Cotton variety CB - 9 was used in this experiment as it is a widely cultivated variety. Then four hundred seeds were selected randomly for laboratory seed health study. Collected seeds were sterilized with 1% Clorox (NaOCI) for 5 minutes and rinsed with sterilized water for 3 minutes. Seed germination was determined by the blotter method (ISTA, 1996). Ten seeds were placed on 4 layers of moist blotter paper in 5 cm petridishes maintaining uniform distance between them. Each of the plates was incubated in 25 ± 4°C temperature for 7 days in incubation chamber with an alternation of twelve hours light and dark. After 7 days of incubation, plates were collected and examined under stereomicroscope for primary identification of the Pathogenic organism(s). Then the identified fungi were transferred to PDA plates for proper sporulation and purification. Hyphaltip culture method was used to make the pure culture of the fungi. Seeds obtained from the field experiment were also tested under same procedure described before following ISTA (1996) rules in order to find out seed borne boll rot pathogens present in them to determine the efficacy of different treatments to subdue the engender of cotton boll rot. The difference between of pathogenic presence in two different seeds of the same cotton variety was then calculated.
Experimental site and duration: The field experiment was carried out during the period 30th May to November, 2013 in the experimental field in Sher-e-Bangla Agricultural University, Dhaka. In-vitro experiment was conducted in the Seed Pathology Laboratory and the M.S. Laboratory of the department of plant pathology of Sher-e-Bangla Agricultural University, Dhaka. The selected field for this experiment was properly ploughed and proper doses of required fertilizers were applied to the field. In the field experiment thirty plots were prepared for different treatments. Each plot was 3 meters in length and 2 meters in width where row to row distance was 2.8 m and plot to plot distance was 0.5 m. The total area was covered by 511.2 m2.
Isolation of seed borne fungi from incubated seeds: Fungi grown over the incubated seeds were aseptically transferred on to PDA medium with the help of a sterile needle and the PDA plates were kept in incubation at 25±2°C and 12 hours alternating cycle of light and darkness for 7 days. Purification was done by re-culturing fungi identified on the basis of their characteristics under compound microscope. These fungi were identified following the keys of Kamal and Khan (1964) and Kuch (1986) .
First, chemical suspensions were prepared as per following concentration, 0.4% for the fungicides viz: Cupravit 50 WP and Mancozeb 80 WP and 1 ppm for the antibiotic, viz, Streptomycin sulphate. A fungal mycelial block was cut from a 7 days old fungal culture and transferred on a PDA. An in-vitro evaluation was conducted to find out the effect of chemicals against the seed borne fungi of cotton on PDA following well method. Discs of mycelia (5 mm diameter) from each of the isolated fungi were cut from the edge of the actively growing fungal colony with a cork borer. One mycelialdisc of each fungus was placed on the edge of each PDA plate and simultaneously on the other side a 5 mm well was prepared and on that well 80 μl of chemical suspension was poured and these plates was incubated at 25±2°C for 7 days. In case of the control plate, only the fungal mycelial block was placed without any chemical. After 7 days of incubation, radial mycelial growth of control plate and plates with fungicides were measured in diameter. The following formula (Kantwa et al., 2014) was used to determine the inhibition zone of fungal myecelia.
% inhibition = {(C-T)/C}× 100
C = Radial growth of control plates.
T = Radial growth of fungicide and antibiotic treated plates.
Seed treatment: Total required amount of seed for the field experiment was separated and divided in to three equal parts. Then one part was treated with Cupravit 50 WP @ 0.4%, another part was treated with a combination of Cupravit 50 WP @ 0.4% and Mancozeb 80 WP @ 0.4% and third part of the seeds were treated with antibiotic Streptomycin sulphate @ 0.1%. To treat the seeds with fungicides, first required amount of seed were kept in a Petri dish and then the fungicide was added there. Then the Petridish was covered with the lid and it wa s shacked thoroughly for a few minutes so that the fungicide covers total surface of the seed coat. To treat seeds with antibiotic first, a regular bottle was filled with 100 ml sterile distilled water and 1 gm streptomycin sulphate was mixed to it. Then s elected seeds were poured in the bottle and the bottle cap was attached. All three treated seed items were kept overnight till the next morning as it was the sowing day. In case of control plot, seeds were treated with sterile distilled water only.
Collection of diseased bolls: Infected cotton bolls those showed ostensible identical symptoms depicted by the previous onerous researches were collected from experimental field. The visible suspicious symptoms of the disease were recorded and disease was identified based on the symptoms (Hillocks, 1992). To prevent from being dried, collected bolls were kept in polythene bag immediately after collection. Then these samples were taken to the plant pathology laboratory, Sher-e-Bangla Agricultural University. Collected bolls were wrapped with two layers of brown paper and kept in refrigerator at 4°C until isolation of the fungi was done.
Isolation of causal organisms and tissue planting method: The pathogens associated with boll rot were isolated by following tissue planting method (Tuite, 1969). The parts of bolls associated with disease were cut in to small pieces and surface sterilized with 0.1% Clorox (NaOCI) for 3 minutes and washed for three times in distilled and sterilized water. Then it was placed on mo ist filter papers. Two pieces of filter papers were dipped in sterile water to keep it moist. The covered petridishes containing the specimens were brought in the seed pathology laboratory and kept under incubation for three days. After incubation those plates were observed under stereomicroscope for the primary identification of the organisms (fungi). Th en the fungi were transferred to PDA plate for proper sporulation and purification.
Treatments: Ten treatments were selected for this experiment, which were 1. T1: Seed treatment with Cupravit 50 WP @ 0.4%, 2.T2: Seed treatment with Cupravit 50 WP @ 0.4% + Mancozeb 80 WP @ 0.4%, 3.T3: Seed treatment with Streptomycin sulphet @ 0.1% , 4.T4: Seed treatment with Cupravit 50 WP @ 0.4 % + Foliar spray with Cupravit 50 WP @ 0.4 %, 5. T5: Seed treatment with Cupravit 50 WP and Mancozeb 80 WP both @ 0.4 % + Foliar spray with Cupravit 50 WP and Mancozeb 80 WP both @ 0.4 %,6. T6: Seed treatment with Streptomycin sulphate @ 0.1% + foliar spray with Streptomycin sulphate @ 0.1 %, 7. T7: Foliar spray with Cupravit 50 WP @ 0.4 %, 8. T8: Foliar spray with Cupravit 50 WP @ 0.4 + foliar spray with Mancozeb 80 WP @ 0.4 %, 9. T9: Foliar spray with Streptomycin sulphate @ 0.1 % and 10.T10: Control.
Foliar application: After formation of bolls in the cotton plants, treatments were randomly assigned to different plots were applied to them for total f our times with a certain interval. These treatments were applied, both seed and foliar spray or only foliar spray. The treatments associated with seed treatment were done prior to the sowing of cotton seeds in the field.
Data recording and statistical analysis: Data was recorded on leaf spot incidence, severity of leaf spot in PDI, boll rot incidence, and different yield contributing characters (number of branches/plant, number of leaves per plant, number of bolls per plant, plant height, weight of bolls, yield). Ten treatments with three replications were used following Randomized Block Design (RCBD). Data were analyzed for ANOVA using MSTAT-C program (MSTAT, 1991). Least significant difference (LSD) were performed to determine the level of significant differences and to separate the means within the parameters (Gomez and Gomez, 1984).