EFFECT OF ROTATION, ORGANIC INPUTS AND TILLAGE ON CROP PERFORMANCE AND SOIL QUALITY IN CONVENTIONAL AND LOW-INPUT ROTATIONS IN CENTRAL IOWA

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1 EFFECT OF ROTATION, ORGANIC INPUTS AND TILLAGE ON CROP PERFORMANCE AND SOIL QUALITY IN CONVENTIONAL AND LOW-INPUT ROTATIONS IN CENTRAL IOWA BY PATRICIA ANN LAZICKI THESIS Submitted in prtil fulfillment of the requirements for the degree of Mster of Science in Nturl Resources nd Environmentl Sciences in the Grdute College of the University of Illinois t Urbn-Chmpign, 2011 Urbn, Illinois Adviser: Associte Professor Michelle Wnder

2 ABSTRACT Even though the benefits of low externl input (LEI) cropping systems to crops nd soils re well recognized the specific links between cropping prctices nd ssocited soil qulity nd crop responses re not yet cler. In the Mrsden plots in centrl Iow, crop yields nd input use efficiency hve been incresed by the use of longer nd more diversified rottions nd reduced chemicl inputs. In this work we smpled roots nd soil prmeters t multiple dtes nd two depths in ll cropping phses, in order to quntify chnges in physicl, chemicl nd biologicl soil qulity indictors nd root responses ssocited with tillge nd cropping fctors in conventionl nd two LEI rottions of different lengths nd including different legume species. Improvements in soil qulity indictors nd plnt productivity were expected to be driven by the mount nd plcement of orgnic residues nd to fluctute with tillge nd cropping phse. On system bsis, prticulte orgnic crbon (POM-C) nd potentilly minerlizble nitrogen (PMN) were incresed in both LEI rottions reltive to two yer (2-yr) corn (Ze mys L.) -soyben (Glycine mx L.) rottion. Biologiclly lbile orgnic mtter frctions were highly strtified in the 2-yr rottion compred to the LEI rottions nd the lower depth of the 2-yr rottion ws consistently depleted. Corn roots followed similr pttern, being concentrted in the top depth in the 2-yr rottion while more fully exploring the profile in the LEI rottions. Low C:N rtios in the soyben roots in the LEI rottions suggest greter N vilbility in the LEI soyben phse. Soil prmeters did not differ between LEI rottions even though the 3-yrrottion included red clover (Trifolium prtense L.) insted of lflf (Medicgo stiv L.), shorter rottion length, nd significntly greter men nnul orgnic inputs thn did the 4-yr rottion. Corn yield in the 3-yr LEI rottion ws significntly higher thn tht chieved in the 2-yr conventionl rottion, nd soyben yield in the 4-yr rottion ws higher thn tht in the 2-yr rottion. Sesonl smpling showed tht 1) soil prmeters fluctuted during the growing seson but did not increse in response to prticulr cropping phses nd 2) tht the strtifiction observed in the 2-yr rottion ws consistent over time for both corn nd soyben. The ii

3 prctice most responsible for incresing soil qulity nd plnt performnce in the LEI rottions ppered to be the deep incorportion of compost nd green mnures prior to corn production. This prctice benefited both corn nd soyben, primrily by incresing the mount nd distribution of nutrients vilble to roots s evidenced by greter POM-C nd PMN levels in the subsoil. iii

4 TABLE OF CONTENTS LIST OF TABLES... v LIST OF FIGURES... vi INTRODUCTION... 1 METHODS... 6 RESULTS DISCUSSION CONCLUSIONS REFERENCES TABLES FIGURES iv

5 LIST OF TABLES Tble 1. Summry of cropping system mngement Tble 2. Estimted verge of yerly crbon (C) nd nitrogen (N) inputs to ech crop phse Tble 3. Tble 4. Anlysis of vrince of soil prmeters nd strtifiction rtios on soils from spring 2009 nd Soil vrible mens nd strtifiction rtios for soils collected in spring 2009 nd 2010 from 0-10 cm nd cm depths Tble 5. Corn nd soyben boveground biomss, men yield, mesured in Fll Tble 6. Pre-plnned contrsts for root-length density in summer, conducted s one-tiled t- tests Tble 7. Mens nd differences in summer soyben root C:N rtios Tble 8. Anlysis of vrince of soil nd root prmeters nd strtifiction rtios from the 2009 growing seson Tble 9. Soil nd root vrible mens nd strtifiction rtios for soils collected in the 2009 growing seson Tble 10. Crop(system) by seson interctions in wter-filled pore spce (WFPS) nd prticulte orgnic mtter C (POM-C) Tble 11. Multiple regression models for ggregte stbility in spring, summer, nd fll of 2009 nd spring of v

6 LIST OF FIGURES Figure 1. Conceptul frmework by which soil nd plnt prmeters from the previous crop (Y-1) re considered both s cusl gents contributing to the soil environment for the subsequent crop (Y) nd s endpoints ssessing the performnce of the system under crop Y Figure 2. Root length density in summer of Figure 3. Physicl indictors of soil qulity by rottion nd depth for ech crop, verged over the 2009 growing seson Figure 4. Soil chemicl nd biologicl indictors plotted by rottion nd depth for ech crop vi

7 Introduction Low externl input (LEI) cropping systems im to reduce relince on chemicl pesticides nd fertilizers by using longer, more diverse rottions, green mnures nd composts, nd focused pplictions of chemicl inputs to supply nutrients nd control insect pests, weeds nd diseses (Bll et l., 2005; Drinkwter nd Snpp, 2007). Such systems im to improve yield nd system efficiency by coupling plnt needs with soil processes to support those needs insted of supplying nutrients with chemicl fertilizers nd reducing competition with pesticides s is done in conventionl systems (Liebmn et l., 2008). When weeds re well controlled, cerel crops grown in LEI or orgnic systems cn produce yields comprble to or higher thn those chieved in simpler nnul crop systems receiving greter chemicl inputs (Liebhrdt et l., 1980; Delte nd Cmbrdell, 2004; Tesdle et l., 2007; Posner et l., 2008; Coulter et l., 2011). While this rottion effect ssocited with length nd crop diversity is prtly ttributble to pest nd disese suppression (Smith et l., 2008), mny rgue these benefits re derived from improvements in soil qulity, becuse increses in the quntity nd or qulity of soil orgnic mtter re frequently observed in such systems (Wnder et l., 1994; Bll et l., 2005, Krlen et l., 2006; Drinkwter nd Snpp, 2007), nd improved crop yields hve been shown to be correlted to increses in soil qulity indictors (Sprgo et l., 2011). Longer rottions (Delte nd Cmbrdell, 2004; Coulter et l., 2011), mnure (Sinju et l., 2008), nd cover crops (Wnder et l., 1994) re known to be beneficil, but the optiml length of rottion, rte nd frequency of mnure ddition, or species nd frequency of cover crops re not known. Most studies of diversified frming systems tend to focus on yield outcomes or soil qulity s endpoints, nd do not ttempt to evlute the processes by which mngement prctices chnge soil qulity to relte those chnges to plnt performnce or how those chnges in plnt response cycle bck to lter the soil system. 1

