Microbial Life Inside Posidonia Seeds: Beneficial Endophytes and Implications for Marine Plant Health

15 P l a n t – m i c r o b e i n t e r a c t i o n s a r e k e y d r i v e r s o f p l a n t h e a l t h a n d e c o s y s t e m f u n c t i o n -16 ing, yet th e ir r oles in m a rine e nvir o n m e nts re m a i n po orly u nders to od. T he seagr ass 17 Po si d on i a oc e a n ic a , a fo un da t i on s p ec ie s i n th e M e di te r r a nean S ea, fo r ms c ompl e x a ss oc i -18 ati ons with m i cr obi a l co m m u nitie s th at infl uen ce its deve l op m e nt a nd stress tolera nc e. 19 H e r e , w e p r o v i d e t h e f i r s t ev i d e n c e o f c u l t u r a b l e b a c t e r i a l a n d f u n g a l e n d o p h y t e s i n h a b -20 iting P. oc e an ic a s e e d s c o l l e c t e d f r o m c e n t r a l M e d i t e r r a n e a n , a r e g i o n r e p r e s e n t i n g a m a -21 jor c enter o f t he spe c i es’ geneti c div ers ity . Usin g t wo dif fe r e nt m a rin e cultu re m e d ia , w e 22 is ola ted a div ers e as se m b l age o f end ophytes, pred om in a ntly a f filiat e d w ith 23 M a ri nom ona s , Ce le rin at a nt imo n as , Vi br io , Ha lom ona s , Ko c u ri a , B ac ill us, Me t a b a c i l l u s , 24 L ysob act er , an d Au re imo n as , al on g with t he f un gi Pae ci lom yc es ma xim us a n d 25 Ha loph ytop htho ra s p. M os t b a c teri al is o l at es displaye d pl ant gro wt h – pr om o ting (PGP) 26 t ra i t s s uch a s i n do l e -3 -ac e t ic aci d pro d uct i o n an d n i tr o ge n f i xa t i o n , su ppo rt i n g t he i r p o -27 t e n t i a l c o n t r i b u t i o n t o s e e d g e r m i n a t i o n a n d e a r l y s e e d l i n g e s t a b l i s h m e n t . T h e d e t e c t i o n 28 of Ca n dida t us C e le r i na tan t imona s ne p tuna , a n i t ro g en - f i xin g s ymb io n t p re v i ou s l y d e -29 sc ri b e d in P. o ce a nic a r oo t s, suggests a p oss ible r oute of verti c al tr a ns m ission. Altho ugh 30 f u ngal end op hytes we r e less f re q u e nt, th e ir pr e s e n c e in di c a t e s t hat P. oc e a n i c a s e e d s m a y 31 s e r ve a s a reser voir o f both bene fi c i al a nd pot enti a lly p at hog e ni c t axa . T hese findings 32 exp and o ur u nders t anding of th e P. o c e anic a h o l o b i o n t , h i g h l i g h t t h e r o l e o f s e e d s i n t h e 33 pers isten c e a nd diss emi n atio n o f endo p hyti c co m m u nitie s a nd l a y t he gr ou nd w ork f or 34 the b iot e chn ol ogi cal us e o f s e ed- associ a ted mi c r ob es i n m ari ne pl a nt rest o r a ti o n a nd 35 c o nserv ati on, a nd i n c r op str e s s tole r an c e. 36 37 Keywor ds : se a gr as s; Po sido n ia oc ean ica ; end o phy t e s ; m a rin e m i cr obi ot a ; P l a n t g r o w t h 38 prom ot i ng ba cte r ia; Hal oph ytop htho ra; C el erin ata n ti mo na s ; M ar ino m o n as ; V ib ri o ; K ocur ia 39 40 1. Introduction 41 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 2 of 36 S ev e r a l s p ec ie s of e n dop hy t ic bac t e r ia , ac t in omyc e t e s , f un g i , a rc haea , an d p r o ti st s 42 c ol oniz e i nter n a l pl a nt tissues, e s ta blishi ng m u tu alist ic int er a c ti ons t h at co nf er v ari ous 43 b e ne fi t s t o t h e i r ho s t s ( C ompan t e t a l. 20 13 ; Har d o im e t a l . 201 5). T he se po s i t i v e e ffec t s o n 44 pl ant gr o wth and r e s istan ce t o bi oti c a nd a bi oti c str e s ses arise b o th dir ectly ( fr o m m i c r o-45 bial m et a bo li c a ctivities suc h as nitrog en fixa t ion, phos p h a te sol ubiliz atio n, h o r m on e 46 pr od u c ti on, and s ynt hesis of b io c ide s o r anti mi c ro bi a l co m p ou nds ) and indi r ectly, 47 thr o ugh t h e m o dul ati on o f pl a nt p hysiol ogy, nutr ient b al an c e, or b y o c cu pying t he e co-48 logi cal ni ch es of p a t hoge ns, th ereby en h an cin g str e s s t o lera n c e ( C o m p ant e t al. 2 01 3 ; 49 San t o yo e t a l . 20 16; K har e e t a l . 20 18; Pac if ic o e t a l . 20 19; A l i e t a l . 2 022 ). T h e m o s t a d -50 v a n c ed s tudie s o n pl ant – m i cr oor ga nis m inter a cti o ns h av e re veale d a w ide r a ng e of po-51 tenti al b i ote ch n ologi cal a ppli c a ti o ns f or c rop im p r o vem e nt a nd envir on m e nt al pr ote ction 52 o r re st o ra t io n (C omp an t e t a l. 2 019) . 53 P lant en doph ytes c an be tr a ns mitte d hor i zo nt ally t hr o ug h th e en viro n m e nt (vi a s oil 54 or bet w e en pl a n t s), v erti c a lly thr o ugh s eeds and p o llen, or vi a a m ixed t r ans m iss io n 55 mod e ( F ran k e t a l . 2017 ; N e l s on 20 18) . V e r t i ca l tra n s mi s si o n e n s u re s t he i nhe ri t a nce o f 56 ben e f ici al endo p hyte s exhibiting pl ant gr o wt h-pr om oti ng (P GP ) tr aits in s e edlin g s, con-57 t ri b u t i n g t o do rmanc y r e l e a s e, e nhanc e d g e rmina t io n a n d g r owth , a n d p r o tec t i on a g a in s t 58 bioti c a nd a b ioti c stress ors (Tr uy e ns et al. 2 01 5 ; Nelso n 20 1 8) . 59 T h e M e diterr an e an se ag r a s s P o s i d oni a oc e an i c a (L. ) De lile is t he d o m in ant m a rin e 60 a ngi osper m i n t he M e diterr a n e an S e a, where it f or ms e xte n s ive co astal m e a d o ws th a t 61 s u p p o r t h i g h bi o d i v e r s i t y . T h e s e m e a d o w s p r o v i d e c r u c i a l e c o s y s t e m s e r v i c es , i n c l u d i n g 62 c a rb on s equestr a tio n, oxyge n pr o d u ctio n, bior e m edi a ti on, nursery h ab it at a nd s he l ter f or 63 f i s h , a n d p r o t e c t i o n a g a i n s t c o a s t a l e r o s i o n ( S c a n u e t a l . 2 0 2 2 ) . O v e r t h e p a s t d e c a d e , 64 ho we v er , th e distributio n and de nsity o f P. oc e an ic a m e a dows h a v e dr am ati ca lly d eclined 65 due to clim a te ch a nge a nd i n c r easing h u m a n e xploit ati o n of c o a s t al a r eas (T eles ca et al. 66 201 5; Sc anu e t a l . 2 022 ). Na tu ra l l y , mea do w e xpa n si o n occ u r s s l o w l y t h rou g h th e p r o l if -67 er atio n o f l ater al b uds a nd veget a tiv e fr a g m e nt disp ers a l (M ar b à 19 9 8; D i C arl o e t al. 68 200 5). Lo n g - d i st a nce d i s p e r s a l de pe nd s on f loa t in g f r ui ts ca r r ie d b y mar i ne cu r re n t s, 69 w h ich re l ea s e la rge , f l e s h y s e e ds u pon r ip en i n g ( Ruo cc o e t a l . 202 4). U n de r un f a vo ra b l e 70 c o nditio ns, la r g e a m ou nts o f see ds are ca st on be ac h e s , w he re t he y r a pidly lo s e vi ability, 71 u n l e s s p ro t ec te d by fru i t enc a s in g o r r e tr i e v e d b y t i de s (S u t e ra e t a l . 2024 ). Fo l l o win g 72 dis turba n ce eve nts, P. o c e anic a g r ass l a nds exhib it a lo w n a t ur a l re c over y rat e 73 (Ba da lame n t i e t a l . 20 11; Ca s t e j ó n - S i lv o a n d T e r r a do s 2 021 ) . R e s t o r a t io n p ro g ram s h av e 74 been i niti ated to r e cov er degr a ded s ea gr ass habit ats thr ough t he tr ansp la nt ati on of cut-75 t i n g s f r o m d o n o r m e a d o w s o r s e e d l i n g t r a n s p l a n t ( C a l v o e t a l . 2 0 2 1 ; B a c c i e t a l . 2 0 2 5 ) . 76 Alth oug h s till li mited b y t he av aila bility o f c l o n al m a t e r i a l and h u m an distur b a n c e s , 77 t h e s e e ff o r ts ha ve y i e l d e d e nc ou ra g in g r e s u l t s ( C a lv o e t a l. 20 21; Bac ci e t a l . 2 025) . A 78 s e ed-based pr o p agati on str at e gy is m o re con ve nient t h an c utting tr a nspl a nt, and it pr e -79 s e r v e s g e n e t i c d i v e r s i t y a n d e xi s t i n g m e a d o w s . I n a d d i t i o n , a r e c e n t s t u d y d e v e l o p e d a 80 f easible pr oto col f or long-ter m s t or ag e o f P. oc e a n i c a s eeds, allo wi n g s cal ab l e pro p agul e 81 pr od u c ti on and tr a ns pl ant ( Suter a e t al. 2 0 25 ). 82 En do ph y t e s a r e kno w n t o p la y k ey r o l e s i n nu t r i t i o n an d de f e n se pr oc e s se s ( E s p i -83 n o s a e t a l . 20 10; G a rc ia s- B on e t e t a l . 20 16 ; Moh r e t a l . 2 021 ; T o r ta e t a l . 20 22). A re ce n t 84 st u d y on t h e A u st ra l ia n s ea g ra ss H a lop hila ov alis r e v e a led a co m pl ex endo phyti c 85 m i cr obi ot a ass o c i ated w ith repr odu c ti ve tiss ue s (flowe rs , fr uits , and s e eds) , in cl u ding a 86 c ore o f b a cteri al spe cies wit h P G P tr aits a nd nitr ogen-f ixi ng ca p a bi lities c onser ve d a cr oss 87 o rg an s ( Ta rq u in i o e t a l . 202 1). I n P. oce anic a , the e nd o phyti c m i c r o bi al c om m u ni ty h as 88 been c h ar acteri z e d us ing m o le cul ar / m et agen o m i c a p pr o a ches (G ar c i as-Bon et e t al. 20 1 2, 89 201 6) an d c u l t u rin g me t ho d s ( E sp i no s a et a l . 201 0; B la n che t e t a l . 2017 ; To r ta e t a l . 2 022 ). 90 Ga rc ia - Bo ne t et a l . ( G a r c i a s - B o n e t e t a l . 2 0 1 2 ) s h o w e d d i f f e r e n t c o mp o s i t i o n s o f P. o ce an ic a 91 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 3 of 36 e n d o p h y t i c c o m m u n i t y a m o ng p l a n t o r g a n s ( r o o t s , r h i z o m es , a n d l e a v e s ) . H o w e v e r , t h e 92 pr e s en ce o f m i c r ob i al end op hytes in see d s r em ai ns un explor e d. 93 I n this stu dy , w e a p plied cult urin g tec hn iqu e s to i s ola t e the e nd o ph yti c mi c ro biot a 94 as so c i at ed with s urf a ce-steriliz ed P. oc e an ic a see ds , with t he ai m of ide ntifyi ng ve r tic ally 95 tr ans mitted mi c ro org anis ms pot e nti ally be ne fi c i a l f or se e d ger m i n a ti on and se edling 96 devel op m e nt. End o p hyti c isol a t e s f r o m P. o c ean ica s e eds cou l d be expl oite d to ex t e nd t h e 97 s he lf l if e o f pr o p ag ati o n m a teri al used i n futur e restor atio n pr ogr a m s. Furt her m o re, the 98 a v ail a bility o f c ultu r able mi c r obi a l isola te s r e pr e s e nts an i m p ort a nt first st e p towa rd 99 elu ci d ati ng the co m p le x i nter a ctio ns bet we e n end op hytes a nd m ar i n e an g ios p e r ms , a nd 100 s upp orts the de velop m e nt o f n o ve l m o d el syst ems de s igne d to s i m ul a te pl a nt –m icr ob e 101 inter a cti o ns u nde r m ar ine co nditio ns at t he l a b or at ory sc al e . C ultur able endo p hyt es iso-102 lated fr o m pla nts gr owing i n e xtre m e en vir onm e nts, su c h a s seagr ass e s , m ay a ls o p r e s e nt 103 biote ch nol ogi cal p ote nti al t o en h an c e s tress tolera n ce to c ro ps. 104 2. M aterials and M ethods 105 2 .1. P lan t M a t e r ia l Coll ec tio n and s t er il iza tion 106 F ro m ha lf Ma y to e a rl y Ju ne 20 21, 4 4 P. oc e ani c a se eds, still co m p le tely en cl osed by 107 fr u i t s , br oug ht n atur a lly a s h o r e by t he c ur re nt, were h ar ve s te d in fi ve dis tin c t l o cati o ns 108 on t he S i cili an c oas t: C orni n o ( 38 . 09 3 N, 1 2.6 6 1 E ) ( 1 0 see ds ), Eri ce ( 3 8. 0 46 N, 1 2 . 54 8 E ) ( 5 109 se e d s ), a n d Ma rs a la (3 7.74 0 N , 12 .47 2 E ) (9 s ee ds ) a lo n g t he w e s t e r n c oa s t ; Sa n N i co la 110 ( 3 8. 00 9 N, 13. 6 24 E ) ( 8 s e eds) in t he n o r t he r n co ast, in t he Tyr r heni a n Sea; an d S ci a cc a 111 ( 3 7 . 5 0 4 N, 1 3 . 0 6 3 E ) ( 1 2 s e ed s ) , i n t h e s o u t h e r n c o a s t , i n t h e S t r a i t o f S i c i l y ( F i g u r e 1 ) . T h e 112 fr u i t s we r e t r a n spo r t e d to t h e la bo ra to r y , wa s h e d in st e ri l e a r t if ic ia l s e awa te r (AS W , 3 .8% 113 s al inity) to r e m o v e pl ant an d s an d r esidues, then stor e d indi vidu ally f or 2 4- 48 h in 1 5 m L 114 of A S W at 4° C b ef ore p r o cessing. U nd e r a s epti c co nditio ns, fr u i t s we r e dis sec t ed with a 115 s t e r i l e b l a d e t o e xt r a c t s e e d s . E a c h s e e d w a s s u r f a c e d i s i n f e c t e d t o i n a c t i v a t e t h e m i c r o -116 or ga nis ms present on t he s urf a ce by s e q u enti a l i m m e rsion f or 2 mi n in 70 % (v/ v ) e th a nol, 117 3 m i n in 7% (v/ v ) sodiu m hy p o chlorite ( 10 % a ctiv e c hlori ne) , 1 mi n in 7 0 % ( v/ v) e t h a nol, 118 f o ll ow ed by t hre e r inses o f 2 m i n in s terile dis tille d w ate r ( S DW) . 119 120 121 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 4 of 36 122 Fi gure 1: Lo ca tio ns, a lo ng th e S i cilian c o a s t, wher e cast fr uits w ere c ollect e d. S h a de d ar-123 e as indi cate th e a p pr o xi m a te c urr ent dis tributio n o f P. o ce a n ic a m ea d ows ( ad apt e d fr om 124 C a lv o et a l. 20 1 0) . 125 2 .2. E ndo phy te i s o latio n 126 E ac h s ur f a ce-disinfe c t ed see d was wei g hed (A p pe ndix 1) an d lo ngitudi n all y cut 127 with a s terile bl a de . H a lf s eed was t he n g r ou nd in 3 m L o f S D W und er asepti c cond itions . 