BIOTROPIA Vol. 29 No. 1, 2022: 28 - 38 DOI: 10.11598/btb.2022.29.1.1584 28 CHARACTERIZATION AND POLYPHASIC IDENTIFICATION OF NOVEL RHIZOBACTERIA STRAIN ISOLATED FROM SAND DUNES ECOSYSTEM KETUT ARTE WIDANE1, ANNISAA WIDYASARI2 AND ENDAH RETNANINGRUM3* 1Undergraduate Student, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia 2Graduate Student, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia 3Microbiology Laboratory, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia Received 9 April 2021 / Accepted 3 December 2021 ABSTRACT The coastal sand dune ecosystem at the Parangtritis Coast of Yogyakarta, Indonesia has unique characteristics such as low moisture sandy soil, high salinity and low nutrient content. Fimbristylis cymosa is one of the plant species having the capability to survive in that unique ecosystem. In this study, rhizobacteria isolated from the rhizosphere of F. cymosa were isolated to be further analyzed on their phosphate solubilizing and antagonistic properties against Fusarium oxysporum which cause the Wilt disease. The isolates of Phosphate Solubilizing Rhizobacteria (PSR) having the most potential capabilities were then polyphasically identified based on phenotypic and genotypic characters followed by 16S rDNA sequencing. The results showed that four PSR isolates (I8, I11, I12 and I24) have high phosphate dissolution indices. The highest indices were observed in isolates I11 (3.08) and I12 (3.44), respectively. Analysis of the dual plate experiments for PSR I11 and PSR I12 isolates against the growth of F. oxysporum also showed quite high inhibitory activities, i.e., isolate PSR I11 was 42.40%, while isolate PSR I12 was 42.08%. The two isolates were polyphasically identified as Burkholderia dolosa. This study clearly showed that PSR I11 and PSR I12 isolates are very potential and prospective to be used as marginal land inoculants and as providers of phosphorus. This study also showed that the isolates are useful as biocontrol agents against F. oxysporum in plants. Keywords: inhibitory activity, phosphate dissolution index, phosphorus, polyphasic identification, sandy soil INTRODUCTION Coastal sand dunes are Aeolian landforms commonly found in coastal areas at all latitudes in the world. Indonesia has coastal sand dunes located in the southern part of Java Island, extending from the southern coast of West Java Province to Yogyakarta Province. The most significant sand dunes formation occurs in the Parangtritis Coastal Area, located on the southern coast of Yogyakarta Province. The sand dunes ecosystem has a high temperature, relatively little vegetation, strong winds, high salt content and very low groundwater content, making it a dry and infertile area (Mahdavi & Bergmeier 2016; Campos et al. 2020). In general, plants are difficult to grow in dry and less fertile areas. However, there are several groups of plants that can survive in the sand dunes habitat. Among plants that survived the sand dunes habitat on Yogyakarta’s southern coast is Fimbristylis cymosa, a family of Cyperaceae, which grows well and is widespread along with the coastal sand dunes habitat. The growth and development of F. cymosa in the coastal sand dunes habitat are strongly influenced by the availability of essential soil macronutrients. One of the main macronutrients supporting the growth and development of F. cymosa is phosphorus. Phosphorus is among minerals needed at 0.2% dry weight of F. cymosa, as a component of nucleic acids (DNA and RNA) for conserving energy. Plants absorb phosphorus in the form of phosphate anions (PO4 - or PO4 2-) which are dissolved in groundwater. However, phosphate anions have *Corresponding author, email: endahr@ugm.ac.id Novel rhizobacteria strain isolated from sand dune ecosystem – Ketut Arte Widane et al. 29 a high tendency to bind with calcium, aluminum, and iron in the soil, which makes them insoluble and easily settled so that they cannot be absorbed by plants, including F. cymosa (Yang et al. 2021). Undissolved phosphorus in the soil can be dissolved by the Phosphate Solubilizing Bacteria (PSB) group. A study conducted by Liu et al. (2015) in calcareous soil found PSB genera Bacillus, Pseudomonas, Rhizobium and Acinetobacter. Pastore et al. (2020) reported PSB genera Burkholderia, Bacillus, and Pseudomonas in forest soils. In the mangrove forest rhizosphere, Teymouri et al. (2016) observed the existence of PSB genera Bacillus, Pseudomonas and Acinetobacter. Rasul et al. (2019) obtained PSB genera Pseudomonas and Bacillus from rhizospheric paddy field soil, while the PSB genus Paenibacillus was found from wheat rhizosphere (Cherchali et al. 2019). In soil environments, there are also soilborne pathogens causing plants infection. One species of a soil-borne pathogen is Fusarium oxysporum causing the Fusarium Wilt disease in various types of agricultural crops within the Solanaceae family, such as tomatoes, potatoes, and chilies (Ávila et al. 2019; Srinivas et al. 2019; Chowdhury