Effectiveness of Amaranthus retroflexus L. aqueous extract in preventing iron chlorosis of pear trees (Pyrus communis L.)

Abstract

The aim of this study was to evaluate the effectiveness of soil-applied aqueous extract of Amaranthus retroflexus L. in preventing lime-induced iron (Fe) chlorosis of pear trees (Pyrus communis L.). Tree growth, nutritional status, yield and fruit quality were also assessed. The aqueous extract was obtained by soaking dried and ground canopy (epigeal part) of spontaneous A. retroflexus plants in tap water. A. retroflexus extract was chosen because of its ability to solubilize Fe from calcareous soil, which was found to be 100-fold higher than deionized water alone. Two experiments were carried out (controlled environment and commercial field conditions) where soil-applied aqueous extract of A. retroflexus alone or mixed with iron sulfate (FeSO₄) was compared with synthetic Fe-chelate and an untreated control. Soil-applied aqueous extract of A. retroflexus increased shoot length, leaf SPAD and total plant biomass in controlled environment. In the commercial orchard control trees showed severe leaf Fe-chlorosis symptoms effectively prevented by Fe-chelate. The supply of A. retroflexus aqueous extract improved Fe nutrition of trees, particularly when enriched with FeSO₄. Fe-chelate increased tree yield but decreased fruit weight, leaf potassium (K) and manganese (Mn) concentration. At harvest, all strategies raised fruit soluble solid concentration compared to the untreated control. Results showed that soil-applied A. retroflexus aqueous extract improved Fe nutritional status of pear trees, probably because of the natural Fe chelating capacity of the compounds released by its tissues.

Keywords:

• C. oblonga
• Fe-deficiency
• pigweed
• soil extractable Fe
• vegetal extract.

Introduction

Lime-induced iron (Fe) chlorosis represents the most important nutritional disorder of susceptible fruit crops especially when cultivated on alkaline-calcareous soils (Rombolà and Tagliavini 2006) which represent approximately 39% of world soils (Çelik and Katkat 2010). Fe chlorosis in crops occurs mainly as a consequence of a scarce solubility of mineral Fe sources in the soil (Römheld and Nikolic 2007) and of a reduced Fe uptake by the symplast, induced by the soil active lime fraction (Tagliavini and Rombolà 2001). Fe-deficiency decreases leaf photosynthetic pigment concentrations, especially chlorophyll (Chl) (Abadía and Abadía 1993). Likewise, light absorption, photosystem II and Rubisco carboxylation efficiencies are negatively affected in Fe-deficient plants (Larbi et al. 2006). Furthermore, recent findings pointed out physiological alterations of leaf morphology, stomatal control, xylem vessel morphology, leaf hydraulic conductance and leaf water potentials of chlorotic peach leaves, indicating that Fe deficiency implies pronounced disturbances in leaf water relations (Fernández et al. 2008; Eichert et al. 2010). Fe chlorosis depresses yield and fruit quality, decreases tree vigor and shortens orchard productive lifetime (see Tagliavini and Rombolà 2001; Álvarez-Fernández et al. 2006 for reviews).

In alkaline-calcareous soils, when quince (Cydonia oblonga Mill.) is adopted as rootstock, pear is one of the species most susceptible to Fe chlorosis (Rombolà and Tagliavini 2006). Although synthetic Fe-containing compounds such as Fe-chelate (e.g., Fe-EDDHA) are usually effective in overcoming Fe-deficiency disorder (Lucena 2003, 2006), they induce a short-lasting re-greening effect, are expensive (Tagliavini and Rombolà 2001) and represent a risk of water table contamination. In fact, soil-applied Fe-chelates are scarcely degradable (Nörtemann 1999), easily leached out of the root zone (Rombolà et al. 2002) and may induce toxic effects in soil microorganisms (i.e., mycorrhiza) (Grčman et al. 2001). Current agriculture requires sustainable and cost-effective alternative strategies to overcome lime-induced Fe chlorosis on susceptible crops, but no single sustainable approach has been found, making it one of the most complex nutritional deficiencies of fruit tree crops (Pestana et al. 2003).

Preliminary reports indicate that soil-applied aqueous extracts of some herbaceous species (e.g., Urtica dioica L., Amaranthus retroflexus L. ) may prevent Fe chlorosis symptoms in potted pear trees (Rombolà et al. 2001) also improving, at low concentrations, the nutritional status and vegetative growth (Sorrenti et al. 2011). In addition, Amaranthus spp. (commonly pigweed) residues mixed with exogenous iron sulphate (FeSO₄) were more effective than Fe-EDDHA in reducing Fe chlorosis and increasing plant dry matter and grain yields in sorghum plants grown on severely Fe-deficient soil (Matocha and Pennington 1982; Matocha 1984; Mostaghimi and Matocha 1988).

