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A practicable method for zinc enrichment in lettuce leaves by the endophyte fungus Piriformospora indica under increasing zinc supply
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A practicable method for zinc enrichment in lettuce leaves by the endophyte fungus Piriformospora indica under increasing zinc supply Akbar Padash a , Saleh Shahabivand b,∗ , Farhad Behtash a , Ahmad Aghaee b a b
Department of horticulture, Faculty of Agriculture, University of Maragheh, Maragheh, Iran Department of Biology, Faculty of Science, University of Maragheh, Maragheh, Iran
a r t i c l e
i n f o
Article history: Received 3 June 2016 Received in revised form 26 October 2016 Accepted 28 October 2016 Available online xxx Keywords: Enrichment Lettuce Piriformospora indica Zinc
a b s t r a c t Zinc (Zn) deficiency is well-documented public health issue and a significant factor hampering to crop production. There is a close overlap between soil deficiency and human deficiency of Zn, indicating a need for increasing Zn content in crops and vegetables. In this study, the effects of endophytic fungus Piriformospora indica and Zn treatment on growth parameters, chlorophyll amounts, and Zn and some other micronutrient concentrations in Lactuca sativa cv. Siyahoo plants were investigated. The experiment was carried out including two treatments (P. indica inoculation and non-inoculation), each having four Zn concentrations (0, 2.5, 5 and 10 mg/l Zn added to full-strength Hoagland solution) in a sand culture substrate. By increasing Zn concentration in sand substrate, growth parameters, chlorophyll content and leaf Zn concentration were significantly increased from 34.1 to 185.1 g/g DW, whereas leaf iron (Fe) concentration was decreased. Significant increase in various parameters including growth, chlorophyll content and leaf Zn concentrations (7.6 fold) were observed in plants inoculated with P. indica under supplemented concentration of 10 mg/l Zn. Results from this study indicate that P. indica inoculation with Zn supplementation can help in Zn enrichment in lettuce plants. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Two-thirds of the world population is at risk of deficiency in one or more essential mineral elements (Stein, 2010) and micronutrient malnutrition has been designated as the most serious challenge to humanity especially in developing countries (Bouis et al., 2011). The mineral elements most commonly lacking in human diets are iron (Fe) and zinc (Zn) (Stein, 2010). Micronutrient zinc is an essential metal for metabolism in plants, animals and humans, and it is as an integral component in over 200 enzymes (Alloway, 2009). This trace element is necessary for development, reproduction and signaling due to its structural, catalytic and activating functions (Roohani et al., 2013). Any sustainable attempt to improve the zinc quality of crops should include a focus on the plant-soil interaction, because Zn deficiency usually appears simultaneously in humans, livestock and crops as a consequence of low Zn concentrations in soil (Lehmann et al., 2014). Reduced Zn bioavailability, due to the low mobility of zinc in soil solution, is a widespread problem espe-
∗ Corresponding author at: University of Maragheh, Madar Square, Golshahr, Maragheh, Iran. E-mail addresses: [email protected], [email protected] (S. Shahabivand).
cially in calcareous soils of arid and semiarid regions (Broadley et al., 2007). Biofortification, the process of enriching the nutrient content of staple crops, provides a sustainable solution to zinc deficiency and its malnutrition in the world (Jeong and Guerinot, 2008). Biofortification involves the fortification of crops at source to accumulate nutritionally important minerals, therefore avoiding the need to fortify processed food products (Prasanna et al., 2016). The large quantities of the chemical fertilizers that are commonly used to obtain high yields in many crops compromise on human and animal health and pollute the environment (Prasanna et al., 2016). The various microorganisms represent a promising input, which possess an array of mechanisms to sequester macroand macronutrients from soil or water, which can be also made available to plants (Nain et al., 2010; Mäder et al., 2011). A practicable tool to improve micronutrient concentrations in crops could be the use of soil microorganisms such as arbuscular mycorrhizal and endophytic fungi. In this case, Mäder et al. (2011) found a substantial increase in Zn and Mn concentrations in wheat through the use of natural arbuscular mycorrhizal fungi consortium. Ortas et al. (2011) also indicated that inoculation with various mycorrhizal species increased Zn shoot concentrations in pepper plants. Root endosymbiotic fungus Piriformospora indica, with similarities to arbuscular mycorrhizal fungi, belongs to new family Sebacinaceae from Hymenomycetes class, Basidiomycota. This fun-
http://dx.doi.org/10.1016/j.scienta.2016.10.040 0304-4238/© 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: Padash, A., et al., A practicable method for zinc enrichment in lettuce leaves by the endophyte fungus Piriformospora indica under increasing zinc supply. Sci. Hortic. (2016), http://dx.doi.org/10.1016/j.scienta.2016.10.040
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gus is easily cultivable, lacks host specificity and interacts with roots of many different plants such as mono- and dicotyledon plants, mostly in an endophytic pattern (Varma et al., 2001). It forms pear-shaped inter- and intracellular chlamydospores within the cortex of the colonized roots and in the rhizosphere zone, but it does not invade the endodermis and the aerial parts of the plants (Varma et al., 2012). Some studies showed that P. indica promoted growth and nutrient uptake, allowed plants to survive under biotic and abiotic stresses, and stimulated early flowering and seed production (Yadav et al., 2010; Das et al., 2012). Lettuce, the main leafy horticultural crop, is a vegetable of great consumption in most countries of the world and can be produced under different temperature ranges, which allows it to be marketed throughout the year (Jordão et al., 2007). Lettuce has a high nutritional value because it is a good source of vitamin A, B6, C and K, and micre- and macronutrients Fe, K, Mn, Ca and Mg. This vegetable is one of the most efficient species in metal absorption (Jordão et al., 2007). It has been reported that the lettuce leaves have enhanced concentration of Cu, Mn, Ni, Pb and Zn with increase in doses of composted urban soil waste (Jordão et al., 2007). The application of microbial biofertilizers needs to be advocated as a possible strategy to increase the nutrient concentrations in edible crops and thus combating malnutrition. There is scarce information about the effects of various microorganisms along with micronutrient treatment on the biofortification of vegetable crops. Therefore, to assess the role of P. indica and Zn application on biofortification of lettuce plants, we investigated the effects of this endophytic fungus and Zn treatment on growth parameters, Zn and some other micronutrient concentrations and chlorophyll content in leaves of Lactuca sativa cv. Siyahoo under greenhouse conditions. 2. Materials and methods 2.1. Experimental design A/4 × 2 factorial randomised block design including two P. indica treatments (with or without fungus inoculation) and 4 Zn levels (1, 2.5, 5 and 10 mg/l Zn added to full-strength Hoagland solution) were tested in five replicates to investigate the effect of inoculation. Thus, there were 8 treatment combinations replicated five times.
