Essay: The effect of arbuscular mycorrhizal fungi and potassium silicate in alleviating the adverse impact of salt stress in Thevetia peruviana

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The current investigation was carried out to evaluate the effect of arbuscular mycorrhizal (AM) fungi and potassium silicate (K-Si) in alleviating adverse impact of salt stress in Thevetia peruviana. The plants were irrigated with water salinity (0, 4.7, 9.4 and 14.1 dSm-1 sodium chloride (NaCl) with and without application of AM-fungi or K-Si. Potassium silicate had a stimulating effect on plant height, branches and leaves number, whereas, AM fungi gave the thickest plants and heaviest fresh and dry weight of shoots and roots.
Salt stress caused significant decrease in chlorophyll and carotenoid contents, however, the application of AM fungi and K-Si restored the pigments content in salt-affected plants and that might interpret the overproduction of total carbohydrates which are supplied mainly through the process of photosynthesis. Proline, phenol and phenol/Indol ratio were increased with increasing concentration of NaCl, but lower accumulation has been reported in plants treated with any of the anti-salinity. Maximum soil enzymatic activities, in rhizospheric soil were recorded with unstressed plants and receiving inoculation of AM fungi followed by that recorded for plants treated with K-Si and minimum activity were recorded in soil under no inoculation and stressed plants. NaCl stress also showed decrease in different enzymes activities (nitrogenase, dehydrogenase and phosphatase), and number of AM spores, further increase was observed in plants treated with AM fungi.

The applied salinity levels caused gradual increases in Na+ and Cl percentages accompanied by gradual decreases in N, P, Ca and K percentages as well as ratios of K+ / Na+ and Ca+2/ Na+ relative to the control plants (unstressed). Concerning K-Si and AMF effects on mineral content, it was found increases in the percentages of P, K and Ca as well as ratios of K+ /Na+, Ca+2/Na+ ratio and N relative to unstressed plants. K+ /Na+ ratio in plants under saline conditions is considered as one of the important selection criteria for salt tolerance.
Results indicated that K-Si and AM fungi treatments improved the endogenous nutrients, growth parameters attributes under different levels salinity stress.

Keyword: Thevetia peruviana- salinity – potassium silicate – K-Si – arbuscular mycorrhizal fungi- salt stress ‘ AMF- K/Na


Thevetia peruviana is a plant of tropical, a fast-growing small tree and because of its beautiful flowers and slender leaves very decorative effect. Its flowers have different colors, yellow, white and orange. The tree blooms, the leaves are linear-lanceolate, glossy and green almost all year round. They are coated with a waxy layer to reduce water loss. Its considered as an ornamental tree. Thevetia peruviana plants can succeed in rather poor and dry soils. It can tolerate shorter dry periods and moderately saline soils. Its best grows and flowers in full sun or light shade. T. peruviana contains a number of phytoconstituents which reveals its uses for different therapeutic purposes. The individual parts of plant can be used for the treatment of different disorders in human being such as, liver toxicity, diabetes, fungal infection, inflammation, microbial infection, pyrexia and to relieve pain. Furthermore, so much work is required with the thevetia to investigate the mechanism of actions with other therapeutic activities (Singh Kishan et al. 2012 and Ramos-Silva et al. 2017).

