A Phloem-Feeding Insect Transfers Bacterial Endophytic Communities between Grapevine Plants.

ORIGINAL RESEARCH published: 15 May 2017 doi: 10.3389/ micb.2017.00834 F on ie s in Mic obiology | www. on ie sin.o g 1May 2017 | Volume 8 | A icle 834 Edi ed by: Lau e Weisskop , Uni e si y o F ibou g, Swi ze land Re iewed by: Da id Bal us, Uni e si y o A izona, USA Ra aella Bales ini, Consiglio Nazionale Delle Rice che, I aly *Co espondence: Sebas iàn Lòpez-Fe nàndez [email p o ec ed] Special y sec ion: This a icle was submi ed o Plan Mic obe In e ac ions, a sec ion o he jou nal F on ie s in Mic obiology Recei ed: 24 Janua y 2017 Accep ed: 24 Ap il 2017 Published: 15 May 2017 Ci a ion: Lòpez-Fe nàndez S, Mazzoni V, Ped azzoli F, Pe o I and Campisano A (2017) A Phloem-Feeding Insec T ans e s Bac e ial Endophy ic Communi ies be ween G ape ine Plan s. F on . Mic obiol. 8:834. doi: 10.3389/ micb.2017.00834 A Phloem-Feeding Insec T ans e s Bac e ial Endophy ic Communi ies be ween G ape ine Plan s Sebas iàn Lòpez-Fe nàndez1, 2, 3*, Vale io Mazzoni1, Fede ico Ped azzoli4, Ila ia Pe o 1, 5 and And ea Campisano1 1Resea ch and Inno a ion Cen e, Fondazione Edmund Mach, San Michele all’Adige, I aly, 2In ec ion Biology Depa men , Ins i u e o Mic obiology, Technische Uni e si ä B aunschweig, B aunschweig, Ge many, 3Depa men Mic obial D ugs, Helmhol z Cen e o In ec ion Resea ch, B aunschweig, Ge many, 4Technology T ans e Cen e, Fondazione Edmund Mach, San Michele all’Adige, I aly, 5Cen e Ag icul u e Food En i onmen , Uni e si y o T en o, T en o, I aly Bac e ial endophy es colonize he inne issues o hos plan s h ough he oo s o h ough discon inui ies on he plan su ace, including wounds and s oma a. Li le is known ega ding a possible ole o insec s in acqui ing and ansmi ing non-phy opa hogenic mic oo ganisms om plan o plan , especially hose endophy es ha a e bene icial symbion s p o iding plan p o ec ion p ope ies and homeos a ic s abili y o he hos . To unde s and he ecological ole o insec s in he ansmission o endophy ic bac e ia, we used eshly ha ched nymphs o he Ame ican sap- eeding lea hoppe Scaphoideus i anus ( ec o ) o ans e mic oo ganisms ac oss g ape ine plan s. A e con ac wi h he ec o , sink plan s we e colonized by a complex endophy ic communi y domina ed by P o eobac e ia, highly simila o ha p esen in sou ce plan s. A simila bac e ial communi y, bu wi h a highe a io o Fi micu es, was ound on S. i anus. Insec s eeding only on sink plan s ans e ed an en i ely di e en bac e ial communi y domina ed by Ac inobac e ia, whe e Mycobac e ium sp., played a majo ole. Despi e he ac ha insec s dwelled mos ly on plan s ems, he bac e ial communi ies in plan oo s esembled mo e closely hose inside and on insec s, when compa ed o hose o abo e-g ound plan o gans. We p o e he e he po en ial o insec ec o s o ans e en i e endophy ic bac e ial communi ies be ween plan s. We also desc ibe he ole o plan s and bac e ial endophy es in es ablishing mic obial communi ies in plan - eeding insec s. Keywo ds: endophy es, py osequencing, molecula ecology, insec s, g ape ine INTRODUCTION Plan s a e open sys ems ha cons an ly acqui e wa e and nu ien s om he soil and in e ac wi h he as biological di e si y o he su oundings (Médiène e al., 2011). This di e si y encompasses o he plan s, animals (i.e., p o ozoa, annelids, nema odes, a h opods, and e eb a es) and mic oo ganisms. The complex in e ac ion among hese di e se playe s in luences c op heal h and p oduc i i y (A angana e al., 2014). A be e unde s anding o he ou come o hese in e ac ions is c ucial o imp o ing sus ainable c op managemen and a he same ime o iden i ying new app oaches o pes managemen . Insec s and o he in e eb a es can ansmi di e se mic obial plan pa hogens (e.g., i uses, phy oplasmas, ungi, and bac e ia). Mos insec ec o s belong o he Hemip e a, an o de Lòpez-Fe nàndez e al. An Insec T ans e s Bac e ial Endophy es in G ape ine cha ac e ized by pie cing and sucking mou hpa s ha enable eeding om phloem o xylem essels and, consequen ly, allow hem o acqui e and ansmi phy opa hogens. Fo example plan hoppe s and lea hoppe s can ansmi nume ous phy oplasmas, i uses and bac e ia (Ha is and Ma amo osch, 1980). The ansmission o insec -bo ne pa hogens and he ecological ole o insec s as ec o s o pa hogenic mic oo ganisms ha e been deeply s udied in nume ous c ops (Wein aub and Beanland, 2006). Mechanis ically, he e a e simila i ies (modes o acquisi ion and deli e y) in he insec -media ed ansmission o indi idual mu ualis s and pa hogens be ween plan s (B igh and Bulghe esi, 2010; Pè ez-B ocal e al., 2013). Howe e , li le is known abou he e ec s o ansmi ing en i e communi ies o mu ualis symbion s and he implica ions o his ansmission in plan hos i ness. In addi ion, in o ma ion ega ding he po en ial use o ansmi ed mu ualis s as a p ophylac ic ool in plan p o ec ion and he ecological implica ions o a possible na u al inocula ion wi h such mic oo ganisms by phloem- eeding insec s is lacking. We chose he Ame ican g ape ine lea hoppe , Scaphoideus i anus (Hemip e a: Cicadellidae), as insec model because his species has been la gely s udied as ec o o he la escence do ée phy oplasma (FDP). S. i anus is mono ol ine and specialis