POST GRADUATE SCHOOL
ICAR-INDIAN AGRICULTURAL RESEARCH INSTITUTE
NEW DELHI – 110012
OUTLINE OF RESEARCH WORK
1. Name of the student: Kiran K. R
2. Roll No.: 10536
3. Discipline: SSAC
4. M.Sc. /PhD: Ph.D.
5. Date of Joining P.G. School: 1stAugust, 2014
6. Major Field: SSAC
7. Minor Fields: Environmental Science,
Agricultural Physics
8. Proposed title of thesis:“MOBILIZATION OF SOIL IRON TO MINIMIZE IRON DEFICIENCY CHLOROSIS OF SOYBEAN (Glycine max(L.) Merr.) UNDER AMBIENT AND ELEVATED CO2 AND TEMPERATURE CONDITIONS”.
Introduction and Justification
Soybean is relatively new crop to India, emerged as number one among oil seed crop grown in an area of 10.91 m ha with total production of 12.15 mt. The total area and production of soybean in the world were 118.14 mha and 318.80 mt, respectively (USDA, 2016). Soybean is a crop of considerable importance to India. We have the potential to double soybean production in the next decade through improved technologies and adequate management practices.India is supporting soybean cultivation for nutritional security, however biotic and abiotic stress thwarts expansion in area and productivity(NAAS, 2014).
It was projected that ambient atmospheric CO2 is expected to increase from current 400 mol mol-1 to 540-958 mol mol-1 by 2100. It was also predicted that mean average surface air temperature is expected to increase by 3 to 4 0 C during this period (IPCC, 2013). The increasing CO2 content of the atmosphere coupled to the soil atmosphere dissolve the CaCO3 thereby increase carbonate and bicarbonate ion activity (Emmerich, 2003).
Iron is the fourth most abundant element in earth's crust (after oxygen, silicon and aluminum), but the solubility and bioavailability is usually very low especially under neutral and high pH conditions, making iron unavailable to plants in alkaline and calcareous soils. Iron solubility is affected by many factors of which, amorphous Fe (III) oxide is of considerable importance, solubility of which follows the order Fe(OH)3 (amorphous) ˃ Fe(OH)3 (soil) ˃ Fe2O3(maghemite) ˃ FeOOH (lepidocrocite) ˃ Fe2O3 (haematite) ˃ FeOOH (goethite). Freshly precipitated amorphous Fe(OH)3has 3630 time more solubility than goethite (Lindsay and Schwab, 1982). Most of the iron present in earth crust constitutes Fe3+ form; infact Fe2+form is more important from the plant nutrition point of view (USEPA, 2003). The ferrous form is relatively soluble, but is readily oxidized to ferric form under well aerated condition which later precipitates out and renders it unavailable. Although the total content of iron is high in soils, all plants experience iron deficiency resulting from limited availability of soluble iron.
Iron deficiency can be considered as limiting factor for growth and productivity of soybean especially at high pH condition. Iron deficiency chlorosis (IDC) causes yield losses in many crops. It is an abiotic stress that impacts 30% of the world's agricultural land (Mori, 1999).All India Co-ordinated Research Project on Micronutrients in Soils and Plants reveals that 13% of Indian soils are deficient in iron. Moreover, utilization efficiency of applied iron is less than 2%.Accordingly, plants have developed iron-acquisition strategies to assist them in solubilizing iron in soil and subsequently obtaining adequate amounts of iron to stay alive (Mori, 1999).All plants, except grasses, acquire Fe2+ through Strategy I mechanism. Strategy I plants induce an Iron-Stress Response (ISR) under conditions of iron-deficiency stress, which involves increase in acidification of the rhizosphere, release of reductants, and organic acids at the root surface. Release of proton coupled with reduction in pH in the rhizosphere facilitates the uptake of iron from soil. All these activities facilitate reduction of Fe3+ to Fe2+by roots (Romheld and Marchner, 1984; Roberts et al., 2004). Research work addressing iron nutrition by plant under changing climate scenario is very scanty.
