Vol. 44, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1982, p. 965-9710099-2240/82/100965-07$02.00/0Copyright © 1982, American Society for Microbiology

Nodulation of Pole Bean (Phaseolus vulgaris L.) byRhizobium Species of Two Cross-Inoculation Groups

ARYA K. BAL,' S. SHANTHARAM,2 AND PETER P. WONG2*Department ofBiology, Memorial University of Newfoundland, St. John's, Newfoundland, Canada AIB 3X91

and Division of Biology, Kansas State University, Manhattan, Kansas 665062

Received 27 April 1982/Accepted 14 June 1982

Physiology and morphology of pole bean (Phaseolus vulgaris L. cv KentuckyWonder) root nodules induced by two Rhizobium species of different cross-inoculation groups have been compared. Root nodules induced by Rhizobium sp.127E15, which is a strain of the cowpea group Rhizobium, were pinkish, hadirregular shapes, and were only partially effective. Their peak acetylene reductionactivity was 4.36 ,umol of C2H4 formed per g offresh nodules per h at 30 days afterinoculation. The effective nodules induced by Rhizobium phaseoli 127K14, whichis a strain of the bean group Rhizobium, were dark red, spherical, and showedpeak acetylene reduction activity of 15.95 ,umol of C2H4 formed per g of freshnodules per h at 15 days after inoculation. The partial effectiveness of 127E15-induced nodules was associated with fewer infected cells, a delay in the increaseof bacteroid population within the host cells, abundance of cytoplasmic vesicles inthe host cells, more bacteroids within a membrane envelope (peribacteroidmembrane), and the inability of bacteroids to completely fill up the hostcytoplasmic space. The 127K14-induced nodules were fully mature, with hostcells filled with bacteroids by 12 days after inoculation. In contrast, the 127E15-induced nodules did not reach a similar developmental stage even 30 days afterinoculation.

Pole bean (Phaseolus vulgaris L.) and limabean (Phaseolus lunatus L.) are of the samegenus, but they are nodulated by Rhizobiumspecies of two different cross-inoculation groups(1). Effective root nodules (able to fix N2 suffi-ciently to support plant growth) of pole bean areinduced by Rhizobium phaseoli 127K14, a strainof the bean group Rhizobium, and effective rootnodules of lima bean are induced by Rhizobiumsp. 127E15, a strain of the cowpea group Rhizo-bium. Rhizobium sp. 127E15, however, also canpromiscuously induce partially effective rootnodules (able to fix only little N2) on pole bean,whereas R. phaseoli 127K14 cannot induce anynodules on lima bean (24).We have shown earlier that Rhizobium sp.

127E15 elicits specific host responses from polebean during their initial contact (19). Inoculationof pole bean with Rhizobium sp. 127E15 causedroot hair tip curling and swelling and infectionthread formation. The initial interactions be-tween Rhizobium sp. 127E15 and pole bean weresimilar to that of R. phaseoli 127K14 and polebean. Rhizobium sp. 127E15 elicited similar spe-cific host responses from lima bean, but R.phaseoli 127K14 did not. In this report, the N2fixation (acetylene reduction) activities and mor-phologies of developing pole bean nodules in-

duced by R. phaseoli 127K14 and Rhizobium sp.127E15 are compared. Our results give furtherinsight into how a legume interacts with Rhizobi-um species of two different cross-inoculationgroups.

MATERIALS AND METHODS

Pole beans (Phaseolus vulgaris L. cv KentuckyWonder) were purchased from Burpee Seed Co.,Clinton, Iowa. Rhizobium phaseoli 127K14 and Rhizo-bium sp. 127E15 were gifts of Joe Burton, The Nitra-gin Co., Milwaukee, Wis.

