P-solubilizing Microorganisms from The Poteran Island, Madura

P-solubilizing Microorganisms from The Poteran Island, Madura M.Shovitri 1*, N.D. Kuswytasari and E. Zulaika 2 1 Department of Biology, InstitutTeknologiSepuluhNopember, Surabaya, Indonesia 2 Department of Biology, InstitutTeknologiSepuluhNopember, Surabaya, Indonesia * Corresponding author s Tel:+62-85785616896, E-mail: maya@bio.its.ac.id ABSTRACT The Poteran Island faces natural problem of dryness and less fertile crop soil. It is containing more insoluble phosphate (P), while plant absorbs P in a soluble P form. P is one essential macronutrient that influences ex. crop maturity and production, disease resistance and energy conservation. This study was aimed to explore the existing indigenous phosphate-solubilizing microorganisms (PSM) in the island to be applied back as biofertilizer. As a comparison, a commercial liquid bioactivator was chosen and treated as the same.this study successfully isolated, purified, characterized and measured their ability to solubilize P. They were 4 fungal isolates tended to be as Aspergillussp (coded as A, B, C and D) and 12 bacterial isolates which were based on their chemical character tended to be Aeromonas sp (TA2 and TA5), Enterobacteriaceae (TA6), Corynebacterium sp (TA 7 and TA16), Bacillus sp (TA9, TA10, TA11 and TA13), Providencia sp (TA 12), Pseudomonas sp (TA14) and Staphylococcus sp (TA17). After 3-4 days incubated in a Pikovskaya medium, fungal isolate A, B, C and D showed index P solubility number of 15, 13, 6 and 8 respectively. But, bacterial isolates needed a longer incubation time (up to 10 days) to produce a clear zone around the growth colony. Bacterial isolates gave average P-solubility index about 30. The high index were 76 and 60 from isolate TA17 (Staphylococcus sp) and TA14 (Pseudomonas sp). As a comparison, 6 fungal isolates were found in a commercial liquid bioactivator with less P-solubility index of 5-10; which were Aspergillus flavus (O1), Aspergillus niger (O2), Trichoderma sp1 and sp2 (O3 and O4), and Rhizopus sp (O5). Keywords : P-solubilizing microorganisms, Poteran village INTRODUCTION Phosphorus (P) is an important element for plants, as it plays a significant role in all major metabolic plant processes for instance photosynthesis, crop maturity and production, disease resistance and energy conservation (Khan et al. 2010). Mostly P is abundant in soils in both inorganic and organic forms, but it is in an unavailable form for root uptake (Sharma et al 2013). P-solubilizing microorganisms (PSM) are able to enhance P availability to plants by releasing organic acids, solubilization and mineralization. Microbial population in soil is about 1 to 50 % consisted of P- solubilizng bacteria (PSB) and only 0.1 to 0.5 % of P- solubilizing fungi (PSF) (Chen et al., 2006). Poteran Island is one island in Madura. The island faces natural problem of dryness and less fertile crop soil. Since naturally soil contains PSM, this study was aimed to explore the existing indigenous PSM in the island to be applied back as biofertilizer to increase the crop soil fertility. 18-22 August 2014, Korea University, Seoul, Korea Page 1

