargement of root-lets can be induced by treating legumes with 2-bromo-3,5-dichlorobenzoic acid (1). In addition,rhizobia may be modified genetically so that theyare able to induce the formation of tumors; thus,Rhizobium leguminosarum has been reported tobe transformed into -a tumor-inducing organismby means of deoxyribonucleic acid (DNA) ob-tained from Agrobacterium tumefaciens (6).

This paper presents results showing the effectof inoculating leguminous plants of four cross-inoculation groups with a culture of non-nodulat-ing R. trifolii and with irradiated cultures of twonodulating Rhizobium strains.

I Present address: Department of Agricultural Chemistry andSoils, University of Arizona, Tucson, Ariz. 85721.

MATERIALS AND METHODSR. meliloti 5, R. trifolii 205, R. trifolii 0435, and

Rhizobium strain 899 which nodulates Lotus cornicu-latus were inoculated onto appropriate plants and thenreisolated from the root nodules in clonal form. Strain0435 and bacteriophage 5000 were provided by P. S.Nutman, strain 899 by 0. N. Allen, and a virulentstrain of A. tumefaciens by R. S. Dickey. The bacteriawere grown in yeast extract-mannitol medium (YEM),which contained 10 g of mannitol, 1.0 g of yeast ex-tract, 0.80 g of K,HPO4, 0.20 g of KH2PO4, 0.20 g ofMgSO4.7H20, 0.10 g of CaCl2.2H20, and 0.10 g ofNaCl in 1.0 liter of deionized, distilled water. The yeastextract-glucose medium (YEG) contained glucose inplace of mannitol. Bacteriophage 5000 was propagatedon R. trifolii 0435, and it failed to lyse the other rhi-zobia.

Seeds of Trifolium pratense and T. repens were ver-nalized by holding at 4 C for 24 hr. To sterilize the sur-face of the seeds, they were shaken in 0.2% Formalin in95% ethanol for 30 min and then in 0.2% HgCl2 inwater for 30 min. The seeds were then washed six timesin sterile distilled water and allowed to imbibe water bystanding in the last wash for 3 to 4 hr. The effectivenessof the sterilization was determined by incubatingtreated seeds and germinated seedlings on solid andliquid YEM and YEG media for 4 days at room tem-perature and examining for the appearance of microbialgrowth. Seedlings were cultivated on seedling mediumprepared by the method of Fahraeus (4).To test the nodulating capacities of various rhizobia,

surface-sterilized seeds were germinated on the solidi-fied seedling medium. Young seedlings were gentlytransferred to fresh medium when their roots wereabout I mm long, and they were inoculated with a fewdrops of a washed Rhizobium suspension containing 107

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cells/ml. In some nodulation tests, the young seedlingswere transferred to a sheet of cellulose dialyzing mem-brane covering the surface of the seedling agar, and theroots were allowed to develop to 3 to 4 cm in lengthbefore they were transferred to plates containing seed-ling medium previously inoculated with 0.2 ml of awashed suspension of the test rhizobium. Inoculatedseedlings were incubated at 26 C under cool, white flu-orescent lights with a light intensity of 275 ft-c. Theplants were illuminated for 14 hr each day for a total of3 weeks. The petri dishes were covered with a sheet ofclear polyethylene to minimize evaporation.

The bacteria were treated with ultraviolet (UV) lightby placing open petri dishes containing the cells 33 cmfrom a 12-w germicidal tube (wave length, 253.7 nm).Under these conditions, the times of treatment to yield1% surviving cells were 2 min for R. meliloti 5 and R.trifolii 205 and 3 min for Rhizobium 899, starting withan initial density of about 108 cells/ml.To obtain antibiotic-resistant bacteria, UV-treated

cultures of R. trifolii 205 and Rhizobium 899 were firstincubated in YEM medium for 18 hr at room tempera-ture in the dark, and portions of the cultures were thenplated onto YEM agar containing 100 ug of strepto-mycin or kanamycin per ml. Ethyl methane sulfonatewas used to obtain mutants of R. meliloti 5 that wereresistant to 500 ,ug of penicillin per ml; for this purpose,a drop of undiluted ethyl methane sulfonate was ap-plied to the edge of a plate of YEM agar inoculatedwith the bacteria and containing the antibiotic. Plateswere incubated for 5 days at room temperature, andantibiotic-resistant colonies were replated onto a me-dium containing the identical antibiotic at the sameconcentration. The antibiotic-resistant mutants retainedtheir nodulating capacities, and they failed to grow onYEM containing either of the antibiotics tolerated bythe mutants derived from the other two Rhizobiumstrains. Thus, the mutants could be readily differ-entiated in vitro.To isolate bacteria from the nodules, roots were