8 As generliztion, the use of diversified rottions nd ddition of niml nd green mnures cn lter system efficiency by chnging the quntity, degree of incorportion nd type of mteril entering the soil, nd with this, presumbly by ltering the decy environment (Goverts et l., 2009). How LEI system orgnic mtter (OM) quntity, incorportion nd type differ from conventionl rottions depends on the system. Use of mnures nd composts cn increse the quntity of OM dded in LEI rottions. Greter OM ddition is often ssocited with incresed soil OM (SOM) (i.e. Buynovsky nd Wgner, 1998; Aoym et l., 1999; M et l., 2011). The presence of SOM is known to promote good soil physicl, chemicl nd biologicl properties, incresing nutrient retention nd system efficiency (Drinkwter nd Snpp, 2007), nd studies which observe soil qulity under different rtes of residue ddition hve found higher rtes of residue return to be ssocited with higher SOM, lower bulk density, higher ggregte stbility nd higher microbil biomss (i.e. Krlen et l., 1994, Frnzluebbers nd Brock, 2006). However, SOM does not lwys increse with greter OM dditions, prticulrly in very high orgnicmtter soils (i.e. Gulde et l., 2008, Chung et l., 2010) or under moldbord plow tillge (Moebius-Clune et l., 2008). In ddition to chnging the quntity of inputs LEI rottions lter the pttern of input distribution nd with this the intensity of soil disturbnce. Studies tht emphsize the role of crop nd mnure -bsed inputs overlook the fct tht, in the Midwestern United Sttes, to the extent tht the LEI rottion ims to reduce externl chemicls, these prctices often require full-inversion tillge to completely kill the green mnure crop, reduce weed competition, nd incorporte mnures to promote minerliztion (Peigné et l., 2007). The negtive effects of tillge on soils nd soil orgnic mtter re well known (i.e. Frnzluebbers, 2010). How tillge-bsed disturbnce intercts with other prctices, including mnure dditions nd different crops, is less well understood. Moldbord plowing, dominnt form of inversion tillge, intercts with the other mngement prctices by ffecting orgnic 2

9 input minerliztion rte nd plcement. It cuses fster minerliztion of SOM by breking up protective structures (Six et l., 2002; Chung et l., 2008), incresing ertion (Topp et l., 2000) nd putting residues in closer contct with the soil (Frnzluebbers et l., 1998). The fster minerliztion rtes typiclly ssocited with tillge provide more N to the growing crop but cn lso degrde soil qulity, prticulrly in the surfce soil (Peigné et l., 2007; Frnzluebbers, 2010). The effect of tillge vries by the degree to which the OM is protected nd the ctivity of the microbil community; soils with very strong structure (Yoo nd Wnder, 2008), in cooler climte (Angers et l., 1997) or whose prior OM concentrtion exceeds the protective cpcity of the soil (Vndenbygrt, 2003) my not lose SOC when subjected to n increse in tillge. Tillge lso, lters structure nd moisture interctions in wys tht influence decomposition; for exmple, orgnic mtter my decy fster in surfce of less-tilled soils where wter is limiting, since less-tilled soils tend to hold more moisture (Frnzluebbers nd Arshd, 1996). There my be other importnt depth-bsed effects of tillge tht re overlooked. Since moldbord plow tillge tends to plce residues towrds the bottom of the plow depth (Allmrs et l., 1996)) soil qulity nd C storge cn, in cool climtes nd hevy soils, be improved overll by C ccumultion t or below the depth of plowing (Angers nd Eriksen-Hmel, 2008). Tillge lso hs direct positive structurl effects, temporrily loosening compcted or hevy soils. If soil strength is limiting, tillge in this cse my promote OM ccumultion by breking up physicl impednce to plnt root growth, leding to root prolifertion. It is importnt to deepen our understnding of the conditions under which tillge degrdes or improves soil qulity. A useful tool for looking t the effects of tillge on the soil environment is the strtifiction rtio; tht is, the vlue of soil qulity in the surfce soil divided by its vlue in the subsurfce (Frnzluebbers, 2002). Becuse tillge lters OM distribution nd decy environments, n expression of the depth distribution my be more useful thn vlue for the totl soil profile (Frnzluebbers, 2002). 3

10 An importnt fctor which intercts with the quntity nd plcement of orgnic mteril is the type of mteril dded. LEI rottions frequently include species with extensive root systems, which contribute to soil qulity through their influence on the decy environment nd their intrinsic chrcteristics. Living roots contribute to improvements in soil structure nd OM protection by drying the soil (Hllett et l., 2009), creting richer microbil environment, prticulrly for fungi (Hynes nd Bere 1997) nd providing durble physicl support round which ggregtes cn form nd stbilize (Odes nd Wters, 1991; Puget nd Drinkwter, 2001). Roots lso hve been shown to be more reclcitrnt in soil thn shoots (Rsse et l., 2005) nd hve been shown to contribute disproportionte mount of soil orgnic C (Blesdent nd Blbne, 1996; Kong nd Six, 2010). These findings suggest tht extensively rooted crops such s lflf, smll grins nd clover hve n importnt soil-building function in the rottion, which my counterct the destructive effect of tillge (Sprgo et l., 2011). In ddition, incorportion of composted mnure, which is highly reclcitrnt (Abiven et l., 2007), hs been shown to increse the number nd stbility of soil pores (Bll et l., 2005), my hve mild but long-lsting ggregtive effect (Abiven et l., 2009) nd contribute gretly to the soil lbile frction (Poudel et l., 2002; Gulde et l., 2008). The LEI system prctices of dding composts nd more roots to the soil, whether by promoting soil structure nd ggregte protection or by their own longer men residence time, re expected to hve observble effects on soil structure nd OM content, nd overll nutrient cycling efficiency (Puget nd Drinkwter, 2001; Bll et l., 2005; Nyirnez et l., 2010). The gol of this study ws to determine which if ny of the mechnisms by which LEI mngement is predicted to improve soil qulity re importnt in the finely-textured, high OM soils of Mrsden Frm Cropping Systems Experiment. The tril, locted ner Ames, Iow ws strted in 2002 nd consists of conventionlly mnged 2-yer rottion (corn-soyben), s well s 3-yer (cornsoyben-ot + red clover) nd 4-yer (corn-soyben-ot + lflf-lflf) rottions. Composted beef mnure is incorported into the 3-yr nd 4-yr rottions in fll before corn. This site provides n 4

