Predictive Factors for the Suppression of Fusarium Wilt of Tomato in Plant Growth Media

Bo e o, C., 1, Phy opa hology P edic i e Fac o s o he Supp ession o Fusa ium Wil o Toma o in Plan G ow h Media Celia Bo e o, Mª Isabel T illas, José O do ás, Julio C. Tello, Manuel A ilés Fi s , hi d and i h au ho s: Dep . Ciencias Ag o o es ales, Uni e sidad de Se illa, C a. U e a, Km. 1. 41013 Se illa, Spain. Second au ho : Dep . Biología Vege al, Uni e si a de Ba celona, A da Diagonal, 645, 08028 Ba celona, Spain. Fou h au ho : Dep . P oducción Vege al, Uni e sidad de Alme ía, C a. Sac amen o, s/n, 04120 La Cañada de San U bano, Alme ía, Spain. Co esponding au ho : [email p o ec ed] Bo e o, C., 2, Phy opa hology ABSTRACT Fusa ium wil s a e economically impo an diseases o which he e a e no e ec i e chemical con ol measu es. Howe e , biological con ol and e ili y managemen a e becoming e icien al e na i es o con olling his disease. G ow h media o mula ed wi h compos s ha a e able o supp ess Fusa ium wil o oma o p o ide a con ol sys em ha in eg a es bo h s a egies. The aim o his s udy was o p edic Fusa ium wil supp ession o g ow h media using abio ic and bio ic a iables. G ape ma c compos was he mos e ec i e medium used o supp ess Fusa ium wil . Co k compos was in e media e, and ligh pea and expanded e miculi e we e he mos conduci e g ow h media. The g ow h media e alua ed we e in a pH ange o 6.26 o 7.97. Bo h compos s had high β-glucosidase ac i i y. When pH and β-glucosidase ac i i y we e aken in o accoun as p edic i e a iables, mo e han 91% o he a ia ion in se e i y o Fusa ium wil was explained. This ela ionship illus a es he e ec o nu ien a ailabili y and he deg ee o mic obios asis, wo key ac o s in his pa hosys em. Mic obial popula ions in ol ed in supp essi eness we e celluloly ic and oligo ophic ac inomyce es, ungi, and he a ios celluloly ic ac inomyce es/celluloly ic bac e ia, oligo ophic bac e ia/copio ophic bac e ia, and oligo ophic ac inomyce es/oligo ophic bac e ia. Based on communi y le el physiological p o iles, di e en communi y s uc u es we e e iden among g ow h media e alua ed. Keywo ds: Fusa ium oxyspo um . sp. lycope sici, Lycope sicon esculen um, mic obial ac i i y, pH, communi y le el physiological p o iles, Biolog, biological con ol Bo e o, C., 3, Phy opa hology Fusa ium wil diseases, caused by pa hogenic o mae speciales o he soil-inhabi ing ungus Fusa ium oxyspo um (Sacc.) Snd. & Hans., cause se e e losses in a wide a ie y o c op plan s including oma o (Lycope sicon esculen um Mill.). This pa hogen occu s h oughou mos oma o g owing a eas and de as a es he c op. Fusa ium- esis an cul i a s ha e been a ailable o decades and p o ide some deg ee o con ol; howe e , he occu ence and de elopmen o new pa hogenic aces is a con inuing p oblem. The e a e no comme cially accep able oma o cul i a s wi h adequa e esis ance (28). In addi ion, he esu gen in e es in plan ing hei loom and o he suscep ible oma o cul i a s has inc eased he incidence o Fusa ium wil . E ec i e soil ungicide ea men s o his disease a e also una ailable. Di icul ies in con olling Fusa ium wil ha e s imula ed he sea ch o biocon ol sys ems. The na u e o soils and compos s ha a e supp essi e o Fusa ium wil is he esul o complex in e ac ions be ween he abio ic cha ac e is ics o he media and mic obial popula ions (1, 2, 21, 60). Nume ous biocon ol agen s ha e been iden i ied in hese soils and compos s (21, 60). Un o una ely, he in oduc ion o biocon ol agen s has p o ided inconsis en esul s (59). G ow h media o mula ed wi h compos s such as ha dwood ba k (9, 55), pine ba k (8, 42, 44), popla ba k (16), wood sha ings (8), co k (54), g ape ma c (54), oli e pumice (44), ca le manu e (45), sewage sludges (11), and e micompos (16, 51, 52) can supp ess Fusa ium wil . Se e al o hese ha e been used success ully o consis en biological con ol, pa icula ly o con aine ized c ops (21, 22). The mic obiological na u e o Fusa ium wil -supp essi eness o mos o hese compos - amended g ow h media has been demons a ed (8, 9, 11, 16, 22, 42, 43, 45, 47, 51, 54, 55). The ole o nu i ional ac o s in wil -supp essi eness has also been sugges ed (11, 22, 42, 55). Fo some compos s, i has been demons a ed ha ungi oxic subs ances a e no in ol ed (43, 45, 47, 51). Bo e o, C., 4, Phy opa hology The main pu pose o his s udy was o de e mine he ela ionship be ween bio ic and abio ic p ope ies o g ow h media and he se e i y o Fusa ium wil in o de o p edic disease supp essi eness o g ow h media. In addi ion, nu ien a ailabili y, nu ien s a us o plan s and mic obial di e si y we e e alua ed a he conclusion o he expe imen s in o de o unde s and possible mechanisms in ol ed in he supp essi e phenomena. Fo his pu pose, wo Fusa ium supp essi e compos