Effect of Azotobacter sp. and N Fertilizer on the Growth of Oil Palm Seedling Inoculated with Ganoderma sp

Excess application of nitrogen fertilizer causes soil nutrient imbalances and reduce the number and soil microbial diversity. This condition could induce soil born dieseses such basal stem rot caused by Ganoderma sp. A study was conducted to enhance the plant tolerance to Ganoderma sp. through introduction of microbial community especially non-symbiotic N fixing bacteria, Azotobacter sp. Plant materials used were 4 months old of germinated oil palms, while Ganoderma sp. was isolated from Bekri, Lampung. There are seven treatments tested i e Azotobacter sp. + Ganoderma sp. (+A+G), Azotobacter sp. + 30% N fertilizer of recommended dosage + Ganoderma sp. (+A+30N+G), Azotobacter sp. + 60% N fertilizer of recommended dosage + Ganoderma sp. (+A+60N+G), Azotobacter sp. + 100% N fertilizer of recommended dosage + Ganoderma sp. (+A+100N+G), 100% N fertilizer of recommended dosage + Ganoderma sp. (+100N+G), Ganoderma sp. inoculation (positive control, +G), and non-inoculated Ganoderma sp. (negative control, -G). The result showed that Azotobacter sp. enhanced the height of plant inoculated with Ganoderma sp. when accompanied with N fertilizer of 30 to 100% of recommended dose. Moreover, +A+100N+G seedling had significantly higher fresh and dry weight of shoot compared to those of +G seedling or +100N+G seedling.


INTRODUCTION
Nitrogen is the highest amount of nutrients added as fertilizer in soil compared to other macro nutrients such as P and K. Long term and high dose of N fertilizers, especially in the form as urea, is believed to change the soil nutrient balance and soil characteristics. The development of nutrient imbalances could cause various problems including decrease in soil bearing capacity against soil pathogens. This resulted in decline of population of microbial antagonists that play a role in the control of soil borne pathogens. In general, N fertilization promotes vegetative growth, lowering the concentration of carbon-based secondary compounds (phenol, terpenoids) and increasing N compounds such as alkaloids (Rasmussen et al. 2008). In addition, excess of nitrogen input could decrease the defense of plants. Nitrogen enrichment can increase growth, and differently affect the shoot and root defense (Rasmussen et al. 2008;Jamieson et al. 2012). Excessive N fertilization can lead to 40% endophytic depletion and can increase the attack of certain plant diseases. Olese et al. (2003) suggests that increased N fertilizers may increase the attack of powdery mildew on wheat crops. However, excessive N fertilization impacting plant disease attacks and also has an impact on environmental pollution.
Azotobacter sp. is known as non-symbiotic N-fixing bacteria, Gram negative, obligate aerobic, and soil dwelling. Its role in N fixation is extensively researched because it can associate with various plant species. Besides known as N fixers, this bacterium is also known as a producer of IAA hormone which can enhance the growth of plant roots. Romero et al. (2013) suggests that this bacterium can survive in dry conditions because of its ability to form cysts that are controlled by the sigma factor RpoS. In addition, it is expected that application of Azotobacter improve the quality of oil as has been done on the plant Brassica carinata cv. Peela Raya. Addition of Azospirillum and Azotobacter in combination with a half-dose of chemical fertilizers enhance the growth, yield and quality of Ethiopian mustard oil (Nosheen et al. 2013).
In oil palm plantations at this time, there is a very important disease known as basal stem rot caused by Ganoderma sp. Widiastuti et al. (2013) reported that there was a decline in microbial diversity, especially in area with severe attack of Ganoderma sp. However, in all samples taken from selected area, Azotobacter sp. Were consistently found. Azotobacter associations with plants are thought to alter the nutrient status of N crops as well as exudation of plant roots in both the number and components of exudates which may further alter the rhizosphere microbial community (Lakshmanan et al. 2014). This change can be through changes in the physical properties of soil chemistry and or indirectly affect the microbial community of antagonists (Kloepper et al. 2004). Research on the characterization of Azotobacter sp. of various habitats have been carried out including estate crops (Widiastuti et al. 2010). This study aims to determine the effect of microbial community improvement through inoculation of Azotobacter bacteria on the growth performance of oil palm inoculated with Ganoderma sp.