11 opportunity to explore plnt-soil reltions in LEI systems tht produce corn nd soyben yields which equl or exceed those of their conventionl counterprts (Liebmn et l., 2008). Previous work suggests improved soil-bsed differences my be responsible but does not yet indicte chnges in totl SOC or N (Lmmerding nd Wnder 2005, M. Liebmn, personl communiction, dt not shown). The objectives of this study were to: 1) quntify the effects of cropping system nd component prctices (rottion length, residue nd mnure dditions, nd tillge) on soil qulity nd productivity nd 2) evlute the influences of specific crops nd mngement events soil response nd the helth of the following crop. By smpling ll crops in ll rottions t two depths in the spring of 2009 nd 2010, we imed to describe long term differences which hd developed between systems s result of the different mngement pckges. In ddition, we smpled these rottions on multiple dtes in the cropping seson, to describe to wht extent ech crop growth environment ws legcy of the preceding crop s growth nd decy chrcteristics nd to cpture the impct nd durtion of specific mngement events (Figure 1). Improvements in soil qulity indictors nd plnt productivity were expected to be driven less by the mount thn the degree of incorportion nd type of orgnic residues. Despite their more extensive tillge, in these hevy-textured soils LEI plots were expected to preserve greter OM thn the 2-yr plots. Attendnt structurl benefits were expected to include greter overll %WSA nd decresed bulk density. Decresed bulk density ws expected to be seen prticulrly under the recently-tilled corn. This difference ws expected to be correlted with incresed root length. Orgnic mtter nd ggregte stbility were expected to be significntly incresed under smll grins nd forge legume crops, offsetting the negtive effects of tillge. 5

12 Methods Timing nd extent of smpling To quntify inputs in order to test both system nd temporl hypotheses we smpled bovend below-ground biomss in fll 2009 nd generted rough C budgets nd estimtes of externl N entering ech system. Crbon inputs to crop were clculted s the sum of mesured C from the biomss nd from the fll root mss from the crop preceding it in the rottion. This method ssumes the 2009 vlues re representtive. Dt bsed on smples tken on single dte were used to evlute plnt productivity; corn nd soyben biomss nd grin from fll of 2009, ot grin from just before it ws hrvested, nd roots from midsummer of 2009, t which point corn, soy nd ot roots were expected to be round their mximum. Soil smples tken in spring 2009 nd 2010 were used in order to fulfill our first objective, to quntify the effects of cropping system nd component prctices on overll system soil physicl, chemicl nd biologicl indictors nd plnt productivity. By considering two dtes we were ble to ensure tht observed differences were not merely plot effects. Soil smples tken in spring, summer nd fll of 2009 were used to trck temporl chnges in the soil nd to test hypotheses bout the temporl or sptil ptterns of ddition or disturbnce ssocited with prticulr prctices or crops. By observing sesonl dynmics, we were ble to show the durtion of effects, nd were ble to ensure tht observed ptterns were true over the whole growing seson nd not just spring. Site description The Mrsden Frm Cropping Systems Experiment is rndomized complete block experiment with four replictions locted in Iow Boone County, Iow (42 01 N; W; 333m bove se level). Averge nnul precipittion is 723 mm (Liebmn et l., 2008), lthough in 2008, the site received 1100mm (Ntionl Wether Service, which qulified s enough rin to 6

13 cuse 100 yer flood. Soils vry cross the experimentl site nd consist of pproximtely equl proportions of Clrion lom (fine-lomy, mixed, superctive, mesic, Typic Hpludolls, 2-5% slope), Nicolet lom (fine-lomy, mixed, superctive, mesic, Aquic Hpludolls, 1-3% slope) nd Webster silty cly lom (fine-lomy, mixed, superctive, mesic, Typic Endoquolls, 0-2% slope) (Liebmn et l., 2008). Detils on site history nd mngement re found in Liebmn et l., 2008 nd Cruse et l., Ech of four blocks re seprted by 15.2 m grss border nd divided into nine 85x18 m strips such tht ech crop in ech rottion ws represented once in ech block in ech yer (Tble 1). The soil is chisel-plowed to 15 cm in the fll fter corn hrvest nd ure is spred in the spring before corn plnting t rte of 100 kg N h -1. In the3-yr rottion corn nd soyben were followed by yer of smll grins (triticle ( Triticosecle Wittmck) until 2005 but subsequently ot (Aven stiv L.) interseeded with red clover. The ot crop is hrvested for grin, the strw is bled nd tken off, nd in the fll the red clover is moldbord plowed to 20 cm s green mnure long with 15.7 Mg h -1 (fresh weight bsis) composted beef cttle (Bos turus) mnure contining on verge 128 kg N h -1, to provide nutrition for corn the following yer. The 4-yr rottion consists of corn nd soyben followed by yer of ots nd lflf. In the third yer ots re hrvested s in the 3-yr rottion nd the lflf is mowed once nd continues on to second yer. The second-yer lflf is hrvested for hy three or four times, nd plowed under s green mnure in November long with composted mnure, s in the 3- yr rottion. As moldbord plowing is lwys done in conjunction with mnure ddition, their effects re confounded. For corn in ll systems, dditionl inorgnic N is side-dressed in erly June if needed in response to lte spring soil nitrte test tken to 30 cm. Lower thresholds re used in the conventionl rottion. Side-dress N rnged from 0 kg N h -1 to high of 100 kg N h -1 in 2008, the flood yer. Thresholds used vry but re higher in the LEI rottions thn in the conventionl. Both P nd K re periodiclly pplied in ll rottions s needed in response to stndrd soil tests. 7

