# Antioxidant Enzyme Activities and Secondary Metabolite Profiling of Oil Palm Seedlings Treated with Combination of NPK Fertilizers Infected withGanoderma boninense

Authors: Mahbod Sahebi; Mohamed M. Hanafi; Hasmah Mohidin; M. Y. Rafii; Parisa Azizi; Abu Seman Idris; A. Fariz; Rambod Abiri; Sima Taheri; Mehdi Moradpoor
Journal: BioMed Research International
 (2018)

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## Abstract

Oil palm (Elaeis guineensis Jacq) is one of the major sources of edible oil. Reducing the effect ofGanoderma,main cause of basal stem rot (BSR) on oil palm, is the main propose of this study. Understanding the oil palm defense mechanism againstGanoderma infection through monitoring changes in the secondary metabolite compounds levels before/after infection byGanoderma under different fertilizing treatment is required. Oil palm requires macro- and microelements for growth and yield. Manipulating the nutrient for oil palm is a method to control the disease. The 3-4-month-old oil palm seedlings were given different macronutrient treatments to evaluate induction of defense related enzymes and production of secondary metabolite compounds in response toG. boninenseinoculation. The observed trend of changes in the infected and uninfected seedlings was a slightly higher activity for β-1,3-glucanases, chitinase, peroxidase, and phenylalanine ammonia-lyase during the process of pathogenesis. It was found that PR proteins gave positive response to the interaction between oil palm seedlings andGanoderma infection. Although the responses were activated systematically, they were short-lasting as the changes in enzymes activities appeared before the occurrence of visible symptoms. Effect of different nutrients doses was obviously observed among the results of the secondary metabolite compounds. Many identified/unidentified metabolite compounds were presented, of which some were involved in plant cell defense mechanism against pathogens, mostly belonging to alkaloids with bitter-tasting nitrogenous-compounds, and some had the potential to be used as new markers to detect basal stem rot at the initial step of disease.


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## Body

## 1. Introduction

Oil palm (Elaeis guineensis Jacq.) is one of the world’s sources of edible oil produced largely in Indonesia and Malaysia. Beside the palm oil and kernel oil, various products such as paper, pulp, particle boards, fertilizer, energy, and biofuels are derived from fiber, empty fruit bunches, and trunk of oil palm [1, 2]. Annually, the increasing demands for the oil palm products lead to expanding the cultured area of this tree in the major producing countries [3, 4].Basal stem rot (BSR) disease caused byGanoderma boninenseis a serious threat for oil palm [5]. Reportedly, between 30 and 70% of oil palm production is usually lost because of BSR during repeating planting cycles [6]. TheGanodermaspp. attack the roots of palm, then spread slowly to the stem’s bole, and cause dry rot that stops nutrient uptake and transport [7]. This infection leads to decrease in the ability of fruits producing and in the sever levels causes collapse of the trunk. Fruiting bodies ofGanodermaon the oil palm are not detectable till more than half of the internal tissues would be rotted [5]. Currently, research on biochemical and molecular aspects of oil palm at the early stage of the BSR disease is ongoing to understand interactions between palm andGanoderma. In this regard, any changes in defense-responsive genes, transcription factors, proteins, and metabolite compounds should be monitored between healthy andGanoderma-inoculated oil palm seedlings.Plants have to develop their chemical-defense metabolites to stand unfavorable conditions and survive. These chemical-defense metabolites can be either resistance against pathogen or tolerance towards abiotic stress [8]. Therefore, induction of defense metabolite compounds in oil palm leaves may involve in tolerance of tree and acts as defense mechanism againstGanoderma[9]. Reportedly, the amount of secondary metabolite compounds in plants is affected by Phosphate (P) and Potassium (K) fertilizers [10–12]. It was proposed that production of cellulose, starch, and non-nitrogen containing secondary metabolite compounds and some of important bioactive compounds would be correlated and increased in well-fertilized crops [13]. Results of another study showed that application of combined biofertilizers, NPK fertilizer, and vermicompost obviously effected biochemical and morphophysiological responses of mustard plant. All these changes lead to synthesis and accretion of some secondary metabolite compounds; also some pigment contents, such as proline, sugar, and chlorophyll in plant leaves, resulted in regulating plant osmosis and growth improvement [14].Effects of N, P, and K fertilizing on resistance of oil palm againstGanoderma via induction of fungi associated proteins need to be more considered. Comparison of metabolite compounds between NPK treated and untreated palms leaf extract is one of the methods to reveal the biochemical pathways induced by BSR disease. In the current research LC-MS as a metabolite profiling technique was applied to identify different metabolite compounds from leaves of two groups of NPK treated and untreated oil palm seedlings as another objective of this study. Profiling of these metabolite compounds will provide strong possibility to be used as biomarker discovery of BSR disease as well as understanding enhanced resistance of oil palm againstGanoderma [15].


## 2. Material and Methods

### 2.1. Site and Plant Material

The 3-4-month-old oil palm seedlings were obtained from the Federal Land Development Authority (FELDA) Agriculture Services Sdn Bhd, Sungai Tekam, Jerantut, Pahang, Malaysia. Seedlings were given treatments as presented in Tables1 and 2 to evaluate induction of defense related enzymes as a response to macronutrients application onG. boninenseinoculated and healthy oil palm seedlings. The experiment was conducted at the experimental farm (2°59′20.56″N, 101°42′44.42″E) under nursery shade house conditions. The experiment was designed as a split-plot withGanoderma infected palms and uninfected palms as main plots and ten fertilizers treatment rates as subplots. Each main plot consisted of 500 healthy uninfected palms (−G) as control and 500Ganoderma (+G) infected palms. The treatment comprised low, optimum, and high level for N, P, K nutrition with respective element in mixed fertilizers. The experiment was conducted using a randomized complete block design (RCBD), with five replications of ten seedlings per treatment. The nutrient levels of fertilizer were represented by N1, P1, and K1 for “low”; N2, P2, and K2 for “optimum”; and N3, P3, and K3 for “high” level. The recommended optimum level (referred to as N2, P2, and K2) applied to the oil palm seedlings was formulated based on the best vegetative growth performance obtained from the previous experiment. Fertilizer treatment was applied once every four weeks and the other elements, such as magnesium and micronutrients serve as basic fertilizer (BF). The composition of different fertilizer treatments used is presented in Table 1.Table 1
Fertilizer treatment levels for healthy and (+G) seedlings inoculated withG. boninense.
T1 = seedling treated withN1∗ (N low) + BF T2 = seedling treated withN3∗ (N high) + BF T3 = seedling treated withP1∗∗ (P low) + BF T4 = seedling treated withP3∗∗ (P high) + BF T5 = seedling treated withK1∗∗∗ (K low) + BF T6 = seedling treated withK3∗∗∗ (K high) + BF T7 = seedling treated with N1P1K1 (N low, P low, K low) + BF T8 = seedling treated with N2P2K2 (N optimum, P optimum, K optimum) + BF T9 = seedling treated with N3P3K3 (N high, P high, K high) + BF T10 = seedling treated with compound fertilizer (CF) Note. P   ∗ and K optimum, N∗∗ and K optimum, N∗∗∗ and P optimum. All treatments with BF = basic fertilizer 2% MgO (source from kieserite) + micronutrients (TE). In the common commercial name (CF), where N stands for % N (12%), P % for P2O5 (12%), K for % K2O (17%), and Mg for % MgO (2%) + TE.Table 2
Fertilizer and rates applied in the nursery trials.
Month  Urea   TSP   MOP N1 N2 N3 P1 P2 P3 K1 K2 K3 g/palm  4 0.69 0.99 1.29 0.693 0.99 1.29 1.15 1.64 2.13 5 0.80 1.14 1.48 0.798 1.14 1.48 1.34 1.92 2.50 6 0.88 1.26 1.64 0.882 1.26 1.64 1.57 2.24 2.91 7 0.97 1.38 1.79 0.966 1.38 1.79 1.74 2.48 3.22 8 1.03 1.47 1.91 1.029 1.47 1.91 1.90 2.72 3.54 9 1.11 1.59 2.07 1.113 1.59 2.07 2.07 2.96 3.85 10 1.20 1.71 2.22 1.197 1.71 2.22 2.24 3.20 4.16 11 1.26 1.80 2.34 1.26 1.8 2.34 2.38 3.40 4.42 12 1.34 1.92 2.50 1.344 1.92 2.50 2.49 3.56 4.63 13 1.41 2.01 2.61 1.407 2.01 2.61 2.63 3.76 4.89 14 1.47 2.10 2.73 1.47 2.1 2.73 2.74 3.92 5.10  Total 12.16 17.37 22.58 12.16 17.37 22.58 22.26 31.80 41.34 Note. Application of fertilizer in eleven months of experiment for growth analysis of oil palm seedlings. Amount of fertilizer needed for 1 replication for each level (g); TSP: triple superphosphate, MOP: muriate of potash. Level N1, P1, K1 (30%) less than N2, P2, and K2. Level N3, P3, and K3 (30%) more than N2, P2, and K2.The sources of macronutrients were straight fertilizer consisting of urea [CO(NH2)2] for N (46% N), triple superphosphate [Ca(H2PO4)2 H2O] for P (46% P2O5), and muriate of potash for K (60% K2O). The basic fertilizers (BF) were referred to as the other elements and they include MgO (source from kieserite) and trace elements as recommended in commercial production pocket guide in nursery stage [16]. The control, T10, was the granular compound fertilizer (CF), commonly used commercial fertilizer, where N stands for % N (12%), P for % P2O5 (12%), K for % K2O (17%), and Mg for % MgO (2%).


### 2.2. Experimental Design and Treatment

Based on the pathological performances and occurrence of diseases incidence responses, treatments T2, T5, T6, T8, and T10 were selected to be further evaluated of their enzymes activities. Treatments T2, T5, T6, T8, and T10 were arranged in a randomized complete block design with five replications of 20 seedlings per experimental unit withG. boninenseinfected and healthy palms for the detection test. Biochemical investigations were performed using three seedlings per treatment at weekly interval. Plants were destructively exercised at time course of being artificially inoculated byG. boninenseand collected at week 1, 4, 8, and 12 after inoculation (WAI) according to the treatment application and inoculation method.


### 2.3. Biochemical Analysis Assays

The random excised tips of primary root tissues were gently washed with distilled water, wrapped with aluminum foil, and maintained in liquid nitrogen. After reaching the laboratory, the root tissues were weighed, before being frozen, and stored at −80°C for further analysis. The activity of four enzymesβ-1,3-glucanase, chitinase, PAL, and POX was measured quantitatively using UV-1700 Pharmaspec, UV-VIS Spectrophotometer, Shimadzu Corporation, Japan.


### 2.4.β-1,3-Glucanase Activity

The activity ofβ-1,3-glucanase was determined according to the Nelson Somogyi method [17, 18], with slight modifications. The β −1,3-glucanase was assayed by measuring the rate of reducing sugar production with laminarin (Sigma Aldrich) as the substrate. Glucose was used as the standard and the activity was expressed as U mL−1 in unit−1 g tissue.


### 2.5. Chitinase Activity

The activity of chitinase was determined according to Tonon et al. with slight modifications [19]. Chitinase activity was assayed by measuring the rate of N-acetylglucosamine production using chitin (Sigma Aldrich) as the substrate. Chitinase activity was expressed as U mL−1 with N-acetylglucosamine as the standard.


### 2.6. Phenylalanine Ammonia-Lyase Activity

The activity of PAL was determined following the method of Chmielowska et al. with a slight modification [20]. The PAL activity was expressed in enzyme units (U) defined as μmol oftrans-cinnamic acid formed per minute, per mL of solution.


### 2.7. Peroxidase Activity

Peroxidase (POX) activity was measured following the modifications applied to the protocol of Chmielowska et al. [20]. The blank consisted of reaction mixture without enzyme extract. The POX activity was expressed in enzyme units (U) defined as μmol of DAB oxidized per minute, per mL of solution.


### 2.8. Data Analysis

Data were compared by the analysis of variance (ANOVA) using SAS 9.3. When significant, means were compared by Duncan’s Multiple Range Test (DMRT) at the 0.05 significance level. At-test was performed for each treatment to compare infected and uninfected seedlings for plant biomass and for content in roots. All the assays were performed in triplicate using primary roots of noninoculated (healthy) and inoculated seedlings.


### 2.9. Metabolite Compounds Profiling

Based on the existence of diseases incidence responses, samples with the same treatments used for biochemical analysis were selected for this part of experiment. Aliquots of 500 mg oil palm leaf powder were extracted with 1.5 ml of 99.875% methanol acidified with formic acid 0.125%. The extracts were solicited, centrifuged, and filtered through a 0.2μm polytetrafluoroethylene (PTFE) filter. For each accession, two biological replicates were prepared, resulting in total of 40 extracts. To check the technical variation, including extraction, sample analysis, and data processing, quality control data were prepared by pooling leaf material of several randomly chosen accessions, extracted using the same procedure and injected after every 10 accession sample extract. All extracts were analyzed using reversed phase liquid chromatography with Mass-Spec (LC-MS) system, using C-18 reversed phase chromatography and negative electrospray ionization. About five μm of the extract was injected and separated using a binary gradient of ultrapure water and acetonitrile, both acidified with 0.1% formic acid, with flow rate of 0.19 mL/min.


## 2.1. Site and Plant Material

The 3-4-month-old oil palm seedlings were obtained from the Federal Land Development Authority (FELDA) Agriculture Services Sdn Bhd, Sungai Tekam, Jerantut, Pahang, Malaysia. Seedlings were given treatments as presented in Tables1 and 2 to evaluate induction of defense related enzymes as a response to macronutrients application onG. boninenseinoculated and healthy oil palm seedlings. The experiment was conducted at the experimental farm (2°59′20.56″N, 101°42′44.42″E) under nursery shade house conditions. The experiment was designed as a split-plot withGanoderma infected palms and uninfected palms as main plots and ten fertilizers treatment rates as subplots. Each main plot consisted of 500 healthy uninfected palms (−G) as control and 500Ganoderma (+G) infected palms. The treatment comprised low, optimum, and high level for N, P, K nutrition with respective element in mixed fertilizers. The experiment was conducted using a randomized complete block design (RCBD), with five replications of ten seedlings per treatment. The nutrient levels of fertilizer were represented by N1, P1, and K1 for “low”; N2, P2, and K2 for “optimum”; and N3, P3, and K3 for “high” level. The recommended optimum level (referred to as N2, P2, and K2) applied to the oil palm seedlings was formulated based on the best vegetative growth performance obtained from the previous experiment. Fertilizer treatment was applied once every four weeks and the other elements, such as magnesium and micronutrients serve as basic fertilizer (BF). The composition of different fertilizer treatments used is presented in Table 1.Table 1
Fertilizer treatment levels for healthy and (+G) seedlings inoculated withG. boninense.
T1 = seedling treated withN1∗ (N low) + BF T2 = seedling treated withN3∗ (N high) + BF T3 = seedling treated withP1∗∗ (P low) + BF T4 = seedling treated withP3∗∗ (P high) + BF T5 = seedling treated withK1∗∗∗ (K low) + BF T6 = seedling treated withK3∗∗∗ (K high) + BF T7 = seedling treated with N1P1K1 (N low, P low, K low) + BF T8 = seedling treated with N2P2K2 (N optimum, P optimum, K optimum) + BF T9 = seedling treated with N3P3K3 (N high, P high, K high) + BF T10 = seedling treated with compound fertilizer (CF) Note. P   ∗ and K optimum, N∗∗ and K optimum, N∗∗∗ and P optimum. All treatments with BF = basic fertilizer 2% MgO (source from kieserite) + micronutrients (TE). In the common commercial name (CF), where N stands for % N (12%), P % for P2O5 (12%), K for % K2O (17%), and Mg for % MgO (2%) + TE.Table 2
Fertilizer and rates applied in the nursery trials.
Month  Urea   TSP   MOP N1 N2 N3 P1 P2 P3 K1 K2 K3 g/palm  4 0.69 0.99 1.29 0.693 0.99 1.29 1.15 1.64 2.13 5 0.80 1.14 1.48 0.798 1.14 1.48 1.34 1.92 2.50 6 0.88 1.26 1.64 0.882 1.26 1.64 1.57 2.24 2.91 7 0.97 1.38 1.79 0.966 1.38 1.79 1.74 2.48 3.22 8 1.03 1.47 1.91 1.029 1.47 1.91 1.90 2.72 3.54 9 1.11 1.59 2.07 1.113 1.59 2.07 2.07 2.96 3.85 10 1.20 1.71 2.22 1.197 1.71 2.22 2.24 3.20 4.16 11 1.26 1.80 2.34 1.26 1.8 2.34 2.38 3.40 4.42 12 1.34 1.92 2.50 1.344 1.92 2.50 2.49 3.56 4.63 13 1.41 2.01 2.61 1.407 2.01 2.61 2.63 3.76 4.89 14 1.47 2.10 2.73 1.47 2.1 2.73 2.74 3.92 5.10  Total 12.16 17.37 22.58 12.16 17.37 22.58 22.26 31.80 41.34 Note. Application of fertilizer in eleven months of experiment for growth analysis of oil palm seedlings. Amount of fertilizer needed for 1 replication for each level (g); TSP: triple superphosphate, MOP: muriate of potash. Level N1, P1, K1 (30%) less than N2, P2, and K2. Level N3, P3, and K3 (30%) more than N2, P2, and K2.The sources of macronutrients were straight fertilizer consisting of urea [CO(NH2)2] for N (46% N), triple superphosphate [Ca(H2PO4)2 H2O] for P (46% P2O5), and muriate of potash for K (60% K2O). The basic fertilizers (BF) were referred to as the other elements and they include MgO (source from kieserite) and trace elements as recommended in commercial production pocket guide in nursery stage [16]. The control, T10, was the granular compound fertilizer (CF), commonly used commercial fertilizer, where N stands for % N (12%), P for % P2O5 (12%), K for % K2O (17%), and Mg for % MgO (2%).


