Microbial community dynamics and function associated with rhizosphere over periods of rice growth

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1 Miroil ommunity ynmis n funtion ssoite with rhizosphere over perios of rie growth Q. Hussin 1,2, G.X. Pn 1, Y.Z. Liu 1, A. Zhng 1, L.Q. Li 1, X.H. Zhng 1, Z.J. Jin 1 1 Institute of esoures, Eosystem n Environment of Agriulture, Nnjing Agriulturl University, Nnjing, P.. Chin 2 Deprtment of Soil Siene n Soil Wter Conservtion, Pir Mehr Ali shh, Ari Agriulture University, wlpini, Pkistn ASTACT A fiel experiment ws onute to illustrte the ifferent egree n ynmis of miroil ommunity struture n funtion in the rhizosphere ross four growing stges (efore plnttion n three growth stges) using omintion of iohemil (enzyme ssy n miroil iomss ron) n moleulr pprohes of qpc n PC-DGGE (polymerse hin retion-enturing grient gel eletrophoresis). ie plnt ultivtion promote higher enzyme tivities (invertse n urese), miroil iomss ron (C mi ), teril (16S rna) n fungl (ITS rna) genes unnes in the rhizosphere ompre to unplnte soil. Prinipl omponent nlyses of PC-DGGE profile lso revele tht strutures of teril n fungl ommunities of rie plnte soil were well istint from unplnte soil. Moreover, enzyme tivities showe signifint positive orreltion with the totl miroil iomss in the rhizosphere throughout growth stges of rie plnt. eltive fungl: teril rtios were signifintly higher in rie plnte soil ompre to unplnte soil, suggesting rie plnttion enhne the fungl ommunity in the rie rhizosphere environment. These results further suggest signifint linkge etween the miroil ommunity ynmis n funtion in the rhizosphere ssoite with rie plnt over time. Keywors: Oryz stiv; temporl effet; sptil effet; symiosis of plnts n soil miroflor; enzyme tivities; fiel eosystems ie eosystems remin flooe through mjor prt of the ropping perio n re istintive from upln soils in severl physiohemil n iologil properties. Therefore, flooe rie fiels eme moel system to stuy soil miroil eology (Leisk et l. 2). In plnte soil the rie renhymtous tissue is lso responsile for the lekge of oxygen reting n oxi rhizosphere within the noxi ulk soil (evseh et l. 1999). Moreover, plnt rhizoeposition in the rhizosphere results in inrese miroil popultion size n ommunity strutures istint from tht in ulk soil (is et l. 26). Hene, oxygen n ron-relesing erenhymtous rie plnts my ffet the unne, strutures n funtion of the miroil ommunity in the rhizosphere of wterlogge py soil. The hnges in overll miroil ommunity strutures n omposition n e orrelte with hnges in ertin funtions oth uner ontrolle lortory onitions (Avrhmi et l. 23) n in the fiel (Mrshner et l. 21). ut our unerstning out the linkge etween miroil ommunity struture n funtion in the rhizosphere of rie rop is poorly unerstoo. A signifint reserh ws one out the influene of plnt on the ynmis of teril ommunities se on 16S rna nlysis (Mrshner et l. 21, Smll et l. 21). Few stuies investigte the strutures n eosystem funtioning of fungl ommunities in the py soil; to our knowlege, none hve investigte the reltive fungl n teril unnes in rhizosphere ross rie growth stges uner fiel onitions. The ojetives of this stuy were therefore (i) to evlute the linkge etween totl miroil iomss n funtion ross ifferent growing stges; (ii) to ompre the strutures of teril n fungl ommunities in rie plnte soil n unplnte soil; (iii) to ssess the reltive unnes of fungl n teril popultions in plnte soil n unplnte soil t vrious growth stges. Supporte y the Ntionl Siene Fountion of Chin, Projet No PLANT SOIL ENVION., 58, 212 (2):

