Isotopes in plant nutritional studies

WEL COME

UTILIZATION OF ISOTOPES IN PLANT NUTRITIONAL STUDIES

Seminar-Ion

Veerendrapateel, L.Snr. M.Sc.(Agri)PG15AGR7121

Sequence of presentation: Introduction

Isotopes- Definition and types of isotopes

Application of isotopes• In Agriculture• In plant nutrition

Research papers

Conclusion

Introduction

An atom….

What is an Isotope….? Atoms of element with different numbers of

neutrons are called "isotopes" of that element

+

+

+

+

+

+

NucleusElectrons

Nucleus

Neutron

Proton

Carbon-12

Protons 6Electrons 6Neutrons 6

Nucleus

Electrons

Carbon-14

Protons 6Electrons 6Neutrons

8

+

+

+

+

+

+

Nucleus

Neutron

Proton

The existence of isotopes was first suggested in 1913 by

Frederick Soddy- Radio chemist England

Isotopes

Stable Unstable

No. Of protons < no of neutrons No. Of protons > no of neutrons

Unstable atoms have an excess of energy or mass or both

Undergo radioactive decay- ionizing radiation

Do not undergo radioactive decay

eg; 15N, 31P, 18O, 12C etc..eg; 32P, 11C etc..

Table 1: Radiations produced by unstable isotope

Type Of Radiation

Description Electrical Charge

Relative Damage To Living Cells

- Radiation Fast moving particles containing two protons and two neutrons

Positive Most damaging

- Radiation Fast moving electrons Positive Or Negative

Most damaging

- Radiation Electromagnetic radiation, like light but with A much shorter wavelength and higher energy

None Least damaging

Applications of IsotopesMedicine ArcheologyIndustrial uses- Na-24 is used to detect the leakage of underground pipes, in the smoke detectors, help to control the thickness of plastic, paper and metal sheets in respective industriesEnergy- Radioisotope thermoelectric generators (RTGs)Agricultural uses

Americium -241

Application in AgricultureIsotopes are used widely in the field of

Crop improvement Soil fertility and Plant nutrition Irrigation and Water management Insect pest control Livestock production and health Chemical residue and pollution Food preservation Soil conservation

Application in Soil fertility and Plant nutrition

• Fertilizer use efficiency & uptake of fertilizers• Ions mobility in soil and plants• Metabolism of nutrients by plants.• Biological nitrogen fixation• Rate of soil erosion and soil formation.• Nutrient turnover in soil

• Genotypic difference in nutrient uptake and use• Recovery of nutrient from crop residues.• Nitrogen gaseous losses (volatilization and

denitrification)• Degradation of nutrient among plant parts• Tolerance of plant for salinity and drought

Measurement of fertilizer use efficiency

1) The classical or conventional method based on yield.2) Methods based on nutrient uptake:

1. Differential method: Indirect method2. Isotopic method: Direct measurement of uptake from the applied fertilizer through the use of isotopes.

Using labeled fertilizer with isotope 15N and radioactive isotopes 32P or 33P etc.

Isotopic labeling…?Technique to track the passage of an isotope

through a reaction, metabolic pathway.George De Hevesy- 1943

• A radioactive compound is introduced into a living organism and the radio-isotope provides a means to construct an image showing the way in which that compound and its reaction products are distributed around the organism

In this technique, one/more of the atoms of the molecule of interest is substituted for an atom of the same chemical element, but of a different isotope (radioactive isotope used in radioactive tracing).

Used in chemistry and biochemistry: understand chemical reactions and interactions.

Ways to detect the presence of labelling isotopes

1. Mass- Mass spectroscopy2. Vibrational mode- IR-spectroscopy3.Radioactive decay- Nuclear magnetic resonance(NMR), Autoradiographs of gels- Gel electrophoresis Liquid scintillation Geiger-Mueller (GM) counters

Stable Isotope Analysis (SIA) - 15N, 13C, 18O and 34S

In stable isotope analysis, milligram amounts of samples are combusted or pyrolysed at high temperature. After suitable preparation the measurable gases (N2, CO2, CO or SO2) are separated on a chromatography column. The gas species of different masses are subsequently measured by mass spectroscopy

MASS SPECTROMETER

Liquid scintillation

https://www.perkinelmer.com.cn/CMSResources/Images/46-148778lsc_principle.JPG

Geiger-Mueller (GM) tube, also called as GM counter.