## 2.2. Experimental Design and Treatment

Based on the pathological performances and occurrence of diseases incidence responses, treatments T2, T5, T6, T8, and T10 were selected to be further evaluated of their enzymes activities. Treatments T2, T5, T6, T8, and T10 were arranged in a randomized complete block design with five replications of 20 seedlings per experimental unit withG. boninenseinfected and healthy palms for the detection test. Biochemical investigations were performed using three seedlings per treatment at weekly interval. Plants were destructively exercised at time course of being artificially inoculated byG. boninenseand collected at week 1, 4, 8, and 12 after inoculation (WAI) according to the treatment application and inoculation method.


## 2.3. Biochemical Analysis Assays

The random excised tips of primary root tissues were gently washed with distilled water, wrapped with aluminum foil, and maintained in liquid nitrogen. After reaching the laboratory, the root tissues were weighed, before being frozen, and stored at −80°C for further analysis. The activity of four enzymesβ-1,3-glucanase, chitinase, PAL, and POX was measured quantitatively using UV-1700 Pharmaspec, UV-VIS Spectrophotometer, Shimadzu Corporation, Japan.


## 2.4.β-1,3-Glucanase Activity

The activity ofβ-1,3-glucanase was determined according to the Nelson Somogyi method [17, 18], with slight modifications. The β −1,3-glucanase was assayed by measuring the rate of reducing sugar production with laminarin (Sigma Aldrich) as the substrate. Glucose was used as the standard and the activity was expressed as U mL−1 in unit−1 g tissue.


## 2.5. Chitinase Activity

The activity of chitinase was determined according to Tonon et al. with slight modifications [19]. Chitinase activity was assayed by measuring the rate of N-acetylglucosamine production using chitin (Sigma Aldrich) as the substrate. Chitinase activity was expressed as U mL−1 with N-acetylglucosamine as the standard.


## 2.6. Phenylalanine Ammonia-Lyase Activity

The activity of PAL was determined following the method of Chmielowska et al. with a slight modification [20]. The PAL activity was expressed in enzyme units (U) defined as μmol oftrans-cinnamic acid formed per minute, per mL of solution.


## 2.7. Peroxidase Activity

Peroxidase (POX) activity was measured following the modifications applied to the protocol of Chmielowska et al. [20]. The blank consisted of reaction mixture without enzyme extract. The POX activity was expressed in enzyme units (U) defined as μmol of DAB oxidized per minute, per mL of solution.


## 2.8. Data Analysis

Data were compared by the analysis of variance (ANOVA) using SAS 9.3. When significant, means were compared by Duncan’s Multiple Range Test (DMRT) at the 0.05 significance level. At-test was performed for each treatment to compare infected and uninfected seedlings for plant biomass and for content in roots. All the assays were performed in triplicate using primary roots of noninoculated (healthy) and inoculated seedlings.


## 2.9. Metabolite Compounds Profiling

Based on the existence of diseases incidence responses, samples with the same treatments used for biochemical analysis were selected for this part of experiment. Aliquots of 500 mg oil palm leaf powder were extracted with 1.5 ml of 99.875% methanol acidified with formic acid 0.125%. The extracts were solicited, centrifuged, and filtered through a 0.2μm polytetrafluoroethylene (PTFE) filter. For each accession, two biological replicates were prepared, resulting in total of 40 extracts. To check the technical variation, including extraction, sample analysis, and data processing, quality control data were prepared by pooling leaf material of several randomly chosen accessions, extracted using the same procedure and injected after every 10 accession sample extract. All extracts were analyzed using reversed phase liquid chromatography with Mass-Spec (LC-MS) system, using C-18 reversed phase chromatography and negative electrospray ionization. About five μm of the extract was injected and separated using a binary gradient of ultrapure water and acetonitrile, both acidified with 0.1% formic acid, with flow rate of 0.19 mL/min.


