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No description available.

Research detailed in this thesis investigated the generation of Nitric Oxide (NO) and its role in the pathogenesis of the rice blast fungus Magnaporthe oryzae. Two putative nitric oxide synthase genes and single copy nitrate and nitrite reductase genes were identified as potential sources of NO in M. oryzae. Single and double gene disrupted mutants were generated and their phenotypes assessed. Detection of NO is problematic. Herein, a fluorescent plate reader assay was developed, exploiting the NO sensitive dye DAR-4M AM and the NO scavenger PTIO, to compare wildtype NO generation with the mutant strains. All strains were assessed for infection-related development on an artificial surface inductive to appressorium formation and maturation in the wildtype strain. Appressorium formation in the presence of PTIO and the NO donor DETANONOate was recorded for all strains on this surface. The pathogenicity of the wildtype and mutant strains were assessed, in terms of their ability to infect rice and barley plants. Finally, the capacity of each strain to metabolise nitrogen was evaluated to confirm the disruption of the nitrate and nitrite reductase genes. Collectively, the data demonstrates that the plate reader assay provides robust evidence for the generation of NO in M. oryzae. However, none of the various mutant strains showed a reduction in NO emission during germling morphogenesis. However, they exhibited significantly different infection-related development on an inductive artificial surface as compared with the wildtype strain. Moreover, exogenous application of PTIO to the wildtype strain provided evidence for NO and its involvement in germination and appressorium development. No significant differences in pathogenicity were observed between the wildtype and mutant strains, indicating the disrupted genes are dispensable for pathogenesis. The nitrate and nitrite reductase genes were found to be essential for nitrate assimilation. In summary, this work provides the most robust evidence for the generation of NO in fungi to- date, but the molecular mechanism underpinning the generation of NO remains elusive.

The 'C4 rice project' aims to introduce the C4 photosynthetic mechanism into C3 rice plant in order to boost biomass and grain yield to cope with the predicted food demand and adverse environmental conditions in the future. As a first step, identification of rice mutants with novel photosynthetic traits conferring increased photosynthetic capacities is an utmost requirement in order to improve biomass production. One approach is to screen large numbers of rice mutants to achieve this goal. This would confirm whether the rice genome possesses the required 'plasticity' to introduce C4-like traits. Two traits are ubiquitous in the C4 syndrome and can be introduced into high throughput screens: anatomically they have higher leaf vein densities and physiologically they have lower CO2 compensation points (r). As a first step, a systematic screening experiment was conducted to identify C4 properties in a gamma-induced, EMS-induced mutant populations of IR64. The major objectives are to develop reliable rapid screening techniques and to identify C4-like photosynthetic traits in rice. A mass-screening chamber with low-C02 treatment (1000 seedlings per week) was used. Rice seedlings at 5th leaf stage were exposed to 60 ppm CO2 for the first day and 30 ppm for the remaining three days. Following this, putative candidates were identified by measuring chlorophyll using SPAD method by a SPAD depletion % (SPD%) calculation . Phenotypic responses of the mutants were compared with wild type IR64 and with the Cr C4 intermediate Panicum milioides. Initial SPD% results showed a distinct gap between parental wild type IR64 and P. milioides, means ranging from 65% to 11% respectively, whereas in the mutant lines SPD% spanned 0 to 100%, suggesting considerable phenotypic variation in response to low CO2 in the mutant population. Mutants with lower SPD% «10%) were identified as 'Chlorophyll retention mutant' (CRM) variants under low CO2 stress. Up to now, 1909 mutant lines have been screened for low SPD% under 30 ppm CO2 and 28 CRM lines from M4 families were identified as putative candidates with tolerance to low CO2 were self seeded and produced Ms generation lines to determine the genetic control of the altered response to low CO2. Ms putative mutants were subject to physiological measurements. Leaf gas exchange analyses with the U-6400 XT portable photosynthetic system were performed for CO2 compensation point (r) carboxylation efficiencies (CE) and CO2 assimilation rates at ambient levels (A400). A comprehensive profile of r in wild type rice plants were revealed, In early growth stages (8st to 11th leaf) rice displayed an increased r of above 50 J.!mol mol-1 , whilst leaves produced in the reproductive stages (lih ,13th , 14th and Flag leaves, FL) showed a sharp decline in r and reached below 50 J.!mol mor1 with the lowest r seen in FL. With the analysis of rice CRM lines, it was shown that three mutant lines had significantly declined r in early growth stages ( CRM 27, CRM 29 and CRM 65) than the WT, whilst further analysis with CRM 29 showed a sharp decline in r (43 J.!mol mol-1 ) in FLs. Additional photosynthetic analysis indicated CE and A400 were superior in these lines compared to WT. Photosynthetic correlation studies was shown a negative correlation between rand CE and A400 suggesting lowering r in rice is beneficial in terms of improving rice photosynthetic efficiencies in CRM lines. Detailed anatomical investigations were carried out in CRM 29 FLs which displayed significantly increased mesophyll cell plan area and number of lobes per cell than in WT. Biomass analysis suggested these three mutant lines had superior ability in producing AGDM, panicle weight than the control plants, suggests moderately lowering r with improved anatomical traits in rice may be associated with greater biomass and grain yield in CRM lines. In conclusion, it is optimistic to produce an intermediate C4-photosynthetic rice plant that can out-yield current wild types before successfully incorporating a complete functioning C4 pathway into rice. Significant changes may be achieved by targeting existing metabolic processes and leaf anatomy in rice. There is a need to search the genes which determine these novel photosynthetic and anatomical features in leaves of rice. Thus a co-ordinated attempt of plant physiologist, geneticists, and the molecular biologists is required. ii

