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Comparative proteomic analysis of oil palm leaves infected with Ganoderma boninense revealed changes in proteins involved in photosynthesis, carbohydrate metabolism, and immunity and defense

Jeffery Daim LD, Ooi TE, Ithnin N, Mohd Yusof H, Kulaveerasingam H, Abdul Majid N, et al.

Electrophoresis, 2015 Aug;36(15):1699-710.
PMID: 25930948 DOI: 10.1002/elps.201400608

The basidiomycete fungal pathogen Ganoderma boninense is the causative agent for the incurable basal stem rot (BSR) disease in oil palm. This disease causes significant annual crop losses in the oil palm industry. Currently, there is no effective method for disease control and elimination, nor is any molecular marker for early detection of the disease available. An understanding of how BSR affects protein expression in plants may help identify and/or assist in the development of an early detection protocol. Although the mode of infection of BSR disease is primarily via the root system, defense-related genes have been shown to be expressed in both the root and leafs. Thus, to provide an insight into the changes in the global protein expression profile in infected plants, comparative 2DE was performed on leaf tissues sampled from palms with and without artificial inoculation of the Ganoderma fungus. Comparative 2DE revealed that 54 protein spots changed in abundance. A total of 51 protein spots were successfully identified by LC-QTOF MS/MS. The majority of these proteins were those involved in photosynthesis, carbohydrate metabolism as well as immunity and defense.
Optimization of Protein Extraction and Two-Dimensional Electrophoresis Protocols for Oil Palm Leaf

Daim LD, Ooi TE, Yusof HM, Majid NA, Karsani SA

Protein J, 2015 Aug;34(4):304-12.
PMID: 26263918 DOI: 10.1007/s10930-015-9626-x

Oil palm (Elaeis guineensis) is an important economic crop cultivated for its nutritional palm oil. A significant amount of effort has been undertaken to understand oil palm growth and physiology at the molecular level, particularly in genomics and transcriptomics. Recently, proteomics studies have begun to garner interest. However, this effort is impeded by technical challenges. Plant sample preparation for proteomics analysis is plagued with technical challenges due to the presence of polysaccharides, secondary metabolites and other interfering compounds. Although protein extraction methods for plant tissues exist, none work universally on all sample types. Therefore, this study aims to compare and optimize different protein extraction protocols for use with two-dimensional gel electrophoresis of young and mature leaves from the oil palm. Four protein extraction methods were evaluated: phenol-guanidine isothiocyanate, trichloroacetic acid-acetone precipitation, sucrose and trichloroacetic acid-acetone-phenol. Of these four protocols, the trichloroacetic acid-acetone-phenol method was found to give the highest resolution and most reproducible gel. The results from this study can be used in sample preparations of oil palm tissue for proteomics work.
An effective placental cotyledons proteins extraction method for 2D gel electrophoresis

Tan NJ, Daim LD, Jamil AA, Mohtarrudin N, Thilakavathy K

Electrophoresis, 2017 03;38(5):633-644.
PMID: 27992069 DOI: 10.1002/elps.201600377

Effective protein extraction is essential especially in producing a well-resolved proteome on 2D gels. A well-resolved placental cotyledon proteome, with good reproducibility, have allowed researchers to study the proteins underlying the physiology and pathophysiology of pregnancy. The aim of this study is to determine the best protein extraction protocol for the extraction of protein from placental cotyledons tissues for a two-dimensional gel electrophoresis (2D-GE). Based on widely used protein extraction strategies, 12 different extraction methodologies were carefully selected, which included one chemical extraction, two mechanical extraction coupled protein precipitations, and nine chemical extraction coupled protein precipitations. Extracted proteins were resolved in a one-dimensional gel electrophoresis and 2D-GE; then, it was compared with set criteria: extraction efficacy, protein resolution, reproducibility, and recovery efficiency. Our results revealed that a better profile was obtained by chemical extraction in comparison to mechanical extraction. We further compared chemical extraction coupled protein precipitation methodologies, where the DNase/lithium chloride-dense sucrose homogenization coupled dichloromethane-methanol precipitation (DNase/LiCl-DSH-D/MPE) method showed good protein extraction efficiency. This, however, was carried out with the best protein resolution and proteome reproducibility on 2D-gels. DNase/LiCl-DSH-D/MPE was efficient in the extraction of proteins from placental cotyledons tissues. In addition, this methodology could hypothetically allow the protein extraction of any tissue that contains highly abundant lipid and glycogen.
Fulltext Differential abundance analysis of mesocarp protein from high- and low-yielding oil palms associates non-oil biosynthetic enzymes to lipid biosynthesis

Ooi TE, Yeap WC, Daim LD, Ng BZ, Lee FC, Othman AM, et al.

Proteome Sci, 2015;13:28.
PMID: 26617468 DOI: 10.1186/s12953-015-0085-2

BACKGROUND: The oil palm Elaeis guineensis Jacq. which produces the highest yield per unit land area of the oil crops is the most important commercial oil crop in South East Asia. The fleshy mesocarp of oil palm fruit, where oil is mostly derived from, contains up to 90 % dry weight of oil (one of the most concentrated in plant tissues). Hence, there is attention given to gain insights into the processes of oil deposition in this oil rich tissue. For that purpose, two-dimensional differential gel electrophoresis (DIGE) coupled with western assays, were used here to analyze differential protein levels in genetically-related high-and low-yielding oil palm mesocarps.
RESULTS: From the DIGE comparative analysis in combination with western analysis, 41 unique differentially accumulated proteins were discovered. Functional categorization of these proteins placed them in the metabolisms of lipid, carbohydrate, amino acids, energy, structural proteins, as well as in other functions. In particular, higher abundance of fructose-1,6-biphosphate aldolase combined with reduced level of triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase may be indicative of important flux balance changes in glycolysis, while amino acid metabolism also appeared to be closely linked with oil yield.
CONCLUSIONS: Forty-one proteins in several important biological pathways were identified as exhibiting differential in abundance at critical oil production stages. These confirm that oil yield is a complex trait involving the regulation of genes in multiple biological pathways. The results also provide insights into key control points of lipid biosynthesis in oil palm and can assist in the development of genetic markers for use in oil palm breeding programmes.
Fulltext Preparation of Chitosan-Hexaconazole Nanoparticles as Fungicide Nanodelivery System for Combating Ganoderma Disease in Oil Palm

Maluin FN, Hussein MZ, Yusof NA, Fakurazi S, Idris AS, Zainol Hilmi NH, et al.

Molecules, 2019 Jul 08;24(13).
PMID: 31288497 DOI: 10.3390/molecules24132498

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

Maluin FN, Hussein MZ, Yusof NA, Fakurazi S, Idris AS, Hilmi NHZ, et al.

Int J Mol Sci, 2019 May 07;20(9).
PMID: 31067720 DOI: 10.3390/ijms20092247

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

Maluin FN, Hussein MZ, Azah Yusof N, Fakurazi S, Idris AS, Zainol Hilmi NH, et al.

J Agric Food Chem, 2020 Apr 15;68(15):4305-4314.
PMID: 32227887 DOI: 10.1021/acs.jafc.9b08060

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.