Compost Tea Induces Growth and Resistance against Rhizoctonia solani and Phytophthora capsici in Pepper

ag onomy A icle Compos Tea Induces G ow h and Resis ance agains Rhizoc onia solani and Phy oph ho a capsici in Peppe Ana Isabel González-He nández , M. Belén Suá ez-Fe nández , Rod igo Pé ez-Sánchez , Ma íaÁngeles Gómez-Sánchez and Ma ía Remedios Mo ales-Co s *   Ci a ion: González-He nández, A.I.; Suá ez-Fe nández, M.B.; Pé ez-Sánchez, R.; Gómez-Sánchez, M.Á.; Mo ales- Co s, M.R. Compos Tea Induces G ow h and Resis ance agains Rhizoc onia solani and Phy oph ho a capsici in Peppe . Ag onomy 2021,11, 781. h ps://doi.o g/10.3390/ ag onomy11040781 Academic Edi o s: Mi ko Cucina and Luca Regni Recei ed: 25 Ma ch 2021 Accep ed: 14 Ap il 2021 Published: 16 Ap il 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). Facul y o Ag icul u al and En i onmen al Sciences, Uni e si y o Salamanca, A da. Filibe o Villalobos, 119, 37007 Salamanca, Spain; [email p o ec ed] (A.I.G.-H.); [email p o ec ed] (M.B.S.-F.); od igope [email p o ec ed] (R.P.-S.); [email p o ec ed] (M.Á.G.-S.) *Co espondence: [email p o ec ed] Abs ac : Compos eas (CTs) a e o ganic solu ions ha cons i u e an in e es ing op ion o sus ainable ag icul u e. Those ha come om ga den was e ha e been applied in i o and in i o on peppe plan s o de e mine i s supp essi e e ec agains bo h Phy oph ho a capsici and Rhizoc onia solani. The s udied CT showed ele an con en in NO 3− , K 2 O, humic acids, and mic oo ganisms such as ae obic bac e ia, N- ixing bac e ia, and ac inobac e ia, which play a ole in plan g ow h and esis ance. This ich abundance o mic obio a in he CT induced a educ ion in he ela i e g ow h a e o bo h P. capsici and R. solani (31.7% and 38.0%, espec i ely) in in i o assays compa ed o con ol. In addi ion, CT-i iga ed plan s displayed inc eased g ow h pa ame e s and showed he i s open lowe one week be o e hose ea men s wi hou CTs, which sugges s ha i s applica ion ad anced he c op cycle. Conce ning pa hogen in ec ion, damage caused by bo h pa hogens became mo e appa en wi h a one-week inocula ion compa ed o a ou -week inocula ion, which may indica e ha a mic obiological and chemical balance had been eached o cope wi h bio ic s esses. Based on hese esul s, we conclude ha CT applica ion induces plan g ow h and de ense in peppe plan s agains P. capsici and R. solani because o i s ele an soluble nu ien con en and mic obio a ichness, which p o ides a no el poin o plan nu i ion and p o ec ion in ho icul u al c ops. Keywo ds: Capsicum annuun L.; compos ea; bio-s imulan ; plan nu ien s; biocon ol; pa hogens 1. In oduc ion La ge amoun s o bio-was e p oduced e e y yea and should be managed and al- o ized o p o ide a well-humi ied ma e ial employable in ag icul u e. The e o e, ecycling o ganic was e as o ganic e ilize is a ele an s a egy o sus ainable c op p oduc ion. The applica ion o hese e ilize s o he soil could be applied as compos , g een manu e, a m ya d manu e, o ce eal esidues. Some o his biowas e-biosolids, p uning esidues, g een was e, sewage sludge-displayed good esul s when p ope ly compos ed. [ 1 ]. This ma e ial is ob ained h ough compos ing, a con olled bioxida i e p ocess ha equi es p ope humidi y, ae a ion, and he e ogeneous solid o ganic subs a es [ 2 ]. Mo eo e , aking in o accoun he high a iabili y o he pa ame e s ha cha ac e ize he di e en compos s (pH, elec ical conduc i i y, C- o-N a io and nu ien con en ), i is necessa y o know hei physicochemical and biological p ope ies when applying hem o soil o subs a e [ 3 ]. Ne e heless, compos is a sou ce o mac o- and mic onu ien s no only o plan s bu also o he mic oo ganisms ha suppo soil heal h by se ing as a quick and easily a ailable sou ce o ca bon [4]. Compos om g een was e seems o p esen a lowe isk o oxici y han do o ganic ones, which con ain di e en compounds o mic oo ganisms such as hea y me als, pol- lu an s, i uses, o ecal coli o ms [ 3 , 5 ]. In addi ion, se e al au ho s ha e shown he s imulan and supp essi e e ec o di e en compos applica ions in many c opping Ag onomy 2021,11, 781. h ps://doi.o g/10.3390/ag onomy11040781 h ps://www.mdpi.com/jou nal/ag onomy Ag onomy 2021,11, 781 2 o 12 sys ems agains se e al pa hogens such as Fusa ium oxyspo um,Ve icillium dahliae, and Rhizoc onia solani [6,7]. Mo eo e , compos eas (CTs) a e o ganic liquid p oduc s ha come om he mix u e o ma u e compos wi h ap wa e in 1:5 o 1:10 ( / ) a ios o a speci ic pe iod o incuba- ion [ 8 , 9 ]. Se e al ac o s a ec CT quali y, such as he compos ype, compos - o-wa e a io, and ae a ion, which al oge he modula e he de elopmen o bene icial mic oo gan- isms [ 10 – 12 ]. Fu he mo e, CTs a e composed o soluble nu ien s, and use ul compounds and mic oo ganisms (bac e