POTENT PHOSPHATE-SOLUBILIZING BACTERIA ISOLATED FROM DIPTEROCARPS GROWN IN PEAT SWAMP FOREST IN CENTRAL KALIMANTAN AND THEIR POSSIBLE UTILIZATION FOR BIOREHABILITATION OF DEGRADED PEATLAND Irnayuli R. Sitepu1, Yasuyuki Hashidoko2, Erdy Santoso1 and Satoshi Tahara2 1
Laboratory of Forest Microbiology, Forest and Nature Conservation Research and Development Centre, Ministry of Forestry, Indonesia. Jalan Gunung Batu No. 5, Bogor 65145, Indonesia. 2 Laboratory of Ecological Chemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-Ku, Sapporo 060-8589, Japan. Emails:
[email protected];
[email protected] SUMMARY Seventy one bacteria associated with dipterocarp seedlings and saplings were isolated from peat soil at Nyaru Menteng Arboretum and Palangkaraya University Nursery, Central Kalimantan, Indonesia. These bacteria were screened for their capacity in solubilizing inorganic and mineralizing organic phosphate using modified NBRIP media in vitro. Most isolates were able to solubilize Ca3(PO4)2 with various solubilization index in the first screening. Secondary screening using P sources with lower pH, further grouped these bacteria into: (1) solubilizing inorganic Ca3(PO4)2 with pH 4.5: Erwinia sp. CK10, Roseateles sp. CK15, Rhizobium sp. CK19, Enterobacter sp. CK23, and Erwinia sp. CK24.; (2) mineralizing organic C6H6(OPO3H2)6 with pH 6.0: Roseateles sp. CK15, Rhizobium sp. CK19, Enterobacter sp. 23CK, Erwinia sp. CK24, NI CK53, and NI CK54; (3) solubilizing and mineralizing both P sources: Roseateles sp. CK15, Rhizobium sp. CK19, Enterobacter sp. CK23, and Erwinia sp. CK24. Test of P-solubilizing/-mineralizing efficiency to promote plant growth under glasshouse conditions will be conducted prior to field utilization for biorehabilitation purposes. Keywords: deforestation, rhizobacteria
bioreforestation,
screening,
plant
growth-promoting
INTRODUCTION Deforestation activities in Indonesia have led to enormous consequences of loss of ecosystems and biodiversity. In Kalimantan island alone, lowland forests dominated by dipterocarp species have decreased by more than 56% or about 2.6 million hectares (Butler, 2005). The native Dipterocarpaceae plays an important role in Indonesian forests because they are mother trees for thousands of animals and plant species, and the livelihood of local people. Seedlings and saplings transplanted into these degraded areas usually suffer low survival rate owing to typically acidic soil, the presence of toxic elements, and severe leaching that leads to limited nutrient availability. The usual practice of reforestation based solely on transplants with standard chemical inputs is often ineffective and ecologically unsustainable. These complex issues along with the ongoing deforestation urge us to find a practical solution for growth promotion of trees for reforestation. Phosphorous is the most limiting nutrient in tropical soil, only 0.1% of the total P present is available to the plants because of its chemical bonding and low solubility (Tilak et al., 2005). However, many soil microorganisms have the ability to solubilize and
