Influence of mycorrhizal inoculation and source of phosphorus on growth and nutrient uptake of pepper (Capsicum annuum L.) in calcareous soil.

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Volume 28, Article 5

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E. AISSA 

A. MOUGOU

K. KOUKI KHALFALLAH

National Agronomic Institute of Tunisia

Abstract - A greenhouse experiment was carried out during the spring–summer 2014 to examine that arbuscular-mycorrhizal (AM) can enhance growth of pepper plant (Capsicum annuum L.) supplied with Rock Phosphate (RP). Arbuscular mycorrhizal (+AM) and non AM pepper plants were grown in a calcareous soil with three fertilization regimes (without any phosphate amendment, supplied with Triple super phosphate (TSP) and supplied with RP). Mycorrhizal inoculation increase significantly plant biomass, height, leaves area and stems diameter for all fertilization regimes. The uptake of phosphorus was influenced by AM inoculation, hence inoculated plant supplied by TSP and RP had statically similarly and higher concentration of P in leaf tissue than unfertilized plants and non inoculated plants amended with P fertilizers. A slight increase of available P was registered in soil around roots of inoculated plant amended with RP.

Keywords: Gafsa Rock Phosphate, Arbsucular mycorrhizal fungi, Capsicum annuum L.

1. Introduction

Phosphorus (P) is an essential macronutrient for plant growth. In soil, Phas a low biodisponibility, consequently it’s considered as a limit factor for crop development. The low availability of P in calcareous soil, characterized by high amount of CaCO₃, is due to the precipitation reaction of P with calcium (Leytemand and Mikkelson 2005). To increase the uptake and the availability of P in such condition of P deficiency, many strategies were involved by plants (Marschner 1995). Arbuscular mycorrhizal (AM) symbiosis between soil mycorrhizal fungi and roots of 80% of terrestrial plant species is one of the most common of these strategies. From more 450 million years (Smith and Read 2008) this obligate symbiosis provides low mobile nutrient to the plant (particularly phosphorus P) and takes organic carbon from host plant (Smith and Smith 2012).

Arbsucular mycorrhizal fungi form an extensive extraradical mycelium that uptake nutrients from soil and a symbiotic interfaces within cortical cells of root involved in transfer nutrient. The effectiveness of AM in the transfer and deliverance of P to plant leads to a better P nutrition and higher growth of mycorrhizal plants (smith and smith 2012). arbsucular mycorrhizae can also access effectively to the poorly available P sources. In fact, many studies showed that AM increase the growth of plants amended with rock phosphate (Guissou 2001; Duponnois 2005; Najjar et al 2012).

In the present work, we studied the impact of AM fungi inoculation by Rhizophagus irregularis (R. irregularis) on the growth of pepper (Capsicum annuum L) amended with Gafsa rock phosphate (RP) and triple super phosphate (TSP).

2. Materials and methods

   1. Plant material, growth conditions and experimental design

Treatments were defined by a factorial combination of three type of fertilization (Triple Super Phosphate (TSP), Gafsa Rock Phosphate (RP) and control (no phosphate fertilizer) and two mycorrhizal treatments: with mycorrhizae (+AM) or without mycorrhizae. Treatments were arranged in a randomized complete-block design with 12 replications for each treatment.

The experiment was conducted under greenhouse conditions at National Agronomic institute of Tunisia. Pepper seeds (Capsicum annuum L. cv Anahaim) were surface sterilized with 1:3 (v/v) of 2.5% sodium hypochlorite solution for 10 minutes, rinsed four times with distilled water and sowed in a peat substrate on April 2^th 2014. Seedlings were transplanted at the four-true-leaf stage into pots (diameter 10 cm, height 12 cm) containing 4 kg of autoclaved (20 min, 120°C) mixture of soil and sand (2:1). Soil was collected from the experimental site at National Agronomic institute of Tunis. The physicochemical characteristics of the mixture of soil were as follows: sand 54%, silt 32%, clay 11%, pH 7.9, 0.6% organic matter, 20% total CaCO₃, 7% active CaCO₃, 5 ppm available P (olsen), 450 ppm exchangeable Ca.

