Silicon promotes nodule formation and nodule function in symbiotic cowpea (Vigna unguiculata)

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1 New Phytol. (1999, 142, Silicon promotes nodule formtion nd nodule function in symiotic cowpe (Vign unguicult A. NELWAMONDO AND F. D. DAKORA* Botny Deprtment, University of Cpe Town, Privte Bg, Rondeosch 771, South Afric Received 16 July 1998; ccepted 18 Ferury 1999 SUMMARY Applying silicon in the form of metsilicic cid or silicic cid tobrdyrhizoium-infected, hydroponiclly grown cowpe seedlings resulted in significnt (P 5 increse in the numer of nodules, nodule dry mtter, nd nitrogen fixed on per plnt sis. Totl dry mtter of plnts incresed with silicon supply, nd the differences were significnt (P 5 t the higher silicon concentrtions. Cowpe plnts cultured in snd were lso ssessed for their response to silicic cid. Nodule numer nd nodule mss incresed with silicon supply to snd cultured plnts, though nitrogen fixtion ws unltered. Although silicon is not essentil for growth of cowpe, it is importnt for nodule formtion nd nodule functioning in hydroponiclly grown plnts. Consequently, dt collected nd conclusions drwn from erlier glsshouse experiments, which hve excluded silicon from nutrient solutions, my e flwed. Future studies on nodultion nd nitrogen fixtion using legumes in liquid culture must therefore include silicon s nutrient element. Key words: silicon, cowpe, nodultion, N fixtion, nutrient solution. INTRODUCTION Most glsshouse studies involving legumes hve een conducted using vrious nutrient solutions for growth of plnts in liquid nd snd culture (Smith et l., However, these solutions usully lck silicon. Whether the omission of silicon s nutrient ffected the qulity of dt collected, nd or the conclusions drwn, is still open to question, nd recent report suggests tht silicon stimultes growth of plnts (Epstein, With legumes, nodultion nd nitrogen (N fixtion is dependent on n dequte supply of oth mcro- nd micro-nutrients (Munns, 1977; Smith, These nutrients re not only essentil for the symiotic interction ut lso for the host plnt nd its microil prtner (Munns, 1977; Smith, Although silicon is n element tht occurs undntly in soil solution nd in the dry mtter of mny plnts (Epstein, 1994, its role in the symiotic process hs not een ssessed. In non-legume species, such s rice, rley nd eet, silicon hs een shown to e essentil for oth plnt growth nd incresed yields (Okud & Tkhshi, 1965; Deren et l., 1994; Winslow et l., In legumes such s common en nd *Author for correspondence (fx ; e-mil Dkor otzoo.uct.c.z. soyen, silicon is reported to increse totl iomss, root length, nd root mss when supplied in the root medium (Horst & Mrschner, 1978; Miyke & Tkhshi, In legumes which hve the potentil to form nodules this increse in root length nd root mss with dded silicon would imply n increse in the numer of potentil infection sites for rhizoil invsion, which would in turn, led to incresed nodultion nd N fixtion. Unfortuntely, these studies on silicon nutrition of the two nodulting legumes (Horst & Mrschner, 1978; Miyke & Tkhshi, 1985 focus only on plnt growth nd not on its symiotic performnce. The purpose of this study ws to exmine the symiotic response of cowpe to silicon nutrition under glsshouse conditions using oth liquid nd snd culture. MATERIALS AND METHODS The effects of metsilicic cid nd silicic cid on growth nd symiotic performnce of cowpe in liquid culture Seeds of cowpe (Vign unguicult L. Wlp. cv 8586 were sown in freely drining snd, inoculted with pet-sed inoculnt of Brdyrhizoium strin CB756, nd left to germinte in nturlly lit glsshouse during the summer in Cpe Town. At 7

2 464 A. Nelwmondo nd F. D. Dkor d fter plnting, the seedlings were rinsed, nd cultured hydroponiclly in N-free modified Hoglnd nutrient solution (Hewitt, 1966, using 3 5 l pots. The solution ws djusted to contin different concentrtions of silicon, either s metsilicic cid or silicic cid. The concentrtions used were, 4, 8,, nd 8 g l for metsilicic cid nd, 48, 96, 24, 48 nd 96 g l for silicic cid. The nutrient solution ws erted y uling with ir nd chnged fter every 3 d. Four replicte pots were used for ech tretment with four plnts per pot. The plnts were hrvested 36 d fter plnting (DAP. Dt collected included nodule numers, nodule dry weights, nd totl iomss. The