128 P re l imina r y t e n - f o l d se ri a l d i lu t i on a s say s we r e pe rfo r me d on a s ub sa mpl e o f te n s ee d s to 129 as sess the mi cro bi al lo ad o f th e h o m o g e n at e s . U ndiluted s e e d h o m oge n ate ( 1 00 µL ) w e r e 130 pl ated on s a lin e Nutri e nt Ag a r pl a tes (N A: 3 g yeast e xtr a c t, 5 g pe pto ne , 2 0 g N aC l, 1 5 g 131 Ag ar, 20 0 m L ASW, S D W u p to 1 L, p H 6.8- 7. 2) , a nd salin e St a r c h Yeas t e xtr a ct Glu co s e 132 Ag ar pl a tes ( S GY, 15 g s t ar ch, 5 g glu c o s e, 5 g ye a s t e xtr a c t, 2 0 g N a Cl, 20 g Ag ar, 20 0 m L 133 A SW, S DW up to 1 L, pH 6.8- 7. 2) . All m e dia were a ut ocl a v ed f or 2 0 m i n at 12 1 °C bef ore 134 us e . To co nfir m s ur f a ce dis inf ecti on, a li qu ots o f t he wat er us e d i n th e fin a l rins e w e r e 135 pl ated o nt o th e sa m e m edi a ( n eg a tiv e co ntrol pl ate s ). P lat es we r e th en i ncu b ate d at 26  ° C 136 i n t h e d a r k a n d o b s e r v e d d a i l y u p t o t w e n t y d a y s . T o t a l c o l o n i e s w e r e c o u n t e d , a n d t h e 137 co lo n y - f o r min g un i t ( C FU) / g o f f re sh m ate r ia l wa s ca lcu la t e d fo r bo th me d ia ac co rd i n g to 138 Silv a et al. (d a Sil v a e t al . 2 0 13 ) (A p pendi x 1 ) . 139 M i c ro bi al co l o nies wer e initi ally gro up ed ac co rding t o colo ny m or p hol ogic al fe a -140 tu re s (sh a p e , c olor , elev atio n, surf a c e , m a r gin , a e r i a l m y c e liu m , p resen ce of exud a t e, and 141 g r o w t h r a t e ) a n d m i c r o s c o p i c s i m i l a r i t y ( A p p e n d i x 2 ) , a n d a t l e a s t t w o r e p r e s e n t a t i v e 142 c o l o n ie s o f e a c h m o r p h o t y p e f o r e a c h o r g a n a n d h o s t s a m p l e w er e s el e c t e d a n d p u r i f i ed 143 st r e a k in g ag a r p la t e s . Pu r e b ac te ri a l co lon i e s we re cu l t u re d in 5 mL o f t h e c o rre spo n d in g 144 b r o t h , i n c u b a t e d a t 2 8 ± 2 ° C f o r 2 4 h . F i v e h u n d r e d m i c r o l i t e r s w e r e s t o r e d a s p u r e c u l -145 t u r e s a t − 8 0 ° C i n g l y c e r o l ( 5 0 % v / v ) . T h e r e m a i n i n g v o l u m e o f t h e c e l l c u l t u r e w a s p e l -146 leted b y c entrif uge at 2 , 6 00 r c f f o r 10 m i n ute s a nd used f or D NA extr a cti on. 147 2. 3 . En d op h yt e mo le cul ar ide nt if ic at i on 148 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 5 of 36 M o l e c u l a r i d e n t i f i c a t i o n w a s p e r f o r m e d t h r o u g h s e q u e n c e a n a l y s i s o f 149 P CR -amp l i f ie d b ac t e ria l 16S rDN A g en e s an d f un ga l i n te r na l t r a n s c ri b e d spa ce r ( I TS1 , 150 5. 8 S, IT S 2) r D N A r eg io ns . 151 Ba c te r i a l pellets w ere r e s uspe nded in 50 0 µ L o f Tr i s -EDT A b uf fer ( TE, p H 8. 0 ) a nd en-152 z y m a t i c a l l y l y s e d w i t h P r o t e i n a s e K ( 2 0 m g / m L ) f o r 6 0 m i n u t e s a t 3 7 ° C . G e n o m i c D N A 153 w as p urified using t he p he no l/ c hl or of o r m/iso a m yl a l coh ol ( 2 5: 2 4: 1) m eth od ( Wils o n 154 1 9 97 ) a nd r e s usp en de d in 1 00 µL TE. B a ct e r ial endo phyt es we r e ide ntifi e d by seq u e n ci ng 155 ~15 00 b p o f th e 16S rDNA w i t h th e un iv er s a l p r ime r s 27 f an d 1 492 r f o l l ow i ng th e or i g i na l 156 PC R prot o col (L a n e 1 9 91 ) usin g the D re am T a q DN A P ol y m er ase ( 5 U/µL, T he r m o Fisher 157 S cientifi c ). 158 Pu r e f un ga l c u l t u r e s w er e o b ta in e d o n SG Y p la te s i nc uba t e d fo r se v en da y s a t 28 ± 2 159 ° C. Th e a e ri al m y c eli u m ( 2 00 µg ) of e ac h selected fu ng a l c ol o ny w as s c r a ped, pl ac e d i n 160 cl ean t ube s a n d disr upted with tu ngsten ca rbide bea d s i n a T i s su e L y s e r I I (Qia g e n) s e t a t 161 3 0 H z f or 2 m in. F u ngal D N A w a s extr act ed f ollo wi ng a Cetyl tri m eth yl a m m oniu m 162 br om ide ( CTAB ) pr oto c o l ( Doyl e a nd Do yle 19 8 7). a nd resusp ende d in 1 0 0 µ L TE. D N A 163 ampl i f ica t io n wa s pe rfo rme d u s i n g t he I T S 5 /I T S4 p r ime r s e t ( Wh i t e 19 90), f o l lo wi n g th e 164 o r i g i n a l P C R p r o t o c o l w i t h a n a n n e a l i n g t e m p e r a t u r e o f 5 6 ° C , a n d b y a m p l i f i c a t i o n o f 165 ~ 1 00 0 bp- fr ag m ent of t he L arg e Sub unit ( LS U) of t he nu c l e a r rib o s o m al RN A gen e, us ing 166 the p ri m e r s L R 0 R ( 5 ’- AC C CGC TGA A CTTA AGC- 3 ’) and LR 5 167 ( 5’-TC CT GAG G G AA ACTT CG- 3 ’) f o llo wing th e o rigin al PC R pr oto col (Vilg a ly s and 168 Hester 1 9 90 ) ,using t he Dr e a m T aq D N A Po ly m er as e ( 5 U/ µL, Th er m o Fisher S c i entifi c , 169 US A ) . 170 T h e a m p lified pr odu cts we r e pur ifi e d and s equ e n c ed by E uro fins G e no m ics 171 ( Eb ers be r g , Ge r m a ny ). B act eri a l and f u ng a l ide ntifi cati on w as per for m e d usin g the 172 h i g h e s t i d e n t i t y s c o r e f r o m B L A S T 2 . 1 5 . 0 s o f t w a r e o f t h e N a t i o n a l C e n t e r f o r B i o t e c h -173 nol ogy I n for m ati o n ( N CBI ) a g ai nst the NR d a t a b ase. Sequ en ces w ere d e p os ite d in th e 174 G en Ba n k da ta ba s e u n de r a cc e s si o n n umbe r s OQ8 745 3 0, OQ8 74 5 31, OQ8 747 04, 175 OQ8 74 7 05, O Q88 3 73 2-OQ 883 77 3 ( Ta b l e 1) . A pe rc en ta ge i d e n ti ty > 98% w i th NC B I se -176 qu en ces w as a c ce pte d f or spe c i es-leve l identifi c atio n, while id entities bet w e en 9 5 % a n d 177 9 7 % were cl assif i ed at th e gen us le vel ( S t a c k ebr a ndt e t al. 20 2 2) . 178 179 2 .4 D e t e rm in a tio n o f P lan t Gro w th P rom otio n P rop ert i es a nd E nzy ma t i c Ac t i v it ie s o f 180 B ac teria l Iso la tes 181 T h e p r o d u c t i o n o f i n d o l e a c e t i c a c i d ( I A A ) w a s e v a l u a t e d u s i n g a c o l o r i m e t r i c t e c h -182 niq ue by in c ub ating 3 m L o f a l i q u i d c u l t u r e i n N u t r i e n t B r o t h ( N B : 3 g y e a s t e xt r a ct , 5 g 183 p e p t o n e , 2 0 g N a C l , 2 0 0 m L A S W , S D W u p t o 1 L , p H 6 . 8 - 7 . 2 ) w i t h L - t r y p t o p h a n ( 1 0 0 184 m g / L ) , t h e I A A p r e c u r s o r , a t 2 8 ° C f o r 7 2 h in sh aki ng co nditio n at 1 20 r p m . C u ltures 185 w e r e c e n t r i f u g e d f o r 5 m i n a t 1 5 , 0 0 0 × g a n d s u p e r n a t a n t s t r a n s f e r r e d to gl a s s tube s . 186 C o l or c h a n g i n g t o p i n k ( p o s i t i v e r e a ct io n ) w a s v er i f i ed f o l l o w i n g t he a ddition o f 4 mL of 187 Sa l ko w sk i ’ s r eag e n t (G o r don an d We b er 19 51). T h e amou n t o f IA A wa s ca lc u lat e d b y 188 mea su r i n g th e a b s o r banc e a t 535 nm i n a sp e c t r oph o tome te r (AS Y S U V M-340 Mic ro p la te 189 Re a d e r , Bio chr o m , Ltd., C am br i d ge, UK), a cc ordi ng to a s tand ard cur ve a p pro p riately 190 re a l iz e d . 191 S i d e ro pho r e s p ro duc t i on wa s e va lua t e d i n Ch ro me A zu ro l S ( CAS) a ga r p la t es 192 (S chw y n an d N e i l an d s 1 987 ) . Aliq u o ts ( 1 00 µL ) o f b ac te r i a l c ultur es were in oc ul a te d in 193 t h e w e l l s o f t h e C A S p l a t e s a n d inc u ba t e d f o r 7 da y s a t 28 ° C in da rk n e s s. Ba c t e ri a tha t 194 pr od u ced sidero p hor e s sho we d a n or ang e hal o. 195 P hos p h a te sol ubili zatio n w as d e t e r m in e d by t he prese n ce o f a tr ans p a r e nt h al o 196 a r o u n d t h e b a c t e r i a l c u l t u r e s ( 1 0 0 µ L ) i n o c u l a t e d i n w e l l s o f N B R I P p l a t e s ( p h o s p h a t e 197 g r o w t h m e d i u m f r o m t h e N a t i o n a l I n s t i t u t e o f B o t a n i c a l R e s e a r c h ) a f t e r 7 d a y s a t 2 8 ° C 198 (N au t i ya l 199 9). 199 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 6 of 36 Nitr ogen fixation w as ass e s sed by platin g strains in a nit r og e n-fr e e m ini m u m m e -200 d i u m ( N F B ; ( D ö e b e r e i n e r 1 9 9 5 ) ) a n d i n c u b a t i n g f o r 5 d a y s a t 2 8 ° C . B a c t e r i a l g r o w t h i n -201 dic at e d th e po s sible ability of t he b ac teri a to fix a t m os p her ic nitr ogen. 202 T h e f o r m a t i o n o f b i o f i l m s w a s d e t e r m i n e d b y c h e c k i n g t h e a d h e s i o n c a p a c i t y o f t h e 203 b a c t er i a i n m i c r o p l a t e s w i t h 1 2 w el l s i n N B a t 2 8 ° C f o r 4 d a y s . A f t er i n c u b a t i o n , t h e b i o -204 f i l m f o r m a t i o n w a s o b s e r v e d i n t h e s u r f a c e a n d / o r b o t t o m o f t h e w e l l s . T h e n , e a c h w e l l 205 w as s tai n ed f or 2 0 m in w ith 0. 0 1% cryst al vi ole t (d el C astillo et al. 2 0 12 ) to ev a l u at e the 206 pr e s en ce o f a ri ng of bi ofil m a r ound w ell w alls. 207 T h e AC C-de am in ase a cti vi t y w a s per f o r med a s des cribe d by P en rose a nd G li ck 208 (2003 ) ( Pen r o s e an d G l ic k 20 03) . B r i ef ly , b ac t e r ia l c u l tu r e s w e r e i nc u ba t e d i n a s a l t s 209 m ini m a l br oth added wit h 5 m M 1- a m i n ocy c l o pro p a ne - 1 - c arb oxyli c a cid ( A C C ) f or 2 4 h 210 at 2 8 °C in s haki ng c onditi on at 12 0 rp m . The α-ke t obutyri c ac id pr odu ced was qu a ntifie d 211 us ing a sta nd ard cur ve with k n ow n co n ce ntr atio ns by m e asuri ng ab s orpti on at 5 40 n m in 212 a sp e c tr op h oto m et e r . A C C-de am i n ase a ctivity w a s expressed a s µ m o les of 213 α -ke tob utyr ate pe r m g of pr ot e in per h ou r. 214 E nzy m a ti c a c t ivities w e re d ete r m i ne d o n pl ates in c uba ted a t 2 8 °C f or 7 d a ys , ob-215 s e r ving th e f or m atio n o f h al o ar ou nd th e ba cte r ial bi o m ass. DN A s e a cti vity was de ter-216 m in ed by stre akin g the isol ates o n DNA se agar pl ates ( S ch arl ab, Sp ain ) r eve a led with 1 217 M HCl. Am yl ase a ct i vity w a s p erf or m ed o n st ar c h a g ar plates ( S ch arl ab, B ar c e lo n a, 218 S p a i n ) a n d r e v e a l e d w i t h 1 0 m L l u g o l ( P a n r e a c A p p l i c h e m , S p a i n ) . P r o t e a s e a n d l i p a s e 219 a cti vitie s wer e as sessed in casein ag ar a nd T we e n 8 0 m edi a, r e s pec tive ly, a s de s c ribed by 220 H a r l e y a n d P r e s c o t t ( 2 0 0 2 ) ( H a r l e y a n d P r e s c o t t 2 0 0 2 ) . P e c t i n a s e a n d c e l l u l a s e a c t i v i t i e s 221 wer e e xa m i ne d as de s c r ibed by Elb e lt ag y et a l. ( E l b e l ta gy e t a l . 200 0) . Fo r p ec t ina s e ac -222 tivity, s trains pl at ed on a m m o n iu m m in er al agar pl a tes were re vea led with 2 % C TAB , 223 a n d p o s i t i v e b a c t e r i a s h o w e d a h a l o . F o r c e l l u l a s e a c t i v i t y , s t r a i n s w e r e p l a t e d o n s o l i d 224 m ini m a l m ediu m sup ple m e nte d with 0 . 2 % ye ast extr act and 1 % car bo x ym et hyl c el lulose. 225 Pl ates wer e de ve l op ed by co ve ring t he pl ate wi t h 1 m g / m L C o ng o Red s ol u ti on ( S ig-226 m a -Aldri c h, U SA) f or 15 m in and de colori zi ng wit h 1 M N aCl f or 20 m i n. Fin ally, 227 c h i t i n a s e a c t i v i t y w a s p e r f o r m e d a s d e s c r i b e d b y M e s a et a l . ( M e sa e t a l . 20 15) on a g ar 228 pl ates cont ai ning m ini m a l m e di u m su pp le m ent e d with colloid a l c hitin. 229 230 2 .5 Data a naly sis 231 A no n-p ar a m etr i c a p pro a ch was a d o pte d to a n alyse th e cultur abl e end op hyte o c -232 c urr en c e in seed m a te r i a l on NA and SGY medi a . To i nv e s tiga te t he di ff ere n ces in CF U/g, 233 a bin o m i al sign n o n-p ar a m e tri c te s t w a s ad opt ed (Higgi ns 2 0 04 ) a nd i m ple m e nted in 234 M icros o f t E xc e l (Mi cr oso ft Of fi ce 3 65 ). 235 T o obt ain in for m ati o n a b out th e f un c tio nal di ve r sity o f f u ngal str a ins , t he FU N G ui ld 236 dat a b a s e w as used t o esti m at e t he tro p hi c m od e s and g uilds o f f ungi ( Ng uyen et a l . 20 1 6) . 237 T h i s t o o l a l l o w s t o p r e d i c t t h e f o l l o w i n g p r i m a r y f u n g a l l i f e s t y l e s : p a t h o t r o p h : r e c e i v i n g 238 nut rients at t he expens e of th e h ost ce lls and c a usin g dise a s e ; s apr otr o ph: re ceiving nu-239 t r i e n t s b y b r e a k i n g d o w n d e a d h o s t c e l l s ; s y m b i o t r o p h : r e c e i v i n g n u t r i e n t s b y e xc h a n g -240 ing reso ur c es with h os t cells. 241 T h e V enn diag r a ms w ere c ons tru ct e d m an u ally or us ing t he w ebt o o l : 242 htt ps://bi oinf or m ati cs.ps b .ug e nt.b e/ webt ools/V enn /. 