Investigating the role of lipid mobilisation and metabolism in the rice blast fungus Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae infects plants by developing a specialised infection structure known as an appressorium. In M. oryzae the appressorium is a melanin-pigmented cell with a reinforced cell wall, allowing the cell to generate enormous internal turgor to enable penetration of the plant tissue by a narrow penetration hypha. Previously it has been shown that mobilisation of lipid droplets to the nascent appressorium is essential for successful plant infection. In this thesis, I describe a series of studies that have identified and characterised genes associated with infection-associated lipid metabolism in M. oryzae, including the role of fatty acid β-oxidation, acetyl-CoA transport and metabolism and regulation of lipid body breakdown. First, I report identification of FAR1 and FAR2, which encode putative Zn2-Cys6 binuclear proteins that appear to act as transcriptional regulators of lipid metabolism. Deletion mutants of M. oryzae FAR1 and FAR2 were deficient in growth on long chain fatty acids. In addition Δfar1 mutants were unable to grow on acetate as a sole carbon source. FAR1 and FAR2 affect the expression of genes involved in fatty acid β-oxidation, acetyl-CoA translocation, peroxisomal biogenesis, the glyoxylate cycle and acetyl-CoA synthesis. Next, I functionally characterized the CAR1, CAR2, CAR3 and CAR4 genes, which encode enzymes involved in carnitine biosynthesis, which is required for translocation of acetyl-CoA between mitochondria, peroxisomes and the cytoplasm. Only a sub-set of carnitine biosynthetic enzymes was necessary for growth on fatty acids and lipids by M. oryzae, but redundancy was also apparent in carnitine biosynthesis, because CAR1, CAR2, CAR3 and CAR4 were dispensable for pathogenicity, while the carnitine acetyltranferase, PTH2, is essential for rice blast disease. To investigate the role of the appressorium acetyl-CoA pool in more detail, I functionally characterized the acetyl-CoA synthetase gene, ACS2 and ACS3, and CRC1, which encodes the mitochondrial carnitine carrier, both of which are highly expressed during appressorium development and appear to play a role in appressorium physiology. Finally, to understand the onset of lipid droplet degradation in more detail, I characterised a putative perilipin, encoded by CAP20, which localizes specifically to the periphery of lipid droplets. Perilipins are known to play roles in lipid droplet mobilisation and lipase accessibility. Consistent with this idea, M. oryzae mutants lacking CAP20, were severely affected in fungal virulence due to impaired appressorium function. When considered together, the results presented in this thesis suggest that lipid body mobilisation and acetyl-CoA metabolism are fundamental processes required for appressoria to function correctly and cause rice blast disease

Duplex PCR assay for the species-specific detection of the marine pathogen, vibrio alginolyticus, using dnaJ and ompK genes

Vibrio alginolyticus, is an important opportunistic pathogen for worldwide aquatic animals and marine environment. However, the microbiological methods like culture-based diagnosis and biochemical identification of V. alginolyticus is time consuming and unspecific. Thus, the aim of the study was to develop a duplex PCR assay for the species-specific identification of V. alginolyticus. To evaluate PCR specificity, this assay directed toward the ompK-virulence gene and dnaJ gene tested on six Vibrio species and three non- Vibrio species. Two specific bands with the expected sizes of 846 bp and 144 bp, respectively, were produced in isolates belong to V. alginolyticus and only one band were produced by others Vibrio species, 846 bp for ompK gene indicating a high specificity of duplex PCR assay. The sensitivity test of duplex PCR was detected by using different cells concentration of V. alginolyticus. The detecting capability of the duplex PCR from crude DNA was at 102 and 103 cells/mL. The sensitivity and efficacy of the assay were clarified using artificially infected Artemia and water culture which a clear PCR bands of 846 bp and 144 bp were generated from Artemia homogenates and water culture infected with V. alginolyticus. Our results showed that this newly developed duplex PCR would offer an accuracy and ideal tool for species-specific detection of V. alginolyticusin preventing disease outbreak in marine aquaculture