avoid salinity. Zn treatment was given as zinc sulfate (ZnSO4 , H2 O). All chemicals and reagents used in this study were of reagent grade and purchased from Sigma-Aldrich Co. and Merck Ltd. 2.3. Plant growth measurements After 35 days of planting, lettuces were harvested by cutting the shoots at the soil surface and the roots were carefully separated from the sandy substrate. The shoots and roots were rinsed with distilled water, wiped with tissue paper and weighted. The shoot and roots were separated, then shoot height and leaf number per plant were determined, and were finally dried at 75 ◦ C for 48 h in order to determine the dry weights and micronutrient concentrations. Some root segments were placed in 50% ethanol in order to determine the root colonization. 2.4. Root staining and measurement of root colonization Root staining to determine the colonization of endophytic fungus followed using the procedure of Phillips and Hayman (1970). The roots of the sampled plants were heated for 5 min in 10% KOH solution and then washed under running tap water thrice. Root samples were acidified with 1% HCl for 1 min and then immersed in 20% trypan blue staining solution and were heated for 10 min. From the stained samples, 30 root segments (1 cm long) per plant were cut and observed with a light microscope (Olympus BH-2) at 20× . Root colonization was determined according to the gridline intersection method described by Giovannetti and Mosse (1980). In this technique, the percentage of root colonization per plant was determined by dividing the total number of colonized root fragments (either with arbuscules, vesicles, or hyphae) by the total number of root pieces examined ×100. 2.5. Micronutrient determination The dried plant samples finely ground (0.1 g) and were digested with a mixture (7:1, v/v) of HNO3 and HClO4 (Zhao et al., 1994). Zn, Fe, Cu and Mn concentrations in digested solutions were determined using an atomic absorption spectrophotometer (Shimadzu, Japan). 2.6. Chlorophyll content determination
2.2. Plant and fungal material and growth conditions Seeds of lettuce (Lactuca sativa cv. Siyahoo) were obtained from Seed and Plant Breeding Institute in Karaj, Iran. Lettuce seeds of cultivar Siyahoo were surface-sterilized with 70% ethanol for 2 min followed by 5 min in NaOCl solution (0.75% Cl), and finally washed six times with sterile water. The seeds were placed on moist filter paper at 4 ◦ C for 2 day in order to synchronize germination. Two days after germination, the seedlings were transferred into pots (two seedlings per each pot) with a diameter of 25 cm and a depth of 40 cm, filled with 10 kg of sterilized 2:1 (v/v) mixture of sand and perlite (sand culture substrate) and then transferred into greenhouse at 22/18 ◦ C day/night cycle, 60–70% relative humidity, and a photoperiod of 14 h. P. indica was cultured in Petri dishes on a Hill & Käfer medium (Hill and Käfer, 2001). The plates were placed in a growth chamber at 29 ± 1 ◦ C in dark for 2 weeks. To impose the fungal treatment, one fungal plug of 10 mm in diameter was placed at a distance of 1 cm below the roots of 10-days-old lettuce seedlings. Non-P. indica treatments received the same weights of autoclaved P. indica inoculum. Plants were continuously irrigated (200 ml daily) with full-strength Hoagland nutrient solution supplemented with Zn concentrations of 0, 2.5, 5 and 10 mg/l to near field capacity. Also, pots received 600 ml of tap water (once every 3 days) in order to
Based on Arnon’s method (1967) the youngest fully expanded leaves were used for chlorophyll a (Chl a) and chlorophyll b (Chl b) analyses. Fresh tissue samples (0.1 g) were homogenized in 80% acetone, centrifuged at 4000 rpm for 20 min. The optical density of the supernatant was read at 663 and 645 nm wavelengths for Chl a and Chl b, respectively. 2.7. Statistical analysis Two-way ANOVA was performed on all experimental data using IBM SPSS version 19 software (Chicago, USA). The variance was related to the main factors (P. indica and Zn supply) and to the interaction between them. The differences between means were determined using Duncan’s Multiple Range Test at 0.05 and 0.01 probability level. 3. Results 3.1. Root colonization and growth parameters The results of this study indicated that by increasing Zn concentration in sand substrate, root colonization was significantly (P < 0.01) decreased and the lowest levels of root colonization was
Please cite this article in press as: Padash, A., et al., A practicable method for zinc enrichment in lettuce leaves by the endophyte fungus Piriformospora indica under increasing zinc supply. Sci. Hortic. (2016), http://dx.doi.org/10.1016/j.scienta.2016.10.040
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Table 1 The effects of P. indica and Zn treatment on root colonization, shoot fresh and dry weights, shoot height and number of leaf in lettuce. Fungal treatment
Zn treatment (mg/l)
Root colonization (%)
Shoot fresh weight (g/plant)
Shoot dry weight (g/plant)
Shoot height (cm)
Leaf number (No/plant)
−P
0 2.5 5 10 0 2.5 5 10
0±0e 0±0e 0±0e 0±0e 61.3 ± 1.45 a 55 ± 1.73 b 46 ± 1.73 c 42 ± 1.52 d
65.6 ± 2.33 c 66.3 ± 3.48 c 71.6 ± 6.0 bc 85.6 ± 2.33 b 85 ± 8.38 b 84.6 ± 8.08 b 161.6 ± 6 a 171 ± 1.52 a
7.6 ± 0.24 d 7.6 ± 0.38 d 8.3 ± 0.63 cd 10 ± 0.28 c 9 ± 0.92 c 9.8 ± 1 c 19.2 ± 0.60 b 26.2 ± 32 a
22 ± 0.57 22.6 ± 1.33 24 ± 0.57 25.3 ± 0.88 25.3 ± 0.33 26 ± 0.57 26.6 ± 1.76 25.6 ± 1.45
11.6 ± 0.88 c 14 ± 0.66 b 14.3 ± 0.88 b 16.3 ± 0.88 b 15.3 ± 0.33 b 17.3 ± 0.33 b 18.3 ± 0.33 b 23.6 ± 0.66 a
0±0 51 ± 2.38
77.3 ± 3 125.7 ± 12.64
8.4 ± 0.34 16.3 ± 0.60
23.5 ± 0.54 b 26 ± 0.52 a
14 ± 0.61 18.6 ± 0.94
30.6 ± 13.72 27.5 ± 12.32 23 ± 10.31 21 ± 9.41
75.3 ± 5.81 75.5 ± 5.68 116.6 ± 20.48 128.6 ± 19.12
8.7 ± 0.65 8.7 ± 0.68 13.8 ± 2.47 18.1± 3.62 1
23.6 ± 0.8 24.3 ± 0.98 25.3 ± 1.02 25.5 ± 0.76
13.5 ± 0.92 15.6 ± 0.88 16.3 ± 0.98 20 ± 1.71
+P
Main effect −P +P Main effect 0 2.5 5 10 P Zn P × Zn
**
**
**
**
**
**
**
**
**
**
**
**
ns ns
**
−P: non-inoculation (control), +P: P. indica. Values are mean ± SE, n = 5. The same letter within each column indicates no significant difference among treatments using Duncan’s Multiple Range Test. ns: not significant. * P < 0.05, ** P < 0.01.
observed under 10 mg/l Zn in sand culture (Table 1). Data from Table 1 showed that in the both colonized and non-colonized plants with P. indica, the growth parameters including shoot fresh weight, shoot dry weight and leaf number per plant were significantly increased by increasing Zn concentration in the sand culture substrate. The maximum and minimum levels of these growth parameters were found in endophyte fungus inoculated lettuces at 10 mg/l Zn and non-inoculated plants at 0 mg/l Zn, respectively. The application of Zn in culture substrate did not influence on shoot height, whereas P. indica inoculation increased shoot height in comparison to control plants (Table 1).