2 MATEIAL AND METHODS Site esription n experiment lyout. The experiment ws rrie out t rie frm lote in Yixing City, Yifeng Villge ( 'N, 'E), Jingsu Provine, Chin. Derive from lustrine eposit, the soil ws typil high-yieling py soil lssifie s hyrogri Stgni Anthrosols n n enti Hlpuept oring to Soil Survey Stff, USA. The si properties of the stuie top soil ( 1 m) re mentione in Tle 1. One month ol seelings of rie (Oryz stiv), Wugeng13 ultivr, were plnte in rnomly selete plots t plnt ensity of 2 plnts/m 2. The experiment use rnomize lok esign with three replite plots. The size of eh plot ws 4 m 5 m. The fiel ws kept most of the time flooe with irrigtion wter. No hemils were pplie for plnt protetion n the plots were weee y hn. hizosphere n ulk soil smples were ollete on 45 ys fter plnting (tillering stge-s1), 81 ys fter plnting (grin filling stge-s2) n 17 ys fter plnting (ripening stge-s3). Unplnte ulk soil (S) ws lso ollete efore rie plnttion from eh plot to investigte the effet of the rie plnt ultivtion on the miroil ommunities. Soil smpling. For smpling of rhizosphere soil, ten rie plnts with root-soil systems were rnomly slie own 1 m eep in the mile from eh plot n shken up in plsti gs until pproximtely four-fifths of the initil weight (1 g) ws in the g; this portion ws onsiere s ulk soil. The remining fifth tht ws still tthe to the root system n ws onsiere s rhizosphere soil (utler et l. 23). The rhizosphere n orresponing ulk soil smples of the sme plot t the sme plnt growth stge were susequently poole to mke one omposite smple, respetively. Soil smples were sieve (< 2 mm) n store t 4 C for nlysis of miroil iomss C, N n enzymes tivity n t 2 C for DNA extrtion, n prt of sieve soil smples ws ir rie for physil n hemil nlyses. Miroil iomss C n N etermintion. Miroil iomss ron (C mi ) ws etermine y the fumigtion-extrtion metho s esrie y Vne et l. (1987). C mi ws lulte from (extrtle ron/.45, where extrtle ron = (C extrte from fumigte soil) (C extrte from non-fumigte soil). Miroil iomss N (N mi ) ws estimte s (extrtle nitrogen)/.54, where extrtle nitrogen = (totl N extrte from fumigte soils) (totl N extrte from non-fumigte soils). Enzyme tivity ssy. Enzyme tivities were etermine on fresh moist sieve (< 2 mm) soils within 15 2 ys from the olletion of the smples. Two enzymti tivities (invertse n urese) were nlyze oring to the protools esrie y Gu et l. (29). In se of invertse, 5 g of soil (< 2 mm) ws mixe with.2 ml of toluene n 5 ml of phosphte uffer (ph 5.5) in 5-mL Erlenmeyer flsks, then 15 ml of the 8% surose solution ws e, n the flsks were swirle for few seons. The flsks were overe with stopper n ple in n inutor t 37 C for 24 h. After inution, solution ontents were psse through filter pper. The 1 ml of filtrte ws pipette into 5 ml test tue n 3 ml 3, 5-initrosliyli i monohyrte solution ws e. Then ll tues were ple into oiling wter th for 5 min n llowe to ool t room temperture with tp wter. Further, the finl volume of the eh solution ws me up to 5 ml with H 2 O. The gluose proue ws etermine olorimetrilly t 58 nm n expresse s mg gluose/g soil/24 h. Urese tivity ws etermine using 5 g of soil (< 2 mm) tht ws mixe with 1 ml of toluene in 5-mL Erlenmeyer flsk, n llowe to stn for 15 min. Then 1 ml 1% ure solution n 2 ml itrte uffer (ph 6.7) were e n mixe well. The flsks were overe n ple in n inutor t 37 C for 24 h. After inution, soil