Expression of Genes Encoding Manganese Peroxidase and Laccase of Ganoderma boninense in Response to Nitrogen Sources, Hydrogen Peroxide and Phytohormones

Ganoderma produces lignolytic enzymes that can degrade the lignin component of plant cell walls, causing basal stem rot to oil palms. Nitrogen sources may affect plant tolerance to root pathogens while hydrogen peroxide (H2O2), salicylic acid (SA) and jasmonic acid (JA) play important roles in plant defense against pathogens. In this study, we examined the expression of genes encoding manganese peroxidase (MnP) and laccase (Lac) in Ganoderma boninense treated with different nitrogen sources (ammonium nitrate, ammonium sulphate, sodium nitrate and potassium nitrate), JA, SA and H2O2. Transcripts encoding MnP and Lac were cloned from G. boninense. Of the three GbMnP genes, GbMnP_U6011 was up-regulated by all nitrogen sources examined and H2O2 but was down-regulated by JA. The expression of GbMnP_U87 was only up-regulated by JA while GbMnP_35959 was up-regulated by ammonium nitrate but suppressed by sodium nitrate and down-regulated by H2O2. Among the three GbLac genes examined, GbLac_U90667 was up-regulated by ammonium nitrate, JA, SA and H2O2; GbLac_U36023 was up-regulated by JA and H2O2 while GbLac_U30636 was up-regulated by SA but suppressed by ammonium sulphate, sodium nitrate, JA and H2O2. Differential expression of these genes may be required by their different functional roles in G. boninense.


Introduction
Palm oil production is threatened by a fungal disease known as basal stem rot (BSR) which is mainly caused by Ganoderma boninense. Infected oil palms may not show obvious symptoms at the initial stage of infection. The foliar symptoms expressed by infected oil palms include unopened spear leaves, yellowing leaves, small canopy and flattened crown. The lower stem rotted at the advanced stage of BSR with the emergence of basiodiocarps [1].
Ganoderma species are white rot fungi which can degrade lignin by producing extracellular ligninolytic enzymes including manganese peroxidase (MnPs; EC 1.11.1.13) and laccase (Lac; EC 1.10.3.2) [2,3]. MnP oxidizes Mn 2+ to Mn 3+ and nonphenolic aromatic compounds with high oxidation-reduction potentials such as lignin [4]. Lac, which is catalyzed by the redox ability of copper ions, can also oxidize nonphenolic substrates with high oxidation-reduction potentials, concomitantly with the reduction of oxygen to water [5][6][7]. Purified and well characterized Lac enzymes from different Ganoderma strains have been Three replicates were prepared for each treatment and control. The flasks were kept in the dark as static cultures at 30 • C. The mycelia of G. boninense were harvested at 6 days post-inoculation (dpi).
For phytohormone and H 2 O 2 treatments, 35 mL potato dextrose broth (PDB) was prepared in each 125 mL-conical flask and inoculated with eight mycelial discs. The medium was supplemented with 1 mM salicylic acid (SA) (Sigma-Aldrich, St. Louis, MO, USA), or 1 mM jasmonic acid (JA) (Sigma-Aldrich, USA) or 1 mM H 2 O 2 . Untreated G. boninense in PDB was used as control. Three replicates were prepared for each treatment and control. All treatments were kept in the dark and incubated as static cultures at 30 • C for 4 days. The mycelia of G. boninense were harvested at 4 dpi. The amount of phytohormones was determined based on [24][25][26] while the amount of H 2 O 2 was determined based on [28].
The mycelia were collected by filtration using Whatman filter paper No. 1, rinsed with distilled water to remove the remaining medium and dried by pressing on filter paper. The mycelia collected were frozen in liquid nitrogen and stored at −80 • C for RNA extraction.

