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439 and became 15 b 2009 Society of Chemical Industry c ity of Kentucky, Lexington, KY, USA Abel manufacture, were tested for Although these methods may sometimes they generally are not consistent or effective 10–12 sp.) manufacture 14 6,7,13,14 oleifera ober 2009 Published online in Wiley Interscience: 18 December 2009 Correspondenceto:Daniel A Potter,S-225AgricultureScienceBldgN.,University of Kentucky, Lexington, KY 40546-0091, USA. E-mail: [email protected] Department of Entomology, Univers USDA-ARS Natural Products Utilization Research Unit, University ofUniversity, Mississippi, MS, USA Department of Plant and Soil Sciences,USA University of Kentucky, Lexington, KY, During the 1890s, an earthworm management method based No pesticides are labeled for earthworm control in the United ∗ c a b f courses and sports fields when they disrupt the playability, on a botanical expellant was developed in England enough to be relied upon by green-keepers,adversely are impractical affect or can the turf.switching Physical or removal dragging of is castsbenefit. laborious and by provides brushing, only temporary States. Research on alternative methods for managing earthworms and casts has focused on soil acidification, clipping removalfood to limit resources oraggregates. topdressing with angular sands or abrasive reduce casting, attract robins, starlingshazard and at airports. other birds, increasing bird strike . Currently, no pesticides are labeled for earthworms in the turf. The fate of expelled worms, methods for removing them earthworms in grassy strips alongside runways to reduce bird wing or sweeping removed expelled earthworms from putting 2 Kumudini M Meepagala lts from a mix of saponins. TSPs did not reduce the abundance of Agrostis stolonifera a Camellia , followed by irrigation, quickly expelled earthworms from the soil. A single 2 − Earthworm activity also 1,2 ; saponins; Lumbricidae; facilitating decomposition c 3 Earthworms in grass aprons of Carl T Redmond, 6,7 ∗ a They blunt mower blades set at low cutting : 439–446 www.soci.org Casting occurs when worms deposit their 9 66 Casts also provide ideal seedbed conditions for 5–8 6 4 2010; vermicide;

2009 Society of Chemical Industry Earthworms can be a serious problem, however, when their c heights, and may bebe so mowed numerous without thatsteel first drag. putting dispersing Casting greens activity the is cannot periods especially casts prolific in using during autumn cool, a and moist spring. brush or application reduced casts by 80–95% for at least 5 weeks. Mo green surfaces. Most expelledguided earthworms fractionation burrowed confirmed down the vermicidal whenbeneficial soil activity transferred arthropods, resu to nor did untreated they turf, control black but cutworms few or white survived.CONCLUSION: grubs Bioassay- in TSPs treated are turf. anclosely effective mowed turfgrass. botanical They vermicide might that also be could used be to useful suppress for selectively managing earthworm casts on strike hazard at airports. Keywords: casts disrupt theplaying playability surfaces. and aesthetics of closely mowed weed establishment. and recycling ofexcessive thatch. organic matter and alleviating build-up of Pest Manag Sci Casts result inaffect uneven, ball muddy roll. playingtraffic, surfaces When they and smeared may adversely orwater smother infiltration. compacted the by grass tires and or cause foot problems with 1INTRODUCTION Earthworms (Oligochaeta: Lumbricidae) play a vitaland role in managed natural grasslands,activity where stimulates their burrowing microbialavailability, breaks and activity, down and feeding mixes increases surfaceenhances litter infiltration of plant into water the and gases. soil nutrient and BACKGROUND: Earthworm casts are a worldwide problem on gol Abstract aesthetics and maintenance ofUnited closely States. mowed playing seedexpelling surfaces earthworms pellets and (TSPs), reducing casts a onand creeping saponin-rich impacts bentgrass on byproduct pest and of beneficial arthropods were also evaluated. RESULTS: Application of TSPs at 2.93 kg 100 m byproduct of tea oil ( Daniel A Potter, and David W Williams Lumbricidae) in turfgrass using a natural (www.interscience.wiley.com) DOI 10.1002/ps.1896 Managing earthworm casts (Oligochaeta: Research Article Received: 27 August 2009 Revised: 22 October 2009 Accepted: 26 Oct soil-richfecalmatterassmallmounds,orcasts,onthesurface. benefits lawns, golf courses, sports fieldssites and by other managed alleviating turf soil compaction, runways and taxiways or moving onto pavement after rains may . , 2 − et al : 439–446 5m,with . 66 1 respectively, C for 1 week. 1 × ◦ − 5 . ), plus untreated 2010; 2 − seed meal that had been Pest Manag Sci C, and there had been no rainfall ◦ inner portion of each plot within 1 h 2 C. oleifera area was delineated in the center of each 2 1 m plots) at three rates, 28, 56 and 112 g m × Tea seed cake pellets (3.2 mm diameter, 5–8 mm long) Additional trials were initiated in April 2008 to evaluate TSPs The viability of the expelled earthworms was assessed in two 0.5 m untreated buffer zones betweenswept all plots. with The corn site was husk hand break brooms up and immediately remove before already present treatmentmedium casts. or Treatments to high were rates of low, TSPs (33, 66 and 132 g plot plot by running athen string irrigated (1.25 grid cm) from andof the ten the persons perimeter. plots who were The steppedsurfacing only patrolled turf in within by border was the a areas. All team 1 earthworms m formulated from fresh harvested and processed as describedwere obtained earlier, from without Eric Lee additives, (DepartmentUniversity of of Biology, Hong The Kong) through Chinese International Turfgrass Services, Newnan, GA. Powdered tea seed2007, meal was but, also because tested the intea autumn powder seed was pellet awkward (TSP) toAnalysis formulation apply, by University only was of the used Kentucky Division in ofFeed Regulatory subsequent and Services, trials. Fertilizer Laboratory indicated1.2%totalnitrogen,1.3%solublepotash,0.6%availablephosphate that the TSPs contained and smaller percentages of calcium (0.19),(0.11) magnesium and (0.16), trace iron metals. 