Pole bean growth conditions. Bean seeds were sur-face sterilized by immersion in 75% (vol/vol) ethanolfor 10 min, 1% NaOCl (wt/vol) for another 10 min, andwashing thoroughly with sterile H20.Wide-mouth (6.5-cm diameter) 900-ml mason jars

were used for pole bean cultivation. Each jar was filledto the neck with a mixture of 50% vermiculite and 50%perlite. The jar then received 450 ml of a nutrientsolution free of combined nitrogen (14), and its mouthwas covered with a paper autoclave bag (10 cm indiameter and 23 cm in length), purchased from ArthurH. Thomas Co., Philadelphia, Pa. The jars wereautoclaved for 1.5 h at 121°C.The sterilized seed were planted in the jars by

embedding them with a pair of forceps about 2 cmbelow the surface of the vermiculite-perlite mixture(four seeds per jar). Three days later, the jars wereinoculated with approximately 109 cells of either R.

965

on Septem

ber 11, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

966 BAL, SHANTHARAM, AND WONG

phaseoli 127K14 or Rhizobium sp. 127E15. Rhizobiawere cultured in a yeast extract-mannitol medium (14)to late log phase before being used as inoculants. Thejars were placed in growth chambers (light intensity,24,000 lx; photoperiod, 16 h of light and 8 h ofdarkness; day temperature, 27°C; night temperature,22°C). Six days after planting, paper autoclave bagswere removed from the jars and covered with sterilepolypropylene mason jar caps (Arthur Thomas). Eachcap had a 2.4-cm hole in the center. Two seedlingswere gently guided through the hole with a pair offorceps. The hole was plugged, with the seedlingsprotruding through the side of the sponge plug. Start-ing 2 weeks after planting, each jar received 100 to 150ml of half-strength nutrient solution weekly until theexperiments were terminated. The entire procedure ofplanting seeds, inoculating, handling seedlings, andadding nutrient solution was performed aseptically in alaminar flow hood. Rhizobium sp. 127E15 and R.phaseoli 127K14 in crushed nodules were identified byserological agglutination test (21). Rabbit antiseraagainst each Rhizobium strain were obtained as de-scribed by Vincent (21).The purity of the Rhizobium sp. 127E15 cultures

was ascertained by the fact that rabbit antiserumagainst this strain did not cross-react with R. phaseolistrain 127K12, 127K14, or 127K35. Similarly, antise-rum against R. phaseoli 127K14 did not cross reactwith Rhizobium sp. 127E15. Cultures of Rhizobium sp.127E15 were also routinely plated on mannitol-yeastextract medium to make sure that they were notcontaminated with R. phaseoli. Since Rhizobium sp.127E15 is a slow-growing strain and R. phaseoli is a

fast-growing Rhizobium, the large variation in the sizeof the colonies indicates a mixed culture. Further-more, Rhizobium sp. 127E15 has been subculturedrepeatedly in our laboratory for over 4 years. If it is amixed culture with R. phaseoli, then after more than 4years one would expect mainly the fast-growing R.phaseoli in the culture. Up to now, our Rhizobium sp.127E15 cultures still exhibit the characteristics of aslow-growing strain.

Nitrogen fixation activity of root nodules. N2 fixationactivity was assayed by the C2H2 reduction technique(8). Upper 4-cm sections of nodulated roots at differentstages of growth (7, 12, 15, 21, 30, and 38 days afterinoculation) were placed in 38-ml serum bottles, whichwere sealed with rubber serum stoppers. C2H2 was

injected into the bottles to 0.1 atm (10.1 kPa). Thebottles were incubated for 1 h at 30°C. The C2H4produced was measured by gas chromatography. Afterbeing assayed, nodules were detached from the rootsand weighed. C2H2 reduction activity was expressedas micromoles of C2H4 produced per gram of freshnodules per hour.