## 3. Results

### 3.1. Biochemical Analysis Assays

#### 3.1.1. Glucanase Activity

Glucanase enzyme activity showed high significant difference (p<0.05) between theGanoderma infected seedlings and control from week 1 to week 12 with t-value of 10.43, 6.74, 11.06, and 8.66 U mL−1 at 1 to 12 WAI, respectively (Table 3). The relatively low content of the uninfected seedlings is a reflection of the relatively low resistance towardsG. boninense in the nutrient level studied. The time of defense related response induction was rapid, within week 1 and week 8, for β-1,3-glucanase in all the treatments (Table 3). Significant increase (p<0.001) in glucanase activity onG. boninense-RWB seedlings was detected in most of the treatments at one WAI. However, T6 and T2 recorded great differences between control and +G inoculated oil palm seedlings while T5, T8, and T10 recorded slight changes among the treatments. By week 8, glucanase activity of T6 was dramatically higher, about 5-fold higher than untreated seedlings (Figure 1), as a sign of activated glucanase enzyme activity. However, β-1,3-glucanases are induced not only by pathogen infection, but also by other factors. Except for T10, at 12 WAI, the glucanase activity of allG. boninense seedlings of T2, T5, T6, and T8 was decreased gradually compared to the other treatments. However, result of OD recorded at 1, 8, and 12 WAI for glucanase activity of theG. boninense-RWB seedlings was notably higher than the control.Table 3
Effect of N, P, and K nutrition and different enzyme activities of healthy andGanoderma infected oil palm seedlings.
Week after inoculation Ganoderma infected seedlings (UmL−1) Control (Um L−1) t value T2 T5 T6 T8 T10 Glucanase activity Effect of N, P2O5, and K2O nutrition on glucanase activity  1WAI 10.097 ± 0.96 5.74 ± 0.33 10.43 s ∗ 9.93 a 7.4 b 10.2 a 7.2 b 4.9 c 4WAI 2.397 ± 0.35 2.5 ± 0.47 6.74 s ∗ 3.3 a b 3.90 a 1.65 c 2.16 b c 1.25 c 8WAI 16.837 ± 1.5 10.44 ± 1.15 11.06 s ∗ 17.78 a 9.75 d 16.2 b 14.56 c 9.9 d 12WAI 3.197 ± 0.36 4.188 ± 0.69 8.66 s ∗ 3.48 b c 2.35 c 3.9 b 2.9 b c 5.87 a  Chitinase activity Effect of N, P2O5, and K2O nutrition on chitinase activity  1WAI 4.53 ±0.39 2.67 ± 0.46 11.54 s ∗ 3.18 4.07 4.27 3.93 2.57 4WAI 1.57±0.26 1.96 ± 0.33 5.89 s ∗ 1.50 1.22 2.23 1.73 2.16 8WAI 6.37± 0.76 4.36 ± 0.52 8.34 s ∗ 3.80 c 3.67 c 3.68 a 6.03 a b 5.65 b 12WAI 3.28± 0.63 2.28 ± 0.39 5.16 s ∗ 2.32 b 2.16 b 4.97 a 2.75 b 1.73 b  PAL activity Effect of N, P2O5, and K2O nutrition on PAL activity  1WAI 10.9 ±0.89 8.90 ± 0.64 13.87 s ∗ 6.67 c 8.85 b c 11.30 a b 10.25 a b 12.45 a 4WAI 12.08 ± 1.27 8.96 ± 0.77 11.58 s ∗ 7.75 c 7.50 c 16.15 a 9.65 b c 11.55 b 8WAI 8.86 ± 0.90 5.66 ±0.48 11.72 s ∗ 5.25 b 8.00 a b 8.35 a 7.15 a b 7.55 a b 12WAI 10.28 ± 1.33 4.4 ± 0.46 9.47 s ∗ 4.10 b 4.55 b 10.81 a 8.88 a 8.37 a  POX activity Effect of N, P2O5, and K2O nutrition and BSR on POX Activity  1WAI 20.65 ± 1.42 11.64 ± .91 12.82 s ∗ 15.55 b 11.80 c 15.75 b 19.26 a 18.35 a b 4WAI 13.52 ± 1.74 9.96 ± 1.51 6.60 s ∗ 4.20 c 13.90 a b 15.25 a 12.25 b 12.1 b 8WAI 6.86 ± 0.85 6.8 ±0.40 16.74 s ∗ 6.50 a 5.35 a 7.35 a 6.95 a 8.00 a 12WAI 12.86 ± 1.43 14.82 ± .94 15.74 s ∗ 11.50 c 15.35 a b 17.35 a 11.95 b c 13.05 b c Note. values are the means of three replicates;  s∗ indicates highly significant according to T-test at α′=0.05. WAI: week after inoculation; all the t-values are highly significant at 0.001%. Means with the same letter at a given parameter are not significantly different according to Duncan’s Multiple Range Test (DMRT) at α=0.05; values are the means of three replicates ± standard error. Treatments: T2 = N3+ BF + Ganoderma; T5 = K1 + BF + Ganoderma; T6 = K3 + BF + Ganoderma; T8 = N2P2K2 + BF + Ganoderma; T10 = control (compound fertilizer) + BF + Ganoderma.Figure 1
Effect of different levels of N, P, and K on glucanase activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Chitinase Activity. A highly significant difference was found between the chitinaseGanoderma treatments and control from week 1 to week 12 with t-value of 11.54, 5.89, 8.34, and 5.16 U mL−1 for week 1 to week 12, respectively (Table 3). Although the level of chitinase increased in theGanoderma infected seedlings during the study duration, it appeared that it was not sufficiently high, had not reached effective levels, or was too late to stop the fungal from growth.Significant differences were observed at 8 to 12 WAI with T6 and T8 recording markedly higher enzyme activities compared to other treatments. Relatively low levels of chitinase were detected initially at 4 WAI for all different treatments and only began to produce higher levels of the enzyme at week 8 in both T6 and T8 and at week 12 in T6 when they were subjected toGanoderma attack. Although, relatively high levels of chitinase were observed in most treatments at 1 WAIT, it does not seem to be vital for plants at the initial time period of treatment. The present results indicated that time of defense related response induction was at weeks 8 and 12 in T6 and at week 8 in almost all the treatments except T2 and T5. Treatments T6 and T8 at 8 WAI recorded the highest values of the chitinase activity. However, T2 recorded lower chitinase activity for +G inoculated seedlings treatments when compared to T6 and T8 (Figure 2). The low chitinase activity in T2 may be the cause of defense failure in the event ofGanodermainfection. All uninoculated seedlings from the uninoculated treatment, recorded lesser chitinase activity as compared toGanoderma-inoculated treatment, which revealed a positive relationship at p<0.05 between chitinase activity andG. boninense infected seedlings.Figure 2
Effect of different levels of N, P2O5, and K2O on chitinase activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Phenylalanine Ammonia-Lyase Activity. High significant difference was found between the PALGanoderma treatment and control from week 1 to week 12 with t-value of 13.87, 11.58, 11.72, and 9.47 for week 1 to week 12, respectively (Table 3). The PAL activity measured in the roots of infected palms revealed highly significant differences among treatments (p<0.0001). The highest activity was observed in the infected roots of T6, followed by T8 and T10 with no significant difference between them, while the lowest was detected in T2. This could indicate that at this stage more phenolics including lignin were synthesized in T6, T8, and T10 and are more tolerant compared to T2 (Figure 3).Figure 3
Effect of different levels of N, P2O5, and K2O on PAL activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Peroxidase Activity. High significant difference was found between theGanodermatreatment and control from week 1 to week 12 with t-value of 12.82, 6.60, 16.74, and 15.74, respectively. Higher POX was expressed in T8 at 1 WAI, but T6 showed higher POX activities from 8 WAI to 12 WAI. The lowest POX activity in the root was in sample T2 (Table 3). The results obtained have shown that T6 significantly gave high value for enzyme POX activity. However, at 1 WAI, T8 was the highest followed by T10, an indication of a fast and rapid induction of POX at the beginning of infection. However, at 4, 8, and 12 WAI, T6 gave the highest POX activity and was significantly higher in all infected root tissues. At 8 WAI, all POX activities were not significantly different among theGanoderma infected root tissues for POX enzymes. Glucanase, POX (weeks 1 and 4), and PAL activities were stimulated by theGanoderma infection within 1 week, recurring at week 8, and chitinase within week 8 after treatment application (Figure 4).Figure 4
Effect of different levels of N, P2O5, and K2O on POX activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Metabolite Compounds of Uninfected and Infected Oil Palm Seedlings. Several metabolite compounds were detected in uninfected and infected seedlings treated with different levels of N, P, and K fertilizer. However, only known compounds are listed here to study more details and find interaction between effect of pathogen and fertilizer (Table 4). There was a big difference observed in the number of released metabolite compounds between uninfected/infected samples with different N, P, and K treatments most probably due to alteration in antioxidant enzyme activities. For instance, forty compounds were obtained from uninfected sample T1 with basal fertilizer (BF) and low N fertilizer, while only three compounds were extracted from the same fertilized sample T1 infected withGanoderma. One and five compounds were observed, respectively, from uninfected and infected sample T2 with BF and high N fertilizer. One compound was obtained from uninfected sample T3 with basic fertilizer (BF) and low P fertilizer, while 18 compounds were extracted from the same fertilized sample T3 infected withGanoderma. One and 13 compounds were observed, respectively, from uninfected and infected sample T4 with BF and high P fertilizer. Two compounds were obtained from uninfected sample T5 with basic fertilizer (BF) and low K fertilizer, while nine compounds were extracted from the same fertilized sample T5 infected withGanoderma. Four and two compounds were observed, respectively, from uninfected and infected sample T6 with BF and high K fertilizer.Table 4
Mass spectrometry for each compound and its molecular structure observed in different N, P, K combination ofGanoderma inoculated (+) and uninoculated oil palm seedlings.
Sample Ganoderma RT m/z Mass Name Molecular formula T1 (−) 0.87 175.119 174.112 Arginine C6 H14 N4 O2 T1 (−) 0.92 104.107 103.1 2-Amino-3-methyl-1-butanol C5 H13 N O T1 (−) 1 293.066 254.101 Dyphylline C10 H14 N4 O4 T1 (−) 1.01 203.054 180.065 Theobromine C7 H8 N4 O2 T1 (−) 1.02 435.062 180.065 4-Hydroxylevamisole glucuronide C17 H20 N2 O7 S T1 (−) 1.13 220.134 180.065 Meperidinic acid C13 H17 N O2 T1 (−) 1.2 268.155 180.065 Metoprolol acid C14 H21 N O4 T1 (−) 1.24 206.119 180.065 Normeperidinic acid C12 H15 N O2 T1 (−) 1.25 282.171 180.065 Benzenepropanoic acid, 4-[2-hydroxy-3- C15 H23 N O4 [(1-methylethyl)amino]propoxy]-(9CI) ASL 8123 T1 (−) 1.93 417.18 180.065 S-adenosylmethionine C15 H23 N6 O5 S T1 (−) 1.96 362.209 180.065 Phe Pro Val C19 H27 N3 O4 T1 (−) 2.18 234.15 180.065 Normeperidine C14 H19 N O2 T1 (−) 2.23 409.185 180.065 Asn Met Gln C14 H25 N5 O6 S T1 (−) 2.31` 409.185 180.065 Asn Met Gln C14 H25 N5 O6 S T1 (−) 2.89 480.226 180.065 Gln Phe Trp C25 H29 N5 O5 T1 (−) 3.17 236.166 180.065 5,7,9,11,13-tetradecapentaenoic acid C14 H18 O2 T1 (-) 3.23 480.224 180.065 Gln Phe Trp C25 H29 N5 O5 T1 (−) 3.47 601.154 180.065 Rhoifolin C27 H30 O14 T1 (−) 4.71 393.19 180.065 Trp Gly Asn C17 H21 N5 O5 T1 (−) 4.89 541.263 180.065 17-Alpha, 21-dihydroxy-11,20-dioxo-5-beta-pregnan- C27 H40 O11 3-alpha-yl-beta-d-glucuronide T1 (−) 5.26 395.205 180.065 Tyr Gly Arg C17 H26 N6 O5 T1 (−) 9.75 246.243 180.065 4,8-Dimethyl-dodecanoic acid C14 H28 O2 T1 (−) 12.1 274.275 180.065 C16 sphinganine C16 H35 N O2 T1 (−) 12.1 274.275 180.065 C16 sphinganine C16 H35 N O2 T1 (−) 12.2 230.249 180.065 Myristaldehyde C14 H28 O T1 (−) 12.2 318.301 180.065 Phytosphingosine C18 H39 N O3 T1 (−) 12.2 288.291 180.065 C17 sphinganine C17 H37 N O2 T1 (−) 12.4 288.291 180.065 C17 sphinganine C17 H37 N O2 T1 (−) 12.6 272.259 180.065 2R-aminohexadecanoic acid C16 H33 N O2 T1 (−) 12.8 288.291 180.065 C17 Sphinganine C17 H37 N O2 T1 (−) 13.2 288.291 180.065 C17 Sphinganine C17 H37 N O2 T1 (−) 13.3 244.265 180.065 9-Pentadecen-1-ol C15 H30 O T1 (−) 15 272.296 180.065 14-Methyl-8-hexadecen-1-ol C17 H34 O T1 (−) 15.5 272.296 180.065 14-Methyl-8-hexadecen-1-ol C17 H34 O T1 (−) 16.2 599.412 180.065 Mytiloxanthin C40 H54 O4 T1 (−) 21.3 599.412 180.065 Mytiloxanthin C40 H54 O4 T1 (−) 21.5 585.433 180.065 Myxol C40 H56 O3 T1 (−) 21.5 601.427 180.065 Capsanthin 3,6-epoxide C40 H56 O4 T1 (−) 21.8 429.374 180.065 25-Hydroxy-26,27-dimethylvitamin D3/ C29 H48 O2  25-Hydroxy-26,27-dimethylcholecalciferol T1 (−) 22.1 167.01 180.065 6-Hydroxy-2-hexynoic acid C6 H8 O3 T1 (+) 8.79 213.145 180.065 meglumine C7 H17 N O5 T1 (+) 17.8 248.198 180.065 Leu Val C11 H22 N2 O3 T1 (+) 17.8 192.135 180.065 Lupinine C10 H19 N O T2 (−) 21.92 125.039 248.064 Asp Asp C8 H12 N2 O7 T2 (+) 1.06 300.148 299.14 Metoclopramide C14 H22 Cl N3 O2 T2 (+) 1.17 300.148 299.141 Metoclopramide C14 H22 Cl N3 O2 T2 (+) 2.28 409.187 408.18 Trp Phe Gly C22 H24 N4 O4 T2 (+) 5.24 395.208 356.246 Dopexamine C22 H32 N2 O2 T2 (+) 17.77 243.139 242.132 6E,8E,14E-hexadecatriene-10,12-diynoic acid C16 H18 O2 T3 (−) 22.09 102.971 64.0082 Ethyl chloride C2 H5 Cl T3 (+) 0.26 257.975 219.011 4-Amino-3-(5-chlorothien-2-YL) Butanoic acid C8 H10 Cl N O2 S T3 (+) 0.97 293.066 