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2 Author's personl copy Applied Soil Ecology 70 (2013) Contents lists ville t SciVerse ScienceDirect Applied Soil Ecology journ l h om ep ge: Vermicompost from iodegrded distilltion wste improves soil properties nd essentil oil yield of Pogostemon clin (ptchouli) Benth Rkshpl Singh, Rshmi Singh, Sumit K. Soni d, Shivesh P. Singh, U.K. Chuhn c, Alok Klr d, Biology Centrl Fcility, CSIR-Centrl Institute of Medicinl nd Aromtic Plnts, P.O. CIMAP, Lucknow , Indi Govt. P.G. College, Stn, (M.P.), Indi c School of Environmentl Biology, A.P.S. University, Rew, (M.P.), Indi d Microil Technology Deprtment, CSIR-Centrl Institute of Medicinl nd Aromtic Plnts, P.O. CIMAP, Lucknow , Indi r t i c l e i n f o Article history: Received 5 Septemer 2012 Received in revised form 17 April 2013 Accepted 21 April 2013 Keywords: Ptchouli Bioinoculnts enriched-vermicompost Nutrient sttus Essentil oil yield s t r c t Pre-tretment of ptchouli lignocellullosic distilltion wste with consortium of microes viz. Trichoderm hrzinum ATCC PTA-3701, Pseudomons monteilii HQ995498, Bcillus megterium ATCC nd Azotocter chroococcum MTCC 446 significntly enhnced the io-degrdtion of cellulose, hemicelluloses nd lignin y 58.44%, 29.44% nd 65.23%, respectively nd improved the yield of vermicompost y 15%. Appliction of ioinoculnt-enriched vermicompost (BEVC) in ptchouli reduced the percent disese index (PDI) of Rhizoctoni root-rot y 36.36% nd improved the essentil oil yields y 59.74%, 37.70% nd s compred to plots receiving untreted (not enriched) vermicompost (VC), ioinoculnt-enriched compost (BEC) nd chemicl fertilizers (CF), respectively. Furthermore, BEVC ppliction resulted in mrked improvement in physicl (ulk density nd wter holding cpcity) nd chemicl properties (ph, percent orgnic cron, ville N, P nd K) of the soil. In conclusion, BEVC from distilltion wste cn e sfely utilized s io-orgnic input unmiguously prticulrly in situtions where the use of chemicl fertilizers nd pesticides is restricted like orgnic griculture Elsevier B.V. All rights reserved. 1. Introduction In Indi, out three million tons of distilltion wste (plnt mteril left fter extrction of essentil oil) is generted nnully. This wste rots in the fields during rins, posing sequel of environmentl nd helth prolems (Klr et l., 2002). The frm generted gro-wste is used in lndfill nd smll mount is used s fuel (Kumr nd Shwet, 2011). Disposl of solid wste hs ecome mjor prolem due to shortge of dumping sites nd strict environmentl lws. As result, emphsis is now on eroic composting, tht converts wste into orgnic mnure, rich in plnt nutrients nd humus (Shrm et l., 1999). Vermicomposting is process of ccelerted rekdown of orgnic lignocellulosic wstes y comined ction of microorgnisms (especilly cteri nd fungi) nd erthworms in mesophillic environment (Dominguez, 2004). The resultnt vermicompost cn e utilized s plnt growth medi or soil conditioner (Edwrds nd Arncon, 2004; Singh et l., 2012). Biodegrdtion nd ioconversion of lignocellulosic wstes Corresponding uthor. Tel.: ; fx: E-mil ddress: lok.klr@yhoo.com (A. Klr). hs greter importnce in production of qulity vermicompost rich in eneficil microes, essentil plnt nutrients, humus (Singh nd Shrm, 2002) nd simultneous reduction in vermicomposting period y ccelerting the iodegrdtion process (Kumr nd Shwet, 2011). Lignocelluloses re composed of 30 56% cellulose, 10 27% hemicelluloses, 3 30% lignin, nd % protein (Emtizi et l., 2001) though their composition my vry with plnt. Though most of the orgnic wstes cn e converted into nutrient rich vermicompost, utiliztion of distilled wste is etter option (Klr et l., 2007). Delignifiction of lignocellulosic mteril y fungi nd cteri improves the digestiility of griculturl wstes, wood or strw for niml feed nd reduces cost for pulp nd pper industries (Sinegni et l., 2005; Hu nd Yu, 2005; Johnsson nd Nymn, 1993; Vres et l., 1995). Pre-tretment with cteril inoculnts such s Bcillus shckletonni, Streptomyces thermovulgris, Ureicillus thermosphericus nd ligno-cellulolytic fungus like Trichoderm spp. re generlly used to improve the composting process y reducing the cellulose nd lignin levels of the lignocellulosic wste (Pérez et l., 2002; Vrgs-Grcí et l., 2007; Chndr et l., 2009,, 2010). Few reports suggest tht wste (wood wste, sugr cne wste, rw olive pomce, pepper plnt wste nd crop residues) decomposition cn e esily chieved y microil /$ see front mtter 2013 Elsevier B.V. All rights reserved.

