Acta agriculturae Slovenica, 118/1, 1–8, Ljubljana 2022 doi:10.14720/aas.2022.118.1.2262 Original research article / izvirni znanstveni članek Isolation of phosphate solubilizing bacteria from root rhizosphere to sup- plement biofertilizer Thi Thuy NGUYEN 1, The Anh LUU 2, Quang Trung DO 2, 3 Received June 28, 2021; accepted December 17, 2021. Delo je prispelo 28. junija 2021, sprejeto 17. decembra 2021 1 Institute of Geography, Vietnam Academy of Science and Technology, Ha Noi, Vietnam 2 Central Institute for Natural Resources and Environmental Studies, Vietnam National University, Ha Noi, Vietnam 3 Corresponding author, e-mail: trungcnsinh@gmail.com Isolation of phosphate solubilizing bacteria from root rhizo- sphere to supplement biofertilizer Abstract: In soil, a large amount of supplemented phos- phorus (P) are immediately transferred into insoluble forms and only 0.1 % of them is available for plant uptake. Therefore, exploring naturally occurring phosphate-solubilizing micro- organisms is an essential activity to exploit them in reducing mineral phosphorus added to agricultural soils. In this study, we screened and isolated 7 bacteria that solubilized phosphate at different phosphate solubilization indexes, ranging from 4.2 to 226.1. Of them, the most efficient isolate is PSB31, which solubilized tri calcium phosphate (Ca3(PO4)2 at a rate of 962 mg l-1 and molecularly identified as Bacillus sp. (in: Bacteria) strain IMAU61039. This bacterial strain generated the low superna- tant pH and the phosphatase, which are involved in the phos- phorus solubilization mechanism. Furthermore, greenhouse experiments showed that tomato seedlings grown in PSB31-in- oculated soil contained higher P amount and had much higher biomass than those plants grown in soil without PSB31 addi- tion. These results suggest that the PSB31 strain has potential use as a biofertilizer. Key words: phosphates-solubilizing bacteria; plant growth promoting bacteria; biofertilizer; tomato; phosphatase Izolacija bakterij, ki sproščajo fosfat iz rizosfere kot nadome- stek biognojilom Izvleček: V tleh se velike količine dodanega fosforja hitro spremenijo v netopne oblike tako, da ostane rastlinam raspo- ložljivega le okrog 0,1  %. Zaradi tega je izkoriščanje v naravi prisotnih fosfat sproščajočih mikroorganizmov nepogrešlji- va aktivnost, ki omogoča njihovo uporabo in zmanjšujej do- dajanje mineralnega fosforja v kmetijska tla. V raziskavi smo preverili in izolirali 7 bakterij, ki sproščajo fosfat z različnimi indeksi sproščanja od 4,2 do 226,1. Med njimi je bil najučiko- vitejši izolat PSB31, ki je sproščal tri kalcijev fosfat (Ca3(PO4)2 v velikosti 962 mg l-1, na osnovi molekularnih testov določen kot IMAU61039 soj bakterije iz rodu Bacillus. Ta soj bakterije je generiral nizek pH v raztopini in fosfataze, ki so vključene v mehanizem sproščanja fosforja. Nadalje je poskus v rastlinja- ku pokazal, da so vsebovale sejanke paradižnika, ki so rastle v tleh inokuliranih z izolatom PSB31 večjo vsebnost fosforja in mnogo večjo biomaso kot tiste, ki so rastle v tleh brez dodatka PSB31. Izsledki nakazujejo, da bi se izolat PSB31 lahko upora- bljal kot biognojilo. Ključne besede: fosfor sproščajoče bakterije; rast rastlin vzpodbujajoče bakterije; biognojila; paradižnik; fosfataze 2 Acta agriculturae Slovenica, 118/1– 2022 T. T. NGUYEN et al. 1 INTRODUCTION Phosphorus is an essential macronutrient that to- gether with others such as C, N, plays an important role in the normal growth of a plant. In agriculture, chemi- cal fertilizer was used to supplement the phosphorus source to promote plant development and increase crop yield. However, it is reported that only a small propor- tion of phosphorus in provided fertilizer is available for plant uptake, and 95  %–99  % of it were insoluble, im- mobilized, or precipitated under effect of environmental factors such as soil pH (Khan et al., 2009). Subsequently, the proportion of insoluble phosphates deposited