Vol. 9(5), pp. 36-44, October 2017 DOI: 10.5897/JABSD2017.0294 Article Number: D10CC7366409 Journal of Agricultural Biotechnology and ISSN 2141-2340 Copyright ©2017 Sustainable Development Author(s) retain the copyright of this article http://www.academicjournals.org/JABSD

Full length Research Paper

Improvement of tree growth in salt-affected soils under greenhouse conditions using a combination of peanut shells and microbial inoculation

Dioumacor Fall1,2,3*, Niokhor Bakhoum2, Fatoumata Fall2,3, Fatou Diouf2,3, Mathieu Ndigue Faye2, Cheikh Ndiaye2, Valérie Hocher4 and Diégane Diouf2,3,5

1Institut Sénégalais de Recherches Agricoles, Centre National de Recherches Forestières, Route des Pères Maristes, Dakar-Sénégal. 2Laboratoire Commun de Microbiologie, Centre de Recherche de Bel-Air, Dakar-Sénégal. 3Laboratoire Mixte International Adaptation des Plantes et Microorganismes Associés Aux Stress Environnementaux, Dakar-Sénégal. 4Laboratoire des Symbioses Tropicales et Méditerranéennes, Institut de Recherche pour le Développement, Campus International de Baillarguet, 34398 Montpellier, France. 5Département de Biologie Végétale, Université Cheikh Anta Diop de Dakar, Dakar-Sénégal.

Received 1 August, 2017; Accepted 22 September, 2017

This study aimed at selecting an effective microbial inoculum to enhance the performance of senegal, seyal and Prosopis juliflora and assessing the combination effect of microbial inoculation and peanut shells amendment on their growth under salinity in greenhouse conditions. In the first experiment, seedlings were individually cultivated in plastic bags containing non-sterile sandy soil. Seedlings were inoculated at transplantation with rhizobial and arbuscular mycorrhizal fungi (AMF) strains. Four inoculation treatments were performed: control, inoculation with rhizobia, inoculation with AMF and dual-inoculation with rhizobia and AMF. After one month, seedlings were gradually watered with four saline solutions (0, 86, 171 and 257 mM NaCl) for 4 months. ANOVA showed that inoculation treatments significantly increased seedlings growth particularly in saline conditions and the best performance was obtained with dual inoculation. In the second experiment, seedlings were grown under the same experimental conditions on a mixture of non-sterile sandy soil and 6 tha-1 (169.56 g per bag) of peanut shells var 73-33. Results showed that inoculation, peanut shells and their combination significantly improved seedlings growth. The higher performance was obtained with the combination of microbial inoculation and peanut shells.

Key words: Mycorrhiza, rhizobia, organic amendment, , , Prosopis juliflora, salt tolerance.

INTRODUCTION

Soil salinity is a worldwide environmental problem, which and dispersion of clays, as well as breaking soil negatively affects soil properties (Sumner, 2000; Kahlown aggregates. Consequently, both water infiltration and and Azam, 2003). Salt-affected soils contain high water-holding capacity could be reduced, which leads to exchangeable Na+ percentage, which leads to swelling decreased growth and productivity, particularly in Fall et al. 37