et al. 2020). The wilting phenomenon that occurred in those crops due to Fusarium Wilt disease resulted in a significant reduction in crop yields and has been a major problem for agriculture worldwide. The use of chemical compounds for treating the Wilt disease has adverse effects on non-target organisms. Therefore, it is pertinent to find biocontrol agents which is considered safer for the environment. In soil environments, Phosphate Solubilizing Bacteria (PSB) are reported to be more dominantly existing in the rhizosphere. In addition, PSB from the rhizosphere is known to produce active metabolites compared to other sources and is able to increase plant growth and development. PSB originating from extreme environments such as sand dunes, might have specific characteristics as a biocontrol agent against F. oxysporum (Ahluwalia et al. 2021; Rasool et al. 2021) Therefore, this study aimed to: 1. isolate and characterize Phosphate Solubilizing Bacteria (PSB) from the rhizosphere of F. cymosa living in sand dunes habitat; and 2. screen the capability of isolated PSB as a biocontrol agent against F. oxysporum. MATERIALS AND METHODS Rhizosphere Soil Samples and Isolate of F. oxysporum Rhizosphere soil samples from the planting area of F. cymosa were taken and collected from 8 different sampling points along with the coastal sand dune habitat in the Parangtritis Coastal Area, Yogyakarta. The roots of the F. cymosa were carefully extracted with a shovel. Soil attached to the roots was collected in sterile plastic containers as rhizosphere soil samples. The samples were then stored at 4 oC for further isolation of Phosphate Solubilizing Bacteria (PSB). The isolate of Fusarium oxysporum was obtained from the culture collection of the Microbiology Laboratory, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia and was grown in Potato Dextrose Agar (PDA) medium at 30 oC for 10 days. PSB Isolation and Phosphate Dissolution Index Analysis Ten (10) g of rhizosphere soil samples were poured into a 50 mL Erlenmeyer and suspended in 90 mL of sterile distilled water, then shaken using a vortex at a speed of 200 rpm for 30 min. The sample suspension was diluted in a series of up to 10-4 dilutions. A total of 0.1 mL of the respective 10-3 and 10-4 diluted sample suspensions was inoculated on the National Botanical Research Institute's Phosphate (NBRIP) agar medium by means of the spread plate method, then incubated at 30 oC for 48 - 72 hours. The NBRIP medium consists of 1% glucose, 0.5% Ca3(PO4)2, 0.5% MgCl2, 0.01% (NH4)2SO4, 0.0.5% MgSO4.7H2O, and 0.02% KCl (Nautiyal 1999). PSB isolates having a clear zone around the colony were then re-isolated as PSR, and purified on an NBRIP medium. In order to measure the phosphate dissolution index, the PSR isolates were re- cultured in NBRIP liquid medium and incubated at 30 oC for 48 h. Forty (40) μL of the incubated culture was diluted until reaching a colony having cell number 108 CFU/mL. The liquid culture was then poured into a Petri dish containing NBRIP agar medium by means of the pour plate method and incubated at 30 oC for 48 h. BIOTROPIA Vol. 29 No. 1, 2022 30 The formed clear zone and the size of the bacterial colony were calculated. The phosphate dissolution index was determined with the following formula developed by Haile et al. (2016): PSI = (HZD + CD) CD where: PSI = Phosphate Solubilization Index HZD = Halo Zone Diameter (mm) CD = Colony Diameter (mm) Antagonistic Activity of PSR Against F. oxysporum In vitro test of PSR antagonist activity against F. oxysporum was carried out by using the dual plate experiments method according to Zhang et al. (2017) with minor modifications. Potato Dextrose Agar (PDA) medium was prepared on a 9 cm diameter Petri dish, followed by making a 5-mm diameter well in the PDA medium at the center of the Petri dish by using a cork borer. The other four 5-mm diameter wells were made at a distance of 2 cm from the well located at the center of the Petri dish and from each other. F. oxysporum mycelium with a diameter of 5 mm was taken from the outermost part of the 10- day-old fungal colony and was inoculated into the well at the center of the Petri dish containing PDA medium by using an inoculation needle. A total of 8 µL of PSR isolate suspension with a cell number of 109 CFU/mL was inoculated into the other four wells in the same Petri dish and then incubated at 30 °C for 10 days. The control agar plates were then prepared with the same agar medium without inoculating the PSR isolates suspension. At the end of the incubation time, the diameter of F. oxysporum colonies was measured diagonally, vertically and horizontally. The inhibition percentage of PSR isolates against F. oxysporum was determined using the following formula of Royse and Ries (1978): IP = (R1 - R2) x 100% R1 where: IP = Inhibition Percentage R1 = Diameter of F. oxysporum colonies on the control plate R2 = Diameter of F. oxysporum colonies on the treated plate Polyphasic Identification