Isolation, Identification, and Characterization of Phosphate-Solubilizing Bacteria from Tunisian Soils

Ci a ion: Am i, M.; Rjeibi, M.R.; Ga ouni, M.; Ma eus, D.M.R.; Asses, N.; Pinho, H.J.O.; Abbes, C. Isola ion, Iden i ica ion, and Cha ac e iza ion o Phospha e- Solubilizing Bac e ia om Tunisian Soils. Mic oo ganisms 2023,11, 783. h ps://doi.o g/10.3390/ mic oo ganisms11030783 Academic Edi o : Ch is ophe B. Blackwood Recei ed: 24 Janua y 2023 Re ised: 8 Ma ch 2023 Accep ed: 16 Ma ch 2023 Published: 18 Ma ch 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). mic oo ganisms A icle Isola ion, Iden i ica ion, and Cha ac e iza ion o Phospha e-Solubilizing Bac e ia om Tunisian Soils Ma wa Am i 1,2 , Mohamed Ridha Rjeibi 3,4, Ma wa Ga ouni 2, Dina M. R. Ma eus 5,* , Ned a Asses 2,6, Hen ique J. O. Pinho 7and Chaabane Abbes 1 1Labo a o y o Resou ces Syl o-Pas o al, Ins i u e Syl o-Pas o al o Taba ka (ISPT), Uni e si éde Jendouba, Isp.Taba ka BP. n◦345, Taba ka 8110, Tunisia 2Labo a o y Resea ch o Science and Technology o En i onmen al (LRSTE), Highe Ins i u e Science and Technology En i onmen al ÀBo j Céd ia, and Facul y o Sciences o Bize e (FSB), Uni e si éde Ca hage, BP-1003, Hammam-Li 2050, Tunisia 3Labo a oi e de Pa asi ologie, École Na ionale de Médecine Vé é inai e de Sidi Thabe , Uni e si éde La Manouba, Sidi Thabe 2020, Tunisia 4Labo a oi e de Pa asi ologie, Ins i u de La Reche che Vé é inai e de Tunisie, Uni e si éde Tunis El Mana , 20 Rue de Jebel Lakdha , La Rab a, Tunis 1006, Tunisia 5Techn&A , Cen e o Technology, Res o a ion and A Enhancemen , Ins i u o Poli écnico de Toma , Es ada da Se a, 2300-313 Toma , Po ugal 6Labo a o y o Ecologies and Mic obial Technology (LETMI), Na ional Ins i u e o Applied Science and Technology (INSAT), Uni e si éde Ca hage, 2 Boule a d de La Te e, B.P. 676, Tunis 1080, Tunisia 7 Ci2, Sma Ci ies Resea ch Cen e , Ins i u o Poli écnico de Toma , Es ada da Se a, 2300-313 Toma , Po ugal *Co espondence: [email p o ec ed] Abs ac : Soil mic oo ganisms play an impo an ole in main aining na u al ecological balance h ough ac i e pa icipa ion in ca bon, ni ogen, sul u , and phospho ous cycles. Phospha e-solubilizing bac e ia (PSB) a e o high impo ance in he hizosphe e, enhancing he solubiliza ion o ino ganic phospho us complexes in o soluble o ms a ailable o plan nu i ion. The in es iga ion o his species o bac e ia is o majo in e es in ag icul u e, as hey can be used as bio e ilize s o c ops. In he p esen s udy, 28 isola es o PSB we e ob ained a e he phospha e en ichmen o soil samples om i e Tunisian egions. Fi e PSB species we e iden i ied by 16S RNA gene sequencing including Pseudomonas luo escens,P. pu ida, and P. aiwanensis,S eno ophomonas mal ophilia, and Pan oea agglome ans. Solid and liquid Piko skaya’s (PVK) and Na ional Bo anical Resea ch Ins i u e’s (NBRIP) media con ain- ing insoluble icalcium phospha e we e used o he e alua ion o he phospha e solubiliza ion abili y o he bac e ial isola es by wo me hods: isual e alua ion o he solubiliza ion zone a ound colonies (halo) and de e mina ion o solubilized phospha es in liquid medium by he colo ime ic me hod o he anado-molybda e yellow. Based on he esul s o he halo me hod, he isola e o each species ha showed he highe phospha e solubiliza ion index was selec ed o e alua ion o phospha e solubiliza ion by he colo ime ic me hod. In he liquid media, he bac e ial isola es showed phospha e solubiliza ion anging om 535.70 o 618.57 µ g mL −1 in he NBRIP medium, and 374.20 o 544.28 µ g mL −1 in he PVK medium, wi h he highes alues p oduced by P. luo escens. The bes phospha e solubiliza ion abili y and highe educ ion in b o h pH, which indica es highe o ganic acid p oduc ion, we e achie ed in NBRIP b o h o mos o he PSB. S ong co ela ions we e obse ed be ween he a e age capabili y o PSB o solubilize phospha es and bo h he pH and o al phospho ous con en in he soil. The p oduc ion o he ho mone indole ace ic acid (IAA), which can p omo e plan g ow h, was obse ed o all i e PSB species. Among hem, P. luo escens ob ained om he o es soil o no he n Tunisia showed he highes p oduc ion o IAA (50.4 ± 0.9 µ g mL −1 ). Keywo ds: indole ace ic acid; phospha e-solubilizing bac e ia; phospha e solubiliza ion index; icalcium phospha e Mic oo ganisms 2023,11, 783. h ps://doi.o g/10.3390/mic oo ganisms11030783 h ps://www.mdpi.com/jou nal/mic oo ganisms Mic oo ganisms 2023,11, 783 2 o 14 1. In oduc ion Phospho us (P) is conside ed he mos impo an elemen in plan nu i ion, a e ni ogen [ 1 ]. I ep esen s an essen ial componen in all main me abolic pa hways o plan s such as pho osyn hesis, signal ansduc ion, and espi a ion [ 2 ]. Howe e , he concen a ion o soluble P in soil is usually e y low (400–1260 mg kg −1 ) [ 3 ], which equi es he use o e ilize s o ensu e highe c op p oduc i i y o espond o he inc easing demand because o he con inuous g ow h o he wo ld popula ion. Fo his eason, he o e all need o P e ilize s has been inc easing by 2.5–3.0% pe yea [4]. Con en ional chemically ob ained e ilize s include a ious o ms o P, such as iple supe phospha e, mono-ammonium phospha