Electron microscopy. Nodules harvested from upper

4-cm sections of the roots at different times duringdevelopment (7, 12, 15, 21, and 30 days after inocula-tion) were sliced and fixed in a mixture of paraformal-dehyde and glutaraldehyde dissolved in Sorensen buff-er, pH 7.2 (10) for a period of 1 h at room temperature.After a thorough washing in buffer, the tissue sliceswere treated with 1% buffer OS04 solution for 1 h at4°C. The tissue slices were then dehydrated in anethanol series and embedded in Spurr medium (20).Sections of 1- to 1.5-p.m thickness were cut with glassknives and stained with toluidine blue. Ultrathin sec-

TABLE 1. Acetylene reduction activities of polebean root nodules induced by R. phaseoli 127K14

and Rhizobium sp. 127E15p.mol of C2H4 formed/g of fresh

Nodule age nodules per ha(Days after Induced by R. Induced byinoculation) phaseoli 127K14 Rhizobium sp. 127E15

7 0.30 ± 0.09 012 5.06 ± 0.47 0.08 ± 0.0215 15.95 ± 3.10 0.79 ± 0.0521 14.37 ± 1.86 3.04 ± 0.1030 10.96 ± 2.96 4.36 ± 0.4538 5.40 ± 0.25 4.21 ± 0.90

a Data are expressed as the average values of tripli-cate samples followed by the standard deviation fromthe mean.

tions cut with a diamond knife were stained withuranyl acetate and lead citrate. Observations werecarried out with a Zeiss 109 electron microscope.

RESULTSRhizobium sp. 127E15 and pole bean interact-

ed to form root nodules (127E15 nodules), butthe nodules were only partially effective, asshown by their acetylene reduction activity (Ta-ble 1). The peak specific acetylene reductionactivity of 127E15 nodules was only 27% of thatof R. phaseoli 127K14-induced pole bean nod-ules (127K14 nodules). The effective 127K14nodules were dark red and were mostly spheri-cal with distinct white bands (see Fig. 3a),whereas the partially effective 127E15 noduleswere pinkish and had irregular shapes, somespherical and some shaped like a top (see Fig.4a). In both cases, each plant had about 100nodules.

Pole bean plants nodulated by R. phaseoli127K14 had green leaves and showed no sign ofN deficiency throughout the duration of theexperimemt. Plants nodulated by Rhizobium sp.127E15 had pale yellow leaves and showed Ndeficiency symptoms.

Histological analysis of the bacteroid zonesrevealed considerable lag in the developmentalsequence of 127E15 nodules. At 12 days afterinoculation, approximately 40% of the 127K14nodule cells were swollen and were completelyfilled with bacteroids (Fig. 1). In contrast, onlyabout 20% of the 127E15 nodule cells of thesame age were swollen, and they were filled withfewer bacteroids (Fig. 2). The uninfected cellsaccumulated starch in both 127K14 and 127E15nodules (Fig. 1 and 2). At this stage, infectionthreads were seen protruding into the 127E15nodule cells (Fig. 2). Infection threads were alsorevealed by electron micrograph (see Fig. 7).Although not shown, infection threads also wereseen in 127K14 nodules.No significant structural changes occurred in

APPL. ENVIRON. MICROBIOL.

on Septem

ber 11, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

BEAN NODULES INDUCED BY TWO RHIZOBIUM SPP. 967

4a

3 3i

FIG. 1-4. 1 and 2. Photomicrographs of toluidine blue-stained sections of 127K14 and 127E15 nodules,respectively, 12 days after inoculation. Note the presence of starch in uninfected cortical cells of both types ofnodules and the presence of very few bacteroids in swollen infected cells and an infection thread (arrow) in the127E15 nodule. 3 and 4. Photomicrographs of toluidine blue-stained sections of 127K14 and 127E15 nodules,respectively, 30 days after inoculation. Figures 3a and 4a show the respective nodule morphologies.

the 127K14 nodules between 12 and 30 daysafter inoculation (Fig. 1 and 3). The 127E15nodule cells showed an increase in bacteroidpopulation during the same period, although thecells were still not completely filled with bacte-roids and the ratio of infected to uninfectednodule cells did not increase (Fig. 2 and 4).Starch disappeared from the uninfected cells inboth types of nodules (Fig. 3 and 4).