in the soil are increased (Morales et al., 2011). Therefore, the best strategy in crop management is limit the addition of phosphorus into soil under chemical fertilizer form; increase the agents converting P reserved in the soil; and reclaim the chemically-bound P from insoluble form (Cordell et al., 2009). Although there are many solutions to balance the input/output ratio, identification of the least risky alternatives to traditional practices is carrying out. Among those, using phosphate-solubilizing bacteria (PSB) that can solubilize the insoluble phosphate is an emerging solution. Phosphate-solubilizing bacteria (PSB) living in the root rhizosphere present the ability in reversing the in- soluble phosphate into a soluble form easily used by plants. Chen et al. (2006) demonstrated the low molecu- lar mass organic acids produced by PSB played role in dissolving the phosphate complexed minerals. In another study, Vyas and Gulati (2009) showed that PSB-produced low molecular mass organic acids also chelated the cati- ons that formed complexes with P ions (PO4 3-) to release P directly into the rhizosphere soil. These results demon- strated soil PSB could release P from the insoluble forms to increase soil fertility. In addition, some PSB genera (such as Arthrobacter, Burkholderia, Beijerinckia, Erwin- ia, Bacillus, Rhizobium, Pseudomonas, and Mesorhizo- bium) have been exploited as soil inoculants to promote plant growth and subsequently increase the yield (Kumar et al., 2017). Hence, using PSB as a biofertilizer was determined as an alternative for expensive and environmentally dam- aging fertilizers in the future. Despite the role in solubi- lizing insoluble P and promoting plant development, the application of PSB as a bio-fertilizer is still needed fur- ther studies due to the composition or variation in soils and bacterial community (Barea, 2015). Thai Binh province play important role in providing agricultural products for market in Vietnam. However, the overuse of chemical fertilizer is one of major factors which has caused soil pollution in Thai Binh. Therefore, development of biofertilizer plays a key role for the sus- tainable agriculture in this province. Hence, we aimed to isolate and characterize high phosphorus-solubilizing bacteria from agricultural soil collected in Thai Binh, and also investigated their potential in developing these strains as biofertilizer. 2 MATERIAL AND METHODS 2.1 ISOLATION OF BACTERIA WITH PHOSPHO- RUS-SOLUBILIZING ABILITY The soil samples were collected from the field grown maize, rice, and tomato in Thai Binh (Vietnam) on December 14, 2020. Different locations in Thai Binh province were located including Tien Hai (20°24’27.7”N 106°31’00.1”E) and Kien Xuong (20°22’54.0”N 106°23’43.2”E), which provided more than 80% agricul- tural products (rice, maize and tomato) for the market in Vietnam. Sampling procedure is carried out according to TCVN 4046-1985 (TCVN 4046 – 85, 1985) as follows: soil samples were taken according to the diagonal or zig- zag rule depending on the topography of the land. Each site took from 15 to 20 samples, each sample was about 0.5 kg and the samples were mixed to be represented by the diagonal rule of about 0.5 kg. PSB was isolated from soil samples by serially dilut- ing up to 10−10 by sterilized water and inoculating in Pik- ovskaya’s agar (PVK) agar medium by pour plate method (Cao et al., 2018). For control, only sterilized water was used. The incubation of all plates was done at 30 °C for 7 days. After 7 day of incubation, the bacteria generated a clear zone around colonies were identified as strains hav- ing the phosphorus-solubilizing ability. Then, the isolat- ed single colonies presenting a clearing zone around were transferred onto new PVK plates. The strains generated the highest clearing zones are considered as potential PSB and were selected for the next experiments. 