14 Plnt smpling nd nlysis To quntify crop C input to the soil nd to evlute reltive crop growth, hrvested portions nd boveground stover were obtined from ll crops in In fll directly before soyben hrvest nd fter corn hd dried down, corn grin nd stover were collected in fll from four representtive corn plnts ner the center of the field, nd soyben grin nd stlks, pods nd leves were smpled from two m 2 smple res ner the north nd south ends of ll plots. In October before plow-down, boveground biomss smples of lflf nd red clover were tken from four 0.25 m 2 smple res. Plnt smples were dried t 40 o C nd ground before totl C nd N were determined by dry combustion. Grin removed from the corn nd soyben crops ws dried nd weighed. Grin C nd N were not mesured, but were estimted using vlues from relted hybrids grown on neighboring reserch plots. Vlues for mnure weight, C nd N from 2008 were lso obtined from the Mrsden field stff. C inputs for ech plot in crop (Y ) were clculted s C s: To estimte the distribution of inputs within the soil we ssumed tht moldbord plowing distributed 50% of the residues in the 0-10cm depth nd 50% of the residues in the 10-20cm depth. This is consistent with 13 C lbeling studies of residues buried by moldbord plowing conducted by Wnder nd Yng (2000). Some studies show up to 80% of residues being deposited in the 10-20cm depth fter moldbord plowing (i.e. Allmrs et l. 1996), but equl mixing ws ssumed s being the most conservtive for comprisons between chiseled nd moldbord plowed systems. To derive N fixtion estimtes, we obtined dt from the field stff on biomss nd N content of the hrvested lflf. As the flooding in 2008 resulted in poor stnd of lflf in the second-yer lflf plots, we used verge biomss nd N mesurements from to estimte typicl 8

15 removl rtes. For leguminous crops we used conservtive estimtes (N-fixed= 41% of lflf or red clover boveground biomss N nd 41% soyben grin N) from recent study estimting N-fixtion in the Mississippi vlley (Russelle nd Birr 2004). We clculted verge nnul externl input N s: A wekness of this method is tht N-fixtion rtes re highly vrible even within the sme climte, nd re likely to differ by system. The 4-yr rottion would be most ffected by errors, in which three out of the four crop yers involve N inputs by fixtion. Root smpling nd nlysis To evlute the influence of root inputs we mesured root growth prmeters root length, dimeter nd qulity tht should be directly ffected by chnges in soil nd hve the potentil to lter the environment by direct dditions nd through indirect effects on structure. Tht is, soil nd crop chrcteristics were not only mesured s indictors of system function, but lso s potentil mechnistic ctors. Composite smples of four soil cores (5.08 cm dimeter) were obtined from 0-10 nd cm depths. Cores were tken from between crop-row position in the ot nd lflf plots nd 15 cm from the crop-row in the corn nd soyben plots. Smples from soil under ot were tken in lte June just before the ot hrvest. For ll other crops, cores were tken in lte July. Cores from ll crops were retken in lte September in similr mnner. Root cores were put on ice fter collection nd kept t 4 o C until nlysis within month from the time of collection. For both smpling dtes, root length, dimeter nd C nd N content were determined. Root smples were elutrited to seprte ll low-density mteril from soil, nd non-root debris ws identified nd discrded. Clen root smples were stored in 50% ethnol t 4 o C. Root length nd dimeter were mesured on n Epson 1680 scnner (Epson 9

16 Americ Inc., Long Bech, CA, USA) utilizing WinRhizo root scnning softwre (WinRhizo, Regent Instruments, Québec, Cnd). After scnning, root smples were dried, weighed, nd ground to powder for C nd N nlysis by dry combustion on Costech Elementl Anlyzer (Model ECS 4010, Costech Anlyticl Technologies, Inc. Vlenci, CA). Root exudtes were estimted s 0.65*mesured summer root C, fter Bolinder et l. (2007). Results were used to estimte root length nd root biomss. This method, while sufficient for compring verge root length nd dimeter between crops nd rottions, likely underestimtes root C. Extrpoltion to field scle estimtes will therefore be lower thn ctul vlues, prticulrly for tp-rooted plnts. The influence of this underestimtion of root inputs would be worst in the 4-yr rottion, in which three of the four crop-yers included tp-rooted species. Soil smpling nd nlysis We wished to exmine if soil qulity, bility to retin orgnic mtter nd system efficiency were improved under the low-input rottions, prticulrly towrds the end of the rottion under the hy crops. To quntify mngement effects on the soil we mesured vriety of physicl, chemicl nd biologicl soil qulity prmeters: bulk density (BD), wter filled pore spce (WFPS), whole soil C nd N, prticulte orgnic mtter C (POM-C), potentilly minerlizble N (PMN), nd fluorescein dicette hydrolysis rte (FDA). These ssys hve been shown to be sensitive to mngement (Schnurer nd Rosswll, 1982; Frnzluebbers nd Arshd, 1996; Mrriott nd Wnder, 2006; Krlen et l., 2006) nd soil function (Wnder, 2004). Wet ggregte stbility nd wter filled pore spce re mesures of physicl qulity tht re relted to soil s bility to protect SOC from decy. The WFPS hs been shown to correlte well with OM minerliztion cross wide rnge of soils (Linn nd Dorn, 1984) nd to be sensitive to tillge (Frnzluebbers nd Arshd, 1996), while BD is relted to the plnt roots bility to penetrte the soil. We took soil smples from 0-10cm nd 10-20cm depths to document different 10

17 ptterns of residue distribution nd the ssocited vrition in soil qulity tht were expected to result (Wnder nd Yng, 2000). Soil cores from 0-20cm were obtined from ech plot four times in with 5.08 cm dimeter splittble soil probe. Two cores ech were tken 10m from the north nd south ends of ech plot nd composited to obtin representtive smple. All the corn, ot nd lflf plots were smpled in April 2009 fter spring soil disturbnce; this ws directly fter corn plnting nd few weeks fter ot nd lflf plnting. Soyben plots were smpled in erly My, directly fter plnting. All plots were smpled lte July 2009 t pproximtely the time of mximum corn nd soyben root development, nd gin in lte September 2009, just before soyben hrvest nd well before ny fll tillge. In April nd My 2010, cores were tken s in For ll smpling dtes, cores were divided in the field into 0-10cm nd 10-20cm segments, plced on ice for trnsport to the lb nd kept t 4 o C. Physicl nlyses Within seven dys of collection, cores for soil nlysis were weighed for bulk density nd sieved while fresh through n 8mm sieve. Next, 10-20g of fresh soil ws weighed nd oven-dried for 24 hr. t 105 o C to determine moisture content. Bulk density ws clculted s oven-dried soil weight corrected for rocks nd lrge residues. All soil chemicl, physicl nd biologicl nlyses described below were corrected for bulk density nd reported on volume bsis. Wter-filled pore spce (WFPS) ws clculted s: From portion of ir-dried <8mm soil ll visible residues >1 mm were hndpicked out with tweezers to yield soil frction to be used to mesure structurl, chemicl nd physicl chrcteristics. A portion ws dry sieved between 2mm nd 1mm sieves nd the resulting 1-2mm frction ws reserved 11