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mineralize P from inorganic and organic pools of total soil P, making the element available for plants. The objective of this study was to investigate bacteria isolated from dipterocarps for their P-solubilizing and/or mineralizing capacity, and their possible utilization for sustainable P supplies to support plant growth in degraded lands. This study is a part of our wider efforts to restore degraded forest areas, with Indonesia as a model site, by using local biologically potential plant growth-promoting bacteria (PGPR) toward dipterocarps. MATERIALS AND METHODS Isolation and identification of bacteria Seventy one bacteria were isolated from root and soil of dipterocarps of the lowland Nyaru Menteng Arboretum located at 2° 43`49``S; 111°, 38`54``E and the Nursery of Center for International Cooperation in Sustainable Management of Tropical Peatland (CIMTROP), the University of Palangkaraya, Central Kalimantan, Indonesia. Bacteria were cultured in an N-free Winogradsky’s mineral mixture with 1% sucrose solidified with 0.3% gellan gum as gel matrix with pH 5.6 – 6.2 (Hashidoko et al., 2002). Molecular identification of the bacteria were based on 16S rRNA gene sequencing followed a description by Weisburg et al. (1991). The amplified product was sequenced using BigDye Terminator v3.1 cycle sequence kit (Applied Biosystems, Foste City, USA) with 4 choices of primers: 926F, 518R, 1112F, and/or 1080RM. Sequence homology was sought for using BLASTN online DNA database of NCBI (National Center for Biotechnology Information). Screening of phosphate solubilization activity A plate assay used a National Botanical Research Institute’s Phosphate medium (NBRIP) (Nautiyal, 1999) with different sources of P. (1): Initial screening for all 71 strains used NBRIP medium with Ca3(PO4)2, solidified with 1.5% agar at pH 7.0. (2): The secondary screening for strains that had a solubilization index (SI) ≥3, used different sources of inorganic and organic P with lower pH and only 1/10 strength of N source from the original amount for NBRIP. The P sources and pH of the three media were Ca3 (PO4)2 at pH 4.5, C6H6(OPO3H2)6 (phytic acid) at pH 6.0, and AlPO4 (+ CaCl2) at pH 4.3, respectively. CaCl2 served as a transporter agent to assist solubilization of AlPO4 by bacteria (Rodríguez & Fraga, 1999) in the NBRIP medium. The solubilization index followed Premono et al. (1996) (Figure 1). Data were subjected to statistical analysis. Phosphate Solubilization Index= A/B A= total diameter (colony + halo zone) B= diameter of colony
B A Figure 1 Calculation of Phosphate Solubilization Index. RESULTS Most strains were able to solubilize Ca3(PO4)2 with various indexes of solubilization at 4 days (Table 1). Three isolates, Frateuria sp. CK14, Paenibacillus spp. CK57 and CK58, grew on the medium but had no halo zone. Ten strains, Sphingomonas spp. CK20 and CK25, Rhizobium sp. CK27, Burkholderia spp. CK30, CK33, CK37, CK38, and CK71,
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Bacillus spp. CK39 and CK50 were unable to grow in NBRIP’s medium at pH 7.0. Solubilization index (SI) of 1.00 was obtained from bacteria with solubilization zone as wide as the colony diameter. Erwinia spp. CK10 and CK24, Enterobacter sp. CK23, Roseateles sp. CK15, Rhizobium sp. CK19, Burkholderia sp. CK52, and 4 unidentified strains, NI CK36, NI CK42, NI CK53, and NI CK54, the most efficient P solubilizers on 4, 6, and 8 days, were selected for secondary screening (Table 1). Table 1 Solubilization of Ca3(PO4)2 on NBRIP’s media at 4, 6, and 8 days after inoculation Strain Chromobacterium sp. CK8 Roseateles sp. CK15 Azospirillum sp. CK26 Stenotrophomonas sp. CK34 Rhizobium sp. CK19 Bacillus sp. CK41 Pandoraea sp. CK60 Pseudomonas sp. CK63 Serratia sp. CK67 Klebsiella sp. CK6 Klebsiella sp. CK17 Klebsiella sp. CK40