2.2 Earthwormand expulsion casting by tea reduction seed after cake, worm the fate removal of worms A preliminary trialfeasibility conducted of in using teaTwo autumn formulations, seed 2007 powdered cake tea evaluated seedapplied for to cake small the earthworm (1 and management. TSPs, were each there was active casting in theDifferent weeks portions before of and the during green the were trials. used for each trial. for earthworm expulsion, thethe fate efficacy of in worms reducing that casts. surfaced Plot and size was 1 on 4 October. At that time thereone was only replicate enough with material to powder treat were and two broadcast with by TSPs.two The gloved formulations directions, hand, and evenlywhole activated covering site. Worms with each began surfacing 1.25 plot within cm aexpelled few in within of minutes; 30 all min irrigation those were collected toplot, into counted separate the and cups weighed. for each or irrigation forwere the broadcast, previous a 1 72 m h. Immediately after the TSPs ways. First, their abilitydetermined to by burrow immediately transferring down 20 into individualstreated untreated from plot each turf to was one10.2 of cm in 20 height) that PVC had been enclosures arrangedof on the (39 an same cm untreated putting section green in in the diameter sameevenly RCB by layout. placed The worms on were the untreatedthat turf, and failed dead to or burrow moribundsamples down ones within of 2 h 20 were947 mL counted. surfaced Additional polypropylene worms containersMagic worm per bedding containing with 10 plot g 100 Magic g wormAmherst food were (Magic Junction, moistened Products, WI) transferred and to held in darkness at 24 were hand removed and processed as described below. Surviving earthworms then were examined, counted and weighed. Very few worms surfacedwere from dug with the a untreated spade from plots, an so untreated area samples at the edge of the controls, arranged in afive randomized replications. complete Treatments blockconditions were were (RCB) cloudy applied with and on 13 9 April; weather corresponding to 1.46, 2.93 or 5.86 kg 100 m . 1 21 − Tea Poa 23 www.soci.org DA Potter Numerous 2009 Society of Chemical Industry c Bassia latifolia 23,26–28 Tea seed cake, like per growing season. 2 24,25 − 29 m stand of ‘Penncross’ × L. and annual bluegrass The process involved applying Mowrah meal is no longer sold in 16,17 18,19 seeds, fruits and oil are rich in antioxidants Agrostisstolonifera Abel. (Theaceae), is used extensively for cooking Mowrah meal contains sapo-glucoside mowrin and for a total of 1.45 kg N 100 m Camellia 2 Other expellant materials, some of which (e.g. mercuric 6,16 − L. (species distribution, by visual estimation, 75 and 25% 20–22 15–18 Tea pressed from seeds of the oil plant, This study sought to determine if tea seed cake could be used as the United States, nor is it labeled for earthworm management. Camellia oleifera in and elsewhereof in eastern soaps, Asia, , anditems. in ointments, the hair manufacture care products and other The site had a history of high numbers of earthworm casts, and mowrah meal, contains triterpene saponins. seeds, harvested mainly in Septemberwere obtained and from October, wild traditionally plants, althoughcommon cultivation is (Lee, becoming E.,crushed private for communication). oil extraction, After the residuetea the is seed bound seeds ‘cakes’ together, for forming are easymay transportation. Tea be seed cake ground orused to residue in a Asia powdereliminate as predatory or an fish formed in organic prawn into molluscicide ponds. and, pellets at that high are rates, to Chinese websites (e.g. http://www.camellia-oil.com)also claim eliminate it earthworms will from golfsearch courses of and the sports scientific fields.concerning A literature, however, or found data nomanagement. references supporting tea seed cake fora botanical pesticide earthworm for managing earthworms and reducing casts on turf playing surfaces. Ratesworms of and application, methods for the removing fate them, the of effectsand expelled on beneficial other arthropods pests and the duration ofduring casting autumn suppression and spring casting periodsthe were investigated, active and fraction wascake identified. is Results well suited indicate for thaton selective golf tea courses management and sports seed of fields. It earthworm could also casts bebird-attracting useful earthworm for reducing populations at airports. Fungicides were applied curatively forand control dithiopyr of was fungal applied diseases, as crabgrass control at 0.07 kg AI ha www.interscience.wiley.com/journal/ps 22.1 MATERIALS AND METHODS General methods Most field trials were conducted on a 29 creeping bentgrass annua respectively) established in 1978 andputting managed as green a at push-up golf theFacility, Spindletop University Research of Farm, Lexington. Kentuckysilt The Turfgrass loam soil Research is (fine, a mixed, Maury mesictype typic is Paleudalf) homogeneous with and pH original, 6.3.been and The applied. no soil sand The topdressing turf had height was mowed of 5 cut times andto per irrigated prevent week from at visible 4.0 a stress.applied mm permanent Nitrogen in sprinkler September, fertilizer system October (urea:N and 46–0–0) 100 November m at was 0.48 kg actual and have long been used in Chinese traditional medicine. widely used on golf courses. chloride) were highlythe toxic, early were twentieth century, also butmowrah used proprietary on products meal containing golf aseffective. courses their in active agent were probably the most mowrah meal, derivedRoxb. from after the seeds oilit, of had causing been the earthworms pressed to tree out, comeraked and to into liberally the piles, watering shoveled surface. into Thesite. wheelbarrows worms and were hauled off the other triterpene saponins.