ia, ac inomyce es, ilamen ous ungi, oomyce es, and yeas s) ha ha e a syne gic e ec on supp essing disease and p omo ing plan g ow h [ 13 – 15 ]. Sol- uble nu ien s, plan -g ow h egula o s, and humic acids ha e been p e iously desc ibed as media o s in plan disease supp ession [ 16 , 17 ]. Ne e heless, se e al au ho s ha e no ed ha among he mos abundan mic oo ganisms in hese ex ac s a e plan -g ow h p omo - ing hizobac e ia (PGPR), which in luence plan g ow h h ough di e en mechanisms (e.g., ni ogen ixa ion, nu ien solubiliza ion, g ow h ho mones elease, and enzymes) ha p oduce a be e nu ien a ailabili y [ 18 ]. These mechanisms seem o be in ol ed in plan -induced esis ance, making plan s able o cope wi h subsequen s esses [ 19 ]. In sho , CTs om g een was e seem o display he bes supp essi e e ec s agains a wide ange o di e en plan disease pa hogens [ 20 ]. Fo his eason, he employmen o hese ex ac s as a po en ial eco- iendly al e na i e o e ilize s and syn he ic ungicides has been ising in ecen yea s due o hei bene icial e ec s on c ops [ 20 – 22 ], bu u he esea ch is s ill equi ed. Peppe (Capsicum annuum L.) is an impo an ho icul u al c op wo ldwide o i s nu i- ional and medicinal alue, and has an annual p oduc ion o mo e han 38,000,000 ons [23,24]. The peppe ui is widely consumed o i s ichness in i amins, mine als, and nu ien s, as well as in phy ochemicals like ca o enoids, capsaicinoids, la onoids, asco bic acid, and ocophe ols [ 25 ]. Mo eo e , peppe p oduc ion could be comp omised by di e en soilbo ne pa hogens such as Phy oph ho a capsici and Rhizoc onia solani.P. capsici, one o he mos economically des uc i e soil-bo ne pa hogens, causes oo o and olia , s em and ui bligh o he peppe plan [ 26 ]. Mo eo e , he ungus R. solani can also p oduce seed decay, p e- and pos -eme gence damping-o , wi e s em, lea decay, oo o , and hypoco yl o ap oo wi h nec o ic spo s on he peppe [ 27 , 28 ]. The end owa ds inc eas- ingly o ganic ag icul u e has esul ed in he need o ind new en i o- iendly compounds o con ol plan diseases. In his con ex , we hypo hesized ha he applica ion o a nu ien - and mic obial- ich CT ob ained om g een was e ma u e compos would imp o e plan g ow h and media e plan de enses. Thus, he main objec i e o his wo k was o de e - mine he bio-s imulan and supp essi e e ec o he ex ac agains P. capsici and R. solani soil-bo ne pa hogens. 2. Ma e ials and Me hods 2.1. Compos Tea P epa a ion The compos ea (CT) came om he compos ing o g een and p uning was es ca ied ou in a ga den cen e loca ed in Salamanca (Spain) (40 ◦ 57 0 23 00 N; 5 ◦ 41 0 8 00 W, 775 m a.s.l.). This p ocess was pe o med in ae a ed piles o 15 × 2 × 2 m o 180 days. Piles we e u ned wice pe week o eigh weeks and once a week du ing he es o he bio-oxida i e p ocess. Mo eo e , he mois u e o he piles was con olled once a week. The ma u e compos was ob ained unde ambien condi ions in Ma ch. Then, compos was mixed wi h ap wa e in a a io o 1:5 ( / ) in polye hylene non-deg adable 1000 L con aine s a oom empe a u e o a b ewing pe iod las ing i e days. Wa e had been p e iously ae a ed o 8 h o educe he numbe o chlo ines. This mix u e was ae a ed o i e hou s e e y day wi h a pump. Then, i was il e ed wi h a double-laye ed cheeseclo h, and he ae a ed CT was s o ed in a da k con aine un il use. Ag onomy 2021,11, 781 3 o 12 2.2. Chemical and Mic obiological P ope ies o he Compos Tea The chemical p ope ies o 9 CT samples we e di ec ly analyzed. The pH, elec ical con- duc i i y (EC) and C- o-N a io we e de e mined as desc ibed by Mo ales-Co s e al. [ 29 ]. Fu he mo e, assimilable nu ien con en s (NO 3− , PO 43− , K 2 O, SO 42− , Ca 2+ and Mg 2+ ) we e analyzed wi h he nu ien analysis pho ome e HANNA HI 83225. Finally, humic acids we e de e mined ollowing he alkali-acid me hod desc ibed by Pan e al. [17]. Mic obial analysis o he CT was es ima ed using he se ial dilu ion sp ead pla e me hod. To de e mine he mic obial popula ion, di e en selec i e cul u e media and CT dilu ions we e used o mic oo ganism isola ion: nu ien aga and 10 −3 dilu ion o o al ae obic bac e ia, Ashby medium, and 10 −2 dilu ion o N- ixing bac e ia, ISP-2 medium and 10 −1 dilu ion o ac inobac e ia and modi ied po a o dex ose aga medium bu no dilu ion o o al ungi and T ichode ma ssp. quan i ica ion [ 30 – 33 ]. Then, pla es we e inocula ed by deposi ing on he aga su ace 0.1 mL o he CT dilu ion, which was sp ead on he media su ace wi h s e ile glass beads. Mo eo e , non-inocula ed pla es we e included as a nega i e con ol. Pe i dishes we e incuba ed in he da k a 28 ◦ C o 3 o 15 days, depending on he medium. A e his ime, colony- o ming uni s (CFU) we e coun ed o es ima e he cul i able mic oo ganism’s popula ion. This expe imen was conduc ed