Inoculated plants received a commercial mycorrhizal inoculum carrying R. irregularis (50 spores g⁻¹) by mixing 2L germination substrate with 84 g of inoculum and at the transplantion inoculum was added in the hole transplanting at a rate of 1,5 g per plant. P₂O₅ content of phosphate fertilizers were 28.6% for PR and 45% for TSP Phosphorus was applied before transplanting by mixing 300 mg Kg^-1 of P₂O₅ to the mixture soil as Gafsa Rock Phosphate for RP treatments and TSP for TSP treatments. Plants were irrigated with a nutrient solution containing: 14.0 mM N–NO3, 1.6 mM S, 6.0 mM K, 4.5 mM Ca, 1.5 mM Mg, 20 μM Fe, 9 μM Mn, 0.3 μM Cu, 1.6 μM Zn, 20 μM B, and 0.3 μM Mo.

2. Data collection and analysis

To determine chlorophyll index, a chlorophyll meter (Minolta SPAD-502) was used to take SPAD values (SPAD units). Measures were taken from the fully expanded functional leaves (the fourth from the apex) four times in the growing cycle (25, 38, 56 and 70 days after transplanting).

At the end of experiment (71 days after transplanting), plants were separated into stems, leaves, and roots. Leaves area was determined by LI-3000 A planimeter and stems diameter by Art. C041/150 electronic digital caliper. Shoot and roots were separately washed with distilled water to remove any adhering debris. Subsamples of roots were saved for assessment of AM-fungi root colonization. All plant tissues were dried in a forced-air oven at 75° C for 72 h for biomass determination. Dried leaves were ground and 0.5 g of the dried tissues was analyzed for the following macronutrients: N, P, K, Ca, Mg. N concentration of leaf. N was determined after mineralization with sulfuric acid using Kjeldahl method (Bremner, 1965). P, K, Ca, Mg, concentrations were determined after dry-ashing at 400°C for 24 h and dissolving the ash in HNO₃ 1:20. P were determined by the molybdenum blue method (Murphy and Riley, 1962) whereas K, Ca were determined by flame photometry and Mg by atomic adsorption spectrometry.

3. Root Colonization

A fraction of the roots samples were cleared with 10% KOH at 90°C, acidified in 2% HCl for 5 minutes and stained with 0.05% trypan blue in lactophenol as described by Phillips and Hayman (1970), and examined microscopically for AMF colonization by determining the percentage of root segments containing arbuscules + vesicles using a gridline intercept method (Giovannetti and Mosse, 1980).

4. Soil extraction and analysis

At the end of the experiment, soil samples from pots of each treatment were analyzed for changes in soil-available P and pH. A composite sample from each pot was collected and air-dried for 3 days. The soil-available P was determined using sodium bicarbonate extractable phosphorus colorimetric method (Olsen et al, 1954). The soil pH was determined using a glass electrode in a 1:2.5 (v/v) soil–water.

5. Statistical analysis

All data were statistically analyzed by ANOVA using the SPSS software package (SPSS 10 for Windows; SPSS, 2001). Duncan’s multiple range test was performed at p = 0.05 on each of the significant variables measured

3. Results

   1. Roots colonzation

Type of fertilization had a significant effect on the rate of roots mycorhization (figure 1). Plants that received RP had higher mycorrhization (81,2%) than plant fertilized with TSP (22%). Plants without phosphate fertilizer showed a rate of mycorhization significantly similar to that observed in roots of RP treatment (78,5%).

2. Growth parameter

71 days after transplantation, growth parameter (Table 1) were highly influenced by type of P fertilization and AM inoculation. Plant of P0 and RP treatment had a poor and statically similar growth parameter. In comparison to non inoculated plants, AM inoculation increased plant’s high, leaves area, leaves number and diameter of stem respectively by 40, 77, 85 and 24% for unfertilized plants and by 61, 86, 92 and 41% for plants supplied with RP. Inoculated plants of TSP treatment had statically similar high and leaves number to non mycorrhizal plants but had higher leaves area and stem diameter respectively by 13% and 30%. In comparison with TSP treatment, Plants of RP +AM had higher plant and produced more leaves but produced less flowers.

3. Biomass accumulation

Shoot dry weight was highly affected by phosphorous fertilization and mycorrhizal inoculation and their interaction (Table 2). TSP+AM treatment showed the highest Shoot dry weight (4.01 g plants^-1). Inoculation had increased shoot dry weight of P0 +AM, TSP+AM and RP+AM treatment respectively by 397, 20 and 772%. Plant of RP+AM treatment had the same shoot DW as non inoculated plant of TSP treatment. There was no significant difference between shoot DW of non inoculated plants and RP treatment. Mycorrhizal inoculation enhanced root biomass by 68, 86 and 39% respectively for P0 plants, plants fertilized with RP and plants fertilized with TSP.