3.2. Chlorophyll a, chlorophyll b and total chlorophyll contents Presence of P. indica and Zn treatment significantly elevated chlorophyll a content (P < 0.05 and P < 0.01, respectively), but had not positive effects on chlorophyll b content in lettuce plants in compare to control (Table 2). In the case of total chlorophyll, a significant increase was observed under 10 mg/l Zn in comparison with other Zn concentrations in culture substrate. Also, maximum level of total chlorophyll was found in P. indica-inoculated plants at 10 mg/l Zn (Table 2).
3.3. Micronutrients analyses P. indica inoculation significantly increased the levels of Zn (by 44%, P < 0.01) and Mn (by 34%, P < 0.01), whereas it had no significant influence on Fe and Cu in leaves of lettuce plants in compare to control (Table 3). By increasing Zn concentrations in culture substrate, the levels of Zn were significantly increased (P < 0.01), but Fe levels were significantly decreased (P < 0.01) in lettuces leaf tissue (Table 3). Also, the minimum level of Cu in leaf tissue (60.4 g/g DW) was shown at the highest level of Zn exposure (10 mg/l Zn). The results from Table 3 showed that highest and lowest levels of Zn in leaves (259.8 and 34.1 g/g DW, respectively) were found under fungal inoculation at 10 mg/l Zn and under non-inoculation condition at 0 mg/l Zn, respectively. Also, the maximum and minimum levels of Fe in leaf tissues were under P. indica-inoculated plants at 0 mg/l Zn and fungus-inoculated plants at 10 mg/l Zn, respectively.
4. Discussion Data from our experiment showed that fungal colonization was significantly reduced with an increase in Zn concentration in sand substrate. We have not found reports of increased or reduced root colonization by P. indica under increasing Zn concentrations in different plants. In the earlier studies, a reduction in mycorrhizal colonization with increased Zn supply was observed in tomato genotypes (Watts-Williams and Cavagnaro, 2012; Watts-Williams et al., 2013). Pawlowska and Charvat (2004) showed that spores of various arbuscular mycorrhizal fungi differed noticeably in their sensitivities to the Cd, Pb and Zn exposures (ranging from 0 to 10 mM), and symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. They concluded that an increase in metal sensitivity in fungi spores may indicate that the cellular system responsible for metal buffering in fungi is saturated with metal ions and subsequent exposure to elevated metal concentrations results in toxicity. This decrease in colonization with increasing Zn concentrations may be due to the negative effects of toxic Zn supply on rates of spore germination and/or hyphal growth. Irrespective of the reduction in colonization by increasing Zn concentration, P. indica formed was still functional. In the present work we have studied the impact of endophytic fungus P. indica on growth parameters including shoot fresh weight, shoot dry weight, shoot height and leaf number per plant in lettuce plants under increasing Zn concentration in sand substrate. One considerable – feature of this fungus is its ability to colonize and benefit a variety of unrelated host plants and this has led to the promotion of P. indica as a putative biofertilizer (Waller et al., 2005). We found that P. indica-colonized lettuces showed an increase in biomass production as compared with non-colonized plants. Similar results have also been found by Kumar et al. (2009) in the interaction between maize and P. indica, Sun et al. (2010) in the symbiosis between P. indica and chinese cabbage, and Shahabivand et al. (2012) in wheat plants under P. indica symbiosis. It has been demonstrated that this endophytic fungus may increase host fitness and competitive abilities by increasing growth rate through evolving biochemical pathways to produce plant growth hormones such as indole-3-acetic acid and cytokinins or enhance the uptake of nutrients especially P and N by the host plant (Shahabivand et al., 2012).
Please cite this article in press as: Padash, A., et al., A practicable method for zinc enrichment in lettuce leaves by the endophyte fungus Piriformospora indica under increasing zinc supply. Sci. Hortic. (2016), http://dx.doi.org/10.1016/j.scienta.2016.10.040
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Table 2 The effects of P. indica and Zn treatment on chlorophyll a, chlorophyll b and total chlorophyll contents in lettuce. Fungal treatment
Zn treatment (mg/l)
Chlorophyll a (mg/g FW)
Chlorophyll b (mg/g FW)
Total chlorophyll (mg/g FW)
−P
0 2.5 5 10 0 2.5 5 10
7.9 ± 1.05 7.4 ± 1.04 8.6 ± 0.7 12 ± 0.83 8.6 ± 0.85 5.5 ± 0.77 10.3 ± 0.4 16 ± 2
8.3 ± 0.64 9.3 ± 1.36 9.2 ± 1.47 7.7 ± 0.72 7.5 ± 1.71 8.1 ± 0.86 7 ± 0.54 10 ± 0.83
16.3 ± 1.34 b 16.7 ± 1 b 18 ± 1.30 b 19.8 ± 1.56 b 16.1 ± 1.1 b 16.6 ± 0.81 b 17.2 ± 0.22 b 26 ± 1.8 a
9 ± 0.68 b 10.8 ± 1.04 a
8.6 ± 0.5 8.1 ± 0.57
17.7 ± 0.7 19 ± 1.32
8.3 ± 0.62 b 8 ± 0.63 b 9.5 ± 0.51 b 14 ± 1.31 a
8 ± 0.84 8.7 ± 0.77 8.1 ± 0.88 8.8 ± 0.70
16.2 ± 0.77 16.6 ± 0.57 17.6 ± 0.62 22.8 ± 1.8
*
ns ns ns
ns
+P
Main effect −P +P Main effect 0 2.5 5 10 P Zn P × Zn
**
ns
** *
−P: non-inoculation (control), +P: P. indica. Values are mean ± SE, n = 5. The same letter within each column indicates no significant difference among treatments using Duncan’s Multiple Range Test. ns: not significant. * P < 0.05. ** P < 0.01. Table 3 The effects of P. indica and Zn treatment on Zn, Fe, Cu and Mn concentrations in leaves of lettuce. Fungal treatment
Zn treatment (mg/l)
Zn concentration (g/g DW)
Fe concentration (g/g DW)
Cu concentration (g/g DW)
Mn concentration (g/g DW)
−P
0 2.5 5 10 0 2.5 5 10
34.1 ± 8.64 f 86.7 ± 6.74 e 97.2 ± 2.3 de 185.1 ± 6.64 b 56.8 ± 3.11 e 122.7 ± 10.42 cd 141.7 ± 10.03 c 259.8 ± 5.65 a
260.5 ± 19.44 bc 268.4 ± 12.8 bc 272 ± 6.57 bc 256 ± 7.56 c 325.3 ± 13 a 290.5 ± 8.61 b 261.08 ± 7.22 bc 223.04 ± 11.54 d
63.1 ± 1.1 65.8 ± 0.75 65.6 ± 1.13 60.8 ± 0.46 64.8 ± 20.6 65 ± 1.47 66.5 ± 0.25 60 ± 1.56
103.1 ± 3.26 102.6 ± 1.33 98.8 ± 10.11 105.3 ± 8.86 135 ± 2.30 148.7 ± 12.17 138.5 ± 13.45 131.03 ± 16.37
100.8 ± 16.58 145.2 ± 22.34
264.2 ± 5.72 275 ± 12.17
63.8 ± 0.72 64.1 ± 5.4
102.5 ± 3.04 b 138.3 ± 5.6 a
45.4 ± 6.53 104.7 ± 9.77 119.4 ± 10.97 222.4 ± 17.16
293 ± 17.86 279.4 ± 8.48 266.5 ± 5 239.5 ± 9.62
63.8 ± 10.15 a 65.4 ± 0.76 a 66 ± 1.36 a 60.4 ± 0.75 b
119.01 ± 7.32 125.7 ± 11.66 118.7 ± 11.64 118.1 ± 10.12
**
ns
ns
**
**
**
**
*
**
ns
ns ns
+P
Main effect −P +P Main effect 0 2.5 5 10 P Zn P × Zn
−P: non-inoculation (control), +P: P. indica. Values are mean ± SE, n = 5. The same letter within each column indicates no significant difference among treatments using Duncan’s Multiple Range Test. ns: not significant. * P < 0.05. ** P < 0.01.