using i e eplica ions. 2.3. In Vi o Assays An ini ial in i o es was pe o med o de e mine he supp essi e e ec o he CT agains he pa hogens R. solani and P. capsici. The ela i e g ow h o bo h pa hogens was measu ed in Pe i dishes o 90 mm in diame e , p epa ed wi h s e ilized PDA a 60 ◦ C wi h he CT added a e s e iliza ion. Once he medium solidi ied, a 5-mm diame e PDA plug o R. solani o P. capsici was inse ed in o he cen e o he Pe i pla es. The CT- o-PDA ela ion was 1:10. Fu he mo e, con ols o each s ain we e included in s e ilized wa e and a PDA medium o assess hei g ow h in he absence o he CT. The g ow h diame e o he pa hogens (mm) was assessed a se en days a e inocula ion. In he end, he RGR was calcula ed ( ela i e g ow h a e o he pa hogen in compa ison o he g ow h o he colony wi hou he CT) [ 34 ]. The essay was ca ied ou including i e eplica ions pe ea men . Pe i dishes we e main ained in he da k a 24 ◦ C o 7 days. This expe imen was epea ed h ee imes. The P. capsici s ain p oceeded om a selec ion o Neike (Basque Cen e o Ag icul u al Resea ch) and Rhizoc onia solani s ain om he pa hogen collec ion o he Regional Cen e o Pes and Diseases Diagnosis, Jun a de Cas illa y León. 2.4. In Vi o Assays Fo in i o assays, on May 3, peppe seeds (Capsicum annuum c . Mo ón de Con- se a 4) we e ge mina ed in a s e ile subs a e o med by e miculi e. When he plan s had ou lea es (30 days a e sowing), hey we e ans e ed o 15 × 12 × 15 cm po s con aining a s e ile mix o blond pea (50%), sandy, acid pH, and nu ien -poo soil (37.5%, om Ledesma-Salamanca) and e miculi e (12.5%) as a e y nu i i e-de icien subs a e. The main cha ac e is ics o he subs a e we e: pH 6, 70 mg L −1 N, 71 mg L −1 P 2 O 5 , 150 mg L−1K2O, 35% o o ganic ma e , 0.6 dS m−1EC. The expe imen was ca ied ou in a g eenhouse unde he ollowing condi ions: empe a u e 24 ◦ C day, 18 ◦ C nigh and 80% ela i e humidi y. Ten di e en ea men s we e conside ed depending on he CT supply (40 mL was supplied weekly and indi idually by i iga ion), pa hogen inocula ion, and inocula ion ime: T1 (CT), T2 (CT + R. solani inocula ion one week a e ansplan ing), T3 (CT + P. capsici inocula ion one week a e ansplan ing), T4 (Con ol plan s inocula ed wi h R. solani one week a e ansplan ing), T5 (Con ol plan s inocula ed wi h P. capsici one week a e ansplan ing), T6 (CT + R. solani inocula ion ou weeks a e ansplan ing), T7 (CT + P. capsici inocula ion ou weeks a e ansplan ing), T8 (Con ol plan s inocula ed wi h R. solani ou weeks a e ansplan ing), T9 (Con ol plan s inocula ed wi h P. capsici ou weeks a e ansplan ing) and T10 (Con ol plan s wi hou in ec ion) (Figu e 1). Fo pa hogen inocula ion, he ice g ain Ag onomy 2021,11, 781 4 o 12 me hod was ca ied ou [ 35 ]. Six inocula ed ice seeds we e placed pe po . Mo eo e , o de e mine he e ec o CT applica ion, he ollowing plan g ow h and p oduc ion pa ame e s we e de e mined: s em diame e measu ed wi h a digi al calipe , chlo ophyll con en wi h a chlo ophyll me e Minol a SPAD-502 (bo h we e measu ed a mid-cycle), d y weigh o he oo sys em, and an ae ial pa (be o e being weighed, he plan s we e d ied in a P-Selec a-210 o en a 65 ◦ C o 48 h), lowe ing da e, ui weigh , and amoun o ui pe plan . The o al d y biomass p oduced pe plan was calcula ed as he sum o he d y ui , oo , and ae ial pa s. Weigh s we e de e mined by using a p ecise balance Sa o ius BL150S. Fu he mo e, he pa hogen incidence was also de e mined ollowing a speci ic symp om scale (1–5). The expe imen al design was a andomized comple e-block design wi h eigh plan s pe ea men . This essay was ca ied ou wice du ing 2017 and 2018. Ag onomy 2021, 11, x FOR PEER REVIEW 4 o 12 ansplan ing), T9 (Con ol plan s inocula ed wi h P. capsici ou weeks a e ansplan - ing) and T10 (Con ol plan s wi hou in ec ion) (Figu e 1). Fo pa hogen inocula ion, he ice g ain me hod was ca ied ou [35]. Six inocula ed ice seeds we e placed pe po . Mo eo e , o de e mine he e ec o CT applica ion, he ollowing plan g ow h and p o- duc ion pa ame e s we e de e mined: s em diame e measu ed wi h a digi al calipe , chlo ophyll con en wi h a chlo ophyll me e Minol a SPAD-502 (bo h we e measu ed a mid-cycle), d y weigh o he oo sys em, and an ae ial pa (be o e being weighed, he plan s we e d ied in a P-Selec a-210 o en a 65 °C o 48 h), lowe ing da e, ui weigh , and amoun o ui pe plan . The o al d y biomass p oduced pe plan was calcula ed as he sum o he d y ui , oo , and ae ial pa s. Weigh s we e de e mined by using a p ecise balance Sa o ius BL150S. Fu he mo e, he pa hogen incidence was also de e - mined ollowing a speci ic symp om scale (1–5). The expe imen al design was a andom- ized comple e-block design wi h eigh plan s pe ea men . This essay was ca ied ou wice du ing 2017 and 