Microbial consortium increases maize productivity and reduces grain phosphorus concentration under field conditions

O iginal a icle Mic obial conso ium inc eases maize p oduc i i y and educes g ain phospho us concen a ion unde ield condi ions Inês Pacheco a , Rodol o Fe ei a a , Pa ícia Co eia a,b , Luís Ca alho a,b , Te esa Dias a, ⇑ , C is ina C uz a a Cen e o Ecology, E olu ion and En i onmen al Changes (cE3c), Faculdade de Ciências, Uni e sidade de Lisboa, Campo G ande, 1749-016 Lisboa, Po ugal b Soil i ae Lda., Tec Labs – Cen o de Ino ação Campus da Faculdade de Ciências, Uni e sidade de Lisboa, Campo G ande, 1749-016 Lisboa, Po ugal a icle in o A icle his o y: Recei ed 28 July 2020 Re ised 23 Sep embe 2020 Accep ed 27 Sep embe 2020 A ailable online 8 Oc obe 2020 Keywo ds: A buscula myco hizal ungi G ain phy a e Mic obial conso ium Phospha e solubilizing bac e ia Phospho us acquisi ion e iciency Plan in e nal phospho us use e iciency abs ac Backg ound: The use o mic obes ha imp o e plan phospho us (P) use e iciency is an a enue o boos c op yields while alle ia ing en i onmen al impac s. We es ed h ee mic obial inoculan s (Rhizoglomus i egula e alone – designa ed AMF; Pseudomonas pu ida alone – designa ed PSB; and R. i egula e and P. pu ida in conso ium – designa ed AMF+PSB), combined wi h chemical e ilize s, in an in ensi e maize ag icul u al sys em. Resul s: As hypo hesized: (i) despi e he na i e soil mic obial communi y and he applica ion o P e il- ize , he mic obial inoculan s enhanced plan P up ake om he soil by 14–60%, and consequen ly imp o ed P acquisi ion e iciency; (ii) PSB and AMF+PSB plan s p oduced ±50% mo e biomass pe uni o P aken up, and consequen ly enhanced plan in e nal P use e iciency (i.e. he biomass p oduced pe uni o P); and (iii) he combined inocula ion o AMF and PSB p o ided he bes esul s in e ms o p oduc i i y and P use e iciency. Fu he , he mic obial inoculan s al e ed P alloca ion wi hin he plan , educing g ain P concen a ion. Conclusion: By es ing he mic obial inoculan s unde ield condi ions, ou s udy clea ly shows ha he mic obial conso ium (AMF+PSB) inc eased maize p oduc i i y, and a he same ime imp o ed P use e i- ciency. Fu he , he use o hese mic obial inoculan s was shown o be compa ible wi h con en ional ag icul u al managemen p ac ices. Ó2020 The Au ho (s). Published by Else ie B.V. on behal o King Saud Uni e si y. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). 1. In oduc ion Phospho us (P) is an essen ial mac onu ien o all li e o ms, which g ea ly limi s eco- and ag osys ems’ p oduc i i y. To mee he needs o a g owing human popula ion and hei changing con- sump ion pa e ns, ag icul u al p oduc ion was d as ically in ensi- ied and so was he use o P e ilize s (Childe s e al., 2011). Rock phospha e (a high-quali y P sou ce used in con en ional ag icul- u e) is a non- enewable ini e esou ce whose ese es a e quickly being o e exploi ed, and he e o e p ices a e expec ed o inc ease d ama ically in he nea u u e (Co dell and Whi e, 2011; Reijnde s, 2014; Dias e al., 2015). Despi e his o eseen limi a ion o he use o P e ilize s, hei applica ion keeps ising (Shephe d e al., 2016) as ood demand is o ecas ed o double by 2050. Fu - he , 60–90% o he P applied o soils as e ilize is apidly immo- bilized, making i una ailable o plan s (Richa dson and Simpson, 2011; Es ada e al., 2013; Melo e al., 2016). Consequen ly, P accu- mula es in ag icul u al soils and some con ain mo e P han ecom- mended (e.g. Eu opean soils), bu o ensu e high p oduc i i y P