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Determination of Nutrients by SEM-EDS and ICP-OES in Mexican Pine Pinus greggii var. greggii



Rebeca Casique-Valdés, Fernando Galindo-García, Willian Narvaez-Ortiz, Rosalinda Mendoza-Villarreal and Eduardo San Martin-Martinez
 
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ABSTRACT

Background and Objective: Pinus greggii var. greggii, an endemic species of northern Mexico, adapts to nutritionally poor soil conditions. Despite its importance, the information on the nutritional needs of this species is scarce. This study evaluates the applicability of the X-ray Energy Dispersion Spectroscopy method (SEM-EDS) versus the Inductively Coupled Plasma Spectroscopy (ICP-OES) towards the elemental analysis of P. greggii grown in conventional management with Peat Moss (PM) and traditional management with forest soil (TM). Materials and Methods: Nutrient determination, in most cases, is performed by wet digestion, a process that becomes laborious and dangerous for the person who develops the laboratory technique. Six consecutive samples were taken every 25-35 days to analyze by SEM-EDS and ICP-OES methods. A t-student analysis and Pearson correlation were performed using the P. greggii nutrient percentage results. Results: It was demonstrated, that the nutritional absorption in both detection methods showed a highly significant Pearson correlation for P, K, Ca and S nutrients. Seedlings grown in PM had higher dry biomass content than results found in TM. The substrate based on PM induced higher percentages of mineral absorption against TM. Conclusion: The elemental composition of P. greggii seedlings can be evaluated more precisely via SEM-EDS technique, avoiding the use of corrosive materials, which damages the environment, when the ICP-OES analysis technique is used.

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  How to cite this article:

Rebeca Casique-Valdés, Fernando Galindo-García, Willian Narvaez-Ortiz, Rosalinda Mendoza-Villarreal and Eduardo San Martin-Martinez, 2021. Determination of Nutrients by SEM-EDS and ICP-OES in Mexican Pine Pinus greggii var. greggii. Asian Journal of Plant Sciences, 20: 67-79.

DOI: 10.3923/ajps.2021.67.79

URL: https://scialert.net/abstract/?doi=ajps.2021.67.79
 
Received: August 20, 2020; Accepted: November 10, 2020; Published: December 15, 2020


Copyright: © 2021. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

An alternative to restore highly degraded soils is to establish plantations where survival success depends mainly on the use of plants that are adaptable to poor nutrient-soil1. Pinus greggii is an endemic Mexican pine that adapts to nutritional scarce soil conditions that are shallow and slightly alkaline, with a pH range of 7.0-8.0; this species has a great potential for forest plantations to help the recovery of eroded soils2.

Most nurseries in Mexico use forest soil for P. greggii production, as the main component of the growing media3, which is dense and often creates a compacted and poorly drained system inside the container4. More than 60% of nursery-grown seedlings are obtained under the traditional production method, by shoot transplant using forest soil in polybags. In Mexico, information on the nutritional demands of most forest species, specifical endemics, is meager; that delays the nutritional diagnosis and the initiation of nutrition-related programs and results in erroneous applications of fertilizer, involving economic losses and environmental degradation5. The determination of nutrients found in pine can be carried out by different techniques, among them, the most commonly used is the nitrogen determination by wet digestion, by the method of Kjeldahl6. Other macronutrients and micronutrients concentrations are determined by ICP-OES7, or atomic absorption spectrometry8. These wet digestion procedures need the use of dangerous concentrated acids and time-consuming processes that can lead to systematic errors and failures9,10. The aim of the present work was, to study the applicability of the X-ray Energy Dispersive Spectroscopy (SEM-EDS) in comparison with Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) methods. To study the elemental analysis of P. greggii var. greggii grew in traditional soil management (TM) and conventional management with Peat Moss (PM) in the greenhouse.

MATERIALS AND METHODS

Study area: The experimental design with pine seedlings was conducted in the Horticulture department of the University Autonomous Agrarian Antonio Narro, during the months of February-October, 2016, during this time the destructive samplings were carried out. Nutrient determination tests were carried out during the months of February, 2017 to June, 2017 at the biomaterials laboratory of CICATA-IPN.

Substrates: For conventional management, a mixture of peat moss and perlite was used in a 1:1 ratio (PM). In the case of traditional management, forest soil of horizon A (0-25 cm) (101° 1'37.32 W, 25° 20'44.68 N), silt and clay at 2:1:1 ratio (TM) was used. Both substrates were sterilized for 1 h in an autoclave at 6.803 kg pressure. The mixture of peat moss and perlite was fertilized with osmocote 14-14-14® (1 kg m–3)11. The forest soil mixture was not fertilized.

Plant material and culture conditions: Pinus greggii seeds were obtained from cones of the Cañón de Caballos stand, located in Saltillo, Coahuila, México (100° 54'46.61 W and 25° 14'47.13 N). Seeds were treated with hydrogen peroxide at 2% for 4 hrs and seeded into 77 germination trays cells previously filled with autoclaved peat moss and perlite (1:1). Trays were kept in a greenhouse under a 70% sunblock shade cloth for two months, before transplanting. Irrigation was every three days and kept at 30-35°C during the day and 20-25°C at night. The natural daytime length was approximately 14-16 hrs during the months of November-January (2016 and 2017).