Solubilization index 4 day 6 day a 1.42 1.25
Strain a
8 day a 1.28
Erwinia sp. CK12
Solubilization index 4 day 6 day b 2.35 2.96
b
8 day 2.88
b
5.33
d
6.31
cd
6.00
cd
Klebsiella sp. CK18
2.75
bc
3.00
b
2.86
b
2.07
b
1.94
ab
2.00
ab
5.67
d
7.00
d
6.00
cd
1.00
a
1.00
a
1.00
a
Enterobacter sp. CK23 Erwinia sp. CK24
3.05
bc
3.84
bc
3.52
bc
3.82
c
3.77
bc
3.85
bc
Frateuria sp. CK1
2.31
b
1.65
ab
2.08
ab
2.67 1.85
b
2.11 1.87
bc
2.32 1.94
ab
Frateuria sp. CK2 Frateuria sp. CK7
1.56 1.00
ab
1.25 1.00
a
1.19 1.00
a
1.00
a
1.13
a
1.07
a
Frateuria sp. CK29
1.79
ab
2.19
ab
2.73
b
1.33 1.00 1.43
ab
1.53 1.00 1.48
a
1.69 1.00 1.39
ab
1.43 2.73 1.50
ab
1.59 3.33 1.42
ab
2.00 3.67 1.32
ab
2.24
b
2.00
bc
1.95
ab
Frateuria sp. CK49 Erwinia sp. CK10 Enterobacter sp. CK13 Enterobacter sp. CK64 Enterobacter sp. CK69 NI CK3
1.29
bc
1.36
a
1.30
a
bc
a ab
b
ab
a a
ab
ab
a ab
b
a
bc ab
a
a
b a
a
a
bc a
a
1.00 Burkholderia sp. 2.00 2.17 2.48 1.00 1.00 CK9 b ab ab b ab b Burkholderia sp. 2.09 2.23 2.17 2.44 2.31 2.54 CK11 ab ab ab ab a a Bukholderia sp. 1.62 1.97 2.03 NI CK4 1.24 1.16 1.04 CK28 ab ab ab ab a a Burkholderia sp. 2.05 2.07 2.13 NI CK5 1.67 1.33 1.27 CK31 a a bc b b a Burkholderia sp. 1.00 1.00 1.00 NI CK16 2.71 2.94 2.89 CK32 ab ab b b ab ab Burkholderia sp. 2.00 2.40 2.70 NI CK21 2.09 1.94 2.15 CK43 ab ab ab ab ab a Burkholderia sp. 1.50 1.82 2.31 NI CK22 1.58 1.64 1.55 CK44 b ab b a a a Burkholderia sp. 2.11 2.35 2.64 NI CK35 1.00 1.00 1.00 CK45 ab a a b b bc Bukholderia sp. 1.23 1.46 1.50 NI CK36 2.44 2.69 3.44 CK46 ab a a bc b b Bukholderia sp. 1.22 1.52 1.50 NI CK42 2.92 2.92 3.08 CK47 a a a cd d cd Burkholderia sp. 1.04 1.00 1.00 NI CK53 5.00 6.64 6.08 CK51 bc b bc cd Cd cd Burkholderia sp. 3.50 3.46 3.50 NI CK54 4.56 5.70 5.67 CK52 ab a a ab a a Burkholderia sp. 1.15 1.14 1.04 NI CK55 1.17 1.17 1.00 CK56 b bc b bc ab ab Burkholderia sp. 2.14 2.29 2.60 NI CK61 1.66 1.93 2.06 CK59 bc ab ab bc a a Burkholderia sp. 1.73 2.06 2.29 NI CK65 1.32 1.48 1.46 CK62 b bc ab a a a Burkholderia sp. 2.44 2.40 2.09 NI CK70 1.00 1.00 1.00 CK66 ab ab ab Burkholderia sp. 2.00 1.96 2.07 CK68 NI: Unidentified bacteria. Value is average of triplicate. SI values followed by the same letter in the same column are not significantly different at (P0.05) by Fisher’s pairwise comparison.
1 cm
b) a) Figure 3 Phosphate solubilization by Roseateles sp. CK15 on modified NBRIP medium shown by clear zone (halo) around the colony. (a): Ca3(PO4)2 at pH 4.0, 4 days after the inoculation, (b): phytic acid at pH 4.0, 3 days after the inoculation.
DISCUSSION This study suggested P-solubilizing/-mineralizing bacteria can be classified into four groups, as follows: 1. Solubilizing inorganic Ca3(PO4)2 with pH 4.5: Erwinia sp. CK10, Roseateles sp. CK15, Rhizobium sp. CK19, Enterobacter sp. CK23, and Erwinia sp. CK24. 4
2. 3. 4.
Mineralizing organic C6H6(OPO3H2)6 with pH 6.0: Roseateles sp. CK15, Rhizobium sp. CK19, Enterobacter sp. 23CK, Erwinia sp. CK24, NI CK53, and NI CK54. Solubilizing and mineralizing both P sources: Roseateles sp. CK15, Rhizobium sp. CK19, Enterobacter sp. CK23, and Erwinia sp. CK24. Unable to grow on media containing both of the P sources: Burkholderia sp. CK52, Erwinia spp. CK10 and CK24, NI CK36, and NI CK42.