e ilize s con inue o be applied. This excess use o P e ilize s causes se e e nega i e en i onmen al impac s (Reijnde s, 2014; Childe s e al., 2011), namely eu ophica ion o wa e bodies (Hua e al., 2016). This global P pa adox c ea es an u gen need o cleane ag onomic p ac ices capable o boos ing c op yields while imp o ing P use e iciency. Imp o ing P use e iciency in ag icul u e can be achie ed by inc easing plan p oduc ion main aining a gi en a e o P e ilize o by p oducing he same wi h lowe P-inpu s (Rose e al., 2013; Richa dson e al., 2009). The imp o emen o P use e iciency could be accomplished by imp o ing plan in e nal P use e iciency (IPUE) and/o inc easing P up ake om he soil (P-acquisi ion e i- ciency; PAE) (Rose and Wissuwa, 2012; Veneklaas e al., 2012; h ps://doi.o g/10.1016/j.sjbs.2020.09.053 1319-562X/Ó2020 The Au ho (s). Published by Else ie B.V. on behal o King Saud Uni e si y. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). ⇑ Co esponding au ho a : Faculdade de Ciências da Uni e sidade de Lisboa, Edi ício C2, Piso 5, sala 2.05.37, Campo G ande, 1749-016 Lisboa, Po ugal. E-mail add ess: [email p o ec ed] (T. Dias). Pee e iew unde esponsibili y o King Saud Uni e si y. P oduc ion and hos ing by Else ie Saudi Jou nal o Biological Sciences 28 (2021) 232–237 Con en s lis s a ailable a ScienceDi ec Saudi Jou nal o Biological Sciences jou nal homepage: www.sciencedi ec .com Wang e al., 2010). A combina ion be ween hese wo s a egies is desi able in bo h high- and low-inpu ag icul u al sys ems (Rose and Wissuwa, 2012; Heue e al., 2017). Since plan IPUE has se - e al de ini ions (e.g., g ain yield pe uni o P e ilize applied; plan biomass pe P p esen in speci ic issues), i is no clea how o imp o e i (Rose and Wissuwa, 2012). By con as , s a e- gies o imp o e plan PAE include molecula plan b eeding, engi- nee ing ansgenic plan s and inocula ing bene icial mic obes such as plan g ow h-p omo ing hizobac e ia (PGPR) and myco hizae (Ramaeke s e al., 2010). So a , plan b eeding and gene ic engi- nee ing ha e no gua an eed highe PAE, and hei impac on soil conse a ion, quali y and biodi e si y ha e no been ho oughly explo ed (Rose e al., 2013). By con as , inocula ing bene icial mic obes, combined wi h echnologically ad anced ag icul u al p ac ices, is becoming an impo an a enue o imp o e PAE, and consequen ly P use e iciency. Bene icial mic obes (e.g. in he o m o bio e ilize s) can con- ol nu ien bioa ailabili y in he soil by es ablishing mo e com- plex in e ac ions wi h he soil s uc u e, and a e mo e e icien a lowe nu ien le els han a highe (Wa on e al., 2015; Wel in e al., 2018; Dias e al., 2015). Indeed, mic obes play a key ole in he P cycle and among he wide di e si y o soil mic obes, a buscu- la myco hizal ungi (AMF) and phospha e solubilizing bac e ia (PSB) a e di ec ly in ol ed in P u no e and plan P acquisi ion (Zhang e al., 2014). Fu he , plan s na u ally in e ac wi h bo h AMF and PSB (Owen e al., 2015). Many s udies poin ou he ben- e icial e ec s o AMF (Smi h and Read, 2008) and/o PSB inocula- ion (Zhang e al., 2014, 2016; Melo e al., 2018; O doñez e al., 2016) o plan g ow h, P up ake and as a biological ool o eco- es o a ion (Wahid e al., 2016). Howe e , mos o hese bene icial e ec s we e obse ed in mic ocosm expe imen s o in s e ile soil (Rod iguez and Sande s, 2015). These con olled expe imen s did no ake in o accoun how he na i e soil mic obial communi y shapes AMF’s and PSB’s impac on hos plan pe o mance. The e- o e, AMF and/o PSB inocula ion has o be es ed unde