Sampling: The substrates were distributed in 10×20 cm polybags. Two hundreds bags were filled with PM and the other 200 with TM. Pinus greggii seedling was transplanted on each polybag for a total of 400 seedlings. Tap water was adjusted to a 5.8 pH using nitric acid to irrigate plants every five days. Seedlings were covered with a 70% sunblock shade cloth in the same environmental conditions as described above. Six consecutive samplings of four replicates each (three plants as an experimental unit) per treatment (PM and TM), were taken every 25-35 days for analysing, by removing them from the bag for the following eight months. Samples were taken after 85 days of being transplanted inside the polybags for eight months (210 days from February to October, 2016). Seedlings were separated into the root and shoot with for a total of 12 plants (12 roots and 12 shoots) yielding 72 plants (144 roots and 144 shoots) per treatment. Roots and shoots were cleaned with tap water to remove any remaining substrate and rinsed with distilled water. They were placed into separate paper bags afterward and dried in an oven at 52°C for 24-72 hrs. The dried biomass was weighed.

Elemental analysis by SEM-EDS
Sample preparation: For the elemental analysis, two repetitions per treatment (three plants per experimental unit) with a total of 6 dried plants (shoot and root separately), were ground from each consecutive sample. Approximately 1 g of milled sample was taken into a crucible and calcined in a muffle furnace at 500°C for 3 hrs according to Lohse12.

After the time in the furnace, the crucibles were coolled at room temperature. The ash was weighed and placed into a 1 cm of the diameter sample holder. The ash was retained on a conductive double-sided carbon tape (NEM TAPE Nisshin Co., Ltd., USA). Samples were not coated.

Two spectrum readings of the two replicas were measured for the elements N, P, Mg, K, Ca and S using X-ray Dispersive Energy (EDS) Spectroscopy. The SEM-EDS equipment was an INCAx-sight model 7582 (Oxford Instruments, Oxfordshire, England) with a resolution of 5.9 keV at 137 eV and a detection area of 10 mm2 coupled with SEM scanning electron microscope column JEOL JSM-639OLV Noran Six (Jeol Ltd., Japan). The electron beam emission was generated from a tungsten filament with a spectrum range of 0-20 KeV, 11 mm of Work Distance (WD), voltage acceleration of 20 kV, electron beam diameter 60-70 SS (SpotSize) and a magnification of 5000x, the exposure for each spectrum was 120 s.

The spectrum showed the percentage of each mineral found in the ash. With this data, the percentage of each mineral in the plant was extrapolated with the amount of dry weight taken to produce ash.

Elemental analysis by ICP-OES: For elemental analysis by ICP-OES, four repetitions per treatment (three plants per experimental unit) separated into the shoot and root, were ground from each consecutive sample, 1 g of the milled sample was taken and calcined as previously described. Thereafter 10 mL of nitric acid (65%) was added to each crucible and boiled. Once the sample was cold, 1 mL of hydrogen peroxide (30%) was added and simmered13. The solution was coolled and filtered using Whatman® 42 paper in a 25 mL volumetric flask. The crucible and filter paper were washed with deionised water (Milli-Q 18.2 MΩ cm; Millipore, Bedford, MA, USA) and the sample was diluted to the mark with deionised water and mix. The sample was analysed by ICP-OES Optima 8300 (Perkin Elmer, Inc., PA, USA) for Mg, P, Ca, S and K14.

Nitrogen determination: Nitrogen in roots and shoots was quantified using the micro-Kjeldahl procedure15. Briefly, 0.5 g of the ground sample from each replicate was placed in a digestion tube with 0.4 g of CuSO4, 3 g of anhydrous Na2SO4 and 10 mL of concentrated H2SO4 according to Vanden-Heuvel16. The sample was processed in the micro-Kjeldahl distillation unit (Rapid Still II, 65200, Labconco). Digestion was completed when the solution turned colour green. Two aliquots from the digestion (between 6-11 mL approx.) were taken and the evolved ammonia was trapped in HCl 0.1 N.

Statistical analysis: The comparisons of dried biomass between both treatments (PM and TM), were performed with the t-student test. Plant nutrient percentages determined by both techniques (ICP-OES and SEM-EDS), were performed with the t-student test and Pearson correlation (trials from all samples were used). Charts of the nutrient extraction curves were obtained with Sigma Plot 11.0. The analyses were conducted with the statistical software R, version 3.3.017 (p<0.05).

RESULTS

Dried biomass of P. greggii var. greggii: The dry biomass of both shoot and root was greater (p<0.05) in plants grown in the peat-based (PM) media (Table 1). It was shown that after 210 days of transplantation (300 days after seed germination), the dry mass weighed 3.491±0.52 g shoot–1 in PM compared to 0.662±0.14 g shoot–1 in TM. Values of 1.1242±0.27 g root–1 in PM compared to 0.3038±0.16 g root–1 in TM were obtained.