of PSR Isolates Polyphasic identification of PSR isolates was determined based on a combination of phenotypic and genotypic characters. Phenotypic characters of the observed isolates, including the morphological and biochemical characters, were determined by referring to Bergey's Manual of Systematic Bacteriology (Brenner et al. 2005). The morphological characters were observed based on cell and colony morphologies. The cell morphology of PSB isolates was observed based on the shape and characteristics resulting from the gram staining procedure. Meanwhile, colony morphology of PSB isolates was observed based on the shape, color, margins, elevation, internal structure and optical features of the colony. The biochemical characters were determined based on several tests, such as the motility, catalase, oxidase tests, glucose, sucrose, lactose fermentation tests, urease and Voges-Proskauer (VP) tests. The isolates were then characterized genotypically based on the 16S rDNA nucleotide sequence. Total genomic DNA of PSB isolates was extracted using the Quick- DNATM Fungal/Bacterial Miniprep Kit (Zymo Research, USA). PCR amplification of the 16S rDNA gene of the PSB isolates was analyzed by using 27F primers (5’-AGAGTTTGATCCT GGCTCAG-3’) and 1492R (5’- GGTTACCTT GTTACGACTT-3’) (Hossain et al. 2020). The PCR amplification was performed using a DNA thermal cycler (Bio-Rad, USA) with the following program: pre-denaturation at 95 °C for 30 min, followed by 30 cycles consisting of denaturation at 95 °C for 30 sec, annealing at 57 °C for 1 min, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min. The PCR products were purified and sequenced. The nucleotide sequences were then compared with DNA sequence databases (GenBank) through the BLAST program (BLASTn) at the National Biotechnology Information Center (NCBI). A phylogenetic tree was constructed through the maximum likelihood method using Molecular Evolutionary Genetics Analysis X (MEGA version X). Statistical Analysis Data were statistically analyzed using the one- way variance analysis (ANOVA) followed by the Duncan Multiple Range Test (DMRT) approach for testing the mean differences. At P < 0.05, Novel rhizobacteria strain isolated from sand dune ecosystem – Ketut Arte Widane et al. 31 the differences were considered significant. Results are expressed as means of three replicates ± standard deviation. The software used was IBM SPSS Statistics version 21. RESULTS AND DISCUSSION PSR Isolate and Phosphate Solubilization Index A total of 26 bacteria were isolated from F. cymosa rhizosphere soil samples in the sand dunes habitat of Parangtritis Coastal Area, Yogyakarta, Indonesia. Of the 26 rhizobacterial isolates, 4 rhizobacterial isolates were selected as PSR based on the clear halo formation around the colony in the NBRIP medium containing tricalcium phosphate. The clear halo was formed due to the production of organic acids or polysaccharides as a result of the phosphatase enzyme activity of the PSR isolate (Paul & Sinha 2013; Behera et al. 2017). The phosphate solubilization indices of each PSR isolate after 10 days of incubation are presented in Table 1 (Widane et al. 2018). The highest phosphate solubilization index of 3.44 was obtained from PSB I12. Meanwhile, the lowest phosphate solubilization index of 3.00 was obtained from PSB I24. A previous study by Teymouri et al. (2016) reported the phosphate solubilization index of 3.5 which was obtained from Bacillus sp. found in the rhizosphere of mangrove forest. Mardad et al. (2013) reported the phosphate solubilization index of 3.5 which was obtained from Enterobacter hormaechei found in the phosphate rock deposits. In contrast, several other researchers reported a lower-than-three phosphate solubilization index, such as those obtained from Pseudomonas (2.6) and Acinetobacter (2.0) found in the rhizosphere of forest (Teymouri et al. 2016). The Paenibacillus genus from the wheat rhizosphere was reported to have a very low phosphate solubilization index of 1.32 (Cherchali et al. 2019). Pseudomonas and Serratia obtained from the rhizosphere of Allium cepa L. were also reported to have low phosphate solubilization indices of 2.0 and 2.1, respectively (Blanco-Vargas et al. 2020). Table 1 Phosphate solubilization index of PSR isolates obtained from the F. cymosa rhizosphere after 10 days of incubation No PSR Isolate Phosphate solubilization index 1 I8 3.04 ± 0.51bcd 2 I11 3.08 ± 0.38cd 3 I12 3.44 ± 0.19d 4 I24 3.00 ± 0.43abcd Notes: The index is presented as mean ± SD; Numbers followed by the same letter in a column do not show a significant difference according to the Duncan test (DMRT) at P < 0.05. Different values of phosphate solubilization index among PSR isolates are closely related to organic acids production, causing a pH decrease in bacterial cells and their environment, leading to proton substitution in the phosphate mineral and P release (Ludueña et al. 2018; Rasul et al. 2021). Organic acids produced by