MATERIAL AND METHODS
The experiment was conducted in Microbiology Laboratory and Greenhouse of Indonesian Research Institute for Biotechnology and Bioindustry (IRIBB). Planting materials used were germinated oil palm seeds from Indonesian Oil Palm Research Institute (IOPRI), Medan, grown for 4 months in sterilized sand medium, before they were transplanted in sterilized soil medium obtained from Ciomas, Bogor. Sterilization was done by autoclave for 60 minutes at 121 °C and pressure 1.2 atm. Planting medium was filled in polybags 30x40 cm 2 in size. N fertilizer was given in the form of urea with a dose of 5 g referred as 100% standard. Ganoderma sp. was inoculated following the method of Widiastuti et al. (2011) prior to inoculation of Azotobacter sp. Inoculant of Azotobacter sp. with the concentration of 10 8 colony forming units (cfu) in 10 mL water for each seedlings, applied at the beginning of planting the seeds, while the application of N fertilizer had been adjusted to the standard time of fertilization that is every 2 weeks (Lubis 2000). Plant maintenance had been done by watering using tap water every day. Visual symp-toms of Ganoderma attack and seedling growth, nutrient N content of plants and soil were observed at 8-month after planting.

RESULT AND DISCUSSION
Gram Characterization and Biochemical Properties of Azotobacter sp. Gram staining showed that Azotobacter sp. is Gram-negative bacteria and SEM observation had revealed that the isolate produced polysaccharides. Mandal et al. (2008) suggest this isolate is oval in shape that the formed polysaccharides are useful for protecting the activity of nitrogenase from reactive oxygenase, thereby increasing the activity of fixation. According to Samiran et al. (2012) exopolysacharida applications produced by Azotobacter sp. can be useful as growth promoting substances, increase plant resistance to drought stress, improve soil aggregates and increase soil microbial community interactions to inhibit pathogens. In addition, Azotobacter sp. also produce cyst at dormant stage. Based on the analysis reduction of acetylenic (ARA) activity of nitrogenase Azotobacter sp. used in this study was 0.727 μmol hour-1.