ield con- di ions and ‘no mal’ managemen p ac ices o a gi en c op. Ou objec i e was o es , unde ield condi ions, he e ec o h ee mic obial inoculan s (wi h po en ial o become bio e ilize s: AMF alone, PSB alone and AMF+PSB) on imp o ing P use e iciency in an in ensi e ag icul u al sys em. We ocused on a ce eal c op, maize (Zea mays L.) which p o ides 15% o he wo ld’s p o ein and 20% o he wo ld’s calo ies (Nuss and Tanumiha djo, 2010), and is e y P demanding (e.g. e ilize doses commonly ange om 10 o 250 kg ha 1 in Eu ope) (de Va ennes, 2003). Fu he , as in o he ce eals, 60–85% o he P acqui ed by maize plan s is alloca ed o seeds (i.e., g ains) in he o m o phy a e, which is essen ial o seed ge mina ion and seedling igou (Yamaji e al., 2017; Rose e al., 2013). Howe e , phy a e canno be diges ed by humans o o he monogas ic animals, so ha all P in he o m o phy a e esul s in la ge quan i ies o P in animal exc emen s, g ea ly con- ibu ing o eu ophica ion (Nese and Co dell, 2012; Co dell and Whi e, 2011). I also dec eases o he nu ien s’ abso p ion because phy ic acid binds wi h ions, such as zinc, calcium, i on and magne- sium (Hu ell e al., 2003) esul ing in e y insoluble sal s wi h poo bioa ailabili y. Reducing P g ain concen a ion can imp o e g ain quali y by inc easing i s nu i ional alue and diges ibili y, and educe he en i onmen al impac s associa ed wi h P losses h ough exc e a (Veneklaas e al., 2012). The e o e, in sol ing he P pa adox, seeds P concen a ion should be educed wi hou com- p omising plan g ow h o igou (Rose e al., 2013; Yamaji e al., 2017). As bo h AMF (Smi h and Read, 2008) and PSB (Melo e al., 2018; O doñez e al., 2016; Zhang e al., 2014, 2016) enhance P bioa ail- abili y o plan s, we hypo hesized ha despi e he na i e soil mic obial communi y and he applica ion o P e ilize , he mic o- bial inoculan s would enhance plan P up ake om he soil and, consequen ly imp o e PAE. Besides he well-known imp o emen in P bioa ailabili y, AMF and PSB can al e plan g ow h and nu i- ion (Melo e al., 2018) so ha we hypo hesized ha inocula ed plan s would p oduce mo e biomass pe uni o P aken up, and consequen ly enhance plan IPUE, conside ed as he a io be ween biomass and i s P con en (Veneklaas e al., 2012; Rose e al., 2011; Rose and Wissuwa, 2012; an de Wiel e al., 2016). As a esul o enhanced plan IPUE, biomass P concen a ion ends o dec ease, including ha o he g ain (e.g. educed phy a e concen a ion). Finally, as AMF p oduce ex ensi e ex a adical hyphae in he soil, which a e a habi a o o he mic obes, and coope a ion be ween AMF and PSB has been shown in i o (Zhang e al., 2016, 2018) we hypo hesized ha he combined inocula ion o AMF and PSB would p o ide he bes esul s in e ms o p oduc i i y and P use e iciency. 2. Ma e ial and me hods 2.1. Expe imen al design Ou expe imen consis ed o 1 ac o : mic obial inocula ion. The design was a comple e andomized block design o 3 blocks, each con aining 4 pa cels, one o each ea men : con ol, AMF inocula- ion (designa ed AMF), PSB inocula ion (designa ed PSB) and com- bined inocula ion o AMF and PSB (designa ed AMF+PSB). Each pa cel had an a ea o 2.25 m 2 and each block had a o al a ea o 27 m 2 (3 9 m). To emo e bounda y e ec s, plan s wi hin in an a ea o 5.4 m 2 in bo h he eas and wes side o he expe imen al a ea we e conside ed plan gua d ows and we e no sampled. The AMF inoculum consis ed o p opagules and spo es o Rhi- zoglomus i egula e, which was pu chased om Symbiom (h ps://www.symbiom.cz/en). AMF p opagules and spo es we e isola ed and coun