Which stage of nodule initiation in Lupinus angustifolius L. is sensitive to iron deficiency?

Transcription

1 New Phytl. (99), 7, Which stage f ndule initiatin in Lupinus angustiflius L. is sensitive t irn deficiency? BY CAIXIAN TANG\ ALAN D. RBSNi AND MICHAEL J.DILWRTH^ Sil Science and Plant Nutritin, Schl f Agriculture, The University f Western Australia, Nedlands, WA 6009, Australia 2 Schl f Bilgical and Envirnmental Sciences, Murdch University, Murdch, WA 650, Australia {Received 5 April 990; accepted 2 ctber 990) SUMMARY Transfer experiments in slutin culture were cnducted t establish the stage f ndulatin in lupins {Lupinus angustiflius L. cv. Yandee) mst sensitive t irn deficiency. In all experiments, irn deficiency had a much greater effect n ndule number than n hst plant grwth. Irrespective f the irn treatment f either the Bradyrhizbiuni r the lupin plant prir t inculatin, plants receiving 2-5 /tm irn after inculatin successfully frmed ndule initials and ndules while thse receiving 0-05 //M irn almst cmpletely failed t initiate ndules. Thus, the preventin f ndulatin by irn deficiency is nt a cnsequence f either an inadequate number f infective bradyrhizbia surviving in the slutin r an alteratin in the irn status f the hst rt. Supply f 2-5 fim irn fr 4 d r mre after inculatin prduced a similar number f ndule initials and ndules as did cntinuus supply f 2-5 IIM irn. Delaying supply f 2-5 (im. irn fr 3 d r less after inculatin did nt delay r prevent ndule initiatin and frmatin. ne-day expsure f plants t 2-5//,M irn n day 4 after inculatin induced the highest number f ndules f any d treatment althugh this shrt expsure was nt enugh t allw the full cmplement f ndules t frm. Hence, the impairment f the ndulatin prcess by irn deficiency can be attributed t the preventin f a step at day 4, the stage just befre ndule initials are frmed. A further study was cnducted t examine the effect f irn n ndule frmatin by using a vertical split-rt technique in which Bradyrhizbiuni sp. {Lupinus) was added t the upper cmpartment. Cmpared t plants receiving 0-05 jim irn in bth cmpartments, plants receiving 0-05 //.M in the upper and 5 fim irn in the lwer cmpartments had nly a slightly higher cncentratin f irn in the crtex f the upper part f the rt. Furthermre, supplying 5 //.M irn t the lwer part f a rt did nt permit ndulatin n the upper part f the rt receiving 0-05 //M irn. Lw cncentratin f irn in the crtex f rts may limit ndule frmatin. Key wrds: Lupinus angustiflius, irn deficiency, ndule initiatin, vertical split-rt, irn translcatin. lupins must therefre ccur at an early stage f INTRDUCTIN ^^^^^^ initiatin. In lupins, irn appears t be required in greater amunts fr ndule frmatin than fr hst plant grwth (Tang, Rbsn & Dilwrth, 990a) and the effect f irn n ndule frmatin is direct (Tang, Rbsn & Dilwrth, 9906). Irn deficiency markedly depressed the number f ndule initials and thereafter the number f visible ndules. initiatin seems t be mre sensitive than subsequent ndule develpment t irn deficiency. Fr irnsufficient plants, appearance f ndule initials cmmenced n day 5 after inculatin and was cmpleted within a further 3 d (Tang et al., 9906). The impairment f ndulatin by irn deficiency in In a hrizntal split-rt experiment. Tang et al. (9906) fund that ndule initials and ndules were frmed nly n that half f the rt system expsed t a high cncentratin f irn. Irn was nt translcated frm the half f the rt supplied with a high cncentratin f irn t the ther half f the rt, receiving a lw cncentratin f irn. In additin, irn applied t leaves as fliar sprays was nt translcated t the rts and ndules were nt prduced (Tang et al., 9906). These results did nt specify whether irn within the rts r in the external slutin is required fr ndule frmatin, The aim f the wrk described here was t