s, which can be used as plan g ow h media, we e compa ed wi h wo s anda d g ow h media. MATERIALS AND METHODS Plan g ow h media. Two esidues om ag icul u al and indus ial was es we e compos ed and e alua ed o Fusa ium wil supp ession: co k compos (CC) om co k (Que cus sube L.) p oduc ion and g ape ma c compos (GMC) om he alcohol-p oducing indus y (g ape skins, seeds and s ems). Bo h was es we e compos ed as desc ibed elsewhe e (54). Compos s we e compa ed o Sphagnum ligh pea (P) (Klasmann, Valinex, Palle e , Spain) and expanded e miculi e (V) (Ve miculi a y de i adas, Gijón). Pea was neu alized wi h 4 g pe li e CaCO3. To de e mine he ole o mic o lo a in supp ession, he wo compos s also we e hea ed o 60ºC o 6 days, (CC-60, GMC-60). Assessmen o disease se e i y. Disease supp essi e p ope ies o plan g ow h media we e measu ed by a Fusa ium wil bioassay desc ibed elsewhe e (11). To s anda dize ini ial condi ions, he g ow h media we e incuba ed a a wa e ension o 1000 Pa (adjus ed o weigh ) o 14 days a 25ºC. Liquid cul u e, 500 ml 1% mal ex ac (Sigma Chemical Company, S Louis, MO), o Fusa ium oxyspo um . sp. lycope sici ace 2, isola e RAF 70 ob ained om an in ec ed oma o plan s (FOL-2) was p epa ed and g own wi h con inuous Bo e o, C., 5, Phy opa hology agi a ion (130 pm) o 10 days a 25ºC. Conidia we e eco e ed by cen i uga ion a 5,000 pm, o 15 min, (Selec a Medi ige BL-S, Ab e a, Spain) and insed wice wi h s e ile dis illed wa e . The concen a ion o conidia was de e mined wi h a hemocy ome e . The six plan g ow h media we e in es ed wi h FOL-2 (105 mic oconidia/cm3 plan g ow h medium), mixed igo ously and pou ed in o 9-cm-diam. plas ic po s (330-cm3 olume). Po s o g ow h media wi hou FOL-2 we e p epa ed as con ols. Fou oma o seedlings (1 o 2 ue lea s age) g own in s e ile e miculi e we e ansplan ed in o each po . Plan s we e i iga ed as needed and e ilized wi h a nu ien solu ion con aining: 0.5 g/li e Pe e ’s olia eed 27-15-12 (Sco s, Hee len, The Ne he lands), 0.6 g/li e CaCl2 and 0.696 g/li e MgSO4.7H2O (pH 5.68). Plan s we e g own in a g ow h chambe (25ºC, 280 μE/m2/s PAR in ensi y, 16:8 h ligh : da k pho ope iod and 60% RH). Bioassays we e epea ed a leas h ee imes wi h ou plan s pe po and i e po s pe ea men . T ea men s we e a anged in a andomized block design. Disease se e i y was moni o ed a 2-day in e als o 25 days a e plan ing, and was sco ed based on a symp om se e i y scale whe e: 0 = asymp oma ic plan s; 1 = weakly in ec ed plan s (≤ 50% o lea es chlo o ic o wil ed); 2 = highly in ec ed plan s (> 50% o lea es wil ed bu plan s no dead) and 3 = dead plan s (11). Disease se e i y was exp essed as p opo ion o he maximum possible disease se e i y. A each disease assessmen , he mean o disease se e i y pe po was calcula ed. The a ea-unde - he-disease-p og ess-cu e-s anda dized (AUDPCs) pe po was calcula ed by disease se e i y in eg a ed be ween symp oms onse and bioassay inal ime and di iding by he o al du a ion (days) o he epidemic in each bioassay, in o de o compa e he a ious bioassays, which had a a ie y o epidemic du a ions. A he end o he bioassays, mean alues pe po ( ou plan s) o he ela i e-leng h-o - he-s em-wi h-b own- Bo e o, C., 6, Phy opa hology xylem (RLSBX), and he esh and d y weigh o lea es and s ems means pe po we e eco ded. These alues pe po we e conside ed as eplica es o each obse ed a iable. Physical and physico-chemical p ope ies o g ow h media. The bulk densi y (BD) o he plan g ow h media was de e mined acco ding o De Boo e al. (13). Elec ical conduc i i y (EC) and pH o media we e measu ed in a wa e ex ac (2:1; ol / ol), as desc ibed by Bun (6) and Gab iëls e al. (15), espec i ely. Physical and chemical a iables o he g ow h media we e de e mined a he beginning o he bioassays. The pH also was measu ed a he end o each bioassay. Th ee samples we e analyzed o each g ow h medium. Nu ien a ailabili y and nu ien s a us o plan s. Plan g ow h media samples (50 g) we e collec ed om he po s and ex ac ed wi h ammonium ace a e 0.5 M (pH 4.65). Plan samples (0.25 g d y weigh ) om all ea men s we e incine a ed a 550ºC o 8 h and dissol ed in 20 ml 1N HCl, il e ed as necessa y and s o ed a 4ºC un il analysis. Calcium, magnesium, i on, coppe , manganese and zinc we e de e mined by a omic abso p ion spec oscopy (63) and sodium and po assium we e analyzed by a omic emission spec ome y (63). Phospho ous was measu ed wi h colo ime y (37), ni a e (plan g ow h media) wi h e lec ome y (RQ lex Me ck, Da ms ad , Ge many) (50) and bo on (plan s) wi h colo ime y (31). To al ni ogen in plan s was measu ed wi h Kjeldahl diges ion, modi ied o include ni a es, and ammonium was subsequen ly de e mined wi h colo ime y (36). All analyses we e pe o med wi h h ee samples o plan s and wo samples