several PSRs include gluconic, glycolic, oxalic, malonic and succinic acids. Gluconic acid is an organic acid having a role as a phosphate solvent (Joe et al. 2018; Santos-Torres et al. 2021), which is useful in providing P minerals needed for plant growth (Valetti et al. 2018; Sahandi et al. 2019). PSR I11 and PSR I12 isolates have a high ability to dissolve phosphate in vitro. Therefore, these two isolates are the potential to be developed as agents for providing P for plants. Antagonistic Activity of PSR Isolates Against F. oxysporum PSR I8, PSR I11, PSR I12, and PSR I24 isolates were tested in-vitro for their antagonistic activity against F. oxysporum by using the dual plate technique. The results showed a significant inhibitory effect on the growth of F. oxysporum mycelium (Fig. 1), which was clearly seen by comparing the inhibitory effect of the four isolates with the control against the growth of F. oxysporum. The four PSR isolates clearly caused a smaller colony size of F. oxysporum compared to that in the control treatment. The colony diameter of F. oxysporum, however, was different among the four PSR isolates, i.e., 44.75 mm was observed in PSR I11 isolate, 45.00 mm in PSR I12, 53.00 mm in PSR I24 and 62.50 mm in PSR I8 (Widane et al. 2018). BIOTROPIA Vol. 29 No. 1, 2022 32 A B C D Figure 1 Antagonistic activity of PSR isolates against F. oxysporum observed in-vitro after 14 days incubation period Notes: A = PSR I8; B = PSR I12; C = PSR I11; D = PSR I24. In addition, our study showed that PSR isolates also caused thinner F. oxysporum mycelium growth compared to that in the control treatment. This explains the inability of F. oxysporum to grow well in the presence of PSR isolates. The graph of the inhibition percentage of PSR isolates against F. oxysporum is presented in Figure 2 (Widane et al. 2018). The dual culture method applied in this study allows direct in-vitro interactions between PSR isolates and F. oxysporum. These interactions can be in the form of competition for space or for nutrients from the growth medium, leading to growth inhibition of F. oxysporum. Growth inhibition can also be caused by metabolite compounds produced by PSR isolates. Several metabolite compounds are reported to have the most effective antagonistic activity in inhibiting the growth of plant pathogens in the form of antibiotics, siderophores and bacteriocins (Khedhera et al. 2021). Bacteria can produce hydrolytic enzymes such as protease, lipase, chitinase and glucanase which can lyse fungal cells so that they have antagonistic activity against plant pathogenic fungi (Cui et al. 2019; Lau et al. 2020). Our study also showed that the PSR I11 isolate was the most effective at inhibiting the growth of F. oxysporum with an inhibition percentage of 42.40%, while the PSR I12 isolate had an inhibition percentage of 42.08% which is not significantly different from that of PSR I11. Lower inhibition percentages were observed for PSR I24 and PSR I8, i.e., 31.82% and 19.58%, respectively (Fig. 2). Figure 2 Inhibition percentage value of PSR isolates against F. oxysporum after 14 days of incubation period Novel rhizobacteria strain isolated from sand dune ecosystem – Ketut Arte Widane et al. 33 Several studies have also reported that bacteria isolated from the rhizosphere are able to protect plants from fungal pathogens (Babu et al. 2015; Panda et al. 2016). Bacillus amyloliquefaciens from the rhizosphere of apple trees were reported to have inhibition capability against F. oxysporum with an inhibition percentage of 35% (Guleria et al. 2016). Meanwhile, research conducted by Jangir et al. (2018) showed that Bacillus sp. from the rhizosphere of tomato plants had antagonistic activity against F. oxysporum with higher inhibition percentage (70%) and therefore, is potential as a biocontrol agent against Fusarium Wilt disease in tomato plants. Karthika et al. (2020) also isolated Bacillus cereus from the rhizosphere of a tomato plant, which has an inhibition percentage of 66%. Our study further showed that PSR I11 and PSR I12 isolates have the capability of phosphate solvent and have high antagonistic activity against F. oxysporum. Polyphasic Identification of PSR Isolates Observation of the morphological characters of PSR I11 and PSR I12 showed different colony morphology forms, i.e., circular and irregular, respectively (Table 2). However, the two isolates have the same colony color, elevation, internal structure, optical features and shape, such as cream, entire, convex, smooth, translucent and rod-shaped (Table 2). Biochemical properties of the two isolates, PSR I11 and PSR I12 showed similarities in terms of motility, production of catalase and oxidase enzymes and positive reaction toward the Voges-Proskauer test. Different reactions occurred when being tested in a medium containing urea, showing that PSR I11 isolate is able to produce urease, while PSR I12 isolate is not able to produce urease (Table 3). Comparing the results from the