MATERIALS AND METHODS Isolation of phosphate solubilizing fungi and bacteria and their P solubilizing assay Soil samples were collected from Talango and Poteran village in the Poteran Island. Approximately 50g of soil sample was taken from the upper 10-30 cm of the soil profile with a plastic core aseptically. The soil ph was7-8. Soil from Talango village was typically clay, red and humid, while it was rocky from Poteran village. The serially diluted soil samples were directly plated on a Pikovskaya medium (ph 6.8 7.0) containing 2.5 mg tricalcium phosphate as sole phosphorus source for selectively screening (Nautiyal et al., 2000). After 48 hours of incubation time at a room temperature, the single growth PSB colony was taken and transferred onto a fresh same medium. The bacterial colony was continue transferred each 24 hours of incubation time, until a pure bacterial culture was obtained. A single pure bacterial colony was then plated on a fresh same medium for 10 days to develop clear zone around the growth colony. For fungi, the serially diluted soil samples were first plated on a Potatoes Dextrose Agar containing 100 mg Chloramphenicol and incubated for 3-7 days. After doing purification by transferring a single chosen colony, a pure fungi culture was then separately plated on Pikovskaya medium (ph 6.8 7.0) containing 2,5 gram Ca3(PO4)2 or AlPO4 or P2O5 and incubated for 72 hours to develop clear zones around colony. As a comparison, a commercial liquid bioactivator was also treated as well as Talango soil sample to isolate and characterize fungi, and measure their P-solubizing ability. Phospat solubilizing index was measured following a simple equation below : diameter of clear zone diameter of colony X 100 diameter of colony Bacterial and fungi identification and characterization Serial biochemical characterization was tested to determine bacteria following Cappucino and Sherman (2001) and Harley and Prescott (2002) methods. The examined characters were Gram staining, endospore detection, oxygen need; carbohydrate fermentation of glucose, lactose and mannitol; catalase; hydrogen sulphide production; motility; indole; metyl red; Voges-Proskauer; citrate and urease production. The Bergey s manual of determinative bacteriology (Holt et al., 1994) was used for bacteria determination and identification. Fungi identification was performed following steps of Illustrated Genera of Imperfecti Fungi (Barnett, 1969), Pictorial Atlas of Soil and Seed Fungi Morphologies of Cultured Fungi and Key to Species (Watanabe, 2002), and Fungi and Food Spoilage (Pitt and Hocking, 2009) RESULTS AND DISCUSSION Practically chemical P fertilizer is added to soil. But long term use give impacts on the environment as eutrophication, soil fertility depletion and P precipitation in soil. PSM lately is the best eco-friendly means for P nutrition of crop (Sharma et al, 2013), as they are able to convert insoluble P to an accessible P form for increasing soil fertility and plant yield (Chen et al, 2006). PSM mostly are present in the rhizosphere (Vazquez et al, 2000), especially in in agricultural soil (Yahya and Azawi, 1998). But anyhow their population depends on different soil condition of physical and chemical properties, organic matter, and P content, as well cultural activities (Kim et al., 1998). This present study successfully isolated, purified and characterized 12 soil bacteria from Talango village which had ability to use tricalcium phosphate as sole phosphorus. Based on their biochemical 18-22 August 2014, Korea University, Seoul, Korea Page 2

characters (Fig.1) and its comparison to those in the Bergey s manual (Holt et al., 1994), they were Gram positive basil isolates most probably affiliated to genus Bacillus (TA9, TA10, TA 11 and TA13) and Corynebacterium (TA7 and TA16); the Gram negative basil to genus Pseudomonas (TA14), Aeromonas (TA2 and TA5), Enterobacteriaceae (TA6 and TA12); and Gram positive coccus to genus Staphylococcus (TA17). Their P solubilizing activity was measured by P-solubility index presented in Fig 2. In average their index is around 30, in which the high 5 index numbers of 76, 61, 60 and 48 were showed particularly by isolate TA17 (Staphylococcus), Pseudomonas (TA14), Enterobacteriaceae (TA12), Bacillus (TA13) and Corynebacterium (TA7). This result indicated Talango village soil was also populated by P-solubilizing bacteria (PSB), while genus Bacillus, Pseudomonas and Enterobacter had been reported as powerful PSB (Chung et al, 2005; Chen et al, 2006; Sharma et al, 2013). Fig.1. Dichotomy biochemical characters of bacterial isolates from the Talango village and cell of Corynebacterium (TA7) and Enterobacteriaceae (TA12). 18-22 August 2014, Korea University, Seoul, Korea Page 3

Isolate code P-solubility Index Fig.2. P-solubility index of isolated bacteria Fig.3. Macroscopic colony (A) and Conidiosphore (B) of Aspergillus sp2 There were only 4 fungi isolates from the Talango village. This was relevant to Chen et al (2006) that mentioned P-solubilizing fungi constitute about 0.1 0.5% of total fungal populations in soil. Based on their characters, they were tended to affiliate to genus Aspergillus (Fig 3). After 3-4 days incubation time on room temperature, the clear zone around their colonies appeared (Fig. 4). Table 1 showed the P- solubility index. Table 1. P-solubility index for 4 isolates of Aspergillus Isolate ɵ colony ɵ clear zone Index sol-p Fig.4. Clear zone around the growth colony of Aspergillus sp2 A 3,49 4,01 15 B 3,1 3,51 13 C 3,41 3,62 6 D 3,86 4,16 8 Strains Penicillium and Aspergillus fungi are the most powerful PSM9 (Whitelaw, 2000; Khan and Khan 2002). Our results showed that the fungi isolates (Table 1) were faster than bacterial isolates (Fig. 3) in indicating clear zone, and even greater in the P-solubility index. Many study explained this results, e.x P-solubilizing fungi do not lose the P dissolving activity upon repeated sub culturing under laboratory conditions as occurs with the P-solubilizing bacteria and the P-solubilizing fungi produce more acids than bacteria and consequently exhibit greater P-solubilizing activity (Sharma et al, 2013). As a comparison to those fungi activity, this study also did fungi isolation, identification and P- solubility assay from a commercial liquid bioactivator. This activator contained 6 different fungi isolates (Fig 5), which were Aspergillus flavus, A. niger, Tricoderma sp1, Trichoderma sp2, Rhizophus sp and Penicillium sp. They were also reported as the most powerful PSM (Sharma et al, 2013). Even the activator contained those mixture cultures, but their P-solubizing activity was even lower than it performed by a single fungi isolated from the Talango village. This remains an excellent prospectus 18-22 August 2014, Korea University, Seoul, Korea Page 4