o plan g ow h media om one o he h ee bioassays, a he end o he expe imen . Bo e o, C., 7, Phy opa hology Biological cha ac e is ics o g ow h media. All de e mina ions we e pe o med in incuba ed g ow h media a he beginning o bioassays, excep o mic obial di e si y. Mic obial biomass. Th ee echniques we e used o assess mic obial biomass: i) phospha es om phospholipids, (10) wi h diges ion as desc ibed by Van Veldho en and Mannae s (57) and phospha e de e mina ion wi h colo ime y (37); ii) umiga ion-ex ac ion, based on he es ima ion o cell ca bon by ex ac ion wi h saline solu ion o umiga ed and non umiga ed samples and hei con e sion o biomass (46); and iii) ac idine o ange di ec coun ing (AODC) echnique which was pe o med by coun ing cells om an aqueous ex ac ion wi h an epi luo escence mic oscope (30). The aqueous ex ac ion was il e ed h ough 0.2 μm po e-size black polyca bona e memb anes (Isopo e, Millipo e Ibe ica S.A., Mad id, Spain). Coun ing was pe o med a 1,250 x wi h a minimum o 400 cells pe il e . Con ols we e coun ed also in s e ile wa e . Th ee samples we e analyzed o each plan g ow h medium excep o hose ha we e hea ed. Mic obial ac i i y. Mic obial ac i i y was es ima ed by measu ing β-glucosidase ac i i y. This me hod is based on colo ime ic de e mina ion o he p-ni ophenol eleased by β- glucosidase when he plan g ow h medium was incuba ed wi h p-ni ophenil-β-D- glucoside (pH 6.0). The p-ni ophenol eleased was ex ac ed by il a ion and de e mined colo ime ically. β-glucosidase ac i i y was measu ed acco ding o Bandick and Dick (4). Two samples we e analyzed o each plan g ow h medium. Mic obial di e si y. The densi y o cul i able g oups o bac e ia and ungi associa ed wi h biocon ol phenomena was de e mined by dilu ion pla ing on semi-selec i e media acco ding o Tui e e al. (56) wi h modi ica ions. Samples we e aken om he hizosphe e a he end o bioassays. Plan g ow h media (5 o10 g) we e suspended in 250 Bo e o, C., 8, Phy opa hology ml o 0.1% sodium py ophospha e. The suspension was shaken and en old dilu ion se ies we e p epa ed wi h 0.1% wa e aga . Suspensions we e pipe ed on o h ee pla es pe cul u e medium and dilu ion. Fou o i e dilu ions pe se ies we e placed on pla es. Fo isola ion o copio ophic bac e ia, Bacillus spp., luo escen Pseudomonas spp., oligo ophic and celluloly ic bac e ia and ac inomyce es, 100 μg o cycloheximide was subs i u ed o 10 μg o benomyl pe ml (Ene gía e Indus ias A agonesas, S.A., Mad id) and 0.3 μl o P e icu (P opamoca b, 72.2%, Sche ing, Alcáce , Spain) pe ml. Fungal coun s we e made on po a o dex ose aga amended wi h 1,000 ppm o Te gi ol-7 (Fluka Chemie AGB, Buchs, Swi ze land) and 50 μg o oxy e acycline hyd ochlo ide (Sigma Chemical Company, S Louis, MO) (10). Da a o popula ion densi y was no malized by na u al log (Ln) ans o ma ions. Analyses we e pe o med wice wi h one sample pe each con ol and each in es ed plan g ow h medium om each o he wo bioassays. Communi y le el physiological p o iles (CLPPs). Eco-mic opla es (Biolog Inc., Haywa d, CA) we e used o de e mine di e ences in physiological abili ies o he mic obial communi ies om di e en plan g ow h media and he simila i ies be ween hese communi ies and he pa hogen. The mic opla es con ained 96 wells wi h 31 ca bon sou ces plus con ol, wi h h ee eplica es each, as has been p oposed o communi y cha ac e iza ion in en i onmen al samples (25). The g ow h media ex ac ion me hod was pe o med acco ding o Insam e al. (26). Popula ion cell densi y was de e mined by AODC o s anda dize he inoculum densi y. The densi y o FOL-2 conidia in suspension was de e mined by hemocy ome e . Fi e eco-mic opla es we e inocula ed o plan g ow h medium suspensions and h ee o FOL-2 conidial suspension. The mic opla es we e incuba ed in da kness a 25ºC o 5 days. Colo o ma ion was measu ed wi h a mic opla e eade (Labsys em Mul iskan MS, Helsinki, Finland) a 590 nm and eadings we e made a Bo e o, C., 9, Phy opa hology 12-h in e als. Fo he analysis, he a e age abso bance o he con ol wells in each pla e was sub ac ed om ha o wells con aining he same C-sou ce. Nega i e alues we e conside ed as ze o. Mic obial communi y di e si y was calcula ed wi h hese da a using Shannon (48) and Gini (19) indices o compa e unc ional di e si y o mic obial communi ies. Fo u he analysis o Biolog da a, a Riemann’s sum o each C-sou ce was calcula ed om all eadings aken du ing he 5 days o incuba ion (49). The ans o med da a we e subjec ed o p incipal componen analysis (PCA) (17), and dis ances in Euclidean space om he i s i e p incipal componen s we e calcula ed (24). S a is ical analysis. Da a collec ed om all ials we e analyzed wi h S a g aphics Plus, Ve sion 6 (SGS, 1999). The e ec o g ow h medium on AUDPCs, pH, EC, bulk densi