Effect of Mycorrhizal Symbiosis on the Development of the Canary Island Tomato Variety “Manzana Negra” under Abiotic Stress Conditions

Abstract

Tomato production in the Canary Islands has significantly decreased in recent years due to the presence of parasites and pathogens, poor-quality irrigation water, lack of infrastructure modernization, and increased competition. To address this issue, local varieties with better agro-climatic adaptation and organoleptic characteristics have been cultivated. These varieties show their maximum potential under an agro-ecological cultivation system, where the beneficial micro-organisms of the rhizosphere (in general) and mycorrhizal fungi (in particular) have a positive influence on their development, especially when the plants are subjected to biotic or abiotic stresses. Irrigation water in Canary Islands tomato cultivation comes from groundwater sources with moderate levels of sodium and chlorides or sodium and bicarbonates. This study evaluated the response of mycorrizal plants of the local tomato variety “Manzana Negra” under abiotic stress conditions due to the presence of chlorides and bicarbonates. Two tests were carried out with mycorrhizal and non-mycorrhizal plants. In the first one, 0, 75, and 150 mM NaCl solutions were applied. In the second, the nutrient solution was enriched with sodium bicarbonate at doses of 0, 2.5, 5, 7.5, 10, and 12.5 mM. Presence of native mycorrhizae improved the growth and nutrition of plants affected by irrigation with saline and alkaline water containing chloride and sodium carbonate. Symbiosis produced statistically significant increases in all plant-development-related variables (stem length and diameter; fresh and dry weight) in all bicarbonate concentrations. However, the results with the application of sodium chloride do not seem to indicate a positive interaction in most of the analytical parameters at 150 mM NaCl concentration. The mycorrhizal inoculation with local fungi can be interesting in the production of seedlings of this tomato variety in situations of moderate salinity, especially under bicarbonate stress conditions.

1. Introduction

The Canary Island archipelago, made up of eight islands, is located off the north-west coast of Africa, between latitudes 27°37′ and 29°25′ N and longitudes 13°20′ and 18°10′ W (Figure 1). Since the end of the 19th century, tomato cultivation has been important for the economy of the islands. The tomato (Solanum lycopersicum L.) is well adapted to the islands’ soil and climatic conditions, which has made it an important part of the archipelago’s economy and a characteristic element of the landscape and culture. As a result, cultivation increased until production reached over 300,000 tonnes at the end of the 20th century. Since then, there has been a gradual decline in production, mainly due to the salinisation and desertification of the land, the increase in pests and diseases, and the cultivation of other, more competitive crops [1].

Of the processes responsible for soil degradation in the Canary Islands, salinisation and sodification affect 20% of the territory [2]. This is not only due to natural causes; irrigated agriculture has aggravated the situation through the indiscriminate use of very low-quality saline water. In many cases, this is the only water available, owing to the continuous overexploitation of aquifers, which has led to marine intrusion in coastal wells [3].

The mobility of salts and sodium in soil is high, making them susceptible to removal by excess water, a process known as salt washing. In the Canary Islands, traditional irrigation systems that used large volumes of water and rainfall facilitated flushing, which solved the problems of salinisation and sodification. However, the use of high-frequency localized irrigation systems, which use very low water volumes, does not facilitate the removal of salts from the surface horizons of the soil. Instead, it only relocates them, leading to a concealed salinization of agricultural soils in the medium term [3].

The groundwater in the Canary Islands is classified into three types: sodium chloride, sodium bicarbonate, and calcium-magnesium bicarbonate [3]. Groundwater extracted from wells is typically found in the arid coastal areas of Lanzarote, Fuerteventura, South Tenerife, and Gran Canaria. The largest tomato-producing areas in the Canary Islands are in Gran Canaria, where the groundwater contains sodium chloride. The quality of the water in galleries is better, but it is bicarbonated. The water is either sodic when the aquifer saturates alkaline rocks such as phonolites and ignimbrites or calcic and magnesic when it saturates basaltic rocks.

