Nodulation, biomass production, and diversity of ...

Revue des Régions Arides -Numéro spécial- Actes du séminaire international : Gestion des ressources et applications biotechnologiques en aridoculture et cultutres oasiennes : Perspectives pour la valorisation des potentialités du Sahara

2006

Nodulation, biomass production, and diversity of rhizobia nodulating chickpea under water deficiency Samir BEN ROMDHANE, Mohamed Elarbi AOUANI, & Ridha MHAMDI Laboratoire Interactions Légumineuses- Microorganismes, Centre de Biotechnologie de Borj-Cedria, BP 901, Hammam-lif 2050, Tunisie E-mail : [email protected] SUMMARY The effect of water stress on nodulation, biomass production, and competition for nodule occupancy was evaluated in four different soils with two chickpea cultivars, cv. Amdoun I and cv. Chetoui. Plants were grown in plastic pots in the greenhouse. Two treatments were considered; a control treatment which received water three times per week and a water deficient treatment which was irrigated only one time a week. Results showed that water deficiency significantly decreased the nodule number and the shoot dry weight for both cultivars. Nodulating bacteria were isolated and characterized by PCRRFLP of 16S rDNA. The results showed that water deficiency affected the diversity of nodulating rhizobia. The nodulation by Mesorhizobium mediterraneum was inhibited while nodulation by Sinorhizobium meliloti seemed to be favoured. Analysis for salt tolerance showed that chickpea was selectively nodulated by salt tolerant rhizobia under water deficiency. Key words: Cicer arietinum, drought, genetic diversity, salt tolerance, Mesorhizobium mediterraneum, Sinorhizobium meliloti RESUME Nodulation, production de biomasse, et diversité des rhizobia nodulant le pois chiche sous déficience hydrique L’effet du stress hydrique sur la nodulation, production de biomasse, et la compétitivité pour l’occupation nodulaire a été évalué dans quatre sols différents et avec deux cultivars de pois chiche, cv. Amdoun I et cv. Chetoui. Les plantes ont été développées dans des pots en plastique et sous serre. Deux traitements ont été considérés; un traitement controle qui reçoit l’eau trois fois par semaine et un traitement déficient en eau qui est irrigué une fois par semaine. Les résultats montrent que la déficience hydrique entraîne une diminution significative dans le nombre de nodules et dans le poids de matière sèche aérienne pour les deux cultivars. Les bactéries nodulantes ont été isolées et caractérisées par PCR-RFLP de l’ADNr 16S. Les résultats montrent que la déficience hydrique affecte la diversité des rhizobia nodulant. La nodulation par Mesorhizobium mediterraneum a été inhibée par contre celle engendrée par Sinorhizobium meliloti semble être favorisée. L’analyse pour la tolérance au sel montre que le pois chiche a été sélectivement nodulé par les rhizobiums tolérants au sel sous déficience hydrique. Mots clés: Cicer arietinum, sécheresse, diversité génétique, tolérance au sel, Mesorhizobium mediterraneum, Sinorhizobium meliloti 1. INTRODUCTION Chickpea (Cicer arietinum L.) is one of the most important grain legumes traditionally cultivated in deprived areas and saline soils (Rao et al., 2002). The agronomical importance of chickpea is based on its high protein concentration 25–29% (Hulse, 1991) for the human and animal diet, being used more and more as an alternative protein source. In the Mediterranean area, a substantial part of the year passes with little or no rainfall, and in the absence of irrigation, agricultural soils dry out. In the absence of the host plant, this desiccation has been recognized for a long time as a potential stress on rhizobial survival. This stress is very often associated with high temperatures. The mechanism relating soil water loss to the numbers of rhizobia surviving desiccation is just beginning to come to light and reviews concerning the survival of rhizobia in soil have been proposed (Zahran et al., 2001). Most of Tunisia lies in the semiarid, arid and Saharan climatic zones and the annual rainfall in these areas varies from 300 to less than 100 mm. Chickpea is one of the main grain legumes used for human 1273