The aim of this study was to test the hypothesis that soil-applied aqueous extract of A. retroflexus can be effective in preventing Fe chlorosis of pear trees. The effect of the treatments on tree growth, nutritional status, yield and fruit quality were also evaluated. To achieve our goals two experiments were set including controlled and field environment growth conditions under field conditions. Moreover, a preliminary lab test was carried out to evaluate the ability of A. retroflexus aqueous extract as a natural soil Fe eluent.

Materials and Methods

Soil solution extractable Fe

Samples of an alkaline-calcareous soil, where no Fe sources were applied in the previous years, were collected (5–30 cm depth), oven dried (105°C) and ground (1-mm mesh). Soil Fe concentration was measured after extraction with deionized water, aqueous extract of A. retroflexus (in deionized water) and diethylenetriamine-pentaacetic-acid (DTPA) (Lindsay and Norvell 1978) as eluents (5 replicates each). The aqueous extract of A. retroflexus was prepared at a rate of 3.6 g dry weight (dw) L⁻¹, whereas the DTPA solution was obtained by mixing 0.005 M DTPA, 0.01 M calcium chloride (CaCl₂) 0.1 M and triethanolamine, then the pH was adjusted to 7.3 ± 0.05 by 0.5 M hydrochloric acid (HCl). Sixty mL of each eluent were added to 30 g dw of soil, shaken for 4 h, then filtrated. The Fe concentration in the soil solution was determined by AAS (Varian AA200, Mulgrave, Victoria, Australia).

Controlled environment experiment

A 2-year experiment (2007–08) was performed outdoors at the experimental station of the University of Bologna (in Cadriano, Bologna, 44°33′ N, 11°25′ E) on 1-year old micropropagated pear (Pyrus communis L.) trees cv. Abbé Fétel grafted on quince (C. oblonga Mill.) Sydo grown in 33-L pots in a heavy alkaline-calcareous soil (75% of total lime) mixed with sand at a ratio of 2:1, respectively. Trees were set in a trench and covered with shade netting while the pots were protected with a reflective radiant barrier insulating film to prevent solar overheating. Trees were trained as in 3 shoots per plant and in summer watered daily, by microirrigation, with tap water to return the evapo-transpirated rate.

Canopies (the whole aerial part) of wild A. retroflexus plants were collected from different fields in the vicinity of our Research Station. The biomass was dried (65°C), ground (0.2-mm mesh) then analyzed (4 replicates) for Fe, nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), manganese (Mn), zinc (Zn) and copper (Cu) concentration. According to US EPA Method 3052 (Kingston 1988), 0.5 g of dry matter were mineralized by 8 mL of nitric acid (65%) and 2 mL of hydrogen peroxide (30%) at 180°C in an Ethos TC microwave labstation (Milestone, Bergamo, Italy). Mineral concentrations were determined by atomic absorption spectrophotometry (AAS) (Varian AA200, Mulgrave, Victoria, Australia). Prior to K, Ca and Mg readings, lanthanum chloride (LaCl₃ at 10%) and cesium chloride (CsCl at 5%) were added to the sample at ratios of 20% and 4%, respectively. Total N concentration was determined by the Kjeldahl method (Schuman et al. 1973), by mineralization of 1 g of sample with 12 mL of a 95:5 (v:v) H₂SO₄:H₃PO₃ mixture, at 420°C, for 180 min and subsequent distillation with 32% (v:v) NaOH and titration with 0.2 M HCl. Phosphorus was spectrophotometrically quantified at 700 nm, through extract mineralization (Saunders and Williams 1955) of 0.5 g of the sample with 96% (v:v) sulphuric acid and 35% (v:v) oxygen peroxide, and subsequent neutralization with 0.1 M NaOH enriched with 0.1 M ascorbic acid, 32 mM ammonium molybdate, 2.5 M sulphuric acid and 3 mM potassium antimonyl tartrate to develop a phospho-molybdic blue color.

In a completely randomized block design, with 5 replicates (single trees) the following soil-applied treatments were compared: (a) commercial Fe-chelate (4.16 g L⁻¹ of commercial Fe-EDDHA with a Fe content of 5.5%); (b) Iron (II) sulphate heptahydrate (FeSO₄.7H₂O) (543 mg L⁻¹); (c) aqueous extract of A. retroflexus at a rate of 15 g (dw)L⁻¹ (LR); (d) aqueous extract of A. retroflexus at a rate of 30 g (dw)L⁻¹ (HR); (e) aqueous extract of A. retroflexus (HR) mixed with FeSO₄.7H₂O (543 mgL⁻¹). The aqueous extract was prepared by macerating in tap water (pH 7.4; Fe < 0.08 mgL⁻¹) the powder of A. retroflexus at least for 24 h before its application, maintaining the suspension at room temperature and in the dark. Each treatment was applied at weekly intervals starting from bud burst at a rate of 200 mL plant⁻¹, 10 and 6 times in the first and second year, respectively. Untreated control trees, included in the experimental design, received 200 mL plant⁻¹ of tap water coincident with the other applications.

