Role of arbuscular mycorrhizal fungi in alleviating the adverse effects of acidity and aluminium toxicity in zucchini squash

Scientia Horticulturae 188 (2015) 97–105

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Role of arbuscular mycorrhizal fungi in alleviating the adverse effects of acidity and aluminium toxicity in zucchini squash Youssef Rouphael a , Mariateresa Cardarelli b , Giuseppe Colla c,∗ a

Department of Agricultural Sciences, University of Naples Federico II, Via Università 100, 80055 Portici, Italy Consiglio per la Ricerca in Agricoltura e l’analisi dell’economia agraria, Centro di ricerca per lo studio delle Relazioni tra Pianta e Suolo, Via della Navicella 2-4, 00184 Roma, Italy c Department of Agriculture, Forestry, Nature and Energy, University of Tuscia, Via San Camillo De Lellis snc, 01100 Viterbo, Italy b

a r t i c l e

i n f o

Article history: Received 2 January 2014 Received in revised form 18 March 2015 Accepted 19 March 2015 Available online 7 April 2015 Keywords: Acid soil Aluminium Arbuscular mycorrhizal Cucurbita pepo L. Nutritional status pH level

a b s t r a c t The aim of the current research was to assess whether arbuscular mycorrhizal (AM) inoculation would give an advantage to overcome acidity and aluminium (Al) toxicity problems and to study the changes induced by AM at agronomical and physiological level. A greenhouse experiment was carried out, to determine yield, growth, fruit quality, SPAD index, electrolyte leakage, and mineral composition of zucchini squash (Cucurbita pepo L.) inoculated (+AM) and noninoculated (−AM) with arbuscular mycorrhizal and cultured in pots filled with quartziferous sand. Plants were supplied with nutrient solutions having different pH and aluminium concentration (pH 6.0, pH 3.5 or pH 3.5 + Al). The low pH treatment had the same nutrient composition plus HCl, whereas the aluminium treatment (pH 3.5 + Al) was induced by adding 1.0 mM of AlCl3 ·6H2 O. The AM root colonization was higher in the control treatment (pH 6.0; 48.5%), followed by pH 3.5 (23.4%), and finally in pH 3.5 + Al treatment (17.1%). Significant depression of yield, biomass, SPAD index, leaf area, N, P, Mg, Fe, and Zn concentration in leaf tissue was observed in response to low pH level with more detrimental effects with pH 3.5 + Al. The inoculated plants under acidity and Al conditions had higher total, marketable yield, and total biomass than noninoculated plants. Zucchini-fruit quality, in particular fruit dry matter, total soluble-solids content, P, and Fe concentration, was improved by mycorrhizal colonization. Mycorrhized zucchini plants grown under acidity and Al conditions had a higher macronutrient concentration in leaf tissue compared to noninoculated plants. The better crop performance in inoculated plants were related to the capacity of maintaining higher SPAD index, lower electrolyte leakage, and better nutritional status (high N, P, K, Ca, Mg, Fe, Zn, and B and low Al accumulation) in response to Al stress with respect to −AM plants. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Soil acidity is a major limitation to agricultural production throughout the world and one of the main causes of aluminium (Al) stress situations, with acidic soils representing about 50% of the total arable lands (Panda et al., 2009). Soil acidification is a natural process that is accelerated by some crop production practices. The main causes of soil acidity include (i) organic matter decay which produces hydrogen ions (H+ ), (ii) crop absorption of limelike elements such as mono and bivalent cations (K+ , Ca2+ , Mg2+ ), and (iii) the use of ammoniacal fertilizers; all these processes have

∗ Corresponding author. Tel.: +39 0 761 357536; fax: +39 0 761 357453. E-mail address: [email protected] (G. Colla). http://dx.doi.org/10.1016/j.scienta.2015.03.031 0304-4238/© 2015 Elsevier B.V. All rights reserved.

a strong and direct impact on soil acidity (e.g. pH) (Martens, 2001; Tang and Rengel, 2003). When soil pH is above 5.5, Al is present as harmless oxides and aluminosilicates (Ma et al., 2001); however, as pH drops below 5.5, Al is solubilized into the toxic trivalent cation Al3+ , limiting thereby plant productivity on such soils (Watanabe and Okada, 2005; Seguel et al., 2013). Increased concentrations of Al (for many plant species as low as 1–2 ppm) inhibit root cell division by destroying the cell structure of the root apex, resulting in poor root growth and development, drought susceptibility, altered nutrient uptake and translocation by plants (Foy, 1983; Seguel et al., 2013). For instance, Macklon et al. (1994), and Cumming and Ning (2003), observed that Al decreases inorganic phosphorus (Pi ) availability by forming Al–Pi precipitates in the rhizosphere and limits phosphorus uptake and translocation within plants. In addition, Al interferes with the bivalent cations (e.g. Ca2+ and Mg2+ )