on g ape ine, which means ha i li es and eeds on g ape ine om ha ched nymphs o adul s (Chuche and Thié y, 2014). The li e cycle o he insec begins in summe wi h he egg laying in he ba k o woody s ems o g ape ine, ollowed by a win e diapause wi h g adual ha chings occu ing om May o ea ly Augus . Nymphs ( i e ins a s) emain mos o he ime on he abaxial side o lea es o he plan hey ha ched on. Unde labo a o y condi ions, a a empe a u e o 23–25◦C, he ime lapse om egg ha ching o adul hood is ∼30 days. The adul s can li e o se e al weeks and emales su i e on a e age 60 days (Je mini e al., 2015). S. i anus is mainly a phloem eede , al hough mou h s yle s can e enly pie ce bo h phloem and xylem essels (Chuche e al., 2011). While eeding, he insec can acqui e FDP ha can be hen ansmi ed o o he g ape ines in a pe sis en -p opaga i e manne (Foissac and Wilson, 2009). The ansmission p ocess includes an incuba ion pe iod o abou 1 mon h du ing which phy oplasmas mul iply, mos ly in he o e- and mid-gu , and accumula e in he sali a y glands un il hey each a densi y ha pe mi s ansmission (Chuche and Thié y, 2014). The e iciency o FDP acquisi ion is co ela ed wi h phy oplasma i e in he sou ce plan (Gale o e al., 2016). The ansmission is non- anso a ial, which means ha newbo n nymphs do no ca y he mic oo ganism, bu a he hey acqui e i om in ec ed plan s. S. i anus engages in mul iple symbioses wi h bac e ia, including Ca dinium sp., Asaia sp., and yeas -like endosymbion s (Sacchi e al., 2008). Endophy es asymp oma ically colonize he inne issues o plan s (Schulz and Boyle, 2006). Plan coloniza ion mechanisms o bac e ial endophy es a e complex and symbiosis genes in he genomes o he mic obe, in e -kingdom signaling be ween he plan and he bac e ium and plan immuni y may play impo an oles in i , as is he case in many o he plan -mic oo ganisms in e ac ions (Iniguez e al., 2005; Reinhold-Hu ek and Hu ek, 2011; Kusa i e al., 2015). The coloniza ion o he plan may esul in e ec s ha span om plan g ow h p omo ion by ni ogen ixa ion (San oyo e al., 2016) o an agonis ic p ope ies agains plan pa hogens (Rabha e al., 2014) and syn hesis o exogenous plan ho mones ha media e de elopmen al p ocesses in he plan (Khan e al., 2012). Coloniza ion o bac e ial endophy es is issue- speci ic (Quad -Hallmann e al., 1997). While many endophy ic bac e ia can in ec and colonize he plan issues h ough he oo s and mo e up o he s ems (Compan e al., 2008, 2013), some endophy es a e known o pene a e he lea es o he plan , possibly h ough s oma a (Compan e al., 2010). In addi ion, e ical ansmission o endophy es has also been demons a ed by he ac ha colonized seeds can be a majo sou ce o he plan ’s endomic obiome (T uyens e al., 2015). The use o endophy es o disease biocon ol has been pos ula ed in di e se symbiosys ems and he e ec i eness o endophy es o plan p o ec ion and plan g ow h p omo ion has been demons a ed (Me cado-Blanco and Lug enbe g, 2014). Howe e , he ansmission o plan s o bene icial bac e ia by insec s is s ill poo ly unde s ood. E idence sugges s he ansmission o endosymbion s o S. i anus (namely Asaia sp. and Ca dinium sp.) h ough eeding (Gonella e al., 2015). These mic oo ganisms can also be ans e ed om insec o insec by he ene eal ou e, du ing copula ion and hen om insec o plan by eeding. Whe he o no hese symbion s can su i e as endophy es o plan s is s ill unclea . In addi ion, epo s show he ho izon al ansmission o a common bac e ial endophy e, Me hylobac e ium mesophilicum, o Ca ha an hus oseus plan s h ough he lea hoppe Bucephalogonia xan hophis (Gai e al., 2009). In his wo k, he bac e ium isola ed as an endophy e om ci us plan s was ans o med wi h an enhanced G een Fluo escen P o ein (eGFP)-encoding plasmid, and hen ans e ence expe imen s we e se up whe e he bac e ium was acked wi h he eGFP signal inside he plan s and in he insec . The ansmission o endophy es by insec s is a p omising subjec o s udy, no only because i may allow he econs uc ion o an impo an s ep in hei ecology, bu also because i may enable he e icien deli e y o bene icial mic oo ganisms o c ops. Fo his eason, he aim o his wo k was o assess he ansmission by S. i anus o he endophy ic bac e ial communi y om g ape ine plan s na u ally colonized by endophy es o mic op opaga ed, bac e ia- ee g ape ine plan le s. Using 454 sequencing and qPCR assis ed acking o endophy es, he s uc u e o he endophy ic bac e ial communi y was elucida ed. In addi ion, he ole o S. i anus as ec o o bac e ial endophy ic communi ies was es ablished. Mo eo e , he e ec o plan and insec hos s in endophy ic communi y s uc u e e ealed in e ac ions in he i-pa i e sys em (sou ce plan , sink plan and insec ec o ). MATERIALS AND METHODS Plan Ma e ial Fou 2-yea -old g ape ine (Vi is ini e a L.) plan s (c . Pino noi g a ed on Kobe 5BB) we e g own unde g eenhouse- con olled condi ions a 24 ±1◦C, 70 ±10% ela i e humidi y (RH) and a pho ope iod o 16L:8D h. Plan s we e g own in po s on an o ganic plan subs a e and we e no ea ed F on ie s in Mic obiology | www. on ie sin.o g 2May 2017 | Volume 8 | A icle 834 Lòpez-Fe nàndez e al. An Insec T ans e s Bac e ial Endophy es in G ape ine wi h any pes icides o he en i e cou se o he expe imen