ed o co espond o 2.5x10 6 AMF spo es ha 1 , in ag eemen wi h he ecommenda ions o comme cial p oduc s ials (Cozzolino e al., 2013). A he ime o sowing (T0), AMF inoculum was sp ead manually on he soil along he plan ing u - ows co esponding o AMF and AMF+PSB ea men s. The PSB inoculum consis ed o Pseudomonas pu ida (and he espec i e cul u e medium) which had been isola ed om a Po - uguese ag icul u al soil and belongs o he Soil i ae (h ps://soil- i ae.com) PGPR collec ion. These bac e ia we e cha ac e ized as PSB due o i s capaci y o solubilize i-calcium phospha e and phy- a e in i o. PSB was inocula ed o e he a ea co esponding o PSB and AMF+PSB ea men s wi hin he ideal dose ange epo ed by Bashan (1986): 1.5 10 7 CFUs plan 1 , which co esponds o 10 8 CFUs pa cel 1 and 10 12 CFUs ha 1 . The cul u e b o h con aining he bac e ia was dilu ed o achie e he desi ed concen a ion (10 12 CFUs ha 1 ) and was applied manually oge he wi h he seeds along he plan ing u ows. A second inocula ion o PSB (10 12 CFUs ha 1 ) was pe o med 15 days a e sowing (Rakiami e al., 2019; Cip iano e al., 2016). AMF+PSB was a combina ion o he wo mic obial inoculan s using he same doses. Con ol pa cels we e no inocula ed. 2.2. Field si e and a ming p ac ices This expe imen was conduc ed in a a m, loca ed in Lou inhã, Lisboa, Po ugal (39°16 0 32.3 0 ’N9°17 0 27.4 0 ’ W), om ea ly June o la e Sep embe 2016 (110 days). Daily mean ai empe a u e was 24 °C anging be ween 16 and 32 °C, ela i e humidi y anged be ween 53 and 68%, acco ding o Ins i u o Po uguês do Ma e da A mos e a (IPMA). These alues ep esen he a e age ob ained om si e equidis an me eo ological s a ions. The soil had a coa se sand ex u e, 0.8% o o ganic ma e , pH (H 2 O) 6.3 and ex ac able P (Egné -Riehm me hod) o 442 ppm Inês Pacheco, R. Fe ei a, Pa ícia Co eia e al. Saudi Jou nal o Biological Sciences 28 (2021) 232–237 233 (analysis pe o med by Labo a ó io de Solos e Plan as, UTAD, Po - ugal, 2016). The expe imen al ield is cha ac e ized by a egime o in ensi e ag icul u e, wi h o a ion: cabbages, po a oes and occasionally maize. Field is e ilized wi h backg ound e ilize and op- d essing e ilize . All cul u es a e i iga ed h ough a dispe sion sys em. Zea mays L., cul i a Since e (Syngen a) can be used o o age and g ain p oduc ion. Seeds we e hand sowed on he 4 h o June 2016. Plan s we e g own 0.75 m apa be ween ows and wi h 0.20 m spacing along he ow, he equi alen o ~67 000 plan s ha 1 (da Sil a, 2013). Be o e sowing, a basal e iliza ion o 16 kg NO 3  , 48 kg NHO 4 + ,96kgP 2 O 5 and 96 kg K 2 Oha 1 was applied. This was ollowed by a op-d essing e iliza ion, 6 weeks a e sowing, o 60 kg NHO 4 + , 180 kg NH 2 CONH 2 ,96kgP 2 O 5 and 120 kg K 2 Oha 1 (acco ding o he ecommenda ions o ADP Fe ilizan es). 2.3. Sampling and analysis The expe imen was comple ed when plan s eached physio- logical ma u i y and g ains we e on he milk o dough phenolog- ical phase. A ha es (22 h Sep embe ), plan s we e manually cu wi h loppe s. Ha es was limi ed o a cen al sampling a ea o 1.35 m 2 pe pa cel o 16.2 m 2 , wi h all bo de plan s being excluded as bo de plan s end o be mo e igo ous and mo e p o- duc i e han hose ha g ow inside he expe imen al uni s, due o he smalle e ec o compe i ion be ween plan s and di e en ligh exposu e. Maize abo eg ound issues we e classi ied as plan shoo . The numbe o ille s pe plan was eco ded and shoo s we e sepa- a ed in culm (lea es included) and ea maize (cobs and g ain) and hen weigh ed. F esh weigh (Fw) o plan s om each pa cel was measu e