Pea roots affect immobilisation and solubilisation of phosphorus depending on genotype, stage and phosphorous source

Pea oo s a ec immobilisa ion and solubilisa ion o phospho us depending on geno ype, s age and phospho ous sou ce HANNU AHOKAS , EILA HEIKKIL Ä and LEENA RAMSTEDT MTT – Ag i ood Resea ch Finland, BEL, ET-house, Jokioinen, Finland Ahokas, H., Heikkil ä , E. and Rams ed , L. 2011 . Pea oo s a ec immobilisa ion and solubilisa ion o phospho us depending on geno ype, s age and phospho ous sou ce. – He edi as 000 : 1–8. Lund, Sweden. eISSN 1601-5223. Recei ed 16 Feb ua y 2010. Accep ed 5 May 2011. To assess he e i ciency o pea oo s o mobilize a ailable phospho us (P) om P compounds we subjec ed a ious pea geno ypes o a pos - ea men me hod. Axenic seedlings we e aised on P-de i cien semisolid syn he ic medium using con ol blanks wi hou a plan o he wise ea ed in he same way. AlPO 4 , CaHPO 4 , FePO 4 , apa i e and mea -bone-meal (MBM) we e es ed. A geno ype was es ed om 1-day h ough 15-days o g ow h. The e we e di e ences be ween he compounds (p ⬍ 0.001). P was dissol ed om CaHPO 4 wi h appa en maxima a 72-h in e als and o a signi i can ly lesse ex en om MBM. Wi h AlPO 4 , FePO 4 and apa i e, he oo s did no show a dissol ing e ec , bu , on he con a y, signi i can ly immobilised P. In each case a co ela ion wi h an inc ease in acidi y, H ⫹ (p ⬍ 0.001) was obse ed. The co ela ion was nega i e in he AlPO 4 , FePO 4 and apa i e se ies. A CaHPO 4 ea men combined wi h apa i e o MBM signi i can ly dec eased solubili y o P om ha o CaHPO 4 singly. Tes s wi h six addi ional geno ypes showed ha all solubilised P om CaHPO 4 , some o a signi i - can ex en om apa i e, MBM o sligh ly om FePO 4 , bu none om AlPO 4 . The accumula ion o nea ly wa e -insoluble alu- minium and i on phospha es in i eld and i gin soils is pa ly explainable by he immobilisa ion h ough he oo ac ion on P, which we ha e ound also wi h o he plan species. The oo esponses mus also ha e ecophysiological unc ions dis inc om P acquisi ion. Hannu Ahokas, MTT – Ag i ood Resea ch Finland, BEL, ET-house, FI – 31600 Jokioinen, Finland . Email: [email p o ec ed] The esou ces o e ilize phospha es a e declining globally ( VANCE e al. 2003). I is, he e o e, impe a i e o unde s and he P (phospho us) acquisi ion o c ops. O all he nu ien elemen s, soil P is he mos immobile, inaccessible and una ailable one ( HOLFORD 1997). Wha li le na i e P he e is in ge mina ing pea ( Pisum sa i um ) g ains is a mino and sho ly exhaus ed sou ce. The mean P con en o d y pea g ains was ound o be only 0.37 o 0.38% ( SCHERZ and KLOOS 1981; RASTAS e al. 1989). The main P ese oi s in he pea g ains a e nucleic acids and inosi ol phospha es. All o he g ain P is p obably no in he o m suscep ible o be mobilized a ge mina ion. Abou 80% o he inosi ol phospha es, he appa en s o age P in pea, we e deg aded in 8 days a e ge mina ion ( HONKE e al. 1998). Ac i e means o P acquisi ion a e supposed o begin al eady in ge mina ion in associa ion wi h ela i ely young sec ions o oo s. We ( AHOKAS and MANNINEN 2001) ha e ound ha such ac i i ies s a on he 4 h day o ge mina ion in ba ley ( Ho deum ulga e ). Case s udies in Finland ( SAARELA e al. 2003, 2004) show ha insoluble phospha es de i ed om e ilize s seem o ha e accumula ed in soils. Fe ilize s con ain phospha e in he o m o CaHPO 4 . Aluminium phospha e o i on phospha e, o bo h, a e known o accumula e in Finland in pea soils ( VALMARI 1970; NIEMINEN and J ARVA 1996), i gin mine al soils ( KAILA 1963) and i eld soils ( KAILA 1963, 1964; HARTIKAINEN 1989; SAARELA e al. 2003) c ea ing a soil sedimen o P. The mobilisa- ion o his esou ce is o in e es he e. I seems ha he accumula ed sedimen s o P in he i eld soil a e no used in plan p oduc ion. In he oo s o ba ley, aluminium is abso bed by cell walls and oo su aces and p ecipi a e phospha e he e ( CLARKSON 1967), a