3 Author's personl copy R. Singh et l. / Applied Soil Ecology 70 (2013) inocultion prior to vermicomposting (Kumr et l., 2010; Kumr nd Shwet, 2011; Hdddin et l., 2009; Vrgs-Grcí et l., 2007; Singh nd Shrm, 2002). The resultnt vermicompost hs dvntges in terms of higher mounts of essentil nutrients (N, P nd K) nd useful microes (Singh nd Shrm, 2002; Kumr nd Shwet, 2011). Biodegrdtion cn lso e hstened y synthesis of uxins like indole cetic cid nd gierellins; vitmins like thimine, rioflvin, pyridoxin, cynocolmin, nicotinic nd pntothenic cid nd microes y the A. chroococcum, which my influence the growth of other inoculted microflor (Su Ro, 1982). This process holds promise s n gro-wste mngement for effective recycling of orgnic wstes to the soil nd eco-friendly wy of converting wste into nutrient-rich composts (Bno et l., 1987). Vermicompost with desirle esthetics, high level of plnt growth hormones nd soil enzymes improves microil popultion. It tends to hold more nutrients over longer periods with no or miniml dverse impcts on environment (Ndegw nd Thomson, 2001). It cn lso e used s ioremedil mesure to reclim prolem soils, especilly cid soils, ecuse of the ner-neutrl to lkline ph of vermicompost nd its ility to suppress lile luminium (Mitchell nd Alter, 1993). Ptchouli oil rnks high mong essentil oils (Rekh et l., 2009). There is no synthetic replcement for this oil, which further enhnces its vlue (Rmchndr et l., 2002). Production of ptchouli oil in Indi is low (20 t nnum 1 ) nd to meet its domestic demnd Indi nnully imports over 200 t oil from Indonesi, Mlysi nd Singpore (Jhunjhunwll, 2006). Becuse of the high domestic demnd, the cultivtion of ptchouli is picking up nd s result, it is expected tht higher mount of ptchouli distilltion wste would e generted t frmer s field. The growers of ptchouli crop cn e further enefited if the ptchouli distilled wste is rpidly converted into enriched vermicompost. In generl, conversion of ptchouli distilltion wstes into vermicompost using vermitechnology requires dys (Klr et l., 2002). Further, vermicompost produced from distilltion wste hs een found to e n effective crrier of vrious ioinoculnts (Klr et l., 2010; Singh et l., 2012). Considering the uses nd demnd of ptchouli oil, there is need to develop complete gro-technology (cultivtion nd wste utiliztion) pckge for this crop to improve the yields nd soil helth nd reduce the gp etween demnd nd supply. An effort ws mde to produce n enriched orgnic fertilizer y pre-treting the distilltion wste with efficient ioinoculnts useful s cellulse producers/growth promoters nd suppressors of soil-orne pthogens into the vermicomposting process. The ojective of the present study ws to produce ioinoculnts-enriched vermicompost (BEVC) utilizing ptchouli de-oiled wste which prt from eing rich orgnic source of nutrients nd eneficil microes, would suppress mny soil-orne diseses including Rhizoctoni root-rot/wilt cusing extreme losses in ptchouli, improve iomss yields nd helth of soil. 2. Mterils nd methods 2.1. Microil cultures CSIR-Centrl Institute of Medicinl nd Aromtic Plnts (CSIR- CIMAP), Lucknow, Indi, hs lrge collection of ioinoculnts which hve een screened for their growth promoting nd disese suppressing ctivities in medicinl nd romtic plnts (MAPs). Four microil isoltes: () n efficient growth promoter Pseudomons monteilii ( ntive strin CRC1) (HQ995498, MTCC9796) (Singh et l., 2013) nd lso cellulse producer [(cler re of 18 mm on M9 medium gr mended with 10 g croxy methyl cellulose (CMC) nd 1.2 g yest extrct/l]; () P soluilizer Bcillus megterium (ATCC 14581); (c) n efficient N fixer Azotocter chroococcum (MTCC 446); (d) Trichoderm hrzinum (ATCC no. PTA-3701) [ strin developed y CSIR-CIMAP useful s growth promoter in ptchouli (Puttnn et l., 2010) nd n efficient cellulse producer (cler re of 12 mm on M9 medium gr with CMC) (Chndr et l., 2009,)], found promising in our preliminry screening for growth promotion nd iomss production of ptchouli (unpulished dt), were used in vermicomposting process utilizing distilltion wste of ptchouli Selection of comptile ioinoculnts Comptiility test etween Bcillus nd fluorescent Pseudomons isoltes ws performed on nutrient gr (NA, M001, HiMedi, Mumi, Indi) medium. Bcillus colonies were streked on the centre of the plte, followed y sprying of 24 h old culture of fluorescent Pseudomons nd Azotocter using n tomizer in different Petri dishes (Jin et l., 2011). The sme test ws performed with Pseudomons nd Azotocter individully streked on the centre of the Petri dish. No zone of inhiition ws oserved t the point of intersections indicting the comptiility mong the strins. All the three strins were further checked for their comptiility with Trichoderm isolte on potto dextrose gr (PDA, M096, HiMedi, Mumi, Indi) medium. A 5 mm dimeter mycelil plug from ctively growing culture of Trichoderm ws kept t the centre of the plte nd Bcillus, Azotocter nd Pseudomons isoltes were streked on either side. Overgrowth of Trichoderm on cteril streks without zone of inhiition showed comptiility of Trichoderm with cteril isoltes. Bsed on their comptiility, T. hrzinum (ATCC no. PTA-3701), cteril strins P. monteilii (HQ995498, MTCC9796), B. megterium (ATCC 14581) nd A. chroococcum (MTCC 446) were selected for iodegrdtion of ptchouli distilltion wstes. All the four ioinoculnts, found promising in the present study, re kept t CSIR-CIMAP-Microil Culture Collection Repository for further use Moleculr chrcteriztion (16S rrna) of selected cterium (CRC1) Bcteril genomic DNA ws isolted from overnight grown cells using stndrd procedures (Chchty nd Sulnier, 2000; Awsthi et l., 2011). 16S rdna mplifiction nd their sequence nlysis of newly isolted strin CRC1 ws crried out s descried erlier y Singh et l. (2013) using the nucleotide BLAST ( Further 16S rrna gene sequence of strin CRC1 ws sumitted to NCBI Gennk (HQ995498) Experimentl setup for in vitro nd pot study Multipliction of ioinoculnts T. hrzinum ws mss multiplied on potto dextrose roth t 28 ± 2 C for 7 dys. Mycelil mt ws seprted from roth, homogenized nd suspended in 500 ml of 100 mm of phosphte uffer to chieve colony forming unit (CFU) of ml 1 uffer suspension. The cteril cultures P. monteilii, B. megterium, nd A. chroococcum were multiplied in nutrient roth (A. chroococcum on Jensen s roth) for 36 hours t 210 rpm in n incutor shker t 30 C. The cteril suspensions were centrifuged t 8000 rpm for 10 minutes. The superntnts were discrded nd the pellets contining cteril cells were suspended in 100 mm phosphte uffer. The CFU in this suspension ws ml 1 for P. monteilii, ml 1 for B. megterium nd ml 1 for A. chroococcum.