Among the abiotic stresses, salinity is one of the main factor that contributes to desertification of arid lands. Soil salinization has become a great challenge for rehabilitation of range lands and plant productivity (Alqarawi et al. 2014). Salinity causes nutritional disorders in plants which lead to deficiencies of several nutrients and drastically increasing Na+ levels in the plant cells (Iqbal and Ashraf, 2013).Thus, salinity stress affects the plant ability to uptake water in the root zone through decreasing the water potential of the soil (Sabir et al., 2009). This deficiency in available water under saline condition raises the potential of cells to be dehydrated which is a result of the osmotic stress caused by salinity. The higher ratios of toxic ions like Na+ and Cl damage the balance between ions through reducing the plant ability to absorb other ions like K+, Ca2+, and Mn2+ (Hasegawa et al., 2000). In this regard, the application of AM fungi as a biological method could improve plant growth and tolerance through helping the plants to mitigate the negative the adverse effects of salinity ( Abdel-Fattah and Asrar, 2012; Asrar et al., 2014).
Plants can overcome salinity by interacting with beneficial soil microorganisms such as arbuscular mycorrhiza fungi (AMF). Mycorrhiza symbiosis is a close association with the roots of most plants, from which both partners benefit: carbon compounds are supplied by the plant to the fungus, which provides the plant with mineral nutrients, mainly phosphate. AM symbiosis is able to increase plant growth under different environmental stresses (Garg and Chandel, 2015). The result is improved growth of the plant, and completion by the fungus of its life cycle. AM-fungi is known to exist in saline soil, and participates in the plant growth and development, and also improves the plant tolerance against biotic and abiotic stress (Abdel-Fattah et al., 2010) by regulating the physiological and biochemical process of plants (Fernanda et al., 2012).

AM fungi acts as growth regulator and mitigate the harmful effects of plants exposed to salt stress. Its play a key role in alleviating the toxicity induced by salt stress thus normalizing the uptake mechanism in plants by supplying the essential nutrients. The plant recovers the water balance machinery, enhancing their tolerance capacity, and thereby enduring the salt stress (Bhosale and Shinde 2011). Heikham et al. (2009) suggested many physiological parameters which could be responsible for alleviation of the harmful effects of salinity on plants upon inoculation with AM :
1) maintenance of a high K/Na ratio, 2) improved acquisition of nutrients such as N, P, Mg and Ca; 3) extended accumulation of proline, GB, polyamines, and carbohy-drates, 4) enhanced activation of antioxidant enzymes, 5) increased chlorophyll content and higher rates of photosynthesis, 6) improved integrity and stability of cell membranes, 7) higher hydraulic permeability and improved water status, 8) increased number of nodules and nitrogen fixation by legumes, 9) molecular changes, such as enhanced expression of the plasma membrane intrinsic protein (PIP) gene, expression of two Na+/H+ antiporters, and expression of genes encoding.
Silicon (Si) is a beneficial element as it improves growth, confers rigidity, strength and enhances plant tolerance to various abiotic stresses (Meena et al. 2014;Abbas et al. 2015). It is always combined with other elements, usually forming silicates and oxides (Gunes et al. 2007). Plant roots generally take up Si in the form of silicic acid which then translocate to the shoot, where it is polymerized to form silica gel (SiO2nH2O) (Zhu and Gong 2014). Exogenous application of Si has been reported to induce favorable effects on plant growth under abiotic stresses (Balakhnina et al. 2015). Many studies have indicated the role of Si in alleviating salt-stress have been attributed to reduced uptake and translocation of Na+ to shoots (Al-Aghabary et al. 2004), maintenance of plant’water relations (Gong et al. 2006), which in turn, contributes to salt dilution (Romero-Aranda et al. 2006). Thus the objective of this study was to investigate of K-silicate or AM-fungi mediated salinity tolerance mechanism for developing Thevetia peruviana resistance to salt stress.
Experimental design:
To achieve the aforementioned target, a pot experiment was conducted at the Nursery of the Department of Ornamental and landscape Gardening Research during two successive summer seasons (2015 and 2016). Seedlings of Thevetia peruviana (60 cm height of plant) were obtained from a commercial nursery, where healthy seedlings of uniform size were selected and transplanted into pots (30 cm diameter) which contain 7 kg of soil (60.7 % clay, 25.8% silt and 13.5% sand). After 3 weeks of transplanting seedling, salt was applied as a water solution of sodium chloride concentrations (0, 4.7, 9.4 and 14.1 dSm-1 NaCl respectively, where 0 dSm-1 means no added NaCl) with and without AM or K-Si inoculations. All pots were irrigated to reach field capacity with saline solutions to maintain the level of salinity after transplanting. Plants were received NPK (Krystalon 19-19-19) at the rate of 2g pot-1 with other treatments after two weeks from transplanting then one monthly interval during the growth seasons. Plants were supplemented twice a week with 500 mL of a salinity solution per pot. Each experiment included 4 salinity levels and two antisalinity application {arbuscular mycorrhiza (AM) fungi, and potassium silicat (K-Si)} plus control without treatment application (12 treatments) replicated 3 times in a completely randomized design.

