Effect of Zinc-Phosphorus Interaction on Corn Silage Grown on Sandy Soil

This study investigated the response of corn silage to different combinations of zinc (Zn) and phosphorus (P) soil supply when grown in sandy soil. The soil was naturally poor in extractable Zn and rich in plant-available P. The experiment was conducted in outdoor containers. The treatments consisted of soil supply combinations of 3 levels of Zn (0, 5 and 10 mg Zn kg−1 of dry soil) and 4 levels of P (0, 12, 36 and 72 mg P2O5 kg−1 of dry soil). The results showed the absence of a significant effect (at p ≤ 0.05) of Zn-P interaction on plant growth, plant mineral content or total aerial dry weight at harvest. P application depressed Zn shoot content, and conversely, Zn supply slightly reduced P shoot content. The total aerial dry weight at harvest was not enhanced by P application. However, it was significantly increased by Zn supply of 5 mg·kg−1 only for the highest P (72 mg·kg−1) application (at p ≤ 0.05). This increase was around 15% compared to no Zn soil supply. It was especially linked to kernel dry weight and particularly to pollination rate. For the highest level of P supply, Zn applications significantly enhanced (at p ≤ 0.05) the kernel dry weight and the pollination rate by 22.1% and 38.4% respectively, compared OPEN ACCESS Agriculture 2015, 5 1048


Introduction
Zinc (Zn) deficiency is a common nutritional constraint for crop production, particularly cereals.As is well documented, corn is known to be very sensitive to Zn deficiency stress [1,2].This deficiency was reported in sandy soils, waterlogged soils and soils with high phosphorus (P) content [3].It is reported that P fertilization may aggravate Zn deficiency in many crops [1,4,5].In this regard, a diminishing effect of P fertilization on plant Zn content was reported in corn [6], in wheat [7,8], and in dwarf bean [9].The Zn-P interaction was explained by numerous reasons such as the dilution of Zn due to the enhancement of plant growth with P, the low translocation of Zn from root to shoot due to the interference with P, and the reduction of Zn availability due to the interaction of Zn with P in soil [1,10].In a recent study, Ova et al. [7] found out that Zn and P interaction in wheat depends on mycorrhiza development, which is reduced under high soil P content.Such reduction minimized the Zn plant absorption.On the other hand, other responses from corn to Zn and P supply have been reported.For example, Brown et al. [11] found a mutual depressing effect from Zn and P on corn.Another example is Mallarino and Webb [12], who observed that continuous P fertilization at high rates did not aggravate Zn deficiency in corn.Similarly, a positive relationship between Zn and P content was noted in corn grown on a calcareous soil [5] and on oilseed rape [13].
In the sandy soil of the Loukkos area (Northwestern Morocco), poor in Zn and relatively rich in P, adequate Zn soil supply corrects Zn deficiency and enhances silage yield [14].Furthermore, it appears that soil P application enhances corn growth even if this soil is naturally rich in P.However, such application seems to intensify the visual Zn deficiency symptoms, exhibited as white areas between the midrib and the margin of leaves.Despite the numerous studies undertaken to explain Zn-P interaction in corn, the magnitude of this interaction in silage yield and its specific mechanism are still not clear.The current work is undertaken to test the interactive effect of differential rates of Zn and P supply on growth, on mineral content, and on biomass production of corn silage grown on sandy soil.

Experimental Soil
Sandy soil samples were collected from the sandy area of the Loukkos perimeter (34,96° N, 6,21° W, Northwestern Morocco).The soil was air dried, sieved and homogenized.The soil is sandy (87.3% sand, 8% of clay and 5.6% of silt), with a low Di-Ethylene Triamine Penta Acetic acid (DTPA) extractable Zn (0.23 mg•kg −1 ) and a relatively high Olsen P content (38 mg•kg −1 of P2O5).It is not calcareous and has a pH of 6.1.The other basic soil chemical properties are presented in Table 1.