9. Previous work done:
Iron chlorosis affects many cereals and pulses grown in high pH and calcareous soils (Hansen et al., 2004).As the pH increases, iron solubility decreases, which leads to the development of IDC on calcareous soils with a pH range of 7.5 to 8.5 (Lindsay and Schwab, 1982).Due to the stability and insolubility of ferric oxides at neutral pH, soluble Fe3+ in aerobic environments are limited to a theoretical concentration of 10-17 M (Neilands et al., 1987), which is well below the 10-9 to 10-4 M needed by plants for optimal growth (Mori, 1999).
Soil scientists have used natural organic chelates in the mobilization of iron from soils (Powell and Szaniszlo, 1982).Iron concentration in soil solution is often higher than that expected from chemical equilibria equations of soil Fe minerals which were mainly attributed to the presence of organic molecules exhibiting various extents of Fe-chelation abilities (Siebner-Freibach et al., 2004). By virtue of high affinity of carboxylate group for soil solution Fe (III) and Fe (III) hydrous oxide, organic matter is thermodynamically able to reduce Fe (III), which is frequently observed in natural water, soils and sediments (Pedersen, 2006). The main storage protein of iron in plant is a heat stable, non-heam storage protein, called ferritin, an assemblage of thousands of iron and oxygen atoms inside a protein nanocage (Lonnerdalet al. 2006).
Soybean genotype differ in their ability to tolerate IDC, and this resistance is controlled genetically (Weiss, 1943).Environmental factor alone did not have a significant effect on iron level in plant, but genotype by environment (G x E) interaction was highly significant (Oikeh et al., 2003).One-hundred and eight SSR markers genetically linked to eight QTLs on eight molecular linkage groups (MLGs) previously identified for IDC were tested in a breeding population evaluated for IDC resistance on calcareous soils and shown that Satt481 was associated to IDC resistance across environments (Charlson et al., 2004). Several genetic mechanisms and quantitative trait loci have been identified for IDC resistance (Cianzio and Fehr, 1980, 1982; Lin et al., 1997, 2000).
Yields of most agricultural crops will increase under elevated CO2 by 15 to 41% for C3 crops and 5 to 10% for C4 crops (Cure, 1985; Kimball, 1983; IPCC, 2007; Lotze-Campen and Schellnhuber, 2009). C3 grains and legumes have lower concentrations of iron when grown under elevated atmospheric CO2 concentration (Myers et al., 2014). Recent studies have shown that occurrence of significant changes in soil inorganic carbon (SIC) pools of different soil types linked to climatic variation (Landi et al., 2003). The increasing CO2 content of the atmosphere coupled to the soil atmosphere dissolve the CaCO3 thereby increase carbonate and bicarbonate ion activity (Emmerich, 2003).
10. Research Gap
The challenges of the next century are to be tackled with our limited resources. In recent times, the yield potential of crop varieties has already reached a plateau owing to several constraints. The most important among them is the deficiency of mineral nutrients.Iron Deficiency Chlorosis (IDC) is an abiotic stress that limits crop production especially in calcareous soils and high pH soils. Therefore, judicious use of resources including adoption of iron efficient varieties, mobilization of soil iron in conjunction with soil amendment and microorganisms, evolving technologies which are cost effective and resource saving is essential.Further, research work addressing iron nutrition by plant under changing climate scenario is very scanty. There is a lacuna exist in understanding interaction between genotype and soil amendment and iron mobilizing microorganism with CO2 and temperature
Keeping all these in mind, a research will be conducted with the title “Mobilization of soil iron to minimize iron deficiency chlorosis of soybean (Glycine max (L.) Merr.) under ambient and elevated CO2and temperature conditions”.