4 Author's personl copy 50 R. Singh et l. / Applied Soil Ecology 70 (2013) Tle 1 Chemicl nlysis of ptchouli distilltion wste prior to inocultion of ioinoculnts. Prmeter ph 7.01 TOC 39.2 TKN 1.90 TP 0.21 TK 0.70 Cellulose 54.5 Hemicellulose 24.1 Lignin 18.3 All vlues re men of five oservtions; ll vlues re given in percentge except ph Collection of distilltion wste of ptchouli nd enrichment with ioinoculnts The distilltion wste of ptchouli ws collected from field distilltion unit of CSIR-CIMAP, Reserch Centre, Bnglore, which contined 85 ± 5% moisture. The ptchouli distilltion wste hd ph of 7.0 ± 0.1, totl orgnic cron (TOC) of 39.2 ± 1.0%, totl Kjeldhl nitrogen (TKN) of 1.9 ± 0.1%, totl phosphorus (TP) of 0.21 ± 0.01% nd totl potssium (TK) of 0.70 ± 0.1% (Tle 1) on dry weight sis. Distilltion wste (1 kg), trnsferred into utoclvle HiDispo Bgs (HiMedi, Mumi, Indi, size: 50 cm 35 cm) ws sterilized for 1 hour. Bioinoculnts were trnsferred septiclly (10 ml culture 1 kg 1 distilled wste) in their six respective tretments nmely T. hrzinum, P. monteilii, B. megterium, A. chroococcum, T. hrzinum + P. monteilii + B. megterium + A. chroococcum nd un-inoculted control. Inoculted sustrte with different ioinoculnt tretments nd n un-inoculted control were replicted five times nd incuted for 7 dys t 28 ± 2 C in B.O.D incutor. Bioinoculnts popultion ws enumerted in their respective tretments on semi selective medium using seril dilution technique. Popultion (CFU g 1 inoculted sustrte) of vrious ioinoculnts s oserved fter 7 dys incution ws: T. hrzinum ( ), P. monteilii ( ), B. megterium ( ) nd A. chroococcum ( ) while T. hrzinum + P. monteilii + B. megterium + A. chroococcum hd T. hrzinum ( ), P. monteilii ( ), B. megterium ( ) nd A. chroococcum ( ). Lter, inoculted sustrte ws mixed (1:10 w/w) with freshly distilled, ut cooled wste of ptchouli nd the mixture ws sujected to vermicomposting nd composting (without worms) process for 80 dys. Generlly, the commercil distilltion of ptchouli her is crried out for 6 8 h t high stem pressure (30 psi) which completely sterilizes the mteril. If inoculted immeditely fter removl of cooled de-oiled her, the wste cn e considered for growing ioinoculnts cultures. The experiments were conducted twice to confirm the results ut only men dt of two experiments hve een discussed Collection nd multipliction of erthworms Erthworms (Eiseni fetid), otined from vermicomposting unit of CSIR-CIMAP, Lucknow, Indi, were mss multiplied in cow dung. Cow dung ws dded s worm-edding mteril together with the ptchouli distilltion wste Vermicomposting in pot conditions The io-inoculted nd un-inoculted sustrte of ech replicted tretment ws kept individully in seprte sterile erthen pots (dimeter 30 cm) in completely rndomized design. There were five replictes. The pots were kept in glss house t mient temperture (28 32 C). To keep the mteril moist, sprinkling of sterile distilled wter ws done with fine rosecn on lternte dys. The epigeic species of erthworm (25 numers of dult clitellte E. fetid) ws introduced, in ech pot. The sustrte ws turned-over every week to void thermophillic stge. The pots were hrvested when the compost ws redy y its physicl ppernce, s judged y development of drk rown to lck uniform grnulr structure. Wtering ws stopped t this stge. Two dys lter, the vermicompost/compost ws removed from the pot long with worms nd uniformly spred on plstic sheet under shde. The totl iomss of vermicompost ws determined fter sieving (2 mm sieve) the produced vermicompost nd the numer of dults worms seprted were counted. Smples were collected from sieved vermicompost (out 10 g) of ech replicted tretment for chemicl nd iologicl nlysis on dry weight sis Field study Ptchouli vr. Johore nursery ws rised from terminl stem cuttings (5 months old crop) in polyethylene gs (7.5 cm 14 cm) filled with mixture of soil nd snd in the rtio 1: 1 (v/v). The experiment ws conducted t CSIR-CIMAP, Reserch Centre, Bnglore, Indi. Bnglore is locted t ltitude N, longitude E nd t n ltitude of 930 m ove men se level. The climte is semi-rid tropicl. The soil ws red sndy lom (Kndiustlf) with ph of 6.1, electricl conductivity 0.05 ds m 1, wter holding cpcity 46%, ulk density 1.61 g cm 3, percent orgnic cron 0.45, ville N 175 kg h 1, Olsen s P 2 O kg h 1 nd exchngele K 2 O kg h 1. The ioinoculnts-enriched vermicompost (BEVC) nd ioinoculntsenriched compost (BEC), which contined four ioinoculnts (T. hrzinum, P. monteilii, B. megterium nd A. chroococcum), overll found superior oth in terms of its nutrient sttus nd ioinoculnts popultion s compred to vermicompost/compost produced y individul ioinoculnt, ws used in the field experiment in the present study. The field trils were composed of five tretments: BEVC, BEC, vermicompost (VC), recommended dose of chemicl fertilizers (CF) nd control replicted five times dopting rndomized complete lock design (RCBD). The initil soil smples were collected to determine the initil levels of ioinoculnts popultion. The 45-dy old rooted cuttings in the nursery were trnsplnted with spcing of 60 cm 45 cm in 3.6 m 3.6 m rised eds in fields continuously cultivted with ptchouli crop for lst three yers where Rhizoctoni root-rot/wilt ws consistent prolem (Nrynpp et l., 1984). The ptchouli crop ws mintined in the sme plots with sme tretments for the period of 2 yers. In tretments involving BEVC, BEC, VC