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.