In this study, inoculation of lettuces with P. indica increased chlorophyll a content in comparison with non-inoculated plants. Similar findings have also been observed by Sun et al. (2010) in chinese cabbage leaves and Shahabivand et al. (2012) in wheat. On the other hand, Zn treatment increased chlorophyll a and total chlorophyll contents, and highest level of these parameters were achieved in P. indica-inoculated plants under 10 mg/l Zn. Zn plays a major role in chlorophyll development and function, of which most important are the Zn-dependent activity of SPP peptidase and the repair process of photosystem II by turning over photodamaged D1 protein (Hansch and Mendel, 2009). Zarrouk et al. (2005) indicated a positive correlation of Zn concentrations with chlorophyll content in plants. Abadi and Sepehri (2015) observed that contents of chlorophyll and carotenoid pigments in wheat plants colonized
with P. indica were significantly higher than the control ones under Zn deficiency and Zn sufficiency conditions. They concluded that improved nutritional status of Zn in P. indica-colonised plants can lead to increase in carotenoid and chlorophyll contents and consequently specific stimulation of photosynthetic capacity through stomatal conductance (Abadi and Sepehri, 2015). These results indicate the positive influence of P. indica-plant interaction and Zn application on photosynthetic apparatus of lettuce plants. A number of strategies have been used for enrichment of Zn in edible parts of crops which among them fertilizer management can be mentioned. Judicious application of fertilizer Zn helps increase crop production as well as it helps enrichment of Zn in plant organs including leaves and grains (Sharma et al., 2013). The results from Table 3 revealed that by increasing Zn concentrations in sand sub-
Please cite this article in press as: Padash, A., et al., A practicable method for zinc enrichment in lettuce leaves by the endophyte fungus Piriformospora indica under increasing zinc supply. Sci. Hortic. (2016), http://dx.doi.org/10.1016/j.scienta.2016.10.040
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strate, the levels of Zn were significantly increased in lettuce leaf. Watts-Williams et al. (2013) showed that shoot and root Zn concentrations were significantly higher at high Zn addition treatment than at low Zn addition treatment in genotypes of tomato. The lettuce plants grown in soil amended with vermicompost enriched with Zn showed a 17 times higher leaf Zn concentration compared to those grown in vermicompost (Jordão et al., 2006). In our results, presence of P. indica, a mycorriza-like fungus, significantly increased Zn levels in lettuces leaf. As regards to the low mobility of Zn, Fe, Cu and Mn in the soil, uptake of these metal nutrients by roots is diffusion limited. The higher the density of extraradical hyphae in soil, the higher the absorption surface, the shorter distance these metals have to diffuse, and the more effectively mycorrhizal plants will absorb these low mobility metal nutrients (Liu et al., 2000). The positive role of mycorrhizal fungi inoculation on Zn uptake for several plant species has been addressed by several researchers (Cavagnaro, 2008; Ortas et al., 2011). In wheat seedlings, inoculation of P. indica produced the most levels of shoot Zn under the Zn deficiency and sufficiency conditions to compare with other two microbial treatments (Abadi and Sepehri, 2015). Our experiment clearly demonstrates that P. indica confer a benefit to plants, in terms of leaf Zn concentration, under different levels of Zn (especially at highest level) in sand culture. Enhanced Zn nutrition is thought to be one of the major mechanisms which plant growth promoting microorganisms (PGPMs) such as arbuscular mycorrhizal and endophyte fungi can improve their host plants (Saravanan et al., 2011). Inoculation of lettuce plants by P. indica caused an increase (maximum level) on leaf Fe concentration under 0 mg/l Zn in comparison to non-inoculated plants under 0 mg/l Zn (Table 3). This is in agreement with those of Abadi and Sepehri (2015), who reported that maximum shoot Fe concentration under Zn sufficiency conditions were registered by inoculation of P. indica in wheat. On the other hand, by increasing Zn concentrations in sand culture, Fe content in leaf tissue was considerably decreased. Samreen et al. (2013) observed that Zn application has an adverse effect on Fe contents and Fe uptake in Mungbean plants. This reduction in Fe content may be due to competitive interactions with Zn which probably occur at the absorption sites of plant roots. In grape plants, Fe concentration in shoot exhibited a significant reduction under excess Zn levels (Yang et al., 2011). Also, due to growth promotion that accrued in inoculated plants, the phenomenon could have dilution effects on Fe concentrations, therefore in future studies it is suggested that along with Zn enhancements in presence of P. indica, other micronutrients such as Fe and Mn add at supplementary concentrations. The presence of P. indica significantly elevated leaf Mn concentration whereas, Zn treatment had no significant effect on Mn concentration in the leaves of lettuces compared to control. Mycorrhizal inoculation increased the content of micronutrient Mn compare to non-inoculated ones in lettuce plants (Sanmartín et al., 2014). Gosal et al. (2010) showed that micronutrient acquisition (Cu and Mn) was highly improved by inoculation of P. indica in Chlorophytum sp. plants. In olive plantlets, mycorrhization induced an increase in the percentage of Mn translocated to the leaves (Bati et al., 2015). This increase in leaf Mn content could be related to increased photosynthetic activity in the mycorrhizal plants, since Mn is a constitutive element of the water-splitting system of photosystem II (PSII), which provides the necessary electrons for photosynthesis in the thylakoid membranes (Enami et al., 2008). In conclusion, our results showed that presence of P. indica and Zn treatment increased growth, chlorophyll content and nutrient acquisition especially Zn in leaves of lettuce plants. Because, P. indica unlike mycorrhizal fungi can easily be propagated on large scale in the absence of a host plant, we suggest the consideration
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of this fungus along with Zn application as tools for agronomic (horticultural) biofortification of Zn in leaves of lettuce plants. References Abadi, V.A.J.M., Sepehri, M., 2015. Effect of Piriformospora indica and Azotobacter chroococcum on mitigation of zinc deficiency stress in wheat (Triticum aestivum L.). Symbiosis 69, 9–19. Alloway, B.J., 2009. Soil factors associated with zinc deficiency in crops and humans. Environ. Geochem. Health 31, 537–548. Arnon, A., 1967. Method of extraction of chlorophyll in the plants. Agron. J. 23, 112–121. Bati, C.B., Santilli, E., Lombardo, L., 2015. Effect of arbuscular mycorrhizal fungi on growth and on micronutrient and macronutrient uptake and allocation in olive plantlets growing under high total Mn levels. Mycorrhiza 25, 97–108. Bouis, H.E., Hotz, C., McClafferty, B., et al., 2011. Biofortification: a new tool to reduce micronutrient malnutrition. Food Nutr. Bull. 32 (Suppl. (1)), 31S–40S. 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(Eds.), Biofortification of Food Crops. Springer, India, pp. 249–262. Roohani, N., Hurrell, R., Kelishadi, R., Schulin, R., 2013. Zinc and its importance for human health: an integrative review. J. Res. Med. Sci. 18, 144–157. Samreen, T., Shah, Humaira, Ullah, H.U., Javid, S., 2013. Zinc effect on growth rate, chlorophyll, protein and mineral contents of hydroponically grown mungbeans plant (Vigna radiata). Arab. J. Chem., SPECIAL ISSUE. Sanmartín, C., Garmendia, I., Romano, B., Díaz, M., Palop, J.A., Goicoechea, N., 2014. Mycorrhizal inoculation affected growth, mineral composition, proteins and sugars in lettuces biofortified with organic or inorganic selenocompounds. Sci. Hort. 180, 40–51. Saravanan, V.S., Kumar, M.R., Sa, T.M., 2011. Microbial zinc solubilisation and their role on plants. In: Maheshwari, D.K. (Ed.), Bacteria in Agrobiology: Plant Nutrient Management. Springer, Berlin, pp. 47–63.
