Journal of Agriculture and Life Sciences ISSN (Print), (Online) Vol. 4, No. 1, June 2017

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1 Journl of Agriculture nd Life Sciences ISSN (Print), (Online) Vol. 4, No. 1, June 217 Effects of Chemicl Form nd Nitrogen Concentrtion of Fertilizer nd Wter Culture Medium ph on Growth nd Root System Development of Alflf (Medicgo stiv L.) Disuke Hirose Fculty of Environmentl Horticulture Minmikyushu University Tteno-cho Miykonojo-shi, Miyzki, , Jpn Abstrct Effects of differences in nitrogen concentrtion nd wter culture medium ph on growth nd root system development of lflf grown in plots fertilized with mmonium nitrogen or nitrte nitrogen were investigted. Differences in dry weight, totl root length, nd men lterl root length were observed between mmonium nd nitrte plots t nitrogen concentrtions of 2 nd 3 ppm nd ph 6.5. However, no difference ws observed when the ph ws shifted in the cid or lkline direction. Differences in the number of roots between mmonium nd nitrte plots were only observed t nitrogen concentrtion of 2 ppm nd ph 6.5. Moreover, no significnt difference ws observed when the ph ws shifted in the cid or lkline direction. These results indicted tht the high growth-promoting effect of mmonium nitrogen nd nitrte nitrogen on growth nd root system development observed t nitrogen concentrtions of 2 nd 3 ppm nd ph 6.5 re lost when the medium ph is shifted in either the cid or lkline direction. Keywords: Alflf, Growth, Nitrogen Fertilizer, Root System, Wter Culture Medium ph. 1. Introduction In studies to dte, it hd been reported tht ppliction of mmonium nitrogen is superior to nitrte nitrogen in improving erly growth nd the first yer yield nd qulity of lflf seedlings (Hirose et l., 1994; Hirose 1997). It ws lso clrified tht these results were prtly due to differences in root system development (Hirose 23, 25). In ddition, during erly growth of lflf, ppliction of mmonium nitrogen ws superior in improving growth nd root system development t nitrogen concentrtion of 2 ppm nd wter culture medium ph of 6.5, wheres t nitrogen concentrtions higher thn 3 ppm, no significnt difference in growth ws observed. By contrst, ppliction of nitrte nitrogen ws superior in improving root system development (Hirose 26). However, it hs been reported tht growth nd yield of lflfre esily ffected by wter culture medium ph, s both re reduced when cidity of the medium increses (Jo et l.,198, 198b, 1981; Sto et l., 1984; Fgeri et l., 1989; Peters et l., 25) nd root length shortens s ph decreses (Fox et l., 1955; Rechcigl et l., 1986). Hence, the result indicting tht the chemicl form of fertilizer nitrogen tht is superior in improving the growth nd root system development chnges depending on the nitrogen concentrtion of the wter culture medium my not be pplicble to cses when wter culture medium ph is chnged. This reserch ws performed to clrify the influence of differences in wter culture medium ph, in ddition to chemicl form nd concentrtion of fertilizer nitrogen, on growth nd root system development of lflf by djusting wter culture medium ph using utomtic ph djusters nd pplying mmonium nitrogen nd nitrte nitrogen. 2. Mterils nd Methods 2.1. Vriety nd culturing method of lflf This experiment ws conducted in glsshouse using Tchiwkb vriety lflf. On April 12, 21, lflf ws seeded in continers ( cm) filled with expnded vermiculites nd rised with distilled wter lone until April