arid and semi-arid regions (Paul, 2012; Shahbaz and Vachellia seyal (Syn. Acacia seyal) and Prosopis juliflora Ashraf, 2013). are multi-purpose forest legumes widely found in arid and Revegetation of salt-affected soils is very challenging semi-arid zones. They have huge potential in agroforestry because, in addition to their high Na+ content, they are systems, fuel wood production, forage, medicinal deficient in nutrients such as nitrogen (N), phosphorus products and gum production except for P. juliflora (Von (P) and devoid of organic matter (OM) (Asma et al., Maydell, 1986). Thus, the rehabilitation of salt-affected 2009). However, their physical, chemical and biological soils with these species would allow local populations to properties can be improved with the application of OM benefit from their ecosystem services. Thus the (Wang et al., 2014). These soils can be improved by objectives of this study are to; select an effective providing a source of calcium (Ca2+) to replace excess microbial inoculum (rhizobial alone, mycorrhizal alone or sodium (Na+) from the cation exchange sites (Qadir et al., dual inoculation) to enhance the performance of S. 2007). Therefore, the application of OM rich in calcium for senegal, V. seyal and P. juliflora under salt conditions soil remediation is important for sustainable land use and and evaluate the combined effect of microbial inoculation plant productivity under salinity (Choudhary et al., 2004; and peanut shells amendment on seedling growth of Wong et al., 2009). Recent studies show that peanut these species in salty soils under greenhouse conditions. shells of variety 73-33 contains high proportion of calcium and could be used to improve saline soils characteristics (Fall, 2016). MATERIALS AND METHODS In addition to their role in improving soil properties, enhancing plant salt tolerance facilitates growth in saline Plant material conditions. The role of biofertilizers such as rhizobia (Diouf et al., 2005; Thrall et al., 2008), Frankia (Diagne et Seeds of S. senegal, V. seyal and P. juliflora collected in 2013, al., 2013; Ngom et al., 2016), arbuscular mycorrhizal respectively, at Dahra and Ndiaffate for V. seyal and P. juliflora, fungi (Evelin et al., 2009; Kohler et al., 2010) and plant were provided by the Centre National de Recherches Forestières growth-promoting rhizobacteria (Paul and Nair, 2008; (CNRF) of the Institut Sénégalais de Recherches Agricoles (ISRA). Kohler et al., 2010) in tolerating salt stress has Seed scarification and pre-germination were done as described by Fall et al. (2009). been well established in many studies. The bacterial- mycorrhizal-legume tripartite symbiosis is currently being suggested as a possible solution to reforestation (Kohler Rhizobial and fungal inocula preparation et al., 2010 ; Diagne et al., 2013; Soliman et al., 2014;

Ngom et al., 2016; Zhu et al., 2016). Pre-germinated seedlings were transplanted into plastic bags (12 Biological , which consist of cm × 25 cm) filled with non-saline and none sterile soil (Table 1) assimilation of atmospheric N in form of organic collected from Sadioga (Centre of Senegal Peanut Basin, compounds by rhizobia or Frankia, is a sustainable 16°23’18’’W; 14°03’53’’N) at 0 to 25 cm depth. For rhizobial inocula, source of N in cropping systems, as fixed N can be used two strains selected for their symbiotic infectivity and effectiveness directly by plants (Jensen and Hauggaard-Nielsen, 2003; in controlled conditions were used for each species. The strains ORS 3574 and ORS 3593 were used for S. senegal (Bakhoum et Garg and Geetanjali, 2007). Phosphorus availability is al., 2012), whereas ORS 3356 to ORS 3359 and Pj 34 and Pj 36 another limiting factor in salt-affected soils. Due to an were used for V. seyal (Diouf et al., 2010) and P. juliflora (Diagne extended network of fine hyphae, the arbuscular and Ingleby, 2003), respectively. Each strain was cultured in glass mycorrhizal fungi (AMF) can considerably improve the flasks containing liquid yeast extract mannitol medium (Vincent, uptake of mineral nutrients particularly P and water in 1970) at 30°C for an orbital shaker. For each plant species, the two host plants (Aggarwal et al., 2011). AMF help plants to individual cultures were adjusted to contain approximately the same rhizobial cells number. These cultures were mixed in equal alleviate salt stress by enhancing mineral nutrition to proportions (v/v) to obtain the rhizobial inoculant (approximately 109 compensate for nutrient deficiencies, compensating for cells/ml). For mycorrhizal inocula, a mixture of three AMF species nutritional imbalances, improving plant water status, and Glomus fasciculatum (Thaxter sensu Gerdemann DAOM 227 130), reducing salt uptake into the host plant (Weissenhorn, Rhizophagus irregularis (Schenck and Smith DAOM 197 198) and 2002). Glomus aggregatum (IR 27) were used. They were obtained from The improvement of soil physical, chemical and the LCM collection (Laboratoire Commun de Microbiologie, IRD/ISRA/UCAD, and Dakar, Senegal). Equal amounts of each biological properties and the strengthening of plant’s salt AMF inoculum were mixed to obtain a composite AM fungal tolerance would contribute to better reforestation of salt- inoculant with an average of 40 spores/g of soil and 80% colonized affected areas. Senegalia senegal (Syn. Acacia senegal), mycorrhizal (Ndoye et al., 2015).

*Corresponding author. E-mail : [email protected], [email protected]. Tel: +221 77 532 37 91; +221 33 849 33 02.

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 38 J. Agric. Biotech. Sustain. Dev.

