Thesis

Exploring the potential of wheat (Triticum aestivum L.
) cultivation on NaHCO3
extractable phosphorus in soil
By
IQRA ZAFAR KHAN
M.Sc. (Hons.) Department of Agronomy (UAF)
A dissertation submitted in partial fulfillment of the requirements for the degree of
MASTER OF PHILOSOPHY
IN
AGRONOMY
UNIVERSITY OF AGRICULTURE,
FAISALABAD
2021
1
DEDICATED TO
HAZRAT MUHAMMAD
(PEACE BE UPON HIM)
AND
MY PARENTS
WHO’S PRAYERS ARE KEY BEHIND
MY SUCCESS
2
ACKNOWLEDGMENT
To esteem the Highness of Almighty Allah, I feel myself inept as my words have lost their
expressions, knowledge is lacking and diction is too short to express gratitude in the
rightful manner to the blessings and support of Allah Almighty whose help had flourished
my ambitions and helped me to attain goals. Quivering hands feel mortified to hunt for
words of praise for Holy Prophet Muhammad (P.B.U.H.) for enlightening our lives with the
faith in Allah, selecting course of contented conscience, converging all
His kindness and mercy upon him.
The work presented in this manuscript could never have attained its final shapes without
the suggestions and guidance of my respectable supervisor Professor Dr. Imran Ashraf,
Department of Agronomy, University of Agriculture, Faisalabad. I will be ever grateful to
him for his guidance and constructive suggestion.
I am thankful to the thesis supervisory committee that includes Prof. Dr.
Muhammad Sana Ullah, Institute of Soil and Environmental Sciences, University of

Agriculture, Faisalabad and Dr. Hafeez ur Rehman, Department of Crop Physiology,
University of Agriculture, Faisalabad. Their valuable feedback helped me to improve the
dissertation in many ways.
No dedication could ever be completed without expressing my humble obligation to my

sweet parents and siblings who always longed for my successful and happy life. Their
endless efforts and best wishes sustained me at all stages of my life and encouraged me
for achieving my ideas.
3
IQRA ZAFAR KHAN
4
Abstract
Phosphorus is an important plant macronutrient. Plant growth in low P soils is a

primary limitation in most soils of the world because phosphorus is tightly bound to
soil components. This project aims to know the effective conditions of extraction
such as the period of shaking, the concentration and pH of NaHCO 3 solution on the
amount of phosphorus extracted from the soil and its correlation with growth
parameters of wheat plants will be investigated. Because the Olsen approach has
gained a reputation as the best and cheapest method for mild acidic to basic soils
over the years, it is recommended. Therefore, A pot experiment will be conducted to
investigate the effect of phosphorus uptake at the Agronomic Research Area,
University of Agriculture Faisalabad during 2021. Phosphorus level will be taken
as 10 mg P/kg sand (P1), 10mg P+90 mg apatite P/kg sand ( P2) and 100 mg P/kg
sand (P3). Equal nutrient solution will be ap p l i e d with one weak interval. 2 kg
sand will be filled in each pot with 10 seeds per pot. Experiment will be carried out
under completely randomized design (CRD) with factorial arrangement and having
three replication treatments. Data regarding the release of organic acids on
phosphorus uptake, root exudates, soil P content, plant P content, soil pH and
biomass component of wheat will be determined by following standard
methodologies. Data will be statistically analyzed by using Fisher’s analysis of
variance procedure. Least significant difference (LSD) test at 5% level of
significance will be used to differentiate the mean of all treatments.
INTRODUCTION
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The plants have always been a key for survival as they are vital to meet the worlds
food needs. They provide over 65% of food protein and more than 80% of food energy
globally. The plant products directly contribute about 82% of the total world food production
harvested from land resources while animals and marine products contribute only 17%.
Among four major groups of plant species; cereal grain contribute the largest and the most
important single group of foods and play a dominant role in the total world food supply.
(FAO, 2001).
Wheat (one of the leading cereal) has been cultivated since prehistoric times and is
among the oldest mans crop. It was originally domesticated (almost 1000 years ago) in a hilly
region of Southwestern Asia (near East) called the fertile crescent. The area is bordered on
one side by the Tigris-Euphrates basin and on the other side by the mountains of what are
now Iran, Turkey, Syria and Jordan. It rapidly become the most important cereal and is still
second most produced cereal after maize. The modern wheat, the hexaploid (Triticum
aestivum L.) is the most cultivated species of wheat in the world.
