Oil palm is a major agricultural commodity in Southeast Asia, particularly in Malaysia and Indonesia. Its productivity, however, is being jeopardised by climate change and disease outbreaks, particularly basal stem rot (BSR) caused by Ganoderma boninense. Monoculture practices exacerbate disease spread, calling for innovative and sustainable solutions. Beyond current integrated plant management (IPM) practices, developing disease-tolerant plant material could enhance disease management. For that purpose, efforts have been initiated to identify potential genetic markers associated with G. boninense tolerance; include in vitro screening (IVS) using oil palm clonal material is employed, where artificial infection is conducted under a controlled environment. However, the reliability of such studies depend on accurate quantification of gene expression, which requires the use of reliable reference genes (RGs). Quantitative PCR (qPCR) is a powerful tool for measuring gene expression levels; however, reliable normalisation depends on RGs that maintain consistent expression across tissues and experimental conditions. Consistent RG expression is essential for minimising technical and biological variation, ensuring that observed changes in target gene expression reflect true biological responses rather than variation arising from the normalisation process. Therefore, this project evaluated ten potential RGs selected from the literature for gene expression analysis in young oil palm ramets under controlled G. boninense-infected conditions. Among the ten RGs evaluated, ubi and fbox were consistently identified as the most stable and reliable RGs for in vitro expression analysis under controlled disease-stressed conditions, based on geNorm, NormFinder, BestKeeper, and RefFinder analyses. In contrast, gapdh was unsuitable as an RG under these conditions. The RGs identified and validated in this study are anticipated to enhance the accuracy of gene expression analysis in G. boninense-infected oil palm ramets under controlled in vitro conditions. This approach will be instrumental in identifying potential defence-related genes associated with G. boninense tolerance through comprehensive gene expression profiling.
The exploration of natural plant sources for functional ingredients has gained significant attention in food science, with emphasis on bioactive compounds that can enhance human health. Crescentia cujete L. (calabash tree) is traditionally valued in folk medicine, yet the safety of its bark extracts for functional food applications remains underexplored. This study evaluated the safety performance of hot water and ethanol extracts of C. cujete bark using the zebrafish embryo toxicity (ZFET) model, an established in vivo tool for early toxicological screening. Fertilised zebrafish embryos were exposed to graded concentrations of each extract from 0 to 120 hr post-fertilisation (hpf), and endpoints including survival, hatching, heart rate, and morphological development were monitored. The hot water extract demonstrated a significantly higher median lethal concentration (LC50) of 9.69 mg/mL, while the ethanol extract exhibited a markedly lower LC50 value of 0.42 mg/mL (p<0.001), suggesting higher relative safety of the aqueous preparation. Dose-dependent reductions in survival and hatching rates were observed, with ethanol extracts producing more severe developmental delays and cardiac abnormalities. Heart rate disturbances, pericardial edema, and axial malformations were most prominent at concentrations near the ethanol LC50 value, whereas the hot water extract induced only mild effects at higher concentrations. No significant changes in heart rate were observed with the hot-water extract, whereas significant reductions were observed at higher concentrations of the 50% ethanol extract (p<0.05). These findings indicate that the extraction solvent significantly influences the safety profile of C. cujete bark. The hot water extract demonstrated lower developmental toxicity in the zebrafish embryo model and warrants further investigation through phytochemical characterisation and mammalian toxicity studies. By establishing the first zebrafish-based safety evaluation of C. cujete bark, this study highlights the importance of integrating toxicological validation in the development of novel plant-derived functional ingredients.
Potassium (K) is one of the macronutrients essential for plant growth; however, most soil K exists in insoluble mineral forms that are not readily available for plant uptake. The use of potassium-solubilizing bacteria (KSB) offers a sustainable approach to enhance K bioavailability in agricultural systems. This study aimed to isolate, characterize, and identify KSB from the rhizosphere of seven local rice varieties cultivated in Lima Puluh Kota Regency, West Sumatra, Indonesia. Total culturable heterotrophic bacterial populations were enumerated using nutrient agar, and one morphologically distinct colony from each sample was purified for further analysis. K-solubilizing activity was evaluated on Aleksandrov agar by measuring the dissolution index (DI) and in liquid Aleksandrov medium supplemented with K-feldspar powder. All seven isolates formed clear halo zones on solid medium. The highest DI value was observed in isolate KSP (2.2), followed by KKP and KJG, while KRK exhibited the lowest DI. In liquid medium, soluble K release ranged from 0.95 to 1.95 mg L⁻¹, with KSP demonstrating the highest solubilization capacity. Statistical analysis revealed significant differences among isolates (p<0.05). Molecular identification based on 16S rRNA gene sequencing indicated that the isolates belonged to Lysinibacillus xylanilyticus, Bacillus velezensis, Acinetobacter johnsonii, Pseudarthrobacter phenanthrenivorans, Klebsiella aerogenes, Paenarthrobacter nicotinovorans, and Enterobacter cloacae. These findings demonstrate the diversity and functional potential of indigenous KSB associated with rice rhizosphere soils and support their prospective application as biofertilizer candidates for sustainable rice production.
Small open reading frames (sORFs) are emerging as key players in bacterial gene regulation, yet their roles remain underexplored in many pathogens. This study investigates the regulatory landscape of sORFs in Group A Streptococcus (GAS) by integrating transcriptomic data and weighted gene co-expression network analysis (WGCNA). We focused on the functional consequences of inactivating the Type 1 Restriction-Modification (RM) system, specifically the hsdM subunit, which is a known barrier to genetic transformation in GAS. By reanalysing the transcriptomic profile of the hyper-transformable 854ΔhsdM strain, we identified 28 sORFs localised within significantly altered co-expression modules. Notably, sORF_00369 (associated with fatty acid biosynthesis) and sORF_00282 (linked to ABC transport) were significantly downregulated. These findings suggest that the inactivation of the RM system, while improving transformation efficiency, may trigger a targeted shift in sORF-mediated metabolism and membrane integrity. Our results propose that sORFs are candidate regulators within the epigenetically mediated regulatory networks of GAS, providing new insights into how genetic recalcitrance and metabolic fitness might be linked. This in silico study lays a theoretical foundation for potentially targeting sORFs in antimicrobial strategy development and optimising GAS as a chassis for synthetic biology.