Enzyme Assays
Secreted enzymes were collected from liquid cultures of G. boninense by centrifugation at 10,621 g for 30 min at 4 • C. Crude enzymes in the clear supernatants contained were used for enzyme assays. The activity of MnP (EC 1.11.1.13) was determined based on [31] in triplicate. The reaction mixture contained 0.5 M sodium succinate buffer (pH 4.0), 1 mM of manganese sulphate (MnSO 4 ), 0.1 mM H 2 O 2 , 1 mM guaiacol (Acros Organics, Fair Lawn, NJ, USA) as enzyme substrate and 500 µL crude enzyme. Absorbance reading was measured at 465 nm. The molar absorption coefficient at 465 nm (E 465 ) at 12.1 mM −1 cm −1 was used for the calculation of enzyme activity. Laccase (EC 1.10.3.2) enzyme activity was determined as described by [32] in triplicate. The assay mixture contained 1 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) (Sigma-Aldrich, USA), 100 mM sodium acetate buffer (pH 5.0) and 500 µL cell-free crude enzyme. Absorbance reading was measured at 420 nm. The molar absorption coefficient at 420 nm (E 420 ), at 36.0 mM −1 cm −1 was used for the calculation of enzyme activity. One unit (U) of enzyme activity is defined as the amount of enzyme needed to transform 1 µmol of product in one minute under the assay conditions. Differences between the means were analyzed using analysis of variance (ANOVA). Significant differences among mean values (at p < 0.05) were estimated using Duncan's multiple range test.

Inoculation of Oil Palms with G. boninense in the Presence of Different Nitrogen Sources
Rubber wood blocks (6 cm × 6 cm × 6 cm) colonized by G. boninense were used for artificial inoculation of three-month-old Dura x Pisifera GH500 oil palm seedlings (Sime Darby Seeds and Agricultural Services Sdn. Bhd., Banting, Malaysia). Treatment 1 consisted of uninfected oil palm seedlings treated with different nitrogen sources while Treatment 2 consisted of G. boninense-infected oil palm seedlings (infected by using sitting technique as described in [33]) treated with a once-off treatment of 250 mL of 50 mM ammonium sulphate, 50 mM ammonium nitrate, 50 mM sodium nitrate and 50 mM potassium nitrate at the beginning of the experiment. Three replicates were used for each treatment. Disease symptoms of oil palm seedlings were evaluated weekly based on a modified disease severity scores for 24 weeks as shown in Table S1. The disease severity index (DSI) was calculated as mentioned in [34], where DSI (%) = ( (score frequency × disease severity scores))/((total number of plants) × (maximal disease severity scores)) × 100.

Total RNA Extraction and RACE-PCR
Total RNA was extracted by using a cetyl trimethylammonium bromide (CTAB)-method [35] followed by treatment with DNase I (New England Biolabs, Hitchin, UK) according to the manufacturer's instructions prior to cDNA synthesis. The first-strand cDNA was synthesized according to the protocol of SMART RACE cDNA amplification kit (Clontech, Mountain View, CA, USA). Amplification of the 5 , 3 , or both ends, of transcripts was conducted by using RACE-PCR according to the manual (Clontech, USA). The cycling conditions for the PCR program were 30 cycles of 94 • C for 30 s, 68 • C for 30 s and 72 • C for 3 min. The PCR products obtained were analyzed on 1.5% (w/v) Tris-acetate-EDTA (TAE) agarose gel followed by gel purification using MEGAquick-spin Plus Total Fragment DNA Purification Kit (iNtRON, Seongnam-Si, Korea). The RACE-PCR products with A-overhang were cloned into yT&A vector (Yeastern Biotech, New Taipei City, Taiwan). The ligated PCR products were transformed into 100 µL of Escherichia coli DH5α. The plasmid DNA was confirmed by colony PCR for restriction enzyme analysis before Sanger sequencing.

Quantitative Reverse Transcription-PCR (qRT-PCR)
Gene expression analysis was conducted for G. boninense samples from treatments with different nitrogen sources in Czapek-Dox broth for 6 days, and from treatments with JA, SA and H 2 O 2 in PDB collected at 4 dpi; compared with respective control treatments. Affinity Script qPCR cDNA Synthesis Kit (Stratagene, La Jolla, CA, USA) was used to synthesize cDNA from DNase-treated RNA samples. Real time PCR was performed in iQ5 iCycler Thermal Cycler (Bio-Rad, Hercules, CA, USA) with Brilliant SYBR Green QPCR Master Mix (Stratagene, USA). The primers used are shown in Table  S3. The relative transcript abundance of each target gene was normalized to that of housekeeping genes (α-tubulin and β-tubulin; [39]). Differential gene expression was considered as significant when relative abundance of transcript in treated G. boninense to that in untreated G. boninense in either Czapek-Dox broth (control for nitrogen treatments) or PDB (control for JA, SA and H 2 O 2 treatments), was ≥2-fold (up-regulation) or ≤0.5-fold (down-regulation) [40].

