Induced expression of Ganoderma boninense Lanosterol 14α-Demethylase (ERG11) during interaction with oil palm.

BACKGROUND: The basidiomycete fungus, Ganoderma boninense is the main contributor to oil palm Basal Stem Rot (BSR) in Malaysia and Indonesia. Lanosterol 14α-Demethylase (ERG11) is a key enzyme involved in biosynthesis of ergosterol, which is an important component in the fungal cell membrane. The Azole group fungicides are effective against pathogenic fungi including G. boninense by inhibiting the ERG11 activity. However, the work on molecular characterization of G. boninense ERG11 is still unavailable today. METHODS AND RESULTS: This study aimed to isolate and characterize the full-length cDNA encoding ERG11 from G. boninense. The G. boninense ERG11 gene expression during interaction with oil palm was also studied. A full-length 1860 bp cDNA encoding ERG11 was successfully isolated from G. boninense. The G. boninense ERG11 shared 91% similarity to ERG11 from other basidiomycete fungi. The protein structure homology modeling of GbERG11 was analyzed using the SWISS-MODEL workspace. Southern blot and genome data analyses showed that there is only a single copy of ERG11 gene in the G. boninense genome. Based on the in-vitro inoculation study, the ERG11 gene expression in G. boninense has shown almost 2-fold upregulation with the presence of oil palm. CONCLUSION: This study provided molecular information and characterization study on the G. boninense ERG11 and this knowledge could be used to design effective control measures to tackle the BSR disease of oil palm.

Improved Growth Performance of Elaeis guineensis Jacq. Through the Applications of Arbuscular Mycorrhizal (AM) Fungi and Endophytic Bacteria.

Agrochemical application in the oil palm industry has been estimated to be the largest component amounting to almost 30% of the operational costing. Therefore, there is a huge pressure in the oil palm cultivation to exercise sustainable practices, preferably using cheaper alternatives such as biofertilizers and organic substrates. This study investigates the effect of arbuscular mycorrhizal (AM) fungi and endophytic bacteria applied independently and in combination on oil palm growth and nutrient uptake. Greenhouse and field studies were conducted with plant responses assessed through growth parameters. Greenhouse plants were significantly stimulated by AM fungi, Rhizophagus intraradices UT126 (M1), through single microbe application. An increase of 36% in leaf area was noted in M1 plants while the calcium (Ca) uptake in leaves increased by 11%. There was no significant improvement in overall nitrogen, phosphate, and potassium (NPK) uptake but a significant improvement of Ca measurement in greenhouse and field was observed in the leaves. The predicted synergism between mixed inocula of M1 and R. clarus BR152B (M2) on vegetative growth was not observed, suggesting the probability of interspecies incompatibility that requires further investigation. Growth readings in plants treated with the combination of M1-M2 and Pseudomonas aeruginosa UPMP3 in the field were highest without a significant difference as compared to single application of M1. The difference in readings for field and greenhouse may have been influenced by other external factors such as soil type, rhizospheric microbial community, and climate, and therefore requires further elucidation. These findings suggest R. intraradices UT126 as a promising biostimulant candidate in sustainable agronomic practices especially in the nursery practices.

Hexaconazole-Micelle Nanodelivery System Prepared Using Different Surfactants for Ganoderma Antifungal Application.

Reports on fungicide-based agronanochemicals in combating disastrous basal stem rot disease in the oil palm industry are scant. Herein, we describe the potential of fungicide nanodelivery agents based on hexaconazole-micelle systems produced using three different surfactants; sodium dodecylbenze sulfonate (SDBS), sodium dodecyl sulfate (SDS) and Tween 80 (T80). The resulting nanodelivery systems were characterized and the results supported the encapsulation of the fungicide into the micelles of the surfactants. We have investigated in detail the size-dependent effects of the as-synthesized micelles towards the inhibition growth of Ganoderma Boninense fungi. All the nanodelivery systems indicate that their size decreased as the surfactant concentration was increased, and it directly affects the fungal inhibition. It was also found that Tween 80, a non-ionic surfactant gave the lowest effective concentration, the EC50 value of 2, on the pathogenic fungus Ganoderma boninense compared to the other anionic surfactants; SDBS and SDS. This study opens up a new generation of agronanofungicide of better efficacy for Ganoderma disease treatment.