11. Objectives
We undertake an investigation to minimize IDC by studying IDC tolerance and iron acquisition strategies by soybean genotypes. The specific objectives of the study are:
1.To study the basis of iron deficiency chlorosis in soybean genotypes
2. Evaluation of effectiveness of different strategies to mobilize soil iron and its impact on iron deficiency chlorosis tolerance by soybean genotypes
3. To study the effect of iron mobilization strategy inenhancing bioavailability of iron to soybean genotypes under ambient and elevated CO2 and temperature conditions
12. Programme of Research Work
Objective 1
To study the basis of iron deficiency chlorosis in soybean genotypes
12.1.1 Identification of iron efficient and inefficient genotypes of soybean with the help of hydroponics
Methodology:Hydroponics using Hoagland solution
Composition of Hoagland solution
N:210 mg L-1, K:235 mg L-1, Ca: 200 mg L-1, P: 31 mg L-1, S: 64 mg L-1, Mg: 48 mg L-1, B: 0.5 mg L-1, Mn: 0.5 mg L-1, Zn: 0.05 mg L-1, Cu: 0.02 mg L-1, Mo: 0.01 mg L-1
Experimental material:Approximately 50 Soybean genotypes
Treatment
T1: Fe sufficient Hoagland solution(5mg L-1soluble iron)
T2: Fe deficient Hoagland solution conditions
T3: Fe deficient Hoagland solution + Application of soluble source of Fe (5 mg L-1 Fe in the form of FeEDTA) after 10 days of planting
T4: Fe deficient Hoagland solution + Application of insoluble source of Fe (15mg L-1 total Fe in the form of Amorphous iron oxide) after 10 days of planting
(Fe deficient Hoagland nutrient solution should contain trace of soluble iron so as to facilitate growth of plant in these treatments)
No.of replication: 3
Observations to be recorded:
Root and shoot length
Fe accumulation in root and shoot
Root and shoot dry weight
Total chlorophyll content
Leaf greenness using SPAD 502
Tolerance Index
Iron deficiency recovery index
12.1.2 Screening of genotypes for total carbon exudation using 14CO2
Fourteen days old seedlings with two fully expanded trifoliate leaves will be placed individually in 100 mL Erlenmeyer flasks containing 50 mL of basal nutrient solutionwith sufficient and low-Fe. An air-tight chamber (length 70 cm x breadth 35 cm x height 65 cm) will be fabricated with two Teflon inlet tubes.The inlet tube (1 cm diameter) should be long enough to reach the floor of the chamber below which a petri dish should be placed. An aeration tube (0.5 cm diameter) will also maintained at plant canopy level to circulate air within the chamber using an air pump. Sufficient quantity of NaH14CO3solution (procured from Bhaba Atomic Research Center, Mumbai, India) with specific activity of 185 kBq will pour in the Petri dish and chamber will besealed using silicon paste. Addition of 1.0 N HCl to NaH14CO3solution using 10 mL syringe through the inlet tube allow complete evolution of 14CO2into the chamber. The inlet tube is then closed by a clamp. Plants will be illuminated under 350µmol m-2 s-1 PAR and incubate in 14CO2environment for an hour to facilitate assimilation of 14CO2. After 1 h, unassimilated 14CO2 in the chamber is quenched by adding 15 mL of saturated potassium hydroxide solution through the inlet tube into the petridish. Chamber will open after half an hour and plants should beshifted to controlled environment chambers with growth conditions as mentioned above. The root exudates will be collected at three time intervals viz. 24, 48 and 96 h after exposure of plants to 14CO2. Roots will be rinsed in double distilled water (pH 4.5) and then submerged in 50 mL of 0.5 mM CaCl2 solution (pH 4.5) for 4 h. After collection, the plants musthave returned to the respective normal and Fe-stress condition till the next collection (Dong et al. 2004). Sampling will be done by withdrawing 100 µl of aliquot from each flask and pipetting into scintillation vials. Before every sampling, care should be taken to make up the volume of CaCl2solution in each flask to 50 mL. The samples in vial will be evaporated at 500C in a hot air oven followed by addition of 10 mL of scintillation mixture (Cocktail O, SRL). At the end of 96 h, plants are taken out of the conical flask, blotted dry and separated into root and shoot. Fresh weights of roots will be recorded for each genotype. The 14C counts will be measured using liquid scintillation counter (Packard 1900 Tricarb). The total 14C exudation is expressed as disintegration per minute (dpm) per g-1 root-1. The relative total 14C exudation and relative total biomass should be calculated as follows:
Relative total root exudation (%) = Total 14C root exudation at low Fex 100
Total 14Croot exudation at sufficient Fe
Relative total biomass (%) = Total biomass at low Fe x 100
Total biomass at sufficient Fe
Based on the relative values for total 14C exudation and biomass, fifty genotypes will be categorized into fourgroups as:
(1)Fe-Efficient and responsive (FeER)
(2) Fe-Efficient and non-responsive (FeENR)
(3) Fe-Inefficient and responsive (FeIR)
(4) Fe-Inefficient and non-responsive (FeINR).