2 244 C. Tang, A. D. Rbsn and M. J. Dilwrth examine whether ndule initiatin might be restricted by inadequate numbers f bradyrhizbia r uninfective frms resulting frm a lw cncentratin f irn in the plant grwth medium r, alternatively, whether the irn status f the plant rt at inculatin might limit ndule initiatin. In additin, we reprt the effects f impsing a chsen level f irn at particular times during the ndulatin prcess n the number and psitin f the subsequent ndules. Finally, we examine the hypthesis that the effect f irn n ndule frmatin is external, using a technique where upper and lwer segments f a rt system were supplied with different cncentratins f irn. MATERIALS AND METHDS Transfer experiments All experiments were cnducted in a glasshuse, in slutin culture with rts maintained at C. Unifrm-sized seeds f Lupinus angustiflius L. cv. Yandee were germinated n a stainless-steel screen sitting ver an aerated slutin f 600 /im CaClg and 2 fim H3B3 fr 4-7 d. Seedlings were then transplanted t 5 pts cntaining cntinuusly aerated nutrient slutin. The cmpsitin f the basal nutrient slutin was as previusly described (Tang et al., 990«). Irn was applied as the ferric mnsdium salt f ethylenediamine tetraacetic acid (Fe"'NaEDTA) at cncentratins f 0-05 //,M (inadequate) r 2-5 //,m (adequate). A dense suspensin Figure. A vertical split-rt system fr grwing Lupinus angustiftius. A, upper cmpartment; B, lwer cmpartment; C, putty; D, lid; E, aeratin system. f Bradyrhizbium sp. {Lupinus) WU 425 frm a 5- r 6-d-ld culture (Tang et al., 990a) was added t the slutin at a rate f 3 X l'^ cells ml'^ The ph f the slutin was adjusted daily t 5'5 with 0- M KH. Nutrient slutins were changed every secnd day except where transfers were made. In all experiments day 0 is the day that plants first came in cntact with Bradyrhizbium. Subsequent treatments f plants in individual experiments are summarized diagrammatically in Tables -5. initials were examined as described by Tang et al. (990a). Vertical split-rt experiment Lupin seeds were germinated n a stainless steel screen ver an aerated slutin f 600 fim CaClj and 2/^m H3B3 fr 5 d. Ten seedlings were then transferred t 5 plastic pts cntaining basal nutrient slutin and 0-05 /J,M irn. As the rts f lupins can be infected by bradyrhizbia nly n a very small prtin f the rt just behind rt tips, and the sensitive stage f ndule frmatin is at day 4 after inculatin, an inculum f Bradyrhizbium was added t the slutin fr 2 d befre irn treatments were applied. ne seedling was finally transplanted int eacb split-rt system which cnsists f tw differently-sized cylindrical cmpartments (Fig. ). The upper cmpartment was psitined in tbe lwer cmpartment. The vlumes f the upper and the lwer cmpartments were 460 and 570 ml respectively. The plants were supprted with a lid sitting ver the upper cmpartment. Tbe grwing rt was allwed t prtrude by 2 cm thrugh a hle at the bttm f tbe upper cmpartment int the slutin f tbe lwer cmpartment. Tbe rt parts in the tw cmpartments were kept separate witb Terstat putty (Tersn GmbH, Heidelberg, West Germany). The