in he ield wi h a ield weighing scale (Ke n CXB). A sub-sample o 3 plan s pe pa cel ( andomly chosen) we e d ied o cons an mass a 65 °C and he d y weigh (Dw) o shoo s (shoo biomass), culm and cobs (s o e biomass), and g ain (g ain biomass) we e eco ded (p ecision ±0.01 g, model PGW 3502e digi al balance). D ied g ain and h ee lea es om each plan we e g ound using a mill o sphe es (Re sch MM 2000). G ound samples we e used o de e mine g ain and lea P concen a ion, using an Op ical Emission Spec oscopy a e acid diges ion (Huang and Schul e 1985) (analysis pe o med by Cen o de Eda- ología y Biología Aplicada del Segu a – CEBAS-CSIC – Mu cia, Spain 2016). To cha ac e ize he soil, samples we e collec ed om ba e soil (composi e sample, n = 5) un il a maximum dep h o 15 cm. Sam- ples we e ai -d ied and hen analysed o chemical and physical p ope ies: ex ac ed P (Egné -Riehm me hod), o ganic ma e quan i ica ion and pH (H 2 O) (analysis pe o med by Labo a ó io de Solos e Plan as, UTAD, Po ugal, 2016). 2.4. Calcula ions and s a is ics The a e age Fw o culm and ea maize o each pa cel and he a e age shoo Dw o he 3 sub-samples we e ep esen ed in onne pe hec a e ( ha 1 ) by mul iplying he a e age pa cel alue o Fw o Dw by he numbe o plan densi y used pe hec a e (~67 000 plan s). Shoo Fw pe hec a e was de ined as g een o age and shoo Dw as he biomass. The d y ma e con en o he g een o age (%) was ob ained by mul iplying plan biomass (kg) pe 100 and di iding by he g een o age (kg). Phospho us acquisi ion e iciency (PAE) which ep esen s he amoun o P aken up pe plan (Wang e al., 2010; Vandamme e al., 2016) was e alua ed h ough shoo P ex ac ion and P e il- ize eco e y e iciency, as ollows: P ex ac ion ¼BiomassðDw issueÞ P issue concen a ion Plan densi y ð2:1Þ P e ilize eco e y e iciency ¼P ex ac ionðshoo Þ P applied in he ield ð2:2Þ P ex ac ion (kg ha 1 ) e lec s he o al P con en in plan issues: shoo , s o e and g ain. This was calcula ed by combining g ain (g) and s o e biomass (g) wi h espec i e P concen a ion (g P/100 g plan ) o each ea men and was es ima ed o a hec a e by mul iplying he esul wi h plan densi y, as depic ed in o mula (2.1). We used lea concen a ion o es ima e s o e concen a ion (Ca aco and Calou o, 2006). Shoo P ex ac ion was ob ained h ough he sum o g ain and s o e P ex ac ion, while shoo con- cen a ion was ob ained by di iding shoo P ex ac ion by shoo biomass. P e ilize eco e y e iciency (kg ha 1 ) e lec s plan ’s abili y o acqui e nu ien s applied o he soil (Baliga e al., 2001) and was calcula ed as shoo P ex ac ion (kg) di ided by he amoun o P e ilize applied (kg ha 1 ), as depic ed in o mula (2.2). Plan In e nal Phospho us U iliza ion E iciency (IPUE) was e alua ed h ough he amoun o biomass (Shoo , S o e o G ain Dw) p oduced pe uni o P p esen in he shoo (shoo P ex ac- ion) (Rose and Wissuwa, 2012), as ollows: IPUE ¼BiomassðDw issueÞ P ex ac ionðshoo Þð2:3Þ IPUE (kg Dw kg P ex ac ed ha 1 ) was exp essed in kg o issue bio- mass p oduced pe kg o P p esen in he shoo in a hec a e, o mula (2.3). The e ec o he inoculan s on maize pe o mance was es ed using one-way analysis o a iance (ANOVA). Di e ences among ea men means we e de e mina e by Tukey es (p 0.05). In all cases, p elimina y analyses we e pe o med o ensu e ha he e was no iola ion o s a is ical assump ions (including he Le ene’s es o check o homogenei y o a iances). SPSS ( e sion 250, IBM, Inc., Chicago, IL, USA) was used o all hese analyses. G aphs we e de eloped wi h G aphPad P ism e sion 6 (G aphPad So - wa e, San Diego, CA). 