s. These plan s a e he ea e e e ed o as “sou ce” (SRC), since hey hos he ypical complex mic obial communi y o plan s g own unde na u al condi ions (Campisano e al., 2014a). A o al o 35 in i o axenically mic op opaga ed g ape ine plan le s c Pino noi clone I-SMA 185 Co e —[Associazione cos i u o i i icoli i aliani (ACOVIT); Co e i, 1992] we e p epa ed. Homogeneous (mean weigh =0.409 g; s anda d de ia ion =0.058 g) and coe al heal hy plan le s wi h a leas h ee lea es we e selec ed o he expe imen s. B ie ly, he plan le s we e mic op opaga ed in cylind ical glass ubes on comple e Mu ashige-Skoog (MS) medium pH 5.6 supplemen ed wi h 3% suc ose and 0.6% mic oaga (Duche a biochemie, The Ne he lands). Explan s wi h one node and in e node we e incuba ed in a g ow h chambe o 51 days a 21 ±1◦C, 16L:8D h pho ope iod and a pho on i adiance o 50 µm s/m2. These in i o plan le s a e he ea e e e ed o as “sink” plan s (SNK) and hey ep esen he plan s whe e he bac e ial communi y will be deli e ed. To u he exclude any bac e ial p esence in he issues, 10 o hese 35 SNK we e used as con ols. Insec s S. i anus eggs o igina ed om 2-yea -old g ape ine canes collec ed om o ganic a ms in No he n I aly (Villazzano, T en o, I aly, 46◦05′N, 11◦14′E) du ing he i s week o Decembe 2014 and s o ed in a cool chambe (4 ±1◦C). S a ing om he beginning o Ap il 2015, bundles o canes (0.5 kg) we e weekly placed inside plas ic boxes con aining humid Pe li e (Pe li ech, I aly) in a clima e chambe (24 ± 1◦C, 16L:8D h pho ope iod, 75% RH) whe e, a e 30–60 days, eggs g adually ha ched. F eshly ha ched nymphs (IN) we e emo ed daily and gen ly ans e ed o a SRC using a suc ion aspi a o . Expe imen al Design The ansmission expe imen (Figu e 1) was ca ied ou independen ly ou imes, using new plan s and insec s. Fou SRC we e kep unde cons an en i onmen al condi ions as men ioned abo e. Then, 96 IN we e placed and con ined on o ou well de eloped SRC lea es, whe e es ic ed a eas we e delimi ed by small cages ( ou cages pe plan , wi h six INs each o a o al o 24 insec s pe plan ) made ou o a mesh slee e (250 µm mesh size) and a suppo ing plas ic cylind ical s uc u e (ø = 10 cm; h =20 cm). The IN we e le eed and g ow o 14 days un il hey eached a s age be ween he hi d and ou h nymphal ins a . Then, ou o he 96 IN p e iously ans e ed, 48 indi iduals we e collec ed om he SRC (12 IN pe SRC) and ans e ed o 16 SNK ( ou SNK wi h h ee IN pe each SRC). In addi ion, an insec - ee SNK pe each SRC was included as a s e ili y con ol o he eplica e. Be o e ans e ing he IN, su ace o he MS medium suppo ing he mic op opaga ed SNK was o e laid wi h 1 ml o s e ile mel ed pa a in (Sigma-Ald ich, Ge many) in o de o p e en he con amina ion o he g ow h medium and he oo s by mic oo ganisms ca ied by IN. In his way, we could assu e ha no con ac be ween he oo s o he g ow h medium was aking place. Once ans e ed o he SNK, IN we e allowed o eed o 10 days a 21◦C, 16L:8D h pho ope iod and a pho on i adiance o 50 µm s/m2un il hey we e i h ins a nymphs o adul s. As con ol, i e SNK (CTRLSNK) we e each in es ed wi h i e eshly ha ched nymphs (CTRLIN) ha had no been p e iously ea ed on SRC, bu we e eeding only on SNK (Figu e 1B). In addi ion, om he emaining en SNK, i e we e used o p obe o bac e ial DNA in he plan ’s issues. To al plan DNA was ex ac ed using he me hod p e iously desc ibed (Campisano e al., 2014a) and he ex ac ed DNA was ampli ied using he p ime pai 799F/1520R (Yousa e al., 2014). Since he i e es ed plan s we e PCR nega i e (no ampli ica ion o bac e ial 16S DNA gene), hey we e conside ed bac e ia- ee. Al hough p ime s 799F FIGURE 1 | Expe imen al design. (A) Se up o endophy e ansmission expe imen s h ough Scaphoideus i anus—Tes s. (1) Sou ce plan s (SRC) we e in es ed wi h insec s (IN); (2) insec s we e placed on sink plan s (SNK); (3) insec s and sink plan s we e incuba ed; (4) su aces o insec s we e washed (INSRUF); (5) insec s, oo s (ROOTSNK), and s ems (STEMSNK) o sink plan s we e sepa a ed. Su ace s e iliza ion was pe o med be o e each DNA ex ac ion s ep. (B) Se up o endophy e ansmission expe imen s h ough S. i anus—Con ols. (1) Con ol insec s (CTRLIN) we e le o ha ch on g ape ine unks; (2) con ol insec s we e placed in con ol sink plan s (CTRLSNK); (3) con ol insec s and con ol sink plan s we e incuba ed, (4) con ol insec s, con ol oo s (CTRLROOT), and con ol s ems (CTRLSTEM) we e sepa a ed. Su ace s e iliza ion was pe o med be o e each DNA ex ac ion s ep. F on ie s in Mic obiology | www. on ie sin.o g 3May 2017 | Volume 8 | A icle 834 Lòpez-Fe nàndez e al. An Insec T ans e s Bac e ial Endophy es in G ape ine and 1520R a e a uni e sal pai o 16S DNA ampli ica ion, s ill some p oka yo es migh no ha e been de ec ed. Thus, he las i e SNK plan le s we e used o con ol mic obial con amina ion inside he issues. SNK we e incuba ed unde he same condi ions wi hou IN. Then, plan s we e c ushed in a s e ile mo a wi h 1 ml phospha e bu e saline 1X, pH 7.2, and he esul ing ex ac was pla ed on Lu ia-Be ani aga (LBA; Sigma Ald ich, Ge many) and incuba ed a 30◦C o 5 days, a e which no g ow h was eco ded. A e he incuba ion pe iod, all SNK and IN we e asep ically emo ed om he glass ubes. SNK we e cu in o s ems (STEMSNK) and