Experimental Design and Crop Management
Containers (0.42 m length, 0.25 m width and 0.16 m depth) were filled with 20 kg of air dried sandy soil.5 seeds of maize (cv.Panama) were sown in each container.Thinning was done 7 days after emergence to keep one plant per container.The experiment was conducted in outdoor conditions.During the growing season (May to August 2014), the average maximum and minimum temperatures were 33 °C and 17 °C, respectively.Also, no precipitation was recorded during this period.Watering was done whenever required and the soil was irrigated up to its field capacity.
The treatments consisted of different soil supply combinations of 3 levels of Zn (0 or no Zn supply; 5 and 10 mg Zn kg −1 of dry soil) and 4 levels of P (0 or no P supply; 12; 36 and 72 mg P2O5 kg −1 of dry soil).Zn was supplied as a solution of Zn sulfate (ZnSO4•7H2O) and P as a solution of di-ammonium phosphate (DAP).These treatments were split among 3 different times during the growing season: (i) 50% immediately after sowing; (ii) 25% at 4-5 leaf stage; (iii) and 25% at 8-9 leaf stage.The experimental design was a randomized complete block with 4 replications.
The soil was also supplied during the growing season with 371 kg•ha −1 of nitrogen (N) as ammonium nitrate, 300 kg•ha −1 of K2O as soluble potassium sulfate, 1.6 kg•ha −1 of manganese (Mn) as manganese sulfate, 2.5 kg•ha −1 of copper (Cu) as copper sulfate and 2 kg•ha −1 of boron (B) as boron sulfate.In order to assure a homogeneous N fertilization for all treatments, an equivalent amount of the difference between the ammonium brought by DAP for the highest P supply and the other P treatments was applied as ammonium sulfate.
Fungal disease (Helminthosporium) was controlled by one application of flusilazole at the 7-8 leaf stage.Each container was equipped with a leaching system in order to reuse the leaching solution.

Measurements
Stem height, stem diameter and leaf area per plant were determined at harvest, which was done approximately at shoot moisture content of 66%.The leaf area was measured using Formula (1) reported by Mokhtarpour et al. [15] for corn: where L, W, and n are leaf length, leaf greatest width, and last leaf of corn, respectively.The harvest was done on August, 2014.Plants were separated into stem, leaves and ear.The ear was separated into kernels, husks and cob.These plant's parts were oven dried at 70 °C until constant weight and dry weights were recorded.Also, different kernels yield components were determined: (i) number of kernels per ear; (ii) pollination rate using Formula ( 2 In order to determine mineral contents including Zn, P, K, N, Mg, Mn and Cu in shoot and in root at harvest, all fractions of aerial plant's parts and roots were ground to pass through a screen with 0.25 mm openings.To note, the plant roots were rubbed by hand and washed several times with tap water and finally with dionized water to remove soil particles before being ground.2 g of the ground material, for each treatment's replication, was digested with a tri-acid mixture (nitric, perchloric, and sulfuric acids).Then, the digested material was analyzed for Zn, Mn and Cu using an atomic absorption spectrophotometer (Varian AA 240 Fast Sequential; air acetylene flame, Varian Technologies, Victoria, Australia).Concerning P, N, Mg and K contents, 0.6 g of the ground material was digested with a di-acid mixture (salicylic and sulfuric acids).Then, the digested material was analyzed for P, Mg and K using an inductively coupled plasma spectrophotometer (iCAP 7400 DUO, Thermo Scientific, China) while N content was determined colorimetrically on a Skalar San ++ autoanalyzer (Skalar Analytical B.V., Breda, Netherlands) according to the Skalar standard method.
A soil sample from each treatment's replication was taken to determine its content in terms of extractable Zn and assimilable P after harvest.It was oven dried at 40 °C and ground to pass a 2 mm sieve.The exchangeable Zn was determined by DTPA extractant method [2] using the atomic absorption spectrophotometer mentioned above.The available P was extracted with Sodium bicarbonate (NaHCO3) [16] and analyzed colorimetrically using an UV-Visible spectrophotometer (Cary 50 Conc, Varian Technologies, Victoria, Australia).

Statistical Analysis
Experimental data were subjected to analysis of variance (ANOVA) in order to evaluate the significance of the treatments and their interactions effect (p ≤ 0.05 level).Differences between means of different combinations were compared by Duncan's multiple range test (p ≤ 0.05 level).All statistical analyses are performed using the SPSS software (Version 17.0, SPSS Inc., Chicago, IL, USA).

