INTRODUCTION Uganda is a country whose agro-climatic conditions can support a wide range of horticultural crops.

Passion fruit is one of the populars, nevertheless knowledge on its effective propagation, yield and diseases is greatly lacking. (Mwaku, A.1990) Passion fruits belong to the Passiflora.sp, they are one of the most important fruits grown in Uganda. With 16 passion fruit growing districts mainly in the lake basin districts of; Tororo, Iganga, Mukono, Kampala, Mpigi and Rakai, Central; Luwero, Mubende, Kiboga, West; Kasese, Kabarole, Hoima and East; Mbale. In Uganda collar rot has become an economically important disease, it is caused. (Emechebe, A.M 1976) Edible passion fruit varieties in Uganda Purple passion fruit; Passiflora edulis Sim. Bears ripe purple fruits, short-lived perennial, densely covered with dark green, deeply lobed leaves with elongated tendrils in their axils on jointed stalks. Fully ripe fruits are round to oval in shape 4-5cm in diameter with hard, leathery, purple skin. Fruit cavity contains numerous small, black seeds surrounded by a yellowish, aromatic pulp which has a pleasantly acidic flavour, several strains occur varying mainly in fruit size. Yellow or Golden passion fruit; Passiflora edulis var flavicarpa Yellow or golden passion fruit presumed to have originated in Queensland, Austria, possibly a hybrid between purple passion fruit and an unknown species introduced into Uganda in 1970. Yellow on maturity and larger than purple passion fruit, more vigorous and thrives better in heavy rainfall areas. Some shows signs of unfruitfulness which is attributed to the protanderous nature of flowers i.e. stamens ripen before the stigma. Fruits are usually spherical, when immature, the rind is mottled with white spots but ripe fruits are pale golden or yellow coloured. This passion fruit has more aromatic and acidic pulp than purple. Hard shelled passion fruit; Passiflora maliformis Or calabash, relatively small fruit 4cm in diameter with light green to brownish skin and very hard rind enclosed within three large cream-coloured bracts. Seeds are small and pulp grayishyellow. Flavour is inferior to purple passion fruit. Immne to several diseases and insect pests of Passiflora.spp. Granadilla; Passiflora quadrangularis A fruit which has adapted well to most parts of Uganda at high temperatures and high humidity optimums. Under dry atmospheric conditions the flowers produce little or no pollen and fruit set is poor. The fruit is oval in shape 15-22cm long, it is initially green in colour-yellow at maturity when the flesh becomes soft. It has a characteristic soft rind which is approximately 4cm thick. The main difference from other species is that the seeds and pulp are edible. Kawanda hybrid passion fruit- Purple x Yellow

Preliminary investigations started at Kawanda Research Center in 1970, 3 cultivers namely Kiisi, New Guinea and Mubende local (all of which are purple passion fruits-P.edulis. Sim) succumbed to brown spot disease. Yellow passion fruit Passiflora edulis var flavicarpa showed a high degree of resistance. So Mubende local which somewhat had resistance to the disease was crossed with yellow species with the aim of producing a variety which combined both the quality of the purple with the brown spot resistance of the yellow. (Emechebe, A.M 1976) Collar rot disease One of the commonest diseases of passion fruit is the fusarium wilt also known as collar rot, caused by Fusarium oxysporum. f.sp passiflorae. Symptoms of the disease include wilting of the leaves, the collar region of affected plants also turns brownish, vertically cracks and the vines wilt followed by a complete collapse of the plant. On dissection of the infected stem, vascular tissues show a brown discolouration. Fusarium wilt is a disease particularly for local purple and Kawanda hybrids, the soil borne fungus attacks the rooting system of affected plants. The disease is then spread upwards along the stem and one can easily recognize brown patches scattered on the stem. Since attacks originates from the roots, translocation of water and minerals from the soil are interfered with and the plant eventually dies. Origin of the fungus The fungus is seed borne or soil borne. It may become established in many types of soil. Fungus produces resting spores called chalmydospores which can survive in soil indefinitely even with no host plant. Spores can also survive in fibrous roots of weeds i.e. Amaranthus, Digitata and Malva. The fungus is spread by movement of infested soil or infected plant parts. Conditions favourable for the fungus For survival the fungus needs high nitrogen levels in the soil, soil moisture stress, high soil temperatures and Fusarium infested soils. Fungal identification Is made using conidia size, length/width ratio, shape, septation and structure of top and foot cells, nature of aerial mycelium, pigmentation/spore colour, presence of chlamydospores, sporodochia and promates, growth rate on Potato Dextrose Agar (PDA). (Wollen, W.1986/ Emechebe, A. 1976) Aerial mycelium have both microconidia and macroconidia. Phialides are long, septate and branched conidiophores. Shape of macroconidia; they are slightly bent, sausage or cylindrical shapped with round apical cells slightly bicked and marked foot cells.