Sequence Analysis of GbMnPs and GbLacs in G. boninense
Since G. boninense is a white rot fungus which can degrade lignin, it is crucial to investigate the regulation of genes encoding lignolytic enzymes that contribute to BSR. In this study, the transcript sequences for GbMnP and GbLac, that were previously found to be present in infected oil palm [15], were successfully isolated by RACE-PCR. These sequences shared high identities with those from Ganoderma applanatum or G. lucidum with an E-value of 0. The signal peptide and cleavage site were predicted in the predicted amino acid sequence of these GbMnPs and GbLacs (Figures 1a and 2a). Since MnP and Lac are extracellular enzymes [7,11], the signal peptide may facilitate the secretion of these lignin degrading enzymes. The distal histidine, distal arginine (as acid-base catalyst), proximal histidine, H-bonded aspartate (which lowers redox potential of heme iron) and manganese binding sites (for manganese binding which enhances MnP activity) [7] were conserved in GbMnPs (Figure 1a). The GbMnPs were clustered together with sequences from other Ganoderma spp. in a separate group from the fungal LiP clade (Figure 1b). Four conserved copper-binding domains where copper binds to enhance Lac activity were identified in GbLacs [11]. GbLacs were clustered together with Lacs from other Ganoderma spp. (Figure 2b). GbLac_U36023, which has a longer C-terminus, was found to be distinct from the other two GbLacs. Since the enzyme activities of the treated fungal culture may reflect the effects of a treatment on the levels of MnP and Lac, we also measured the MnP and Lac activities in the G. boninense in vitro cultures ( Figure 3) used for transcript profiling of GbMnPs and GbLacs. However, the measured enzyme activity in each culture may be contributed by more than one enzyme isoform and cannot be separated individually and might thus not necessarily agree with the transcript abundance of individual genes. In addition, we also investigated effects of nitrogen sources on the DSI of oil palm (Figure 4) caused by G. boninense. Meanwhile, the effects of JA and SA on the DSI of oil palm caused by G. boninense was inferred from the results of another in-house study which showed that the infected oil palm seedlings treated with JA and SA had no significant difference in disease symptoms compared with the untreated oil palm seedlings (unpublished data). Figure 3 shows the enzyme activities of MnP and Lac in G. boninense treated with different nitrogen sources, phytohormones and H 2 O 2 . Although the mean values of MnP enzyme activity were increased by sodium nitrate, potassium nitrate and ammonium nitrate in G. boninense at 6 dpi ( Figure 3a), only the increase of the mean value of MnP activity in G. boninense treated with sodium nitrate was statistically significant (p < 0.05). Treatment with JA, SA and H 2 O 2 did not cause any significant changes to the mean values of MnP enzyme activity of treated G. boninense at 4 dpi ( Figure 3c). Only treatments with ammonium sulphate and JA elevated the mean values of Lac enzyme activity in G. boninense (Figure 3b,d). Our results demonstrated that the enzyme activity of MnP from G. boninense was elevated by different nitrogen sources, whereby sodium nitrate had significant effect on the enzyme activity of MnP while ammonium sulphate caused significant increase to the enzyme activity of Lac. Figure 4a shows that the untreated oil palm seedlings have the highest DSI (67%) compared to infected oil palms treated with different nitrogen sources, respectively. Our findings concurred with previous findings that reported that low nitrogen content may enhance delignification while high nitrogen concentrations inhibit lignin degradation [17]. We also found that infected oil palm seedlings treated with ammonium nitrate had a lower DSI compared to those in other treatments and untreated oil palm seedlings ( Figure 4) although ammonium sulphate was shown to be able to increase Lac enzyme activity in the in vitro culture of G. boninense. Despite sodium nitrate being shown to be able to increase MnP enzyme activity in the in vitro culture of G. boninense, the oil palm seedlings treated with sodium nitrate have a lower DSI (i.e., 50%) compared to the untreated oil palm seedlings. Our findings suggested that the interactions of oil palm seedlings and G. boninense may affect the enzyme activity of lignolytic enzymes, and the outcomes may differ from those inferred from in vitro cultures that did not interact with the plant host. The expression of GbMnPs and GbLacs in the infected oil palm seedlings treated with different nitrogen sources warrants further analysis in the future.