440 441 1 − year 1 2mplots − × Agrotis ipsilon ). Control enclosures 2 − C. Expelled worms were ◦ enclosed area) were driven 2 shipped from a commercial insectary www.interscience.wiley.com/journal/ps A. ipsilon -tests. t Six pairs of polyvinyl chloride (PVC) cylinder enclosures (39 cm One plot of each pair was then mowed once with a Toro Impact on white grubs was assessed by driving ten pairs of diameter, 10.2 cm height, 0.12 m (Hufnagel)(Lepidoptera:Noctuidae),androot-feeding whitegrubs (Coleoptera: Scarabaeidae). Thepopulations effects were also on evaluated. soil microarthropod 2.5 Potentialarthropods impact onGiven pest the insects anecdotal and reportsactivity, non-target that the tea soil authors seedmanaging sought cake earthworms and has to casts insecticidal might determine alsoturfgrass control if two common pests: applying foliage-feeding TSPs black for cutworms, Greensmaster 3150 riding mower (ToroMN) Company, with Bloomington, nine-bladed cuttingand the units number set of at markedrecorded. worms 4 The mm removed second from height each setrotary of plot broom of cut, sweeper was plots (model 45-0320-062; was Agri-Fab,pulled Sullivan, swept by IL) with a an Gatorbrushes were Agri-Fab utility adjusted vehicle to contact (John the surfacesweeping Deere, and width had Lathrop, an of effective CA). 1.1 The remaining m. were The numbers recordeda of after second marked the earthworms pass fromearthworms first removed the pass was opposite compared and between direction.by paired the again The two after percentage methods of left on the plots, and the followingdead morning or there were moribund numerous earthworms onof the 15 turf earthworms surface. on The eachsmall locations plot spot were of then orange markedcadaver. turf by marking The dabbing paint worms a on themselvesone the were plot grass not where beside only disturbed, the threeearthworms except worms from for other had treated surfaced, plots so were placed 12 on additional that plot. from urea, irrigated from a permanent sprinklerdrought system stress to prevent and mowed atfive 16 third-instar mm 3 timeswere per week. added Twenty- to eachthose enclosure few in larvae the5 morning that min on were had 13 replaced. not May,for The 5 and burrowed h, cutworms into then wereaforementioned TSPs the allowed high were turf to rate applied establish (131.5 within to g one 1.5 ring m of each pair at the about 7 cm deep intoturf adjacent a to the stand main study of site. The ‘Penncross’ turfto was creeping managed golf similarly bentgrass course fairways, i.e. fertilized with 146 kg N ha 25 cm diameter, 20 cm high beveled PVC rings 17 cm deep into a werenottreated.Irrigation(1.25 cm)wasthenappliedtothewholesite, and pieces of meshthe wire enclosures hardware to cloth prevent wereafter bird 72 secured predation. h over by Survival sampling was eachUltra enclosure dishwashing assessed detergent by (Proctor soap and drench Gamble, [10 Cincinnati,per OH) mL 7.6 Joy L water], which causes healthy cutworms to surface. 16, 23 and 30 DAT, averagedplots per were plot, not and mowed analyzed for as 24 above. h The before each count. 2.4 RemovalofexpelledearthwormsbymowingorsweepingTwo standardsweeping, green-keeping were evaluated for removing practices, TSP-expelled earthworms from putting mowing green surfaces. and Six pairs of rotary replicated 2 were treated with theafternoon, medium followed by rate 1 of cmof of TSPs treatment irrigation. on were Conditions sunny 16 at and April the about time in 20 late portion 2 2009 Society of Chemical Industry c -tests within sample t 5 m plots were marked on . 29 standard error (SE). Statistical 1 ± × 5 . 05) for comparing treatment means with . 5 m plots, separated by 0.5 m buffers, were . : 439–446 0 1 portion of each plot were counted by two 66 = × 2 5 α . 2010; of each plot in the main trial at 1, 8, 15 and 35 days 2 Two independent observers counted the fresh casts on the Another trial conducted on 24 April compared the propensity Analysis of variance (ANOVA) for an RCB was used to test for Another trial tested TSP effectiveness for casting reduction in Managing earthworm casts on putting greens www.soci.org marked on a previouslyplacing unused them portion where ofobserved. similar the All numbers push-up casts green, oftreatment. were abundant Weather removed casts conditions withwas were were the sweep same similar as brooms described in and before that Section 2.2, methodology surfaced except were that earthworms lefton on the the plots. inner The 1 numbers m of fresh casts previously unused portions of the push-uppretreatment green cast and counts. blocked One by plot66 of g each of pair TSPs was per treated7 plot, with October. followed Expelled by earthworms about were 1.25were cm left not of removed on because irrigation, the they on had plots. been Oldand compacted irrigation. by casts The numbers mowing of fresh casts on the inner 1 m inner 1 m Pest Manag Sci of expelled earthworms to burrow backuntreated down turf, into an treated important versus question ifremoved worms from are not treated physically areas,transplanted to or golf alternatively, roughs are orof other plots collected were non-treated marked, and sites. and one Five randomlywas selected pairs treated plot with of the each medium pair rate of TSPsenclosures, as as described above, earlier. were PVC placed onturf. the treated Irrigation and was non-treated applied, andnear worms enclosures that on surfaced the within treatedTen representative plots and individuals then were were placed on temporarily the turf removed. within surface each enclosure and observed. Theon numbers that the remained surface, orwere recorded that after 1 had h. partially or totally burrowedtreatment down, effects on each variable inDunnett’s the above test trials, ( followed by same green and used as controlsassays. for Finally, the all enclosure additional and worms container into that separate surfaced containers were for collected weighed. each plot, counted and collectively the control, and polynomial contrasts to test foreffects linear of or quadratic rate. Counts werethe log assumptions of transformed normality as and required homogeneity of toare variances. meet Data reported as originalanalyses means were done with Statistix 8. 