e, di-ammonium phospha e, and ammonium polyphospha e. The majo p oblem wi h he applica ion o chemical e ilize s is ha a la ge pa o he soluble o m o ino ganic phospha e applied o he soil is apidly immobilized and becomes una ailable o plan s [ 5 ]. Addi ionally, highe a ailabili y o P in he soil can educe he assimila ion o hea y me als, such as cadmium, mi iga ing he isk o nega i e e ec s on plan g ow h as well as animal and human heal h h ough he ood chain [6]. The sca ci y o phospho ous wo ld ese es is ano he essen ial aspec ha mo i a es he sea ch o solu ions o p e en he low a ailabili y o phospho ous in soils as a esul o i s immobiliza ion. Wo ld phospho ous ese es a e being deple ed because o he use o P in se e al indus ies, bu mainly o he p oduc ion o e ilize s [ 7 ]. In addi ion o he deple- ion o phospho us esou ces by he phospha e- and phospho ic acid-p oducing indus ies, which a e ele an in Tunisia, he p oduc ion p ocesses gene a e la ge amoun s o was e ha a e c ea ing se ious en i onmen al p oblems. This issue is e idenced in he Gul o Gabes, in sou he n Tunisia [ 8 ]. The e o e, he use o s a egies o educe e ilize consump- ion, such as imp o ing hei e ec i eness h ough highe solubiliza ion o phospha es in soils, can con ibu e o educing he nega i e impac s o phospho us indus ies. Solubiliza ion o mine al phospha es is an impo an abili y exhibi ed by some mi- c oo ganisms, mo i a ing he s udy o hei solubilizing capabili y [ 9 ]. The phospha e- solubilizing bac e ia (PSB) a e p edominan o e o he phospho us-solubilizing mic oo gan- isms and play an impo an ole in biogeochemical phospho us cycling in bo h e es ial and aqua ic en i onmen s [10]. PSB can sol e he p oblem o he abili y o solubilize soil-insoluble phospha es h ough di e en mechanisms, such as he sec e ion o o ganic acids, he p oduc ion o enzymes, and he exc e ion o side opho es [ 11 ]. In addi ion, PSB can p omo e plan g ow h h ough he p oduc ion o ho mones such as auxins, cy okinins, and gibbe ellic acid, as well as e hylene, hyd ogen cyanide, and side opho es [ 12 ]. PSB’s con ibu ion o ni ogen ixa ion and esis ance o soil pa hogens is also epo ed as p omo ing plan g ow h [13]. PSB a e pa o he plan g ow h-p omo ing hizobac e ia and can solubilize di e - en ino ganic phospha e compounds, such as icalcium phospha e and o he mine al phospha es, allowing o a educ ion in he use o syn he ic e ilize s in ag icul u e [ 14 ]. The e o e, he use o PSB as a bioinoculan is conside ed an ecological and sus ainable app oach [15]. Conside ing ha he solubiliza ion o phospha es in e ilize s and o he phospho ous compounds al eady p esen in he soil is a con ibu ion o he sus ainable use o esou ces, he pu pose o his wo k was o s udy a ious species o PSB ound in di e en Tunisian soils and o assess he s ains which a e he mos e ec i e in he solubiliza ion o phospha es. In addi ion, as some bac e ia we e epo ed o p oduce ho mones and o he compounds ha p omo e plan g ow h, and he po en ial o he isola ed bac e ia o p oducing he ho mone indole ace ic acid was also e alua ed. S udying he PSB in he Tunisian soils has wo main jus i ica ions: Tunisia is a ele an example o he de elopmen o sus ainable managemen o phospho ous compounds, hus mi iga ing he en i onmen al impac o phospha e p oduc ion; and using locally a ailable PSB al eady adap ed o Tunisian soil ypes is a way o expand hei applica ion in he coun y. Mic oo ganisms 2023,11, 783 3 o 14 2. Ma e ials and Me hods 2.1. Soil Samples 2.1.1. Si e Desc ip ion A se ies o sampling was ca ied ou in wo ypes o soils ( o es soil and ag icul- u al soil) in he a ea o Taba ka nea a hyd o he mal sou ce in Aïn D aham (Hammam Bou guiba). The si e loca ion and cha ac e is ics a e shown in Table 1. Table 1. Geog aphical loca ion and cha ac e is ics o he sampling si es. Si e Loca ion Clima e A e age Tempe a u e Typical Mon hly P ecipi a ion Main Cul u e Type 1N: 36◦46012.000 E: 008◦34044.500 Wa m empe a e 18.6 ◦C 1–11 mm Hackbe y ees Fo es 2N: 36◦46010.700 E: 008◦34041.200 Wa m empe a e 18.6 ◦C 1–11 mm Que cus sube Fo es 3N: 36◦46012.600 E: 008◦34042.600 Wa m empe a e 18.6 ◦C 1–11 mm Hackbe y ees Fo es 4N: 36◦58013.300 E: 008◦52034.900 Ho 24.0 ◦C 1–6 mm Oli e ees Ag icul u e 5N: 36◦58012.800 E: 008◦52034.700 Ho 24.0 ◦C 1–6 mm Pine ees Fo es 2.1.2. Sampling Design Th ee samples we e collec ed a each loca ion. The samples we e aken a a dep h o 15 cm wi h a hand auge and placed in s e ile poly hene bags wi h app op ia e labeling, s o ed in a coole , and b ough o he labo a o y o u he analysis. 2.1.3. De e mina ion o Soil Physical and Chemical Pa ame e s In he labo a o y, he samples we e homogenized, sie ed ( h ough a 2 mm mesh sc een), and s o ed a 4 ± 0.1 ◦ C in a e ige a o . The samples we e hen subjec ed o a ious analyses, such as pH, o ganic ma e (OM), o al ca bon (C To al ), o al ni ogen (NTo al), o al phospho us (PTo al), and ex u e de e mina ion. The pH was measu ed a e suspending 20 g o soil in 40 mL o dis illed wa e . The OM was de e mined by sample weigh loss on igni ion a 600 ◦ C, a e emo al o mois u e by d ying a 60 ◦ C o 24 h. The C To al was es ima ed by di iding he OM alue by 1.724 [ 16 ]. The N To al was de e mined using he Kjeldahl me hod ollowing he mine aliza ion o soil samples a 400 ◦ C a e he addi ion o sul u ic acid and cup ic sul a e. The P To al was de e mined by he anado-molybda e yellow me hod a e he diges ion o soil samples wi h hyd ochlo ic acid. The soil ex u e analysis was pe o med acco ding o Robinson’s pipe e me hod as desc ibed by Mwendwa [17]. 