Seed collection and soil preparation
The experiment was carried out during kharif season from March to May 2017 and March to June 2018 in the nethouse of Soil Science Division, BARI, Joydebpur, Gazipur. Seeds of mungbean (BARI Mung-6) were collected from Pulses Research Centre, BARI, Gazipur. Bulk soil was collected from the bank of Turag river at Kodda, Gazipur. Soil was mixed with cowdung (Soil: Cowdung = 5:1) for use in the pot experiment. Each pot (28 cm in diameter and 23 cm in height) was filled with 8-kg soil leaving upper 3 inches of pot vacant to facilitate watering.
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 by modified Kjeldahl method (Jackson, 1962), and Ca, K & Mg 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, P by Modified Olsen method (Olsen et al., 1954) and S by CaH4(PO4)2, H2O extraction method (Chesnin and Yien, 1950).
Fertilizer application
Chemical fertilizers @ 23 kg N ha-1 from urea, 25 kg P (100%) ha-1 from TSP, 35 kg K ha-1 from MoP, 8 kg S ha-1 from gypsum, 2.25 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 and the remaining half was applied after 10 days of sowing. There were three P levels: 50%, 75% and 100% P. Peat based rhizobial inoculum (BARI RVr-403) was used in this experiment @ 50 g kg-1 seed and in that case N fertilizer was not used. The population density of used inoculum was above 108 cfu g-1 inoculant.
Preparation of salinity solution and mycorrhizal inoculum
Required concentrations of salinity were prepared according to New South Wales (NSW), Australia and applied three times during the experimentation period (Before seed sowing, 25 DAS and 35 DAS to maintain salinity level at 4 dSm-1). The arbuscular mycorrhizal inoculum was prepared from the roots and rhizosphere soils of Sorghum. Mycorrhizal species was originally isolated from different AEZs using the wet sieving and decanting method (Gerdemann and Nicolson, 1963). The spores mixed soil was put in the bed of sorghum plants to multiply for 6 months in the nethouse 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. Dominant mycorrhizal spores were from the genera Glomus, Acaulospora, Gigaspora and Scutellospora. The soil-based AM fungal inoculum containing 150 g of rhizosphere soil (approximate 183 ± 20 spores/100 g soil) along with infected sorghum root fragments with a minimum infection level was inoculated to each mycorrhizal pot. Different mycorrhizal spores identified in the Soil Microbiology Laboratory, Soil Science Division, BARI. The mycorrhizal inoculum was 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 AM colonization of plant roots.
Identification of AM fungal spore
AM fungal spore, single spore or sporocarps were easily picked up from the Petridis by filter paper with the help of syringe or fine point camel brush and mounted on a glass slide with a drop of polyvinyl lactophenol (PVL) and a cover slip was placed. Subsequently, recovered spores were identified with the help of a Manual and different taxonomic keys proposed by different workers (Schwarzott et al., 2001). Spore morphology, size, shape and peridium of spore, sporocarps colour, wall ornamentation, subtending hyphae and mode of attachment are considered for identification of spore or sporocarps.
Design of experiment and treatments
The experiment was designed in CRD with 10 treatments and 4 replications. There were 10 treatments viz. T1: Control (Not absolute 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. Ten seeds were sown in each pot beneath 1 cm soil depth. After collecting germination percentage data, 6 vigorous seedlings were kept in each treatment and other seedlings were removed from the pot. Three plants of each were collected for nodulation and colonization data and rest three plants were kept finally in each pot for yield and yield contributing measurements.
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 (28 ± 2oC). 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.
Crop harvest
Mungbean was 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 recorded. Other parameters like nodule number, nodule weight and root infection (%) were measured at the time of 50% flowering stage of mungbean.
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 by 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.
Statistical analysis
Data were statistically analyzed using Analysis of Variance (ANOVA) following CropStat package while the all pair comparisons were done by Statistix 10.