Identification of Oil Palm's Consistently Upregulated Genes during Early Infections of Ganoderma boninense via RNA-Seq Technology and Real-Time Quantitative PCR.

Basal stem rot (BSR) disease caused by pathogenic fungus Ganoderma boninense is a significant concern in the oil palm industry. G. boninense infection in oil palm induces defense-related genes. To understand oil palm defense mechanisms in response to fungal invasion, we analyzed differentially expressed genes (DEGs) derived from RNA-sequencing (RNA-seq) transcriptomic libraries of oil palm roots infected with G. boninense. A total of 126 DEGs were detected from the transcriptomic libraries of G. boninense-infected root tissues at different infection stages. Functional annotation via pathway enrichment analyses revealed that the DEGs were involved in the defense response against the pathogen. The expression of the selected DEGs was further confirmed using real-time quantitative PCR (qPCR) on independent oil palm seedlings and mature palm samples. Seven putative defense-related DEGs consistently showed upregulation in seedlings and mature plants during G. boninense infection. These seven genes might potentially be developed as biomarkers for the early detection of BSR in oil palm.

Enhanced polyethylene glycol (PEG)-mediated protoplast transformation system for the phytopathogenic fungus, Ganoderma boninense.

The basidiomycete fungus, Ganoderma boninense, has been identified as the main causal agent of oil palm basal stem rot (BSR) disease which has caused significant economic losses to the industry especially in Malaysia and Indonesia. Various efforts have been initiated to understand the disease and this plant pathogen especially at the molecular level. This is the first study of its kind on the development of a polyethylene glycol (PEG)-mediated protoplast transformation system for G. boninense. Based on the minimal inhibitory concentration study, 60 µg/mL and above of hygromycin were effective to completely inhibit G. boninense growth. Approximately 5.145 × 107 cells/mL of protoplasts with the viability of 97.24% was successfully obtained from G. boninense mycelium tissue. The PEG-mediated G. boninense protoplast transformation using 1 µg of transformation vector, 25% of PEG solution, 10 min of pre-transformation incubation, and 30 min of post-transformation incubation has improved the transformation rate as compared with the previous reported protocols for other basidiomycete fungi. Optimization of four transformation parameters has improved the transformation efficiency of G. boninense from an average of 2 to 67 putative transformants. The presence of hygromycin phosphotransferase (hpt) and enhanced green fluorescent protein (eGFP) genes in the putative transformants was detected by PCR and verified by gene sequence analysis. Southern hybridization result further confirmed the integration of hpt gene in G. boninense transformants, and the green fluorescent signal was detected in the G. boninense transformants under the microscopic analysis. The establishment of this transformation system will accelerate the gene function studies of G. boninense especially those genes that may contribute to the pathogenesis of this fungus in oil palm.

EgJUB1 and EgERF113 transcription factors as potential master regulators of defense response in Elaeis guineensis against the hemibiotrophic Ganoderma boninense.