2.3 Castingremoval reduction byA trial initiated tea on 19 April 2008 seed testedreducing if casts TSPs cake would if be effective the in without expelledremoved. earthworms Ten were worm 1 not immediately after treatment (DAT). Countsthe were green always taken was 1–2 mowed,accumulate. days to The after two allow observers’ atlog counts least transformed and were one examined averaged by night ANOVA per forfollowed for plot, repeated by measures, ANOVA casts for to main effectstreatment and trends polynomial on contrasts each for evaluation date. dates. autumn 2008. Six pairs of 1 independent observers at 2 and 9 DAT.for The 24 plots h were before not counting. mowed The two observers’ countsto were provide averaged a single value per plot, andrepeated then measures, analyzed followed by by ANOVA two-sample for of each plot were counted by two independent observers at 2, 9, . L M µ sp. et al spp., : 439–446 Lumbricus 66 L) was added Gypsophila µ sodium chloride) 2010; Aporrectodea M 05). . 0 respectively. Individual L) of each solution were < 2 µ P − Pest Manag Sci 10 m sodium chloride) were prepared; SE numbers of worms expelled ± M ± using commercially available 9and56 C NMR spectra of the active samples were 13 ± 30,31 saponins in 0.9 Thousands of these earthworms were expelled by 1 − 32 13, 55 Hand 1 L. The powder formulation was dusty, prone to drift ± were expelled from the non-replicated plots treated with the 2 All rates of TSPs also greatly reduced the numbers of earthworm In April 2008, all rates of TSPs followed by irrigation rapidly Saponin concentration in the TSPs was estimated from its − casts for(Fig. at 1). Repeated-measures least ANOVA indicated 35 significant days effects when counting was terminated then aliquots (0, 5, 10, 25,added 50, to 75 separate or wells 100 ofplates 96-well, (Costar, V-bottom, Cambridge, uncoated MA). Corresponding titration amounts of 0.9 the treatments, as opposed to very few night crawlers, expelled a large numbersurfaced in of response to earthworms, irrigation alone whereasby (Table 1). almost the Worms none expelled twodown highest when placed rates ontheir showed untreated plots impaired survival on ability when thealso same to compromised transferred green, burrow (Table and to 1).appeared flaccid, Many clean with reddish of worm cankers. the Allvariables of bedding affected the showed aforementioned earthworms was significantapplication linear rate (polynomial and contrasts, quadratic response to terrestris and awkward to apply,trials. so only TSPs were used in subsequent (soapwort) saponins (Sigma #1252;as Sigma-Aldrich, St a Louis, standard. MO) Forand 1 standard mg curves, mL four solutions (0.2, 0.33, 0.5 in each well. TSPs were defattedwith with hexane ethanol, and with extracted twice thethe solvent precipitate evaporated was under resuspendedserial nitrogen, dilutions in of and the 0.9 TSP m extract (0–67% sodium in chloride. 0.9 Ten 3RESULTS 3.1 Earthwormand expulsion casting by tea reduction seed after cake, worm the fate removal of worms Both the powdered tea seednumber cake of and earthworms the in TSPsthe the expelled October push-up a 2007 green. large preliminary Wormsa trial began on few surfacing minutes fromm after treated irrigation. plots Totals of 93, 63 and 61 worms sodium chloride buffer were added to bring the volume to 100 were added to well plates. Freshto rabbit blood each (100 well; platesread with after TSP 4 h extracthemolysis. by or determining The saponin the saponin standard thresholdestimated were content by required comparing of to hemolysis thresholds three induce ofthe TSP samples standards. extract with of TSPs was earthworms were not identified, butsite the is species dominated complex by atthat this make smaller shallow, semi-permanent species, burrows and mainly depositthe casts on surface. hemolytic activity obtained on 400 and 600 mHz instruments. low, medium and hightreated rates with of powder. TSPs,were For 59 mean the two replicates hexane, 100% ethyl acetatewith and 20% 0.5% methanol (v/v) insimilar ethyl water. fractions acetate, Fractions according to ofafford the 18 six TLC mL fractions. profile were were Eachidentify collected, combined fraction the to and was active testedthe fraction. in highest Two the activity. These bioassay fractions twosilica to were fractions gel were column found further chromatography to purified to(112 by afford have mg). an off-white powder ) 2 − spp.) www.soci.org DA Potter L., near the 2009 Society of Chemical Industry c Cyclocephala sp.) dug at the main Poa pratensis l until sorted. Predominant rther, the methanol extract was Apporectodea 05) were used to compare means for . 