oo s (ROOTSNK); CTRLSNK we e likewise cu in o CTRLROOT and CTRLSTEM samples. IN we e washed wi h dis illed s e ile wa e by ho oughly o exing in o de o dislodge he majo i y o su ace-adhe ing bac e ia. The bac e ial cells in he washing wa e (INSURF) we e pelle ed by cen i uga ion a 13,000 pm on a able op cen i uge and s o ed a −20◦C be o e ex ac ing he DNA. All SRC, IN, CTRLIN, STEMSNK, ROOTSNK, CTRLSTEM, and CTRLROOT we e hen su ace-s e ilized by successi e washing in 98% e hanol o wo min, 4% sodium hypochlo i e o 2 min and 70% e hanol o 2 min as desc ibed p e iously (Panche e al., 2012), and hen insed h ee imes wi h dis illed s e ile wa e . The wa e om he inal washing s ep o all samples was pla ed on LBA and incuba ed o 5 days a 30◦C o check o mic obial g ow h as a p oxy o su ace disin ec ion e icacy. DNA Ex ac ion, 16S DNA Ampli ica ion, and Py osequencing A e s e iliza ion, SRC, IN, CTRLIN, STEMSNK, ROOTSNK, CTRLSTEM, and CTRLROOT we e asep ically ans e ed o s e ile s ainless s eel capsules con aining s eel beads. The ma e ial was ozen in liquid ni ogen o 5 min and c ushed in a Re sch MM200 issue lyse (Qiagen, The Ne he lands) o 2 min a a equency o 25 he z. The esul ing powde was weigh ed and hen deoxy ibonucleic acids we e ex ac ed using he Fas DNATM SPIN Ki o Soil (MP, Uni ed S a es) acco ding o manu ac u e ’s ins uc ions. DNA om he INSURF samples was ex ac ed wi h he same ki a e pelle ing and suspending he cells in ex ac ion bu e be o e wo kup. DNA was hen quan i ied in an UV-VIS nanod op 8,000 spec opho ome e (The mo Fische Scien i ic, Uni ed S a es) and PCR-ampli ied using he p ime pai 799F (AACMGGATTAGATACCCK) and 1520R (AAGGAGGTGATCCAGCCGCA) a ge ing he V5–V9 16S DNA hype a iable egions wi hou ampli ica ion o plas id DNA. These p ime s bea 454 adap o s and a sample-speci ic ba code on he o wa d p ime . PCR was pe o med using he Roche high ideli y Fas S a PCR sys em (Roche, Swi ze land) in a inal olume o 25 µl. The ollowing olumes, eagen s and concen a ions we e used: 2.5 µl ampli ica ion bu e 10X, 5 µl MgCl225 mM, 0.5 µl e e se p ime 10 µM, 0.5 µl o wa d p ime 10 µM, 2.5 µl dNTPs 25 mM, 1 µl DMSO, 2.5 Bo ine se um albumin (BSA) 10 mg/ml, 0.4 µl HI-FI Taq polyme ase 5 U/µl and wa e . DNA was adjus ed o an ini ial concen a ion o 3 ng/µl and o some samples dilu ions o 1:10 we e used in o de o ob ain op imal ampli ica ion. Thi y cycles o PCR we e ca ied ou acco ding o he manu ac u e ’s ins uc ions wi h condi ions o ampli ica ion as ollows: 5 min o ini ial dena u a ion a 95◦C, 30 s a 95◦C, 1 min o annealing a 53◦C, 2 min o ex ension a 72◦C, and a inal ex ension s ep 10 min a 72◦C. PCR p oduc s we e sepa a ed in a 1.5% aga ose gel s ained wi h SYBR R Sa e DNA Gel S ain (The mo Fishe Scien i ic, Uni ed S a es), and isualized on a Gel Doc XR+ sys em (BiO-RAD, Uni ed S a es). The app op ia e ampli ica ion bands we e excised om he gel. DNA was eco e ed using he Pu eLink Quick gel ex ac ion Ki (The mo Fishe Scien i ic, Uni ed S a es) acco ding o manu ac u e ’s ins uc ions. Th ee di e en ampli ica ions o each sample we e pe o med and he PCR p oduc s we e pu i ied om gel and pooled oge he o py osequencing. Amplicons we e quan i ied wi h quan i a i e PCR using he lib a y quan i ica ion ki Roche 454 Ti anium (KAPA Biosys ems, Uni ed S a es) and pooled in equimola a io in he inal amplicon lib a y. Py osequencing was ca ied ou on he Roche GS FLX+sys em using he new XL+ chemis y dedica ed o long eads o up o 800 bp, ollowing he manu ac u e ’s ecommenda ions. Bac e ial 16S DNA Amplicon Demul iplexing and S a is ical Analysis Ou pu s om he 454 py osequencing we e analyzed using he “Quan i a i e Insigh s in o Mic obial Ecology (QIIME)” pipeline, e sion 1.9.0 (Capo aso e al., 2010b). The analysis consis ed o decoding he sequence lowg am iles (SFF) and p oducing as a and quali y iles wi h which leng h o sequences and quali y o eads we e checked. Amplicon sequences we e demul iplexed (assigned o sample pools) acco ding o hei ba coded p ime . Only bac e ial sequences a leas 300 n long we e e ained. Sequences we e unca ed when he quali y sco e in a 50 n long sliding window wen below 25. Chime ic PCR p oduc s we e iden i ied using USEARCH 6.1.544 (Edga , 2010). Ope a ional axonomic uni s (OTUs) we e picked using a h eshold iden i y o 97% and he G eengenes da abase, Augus 2013 e sion (DeSan is e al., 2006). USEARCH clus e seeds we e used as ep esen a i es o OTUs, while axonomy was assigned using USEARCH and he G eengenes da abase as a empla e. Sequences assigned o chlo oplas s and mi ochond ia we e emo ed. OTUs ep esen ed by only one o wo eads (single ons and double ons) we e emo ed om he OTU ables. The amplicons we e hen aligned de no o using pynas (Capo aso e al., 2010a) and he alignmen was used o gene a e a phylogene ic ee. F om py osequencing we ob ained 1,404,963 eads om he whole se o samples, wi h a median o 13,271 eads pe sample. A e he i s quali y con ol s eps whe e we emo ed sho sequences (<200 n ), mis-sequenced agmen s and mu a ed amplicons, only 1,024,657 sequences we e le . Following emo al o chime ic sequences using he Usea ch algo i hm, 871,497 emained as non-chime ic sequences. He e, a maximum o 31,039 sequences o IN