Genes 2020, 11, 1263 6 of 15 sites (for manganese binding which enhances MnP activity) [7] were conserved in GbMnPs ( Figure  1a). The GbMnPs were clustered together with sequences from other Ganoderma spp. in a separate group from the fungal LiP clade (Figure 1b). Four conserved copper-binding domains where copper binds to enhance Lac activity were identified in GbLacs [11]. GbLacs were clustered together with Lacs from other Ganoderma spp. (Figure 2b). GbLac_U36023, which has a longer C-terminus, was found to be distinct from the other two GbLacs.    enzyme activity in each culture may be contributed by more than one enzyme isoform and cannot be separated individually and might thus not necessarily agree with the transcript abundance of individual genes. In addition, we also investigated effects of nitrogen sources on the DSI of oil palm (Figure 4) caused by G. boninense. Meanwhile, the effects of JA and SA on the DSI of oil palm caused by G. boninense was inferred from the results of another in-house study which showed that the infected oil palm seedlings treated with JA and SA had no significant difference in disease symptoms compared with the untreated oil palm seedlings (unpublished data).     Although the production of lignolytic enzymes from white-rot basidiomycetes was reported to be suppressed upon application of nitrogen fertilizer [16], we found that only the mean value of Lac enzyme activity in potassium nitrate-treated G. boninense was lower than that of the untreated G. boninense but the difference was non-significant (Figure 3b). Although G. lucidum grown in high-nitrogen was demonstrated to produce a higher Lac level in shaken cultures than that grown in low-nitrogen by a previous study [19], our results showed that only 50 mM ammonium sulphate was able to increase the Lac enzyme activity in G. boninense. Our findings suggested that the source of nitrogen may play a role in affecting the enzyme activity of Lac being produced.
Growth suppression of G. boninense by 150 parts per million (ppm) (which is approximately 1 mM), or higher concentrations of SA, accompanied by mycelial growth recovery was previously documented [24]. The growth of G. boninense was also found to be completely inhibited on media containing 5 mmol benzoic acid (the precursor of SA) or SA, while the addition of 1 mmol SA inhibited MnP and Lac up to 72 and 64% [23]. Although we found that 1 mM SA could decrease the mean values of MnP and Lac enzyme activities in SA-treated G. boninense, the decreases were shown to be non-significant. Our finding suggested that 1 mM SA might be able to suppress mycelial growth or inhibit the activities of lignolytic enzymes, but had non-significant effects on the enzyme activity of both MnP and Lac in G. boninense.
Our current study found that G. boninense treated with 1 mM JA had increased Lac enzyme activity but not MnP enzyme activity. Previously, 254 µM MeJA was found to be able to induce ganoderic acid biosynthesis in G. lucidum [41] and the expression of genes involved in ganoderic acid biosynthesis in response to 50-200 µM MeJA was profiled in G. lucidum [42]. Although the amounts of JA at millimolar concentrations were found to be effective against Fusarium wilt in faba bean [43] and plant brownhopper in rice [44], 50-200 ppm JA was reported to improve the growth of G. boninense [24]. Our findings indicated that JA may have different effects to the growth of G. boninense and lignolytic enzyme activities. Since lignolytic enzymes are involved in lignin degradation, we expected the hemibiotrophic fungus to have a higher activity of these enzymes at its necrotrophic phase, possibly coinciding with the JA-dependent defense in the plant host. Although we showed a higher enzyme activity of Lac in G. boninense in vitro culture treated with JA, it is unknown whether endogenous plant JA, which mediates the wound-induced defense response [22], could also elevate a higher Lac enzyme activity in G. boninense.