0 < P -tests ( t . The aqueous layer was lyophilized, and the butanol 1 − Impact of TSPs on soil arthropods in the creeping bentgrass TSP impact on soil microarthropods in fairway-cut creeping For the bioassay-guided fractionation, crushed TSPs (500 g) 2.6 Determination ofBioassay-guidedextractionandfractionationweredonetoidentify earthworm-active components in TSPs the active vermicidal components in thefreshly TSPs. The collected bioassays earthworms used ( treated and control plots in thissection. assay and others described in this study and placed ondishes,with3–5wormsperplateand4–6replicatespertreatment. moist filter paper inEach 90 trial included mm a diameter positive control petri (aground suspension of with 187 a mg TSPs mortarwhich and pestle corresponded and to dissolved the in medium 32 rate mL (65.8 water), g TSPs 2.24 m were dug from untreated roughswere of placed a in each local enclosure golf on48 course; 2 h to 14 May. establish before grubs The one enclosure grubs of were eachthe pair allowed was high treated with rate of TSPs,The followed turf by and irrigation as soilexamined on described 13 within May, above. and each remaining grubs were enclosurewas counted. assessed Viability was by broken placing grubsmoss apart on and and the recording surface the of percentagesurface, moistened the able normal to response, burrow within 10 beneath min. the push-up green wasdiameter, 5.08 assessed cm bythe deep) first pulling of April grass two4 2008 weeks and trial cores after application, soil (Section cut (5.08 intothe 2.2). cm from eight chances The smaller of each pieces, microarthropods samples to being plot reduce and were trapped extracted in in taken the for drying 72 soil, hInstruments, in Uxbridge, Tullgren Middlesex, UK) funnels equipped (Burkhardt withSamples 25 Agronomic W were bulbs. stored intaxa, 75% mainly ethano oribatid andwere mesostigmatid counted with mites a binocular and microscope Collembola, treatments and compared as among described for the earthworm counts. bentgrass was assessed inblack cutworm the trial. same Paired PVC stand enclosuresset (39 as on cm the that diameter) turf, were and used the for highenclosure of rate the each of pair pellets on was 10 applied May, withinto followed one by the 1.25 cm whole of irrigation site.each Two plot after cores 72 of h and grass extractedabove. in and Paired Tullgren soil funnels as were described pulled from other study sites. Third-instar masked chafers ( used in field trials, and a waterdiluted control. in Extracts and distilled fractions were waterearthworms, and which applied were via observed pipetteagitation, for curling) directly immediate and prodded to response to the evaluate (e.g. mortalityafter or 1 morbidity and 2 h. wereextractedtwicewith1.5 Lmethanolbystirringthesuspensionfor 2 h.filter The paper, extracts andextract were the which solvent filtered was wasafter through screened, evaporated extraction, Whatman along for to No. activity.partitioned with yield between water Fu and 53 butanol 1 the (1 g : 1), residual using of 2.5 30 g mL powder butanol www.interscience.wiley.com/journal/ps stand of ‘Midnight’ Kentucky bluegrass, soluble fraction was evaporated,and resuspended assayed. The in active distilled butanol-solubleextract water fraction was of run the on methanol a gravityactivity column to confirmed purify by the bioassay. compound, This with to active fraction silica was subjected gelflash column column chromatography chromatography system using by 50% ethyl means acetate in of a Biotage

442 443 2 − 001; 9, df . . 0 0m . 10 1 < ± P =− 4 . 8, caused them t = in situ 93, df . 9 respectively; 2 =− 07). Sweeping a second time 9 for mowing and sweeping . − . t 0 1 8 worms still present. The one . = 1 7m ± . P 2 0 ± . 9; ± 8 . 6 = . www.interscience.wiley.com/journal/ps respectively; 2 04; df − . 7 versus 9 . 2 0 C, with an average low for the preceding 8 days of versus 15 ) on 7 October 2008, with expelled worms left on the ◦ = 2 ± 2 24 m − t − 2 6.7 001). The night preceding 28 April was unseasonably . ± . 0 = Reduction in earthworm casts following application of TSPs 2m . < 0 C), which probably accounts for the relatively low numbers P ◦ ± 4 8, . in a perpendicularincreasing direction removed the a cumulative48%, few removal leaving additional from a worms, mean thosereplicate for of plots the sweep 7 treatment to on whichplaced about earthworms to had augment been their numbers wasbecause excluded the from sweeper the was analysis much moreearthworms effective in (13/15 removing removed those bydead the earthworms first that pass) had thanthat not in been removing secretions disturbed. from It earthwormsto was that adhere observed died to thesweeper than grass individuals that and had be been moved. less efficiently dislodged by3.4 the Potentialarthropods impact onApplication pest insects of andthe TSPs non-target abundance followed soil ofbentgrass by black or of irrigation cutworms masked in(Table chafer failed 2). Larvae grubs fairway-height sampled from to in the creeping treated Kentucky plots were reduce vigorous bluegrass and turf analysis on log-transformedalmost no data). casts on At the(0 treated 9 plots compared DAT with the there controls were still respectively ( surface. 11.7 versus 120 Figure 2. (2.93 kg 100 m 3.2 Castingremoval reduction byThe tea application of seedof TSPs earthworm cake on casts, 19 without evenphysically removed. April when At worm 2 DAT expelled greatly the numbers earthworms of reducedwere casts were on reduced treated numbers by plots not 99% compared with the controls (1 of casts on the control plots at theearly 9 October DAT was evaluation. Application similarly in effective, reducing85.8, casts 87.4 by and 98.3, 89.7, 82.8%(Fig. after 2). 2, 8, 16, 23 and 30 days3.3 respectively RemovalofexpelledearthwormsbymowingorsweepingA single pass with theabout greens mower or 66 sweeper 1 andpresent DAT removed on 40% the of puttingworms green the (out surface. of dead Theaveraged 15) numbers and 5 remaining of moribund marked on earthworms the plots after a single pass = cool (low 0 . 26 ∗ ∗ 1 05). . ± 0 72; df 76; df 2) 6) 10) 17) . . 