Soil characteristics:

Some physical and chemical properties of the experimental soil and water used were determined according to the standard methods undertaken by Black et al., (1965); Chapman and Pratt (1961), Page et al., (1982), and the obtained data are presented in Table (1).
Table (1): Some physical and chemical characteristics of the studied soil
Soil characteristics Value Soil characteristics Value
Particle size distribution%: Soluble cations (soil paste mmolecL-1):
Sand 13.5 Ca2+ 2.73
Silt 25.8 Mg2+ 1.15
Clay 60.7 Na+ 1.85
Textural class Clay K+ 0.60
Soil chemical properties: Soluble anions (soil paste mmolecL-1):
pH (soil paste extract) 8.15 CO32- 0.00
CaCO3 % 3.15 HCO3- 1.50
Organic matter % 1.12 Cl- 2.05
ECe (dS/m, soil paste extract) 0.69 SO42- 3.00
Soil physical properties:
Bulk density g cm-3 1.37 Soil moisture at wilting point % 8.69
Soil moisture at field capacity % 21.00 Avail. Water % 12.31
Available Nutrients mg kg-1
N P K Cu Fe Mn Zn
27.40 4.12 374.50 0.65 3.84 0.95 0.78

Ant-salinity applications

Arbuscular Mycorrhizal Fungi (AMF) mycorrhizal pot treatments received the AMF by placing 200 g of mycorrhizal inoculum as soil application at the transplanting time. The AM inoculum consisted of soil AM colonized roots, rhizosphere soil having extramatrical mycelium and spores (approximately, 250-300 spores 100g-1 of soil) were used for each mycorrhizal pot treatments. The inoculum was placed five cm below each plant.
Potassium silicate(K-Si)The plants were treated with potassium silicate (11:25% K2O:SiO2) at rate of 5 ml pot-1as soil application at the transplanting time.
Growth measurement and biochemical analysis
In both growing seasons, samples were selected randomly and the following growth characters were recorded:
Plant height (cm); branches number plant-1; leaves number plant-1; shoots fresh and dry weight (g); Stem diameter (mm) and leaf area (cm2)
Roots length (cm) and roots fresh dry weight (g).
Quantification of the Number AM Spores was done using the Adholeya and Gaur ‘Grid Line Intersect Method’ (1994).
Determination of Photosynthetic Pigments were determined in representative fresh leaves samples. Chlorophyll and carotenoids were extracted and determined according to Lichtenhaler and Wellburn (1983) method.
Bio-chemical Composition
Proline: was determined according to the method of Petters et al. (1997).
Phenols and indoles in leaves were determined according to A.O.A.C. (2000) and Larsen et al. (2006), respectively.
Total carbohydrates: was determined according to Herbert et al. (2005)
The enzymes activity: nitrogenase (”mole C2H4.g dry nodule-1day-1), dehydrogenase (”g TPF.g dry and phosphatases (”g.g dry soil-1) were determined according to the methods described by Somasegaran and Hoben, (1994), Skujins (1976) and Tabatabai and Bremner (1969), respectively.
Crude protein percent calculate as, nitrogen was multiplied by 6.25 (A.O.A.C., 2000).
Mineral Content
Nitrogen was determined in plants with sulfuric acid using the semi-micro Kjeldahl method Walinga et al.(1995). Phosphorous was determined by colorimetric method (A.O.A.C., 2000). Potassium and calcium in shoots were determined using flame photometer apparatus according to Walinga et al (1995).
The experiment was laid as completely randomized in a factorial design, the obtained data were statistically analyzed using LSD test at 5% (Mead et al, 1993).
Vegetative Growth