Tree Fe chlorosis incidence was monitored by an estimation of the leaf Chl concentration made by a hand-held Chl meter (SPAD 502, Minolta Co. LTD, Osaka, Japan). Shoot length and leaf Chl (measured on 3 apical expanded leaves per shoot) were recorded monthly. At the end of the second season 20 random fully expanded leaves per tree were collected and used to determine leaf area with a portable area meter (LI 3000, Li-Cor Inc., Lincoln, Nebraska, USA). Petioles were removed, then leaf laminas were washed in a 0.1 N HCl solution supplemented with a surfactant (Tween 20) at a rate of 1 mL L⁻¹ as indicated by Álvarez-Fernàndez et al. (2001), rinsed three times in deionized water, oven-dried (65°C), and milled (0.2-mm mesh). Macro- (N, P, K, Ca, Mg) and micro- (Mn, Fe, Cu, Zn) nutrient concentration was determined as previously described for A. retroflexus tissues. Specific leaf weight (SLW) was obtained by dividing leaf dry weight by leaf area. Thereafter, 4 trees per treatment were harvested and divided into fine and coarse roots, rootstock, stem, shoots and leaves. The different organs were oven-dried and weighed. From each pot, soil samples were collected at the end of the experiment and total macro- and micronutrient concentration was determined as described.

Field conditions experiment

A 2-year (2009–10) field trial was carried out in a mature commercial orchard of pear (Pyrus communis L.) cv. Abbé Fétel grafted on quince (C. oblonga Mill.) BA 29 planted in 1996 with a frame of 3.5 × 1.3 m (2.198 trees ha⁻¹) and pollinated by trees of the cv. Bartlett. The orchard was located in the South-Eastern Po Valley (in Massalombarda, Ravenna, 44°46′ N, 11°83′ E; 13 m above sea level) on a clay soil, classified as Sant’Omobono 2 (Inceptisols, Udifluventic Haplustepts) according to regional criteria based on Soil Taxonomy (USDA 2010), characterized by a sub-alkaline pH (7.7), low content of organic matter (1.3 g kg⁻¹), moderate-high content of active lime (7.4%) and low DTPA Fe (9 mg kg⁻¹) concentration. The climate of the area is classified as temperate sub continental with cold winter and humid warm summer. During the experiment, the air temperatures were on average 25.3°C in summer and 3.4°C in winter, while annual precipitation ranged between 600 and 780 mm, mainly concentrated in the spring and autumn.

Trees were trained as in a palmette system, drip-irrigated with the alleys maintained with spontaneous grass species whereas the tree rows were herbicided (glufosinate ammonium) twice per year. Trees were not thinned and were managed in terms of nutrition, pruning, irrigation as well as pest and disease following the regional advisories for Integrated Crop Management (ICM 2009). Trees did not receive exogenous Fe input for at least 5 years prior the beginning of our trial, and leaf Fe-deficiency symptoms were often observed. In a completely randomized block design with 5 replicates of 5 trees each, arranged in 2 tree rows, the following soil-applied treatments were compared: a) untreated control; b) Fe-chelate (30 g tree⁻¹*year of commercial Fe-EDDHA with a Fe content of 5.5% in the ortho-ortho isomer) split in 2 applications and supplied by fertigation from bud burst; c) aqueous extract of A. retroflexus at a rate of 3.6 g (dw) L⁻¹ and d) aqueous extract of A. retroflexus at a rate of 3.6 g L⁻¹ enriched with FeSO₄ · 7H₂O at a rate of 0.54 g L⁻¹. From bud burst, the aqueous extract of A. retroflexus was applied biweekly 5 times per year as a water suspension (5 L plant⁻¹). In each experimental plot, only the central 3 trees were used for data collection, while consecutive plots along the row were separated by at least 2 untreated trees. In both seasons, leaf Chl was recorded in summer by a hand-held Chl meter (SPAD 502, Minolta Co. LTD, Osaka, Japan) on 30 apical leaves per tree.

In addition, 20 random fully expanded leaves per tree were sampled from shoots. Collected leaves were immediately closed into polyethylene bags and transported to the laboratory in a portable refrigerator. Thereafter, leaf area, SLW and mineral concentration were determined as previously described. At commercial harvest, yield, fruit number and size per tree were determined. A sample of 20 healthy fruits per plot was used to evaluate fruit quality parameters. Fruit firmness was measured individually on the two opposite faces of peeled fruits by a hand pressure tester, FT 011 (EffeGi, Ravenna, Italy), fitted with an 8-mm diameter plunger. Fruit soluble solid concentration (SSC), as a means to evaluate total sugars concentration, was determined on a 20-fruit juice by a digital refractometer (Digital Refractometer PR-1, Atago, Tokio, Japan), while 10 mL of juice were added to 30 mL of deionized water and titrated with 0.1 N NaOH to a endpoint of pH 8.1 for titratable acidity measurement (expressed as malic acid) by a Compact Titrator I (Crison, Barcelona, Spain). Data were submitted to analysis of variance according to a complete randomized block design, and when treatment effect was statistically significant means were separated by the Least Significant Difference (LSD) test (P ≤ 0.05).