shoots, roots nd nodules of plnts were oven dried t 8 C, weighed, finely ground, nd digested for N determintion y the Kjedhl technique. The mount of N fixed ws estimted s the difference etween plnt totl N nd seed N nd specific N -fixing ctivity clculted s mg N fixed per g nodule dry mtter (DM. The effects of exogenous supply of silicon on cowpe growing in snd culture Snd is unwethered silic mteril. Plnts growing in snd culture cn therefore potentilly otin silicon from the snd used s the rooting medium (Brdy, 199. An experiment ws therefore conducted to determine whether, s in the hydroponic system, plnts growing in snd culture require n exogenous supply of silicon for growth. Seeds of cowpe were sown in freely drining snd contined in 3 5 l plstic pots nd inoculted with Brdyrhizoium strin CB756. After germintion, seedlings were thinned to four per pot nd plnts exposed to silicic cid s indicted. Four pots were used per tretment. The plnts were grown under the glsshouse conditions lredy descried nd ech silicon concentrtion ws pplied four times week. Accumulted slts from frequent nutrient ppliction were flushed out once week. The plnts were hrvested t 36 DAP, nd nlysed for growth nd symiotic performnce. Sttisticl nlysis The effects of different silicon concentrtions on growth nd symiotic performnce of cowpe were nlysed sttisticlly y one-wy ANOVA using the STATISTICA pckge. RESULTS Growth nd symiotic response of hydroponiclly cultured cowpe plnts to metsilicic cid (H SiO Supplying metsilicic cid to cowpe significntly (P 5 incresed nodule numer per plnt in ll tretments except the control nd 4 g l (Fig. 1. Nodultion in silicon-treted plnts lso vried Nodules per plnt c c c Concentrtion of silicon (g l 1 Fig. 1. Effects of the concentrtion of silicon on nodultion of 36-d-old cowpe plnts. Plnts grown hydroponiclly nd supplied with H, grown hydroponiclly nd supplied with H, nd grown in snd nd supplied with H. Verticl lines on rs represent SD (n 4. Different letters show significnt differences t P 5 using one-wy ANOVA. significntly with the concentrtion of metsilicic cid in the root medium. Nodule numer per plnt ws significntly (P 5 higher from 8 to gl ut not t 96 g l (Fig. 1. Nodule DM followed similr pttern to tht of nodule numer, with the highest mss found in plnts receiving 8 g l of metsilicic cid (Figs 2, 3. The totl DM of plnts lso incresed with silicon supply in the nutrient solution (Fig. 3, ut this ws significnt only t the higher silicon concentrtions. Consequently, the mount of N fixed per plnt ws significntly (P 5 greter in ll plnts receiving metsilicic cid except in those given 4 g l (Tle 1. However specific N -fixing ctivity in nodules ws the sme in ll tretments except t the highest silicon concentrtion, where it differed significntly (Tle 2. Growth nd symiotic response of hydroponiclly cultured cowpe plnts to silicic cid Plnts grown with silicic cid s the min source of silicon showed similr response to those provided

3 Silicon nd N fixtion in cowpe 465 Nodule DM (g per plnt c c c c Concentrtion of silicon (g l 1 Fig. 2. Effects of the concentrtion of silicon on nodule dry mtter (DM in 36-d-old cowpe plnts. Plnts growth hydroponiclly nd supplied with H, grown hydroponiclly nd supplied with H, nd grown in snd nd supplied with H. Verticl lines on rs represent SD (n 4. Different letters show significnt differences t P 5 using one-wy ANOVA. with metsilicic cid. The numer of nodules per plnt incresed mrkedly (P 5 with incresing silicon supply (Fig. 1. Nodule DM lso showed n increse which ws significnt t higher concentrtions of silicic cid (Fig. 2. As with metsilicic cid, totl DM of plnts lso incresed mrkedly (P 5 in the presence of higher concentrtions of silicic cid (Fig. 3. Specific N -fixing ctivity ws generlly the sme for ll tretments except t 48 g l (Tle 2. Growth nd symiotic response of snd-cultured cowpe plnts to silicic cid (H SiO Applying silicic cid to snd-grown cowpe plnts promoted n increse in nodule numer, especilly t the higher concentrtions (Fig. 1c. Consequently, nodule DM per plnt lso incresed with incresing silicon supply to the roots (Fig. 2c. However, plnt growth in snd culture ws not stimulted y silicic cid (Fig. 3c. The mount of N fixed ws not c Totl DM (g per plnt Concentrtion of silicon (g l 1 Fig. 3. Effects of the concentrtion of silicon on totl plnt dry