2 Published by Center for Promoting Ides, USA Copyright The Author(s) The lflf seedlings were subsequently trnsplnted to continers ( cm), 35 individuls per continer, to strt wter culture, which ws conducted in the sme mnner s reported previously(hirose 23, 2). The wter culture medium ws completely replced every five dys Setting test plots Tretments with nitrogen concentrtions of 2, 3, or 4 ppm (ingredient content in ll cses) in wter culture medium ph tht ws djusted to 5.5 ±.1 (hereinfter referred to s 2 ppm ph 5.5 tretment, 3 ppm ph 5.5 tretment, nd 4 ppm ph 5.5 tretment, respectively), 6.5 ±.1 (hereinfter referred to s 2 ppm ph 6.5 tretment, 3 ppm ph 6.5 tretment, nd 4 ppm ph 6.5 tretment, respectively), or 7.5 ±.1 (hereinfter referred to s 2 ppm ph 7.5 tretment, 3 ppm ph 7.5 tretment, nd 4 ppm ph 7.5 tretment, respectively) were prepred by pplying either mmonium sulfte (mmonium plot) or sodium nitrte (nitrte plot) (totl plots = 18). Two continers were used per plot nd the ph of ech plot ws djusted using n utomtic ph djuster Preprtion of utomtic ph djusters Ech utomtic ph djuster ws prepred by connecting ph sensor (IS-GFV-TV: Iwki Co. LtD., Tokyo, Jpn) nd n electromgnetic metering pump, for feeding of 2N hydrochloric cid nd 2N sodium hydroxide (EH/R- AE: Iwki Co. LtD., Tokyo, Jpn), to n utomtic ph controller (PH-7P model: Iwki Co. LtD., Tokyo, Jpn). 2.4.Timing of collection of mterils nd their seprtion into orgns Mterils were collected from six individuls per plot (three individuls per continer) every ten dys between dys 3 nd 6 fter sowing (totl collections = 4). The collected individuls were seprted into leves, stems, nd roots. Immeditely fter collection, the leves nd stems were ir-dried for two dys t 8 C. The roots were initilly stored in FAA solution (ethnol: distilled wter: cetic cid: formlin = 9:9:1:1 [v/v]), nd then tken out s required to mesure tproot nd lterl root lengths nd the number of lterl roots. After mesurement, the mterils were seprted into tproots nd lterl roots nd ir-dried in the sme mnner s for leves nd stems. After drying, dry weight of ech orgn ws mesured Mesuring methods of tproot nd lterl root lengths nd the number of lterl roots Tproot nd lterl root lengths nd the number of lterl roots were mesured using n imge nlysis method (Kimur et l., 21; Kimurnd Ymski, 23) with 3 dpi resolution Sttisticl nlysis Anlysis of vrince ws pplied to dry weight, totl root length, nd number of lterl roots to clrify the influence of chemicl form nd concentrtion of fertilizer nitrogen nd wter culture medium ph on these mesurements in more detil. 3. Results Figure 1 shows chnges in dry weight of ech orgn in ech plot during the experimentl period. At nitrogen concentrtion of 2 ppm, mmonium nd nitrte plots exhibited no significnt difference between ph 5.5 nd 7.5 tretments. On the other hnd, fter ph 6.5 tretment, the dry weights were significntly hevier in the mmonium plots on dy 5 fter sowing nd therefter, nd mmonium plots exhibited pproximtely 1.2 times higher vlues thn nitrte plots on dy 6. In 3 ppm ph 5.5 nd 7.5 tretment plots, no significnt difference ws observed between mmonium nd nitrte plots. In ph 6.5 tretment plots, dry weights of the nitrte plots were higher thn those of the mmonium plots on nd fter dy 4 nd exhibited pproximtely 1.9 times higher vlues on dy 6. At 4 ppm, mmonium nd nitrte plots exhibited no significnt differences for ll tretments. 25

3 Journl of Agriculture nd Life Sciences ISSN (Print), (Online) Vol. 4, No. 1, June ppm ph ppm ph ppm ph Dry weight (mg plnt 1 ) A B A B A B A B A B A B A B A B A B A B A B A B 2 ppm ph ppm ph ppm ph 6.5 b b b b b A B A B A B A B A B A B A B A B A B A B A B A B 5 2 ppm ph ppm ph ppm ph A B A B A B A B A B A B A B A B A B A B A B A B Dys fter sowing A : NO 3 B : NH 4 Tproot Lterl root Stem Lef Figure 1: Effects of chemicl form nd concentrtion of nitrogen fertilizer nd wter culture medium ph on dry weights of orgns of lflf seedlings.totl weights (full column heights) with different letters on the sme smpling dy in the sme nitrogen concentrtion nd wter culture ph indicte significnt difference t 5% level. Figure 2 shows chnges in tproot lengths in ech plot. In 2 ppm ph 5.5 tretment plots, on dy 6 fter sowing, lengths were significntly higher in mmonium plots thn in nitrte plots. On the other hnd, neither mmonium nor nitrte plots exhibited significnt differences under other tretment conditions. 26