During the early development of 127K14 nod-ules (7 days after inoculation), cytoplasmic vesi-cles were found to concentrate around bacte-roids surrounded by membrane envelope (peri-bacteroid membrane) (Fig. 5). As the 127K14nodules developed further, cytoplasmic vesicles

disappeared, and each peribacteroid membranecontained one or two bacteroids (Fig. 6). Incontrast, during the early development of127E15 nodules (12 days after inoculation), nu-merous cytoplasmic vesicles were found aroundthe peribacteroid membrane containing bacte-roids (see Fig. 8). These vesicles were seen tofuse with the peribacteroid membrane (see Fig. 9and 10). The number of bacteroids within eachperibacteroid membrane increased from one ortwo at 12 days after inoculation (Fig. 7-11) to asmany as nine at 15 days after inoculation (Fig.12). The number increased further. By 21 days,18 to 20 bacteroids could be counted.Rhizobium sp. 127E15 formed effective nod-

VOL. 44, 1982

on Septem

ber 11, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

968 BAL, SHANTHARAM, AND WONG

'-, i..f641-

vw~-A

Vi- ,c"m V ItI

'4,

4

FIG. 5 and 6. 5. Electron micrograph of an infected 127K14 nodule cell, 7 days after inoculation, showing abacteroid (b) surrounded by peribacteroid membrane with a few fusing vesicles (arrow) near the nucleus (N). 6.Electron micrograph of an infected 127K14 nodule cell, 12 days after inoculation, showing characteristic vacuole(v) near the nucleus (N) and cytoplasm filled with bacteroids. One bacteroid (b) per peribacteroid membrane isseen in general, but in some areas there are two per membrane. Insert is a photomicrograph of a toluidine blue-stained thick section, showing closeness and crowded packing of bacteroids.

APPL. ENVIRON. MICROBIOL.

on Septem

ber 11, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

BEAN NODULES INDUCED BY TWO RHIZOBIUM SPP. 969

Izm

F. I# .

. tiI*,-4bI-

't'r -94.

(..I' -

>IW -'s't*6 It

, n I

_ .

FIG. 7-10. 7. Electron micrograph of 127E15 nodule showing infection threads (IT), 12 days after inoculation.8. Electron micrograph showing concentration of numerous vesicles (arrow) around a bacteroid surrounded byperibacteroid membrane in an infected 127E15 nodule cell, 12 days after inoculation. 9 and 10. Electronmicrographs of 127E15 nodule, showing different stages of fusion of the vesicles (arrows) with the peribacteroidmembrane, 12 days after inoculation.

",>.. sj

VOL. 44, 1982

'allll',.J.".". olk"IVI. (. -It.

I;14 7- )kV . I

r

I.

4 %'.Q

VAI

16

on Septem

ber 11, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

970 BAL, SHANTHARAM, AND WONG

FIG. 11 and 12. Electron micrographs of 127E15 nodules, showing two bacteroids (b) per peribacteroidmembrane 12 days after inoculation (Fig. 11) and a multiple number 15 days after inoculation (Fig. 12).

ules on lima bean. Nodulated lima bean plantshad green leaves and showed no N deficiencysymptoms. The nodules had peak specific acety-lene reduction activity of 11.15. Histology of thenodules revealed no anomalous developmentsimilar to that of pole bean 127E15 nodules (la).

