An experiment was conducted in undrained poly bags under glasshouse conditions to quantify the N2 fixing capacity (15N isotope dilution method) of plant growth promoting rhizobacteria (Azospirillum and Bacillus spp.) in association with oil palm seedlings. Effects of inoculation on nutrient uptake and plant growth promotion will also be observed. The experiment was arranged in a randomized complete block design with five replications and harvested at 390 days after planting. The treatments involved were: 1) killed Azospirillum brasilense (Sp 7), 2) killed Sp 7; + inorganic-Ni, 3) Sp 7, 4) A. lipoferum (CCM 3863), 5) locally isolated rhizobacteria UPMB 10, and 6) UPMB 13 inoculation. Results showed that inoculation of the rhizobacteria could contribute up to 20-50% of the total nitrogen requirement of the host plant through N2 fixation process. Besides that, the inoculation process had also stimulated accumulation of nutrient and plant growth (tops and roots) comparable to the control with full inorganic nitrogen (Ni) fertilization after 390 days of growth.
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Recently there has been considerable interest in diazotrophic rhizobacteria such as Acetobacter diazotrophicus, Herbaspirillum spp., Azoarcus spp. and Azospirillum spp. which colonize the exterior and interior of sugarcane, rice and palm trees (Reis et al., 2000). The rhizobacteria have been reported as being important for establishment and growth of the host through associative N2 fixation (biofertilizer) and plant growth enhancement effects (bioenhancer)(Tsimilli et al., 2000). The biological N2 fixation process provides an opportunity to reduce application of synthetic nitrogenous fertilizer, save cost and potentially increase crop production (Cocking, 2000). Elimination or substantial reduction of N fertilizer is considered as a key factor in the development of environmentally friendly agricultural system (Reis et al., 2000). Findings by Malik et al. (1997) have suggested that rhizobacterial-rice association may acquired nearly 70% of the N requirement from atmospheric N2, thus conserving soil fertility by reducing the plants reliance on soil N. Exploitation of this associative N2 fixing rhizobacteria on oil palm seedlings can potentially make the seedling production be more sustainable and profitable (Dobereiner and Baldani, 1998; Shamsuddin et al., 2000). Thus, this experiment was conducted: 1) to estimate the total amount of N2 fixed by Azospirillum spp. and locally isolated rhizobacteria in association with oil palm seedlings, 2) to observe effects of inoculation on the accumulation of essential nutrients in plant tissues and 3) to observe effects of rhizobacterial inoculation on growth and development (tops and roots) of the host plants.
Materials and methods
An experiment was conducted in undrained polybag (to prevent leached out of applied 15N solution and fertilizer) with Selangor series soil (Typic Sulfic Tropaquept, pH 4.2) at 8 kg/polybag and was planted with newly germinated oil palm seed. The soil was maintained at an appropriate field capacity (28% moisture) daily. N-free fertilizer was applied monthly in the form of P2O5, K2O and MgO (reformulated based on the equivalent rate of NPK fertilizer required) (Foo and Mat, 1995) (Table 1). The 15N labeled solution enriched with 10%15N atom excess (a.e.) (14.92 g (15NH4)2.SO4)/6 L distilled water) was applied for labeling the soils for N2 fixation analyses by 15N isotope dilution method. The 15N solution was applied at 100 mL/polybag for all of the inoculation treatments, which is equivalent to 0.053 g N/polybag (20 kg N ha-1) at 130 and 260 days after planting (Hashim and Zaharah, 1994).
Two bacterial cultures of Azospirillum spp. were used for inoculation; A. brasilense (Sp 7) and A. lipoferum (CCM 3863), which were obtained from Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA), Brazil and Czechoslovakian Collection of Microorganism, Republic of Czechoslovakia. Two locally isolated rhizobacteria (Bacillus spp. UPMB 10 and Bacillus spp UPMB 13) provided by Soil Microbiology Laboratory, Universiti Putra Malaysia were also tested.
|Table 1:||Basal fertilizer rate based on recommended fertilizer applications for oil palm seedlings|
|**Supplied only to the control treatment (Sp 7 k +Ni)|
The strains were sub-cultured in 100 mL nutrient medium/flask and shaken continuously for 48 h (150 rpm, 28°C). The treatments applied in this experiment were as follows: 1) Control 1 (+ killed Sp 7, non-sterile soil (nss); autoclaved at 121°C for 20 minutes (Sp 7 k nss) - a non-fixing reference)(Malik et al., 1987; Dobbelaere et al., 1999), 2) Control 2 (+ killed Sp 7 nss, + inorganic-N (Sp 7 k +Ni)), 3) Azospirillum brasilense (Sp 7) inoculation; nss, 4) A. lipoferum (CCM 3863) inoculation; nss, 5) UPMB 10 inoculation; nss and 6) UPMB 13 inoculation; nss, arranged in a randomized complete block design with five replications. Inoculation of the seedlings with respective treatment was carried out after the plants emerged and at two monthly interval with 20 mL inoculum (approx. 108 cfu mL-1) per polybag.