removed from the plant and agitated gently in steriledistilled water to dislodge adhering debris. A portion ofroot bearing a well-defined nodule was placed in a tubecontaining 10 ml of 0.2% aqueous HgCl2, and the tubewas agitated vigorously. The root segment was thenwashed six times with sterile distilled water. Excess roottissue was trimmed away from the nodule, which wasthen transferred to a sterile tube containing 0.2 ml ofsterile distilled water and a glass rod. The nodule wascrushed under the rod until the tissue appeared to bewell macerated, and a loopful of the nodule extract wasplated on YEM. The same procedure was used in at-tempts to isolate bacteria from nodule-like structures.The effectiveness of the surface sterilization of noduleswas determined by treating 2.0-cm long nodule-freeroot segments in a similar manner.To obtain non-nodulating rhizobia, a culture of nodu-

lating R. trifolli 0435, which is resistant to 100 Mg ofstreptomycin per ml and sensitive to phage 5000, wastreated with UV for 2 min, plated on YEM, and incu-bated for 5 days at room temperature. Colonies whichwere visibly less gummy than colonies of the parent cul-ture were transferred five consecutive times on YEMagar and finally into YEM broth, using single colony

isolates fbr each transter. The cells from the 3-daybroth cultures were washed and inoculated onto 50seedlings each of T. pratense, T. subterraneum, T. fra-giferum, and T. hirtum growing on seedling agar. Theseedlings were examined for nodules after 3 weeks. Astrain of Rhizobium incapable of nodulating Trifoliumspecies was obtained in this way. This isolate grew onYEM agar containing 100 Mg of streptomycin per ml,was lysed by bacteriophage 5000, and was antigenicallysimilar, on the basis of the immunodiffusion technique,to the original nodulating strain of R. trifolii 0435.

RESULTSFive clones of streptomycin-resistant R. trifolii

205, penicillin-resistant R. meliloti 5, and kana-mycin-resistant Rhizobium 899 were irradiatedwith UV and inoculated onto 10 seedlings each ofMedicago sativa, Melilotus alba, T. repens, T.pratense, Alysicarpus vaginalis, and L. cornicula-tus, plants representing four cross-inoculationgroups. No nodules developed on these plantsexcept on those legumes inoculated with thehomologous rhizobia. However, tumor-like pro-tuberances were found on seedlings of A. vagin-alis inoculated with a UV-treated culture of ei-ther streptomycin-resistant R. trifolli 205 or kan-amycin-resistant Rhizobium 899.The aberrant root growths were generally

about 1.0 mm long and 0.5 mm in diameter, orsometimes larger, and they were superficially dis-tinguishable from nodules by their irregular, dis-torted shape and loose, easily fragmented struc-ture. Some of the protuberances exhibited an ir-regular, convoluted surface, whereas a fewshowed small outgrowths on the surface. Most ofthe swollen structures were located at the junc-tion of lateral and main roots, but a few wereobserved to occur on main roots at some distancefrom the lateral root junctions.These odd morphological entities were sepa-

rated from the remainder of the root tissue,mounted in a drop of 0.1% aqueous methyleneblue on a microscope slide, and pressed under acover glass. Microscopic examination revealedthat the structures appeared to be composed ofplant tissue originating from the epidermal layersof the root and not, as in nodules, the root cortex.By contrast with nodules, they also had no dis-tinct meristematic center, and cytologically theyappeared to be free of bacteria. Furthermore, noviable organisms could be isolated from theseroot structures if they were surface sterilized with0.2% HgCl2, a procedure commonly used to iso-late rhizobia from nodules. Hence, the root pro-tuberances are not nodules but rather shouldprobably be regarded as tumors.Tumors on A. vaginalis seedlings that were