18 for wter-stble ggregte nlysis. The percentge of soil contined in wter-stble ggregtes (%WSA) were mesured on ir-dried soils from 2009 nd 2010 using the stndrd wet sieving method dpted from Kemper nd Roseneu (1986). Briefly, 10g of dry ggregtes between 1-2mm in dimeter were poured in single lyer on top of 250 m sieve, which ws in turn plced on 53 m sieve. Aggregtes were slked by just submerging them in wter for five minutes, fter which the sieves were plced on mchine which moved them up nd down through column of wter t constnt rte for ten minutes. After sieving, mteril on top of ech sieve ws quntittively wshed into 53 m nylon mesh, secured with clip, dried t 60 o C for 24 hr., nd weighed. From ech ggregte size frction, the snd nd POM were extrcted by quntittively trnsferring sieved mteril into 40 ml Nlgene bottles nd dispersing, drying nd weighing the <53 m frction s described for POM below. Snd-free mcroggregte nd microggregte concentrtions were clculted s the weight of the ggregte frction less the weight of the snd nd POM frction left following dispersion. As the mcroggregte frction ws more meningful to our nlyses, %WSA used henceforth refers to the mcroggregte frction. Soil texture ws nlyzed on soils from the summer of 2009 only, using the stndrd hydrometer method (Gee nd Buder, 1986). Chemicl nd biologicl nlyses The remining soil ws mechniclly crushed to pss through 2mm sieve for use in totl soil crbon (C) nd nitrogen (N), prticulte orgnic mtter C nd N (POM-C nd POM-N), fluorescein dicette hydrolysis, (FDA, mesure of heterotrophic microbil ctivity) nd potentilly minerlizble nitrogen (PMN) determintion. In fll 2009 only, soils were finely ground nd nlyzed for totl C nd N by dry combustion. Orgnic C ws obtined by subtrcting inorgnic C vlues determined for ech plot fter Bundy nd Bremner (1972). Soil POM-C nd N (concentrtion of orgnic C nd N in the size frction 12

19 between 53 m nd 1000 m) concentrtion ws determined s described by Mrriott nd Wnder (2006). The POM-C frction ws determined by dry combustion. The POM frction s found to contin crbontes nd thus, we developed pproprite correction fctors by removing crbontes by fumigtion s described by Hrris et l. (2001) using soils collected in spring These correction fctors were bsed on difference where the percent of C in crbontes ws ssumed to equl C in the uncorrected minus the corrected smples nd pplied to other dtes. Soil PMN ws determined with n nerobic incubtion, s described by Drinkwter et l. (1996) with some modifictions. Three sets of 5g smples of ir-dried soil ground to pss through 2mm sieve were plced in 50mL centrifuge tubes. Next, 50mL 2 mol L - 1 KCl were dded to one set to quntify the mount of NH 4 -N originlly present in the soil. Smples were shken for 1 hr. on reciprocl shker nd vcuum-filtered through Whtmn No. 42 filter pper, nd nlyzed colorometriclly for NH 4 -N fter Sims et l. (1995). To the other two replicte sets, 10mL deionized wter ws dded nd incubted for 7 dys t 40 o C. After one week, smples were mixed with 40 ml 2.5 mol L - 1 KCl solution, shken, before the solution ws filtered nd nlyzed s in the first set of smples. Enzyme ctivity ws determined by the fluorescein dicette (FDA) hydrolysis method, which represents microbil enzymtic ctivity, modified from tht proposed by Schnurer nd Rosswll (1982) s improved by Adm nd Duncn (2001). Duplicte 1.00 g ir-dried <2 mm soil smples plced in 40mL Nlgene centrifuge tubes nd shken with 20mL phosphte buffer (60 mmol L -1, ph 7.6) nd either 0.50ml FDA solution (4.8 mmol L -1 ) or 0.50mL cetone in tightly cpped bottles t 200 RPM on n orbitl shker t o C. After 5 hr. smples were removed from the shker nd the rection ws stopped by the ddition of 20mL cetone. Smples were centrifuged for six minutes t n ngulr velocity of 7000 RPM nd extrcted through Whtmn No. 42 filter pper. Color development in the filtrte ws determined vi spectrophotometer t 490 nm ginst stndrd curve. Soil FDA hydrolysis ws 13

20 clculted s the concentrtion in the FDA ddition smples less the concentrtion in the cetone blnks, expressed s mg FDA hydrolyzed per g of soil nd per hour of incubtion. Sttisticl nlyses To test the hypothesis tht improvements in soil qulity indictors nd plnt productivity would be driven by the mount nd plcement of orgnic residues, plnt nd soil fctors nd input C were compred between rottions. Differences between rottions were nlyzed s rndomized complete block design with four replictions using PROC MIXED in the SAS system (SAS Institute, Cry, NC). In order to compre rottions under the sme crop, we regrded ech crop within ech rottion s tretment, giving the clss vrible crop(system) with nine levels. Other clss vribles were block, depth nd yer when pproprite. Block nd its interctions run s rndom vribles nd the others s fixed vribles. Rottion comprisons were mde with estimte sttements between the verge of ll crops in one system nd the verge of ll crops in the other. All soil vribles except totl soil orgnic C (SOC) were nlyzed using dt from spring 2009 nd Totl SOC nd C nd N inputs were nlyzed using fll 2009 dt. Root length, dimeter nd qulity were compred using dt from summer Percentge of cly prticles (<2 m) were used s covrite when necessry, s the blocking did not entirely remove texture differences between rottions. Covrite interctions were removed from the model if they were insignificnt t p>0.05 nd clss vrible interctions were removed if they were insignificnt t p>0.35. Dt were trnsformed when necessry to meet ssumptions of residul normlity nd homogeneity of vrince. The squre root trnsformtion ws performed on the POM-C, PMN, FDA nd root length vribles, nd the nturl log trnsformtion on the strtifiction rtios for POM-C, PMN, FDA, nd %WSA. Reported vlues re bck-trnsformed LSMEANS, nd rottion mens for trnsformed vribles re verges of bck-trnsformed crop(system) LSMEANS. Differences in 14