samples and a minimum o 57 sequences o CTRLIN samples we e ob ained, and a mean o 12,939 sequences o all he samples. F on ie s in Mic obiology | www. on ie sin.o g 4May 2017 | Volume 8 | A icle 834 Lòpez-Fe nàndez e al. An Insec T ans e s Bac e ial Endophy es in G ape ine Fo clus e ing OTUs, we picked a ep esen a i e se o sequences ha u he ep esen ed he OTU wi h 97% accu acy, esul ing in 2,005 g ouped sequences a ailable o analysis. Some o he sequences ob ained we e ound o be o plan na u e (plas id sequences) and we e emo ed, leading o a inal coun o 1,923 sequences. F om hose, we emo ed he sequences ha we e ep esen ed in <1% o he o al popula ion, ob aining an OTU able wi h a o al o 447 OTUs ha we e de ined as clus e s composed o h ee o mo e sequences. Alpha- and be a-di e si y we e es ima ed on mul iple OTU ables a e ied o 1,300 eads (conside ing he sample wi h he lowes numbe o eads). Alpha-di e si y di e ences we e es ed o s a is ical signi icance using 999 Mon e Ca lo pe mu a ions and he p- alue ob ained co ec ed using he Bon e oni co ec ion o mul iple compa isons. Be a-di e si y was compu ed using he phylogene ic unweigh ed UniF ac dis ances. PCoA plo s ende ing sample dis ances we e isualized using Empe o and u he d awn in R. A K uskal–Wallis es was used o assess i he di e en ial dis ibu ion o OTUs and axa was s a is ically signi ican o all he a iables analyzed. The mul i a ia e es ANOSIM o de ec di e ences be ween g oups o samples was used as implemen ed in QIIME. T ansmission and Quan i ica ion o Endophy es h ough qPCR To quan i y he bac e ia ans e ed by S. i anus ac oss plan s, we used a simila se ing o he one desc ibed abo e. In his case, he sou ce o inoculum is no he SRC, bu a bac e ial cell suspension o cul i able endophy es isola ed om g ape ine unks in a p e ious wo k. These bac e ia we e classi ied as En e obac e ludwigii EnVs6, E. ludwigii EnVs2, and Pan oea agans PaV 9 (Campisano e al., 2015; Lòpez-Fe nàndez e al., 2015). B ie ly, hese bac e ial endophy es we e ans o med wi h he eGFP encoding plasmid pMP4655 (Bloembe g e al., 2000) as ollows: bac e ia we e g own on LBA o 48 h a 30◦C. Then, 2 ml o supe op imal b o h amended wi h suc ose (SOC) we e inocula ed wi h a single colony and incuba ed o 24 h a 30◦C and 160 pm (Hanahan, 1983). Aliquo s o 400 µl o his s a e cul u e we e inocula ed in o 40 ml o SOC b o h and hen incuba ed o u he 24 h a 30◦C and 160 pm. Cells we e hen cen i uged a 45,895 pm o 15 min a 4◦C, and subsequen ly suspended in elec opo a ion bu e (glyce ol 10%, dis illed s e ile wa e main ained a <4◦C) o plasmid inse ion in o he bac e ial cells. Th ee washing s eps we e pe o med wi h elec opo a ion bu e , educing in hal es he esuspension olume. A he end, aliquo s o 50 µl o bu e ed imme sed (compe en ) bac e ia we e dispensed in ubes and kep a −80◦C. Bac e ial cells we e hen gen ly mixed wi h 1 µg o he plasmid and incuba ed on ice o 30 min. La e , he mix u e was ans e ed o 0.2 cm elec opo a ion cu e es (Bio ad, Uni ed S a es) and elec opo a ed a 1,500 mV, 25 µF, and 200 . Cells we e immedia ely imme sed in 800 µl o SOC and incuba ed a 30◦C and 160 pm o 2 h. Cul u es we e cen i uged and hal o he olume disca ded. Then, cells we e suspended in he emaining olume and pla ed on o LBA supplemen ed wi h e acycline (20 µg/ml). T ans o man s we e con i med by ampli ying he esis ance ma ke casse e e A/R, p esen in he plasmid, wi h p ime s di ec ed owa d he gene, as p e iously epo ed (Mølle e al., 2016). Endophy ic cells bea ing he pMP4655 we e g own on LB o 24 h and cell densi ies we e adjus ed o 3 ×107CFU/ml. Then, cells we e cooled down on ice and washed h ee imes wi h PBS 1X, pH 7.2. A e he las washing s ep, cells we e e- suspended in 200 µl o a T is-EDTA-suc ose pH 8.0 solu ion (TES: T is 10 mM, EDTA 1 mM, suc ose 5% w/ ) and dis ibu ed in he lids o bo omless ( eplaced by a co on plug) 1.5 ml plas ic ubes (Eppendo , Ge many). Lids we e co e ed wi h one laye o s e ile pa a ilm (Bemis NA, Uni ed S a es). S. i anus indi iduals we e ea ed as desc ibed abo e. Insec s we e ans e ed o plas ic ubes wi h he lids hanging upside down, and le o eed on he eGFP- agged bac e ia o 5 days. In his se ing, insec s punched he pa a ilm laye on he lid, eleasing and eeding om he bac e ia- ich TES solu ion. A e eeding, insec s we e ans e ed o in i o mic op opaga ed g ape ine plan le s, as desc ibed in he expe imen s abo e. Pe each bac e ium es ed we pe o med one se o ou eplica es consis ing o ou plan s in es ed wi h h ee insec s pe plan plus one nega i e con ol whe e no insec s we e ans e ed. Replica es we e incuba ed o 5 days. A he end o he incuba ion, he insec s, oo s and s ems we e collec ed sepa a ely. DNA om he insec s and plan s was ex ac ed using he NucleoSpin R Plan II ki ( o ROOTNSK, STEMSNK, CTRLROOT, and CTRLSTEM) and he Nucleospin R Tissue ( o IN and CTRLIN) acco ding o manu ac u e ins uc ions (Mache ey-Nagel, Ge many). DNA was quan i ied as desc ibed abo e. Bac e ial DNA, including he pMP4655 eGFP encoding plasmid, was quan i ied on a Roche Ligh Cycle R 480 Real- Time PCR (Roche, Swi ze land) wi h he pla inum SYBR G een qPCR supe Mix-UDG (The mo Fishe Scien i ic, Uni ed S a es). The ollowing ampli ica ion p o