4 Published by Center for Promoting Ides, USA Copyright The Author(s) ppm ph ppm ph ppm ph Length of tproot (cm plnt -1 ) ppm ph ppm ph ppm ph ppm ph ppm ph ppm ph Dys fter sowing NO3 3 NH4 4 4 ppm Figure 2: Effects of chemicl form nd concentrtion of nitrogen fertilizer nd wter culture medium ph on the length of tproot of lflf seedlings. Verticl br indictes ±SE. on the sme smpling dy in the sme nitrogen concentrtion nd wter culture ph indicte significnt difference t 5% level. Figure 3 shows chnges in totl root length in ech plot. In 2 ppm ph 5.5 nd 7.5 tretment plots, nitrte nd mmonium plots exhibited no significnt differences. In ph 6.5 tretment plots, the totl root length in mmonium plots ws higher thn tht in nitrte plots on dy 5 fter sowing nd therefter, nd pproximtely 1.3 times higher on dy 6. At 3 ppm, mmonium nd nitrte plots exhibited no significnt differences in ph 5.5 nd 7.5 tretments, but the vlue becme pproximtely 1.3 times higher in nitrte plots with ph 6.5 tretment on dy 6 fter sowing. No significnt differences were observed between mmonium nd nitrte plots in ll ph tretments t 4 ppm. 27

5 Journl of Agriculture nd Life Sciences ISSN (Print), (Online) Vol. 4, No. 1, June ppm ph ppm ph ppm ph Totl length of roots (m plnt -1 ) ppm ph ppm ph ppm ph ppm ph ppm ph ppm ph Dys fter sowing NO3 3 NH4 4 4 ppm Figure 3: Effects of chemicl form nd concentrtion of nitrogen fertilizer nd wter culture medium ph on the totl length of roots of lflf seedlings. Verticl br indictes ±SE. nd on the sme smpling dy in the sme nitrogen concentrtion nd wter culture ph indicte significnt difference t 5 nd 1% levels, respectively. Figure 4 shows chnges in the number of lterl roots in ech plot. Ammonium nd nitrte plots exhibited no significnt differences under 2 ppm ph 5.5 nd 7.5 tretments. In ph 6.5 tretment plots, the number of lterl roots in mmonium plots ws pproximtely 1.3 times greter thn tht in nitrte plots on dy 6 fter sowing. No significnt differences were observed between mmonium nd nitrte plots in ll ph tretments t 3 nd 4 ppm. Tble 1 shows chnges in specific root length of tproot (tproot length/tproot weight). Both nitrte nd mmonium plots exhibited the highest vlues on dy 3 fter sowing in ll tretments, nd the vlues subsequently tended to decrese s the plnts grew. As per the results of dy 3 fter sowing, nitrte plots exhibited higher vlues thn mmonium plots in 2 ppm ph 7.5 tretments, wheres both plots exhibited no significnt differences in ph 5.5 nd 6.5 tretments. In 3 ppm ph 5.5 nd 6.5 tretment plots, nitrte plots exhibited higher vlues thn mmonium plots; however, no significnt differences were observed between the two plots in ph 7.5 tretment. 28

6 Published by Center for Promoting Ides, USA Copyright The Author(s) At 4 ppm, mmonium plots nd nitrte plots exhibited higher vlues in ph 5.5 tretment nd ph 6.5 tretment, respectively, but the two plots exhibited no significnt differences in ph 7.5 tretment. 1 2 ppm ph ppm ph ppm ph Number of lterl roots (number plnt -1 ) ppm ph ppm ph ppm ph ppm ph ppm ph ppm ph Dys fter sowing NO3 3 NH4 4 4 ppm Figure 4 : Effects of chemicl form nd concentrtion of nitrogen fertilizer nd wter culture medium ph on number of lterl roots of lflf seedlings. Verticl br indictes ±SE. on the sme smpling dy in the sme nitrogen concentrtion nd wter culture ph indicte significnt difference t 1% level. 29

7 Journl of Agriculture nd Life Sciences ISSN (Print), (Online) Vol. 4, No. 1, June 217 Tble 1: Effects of chemicl form nd cocentrtion of nitrogen fertilizer nd wter culture ph on specific root length of tproot of lflf seedlings. Dys fter sowing 2ppm 3ppm 4ppm NO 3 NH 4 NO 3 NH 4 NO 3 NH 4 (m g -1 ) ph b b b ph b b ph b b Mens with different superscripts (-b) within the sme dy, nitrogen concentrtion nd wter culture ph re significntly different t 5% level. Tble 2 shows the men length of lterl roots. As per the results on dy 6 fter sowing, mmonium nd nitrte plots exhibited no significnt differences