Element Stable isotope

Radio isotope

Typical applications

Carbon 12C 14C Photosynthesis, SOM studies, Carbon balance

Hydrogen 1H 3H Water movement, Biochemical studies

Oxygen 18O, 16O 15O, 13O Photosynthesis, Respiration, HydrologyPotassium 39K 42K Ion uptake mechanism,Magnesium 24Mg 28Mg Movement in plantSulfur 32S 35S Availability from soil, uptake from soil and airIron 56Fe 59Fe Soil erosion, movement in soil and plantsChlorine 35Cl 36Cl

37ClSolute movement in soil, Herbicidal effects on life forms

Cesium 133Cs 134Cs137Cs

Soil erosion (sediment movement and deposition)

Boron 11B, 10B 12B Foliar absorption, Soil moisture studiesMolybdenum 96Mo 99Mo Plant nutrition

Table 2. Principal isotopes used in soil-plant studies

Zapata, 1990

PHOSPHORUS

Isotope Natural abundance (atom %) Typical applications

13N Trace N- fixation Denitrification.

14N 99.63 14N enriched materials for single season FUE

15N 0.368

FUE, biological N fixation N transformation in soils (N-cycling) Animal nutrition studies. Nitrate pollution in groundwater.

Isotope Half life (days) Typical applications

32P 14.26 Fertilizer use efficiency Residual P fertilizer studies Root activity pattern of crops

33P 25.34

Root autoradiography Double labelling for root. Activity pattern of crops. Fertilizer use efficiency.

NITROGEN

Zapata, 1990

Principle in use of Labelled fertilizer

“For a known constant amount of radioactivity, the specific activity is proportional to the total amount of test element present in the system”.

Specific activity:

Specific activity of standard:

Specific activity of Sample:

100

% of nutrient derived from the fertilizer:

% Ndfs = 100- %Pdff% of nutrient derived from the soil:

Fertilizer nutrient uptake : Total nutrient uptake

100

:

For example: In a pot culture experiment• 32P labelled fertilizer containing 100 mg P with an activity of

5000 k Bq is added.• An aliquot of a plant sample containing 50 mg P gives an

activity of 500 k Bq• Specific activity of standard or fertilizer P: 5000/100= 50 k Bq

mg-1

• Specific activity of sample: 500/50= 10 k Bq mg-1

• = 10/50 ×100 = 50%• 100-50 = 50%• % utilization of fertilizer nutrient = 50 ×50/100= 2.5

• For stable 15N same principle applies, instead of “specific activity” the term “% 15N atom excess is used”

** since the amount of sample is expressed as %15N atom excess over the natural abundance of 0.3663. i.e. 14N= 99.63%, 15N= 0.366%(subtracting 0.3663 from the determination of 15N abundance to obtain 15N atom excess).

% Ndff X 100

• In a field experiment 70 kg N/ha in the form of 15N labelled urea fertilizer with 1.566 % 15N excess was applied to a crop

• The crop was harvested with a dry matter yield of 4 tons/ha and plant sample had 0.566 % N abundance and 3% total N.

• % 15N atom excess plant = 0.566 - 0.366 = 0.20• % 15N atom excess fertilizer = 1.566 - 0.366 = 1.20• % Ndff: 0.2/1.2 ×100 =16.66• % Ndfs = 100 – 16.66= 83.33• Total N yield = 4000 x 3/100= 120 kg N/ha• Fertilizer N yield = 16.66/100x 120 = 19.99kg N/ha• % Fertilizer N utilization = 19.99/70 x 100 = 28.56 %

Advantages of isotope study1.With the help of radioisotopes we can easily locate the

presence of a single atom and molecule and their movement.

2.Very small quantities of labelled nutrients can be accurately measured in presence of large quantities of other nutrients.

3.Tracer technique enables one in tracing those elements taken by the plants accurately and precisely.

4.It also helps to study accurately the interaction among the mineral nutrients

5. You can label specific atoms (say carbon-1 in glucose) to follow where each one goes.

6. A radioactive molecule is chemically exactly like the unlabelled form. Thus, it will behave just like the unlabeled form so you dont have to worry about effects due to the labelling itself.

7. Since carbon, hydrogen and phosphorus can be easily purchased in radioactive forms, you can make any biomolecule in a radioactive form.