The enzyme activities of both MnP and Lac in G. boninense treated with at 1 mM H 2 O 2 , were not significantly different from that of the untreated culture. The optimum amount of H 2 O 2 to be applied to white rot fungi to induce lignolytic activities is not known. However, 6-8 mM H 2 O 2 were reported to be sufficient to induce the hypersensitive response in soybean suspension cultures [45] while 0.67-60 µmol H 2 O 2 (gFW) −1 was recorded for leaf tissues from different plants [46,47] (whereby 60 µmol H 2 O 2 (gFW) −1 is close to 100 mM on tissue water basis [46]). Although a previous study [48] had indicated that a much lower amount of H 2 O 2 was required to suppress fungal development and suggested that lower amounts of H 2 O 2 and possibly phytohormones could be of biological significance, higher amounts of H 2 O 2 in millimolar concentration were found to stimulate MnP activity in white rot fungi monocultures [26]. The effects of different concentrations of SA, JA and H 2 O 2 on the enzyme activity of MnP and Lac by G. boninense could be investigated in future.

Transcript Abundance of MnPs and Lacs in G. boninense Treated with Different Nitrogen Sources, Phytohormones and H 2 O 2
Since G. boninense possesses MnPs and Lacs that may have different functions and are regulated by different conditions, the relative transcript abundance of GbMnPs and GbLacs in response to different nitrogen sources, phytohormones and H 2 O 2 was measured in this study. Of the three GbMnPs, the transcript abundance of GbMnP_U6011 showed significant changes (>2-fold) in G. boninense treated with different nitrogen sources (i.e., 108-fold in potassium nitrate, 43-fold in ammonium sulphate, 7.36-fold in sodium nitrate and 4.09-fold in ammonium nitrate; Figure 5b); and GbMnP_U35959 showed an up-regulation of 2.90-fold in G. boninense grown in medium with ammonium sulphate in relative to that in G. boninense grown in Czapek-Dox broth (Figure 5c). GbMnP_U35959 was not expressed in G. boninense grown in Czapek-Dox broth with sodium nitrate (Figure 5c). As for GbLacs, the transcription of GbLac_U30636 was suppressed in Czapek-Dox broth with ammonium sulphate and potassium nitrate, respectively; (Figure 5d) while the transcript abundance of GbLac_U90667 showed an increase of 2.41-fold in G. boninense grown in Czapek-Dox broth with ammonium nitrate in relation to that in G. boninense grown in Czapek-Dox broth (Figure 5f). We found that the enzyme activities of MnP and Lac in G. boninense did not necessary reflect the expression of individual genes encoding GbMnPs and GbLacs in G. boninense because the enzyme activities were contributed by all MnP and Lac enzyme isoforms in the samples while the real-time PCR analysis only measured the transcript abundance of individual genes. In addition, enzyme activities could also be affected by protein abundance that was regulated at post-transcriptional, translational and post-translational levels. Although the G. boninense in vitro cultures treated with sodium nitrate had a higher MnP activity, the gene expression experiments showed that none of the GbMnP genes analyzed was up-regulated by sodium nitrate. Similarly, none of the GbLac genes analyzed was up-regulated by ammonium sulphate although the treated G. boninense in vitro culture had a higher Lac activity. In fact, sodium nitrate and ammonium sulphate suppressed the transcription of GbMnP_U35959 (Figure 5c) and GbLac_U30636 (Figure 5d), respectively.
JA treatment of G. boninense increased the transcript abundance of GbMnP_U87 by 2.23-fold but SA and H 2 O 2 treatments of G. boninense did not cause any significant changes in its gene expression compared to that of G. boninense grown in PDB (Figure 5g). The JA level is normally elevated in wounded host plants due to physical injury or damages inflicted by herbivores, insects and necrotrophic pathogens [22]. The expression of GbMnP_U6011 was up-regulated 3.54-fold in G. boninense treated with H 2 O 2 but the transcript abundance of GbMnP_U6011 in G. boninense treated by JA was only 0.45-fold of that in PDB (Figure 5h). GbMnP_U35959 was suppressed in G. boninense treated with SA while its transcript abundance in G. boninense treated by H 2 O 2 was 0.35-fold of that in G. boninense grown in PDB (Figure 5i). Our findings showed that the transcription of GbLac_U30636 was suppressed in G. boninense treated with JA and H 2 O 2 while its transcript abundance in G. boninense treated with SA was increased by 2.5-fold (Figure 4j). Meanwhile, the transcript level of GbLac_U36023 was increased 5.59-fold and 2.55-fold in G. boninense by JA and H 2 O 2 , respectively; but was reduced significantly by SA (Figure 5k). GbLac_U90667 was up-regulated 16.74-fold in G. boninense by JA, 2.64-fold by SA and 3.93-fold by H 2 O 2 (Figure 5l). Coinciding with the elevation of Lac enzyme activity by JA, the gene expression of GbLac_U36023 and GbLac_U90667 was also up-regulated by JA. These genes may possibly encode for two of the Lac enzyme isoforms that contributed to the increase of Lac enzyme activity in JA-treated G. boninense.