identification of the morphological and biochemical characters with the characters described in Bergey’s Manual of Determinative Bacteriology, it is concluded that PSR I11 and PSR I12 isolates belong to the genus Burkholderia (Brenner et al. 2005). Table 2 Morphological characters of PSR isolates from rhizosphere of F. cymosa No Morphological characters PSR isolate I11 I12 1. Colony morphology Shape Circular Irregular Color Cream Cream Margin Entire Entire Elevation Convex Convex Internal structure Smooth Smooth Optical feature Translucent Translucent 2. Cell morphology Shape Rod-shaped Rod-shaped Gram properties Negative Negative Tabel 3 Biochemical characters of two PSR isolates from rhizosphere of F. cymosa PSR isolate No Biochemical characters I11 I12 1 Motility test + + 2 Catalase test + + 3 Oxidase test + + 4 Glucose fermentation - - 5 Sucrose fermentation - - 6 Lactose fermentation - - 7 Urease + - 8 Voges-proskauer (VP) test + + BIOTROPIA Vol. 29 No. 1, 2022 34 Burkholderia spp. is reported of having the ability to dissolve phosphate and at the same time has antagonistic activity against F. oxysporum. Simonetti et al. (2018) isolated Burkholderia ambifaria from the rhizosphere of barley having a role as phosphate solvents and antagonistic properties against F. oxysporum. Burkholderia cepacia and Burkholderia contaminans with similar capabilities were isolated from the rhizosphere of maize (Zhao et al. 2014; Tagele et al. 2018). Burkholderia sp. was also obtained from the rhizosphere of Juçara palm (Euterpe edulis Mart) (de Castilho et al. 2020). In addition to the plant rhizosphere, Burkholderia spp. with the same properties is also obtained from the root nodules of fenugreek (Trigonella foenum- graecum L.) (Kumar et al. 2017). B. contaminans were also existed at the nodule of the common bean (Phaseolus vulgaris) (Tapia-García et al. 2020). Apart from dissolving phosphate and having antagonistic properties against F. oxysporum, B. contaminans also has nitrogenase properties (Silva et al. 2012). Both PSR isolates were also genotypically identified using the 16S rDNA genetic marker. Based on the 16S rDNA gene sequencing, the PSR I11 and PSR I12 isolates had similarities with Burkholderia dolosa strain LMG 18943B with a similarity index of 99.51% and 99.44%, respectively (Table 4). The two PSR isolates were categorized in the same species because the similarity percentage in the 16S rRNA gene sequences was both ≥ 99% (Schlaberg et al. 2012). Phylogenetic trees based on 16S rDNA sequences of PSR I11 and PSR I12 isolates were reconstructed by using comparative sequences with the in-group and out-group categories obtained from NCBI (Table 4). Some of the relatively close sequences of Burkholderia spp. included Burkholderia dolosa strain LMG 18943 (Yarza et al. 2013), Burkholderia latens strain R-563 (Vanlaere et al. 2008), Burkholderia multivorans strain Struelens (Bauernfeind et al. 1999), Burkholderia vietnamiensis strain LMG 10929 (LiPuma et al. 1999), Burkholderia vietnamiensis strain TVV75 (Viallard et al. 1998), Burkholderia metallica strain R-16017 (Vanlaere et al. 2008), Burkholderia territorii strain LMG 28158 (De Smet et al. 2015) and Burkholderia seminalis strain R-2419 (Vanlaere et al. 2008) as the in-group. Meanwhile, Alcaligenes faecalis subsp. parafaecalis strain G 16S was used as the out-group because the species is a class- level classification group Betaproteobacteria with Burkholderia spp. In addition, Alcaligenes sp. has the ability to dissolve phosphate and antibiosis properties against F. oxysporum (Rasool et al. 2021). Reconstruction of the phylogenetic tree was carried out using the maximum likelihood (ML) method with a substitution model (Tamura-Nei 93, G: gamma-distributed) and a bootstrap of 1,000 replications (Fig. 3). This phylogenetic tree shows that both strains form clusters with Burkholderia dolosa strain LMG 18943 in 83% bootstrap replications; therefore, reconstruction of these phylogenic trees can be trusted (Hillis & Bull 1993). The phylogenetic tree reconstruction of the two isolates was also in accordance with the BLAST results, which indicated that the two isolates could be identified as Burkholderia dolosa. In addition, we already submitted the 16S rDNA sequences of both strains (B. dolosa I11 and B. dolosa I12) to the GenBank, with accession numbers of OK083732 and OK083731, respectively https://www.ncbi.nlm.nih.gov). Tabel 4 Identify sequences of PSR isolates from the rhizosphere of F. cymosa based on the GenBank data by using BLAST Isolate Accession number Species of PSR homolog Identity Query cover Reference PSR I11 NR_104973.1 Burkholderia dolosa strain LMG 18943 99.51 % 99% Yarza et al. (2013) NR_042632.1 Burkholderia latens strain R-5630 99.30 % 99% Vanlaere et al. (2008) NR_029358.1 Burkholderia multivorans strain Struelens 99.09 % 99% Bauernfeind et al. (1999) NR_041720.1 Burkholderia vietnamiensis strain LMG 10929 99.09 % 99% LiPuma et al. (1999) NR_118872.1 