In areas with an optimal climate for tomato cultivation, salinity is a serious problem, not only for establishing new plantations but also for maintaining high production levels [4]. Although tomatoes are generally moderately tolerant to salinity, there are considerable differences between cultivars when there is an excess of salt in the soil. This can affect vegetative growth. Indeed, it can result in a reduction in the length and dry weight of the stems, as well as the fresh and dry weight of the roots [5,6] and the number and size of the fruits [7,8,9]. It has also been found that the addition of bicarbonate and sodium chloride in irrigation water produces more negative effects than sodium chloride alone [10]. The decrease in biomass observed when plants are irrigated with bicarbonate may be attributed to the high pH resulting from the dissolution of bicarbonate ions. This high pH level may negatively impact nutrient uptake [11,12].

The ability of arbuscular mycorrhizal (AM) fungi to enhance plant tolerance to abiotic stress conditions has been widely acknowledged [5,6,13,14,15,16,17,18,19,20,21,22]. Manipulating these fungi in sustainable agricultural systems can be crucial for improving soil quality and crop productivity under unfavourable soil and climatic conditions [13]. Several authors have highlighted the ability of AM fungi to increase crop tolerance to soil salinity, decrease agricultural productivity losses, and generally benefit agricultural production [14,15,16,17,18,19,20,21,22]. In the case of tomatoes, numerous studies have demonstrated the potential of AM fungi to provide plants with greater resistance to salt stress in arid and semi-arid regions. This can lead to improved plant nutrition and growth and, therefore, better yields and productivity, as it allows the size and efficiency of the roots, the leaf area index, and the low biomass to adapt in drought conditions and improves photosynthetic efficiency, stomatal conductance, and leaf water relations in saline conditions [5,6,9,16,23,24,25,26,27,28,29].

This study aims to evaluate the effects of the local arbuscular mycorrhizal fungus Funneliformis mosseae on a traditional Canary Island tomato variety “Manzana Negra” under abiotic stress caused by high concentrations of sodium chloride and sodium bicarbonate in the soils of the Canary Islands.

2. Materials and Methods

Two trials with increasing salt concentrations, one with sodium chloride and the other with sodium bicarbonate, were carried out on mycorrhizal tomato plants under controlled growing conditions.

2.1. Plant Material: Host Plant

Both experiments used the “Alajeró” entry of the traditional Canary Island tomato variety “Manzana Negra” (

Table 1. Characteristics of the traditional tomato variety Manzana Negra [30].

Code Bank	Varietal Type	Origin	Growth	Shape of the Fruit	Average Fruit Weight (g)	Greenback Intensity
CCBAT 01899	Marmande	Alajeró La Gomera	Semi-determined	Flattened slightly	200–210	Slight

) from the collection of the Centro de Conservación de la Biodiversidad Agrícola de Tenerife (CCBAT, Bank Code ESP0172), an organic unit of the Cabildo Insular de Tenerife [30]. The seeds were previously disinfected in a 0.5% sodium hypochlorite solution for one minute and then rinsed with sterile distilled water.

2.2. Substrate and Containers, Inoculation, and Growing Conditions in the Semi-Fertilised Phase

2.2.1. Substrate and Containers

The substrate used was a mixture of corrected blond peat TKS1-Instant (Sphaghum-Torf^® Klasmann-Deilmann GMBH, Geeste, Germany), fine black picón (volcanic ash), and soil with low P content, in a 1:1:1 ratio. The substrate, whose physical–chemical characteristics can be found in

Table 2. Physico-chemical properties of the substrate used in the tests.

General Prop.		Exchangeable Cations		Nutrients		Soil Solution Cations and Ions
pH	7.14	CIC (cmolc Kg−1)	28.80	P (ppm)	14.51	Ca (meq L−1)	1.73	CO3 (meq L−1)	0.00
CEC (dS.m−1)	0.38	Ca (cmolc Kg−1)	11.93	Fe (ppm)	7.15	Mg (meq L−1)	0.82	HCO3 (meq L−1)	0.46
OM (%)	0.73	Mg (cmolc Kg−1)	5.78	Mn (ppm)	4.10	Na (meq L−1)	1.49	Cl (meq L−1)	0.65
		Na (cmolc Kg−1)	1.09	Zn (ppm)	15.45	K (meq L−1)	0.16	S-SO4 (meq L−1)	2.55
		K (cmolc Kg−1)	1.69	Cu (ppm)	0.52			N-NO3 (meq L−1)	8.93
				N (ppm)	525.84

OM: organic matter; CEC: cation exchange capacity; cmolc Kg⁻¹: centimoles of load per kilogram of soil.