The observations undertaken at harvest (D390) were; rates of N2 fixation by the inocula tested, uptake of essential nutrient (N, P, K, Ca and Mg) and top and root growth of the inoculated host plants. At harvest (D390), the plant samples were prepared for total N and 15N analysis by semi-micro Kjeldahl methods (Bremner, 1996). Dilution of the 15N was analyzed by emission spectrometer (NOI-6PC). The 15N abundance found in the plant tissue was corrected for the %15Na.e. present in the atmosphere (0.3663 % 15N a.e.). The proportion of N derived from the atmosphere (%Ndfa) and total N2 fixation were calculated using the following equation:
|N2 fixed (mg)||=||[(%Ndfa)(total N content)/100] x 1000|
An analysis of P was conducted by Technicon autoanalyzer (2nd Ed.), while for K, Ca and Mg were analyzed by Atomic Absorption Spectrophotometer (Perkin Elmer 5100 PC). (Rennie, 1980; Boddey et al., 1983; Giller et al., 1986).
RESULTS AND DISCUSSION
The 15N dilution technique has been used widely for quantification of biologically fixed nitrogen in legumes (Chalk, 1985) and associative nitrogen fixation in grasses
|Table 2:|| |
Percentage of N2 derived from atmosphere (%Ndfa) and N2 fixed (mg N plant-1) of inoculated oil palm seedlings at D390 in Selangor series soil
|Means with the same letters are not statistically significant at 5% level |
*Estimated based on Sp 7 k (nss) reference plants
|Table 3:||Effects of rhizobacteria inoculation on uptake of N, P, K, Ca and Mg of oil palm seedlings at D390 in Selangor series soil|
|Means with the same letters are not statistically significant at 5% level|
|Table 4:||Effects of rhizobacteria inoculation on plant growth and development of oil palm seedlings at D390 in Selangor series soil|
|Means with the same letters are not statistically significant at 5% level|
This experiment has proven that association of rhizobacteria (Azospirillum and locally isolated Bacillus spp.) with oil palm seedlings could successfully contribute fixed N2 for the host plants (20-30%Ndfa; 300-500 mg N plant-1; 0.50-0.60%15N a.e) (Table 2). Similar studies using 15N isotope dilution which was conducted by Urquiaga et al. (1992) and Boddey et al. (1995) have also shown that inoculated brazilian sugar cane with Azospirillum can fix substantial amount of N2 up to 70% of their N-requirement (150 kg N fixed ha-1 year-1). A number of tropical forage grasses including Brachiaria humidicola, B. decumbens, Paspalum notatum and Panicum maximum have shown relatively high N2 fixation rates by the associated N2 fixer and may derive up to 40% of their N-needs (Boddey, 1987). Malik et al. (1997) have also reported the N2 fixation ability of Azoarcus in association with kallar grass and contributed 26% of its N content from fixation. Another report by Dobereiner (1997) has shown that associative diazotrophic microorganisms could contribute at least 20-40% of the plant N requirement of several non-leguminous crops through N2 fixation process. According to Dobereiner (1997), development of N2 fixation process on oil palm would increase the net bio-energy yield by eliminating the large fossil energy input inherent in the use of N fertilizer. The technology hopefully will reduce input cost, increase the energy balance and diminish the negative environmental consequences on the use of excessive N fertilizer.
The inoculation process also showed significant effects on total uptake of N, P and K of the host plants (Table 3). Increment in N content was related to the N2 fixation process by the inocula tested (Sp 7, CCM 3863 and UPMB 10). Highly accumulation of P and K was also shown in the inoculated host plants and could be related indirectly to the inoculation effects through enhanced essential nutrient uptake by stimulation of root growth and development of the host plants (root dry weight and volume) (Table 4). Inoculation of Sp 7 and UPMB 10 had enhanced root dry weight (37%), volume (41%) and chlorophyll content of the host plants compared to the control (Sp 7 k). This is in agreement with earlier findings by Lin et al. (1983), Rai and Hunt (1993) and Bashan and Holguin (1997), that Azospirillum brasilense inoculation could improve ion uptake and contributed to significant elevation of plant growth. Saad et al. (1999) have shown that, inoculated sweet potato (Ipomea batatas) with Azospirillum produced similar or higher root yield, vigorously vegetative growth, and higher N content in the roots and leaves compared to uninoculated plants given normal rate of N fertilizer.
Bashan (1998) reported that inoculation of A. brasilense and A. lipoferum 1842 would increase the root hair formation and produced more lateral roots of wheat. Similar response of inoculation on root growth and development of soybean was also highlighted by Molla et al. (2001), where single or co-inoculation of Sp 7 with Bradyrhizobium (UPMR 48) had significantly stimulated higher root dry weight, root volume, specific root length, total root length and shoot dry weight of the host plant compared to the control (without any inoculation). It was proposed earlier by Bashan et al. (1990) that enhancement of mineral uptake by plants should result in an increased accumulation of both dry matter and minerals in the stem and leaves of the plants. Positive response of the inoculation process in promoting plant growth was related to the fixation capacity of the inocula tested.
The experiment indicated that the rhizobacterial strains especially Sp 7, CCM 3863 and UPMB 10 are potential biofertilizer with an average of 30% Ndfa (500 mg N plant-1). The inoculation process had also stimulated essential nutrient accumulation especially N, P and K and considered as a bioenhancer. As a PGPR the inocula had enhanced root dry weight, volume and chlorophyll content of the host plants. These strains are suitable and could be recommended for oil palm seedlings production and more in-depth studies are necessary to observe the efficiency of the inoculum on immature and mature oil palm in the field.
The authors are indebted to Universiti Putra Malaysia (UPM), Malaysian Institute for Nuclear Technology Research (MINT), Federal Land Development Agency (FELDA) for technical assistance and advice and Ministry of Science, Technology and Environment for the research funding (IRPA Program No. : 1-07-05-049).
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