Rhizobium strain identification in Arachis hypogaea nodules by enzyme-linked immunosorbent assay (ELISA)' B. K I S H I N E V S K Y L)i~~i,siotr of'Lc,,qi~r)~c, f/roe~ii/(i/iotr, A g ~ ~ i c ~ i r I R(~.soe~~~clr t ~ ~ r o / Ot~g(rrri:(itiot~,Ti/(, I'II/(.(III~Cc,~rf(,t.,BPI Ihi,ilil?~cells from encl of the nodule. All nodules obtained from plants strain were mixed together in paired combination inoculated with the Rhi:oDiii~n strains gave strong at a ration of I: I (Table 4). Nine plants sown i~ positive ELISA reactions with homologous anti- sterile soil were inoculated with each Rhizohiirn sera. Also the small-sized nodules, weighing only mixture and ~lsi~ally 10 nodules were excised fron 2-8mg, gave consistently positive colorimetric each plant. Single nodules were tested against al reactions (mean = A,,, , > 0.50). Root tissues of three antisera to identify the antigens present i uninoculated plants did not interfere with the re- these nodules. sults of the ELISA test. Nodules from plants inIt was found that for all mixtures tested, one c oculated with serologically unrelated strains failed the strains in each pair formed most of the nodule to react in the ELISA test with heterologous anti- examined. Thus, strain 3847A, which was able t sera. form effective nodules when used as the sole ir The specificity and sensitivity of ELISA for de- oculant. formed only 4% nodules when used i tecting nodule antigens were investigated using combination with strain 5/70. In all cases the effec single-strain nodule samples that were collected in tive strain dominated the ineffective 944C corr bulk. Even 0.4mg fresh weight of nodule tissue petitor. When the combination of strains 5/70 an

I

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K I S H I N E V S K Y A N D BAR-JOSEPH

.

.

Fresh

weight

of

nadules

(rng/rnll

FIG. 2. ELISA absorh:~nce values ~~singUlti:ohici~~~ antibodies ;~g;rinstV;~I.~OLIS weights of notlule tissue taken h-om .-!rtrc~lris Irypogtrc~oplanlsgrown in sterile soil. Each point i s the mean of five I-eplicateh. Plateco:tting;lncl conjugate application were ;IS clesc~ibedin Fig. I. (11) Antibotly to strain 5/70 tested against notlule antigens o f stririns 5/70 ( 0 - 0 ) . 944C ( A - A ) . and 3847A (0-0). ( b ) Antibocly to strain 3847A tested against nodule antigens of str;rins 3847A ( 0 - 0 ) . 944C ( A - A ) . kind 5/70 ( 0 - 0 ) . (c.) Antibotly to strain 944C tested against nodule antigens of strains 934C ( 0 - 0 ) . 5/70 ( A - A ) . ant1 3847A .

. .

.

.

(0-0).

. .

.

TABLE3. The detection o f antigens in peanut nodules on ~~nsterilized soil

Inoculum strains

Number o f nodules tested 35 72 81 81

Natural soil 5/70 3847A 944C

Number o f nodules which gave positive E L l S A reactions with antisera 5/70

3847A

944C

12 72(100x)

-

-

77(95m

-

-

75(92x)

2

-7

4

T ~ L 4. E The detection o f antigens in peanut nodules, inoculated with combinations o f strains 5/70, 280A, R283A, 3847A, and 944C

Inoculum strains

Numbcr o f nodules from which one o r both inoculum strains were detected

A+B 5/70+ 3847A 5/70+ 944C R283A, 3847A R283Al +944C 2YOA 3847A 280A 944C

A 78 76 69 79 65 78

+

+ +

B 3 I2 15 8 12 10

Nodules not reacting with any o f the antisera used

A and B 9

-7 6 3 13 2

944C was used for the mixed inoculum. only 15.8% of the nodules tested contained strain 944C alone. Similar I-esults were obtained with all other strain combinations examined. Assuming that competieness of R11i~06ilit?lstrains is based on the number of nodules formed, strains 5/70 and 280A were the most competitive. The ability of I hizobia strains to compete successfully with other strains for nodule sites has been demonstrated several times (Caldwell 1969; Labandera and Vincent 1975; Robinson 1965). Among the 90 I-ep~esentative nodules examined for

-

23

-1

each pail of strains, most contained one strain. However. plants inoculated with equal numbers of each stsain developed some nodules that contained both strains. The present results confirm those of Lindemann e t a / . (1974) for soybean and Pinto ~ t a l . (1974) for M ~ d i c ~ l gand o clover, showing that some of the nodules may contain more than one strain of Rlli~ohi1it71. Discussion Various fol-ms of enzyme-linked irnmunosorbent assay (ELISA) are increasingly used in clinical bactcl-iology (Nassau et nl. 1976; Su~ndel-sand Clinard 1976) and immunology (Maolini and Masseyeff 1975), where they are rcported to have sensitivities comparable to I-adioirnrnunoassay techniclues (Engvall and Perlmann 1971, 1972). Our results showed that ELISA can provide rapid and sensitive identification of peanut Rllizobilit?~ stsains, both in pu1.e cultures and in single root nodules. The ELlSA technique enabled the detection of Rl1i~obii1171 strains in suspensions containing lo4-10' cells/ml while the sufficient concentration to ensure detectable bands in imrni~nodiffusion technique is 10'4 x 101° cells/rnl (Dudman and Brockwell 1968; Vincent 1970) and 1-10 x 10"

