Identifying characteristics of Verticillium wilt suppressiveness in olive mill composts

A ilés, Plan Disease 1 TITLE 1 Iden i ying cha ac e is ics o Ve icillium wil supp essi eness in oli e mill compos s 2 AUTHORS 3 Manuel A ilés and Celia Bo e o 4 Dep . Ciencias Ag o o es ales, Escuela Técnica Supe io de Ingenie ía Ag onómica, Uni e sidad 5 de Se illa, C a. U e a km 1, 41013 Se illa, Spain. e-mail: a [email protected] and cbo [email p o ec ed] 6 Co esponding au ho : Celia Bo e o. Tel: +34 954486457. Fax: +34 954486436. e-mail: 7 [email p o ec ed] 8 ABSTRACT 9 The aims o his s udy we e o assess he po en ial supp essi e e ec s o di e en oli e mill 10 compos s on Ve icillium wil and o elucida e he supp essi e mechanisms. To his end, ou oli e 11 mill compos s om di e en c op a eas wi h wo ma u a ion le els we e selec ed. A e conduc ing 12 he Ve icillium wil bioassays in co on, he supp essi e e ec was obse ed in only one compos . 13 Compos ma u a ion le el did no a ec disease de elopmen . The s anda dized a ea-unde - he-14 disease-p og ess-cu e- and mic oscle o ia concen a ion we e associa ed wi h low API-ZYM 15 enzyma ic di e si y, β-glucosidase ac i i y, pH and high elec ical conduc i i y (EC). To assess 16 he na u e o supp essi eness in he sup essi e compos , addi ional bioassays we e pe o med 17 wi h h ee ea ed compos -amended g owing media (N-supplemen ed, au ocla ed and hea 18 ea ed a 60ºC o 6 days). Supp essi eness was pa ially educed wi h hea ea men s, whe e 19 N-ace yl-β-glucosaminidase ac i i y disappea ed. In his compos high oligo ophic ac inomyce e 20 popula ions we e associa ed wi h disease educ ion. The e o e, plan g ow h media amended wi h 21 di e en oli e mill compos s do no always show supp essi eness agains Ve icillium wil . 22 Enzyma ic di e si y, β-glucosidase ac i i y, pH and EC may be su icien o p edic whe e oli e 23 A ilés, Plan Disease 2 mill compos plan g ow h media will be e ec i e in educing Ve icillium wil and mic oscle o ia 24 concen a ion. Gene al and speci ic supp essi eness a e in ol ed in he mechanism o compos 25 supp ession. 26 Keywo ds: Plan g ow h media, oli e mill compos , Ve icillium dahliae, supp essi eness 27 28 Ve icillium wil is cu en ly conside ed a majo soilbo ne disease h ea ening many c ops 29 wo ldwide and un o una ely, he e a e limi ed chemical con ol op ions (López-Escude o and 30 Me cado-Blanco, 2011). In Andalusia, sou he n Spain, his disease causes se e e p oblems in 31 bo h young and old oli e o cha ds and has sp ead o all he o he majo oli e-g owing a eas in 32 Spain (Jiménez-Díaz e al., 2012). Ve icillium wil o oli e and co on a e e y simila 33 pa hosys ems. Ve icillium dahliae isola es in ec ing bo h c ops a e adi ionally classi ied in o 34 de olia ing and non-de olia ing pa ho ypes. Thus, an isola e eco e ed om co on o oli e 35 displays c oss- i ulence in bo h c ops (López-Escude o and Me cado-Blanco, 2011). The p ima y 36 o ms o V. dahliae dispe sion a e he es ablishmen o new oli e o cha ds on o me co on ields 37 and pa hogen in ec ion o suscep ible c ops nea by (Jiménez-Díaz e al., 2012; López-Escude o 38 and Me cado-Blanco, 2011). P e en i e and/o biological con ol s a egies mus be de eloped o 39 educe his disease. 40 Spain is he main oli e ui and oli e oil p oduce in he wo ld, accoun ing o nea ly 40% o 41 p oduc ion (FAO, 2015). Andalusia is he la ges oli e cul i a ion a ea (61%) and accoun s o 42 83% o he o al na ional p oduc ion o oli es o oil p oduc ion (Minis e io de Ag icul u a, 43 Alimen ación y Medio Ambien e, 2015). Nowadays, a wo-phase cen i uga ion sys em is he mos 44 commonly used oli e oil ex ac ion me hod in Spain. This sys em gene a es oli e oil plus a semi-45 solid was e, known as oli e mill was e o alpe ujo (Mo illo e al., 2009), which ep esen s 90% o 46 oli e weigh (Minis e io de Medio Ambien e, 2007). 47 A ilés, Plan Disease 3 Oli e mill was e is no ecommended o use as an o ganic soil amendmen in oli e o cha ds due 48 o i s acidic pH, po en ial phy o oxici y and he possibili y o soil con amina ion wi h V. dahliae 49 (Jiménez-Díaz e al., 2012; Mo illo e al., 2009). The e is inc easing in e es in compos ing as a 50 sus ainable s a egy o ecycle his was e o ag icul u al pu poses. To achie e his, due o i s 51 semi-solid consis ency and low po osi y, he esidue should be mixed wi h bulking agen s 52 (Chowdhu y e al., 2013; Mo illo e al., 2009). The ype o bulking agen , he a io o oli e mill 53 was e o bulking agen and he compos ing me hod used may a y. Many ypes o bulking agen s 54 exis , including oli e lea es, co on gin ash, ice hulls, sheep manu e and g ape s alks (Al ano e 55 al., 2011, 2009; Bo e o e al., 2009; Cayuela e al., 2008; Mo eno e al., 2016). A e compos ing, 56 non-phy o oxic o ganic ma e - ich ma e ials ee o pa hogens a e ob ained (Mo illo e al., 2009; 57 P incipi e al., 2001), and hese ma e ials a e sui able o use as o ganic soil amendmen s and 58 plan g owing media (Caballe o e al., 2009). I is pa icula ly signi ican ha compos ing can be 59 an e icien way o educing V. dahliae p opagules (Noble and Robe s, 2004). 60 Some compos s ha e been shown o ha e supp essi e e ec s on di e en phy opa hogens, 61 including V. dahliae, when added o plan g ow h media o soils (Bonanomi e al., 2010; Li e ick 62 e al., 2004; Noble and Co en y, 2005). In pa icula , he supp essi e e ec s o some oli e mill 63 compos s on Ve icillium wil disease ha e been obse ed in oma o (Al ano e al., 2011; 64 A iagada e al., 2012; Vi ullo e al., 2013), eggplan (Maland aki e al., 2008; Ma kakis e al., 65 2008; Papaso i iou e al., 2013), and co on (Cas año and A ilés, 2013). Also, oli e mill ex ac s 66 can inhibi V. dahliae g ow h in aga cul u e media (Al ano e al., 2011; A iagada e al., 2012; 67 Lima e al., 2008) and educe mic oscle o ia concen a ion in soils o plan g ow h media 68 (Cas año and A ilés, 2013; Lima e al., 2008; Vi ullo e al., 2013). 