4. Chlorophyll Index

For the four measuring date, SPAD value was influenced by the different treatments (Table 3). From the first date, SPAD values were low and statically similar for control and PN treatment and recorded an important fall at the 55 days after transplantation (DAT). This fall continues at 70 DAT and final value measured were respectively 26.6 and 27.5. For P0+M treatment, SPAD values were statically similar to value of both P0 and RP treatment for the tow first date of measurement. But from 56 day after transplantation, SPAD value were maintained and varied from 47.5 at 38 day after transplantation to 49.1 at the last date of measurement. From 38 day after transplantation, chlorophyll index of TSP+AM was the highest (50.2; 55.6 and 60.3 respectively at 38, 55 and 70 day after transplantation) whereas value of SPAD index of PN+AM and TSP treatments were statically similar at this three dates of measurement.

5. Mineral composition

Mycorrhizal inoculation improved significantly the P concentration in leaves for both TSP and RP treatments (Table 4). P concentration was statically similar for TSP+M and RP +M treatment. Potassium (K) statute was significantly increased for of P0+M and RP+M treatment but there is no effect of AM inoculation on the uptake of K by plants supplied by TSP. we observed high concentration of Ca in P0 and RP treatment and low concentration in leaves of plants amended with TSP. For N and Mg concentration, there is no statistical difference between treatments.

6. Phosphorous and pH values

The highest concentration of available phosphorous (P_av) was measured in soil of treatment received TSP as P source (table 5). Application of RP did not show a significant increase in comparison with P0. Also P0+ AM did not show a significant increase of Pdl whereas we observed a significant increase of P_av measured in soil of PN + AM treatment. Beside, pH value was lower for control +AM and RP +AM treatments. No significant difference was detected for control, RP and TSP treatment in except of TSP +AM treatment which had a significant slight decrease for the pH value.

3. Discussion

For control, Triple Super Phosphate and Rock Phosphate amendment, pepper plant showed a positive response to mycorrhizal inoculation. Dry weight, plant height, stem diameter and leaves area were higher in inoculated plants for each P treatment. Plants amended with TSP showed the highest growth because of immediately release of Pi in soil solution from TSP. Our results showed that mycorrhizal plant had better growth than non mycorrhizal plant of TSP treatment. Jujube plant inoculated by Rhizophagus irregularis and fertilized with different levels of TSP had a higher DM than non inoculated plant (Guissou 2001). Tanwar et al. (2013) observed that mycorrhizal inoculation promote growth of pepper in different level of TSP supply. The increase of growth by mycorrhizal inoculation was more pronounced in pepper plant fertilized with rock phosphate. In RP treatment, non inoculated plant had no growth response due to the low solubility of RP in calcareous soil. The very low effectiveness of RP in non acidic soil is very well documented (Chien & Menon 1995). Pepper inoculated by AM fungi showed a positive response into RP application for biomass accumulation. This is an accordance with other results on jujube (Guissou 2001), acacia ((Duponnois et al. 2005), bean and maize (Najjar et al. 2012)

Our results showed that SPAD index was maintained for TSP, TSP+M, RP+M and Control +AM and dropped for non inoculated plant of RP treatment and control. SPAD index is commonly used to estimate leaf chlorophyll content of pepper plant (Zhu et al 2012; Díaz-Pérez, 2013 ). The SPAD–502 meter readings is very well correlated to the chlorophyll content of pepper leaves with high coefficient of correlation (Estrada-Luna & Davies 2003; Medeir 2007 ). It was reported that AM fungi increases total chlorophyll content in pepper plants (Olsen et al. 1999; Estrada and Davies 2003; Demir 2004). In the study of Çekiç et al. (2012), chlorophyll content of pepper plants inoculated with Rhizophagus irregularis was higher than that of non inoculated plants. Tanwal and Aggarwal (2013) showed that the increase of chlorophyll content in leaves of pepper plants inoculated with AM was associated to the increase of photosynthetic rate.