254.103 Dyphylline C10 H14 N4 O4 T3 (+) 0.98 219.028 180.065 Theobromine C7 H8 N4 O2 T3 (+) 1 277.0914 254.102 Dyphylline C10 H14 N4 O4 T3 (+) 1.01 203.055 180.065 Theobromine C7 H8 N4 O2 T3 (+) 1.03 435.061 396.097 4-Hydroxylevamisole glucuronide C17 H20 N2 O7 S T3 (+) 2 362.244 344.21 Oxyphencyclimine C20 H28 N2 O3 T3 (+) 2.27 409.187 408.18 Trp Phe Gly C22 H24 N4 O4 T3 (+) 2.9 480.227 479.219 Gln Phe Trp C25 H29 N5 O5 T3 (+) 3.24 480.226 479.219 Gln Phe Trp C25 H29 N5 O5 T3 (+) 3.63 264.161 246.127 Santonin C15 H18 O3 T3 (+) 5.26 395.207 377.174 Gln Lys Cys C14 H27 N5 O5 S T3 (+) 12.07 274.275 273.268 C16 Sphinganine C16 H35 N O2 T3 (+) 12.49 409.16 370.197 Lys His Ser C15 H26 N6 O5 T3 (+) 16 243.134 242.126 Hydroxyamobarbital C11 H18 N2 O4 T3 (+) 18.2 419.243 418.236 Met Arg Leu C17 H34 N6 O4 S T3 (+) 18.22 419.279 418.272 Simvastatin C25 H38 O5 T3 (+) 18.23 375.253 374.245 Digitoxigenin C23 H34 O4 T4 (−) 16.01 243.133 242.126 Hydroxyamobarbital C11 H18 N2 O4 T4 (+) 0.99 277.091 254.102 Dyphylline C10 H14 N4 O4 T4 (+) 1.01 138.058 120.025 Sulfolane C4 H8 O2 S T4 (+) 1.95 362.243 344.209 Oxyphencyclimine C20 H28 N2 O3 T4 (+) 2.25 409.186 408.178 Trp Phe Gly C22 H24 N4 O4 T4 (+) 2.86 480.226 479.218 Gln Phe Trp C25 H29 N5 O5 T4 (+) 3.2 480.226 479.219 Gln Phe Trp C25 H29 N5 O5 T4 (+) 3.44 601.156 578.166 Rhoifolin C27 H30 O14 T4 (+) 5.23 395.207 377.173 Gln Lys Cys C14 H27 N5 O5 S T4 (+) 12.06 274.275 273.268 C16 sphinganine C16 H35 N O2 T4 (+) 12.18 318.3 317.292 Phytosphingosine C18 H39 N O3 T4 (+) 12.48 409.163 386.174 Lys Cys His C15 H26 N6 O4 S T4 (+) 18.19 419.279 418.271 Simvastatin C25 H38 O5 T4 (+) 21.49 585.433 584.426 Myxol C40 H56 O3 T5 (-) 22.61 179.988 141.025 5-Acetyl-4-methylthiazole C6 H7 N O S T5 (−) 22.93 ― 141.025 5-Acetyl-4-methylthiazole C6 H7 N O S T5 (+) 1.35 167.01 128.047 6-Hydroxy-2-hexynoic acid C6 H8 O3 T5 (+) 17.76 248.196 230.162 Leu Val C11 H22 N2 O3 T5 (+) 17.77 192.136 169.147 Lupinine C10 H19 N O T5 (+) 21.45 411.2875 410.2796 24,25-epoxy-1 alpha-hydroxy-22,22 C27 H38 O3 T5 (+) 21.46 343.2247 320.2348 (+/−)14,15-EpETrE C20 H32 O3 T5 (+) 21.48 479.3497 456.3595 Testosterone undecanoate C30 H48 O3 T5 (+) 21.65 343.2245 320.2353 (+/−)14,15-EpETrE C20 H32 O3 T5 (+) 21.66 411.2861 388.297 1α,25-Dihydroxy-23,24-dinorvitamin D3 C25 H40 O3 T5 (+) 21.94 125.0381 248.0616 Dapsone C12 H12 N2 O2 S T6 (−) 17.77 248.1973 230.1635 Leu Val C11 H22 N2 O3 T6 (−) 17.78 243.1331 242.1258 Hydroxyamobarbital C11 H18 N2 O4 T6 (−) 21.56 839.4909 821.4566 Tacrolimus metabolite M-VIII C43 H67 N O14 T6 (−) 21.65 498.9004 497.8933 Perflubron C8 Br F17 T6 (+) 21.66 498.9008 497.8937 Perflubron C8 Br F17 T6 (+) 21.95 125.0378 248.0611 Dapsone C12 H12 N2 O2 S T7 (−) 1.2 179.9883 141.0251 5-Acetyl-4-methylthiazole C6 H7 N O S T7 (−) 17.77 248.1964 230.1627 Leu Val C11 H22 N2 O3 T7 (−) 21.65 498.9009 497.8938 Perflubron C8 Br F17 T7 (+) 17.51 301.1413 278.152 Phthalic acid mono-2-ethylhexyl Ester C16 H22 O4 T7 (+) 17.77 192.1362 169.1473 Lupinine C10 H19 N O T7 (+) 17.77 248.2013 247.194 Lycopodine C16 H25 N O T7 (+) 20.14 282.2793 281.2721 Oleoyl amine C18 H35 N O T7 (+) 21.44 411.2876 388.298 1α-hydroxy-21-nor-20-oxavitamin D3 C25 H40 O3 T7 (+) 21.45 343.2242 320.235 (+/−)14,15-EpETrE C20 H32 O3 T7 (+) 21.62 343.224 320.2347 (+/−)14,15-EpETrE C20 H32 O3 T7 (+) 21.64 411.2862 388.297 1α,25-Dihydroxy-23,24-dinorvitamin D3 C25 H40 O3 T7 (+) 22.15 167.011 128.0479 6-Hydroxy-2-hexynoic acid C6 H8 O3 T7 (+) 22.74 179.9885 141.0251 5-Acetyl-4-methylthiazole C6 H7 N O S T8 (−) 16.01 243.1334 242.1261 Hydroxyamobarbital C11 H18 N2 O4 T8 (−) 17.77 243.1333 242.126 Hydroxyamobarbital C11 H18 N2 O4 T8 (+) 17.76 248.1967 230.1631 Leu Val C11 H22 N2 O3 T8 (+) 17.77 192.1347 174.1013 Val Gly C7 H14 N2 O3 T8 (+) 21.61 395.2916 356.3286 (+)-3-hydroxy behenic C22 H44 O3 T8 (+) 21.67 909.5463 886.5559 Glc-GP(18:0/20:4(5Z,8Z,11Z,14Z)) C47 H83 O13 P T8 (+) 21.93 125.0388 248.0632 Dapsone C12 H12 N2 O2 S T9 (−) 0.92 104.1065 103.0992 2-Amino-3-methyl-1-butanol C5 H13 N O T9 (−) 0.97 176.0109 137.0479 p-aminobenzoic acid C7 H7 N O2 T9 (−) 0.98 293.0644 254.1012 Dyphylline C10 H14 N4 O4 T9 (−) 0.99 160.0375 137.0483 3-Pyridylacetic acid C7 H7 N O2 T9 (−) 1.02 203.0545 180.0654 Theobromine C7 H8 N4 O2 T9 (−) 1.03 435.0635 396.0998 4-Hydroxylevamisole glucuronide C17 H20 N2 O7 S T9 (−) 1.05 265.0252 226.0621 Anthralin C14 H10 O3 T9 (−) 1.13 230.0935 191.1302 Diethyltoluamide C12 H17 N O T9 (−) 1.19 287.1111 264.122 1-(5-Ketohexyl)-3-methylxanthine C12 H16 N4 O3 T9 (−) 1.21 268.1553 267.1479 Metoprolol acid C14 H21 N O4 T9 (−) 1.26 282.1712 281.1639 Benzenepropanoic acid C15 H23 N O4 T9 (−) 1.98 362.2078 361.2005 Phe Val Pro C19 H27 N3 O4 T9 (−) 2.03 362.242 344.2085 Oxyphencyclimine C20 H28 N2 O3 T9 (−) 2.28 411.2 388.2101 Methyl 10,12,13,15-bisepidioxy- C19 H32 O8  16-Hydroperoxy-8E-octadecenoate T9 (−) 2.28 409.185 391.1507 Asn Met Gln C14 H25 N5 O6 S T9 (−) 2.45 621.217 620.2091 Diethylstilbestrol diglucuronide C30 H36 O14 T9 (−) 2.89 480.225 479.2173 Gln Phe Trp C25 H29 N5 O5 T9 (−) 3.23 480.225 479.2173 Gln Phe Trp C25 H29 N5 O5 T9 (−) 3.5 601.155 578.1652 Rhoifolin C27 H30 O14 T9 (−) 5.2 395.174 377.1404 Ofloxacin-N-oxide C18 H20 F N3 O5 T9 (−) 5.26 395.206 377.1723 Gln Lys Cys C14 H27 N5 O5 S T9 (−) 5.69 205.062 182.0728 9-Hydroxyfluorene C13 H10 O T9 (−) 12.06 274.275 273.2672 C16 sphinganine C16 H35 N O2 T9 (−) 12.19 318.3 317.2924 Phytosphingosine C18 H39 N O3 T9 (−) 12.49 409.13 408.1232 Ichthynone C23 H20 O7 T9 (−) 16.17 599.411 598.4032 Mytiloxanthin C40 H54 O4 T9 (−) 20.08 797.52 774.5304 1,2 di-(9Z,12Z,15Z-octadecatrienoyl)-3- C45 H74 O10  O-beta-D-galactosyl-sn-glycerol T9 (−) 21.5 585.433 584.425 Myxol C40 H56 O3 T9 (−) 21.5 601.427 600.4195 Capsanthin 3,6-epoxide C40 H56 O4 T9 (−) 21.69 646.254 645.2443 Acarbose (Glucobay) C25 H43 N O18 T9 (−) 21.49 607.29 568.3266 (3a,5b,7a)-23-carboxy-7-hydroxy-24-norcholan-3-yl, C30 H48 O10  b-D-glucopyranosiduronic acid T10 (−) 0.97 293.065 254.1022 Dyphylline C10 H14 N4 O4 T10 (−) 1.03 265.025 226.0623 Anthralin C14 H10 O3 T10 (−) 1.17 287.112 264.1228 1-(5-Ketohexyl)-3-methylxanthine C12 H16 N4 O3 T10 (−) 2 362.243 344.2091 Oxyphencyclimine C20 H28 N2 O3 T10 (−) 2.29 411.2 410.1925 Flumethasone C22 H28 F2 O5 T10 (−) 2.29 409.185 408.1781 Trp Phe Gly C22 H24 N4 O4 T10 (−) 2.45 621.218 620.2108 Diethylstilbestrol diglucuronide C30 H36 O14 T10 (−) 2.92 480.225 479.2179 Gln Phe Trp C25 H29 N5 O5 T10 (−) 3.25 480.225 479.2187 Gln Phe Trp C25 H29 N5 O5 T10 (−) 3.64 264.159 246.1251 Santonin C15 H18 O3 T10 (−) 5.23 395.207 377.1738 Gln Cys Lys C14 H27 N5 O5 S T10 (−) 12.19 318.302 317.2943 Phytosphingosine C18 H39 N O3 T10 (−) 12.72 288.291 287.2833 C17 sphinganine C17 H37 N O2 T10 (−) 13.14 288.29 287.2824 C17 sphinganine C17 H37 N O2 T10 (−) 14.93 316.321 298.2873 11-Methyl-octadecanoic acid C19 H38 O2 T10 (−) 14.97 272.295 254.2608 14-Methyl-8-hexadecen-1-ol C17 H34 O T10 (−) 15.51 304.286 286.2515 17-Hydroxy-heptadecanoic acid C17 H34 O3 T10 (−) 16.17 599.409 598.4011 Mytiloxanthin C40 H54 O4 T10 (−) 17.76 248.197 230.1627 Leu Val C11 H22 N2 O3 T10 (−) 21.31 597.238 574.2481 p-hydroxy atorvastatin C33 H35 F N2 O6 T10 (−) 21.48 585.433 584.4252 Myxol C40 H56 O3 T10 (−) 21.63 607.287 568.3253 (3a,5b,7a)-23-carboxy-7-hydroxy-24-norcholan-3-yl, C30 H48 O10 b-D-Glucopyranosiduronic acid T10 (−) 21.69 469.368 468.36 1α,25-Dihydroxy-26,27-dimethyl-22,22,23,23- C31 H48 O3 tetradehydro-24a,24b-dihomovitamin D3 T10 (−) 21.76 469.368 468.357 1α,25-Dihydroxy-26,27-dimethyl-22,22,23,23 C31 H48 O3 -tetradehydro-24a,24b-dihomovitamin D3 T10 (−) 21.77 429.375 428.367 25-hydroxy-26,27-dimethylvitamin D3/ C29 H48 O2 25-hydroxy-26,27-dimethylcholecalciferol T10 (−) 22.48 607.287 606.277 Trandolapril glucuronide C30 H42 N2 O11 T10 (+) 1.04 265.027 226.064 Anthralin C14 H10 O3 T10 (+) 1.07 300.148 299.141 Metoclopramide C14 H22 Cl N3 O2 T10 (+) 1.17 300.147 299.14 Metoclopramide C14 H22 Cl N3 O2 T10 (+) 1.98 362.245 344.211 Oxyphencyclimine C20 H28 N2 O3 T10 (+) 2.27 409.187 408.18 Trp Phe Gly C22 H24 N4 O4 T10 (+) 5.22 395.208 377.175 Gln Cys Lys C14 H27 N5 O5 S T10 (+) 11.98 274.254 273.246 10Z,13Z,16Z-nonadecatrienenitrile C19 H31 N T10 (+) 12.15 230.248 212.214 Myristaldehyde C14 H28 O T10 (+) 12.17 318.3 317.293 Phytosphingosine C18 H39 N O3 T10 (+) 17.76 243.139 242.131 6E,8E,14E-Hexadecatriene-10,12-diynoic acid C16 H18 O2 T10 (+) 17.87 243.138 242.131 6E,8E,14E-Hexadecatriene-10,12-diynoic acid C16 H18 O2 T10 (+) 18.22 419.281 418.273 Simvastatin C25 H38 O5 T10 (+) 21.29 645.275 606.312 1,25-Dihydroxy-20S-21-(3-hydroxy-3-methylbutyl) C32 H44 F6 O4 -23-yne-26,27-hexafluorovitamin D3 T10 (+) 21.7 646.258 645.249 Acarbose (Glucobay) C25 H43 N O18Three compounds were obtained from uninfected sample T7 with basic fertilizer (BF) treated with N1P1 K1 (N low, P low, K low), while ten compounds were extracted from the same fertilized sample T7 infected withGanoderma. Two compounds were obtained from uninfected sample T8 with basal fertilizer (BF) treated with N2P2 K2 (N optimum, P optimum, K optimum), while five compounds were extracted from the same fertilized sample T8 infected withGanoderma. Thirty-one compounds were obtained from uninfected sample T9 with basic fertilizer (BF) treated with N3P3 K3 (N high, P high, K high), whereas unknown compounds were identified from the same fertilized sample T9 infected withGanoderma. Finally, twenty-six compounds were obtained from uninfected sample T10 with basic fertilizer (BF) and 14 compounds were extracted from the same fertilized sample T10 infected withGanoderma.The number of detected known metabolite compounds differs among infected and uninfected samples. For instance, the number of known compounds extracted from uninfected sample T1, T6, T9, and T10 was higher than that from infected ones, while the number of compounds extracted from infected samples T2, T3, T4, T5, T7, T8 was higher than that from uninfected ones.The dendrogram and fan plot produced by hierarchal cluster analysis (HCA) of the different fertilized oil palm seedlings, based on a common molecular mass deduced from the recordedm/z value and similar retention time (RT) and peak shape (Figures 5 and 6), provided a more detailed view on the relationships between treatments. The HCA distinguished two major clusters for treatments denoted as A and B. Cluster A consist of [T2, T6, T8, T10] with the higher enzyme activities among other treatments. Cluster B contained two subgroups: one consisting of [T1, T3, T9] and a second one consisting of [T4, T5, T7]. The HCA of the set of 190 metabolite compounds revealed the presence of several metabolite groups, arbitrarily denoted with A to D, characterized by their particular expression pattern across the ten treatments (Figure 6). Although there is no direct evidence to show the extent of plant enzymes contributing to the resistance at the infection site because they are interacted by various factors and encoded by multigene family, a previous considerable research provided corroborative evidence that plant chitinase and β-1,3-glucanases in plant self-defense mechanism (Figure 7) illustrate how optimum NPK nutrients might either mutate the production of glucanase, chitinase, POX, and PAL activities or be involved in secondary metabolite profiling for basal resistance in oil palm.Figure 5
Heatmap of 190 metabolite compounds in 10 types of NPK treatment oil palm seedling. A color matrix represents the mean values of the metabolite compounds in two biological replicates of oil palm. Characterization of the underlying metabolites is presented in Table4.Figure 6
Fan plot showing all metabolite compounds of oil palm in both forms of control (−G) and inoculated withGanoderma (+G), under different NPK treatments. Four different groups A, B, C, and D represent them/z mean values of the metabolite compounds including flavonoids, phenylpropanoids, acyclic diterpenoids, and fatty acid derivative.Figure 7
Schematic model of oil palm,Ganoderma boninense, fertilizer regime interaction focusing on the role of plant lytic enzymes (Glycoforum: http://www.glycoforum.gr.jp). This schematic model indicates the contribution of PR proteins in response to macronutrients nutrition in oil palm withGanoderma infection. Plants produce β-1,3-glucanases, chitinase, POX, and PAL during the process of pathogenesis for self- defense, and pathogens struggle against them. Application of NPK nutrients, especially K fertilizing, might either act as elicitors triggering the production of glucanase, chitinase, POX, and PAL activities or be involved in secondary metabolite profiling associated with induced resistance.