Nutrient determination by SEM-EDS and ICP-OES: The percentage of the nutrient concentration is given in an average of four replicates for each consecutive sample in shoots (Table 2) and roots (Table 3). It is important to recall that N determination was made with micro Kjeldahl method instead of ICP-OES.

The obtained percentages from shoots samples show that there are no statistical differences between the evaluated analytical methods (ICP-OES and SEM-EDS) for P, S and Mg concentration evaluated at the final sample. A highly significant difference was found between| micro-Kjeldahl and SEM-EDS methods for N concentration, however, in TM substrate, no differences were shown for the first and final sampling. The highest N percentage was obtained during the final sample by micro Kjeldahl technique in PM substrate with 13.29±2.07% of N, in comparison to 1.66±0.24% in SEM-EDS. For evaluation on the TM substrate, an N concentration of 1.52±0.98% was obtained by micro-Kjeldahl in comparison to 0.27±0.12% by the SEM-EDS. The phosphorus percentage in shoots grown in PM substrate, varied from 0.50±0.28% analysed by SEM-EDS and 0.53±0.14% by the ICP-OES method at the final sample. This percentage was 25-50 times lower in TM substrate results (0.01-0.02%) (Table 2). It was demonstrated that P. greggii shoots absorbed more nutrients from PM substrate than TM.

The concentration of nutrients analyzed in the roots of P. greggii is shown in Table 3. Nitrogen concentration was analyzed using the SEM-EDS and micro-Kjeldahl techniques; It was observed that there were no statistical differences between the evaluated methods except for the last sample (p<0.05) cultivated in TM.

Table 1: Dried biomass of Pinus greggii var. greggii as affected by the substrate at varying days after transplant sampling dates
Treatment
Shoot/root
60 DAT (g)
90 DAT (g)
120 DAT (g)
150 DAT (g)
180 DAT (g)
210 DAT (g)
(PM)
Shoot
0.23
0.517
0.912
2.823
2.536
3.491
(TM)
0.151
0.164
0.292
0.699
0.352
0.662
p-value
p<0.05
p<0.001
p<0.01
p<0.001
p<0.001
p<0.001
(PM)
Root
0.159
0.2191
0.367
0.8215
0.81328
1.1242
(TM)
0.148
0.1545
0.1419
0.326
0.1711
0.3038
p-value
0.661
0.058
p<0.05
p<0.001
p<0.001
p<0.01
PM: Peat moss: perlite (1:1), TM: Forest soil, sand and silt (2:1:1), DAT: Days after transplant, n: 12 seedlings


Table 2: Average shoot nutrient concentration in P. greggii seedlings of all the trials grown on two substrates after 210 days of transplant
PM (%) TM (%)
Nutrient
Sample
SEM-EDS
ICP-OES
SEM-EDS
ICP-OES
N2
1
0.08±0.02
0.48±0.05
**
0.09±0.02
0.17±0.07
NS
2
0.09±0.019
1.56±0.26
**
0.11±0.05
0.51±0.14
**
3
0.13±0.024
1.94±0.44
**
0.15±0.02
1.45±0.19
**
4
0.75±0.19
5.61±1.22
**
0.23±0.02
0.88±0.01
**
5
0.34±0.10
4.69±1.50
*
0.08±0.009
0.94±0.44
*
6
1.66±0.24
13.29±2.07
**
0.27±0.12
1.52±0.98
NS
P
1
0.05±0.02
0.03±0.006
NS
0.02±0.006
0.02±0.006
NS
2
0.06±0.01
0.04±0.006
*
0.02±0.01
0.02±0.01
NS
3
0.09±0.01
0.04±0.009
**
0.03±0.01
0.02±0.004
NS
4
0.46±0.06
0.48±0.11
NS
0.01±0.002
0.03±0.003
**
5
0.26±0.05
0.12±0.03
*
0.004±0.001
0.01±0.001
**
6
0.50±0.28
0.53±0.14
NS
0.01±0.003
0.02±0.01
NS
K
1
0.10±0.01
0.10±0.01
NS
0.09±0.03
0.09±0.028
NS
2
0.15±0.03
0.13±0.01
*
0.13±0.06
0.11±0.06
NS
3
0.26±0.03
0.20±0.04
NS
0.23±0.02
0.13±0.02
**
4
1.03±0.20
0.91±0.38
NS
0.34±0.05
0.23±0.01
*
5
0.77±0.21
0.60±0.16
NS
0.09±0.003
0.09±0.01
NS
6
2.94±0.52
2.02±0.43
*
0.27±0.13
0.17±0.07
NS
Ca
1
0.129±0.029
0.088±0.01
NS
0.13±0.04
0.12±0.03
NS
2
0.109±0.01
0.07±0.012
*
0.22±0.10
0.17±0.08
NS
3
0.13±0.01
0.11±0.02
NS
0.20±0.04
0.22±0.03
NS
4
1.38±0.29
1.68±0.63
NS
0.39±0.01
0.31±0.01
**
5
0.47±0.14
0.28±0.07
NS
0.19±0.03
0.12±0.01
**
6
1.64±0.14
1.06±0.29
*
0.40±0.20
0.24±0.10
NS
S
1
0.03±0.006
0.03±0.006
NS
0.04±0.01
0.038±0.01
NS
2
0.02±0.003
0.02±0.006
NS
0.07±0.08
0.04±0.02
NS
3
0.02±0.008
0.03±0.006
NS
0.12±0.01
0.05±0.008
**
4
0.52±0.18
0.69±0.34
NS
0.09±0.01
0.078±0.007
NS
5
0.06±0.02
0.07±0.02
NS
0.09±0.01
0.029±0.003
**
6
0.20±0.08
0.34±0.07
NS
0.08±0.04
0.06±0.02
NS
Mg
1
0.047±0.01
0.03±0.006
NS
0.06±0.01
0.04±0.01
NS
2
0.047±0.005
0.03±0.004
*
0.05±0.02
0.05±0.02
NS
3
0.05±0.008
0.044±0.009
NS
0.05±0.01
0.06±0.008
NS
4
0.23±0.09
0.24±0.05
NS
0.12±0.01
0.09±0.007
*
5
0.21±0.05
0.11±0.02
*
0.03±0.006
0.03±0.002
NS
6
0.53±0.13
0.51±0.06
NS
0.06±0.02
0.06±0.03
NS
PM: Peat moss: perlite (1:1), TM: Forest soil, sand and silt (2:1:1), N2: Nitrogen, micro Kjeldahl method was used instead ICP-OES. t-student analysis, n: 4 replicates, **p<0.01, *p<0.05