2.2. Isola ion and Pu i ica ion o Phospha e Solubilizing Bac e ia As a i s s ep o isola e he PSB, 10 g o sample soil was added o 140 mL o phospha e selec i e media (90 mL o s e ile wa e and 50 mL o nu ien b o h con aining insoluble sou ces o P such as AlPO 4 , FePO 4 , and phy a e), and hen we e agi a ed a 130 pm in an o bi al shake a 30 ◦ C o 7 days [ 18 ]. F om his suspension, a dilu ion se ies anging om 10 −1 o 10 −5 was ca ied ou . A olume o 1 mL o each dilu ion was hen sp ead on a pla e coun aga medium PCA (con aining 5 g/L o yp one, 2.5 g/L yeas ex ac , 1 g/L glucose, and 12 g/L aga ) (su ace seeding) and 1 mL was placed a he bo om o a Pe i dish (deep seeding). A e incuba ion a 30 ◦ C o 24 h, PSB we e isola ed om soil samples by se ial dilu ion using pla ing on PCA medium. This s ep was ca ied ou by successi e subcul u ing un il a homogeneous cul u e was ob ained and all he colonies became iden ical. Mic oo ganisms 2023,11, 783 4 o 14 2.3. Iden i ica ion o he S udied Isola es The ollowing mo phological cha ac e is ics we e used o he iden i ica ion o isola ed bac e ia: shape, size, mobili y, p esence o absence o spo es, and G am s aining. The molecula iden i ica ion o isola ed bac e ia was pe o med a e DNA ex ac ion ca ied ou by he mal lysis. F om a young cul u e o 24 h on aga medium, colonies we e suspended in an Eppendo ube wi h 200 µ L o s e ile dis illed wa e , incuba ed a 100 ◦ C o 10 min, and inally cen i uged a 14,000 pm o 10 min. The supe na an con aining he DNA was collec ed and s o ed a −20 ◦C un il he PCR analysis. APCRwas unusinguni e salp ime s( o wa dEU49 [5 0 -TTAACACATGCAAGTCGAACGG- 3 0 ], e e seEU1070 [5 0 -GGACTTAACCCAACATCTCACGA-3 0 ]) ha ampli y a 1021 bp sequence o 16S RNA [ 1 ]. The eac ion was pe o med in a inal olume o 25 µ L, con aining 1×PCR bu e , 1.5 mM MgCl 2 , 0.3 mM dNTPs and 1.4 uni s o Taq DNA Polyme ase (Biobasic Inc., Ma kham, ON, Canada), 10 pmol o each p ime , and 1 µL o DNA empla e. The he mocycling was pe o med wi h a he mocycle (ESCO Swi MaxP o, Ho sham, PA, USA ) and s a ed wi h an ini ial dena u a ion o 5 min a 94 ◦ C, ollowed by 30 cycles (94 ◦ C o 60 s, 55 ◦ C o 60 s and 74 ◦ C o 60 s) and a inal ex ension a 74 ◦ C o 5 min. PCR p oduc s we e sepa a ed by elec opho esis in 1.2% aga ose gel o e alua e he size o he amplicons. All posi i e amplicons ob ained wi h he p ime s EU49 /EU1070 we e pu i ied wi h he Wiza d SV gel and PCR clean-up sys em (P omega, Madison, WI, USA) acco ding o he manu ac u e ’s ins uc ions. Pu i ied DNA agmen s we e sequenced in bo h di ec ions using he same p ime s as o PCR. The eac ions we e pe o med using a con en ional Big Dye Te mina o cycle sequencing- eady eac ion ki (Applied Biosys ems, Fos e Ci y, CA, USA) wi h an ABI3730XL au oma ed DNA sequence. The ob ained 16S RNA gene sequences we e edi ed using he Ch omasP o so - wa e ( e sion 1.7.4). The pai wise nucleo ide pe cen iden i y o he new sequences was calcula ed using MEGA 6.1 so wa e [ 19 ]. Phylogene ic ees we e cons uc ed by he neighbo -joining me hod. Dis ances we e es ima ed by he Tamu a-Nei me hod [20]. 2.4. Solubiliza ion o Phospha es The bac e ia isola es we e es ed o hei abili y o solubilize phospha e acco ding o he me hods o Nau iyal [ 21 ] and Piko skaya [ 22 ] using he NBRIP and PVK media. An imp o ed Piko skaya’s phospha e g ow h (PVK) solid medium con aining blue b o- mophenol, which p oduced yellow-colo ed halos a ound he colonies due o o ganic acid p oduc ion, was de eloped by Gup a e al. [ 23 ], and is commonly used in he sc eening o PSB [ 9 ]. Nau iyal [ 21 ] de eloped he Na ional Bo anical Resea ch Ins i u e’s phospha e g ow h liquid medium (NBRIP) comp ising glucose, 5.0 g/L o icalcium phospha e (TCP), magnesium chlo ide hexahyd a e, magnesium sul a e hep ahyd a e, po assium chlo ide ammonium sul a e, and dis illed wa e , and his medium is widely used o he e alua ion o phospha e solubiliza ion [9,24]. The abili y o bac e ia o solubilize he TCP was de e mined by measu ing he solubi- liza ion halo diame e a ound he colonies a e he inocula ion o esh bac e ial suspension on he PVK and NBRIP aga media. The solubiliza ion index (SI) was calcula ed a e 7, 14, and 21 days o incuba ion a 28 ±2◦C using he ollowing o mula [25]: SI =CD +HD CD whe e CD is he colony diame e , and HD is he halo zone diame e . The phospha es’ solubilizing capaci y o he PSB isola es was also e alua ed based on he colo ime ic measu emen o he concen a ion o solubilized phospha es in liquid medium. Fi y mL o liquid medium (NBRIP o PVK) supplemen ed wi h 0.5% TCP was inocula ed wi h 200 µ L o esh bac e ial suspension wi h an op ical densi y o 0.8, co esponding o 5 × 10 8 CFU/mL, and incuba