y, nu ien a ailabili y, nu ien s a us in plan s and biological cha ac e is ics o con aine medium we e analyzed wi h ANOVA. Signi ican means we e compa ed by Tukey’s whole signi ican di e ence es (P = 0.05). O e all ela ionships be ween AUDPCs and con inuous measu ed a iables we e analyzed wi h eg ession analysis. RESULTS Supp essi eness o plan g ow h media. Fusa ium wil was supp essed mos e ec i ely wi h GMC, wi h mo e han a 92% educ ion in AUDPCs and RLSBX han in pea o e miculi e. Media CC, GMC-60 and CC-60 we e in e media e and P and V we e he mos conduci e g ow h media (Fig. 1A). The hea ed compos s had educed supp essi e abili y; howe e , GMC-60 main ained mode a e le els o supp essi eness compa ed wi h P and V (Fig. 1A, B). Medium CC-60 was no di e en om P o disease se e i y measu ed by RLSBX (Fig 1B). A an inoculum concen a ion o 1x104 mic oconidia / cm3 plan g ow h Bo e o, C., 16, Phy opa hology Py enochae a lycope sici. O he s ong nega i e co ela ions ela ed o supp essi e phenomena ound in his s udy in ol ed oligo ophic ac inomyce es, oligo ophic ac inomyce es / oligo ophic bac e ia and oligo ophic bac e ia / copio ophic bac e ia, Tui e e al. (56) ound highe oligo ophic popula ions in g ow h media ha we e supp essi e o , a he han conduci e o, R. solani. Oh a and Ha o y (41) sugges ed ha oligo oph li e is associa ed wi h cellulose decompose s on o ganic deb is. Ou esul s ega ding he nega i e co ela ion o ungi popula ions and AUDPCs a e also in ag eemen wi h hose o o ganic household was e compos s supp essi e o R. solani (56). On he o he hand, Ku e e al. (32) ound no di e ences in o al numbe s o ungi o ha dwood ba k compos s ha we e supp essi e and non-supp essi e o Rhizoc onia damping-o ; howe e , he e we e quan i a i e di e ences in he ela i e abundance o ce ain axa which sepa a ed supp essi e om conduci e media amended wi h ha dwood ba k compos . The high popula ion densi ies o Bacillus spp. associa ed wi h he wo compos s, ei he na u al o hea ed (Table 7), may accoun o he supp essi eness in ou compos s. Simila ly in a s udy on po ing mix amended wi h chicken manu e, high popula ions densi ies o Bacillus spp. we e associa ed wi h he su i al o seedlings in es ed wi h Phy oph ho a cinnamomi (3). The high popula ion densi ies o luo escen Pseudomonas spp. in CC (con ols) may be in ol ed in he supp essi eness o his ma e ial (2, 14). The communi y s uc u es o he plan g ow h media s udied we e di e en a he beginning o he bioassays, based on communi y le el physiological p o iles, he simila ca abolic po en ial o CC mic obial communi ies and FOL-2, oge he wi h he lowe di e si y index and he highe β-glucosidase ac i i y and AOCD mic obial biomass, sugges a high compe i ion o C-sou ces. These esul s, along wi h he low le el o disease supp ession o hea ed CC, sugges ha con ol is biological, and basically conce ns compe i ion be ween Bo e o, C., 17, Phy opa hology F. oxyspo um and endogenous mic obial communi ies. Al hough he high biological di e si y ound in e miculi e is no ewo hy, i s e y low mic obial ac i i y and biomass should no be o e looked. The high di e si y ound o his ino ganic ma e ial can be explained by he ac ha e miculi e is almos o ally s e ile p io o use. The p ima y sou ce o mic oo ganisms in his medium is p obably handling p ocesses, al hough ae ial ansmission may also play a ole (7). Highe di e si y canno explain disease con ol in his s udy, as e idenced by he Shannon di e si y index be ween he conduci e pea and he supp essi e g ape ma c compos and he lowe 1-G and Shannon index o he supp essi e co k compos wi h espec o pea . Thus, ou esul s a e in ag eemen wi h hose o Boehm e al. (5) o con ol o Py hium oo o , bu no wi h hose o Ni a (39) o Toyo a e al. (53), whe e lowe di e si y was associa ed wi h conduci eness o b own s em o o bean and Fusa ium wil o adish, espec i ely. In conclusion, he s ong ela ionship ound a he beginning o he bioassays o AUDPCs wi h pH o g ow h media and β-glucosidase ac i i y o mic obial popula ions suppo s i s p edic i e alue. The pH e lec ed he a ailabili y o ino ganic nu ien s, in his mul iple co ela ion. Fo Fusa ium wil con ol, he impo ance o a ailabili y o speci ic nu ien s is demons a ed by e ili y managemen (27) and by he biocon ol induced o Fe compe i ion by luo escen Pseudomonas (2, 14). Based on ou esul s, mic obial compe i ion (measu ed as β-glucosidase ac i i y), especially o he C-sou ce p esen in he media and oo exuda es (33, 38, 60), plays a signi ican ole in disease supp ession. The mic obial communi y ac s as a nu ien sink ha is independen o he mic obial communi y s uc u e in his s udy. Fu u e esea ch may p o ide addi ional in o ma ion ha would allow Bo e o, C., 18, Phy opa hology us o gene alize he ela ionships be ween