243 244 3. R e su lt s 245 3 .1 En do phyte i so lat io n 246 T h e n u m be r of ba c te r i a l and fu ng a l c olonies on N A a nd SG Y m e di a in cre ased o ver 247 the in cub a tio n p eriod, with m o s t colo nie s a pp e a r in g wit hin th e fir st 1 0 d ays. N o c o lo nies 248 w e r e o b s e r v e d o n a n y o f t h e n e g a t i v e c o n t r o l p l a t e s . S i m i l a r l y , n o g r o w t h w a s o b t a i n e d 249 f ro m s o m e seed s am pl es reg ardl ess of col le c tio n site and m edia (Figur e 2) . Th e abs en ce of 250 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 7 of 36 c ol oni es o n b ot h N A a nd S G Y plates w as m ost likely d ue to th e l ow mi cr obi al 251 conc en t ra t io n s t yp ica l l y a s soc ia t e d w i th p lan t e n dop hy t e s . Co lo n y - fo rmin g un i t s w e r e 252 obs e rv ed fro m 30 se ed s am ples on SG Y p lates ( 68 % o f see ds ) an d 2 1 s e ed s a m pl e s on N A 253 p l a t e s ( 4 8 % ) ( F i g u r e 2 , A p p e n d i x 1 ) . C F U s w e r e r e c o v e r e d o n b o t h m e d i a f r o m 1 9 s e e d 254 sa mpl e s (43%) , e xc l u si ve l y f rom SG Y in 11 samp l e s ( 2 5%), a n d e x cl u s i v e l y fr om N A in 2 255 sa mpl e s (5%) ( F i gu r e 2 , App e n d i x 1 ) . Twe l v e ou t of 44 samp le s (27 %) sho w e d no co lo n y 256 gr owth o n e ith er m edi u m (Fig ure 2 ). 257 258 259 260 Fi gure 2: P e r c e n t a g e a n d i d e n t i t y o f s e e d s y i e l d i n g c o l o n i e s o n S G Y a n d N A m e d i a , o r 261 no ne . 262 263 T h e hig he s t tot a l en do ph yte c on c e nt r ati ons , express e d as CFU /g, w er e 3.6 2 × 1 0² on 264 S G Y a n d 3 . 3 9 × 1 0 ² o n N A ( A p p e n d i x 1 ) . S t a tistic al a n a lys i s r e ve aled n o signifi c ant 265 dif fe r e n c e i n t ot a l e nd op hy t e con c entr a ti ons b etw e e n t he t w o m edi a at a con fiden c e le ve l 266 ( 1 –α ) ≥ 0 . 9 5 . 267 268 3 .2 M o le cu la r ide n ti fi catio n o f c u lt u rab le end op hyte s 269 B a s e d o n p hen otypi c c har a ct e r istics, 42 b acter i a l an d two f u ng al is olates wer e 270 s e lect e d ( A p pe ndix 2 ) and id e ntifi ed th roug h seq uen c e an a l ysis . Ba c t e ri al 16 S rD N A 271 se q ue nc e s r an ge d f rom 754 t o 144 6 b p i n l e n g t h, wh i l e f un ga l I TS an d L S U r e g i on s 272 ra n g ed fro m 54 1 t o 9 10 bp an d 862 t o 10 69 b p , re spe c t i ve l y (T a b l e 1 ). Th e i den t i f i e d 273 end op hyti c ba c te r ial is ol a tes be l o nge d t o thr ee p hyla — Pr oteob a cteri a, Fir m i cu t e s , and 274 A cti no b a cteri a — a nd w ere a s so ciat ed wi th ten di ff ere nt ge ne r a (T abl e 1). Most is ola tes 275 w e r e g r o u p e d i n t o t h r e e d o m i n a n t g e n e r a : Vibr io , Sta phy lococ c us , a n d Ma r i n o m o na s , 276 fo l lo we d b y Au rei m on as , B ac i llu s , C el eri na ta nt im ona s , Hal omo na s , Ko c u r ia , Lysob acte r, and 277 M e ta bacill u s (Fig ur e 3) . 278 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 8 of 36 279 280 Fi gure 3: Dist ributio n of b a c teri al f am ilie s isolated f ro m s eeds. 281 282 I n ter ms of s equ en ce identity, 78. 6 % of t h e is o lates sho we d hig h simil arity ( ≥ 98 % ) t o 283 16S rDNA s e qu en ce s a va i la b l e in G e nB an k ( Tab l e 1) . N in e Vibr io i s o l a t e s , h o w e v e r , 284 exhibit e d lo we r ide ntity v a l ues ( <9 7 %) wi t h r ef e r en ce s e q ue n ces i n G e nB ank (T abl e 1) . 285 Sp e c i fi c a lly, fi ve isol at e s (Vsp -PoC 2, V sp -PoE 2, Vsp -P o E 4, Vs p-P oM 1 0, a nd Vsp - PoS C 1 ) 286 wer e ide nti cal and ass oci ate d wit h Vi br io ru b er s tr ain H MF 8 00 4 (G enBa nk ac c ess io n 287 KY 04 740 9, 9 6 .63 %). Th e r ema in i n g f ou r Vi b r i o is ol a t es (V sp-P oC 8 , Vs p- Po M 7, 288 V sp-PoS C 3, V sp-P o SC 1 1) we r e ide nti c a l and a s so ciated with Vi b r i o s p. str a in P CEP 2 8 289 (G e n Ba n k MK 2 942 32, 96. 99%) . Th e se tw o g ro up s of Vib rio isol ates sh are d 95. 96 % id e ntit y 290 with e ac h o th er. 291 I n t ot al, t wo f ung a l colo nie s wit h d istinct gr o wth r a t e s and m o rp h olo gi c a l 292 c h ar a cteristi cs w e re isol at ed fro m t w o s e ed s a m pl es c ollecte d in Sa n Ni c o l a a nd Eri ce, 293 r e s pecti ve ly ( Ap pendix 2). Th e is ol a tes P m- P oE 1 a nd H psp-P oS N 1 sh ow ed 1 0 0% i de nt i t y 294 with f un g al I T S s e q ue n c es de posited in G e nB a nk, c orr espo ndi ng to P aec i l om yc e s m ax i m u s 295 st r a i n F RR 3 793 (G e n Ban k MW64 21 6 9) a nd Ha lop hyto p hth ora sp . M H J - 2021 e i so la t e B D 638 296 ( GenB ank OK 0 4 10 0 4) , r espe c ti vely (T a bl e 1). L S U se quen c e a n alysis f urt her c onf ir me d 297 s pecies ide ntifi cati on, sho wi ng 99. 3 % i d entity b etw e e n P m -P oE 1 and P. m ax i mus s t r a i n 298 CBS3 72 . 70 ( Ge n Ban k MH87 146 7 0) , a nd 10 0% i d en t i t y b e tw e e n H p s p - PoSN1 a nd 299 Ha loph ytop htho ra s p . MHJ -20 21e i s o la t e BD63 8 (G e n Ban k O K033 60 1 ). 300 301 3. 3 PG P p r op e rt ie s: Pl ant Gr o w t h Pr o m ot in g T r a i ts a n d e nz y m a t ic ac t i vit ie s 302 A tot al of 2 2 ba c te r ial is olates ( F ig ur e A 1) —select ed b a s e d on t heir hig her i n vit r o 303 g r o w t h c a p a c i t y — w e r e c h a r a c t e r i z e d t o a s s e s s t h e p r e s e n c e o f p l a n t g r o w t h - p r o m o t i n g 304 ( P G P ) tr aits a nd enz y m a ti c a c ti vities (T ab le s 2 a nd 3 ). 305 All is o l ates exhibi t e d at le ast on e of th e tested pro perti es M o s t isolat es ( 7 7 % ) di s-306 pl ayed b et ween f o ur a nd e ig ht tr aits s i m ulta neo usly. Only on e isol a t e (Vt -PoC 4) did not 307 s how an y P G P tr ait, and o ne ( A a-P oSC 9) no e nzy m a ti c a ctivity. 308 T h e m os t fr equ ently obser ved PGP ch a ra c te r i s tic s w ere bio fil m for m a tio n ( 8 6 % ) , 309 si d e ro ph o re p ro d uc t ion (77%), an d in d o le - 3 -a ce t ic ac i d ( IAA ) p ro duc t i on (36%) , fol l o w e d 310 by nitrog en fixati on ( 22 % ), a nd p h osph at e s olub ili z atio n ( 1 8 % ) (Fi gu r e 4 A ) . Mos t is ola tes 311 f o r m ed bi ofil ms pr edo m i n antly at t he b ot to m o f t he wells (T a ble 2 ). Is ol ate H ms p-Po S N 7 312 ( Ha l om on as sp.) e xhibite d t he hig hest s ide r oph or e pr odu c ti o n, as indic ated by th e h alo 313 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 9 of 36 dia m et e r on C AS m ediu m , w h ile isol ate Vn-Po S N8 ( V ibr i o nit r i fi gil is ) was t he m o st effi-314 c i en t I A A p r o d u c e r . No n e o f t h e b a c t er i al i s o l a t es e xh i b i t e d A C C d e a m i n a s e a c t i vi t y 315 und er in vitro conditi ons. 316 A m o ng t he six e n zym a ti c a ctivities te s t e d, D N ase ( 6 4% ) was th e m ost pr ev alent , 317 fo l lo we d b y p ro t e a se (45%), a my la s e (36%) , ce l l u la s e (32%), l i pa se (18%) a n d p e ct i na se 318 ( 1 3 % ) (Fig ure 4 B a nd T abl e 3). T he str on gest a m yl ase and ce llul ase a ctivities wer e bo t h 319 o b s er v e d i n i s o l a t e V s p - P o M 1 0. N o n e o f t h e b a c t er i a l i s o l a t es exh i b i t e d c h i t i n as e a c t i vi t y 320 und er in vitro conditi ons. 321 322 323 324 Fi gure 4: F r e q u e n c i e s o f P G P t r a i t s ( A ) a n d e n z y m a t i c ( B ) a c t i v i t i e s a m o n g t h e 2 2 b a c t e -325 r ia l is o l ates. Cellul a s e a nd pec tin a s e a ctiv ities were gr ou pe d tog e th er. 326 327 3 .4 T r o phic m od e o f e ndo phyti c f u ngi 328 Fung al t a xo no m ic and fu n cti onal a nalyse s us i ng F UNG uild c l ass i f ie d t he t w o fu ng a l 329 is ola tes, P m- PoE 1 a nd Hp sp-Po S N1, int o dis tinc t put ati ve tro phi c m o des. P . m a xim u s wa s 330 as signe d to th e p at hotr o ph –s a pr otro p h – sy mbiotr o ph m od e, w he r eas H alo phy t o p h thora 331 s p . w a s c a t e g o r i z e d a s a s a p r o t r o p h . T h e f o r m e r t r o p h i c m o d e i s p r i m a r i l y a s s o c i a t e d 332 with endo p hytes, cl a vici pita ce ous fu ngi, pl a n t p at hog e ns , a nd b oth pl a nt and w oo d 333 s apr otr op hs; w hile t he l atter is m ai nly r epr e s ented by pl a nt and wo od s ap rotr op h s. 334 335 4. D iscu ssion 336 T h e p os itive eff e c ts of pl ant – m i cr oor ga nis m inter actio ns, widely d em o nstr a t e d in 337 t e r re st r i a l a g ro ec o sy st e ms ( Compan t e t a l. 20 19; Paci fic o e t a l . 201 9) , a r e now i nc rea si n g l y 338 being r ec o gn i z ed i n m a rin e en vir on m ent s , s upporte d b y the g r ow i ng b ody o f kn o w ledge 339 on t he se agr ass hol obi ont ( U g arelli e t al. 2 01 7 ; T ar q uin i o et a l. 20 1 9). E arly e xp e r i me nts 340 with epip hyti c m i c r o org a nis ms a s so ciat ed with t he veget ativ e o r g a ns of P. oc e a n ic a re -341 ve aled th e ir ab ility t o sti mul at e pl ant d ev el o p m ent and in flu e n c e t he str u ctur e of th e 342 epip hyt e c o m m u nity (C e ldr á n et al. 2 01 2 ). M ore r e c e ntly, p ositive e ff ects on pl ant de -343 vel op m ent we re a ls o as cribed to endo p h yte s (M oh r et a l. 2 0 21 ) . G ro w ing e viden c e indi-344 c at e s pl a nt - a s so c i ated m i c r o b es as ke y f a c t o r s en h an ci ng devel op m ent a nd s tress t o ler-345 ance i n P. oc ean ica m e a d o w s . H o w e v e r , i t i s n o t k n o w n h o w t h e s e b e n e f i c i a l g u e s t s a r e 346 tr ans mitted a m ong i ndivid u a l pl ants . While r hi zo m es c a n e xt end l a ter ally a n d inter-347 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 10 o f 3 6 c o nn ect pl ants wit hin t he s am e m e ado w, it is still a n ope n qu estion wh e t her differ e nt 348 m e a d o w s a r e c h a r a c t e r i z e d b y s i m i l a r e p i - a n d e n d o p h y t e s , a n d h o w t h e s e a r e t r a n s -349 m itt e d thr o ugh ge n e r atio ns. In terr e s trial pl a nts, it is know n t h at s e eds ca n a ct as a 350 t r a n s - g en e r a t i o n al r es er v o i r of e n d o p h y t es ( T r u y e n s e t al . 2 0 1 5 ) . I n P. oc e a n ic a , h o wev e r, 351 n o s t u d y h a d e v e r d e s c r i b e d t h e p r e s e n c e o f e n d o p h y t e s . I n o u r w o r k , w e p r e s e n t t h e 352 divers e cult ur able b a cteri al a nd f un g a l e ndo p hyti c c o m m u nity is ola t ed fr o m P. o ce an ic a 353 s e eds c ollecte d i n diff ere nt Si c ili a n lo c at ions . Lo c ated i n the c entr e o f th e Me d i t err ane a n, 354 a t t he i n t e r se c t ion of t h e w e s t e rn a n d Le van t i ne g e n e poo l s, S ic i l ia n mea d ows p r e se n t t he 355 high est g en eti c bio diversity ( A r n au d-H a ond e t al . 20 0 7). B y sa m p ling se e ds al o ng th e 356 d i f f er en t q u a d r a n t s o f t h e Si c i l i a n c o a s t s , w e a i m ed t o f u r t h er m a xi m i s e t h e p o t e n t i al r e-357 c o ve r y o f m i cr obi a l e nd op hyte c o m m unities. Sin ce t he seeds we r e c o lle c t ed fr om 358 s t r a n d e d f r u i t s , i t w a s n o t p o s s i b l e t o t r a c e e a c h s e e d b a c k t o a s p e c i f i c m o t h e r p l a n t o r 359 m e a dow. H o w ev e r , it is like ly th a t th e f ruits h ad d et a ched fr o m n e arby m e a dows . 360 W h e the r a m ot her pl a nt co nsis tently tr a n s mits th e sa m e e nd o p hyti c co m m u nity t o all its 361 s e eds r em ai ns an o pe n qu e s tio n. 362 O ur pri m a ry objecti ve w a s to isol ate cultur abl e e nd o p hyti c m i cr oo rg anis ms t h a t 363 c o uld enh a n ce t he gr owt h and s tress tole ra n ce of s eagr ass pr o p a g atio n m a t eri al, w ith th e 364 a i m o f f a c i l i t a t i n g r e s t o r a t i o n e f f o r t s . D u e t o t h e l a c k o f s t a n d a r d i z e d p r o t o c o l s f o r i s o -365 latin g e nd ophytes fro m P. o c e anic a s e eds , w e used tw o cult ur e m edi a re co m m e n de d i n 366 the liter a tur e for m a ri ne mi cro bi al isol a tion (K ik i 20 1 6) . B a s e d on C FU /g v alu es, bo t h 367 m edi a pr oved ef f ectiv e f or isol ating s eed-ass oci ated e ndop hytes. N onet hele s s, so me 368 s am p l es yi elde d no C FUs on eith e r m edi u m , like ly d ue t o eit her a l ow m i cr obi al l o a d or 369 s e l e c t i v e l i m i t a t i o n s o f t h e m e d i a — p a r t i c u l a r l y S G Y , w h i c h h a s a m o r e c o m p l e x f o r m u -370 latio n (Kiki 2 0 16 ). Isol atio n e f fi cien cy lik e ly de pends n o t only o n cultur e c o nditions a nd 371 dis infecti on pr ot o c o ls but a ls o on th e i n he r e nt r arity, un even dis tributio n, a nd v a r ie d 372 gr owth ca p a c ities of end op hytes u nde r asepti c c o n ditions (R u ngj i nd a m a i a nd J o n es 373 2 0 24 ) . Fut ure is o l ati on ef fort s c ould be nef i t fr om th e us e of s e l e c t ive, e nri c hed, a nd 374 oligotr op hi c m e di a to in cr ease r e cov ery r at e s a nd ca pt ur e slo w-gro win g t axa. 375 A s ig ni fi c ant nu m ber of s e eds did n ot yi eld colo nies on pl a t es. H o wever, this d oes 376 not ne cess a r ily in di c ate s e e d sterility, a s our study fo c used e xclus ive ly on t he c ult ur able 377 f r acti on of t h e m i c r o bi ot a. F ur t he r m or e , we only te s te d t wo g e n er alist medi a, w hi ch m a y 378 not su p port t he gro wt h o f m o r e s pe c i al iz e d m i cr obi a l spe cies. An a dditio n al le v el of 379 c o m pl e xity m ight d e r ive fr o m t he c o m pe titio n am o ng s p ecie s in th e sa m e pl a te . A l-380 tho ug h we st reaked colo nies i n f re s h pl at e s m ultiple ti m es t o is ol a te pur e s pec i e s , w e 381 cann o t e xc l u d e t ha t in th e o ri g i na l homog e na te , t he p re se nc e of s ome mic r ob e s i nh i b i t e d 382 the gr owt h o f ot hers. Th eref o r e , f urth er c om ple m ent a ry m et a g en o m ic appr o ac h e s will be 383 ne cess ary t o det e c t t he pr e s en ce o f u n cultu r ab le or e lus ive m i cr o o r ga nis ms . 