Effect of nitrogen fertilization on the symbiotic rhizobia/Common bean (Phaseolus vulgaris L.)

S. KOUKI; B. SIFI

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Volume Spéciale - Conférence IABC 2015, Article 19	[Volume Spéciale - Conférence IABC 2015, Article 19]	729 kB

S. KOUKI^{1, 2*}

B. SIFI ¹

¹Laboratoire des Sciences et Techniques Agronomiques, Institut National de la Recherche Agronomique de Tunisie, Rue. Hédi Karray 2049 Ariana, Tunisie.

² Institut National Agronomique de Tunisie, 43, Avenue Charles Nicolle, 1082 Tunis, Tunisie.

Abstract - Nitrogen is an essential component of proteins. This element is one of the most determining factors of agricultural production. In legumes, the nitrogen fertilizer intake ensures a good start growth until the formation of nodules. The contribution is made minimum amount to avoid the depressive effect of mineral nitrogen on nodulation. The test was conducted in greenhouse cultivation condition. The variety of common bean (Phaseolus vulgaris L.) Coco Blanc used and the two rhizobial strains Ar02 and CIAT899 the most efficient in the collection were selected for the study of their effectiveness combined with the contributions of different nitrogen rates (0mM, 0.5mM, 2mM to 6mM). The measurement parameters focused on the nodulation, growth, biomass production and nitrogen content. The results showed that the dose of 2 mM nitrogen, was dose nitrate threshold beyond which there is an inhibition of nodulation of common bean plants inoculated with the two strains tested. Thus, this dose can be regarded as stimulating the growth of stater and biological nitrogen fixation in the presence of rhizobia strain.

Keywords: biomass; common bean; nitrogen; nodulation; rhizobia; symbiosis.

Introduction

Common bean (Phaseolus vulgaris L.) is the world's most important grain legumes for direct human consumption, they comprise 50% of the grain legumes consumed worldwide (Broughton et al. 2003; Graham et al. 2003). Environmental constraints such as high soil acidity and low soil nitrogen considerably limit common bean production, in particular in the Mediterranean and tropical zones (Graham et al. 2003). Moreover, in some previous studies on the mineral requirements of the symbiotic process between common bean roots and rhizobia, the absence of nitrogen limited growth of Anadenanthera colubrina and Pinus rigida but nitrogen mineral addition inhibited the nodulation (Camela and Lazara 2004). Furthermore, experiments under controlled conditions showed that N content and biomass of common bean plants were increased in response to adequate P nutrition (Vadez et al.2001). Consequently, combining adequate nutrient fertilization, especially N and P and efficient native rhizobia may have a synergitic effect in order to improve symbiotic nitrogen fixation (SNF) and then production of common bean considered as a weak nodulating legume species (Abdi et al. 2014). Symbiotic nitrogen fixation is important for the production of legumes and the sustainability of agriculture. Several environmental conditions limit the growth of the N2-fixing legumes. Adverse conditions can lead to important reductions in their productivity and their subsequent agronomic and environmental roles. Major environmental limitations faced by legumes, and their rhizobial symbiotic partners, include nutrient deficiencies or disorders. Several of the nutrients that are essential for growth of plants or bacteria play specific roles in symbiotic N2 fixation through their effects on nodulation and/or the N2 fixation process (O’Hara 2001). Deficiencies of these nutriments can specifically reduce the number and size of nodules, and the amount of N2 fixed. Thus, the aim of this work was to study the effect of N fertilization on rhizobia strain efficiency and it impact on symbiotic nitrogen fixation, nodulation, plant growth, biomass nitrogen content under greenhouse condition.

2. Materials and methods

2.1. Biological material and experimental treatments

The study was carried out on common beans (Phaseolus vulgarisL.) variety, Coco Blanc frequently cultivated in the Tunisia. Common bean seeds are sterilized with calcium hypochlorite (6.7%) for 15 min and then washed carefully in 4 changes of sterile distilled water. Thereafter, seeds were germinated for three days in Petri dishes containing sterile moistened blotting paperand the seeds were transferred in pots containing sterile perlite. Reference rhizobia CIAT899 and local rhizobia Ar02 was provided by the National Institute of Agronomic Research in Tunisia (INRAT, Tunisia). Rhizobial inoculants, prepared as a liquid culture in YEM medium, were applied by soaking seedlings for 30 minutes in the inoculants prior to transplanting. The trial was conducted in a greenhouse with increasing concentrations of nitrate (0mM, 0.5 mM, 2 mM and 6 mM). The test comprises twelve treatments each treatment was repeated 4 times. Pre-germinated seeds were sown in pots containing sterile perlite and placed according to the following treatments: T1: 0 mM N (control); T2: 0 mM N+ Ar02; T3: 0 mM N+ CIAT899; T4: 0.5 mM N; T5: 0.5 mM N+Ar02; T6: 0.5 mM N + CIAT899; T7: 2 mM N; T8: 2 mM N + Ar02; T9: 2 mM N + CIAT899; T10: 6 mM N; T11: 6 mM N + Ar02; T12: 6 mM N + CIAT899. Irrigation was performed at 40 ml per pot 2 times a week with a nutrient solution devoid of nitrogen. (Vincent 1970). The measurement of parameters of the nodulation bearing on the number and nodule biomass, growth (aerial parts and root length) and (shoot and root biomass) were determined in flowering stage.

2.2. Harvest and data analysis

For greenhouse trials, four plants for each treatment were harvested at the early flowering stage. Nodules were then removed from the roots and the plants were separated into shoots and roots and dried in an oven at 70 °C for 72 h. After dry weight measurements, shoots of each sample were ground individually and the N content was measured using the Kjeldahl procedure. Complete systems with nodules of four plants were collected. Then, after rinsing them carefully, the roots and shoots of each plant were placed in paper bags. Symbiotic parameters (nodule number and nodule dry weight), shoots and roots dry weight, aerial parts and root length, nitrogen content (%N/Pl) at flowering stage were measured on each plant individually.

2.3. Statistical analysis

The experimental design was a randomized complete block. Statistical analysis was performed by the SPSS 11.5 software. The data were analysed using ANOVA and subsequent comparison of means was performed using the Fisher’s LSD test at p < 0.05.

3. Results and discussion

3.1. Number and nodule biomass

Statistical analysis of the results showed that excess mineral nitrogen in the medium inhibited the nodulation in number and biomass with both strains Ar02 and CIAT899 with significant differences between nitrogen treatments (Fig.1).