Since G. boninense is a hemibiotrophic pathogen which is biotrophic at the initial stage and turns necrotrophic at a later infection stage, the endogenous SA and JA levels in the host plants may fluctuate in response to the feeding modes or infection of the pathogen. It is logical that different GbMnPs and GbLacs are present in G. boninense and each of them was differentially expressed in G. boninense in response to JA and SA in the host plants and functional at different infection stages. Although SA and JA are involved in plant defense against biotrophic and necrotrophic pathogens, respectively; while reactive oxygen species (including H 2 O 2 ) are involved in both plant signaling and defense [22], it is unknown whether plant endogenous SA and JA could regulate the gene expression of GbMnP and GbLac genes as in in vitro cultures. respectively; while reactive oxygen species (including H2O2) are involved in both plant signaling and defense [22], it is unknown whether plant endogenous SA and JA could regulate the gene expression of GbMnP and GbLac genes as in in vitro cultures.  The gene expression profile demonstrated that GbMnP_U6011, GbLac_U36023 and GbLac_U90667 was upregulated by H 2 O 2 while the expression of GbMnP_U35959 and GbLac_U30636 was down-regulated and suppressed by H 2 O 2 , respectively; suggesting that individual GbMnPs and GbLacs may respond to H 2 O 2 differently although H 2 O 2 is required for the catalytic activities of both enzymes [28,30]. Despite the up-and down-regulation of these genes by H 2 O 2 , the enzyme activities of MnP and Lac were not significantly increased or decreased by H 2 O 2. It is also noteworthy that the response of these GbMnP and GbLac genes to H 2 O 2 was not always in line with that of JA or SA. Since H 2 O 2 is a common mediator of both abiotic and biotic stresses, and can be degraded by plant enzymes including peroxidases and catalases [49], the effects of H 2 O 2 on the gene regulation of GbMnPs and GbLacs in oil palm-G. boninense interaction warrant further investigation. In addition, the interactions of H 2 O 2 with various phytohormones [50] may further complicate the gene expression profiles of these genes. The transcription regulation of these genes is summarized in Figure 6.
Genes 2020, 11, x FOR PEER REVIEW 12 of 15 The gene expression profile demonstrated that GbMnP_U6011, GbLac_U36023 and GbLac_U90667 was upregulated by H2O2 while the expression of GbMnP_U35959 and GbLac_U30636 was down-regulated and suppressed by H2O2, respectively; suggesting that individual GbMnPs and GbLacs may respond to H2O2 differently although H2O2 is required for the catalytic activities of both enzymes [28,30]. Despite the up-and down-regulation of these genes by H2O2, the enzyme activities of MnP and Lac were not significantly increased or decreased by H2O2. It is also noteworthy that the response of these GbMnP and GbLac genes to H2O2 was not always in line with that of JA or SA. Since H2O2 is a common mediator of both abiotic and biotic stresses, and can be degraded by plant enzymes including peroxidases and catalases [49], the effects of H2O2 on the gene regulation of GbMnPs and GbLacs in oil palm-G. boninense interaction warrant further investigation. In addition, the interactions of H2O2 with various phytohormones [50] may further complicate the gene expression profiles of these genes. The transcription regulation of these genes is summarized in Figure 6.