69 His o ically, compos supp essi eness has been di ided in o wo majo ca ego ies: gene al and 70 speci ic. Gene al supp ession is ela ed o he sum o he ac i i ies o he o e all mic obial 71 biomass, and speci ic supp ession is a consequence o he ac i i ies o a educed numbe o 72 A ilés, Plan Disease 4 mic oo ganism popula ions (Al ano e al., 2011; A ilés e al., 2011; Hoi ink and Boehm, 1999; 73 S one e al., 2004). The abio ic en i onmen (ae a ion, pH, mine al composi ion, e c) is also 74 ele an in explaining supp essi eness (A ilés e al., 2011; Te mo shuizen and Jege , 2008). I 75 supp essi eness is elimina ed by pas eu iza ion, biocides o ha she ea men s like au ocla ing, 76 hen he supp essi eness is due o biological ac o s (Welle e al., 2002). In con as o speci ic 77 supp ession, gene al supp ession is usually eco e ed a e s e iliza ion due o he apid 78 coloniza ion o mic oo ganisms (Te mo shuizen and Jege , 2008). Ano he cha ac e is ic o 79 gene al supp ession is ha he numbe o pa hogen p opagules does no decline apidly in 80 supp essi e media (Hoi ink e al., 1993). 81 Ve icillium dahliae can be anspo ed long dis ances in in ec ed plan ing s ock and/o in es ed 82 po ing soil, and hus can be in oduced in pa hogen- ee oli e-g owing a eas. The sp ead o he 83 pa hogen can be u he enhanced i oli e nu se ies a e es ablished in V. dahliae-in es ed a eas 84 and he asymp oma ic hos plan s dis ibu ed ac oss he oli e p oduc ion egion (Jiménez-Díaz e 85 al., 2012). Compos applica ion in he ield can be expensi e due o he la ge quan i ies equi ed; 86 howe e , i s applica ion in nu se ies is easie and mo e p ac ical han in la ge-scale ield 87 p oduc ion (Maland aki e al., 2008). Thus, oli e mill compos ob ained a e a s ong he mophilic 88 phase ollowed by ex ensi e ecoloniza ion o mesophilic mic oo ganisms would be ee o 89 pa hogens and could con e Ve icillium wil supp essi eness o he plan g ow h media used in 90 oli e nu se ies, e en a e ansplan ing in open ields (Hoi ink e al., 1999; Noble and Robe s, 91 2004). Addi ionally, he inco po a ion o oli e mill compos in plan g ow h media would gi e alue 92 o his esidue. 93 The i s objec i e o his wo k was o s udy he po en ial supp essi e e ec s o di e en oli e mill 94 compos s on Ve icillium wil . Fo his pu pose, comme cial oli e mill compos s ob ained om 95 di e en sou ces and ha ing di e en ma u a ion le els we e s udied. The second objec i e was 96 o elucida e he p ima y mechanisms explaining hese supp essi e e ec s. 97 A ilés, Plan Disease 5 98 MATERIALS AND METHODS 99 Oli e mill was e compos s and plan g ow h media. Fou oli e mill compos s we e ob ained 100 om ou companies in di e en oli e p oduc ion a eas in Andalusia. These compos s 101 encompassed he usual a iabili y ound in hese ypes o p oduc s (i.e. bulk densi y, pH, o ganic 102 ma e , e c.), and we e named OC1, OC2, OC3 and OC4 (Supplemen a y Table S1). The 103 compos s ep esen ed wo ma u a ion le els, wi h cu ing phases o ei he ou mon hs o one 104 yea , and each con ained sou ce ma e ials speci ic o each company (Supplemen a y Table S1). 105 Fo each ype o compos and ma u a ion le el, wo subsamples we e collec ed and assessed o 106 a numbe o p ope ies including: bulk densi y (BD) acco ding o de Bood e al. (1974), o ganic 107 ma e ollowing he Eu opean S anda d (EN 13039, 2011), o al C and N by he Dumas me hod 108 desc ibed in UNE-EN 13039 (AENOR, 2002), elec ical conduc i i y (EC) and pH. The la e wo 109 p ope ies we e de e mined in 1:5 / compos :wa e ex ac s. 110 To educe salini y, he compos s we e lushed wi h h ee olumes o wa e pe olume o compos 111 (Sulli an and Mille , 2001) and hen mixed a a a io o one pa compos o wo pa s pea 112 subs a e. The comme cial pea subs a e used (Kekkilä Ibe ia S.L., Valencia, Spain) was 113 composed o a mix u e o ligh and b own pea (50:50 / ) sligh ly amended wi h a NPK e ilize 114 and pH co ec ed. These plan g ow h media o mula ed wi h he di e en compos s we e 115 designa ed OC1-GM, OC2-GM, OC3-GM and OC4-GM and compa ed wi h pea subs a e as he 116 s anda d plan g ow h medium (con ol). The pea used as he con ol was en iched wi h 0.17 g / 117 li e o NH4H2PO4 (12% N and 61% P2O5, Fe ibe ia, Mad id, Spain), 0.52 g / li e o NH4NO3 118 (33.5% N, Fe ibe ia, Mad id, Spain), and 6.15 g / li e o K2SO4 (50% K2O, Compo Ag icul u a 119 S.L., Ba celona, Spain) in o de o achie e nu ien a ailabili y compa able o he o he compos 120 A ilés, Plan Disease 6 amended plan g ow h media. To s anda dize he ini ial condi ions, all plan g ow h media we e 121 incuba ed in 7-li e bags a 25ºC o 14 days (Inba e al., 1991) wi h 40% wa e by olume. 122 Oli e mill compos bioassays. Fou bioassays we e pe o med o s udy he po en ial 123 supp essi e e ec s o he di e en compos amended plan g ow h media on Ve icillium wil . Two 124 bioassays we e pe o med wi h sho -ma u ed compos s (compos s ma u ed o ou mon hs) and 125 wo wi h long-ma u ed compos s (compos s ma u ed o one yea ). Each bioassay was a anged 126 in a andomized block design wi h 4 compos ypes x 3 blocks x 5 inocula ed po s ( eplica ions) 127 o a o al o 60 po s. In addi ion, in each bioassay h ee po s o each plan g ow h medium we e 128 no inocula ed and se ed as he nega i e check in o de o e alua e compos e ec s on plan 129 g ow h in he absence o disease, esul ing in an addi ional 36 con ol po s o a o al o 96 po s 130 pe bioassay. 131 The supp essi e capaci y o oli e mill compos agains Ve icillium wil was e alua ed in co on 132 plan s c . Ca lo a (Eu osemillas, S.A., Có doba, Spain). Co on bioassays a e as e han hose 133 o oli e, and acili a e s anda dized plan de elopmen . The simila i y o bo h co on and oli e 134 pa hosys ems makes his hos subs i u ion possible (López-Escude o and Me cado-Blanco, 135 2011). 136 Each eplica ion consis ed o one 0.8 li e po wi h one co on plan de i ed om h ee seeds ha 137 we e p e-ge mina ed, sown in indi idual po s and hinned a e one week. The plan s we e g own 138 in a g ow h chambe se a 25ºC (day) and 23ºC (nigh ), wi h 14 h ligh , 10 h da k, and d ip-139 i iga ed. The plan s we e e ilized wi h 1 g / li e o Pe e s olia eed 27-15-12 (N-P-K, Sco s, 140 Hee len, The Ne he lands) on a weekly basis. 