2.2 DETERMINE PHOSPHATE SOLUBILIZING ACTIVITY OF BACTERIA ON AGAR MEDIUM After 7 days of incubation, the clearing zones around single colonies on the reinoculated plates were measured and validated (Sharon et al., 2016): Phosphate solubilizing index (PSI) = [(colony diam- eter + clearing zone)/ colony diameter] x 100 3Acta agriculturae Slovenica, 118/1 – 2022 Isolation of phosphate solubilizing bacteria from root rhizosphere to supplement biofertilizer 2.3 DETERMINE PHOSPHATE SOLUBILIZING ACTIVITY OF BACTERIA IN PVK LIQUID MEDIUM The P solubilizing efficiency of the isolates was in- vestigated by growing in the PVK liquid medium. 200 µl of selected isolate was cultured in 9.8 ml of PVK medi- um with 0.5 % Ca3(PO4)2 (w/v) on the shaker at 30 °C. The culture was collected and centrifuged to obtain the supernatant using to determine the solubilized P by va- nadomolybdate method (Pearson, 1976). Briefly, 1 ml of supernatant (distilled water for the blank) was trans- ferred into a clean cuvette. Then, added 0.25 ml of vana- date-molybdate reagent and mixed well by pipetting up and down several times. After 10 minutes, placed the cu- vette with sample into the UV/VIS spectrophotometers (METTLER TOLEDO, USA) and measured. The efficiency of the bacteria in solubilizing the in- soluble phosphorus compound was identified as the per- cent of the total phosphorus presenting in the medium. The culture pH was also measured by using a benchtop pH meter (Mettler Toledo, USA). All experiments were performed in triplicates. 2.4 DETERMINE PHOSPHATE SOLUBILIZING ACTIVITY OF BACTERIA IN A POTTING SAND MATRIX The P solubilization efficacy of the isolates was also investigated in a less-nutrient environment like acid- washed and sterilized sand. The experiment was pre- pared in triplicates as follows: 9 g of treated sand were added into a 15 ml tube containing a 5 ml reaction. Af- ter mixing well the reaction mixture, added 1 ml of PVK media (5 % Ca3(PO4)2) inoculating with or without PSB strain (2 x 106 cfu ml-1) into the reaction. Then, the in- oculated samples were kept in the incubator at 30 oC for 24 h. After incubation, adding the distilled water into the sample to make a final volume of 10 ml was done before shaking it at 200 rpm for 1 h, and followed by centrifuga- tion at 3,500 rpm. After that, the supernatant was col- lected by filtrating the culture media through a 0.45 μm filter. Then, the amount of released P was identified by vanadomolybdate method (Pearson, 1976). 2.5 PHOSPHATASE ENZYMATIC ASSAY Phosphatase was explored by using the method de- scribed by Tabatabai and Bremner (1969). Briefly, the selected strain was grown in a 250 ml conical flash con- taining 100 ml broth PVK medium for 80 h. In every 5 h, the culture was taken and removed the bacterial cells by centrifuging at 10,000 rpm for 10 min at 4 °C. After that, 1ml of cell-free supernatant was mixed with 4 ml of modified universal buffer (pH 6.5). Then added 1 ml of 0.025 mM disodium p-nitrophenyl phosphate (tetrahy- drate) into this mixture. The solution was mixed well and incubated at 37 °C for 1 h. After 1 hour incubation, 4 ml of 0.5 M NaOH and 1 ml of 0.5 M CaCl2 was added to stop the reaction. The solution was then filtered through Whatman No. 42 filter paper. The filtered solution was used to measure the concentration of p-nitrophenol by using the UV/VIS spectrophotometers (Mettler Toledo, USA) at 420 nm. The values were identified on the stand- ard curve. Each measurement was done in triplicate. The standard curve was obtained by serially dilut- ing the standard p-nitrophenol solution. The control was also prepared as above procedure but the additions of 0.5M CaCl2 and 0.5M NaOH was applied before the ad- dition of 1 ml of 0.025 mM disodium p-nitrophenyl. The amount of enzyme that used to release 1 μmol of p-nitro- phenol ml-1 min-1 from di-Na  p-nitrophenyl phosphate (tetrahydrate) under the assay condition was defined as one unit (U) of phosphatase activity. 