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and animal nutrition in Tunisia and is grown under rain-fed and irrigated conditions. Drought imposed by uncertain and scarce precipitation or sporadic irrigation reduces nitrogen fixation and yield of this legume. Over the past 20 years, chickpea cultivation, as well as other grain legumes, has decreased in favour of continuous cereal crops. In Tunisian farming systems, yields of chickpea are lower and generally more variable than those of cereal crops due to the effect of drought. Drought influences persistence and survival of rhizobia in the soil and can limit both nodulation and nitrogen fixation (Barnet et al., 1988; Sprent and Zahran, 1988; Graham, 1992). Drought directly affects nodule activity and function (Albrecht et al., 1984; Davey and Simpson, 1990) and also limits nodulation through its effects on root-hair colonization and infection by rhizobia (Singleton and Bohlool, 1984; Zahran and sprent, 1986; Sprent and Zahran, 1988). Intermittent drought can severely reduce nitrogen fixation (Bacana et al., 1986; Davey and Simpson, 1989), whereas prolonged drought may increase rates of nodules senescence (Sprent et al., 1983; Sutton, 1983). Nodules may be shed from legume roots by drought-induced senescence, and resumption of nitrogen fixation depends on formation of new nodules (Sprent and Zahran, 1988; Sinclair et al., 1988). Although the widespread prevalence of drought in Tunisia may have selected for drought-tolerant rhizobial strains, no information is available concerning the effect of drought on nodulation and symbiotic nitrogen fixation of chickpea in Tunisia. As a result, the present study was undertaken to evaluate the influence of water stress on nodulation, biomass production and diversity of rhizobia nodulating chickpea in different soil samples under greenhouse conditions. 2. MATERIALS AND METHODS 2.1. Water deficiency under soil conditions Four soil samples originating from the north of Tunisia (Menzel Bourguiba and Mateur), Ben Mustapha, Maghraoui, Mastouri and Tijeni Tej, were used. The four sites belonged to the sub-humid stage and had been cultivated with cereals in rotation with three main grain legumes, faba bean, chickpea and common bean. These soils were non-saline and slightly alkaline. The soil samples were distributed in 3 litre plastic pots (2 kg per pot) and transferred to the glasshouse. Two chickpea cultivars, Amdoun I and Chetoui, were used in this study. Two pre-sterilized seeds were sown in each pot. Two treatments were considered, a control treatment which received 150 ml of water three times per week and a water deficient treatment which received 150 ml one time per week. At flowering, the root systems were washed, nodules were picked and counted for each plant and stored in small vials containing dehydrating powder (CaCl2) for later rhizobia isolation and characterization.. Shoots were oven dried at 70°C for 48 h and weighted. Ten individual plants were considered for each treatment. Statistical analysis was done using the STATISTICA software by the ANOVA/MANOVA program. The HSD test (P < 0.05) was used for means comparison. 2.2. Isolation and characterization of rhizobia Rhizobia were isolated from root nodules of chickpea according to standard procedures as previously described (Mhamdi et al., 1999). One isolate was kept from each nodule. Nodules produced on the water-deficient plants were all used. PCR-RFLP of 16S rRNA genes was conducted on cells treated with proteinase-K using primers fD1 and rD1 (Weisburg, 1991) as previously described (Mhamdi et al., 2002). Digestion was performed by five endonucleases, MspI, NdeII, CfoI, HaeIII and RsaI. Species assignation was done according to 16S rDNA typing and comparison with the published database of mapped restriction sites in the 16S rRNA genes of rhizobia (Laguerre et al., 1997). 2.3. Salt tolerance Rhizobia isolated from the two treatments for both cultivars were evaluated for their tolerance to salinity. Salt tolerance of the different isolates was assessed in yeast-extract mannitol broth (YEM) supplemented with different concentrations of sodium chloride going from 0.2 to 3% (w/v). Rhizobia were taken from cultures stored in 20% glycerol at –80°C and transferred to YEM slants. Transfers were made from the slants to YEM broth and inoculated flasks were incubated on a rotary shaker at 150 rev min-1 and 28°C. At mid-exponential growth phase, the equivalent of 20 µl of broth culture for OD620nm =1 was transferred to 30 ml flasks containing 5 ml of YEM broth with varying concentrations 1274


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of NaCl. Three replicates were considered for each test. The ability to grow was checked visually after incubation at 28°C under orbital agitation.

3. RESULTS 3.1. Effect of water stress on nodulation The water deficiency affected the number of nodules induced on both cultivars (Table 1). Table 1: Effect of water deficiency on nodule number of two chickpea cultivars cultivated on soil samples in the greenhouse. Values are means of 10 replicates. Tijeni Tej Ben Mustpha Mastouri Maghraoui T0 WD T0 WD T0 WD T0 WD AmdounI 7 2* 4 1,2* 1,7 1,2 3,7 0* Chetoui 9 2* 0,6 0,4 3,5 1* 5,3 0* T0, control treatment; WD, water-deficient treatment; *, the difference between the control and the water-stressed plants is significant (p