Characterization of an azo-dye-degrading white rot fungus isolated from Malaysia

Sixty-three local white-rot fungi were isolated from soil and wood samples on potato dextrose agar (PDA). All these isolates were screened for their ability to degrade 4 textile azo dyes;Ponceau 2R (C.I. 16450), Orange G (C.I. 16230), Direct Blue 71 (C.I. 34140) and Biebrich Scarlet (C.I. 26905). Out of 40 isolates that gave positive results, only 1 promising isolate which completely degrades all 4 dyes in the minimum amount of time was selected for further investigation. This isolate was sourced from University Putra Malaysia (UPM) Serdang campus.The isolate was tentatively identified as Coriolopsis sp. Strain arf5 based on the analysis of the internal transcribed spacer (ITS) region. Nutritional studies on defined solid medium showed that this isolate was only able to degrade the 4 azo dyes under nitrogen-limiting conditions and an additional carbon source (glucose) need to be added to provide sufficient energy for the degradation to occur. Various parameters were optimized

Bioremoval of molybdenum from aqueous solution

Molybdenum is very toxic to ruminants with level as low as 2 parts per million can cause severe scouring. Its contamination of waters and soils in agricultural areas needs novel removal technology. In this work we demonstrated a novel method of molybdenum removal from aqueous solution using the dialysis tubing method coupled with molybdenum-reducing activity of Serratia sp. strain Dry5. The enzymatic reduction of molybdenum is molybdenum blue, a colloid that does not pass through dialysis tubing. The calculated maximal rate of molybdenum blue production (VMoblueMax) was 0.264±0.034 mM (Mo-blue h)-1 and the concentration of molybdate resulting in the half-maximal rate of reduction (KMo) was 21.78±3.89 mM molybdate and the specific maximal rate of Mo-blue production was approximately 80 mM (Mo-blue.hr.mg cells)-1 indicating an efficient system with high tolerance towards molybdenum

Vibrio harveyi protease deletion mutant as a live attenuated vaccine candidate against vibriosis and transcriptome profiling following vaccination for Epinephelus fuscoguttatus

Grouper aquaculture industries have a high risk of being inflicted by bacterial diseases such as vibriosis. Various types of vaccines for vibriosis have been studied throughout the years, yet the potential of live attenuated vaccines remains unsubstantial. Correspondingly, this study attempts to develop a Vibrio harveyi protease deletion mutant into a live attenuated vaccine candidate against vibriosis for Epinephelus fuscoguttatus. Site-directed mutagenesis (SDM) and allelic exchange replacement techniques are employed to synthesize genetically attenuated V. harveyi strain MVh-vhs. The evaluation on safety levels showed that MVh-vhs strain is safe when tested on E. fuscoguttatus. A 100% survival rate with no sign of vibriosis is indicated in fish challenged with the attenuated strain. In contrast, fish challenged with the parental strain showed obvious clinical signs of vibriosis. The median lethal dosage (LD50) of fish challenged with the parental strain is found at 10⁶ CFU/fish. A single dose IP administration of the attenuated strain at 10⁵ CFU/fish following a bacterial challenge at dose 10⁸ CFU/fish is done 4 weeks post vaccination. The vaccinated fish show 52% relative percentage survival (RPS). The transcriptomic profiling following vaccination evoked the regulation of autophagosome pathway and the coagulation and complement cascade pathways as well as antigen processing and presentation pathways. As a conclusion, the V. harveyi attenuated strain MVh-vhs has significant potential to be applied as a live vaccine candidate against vibriosis for E. fuscoguttatus

Purification and properties of polygalacturonase associated with the infection process of Colletotrichum truncatum CP2 in chilli

In this study, polygalacturonase enzyme produced by Colletotrichum truncatum CP2 was partially purified by aqueous two-phase system and the properties of this enzyme was characterized. The highest yield (57.4%) and purification fold (5.1) was obtained using 22% PEG 6,000/15% sodium citrate comprising crude load of 16% (w/w) at pH 7.0 with addition of 1.0% (w/w) sodium chloride. The partially purified PG remained active over a wide range of pH (2.5-6.0) and the optimum activity was obtained at pH 5.0. Incubation of the partially purified PG at 40 and 50 °C for 30 min caused the activity of PG to decrease up to 20% and 40%, respectively. However, no significant changes in the activity when the enzymes were incubated up to 4 h at 40 and 50 °C. The results from this study suggested that ATPS comprising of PEG and sodium citrate could be potentially used as an alternative method for purification of PG

Evaluation of Trichoderma asperellum for inhibiting growth of Fusarium oxysporum f. sp. lycopersici and enhancing growth of tomato and fruit quality

