Alternaria alternata (Accession Number: 1200) was collected from the Fungal Culture Bank of Pakistan (FCBP), Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan. For the multiplication of fungus, under aseptic conditions fungus was placed on Potato Dextrose Agar (PDA) media. Then incubated at 28˚C for seven days for fungal growth. The growth of fungus obtained on PDA media was re cultured. Culturing and sub culturing was continued until the pure culture was obtained. The pure culture of fungus was stored at 4˚C and periodically cultured. For slide preparation, Small amount of lacto phenol was placed on glass slide with the help of dropper. With the help of sterilized needle, fungus was placed on slide and covered with coverslip with the care that no air bubble was formed. The slide was observed under microscope and photographs were taken.

Three varieties of sesame seeds TS 3, TS 5 and SG 27 were collected from the Oil Seed Program of National Agriculture Research Centre (NARC). Seeds were surface sterilized with 2 percent sodium hypochlorite (NaOCl) for 2 minutes and then rinsed three times with distilled water. Potting mixture contains sand, clay and Farm yard manure with the ratio of 2:1:1. Sesame seeds were sowed in pots with ten replicate of each variety. Each pot contains four to six seeds. Plants were watered regularly. After 40 days of sowing, when the seedlings were at six to eight leaf stage conidial suspension of Alternaria alternata was inoculated. For the preparation of conidial suspension, 5 ml of sterilized water were added in 15 days old culture plate of pathogen and conidia were detached with tooth brush and suspension was collected in sterilized beaker. The number of conidia were counted with the help of hemacytometer and adjusted to 2 × 10³ conidia per ml [30] .

Pathogens were inoculated by spray method. Spray was done in evening and inoculated plants were covered with plastic bags for 24 hours after inoculation and placed under darkness to provide maximum humidity for penetration of fungus into host cells. The plants sprayed with sterilized distilled water were served as control. When artificially inoculated plants showed the typical symptoms of disease, their histopathology was done.

For histopathological study, following steps were performed [31] .

On PDA media, rapid growth of Alternaria alternata was observed (Figure 1). Maximum growth of fungus occurred in fifteen days at a temperature of 26˚C - 28˚C. At earlier stages, the colour of colony was grey whitish but at maturity its colour became brownish black. Fungus forms a compact mass like a carpet. The shape of the colony was circular and margins were smooth, entire or irregular. Fungus grows in all directions equally from the centre without the formation of concentric rings. These results are supported by the [34] - [38] .

Microscopic examination of Alternaria alternata shows that the mycelium of the fungus was branched, multicelled, septate, dense aerial and golden brown in colour (Figure 2). Hyphae were branched, septate, golden brown in colour. Initially the hyphae were thin, later it became thick on maturity. Alternaria alternata possess a septate conidia which were produced singly or in short or long chains called conidiophore. Conidiophores was simple, short, straight, branched or unbranched, septate, slightly swollen at the apex, present alone or combine to form group or clusters and golden brown in colour. Conidia was small, multicellular, branched or unbranched, brown in colour, vary in shape and size, may be ovoid, obclavate, and obpyriform or ellipsoidal, smooth walled, broadest near the base and tapered gradually to form conical or cylindrical beak. Conidia possess three to five

transverse and one to two longitudinal septa, possess a smooth wall. The length of conidia was 3 - 5 times more than the width. These result are largely resembles with [39] - [43] .

Light microscopic examination of uninfected (control) stem of three varieties TS 3, TS 5 and SG 27 of sesame (Sesamum indicum L.) showed the same structure (Figure 3). Histologically, the sesame stem consist of non- vascular and vascular tissues. Non vascular tissues consist of trichrome, epidermis, hypodermis and cortical parenchyma. Vascular bundles contain xylem and phloem. Epidermis was the outer most single layer of stem consisting of closely packed living cells. Massive growth of trichrome was present on the surface of epidermis which was unicellular or multicellular outgrowth of epidermis. Beneath the epidermis, hypodermis was present which consist of two to three layers of cells. Cortex was present next to hypodermis which was multi-layered parenchyma cells. Cells of cortex were separated by intercellular spaces. In discontinuous manner, fiber cells were also present. Vascular tissue contains xylem and phloem. Phloem consists of primary and secondary form, present on the top of xylem. Xylem was inside to the phloem consisting of primary xylem and secondary xylem. Cambium was present between xylem and phloem. The arrangement of vascular tissue was as follow. Primary phloem, secondary phloem, cambium, secondary xylem and the innermost is primary xylem. Pith was composed of parenchyma cells and occupies the centre position; some cells of pith contain food reserves. This result is largely resembled with earlier study reported by [44] .

Initial 8 - 10 days of inoculation of pathogen on sesame plant, no visible symptoms or change was appeared on plant. Ten days after infection, minute brown colour spots were appeared at inoculation site which became enlarge later. Initially the colour of spot was brown but later it became darker. At the same time, small lesions surrounded by the yellow halos appeared which enlarge to form large necrotic areas with concentric rings. At initial stage, the shapes of spots were round but later it became irregular. The plants which were used as a control and sprayed with distilled water showed no such symptoms of infection. Same symptoms were recorded

due to Alternaria spp. in tobacco [23] , pomegranate [45] , soybeans [46] , California pistachios [47] , cowpea [26] and in citrus fruit [48] .

Fungus in infected tissues was stained as dark bluish black with toluidine blue O staining. Control did not show any such structure treated with same stain. In the present study, artificial inoculation of Alternaria alternata was done by using spray method. When the symptoms became visible in three varieties TS 3, TS 5 and SG 7 of sesame seedling, their tissues were studied after 10, 15, 20, 25 and 30 days after inoculation to understand the mechanism of fungal infection and penetration in infected sesame plant as well as to determine the histological changes in sesame cells infected with Alternaria alternata. 10 days after infection when the symptoms became visible by naked eye as a minute brown spot, microscopic examination of their cross section (CS) showed the presence of fungus in epidermis, hypodermis and in cortical parenchyma cells (Figure 4). Previous studies shows that this fungus enters in Minneola tangelo through epidermis and penetrate in to the parenchyma tissue [25] . Another study on Withania somnifera by [49] revealed the presence of hyphal mat of Alternaria alternata on beneath the epidermis, from there; it spread in to the all tissue of plant. 15 days after infection when the spot became larger in size, the CS of three verities of sesame seedling showed that the fungus entered in vascular bundle from cortical parenchyma tissue (Figure 5). Fungus moves through the intercellular spaces of cortex and enter in vascular bundle (phloem and xylem). 20 days after infection, CS of three varieties of sesame seedlings showed that the fungus was present in epidermis, hypodermis, cortical parenchyma, and phloem and xylem tissue (Figure 6). Fungus completely overlapped the vascular tissue. Due to excessive replication of fungus with in the plant it completely covers the vascular tissue. These results are very much similar with [50] , he reported the shrinkage of vascular bundle with the decrease in the diameter of vessels due to the infection of A. alternata on mango bark. 25 days after infection excessive growth of fungus was present in epidermis, hypodermis, cortical parenchyma, vascular bundle and pith. Fungus completely overlapped all the tissues of sesame stem (Figure 7). 30 days after infection sesame seedlings showed a complete disorganization of tissue (Figure 8). Fungus completely overlapped all the tissue of plant including epidermis, hypodermis, cortical parenchyma, vascular bundle and pith. When fungus growth was most abundant in every tissue it resulted in breakdown of tissue. Previous studies showed that due to fungus host interaction complete disorganization of cells of host plant was recorded without the differentiation of any tissue [27] [28] .