384 T h e b ac teri al i s ol a tes ide ntified in P. o ce a n ic a s eeds larg el y bel ong to t he s a m e ph y la 385 r e p o r t e d i n s e e d s o f t h e s e a g r a s s H alo ph ila o va li s ( T a r q u i n i o e t a l . 2 0 2 1 ) , s u g g e s t i n g t h a t 386 unr e l ated se ag r a s s s pecies—b elongi ng to line ages th a t diverged a p pro xim a tely 30 m il-387 l i o n ye a rs a g o an d in d e p en d e n tl y ada p te d t o mar in e en v i ronme n ts ( L ee e t a l . 2018) may 388 hos t si mila r end o phy ti c co m m u nities. N ot a bly, o ur s tudy is the firs t to report th e pres-389 en ce of b ot h O om y c ot a a nd As co m y cot a in se agr ass s e eds. Our P G P assay r esults s ug gest 390 th at P. oc e a n ic a s eeds, in additi on t o co n taining a large a m o u nt of r e s e r ves with i n th e 391 end osper m , also r ec ruit a nd verti c a lly t r ans mit mi c r oorg anis ms c ap abl e of su pp or ting 392 ger mi n ati on and e arly see dli ng de ve l op m ent , t hr o ugh h or m o n e pr od u c t i o n or b y f a c ili-393 tatin g th e a cq uis ition of essenti al n u trient s suc h as ir on a nd nitrog e n. 394 A m o ng t he b act e r i a l gen er a identif ie d i n t his s tudy, Ma r i n o m o na s , 395 Ce l er i nata n timo nas , and Vib rio ar e c o m m o n l y f o u n d i n a q u a t i c a n d m a r i n e e n v i r o n m e n t s . 396 Ma r i n o m o n a s h a s pr evi o u sly been r e p ort ed as an e nd op hyte i n P. o c e a n ic a , an d sever al 397 sp e c i e s , i nc lu d i ng M. r h i zo ma e , are kn ow n to be abu nd a nt within P. o ce an ic a t i s s u e s ( E s -398 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 11 o f 3 6 pin os a et al. 2 01 0 ; L u cas-E l ió et al. 2 01 1 ; Gar ci a s -Bon e t e t al. 2 0 12 ). Gr o wth-pr o m o ting 399 Ma r i n o m o n a s spe c i es h ave a lso bee n de tected i n t he cor e m i cr obi om e o f th e se agr ass 400 H a loph ila ov ali s s e e d s ( T a r q u i n i o e t a l . 2 0 2 1 ) . G i v e n t h e r e p o r t e d p o s i t i v e e f f e c t s o f 401 Ma r i n o m o n a s on see dli ng gr o wt h ( C eldr á n e t al. 20 1 2) and the strong I AA pr od u ction 402 obs e rv ed in o ur M. r h i zo ma e is o l ate Mr-P o SC 8, our findi ngs s upp ort the hypot hesis of a 403 cl ose pl ant – m i c r o b e a s so ciati on t h a t m a y b e sus tained t hr oug h ve r tic al tr a ns miss ion. 404 R ecently, M o hr et a l . ( M ohr e t a l. 2 0 21 ) de m ons trat ed th e po s itive o ut c o m e s o f t he 405 s y mbi oti c asso ci a tio n bet we e n P. o c e a n ic a a nd t he n it r ogen-fixing end op hyte C and i d atus 406 C e leri n at a nti m on as ne ptu n a , w hi c h c olo nizes the pl ant r oots . O th er nitr oge n-fixing s pe -407 ci e s , in c l uding C . y am al e ns is a n d C . dia zot roph ica , h a v e b e e n i s o l a t e d f r o m t h e s e a g r a s s 408 Th a l ass i a h em p ri c h ii a nd fr om e s tu ar ine grasses su ch as Sp a rt in a a lt er nif l or a a n d Juncu s 409 r o em eria n u s , r espe c ti vely ( Cr a m er e t al . 20 1 1; M o hr et a l. 2 02 1 ) . As with terr e s trial pl ants , 410 these s ym bi oti c r e l atio nships e n a ble m a r ine sp e c ies t o t hr iv e i n nitrog e n-po o r , o li gotro-411 phi c envir o n m e nts, s u ch as s e agr ass m ead o ws a nd salt m a rsh es. Our is olate Cn- Po S N 8 412 sh o w e d 98 . 93% n uc le o ti de i d e n t i t y w i t h a ~ 140 0 bp f r a gmen t o f th e Ca nd id atu s C . n e p -413 t un a g e nome ( G e nBa n k O U46 127 6.1 , reg i o n 4 265 5 8– 425198 ) r e t r i ev e d fr om P. o ce an ic a 414 r o o t s i n t h e E l b a / P i a n o s a a r e a . W e o n l y f o u n d C e leri na ta nt im o na s i n o n e l o c a l i t y , S a n N i -415 c ol a , l o c a ted o n the so uth ern Tyr r heni a n S e a , while it w as abse nt in seeds collect e d i n 416 west Si cily and al ong t he S t r ai t o f Sicily. C. ne p tuna w as origin ally f ou nd in r oot s of P. 417 oc e a n ica m ea do ws f r o m t he T usc an ar c h ipe l ag o, in n ort her n Tyrr heni a n. It is th eref o r e 418 i n t r i g u i n g t o q u e s t i o n w h e t h e r t h i s i m p o r t a n t e n d o s y m b i o n t i s r e s t r i c t e d t o m e a d o w s o f 419 the Tyrr h eni an S e a , p o s sibly to the western ge ne ti c po ol of P. o ce an ic a , o r i t s p r e s e n c e i s 420 widespr ea d i n t he w h o le b a s i n. Its prese nc e i n P. oc e anic a s e e d s s u g g e st s a po te n t ia l r ou t e 421 of verti c a l tr ans mission , like ly refl ectin g its ben e fi ci a l role f or th e h os t (M ohr e t al. 20 2 1) . 422 Alth oug h we w ere u n able to e valu at e th e PG P tr a its of this is ola te du e to its f a ilur e to 423 s urvive lo ng - te r m sto r age at – 80 ° C— p os sibly bec a use it m a y r e q uire m eso phi lic and 424 p art ially an a e r obi c gr o wth co nditio ns ( Cr am er et al. 2 0 11 ) —w e su c cess fully identifi e d 425 oth er nitr o g en-fixi ng b a ct e ri a in se eds collec t ed f r om di ff ere nt Si c ili an sites: Aur ei mon as , 426 M e ta bacill u s , a nd di ff ere nt Vi br io isol at e s o bt ai ned in this study displ aye d nitroge n -fixin g 427 capa ci t y . 428 I n t his study, we ide ntifie d fi ve Vi br io s e qu enc e g ro up s c o rr e spon d i n g t o kn ow n 429 s pecies n ot pre vio usly rep orted a s se ag r a s s end op hyte s , a nd ni ne isol ate s gro up e d into 430 t w o se qu enc e g r ou p s w i t h <9 7% i de n t i t y t o Vib r i o 1 6S r D N A seque n ces av ail able in th e 431 Ge nB ank. Furt her m u ltiloc us s e qu en ce typing (ML ST) will b e nec essary to d e t e r m i ne th e 432 p r e c i s e t a xo n o m i c s t a t u s o f t h e s e i s o l a t e s , w h i c h m a y r e p r e s e n t t w o n o v e l s p e c i e s ( J i a n g 433 et al. 2 02 2 ) . Nu m e ro us s tudie s h a ve r epor ted asso ci a tio ns bet ween Vi b r i o s p p . a n d ma r i n e 434 m i c r o - a n d m a c r o a l g a e ( S a m p a i o e t a l . 2 0 2 2 ) . S o m e Vi b r i o s p e c i e s a r e a l s o k n o w n t o 435 c ol oniz e se a gr a s s ca n opies (e.g . , Zos t er a s pp.) , c o ntributi ng t o t he r e du c tion of mi c robi a l 436 lo ads in s urro undi ng w a te r b o dies ( Reus ch et al. 20 2 1). Additio n al ly, e nd op hy t ic Vi b ri o 437 s p p . h a v e b e e n i s o l a t e d f r o m t h e m e r c u r y - t o l e r a n t h a l o p h y t e Ha l imi on e po rtu l ac oi de s 438 ( F id algo et al . 20 1 6). A s ynergis tic c o m bin atio n of Vib rio a est ua ri an u s ( a nitr oge n-fixing 439 ba ct e ri u m ) an d Vi b ri o pr o t e ol yt icu s ( a p h o sph ate-solubili z i ng s p ecie s ), b ot h prese nt in th e 440 m a n gr ov e rhi z osp her e , signifi ca ntly en h an c e d gr owt h in t he h al op hy t e S a li cor nia ( B a -441 s han et al . 20 0 0). Alth o ugh kn owl e dge a b out t he r ole of Vib r i o s p p . a s p l a n t e n d o p h y t e s 442 re ma in s l imi t e d , s e v e ra l spe c i e s i de n t if i e d in th i s s tu d y ha v e a l r ead y b e en a s soc ia t e d 443 with pl a nt-be n efi c i al tr aits, p arti cul ar ly t hos e li nke d t o nutr ient a cq uisition ( Hu a n g et al. 444 2 0 2 1 ) . H e r e , w e c o n f i r m e d t h e p r e s e n c e o f m u l t i p l e P G P p r o p e r t i e s i n n e a r l y a l l Vi b ri o 445 is ola tes obt ai ne d fr o m P. o c e a n ic a seeds, with V . eu rop ae us a n d V . z h u g e i s t a n d i n g o u t a s 446 the m ost pro m ising c an did ates. 447 M ost of t he re m ai ning c ultur a bl e b a c t eria isol ate d fr o m P. oc e ani c a s e e d s h a v e p r e -448 vio us l y be e n ide ntified i n pl ant spe cies a da pte d to e xt rem e en vir on m e nts, su c h as sali ne 449 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 12 o f 3 6 or co nt a m in ated s oils. Ha l om on a s sp., St ap h yl o c o c cus w a rner i , S . epid erm idi s, an d Ko cur i a 450 pal u str i s h a ve b e en r e p or te d as m e m bers of t he end oph yti c mi cr obiot a o f th e h al op hyte 451 Se id li t z i a r os m ar inus , w hi ch gro ws in s alin e soils ( Shur ig i n et a l . 2 02 0 ). 452 H al otole r ant str ains of M et a ba c i ll us in d ic us a n d S. w a rne r i e xhibiting m ulti ple P G P 453 t r a i t s w e r e a l s o i s o l a t e d f r o m t h e r h i z o s p h e r e o f t h e h a l o p h y t e Se suv i um p or t ula c a str um 454 (Jo hn e t a l . 2 023) . Ha lom ona s spp . a r e f re q u en t l y r ec ov e re d f rom ro o t s , l ea ve s ( Z ha n g e t a l. 455 2 0 18 ; Ke ar l e t al. 2 0 19 ) , and s eeds o f h a lo phyti c s peci e s ( W a ng et al. 2 0 21 ) . T he ir a bility t o 456 ad a pt t o h a rs h e n viro n m ent al co nditions is largely attrib uted t o s peci fi c g en om i c feat ures 457 (Chau d h r y e t a l . 201 7; A l i e t a l . 2 022 ). F o r i n s tanc e , Zha n g et al . ( Zh an g e t a l . 2 020 ) 458 s howed th a t e n do p hyti c Ha lom ona s s t r a i n s r e s p o n d t o s a l t s t r es s b y m od u l a t i n g o v er 8 0 0 459 gen e s in vo l ved in c ell ul a r and m et ab o li c pro cess e s . O ur results ar e fully co ns isten t w ith 460 the liter at ure, co nfir m i ng th e pr esen ce o f be nefi ci a l t r aits in Ha lom ona s a n d Ko c u r i a i s o -461 lates obt ai ne d fr om seeds o f a m a ri ne ph aner og a m . T he Koc ur ia i s o l a t e K p - P o C 9 i d e n t i -462 f ie d i n t his st udy bel ongs to a gro u p of endo p hytes ch ar a cteri z ed by a lim ited n u m b e r of 463 k n o w n b e n e f i c i a l t r a i t s . H o w e v e r , w e d o n o t e xc l u d e t h a t i t s r e c r u i t m e n t i n t o t h e s e e d 464 m i cr obi o m e m a y be link ed to ot he r a d v a nt ag e ous pr operties. I n f ac t, so m e Ko cu ria s p e -465 ci e s h a ve been ide ntified as a r seni c -r e s istant e ndo p hy tes, potenti ally s u pp orti n g h ost 466 pl ants i n envir o n m e nts c ont a m in a te d wit h ar senic and ot her h ea vy m et a ls (R o m án - Po n c e 467 et al. 20 1 6; Z ac a r i a Vit al e t al. 2 01 9 ). K. pa lus tr is i s o l a t e d f r o m P. o ce ani c a e xhibite d hig h 468 s e q u e n c e i d e n t i t y w i t h t h e a r s e n i c - r e s i s t a n t N E 1 R L 3 s t r a i n f r o m Sp h ae r alc e a an gu s ti f ol i a 469 gr owing i n m et a l- co nt am i n ated s oils ( Za ca ri a V it a l et al. 2 0 19 ). It is possible t hat its 470 p r e s e n c e i n s e a g r a s s s e e d s c o u l d a s s i s t s e e d l i n g s i n c o l o n i z i n g p o l l u t e d s i t e s . F u r t h e r -471 m o r e , t his s pecies m a y of f e r a dditio nal be n efits thr oug h t he pr odu c ti o n of bi o acti ve 472 compo un d s w i t h fun g ic i da l act i v i ty , a s re po r t e d f o r K. p al u st ri s s t r a i n s i s ol a t e d f r o m m a-473 r ine s pon ges ef fec tive ag a i ns t F u sa r i u m o xy sp o r u m ( S e t iaw an e t a l. 2 022 ) . 474 Alth oug h Sta phy lococ c us s p p . a r e p r i m a r i l y k n o w n a s p o t e n t i a l h u m a n a n d a n i m a l 475 p ath og e ns , t he y h a v e als o bee n fr e qu e ntl y isolat ed fr o m ve g et a tiv e org ans ( Sur ette et al. 476 2 0 03 ; C hiellini e t al. 2 01 5 ; Ga briele et al. 2 0 2 2 ) a nd seeds (Liu et al. 2 0 13 ; F add e tta et al. 477 202 1) of va r i ou s p lan t s . Ac cor d i n g t o th e l i t e ra tu re , S tap hylo coccu s s t r a i n s a d a p t e d t o a n 478 end op hyti c lif estyle diff er f r o m h u m an strains due t o th e pr e s e n c e of gene c l us te r s in-479 v o lv e d i n t he DNA dam ag e ca u se d b y ROS a cc umu la t ion an d U V ra d ia t i on (Chau d h ry 480 a n d P a t i l 2 0 1 6 ) . I n t h e c a s e o f o u r i s o l a t e s , d u e t o t h e l a c k o f c o m p l e t e g e n o m i c d a t a a n d 481 i n f o r m a t i o n o n t h e p r e s e n c e o f v i r u l e n c e f a c t o r s , w e d e c i d e d t o e xc l u d e t h e m f r o m P G P 482 tr ait an alyses, as no i m m edi ate bio t e c hno logi ca l a ppli cati o n c an be f oresee n for P. o ce -483 an ic a or agri c ultur al c ro ps . 484 Ba c ill u s a n d M e t ab ac i llus in c l ude m an y well-know n pl a nt e nd o ph ytes (Z h ang et al. 485 201 8; Y in e t a l . 