Abstract

Materials and Methods

The experiment was conducted in a field at Tangkah Estate, Sime Darby Plantation Berhad (formerly Golden Hope Plantation Berhad), Tangkak, Johor, in southern Peninsular Malaysia (2°21′ N, 102°40′ E). Some chemical properties of the 0–15 cm layer of the Bungor soil (Ultisol) in the experimental area are presented in Table 1. Fourteen-month-old GH500 cloned oil palms were allowed to establish for 5 months after transplantation in the field in a triangular planting pattern of 8 m by 8 m (Fig. 1). The upkeep and maintenance of the trial plots included a normal estate manuring schedule of inorganic straight fertilizers, comprising N as ammonium sulfate, P as Christmas Island Rock Phosphate, K as muriate of potash, Mg as kieserite and B as borate (17). Straight fertilizer is a fertilizer that contributes a single nutrient to the crops.

Object name is 27_257f1.jpg" title="Click on image to zoom" class="tileshop" src="/pmc/articles/PMC4036051/bin/27_257f1.jpg"/>

Open in a separate window

Fig. 1

A plot with 16 recording oil palms (including 2 randomly selected palms receiving ¹⁵N-labeled fertilizer) and two outermost rows serving as a buffer. The buffer oil palms help to prevent cross-contamination between plots. They were treated the same as the recording palms in the ¹⁵N isotopic microplot. Recording palms were also used to conduct vegetative growth measurements (data not presented in the present study).

Bacillus sphaericus UPMB-10, isolated in Malaysia from oil palm roots (2), was subcultured on tryptic soy agar (TSA) (Merck KGaA Germany) to produce a pure mother culture for inoculum production. The carrier-based inoculum of UPMB-10 strain was prepared by transferring 1.0 mL of a 24-h culture (≥1×10⁹ cfu mL⁻¹) to 100 mL tryptic soy broth (TSB) (Merck KGaA) contained in 500 mL flasks. The flasks were incubated on an orbital shaker at 28±2°C for 24 hours and the number of viable cells in the culture was determined by the spread-plate method on TSA after 24-h incubation. An aliquot (30 mL) of broth culture was withdrawn from the flask when the cell concentration reached 10⁹ cfu mL⁻¹ and was injected through a puncture into a double layered gamma-irradiated (50 kGy) polyethylene bag containing a mixture of (a) ground oil palm frond with the following compositions: 492 mg kg⁻¹ total N, 54 mg kg⁻¹ available P, 30 mg kg⁻¹ available K, 4 mg kg⁻¹ available Ca and 1 mg kg⁻¹ available Mg, and (b) a commercial peat-based organic fertilizer with the following compositions: 253 mg kg⁻¹ total N, 389 mg kg⁻¹ available P, 172 mg kg⁻¹ available K, 536 mg kg⁻¹ available Ca and 126 mg kg⁻¹ available Mg. The polyethylene bag contained 1,500 g of (a) and 500 g of (b) in a homogenous mixture. The inoculated bags were incubated at room temperature (28±2°C) for 2 weeks before use. The cultures were then checked for quality by the spread-plate method. The minimum population of strain UPMB-10 was ≥10⁹ cfu g⁻¹ during field inoculation.

In the field, the plants were laid down in randomized complete block design with 4 treatments and 4 replicates, as shown in Table 2. The (Uninoculated−N_i+¹⁵N_i) and (Uninoculated+100% N_i+¹⁵N_i) treatments served as negative and positive controls, respectively, and also as a benchmark for deficient N (negative control) and optimum N (positive control). The (Inoculated+67% N_i+¹⁵N_i) treatment involved inoculation with B. sphaericus strain UPMB-10 inoculum. The (Uninoculated+67% N_i+¹⁵N_i) control treatment had similar N_i (67%) to the inoculated treatment. All uninoculated treatments were provided with killed inoculum (gamma-irradiated at 50 kGy) per palm. ‘100% N_i’ and ‘67% N_i’ refer to the full recommended inorganic N fertilizer regime (17) and 67% of the full N fertilizer regime, respectively.

