Effects of rhizobia inoculation and molybdenum application on nodulation, N uptake and yield of peanut (Arachis hypogaea L.)

A field experiment was conducted in two sites of the humid forest zone of the Centre Region of Cameroon (Yaounde and Bokito), to study the interactive effect of rhizobia inoculation and Molybdenum (Mo) application on nodulation and yield of peanut (Arachis hypogaea L.). The experiment was laid out in a randomized complete block design with four replications. The treatments consisted of control (C), Rhizobium inoculation (R), Mo application (Mo) and a combination R+Mo. The results showed that Molybdenum and rhizobia inoculation had a significant effect on the yield of peanut at both sites. The inoculation with rhizobia showed a remarkable effect on the nodulation of the fallow of Yaounde (67. 73%) and not on the mixedfarm in Bokito (55. 43%) at P = 0.05. Nodulation was also stimulated through the combination of rhizobia inoculation with molybdenum application with increment of 111.26% and 50.44% nodule dry weight respectively for Yaounde and Bokito sites. The application of molybdenum alone improved significantly (P = 0.05) seed yield of peanut in both sites. However the increment was high in Yaoundé site (174%) as compared to Bokito site (10%). It stands out clearly that rhizobia inoculation combined with molybdenum application greatly enhanced biological N-fixation of peanut. The effect of these treatments on yield and the nitrogen uptake of peanut were much more remarkable on the fallow of Yaounde than the mixed-farm of bokito. It is therefore possible to greatly stimulate the biological Nfixation and the nitrogen uptake of peanut through symbiosis with the native rhizobia of the soil alone or after inoculation. * Corresponding Author: Marie Solange Mandou  solangemandou@yahoo.fr International Journal of Agronomy and Agricultural Research (IJAAR) ISSN: 2223-7054 (Print) 2225-3610 (Online) http://www.innspub.net Vol. 11, No. 1, p. 103-113, 2017 Int. J. Agron. Agri. R. Mandou et al. Page 104 Introduction Peanut (Arachis hypogaea L.) is one of the principal economic crops of the world and ranks 13th among the food crops (Kabir et al., 2013). In Cameroon it is the main cultivated grain legume in all the agro ecological zones. 250,000 ha are under its cultivation with half of the area concentrated in the northern part of the country (Ntoukam et al., 1996). Peanut is an important source of protein for human nutrition and income for the farmers. However its average yield in Cameroon is still low with 1.40 t ha-1 (FAOSTAT, 2014). The poor yields are partly due to poor soil fertility of acidic soils with low nutrient content including nitrogen (Zahran, 1999) and molybdenum (Liebenberg, 2002). Nitrogen is a critical limiting element for the growth and development of most plants (Bambara and Ndakidemi, 2010). It is the most affected due to its high uptake, vulnerability to leaching, losses in gaseous form and through crop harvest (Stoorvogel et al., 1993) and has been previously identified as the main soil fertility constraint for crop yields (Bationo and Mokyunwe (1991). Then supply to plants is mostly done through the application of mineral fertilizers which are costly and not available to poor resource farmers such as those found in Africa (Bado, 2002). Also, its production requires high amounts of non-renewable fossil energy resulting to the release of greenhouse gases (Jensen et al., 2012). However, biological N-fixation (BNF), a key resource of N may represent one of the possible solutions and a great possibility for sustainable production of grain legumes (Mfilinge et al., 2014). The BNF is an alternative to the use of expensive inorganic fertilizers. This process can reduce the need for N fertilizers, resulting in an economy estimated to 3billion US dollars per cropping season (Nicolas et al., 2006). Peanut, as leguminous plant, in symbiotic relationship with Rhizobium, has the ability to convert nitrogen from the air into the soil and transform it into ammonium (NH4). According to Giller (2001), the quantities of nitrogen fixed by peanut vary from 21 to 206 kg Nha-1. However, soil nutrient deficiencies can limit the BNF in several agricultural soils and subsequently reduce the yield potential (Bordeleau and Prévost, 1994; Zahran, 1999; Hussein, 2000; Paudyal et al., 2007). In addition, nutrient limitations in the production of grain legume results not only from the deficiency of macroelements such as nitrogen, phosphorus, potassium and sulfur (Jamal, 2010), but also from the deficiency of microelements such as molybdenum (Mo), boron and iron (Rahman, 2008). Molybdenum is one of the micronutrients required for plant growth and development. It constitutes part of the enzyme nitrogenase which is essential for the conversion of atmospheric N2 to ammonia NH3. Mo deficiencies are therefore much more pronounced in legumes (Bailey and Laidlaw, 1999) as compared to non-leguminous plants. Symbiotic bacteria require more Mo than host plant for N2 fixation (O’hara et al., 1988) and thus, the supply of this element to bacteroids is an important process in the maintenance of BNF in legumes. In Cameroon 80 % of the arable lands are acidic with low Mo content (Thé, 2000). Since peanut is a legume, it is highly susceptible to Mo deficiency when grown in acidic soils and subsequently affects its ability to nodulate and to biologically fix N. Although yield increase is observed following inoculation with selected strains of rhizobia, there is little or no data on yield response of peanut in legumes mix-cropping. In addition, the application of molybdenum in these conditions has been seldom reported in Cameroon. This study therefore aimed at investigating the effects of seed rhizobia inoculation and/or molybdenum application on peanut nodulation, yields and N-uptake in two contrasting soils in the humid forest zone of Cameroon. Materials and methods Experiments were carried out in two location sites of the humid forest zone in the Centre Region of Cameroon: Yaounde with a red clayey soil and Bokito with a gray sandy soil. The site of Yaounde (3°50’N, 11°30’E) was a three years fallow dominated by Chromolaena odorata and Pennisetum purpeureum Int. J. Agron. Agri. R. Mandou et al. Page 105 whereas that of Bokito (4°34’N, 11°6’E) was a mixed farm with a long history of legume cropping such as peanut and cowpea. Soil chemical characteristics of both sites are presented on the table 1. Experimental design and treatments The experiment followed a complete randomized bloc design with four treatments: C (Control: no inoculation and no Mo application), R (inoculation with rhizobium), Mo (Mo application) and R+Mo (the combination of rhizobia inoculation and Mo application). Each treatment was replicated four times. The basic plot size was 8 m2 and plots were separated by 0.5 m to prevent contamination. The peanut seeds of the A26 variety obtained from the Institute of Agricultural Research and Development (IRAD) were used in this experiment. Planting was done during the rainy season in the month of April. For Mo and Mo+R treatments, seeds were soaked overnight in the solution of ammonium molybdate (53% of Mo) at the rate of 1kg of Moha-1. The C and R treatment seeds were soaked overnight in distilled water. The rhizobium inoculant used was “Cynthia T” and consisted of a charcoal carrier Bradyrhizobium spp (108 cells/g of inoculant) isolated from Cameroonian soils. It was provided by the Laboratory of Microbiology, Biotechnology Centre, University of Yaounde I. The inoculant was coated to R and R+Mo treatment seeds (1kg inoculant per hectare) using powder milk as a sticking agent and the inoculated seeds were air-dried 1 hour before sowing. To avoid contamination, all Rhizobium uninoculated treatments were sown first. Sowing was carried out manually at 25 cm between rows, 20cm between plants (240000 plants per hectare) and at depth 2–4 cm. One seed was introduced in each planting hole and no nitrogen fertilizer was applied throughout the study period. Data collection Estimation of viable rhizobia in soils An experiment was set up in a growth chamber (Laboratory of Soil Microbiology, Biotechnology Centre, University of Yaounde I) to estimate the total viable rhizobia in the experimental soils using plant infection method (Most Probable Number [MPN] technique). Soil samples collected from Bokito and Yaounde sites (0-20 cm) were stored at 4°C overnight. Serial dilutions (ten-fold dilution) were prepared as described by Somasegaran and Hoben (1994) with 100g of soil for the first dilution step. The dilution was up to 10-6 level. A diluent solution was prepared by dissolving 0.125 g KH2PO4 and 0.05 g MgSO4.7H2O in 1000 ml of distilled water. The host plants, Macroptilium atropurpureum, were cultured in tubes containing a sterilized mixture of sand and vermiculite (1:3 (v/v)). Seeds were scarified and surface sterilized in concentrated sulfuric acid (H2S04) for 4 minutes, rinsed several times with sterile distilled water and pre-germinated in petri dishes filled with agarose solution (2%). Upon emergence of the radicle, one pre-germinated seed was transferred aseptically to each tube. A week later, 1 ml of each level of dilution in four replicates was inoculated onto the root zone of cultured plants. Plants were watered with sterile nitrogen freenutrient solution (Vincent, 1970). They were then scored for nodulation 28 days after inoculation and the indigenous rhizobia was determined using the Fisher and Yate table (Vincent, 1970). Nodulation and biomass assessment Ten plants from the inner rows were harvested randomly in each plot (total of 40 plants per treatment) at 55days after sowing to record the nodulation status and biomass of A. hypogaea. The plants were carefully dug out with their entire root system, washed with tap water and their nodules picked and counted. Nodules were then excised and the number with pink pigmentation were recorded. The nodules with pink color were considered efficient. The efficiency of nodulation was calculated