Effects Of Biofertilization On Nodulation, Nitrogen And Phosphorus Content And Yield Of Pigeon Pea (Cajanus Cajan)

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1 Advances in Environmental Biology, 5(9): , 2011 ISSN This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Effects Of Biofertilization On Nodulation, Nitrogen And Phosphorus Content And Yield Of Pigeon Pea (Cajanus Cajan) 1 Awad Galal Osman, 2 Ahmed Mohammed Elhassan Rugheim and 3 Elkheir Mohammed Elsoni 1 The National Centre for Research, Khartoum. 2 Ommdurman Islamic University, Ommdurman, Sudan. 3 Ministry of Agriculture,Algazeira State,Sudan. Awad Galal Osman, Ahmed Mohammed Elhassan Rugheim and Elkheir Mohammed Elsoni: Effects Of Biofertilization On Nodulation, Nitrogen And Phosphorus Content And Yield Of Pigeon Pea (Cajanus Cajan). ABSTRACT A field experiment was conducted at Elbagair area, central Sudan, for two successive seasons 2009/ /2011 to study the effects inoculation with five introduced or locally isolated Rhizobium or Bradyrhizobium strains each alone or with Bacillus megatherium var. phosphaticum strain (BMP) alone, and combination of each rhizobial strain with Bacillus megatherium var. phosphaticum strain (BMP) on the symbiotic properties, and yield of pigeon pea (Cajanus cajan). Rhizobium and BMP inoculation separately significantly (P 0.05) increased nodulation, nodules dry weight, root dry weight, shoot dry weight, nitrogen and phosphorus content in shoot and seed yield of pigeon pea plants in comparison to the un-inoculated control. Rhizobium and BMP co-inoculation significantly (P 0.05) increased nodulation, nodules, root and shoot dry weights nitrogen and phosphorus content in shoot and seed yield. The results of this study revealed the need for biological fertilizers with compatible effective strains for application to inoculate pigeon pea, especially this crop is cultivated in Sudan without fertilization. Key words: Pigeon Pea, Rhizobium, Bacillus, Inoculation. Introduction The legumes and grains families are by far the world s most important sources of food, grains supply starch while legumes which include bean, peas supply protein and fats [14]. Legumes are very important not only as food crops but possess high propensity to grow in depleted soils thereby serving as a medium of fertilizing succeeding crops through their unique symbiotic capability with nitrogenfixing Rhizobium bacteria which are inhabited in root nodules of the legumes, and the nitrogen balance in the soil is thereby preserved [20]. Pigeon peas are an important food in developing tropical countries. An excellent source of protein, the seeds (and sometimes the pods) are eaten as a vegetable, as a flour additive to other foods, in soups, and with rice [3]. The performance of pigeon pea globally is in an upward trend in terms of area and production from 2.86 million hectares and 1.96 million tons in 1980 to 4.63 million hectares and 3.46 million tons in 2006, respectively. [8]. Pigeon pea forms root nodules in association with Rhizobium sp. bacteria and is capable of fixing 41 to 280 kg/ha of nitrogen [24]. Preparations of the leaves are used to treat jaundice, inflammation, and sores of the mouth [23]. In spite of the importance and benefits of this crop it does not enjoy global popularity, hence there is limited information available on this plant. Most of the rhizobia inoculation studies done in Sudan have concentrated on faba bean, groundnut, alfalfa, guar, chickpea and common bean [5] with little focus on such crops as cowpea, fenugreek and pigeon pea. One of the major problems that limit economically successful agricultural production worldwide is poor soil fertility[35,36,37,38]. Therefore, addition of fertilizers is necessary to correct poor soil fertility by supplying nutrients needed for optimum crop growth [7]. Chemical fertilizers became the target for criticisms mainly because of the heavy use in the world, where they were suspected of having adverse impact on the environment through nitrate leaching, eutrophication, greenhouse gas emissions and heavy metal uptakes by plants. Consequently, fertilizer use was identified as a harmful to the environment. Biofertilizer is a substance which contains living microorganisms which, when applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of the plant and promotes growth by increasing the supply or Corresponding Author Awad Galal Osman, The National Centre for Research, Khartoum, Sudan. agosman@hotmail.com