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1.542

CAN. J. MICROBIC

cells/ml in the agglutination test (Vincent 1970). Similar high sensitivity of ELISA was revealed for several plant virus antigens (Clark and Adams 1977). RI1i:7oOi1etl1strains could be detected both in heated and in unheated cell suspensions, but ELISA values of the heated culture antigen were significantly higher than those obtained for the unheated suspensions, probably because of the release of thermostable antigens from the killed cells. The sel-ological specificity of ELISA generally follows that of the somatic agglutination and imniunodiffi~siontests. With the exception of strain 3844C, no common antigens were found in strains belonging to different sel-ological groups. The ELISA test with unheated antigens of strain 3844C (Table 2) indicates that this strain shared antigens with strains (5170, 280A, 23/70, and R283A) belonging to another sel-ological group. Common antigens were also found by immunodiffusion of different slow-growing rhizobia strains tested against antisel-a to three strains of Rhi:7ohirttn ,jcrpotliccoll (Vincent ~t (11. 1973). The cultul-ed cells and bacterial components of the nodules of all Rllizohilitlz strains examined have been found to be closely I-elatedantigenically. ELISA tests of single nodules from peanut plants inoculated with different RllizoOicrtn strains on sterilized soil also revealed a similar pattern of serogroup I-elatedness and no false reactions appeared with root tissues or with nodules detached from plants inoculated with serologically unrelated strains. Parker and Grove (1970) have found it possible to identify rhizobia directly from small nodules using a microagglutination method. However, this technique has the limitation that each crushed nodule may be tested only against a single antiserum. The high sensitivity of the ELISA (Fig. 20, 19, c ) nieans that only very small amounts of serum and antigens are required, since both the serum and antigen can be greatly diluted for the assay. Thus, it is possible to carry out microplate ELISA tests on a large scale and each cl-ushed nodule may be tested against sevel-al antisera. In dilutions of suspensions prepared from combined samples of several nodules, positive ELISA values (A,,, = 0.35-0.62) were obtained with 0.4 mg/ml fresh weight of nodule tissues. Even the smallest nodules, weighing only 2-8 mg, contained enough antigen for the performance of ELISA tests. Using agglutination and immunodiffusion techniques, such small amounts of antigen would probably remain undetected. The data obtained from the mixed infection experiment demonstrate the ~~sefulness of ELISA in

studies of conipetition between Rl7irokiertll strains for nodule formation. The microplate method is suited to accul-ate large-scale testing of samples. such us might be required in studies of Rl1i:ohiect71 colonization and nodulation efficiency undel- different ecological conditions. Acknowledgements We thank Dr. J . Schiffmann and Mrs. Rina Lobel fot supplying peanut rhizobiuni strains 5/70. 280A, R283A. and 23/70 and Drs. N . Seligman, D. Gurfel. and Mr. D. Lachover for their encouragement, criticism, and helpfill suggestions in this manuscript's preparat ion.