cmpartments were filled witb basa! nutrient slutin and irn treatments were impsed at transplanting: 5 //M irn (as Fe'"NaEDTA) fr an adequate level and 0-05 /im irn fr a deficient level. A dense suspensin f Bradyrhizbium WU425 (Tang et al., 990a) was added t the upper cmpartment f tbe system at a density f 3 x 0'^ cells ml~\ prviding tbe fur treatments (Table ), eacb f which bad five replicates. Tbe slutin was left uncbanged fr 3 d after whicb it was renewed every third day withut further additin f bradyrbizbia. Tbe ph f the slutin was adjusted t 5-5 witb 0- M KH twice a day thrugbut tbe experiment. Plants were harvested 5 d after irn treatments had been applied. T remve dust, whle sbts were rinsed in deinized water, wasbed in 'l M HCl and again rinsed in deinized water. Rts were rinsed in deinized water twice. Plant materials were separated int tbe yungest fully expanded leaf blade (YEB), sbts, crtex f the taprts, stele f the

3 Ndide initiatin sensitivity t irn deficiency in L. angustiflius 245 Table. FJfects f irn applied t bradyrhizbial suspensins in slutin culture befre and after intrductin f Lupinus angustiflius. Preinculatin treatments were impsed n the fully grwn inculum f Bradyrbizbium WU 425 in the nutrient slutin fr 2 d Planting cunting s plant Day 0 Day 3 Tp fresh weight (g plant"') Day 3 Rt fresh weight Day b a 2a 0-2 b 36a 39a l-75a l-99a l-79a 2-23 b 2-53a 2-57 a The hrizntal scale is time in days. Symbls n the hrizntal scale are: 0, the perid f plant cntact with bradyrhizbium; Q, 0-05 /;M irn in slutin;, 2-5 /(M irn in slutin;, additin i Bradyrhizbium t the slutin; 9, the time f ndule appearance. number was analysed as lgj^. Values in each clumn fllwed by the same letter are nt different at P = Table 2. Fffect f adequate supply f irn, befre and after inculatin, n ndule frmatin n Lupinus angustiflius. Preinculatin treatments were impsed n 5-d-ld seedling fr 5 d Planting Cunting ndule initiates initials cunting plant"' i I i s plant"' Fresh weight f sht (g plant"') Fresh weight f rt (g plant"') Day 6 Day 9 Day 2 Day IS Day 2 Day IS Day 2 Day IS b 3a 3 a 4a lc 4b 6a 8a lb 4b 27 a 27 a 4c 3b 7a 76 a b62a b68a b77a ^76a b95a 2^00 a 2^0 a 2.4a The hrizntal scale is time in days. Symbls n the hrizntal scale are: 0, the perid f inculatin with Bradxrhizbium; D, ^S /fm irn in slutin;, 2^5 //M irn in slutin; ;j;, additin ai Bradyrhizbium t the slutin; 0, the time f ndule appearance. Number f ndule initials and ndules was analysed as lgj,, and data in each clumn with a cmmn letter are nt significantly different at P = ^S. 2^5 a 2^43 a 2^69 a taprts, lateral rts and ndules. All plant tissues were ven-dried at 68 C. Ttal irn cncentratin in tissues were measured using atmic absrptin spectrplitmetry after digesting plant materials in a 4: mixture (by vlume) f cncentrated nitric and perchlric acids (Jhnsn & Ulricb, 959). RESULTS General effects f irn-deficiency In all transfer experiments, plants grwn in slutin cntaining 0^05 //M irn sbwed fliar symptms f irn deficiency abut 2 wk after germinatin. Tbe symptms f irn deficiency were delayed fr 3-6 d in tbe case f plants transferred frm 2^5 /<M irn t 0^05 //M (treatments described in Tables 2, 3 and 5), but n symptms were bserved fr plants cntinuusly given 2^5 fim irn. Sligbt nitrgen deficiency ccurred in all plants during the late stages t all experiments. Tbe fresh weight f shts was nt affected by irn treatments in any experiment (Tables -5). Tbe fresb weigbt f rts expsed t 0-05 //M irn r expsed t 2-5 fim irn fr nly d was less tban that f tbse grwn cntinuusly witb 2^5 /<M irn at tbe latest harvest but nt at earlier harvests (Tables -5). Under irn-sufficient cnditins, ndules appeared 8-9 d after inculatin. s were frmed n a narrw regin f rts just bebind the rt tips at tbe time f inculatin. Under irndeficient cnditins, many brwn pints were clearly bserved n the narrw regin f rts just bebind tbe rt tips at the time f inculatin. Treatment f Bradyrbizbium tvith irn prir t inculatin. Whether r nt Bradyrhizbium cells were supplied with 2^5 //M irn in slutin fr 2 d befre plants were intrduced, the lupins subsequently receiving 2^5 //M irn prduced ver 0 and 30 ndules eacb at

4 246 C. Tang, A. D. Rbsn and M.J. Dilwrth Table 3. Effect f withdrawal f adequate irn r delayed supply f adequate irn n the number f ndule initials and ndules, and grwth f Lupinus angustiflius Cunting ndule Planting initials cunting i I t t t initials plant' Day 8 s plant ' Day 2 Day 6 Fresh weight f sht (g plant" ') Day 2 Day 6 Fresh weight f rt Day 2 Day 6 2 b 3 b 2b 7a 2a 2a lb 2b 2b 2a 20 a 26 a 4b 4b Sb 26 a 45 a 38a -47 a l-54a l-57a l-54a 59a -7 a 2-03 a l-77a -74 a 70 a 2-08 a -95 a -5 a l-63a -58 a l-55a l-53a l-79a 2-23 ab 2-4b 2-00 b 2-25ab 2-80 a 2-S5a The hrizntal scale is time in days. Symbls n the hrizntal scale are: 0, the perid f inculatin with Bradyrhizbiuni \, 0-05 /IM irn in slutin;, 2-5 //M irn in slutin; \, additin i Bradyrhizbiuni t the slutin. Number f ndule initials and ndules was analysed as lgj^, and data with a cmmn letter are nt different at P = Table 4. Effects f delayed supply f adequate irn n ndulatin and grwth f Lupinus angustiflius at day 5 after inculatin t Planting cunting s plant ' psitin* (cm) Tp fresh weight Rt fresh weight 35a 38a 37 a 24 a 9b lc 0-5 c ll-8c 2-5c 6-5b 8-3ab 2-3a na na 2-8a -97 a 2-07 a 2-20a 2-5a 96a l'80a 3-47 a 3-26 a 3-36a 3-44 a 3-56a 3-07 a 2-85 a The hrizntal scale is time in days. Symbls n the hrizntal scale are: 0, the perid f inculatin with Bradyrhizbium; D, 0-05 /im irn in slutin;, 2-5 //,M irn in slutin: J;, additin f Bradvrhizbittm t the slutin; %, the time f ndule appearance. psitin refers t the distance between hypctyl f rts and the site f earliest ndules frmed n the rts ; Data were the average f 0 plants. na, nt applicable since plants in these tw treatments almst cmpletely failed t ndulate. number (day 5 after inculatin) was analysed as lgj(. Values in each clumn fllwed the same letter are nt different at P = ()-()5. days 0 and 3, respectively. Hwever, lupins failed t ndulate if they received 005 /tm irn thrughut (Table ). Treatment f plants with irn prir t inculatin Plants cntinuusly given 2-5 //M irn, and plants given 0-05 //M irn befre but 2-5 //M irn after inculatin, prduced a similar number f ndule initials and ndules. These numbers were much greater than thse n plants either receiving 0-05 /im irn cntinuusly r receiving 2-5 jim irn befre but 0 05 [im irn after inculatin. Plants receiving 2-5 //,M irn befre but 0-05 fim irn after inculatin had slightly mre ndules than thse cntinuusly receiving 0'05 //M irn (Table 2). Hwever, n plants given 0'05 //M irn after 2 5 /i.m irn, ndules were distributed in a very narrw regin f the taprt, with 90% f ndules within 4 cm f the tpmst ndule, while plants receiving 2 5 fim irn after inculatin had 50% f their ndules mre than 4 cm belw the tpmst ndule.