The abilities of bacteria to grow and solubilize/mineralize both of the P sources on the NBRIP media suggested that the peat swamp ecosystem in Central Kalimantan is rich in such rhizobacteria that effectively utilize unavailable P. The P solubilization mechanisms may involve 1): solubilization of inorganic P by organic acid bio-synthesized by soil microorganisms, and 2): decomposition of organic P by acid-phosphatase and/or phytase. The capacity to mineralize phytic acid more rapidly than the inorganic Ca3(PO4)2 is because the bacteria were isolated from the rhizosphere of peat soil which is known to contain a high amount of phytic acid. Dipterocarps are unable to uptake phytic acid directly, so P for the tree-growth may be highly dependent upon mineralized phytic acid. In the screening assay, unexpectedly, many bacteria showed the ability to solubilize P in the forms of Ca3(PO4)2, suggesting peat swamp forest is a potent source for inorganic Psolubilizing rhizobacteria. It was reported that only 0.1% of the total phosphorous from soil is available to plants (Peix et al., 2001; Tilak et al., 2005) and available P is immediately depleted around the root zone owing to continued plant uptake (Smith et al., 2003). Hence, rhizoplane bacteria possessing abilities to solubilize P are likely to play an important role in P uptake in the ecosystem. CONCLUSION We demonstrated that many of the bacteria had P-solubilizing/-mineralizing properties and the ability was not exclusive to specific genera, suggesting the importance of preliminary screening in vitro for a wide range of bacteria to characterize their potent P-solubilizing/mineralizing trait. In vitro assay is a rapid and easy approach to test abilities of bacteria, however, this in vitro potential needs to be further tested by in vivo and in natural field conditions to confirm their actual P-solubilizing and/or –mineralizing properties. Incorporation of PGPR isolated from native trees for seedling preparation in the nursery is one alternative approach for ecological and economical benefits, in particular minimization of the excessive inputs of chemical fertilizers to the environment. This is probably the pioneer study to reveal that lowland dipterocarp forests in Central Kalimantan are a good source of diverse genera of highly potential P-solubilizers/-mineralizers. ACKNOWLEDGEMENTS This study was financially supported by a RITE (Research Institute of Innovative Technology for the Earth) project, 2003 (to Y.H.), grant-in-aid (no. 16208032 to Y.H.) by JSPS (Japan Society for the Promotion of Science), Monbukagakusho, and JSPS-LIPI Core University Program on Environmental Conservation and Land-use Management of Wetland Ecosystem in Southeast Asia. CIMTROP was appreciated for Central Kalimantan sampling: Dr. Suwido Limin, Erna Shinta, and Sampang. REFERENCES Butler, T. (2005) Deforestation in Borneo, Kalimantan at the crossroads: Dipterocarp Forest and the Future of Indonesian Borneo. www.mongabay.com edition April 17, 2005.
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Hashidoko, Y., Tada, M., Osaki, M., and Tahara, S. (2002) Soft gel medium solidified with gellan gum for preliminary screening for root-associating, free-living nitrogen-fixing bacteria inhabiting the rhizoplane of plants. Bioscience Biotechnology Biochemistry 66, 2259–2263. Nautiyal, C.S. (1999) An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiology Letters 170, 265-270. Peix, A., Rivas-Boyero, A. A., Mateos, P. F., Rodríguez-Barrueco, C., Martínez-Molina, E. and Velázquez, E. (2001) Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhizobium mediterraneum under growth chamber conditions. Soil Biolology and Biochemistry 33, 103-110. Premono, M.E., Moawad, A.M. and Vlek, P.L.G. (1996) Effect of phosphate-solubilizing Pseudomonas putida on the growth of maize and its survival in the rhizosphere. Indonesian Journal of Crop Science 11, 13-23. Rodríguez, H. and Fraga, R. (1999) Phosphate solubilizing bacteria and their role in plant growth promotion. Research review paper. Biotechnology Advance 17, 319–339. Smith, S.E., Smith, F.A. and Jakobsen, I. (2003) Mycorrhizal fungi can dominate phosphate supply to plants irrespective of growth responses. Plant Physiology 133, 16-20. Tilak., K.V.B.R., Ranganayaki, N., Pal, K.K., De, R., Saxena, A.K., Nautiyal, C.S., Mittal, S., Tripathi, A.K. and Johri, B.N. (2005) Diversity of plant growth and soil health supporting bacteria. Current Science 89, 136-150. Weisburg, W. G., Barns, S. M., Pelletier, D. A. and Lane, D. J. (1991) 16S ribosomal DNA amplification for phylogenic study. Journal of Bacteriology 173, 697-707.
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