solution ontents were psse through filter pper. Moreover, 3 ml of liquot from filtrte ws e into 5-mL test tue long with 4 ml soium phenol solution n 3 ml soium hypohlorite solution n tues were swirle well for mixing. After 2 min, the finl volume of eh solution ws me to 5 ml with H 2 O. The relese mmonium ws mesure olorimetrilly t 578 nm n expresse s mg NH 4 -N proue/g soil/24 h. Control tests with utolve soils were rrie out to evlute the spontneous or ioti trnsformtion of sustrtes. All hemil etermintions n enzymti tivities were etermine in triplite. Tle 1. si physio-hemil properties of soil ( 1 m) ph (H 2 O) CEC (mol + /kg) Cly (<.2mm) (g/kg) ulk ensity (g/m 3 ) Totl ron (g/kg) Totl nitrogen (g/kg) Alkli-relesle nitrogen (mg/kg) PLANT SOIL ENVION., 58, 212 (2): 55 61

3 Tle 2. Primer sets n therml profiles use for the DGGE n qpc nlysis Trget gene 16S rna (teri) 18S rna (Fungi) 16S rna (teri) ITS rna (Fungi) Primer set Size (p) 338-F-GC NS1 Fung-GC F ITS-F 5.8s- 35 Therml yling profile 94 C (5 min); 35 yles of 94 C (6 s), 55 C (6 s), n 72 C (6 s) 94 C (5 min); 35 yles of 94 C (6 s), 58 C (6 s), n 72 C (6 s) 95 C (5 min); 4 yles of 94 C (3 s), 55 C (6 s), n 72 C (6 s) 95 C (5 min); 4 yles of 94 C (3 s), 53 C (6 s), n 72 C (6 s) eferene Muyzer et l. (1993) My et l. (24) Fierer et l. (25) Fierer et l. (25) primer set use for DGGE (enturing gel grient eletrophoresis); primer set use for qpc (quntittive polymerse hin retion) Moleulr n sttistil nlysis. Three DNA extrtions of eh soil smple (.5 g) from the sme replite fiel plot were me using PowerSoil TM DNA Isoltion Kit (Mo io Lortories In., Crls, USA) following the mnufturer s instrutions. eltive teril n fungl unnes were estimte using rel time PC (qpc), with primers n the therml yling onitions esrie in Tle 2. The DNA onentrtion ws mesure t 26 nm using UV spetrophotometer (io Photometer, Eppenorf, Germny). Eh qpc retion ws performe in 25 µl volume ontining 1 ng of DNA,.2 mg/ml SA,.2 µmol of eh primer n 12.5 µl of SY premix EX Tq TM (Tkr Shuzo, Shing, Jpn). The size of PC prout ws onfirme y melting urve nlysis n eletrophoresis in 1.5% grose. A plsmi ontining trget region of teril (16S rna) n fungl (ITS rna) gene ws use to onstrut stnr urve. The stnr urves were generte using triplite 1:5 seril ilutions of plsmi DNA rnge from to opies for teril 16S rna gene n to opies of templte for fungl ITS rna gene per ssy, respetively. Denturing grient gel eletrophoresis (DGGE) ws performe to ssess the teril n fungl ommunities struture using 338F-GC n 518 primers set (Muyzer et l. 1993), n the NS1 n Fung-GC (My et l. 21), respetively. For DGGE nlysis, PC prouts were seprte on 8% (w/v) polyrylmie gels (rylmieisrylmie [37.5:1]) ontining enturing grients of 35% to 7% for teri n 1% to 4% for fungi using the io- D-Coe universl muttion etetion system. A 1% enturnt ws efine s 8% rylmie ontining 7 mol/l ure n 4% eionise formmie. DGGE ws performe using 2 µl of the PC prout in 1 TAE uffer t 6 C, 2 V for 5 min, then 1 V for 12 h (teri) n 14 V for 5.5 h (fungi). Gels were stine with silver stining n were snne with gel oument system (io-, Herules, USA). All sttistil nlyses were performe using Minit v The signifine level ws fixe t P <.5. Vrition in enzyme tivities n totl miroil iomss in rhizosphere ross rie growth stges. Soil enzyme tivities re often use s inies of miroil funtion n soil fertility n re gretly ffete y qulity n quntity of root exutes epening on the plnt speies, ultivr, plnt