BACKGROUND: Hemibiotrophic pathogen such as the fungal pathogen Ganoderma boninense that is destructive to oil palm, manipulates host defense mechanism by strategically switching from biotrophic to necrotrophic phase. Our previous study revealed two distinguishable expression profiles of oil palm genes that formed the basis in deducing biotrophic phase at early interaction which switched to necrotrophic phase at a later stage of infection. RESULTS: The present report is a continuing study from our previous published transcriptomic profiling of oil palm seedlings against G. boninense. We focused on identifying differentially expressed genes (DEGs) encoding transcription factors (TFs) from the same RNA-seq data; resulting in 106 upregulated and 108 downregulated TFs being identified. The DEGs are involved in four established defense-related pathways responsible for cell wall modification, reactive oxygen species (ROS)-mediated signaling, programmed cell death (PCD) and plant innate immunity. We discovered upregulation of JUNGBRUNNEN 1 (EgJUB1) during the fungal biotrophic phase while Ethylene Responsive Factor 113 (EgERF113) demonstrated prominent upregulation when the palm switches to defense against necrotrophic phase. EgJUB1 was shown to have a binding activity to a 19 bp palindromic SNBE1 element, WNNYBTNNNNNNNAMGNHW found in the promoter region of co-expressing EgHSFC-2b. Further in silico analysis of promoter regions revealed co-expression of EgJUB1 with TFs containing SNBE1 element with single nucleotide change at either the 5th or 18th position. Meanwhile, EgERF113 binds to both GCC and DRE/CRT elements promoting plasticity in upregulating the downstream defense-related genes. Both TFs were proven to be nuclear-localized based on subcellular localization experiment using onion epidermal cells. CONCLUSION: Our findings demonstrated unprecedented transcriptional reprogramming of specific TFs potentially to enable regulation of a specific set of genes during different infection phases of this hemibiotrophic fungal pathogen. The results propose the intricacy of oil palm defense response in orchestrating EgJUB1 during biotrophic and EgERF113 during the subsequent transition to the necrotrophic phase. Binding of EgJUB1 to SNBE motif instead of NACBS while EgERF113 to GCC-box and DRE/CRT motifs is unconventional and not normally associated with pathogen infection. Identification of these phase-specific oil palm TFs is important in designing strategies to tackle or attenuate the progress of infection.

Residual analysis of chitosan-based agronanofungicides as a sustainable alternative in oil palm disease management.

The nanoformulations of pesticides have shown great interest from many parties due to their slow release capability and site-specific delivery. Hence, in this work, a new nanoformulation of a fungicide, namely chitosan-hexaconazole nanoparticles with a mean diameter size of 18 nm was subjected to the residual analysis on oil palm tissue, leaf and palm oil (crude palm oil and crude palm kernel oil) using a quick, easy, cheap, effective, rugged and safe (QuEChERS) method coupled with the gas chromatography-micro electron capture detector (GC-µECD). The chitosan-hexaconazole nanoparticles were applied using the trunk injection method at 4.5 g a.i./palm (standard single dose) and 9.0 g a.i./palm (double dose). The fungicide residue was analyzed at 0 (6 h after application), 1, 3, 7, 14, 30, 60, 90, and 120 days after treatment. The palm oil matrices; the crude palm oil (CPO) and crude palm kernel oil (CPKO) were found to be residue-free. However, it was observed that high accumulation of the fungicide in the stem tissue and leaf after the treatment using the chitosan-hexaconazole nanoparticles, which is good for better bioavailability for the treatment of the fungi, Ganoderma boninense. The dissipation kinetic at double dose treatment in the tissue and leaf was found to govern by the second-order kinetic with half-lives (t1/2) of 383 and 515 days, respectively.

Cytoprotection, Genoprotection, and Dermal Exposure Assessment of Chitosan-Based Agronanofungicides.

Health risks which result from exposure to pesticides have sparked awareness among researchers, triggering the idea of developing nanoencapsulation pesticides with the aim to enhance cytoprotection as well as genoprotection of the pesticides. In addition, nanocapsules of pesticides have slow release capability, high bioavailability, and site-specific delivery, which has attracted great interest from researchers. Hence, the objective of this work is to synthesize a nanoformulation of a fungicide of different sizes, namely, chitosan-hexaconazole nanoparticles (18 nm), chitosan-dazomet nanoparticles (7 nm), and chitosan-hexaconazole-dazomet nanoparticles (5 nm), which were then subjected to toxicological evaluations, including cytotoxicity, genotoxicity, cell death assay, and dermal irritation assays. Incubation of chitosan-based nanofungicides with V79-4 hamster lung cell did not reveal cytotoxicity or genotoxicity, potentially suggesting that encapsulation with chitosan reduces direct toxicity of the toxic fungicides. Meanwhile, pure fungicide revealed its high cytotoxic effect on V79-4 hamster lung cells. In addition, dermal exposure assessment on rabbits revealed that chitosan-hexaconazole nanoparticles are classified under corrosive subcategory 1C, while chitosan-dazomet nanoparticles are classified under corrosive subcategory 1B. Moreover, both chitosan-hexaconazole nanoparticles and chitosan-dazomet nanoparticles are classified as causing mild irritation.