, was previously sifted through a 5 mm sieve and disinfected in a steam autoclave for 1 h for three consecutive days. The characteristics of the water used for irrigation are shown in

Table 3. Chemical properties of water used for irrigation.

General Properties		Cations Present		Ions Present
pH	8.67	Ca (meq L−1)	1.73	CO3 (meq L−1)	0.00
CEC (dS.m−1)	0.79	Mg (meq L−1)	0.82	HCO3 (meq L−1)	0.46
		Na (meq L−1)	1.49	Cl (meq L−1)	0.65
		K (meq L−1)	0.16	S-SO4 (meq L−1)	2.55
				N-NO3 (meq L−1)	8.93

. The seedlings were grown in rigid expanded polystyrene (EPS) multipot trays with 15 cells of 300 cm³ volume each, without a bottom.

2.2.2. Inoculation

The plants were inoculated with a mixture of rhizospheric soil and sorghum roots (Sorghum bicolor var. sudanense) colonised by the arbuscular mycorrhizal (AM) fungus Funneliformis mosseae ‘raw inoculum’. The inoculum had a 72% colonisation rate and a richness of 100 spores per 100 g of soil. This was achieved by filling each alveolus of the multipots 2/3 full with substrate, adding 12 cm³ of crude inoculum to each alveolus, and then topping up with substrate. The plants were inoculated with mycorrhizal fungi during sowing. Non-mycorrhized plants were sown by filling each multipot alveolus completely with substrate. In both cases, the seeds were buried to the same depths using forceps.

2.2.3. Growing Conditions in the Seedbed

The trials were conducted under semi-controlled conditions in a mesh greenhouse with a polycarbonate cover. Irrigation was carried out according to the water requirements of the crop and Hewit nutrient solution was used as fertiliser [31].

2.3. Experimental Conditions Specific to Each Test

The experimental conditions and details of each of the experiments are given below.

2.3.1. Trial 1: Effect of Mycorrhizal Symbiosis on Sodium Chloride Stress

At the end of the seedling phase, which lasted six weeks, eight randomly selected seedlings per treatment were separated to check their growth and the degree of mycorrhizal colonisation (pre-experiment lifting) (Figure 2).

Experimental Design

The trial comprised a full factorial design with two factors—mycorrhiza (AM and Control) and sodium chloride (0 mM, 75 mM and 150 mM)—resulting in six treatments. A total of 270 seeds were sown, with half inoculated with a local AM fungus, Funneliformis mosseae (AM), and the other half left without inoculum. The sodium chloride factor was applied using 0, 75, and 150 mM NaCl solutions. The solutions were made with irrigation water, with the 0 mM dose consisting of irrigation water without added NaCl. This was performed depending on the salt treatment.

Substrate and Containers in the Experimental Phase

After the seedling phase, 42 seedlings per treatment were selected and transplanted into 17 cm diameter pots with a capacity of 2 L. The substrate used on this occasion was a previously washed mixture of fine black picón (volcanic ash) and soil with a low P content, in a 1:1 ratio, sifted through a 1 mm sieve.

Growing Conditions in the Experimental Phase

A weekly treatment with Bacillus thuringiensis was used to prevent damage by the Tuta absoluta moth and a treatment with wettable sulphur (Rubigán 0.5 cc/L) was applied to prevent powdery mildew attacks. The plants were kept under the same irrigation and phytosanitary conditions for three weeks to ensure their adequate development. From that point onwards, salt treatments were conducted by irrigating with salt solutions at the indicated concentrations three times a week. Four weeks later, the second lifting was performed, where eight seedlings per treatment were randomly selected as the experimental unit, with each unit consisting of one plant.

2.3.2. Trial 2: Effect of Mycorrhizal Symbiosis on Stress Caused by Sodium Bicarbonate

At the end of the five-week seedling phase, we randomly selected eight seedlings per treatment to assess their growth and degree of mycorrhizal colonization. This was performed using the rooted seedlings prior to the experiment, as shown in Figure 3.