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Use of known antagonists and antagonistic, endophytic bacteria was investigated to screen a successful antagonist for the fungal strains <i>R. solani</i> AG 2-1, <i>R. solani</i> AG 4 and <i>R. solani</i> AGSL01.   Reputed antagonists used in the study; <i>Bacillus subtilis </i>MBI 205,  <i>B. subtilis</i> MBI 600, <i>Pseudomonas fluorescens</i> B5 and <i>P. corrugate</i> R. 117 were capable of suppressing the fungus in dual cultures. Manipulation of soil factors to reduce pre-plant density of <i>R. solani</i> sclerotia involved a laboratory experiment followed by two field experiments.  In the laboratory study, the effect of constant soil temperatures was investigated by incubating sclerotia at 30, 35, 40, 45 and 50°C.  Sclerotia were counted into lots of 100, placed in polyester mesh bags (85 μm pore size, 10 x 10 cm dimension) at depths of 5 and 10 cm in rice field soils contained in plastic containers (20x20x18 cm).  Total loss of viability was observed on day 1, day 8 and day 28 at 45°C, 40°C and 35°C, respectively.  Loss was even detected within 6.00 h at 50oC. Field experiments of soil solarization (carried out at The Regional Agricultural Research Institute (RARI), Bombuwela, Sri Lanka) were conducted during the fallow periods between the two main cropping seasons in 2003.  During the trials, the effect of polythene mulching, straw incorporation and their combined effect on the viability of sclerotia were investigated.  Sclerotia (lots of 100) were buried at depths of 5 and 10 cm and the soil temperature was recorded at 8.00, 11.00, 14.00 and 17.00h daily at both depths.  During the field trials, at both depths, the effect of treatment over time was noted on percentage recovery and viability of sclerotia (<i>p</i><0.001).  The results showed a drop in mean percentage recovery of less than 50% during the first week of both trials.  Germination was markedly reduced to less than 10% by the first week in all the treatments.  Depth of burial and straw incorporation had no effect.  In solarized plots, a significant increase in soil temperature at both soil depths was observed between 14.00 and 17.00h.  Average temperatures of 40°C and 34°C were observed for solarized and non-solarized plots, respectively.  The study therefore has identified a practical, low cost and environmentally friendly method of control, and the use of polythene five weeks prior to rice seed sowing is recommended to minimise sheath blight and other diseases caused by similar soil-borne fungal pathogens.