2018. Figu e 1. Summa y o ea men s. 2.5. S a is ical Analyses S a is ical analyses we e ca ied ou by one-way analysis o a iance in S a g aphics Cen u ion XVIII so wa e (S a is ical G aphics Co p., Rock ille, MD, USA). Resul s we e p esen ed as means wi h s anda d e o s, and o compa ison he Tukey’s Hones Signi - ican Di e ence (HSD) es wi h a 95% con idence in e al (p < 0.05) was used. 3. Resul s 3.1. Compos Tea P ope ies The s udied cha ac e is ics (pH, EC, C- o-N a io, assimilable mac o and mic onu i- en s, and humic acids) a e shown in Table 1. Highligh ed a e he high NO3− and K2O con- cen a ions (2240.4 and 2851.2 ppm, espec i ely) and he low C- o-N a io o he ex ac , as well as he ele an humic acids amoun Table 1. Chemical cha ac e is ics o he s udied CT. Resul s a e indica ed as mean ± s anda d de i- a ion. pH EC (dS/m) C/N NO3− (ppm) P2O5 (ppm) K2O (ppm) SO42− (ppm) Ca2+ (ppm) Mg2+ (ppm) Humic Acids (mg/L) 7.16 ± 0.15 1.2 ± 0.14 7.1 ± 0.2 2240.4 ± 225 61.4 ± 25 2851.2 ± 188 43 ± 20 280 ± 17 20 ± 14 198 ± 31 Figu e 1. Summa y o ea men s. 2.5. S a is ical Analyses S a is ical analyses we e ca ied ou by one-way analysis o a iance in S a g aph- ics Cen u ion XVIII so wa e (S a is ical G aphics Co p., Rock ille, MD, USA). Resul s we e p esen ed as means wi h s anda d e o s, and o compa ison he Tukey’s Hones Signi ican Di e ence (HSD) es wi h a 95% con idence in e al (p< 0.05) was used. 3. Resul s 3.1. Compos Tea P ope ies The s udied cha ac e is ics (pH, EC, C- o-N a io, assimilable mac o and mic onu i- en s, and humic acids) a e shown in Table 1. Highligh ed a e he high NO 3− and K 2 O concen a ions (2240.4 and 2851.2 ppm, espec i ely) and he low C- o-N a io o he ex ac , as well as he ele an humic acids amoun Table 1. Chemical cha ac e is ics o he s udied CT. Resul s a e indica ed as mean ±s anda d de ia ion. pH EC (dS/m) C/N NO3− (ppm) P2O5 (ppm) K2O (ppm) SO42− (ppm) Ca2+ (ppm) Mg2+ (ppm) Humic Acids (mg/L) 7.16 ± 0.15 1.2 ±0.14 7.1 ± 0.2 2240.4 ± 225 61.4 ±25 2851.2 ± 188 43 ±20 280 ± 17 20 ±14 198 ±31 Conce ning he mic obiological analysis o he CT, he mos abundan g oup o mic oo ganisms was o al ae obic bac e ia ollowed by he N- ixing bac e ia and ac i- nobac e ia (Figu e 2). Mo eo e , T ichode ma spp. and ungi we e ound be ween 2.7 and 8.7 ×102c u/mL, espec i ely. Ag onomy 2021,11, 781 5 o 12 Ag onomy 2021, 11, x FOR PEER REVIEW 5 o 12 Conce ning he mic obiological analysis o he CT, he mos abundan g oup o mi- c oo ganisms was o al ae obic bac e ia ollowed by he N- ixing bac e ia and ac inobac- e ia (Figu e 2). Mo eo e , T ichode ma spp. and ungi we e ound be ween 2.7 and 8.7 × 102 c u/mL, espec i ely. Figu e 2. Mic obial popula ions in g een was e compos ea. 3.2. Compos Tea E ec in In Vi o Assays agains Rhizoc onia solani and Phy oph ho a capsici To s udy in i o g ow h o he plan pa hogens R. solani and P. capsici in esponse o applica ion o he CT, he ela i e g ow h a e o each pa hogen was calcula ed. Acco ding o his pa ame e , he ela i e g ow h a es o bo h R. solani and P. capsici pa hogens we e 38% and 31.7%, espec i ely, in compa ison o he con ol ea men (Figu e 3 and Table 2). Figu e 3. E ec o CT on he ela i e g ow h a e o R. solani and P. capsici in compa ison o con ol ea men . Table 2. Rela i e g ow h a e o R. solani and P. capsici in CT ea men . Resul s a e indica ed as mean ± s anda d de ia ion. Pa hogen RGR (%) Rhizoc onia solani 38.0 ± 4.2 Phy oph ho a capsici 31.7 ± 4.6 3.3. Compos Tea E ec in In Vi o Assays agains Rhizoc onia solani and Phy oph ho a capsici As shown in Table 3, he ea men s wi h compos ea (T1, T2, T3, T6 and T7) ad- anced he lowe ing da e o peppe plan s. CT ea men (T1) also p oduced an inc ease in he diame e o he s em in compa ison o he con ol (T10). Likewise, he measu emen o his pa ame e also indica ed signi ican di e ences in T6 and T7 (CT + R. solani o P. Figu e 2. Mic obial popula ions in g een was e compos ea. 3.2. Compos Tea E ec in In Vi o Assays agains Rhizoc onia solani and Phy oph ho a capsici To s udy in i o g ow h o he plan pa hogens R. solani and P. capsici in esponse o applica ion o he CT, he ela i e g ow h a e o each pa hogen was calcula ed. Acco ding o his pa ame e , he ela i e g ow h a es o bo h R. solani and P. capsici pa hogens we e 38% and 31.7%, espec i ely, in compa ison o he con ol ea men (Figu e 3and Table 2). Ag onomy 2021, 11, x FOR PEER REVIEW 5 o 12 Conce ning he mic obiological analysis o he CT, he mos abundan g oup o mi- c oo ganisms was o al ae obic bac e ia ollowed by he N- ixing bac e ia and ac inobac- e ia (Figu e 2). Mo eo e , T ichode ma spp. and ungi we e ound be ween 2.7 and 8.7 × 102 c u/mL, espec i ely. Figu e 2. Mic obial popula ions in g een was e compos ea. 3.2. Compos Tea E ec in In Vi o