Results

Soil solution extractable Fe

As expected, DTPA extracted a higher amount of Fe from the soil than the other two eluents (Table 1). Nevertheless, the A. retroflexus aqueous extract increased significantly by more than 100-fold the amount of Fe solubilized from the soil compared to deionized water.

Table 1. Soil extractable iron (Fe) concentration in deionized water; aqueous extract of Amaranthus retroflexus L. and diethylenetriamine-pentaacetic-acid (DTPA) used as Fe eluents

Eluents	Soil extractable Fe concentration (mg kg^−1)
Deionized water	0.002c
Aqueous extract of A. retroflexus	0.242b
DTPA	67.6a
Significance	***
***effect of eluents significant at P ≤ 0.001. In the same column, means followed by the same letter are not statistically different (P ≤ 0.05, LSD Test).

Potted conditions

Macro and micronutrient concentrations of the A. retroflexus tissues used in the experiment were 12 ± 2, 2.9 ± 0.1, 52 ± 9, 21 ± 1, 4.3 ± 0.1 g kg⁻¹ (dw) for N, P, K, Ca and Mg respectively and 236 ± 17, 35 ± 3, 129 ± 3, 46 ± 3 mg kg⁻¹ (dw) for Fe, Mn, Cu and Zn respectively. During the first season, trees did not show leaf Fe chlorosis symptoms and high SPAD values were recorded without significant differences among treatments (data not shown). However, the supply of aqueous extract of A. retroflexus (HR) significantly increased shoot length in comparison with the control, FeSO₄ and A. retroflexus (LR) (Table 2), while other treatments were statistically similar to the untreated control. In the second season severe leaf chlorosis appeared in control plants (Table 2). These symptoms were effectively prevented by soil-applied Fe-chelate which induced the highest SPAD and, to a lesser extent, by A. retroflexus aqueous extract, which was more effective at a high rate and when enriched with FeSO₄ (Table 2). The A. retroflexus aqueous extract (LR) increased leaf Chl content only in June, whereas FeSO₄ alone was ineffective in preventing Fe chlorosis occurrence (Table 2). Leaf area, leaf weight and SLW were not affected by treatments (data not shown). A. retroflexus aqueous extract increased leaf N concentration in comparison to Fe-chelate (Table 3). When applied at the high rate, the aqueous extract induced the highest leaf N concentration, while at the low rate and in combination with FeSO₄, it did not show a statistical difference from the untreated plants (Table 3). A similar response was observed for leaf K concentration (Table 3). Leaf Mn concentration was considerably reduced in plants treated with Fe-chelate compared to other treatments (Table 3). No treatment effect was observed for leaf P, Ca, Mg, Fe, Cu, Zn (Table 3) and soil mineral concentration at the end of the trial (data not shown).

Table 2. Effect of treatment on shoot length (2007) and leaf chlorophyll (Chl) (controlled environment, 2008)

	Shoot length (cm shoot^−1)			Leaf Chl (SPAD)
Treatment	June	July	August	June	July	August
Control	48.9c	86.1b	96.1b	13.5d	17.2c	27.0b
Fe-chelate	57.3abc	99.3ab	102.7ab	25.1a	32.2a	38.8a
FeSO4	51.4bc	81.2b	84.3b	16.8cd	22.1b	33.8ab
A. retroflexus + FeSO4	60.9ab	100.5ab	103.5ab	20.1abc	22.8b	38.2a
A. retroflexus HR	63.0a	113.9a	117.7a	22.2ab	21.9b	38.1a
A. retroflexus LR	49.1c	90.4b	94.9b	19.8bc	17.3c	33.7ab
Significance	**	**	***	***	***	**
** and *** effect of treatment significant at P ≤ 0.01 and P ≤ 0.001, respectively. In the same column, means followed by the same letter are not statistically different (P ≤ 0.05, LSD Test). Fe, iron; FeSO4, iron sulphate; A. retroflexus, Amaranthus retroflexus L.; A. retroflexus HR, aqueous solution at 30 g (dry weight, dw) L^−1; A. retroflexus LR, aqueous solution at 15 g (dw) L^−1.