5 initiati?! sensitivity t irn deficiency iti L. angustiflius Days after inculatin Figure 2 Effects n the number f ndules n Lupinus august iflius f delaying supply f 2'5 /(M irn fr 0 ( ), 3 (A), 4 (), 5 ( )', 6 ( ) r 8 (A) d after inculatin. Cntrl plants received a cntinuus supply f 0-05 //M irn ( ). Withdraival f irn r delayed supply f adequate irn Plants given 2-5 //M irn fr 4 d fllwing inculatin prduced a similar number f ndule initials and ndules t thse receiving 2-5 fim irn cntinuusly. Hwever, supplying 2-5 //M irn fr nly r 2 d markedly decreased the number f ndule initials and ndules; plants almst failed t ndulate, and were similar t thse given 'S fim irn cntinuusly. Delaying the supply f 2'5 /tm irn fr d after inculatin did nt decrease the number f ndule initials and ndules (Table 3). Plants supplied with cntinuus 2-5//M irn prduced ndules 8 d after inculatins and prduced mre than 30 ndules (Fig. 2, Table 4). Hweyer, delaying supply f 2'5 /tm irn fr 4, 5 and 6 d fllwing inculatin delayed the subsequent appearance f ndules by, 2 and 3 d respectively. Delaying supply f 2-5 //M irn fr 8 d almst cmpletely prevented ndule frmatin; plants behaved similarly t thse receiving a cntinuus supply f 0-05 //M irn. In cntrast, delaying supply f 2-5 /tm irn fr 3 d did nt delay ndule appearance and did nt decrease ndule number (Fig. 2). number was decreased when supply f 2-5 /(M irn was delayed fr 5 d r mre, and ndules ccurred lwer n the taprt when supply f 2-5 //M irn was delayed fr 4 d r mre fllwing inculatin (Table 4). Shrt expsures t adequate irn Where plants were given 25 //M irn nly fr d rather than cntinuusly, ndule appearance was delayed and ndule frmatin was almst prevented. An exceptin t this behaviur was seen in plants expsed t 2-5 //M irn fr 24 h n day 4 after inculatin. In these plants ndule appearance was nt delayed and they induced mre ndules than plants receiving 25 pim irn n any ther single day. Hwever, the number f ndules was still less than that n plants receiving a cntinuus supply f 2-5 //M irn (Table 5). Irn supply t different parts f the split-rt system Slight chlrsis f yung leaves was bserved in plants receiving 005 //M irn in bth cmpartments n day 6 after treatments had been impsed. In these plants, this symptm persisted thrughut the experiment, but did nt appear fr any ther treatment. The fresh weights f shts and rts, the number f lateral rts and the lengths f taprts were unaffected by the irn treatments (Table 6). Table 5. Effects f shrt expsure t adequate irn n ndulatin and grwth f Lupinus angustiflius inculated with Bradyrhizbium Planting L L I 8 \ cunting \ \ \ s Day a 0-8 a plant"' Day c 0-4 b l-2b 0-4 b 0 4b 0- c 90a Day c 0-7 c 2-2b l-6b l-2b 0-5 c 35-a Tp fresh weight Day 5-80 a -97 a -95 a 2-03 a -92 a 2-03 a 2-8a Rt fresh weight Day c 2-92bc 2-85 c 3-05 be 2-97bc 3-3b 3-47 a The hrizntal scale is time in days. Symbls n tbe hrizntal scale are: 0, the perid f inculatin with Bradyrhizhiuiii; D, 0-05 //M irn in slutin ;, 2-5 /im irn in slutin :, additin f Bradyrhizbium t the slutin ; 9, tbe time f ndule appearance. number was analysed as lgi,,. Values in each clumn fllwed by tbe same letter are nt different at P = 0-05.

6 ( 248 C. Tang, A. D. Rbsn and M.J. Dilwrth.42 I SP II "- a g u.s J "a "3 '«M cd Jd 3 '^ cd 3 u fn 00 rn ^. l T., fn 0) T-H m (N r <N l c6 I 00 * " * sd fn 50 fn T ' CN CM 04 (N S-i (N fn dv in in in >0 N ir n m ^ 0) 00 (N I) tn C r d c d v 0) d d fn 0) t/) c l II cd lu c cd u igni:.3. "a. li Rts directly expsed t an adequate level f irn (5 //M) in slutin in the upper cmpartment had large number f ndules, while thse grwn in the slutin with a deficient level (0-05 //.M) almst failed t ndulate, irrespective f the cncentratin f irn in the slutin surrunding the ther part f the rt system (Table 6). Because the lwer 2 cm segment f the rt had been pretreated with bradyrhizbia, ndules were als frmed n the lwer part f the rts expsed t 5 //M irn. Plants grwn in 'S /im irn in bth cmpartments had an irn cncentratin in YEB belw the critical level f 65 /ig (g d wt)~' required fr chlrphyll synthesis (Tang et al., 990). Plants grwn in the ther three treatments had irn