141 An isola e o Ve icillium dahliae de olian pa ho ype (isola e V25) ob ained om a diseased oli e 142 ee was used as he pa hogen. The isola e was g own on PDA wi h s ep omycin sulpha e (0.05 143 g / li e ). The pla es we e sc aped wi h wa e , il e ed h ough cheeseclo h, and he conidia we e 144 A ilés, Plan Disease 7 coun ed wi h a hemocy ome e . Twel e days a e sowing, each po was placed in a plas ic bag 145 which, in u n, was inse ed in o an emp y po . The inoculum was adjus ed o 5x106 conidia pe ml 146 o solu ion based on p e ious p elimina y assays. A 400 ml aliquo o inoculum solu ion was 147 added o each po as a d ench, and allowed o in use h ough he g ow h media o one hou . The 148 po s we e hen d ained by emo ing he plas ic bag om a ound each po and incuba ed a 90% 149 ela i e humidi y o 3 days in he da k. The con ols we e ea ed in he same way using wa e . 150 Disease se e i y was sco ed wice a week based on a symp om se e i y scale whe e: 0 = 151 asymp oma ic plan s; 1 = sligh ly symp oma ic plan s (1 o 33% o lea es a ec ed); 2 = 152 mode a ely symp oma ic plan s (34 o 66% o lea es a ec ed); 3 =se e ely symp oma ic plan s 153 (67 o 99% o lea es a ec ed bu plan s no dead); and 4 = dead plan s (Beja ano-Alcáza e al., 154 1995). Each sco e was con e ed o he midpoin o he co esponding disease se e i y ange 155 p io o using pa ame ic analyses. The s anda dized a ea-unde - he-disease-p og ess-cu e 156 (AUDPCs) pe po was calcula ed om he disease se e i y alues by he apezoidal in eg a ion 157 me hod be ween he onse o symp oms and he ending ime o he bioassay di ided by he o al 158 du a ion (days) o he epidemic in each bioassay, in o de o compa e he a ious bioassays, in 159 which he du a ion o he epidemic a ied (Campbell and Madden, 1990). The disease se e i y a 160 he end o bioassays and he AUDPCs da a we e used o u he analyses. 161 The bioassays ended when he majo i y o he inocula ed plan s g own in he unamended pea 162 media we e dead (abou wo mon hs). A he end o he bioassays, esh shoo weigh was 163 eco ded, as well as d y weigh a e d ying in a o ced-ai o en un il a cons an weigh was 164 ob ained. 165 In o de o quan i y he concen a ion o V. dahliae mic oscle o ia in he hizosphe e a he end o 166 he bioassays, hizosphe ic and non- hizosphe ic g ow h media we e sepa a ed ollowing Dhing a 167 and Sinclai (1995). Rhizosphe e samples om i e po s o each compos amendmen , block and 168 A ilés, Plan Disease 8 bioassay we e collec ed, combined, c ushed, mixed and ai -d ied o 4 weeks. Fo we sie ing 169 analysis (Ha is e al., 1993), 25 g o each plan g ow h medium was suspended in 250 ml o 170 dis illed wa e and shaken o 1 h a 270 pm in an o bi al shake . The suspension was hen 171 washed h ough nes ed 150 and 36 μm sie es wi h ap wa e , and he ma e ial on he 36 μm 172 sie e was eco e ed and made up o 100 ml wi h dis illed wa e . Aliquo s o 1-ml suspension 173 we e pla ed on modi ied soil ex ac aga medium (Ha is e al., 1993) wi h 10 pla es pe sample. 174 The pla es we e incuba ed a 22 °C in he da k o 2 weeks and he plan g ow h medium 175 esidues we e washed away wi h dis illed wa e . The pla es we e hen d ied and incuba ed o an 176 addi ional 2 o 3 weeks, a e which he V. dahliae colonies we e coun ed. 177 Physical and chemical p ope ies o he plan g ow h media. Bulk densi y, pH and EC o he 178 g ow h media we e measu ed on sub-samples collec ed om each o he o mula ed and 179 incuba ed plan g ow h media used in each o he bioassays. Elec ical conduc i i y and pH we e 180 measu ed in 1:5 / wa e ex ac s, acco ding o Hand eck and Black (2002), in wo samples o 181 each plan g ow h medium, and block, bioassay and mean alues pe block we e used o 182 s a is ical analysis. Bulk densi y was also de e mined o each o hese samples, ollowing De 183 Bood e al. (1974). 184 To assess phy o oxici y, a seedling g ow h es was pe o med di ec ly in he plan g ow h media. 185 P io o conduc ing he bioassays, h ee bags ( eplica ions) pe plan g ow h medium we e 186 p epa ed and incuba ed o 11 days, unde he condi ions p e iously desc ibed, and h ee 187 subsamples collec ed om each bag we e es ed o phy o oxici y ollowing he modi ied me hod 188 o O ega e al. (1996). Fo his es , 20 ml olume sample o he plan g ow h media was placed 189 in 9 cm diame e Pe i dishes, we ed wi h dis illed wa e , and en le uce (Lac uca sa i a) seeds 190 we e sown in each dish (3 Pe i dishes pe plan g ow h medium and bag). A e sowing, he 191 dishes we e placed in an incuba o a 20ºC in he da k o one week. Subsequen ly, he 192 ge mina ed seeds we e coun ed (G) and adicle g ow h (L) measu ed. The ge mina ion index (GI) 193 A ilés, Plan Disease 9 was calcula ed acco ding o he o mula GI = G/Go x L/Lo x 100, whe e Go and Lo a e he 194 ge mina ion pe cen age and adicle g ow h o he con ol, espec i ely (Zucconi e al., 1985). The 195 ge mina ion index can assess bo h low oxici y, which mainly a ec s oo g ow h, and high 196 oxici y, which a ec s seed ge mina ion (Selim e al., 2012). 