Table 3. Effect of treatment on leaf mineral concentration at the end of the experiment (controlled environment)

	Macronutrients (g kg^−1)					Micronutrients (mg kg^−1)
Treatment	N	P	K	Ca	Mg	Fe	Mn	Cu	Zn
Control	10.7bc	1.6	6.1b	13.5	3.7	54.0	26.5ab	6.6	52.7
Fe-chelate	9.9c	1.1	6.3b	12.2	3.8	68.7	9.6c	4.9	36.5
FeSO4	11.5bc	1.2	5.5b	15.1	4.7	56.0	29.7ab	5.4	34.0
A. retroflexus + FeSO4	13.0ab	1.5	8.2ab	16.8	3.8	58.4	32.6a	7.7	68.0
A. retroflexus HR	13.7a	1.7	10.5a	15.0	3.2	39.7	21.5b	8.0	47.1
A. retroflexus LR	11.9ab	1.7	7.4ab	13.3	4.1	53.8	29.2ab	7.3	50.4
Significance	*	ns	*	ns	ns	ns	**	ns	ns
ns, * and ** effect of treatment not significant or significant at P ≤ 0.05 and P ≤ 0.01, respectively. In the same column, means followed by the same letter are not statistically different (P ≤ 0.05, LSD Test). N, nitrogen; P, phosphorus; K, potassium; Ca, calcium; Mg, magnesium; Fe, iron; Mn, manganese; Cu, copper; Zn, zinc; FeSO4, iron sulphate; A. retroflexus, Amaranthus retroflexus L.; A. retroflexus HR, aqueous solution at 30 g (dry weight, dw) L^−1; A. retroflexus LR, aqueous solution at 15 g (dw) L^−1.

Total plant dry weight was significantly enhanced by the application of Fe-chelate and aqueous extract (HR) and in combination with FeSO₄ (Table 4), while root dry weight was similar for all treatments (Table 4).

Table 4. Effect of treatment on root (coarse + fine) and total tree dry weight at the end of the experiment (controlled environment)

Treatment	Root dry weight (g plant^−1)	Tree dry weight (g plant^−1)
Control	103	339b
Fe-chelate	118	396a
FeSO4	97	331b
A. retroflexus + FeSO4	125	392a
A. retroflexus HR	106	359a
A. retroflexus LR	110	342b
Significance	ns	*
ns and *effect of treatment not significant or significant at P ≤ 0.05, respectively. In the same column, means followed by the same letter are not statistically different (P ≤ 0.05, LSD Test). Fe, iron; FeSO4, iron sulphate; A. retroflexus, Amaranthus retroflexus L.; A. retroflexus HR, aqueous solution at 30 g (dry weight, dw) L^−1; A. retroflexus LR, aqueous solution at 15 g (dw) L^−1.

Field conditions

In both seasons, pronounced leaf Fe chlorosis symptoms appeared in untreated trees (Table 5). All strategies were effective in significantly increasing leaf SPAD compared to control plants and, in detail, Fe-chelate induced the highest values (Table 5) followed by the supply of the aqueous extract of A. retroflexus, particularly when enriched with FeSO₄ (Table 5). Leaf dry weight decreased only in control plants while no differences were observed in leaf area and SLW (data not shown). Fe-chelate treated plants showed a significant increase in leaf Fe and a decrease in leaf K, Mn and Zn concentration (Table 6). The described effect on leaf K, Fe and Mn concentration was observed in both seasons (only data from 2009 are reported). Also, the supply of aqueous extract reduced leaf K concentration with respect to the concentration in the control plants (Table 6). Leaf P was negatively affected by application of the A. retroflexus extract enriched with FeSO₄ compared to the extract alone (Table 6). Soil-applied synthetic Fe significantly increased yield compared to untreated plants (Table 7), whereas both treatments based on the aqueous extracts showed intermediate crop load not statistically different from control and Fe-chelate (Table 7). Fruits from untreated trees were bigger in the first season (Table 7), and only in 2010 did the aqueous extract enriched with FeSO₄ reduce fruit weight compared to untreated and soil-applied Fe-chelate trees (Table 7). At harvest, no differences were recorded in fruit firmness and titratable acidity, but all strategies were effective in increasing the soluble solid concentration (°Bx), during the first season in particular (Table 8).

Table 5. Effect of treatment on leaf chlorophyll (Chl) (field conditions) recorded in July 2009 and 2010

	Leaf Chl (SPAD)
Treatment	2009	2010
Control	20.9d	15.5d
Fe-chelate	44.0a	31.6a
A. retroflexus	29.3c	20.7c
A. retroflexus + FeSO4	31.9b	22.8b
Significance	***	***
***effect of treatment significant at P ≤ 0.001. In the same column, means followed by the same letter are not statistically different (P ≤ 0.05, LSD Test). Fe, iron; FeSO4, iron sulphate; A. retroflexus, Amaranthus retroflexus L.