2.6 MOLECULAR IDENTIFICATION OF PSB31 STRAIN The total DNA of strain PSB31 was extracted using a Rapid Bacteria Genomic DNA Isolation Kit (Biobasic, Canada) as per the kit instructions. The PCR amplifica- tion of 16S rDNA was done with the extracted DNA by using the universal primers 27 F (5′-AGA GTT TGA TCC TGG CTC AG-3′), and 1492 R (5′-TAC GGT TAC CTT GTT ACG ACT T-3′) (Mohamed et al., 2018). The amplification was done in a GeneAmp PCR System 2700 thermocycler (Applied Biosystems, CA, USA) using the following program: 95 ˚C for 5 min; 30 cycles at 95 ˚C for 30 s, 55˚C for 30 s, and 72˚C for 90 s; and 72 ˚C for 7 min. The fragment of 16S rDNA sequences (1.5 kb) was obtained by running the PCR product on the 1 % agarose gel in an electrophoresis tank. Then the expected band was cut and purified by using the QIAquick PCR Purifi- cation Kit (Qiagen, USA). The purified 16S rDNA frag- ment was sequenced by Fisrt Base Company (Singapore). The obtained sequence was blasted on NCBI to identify the species. The sequences with high similarity (more than 99 %) were used for multiple cluster alignment and phylogenetic analysis on MEGA software (v.7.2). The nucleotide sequence data reported in this pa- per deposited on GenBank with the accession numbers is OL753109. 4 Acta agriculturae Slovenica, 118/1 – 2022 T. T. NGUYEN et al. 2.7 GREENHOUSE TESTING The ability of selected PSB strains in promoting plant growth was investigated by pot experiments under greenhouse condition (with a temperature of 30°C and constant humidity of 85-95%) at the VNU Central Insti- tute for Natural Resources and Environmental Studies, Ha Noi, Vietnam. The sand matrix was pretreated by washing with 0.1 M hydrochloric acid (HCl). Then, the acid-washed sand was submerged in the 0.1 M HCl for another 24 h. After that, the submerged sand was drained and washed three times with DI water. The pH of the sand was adjusted to 7-7.5. Finally, this sand was autoclave at 121 ˚C for 15 min. The sterilized sand was used as the potting medium. In this pot experiment, the insoluble form of P used is Ca3(PO4)2, which was added into the potting matrix as a P source. Tomato seeds (Lycopersicon esculentum ‘Thuan Dien’) were used as an indicator and were surface-ster- ilized by using alcohol and Javen solution as described by Li et al. (2017). After that, the sterilized seeds were germinated on agar plates, which were covered by the aluminum foil for 3 days at room temperature. Homog- enous seedlings were chosen for further experiments, some of which were covered with selected bacterial strain by dipping their roots for 30 min in bacterial culture (OD = 1). Then, four bacterized seedlings were planted in each plastic pot, which was kept in a greenhouse with long- day condition. Each treatment was repeated in triplicate. The experimental treatments were: (T1) tomato seedlings inoculated with selected bacteria; (T2) non-in- oculated tomato seedlings were considered as a negative control; and (T3) the positive control is the seedlings that were not bacterized but were regularly watered with the solution added 0.25 mM KH2PO4. All pots were provided the macro and micronutrients by watering them with 30 ml of the nutrient solution only or added KH2PO4, where applicable. The irrigated nutrient solution was referenced from Li et al. (2017) and consists of 0.65 mM MgSO4, 2 mM NH4NO3, 2 mM CaCl2, 0.75 mM K2SO4, 0.1 mM KCl, 0.25 mM KH2PO4, 0.2 mM Fe-EDTA, 1 × 10 −3mM MnSO4, 1 × 10−3mM ZnSO4, 1 × 10−4mM CuSO4, and 5 × 10−6 mM (NH4)6Mo7O24, 1 × 10 −3mM H3BO3. The seedlings were grown in three weeks until har- vested. The root samples were removed from the adher- ing soil by washing with sterile water. The measurement of shoot and root length was carried out for three plants. After that, the plant samples were dried in the oven for 30 min at 105 °C to inactivate the enzyme, then reduced the temperature to 65 °C and kept at that temperature until the plant weight is constant. The weight of the dried plant was recorded and analyzed. Finally, the oven-dried samples were powdered and then digested by an H2SO4-H2O2 mixture at 370 °C. The vanadomolybdate method (Hanson, 1950) was applied to identify the P amount in the solution. 