ed a 28 ± 2 ◦ C o 7 days on a o a y shake a 180 pm [ 26 ]. A e wa ds, he cul u es we e cen i uged a 10,000 pm o 10 min, and he Mic oo ganisms 2023,11, 783 5 o 14 supe na an was used o he quan i ica ion o solubilized phospho us by he colo ime ic me hod o anado-molybda e yellow a 430 nm [ 27 ]. The pH o he b o h medium was also measu ed wi h a digi al pH me e (Jenway 3510) a h ee and se en days o incuba ion. The wo me hods desc ibed abo e we e applied sequen ially: i s , all isola es om soil samples we e sc eened o hei SI; hen, he isola e o each species ha showed be e e ec i eness (SI ≥ 2) was es ed by he colo ime ic me hod. All expe imen s we e ca ied ou in iplica e o each isola e. 2.5. De e mina ion o he P oduc ion o Indole Ace ic Acid PSB we e assayed o hei capaci y o p oduce indole ace ic acid (IAA) in Lau ia Be ani (LB) medium con aining L- yp ophan as a p ecu so (10 g/L), yeas ex ac (5 g/L), and NaCl (10 g/L). Fi y ml o LB medium was inocula ed wi h one mL o bac e ial suspension (10 9 CFU/mL) and incuba ed a 28 ± 2 ◦ C o 4 days in a shaking incuba o a 180 pm. Then, 4 mL o Salkowski eagen (p epa ed om 50 mL o 35% pe chlo ic acid and 1 mL o 0.5 M FeCl 3 solu ion) was added o 1 mL o he supe na an ob ained by cen i uging he bac e ial suspension a 6000 pm o 30 min [ 28 ]. The abili y o p oduce IAA was indica ed by he appea ance o pink colo a e he addi ion o Salkowski eagen . Abso bance was measu ed a 530 nm and he quan i y o IAA was de e mined om a s anda d cu e and exp essed as µ g/mL. These expe imen s we e ca ied ou in iplica e o each isola e. 2.6. Da a Analysis The expe imen al da a we e o ganized and analyzed using Mic oso Excel ® sp ead- shee so wa e, e sion 2206, 64 bi . This sp eadshee so wa e was also used o calcula e he means and he 95% con idence in e als o phospha e-solubilizing ac i i y, pH, and IAA p oduc ion. IBM SPSS ® so wa e package e sion 28 was used o pe o m Pea son’s co ela ions, Analysis o Va iance (ANOVA), and pos hoc compa ison es s wi h a con idence le el o 95% ( α = 0.05). ANOVA F- es es was pe o med o de e mine he signi icance o he di e ences be ween he means o phospha e-solubilizing ac i i y, pH, and IAA p oduc ion, and Tukey’s HSD pos hoc me hod was applied o iden i y homogeneous subse s. Pea son co ela ion analysis was pe o med o de e mine he co ela ion be ween PSB’s phospha e- solubilizing ac i i y and he soil’s physicochemical p ope ies. 3. Resul s and Discussion 3.1. Physical and Chemical Cha ac e is ics o Soil Samples The physical and chemical cha ac e is ics o he soil samples a e shown in Table 2. All si es had soil wi h a sandy ex u e and almos neu al pH. The soil om si e 5 had he lowes o ganic ma e and ca bon con en . The phospho us was is simila o all soils and close o he alue o 70 ppm. Table 2. Physical and chemical cha ac e is ics o soil samples. Si e Type Tex u e pH O ganic Ma e (%) To al Ca bon (%) To al Ni ogen (mg/L) To al Phospho ous (ppm) 1 Fo es Sandy clay 7.05 13.21 7.65 0.52 67.19 2 Fo es Sandy loam 6.50 7.02 4.06 0.39 72.51 3 Fo es Sandy loam 6.56 11.27 6.49 0.47 68.86 4 Ag icul u e Sandy loam 6.90 4.38 2.53 0.30 66.12 5 Fo es Sandy 6.60 1.07 0.62 0.28 66.69 3.2. Cha ac e iza ion o he Isola es Twen y-eigh bac e ial colonies we e isola ed and hei mac oscopic and mic oscopic cha ac e is ics we e de ined. Isola ed colonies had pink and yellowish colo s. All bac e ia had s em o m and G am-nega i e bac e ial g oup iden i y, and hey we e mobile and Mic oo ganisms 2023,11, 783 6 o 14 spo e- ee. Mei eles e al. [ 29 ] epo ed ha his ype o bac e ia is mos abundan in soils. Acco ding o Pa en and Glick [ 30 ], hese od-shaped, mo ile, non-spo e- o ming bac e ia wi h G am-nega i e s ains usually belong o he genus Pseudomonas, which is p edominan in he hizosphe e. 3.3. Molecula Iden i ica ion and Phylogene ic Analyses The molecula iden i ica ion o 28 isola es con i med he p esence o i e species belonging o h ee gene a, namely Pseudomonas (P. luo escens,P. pu ida, and P. aiwanensis), Pan oea agglome ans, and S eno ophomonas mal ophilia (Figu e 1). The inding o hese i e species o PSB is in line wi h he p e ious esul s s a ing ha bac e ia belonging o he gene a Pseudomonas,En e obac e , and Bacillus [ 31 ], Pan oea [ 32 ], in addi ion o some species o he gene a Rhizobium and A h obac e [ 33 , 34 ], a e powe ul PSB. Biswas e al. [ 31 ] men ioned ha Pseudomonas spp., Bacillus spp., and Rhizobium spp. a e among he mos e ec i e gene a o solubilize phospha es. In addi ion, s ains o he genus Pan oea ha e been speci ied as solubilizing agen s. Singh and Jha [ 35 ] also demons a ed ha S eno ophomonas mal ophilia is a powe ul species in he solubiliza ion o P, which makes i possible o elease phospha e om insoluble g anules such as icalcium phospha e o o he na u al phospha es. Mic oo ganisms 2023, 11, x FOR PEER REVIEW 6 o 15 Si e Type Tex u e pH O ganic Ma e (%) To al Ca bon (%) To al Ni ogen (mg/L) To al Phospho ous (ppm) 1 Fo es Sandy clay 7.05 13.21 7.65 0.52 67.19 2 Fo es Sandy loam 6.50 7.02 4.06 0.39 72.51 3 Fo es Sandy loam 6.56 11.27 6.49 0.47 68.86 4 Ag icul u e Sandy loam 6.90 4.38 2.53 