In this work, inoculated plant amended with TSP and RP had the highest P status. Mycorrhizal plants of TSP treatment had better P status than non mycorrhizal plants even there is no significant difference between soil levels of Pi of these tow treatments at the end of the experiment. In fact the amount of Pi around roots decrease rapidly thus P concentration around hyphae of mycorhhizae is higher than that near roots. This efficiency in P uptake of mycorrhizal plants amended with TSP was observed with only 20% of mycorrhization. This low percentage is contributed to the decrease of AM colonization at High concentration of Pi in soil solution (Nagy et al. 2009; Balzergue et al. 2011). For control and RP treatment, AM colonization was high due to the low initial amount of available Pi in the soil and the low availability of Pi from RP that prevent the inhibition of mycorrhizal association. For mycorrhizal inoculated treatement, plants amended with RP had high leaves P content. Moreover, the final amount of Pi in soil was slightly higher in RP treatment than in control, control+ AM, and RP treatment. We suggested that AM fungi can solubilize locally RP than absorb and transfer quickly the Pi solubilized to the plant host. The contribution of RP in P nutrition of mycorrhizal plants was demonstrated by Toro et al. (1997). They used radioactive ³²P and showed that plants inoculated with Rhizophagus irregularis had lower specific activity (³²P/³¹P) than non inoculated plants, suggesting that mycorrhizal plant used RP as source of P not available for non mycorrhizal plant. Moreover, our measurement showed that the slight increase of P_av in mycorrhizal treatment of the control and RP amendment was associated with a low drop of the pH. This decrease of pH could be due to the release of organic acids and protons (Najjar et al. 2012) but mechanisms by which AM symbiosis can mobilize P from RP is still not very well understood (Bolan 1991; Antunes et al. 2007; Smith and Smith 2012).

The uptake of K⁺ was improved with AM inoculation. Smith et al. (1981) observed that the decrease of P availability in soil leaded to an increase of K⁺ content in mycorrhizal Trifolium. Satter et al. (2006) showed that AM fungi improve P and K uptake for acacia seedling amended with Gafsa RP. Olsson et al.( 2008) reported a strong correlation between K⁺ and P during mycorrhizal symbiosis with Rhizophagus irregularis. According to Garcia and Zimmermann (2014), K⁺ is involved in the homeostasis and transfer of inorganic phosphate in the mycorrhizal association.

3. Conclusions

Mycorrhizal inoculation increased significantly plant biomass, height, leaf area and stem diameter for each phosphate treatment. The uptake of phosphorus was influenced by AM inoculation. Inoculated plant supplied by Triple Super Phosphate and Rock Phosphate had statically similarly and the highest growth. Moreover, our measurement showed that the slight increase of P_av for mycorrhizal treatment of the control and RP amendment was associated with a low drop of the pH. We can conclude that AM can access effectively to the RP, thus mycorrhizal plant used RP as source of P not available for non mycorrhizal plant.

ACKNOWLEDGMENTS

We would like to thank the Groupe Chimique Tunisien for providing Gafsa Rock Phosphate and Italpollina Company for providing propagules of Rhizophagus irregularis

3. References

Antunes, P. M., Schneider, K., Hillis, D., Klironomos, J. N. (2007) Can the arbuscular mycorrhizal fungus Glomus intraradices actively mobilize P from rock phosphates? Pedobiologia, 51(4), 281–286.

Arcand, M. M., Schneider, K. I. M. D. (2006) Plant- and microbial-based mechanisms to improve the agronomic effectiveness of phosphate rock : a review, 78, 791–807.

Balzergue C, Puech-Pages V, Becard G, Rochange SF, (2011) The regulation of arbuscular mycorrhizal symbiosis by phosphate in pea involves early and systemic signalling events. J Exp Bot 62: 1049–1060

Bolan, N. S. (1991) A critical review on the role of mycorrhizal fungi in the uptake of phosphorus by plants. Plant and Soil, 134(2), 189–207.

Çekiç F.O., Ünyayar S. , Ortaş I., (2012) Effects of arbuscular mycorrhizal inoculation on biochemical parameters in Capsicum annuum grown under long term salt stress. Turk J Bot 36: 63-72

Chien, S. H., Menon, R. G. (1995) Factors affecting the agronomic effectiveness of phosphate rock for direct application. Fertilizer Research, 41(3), 227–234.