197 A ungi oxici y assay was pe o med o de ec inhibi ion o V. dahliae mycelial g ow h in each 198 plan g ow h medium amended wi h ei he sho - and long-ma u ed compos . Sub-samples 199 collec ed om he same bags used o phy o oxici y measu emen we e d ied a oom 200 empe a u e, milled and sie ed a 250 μm. As desc ibed by Kokalis-Bu elle and Rod íguez-201 Kábana (1994), he cul u e media we e p epa ed wi h each milled plan g ow h medium by 202 suspending 100 g o his ma e ial and 15 g o aga in one li e o deionized wa e . Con ol pla es 203 wi hou he plan g ow h medium (wa e aga ) we e also p epa ed. The media we e au ocla ed a 204 105ºC o 30 min. The pla es we e hen inocula ed wi h a 5-mm-diame e plug o V. dahliae aken 205 om he leading edge o a 6-day-old cul u e. Twen y eplica e pla es pe ea men and eplica ion 206 (n=60) we e incuba ed a 25ºC in he da k. A e 20 days, adial ungal g ow h was e alua ed and 207 he pe cen age o g ow h inhibi ion (PGI) calcula ed as ollows: 208 PGI = (G ow h in con ol – G ow h in ea men ) x 100 / G ow h in con ol 209 210 Biological cha ac e is ics o he plan g ow h media. Biological analyses we e pe o med on 211 sub-samples collec ed om incuba ed g ow h media a he beginning o each bioassay p io o 212 being di ided in o he po s ( h ee bags ( eplica ions) pe plan g ow h medium). The enzyma ic 213 ac i i ies in he plan g ow h media we e de e mined using he API-ZYM sys em (BioMe eux SA, 214 Ma cy-l’E oile, F ance). Wi h he API-ZYM sys em, semi-quan i a i e e alua ions we e pe o med 215 on he ac i i ies o 19 hyd oly icenzymes [alkaline phospha ase, es e ase (C4), es e ase–lipase 216 (C8), lipase (C14), leucine a ylamidase, aline a ylamidase, cis ine a ylamidase, ypsin, α-217 A ilés, Plan Disease 16 O he cul u al mic oo ganisms, he popula ion o Bacillus spp. and he a io o celluloly ic 364 ac inomyce es / celluloly ic bac e ia we e he highes in g ow h medium hea ea ed a 60ºC. 365 Hea ing a 60ºC seemed o a o copio ophic bac e ia, bu he concen a ion o hese popula ions 366 did no di e signi ican ly om he un ea ed OC3s-GM. Ne e heless, copio ophic bac e ia 367 de eloped highe popula ions in OC3s-GM-H han in OC3s-GM-A (Table 8). 368 Fo OC3s-GM ea men s, he bes MLR model was ound when oligo ophic ac inomyce es and 369 Bacillus spp. popula ions we e aken in o accoun as p edic i e a iables. This model explained 370 86% o he a ia ion in Ve icillium wil disease se e i y ( 2 =0.86, P < 0.001; Supplemen a y Fig. 371 2A). These a iables we e also included in he bes model o es ima ing mic oscle o ia 372 concen a ion in he hizosphe e a he end o bioassays. This model explained 74% o he 373 a ia ion in a Napie ian loga i hm o he mic oscle o ia concen a ion ( 2 = 0.74, P < 0.01; 374 Supplemen a y Fig. 2B) (bo h dependen a iables a e co ela ed). Final Ve icillium wil se e i y 375 explained 84.5% o he a ia ion in a Napie ian loga i hm o he mic oscle o ia concen a ion 376 (Se e i y = 18.17 + 15.04 x ln mic oscle o ia concen a ion; 2 = 0.85, P < 0.001) meaning ha as 377 disease se e i y inc eased so did he concen a ion o mic oscle o ia enume a ed om he 378 hizosphe e. 379 DISCUSSION 380 The e is e idence ha ce ain oli e mill compos s a e supp essi e o Ve icillium wil , educe 381 mic oscle o ia concen a ion o inhibi mycelial g ow h (Al ano e al., 2011; A iagada e al., 2012; 382 Cas año and A ilés, 2013; Lima e al., 2008; Maland aki e al., 2008; Ma kakis e al., 2008; Vi ullo 383 e al., 2013; Yildiz and Benlioglu, 2010). Howe e , ou esul s demons a e ha no all oli e mill 384 compos s a e supp essi e o Ve icillium wil when used as an amendmen in plan g ow h media; 385 hey can e en exace ba e disease compa ed o pea alone. In hese bioassay ials, only he pea 386 o mula ed wi h OC3 compos , ega dless o ma u a ion le el, was supp essi e o Ve icillium wil 387 A ilés, Plan Disease 17 when compa ed o pea , an e ec which was ela ed o a lowe concen a ion o mic oscle o ia a 388 he end o bioassays. On he o he hand, compos ma u a ion did no a ec disease 389 supp essi eness, despi e he ac ha sho ma u ed compos s we e shown o ha e a lowe 390 mic oscle o ia concen a ion a he end o bioassays han long ma u ed compos s. The 391 ela ionship be ween he compos cu ing du a ion and disease supp ession was no always clea . 392 The e is e idence o dec eased supp essi eness in compos wi h inc eased age o 393 Phy oph ho a spp. (Danon e al., 2007), Py hium spp. (Da by e al., 2006; S one e al., 2001), and 394 Scle o ium ol sii (Danon e al., 2007). Howe e , e idence also exis s o an inc eased 395 supp essi e e ec in mo e ma u ed compos , in he case o Fusa ium wil on melon (Saadi e al., 396 2010), and Rhizoc onia solani on cucumbe (T illas e al., 2006; Tui e e al., 1998). Thus, he 397 e ec s o ma u a ion on compos supp essi eness seem o depend on he na u e o he 398 compos ed ma e ials and he pa hosys em (A ilés e al., 2011; Hoi ink and Boehm, 1999). 399 Fo he ou oli e mill compos s s udied in hese bioassays, he explica i e ac o s o Ve icillium 400 wil disease se e i y and he inal hizosphe e mic oscle o ia concen a ion, we e de e mined o 401 be API-ZYM Shannon’s di e si y index, β-glucosidase ac i i y and pH (nega i ely ela ed) and EC 402 (posi i ely ela ed) based on he bes MRL models. In he p esen expe imen al design, disease 403 se e i y and mic oscle o ia concen a ion we e conside ed dependen a iables, because V. 404 dahliae inocula ion was ca ied ou in plan g ow h media ee om his pa hogen. Papaso i iou e 405 al. (2013) also p oposed ha he supp essi e e ec o oli e mill compos on V. dahliae on 406 eggplan was due o abio ic and bio ic ac o s. In o de o explain he possible biological meaning 407 o hese p edic i e ac o s and he complex mechanism in ol ed, i is impo an o discuss he 408 biological e ec o each ac o . Cas año and A ilés (2013), also wo king wi h di e en compos s 409 o mula ed as plan g ow h media, ound nega i e co ela ions be ween oli e and co on 410 Ve icillium wil disease se e i y, β-glucosidase ac i i y and pH. Fo g een manu e amendmen s, 411 Ochiai e al. (2008) included mic obial ac i i y, pH and inoculum densi y in hei bes MLR model 412 A ilés, Plan Disease 18 o Ve icillium wil disease in po a o. Ne e heless, in his eg ession model, pH had a posi i e 413 ela ion o disease se e i y. The di e ence in he di ec ion o hese ela ionships wi h he pH 414 could be due o he ange o pH s udied. In he p esen wo k, he pH o plan g ow h medium 415 amended wi h oli e mill compos s anged om 7.64 o 8.09, while in Ochiai e al. (2008) he pH 416 ange was om 5.2 o 7.5. The e o e, in acid o neu al media, pH inc ease seems o a o 417 Ve icillium wil while in alkaline media, such as plan g ow h media wi h oli e mill compos s, 418 dec easing pH seemed o a o his disease and is consis en wi h o he esea ch whe e 419 Ve icillium wil o oma o and co on inc eased in acidic soils limed o inc ease pH (Jones e al., 420 1971; Shao and Foy, 1982). Fu he mo e, ce ain ac inomyce es, which a e V. dahliae 421 an agonis s, a e mo e an agonis ic in alkaline pHs (Abd-Allah, 2001; Bonja and Aghighi, 2005). 