## 3.1. Biochemical Analysis Assays

### 3.1.1. Glucanase Activity

Glucanase enzyme activity showed high significant difference (p<0.05) between theGanoderma infected seedlings and control from week 1 to week 12 with t-value of 10.43, 6.74, 11.06, and 8.66 U mL−1 at 1 to 12 WAI, respectively (Table 3). The relatively low content of the uninfected seedlings is a reflection of the relatively low resistance towardsG. boninense in the nutrient level studied. The time of defense related response induction was rapid, within week 1 and week 8, for β-1,3-glucanase in all the treatments (Table 3). Significant increase (p<0.001) in glucanase activity onG. boninense-RWB seedlings was detected in most of the treatments at one WAI. However, T6 and T2 recorded great differences between control and +G inoculated oil palm seedlings while T5, T8, and T10 recorded slight changes among the treatments. By week 8, glucanase activity of T6 was dramatically higher, about 5-fold higher than untreated seedlings (Figure 1), as a sign of activated glucanase enzyme activity. However, β-1,3-glucanases are induced not only by pathogen infection, but also by other factors. Except for T10, at 12 WAI, the glucanase activity of allG. boninense seedlings of T2, T5, T6, and T8 was decreased gradually compared to the other treatments. However, result of OD recorded at 1, 8, and 12 WAI for glucanase activity of theG. boninense-RWB seedlings was notably higher than the control.Table 3
Effect of N, P, and K nutrition and different enzyme activities of healthy andGanoderma infected oil palm seedlings.
Week after inoculation Ganoderma infected seedlings (UmL−1) Control (Um L−1) t value T2 T5 T6 T8 T10 Glucanase activity Effect of N, P2O5, and K2O nutrition on glucanase activity  1WAI 10.097 ± 0.96 5.74 ± 0.33 10.43 s ∗ 9.93 a 7.4 b 10.2 a 7.2 b 4.9 c 4WAI 2.397 ± 0.35 2.5 ± 0.47 6.74 s ∗ 3.3 a b 3.90 a 1.65 c 2.16 b c 1.25 c 8WAI 16.837 ± 1.5 10.44 ± 1.15 11.06 s ∗ 17.78 a 9.75 d 16.2 b 14.56 c 9.9 d 12WAI 3.197 ± 0.36 4.188 ± 0.69 8.66 s ∗ 3.48 b c 2.35 c 3.9 b 2.9 b c 5.87 a  Chitinase activity Effect of N, P2O5, and K2O nutrition on chitinase activity  1WAI 4.53 ±0.39 2.67 ± 0.46 11.54 s ∗ 3.18 4.07 4.27 3.93 2.57 4WAI 1.57±0.26 1.96 ± 0.33 5.89 s ∗ 1.50 1.22 2.23 1.73 2.16 8WAI 6.37± 0.76 4.36 ± 0.52 8.34 s ∗ 3.80 c 3.67 c 3.68 a 6.03 a b 5.65 b 12WAI 3.28± 0.63 2.28 ± 0.39 5.16 s ∗ 2.32 b 2.16 b 4.97 a 2.75 b 1.73 b  PAL activity Effect of N, P2O5, and K2O nutrition on PAL activity  1WAI 10.9 ±0.89 8.90 ± 0.64 13.87 s ∗ 6.67 c 8.85 b c 11.30 a b 10.25 a b 12.45 a 4WAI 12.08 ± 1.27 8.96 ± 0.77 11.58 s ∗ 7.75 c 7.50 c 16.15 a 9.65 b c 11.55 b 8WAI 8.86 ± 0.90 5.66 ±0.48 11.72 s ∗ 5.25 b 8.00 a b 8.35 a 7.15 a b 7.55 a b 12WAI 10.28 ± 1.33 4.4 ± 0.46 9.47 s ∗ 4.10 b 4.55 b 10.81 a 8.88 a 8.37 a  POX activity Effect of N, P2O5, and K2O nutrition and BSR on POX Activity  1WAI 20.65 ± 1.42 11.64 ± .91 12.82 s ∗ 15.55 b 11.80 c 15.75 b 19.26 a 18.35 a b 4WAI 13.52 ± 1.74 9.96 ± 1.51 6.60 s ∗ 4.20 c 13.90 a b 15.25 a 12.25 b 12.1 b 8WAI 6.86 ± 0.85 6.8 ±0.40 16.74 s ∗ 6.50 a 5.35 a 7.35 a 6.95 a 8.00 a 12WAI 12.86 ± 1.43 14.82 ± .94 15.74 s ∗ 11.50 c 15.35 a b 17.35 a 11.95 b c 13.05 b c Note. values are the means of three replicates;  s∗ indicates highly significant according to T-test at α′=0.05. WAI: week after inoculation; all the t-values are highly significant at 0.001%. Means with the same letter at a given parameter are not significantly different according to Duncan’s Multiple Range Test (DMRT) at α=0.05; values are the means of three replicates ± standard error. Treatments: T2 = N3+ BF + Ganoderma; T5 = K1 + BF + Ganoderma; T6 = K3 + BF + Ganoderma; T8 = N2P2K2 + BF + Ganoderma; T10 = control (compound fertilizer) + BF + Ganoderma.Figure 1
Effect of different levels of N, P, and K on glucanase activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Chitinase Activity. A highly significant difference was found between the chitinaseGanoderma treatments and control from week 1 to week 12 with t-value of 11.54, 5.89, 8.34, and 5.16 U mL−1 for week 1 to week 12, respectively (Table 3). Although the level of chitinase increased in theGanoderma infected seedlings during the study duration, it appeared that it was not sufficiently high, had not reached effective levels, or was too late to stop the fungal from growth.Significant differences were observed at 8 to 12 WAI with T6 and T8 recording markedly higher enzyme activities compared to other treatments. Relatively low levels of chitinase were detected initially at 4 WAI for all different treatments and only began to produce higher levels of the enzyme at week 8 in both T6 and T8 and at week 12 in T6 when they were subjected toGanoderma attack. Although, relatively high levels of chitinase were observed in most treatments at 1 WAIT, it does not seem to be vital for plants at the initial time period of treatment. The present results indicated that time of defense related response induction was at weeks 8 and 12 in T6 and at week 8 in almost all the treatments except T2 and T5. Treatments T6 and T8 at 8 WAI recorded the highest values of the chitinase activity. However, T2 recorded lower chitinase activity for +G inoculated seedlings treatments when compared to T6 and T8 (Figure 2). The low chitinase activity in T2 may be the cause of defense failure in the event ofGanodermainfection. All uninoculated seedlings from the uninoculated treatment, recorded lesser chitinase activity as compared toGanoderma-inoculated treatment, which revealed a positive relationship at p<0.05 between chitinase activity andG. boninense infected seedlings.Figure 2
Effect of different levels of N, P2O5, and K2O on chitinase activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Phenylalanine Ammonia-Lyase Activity. High significant difference was found between the PALGanoderma treatment and control from week 1 to week 12 with t-value of 13.87, 11.58, 11.72, and 9.47 for week 1 to week 12, respectively (Table 3). The PAL activity measured in the roots of infected palms revealed highly significant differences among treatments (p<0.0001). The highest activity was observed in the infected roots of T6, followed by T8 and T10 with no significant difference between them, while the lowest was detected in T2. This could indicate that at this stage more phenolics including lignin were synthesized in T6, T8, and T10 and are more tolerant compared to T2 (Figure 3).Figure 3
Effect of different levels of N, P2O5, and K2O on PAL activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Peroxidase Activity. High significant difference was found between theGanodermatreatment and control from week 1 to week 12 with t-value of 12.82, 6.60, 16.74, and 15.74, respectively. Higher POX was expressed in T8 at 1 WAI, but T6 showed higher POX activities from 8 WAI to 12 WAI. The lowest POX activity in the root was in sample T2 (Table 3). The results obtained have shown that T6 significantly gave high value for enzyme POX activity. However, at 1 WAI, T8 was the highest followed by T10, an indication of a fast and rapid induction of POX at the beginning of infection. However, at 4, 8, and 12 WAI, T6 gave the highest POX activity and was significantly higher in all infected root tissues. At 8 WAI, all POX activities were not significantly different among theGanoderma infected root tissues for POX enzymes. Glucanase, POX (weeks 1 and 4), and PAL activities were stimulated by theGanoderma infection within 1 week, recurring at week 8, and chitinase within week 8 after treatment application (Figure 4).Figure 4
Effect of different levels of N, P2O5, and K2O on POX activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Metabolite Compounds of Uninfected and Infected Oil Palm Seedlings. Several metabolite compounds were detected in uninfected and infected seedlings treated with different levels of N, P, and K fertilizer. However, only known compounds are listed here to study more details and find interaction between effect of pathogen and fertilizer (Table 4). There was a big difference observed in the number of released metabolite compounds between uninfected/infected samples with different N, P, and K treatments most probably due to alteration in antioxidant enzyme activities. For instance, forty compounds were obtained from uninfected sample T1 with basal fertilizer (BF) and low N fertilizer, while only three compounds were extracted from the same fertilized sample T1 infected withGanoderma. One and five compounds were observed, respectively, from uninfected and infected sample T2 with BF and high N fertilizer. One compound was obtained from uninfected sample T3 with basic fertilizer (BF) and low P fertilizer, while 18 compounds were extracted from the same fertilized sample T3 infected withGanoderma. One and 13 compounds were observed, respectively, from uninfected and infected sample T4 with BF and high P fertilizer. Two compounds were obtained from uninfected sample T5 with basic fertilizer (BF) and low K fertilizer, while nine compounds were extracted from the same fertilized sample T5 infected withGanoderma. Four and two compounds were observed, respectively, from uninfected and infected sample T6 with BF and high K fertilizer.Table 4
Mass spectrometry for each compound and its molecular structure observed in different N, P, K combination ofGanoderma inoculated (+) and uninoculated oil palm seedlings.
Sample Ganoderma RT m/z Mass Name Molecular formula T1 (−) 0.87 175.119 174.112 Arginine C6 H14 N4 O2 T1 (−) 0.92 104.107 103.1 2-Amino-3-methyl-1-butanol C5 H13 N O T1 (−) 1 293.066 254.101 Dyphylline C10 H14 N4 O4 T1 (−) 1.01 203.054 180.065 Theobromine C7 H8 N4 O2 T1 (−) 1.02 435.062 180.065 4-Hydroxylevamisole glucuronide C17 H20 N2 O7 S T1 (−) 1.13 220.134 180.065 Meperidinic acid C13 H17 N O2 T1 (−) 1.2 268.155 180.065 Metoprolol acid C14 H21 N O4 T1 (−) 1.24 206.119 180.065 Normeperidinic acid C12 H15 N O2 T1 (−) 1.25 282.171 180.065 Benzenepropanoic acid, 4-[2-hydroxy-3- C15 H23 N O4 [(1-methylethyl)amino]propoxy]-(9CI) ASL 8123 T1 (−) 1.93 417.18 180.065 S-adenosylmethionine C15 H23 N6 O5 S T1 (−) 1.96 362.209 180.065 Phe Pro Val C19 H27 N3 O4 T1 (−) 2.18 234.15 180.065 Normeperidine C14 H19 N O2 T1 (−) 2.23 409.185 180.065 Asn Met Gln C14 H25 N5 O6 S T1 (−) 2.31` 409.185 180.065 Asn Met Gln C14 H25 N5 O6 S T1 (−) 2.89 480.226 180.065 Gln Phe Trp C25 H29 N5 O5 T1 (−) 3.17 236.166 180.065 5,7,9,11,13-tetradecapentaenoic acid C14 H18 O2 T1 (-) 3.23 480.224 180.065 Gln Phe Trp C25 H29 N5 O5 T1 (−) 3.47 601.154 180.065 Rhoifolin C27 H30 O14 T1 (−) 4.71 393.19 180.065 Trp Gly Asn C17 H21 N5 O5 T1 (−) 4.89 541.263 180.065 17-Alpha, 21-dihydroxy-11,20-dioxo-5-beta-pregnan- C27 H40 O11 3-alpha-yl-beta-d-glucuronide T1 (−) 5.26 395.205 180.065 Tyr Gly Arg C17 H26 N6 O5 T1 (−) 9.75 246.243 180.065 4,8-Dimethyl-dodecanoic acid C14 H28 O2 T1 (−) 12.1 274.275 180.065 C16 sphinganine C16 H35 N O2 T1 (−) 12.1 274.275 180.065 C16 sphinganine C16 H35 N O2 T1 (−) 12.2 230.249 180.065 Myristaldehyde C14 H28 O T1 (−) 12.2 318.301 180.065 Phytosphingosine C18 H39 N O3 T1 (−) 12.2 288.291 180.065 C17 sphinganine C17 H37 N O2 T1 (−) 12.4 288.291 180.065 C17 sphinganine C17 H37 N O2 T1 (−) 12.6 272.259 180.065 2R-aminohexadecanoic acid C16 H33 N O2 T1 (−) 12.8 288.291 180.065 C17 Sphinganine C17 H37 N O2 T1 (−) 13.2 288.291 180.065 C17 Sphinganine C17 H37 N O2 T1 (−) 13.3 244.265 180.065 9-Pentadecen-1-ol C15 H30 O T1 (−) 15 272.296 180.065 14-Methyl-8-hexadecen-1-ol C17 H34 O T1 (−) 15.5 272.296 180.065 14-Methyl-8-hexadecen-1-ol C17 H34 O T1 (−) 16.2 599.412 180.065 Mytiloxanthin C40 H54 O4 T1 (−) 21.3 599.412 180.065 Mytiloxanthin C40 H54 O4 T1 (−) 21.5 585.433 180.065 Myxol C40 H56 O3 T1 (−) 21.5 601.427 180.065 Capsanthin 3,6-epoxide C40 H56 O4 T1 (−) 21.8 429.374 180.065 25-Hydroxy-26,27-dimethylvitamin D3/ C29 H48 O2  25-Hydroxy-26,27-dimethylcholecalciferol T1 (−) 22.1 167.01 180.065 6-Hydroxy-2-hexynoic acid C6 H8 O3 T1 (+) 8.79 213.145 180.065 meglumine C7 H17 N O5 T1 (+) 17.8 248.198 180.065 Leu Val C11 H22 N2 O3 T1 (+) 17.8 192.135 180.065 Lupinine C10 H19 N O T2 (−) 21.92 125.039 248.064 Asp Asp C8 H12 N2 O7 T2 (+) 1.06 300.148 299.14 Metoclopramide C14 H22 Cl N3 O2 T2 (+) 1.17 300.148 299.141 Metoclopramide C14 H22 Cl N3 O2 T2 (+) 2.28 409.187 408.18 Trp Phe Gly C22 H24 N4 O4 T2 (+) 5.24 395.208 356.246 Dopexamine C22 H32 N2 O2 T2 (+) 17.77 243.139 242.132 6E,8E,14E-hexadecatriene-10,12-diynoic acid C16 H18 O2 T3 (−) 22.09 102.971 64.0082 Ethyl chloride C2 H5 Cl T3 (+) 0.26 257.975 219.011 4-Amino-3-(5-chlorothien-2-YL) Butanoic acid C8 H10 Cl N O2 S T3 (+) 0.97 293.066 254.103 Dyphylline C10 H14 N4 O4 T3 (+) 0.98 219.028 180.065 Theobromine C7 H8 N4 O2 T3 (+) 1 277.0914 254.102 Dyphylline C10 H14 N4 O4 T3 (+) 1.01 203.055 180.065 Theobromine C7 H8 N4 O2 T3 (+) 1.03 435.061 396.097 4-Hydroxylevamisole glucuronide C17 H20 N2 O7 S T3 (+) 2 362.244 344.21 Oxyphencyclimine C20 H28 N2 O3 T3 (+) 2.27 409.187 408.18 Trp Phe Gly C22 H24 N4 O4 T3 (+) 2.9 480.227 479.219 Gln Phe Trp C25 H29 N5 O5 T3 (+) 3.24 480.226 479.219 Gln Phe Trp C25 H29 N5 O5 T3 (+) 3.63 264.161 246.127 Santonin C15 H18 O3 T3 (+) 5.26 395.207 377.174 Gln Lys Cys C14 H27 N5 O5 S T3 (+) 12.07 274.275 273.268 C16 Sphinganine C16 H35 N O2 T3 (+) 12.49 409.16 370.197 Lys His Ser C15 H26 N6 O5 T3 (+) 16 243.134 242.126 Hydroxyamobarbital C11 H18 N2 O4 T3 (+) 18.2 419.243 418.236 Met Arg Leu C17 H34 N6 O4 S T3 (+) 18.22 419.279 418.272 Simvastatin C25 H38 O5 T3 (+) 18.23 375.253 374.245 Digitoxigenin C23 H34 O4 T4 (−) 16.01 243.133 242.126 Hydroxyamobarbital C11 H18 N2 O4 T4 (+) 0.99 277.091 254.102 Dyphylline C10 H14 N4 O4 T4 (+) 1.01 138.058 120.025 Sulfolane C4 H8 O2 S T4 (+) 1.95 362.243 344.209 Oxyphencyclimine C20 H28 N2 O3 T4 (+) 2.25 409.186 408.178 Trp Phe Gly C22 H24 N4 O4 T4 (+) 2.86 480.226 479.218 Gln Phe Trp C25 H29 N5 O5 T4 (+) 3.2 480.226 479.219 Gln Phe Trp C25 H29 N5 O5 T4 (+) 3.44 601.156 578.166 Rhoifolin C27 H30 O14 T4 (+) 5.23 395.207 377.173 Gln Lys Cys C14 H27 N5 O5 S T4 (+) 12.06 274.275 273.268 C16 sphinganine C16 H35 N O2 T4 (+) 12.18 318.3 317.292 Phytosphingosine C18 H39 N O3 T4 (+) 12.48 409.163 386.174 Lys Cys His C15 H26 N6 O4 S T4 (+) 18.19 419.279 418.271 Simvastatin C25 H38 O5 T4 (+) 21.49 585.433 584.426 Myxol C40 H56 O3 T5 (-) 22.61 179.988 141.025 5-Acetyl-4-methylthiazole C6 H7 N O S T5 (−) 22.93 ― 141.025 5-Acetyl-4-methylthiazole C6 H7 N O S T5 (+) 1.35 167.01 128.047 6-Hydroxy-2-hexynoic acid C6 H8 O3 T5 (+) 17.76 248.196 230.162 Leu Val C11 H22 N2 O3 T5 (+) 17.77 192.136 169.147 Lupinine C10 H19 N O T5 (+) 21.45 411.2875 410.2796 24,25-epoxy-1 alpha-hydroxy-22,22 C27 H38 O3 T5 (+) 21.46 343.2247 320.2348 (+/−)14,15-EpETrE C20 H32 O3 T5 (+) 21.48 479.3497 456.3595 Testosterone undecanoate C30 H48 O3 T5 (+) 21.65 343.2245 320.2353 (+/−)14,15-EpETrE C20 H32 O3 T5 (+) 21.66 411.2861 388.297 1α,25-Dihydroxy-23,24-dinorvitamin D3 C25 H40 O3 T5 (+) 21.94 125.0381 248.0616 Dapsone C12 H12 N2 O2 S T6 (−) 17.77 248.1973 230.1635 Leu Val C11 H22 N2 O3 T6 (−) 17.78 243.1331 242.1258 Hydroxyamobarbital C11 H18 N2 O4 T6 (−) 21.56 839.4909 821.4566 Tacrolimus metabolite M-VIII C43 H67 N O14 T6 (−) 21.65 498.9004 497.8933 Perflubron C8 Br F17 T6 (+) 21.66 498.9008 497.8937 Perflubron C8 Br F17 T6 (+) 21.95 125.0378 248.0611 Dapsone C12 H12 N2 O2 S T7 (−) 1.2 179.9883 141.0251 5-Acetyl-4-methylthiazole C6 H7 N O S T7 (−) 17.77 248.1964 230.1627 Leu Val C11 H22 N2 O3 T7 (−) 21.65 498.9009 497.8938 Perflubron C8 Br F17 T7 (+) 17.51 301.1413 278.152 Phthalic acid mono-2-ethylhexyl Ester C16 H22 O4 T7 (+) 17.77 192.1362 169.1473 Lupinine C10 H19 N O T7 (+) 17.77 248.2013 247.194 Lycopodine C16 H25 N O T7 (+) 20.14 282.2793 281.2721 Oleoyl amine C18 H35 N O T7 (+) 21.44 411.2876 388.298 1α-hydroxy-21-nor-20-oxavitamin D3 C25 H40 O3 T7 (+) 21.45 343.2242 320.235 (+/−)14,15-EpETrE C20 H32 O3 T7 (+) 21.62 343.224 320.2347 (+/−)14,15-EpETrE C20 H32 O3 T7 (+) 21.64 411.2862 388.297 1α,25-Dihydroxy-23,24-dinorvitamin D3 C25 H40 O3 T7 (+) 22.15 167.011 128.0479 6-Hydroxy-2-hexynoic acid C6 H8 O3 T7 (+) 22.74 179.9885 141.0251 5-Acetyl-4-methylthiazole C6 H7 N O S T8 (−) 16.01 243.1334 242.1261 Hydroxyamobarbital C11 H18 N2 O4 T8 (−) 17.77 243.1333 242.126 Hydroxyamobarbital C11 H18 N2 O4 T8 (+) 17.76 248.1967 230.1631 Leu Val C11 H22 N2 O3 T8 (+) 17.77 192.1347 174.1013 Val Gly C7 H14 N2 O3 T8 (+) 21.61 395.2916 356.3286 (+)-3-hydroxy behenic C22 H44 O3 T8 (+) 21.67 909.5463 886.5559 Glc-GP(18:0/20:4(5Z,8Z,11Z,14Z)) C47 H83 O13 P T8 (+) 21.93 125.0388 248.0632 Dapsone C12 H12 N2 O2 S T9 (−) 0.92 104.1065 103.0992 2-Amino-3-methyl-1-butanol C5 H13 N O T9 (−) 0.97 176.0109 137.0479 p-aminobenzoic acid C7 H7 N O2 T9 (−) 0.98 293.0644 254.1012 Dyphylline C10 H14 N4 O4 T9 (−) 0.99 160.0375 137.0483 3-Pyridylacetic acid C7 H7 N O2 T9 (−) 1.02 203.0545 180.0654 Theobromine C7 H8 N4 O2 T9 (−) 1.03 435.0635 396.0998 4-Hydroxylevamisole glucuronide C17 H20 N2 O7 S T9 (−) 1.05 265.0252 226.0621 Anthralin C14 H10 O3 T9 (−) 1.13 230.0935 191.1302 Diethyltoluamide C12 H17 N O T9 (−) 1.19 287.1111 264.122 1-(5-Ketohexyl)-3-methylxanthine C12 H16 N4 O3 T9 (−) 1.21 268.1553 267.1479 Metoprolol acid C14 H21 N O4 T9 (−) 1.26 282.1712 281.1639 Benzenepropanoic acid C15 H23 N O4 T9 (−) 1.98 362.2078 361.2005 Phe Val Pro C19 H27 N3 O4 T9 (−) 2.03 362.242 344.2085 Oxyphencyclimine C20 H28 N2 O3 T9 (−) 2.28 411.2 388.2101 Methyl 10,12,13,15-bisepidioxy- C19 H32 O8  16-Hydroperoxy-8E-octadecenoate T9 (−) 2.28 409.185 391.1507 Asn Met Gln C14 H25 N5 O6 S T9 (−) 2.45 621.217 620.2091 Diethylstilbestrol diglucuronide C30 H36 O14 T9 (−) 2.89 480.225 479.2173 Gln Phe Trp C25 H29 N5 O5 T9 (−) 3.23 480.225 479.2173 Gln Phe Trp C25 H29 N5 O5 T9 (−) 3.5 601.155 578.1652 Rhoifolin C27 H30 O14 T9 (−) 5.2 395.174 377.1404 Ofloxacin-N-oxide C18 H20 F N3 O5 T9 (−) 5.26 395.206 377.1723 Gln Lys Cys C14 H27 N5 O5 S T9 (−) 5.69 205.062 182.0728 9-Hydroxyfluorene C13 H10 O T9 (−) 12.06 274.275 273.2672 C16 sphinganine C16 H35 N O2 T9 (−) 12.19 318.3 317.2924 Phytosphingosine C18 H39 N O3 T9 (−) 12.49 409.13 408.1232 Ichthynone C23 H20 O7 T9 (−) 16.17 599.411 598.4032 Mytiloxanthin C40 H54 O4 T9 (−) 20.08 797.52 774.5304 1,2 di-(9Z,12Z,15Z-octadecatrienoyl)-3- C45 H74 O10  O-beta-D-galactosyl-sn-glycerol T9 (−) 21.5 585.433 584.425 Myxol C40 H56 O3 T9 (−) 21.5 601.427 600.4195 Capsanthin 3,6-epoxide C40 H56 O4 T9 (−) 21.69 646.254 645.2443 Acarbose (Glucobay) C25 H43 N O18 T9 (−) 21.49 607.29 568.3266 (3a,5b,7a)-23-carboxy-7-hydroxy-24-norcholan-3-yl, C30 H48 O10  b-D-glucopyranosiduronic acid T10 (−) 0.97 293.065 254.1022 Dyphylline C10 H14 N4 O4 T10 (−) 1.03 265.025 226.0623 Anthralin C14 H10 O3 T10 (−) 1.17 287.112 264.1228 1-(5-Ketohexyl)-3-methylxanthine C12 H16 N4 O3 T10 (−) 2 362.243 344.2091 Oxyphencyclimine C20 H28 N2 O3 T10 (−) 2.29 411.2 410.1925 Flumethasone C22 H28 F2 O5 T10 (−) 2.29 409.185 408.1781 Trp Phe Gly C22 H24 N4 O4 T10 (−) 2.45 621.218 620.2108 Diethylstilbestrol diglucuronide C30 H36 O14 T10 (−) 2.92 480.225 479.2179 Gln Phe Trp C25 H29 N5 O5 T10 (−) 3.25 480.225 479.2187 Gln Phe Trp C25 H29 N5 O5 T10 (−) 3.64 264.159 246.1251 Santonin C15 H18 O3 T10 (−) 5.23 395.207 377.1738 Gln Cys Lys C14 H27 N5 O5 S T10 (−) 12.19 318.302 317.2943 Phytosphingosine C18 H39 N O3 T10 (−) 12.72 288.291 287.2833 C17 sphinganine C17 H37 N O2 T10 (−) 13.14 288.29 287.2824 C17 sphinganine C17 H37 N O2 T10 (−) 14.93 316.321 298.2873 11-Methyl-octadecanoic acid C19 H38 O2 T10 (−) 14.97 272.295 254.2608 14-Methyl-8-hexadecen-1-ol C17 H34 O T10 (−) 15.51 304.286 286.2515 17-Hydroxy-heptadecanoic acid C17 H34 O3 T10 (−) 16.17 599.409 598.4011 Mytiloxanthin C40 H54 O4 T10 (−) 17.76 248.197 230.1627 Leu Val C11 H22 N2 O3 T10 (−) 21.31 597.238 574.2481 p-hydroxy atorvastatin C33 H35 F N2 O6 T10 (−) 21.48 585.433 584.4252 Myxol C40 H56 O3 T10 (−) 21.63 607.287 568.3253 (3a,5b,7a)-23-carboxy-7-hydroxy-24-norcholan-3-yl, C30 H48 O10 b-D-Glucopyranosiduronic acid T10 (−) 21.69 469.368 468.36 1α,25-Dihydroxy-26,27-dimethyl-22,22,23,23- C31 H48 O3 tetradehydro-24a,24b-dihomovitamin D3 T10 (−) 21.76 469.368 468.357 1α,25-Dihydroxy-26,27-dimethyl-22,22,23,23 C31 H48 O3 -tetradehydro-24a,24b-dihomovitamin D3 T10 (−) 21.77 429.375 428.367 25-hydroxy-26,27-dimethylvitamin D3/ C29 H48 O2 25-hydroxy-26,27-dimethylcholecalciferol T10 (−) 22.48 607.287 606.277 Trandolapril glucuronide C30 H42 N2 O11 T10 (+) 1.04 265.027 226.064 Anthralin C14 H10 O3 T10 (+) 1.07 300.148 299.141 Metoclopramide C14 H22 Cl N3 O2 T10 (+) 1.17 300.147 299.14 Metoclopramide C14 H22 Cl N3 O2 T10 (+) 1.98 362.245 344.211 Oxyphencyclimine C20 H28 N2 O3 T10 (+) 2.27 409.187 408.18 Trp Phe Gly C22 H24 N4 O4 T10 (+) 5.22 395.208 377.175 Gln Cys Lys C14 H27 N5 O5 S T10 (+) 11.98 274.254 273.246 10Z,13Z,16Z-nonadecatrienenitrile C19 H31 N T10 (+) 12.15 230.248 212.214 Myristaldehyde C14 H28 O T10 (+) 12.17 318.3 317.293 Phytosphingosine C18 H39 N O3 T10 (+) 17.76 243.139 242.131 6E,8E,14E-Hexadecatriene-10,12-diynoic acid C16 H18 O2 T10 (+) 17.87 243.138 242.131 6E,8E,14E-Hexadecatriene-10,12-diynoic acid C16 H18 O2 T10 (+) 18.22 419.281 418.273 Simvastatin C25 H38 O5 T10 (+) 21.29 645.275 606.312 1,25-Dihydroxy-20S-21-(3-hydroxy-3-methylbutyl) C32 H44 F6 O4 -23-yne-26,27-hexafluorovitamin D3 T10 (+) 21.7 646.258 645.249 Acarbose (Glucobay) C25 H43 N O18Three compounds were obtained from uninfected sample T7 with basic fertilizer (BF) treated with N1P1 K1 (N low, P low, K low), while ten compounds were extracted from the same fertilized sample T7 infected withGanoderma. Two compounds were obtained from uninfected sample T8 with basal fertilizer (BF) treated with N2P2 K2 (N optimum, P optimum, K optimum), while five compounds were extracted from the same fertilized sample T8 infected withGanoderma. Thirty-one compounds were obtained from uninfected sample T9 with basic fertilizer (BF) treated with N3P3 K3 (N high, P high, K high), whereas unknown compounds were identified from the same fertilized sample T9 infected withGanoderma. Finally, twenty-six compounds were obtained from uninfected sample T10 with basic fertilizer (BF) and 14 compounds were extracted from the same fertilized sample T10 infected withGanoderma.The number of detected known metabolite compounds differs among infected and uninfected samples. For instance, the number of known compounds extracted from uninfected sample T1, T6, T9, and T10 was higher than that from infected ones, while the number of compounds extracted from infected samples T2, T3, T4, T5, T7, T8 was higher than that from uninfected ones.The dendrogram and fan plot produced by hierarchal cluster analysis (HCA) of the different fertilized oil palm seedlings, based on a common molecular mass deduced from the recordedm/z value and similar retention time (RT) and peak shape (Figures 5 and 6), provided a more detailed view on the relationships between treatments. The HCA distinguished two major clusters for treatments denoted as A and B. Cluster A consist of [T2, T6, T8, T10] with the higher enzyme activities among other treatments. Cluster B contained two subgroups: one consisting of [T1, T3, T9] and a second one consisting of [T4, T5, T7]. The HCA of the set of 190 metabolite compounds revealed the presence of several metabolite groups, arbitrarily denoted with A to D, characterized by their particular expression pattern across the ten treatments (Figure 6). Although there is no direct evidence to show the extent of plant enzymes contributing to the resistance at the infection site because they are interacted by various factors and encoded by multigene family, a previous considerable research provided corroborative evidence that plant chitinase and β-1,3-glucanases in plant self-defense mechanism (Figure 7) illustrate how optimum NPK nutrients might either mutate the production of glucanase, chitinase, POX, and PAL activities or be involved in secondary metabolite profiling for basal resistance in oil palm.Figure 5
Heatmap of 190 metabolite compounds in 10 types of NPK treatment oil palm seedling. A color matrix represents the mean values of the metabolite compounds in two biological replicates of oil palm. Characterization of the underlying metabolites is presented in Table4.Figure 6
Fan plot showing all metabolite compounds of oil palm in both forms of control (−G) and inoculated withGanoderma (+G), under different NPK treatments. Four different groups A, B, C, and D represent them/z mean values of the metabolite compounds including flavonoids, phenylpropanoids, acyclic diterpenoids, and fatty acid derivative.Figure 7
Schematic model of oil palm,Ganoderma boninense, fertilizer regime interaction focusing on the role of plant lytic enzymes (Glycoforum: http://www.glycoforum.gr.jp). This schematic model indicates the contribution of PR proteins in response to macronutrients nutrition in oil palm withGanoderma infection. Plants produce β-1,3-glucanases, chitinase, POX, and PAL during the process of pathogenesis for self- defense, and pathogens struggle against them. Application of NPK nutrients, especially K fertilizing, might either act as elicitors triggering the production of glucanase, chitinase, POX, and PAL activities or be involved in secondary metabolite profiling associated with induced resistance.