Assays agains Rhizoc onia solani and Phy oph ho a capsici To s udy in i o g ow h o he plan pa hogens R. solani and P. capsici in esponse o applica ion o he CT, he ela i e g ow h a e o each pa hogen was calcula ed. Acco ding o his pa ame e , he ela i e g ow h a es o bo h R. solani and P. capsici pa hogens we e 38% and 31.7%, espec i ely, in compa ison o he con ol ea men (Figu e 3 and Table 2). Figu e 3. E ec o CT on he ela i e g ow h a e o R. solani and P. capsici in compa ison o con ol ea men . Table 2. Rela i e g ow h a e o R. solani and P. capsici in CT ea men . Resul s a e indica ed as mean ± s anda d de ia ion. Pa hogen RGR (%) Rhizoc onia solani 38.0 ± 4.2 Phy oph ho a capsici 31.7 ± 4.6 3.3. Compos Tea E ec in In Vi o Assays agains Rhizoc onia solani and Phy oph ho a capsici As shown in Table 3, he ea men s wi h compos ea (T1, T2, T3, T6 and T7) ad- anced he lowe ing da e o peppe plan s. CT ea men (T1) also p oduced an inc ease in he diame e o he s em in compa ison o he con ol (T10). Likewise, he measu emen o his pa ame e also indica ed signi ican di e ences in T6 and T7 (CT + R. solani o P. Figu e 3. E ec o CT on he ela i e g ow h a e o R. solani and P. capsici in compa ison o con ol ea men . Table 2. Rela i e g ow h a e o R. solani and P. capsici in CT ea men . Resul s a e indica ed as mean ±s anda d de ia ion. Pa hogen RGR (%) Rhizoc onia solani 38.0 ±4.2 Phy oph ho a capsici 31.7 ±4.6 3.3. Compos Tea E ec in In Vi o Assays agains Rhizoc onia solani and Phy oph ho a capsici As shown in Table 3, he ea men s wi h compos ea (T1, T2, T3, T6 and T7) ad anced he lowe ing da e o peppe plan s. CT ea men (T1) also p oduced an inc ease in he diame e o he s em in compa ison o he con ol (T10). Likewise, he measu emen o his pa ame e also indica ed signi ican di e ences in T6 and T7 (CT + R. solani o P. capsici inocula ion ou weeks a e ansplan ing) compa ed o T10. On he o he hand, al hough he e we e no signi ican di e ences, hose ea men s inocula ed wi h R. solani and P. capsici one week a e ansplan ing (T4 and T5) showed lowe means han he con ol did o diame e and chlo ophyll con en pa ame e s. Ag onomy 2021,11, 781 6 o 12 Table 3. Plan g ow h pa ame e s o peppe plan s g own unde he di e en ea men s. T ea men s Days o Flowe ing (a e T ansplan ing) Plan Heigh (cm) S em Diame e (mm) Chlo ophyll Con en (SPAD Uni s) T1 40.1 a 31.2 abc 8.97 a 37.6 a T2 39.2 a 33.0 ab 7.72 bc 40.9 a T3 39.7 a 31.0 abc 9.10 a 38.7 a T4 48.6 b 26.5 c 7.80 bc 37.1 a T5 49.0 b 30.0 bc 7.65 c 39.4 a T6 40.1 a 30.7 abc 9.22 a 39.2 a T7 41.0 a 36.2 a 8.92 a 38.5 a T8 48.2 b 33.5 ab 8.60 ab 41.6 a T9 48.7 b 33.2 ab 8.40 abc 38.6 a T10 49.2 b 29.7 bc 7.92 bc 39.8 a Dis inc le e s in he same column indica e s a is ically signi ican di e ences among ea men s as de e mined by Tukey’s hones signi ican di e ence (HSD) (p< 0.05). To examine whe he he obse ed changes in g ow h we e ela ed o he CT applica ion and R. solani and P. capsici diseases, he o al amoun o ui s, plan d y weigh , and pa hogen incidence we e measu ed (Table 4). Plan s i iga ed wi h he CT (T1) displayed he bes esul s in he o al weigh o peppe ui , being wo imes highe han hose o T10. Mo eo e , CT- ea ed and -inocula ed ea men s (T2, T3, T7, and T8) sligh ly imp o ed he ae ial biomass pa ame e s compa ed o he con ol (wi hou signi ican di e ences). Plan s inocula ed wi h R. solani o P. capsici one week a e ansplan ing (T4 and T5) showed a educ ion in shoo and oo d y weigh , while plan s o T5 also p esen ed a dec ease in o al ui weigh . Table 4. F ui p oduc ion, plan d y weigh and R. solani and P. capsici incidence on peppe plan s unde CT applica ion. T ea men s To al F ui Numbe To al F ui Weigh (g) Mean o F ui Weigh (g) Roo D y Weigh (g) Shoo D y Weigh (g) Pa hogen Incidence T1 5 322.19 64.44 a 8.71 ab 5.25 b 0 T2 6 219.25 36.54 bc 7.82 b 5.59 b 0 T3 6 249.58 41.60 b 8.94 ab 6.07 ab 0 T4 4 171.21 42.80 b 4.33 c 4.01 c 2 T5 7 154.44 22.06 d 3.70 cd 3.55 c 3 T6 4 182.07 45.52 b 11.60 a 5.98 ab 0 T7 5 228.38 45.68 b 10.59 a 6.42 a 0 T8 7 209.38 29.91 c 6.51 bc 4.98 b 1 T9 4 147.13 36.78 bc 4.98 c 4.55 bc 2 T10 6 162.23 27.04 c 8.66 ab 4.95 b 0 Dis inc le e s in he same column indica e s a is ically signi ican di e ences among ea men s as de e mined by Tukey’s hones signi ican di e ence (HSD) (p< 0.05). Conce ning pa hogen incidence, i should be poin ed ou ha collapse only occu ed in one plan o he T5 ea men . Fu he mo e, mos o he ea men plan s showed di e en symp oms such as black oo s, na ow neck, and a hin s em (g ade 3 a ack se e i y) and hose plan s o T4 displayed a g ade 2 pa hogen a ack. Mo eo e , T8 and T9 (R.solani and P.capsici inocula ed 4 weeks a e ansplan ing) gene a ed less damage han inocula ions a week a e ansplan ing (T4 and