Deniz A.(2004) Influence of Arbuscular Mycorrhiza on Some Physiological Growth Parameters of Pepper, 28, 85–90.

Díaz-Pérez, J. C. (2013) Bell pepper (Capsicum annum L.) crop as affected by Shade level: Microenvironment, plant growth, leaf gas exchange, and leaf mineral nutrient concentration. HortScience, 48(2), 175–182.

Duponnois, R., Colombet, A., Hien, V., & Thioulouse, J. (2005) The mycorrhizal fungus Glomus intraradices and rock phosphate amendment influence plant growth and microbial activity in the rhizosphere of Acacia holosericea. Soil Biology and Biochemistry, 37(8), 1460–1468.

Estrada-Luna, A., Davies, F. T. (2003) Arbuscular mycorrhizal fungi influence water relations, gas exchange, abscisic acid and growth of micropropagated chile ancho pepper (Capsicum annuum) plantlets during acclimatization and post-acclimatization. Journal of Plant Physiology, 160, 1073–1083.

Garcia K.,Zimmermann S.D., (2014) The role of mycorrhizal associations in plant potassium nutrition. Front. Plant Sci. 5:337.

Giovannetti M, Mosse B. 1980. An evaluation of techniques for measuring vesicular-arbuscular infection in roots. New Phytologist 84, 489–500.

Guissou, T., BA, A.M., Guinko, S., Plenchette, C., Duponnois, R., (2001) Mobilisation des phosphates naturels de Kodjari par des jujubiers (Ziziphus mauritiana Lam.) mycorhize´s dans un sol acidifie´ avec de la tourbe. Fruits 56, 261–269

Murphy, J., and Riley, J.P., (1962) A modified single-solution method for the determination of phosphorus in natural waters: Analytica Chimica Acta, v. 27, p. 31-36.

Najjar G., Godlinski F., Vassilev N., Eichler-Löbermann B. (2012) Dual inoculation with Pseudomonas fluorescens and arbuscular mycorrhizal fungi increases phosphorus uptake of maize and faba bean from rock phosphate Landbauforschung - vTI Agriculture and Forestry Research 62(3):77-82

Nagy R, Drissner D, Amrhein N, Jakobsen I, Bucher M. (2008) Mycorrhizal phosphate uptake pathway in tomato is phosphorus-repressible and transcriptionally regulated. New Phytologist 181, 950–959.

Olsson, P. A., Hammer, E. C., Wallander, H., and Pallon, J. (2008) Phosphorus availability influences elemental uptake in the mycorrhizal fungus Glomus intraradices, as revealed by particle-induced X-ray emission analysis. Appl. Environ. Microbiol. 74, 4144–4148

Olsen S., Cole C., Watanabe F., Dean L. (1954) Estimation of available phosphorus in soils by extraction with sodium bicarbonate. USDA Circular Nr 939, US Gov. Print. Office, Washington, D.C

Olsen J.K., Schaefer J.K., Hunter M.N. (1999) Effects of a network of mycorrhizae on capsicum (Capsicum annuum L.) grown in the field with five rates of applied phosphorus. Aust J Agric Res 50: 239-252,.

Smith, S. E., Smith, F. A., and Nicholas, D. J. D. (1981) Effects of endomcorrhizal infection on phosphate and cation uptake by Trifolium subterraneum. Plant Soil 63, 57–64

Smith S.E., Read D.J. (2008) Mycorrhizal Symbiosis, Ed 3. Academic Press, New York

Smith, S. E., & Smith, F. A. (2012) Fresh perspectives on the roles of arbuscular mycorrhizal fungi in plant nutrition and growth, 104, 1–13.

Toro, M., Azcon, R., Barea, J.M. (1997) The use of isotopic dilution techniques to evaluate the interactive effects of Rhizobium genotype, mycorrhizal fungi, phosphate-solubilizing rhizobacteria and rock phosphate on nitrogen and phosphorus acquisition by Medicago sativa. New Phytol. 138, 265–273.

Zhu, J., Peng, Q., Liang, Y., Wu, X.,Hao, W. (2012) Leaf Gas Exchange, Chlorophyll Fluorescence, and Fruit Yield in Hot Pepper (Capsicum anmuum L.) Grown Under Different Shade and Soil Moisture During the Fruit Growth Stage. Journal of Integrative Agriculture, 11(6), 927–937.