IOG PROJECT PROGRESS REPORT 2012 TITLE: PHYTONUTRIENTS ND GENOMICS OF ORGNIC TOMTOES: SOIL FERTILITY ND/OR PLNT DEFENSE PRINCIPL INVESTIGTORS ND COOPERTORS: PIS Preston ndrews nd mit Dhingr Coopertors Rick Shrpe, rtemus Hrper nd Derek Jiwn Deprtment of Horticulture, Wshington Stte University, Pullmn, W 99164 6414 KEY WORDS: ntioxidnts, phid, scorbic cid, conventionl, fertilizer, fruit, genomics, herbivory, lycopene, minerl elements, nitrogen, orgnic, pests, phenolics, phytochemicls, phytonutrients, plnt defense, soil fertility STRCT In previous studies, we showed tht pples, strwberries, nd tomtoes grown with orgnic nitrogen sources hd higher phytochemicl contents thn those fertilized with redily vilble sources of nitrogen. While soil bsed crbon/nitrogen my be the principl contributor to these differences, it is possible tht incresed pest herbivory in orgnic crops stimultes gene expression ptterns tht promote the synthesis of defense relted phytochemicls. In this study we re testing the reltive contribution of soil fertility mngement nd phid herbivory on phytonutrients in tomtoes nd gene expression differences mong these tretments. We found tht overll yield nd verge fruit size were lrger in conventionlly (CON) grown plnts, but fruit from orgniclly (ORG) grown hd greter dry mtter nd soluble solids concentrtion. ORG grown plnts lso hd 4 fold lower phid popultion densities thn CON grown plnts. Herbivory incresed the hydrophilic ntioxidnt cpcity in both immture nd ripe ORG fruit, but not in CON fruit. However, herbivory decresed the ntioxidnt cpcity in leves regrdless of fertility tretment. Ripe ORG fruit lso hd higher concentrtions of phenolic compounds, lycopene, nd vitmin C thn CON fruits, ll of which contribute to pest nd stress relted defense responses. Lef N, s well s P, ws higher in CON grown plnts, which could hve contributed to the greter phid densities on CON plnts. Lef C, Mg nd S were higher in ORG plnts. Lef C:N rtios were higher in ORG plnts, thus fvoring the synthesis of C bsed compounds, like phenolics, scorbic cid, nd crotenoids. Overll gene expression in ripe fruit ws rmped up t certin loci in ORG grown plnts. Our initil investigtion of these differences is focusing on three mjor defense relted metbolic pthwys (scorbic cid, crotenoid, nd phenylpropnoid). The observed trde off between yield/fruit size nd ccumultion of dry mtter nd defense relted phytochemicls fvors protective mechnisms ginst rthropod herbivory nd results in ensuing improvements in nutritionl qulity for humn consumption. Differentil responses by specific plnt prts t different developmentl stges indictes tht there is dynmic reltionship between rhizosphere microbiology nd plnt roots tht result in gene expression ptterns tht promote lloction of photosyntheticlly derived resources to the synthesis of defense relted phytochemicls. PROJECT DESCRIPTION In 2010 11 we grew Oregon Spring, determinte tomto cultivr, in pots in greenhouse in either n conventionl (SUNGRO Sunshine Mix #1, LC1) or orgnic (LC1:Whitney Frms Orgnic Plnting compost:topsoil (75:20:5) soil medi nd fertilized with either conventionl (Peters Professionl 20 10 20 Pet Lite Specil nd superphosphte) or orgnic [Westbridge iolink Orgnic ll Purpose 5 5 5, Micronutrient (2% Fe, 2% Mn, 3% Zn), nd Cl Plus (6% C)] liquid fertilizers. Hlf of ech fertility tretment ws grown in in the presence (+phid) or bsence ( phid) of introduced green pech phids, which were rered in the Entomology greenhouse. The phids were contined on ech plnt by exclusion cges mde of fine mesh fbric nd seled to prevent migrtion of phids from plnt to 1