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A practicable method for zinc enrichment in lettuce leaves by the endophyte fungus Piriformospora indica under increasing zinc supply Akbar Padash a , Saleh Shahabivand b,∗ , Farhad Behtash a , Ahmad Aghaee b a b
Department of horticulture, Faculty of Agriculture, University of Maragheh, Maragheh, Iran Department of Biology, Faculty of Science, University of Maragheh, Maragheh, Iran
a r t i c l e
i n f o
Article history: Received 3 June 2016 Received in revised form 26 October 2016 Accepted 28 October 2016 Available online xxx Keywords: Enrichment Lettuce Piriformospora indica Zinc
a b s t r a c t Zinc (Zn) deficiency is well-documented public health issue and a significant factor hampering to crop production. There is a close overlap between soil deficiency and human deficiency of Zn, indicating a need for increasing Zn content in crops and vegetables. In this study, the effects of endophytic fungus Piriformospora indica and Zn treatment on growth parameters, chlorophyll amounts, and Zn and some other micronutrient concentrations in Lactuca sativa cv. Siyahoo plants were investigated. The experiment was carried out including two treatments (P. indica inoculation and non-inoculation), each having four Zn concentrations (0, 2.5, 5 and 10 mg/l Zn added to full-strength Hoagland solution) in a sand culture substrate. By increasing Zn concentration in sand substrate, growth parameters, chlorophyll content and leaf Zn concentration were significantly increased from 34.1 to 185.1 g/g DW, whereas leaf iron (Fe) concentration was decreased. Significant increase in various parameters including growth, chlorophyll content and leaf Zn concentrations (7.6 fold) were observed in plants inoculated with P. indica under supplemented concentration of 10 mg/l Zn. Results from this study indicate that P. indica inoculation with Zn supplementation can help in Zn enrichment in lettuce plants. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Two-thirds of the world population is at risk of deficiency in one or more essential mineral elements (Stein, 2010) and micronutrient malnutrition has been designated as the most serious challenge to humanity especially in developing countries (Bouis et al., 2011). The mineral elements most commonly lacking in human diets are iron (Fe) and zinc (Zn) (Stein, 2010). Micronutrient zinc is an essential metal for metabolism in plants, animals and humans, and it is as an integral component in over 200 enzymes (Alloway, 2009). This trace element is necessary for development, reproduction and signaling due to its structural, catalytic and activating functions (Roohani et al., 2013). Any sustainable attempt to improve the zinc quality of crops should include a focus on the plant-soil interaction, because Zn deficiency usually appears simultaneously in humans, livestock and crops as a consequence of low Zn concentrations in soil (Lehmann et al., 2014). Reduced Zn bioavailability, due to the low mobility of zinc in soil solution, is a widespread problem espe-
∗ Corresponding author at: University of Maragheh, Madar Square, Golshahr, Maragheh, Iran. E-mail addresses: [email protected], [email protected] (S. Shahabivand).
cially in calcareous soils of arid and semiarid regions (Broadley et al., 2007). Biofortification, the process of enriching the nutrient content of staple crops, provides a sustainable solution to zinc deficiency and its malnutrition in the world (Jeong and Guerinot, 2008). Biofortification involves the fortification of crops at source to accumulate nutritionally important minerals, therefore avoiding the need to fortify processed food products (Prasanna et al., 2016). The large quantities of the chemical fertilizers that are commonly used to obtain high yields in many crops compromise on human and animal health and pollute the environment (Prasanna et al., 2016). The various microorganisms represent a promising input, which possess an array of mechanisms to sequester macroand macronutrients from soil or water, which can be also made available to plants (Nain et al., 2010; Mäder et al., 2011). A practicable tool to improve micronutrient concentrations in crops could be the use of soil microorganisms such as arbuscular mycorrhizal and endophytic fungi. In this case, Mäder et al. (2011) found a substantial increase in Zn and Mn concentrations in wheat through the use of natural arbuscular mycorrhizal fungi consortium. Ortas et al. (2011) also indicated that inoculation with various mycorrhizal species increased Zn shoot concentrations in pepper plants. Root endosymbiotic fungus Piriformospora indica, with similarities to arbuscular mycorrhizal fungi, belongs to new family Sebacinaceae from Hymenomycetes class, Basidiomycota. This fun-
http://dx.doi.org/10.1016/j.scienta.2016.10.040 0304-4238/© 2016 Elsevier B.V. All rights reserved.
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gus is easily cultivable, lacks host specificity and interacts with roots of many different plants such as mono- and dicotyledon plants, mostly in an endophytic pattern (Varma et al., 2001). It forms pear-shaped inter- and intracellular chlamydospores within the cortex of the colonized roots and in the rhizosphere zone, but it does not invade the endodermis and the aerial parts of the plants (Varma et al., 2012). Some studies showed that P. indica promoted growth and nutrient uptake, allowed plants to survive under biotic and abiotic stresses, and stimulated early flowering and seed production (Yadav et al., 2010; Das et al., 2012). Lettuce, the main leafy horticultural crop, is a vegetable of great consumption in most countries of the world and can be produced under different temperature ranges, which allows it to be marketed throughout the year (Jordão et al., 2007). Lettuce has a high nutritional value because it is a good source of vitamin A, B6, C and K, and micre- and macronutrients Fe, K, Mn, Ca and Mg. This vegetable is one of the most efficient species in metal absorption (Jordão et al., 2007). It has been reported that the lettuce leaves have enhanced concentration of Cu, Mn, Ni, Pb and Zn with increase in doses of composted urban soil waste (Jordão et al., 2007). The application of microbial biofertilizers needs to be advocated as a possible strategy to increase the nutrient concentrations in edible crops and thus combating malnutrition. There is scarce information about the effects of various microorganisms along with micronutrient treatment on the biofortification of vegetable crops. Therefore, to assess the role of P. indica and Zn application on biofortification of lettuce plants, we investigated the effects of this endophytic fungus and Zn treatment on growth parameters, Zn and some other micronutrient concentrations and chlorophyll content in leaves of Lactuca sativa cv. Siyahoo under greenhouse conditions. 2. Materials and methods 2.1. Experimental design A/4 × 2 factorial randomised block design including two P. indica treatments (with or without fungus inoculation) and 4 Zn levels (1, 2.5, 5 and 10 mg/l Zn added to full-strength Hoagland solution) were tested in five replicates to investigate the effect of inoculation. Thus, there were 8 treatment combinations replicated five times.