T5) conside ing he damage se e i y o g ade 1 and 2, espec i ely. In addi ion, i should be no ed ha he e we e signi ican di e ences among he inocula ed P. capsici plan s one week a e ansplan ing unde CT and con ol condi ions (T3 and T5, espec i ely). Fu he mo e, he e we e no di e ences be ween CT- ea ed and inocula ed wi h R. solani plan s one week a e ansplan ing (T2) and hose Ag onomy 2021,11, 781 7 o 12 ea ed and inocula ed ou weeks a e ansplan ing (T6), while he e we e signi ican di e ences be ween T2 and T4. The o al d y biomass p oduced pe plan and ea men a e shown in Figu e 4. T ea men s wi h he CT (T1, T2, T3, T6, and T7) inc eased he o al biomass o plan s. This e ec was signi ican when CT (T1) and con ol wi hou CT applica ion (T10), we e compa ed. The CT applica ion p oduced an 36% inc ease in biomass o e con ol. Plan s inocula ed wi h P. capsici one week a e ansplan ing (T5) displayed a 50% educ ion in biomass p oduc ion wi h espec o hose inocula ed and ea ed wi h he CT (T3). Ag onomy 2021, 11, x FOR PEER REVIEW 7 o 12 T4 4 171.21 42.80 b 4.33 c 4.01 c 2 T5 7 154.44 22.06 d 3.70 cd 3.55 c 3 T6 4 182.07 45.52 b 11.60 a 5.98 ab 0 T7 5 228.38 45.68 b 10.59 a 6.42 a 0 T8 7 209.38 29.91 c 6.51 bc 4.98 b 1 T9 4 147.13 36.78 bc 4.98 c 4.55 bc 2 T10 6 162.23 27.04 c 8.66 ab 4.95 b 0 Dis inc le e s in he same column indica e s a is ically signi ican di e ences among ea men s as de e mined by Tukey’s hones signi ican di e ence (HSD) (p < 0.05). The o al d y biomass p oduced pe plan and ea men a e shown in Figu e 4. T ea - men s wi h he CT (T1, T2, T3, T6, and T7) inc eased he o al biomass o plan s. This e ec was signi ican when CT (T1) and con ol wi hou CT applica ion (T10), we e compa ed. The CT applica ion p oduced an 36% inc ease in biomass o e con ol. Plan s inocula ed wi h P. capsici one week a e ansplan ing (T5) displayed a 50% educ ion in biomass p oduc ion wi h espec o hose inocula ed and ea ed wi h he CT (T3). Figu e 4. To al d y biomass p oduced pe peppe plan and ea men . T1: CT (compos ea); T2 and T3: CT + pa hogen inocula ed one week a e ansplan ing; T4 and T5: pa hogen inocula ed one week a e ansplan ing; T6 and T7: CT + pa hogen inocula ed ou weeks a e ansplan ing; T8 and T9: pa hogen inocula ed ou weeks a e ansplan ing; T10: con ol. Di e en le e s abo e he columns indica e signi ican di e ences (p<0.05). 4. Discussion The p esen wo k showed ha g een was e CT no only p omo es plan de elopmen , bu also p o ec s plan s when exposed o R. solani and P. capsici pa hogens a di e en imes om ansplan ing. In his s udy, a CT coming om g een was e was es ed. Fi s , he chemical p ope ies o his ex ac we e s udied, and he esul s showed ha no only did he CT dose play a ele an ole in plan g ow h and plan esis ance, bu i also imp o ed soil quali y [36,37]. The pH and EC we e signi ican ly lowe han hose ob ained by o he au ho s [9,38]. The esul o he C- o-N a io showed a educ ion wi h espec o he o iginal compos (11.4) [29], which indica ed a low C amoun in ela ion o he N ex ac ed in he CT. This low a io poin s o he ex ac ’s high s abili y and conse a ion. Nu ien analyses e ealed ha ni ogen (N) and po assium (K) we e well p esen ed in his CT, making eas an in e - es ing sou ce o e ilize o c op applica ion. Fu he mo e, high K concen a ion could also be bene icial o plan esis ance agains bo h R. solani and P. capsici in ec ions, since Am mann e al. [39] w o e ha K applica ion ends o educe ungal diseases. In addi ion, 54.2 a 39.3 b 44 b 29.5 c 22.2 39.8 b 43.9 b 35 bc 26.1 c 35.8bc 0 10 20 30 40 50 60 70 (T1) CT (T2) CT+ R.solani1 (T3) CT+ P.capsici1 (T4) R.solani1 (T5) P.capsici1 (T6) CT+ R.solani4 (T7) CT+ P.capsici4 (T8) R.solani4 (T9) P.capsici4 (T10) Con ol To al D y Biomass p oduced pe peppe plan (g) Figu e 4. To al d y biomass p oduced pe peppe plan and ea men . T1: CT (compos ea); T2 and T3: CT + pa hogen inocula ed one week a e ansplan ing; T4 and T5: pa hogen inocula ed one week a e ansplan ing; T6 and T7: CT + pa hogen inocula ed ou weeks a e ansplan ing; T8 and T9: pa hogen inocula ed ou weeks a e ansplan ing; T10: con ol. Di e en le e s abo e he columns indica e signi ican di e ences (p< 0.05). 