2 022 ). Man y B a c illu s s p p. ar e alr eady used i n co m m er c i al biofor m u l atio ns 486 due to th e ir bro ad pl ant-ben e f i c i al e ff ec ts, includi ng pr ote c ti o n a g a ins t p at hog ens and 487 s tim ul ation o f pl ant gr o wth (Lo pe s e t al. 20 1 8) . In a dditio n to t he s e g ener a, t he pr e s e n c e 488 of Lysob act er i n P. oc e anic a s e e d s i s p a r t i c u l a r l y n o t e w o r t h y d u e t o i t s p o t e n t i a l a s a b i o -489 logi cal c ontr ol ag e nt (B C A). Lysob ac te r s p p . a r e p a r t o f c o m p l e x m i c r o b i a l c o m m u n i t i e s 490 as so c i at ed with soil and pl a nts a nd are c ap a ble o f c ol o ni z ing a wid e r ang e of extre m e 491 e n v i r o n m e n t s , i n c l u d i n g a q u a t i c s y s t e m s ( ( B r e s c i a e t a l . 2 0 2 0 ) . S o m e e n d o p h y t i c 492 L ysob act er str ains h ave alr e a d y dem o nstr a t ed B C A a cti v it y ag ainst s ever al pl a nt p a t ho-493 gens thr o ug h th e pr odu c tion o f lyti c en z y me s and antibi oti c c o m p ounds ( Dr e nk e r et al. 494 2 0 23 ; T u et al. 2 02 3 ). Ou r s tudy c o n fir ms t he p r e s e n c e of th ese t hr ee g e n er a as end op h yt es 495 in a m a rin e pl ant and pl a ces th em a m o n g the is olates wit h t he hig he s t n u m b e r o f P G P 496 p r o p er t i es . 497 Few A ur ei mon as spe cies h a ve b een r ep ort ed as pl ant e ndo p hyte s . A . en do p hy t i c a was 498 i s o l at e d i n C h i n a f r o m t h e h al o p h y t e A n ab as is e l a t i o r ( L i u e t a l . 201 6) , wh i l e A. a l t a mir en si s 499 w as re co ve r ed fr o m le a v e s of as h tre e s toler a nt t o d i eb a ck c a used by Hy m e noscyph us 500 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 13 o f 3 6 fr ax in eus ( B e c k e r e t a l . 2 0 2 2 ) . C o m b i n i n g f u n c t i o n a l g e n o m e a n a l y s i s a n d i n o c u l a t i o n a s -501 sa y s, B e c ke r et al . ( B ec k e r e t a l . 2022 ) de mon s t r a t e d tha t th i s s p ec i e s may co n t rib u t e to 502 in cre a s e d pla nt r e s ili en c e . O ur fin dings pr ovide a dditio n a l e viden c e f o r t he pr e s e n c e of 503 A urei m on as as a pl a nt e ndop hyte with b en efi c i al pr op erties, incl uding nitr oge n -fixin g 504 c a p ac ity. How ever, we ca n not e xclud e a pote nti al ant ag onisti c r ole, whi c h will r eq uire 505 f urt her i nvest i gati on t hr oug h sp ecifi c bio assays. 506 R ecent s tudies ha v e re p orted sev e r al end op hy ti c f un g a l s pecies associ a t ed wit h th e 507 veg e t ative or g ans of P. o ce a ni c a in th e ce n tra l Me d i t e rran e an ( To rta e t a l . 20 15, 20 22; 508 Vo hn í k e t a l . 20 16, 20 19) . Ho we ve r , onl y a l i m i te d numb e r o f cu l t u rab l e fun ga l a n d 509 oo m y cot e spe cies w ere r ec o ve red fr o m s e eds in t he pr e s e nt study. Th e tw o g ener a id e n-510 tif ie d h ad pre vious ly b ee n i s ol a ted fro m s urf a c e-sterili zed P. o ce a ni c a t i s s u e s ( T o r t a e t a l . 511 202 2). 512 Fung al g e ner a m os t fr equ ently d e te ct e d a s en d op hytes in ro ots and rhi zo m es— su c h 513 as Pos ido n io myce s , Lu lw o an a , Oc h r o c on i s , Peni ci ll iu m , and m e m bers o f th e X y l ar ia c eae 514 fami l y ( T o r ta e t a l . 20 15, 2022 ; Vo hn í k e t a l . 20 19; V oh n í k 202 2)—w e r e n o t i s o la te d f ro m 515 s e e d s . A c c o r d i n g t o T o r t a et a l . ( To r ta e t a l . 2 022 ), f un ga l e n dop h y te d i s t ri b u t i o n va r i e s 516 a m o n g plant org a ns i n P. o ce ani c a , wit h h ighe r col o niz atio n in b as al str u c tur es ( r o o t s a n d 517 r hi z o m es ) a nd o nly s po r adi c prese n ce i n le a ves. B a s e d on o u r findi ngs, w e ca n not ex-518 c lu d e t ha t fu n ga l en d o ph y t e s a re na tu ra ll y sc arc e i n se e d s o r t ha t th e y e xh i b i t a sp ec ie s 519 c o m p ositio n distin ct fr o m ot her pl ant o r gans , p art ic ul arly t he b as al o ne s . G ive n t he i m-520 por t ant r ole o f r oot-as so c i ate d f ungal end op hytes in m ai nt ai ning pl a nt h ealth (Tort a et al. 521 2 0 22 ) , f urth er rese ar ch is need e d t o deter m in e t he ti m ing and m od e o f r o ot c o l o ni z a t ion. 522 Pa e c i lo my ces s p e c i e s a r e f o u n d i n a w i d e r a n g e o f t e r r e s t r i a l e n v i r o n m e n t s . W h i l e 523 m a n y a r e k n o w n a s b i o l o g i c a l c o n t r o l a g e n t s a g a i n s t p e s t s a n d p a t h o g e n s , o t h e r s h a v e 524 also been d es c r ibe d as PG P fu ngi ( M ore n o-Ga vír a e t a l. 20 2 0). S o m e str a i ns of P . m a xim u s 525 (s y n . P. f or mo sus ; (H ou b r a k en e t al. 2 0 20 ) h ave been linked to t r e e decli ne s in b oth n a tu r al 526 an d cu l t i v at e d fo r e s t s (S a be r na sab e t a l . 2019 ; Oz an e t a l . 20 22) . Th i s s p ec i e s i s c urr e n t l y 527 c o n s i d e r e d a c o m p l e x c o m p r i s i n g a t l e a s t t h r e e c r y p t i c t a xa ( H o u b r a k e n e t a l . 2 0 2 0 ) , p o -528 tenti ally sh owi ng a d u a l r ole i n pl a nts — a c tin g as bene fi c i al e nd op h ytes by pr o m oti ng 529 gr owth thr ou g h h or m o ne prod u cti o n ( I AA and GA) a nd in c r e asing tol e r an ce t o abi oti c 530 s t r e s s es , e s p e c i a l l y h e a v y m et a l s ( B i l al e t a l. 2 0 1 7 ) . T h er e f o r e, t h e p r es e n c e o f P. m ax i m us 531 in P. oc e a n ic a see ds m a y co nf er d e v elo p m ent al a dv a nt a g es a nd r e du c e t he n e g ativ e ef fects 532 o f h ea v y meta l co n tamina t io n. 533 Th e g en u s Ha lop hyto phth ora inc l u de s sp e c ie s r e po r t ed a s sa p ro t ro ph s o r pat ho g en s 534 in m a n gr ov e f or ests , la g oo ns, e s tu ari e s , salt m ar s he s , and m a ri ne e cos yste ms (M ai a et al. 535 202 2). Ha lop hyto phth ora sp. Z os te r a in fe ct io n i n th e s eagr ass Zos t er a ma r i na h a s b e e n a s -536 s oc i at e d wit h r e d u ce d seed vi ab ility a n d seedli ng gro wth , p osing a thre at to c o a s ta l 537 e co s yste m restor ati on (Go vers et al. 20 1 6). R ecently, thr ee Hal oph ytop htho ra sp e c ie s ha ve 538 b e e n i so la t e d i n r o t ti n g P. oce a n ic a s e e d s an d p o s t u la te d to be t he cau sa t i ve a g e n t s of ea rl y 539 s e ed m ort ality in t hi s s e agr ass ( A l ag n a e t a l. 2 0 24 ). W h e n c ast s e e ds a r e c ollect e d a nd 540 p lac e d in aq uar ia , a f r a ct i on o f th e s e , ab o u t 10 -45%, c ov e r i n w h i t i sh mo u l d an d ro t . 541 Tr eat m e nt with a nti fu ng a l c hem i c als w as tested as a m e a n t o c o ntr ol in fe c t io n ( Al agn a e t 542 a l . 202 4) . Ou r f in d i n g s in d ica te t hat th i s s ap ro t r op h ma nag e s to s p rea d , s pat ia l l y a n d 543 thr o ugh g e n er atio ns, as a see d endo p hy te , and t h a t pr oto cols of s e ed disinf ecti on are 544 likely no t able t o p reve nt inf ecti on b y t his fungus. I nterestingly, sin c e we f o u nd 545 Ha loph ytop htho ra o n l y i n f e w s e e d s , i t i s p o s s i b l e t h a t t h e f r a c t i o n o f s e e d s t h a t r o t i n t h e 546 f i r s t w e e k s a f t e r c o l l e c t i o n i s a l r e a d y c a r r y i n g t h e f u n g u s a s e n d o p h y t e , a n d i s t h e r e f o r e 547 “d o o m e d ”. 548 C l arifyi ng th e pot enti a l p ath o g eni c ity o f Ha lop hyto p hth ora s p . a n d P. ma xi m us i n 549 M e diterr a ne an s eagr ass e s will req uire a dditi on al resea r c h, incl uding isol ation fr om 550 s y m p t o m a t i c p l a n t s a n d e xp e r i m e n t a l i n o c u l a t i o n o f s e e d s a n d s e e d l i n g s u n d e r c o n -551 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 14 o f 3 6 tr olle d conditi ons. Furt her m o re, field studies co m bi ning c ultur e-depend ent a n d me -552 tag en om i c ap pr o ac h e s a r e n eed e d t o in v estigate th e spr ead o f fu ng al end op hytes vi a P. 553 oc e a n ica see ds a nd t o a s sess their role i n t he e pide m i olo g y o f pot e nti al pl ant p at h o ge ns . 554 A m o ng t he lo ng-te r m c o nseq uen c e s of cli m ate ch a nge , t he de c li ne i n t he a vail abili t y 555 a nd qu ality of w a t er b odies r e s o u r c es r epr e s ents on e o f th e m ost criti c al ch allen ges for 556 glob al a gri c ulture. W at er is a n indis pens able c o m p o nent o f food pr odu c tio n; a cc o r ding t o 557 U N and F A O e s ti m a tes, a p pro xi m ately 3,0 0 0 - 5 , 0 00 litres of w at e r a r e r eq uired t o satisfy 558 the d aily die t ar y needs o f a singl e p erso n. Furt he r m or e, th e G l ob a l Risks R e p ort o f t he 559 W orld E c ono m i c For u m identifies w at er c r ises as the thir d m o s t sev e r e glo b al th re a t in 560 t e r m s o f i m p a c t o n h u m a n i t y ( P a t h i r a n a a n d C a r i m i 2 0 2 4 ) . I n t h i s s c e n a r i o , s a l i n i t y h a s 561 e m erge d a s o ne of t he m a j o r ab i oti c con str ain t s li miting cr o p pr od u c tivity a nd q u ality. 562 The Fo od a nd Agri c ulture Org ani z ati on (F AO 20 2 4) e s ti m at e s th at ar ou nd 1 . 4 billio n 563 h e c t a r e s - o v e r 1 0 % o f t h e g l o b a l l a n d s u r f a c e - a r e a l r e a d y a f f e c t e d b y s a l i n i z a t i o n , w i t h 564 a n additio n al on e b illion he ct ares a t i m m in ent risk du e t o c li m a te ch a ng e and unsus-565 tai n able m a n age m ent pr acti ces. O ur fi nd ings hig hlight t h at end op hyti c m ic ro org a nis ms 566 as so c i at ed with P. o ce a ni ca re prese nt a v al uabl e and l a r gely unt a p pe d sou rc e of 567 pl ant-be nefi ci a l m i c ro be s . Cultiv a ble e nd op hy t e s isol at e d fr o m P. oc e a n ic a d i s p l a y t r a i t s 568 pot enti ally lin ke d t o s al inity tol e r an c e , su c h as th e prod u cti on o f pl a nt 569 g r o w t h – p r o m o t i n g s u b s t a n c e s a n d a l l o w t o g u a r a n t e e n i t r o g e n f i xa t i o n e v e n i n s i t u a -570 tions o f s a li ne str e s s. The us e of t he s e e ndo p hyte s as bioin o cul a nts i n te r restri al cr o ps 571 c o uld ther ef ore c ontrib ute t o e nh a nci ng pl ant r e s ilie n c e u nder s al in e c onditi on s, im-572 pr o v in g b oth pr odu c ti vity a nd s ustai n ab i lity in salt- af fe c t ed soils. 573 5. Conc l us i ons 574 T h is s tudy u nr a ve ls t he m ec h anis ms of tra nsgener ati on al and sp ati al dif fusio n of 575 e n do ph y t ic com mun i t i e s in P. oc e an ic a m e a d o w s . I t a l s o e xp a n d s t h e l i s t o f c u l t u r a b l e 576 end op hyte s fr om t his s pecies, pr ovidi n g a th or oug h c h ar a cteri z a ti o n o f t he ir p ut ative 577 ben e f ici al ser vi ces. M os t of th e is olated mi c r ob i al sp e cie s exhibit tr aits ass o c i ate d with 578 pl ant nutr ition a nd gr owt h pro m o tio n . In additio n , is ol a te sele ctio n re veal e d the 579 c o-o cc ur re n c e of dif fere n t m ic ro bi al sp ec ies withi n t he s am e se e d, p ote nti a lly c ap abl e of 580 per for m ing co m p lem e nt ary or overl a ppi ng f u n ctions . Th e ir i m p a ct on see dling deve l-581 op m e nt and p at h ogen resis ta nce will be f urth er e v al u ate d t hr oug h in pl a nt a tr ials und er 582 c o ntrolle d c o nditio ns, l ay ing th e g ro und wor k for th eir pot e nt ial us e i n s tr ate gi es ai m e d 583 at pr ote c ti ng a nd resto r i ng this d o mi n ant Mediterr a n ea n se a gr as s. Additio n ally, the 584 s trains i s ol a ted an d c h a r ac te r i zed in this w ork ar e fully cu l t ur a b le i n vitr o , allo wing a 585 s cal able a ppli cati o n as in o c ula nt in ot he r plant s peci e s , s u c h as ot he r se agr as ses, but a ls o 586 terr e s trial cro ps , p ote nti a lly e nh an ci ng t o ler an ce t o s tress s uch as salin e s oils. 587 588 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 15 o f 3 6 Author Contributions: 589 Da l ila C r u c it ti : Co n c ep t u a liz at i o n ( e q ua l ); me t h od o lo g y ( l ead ); v a l i dat io n (l ea d) ; f o rm a l a n a ly s i s 590 (le a d ); inves ti g a t ion (le a d ); r esource s (s u pp o rtin g ); d at a c u r at ion (le ad); w ri t ing —o rig i n a l dr aft 591 pre pa r a t io n ( le ad); wr i ting—re view a nd ed i ting (le ad) ; vi s ua li za ti o n (equ a l) . 592 Alb e r t o Sute r a: In vest ig a t i on (e qua l) ; re s ource s (equal) ; v isuali z ati on (suppor ting). 593 Rober to D e M ic h e le : Conce p tu a liz ati on (su ppo r tin g) ; f orm al an a lysis ( equal) ; re s ou r ces (le ad ); 594 w riting — or igina l dr a f t pre par ati on (e q u a l); writing— rev iew an d ed it ing (equ al); v isuali z ati on 595 (le a d ); p r o jec t adminis tr a ti o n (suppor ting ); fu nd ing acqu isit i o n ( le ad). 596 Frances c o Ca rimi: Res o urc es (supp o r ting ); w ritin g— re vie w a n d e d it i ng (equ a l) ; visu a l iz at ion 597 (e qu a l) ; pr ojec t a d minis tr ati on (le ad) ; fu nding acqu is iti on ( e q ua l) . 598 St ef ano B ar o ne : Forma l ana ly s is (sup por t ing ); d at a cur ati on (s u ppor tin g ). 599 Fa b i o B a dal a men ti : Re so urc es (equ al) ; r e vie w a nd e di tin g (s u ppor ting); pro ject a dmini str a t i on 600 (e qu a l) . 601 D a v ide P a c if ico: Conce p tu a liz ati on ( le a d ); m ethod ol o gy (equ al) ; v ali d a t i on (e qual) ; da t a c u r ati on 602 (e qu a l) ; writ ing — orig inal dr a f t pre pa r at i on ( equa l ); wr itin g—re view a n d ed it ing (e qua l) ; visu ali-603 z ati on (e qual) . 604