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Y. Rouphael et al. / Scientia Horticulturae 188 (2015) 97–105

assimilation in plants (Lux and Cumming, 2001; Cumming and Ning, 2003). These effects result in nutrient deficiencies in plants, consequently reducing plant performance. In order to achieve a better crop yield on acid soils, growers are recommended to apply lime to increase the soil pH, and thus to mitigate the phytotoxic effect of Al. However, soil has a huge buffering capacity that is able to reduce the effects of lime, making this practice unsustainable as it is time and economically unfeasible (Nawrot et al., 2002; Seguel et al., 2013). An alternative way to mitigate the negative effects of acid soil on crop production is the use of Al tolerant/resistant genotypes that sustain acceptable yields on these soils (Seguel et al., 2013). However, the long-term needed to produce Al-tolerant varieties and the nature of the genetically complex tolerance of aluminium stress (Nawrot et al., 2002; Seguel et al., 2013), make this task extremely difficult. Consequently, searching for strategies that will generate improved tolerance to aluminium stress in plants is a priority. As a rapid alternative to the relatively slow breeding methodology aimed to overcome the problems caused by aluminium toxicity in acidic soils, inoculation with arbuscular mycorrhizal (AM) fungi could be a promising tool, for reducing the detrimental effect of external pH and aluminium toxicity on crop performance. AM are beneficial associations between soil fungi and plant roots, related with 83% of higher plants (Ma et al., 2001). The association of AM fungi with plant roots alters plant–soil interactions and enhances plant growth under stressful edaphic conditions (Smith and Read, 1997). AM fungi have been demonstrated to enhance soil structure (Miller and Jastrow, 2000; Rillig and Mummey, 2006), improve macro and micronutrient uptake and translocation by plants (Clark and Zeto, 2000; Rouphael et al., 2010), overcome the detrimental effect of salinity (Colla et al., 2008), alkalinity (Cardarelli et al., 2010; Rouphael et al., 2010), and heavy metals (Miransari, 2010), improve drought tolerance (Sanchez Blanco et al., 2004), suppresses root knot nematode (Zhang et al., 2008), maintain and restore soil health and fertility (Jeffries et al., 2003). The former positive effects of AM are generally attributed to the extension of the host’s root apparatus (e.g. up to seven times), penetration of substrates and excretion of enzymes by infected AM fungi roots and/or hyphae (Marschner, 1995; Smith and Read, 1997; Cartmill et al., 2008). AM fungi could be also considered an effective tool in the protection of root apparatus from Al phytotoxicity in acidic soils (Marschner, 1995). In fact, Danielson (1985), Clark (1997), and Cumming and Ning (2003), reported that AM fungi are widely established in low pH soils and colonization of plant roots by AM often, but not always enhances the crop performance on such soils by improving nutrient uptake and assimilation under Al stress conditions. Despite the number of reports demonstrating that AM fungal colonization ameliorates Al toxicity in plants (Mendoza and Borie, 1998; Lux and Cumming, 2001; Cumming and Ning, 2003; Kelly et al., 2005; Dudhane et al., 2012; Seguel et al., 2012), to our knowledge no published data are available on the effect of AM fungi inoculation under low pH and in the presence of Al on agronomical and physiological responses of vegetable crops, in particular zucchini squash (Cucurbita pepo L.), widely grown worldwide under open-field and greenhouse conditions. We hypothesize that arbuscular mycorrhizal inoculation would give an advantage to overcome acidity and aluminium toxicity problems. To verify this hypothesis, zucchini plants inoculated (+AM) or noninoculated (−AM) with AM fungi were grown in sand culture with nutrient solution having different pH and aluminium concentration (pH 6.0, pH 3.5 or pH 3.5 + Al). +AM and −AM plants were compared in terms of yield, biomass production, fruit quality, SPAD index, electrolyte leakage, and mineral composition.

2. Materials and methods 2.1. Growth conditions, treatments, and arbuscular mycorrhizal inoculation The experiment was carried out during the growing season of 2013 in a polyethylene greenhouse situated on the experimental farm of Tuscia University, Central Italy (42◦ 25 N, 12◦ 08 E). The greenhouse was maintained at daily temperatures between 16 ◦ C and 33 ◦ C, and day/night relative humidities of 55%/85%, respectively. Zucchini seeds (Cucurbita pepo L. cv. Tempra) (Società Agricola Italiana Sementi—SAIS, Cesena, Italy) were germinated in a 2:1 (v:v) mixture of BRILL-Type 3 propagation substrate (Gebr. Brill Substrate GmbH & Co. KG, Georgsdorf, Germany) and sand on February 25, 2013. The seedlings were transplanted 14 days (March 11) after sowing, into plastic pots (diameter 30 cm, height 30 cm) containing 17.7 L of quartziferous sand. Pots were disposed in double rows on the greenhouse floor at a plant density of 1.33 plants m−2 . The greenhouse experiment was laid out in a factorial combination of three nutrient solutions having different pH and aluminium concentration (pH 6.0, pH 3.5, or pH 3.5 + 1 mM Al), and two mycorrhizal treatments (with AM, +AM or without AM, −AM). Treatments were organized in a randomized complete-block design with four replicates. Each plot consisted of six plants. Prior to transplanting, half of the pots received a commercial mycorrhizal inoculum carrying Rhizophagus irregularis (formerly Glomus intraradices) and Funneliformis mosseae (formerly Glomus mosseae) (Aegis Microgranule, Italpollina S.p.A., Rivoli Veronese, Italy) by mixing 15 L of inoculum containing bulking material composed of 95% minerals as granular carriers, plus root fragments and spores (25 spores mL−1 of R. irregularis and 25 spores mL−1 of F. mosseae) per m3 of substrate. 2.2. Nutrient solution management The nutrient solution used in this study was a modified Hoagland and Arnon formulation, with the following composition: 13.0 mM N NO3 − , 1.6 mM S, 0.3 mM P, 4.3 mM K, 4.0 mM Ca, 1.3 mM Mg, 20 ␮M Fe, 9 ␮M Mn, 0.3 ␮M Cu, 1.6 ␮M Zn, 20 ␮M B, and 0.3 ␮M Mo. The pH of the nutrient solution was 6.0. The low pH treatment (pH 3.5) had the same nutrient composition plus HCl which was added to maintain the pH at 3.5. The HCl was added to the nutrient solution to simulate the effects of acidity. The aluminium treatment (pH 3.5 + Al) was induced by adding 1.0 mM of AlCl3 ·6H2 O. The acid treatments (pH 3.5 and pH 3.5 + Al) initialized 14 days after transplanting. All nutrient solutions were prepared using de-ionized water. Nutrient solution was pumped from independent reservoirs through a drip-irrigation system, with one emitter per plant (2 L h−1 ). Irrigation scheduling was achieved using electronic lowtension tensiometers (four tensiometers per experimental unit, LT-Irrometer; Riverside, CA, USA) that control irrigation based on substrate matric potential (Kiehl et al., 1992). Five to 15 fertigations were applied per day, each of 2–3 min duration. The duration of irrigations was increased so as to drain 35% of the nutrient solution from the bottom of the pots (Rouphael et al., 2006). 2.3. Yield and growth measurements From April 15 to May 22, the marketable fruit weight and number were recorded on six plants per experimental unit. Fruits were harvested when they reached a marketable length higher than 12 cm. Fruits measuring less than 12 cm or deformed were considered nonmarketable (Rouphael et al., 2004; Rouphael and Colla, 2005). At the end of the experiment (May 22, 72 days after transplanting), six plants per experimental unit were separated into