Table 6. Effect of treatment on leaf mineral concentration in 2009 (field conditions)

	Macronutrients (g kg^−1)					Micronutrients (mg kg^−1)
Treatment	N	P	K	Ca	Mg	Fe	Mn	Cu	Zn
Control	18.8	17.2ab	16.9a	13.5	5.2	49.7b	40.2a	101.7	43.7a
Fe-chelate	20.0	15.4ab	10.3c	12.9	4.9	85.5a	32.3b	103.9	33.7b
A. retroflexus	19.7	18.8a	14.3b	14.2	5.3	50.5b	42.3a	94.0	41.9a
A. retroflexus + FeSO4	19.6	13.4b	13.4b	13.7	5.3	47.3b	43.3a	95.0	39.8a
Significance	ns	*	***	ns	ns	**	*	ns	**
ns, *, ** and *** effect of treatment not significant or significant at P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001, respectively. In the same column, means followed by the same letter are not statistically different (P ≤ 0.05, LSD Test). N, nitrogen; P, phosphorus; K, potassium; Ca, calcium; Mg, magnesium; Fe, iron; Mn, manganese; Cu, copper; Zn, zinc; A. retroflexus, Amaranthus retroflexus L.; FeSO4, iron sulphate.

Table 7. Effect of treatment on yield and fruit weight in 2009 and 2010 (field conditions)

	2009		2010
Treatment	Yield (kg tree^−1)	Fruit weight (g fruit^−1)	Yield (kg tree^−1)	Fruit weight (g fruit^−1)
Control	20.4b	220.9a	11.5b	273.1a
Fe-chelate	27.4a	196.9b	17.2a	265.3a
A. retroflexus	24.4ab	194.9b	13.5ab	259.6ab
A. retroflexus + FeSO4	24.8ab	188.8b	13.0ab	244.9b
Significance	*	*	*	**
* and ** effect of treatment significant at P ≤ 0.05 and P ≤ 0.01, respectively. In the same column, means followed by the same letter are not statistically different (P ≤ 0.05, LSD Test). Fe, iron; A. retroflexus, Amaranthus retroflexus L.; FeSO4, iron sulphate.

Table 8. Effect of treatment on fruit firmness, soluble solid content (SSC) and titratable acidity at commercial harvest in 2009 and 2010 (field conditions)

	2009			2010
	Firmness	SSC	Titratable acidity	Firmness	SSC	Titratable acidity
Treatment	(kg)	(°Brix)	(g L^−1 malic acid)	(kg)	(°Brix)	(g L^−1 malic acid)
Control	5.33	12.9b	1.41	5.44	12.8b	1.72
Fe-chelate	5.30	14.2a	1.82	5.46	13.6a	2.28
A. retroflexus	5.18	14.1a	1.45	5.51	13.0ab	1.76
A. retroflexus + FeSO4	4.96	14.4a	1.60	5.60	13.5a	1.94
Significance	ns	*	ns	ns	*	ns
ns and * effect of treatment not significant or significant at P ≤ 0.05, respectively. In the same column, means followed by the same letter are not statistically different (P ≤ 0.05, LSD Test). Fe, iron; A. retroflexus, Amaranthus retroflexus L.; FeSO4, iron sulphate.

Discussion

The most visible and recognizable symptom of Fe-deficiency in most of the susceptible crops is interveinal yellowing starting from apical leaves (Rombolà and Tagliavini 2006), while in pear leaf Fe chlorosis appearance is atypical, since it often occurs in the whole leaf lamina, including veins (Abadía et al. 1999). In our experiments, potted untreated trees showed growth depression prior to the typical chlorosis of the leaves. At the beginning of the experiment, plants likely could have accounted for Fe from perennial organs that contributed, partially, to satisfy Fe requirements. In fact, before transplanting to calcareous soil, trees were grafted in the nursery, grown on a sub-acid substrate and adequately fertilized by mineral sources. However, it has been suggested that growth reduction is a response of the Fe-deficiency caused by a sequestration of this nutrient in the apoplast and consequent insufficient supply of physiologically available Fe to the leaves (Kosegarten et al. 2001; Gruber and Kosegarten 2002). We believe that in our experimental conditions, Fe in potted trees was sufficient to prevent leaf chlorosis but insufficient to avoid a growth reduction. Similar results were observed on maize, sunflower (Masalha et al. 2000) peach (Shi et al. 1993) and grapevine (Gruber and Kosegarten 2002), indicating that from an agronomical standpoint, the reduction of tree growth is an important index for an early diagnosis of Fe chlorosis, the occurrence of which anticipates leaf discoloration.

Afterwards, leaf chlorosis symptoms clearly appeared in control plants in the second season of experiment. These symptoms were effectively prevented by soil-applied A. retroflexus aqueous extract as a result of the improved soil Fe availability and consequent Fe uptake. In field conditions, although Fe-chelates were the most effective strategy for preventing Fe deficiency, aqueous extract of A. retroflexus raised leaf SPAD in both years compared to untreated control plants. This response is in agreement with the results obtained in the controlled environment. It is well known that Fe availability in the soil strongly depends on the dynamic equilibria among oxide, hydroxide, ionic and complexed (chelated) Fe fractions, the latter representing more than 95% of the soluble Fe (Varanini and Pinton 2006). Thus, in the rhizosphere, soluble Fe sources available for plant uptake are mainly a mixture of Fe-complexes with organic ligands such as organic acids, phenolic compounds, phytosiderophores (Römheld and Marschner 1986), microbial siderophores (Shenker et al. 1992) as well as fractions of water extractable humified organic matter (Varanini and Pinton 2006).