pH and β-glucosidase ac i i y as p edic i e a iables o he supp essi eness o o he g ow h media and o he Fusa ium wil diseases. ACKNOWLEDGEMENTS This esea ch was suppo ed by g an IFD97-1322-C04 om he Comisión In e minis e ial de Ciencia y Tecnología (P oyec os I+D del P og ama FEDER) and by g an AGL2002- 04313-C03 om he Minis e io de Ciencia y Tecnología (P oyec os I+D del Plan Nacional de In es igación Cien í ica, Desa ollo e Inno ación Tecnológica) o Spain. We hank S. Cas illo, M. C. Mau i and M .J. In an es o excellen echnical assis ance and D . H. A. J. Hoi ink o kindly e iewing he manusc ip . LITERATURE CITED 1. Alabou e e, C. 1986. Fusa ium-wil supp essi e soils om he Châ eau ena d egion: e iew o a 10-yea s udy. Ag onomie 6:273-284. 2. Alabou e e, C., Hoepe , H., Lemanceau, P., and S einbe g, C. 1996. Soil supp essi eness o diseases induced by soilbo ne plan pa hogens. Pages 371-413 in: Soil Biochemis y, Vol 9. G. S o zky, and J. M. Bollag, eds. Ma cel Dekke , Inc, New Yo k. 3. A yan ha, I. P., C oss, R., and Gues , D. I. 2000. Supp ession o Phy oph ho a cinnamomi in po ing mixes amended wi h uncompos ed and compos ed animal manu es. Phy opa hology 90:775-782. 4. Bandick, A. K., and Dick, R. P. 1999. Field managemen e ec s on soil enzyme ac i i ies. Soil Biol. Biochem. 31:1471-1479. 5. Boehm, M. J., Wu, T., S one, A. G., K aakman, B., Ianno i, D. A., Wilson, G. E., Madden, L. V., and Hoi ink, H. A. J. 1996. C oss-Pola ized magic-angle spinning 13C nuclea magne ic esonance spec oscopic cha ac e iza ion o soil o ganic ma e ela i e o cul u able bac e ial species composi ion and sus ained biological con ol o Py hium oo o . Appl. En i on. Mic obiol. 63:162-168. Bo e o, C., 19, Phy opa hology 6. Bun , A. C. 1988. Media and Mixes o Con aine -G own Plan s: a Manual on he P epa a ion and Use o G owing Media o Po Plan s. 2nd ed. Academic Di ision o Unwin Hyman L d. London. 7. Ca lile, W. R., and Wilson, D. P. 1991. Mic obial ac i i y in g owing media - A b ie e iew. Ac a Ho icul . 294:197-206. 8. Cebolla, V., and Pe a, J. 1983. Supp essi e e ec s o ce ain soils and subs a es agains Fusa ium wil o ca na ion. Ac a Ho icul . 150:113-119. 9. Che , D. G., Hoi ink, H. A. J., and Madden, L. V. 1983. E ec s o o ganic componen s in con aine media on supp ession o Fusa iu n wil o ch ysan hemum and lax. Phy opa hology 73:279-281. 10. Chen, W., Hoi ink, H. A. J, Schmi henne , A. F., and Tuo inen, O. H. 1988. The ole o mic obial ac i i y in supp ession o damping-o caused by Py hium ul imum. Phy opa hology 78:314-322. 11. Co xa e a, L., T illas-Gay, M. I., S einbe g, C., and Alabou e e, C. 2002. Use o sewage sludge compos and T ichode ma aspe ellum isola es o supp ess Fusa ium wil o oma o. Soil Biol. Biochem. 34:467-476. 12. Da e , P., Messiaen, C. M., and Rieu , P. 1996. In e p e a ion des mani es a ions hi e nales de la usa iose de la oma e en A ique du no d, a o isées pa la p esence de sels dan les eaux d’i iga ion. P oc. Fi s Cong. Medi e . Phy opa hol. Union: 407-416. 13. De Bood , M., Ve donk, O., and Cappae , I. 1974. Me hod o measu ing he wa e elease cu e o o ganic subs a es. Ac a Ho icul . 37:2054-2062. 14. Duij , B. J., Meije , J. W., Bakke , P. A. H. M., and Schippe s, B.1993 Side opho e-media ed compe i ion o i on and induced esis ance in he supp ession o Fusa ium wil o ca na ion by luo escen Pseudomonas spp. Ne h. J. Pl. Pa h. 99:277-289. 15. Gab iëls, R., Van Kei sbulck, W., and Ve donck, O. 1991. Re e ence me hod o physical and chemical cha ac e iza ion o g owing media: an in e na ional compa a i e s udy. Ac a Ho icul . 294:147-160. 16. Ga ibaldi, A. 1988. Resea ch on subs a es supp essi e o Fusa ium oxyspo um and Rhizoc onia solani. Ac a Ho icul . 221:271-277. 17. Glimm, E., Heue , H., Engelen, B., Smalla, K., and Backhaus, H. 1997. S a is ical compa isons o communi y ca abolic p o iles. J. Mic obiol. Me h. 30:71-80. 18. Hand eck, K. and Black, N. 1991. G owing Media o O namen al Plan s and Tu . New Sou h Wales Uni e si y P ess, Kensing on, Aus alia. Bo e o, C., 20, Phy opa hology 19. Ha ch, B. D., Co ell, R. L., Meech, W., Ki kby, C. A., and Pankhu s , C. E. 1997. Using he Gini coe icien wi h BIOLOG subs a e u ilisa ion da a o p o ide an al e na i e quan i a i e measu e o compa ing bac e ial soil communi ies. J. Mic obiol. Me h. 30:91-101. 20. Hoi ink, H. A. J., and Boehm, M. J. 1999. Biocon ol wi hin he con ex o soil mic obial communi ies: a subs a e-dependen phenomenon. Annu. Re . Phy opa hol. 37:427-446. 21. Hoi ink, H. A. J., Boehm, M. J., and Hada , Y. 1993. Mechanisms o supp ession o soilbome plan pa hogens in compos -amended subs a es. Pages 601-621 in: Science and Enginee ing o Compos ing: Design, En i onmen al, Mic obiological and U iliza ion Aspec s. H. A. J. Hoi ink, and H. M. Keene , eds. Renaissance Publica ions, Wo hing on, OH. 22. Hoi ink, H. A. J., Inba , Y., and Boehm, M. J. 1991. S a us o compos -amended po ing mixes na u ally supp essi e o soilbo ne diseases o lo icul u al c ops. Plan Dis. 75:869-873. 