Y. Rouphael et al. / Scientia Horticulturae 188 (2015) 97–105

2.4. Fruit quality analysis Eight fruits of each experimental unit were analysed for quality parameters. Fruit shape index (SI), was determined as the ratio of width to length. Zucchini fruit colour was measured using a Minolta CR-200 chromameter (Minolta Camera Co. Ltd, Osaka, Japan) with an 8 mm measuring aperture in CIE L* , a* , b* mode with CIE standard illuminant C. The instrument was calibrated with a Minolta standard white reflector plate before sampling. The readings were taken in the mid part of the upper and lower surface of zucchini squash. The total soluble-solids (TSS) content was measured by an Atago N1 refractometer (Atago Co. Ltd., Japan) and expressed as ◦ Brix. Acidity was determined by potentiometric titration with 0.1 M NaOH up to pH 8.1. Results were expressed as percentage citric acid in the juice. The fruit dry matter (DM) and the pH of the fruit juice (pH meter, HI-9023; Hanna Instruments, Padova, Italy) were also measured.

60 -AM +AM

a

50

Root colonization (%)

leaves, stems, and roots. Roots were rinsed with water from sand, and subsamples were saved for the determination of AM fungi root colonization. Dry matter of each plant tissue was measured after oven-drying at 80 ◦ C for 72 h. Shoot biomass was equal to the sum of leaves and stems. Root-to-shoot ratio was calculated on dry weight basis, whereas leaf area (LA) was measured with an electronic area meter (Delta-T Devices Ltd., Cambridge, UK).

99

40

30

b 20

b

10

0

c

c

pH 6

pH 3.5

c pH 3.5 + Al

Fig. 1. Effects of arbuscular mycorrhizal inoculation, solution pH and aluminum concentration on root colonization. Different letters indicate significant differences according to Duncan’s test (p = 0.05). Values are the means of four replicate samples. +AM, −AM, mycorrhizal and non-mycorrhizal plants, respectively.

coupled plasma spectrophotometer (ICP Iris; Thermo Optek, Milano, Italy; Isaac and Johnson, 1998).

2.8. Statistical analysis 2.5. Arbuscular mycorrhizal fungi root colonization Root samples were cleared with 10% KOH, stained with 0.05% trypan blue in lactophenol as described by Phillips and Hayman (1970), and microscopically examined for AM fungi colonization by determining percentage of root segments containing arbuscules + vesicles using the well known gridline intercept method (Giovannetti and Mosse, 1980).

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

3. Results 3.1. Root colonization

2.6. SPAD index and electrolyte leakage determination On May 13 (64 days after transplanting), a chlorophyll meter (SPAD-502, Minolta corporation, Ltd., Osaka, Japan) was used to take readings from the fully expanded leaves. Fifteen leaves per experimental unit were randomly measured and averaged to a single SPAD value for each treatment. Electrolyte leakage (EL) was determined as described by Lutts et al. (1995). Briefly, six randomly chosen plants per treatment were taken and cut into 1-cm segments. Leaf samples were rinsed with distilled water, and then placed in stoppered glass vials containing 10 mL of deionized water. The samples were incubated at room temperature (25 ◦ C) on a shaker for one day. Electrical conductivity of the bathing solution (EC1 ) was measured directly after incubation. The same samples were then placed in an autoclave at 120 ◦ C for 20 min and a second measurement of the EC (EC2 ) was made after cooling the solution. The EL expressed as percentage was calculated as the ratio of EC1 /EC2 . 2.7. Mineral analysis Dried leaf and fruits were ground and 0.5 g of the dried tissues was analysed for following macro and micro nutrients: N, P, K, Ca, Mg, Fe, Cu, Zn, Mn, and B. Nitrogen was determined by the Kjeldahl method (Bremner, 1965) after mineralization with H2 SO4. Phosphorus, K, Ca, Mg, Fe, Cu, Zn, Mn, and B concentrations were determined by dry ashing at 400 ◦ C for 24 h, dissolving the ash in HNO3 1:20 v/v and assaying the solution obtained by an inductively

At the end of the experiment, the AM root colonization was significantly affected by nutrient solution (S; p < 0.05), mycorrhizal treatment (M; p < 0.001), and S × M (p < 0.001) interaction. No AM-fungi colonization was observed in roots of noninoculated plants while in the +AM treatments (Fig. 1), the percentage of AM infection varied significantly among plants grown under different nutrient-solution pH and aluminium concentration. Among nutrient solution treatments, the percentage root colonization was higher in the control treatment (pH 6.0) followed by pH 3.5, and pH 3.5 + Al treatments (Fig. 1).