avoid salinity. Zn treatment was given as zinc sulfate (ZnSO4 , H2 O). All chemicals and reagents used in this study were of reagent grade and purchased from Sigma-Aldrich Co. and Merck Ltd. 2.3. Plant growth measurements After 35 days of planting, lettuces were harvested by cutting the shoots at the soil surface and the roots were carefully separated from the sandy substrate. The shoots and roots were rinsed with distilled water, wiped with tissue paper and weighted. The shoot and roots were separated, then shoot height and leaf number per plant were determined, and were finally dried at 75 ◦ C for 48 h in order to determine the dry weights and micronutrient concentrations. Some root segments were placed in 50% ethanol in order to determine the root colonization. 2.4. Root staining and measurement of root colonization Root staining to determine the colonization of endophytic fungus followed using the procedure of Phillips and Hayman (1970). The roots of the sampled plants were heated for 5 min in 10% KOH solution and then washed under running tap water thrice. Root samples were acidified with 1% HCl for 1 min and then immersed in 20% trypan blue staining solution and were heated for 10 min. From the stained samples, 30 root segments (1 cm long) per plant were cut and observed with a light microscope (Olympus BH-2) at 20× . Root colonization was determined according to the gridline intersection method described by Giovannetti and Mosse (1980). In this technique, the percentage of root colonization per plant was determined by dividing the total number of colonized root fragments (either with arbuscules, vesicles, or hyphae) by the total number of root pieces examined ×100. 2.5. Micronutrient determination The dried plant samples finely ground (0.1 g) and were digested with a mixture (7:1, v/v) of HNO3 and HClO4 (Zhao et al., 1994). Zn, Fe, Cu and Mn concentrations in digested solutions were determined using an atomic absorption spectrophotometer (Shimadzu, Japan). 2.6. Chlorophyll content determination
2.2. Plant and fungal material and growth conditions Seeds of lettuce (Lactuca sativa cv. Siyahoo) were obtained from Seed and Plant Breeding Institute in Karaj, Iran. Lettuce seeds of cultivar Siyahoo were surface-sterilized with 70% ethanol for 2 min followed by 5 min in NaOCl solution (0.75% Cl), and finally washed six times with sterile water. The seeds were placed on moist filter paper at 4 ◦ C for 2 day in order to synchronize germination. Two days after germination, the seedlings were transferred into pots (two seedlings per each pot) with a diameter of 25 cm and a depth of 40 cm, filled with 10 kg of sterilized 2:1 (v/v) mixture of sand and perlite (sand culture substrate) and then transferred into greenhouse at 22/18 ◦ C day/night cycle, 60–70% relative humidity, and a photoperiod of 14 h. P. indica was cultured in Petri dishes on a Hill & Käfer medium (Hill and Käfer, 2001). The plates were placed in a growth chamber at 29 ± 1 ◦ C in dark for 2 weeks. To impose the fungal treatment, one fungal plug of 10 mm in diameter was placed at a distance of 1 cm below the roots of 10-days-old lettuce seedlings. Non-P. indica treatments received the same weights of autoclaved P. indica inoculum. Plants were continuously irrigated (200 ml daily) with full-strength Hoagland nutrient solution supplemented with Zn concentrations of 0, 2.5, 5 and 10 mg/l to near field capacity. Also, pots received 600 ml of tap water (once every 3 days) in order to
Based on Arnon’s method (1967) the youngest fully expanded leaves were used for chlorophyll a (Chl a) and chlorophyll b (Chl b) analyses. Fresh tissue samples (0.1 g) were homogenized in 80% acetone, centrifuged at 4000 rpm for 20 min. The optical density of the supernatant was read at 663 and 645 nm wavelengths for Chl a and Chl b, respectively. 2.7. Statistical analysis Two-way ANOVA was performed on all experimental data using IBM SPSS version 19 software (Chicago, USA). The variance was related to the main factors (P. indica and Zn supply) and to the interaction between them. The differences between means were determined using Duncan’s Multiple Range Test at 0.05 and 0.01 probability level. 3. Results 3.1. Root colonization and growth parameters The results of this study indicated that by increasing Zn concentration in sand substrate, root colonization was significantly (P < 0.01) decreased and the lowest levels of root colonization was
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Table 1 The effects of P. indica and Zn treatment on root colonization, shoot fresh and dry weights, shoot height and number of leaf in lettuce. Fungal treatment
Zn treatment (mg/l)
Root colonization (%)
Shoot fresh weight (g/plant)
Shoot dry weight (g/plant)
Shoot height (cm)
Leaf number (No/plant)
−P
0 2.5 5 10 0 2.5 5 10
0±0e 0±0e 0±0e 0±0e 61.3 ± 1.45 a 55 ± 1.73 b 46 ± 1.73 c 42 ± 1.52 d
65.6 ± 2.33 c 66.3 ± 3.48 c 71.6 ± 6.0 bc 85.6 ± 2.33 b 85 ± 8.38 b 84.6 ± 8.08 b 161.6 ± 6 a 171 ± 1.52 a
7.6 ± 0.24 d 7.6 ± 0.38 d 8.3 ± 0.63 cd 10 ± 0.28 c 9 ± 0.92 c 9.8 ± 1 c 19.2 ± 0.60 b 26.2 ± 32 a
22 ± 0.57 22.6 ± 1.33 24 ± 0.57 25.3 ± 0.88 25.3 ± 0.33 26 ± 0.57 26.6 ± 1.76 25.6 ± 1.45
11.6 ± 0.88 c 14 ± 0.66 b 14.3 ± 0.88 b 16.3 ± 0.88 b 15.3 ± 0.33 b 17.3 ± 0.33 b 18.3 ± 0.33 b 23.6 ± 0.66 a
0±0 51 ± 2.38
77.3 ± 3 125.7 ± 12.64
8.4 ± 0.34 16.3 ± 0.60
23.5 ± 0.54 b 26 ± 0.52 a
14 ± 0.61 18.6 ± 0.94
30.6 ± 13.72 27.5 ± 12.32 23 ± 10.31 21 ± 9.41
75.3 ± 5.81 75.5 ± 5.68 116.6 ± 20.48 128.6 ± 19.12
8.7 ± 0.65 8.7 ± 0.68 13.8 ± 2.47 18.1± 3.62 1
23.6 ± 0.8 24.3 ± 0.98 25.3 ± 1.02 25.5 ± 0.76
13.5 ± 0.92 15.6 ± 0.88 16.3 ± 0.98 20 ± 1.71
+P
Main effect −P +P Main effect 0 2.5 5 10 P Zn P × Zn
**
**
**
**
**
**
**
**
**
**
**
**
ns ns
**
−P: non-inoculation (control), +P: P. indica. Values are mean ± SE, n = 5. The same letter within each column indicates no significant difference among treatments using Duncan’s Multiple Range Test. ns: not significant. * P < 0.05, ** P < 0.01.
observed under 10 mg/l Zn in sand culture (Table 1). Data from Table 1 showed that in the both colonized and non-colonized plants with P. indica, the growth parameters including shoot fresh weight, shoot dry weight and leaf number per plant were significantly increased by increasing Zn concentration in the sand culture substrate. The maximum and minimum levels of these growth parameters were found in endophyte fungus inoculated lettuces at 10 mg/l Zn and non-inoculated plants at 0 mg/l Zn, respectively. The application of Zn in culture substrate did not influence on shoot height, whereas P. indica inoculation increased shoot height in comparison to control plants (Table 1).
