Pathogenicity of monokaryotic and dikaryotic mycelia of Ganoderma boninense revealed via LC-MS-based metabolomics.

This study compared the pathogenicity of monokaryotic (monokaryon) and dikaryotic (dikaryon) mycelia of the oil palm pathogen Ganoderma boninense via metabolomics approach. Ethyl acetate crude extracts of monokaryon and dikaryon were analysed by liquid chromatography quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS) coupled with multivariate data analysis using MetaboAnalyst. The mummichog algorithm was also used to identify the functional activities of monokaryon and dikaryon without a priori identification of all their secondary metabolites. Results revealed that monokaryon produced lesser fungal metabolites than dikaryon, suggesting that monokaryon had a lower possibility of inducing plant infection. These findings were further supported by the identified functional activities. Monokaryon exhibits tyrosine, phenylalanine, and tryptophan metabolism, which are important for fungal growth and development and to produce toxin precursors. In contrast, dikaryon exhibits the metabolism of cysteine and methionine, arginine and proline, and phenylalanine, which are important for fungal growth, development, virulence, and pathogenicity. As such, monokaryon is rendered non-pathogenic as it produces growth metabolites and toxin precursors, whereas dikaryon is pathogenic as it produces metabolites that are involved in fungal growth and pathogenicity. The LC-MS-based metabolomics approach contributes significantly to our understanding of the pathogenesis of Ganoderma boninense, which is essential for disease management in oil palm plantations.

Medium composition optimization and characterization of polysaccharides extracted from Ganoderma boninense along with antioxidant activity.

Ganoderma is a well-known medicinal macrofungal genus, of which several species have been thoroughly studied from the medicinal perspective, but most species are rarely involved in. In this study, we focus on the polysaccharides extracted from Ganoderma boninense and their antioxidant activity. Ganoderma boninense is a serious pathogen of oil palms that are cultivated commercially in Southeast Asia. Response surface methodology was conducted to optimize the liquid medium composition, and the mycelia biomass reached 7.063 g/L, that is, 1.4-fold compared with the seed medium. The crude and purified polysaccharides extracted from the fermentation broth showed well 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical scavenging abilities, and the scavenging abilities of purified polysaccharides reached 94.47 % and 99.88 %, respectively. Six fractions of polysaccharides were extracted and purified from fruiting bodies, mycelia and fermentation broth separately with the elution buffers of distilled water and 0.1 M NaCl solution. Generally, the polysaccharides from fruiting bodies showed stronger protective effect on H2O2-induced HepG2 cell oxidative damage than other fractions. A total of five to seven monosaccharides were identified in the six fractions of polysaccharides. The correlation analysis revealed that the content of fucose was significantly correlated with the antioxidant activity of polysaccharides, while xylose showed negative correlation results. In summary, the polysaccharides from G. boninense have a potential to be used as natural antioxidants.

Comparison of Ganoderma boninense Isolate's Aggressiveness Using Infected Oil Palm Seedlings.

Basal stem rot incidence caused by a white-rot fungus, Ganoderma boninense, is the major disease of oil palm in Southeast Asia. The rate of disease transmission and host damage are affected by variations in pathogen aggressiveness. Several other studies have used the disease severity index (DSI) to determine G. boninense aggressiveness levels while verifying disease using a culture-based method, which might not provide accurate results or be feasible in all cases. To differentiate G. boninense aggressiveness, we employed the DSI and vegetative growth measurement of infected oil palm seedlings. Disease confirmation was performed through scanning electron microscopy and molecular identification of fungal DNA from both infected tissue and fungi isolated from Ganoderma selective medium. Two-month-old oil palm seedlings were artificially inoculated with G. boninense isolates (2, 4A, 5A, 5B, and 7A) sampled from Miri (Lambir) and Mukah (Sungai Meris and Sungai Liuk), Sarawak. The isolates were categorized into three groups: highly aggressive (4A and 5B), moderately aggressive (5A and 7A), and less aggressive (2). Isolate 5B was identified as the most aggressive, and it was the only one to result in seedling mortality. Out of the five vegetative growth parameters measured, only the bole size between treatments was not affected. The integration of both conventional and molecular approaches in disease confirmation allows for precise detection.

Assessment of genetic diversity and population structure of oil palm (Elaeis guineensis Jacq.) field genebank: A step towards molecular-assisted germplasm conservation.