nd CF, the recommended dose of totl N requirement (66 kg h 1 hrvest 1 ) ws supplied through their respective vermicompost/compost nd therefore different mounts of vermicompost/compost were dded depending on their N content. However, the chemicl fertilizer treted plots received recommended dose of fertilizers N P K (66:50:50 kg h 1 hrvest 1 ) through ure, single super phosphte nd murite of potsh. The ure ws pplied in two split doses every lternte month. Ten plnts were rndomly tgged for growth/severity of root-rot oservtions from ech plot nd the men vlue of ten plnts were tken for sttisticl nlysis from ech plot. Plnt height nd numer of primry rnches were recorded t the time of hrvesting. Re-trnsplnting (three times) of the ptchouli rooted cuttings ws done following sme methods during the period of 2 yers. The hrvesting of the crop ws done fter dys of trnsplnting nd iomss yield ws recorded. Severity of stem/root-rot disese ws mesured on 0 4 scle of Kesvn nd Chowdhry (1977) where 0 = no symptoms, 1 = 1 25%, 2 = 26 50%, 3 = 51 75% nd 4 75% stem/root ffected y rot. Bsed on the root disese symptoms score of ech tretment, the percentge disese index (PDI) ws clculted s follows:pdi = ( Sum of numericl grding recorded Numer of roots oserved highest numericl rting) 100

5 Author's personl copy R. Singh et l. / Applied Soil Ecology 70 (2013) Physicl, chemicl nd microiologicl nlysis The rhizosphere soil smples of ech crop were collected fter hrvesting from depth of 0 15 cm t 5 rndom points djcent to plnt roots with the help of soil uger for ech replicted tretments for period of 2 yers. Pooled soil smples (5 rndom points) were stored in seled plstic gs under refrigertion (4 C) prior to ssy of chemicl nd microiologicl properties of soil. Undistured soil cores collected from ech plot with soil smpling rings of known volume were weighed nd then dried in n oven nd reweighed for ulk density ssys (Liu et l., 2007). Soil wter holding cpcity ws determined s the method descried y Smpson nd Allen (2000). The ph ws determined in 1:10 (w/v) vermicompost/distilltion wste/rhizo-soil:wter suspension. The ptchouli distilltion wste (efore nd fter inocultion of ioinoculnts) nd BEVC were nlyzed on oven dry sis for percent totl orgnic cron [TOC (%)] y Wlkley-Blck method (Nelson nd Sommers, 1996) nd totl Kjeldhl nitrogen (TKN), totl phosphorus (TP) nd totl potssium (TK) y the methods descried y Jckson (1973). Soil smples were nlyzed for TOC (%), ville N (kg h 1 ), ville P (Olsen s P 2 O 5, kg h 1 ) nd ville K (exchngele K 2 O, kg h 1 ) following Jckson (1973). Cellulose, hemicellulose nd lignin were frctionted sequentilly y Dutt s method (1981). Distilltion wste/ioinoculnts-eriched vermicompost/vermicompost/rhizo-soil were used for the determintion of microil lod (CFU g 1 ) in triplicte on dry weight sis. Trichoderm, Pseudomons, Bcillus nd Azotocter popultions in the distilltion wste/vermicompost/rhizo-soil were determined y seril dilution technique (10 3 folds for fungi nd 10 4 folds for cteri) in 0.85% NCl (w/v) (Denin, 1963) under in vitro conditions using Trichoderm selective medium (Eld nd Chet, 1983), King s B medium (King et l., 1954), Bcillus isoltion nd cultivtion medium for P soluilizers (Pikovsky, 1948) nd Jensen s medium (Jensen nd Petersen, 1954), respectively Essentil oil estimtion of her A smple of the her ws shde dried for three dys y spreding in shde nd the oil content ws determined using Clevenger s pprtus (Lngenu, 1948). The oil smples were nlyzed for mjor constituents using Vrin CP 3800 gs chromtogrph. The chromtogrph ws fitted with CP 5 SIL 30 m 0.25 mm column nd progrmmed 100 C (2 C), 8 C, 200 C (3 min.). The crrier gs ws nitrogen t flow rte of 0.4 ml min 1 nd the injector nd the flme ioniztion detector were mintined t 250 C nd 300 C, respectively. 0.2 l of smples were injected with split rtio of 1:80. Peks were identified y co-injection with uthentic pure smples. The percentges of the min components of ptchouli oil, nmely, -ptchoulene, cryophyllene, -guiene, seychellene, - -ptchoulene, -ulnesene nd ptchouli lcohol were determined Sttisticl nlysis The collected dt were sujected to sttisticl nlysis y nlysis of vrince method (ANOVA), suitle to completely rndomized design (CRD) for pot experiment nd rndomized complete lock design (RCBD) for field experiment, with the help of softwre ASSISTAT Version 7.6 et (2012). The experimentl dt of the two trils for pot experiments nd four trils for field experiments hd similr vrince vlues, so the dt were comined for further nlyses. Significnt differences mong tretments were sed on the F-test in ANOVA nd tretment mens were compred using lest significnt difference (LSD) t P The stndrd error (SE) of the men in verticl r chrts ws computed with Tle 2 Anlysis of cellulose, hemicelluloses nd lignin left in the vermicompost hrvested fter 80 dys. Tretments Cellulose (%) Hemicellulose (%) Lignin (%) THVC 24.15cd 16.05ef 4.21d THC 24.82c 16.82de 4.88cd PMVC 23.08d 17.05de 4.11d PMC 23.78cd 17.85cd 4.45cd BMVC c 5.01c BMC AZVC cd 5.18c AZC c 5.45 BEVC 14.74e 14.1g 3.14e BEC 15.21e 14.97fg 4.12d VC C THVC = Trichoderm hrzinum enriched vermicompost, THC = Trichoderm hrzinum enriched compost, PMVC = Pseudomons monteilii enriched vermicompost, PMC = Pseudomons monteilii enriched compost, BMVC = Bcillus megterium (P soluilizer) enriched vermicompost, BMC = Bcillus megterium enriched compost, AZVC = Azotocter enriched vermicompost, AZC = Azotocter enriched compost, BEVC = Bioinoculnts (TH + PF + BM + AZ) enriched vermicompost, BEC = Bioinoculnts (TH + PF + BM + AZ) enriched compost, VC = un-inoculted microe vermicompost, C = un-inoculted microe nd erthworm compost; Averge of two trils (ten oservtions for ech tretment) during the 2-yer period; the men vlues in verticl columns followed y the sme letters do not differ sttisticlly etween themselves t P Sigm Plot 11 ( The results nd discussion re sed on the verge of the trils during the 2-yer period. 