Experimental Design

The experimental design comprised a full factorial with two factors—mycorrhiza (AM and Control) and sodium bicarbonate (0 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM, and 12.5 mM)—resulting in a total of 12 treatments. For this, 240 seeds were sown, half of them inoculated with a local AM fungus, Funneliformis mosseae (AM), and the other half without inoculum. Sodium bicarbonate was applied to irrigation water at concentrations of 2.5 mM, 5 mM, 7.5 mM, 10 mM, and 12.5 mM (with 0 mM being the control group).

Substrate and Containers in the Experimental Phase

After the seedling phase, 20 seedlings per treatment were selected and transplanted into 17 cm diameter pots with a capacity of 2 L. The substrate used was the same as that in the previous experiment.

Growing Conditions in the Experimental Phase

A weekly treatment with Bacillus thuringiensis was used to prevent damage by the Tuta absoluta moth and a treatment with wettable sulphur (Rubigán 0.5 cc/L) was applied to prevent powdery mildew attacks. To ensure adequate development of the plants, they remained under the same irrigation and phytosanitary treatment conditions as those described above for the seedbed for three weeks. After that, bicarbonate treatments were started, which consisted of irrigation with the sodium bicarbonate solutions at the concentrations previously indicated, three times a week. Nine weeks later, the seedlings were lifted, separating 12 randomly selected seedlings per treatment, where the experimental unit consisted of one plant.

Experimental Variables

After the seedling stage, ten seedlings per treatment were randomly selected to assess their development and degree of mycorrhizal colonization prior to the experiment. The following parameters were measured:

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Fresh and dry weights of the aerial part and of the root system (g). To determine the dry matter, it was dried in a hot air oven at 70 °C until constant weights were achieved.

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Plant length (cm).

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The presence of mycorrhizal fungus was determined by calculating the percentage of colonisation using the Phillips and Hayman technique [32], which was modified by Koske and Gemma [33]. To determine the percentage of colonisation, 10 pieces of 1 cm length of stained root were observed under a microscope following the technique of Brundrett et al. [34].

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The relative mycorrhizal dependence (RMD) [35] is a numerical expression of the degree to which plants require mycorrhization to achieve maximum growth or yield at a given fertility level. The formula proposed by Plenchette et al. [36] was used to calculate RMD.

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The relative water content (RWC) was calculated as a percentage using the method of Barrs and Weatherley [37]. A leaf lobe was taken from each plant, previously weighed, and placed in a falcon tube on a piece of cotton wool saturated with distilled water so that the cut part touched the cotton wool. The samples were kept in a refrigerator at 4 °C for 24 h. Each leaf piece was weighed again to determine the turgid weight and then dried in an oven at 70 °C to determine the dry weight. Finally, the relative water content was determined using the formula: [(initial weight − dry weight)/(turgid weight − dry weight)] × 100.

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The relative electrolyte loss (REL) (%) was determined using the method described by Verslues et al. [38]. To achieve this, one leaf lobe of each plant, which had been previously weighed, was placed in a falcon tube containing 30 mL of milli-Q water. The electrical conductivity of the water was measured prior to immersion. After 24 h at room temperature, the electrical conductivity of each sample was measured. The samples were autoclaved to release all the electrolytes present in the lobe. After cooling, the electrical conductivity was measured again using the formula: [(initial EC − EC water mili-Q)/(final EC − EC water mili-Q)] × 100.

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The concentration of photosynthetic pigments (mg cm²) was determined by extracting eight leaf discs from each plant using a punch of known diameter. The discs were then placed in glass tubes containing 4 mL of pure acetone extractant and left for 24 h at 4 °C. To find out which was the best extractant, a previous study was carried out with all those used by Lichtenthaler [39], obtaining pure acetone as the best extractant in the case of the traditional tomato variety ‘Manzano negro’. Therefore, the absorbance was then measured at the 3 wavelengths corresponding to pure acetone (λ1 = 661.6; λ2 = 644.8; λ3 = 470). The concentration of photosynthetic pigments (mg cm²) was determined by extracting eight leaf discs from each plant using a punch of known diameter. The discs were then placed in glass tubes containing 4 mL of pure acetone extractant and left for 24 h at 4 °C. The concentrations of chlorophyll a (C_a), chlorophyll b (C_b), chlorophyll a + b (C_a+b), and carotenes were calculated using the formulae described for this extractant [39]. In detail, the formulas used were:

C_a = 11.24A_661.6 − 2.04A_644.8;

C_b = 20.13A_644.8 − 4.19A_661.6;

C_a+b = 7.05A_661.6 − 18.09A_644.8;

Carotenes = (1000A₄₇₀ − 1.9C_a − 63.14C_b)/214.