formed in response to infection with both variant

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bacterial strains were excised from the roots andcrushed in 0.5 ml of sterile distilled water. Thesetumors were not surface sterilized. The crushedmaterial was plated on YEM agar, and the plateswere incubated for 5 days at room temperature.Numerous colonies grew on the medium. Theidentities of 15 randomly selected colonies were

verified by assessing their ability to grow on

YEM containing 100 jig of either kanamycin or

streptomycin per ml and also by performing a

slide agglutination test on each colony. All 15clones derived from the tumor of A. vaginalisinoculated with the UV-treated culture of R. tri-folii 205, a strain not sensitive to streptomycin,were found to be resistant to 100 ,ug of strepto-mycin per ml and gave a positive agglutina-tion test only with R. trifolii-specific antiserum.The 15 clones derived from the tumor of A. vag-

inalis inoculated with a UV-treated culture ofRhizobium 899, a kanamycin-resistant organism,were resistant to 100 ,g of kanamycin per ml andgave a positive agglutination test only with Rhizo-bium 899-specific antiserum. It thus appears thatthe rhizobia isolated from tumors were identicalto the bacteria used in the original inoculum ap-

plied to the plant.To determine whether all 15 clones of rhizobia

isolated from the non-surface-sterilized tumorscould induce the formation of analogous growthson A. vaginalis and still nodulate the homologouslegume, each isolate was inoculated directly ontoappropriate seedlings. The same 15 clones origi-nating from two different rhizobia were alsotransferred to YEM, and washed cells from theresulting cultures were inoculated onto a secondset of seedlings. In this way, rhizobia which origi-nated from streptomycin-resistant R. trifolii 205were inoculated onto 20 seedlings of T. pratenseand A. vaginalis, and suspensions containing rhi-zobia derived from kanamycin-resistant Rhizo-bium 899 were applied to 20 seedlings of L. cor-niculatus and A. vaginalis. The seedlings were

examined after 3 weeks of incubation. Some ofthe rhizobia isolated from tumors on A. vaginalisindeed had tumorogenic activity on seedlings ofA. vaginalis (Table 1). After one subculture inYEM, the tumorogenic but not the nodulatingcapacity was lost by the R. trifolii clones thatinitially had both of these capacities. By contrast,tumorogenic clones of Rhizobium 899 did notlose this facility after subculture, and curiouslyenough, a greater proportion of the 15 originalRhizobium 899 clones were found to be tumoro-genic after subculture than before. Apparently,strains of Rhizobium 899 which were formerlyunable to elicit any plant response developedtumorogenic and nodulating capacities after sub-culture in artificial media. Except for one clone

of Rhizobium 899, all of the rhizobia inducingtumors on A. vaginalis were also able to formnodules on their homologous hosts.

Because A. tumefaciens is able to induce tu-mors on a variety of non-legumes, it was of in-terest to know whether this organism could alsobring about the formation of tumors on the rootsof legumes. A washed cell suspension containingabout 107 cells/ml was prepared from a virulentstrain of A. tumefaciens grown 3 days at roomtemperature in YEM; a 0.25-ml amount of sus-pension was inoculated onto 10 seedlings each ofT. pratense, T. repens, M. alba, M. sativa, L.corniculatus, and A. vaginalis which had beengerminated aseptically and placed on seedlingagar. After a 3-week growth period, the roots ofseedlings of A. vaginalis were stunted and boretumors similar to those induced by the rhizobia,and the epidermal layers of the roots showed lon-gitudinal splitting. The roots of T. pratense werealso stunted, the plants exhibited greater lateralroot development than uninoculated controls, andthe root surface was split. Roots of T. repens, M.alba, M. sativa, and L. corniculatus exposed toA. tumefaciens and uninoculated plants werenormal.To determine whether the non-nodulating var-

iant derived from R. trifolii 0435 could provokethe formation of tumors on plants of cross-inoc-ulation groups other than the clover group, awashed cell suspension containing 107 cells/mlwas prepared from a culture grown in YEM for 3days, and 0.25 ml was inoculated onto 20 asepti-cally grown seedlings of M. sativa, T. pratense,TABLE 1. Nodulation and tumorogenesis by isolates

derivedfrom tumors on Alysicarpus vaginalisNo. of isolates causing a

plant response

Isolates from Isolates fromRhizobium 899- R. trifolii 205-

Influence on induced tumorsa induced tumorsaplants

No After No Aftersub- sub- sub- sub-

culture culture culture cultureon on on on

YEM YEM YEM YEM

Nodules on homologoushost and tumors on A.vaginalis° ........... 4 1 1 2 0

Only nodules on homolo-gous host ........... 4 4 13 15

Only tumors on A. vagin-alis ................ I 0 0 0

No effect 6 0 0 0

aFifteen isolates of each group tested.I Homologous hosts are Lotus corniculatus for Rhi-

zobium 899 and Trifolium pratense for R. trifolii 205.c Yeast extract-mannitol medium.