3.2. Chlorophyll a, chlorophyll b and total chlorophyll contents Presence of P. indica and Zn treatment significantly elevated chlorophyll a content (P < 0.05 and P < 0.01, respectively), but had not positive effects on chlorophyll b content in lettuce plants in compare to control (Table 2). In the case of total chlorophyll, a significant increase was observed under 10 mg/l Zn in comparison with other Zn concentrations in culture substrate. Also, maximum level of total chlorophyll was found in P. indica-inoculated plants at 10 mg/l Zn (Table 2).
3.3. Micronutrients analyses P. indica inoculation significantly increased the levels of Zn (by 44%, P < 0.01) and Mn (by 34%, P < 0.01), whereas it had no significant influence on Fe and Cu in leaves of lettuce plants in compare to control (Table 3). By increasing Zn concentrations in culture substrate, the levels of Zn were significantly increased (P < 0.01), but Fe levels were significantly decreased (P < 0.01) in lettuces leaf tissue (Table 3). Also, the minimum level of Cu in leaf tissue (60.4 g/g DW) was shown at the highest level of Zn exposure (10 mg/l Zn). The results from Table 3 showed that highest and lowest levels of Zn in leaves (259.8 and 34.1 g/g DW, respectively) were found under fungal inoculation at 10 mg/l Zn and under non-inoculation condition at 0 mg/l Zn, respectively. Also, the maximum and minimum levels of Fe in leaf tissues were under P. indica-inoculated plants at 0 mg/l Zn and fungus-inoculated plants at 10 mg/l Zn, respectively.
4. Discussion Data from our experiment showed that fungal colonization was significantly reduced with an increase in Zn concentration in sand substrate. We have not found reports of increased or reduced root colonization by P. indica under increasing Zn concentrations in different plants. In the earlier studies, a reduction in mycorrhizal colonization with increased Zn supply was observed in tomato genotypes (Watts-Williams and Cavagnaro, 2012; Watts-Williams et al., 2013). Pawlowska and Charvat (2004) showed that spores of various arbuscular mycorrhizal fungi differed noticeably in their sensitivities to the Cd, Pb and Zn exposures (ranging from 0 to 10 mM), and symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. They concluded that an increase in metal sensitivity in fungi spores may indicate that the cellular system responsible for metal buffering in fungi is saturated with metal ions and subsequent exposure to elevated metal concentrations results in toxicity. This decrease in colonization with increasing Zn concentrations may be due to the negative effects of toxic Zn supply on rates of spore germination and/or hyphal growth. Irrespective of the reduction in colonization by increasing Zn concentration, P. indica formed was still functional. In the present work we have studied the impact of endophytic fungus P. indica on growth parameters including shoot fresh weight, shoot dry weight, shoot height and leaf number per plant in lettuce plants under increasing Zn concentration in sand substrate. One considerable – feature of this fungus is its ability to colonize and benefit a variety of unrelated host plants and this has led to the promotion of P. indica as a putative biofertilizer (Waller et al., 2005). We found that P. indica-colonized lettuces showed an increase in biomass production as compared with non-colonized plants. Similar results have also been found by Kumar et al. (2009) in the interaction between maize and P. indica, Sun et al. (2010) in the symbiosis between P. indica and chinese cabbage, and Shahabivand et al. (2012) in wheat plants under P. indica symbiosis. It has been demonstrated that this endophytic fungus may increase host fitness and competitive abilities by increasing growth rate through evolving biochemical pathways to produce plant growth hormones such as indole-3-acetic acid and cytokinins or enhance the uptake of nutrients especially P and N by the host plant (Shahabivand et al., 2012).
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Table 2 The effects of P. indica and Zn treatment on chlorophyll a, chlorophyll b and total chlorophyll contents in lettuce. Fungal treatment
Zn treatment (mg/l)
Chlorophyll a (mg/g FW)
Chlorophyll b (mg/g FW)
Total chlorophyll (mg/g FW)
−P
0 2.5 5 10 0 2.5 5 10
7.9 ± 1.05 7.4 ± 1.04 8.6 ± 0.7 12 ± 0.83 8.6 ± 0.85 5.5 ± 0.77 10.3 ± 0.4 16 ± 2
8.3 ± 0.64 9.3 ± 1.36 9.2 ± 1.47 7.7 ± 0.72 7.5 ± 1.71 8.1 ± 0.86 7 ± 0.54 10 ± 0.83
16.3 ± 1.34 b 16.7 ± 1 b 18 ± 1.30 b 19.8 ± 1.56 b 16.1 ± 1.1 b 16.6 ± 0.81 b 17.2 ± 0.22 b 26 ± 1.8 a
9 ± 0.68 b 10.8 ± 1.04 a
8.6 ± 0.5 8.1 ± 0.57
17.7 ± 0.7 19 ± 1.32
8.3 ± 0.62 b 8 ± 0.63 b 9.5 ± 0.51 b 14 ± 1.31 a
8 ± 0.84 8.7 ± 0.77 8.1 ± 0.88 8.8 ± 0.70
16.2 ± 0.77 16.6 ± 0.57 17.6 ± 0.62 22.8 ± 1.8
*
ns ns ns
ns
+P
Main effect −P +P Main effect 0 2.5 5 10 P Zn P × Zn
**
ns
** *
−P: non-inoculation (control), +P: P. indica. Values are mean ± SE, n = 5. The same letter within each column indicates no significant difference among treatments using Duncan’s Multiple Range Test. ns: not significant. * P < 0.05. ** P < 0.01. Table 3 The effects of P. indica and Zn treatment on Zn, Fe, Cu and Mn concentrations in leaves of lettuce. Fungal treatment
Zn treatment (mg/l)
Zn concentration (g/g DW)
Fe concentration (g/g DW)
Cu concentration (g/g DW)
Mn concentration (g/g DW)
−P
0 2.5 5 10 0 2.5 5 10
34.1 ± 8.64 f 86.7 ± 6.74 e 97.2 ± 2.3 de 185.1 ± 6.64 b 56.8 ± 3.11 e 122.7 ± 10.42 cd 141.7 ± 10.03 c 259.8 ± 5.65 a
260.5 ± 19.44 bc 268.4 ± 12.8 bc 272 ± 6.57 bc 256 ± 7.56 c 325.3 ± 13 a 290.5 ± 8.61 b 261.08 ± 7.22 bc 223.04 ± 11.54 d
63.1 ± 1.1 65.8 ± 0.75 65.6 ± 1.13 60.8 ± 0.46 64.8 ± 20.6 65 ± 1.47 66.5 ± 0.25 60 ± 1.56
103.1 ± 3.26 102.6 ± 1.33 98.8 ± 10.11 105.3 ± 8.86 135 ± 2.30 148.7 ± 12.17 138.5 ± 13.45 131.03 ± 16.37
100.8 ± 16.58 145.2 ± 22.34
264.2 ± 5.72 275 ± 12.17
63.8 ± 0.72 64.1 ± 5.4
102.5 ± 3.04 b 138.3 ± 5.6 a
45.4 ± 6.53 104.7 ± 9.77 119.4 ± 10.97 222.4 ± 17.16
293 ± 17.86 279.4 ± 8.48 266.5 ± 5 239.5 ± 9.62
63.8 ± 10.15 a 65.4 ± 0.76 a 66 ± 1.36 a 60.4 ± 0.75 b
119.01 ± 7.32 125.7 ± 11.66 118.7 ± 11.64 118.1 ± 10.12
**
ns
ns
**
**
**
**
*
**
ns
ns ns
+P
Main effect −P +P Main effect 0 2.5 5 10 P Zn P × Zn
−P: non-inoculation (control), +P: P. indica. Values are mean ± SE, n = 5. The same letter within each column indicates no significant difference among treatments using Duncan’s Multiple Range Test. ns: not significant. * P < 0.05. ** P < 0.01.