The data presented refer to cm² of leaf area.

-

The leaf nutrient content (mg g⁻¹) was also determined. P, Na, K, Ca, Mg, and Fe were analysed via mineralisation through incineration of the leaves at 450 °C and extraction with hot dilute hydrochloric acid. P was determined through colourimetry on the Technicon AAII Auto-analyser (SEAL Analitical, Rijen, The Netherlands) by forming the vanadomolybdophosphoric acid complex. Na, K, Ca, Mg, and Fe were determined through atomic absorption spectrophotometry using specific lamps for each element. Potentiometric titration was used to determine the water-soluble chlorides. The determination of N was carried out through wet mineralisation and colorimetric detection of the formation of the ammonium salicylate complex in the Technicon AAII Autoanalyser.

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The electrical conductivity of the substrate was determined in saturated paste extract (dS.m⁻¹).

2.4. Analysis of the Data

Statistical analysis was conducted using the Statistical Package for Social Sciences (SPSS v.17) for Microsoft Windows™. Homoscedasticity was assessed using Levene’s test, and normality was assessed using the Kolmogorov–Smirnov and Shapiro–Wilk tests. The statistical tests performed included multifactorial analysis using the General Linear Model and single-factor analysis by comparing groups using analysis of variance (Student’s t-test and ANOVA). If significant differences were found, means were compared using post hoc analysis. In cases where the normality and homoscedasticity requirements were not met, a comparison between groups was performed using non-parametric Kruskal–Wallis analysis. As mycorrhizal colonisation is expressed as a percentage, the arc-sine transformation ((×/100)1/2) was applied to meet the parametric requirements of normality.

3. Results

3.1. Effect of Mycorrhizal Symbiosis on Salt Stress

The pre-experiment survey revealed that plants inoculated with AM fungi exhibited mycorrhizal colonisation in their roots. Additionally, a significant increase in seedling development was observed, as evidenced by the length and weight of both the aerial and root parts of the mycorrhizal plants in comparison to the control group. These plants also demonstrated high levels of mycorrhizal dependence.

At the end of the trial, the electrical conductivity (EC) of the substrate significantly increased with the application of more concentrated doses of salt, regardless of the presence or absence of AM fungi on the plants (see Figure 4). The substrate was observed to have been salinized by the irrigation water used (EC 0.79 dS.m⁻¹), as it originally had an EC of 0.38 dS.m⁻¹. By the end of the experiment, the mean values had reached 2.2 dS.m⁻¹. In the 75 mM and 150 mM NaCl treatments, the electrical conductivity (EC) was 14.2 and 24.9 dS.m⁻¹, respectively. This occurred regardless of the presence of arbuscular mycorrhizal (AM) fungi.

Table 4. F-value of ANOVA analysis of aerial, root, and total length; stem diameter; fresh and dry weight; and aerial, root, and total weight, explained using the factors ‘mycorrhiza (AM)’ and ‘sodium chloride (NaCl)’.

	Length (cm)			Diám.	Fresh Weight (g)			Dry Weight (g)
	Aerial	Root	Total		Aerial	Root	Total	Aerial	Root	Total
AM	7.31 **	0.00 (ns)	23.52 ***	8.56 **	9.95 **	6.27 *	10.64 **	9.23 **	5.82 *	10.21 **
NaCl	8.72 ***	8.89 ***	17.95 ***	27.09 ***	4.58 *	35.76 ***	7.86 **	12.60 ***	22.24 ***	15.67 ***
AM × NaCl	9.57 ***	0.95 (ns)	3.98 *	0.79 (ns)	1.32 (ns)	0.94 (ns)	1.37 (ns)	1.10 (ns)	1.11 (ns)	1.17 (ns)

AM: mycorrhizal inoculation; NaCl: dose of NaCl applied; ns: not significant. * p < 0.05; ** p < 0.01; *** p < 0.001.

shows that the local mycorrhizal fungus led to a significant increase in development, while the applied salt concentrations caused a decrease. The interaction between both factors was only statistically significant for aerial length and total length.