cncentratins in YEB abve this critical level. Hwever, plants treated with 5 /<M irn in the lwer r bth cmpartments had greater irn cncentratins in YEB than thse in plants treated with 5 JUM irn in the upper cmpartment nly. Cncentratins f irn in whle shts fllwed a similar trend t thse in YEB (Table 7). The cncentratins f irn in the crtex f taprts, lateral rts r whle rts f the upper part expsed t 5 /im irn were mre than twice as high as thse in the same tissues expsed t 0-05 /JM irn. The upper part f the rt f plants given 0-05 fim irn in the upper cmpartment and 5 /IM in the lwer cmpartment had a slightly higher cncentratin f irn in the crtex than that f plants given 0'05 /im irn in bth cmpartments. The cncentratins f irn in the stele f the upper part f the rts were higher when the lwer r bth cmpartments received 5 fim irn than when the upper cmpartment received 5 /im r when bth cmpartments received 0'05 /JM irn. Irn cncentratins in the crtex were much higher than thse in the stele fr all treatments (Table 7). DISCUSSIN The ndulatin prcess f L. angustiflius is apparently mst sensitive t irn deficiency 4 d after inculatin with Bradyrhizbium, just befre ndule initials are beginning t be frmed (Tang et al., 9906). It is pssible therefre that impairment f ndulatin by irn deficiency is due either t a limiting effect n the stimulatin f cell divisin in hst rts by Bradyrhizbium, which nrmally establishes the meristem f ndule initials, r t inhibitin f the prliferatin f Bradyrhizbium within the rts. Hwever, this has nt been firmly established. The limitatin f ndule frmatin des nt seem t be caused by a limited number f Bradyrhizbium in irn-deficient slutin. Bradyrhizbia supplied at a rate f 3 x 0"' cells ml"', incubated fr 2 d with either inadequate irn r adequate irn, culd successfully ndulate lupins, as lng as adequate irn

7 initiatin sensitivity t irn deficiency in L. angustiflius 249 Table 7. Effect f irn supply n irn cncentratins {/Jg g ^ d. ivt) in the yungest fully expanded leaf blades (YEB), shts, rts and ndules f Lupinus angustiflius grwn in the vertical split-rt system Cncentratins f irn in culture slutin (//,M) UC LC s = 0-05) LSD (P YEB Shts Upper Rt crtex cmpartment (UC) Rt stele Lateral rt Whle rt * 3 cm segment f rts frm the putty that separated tw cmpartments, f Insufficient sample. s t Lwer cmpartment (LC) Rt belw 3-cni 3-cm segment * segment Whle rt was applied after inculatin (Table ). Althugh irn deficiency might have affected the grwth r survival f the intrduced bradyrhizbia, the numbers f surviving cells were still adequate fr successful ndutatin. In field cnditins, 'Hara et al. (988) fund that the numbers f peanut Bradyrhizbium sp. in the rhizsphere f peanuts were nt limited in an irn-stress sil in which seedlings develped symptms f irn deficiency. Internal irn status f hst rts may nt be an imprtant factr in limiting ndule frmatin. Pretreating plants with adequate irn fr 5 d allwed the frmatin f nly 5-22 % f the ndule initials and ndules fund n plants given adequate irn after inculatin. The ccurrence f slightly mre ndule initials and ndules n the pretreated plants than n thse receiving inadequate irn culd be due t the presence f irn remaining in tlie applast after irn was withdrawn frm the slutin. Lupins culd ndulate successfully if adequate irn was applied fr 4 d r mre after inculatin. This perid f adequate irn supply seems t be enugh t establish ndule initials. s develped frm these ndule initials even if n mre irn was supplied. In a previus study. Tang et al., (9906) fund that the respnse f ndule number t irn supply had the same pattern as that f ndule initials and suggested that ndules culd develp successfully frm ndule initials in the rts under cnditins f bth irn-deficiency and irn-sufficiency. Delaying supply f adequate irn fr 3 d r less after inculatin neither decreased ndule number nr delayed ndule appearance, pssibly indicating that irn deficiency did nt limit rt clnizatin and/r infectin by Bradyrhizbium. In the present study, many brwn pints were bserved n the taprt f irn-deficient plants. If these are infectin sites that die frm irn deficiency, the frmatin f ndule initials culd