study to find out a powerful mixer cultures of PSM consisted of bacteria and fungi. There must be a synergistic relationship between PSM and plants; PSM provide soluble phosphate and plants supply carbon compounds. Anyhow laboratory results when it is applied in vitro field trials most probably will be hampered by environmental variables of salinity, ph, moisture, temperature and climatic conditions of the soil. This is also an important consideration, a further study of PSM activity in correlation with these environmental factors need to be solved out before PSM application as a biofertiliser. Fig.5. Isolate fungi from the a commercial bioactivator and their P-solubility index CONCLUSION The indigenous PSM from the Talango village of The Poteran Island were fungi Aspergillus and bacteria of Bacillus, Staphylococcus, Pseudomonas, Corynebacterium and Enterobacteriaceae. Further study to improve their performance and to mix a microbial combination of those PSM is necessary to carry out to figure out the synergetic interactions in between in the level of an applied study. ACKNOWLEDGEMENT We would like to thank to ITS for the financial support with contract number 013674.175/IT2.7/PN.08.01/2013, to our student Hefdyah, Jean Ishendy Putera Akbar and Adyanti Kharismasari Sutoro for their hard lab works, to our colleagues and lab Microbiology and Biotechnology for their nice and warm environment during the research. REFERENCES Barnett, H. L. 1969. Illustrated Genera of Imperfect Fungi. Virginia: Burgess Publishing Company. Cappucino, J.G. and Sherman, N. 2001. Microbiology: A Laboratory Manual, Benjamin Cummings Publishing : United State of America Chen, Y.P. P.D. Rekha, A.B. Arun, F.T. Shen,W.-A. Lai, C.C. Young. 2006. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Elsevier. Applied Soil Ecology 34:33 41 18-22 August 2014, Korea University, Seoul, Korea Page 5

Chung H, Park M, Madhaiyan M, Seshadri S, Song J, Cho H, Sa T (2005) Isolation and characterization of phosphate solubilizing bacteria from the rhizosphere of crop plants of Korea. Soil Biol Biochem 37:1970 1974 Harley, J.P. and Prescott, L.M. 2002. Laboratory Exercises in Microbiology 4 th Edition. The McGraw Hill Companies : New York Holt, J.G. and Krig, N.R., Sneath, p., Staley, J., Williams, S. 1994. Bergeys Manual Of Determinative Bacteriology 9 th Edition. Lipincott Williams and Wilkins Company : Philadelphia USA. Kim KY, Jordan D, McDonald GA (1998) Enterobacter agglomerans, phosphate solubilizing bacteria, and microbial activity in soil: effect of carbon sources. Soil Biol Biochem 30:995 1003. Khan MR, Khan SM (2002) Effect of root-dip treatment with certain phosphate solubilizing microorganisms. Bioresour Technol 85(2):213 215 Khan MS, Zaidi A, Ahemad M, Oves M, Wani PA (2010) Plant growth promotion by phosphate solubilizing fungi current perspective. Arch Agron Soil Sci. 56:73 98 Pitt, J. I. and A. D. Hocking. 2009. Fungal and Food Spoilage. New York: Springer Science + Business Media. Sharma, S.B, Riyaz Z Sayyed, Mrugesh H Trivedi and Thivakaran A Gobi. 2013. Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. SpringerPlus 2:587. Vazquez, P., G. Holguin, M. Puente, A. E. Cortes and Y. Bashan. 2000. Phosphate solubilizing microorganisms associated with the rhizosphere of mangroves in a semi arid coastal lagoon. Biol. Fert. Soils 30:460-468. Watanabe, T. 2002. Pictorial Atlas of Soil and Seed Fungi: Morphologies of Cultured Fungi and Key to Species 2 nd ed. Florida: CRC Press LLC. Whitelaw, M. A. 2000. Growth promotion of plants inoculated with phosphate solubilizing fungi. Adv. Agron. 69:99-151. Yahya, A. and S. K. A. Azawi. 1998. Occurrence of phosphate solubilizing bacteria in some Iranian soils. Plant Soil 117:135-141. 18-22 August 2014, Korea University, Seoul, Korea Page 6