3. Resul s 3.1. E ec o he mic obial inoculan s on maize p oduc i i y Plan inocula ion wi h PSB alone (PSB) and in combina ion wi h AMF (AMF+PSB) p omo ed o age p oduc i i y (i.e. g een o age Fw) compa ed o he con ol plan s (inc emen s o 41% and 48%, espec i ely). Inocula ion wi h AMF alone (AMF) ailed o inc ease o age p oduc i i y (ANOVA g een o age p oduc i i y F3,6 = 9.90, P0.01) (Fig. 1). The mic obial inoculan s did no a ec Fw pa i- ioning be ween culm and ea s (ANOVA a io Ea /Culm F3,6 = 0.82, P > 0.05). Al hough no signi ican , inocula ion wi h PSB alone (PSB) and in combina ion wi h AMF (AMF+PSB) ended o p oduce bigge plan s ela i ely o he con ol (inc emen o 53% and 65%, espec- i ely) (ANOVA biomass F3,6 = 3.08, P > 0.05) (Table 1). Mic obial inoculan s did no change he d y weigh pa i ioning be ween s o e and g ain (ANOVA a io G ain/S o e Dw F3,6 = 0.25, P > 0.05), no e en he Dw o each componen , when conside ed sepa a ely (ANOVA S o e Dw F3,6 = 2.77, P > 0.05; G ain Dw F3,6 = 3.54, P > 0.05). 3.2. E ec o he mic obial inoculan s on P use PSB inc eased P acquisi ion e iciency (PAE) by inc easing P con en (Table 1), while AMF+PSB inc eased plan in e nal P use Inês Pacheco, R. Fe ei a, Pa ícia Co eia e al. Saudi Jou nal o Biological Sciences 28 (2021) 232–237 234 e iciency (IPUE – Fig. 2). Only inocula ion wi h PSB alone (PSB) inc eased shoo P ex ac ion: PSB plan s had 60% mo e P in hei shoo s (s o e and g ain) when compa ed o con ol plan s (ANOVA shoo P ex ac ion F3,6 = 4.88, P 0.05 – Table 1). Pa o his ex a P was alloca ed in o he s o e , as s o e om PSB plan s had app oxima ely 31% mo e P in hei shoo s han con ol plan s (ANOVA s o e P ex ac ion F3,6 = 8.56, P 0.05). By con- as , g ain P ex ac ion showed no di e ences be ween ea men s (ANOVA g ain P ex ac ion F3,6 = 0.36, P > 0.05). Thus, he di e - ences in shoo P ex ac ion e lec ed he di e ences in s o e P con en . Only inocula ion wi h PSB alone (PSB) imp o ed P e il- ize eco e y e iciency compa ed o con ol (ANOVA P e ilize eco e y e iciency F3,6 = 4.88, P 0.05). Only plan s inocula ed wi h AMF+PSB p oduced mo e shoo biomass (including g ain) pe uni o P p esen in he shoo , when compa ed o he plan s ea ed wi h he o he inoculan s (ANOVA IPUEShoo F3,6 = 12.82, P 0.01; IPUEG ain F3,6 = 13.91, P 0.01) (Fig. 2). The e o e, AMF+PSB enhanced plan s IPUE. 3.3. P concen a ion in plan issues Only AMF+PSB plan s had signi ican ly lowe shoo P concen a- ion han es o he plan s (ANOVA P concen a ion shoo F3,6 = 17.13, P 0.01). Fu he , no di e ence in shoo P concen a- ion was de ec ed be ween con ol, AMF and PSB plan s (Table 2). The mic obial inoculan s had no e ec on s o e P concen a ion (ANOVA P concen a ion s o e F3,6 = 9.60, P 0.01) bu AMF +PSB plan s had lowe s o e P concen a ion han AMF and PSB plan s. Las ly, con ol plan s had he highes P concen a ion in he g ain (ANOVA P Concen a ion G ain F3,6 = 64.58, P 0.001). Among inocula ed plan s, AMF+PSB had lowe g ain P concen a- ion han AMF inocula ed plan s. PSB e ec did no di e om AMF and AMF+PSB. The mic obial inoculan s we e able o educe g ain P concen a- ion (Table 2) and consequen ly g ain phy a e. Al hough, his di - e ence was no de ec ed in he s o e , AMF+PSB ea ed plan s had lowe s o e P concen a ion han plan s ea ed wi h he o he inoculan s. O e all, AMF+PSB plan s we e he mos e icien in educing shoo P concen a ion, a dec ease o abou 30% com- pa ed o con ol plan s. 4. Discussion 4.1. Imp o ing plan p oduc i i y The alues o p oduc i i y we ob ained (Fig. 1) a e wi hin