Isloation of Alternaria and Fusarium spp., their Pathogenesis and Pathogenesis Related (PR) Protiens Associated with Sesame (Sesamum indicum L.)

Abstract

Sesame is one of the oldest and an important oil seed crops of the world known to human being, holding special importance due to the high-quality oil produced from the seed. Sesame seeds contain 48-60% oil and 18-23.5% proteins. It is very valued cash crop because of the little expenses of production and great market value. Pakistan ranks fourteenth amongst the major sesame producing countries of the world. In Pakistan, sesame is grown in all four provinces. The Punjab province covers 28.5% of the land area of Pakistan but contributes almost 90% to total sesame production in the country, and is considered a major sesame producing area of South Asia. Despite the multiple uses of sesame, it’s farming in Pakistan is highly disappointing because of the low yields per hectare. The demand to supply ratio of edible oil is raising with the alarming rate of human population increase in Pakistan, but the generation of edible oil is declining each year. There is a wide gap, therefore, between production and consumption of edible oil in the country. The sesame crop is subjected to various biotic and abiotic stresses in all growth stages. Low yields of sesame in Pakistan may be attributed to attacks by various pathogens, amongst which two fungal pathogens predominate: Alternaria and Fusarium, which are responsible for blight and wilt diseases, respectively. Even with low yields and high demand for edible oil, no concerted efforts have been made on this highly valuable cash crop with respect to these two major pathogens. To address this lack of effort, there was an urgent need to explore the plant-pathogen interactions between sesame and Alternaria and Fusarium. The aim of the work presented in this thesis, therefore, was to study the incidence of Alternaria and Fusarium species on sesame seeds and plants, to determine their virulence, and to provide an evaluation of the potential for pathogenesis-related proteins to be used in examining the acquisition of systemic acquired resistance in response to these destructive pathogens. The prevalence of Alternaria and Fusarium species associated with sesame seeds from crops in the Punjab province was determined to illustrate the contributions of these pathogens to yield losses in production in South Asia. For this purpose, 105 seed samples were collected directly from the fields of major sesame producing areas in the Punjab and subjected to the standard blotter paper method for the isolation of fungi. A total of 428 isolates of Alternaria and 520 isolates of Fusarium were recovered and grouped into 36 and 23 distinct taxonomic groups on the basis of growth patterns and morphology in vitro. Three isolates of Alternaria (A13, A47, A215) and three of Fusarium (F01, F98, F153) were chosen as representative of the most common morphological groups recovered from seed, and identified using molecular methods based on DNA barcoding using universal (ITS) and species-specific primers. The identities of these isolates of Alternaria and Fusarium were confirmed as Alternaria alternata and Fusarium proliferatum based on sequencing data obtained major allergen gene (Alt a 1) and translation elongation factor (TEF-1α), respectively, after initial identification using internal transcribed spacer (ITS) region of ribosomal DNA of both genera. This basic work on seed testing demonstrated that seeds can be badly contaminated by pathogenic fungi so the detection of seed-borne pathogens and their accurate identification is an important step in crop disease management. The pathogenicity and virulence of the identified isolates of Alternaria and Fusarium on sesame seeds and plants was investigated using both fungi and culture filtrates. Sesame seeds and intact sesame plants were inoculated or treated to confirm the effects of the pathogens on germination, seedling growth and overall plant health. All isolates tested were pathogenic to sesame, but A13 and F01 were the most virulent, causing the lowest germination and highest disease incidence (DI) and disease severity index (DSI). Culture filtrates of A13 and F01 also reduced seed germination and vigor of sesame seedlings in the absence of fungal cells. Typical symptoms of blight or wilt appeared after application of spore suspensions of these isolates on sesame plants. Alternaria isolates caused foliar blight, leading to browning and defoliation. Fusarium isolates induced wilting symptoms on the plants, with yellowing of leaves, and internal staining of stem and roots, ultimately leading to disintegration. The inoculations confirmed that Alternaria and Fusarium are capable of causing leaf blight and wilt diseases in sesame, and may be amongst the causes of sesame yield losses in the Punjab. The possibility of using exogenous application of chemical and biological elicitors of plant defense responses, for example salicylic acid, methyl jasmonate and Pseudomonas syringae for induction of systemic acquired resistance (SAR) against Alternaria and Fusarium was determined. Induction of SAR was evaluated through assessing the accumulation of Pathogenesis-Related protein family 1 (PR1) by western blotting and determining metabolomic changes in the plants following treatments. Total protein content of extracted plant samples was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunological detection of PR1 proteins. For investigation of metabolomic changes in primed and control plants, non-targeted metabolites were extracted and metabolite profiling was carried out by Linear Trap Quadrupole (LTQ) mass analyzer attached to Surveyor HPLC system using Flow Infusion Electrospray – Mass Spectrometry. The work demonstrated clearly that SAR occurred in sesame. The mechanisms of SAR included accumulation of PR1 proteins, biomarkers of the phenomenon in primed plants. The metabolomic changes associated with SAR were to metabolites involved in lipid/sterol metabolism, the TCA cycle and the defense based phenylpropanoid pathway. The work presented in this thesis demonstrated unambiguously that species of Alternaria and Fusarium infect sesame in the Punjab, Pakistan, and some isolates of these species are of high virulence. The finding that SAR occurs in sesame provides a potential novel and environmentally-friendly method for the control of fungal pathogens in integrated crop management systems.