plnt. Thus, there were totl of four tretments (CON/+phid, CON/ phid, ORG/+phid, ORG/ phid) grown in rndomized complete six block experimentl design under 14 hour dylength with supplementl lighting (1000 W metl hlide) t 21.1 23.2 o C dy/18.3 20 o C night tempertures. The greenhouse experiment ws continued until ech tomto plnt hd produced t lest six, completely red ripe fruits, however, most plnts produced more thn 10 ripe fruits. Ech red ripe fruit ws hrvested sequentilly s it reched mturity, nd then weighed nd its soluble solids mesured with refrctometer. smple of pericrp tissue from the equtor of ech fruit ws chopped, frozen in liquid nitrogen, nd stored in 80 o C freezer. fter the minimum number of red ripe fruits were hrvested from ech plnt, the remining unripe fruits were hrvested, their stge of development recorded (i.e. immture green, breker, turning, pink nd light red), their individul weights mesured, nd then pericrp tissue ws chopped, frozen, nd stored s for the ripe fruit. fter ll fruits were removed, the vines were destructively hrvested nd the fresh nd dry weights of leves, stems, nd roots from ech plnt were determined. Totl fruit yields nd yield efficiency (fruit yield per unit vine weight) were clculted. Smples of lef tissue ws frozen in liquid nitrogen nd stored for minerl nlysis, biochemicl ssys, nd genomic nlysis. The potting medi ws collected t the conclusion of the experiment for future minerl nlysis. phid counts of lef sub smples from ech plnt were used to estimte the totl phid infesttion on ech plnt. Since some of the cged plnts hd become infested with white flies fter the green pech phids were introduced nd pest control could no longer be used, sticky trps were plced within ech cge in order to estimte the reltive mount of white fly infesttion. White fly counts were used s covrites in the sttisticl nlysis, but they never hd significnt effect on tretment vribles. Ripe pericrp tissue from individul fruits of different weights ws nlyzed for totl phenolics, lycopene, nd totl, hydrophilic nd lipophilic Trolox Equivlent ntioxidnt Cpcity (TEC). Lef tissue ws pooled cross pirs of greenhouse blocks nd sent to the University for Idho nlyticl Sciences Lbortory for minerl nlysis. Ripe pericrp nd lef tissues were pooled cross pirs of greenhouse blocks for isoltion of totl RN. Pooled smples were pulverized using freezer mill. Totl RN ws isolted using custom modifictions of the Qigen RNEsy plnt RN isoltion kit. Qulity control nlysis ws performed on ech RN smple followed by extensive moleculr biology mnipultions to prepre sequencing redy cdn templte. riefly, ech RN smple ws converted to cdn, shered using nebulizer, polished using T4 DN polymerse, ligted to brcoded sequencing oligonucleotides nd checked on bionlyzer for qulity. The smples re currently in the wit list for generting quntittive trnscriptome dt from ech smple. OUTPUTS Work Completed s previously reported, totl yields were higher for the conventionl (CON) fertility tretment thn the orgnic (ORG) tretment, but this difference ws due to yields of immture green fruits tht remined t the end of the greenhouse experiment nd not to red ripe fruits hrvested during the experiment. There were no differences in fruit yields due to phid tretment. The verge weight of red ripe fruit ws significntly greter (P<0.001) for the CON tretment (121 g) thn the ORG tretment (99 g), with no effect of the phid tretments. There were no interctions between soil fertility nd phid tretments for fruit or vine growth mesurements. Commercilly importnt were differences in fruit dry mtter nd soluble solids, with ripe ORG fruit hving 7.3% nd 19.4% greter dry mtter nd soluble solids, respectively, thn CON fruit. One of the most striking findings ws tht the ORG/+phid tretment hd more thn four fold lower phid popultion density thn the CON/+phid tretment, lthough these differences were 2
