Genetic Diversity and Potential of Soybean (Glycine Max. L.) Brady(rhizobial) populations of Rawalakot Soils for Plant Growth Promotion

Abstract

This study was conducted to examine the variability of soybean nodulation and growth in relation to elevation and soil properties across the slopping uplands of the Himalayan region of Rawalakot Azad Jammu and Kashmir (AJK), Pakistan in order to find efficient native N2 fixing bacteria adapted to local soil and climatic characteristics. Soils from twenty two different sites with variable altitude were collected and analyzed for different physico-chemical characteristics including the quantitative estimation of rhizobium population through spread plate method. Soybean cultivar William-82 was grown in these soils under greenhouse conditions for determining the nodulation potential (number and mass) and plant growth characteristics. Morphology of the nodules was observed through optical and transmission electron microscopy. Soil altitude ranged from 855 m to 3000 m while organic matter content varied between 0.8% to 3.5% and pH from 6.0 to 8.1. The number of nodules per plant varied from 7 to 40 (CV 38%) suggesting site/location as an important factor contributing towards rhizobia population and impacting root nodulation. Results showed a substantial variability between sites and this is likely to be due to inter/intra species diversity, as well as changes in microbial community composition/structure. Root nodules bacteria were isolated from soybean grown in soils of 22 different sites of subdivision Rawalakot. A total of 37 strains were isolated on YEMA medium and purified. All isolates were found Gram negative except NR15, NR30 and NR32. The isolates were further characterized for phosphate solubilization, indole acetic acid production and nitrogen fixation. Out of 37 strains, twenty eight were able to produce IAA while nine did not. The maximum IAA of 13.20 μg/mL was produced by the strain NR30 followed by 13.0 μg/mL by NR19, and 11.20 μg/mL by NR35 and NR4. Similarly, twenty nine strains solubilized P, while eight isolates were xxii not able to solubilize P in the culture medium. The maximum P solubilization of 15.56 μg/mL was recorded for strain NR15 followed by 12.52 μg/mL and 10.52 μg/mL for the strains NR35 and NR25, respectively. Five strains (NR15, NR19, NR29, NR30 and NR32) did not nodulate the host upon re-inoculation. Nitrogen fixing capacity of the strains was determined by acetylene reduction assay (ARA). A significant higher ARA (729 n moles/plant/hr) was recorded for NR20 followed by NR22 (697 n moles/plant/hr) and NR25 (680.7 n moles/plant/hr). Out of 37 bacterial strains isolated, 33 strains were sequenced and were submitted at Genbank data base and accession numbers were obtained. Out of 33 sequenced bacterial strains, 28 nodules forming rhizobial strains belong to Bradyrhizobium genera. Fourteen soybean root nodule forming strains were identified as Bradyrhizobium japonicum specie by showing the maximum similarity with Bradyrhizobium japonicum. Six nodules forming strains were identified as Bradyrhizobium elkanii specie. Three nodules forming strains belong to Bradyrhizobium yuamingense specie. Strains NR24 were identified as Bradyrhizobium liaoningense specie and four nodule forming strains were identified as Bradyrhizobium species on the basis of maximum sequence similarity. The five non-nodules forming strains were belong to five different genera. The bacterial strain NR15 was identified as Microbacterium specie, strain NR19 as Agrobacterium rhizogens, strain NR29 as Rhizobium specie, NR30 as Paenibacillus specie and NR32 as Bacillus specie on the basis of maximum sequence similarity. The results of the study demonstrate the potential of indigenous Bradyrhizobium strains for P soluibilization, N2 fixation and phytohormone production those could be used in biofertilizers. Seven indigenous Bradyrhizobium strains, one exotic strain TAL-102, an un-inoculated control and three N fertilizer rates (urea) i.e. 25, 50 and 100 kg N ha-1 were tested on soybean variety William-82 in pots and under field conditions in the mountain region of Rawalakot Azad xxiii Jammu and Kashmir, Pakistan in 2009 and 2010. Results showed that Bradyrhizobium inoculation accelerated plant growth by increasing shoot length (26-47%), root length (45-73%) and shoot dry weight (58-104%). Seed yield in the control was 861 kg ha-1 that significantly increased to 1450–2072 kg ha-1 following inoculation with Bradyrhizobium strains. Seed yields obtained with the indigenous NR20 and NR22 strains was 24 and 28% higher than that obtained with the exotic strains TAL-102. Number of nodules, nodules dry weight (mass) and ARA (N2 fixation) under Bradyrhizobium strains were significantly higher i.e. 55–123%, 94–178%, and 38–103%, respectively than that recorded under the un-inoculated control. However, higher N fertilization rate (N100) depressed nodulation and N2 fixation. Both Bradyrhizobium inoculation and N fertilization significantly increased oil and protein content of seed compared to the uninoculated control. Similar results were obtained under pot experiments. Bradyrhizobium inoculation and N fertilization in general had no significant effect on the saturated fatty acids (palmitic and stearic) whereas unsaturated fatty acids (linoleic acid and oleic acid), significantly increased compared to un-inoculated control. The mineral nutrient content N, P, and K and their accumulation in seed showed a 2-3-fold increase in response to applied strains and N fertilizer. Results demonstrated a significant variation in the symbiotic effectiveness and yield potential characteristic of tested strains showing that inoculation response was site specific. Two indigenous strains NR20 and NR22 were found highly efficient and displayed superiority over the exotic strain TAL-102. Pot and field experiments were conducted to observe the residual effects of Bradyrhizobial inoculation of soybean for two consecutive years on succeeding wheat crop. Wheat was grown on the same pots and field in which soybean was grown for two consecutive years 2009 and 2010. The pots and seed beds used for growing soybean were not disturbed after xxiv harvesting soybean and were manually prepared by spade. The treatments comprised of a control, three N fertilization rates (i.e. 100 kg N ha-1, 50 kg N ha-1, 25 kg N ha-1) and eight Bradyrhizobial inoculation treatments of previous soybean crop. The inoculation strains were not applied to wheat crop. The treatments were assigned to respective pots and plots where soybean was sown accordingly. Wheat variety Shafaq-2006 was used as test crop. Results of the field study revealed that highest plant height, root length, leaf surface area and chlorophyll contents of wheat were recorded in N100 followed by N50. Similarly spike length, 1000 seed weight and biological yield of wheat significantly increased by the application of 100 kg N ha-1 followed by 50 kg N ha-1. Among the inoculation treatments of previous soybean crop, all treatments significantly increased the growth and yield parameters of wheat over control but statistically at par with that recorded in N25 (25 kg N ha-1). The maximum grain yield of 2365 kg ha-1 was recorded in N100 followed by yield of 2102 kg ha-1 under N50 and the difference between these two was also significant. Among different inoculation treatments, the highest yield of 1844 kg ha-1 was recorded in NR22 which was at par with NR20, NR18 and N25. Seed protein and NPK uptake was also significantly affected by N fertilization and inoculation treatments. Response of growth and yield attributes to different treatments in greenhouse experiment was same to that observed under field conditions. The results of the study clearly demonstrate that the residual effect of inoculation to soybean crop was almost equivalent to 25 kg N ha-1 and inclusion of soybean in rotation could save about 25 kg N ha-1 for succeeding wheat crop. Overall results of this study revealed a marked variation in soil properties and microbial indices among soils collected from different sites of Rawalakot. A diversity in soybean nodulating bradyrhizobia were recognized having varied potential in symbiotic effectiveness and PGP activities and these bradyrhizobia can be best utilized in biofertilizers in Pakistan.