0.30 66.12 5 Fo es Sandy 6.60 1.07 0.62 0.28 66.69 3.2. Cha ac e iza ion o he Isola es Twen y-eigh bac e ial colonies we e isola ed and hei mac oscopic and mic oscopic cha ac e is ics we e de ined. Isola ed colonies had pink and yellowish colo s. All bac e ia had s em o m and G am-nega i e bac e ial g oup iden i y, and hey we e mobile and spo e- ee. Mei eles e al. [29] epo ed ha his ype o bac e ia is mos abundan in soils. Acco ding o Pa en and Glick [30], hese od-shaped, mo ile, non-spo e- o ming bac e ia wi h G am-nega i e s ains usually belong o he genus Pseudomonas, which is p edomi- nan in he hizosphe e. 3.3. Molecula Iden i ica ion and Phylogene ic Analyses The molecula iden i ica ion o 28 isola es con i med he p esence o i e species be- longing o h ee gene a, namely Pseudomonas (P. luo escens, P. pu ida, and P. aiwanensis), Pan oea agglome ans, and S eno ophomonas mal ophilia (Figu e 1). The inding o hese i e species o PSB is in line wi h he p e ious esul s s a ing ha bac e ia belonging o he gene a Pseudomonas, En e obac e , and Bacillus [31], Pan oea [32], in addi ion o some spe- cies o he gene a Rhizobium and A h obac e [33,34], a e powe ul PSB. Biswas e al. [31] men ioned ha Pseudomonas spp., Bacillus spp., and Rhizobium spp. a e among he mos e ec i e gene a o solubilize phospha es. In addi ion, s ains o he genus Pan oea ha e been speci ied as solubilizing agen s. Singh and Jha [35] also demons a ed ha S eno- ophomonas mal ophilia is a powe ul species in he solubiliza ion o P, which makes i possible o elease phospha e om insoluble g anules such as icalcium phospha e o o he na u al phospha es. Figu e 1. Pa ial sequence 16S RNA gene phylogene ic ee o Pseudomonas ( A – C ), Pan oea agglome ans (D) , and S eno ophomonas (E) species. (Species desc ibed in his s udy a e indica ed wi h a black squa e). 3.4. Composi ion o PSB Communi y Among isola ed PSB, P. luo escens domina ed, ollowed by S. mal ophyla, P. pu ida , P. aiwanensis , and P. agglome ans (Figu e 2). Simila esul s we e ob ained by Mei eles e al. [29] , s a ing ha he genus Pseudomonas is es ima ed as he dominan PSB in soils. Mic oo ganisms 2023,11, 783 7 o 14 Mic oo ganisms 2023, 11, x FOR PEER REVIEW 7 o 15 Figu e 1. Pa ial sequence 16S RNA gene phylogene ic ee o Pseudomonas (A–C), Pan oea agglom- e ans (D), and S eno ophomonas (E) species. (Species desc ibed in his s udy a e indica ed wi h a black squa e). 3.4. Composi ion o PSB Communi y Among isola ed PSB, P. luo escens domina ed, ollowed by S. mal ophyla, P. pu ida, P. aiwanensis, and P. agglome ans (Figu e 2). Simila esul s we e ob ained by Mei eles e al. [29], s a ing ha he genus Pseudomonas is es ima ed as he dominan PSB in soils. Figu e 2. Rela i e abundance o he di e en species o PSB in he s udied Tunisian soils. 3.5. Calcula ion o he Phospha e Solubiliza ion Index All isola ed colonies we e sc eened o phospha e solubiliza ion on solid media. The phospha e solubiliza ion abili y is ma ked by he o ma ion o anspa en halos a ound he bac e ial colony (on he solid PVK and NBRIP media), supplemen ed wi h TCP as he only sou ce o phospho us. The isola es showed a clea halo zone a ound hei colonies, which could be he esul o he p oduc ion o o ganic acids o polysaccha ides, o he ac i i y o phospha ase enzymes [10]. The a e age SI o he mos e ec i e isola e o each specie is p esen ed in Table 3. Table 3. Phospha e solubiliza ion index o PSB isola es (mean ± con idence in e al, n = 3). Same le e s indica e ha he di e ence be ween he means is no s a is ically signi ican . PSB SI in NBRIP SI in PVK Pseudomonas pu ida 3.53 ± 0.05 a 2.14 ± 0.03 e Pseudomonas luo escens 3.82 ± 0.05 c 3.51 ± 0.05 a Pseudomonas aiwanensis 3.64 ± 0.05 a 3.12 ± 0.03 S eno ophomonas mal ophilia 2.83 ± 0.02 d 2.31 ± 0.02 b Pan oea agglome ans 2.35 ± 0.02 b 2.98 ± 0.03 g The di e ence in he SI means was s a is ically signi ican among he esul s ob ained o bo h NBRIP and PVK media (based on he one-way ANOVA, p < 0.01). Mo eo e , SI o he same isola es on di e en media we e also signi ican ly di e en (p < 0.01). This obse a ion con i ms ha he halo me hod should be limi ed o a p elimina y sc eening o PSB, al hough being a p ac ical s ep because o i s i s easy and as applica ion [10]. Fo his eason, he isola ed and pu i ied bac e ia we e also assayed in a liquid medium wi h a p ecise quan i ica ion o soluble phospho us eleased o alida e hei ac i i y. S ill, SI can be a basis o a p elimina y compa ison o he i e isola es’ abili y o sol- ubilize TCP. Thus, he isola es o he genus Pseudomonas seem o be he mos e icien o solubilizing solid phospha es among he es ed isola es, al hough di e ences among hem we e no s a is ically signi ican based on he one-way ANOVA. The solubiliza ion o Figu e 2. Rela i e abundance o he di e en species o PSB in he s udied Tunisian soils. 