Morpho-physiological Characteristics of Basil (Ocimum basilicum L.) under NaCl-stress and Rhizophagus fasciculatum as NaCl-stress Mitigator

Salinity stress is one of the main problems limiting growth and development of cultivated species. The objective of this study was to assess NaCl-stress basil plants (Ocimum basilicum L.) cv. ‘Nufar’ and to determine whether the mitigating effect of an arbuscular mycorrhizal fungus strain (AMF). A completely randomized factorial design was used considering three NaCl concentration (0, 50 and 100 mM) as factor 1 and presence or absence of AMF (0 and 10 g of inoculum) as factor 2, with four replicates per treatment and four plants per repetition. The assessed response variables were, fresh and dry of aerial part and root, root length, leaf area, relative water content, water potential, plant height, number of spores and mycorrhizal colonization percentage after 20 and 50 days (T20, T50) of the experiment. The results showed greater values in all variables in the control group (0 mM NaCl) than in plants inoculated with R. fasciculatum with T20 and T50; although values decreased as NaCl concentration increased; the tendency to increase was maintained even in at 50 and 100 mM of NaCl with AMF with respect to 50 and 100 mM NaCl without AMF. The AMF colonization percentage decreased as NaCl concentration increased. Nonetheless, the development and growth response for all variables in the inoculated plants with AMF was greater vs noninoculated, which suggests that basil plant inoculation with AMF has a positive effect in mitigating NaCl stress.


Introduction
Salinity is one of the main abiotic factors that worldwide negatively affect agriculture production (Iqbal et al., 2015).The increase of salt concentration in soil is an abiotic factor that stresses plants and decreases their capacity to absorb water, limiting important metabolic processes, affecting osmotic equilibrium, nutrient absorption, hydraulic and stomatal conductivities, raw photosynthesis rate and intracellular CO2 concentration, which directly damages plant development (Batista-Sánchez et al., 2017).Salinity stress affects water absorption in plants in the radicle area because of a decrease of osmotic potential and consequently plant soil hydration uptake (Batista-Sánchez et al., 2015).It is estimated that more than 800 million hectares have been affected by salinity around the world, which represents a loss of more than 12 billion USD annually for the agricultural industry (Khalig et al., 2014).In México, the salinization of agricultural soil affects 3.2% of its territory (SEMARNAT, 2009), with salinity problems being more frequent in dry lands where water used for irrigation is rich in salts, thus consequently causing further and progressive soil deterioration (Zamudio-González et al., 2004).
Arbuscular mycorrhizal fungi (AMF) are plant root symbionts, considered to be the cornerstone of mutualism in terrestrial ecosystems (Wimmer et al., 2016).Studies have demonstrated that AMF are capable of promoting plant growth while providing increased tolerance to soil salinity in many plants species by a use of diverse mechanisms, such as favoring nutrient acquisition, plant growth hormone production, rhizosphere development as well as improving

Experimental design
A completely randomized design with factorial arrangement was used, considering three NaCl concentrations (0, 50 and 100 mM) as factor one and the presence or absence of AMF R. fasciculatum (control and 10 g of inoculum) as factor two.The factorial arrangement was 3×2 treatments with four replicates per treatment and four plants per repetition.

Experimental conditions
Seeds were previously disinfected by 5-min immersion in a calcium hypochlorite solution containing 5% of active chlorine and subsequently washed with distilled and sterilized water.The seeds were seeded in polystyrene 200well trays with the commercial substrate previously described.To maintain substrate humidity, subsequently after sowing, daily irrigation was applied to achieve a homogeneous emergence.When seedlings reached an average height of 15 cm, they were transplanted in 1-kg plastic pots with the same sterilized commercial substrate according to the label, placing one plant per pot with daily application of 250 mL of water with 0.35 dS m -1 , pH 6.7 and nutritional solution according to Samperio (1997) and modified in P content according to Swift (2002).

Spore content in the inoculum and mycorrhizal colonization percentage
Inoculation of R. fasciculatum was performed at the moment of transplanting, applying 10 g of AMF to each seedling, equivalent to an average content from 50-70 spores per gram of the product.The colonization percentage was assessed 50 days subsequent to NaCl treatment.Previous to inoculation, the spores were extracted following the wet sieving and decanting method described by Daniels and Skipper (1982) and modified by Utobo et al. (2011).Colonization percentage was calculated by the methodology described by Hashem et al. (2014) through the following formula: Colonization percentage = Total colonized segments/Total studied segments × 100.