mtter (DM in 36-d-old cowpe plnts. plnts grown hydroponiclly nd supplied with H, grown hydroponiclly nd supplied with H, nd grown in snd nd supplied with H. Verticl lines on rs represent SD (n 4. Different letters show significnt differences t P 5 using one-wy ANOVA. ffected y ppliction of silicic cid to the roots (Tle 1. DISCUSSION Contrry to the generlized notion tht silicon is not importnt for plnt growth (Epstein, 1994, the totl iomss of nodulted cowpe incresed significntly (P 5 when silicon ws supplied to hydroponiclly grown plnts (Fig. 3,. Nevertheless, the view tht silicon is not needed for plnt growth hs led to the exclusion of this element from most nutrient solutions used in glsshouse experiments (Smith et l., The incresed iomss in silicon-fed cowpe is consistent with the findings of other studies which show tht exogenous supply of silicon cn promote growth, nd even increse yields of certin griculturl crop plnts (Okud & Tkhshi, 1961; Horst & Mrschner, 1978; Miyke & Tkhshi, 1985, 1986; M & Tkhshi, As n extreme cse, vegetles such s cucumer fil to grow in the sence of silicon (Miyke & Tkhshi, 1983, indicting tht this element is n

4 466 A. Nelwmondo nd F. D. Dkor Tle 1. Effect of ppliction of silicon on the mounts of N fixed in 36- d-old nodulted cowpe plnts grown in liquid culture with metsilicic cid or in snd culture with silicic cid Silicon concentrtion (g l N fixed (mg per plnt Snd culture Snd culture c c Vlues followed y dissimilr letters re significntly different t P 5 using one-wy ANOVA. Men SD (n 4. Tle 2. Effect of silicic cid on specific N -fixing ctivity of 36-d-old nodulted cowpe plnts grown hydroponiclly with metsilicic cid or in snd culture with silicic cid Silicon concentrtion (g l mg N fixed per g Nodule DM Snd culture Snd culture Vlues followed y dissimilr letters re significntly different t P 5 using one wy ANOVA. Men SD (n 4. Silicon concentrtion in liquid culture is the sme s in snd culture, ut the ctul mounts used differ ecuse of the molr mss of silicon source. essentil nutrient for these species. Mny uplnd rice ecotypes lso show significnt decreses in yield with indequte silicon supply in cid soils (Deren et l., 1994; Winslow et l., The significnt (P 5 increse in totl DM of silicon-treted symiotic cowpe plnts (Fig. 3, therefore suggests tht while silicon my not e essentil for growth of this species, it is nevertheless importnt for promoting incresed iomss. However whether this increment in growth of cowpe trnsltes into higher pod yields, s oserved in soyen (Miyke & Tkhshi, 1985, ws not tested. The promotion of nodule formtion y silicon in hydroponiclly grown cowpe plnts (Fig. 1, hs close prllel with the effect of phosphorus (P, oron (B, molydenum (Mo nd other nutrient elements on symiotic legumes (Isrel, 1987; Le Roux, 1994; Muofhe, The mounts of N fixed lso incresed with silicon ppliction to plnts (Tle 1 s did tissue concentrtion of silicon (Nelwmondo A, & Dkor FD, unpulished, finding consistent with the effects of vrious other essentil elements on the symiotic process (Munns, 1977; Smith, Although N fixtion ws not mesured in n erlier study (Miyke & Tkhshi, 1985, the N content of leves, stems nd roots of soyen ws consistently higher in the nodulting isoline thn in the non-nodulting isoline when silicon ws provided. This grees with our dt. The stimultory effect of P on the symiotic performnce in legumes hs een ttriuted to mny fctors including incresed production of compounds tht induce nodultion genes (Dkor & Le Roux, Clcium nd mgnesium re lso known to increse the iosynthesis of isoflvonoid nodultion signls in symiotic legumes (Richrdson et l., 1988; Dkor & Phillips, However, it remins to e determined whether the increse in nodule formtion tht follows supply of silicon to cowpe is consequence of greter signl production. The incresed root mss oserved with silicon ppliction to cowpe (dt not shown could doule the numer of infection sites on root hirs, ultimtely leding to enhnced invsion of rhizoium nd incresed