p ocess which may con ibu e o he insoluble phospha e in pos -ha es soils g own wi h ba ley. This s udy is an ex ension o he p e ious one on ba ley ( AHOKAS e al. 2007). The aim o he s udy was o assess gene ic a ia ion using di e en pu a i e phospho us sou ces wi h pea. Because P a ailabili y in he o m o annually added e ilize ee om pollu ing elemen s is becoming mo e expensi e, i may be p uden o s a b eeding c ops wi h p ope ies which u ilize he soil-bound esou ces o P o bone meal, a ecyclable P sou ce. The mea bone meal p oduc s udied he e was accep ed by EU o be used as a e ilize . I was hough o ha e abou 40% o i s P a ail- able o c ops ( KALLAMA and UUSIHONKO 2006). The apa i e ype s udied he e has been ied as a P sou ce in o ganic cul u e ( SEURI e al. 2001). He edi as 148: 85–92 (2011) © 2011 The Au ho s. This is an Open Access a icle. DOI: 10.1111/j.1601-5223.2011.02177.x 86 H. Ahokas e al. He edi as 148 (2011) MATERIAL AND METHODS Plan s and g owing medium B eeding lines and old cul i a s o he pea we e selec ed wi hou p e ious in o ma ion o hei P up ake p ope ies. The pea line L1703 was ob ained om D . S ig Blix , Weibullshom Plan B eeding Ins i u e, Sweden. I is a selec ion o c . ‘ Vinco ’ ( MURFET 1967). The o igin o he line V84 – 77 was desc ibed p e iously ( AHOKAS 1987); i is in p inciple he geno ype o L200 in BLIXT ’ s (1972) collec ion. The line 93 – 893 is o hyb id o igin ( Pisum ela ius ⫻ Ville) ⫻ (V84 – 77 ⫻ Ville) and o F 8 o la e gene a ion c ossed and b ed by one o us (H. Ahokas). The old Finnish pea en ies, Linie on No d (K – 2402), he land ace lines, Linie on Lande bse (K – 2406), and Sou h-Wes Finnish (K – 3668) and c . ‘ Kelle ä ’ (K – 2403) we e ob ained om he N. I. Va ilo All-Russian Scien- i i c Resea ch Ins i u e o Plan Gene ic Resou ces, S . Pe e sbu g, Russia. The seeds we e p oduced in g een- house. To educe possible gene ic a ia ion wi hin he accessions, single plan o igins we e used. The seeds we e usually picked om ma u e pods s o ed unopened wi h s e ile o ceps, pencil-ma ked, weighed and su ace s e ilized and washed in s e ile wa e se ies as desc ibed ( AHOKAS and MANNINEN 2001; AHOKAS e al. 2007), how- e e , using he acuum ea men only in he Linie on No d se ies, because he ge mina ion in some o he pea lines appea ed o be impai ed by he acuum s ep du ing he s e ilisa ion in 0.01% HgCl 2 . The plan s, single pe a 14-ml polyp opylene ube (Falcon 2059, USA), we e g own o 1 o 15 days on abou 7 ml o medium made semisolid wi h 0.6% aga o abou 42 mg pe ube (Plan Cell Cul u e Reagen s, A – 1296, Sigma, USA) ha ing he ino ganic cons i uen s o Ba ley Medium II ( NORSTOG 1973) bu lacking phospha e he plan s being subjec ed o P s a a ion bu supplied wi h o he mac o and mic o mine als o Ba ley Medium II. The sal s we e o analy i- cal (Me ck, Ge many) o Plan Cell Cul u e Reagen s g ade (Sigma, USA). The masses o bo h he emp y ube be o e he expe imen and he medium we e eco ded. In calcula ions, 1 g o he medium was aken as 1 ml. A leas wo con ol ubes wi hou any plan we e included o each sample se ies o he di e en P compounds o de e - mine he ze o le el. The daily se ies comp ised o 12 o 24 ubes. The na u al pH (in mean 5.74) o each con ol medium (wi hou a plan ) a he sampling day was used as he e e ence o he calcula ions o he acidi i ca ion by he plan . Fo he pH measu emen s wi h pH-Me e CG840 (Scho , Ge many), he plan less blank medium was mixed wi h a s e ile glass od and he elec ode (Scho N 6180) was inse ed midway in he medium in he ube. The pH measu ing appa a us was calib a ed be o e each se ies o measu emen s. The elec ode solu- ion was changed once a mon h. The pH was exp essed as mic o-equi alen l – 1 o H ⫹ . The expe imen s we e done in an ai -condi ioned g eenhouse be ween la e au umn and ea ly summe wi h nigh minima o 9 – 13 ° C and day maxima o 20 – 25 ° C a he plan le el. Addi ional ligh was