GanoCare® Improves Oil Palm Growth and Resistance against Ganoderma Basal Stem Rot Disease in Nursery and Field Trials.

Basal stem rot (BSR) caused by Ganoderma boninense is a major threat to sustainable oil palm production especially in Southeast Asia and has brought economic losses to the oil palm industry around the world. With no definitive cure at present, this study introduces a new fertilizer technology called GanoCare®, as an effort to suppress BSR incidence in oil palm. Experiments were carried out to evaluate the effect of GanoCare® on growth, physiology, and BSR disease suppression using sitting technique in the oil palm nursery stage. A follow-up using similar treatments was carried out in the field to test on severity of Ganoderma using baiting technique under natural condition. Treatments tested were 10 g/month and 30 g/three months given as pretreatment only or continuous treatment. Results showed that GanoCare® increased the height, bulb diameter, leaf area, chlorophyll content, photosynthesis rate, and fresh and dry weight of the leaf, bole, and root of oil palm seedlings in the nursery trial. Seedlings treated with GanoCare® exhibited reduced percentage of disease severity, incidence, and dead seedlings, compared to the control. In nursery and field, lowest percentage of dead seedlings due to Ganoderma was found in seedlings given combination of pretreatment and continuous treatment of 30 g/three months (T4) with 5.56 and 6.67%, while control seedlings significantly marked the maximum percentage of 94.45 and 93.33%. The most successful treatment in both nursery and field was T4 with disease reductions of 77.78 and 82.36%, respectively, proving that nutrients contained in GanoCare® are essential in allowing better development of a strong defense system in the seedlings.

Phytotoxicity of chitosan-based agronanofungicides in the vegetative growth of oil palm seedling.

Although fungicides could be the best solution in combating fungal infections in crops, however, the phytotoxic level of fungicides to the crops should be tested first to ensure that it is safe for the crops. Moreover, nanocarrier systems of fungicides could play a significant role in the advancement of crop protection. For this reason, chitosan was chosen in the present study as a nanocarrier for fungicides of hexaconazole and/or dazomet in the development of a new generation of agronanofungicides with a high antifungal potent agent and no phytotoxic effect. Hence, the encapsulation of fungicides into the non-toxic biopolymer, chitosan was aims to reduce the phytotoxic level of fungicides. In the present study, the in vivo phytotoxicity of chitosan-fungicides nanoparticles on the physiological and vegetative growth of oil palm seedlings was evaluated in comparison to its pure fungicides as well as the conventional fungicides. The results revealed the formation of chitosan-fungicides nanoparticles could reduce the phytotoxic effect on oil palm seedlings compared to their counterparts, pure fungicides. The chitosan-fungicides nanoparticles were seen to greatly reduce the phytotoxic effect compared to the conventional fungicides with the same active ingredient.

Chitosan-Based Agronanofungicides as a Sustainable Alternative in the Basal Stem Rot Disease Management.