2.8 STATISTICAL ANALYSIS All experiments were repeated three times, the re- sults were presented as mean values with ±SD. Tukey’s honestly significant difference (HSD) method in SPSS (version 17) was applied to compare the means in all ex- periments. 3 RESULTS AND DISCUSSION 3.1 ISOLATION OF PHOSPHATE-SOLUBILIZING BACTERIA The overuse of chemical fertilizer caused the in- crease of insoluble P in the soil leading to many problems for humans and other living creatures. Hence, soil-iso- lated microorganisms having the ability in solubilizing phosphorus is emerging as an alternative to chemical fertilizer because of their environment-friendly nature. The results showed that a total of 7 colonies grown and generated a circular clearing zone on PVK medium was obtained. Among obtained colonies, five of them (PSB11 to PSB51) were from soil grown maize while only one colony was observed for soil grown rice (PSB61) and tomato (PSB71). The results were presented in Table 1 and illustrated in Figure 1. Figure 1: Insoluble phosphate solubilization studies on Pikovs- kaya’s agar (PVK): PSB31 show efficient phosphate-solubilizing isolate (large haloes), whereas three others show weak (PSB41 and PSB21) or no activity (PSB62, no visible halo) 5Acta agriculturae Slovenica, 118/1 – 2022 Isolation of phosphate solubilizing bacteria from root rhizosphere to supplement biofertilizer (Accession number: MF803700.1). The Bacillus sp. have been reported as phosphorus solubilizers (Kumar et al., 2017; Mohamed et al., 2018). 3.3 FACTORS AFFECTED TO PHOSPHATE SOLU- BILIZATION OF PSB31 3.3.1 pH The results also showed that the highest amount As can be seen from Table 1, the PSB31 strain pro- duced the largest clearing zone, with a PSI of 226.1. On the other side, the lowest index value was 4.2 produced by PSB11 isolate. The results also present the un-correlation between the PSI and the insoluble phosphate solubiliza- tion ability of the other isolates including PSB51, PSB61, and PSB71, those were isolated from soil grown maize, rice, and tomato, respectively. These results were demon- strated by a study of Sharon et al. (2016), who reported the solubilization of Ca3(PO4)2 of microbial communities living in the rhizosphere of potato roots is higher than the one produced by microbes in the rhizosphere of to- mato roots. In addition, the results showed that four of the seven isolates (PSB11, PSB21, PSB31, PSB41) pro- duced colonies that were opaque and bright yellow while the colonies formed by the other three isolates (PSB51, PSB61, PSB71) were opaque and white (Table 1). These results suggest the variation in phosphate solubilization of the isolates could be due to the differences of microbial communities that were strongly affected by soil proper- ties and plant species (Sharon et al., 2016). The explain was strengthened by the discovery of Grayston et al. (1998), in which the microbial diversity in the rhizos- phere was highly affected by metabolites exuded by dif- ferent plant species into the rhizosphere such as amino acids, carbohydrates, and carboxylic acids. 3.2 IDENTIFICATION OF BACTERIAL STRAIN PSB31 The 16S rRNA gene sequences of the PSB31 strain was blasted against the one of the other microorganisms on the NCBI (Figure 2). As can be seen from Figure 2, the PSB31 strain closed to the Bacillus sp. strain IMAU61039 PSB isolates Phosphate solubilizing index (Agar)a Final pH of PVK Liq. Med. Color of colonies Soluble P (mg l-1)b PVK medium only 0 6.5 N/A 11.12 ± 4.5a PSB11 4.2 5.5 Yellow 53 ± 7.5b PSB21 13.8 5 Yellow 63 ± 9.2c PSB31 226.1 4.5 Yellow 962 ± 11.3f PSB41 78.3 4.5 Yellow 303 ± 6.3d PSB51 67.5 4.5 White 313 ± 7.2e PSB61 73.4 4.5 White 301 ± 8.2d PSB71 91.1 4.5 White 310 ± 9.1e Table 1: Biochemical properties of isolated PSB strains a