in ph 5.5 nd 7.5 tretments t nitrogen concentrtions of 2 nd 3 ppm. However, in the ph 6.5 tretment plots, the length in mmonium plots ws higher t 2 ppm, wheres conversely t 3 ppm, the length in nitrte plots ws higher. Ammonium nd nitrte plots exhibited no significnt differences in ll tretments t 4 ppm. Tble 2: Effects of chemicl form nd cocentrtion of nitrogen fertilizer nd wter culture ph on men length of lterl root of lflf seedlings. Dys fter sowing 2ppm 3ppm 4ppm NO 3 NH 4 NO 3 NH 4 NO 3 NH 4 (cm root -1 ) ph b ph b b b b b ph Mens with different superscripts (-b) within the sme dy, nitrogen concentrtion nd wter culture ph re significntly different t 5% level. 3

8 Published by Center for Promoting Ides, USA Copyright The Author(s) Tble 3 shows the results of nlysis of vrince on dry weight, totl root length, nd number of lterl roots. The chemicl form of nitrogen gretly ffected the dry weight only on dy 6. Nitrogen concentrtion nd wter culture medium ph significntly influenced dry weight on dy 3 fter sowing nd therefter. In ll cses, chemicl form of nitrogen nd nitrogen concentrtion of wter culture medium ffected dry weight on dy 5 fter sowing; nitrogen concentrtion nd wter culture medium ph ffected dry weight on dy 3 fter sowing nd therefter; nd chemicl form of nitrogen, nitrogen concentrtion, nd wter culture medium ph ffected dry weight on dys 3 nd 5 fter sowing, in highly interctive mnner. Tble 3: Anlysis of vrince(anova) of dry weight, totl length of roots nd number of lterl roots of lflf seedlings. Dys fter sowing Dry weight nitrogen concentrtion (A) wter culture ph (B) nitrogen form (C) A B B C ns ns A C ns ns Totl length of roots nitrogen concentrtion (A) wter culture ph (B) ns nitrogen form (C) ns ns A B ns B C ns ns ns A C ns ns ns Number of lterl roots nitrogen concentrtion (A) wter culture ph (B) nitrogen form (C) A B B C ns ns ns A C ns ns ns, nd indicte no significnt, significnt difference t 5% level nd 1% level, respectively. Chemicl form of nitrogen nd nitrogen concentrtion of the wter culture medium gretly influenced totl root length on dy 6 fter sowing nd on dy 3 fter sowing, respectively. On the other hnd, wter culture medium ph exhibited gret influence t every time point except on dy 4 fter sowing. In ddition, chemicl form of nitrogen nd nitrogen concentrtion of the wter culture medium ffected totl root length on dy 5 fter sowing nd therefter in n interctive mnner. Chemicl form of nitrogen nd wter culture medium ph exhibited the interction only on dy 6 fter sowing. Nitrogen concentrtion nd wter culture medium ph exhibited high interction on dy 4 fter sowing nd therefter. Furthermore, chemicl form of nitrogen, nitrogen concentrtion, nd wter culture medium ph influenced totl root length in n interctive mnner only on dy 6 fter sowing. Chemicl form of nitrogen exhibited no significnt influence on the number of lterl roots t ll the significnt interctions in ffecting time points. Nitrogen concentrtion of wter culture medium gretly influenced lterl root number on dy 3 fter sowing nd therefter. Wter culture medium ph gretly influenced lterl root number on dy 4 fter sowing nd therefter. 31

9 Journl of Agriculture nd Life Sciences ISSN (Print), (Online) Vol. 4, No. 1, June 217 In ddition, chemicl form of nitrogen nd nitrogen concentrtion of wter culture medium, nd chemicl form of nitrogen nd wter culture medium ph exhibited no significnt interctions in ffecting the number of lterl roots t ll the time points. On the other hnd, nitrogen concentrtion nd wter culture medium ph begn to show the interction on dy 3 fter sowing nd therefter, nd high influence ws observed especilly on dy 5 fter sowing nd therefter. Furthermore, chemicl form of nitrogen, nitrogen concentrtion, nd wter culture medium ph intercted t high level only on dy 6 fter sowing. No dherences of root nodules were observed in ny plots. 