supplied wi h high-p essu e sodium lamps (Philips SON – T AGRO 400, Belgium) o gua an ee a 16-h day- leng h, being u ned on and o by a pho osenso ou side he g eenhouse du ing he illumina ion pe iod. A di e - en pe iods o he yea , he se ies we e subjec ed o he no he n la i ude o abou 61 ° wi h a ying na u al ligh in ensi ies and spec al dis ibu ions. Tes ing he phospho ous compounds CaHPO 4 ( ⫽ CaP) (BDH 27598, UK), FePO 4 ( ⫽ FeP) (Me ck 103923, Ge many), AlPO 4 ( ⫽ AlP) (Ald ich 34,145 – 2, Ge many) and Siilinj ä i Apa i e (Kemi a G owHow, Finland) we e g ound in a mo a , washed a leas i e imes du ing a day wi h s e ile Milli-Q wa e using 10 olumes o mo e, collec ed by cen i uga ion and d ied a 62 – 65 C o h ee days on Wha man 1 (UK) i l e pape and s o ed exsicca ed. The g ound mea bone meal ( ⫽ MBM) (Honkajoki Oy, Finland) was used as such. The MBM has been subjec ed o au ocla ing a 133 C o 20 min in he p ocess o he ac o y and was s o ed a – 20 ° C du ing he expe imen . The P con en o MBM is indica ed o be 5.5 %. Siilinj ä i Apa i e ep esen s l uo- oapa i e ( NEUVONEN 1960) wi h a epo ed o al P con en o 14% and wi h wa e soluble P o 0% and wi h o iginal pa icle size ac ions o ⬍ 0.06 mm 40%, ⬍ 0.1 mm 60 % and ⬍ 0.3 mm 95 % ( ANONYMOUS 2003). Each compound con aining phospho us was weighed (10 mg) on pa ly cu 1-ml pipe o ips in ad ance used o inse he compound on he medium in he g ow h ube. A e he plan s we e pulled ou , he semisolid medium mixed and he pH measu ed, he abili y o he medium in each ube o dissol e phospho us om CaP, FeP, AlP, apa i e o MBM was de e mined. Two pe sons wo ked oge he wi hou any delays un il he i nal sample was ob ained o he P de e mina ion. A ube wi h he medium was mixed wi h he 10 mg compound wi h a glass od o 30 s, he ubes we e capped, s uck by hand o lowe he medium in he ube and incuba ed in a 25 ° C wa e ba h o 1 h. A e 15 s igo ous o exing, he ubes we e cen i- uged a abou 9500 g in an angle o o (Beckman JA – 20.1, USA) o 10 min a ⫹ 8 ° C se ing. The supe na an was collec ed wi h wide-po e 1-ml ips (Geno-DNA- ip, Finnpipe e, Finland) on a pipe o in wo chilled 1.5-ml Eppendo (Ge many) ubes. The ubes we e cen i uged a abou 15 000 g in a So all FA-MICRO o o (USA) o 5 min a 8 ° C se ing, and he supe na an s pooled usu- ally o make ⬎ 1.5 ml by pou ing o a p e-chilled 2-ml ube. The samples we e s o ed in ice and de e mined wi hin a ew hou s o ozen ( – 20 ° C) o de e mina ion la e . In a He edi as 148 (2011) Pea geno ypes, oo s and phospho us solubilisa ion 87 ep esen a i e o he oldes pea cul i a (No d) eleased in Finland in 1904 ( AHOKAS 2000). Linie on No d was selec ed om i in abou 1910 when e y li le a i i cial e ilize s we e used. Linie on No d also ge mina ed uni o mly unde he expe imen al condi ions. The mean pH le el o he con- ol medium was ound o be 5.74, bu he oo -induced acidi i ca ion exp essed as he inc ease o equi alen s o H ⫹ ml – 1 was s onges a oo b anching in be ween 8- and 10-days samples declining la e unde he es condi ions (Fig. 1). The acidi i ca ion and p ima y oo elonga ion showed a hy hm o 72 h du ing he 9-days pe iod (Fig. 1), a e which also he seconda y oo s s a o play a ole. S a is ically, he i e es ed compounds showed highly signi i can di e ences o e he 15-days pe iods: χ 2 ⫽ 305.9, p ⬍ 0.001. A e a 6-days pe iod all he es ed compounds seem o di e se om he p e ious solubilisa ion alues. CaP, howe e , showed an ea lie esponse, wi h i s i s peak in 3-days, he second in 6-days and he hi d in 9-days samples. A e ha , he ac i i ies o he seconda y oo s may ha e become o e whelming and he condi ions o de elopmen may also be s ess ul. CaP and MBM showed highly signi i can (p ⬍ 0.001) posi i e co ela ion be ween acidi ying o he medium and abili y o solubilise phospho us, while AlP, apa i e and FeP showed highly signi i can (p ⬍ 0.001) nega i e co ela ion (Table 1). Phospho ous compounds in combina ion wi h CaP Fo u he s udies, he 8-days sample age was selec ed o ep esen a s age no much ye a ec ed by he seconda y oo s o by he pu a i e s esses caused by he minu e space o he plan . The e ec o he indi idual phospho- ous compound oge he wi h a 10 mg aliquo o CaP and singly was es ed in a se ies a he 8-day samples. The esul s a e gi en in Table 2. FeP, AlP and CaP, 10 mg each wi h an addi ional 10 mg o CaP did no change he amoun o P solubilised om ha o he single 10 mg dose o CaP, while apa i e signi i can ly a p ⬍ 0.01 and MBM a p ⬍ 0.05 lowe ed he amoun o P solubilised compa ed wi h he single (10 mg) samples o CaP. Geno ypic e ec s The esul s wi h six pea geno ypes a hei e i ciency o make P soluble om he i e es ed compounds we e de e mined wi h 8-days seedlings bo h on single plan basis and co ec ed o he di e ences in single-seed masses (Table 3, 4). The esul s o he unco ec ed and co ec ed alues we e, howe e , cohe en . Wi h AlP all he geno ypes consis en ly p ecipi a ed P compa ed wi h he con ols wi hou a plan , e en hough L1703, 93 – 893 and Kelle ä p ecipi a ed somewha less. Signi i can di - e ences we e ound, in pa icula , i co ec ed o he seed mass. This mos ly mo es L1703 wi h i s la ge seeds joined es , bo h 10 mg aliquo s o AlP, apa i e, CaP, FeP o MBM we e mixed oge he wi h 10 mg o CaP simul a- neously and he sample p ocessed o he wise as hose wi h indi idual phospho ous compounds. Phospha e de e mina ion Phospha e was de e mined in 1.5-ml samples o he supe - na an in 14-ml Falcon 2059 ubes in he me hod o DICK and TABATABAI (1977) in a i nal olume o 10 ml. Bo h he samples and con ol blanks wi hou plan s we e ead wi h a spec opho ome e (HP 8452A, Ge many) a 700 nm agains wa e . The eadings we e co ec ed o olumes o he o iginal samples. In case ha less han 1.5 ml o he supe na an was eco e ed, he olume was co ec ed by calcula ion. Fo a s anda d plo o phospha e, dilu e K 2 HPO 4 solu ion was added o he ube wi h he same aga medium, Ba ley Medium II (see abo e), mixed o 30 s wi h a glass od, incuba ed in a 25 ° C wa e ba h o 1 h, cen i uged as he samples, and hen de e mined and calcula ed on he mass basis as wi h he sample ubes. S a is ics The obse ed alues we e calcula ed on medium bases wi hou seed mass co ec ion o co ec ed o a s anda d g ain mass o 200 mg, which was app oxima ely he mean single pea mass in Linie on No d. To ci cum en any e o s caused by nonno mali y, he nonpa ame ic s a is ics o p obabili y (p), K uskal-Wallis one-way ana- lysis o a iance (H), Spea man ank co ela ion ( s ) and F iedman wo-way analysis o a iance ( χ 2 ) we e used and he signi i cances de e mined acco ding o SIEGEL (1956). SEM means he s anda d e o o he mean, and n, he numbe o samples. RESULTS The alues shown a e di e ences be ween he samples and he con ol blanks ob ained wi h media wi hou a plan (Ba ley Medium II). The alues can a y om posi i e (solubilising) o nega i e (immobilising). In many cases his makes he s anda d e o o he mean (SEM) alues nume ically appa en ly high wi h ega d o he nume al o he means. The alues a e p esen ed on he μ mol phospha e pe ml o he medium o en bo h wi hou and wi h a co ec- ion o he seed mass. Wi hin a geno ype such co ec ion o seed mass has a ma ginal e ec , bu be ween geno ypes he co ec ion is meaning ul especially because L1703 has nea ly double he single-seed mass o he o he s. Time se ies om 1-day ge minan s o 15-days seedlings The esponses o seedlings in he ime se ies we e s udied wi h he pea line Linie on No d. The line was chosen as a 88 H. Ahokas e al. He edi as 148 (2011) Wi h FeP, L1703 and 93 – 893 showed posi i e alues, he o he geno ypes p ecipi a ing P and wi h signi i can di - e ences (0.02 ⬎ p ⬎ 0.01). The geno ypes L1703 and 93 – 893 also showed posi i e alues wi h MBM, he o he geno ypes being nega i e hough nea ly ze o, gi ing no s a is ical signi i cances. owa ds he ze o alue. Highly signi i can di e ences (p ⬍ 0.001) we e obse ed wi h apa i e as he sou ce, whe e L1703 and V84 – 77 showed solubilising p ope ies, he o he geno ypes showing consis en ly p ecipi a ion o P. F om CaP all he geno ypes could make soluble P, he e being no signi i can di e ences be ween he geno ypes. Phospha e solubilisa ion x 24 h –PO4 3– mmol ml–1 –4 –2 0 2 4 6 8 10 MBM Apa i e FeP AlP CaP Mean p ima y oo elonga ion x 24 h cm 0 2 4 6 8 10 12 Mean acidi y depa u e ( he con ol medium, pH 5.7 = 0) x 24 h 0246810121416 0246810121416 0 2 4 6 8 10 12 14 16 mEq l–1 –16 –14 –12 –10 –8 –6 –4 –2 0 2 Figu e 1. Resul s on soluble phospha e quan i ies om di e en phospho ous subs ances in axenic media g own o 1 o 15 days in i o wi h pea, Linie on No d. Values we e co ec ed o he ue g ain weigh o co espond esul s wi h a 200 mg g ain. – Top plo : phospha e solubilisa ion o immobilisa ion (nega i e alues) ela i e o con ol blanks wi hou a plan sampled o each MBM, apa i e, FeP, AlP and CaP. – Cen al plo : he elonga ion o he p ima y oo (mean ⫾ SEM) o all samples. – Bo om plo : acidi i ca ion in mic o-equi alen s pe li e o he media ela i e o con ol blanks wi hou a plan (mean ⫾ SEM) o all samples. The change om 0 o –10 means an acidi i ca ion o one pH uni om pH 5.7. He edi as 148 (2011) Pea geno ypes, oo s and phospho us solubilisa ion 89 mimics he clus e - oo ini ia ion de ec ed in many species unde low P in he soil. Doubling he amoun o CaP om 10 o 20 mg did no a ec he amoun o phospha e solubilised (Table 2) sug- ges ing ha he solubili y is dependen on he quan i y o one o mo e speci i c subs ances sec e ed om he oo s and ha he amoun o he es ed P subs ances was no a e limi ing. The inabili y o some plan species o use P om calcium phospha e was shown o ende hem calci uge ( TYLER 1994; ZOHLEN and TYLER 2004). The bes a ailabil- i y o P om CaP ound in his s udy sugges s ha pea is a he a calcicole han calci uge. The e we e signi i can lowe ing in e e ences by apa i e and MBM wi h he a ailabili y o phospha e om CaP in he joined expe i- men s (Table 2). I such a phenomenon would occu in he soil en i onmen oo, mixing o di e en ypes o P e i- lize s, a leas CaP wi h apa i e migh be was e ul o P in he pea oo en i onmen . In he case o apa i e as e i- lize , including Siilinj ä i Apa i e, he i eld ials wi h annual ba ley in o ganic a ming ( SEURI e al. 2001) co obo a e he i nding ha P o apa i e is una ailable o poo a ailable o ba ley. This was con i med wi h 7 o he ba ley geno ypes wi h an in i o me hod ( AHOKAS e al . 2007). In soil, he es ed compounds would be subjec ed o biological leaching. Then i is, howe e , mo e likely ha he o ganisms making P a ailable p e e en ially ake up he soluble P by hei own cells a he han supply i o o he species. Solubilisa ion o P om FeP, AlP and apa i e by pea showed a nega i e co ela ion wi h he acidi i ca ion o he medium (Table 1). SUBBARAO e al. (1997) ound ha he pH o he oo exuda es o pigeon pea ( Cajanus cajan ) did no o co ela e wi h he P solubilisa ion, bu hey asc ibed he p ope y o he chela ing compound eleased by oo s. E iden ly he sec e ion o o ganic acids ha is a ibu ed o pa icipa e in he mobilisa ion o P in he hizosphe e ha e, o mainly ha e o he unc ions o he DISCUSSION The me hod o P de e mina ion used is insensi i e o o ganic phospha es ( DICK and TABATABAI 1977). I is he e o e sui able o accu a ely de e mina e any ino - ganic P ha has p e iously been in con ac wi h he seed- ling o he es plan and allows also using he o ganic MBM as a es sou ce o P. The li le amoun , abou 42 mg pe sample o aga o plan cell cul u e pu i y also appea ed in he con ol blank ubes and he ubes o he phospha e s anda d and i s possible e ec s a e, he e o e, ully con olled. I is appa en ha only he ela i ely new oo s a e e ec i e a dissol ing phospha e con aining compounds in he soil. Unde he p esen es condi ions he abili y pe