VIRULENCE ANALYSIS OF XANTHOMONAS CAMPESTRIS PV. SESAMI AND PSEUDOMONAS SYRINGAE PV. SESAMI THE CAUSAL ORGANISMS OF SESAME (SESAMUM INDICUM L.) BACTERIAL BLIGHT

Abstract

Sesame (Sesamum indicum L.) locally called as til is an important conventional oilseed crop of Pakistan. Pakistan ranks 14th among major sesame producing countries in the world. Pakistan is facing a chronic shortage in edible oil and the situation is getting serious with alarmingly explosion of population. Its indegenious production is below the utilization level and there exists wide gap between production and utilization. Sesame crop is subjected to various abiotic and biotic stresses in all stages of growth. Two prominent bacterial pathogens associated with sesame are bacterial blight caused by Xanthomonas campestris pv. sesami (Xcs) and bacterial leaf spot caused by Pseudomonas syringae pv. sesami (Psse). These pathogens are responsible for sesame production constraints during monsoon season. Despite the shortage of edible oil, no profound efforts have been made on this important oilseed crop with reference to diseases. To handle the shortage of edible oil, there was an urgent need to explore the basic information on host pathogen interaction. The present work consisted of five experiments. The first study was the standardization of mass culturing of stored Xcs and Psse isolates to enhance their virulence and confirmation of their ability to induce hypersensitive reaction. All isolates were revived on non host plant and confirmation was made on the basis of pigmentation they produced in their respective media and hypersensitive test was performed in tomato and potato plants. The second study was conducted to analyse the virulence of virulent isolates in vitro by comparing symptoms induction and bacterial multiplication in different genotypes. Plants were inoculated by pin prick method and were monitored daily for symptoms development and measurements of lesions were taken until fully symptoms induction. Bacterial populations were determined by counting bacterial colonies. Psse isolates showed necrotic lesions (chl+) surrounded by halos as well as only black necrotic lesions (chl-). Size of the lesions and bacterial population between chl+ and chl- was the same and at maximum at 7 DAI in susceptible genotypes, while tolerant showed delayed in reaction. Similar mode of lesions expansion and rate of bacterial growth between chl+ and chl- isolates of Psse indicated that the virulence factor involved in symptomatology function as pathogenicity factor and only contributed to induction of chlorotic producing symptoms for Psse. Water soaking to blight symptoms along with maximum bacterial growth in all the susceptible and moderately susceptible genotypes by Xcs was recorded at 12 DAI. The third study was conducted to confirm process of infection of these bacterial pathogens in susceptible and tolerant genotypes by light microscopy. Inoculation was done by Injection method (IM) and Bacterial suspension dip method (BSDM). Xcs colonized tracheary elements of xylem vessels through intercellular spaces of the spongy parenchyma at 7 DAI and bacterial masses were identified as dark blue infected structures using toluidine blue O stain. Blight symptoms by Xcs were reported to be due to the blockage of nutrients and water flow. Psse showed thining and disruption of mesophyll tissues on the appearance of chlorotic symptoms 3-4 DAI. There were only empty spaces of tissues were observed 7 DAI. Overall the infection was same but delayed in tolerant genotypes. Disruption of mesophyll tissues might be due to the action of chlorosis producing toxin (coronatine) that degraded chloroplast membrane of host tissues. The forth study was conducted to detect the virulence factors of Xcs and Psse using suitables bioassays such as antibacterial test, induction of potato hypertrophic outgrowth and seedlings assay. Xcs and Psse (chl-) isolates showed zone of inhibition. The zone of inhibition produced by chl- isolates showed that chl- was not the defective mutant of chl+ isolates as reported in third study, but this test confirmed that these isolates produced another class of toxin that showed antibacterial activity. Induction of hypertrophic outgrowth in potato tuber and seedlings inhibition from culture filtrate of chl+ isolates of Psse confirmed that the toxin produced by these isolates was similar to phytotoxin coronatine (a polyketide molecule) and it might mimics the action of one of the phytohormones. The fifth study was conducted to extract the virulence factors as well as their purification and identification was also performed. Identification was made on the basis of reference data. Crude extracts of acetone preparation of Xcs and Psse (chl-) isolates were concentrated on silica TLC plates. Further purification was carried out by HPLC and TLC. The toxic aciticity eluted from the HPLC column after 10 min corresponding with single active peak showed antibacterial activity. Reverse phase HPLC of chl- isolates extracted partially purified produced an elution pattern like reported in mangotoxin from Pss strain UMAF0158. Acetone praperation of cell free culture filtrates of virulent Xcs also showed active peaks having phytotoxic activity obtained from the HPLC column after 10 min.