Disadvantages:

1. Radioisotopes are rather expensive.2. Radioisotopes are hazardous and must be handled

with extreme care. By the same token, they present a disposal hazard.

3. Some radioisotopes (like P-32 and I-125) have short half-lives, so have to be used quickly.

Research Findings

The application of isotopic (32P and 15N) dilution techniques to evaluate interactive effect of

phosphate solubilizing rhizobacteria, mycorrhizal fungai and Rhizobium to improve the agronomic efficiency of of rock phosphate

for legume crops

Barea et al. (2002)

Institute of Systematica and Ecology, Cuba

Pot culture experiment

Soil Properties

pH : 6.8

Organic carbon (%)

: 0.8

Total N (mg kg-1) : 2600

Avail. P (mg kg-1) : 15

Exch Ca. (meq l-1) : 10

Material and methods

• Pot culture- Factorial RCBD• Treatments: 8, Replications: 5• 4 microbial treatments

1. Rhizobium inoculation- Rhizobium meliloti (WTGR4 isolate)2. Arbuscular Mycorrhiza(AM) inoculation- Glomus mosseae. 3. PSB inoculation(RB)- Enterobacter sp4. AM+ RB inoculation

• 2 Chemical treatments1. Unammended control- without P application2. Rock phosphate application: 100 mg per pot (11.4% total P)

Plant selected: Alfalfa (Medicago satia L.,)

• Plants were fertilized (5 ml wk−1 pot−1)with a basal nutrient solution (lacking N and P) in the following amounts

• After 10 d of plant growth each pot received a solution of (15NH4)2SO4 with 10% 15N atom excess, which supplied 2 mg N kg−1 soil. An aliquot containing 1850 K Bq 32P pot−1 was added to obtain sufficient activity in the plant material.

Nutrients Conc. (μmol kg−1) Nutrients Conc. (μmol kg−1)

K2SO4

MgSO4.7H2OMnSO4.H2OCuSO4.5H2O

20082029118100

ZnSO4.7H2O CaCl2.6H2OH3BO3

NaMoO4.2H2O

3521812

• Plants were harvested after 55 d of growth.• The N isotopic composition of plant was determined by

using an automated N analyzer- continuous-flow isotope ratio mass spectrometer (ANA–MS method).

• The 32P activity in the plant material was measured by liquid scintillation counting of the 32P expressed in Bq.

• The specific activity of P was then calculated by considering the radioactivity per amount of total P content in the plant and expressed in Bq mg P−1

Table 3. Effect of treatments on shoot weight of alfalfa plants

Shoot dry weight (mg pot-1)

Microbial treatment

Chemical treatments

Control Rock phosphate

Rhizobium (WT)

Control 269 390

Rhizobacteria (RB) 290 535

Mycorrhiza (AM) 405 570

RB+ AM 560 618

Barea et al. (2002)

Table 4. Effect of microbial treatment on shoot N content and atom percent 15N excess in alfalfa plants

Microbial treatment

Chemical treatments

Control Rock phosphateN

content(mg pot-

1)

15N % a.e N content (mg pot-1)

15N % a.e.

Rhizobium (WT)

Control 12.8 0.90 15.1 0.93Rhizobacteria (RB) 12.1 0.81 25.3 0.65

Mycorrhiza (AM) 20.1 0.60 27.8 0.62

RB+ AM 29.6 0.59 31.2 0.5015N : 10% atom excess which supplies 2 mg N kg-1 32P: 1850 K Bq 32P pot-1

Barea et al. (2002)

Microbial treatment

Chemical treatments

Control Rock phosphate

P content (mg pot-1)

32P/TotalP

content (mg pot-

1)

32P/Total

Rhizobium (WT)

Control 0.39 2200 0.65 1083

Rhizobacteria (RB) 0.34 2080 1.10 900

Mycorrhiza (AM) 0.81 1333 1.75 650

RB+ AM 1.01 1117 1.85 580

Table 5. Effect of microbial treatment on shoot P content and specific activity in alfalfa plants

Barea et al. (2002)

Fig 1. Effect on dry matter

Barea et al. (2002)

Fig 2. Effect on accumulation of N and P (g 10m-2)

Barea et al. (2002)

Assessment of FUE under enhanced crop intensity of vegetables due to intercropping

using 15N and 32P labelled fertilizers.