All solubilization rates were measured from cultures grown for 24 h in a liquid medium b Data are means ± SE of three independent biological replicates. Bearing different letters in the same row are significantly different from each other according to the least significant difference (LSD) test (p < 0.05) Figure 2: A neighbor-joining tree shows the phylogenetic relationships among 16S rDNA sequences of PSB31 and their closely related sequences from NCBI. The scale bar indicates evolutionary distance 6 Acta agriculturae Slovenica, 118/1 – 2022 T. T. NGUYEN et al. of soluble P (962 ± 11.3 mg L-1) was calculated for the PSB31 culture, while the lowest one (53 ± 7.5 mg l-1) was observed in PSB11 culture (Table 1). Furthermore, the results also indicated that the decrease of pH of final filtrate from isolates compared to the control (Table 1). This phenomenon was reported by some previous stud- ies which demonstrated that the organic acid produc- tion of PSB reduced the medium pH facilitating phos- phate solubilization (Sharon et al., 2016; Mohamed et al., 2018). Moreover, our results also showed the supernatant pH was uncorrelated with soluble P presented in the cul- ture of PSB31. This could be due to the calcium ion freed from the linkage with PO4 3- by PSB31 strain neutralized the produced acids during the experiment (Nelofer et al., 2015). These results suggest the mechanism of P solu- bilization by PSB was not only produced low molecular acids but also generated others factors such as hydrolytic enzymes. 3.3.2 Phosphatase activity The result of the phosphatase experiment showed that the insoluble Ca3(PO4)2 was completely solubilized in the solution containing 1 ml of supernatant from PSB31 strain after 48 h of incubation was indicated by the color change of culture from milky to transparent. The result suggested that the PSB31 strain produced phosphatase to degrade the Ca3(PO4)2 in the medium (Figure 3). The en- zymatic activities rapidly increased from 0 to 18 UI after about 25 hours of incubation. After that, the enzymatic activities were slightly increased and reached 20.2 UI af- ter 50 hours of incubation before dropped out to 17.3 UI after 55 hours. Then the enzymatic activities were slightly decreased to 16.1 UI at the end of the experiment. The current study also showed the presence of phos- phatase in the supernatant of PSB31 with amounts that were higher than the results of previous studies (Men- doza-Arroyo et al., 2020). All in all, the result suggested the PSB31 strain is a very promising agent that could be used to solubilize the phosphorus compound in the soil to increase the P availability for crops. 3.4 PSB31 ENHANCED THE GROWTH OF TO- MATO SEEDLINGS The results of greenhouse experiments clearly showed that PSB31 was able to promote tomato growth under the stress of nutrient conditions. The results were illustrated in Figure 4A and presented in Figure 4B. As can be seen from Figure 4B, tomato seedlings supple- mented with both PSB31 and Ca3(PO4)2 (T2) had a great- er increase of root and shoot length than those with no bacteria + Ca3(PO4)2 + KH2PO4 (T3) and with no bacte- ria + Ca3(PO4)2 (T1). The result also presented a maximum quantity of shoot length for the seedlings that were not bacterized but were regularly watered with the P solution (posi- tive control). Furthermore, the results also showed the tomato seedlings received both insoluble Ca3(PO4)2 and PSB31 strain had the fresh and dry mass moderately higher than the negative control but slightly lower than the positive control (Table 2). It was consistent with re- ports in which PSB when applied into soil could enhance significantly the development and phosphate uptake in many crop species (Kumar et al., 2017; Mendoza-Arroyo et al., 2020). Additionally, the results of the experiment for pot- ting medium indicated that PSB31 could solubilize Ca3(PO4)2 in sand increasing the soluble P in the sand matrix to 106.7 ± 3.5 mg l-1. This could be