21 mens were ssessed using Tukey s test, nd were considered to be significnt t p<0.05 nd mrginlly significnt t p<0.10. Differences in written contrsts were ssessed using stepdown bonferroni djustment. To explore the differentil effect of rottions on depth distribution, for ech soil nd root vrible we clculted strtifiction rtio between the depths for ech plot i s The strtifiction rtios were nlyzed s described bove. For our first hypothesis, significnt difference between smpling yers ws not considered to be importnt, prticulrly for the physicl indictors, since smples were tken in different plots where texture is known to vry nd under different climctic conditions. However, interctions with yer were used to ssess vribility in crop(systems), depths, nd crop(system) by depth interctions, nd especilly to determine if rnkings nd behvior ptterns remined in the sme despite the chnge of plot nd climte. To test our second hypothesis on the influences of specific crops on plnt nd soil response, we performed ANOVAs in PROC MIXED s before, using for the soil vribles dt from spring, summer nd fll of Smples collected from the sme plots on different dtes were not nlyzed s repeted mesures, s the covrince structure met independence ssumptions. In order to evlute the ssocition of soil ggregtion with other soil fctors, we used the RSQUARE nd CP model selection procedures in PROC REG of the SAS system to identify pproprite terms, nd modeled them using PROC REG, using djusted R 2 vlues to ssess the percent of vribility in %WSA tht could be explined by the chosen fctors. In order to evlute the effect of soil physicl structure on root growth, we regressed summer root length nd verge dimeter ginst summer bulk density for ech crop species using PROC REG in the SAS system. All ssumptions were met. 15

22 Results System inputs Averge yerly C inputs were significntly different in ll three systems nd rnked 3-yr>2-yr>4- yr (Tble 2). The 0-10cm depth received significntly more input C thn the 10-20cm depth in ll rottions. Subsoil C dditions were 3, 13, nd 18% of totl dditions in the 2-yr, 4-yr nd 3-yr rottions with the conservtive ssumption tht moldbord plowing mixed residues eqully between the depths. If, s hs been suggested in some studies, up to 80% of moldbord plowed residue ends up below 10cm, up to 19% nd 30% of the totl C inputs in the 4-yr nd 3-yr rottions my be dded to the 10-20cm depth. The lrgest C inputs were to the soyben yer (from the corn residues), while the second-yer lflf stnd hd the lowest C inputs. The 3-yr nd 2-yr rottions hd similr verge nnul C input to the 0-10cm depth, while tht of the 4-yr rottion received significntly lower C inputs thn both. The verge differences were lrgely ttributble to differences in the frequency of corn in the rottion nd the bsence of surfce residues derived from the lflf. In the 10-20cm depth input C to the 3-yr nd 4-yr rottions ws over 600% nd 200%, respectively, tht of the 2-yr rottion. This difference is due to the lrge inputs to the 10-20cm depth from moldbord plowing before the corn yer. About g C cm -3 ws dded s composted mnure before corn in the 3-yr nd 4-yr rottions, n nnul verge of 23g C m -2 nd 17g C m -2 respectively, bout 33% nd 56% of the men C inputs to their 10-20cm depths.. Estimted nnul N inputs differed mong ll three rottions (p<0.0001), with the 3-yr nd 4-yr rottions receiving 24% nd 40% less externl N respectively, thn the 2-yr rottion for the 2009 seson (Tble 2). Indictors of soil qulity Prticle size nlysis Texture vried considerbly cross the experimentl sites. Soils were loms nd silty cly loms, with snd ( m), silt (20-53 m) nd cly (<2 m) concentrtions rnging from 21 to 46%, 30-47% 16

23 nd 19-35%, respectively. The 10-20cm depth hd more fine prticles thn the 0-10cm depth nd the 3- yr nd 4-yr rottions hd higher concentrtion of fines thn the 2-yr rottion (dt not shown). Texture ws therefore used s covrite for the other nlyses. Physicl indictors: bulk density, percent wter-filled pore spce nd wet ggregte stbility There were no system-bsed differences for ny of the physicl nlyses when differences mong rottions were mde cross both depths. Spring bulk density vried between depths nd yers (Tble 3), being higher in the 10-20cm depth thn the 0-10cm depth. Bulk density tended to be low, between 0.8 nd 1.2g cm -3 in the 0-10cm depth nd between g cm -3 in the cm depth, nd did not differ between systems (Tble 4). The lck of yer interctions shows the results to be stble cross both smpling yers. Crop(system), depth nd yer, nd the interction between crop(system) nd depth were ll strongly significnt for spring WFPS (Tble 3). The 10-20cm depth hd higher WFPS thn the 0-10cm depth. Overll the 2-yr rottion hd significntly higher WFPS thn the 4-yr rottion, nd mrginlly higher thn the 3-yr; however, these differences were only significnt in the 0-10cm depth (Tble 4). Agin, no yer interction ws observed. Spring %WSA showed significnt differences between depths, the interction between crop(system) nd depth, yer, nd the interction between depth nd yer. The 10-20cm depth hd significntly higher %WSA thn the 0-10cm depth. There ws no overll difference between rottions, but t the 0-10cm depth the 3-yr rottion hd lower %WSA thn the 2-yr rottion (Tble 4). Spring of 2010 hd greter %WSA thn spring of 2009, but only in the 0-10 cm depth (dt not shown). 17