only notbly significnt t P=0.054 (Fig. 1). The mesured popultion density of the CON/+phid tretment would exceed the IPM ction threshold for commercil greenhouse tomto production nd would necessitte use of control mesures (e.g. pesticides) to reduce phid popultions, wheres the ORG/+phid tretment would not require such mesures. Figure 1. Green pech phid popultion density on tomto leves with conventionl (CON) or orgnic (ORG) soil fertility nd infested with phids (i.e. +phids). This difference in phid popultions my be due to incresed phytochemicl defenses (e.g. phenolics) in the orgniclly fertilized plnts. Indeed, totl phenolics were higher in the ripe ORG fruit thn in the CON fruit (Tble 1), however, lef phenolics, where most phid feeding occurs, ws not different between the fertility tretments. Vitmin C nd lycopene concentrtions in ripe fruit were lso greter in the ORG fertility tretment (Tble 1), but there were no differences between phid tretments. lthough there were no differences in lef ntioxidnt cpcity between fertility tretments, leves of +phid plnts hd lower Trolox Equivlent ntioxidnt Cpcity (TEC) thn plnts without phids (4.93 vs. 4.17 mmol Trolox/g FW, P=0.014), indicting tht they were under greter biotic stress. Orgniclly fertilized plnts with phids present (ORG/+phids) hd higher TEC (especilly for the hydrophilic frction) in both immture green nd red ripe fruit thn CON/+phid fruit (Fig. 2), indicting tht fruit in ORG fertility tretment were better protected ginst oxidtive stress thn were the CON fruit. Tble 1. Totl phenolics, vitmin C, nd lycopene concentrtions of red ripe tomto fruit under conventionl (CON) or orgnic (ORG) soil fertility. There were no significnt effects of phid tretment or interctions between fertility nd phid tretments. Phytochemicls (per g FW) CON ORG P vlue Totl phenolics (mg gllic cid equiv.) 0.24 0.28 0.0002 Vitmin C ( g scorbic cid) 14.3 16.3 0.004 Lycopene (( g lycopene) 3.78 4.22 0.002 b b b b Immture green fruit Red ripe fruit 3
Figure 2. Hydrophilic nd lipophilic Trolox Equivlent ntioxidnt Cpcity (TEC) of immture green nd red ripe tomto fruit under conventionl (CON) or orgnic (ORG) soil fertility nd infested with green pech phids (+phids) or without phids ( phids). There were lso higher levels of lef C, Mg nd S in the ORG fertility tretment, but lower levels of C, N nd P thn in leves of the CON fertility tretment (Tble 2). The lef C:N rtio ws higher under ORG fertility mngement thn CON fertility, likely providing more crbon bckbones for the synthesis of defense nd stress relted phytochemicls, such s, phenolics nd ntioxidnts. There were no differences in lef minerls due to phid tretment. Tble 2. Tomto lef minerl concentrtions under conventionl (CON) or orgnic (ORG) soil fertility. Lef minerl elements (units) CON ORG P vlue Crbon (% DW) 38.3 34.8 <0.0001 Nitrogen (% DW) 5.17 4.15 0.0003 Crbon:nitrogen rtio 7.44 8.41 0.005 Phosphorus (mg/g DW) 14,167 11,833 0.03 Potssium ( g/g DW) 58,500 64,500 0.08 Clcium ( g/g DW) 39,500 47,000 0.001 Mgnesium ( g/g DW) 8,467 9,750 0.02 Sulfur ( g/g DW) 16,167 36,000 <0.0001 preliminry snpshot of gene expression ptterns (i.e. trnscript reds) in red ripe fruit tht mpped to chromosomes 1 nd 2 showed tht certin genes in the ORG plnts hd up to 200 fold higher expression frequency thn those genes in CON plnts (Fig. 3). Initil investigtion of gene expression in three metbolic pthwys tht contribute to defense relted processes will focus on the scorbic cid pthwy, the crotenoid pthwy in which lycopene nd crotene re synthesized, nd the phenylpropnoid pthwy in which flvonoids, nthocynins, nd other phenolic compounds re synthesized. Chromosome 1 Chromosome 2 Fold Difference 4