8 . ± ± ± ± 3 )differ 2 3 versus < 01; one- ∗ . . 1 = = 44( 46( P 0 % survival 01 for each F 2009 Society of Chemical Industry F . after 1 week ± 0 respectively. = c 2 . 2 001, except for < . P − ∗ 0 P ∗ medium SE) numbers (out of 0) 95( 4) 71( 10) 6) < 4.7; ± ± ± ± ± P 3, 12; 001), date ( Viability in untreated = . 94( 84( 74 ( 0 = %ableto 01). , total mass and viability of . 2 burrow down 0 < P < 001), with linear and quadratic a . 30; df ∗ ∗ ∗ 2 . P 0 − 4) 5) 8) 0) 100( 3 8; ± ± ± ± < 3, 16; = = P 20( 29( 32( = t ∗ ∗ ∗ 0, 10.2, 6.0 and 6.1 for number, mass, % able 3, 12; . 8, 27.0, 29.2 and 159 for 1, 8, 15 and 35 28; df 6; df . . . 5 : 439–446 48) 31) 38) 0) 0( 3 = SE) number per 1 m -test with correction for unequal variances). ± ± ± ± 39 46 = 66 t = ± F t = = Worms expelled m 5, respectively, for worms placed on treated or 05). Reductions were also significant within each . . F F 0 05) and treatment by date interaction ( 2010; 0 . )NumberMass(g) 0 2 Mean ( ± < − 0 < P Reduction in earthworm casts following TSP application on 9 . 4 for expelled worms transferred to treated versus un- 05). . P . 0 0 ± = ANOVA results: 9, 46; 3, 9; In the second reburial trial, expelled earthworms were more 8 29.2 216( 14.6 172( to burrow and %variable. survival Within respectively; df columns, means marked with an asterisk ( significantly from theα corresponding control mean (Dunnett’s test, 58.4a 229( 00( Rate (g/m Table 1. expelled earthworms following thecreeping application bentgrass green, of April TSPs 2008 to a push-up . Managing earthworm casts on putting greens www.soci.org tailed, two-sample treated turf respectively ( 1 sample date ( Reductions in casts from the low, medium and89.6, high rates 99.3 averaged and 100% respectively atrespectively 15 at DAT, 35 and DAT. 85.4, 98 and 99% likely to die on the surface if leftto on untreated treated plots on plots the than same if green. transferred Mean ( 10) remaining wholly exposed after 30 min were 6 effects for rate (polynomial contrasts; all DAT respectively; df = the quadratic trend at day 8, which was significant at untreated plots ( versus 3 = Pest Manag Sci If expelled worms thatincluded, had then the burrowed numbers that only had failed part-way to rebury down were 6 were for treatment ( Figure 1. April 2008 and physicaland removal low rates of of worms TSPs were that 5.86, surfaced. 2.93 and High, 1.46 medium kg 100 m . 23 No No and 24,42 et al but a 24 22 16,17 22 Also, the : 439–446 Saponins but effects 66 6,16 Monomorium 24,25 Mill., exhibited 30,39 fruit hulls bioassay-guided 2010; Although the tea 36 35 Vaill. 5 cm) needed to activate C. oleifera . seeds, leaves and . 8 1 ≤ Ethanolic extract of tea seed 38 98% reduction in casts by 2–3 Pest Manag Sci 37 a known carcinogen, and more Opuntia vulgaris > 33 Camellia but higher than reported concentrations seed residue is reputed to protect crops from 34 Pheretima posthuma Tea seed saponins degrade naturally in the )thatprovided saponins to earthworms were found, but saponins 2 − 40,41 oil manufacture and should be relatively inexpensive Camellia L., workers on filter paper. seeds after the oil is extracted, is an effective botanical Camellia Camellia In China, It was found, however, that a rate of TSPs that expelled and Several forms of saponins, which contribute to the bitterness of Plant-derived toxicants, including tea seed saponins, are used 10% in the seeds, TSPs would lessen risks of run-off from treated sitesthe compared profuse with watering that was needed with mowrah meal. literature search found no published efficacyIn data for laboratory such tests, usage. saponinspopulation refined growth from of tea twoand concentrated seed sprays spider also reduced suppressed oviposition mite and killed (Tetranychidae) larvae and nymphs, species, but not adult mites. act on thedeprivation. respiratory epithelium of fish gills, causing oxygen DAT, and 83–98% reductionthe after rate 4–5 at which weeks, mowrah is meal 60% was used lower in than the past. fractionation indicates that the vermicidalfrom activity a of mix TSPs results ofout closely structure related elucidation saponins. work to The identity authors those specific are saponins. carrying certain aphids, scales, leafhopper, beetles and caterpillars, cake also had contact activity against Pharoah ant, pharaonis killed earthworms did not measurably impactmaskedchafergrubsorsoilmicroarthropodsincreeping on black cutworms, bentgrass turf. This indicates that TSP components either lack contactagainst activity those arthropods or else did not reach the thatchhigh and soil enough at concentration for activity todeter be feeding expressed. by Saponins various non-adapted arthropods, tea, have been isolated from Sublethal exposure can, however, have chronic effects on decapod 4 DISCUSSION This study indicates that tea seed cake,C. the oleifera saponin-rich residue of vermicide that could be usedand selectively their to casts manage on earthworms golf(2.93 courses kg and 100 sports m fields. The rate of TSPs Thepresentestimateof12%saponinsintheTSPsusedinthisstudy is similar to reports of 13.5% saponins in environment in 2–10 days,toxic to shrimps, and crabs and other at food organisms low in the ponds. concentrations are less plant also produces polyphenols and alkaloids, relatively light amount of irrigation ( phytotoxicity or turf discolorationTSPs that was were applied. apparent TSPs were at readilyof available the as a rates byproduct of to market and use. TSPs could alsoin be used ecological to sample studies, earthworms whichdrenching with in dilute the formalin, past have typically involved previously published studies concerning the toxicology of tea