Oil palm (Elaeis guineensis) germplasm is exclusively maintained as ex situ living collections in the field for genetic conservation and evaluation. However, this is not for long term and the maintenance of field genebanks is expensive and challenging. Large area of land is required and the germplasms are exposed to extreme weather conditions and casualty from pests and diseases. By using 107 SSR markers, this study aimed to examine the genetic diversity and relatedness of 186 palms from a Nigerian-based oil palm germplasm and to identify core collection for conservation. On average, 8.67 alleles per SSR locus were scored with average effective number of alleles per population ranging from 1.96 to 3.34 and private alleles were detected in all populations. Mean expected heterozygosity was 0.576 ranging from 0.437 to 0.661 and the Wright's fixation index calculated was -0.110. Overall moderate genetic differentiation among populations was detected (mean pairwise population FST = 0.120, gene flow Nm = 1.117 and Nei's genetic distance = 0.466) and this was further confirmed by AMOVA analysis. UPGMA dendogram and Bayesian structure analysis concomitantly clustered the 12 populations into eight genetic groups. The best core collection assembled by Core Hunter ver. 3.2.1 consisted of 58 palms accounting for 31.2% of the original population, which was a smaller core set than using PowerCore 1.0. This core set attained perfect allelic coverage with good representation, high genetic distance between entries, and maintained genetic diversity and structure of the germplasm. This study reported the first molecular characterization and validation of core collections for oil palm field genebank. The established core collection via molecular approach, which captures maximum genetic diversity with minimum redundancy, would allow effective use of genetic resources for introgression and for sustainable oil palm germplasm conservation. The way forward to efficiently conserve the field genebanks into next generation without losing their diversity was further discussed.

Leaf transcriptomic signatures for somatic embryogenesis potential of Elaeis guineensis.

KEY MESSAGE: Potentially embryogenic oil palms can be identified through leaf transcriptomic signatures. Differential expression of genes involved in flowering time, and stress and light responses may associate with somatic embryogenesis potential. Clonal propagation is an attractive approach for the mass propagation of high yielding oil palms. A major issue hampering the effectiveness of oil palm tissue culture is the low somatic embryogenesis rate. Previous studies have identified numerous genes involved in oil palm somatic embryogenesis, but their association with embryogenic potential has not been determined. In this study, differential expression analysis of leaf transcriptomes from embryogenic and non-embryogenic mother palms revealed that transcriptome profiles from non- and poor embryogenic mother palms were more similar than highly embryogenic palms. A total of 171 genes exhibiting differential expression in non- and low embryogenesis groups could also discriminate high from poor embryogenesis groups of another tissue culture agency. Genes related to flowering time or transition such as FTIP, FRIGIDA-LIKE, and NF-YA were up-regulated in embryogenic ortets, suggesting that reproduction timing of the plant may associate with somatic embryogenesis potential. Several light response or photosynthesis-related genes were down-regulated in embryogenic ortets, suggesting a link between photosynthesis activity and embryogenic potential. As expression profiles of the differentially expressed genes are very similar between non- and low embryogenic groups, machine learning approaches with several candidate genes may generate a more sensitive model to better discriminate non-embryogenic from embryogenic ortets.

Genetic Diversity and Demographic History of Ganoderma boninense in Oil Palm Plantations of Sarawak, Malaysia Inferred from ITS Regions.

Ganoderma boninense causes basal stem rot (BSR) and is responsible for substantial economic losses to Southeast Asia's palm oil industry. Sarawak, a major producer in Malaysia, is also affected by this disease. Emergence of BSR in oil palm planted on peat throughout Sarawak is alarming as the soil type was previously regarded as non-conducive. Phylogenetic analysis indicated a single species, G. boninense as the cause of BSR in Sarawak. Information on evolutionary and demographic history for G. boninense in Sarawak inferred through informative genes is lacking. Hence, a haplotype study on single nucleotide polymorphisms in internal transcribed spacers (SNPs-ITS) of G. boninense was carried out. Sequence variations were analysed for population structure, phylogenetic and phylogeographic relationships. The internal transcribed spacers (ITS) region of 117 isolates from four populations in eight locations across Sarawak coastal areas revealed seven haplotypes. A major haplotype, designated GbHap1 (81.2%), was found throughout all sampling locations. Single nucleotide polymorphisms were observed mainly in the ITS1 region. The genetic structure was not detected, and genetic distance did not correlate with geographical distance. Haplotype network analysis suggested evidence of recent demographic expansion. Low genetic differences among populations also suggested that these isolates belong to a single G. boninense founder population adapting to oil palm as the host.

High density SNP and DArT-based genetic linkage maps of two closely related oil palm populations.