4. Discussion The p esen wo k showed ha g een was e CT no only p omo es plan de elopmen , bu also p o ec s plan s when exposed o R. solani and P. capsici pa hogens a di e en imes om ansplan ing. In his s udy, a CT coming om g een was e was es ed. Fi s , he chemical p ope ies o his ex ac we e s udied, and he esul s showed ha no only did he CT dose play a ele an ole in plan g ow h and plan esis ance, bu i also imp o ed soil quali y [ 36 , 37 ]. The pH and EC we e signi ican ly lowe han hose ob ained by o he au ho s [ 9 , 38 ]. The esul o he C- o-N a io showed a educ ion wi h espec o he o iginal compos (11.4) [ 29 ], which indica ed a low C amoun in ela ion o he N ex ac ed in he CT. This low a io poin s o he ex ac ’s high s abili y and conse a ion. Nu ien analyses e ealed ha ni ogen (N) and po assium (K) we e well p esen ed in his CT, making eas an in e es ing sou ce o e ilize o c op applica ion. Fu he mo e, high K concen a ion could also be bene icial o plan esis ance agains bo h R. solani and P. capsici in ec ions, since Am - mann e al. [ 39 ] w o e ha K applica ion ends o educe ungal diseases. In addi ion, he high con en o humic acids (10.3% o d y ma e ) could explain he posi i e e ec o he ob ained CT on plan g ow h p omo ion and esis ance. Besides, he applica ion o humic acids enhances phenolic and la onoid accumula ion and imp o es yields in Cicho ium in ybus plan s [ 40 ]. Se e al au ho s ha e linked humic subs ances as compounds ha ing an auxin-like ac i i y ha induces ni a e me abolism o plan g ow h [ 41 , 42 ]. In addi ion, Mo ales-Co s e al. [ 9 ] explained ha he g ow h e ec o CT could be no only explained by humic acid con en , bu also by nu ien concen a ion, phy oho mones, and he p esence o bene icial mic oo ganisms. The e o e, he mic obio a in a CT also play a undamen al ole in he abili y o hese ex ac s o supp ess plan diseases o p omo e Ag onomy 2021,11, 781 8 o 12 g ow h [ 13 ]. Bac e ial communi ies ( o al ae obic bac e ia and N- ixing bac e ia) we e he p edominan mic oo ganisms in his ae a ed CT. These esul s ollow Kim e al. [ 43 ], who obse ed simila popula ion densi ies o cul u able bac e ia in he mix u e o o i- en al medicinal he b compos and e micompos ea du ing incuba ion. Fu he mo e, Li e al. [ 44 ] showed analogous esul s in mic obial popula ions in maize s aw CT, ha ing he mos o al bac e ia mic obial popula ions, ollowed by ac inomyce es and o al ungi. I is clea ha mic obial communi ies ound in a CT can be indi ec ly esponsible o g ow h imp o emen wi h espec o he con ol because o hei implica ion o he a ailabili y o nu ien s as p e iously sugges ed by Hamid e al. [ 19 ], especially due o K + con en in he s udied CT. Conce ning he inhibi o y e ec o he CT on he s udied ungal pa hogens, in i o assay esul s showed ha he applica ion o CT o PDA (1:10) signi ican ly dec eased he g ow h a e o R. solani and P. capsici in compa ison wi h he con ol ea men . Acco ding o hese esul s, Dionne e al. [ 45 ] showed ha he CT signi ican ly educed he g ow h o he mycelium o R. solani. This educ ion seems o be ela ed o he high mic obio a popula ion, since in ou expe imen he s e ilized CT did no show his di ec supp es- si e e ec (da a no shown). Hence, CT-en iched media displayed a la ge numbe o mic oo ganisms ha could di ec ly ep ess ungi g ow h. In addi ion o his s a emen , se e al au ho s ha e p e iously w i en ha mic obial popula ions a e equi ed o de e - mine he supp essi e e ec o he CT [ 36 , 46 ]. Mo eo e , he CT con ains di e en species o he gene a T ichode ma,Penicillium,Aspe gillus,Bacillus,En e obac e ,Rhizobac e ia o Pseudomonas spp., among o he s, which could ha e he abili y o con ol pa hogens and s imula e plan g ow h [47–50]. The e ec o a CT on plan g ow h and esis ance agains P. capsici and R. solani was es ed. The plan s g own unde CT ea men (T1) displayed he bes esul s in biomass and in he o al and a e age weigh o ui . These esul s a e in conco dance wi h hose indica ed by Ros e al. [ 51 ], who poin ed ha he olia applica ion o CT inc eases he yield and quali y o baby spinach plan s. This could be explained by he con ibu ion o nu ien con en , humic acids, and mic obial popula ion in g ow h p omo ion [ 52 ]. In addi ion o his s a emen , Ree e e al. [ 53 ] indica ed ha he nu ien con en in a CT could supplemen o subs i u e o o he ypes o e ilize s. Mo eo e , i should be poin ed ou ha he CT applica ion ca ied ou in his ial does no p o ide all he necessa y nu ien s o he peppe c op, since he con ol plan s showed nu i ional de iciencies om he middle o he cycle. The e o e, a supplemen a y e iliza ion is equi ed o co e he nu i ional equi emen s o peppe plan s o ge a high yield. The e o e, he combina ion o mine al and o ganic componen s should be conside ed o educe he chemical esou ces applica ion. In ela ion o his s a emen , se e al au ho s es ed he combina ion compos o mine al solubilizing mic oo ganism wi h e ilize s as a good solu ion o inc ease he a ailabili y o nu ien s [54–56]. I is ele an o he poin ha he i s lowe o he CT- ea ed plan s (T1, T2, T3, T6, and T7) s a ed opening one week be o e hose wi hou CT ea men (Table 3), which indica es ha applica ion o he CT ad anced he c op cycle. Simila esul s we e obse ed in po a o plan s ea ed wi h ga den was e CT [ 57 ]. Fu he mo e, peppe plan s inocula ed wi h P. capsici one