422 As o EC, some au ho s ha e obse ed ha i iga ing wi h saline wa e boos ed V. dahliae 423 in ec ions in po a o, pis achio and co on (Bes i, 1981; Kau man e al., 1990; Mohammadi e al., 424 2007). O he wo ks ha e demons a ed ha salini y inc eases oo and shoo coloniza ion by V. 425 dahliae and, and consequen ly, he disease se e i y (Le in e al., 2007; Saada mand e al., 2008; 426 Pascual e al., 2009). These au ho s also sugges ed salini y as a possible cause o he 427 enhancemen o Ve icillium wil in peppe u he con i ming he impo an ole o EC in 428 inc easing Ve icillium wil de elopmen simila o he MLR models p oposed he e. 429 Compos amendmen s ha e been shown o a ec mic obial communi y s uc u e and composi ion 430 and can enhance mic obial di e si y (Bonilla e al., 2012; D’Hose e al., 2014; Saison e al., 431 2006). Disease supp essi eness is o en ela ed o o e all inc eases mic obial biomass and 432 ac i i y in plan g ow h media o soils, which c ea e a compe i i e en i onmen which is 433 dele e ious o he pa hogen (Bonanomi e al., 2010; Bonilla e al., 2012; Hada and 434 Papadopoulou, 2012; La kin, 2015). La kin e al. (2011) wo king wi h g een manu es, ound high 435 di e si y (Shannon´s index) and he highes mic obial ac i i y in he ea men wi h he lowes 436 se e i y in Ve icillium wil on po a o. In he same sense, o he au ho s wo king wi h o ganic soil 437 A ilés, Plan Disease 19 amendmen s ha e ound nega i e co ela ions be ween mic obial ac i i y and he disease 438 se e i y o Ve icillium wil on po a o (Conn and Laza o i s, 1999; Da is e al., 1994, 1996). 439 Con e sely, wi h compos s o mula ed as plan g ow h media, Te mo shuizen e al. (2006) ound 440 a posi i e co ela ion be ween espi a ion and disease se e i y o Ve icillium wil on eggplan , bu 441 we e unable o clea ly explain his ela ionship. 442 Rega ding o he p ope ies e alua ed in his s udy, he low ungi oxici y obse ed in he 443 supp essi e medium OC3-GM compa ed o he o he media did no seem o be an impo an 444 ac o o supp essi eness, gi en ha he medium wi h he highes ungi oxici y, OC2-GM, also 445 showed he highes AUDPCs alue. On he o he hand, he high le el o lysogenic enzyma ic 446 ac i i y ound in OC3-GM could be associa ed wi h ungal cell wall deg ada ion. Cell wall-447 deg ading enzymes a e in ol ed in he an agonis ic ac i i y o biocon ol agen s agains 448 phy opa hogenic ungi (Elad, 1985; Gaje a, 2012; Madi e al., 1997). 449 To elucida e he main mechanisms explaining he supp essi e e ec s o OC3s-GM, i was al e ed 450 wi h ea men s designed o dis up he mic obial communi y and assess he ole o ni ogen on 451 disease supp ession. The p edic i e ac o s o supp essi eness ob ained om he ini ial 452 bioassays conduc ed using he g ow h media amended wi h ou oli e compos s do no 453 necessa y co espond o he explica i e supp essi e p ope ies obse ed om he bioassays 454 conduc ed wi h OC3s-GM. The mic obial communi ies o he di e en OC3s-GM ea men s we e 455 igge ed by a esponse o dis u bances c ea ed h ough he hea ea men o ni ogen 456 applica ion. The same o ganic ma e composi ion can show di e en unc ional abili ies 457 depending on he mic obial communi y i ha bou s ( an B uggen and Semeno , 2000). The 458 a ying Ve icillium wil se e i y be ween he hea ea men s could be due o hei di e en e ec s 459 on he mic obial communi y, wi hou disca ding possible mino changes in he o ganic ma ix. 460 A ilés, Plan Disease 20 Ni ogen supplemen a ion did no a ec supp essi eness, a he , i con ibu ed sligh ly o 461 inc eased mic oscle o ia concen a ion. An inc emen in ni ogen a ailabili y can help 462 mic oscle o ia o o e come ungis asis (G een and Papa izas, 1968). The e o e, his loss o 463 ungis asis could allow mic oscle o ia o h i e in he p esence o he hos plan , which would 464 explain he inc ease in concen a ion obse ed. In any case, a possible low a ailabili y o ni ogen 465 in un ea ed OC3s-GM as a cause o supp essi eness is uled ou , gi en ha N-supplemen ed 466 OC3s-GM-N main ains he same le el o supp essi eness. 467 Fo he di e en ea men s o supp essi e OC3s-GM, bo h hea ea men s (60ºC hea ea ed 468 and au ocla ed) caused an impo an d op in supp essi eness, bu hea ing a 60ºC mos 469 signi ican ly inc eased disease se e i y. Ne e heless, he hea ea men s we e s ill supp essi e 470 in compa ison o unamended pea , despi e showing a simila mic oscle o ia concen a ion a he 471 end o bioassays. In con as , he un ea ed OC3s-GM medium showed e y low mic oscle o ia 472 concen a ion. The e o e, he supp essi eness and he absence o a educ ion in mic oscle o ia in 473 hea ea men s compa ed o pea could indica e ha un ea ed OC3s-GM supp essi eness is 474 due o he p esence o bo h gene al and speci ic supp essi eness. Al ano e al. (2011) also 475 sugges ed gene al and speci ic supp essi eness in his kind o compos agains Py hium ul imum 476 and Fusa ium oxyspo um .sp. lycope sici in oma o. The di e en ial supp essi e e ec o 60º C 477 hea ea men compa ed o un ea ed OC3s-GM seems o indica e ha mesophilic he mo-478 ole an axa a e no in ol ed in speci ic supp ession in his compos . The e o e, he ela i ely 479 highe Bacillus spp. concen a ion obse ed in OC3s-GM-H compa ed o he o he ea men s 480 p o es ha hese mic oo ganisms, which a e usually desc ibed as biocon ol agen s, do no play 481 a majo ole in he speci ic supp essi eness o his plan g ow h medium. On he o he hand 482 inc eased celluloly ic ac inomyce es compa ed o celluloly ic bac e ia in OC3s-GM-H seem o be 483 ela ed o dec eased supp essi eness, which con adic s obse a ions by Tui e e al. (1998) o 484 Rhizoc onia solani in household was e compos s and Bo e o e al. (2004) o oma o Fusa ium 485 A ilés, Plan Disease 21 wil in o he ag o-indus ial was e compos s. The highe mean