3.2. Yield and yield components In mycorrhizal (+AM) and nonmycorrhizal (−AM) plants, total and marketable yields decreased sharply in response to pH 3.5 + Al. The lowest marketable production observed on plants treated with aluminium in comparison to low pH and control (pH 6.0) treatments was mainly attributed to a reduction in both the number of zucchini fruits per plant and the mean weight (Table 1). Moreover, with pH 3.5 and pH 3.5 + Al, the percentage of yield reduction in comparison to control was significantly lower in +AM than −AM plants, with the highest yield reduction recorded with pH 3.5 + Al in comparison to those recorded with pH 3.5 treatment (Table 1). Irrespective of nutrient solution pH level and aluminium concentration, the marketable yield was significantly higher by 14.7% in zucchini plants supplied with AM inoculum (avg. 3.9 kg plant−1 ) in comparison to the noninoculated plants (avg. 3.4 kg plant−1 ).

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Table 1 Effects of arbuscular mycorrhizal (AM) inoculation, solution pH and aluminum concentration on total (T), marketable (M), unmarketable (NM) yield, marketable fruit mean weight and fruit number of zucchini plants. Yield (kg plant−1 )

AMa

Solution

−AM +AM Mean −AM +AM Mean −AM +AM Mean

pH 6.0

pH 3.5

pH 3.5 + Al

Significanceb Solution (S) Mycorrhizae (M) S×M

Marketable fruit

T

M

NM

Mean weight (g)

Number (no. plant−1 )

4.74 4.86 4.80 a 4.12 (13) 4.69 (3) 4.41 a 1.99 (58) 2.83 (42) 2.41 b

4.55 4.66 4.61 a 3.90 (14) 4.57 (2) 4.24 a 1.89 (58) 2.60 (44) 2.25 b

0.20 0.20 0.20 0.22 0.12 0.17 0.10 0.23 0.17

210.9 195.5 203.2 a 211.7 218.2 215.0 ab 182.5 190.4 186.5 b

21.5 23.8 22.7 a 19.0 21.1 20.1 b 10.5 13.9 12.2 c

***

***

***

**

ns

ns ns

**

ns

ns ns ns

**

*

ns

Values are the means of four replicate samples. The percentage of reduction in low pH-and aluminium treatments with respect to the control (pH 6) is reported in parenthesis. a +AM, −AM, mycorrhizal and non-mycorrhizal plants, respectively. b ns, * , ** , *** Nonsignificant or significant at p < 0.05, 0.01 or 0.001, respectively.

3.3. Biomass measurements

plants treated with both low pH solution treatments (pH 3.5 and pH 3.5 + Al; Table 2).

Leaves, fruits, roots and total dry weight were significantly affected by mycorrhiza and nutrient solution with no interaction of these two variables, whereas the root-to-shoot ratio was significantly affected by mycorrhiza, nutrient solution and their interaction (Table 2). When averaged over AM treatments, the highest total biomass dry weight was recorded in the pH 6.0 treatment (avg. 616 g plant−1 ), followed by plants treated with pH 3.5 (avg. 521 g plant−1 ), and finally on zucchini plants treated with pH 3.5 + Al (avg. 367 g plant−1 ). The leaf, and fruit dry weight were 36%, and 76% higher with plants grown at the low pH (3.5) and pH 6.0 treatments (avg. 226.8, and 247.1 g per plant, respectively) compared to plants grown at pH 3.5 + Al (avg. 166.5, and 140.2 g per plant, respectively), while the roots dry weight was 78% higher in the control treatment (65.9 g per plant) in comparison to those recorded in low pH-and aluminium treatments (avg. 37.1 g per plant; Table 2). Moreover, when averaged over nutrient solution pH level and aluminium concentration the effect of the AM on leaves, roots, and total dry weight was less marked with an increasing of 16.2%, 20.5%, and 12.5%, respectively, of inoculated (avg. 222.2, 51.0, and 530.6 g per plant, respectively) than noninoculated zucchini plants (avg. 191.2, 42.3, and 471.5 g per plant, respectively; Table 2). The lowest root-to-shoot ratio was recorded in noninoculated

3.4. Fruit quality No significant difference was observed for the fruit juice pH (avg. 6.90), the fruit acidity concentration (avg. 0.051%; Table 3), and the colour parameter a* (greenness) (avg. −9.3; data not shown). The fruit shape index (SI) and the colour parameter L* (lightness) were only influenced by the nutrient solution with the highest values recorded with plants treated with pH 3.5 + Al (0.21, and 46.1, respectively) compared to the pH 3.5 and pH 6.0 treatments (avg. 0.18, and 49.0, respectively). AM enhanced the fruit DM and TSS of zucchini fruits, with the highest values recorded on inoculated (avg. 5.7%, and 5.0 ◦ Brix, respectively) compared to noninoculated plants (avg. 5.2%, and 4.7 ◦ Brix, respectively; Table 3). Finally, the higher b* values were recorded with −AM (avg. 28.7), in comparison to +AM plants (avg. 25.1; data not shown). 3.5. Leaf area, SPAD index and electrolyte leakage When averaged over mycorrhizal treatments, the highest LA was recorded in the control treatment (avg. 3.6 m2 plant−1 ), followed by plants treated with pH 3.5 (avg. 3.2 m2 plant−1 ),