Expected output:Identification of iron efficient and inefficient genotypes
12.1.3 Evaluation of relative abundance of low molecular weight organic acids in root exudates of iron efficient and inefficient genotypes under iron deficiency stress
Methodology:
Exudation of carboxylic acids into the rhizosphere willbe consider as the root exudate while those inthe root apices represents the internal concentration. Out of 50, four genotypes representing all thefour categories will be selected and grow in nutrient solution with similar Fe levels as previously mentioned. Rootexudates will be collected from 14 days old plants following the method described by Dong et al. (2004). Plants with two fully expanded trifoliate leaves will be removed fromnutrient solution and rinse the root with double distilled water.After blotting excess water, roots will be immersed in 50 mL of 0.5 mM CaCl2solution (pH 4.5) in a conical flask. The whole set-up is set aside in growth chamberand keep for 4 h. The exudate will elute through Amberlite resinIR 120 (H) filled in a glass column (10 cm 91.8 cm) and filtered through 0.4 µm filter before injecting into high pressure liquid chromatography (Agilent Technologies, 1200 Infinity Series). To measure root internal concentrations of carboxylic acids, 0.1 g root apices (approximately 1 cm from the tip)will be collected and homogenize in 0.2 mL of 0.1 M HCl using pestle-mortar. To this, 1.8 ml of double distilledwater shouldhave added and transfer the contents to 2 ml centrifuge tubes. The samples will be incubated at 800C in water bath for 40 min, cool to room temperature and centrifuged at12,000 x g for 10 min. Supernatant will be collected in a fresh tube and 1 ml is diluted to 5 mlbefore characterization and quantification of carboxylic acids using HPLC.
Observations to be recorded:
•Low molecular weight organic acid in the root exudates such as citric, tartaric, oxalic and malic acids (µmol plant-1 day-1)
Expected output: Relative preponderance of low molecular weight organic acids in the root exudate will be obtained.
Objective 2
Evaluation of effectiveness of different strategies to mobilize soil iron and its impact on IDC tolerance by soybean genotypes
Methodology: Pot culture experiment with soybean crop. The soil will be treatedwith various treatments as given below
Design: RCBD
Treatments
Genotype: Fe efficient and responsive soybean genotype
Absolute control: RDF of all nutrient without iron
T1: RDF+FeSO4 at 25 mg pot-1
T2: RDF+SPM at 5 gpot-1
T3: RDF+ Partly decomposed SPM by Aspergillus niger at 5gpot-1
T4: RDF+Partly decomposed SPM by Aspergillus niger at 5g pot-1+ Plant inoculation of Piriformospora indica
T5: RDF+Partly decomposed SPM by Aspergillus niger at 5 g pot-1+ Plant inoculation with AM Fungi
T6: RDF+Pyrite at 20 mg pot-1
T7: RDF+Pyrite at 20 mg pot-1+ Plant inoculation of Piriformospora indica
T8: RDF+Pyrite + AM Fungi
T9: RDF+ Citric acid at 1mmol kg-1 soil-1
T10 : RDF + Siderophore producing bacteria (Pseudomonas fluorescens)
(SPM-Sulphitation press mud, IDC- Iron deficiency chlorosis, RDF- Recommended Dose of Fertilizer)
Observations to be recorded:
Soil observations at two stages 30 DAS and at harvest
No. of soil samples: 11 (treatment)x2(stages)x2(replication)=44
•pH and CaCO3 content in soil
•Available Fe, Mn, Cu and Zn content in soil