BEKGI:KSEN. F. J. 1961. The growth o f R l ~ i : o h i o ~in ~ synthetic media. A~rst.J. Biol. Sci. 14: 349-360. BOHLOOI.. B . B.. and E . L. SCHRIIIYI.. 1973. ['el-sistence and competition aspects o f R l r i : o l ~ i r ~ r t ~ , j ( ~ohse~.ved ~ ~ o ~ ~ iinc ~ soil ~~ by iiiirnl~nofl~~orescence microscopy. Proc. Soil Sci. Soc. Am. 37: 561-564. CALDWELL.B. E. 1969. I n i t i ~competition ~l o f ~.oot-nodulehacteria on soybe~insin ;I lield environment. Agron. J. 61: 813-815. CI-AKK. M. F.. :rnd A . N . An.\hts. 1977. Char;tcte~.islics o f the microplate method o f enzyme-linked irnm~~noso~.hent assay for the detection o f plant vi~.uses.J. Gen. Virol. 34: 475-183. DUDMAN.W. F. 1977. Serological niethodsancl their application to dinitrogen-fixing organisms. 111 A treatise o n dinitl.ogen fixation. Section I V . Agl.onomy and Ecology. Etlirc.(l hy K. W. F. Hardy and A. U. Gibson. Wiley and Sons. New York. pp. 487-508. I>uI)~I.&N. W. F.. and J. BKOCKWELL. 1968. Ecological sti~dies o f root-nod~~le hactel-i;~introduced into field envi~.onments.I. A SLII-vey o f field performance o f clover inoculants hy gel immune tliffi~sionserology. Aust. J. Agric. Kes. 19: 739-747. ENGVALL, E., and P. PEKLM,\NN. 1971. Enzyme-linked im. munosorbent assay (EL.ISA). Qu:untit>itive assay o f im. m~inoglohulinG. Imrn~lnochemihtry.8: 871-874. -1972. Enzyme-linked immunosorhent ;1\21y. EL-ISA 111. Quantitation o f specilic antihotlies hy enzyme-l;ibelec nntiimm~~noglobulin in antigen-coated tubes. J. I m r n ~ ~ n o 109: 129-135. JOHNS-1.0~. A. W. B.. and J . E. BEKINGER. 197.5. Iclenlificatiol o f the Rlli;ohirrt~r strains in pea root nodules sing genetic marke~.s.J. Gen. Microbial. 87: 343-350. LAB,\XI~ERA, C. A.. and J. M . VINCENI. 1975. Cornpetitior between ;In introducecl stl->\inand native Ur~~guaynn strains o Rlri:ohirr~,l tr(fi)lii. Plant Soil, 42: 327-347. LINDEAIANN, W. C.. E. L. SCHXIIDT, and G. E. HAM. 1974 Evidence for. double infection within soybean nodules. Soi Sci. 118: 274-279. LOBEL.K., and J. SCHIFFMAKN. 1975. 'Testing o f peanu nitrogen-lixing bacteria fol- their symbiotic effectivity. Sci entific Activities 1971-1974. lnstit~lteo f Field and Gtrrde Crops. ARO, 'The Volcani Center. Bet D:igan. Isr'nel. pp 214-215. MAOLINI, R., nncl R. M\SSEYEFF.1975. A sandwich method u enzymoin~m~~nosassny. I. Applic;ition to i.at and hilma alpha-fetoprotein. J. I r n m ~ ~ n oMethods. l. 8: 223-234. MEANS.M . , H . W. JOHNSON.and R. A. D.4.r~. 1964. Quic se~.ological method of classifying strains o f R1zi:ohilrl jtrl~o~riclrrtr in nodules. J. Bactei-iol. 87: 547-553.

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ER A.,ilnd , W . F. D U D M A N 1973. . Evalu;~tionof N..\ssAu. E., E. R. PARSONS, and G. D. JOHNSON. 1976. The S H W I N G H A ME. detection of i~ntibodiesto Myc~ol)trc./rricr,,(1rrbe1~c.111o.si.s by spectinomycin resistance as a marker fol- ecological studies micl-oplate enzyme-linked irnmunosor.ben~ ;\.;say (ELISA). spp. J. Appl. Bi~cteriol.36: 263-271. with Rl~izol~irrttr VANS C I I R E V ED. N , A. 1959. Effects of added sugars and nitroTubercle, 57: 67-70. P A R K E RC. , A,. ilnd P. L. GROVE.1970. Rapid se~.ologic;~l gen on nodulation of leg~lrnes.Plant Soil, 11: 93-1 12. J. M . 1970. A nianual fol- the pr;~ctical study of the identification of rhizobia in small nodules. J . Appl. B;~cteriol. VINCEN-rr, ~.ootnodule bacteria. Blackwell Scientific Publications. Ox33: 248-252. ford. I'IN~IO, C . M., P. Y. YAO,and J. M. VINCENT. 1974. Nodulating E Y . V. SKRDLETA. 1973. conipetitiveness amongst strains of Rlrizohirr~tr~tleli/otiand VINCENT,J. M., B. H U ~ V I P H R and R. ~rjfolii.Aust. J . Agric. Res. 25: 317-329. Group antigens in slow-gl-owing I-hizobia. AI-ch. Microbiol. 89: 79-82. ROBINSON, A. C. 1969. Competition between effective and inA,, A. BAR.I-LE.I--I-, D. E . B I D W E L LM. , F. CLARK, and effective strains of Rlii:obir~ttl /r(fblii in the nodulation of VOLLER, A. N. ADAMS.1976. The detection of vi~.usesby enzymeT~.ifbliotrr.srrb/rr~.trtrerrt~l. Aust. J . Agric. Res. 20: 827-841. linked immunosorbent assay (ELISA). J. Gen. Virol. 33: SAUNDI~R G.S ,C., and E. H. C L I N A R D1976. . Rapid micro165-167. method of screening for antibodies to disease agents sing the indirect enzyme-labeled antibody test. J. Clin. Microbiol. 3: 604-608.

Rhizobium strain identification in Arachis hypogaea nodules by enzyme-linked immunosorbent assay (ELISA).

Rhizobium strain identification in Arachis hypogaea nodules by enzyme-linked immunosorbent assay (ELISA)' B. K I S H I N E V S K Y L)i~~i,siotr of'Lc,...
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