Table 1. Physical and chemical characteristics of soil collected at peanut shells alone, plants dual inoculated alone, and plants with Sadioga (Central part of Senegal) in non-saline zone. peanut shells and dual inoculated. For the supply of peanut shells, they were crushed and mixed with soil at dose of 6 tha-1 Parameter Value corresponding to 169.56 g per bag (12 cm × 25 cm). Chemical characteristics of peanut shells variety 73-33 are presented in Table Physical characteristic 2. Pre-germinated seedlings were transplanted into plastic bags Clay (%) 05.5 with the different treatments. For microbial inoculation, the inocula Silt (%) 11.5 (rhizobial and AMF) were prepared as described above for each Sand (%) 83.0 plant species. Seedlings were dual inoculated at transplantation with 5 ml of rhizobial suspension and 20 g of AMF inoculants. Salt stress was gradually applied one month later and plants were Chemical characteristic arranged in a randomized complete block as described above. The experiment was carried out in greenhouse conditions (30°C / 25°C pH H2O 5.5 -1 day / night, 16 h photoperiod) at the LCM-Laboratoire Commun de Electrical conductivity (at 25°C) µScm 27 Microbiologie IRD/ISRA/UCAD in Dakar-Senegal (certified ISO Salinity (‰) 0.00 9001: 2015). Four months after salt stress application, the plant Total nitrogen (%) 0.05 height, collar diameter, shoot (leaves + stems), and total dry weight were evaluated as described above. The number of nodules Total carbon (%) 0.56 per plant was counted. For the assessment of mycorrhizal root -1) Total phosphorus (mgkg 52.00 colonization (MRC), a subsample of total root mass were cleared at Calcium (Ca) (méq%) 0.78 90°C for 30 min in 10% KOH and stained with 0.05% trypan blue Magnesium (Mg) (méq%) 0.25 (Phillips and Hayman, 1970). MRC (corresponding to the proportion of cortex colonized by AMF) was evaluated microscopically using Sodium (Na) (méq%) 0.09 the notation scale described by Trouvelot et al. (1986). Potassium (K) (méq%) 0.15 Cationic exchange capacity (méq%) 2.99 Statistical analyses

Three replicates (three seedlings) per treatment were used for Seedlings inoculation statistical analyses. Two-way-ANOVA analysis was used to assess the effect of salinity, microbial inoculation and their interaction on For each salinity level, four inoculation treatments were performed: measured variables in the first experiment. In each experiment, non-inoculated or control plants, plants inoculated with rhizobia, Student-Newman-Keuls´s post-hoc test was used to determine plants inoculated with AMF and plants dual-inoculated with rhizobia significant differences (P ≤ 0.05) among treatments for various and AMF. Seedlings were inoculated at transplantation with 5 ml of traits. All analyzes were carried out with XLSTATTM software rhizobial suspension for rhizobial inoculation alone, 20 g of AMF for package (version 2009, Addinsoft, Paris, France). mycorrhizal inoculation alone and 5 ml of rhizobial suspension and 20 g of AMF for dual inoculation. The control plants received equal amounts of autoclaved inocula. RESULTS

Salt stress and experimental design Selection of microbial inoculum to improve S. senegal, V. seyal and P. juliflora plants growth under Salt stress was performed one month after transplantation. Plants salinity were gradually exposed to NaCl in order to minimize any salinity shock. Four NaCl concentrations (0, 86, 171 and 257 mM) were tested. NaCl concentrations were increased by 43 mM per day until The ANOVA showed that salinity stress and inoculation the required final concentration was reached. The electrical had a significant (P < 0.05) effect on plant growth conductivity of the leachate from representative pots was monitored parameters (height, collar diameter, shoot and root dry regularly with a salinometer (Digit 100 ATC Salinity pocket weight) except for the height and collar diameter of V. refractometer, CETI, Optical Instruments, Belgium) to ascertain seyal (Table 3). In the same way, the interaction between actual NaCl concentrations within the rooting medium. Plants were arranged in a randomized complete block with two factors (salinity salinity and inoculation had a significant effect on these and inoculation). Each treatment was replicated ten times. Four parameters except for height and collar diameter of S. months after salt stress application, plant height and collar were senegal and P. juliflora. Results showed that salinity measured. The plants were harvested and the shoot (leaves + stem reduced the growth (height, collar diameter, shoot and + branches) and root dry biomass were evaluated. root dry weights) of S. senegal, V. seyal and P. juliflora