Stem Height, Stem Diameter and Leaf Area at Harvest
The stem height and the leaf area were not significantly influenced by Zn-P interaction.The significant effect of Zn application on stem height was observed only for the highest P supply (72 mg•kg −1 ).At this level of P, Zn supply resulted in a significant increase around 5.8% compared to no Zn supply (Table 2).This significant effect can be explained by the depressing effect of high P soil content in the reduction of Zn availability [1,10].Many authors reported the role of Zn in stem stretching through its involvement in the metabolism of the Indole 3 Acetic acid as a growth regulator [17].However, the leaf area did not show a significant increase in terms of Zn application for all P supply levels.On the other hand, a positive response from stem height and leaf area to P application was recorded only at 5 mg•kg −1 of Zn.Such a behavior was not understood and is reason for further investigation.At this level of Zn, the highest P supply (72 mg•kg −1 ) resulted in increases of around 7.08% and 7.4% in stem height and in leaf area respectively compared to no P application.The positive effect of P supply can be explained by the key role of this macro-nutrient in enhancing cell division activity which stimulates growth parameters [18].Besides, no obvious stem diameter response was observed even though significant effects of Zn-P interaction and P were recorded (Table 2).

Shoot Dry Weight and Partitioning into Ear, Stem, Leaves and Kernels
The total aerial dry weight at harvest, which is equivalent to silage yield, was not influenced by Zn-P interaction.However, it was significantly enhanced by Zn supply only for the highest P (72 mg•kg −1 ) application.This increase was around 15% at Zn application of 5 mg•kg −1 compared to no Zn soil application (Table 3).This significant response can be explained by the depressing effect of high P soil content in the reduction of Zn availability [1,10].The beneficial effect of Zn in biomass production was reported in corn [14,19,20], in wheat [21], and in tomato [22].Such an effect can be explained by the role of Zn in the metabolism of the β-carbonic anhydrase as a crucial enzyme involved in photosynthesis [23].On the other hand, no significant effect from P supply was recorded.This can be explained by the adequate native Olsen P in soil (38 mg•kg −1 ).Therefore, P application can be avoided in this kind of soil.
The shoot dry matter partitioning showed that ear and particularly kernel dry weights were significantly enhanced by Zn supply only for the highest P (72 mg•kg −1 ) application.The increase of kernel dry weight was around 38.43% compared to no Zn soil supply.Furthermore, the biomass production response was particularly related to the kernel dry matter, which represents around 34% of the total shoot dry weight.This result agreed with our previous findings on corn responses to Zn soil supply [14,20] and to Zn foliar spray [19].Concerning stem and leaves dry matter, they did not exhibit significant responses towards P and Zn supply.Table 3.Effect of zinc and phosphorus supply on shoot dry weight of corn silage at harvest and its partitioning into stem, leaves, ear and kernels.

Kernels Yield Compounds
As can be seen in Table 4, the response of kernel dry weight was particularly related to the response of the pollination rate.For no Zn supply, high P application (72 mg•kg −1 ) induced a significant decrease of 21.50% in the pollination rate compared to no P supply.Also, at the highest level of P, Zn applications significantly enhanced the pollination rate, by around 22.14% compared to no Zn supply.Such results showed the depressing effect of high levels of P application without Zn supply on the pollination rate.The crucial role played by Zn in pollination was reported in previous studies conducted in the same sandy soil [14,19,20].This role is particularly linked to the enhancement of male fertility [24].The positive response of the pollination rate resulted in an increase in the number of kernels per ear.This latter not only elevated the biomass production but also enhanced the silage quality through its content of kernels.On the other hand, 1000 kernel dry weight did not significantly respond to the studied treatments.Table 4. Effect of zinc and phosphorus supply on the number of kernels per ear, pollination rate and 1000 kernel dry weight of corn silage at harvest.Data are the means ± standard error (n = 4).For each measured parameter, means without common letter are significantly different (at p ≤ 0.05), according to Duncan test.*Significant at 5% probability level; n.s.not significant at 5% probability level.