The Growth of Oil Palm Seedling
The performance of oil palm seedlings growth after 8 months is presented in Figure 1 while the rooting of the plants is presented in Figure 2. The result of statistical analysis shows that the height of oil palm seedlings +G lower than that of -G although not significantly different (Table 1). The application of Azotobacter sp. in addition to 30%N, +60%N, and +100%N significantly yield higher palm seedling growth compared to those +G, while +A+G did not yield significantly higher growth compared to those +G. These results indicate that inoculation of     Ganoderma sp. slightly inhibited the growth ofseedling height and N nutrient adequacy did not seem to be fulfilled from the fixation of N Azotobacter sp. alone but should be combined with addition of N fertilizer. Better growth of plants inoculated with Azotobacter sp. and N fertilizer appears to be able to increase plant resistance to Ganoderma sp attack. In addition, Basidiomycetes among which Ganoderma sp. is within this Division, known as a major agent in the decomposition of cell wall polymers. Laboratory test results indicate that an increasing of inorganic N concentration can suppress the transcription of fungi genes necessary for lignin and lignocellulose metabolism (Li et al. 1994) and may even lead to a change in the composition of the fungi basidiomisetes (Edwards et al. 2011). These results indicate that N fertilization may inhibit Ganoderma sp. activity. Further Elsenlord et al. (2014) suggested that the presence of N in the soil significantly reduced the population and the diversity of genes involved in depolymerization of starch (12%), hemicellulose (16%), cellulose (16%) and chitin 15% and 16% lignin which generally had an effect on weathering and accumulation of organic matter.
However, this possibility does not occur in the presence of N fixation due to fixation by Azotobacter which is different in addition to other functions of Azotobacter sp. Rasmussen et al. (2008) suggested that in Lolium perenne plants infected with endophytic nitric microbes showed a decrease in some amino and magnesium acids, while the water-soluble carbohydrate content, fats, some organic acids and chlorogenic acids, increased. On the other hand, the application of N fertilizer causes an increase in organic and lipid acids while water-soluble carbohydrates, kholorogenic acids, and fiber decreases.
These results indicate thar there is differences in metabolism between N fertilized plants and infected endophytic fungi. Widiastuti et al. (2013) showed that there was low correlation between population of Azotobacter sp. and the attack rate of Ganoderma sp. among the observed microbial parameters analyzed such as total microbes, total fungi, phosphate solubilizing bacteria and total cellulolytic fungi. In addition to N fixers Azotobacter sp. also produces indole acetic acid hormone known as plant growth promoting (Widiastuti et al. 2010). With the presence of Azotobacter sp. the plant gets sufficiency supply of nitrogen and IAA that promote plants to survive Ganoderma sp. attacks. This can be seen in Table 1, the height of palm fertilized with N 100% of recommended dosage was 30.75 cm while the treatment of N fertilizer at various doses combined with Azotobacter sp. causing an increase in plant height of 2-4 cm. The addition of N 100% fertilizer alone (+N100+G) resulted in seedlings heights that are not significantly different from those given by Azotobacter sp. only (+A+G). This result indicates that the supply of N from fixation by Azotobacter sp. had been sufficient to support the superior performance of oil palm seedlings.
The seedlings of oil palm treated with +A+N30-100 that are inoculated with Azotobacter sp. accompanied by N fertilizer appears to show leaf breakage ( Figure  1). These results are thought to be due to the precence of IAA hormone produced by Azotobacter sp. which is not found in the application of N fertilizer (urea) alone, especially at low doses of N fertilizer. Although, the number of leaves was not different in all treatments tested (Table  1), however, +N100+G gave the highest number of leaves, while +A+G yielded the lowest number of leaves. The relatively short observation period may not be sufficient to show the effect of treatments to the number of leaves.
Treatment of +A+N30 and +A+N60, yielded fresh and dried shoot biomass unsignificantly different from those +G, but on the +A+100N+G resulting in fresh and dry biomass weight significantly higher than that +G. Between 30% doses and 60% of N fertilizer there was no significant difference in fresh and shoot biomass weight compared to those 100N dose. These results suggest that a higher dose of N may be required to produce fresh and dry weight of plant infected with Ganoderma sp.
The performance of oil palm seedlings is presented in Figure 2. In general, oil palm seedlings did not show symptoms of Ganoderma sp. Infection such as decay, however, inoculation of Ganoderma sp. reduced the development of rooting. Azotobacter inoculation alone results in better rooting but the best rooting treatment is +A+30N+G and +A+60N+G. Addition of Azotobacter sp. does not significantly affect fresh and dry weight of roots biomass but there is a tendency that all Azotobacter sp. application results in higher fresh roots biomass compared to +N100+G. These results indicate that the application of N fertilizer inhibits root growth. Application of Azotobacter sp. seems to promote root growth, thus decreasing the need of N fertilizer. The optimum N fertilizer is 60%.

Soil and Leaf nutrient Status as well as Azotobacter Populations in Rhizosphere of Oil Palm Seedlings
Population of Azotobacter sp. in soil with the inoculation treatment of Ganoderma sp. was higher than without inoculation of Ganoderma sp. It is generally shown that Azotobacter sp. were found in all rhizosphere of oil palm seedlings, although the highest population was found in +A+G treatment. The cause of high population in this treatment can not be explained using available data in this study.
The result of N soil analysis indicated that the soil N content in +G treatment is significantly higher than that of -G. The cause of this can not be explained in this study. The same results were found on soil in oil palm seedlings of +A+30N+G, +A+60N+G, and +A+100N+G compared to that -G. However, the soil N content in these plant rhizosphere was not significantly different compared to those +G treatment.
Decrease in dosage of N fertilizer up to 70% but accompanied by Azotobacter sp. application did not cause any significant difference to soil N levels. These results indicate that there is influence of Azotobacter sp. although this influence is not significant. Inoculation of Azotobacter sp. seemed to cause an increase in soil N as a result of N 2 fixation activity by Azotobacter sp. However, it seems that N 2 fixation activity did not give the same influence on soil N levels as that of 100% N fertilizer application.
The result of N leaf analysis showed that N content of leaf of oil palm seedlings +G were not significantly different to -G. These results suggest that inoculation of Ganoderma sp. had no effect on leaf N content. However, +A+100N+G, +A+60N+G, and +A+30N yield signifi-