The rise of environmental and health concerns due to the excessive use of the conventional fungicide urges the search for sustainable alternatives of agronanofungicides where the latter is aimed to enhance plant uptake and minimize the volatilization, leaching, and runoff of fungicides. With this in mind, fungicides of hexaconazole and/or dazomet were encapsulated into chitosan nanoparticles for the formulation of chitosan-based agronanofungicides. In the present study, chitosan nanoparticles (2 nm), chitosan-hexaconazole nanoparticles (18 and 168 nm), chitosan-dazomet nanoparticles (7 and 32 nm), and chitosan-hexaconazole-dazomet nanoparticles (5 and 58 nm) were synthesized and used as potent antifungal agents in combating the basal stem rot (BSR) disease caused by Ganoderma boninense in which they were evaluated via an artificial inoculation of oil palm seedlings with the rubber woodblock, which was fully colonized with the fungal Ganoderma boninense mycelium. The results revealed that chitosan nanoparticles could act as dual modes of action, which are themselves as a biocide or as a nanocarrier for the existing fungicides. In addition, the particle size of the chitosan-based agronanofungicides plays a crucial role in suppressing and controlling the disease. The synergistic effect of the double-fungicide system of 5 nm chitosan-hexaconazole-dazomet nanoparticles can be observed as the system showed the highest disease reduction with 74.5%, compared to the untreated infected seedlings.

Comprehensive studies on optimization of ligno-hemicellulolytic enzymes by indigenous white rot hymenomycetes under solid-state cultivation using agro-industrial wastes.

The disposal of oil palm biomass is a huge challenge in Malaysian oil palm plantations. The aim of this study was to develop efficient solid-state cultivated (SSC) ligno-hemicellulolytic bio-degrader formulations of indigenous white-rot hymenomycetes (Trametes lactinea FBW and Pycnoporus sanguineus FBR) utilizing oil palm empty fruit bunches (EFB), rubber wood sawdust (SD) and vermiculite (V) either alone or in combination as substrates. Based on significant laccase (849.40 U mg-1 protein), xylanase (42.26 U g-1 protein) and amylase (157.49 U g-1 protein) production, SD+V (T5) and V (T3) were the optimum substrates for SSC of T. lactinea FBW. Whereas, utilizing EFB (T1) substrate for SSC of P. sanguineus FBR enhanced the production of MnP (42.51 U mg-1 protein), LiP (103.20 U mg-1 protein) and CMCase (34.39 U g-1 protein), enzymes. Apparently, this is the first study reporting on the protein profiles by T. lactinea FBW, producing two isoforms of un-purified laccase (~55 and 70 kDa) and MnP (~40 and 60 kDa) and a CMCase band (~60 kDa) during SSC on SD+V (T5) substrate. Interestingly, this is also the first report to document a single isoform of un-purified laccase (~50 kDa), MnP (~45 kDa), CMCase (~60 kDa) and xylanase (~55 kDa) by P. sanguineus FBR during SSC on empty fruit bunches substrate. The computed Principal Component Analysis (PCA) Biplot analysis elucidated the relationship between the solid substrate compositions, the hymenomycete strain, ligno-hemicellulolytic enzyme profiles, and cultivation time. Therefore, it is suggested to use PCA as a tool for multivariate analysis method for comprehensive selection and optimization of ligno-hemicellulolytic enzyme cocktails by the indigenous white rot hymenomycetes. These non-toxic (acute oral toxicity) formulations are safe to be used in field applications to efficiently degrade oil palm trunks and root mass that had been felled, chipped or pulverized under zero burning waste management program. This study could also serve as an alternative method for efficient utilization of agro-industrial waste as substrates for the development of cost-effective bio-degraders formulations for agro-waste management.

Preparation of Chitosan-Hexaconazole Nanoparticles as Fungicide Nanodelivery System for Combating Ganoderma Disease in Oil Palm.

Fungicide is used to control fungal disease by destroying and inhibiting the fungus or fungal spores that cause the disease. However, failure to deliver fungicide to the disease region leads to ineffectiveness in the disease control. Hence, in the present study, nanotechnology has enabled the fungicide active agents (hexaconazole) to be encapsulated into chitosan nanoparticles with the aim of developing a fungicide nanodelivery system that can transport them more effectively to the target cells (Ganoderma fungus). A pathogenic fungus, Ganoderma boninense (G. boninense), is destructive to oil palm whereby it can cause significant loss to oil palm plantations located in the Southeast Asian countries, especially Malaysia and Indonesia. In regard to this matter, a series of chitosan nanoparticles loaded with the fungicide, hexaconazole, was prepared using various concentrations of crosslinking agent sodium tripolyphosphate (TPP). The resulting particle size revealed that the increase of the TPP concentration produced smaller particles. In addition, the in vitro fungicide released at pH 5.5 demonstrated that the fungicide from the nanoparticles was released in a sustainable manner with a prolonged release time up to 86 h. On another note, the in vitro antifungal studies established that smaller particle size leads to lower half maximum effective concentration (EC50) value, which indicates higher antifungal activity against G. boninense.