Higher concentrations of N were detected using the micro-Kjeldahl (1.13±0.35%) than SEM-EDS (0.41±0.09%). Samples 1-4 had greater statistical differences between techniques (p<0.01) of N concentrations when they were cultured in PM. No significant differences were found in samples 5 and 6.

The highest concentration of N was observed in sample 6 of the PM substrate (1.62±2.02 SEM-EDS, 4.21±1.14 micro-Kjeldahl).

After 210 days of transplantation, the roots that were cultivated in PM substrate presented P values of 0.51±0.60% obtained by ICP-OES and 0.61±0.52% by the SEM-EDS method; roots grown in PM substrate absorbed sixty times more P concentration than those grown in TM (Table 3).

Table 3: Average root nutrient concentration (%) in P. greggii seedlings of all the trials grown on two substrates after 210 days of transplant
PM (%) TM (%)
Nutrient
Sample
SEM-EDS
ICP-OES
SEM-EDS
ICP-OES
N2
1
0.09±0.03
0.45±0.08
**
0.18±0.05
0.46±0.27
NS
2
0.07±0.03
0.50±0.06
**
0.19±0.03
0.37±0.13
NS
3
0.29±0.10
0.78±0.23
*
0.20±0.05
0.28±0.03
NS
4
0.55±0.07
1.41±0.19
**
0.28±0.11
1.07±0.80
NS
5
1.21±0.47
1.76±0.22
NS
0.28±0.04
0.50±0.27
NS
6
1.62±2.02
4.21±1.14
NS
0.41±0.09
1.13±0.35
*
P
1
0.07±0.04
0.04±0.01
NS
0.04±0.01
0.03±0.01
NS
2
0.07±0.03
0.04±0.02
NS
0.06±0.02
0.03±0.01
NS
3
0.24±0.15
0.14±0.05
NS
0.05±0.06
0.03±0.007
NS
4
0.39±0.10
0.20±0.02
*
0.03±0.01
0.028±0.01
NS
5
0.58±0.29
0.46±0.15
NS
0.04±0.01
0.027±0.003
*
6
0.61±0.52
0.51±0.60
NS
0.01±0.019
0.02±0.009
NS
K
1
0.07±0.02
0.08±0.03
NS
0.11±0.029
0.08±0.035
NS
2
0.07±0.01
0.08±0.04
NS
0.15±0.06
0.08±0.02
NS
3
0.34±0.15
0.23±0.08
NS
0.10±0.05
0.06±0.009
NS
4
0.19±0.05
0.16±0.02
NS
0.15±0.01
0.09±0.02
*
5
0.37±0.13
0.50±0.13
NS
0.08±0.02
0.09±0.01
NS
6
0.44±0.45
0.34±0.38
NS
0.05±0.02
0.07±0.02
NS
Ca
1
0.16±0.06
0.11±0.03
NS
0.37±0.11
0.32±0.16
NS
2
0.13±0.05
0.10±0.03
NS
0.44±0.08
0.37±0.10
NS
3
0.44±0.17
0.40±0.15
NS
0.35±0.09
0.37±0.06
NS
4
1.27±0.11
0.67±0.10
**
0.81±0.24
0.69±0.18
NS
5
2.99±1.68
1.80±0.71
NS
0.60±0.16
0.43±0.05
NS
6
3.48±4.11
3.32±4.2
NS
1.01±0.31
0.78±0.23
NS
S
1
0.02±0.005
0.02±0.004
NS
0.06±0.03
0.03±0.01
NS
2
0.012±0.002
0.02±0.01
NS
0.03±0.01
0.03±0.01
NS
3
0.12±0.06
0.12±0.04
NS
0.03±0.01
0.04±0.006
NS
4
0.61±0.03
0.24±0.07
**
0.14±0.04
0.11±0.02
NS
5
1.95±1.12
0.82±0.32
NS
0.07±0.01
0.05±0.007
NS
6
2.56±3.41
1.70±2.19
NS
0.07±0.03
0.11±0.03
NS
Mg
1
0.03±0.01
0.03±0.01
NS
0.08±0.02
0.04±0.02
NS
2
0.03±0.01
0.02±0.01
NS
0.09±0.02
0.05±0.01
*
3
0.06±0.02
0.06±0.02
NS
0.05±0.04
0.04±0.007
NS
4
0.09±0.06
0.06±0.01
NS
0.11±0.05
0.07±0.02
NS
5
0.13±0.14
0.16±0.03
NS
0.07±0.009
0.05±0.006
NS
6
0.10±0.09
0.16±0.18
NS
0.05±0.03
0.05±0.01
NS
PM: Peat moss: perlite (1:1), TM: Forest soil, sand and silt (2:1:1), N2: Nitrogen, micro Kjeldahl method was used instead ICP-OES. t-student analysis, n: 4 replicates, **p<0.01, *p<0.05