In this study, inoculation of lettuces with P. indica increased chlorophyll a content in comparison with non-inoculated plants. Similar findings have also been observed by Sun et al. (2010) in chinese cabbage leaves and Shahabivand et al. (2012) in wheat. On the other hand, Zn treatment increased chlorophyll a and total chlorophyll contents, and highest level of these parameters were achieved in P. indica-inoculated plants under 10 mg/l Zn. Zn plays a major role in chlorophyll development and function, of which most important are the Zn-dependent activity of SPP peptidase and the repair process of photosystem II by turning over photodamaged D1 protein (Hansch and Mendel, 2009). Zarrouk et al. (2005) indicated a positive correlation of Zn concentrations with chlorophyll content in plants. Abadi and Sepehri (2015) observed that contents of chlorophyll and carotenoid pigments in wheat plants colonized
with P. indica were significantly higher than the control ones under Zn deficiency and Zn sufficiency conditions. They concluded that improved nutritional status of Zn in P. indica-colonised plants can lead to increase in carotenoid and chlorophyll contents and consequently specific stimulation of photosynthetic capacity through stomatal conductance (Abadi and Sepehri, 2015). These results indicate the positive influence of P. indica-plant interaction and Zn application on photosynthetic apparatus of lettuce plants. A number of strategies have been used for enrichment of Zn in edible parts of crops which among them fertilizer management can be mentioned. Judicious application of fertilizer Zn helps increase crop production as well as it helps enrichment of Zn in plant organs including leaves and grains (Sharma et al., 2013). The results from Table 3 revealed that by increasing Zn concentrations in sand sub-
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strate, the levels of Zn were significantly increased in lettuce leaf. Watts-Williams et al. (2013) showed that shoot and root Zn concentrations were significantly higher at high Zn addition treatment than at low Zn addition treatment in genotypes of tomato. The lettuce plants grown in soil amended with vermicompost enriched with Zn showed a 17 times higher leaf Zn concentration compared to those grown in vermicompost (Jordão et al., 2006). In our results, presence of P. indica, a mycorriza-like fungus, significantly increased Zn levels in lettuces leaf. As regards to the low mobility of Zn, Fe, Cu and Mn in the soil, uptake of these metal nutrients by roots is diffusion limited. The higher the density of extraradical hyphae in soil, the higher the absorption surface, the shorter distance these metals have to diffuse, and the more effectively mycorrhizal plants will absorb these low mobility metal nutrients (Liu et al., 2000). The positive role of mycorrhizal fungi inoculation on Zn uptake for several plant species has been addressed by several researchers (Cavagnaro, 2008; Ortas et al., 2011). In wheat seedlings, inoculation of P. indica produced the most levels of shoot Zn under the Zn deficiency and sufficiency conditions to compare with other two microbial treatments (Abadi and Sepehri, 2015). Our experiment clearly demonstrates that P. indica confer a benefit to plants, in terms of leaf Zn concentration, under different levels of Zn (especially at highest level) in sand culture. Enhanced Zn nutrition is thought to be one of the major mechanisms which plant growth promoting microorganisms (PGPMs) such as arbuscular mycorrhizal and endophyte fungi can improve their host plants (Saravanan et al., 2011). Inoculation of lettuce plants by P. indica caused an increase (maximum level) on leaf Fe concentration under 0 mg/l Zn in comparison to non-inoculated plants under 0 mg/l Zn (Table 3). This is in agreement with those of Abadi and Sepehri (2015), who reported that maximum shoot Fe concentration under Zn sufficiency conditions were registered by inoculation of P. indica in wheat. On the other hand, by increasing Zn concentrations in sand culture, Fe content in leaf tissue was considerably decreased. Samreen et al. (2013) observed that Zn application has an adverse effect on Fe contents and Fe uptake in Mungbean plants. This reduction in Fe content may be due to competitive interactions with Zn which probably occur at the absorption sites of plant roots. In grape plants, Fe concentration in shoot exhibited a significant reduction under excess Zn levels (Yang et al., 2011). Also, due to growth promotion that accrued in inoculated plants, the phenomenon could have dilution effects on Fe concentrations, therefore in future studies it is suggested that along with Zn enhancements in presence of P. indica, other micronutrients such as Fe and Mn add at supplementary concentrations. The presence of P. indica significantly elevated leaf Mn concentration whereas, Zn treatment had no significant effect on Mn concentration in the leaves of lettuces compared to control. Mycorrhizal inoculation increased the content of micronutrient Mn compare to non-inoculated ones in lettuce plants (Sanmartín et al., 2014). Gosal et al. (2010) showed that micronutrient acquisition (Cu and Mn) was highly improved by inoculation of P. indica in Chlorophytum sp. plants. In olive plantlets, mycorrhization induced an increase in the percentage of Mn translocated to the leaves (Bati et al., 2015). This increase in leaf Mn content could be related to increased photosynthetic activity in the mycorrhizal plants, since Mn is a constitutive element of the water-splitting system of photosystem II (PSII), which provides the necessary electrons for photosynthesis in the thylakoid membranes (Enami et al., 2008). In conclusion, our results showed that presence of P. indica and Zn treatment increased growth, chlorophyll content and nutrient acquisition especially Zn in leaves of lettuce plants. Because, P. indica unlike mycorrhizal fungi can easily be propagated on large scale in the absence of a host plant, we suggest the consideration
5
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Please cite this article in press as: Padash, A., et al., A practicable method for zinc enrichment in lettuce leaves by the endophyte fungus Piriformospora indica under increasing zinc supply. Sci. Hortic. (2016), http://dx.doi.org/10.1016/j.scienta.2016.10.040