he ange epo ed o his a ie y (Ca aco and Calou o, 2006), hus alida ing he da a ob ained in his ield ial applying ‘no mal’ managemen p ac ices o maize. As hypo hesized, and in ag ee- men wi h o he s udies (Owen e al., 2015; O doñez e al., 2016), inocula ion wi h PSB alone (PSB) o in combina ion wi h AMF (AMF+PSB), enhanced plan p oduc i i y unde ield condi- ions. The mic obial e ec was signi ican o g een o age (c op Fw – Fig. 1) bu no o biomass (Table 1), sugges ing ha he mic obes alone o in combina ion p omo ed di e en e ec s on plan wa e up ake and wa e sa ing s a egies (Richa dson e al., 2011). Non-exclusi ely, he inc eased p oduc i i y we obse ed may be ela ed wi h changes in plan ho monal balance induced by plan -mic obial in e ac ion (Nadeem e al., 2014). I may also e lec he in luence o he inoculan s in delaying plan de elop- men as in he case o ce eals wa e de ici is a main ac o igge - ing and accele a ing g ain p oduc ion (de Va ennes, 2003). 4.2. Imp o ing P use e iciency while educing g ain P concen a ion P oducing plan s wi h lowe P concen a ions is a way o achie e highe plan IPUE (Veneklaas e al., 2012; Rose e al., 2011). Howe e , i P concen a ion alls below ha ecommended o egula plan g ow h and de elopmen (i.e. 0.2–0.5%) (de Va ennes, 2003), P de iciency occu s. In ou expe imen , all inocu- la ed plan s showed shoo P concen a ions (Table 2) simila o he ecommended ones showing ha he mic obial inoculan s we e able o educe P concen a ion wi hou igge ing P limi a ion. Ano he a ge is educing g ain P concen a ion and conse- quen ly phy a e (Yamaji e al., 2017; Rose e al., 2013). Indeed, he mic obial inoculan s changed P alloca ion wi hin he plan , esul ing in lowe g ain P concen a ion (Table 2). The amoun o phy a e p esen in plan seeds and g ains anges om 0.5 o 5% o Dw and ideally i should be educed o 0.025% o Dw o less o minimize P losses (Hu ell e al., 2003). Al hough PSB inocula ion was no he mos e icien ea men a imp o ing p oduc i i y, PSB inocula ed plan s we e he mos e icien a expo ing P o he shoo as shown by s o e P (Table 1) con en . Mos P in he s o e is in he phospha e o m, which is essen ial o li es ock nu i ion (Richa dson e al., 2011). Since P con en is usually low in o ages and/o li es ock ha e low P assimila ion e iciencies (due o su plus o phy a e), die a y P supplemen s a e o en equi ed (Sha pley e al., 2000). The e o e, by p oducing eed o Fig. 1. E ec o he mic obial inoculan s on maize g een o age p oduc i i y. S acked ba s (g een and yellow) show he esh weigh o ege a i e (culm) and ep oduc i e (ea ) s uc u es. Pe cen ages on op o he ba s show he e ec in maize g een o age p omo ed by he espec i e inoculan when compa ed o con ol plan s. Di e en le e s show signi icance a 5% le el ( o o al g een o age p oduc i i y), acco ding o Tuckey’s HSD es . Ba s a e he mean ± SD (n = 3 plo s). Table 1 E ec o he mic obial inoculan s on biomass, P ex ac ion a shoo , s o e (culm and cobs), and g ain le el and P e ilize eco e y e iciency. Fo each line, di e en le e s (mean ± SD o 3 sampling plo s, n = 3) show signi icance a 5% le el (no le e s mean no signi ican ), acco ding o Tuckey’s HSD es . T ea men Con ol AMF PSB AMF+PSB Biomass ( Dw ha 1 ) 28.8 ± 5.3 35.1 ± 9.0 44.1 ± 7.7 47.6 ± 9.0 Shoo P ex ac ion (kg ha 1 ) 85.5 ± 8.8 b115.4 ± 11.3 ab 136.8 ± 5.6 a98.4 ± 7.8 ab S o e P ex ac ion (kg ha 1 ) 77.8 ± 10.9b84.2 ± 10.6 ab 102.2 ± 6.4 a68.4 ± 7.4b G ain P ex ac ion (kg ha 1 ) 25.6 ± 5.7 18.3 ± 0.8 22.0 ± 2.9 21.4 ± 0.9 P e ilize eco e y e iciency (kg P con en shoo kg 1 P 