NaCl treatments
One week after transplanting, NaCl was gradually applied according to the proposed experimental design and the methodology of Murillo-Amador et al. (2007), applying daily 250 mL of the corresponding solution to each pot.This amount was enough to drain and avoid NaCl accumulation in the substrate.This was confirmed when electrical conductivity (CE) and pH of drained water were measured and then compared with CE and pH of the original NaCl concentration during the experimental period.

Morphometric variables
The first and second biomass production measurement was performed 20 and 50 days after applying the NaCl treatments.The samples collected were transferred to the plant physiology laboratory.The leaf area (cm 2 ) was determined by integrated leaf area (Li-Cor ® , Model-LI-3000A, series PAM 1701, USA).Fresh and dry biomass (g) soil conditions (Elhindi et al., 2017).Research on AMF efficiency in increasing plant halotolerance has been performed with horticulture (Abdel-Latef and Chaoxing, 2011) and industrial (Yamato et al., 2008) cultivation, while scarce reports exist on aromatic species.From a scientific and technological point of view, it is important to study these microorganisms and their mitigating affects on salinity stress in aromatic species, such as basil and others of commercial importance.
Basil (Ocimum basilicum L.) is an aromatic plant of the family Lamiaceae that is native to southern Asia and is widely distributed in tropical and subtropical regions (Briseño et al., 2013).It is a plant species considered to have important applications in medicine and culinary areas (Makri and Kintzios, 2008).Basil has different therapeutic applications, such as the ability to reduce blood pressure and glucose and cholesterol levels in blood, also it has antiinflammatory and anti-stress properties (Tomar et al., 2010).The cultivation of this species generates important economical income for farmers, but salinity in irrigation water and soil limit production, especially of the varieties sensitive to salinity.The objective of this study was to assess the mitigating effect of arbuscular mycorrhizal fungus (Rhizophagus fasciculatum) strain in 'Nufar' cv. in basil plants subjected to NaCl-stress during the initial growth stage.

Study area
This study was developed at the experimental field of Centro de Investigaciones Biológicas del Noroeste (CIBNOR) located 24° 08' 10.03 N and 110° 25' 35.31W northwest of La Paz, Baja California Sur, Mexico at 7 masl.The experiment was done under a white anti-aphid meshhouse with 30% shade (55 mesh) and cover with a second black mesh with 35% shade (20 mesh).The experimental site has a type Bw (h') hw (e) climate, characterized as semiarid with xerophilous vegetation (García, 2004).During the experimental time, the average, maximum and minimum temperatures were 29, 30 and 20 °C, respectively; average relative humidity was 69%, dewpoint 22 °C, showing a total precipitation of 14.6 mm and average solar radiation of 293.3 W m 2 .These variables were recorded daily using a portable meteorological station (Vantage Pro2 ® Davis Instruments, U.S.A.) placed inside the mesh house.

Genetic material
The seeds used were from basil 'Nufar' cv. which were provided by the Vis Seed Company, Inc. (http://www.visseed.com/)(Arcadia, California, USA).The arbuscular mycorrhizal fungus (AMF) was a commercial strain of Rhizophagus fasciculatum which was provided by from Republic of Cuba.This strain contains about 50 to 70 spores per gram.The morphological strain description showed globose shape, hyaline colour to pale cream and could reach a run travel from 60 to 110 micrometers.The number of pairs constituted three layers (Rodríguez et al., 2004).
were measured with an analytical balance (Mettler ® Toledo, Model AG204, USA).Dry biomass was obtained by placing leaves and stems in paper bags that were introduced into an oven (Shel-Lab ® , Model FX-5, series-1000203, USA) at a temperature of 70 °C until a constant weight (approximately 72 h).Total biomass crop was performed at day 50, which included the collection of the complete plant and separation of leaves, stems and roots.The plant height (cm), root length (cm), fresh and dry biomass (g) of the aerial part (leaves and stems), root and foliar area (cm 2 ) were measured.