This re se arch w as fund ed b y the pr oj e c t M a rine H az a r d , PON 03P E_ 00 20 3_ 1 605 (Minist ry o f E du c at ion , Un iv ers ity a n d Rese a r c h, MUR, I t a l y 606 Data availability: The d at a se ts gener at ed and an aly z e d du ring the curre nt stud y are av a ila b le in 607 the G enB a n k r ep o s i tory with th e acc ess i ons li sted in T able 1 . 608 Conflicts of Interest: No n e d ec la red 609 Ethics statement: No n e re q u i red 610 611 Re fe re nces 612 A l a g n a A , G i a c a l o n e V M , Z e n o n e A , et a l ( 2 0 2 4 ) T a n n i n s a n d c o p p e r s u l p h a t e as a n t i m i -613 cr obi al ag ents to pr ev e nt c ont a m in ati o n of P o si d oni a o ce a ni c a s e edling culture for r est o r a -614 t i o n p u rpo se s. F r on t P lan t S c i 1 5:14 333 58. h t t p s: / / do i . o rg /10 .3 389 /fp l s. 2 024 .143 3358 615 A l i B , H a f e e z A , J a v e d M A , e t a l ( 2 0 2 2 ) R o l e o f e n d o p h y t i c b a c t e r i a i n s a l i n i t y s t r e s s 616 a m eli o r a t ion by physiol ogi cal a nd m o le c ul ar m e c ha nis ms of de fens e : A co m pr e h ensive 617 re v i ew . S o u th A f r ican J ou r na l o f Bo ta n y 151 :33– 46. 618 h t tp s : / / do i . o r g /1 0 .1 016/ j . sa jb .2 022 .09 . 036 619 Ar n aud- H aond S , M ig li a c cio M, Di az-A l me l a E, et al ( 2 0 07 ) V i cari a n ce p att erns in the 620 M e d i t e r r a n e a n S e a : e a s t – w e s t c l e a v a g e a n d l o w d i s p e r s a l i n t h e e n d e m i c s e a g r a s s 621 Po si d on i a o ce ani c a . J B i og e o g r 34 :9 63–976 . h t t p s : / / do i . o rg /10 .111 1/ j.1 365 -269 9.20 06. 016 71. x 622 Ba c ci T, Sc a rdi M, T om as e llo A , V ali ant e LM, Pi a zz i L, C al v o S, B ad a l a m enti F, D i nu z z o 623 F , R a i m o n d i V , A s s e n z o M , C e c c h i E , P e n n a M , B e r t a s i F , P i a z z i A , L a P o r t a B ( 2 0 2 5 ) 624 Lon g-ter m resp onse o f P osi d o ni a oc e a n ic a m ea do w r e s to r a t ion at th e p o pul ati o n a n d pl ant 625 level : i m pli ca tio ns f or m a na ge m ent d eci sions. 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It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 16 o f 3 6 h alot ole r ant Azo s p i ri ll um s pp. Biol Fe r til S oils 32: 2 65 – 27 2 . 633 h t tp s : / / do i . o r g /1 0 .1 007/ s00 374 0 00 0246 634 B e c k e r R , U l r i c h K , B e h r e n d t U , e t a l ( 2 0 2 2 ) G e n o m i c C h a r a c t e r i z a t i o n o f Au re imo n as 635 al ta mir en s i s C 2P 0 0 3— A S p e ci f i c M em ber of t he Mi c r obi o m e of Fr a xinu s e xc el si o r T r e e s 636 T o l e ran t t o A s h D i e bac k . P lan t s 1 1 :1– 17. h t t p s: / / do i . o r g /10 .3 390 /p lan t s 1124 3 4 87 637 Bilal S, Kh a n A L, Sh a h za d R, et al ( 20 1 7) End o phyti c P aec il om yc e s f o r m o su s L H L 1 0 A u g -638 men t s Gl y ci ne ma x L. A d a pt ati on to N i-c ont a m in atio n t hr ough af fecti ng e ndog e n ous 639 phyt o hor m o n es and oxid ati ve stress . Front P la nt Sci 8 : 87 0, 1 –1 7 . 640 h t tp s : / / do i . o r g /1 0 .3 389/fp l s . 20 17. 008 70 641 B l a n c he t E , P r a d o S , S t i e n D , e t al ( 2 0 1 7 ) Q u or u m s e n s i n g a n d q u o r u m q u e n c h i n g i n t h e 642 M e diterr a ne an S e ag r ass P o s id oni a o c e ani c a m i c r o b i o t a . F r o n t M a r S c i 4 : 1 – 1 0 . 643 h t tp s : / / do i . o r g /1 0 .3 389/fma rs .2 017. 002 18 644 B r e s c i a F , P e r t o t I , P u o p o l o G ( 2 0 2 0 ) Lyso bact e r. I n: Be nef ici al Mi c r o b es in Agr o-E cology : 645 Ba c te r i a a nd Fu ngi. Ac a d emi c Pr e s s, 31 3- 3 38 646 C a l v o S , C a l v o R , L u z z u F , e t a l ( 2 0 2 1 ) P e r f o r m a n c e a s s e s s m e n t o f Po sid o nia oc ean i c a ( L . ) 647 Delile r estor a tio n e xp eri m ent o n dead m a t te t wel ve ye ar s a fte r pl anti ng—s tru ct ur al and 648 fun c t i ona l mea do w f ea tu re s. 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Ma r Ec o l Pr o g S e r 456 :21– 27. 656 h t tp s : / / do i . o r g /1 0 .3 354/me p s0967 2 657 C h aud hr y V, P atil PB ( 20 1 6) G e n o mi c i nv estigati on r ev eals ev oluti on and lif e s tyle ad-658 ap ta t i on of e n do ph y t ic St a p hy l oc occ us e pi de r m i dis . Sci R e p 6 :1– 11. 659 h t tp s : / / do i . o r g /1 0 .1 038/ s r ep19 263 660 C h a u d h r y V , S h a r m a S , B a n s a l K , P a t i l P B ( 2 0 1 7 ) G l i m p s e i n t o t h e g e n o m e s o f r i c e 661 end op hyti c ba ct e r i a : D ive rsity a nd distr ib ut ion of Fir m i cut e s . Fr ont Mi c r obi ol 7: 2 11 5 . 662 h t tp s : / / do i . o r g /1 0 .3 389/fmic b .2 016 .021 1 5 663 C hi e llini C, M a id a I, E m ili ani G, e t al ( 2 0 15 ) E nd o p hyti c a nd r hi z o s ph e r i c b act e r i a l 664 c o m m u nitie s isol ate d f ro m t he m edi ci n a l pl ants E chi na cea p urp u rea a n d Echi na c ea 665 a ng u s t ifo l ia . I nter n atio nal Mi cr obio l ogy 1 7: 165–174. h t t p s: / / do i .o r g /10 .2 436 /20. 150 1 . 01 .21 9 666 C o m p a n t S , B r a d e r G , M u z a m m i l S , e t a l ( 2 0 1 3 ) U s e o f b e n e f i c i a l b a c t e r i a a n d t h e i r s e c -667 ond a ry m et a b olites to c o ntr ol gra pe vine p at h o ge n dise a s e s . Bio C ontr ol 58: 4 35 – 45 5 . 668 h t tp s : / / do i . o r g /1 0 .1 007/ s10 526 -012 -9479 -6 669 C o m p a n t S , S a m a d A , F a i s t H , S e s s i t s c h A ( 2 0 1 9 ) A r e v i e w o n t h e p l a n t m i c r o b i o m e : 670 Ec ol og y, f u ncti o ns, a nd e m e r ging tre n d s in mi cr obi a l appli c a t io n. J A d v Re s 1 9 :2 9 –3 7 . 671 h t tp s : / / do i . o r g / 10 .1 016 /j . ja re .201 9.0 3.00 4 672 C r a m e r M J , H a g h s h e n a s N , B a g w e l l C E , e t a l ( 2 0 1 1 ) C el e r in at an ti mon as d i az ot r op hic a g e n . 673 n o v . , s p . n o v . , a n i t r o g en - f i xi n g b a c t er i u m r e p r es e n t i n g a n ew f a m i l y i n t h e 674 Gam ma pro te oba c t e r ia , C e le r i na ta n t imona da ce ae fam. no v . I n t J S y st E v o l Mi c ro b io l 675 61:1 053 –10 6 0. h t t p s: / / do i . o rg /10 .1 099 / i j s. 0. 01 790 5-0 676 da S ilv a N, T ani w aki M H, Ju n queri a VCA, et a l ( 2 01 3 ) M ic ro biol og i c al exa m in ation 677 m et h ods of f ood a n d w at e r : a l abor a t or y m a n u al. 2 nd Editio n. C RC Pr e s s, 1 - 46 9 . 678 h t tp s : / / do i . o r g /1 0 .1 201/9 781315 16 5 011 679 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 17 o f 3 6 del C astillo I, He r n á nde z P, L af u ente A , e t al ( 2 01 2 ) Self-bi ore m edi at ion of 680 c ork-pr o c e s sing was te w at e r s by ( chl or o) ph en ol-de gr adi ng b act eri a i m m o bilised o nto r e -681 si d u a l co r k pa rt ic l e s . Wa t e r R e s 46 :1 723 –1 7 34. h t tp s: / / d o i . o rg / 1 0.1 016 / j .wa t r e s .2 011 .12. 038 682 Di C a r lo G , B a d al am e nti F, Je nsen A C, et al ( 2 0 05 ) C olonis a t i o n pr o cess of veget ative 683 fra g men t s o f Po sid o nia oc e a ni ca ( L .) 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Sc i R e p 11 :1–1 2. h t tp s : / / d o i .o r g /10 .10 38/ s415 98 -0 21 -8 639 9-5 704 FA O ( 2 0 24 ) Glob al st atus o f salt- af fe cted soils - Main r ep o r t. FA O , Ro m e 705 F i d a l g o C , He n r i q u e s I , R o c h a J , et a l ( 2 0 1 6 ) C u l t u r a b l e e n d o p h y t i c b a c t e r i a f r o m t h e s al t 706 mar sh p lan t H ali m ion e po r t u l acoid es : phyl ogeneti c d ivers ity, f un c ti o n al ch ar a ct e ri z ati on, 707 an d in flu e nce o f me ta l( l o i d ) c on tami na ti o n . En v i r o nmen ta l Sc i enc e an d Po l l u t io n Re -708 se a rch 2 3:1 0200 –1 0 214 . h t tp s : / / d o i .o r g /10 .100 7/ s113 56 -0 16 -62 08-1 709 Fr ank A C, Gu z m á n J P S, Sh ay JE ( 20 1 7) Tr ans miss i on o f b a ct e ri al en do ph y tes. M ic r o or-710 g an i sms 5 :70 , 1– 21. h t t p s: / / do i . o r g /10 .3 39 0 /micr oo r gan i sms504 007 0 711 G abrie l e M , Vit ali F, C he l u c c i E, Chi ell ini C ( 20 2 2) C h ar a cteri z ati on o f th e C ultiv able 712 Endo p hyti c B a c t eri al C om m u nity of See ds a nd S pr o uts of Ca n nab is sa t i va L. a n d Per-713 sp e c t i ve s f o r t he A p p l ica t ion a s B io s t imu lan t s . Mic ro o r gan i sms 10 : 174 2, 1– 20. 714 h t tp s : / / do i . o r g /1 0 .3 390/mi cr oo r gan i sms100 917 42 715 Ga rc ia s- B o n e t N , A r r ie ta JM, de S an tana C N , e t a l (20 12) En do ph y t ic bac te r ia l co m mu -716 nity o f a Me d i t e r r a n e a n m ar in e angi osp er m ( P o si d oni a oc e ani c a ) . 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Pr o c e edi ngs Roy a l 725 Soc i e t y B 2 83: 201 60 8 12: h t t ps : / / do i . o rg /1 0 .1 098 /rs p b .20 16. 081 2 726 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 18 o f 3 6 H ard oi m PR, va n O ve r be e k L S, Be r g G , et al ( 2 0 15 ) Th e Hidde n W orld w ithi n Pl ants : 727 Ec ol og i c al and E vol utio n a r y Co nsider a tions f or Def ining Fu n cti o ni ng of Mi c robi a l 728 Endo p hytes. Mi cr obi olo gy a nd Mole cul ar Biology Re views 79: 2 93 – 32 0 . 729 h t tp s : / / do i . o r g /1 0 .1 128/mmbr. 000 50 -14 730 H arley JP , Pr esc ott LM ( 2 0 02 ) L ab or at or y Exe r c is e s in Mi c r ob iol ogy, Fifth Editio n. 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J o urn al of Pl a nt P at hology 1 0 4: 1 16 5 . 800 h t tp s : / / do i . o r g /1 0 .1 007/ s42 161 -022 -0114 2 -x 801 P a cifi co D, S qu a r ti ni A, C ru citti D, et a l ( 2 01 9 ) T he Role of t he E nd op hy t ic Mi c r ob i o m e in 802 t h e G rap e v i ne Re sp on s e t o Env i r onmen ta l T ri g g e r s. F ron t Pl an t Sc i 10: 125 6, 1– 15 . 803 h t tp s : / / do i . o r g /1 0 .3 389/fp l s . 20 19. 012 56 804 P athir a n a R, C ari m i F ( 2 0 24 ) Pl ant Bi otec h nol o gy —A n I ndisp ens able T ool f or Cr op I m-805 p rov emen t . P lan t s 13 (8) : 11 33, 1 -18 . h t tp s: / /d o i .o r g /10. 339 0/p la n t s130 811 33 806 Pe nros e DM, Gli ck BR ( 2 00 3 ) Me t h ods f or is ol a ting a nd c h ar acte ri zi ng A C C 807 d eamina se -co n ta in in g p lan t g r o w t h - p r o mot i n g rh izo b ac te ri a . 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It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 20 o f 3 6 R uoc co M, L a co r at a G , P alat ell a L , Pro ve ra I, Z en on e A, M arti nez M, Dat ol o E, P a z za gli a 818 J , G i a cal o ne VM, B ad al a m enti F , Pr o ca c ci ni G ( 2 0 24 ) M ov e m ent E cology of a C oas ta l 819 Fou nd ati on S e a gr a s s S peci e s : I nsig hts Fro m Gen e t ic Dat a a n d O ce anogr a p hi c Mo delling. 820 D i ve rs i t y a n d D i s t ri b u t io n s 3 1 :e 139 44, 1 -18. h t tp s: / / d o i . o rg /10.1 111 / d d i .139 44 821 Sa be rna s a b M, J a m al i S , Ma re fa t A , A b ba s i S (2019 ) M o l ec u la r a n d Pa th o ge n ic Cha rac -822 t e r i st i c s o f P a ec il o my ce s f orm o su s , a N ew C a us a l Ag e nt o f Oa k Tre e Dieb a c k i n Ir an . Forest 823 Sc i e nce 65 :743 –75 0. h t t p s : / / do i .o r g /1 0.10 93 /fo r s c i/ f xz 045 824 Sampa i o A, S i l va V, Po e ta P , A on of r i e s e i F (202 2) Vib rio s p p. : Life Str a tegie s , E c ol ogy, and 825 R isks in a C h a ng ing E nv iron m e nt. Div e r sity (B asel) 14: 1 –2 6 . 826 h t tp s : / / do i . o r g /1 0 .3 390/d1 402 009 7 827 S a ntoy o G, M or eno- H a gelsieb G, de l C ar m e n Oro z co- M o s qu e d a M, Gli c k BR ( 2 01 6 ) Pl ant 828 gr owth-pr om oti ng ba cter i a l end op hytes. Mi c r obiol Re s 1 8 3 : 9 2 –9 9 . 829 h t tp : //d x. do i . or g /1 0 .10 16/j .micr e s .20 15. 11 .0 08 830 Sca nu S , P ia zzo l l a D, B o namano S , e t a l (20 22) Ec o nomic Eva l uat i o n o f Pos ido nia o c ea ni ca 831 Ec osyst em S er vi ce s al ong t he It ali a n C o ast. S us t a in a bility ( S wit ze r la nd) 14: 1 –1 7 . 832 h t tp s : / / do i . o r g /1 0 .3 390/ s u14 01 0 489 833 S c h w yn B, N e il ands JB ( 1 9 87 ) Uni v ers a l c hem i c a l ass ay f or t h e det ectio n an d det e r m in a -834 t i o n o f s i d e ro pho r e s . A na l B i oc hem 160 :47– 56. 