Burkholderia vietnamiensis strain TVV75 99.22% 98% Viallard et al. (1998) NR_042636.1 Burkholderia metallica strain R-16017 98.95% 99% Vanlaere et al. (2008) NR_136496.1 Burkholderia territorii strain LMG 28158 98.95% 99% De Smet et al. (2015) NR_042635.1 Burkholderia seminalis strain R-2419 98.74% 99% Vanlaere et al. (2008) PSR I12 NR_104973.1 Burkholderia dolosa strain LMG 18943 99.44 % 99% Yarza et al. (2013) NR_042632.1 Burkholderia latens strain R-5630 99.16 % 99% Vanlaere et al. (2008) NR_029358.1 Burkholderia multivorans strain Struelens 99.09 % 99% Bauernfeind et al. (1999) NR_041720.1 Burkholderia vietnamiensis strain LMG 10929 99.09 % 99% LiPuma et al. (1999) NR_118872.1 Burkholderia vietnamiensis strain TVV75 99.43% 98% Viallard et al. (1998) NR_042636.1 Burkholderia metallica strain R-16017 98.95% 99% Vanlaere et al. (2008) NR_136496.1 Burkholderia territorii strain LMG 28158 98.88% 99% De Smet et al. (2015) NR_042635.1 Burkholderia seminalis strain R-2419 98.81% 99% Vanlaere et al. (2008) Novel rhizobacteria strain isolated from sand dune ecosystem – Ketut Arte Widane et al. 35 Figure 3 Phylogenetic tree based on 16S rDNA sequences constructed by the maximum likelihood method Note: Numerals at the nodes indicate the bootstrap value (%) derived from 1,000 replication. CONCLUSION PSR I11 and PSR I12 bacterial isolates obtained from the rhizosphere of F. cymosa in the sand dunes habitat of Parangtritis, Yogyakarta, Indonesia showed the capability to dissolve phosphate. The two isolates also showed an inhibition percentage of more than 40%, and therefore, can be used as biocontrol agents against F. oxysporum. PSR I11 and PSR I12 isolates were polyphasically identified as Burkholderia dolosa. Since the two isolates were obtained from F. cymosa living in sand dunes habitat, which is an extreme environment, this study can be further developed into determining the possibility of obtaining these two isolates in other marginal lands, to be used as phosphate solvent and biocontrol agents against F. oxysporum. Further research is also suggested to determine the ability of these two isolates for procuring phosphorous mineral supply and as biocontrol agents against F. oxysporum in plants, especially Solanaceae family, plants both in polybags and agricultural land. ACKNOWLEDGMENTS The authors would like to thank the Laboratory of Microbiology and Falitma, Faculty of Biology, Universitas Gadjah Mada for providing facilities and media during this research. REFERENCES Ahluwalia O, Singh PC, Bhatia R. 2021. A review on drought stress in plants: Implications, mitigation and the role of plant growth promoting rhizobacteria. Res Environ Sustain 5:100032. Ávila AC, Ochoa J, Proaño K, Martínez MC. 2019. Jasmonic acid and nitric oxide protects naranjilla (Solanum quitoense) against infection by Fusarium oxysporum f. sp. quitoense by eliciting plant defense responses. Physiol Mol Plant Pathol 106:129-36. Babu AN, Jogaiah S, Ito S, Nagaraj AK, Tran LP. 2015. Improvement of growth, fruit weight and early blight disease protection of tomato plants by rhizosphere bacteria correlated with their beneficial traits and induced biosynthesis of antioxidant peroxidase and polyphenol oxidase. Plant Sci 231:62-73. Bauernfeind A, Schneider I, Jungwirth R, Roller C. 1999. Discrimination of Burkholderia multivorans and Burkholderia vietnamiensis from Burkholderia cepacia genomovars I, III, and IV by PCR. J Clin Microbiol 37 (5):1335-9. Behera BC, Yadav H, Singh SK, Sethi BK, Mishra RJ, Kumari S, Thatoi H. 2017. Alkaline phosphatase activity of a phosphate solubilizing Alcaligenes faecalis, isolated from mangrove soil. Biotechnol Res Innov 1:101-110. Blanco-Vargas A, Rodríguez-Gacha LM, Sanchez-Castro N, Garzon-Jaramillo R, Pedroza-Camacho LD, BIOTROPIA Vol. 29 No. 1, 2022 36 Poutou-Pinales RA, …, Pedroza-Rodríguez AM. 2020. Phosphate-solubilizing Pseudomonas sp., and Serratia sp., co-culture for Allium cepa L. growth promotion. Heliyon 6:e05218. Brenner DJ, Krieg NR, Staley JR. 2005. Bergey’s Manual Systematic Bacteriology, New York (US): Springer. p. 52-150. Campos CA, Suarez MG, Laborde J. 2020. Analyzing vegetation cover-induced organic matter mineralization dynamics in sandy soils from tropical dry coastal ecosystems. Catena 185:104264. Cherchali A, Boukhelata N, Kaci Y, Abrous-Belbachir O, Djebbar R. 2019. Isolation and identification of a phosphate-solubilizing Paenibacillus polymyxa strain GOL 0202 from durum wheat (Triticum durum Desf.) rhizosphere and its effect on some seedlings morphophysiological parameters. Biocatal Agric Biotechnol 19:101087. Chowdhury SK, Majumdar S, Mandal V. 2020. Application of Bacillus sp. LBF-01 in Capsicum annuum plant reduces the fungicide use against Fusarium oxysporum. Biocatal Agric Biotechnol 27:101714. Cui W, He P, Munir S, He P , Li X , Li Y, …, He Y. 2019. Efficacy of plant growth promoting bacteria Bacillus amyloliquefaciens B9601-Y2 for biocontrol of southern corn leaf blight. Biol Control 139: 104080. da Silva K, Cassetari AD, Lima AS, Brandt ED, Pinnock E, Vandamme P, Moreira FMD. 2012. Diazotrophic Burkholderia species isolated from the Amazon region exhibit phenotypical, functional and genetic diversity. Syst Appl Microbiol 35:253-62. de Castilho CL, Longoni L, J Sampaio J, Lisboa BB, Vargas LK, Beneduzi A. 2020. The rhizosphere microbiome and growth-promoting rhizobacteria of the Brazilian juçara palm. Rhizosphere 15:100233. De Smet B, Mayo M, Peeters C, Zlosnik JE, Spilker T, Hird TJ, …, Vandamme P. 2015. Burkholderia stagnalis sp. nov. and Burkholderia territorii sp. nov., two novel Burkholderia cepacia complex species from environmental and human sources. Int J Syst Evol Microbiol. 