plants (Table 4) whereas microbial inoculation Evaluation of the combination of peanut shells and microbial significantly increased plant growth. inoculation on plants growth under salinity This positive effect of microbial inoculation depends on NaCl concentration, the species and type of After selection of the best inoculum (dual inoculation), the combined microorganism used as inoculum. No significant effect of effect of microbial inoculation and peanut shell were evaluated rhizobial, mycorrhizal or dual inoculation was observed under the same experimental conditions. Four NaCl concentrations (0, 86, 171 and 257 mM) were studied. For each species and each on the collar diameter of S. senegal seedlings and the salinity level, four treatments with ten replicates were performed: height of V. seyal regardless of NaCl concentrations control plants without inoculation and peanut shells, plants with (Table 4). A significant effect of AMF inoculation and dual Fall et al. 39

Table 2. Chemical characteristics of peanut shells (var 73-33) used in the study.

N total (%) C total (%) C/N P total (gkg-1) Ca (gkg-1) K (gkg-1) Na (gkg-1) Cl (gkg-1) 1.00 46.24 46 0.61 6.9 4.73 1.4 1.7

Table 3. Significance level from two-way ANOVA analysis testing the effects of salinity and inoculation on height, collar diameter (Col. diam.), shoots dry weight (SDW) and root dry weight (RDW) of S. senegal, V. seyal and P. juliflora plants grown on non-sterile soil for four months under greenhouse conditions.

Species Factor Height Col. diameter SDW RDW Salinity S (p<0.0001) S (p<0.0001) S (p<0.0001) S (p<0.0001) S. senegal Inoculation S (p=0.001) NS (p=0.381) S (p<0.0001) S (p<0.0001) Salin*Inoc NS (p=0.480) NS (p=0.285) S (p<0.0001) S (p<0.0001)

Salinity S (p<0.0001) S (p<0.0001 S (p<0.0001) S (p<0.0001) V. seyal Inoculation NS (p=0.590) NS (p=0.574) S (p<0.0001) S (p<0.0001) Salin*Inoc S (p=0.048) S (p=0.002) S (p<0.0001) S (p=0.001)

Salinity S (p<0.0001) S (p<0.0001) S (p<0.0001) S (p<0.0001) P. juliflora Inoculation S (p<0.0001) S (p=0.001) S (p<0.0001) NS (p=0.847) Salin*Inoc NS (p=0.524) NS (p=0.200) S (p<0.0001) S (p=0.039)

Salin*Inoc = interaction between salinity and inoculation. S = Significant, NS = Not significant.

inoculation were observed on collar diameter and height treatments was more pronounced with an increase in in P. juliflora plants. No significant difference was NaCl concentration. The best performance was observed observed between AMF inoculation alone and dual with the combination of peanut shells and microbial inoculation except for the collar diameter of V. seyal and inoculation, irrespective of species. Collar diameter of S. the height of S. senegal plants grown at 257 mM of NaCl. senegal increased by 21, 34 and 126%, respectively, with An increase of 20 and 37% were observed with dual microbial inoculation, peanut shells alone and inoculation on collar diameter of V. seyal and height of S. combination peanut shells and inoculation at 257 mM senegal seedlings at 257 mM NaCl, respectively. NaCl whereas increases of 55, 162 and 169% in the For biomass production, results showed that height of P. juliflora were observed with the same inoculation with rhizobia, AMF or dual inoculation treatments. The same trend was obtained on collar significantly increased shoot dry weight (SDW) and root diameter and height of V. seyal plants. All treatments dry weight (RDW) of seedlings for all species. However, significantly increased shoot dry biomass under salinity, there were no significant differences between inoculation except inoculation in S. senegal at 86 mM NaCl (Table treatments, the best growth was obtained with dual 5). No significant difference was observed between inoculation. For S. senegal, SDW increased by 23, 26 peanut shells alone and its combination with microbial and 49%, respectively with rhizobial, AM fungal and dual inoculation. However, the best aerial growth (SDB) was inoculation at 257 mM NaCl. RDW of V. seyal increased obtained with combination peanut shells and microbial by 11, 64 and 67%, respectively with rhizobial, AM fungal inoculation. SDW of S. senegal plants was increased by and dual inoculation at the same NaCl concentration 133, 187 and 267%, respectively, with microbial (Table 4). inoculation alone, peanut shells alone and their combination at 257 mM NaCl. The same trend was observed in V. seyal with an increase of 126, 409 and Combined effect of microbial inoculation (dual 417%, respectively, and in P. juliflora with 417, 1280 and inoculation) and peanut shells application on plant 1367% at the same NaCl concentration (Table 5). Peanut growth under salinity shells alone and its combination with microbial inoculation significantly increased root dry weight Results showed that microbial inoculation, peanut shells especially at high level of NaCl concentrations (171 and or the combination of both improved the growth of S. 257 mM). The best root growth was obtained with the senegal, V. seyal and P. juliflora plants under salinity combination of peanut shells and microbial inoculation for conditions (Table 5). The positive effect of these all species. 40 J. Agric. Biotech. Sustain. Dev.