24 Soil orgnic mtter When normlized for bulk density nd clculted on volume bsis (mg SOM cm -3 ), SOM stocks were found to be higher in the 10-20cm thn the 0-10cm depth in ll rottions (Tble 4). However, when nlyzed on weight bsis (g SOM g -1 ) the 0-10cm depth hd higher concentrtion (dt not shown). No system differences were observed t either depth. This is consistent with nnul nlyses done on soils tken from the 0-20cm depth since 2002, which show considerble yerly fluctutions but no significnt overll chnge in SOM content (M. Liebmn, personl communiction, dt not shown). As bulk density ws not mesured in these yers, trends in SOM my be msked by chnges in the effective depth of smpling. Chemicl nd biologicl indictors: prticulte orgnic mtter C, potentilly minerlizble N, enzymtic ctivity Spring POM-C vried significntly by crop(system), depth, the interction of crop(system) nd depth, nd the interction of depth nd yer (Tble 3). Averged cross both depths, both the LEI rottions hd higher men POM-C thn the 2-yr rottion (Tble 4). This difference ws result of the strong strtifiction in the 2-yr rottion, which ws bsent in both LEI rottions (Tble 4). The POM-C content of the 2-yr rottion, while not differing from the 3-yr rottion nd higher thn the 4-yr rottion in the 0-10cm depth, ws gretly depleted in the 10-20cm depth compred to both LEI rottions (Tble 4). On verge, POM-C ws greter in the 0-10cm depth thn in the 10-20cm depth ; however, this difference ws only significnt in the 2-yr rottion. The POM-C ws greter in the spring of 2009 thn 2010, but only t the 0-10cm depth (dt not shown). Distribution of potentilly vilble N mirrored tht of POM-C. For PMN s well, depth nd the interction between depth nd crop(system) were strongly significnt due to the 2-yr rottions depletion in the 10-20cm depth (Tble 3, 4). Overll, the 3-yr rottion hd significntly nd the 4-yr 18

25 rottion mrginlly significntly greter PMN thn the 2-yr rottion (Tble 4). Unlike for POM-C, concentrtions did not differ mong the rottions t the 0-10cm depth. In enzymtic ctivity (FDA) the strong effect of depth nd interction of depth with crop(system) ws gin present, long with yer effect (Tble 3). This is not surprising given the different moisture conditions t time of smpling in the two yers. While the 2-yr rottion hd lower FDA tht either of the LEI rottions in the 10-20cm depth, it ws higher thn either in the 0-10cm depth nd there ws no overll difference between the rottions. The FDA strtifiction rtio for the 2-yr rottion, while still significntly greter thn for either of the LEI rottions, ws less thn for POM-C or PMN (Tble 4) Comprtive plnt response Root prmeters Root prmeters were nlyzed from the midsummer smpling only. Summer RLD differed significntly by depth nd the depth by crop(system) interction (Tble 3). Higher verge RLD ws observed in the 4-yr rottion thn in the 2-yr rottion overll (Tble 4). Agin, vlues for the 2-yr rottion in the 10-20cm depth were significntly lower thn those in the LEI rottions (Tble 4). The strtifiction rtio in the 2-yr rottion ws higher thn in the 4-yr rottion, but not the 3-yr (Tble 4). Ptterns in the corn roots prlleled the distribution of soil lbile C, in tht RLD in the 2-yr ws more strtified thn tht observed in the 3-yr nd 4-yr rottions (Figure 2). The sme pttern ws suggested in the soyben roots, but depth-bsed differences were not significnt. Pre-plnned contrsts to determine differences in corn RLD between rottions reveled the root length of 2-yr corn to be equl to tht of the 3-yr corn but significntly less thn tht of 4-yr corn (Tble 6). There were no rottion differences between soyben root lengths. Averge RLD in the ots, lflf nd red clover plots ws higher thn tht in corn or soybens (Tble 6). While root C:N did not significntly differ by crop(system), contrsts reveled the root C:N rtio of the 4-yr soybens to be significntly lower thn 19

26 tht of the 2-yr soybens (Tble 7). Regression nlysis reveled no liner reltionship between bulk density nd either RLD or verge dimeter (Tble 6). Corn nd soyben productivity Grin yields in 2009 in the LEI rottions were somewht higher thn those chieved in the 2-yr rottion (Tble 5). Averge 3-yr nd 4-yr corn yields were 10% nd 8% higher thn conventionl yields, respectively, while 3-yr nd 4-yr soyben yields were 13% nd 18% higher (Tble 5). The difference ws mildly significnt in the 3-yr corn (p=0.09) nd the 4-yr soybens (p=0.09). These yields re consistent with those seen in previous yers. Hrvest index, clculted s grin dry weigh/totl boveground plnt dry weight, did not differ mong the rottions (dt not shown). Sesonl differences in soil qulity Soil physicl indictors: bulk density, percent wter-filled pore spce nd wet ggregte stbility Sesonl nlysis of physicl indictors BD, WFPS nd %WSA showed strong depth-bsed differences nd strong fluctutions through the growing seson (Tble 8). However, no crop or crop(system)-bsed differences were observed in ny of the three indictors when verged over ll depths nd sesons (Tble 9). Vlues for ll three mesures observed in the 10-20cm depth were greter thn those seen in the 0-10cm depth. Overll, BD nd WFPS were highest in spring, declined in summer nd rose gin in fll. Conversely, %WSA rose from spring to summer, nd declined shrply in fll. While crop(systems) did not differ overll, vribles behved differently t different depths for ll physicl indictors (Figure 3). For corn nd soybens the 0-10cm depth hd higher BD nd WFPS thn the 10-20cm depth, but this difference ws not significnt for the ot, red clover or lflf. When 20

27 depth differences in %WSA were compred for individul crop(systems), s well, ggregtion in the 0-10cm depth ws only significntly lower thn tht in the 10-20cm depth for the 3-yr nd 4-yr soybens. For ll three indictors, sesonl behvior differed t different depths. For BD, the increse from summer nd fll ws due to n increse the 10-20cm depth. Similrly the difference between spring nd fll WFPS ws observed in the 10-20cm depth but not the 0-10cm depth. An increse in %WSA from spring to summer ws seen only in the 0-10cm depth. For ll three physicl indictors, strtifiction rtios were more vrible in the 3-yr rottion thn in the other systems. In the 3-yr rottion, the rtio of BD, WFPS nd %WSA in the surfce to subsurfce soil ws consistently lower in the soybens thn in the ots or the corn (Tble 9). This pttern ws not observed in the other rottions. In the 4-yr rottion, WFPS ws more strtified in the corn thn in the ots or the lflf. For WFPS strong three-wy interction with crop(system), depth nd seson ws observed. For ll cropping phses besides the mixed ot nd legume yers in the 3-yr nd 4-yr rottions, WFPS rose between summer nd fll. However, in the ot nd red clover phses WFPS did not rise gin in fll, nd in the ot nd lflf phse it rose in fll but did not rech the sme level it hd in spring (Tble 10). For both LEI systems, this ws only true in the 10-20cm depth (dt not shown). Model selection procedures in PROC REG used to identify the soil vribles tht best predicted %WSA showed tht in the 0-10cm depth enzymtic ctivity, whole soil N nd cly were positively relted to ggregte stbility nd tht WFPS ws negtively relted (Tble 11). At the lower depth, whole soil N nd cly were gin chosen; these vribles nd POM C:N were positively ssocited with stbility t tht depth. The model in the cm depth ccounted for more of the vribility thn ws ccounted for in the 0-10 cm depth (Tble 11). When summer %WSA ws modeled by itself, with root length s one of the vribles given to the RSQUARE procedure, the procedure still identified texturl vribles s being importnt, long with mesure dynmic C PMN or FDA. 21