Wheat is an excellent health building food (Kumar et al., 2011) and is an excellent
source of minerals, dietry fiber, protein and B-group vitamins although nutritional
composition of wheat grain might be influenced by environmental conditions. The
adaptability and high yields of wheat are considered key to its dominance in temperate world,
but unique properties of wheat flour dough has given it a clear advantage over other
temperate crops. Wheat dough can be processed into a range of breads, cakes, biscuits and
other baked products and processed foods like noodles and pasta. The predominant
carotenoids present in wheat is Lutein (Abdel-Aal, 2007) which along with zeaxanthin, is
vital for the health of human skin and human eyes. The wheat bran and wheat germ are
responsible for protection against heart diseases, constipation, disease of the colon called
diverticulum, ischemic, appendicitis, diabetes and obesity (Kumar et al., 2011).
In Pakistan, wheat is cultivated on largest acreages in almost every part of the country
and is staple diet of population of the country . Average per capita consumption of wheat in
Pakistan stands approximately at 125 kg per anum, which works out to be over 20 million
tons national requirement per anum. Wheat contributes 21.73 percent to value added in
agriculture and 4.20 percent to GDP in the country . In 2019-2020, wheat was cultivated on an area
6
of 8.371million hectares with a production of 25,200 thousand tons of wheat (Pakistan Economic
Survey .2019- 2020).
The main causes of low productivity of wheat are poor weather conditions during
crop growth, unavailability of fertilizer and other pesticides at the proper time. Other causes
etinvasion, parasite attack and the selection of inappropriate cultivars in certain environments
(Badmus and Ariyo, 2011).
Plants are unable to complete their life cycle in the absence of essential nutrients.
Phosphorus ranks second among macro essential nutrients. It is the essential part of the
energy compounds (ATP and ADP) and needed for cell division, photosynthesis and
respiration that are growth and development affecting processes of plant (Khan et al ., 2014).
Phosphorus is not only needed for plant metabolic processes but also required for growth of
roots, earlier flowering seed formation and strength of straw in cereal (Gupta, 2003). Its
availability to the plant in soils of arid and semiarid regions is a serious issue because the
soils are about 80-90% deficient in available phosphorus. On average, soils comprise a
minute amount of phosphorus (0.02-0.5%) out of which approximately 0.1% is avaailable to
plants (Zhou et al., 1992). For plant uptake, phosphorus is available in very minute quantity
in soil (Memon et al., 1992) due to its fixation in soil layer (Khan et al., 2009; Wang et al.,
2009; Shenoy and Kalagudi, 2005) with calcium (Ca3PO4)2, aluminium (Al3PO4 and iron
(FePO4), which can be transformed to soluble form with the help of phosphorus solubilizing
organisms (Sharma et al., 2013; Khan et al., 2009; Song et al., 2008; Gupta et al., 2007).
About 80% of added phosphate fertilizer become unavailable to plants due to precipitation
reactions in soil. That is why phosphorus recovery efficiency does not exceed 20% globally
(Qureshi et al., 2012).
Fertilizer plays an important role to exploit the inherited potential of a crop cultivar.
Phosphorus is an important plant macronutrient that is required for growth and
development. Natural risks, as well as extensive vegetable and other crop production, cause
for natural phosphate content in the soil is being depleted. Therefore, to combat this
depletion, phosphate fertilizers have been utilized to add to the soil's fertilizer.
Phosphorus is a major plant nutrient that has been recognized to enhance root
growth (Mohammad et al., 1998). It concluded that P itself plays a major role in the
7
morphogenetic and leaf broaden processes (Fredeen et al., 1989; Rodríguez et al., 1998).
So, similarly it stimulates root growth by release of organic acids. Phosphorus also plays a
chief role in energy transfer processes in cell, photosynthesis and respiration (Jastrzebska et
al., 2015)
Phosphorus scarcity is a crucial element that threatens crops in low efficient farming
systems especially in the midland region, where soil is rich with Ca 2+ion (Vance et al.,
2003). In P deficit conditions, plants respond to this condition by developmental and
metabolic responses to cope with internal P level within plants and external soil P
possibility. These responses involve change in root framework and morphology (Yuan and
Liu, 2008) and increase in the synthesis and release of phosphates enters the rhizosphere to
boost soil P availability (Li et al., 2011). Because Olsen's approach has been applied in
both acid and neutral soils, it is based on phosphorus solubility in calcareous soils. In this
method, phosphate is replaced by carbonate, bicarbonate and hydroxide ions (Barrow and
Shaw, 2001). In this approach, high pH soil solution activity of Ca2+ results in dissolution
of Ca-P, Fe, and Al-P compounds in acid and calcareous soils by precipitating as calcium
carbonate. (Demetz and Insam, 1999). NaHCO3 functions as an extractant by removing
organic, inorganic, and labile solid-phase P compounds such as phosphate adsorbed mildly
soluble calcium phosphate precipitates, free lime, and phosphate loosely adsorbed to Al
and Fe oxides and clay minerals through the ion effect and pH. NaHCO3, which is easily
hydrolyzed to inorganic P, can also be used to eliminate labile organic P forms. (Demetz
and Insam, 1999).