Vegetative growth of Thevetia peruviana was more superior under (0 dSm-1 NaCl) as compared with plants grown under salt stress conditions (Tables 2,3 and 4). On the contrary, with the increase of salinity stress levels the vegetative growth characters of Thevetia peruviana expressed as plant height, stem diameter, number of branches (Table 2), number and area of leaves and plant fresh and dry weights (Tables 3 and 4) were significantly decreased mainly at the highest salinity level (14.1 dSm-1) in both studied seasons. Anti-salinity treatments (Mycorrhiza and potassium silicate) improved all mentioned plant vegetative growth characters affected by salt stress compared with untreated plants (control). It was shown that AM-fungi
Table (2): Effect of applied treatments on plant height, stem diameter and branches number of T.peruviana irrigated with different levels of water salinity.
Salinity levels
(dSm-1) A
First season Second season
Treatments (B)
control K-Si AMF Mean control K-Si AMF Mean
Plant height (cm)
0.0 98.75 110.30 107.25 105.40 109.90 117.80 116.10 114.60
4.7 82.00 93.17 82.92 86.03 85.58 104.10 100.60 96.75
9.4 78.33 83.00 81.58 80.97 80.25 84.75 83.33 82.78
14.1 73.75 80.92 80.08 78.25 79.08 82.08 81.75 80.97
Mean 83.21 91.83 87.96 88.71 97.18 95.43
LSD at 5% A: 0.42 B: 0.36 AB: 0.72 A: 0.50 B: 0.43 AB: 0.86
Stem diameter (mm)
0.0 14.94 17.09 18.57 16.87 15.64 16.29 19.74 17.22
4.7 13.58 14.57 15.10 14.42 14.77 16.71 17.37 16.28
9.4 12.29 13.94 14.27 13.50 12.70 15.58 16.96 15.08
14.1 11.08 12.34 13.04 12.15 11.55 15.74 15.11 14.13
Mean 12.97 14.48 15.24 13.66 16.08 17.30
LSD at 5% A: 0.031 B: 0.027 AB: 0.054 A: 0.99 B: 0.86 AB: 1.72
Branches number plant-1
0.0 10.17 11.67 11.00 10.95 11.33 12.33 11.93 11.86
4.7 9.50 11.50 10.00 10.33 10.67 12.00 11.33 11.33
9.4 8.73 10.83 9.33 9.63 9.43 11.67 10.64 10.58
14.1 8.50 9.17 8.93 8.94 8.94 10.50 9.83 9.76
Mean 9.23 10.79 9.87 10.09 11.63 10.93
LSD at 5% A: 0.21 B: 0.18 AB: 0.36 A: 0.15 B: 0.13 AB: 0.27
K-Si: Potassium silicate AMF: Arbuscular mycrrohizae fungi

application significantly mitigated the effects of low salt stress (4.7 dSm-1) and moderately counteracted the destructive effects of high salt stress level (9.4 and 14.1 dSm-1) of most vegetative growth parameters followed by K-Si treatment. The same trend was found in the two studied seasons. Such results are in harmony with other previous investigations who demonstrated that, decreases in vegetative growth as a result of increasing concentration of salt irrigated water, El-Zait (2011) on Conocarpus erectus plant, Kafi and Rahimi (2011) on Portulaca olercea plant and El-Sayed (2013) on Moringa oleifera plant. As demonstrated in result tables application of K-Si had a stimulating effect on the plant growth, improved plant height, leaf area, branches and leaves number, whereas (AM) gave the thickest plants and heaviest fresh and dry weight of shoots and roots, Manivannan et al (2015) on Zinnia elegans. Watfa (2009) confirmed that AM fungi gave a positive overall impact on plant biomass under salt stress conditions, with both shoot and root dry weights being significantly greater in mycorrhizal than in nonmycorrhizal on Ceratonia siliqua L.and Garg and Bhandari (2016) on Cicer arietinum plant, reported that the silicon and arbuscular mycorrhiza increase the growth parameters under salinity stress.

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