3. Results S rrna gene sequence nlysis-sed identifiction of selected cteri The BLAST nlysis of 16S rrna gene sequence of isolte CRC1 reveled tht the cterium elongs to the genus Pseudomons. The isolte CRC1 hs shown mximum similrity (99%) with Pseudomons monteilii strin WAPP53 (Accession no. FJ905913), therefore isolte CRC1 ws designted s P. monteilii (Singh et l., 2013). The 16S sequence of isolte CRC1 ws sumitted to Gennk (NCBI) under the Accession numer HQ The identifiction of strin CRC1 ws lso confirmed y CSIR-IMTECH (Institute of Microil Technology), Chndigrh, Indi, nd the cterium is deposited with MTCC (Microil Type Culture Collection nd Gene Bnk n interntionl Depositry Authority) s strin no Pre-tretment of de-oiled lignocellulosic ptchouli wste for rpid production of enriched vermicompost in pot conditions The initil composition of ptchouli de-oiled wste is presented in Tle 1. Preliminry experiments indicted tht inocultion of distilltion wste with plnt growth promoting/cellulse producing microes like T. hrzinum, P. monteilii, B. megterium nd A. chroococcum yield vermicompost rich in the respective microe (un-pulished dt). Further, it ws oserved tht vermicomposting of distilltion wstes of ptchouli pre-inoculted with ll four microes (T. hrzinum, P. monteilii, B. megterium nd A. chroococcum) together resulted in fster decomposition of lignocellullosic wste in terms of cellulose, hemicelluloses nd lignin io-degrdtion y 58.44%, 29.44% nd 65.23%, respectively (Tle 2), resulting in higher yields (15%) of grnulr vermicompost recovered through 2 mm sieves (Fig. 1) nd higher popultion (21%) of erthworms (dt not presented). The ph of BEVC ws ner neutrl (7.02). As result of higher degrdtion in BEVC, the totl orgnic cron (TOC) ws significntly reduced y 21% s compred to VC (Tle 3). Also, totl Kjeldhl nitrogen (TKN), totl

6 Author's personl copy 52 R. Singh et l. / Applied Soil Ecology 70 (2013) Vermicompost/compost (kg pot -1 ) c h f l de THVC THC PMVC PMC BMVC BMC AZVC AZC BEVC BEC Fig. 1. Effect of pre-tretment of ioinoculnts for the production of enriched vermicompost/compost. Error rs re presented s stndrd error of men (±SE), Different letters ove the error rs show significnt difference t P 0.05, THVC = Trichoderm hrzinum enriched vermicompost, THC = Trichoderm hrzinum enriched compost, PMVC = Pseudomons monteilli enriched vermicompost, PMC = Pseudomons monteilii enriched compost, BMVC = Bcillus megterium (P soluilizer) enriched vermicompost, BMC = Bcillus megterium enriched compost, AZVC = Azotocter enriched vermicompost, AZC = Azotocter enriched compost, BEVC = Bioinoculnts (TH + PF + BM + AZ) enriched vermicompost, BEC = Bioinoculnts (TH + PF + BM + AZ) enriched compost, VC = un-inoculted microe vermicompost, C = un-inoculted microe nd erthworm compost. phosphorus (TP) nd totl potssium (TK) incresed significntly in BEVC y 20%, 32% nd 3%, respectively s compred to VC (Tle 3) Effect of BEVC, BEC, VC nd CF on soil helth, incidence of Rhizoctoni root-rot/wilt, growth nd yield of ptchouli under field conditions The initil popultion (CFU g 1 soil) of Azotocter, P. monteilii, B. megterium nd T. hrzinum in the experimentl plots ws , , nd , respectively. Tle 3 Production nd chemicl nlysis of enriched vermicompost/compost hrvested fter 80 dys. j d i VC g C m Plnt height/spred (cm) c Plnt height (cm) Plnt spred (cm) Numer of rnches BEVC BEC VC CF Soil only Fig. 2. Effect of enriched vermicompost/compost/chemicl fertilizers on growth chrcteristics of ptchouli in field conditions. Error rs re presented s stndrd error of men (±SE), Different letters ove the error rs show significnt difference t P 0.05, BEVC = Bioinoculnts-enriched vermicompost, BEC = Bioinoculnts-enriched compost, VC = Vermicompost, CF = Chemicl fertilizers. Plnt height enhnced significntly (15.2%) in plots treted with BEVC nd BEC/CF (13.7%) treted plots compred to VC treted plots (Fig. 2). The highest plnt spred ws oserved with BEVC (24.7%) followed y BEC (9.8%) nd CF (6.7%) treted plots compred to VC treted plots (Fig. 2) Numer of rnches were significntly higher in BEVC (22.5%) nd BEC (15.1%) treted plots compred to CF nd VC treted plots (Fig. 2). Over ll, plnt height, plnt spred nd numer of rnches incresed in ll the tretments (BEVC, BEC, VC nd CF) over control. The percent disese index (PDI) of Rhizoctoni root-rot/wilt rnged from 7 13% in vrious tretments nd ws significntly lower in plots receiving BEVC nd BEC; the severity of Rhizoctoni root-rot/wilt in BEVC treted plots ws reduced y 48% nd 61% compred to VC nd CF treted plots, respectively (Fig 3). VC lso reduced the disese severity y 26% compred to CF treted plots (Fig. 3). The shde dried her yield of ptchouli ws significntly higher (54%) in BEVC treted plots compred to VC lone plots nd 32% higher thn CF