5 Silicon nd N fixtion in cowpe 467 symiotic performnce. Additionlly, n increse in root surfce re would result in more efficient nutrient uptke, which could in turn, improve plnt growth. In the field, nodulting legumes proly enefit in ll of these different wys from the sorption nd ssimiltion of silicon in soil solution. Nodule numer nd nodule DM, though not N fixed, were stimulted y provision of silicic cid to snd-cultured plnts (Figs 1c, 2c, Tle 1. Since silicon is ville in sufficient quntities in soil solution for uptke y plnts (Epstein, 1994, it is unlikely tht studies involving the use of soil culture would require exogenous supplement of silicon. The lck of ny effect of exogenous silicon on growth, specific N -fixing ctivity or N fixtion in sndcultured plnts lso suggests possile relese of silicon from the snd itself. This study is significnt ecuse silicon hs so fr een excluded from ll conventionl nutrient solutions used in glsshouse experiments with plnts. Our findings show tht exclusion of silicon would hve n dverse effect on growth of hydroponiclly cultured plnts, nd consequently my hve ffected the qulity nd perhps interprettion of the dt collected in erlier experiments. Future studies on legume nodultion nd N fixtion in liquid culture must therefore include silicon s nutrient element. Our dt demonstrte tht, though not essentil for cowpe growth, silicon is importnt for the symiotic process in this species However, how nd where silicon exerts its influence on nodultion nd N fixtion is yet to e determined. ACKNOWLEDGEMENTS This work ws supported y funds from the Foundtion of Reserch nd Development, Pretori, nd the University Reserch Committee, University of Cpe Town. REFERENCES Brdy NC The nture nd properties of soils, 1th edn. New York, USA: McMilln Pulishing Compny. Dkor FD, Le Roux RJ Phosphorus nutrition lters root flvonoid content, nitrogen fixtion nd phosphorus prtitioning in cowpe. In: Tihonovich IA, Provorov NA, Romnov VI, Newton WE, eds. Nitrogen fixtion: fundmentls nd pplictions. London, UK: Kluwer Acdemic Pulishers. Dkor FD, Phillips DA Diverse functions of isoflvonoids in legumes trnscend nti-microil definitions of phytolexins. Physiologicl nd Moleculr Plnt Physiology 49: 1. Deren CW, Dtnoff LE, Snyder GH, Mrtin FG Silicon concentrtion, disese response, nd yield components of rice genotypes grown on flooded orgnic histosols. Crop Science 34: Epstein E The nomlly of silicon in plnts. Proceedings of the Ntionl Acdemy of Sciences, USA 91: Hewitt EJ Snd nd wter culture methods used in the study of plnt nutrition, 2nd revised edition. Commonwelth Bureu of Horticulturl nd plnttion Crops, Est Mlling. Technicl Communiction No. 22. Frnhm Royl, UK: Commonwelth Agriculturl Bureu. Horst WJ, Mrschner H The effect of silicon on mngnese tolernce of en plnts (Phseolus vulgris L.. Plnt Soil 5: Isrel DW Investigtion of the role of phosphorus in symiotic dinitrogen fixtion. Plnt Physiology 84: Le Roux RJ Honours thesis. Botny Deprtment, University of Cpe Town, South Afric. M JF, Tkhshi E Interction etween clcium nd silicon in wter-cultured rice plnts. Plnt Soil 148: Miyke Y, Tkhshi E Effect of silicon on the growth of solution-cultured cucumer plnt. Soil Science Plnt Nutrition 29: Miyke Y, Tkhshi E Effect of silicon on the growth of soyen plnts in solution culture. Soil Science Plnt Nutrition 31: Miyke Y, Tkhshi E Effect of silicon on the growth nd fruit production of strwerry plnts in solution culture. Soil Science Plnt Nutrition 32: Munns DN Minerl nutrition nd the legume symioses. In: Hrdy RWF, Gison AH, eds. A Tretise on Dinitrogen Fixtion. Section iv. New York, USA: John Wiley nd Sons, Muofhe ML Effects of oron nutrition nd cotyledon mnipultion on symiotic performnce of Bmr groundnut (Vign suterrneum L.. Honours thesis, Botny Deprtment, University of Cpe Town, South Afric. Okud A, Tkhshi E Studies on the physiologicl role of silicon in crop plnts. I. Discussion on the silicon deficient culture method. Journl of Science nd Soil Mnure 32: Okud A, Tkhshi E The role of silicon. In: The minerl nutrition of the rice plnt, IRRI. Bltimore, MD, USA: Johns Hopkins Press, Richrdson AE, Djordjevic MA, Rolfe BG, Simpson RJ Effects of ph, C nd Al on the exudtion from clover seedlings of compounds tht induce the expression of nodultion genes in Rhizoium trifolii. Plnt Soil 19: Smith FW Minerl Nutrition of Legumes. In: Vincent JM, ed. Nitrogen fixtion in legumes. Sydney, Austrli: Acdemic Press, Smith GS, Johnston CM, Cornforth IS Comprison of nutrient solutions for growth of plnts in snd culture. New Phytologist 94: Winslow MD, Okd K, Corre-Victori F Silicon deficiency nd the dpttion of tropicl rice ecotypes. Plnt Soil 188: