Diversity of Arbuscular Mycorrhizal Fungus Populations in Heavy-Metal-Contaminated Soils

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1999, p Vol. 65, No /99/$ Copyright 1999, Americn Society for Microbiology. All Rights Reserved. Diversity of Arbusculr Mycorrhizl Fungus Popultions in Hevy-Metl-Contminted Soils C. DEL VAL,* J. M. BAREA, AND C. AZCÓN-AGUILAR Deprtmento de Microbiologí del Suelo y Sistems Simbióticos, Estción Experimentl del Zidín, CSIC, Grnd, Spin Received 17 August 1998/Accepted 22 November 1998 High concentrtions of hevy metls hve been shown to dversely ffect the size, diversity, nd ctivity of microbil popultions in soil. The im of this work ws to determine how the diversity of rbusculr mycorrhizl (AM) fungi is ffected by the ddition of sewge-mended sludge contining hevy metls in long-term experiment. Due to the reduced number of indigenous AM fungl (AMF) propgules in the experimentl soils, severl host plnts with different life cycles were used to multiply indigenous fungi. Six AMF ecotypes were found in the experimentl soils, showing consistent differences with regrd to their tolernce to the presence of hevy metls. AMF ecotypes rnged from very sensitive to the presence of metls to reltively tolernt to high rtes of hevy metls in soil. Totl AMF spore numbers decresed with incresing mounts of hevy metls in the soil. However, species richness nd diversity s mesured by the Shnnon-Wiener index incresed in soils receiving intermedite rtes of sludge contmintion but decresed in soils receiving the highest rte of hevy-metl-contminted sludge. Reltive densities of most AMF species were lso significntly influenced by soil tretments. Host plnt species exerted selective influence on AMF popultion size nd diversity. We conclude bsed on the results of this study tht size nd diversity of AMF popultions were modified in metl-polluted soils, even in those with metl concentrtions tht were below the upper limits ccepted by the Europen Union for griculturl soils. In recent yers severl studies hve shown the hrmful effects of metls on microbil diversity nd ctivity in soil (8, 10, 28). The ccumultion of metls in soils t high concentrtions cn be due to nthropogenic ctivities such s the ppliction of sewge sludge. This prctice hs been widely used for nutrient recycling nd is ccepted for wste disposl in griculturl soils (32). However, the ddition of sludge considerbly increses the mount of hevy metls in soil, cusing chnges in soil properties which could be toxic for soil microorgnisms (10). The primry chemicl chnge in soil is cidifiction, which increses the vilbility of metl in the soil solution to toxic levels which cn persist for extremely long periods of time. In spite of this, rtes of 50 to 100 kg of dry mtter per hectre per yer re currently pplied to griculturl soils. Thus, the contribution of sewge sludge to the overll input of hevy metls in soils is considerble. In this context, there is incresing concern bout the possible side effects on microbil popultions, especilly fter long-term sludge pplictions to cidic soils. Soil microorgnisms re known to ply key role in the mobiliztion nd immobiliztion of metl ctions, thereby chnging their vilbility to plnts (6). Arbusculr mycorrhizl fungi (AMF) re soil microorgnisms tht estblish mutul symbioses with the mjority of higher plnts, providing direct physicl link between soil nd plnt roots (3). AMF occur in lmost ll hbitts nd climtes (4), including in disturbed soils such s those derived from mine ctivities (9), but soil degrdtion usully produces chnges in the diversity nd bundnce of AMF popultions (21, 23, 27). Mycorrhizl fungl popultions re criticl during nd fter soil disturbnce becuse of their role in the estblishment nd survivl of plnts * Corresponding uthor. Miling ddress: Depto. Microbiologí del Suelo y Sistems Simbióticos, Estción Experimentl del Zidín, CSIC, Profesor Albred 1, Grnd, Spin. Phone: Fx: E-mil: corlvm@eez.csic.es. (18, 30). Thus, chnges in the diversity of their popultion produced by the ppliction of high mounts of metl re expected to interfere with the possible beneficil effects of this symbiotic ssocition, since reestblishment of AMF popultions is slow (12). However, only few studies hve been crried out involving interctions between AMF nd metls s source of soil disturbnce. Most of the results lredy obtined derive from lbortory nd pot experiments, with metl slts used s the source of hevy metls, which re not very representtive of nturl field conditions, under which metls usully ccumulte in less-vilble chemicl form. Hevy metls cn dely, reduce, nd even completely eliminte AM coloniztion nd AMF spore germintion in the field (14), nd negtive correltion between Zn concentrtions nd AM coloniztion hs been reported in soil treted with urbnindustril sludge (7). In other studies, however, the ddition of metl-contining sludge did not significntly ffect AM development under field conditions (2), probbly becuse different AMF ecotypes cn exhibit different degrees of metl tolernce (26). Thus, reltively high rte of mycorrhizl coloniztion cn be found in plnts growing in very polluted soils (31). A higher tolernce to Cu, Zn, Cd, nd Pb of indigenous fungi from sludge-polluted sites in comprison to those of reference isoltes from unpolluted soils hs been described previously (13, 15). To our knowledge, no studies hve been reported on the long-term effects of incresing concentrtions of sewge sludge on the diversity of mycorrhizl propgules or on the influence of the host plnt on AM fungl diversity in hevymetl-polluted soils. Our im in this study ws to determine how AM fungl diversity is ffected by the ddition of sewge-mended sludge in long-term experiment compred with tht of the pproprite noncontminted control soils. Since the host plnt cn ffect the structure nd composition of the AMF popultion, four different plnts with different life cycles nd growth strtegies were used. 718

2 VOL. 65, 1999 AMF POPULATIONS IN HEAVY-METAL-CONTAMINATED SOILS 719 TABLE 1. Chemicl nd physicochemicl chrcteristics of the test soils Sludge tretment Orgnic mtter (%) NH 4 NO 3 P b (mg/liter) Totl P c (mg/kg) ph Free-ction concn in the soil solution ( g/liter 1 ) Totl metl concn d (mg/kg of soil) Zn 2 Cd 2 Zn Cd Cu Ni Pb Unsludged m 3 h 1 yr 1, low metl m 3 h 1 yr 1, high metl , m 3 h 1 yr 1, low metl , , m 3 h 1 yr 1, high metl , , Adpted from reference 29 with permission from the publisher. b NH 4 NO 3 -extrcted P. c Aqu regi-extrcted P. d Aqu regi-extrcted metls. MATERIALS AND METHODS Experimentl site. A long-term sewge sludge field experiment site locted t the Federl Reserch Centre for Agriculture in Brunschweig (Germny) ws estblished in The experimentl site ws formerly (40 yers go) woodlnd nd ws lter converted into rble soil. The soil (silty lom) contins 5% cly, 50% silt, nd 45% snd, with ph rnging from 5.3 to 6.0 (10). The sewge sludge ws obtined from locl sewge works, but ws rther low in hevy metl (low-metl sludge), s it ws received from the tretment plnt. Portions of the sludge were contminted by dding wter-soluble chlorides (10) of the hevy metls Pb, Cd, Cr, Cu, Ni, Hg, nd Zn in order to obtin higher metl concentrtion (high-metl sludge) nd were incubted for 6 weeks. Five different tretments were pplied to the experimentl plots s follows: inorgnic fertilizer t 180 kg of N h 1 yer 1, low-metl sludge t 100 m 3 or 300 m 3 h 1 yer 1, nd high-metl sludge t 100 m 3 or 300 m 3 h 1 yer 1. Ech tretment ws replicted four times. The sludges were pplied nnully from 1980 to 1990, nd the chnges induced in soil properties re recorded in the study of Chudri et l. (10). However, the highest mounts of hevy metls in the experimentl plots were still within the upper limits ccepted by the Europen Union for metl concentrtions in soils receiving sewge sludge. The min chrcteristics of the test soil, in which the rnge of hevy-metl concentrtions ws creted by the combintion of different doses nd types of sludge, re shown in Tble 1 (29). Soil smpling. The study ws crried out with soil from plots receiving the different tretments. At smpling (June, 1996), mize plnts were growing t the experimentl site. Spring rpe hd been grown before mize. Five cores of the rble-soil lyer (0 to 20 cm) were rndomly tken from mize rhizosphere in the centrl prt of ech replicte plot to void edge effects. Smples were then pooled, thoroughly mixed, sieved through 4-mm-pore-size mesh, nd stored t 4 C until used. Multipliction of AMF: estblishment of trp cultures. The nturl mycorrhizl potentil in the soil smples in terms of both the number of AMF spores nd the AM coloniztion levels ws very low, mking the study of diversity difficult. In order to increse the popultion of the indigenous AM fungi, trp cultures were estblished for ll the different tretments nd replictes. Four trp plnts were used s follows: Sorghum bicolor (L.) Moench (sorghum), Allium porrum (L.) (leek), Trifolium repens (L.) (clover), nd Timus vulgris (L.) (thyme). Seeds of these test plnts were surfce sterilized, pregerminted, nd trnsplnted fter emergence into 2-liter pots contining soil from ech experimentl plot. Four replictes per soil tretment nd host plnt were estblished. Plnts were grown in greenhouse, with tempertures rnging from 18 to 25 C nd reltive humidity rnging from 80 to 60%. Plnts were wtered every 3 dys with tp wter nd fertilized once fortnight with Long Ashton nutrient solution lcking phosphorus (19). After 6 months, the pot cultures were smpled by tking up to 100 g of soil nd roots mixture tht ws stored t 4 C until used. The smple size ws previously determined s the minimum mount required to study AMF diversity, since it contined complete representtion of ll the different morphotypes present in the soil. AMF extrction nd identifiction. AMF spores were isolted from 100 g of soil by the wet sieving nd decnting method, followed by sucrose centrifugtion (34). After centrifugtion, the superntnt ws poured through 50- m-pore-size mesh nd quickly rinsed with tp wter. Spores were counted with Doncster dish under the dissecting microscope nd grouped ccording to morphologicl chrcteristics. Permnent slides were prepred for ech different spore morphotype with both polyvinyl-lcohol nd polyvinyl-lcohol plus Melzer s solution (1:1). After the uniformity of the morphologicl groups ws confirmed under the opticl microscope, the different morphotypes were identified to the genus level nd, when possible, to the species level. Spore identifiction ws bsed minly on spore size nd color, wll structure, nd hyphl ttchment (20, 33, 36). With the dt obtined, severl indices were clculted s follows: richness (R number of species found in the smple), reltive bundnce of ech species in ech plot, clculted s (n i /N j ) 100, where n i number of spores tht belong to species i nd N j totl number of spores in the site. Mycorrhizl fungl diversity ws clculted by using the Shnnon-Wiener index, which combines two components of diversity, species richness nd evenness of individuls mong the species (24). Sttisticl nlysis. A two-wy nlysis of vrince (ANOVA) ws used to evlute the effect of the different host species nd soil tretments on totl spore number, species richness, nd diversity of the AM fungi. Reltive densities were rcsine-squre-root trnsformed before the two-wy ANOVA ws pplied to evlute the effects of the different tretments on the densities of the AM fungl species present in the plots. ANOVA ws followed by Duncn s test when pproprite. RESULTS Six AM fungl species belonging to the genus Glomus were found in rhizosphere smples from the different experimentl trp plnts nd soil tretments s follows: G. clroideum, G. mossee, nd four dditionl, unidentified species numbered III to VI. Totl AMF spore number decresed significntly with incresing mounts of hevy metls in soil, from 550 spores (per 100 g of dry soil) in the control plot to 30 spores in the 300 m 3 h 1 yer 1 contminted sludge (Fig. 1). In the 100 m 3 h 1 yer 1 uncontminted nd contminted sludge plots, the number of spores verged 330 nd 230 per 100 g of soil, respectively. This vlue decresed to 110 nd 30 spores in the soils dded t rte of 300 m 3 h 1 yer 1. Host plnt lso hd significnt effect on the totl AMF spores produced in the rhizosphere, Sorghum bicolor being the trp plnt tht produced AMF spores most effectively (Fig. 1). Both species richness nd the Shnnon-Wiener index rting incresed t intermedite levels of soil contmintion (100 m 3 TABLE 2. F nd P vlues from ANOVA tests of soil tretment, host plnt effects, nd their interction on the reltive spore densities of AMF species Source of vrition Glomus species Soil tretment Host plnt Soil plnt interction F P F P F P Glomus sp. III G. mossee G. clroideum Glomus sp. IV Glomus sp. V Glomus sp. VI F is the between tretment vrince/within tretment vrince rtio nd provides vlue for the equlity of tretment mens. The P vlue obtined for ech F, ccording to the corresponding degree of freedom, s follows (soil tretment, 4; host plnt, 3; soil plnt interction, 12), indictes the probbility tht differences between tretment mens re due to smpling error.

3 720 DEL VAL ET AL. APPL. ENVIRON. MICROBIOL. FIG. 1. Overll effect of soil tretments nd host plnts on the totl number of AMF spores (per 100 g of dry soil), species richness, nd diversity, mesured by the Shnnon Wiener index, of AMF popultions. For ech grph, brs with different letters indicte significntly different mens (P 0.01) by Duncn s test. Host plnt bbrevitions: Ap, A. porrum; Sb, S. bicolor; Tr, T. repens; Tv, T. vulgris. Soil tretment bbrevitions: C, control soil; 100 m 3 nd 300 m 3 low-metl-sludge soil, 100 nd 300 m 3 h 1 yer 1 sludge-mended soil, respectively. h 1 yer 1 nd 300 m 3 of low-metl sludge), decresing t the highest contmintion level (300 m 3 h 1 yer 1 contminted sludge) (Fig. 1). Host plnts lso exerted differentil effect on AMF diversity, with A. porrum nd S. bicolor promoting significntly higher levels of diversity in their rhizospheres thn those produced by T. vulgris nd T. repens (Fig. 1). Reltive densities of ll AMF species were significntly influenced by soil sludge tretments. Four species, minly Glomus sp. III, were significntly influenced by the host plnt, nd ll Glomus species except Glomus sp. IV were significntly influenced by the soil plnt interction (Fig. 2 nd Tble 2). The composition of the AM fungl popultion in the vrious host plnts rhizospheres, s ffected by the different soil tretments, is recorded in Fig. 2. Glomus sp. III ws the most common AMF species in the rhizospheres of A. porrum nd S. bicolor in unpolluted soils, but its popultion decresed shrply with incresing metl content in soils. At higher rtes of metl sludge contmintion, G. clroideum seemed to be the AMF species with the best bility to sporulte, becoming the most common species in the rhizospheres of both plnts. Both T. repens nd T. vulgris were colonized minly by G. clroideum, even in the unpolluted soil. G. clroideum nd Glomus sp. V were the most common fungi in the rhizospheres of ll host plnts growing in soils treted with 300 m 3 h 1 yer 1 of contminted sludge. The effects of the interction between soil tretment nd host plnt on the diversity of the AMF species nd the richness of the AMF species, s mesured by the Shnnon-Wiener index, re recorded in Tbles 3 nd 4, respectively. Both response vribles decresed significntly in the soil mended with the highest rte of contmintion. Different host plnts showed similr trends with regrd to the chnges they induced in species richness. The low diversity promoted by T. repens nd T. vulgris in their rhizospheres is remrkble, however. The overll effects of soil contmintion nd host plnt on the AMF popultion re illustrted in Fig. 2. As indicted bove, Glomus sp. III ws very sensitive to the presence of metls in soil, nd its propgules prcticlly disppered in the most contminted soil, while G. clroideum mintined similr reltive density in ll soils independent of sludge tretment. G. mossee showed nother pttern, incresing its density t intermedite rtes of contmintion. It is noteworthy

4 VOL. 65, 1999 AMF POPULATIONS IN HEAVY-METAL-CONTAMINATED SOILS 721 FIG. 2. Reltive bundnce of the vrious AMF species in the rhizospheres of different host plnts grown in smples of field soil with different sludge ppliction tretments. Br height represents the men of the totl number of AMF spores for ech tretment per 100 g of dry soil (the sum of the numbers of spores of ech species in ech tretment). Vlues for single fungl species, represented by vrious shding ptterns within brs, re not cumultive. Brs with the sme letter do not differ significntly (P 0.001) by Duncn s test. Host plnt bbrevitions: Ap, A. porrum; Sb, S. bicolor; Tr, T. repens; Tv, T. vulgris. Soil tretment bbrevitions: c control; t4 nd t6, 100 nd 300 m 3 h 1 yer 1 of noncontminted sludge, respectively; t5 nd t7, 100 nd 300 m 3 h 1 yer 1 of contminted sludge, respectively. tht Glomus sp. III ws bundnt in A. porrum nd S. bicolor rhizospheres, since it ws lmost bsent from the rhizosphere of T. repens. A negtive correltion (P 0.001) ws shown between the totl number of AMF spores nd soil metl content (Tble 5). For metls, both the totl mount of Zn nd the free Zn ction concentrtion determined in the soil solution gve the lowest correltion coefficients with totl number of spores, which lso correlted negtively with the P content of the soil (both totl nd vilble) nd positively with soil ph. Glomus sp. III ws negtively correlted with the totl content of ll the metls studied (Ni, Cu, Cd, Pb, nd Zn), corroborting its sensitivity to the presence of hevy metls (Tble 5). Glomus sp. V, however, did not show significnt correltion with the content of the metls studied, except for the free Zn ctions in the soil solution (Tble 5). DISCUSSION Long-term sludge ppliction with incresing concentrtions of hevy metls produced significnt decrese in both the size nd diversity of AMF popultions in soil. The totl number of AMF spores strongly decresed with the ddition of incresing mounts of hevy metls, but the AMF propgules never disppered completely in soils mended with the highest rtes of sludge, suggesting certin dpttion of these indigenous AMF to such environmentl stress. Notbly, the totl number of AMF spores correlted TABLE 3. Diversity of AMF popultions by the Shnnon-Wiener index s ffected by soil mendment nd different types of host plnts Vrible Men for indicted host plnt A. porrum S. bicolor T. repens T. vulgris Unsludged 2.13 bcd 1.61 bcd 0.39 d 0.00 d Low metl, 100 m 3 h 1 yr b 2.03 bcd 1.77 bcd 2.25 bcd High metl, 100 m 3 h 1 yr b 2.69 bcd 1.23 cd 2.63 bcd Low metl, 300 m 3 h 1 yr d 0.33 d High metl, 300 m 3 h 1 yr d 1.08 d 0.32 d 0.25 d Mens followed by the sme letter did not differ significntly (P 0.01) by Duncn s test.

5 722 DEL VAL ET AL. APPL. ENVIRON. MICROBIOL. TABLE 4. Species richness index scores of AMF popultions s ffected by soil mendment nd different types of host plnts Vrible Men for indicted host plnt A. porrum S. bicolor T. repens T. vulgris Unsludged 3.0 bcd 3.0 bcd 1.3 ef 1.0 f Low metl, 100 m 3 h 1 yr bcd 3.3 bc High metl, 100 m 3 h 1 yr bcd 3.0 bcd 2.8 bcde 3.3 bc Low metl, 300 m 3 h 1 yr b cdef 1.5 def High metl, 300 m 3 h 1 yr bcdef 1.8 cdef 1.3 ef 1.3 ef Mens followed by the sme letter did not differ significntly (P 0.01) by Duncn s test. negtively with the metl (Zn, Cu, Cd, nd Ni) content of the soils, but the correltion coefficient ws higher for the concentrtion of free ctions (Zn 2 nd Cd 2 ) in the soil solution. When present in excess, these ions re generlly ssumed to be the chemicl species tht re tken up by nd re toxic to soil microbes (16). In previous studies on AMF nd Cu (17) nd Zn nd Cd (39), no correltion ws found between the concentrtion of these metls in sludge-mended griculturl soils nd AMF popultions. Despite their vilbility, methods to mesure free-ion ctivity in the soil solution hve rrely been used in studies relting to hevy metl nd AMF. The use of such methods s reference for comprison would probbly help elucidte the reson for the discrepncies found between different studies. AMF popultion size lso correlted negtively with the P content of the soil, result tht is well documented (35). Species richness nd diversity s mesured by the Shnnon- Wiener index incresed t moderte levels of soil contmintion. This increse in AM propgule diversity could be fungl stress response whereby fungl ecotypes better dpted to unpolluted soil but ffected t intermedites rtes of contmintion llow other fungi, probbly less competitive in nonstressed soils but better dpted to hevy metls, to colonize the roots nd complete their life cycles. Thus, the number of fungl ecotypes in these soils cn be incresed. However, t the highest levels of soil pollution, both indices diminished shrply. This my hve resulted from fungitoxic effect of metls, cusing certin AMF species inbility to colonize the root system nd/or to multiply in the rhizosphere. Only AMF species better dpted to the disturbnce produced by the ddition of metls would overcome the stress sitution nd complete their life cycles. A similr response model concerning diversity, suggested for other microbil groups (37), my hold true for Rhizobium leguminosrum bv. trifolii from the sme experimentl field in Brunschweig; the reltionship between genetic diversity within popultions nd hevy-metl stress in soils my led to n increse in diversity with moderte metl loding, followed by shrp decrese t higher levels of stress (16). These chnges in genetic diversity my be crucil in determining the response of popultion to chnging conditions. However, genetic diversity studies hve not yet been described for AM fungi; thus, it is not possible to relte the phenotypicl chnges found in the present study to chnges in the genetic structure of the AMF popultion. Glomus sp. III spore density ws very much influenced by both soil tretments nd host plnt species. This fungl ecotype ppers to be very sensitive to incresing concentrtions of metls in soil, disppering lmost completely in the most polluted soil. At intermedite rtes of contmintion, Glomus sp. III ws replced by other species, such s G. clroideum. In fct, G. clroideum mintined similr reltive density levels in ll tretments, showing higher degree of tolernce to hevy metls thn the other fungl species present in the soil, s hs been described for other AMF ecotypes (13, 15, 25, 38). Glomus sp. V ws preferentilly found in soils with the highest level of contmintion, indicting low competitiveness of this fungus in the bsence of the stress sitution to which it is well dpted. Mjor differences mong the species in terms of both numbers of spores nd tolernce to metls suggest tht fungi follow different strtegies to estblish symbiosis nd probbly revel differences in functioning. The host plnt-medited effect on the composition nd diversity of the AM fungl community is noteworthy. In prticulr, the high diversity promoted by S. bicolor nd A. porrum contrsts with the poor levels induced by T. repens nd T. vulgris. S. bicolor ppered to be good host for spore production, possibly becuse of the higher root growth rte of this plnt species, which cn fcilitte further contct with most AM fungi present in the soil. In contrst, in the rhizosphere of T. vulgris, species with very slow growth rte nd poorly developed root system, AMF popultion size nd diversity were very low. These results corroborte the key role of the host plnt s selective force in mintining specific popultions of these ecologiclly obligte fungl symbionts (1, 5, 22). The root growth rte seems criticl to llow coloniztion by certin AM fungi; thus, the present results provide new insights into the specificity concept in rbusculr mycorrhizs. The resons underlying stress-relted chnges in the diversity of AMF popultions, prticulrly those due to the presence of hevy metls, re not completely understood. It is well known tht hevy metls cnnot be chemiclly degrded. Therefore, remedition of metl-polluted soils is limited minly to immobiliztion, for exmple by phytostbiliztion, TABLE 5. Correltion coefficients d for severl prmeters of the test soils nd some chrcteristics of the AMF spore popultions Vrible Totl no. of AMF spores Spore density Glomus sp. III Glomus sp. V ph 0.939** NS 0.942** P totl 0.971** NS 0.910* b P vilble 0.959** NS 0.886* NO 3 -N NS NS 0.970** Zn totl 0.978*** 0.931** NS Zn 2 c 0.957*** 0.884* 0.890* Cd totl 0.905* 0.951** NS Cd 2 c 0.922** 0.894* NS Ni totl 0.897* 0.971** NS Pb totl NS 0.941** NS Cu totl 0.944** 0.952** NS Extrcted with qu regi. b Extrcted with NH 4 NO 3. c Free in soil solution (29). d, significnt t P 0.05;, significnt t P 0.01;, significnt t P 0.001; NS, not significnt.

6 VOL. 65, 1999 AMF POPULATIONS IN HEAVY-METAL-CONTAMINATED SOILS 723 which consists of promoting plnt growth to reduce or eliminte the biovilbility of metls (11). In this context, AMF constitute n importnt functionl component of the soil-plnt system tht is criticl for sustinble productivity in stressed soils (3). A better understnding of the mechnisms behind these chnges in AMF diversity, nd prticulrly of those on which AMF dpttion nd tolernce to metls re bsed, is importnt, since such n understnding could fcilitte the mngement of these soil microorgnisms for restortion nd/or bioremedition progrm. ACKNOWLEDGMENTS We thnk the Europen Commission (project ENVIRONMENT EV5V-0415) for supporting this reserch. We lso thnk B. Knight nd S. P. McGrth, from IACR-Rothmsted (Hrpenden, United Kingdom) for supplying the soil chemicl nlyses nd specition dt nd Custodi Cno for vluble technicl support. REFERENCES 1. Allen, B. E., M. F. Allen, D. J. Helm, J. M. Trppe, R. Molin, nd E. 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