65(7): 2265-71. Guleria S, Walia A, Chauhan A, Shirkot CK. 2016. Molecular characterization of alkaline protease of Bacillus amyloliquefaciens SP1 involved in biocontrol of Fusarium oxysporum. Int J Food Microbiol 232:134-43. Haile D, Firew Mekbib F, Assefa F. 2016. Isolation of phosphate solubilizing bacteria from white lupin (Lupinus albus L.) rhizosphere soils collected from Gojam, Ethiopia. J Fertil Pestic 7(2):1000172. Hillis DM, Bull JJ. 1993. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol 42(2):182-92. Hossain MI, Mizan MFR, Ashrafudoulla M, Nahar S, Joo HJ, Jahid IK, …, Ha SD. 2020. Inhibitory effects of probiotic potential lactic acid bacteria isolated from kimchi against Listeria monocytogenes biofilm on lettuce, stainless-steel surfaces, and MBEC™ biofilm device. LWT-Food Sci Technol 118: 108864. Jangir M, Pathak R, Sharma S, Sharma S. 2018. Biocontrol mechanisms of Bacillus sp., isolated from tomato rhizosphere, against Fusarium oxysporum f. sp. lycopersici. Biol Control 123:60-70. Joe MM, Deivaraj S, Abitha Benson A, Henry AJ, Narendrakumar G. 2018. Soil extract calcium phosphate media for screening of phosphate- solubilizing bacteria. Agric Nat Resour 52:305-308. Karthika S, Midhun SJ, Jisha MS. 2020. A potential antifungal and growth-promoting bacterium Bacillus sp. KTMA4 from tomato rhizosphere. Microb Pathog 142:104049. Khedhera SB, Mejdoub-Trabelsib B, Tounsia S. 2021. Biological potential of Bacillus subtilis V26 for the control of Fusarium wilt and tuber dry rot on potato caused by Fusarium species and the promotion of plant growth. Biol Control 152:104444. Kumar H, Dubey RC, Maheshwari DK. 2017. Seed- coating fenugreek with Burkholderia rhizobacteria enhances yield in field trials and can combat Fusarium wilt. Rhizosphere 3:92-9. Lau ET, Tani A, Khew CY, Chua YQ, Hwang SS. 2020. Plant growth-promoting bacteria as potential bio- inoculants and biocontrol agents to promote black pepper plant cultivation. Microbiol Res 240:126549. LiPuma JJ, Dulaney BJ, McMenamin JD, Whitby PW, Stull TL, Coenye T, Vandamme P. 1999. Development of rRNA-based PCR assays for identification of Burkholderia cepacia complex isolates recovered from cystic fibrosis patient. J Clin Microbiol 37(10):3167-70. Liu Z, Li YC, Zhang S, Fu Y, Fan X, Patel JS, Zhang M. 2015. Characterization of phosphate-solubilizing bacteria isolated from calcareous soils. Appl Soil Ecol 96:217-24. Ludueña SLM, Anzuay MS, Angelini JG, McIntosh M, Becker A, Rupp O, …, Taurian T. 2018. Strain Serratia sp. S119: A potential biofertilizer for peanut and maize and a model bacterium to study phosphate solubilization mechanisms. Appl Soil Ecol 126:107-12. Novel rhizobacteria strain isolated from sand dune ecosystem – Ketut Arte Widane et al. 37 Mahdavi P, Bergmeier E. 2016. Plant functional traits and diversity in sand dune ecosystems across different biogeographic regions. Acta Oecol 74:37-45. Mardad IA, Serrano A, Soukri A. 2013. Solubilization of inorganic phosphate and production of organic acids by bacteria isolated from a Moroccan mineral phosphate deposit. Afr J Microbiol Res 7(8):626- 35. Nautiyal CS. 1999. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 170:265-70. Panda B, Rahman H, Panda J. 2016. Phosphate solubilizing bacteria from the acidic soils of Eastern Himalayan region and their antagonistic effect on fungal pathogens. Rhizosphere 2:62-71. Pastore G, Kernche S, Spohn M. 2020. Microbial solubilization of silicon and phosphorus from bedrock in relation to the abundance of phosphorus-solubilizing bacteria in temperate forest soils. Soil Biol Biochem 151:108050. Paul D, Sinha SN. 2013. Isolation of phosphate solubilizing bacteria and total heterotrophic bacteria from river water and study of phosphatase activity of phosphate solubilizing bacteria. Adv Appl Sci Res 4:409-12. Rasool A, Mir MI, Zulfajri M, Hanafiah MM, Unnisa SA, Mahboob M. 2021. Plant growth promoting and antifungal asset of indigenous rhizobacteria secluded from saffron (Crocus sativus L.) rhizosphere. Microb Pathog 150:104734. Rasul M, Yasmina S, Zubaira M, Mahreena N, Yousaf S, Arifa M, …, Mirza MS. 2019. Phosphate solubilizers as antagonists for bacterial leaf blight with improved rice growth in phosphorus deficit soil. Biol Control 136:103997. Rasul M, Yasmin