Table 4. Height (cm), collar diameter (mm), shoot dry weight (g plant-1) and root dry weight (g plant-1) of S. senegal, V. seyal and P. juliflora plants grown on non-sterile inoculated with rhizobia and/or mycorrhiza, and exposed for four months to four salinity levels under greenhouse conditions.

Salinity Inoculation S. senegal V. seyal P. juliflora (mM NaCl) treatment Height Diameter SDW RDW Height Diameter SDW RDW Height Diameter SDW RDW Control 23.6a 3.55a 0.53a 1.34a 30.2a 2.12a 0.47a 1.55a 36.9a 2.32a 0.69a 0.39a Rhizobia 23.8a 3.48a 0.59a 1.37a 31.2a 1.90a 0.69b 1.86a 37.0a 2.35a 0.73a 0.49a 0 AFM 24.0a 3.81a 0.80b 1.61a 32.5a 2.05a 0.79b 2.49b 37.5a 2.63a 1.00b 0.48a Rhizo+AFM 24.2a 3.50a 1.10c 2.12b 35.3a 1.77a 0.85b 1.88a 39.2a 2.38a 1.35c 0.54b

Control 19.4a 3.27a 0.47a 1.30b 26.8a 1.57ab 0.27a 0.60a 23.3a 1.65a 0.34a 0.23a Rhizobia 20.7a 3.38a 0.56b 0.84a 24.8a 1.31a 0.37a 0.99b 25.6ab 1.97b 0.42a 0.25a 86 AFM 19.9a 3.38a 0.59b 1.55c 28.1a 1.83b 0.49b 1.03b 27.2b 1.84ab 0.49b 0.26a Rhizo+AFM 23.7a 3.40a 0.67c 1.32b 28.7a 1.91b 0.56b 1.14b 29.7b 1.96b 0.57b 0.28a

Control 19.1a 3.13a 0.41a 0.56a 22.9a 1.38a 0.16a 0.40a 20.9a 1.53a 0.25a 0.16a Rhizobia 20.4a 2.99a 0.53b 0.70b 23.1a 1.47a 0.25b 0.49a 25.4b 1.76ab 0.42b 0.24b 171 AFM 20.8a 3.05a 0.55b 0.74b 24.2a 1.37a 0.27b 0.64b 25.1ab 1.83b 0.41b 0.25b Rhizo+AFM 22.8a 3.04a 0.58b 0.79b 27.4a 1.54a 0.28b 0.65b 27.3b 1.83b 0.50b 0.25b

Control 16.0a 2.76a 0.35a 0.56a 25.0a 1.14ab 0.14a 0.36a 20.5a 1.50a 0.25a 0.13a Rhizobia 20.1ab 2.87a 0.43b 0.64a 27.3a 1.25ab 0.20ab 0.40a 22.4ab 1.65a 0.39b 0.21b 257 AFM 19.0a 2.79a 0.44b 0.80b 22.9a 1.06a 0.26b 0.59b 23.3ab 1.83b 0.40b 0.21b Rhizo+AFM 21.9b 2.59a 0.52c 0.75b 25.8a 1.37b 0.27b 0.60b 26.2b 1.76b 0.48b 0.20b

Diameter.= collar diameter; SDW = Shoot Dry Weight; RDW = Root Dry Weight; AFM = Arbuscular Mycoorhizal Fungi; Rhizo= Rhizobia. For each NaCl concentration, values in column sharing the same letter comparing microbial inoculations are not significantly different at P<0.05 (Student-Newman-Keuls test). Each value is the mean of three replicates.