PSB31 strain generated phosphatase or low molecular organic acids to converted the phosphate from insoluble to soluble form that provided a P balance for plant development result- ing in plant growth promotion (Mendoza-Arroyo et al., 2020). These results suggest the potential application of PSB31 as a biofertilizer for sustainable agriculture. As can be seen from Table 2, tomato seedling inocu- lated with PSB31 strain had the fresh and dry shoot weights 2 times higher than the one that grew in sand mixed with the Ca3(PO4)2 but lower than the one supported with sol- uble P. These results were similar to the one of Lee et al. (2020), in which the Arabidopsis thaliana (L.) Heynh. seed- lings bacterized with Bacillus subtilis (Ehrenberg  1835) Cohn 1872 strain L1 via the roots had a considerable in- crease in plant mass. These results suggested a possible role of PSB31 strain in the process of assimilation by pho- Figure 3: Phosphatase profile of phosphorus-solubilizing bacteria PSB31 for 72 h of incubation and 5 g l-1 Ca3(PO4)2 in the medium 7Acta agriculturae Slovenica, 118/1 – 2022 Isolation of phosphate solubilizing bacteria from root rhizosphere to supplement biofertilizer tosynthesis, subsequently, the plant mass improvement (Wu et al., 2019). Moreover, the higher P amount measured in bacte- rized seedlings (0.31 %) compared to the one grown in the sand with only Ca3(PO4)2 (0.15 %) indicated that the PSB31 strain functioned in releasing the soluble P from Ca3(PO4)2 enhancing P uptake of seedlings. The results also presented the P amount in seedling grown in pot wa- tered with nutrient containing P was the highest among treatments with 0.35 % (Fig. 4B). These results suggested strain PSB31 could enhance P uptake in the bacterized seedlings led to the high amount of water in plant con- tributing to biomass formation. These results endorsed that the optimistic result of PSB31 on crop yield due to the increase of nutrients uptake (predominantly phos- phorus). 4 CONCLUSIONS The soil microorganisms capable of converting in- soluble P to soluble P are being explored as an environ- mentally friendly agent to promote plant development and subsequently increasing yields. The results showed that among 7 isolates, strain PSB31 has good phosphate solubilization activity and promoted the growth of  to- mato seedlings under phosphate limiting conditions. This PSB31  strain had been identified belonging to Ba- cillus sp. (in: Bacteria) strain IMAU61039. All of these results suggested the PSB31 strain could be potentially used as a microbial biofertilizer candidate for commer- cial applications in the future. 5 ACKNOWLEDGEMENT This article was supported by the Vietnam Acad- emy of Science and Technology, Vietnam, code: VAST05.06/20-21. 6 REFERENCES Barea, J.M. (2015). Future challenges and perspectives for ap- Figure 4: Plant growth promotion activity by phosphate-solubilizing bacteria. A. Illustration for plant growth. B. Length of root and shoot of plant under different conditions. T1: no bacteria + Ca3(PO4)2, T2: with PSB31+ Ca3(PO4)2, T3: no bacteria + Ca3(PO4)2 + KH2PO4 Treatment Root mass (g) Shoot mass (g) Total P (%)Fresh Dry Fresh Dry No bacteria + Ca3(PO4)2 0.153 ± 0.057 a 0.022 ± 0.002a 0.378 ± 0.006a 0.044 ± 0.003a 0.15 PSB31 + Ca3(PO4)2 0.425 ± 0.223 a 0.025 ± 0.007a 0.608 ± 0.027b 0.089 ± 0.013b 0.31 No bacteria + Ca3(PO4)2 + KH2PO4 0.492 ± 0.257 a 0.26 ± 0.004a 0.697 ± 0.034b 0.091 ± 0.032b 0.35 Table 2: Effect bacterial isolates on plant biomass production Data are means ± SE of three independent biological replicates. Bearing different letters in the same row are significantly different from each other according to the least significant difference (LSD) test (p < 0.05) 8 Acta agriculturae Slovenica, 118/1 – 2022 T. T. NGUYEN et al. plying microbial biotechnology in sustainable agriculture based on a better understanding of plant-microbiome inter- actions. 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