seed cake or isolated from stems of a cactus, recently with powdered mustard seed. > in China, Taiwan,eradicate undesirable Thailand fish in and shrimp farming other ponds. East-Asian countries to of TSPs on feeding by cutworms or grubs werepresent not study. evaluated in Samples the suggest that thesports use fields of would TSPs not on disrupt greensthan beneficial or earthworms. soil invertebrates other in Thai and Japanese tea seeds (5.2 and 7.2% respectively). dose-dependent activity, including paralysisIndian earthworm and death, on the Cy- a ) P ( www.soci.org DA Potter t 0.3 (0.40) 1.7 (0.06) 1.4 (0.23) 0.1 (0.93) − − − − 2009 Society of Chemical Industry CNMR)and ) and natural c 13 SE) ± 18.5) 0.7 (0.52) 0.9) 5.8) 1.3 (0.25) 6.5) 1.1 (0.34) 0.4) 8.1) 6.1) 6.0) 0.8 (0.49) 14.0) 0.0 (1.0) 0.2) 1.0 (0.38) 0.4) 1.2 (0.28) 0.5) 1.6 (0.19) 1.0) 0.7 (0.50) 1.3) 1.8 (0.13) ± ± ± ± ± ± ± ± ± ± ± ± ± ± 5, 4 and 5 for pest insects c = Agrotis ipsilon tablished in each plot, treated c reen or fairway turf respectively. roarthropods in creeping bentgrass 12.9) 104.6( 0.8) 20.8( 3.4) 59.5( 6.1) 25.8( 0.7) 12.2( 13.5) 38.3( 2.5) 25.8( 11.3) 55.0( 1.4) 0.2( 1.0) 0.4( 1.1) 1.2( 0.5) 2.5( 2.1) 17.4( 2.6) 1.6( C NMR. Based on hemolytic activity, ± ± ± ± ± ± ± ± ± ± ± ± ± ± 13 Mean number per sample ( H NMR) in the NMR spectra, unambiguous 1 b spp.) and black cutworms ( Absence of TSP effect on implanted white grubs ( -tests, treated versus control; df t For masked chafers, 14 grubs were es Paired Counts based on Tullgren funnel extractions of two 5.08 cm diameter, -tests. Soil microarthropods, push-up green Soil microarthropods, fairway-height turf Midge larvae 1.6( Protura 1.6( Diplura 3.0( Midge larvae 3.5( aphids 3.3( Root aphids 7.6( Black cutworms 20.5( Collembola 37.0( Collembola 30.2( Mesostigmatid mites 69.0( Mesostigmatid mites 34.6( c a b Oribatid mites 55.0( Oribatid mites 119.0( Pest insects Masked chafer grubs 10.8( Taxons sampled Treated Control Table 2. clocephala turf populations of non-target soil mic 5.08 cm deep cores of soil and thatch.plots Putting sampled green 4 and fairway-height weeks or 72 h after treatment respectively. and soil microarthropods in push-up g and sampled 9 DAT. Blackinstars, cutworm treated and plots sampled were after infestedt 72 with h. 25 Data third analyzed by two-sample the crude TSP material contained an estimated 12% saponins. www.interscience.wiley.com/journal/ps 3.5 Determination of earthworm-activeThe components in methanol TSPs extract ofrapid crawling, TSPs curling caused with immediateproduction) a and irritation whipping 100% (e.g. motion, morbidity or copiousearthworms mortality mucus of within topically 2 treated h. Thestandard. same The effects occurred residualnot with powder the active. after TSP Subsequent methanolbutanol-soluble bioassays fraction extraction showed of was high thetreated methanol activity earthworms extract, in with showingproduction, the topically followed immediate by cuticle lesions, irritation apparent cell lysis,of and leakage blood mucus and 100%this mortality active fraction within were typical 2–3 for saponins. h.highly Tea saponins oxygenated The contain aglycones. NMR Owing spectra toNMR of extensive resonances overlap for in the oxygenated carbons (in the appeared normal. Similarly, TSP application to creepingmaintained bentgrass as a push-up green, or atthe fairway abundance height, of did any not of affect the majoreither taxa setting of (Table soil 2). microarthropods in identification of aglycones waslocate not at least possible. three The sugar units,for authors evidenced anomeric by could carbons signals in responsible the the proton regions (

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J Econ Entomol 5 Internat Turfgrass Bull, Sports Turf Res Inst Trends in New Crops and New Uses Madhuca latifolia Camellia oleifera JTurfgrassSci JSportsTurfResInst Care of the Green Earthworms: Their Ecology and Relationships with Soils and Land . Academic Press, Sydney, Australia (1985). :61–65 (1996). :235–242 (1980). :797–802 (2005). :115–116 (1924). Lumbricus terrestris edition, Chapman and Hall, London, UK (1996). characteristics of winter sports pitches. (1981). thatch by earthworms (Oligochaeta:invertebrates. Lumbricidae) and other soil Use casting. in sports turf areas: a review. practices on earthworm castingTurfgrass Soc on Res J golf course fairways. structureonfiveEnglishgolfcourses. (2008). Earthworm Ecology: from Darwin toChapman Vermiculture and Hall, London, UK, pp. 107–122 (1983). ( application of the fungicide benomyl. 10th Ann Meeting Bird(2008). Strike Committee USA/Canada use of acidifying fertilizerfairways. on rates of earthworm casting on golf 34 Triterpene saponins with(the gastroprotective seeds effects of from tea seed fishes in shrimp farms19 by utilization of seed. trai P, The toxicity ofAsian Fisheries five Sci native Thai plants to aquatic organisms. (2006). Am Camellia Yearbk Whipkey A. ASHS Press, Alexan tea seed ( (1981). control, Transport Canada, TP 13029 (1998). investigations into earthworm c levels, in of earthworm casts in10 golf course turf. mowrah ( 4 1830–1952. (2002). chemistry of bio-chemical and physiologicalglucoside obtained properties from of seeds a of sapo- Biochemical J 3 Baker SW, The effect of earthworm activity on the drainage 4 Potter DA, Powell AJ and Smith MS, Degradation of turfgrass 2 Edwards CA and Bohlen PJ, 5 Jefferson P, Studies on the6 earthworms of Kirby EC turf. and C. Baker Earthworms SW, Earthworm and populations, casting and control 1LeeKE, 7 Backman PA, Miltner ED, Stahnke GK and Cook TW, Effects of cultural 8 Bartlett MD, James IT, Harris JA and Ritz K,9 Earthworm Grant JD, community The activities of earthworms and the fates of seeds, in 10 Tomlin AD, Tolman JH and Thorn GD, Suppression of earthworm 13 Baker SW, Firth SJ and Binns DJ, The effect of mowing regime and the 20 Ruter JM, Nursery production of tea oil 23 Morikawa T, Li N, Nagatomo A, Maysuda H, Li24 X Terazaki and M, Tharnbuppa Yoshikawa P M, and Nakayama Y, Eradication of predatory 25 Chiayvareesajja S, Chiayvareesajja J, Rittibhonbhun N and Wiriyachi- 22 Shanan H and Ying G, The comprehensive utilization of Camellia fruits. 