Physiological variables
Relative water content (RWC) was assessed 20 and 50 days after applying NaCl treatments, following Yamasaki and Dillenburg (1999) method.Leaves were collected from the middle part of each plant to avoid decreasing age effect.The collected samples were transferred to the laboratory where three disks of 3.14 cm 2 of each leaf were cut with a hole puncher (Model DIN ® 7200, México); then, the disks were subsequently weighed to obtain fresh (FW) and turgid (TW) weights.Turgid was obtained by placing the disks in distilled water inside a Petri dish and during this process (imbibition), the disks were weighed periodically after eliminating surface water with absorbent paper.At the end of the imbibition process, the disks were placed in an oven at 80 °C during 48 h to get the dry weight (DW).Fresh, turgid and dry weights were obtained using an analytical balance with 0.0001 g precision (Mettler ® Toledo, Model AG204, USA).The relative water content (RWC %) was calculated using the values of FW, TW and DW using the following equation: Water potential (Ψw) was measured 20 and 50 days after applying the NaCl treatments.The leaves were collected at the most critical hour (12:00 h), considering the interval in which the highest temperatures were recorded during the day.The Ψw (MPa) was determined with a water potential meter (Dewpoint Water Potential Meter Model WP4-T, Decagon ® Devices, USA) for soil and botanical samples (grains, seeds, leaves, plant tissues in general), which functioned according to the dew point principle with sensors, a condensation mirror an infrared light.Starting from the collected leaves, disks of 9.60 cm 2 were obtained by using a hole puncher (Model DIN ® 7200, México), and subsequently each disk was placed inside the equipment bucket.

Statistical analysis
Analyses of variance (ANOVA) and multiple comparison of means (Tukey HSD p = 0.05) were performed using Statistica v. 10.0 for Windows (StatSoft ® , 2011).

Morpho-physiological variables assessed at day 20 after applying NaCl
The analysis of the interaction factors (NaCl×AMF) showed that fresh and dry biomass of the aerial part (FBAP and DBAP) and leaf area (LA) decreased as NaCl concentrations increased, showing highest values at 0 mM NaCl in plants inoculated with AMF and lowest in those subjected to 100 mM of NaCl without AMF; nonetheless, fresh and dry biomass and leaf area increased in 50 and 100 mM of NaCl with AMF compared to 50 and 100 without AMF (Table 1).
The factor interaction analysis showed that water potential (Ψw) values were less negative in plants subjected to 0 mM NaCl and inoculated with AMF and most negative values in those subjected to 100 mM NaCl without AMF.The Ψw showed lowest negative values in 50 and 100 mM of NaCl with AMF compared with 50 and 100 without AMF (Table 1).The effect of applying NaCl on relative water content (RWC) showed a greatest RWC at 0 mM NaCl with AMF but decreased at 100 mM NaCl without AMF (Table 1).

Morpho-physiological variables assessed on day 50 (crop) after applying NaCl
The interaction factors (NaCl×AMF) analysis showed that plant height (PH), root length (RL) fresh and dry biomass of aerial part (FBAP and DBAP), fresh and dry root biomass (FRB and DRB) and leaf area (LA) decreased as NaCl concentrations increased.These variables showed highest values in those plants treated with 0 mM NaCl with AMF and lowest in those plants subjected to 100 mM NaCl without AMF.Water potential (Ψw) had lower negative values at 0 mM followed by 50 mM NaCl, both with AMF while the most negative values were showed at 100 mM NaCl without AMF (Table 2).Relative water content did not show significant differences among NaCl concentrations; although, the highest values were at 0 mM with and without AMF, followed by 50 with and without AMF (Table 2).

Mycorrhizal colonization percentage
The mycorrhizal colonization percentage decreased as NaCl increased, which was highest in control (0 mM) and decreased at 50 and 100 mM NaCl (Table 2).

