P. minus is an aromatic plant, the leaf of which is widely used as a food additive and in the perfume industry. The leaf also accumulates secondary metabolites that act as active ingredients such as flavonoid. Due to limited genomic and transcriptomic data, the biosynthetic pathway of flavonoids is currently unclear. Identification of candidate genes involved in the flavonoid biosynthetic pathway will significantly contribute to understanding the biosynthesis of active compounds. We have constructed a standard cDNA library from P. minus leaves, and two normalized full-length enriched cDNA libraries were constructed from stem and root organs in order to create a gene resource for the biosynthesis of secondary metabolites, especially flavonoid biosynthesis. Thus, large-scale sequencing of P. minus cDNA libraries identified 4196 expressed sequences tags (ESTs) which were deposited in dbEST in the National Center of Biotechnology Information (NCBI). From the three constructed cDNA libraries, 11 ESTs encoding seven genes were mapped to the flavonoid biosynthetic pathway. Finally, three flavonoid biosynthetic pathway-related ESTs chalcone synthase, CHS (JG745304), flavonol synthase, FLS (JG705819) and leucoanthocyanidin dioxygenase, LDOX (JG745247) were selected for further examination by quantitative RT-PCR (qRT-PCR) in different P. minus organs. Expression was detected in leaf, stem and root. Gene expression studies have been initiated in order to better understand the underlying physiological processes.
This study aimed to validate the colonisation capability of endophytic bacteria (EB) isolates, Bacillus cereus EB2 and Pseudomonas aeruginosa EB35, that previously exhibited their potentials as biological control agents (BCAs) against the Ganoderma spp., a pathogen for Ganoderma disease in oil palm. Here, we demonstrated a rapid method to determine the colonisation capacity of the selected EB using oil palm tissue culture plantlets and a green fluorescent protein (GFP) visual marker. Wounded plantlet roots were inoculated with GFP-tagged B. cereus EB2 and P. aeruginosa EB35 while the plantlets without EB inoculation served as controls. The GFP signals appeared as bright green spots or lines in the inoculated GFP-tagged EB cells in root and leaf plantlet tissues, respectively, under the confocal laser scanning microscopy (CLSM) 5 days post-inoculation. In contrast, there was no intense GFP spots in neither the control root nor leaf tissues. The cracks in the roots by wounding facilitated the entry of the GFP-tagged EB cells into root tissues, allowing for endophytically colonisation of the root and above-ground tissues. Subsequent result of polymerase chain reaction (PCR)-GFP analysis further displayed the endophytic nature and early chronological colonisation of the tested EB. This is a preliminary report on root colonisation by a Gram-positive endophyte, B. cereus EB2 and leaf tissues colonisation by both EB isolates as internal colonisers, demonstrating their potential as BCAs to protect oil palm against Ganoderma spp. for a sustainable disease management.
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