835 h t tp s : / / do i . o r g /1 0 .1 016/0 003 -269 7(87 ) 9 0612 -9 836 Seti aw a n A, S e ti aw a n F, Juli asih NL GR, e t al ( 2 02 2 ) Fu ng i cid e A ctivity o f Cultur e Extr a c t 837 fr om Koc u ria pal u str is 1 9C38A 1 a ga in s t Fus ar iu m ox ys po ru m . J our n al o f F ungi 8: 1 –1 1 . 838 h t tp s : / / do i . o r g /1 0 .3 390/ j of803 028 0 839 S h u r i g i n V , E g a m b e r d i e v a D , L i L , e t a l ( 2 0 2 0 ) E n d o p h y t i c b a c t e r i a a s s o c i a t e d w i t h h a l -840 op hyte S eid li tz ia ro sm a r i nu s E hre nb . e x Boiss . fr om s aline s oil o f U z be kist an an d t he ir 841 p la n t b e n ef ic ia l t ra i t s . 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Pl an t an d S o i l 2 53: 381 -3 90 849 Sut er a A , Bo n avir i C , S pi nelli P, et al ( 20 2 4) Fr uit e n casin g preser ves th e dispers al pot e n-850 tial a nd vi ability of str and ed P os i do nia oc e a n ic a s e e d s. Sc i Re p 1 4 :62 18. 851 h t tp s : / / do i . o r g /1 0 .1 038/ s41 598 -024 -5653 6 -x 852 Sut er a A, S pin elli P, P ac ifi co D , e t al ( 2 0 25 ) E xtended s tor age of Po sid on ia oc ean i c a s e e d s 853 a nd s e edlings: A br e a kt hro ug h f or year-r o u nd seagr ass restor a tio n. Biol C ons e r v 854 310 :111 385 . h t t p s : / / do i .o r g /10 .101 6/ j . b i ocon . 202 5.1 1138 5 855 Tar q uini o F, Attl an O , V a nderkli ft M A, e t al ( 2 0 21 ) Distin ct End o phyti c B a c t eri al C o m-856 m u n i t i e s I n h a b i t i n g S e a g r a s s S e e d s . 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It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 21 o f 3 6 T o r t a L , B u r r u a n o S , G i a m b r a S , e t a l ( 2 0 2 2 ) C u l t i v a b l e F u n g a l E n d o p h y t e s i n R o o t s , 864 R h i z o m e s a n d L e a v e s o f Po sido n ia oc eani c a (L .) Delil e a l o ng t he C o a s t of S i cily, It aly. 865 P la n t s 11 :1 139 . h t t p s : / / do i .o r g /10 .339 0/ p lan t s 11 091 1 39 866 T o rta L , Lo P ic col o S , P iazz a G , e t a l (2 015 ) Lul w oa n a sp . , a da rk s ep ta te e nd oph y t e in 867 ro o ts o f Pos ido nia oc ea ni ca (L.) D e lile se a gr as s. Pl ant Bi olo gy 1 7 :5 0 5 - 51 1 . 868 h t tp s : / / do i . o r g /1 0 .1 111/p l b .122 46 869 T r u y e n s S , W e y e n s N , C u y p e r s A , V a n g r o n s v e l d J ( 2 0 1 5 ) B a c t e r i a l s e e d e n d o p h y t e s : 870 Ge ne r a, ve r ti ca l tr ans m iss ion an d inter a c tion with p lants . En vir on Mi cr obiol R ep, 7:1- 1 1 . 871 h t tp s : / / do i . o r g /1 0 .1 111/1 758 -222 9.1 2181 872 T u C- K , Wan g P -H , L ee M- H (2023 ) Th e e n do ph y t ic bac t e r ium Ly so bacte r f irm ic u tima c h us 873 s train 5 - 7 is a pro m ising bi oc o ntrol age nt ag a i n s t r i c e s ee d l i n g d i s e a s e c a u s ed b y Py thi u m 874 ar rhe nom a ne s i n nu r s e r y tra y s. P la n t D i se a s e 107 :10 7 5 -10 86. 875 h t tp s : / / do i . o r g /1 0 .1 094/PD IS -05 - 2 2 -11 95 -R E 876 Ug ar e lli K , C h a k r ab arti S, L a as P, Sti ng l U ( 2 01 7 ) Th e se ag r ass h olobi o nt a nd its 877 m i cr obi o m e . Mi cr oor ga n isms 5: 8 1, 1 –2 8 . 878 h t tp s : / / do i . o r g / do i :10 . 339 0/mic roo r ga n i sms50 40 0 81 879 V i l g a l y s R , H e s t e r M ( 1 9 9 0 ) R a p i d G e n e t i c I d e n t i f i c a t i o n a n d M a p p i n g o f E n z y m a t i c a l l y 880 A m pli fied Rib oso m a l DNA fr o m S e v er a l C rypto c o ccu s S pe c i es. J our n a l o f B a c ter iology 881 172 :8 , 4 238 -424 6. h t tp s : / / do i . o r g/10 .11 28/ jb .172 .8.4 238 -424 6.19 90 882 V oh ník M ( 20 2 2) Ar e lul w or t hio id f ungi dark sept ate e ndo p hyte s of th e d o mi n ant M e d-883 iterr an e an se agr ass Po s i do ni a o c e anic a ? Pl ant Biol ogy 24: 1 27–133. 884 h t tp s : / / do i . o r g /1 0 .1 111/p l b .133 53 885 V o h n í k M , B o r o v e c O , K o l a ř í k M ( 2 0 1 6 ) C o m m u n i t i e s o f C u l t i v a b l e R o o t M y c o b i o n t s o f 886 the Seagr ass P o s i d oni a oc e ani c a in t he No r thw est Mediterr a nea n Sea Are D o m in a t e d by a 887 H i t he r t o Un de sc rib e d P l eo s po ra l e an Da r k Se p ta te En do ph y t e . Mi c r ob Eco l 7 1 :44 2–4 51. 888 h t tp s : / / do i . o r g /1 0 .1 007/ s00 248 -015 -0640 -5 889 Vo hn í k M, Bo r o vec O , K o la ř í ko vá Z , e t a l (2 019) E xte n s i v e samp l in g a n d 890 high-thr ough put s e q ue n c i ng r ev eal Po si d o n iom yc e s a t rico lor ge n . Et s p. N ov. ( A igi a lac eae , 891 Pleosp or al es) as t h e d o m i n ant ro ot m y c obio nt o f t he d om in a nt M edite r r a nea n s e agr ass 892 Po si d on i a o ce ani c a . Myco K e y s 5 5 :59 –86 . h tt p s : / / d o i .o r g /10 .389 7/myc o k ey s. 5 5.3 568 2 893 W a ng H , Na rs ing R ao MP , Ga o Y, e t al ( 2 02 1 ) I ns ig hts into t he e nd o ph yti c b act e r i a l 894 c o m m u n i t y c o m p a r i s o n a n d t h e i r p o t e n t i a l r o l e i n t h e d i m o r p h i c s e e d s o f h a l o p h y t e 895 Su ae d a gla uc a . B MC Mic r o b i o l 21 :1– 16. h t t ps : / / d o i .o rg /1 0.1 186 /s12 866 -021 -0220 6-1 896 W hite T J, Bru ns T , L e e S, e t al ( 1 9 90 ) A m pli fi cati on a nd dire c t s eq ue n c in g o f fu ng al ri-897 b o s o m a l R N A G e n e s f o r p h y l o g e n e t i c s . I n : P C R P r o t o c o l s : A G u i d e t o M e t h o d s a n d A p -898 p l ica t i o n s. Acc ade mic Pr e ss I nc . , 31 5–3 22 899 W ilson K ( 19 9 7) Prep ar ati o n of Gen o m i c DN A fr o m B a c t eri a. Cur r ent Pr ot oc ols in M o-900 l ec ul a r B io l og y 56 : 2.4 . 1 -2 .4 .5 901 Yi n Z, Wan g X , H u Y , e t a l (2 022) M eta ba c il l u s don gyi n ge n s i s s p. n o v . I s R e p re s e n te d b y t he 902 Pl ant Gro wt h-Pro m o ting B ac teri u m BY 2 G2 0 Is o l at ed fr o m S aline-Alkalin e So il a nd En-903 hanc e s t he G row t h o f Zea m ays L . u n de r Sa l t S t r e s s. mS y st e ms 7 :1– 16. 904 h t tp s : / / do i . o r g /1 0 .1 128/ms y s t ems .01 426 -21 905 Z a c a r i a V i t a l T , R o m á n - P o n c e B , R i v e r a O r d u ñ a F N , e t a l ( 2 0 1 9 ) A n e n d o p h y t i c Ko cur i a 906 pal u str i s str ain h a rb oring m ulti ple arse n ate re d u ct ase genes. Ar c h Micr obi o l 907 201 :128 5–1 2 93 . h t t p s : / / do i . or g /10 . 100 7/ s002 03 -01 9 -01 692 -2 908 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 22 o f 3 6 Z h ang J, W a ng P, Ti a n H, et al ( 20 1 8) I dentifi c a t ion of interi or s alt-toler a nt ba ct e r ia f r om 909 ic e pl ant M e s e m brya n them um c r y sta ll in um a nd ev alu a t io n of th e ir pr o m oting eff e c ts . 910 S ymb io s i s 76 : 243 –252. h t t p s : / / do i . o rg /10 .1 0 07/ s131 99 -0 18 -0 551 - 6 911 Z h ang J, W ang P, T ia n H, et al ( 2 02 0 ) T r ans c r ipt om e an alysis of i c e pl a nt 912 gr owth-pr om oti ng end oph yti c ba c te r iu m Ha lom ona s s p . St r ai n M C 1 t o i d e n t i f y t h e g en e s 913 inv olv ed in s alt toler a n ce. Mi cr o org anis ms 8 : 8 8, 1 –1 8 . 914 h t tp s : / / do i . o r g /1 0 .3 390/mi cr oo r gan i sms801 008 8 915 916 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint Tab l e 1: List of t he 4 4 i d entified e n do p hy te s isolate d fr om th e Posidonia oceanica s ee d s wi t h th e c o r re s po n d in g mo le cu la r i de n t i f ica t i on . 917 Is o lat e n a me GenB a n k acc ession numb er Be st BLAST m a tch Perc e n t id e n ti ty (16 S rDN A) Se que nc e l en g th ( bp ) Fa mi l y Ph y l u m I sola t ion loc al ity Aa - P oS C 9 OQ883 73 2 A u re i m onas altam iren sis strain LS1 - 9 6 ( O P8 72 59 5 ) 10 0 13 53 Aurantim onad a ce a e Prot e ob a c ter i a Sci acc a Bs p- P o C 1 OQ883 73 3 B ac i l l u s s p. GD 0 70 6 ( D Q08 45 40 ) 99 .8 6 14 46 B a c i l l a c ea e Firm ic ute s C o rnino Cn-Po SN8 OQ883 73 4 Ca Cele rinatantimonas nept u na isola te 1 (OU 4 61 27 6 ) 98 .9 3 14 08 C el e r i na ta nt i m o na da c ea e Prot e ob a c ter i a San N ic ol a Hmsp -PoS N 7 OQ883 73 5 H al o m on as sp . K M M 35 50 ( A F3 16 14 3) 10 0 10 62 Hal o mona d a ce a e Prot e ob a c ter i a San N ic ol a Kp-Po C9 OQ883 73 6 Kocuria palustris strain MS2- 4 (MN 23 87 49 ) 10 0 14 11 Micrococc ace a e Ac t i no ba c t e r ia C o rnino Lysp-Po M4 OQ883 73 7 Ly so bacte r sp . KS A 20 (GU 04 89 37) 99 .5 11 27 L y s ob a c t era cea e Prot e ob a c ter i a M ars al a Mr-PoS C2 OQ883 73 8 Marinomo n a s r h izo mae str ain IVIA-Po -1 45 (NR_ 11 62 33 ) 10 0 14 00 O c e a n o s p i r i l l a c ea e Prot e ob a c ter i a Sci acc a Mr-PoS C4 OQ883 73 9 Marinomo n a s r h izo mae str ain IVIA-Po -1 45 (NR_ 11 62 33 ) 10 0 14 00 O c e a n o s p i r i l l a c ea e Sci acc a Mr-PoS C8 OQ883 74 0 Marinomo n a s r h izo mae str ain IVIA-Po -1 45 (NR_ 11 62 33 ) 10 0 14 00 O c e a n o s p i r i l l a c ea e Sci acc a Mr-PoS C 10 OQ883 74 1 Marinomo n a s r h izo mae str ain IVIA-Po -1 45 (NR_ 11 62 33 ) 10 0 14 00 O c e a n o s p i r i l l a c ea e Sci acc a Mb i - P oC 1 0 OQ883 74 2 Meta bac i llus in dicu s s train MCS 01 (M Z 1 57 02 1 ) 99 .9 3 14 17 B a c i l l a c ea e Firm ic ute s C o rnino Se - Po E 5 OQ883 74 3 Stap hy loco ccus ep idermidis s train N A 4 (M T 2 70 43 8 ) 10 0 14 10 St a ph y l ococc ace a e Firm ic ute s Er i c e Se - Po S C 5 OQ883 74 4 Stap hy lococ cu s epid e rmid is stra in 8 17 (M T 5 85 52 3 ) 10 0 14 23 St a ph y l ococc ace a e Sci acc a Se - Po S C 7 OQ883 74 5 Staph ylo cocc u s epid erm id is stra i n 81 78 17 (M T 5 85 52 3 ) 10 0 14 04 St a ph y l ococc ace a e Sci acc a Se - PoSN 6 OQ883 74 6 Stap hy loco ccus ep idermidis s train N 2 10 0 75 4 St a ph y l ococc ace a e San N ic ol a .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 2 of 36 Is o lat e n a me GenB a n k acc ession numb er Be st BLAST m a tch Perc e n t id e n ti ty (16 S rDN A) Se que nc e l en g th ( bp ) Fa mi l y Ph y l u m I sola t ion loc al ity (NZ_CP 11 90 47 ) Sp-Po SC 5 OQ883 74 7 S ta ph yloc occus p a steuri s train CT1 (M T 0 72 16 1) 10 0 14 08 St a ph y l ococc ace a e Firm ic ute s Sci acc a Sw-PoE 5 OQ883 74 8 Stap hy lococ cu s w arn eri strain DK1 3 1 (M T 6 42 94 2 ) 10 0 14 03 St a ph y l ococc ace a e Firm ic ute s Er i c e Sw-PoS C5 OQ883 74 9 Stap hy l o cocc u s wa rn eri s train DK1 31(MT 64 29 42 ) 10 0 14 08 St a ph y l ococc ace a e Sci acc a Sw-PoS C9 OQ883 75 0 Stap hy l o cocc u s wa rn eri s train DK1 31(MT 64 29 42 ) 10 0 14 25 St a ph y l ococc ace a e Sci acc a Sw-Po SN 6 OQ883 75 1 Stap hy l o cocc u s wa rn eri s train DK1 31(MT 64 29 42 ) 10 0 80 8 St a ph y l ococc ace a e San N ic ol a Vs p - P o C 2 OQ883 75 2 Vibr i o ru b er strain HMF8 0 04 (KY 0 47 40 9 ) 96 .6 3 13 61 Vib r ion a c e ae Prot e ob a c ter i a C o rnino V sp-P oE2 OQ883 75 3 Vibr i o ru b er strain HMF8 0 04 (KY 0 47 40 9 ) 96 .6 3 13 61 Vib r ion a c e ae Er i c e V sp-P oE4 OQ883 75 4 Vibr i o ru b er strain HMF8 0 04 (KY 0 47 40 9 ) 96 .6 3 13 61 Vib r ion a c e ae Er i c e Vs p -PoM1 0 OQ883 75 5 Vibr i o ru b er strain HMF8 0 04 (KY 0 47 40 9 ) 96 .6 3 13 61 Vib r ion a c e ae M ars al a Vsp-Po SC1 OQ883 75 6 Vibr i o ru b er strain HMF8 0 04 (KY 0 47 40 9 ) 96 .6 3 13 61 Vib r ion a c e ae Sci acc a Vs p - P o C 8 OQ883 75 7 Vi b rio sp. stra in PCE P2 8 (MK2 94 23 2 ) 96 .9 9 13 61 Vib r ion a c e ae Prot e ob a c ter i a C o rnino Vsp-PoM 7 OQ883 75 8 Vi b rio sp. stra in PCE P2 8 (MK2 94 23 2 ) 96 .9 9 13 61 Vib r ion a c e ae M ars al a Vsp-Po SC3 OQ883 75 9 Vi b rio sp. stra in PCE P2 8 (MK2 94 23 2 ) 96 .9 9 13 61 Vib r ion a c e ae Sci acc a Vsp-Po SC1 1 OQ883 76 0 Vi b rio sp. stra in PCE P2 8 (MK2 94 23 2 ) 96 .9 9 13 61 Vib r ion a c e ae Sci acc a Vg-P oM9 OQ883 76 1 Vibrio gi g a ntis stra i n TCA- 07 1 (OQ 07 16 83 ) 99 .7 2 14 23 Vib r ion a c e ae Prot e ob a c ter i a M ars al a Vk -PoS C1 1 OQ883 76 2 Vibr i o k analoa e strain D S 1 90 4-Y1 12 4 (M T 2 69 63 3 ) 10 0 13 89 Vib r ion a c e ae Prot e ob a c ter i a Sci acc a Vn-Po E4 OQ883 76 3 Vibrio nitrif i gil is stra in NFV - 1 (N R_1 81 54 1) 99 .3 4 13 60 Vib r ion a c e ae Prot e ob a c ter i a Er i c e Vn-Po SN8 OQ883 76 4 Vibrio nitrif i gil is stra in NFV - 1 (N R_1 81 54 1) 99 .7 8 13 60 Vib r ion a c e ae San N ic ol a V t-P oC1 OQ883 76 5 Vib rio tritoniu s stra in MA 35 (GU 9 51 70 1) 10 0 14 22 Vib r ion a c e ae Prot e ob a c ter i a C o rnino .