Academic Journal of Science, CD-ROM. ISSN: 2165-6282 :: 04(03):35–40 (2015) ISOLATION AND BIOASSAY SCREENING OF BIOFERTILIZER DIAZOTROPH BACTERIA FROM PADDY FIELD Mieke Rochimi Setiawati, PujawatiSuryatmana and Diyan Herdiyantoro Padjadjaran University, Indonesia Uneef Primadi Food Crops Extention Service of West Java Province, Indonesia Diazotroph bacteria are believed to contribute in increasing N content of rice plants which can finally improve the yield of rice plants. These bacteria can fix N2 in abundant air (78% N) and reduce it to ammonium which plays a role in the formation of plant proteins. Various types of diazotroph bacteria isolated around the roots (rhizosphere) of rice plants as well as roots, stems, and leaves of the rice plant tissue can be used as a biological fertilizer (biofertilizer) to improve rice yields. Isolation of various types of bacteria diazotroph is applied with selective media Ashby, Okon and JNFb. The isolates produced were screened to allow the production of superior diazotroph isolates in increasing plant’s height, dry weight and N content of rice plants. From the rhizosphere of rice plants were obtained 4 isolates of Azotobacter and 3 isolates of Azospirillum that produce high N2 fixation through Acetylene Reduction Activity (ARA) test, while from the roots, stem and leaves of the rice plant tissue were obtained 3 endophytic bacterial isolates diazotroph high of ARA. The ten isolates showed different effects in increasing plant’s height, dry weight and N content of rice plants. Isolates of endophytic diazotroph bacteria is better in increasing plant’s height, dry weight and N content of rice plants compared to those of Azotobacter and Azospirillum. High contribution of endophytic diazotroph bacteria related with N2 which has been fixed was directly implanted into the plant tissue with no missing N such as loss of N in the rhizosphere. Keywords: Diazotroph, Endophytic bacteria, Azotobacter, Azospirillum, Paddy plant. Introduction N Fertilizer is the main fertilizer is most needed by crops including rice plants. Urea and ZA is a form of N fertilizer is generally given to the rice crop. Chemical N fertilizer increased with dose and continuously can cause largely lost because folatilization or washed away by the water and cause unwanted enrichment downstream waters (eutrophication). Due to the low N fertilizer efficiency, only a fraction of N (30-40%) is given by the fertilizer can be used by plants resulting waste fertilizer costs. Another alternative that can utilize other N sources abundant in the air (air contains 78% N) was the use of bacteria diazotroph that live in rhizosfir rice plants and endophytic diazotroph bacteria which live in the tissue of root, stems, and leaves of the rice plants. The bacteria can fix N2 gas from the air through its nitrogenase enzyme activity and converted to ammonia as the base material forming cell proteins. 35 36 Isolation and Bioassay Screening of Biofertilizer ... Diazotroph bacteria commonly found in rhizosfer cereal crops which is some kind of Azotobacter and Azospirillum. Aerobic Azotobacter can still grow in unaerobic paddy rice roots. Rhizofir paddy area is a zone known as the area aerobic oxidative compared to some areas away from the root. Azospirillum is microaerophilic which requires oxygen in small amounts so that it is more adaptive in region that contains little dissolved oxygen. Some species of Azospirillum are endophytic that can be entered into the roots of cereal crops. Various types of bacteria have been known to live fixing N2 in the paddy field, but its ability in N2 fixation vary depending on the type of bacteria and species. Dobereiner (1998) stated that the superior of diazotroph bacterial must have nitrogenase activity more than 50 nmol C2H4 ml-1 h-1 through test of Acetylene Reduction Activity (ARA). By isolating the bacteria diazotroph using selective media are expected to be obtained from various isolates potentially be used as a biofertilizer that is capable of fixing the N2 from the air. To obtain a superior diazotroph bacteria, it has to follow by some various screening the isolates with bioassay method of rice plantlets in gnotobiotic condition. Research Methods Diazotroph bacteria isolated from rice fields located in the village of Cileles Jatinangor of Sumedang District. The diazotroph bacteria nonsimbiotic is isolated from the root zone (rhizosfir) of rice plants, while endophytic diazotroph bacteria isolated from the tissues of roots, stems, and leaves of Ciherang variety rice plants. Selective media is used to isolate the non-symbiotic diazotroph bacteria. The Ashby media is used to isolate the Azotobacter sp., while Okon media is used to isolate Azospirillum sp. Tryptic Soy Agar (TSA) and JNFb used to isolate diazotroph endophytic