Seed collection and Soil preparation:
The experiment was carried out during rabi season from March, 2017 to May, 2017 in the net house of Soil Science Division, BARI, Joydebpur, Gazipur (23° 59'378'' N latitude, 900 24'886'' E longitude and 8.4 m elevation). Seeds of mungbean (BARI Mung-6) were collected from Pulses 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 (28 cm in diameter and 23 cm in height) was filled with approximately 8-kg soil leaving 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 (100g-1 soil) spores of indigenous mixed AM fungal species and the experiment was conducted under unsterilized soil condition.
Soil analysis:
Soil pH was measured by a combined glass calomel electrode (Jackson, 1958). Organic carbon was determined by 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 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.
Fertilizer application:
Chemical fertilizers @ 22.56 kg N ha-1 from urea, 24.45 kg P ha-1 from TSP, 34.27 kg K ha-1 from MoP, 7.95 kg S ha-1 from gypsum, 2.23 kg Zn ha-1 from ZnSO4, 0.81 kg B ha-1 from Boric acid and 0.34 kg Mo ha-1 from Na2MoO4.2H2O were applied (BARC, 2012). Half of urea-N and all other fertilizers were applied as basal during final land preparation and remaining half amount of urea-N were applied after 10 days after sowing.
Preparation of salinity solution and mycorrhizal inoculum:
Different concentrations of salinity were prepared according to New South Walles (NSW) Department of Primary Industries 2005, Australia and applied three times during the experimentation period. First before sowing of mungbean seed, next after 25 DAS and then after 35 DAS.
The arbuscular mycorrhizal inoculum was prepared from the roots and rhizosphere soils of Sorghum. Mycorrhizal species was originally isolated from different AEZ region, using the wet sieving and decanting method. The spores were left to multiply for 6 months on sorghum plants using unsterilized soil, collected from the same site, in the net house of Soil Science Division, BARI. Plants were irrigated with tap water as needed. A mixture of infected sorghum root and soil which contained spores was used as mycorrhizal inoculum. The soil based AM fungal inoculum containing 150 g of rhizosphere soil (approximate 275 ± 20 spores 100g-1 soil) and infected sorghum root fragments with a minimum infection level was inoculated to each mycorrhizal pot. The mycorrhizal inoculum were first placed in each pot at 3-5 cm depth and was covered with a thin soil layer of 1 cm immediately prior to the seed sowing of mungbean to facilitate fungal colonization of plant roots.
Design of experiment and treatments:
The experiment was designed in RCBD with 10 treatments and 4 replications. Ten seeds were sown in each pot at 1 cm soil depth. After collecting germination percentage data the treatment was sustained with 6 vigorous seedlings in mycorrhizal and non-mycorrhizal pot, and the other seedlings were removed from the pot. Then after collecting growth parameters and nodulation data 3 plants were left finally in each pot for yield and yield contributing data. There were ten treatments viz. T1: Control, T2: Arbuscular mycorrhiza (AM) + 50% P, T3: AM + 75% P, T4: AM + 100% P, T5: Rhizobium + 50% P, T6: Rhizobium + 75% P, T7: Rhizobium + 100% P, T8: AM + Rhizobium + 50% P, T9: AM + Rhizobium + 75% P and T10: AM + Rhizobium + 100% P.
Determination of germination percentage:
The germination test was carried out according to ISTA rules (ISTA, 1976). For each treatment, 25 seeds were put into Petri dishes. The Petri dishes were put on a laboratory table at room temperature (25 ± 2°C). After 3 days, normal, abnormal and diseased seeds were counted. Germination of mungbean seed in the laboratory table was 84%. Ten seeds were sown in each pot. After 4, 6, 8 and 10 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.
Crop harvest:
Mungbean were harvested after 62 days of sowing. Different growth parameters like plant height, pods plant-1, seeds pod-1, 100-seed weight, seed yield and stover yield were measured. Other growth parameters, fresh and dry weight of root and shoot, nodule number, nodule weight and root infection (%) were measured at the time of flowering stage of mungbean.
Assessment of spore population density:
Assessment of spore population was done following the Wet Sieving and Decanting Method (Gerdemann and Nicolson, 1963). Soil samples from the rhizosphere of the respective plant species were mixed thoroughly by breaking up any large lumps. Any large unwanted particles such as stone, roots, twigs etc. were removed from the soil. Then 100 g of mixed soil was kept in a bucket (8 litres) and filled three quarters with tap water. The soil with water was agitated by stirring vigorously by hand and washed into the bucket and left to settle for one minute. The suspension was sieved by following the wet sieving and decanting method (Gerdemann and Nicolson, 1963). Two sieves (400 µm and 100 µm mesh) were used throughout the experiment. The supernatant was poured through a 100 µm sieve into the second bucked (10 litres) to avoid the loss of useful materials. After allowing the suspension to settle for one minute, the supernatant was decanted into the 400 µm sieve. This time water was discarded and the material was back washed from the sieve into a beaker (250 mL) with a small quantity of water. The solution with spores was distributed in 4 equal size test tubes evenly and balanced up the tubes with water for equal weight. The tubes were plugged properly and then centrifuged for 4 minutes at 3,000 rpm. The supernatant was poured in test tubes and the test tubes were filled with sucrose solution and stirred vigorously with the round-ended spatula to re-suspend the precipitate. The test tubes were balanced properly to equal weight and they were plugged. Then the plugged test tubes were centrifuged for 15 seconds at 3,000 rpm. After centrifuge, the sucrose supernatant was poured through a 400 µm sieve and rapidly washed with water to remove the sucrose from AM spores by back washing the materials from the sieve into watch glass for observation.
All the AM spores were isolated from the extract with the help of a fine forcep into a watch glass with small quantity of water. The extract, with AM spores, was observed under stereomicroscope and the number of spores was counted. Spore numbers from the three replicates per samples were averaged and the result was expressed as number per 100 g of dry soil basis.
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 cover slip 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 CropStat package while the all pair comparisons were done by Statistix 10.