28 Chemicl nd biologicl indictors: prticulte orgnic mtter C, potentilly minerlizble N, enzymtic ctivity All three chemicl nd biologicl indictors- POM-C, PMN nd FDA- behved similrly to ech other nd confirmed the ptterns observed in the spring system comprisons. All three indictors strongly differed by depth nd the interction of crop(system) with depth (Tble 8). Although for ll indictors, overll mens vlues in the 0-10 cm depth were higher thn those found in the cm depth, differences were only significnt in the 2-yr corn nd 2-yr soybens. Even though not significnt, the 4-yr corn nd soybens tended to hve higher POM-C, PMN nd FDA in the 10-20cm depth thn in the surfce depth (Figure 4). Surprisingly, temporl smpling showed tht POM-C, PMN nd FDA did not differ between crops, nd tht the POM-C depth strtifiction rtios did not chnge with ny of the crop phses of the rottions (Tble 9). Soil POM-C levels did differ over the course of the growing seson, nd seson intercted significntly with crop(system) (Tble 8). Spring POM ws mrginlly higher thn fll POM, lthough the difference ws only significnt in the 3C crop(system) (Tble 10). The difference between spring nd fll ws significnt when verged over ll crops, but only t the 0-10cm depth (dt not shown). Neither PMN nor FDA differed by seson or hd ny significnt sesonl interctions. Averge PMN ws mrginlly higher under ot thn under soyben (Tble 9). Strtifiction rtios for PMN lso remined stble over the course of the rottion in ll rottions. For FDA, however, strtifiction rtios vried somewht within the rottions. In the 2-yr rottion, the corn hd mrginlly greter strtifiction thn the soybens. In the 3-yr rottion, the ots hd mrginlly greter strtifiction thn the soybens, nd in the 4-yr rottion, the lflf hd mrginlly greter strtifiction thn the corn (Tble 9). 22

29 Discussion Objective I Our first objective ws to quntify the effects of cropping system nd component prctices (tillge, residue nd mnure dditions, nd rottion length,) on cumultive chnges in physicl, chemicl nd biologicl indictors nd plnt productivity. Improvements in soil qulity indictors nd plnt productivity were expected to be driven by the mount nd plcement of orgnic residues The yer vrible showed the rndom error ssocited with climctic vribles or input differences in given yer. Significnt differences between spring of 2009 nd 2010 were expected, s smples were tken t different moisture nd temperture, nd hd hd different mounts of inputs. The insignificnt crop(system) by yer nd crop(system)*depth*yer interctions showed tht the differences observed between crop(systems) nd strtifiction ptterns were consistent chrcteristic of the rottions nd conclusions were pplicble to yers beyond those smpled. The significnt depth by yer interction, observed only in POM-C nd %WSA, gve n indiction both of how consistent observed differences between depths were nd the reltive vribility of ech depth. Rottion effect on physicl chrcteristics Our hypothesis of improved soil physicl qulity under the LEI rottions ws not supported. The fct tht bulk density did not differ between rottions despite different tillge nd orgnic input regimes suggests either tht this mesure ws not very sensitive to mngement, or tht dmge due to tillge ws offset by orgnic mtter dditions. Ll (1999) nd Puget nd Ll (2005) found similrly tht tillge hd no effect on bulk density in tillge experiments in Ohio, nd Krlen (2006) noted tht of soil qulity indictors used to chrcterize different rottions in Wisconsin nd Iow, bulk density ws the lest sensitive. It lso my be tht in these very low bulk density soils, the rnge of bulk density is so smll tht chnges re difficult to detect. Soil WFPS, conversely, ws significntly higher in the 2-yr thn in the 23

30 LEI rottions, difference which cme from the lower WFPS in the 0-10cm depth of LEI rottions. The tendency of less-tilled surfce soil to retin greter WFPS is well-documented (i.e. Linn nd Dorn, 1984; Hill et l., 1985; Frnzluebbers nd Arshd, 1996; Chvez et l., 2009), nd my be due to greter proportion of mcropores in conventionlly-tilled soils (Hill et l., 1985). The overll greter spring WFPS in the 2-yr rottion, prticulrly in the 0-10cm depth, suggests tht this effect is occurring in the Mrsden soils. Contrry to wht ws expected, %WSA ws not incresed in the LEI rottions compred to the 2-yr rottion, despite their greter POM-C nd root presence. Rther, %WSA differed minly between depths nd yers like the other physicl indictors. The decresed %WSA in the 0-10cm depth of the LEI rottions is likely ssocited with their more intrusive tillge, which hs frequently been shown to dmge soil surfce structure (i.e. Liebig et l., 2004; Chung et l., 2008), nd would, long with the decresed WFPS, indicte tht tillge to some extent dmged the surfce soil structure. This is especilly true for the 3-yr rottion, which ws the most frequently tilled. However, the 10-20cm depths were like in ll three rottions, suggesting the dmge did not ffect the subsurfce, or ws offset by the dditionl C inputs. The greter vribility in bulk density, WFPS nd %WSA ssocited with the top depth, coupled with the fct tht they re consistently higher in the 10-20cm depth suggests tht ll three mesures incresed with lck of disturbnce. Rottion effect on chemicl nd biologicl chrcteristics Overll, soil chrcteristics did not chnge in mnner tht reflected the pttern of residue C inputs from the previous yer. Although the 3-yr received significntly greter verge nnul dditions to both depths, both LEI rottions behved very similrly in terms of POM, PMN nd FDA, which should be strongly influenced by orgnic mtter. The 2-yr rottion received higher orgnic mtter inputs thn the 4-yr rottion, nd yet ws comprtively depleted in lbile C. Volumetric SOM concentrtion did not 24