## 3.1.1. Glucanase Activity

Glucanase enzyme activity showed high significant difference (p<0.05) between theGanoderma infected seedlings and control from week 1 to week 12 with t-value of 10.43, 6.74, 11.06, and 8.66 U mL−1 at 1 to 12 WAI, respectively (Table 3). The relatively low content of the uninfected seedlings is a reflection of the relatively low resistance towardsG. boninense in the nutrient level studied. The time of defense related response induction was rapid, within week 1 and week 8, for β-1,3-glucanase in all the treatments (Table 3). Significant increase (p<0.001) in glucanase activity onG. boninense-RWB seedlings was detected in most of the treatments at one WAI. However, T6 and T2 recorded great differences between control and +G inoculated oil palm seedlings while T5, T8, and T10 recorded slight changes among the treatments. By week 8, glucanase activity of T6 was dramatically higher, about 5-fold higher than untreated seedlings (Figure 1), as a sign of activated glucanase enzyme activity. However, β-1,3-glucanases are induced not only by pathogen infection, but also by other factors. Except for T10, at 12 WAI, the glucanase activity of allG. boninense seedlings of T2, T5, T6, and T8 was decreased gradually compared to the other treatments. However, result of OD recorded at 1, 8, and 12 WAI for glucanase activity of theG. boninense-RWB seedlings was notably higher than the control.Table 3
Effect of N, P, and K nutrition and different enzyme activities of healthy andGanoderma infected oil palm seedlings.
Week after inoculation Ganoderma infected seedlings (UmL−1) Control (Um L−1) t value T2 T5 T6 T8 T10 Glucanase activity Effect of N, P2O5, and K2O nutrition on glucanase activity  1WAI 10.097 ± 0.96 5.74 ± 0.33 10.43 s ∗ 9.93 a 7.4 b 10.2 a 7.2 b 4.9 c 4WAI 2.397 ± 0.35 2.5 ± 0.47 6.74 s ∗ 3.3 a b 3.90 a 1.65 c 2.16 b c 1.25 c 8WAI 16.837 ± 1.5 10.44 ± 1.15 11.06 s ∗ 17.78 a 9.75 d 16.2 b 14.56 c 9.9 d 12WAI 3.197 ± 0.36 4.188 ± 0.69 8.66 s ∗ 3.48 b c 2.35 c 3.9 b 2.9 b c 5.87 a  Chitinase activity Effect of N, P2O5, and K2O nutrition on chitinase activity  1WAI 4.53 ±0.39 2.67 ± 0.46 11.54 s ∗ 3.18 4.07 4.27 3.93 2.57 4WAI 1.57±0.26 1.96 ± 0.33 5.89 s ∗ 1.50 1.22 2.23 1.73 2.16 8WAI 6.37± 0.76 4.36 ± 0.52 8.34 s ∗ 3.80 c 3.67 c 3.68 a 6.03 a b 5.65 b 12WAI 3.28± 0.63 2.28 ± 0.39 5.16 s ∗ 2.32 b 2.16 b 4.97 a 2.75 b 1.73 b  PAL activity Effect of N, P2O5, and K2O nutrition on PAL activity  1WAI 10.9 ±0.89 8.90 ± 0.64 13.87 s ∗ 6.67 c 8.85 b c 11.30 a b 10.25 a b 12.45 a 4WAI 12.08 ± 1.27 8.96 ± 0.77 11.58 s ∗ 7.75 c 7.50 c 16.15 a 9.65 b c 11.55 b 8WAI 8.86 ± 0.90 5.66 ±0.48 11.72 s ∗ 5.25 b 8.00 a b 8.35 a 7.15 a b 7.55 a b 12WAI 10.28 ± 1.33 4.4 ± 0.46 9.47 s ∗ 4.10 b 4.55 b 10.81 a 8.88 a 8.37 a  POX activity Effect of N, P2O5, and K2O nutrition and BSR on POX Activity  1WAI 20.65 ± 1.42 11.64 ± .91 12.82 s ∗ 15.55 b 11.80 c 15.75 b 19.26 a 18.35 a b 4WAI 13.52 ± 1.74 9.96 ± 1.51 6.60 s ∗ 4.20 c 13.90 a b 15.25 a 12.25 b 12.1 b 8WAI 6.86 ± 0.85 6.8 ±0.40 16.74 s ∗ 6.50 a 5.35 a 7.35 a 6.95 a 8.00 a 12WAI 12.86 ± 1.43 14.82 ± .94 15.74 s ∗ 11.50 c 15.35 a b 17.35 a 11.95 b c 13.05 b c Note. values are the means of three replicates;  s∗ indicates highly significant according to T-test at α′=0.05. WAI: week after inoculation; all the t-values are highly significant at 0.001%. Means with the same letter at a given parameter are not significantly different according to Duncan’s Multiple Range Test (DMRT) at α=0.05; values are the means of three replicates ± standard error. Treatments: T2 = N3+ BF + Ganoderma; T5 = K1 + BF + Ganoderma; T6 = K3 + BF + Ganoderma; T8 = N2P2K2 + BF + Ganoderma; T10 = control (compound fertilizer) + BF + Ganoderma.Figure 1
Effect of different levels of N, P, and K on glucanase activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Chitinase Activity. A highly significant difference was found between the chitinaseGanoderma treatments and control from week 1 to week 12 with t-value of 11.54, 5.89, 8.34, and 5.16 U mL−1 for week 1 to week 12, respectively (Table 3). Although the level of chitinase increased in theGanoderma infected seedlings during the study duration, it appeared that it was not sufficiently high, had not reached effective levels, or was too late to stop the fungal from growth.Significant differences were observed at 8 to 12 WAI with T6 and T8 recording markedly higher enzyme activities compared to other treatments. Relatively low levels of chitinase were detected initially at 4 WAI for all different treatments and only began to produce higher levels of the enzyme at week 8 in both T6 and T8 and at week 12 in T6 when they were subjected toGanoderma attack. Although, relatively high levels of chitinase were observed in most treatments at 1 WAIT, it does not seem to be vital for plants at the initial time period of treatment. The present results indicated that time of defense related response induction was at weeks 8 and 12 in T6 and at week 8 in almost all the treatments except T2 and T5. Treatments T6 and T8 at 8 WAI recorded the highest values of the chitinase activity. However, T2 recorded lower chitinase activity for +G inoculated seedlings treatments when compared to T6 and T8 (Figure 2). The low chitinase activity in T2 may be the cause of defense failure in the event ofGanodermainfection. All uninoculated seedlings from the uninoculated treatment, recorded lesser chitinase activity as compared toGanoderma-inoculated treatment, which revealed a positive relationship at p<0.05 between chitinase activity andG. boninense infected seedlings.Figure 2
Effect of different levels of N, P2O5, and K2O on chitinase activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Phenylalanine Ammonia-Lyase Activity. High significant difference was found between the PALGanoderma treatment and control from week 1 to week 12 with t-value of 13.87, 11.58, 11.72, and 9.47 for week 1 to week 12, respectively (Table 3). The PAL activity measured in the roots of infected palms revealed highly significant differences among treatments (p<0.0001). The highest activity was observed in the infected roots of T6, followed by T8 and T10 with no significant difference between them, while the lowest was detected in T2. This could indicate that at this stage more phenolics including lignin were synthesized in T6, T8, and T10 and are more tolerant compared to T2 (Figure 3).Figure 3
Effect of different levels of N, P2O5, and K2O on PAL activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Peroxidase Activity. High significant difference was found between theGanodermatreatment and control from week 1 to week 12 with t-value of 12.82, 6.60, 16.74, and 15.74, respectively. Higher POX was expressed in T8 at 1 WAI, but T6 showed higher POX activities from 8 WAI to 12 WAI. The lowest POX activity in the root was in sample T2 (Table 3). The results obtained have shown that T6 significantly gave high value for enzyme POX activity. However, at 1 WAI, T8 was the highest followed by T10, an indication of a fast and rapid induction of POX at the beginning of infection. However, at 4, 8, and 12 WAI, T6 gave the highest POX activity and was significantly higher in all infected root tissues. At 8 WAI, all POX activities were not significantly different among theGanoderma infected root tissues for POX enzymes. Glucanase, POX (weeks 1 and 4), and PAL activities were stimulated by theGanoderma infection within 1 week, recurring at week 8, and chitinase within week 8 after treatment application (Figure 4).Figure 4
Effect of different levels of N, P2O5, and K2O on POX activity in oil palm roots. Note. Values are the means of three replicates. Error bars represent the standard error. WAI: week after inoculation.Metabolite Compounds of Uninfected and Infected Oil Palm Seedlings. Several metabolite compounds were detected in uninfected and infected seedlings treated with different levels of N, P, and K fertilizer. However, only known compounds are listed here to study more details and find interaction between effect of pathogen and fertilizer (Table 4). There was a big difference observed in the number of released metabolite compounds between uninfected/infected samples with different N, P, and K treatments most probably due to alteration in antioxidant enzyme activities. For instance, forty compounds were obtained from uninfected sample T1 with basal fertilizer (BF) and low N fertilizer, while only three compounds were extracted from the same fertilized sample T1 infected withGanoderma. One and five compounds were observed, respectively, from uninfected and infected sample T2 with BF and high N fertilizer. One compound was obtained from uninfected sample T3 with basic fertilizer (BF) and low P fertilizer, while 18 compounds were extracted from the same fertilized sample T3 infected withGanoderma. One and 13 compounds were observed, respectively, from uninfected and infected sample T4 with BF and high P fertilizer. Two compounds were obtained from uninfected sample T5 with basic fertilizer (BF) and low K fertilizer, while nine compounds were extracted from the same fertilized sample T5 infected withGanoderma. Four and two compounds were observed, respectively, from uninfected and infected sample T6 with BF and high K fertilizer.Table 4
Mass spectrometry for each compound and its molecular structure observed in different N, P, K combination ofGanoderma inoculated (+) and uninoculated oil palm seedlings.
Sample Ganoderma RT m/z Mass Name Molecular formula T1 (−) 0.87 175.119 174.112 Arginine C6 H14 N4 O2 T1 (−) 0.92 104.107 103.1 2-Amino-3-methyl-1-butanol C5 H13 N O T1 (−) 1 293.066 254.101 Dyphylline C10 H14 N4 O4 T1 (−) 1.01 203.054 180.065 Theobromine C7 H8 N4 O2 T1 (−) 1.02 435.062 180.065 4-Hydroxylevamisole glucuronide C17 H20 N2 O7 S T1 (−) 1.13 220.134 180.065 Meperidinic acid C13 H17 N O2 T1 (−) 1.2 268.155 180.065 Metoprolol acid C14 H21 N O4 T1 (−) 1.24 206.119 180.065 Normeperidinic acid C12 H15 N O2 T1 (−) 1.25 282.171 180.065 Benzenepropanoic acid, 4-[2-hydroxy-3- C15 H23 N O4 [(1-methylethyl)amino]propoxy]-(9CI) ASL 8123 T1 (−) 1.93 417.18 180.065 S-adenosylmethionine C15 H23 N6 O5 S T1 (−) 1.96 362.209 180.065 Phe Pro Val C19 H27 N3 O4 T1 (−) 2.18 234.15 180.065 Normeperidine C14 H19 N O2 T1 (−) 2.23 409.185 180.065 Asn Met Gln C14 H25 N5 O6 S T1 (−) 2.31` 409.185 180.065 Asn Met Gln C14 H25 N5 O6 S T1 (−) 2.89 480.226 180.065 Gln Phe Trp C25 H29 N5 O5 T1 (−) 3.17 236.166 180.065 5,7,9,11,13-tetradecapentaenoic acid C14 H18 O2 T1 (-) 3.23 480.224 180.065 Gln Phe Trp C25 H29 N5 O5 T1 (−) 3.47 601.154 180.065 Rhoifolin C27 H30 O14 T1 (−) 4.71 393.19 180.065 Trp Gly Asn C17 H21 N5 O5 T1 (−) 4.89 541.263 180.065 17-Alpha, 21-dihydroxy-11,20-dioxo-5-beta-pregnan- C27 H40 O11 3-alpha-yl-beta-d-glucuronide T1 (−) 5.26 395.205 180.065 Tyr Gly Arg C17 H26 N6 O5 T1 (−) 9.75 246.243 180.065 4,8-Dimethyl-dodecanoic acid C14 H28 O2 T1 (−) 12.1 274.275 180.065 C16 sphinganine C16 H35 N O2 T1 (−) 12.1 274.275 180.065 C16 sphinganine C16 H35 N O2 T1 (−) 12.2 230.249 180.065 Myristaldehyde C14 H28 O T1 (−) 12.2 318.301 180.065 Phytosphingosine C18 H39 N O3 T1 (−) 12.2 288.291 180.065 C17 sphinganine C17 H37 N O2 T1 (−) 12.4 288.291 180.065 C17 sphinganine C17 H37 N O2 T1 (−) 12.6 272.259 180.065 2R-aminohexadecanoic acid C16 H33 N O2 T1 (−) 12.8 288.291 180.065 C17 Sphinganine C17 H37 N O2 T1 (−) 13.2 288.291 180.065 C17 Sphinganine C17 H37 N O2 T1 (−) 13.3 244.265 180.065 9-Pentadecen-1-ol C15 H30 O T1 (−) 15 272.296 180.065 14-Methyl-8-hexadecen-1-ol C17 H34 O T1 (−) 15.5 272.296 180.065 14-Methyl-8-hexadecen-1-ol C17 H34 O T1 (−) 16.2 599.412 180.065 Mytiloxanthin C40 H54 O4 T1 (−) 21.3 599.412 180.065 Mytiloxanthin C40 H54 O4 T1 (−) 21.5 585.433 180.065 Myxol C40 H56 O3 T1 (−) 21.5 601.427 180.065 Capsanthin 3,6-epoxide C40 H56 O4 T1 (−) 21.8 429.374 180.065 25-Hydroxy-26,27-dimethylvitamin D3/ C29 H48 O2  25-Hydroxy-26,27-dimethylcholecalciferol T1 (−) 22.1 167.01 180.065 6-Hydroxy-2-hexynoic acid C6 H8 O3 T1 (+) 8.79 213.145 180.065 meglumine C7 H17 N O5 T1 (+) 17.8 248.198 180.065 Leu Val C11 H22 N2 O3 T1 (+) 17.8 192.135 180.065 Lupinine C10 H19 N O T2 (−) 21.92 125.039 248.064 Asp Asp C8 H12 N2 O7 T2 (+) 1.06 300.148 299.14 Metoclopramide C14 H22 Cl N3 O2 T2 (+) 1.17 300.148 299.141 Metoclopramide C14 H22 Cl N3 O2 T2 (+) 2.28 409.187 408.18 Trp Phe Gly C22 H24 N4 O4 T2 (+) 5.24 395.208 356.246 Dopexamine C22 H32 N2 O2 T2 (+) 17.77 243.139 242.132 6E,8E,14E-hexadecatriene-10,12-diynoic acid C16 H18 O2 T3 (−) 22.09 102.971 64.0082 Ethyl chloride C2 H5 Cl T3 (+) 0.26 257.975 219.011 4-Amino-3-(5-chlorothien-2-YL) Butanoic acid C8 H10 Cl N O2 S T3 (+) 0.97 293.066 254.103 Dyphylline C10 H14 N4 O4 T3 (+) 0.98 219.028 180.065 Theobromine C7 H8 N4 O2 T3 (+) 1 277.0914 254.102 Dyphylline C10 H14 N4 O4 T3 (+) 1.01 203.055 180.065 Theobromine C7 H8 N4 O2 T3 (+) 1.03 435.061 396.097 4-Hydroxylevamisole glucuronide C17 H20 N2 O7 S T3 (+) 2 362.244 344.21 Oxyphencyclimine C20 H28 N2 O3 T3 (+) 2.27 409.187 408.18 Trp Phe Gly C22 H24 N4 O4 T3 (+) 2.9 480.227 479.219 Gln Phe Trp C25 H29 N5 O5 T3 (+) 3.24 480.226 479.219 Gln Phe Trp C25 H29 N5 O5 T3 (+) 3.63 264.161 246.127 Santonin C15 H18 O3 T3 (+) 5.26 395.207 377.174 Gln Lys Cys C14 H27 N5 O5 S T3 (+) 12.07 274.275 273.268 C16 Sphinganine C16 H35 N O2 T3 (+) 12.49 409.16 370.197 Lys His Ser C15 H26 N6 O5 T3 (+) 16 243.134 242.126 Hydroxyamobarbital C11 H18 N2 O4 T3 (+) 18.2 419.243 418.236 Met Arg Leu C17 H34 N6 O4 S T3 (+) 18.22 419.279 418.272 Simvastatin C25 H38 O5 T3 (+) 18.23 375.253 374.245 Digitoxigenin C23 H34 O4 T4 (−) 16.01 243.133 242.126 Hydroxyamobarbital C11 H18 N2 O4 T4 (+) 0.99 277.091 254.102 Dyphylline C10 H14 N4 O4 T4 (+) 1.01 138.058 120.025 Sulfolane C4 H8 O2 S T4 (+) 1.95 362.243 344.209 Oxyphencyclimine C20 H28 N2 O3 T4 (+) 2.25 409.186 408.178 Trp Phe Gly C22 H24 N4 O4 T4 (+) 2.86 480.226 479.218 Gln Phe Trp C25 H29 N5 O5 T4 (+) 3.2 480.226 479.219 Gln Phe Trp C25 H29 N5 O5 T4 (+) 3.44 601.156 578.166 Rhoifolin C27 H30 O14 T4 (+) 5.23 395.207 377.173 Gln Lys Cys C14 H27 N5 O5 S T4 (+) 12.06 274.275 273.268 C16 sphinganine C16 H35 N O2 T4 (+) 12.18 318.3 317.292 Phytosphingosine C18 H39 N O3 T4 (+) 12.48 409.163 386.174 Lys Cys His C15 H26 N6 O4 S T4 (+) 18.19 419.279 418.271 Simvastatin C25 H38 O5 T4 (+) 21.49 585.433 584.426 Myxol C40 H56 O3 T5 (-) 22.61 179.988 141.025 5-Acetyl-4-methylthiazole C6 H7 N O S T5 (−) 22.93 ― 141.025 5-Acetyl-4-methylthiazole C6 H7 N O S T5 (+) 1.35 167.01 128.047 6-Hydroxy-2-hexynoic acid C6 H8 O3 T5 (+) 17.76 248.196 230.162 Leu Val C11 H22 N2 O3 T5 (+) 17.77 192.136 169.147 Lupinine C10 H19 N O T5 (+) 21.45 411.2875 410.2796 24,25-epoxy-1 alpha-hydroxy-22,22 C27 H38 O3 T5 (+) 21.46 343.2247 320.2348 (+/−)14,15-EpETrE C20 H32 O3 T5 (+) 21.48 479.3497 456.3595 Testosterone undecanoate C30 H48 O3 T5 (+) 21.65 343.2245 320.2353 (+/−)14,15-EpETrE C20 H32 O3 T5 (+) 21.66 411.2861 388.297 1α,25-Dihydroxy-23,24-dinorvitamin D3 C25 H40 O3 T5 (+) 21.94 125.0381 248.0616 Dapsone C12 H12 N2 O2 S T6 (−) 17.77 248.1973 230.1635 Leu Val C11 H22 N2 O3 T6 (−) 17.78 243.1331 242.1258 Hydroxyamobarbital C11 H18 N2 O4 T6 (−) 21.56 839.4909 821.4566 Tacrolimus metabolite M-VIII C43 H67 N O14 T6 (−) 21.65 498.9004 497.8933 Perflubron C8 Br F17 T6 (+) 21.66 498.9008 497.8937 Perflubron C8 Br F17 T6 (+) 21.95 125.0378 248.0611 Dapsone C12 H12 N2 O2 S T7 (−) 1.2 179.9883 141.0251 5-Acetyl-4-methylthiazole C6 H7 N O S T7 (−) 17.77 248.1964 230.1627 Leu Val C11 H22 N2 O3 T7 (−) 21.65 498.9009 497.8938 Perflubron C8 Br F17 T7 (+) 17.51 301.1413 278.152 Phthalic acid mono-2-ethylhexyl Ester C16 H22 O4 T7 (+) 17.77 192.1362 169.1473 Lupinine C10 H19 N O T7 (+) 17.77 248.2013 247.194 Lycopodine C16 H25 N O T7 (+) 20.14 282.2793 281.2721 Oleoyl amine C18 H35 N O T7 (+) 21.44 411.2876 388.298 1α-hydroxy-21-nor-20-oxavitamin D3 C25 H40 O3 T7 (+) 21.45 343.2242 320.235 (+/−)14,15-EpETrE C20 H32 O3 T7 (+) 21.62 343.224 320.2347 (+/−)14,15-EpETrE C20 H32 O3 T7 (+) 21.64 411.2862 388.297 1α,25-Dihydroxy-23,24-dinorvitamin D3 C25 H40 O3 T7 (+) 22.15 167.011 128.0479 6-Hydroxy-2-hexynoic acid C6 H8 O3 T7 (+) 22.74 179.9885 141.0251 5-Acetyl-4-methylthiazole C6 H7 N O S T8 (−) 16.01 243.1334 242.1261 Hydroxyamobarbital C11 H18 N2 O4 T8 (−) 17.77 243.1333 242.126 Hydroxyamobarbital C11 H18 N2 O4 T8 (+) 17.76 248.1967 230.1631 Leu Val C11 H22 N2 O3 T8 (+) 17.77 192.1347 174.1013 Val Gly C7 H14 N2 O3 T8 (+) 21.61 395.2916 356.3286 (+)-3-hydroxy behenic C22 H44 O3 T8 (+) 21.67 909.5463 886.5559 Glc-GP(18:0/20:4(5Z,8Z,11Z,14Z)) C47 H83 O13 P T8 (+) 21.93 125.0388 248.0632 Dapsone C12 H12 N2 O2 S T9 (−) 0.92 104.1065 103.0992 2-Amino-3-methyl-1-butanol C5 H13 N O T9 (−) 0.97 176.0109 137.0479 p-aminobenzoic acid C7 H7 N O2 T9 (−) 0.98 293.0644 254.1012 Dyphylline C10 H14 N4 O4 T9 (−) 0.99 160.0375 137.0483 3-Pyridylacetic acid C7 H7 N O2 T9 (−) 1.02 203.0545 180.0654 Theobromine C7 H8 N4 O2 T9 (−) 1.03 435.0635 396.0998 4-Hydroxylevamisole glucuronide C17 H20 N2 O7 S T9 (−) 1.05 265.0252 226.0621 Anthralin C14 H10 O3 T9 (−) 1.13 230.0935 191.1302 Diethyltoluamide C12 H17 N O T9 (−) 1.19 287.1111 264.122 1-(5-Ketohexyl)-3-methylxanthine C12 H16 N4 O3 T9 (−) 1.21 268.1553 267.1479 Metoprolol acid C14 H21 N O4 T9 (−) 1.26 282.1712 281.1639 Benzenepropanoic acid C15 H23 N O4 T9 (−) 1.98 362.2078 361.2005 Phe Val Pro C19 H27 N3 O4 T9 (−) 2.03 362.242 344.2085 Oxyphencyclimine C20 H28 N2 O3 T9 (−) 2.28 411.2 388.2101 Methyl 10,12,13,15-bisepidioxy- C19 H32 O8  16-Hydroperoxy-8E-octadecenoate T9 (−) 2.28 409.185 391.1507 Asn Met Gln C14 H25 N5 O6 S T9 (−) 2.45 621.217 620.2091 Diethylstilbestrol diglucuronide C30 H36 O14 T9 (−) 2.89 480.225 479.2173 Gln Phe Trp C25 H29 N5 O5 T9 (−) 3.23 480.225 479.2173 Gln Phe Trp C25 H29 N5 O5 T9 (−) 3.5 601.155 578.1652 Rhoifolin C27 H30 O14 T9 (−) 5.2 395.174 377.1404 Ofloxacin-N-oxide C18 H20 F N3 O5 T9 (−) 5.26 395.206 377.1723 Gln Lys Cys C14 H27 N5 O5 S T9 (−) 5.69 205.062 182.0728 9-Hydroxyfluorene C13 H10 O T9 (−) 12.06 274.275 273.2672 C16 sphinganine C16 H35 N O2 T9 (−) 12.19 318.3 317.2924 Phytosphingosine C18 H39 N O3 T9 (−) 12.49 409.13 408.1232 Ichthynone C23 H20 O7 T9 (−) 16.17 599.411 598.4032 Mytiloxanthin C40 H54 O4 T9 (−) 20.08 797.52 774.5304 1,2 di-(9Z,12Z,15Z-octadecatrienoyl)-3- C45 H74 O10  O-beta-D-galactosyl-sn-glycerol T9 (−) 21.5 585.433 584.425 Myxol C40 H56 O3 T9 (−) 21.5 601.427 600.4195 Capsanthin 3,6-epoxide C40 H56 O4 T9 (−) 21.69 646.254 645.2443 Acarbose (Glucobay) C25 H43 N O18 T9 (−) 21.49 607.29 568.3266 (3a,5b,7a)-23-carboxy-7-hydroxy-24-norcholan-3-yl, C30 H48 O10  b-D-glucopyranosiduronic acid T10 (−) 0.97 293.065 254.1022 Dyphylline C10 H14 N4 O4 T10 (−) 1.03 265.025 226.0623 Anthralin C14 H10 O3 T10 (−) 1.17 287.112 264.1228 1-(5-Ketohexyl)-3-methylxanthine C12 H16 N4 O3 T10 (−) 2 362.243 344.2091 Oxyphencyclimine C20 H28 N2 O3 T10 (−) 2.29 411.2 410.1925 Flumethasone C22 H28 F2 O5 T10 (−) 2.29 409.185 408.1781 Trp Phe Gly C22 H24 N4 O4 T10 (−) 2.45 621.218 620.2108 Diethylstilbestrol diglucuronide C30 H36 O14 T10 (−) 2.92 480.225 479.2179 Gln Phe Trp C25 H29 N5 O5 T10 (−) 3.25 480.225 479.2187 Gln Phe Trp C25 H29 N5 O5 T10 (−) 3.64 264.159 246.1251 Santonin C15 H18 O3 T10 (−) 5.23 395.207 377.1738 Gln Cys Lys C14 H27 N5 O5 S T10 (−) 12.19 318.302 317.2943 Phytosphingosine C18 H39 N O3 T10 (−) 12.72 288.291 287.2833 C17 sphinganine C17 H37 N O2 T10 (−) 13.14 288.29 287.2824 C17 sphinganine C17 H37 N O2 T10 (−) 14.93 316.321 298.2873 11-Methyl-octadecanoic acid C19 H38 O2 T10 (−) 14.97 272.295 254.2608 14-Methyl-8-hexadecen-1-ol C17 H34 O T10 (−) 15.51 304.286 286.2515 17-Hydroxy-heptadecanoic acid C17 H34 O3 T10 (−) 16.17 599.409 598.4011 Mytiloxanthin C40 H54 O4 T10 (−) 17.76 248.197 230.1627 Leu Val C11 H22 N2 O3 T10 (−) 21.31 597.238 574.2481 p-hydroxy atorvastatin C33 H35 F N2 O6 T10 (−) 21.48 585.433 584.4252 Myxol C40 H56 O3 T10 (−) 21.63 607.287 568.3253 (3a,5b,7a)-23-carboxy-7-hydroxy-24-norcholan-3-yl, C30 H48 O10 b-D-Glucopyranosiduronic acid T10 (−) 21.69 469.368 468.36 1α,25-Dihydroxy-26,27-dimethyl-22,22,23,23- C31 H48 O3 tetradehydro-24a,24b-dihomovitamin D3 T10 (−) 21.76 469.368 468.357 1α,25-Dihydroxy-26,27-dimethyl-22,22,23,23 C31 H48 O3 -tetradehydro-24a,24b-dihomovitamin D3 T10 (−) 21.77 429.375 428.367 25-hydroxy-26,27-dimethylvitamin D3/ C29 H48 O2 25-hydroxy-26,27-dimethylcholecalciferol T10 (−) 22.48 607.287 606.277 Trandolapril glucuronide C30 H42 N2 O11 T10 (+) 1.04 265.027 226.064 Anthralin C14 H10 O3 T10 (+) 1.07 300.148 299.141 Metoclopramide C14 H22 Cl N3 O2 T10 (+) 1.17 300.147 299.14 Metoclopramide C14 H22 Cl N3 O2 T10 (+) 1.98 362.245 344.211 Oxyphencyclimine C20 H28 N2 O3 T10 (+) 2.27 409.187 408.18 Trp Phe Gly C22 H24 N4 O4 T10 (+) 5.22 395.208 377.175 Gln Cys Lys C14 H27 N5 O5 S T10 (+) 11.98 274.254 273.246 10Z,13Z,16Z-nonadecatrienenitrile C19 H31 N T10 (+) 12.15 230.248 212.214 Myristaldehyde C14 H28 O T10 (+) 12.17 318.3 317.293 Phytosphingosine C18 H39 N O3 T10 (+) 17.76 243.139 242.131 6E,8E,14E-Hexadecatriene-10,12-diynoic acid C16 H18 O2 T10 (+) 17.87 243.138 242.131 6E,8E,14E-Hexadecatriene-10,12-diynoic acid C16 H18 O2 T10 (+) 18.22 419.281 418.273 Simvastatin C25 H38 O5 T10 (+) 21.29 645.275 606.312 1,25-Dihydroxy-20S-21-(3-hydroxy-3-methylbutyl) C32 H44 F6 O4 -23-yne-26,27-hexafluorovitamin D3 T10 (+) 21.7 646.258 645.249 Acarbose (Glucobay) C25 H43 N O18Three compounds were obtained from uninfected sample T7 with basic fertilizer (BF) treated with N1P1 K1 (N low, P low, K low), while ten compounds were extracted from the same fertilized sample T7 infected withGanoderma. Two compounds were obtained from uninfected sample T8 with basal fertilizer (BF) treated with N2P2 K2 (N optimum, P optimum, K optimum), while five compounds were extracted from the same fertilized sample T8 infected withGanoderma. Thirty-one compounds were obtained from uninfected sample T9 with basic fertilizer (BF) treated with N3P3 K3 (N high, P high, K high), whereas unknown compounds were identified from the same fertilized sample T9 infected withGanoderma. Finally, twenty-six compounds were obtained from uninfected sample T10 with basic fertilizer (BF) and 14 compounds were extracted from the same fertilized sample T10 infected withGanoderma.The number of detected known metabolite compounds differs among infected and uninfected samples. For instance, the number of known compounds extracted from uninfected sample T1, T6, T9, and T10 was higher than that from infected ones, while the number of compounds extracted from infected samples T2, T3, T4, T5, T7, T8 was higher than that from uninfected ones.The dendrogram and fan plot produced by hierarchal cluster analysis (HCA) of the different fertilized oil palm seedlings, based on a common molecular mass deduced from the recordedm/z value and similar retention time (RT) and peak shape (Figures 5 and 6), provided a more detailed view on the relationships between treatments. The HCA distinguished two major clusters for treatments denoted as A and B. Cluster A consist of [T2, T6, T8, T10] with the higher enzyme activities among other treatments. Cluster B contained two subgroups: one consisting of [T1, T3, T9] and a second one consisting of [T4, T5, T7]. The HCA of the set of 190 metabolite compounds revealed the presence of several metabolite groups, arbitrarily denoted with A to D, characterized by their particular expression pattern across the ten treatments (Figure 6). Although there is no direct evidence to show the extent of plant enzymes contributing to the resistance at the infection site because they are interacted by various factors and encoded by multigene family, a previous considerable research provided corroborative evidence that plant chitinase and β-1,3-glucanases in plant self-defense mechanism (Figure 7) illustrate how optimum NPK nutrients might either mutate the production of glucanase, chitinase, POX, and PAL activities or be involved in secondary metabolite profiling for basal resistance in oil palm.Figure 5
Heatmap of 190 metabolite compounds in 10 types of NPK treatment oil palm seedling. A color matrix represents the mean values of the metabolite compounds in two biological replicates of oil palm. Characterization of the underlying metabolites is presented in Table4.Figure 6
Fan plot showing all metabolite compounds of oil palm in both forms of control (−G) and inoculated withGanoderma (+G), under different NPK treatments. Four different groups A, B, C, and D represent them/z mean values of the metabolite compounds including flavonoids, phenylpropanoids, acyclic diterpenoids, and fatty acid derivative.Figure 7
Schematic model of oil palm,Ganoderma boninense, fertilizer regime interaction focusing on the role of plant lytic enzymes (Glycoforum: http://www.glycoforum.gr.jp). This schematic model indicates the contribution of PR proteins in response to macronutrients nutrition in oil palm withGanoderma infection. Plants produce β-1,3-glucanases, chitinase, POX, and PAL during the process of pathogenesis for self- defense, and pathogens struggle against them. Application of NPK nutrients, especially K fertilizing, might either act as elicitors triggering the production of glucanase, chitinase, POX, and PAL activities or be involved in secondary metabolite profiling associated with induced resistance.