3.5. Calcula ion o he Phospha e Solubiliza ion Index All isola ed colonies we e sc eened o phospha e solubiliza ion on solid media. The phospha e solubiliza ion abili y is ma ked by he o ma ion o anspa en halos a ound he bac e ial colony (on he solid PVK and NBRIP media), supplemen ed wi h TCP as he only sou ce o phospho us. The isola es showed a clea halo zone a ound hei colonies, which could be he esul o he p oduc ion o o ganic acids o polysaccha ides, o he ac i i y o phospha ase enzymes [ 10 ]. The a e age SI o he mos e ec i e isola e o each specie is p esen ed in Table 3. Table 3. Phospha e solubiliza ion index o PSB isola es (mean ± con idence in e al, n = 3). Same le e s indica e ha he di e ence be ween he means is no s a is ically signi ican . PSB SI in NBRIP SI in PVK Pseudomonas pu ida 3.53 ±0.05 a 2.14 ±0.03 e Pseudomonas luo escens 3.82 ±0.05 c 3.51 ±0.05 a Pseudomonas aiwanensis 3.64 ±0.05 a 3.12 ±0.03 S eno ophomonas mal ophilia 2.83 ±0.02 d 2.31 ±0.02 b Pan oea agglome ans 2.35 ±0.02 b 2.98 ±0.03 g The di e ence in he SI means was s a is ically signi ican among he esul s ob ained o bo h NBRIP and PVK media (based on he one-way ANOVA, p< 0.01). Mo eo e , SI o he same isola es on di e en media we e also signi ican ly di e en (p< 0.01). This obse a ion con i ms ha he halo me hod should be limi ed o a p elimina y sc eening o PSB, al hough being a p ac ical s ep because o i s i s easy and as applica ion [ 10 ]. Fo his eason, he isola ed and pu i ied bac e ia we e also assayed in a liquid medium wi h a p ecise quan i ica ion o soluble phospho us eleased o alida e hei ac i i y. S ill, SI can be a basis o a p elimina y compa ison o he i e isola es’ abili y o solubilize TCP. Thus, he isola es o he genus Pseudomonas seem o be he mos e icien o solubilizing solid phospha es among he es ed isola es, al hough di e ences among hem we e no s a is ically signi ican based on he one-way ANOVA. The solubiliza ion o solid phospha es is a e y common cha ac e is ic in Pseudomonas and ce ain En e obac e iaceae (including P. agglome ans) [ 32 ]. These bac e ia also play a signi ican ole in he soil h ough hei me abolic ac i i ies, o example, he solubiliza ion o complex o ms o phospha e o mo e accessible o ms o plan s and h ough he p ocesses o acidi ica ion, chela ion, and exchange eac ions [36]. The isola es o S eno ophomonas mal ophilia and Pan oea agglome ans showed con adic- o y esul s on SI in he wo solid media, bu he a e age SI o bo h species is signi ica i ely lowe han he SI o he isola es o he Pseudomonas genus. Howe e , isola es o S. mal ophila and P. agglome ans can be conside ed e ec i e PSB. P e iously, Ramos e al. [ 37 ] showed ha S. mal ophilia can g ow on a ious phospha e-selec i e media. The indings o he Mic oo ganisms 2023,11, 783 8 o 14 p esen wo k a e also suppo ed by he esul s o Kuma and Audipudi [ 28 ], which demon- s a ed ha S. mal ophilia AVP27 isola ed om peppe hizosphe e was able o p oduce p ominen a eas o P solubiliza ion on he PVK aga pla e. On he o he hand, Pan oea is a less-s udied genus, bu some o hei species, such as P. agglome ans, ha e al eady been desc ibed as PSB [38]. 3.6. E alua ion o he Phospha e Solubiliza ion in Liquid Media Figu e 3shows he esul s ob ained o he i e isola es in liquid NBRIP and PVK media. Al hough he solubiliza ion le els in liquid media a ied among di e en isola es, all he isola es we e capable o solubilizing TCP as he only sou ce o phospha e in PVK and NBRIP media. The di e ences be ween he mean solubiliza ion abili y in he wo cul u e media a e s a is ically signi ican acco ding o he one-way ANOVA (p< 0.01). Mic oo ganisms 2023, 11, x FOR PEER REVIEW 8 o 15 solid phospha es is a e y common cha ac e is ic in Pseudomonas and ce ain En e obac e- iaceae (including P. agglome ans) [32]. These bac e ia also play a signi ican ole in he soil h ough hei me abolic ac i i ies, o example, he solubiliza ion o complex o ms o phospha e o mo e accessible o ms o plan s and h ough he p ocesses o acidi ica ion, chela ion, and exchange eac ions [36]. The isola es o S eno ophomonas mal ophilia and Pan oea agglome ans showed con a- dic o y esul s on SI in he wo solid media, bu he a e age SI o bo h species is signi i- ca i ely lowe han he SI o he isola es o he Pseudomonas genus. Howe e , isola es o S. mal ophila and P. agglome ans can be conside ed e ec i e PSB. P e iously, Ramos e al. [37] showed ha S. mal ophilia can g ow on a ious phospha e-selec i e media. The indings o he p esen wo k a e also suppo ed by he esul s o Kuma and Audipudi [28], which demons a ed ha S. mal ophilia AVP27 isola ed om peppe hizosphe e was able o p o- duce p ominen a eas o P solubiliza ion on he PVK aga pla e. On he o he hand, Pan oea is a less-s udied genus, bu some o hei species, such as P. agglome ans, ha e al eady been desc ibed as PSB [38]. 