Soltanpur’s method with Other OP method, in which sodium bicarbonate (NaHCO 3)

are use to extract plant available P, is commonly used for calcareous soils where P form
complexes with Ca. In the OP extraction the activity of Ca decreases because it precipitates
out the calcium carbonate (CaCO 3) leaving the plant available portion of P leaving in
solution which is then related to plant response. (Yield, uptake ad vigor)
Repeated applications of P fertilizers to improve crop production have contributed

to environmental problems. When the amount of P supplied exceeds crop removal, P
accumulates in soil (Ma et al., 2008). Excess P in soil could move downward and reach
groundwater, or enter rivers and lakes through surface runoff and soil erosion, which leads
8
to eutrophication of waterbodies (Sharpley et al., 2000). Thus, it is very important to
determine the values of soil Olsen P for maximizing agronomic return of P fertilization at
the same time minimizing adverse effect on the environment.
Growth of plants in low soil P is a primary limitation over half of the earth surface
because phosphorus is tightly bound to soil components like Al and Fe oxides that reduce
its availability to plants (Yano, 1998). In the agriculture system, more P fertilizer is being
used to increase crop yield to cope with increasing population. Extensive application of P
fertilizer should be restricted because resources of phosphorus that are limited in the world
and it is a nonrenewable resource (Van Vuuren et al., 2010). So, a sustainable agriculture
system will need to maximize the more crop output by minimizing the phosphorus input.
This will be facilitated by increasing the uptake efficiency of phosphorus by some
chemicals (Jun et al 2002).
Phosphorous is a major plant nutrient that enhances the root growth (Mohammad et
al., 1998). It is concluded that P (phosphorus) itself plays a major role in the morphogenetic
and leaf broaden processes (Fredeen et al., 1989; Rodriguez et al., 1998). So, similarly it
stimulates root growth by improving crop tolerance in saline culture. Phosphorus also play a
chief role in energy transfer processes in cell, photosynthesis and respiration (Jastrzebska et
al., 2015).
The important criteria of an effective soil test are, that is extracts P. Olsen-P
(NaHCO3 extractants) is considered as one of the best routine test for neutral and calcareous
soils. Because it improves the prediction of available P based on soil properties, development
of P fertilizers availability indices based on soil properties that could b e used in soil fertility
programme and to generate the data for computer use in the near future of soil and plant
phosphorus modeling.
1.1 Objectives
To determine the internal phosphorus requirement of wheat.
Determining the relationship between Olsen P and plant P uptake in soil.
9
10
CHAPTER 2
REVIEW OF LITERATURE
To fulfil the demands of an ever-increasing population, it is critical to create a soil

environment conducive to better crop yields (Hall, 1995). An insufficient availability of
necessary plant nutrients in the soil is a production-limiting issue. Phosphorus (P) is the most
critical nutrient for crop production among all the nutrient elements required by a plant, and
attention is being placed on the optimal use of this nutrient. (Clark, 1990).
2.1. Scope of the problem
Major problem in the maintenance and improvement of soil fertility is by large the
soluble P compounds in the soil, which react rapidly with various soil components, and are
quickly converted to slowly available forms. Surfaces of soil particles can adsorb P. It can be
adsorbed on positive edges of kaolinite clay and on CaCO 3 in calcareous soils. . The reaction
between orthophosphate ions and soil has been a subject of considerable study and controversy.
However, workers agree that the reactions are complex and generally range from true adsorption
to the precipitation without clear delineation between the two mechanisms (Mott, 1970). The
degree of P adsorption progressively decreases with increasing rate of P fertilizer (Reddy et al.,
1999).