DISCUSSIONNodulation of legumes from one cross-inocu-

lation group by rhizobia belonging to anotherhas been well documented (7). Lange (12), forexample, reported promiscuous Rhizobiumstrains which are capable of nodulating legumesof four different cross-inoculation groups. Suchout-of-group nodulations, however, are oftenineffective (22). Furthermore, most ineffectivenodules, whether conditioned by rhizobia orlegumes, are accompanied by arrested noduledevelopment at a very early stage (4, 17), accu-mulation of polysaccharide by host cells (3),early degeneration of rhizobia inside the hostcells (2, 13, 15), or complete failure of rhizobiato develop into bacteroids (6). In these regards,the out-of-group nodulation of pole bean byRhizobium sp. 127E15 is unique. The 127E15nodules appear to have completed, althoughwith some anomalies, the entire developmentalsequence of effective nodules as described byVincent (23).We have shown earlier that 127E15 adheres to

pole bean root hairs and causes root hair tip

curling and swelling (19). It causes the formationof infection threads in root hairs (19) and, asshown here, in the nodule cells. Rhizobium sp.127E15 cells within the infection threads werereleased into the host cells. They multipliedintracellularly and developed into bacteroids.Some nitrogenase activity was expressed, andthe activity persisted for 38 days. The pinkishappearance of the nodules also indicated thatsome leghemoglobins were synthesized.Many factors have been suggested to contrib-

ute to ineffectiveness of root nodules (9, 16, 18),but little is known about the actual basis for thisphenomenon. The partial effectiveness of127E15 nodules seems to associate with someanomalies in the nodule development. The rangeof effectiveness is often related to the amountand persistence of bacteroid-containing noduletissue (5). Since the 127E15 nodule tissue per-sisted over 30 days, fewer infected host cells andfewer bacteroids within the infected cells may beimportant factors contributing to partial effec-tiveness.Although few cytoplasmic vesicles can be

seen in 127K14 nodules and similar observationshave been noted in soybean nodules (11), thesignificance of the abundance of cytoplasmicvesicles in the infected 127E15 nodule cells isnot clear. The fusion of the vesicles with peri-bacteroid membrane may cause the membraneto enlarge. Population of 127E15 increased with-in the enlarged peribacteroid membrane. How

APPL. ENVIRON. MICROBIOL.

on Septem

ber 11, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

BEAN NODULES INDUCED BY TWO RHIZOBIUM SPP. 971

multiple bacteroids within a peribacteroid mem-brane affect effectiveness of the nodules is notknown.

ACKNOWLEDGMENTSThis research was supported by a National Science and

Engineering Research Council of Canada grant to A.K.B. andby U.S. Department of Agriculture Competitive ResearchGrants Program grant no. 78-59-2201-0-1-094-1, National Sci-ence Foundation grant PCM-7904063, and the Kansas Agricul-tural Experiment Station (Biology no. 82-280-j) to P.P.W.

LITERATURE CITED1. Alien, 0. N., and E. K. Alien. 1981. The leguminosae, a

source book of characteristics, uses, and nodulation, p.511-515. University of Wisconsin Press, Madison.

la.Bal, A. K., and P. P. Wong. 1982. Infection process andsloughing off of rhizobial outer-membrane in effectivenodules of lima bean. Can. J. Microbiol. 28:890-896.

2. Bassett, B., R. N. Goodman, and A. Novacky. 1977. Ultra-structure of soybean nodules. II. Deterioration of thesymbiosis in ineffective nodules. Can. J. Microbiol.23:873-883.

3. Bergersen, F. J. 1957. The occurrence of a previouslyunobserved polysaccharide in immature infected cells ofroot nodules of Triolium abiuguum M. Bieb. and othermembers of the Trifolieae. Aust. J. Biol. Sci. 10:15-24.

4. Bergersen, F. J. 1957. The structure of ineffective nodulesof legumes: an unusual type of ineffectiveness and anappraisal of present knowledge. Aust. J. Biol. Sci. 10:233-242.

5. Bergersen, F. J. 1974. Formation and function of bacte-roids, p. 473-498. In A. Quispel (ed.), The biology ofnitrogen fixation. Elsevier/North-Holland Publishing Co.,Amsterdam.

6. Bergersen, F. J., and P. S. Nutman. 1957. Symbiotic ef-fectiveness in nodulated red clover. IV. The influence ofhost factors il and ie upon nodule structure and cytology.Heredity 11:175-184.