growth stge n physiologil sttus of the plnt (Sxen et l. 1999). Enzymes tivities (invertse n urese) inrese with rie plnt t the grin filling stge (S2) n then eline t the ripening stge (S3) (Figure 1). This phenomenon ws lso oserve y Gu et l. (29) in py soil uner fiel onitions. The reue enzyme tivities t lter growth stges of rie plnt my pper ue to the reson tht roots relese less orgni ompouns into soil to support enzyme tivities in py soil (Lu et l. 22). A signifint positive orreltion ws oserve etween enzyme tivities (urese n invertse) n totl miroil iomss (C mi ) in rhizosphere ross ll smpling time points (Tle 3). Severl stuies lso reporte tht hnges in overll miroil ommunity strutures n omposition n e orrelte with the hnges in ertin funtions oth uner ontrolle onitions (Avrhmi et l. 23) n in the fiel (Mrshner et l. 21). Moreover, the temporl vriility of miroil iomss ynmis is omplex n poorly unerstoo in rie soils. Previous stuies revele tht with evelopment of rie plnt, C mi ws inrese (Hoque et l. 21) or erese (eihrt et l. 1997), or no ifferene etween plnte n unplnte soils (Witt et l. 2) ws oserve. In orne with the finings y Lu PLANT SOIL ENVION., 58, 212 (2):

4 () Invertse (mg gluose/g DW/24 h) hizosphere ulk soil () + Urese (mg NH 4 -N/g DW/24 h) no plnt growth stges no plnt growth stges Figure 1. Invertse () n urese () tivities in the rhizosphere n ulk soil t the S1 (tillering), S2 (grin filling) n S3 (ripening) growth stges. Different letters inite signifint ifferenes y ANOVA t P <.5 (n = 3; error rs re ± SD) et l. (22) n Zeng et l. (25), we oserve tht C mi in rhizosphere inrese uring erlier growing perios (S1-tillering n S2-grin filling) n eline t the lter stge (S3-ripening stge, Tle 4). eltive teril n fungl unne in rhizosphere uring growth perios of rie plnt. As C mi mesures the totl miroil iomss in soil, inluing teril, rhel, n fungl omponents, A rel-time PC pproh ws use to further estimte the totl teril popultion (16S rna gene), totl fungl popultion (ITS rna gene) n reltive fungl: teril rtio. The ultivtion of rie rops h strong stimulting effet on the numers of totl teri n fungi in the py soil (Figure 2). The numer of 16S rna n ITS rna gene opies in the rhizosphere were signifintly higher thn in ulk soil ross eh growth stge. This my e ue to the rhizoeposition of rohyrtes from plnt roots inresing miroil growth in omprison with tht in the ulk, phenomenon well known s rhizosphere effet (Smll et l. 21, Mougel et l. 26). It is lso well known tht rie roots relese oxygen through erenhymtous tissue t rtes suffiient to support non-speifi eroi miroil proesses (Arth et l. 1998). On the ontrry, in the ulk soil there oul only e miroil respirtion ut lower oxygen onentrtions. The reltive fungi: teri rtio ws lso use s potentil tool to stuy the stte of soil miroil ommunity in response to ifferent environmentl stresses in vrious eosystems (Fierer et l. 25). Luer et l. (28) rgue tht reltive fungl: teril rtios (rna-qpc se) were not signifintly ifferent ross the ln-use types (hrwoo n pine forests, ultivte n livestok psture lns). Our stuy revele tht the rtio of fungi to teri (rna-qpc se) rnge from.3 to.6 ross ll growth stges, whih inite tht teril popultion ominte the rie eosystem (Figure 2). We oserve non signifint ifferene in fungi to teri rtio ross the rie plnt growth stges, suggeste tht the oserve ptterns were not riven y ny prtiulr miroil group. Although rel-time PC is flexile, simple n rpi promising moleulr tool for the quntifition