Bertrand et al. (1999) observed that P adsorption dominance at low P concentration (fast
initial reaction) and precipitation at high P concentration (slow reaction). Soil properties control
11
dynamics of P in soil. Calcareous soils strongly retain P and consequently establish low P
concentration in soil solution (Sharif et al., 2000). In alkaline calcareous soils, few studies have
been aimed at the relationships between chemical properties and P adsorption of soil or to define
relationships between the adsorption characteristics of the soils.
2.2. Forms of P in soil
The relationship between the different forms of P in the soil-plant system can be represented
by the following scheme.
Non labile –P → Labile –P → P in soil solution → P in plant root
P in Soil (Solid phase) ← (Slowly available) ← (Readily available)
(Fernandez and Warren, 1994)
When soluble P compounds are added to the soil these react rapidly with various soil
components and are quickly converted to slowly available forms creating one of the main
problems relevant to the maintenance and improvement of soil fertility.
2.3. Reactions of P with soil components
The probable reactions of P in calcareous soils are i) precipitation of relatively insoluble

calcium P such as octacalcium phosphate, hydroxyl apatite and carbonate apatite favored by high
calcium activity and high pH, ii) surface precipitation with free calcium carbonate, iii) fixation of
P by clays saturated with calcium. The observation suggests that at low concentration of P
exchanges with a) adsorbed sulphate and silicate, b) with water and hydroxyl of metal hydrous
oxides and edge aluminum of clays. At high concentration, displacing the structural silicates of
clays adsorbs additional P. The increase in P adsorption by structural silicate release over that of
surface exchange reactions is about 50 and 25 % in soils containing kaolinite and allophane
clays, respectively (Sample et al., 1980).
Reaction of P with various soil surfaces may be summarized hereafter.
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a. Surfaces of variable charges including calcite for which Ca 2+ and CO32- are the
potential determining ions and Fe (III) and Al oxides and organic matter for
which H+ or OH- ions determine the charge and potential.
b. Surfaces of constant charges i.e. crystalline clay minerals that interact with P
primarily through the cations in the electrical double layer at their planner
surfaces (Fox and Searle, 1978).
2.4. Adsorption of P
P adsorption capability is an important characteristic of soils that affects the rate and plant
response to P fertilizers. P adsorption delineated from P precipitation in that the suspension’s
initial solution phase composition does not exceed the solubility product of any known P
compound (Fixen and Grove, 1990). Adsorption curves and the equations have been a popular
means of describing P adsorption and proposing P bonding mechanisms (Sample et al., 1980).
2.4.1. Adsorption on CaCO3
The reactivity of calcium carbonate in soils depends upon the specific surface area of the
carbonate and on its total surface area (Rashid and Rowell, 1988). Bertrand et al. (1999) stated
that Ca2+ is dominating ion in soil solution of calcareous soils and it is possible that formation of
less soluble complexes with weak acid anions like orthophosphate is due to unavoidable
dominance of this ion. The dynamics of P is managed by calcite, which strongly holds P and
consequently maintains low P concentration in soil solution. Papini et al. (1999) noted that low
CaCO3 showed a P adsorption upper limit varying from 1.4 to 3.5 mg P kg-1 that was not
modified by further increment of P in solution. In contrast, soil with high CaCO 3 content, P
adsorption increased up to the maximum experimental concentration of P in solution (2 g L -1).
The cation forms a sparingly soluble compound with an anion surface. Precipitation may
follow anion adsorption where the concentration of the cation is high. This is complicated and
there is not yet a clear picture about the surface compounds, even though such compounds will
be of key importance in determining soil solution concentration of the ions concerned. Though
Ca2+ has long been known to enhance P adsorption, the clay-Ca-P linkage model was thus
inappropriate (Velayutham, 1980).
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Ca (H2PO4)2. H2O + 2CaCO3 →→ Ca3(PO4)2 + 2CO2 ↑ + 3H2O
In calcareous soils, P adsorption occurs on soil carbonates, whose impurities result in great
specific surface than that of pure calcite. The adsorbed P sites serve as nucleation points for the
formation of basic Ca–P compounds (White, 1980). Though adsorption is through exchange sites
to dominate the initial reaction of soluble P with calcareous soils at low P concentration,
precipitation of P as Ca-P compounds is the ultimate fate (Barrow, 1980a). Further observations
on Ca enhanced P adsorption in both silicate and oxide clay systems have led to the proposal that
Ca2+ and P sorption are complementary interacting a Ca-P surface complex (Smillie et al., 1987).