Combined effect of microbial inoculation and decreased the number of nodules and MRC of The beneficial effect of dual inoculation can be peanut shells on nodulation and mycorrhizal plants compared to microbial inoculation alone attributed, in particular, to enhancement of P and root colonization of plants and the combination of peanut shells and N nutrients uptake (Smith and Read, 2008) which microbial inoculation. Although no significant are lacking in salty soils (Hu and Schmidhalter, Table 6 showed the number of nodules and the difference was noted, the number of nodules and 2005; Asma et al., 2009), and improved plant mycorrhizal root colonization (MRC) of S. senegal, MRC seemed to be higher in inoculated plants water status (Aggarwal et al., 2011). V. seyal and P. juliflora plants grown on non- compared to inoculated and amended ones. An improvement in P uptake through sterile soil in response to dual inoculation mycorrhizae under salt stress increases the + (rhizobia and AMF), amendment with peanut plant’s ability to reduce the negative effects of Na DISCUSSION - shells and their combination. Results showed that and Cl ions (Feng et al., 2002). Furthermore, all treatments increased nodulation and The results of the study show a positive significant mycorrhizal and rhizobial symbioses often act mycorrhization of plants compared to control effect of all microbial inoculation treatments on synergistically on rate, mineral nutrition plants. plant growth. However, the best growth was and plant growth (Patreze and Cordeiro, 2004; However, peanut shell application significantly obtained with dual inoculation (rhizobia + AMF). Rabie, 2005) which increases plant tolerance of Fall et al. 41

Table 5. Height (cm), collar diameter (mm), shoot dry weight (g plant-1) and root dry weight (g plant-1) of S. senegal, V. seyal and P. juliflora plants grown in non-sterile sandy soil amended with peanut shells and/or inoculated with rhizobia and mycorrhiza, and exposed for four months to four salinity levels under greenhouse conditions.

S. senegal V. seyal P. juliflora Inoculation Salinity (mM NaCl) Diamet Diamet Diamet treatment Height SDW RDW Height SDW RDW Height SDW RDW er er er Control 35.3a 4.30a 1.53a 1.72a 46.0a 2.89a 2.15a 3.31a 50.3a 3.29a 1.97a 0.82a Inoculation 36.0a 4.27a 1.57a 1.95a 49.3a 3.40a 2.74a 4.10ab 58.0a 4.49b 3.60b 1.42ab 0 Peanut shells 58.3b 6.23b 4.04b 2.45a 67.3b 4.06ab 3.96b 5.09ab 91.7b 5.68c 9.89c 1.81ab Ino+Pea shells 57.0b 5.96b 3.93b 2.30a 72.3b 5.03b 5.07c 5.78b 98.3b 5.68c 10.1c 2.28b

Control 22.3a 4.65a 0.85a 1.15a 33.0a 3.19a 0.92a 2.25a 49.0a 3.12a 0.99a 0.68a Inoculation 26.3a 4.46a 0.98a 1.42a 32.5a 3.04a 1.88b 2.69a 56.2a 4.05a 2.97b 0.77a 86 Peanut shells 33.0b 6.26b 2.44b 1.65a 53.3b 3.88b 2.19c 3.62b 97.7b 5.74b 9.73c 1.76b Ino+Pea shells 39.0b 6.39b 2.45b 1.86a 49.0b 4.00b 2.32c 4.87c 96.3b 5.72b 10.32c 1.78b

Control 14.3a 3.73a 0.41a 0.69a 19.0a 3.00a 0.32a 0.94a 30.0a 2.07a 0.50a 0.52a Inoculation 18.3a 4.71b 0.74b 1.00ab 26.7a 3.30a 0.57b 1.48a 46.4b 3.40b 2.64b 0.69b 171 Peanut shells 25.0b 6.20c 1.58c 1.47c 46.3b 3.69a 1.80b 3.17b 95.7c 5.01c 7.32c 1.28b Ino+Pea shells 27.0b 6.79c 1.63c 1.21bc 47.3b 4.11b 1.83b 3.88b 96.3c 5.35c 8.33c 1.30b

Control 12.3a 3.41a 0.30a 0.35a 16.3a 2.30a 0.23a 0.69a 31.7a 2.36a 0.40a 0.45a Inoculation 20.0b 4.12b 0.70b 0.94c 21.7a 2.35a 0.52b 0.62a 49.0a 3.99b 2.07b 0.67a 257 Peanut shells 21.3b 4.58b 0.86bc 0.69b 29.0b 2.99ab 1.17c 1.85b 83.0b 4.63b 5.52c 1.15b Ino+Pea shells 25.0b 7.72b 1.10c 0.74bc 31.3b 3.27b 1.19c 1.94b 85.3b 4.71b 5.87c 0.88b