The length of the aerial parts of the mycorrhizal plants increased significantly in the 0 and 75 mM NaCl treatments compared to non-mycorrhizal plants. However, there was no significant increase when subjected to high salt concentrations (150 mM) (see Figure 5). Root length was significantly affected by increasing salt concentration in the solution, regardless of the presence of mycorrhizal fungus. At the mean dose of salt applied (75 mM), the stem diameter of mycorrhizal plants increased significantly. However, as the dose of NaCl applied increased, it decreased significantly.

In all salt treatments studied, mycorrhizal fungi demonstrated a statistically significant increase in plant biomass, both in terms of fresh and dry weight. However, this effect was not observed at 150 mM NaCl (refer to Figure 6).

The level of mycorrhizal colonisation increased linearly with the amount of salt applied. However, the relative mycorrhizal dependence was higher in the medium salinity treatment and decreased in the 150 mM NaCl treatment (Figure 7). This result demonstrates the effectiveness of the symbiosis at 75 mM added salt and its loss at 150 mM, despite greater colonisation.

In terms of the macronutrient content of the aerial part of the plants, the treatments demonstrated a statistically significant positive effect of mycorrhization on the uptake of nitrogen and potassium but not on phosphorus (see Figure 8).

Leaf Na and Cl content increased significantly with increasing amounts of these elements (Figure 9), with no effect of AM fungi. g⁻¹.

The physiological parameters show significant differences in response to the salt dose added; however, differences due to mycorrhization of the plants were found only for relative water content and relative electrolyte loss (

Table 5. F-value of ANOVA analysis of relative water content (RWC), relative electrolyte loss (REL), and photosynthetic pigments (Chlorophyll A (Chlor A), Chlorophyll B (Chlor B), Chlorophyll A and B (Chlor AB), and Carotenes), explained by the factors ‘mycorrhizae (AM)’ and ‘sodium chloride (NaCl)’.

	RWC	REL	Chlor A	Chlor B	Chlor AB	Carotenos
AM	8.86 **	6.46 *	0.88 (ns)	9.61 **	0.98 (ns)	3.614
NaCl	23.20 ***	171.88 ***	233.05 ***	187.68 ***	573.95 ***	103.16 ***
AM × NaCl	0.26 (ns)	0.591 (ns)	11.09 ***	3.41 *	13.18 ***	1.13 (ns)

AM: mycorrhizal inoculation; NaCl: dose of NaCl applied; ns: not significant. * p < 0.05; ** p < 0.01; *** p < 0.001.

). Relative water content shows a significant decrease at any of the applied salt levels compared to the treatment with no salt added (0 mM) for both mycorrhizal and non-mycorrhizal plants.

The concentrations of the photosynthetic pigments (chlorophyll A, chlorophyll B, chlorophyll A + B, and carotenes) were affected with significant differences with increasing salt dosage (Table 5).

No significant differences were found in the relative water content (RWC) and relative electrolyte loss (REL) at the different doses tested, except for the RWC at the dose of 75 mM salt. The results obtained are not conclusive to establish a relationship between these two rates and the different applied doses of NaCl (Figure 10).

3.2. Effect of Mycorrhizal Symbiosis on Bicarbonate Stress

The pre-experimental survey showed the presence of symbiosis in the plants inoculated with AM fungi. A significant increase in the development of the mycorrhizal seedlings was also observed, as reflected in the length of the aerial part as well as in the fresh and dry weights of the aerial part.

The final evaluation of the experiment showed that the symbiosis produced a statistically significant increase in the variables related to plant development. The application of sodium bicarbonate showed a statistically significant effect on these parameters. There was no interaction between the two factors on these parameters.

The length of the aerial part was significantly longer for mycorrhizal plants (Figure 11) regardless of the dose of bicarbonate applied. The opposite effect was found for root length where increasing doses of bicarbonate applied produced an increase in this parameter (Figure 12), regardless of the establishment of symbiosis (

Table 6. F-value of ANOVA analysis of aerial, root, and total length; stem diameter; aerial, root, and total fresh weight; and aerial, root, and total dry weight, explained by the factors ‘mycorrhiza (AM)’ and ‘sodium bicarbonate (NaHCO₃)’.