Shoot and Root Mineral Content
As shown in Table 5, Zn shoot content was significantly enhanced by Zn supply and was significantly reduced by P. The highest shoot content, around 10 mg•kg −1 , was recorded under no P application combined with Zn supply of 10 mg•kg −1 .The depressed effect of P on Zn shoot content was reported in corn by Takkar et al. [6], in rice by Haldar and Mandar [4] and in wheat by Zhu et al. [8].The Zn uptake exhibited the same response trend as Zn shoot content (DATA not shown).Thus, the phenomenon of Zn dilution due to the enhancement of plant growth with P [11] seems not to be the prominent factor in Zn-P interaction in our case study.On the other hand, Zn shoot content was still below the threshold of 22 mg•kg −1 required in corn [3] for all treatments.Such a result can be explained by the limited soil volume in container which may highly reduce available soil Zn compared to field condition.Concerning P shoot content, it was significantly enhanced by P supply while it was slightly and significantly reduced by Zn supply.However, all treatments had P shoot content around the requested level for corn production (0.1%-0.5%) [25].Also, P shoot content did not exceed the phytotoxic threshold of 2% reported by Ova et al. [7] even with high P supply (72 mg•kg −1 ).These results suggest that Zn and P supply induced a mutual depressed effect on each other's shoot content.A similar result was reported by Ova et al. [7] in wheat and by Brown et al. [11] in corn.Nevertheless, this depressed effect did not significantly affect plant growth and shoot dry matter, but may reduce silage mineral nutrient quality.On the other hand, Zn application reduced Mn shoot content at the highest and the lowest dose of P. It also reduced Cu shoot content at the highest P supply.However, all treatments had required shoot contents in Mn and Cu [26].The depressed effect of Zn supply on Mn and Cu shoot content was reported with Zn foliar spray in corn silage grown in the same sandy soil [19].Also, a significant diminishing effect from P application on K shoot content was recorded.Nevertheless, all treatments showed an adequate K shoot content around 0.7% [26], and no K deficiency symptoms has been revealed.In contrast, no significant implication of Zn and P applications on N shoot content was noticed.
In terms of the root mineral content, no significant effect of Zn-P interaction was recorded.The root Zn content was significantly elevated with increasing Zn supply.Further, no Zn accumulation in the root under high P application was recorded.This result is in contrast with the previous finding by Dwivedi et al. [27] in corn.Also, the P root content was enhanced by P application and no significant effect from Zn supply was exhibited.The absence of Zn and P accumulation in roots suggested that the mutual depressed effect of Zn and P supply on each other's shoot content was not related to their translocation from root to shoot.Such a result can be explained by the effect of P in limiting plant Zn absorption [10].In this regard, Ova et al. [7] found out that the reduction of wheat's Zn absorption is due to the limited mycorrhiza development under high P soil supply.On the other hand, no significant implication of P and Zn supply in root content of N, K, Mg, Mn and Cu was revealed (Table 6).

Residual Soil Content on Exchangeable Zn and on Olsen P
After harvest, the residual soil content in exchangeable Zn was significantly elevated with increasing Zn supply.It was, under 5 and 10 mg•kg −1 of Zn supply and for different P applications, higher than the threshold of 0.8 mg•kg −1 requested for corn production [2].Thus, the hypothesis of Zn sorption to soil components under high P soil content [28] seems unlikely to occur.On the other hand, the Olsen P soil content increased significantly with increasing P supply while no significant effect from Zn supply was recorded (Table 7).

Conclusions
The results of this study have shown the absence of a significant effect (at p ≤ 0.05) from Zn-P interaction on corn grown in sandy soil.However, a mutual depressive effect of Zn and P applications on each other's shoot content was recorded.The diminishing effect of high P supply on Zn shoot content was more pronounced than that induced by Zn on P shoot content.The depressive effect of P supply on Zn shoot content does not seem to be related either to the hypothesis of low translocation of Zn from root to shoot nor to the hypothesis of Zn sorption to soil components under high P soil content.The biomass production of corn silage was significantly enhanced by Zn supply of 5 mg•kg −1 only for the highest P application (72 mg•kg −1 ) (at p ≤ 0.05).However, P supply did not show any additional benefit in terms of biomass production.

Table 1 .
Physicochemical properties of the soil.

Table 2 .
Effect of zinc and phosphorus supply on stem height, stem diameter and leaf area of corn silage at harvest. P

Table 7 .
Effect of zinc and phosphorus supply on residual soil content of exchangeable zinc (DTPA Extraction) and assimilable phosphorus (Olsen) after harvest. P