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 3 of 36 Is o lat e n a me GenB a n k acc ession numb er Be st BLAST m a tch Perc e n t id e n ti ty (16 S rDN A) Se que nc e l en g th ( bp ) Fa mi l y Ph y l u m I sola t ion loc al ity V t-P oC2 OQ883 76 6 Vib rio tritoniu s stra in MA 35 (GU 9 51 70 1) 99 .7 2 14 22 Vib r ion a c e ae C o rnino V t-P oC4 OQ883 76 7 Vib rio tritoniu s stra in MA 35 (GU 9 51 70 1) 99 .7 2 14 22 Vib r ion a c e ae C o rnino Vt-Po SC 6 OQ883 76 8 Vib rio tritoniu s stra in MA 35 (GU 9 51 70 1) 99 .7 2 14 22 Vib r ion a c e ae Sci acc a Vz-Po C5 OQ883 76 9 V i b ri o zh ug e i s t ra i n HB UA S 61 00 1 ( C P0 33 07 8 ) 99 .5 0 13 88 Vib r ion a c e ae Prot e ob a c ter i a C o rnino Vz-Po C6 OQ883 77 0 V i b ri o zh ug e i s t ra i n H B UA S 61 00 1 ( C P0 33 07 8 ) 99 .5 0 13 88 Vib r ion a c e ae C o rnino Vz-P oM10 OQ883 77 1 V i b ri o zh ug e i s t ra i n H B UA S 61 00 1 ( C P0 33 07 8 ) 10 0 13 89 Vib r ion a c e ae M ars al a Vz - P o S C 1 OQ883 77 2 V i b ri o zh ug e i s t ra i n H B UA S 61 00 1 ( C P0 33 07 8 ) 10 0 13 89 Vib r ion a c e ae Sci acc a Vz - P o S C 2 OQ883 77 3 V i b ri o zh ug e i s t ra i n H B UA S 61 00 1 ( C P0 33 07 8 ) 10 0 13 89 Vib r ion a c e ae Sci acc a 918 Is o l a te nam e GenB a n k acc essi o n numb er B e st B L AS T ma t ch Perc en t I de n- tity (ITS, LSU) Sequ e n c e l en g th ( bp ) Fa mi l y C l a ss I sola t ion loc al ity Hp s p - P o S N 1 OQ874 70 5 OQ874 53 1 Ha l o ph y to pht h or a s p . MHJ- 2 02 1 e isolate BD 6 38 (p a rt ial I TS1- 5 , 8S rRNA ge n e , p art i a l IT S2) ( OK0 41 00 4) Halop hyto p h t hora s p . M H J- 20 21e isolate BD 6 81 (L S U R N A gene) ( OK0 33 60 4) 10 0 10 0 91 0 10 69 Peronosp o r ace a e Oo m y c ota San Nic ol a Pm -PoE1 OQ874 70 4 OQ874 53 0 Pa ecilomyces ma x im u s stra in FRR 3 79 3 (p ar t ial I T S 1-5, 8S r R N A g e n e, pa r t i al IT S2 ) ( M W6 42 16 9 ) P a ecilomyces m ax im u s str ain CB S 3 72 . 70 ( LS U RN A g en e) ( M H8 71 47 0) 10 0 99 .3 0 54 1 86 2 Ther m o a s c ace ae As com yco t a Erice 919 920 921 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 4 of 36 Tab l e 2: P G P pr op erties s h o wed b y th e endo p hyte s isol ate d fr om Posidonia oceanica s eeds. 922 Is o late I de n ti ficat i o n I AA p ro- duc ti on Sid eroph or e pr o du c tion Pho s p hat e so lub il i s at ion Ni tr oge n f i xa tio n Bi o fi lm A C C deam i nas e a cti vit y Sur f a ce Bott om Ring Aa -Po SC 9 Aureimonas altamirensis - 0. 4 0.4 + - ++ + - - B sp-PoC 1 Bacillus sp. - 0 . 3 0 . 2 - + + + - - Hms p-Po SN 7 Halomonas sp. 4. 61 2. 5 - - - ++ ++ - Kp-P oC 9 Kocuria palustris - 0 . 1 - - - - - - Lysp- Po M 4 Lysobacter sp. - 0 . 3 - - - ++ - - Mr-PoSC 8 Marinomonas rhizomae 3. 39 - - - - + - - Mb i - P oC 1 0 Metabacillus indicus 0. 51 - - + - ++ - - V sp-PoC 2 Vibrio sp. 0. 98 - - - + + ++ ++ - V sp-PoE 2 Vibrio sp. - 0 . 8 - + + - - - V sp-PoE 4 Vibrio sp. - 0 . 9 - + ++ + - - - Vs p-PoM 1 0 Vibrio sp. 0. 51 0 . 1 - - - + ++ - Vs p-PoM 7 Vibrio sp. - 0 . 8 - + - ++ + + - V s p - Po SC11 Vibrio sp. - 0 . 9 - + ++ + + - - Vg-PoM 9 Vibrio gigantis - 0. 2 - - - + - - Vk - P o S C 1 1 Vibrio kanaloae - - - - - + - - Vn - P o E4 Vibrio nitrifigilis - 0 . 5 0.1 - - + - - Vn-Po S N 8 Vibrio nitrifigilis 5. 9 0 . 7 0.6 - - + - - V t-PoC 4 Vibrio tritonius - - - - - - - - Vt-PoS C 6 Vibrio tritonius - 0 . 2 - - - + - - Vz - Po C 5 Vibrio zhugei 0. 69 0. 3 - - - - - - V z - Po M10 Vibrio zhugei - 0 . 4 - - + - + - Vz - PoS C1 Vibrio zhugei 1. 77 0 . 1 - - - ++ + + - .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 5 of 36 Is o late I de n ti ficat i o n I AA p ro- duc ti on Sid eroph or e pr o du c tion Pho s p hat e so lub il i s at ion Ni tr oge n f i xa tio n Bi o fi lm A C C deam i nas e a cti vit y Sur f a ce Bott om Ring Aa -Po SC 9 Aureimonas altamirensis - 0. 4 0.4 + - ++ + - - +, pr e s en ce of th e activity; -, absen ce o f the acti vity. V alue s of ph os ph ate solu bilisati on and s ide r oph ores pr od u c tio n expr es s t h e d i a m e t e r o f t h e h a l o i n c m . V a l - ues o f I AA pr od u c tio n ar e e xpr essed in m g· L −1 . V al ues o f A C C dea mi n ase a c tivit y a re expr ess e d in µ m ol e s α-ke to butyr at e ·m g pr otein −1 ·h −1 923 924 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 6 of 36 Tab l e 3: En zy m a ti c a c t ivities s howed b y t he e nd o phyt e s isolat e d fr om Posidonia oceanica s e eds. 925 926 Is o late I de n ti fica t io n DN Ase Amyla s e Lip a se Ce llu l a s e Pe ct in a s e Pr o te ase C hiti n a se Aa - P oS C 9 Aureimonas altamirensis - - - - - - - Bsp-P oC 1 Bacillus sp. - - - - - + - Hm sp - P oS N7 Halomonas sp. + - - - - - - Kp - P o C9 Kocuria palustris - + + - - - - - Ly s p - Po M4 Lysobacter sp. + + - - - - ++ - Mr - P oS C 8 Marinomonas rhizomae + + - - - - - Mbi-P oC 10 Metabacillus indicus + ++ + - - - ++ - Vsp-P oC 2 Vibrio sp. + - - - - - - V s p- P o E2 Vibrio sp. - - + ++ - + - V s p- P o E4 Vibrio sp. - - + - ++ ++ - Vs p - P o M10 Vibrio sp. + + ++ - ++ + - + - Vs p-P oM 7 Vibrio sp. + - + + ++ ++ - Vs p-P o S C 11 Vibrio sp. + - - - + +++ - Vg-P oM 9 Vibrio gigantis + + - - - - - .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 7 of 36 Is o late I de n ti fica t io n DN Ase Amyla s e Lip a se Ce llu l a s e Pe ct in a s e Pr o te ase C hiti n a se Vk-P oSC 11 Vibrio kanaloae + - - - - - - Vn - P o E4 Vibrio nitrifigilis + - - + - - - Vn - P o SN8 Vibrio nitrifigilis - - - + + + - - - Vt-P oC 4 Vibrio tritonius - + - - - - - Vt-P oS C 6 Vibrio tritonius + - + - - - - Vz - P o C 5 Vibrio zhugei - ++ + - - - - - Vz - P o M10 Vibrio zhugei + - - + - + - Vz-P o S C 1 Vibrio zhugei + ++ - + ++ - ++ - +, pr e s e n c e of t he a ct i vity; -, a bs en c e o f t h e a c t i vity . .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint F igur e l eg end s 927 928 Fi g u r e 1 : L o cati ons, along th e Si c ili a n co ast, w he r e c ast fr uits we r e c olle cte d. Sh a d e d ar e as i ndi cate th e a p pr oxi m a te c urr ent distr i-929 butio n of P. oceanica mea do w s ( a dap t e d fr om Ca lv o e t a l . 2 010) . 930 931 Fi g u r e 2: Pe rce n ta ge an d i d e n t i t y of s e e ds y i e l d i n g c o l on i e s o n S G Y a n d NA med ia , o r no ne . 932 933 Fi g u r e 3: Distr ib u tion of b acteri al f a m ilie s isolate d fr om s eeds 934 935 Fi g u r e 4 : Fr e q u e n c i e s o f P GP t r ai t s ( A ) a n d e n z y m a t i c ( B ) a c t i v i t i e s a m o n g t h e 2 2 b a c t e r i a l i s o l at e s . C el l u l a s e a n d p e ct i n a s e a c t i v i t i e s 936 we re g ro upe d to g e th e r . 937 938 Fi g u r e A1 : M or ph olog y o f 2 2 b a c t eri al isol ate s c h ar act eri ze d f or pl a nt gr o w t h-pr o m ot ing pr op erties. 939 940 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 2 of 36 App end i ce s 941 942 943 App e nd i x 1. W e ig ht of Posidonia oceanica see ds , tot al colo nie s cou nted a nd C olo ny F or m i ng U nit ( CF U ) /g o f fr e s h m a t eri al o n SG Y a nd N A m e dia 944 945 S e ed name Loc a ti o n Se ed weight (g) To t a l micr obi al c olonie s counte d (pl at e with dilu ti o n z e ro ) CF U /g SGY N A SGY N A S N 1 Sa n N i co la 0.3 27 3 01 20 2 29 5. 84 1 98 .5 4 SN2 S a n Nic ol a 0 .20 5 10 4 6 66 .52 3 . 8 4 SN3 S a n Nic ol a 0 .17 4 0 0 0.00 0 . 0 0 SN4 S a n Nic ol a 0 .31 2 0 0 0.00 0 . 0 0 SN5 S a n Nic ol a 0 .29 3 0 0 0.00 0 . 0 0 SN6 S a n Nic ol a 0 .05 4 2 1 4 0.35 2 . 4 8 SN7 S a n Nic ol a 0 .21 1 10 2 8 7 67 .03 57 . 1 7 SN8 S a n Nic ol a 0 .36 3 0 0 0.00 0 . 0 0 E1 Erice 0 .22 0 1 0 0.68 0 . 0 0 E2 Erice 0 .26 6 2 0 1.63 0 . 0 0 E3 Erice 0 .18 2 0 0 0.00 0 . 0 0 E4 Erice 0 .28 6 35 3 0 30 .46 26 . 1 1 E5 Erice 0 .08 9 7 5 2.02 1 . 4 4 C 1 C o r n i n o 0.3 20 3 76 35 2 36 2. 41 3 39 .2 8 C2 Corni no 0 .29 2 47 3 41 .69 2 . 6 6 C 3 C o r n i n o 0.1 45 2 48 26 0 11 4. 34 1 19 .8 7 C4 Corni no 0 .21 8 6 0 4.06 0 . 0 0 C5 Corni no 0 .18 6 7 0 4.09 0 . 0 0 C 6 C o r n i n o 0.1 63 3 00 30 0 15 4. 60 1 54 .6 0 C7 Corni no 0 .21 7 0 0 0.00 0 . 0 0 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 3 of 36 C 8 C o r n i n o 0.2 79 0 15 0 .0 0 1 2. 76 C9 Corni no 0 .25 8 0 1 0.00 0 . 7 9 C1 0 Corni no 0 .23 9 2 3 1.48 2 . 2 1 SC1 S c iacc a 0 .18 7 0 0 0.00 0 . 0 0 SC2 S c iacc a 0 .22 5 1 0 0.70 0 . 0 0 SC3 S c iacc a 0 .18 9 1 0 0.59 0 . 0 0 SC4 S c iacc a 0 .21 0 2 1 1.31 0 . 6 5 SC5 S c iacc a 0 .19 2 5 3 3.01 1 . 8 0 SC6 S c iacc a 0 .41 0 0 0 0.00 0 . 0 0 SC7 S c iacc a 0 .24 0 2 0 1.48 0 . 0 0 SC8 S c iacc a 0 .38 9 1 2 1.15 2 . 3 0 SC9 S c iacc a 0 .14 8 2 3 0.94 1 . 4 1 SC1 0 S c iacc a 0 .31 6 2 0 1.91 0 . 0 0 SC1 1 S c iacc a 0 .21 2 11 5 0 75 .90 0 . 0 0 SC1 2 S c iacc a 0 .19 5 33 0 20 .14 0 . 0 0 M 1 Ma rs a l a 0 .29 7 0 0 0.00 0 . 0 0 M 2 Ma rs a l a 0 .21 8 0 0 0.00 0 . 0 0 M 3 Ma rs a l a 0 .16 9 0 0 0.00 0 . 0 0 M 4 Ma rs a l a 0 .33 3 1 1 1.00 1 . 0 0 M 5 Ma rs a l a 0 .31 4 1 0 0.95 0 . 0 0 M 6 Ma rs a l a 0 .17 0 0 0 0.00 0 . 0 0 M 7 M a r s a la 0.2 88 2 34 20 3 20 4. 96 1 77 .8 1 M 9 Ma rs a l a 0 .29 7 11 8 9.91 7 . 2 1 M10 Ma rs a l a 0 .31 1 28 3 8 26 .30 35 . 6 9 946 947 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 4 of 36 App e nd i x 2 : M or p hologi ca l ch ar a ct e ri z at ion of th e 44 selec ted e nd o phyt e s is ola te d fro m Posidonia oceanica seeds. 948 949 Isolate Location Colony shape Colony eleva- tion Colony surface Colony margin Colony colour Halo Medium B s p- PoC1 Co r n ino circul ar conv e x smo o th even cre a my no SGY Vt-P oC1 Co r n ino circul ar fl a t smo o th even white, sh in y no SGY Vs p - P o C 2 Co r n ino rhi z o id fl a t smo o th curled c r ea m y c en t e r a n d white ar ou nd no SGY Vt-P oC2 Co r n ino circul ar fl a t smo o th even white, sh in y no SGY Vt-P oC4 Co r n ino circul ar fl a t smo o th even white, sh in y no SGY Vz-Po C5 Co r n ino rhi z o id fl a t smo o th curled cre a my no SGY Vz-Po C6 Co r n ino circul ar fl a t smo o th even cre a my no SGY Vs p - P o C 8 Co r n ino circul ar thin smo o th even b e ig e , op a que no NA Kp-PoC9 Co r n ino circul ar um bonate smo o th even c r e a m y c en t e r a n d w h i t e ar oun d , o p aq ue no NA M bi-PoC10 Co r n ino circul ar conv e x smo o th even cre a my, shiny yes SGY Vsp-Po E2 Erice circul ar fl a t concentr ic wa v y cre a my no SGY Vsp-Po E4 Erice circul ar fl a t concentr ic wa v y c r e a m y c en t e r a n d w h i t e ar o und no SGY Vn-PoE 4 Erice circul ar fl a t smo o th even white, sh in y yes SGY Se - P oE 5 Erice circul ar fl a t smo o th even y e llow - or a n ge no SGY Sw-PoE5 Erice circul ar fl a t smo o th even cre a my no SGY Lysp-Po M4 Ma rs a l a circul ar fl a t smo o th wa v y white, sh in y yes SGY Vsp-PoM7 Ma rs a l a circul ar fl a t smo o th even white, sh in y no SGY, NA Vg-P oM9 Ma rs a l a circul ar um bonate smo o th even w hite, op a que no NA Vz-Po M1 0 Ma rs a l a circul ar fl a t smo o th even white, sh in y no SGY Vs p - P o M 1 0 Ma rs a l a circul ar fl a t smo o th even cre a my yes SGY .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 5 of 36 Isolate Location Colony shape Colony eleva- tion Colony surface Colony margin Colony colour Halo Medium Vsp-Po SC1 Sc i a c c a circul ar fl a t smo o th even cre a my yes SGY Vz - P oS C 1 Sc i a c c a rhi z o id fl a t smo o th curled cre a my no SGY Vz - P oS C 2 Sc i a c c a circul ar fl a t smo o th even white, sh in y no SGY Mr-PoS C2 Sc i a c c a circul ar fl a t concentr ic even cre a my, shiny no SGY, NA Vsp-Po SC3 Sc i a c c a circul ar fl a t smo o th even white, sh in y no SGY Mr-PoS C4 Sc i a c c a circul ar fl a t concentr ic even cre a my, shiny no SGY, NA Sp - P oS C 5 Sc i a c c a circul ar fl a t smo o th even da r k c r e a m y no SGY Se - P oS C 5 Sc i a c c a circul ar fl a t smo o th even y e llow - or a n ge no SGY Sw-PoS C5 Sc i a c c a circul ar fl a t smo o th even cre a my and w hi te no SGY Vt-Po SC 6 Sc i a c c a circul ar fl a t smo o th even c re a m y – w h it e , tra n s- pa r e n t no NA Se - P oS C 7 Sc i a c c a circul ar fl a t smo o th even y e llow - or a n ge no SGY Mr-PoS C8 Sc i a c c a circul ar fl a t concentr ic even cre a my, shiny no SGY, NA Aa - P oS C 9 Sc i a c c a circul ar fl a t smo o th even ye l l ow no NA Sw-PoS C9 Sc i a c c a circul ar fl a t smo o th even cre a my no SGY Mr - Po S C 1 0 Sc i a c c a circul ar fl a t concentr ic even cre a my, shiny no SGY, NA Vk -PoSC11 Sc i a c c a rhi z o id conv e x smo o th curled wh i t e yes SGY Vsp-PoS C1 1 Sc i a c c a circul ar fl a t smo o th even white, sh in y no SGY Sw-PoS N 6 Sa n N i co la circul ar fl a t smo o th even cre a my no NA Se - Po S N 6 Sa n N i co la circul ar fl a t smo o th even ye l l ow no SGY Hmsp -PoS N 7 Sa n N i co la circul ar fl a t smo o th even or an ge no SGY Cn-Po SN 8 Sa n N i co la fil a men tou s fl a t smo o th curled cr e a m y , o pa q u e no NA Vn-Po SN 8 Sa n N i co la circul ar fl a t smo o th even white, sh in y yes NA 950 Isolate Location Colony shape Colony colour Aerial mycelium Exudate Growth rate Medium .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 6 of 36 Pm-Po E1 Erice irr egul ar c ircul ar b row n po w dery t o sue de -lik e no fas t SG Y Hpsp -PoS N 1 Sa n N i co la irr egul ar c ircul ar wh i t e fe ather y no sl o w SG Y 951 952 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint 7 of 36 953 954 955 956 957 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted November 19, 2025. ; https://doi.org/10.1101/2025.11.19.689238doi: bioRxiv preprint