2 availability of primary nutrients to the host plant. [33]. Biofertilizers from microorganisms can replace chemical fertilizers to increase crop production. In principle, biofertilizers are less expensive and are more environmentally-friendly than chemical fertilizers [9]. The most limiting nutrients for plant growth are N and P [30,39,40,41,42]. Although soil may contain vast amounts of either nutrient, mostly are not readily available for plant. Atmosphere consists of about 80% nitrogen as a dinitrogen gas [13]. To be available for plant use, nitrogen gas must be combined with other elements (fixed). The process of nitrogen fixation can be accomplished either biologically by microorganisms or chemically [18]. Nitrogen fixed symbiotically mainly by the association between Rhizobium species and legumes which represents a renewable source of N for agriculture [2]. Phosphorus is an essential element for plant nutrition. It has to be added to the soil as a fertilizer because of its deficiency and low solubility. Phosphorus is found in an insoluble form in soil and hence it is unavailable for plants. However, many microorganisms play a great role in solubilizing the inorganic insoluble phosphates into a soluble form through excretion of various organic acids and also by mineralizing the locked up organic phosphorus through excretion of various enzymes in agricultural soils making readily available forms for plants [6]. This present study was carried out to fill in one of the gaps in nitrogen and phosphorus biofertilizer research in Sudan. Materials And Methods A field experiment was conducted at Elbagair area, central Sudan (Latitude N and longitude E), for two successive seasons 2009/2010 and 2010/2011 in a factorial design with four replicates. In these experiments plants were either inoculated with five introduced or locally isolated Rhizobium or Bradyrhizobium strains each alone or with Bacillus megatherium var. phosphaticum strain (BMP) alone in addition to combination of each rhizobial strain with Bacillus megatherium var phosphaticum strain (BMP). Control plants were kept for comparison. The land was prepared by deep ploughing, harrowing then levelling and ridging. The land was then divided into plots 5 x 4m each. Three to four seeds were placed in a hole on the top of the ridge with 20cm spacing (between holes) and 60cm (between ridges). Plots were immediately irrigated after sowing and then subsequently irrigated at days intervals. The first sampling was carried out after four weeks from sowing, and then sampling was done every two weeks, for eight 2743 weeks. During each sampling, three plants from each plot were carefully dug out of the soil with their roots. Plants representing each treatment in each plot were placed in a paper bag and immediately taken to the laboratory for the following measurements per plant: number of nodules, dry weight of nodules, shoots and roots (after oven drying at 80 C for 48 hours). Plant samples from each treatment were ground and kept in polyethylene bags for nitrogen determination by the microkjeldahl method [1]. Shoot phosphorus content was determined calorimetrically after digestion [11]. Harvest was done at 20 weeks after sowing. Each plot was harvested separately by cutting the plants just above soil level. Plants were then threshed on a large mat, then collected and weighed to determine yield of each plot. Multifactor analysis of variance (ANOVA) was used to determine the effect of different treatments on the measured parameters. Least significance difference was used to compare between means [10]. Results and Discussion Effects of Ttreatments on Nodulation: Table (1) shows that there was poor nodulation on the control plants. Schroder and Cruz Perez [31], reported that nodulation of pigeon pea is frequently poor. Pigeon pea is nodulated by rhizobia classified as cowpea miscellany, which are usually abundant in tropical soils [17]. Rhizobium inoculation significantly (P 0.05) increased nodulation compared to un-inoculated control. The highest nodule number was obtained by Rhizobium strains TAL 1132 in the first season and ENRRI 63 in the second season. In previous studies inoculation of pigeon pea with different rhizobial strains showed significant differences on nodulation [17, 28, and 16]. BMP inoculation as phosphobacterin significantly (P 0.05) increased nodulation at 6 and 8 weeks after sowing compared to un-inoculated control in both seasons. Similar results were reported by Kannaiyan et al. [15] for pigeon pea. Rhizobium and BMP co-inoculation significantly (P 0.05) increased nodule number per pigeon pea plant at 4 and 6 weeks after sowing in the first season. At the second season interactions significantly (P 0.05) increased nodulation at all sampling times except ENRRI 63 and USDA 3472, where the increments were insignificant at 4 weeks after sowing. The best interactions were obtained by Rhizobium strain TAL 209 and phosphobacterin (BMP) in both seasons. Significant increment in nodule number of pigeon pea by coinoculation was reported previously [15].