Kotur et al. (2010)

Indian Institute of Horticultural Research, Bangalore

Soil Properties

Texture : Sandy loam

pH : 5.9

Org. N (%) : 0.3

Avail. N (kg ha-1) : 246

Avail P (kg ha-1) : 15.5

CEC (cmol(p+)kg-

1): 8.7

Table 6. Yield under different solo crops and their combinations

Treatment Capsicum* onion**

Watermelon*Radish**

Okra*Frenchbean*

*(kg/1.8 m2)

Sole Crop 1* 6.3 15.5 5.6

Sole Crop 2** 2.7 8.9 2.4Crop combination$ 4.4 15.6 5.5

S.E.(±) 0.10 0.25 0.14

CD (P=0.05) 0.35 0.88 0.48

*Main crop, ** intercrop, #capsicum-eq in capsicum, onion; watermelon-eq in watermelon, radish; okra-eq in okra, Frenchbean crop combination, $crop combination of respective main and intercrop.

15N urea: 1.0 atom%; 32P labelled superphosphate: Specific activity of 0.2mCi/g of P

Kotur et al. (2010)

Table 7. Fertilizer N utilization under different solo crops and their combinations


Watermelon*Radish**

Okra*Frenchbean**

Fertilizer N utilization (%)

Sole Crop 1* 10.85 23.70 22.76

Sole Crop 2** 25.25 37.16 23.10

Crop combination 6.60 19.12 6.44

S.Em (±) 0.59 0.84 0.82

CD (P=0.05) 2.04 2.92 2.84

Kotur et al. (2010)

Table 8. Fertilizer P utilization under different solo crops and their combinations


Watermelon*Radish**

Okra*Frenchbean**

Fertilizer P utilization (%)

Sole Crop 1* 11.9 4.89 8.24

Sole Crop 2** 7.09 10.43 10.14

Crop combination 6.18 6.13 9.31

S.E.(±) 0.15 0.13 0.23

CD (P=0.05) 0.50 0.44 0.79

Kotur et al. (2010)

Effect of 15N- labelled urea application alone and in combination with FYM/green manure

on rice yield and NUE by rice.

Battacharya et al. (2006)

Division of Soil Science and Agricultural Chemistry, Indian Agricultural Research Institute, New Delhi

Soil PropertiesSoil Type : Typic Haplustept

Texture : Sandy loam

pH : 8.50

EC (dSm-1) : 0.45

Total N (mg kg-1) : 680

O.C. (g kg-1) : 4.4

Avail. N (mg kg-1) : 102

Avail. P (mg kg-1) : 11.2

Avail. K (mg kg-1) : 99.0

CEC (cmol(p+) kg-1) : 9.7

Table 9. Treatment details

S.NoNitrogen

rate (kg ha-1)

Source and notation

1 0 Control2 90 Urea (U)3 120 Urea (U)4 90 2/3 N as urea+ 1/3 N as GM (UG2:1)5 90 1/2 N as urea+ 1/2 N as GM (UG1:1)6 120 2/3 N as urea+ 1/3 N as GM (UG2:1)7 120 1/2 N as urea+ 1/2 N as GM (UG1:1)

8 90 2/3 N as urea+ 1/3 N as FYM (UG2:1)




GM (2.31% N, 0.48% P and 0.61% K); FYM (0.46% N, 0.33% P and 0.44% K)Battacharya et al. (2006)

Table 10. Effect of N levels and its combination with FYM or green manure on yield of rice

SourcesGrain yield(t ha-1) Straw yield (t ha-1)

N levels (kg ha-1)90 120 Mean 90 120 Mean

U 5.78 6.62 6.20 8.61 10.39 9.50UG2:1 5.77 6.66 6.22 8.92 9.72 9.32UG1:1 5.50 6.25 5.88 7.62 9.55 8.59UF2:1 5.32 5.73 5.53 8.72 9.52 9.12UF1:1 5.53 6.17 5.85 7.64 8.52 8.08Mean 5.58 6.29 - 8.30 9.54Control - - 4.08 - - 6.26

CD (P=0.05)

Source NS 0.83Level 0.33 0.52S×L NS NS

15N urea: 5.005% atom excess Battacharya et al. (2006)

N uptake (kg ha-1)