Conclusions
A total of six GbMnP and GbLac transcripts were successfully cloned in this study. Of the three GbMnP genes, GbMnP_U6011 was up-regulated by all nitrogen sources examined and H2O2 but was down-regulated by JA. The expression of GbMnP_U87 was only up-regulated by JA while GbMnP_35959 was up-regulated by ammonium sulphate but suppressed by sodium nitrate and down-regulated by H2O2. Of the three GbLac genes analyzed, GbLac_U90667 was up-regulated by ammonium nitrate, JA, SA and H2O2; GbLac_U36023 was up-regulated by JA and H2O2 while GbLac_U30636 was up-regulated by SA but suppressed by ammonium sulphate, sodium nitrate, JA and H2O2. The transcription regulation of individual GbMnPs and GbLacs may vary according to the nitrogen sources applied rather than the overall nitrogen status. Although in vitro cultures of G. boninense exhibited differential expression of individual GbMnPs and GbLacs in response to JA, SA and H2O2; the transcription regulation of individual GbMnP and GbLac genes and the enzyme activity of MnP and Lac enzyme isoforms in G. boninense interacting with its plant host warrant further investigation to verify their involvement at different infection phases of Ganoderma. Since the gene expression of individual genes and the total enzyme activities of MnP and Lac enzyme isoforms in the same fungal culture did not always agree with each other, enzyme assays of individual enzyme isoforms are necessary to verify their contributions to lignin degradation and BSR in oil palm. The cloning and analysis of GbMnP and GbLac transcripts in this study pave the way for recombinant protein production and enzyme assays of individual enzyme isoforms, as an alternative to purification of fungal enzymes. By identifying the main contributors of the lignolytic activities that cause BSR in oil palm, and through in-depth understanding of the regulation of these lignolytic enzymes, the formulation of nitrogen fertilizers can be modified and treatments with phytohormones and phytohormone/H2O2 inhibitors can be devised to delay or control BSR in oil palms. GbMnP_U87 GbMnP_U6011 GbMnP_U35959 GbLac_U30636 GbLac_U36023 GbLac_U90667 Figure 6. Summary of transcription regulation of genes encoding GbMnP and GbLac. Red, up-regulation ≥2-fold; green, down-regulation ≥2-fold; yellow not differentially expressed and black, suppressed.

Conclusions
A total of six GbMnP and GbLac transcripts were successfully cloned in this study. Of the three GbMnP genes, GbMnP_U6011 was up-regulated by all nitrogen sources examined and H 2 O 2 but was down-regulated by JA. The expression of GbMnP_U87 was only up-regulated by JA while GbMnP_35959 was up-regulated by ammonium sulphate but suppressed by sodium nitrate and down-regulated by H 2 O 2 . Of the three GbLac genes analyzed, GbLac_U90667 was up-regulated by ammonium nitrate, JA, SA and H 2 O 2 ; GbLac_U36023 was up-regulated by JA and H 2 O 2 while GbLac_U30636 was up-regulated by SA but suppressed by ammonium sulphate, sodium nitrate, JA and H 2 O 2 . The transcription regulation of individual GbMnPs and GbLacs may vary according to the nitrogen sources applied rather than the overall nitrogen status. Although in vitro cultures of G. boninense exhibited differential expression of individual GbMnPs and GbLacs in response to JA, SA and H 2 O 2 ; the transcription regulation of individual GbMnP and GbLac genes and the enzyme activity of MnP and Lac enzyme isoforms in G. boninense interacting with its plant host warrant further investigation to verify their involvement at different infection phases of Ganoderma. Since the gene expression of individual genes and the total enzyme activities of MnP and Lac enzyme isoforms in the same fungal culture did not always agree with each other, enzyme assays of individual enzyme isoforms are necessary to verify their contributions to lignin degradation and BSR in oil palm. The cloning and analysis of GbMnP and GbLac transcripts in this study pave the way for recombinant protein production and enzyme assays of individual enzyme isoforms, as an alternative to purification of fungal enzymes. By identifying the main contributors of the lignolytic activities that cause BSR in oil palm, and through in-depth understanding of the regulation of these lignolytic enzymes, the formulation of nitrogen fertilizers can be modified and treatments with phytohormones and phytohormone/H 2 O 2 inhibitors can be devised to delay or control BSR in oil palms.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/11/1263/s1, Table S1: Disease severity scores based on external signs and symptoms of oil palm seedlings treated with G. boninense; Table S2: List of primers used for cloning; Table S3: Primers (forward and reverse) for quantitative real-time PCR.