No significant differences were shown between the evaluated methods for the concentrations of K nutrients; A statistical difference (p<0.05) was only observed in sample four when it was cultivated on a TM substrate with higher results in SEM-EDS (0.15±0.01%) compared to ICP-OES (0.09±0.02%).

Samples at PM substrate present K concentrations of 0.44±0.45 and 0.34±0.38% analyzed by the SEM-EDS and ICP-OES methods respectively, after 210 days of transplantation.

The highest concentrations of nutrient K obtained in the TM substrate were observed in sample 4 (approximately 145 days after transplantation).

No significant differences were shown for Ca concentration between analytical methods, using all root samples on the TM substrate. The highest Ca concentration was present after 210 days of transplantation (1.01±0.31% SEM-EDS, 0.78±0.23% in ICP-OES). Regarding the PM substrate, it was shown that sample 4 had a statistical difference between techniques (p<0.05) with higher calcium results in SEM-EDS (1.27±0.11%) comparing ICP-OES (0.67±0.10%).

The highest Ca results were recorded after 210 days of transplantation (3.48±4.11%, SEM-EDS, 3.32±4.2% ICP-OES). This trend was also observed in S concentrations between the analytical methods that evaluate both substrates.

Image for - Determination of Nutrients by SEM-EDS and ICP-OES in Mexican Pine Pinus greggii var. greggii
Fig. 1(a-b): Nitrogen extraction curve (mg plant–1) in (a) Shoot and (b) Root in Pinus greggii var. greggii seedlings after 210 days after transplant (DAT)
PM: Peat moss: perlite (1:1), TM: Forest soil, sand and silt (2:1:1), SEM-EDS: Energy dispersive X-rays spectroscopy

Sample 4 showed a statistical difference between the techniques (p<0.05) with higher sulfur results in SEM-EDS (0.61±0.03%) comparing ICP-OES (0.24±0.07%).

The roots grown in PM substrate registered S concentrations of 2.56±3.41% in SEM-EDS and 1.70±2.19% in ICP-OES after 210 days of transplantation. The roots grown in TM substrate did not show significant differences between the evaluated methods; the highest results were presented in sample 4 (approximately 145 days after transplantation).

Regarding the concentration of Mg in roots, it was found that there were no significant differences between the techniques, in PM substrate samples. The highest Mg concentration was recorded after 170 days of transplantation (0.13±0.14% SEM-EDS, 0.16±0.03% ICP-OES). Regarding the samples grown on the TM substrate, sample 2 showed a difference in concentration between the techniques (p<0.05), SEM-EDS determined a higher concentration of Mg (0.09±0.02%) compared to ICP-OES (0.05±0.01%).

The uptake of nutrients by seedlings (Fig. 1-6), showed higher concentrations in samples grown in PM treatment than samples grown in TM treatment.

The uptake of N in the shoots in PM substrate after 210 days of transplantation, obtained by the micro-Kjeldahl method was 122.38±4.88 mg shoot–1, the determination by SEM-EDS showed a concentration of 20.22±0.49 mg shoot–1 (Fig. 1a). Regarding N uptake in plants on TM substrate, 9.18±0.416 mg shoot–1 were obtained by micro-Kjeldahl compared to 7.44±0.90 mg shoot–1 in SEM-EDS (Fig. 1a). For nutrient N, higher results were observed by the micro-Kjeldahl method.

Figure 1b demonstrates the N uptake in P. greggii roots. The highest concentration was obtained in PM substrate (23.99±1.59 mg root–1 in micro-Kjeldahl) and 12.44±0.78 mg root–1 in EM-EDS). Regarding TM substrate, the nitrogen uptake varied from 4.30±0.16 mg root–1 in SEM-EDS to 17.52±0.25 mg root–1 in micro- Kjeldahl.