treted plots (Tle 4). There ws significnt difference in shde dry her yield (n increse of 35%) in BEVC treted plots s compred to BEC treted plots (Tle 4). The content of essentil oil vried from 2.20 to 2.30% (on shde dry sis). The yields of essentil oil improved y 59.74% c d c Numer of rnches Tretments TOC (%) TKN (%) TP (%) TK (%) THVC de 1.993cd 0.231c THC cd 1.672f 0.211cd PMVC cd 2.079c 0.239c PMC c 1.779de 0.209cd 0.682e BMVC cd 2.044cd BMC c 1.742ef de AZVC cd c AZC c 2.005cd 0.192d 0.693cd BEVC f BEC ef 2.097c f VC cd 0.227c 0.703c C g 0.127e 0.403g THVC = Trichoderm hrzinum enriched vermicompost, THC = Trichoderm hrzinum enriched compost, PMVC = Pseudomons monteilii enriched vermicompost, PMC = Pseudomons monteilli enriched compost, BMVC = Bcillus megterium (P soluilizer) enriched vermicompost, BMC = Bcillus megterium enriched compost, AZVC = Azotocter enriched vermicompost, AZC = Azotocter enriched compost, BEVC = Bioinoculnts (TH + PF + BM + AZ) enriched vermicompost, BEC = Bioinoculnts (TH + PF + BM + AZ) enriched compost, VC = un-inoculted microe vermicompost, C = un-inoculted microe nd erthworm compost, TOC = Totl orgnic cron, TKN = Totl Kjeldhl nitrogen, TP = Totl phosphorus; TK = Totl potssium, Averge of two trils (ten oservtions for ech tretment) during the 2-yer period; the men vlues in verticl columns followed y the sme letters do not differ sttisticlly etween themselves t P Percent disese index (PDI) c c BEVC BEC VC CF Soil only Fig. 3. Effect of enriched vermicompost/compost/chemicl fertilizers on PDI of ptchouli in field conditions. Error rs re presented s stndrd error of men (±SE), Different letters ove the error rs show significnt difference t P 0.05, BEVC = Bioinoculnts-enriched vermicompost, BEC = Bioinoculnts-enriched compost, VC = Vermicompost, CF = Chemicl fertilizers.

7 Author's personl copy R. Singh et l. / Applied Soil Ecology 70 (2013) Tle 4 Effect of ioinoculnts-enriched vermicompost, compost, vermicompost nd chemicl fertilizers on iomss nd essentil oil yield of ptchouli. Tretments Biomss/essentil oil yield Shde dry her yield (t h 1 ) Essentil oil (%) BEVC BEC 0.95c VC 0.83d 2.22c 18.43c CF c Soil only 0.55e 2.21c 12.16d Essentil oil yield (kg h 1 ) BEVC = Bioinoculnts-enriched vermicompost, BEC = Bioinoculnts-enriched compost, VC = Vermicompost, CF = Chemicl fertilizers; the men vlues in verticl columns followed y the sme letters do not differ sttisticlly etween themselves t P Men dt of four trils during 2-yer period. in plots treted with BEVC s compred to VC (not enriched) nd n increse of 37.70% nd 37.96% when compred with BEC nd CF treted plots, respectively (Tle 4). The qulity of essentil oil ws not ffected in ny of the tretments (dt not presented). The content of ptchouli lcohol, mjor chemicl constituent, rnged from to 48.43%. Other min constituents were seychellene ( %), cryophyllene ( %), -ulnesene ( %), -guiene ( %), -ptchoulene ( %) nd - -ptchoulene ( %). Appliction of BEVC lso improved the physicl sttus of soil with reduction in ulk density y 13.58% (Tle 5). On the other hnd, wter holding cpcity of soil incresed y 8.2% over plots treted with CF (Tle 5). However, differences were not significnt mong BEVC, BEC nd VC treted plots. Another significnt importnt chnge in ph ws oserved in plots treted with BEVC where the soil ph improved from 6.1 to 6.39 (Tle 5). There ws no significnt chnge in ph in plots pplied with CF. Soil fertility sttus like percent totl orgnic cron, ville N, ville P nd ville K were significntly higher in BEVC treted plots; n increse of 19.2 nd 24.9%, 13.5 nd 2.95%, 19.1 nd 26.9%, 14.9 nd 20.3% (Tle 5) compred to VC nd CF treted plots, respectively. The ioinoculnts (Azotocter, P. monteilii, B. megterium nd T. hrzinum) popultions (CFU g 1 soil) were significntly higher in plots receiving BEVC compred to VC nd CF treted plots fter hrvesting of ptchouli crop grown over 2-yer period. The finl popultion of Azotocter, P. monteilii, B. megterium nd T. hrzinum in the BEVC treted rhizosphere fter 2-yer period improved y %, %, % nd %, respectively, compred to VC nd CF treted plots (Tle 6). 4. Discussion Orgnic mnures nd plnt eneficil microes re vitl components for improving soil helth nd yields in griculturl systems. Tle 6 Effect of ioinoculnts-enriched vermicompost, compost, vermicompost nd chemicl fertilizers on rhizospheric ioinoculnts popultion. Tretments Root zone ioinoculnts popultion (CFU 10 4 g 1 soil) Azotocter Pseudomonds P soluilizers Trichoderm BEVC BEC VC 3.8c 4.2c 55c 3.1c CF 2.1d 1.1d 25d 1.1d Soil only 0.5e 0.4e 12e 0.2e BEVC = Bioinoculnts-enriched vermicompost, VC = Vermicompost, CF = Chemicl fertilizers; the men vlues in verticl columns followed y the sme letters do not differ sttisticlly etween themselves t P Men dt of four trils during 2-yer period. Delivering oth the components in sufficient quntities/numers will e definitely eneficil prticulrly in orgnic fields (Singh et l., 2012,) Pre-tretment of de-oiled lignocellulosic ptchouli wste for rpid production of enriched vermicompost Recycling of distilltion wstes of medicinl nd romtic plnts my reduce the need for fossil fuel sed fertilizer, nd help in sustining nd restoring soil fertility in terms of ville nutrients nd mjor physicl nd chemicl chrcteristics of the soil (Klr et l., 2012; Singh et l., 2012). Pre-tretment of crop residues, specilly lignocellulosic