7. Graham, P. H. 1976. Identification and classification ofroot nodule bacteria, p. 99-112. In P. S. Nutman (ed.),Symbiotic nitrogen fixation in plants. Cambridge Univer-sity Press, London.

8. Hardy, R. W. F., D. Holsten, E. K. Jackson, and R. C.Burns. 1968. The acetylene-ethylene assay for nitrogenfixation; laboratory and field evaluations. Plant Physiol.43:1185-1207.

9. Holl, F. B., and T. A. LaRue. 1976. Genetics of legumeplant hosts, p. 391-399. In W. E. Newton and C. J.Nyman (ed.), Proceedings of the First International Sym-posium on Nitrogen Fixation. Washington State Universi-ty Press, Pullman.

10. Karnovasky, M. J. 1965. A formaldehyde-glutaraldehydefixative of high osmolarity for use in electron microscopy.J. Cell Biol. 27:137a-138a.

11. KUne, J. W., and K. Planque. 1979. Ultrastructural studyof the endomembrane system in infected cells of pea andsoybean root nodules. Physiol. Plant Pathol. 14:339-345.

12. Lange, R. T. 1961. Nodule bacteria associated with theindigenous leguminosae of South Western Australia. J.Gen. Microbiol. 26:351-359.

13. MacKenzie, C. R., and D. C. Jordan. 1974. Ultrastructureof root nodules formed by ineffective strains ofRhizobiummeliloti. Can. J. Microbiol. 20:755-758.

14. Manhart, J. R., and P. P. Wong. 1979. Nitrate reductaseactivities of rhizobia and the correlation between nitratereduction and nitrogen fixation. Can. J. Microbiol.25:1169-1174.

15. Morse, B. 1964. Electron microscope studies of noduledevelopment in some clover species. J. Gen. Microbiol.36:49-66.

16. Nutman, P. S. 1969. Genetics of symbiosis and nitrogenfixation in legumes. Proc. R. Soc. London Ser. B.172:417-437.

17. Pankhurst, C. E., A. S. Craig, and W. T. Jones. 1979.Effectiveness of Lotus root nodules. I. Morphology andflavolan content of nodules formed on Lotus peduncula-tus by fast-growing lotus rhizobia. J. Exp. Bot. 30:1085-1093.

18. Pankhurst, C. E., and W. T. Jones. 1979. Effectiveness ofLotus root nodules. II. Relationship between root noduleeffectiveness and in vitro sensitivity of fast-growing Lotusrhizobia to flavolans. J. Exp. Bot. 30:1095-1107.

19. Shantharam, S., and P. P. Wong. 1982. Recognition ofleguminous hosts by a promiscuous Rhizobium strain.Appl. Environ. Microbiol. 43:677-685.

20. Spurr, A. R. 1969. A low viscosity epoxy resin embeddingmedium for electron microscopy. J. Ultrastruct. Res.26:31-43.

21. Vincent, J. M. 1970. A manual for the practical study ofroot-nodule bacteria. Blackwell Scientific Publications,Oxford.

22. Vincent, J. M. 1974. Root-nodule symbiosis with Rhizobi-um, p. 265-341. In A. Quispel (ed.), The biology ofnitrogen fixation. Elsevier/North-Holland Publishing Co.,Amsterdam.

23. Vincent, J. M. 1980. Factors controlling the legume-Rhi-zobium symbiosis, p. 103-109. In W. E. Newton andW. J. Orme-Johnson (ed.), Nitrogen fixation, vol. II.University Park Press, Baltimore.

24. Wong, P. P. 1981. Symbiotic nitrogen fixation by Phaseo-lus vulgaris root nodules induced by Rhizobium sp.127E15, p. 470. In A. H. Gibson and W. E. Newton (ed.),Current perspectives in nitrogen fixation. AustralianAcademy of Science Press, Canberra.

VOL. 44, 1982

on Septem

ber 11, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from