The highest P extraction was recorded after 210 days of cultivation on both substrates; in the shoot, PM samples showed values of 6.79±0.25 mg shoot–1 obtained by ICP-OES and 7.77±0.215 mg shoot–1 obtained by SEM-EDS (Fig. 2a). Plants grown in TM obtained less than 0.46 mg shoot–1 in both techniques (Fig. 2a). In P. greggii roots, values of 11.27±1.08 and 7.12±0.38 mg P root–1 were obtained by SEM-EDS and ICP-OES respectively for PM treatment (Fig. 2b). In TM treatment plants acquired 2.24±0.26 mg P root–1 by ICP-OES compared to 1.52±0.07 mg P root–1 by SEM-EDS (Fig. 2b).

For K element concentration, after 210 days of transplant, seedlings obtained 23.61±2.55 mg K shoot–1 determined by ICP-OES, compared to 34.59±2.84 mg K shoot–1 performed by SEM-EDS, for plants grown on the PM substrate (Fig. 3a). These concentrations were almost ten times lower in TM treatment, where the highest reading was 3.58±0.20 mg K shoot–1 realised by ICP-OES and 2.11±0.20 K shoot–1 by SEM-EDS after 210 days of transplant (Fig. 3a).

Image for - Determination of Nutrients by SEM-EDS and ICP-OES in Mexican Pine Pinus greggii var. greggii
Fig. 2(a-b): Phosphorus extraction curve (mg plant–1) in (a) Shoot and (b) Root in Pinus greggii var. greggii seedlings after 210 days after transplant (DAT)
PM: Peat moss: perlite (1:1), TM: Forest soil, sand and silt (2:1:1), SEM-EDS: Energy dispersive X-rays spectroscopy, ICP-OES: Inductively coupled plasma-optical emission spectrometry


Image for - Determination of Nutrients by SEM-EDS and ICP-OES in Mexican Pine Pinus greggii var. greggii
Fig. 3(a-b): Potassium extraction curve (mg plant–1) in (a) Shoot and (b) Root in Pinus greggii var. greggii seedlings after 210 days after transplant (DAT)
PM: Peat moss: perlite (1:1), TM: Forest soil, sand and silt (2:1:1), SEM-EDS: Energy dispersive X-rays spectroscopy, ICP-OES: Inductively coupled plasma-optical emission spectrometry

Roots grown in PM media absorbed 3.46±0.18 mg K root–1 analyzed by SEM-EDS and 4.17±0.28 mg K root–1 by ICP-OES, versus 4.54±0.24 mg K root–1 by SEM-EDS and 1.41±0.004 mg K root–1 by ICP-OES from plants grown in TM media (Fig. 3b).

The maximum amount of sulfur obtained in PM treatment was 2.15±0.46 mg S shoot–1 (SEM-EDS) and 3.80±0.33 mg S shoot–1 (ICP-OES). Plants grown in TM treatment presented values of 1.68±0.26 mg S shoot–1 determined by SEM-EDS and 1.32±0.08 mg S shoot–1 analysed by ICP-OES (Fig 4a).

The maximum accumulation of S element in the root, from PM samples, was 24.78±1.06 mg S root–1 (ICP-OES method) and 13.20±0.53 mg S root–1 (SEM-EDS) (Fig. 4b).

The amount of Ca was higher in seedlings grown in PM treatment, with values of 18.00±0.76 mg Ca shoot–1 (SEM-EDS) and 11.12±0.81 mg Ca shoot–1 (ICP-OES) compared to TM treatment, 6.84±0.56 mg Ca shoot–1 (SEM-EDS) and 4.54±0.37 mg Ca shoot–1 (ICP-OES) (Fig. 5a).

Image for - Determination of Nutrients by SEM-EDS and ICP-OES in Mexican Pine Pinus greggii var. greggii
Fig. 4(a-b): Sulphur extraction curve (mg plant–1) in (a) Shoot and (b) Root in Pinus greggii var. greggii seedlings after 210 days after transplant (DAT)
PM: Peat moss: perlite (1:1), TM: Forest soil, sand and silt (2:1:1), SEM-EDS: Energy dispersive X-rays spectroscopy, ICP-OES: Inductively coupled plasma-optical emission spectrometry


Image for - Determination of Nutrients by SEM-EDS and ICP-OES in Mexican Pine Pinus greggii var. greggii
Fig. 5(a-b): Calcium extraction curve (mg plant–1) in (a) Shoot and (b) Root in Pinus greggii var. greggii seedlings after 210 days after transplant (DAT)
PM: Peat moss: perlite (1:1), TM: Forest soil, sand and silt (2:1:1), SEM-EDS: Energy dispersive X-rays spectroscopy, ICP-OES: Inductively coupled plasma-optical emission spectrometry