21 Lee C-P and Yen G-C, Antioxidant activity and bioactive compounds of 11 Evaluation of the efficacy of products and12 techniques for airport Seamans bird TW, Bernhardt GE and Steyer D, As the worm turns: 14 Williamson RC and Hong SC, Alternat 15 Lees PW, 16 Oakley RA, Earthworms. 19 Cherian KM, Gandhi VM and Mulky MJ, Toxicological evaluation of 17 Beard JB, The art and invention era in18 the early Moore B, evolution Sowton of SCM, turfs Baker-Young FW and Webster TA, On the in China. ThisExperiment is Station. paper 09-08-020 of the Kentucky Agricultural REFERENCES ) 3 25 . 1 − TSPs could 2009 Society of Chemical Industry for tea seed 46 c 50 L.) is about 6 mg L . With label restrictions 1 − 43,44 The 24 h LC 24,42 corresponding to about 1.1 and 45 ). A typical 3 m deep, 0.3 ha (9000 m 1 − values to bluegill sunfish of 0.00035 and The fungicides benomyl and thiram, which : 439–446 50 Oreochromis niloticus respectively. 66 1 10–12 respectively, − and sometimes used off-label to suppress casting, 1 Most of the earthworms expelled by TSPs, however, 2010; − 6 of finished spray at a medium labeled tank mix rate 33,47 1 − Earthworms in grass aprons of runways and taxiways or moving Before 1978, widespread usage of organochlorine biocides Toxicity of tea seed saponins to fish could be an issue in A disadvantage of earthworm expellants such as mowrah meal Managing earthworm casts on putting greens www.soci.org onto pavement afterattracting rains birds, can increasing becomeoff the an and risk aviation landings, of and hazard birdrolling by over by creating them. strikes slippery during conditions take- for aircraft die on the surface,can where be removed they by desiccateremaining mowing on relatively or plots were quickly sweeping. hardly noticeable Dried and byideal worm the remedy second would cadavers day. provide The economical, long-lasting suppression of casting without killing the earthworms,not yet but been such found. a remedy has golf course pond wouldtea need seed cake to in be order contaminated to by reach 6 54 mg kg L of were formerly usedno to longer control available. earthwormsmodification practices Earthworm recommended on by the control such USA Federal is Aviation Administration’s sites, guidelines for one bird are strike mitigation. of the habitat be useful for that purpose. (e.g. chlordane) againstearthworms turf and casting insect as aused pests side typically effect. on A suppressed numberearthworms turf of pesticides in the 1980s and 1990s were also toxic to its registration for usecourses. in Typical managing earthworm rateseastern casting Asia for on for eradicating tea golf undesirable1.1 seed fish and in saponins shrimp 15 ponds mg or L are cake used in butregistrationsofmostofthosecompoundshavebeencancelled. No pesticides are labeledStates. This for study earthworm indicates control thatany other TSPs method in (e.g. are abrasive the topdressings, far acidifying United fertilizers, moreremoval of effective clippings) than being used toorsportsfields.Withappropriatelabelrestrictionsspecifying manage casts on golf courses buffer zones around water, TSPs or otheroil saponin-rich byproducts manufacture of tea couldexcessive be earthworm casts useful on closely for mowed playing selective surfaces. management of Pest Manag Sci ACKNOWLEDGEMENTS The authors thank A Bixby,Keathley, C R Brady, King, P Burris, AJof J Powell, Contra, S Kentucky) J Vanek, Houle, and C (International for L Turfgrass assisting Williams (University Services)formulations with for and the YTE providing LeeUniversity field of tea (Department Hong Kong) of for seed calling studies, to Biology, the senior cake author’sthe The T attention Chinese use Bowyer of tea seed byproducts for earthworm management By comparison,ingredients lambda-cyhalothrin in pyrethroid or insecticidesgreens, labeled bifenthrin, have for use 96 the h on LC putting active crustaceans, e.g. impaired feeding and growth, shell softeningincreased and susceptibility to pathogens. cake to Nile tilapia ( (11.8 mL product 3.785 L 0.00021 mg L 0.85 mL L specifying buffer zones aroundpossible to ponds use TSPs or with little streams, or it no hazard should to aquatic be organisms. used in the past wasunpleasant that and collection labor of intensive, expelled andproblematic. earthworms disposing was of them could be . et al juveniles. :135–145 : 439–446 2 Aquaculture 66 . 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Res Statistix 9.0 User’s Manual 26 Murakami T, Nakamura J, Matsuda H and Yoshikawa M., Bioactive 27 Murakami T, Nakamura J, Kageura T, Matsuda H and Yoshikawa M, 29 31 Wang W, Zhang Y, Zhu Z, Zhu S, Li Y, Li M, 30 Potter DA and Kimmerer TW, Inhibition of herbivory on young holly 28 Yoshikawa M, Morikawa T, Nakamura S, Li N, Li X and Matsuda H, 33 Raw F, Estimating earthworm populations by using formalin. 35 Pal DK, Sahoo M and Mishra36 AK, Antihelminthic Friedman activity M, of stems Overview of of antibact 32 Potter DA, Buxton MC, Redmond CT, Patterson CG and Powell AJ, 34 De Silva ULL and Roberts GR, Products from tea seeds. 2. Extraction www.interscience.wiley.com/journal/ps

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