3.6. E alua ion o he Phospha e Solubiliza ion in Liquid Media Figu e 3 shows he esul s ob ained o he i e isola es in liquid NBRIP and PVK media. Al hough he solubiliza ion le els in liquid media a ied among di e en isola es, all he isola es we e capable o solubilizing TCP as he only sou ce o phospha e in PVK and NBRIP media. The di e ences be ween he mean solubiliza ion abili y in he wo cul- u e media a e s a is ically signi ican acco ding o he one-way ANOVA (p < 0.01). The phospha e-solubilizing abili y o he es ed isola es in NBRIP liquid medium, wi h TCP as a sou ce o insoluble phospho us, a ied om 535.70 o 618.57 µg mL−1, wi h maximal abili y demons a ed by P. luo escens. In he liquid medium PVK, he solubiliza- ion o TCP by he PSB a ied om 374.20 o 544.28 µg mL−1, and P. luo escens also demon- s a ed he bes solubiliza ion capabili y. Figu e 3. Phospha e solubiliza ion by bac e ial isola es g own in NBRIP and PVK media a 28 ± 2 °C. Ve ical ba s ep esen con idence in e als a a con idence le el o 95% (n = 3). Same le e s indica e ha di e ences among he means is no s a is ically signi ican (Tukey’s HSD pos hoc es ). The highes phospha e solubiliza ion was obse ed in he medium NBRIP o all iso- la es. These esul s a e in acco dance wi h he esul s o Nau iyal e al. [39], who classi ied he liquid NBRIP medium as he bes medium o es ing he phospha e solubiliza ion. Che i e al. [40] and Chibani e al. [41] also epo ed ha NBRIP is he p e e ed liquid medium o phospha e solubiliza ion by he majo i y o hizobac e ia. The isola es o P. luo escens p oduced he highes concen a ion o soluble phospha e in he NBRIP b o h a e 7 days o incuba ion (618.57 µg mL−1). This inding is suppo ed by he esul s ob ained by Gup a e al. [42] and Ahmad e al. [43], showing ha Figu e 3. Phospha e solubiliza ion by bac e ial isola es g own in NBRIP and PVK media a 28 ± 2 ◦ C. Ve ical ba s ep esen con idence in e als a a con idence le el o 95% (n = 3). Same le e s indica e ha di e ences among he means is no s a is ically signi ican (Tukey’s HSD pos hoc es ). The phospha e-solubilizing abili y o he es ed isola es in NBRIP liquid medium, wi h TCP as a sou ce o insoluble phospho us, a ied om 535.70 o 618.57 µ g mL −1 , wi h maximal abili y demons a ed by P. luo escens. In he liquid medium PVK, he solubiliza ion o TCP by he PSB a ied om 374.20 o 544.28 µ g mL −1 , and P. luo escens also demons a ed he bes solubiliza ion capabili y. The highes phospha e solubiliza ion was obse ed in he medium NBRIP o all isola es. These esul s a e in acco dance wi h he esul s o Nau iyal e al. [ 39 ], who classi ied he liquid NBRIP medium as he bes medium o es ing he phospha e solubiliza ion. Che i e al. [ 40 ] and Chibani e al. [ 41 ] also epo ed ha NBRIP is he p e e ed liquid medium o phospha e solubiliza ion by he majo i y o hizobac e ia. The isola es o P. luo escens p oduced he highes concen a ion o soluble phospha e in he NBRIP b o h a e 7 days o incuba ion (618.57 µ g mL −1 ). This inding is suppo ed by he esul s ob ained by Gup a e al. [ 42 ] and Ahmad e al. [ 43 ], showing ha Pseudomonas sp. is an e ec i e phospha e solubilize . The isola es o S. mal ophilia and P. agglome ans also exhibi ed a high le el o phospha e solubiliza ion capabili y in he liquid NBRIP. The alues ob ained a e e en highe han he alues ob ained by Pa el and Sa a [ 44 ] and Son e al. [38] , 362 µg mL−1 o S. mal ophilia and 200 µg mL−1 o P. agglome ans. The solubiliza ion o phospha e by he isola es in he PVK liquid media showed signi - ican di e ences in hei means. Mo eo e , all PSB bu P. aiwanensis showed s a is ically lowe solubili y in PVK medium han in NBRIP medium. In he PVK medium, P. luo escens and P. aiwanensis had a signi ica i ely highe phospha e solubiliza ion capabili y han he o he h ee isola es. Al hough he esul s ob ained in bo h liquid media a e p omising, u he s udies mus be conduc ed in he soil o alida e he e ec i e capabili y o hose PSB in eal ield condi ions. Mic oo ganisms 2023,11, 783 9 o 14 3.7. pH Changes in NBRIP and PVK Liquid Media The solubiliza ion o TCP by PSB was accompanied by a signi ican dec ease in he pH o he cul u e supe na an , bo h in he NBRIP and PVK media (Figu es 4and 5). Mic oo ganisms 2023, 11, x FOR PEER REVIEW 9 o 15 Pseudomonas sp. is an e ec i e phospha e solubilize . The isola es o S. mal ophilia and P. agglome ans also exhibi ed a high le el o phospha e solubiliza ion capabili y in he liquid NBRIP. The alues ob ained a e e en highe han he alues ob ained by Pa el and Sa a [44] and Son e al. [38], 362 µg mL−1 o S. mal ophilia and 200 µg mL−1 o P. agglome ans. The solubiliza ion o phospha e by he isola es in he PVK liquid media showed sig- ni ican di e ences in hei means. Mo eo e , all PSB bu P. aiwanensis showed s a is i- cally lowe solubili y in PVK medium han in NBRIP medium. In he PVK medium, P. luo escens and P. aiwanensis had a signi ica i ely highe phospha e solubiliza ion capa- bili y han he o he h ee isola es. Al hough he esul s ob ained in bo h liquid media a e p omising, u he s udies mus be conduc ed in he soil o alida e he e ec i e capabili y o hose PSB in eal ield condi ions. 3.7. pH Changes in NBRIP and PVK Liquid Media The solubiliza ion o TCP by PSB was accompanied by a signi ican dec ease in he pH o he cul u e supe na an , bo h in he NBRIP and PVK media (Figu es 4 and 5). All alues we e signi ican ly di e en om he ini ial pH (p < 0.01, one-way ANOVA) in NBRIP cul u e medium. The means we e signi ica i ely di e en o he pH a h ee and se en days (p < 0.01), bu we e no signi ica i ely di e en among di e en species (p = 0.18 and 0.19, espec i ely o h ee- and se en-day alues, om he one-way ANOVA). Zhu e