Figure 3. Mp of trnscript reds from chromosomes 1 nd 2 in ripe fruit of tomto plnts grown under CON or ORG soil fertility. Included re loci with >2 fold expression chnge, filtered for <200 fold differentil mpped reds per loci. Publictions, Hndouts, Other Text & Web Products hndout ws distributed during the poster session t CSNR s Sustinble Food, griculture nd Nturl Resources Symposium on Dec. 6, 2012. Outrech & Eduction ctivities 1. L. Gustfson (M.S. student) presented his proposl seminr for this project on Feb. 24, 2011 in Hort 510 (Horticulture Seminr). 2. P. ndrews ws n invited speker t the Interntionl Fruit Tree ssocition s nnul conference in Psco, W on Mrch 2, 2011, where he presented Linking Fruit nd Soil Qulity (Helth). 3. P. ndrews ws guest lecturer in SoilS 101 (Orgnic Grdening nd Frming) on Mrch 24, 2011, where he presented The nutritionl benefits of orgnic food. Is it helthier? 4. L. Gustfson (presenting uthor), P. ndrews, nd. Dhingr presented poster, Phytonutrients nd Genomics of Orgnic Tomtoes: Soil Fertility nd/or Plnt Defense, on this project t the Tilth Producers of Wshington nnul conference in Ykim, W on Nov. 11 13, 2011. 5. L. Gustfson (presenting uthor), P. ndrews, nd. Dhingr presented poster, Phytonutrients nd Genomics of Orgnic Tomtoes: Soil Fertility nd/or Plnt Defense, on this project t the WSU Showcse 2012. 6. P. ndrews presented results from this project s prt of n invited presenttion, Linking Soil Qulity to the Nutritionl Qulity of Fruit, in the symposium, Quntifying the Linkges mong Soil Helth, Orgnic Frming, nd Food, t the S/CSS/SSS nnul meeting on Oct. 22, 2012 in Cincinnti, OH. 7. L. Gustfson, P. ndrews (presenting uthor),. Dhingr, R. Shrpe,. Hrper nd D. Jiwn presented poster, Phytonutrients nd Genomics of Orgnic Tomtoes: Soil Fertility nd/or Plnt Defense, on this project t CSNR s Sustinble Food, griculture nd Nturl Resources Symposium on Dec. 6, 2012. IMPCTS Short Term: The tomto greenhouse experiment nd ll growth, yield, minerl, phytochemicl, nd gene sequencing nlyses re completed. Sttisticl nd genomic mpping nlysis of dt is underwy. Following this we will prepre nd submit mnuscript for publiction, which should led to federl grnt ppliction. Intermedite Term: We expect tht the knowledge gined from this nd subsequent projects on the phytochemicl nd genomic ptterns of horticulturl crops grown under orgnic or conventionl fertility mngement will permit us to determine the genetic bsis for plnt defense mechnisms nd improved nutritionl qulity for humn consumption. It will lso inform the development of more effective frming methods in order to produce more phytonutrient dense crops. Long Term: Gining the stted intermedite term knowledge will llow us develop more effective frming methods nd trgeted genes for crop breeding in order to enhnce griculturl sustinbility nd improve nutritionl qulity of horticulturl food crops for public helth. DDITIONL FUNDING PPLIED FOR / SECURED No dditionl funding hs been pplied for yet. Severl USD competitive grnt progrms would be pplicble for future funding of this reserch, including the Ntionl Institute of Food nd griculture s (NIF) Orgnic Reserch nd Extension Inititive (OREI), Specilty Crop Reserch Inititive (SCRI), or the 5
griculture nd Food Reserch Inititive (FRI) Foundtionl Grnt Progrm. It my be helpful to initilly pply for plnning grnt in order to fcilitte effective project plnning nd development. GRDUTE STUDENTS FUNDED: Luke Gustfson, M.S. (grduted Spring 2012) RECOMMENDTIONS FOR FUTURE RESERCH multi crop, field bsed, interdisciplinry study tht includes horticulturl science, plnt physiology, genomics, nd soil ecology nd microbiology is necessry nd justified. Severl different crop types could be studied, including fleshy fruited nnul (tomto s control), woody perennil (e.g. grpe), root crop (e.g. crrot), nd lefy green crop (e.g. spinch). mong crop types, severl cultivrs could be studied, becuse of the cler cultivr differences for the trits of interest. These studies should be conducted in field bsed environments, idelly humid nd semi rid, in order to determine their relevnce to commercil field conditions. Nevertheless, the focus of subsequent reserch will require compromises mong the rry of crops, cultivrs, nd field environments tht cn fesibly be studied. 6