Characterization and Evaluation of Rhizobacteria Isolated from Peas (Pisum Sativum L.)

Abstract

Pea [Pisum sativum L.] is an important pulse legume crop and has the inherent ability to obtain much of its nitrogen (N) requirement from the atmosphere by forming a symbiotic relationship with Rhizobium bacteria in the soil. Pea is among the most highly efficient N2-fixing crops and may obtain as much as 80 % of its total N requirement from air under good growing conditions. However, for this relationship to occur, the presence of appropriate and efficient Rhizobium bacteria must be present. Pea is widely grown in Khyber Pakhtunkhwa province of Pakistan but no or limited information is available if these soils contain Rhizobium bacteria appropriate for field pea. This project was therefore planned to isolate and evaluate the Rhizobacteria of pea for N2 fixing capabilities and their efficiency in improving pea yields during 2011- 2014. The project was comprised of a survey where 45 farmers’ fields under different agro-climatic conditions of Khyber Pakhtunkhwa were assessed for the presence of Rhizobacteria in the nodules of pea crop. The survey was followed by a series of field experiments where the isolated Rhizobium bacteria were tested for the N2 fixing capabilities and their efficiency in improving yield of pea. The survey included three main pea growing areas viz., Peshawar, Charsadda and Swat. Fifteen farmer's pea fields in each area were selected for the study. At flowering stage, 5-10 pea plants at each field were randomly selected and carefully uprooted for the presence and characterization of Rhizobium bacteria in their nodules. At the same time, composite soil samples at 0-15 cm were collected from each field for site characterization. The Rhizobium bacteria were isolated from the pea of each field and assessed for important characteristics. It was observed that Gram negative characteristics of incubated rhizobia representing the purity of isolates were higher in Swat region (74 %) compared to Peshawar (47 %) and Charsadda (40 %). These Gram negative isolates were regarded as effective and pure as 50 % of these had stable colonies. The high purity and effectivity in Swat could be due to regular pea cultivation coupled with favorable agro-climatic conditions in the area being cool and semiarid in nature. Generally, the soils in all the surveyed areas were heavier in texture, non-saline, mostly alkaline in reactions and deficient in organic fertility. The extractable nitrogen (N), phosphorus (P) and potassium (K) in soils of Swat were relatively lower compared to soils in Peshawar and Charsadda. The soils in Peshawar and Charsadda were almost identical in all major characteristics and were invariably high in organic fertility and major nutrients. Subsequently based on exhibiting characteristics as effective N2 fixers, one pure and viable isolate from each of the three areas were selected and kept to evaluate them for their N2 fixing capabilities and efficiency in increasing yield of pea in alkaline calcareous soil in field experiments in Peshawar valley. These isolates were named as strain-I (isolated from Charsadda area), strain-II (isolated from Peshawar area) and strain-III (isolated from Swat area). x The surve was followed by a series of field trials where the efficiency of selected strains were assessed for nodulation, N2 fixation and yield of pea in alkaline calcareous soil at the research farm of Agriculture University, Peshawar during 2012- 2014. Before testing the selected isolates, a preliminary field experiment was conducted to ensure that the test soil of the experimental site is not deficient in micronutrients (viz., Fe, Mo) which are responsible for N2 fixation and if deficient what would be the response of pea crop (with indigenous Rhizobium) to the application of such micronutrients. In this experiment, two levels each of Mo (1.0 and 2.0 kg ha-1 ) and Fe (2.5 and 5.0 kg ha-1 ) were applied alone or in combination arranged in randomized complete block (RCB) design with three replications. Molybdenum was applied in the form of (NH4)6Mo7O24.4H2O and Fe in the form of FeSO4.7H2O salts. We observed that application of Mo and Fe significantly increased the nodulation, N2 fixation, pods yield and nutrient uptake of pea. Although alone application of both Mo and Fe induced significant (P<0.001) increases in pods yield and nutrient uptake of pea, the effect of combined application of Fe and Mo was more prominent than their alone application. The maximum pods yield of 5458 kg ha-1 and biomass yield of 10315 kg ha-1 were obtained with the combined application of Mo (2.0 kg ha-1 ) and Fe (5.0 kg ha-1 ). Furthermore, this treatment enhanced nodule numbers by 152 %, weight of nodules by 135 %, N2 fixation by 85% and plant N by 50 % compared with the control treatment. After pea harvest, the soil organic matter, mineral N, P and K contents were also improved with Fe and Mo fertilization that could be associated with more N2 fixation, enhanced plant and root growth of plants. These results suggest that the test soil may be having insufficient plant available Mo and Fe for pea crop. This was however surprising as the deficiency of Mo is rarely reported in alkaline soils. The 2nd field experiment was then initiated to compare the effects of Rhizobial isolates which were selected during the survey of pea crops, in the presence and absence of Mo and Fe on nodulation, N2 fixation and pods yield of pea crop in alkaline calcareous soil in Peshawar. The treatments were arranged in two factorial (3 strains x 4 fertilizer treatments) RCB design with three replications. Fertilizer treatments were no Fe or Mo, Fe at 5.0 kg ha-1 , Mo at 2.0 kg ha-1 and Fe (5.0 kg ha-1 ) + Mo (2.0 kg ha 1 ). There was one control treatment with no inoculation and no fertilizer (Fe, Mo). The results showed that all the three Rhizobial strains, both in the presence and absence of Fe + Mo significantly improved the nodulation, N2 fixation and pods yield of pea compared with the control treatment. On average, the Rhizobium strains in the presence of Fe + Mo increased pods yield of pea by 61 %, nodules number by 281 %, weight of nodules by 266 %, N uptake by 92 % and N2 fixation by 161 % over control. The post harvest soil fertility was also improved in the Rhizobial inoculated in the presence of Fe + Mo treatment that could be associated with enhanced plant and root growth and N2 fixation of pea crop. Among strains, the performance of Strain–II which was collected from Peshawar was much better in terms of increasing nodulation, N2 fixation and pods yield of pea compared with Strain-I and Strain-II suggesting that local Rhizobium already acclimatized to local environment is better than newly introduced non-local Rhizobia when used in isolation. These results suggested that both Fe and Mo improved the effectiveness of all the three selected isolates in the prevailing agro climatic conditions of Peshawar. Thus Rhizobial inoculation, preferably with local strains, along with both Fe and Mo is necessary for enhanced yield, nutrient uptake, nodulation and N2 fixation of field pea and for improved soil fertility.