A Potent Antifungal Agent for Basal Stem Rot Disease Treatment in Oil Palms Based on Chitosan-Dazomet Nanoparticles.

The use of nanotechnology could play a significant role in the agriculture sector, especially in the preparation of new-generation agronanochemicals. Currently, the economically important plant of Malaysia, the oil palm, faces the threat of a devastating disease which is particularly caused by a pathogenic fungus, Ganoderma boninense. For the development of an effective antifungal agent, a series of chitosan nanoparticles loaded with a fumigant, dazomet, were prepared using various concentrations of sodium tripolyphosphate (TPP)-2.5, 5, 10, and 20 mg/mL, abbreviated as CDEN2.5, CDEN5, CDEN10, and CDEN20, respectively. The effect of TPP as a crosslinking agent on the resulting particle size of the synthesized nanoparticles was investigated using a particle size analyzer and high-resolution transmission electron microscopy (HRTEM). Both methods confirmed that increasing the TPP concentration resulted in smaller particles. In addition, in vitro fumigant release at pH 5.5 showed that the release of the fumigant from the nanoparticles was of a sustained manner, with a prolonged release time up to 24 h. Furthermore, the relationship between the chitosan-dazomet nanoparticles and the in vitro antifungal activity against G. boninense was also explored, where the nanoparticles of the smallest size, CDEN20, gave the highest antifungal efficacy with the lowest half maximum effective concentration (EC50) value of 13.7 ± 1.76 ppb. This indicates that the smaller-sized agronanoparticles were more effective as an antifungal agent. The size can be altered, which plays a crucial role in combatting the Ganoderma disease. The agronanoparticles have controlled release properties and high antifungal efficacy on G. boninense, thus making them a promising candidate to be applied in the field for Ganoderma treatment.

Molecular characterization of Phytophthora palmivora responsible for bud rot disease of oil palm in Colombia.

Bud rot disease is a damaging disease of oil palm in Colombia. The pathogen responsible for this disease is a species of oomyctes, Phytophthora palmivora which is also the causal pathogen of several tropical crop diseases such as fruit rot and stem canker of cocoa, rubber, durian and jackfruit. No outbreaks of bud rot have been reported in oil palm in Malaysia or other Southeast Asian countries, despite this particular species being present in the region. Analysis of the genomic sequences of several genetic markers; the internal transcribe spacer regions (ITS) of the ribosomal RNA gene cluster, beta-tubulin gene, translation elongation factor 1 alpha gene (EF-1α), cytochrome c oxidase subunit I & II (COXI and COXII) gene cluster along with amplified fragment length polymorphism (AFLP) analyses have been carried out to investigate the genetic diversity and variation of P. palmivora isolates from around the world and from different hosts in comparison to Colombian oil palm isolates, as one of the steps in understanding why this species of oomycetes causes devastating damage to oil palm in Latin America but not in other regions. Phylogenetic analyses of these regions showed that the Colombian oil palm isolates were not separated from Malaysian isolates. AFLP analysis and a new marker PPHPAV, targeting an unclassified hypothetical protein, was found to be able to differentiate Malaysian and Colombian isolates and showed a clear clade separations. Despite this, pathogenicity studies did not show any significant differences in the level of aggressiveness of different isolates against oil palm in glasshouse tests.