week a e ansplan ing (T5) showed a educ ion in ui weigh as well as in s em and oo d y weigh compa ed o he con ol ea men . These plan s also displayed a highe g ade o pa hogen incidence (black oo s, na ow neck, hin s em, lea decay and e y p onounced chlo osis). In addi ion, plan s ea ed wi h CT and inocula ed wi h P. capsici (T3 and T7) showed a educ ion in pa hogen incidence. They inc eased g ow h pa ame e s ( o al ui weigh , a e age ui weigh , d y weigh o he oo , and ae ial pa and o al biomass). This ac may be due o he mic obial popula ions o CT since Ezziyyani e al. [ 58 ] p e iously e ealed ha bo h T ichode ma ha zianum and ac inobac e ia e ec i ely educe he a ack o Phy oph ho a capsici. Mo eo e , ega ding he disease p oduced by R. solani, plan s inocula ed wi h his pa hogen one week a e ansplan ing (T4) displayed g ade 2 pa hogen incidence and a Ag onomy 2021,11, 781 9 o 12 educ ion in oo and s em weigh ela i e o he con ol. In e es ingly, as o P. capsici, plan s p esen ed less pa hogen damage when inocula ed wi h R. solani ou weeks a e ansplan ing (T8) wi h espec o hose inocula ed one week a e ansplan ing (T4). This poin led us o conclude ha CT applica ion had a supp essi e e ec on he a ack o R. solani as shown in in i o cul u es. Mo eo e , se e al au ho s p e iously s udied he e ec o di e en CTs on R. solani con ol in di e en plan species [ 59 , 60 ]. In his line, Mo ales- Co s e al. [ 9 ] con i med he e ec o g een was e CT on he con ol o his pa hogen in po a o plan s. This ac is di ec ly ela ed o he p esence o T ichode ma spp., among o he mic obial an agonis s and pa asi ic o an ibio ic-p oduce mic oo ganisms [ 46 , 61 ], and o indi ec mechanisms inducing sys emic esis ance in plan s [ 19 ]. In his sense, he addi ion o PGPR may enhance auxin p oduc ion in he hizosphe e which is linked o de eloping s ess ole ance in plan s. Consequen ly, he addi ion o CT could minimize s ess in he peppe plan s caused by pa hogens, especially when he applica ion is ca ied ou be o e an a ack. Al oge he , indica ions a e ha his CT could be conside ed as a g een al e na i e o supplemen , o subs i u e o , o he ypes o e ilize s and pes icides. Since peppe is one o he mos impo an human ood c ops, managemen s a egies using CT ob ained om g een was e ( ollowing he Sus ainable De elopmen Goals o he Uni ed Na ions o achie e a be e and mo e sus ainable u u e [ 62 ]) could be ele an in in eg a ed and sus ainable ag icul u e. 5. Conclusions This s udy con i med he posi i e e ec o CT applica ion (based on g een was e) on o al biomass and peppe ui p oduc ion and o i s di ec implica ion o he con ol o pa hogen de elopmen by causing a educ ion o bo h P. capsici and R. solani in in i o and in i o essays. The s udied ex ac p esen ed ele an con en in NO 3− , K 2 O, humic acids, and mic oo ganisms, which seem o play a syne gic ole in plan g ow h and p o ec ion. Mic obio a in he CT induced a educ ion in he ela i e g ow h a e o bo h P. capsici and R. solani (31.7% and 38.0%, espec i ely) in in i o assays compa ed o he con ol. Mo eo e , he e ec on pa hogen con ol was signi ican in P. capsici ea ly a acks in he in i o assays. The CT applica ion p oduced a 36% inc ease in biomass wi h espec o he con ol, and plan s ea ed wi h he CT s a ed lowe ing one week be o e hose ha we e no ea ed, hus indica ing ha he applica ion o he CT ad anced he c op cycle. Despi e i s bio-s imulan ac ion on plan g ow h, CT applica ion in his ial did no p o ide all he necessa y nu ien s o he peppe c op. The e o e, supplemen a y e iliza ion is equi ed o co e he nu i ional equi emen s o ob ain a high yield. Fu he s udies should be conduc ed unde ield condi ions and combined wi h o he e ilize s. Au ho Con ibu ions: Concep ualiza ion, M.R.M.-C. and M.B.S.-F.; Me hodology, M.R.M.-C., R.P.-S. and M.B.S.-F.; So wa e, A.I.G.-H., M.Á.G.-S. and M.R.M.-C.; Valida ion, M.R.M.-C. and A.I.G.-H.; In es iga ion, M.R.M.-C., M.Á.G.-S., A.I.G.-H., R.P.-S. and M.B.S.-F.; Da a Cu a ion, M.R.M.-C., M.Á.G.-S., A.I.G.-H., R.P.-S. and M.B.S.-F.; W i ing—O iginal D a P epa a ion, A.I.G.-H. and M.R.M.-C. ; W i ing—Re iew & Edi ing, A.I.G.-H., M.R.M.-C. and R.P.-S.; P ojec Adminis a ion, M.R.M.-C.; Funding Acquisi ion, M.R.M.-C. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This esea ch was suppo ed by Fe ibe ia and Salamanca Uni e si y 83 Con ac Ag ee- men (G ow-in induc i e e iliza ion). Ins i u ional Re iew Boa d S a emen : No applicable. Acknowledgmen s: The au ho s hank Neike (Basque Cen e o Ag icul u al Resea ch) o he Phy oph ho a capsici s ain pa hogen and o he Regional Cen e o pes and Diseases Diagnosis, Jun a de Cas illa y León, Spain o he Rhizoc onia solani s ain pa hogen. Con lic s o In e es : The au ho s decla e no con lic o in e es .