bacteria. To Screen isolates the generated isolates through bioassay test method, planlet rice plants has to grown in 50 ml of semi-solid media in gnotobiotic fahraeus. A total of 1 ml of each bacterial isolate diazotroph with a density of 1.5 x 108 cfu/ml was injected in the 7 days old rhizosphere rice plantlets. We used 5 tubes of rice plant that were planted in semisolid Fahreus media. Rice plantlets inoculated diazotroph bacterial isolates are grown in the growth chamber (with 12 h expose to light/12 h in dark) for 2 weeks. At the end of the observation we measured its height, dry weight and N content of the rice plants. Results and Discussion Isolation of Bacteria Diazotroph of Wetland Characteristics and morphology of isolates diazotroph bacteria summarized in the table below. Table 1. Morphological characteristics of Beneficial Diazotroph Bacteria Isolates from Wetland No. Type of Bacteria Origin of Isolates Colony Form Colony Colour Cells Form Gram ARA (nmol C2H4 ml-1 hour-1) 1 Azotobacter sp. 1 (A1) Rhizospher e Circular White brownish Coccu s - 101 2 Azotobacter sp. 2 (A2) Rhizospher e Circular White brownish Coccu s - 117 3 Azotobacter sp. 3 (A3) Rhizospher e Circular White brownish Coccu s - 123 4 Azotobacter sp. 4 Rhizospher Circular White Coccu - 226 Mieke Rochimi Setiawati et al. (A4) e brownish 5 Azospirillum sp. 1 (Ap1) Rhizospher e Irregula r White Bacill us - 220 6 Azospirillum sp. 2 (Ap2) Rhizospher e Irregula r White Bacill us - 106 7 Azospirillum sp. 3 (Ap3) Rhizospher e Irregula r White Yellowish Bacill us - 138 8 Endofit 1 (E1) Root Circular White pile Bacill us - 220 9 Endofit 2 (E2) Stem Circular White Yellowish Coccu s + 271 10 Endofit 3 (E3) Leaves Circular White Coccu s - 221 37 s From the rhizosphere of rice plants were obtained 4 isolates of Azotobacter and 3 isolates of Azospirillum that produce high N2 fixation through Acetylene Reduction Activity (ARA) test, while from the roots, stem and leaves of the rice plant tissue were obtained 3 endophytic bacterial isolates diazotroph high of ARA. Dobereiner (1998) stated that the superior of diazotroph bacterial must have nitrogenase activity more than 50 nmol C2H4 ml-1 h-1 through test Acetylene Reduction Activity (ARA). Diazotroph Bacteria Sreening through Bioassay Test High of Rice Plants Rice plant height differences are not significant due to the application diazotroph bacteria Azotobacter sp., Azospirillum sp., and endophytic bacteria. Although rice plant inoculated by endophytic bacteria that isolated from the root and stems of rice plants tends higher than the other. All the plants which is inoculated with diazotroph bacteria are higher than control or without inoculation. Figure 1. Hight plantlets inoculated by diazotroph bacteria after 21 days of incubation Plantlets rice crops which have received treatment inoculation bacterial isolates showed fix N2 diverse growth (Figure 1). Rice plant after inoculated with Azotobacter isolates A4 produces higher plant height compared to plants inoculated with Azotobacter another isolates. While the rice plant height does 38 Isolation and Bioassay Screening of Biofertilizer ... not seem to be affected by the provision of Azospirillum inoculation of Azospirillum isolates AP2 although it seems likely to increase the height of rice plants. Rice plants were inoculated with endophytic bacterial isolates E1 and E2 resulted in higher plant height compared to the inoculated endophytic E3. Rice plants grow taller due to diazotroph bacterial inoculation than another plants because the supply of N produced by bacteria such as reducing N2 from the air. N2 reduction results NH4 given by the nonsymbiotic N2 fixing bacteria in rhizosfir rice plants or directly into the tissues of plants by diazotroph endophytic bacteria activity. Increased rice plant height is indicate activity of endophytic diazotroph bacteria. With the N2 fixation activity in the tissues of plants will increase the intake of N in rice plants, as a result will increase plant growth. This opinion is in line with the results Sevilla et al. (1998) in Pa'dua et al. (2001), which reported a diazotroph bacterial effect on plant growth. Acetobacter diazotrophicus an endophytic diazotroph bacteria associated with sugarcane on soil deficient N. A. diazotrophicus bacterial inoculation showed an increase in wet weight and plant height than the control plants (without inoculation). Dry Weight of Rice Plants Rice planlet were inoculated by diazotroph bacteria showed an increasing in dry weight after 21 days incubation. Rice plantlet were inoculated by diazotroph endophytic bacteria derived from the stem of rice plant were no different on dry weight compare to some of plants inoculated with another diazotroph bacteria. While their dry weight of plants which inoculated by that endophytic