Recordings were made from 16 palms for each of the 16 plots (4 treatments by 4 replicates). Palms in the two outermost rows served as a buffer (Fig. 1). The ¹⁵N-labeled fertilizer used was (¹⁵NH₄)₂SO₄ (ammonium sulfate) with 10.13 atom % ¹⁵N excess (at.%¹⁵N_e) serving as a tracer. The field experiment was initiated by the application of ¹⁵N-labeled fertilizer 5 months after transplanting. Within the 16 recording palms, 2 palms (micro-plot) received labeled ¹⁵N, with 10.13 at.%¹⁵N_e ammonium sulfate at a rate of 1 g N m⁻² (Fig. 1). The ¹⁵N-labeled fertilizer was uniformly applied in liquid form using 2 L distilled water per isotopic plot of 1 m² size. The plots were then covered with black polythene sheets evenly to reduce ¹⁵N-labeled fertilizer loss. A week later, the black polythene sheets at the ¹⁵N isotopic microplots were removed after the inner surface of each sheet was rinsed with water prior to inoculum application (31). The black polythene sheets were used once only for all inoculated and uninoculated ¹⁵N isotopic microplots. Inoculum for the first inoculation was then applied followed by the second inoculation four months later. The (Inoculated+67% N_i+¹⁵N_i) treatment was carried out at a rate of 2 kg inoculum (containing more than 10⁹ cfu g⁻¹B. sphaericus UPMB-10) by raking the surface of soil to a depth of approximately 5 cm within an area of 1 m², and at a rate equivalent to 296 kg ha⁻¹.

Harvesting was carried out 240 days (8 months) after the ¹⁵N-labeled fertilizer application. Four palms from each treatment were harvested destructively, and separated into leaflets, rachis, stem and roots. The major roots were extracted with a backhoe tractor, and the remaining roots were excavated by shoveling and sieving the soil within the area occupied by the harvested palm. Fresh weights and weights of oven-dried (70°C for 72 h) sub-samples were recorded. Samples were ground to pass through 0.5 mm sieves and analyzed for total N by the semi-micro Kjeldahl method (5) and atom %¹⁵N excess using the NOI-6PC emission spectrometer at Malaysian Nuclear Agency, Bangi, Selangor, Malaysia. The ¹⁵N abundance found in palm tissue was corrected for the atom %¹⁵N excess present in the atmosphere (0.3663 at.%¹⁵N_e) (30).

N₂ fixation in the whole palm was calculated from weighted atom excess (WAE) in the inoculated palm (Inoculated+67% N_i+¹⁵N_i) and uninoculated palm (Uninoculated+67% N_i+¹⁵N_i), using the following formula (15):

where AE, TN, Lf, Rc, St and Rt are atom % ¹⁵N excess, total N, leaflets, rachis, stems and roots, respectively.

The % of N derived from atmospheric N (%Ndfa) was then calculated as follows:

The data were statistically analyzed using a linear contrast one-way analysis of variance (ANOVA) followed by Dunnett’s test (SPSS Statistics version 17.0). The data were tested for significance of differences between the experimental treatments.

Results

Discussion

Many researchers consider ¹⁵N isotope dilution to be the most accurate technique to quantify biological N₂ fixation by plants under greenhouse and nursery conditions, and even in the field (2, 11, 12, 20, 25). In our study, the atom % ¹⁵N excess in the whole young oil palm inoculated with B. sphaericus UPMB-10 (Inoculated+67% N_i+¹⁵N_i) was significantly lower than in uninoculated palms (Uninoculated+67% N_i+¹⁵N_i), thus indicating that substantial N₂ fixation occurred. B. sphaericus UPMB-10 inoculation resulted in the acquisition of N from the atmosphere, which contributed 63% (almost two-thirds) of the plant N uptake. This was equivalent to 78 g N fixed palm⁻¹ in the 240 day period of growth or 11.6 kg N ha⁻¹, based on a planting density of 148 palms ha⁻¹. The findings in this study support the previous work of Amir et al.(2), but the 63% proportional contribution of fixed N was even higher than the 28% they had earlier reported. The higher contribution of N by B. sphaericus UPMB-10 inoculation through BNF in the present study could have resulted from the use of solid inoculum application, a strategy adopted after studying the outcomes and suggestions from the previous work of Amir et al.(1, 2). In the present study, field inoculation was conducted using solid substrate inoculum, not as in the earlier reports (1, 2), which used liquid inoculum. The solid substrate inoculum has several added benefits over liquid inoculum as it provides a substantial amount of carbon source, which was lacking in the liquid inoculum. In addition, the solid substrate inoculum was incubated for a minimum of two weeks to allow the inoculum population to reach 1×10⁹ cfu g⁻¹ substrate. Thus, the inoculum in the inoculated treatment in young oil palms would have a high population of B. sphaericus UPMB-10 and more supply of organic substrate to sustain growth and multiply in the soil, and subsequently offer more Ndfa (63%) through N₂-fixing activity. The enhancement of N₂-fixing activity could be due to a higher concentration of carbon sources; namely, starch, hemicellulose and degradation products from beneficial soil microbial interactions (28). In addition, the role of solid substrate in supplying an organic carbon source and other degradation products could also provide ‘shelter’ for bacterial cells from direct sunlight and as a buffer zone for solid substrate-soil surface interactions prior to rhizosphere colonization.