sis ed up o 11 o 12 days, a e which new b anches appea and la gely ake o e he ac i i ies o he p ima y oo in pea. Linie on No d s udied o 1 day h ough 15 days, showed a 3-day (72-h) pe iodici y a solubilising phospha e om CaP somewha associa ed wi h he p i- ma y oo elonga ion a e and sec e ion o acidi y in o he oo en i onmen . This las ed a leas o 3 ⫻ 3 days on he p ima y oo s. The pe iodic appea ance o he solubili- sa ion capabili ies a abou 72 h in e als owa ds CaP Table 1. Coe i cien s o Spea man ank co ela ions be ween soluble phospha e and acidi i ca ion wi h he Linie on No d pea om 1 day h ough 15 days wi hou co ec ion o seed weigh di e ences (c . Fig. 1). Subs ance Spea man ank co ela ion, s Signi i cance, p (d ⫽ 13) AlP – 0.767 ⬍ 0.001 Apa i e – 0.889 ⬍ 0.001 CaP 0.932 ⬍ 0.001 FeP – 0.925 ⬍ 0.001 MBM 0.818 ⬍ 0.001 Table 2. In e e ence o phospho ous compounds wi h he solubilisa ion o P om CaP in media a e eigh days g ow h o he Linie on No d pea. Phospha e solubilised μ mol ml – 1 (Mean ⫾ SEM) Phospha e solubilisa ion om CaP-joined compound s 10 mg CaP singly Compound, 10 mg each es ed singly o joined wi h 10 mg CaP Compound singly n ⫽ 7 Compound joined wi h addi ional 10 mg CaP n ⫽ 7 H Signi i cance, p AlP – 1.19 ⫾ 0.16 1.93 ⫾ 0.32 0.494 ⬎ 0.30 Apa i e – 1.07 ⫾ 0.10 0.51 ⫾ 0.11 8.265 ⬍ 0.01 CaP 2.13 ⫾ 0.30 2.08 ⫾ 0.44 0.510 ⬎ 0.30 FeP – 0.80 ⫾ 0.28 1.72 ⫾ 0.53 0.690 ⬎ 0.30 MBM – 0.50 ⫾ 0.34 1.74 ⫾ 0.55 4.592 ⬍ 0.05 H 20.038 15.395 Signi i cance, p ⬍ 0.001 p ⬍ 0.01 90 H. Ahokas e al. He edi as 148 (2011) He e he unde lying cause was ela ed o exuded pen- anedioic acid ( SHEN e al. 2001). In a po expe imen using neu al soils, syn he ic i on phospha e was ound o se e as P sou ce o ye-g ass ( Lolium pe enne ) ( JOHNSTON and RICHARDS 2003). B oad bean ( Vicia aba ) g own in non-s e ile soil was hough o be capable o use P om i on and aluminium phospha e, e en hough calcium phospha e was he mos a ou able ( LI e al. 2007). The obse a ion ha pea lines L1703 and 93-893 made P soluble om FeP, while he o he ones p ecipi a ed P (Table 2, 3, 4) in he sligh ly acidic pH sugges s ha geno ypic di e ences exis in pea in his cha ac e is ic. In li e a u e, FePO 4 is, in a ca eless way, lis ed among he P sou ces a ailable o plan s in soil. Howe e , close s udies ha e shown ha FePO 4 is eally an una ailable P-sou ce o many plan s, including he Poaceae species maize, so ghum and pea l mille ( AE e al. 1990, 1993; A E and SHEN 2002). Geno ypic a ia ion o P up ake om i on phospha e was ound in pigeon pea ( SUBBARAO e al. 1997) and g oundnu ( WISSUWA and AE 1999). A posi i e co ela ion was ound be ween P solubilising o i on phospha e and o aluminium phospha e in pigeon pea in es s, whe e mic obial pa icipa ion was in e e ed plan and he in e ac ion o plan and oo en i onmen . Mala e was ound o be he majo ca boxyla e ound o be exuded om pea in soil ( NURUZZAMAN e al. 2005). Al-induced ci a e sec e ion by ap and basal oo s o bean ( Phaseolus ulga is ) showed geno ypic di e ences ( SHEN e al. 2004). The sec e ed compounds o he Linie on No d pea unde P-de i ciency p obably ha e Al- and Fe-de oxi i ca ion as one o hei unc ions o he adap a- ion o he soil en i onmen . Ou pos - ea men me hod uses axenic condi ions de oid o in e ac ions wi h o ganisms in he hizosphe e, which a e known o in e e e in P acquisi ion o plan s in soil ( RENGEL and MARSCHNER 2005). SUBBARAO e al. (1997) an es s las ing o weeks wi h he pe ennial pigeon pea on non-s e ile soil. They also ound nega i e P solubilisa ion om i on phospha e a some s ages. Phos- pha es may be made a ailable o plan s wi h he aid o a myco hiza ( BUCHER 2007). Unde i on de i ciency, al al a ( Medicago sa i a ) oo exuda es con ain a u an de i a- i e capable o dissol ing e ic phospha e ( NOGUCHI e al. 1994). Elephan g ass ( Pennise um sp.) was ound o bene i om i on and aluminium phospha e in acid soil in a ashion no explainable wi h hizosphe e acidi i ca ion. Table 3. Ac ions o he oo en i onmen o six pea geno ypes on i e phospho ous compounds ela i e o he con ols wi hou co ec ion o he seed weigh di e ences . Phospha e, μ mol ml – 1 Mean ⫾ SEM [n] Pea AlP Apa i e CaP FeP MBM Kelle ä – 0.48 ⫾ 0.23 [7] – 0.59 ⫾ 0.42 [7] 1.90 ⫾ 0.33 [7] – 0.90 ⫾ 0.31 [7] – 0.16 ⫾ 0.37 [8] L1703 – 0.37 ⫾ 0.17 [5] 2.11 ⫾ 1.24 [7] 2.74 ⫾ 0.59 [9] 0.78 ⫾ 0.71 [8] 0.73 ⫾ 0.50 [8] Linie on Lande bse – 1.14 ⫾ 0.19 [6] – 1.08 ⫾ 0.05 [7] 2.20 ⫾ 0.18 [8] – 1.06 ⫾ 0.15 [7] – 0.32 ⫾ 0.13 [7] Sou h – Wes Finnish – 1.34 ⫾ 0.21 [6] – 0.98 ⫾ 0.15 [7] 2.30 ⫾ 0.39 [8] – 1.13 ⫾ 0.22 [7] – 0.25 ⫾ 0.25 [7] V84 – 77 – 1.08 ⫾ 0.21 [7] 0.15 ⫾ 0.51 [7] 1.50 ⫾ 0.29 [7] – 1.11 ⫾ 0.08 [7] – 0.22 ⫾ 0.32 [7] 93 – 893 – 0.30 ⫾ 0.54 [7] – 0.75 ⫾ 0.33 [6] 2.40 ⫾ 0.48 [7] 0.50 ⫾ 0.59 [9] 0.60 ⫾ 0.64 [8] H 9.697 21.758 5.087 14.874 5.870 Signi i cance 0.10 ⬎ p ⬎ 0.05 p ⬍ 0.001 ns 0.02 ⬎ p ⬎ 0.01 ns Table 4. Ac ions o he oo en i onmen o six pea geno ypes on i e phospho ous compounds ela i e o he con ols wi h seed weigh s s anda dized o 200 mg om he esul s in Table 3 . Phospha e, μ mol ml – 1 Mean ⫾ SEM [n] Pea AlP Apa i e CaP FeP MBM Kelle ä – 0.37 ⫾ 0.18 [7] – 0.60 ⫾ 0.35 [7] 1.52 ⫾ 0.22 [7] – 0.87 ⫾ 0.28 [7] – 0.21 ⫾ 0.31 [8] L1703 – 0.17 ⫾ 0.08 [5] 1.00 ⫾ 0.58 [7] 1.39 ⫾ 0.32 [9] 0.38 ⫾ 0.37 [8] 0.39 ⫾ 0.28 [8] Linie on Lande bse – 1.12 ⫾ 0.20 [6] – 1.02 ⫾ 0.06 [7] 2.07 ⫾ 0.17 [8] – 0.97 ⫾ 0.15 [7] – 0.31 ⫾ 0.12 [7] Sou h – Wes Finnish – 1.14 ⫾ 0.17 [6] – 0.86 ⫾ 0.14 [7] 1.89 ⫾ 0.31 [8] – 0.93 ⫾ 0.18 [7] – 0.21 ⫾ 0.20 [7] V84 – 77 – 1.02 ⫾ 0.22 [7] 0.13 ⫾ 0.43 [7] 1.25 ⫾ 0.23 [7] – 1.14 ⫾ 0.16 [7] – 0.20 ⫾ 0.28 [7] 93 – 893 – 0.30 ⫾ 0.45 [7] – 0.68 ⫾ 0.31 [6] 1.86 ⫾ 0.36 [7] 0.44 ⫾ 0.53 [9] 0.55 ⫾ 0.70 [8] H 13.484 1.312 7.586 19.838 5.372 Signi i cance 0.02 ⬎ p ⬎ 0.01 p ⬍ 0.001 ns 0.01 ⬎ p ⬎ 0.001 ns He edi as 148 (2011) Pea geno ypes, oo s and phospho us solubilisa ion 91 acquisi ion obse ed on he p ima y oo s in Line on No d migh bene i a plan in an unp edic able en i onmen and in he neighbou hood o o he geno ypes o he popula ion whe e he plan happened o ge mina e. On he o he hand, geno ypes e i cien a ce ain means o phospho us acqui- si ion may cons i u e a gene ic load in ela ion o some o he e ec s o he en i onmen . A empo al exp ession, a 72-h hy hm obse ed wi h Linie on No d migh mi iga e any ad e se e ec o he p ope y. Many c ops a e g own as mix u es o geno ypes. Mixed geno ypes ha a e able o acqui e P om di e en sou ces migh p o e o be alu- able in condi ions whe e soil P sou ces a e limi ing and mul iple P subs ances a e ound in he soil. Acknowledgemen s – We wan o hank M . Ka i Valkosaa i (Honkajoki Oy, Honkajoki, Finland) o he sample o mea bone meal and M . Jouko Nieminen (Kemphos Oy, he o me Kemi a G owHow, Siilinj ä i, Finland) o he sample o apa i e. The wo k by Ms. Eila Heikkil ä , MSc, was made possible h ough he auspices o he Minis y o Labou , Finland. REFERENCES Ae, N. and Shen, R. F. 2002. Roo cell-wall p ope ies a e p oposed o con ibu e o phospho us (P) mobiliza ion by g oundnu and pigeonpea. – Plan Soil 245: 95 – 103. Ae, N., A iha a, J., Okada, K. e al. 1990. Phospho us up ake by pigeon pea and i s ole in c opping sys ems o he Indian subcon inen . – Science 248: 477 – 480. Ae, N., A iha a, J., Okada, K. e al. 1993. The ole o piscidic acid sec e ed by pigeonpea oo s g own in an Al i sol wi h low-P e ili y. – De . Plan Soil Sci. 50: 279 – 288. Ahokas, H. 1987. A DNA-liposome ans ec ion p oduc in pea wi h pigmen changes. – Ann. Bo . Fenn. 24: 245 – 250. Ahokas, H. 2000. Impac s on ag icul u al de elopmen by Cons an in Boije, a missiona y and he i s plan b eede in Finland. – Helsinki Uni . P ess. Ahokas, H. and Manninen, M.-L. 2001. Polymo phisms o phospha e acquisi ion pa ame e s in ba ley ( Ho deum ulga e ) land aces: sec e ed acid phospha ase and milieu acidi i ca ion o oo s a e ge mina ion in i o . – Biol. Ag ic. Ho icul . 