23. Hoi ink, H. A. J., Madden, L. V., and Boehm, M. J. 1996. Rela ionships among o ganic ma e decomposi ion le el, mic obial species di e si y and soilbo ne disease se e i y. Pages 237-249 in: P inciples and p ac ice o managing soilbo ne plan pa hogens. R. Hall, ed. A.P.S. P ess, S . Paul, MN. 24. Howa d, P. J. A. 1999. Analysis o in e -sample dis ances om BIOLOG pla e da a in euclidean and simplex spaces. Soil Biol. Biochem. 31:1323-1330. 25. Insam, H. 1997. A new se o subs a es p oposed o communi y cha ac e iza ion in en i onmen al samples. Page 259 in: Mic obial Communi ies. Func ional Ve sus S uc u al App oaches. H. Insam, and A. Rangge , eds. Sp inge , Heidelbe g Ge many. 26. Insam, H., Amo , K., Renne , M., and C epaz, C. 1996. Changes in unc ional abili ies o he mic obial communi y du ing compos ing o manu e. Mic obial Ecol. 31:77-87. 27. Jones J. P., Engelha d A. W., and Wol z S. S. 1993. Managemen o Fusa ium wil o ege ables and o namen als by mac o-and mic oelemen nu i ion. Pages 18-32 in: Soilbo ne Plan Pa hogens: Managemen o diseases wi h mac o-and mic oelemen s. W. A. Engelha d, ed. Ame ican Phy opa hological Socie y, S Paul, MN. 28. Jones, J. B., Jones, J. P., S all, R. E., and Zi e , T. A., eds. 1991. Compendium o oma o diseases. Ame ican Phy opa hological Socie y, S . Paul, MN. 29. Jones, J. P., and Wol z, S. S. 1981. Fusa ium-inci ed diseases o oma o and po a o and hei con ol. Pages 157-168 in: Fusa ium: Diseases, Biology and Taxonomy. P. E. Nelson, T. A. Toussoun, and R. J. Cook, eds. Penn. S a e Uni . P ess, Uni e si y Pa k, PA. Bo e o, C., 21, Phy opa hology 30. Kepne J ., R. L., and P a , J. R. 1994. Use o luo och omes o di ec enume a ion o o al bac e ial in en i onmen al samples: Pas and p esen . Mic obiol. Re . 58:603-615. 31. Ke en, R. 1996. Bo on. Pages: 603-626 in: Me hods o Soil Analysis. D. L. Spa ks, ed. Soil Science Socie y o Ame ica, Inc., Madison, WI. 32. Ku e , G. A., Nelson, E. B., Hoi ink, H. A. J., and Madden, L. V. 1983. Fungal popula ions in con aine media amended wi h compos ed ha dwood ba k supp essi e and conduci e o Rhizoc onia damping-o . Phy opa hology 73:1450- 1456. 33. Lockwood, J. L. 1988. E olu ion o concep s associa ed wi h soilbo ne plan pa hogens. Annu. Re . Phy opa hol. 26:93-121. 34. Ma shall, K. C., and Alexande , M. 1960. Compe i ion be ween soil bac e ia and Fusa ium. Plan Soil 12:143-148. 35. Mondal, S. N., Kagejama, K., and Hyakumachi, M. 1995. Ge minabili y, iabili y, and i ulence o chlamydospo es o Fusa ium solani . sp. phaseoli as a ec ed by he loss o endogenous ca bon. Phy opa hology 85:1238-1244. 36. Mul aney, R. L. 1996. Ni ogen-ino ganic o ms. Pages 123-1184 in: Me hods o Soil Analysis. D. L. Spa ks, ed. Soil Science Socie y o Ame ica, Inc., Madison, WI. 37. Mu phy, J., and Riley, J. P. 1962. A modi ied single solu ion me hod o he de e mina ion o phospha e in na u al wa e s. Analy ica Chimica Ac a 27:31-36. 38. Nelson, E. B. 1990. Exuda e molecules ini ia ing ungal esponses o seeds and oo s. Plan Soil 129:61-73. 39. Ni a, T. 1991. Di e si y o oo ungal lo as: i s implica ions o soil-bo ne diseases and c op g ow h. JARQ-Jpn. Ag . Res. Q. 25:6-11. 40. Nogués, S., Co xa e a, L. Aleg e, L., and T illas, M. I. 2002. Limi a ions o pho osyn hesis in oma o lea es induced by Fusa ium wil . New Phy ol. 154:461- 470. 41. Oh a, H., and Ha o i, T. 1983. Oligo ophic bac e ia in o ganic deb is and plan oo s in a paddy ield soil. Soil Biol. Biochem. 15:1-8. 42. O likowski, L. B. 1983. In luence o subs a um ype and ungicidal ea men on he de elopmen o Fusa ium wil o ca na ion. Ac a Ho icul . 150:127-139. 43. Pe a, A., and Filippi, C. 1987. Con olling o Fusa ium wil in ca na ion wi h ba k compos . Biological Was es 22:219-228. Bo e o, C., 22, Phy opa hology 44. Pe a, J., and Cal e , C. 1989. Supp ession o Fusa ium wil o ca na ion in a compos ed pine ba k and compos ed oli e pumice. Plan Dis. 73:699-700. 45. Reu eni, R., Ra i , M., K asno sky, A., F eiman, L., Medina, S., Ba , A., and O ion, D. 2002. Compos induces p o ec ion agains Fusa ium oxyspo um in swee basil. C op P o . 21:583-587. 46. Rice, W. C., Moo man, B. T., and Bea e, M. 1996. Role o mic obial biomass ca bon and ni ogen and soil quali y. Pages 203-215 in: Me hods o Assessing Soil Quali y. D. L. Spa ks, ed. Soil Science Socie y o Ame ica, Inc., Madison,WI. 47. Se a-Wi ling, C., Houo , S., and Alabou e e, C. 1996. Inc eased soil supp essi eness o Fusa ium wil o lax a e addi ion o municipal solid was e compos . Soil Biol. Biochem. 