3 2744 Table 1: Effects of treatments on nodules number per pigeon pea plant Weeks after sowing Uninoculated Control Rhizobium ENRRI ENRRI TAL TAL USDA Phosphobacterin B ENRRI 63 + B ENRRI 4 + B TAL B TAL B USDA B LSD for Rhizobium LSD for phosphobacterin LSD for Rhizobium x phosphobacterin Effects of Ttreatments on Nodules Dry Weight: Inoculation with Rhizobium significantly (P 0.05) increased pigeon pea nodules dry weight in both seasons compared to un-inoculated control (Table 2). It was reported that rhizobia inoculation improved pigeon pea nodule biomass [4]. BMP inoculation significantly (P 0.05) increased pigeon pea nodule dry weight at 6 and 8 weeks after sowing in the second season compared to un-inoculated control. This result is in accord with the results reported by [15] for pigeon pea. Co-inoculation with Rhizobium strains and BMP strain significantly (P 0.05) increased pigeon pea nodules dry weight in both seasons. The highest increasing in nodule dry weight was obtained by the interaction between strain ENRRI 4 and phosphobacterin at 4 and 8 weeks after sowing in the second season. This finding supports the findings of [15] for pigeon pea. Table 2: Effects of treatments on nodules dry weight (mg/plant) of pigeon pea Weeks after sowing Uninoculated Control Rhizobium ENRRI ENRRI TAL TAL USDA Phosphobacterin B ENRRI 63 + B ENRRI 4 + B TAL B TAL B USDA B LSD for Rhizobium LSD for phosphobacterin LSD for Rhizobium x phosphobacterin Effects of on Root Dry Weight: From the results in (Table 3) Rhizobium inoculation significantly (P 0.05) increased the root dry weight at all sampling times in the first season, at the second season the root dry weight significantly (P 0.05) increased by all strains except ENRRI 4 and USDA 3472 at 8 weeks after sowing compared to uninoculated control. These results are in accord with the results obtained by Mustafa, [19] for pigeon pea. Inoculation with BMP significantly (P 0.05) increased pigeon pea root dry weight at 4 and 6 weeks after sowing in both seasons compared to uninoculated control. This result is in accord with the results obtained by Osman and Abd-Elaziz, [21] for faba bean. Co-inoculation with Rhizobium strains and phosphobacterin significantly (P 0.05) increased the root dry weight compared to un-inoculated control at 4 and 6 weeks after sowing in both seasons. Similar results were reported by Rugheim and Abdelgani [26] for faba bean. Interaction between strain ENRRI 63 and phosphobacterin gave the highest significant increment of pigeon pea root dry weight in the first season compared to un-inoculated control and other treatments.

4 2745 Table 3: Effects of treatments on root dry weight (g/plant) of pigeon pea Weeks after sowing Uninoculated Control Rhizobium ENRRI ENRRI TAL TAL USDA Phosphobacterin B ENRRI 63 + B ENRRI 4 + B TAL B TAL B USDA B LSD for Rhizobium LSD for phosphobacterin LSD for Rhizobium x phosphobacterin Effects of on Shoot Dry Weight: Inoculation with Rhizobium strains ENRRI 4, ENRRI 63 and TAL 209 significantly (P 0.05) increased pigeon pea shoot dry weight at 4 and 6 weeks after sowing in the first season (Table 4). In the second season all strains significantly (P 0.05) increased the shoot dry weight at all sampling times compared to un-inoculated control. Inoculation of pigeon pea by introduced or locally-isolated bacterial strains improved dry matter production [16]. BMP inoculation significantly (P 0.05) increased shoot dry weight in both seasons compared to uninoculated control. This result supports the finding of Kannaiyan et al., [15] for pigeon pea plant dry weight. Co-inoculation with Rhizobium strains and phosphobacterin significantly (P 0.05) increased the shoot dry weight in the second season. At 4 weeks interaction between TAL 1132 and phosphobacterin gave the highest significant increment in shoot dry weight, at 6 weeks the highest result was obtained by ENRRI 63 plus phosphobacterin and at 8 weeks by TAL 209 plus phosphobacterin in the