Concentrations of Ca were higher in roots, when grown in PM treatment, the plant extracted 20.93±2.4 mg Ca root–1 determined by SEM-EDS and 35.88±2.66 mg Ca root–1 by ICP-OES (Fig. 5b). The maximum Ca concentration found in roots grown in TM treatment was 3.63±0.54 mg Ca root–1 by the SEM-EDS method and 8.40±0.50 mg Ca root–1 by the ICP-OES method (Fig. 5b). Figure 6a illustrates the accumulation curve of Mg nutrient. Plants absorbed between 4.5-5.6 mg Mg shoot–1 in PM treatment samples in comparison to 1.3-3.32 mg Mg shoot–1 from TM treatment samples. It is shown in Fig. 6b that roots absorbed 4.71±1.06 mg Mg root–1 at 210 days of transplant in PM obtained by ICP method in comparison with the value 2.13±0.317 mg Mg root–1 obtained by EDS, for the TM treatment, lower results were obtained, 1.21±0.16 and 0.378±0.07 mg Mg root–1 by ICP and EDS methods respectively.

Correlation between SEM-EDS and ICP-OES: Figure 7 contains the Pearson correlation coefficients between SEM-EDS and ICP-OES percentage readings. No correlation was found among the evaluated techniques for nitrogen concentration (-0.28), obtaining a negative correlation (Fig. 7a).

Image for - Determination of Nutrients by SEM-EDS and ICP-OES in Mexican Pine Pinus greggii var. greggii
Fig. 6(a-b): Magnesium extraction curve (mg plant–1) in (a) Shoot and (b) Root in Pinus greggii var. greggii seedlings after 210 days after transplant (DAT)
PM: Peat moss: perlite (1:1), TM: Forest soil, sand and silt (2:1:1), SEM-EDS: Energy dispersive X-rays spectroscopy, ICP-OES: Inductively coupled plasma-optical emission spectrometry

The correlation test was highly significant (p<0.001) between ICP-OES and SEM-EDS techniques in the determination of P (0.759) (Fig. 7b), S (0.56) (Fig. 7c), K (0.55) (Fig. 7d) and Ca (0.79) (Fig. 7e). No significant correlation was observed in the concentration of Mg between the evaluated methods (Fig. 7f) (0.14).

DISCUSSION

The results of dry biomass found in PM substrate samples are similar to those reported by Oliet et al.5, in which seedlings of P. strobus L. produced with slow-release fertilizers (like osmocote® used in PM substrate), ranged from 2.6-2.8 g shoot–1 at 210 days after transplant; in another study, Arteaga-León et al.6 reported shoot dry weight of 1.05 g in P. ayacahuite seedlings after 2 years growth in the control, in TM substrate (used without fertilization), the results were inconsistent against the reported in a greenhouse study, carried by Sigala et al.18, Liu et al.19, where P. pinaster seedlings gained 2.43 g shoot–1 and 0.41 g root–1 of dry biomass from unfertilized plants after 205 days of emergence.

The levels of Ca, K and Mg are much lower in PM substrate compared to TM. The traditional substrate presented 3.92% of organic matter compared to 50% in peat-moss; in forest plantations, these plants are transplanted into poor soil conditions with low organic matter and high pH (7.0-8.3)20.

Nitrogen concentration (%) results, were higher when analysed by the micro-Kjeldahl technique against the SEM-EDS method, the difference could lie in the quantification of total nitrogen determined by SEM-EDS, that passed through ash where the inorganic compounds suffer alteration (melting, decomposition or volatilization)21. It is not clear what is the optimal temperature for obtaining ashes, usually, is carried out in the laboratory at temperatures up to 500°C (norm for determining ash content in wood, Bakisgan et al.22); Although 580-600°C is the temperature range utilized, a significant portion of the inorganic material volatilizes.

Figure 1-6 show the elemental absorption per plant (mg) from each evaluated treatment according to the method of analysis; it can be observed that both techniques follow the same trend, with the exception of nitrogen, as previously mentioned. Higher concentrations were obtained using the micro-Kjeldahl extraction (Fig. 1). These results are similar to those reported by Arteaga-León et al.6, where after 720 days of P. ayacahuite plant emergence, values of 12.28±2.66 mg plant–1 were obtained. The N concentration (%) determined by the micro-Kjeldahl method from shoots grown in PM substrate was 13.29±2.07% and for SEM-EDS method 1.6±0.24%. For seedlings grown in TM substrate, 1.52±0.98% was obtained by the micro-Kjeldahl method while SEM-EDS presented values of 0.27±0.12% N shoot–1.

Albaugh et al.23 states, that the optimal nitrogen range for pine, is between 0.12-1.33%. Therefore, P. greggii has a good amount of N, even after growing up in the forest soil. It is possible to see that after 140 days of transplant, this species of pine, needed almost three times the amount of N for its growth, than what it was observed 120 days after transplant. Deficiencies of this nutritional element will lead to low survival, low drought resistance and low development in the plantation field18.