	Length (cm)			Diam.	Fresh Weight (g)			Dry Weight (g)
	Aerial	Root	Total		Aerial	Root	Total	Aerial	Root	Total
AM	99.58 ***	0.55 (ns)	88.21 ***	2.99 (ns)	94.83 ***	31.02 ***	100.8 ***	110.6 ***	10.98 **	107.1 ***
NaHCO3	0.97 (ns)	7.48 ***	0.66 (ns)	11.01 ***	3.16 *	13.40 ***	4.46 **	3.34 **	4.06 **	3.92 **
AM × NaHCO3	1.15 (ns)	1.30 (ns)	1.54 (ns)	0.46 (ns)	1.12 (ns)	1.64	1.01 (ns)	1.33 (ns)	0.71 (ns)	1.17 (ns)

AM: mycorrhizal inoculation; NaHCO₃: dose of NaHCO₃ applied; ns: not significant * p < 0.05; ** p < 0.01; *** p < 0.001.

).

The fresh and dry weights of the aerial part were significantly higher in mycorrhizal plants in all salt treatments applied (Figure 13 shows the data corresponding to dry weight). The mycorrhizal plants did not show any differences between the sodium bicarbonate treatments, but the plants with roots showed a statistically significant decrease in fresh and dry weight compared to the plants without bicarbonate (0 mM NaHCO₃ treatment), which shows the protective effect of the symbiosis (Figure 13).

The most striking effect produced by the application of sodium bicarbonate on the development of tomato plants of the Manzano Negro variety was a significant increase in plant diameter (Figure 14).

Mycorrhizal colonisation decreased significantly with increasing bicarbonate dose, while its relative mycorrhizal dependency was maintained at the different bicarbonate concentrations (Figure 15).

In this trial, the symbiosis did not have a statistically significant effect on the foliar nutrient content of the plants, except for the 7.5 mM NaHCO₃ dose. At this dose, the nitrogen content was statistically significantly higher, while the phosphorus content was statistically significantly lower (Figure 16).

4. Discussion

4.1. Effect of Mycorrhizal Symbiosis on Salt Stress

The effect of AM fungi against increasing doses of salt on tomato plants is confirmed in this work, which shows an increase in developmental parameters, as described by other authors [5,9,26,27,28,29,40], though it is lower in the roots than in the aerial biomass of the plants [5,9,26,27]. However, Al-Karaki, Al-Karaki et al., and Liu et al. [28,29,40] observed that these benefits disappeared at high doses of NaCl, which also agrees with our results.

Copeman et al. [41] found a linear increase in mycorrhizal colonisation as the amount of salt applied to tomato plants increased, being more evident at EC between 2 and 5 dS.m⁻¹, similar to the results obtained in this experiment. In contrast, other authors found the opposite effect, observing greater colonisation in the treatment with no salt applied [5,26,28,29].

The mineral nutrient content of plant tissues was found to decrease as the electrical conductivity of the substrate increased, as demonstrated by Evelin et al. [42], mainly for P, as it is immobilised with Ca, Mg, and Zn ions [42]. The results of this work confirm that AM fungi have a positive effect on nutrient uptake [43,44,45].

Contrary to the findings of other authors in other crops [46,47], mycorrhization did not increase Cl uptake with increasing dose. The higher concentration of Cl in the substrate justifies the greater accumulation of Cl in mycorrhizal plants observed by the authors, as it leads to an increase in the translocation of this element to the mycorrhizal plants, occurring simultaneously with the carbon exchange between the fungus and the plant. In contrast, the opposite effect was observed by [48], where the symbiosis resulted in a decrease in Cl uptake.

The relative water content of mycorrhizal ‘Manzana Negra’ tomato plants was higher than non-mycorrhizal ones, in agreement with the numerous results showing that mycorrhizal plants maintain a higher water content compared to non-mycorrhizal ones [49,50,51]. This is because AM fungi improve root hydraulic conductivity, maintain a lower osmotic potential due to solute accumulation, and have a higher turgor potential, which makes mycorrhizal plants use water more efficiently than non-mycorrhizal plants [51].