Discussion
Previous studies have been reported that in saline soils, AMF increased nutrient intake in plants, especially P with precipitates of Ca 2+ , Mg 2+ and Zn 2+ ions (Porras-Soriano et al., 2009).In addition to nutritional improvement, AMF benefits physiological processes, such as water absorption when roots hydraulic conductivity increases and improves the osmotic balance of adaptation and carbohydrates composition (Sharifia et al., 2007).Salinity affects plant development from reducing growth due to photosynthesis alteration, enzymatic activity and ionic homeostasis to increasing plant death (Fatma et al., 2014).However, in the present study, a favorable response was observed in basil 'Nufar' cv. which confirmed that AMF inoculation was efficient in mitigating NaCl-stress as was reported by Hashem et al. (2015) and Mendes et al. (2016).In other species such as Cucurbita pepo var.pepo cultivated under drought and salinity conditions, the application of a mixed inoculum and a consortium of six AMF native species of the 1288 Sonoran Desert, improved dry weight of the shoot and root, foliar humidity percentage, water and osmotic potential, and radicle colonization percentage, while also decreasing physiological stress caused by drought and salinity, thus AMF inoculation was an efficient alternative as a mitigate agent in salinity stress (Harris-Valle et al., 2011).In lettuce (Lactuca sativa L.), AMF inoculation also showed a mitigating effect with salinity (Aroca et al., 2013).This effect has been associated to the mitigating effect of AMF because their associations with plants improve functions that improve efficiency of nutrients in the radicle area, starting from an increase in the volume of the soil explored, increase in toxin resistance, translocation and solubilization of essential elements and increase in tolerance to adverse abiotic conditions, such as drought and salinity (Evelin et al., 2013).The results showed in the present study could be attributed to AMF which increased the root contact area with soil, assuring continuity among the absorbent radicle surface and soil solution, optimizing soil interaction with roots (Jurkiewics et al., 2010).According to Hajiboland et al. (2010), the AMF are characterized by forming structures in the shape of a miniature tree in the radicle parenchyma cells, a structure called "arbuscule", which is the exchange site between the plant and fugus.Moreover, the mycorrhizal system is formed by a set of hyphas (mycelium) that are connected to the root tissue and branch out in soil.The mycelium found in soil forms a hypha net capable of interconnecting roots and allowing water and nutrient flux between them.These stimulating AMF functions improved the plant water state, which was observed in the relative water content, which increased in plants basil inoculated with AMF in the present study.
induced in colonized plants contributing to increase their resistance to abiotic tension.These results are similar to those reported previously by Alqarawi et al. (2014) where they pointed out that AMF modified the root architecture, allowing a greater range for water and essential elements in soil than in plants stressed without AMF.Moreover, AMF also mitigated salinity stress by improving absorption and nutrient capture rhizosphere conditions (Parra-Rivero et al., 2014); photosynthetic activity and water use efficiency (Hajiboland et al., 2010); compatible solute accumulation and antioxidant enzyme production (Evelin et al., 2013).Most recently, Elhindi et al. (2017) reported an increase in growth, chlorophyll content, gas exchange, photosynthetic efficiency, proline content, water use efficiency and nutrient absorption in sweet basil plants subjected to salinity stress (5 and 10 dS m -1 ) and inoculated with AMF Glomus deserticola, which favored greater growth, vigor and general cultivation productivity.In other species such as Solanum lycopersicum subject to salinity stress and inoculated with Glomus cubense, Mujica-Pérez and Fuentes-Martínez (2012) observed an increase in plant height, number of flowers and fruit and yield.In Dianthus caryophyllus, an ornamental species, Navarro et al. (2012) reported a beneficial effect in plant growth, number and size of flowers, leaves and concluded that the use of mycorrhizal (Glomus intraradices, GII and GIII) was efficient in mitigating NaClstress (1, 3, and 6 dS m -1 ).In citric plants (Poncirus trifoliata) subjected to NaCl-stress (0 and 100 mM) and inoculated with Glomus mosseae and Glomus vesiforme, an increase in growth was observed in plants inoculated with AMF compared with those in the control group without inoculation (Qiang-Sheng et al., 2010).Different studies maintain and conclude that AMF strains mitigate NaCl stress in different plant species.Some AMF strains that have shown efficiency as NaCl-stress mitigators are Funneliformis caledonius, Funneliformis mosseae and Rhizophagus irregularis, among others with positive effects