of soil miroil ommunities, it hs some importnt limittions. These limittions inlue DNA extrtion is, the vilility of sequene t, n equte preprtion of inhiitor-free trget DNA. However, qpc provies reprouile metho to monitor ifferenes n hnges in miroil popultion size within growth stges. Tle 3. Person orreltion etween enzyme tivities n miroil unnes in rhizosphere ross ll smpling time points (n = 4) Urese Invertse C mi (m/kg) teril 16S rna gene opies/g ry soil Fungl ITS rna gene opies/g ry soil.956* * (.44) (.68) (.27).966* (.34) (.175) (.113) *signifint oeffiients (P <.5) re highlighte in ol font. Vlues in rket show the level of signifine 58 PLANT SOIL ENVION., 58, 212 (2): 55 61

5 Tle 4. C mi (mg/kg), N mi (mg/kg) n C mi /N mi (n = 3; men ± SD) in rhizosphere n ulk soil t ifferent smpling points S S1 S2 S3 C mi ± ± ± ± ± ± ± 13.2 N mi 62.1 ± ± ± ± ± ± ± 2.2 C mi /N mi 9.61 ± ± ± ± ± ± ±.4 S efore plnttion ulk soil; S1 tillering stge; S2 grin filling stge; S3 ripening stge; rhizosphere; ulk soil. Different letters in the sme row inite signifint ifferenes (P <.5) Dynmis of teril n fungl ommunity struture in response to rie evelopmentl stges. The use of moleulr pprohes provies vlule informtion out the hrteristion of nturlly ourring miroil ommunities without ultivtion (Šturs et l. 29). Miroil ommunities in the rhizosphere re ynmi n suseptile to hnges in plnt onitions. In response to hnging root exution ptterns with the evelopment of plnt, the miroil ommunity struture n omposition in the rhizosphere lso hnge with time n vries uring the life yle n with the sesonl response of plnts (Germi n Siilino 21). In the urrent stuy, prinipl omponent nlysis of DGGE profile of the 16S rna gene (teril) n the 18S rna gene (fungl) revele ifferenes in oth the teril () 16S rna gene opies/g ry soil 2.5E+6 2.E+6 1.5E+6 1.E+6 5.E+5.E+ hizosphere ulk soil n fungl ommunity strutures ross ifferent growth stges (Figure 3). We foun tht teril n fungl ommunities of unplnte soil (S) n plnte soil were well seprte, showing the well profoun plnt rhizosphere effet. In ft, the introution of plnts to soil ffets the physilhemil properties n the iologil prmeters of the soil environment lose to the growing roots y ontinuously prouing n exreting orgni ompouns (Hinsinger et l. 26). Houlen et l. (28) performe fiel stuy to explore miroil ommunity struture n tivity in the rhizosphere of three fiel rops (pe, whet n sugr eet) over perio of growth. y using DGGE n IOLOG tehniques, they oserve remrkle temporl shifts in iversity n reltive tivity in rhizosphere teril n fungl ommunities with () ITS rna opies/g ry soil 1.2E+5 1.E+5 8.E+4 6.E+4 4.E+4 2.E+4.E+ no plnt growth stges no plnt growth stges () eltive fungi: teri rtio no plnt growth stges Figure 2. Aunne of totl teri (), totl fungi () n reltive fungi: teri rtio () se on the rel-time PC in rhizosphere n ulk soil t the S (no plnt), S1 (tillering), S2 (grin filling) n S3 (ripening) stges. Different letters inite signifint ifferenes y ANOVA t P <.5 (n = 3; error rs re ± SD) PLANT SOIL ENVION., 58, 212 (2):