SourcesN levels (kg ha-1)

90 120 MeanU 101 134 117UG2:1 110 125 118UG1:1 89 118 104UF2:1 97 114 106UF1:1 100 113 106Mean 100 121 -Control - - 65.6

CD (P=0.05)

Source 9.9Level 6.3S×L 14.0

Table 11. Effect of N levels and its combination with FYM or green manure on total N uptake by rice at harvest


SourcesGrain (%Ndff) Straw (%Ndff)


U 25.1 24.9 25.1 27.3 33.5 30.4UG2:1 23.8 29.0 26.4 22.9 28.3 25.6UG1:1 22.1 22.6 22.4 19.0 22.3 20.7UF2:1 21.5 27.9 24.7 25.7 26.6 26.1UF1:1 20.2 25.8 23.0 22.7 21.7 22.2Mean 22.6 26.1 - 23.6 26.5 -

CD (P=0.05)

Source 2.48 2.06Level 1.57 1.30S×L 3.51 2.91

Table 12. Effect of N levels and its combination with FYM or green manure on %Ndff of rice grain and

straw


SourcesN levels (kg ha-1)

90 120 MeanU 29.3 31.9 30.1UG2:1 43.2 47.5 45.4UG1:1 41.9 41.8 41.9UF2:1 37.8 38.4 38.1UF1:1 47.3 45.9 38.1Mean 39.9 41.1 46.6

CD (P=0.05)Source 3.5Level NSS×L NS

Table 13. Effect of N levels and its combination with FYM or green manure on nitrogen use efficiency by rice


SourcesNO3-N (mg kg-1) NH4-N (mg kg-1)


U 3.20 3.38 3.29 6.77 6.95 6.86UG2:1 3.01 2.58 2.79 6.62 8.82 7.72UG1:1 2.72 3.02 2.87 7.85 8.82 8.34UF2:1 2.69 2.66 2.68 6.34 7.60 6.97UF1:1 2.74 3.38 3.06 7.37 8.19 7.78Mean 2.87 3.00 - 6.99 8.08 -Control - - 2.56 6.58

CD (P=0.05)

Source NS NSLevel NS 2.05S×L NS NS

Table 14. Effect of N levels and in combination with FYM or green manure on NO3-N and NH4-N conc. of rice soil at harvest


Predicting soil organic matter stability in agricultural fields through

carbon and nitrogen stable isotopes

Clercq et al. (2015)

Department of Earth and Environmental Sciences, KU Leuven, Belgium

Material and methods

• Survey and laboratory analysis• Places surveyed: a. Belgium- Boutersem and Gembloux b. Austria- Gross- Enzersdorf and Grabenegg,

Belgium- a. Boutersem

The five treatments sampled for this site are: 1. an unfertilized control, 2. a mineral fertilized control, 3. a three-yearly application of VFG-compost

comprising of 45 tons per hectare4. two yearly applications of VFG compost

comprising of 15 and 45 tons per hectare.

b. Gembloux1. Mineral fertilized control2. Stable manure application

Austria- a. Gross- Enzersdorf

Sampling at this site was taken from:1. Conservation tillage,2. Conventional tillage and3. Permanent grass alley

b. GrabeneggIt was a permanent grass land for 15 years

Isotope analysisCarbon and nitrogen content and their

respective stable isotope ratios were analyzed for the POM fraction and bulk soil with an• Elemental analyzer coupled with a mass

spectrometer• For the protected mineral associated organic

matter fraction (mOM), carbon and nitrogen content were calculated as the difference between the bulk soil and the POM

Concept of relative abundance

For nitrogen standard is the atmospheric air, therefore Rstd= 0.366/99.633= 0.0036For sample =…???

Fig 3.Model for organic matter stability given by Conen et al.(2008)


Data analysis and calculations

Where,m & p - ẟ ẟ ẟ15N for mOM & POM respectivrly

rm & rp- C/N ratio for mOM & POM respectivrlyCm & Cp - Carbon mass for mOM & POM respectivrlyfC & fN – fractions of C & N lost during degradationɳ- relative SOM stability

Fig 4. C/N ratio and ẟ15N signature for the SOC fractions of the experimental sites


Table 15. Carbon concentration(mg/g of dry soil)


Table 16. Relative stability(ɳ) of soil organic carbon


conclusion

Thank you