ocol was used: 1 hold a 50◦C ac i a ion (UDG incuba ion) o 5 min, 1 hold a 95◦C o 5 min ac i a ion, 40 cycles a 95◦C o 30 s o mel ing and a 58◦C o 45 s o annealing and ex ension. An analysis o mel ing cu es a 95◦C o 5 s, ollowed by a down un il 55◦C o 1 min, was pe o med o check o speci ici y o he eac ion. Absolu e quan i ica ion o eGFP gene copies in plan s and insec s was done based on in e pola ion om a s anda d cu e ob ained wi h se ial 10- old dilu ions o he eGFP gene ( om 3 ×106 o 3 ×101eGFP gene copies/µl) in DNA o con ol plan s o insec s, espec i ely. Nucleo ide Sequence Accession Numbe s The sequencing ou pu is deposi ed a he Eu opean Nucleo ide A chi e (ENA a h ps://www.ebi.ac.uk), and can be ound unde he accession numbe ERS1629270, s udy name PRJEB20051. RESULTS Ou da a show ha he whole mic obial communi ies li ing on he plan a e anspo ed be ween plan s by insec ec o s. By eeding and ouching he plan , insec s acqui e a se o mic oo ganisms ha adically di e s om hose hey ha e a F on ie s in Mic obiology | www. on ie sin.o g 5May 2017 | Volume 8 | A icle 834 Lòpez-Fe nàndez e al. An Insec T ans e s Bac e ial Endophy es in G ape ine ha ching. Insec s a e able o ca y and ans e his se o o he plan s hey dwell and eed upon. S uc u e o he Communi y in he Tes ed G ape ine Symbiosys em The ela i e abundances o bac e ial phyla a ied be ween hos s. The con ol samples had a dis inc i ely di e en species composi ion han he es samples (Figu e 2 and Table 1). In he SRC, he bac e ial communi y was mainly composed o P o eobac e ia, whe e he mos abundan classes we e Be a-, Gamma-, and Alpha-p o eobac e ia. The es o he communi y was composed o Ac inobac e ia (a majo i y in he class Ac inobac e ia), Fi micu es (wi h mos membe s a ilia ed wi h he class Bacilli), Bac e oide es ( he classes Sphingobac e iia and Spi ospi ae made he majo i y o he phylum) and, in a smalle p opo ion, Acidobac e ia ( ep esen ed only by he class Solibac e es), and Chlamydiae ( ep esen ed only by he class Chlamydiia). Only a small ac ion o he OTUs could no be assigned o any pa icula axon. The bac e ial communi y o IN was composed mos ly o P o eobac e ia ollowed by Fi micu es, Ac inobac e ia, Bac e oide es, and Acidobac e ia, he la e ep esen ing he leas abundan phylum. The INSURF communi y was simila o he inne bac e ial mic obio a o IN wi h he majo i y o OTUs assigned o P o eobac e ia, ollowed by Fi micu es, Ac inobac e ia, Bac e oide es, and Acidobac e ia. In ou analysis, only one OTU was exclusi ely associa ed wi h he INSURF samples. Using Basic Local Alignmen Sea ch Tool (BLAST), his sequence was assigned o he Sinobac e aceae, a amily ha includes he closely ela ed wa e -sp ing associa ed bac e ium Ne skia sp. This sequence was ne e de ec ed in any plan sample o inside he insec s. Mic obio a o he SNK was mos ly composed o P o eobac e ia, wi h Be a- and Gamma-p o eobac e ia being he mos abundan classes. Del ap o eobac e ia we e also p esen and he es o he phyla had only ew ep esen a i es (Table 1). A u he analysis o he endophy ic communi y composi ion in he abo e- and below-g ound compa men s e ealed di e ences in he wo plan compa men s. P o eobac e ial OTUs in STEMSNK we e highly ep esen ed (in o de o abundance: Be ap o eobac e ia, Gammap o eobac e ia, Alphap o eobac e ia, and Del ap o eobac e ia). Ac inobac e ia, Fi micu es, Acidobac e ia, Bac e oide es, and Chlamydiae we e less abundan . In ROOTSNK, P o eobac e ia we e also he mos abundan OTUs (in o de o abundance: Be a-, Gamma-, Alpha-, and Del a-p o eobac e ia). Ac inobac e ia, Fi micu es, Acidobac e ia, Bac e oide es, Chlamydia, and he candida e clade TM6 we e he leas abundan . In con as , he bac e ial communi y o con ol plan s (CTRLROOT, CTRLSTEM, whe e eshly ha ched insec s had ed wi hou p io con ac wi h SRC) was domina ed by Ac inobac e ia, wi h only a small p opo ion o P o eobac e ia (Be a- and Gamma-, bu no Alpha- p o eobac e ia). The communi y in CRTLIN was also domina ed by Ac inobac e ia. Selec ed Endophy es A e T ansmi ed be ween G ape ine Plan s Fo y OTUs we e ans e ed by IN om SRC o SNK and ound bo h in ROOTSNK and in STEMSNK (Table 2). These we e ne e ound in he CTRLROOT o CTRLSTEM, sugges ing ha hey we e e icien ly ansmi ed om SRC o SNK by IN. In pa icula , among he sequences FIGURE 2 | Rela i e abundance o OTUs assigned a he phylum le el. S acked ba plo s ep esen he pe cen ages o 216 membe s o he OTU able in biom o ma . In he legend, unassigned co espond o OTUs whose axonomy could no be cla i ied a he 97% con idence using he G eengenes da abase. F on ie s in Mic obiology | www. on ie sin.o g 6May 2017 | Volume 8 | A icle 834 Lòpez-Fe nàndez e al. An Insec T ans e s Bac e ial Endophy es in G ape ine TABLE 1 | Rela i e abundance o OTUs in he symbiosys em S. i anus—g ape ine, assigned a phylum and class le el. Phylum Class SRC IN STEMSNK ROOTSNK INSURF CTRLIN CTRLTROOT CTRLSTEM Unassigned Unassigned 4.6 0.9 2.4 1.5 4.0 0.0 0.0 0.1 Acidobac e ia Acidobac e iia 0 0.1 0 0.2 0 0 0 0 DA052 clade 0.3 0.1 0.8 0.5 0.4 0 0 0 Solibac e es 0 0 0 0 0 0 0 0 To al 0.3 0.2 0.8 0.7 0.4 0.0 0.0 0.0 Ac inobac e ia Ac inobac e ia 5.3 4.6 19 13.1 8.2 99.5 94.5 98 The moleophilia 0 0 0.1 0 0 0 0 0 To al 5.3 4.6 