In our lab assay, DTPA was the most effective eluent in the extraction of Fe from the soil; however, the addition of A. retroflexus aqueous extract, used as a natural chelator-like agent, effectively increased the capability for Fe extraction. This effect is probably due to the chelating compounds (e.g., organic acids, aminoacids, bioregulator-like substances, siderophores) released by the Amaranthus spp. tissues during the maceration process. Previous research performed by Matocha (1984), Matocha and Pennington (1982), and Mostaghimi and Matocha (1988), investigated the concept of “Plant-Complexed-Fe.” These authors showed that soil-incorporated Amaranthus spp. residues mixed with a Fe inorganic salt were more effective than Fe-EDDHA in reducing Fe chlorosis and increasing plant dry matter and grain yields in sorghum plants grown on severely Fe-deficient soil. In addition, results obtained by Goos et al. (2001) confirmed that when A. retroflexus tissues were mixed with FeSO₄ and incubated in an alkaline soil, Fe solubility increased more than that with FeSO₄ or pigweed alone. This effect appeared to be fairly persistent, (up to 4 weeks), suggesting that the increased availability of complexed Fe in the soil volume colonized by roots may contribute to Fe uptake. Although the ability of the vegetal extract to solubilize Fe in the soil was relatively low, it should be considered that fruit trees have low Fe requirements. For instance, Abadía et al. (2004) reported that Fe requirements in mature peach trees range between 1 and 2 g of Fe per tree per year. For kiwifruit, total removal of Fe in fruits was estimated around 160 g ha⁻¹ for a fruit load of 30 t ha⁻¹, and for several flesh fruit crops, Fe removals ranged between 1 and 10 g t⁻¹ of harvested yield (Tagliavini et al. 2000b). Thus, even low but constant amounts of soluble Fe in the rhizosphere can fulfil plant needs.

On the other hand, the rate of Fe applied with the A. retroflexus tissues (total of 22.7 mg Fe plant⁻¹ in 2 years in the case of the extract HR) is quite low and the contribution to Fe soil availability was probably negligible. The effectiveness of the A. retroflexus aqueous extract was positively related to the rate of application (in potted conditions). In the same experiment, the addition of FeSO₄ to the A. retroflexus aqueous extract did not improve Fe tree nutrition compared to the extract alone. We enriched the aqueous extract with an exogenous source of Fe with the aim of promoting a weak linkage between Fe and organic compounds released by plant tissues, similar to a natural chelating effect. It is possible that A. retroflexus aqueous extract solubilized enough Fe from soil to make the addition of the Fe salt irrelevant for root uptake. However, in field conditions, the effectiveness of the A. retroflexus was improved when enriched with the inorganic Fe salt, probably because the synergic effect was pronounced in soil with a limited availability of Fe (9 mg kg⁻¹). Another explanation may be related to the soil calcium carbonate (CaCO₃) concentration. In potted conditions the amount of soil CaCO₃ was much higher than in field conditions and it probably contributed to insolubilize the Fe added as an inorganic source in the aqueous extract.

As expected, the application of FeSO₄ alone did not improve Fe nutrition of pear trees. There is a body of literature focusing on the use of inorganic Fe-salts to overcome Fe deficiencies in crops. Although soluble inorganic Fe-salts are a cheap source of Fe, their supply to the soil is quite inefficient and has scarce agronomic value, especially in high pH (e.g., calcareous) soils, even when high rates are applied, due to the rapid transformation of most of the applied Fe into highly insoluble compounds such as Fe(III)-hydroxides (Tagliavini et al. 2000a). The beneficial effects on Fe nutrition induced by soil-applied A. retroflexus (HR and HR in combination with Fe-salt) and Fe-chelate led to an increase in total potted plant dry weight. After two years of the experiment the main contribution to total plant dry biomass was observed in the canopies (shoots, leaves and trunk) while no differences were observed in plant root dry weight. These observations are in agreement with results obtained in peach by Shi et al. (1993) and grapevine by Kosegarten et al. (2002), who reported that insufficient soil Fe supply impaired shoot growth more than root growth. Leaf nutrient concentrations were in the range considered optimal for the cv. Abbé Fétel in the area of our study (Toselli et al. 2002), with the exception of N and Cu, the concentrations of which were sub-optimal in the young plants. The low leaf N concentration could be explained by the timing of leaf sampling, which occurred at the end of the trial, in mid-autumn, when this nutrient was partially translocated from the leaves to the perennial organs through the phloem. However, the highest leaf N and K concentrations were measured in plants fertigated with the highest rate of aqueous extract of A. retroflexus. We believe that the amounts of nutrients available for plant uptake applied as fresh vegetal material were quite low. In addition, those nutrients were in an organic form, for which mineralization, release time and availability for plant uptake are unknown.