le el o API-ZYM ac i i y and 486 di e si y, alkaline phospha ase, es e ase lipase and ypsin ac i i ies in OC3s-GM-H han in 487 OC3s-GM-A mus be due o hese he mo- ole an axa. 488 A e bo h hea ea men s, N-ace yl-β-glucosaminidase ac i i y was no de ec ed and acid 489 phospha ase ac i i y dec eased. The e o e, mic oo ganisms con ibu ing o hese ac i i ies 490 (especially N-ace yl-β-glucosaminidase) could be in ol ed in he speci ic supp essi eness 491 obse ed in his plan g ow h medium. N-ace yl-β-glucosaminidase ac i i y is used as an index o 492 chi inoly ic ac i i y in en i onmen al samples (O’B ien and Colwell, 1987). Chi inoly ic enzymes 493 ha e been conside ed impo an in he biocon ol o soilbo ne pa hogens because o hei abili y 494 o deg ade ungal cell walls (Ha an e al., 1996; Ha man e al., 1993; Inba and Che , 1995; 495 Nguyen e al., 2008). Se e al chi inoly ic enzymes ha e been iden i ied in di e en kinds o 496 mic oo ganisms, including a ious species o S ep omyces (Kinkel e al., 2012; Xue e al., 2013). 497 In he same way, acid phospha ase has been sugges ed o play a ole in cell wall hyd olysis o 498 phy opa hogens (Mon ei o e al., 2010) and mic obial phospha ase ac i i y in he hizosphe e has 499 been demons a ed o enhance plan nu i ion and consequen ly plan de elopmen and heal h 500 (Recena e al., 2015). The bes LMR model ound o explain disease se e i y and mic oscle o ia 501 concen a ion in he di e en ea men s o he supp essi e compos OC3s-GM showed he 502 impo ance o popula ions o oligo ophic ac inomyce es and Bacillus spp. In his compos , 503 oligo ophic ac inomyce es seem o a ou disease supp ession, while Bacillus spp. popula ions 504 did no . Many ac inomyce e axa, such as S ep omyces, a e associa ed wi h plan disease 505 supp ession in many soils (Kinkel e al., 2012; Xue e al., 2013) and a e soil sap ophy es wi h a 506 c ucial ole in nu ien cycling (Kennedy, 1999). On he o he hand, oligo ophic popula ions a e 507 ela ed o soils wi h high mic obial di e si y and supp essi eness ( an B uggen and Semeno , 508 2000). In he same way, in o he plan g ow h media o mula ed wi h compos s, highe 509 oligo ophic ac inomyce es popula ions we e ound ha we e supp essi e o Rhizoc onia solani 510 A ilés, Plan Disease 22 on cucumbe (Tui e e al., 1998) and Fusa ium wil on oma o (Bo e o e al., 2004). In plan 511 g ow h media amended wi h oli e mill compos which was supp essi e o Fusa ium wil on oma o 512 and Py hium ul imum, high ac inomyce e and mic obial qui inoly ic ae obic popula ions we e 513 ound (Al ano e al., 2011). 514 These esul s sugges ha oli e mill compos s demons a ed o be supp essi e o Ve icillium wil , 515 such as OC3, and used o amend nu se y plan g ow h media, can p o ec plan le s om his 516 disease a leas du ing he ansplan phase. The lack o disease symp oms ha de eloped in 517 nonV. dahliae inocula ed con ol ea men s con aining oli e mill compos s indica ed ha hese 518 compos s a e also gene ally ee o his pa hogen. We can conclude ha oli e mill compos s 519 amended plan g ow h media a e no always supp essi e agains Ve icillium wil . Howe e , o 520 hese ou compos sou ces, ma u a ion le el did no a ec disease de elopmen . API-ZYM 521 Shannon’s di e si y index, β-glucosidase ac i i y and pH and EC in oli e mill compos plan 522 g ow h media we e he ac o s ha bes desc ibed Ve icillium wil disease se e i y and 523 hizosphe e mic oscle o ia concen a ion. In he oli e mill compos ha was cha ac e ized as 524 supp essi e (OC3s-GM) gene al and speci ic supp essi eness we e bo h in ol ed. In his 525 compos , N-ace yl-β-glucosaminidase ac i i iy and he oligo ophic ac inomyce e popula ions 526 seemed o be in ol ed in supp essi eness. 527 Acknowledgemen s. This esea ch was suppo ed by g an s om Spain’s Minis e io de Ciencia 528 e Inno ación (AGL2010-21982-C02-01). We would like o hank A. Ga a, S. Cas illo, S. Pé ez and 529 R., Cas año o hei excellen echnical assis ance; D. Delgado and F. Fo cella o kindly e ising 530 he manusc ip and J.M. Ál a ez o inding and supplying he compos s. 531 532 LITERATURE CITED 533 Abd-Allah, E. F. 2001. S ep omyces plica us as a model biocon ol agen . 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Mic obial 773 popula ions esponsible o speci ic soil supp essi eness o plan pa hogens. Annu. Re . 774 Phy opa hol. 40: 309–348. 775 Xue, L., Xue, Q., Chen, Q., Lin, C., Shen, G., Zhao, J. 2013. Isola ion and e alua ion o 776 hizosphe e ac inomyce es wi h po en ial applica ion o biocon ol o Ve icillium wil o 777 co on. C op P o . 43: 231-240. 778 Yildiz, A., Benlioglu, S., 2010. E ec s o soil sola iza ion and some amendmen s o con ol 779 Ve icillium wil in es ablished oli e o cha ds. A ican J. Bio echnol. 9: 6660–6665. 780 doi:10.5897/AJB10.507 781 Zak, J. C., Willig, M. R., Moo head, D. L. and Wildman, H. G., 1994. Func ional Di e si y o 782 Mic obial Communi ies: A Quan i a i e App oach. Soil Biol. Biochem. 26: 1101–1108. 783 Zucconi, F., Monaco, A., Fo e, M. and de Be oldi, M. 1985. Phy o oxins du ing he s abiliza ion 784 A ilés, Plan Disease 33 o o ganic ma e . Pages 73–88 in: Compos ing o Ag icul u al and O he Was es. J. K.R. 785 Gasse ed. Else ie Applied Science, London and New Yo k. 786 787 A ilés, Plan Disease 34 Table 1. E ec o oli e mill compos sou ce and le el o ma u a ion on Ve icillium wil disease se e i y, s anda dized a ea unde disease p og ess 788 cu e (AUDPCs) and mic oscle o ia (MS) concen a ionw. 789 G ow h mediumx Se e i yy (%) AUDPCs ( ange 0 o 1 ) MS concen a ion (MS / g d y weigh ) OC1-GM 82 ± 4.5 ab 0.494 ± 0.040 b 18 ± 5 a OC2-GM 96 ± 2.4 a 0.729 ± 0.029 a 21 ± 4 a OC3-GM 43 ± 5.8 c 0.203 ± 0.036 c 3 ± 1 b OC4-GM 71 ± 5.2 b 0.463 ± 0.043 b 37 ± 11 a pea 84 ± 4.5 ab 0.568 ± 0.041 b 54 ± 11 a Ma u a ionz sho 72 ± 3.8 a 0.436 ± 0.030 a 24 ± 6 b long 74 ± 3.6 a 0.509 ± 0.032 a 30 ± 5 a w Da a ep esen mean ± s anda d e o . Fo each column and plan g ow h medium o le el o ma u a ion ac o , alues ollowed by di e en le e s a e 790 signi ican ly di e en acco ding o ANOVA and Tukey’s Tes (P<0.05). Da a o analysis we e ans o med wi h x2 o Se e i y, x0.67 o AUDPCs and x0.17 o 791 A ilés, Plan Disease 35 mic oscle o ia concen a ion in g ow h medium. Se e i y and AUDPCs we e n=60 o g ow h medium and n=120 o le el o ma u a ion. Mic oscle o ia 792 concen a ion was n=12 o g ow h medium and n=24 o le el o ma u a ion. 793 x OC1-GM: g ow h medium o mula ed wi h OC1 compos and pea 1:2 / ; OC2-GM: g ow h medium o mula ed wi h OC2 compos and pea 1:2 / ; OC3-GM: 794 g ow h medium o mula ed wi h OC3 compos and pea 1:2 / ; OC4-GM: g ow h medium o mula ed wi h OC4 compos and pea 1:2 / . 