Oil palm (Elaeis guineensis Jacq.) is an outbreeding perennial tree crop with long breeding cycles, typically 12 years. Molecular marker technologies can greatly improve the breeding efficiency of oil palm. This study reports the first use of the DArTseq platform to genotype two closely related self-pollinated oil palm populations, namely AA0768 and AA0769 with 48 and 58 progeny respectively. Genetic maps were constructed using the DArT and SNP markers generated in combination with anchor SSR markers. Both maps consisted of 16 major independent linkage groups (2n = 2× = 32) with 1399 and 1466 mapped markers for the AA0768 and AA0769 populations, respectively, including the morphological trait "shell-thickness" (Sh). The map lengths were 1873.7 and 1720.6 cM with an average marker density of 1.34 and 1.17 cM, respectively. The integrated map was 1803.1 cM long with 2066 mapped markers and average marker density of 0.87 cM. A total of 82% of the DArTseq marker sequence tags identified a single site in the published genome sequence, suggesting preferential targeting of gene-rich regions by DArTseq markers. Map integration of higher density focused around the Sh region identified closely linked markers to the Sh, with D.15322 marker 0.24 cM away from the morphological trait and 5071 bp from the transcriptional start of the published SHELL gene. Identification of the Sh marker demonstrates the robustness of using the DArTseq platform to generate high density genetic maps of oil palm with good genome coverage. Both genetic maps and integrated maps will be useful for quantitative trait loci analysis of important yield traits as well as potentially assisting the anchoring of genetic maps to genomic sequences.

Differential proteomic analysis of embryogenic lines in oil palm (Elaeis guineensis Jacq).

UNLABELLED: Oil palm tissue culture is one way to produce superior oil palm planting materials. However, the low rate of embryogenesis is a major hindrance for the adoption of this technology in oil palm tissue culture laboratories. In this study, we use proteomic technologies to compare differential protein profiles in leaves from palms of high and low proliferation rates in tissue culture in order to understand the underlying biological mechanism for the low level of embryogenesis. Two protein extraction methods, namely trichloroacetic acid/acetone precipitation and polyethylene glycol fractionation were used to produce total proteins and fractionated protein extracts respectively, with the aim of improving the resolution of protein species using two-dimensional gel electrophoresis. A total of 40 distinct differential abundant protein spots were selected from leaf samples collected from palms with proven high and low proliferation rates. The variant proteins were subsequently identified using mass spectrometric analysis. Twelve prominent protein spots were then characterised using real-time polymerase chain reaction to compare the mRNA expression and protein abundant profiles. Three proteins, namely triosephosphate isomerase, l-ascorbate peroxidase, and superoxide dismutase were identified to be potential biomarker candidates at both the protein abundant and mRNA expression levels. BIOLOGICAL SIGNIFICANCE: In this study, proteomic analysis was used to identify abundant proteins from total protein extracts. PEG fractionation was used to reveal lower abundant proteins from both high and low proliferation embryogenic lines of oil palm samples in tissue culture. A total of 40 protein spots were found to be significant in abundance and the mRNA levels of 12 of these were assessed using real time PCR. Three proteins namely, triosephosphate isomerase, l-ascorbate peroxidase and superoxide dismutase were found to be concordant in their mRNA expression and protein abundance. Triosephosphate isomerase is a key enzyme in glycolysis. Both l-ascorbate peroxidase and superoxide dismutase play a role in anti-oxidative scavenging defense systems. These proteins have potential for use as biomarkers to screen for high and low embryogenic oil palm samples.

Loss of Karma transposon methylation underlies the mantled somaclonal variant of oil palm.

Somaclonal variation arises in plants and animals when differentiated somatic cells are induced into a pluripotent state, but the resulting clones differ from each other and from their parents. In agriculture, somaclonal variation has hindered the micropropagation of elite hybrids and genetically modified crops, but the mechanism responsible remains unknown. The oil palm fruit 'mantled' abnormality is a somaclonal variant arising from tissue culture that drastically reduces yield, and has largely halted efforts to clone elite hybrids for oil production. Widely regarded as an epigenetic phenomenon, 'mantling' has defied explanation, but here we identify the MANTLED locus using epigenome-wide association studies of the African oil palm Elaeis guineensis. DNA hypomethylation of a LINE retrotransposon related to rice Karma, in the intron of the homeotic gene DEFICIENS, is common to all mantled clones and is associated with alternative splicing and premature termination. Dense methylation near the Karma splice site (termed the Good Karma epiallele) predicts normal fruit set, whereas hypomethylation (the Bad Karma epiallele) predicts homeotic transformation, parthenocarpy and marked loss of yield. Loss of Karma methylation and of small RNA in tissue culture contributes to the origin of mantled, while restoration in spontaneous revertants accounts for non-Mendelian inheritance. The ability to predict and cull mantling at the plantlet stage will facilitate the introduction of higher performing clones and optimize environmentally sensitive land resources.