28:1207-1214. 48. Shannon, C. E., and Wea e , W. 1949. The ma hema ical heo y o communica ion. Uni e si y o Illinois P ess, U bana, IL. 49. Sha ma, S., Piccolo, A., and Insam, H. 1997. Di e en ca bon sou ce u iliza ion p o iles o ou opical soils om E hiopia. Pages 132-140 in: Mic obial Communi ies. Func ional Ve sus S uc u al App oaches. H. Insam and A. Rangge , eds. Sp inge , Heidelbe g Ge many. 50. Sha man, K. V., and Whi ehouse, M. 1993. Ni ogen d awdown index as a p edic o o ni ogen equi emen s o Neph olepis in sawdus media. Sci. Ho ic. (Ams e dam) 54:32-33. 51. Szczech, M. M. 1999. Supp essi eness o e micompos agains Fusa ium wil o oma o. J. Phy opa hol. 147:155-161. 52. Szczech, M., Rondomanski, W., B zeski, M. W., Smolinska, U., and Ko owski, J. F. 1993. Supp essi e e ec o a comme cial ea hwo m compos on some oo in ec ing pa hogens o cabbage and oma o. Biol. Ag ic. Ho ic. 10:47-52 53. Toyo a, K., Kimu a, M., Takechi, S., and Kuninaga, S. 2000. Compa ison o mic obial communi y s uc u e be ween soils supp essi e and conduci e o Fusa ium-wil o adish. Soil Mic oo . 54:69-79. 54. T illas, I., A ilés, M., O do ás, J., Bello, A., and Tello, J.C. 2002. Using compos as a me hyl b omide al e na i e. Biocycle 43:64-68. 55. T illas-Gay, M. I., Hoi ink, H. A. J., and Madden, L. V. 1986. Na u e o supp ession o Fusa ium wil o adish in a con aine medium amended wi h compos ed ha dwood ba k. Plan Dis. 70:1023-1027. 56. Tui e , G., Szczech, M., and Bollen, G. J. 1998. Supp ession o Rhizoc onia solani in po ing mix u es amended wi h compos made om o ganic household was e. Phy opa hology 88:764-773. Bo e o, C., 23, Phy opa hology 57. Van Veldho en, P. P., and Mannae s, G. P. 1987. Ino ganic and o ganic phospha e measu emen s in he nanomola ange. Anal. Biochem. 161:45-48. 58. Waksman, S. A. 1927. P inciples o Soil Mic obiology. The Williams and Wilkins Co., Bal imo e, MD. 59. Wa io , P., Kondu u, K., and Vasude an, P. 2002. Fo mula ion o biological con ol agen s o pes and disease managemen . Pages 421-441 in: Biological Con ol o C ops Diseases. Gnanamanickam, S. S. ed. Ma cel Decke , Inc. New Yo k. 60. Welle , D. M., Raaijmake s, J. M., McSpadden Gga dene , B. B., and Thomashow, L. S. 2002. Mic obial popula ions esponsible o speci ic soil supp essi eness o plan pa hogens. Annu. Re . Phy opa hol. 40:309-348. 61. Wol z, S. S., and Jones, J. P. 1981. Nu i ional equi emen s o Fusa ium oxyspo um: Basis o a disease con ol sys em. Pages 340-349 in: Fusa ium: Diseases, Biology, and Taxonomy. P. E. Nelson, T. A. Toussoun, and R .J. Cook, eds. Penn. S a e Uni . P ess, Uni e si y Pa k, PA. 62. Wo kneh, F., and an B uggen, A. H. C. 1994. Mic obial densi y, composi ion, and di e si y in o ganically and con en ionally managed hizosphe e soil in ela ion o supp ession o co ky oo o oma oes. Appl. Soil Ecol. 1:219-230. 63. W igh , R. J., and S uczynski, T. 1996. A omic abso p ion and Flame Emission Spec ome y. Pages: 65-90 in: Me hods o Soil Analysis. D. L. Spa ks, ed. Soil Science Socie y o Ame ica, Inc., Madison, WI. Bo e o, C., 24, Phy opa hology Fig 1. S anda dized a ea unde disease p og ess cu e (AUDPCs) and ela i e leng h o s em wi h b own xylem (RLSBX) o oma o plan s in six g ow h media. CC: compos ed co k; CC-60: hea ed compos ed co k; P: pea ; V: e miculi e; GMC: g ape ma c compos ; GMC-60; hea ed g ape ma c compos . Disease se e i y scale was 0: asymp oma ic plan s, 1: ≤ 50% a ec ed lea es, 2: > 50% a ec ed lea es, 3: dead plan s. Plan g ow h media we e in es ed wi h Fusa ium oxyspo um . sp. lycope sici ace 2 a he dose o 1 x 105 mic oconidia / cm3. Da a o AUDPCs and RLSBX we e ans o med o analysis wi h he a csine √x. Ba s wi h he same le e a e no signi ican ly di e en acco ding o Tukey's es a P < 0.05. S anda d e o o he mean is indica ed by e ical line. Fig 2. Co ela ion (mul iple) be ween s anda dized a ea unde disease p og ess cu e (AUDPCs), β-glucosidase ac i i y and pH, eco e ed om six plan g ow h media a he beginning o bioassays. These g ow h media we e in es ed wi h Fusa ium oxyspo um . sp. lycope sici ace 2 a 1 x 105 mic oconidia / cm3. CC: compos ed co k; CC-60: hea ed compos ed co k; P: pea ; V: e miculi e; GMC: g ape ma c compos ; GMC-60; hea ed g ape ma c compos . Disease se e i y scale was 0: asymp oma ic plan s, 1: ≤ 50% a ec ed lea es, 2: > 50% a ec ed lea es, 3: dead plan s. Fig 3. O dina e plo o p incipal componen s (PC1 and PC2) o mic obial communi ies o six plan g ow h media a beginning o bioassay and Fusa ium oxyspo um . sp. lycope sici (FOL-2) acco ding o Biolog pa e n. CC: compos ed co k; P: pea ; V: e miculi e; GMC: g ape ma c compos . Bo e o, C., 25, Phy opa hology Plan g ow h medium CC CC-60 P V GMC GMC-60 RLSBX AUDPCs 0.0 0.1 0.2 0.3 0.4 0.5 0.6 aa b d bc c ab a