al. [45] p o ed ha he solubiliza ion o insoluble phospha e depends on a - ious ac o s bu , in pa icula , is accompanied by a decline in pH. In ac , he esul s o he p esen wo k con i med a dec ease in pH alue du ing he TCP solubiliza ion by all i e s ains o PSB, like in he NBRIP medium whe e inal pH alues anged om 4.1 o 4.7. Figu e 4. Va ia ion o pH in NBRIP cul u e medium. Ve ical ba s ep esen con idence in e als a a con idence le el o 95% (n = 3). Same le e s indica e ha di e ences among he means is no s a- is ically signi ican (Tukey’s HSD pos hoc es ). The solubiliza ion o phospha e in PVK medium was also accompanied by a dec ease o he o iginal pH (Figu e 5), as obse ed o he NBRIP medium. Howe e , he mean dec eases o pH obse ed o he i s h ee days o cul i a ion in he PVK medium we e no s a is ically di e en om he ini ial pH o he h ee Pseudomonas species (p = 0.23), being signi ican ly di e en a e se en days (p < 0.01). The inal pH a e he se en days o cul u e anged om 4.3 o 5.4, highe han he pH obse ed o he cul u es in he NBRIP medium. Se e al wo ks epo ed a simila dec ease in he cul u e media pH du ing phospha e solubiliza ion: Pa k e al. [46] and Mule a e al. [47] epo ed ha P. lu escens educed he pH alues o media o 4; Ghosh e al. [48] epo ed a dec ease in he pH om 7 o 4.3 a e ou days o S. mal ophila incuba ion in NBRIP medium; Cos a e al. [49] epo ed a de- c ease o pH 5 a e he cul i a ion o P. agglome ans in PVK medium. Figu e 4. Va ia ion o pH in NBRIP cul u e medium. Ve ical ba s ep esen con idence in e als a a con idence le el o 95% (n = 3). Same le e s indica e ha di e ences among he means is no s a is ically signi ican (Tukey’s HSD pos hoc es ). Mic oo ganisms 2023, 11, x FOR PEER REVIEW 10 o 15 The acidi ica ion o cul u e supe na an s indica ed ha he p oduc ion o o ganic ac- ids seemed o be he main mechanism o phospha e solubiliza ion. Wan e al. [50] e- po ed ha a ious PSB p oduced gluconic acid, 2-ke ogluconic acid, lac ic acid, and ci ic acid du ing he solubiliza ion o insoluble phospha es. Khan e al. [51] and O eino e al. [52] also showed ha he solubiliza ion ac i i y o PSB in he soil is based on he sec e ion o o ganic acids. In addi ion, he p oduc ion o acids o elease phospho us om mine al complexes by a ious gene a o bac e ia including Bacillus, Pseudomonas, En e obac e , Se - a ia, and Azo obac e , in connec ion wi h o he chemical mechanisms such as chela ion eac ions, was also demons a ed by Kisho e e al. [36]. When compa ing he medium acidi ica ion wi h he a e age phospha e concen a ion in liquid media, a s ong nega i e co ela ion was obse ed in PVK medium (Pea son’s co - ela ion coe icien o −0.854, p = 0.065). On he o he hand, a e y weak co ela ion was seen in he NBRIP media (Pea son’s co ela ion coe icien o −0.049, p = 0.938). This unexpec ed esul is in pa allel wi h he obse a ion ha he inal pH alues a e no s a is ically di e en ac oss all i e PSB cul u es in he NBRIP medium, gi ing he hypo hesis ha a pH nea 4 can be a lowe limi alue o he g ow h o he isola ed PSB in phospha e- ich media. How- e e , u u e wo k needs o be ca ied ou o con i m his hypo hesis. Figu e 5. Va ia ion o pH in PVK cul u e medium. Ve ical ba s ep esen con idence in e als a a con idence le el o 95% (n = 3). Same le e s indica e ha di e ences among he means is no s a is- ically signi ican (Tukey’s HSD pos hoc es ). 3.8. De e mina ion o Indole Ace ic Acid P oduc ion Indole ace ic acid p oduc ion was cha ac e is ic o almos all bac e ial isola es. The IAA p oduc ion anged om 31.29 o 50.37 µg mL−1, as shown in Figu e 6. A ela i ely high con en o IAA was ound in he cul u e o P. luo escens (50.37 µg mL−1), ollowed by Pan oea agglome ans and P. pu ida wi h 37.83 and 37.41 µg mL−1, espec i ely. Figu e 5. Va ia ion o pH in PVK cul u e medium. Ve ical ba s ep esen con idence in e als a a con idence le el o 95% (n = 3). Same le e s indica e ha di e ences among he means is no s a is ically signi ican (Tukey’s HSD pos hoc es ). All alues we e signi ican ly di e en om he ini ial pH (p< 0.01, one-way ANOVA) in NBRIP cul u e medium. The means we e signi ica i ely di e en o he pH a h ee and se en days (p< 0.01), bu we e no signi ica i ely di e en among di e en species ( p= 0.18 and 0.19, espec i ely o h ee- and se en-day alues, om he one-way ANOVA). Zhu e al. [ 45 ] p o ed ha he solubiliza ion o insoluble phospha e depends on a ious ac o s bu , in pa icula , is accompanied by a decline in pH. In ac , he esul s o he p esen wo k con i med a dec ease in pH alue du ing he TCP solubiliza ion by all i e s ains o PSB, like in he NBRIP medium whe e inal pH alues anged om 4.1 o 4.7. The solubiliza ion o phospha e in PVK medium was also accompanied by a dec ease o he o iginal pH (Figu e 5), as obse ed o he NBRIP medium. Howe e , he mean dec eases o pH obse ed o he i s h ee days o cul i a ion in he PVK medium we e no s a is ically di e en om he ini ial pH o he h ee Pseudomonas species (p= 0.23), being signi ican ly di e en a e se en days (p< 0.01). The inal pH a e he se en days o cul u e anged om 4.3 o 5.4, highe han he pH obse ed o he cul u es in he NBRIP medium.