4. Discussion It hs been previously reported tht wter culture medium ph between 6. nd 6.5 is most pproprite for growth of lflf(hrd 1979). In the present experiment s well, dry weights were highest in ph 6.5 tretment t ll nitrogen concentrtions in both mmonium nd nitrte plots (Figure 1), indicting the sme trend. In ddition, s per the results of ph 6.5 tretment in mmonium nd nitrte plots, the weight ws highest t 2 ppm in mmonium plots nd 3 ppm in nitrte plots (Figure 1). Thus, nitrogen concentrtion most pproprite for growth differed depending on the chemicl form of the fertilizer nitrogen, both in the present experiment nd previous report (Hirose 26). In ddition, the weight incresed in mmonium nd nitrte plots in 2 ppm ph 6.5 nd 3 ppm ph 6.5 tretments, respectively (Figure 1). This finding indictes tht the chemicl form of fertilizer nitrogen pproprite for growth differs depending on nitrogen concentrtion of the wter culture medium. Totl root lengths were highest in 2 ppm ph 6.5 nd 3 ppm ph 6.5 tretments in mmonium nd nitrte plots, respectively (Figure 3). In ddition, s indicted by the totl root length on dy 6 fter sowing, the length decresed when the wter culture medium ph ws shifted in either the cid or lkline direction in both nitrte nd mmonium plots t ll nitrogen concentrtions, nd differences in length between the two plots disppered t 2 nd 3 ppm (Figure 3). Bsed on these results, we speculted tht shifting ph in either the cid or lkline direction inhibited extension of totl root length t 2 ppm nd 3 ppm, resulting in no observtion of significnt differences in length between the chemicl forms of nitrogen. As indicted by the results of the number of lterl roots on dy 6 fter sowing t nitrogen concentrtion of 2 ppm, the number of roots decresed in both nitrte nd mmonium plots when the ph of the wter culture medium ws shifted in either the cid or lkline direction from 6.5, with higher rte of decrese in the mmonium plots (Figure 4). Therefore, we concluded tht lck of significnt difference in the totl root length between mmonium nd nitrte plots fter ph chnge ws prtly due to decrese in the number of lterl roots. On the other hnd, no significnt difference ws observed in the number of roots between 3 ppm ph 5.5 nd 6.5 tretments in both mmonium nd nitrte plots (Figure 4). However, men lengths of lterl roots in 3ppm ph 5.5 tretment were reduced by pproximtely 25% nd 56% in mmonium nd nitrte plots, respectively, compred with the vlues in the 3ppm ph 6.5 tretment (Tble 3). Therefore, we concluded tht lck of significnt difference in totl root length between mmonium nd nitrte plots in ph 5.5 tretment ws prtly due to significntly higher inhibition of extension of individul lterl roots in nitrte plots compred with tht in mmonium plots. In ddition, the number of roots decresed by pproximtely 6% nd 15% in mmonium nd nitrte plots, respectively, in the 3ppm ph 7.5 tretment compred with the 3ppm ph 6.5 tretment (Figure 4). Bsed on this result, we concluded tht lck of significnt difference in totl root length between nitrte nd mmonium plots in the 3ppm ph 7.5 tretment ws prtly due to higher reduction of the number of lterl roots in nitrte plots compred with mmonium plots. In 2 ppm ph 6.5 tretment plots, totl root length ws higher in mmonium plots thn in nitrte plots (Figure 3). In ddition, men length nd number of lterl roots were lso higher in mmonium plots (Figure 4 nd Tble 2); however, tproot lengths exhibited no significnt difference (Figure 2). Thus, we concluded tht higher totl root lengths in mmonium plots compred with nitrte plots in the 2ppm ph 6.5 tretment were due to differences in lengths of individul lterl roots nd number of lterl roots, s reported previously (Hirose 23). On the other hnd, in 3 ppm ph 6.5 tretment plots, totl root length ws higher in nitrte plots compred with tht in mmonium plots (Figure 3). The men length of lterl roots ws lso higher in nitrte plots, but the number of lterl roots nd tproot length exhibited no significnt difference (Figure. 2 nd 3, Tble 2). Therefore, we concluded tht the greter totl root lengths in nitrte plots compred with tht in mmonium plots in the 3ppm ph 6.5 tretment occurred becuse of the difference in length of individul lterl roots. 32