795 y Disease se e i y scale: 0 = asymp oma ic plan s; 1 = sligh ly symp oma ic plan s (1-33% o lea es a ec ed); 2 = mode a ely symp oma ic plan s (34-66% o 796 lea es a ec ed); 3 =se e ely symp oma ic plan s (67-99% o lea es a ec ed bu plan s no dead); and 4 = dead plan s. Each scale sco e was con e ed o he 797 midpoin o he co esponding disease se e i y ange. 798 z Sho : Compos wi h a cu ing phase o 4 mon hs; Long: Compos wi h a cu ing phase o 1 yea . 799 800 801 A ilés, Plan Disease 36 Table 2. The physical and chemical p ope ies o he oli e mill compos amended g ow h media p io o use in he bioassaysw. 802 G ow h mediumy P ope iesx BD (g ml-1) pH EC (mS cm-1) GI (%) PGI (%) OC1-GM 0.24±0.01 a 7.81±0.12 b 0.82±0.06 bc 84.9±8.1 ab 21.2±0.8 c OC2-GM 0.32±0.05 a 7.64±0.07 b 0.88±0.10 bc 103.4±8.6 a 31.5±0.9 a OC3-GM 0.32±0.05 a 8.09±0.07 a 0.73±0.10 c 83.8±7.9 ab 3.9±1.0 d OC4-GM 0.24±0.02 a 7.87±0.08 ab 0.98±0.13 b 61.4±6.0 b 25.7±1.8 b pea 0.13±0.01 b 5.48±0.05 c 2.55±0.11 a 102.3±10.0 a 1.9±0.9 d Ma u a ionz sho 0.20±0.01 b 7.27±0.19 b 1.05±0.17 a 76.6±4.4 b 19.6±1.2 a long 0.29±0.03 a 7.49±0.18 a 1.33±0.10 a 97.6±6.2 a 14.5±0.7 b w Da a ep esen mean ± s anda d e o . Fo each column and plan g ow h medium o le el o ma u a ion ac o , alues ollowed by di e en le e s a e 803 signi ican ly di e en acco ding o ANOVA and Tukey’s Tes (P<0.05). Da a o analysis we e ans o med wi h x-1.5 o BD, x-0.2 o GI. BD: n=4 o g ow h 804 A ilés, Plan Disease 37 medium and n=8 o le el o ma u a ion. pH and EC: n=12 o g ow h medium and n=24 o le el o ma u a ion. GI: n=18 o g ow h medium and n=36 o le el 805 o ma u a ion. PICR: n=120 o g ow h medium and n=240 o le el o ma u a ion. 806 x BD: Bulk densi y; EC: Elec ical conduc i i y; GI: Ge mina ion Index; PGI: Pe cen age o Ve icillium dahliae micelial g ow h inhibi ion. 807 y OC1-GM: g ow h medium o mula ed wi h OC1 compos and pea 1:2 / ; OC2-GM: g ow h medium o mula ed wi h OC2 compos and pea 1:2 / ; OC3-GM: 808 g ow h medium o mula ed wi h OC3 compos and pea 1:2 / ; OC4-GM: g ow h medium o mula ed wi h OC4 compos and pea 1:2 / . 809 z Sho : Compos wi h a cu ing phase o 4 mon hs; Long: Compos wi h a cu ing phase o 1 yea . 810 811 812 A ilés, Plan Disease 38 Table 3. Oxygen up ake a e (OUR), β-glucosidase ac i i y, API-ZYM mean ac i i y and Shannon’s di e si y index o he oli e mill compos amended 813 g ow h media p io o use in he bioassaysx. 814 G ow h mediumy OUR (mg O2 kg-1 day-1) β-glucosidase ac i i y (μg ρni o enol ml-1h-1) API-ZYM mean ac i i y API-ZYM Shannon’s di e si y index OC1-GM 1880.1±177.3 a 177.1±12.6 ab 1.01±0.08 ab 2.09±0.08 ab OC2-GM 1189.7±150.6 bc 100.1±6.0 c 0.69±0.06 c 1.86±0.07 bc OC3-GM 1308.3±103.3 b 151.3±5.4 b 1.11±0.06 a 2.22±0.06 a OC4-GM 1439.1±160.8 b 201.6±19.8 a 0.90±0.10 b 2.06±0.07 ab pea 1037.7±130.9 c 62.4±9.5 c 0.56±0.06 c 1.68±0.12 c Ma u a ionz sho 1741.7±73.9 a 147.1±14.4 a 0.77±0.05 a 1.87±0.06 a long 1000.3±84.2 b 129.9±8.5 b 0.94±0.06 a 2.09±0.05 a x Da a ep esen mean ± s anda d e o . Fo each column and plan g ow h medium o le el o ma u a ion ac o , alues ollowed by di e en le e s a e 815 signi ican ly di e en acco ding o ANOVA and Tukey’s Tes (P<0.05). n=12 o g ow h medium and n=24 o le el o ma u a ion. 816 A ilés, Plan Disease 39 y OC1-GM: g ow h medium o mula ed wi h OC1 compos and pea 1:2 / ; OC2-GM: g ow h medium o mula ed wi h OC2 compos and pea 1:2 / ; OC3-GM: 817 g ow h medium o mula ed wi h OC3 compos and pea 1:2 / ; OC4-GM: g ow h medium o mula ed wi h OC4 compos and pea 1:2 / . 818 z Sho : Compos wi h a cu ing phase o 4 mon hs; Long: Compos wi h a cu ing phase o 1 yea . 819 820 821 A ilés, Plan Disease 40 Table 4. The API-ZYM enzyma ic ac i i ies o oli e mill compos amended g ow h media p io o use in he bioassaysx. 822 G ow h mediumy Alkaline phospha ase Es e ase (C4) Es e ase lipase (C8) Lipase (C14) Leucine a ylamidase Valine a ylamidase OC1-GM 2.2±0.3 b 2.8±0.1 ab 2.1±0.1 ab 0.1±0.1 b 2.7±0.2 a 0.4±0.1 ab OC2-GM 2.6±0.3 ab 2.3±0.2 bc 1.4±0.1 c 0.2±0.1 b 1.9±0.2 b 0.3±0.1 b OC3-GM 3.3±0.3 a 3.2±0.2 a 2.6±0.1 a 0.8±0.2 a 2.8±0.1 a 0.8±0.1 a OC4-GM 2.3±0.2 b 2.1±0.1 c 1.8±0.1 bc 0.1±0.1 b 2.4±0.2 ab 0.3±0.1 b pea 0.7±0.1c 2.0±0.1 c 1.3±0.1 c 0.0±0.0 b 0.8±0.2 c 0.1±0.1 b Ma u a ionz sho 2.0±0.2 a 2.6±0.1 a 1.8±0.1 a 0.3±0.1 a 2.1±0.2 a 0.3±0.1 a long 2.5±0.2 a 2.3±0.1 a 1.9±0.1 a 0.2±0.1 a 2.1±0.1 a 0.5±0.1 a x Da a ep esen mean ± s anda d e o . Fo each column and plan g ow h medium o le el o ma u a ion ac o , alues ollowed wi h di e en le e s a e 823 signi ican ly di e en acco ding o ANOVA and Tukey’s Tes (P<0.05). n=12 o g ow h medium and n= 24 o le el o ma u a ion. 824 y OC1-GM: g ow h medium o mula ed wi h OC1 compos and pea 1:2 / ; OC2-GM: g ow h medium o mula ed wi h OC2 compos and pea 1:2 / ; OC3-GM: 825 g ow h medium o mula ed wi h OC3 compos and pea 1:2 / ; OC4-GM: g ow h medium o mula ed wi h OC4 compos and pea 1:2 / . 826 z Sho : Compos wi h a cu ing phase o 4 mon hs; Long: Compos wi h a cu ing phase o 1 yea . 827 A ilés, Plan Disease 41 828 Table 4 (Con .). The API-ZYM enzyma ic ac i i ies o oli e mill compos amended g ow h media p io o use in he bioassaysx. 