Table 2 Effects of arbuscular mycorrhizal (AM) inoculation, nutrient solution pH and aluminum concentration on biomass production, partitioning and root to shoot ratio of zucchini plants. Solution

AMa

pH 6.0

−AM +AM Mean −AM +AM Mean −AM +AM Mean

pH 3.5

pH 3.5 + Al

Significanceb Solution (S) Mycorrhizae (M) S×M

Dry weight (g plant−1 )

Root to Shoot

Leaves

Stems

Fruits

Roots

Total

222.9 256.0 239.5 a 205.8 222.8 214.3 a 145.1 187.9 166.5 b

42.0 43.7 42.9 a 37.2 41.6 39.4 ab 26.5 31.5 29.0 b

249.4 274.9 262.2 a 203.3 260.4 231.9 a 110.7 169.7 140.2 b

63.8 68.0 65.9 a 36.0 44.3 40.2 b 27.3 40.8 34.1 b

597.0 634.9 616.0 a 495.1 546.1 520.6 b 322.5 411.0 366.8 c

0.25 a 0.25 a 0.25 a 0.15 d 0.17 bc 0.16 b 0.16 cd 0.19 b 0.18 b

***

*

***

***

***

**

**

ns ns

*

*

**

ns

ns

ns

ns

*

ns

Values are the means of four replicate samples. a +AM, −AM, mycorrhizal and non-mycorrhizal plants, respectively. b ns, * , ** , *** Nonsignificant or significant at p < 0.05, 0.01 or 0.001, respectively.

Y. Rouphael et al. / Scientia Horticulturae 188 (2015) 97–105

101

Table 3 Effects of arbuscular mycorrhizal (AM) inoculation, solution pH and aluminum concentration on fruit shape index (SI), dry matter (DM), total soluble solids (TSS) contents, fruit juice pH and titratable acidity (TA) of fruit pulp of zucchini plants. Solution

AMa

SI

DM (%)

TSS (◦ Brix)

pH

TA (%)

pH 6.0

−AM +AM Mean −AM +AM Mean −AM +AM Mean

0.19 0.19 0.19 b 0.19 0.17 0.18 b 0.21 0.22 0.22 a

5.26 5.66 5.46 4.94 5.55 5.25 5.56 6.00 5.78

4.60 5.25 4.93 4.63 4.95 4.79 4.87 5.20 5.04

6.93 6.93 6.93 6.88 6.96 6.92 6.86 6.87 6.87

0.052 0.055 0.054 0.050 0.052 0.051 0.051 0.048 0.050

***

ns

ns

ns ns

**

*

ns

ns

ns ns ns

ns ns ns

pH 3.5

pH 3.5 + Al

Significanceb Solution (S) Mycorrhizae (M) S×M

Values are the means of four replicate samples. a + AM, −AM, mycorrhizal and non-mycorrhizal plants, respectively. b ns, * , *** Nonsignificant or significant at p < 0.05, or 0.001, respectively.

and finally on zucchini plants treated with pH 3.5 + Al (avg. 2.9 m2 plant−1 ; Table 4). Moreover, the SPAD index was significantly higher by 6.2% in both pH 6.0 and pH 3.5 treatments (avg. 49.9) compared with the pH 3.5 + Al treated plants (avg. 47.0; Table 4). When averaged over nutrient solution pH level and aluminium concentration, the LA and SPAD values were significantly higher by 10.0% and 4.6%, respectively in zucchini plants supplied with AM inoculum (avg. 3.3 m2 plant−1 and 50.0, respectively) in comparison to the noninoculated plants (avg. 3.0 m2 plant−1 and 47.8, respectively). The highest electrolyte leakage value was recorded in noninoculated plants treated with the pH 3.5 + Al treatment (Table 4). 3.6. Mineral composition The concentrations of N, Mg, Fe, and Zn in leaf tissue were negatively affected by decreasing pH and the addition of aluminium in the nutrient solution, whereas an opposite trend was observed for the K concentration (Table 5). Decreasing the pH from 6.0 to 3.5 and the addition of aluminium concentration in the nutrient solution significantly decreased the N, Mg, Fe and Zn concentration with the lowest values of macro elements (N and Mg) recorded with pH 3.5 +Al treatment (Table 5). When averaged over nutrient solution pH and aluminium, the concentration of N, K, Mg, Fe, Zn and B in leaves were significantly higher by 8.2%, 7.5%, 12.3%, 19.6%, 6.1% and 19.1%, respectively in +AM zucchini plants (avg. 39.1, 43.4, and

Table 4 Effects of arbuscular mycorrhizal (AM) inoculation, solution pH and aluminum concentration on the final leaf area (LA), SPAD index, and electrolytic leakage (EL) of zucchini plants. Solution

AMa

LA (m2 plant−1 )

SPAD

EL (%)

pH 6.0

−AM +AM Mean −AM +AM Mean −AM +AM Mean

3.5 3.6 3.6 a 3.1 3.3 3.2 b 2.6 3.1 2.9 c

50.2 51.2 50.7 a 48.6 49.6 49.1 a 44.7 49.2 47.0 b

30.3 d 28.8 d 29.6 c 53.8 b 41.5 c 47.7 b 63.8 a 53.5 b 58.7 a

***

**

***

*

*

**

ns

ns

*

pH 3.5

pH 3.5 + Al

Significanceb Solution (S) Mycorrhizae (M) S×M

Values are the means of four replicate samples. a + AM, −AM, mycorrhizal and non-mycorrhizal plants, respectively. b ns, * , ** , *** Nonsignificant or significant at p < 0.05, 0.01 or 0.001, respectively.