bacteria is different from control. Plant roots is relatively more so allegedly positively correlated with the absorption of nutrients better. According Sturz and Nowak (2000) which is non-pathogenic endophytic microbes have a beneficial effect on the host plant which can increase crop N2 fixation. The distribution of nutrients absorbed by plant roots can apparently accelerated by endophytic bacteria through a shorter path. Plantlets inoculated rice plants diazotroph bacteria after an incubation period of 21 days showed an increase in dry weight varied. A4 Azotobacter isolates showed increased bacterial isolates were higher than another diazotroph bacteria. In the group of bacteria Azospirillum, AP3 isolates showed a high increase. Endophytic bacterial isolates E2 looks produce high dry weight of rice plants compared to another endophytic isolates.The increase in plant biomass are influenced by plant tissue forming components such as proteins, carbohydrates and fats obtained from the reduction of NH4 and photosynthesis result of bacterial fixation N2 where N2 gaseus are abundant in the air. When these bacteria have a high ability to perform photosynthesis and N2 fixation, the dry weight of the plant will grow. Figure 2. The dry weight of rice plants were inoculated diazotroph bacteria after 21 days of incubation Mieke Rochimi Setiawati et al. 39 Nitrogen Content of Rice Plants Rice plants were inoculated with endophytic bacteria isolates from the stems and roots of rice plants led to an increase in N content but there as difference among their N content. While other diazotroph bacteria isolates ere inoculated into rice plants does not cause noticeable increase compared each other but very different compare with control. From the figure below shows that the endophytic bacterial isolates E1 and E2 can increase N content higher than another diazotroph bacterial isolates (A4 Azotobacter and Azospirillum isolates AP2). While the increase in N content of plants as a result of inoculation isolates isolates E2 is similar to E1. Figure 3. N content of rice plants inoculated diazotroph bacteria after 21 days of incubation N content of rice plants in a state of adequacy according to Jones et al. (1991) ranged from 2.60 to 3.20%. Meanwhile, according to Reuter and Robinson (1988) N content in the young rice plants range of 3.0-4.0%. In Figure 3 shows that the A4 Azotobacter isolates showed a high N content while the rice plants were inoculated other Azotobacter isolates produce enough N content. Inoculation AP2 Azospirillum isolates causing considerable N content in rice and better than other Azospirillum isolates. All isolates of diazotroph endophytic produce a high N content, while isolates E1 and E2 resulted in a higher N content than isolates E3. N content in rice plants contributed by bacterial activity fixing N2 where N is supplied directly formed into N plants. N content contributed by diazotroph endophytic bacteria (symbiotic) is greater than the nonsymbiotic bacteria (Azotobacter and Azospirillum) due to N which contributed diazotroph bacterial symbiotic directly supplied to the plant tissues without any loss as well as when it is given in the area of non rhizosphere by bacteria symbiotic. James et al. (2000) states that the potential N donated by diazotroph endophytic bacteria is greater than non endophytic N tethered because nothing is lost. Diazotroph endophytic bacteria colonization in plant tissue can exploit carbon substrates supplied by plants without competing with other microbes. Conclusion 1. We have found 10 isolates diazotroph bacteria from the rhizosphere and tissue of rice plants are Azotobacter, Azospirillum and diazotroph endophytic bacteria, all of them have hight activity of their N2 fixation. 40 Isolation and Bioassay Screening of Biofertilizer ... 2. The diazotroph endophytic bacteria isolated from the stem of rice plant tissues have had hight potential to increase dry weight and N content of rice plants. Acknowledgement Acknowledgements are due to the Directorate General of Higher Education DITLITABMAS that fund this research through research scheme STRANAS with contract number 1046 / UN6.R / PL / 2014 Date May 12, 2014. References 1. 2. 3. 4. 5. Dobereiner, J. 1988. Isolation and Identification of Root Associated Diazotrophs. Plant Soil. 110:207-212. James, E.K., F.L. Olivares, A.L.M. de Oliveira, F.B. dos Reis, Jr., L.G. da Silva, and V.M. Reis. 2001. Further observation on interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions. J. Exp. Bot. 52(357): 747-760. Jones, J.B., Jr., B. Wolf and H.A. Mills.1991. Plant Analysis Handbook. A Practical Sampling Preparation, Analysis and Interpretation Guide. Micro-Macro. Publ. Inc., Athens. Pa’dua, V. L. M., H. P. Masuda, H. M. 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