829 G ow h mediumy Acid phospha ase Naph ol-AS-BI- phosphohyd olase β-Galac osidase α-Glucosidase β-Glucosidase N-ace yl-β-glucosaminidase OC1-GM 4.0±0.3 a 1.2±0.1 ab 0.3±0.1 ab 0.2±0.1 b 1.1±0.1 a 1.3±0.3 a OC2-GM 2.8±0.3 b 0.8±0.1 b 0.1±0.1 b 0.1±0.1 b 0.1±0.1 b 0.2±0.1 b OC3-GM 3.1±0.2 b 0.8±0.1 b 0.3±0.1 ab 0.6±0.1 a 0.9±0.1 a 0.9±0.2 ab OC4-GM 2.9±0.4 b 1.7±0.2 a 0.4±0.1 ab 0.0±0.0 b 1.1±0.5 a 1.3±0.3 a pea 3.5±0.3 ab 0.8±0.2 b 0.6±0.1 a 0.0±0.0 b 0.4±0.1 ab 0.2±0.1 b Ma u a ionz sho 2.9±0.2 a 1.0±0.1 a 0.1±0.1 b 0.1±0.1 a 0.4±0.1a 0.7±0.2 a long 3.6±0.2 a 1.1±0.1 a 0.6±0.1 a 0.2±0.1 a 1.0±0.2 a 0.8±0.2 a x Da a ep esen mean ± s anda d e o . Fo each column and plan g ow h medium o le el o ma u a ion ac o , alues ollowed wi h di e en le e s a e 830 signi ican ly di e en acco ding o ANOVA and Tukey’s Tes (P<0.05). Da a o analysis we e ans o med wi h x0.59 o N-ace yl-β-glucosaminidase. n=12 o 831 g ow h medium and n= 24 o le el o ma u a ion. 832 A ilés, Plan Disease 48 871 Table 8 (Con .). Mic obial ecological-nu ien g oups in ea men s o iden i ied supp essi e oli e mill compos (OC3s-GM) a he end o bioassays . 872 T ea men w Bacillus spp. Tala omyces spp. Pseudomonas spp. Fungi OA/OBx CA/CBy OB/CoBz (CFU/ml g ow h medium x 105) OC3s-GM 16.43 b 0.00 a 0.01 a 4.11 a 0.12 a 0.11 b 1.17 a OC3s-GM-N 6.00 b 0.00 a 0.01 a 1.30 a 0.10 a 0.11 b 2.36 a OC3s-GM-A 24.64 b 0.00 a 0.06 a 3.30 a 0.01 a 0.03 b 8.82 a OC3s-GM-H 72.75 a 0.00 a 0.07 a 2.76 a 0.10 a 0.83 a 0.70 a Pea 1.48 b 0.00 a 0.00 a 1.31 a 0.10 a 0.06 b 1.55 a Wi hin each column, 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 873 ans o med da a wi h log (x) o Fungi, n=3. 874 w OC3s-GM: g ow h medium o mula ed wi h sho ma u a ion OC3 compos and pea 1:2 / ; OC3s-GM-N: OC3s-GM e ilized wi h 0.52 g/L o ammonium 875 ni a e; OC3s-GM-A: OC3s-GM au ocla ed ( wo successi e imes wi hin a 24 h in e al o 1 h a 120 ºC, 1.2 a m.); OC3s-GM-H: OC3s-GM hea ed ( o 60 ºC o 876 6 days). 877 A ilés, Plan Disease 49 x OA/OB: Oligo ophic Ac inomyce es/ Oligo ophic Bac e ia. 878 y CA/CB: Celluloly ic Ac inomyce es / Celluloly ic Bac e ia. 879 z OB/CoB: Oligo ophic Bac e ia/ Copio ophic Bac e ia 880 A ilés, Plan Disease 50 881 Es ima ed mic oscle o ia concen a ion [ln (MS/g)] -1 0 1 2 3 4 Mic oscle o ia concen a ion obse ed [ln (MS/g)] -1 0 1 2 3 4 Line 1:1 B Es ima ed AUDPCs 0.0 0.2 0.4 0.6 0.8 1.0 AUDPCs obse ed 0.0 0.2 0.4 0.6 0.8 1.0 A Line 1:1 882 883 Supplemen a y Fig. S1. A) A ea unde he disease p og ess cu e s anda dized (AUDPCs) 884 es ima ion in g ow h media o mula ed wi h oli e mill compos s as a unc ion o : API-ZYM 885 Shannon’s di e si y index, β-glucosidase ac i i y, pH and elec ical conduc i i y (EC); AUDPCs = 886 13.738 - 1.432 x API-ZYM Shannon’s di e si y index - 0.005 x β-glucosidase ac i i y - 1.239 x pH 887 + 0.002 x EC; R2 = 0.71, P < 0.0001, and coe icien s o each explica i e a iable signi ican a P 888 < 0.01 in he leas signi ican case; and mean absolu e e o = 0.42. Obse ed se e i y da a we e 889 mean alues om each block and di e en oli e mill compos s in ou bioassays, n=44. 890 A ilés, Plan Disease 51 B) Es ima ion o he napie ian loga i hm o mic oscle o ia concen a ion in g ow h media 891 o mula ed wi h oli e mill compos s as a unc ion o : API-ZYM Shannon’s di e si y index, β-892 glucosidase ac i i y, pH and EC; ln (MS/g) = 14.543 - 1.063 x API-ZYM Shannon’s di e si y index 893 - 0.001 x β-glucosidase ac i i y - 1.626 x pH + 0.003 x EC; R2 = 0.68, P < 0.0001, and coe icien s 894 o each explica i e a iable signi ican a P < 0.05 in he leas signi ican case; and mean 895 absolu e e o = 0.64. Obse ed mic oscle o ia concen a ion da a we e mean alues om each 896 block and di e en oli e mill compos s in ou bioassays, n=44. 897 898 899 A ilés, Plan Disease 52 900 Es ima ed se e i y 020 40 60 80 Se e i y obse ed 0 20 40 60 80 A Line 1:1 02 4 0 2 4 Es ima ed mic oscle o ia concen a ion [ln (MS/g)] Mic oscle o ia concen a ion obse ed [ln (MS/g)] B Line 1:1 901 Supplemen a y Fig. S2. A) Ve icillium wil se e i y es ima ion in g ow h media wi h OC3s-GM 902 ea men s as a unc ion o : oligo ophic ac inomyce es, and Bacillus spp. popula ions; Se e i y = 903 16.157 - 4.684 x 10-6 x oligo ophic ac inomyce es + 1.235 x 10-5 x Bacillus spp.; R2 = 0.86, P < 904 0.001, and coe icien s o each explica i e a iable signi ican a P < 0.01 in he leas signi ican 905 case; and mean absolu e e o = 8.96. Obse ed se e i y da a we e mean alues om each 906 assay and di e en ea men s o supp essi e OC3s-GM, n=12. 907 B) Es ima ion o he napie ian loga i hm o mic oscle o ia concen a ion in g ow h media wi h 908 OC3s-GM ea men s as a unc ion o : oligo ophic ac inomyce es, and Bacillus spp. popula ions; 909 ln (MS/g) = -0.338 - 1.341 x 10-7 x oligo ophic ac inomyce es + 6.456 x 10-7 x Bacillus spp.; R2 910 A ilés, Plan Disease 53 = 0.74, P < 0.01, and coe icien s o each explica i e a iable signi ican a P < 0.05 in he leas 911 signi ican case; and mean absolu e e o = 0.73. Obse ed mic oscle o ia concen a ion da a 912 we e mean alues om each assay and di e en ea men s o supp essi e OC3s-GM, n=12 913 A ilés, Plan Disease 54 Supplemen a y Table S1. Cha ac e is ics o oli e mill was e compos s. 914 Compos Company Compos ed ma e ial (% w/w) Ma u a iona P ope iesb BD (g ml-1) OM (%) C/N EC (mS cm-1) pH OC1 O obaena S.A.T., Baena, Có doba, Spain we oli e husks (82%), oli e lea es was e (15%), ce eal s aw (3%) sho 0.29 66.3 15.3 5.1 9.1 long 0.39 68.8 20.5 1.9 9.0 OC2 La Reja S.L., Bobadilla, Málaga, Spain we oli e husks (50%), oli e lea es was e (1%), manu e and s aw (49%) sho 0.32 33.6 11.5 5.4 8.1 long 0.80 26.7 11.7 1.6 7.7 OC3 Vado Oli o S.A., Cazo la, Jaén, Spain we oli e husks (63%), oli e lea es was e (30%), sheep manu e (7%) sho 0.55 55.0 21.4 4.3 8.5 long 0.65 39.8 19.6 2.2 7.5 OC4 Ra ael Alonso S.L., Tabe nas, Alme ía, Spain we oli e husks (94%), oli e lea es was e (3%), chicken manu e (3%) sho 0.64 65.8 13.6 11.4 9.1 long 0.42 57.6 13.7 9.9 8.7 a Sho : Compos wi h a cu ing phase o 4 mon hs; Long: Compos wi h a cu ing phase o 1 yea . 915 b BD: Bulk densi y; OM: O ganic ma e ; C/N: Ca bon/Ni ogen a io; EC: Elec ical conduc i i y. 916 917 918