8.8 mg g−1 DW and 65.8, 42.7, and 61.7 ␮g g−1 DW, respectively) compared with −AM plants (avg. 36.1, 40.6, and 7.8 mg g−1 DW, and 55.0, 40.3 and 51.8 ␮g g−1 DW, respectively). Moreover, a decrease in P and Ca concentration was observed in both stressed plant treatments (pH 3.5 and pH 3.5 + Al), with the lowest concentrations of P and Ca recorded in noninoculated than inoculated plants (Table 5). The concentrations of N, P, Fe, and Zn in fruit tissue were also negatively influenced by decreasing pH and the addition of aluminium in the nutrient solution, whereas an opposite trend was observed for the K concentration (Table 6). Finally, the concentrations of N, P, and Fe in fruits was significantly higher by 8.2%, 10.4%, and 19.1% with +AM (avg. 42.7, and 10.3 mg g−1 DW, and 33.0 ␮g g−1 DW, respectively) compared with −AM zucchini plants (avg. 39.4, and 9.3 mg g−1 DW, and 27.7 ␮g g−1 DW, respectively; Table 6).

4. Discussion 4.1. Yield, growth and fruit quality responses Plant growth in acidic soils is limited by a number of conditions including the excess of aluminium phytotoxicity, and deficiencies of essential macro- and micronutrients (Seguel et al., 2013). Plants behave to low pH and Al concentration in soil or in substrate solution with decreased growth due to the inhibitory effect of low nutrient solution pH and Al on metabolic processes and/or deterioration of root activity/growth and/or nutrient uptake and assimilation (Rohyadi et al., 2004; Kelly et al., 2005; Seguel et al., 2013). Similarly, in the current experiment, a significant decreasing in total and marketable yields, shoot biomass, and root dry matter (Tables 1 and 2) with pH 3.5 and especially with pH 3.5 + Al-treated zucchini plants was recorded, and that effect varied as a function of AM inoculation. Under low pH and Al conditions, yield, biomass production and root dry weight in comparison to control (pH 6.0) were significantly higher in +AM compared with −AM plants. In addition to their role in nutrient acquisition, AM fungi alter relationships between plants and soils metals such as Al (Rufyikiri et al., 2000; Lux and Cumming, 2001; Cumming and Ning, 2003). AM fungi increased total mineral uptake and accumulation by the plants, mostly correlated with increased plant biomass and consequently yield. Therefore, the enhancement of zucchini crop performance by AM fungi can be attributed to increased macro and micronutrient acquisition, particularly, N, P, K, Ca, Mg, Fe, Zn and B. This bears out the suggestion that some AM fungi strains are capable of tolerating stressful conditions at low pH with the presence of

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Table 5 Effects of arbuscular mycorrhizal (AM) inoculation, solution pH and aluminum concentration on major and trace elements of zucchini leaves. Major elements (mg g−1 DW)

Trace elements (␮g g−1 DW)

Solution

AMa

N

P

K

Ca

Mg

Al

Fe

Cu

Zn

Mn

B

pH 6.0

−AM +AM Mean

40.1 42.0 41.1 a

5.1 a 5.2 a 5.2 a

35.9 40.2 38.1 b

28.7 a 30.0 a 29.4 a

9.6 9.9 9.8 a

61.1 c 59.4 c 60.3 b

68.7 82.7 75.7 a

4.4 4.2 4.3

48.3 51.9 50.1 a

183.9 192.2 188.1 a

58.5 63.6 61.1

pH 3.5

−AM +AM Mean

35.6 38.9 37.3 b

4.1 c 4.6 b 4.4 b

40.9 43.9 42.4 a

24.8 b 30.5 a 27.7 a

8.2 8.8 8.5 b

64.8 c 67.6 c 66.2 b

50.9 66.4 58.7 b

4.4 4.3 4.4

37.0 38.8 37.9 b

152.6 180.0 166.3 ab

47.7 60.3 54.0

pH 3.5 + Al

−AM +AM Mean

32.7 36.4 34.6 c

3.6 d 4.5 b 4.5 b

45.1 47.0 46.1 a

20.7 c 28.2 a 24.5 b

5.8 7.8 6.8 c

289.9 a 139.1 b 214.5 a

45.6 48.5 47.1 b

4.7 4.5 4.6

35.6 37.6 36.6 b

139.2 153.0 146.1 b

49.4 61.3 55.4

**

***

***

**

***

***

***

**

ns

**

*

**

*

***

*

*

*

ns

*

ns

***

ns

ns ns

*

ns

ns ns ns

**

*

Significanceb Solution (S) Mycorrhizae (M) S×M

ns

ns

Values are the means of four replicate samples. a + AM, −AM, mycorrhizal and non-mycorrhizal plants, respectively. b ns, * , ** , *** Nonsignificant or significant at p < 0.05, 0.01 or 0.001, respectively.