Diameter.= collar diameter; SDW = Shoot Dry Weight; RDW = Root Dry Weight; Ino = dual inoculation (AFM + rhizobia); Ino+Pea shells= combination of microbial inoculation with peanut shells (6 tha- 1)= For each NaCl concentration, values in column sharing the same letter comparing treatments are not significantly different at P<0.05 (Student-Newman-Keuls test). Each value is the mean of three replicates.

salinity stress (Rabie and Almadini, 2005). Similar conditions. However, the best performance was total carbon, nitrogen, phosphorus, cation results have been obtained on Vigna radiata by obtained with the combination of peanut shells exchange capacity in addition to reducing soil Singh et al. (2011), Acacia mangium and Acacia and microbial inoculation. The positive effect of salinity (Oo et al., 2013; Wang et al., 2014). The auriculoformis (Diouf et al., 2005), Acacia spp. this combination could be due to both results also showed that dual inoculation (rhizobia (Thrall et al., 2008). improvement of soil properties through peanut + AMF), peanut shells and their combination The study further shows that microbial shells amendment (Fall, 2016), mineral nutrition significantly improved the nodulation and inoculation, peanut shells or their combination and water status of plants through microbial mycorrhization of S. senegal, V. seyal and P. significantly improved the growth of S. senegal, V. symbioses (Parniske, 2008; Ollivier et al., 2011). juliflora plants grown under saline conditions. The seyal and P. juliflora plants under salinity Indeed, the application of peanut shells increased low negative effect of peanut shells on seedlings 42 J. Agric. Biotech. Sustain. Dev.

Table 6. Nodules number and mycorrhizal root colonization (%) of S. senegal, V. seyal and P. juliflora plants grown in non-sterile sandy soil amended with peanut shells and/or inoculated with rhizobia and mycorrhiza, and exposed for four months to four salinity levels under greenhouse conditions

S. senegal V. seyal P. juliflora Salinity (mM NaCl) Peanut shells (tha-1) NN MRC NN MRC NN MRC Control 5a 10a 4a 7a 6a 12a Inoculation 30b 35c 17b 38c 26b 70b 0 Peanut shells 11a 24b 9a 17b 10a 20a Ino+ Pea shells 27b 30bc 15b 37c 24b 61b

Control 0a 3a 0a 2a 5a 5a Inoculation 8c 27c 10c 28c 16b 57c 86 Peanut shells 3b 16b 4b 14b 9a 18b Ino+ Pea shells 6bc 25c 7bc 28c 13b 46c

Control 0a 3a 0a 0a 0a 4a Inoculation 4b 19b 7b 23c 10c 37b 171 Peanut shells 1a 9a 2a 7b 5b 10a Ino+ Pea shells 3b 17b 4b 20c 9bc 32b

Control 0a 0a 0a 0a 0a 0a Inoculation 1b 14c 2b 16c 4b 26c 257 Peanut shells 1b 4b 1b 5b 2b 7b Ino+ Pea shells 1b 13c 2b 14c 3b 22c

NN= Number of nodules; MRC= mycorrhizal root colonization; Ino = dual inoculation (AFM + rhizobia); Ino+pea shells = combination of microbial inoculation with peanut shells. For each species, values within a column sharing same letter comparing treatments are not significantly different at P<0.05 (Student-Newman-Keuls test). Each value is the mean of three repetitions.

nodulation and mycorrhization compared to the absence (0 mM) and presence of low (86 mM) strains improved the growth of S. senegal, V. inoculation alone could be explained by the slight NaCl concentrations could be related to the seyal and P. juliflora plants under salinity inhibition of microbial symbiosis caused by presence of native fungi and rhizobia strains in conditions. However, the best growth was addition of peanut shells. soil. obtained with dual inoculation (rhizobia + AMF). Thus, it is known that relatively high soil nutrient The combination of dual microbial inoculation and content such as N and P reduce nodulation peanut shells significantly improved height, collar (Hellsten and Huss-Danell, 2001; Gentili and Conclusion diameter, shoot and root biomass, nodulation and Huss-Danell, 2002, 2003) and mycorrhization in mycorrhization of plants. Indeed, it is well plants (Nouri et al., 2014). Nodulation and The results of this study has shown that microbial established that the behavior of plants under mycorrhization observed in control plants grown in inoculation with rhizobial and mycorrhizal selected greenhouse conditions is crucial for survival after Fall et al. 43

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