10 Published by Center for Promoting Ides, USA Copyright The Author(s) As shown by the results of nlysis of vrince, nitrogen concentrtion nd wter culture medium ph gretly influenced dry weights on dy 3 fter sowing nd therefter, wheres chemicl form of nitrogen exhibited less influence thn nitrogen concentrtion nd wter culture medium ph until dy 5 fter sowing (Tble 3). In ddition, nitrogen concentrtion nd wter culture medium ph ffected dry weight in highly interctive mnner on dy 3 fter sowing nd therefter (Tble 3). Bsed on these results, we concluded tht ppliction mounts of nitrogen fertilizer nd soil ph re more importnt thn the type of nitrogen fertilizer used in grsslnd estblishment of lflf. Ammonium sulfte nd sodium nitrte used in the present study s nitrogen sources contined SO4 nd N ions s coexisting ions, respectively, nd the concentrtion of these ions differed mong the plots. However, these coexisting ions exhibited no significnt growth inhibition in ny of the plots. Therefore, we concluded tht the influence of these co-existing ions on the results of our experiments ws negligible. Becuse lflf is leguminous crop, differences in dherence of root nodule bcterind their nitrogen fixtion re considered to significntly influence the yield nd qulity in ctul cultivtion. Becuse no dherences of root nodule bcteri were observed in ny plots in the present experiments, it is still uncler how the chemicl form nd concentrtion of fertilizer nitrogen nd wter culture medium ph influence root nodule bcteri. However, dherence nd nitrogen fixing ctivity of root nodule bcterire ffected by soil ph (Jo et l., 198) nd nitrte nitrogen content in soil (Kwhr 1986). Therefore, results different from those reported here my be obtined depending on differences in dherence nd nitrogen fixing ctivity of root nodule bcteri, which will be subject for future study. As mentioned bove, growth nd totl root length of lflf were not significntly different between mmonium nd nitrte plots when wter culture medium ph ws shifted in either the cid or lkline direction from 6.5 t nitrogen concentrtions of 2 nd 3ppm in the wter culture medium. The number of roots ws not significntly different between mmonium nd nitrte plots except in the 2ppm ph 6.5 tretment. In generl, wter culture medium ph shifts in the cid or lkline direction when plnts bsorb mmonium nitrogen nd nitrte nitrogen, respectively, nd differences in sensitivity to this wter culture medium ph shift prtly determines which ppliction, mmonium or nitrte nitrogen, is more pproprite for plnt growth (Ohym 1998). In ddition, the bsorption rte of nitric cid into plnts is ccelerted t low ph, wheres tht of mmonium is decelerted (Ohym 1998). Tking these results together, we conclude tht differences in root system development cused by wter culture medium ph shift my be prtly due to differences in bsorption chrcteristics for the two forms of nitrogen. It will be necessry to investigte these points s well s the influence on root nodule bcteri in the future to clrify the reltionship between chemicl form of fertilizer nitrogen nd wter culture medium ph in more detil. 5. Conclusion In the present study, we imed to investigte the effect of culture medium ph nd nitrogen concentrtion on the growth of lflf. For the sme, we nlyzed the development of its root system fter tretment with mmonium nitrogen or nitrte-nitrogen using imge nlysis method for mesuring root length nd the number of lterl roots. In terms of dry weight, totl root length, nd length per individul lterl root, difference between individuls treted with mmonium nitrogen nd those treted with nitrte-nitrogen ws observed t ph 6.5 for 2ppm nd 3ppm nitrogen concentrtions. In terms of the number of lterl roots, the difference between individuls treted with both mmonium- nd nitrte-nitrogen ws observed t ph 6.5 for 2ppm nitrogen concentrtion. These results indicted tht the high growth-promoting effect of mmonium nitrogen nd nitrte nitrogen on growth nd root system development observed t nitrogen concentrtions of 2 nd 3 ppm nd ph 6.5 re lost when the medium ph is shifted in either the cid or lkline direction. Furthermore, djustments in ppliction mounts of nitrogen fertilizer nd soil ph re more importnt thn the type of nitrogen fertilizer used in grsslnd estblishment of lflf. 33