Al and supporting host plant growth (Clark, 1997; Clark and Zeto, 2000). The growth improvement by mycorrhiza often includes a decrease in root-to-shoot ratio, with external AM hyphae in soil effectively replacing the roots. The opposite trend was observed in this greenhouse experiment with AM fungi, which significantly increased the ratio at both pH 3.5 and pH 3.5 + Al. Accordingly, in this case the root/shoot ratio was not a useful indicator of AM effectiveness. The effectiveness of AM fungi seemed to be related to increasing growth in terms of root length and surface (Rohyadi et al., 2004). Good development of the root apparatus system is an important aspect for plants growing in acid soils/growing medium and under Al toxicity conditions because it extends accessibility to water and nutrients (Miyasaka and Habte, 2001), especially that the most evident symptoms of Al-induced damage is the fast inhibition of root growth (Seguel et al., 2013). Furthermore, zucchini-fruit quality, in particular fruit dry matter, total soluble-solids content, P, and Fe concentration (Tables 3 and 6), which are particularly important for consumer satisfaction, was improved by mycorrhizal colonization. The increases

in fruit DM and TSS contents observed in the current work agreed with previous results on zucchini squash and cucumber under alkaline conditions (Cardarelli et al., 2010; Rouphael et al., 2010). 4.2. Physiological behaviour The exposure to acidity (low pH) and Al causes several physiological changes at shoot level, as low pH and high level of Al in soil or in growing-medium solution may interferes with photosynthesis and chlorophyll concentration (Marschner, 1995). Since the response to Al depends on the plant’s ability to neutralize the different effects induced by acidity and Al toxicity, the changes induced with AM fungi in the shoot growth should be also observed at the physiological level. This was indeed the case, since the main physiological changes caused by AM inoculation were observed in the leaves of zucchini squash grown under pH 3.5 and pH 3.5 + Al. The highest yield and biomass in +AM plants were related to the capacity of maintaining higher chlorophyll concentration (e.g. SPAD index) in response to acidity and aluminium stress with

Table 6 Effects of arbuscular mycorrhizal (AM) inoculation, solution pH and aluminum concentration on major and trace elements of zucchini fruits. Solution

AMa

Major elements (mg g−1 DW)

Trace elements (␮g g−1 DW)

N

P

K

Ca

Mg

Al

Fe

Cu

Zn

Mn

B

pH 6.0

−AM +AM Mean

43.3 46.0 44.7 a

11.7 11.6 11.7 a

29.8 29.5 29.7 b

1.9 1.9 1.9

2.0 2.2 2.1

5.3 5.4 5.4

32.3 37.4 34.9 a

2.0 1.9 2.0

24.1 23.0 23.6 a

21.0 19.3 20.2 ab

7.4 7.5 7.5

pH 3.5

−AM +AM Mean

39.1 41.7 40.4 b

8.5 9.8 9.2 b

32.8 34.4 33.6 a

1.8 1.6 1.7

1.8 2.0 1.9

5.8 5.6 5.7

26.6 30.8 28.7 b

1.8 1.9 1.9

19.1 18.1 18.6 b

21.4 23.5 22.5 a

6.6 7.4 7.0

pH 3.5 + Al

−AM +AM Mean

36.0 40.5 38.3 b

7.8 9.5 8.7 b

32.3 33.7 33.0 a

1.4 1.5 1.5

1.7 1.8 1.8

6.1 5.9 6.0

24.2 30.8 27.5 b

2.7 2.2 2.5

13.6 17.9 15.8 c

17.0 19.9 18.5 b

7.7 8.7 8.2

**

**

*

ns

ns

ns ns ns

ns ns ns

*

ns ns

ns ns ns

**

*

ns ns ns

**

*

ns ns

ns ns

ns ns ns

Significanceb Solution (S) Mycorrhizae (M) S×M

Values are the means of four replicate samples. a + AM, −AM, mycorrhizal and non-mycorrhizal plants, respectively. b ns, * , ** , *** Nonsignificant or significant at p < 0.05, 0.01 or 0.001, respectively.

*

ns

Y. Rouphael et al. / Scientia Horticulturae 188 (2015) 97–105

respect to −AM plants. It was found that the leaf area and SPAD index increased by 10%, and 5%, respectively, when plants were supplied with AM inoculum (Table 4). This indicates that +AM plants had a larger photosynthetic area per plant in relative terms, which could be attributed to the carbon cost necessary to maintain AM fungi associations (Wright et al., 1998). The inhibitory effect of aluminium on chlorophyll content has been observed earlier in cucumber (Pereira et al., 2010). In addition to reduced SPAD index, leaf area decreased in response to a decrease in the pH level and the addition of Al in the nutrient solution especially in −AM plants. The restriction of leaf area could be related to the suppressed net CO2 assimilation rate, which will consequently reduces the available assimilates for leaf growth (Seguel et al., 2013). The electrolyte leakage method for assessing cell membrane stability has been widely adopted as a benchmark to differentiate stress susceptible and tolerant species/genotypes and in some cases higher membrane stability could be associated with abiotic stress tolerance in vegetable crops (Rouphael et al., 2008; Colla et al., 2010). The current study demonstrated that +AM plants reduced the amount of ion leakage in pH 3.5 and pH 3.5 + Al stressed zucchini plants indicating that AM fungi has facilitated the maintenance of membrane functions (i.e., semipermeability). Calcium increases structural stability of cell membrane because of electrostatic interactions with membrane phospholipids and proteins and of its role as fundamental component of the cell wall (Borer et al., 2005). Aluminium applications in the nutrient solution reduced root calcium uptake leading to a reduction of cell membrane stability of leaf tissues. However, AM fungi were able to mitigate the detrimental effect of Al on membrane stability by improving the Ca uptake in leaf tissues of zucchini plants. 4.3. Nutrient uptake and accumulation It is well known that nutrient uptake by the plants decreases with increasing aluminium stress (Seguel et al., 2013). The uptake and translocation of plant nutrients is important for the maintenance of homeostasis and biomass production of plants under soil stress conditions, and resistance/tolerance to Al is often, demonstrated in limited perturbations to P, Ca, and Mg acquisition as well as the maintenance of these macronutrient in shoot and root tissues (Andrade et al., 2009). There are many research studies on the suppression of macronutrient uptake by roots following short term exposure to Al (Nichol et al., 1993). Despite the extensive research on Al toxicity, it is still unclear how Al affects nutrients and how the changes in mineral nutrients relate to the toxicity and tolerance of plants, due to the variation among plant species/genotypes and growth conditions (pH, Al concentration, treatment period, etc.). In the present study, a significant reduction of macro- (N, P, Ca and Mg) and microelement (Fe, and Zn) contents of leaf tissue of zucchini squash was found with pH 3.5 and pH 3.5 + Al treatments (Table 5). Numerous research studies have demonstrated that Al interferes with the acquisition of cations in plants (Lindberg and Strid, 1997; Lux and Cumming, 1999, 2001). The observed reduction in Ca and Mg of zucchini shoots with respect to Al concentration of the nutrient solution may be due to the competition of Al with the two bivalent cations for common binding sites or near the root surface, and consequently decreased the uptake of Ca2+ and Mg2+ . Exposure to Al results in decreased Mg2+ concentration in leaf tissue (Table 5). This could be attributed to decreased magnesium absorption brought due to the reduction of root growth or to a direct Al inhibition of magnesium uptake (Rengel and Robinson, 1989). Aluminium may also have stimulated Mg efflux from the roots as reported by Silva et al. (2001) for soybean. Rengel and Robinson (1989) concluded that the ability of Lolium multiflorum