2 O 5 supplied) 0.44 ± 0.07b0.60 ± 0.11 ab 0.71 ± 0.11 a0.51 ± 0.04 ab Inês Pacheco, R. Fe ei a, Pa ícia Co eia e al. Saudi Jou nal o Biological Sciences 28 (2021) 232–237 235 highe P nu i ional alue and g ain wi h lowe phy a e (Table 2), inocula ion wi h PSB could con ibu e o educe P eed supple- men s and, consequen ly, educe P losses in li es ock exc e a. This win-win scena io mee s a me s’ objec i es and is an excellen a gumen o u he s udies wi h his PSB isola e (Pseudomonas pu ida), and possibly he de elopmen o a bio e ilize . The mic obial conso ium AMF+PSB educed shoo P concen a- ion by 30% (Table 2) and p oduced mo e g een o age (Fig. 1) showing ha hese plan s used less P o p oduce mo e g een o age (and ended o p oduce mo e biomass – Table 1) han he o he ea men s. Since AMF+PSB also showed he highes plan IPUE (Fig. 2), i could help s eng hen ood p oduc ion pe uni o a ea, and sus ainably manage he amoun o P e ilize applied in a m- ing sys ems. AMF+PSB is he mos p omising mic obial inoculan o be used in sus ainable ag icul u e, and should be u he es ed combined wi h he 30% educ ion in P inpu s ecommended by he Eu opean Union. This mic obial conso ium (AMF+PSB) could be a key con ibu o in p oducing high alue ood esou ces wi h ze o inc ease in land deg ada ion while educing nega i e en i on- men al impac s. Acco ding o he Eu opean ag onomic ules, hese esul s a e a p omising asse owa ds a bio-economic managemen ag icul u e wi h signi ican en i onmen al epe cussions. 5. Conclusion By es ing he mic obial inoculan s unde ield condi ions, ou s udy clea ly showed ha he mic obial conso ium (AMF+PSB) inc eased maize p oduc i i y, and a he same ime imp o ed P use e iciency and educed g ain P concen a ion. Fu he , he use o hese mic obial inoculan s was shown o be compa ible wi h con en ional ag icul u al managemen p ac ices. Decla a ion o Compe ing In e es The au ho s decla e ha hey ha e no known compe ing inan- cial in e es s o pe sonal ela ionships ha could ha e appea ed o in luence he wo k epo ed in his pape . Acknowledgmen s We a e g a e ul o João Fe ei a o making he expe imen al si e a ailable and o he Re iewe s o hei commen s and sugges ions. Funding This s udy was suppo ed by Po uguese unds h ough Fun- dação pa a a Ciência e a Tecnologia (p ojec s PTDC/AGR- PRO/1852/2014 and UIDB/00329/2020, and a pos doc g an SFRH/BPD/85419/2012 o Te esa Dias) and h ough he p ojec PDR2020-20.2.4-FEADER-055319 (a mas e g an o Inês Pacheco). 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S acked ba s (g een and yellow) ep esen he pa i ion o he a e age IPUE a s o e (culm and cobs) and g ain le el. Each ba ep esen s he mean o 3 sampling plo s ± SD (n = 3). Di e en le e s show signi icance a 5% le el ( o o al IPUE), acco ding o Tuckey’s HSD es . Table 2 E ec o he mic obial inoculan s on P concen a ion o shoo , s o e (culm and cobs) and g ain. Fo each column, di e en le e s (mean ± SD o 3 sampling plo s, n = 3) show signi icance a 5% le el, acco ding o Tuckey’s HSD es . T ea men Shoo [P] (%) S o e [P] (%) G ain [P] (%) Con ol 0.30 ± 0.01 a0.29 ± 0.02 ab 0.32 ± 0.02 a AMF 0.33 ± 0.03 a0.37 ± 0.04 a0.23 ± 0.01 b PSB 0.31 ± 0.02 a0.34 ± 0.03 a0.21 ± 0.03 bc AMF+PSB 0.21 ± 0.03 b0.22 ± 0.04 b0.19 ± 0.01 c Inês Pacheco, R. Fe ei a, Pa ícia Co eia e al. Saudi Jou nal o Biological Sciences 28 (2021) 232–237 236 Nese , T.-S., Co dell, D., 2012. Global phospho us sca ci y: iden i ying syne gies o a sus ainable u u e. J. Sci. Food Ag ic. 92 (1), 2–6. 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