19.1 13.1 8.2 99.5 94.5 98.0 Bac e oide es Bac e oidia 0.1 0 0 0.1 0 0 0 0 Cy ophagia 0 0.1 0.1 0 0 0 0 0 Fla obac e iia 0 0 0 0 0 0 0 0 Sphingobac e iia 0.5 0.2 0 0.1 0 0 0 0 Sap ospi ae 0.5 1 0.1 0.4 0.4 0 0 0 To al 1.1 1.3 0.2 0.6 0.5 0.0 0.0 0.0 Chlamydiae Chlamydiia 0.1 0 0.1 0.1 0.1 0 0 0 To al 0.1 0.0 0.1 0.1 0.1 0.0 0.0 0.0 Elusimic obia Elusimic obia 0 0 0 0 0.1 0 0 0 To al 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 FBP FBP 0 0 0 0 0 0 0 0 To al 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Fi micu es Bacilli 4.5 21.5 12.7 9.7 27.7 0 0 0.1 Clos idia 0.1 0.6 0.1 0.2 0.4 0 0 0 To al 4.6 22.1 12.8 10.0 28.1 0.0 0.0 0.1 Fusobac e ia Fusobac e iia 0 0 0 0 0.1 0 0 0 To al 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.0 Gemma imonade es Gemma imonade es 0 0 0 0 0 0 0 0 To al 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 P o eobac e ia Alpha 13.9 2.7 5.7 4.4 2.1 0.2 0.2 0.1 Be a 36.6 29.5 40.9 45.9 31.8 0.2 4.9 0.1 Del a 0.7 0.3 0.3 0.4 0.5 0 0 0 Gamma 32.7 38.2 17.3 23 24.1 0 0.3 1.7 To al 83.9 70.6 64.3 73.8 58.5 0.4 5.4 1.9 SBR1093 VHS-B5-50 0 0 0 0 0 0 0 0 To al 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 TM6 SJA-4 0 0 0 0.1 0 0 0 0 To al 0.0 0.0 0.0 0.1 0.0 0.0 0.0 0.0 The mi Deinococci 0 0.1 0.1 0 0 0 0 0 To al 0.0 0.1 0.1 0.0 0.0 0.0 0.0 0.0 o he main phyla (i.e., P o eobac e ia, Ac inobac e ia, Bac e oide es, Chlamydiae, and Fi micu es), P o eobac e ia we e he mos highly ep esen ed axon, whe e he mos abundan gene a we e Ag obac e ium,Pa acoccus, Sphingomonas, E winia,Pseudomonas,Lysobac e , and S eno ophomonas. In con as , he mos abundan phyla in CTRLIN we e Ac inobac e ia, especially he gene a Mycobac e ium, F on ie s in Mic obiology | www. on ie sin.o g 7May 2017 | Volume 8 | A icle 834 Lòpez-Fe nàndez e al. An Insec T ans e s Bac e ial Endophy es in G ape ine TABLE 2 | OTUs ansmi ed om sou ce (SRC) o sink plan s (STEMSNK and ROOTSNK) by S. i anus. OTU Phylum Class O de Family Genus Species 1 Ac inobac e ia Ac inobac e ia Ac inomyce ales Unclassi ied Unclassi ied Unclassi ied 2 Ac inobac e ia Ac inobac e ia Ac inomyce ales Co ynebac e iaceae Co ynebac e ium du um 3 Ac inobac e ia Ac inobac e ia Ac inomyce ales Geode ma ophilaceae Unclassi ied Unclassi ied 4 Ac inobac e ia Ac inobac e ia Ac inomyce ales Mic ococcaceae Kocu ia palus is 5 Ac inobac e ia Ac inobac e ia Ac inomyce ales Mic ococcaceae Mic ococcus lu eus 6 Ac inobac e ia Ac inobac e ia Ac inomyce ales Noca dioidaceae Unclassi ied Unclassi ied 7 Ac inobac e ia Ac inobac e ia Ac inomyce ales P opionibac e iaceae P opionibac e ium Unclassi ied 8 Bac e oide es Fla obac e iia Fla obac e iales Fla obac e iaceae Fla obac e ium Unclassi ied 9 Bac e oide es Sap ospi ae Sap ospi ales Chi inophagaceae Sediminibac e ium Unclassi ied 10 Chlamydiae Chlamydiia Chlamydiales Pa achlamydiaceae Unclassi ied Unclassi ied 11 Fi micu es Bacilli Bacillales Bacillaceae Bacillus lexus 12 Fi micu es Bacilli Bacillales S aphylococcaceae S aphylococcus au eus 13 Fi micu es Clos idia Clos idiales Tissie ellaceae Anae ococcus Unclassi ied 14 P o eobac e ia Alphap o eobac e ia Rhizobiales Phyllobac e iaceae Unclassi ied Unclassi ied 15 P o eobac e ia Alphap o eobac e ia Rhizobiales Rhizobiaceae Unclassi ied Unclassi ied 16 P o eobac e ia Alphap o eobac e ia Rhizobiales Rhizobiaceae Ag obac e ium Unclassi ied 17 P o eobac e ia Alphap o eobac e ia Rhodobac e ales Rhodobac e aceae Pa acoccus Unclassi ied 18 P o eobac e ia Alphap o eobac e ia Rhodospi illales Rhodospi illaceae Unclassi ied Unclassi ied 19 P o eobac e ia Alphap o eobac e ia Ricke siales Unclassi ied Unclassi ied Unclassi ied 20 P o eobac e ia Alphap o eobac e ia Sphingomonadales Sphingomonadaceae Unclassi ied Unclassi ied 21 P o eobac e ia Alphap o eobac e ia Sphingomonadales Sphingomonadaceae Kais obac e Unclassi ied 22 P o eobac e ia Alphap o eobac e ia Sphingomonadales Sphingomonadaceae Sphingomonas Unclassi ied 23 P o eobac e ia Be ap o eobac e ia Bu kholde iales Alcaligenaceae Ach omobac e Unclassi ied 24 P o eobac e ia Be ap o eobac e ia Neisse iales Neisse iaceae Unclassi ied Unclassi ied 25 P o eobac e ia Be ap o eobac e ia Neisse iales Neisse iaceae Unclassi ied Unclassi ied 26 P o eobac e ia Be ap o eobac e ia Neisse iales Neisse iaceae Kingella Unclassi ied 27 P o eobac e ia Be ap o eobac e ia Neisse iales Neisse iaceae Neisse ia Unclassi ied 28 P o eobac e ia Be ap o eobac e ia Neisse iales Neisse iaceae Neisse ia cine ea 29 P o eobac e ia Del ap o eobac e ia Unclassi ied Unclassi ied Unclassi ied Unclassi ied 30 P o eobac e ia Del ap o eobac e ia Myxococcales 0319-6G20 Unclassi ied Unclassi ied 31 P o eobac e ia Gammap o eobac e ia Al e omonadales Al e omonadaceae Ma inobac e Unclassi ied 32 P o eobac e ia Gammap o eobac e ia En e obac e iales En e obac e iaceae Unclassi ied Unclassi ied 33 P o eobac e ia Gammap o eobac e ia En e obac e iales En e obac e iaceae E winia Unclassi ied 34 P o eobac e ia Gammap o eobac e ia Legionellales Unclassi ied Unclassi ied Unclassi ied 35 P o eobac e ia Gammap o eobac e ia Pseudomonadales Pseudomonadaceae Pseudomonas ni o educens 36 P o eobac e ia Gammap o eobac e ia Xan homonadales Sinobac e aceae Unclassi ied Unclassi ied 37 P