Seed collection and Soil preparation: The experiment was carried out during rabi season from December 2014 to April 2015 and December 2015 to April 2016 in the net house of Soil Science Division, BARI, Joydebpur, Gazipur (230 59'378'' N latitude, 900 24'886'' E longitude and 8.4 m elevation). Seeds of grasspea (BARI Khesari-1) were collected from Pulse Research Centre, BARI, Gazipur. The silted (sandy clay loam) soils were collected from the bank of Turag river at Kodda, Gazipur mixed with cowdung at 5:1 ratio and was used as the potting media. Each pot (25 cm in diameter and 21 cm in height) was filled with approximately 6-kg soil leaving the upper 3 inches of pot was vacant to facilitate watering. The pH of cowdung was 6.7 and the nutrient contents were: organic matter 14.1%, N 0.8%, P 1.26%, K 0.88%, Ca 1.55%, Mg 0.82%, S 0.62%, Fe 0.25% and Mn 0.112%. The physical and chemical properties of the soil are presented. The soil contained 12 AM (100-1 g soil) spores of indigenous mixed AM fungal species and the experiment was conducted under sterilized soil condition. Soil pH was measured by a combined glass calomel electrode (Jackson, 1958). Organic carbon was determined by the Wet Oxidation Method (Walkley and Black, 1934). Total N was determined by modified Kjeldahl method (Jackson, 1962). Calcium, K and Mg were determined by NH4OAc extraction method (Black, 1965). Copper, Fe, Mn and Zn were determined by DTPA extraction followed by AAS reading. Boron was determined by CaCl2 extraction method. Phosphorus was determined by the Modified Olsen method (Neutral + Calcareous soils) according to Olsen et al. (1954). Sulphur was determined by CaH4(PO4)2.H2O extraction followed by turbidimetric turbidity method with BaCl2.
Chemical fertilizers @ 12.6 kg P ha-1 from TSP, 19.00 kg K ha-1 from MoP and 2.003 kg S ha-1 from gypsum were applied (BARC, 2012). All fertilizers was applied as basal during final land preparation.
Collection of pathogen Sclerotium rolfsii and Rhizobium inoculum: Pathogen Sclerotium rolfsii were collected from Plant Pathology Division, BARI, Gazipur which was grown on nonseed barley. Nonseed barley collected from Plant Breeding Division, BARI, Gazipur. Pathogen Sclerotium rolfsii along with nonseed barley 50 g was used per Sclerotium treatment pot. After disease development, pathogen sclerotia mixed with soil. Rhizobium strain BARI RLs-10 were collected from Soil Microbiology Division, BARI, Gazipur and mixed properly with the seed before sowing.
Experimental design: The experiment was designed in RCBD with eight treatments and four replications. Fifteen seeds were sown in each pot at 1 cm soil depth. The eight (08) treatments were: T1: Arbuscular mycorrhizal fungi (AMF), T2: Rhizobium (R), T3: AMF+Rhizobium, T4: Sclerotium rolfsii, T5: Sclerotium rolfsii+AMF, T6: Sclerotium rolfsii+Rhizobium, T7: Sclerotium rolfsii+AMF+Rhizobium and T8: Control.
Determination of germination percentage: The germination test was carried out according to ISTA rules (ISTA, 1976). For each treatment, 100 seeds were put into Petri dishes. The Petri dishes were put on a laboratory table at room temperature (25 ± 2oC). After eight days, normal, abnormal and diseased seeds were counted. Germination of grasspea seed in the laboratory table was 95%. Fifteen seeds were sown in each pot. After eleven, fifteen, nineteen and twenty three days germinated seeds were observed and counted. Germination percentage was calculated by the following formula: Germination (%) = {(Number of germinated seeds in each pot) / (Total number of seeds sown in each pot) x 100.
Assessment of root colonization infection: The percentage of AM infection was estimated by root slide technique (Read et al., 1976). One hundred root segments were examined for each sample. The stained root pieces were mounted in acidic glycerol on slides and the coverslip was placed and slightly pressed. The roots were observed under microscope. A root segment was considered as positively infected, if it showed mycelium, vesicles and arbuscules or any other combination of these structural characteristics of AM infection. The presence or absence of infection in the root pieces was recorded and the percent infection was calculated as follows: % root colonization = {( Number of AM positive segments) / (Total number of segments scored) x 100.
Statistical analysis: Data were statistically analyzed using Analysis of Variance (ANOVA) following Statistix 10 package.