wste, significntly reduces the composting period nd improve the qulity vermicompost (Singh nd Shrm, 2002; Kumr nd Shwet, 2011). An ttempt ws mde towrds effective recycling of distilltion wste of ptchouli nd further enhncing its vlue y incorporting eneficil microes (T. hrzinum, P. monteilii, B. megterium nd A. chroococcum) during vermicomposting process for production of BEVC; superior in terms of ccelerted iodegrdtion y producing lignocellulolytic enzyme nd richness in oth nutrients nd desirle eneficil microes. The vermicompost/compost hd reltively neutrl ph. This my e due to the secretion of NH 4 + ions nd ctivity of clciferous glnds in erthworms. NH 4 + ions temporrily reduce the pool of H + ions (Himi nd Huht, 1987). The clciferous glnds contin cronic nhydrse which ctlyze the fixtion of CO 2 s CCO 3, therey preventing the fll in ph (Kle et l., 1982). Nik et l. (2008) reported tht pproximtely 31% P. monteilii strins re efficient cellulse producers which could e exploited for the mngement of enormous griculturl wste lying in the field which is supported y the present study. Chndr et l. (2009,) explined the role of six Trichoderm spp. for the rpid iodegrdtion of lignocellulosic distilltion wste. Trichoderm spp., Pseudomons spp. nd Bcillus spp. re known to produce cellulse enzyme (Lynch et l., 1981). Pdmvthimm et l. (2008) oserved fster decomposition of nn leves during vermicomposting process with Eudrilus Tle 5 Effect of ioinoculnts-enriched vermicompost, compost, vermicompost nd chemicl fertilizers on physicl nd chemicl properties ptchouli rhizospheric soil. Tretments Men physicl nd chemicl properties of ptchouli rhizospheric soil ph Bulk density (g 1 cm 3 ) Wter holding cpcity (%) Totl orgnic cron (%) Aville N (kg h 1 ) Aville P (kg h 1 ) Aville K (kg h 1 ) BEVC e BEC d VC c c c c CF 6.08c c 0.497d d c Soil only 6.10c c 0.501d d e d BEVC = Bioinoculnts-enriched vermicompost, VC = Vermicompost, CF = Chemicl fertilizers; the men vlues in verticl columns followed y the sme letters do not differ sttisticlly etween themselves t P Men dt of four trils during 2-yer period.

8 Author's personl copy 54 R. Singh et l. / Applied Soil Ecology 70 (2013) euginee worms. Orgnic wstes frgmented y erthworms hve much greter surfce re nd cn support greter microil ctivity (Edwrds et l., 2010) which in turn my help in production of higher mount of cellulses, hemicellulses, pectinses nd ligninses leding to rpid iodegrdtion of lignocellulosic wste. Grnulr vermicompost, produced from the essentil oil ering crops, re efficient crrier for the N-fixer (Rhizoium meliloti, Rmd 201) since it could mintin higher popultions of CFU g 1 vermicompost for 180 dys (Klr et l., 2010), vlue cceptle nd suitle for inocultion (Smith, 1992; Ben Reh et l., 2007). In n nother study, Singh et l. (2012) oserved tht pre-inocultion of ioinoculnts in vermicompost, s rooting medium for ptchouli cuttings, could e etter crrier for trnsferring propgules of ruculr mycorrhizl (AM) fungi (Glomus ggregtum, G. fscicultum, G. intrrdices nd G. mossee) nd ioinoculnts (P. fluorescens, B. megterium nd A. chroococcum). The optiml performnce of the vermicompost is due to its higher nutrient content nd high wter-holding cpcities, which re the two key chrcteristics of good crriers (Smith, 1992). Stchell nd Mrtein (1984) found n increse of 25% in P content in pper wste sludge, fter worm ctivity. They ttriuted this increse in P content to direct ction of worm gut enzymes nd indirectly y stimultion of microflor during vermicomposting process. Increse in totl phosporus during vermicomposting is proly due to minerliztion of orgnic mtter (Edwrds nd Lofty, 1972) nd CO 2 evolution (Hdddin et l., 2009). Grg et l. (2006) lso demonstrted tht microil flor enhnced the totl potssium; cid production y the micro-orgnisms seems to e prime mechnism for soluilizing the insolule potssium. Orgnic mnures nd plnt eneficil microes re importnt for improving soil helth nd sustinility in griculturl systems nd BEVC rich in oth nutrients nd eneficil microes will e n orgnic input of choice for orgnic frming. Vermicomposts re known to provide slow, lnced nutritionl relese pttern to plnts, prticulrly in terms of relese of plnt ville N, solule K, exchngele C, Mg nd P (Edwrds nd Fletcher, 1988; Edwrds, 2004) which re susequently used y plnts efficiently. Vermicompost ppliction generlly improves the soil environment prticulrly soil ertion, encourging the prolifertion of roots, which in turn drw more wter nd nutrient from distnt res nd helps to introduce nd sustin eneficil micro-orgnisms into the rhizosphere (Pdmvthimm et l., 2008) in sufficient numers for longer period Effect of BEVC, BEC, VC nd CF on soil helth, incidence of Rhizoctoni root-rot/wilt, growth nd yield of ptchouli under field conditions Bioinoculnts-enriched vermicompost (BEVC) ws evluted for chieving higher yields prticulrly in situtions where chemicl inputs re restricted. BEVC ws compred with BEC, VC nd CF continuously for 2-yer involving 4 hrvests. During field study, plnt growth prmeters improved significntly y eneficil io-inoculnts. This hs een successfully shown in industrilly importnt romtic crop like ptchouli (Singh et l., 2012), horticulturl crop like tomto (Al-Krki, 2006) nd in medicinl plnt Coleus forskohlii (Singh et l., 2009). The ppliction of BEVC contining higher mount of nutrients nd eneficil microes (T. hrzinum, P. monteilii, B. megterium nd A. chroococcum) in sufficient numers could hve incresed the nutrient vilility nd lso reduced the disese severity resulting in etter growth nd yield of crop. Puttnn et l. (2010) oserved tht the growth nd yield of ptchouli crop ws dversely ffected in soil lone tretment (without vermicompost) due to low nutrient sttus of soil nd low colonizing ility of microes in poor soil. Therefore, the use of BEVC could e lnced orgnic fertilizer for optimum growth nd her yield of the crop. Singh et l. (2009, 2011, 2012,c) demonstrted tht use of efficient ioinoculnts long with VC or VC lone (5t h 1 ) for reduced disese severity nd higher yields in C. forskohlii. Mishr et l. (2000) hve erlier successfully demonstrted the use of eco-friendly iogents (T. hrzinum, Glomus virens nd G. ggregtum) in controlling ptchouli root-rot. Improvement in yields nd reduction in disese severity with BEVC my e suitle lterntive to chemicl fertilizers nd fungicides which my help in reducing environmentl nd helth hzrds incurred y the use of hrmful chemicls. The success of ioinoculnts depends on the selection of potentil ioinoculnts/ntgonist, method nd mode of ppliction, the right environment nd other fctors (Singh et l., 2012). The efficient ioionculnts pplied long with nutrient rich vermicompost performed well ecuse of the presence of vermicompost which proly provided optiml growing environment (ner neutrl ph, nutrients, good wter holding cpcity) for the ioinoculnts. A reltively lower yield noticed in the present study with VC lone suggests the eneficil effects of dded ioinoculnts. Qulity of essentil oil ws not ffected y the tretments, however, yield of essentil oil improved y the ppliction of enriched vermicompost/compost. The similr trends were oserved during previous study where ptchouli plnts rooted in vermicompost long with microes like AM fungi (Glomus ggregtum, G. fscicultum, G. intrrdices nd G. mossee) nd plnt growth promoters (Pseudomons fluorescens, Bcillus sutilis, B. megterium nd Azotocter) when trnsplnted into pots nd fields yielded significntly higher essentil oil yield (Singh et l., 2012). Bioinoculnts enriched vermicompost could e n excellent source of orgnic fertilizer to sustin productivity nd fertility soil. Willson et l. (2001) demonstrted the use of orgnic mnures for the etter mngement nd sustinility of orgnic soils. Klr et l. (2012) studied the effect of menthol mint vermicompost for the improvement of soil properties. The use of orgnic mendment hs een reported to increse soil orgnic mtter, provide nutrients nd improve microil ctivity (Lee et l., 2004). Higher nutrient concentrtion in ioinoculnts enriched vermicompost (BEVC) might e the reson for supporting threshold popultion of ioinoculnts therey higher vilility of nutrients (N, P nd K) to the test crop ptchouli. The enriched vermicompost could e n excellent source of orgnic nutrients especilly under conditions of orgnic griculture preventing loss of yields generlly oserved in trnsition to orgnic frming prctice (Acs et l., 2007). 5. Conclusion The present study reveled tht the ove ground iomss of ptchouli could e exploited in two wys: (i) for the production of essentil oils (economic prt) nd (ii) the generted de-oiled wste (un-economic prt) cn e further utilized to produce ioinoculnts enriched vermicompost. In this wy, the produced distilltion wste in lrge quntities from the ptchouli crop, which contins mjor essentil nutrients exhusted from the soil, cn e iodegrded fter pre-treting with ioinoculnts (T. hrzinum, P. monteilii, B. megterium nd A. chroococcum) nd susequently vermicomposted with the help of epigeic species of erthworm (E. fetid). The resultnt ioinoculnts enriched vermicompost would e n optiml orgnic io-input for supply of nutrients, enriching the soil with eneficil microes nd improving soil helth. BEVC could significntly reduce the disese nd improve the yields of ptchouli her nd essentil oil. This technology could e referred s zero wste technology where the produced iomss her (fter extrction of essentil oil) is gin recycled (involving eneficil microes nd erthworms) contining the sme mount of essentil nutrients (s only secondry metolites re removed) is dded to the soil fter vermicomposting process. Appliction of enriched vermicompost therefore my reduce the use of chemicl fertilizers

9 Author's personl copy R. Singh et l. / Applied Soil Ecology 70 (2013) nd fungicides nd with improvement in soil ph, ulk density nd wter holding cpcity, ioinoculnts enriched vermicompost my lso reduce the numer of irrigtions further reducing the cost of inputs. Acknowledgments The uthors thnk the Council of Scientific nd Industril Reserch (CSIR), New Delhi, Indi for funding nd Director, CSIR- Centrl Institute of Medicinl nd romtic Plnts, Lucknow, Indi for providing fcilities, Scientist-In-Chrge, CSIR-CIMAP Reserch Centre, Bnglore Indi for fcilities, Dr. K.V.N.S. Srinivs (Phytochemist) for nlyzing ptchouli oil nd Mr. R. Tiwri, Hed, Pulictions for criticlly going through the mnuscript. References Acs, S., Berentsen, P.B.M., Huirne, R.B.M., Converstions to orgnic erle frming in The Netherlnds: A dynmic liner progrmming nlysis. Agric. Syst. 94, Al-Krki, G.N., Nursery inocultion of tomto with rusculr mycorrhizl fungi nd susequent performnce under irrigtion with sline wter. Sci. 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