c 0.0 bc d c A B 0.2 0.4 0.6 Fig 1. Bo e o, C., 32, Phy opa hology Table 5. Nu ien s a us o oma o lea es and s ems g own in six plan g ow h media a he end o bioassay. Plan g ow h media we e in es ed wi h Fusa ium oxyspo um . sp. lycope sici ace 2 a 1 x 105 mic oconidia / cm3 N P K Mg Ca Na Fe Cu (10-2) Zn Mn B (10-5) mg nu ien / g d y plan a Plan g ow h medium b CC 15.588 bc 21.106 bc 24.261 cd 7.964 ab 31.398 a 1.931 b 0.112 cd 1.173 ab 0.071 bc 0.125 bc 4.129 b CC-60 17.248 abc 24.588 a 27.893 bc 6.110 cd 31.237 a 1.930 b 0.157 bc 1.601 a 0.127 a 0.306 a 4.469 ab P 22.775 a 24.567 a 22.483 cd 4.590 d 26.552 ab 2.030 b 0.310 a 1.543 a 0.094 ab 0.120 bc 4.155 b V 21.616 ab 23.235 ab 20.025 d 8.953 a 22.076 b 3.276 a 0.240 ab 1.806 a 0.062 c 0.103 c 4.208 ab GMC 9.728 d 17.849 c 34.268 ab 7.176 bc 25.965 ab 1.166 c 0.093 d 0.897 b 0.072 bc 0.084 d 4.155 b GMC-60 14.357 c 20.603 bc 41.617 a 7.133 bc 27.149 ab 1.507 bc 0.125 cd 1.425 a 0.125 a 0.145 b 4.704 a a Analysis o a iance was pe o med wi h ans o med da a: 1/x o Fe, Cu and Mn and Ln(x) o N o ob ain a cons an a iance. Wi hin each, alues ollowed by di e en le e s a e signi ican ly di e en based on Tukey's es a P < 0.05. b CC: compos ed co k; CC-60: hea ed compos ed co k; P: pea ; V: e miculi e; GMC: g ape ma c compos ; GMC-60; hea ed g ape ma c compos . Bo e o, C., 33, Phy opa hology Table 6. Co ela ion (simple) among AUDPCs and nu ien a ailabili y o plan g ow h media (mg o nu ien s / cm3 plan g ow h medium) and oma o plan s (mg o nu ien s /g d y weigh ) a he end o he bioassay Elemen R2 P b Equa ion Plan g ow h media AUDPCs a Na 81.36 -0.902 * AUDPCs = 0.54 – 2.04 * Na Cu 77.23 0.879 * AUDPCs = -0.006 + 12.96 * √Cu Ca 77.16 -0.878 * AUDPCs = 0.58 – 0.20 * Ln (Ca) Toma o plan s AUDPCs a Fe 98.54 -0.993 *** AUDPCs = 0.65 - 0.06 / Fe N 97.24 -0.986 ** AUDPCs = e ( 1.29 - 45.14 / N ) Cu 93.01 -0.964 ** AUDPCs= e ( 1.85 - 0.05 / Cu ) P 87.69 -0.936 ** AUDPCs = e ( 5.56 - 155.53 / P ) Na 78.74 -0.887 * AUDPCs = e ( 1.02 – 4.68 / Na ) a The six plan g ow h media we e inocula ed wi h Fusa ium oxyspo um . sp. lycope sici ace 2 a 1 x 105 mic oconidia / cm3. b Signi icance le els: P < 0.05 *, P < 0.01 ** and P < 0.001 ***. Bo e o, C., 34, Phy opa hology Table 7. Mic obiological p ope ies o plan g ow h media a he end o he bioassay. Con ol: nonin es ed media and in es ed: Fusa ium oxyspo um . sp. lycope sici ace 2 a 1 x 105 mic oconidia / cm3 x 105 CFU / cm3 plan g ow h medium a G ow h media b CONTROLS ca b c p cb oa ob CC 0.1120 ab 3.111 c 2863 ab 16.17 a 336.3 ab 2.596 b 1329 a 0.3523 bc CC-60 0 b 33.63 b 3686 ab 0.0075 c 1093 a 9.263 b 3519 a 1.993 ab P 0.0373 ab 0.122 d 1902 b 0.0886 bc 227.5 ab 0.8007 c 504.1 a 1.574 ab V 0 b 7.480 d 5956 ab 0.2689 bc 135.2 b 0.0787 d 2299 a 0.5135 bc GMC 4.824 a 19.99 b 3781 ab 0.3026 b 2127 a 44.21 a 2728 a 7.270 a GMC-60 ND c 137 a 9225 a 0.0238 c 402.7 ab 2.878 b 2687 a 0.0923 c G ow h media INFESTED CC 0 b 8.766 ab 6646 1.109 ab 1083 2.651 b 3679 0.9636 b CC-60 0 b 32.07 ab 9311 0.7774 ab 688.0 1.354 b 6206 0.4951 b P 0 b 1.478 bc 4526 0.0290 b 671.4 1.082 b 2089 0.5765 b V 0 b 0.123 c 1799 0.0638 ab 227.0 0.0023 c 1377 0.2788 b GMC 1.599 a 25.31 a 3734 0.2308 ab 512.5 49.24 a 4454 3.706 a GMC-60 0 b 90.62 a 5494 2.5614 a 597.6 1.973 b 4201 2.434 b a ca: celluloly ic ac inomyce es, b: Bacillus spp., c: copio ophic bac e ia, p: luo escen Pseudomonas spp., cb: celluloly ic bac e ia, oa: oligo ophic ac inomyce es, ob: oligo ophic bac e ia, : ungi. Wi hin each, alues ollowed by di e en le e s a e signi ican ly di e en based on Tukey's es a P < 0.05. Analysis o a iance was pe o med wi h ans o med da a in Ln(x). Fo celluloly ic ac inomyce es (2m)-1, m = nº eplica e was added o ob ain no malized da a. b CC: compos ed co k; CC-60: hea ed compos ed co k; P: pea ; V: e miculi e; GMC: g ape ma c compos ; GMC-60; hea ed g ape ma c compos . c ND: no de e mined. Bo e o, C., 35, Phy opa hology Table 8. Co ela ion (simple) among AUDPCs and di e en mic oo ganisms eco e ed om g ow h media (CFU / cm3 plan g ow h medium) Mic oo ganism g oup b R2 P c Equa ion AUDPCs a oa 99.31 0.996 *** AUDPCs = 1/(2.8635+5.8 10-6·oa) oa/ob 99.12 0.996 *** AUDPCs = 1/(2.6899+2586.12·oa/ob) ca 98.50 0.992 *** AUDPCs = 1 / ( 3.694 + 0.172 10-3 ca) ca/cb 98.50 0.992 *** AUDPCs = 1/(3.6939+8829.72·ca/cb) 80.68 -0.898 * AUDPCs = e(-0.6941-0.0000064· ) ob/c 68.90 -0.830 * AUDPCs = e(-0.7444-3.1961· ob/c) a The six plan g ow h media we e in es ed wi h Fusa ium oxyspo um . sp. lycope sici ace 2 a 1 x 105 mic oconidia / cm3. b oa: oligo ophic ac inomyce es, ob: oligo ophic bac e ia, ca: celluloly ic ac inomyce es, cb: celluloly ic bac e ia, : ungi, c: copio ophic bac e ia. c Signi icance le els: P < 0.05 *, P < 0.01 ** and P < 0.001 ***.