Soil-applied Fe-chelate dramatically decreased leaf Mn concentration, either in controlled or field conditions, in agreement with literature. Since the first experiments (in the 1950s) involving soil application of Fe-chelates (e.g., EDDHA), the competitive effect of these synthetic molecules on Mn uptake in a large number of plant species, including herbaceous and perennial species, has been reported (Wallace and Alexander 1973; Ghasemi Fasaei et al. 2003; Sorrenti et al. 2010). This suggests that yearly application of soil Fe-chelates should be integrated with foliar applications of Mn in order to avoid Mn-deficiency. Only the application of synthetic chelate raised leaf Fe concentration of mature trees in both seasons. Nevertheless, it is recognized that soil and plant Fe chemical analyses are usually not related to leaf chlorosis because of the so called ‘‘chlorosis paradox’’ (Römheld 2000) used to indicate that chlorotic leaves often have equal or higher total Fe than green, healthy ones (Katyal and Sharma 1980; Nikolic and Kastori 2000). This discrepancy is related to the localization and binding state of Fe in leaves, that might be precipitated in part in the apoplasm of leaves and be physiologically unavailable (Mengel and Geurtzen 1988; Nikolic and Römheld 2007).

Leaf K concentration was reduced by all strategies in the field trial, in particular by Fe-chelate application. This response could be explained by the differences recorded in tree yield, since fruits represent a strong sink for K (Tagliavini et al. 2000b) and plants with a higher crop load induced a higher translocation of this nutrient to the fruits at the expense of the leaves. In fact, this trend was observed only in 2009 when yield was markedly higher (+44%) than in 2010. The application of Fe-chelates induced the highest fruit yield. However, either in 2009 or 2010, even without significant differences from untreated plants, the application of aqueous extract of A. retroflexus increased yield to values comparable to Fe-chelate strategy. In detail, in 2009, control plants yielded 18%, 17% and 26% less than trees supplied with aqueous extract enriched with FeSO₄, aqueous extract alone and Fe-chelate, respectively; whereas in 2010, the losses were estimated at 8%, 16% and 23%, respectively. The reduction in fruit load in untreated trees may be associated with the decrease of fruit number induced by Fe deficiency during flower development and fruit set, as reported also in other studies (see Tagliavini and Rombolà 2001; Pestana et al. 2003; Álvarez-Fernández et al. 2006, 2011, for reviews). Álvarez-Fernández et al. (2006) reported that Fe-deficiency affects fruit number more than fruit size, while an increase in fruit weight is described in moderate chlorotic peach and pear trees and a decrease is reported only in heavily chlorotic plants (Álvarez-Fernández et al. 2011). In 2009, as a consequence of the reduced number of fruits per tree, chlorotic plants led to a greater fruit weight. It is reasonable to believe that moderate Fe-deficiency played a role similar to a thinning agent.

It is widely reported that Fe chlorosis affects fruit metabolism and, as a consequence, could interfere in the ripening process (see Tagliavini and Rombolà 2001; Pestana et al. 2003; Álvarez-Fernández et al. 2006 for reviews). Delays in fruit ripening have been reported in chlorotic tomato, citrus, peach, pear and nectarine plants (Raese et al. 1986; Sanz et al. 1997; Álvarez-Fernández et al. 2003; Razeto and Valdés 2006). On the other hand, Álvarez-Fernández et al. (2011) observed that Fe chlorosis induced an advanced maturity in pear fruits as measured by a decrease in flesh firmness and an increase in fruit yellowness, while SSC was not affected by Fe-supply. An increase in pear yellowing with Fe deficiency has been described for the pear cvs. Anjou and Bartlett as well as in other green fruits such as kiwi and olives (see Álvarez-Fernández et al. 2006 for references). In our conditions fruit shape, skin color, fruit firmness and titratable acidity at harvest were not different in chlorotic and non-chlorotic trees and they appeared at the same maturity stage. However, Fe-deficiency reduced fruit SSC, probably because of the decreased CO₂ assimilation rate and carbohydrate availability.

Conclusion

This work offers an innovative approach to Fe nutrition management of fruit tree crops. Our results indicate the potential of the soil-applied aqueous extract of A. retroflexus to alleviate symptoms of Fe chlorosis, improving Fe nutrition of pear trees grown in calcareous soil. One of the mechanisms behind this effect is the effectiveness of aqueous extract of A. retroflexus to solubilize Fe present in the soil. The adoption of this strategy could include the option to supply the aqueous extract either by the fertigation (after its filtration) or by intercropping fruit trees with A. retroflexus with a regular incorporation of fresh biomass into the soil.

Acknowledgments

This study was partially supported by Regione Emilia-Romagna (Legislation n. 28/98), coordinated by Vegetal Production Research Center (CRPV) and Organic Producers of Emilia Romagna Association (PROBER). The authors thank the Farm of Alberto Asioli (Massalombarda, RA), for providing the experimental pear orchard.