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cultivars to retain higher affinity for Mg2+ by transport proteins under Al stress conditions might be one of the mechanisms of Al tolerance. Moreover, Al is a potential inhibitor of Ca2+ uptake, and high level of Al often causes Ca2+ deficiency in plants (Rengel and Elliott, 1992). The decrease of Ca2+ could be attribute to either (a) an exchange reaction of Al with Ca2+ in the cell wall (Reid et al., 1995), or (b) the repression of Ca2+ uptake by the direct blockage of Ca2+ channels (Rengel and Elliott, 1992). A reduction in cytoplasmic Ca2+ in tobacco cell cultures exposed to Al was also reported (Jones et al., 1998) and a Ca2+ deficiency in root tip cells of barley coincided with a heavy accumulation of Al (Ishikawa et al., 2003). In all cases, Al induces a disturbance of Ca2+ homeostasis, resulting in the inhibition of cell growth (Rengel, 1992; Ishikawa et al., 2003). Moreover, in terms of leaf nutrient concentration, inoculation of zucchini plants with AM fungi increased the N, P, K, Ca, Mg, Fe, Zn and B content (Table 5). Numerous studies with a variety of plants and AM symbionts have reported mycorrhizal protection of P acquisition in the presence of Al (Seguel et al., 2013 and references cited therein). Rufyikiri et al. (2000), using Musa acuminata colonized by R. irregularis, observed a beneficial effect of the AM symbiosis under two levels of Al concentration (78 and 180 ␮M), where shoot of mycorrhizal plants was significantly higher than in nonmycorrhizal plants and the contribution of the AM fungi to nutrient uptake (including P) and water, was particularly pronounced. These positive effects were associated with a sharp decrease in Al concentration in shoots and roots and a delay in the appearance of Al-induced leaf symptoms. Moreover, as stated earlier, Ca and Mg limitation are typical Al toxicity symptoms in non-mycorrhizal plants (Foy et al., 1978). The AM symbiosis may change these charge-based interactions inside the roots by the absorption of cations through fungal symbiont hyphae and their transfer to the plants (Ryan et al., 2003, 2007). In addition, Borie and Rubio (1999), Rufyikiri et al. (2000) and Lux and Cumming (2001) observed that AM fungi moderated Al-induced reductions in the bivalent cation concentrations (Ca and/or Mg) in roots and shoots and these changes were often related with decreasing in Al accumulation, which would accompany Al chelation in the plant root and rhizosphere. Results of this study also showed that Al concentration in zucchini leaves was lower in inoculated than noninoculated plants grown under aluminium conditions (Table 5). This indicates that Al might be retained in intraradical AM fungal hyphae, chelated in the rhizosphere, or compartmentalized in the root cell vacuoles, thus reducing translocation to the shoots (Seguel et al., 2013). These are possible mechanisms of plant tolerance to metals such as Al, which foster metal retention in the root and rhizosphere and reduce accumulation in shoots (Chen et al., 2004; Zhang et al., 2005). The suppression of Al uptake and translocation to shoots of mycorrhizal zucchini plants is consistent with most previous reports (Mendoza and Borie, 1998; Cumming and Ning, 2003).

5. Conclusions In conclusion, our results demonstrate that in both inoculated and noninoculated plants, yield, shoot and root biomass decreased in response to low pH and aluminium concentration in the nutrient solution and these were accompanied by a significant reductions of macro- (N, P, Ca and Mg) and microelements (Fe, and Zn) in leaf tissue. AM fungi alleviated the detrimental effect of Al on growth and productivity of zucchini plants. Zucchini-fruit quality, in particular fruit dry matter, total soluble-solids content, P and Fe content was also improved by mycorrhizal colonization. Improved nutritional status (higher N, P, K, Ca, Mg, Fe, Zn, B, and lower Al concentration in leaf tissue), higher SPAD index, and the capacity of maintaining

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