S, Yahya M, Breitkreuz C, Tarkka M, Reitz T. 2021. The wheat growth-promoting traits of Ochrobactrum and Pantoea species, responsible for solubilization of different P sources, are ensured by genes encoding enzymes of multiple P-releasing pathways. Microbiol Res 246:126703. Royse DJ, Ries SM. 1978. The influence of fungi isolated from peach twigs on the pathogenicity of Cytospora cincta. Phytopathology 68(4):603-7. Sahandi MA, Mehrafarin A, Badi HN, Khalighi-Sigaroodi F, Sharifi M. 2019. Improving growth, phytochemical, and antioxidant characteristics of peppermint by phosphate-solubilizing bacteria along with reducing phosphorus fertilizer use. Ind Crops and Prod 141:111777. Santos-Torres M, Romero-Perdomo F, Mendoza- Labrador J, Guti´errez AY, Vargas C, Castro- Rincon E, …, Estrada-Bonilla GA. 2021. Genomic and phenotypic analysis of rock phosphate- solubilizing rhizobacteria. Rhizosphere 17:100290. Schlaberg R, Simmon KE, Fisher MA. 2012. A systematic approach for discovering novel, clinically relevant bacteria. Emerg Infect Dis 18 (3):422-30. Simonetti E, Roberts IN, Montecchia MS, Flavio H. Gutierrez-Boem FH, Gomez FM, …, Ruiz JA. 2018. A novel Burkholderia ambifaria strain able to degrade the mycotoxin fusaric acid and to inhibit Fusarium spp. growth. Microbiol Res 206:50-9. Srinivas C, Devi DM, Murthy KN, Mohan CD, Lakshmeesha TR, Singh BP, …, Srivastava RK. 2019. Fusarium oxysporum f. sp. lycopersici causal agent of vascular wilt disease of tomato: Biology to diversity-A review. Saudi J Biol Sci 26:1315-24. Tagele SB, Kim SW, Lee HG, Kim HS, Lee YS. 2018. Effectiveness of multi-trait Burkholderia contaminans KNU17BI1 in growth promotion and management of banded leaf and sheath blight in maize seedling. Microbiol Res 214:8-18. Tapia-García EY, Hernández-Trejo V, Guevara-Luna J, Rojas-Rojas FU, Arroyo-Herrera I, Meza-Radilla G, …, Santos PE. 2020. Plant growth-promoting bacteria isolated from wild legume nodules and nodules of Phaseolus vulgaris L. trap plants in central and southern Mexico. Microbiol Res 239:126522. Teymouri M, Akhtari J, Karkhane M, Marzban A. 2016. Assessment of phosphate solubilization activity of rhizobacteria in a mangrove forest. Biocatal Agric Biotechnol 5:168-72. Valetti L, Liliana IL, Fabra A. 2018. Growth promotion of rapeseed (Brassica napus) associated with the inoculation of phosphate solubilizing bacteria. Appl Soil Ecol 132:1-10. Vanlaere E, Lipuma JJ, Baldwin A, Henry D, De Brandt E, Mahenthiralingam E, …, Vandamme P. 2008. Burkholderia latens sp. nov., Burkholderia diffusa sp. nov., Burkholderia arboris sp. nov., Burkholderia seminalis sp. nov. and Burkholderia metallica sp. nov., novel species within the Burkholderia cepacia complex. Int J Syst Evol Microbiol 58 (PT 7):1580- 90. Viallard V, Poirier I, Cournoyer B, Haurat J, Wiebkin S, Ophel-Keller K, Balandreau J. 1998. Burkholderia graminis sp. nov. a rhizospheric Burkholderia species, and reassessment of [Pseudomonas] phenazinium, [Pseudomonas] pyrrocinia and [Pseudomonas] glathei as Burkholderia. Int J Syst Bacteriol 48 (PT 2):549-63. Widane KA, Retnaningrum E. 2018. Kemampuan bakteri pelarut fosfat dan antipatogen Fusarium oxysporum (layu fusarium) isolat rhizosfer Fimbristylis cymosa R. Br. dari gumuk pasir Pantai Parangtritis Yogyakarta. [The capability of phosphate solubilizing bacteria and antipathogen Fusarium oxysporum isolated from the rhizosphere of Fimbristylis cymosa R.Br. obtained in sand dunes habitat in Parangtritis Coastal Area of Yogyakarta]. Final Research Project of Undergraduate Student. Yogyakarta (ID): Universitas Gadjah Mada. https://pubmed.ncbi.nlm.nih.gov/?term=Tapia-Garc%C3%ADa+EY&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=Hern%C3%A1ndez-Trejo+V&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=Guevara-Luna+J&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=Rojas-Rojas+FU&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=Arroyo-Herrera+I&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=Meza-Radilla+G&cauthor_id=32585580 https://pubmed.ncbi.nlm.nih.gov/?term=Estrada-de+Los+Santos+P&cauthor_id=32585580 BIOTROPIA Vol. 29 No. 1, 2022 38 Yang F, Sui L, Tang C, Li J, Cheng K, Xue Q. 2021. Sustainable advances on phosphorus utilization in soil via addition of biochar and humic substances. Sci Total Environ 768:145106. Yarza P, Sproer C, Swiderski J, Mrotzek N, Spring S, Tindall BJ, …, Rossello-Mora R. 2013. Sequencing orphan species initiative (SOS): Filling the gaps in the 16S rRNA gene sequence database for all species with validly published names. Syst Appl Microbiol 36(1):69-73. Zhang X, Yingying Zhou Y, Yan Li Y, Fu X, Wang Q. 2017. Screening and characterization of endophytic Bacillus for biocontrol of grapevine downy mildew. Crop Prot 96:173-9. Zhao K, Penttinen P, Zhang X, Ao X, Liu M, Yu X, Chen Q. 2014. Maize rhizosphere in Sichuan, China, hosts plant growth-promoting Burkholderia cepacia with phosphate solubilizing and antifungal abilities. Microbiol Res169:76-82.