Shape of the micro conidia; varies from oval, round or even oblong, thick walled chlamydospores can also occur in singles, doubles or chains. RNA interference A naturally occurring post-transcriptional gene silencing phenomenon, evolutionarily conserved and sequence specific. Mechanism Long double stranded RNA molecules enter the cell and are processed by the Dicer Complex resulting in formation of small inhibitory RNA (siRNA). Alternatively, to induce RNAi these small 21-23 bp duplexes are directly delivered into the cell. The siRNA are incorporated into a nuclease-containing multiprotein complex called RISC, which becomes activated upon ATPdependant unwinding of the siRNA duplex by an RNA helicase. The now single stranded siRNA guides the RISC complex to its complementary target mRNA which it then cleaves by endonucleotyltic activity of RISC. While the RISC complex is recovered for futher cycles, the cleaved mRNA molecule is rapidly degraded due to its unprotected RNA ends. Application of RNAi in crop biotechnology Host resistance has long been identified as the most feasible and sustainable way of managing diseases in crops. This is achievable through conventional cross breeding and or genetic modification (GM) approaches. Among these is antifungal protection strategy and RNAi. This inhibits expression of one or more target genes in a phytopathogenic microorganism leading to cessation of infection, growth, development and reproduction and eventual death of the pathogen. (Kwanda library) Challenges in application of RNAi The greatest challenge of RNAi based crop protection strategies is the identification of the right target genes in the pathogenic organism. Secondly the RNAi sequence chosen should share no homology with host genes, this is to ensure that it remains inactive until parasitism occurs by the target pathogen. (Kwanda library) The development of the passion fruit production industry in Uganda has been seriously hindered by the outbreak of collar rot disease. Until now, no appropriate control measures have been developed. Therefore, a study was initiated to develop a sustainable, environment-friendly collar rot control package. The potential of chemical control was assessed. Chemical control was found to be economical only in the case of copperoxychloride applied as a drench at a rate of 60 g/20 l of water. Other fungicides tested were Rovral 70 WP (iprodione) and Cercobin 50 L (thiophanate methyl). To address the environmental issue, use of resistant species was investigated. Two species, Passiflora edulis f. flavicarpa and P. maliformis were found to be partially resistant to collar rot, and can be recommended for use as rootstocks to susceptible P. edulis f. edulis and hybrid clones. (Ssekyewa, C. 1999)

GENERAL OBJECTIVE The main objective of this study is to characterize the fungal pathogen and investigate the invitro antifungal potential of synthetic dsRNA molecules towards a pathogenic fungus of passion fruits. SPECIFIC OBJECTIVE I) Molecular characterization of Fusarium oxysporum. f.sp passiflorae from different bioecologies in Uganda. II) Evaluation of antifungal activity of synthetic dsRNA on spores of Fusarium oxysporum. f.sp passiflorae. STATEMENT OF THE PROBLEM Uganda is a country whose agro-climatic conditions can support a wide range of horticultural crops. Passion fruit is one of the populars, Nevertheless some of the knowledge is lacking on its effective propagation (Kwanda library) in particular about diseases affecting it and their effective control. The development of the passion fruit production industry in Uganda has been seriously hindered by the outbreak of collar rot disease. Until now, no appropriate control measures have been developed. Therefore, a study was initiated to develop a sustainable, environment-friendly collar rot control package (Ssekyewa, C. 1999)

METHODS Study areas These will be selected randomly from the three passion fruit growing regions in Uganda ie West, Central and Eastern. Selected districts will then have randomly selected sample materials taken from plants infected with collar rot. Production and purification of the fungus from plants Diseased stem samples will be collected from the field or isolated from soil using passion fruits as bait. Soil will be put in the fruits which were surface sterilized with 10% sodium hypochlorate for 15 minutes. Inoculated fruits will then be incubated at 28 degrees centgrade for 7 days, while the stems will be incubated at the same temperature but for 3 days. DNA isolation Extraction of DNA from mycelia of fungal colonies will be done using a Bacteria DNA kit. Sterile sea sand will be used to crush a sample of mycelia, cells will then be lysed. An instruction manual will then be used to extract the DNA. Quality of extracted DNA will then be assessed by running the extracted DNA on an Agarose gel by electrophoresis. Bands on the gel will be documented using a gel documentation system. Amplification of target DNA. PCR will be performed using primers specific for the selected Fusarium oxysporum. f.sp passiflorae genes for inhibiton. PCR will involve 10minutes initial denaturation step 90 degrees Celcius for 40 cycles consisting of 1 minute denaturation, 1minute primer annealing at 55 degrees Centigrade and a minute extension at 72 degrees followed by 10 minutes extension step at the same temperature. Amplified products will then be size fractionated inclusive of a marker, by Agarose gel electrophoresis in appropriate buffer at given volts. Gels will be stained with ethidium bromide in an aqueous solution for viewing. Antifungal dsRNA assays Efficiency of the assay will be decided relying on the reduction in number of fungal colonies on PDA. Spore suspensions will be prepared for administration of dsRNA following the appropriate protocol from Vengaza.USA. Optimization of dsRNA To determin the optimum test concentration for the dsRNA to use in spore germination inhibition bioassays, tests will be carried out using dsRNA molecules homologous to essential genes for inhibiton with three different concentrations of dsRNA and a control which will contain no dsRNA.

(Kwanda library) Data analysis Optimization data for the dsRNA assays will be done using ANOVA. Significance of the difference between means of treatments will be compared using Fishers test. Inhibition % = Treatment (Av. No. of colonies) Control (Av. No. of colonies)

REFFERENCES Emechebe,A.M and Makumbi,J. (1976) Nectaria collar and root rot of passion fruit in Uganda. (Plant Disease Report 60:227-231) Horticulture Research Program, (October 1998). Kawanda Agricultural Research Institute: Passion fruit quality evaluation and improvement; Kasese District Case Study Kawanda Agricultural Research Institute Library Ssekyewa, C., Opio, A. F., Swinburne, T. R. , Van Damme P. L. J. & Abubaka, Z. M. Sustainable management of collar rot disease of passion fruits in Uganda. International Journal of Pest Management. Volume 45, Issue 3, 1999