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

Oil palm (Elaeis guineensis Jacq) is one of the major sources of edible oil. Reducing the effect of Ganoderma, main cause of basal stem rot (BSR) on oil palm, is the main propose of this study. Understanding the oil palm defense mechanism against Ganoderma infection through monitoring changes in the secondary metabolite compounds levels before/after infection by Ganoderma 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 to G. boninense inoculation. The observed trend of changes in the infected and uninfected seedlings was a slightly higher activity for ß-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 and Ganoderma 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.

Synthesis of (Hexaconazole-Zinc/Aluminum-Layered Double Hydroxide Nanocomposite) Fungicide Nanodelivery System for Controlling Ganoderma Disease in Oil Palm.

A fungicide, hexaconazole was successfully intercalated into the intergalleries of zinc/aluminum-layered double hydroxide (ZALDH) using the ion-exchange method. Due to the intercalation of hexaconazole, the basal spacing of the ZALDH was increased from 8.7 Å in ZALDH to 29.4 Å in hexaconazole-intercalated ZALDH (HZALDH). The intercalation of hexaconazole into the interlayer of the nanocomposite was confirmed using the Fourier-transform infrared (FTIR) study. This supramolecular chemistry intercalation process enhanced the thermal stability of the hexaconazole moiety. The fungicide loading was estimated to be 51.8%. The nanodelivery system also shows better inhibition toward the Ganoderma boninense growth than the counterpart, free hexaconazole. The results from this work have a great potential to be further explored for combating basal stem rot (BSR) disease in oil palm plantation.

Control of Basal Stem Rot Disease in Oil Palm by Supplementation of Calcium, Copper, and Salicylic Acid.

Continuous supplementation of mineral nutrients and salicylic acid (SA) as foliar application could improve efficacy in controlling basal stem rot (BSR) disease in oil palm seedling. It is revealed from the results that the highest disease severity index (58.3%) was recorded in T8 treatments at 9 months after inoculation. The best disease control was achieved by T7 treatments (calcium/copper/SA [Ca/Cu/SA]) (5.0%) followed by T1 (5.5%), T5 (5.8%), T3 (8.3%), T6 (8.3%), T4 (13.3%), and T2 (15.8%) treatments. Continuous supplementation of Ca/Cu/SA was found to be the most effective in controlling the disease and the high performance liquid chromatography results showed the detection of ergosterol at very low concentration in the treated samples. Moreover, the transmission electron microscopy analysis results clearly indicated that T7 treatment was also enhancing lignification, which was responsible for the thickness of the secondary cell walls and middle lamella compared to untreated samples. It was therefore, concluded that continuous supplementation of minerals nutrients and SA could effectively suppress disease severity by reducing ergosterol activity and also improve the process of lignification in the treated plants. Furthermore, this treatment also managed to delay the onset of BSR symptoms and promote the growth of the seedlings and eventually suppress the BSR disease.

Comparison of Different Protein Extraction Methods for Gel-Based Proteomic Analysis of Ganoderma spp.

Ganoderma species are a group of fungi that have the ability to degrade lignin polymers and cause severe diseases such as stem and root rot and can infect economically important plants and perennial crops such as oil palm, especially in tropical countries such as Malaysia. Unfortunately, very little is known about the complex interplay between oil palm and Ganoderma in the pathogenesis of the diseases. Proteomic technologies are simple yet powerful tools in comparing protein profile and have been widely used to study plant-fungus interaction. A critical step to perform a good proteome research is to establish a method that gives the best quality and a wide coverage of total proteins. Despite the availability of various protein extraction protocols from pathogenic fungi in the literature, no single extraction method was found suitable for all types of pathogenic fungi. To develop an optimized protein extraction protocol for 2-DE gel analysis of Ganoderma spp., three previously reported protein extraction protocols were compared: trichloroacetic acid, sucrose and phenol/ammonium acetate in methanol. The third method was found to give the most reproducible gels and highest protein concentration. Using the later method, a total of 10 protein spots (5 from each species) were successfully identified. Hence, the results from this study propose phenol/ammonium acetate in methanol as the most effective protein extraction method for 2-DE proteomic studies of Ganoderma spp.