first season compared to un-inoculated control and other treatments. Inoculation of pigeon pea seeds with multiple co-inoculant produced maximum plant biomass [32]. Table 4: Effects of treatments on shoot dry weight (g/plant) of pigeon pea Weeks after sowing Uninoculated Control Rhizobium ENRRI ENRRI TAL TAL USDA Phosphobacterin B ENRRI 63 + B ENRRI 4 + B TAL B TAL B USDA B LSD for Rhizobium LSD for phosphobacterin LSD for Rhizobium x phosphobacterin Effects of on Shoot Nitrogen Content: Inoculation with Rhizobium strains significantly (P 0.05) increased pigeon pea shoot nitrogen content at 8 weeks after sowing in the first season compared to un-inoculated control (Table 5). In the second season Rhizobium strain TAL 209 significantly (P 0.05) increased nitrogen content at 8 weeks after sowing compared to un-inoculated control. Osman and Mohamed [22] reported that inoculation of faba bean with Rhizobium strain TAL 1400 constantly resulted in severe increment in N 2 fixation. Inoculation with phosphobacterin significantly (P 0.05) increased nitrogen content at 8 weeks after

5 sowing in both seasons compared to un-inoculated control. This result is in accord with the results obtained by Rugheim and Abdelgani [25] for faba bean. Co-inoculation with Rhizobium strains and phosphobacterin strain significantly (P 0.05) 2746 increased shoot nitrogen content at 8 weeks after sowing in the first season compared to un-inoculated control. Inoculation of pigeon pea seeds with multiple co-inoculant produced maximum total nitrogen [32]. Table 5: Effects of treatments on nitrogen content (%) of pigeon pea shoot Uninoculated Control Rhizobium ENRRI ENRRI TAL TAL USDA Phosphobacterin B ENRRI 63 + B ENRRI 4 + B TAL B TAL B USDA B LSD for Rhizobium LSD for phosphobacterin LSD for Rhizobium x phosphobacterin Effects of on Shoot Phosphorus Content: Inoculation with Rhizobium strains TAL 209, TAL 1132 and USDA 3472 strains significantly (P 0.05) increased pigeon pea shoot phosphorus content at 6 weeks after sowing in both seasons compared to un-inoculated control (Table 6). Rhizobium Strains USDA 3472, ENRRI 63 and ENRRI 4 significantly (P 0.05) increased shoot phosphorus content at 8 weeks after sowing in both seasons. Increments in shoot phosphorus content of faba bean due to Rhizobium inoculation was previously reported by Rugheim and Abdelgani [25]. Phosphobacterin inoculation significantly (P 0.05) increased shoot phosphorus content at 6 weeks after sowing in both seasons and at 8 weeks after sowing in the second season compared to un-inoculated control. Similar result was reported by Hassan and Abdelgani [12] for lablab bean. Co-inoculation with Rhizobium strain TAL 209 and phosphobacterin gave the highest significant shoot phosphorus content at 6 and 8 in the first season, and with strains USDA 3472 and ENRRI 4 in the second season. Hassan and Abdelgani [12] [12] found that Co-inoculation increased shoot phosphorus content of lablab bean. Table 6: Effects of treatments on phosphorus content (%) of pigeon pea shoot Uninoculated Control Rhizobium ENRRI ENRRI TAL TAL USDA Phosphobacterin B ENRRI 63 + B ENRRI 4 + B TAL B TAL B USDA B LSD for Rhizobium LSD for phosphobacterin LSD for Rhizobium x phosphobacterin Effects of on Pigeon pea Yield: All Rhizobial strains except ENRRI 63 and ENRRI 4 in the first season significantly (P 0.05) increased pigeon pea seed yield in both seasons (Table 7). The highest yield in both seasons was obtained by TAL Pulses seeds treated with specific strains of Rhizobium increases the yield through better nodulation [29]. BMP inoculation significantly (P 0.05) increased seed yield in both seasons. Rugheim and Abdelgani [27] found that phosphate solubilizing bacteria individually significantly increased faba bean yield. The effects of interaction between nitrogen fixing and phosphate solubilizing microorganisms on legume crops were reported to be relatively scarce

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