•CO3- and HCO32-concentration
•Organic acids in the root exudates such as citric, tartaric, oxalic and malic acids (µmol plant-1 day-1)
Plant parameters
•Scoring of IDC (30 DAS and 60 DAS)
•Total chlorophyll content (30 DAS and 60 DAS)
•Leaf greenness using SPAD 502 (30 DAS and 60 DAS)
•Root colonized by fungi (30 DAS)
•Root and shoot length (At harvest)
•Fe accumulation in shoot and root (At harvest)
Objective 3
To study the effect of iron mobilization strategy in enhancing bioavailability of iron to soybean genotypes under ambient and elevated CO2 and temperature conditionsExperimental Details:
Methodology: Pot culture experiment
Design:Factorial CRD (Levels of CO2 & temperature, components of treatment identified from objective 2 as factors)
Levels of CO2 and temperature: 1. 400±10mol mol-1, 30 0C/22 0C
2. 610±10 mol mol-1, 350C/26 0C
Experimental set up: Phytotron chamber
Soil type: Fe deficient calcareous soil (or alkali soil)
Variety: Iron efficient and responsive genotype
Observations to be recorded:
Soil analysis:Sampling at two stages (30 DAS and at Harvest)
•pH, EC, OC, CEC and CaCO3 content in soil
•Available Fe, Mn, Cu and Zn content in soil
•CO3- and HCO32-concentration
•Available N, P & K (kg ha-1)
•Organic acids in the root exudates such as citric, tartaric, oxalic and malic acids (µmol plant-1 day-1)
Plant analysis:
•Plant height (30 DAS and at harvest)
•Scoring of iron deficiency chlorosis (if any) (30 DAS and 60DAS)
•Root surface area (cm2 plant-1), root length (cm)(At harvest)
•Fe accumulation in root and shoot (At harvest)
•Total chlorophyll content (30 DAS and 60 DAS)
•Leaf greenness using SPAD 502 (30 DAS and 60 DAS)
•Fe accumulation in root and shoot (30 DAS)
Methods
Parameter / Method / ReferencepH / Potentiometry / Jackson(1973)
EC / Conductometry / Jackson(1973)
CEC / Neutral normal ammonium acetate / Jackson(1973)
Organic C / Wet oxidation method by making use of heat of dilution of sulphuric acid for reaction. / Walkley and Black (1934)
Available N / Alkaline permanganate method / Subbiah and Asija (1956)
Available P / Olsen’s method / Olsen et al. (1954)
Available K / Ammonium acetate method / Hanway and Heidel (1952)
Available sulphur / Turbidometric method / Williams and Steinbers (1959)
Available micronutrient cations / DTPA Extraction / Lindsay and Norvell (1978)
Expected output:Extent of soil iron to minimize IDC of soybean by adopting iron efficient variety, low cost iron source in conjunction with organic acid and iron mobilizing micro-organism will beobtained under ambient and elevated CO2and temperature condition.
Facilities required and their availability
The facilities available in the Division of Soil Science and Agricultural Chemistry, as well as other divisions such as Agricultural Chemicals, Plant Physiologyand National Phytotron facility will be utilised for the study.
Note: (1) Whether Radioactivity is involved in the proposed research work : Yes
(2) Whether Radio-safety badge has been obtained or applied for : Yes
(3) Whether the laboratory in which the work has to be carried out is approved
for radioactivity work? : Yes
Date: Signature of student
Recommended by:
Advisory committee Name and DesignationDivision Signature
Chairman:Dr. R. N.PandeySSAC
Principal Scientist
Co- chairman:Dr. S. C. Datta SSAC