6 S NP S1 S2 S3 Seon omponent (21.2%) Seon Component (21.2%) S S1 S1 S3 S2 S2 S3-3 S NP S1 S2 S3 Seon omponent (2.2%) Seon Component (2.2%) S First Component omponent (52.3%) S1 S2 S1 S3 2.5 S2 S First Component omponent (41.4%) 5. Figure 3. Prinipl omponent nlysis (PCA) of enturing gel grient eletrophoresis (DGGE) profile of teril (, ) n fungl (, ) ommunities in the rhizosphere () n ulk () soil t S (no plnt, NP), S1 (tillering), S2 (grin filling) n S3 (ripening) stges of rie plnt. Soli n hollow symols inite the rhizosphere (); ulk () soil evelopmentl stge for ll plnt speies. Further, pot n fiel experiments onute y Xu et l. (29) inite tht teril ommunities in the soyen rhizosphere hnge with growth stges, n higher numer of DGGE ns oserve in erly reproutive growth stges thn in the other stges. Mougel et l. (26) lso hrterize the teril n fungl ommunity struture in rhizosphere of Meigo truntul t five evelopmentl stges in pot experiment y using the utomte riosoml intergeni sper nlysis (AISA). Similrly, we foun pronoune hnges in the geneti iversity of teril n fungl ommunities uring vegettive stge (tillering- S1) n reproutive stge (ripening-s3) of plnt growth evelopment (Figure 3). The ifferene in miroil ommunities struture etween the growth stges my e ue to weker rhizoeposition uring the reproutive stges thn uring the vegettive stge (Mougel et l. 26). A ertin egree of suession ours in the teril n fungl ommunities strutures of the rhizosphere etween vegettive (tillering) n reproutive (grin filling n ripening) stges. Interestingly, rie plnt growing stges showe no signifint effet on reltive fungl: teril rtio, suggeste tht growth stge i not stimulte ny prtiulr miroil group. However rie plnt stimulte higher reltive fungl: teril rtio ompre to unplnte tretment. This stuy illustrtes the signifine of rie eosystem in ontext of rhizosphere miroil ommunity ynmis n funtion over time. A future reserh is require to explore the eologil signifine of ifferent rie ultivrs in terms of ifferene in miroil ommunities ynmis n possile inrese respirtion rtes. 6 PLANT SOIL ENVION., 58, 212 (2): 55 61

7 Aknowlegements We re grteful to Dr. Dvi Crowley, University of Cliforni (iversie), USA for ritil evlution n English improvement of this mnusript. The first uthor is thnkful to the Higher Eution Commission, Pkistn for grnting sholrship to pursue his PhD stuy in Chin. EFEENCES Arth I., Frenzel P., Conr. (1998): Denitrifition ouple to nitrifition in the rhizosphere of rie. Soil iology n iohemistry, 3: Avrhmi S., Liesk W., Conr. (23): Effets of temperture n fertilizer on tivity n ommunity struture of soil mmoni oxiizers. Environmentl Miroiology, 5: is H.P, Weir T.L., Perry L.G., Gilroy S., Vivno J.M. (26): The role of root exutes in rhizosphere intertions with plnts n other orgnisms. Annul eview Plnt iology, 57: utler J.L., Willims M.A., ottomley P.J., Myrol D.D. (23): Miroil ommunity ynmis ssoite with rhizosphere ron flow. Applie n Environmentl Miroiology, 6: Fierer N., Jkson J.A., Vilglys., Jkson.. (25): Assessment of soil miroil ommunity struture y use of txon-speifi quntittive PC ssys. Applie n Environmentl Miroiology, 71: Germi J., Siilino S. (21): Txonomi iversity of teri ssoite with the roots of moern, reent n nient whet ultivrs. iology n Fertility of Soils, 33: Gu Y., Wng P., Kong C.H. (29): Urese, invertse, ehyrogense n polyphenoloxise tivities in py soil influene y llelopthi rie vriety. Europen Journl of Soil iology, 3: 1 6. Hinsinger P., Plssr C., Jillr. (26): hizosphere: A new frontier for soil iogeohemistry. Journl of Geohemistry Explortion, 88: Houlen A., Timms-Wilson T.M., Dy M.J., iley M.J. (28): Influene of plnt evelopmentl stge on miroil ommunity struture n tivity in the rhizosphere of three fiel rops. FEMS Miroiology Eology, 65: Hoque M., Inuushi K., Miur S., Koyshi K., Kim H.Y., Ok M., Yshi S. (21): iologil initrogen fixtion n soil