18: 385 – 399. Ahokas, H., Sii onen, P. and Rams ed , L. 2007. Phospho us mobilisa ion and immobilisa ion a y in he oo en i onmen o di e en ba ley geno ypes wi h di e en phospha e sou ces. – Biol. Ag ic. Ho icul . 25: 79 – 90. Anonymous 2003. Siilinj ä en apa ii i. ⬍ www.kemi a – g owhow. com/FIN/ i /P oduc s-/Plan ⫹ Nu i ion/O he ⬎ . Blix , S. 1972. Mu a ion gene ics in Pisum . – Ag i Ho ique Gene . 30: 1 – 293. Cla kson, D. T. 1967. In e ac ions be ween aluminium and phospho us on oo su aces and cell wall ma e ial. – Plan Soil 27: 347 – 356. Dick, W. A. and Taba abai, M. A. 1977. De e mina ion o o hophospha e in aqueous solu ions con aining labile o ganic and ino ganic phospho us compounds. – J. En i on. Quali y 6: 82 – 85. Gaa de , T. 1934. Fos o sy ens binding og igj ø ing i jo den. [Binding and elease o phospho ic acid in soil.] – No disk Jo db ugs o skning 16: 197 – 216. by chlo amphenicol ea men ( SUBBARAO e al. 1997). Ou unpublished esul s show ha ba ley in axenic con- di ions is a species unable o using FeP as a P sou ce, bu e iden ly has geno ypes which a e empo a ily capable o mobilizing P om FeP. The six pea geno ypes did no di e in hei abili y o solubilise phospha e om CaP signi i can ly (Table 3, 4) e en hough AHOKAS e al. (2007) ound di e ences among he ba ley geno ypes. I on phospha e is one o he majo deposi ed o ms o P in pea soils in Finland ( VALMARI 1970; NIEMINEN and JARVA 1996) and in i eld soils ha ha e been e ilized ( HARTIKAINEN 1989) wi h a su plus o P since he 1940s ( SAARELA e al. 2003). The P immobilisa ion by oo s om FeP by mos pea geno ypes (Table 2, 3, 4) and by ba ley ( AHOKAS e al. 2007) ag ees wi h he accumula ion in soils whe e oo s also con ibu e o he p ecipi a ion o FePO 4 . This highligh s he soil binding o P ( KAILA 1965) being ei he a cha ac e is ic o he soil o possibly a long-las ing e ec o he ege a ion in he soil. The ci cums ance ha he plan s (pea, ba ley and some o he s) immobilise phospha es om se e al P subs ances making P mo e wa e -insoluble also unde con- di ions o P s a a ion shows ha he oo ac i i ies espon- sible o he phenomenon ha e unc ions en i ely un ela ed o P acquisi ion. Ou esul s ob ained in simpli i ed expe i- men al condi ions suppo he no ion o complex o ganic acid beha iou in soils ( JONES e al. 2003). Roo - eleased o ganic acids ha e seen o be ela ed o phospha e up ake. In gene al hey a e ound o bene i he P supply ( HOCKING 2001; VANCE e al. 2003). The sig- ni i can and posi i e co ela ion obse ed in dissol ing phospha e om CaP o ino ganic P om MBM and acidi i ca ion o he medium sugges ha his is ue wi h Linie on No d wi h hese phospho ous subs ances, bu he ela ionship is he e e se wi h apa i e, AlP and FeP as he sou ce o P, since he acidi y ended o make P una ailable (Table 1). The cu e o phospha e solubili y in he p esence o an equi alen o i on in he solu ion was shown o be going down o pH 2.2 ( GAARDER 1934). P solubili y om FePO 4 was low o e a wide pH ange in a mix u e o sand and e miculi e ( AE e al. 1990, 1993). In he case o apa i e, including Siilinj ä i Apa i e s udied by us, he i eld ials wi h ba ley in o ganic a ming ( SEURI e al. 2001) co obo a e he una ailabili y o poo a ailabi- li y o apa i e P o c ops. Likewise INDIATI and NERI (2004) ound ha an Alge ian ock phospha e (wi h 12.6% o al P) con ibu ed li le o no soluble phospha e in wo di e en soil ypes. The e ec i eness o apa i e, AlP o FeP as seasonal e ilize s o mos pea geno ypes is ques ionable. The peas es ed by us ep esen old, pa ly wild ( Pisum ela ius ) geno ypes. In pea land ace popula ions a high le el o gene ic a ia ion was p obably main ained also in ela ion o he abili y o he plan s o acqui e P om soil. The al e na ing o hy hmic ac i i ies o he modes o P 92 H. Ahokas e al. 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