## 4. Discussion

Induction ofβ-1,3-glucanases and other PR proteins in the plant can also occur due to some components of pathogens or degraded components of pathogens. These elicitors may be components of the cell surface of the pathogen that are released by host enzymes, including fungal β-glucan, chitin, chitosan, glycoproteins, and N-acetyl- chitooligosaccharides [21, 22]. They may also be synthesized and released by the pathogen after it enters the host in response to host signals. In view of the obtained results from chitinase assay in this study, it appears likely that chitinase plays a role in the interaction betweenGanoderma and nutrition of the oil palm seedlings. As pointed out by other researchers [23, 24], the early and massive induction of chitinase activity is generally related to the increased resistance of plant tissues to infection by pathogens. However, at week 12, the chitinases activity was significantly decreased with the increase in time over the study period, an indication that the enzyme activities were short-lasting. This has been supported previously by the fact that defense mechanisms are often activated late in the infection process when the pathogen had colonized the tissue and the plant can no longer benefit from these mechanisms [25].It was reported that after a plant was challenged by potentially pathogenic microorganisms, the plant will respond by effecting changes in the composition and physical properties of its cell walls [26]. Firstly, they can degrade the cell wall of the pathogen or disrupt its deposition, eventually contributing to the pathogen death. Secondly, they can release cell wall fragments that act as elicitors of active host defense response. In addition, the plant will produce secondary metabolites that serve to isolate and limit the spread of the invading pathogen and also limit necrotic lesions at the site of the invasion. These responses are collectively known as hypersensitive reaction. Following infection, PR proteins accumulate in leaves and other organs, where they may comprise more than 10% of the total soluble protein. It was reported that there is a close relationship between glucanase and chitinase activity because it has been proposed that glucanase and chitinase would act synergistically together to affect or inhibit fungal growth either by lysis of the hyphal tips [26] or by interfering with the correct balance between fungal cell wall synthesis and wall hydrolysis during cell wall extension of the hyphal tip [27, 28].Although the level of PR proteins increased in theG. boninenseinfected seedlings for the trial duration period, it appears that they are not sufficient or reach effective levels too late to stop the fungal from growth. This was supported by Van Loon who reported that defense mechanisms are often activated late in the infection process when the pathogen had colonized the tissue and the plant can no longer benefit from these mechanisms [25]. Low level of chitinase activity may also be the factor of high susceptibility among oil palm seedlings toG. boninense infection. Other than that, low level of chitinase activity may lower the hydrolysis effect of glucanase activity since both enzymes work synergistically [26]. With the rapid advancement in genetic transformation, oil palm seedlings resistance toGanoderma infection might be improved by regulation of chitinase gene expression and other genes controlling host resistance [29]. As mention earlier, PAL catalyzes the first committed step in phenylpropanoid metabolism which is vital for the synthesis of phenolic compounds including lignin [30]. Most evidence suggests that PAL activity is correlated not only with lignin biosynthesis, but also with the production of salicylic acid precursors. Salicylic acid is reported to play a central role in genetically determined plant disease resistance [31]. Apart from that, the POX activity is not always associated with lignification [32]. The POXs are mainly involved in hydrogen peroxide (H2O2) scavenging to alleviate oxidative stress caused by reactive oxygen species [20], thereby playing a detoxifying role. Since they are also implicated in tissue growth and differentiation in plants, expression of peroxidases in roots might indicate simple root differentiation activity and not necessarily lignin biosynthesis [33].As has been known, POXs are key enzymes in the cell wall-building process, and it has been suggested that extracellular or wall-bound peroxidases would enhance plant resistance by the construction of a cell wall barrier that may impede pathogen ingression and spread [34]. Peroxidases are also involved in HR and for the polymerization of cell wall components such as lignin, suberin, and extensin, leading to the formation of barriers for infecting pathogens [35]. Even though many peroxidases are found in most plant species and are expressed constitutively, some isozymes appear to be inducible upon pathogen infection.The application of N, P, and K nutrients as observed here may play important role in directly stimulating the inducible compounds in oil palm seedlings. Although this study has also shown that macronutrient application has not stopped the progress ofG. boninense, the infection was delayed. The occurrence of induced resistance subsequently implied enhanced tolerance of oil palm seedlings towards development ofG. boninense and delayed the onset of symptoms. Effect of different nutrients doses was obviously observed among results of secondary metabolite compounds. For instance, only a few but important number of metabolite compounds observed in sample T6 leads to the highest activities of four antioxidant enzymes activities. That shows the significant importance of K fertilizing for oil palm trees among other fertilizers. Although highest N fertilizing of sample T2 led plants to produce different metabolite compounds and consequently caused the highest activities of glucanase activities, it failed for chitinase activities among other treatments, which is most probably due to lack of enough K. Sample T8 with optimum levels of NPK fertilizing and extracted different metabolite compounds showed the highest activities for all antioxidant enzyme activities among other treatments. Samples T1, T3, T4, T5, T7, and T9 that were treated with different levels of fertilizing, produced variety of metabolite compounds mostly in control type (−G), which were not effective enough for plants after inoculation withGanoderma. Extracted metabolite compound as well as results of antioxidant enzyme activities of samples T10 with basic fertilizer (BF) and T8 with optimum level of fertilizers also confirmed the importance of using balanced NPK for oil palm trees. It is difficult to determine exact suppression mechanism with the application of macronutrients since other complex interactions in the soil-plant-pathogens exist. Moreover,G. boninense is notoriously adaptable, and many plant and nutrient factors may interact to contribute to different susceptibility to the oil palm seedlings. On the other hand, role of most metabolite compounds presented in oil palm seedlings under different N, P, and K treatments againstGanoderma attack has not been clear yet, though many of them have been identified in other plants as useful medicinal compounds for human health. Identifying and categorizing these compounds and their effective relation in oil palm seedlings againstGanoderma attack may be used as new markers for the early detection of disease. At the next step, besides explaining the role of micronutrients in antioxidant enzymes activities, identifying the role of extracted secondary metabolite compounds may help to formulate improved fertilizers for oil palm trees. It was revealed that susceptibility may be associated with cell maintenance and development, genes involved in the biosynthesis of lignin and phenolics and genes implicated in oxidative burst, programmed cell death, or hypersensitive responses [36].TheG. boninense disturbs the lignin and other structural components of the cell wall during infection using its enzyme activities [37, 38]. Lignin protects both cellulose and hemicellulose components from fungus enzymatic activities through chemical bonds.Ganoderma attacks the oil palm, and due to the lack of sufficient lignin, the permeability of cell wall increased. Lignin plays an important role in protecting plants from microbial degradation and decay. Also, it limits the plant’s biomass conversion to biofuels. Besides all the positive effects of lignin in plant cell wall like protection of plant against several stress conditions, for the purpose of some investigation, existence of lignin will be undesirable trait. Lignin has an important role in plant defense against pathogen invasion. Lignification is a mechanism for resistance in plants. After pathogen invades the plant, lignin or lignin-like phenolic compound accumulation was shown to occur in a variety of plant-microbe interactions during the plant defense responses. Endogenous enzymes chitinase, β-1,3-glucanases, and lignin content in plant leaves can be used as biochemical markers for identifying plant varieties resistant to fungal infection or other biotic and abiotic factors. Also by transferring pathogenesis-related (PR) proteins, such as chitinases, β-1,3-glucanases genes can induce resistance in plants to various pathogens.During the degradation of cell wall in oil palm, an array of cell wall degrading enzymes are expected to be released byGanodermato ensure successful colonization and degradation of the intact root tissues. The defense mechanism fails if a virulent pathogen, such asG. boninense, avoids triggering, suppresses resistance reaction, or evades the effects of activated defense in oil palm seedlings. Consequently, the infection becomes apparent. Researchers indicated that a disease can be reduced when defense mechanisms are triggered by a stimulus prior to infection by a plant pathogen [25, 39]. Hence, lignin is an obstacle point to protect the inner cell wall against fungus infections [40]. It has been reported that decreasing the susceptibility of plants against fungus has been achieved by increasing lignin in plant cell wall [41]. Therefore, it seems that using these mechanisms may increaseG. boninense tolerance in oil palm. Lignocelluloses refer to plant cell in which lignin is associated closely with cellulose and hemicelluloses of cell wall. Lignin acts as a physical barrier to pathogen attack and, in the xylem tissue of plants, provides a water impervious seal throughout cell walls. Lignin forms chemical bonds directly against enzymatic attack of pathogens and protects more amenable hemicelluloses and cellulose [42]. The polymeric lignin structure involves at least three building units in monomeric form. The term “lignin” refers to a big group of polymers involved in oxidative combinatory of 4-hydroxyphenylpropanoids [43]. Hydroxycinnamyl alcohols, sinapyl alcohol, coniferyl alcohol, and a small amount of p-coumaryl alcohol are involved in the formation of the main composition of the lignin building [40].Mineral nutrition also affects the formation of mechanical barriers in plant tissue. As leaves age, the accumulation of silicon (Si) in the cell walls helps form a protective physical barrier to fungal penetration [44, 45]. Excessively high N levels lower the Si content and increase susceptibility to fungal diseases.The secondary cell wall between the middle lamella and the plasma membrane is deposited with a large amount of lignin. Although the composition of lignin, pectin, hemicelluloses, and cellulose microfibril leads to the production of the impervious and rigid secondary cell wall in plants, lignin is the most effective factor on the secondary cell wall structure. However,Ganoderma needs other sources of food, such as cellulose and pectin rather than lignin, to be able to break down plant. Notwithstanding various factors to increase lignin in plant cell wall, it was reported that the most effective factors of lignin biosynthesis are abiotic and biotic stresses [46]. TheGanoderma boninense obtains its required energy for causing the disease from lignin degradation of cell wall, although Paterson reported that pectin and starch also play a light role in surviving fungi [42]. WhenGanoderma attacks the plants, spore of fungi in favorable condition starts to develop its new cells, and then fungi cells become hyphae that later create mycelium. Mycelium of fungi forms a lump which grows out of the soil when mycelium penetrates into the oil palm cells; it uses the lignin of the cell wall and continues to destruct the plant. WhenG. boninense attacks the oil palm, it tries to reach the lignin components of the cell wall; this period of time in different plants may vary based on cell wall thickness.In a research on the BSR of oil palm, infection symptoms appeared on the roots of inoculated plants throughout 5 months [47]. It was shown that penetration of fungus mycelium into the cell wall of oil palm root surface occurred in both epidermis and exodermises layers; this engagement between fungus and root surface can be limited to the primary contact point or cover root completely [37]. It can be understood that the main and the most important step in controllingGanodermadisease is related to the cell wall thickness, particularly in the first layer. By this way, the fungi mycelium contact with the middle lamella of the plant cell wall will be limited. Thus, the BSR disease rate of oil palm will reduce. It was revealed that aGanoderma colonization may occur along with unwounded root parts and then may develop mostly through the cortex part of the inner cell wall [37].


## 5. Conclusion

This study suggests the involvement of PR proteins in response to macronutrients nutrition in oil palm with and withoutGanoderma infection. The observed trend of changes in the infected and uninfected plants was a slightly higher activity for β-1,3-glucanases, chitinase, POX, and PAL during the process of pathogenesis. The PR proteins gave positive response to the interaction between oil palm seedlings andGanoderma infection. Although the response was activated systematically, they were short-lasting as the changes in enzymes activities appeared before the occurrence of visible symptoms. These enzymatic reactions may be useful as early markers of stress condition in oil palm toGanoderma infection. Though direct evidence for the causal role of these enzymes was lacking, this study has proved that the accumulation of PR in oil palm seedlings infected byGanoderma may be related to systemic acquired resistance (SAR). This is because there were some changes in the PR proteins that can be detected in the roots. This indicated that the increase in the PR proteins was a plant response to infection, not simply a contribution of the hyphal enzymes per se. On the other hand, the metabolite profiling of oil palm offers an improvement to the comprehending of the biosynthetic pathways responses to environmental changes and acts as the platform for further exploration and identification of different metabolite markers for BSR disease detection.However, whether or not the N, P, and K macronutrients application resulted in PR proteins induction, which plays a direct role in the disease resistance, remains unclear and needs to be further investigated. Application of NPK nutrients, especially K fertilizing, might either act as elicitor triggering the production of glucanase, chitinase, POX, and PAL activities or be involved in secondary metabolite profiling associated with induced resistance. Many plant enzymes are involved in defenses reactions against plant pathogens. The results of this study could be useful for developing new strategies with proper nutrition, which may decreaseGanoderma diffusion and its growth rate in peat soil.



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*Source: 1494157-2018-03-12.xml*