miroil iomss ron s influene y free-ir ron ioxie enrihment (FACE) t three levels of nitrogen fertiliztion in py fiel. iology n Fertility of Soils, 34: Luer C.L., Strikln M.S., rfor M.A., Fierer N. (28): The influene of soil properties on the struture of teril n fungl ommunities ross ln-use types. Soil iology n iohemistry, 4: Liesk W., Shnell S., evseh N.P. (2): Miroiology of flooe rie pies. FEMS Miroiology eviews, 24: Lu Y., Wtne A., Kimur M. (22): Contriution of plnt-erive ron to soil miroil iomss ynmis in py rie miroosm. iology n Fertility of Soils, 36: Mrshner P., Yng C.H., Lieerei., Crowley D.E. (21): Soil n plnt speifi effets on teril ommunity omposition in the rhizosphere. Soil iology n iohemistry, 33: My L.A., Smiley., Shmit M.G. (21): Comprtive enturing grient gel eletrophoresis nlysis of fungl ommunities ssoite with whole plnt orn silge. Cnin Journl of Miroiology, 47: Mougel C., Offre P., njr L., Corern T., Gmlero E., oin C., Lemneu P. (26): Dynmi of the geneti struture of teril n fungl ommunities t ifferent evelopmentl stges of Meigo truntul Gertn. v. Jemlong line J5. New Phytologist, 17: Muyzer G., e Wl E.C., Uitterlinen A.G. (1993): Profiling of omplex miroil popultions y enturing grient gel eletrophoresis nlysis of polymerse hin retion-mplifie genes oing for 16S rna. Applie n Environmentl Miroiology, 59: eihrt W., Msrin G., Pre., Doll J. (1997): Miroil ommunities of ontinuously roppe, irrigte rie fiels. Applie n Environmentl Miroiology, 63: evseh N.P., Peersen O., eihrt W., riones A. (1999): Mirosensor nlysis of oxygen n ph in the rie rhizosphere uner fiel n lortory onitions. iology n Fertility of Soils, 29: Sxen D., Flores S., Stotzky G. (1999): Insetiil toxin in root exutes from t orn. Nture, 42: 48. Smll K., Wieln G., uhner A., Zok A., Przy J., Kiser S., oskot N., Heuer H., erg G. (21): ulk n rhizosphere soil teril ommunities stuie y enturing grient gel eletrophoresis: plnt-epenent enrihment n sesonl shifts revele. Applie n Environmentl Miroiology, 67: Šturs P., Uhlík O., Kurzwová V., Kouek J., Ionesu M., Strohlm M., Loveká P., Mek T., Mková M. (29): Approhes for iversity nlysis of ultivle n non-ultivle teri in rel soil. Plnt, Soil n Environment, 55: Vne E.D., rookes P.C., Jenkinson D.S. (1987): An extrtion metho for mesuring soil miroil iomss C. Soil iology n iohemistry, 19: Witt C., iker U., Glii C.C., Ottow J.C.G. (2): Dynmis of soil miroil iomss n nitrogen vilility in flooe rie soil mene with ifferent C n N soures. iology n Fertility of Soils, 3: Xu Y., Wng G., Jin J., Liu J., Zhng Q., Liu X. (29): teril ommunities in soyen rhizosphere in response to soil type, soyen genotype, n their growth stge. Soil iology n iohemistry, 41: Zeng L.S., Lio M., Chen C.L., Hung C.Y. (25): Vrition of soil miroil iomss n enzyme tivities t ifferent growth stges of rie (Oryz stiv). ie Siene, 12: eeive on Novemer 25, 21 Corresponing uthor: Prof. Genxing Pn, Nnjing Agriulturl University, Institute of esoures, Eosystem n Environment of Agriulture, Nnjing, P.. Chin e-mil: pngenxing@yhoo.om.n PLANT SOIL ENVION., 58, 212 (2):

Crop Rotations, Reduced Tillage and N Fertilization Effects on Corn Yields And Aflatoxin Levels

Crop Rotations, Reduced Tillage and N Fertilization Effects on Corn Yields And Aflatoxin Levels Crop Rottions, Redued Tillge nd N Fertiliztion Effets on Corn Yields And Afltoxin Levels J.E. Mtoh nd M. Rihrdson Texs AgriLife Reserh nd Extension Center Stte Hwy Corpus Christi, TX 78- jmtoh@g.tmu.edu