Relative electrolyte loss was clearly affected by the salt treatments, increasing significantly for both mycorrhizal and non-mycorrhizal plants. In contrast, the effect of AM fungi was detrimental at the dose of 150 mM NaCl added, where the relative electrolyte loss increased significantly. However, several authors [14,52,53] working with maize (Zea mays), pole beans (Cajanus cajan), and peppers (Capsicum sp.) observed a significant decrease in the relative permeability of electrolytes in mycorrhized plants. This is because AM fungi allow plants to maintain higher electrolyte concentrations while maintaining the integrity and stability of the membranes, which they achieve by increasing P uptake and increasing antioxidant production [52]. The results obtained at the dose of 150 mM of added salt are probably because, at this salt level, the AM fungus we used are not effective, as previously mentioned for other parameters.

The observed decrease in the concentration of photosynthetic pigments (chlorophyll A, chlorophyll B, chlorophyll A + B, and carotenes) with increasing salt dosage is likely due to the suppression of specific enzymes responsible for the synthesis of photosynthetic pigments at high salinities [54]. In addition, reduced uptake of nutrients (such as Mg) required for chlorophyll biosynthesis also reduces photosynthetic pigment content in salinity-stressed plants [42]. However, AM fungi usually cause an increase in photosynthetic pigments under such conditions [55] due to the suppression of the negative effects caused by salinity [43]; although, in our study, the benefit of AM fungi was only found in chlorophyll B content in plants without added salt (0 mM).

4.2. Effect of Mycorrhizal Symbiosis on Bicarbonate Stress

Similar to the findings of other studies [30], an increase in the dose of bicarbonate applied resulted in a decrease in developmental parameters. This effect may be due to an increase in substrate pH, which can inhibit plant metabolic processes and/or reduce root activity and growth [56], as well as decrease nutrient solubility [12].

The benefits of mycorrhizal symbiosis for plants under bicarbonate stress have been previously described by Cartmill et al. [25]. Similar to our findings, they found no significant interaction between the factors ‘mycorrhizal effect’ and ‘applied bicarbonate’.

High pH levels cause an increase in root cell size in both epidermis and cortex, as well as an increase in intracellular space [57]. This could explain the larger plant diameters with increasing NaHCO₃ concentration.

According to previous work by [58,59], the increase in bicarbonate dose in this experiment resulted in a statistically significant decrease in mycorrhizal colonisation. The authors suggest that this may be due to the toxic effect of high pH levels on mycorrhizal fungi.

The relative mycorrhizal dependence recorded in this work was maintained at different bicarbonate concentrations. However, Cartmill et al. [25] found an increase in mycorrhizal dependence up to a dose of 2.5 mM of added bicarbonate, subsequently decreasing with higher concentrations.

A significant decrease in leaf N and P content with increasing bicarbonate dosage was observed by Cartmill et al. [25], which they attributed to immobilisation caused by the increase in substrate pH resulting from the incorporation of bicarbonate ions. As for the effect of the AM fungi, they showed that the leaf concentrations of N, P, K, and Na were significantly higher in the mycorrhizal plants. However, none of these effects were observed in our experiment, except for the 7.5 NaHCO₃ dose, which increased foliar N.

In order to explore in the future use of the fungus Funneliformis mosseae in the cultivation of the traditional variety “Manzana Negra” in the Canary Islands, it is necessary to evaluate the effect of the fungus on production under real cultivation conditions and the advantages of mycorrhizal inoculation in the presence of pathogenic nematodes.

5. Conclusions

Inoculation with the AM fungus Funneliformis mosseae improved the general development of plants of the Alajeró entry of the local Canary Island tomato variety “Manzana negra” under moderate salt stress produced by sodium chloride (75 mM NaCl). However, high salt concentrations (150 mM NaCl) reduce the effectiveness of inoculation with AM fungi.

The benefits of mycorrhizal symbiosis were also observed in tomato plants subjected to bicarbonate stress, with a general increase in development and biomass at all doses of bicarbonate applied.

The results show the general suitability of mycorrhizal inoculation for the cultivation of this tomato variety in situations of moderate salinity and especially under bicarbonate stress conditions.