eventually be prevented. frmatin was severely restricted where adequate irn was prvided fr nly aid perid. implying that the irn-dependent steps in the prcess f ndule initiatin require mre than d. Nevertheless, plants treated in this way gave a clear indicatin f the stage in the prcess f ndule initiatin that is mst sensitive t irn deficiency. ne-day supply f adequate irn n day 4 after inculatin yielded the highest number f ndules as cmpared t that n any ther day. The results further cnfirm the earlier findings that the mst sensitive stage is at day 4 (Table 3, Table 4). In the vertical split-rt system, ndules were prduced nly n that part f the rt where adequate irn was present, regardless f whether r nt the ther part f the rt r the sht received sufficient irn. The results are in agreement with the finding that the limitatin f ndulatin in lupins by irn deficiency is independent f irn status in the sht r grwth f the hst (Tang et al., 9906). Using the vertical split-rt technique, we expected that irn wuld be translcated frm the lwer part f the rt expsed t a high cncentratin f irn t the upper part f the rt expsed t a lw cncentratin f irn, and high cncentratins f irn in the upper part f the rt wuld be btained. Hwever, ur study shwed that the cncentratin f irn in the upper part f the rt given a lw cncentratin f irn in slutin nly slightly increased when the lwer part f the rt was given a high cncentratin f irn. This increased irn in the upper part f the rt is insufficient fr successful ndule frmatin (Tang et al., 990(7). It cannt therefre categrically be cncluded that the effect f irn n ndule frmatin in lupins is external. In a hrizntal split-rt experiment. Tang et al. (9906) fund that irn was nt translcated frm the irnadequate half f the rt t the irn-inadequate half and that ndules were nly frmed n that half f the rt f lupins where adequate irn was applied. Fliar applicatin f irn did nt increase the cncentratin r cntent f irn in the rts (Tang et al., 9906). Hwever, in crn plants, a prnunced translcatin f irn twards rt tips ccurs in the

8 250 C. Tang, A. D. Rbsn and M.jf. Dilwrth phlem (Kashirad, Marschner & Richter, 973), and fliar-applied irn is als translcated t rts in peanut ('Hara et al., 988) and ther plants (Brwn, Yamaguchli & Leal-Diaz, 965). The mbility f irn in phlem may vary with plant species. ne way t study the effects f internal irn n ndulatin in lupins may be t use seeds with different irn cncentratins. The prcess f ndulatin in many legumes includes the curling f rt hairs and the frmatin f infectin threads thrugh rt hairs. Hwever, in lupin plants these phenmena have nt yet been bserved (Dart, 977; Quispel, 983), and the prcess f lupin ndulatin remains unclear. Hw irn deficiency interferes with events f the sensitive stage is therefre nt understd. ACKNWLEDGMENTS We are grateful t S. P. Lss fr the design f the vertical split-rt system. REFERENCES BRWN, A. L., YAMAGUCHLI, S. & LEAL-DIAZ, J. (965). Evidence fr translcatin f irn in plants. Plant Physilgy 40, DART, P. J. (977) Infectin and develpment f leguminus ndules In: A Treatise n Dinitrgen Fixatin (Ed. by R. W. F. Hardy & W. S. Silver), pp Jhn Wiley & Sns, New Yrk. JHNSN, C. M. & UI.RICH, A. (959). Analytical methds fr use in plant analysis. Califrnia Agriatltural Experiment Statin Bulletin N KASHIRAD, A., MARSCHNER, H. & RICHTER, C. (973). Absrptin and translcatin f ^'Fe frm varius parts f the crn rt. Zeitschrift fiir Pflanzenernahrung iinri Bdenkunde 34, 'HARA, G. W., DILWRTH, M. J., BNKERD, N. & PARKPIAN, P. (988). Irn deficiency specifically limits ndule develpment in peanut inculated with Bradyrbisbium sp. New Phytlgist 08, QUISPEL, A. (983). Dinitrgen-fixing symbisis with legumes, nn-legume angisperms and assciative symbises. In: Encyclpedia f Plant Physilgy (new series), vl. 5 A (Ed. by A. Lauchli & R. L. Bieleski), pp Springer-Verlag. TANG, C, RHSN, A. D. & DILWRTH, M. J. (990a). The rle f irn in ndulatin and nitrgen fixatin in Lupinus angustiflius L. New Phyttgist 4, TANG, C, RBSN, A. D. & DILWRTH, M. J. (9906). A splitrt experiment shws that irn is required fr ndule initiatin in Lupinus angustiflius L. New Phytlgist 5, 6-67.

9