Fusarium wilt is a soil borne disease causing severe losses in tomato plants. The pathogen perseverance in the soil makes the disease difficult to control. Therefore, this study was conducted to examine the efficacy of the biocontrol agent Trichoderma asperellum against Fusarium oxysporum f. sp. lycopersici, causal agent of Fusarium wilt disease of tomato based on in vitro and in vivo conditions. Trichoderma asperellum B1092 inhibited mycelial growth of the pathogen under in vitro condition using poison agar method. As in vivo, formulation of Trichoderma asperellum-enriched oil palm empty fruit bunch was significantly enhanced the growth parameters of tomato seedlings as compared to the control and it also significantly reduced the wilt disease severity. As a conclusion, T. asperellum B1902 may offer the potential for biologically controlling Fusarium wilt of tomato, inducing the growth of tomato seedlings and improve its quality

Characterization of phenol-degrading fungi isolated from industrial waste water in Malaysia

Microorganisms have the ability to degrade phenol. However, in Malaysia, there are lack of study on indigenous microorganisms (fungi) that have the ability to degrade phenol. A total of 141 phenoldegrading fungi isolates were isolated from soil and water samples collected from various industrial areas located in Malaysia. The fungi isolate N12 P6C3 was chosen based on its high efficiency in degrading phenol. The fungi isolate N12 P6C3 isolated from a heavy metal factory, Dungun, Terengganu was able to degrade 700 mg/L of phenol within 6 days and the mycelium growth had increased to 0.25 g. The phylogenetic tree based on the ITS sequence analysis confirmed that the fungal identity was closely related to Penicillium janthinellum strain ATCC 4845. The optimum conditions of this fungus to degrade phenol was attained at temperature of 35°C, ammonium sulphate at 3 g/L, 0.05 g/L of sodium chloride, and pH 6. The ability of P. janthinellum strain N12 P6C3 in the degradation of phenol may provide additional knowledge on locally isolated phenol-degrading fungi which could contribute towards phenol waste management in Malaysia

Heavy metals biomonitoring via inhibitive assay of acetylcholinesterase from Periophthalmodon schlosseri

Acetylcholinesterase (AChE) generally known to be sensitive toward insecticides but its sensitivity toward heavy metals was least reported. Herein, a sensitive assay for heavy metals has been pursued using AChE in a rapid and economic manner. The AChE from a mudskipper, Periophthalmodon schlosseri has been found to be sensitive toward copper > mercury > chromium > and arsenic ions at the sub parts per million levels. Field trial works showed that the assay was applicable in detecting heavy metals pollution from effluents of industrial sites at near real time and verified using ICP-OES and Flow Injection Mercury System (FIMS 400). Furthermore, hierarchical cluster analyses of inhibition profiles were performed, revealing a comparable capability of the AChE compared to the gold standard of Microtox™ method

Recent update on the prevalence of Vibrio species among cultured grouper in Peninsular Malaysia

Vibrio infections are common among marine fish and lead to serious problems in the aquaculture sector. This study reports a recent occurrence of Vibrio species (spp.) isolated from cultured groupers in Peninsular Malaysia using the gyrB and pyrH genes. A total of 147 Vibrio strains were successfully isolated from 77 (64%) groupers using culture method and subjected to gyrB and pyrH sequencing for species identification and confirmation. Results showed that 89% of Vibrio strains were identified and clustered to six groups of Vibrio spp., while 11% were not clustered to any Vibrio spp. using the gyrB sequences. Meanwhile, by analysis of the pyrH sequences all the 147 Vibrio strains (100%) were successfully identified and clustered into 11 groups of Vibrio spp., including the gyrB non‐identified strains. The pyrH gene provides a better resolution for identification of Vibrio spp. compared with the gyrB gene. Thus, the pyrH gene was more suitable for a rapid determination of Vibrio spp. distribution in Peninsular Malaysia. Using the pyrH gene, our study found higher prevalence of Vibrio vulnificus (33%), V. alginolyticus (24%) and V. parahaemolyticus (22%), followed by V. rotiferianus (5%), V. harveyi (3%), V. tubiashii (2%), V. campbellii (2%), V. ponticus (1%), V. diabolicus (1%), V. owensii (1%) and others Vibrio sp. (7%). Thus, the results of this study revealed that the occurrence of pathogenic vibrios among grouper fish is still high in Malaysian aquaculture. In addition, the pyrH gene was proved as a suitable marker for rapid identification of Vibrio species compared with the gyrB gene