Among the plant parts, the accumulation of N in leaflets significantly increased in inoculated oil palms in parallel with the respective increases in %Ndfa values as compared with the uninoculated control palms. This could be related to the high amount of N nutrition required in the leaves for photosynthesis, especially crucial in the early growth stages of oil palm in the field (10).

In this present study, the roots contained 13% of the total N₂ fixed by the whole palm and 14% of the accumulated dry matter. This relatively small proportion of the roots in relation to the whole plant was similar to the 12% reported by Carranca et al.(7) in pea roots and 8–15% reported by Samba et al.(25) for legume roots of Crotalaria species. In both of these studies, roots were omitted from their ¹⁵N evaluations. Nevertheless, due caution is advisable since ignoring the contribution from roots might result in significant underestimation of N₂ fixed in some plants, as has been reported in Acacia sp., Faidherbia albida, Gliricidia sepium, Leucaena leucocephala and Pterocarpus sp. (18, 21, 26, 29). Nevertheless, this does not appear to be the case for oil palm, which is a non-nodulating and non-leguminous crop. Conversely, much of the total dry matter was accumulated aboveground.

The present study indicated that inoculation with B. sphaericus UPMB-10 significantly improved N and dry matter yields of leaflets and rachis of young immature oil palms 8 months after treatment, although this effect was not observed in the stem or roots. The leaflets and rachis together accounted for most of the N and dry weight accumulation of the plants. To secure compelling evidence of biological N₂ fixation, Boddey (4) maintained that associative biological N₂ fixation should include both higher N yield and lower ¹⁵N enrichment of the inoculated plant compared to the uninoculated plant. A significant dry matter or fruit yield increase due to inoculation with diazotrophic organisms should be accompanied by such evidence before it can be confidently concluded that the response is due to N₂ fixation, and not to some other factors. The present study, which showed both higher N yield and lower ¹⁵N enrichment as a result of soil inoculation with B. sphaericus UPMB-10, is in agreement with the criteria suggested by Boddey (4). What was still left to be demonstrated was an increase in fruit yield. Since immature oil palms were used in the present study, the effect of inoculation on the fruit yield was outside the scope of the present study. Nevertheless, nutritional, growth and BNF measurements up to palm maturity (2.5 years after planting) and fruit-bearing stages (3 years after planting) in future studies would provide a clearer picture of the effect of B. sphaericus UPMB-10 inoculation on oil palm cultivation. In addition, the planting density, soil type and trial location would need to be carefully considered in order to reduce the soil and spatial variability factors that could compromise data accuracy (14, 16).

In conclusion, field planting of young immature oil palm in soil inoculated with Bacillus sphaericus UPMB-10 reduced N fertilizer dependence through the supplementation of N by biological nitrogen fixation. The inoculation process contributed N₂ fixation from the atmosphere, which provided 63%, or nearly two-thirds of the total N uptake of the young immature oil palm. Hence, B. sphaericus UPMB-10 has the potential to be formulated for use as a biofertilizer. It is proposed that the experimental period should be extended in future studies until the flowering and fruiting stages of the oil palm to elucidate further the role of the diazotrophic rhizobacterium B. sphaericus UPMB-10 in promoting the growth and fruit yield of oil palm.

Acknowledgements

References