o eobac e ia Gammap o eobac e ia Xan homonadales Xan homonadaceae Lu eimonas Unclassi ied 38 P o eobac e ia Gammap o eobac e ia Xan homonadales Xan homonadaceae Lysobac e Unclassi ied 39 P o eobac e ia Gammap o eobac e ia Xan homonadales Xan homonadaceae S eno ophomonas Unclassi ied 40 TM6 SJA-4 Unclassi ied Unclassi ied Unclassi ied Unclassi ied Go donia,Noca dia,Rhodococcus, and Williamsia. CTRLROOT and CTRLSTEM hos ed a communi y ha esembled ha o he CTRLIN (Table 1). Fo example, he genus Noca dia was de ec ed in all sample ypes, bu i s p e alence was lowes in CTRLROOT samples. Likewise, Rhodococcus and Ae omic obium we e less abundan in CTRLROOT and CTRLSTEM han in CTRLIN. An excep ion was he genus Williamsia, which was mo e abundan in CTRLROOT and CTRLSTEM han in CTRLIN. Endophy ic Communi ies Mo e om S ems o Roo s a e he T ans e P ocess To iden i y coloniza ion dynamics o ansmi ed endophy es, we compa ed he communi y in abo e- and below-g ound pa s o he plan (ROOTSNK s. STEMSNK). In ou expe imen al se up, plan oo s we e ex e nally sepa a ed om he s ems by a pa a in laye placed o a oid su ace con amina ion o he syn he ic medium. Ou da a hus indica e ha se e al OTUs we e de ec ed bo h in STEMSNK and ROOTSNK, including S ep ococcus, S e oidobac e ,Rals onia,Pseudomonas, and Me hylobac e ium. F on ie s in Mic obiology | www. on ie sin.o g 8May 2017 | Volume 8 | A icle 834 Lòpez-Fe nàndez e al. An Insec T ans e s Bac e ial Endophy es in G ape ine ROOTSNK had mo e P o eobac e ia han STEMSNK, while Ac inobac e ia we e mo e abundan in STEMSNK han in ROOTSNK. STEMSNK samples we e domina ed by P o eobac e ia (Be a- Gamma-, Alpha-, and Del a-p o eobac e ia). Ac inobac e ia, Fi micu es, Acidobac e ia, Bac e oide es, and Chlamydiae we e he leas abundan phyla. Simila ly, in ROOTSNK P o eobac e ia ep esen ed mos o he communi y, ollowed by he Ac inobac e ia, Fi micu es, Acidobac e ia, Bac e oide es, Chlamydiae, and he candida e phylum TM6. To e i y o insec ansmission, we also e alua ed he endophy ic communi ies in abo e- and below-g ound pa s o con ol sink plan s (CTRLSTEM and CTRLROOT) in con ac only wi h CTRLIN. The bac e ial communi y o CTRLSTEM and CTRLTROOT, whe e eshly ha ched CTRLIN had ed wi hou p io con ac wi h SRC, was domina ed by Ac inobac e ia wi h a small p opo ion o P o eobac e ia (Be a- and Gamma-, bu no Alphap o eobac e ia we e p esen ). Insec s Change he Communi y S uc u e du ing Passage om Sou ce o Sink Plan s To iden i y shi s in bac e ial communi y composi ion in he ans e ing p ocess, we analyzed di e si y o e e y hos ype and compa ed hei signi icance a a la ge scale (highe axonomic hie a chy o phylum le el) and in some cases a a small scale (genus le el). The la ges di e si y was p esen in ROOTSNK ollowed by STEMSNK. The hi d mos di e se mic obio a was ha o SRC ollowed by IN (Figu e 3). In e ms o ichness, a la ge numbe o new species was de ec ed in SRC (obse ed species and Chao 1 index), hough he numbe o species in IN acqui ed du ing eeding was less han a hal o wha i had been in he SRC (Figu e 3 and Supplemen a y Figu e 1). In addi ion, a iance wi hin samples was la ge in IN as compa ed o SRC, sugges ing di e ences in species composi ion in each sample. In STEMSNK he ichness inc eased conside ably mo e han in he below-g ound pa o he plan , wi h a high a iance wi hin samples, sugges ing ha he communi y ha was p e iously s able in SRC was dis u bed in he SNK a e acquisi ion and ansmission by IN. When looking a Shannon-Wiene and Simpson’s indexes, di e si y was ound o be highe in SRC (abundance o new species is la ge and e en) and lowe in IN (Supplemen a y Table 1A). When he communi y was ans e ed o STEMSNK, i s di e si y inc eased. The s anda d a ia ion wi hin samples inc eased om SRC (0.878) o IN (10.52), sugges ing less e enness pe sample. When passing o he SNK, he s anda d a ia ion dec eased (STEMSNK = 10.52 and ROOTSNK =0.430), poin ing o a eco e y o he communi y when mo ing om he insec o he plan hos . Al hough INSURF had ew new species pe sample, he di e si y was nea o ha o he o he samples, hin ing a he possibili y o an inpu o insec su ace-associa ed bac e ia o he endophy ic communi y (Supplemen a y Table 1A). The di e si y o he con ols di e ed om ha o he ea ed samples. The lowes di e si y and ichness we e eco ded o CTRLIN, CTRLROOT, and CTRLSTEM. Ra e ac ion cu e analysis (Supplemen a y Figu e 1) con i med ha he iches samples we e SRC and he compa men s o he sink plan s (STEMSNK and ROOTSNK), ollowed by he INSURF mic obiome and by he inne mic obiome IN. CTRLROOT we e he poo es samples in e ms o new species because no new OTUs being disco e ed a e a sampling e o o 1,505 sequences/sampling. FIGURE 3 | Alpha di e si y measu es on ansmission expe imen s. Alpha di e si y indexes (Richness: Obse ed species, Chao1; Di e si y: Shannon-Wiene , and Simpson) we e calcula ed on he OTU able a he phylum le el. 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Copy igh © 2017 Lòpez-Fe nàndez, Mazzoni, Ped azzoli, Pe o and Campisano. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) o licenso a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Mic obiology | www. on ie sin.o g 17 May 2017 | Volume 8 | A icle 834