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FIG. 1. Aberrant root structures on rootsold legumes inoculated with a non-nodulatingRhizobium trifolii. (A) A lysicarpus vaginx30; (B) Medicago sativa roots, x20; (C) ction of the main and lateral roots of TrifoliurxlO.

L. corniculatus, and A. vaginalis, representingthe alfalfa, clover, lotus, and cowpea cross-inoc-ulation groups, respectively. Except for L. corni-culatus, all plants bore tumors on their rootswhen examined after 3 weeks. More than 100tumors were found on the 20 seedlings of A. vag-inalis, 5 were found on the seedlings of M. sativa,and 2 on the T. pratense seedlings (Fig. 1). OneA. vaginalis plant bore 20 of these structures.Uninoculated seedlings and L. corniculatus devel-oped normally and showed no abnormal rootgrowths.

DISCUSSIONThe present study shows that rhizobia may

form tumor-like entities on leguminous plants.Most but not all of these strains still possessedthe capacity to form nodules on their homologoushosts. This tumorogenic capacity could be lost bysubculturing some of the rhizobia on laboratorymedia. Bacteria losing their tumorogenic activityretained their nodulating ability, however. Thetumorogenic strain of R. trifolii was able to causethe formation of these aberrant structures notonly on T. pratense, the plant that would be itssymbiont if the strain had not lost its nodulatingcapacity, but also on M. sativa and A. vaginalis;hence, the capacity to induce tumors is not neces-sarily associated with the organism's capacity tonodulate.The number of tumors formed on the legume

seedlings depended on the plant species and pos-sibly on the cross-inoculation groups to which theplants are assigned. Few tumors were found onTrifolium and Medicago species, members of theclover and alfalfa cross-inoculation groups, re-spectively. With some exceptions, these two plantgroups are composed of species of a single genus,and nodulating rhizobia from one group areusually unable to symbiose with legumes of theother (5). No tumors were found on L. cornicula-tus, a species nodulated only by highly specificrhizobia. In contrast, large numbers of tumorswere regularly found on A. vaginalis, a memberof the cowpea cross-inoculation group. The rhi-zobia which symbiose with plants of the cowpeagroup have a wide host range and are able to in-fect hosts of several genera.

ACKNOWLEDGMENTThis research was supported by National Science Founda-

tion grant GB-3687.

LITERATURE CITEDof 3-week-g variant of 1. Allen, E. K., 0. N. Allen, and A. S. Newman. 1953. Pseu-alis roots, donodulation of leguminous plants induced by 2-bromo-

Isroothejun3,5-dichlorobenzoic acid. Amer. J. Bot. 40:429-435.

Mt the junc- 2. Allen, 0. N., and E. K. Allen. 1953. Morphogenesis of theml pratense, leguminous root nodule. Brookhaven Symp. Biol. 6:209-

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3. Bergersen, F. J., and P. S. Nutman. 1957. Symbiotic effec-tiveness in nodulated red clover. IV. The influence of thehost factors i1 and ie upon nodule structure and cytology.Heredity 11:175-184.

4. Fahraeus, G. 1957. The infection of clover root hairs bynodule bacteria studied by a simple glass slide technique.J. Gen. Microbiol. 16:374-381.

5. Fred, E. B., 1. L. Baldwin, and E. McCoy. 1932. Root

nodule bacteria and leguminous plants. University of Wis-consin Press, Madison, Wis.

6. Kern, A. 1965. Untersuchungen zur genetischen Transfor-mation zwischen Agrobacterium tumefaciens und Rhizo-bium spec. II. Vergleichende morphologische, physiolo-gische und biochemische Untersuchungen an der Partnerneiner Transformation. Arch. Mikrobiol. 52:206-223.

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