Image for - Determination of Nutrients by SEM-EDS and ICP-OES in Mexican Pine Pinus greggii var. greggii
Fig. 7(a-f): Scatter plots from correlation between SEM-EDS technique and ICP-OES, (a) Nitrogen concentration (%) * for nitrogen correlation was made with Micro Kjeldahl and SEM-EDS, (b) Phosphorus concentration (%), (c) Sulphur concentration (%), (d) Potassium concentration (%), (e) Calcium concentration (%) and (f) Magnesium concentration (%)

As for the P absorption in P. greggii seedlings, 0.05-0.61% P plant–1 was obtained by SEM-EDS (Table 2, 3). The P is a limiting nutrient for the early growth of P. greggii and the availability of Ca in the substrate may hinder soil P and K uptake24.

The obtained shoot concentration of K plant–1 by both techniques (Fig. 3), is comparable to that it was obtained by Liu et al.19 who reported 31.97±3.05 mg K plant–1 in P. pinaster and higher than referred by Arteaga-León et al.6 in P. ayacahuite (6.40±1.27 mg K plant–1).

Typically, in young conifers, potassium deficiency takes the form of yellowing of terminal needles. This deficiency causes important diameter growth reduction and it also affects height growth, reflected in the dry biomass weight where TM treatment had weight values four to five times lower than PM (Table 1).

A concentration between 0.2-0.3% of sulfur is essential for conifers growth25, concentrations of S in roots grown in PM media, varied from 0.02-2.56% in SEM-EDS and 0.02-1.70% in ICP-OES and roots grown in TM media, varied from 0.03-0.14% analysed by SEM-EDS in contrast to 0.03-0.11% by ICP-OES. The concentrations acquired from TM substrate samples, follow the S concentrations found in conifers, reported by Garrison et al.26.

The standards of Ca ranged between 0.28-1.57% of forest species27. The foliar nutrient concentration was higher that reported Garrison et al.26. The highest extraction of Ca was presented after 210 days of transplant (35.88±2.66 mg Ca root–1) in ICP-OES and 20.93±2.49 mg Ca root–1 in SEM-EDS.

The Mg plant–1 absorption by both described techniques, varied from 0.13-0.18%, compared to that reported by Albaugh et al.23 in some forest species. Figure 6 illustrates the accumulation curve of this nutrient. Shoots absorbed between 4.5-5.6 mg Mg plant–1 (0.17-0.19%) in PM treatment in comparison to 1.3-3.32 mg plant–1 in TM treatment (0.13-0.14%). Garrison et al.26 found concentrations of Mg in conifer species between 0.05-0.09%, the obtained concentrations, fall between the limits of essential macronutrients in conifers reported by Burdon28. Readings by SEM-EDS and ICP-OES methods are consistent with what was achieved by Bai et al.29, where, the concentrations of minerals in SEM-EDS and ICP-OES were evaluated and found no significant differences between techniques, with the exception of K, who reports to be underestimated by ICP-OES.

Marguí et al.9 states, that the SEM-EDS technique can provide accurate concentration data for Al, Fe, Mg, Ca and K. The SEM-EDS method proved to be an efficient tool for the determination of metals in plants since it allows the elimination of the digestion stage and minimizes the mistakes in the recovery of some elements.

Studies of elemental content in P. greggii seedlings are scarce, even less, the use of different techniques to evaluate the nutritional composition in forest plants. Although SEM-EDS analysis is generally used to provide a qualitative proportion of elements present in the samples, it measures the relative abundance of emitted x-rays versus their energy and it is possible to determine the elemental composition of the volume sample30. This study demonstrates that it can also be used to obtain quantitative elemental analysis and has the advantage of being fast and easier. The procedure involves the obtaining of ash, to then be analysed in the equipment. This method avoids dangerous reagents that require high care and individual protection, time and experience.

Although it is known that the ICP-OES technique is more sensitive and precise than SEM-EDS31, Alomari et al.32 reported similar results with SEM-EDS than obtained by the ICP-OES.

CONCLUSION

For the production of P. greggii seedlings, a conventional management substrate (peat-based) is recommended, since plants exhibit a higher mineral absorption concerning plants grown in forest soil. It was demonstrated, that the nutrient uptake in both detection methods (SEM-EDS and ICP-OES) followed the same tendency with a highly significant Pearson correlation for P, K, Ca and S elements. Percentage of nitrogen in the plants were higher when analysed by micro-kjeldahl in comparison with SEM-EDS. It is possible to determine the elemental composition of P. greggii seedlings using the SEM-EDS technique, evading the use of corrosive materials, laborious and tedious procedures, that are used in wet digestion for the mineral extraction by ICP-OES analysis.

ACKNOWLEDGMENT

R. Casique especially thanks to CONACYT for the financial support under scholarship CVU 315450 and CICATA-Legaria research center for their hospitality and contribution towards laboratory experiments.

SIGNIFICANCE STATEMENT

This study discovered the applicability of the SEM-EDS method in the analysis of the concentration of nutrients in plants, which could be beneficial for students and research groups. Because they save time and costs in reagents. The method is friendly to the environment because they avoid the use of dangerous reagents such as hydrochloric, sulfuric and nitric acids, among others.

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