11 Journl of Agriculture nd Life Sciences ISSN (Print), (Online) Vol. 4, No. 1, June 217 References Fgeri, N. K., Bligr, V. C., Wright, R. J. (1989). Growth nd nutrient concentrtions of lflfnd common ben s influenced by soil cidity. Plnt nd Soil, 119, Fox, R. L., Lipps, R. C. (1955). Influence of soil profile chrcteristics upon the distribution of roots nd nodules of lflfnd sweet clover. Agronomy Journl, 47, Hrd, I. (1979). Soil nd fertilizer. In Cultivtion nd use t lucerne. Reserchnd development center for diry frming (Ed.), pp. 94. Tokyo: Meibunshobo. Co. Ltd. Hirose, D., Ttsumi, J., Tsugw, H., Nishikw, K. (1994). Plnt growth, uptke nd prtitioning of nitrogen in lflf seedlings s ffected by the form of 15 N-lbelled combined nitrogen. Jpnese Journl of Crop Science, 63(1), Hirose, D. (1997). Effects of mmonium nd nitrte nitrogen on the yield nd qulity in lflf (Medicgo stiv L.) seedlings. Jpnese Journl of Grsslnd Science, 43(1), Hirose, D. (2). The effects of mmonium nitrogen nd nitrte nitrogen on lflf (Medicgo stiv L.) erly growth nd nitrogen bsorption. Jpnese Journl of Grsslnd Science, 45(4), Hirose, D. (23). Effects of different chemicl combintion forms of nitrogen fertilizer on root system development of lflf (Medicgo stiv L.) seedlings. Jpnese Journl of Grsslnd Science,49(5), Hirose, D. (25) Effects of mmonium nitrogen nd nitrte nitrogen on the lterl root development in lflf seedlings grown wter culture. Jpnese Journl of Grsslnd Science, 51(2), Hirose, D. (26). Effects of chemicl form nd concentrtion of nitrogen fertilizer on the root system development of lflf (Medicgo stiv L.) seedlings. Jpnese Journl of Grsslnd Science, 52(2), Jo, J., Yoshid, S., Kym, R. (198).Growth nd nitrogen fixtion of some leguminous forges grown under the cidic soil conditions. Jpnese Journl of Grsslnd Science, 25(4), Jo, J., Yoshid, S., Kym, R. (198b).Acidity tolernce nd symbiotic nitrogen fixtion cpcity of some vrieties of lflf. Jpnese Journl of Grsslnd Science, 26(2), Jo, J., Yoshid, S., Kym, R. (1981). Influence of soil cidity nd pplied nitrogen on the growth nd chemicl compositions of ldino clover nd lflf. Jpnese Journl of Grsslnd Science, 27(1), Kimur, K., Kikuchi S., Ymski, S. (21) Root length nd dimeter mesurement using NIH imge: ppliction of the line-intercept principle for dimeter estimtion. Plnt nd Soil, 234, Kimur, K., Ymski, S. (23). Accurte root length nd dimeter mesurement using NIH imge: use of Pythgoren distnce for dimeter estimtion. Plnt nd Soil, 254, Kuwhr, M. (1986). The high yield condition nd nitrogen metbolism in soyben. Agriculture nd Horticulture, 61(4), Ohym, T. (1998). Physiologicl effects nd function t roots. In Encyclopedi of root, Editoril Committee of Encyclopedi of root (Ed.), pp Tokyo: Askur publishing Co. Ltd. Peters, J. B., Kelling, K. A., SpethP. E., Offer, S.M. (25). Alflf yield nd nutrient uptke s ffected by ph nd pplied K. Communictions in Soil Science nd Plnt Anlysis, 36, Rechcigl, J. E., Reneu, Jr.R. B., Wolf, D. D., Kroontje,W., Vn Scoyoc, S. W.(1986). Alflf seedling growth in nutrient solutions s influenced by luminum, clcium nd ph. Communictions in Soil Science nd Plnt Anlysis, 17(1), Sto, T., Ski, H., Yus, K.(1984). Study on the orchrdgrss-lflf mixture. Ⅲ. Effects of dolomite nd nitrogen fertilizer ppliction on yield, botnicl composition nd nutrient uptke. Jpnese Journl of Grsslnd Science, 29(4),