in several species, such as Fragaria ananassa Duch that has shown increases in leaves and shoots biomass, root length and biomass (Sinclair et al., 2014).The increase in nutrient absorption such as P and water even in abiotic tension, reduced Na and Cl absorption, affecting movement into the aerial parts (leaves and shoots) of the plant augmented water catchment maintained ionic equilibrium to improve nutrient absorption and stimulated selective catchment (Evelin et al., 2012); increased synthesis and efficiency of some enzymes (Wu et al., 2010) as proline (Ibrahim et al., 2011) and caused osmotic adjustment that maintained turgid pressure in leaves, improving equilibrium among photosynthesis, transpiration, water use efficiency and stomatal conductance in inoculated plants (Evelin et al., 2009).Likewise, AMF have improved rhizosphere soil characteristics and root architecture (Hodge and Storer, 2015).The results of the present study are in agreement with those reported by Al-Khaliel (2010), bsince AMF improved plant development benefiting nutrient absorbance and soil structure; had a direct effect in the quality of the soil while causing aggregates that improve humidity retention, such as increasing soil water potential and favoring water and nutrient absorption.These changes stimulate plant growth, hydraulic conductivity and decrease toxic ion effect induced by salinity (Seema and Garampalli, 2015).The response in basil can be attributed to the combination of physical, nutritional and cellular effects as reported previously by Ruiz-Lozano and Aroca (2010).Other studies have also demonstrated that fungus-host plant symbiosis altered water movement rate within and outward from the plants in other species, affecting water relationships and physiology (Ruiz-Lozano et al., 2006).Moreover, AMF have stimulated physiological mechanisms that increase plant stress tolerance and have the potential to increase growth in normal and induced environmental stress conditions (Abd-Allah et al., 2015a, b).Previous studies showed significantly higher values in plant height, stem diameter, leaf area, total plant leaf number and total dry weight where AMF (Rhizophagus intraradices) was used in Vitis vinífera L. plants subjected to three salinity levels (0.65, 1.56 and 4.68 dS m -1 ) compared with those noninoculated (Khalil, 2013).
As expected, no colonization was observed in those noninoculated basil plants since a sterile substrate were used, which did not allow the development of native species capable of colonizing basil plants.Then, in this study an authentic colonization was observed in the inoculated basil plants; nonetheless, colonization percentage decreased as NaCl increased.This result did not affect the benefit reported by this endophyte species, such as basil, which was evident in the increase in the assessed variables in inoculated AMF basil plants.These results are similar to those reported by Harris-Valle et al. (2011) where they found that mycorrhizal colonization of a mixed native AMF inoculum, decreased physiological stress (drought and salinity) in Cucurbita pepo var.pepo; nevertheless, mycorrhizal colonization decreased as salinity increased, which did not affect plant response in such a condition.According to Rivera et al. (2003) colonization percentage is representative of arbuscular mycorrhizal symbiosis functionality.The fact that inoculated AMF has survived, adapted and established under different habitats, indicates that it has a favorable influence in the rhizosphere and substrate conditions where basil plants were established.In arbuscular mycorrhizal symbiosis no threshold colonization value occurs in roots to produce plant growth improvement.This effect depends on plant and fungal species in symbiosis and specific cultivation conditions.The principle mechanism is generally assumed to be that a greater root colonization rate by the fungus improves AMF effects on growth and plant development (Seema and Garampalli, 2015).

Conclusions
The basil plants inoculated with AMF showed increases in their morpho-physiological variables even under NaClstress conditions, showing a degree of infectivity and effectiveness of the AMF strain in the rhizosphere zone and in the growing conditions.The percentage of colonization decreased as the NaCl concentrations increased, being highest in the control (0 mM) and decreasing in 50 and 100 mM NaCl.As expected, no colonization occurred in those non-inoculated basil plants, because of a sterile substrate was used, which did not allow for the development of native species capable of colonizing the basil plants.The substrate used was suitable both for the development of the AMF strain and for the basil plants, as well as the number of inoculum spores.

Table 2 .
Effect of NaCl-stress on morpho-physiological characteristics of basil plants at 50-days treatment (T20) and inoculated with an arbuscular mycorrhizal fungus (Rhizophagus fasciculatum) strain as NaCl-stress mitigator