This mechanism is distinct from that proposed to explain the role of Ca on P fixation in acid soils
by the displacement and hydrolysis of reactive Al (Traina et al., 1986).
2.4.2. Adsorption on layer silicates
It was reported by He et al. (1991) that labile–P determined as E value increased with
kaolinite and decreased with goethite. Gimsing and Borggaard (2002) evaluated the competitive
adsorption of glyphosate and phosphate on goethite and gibbsite and on illite, montmorillonite
and two kaolinites differing in surface area. The results showed that glyphosate and phosphate
were competing for the adsorption sites, but the degree of competition was dependent upon the
adsorbent. On goethite the competition was very much in favour of phosphate, on gibbsite the
competition was closer, but still phosphate was favoured, while on illite, montmorillonite and
kaolinite the competition was almost equal. The amounts of glyphosate and phosphate, which
could be adsorbed, also depended on the adsorbent: the oxides adsorbed more than the clay
silicates. The amount adsorbed on kaolinite was dependent on the specific surface area. Changes
in the surface area did not affect the competition between glyphosate and phosphate for
adsorption sites. The results indicated that differences among soils of different mineralogical
composition regarding the adsorption of glyphosate and phosphate could be expected.
Penn et al. (2005) worked on mineralogy in relation to phosphorus sorption and dissolved
phosphorus losses in runoff. They concluded that P retention for adsorption and desorption in
separated clay fractions and whole soils was well correlated to Al bearing minerals such as
hydroxy-interlayered-vermiculite (HIV), gibbsite, and amorphous Al. However, P retention was
negatively related to kaolinite content, which was also confirmed by isotherms conducted on
pure clay minerals. Based on the isotherm results, all soils were split into two groups based on
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the ratio of HIV/kaolinite. Soils with a HIV/kaolinite ratio >0.5 had a significantly lower
concentration of dissolved reactive P (DRP) in runoff for a given soil water soluble P level
compared with soils with a ratio <0.5.
2.4.3. Adsorption on hydrous oxides, Fe and Al
Some P is likely adsorbed at the broken edges of the silicate clay lattice by legend exchange
at Al (White, 1980). Singh and Singpuri (1986) reported that oxides of Fe and Al were correlated
significantly with P adsorption maxima. Higher value of P adsorption maxima in case of soil
containing higher content of oxides of Fe and Al might be due to formation of their respective
metal phosphate.
Hydrous oxide surfaces are the principal sites on which phosphate is strongly adsorbed (high
energy surfaces) as H+ and OH- determine the surface charge and potential. The liability of P
adsorbed on an oxide surface depends on the formation of ring structure (Parfitt, 1990). Papini et
al. (1999) stated that soils differed markedly in their ability to hold P. Though P adsorption was
high in all observed soils when P was held by Fe oxides and Al oxides contents dissolved,
reactive P could be found in percolating water in consequence of Fe solubility enhanced by
reducing condition.
A relationship between the iron content in high reactivity form and the adsorption of P
resulting from a decreased P in solution was found. Therefore, after flooding drained condition, P
availability to plants decreased due to adsorption of P with recently precipitated high reactivity
forms of iron oxides (Hernandez and Meurer, 2000). Furthermore, contents of amorphous iron
oxides of the soils increase significantly with the decrease in crystalline iron oxides content
during the flooding process and the maximum capacity for P adsorption increased markedly
while the adsorption P: desorption P ratio decreased (Su-Ling et al., 2001).
Al3+ + H 2PO 4- + 2H 2O ↔ 2H+ + Al(OH)2 H 2 PO 4
(Soluble) (Insoluble)
2.4.4. Adsorption on organic matter
There are two major mechanisms whereby organic matter may affect the detention and
hence availability of P in soils;
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(i) Negatively charged organic matter adsorbs or complexes cations such as Al and
Fe in acid soils (Bloom et al., 1979) and Ca in calcareous soils (Barrow, 1973)
and hence reduces their solution activities and their role in P sorption and
precipitation.
(ii) Organic anions compete with P ions for adsorption sites on P reactive surfaces.
These reactions may be manifested by a decrease in P adsorption capacity or in
high affinity adsorption of P (Lindsay, 1979).
Most probably, the organic matter itself contains available chelates of Al and Fe. Robert
(1969) pointed out that P is initially bonded to anion exchange sites on organic matter and
subsequently transformed into less soluble Fe and Al phosphate.
Lopez-Hernandez et al. (1986) studied the competition of the organic anions (oxalate and
malate) currently present in the rhizosphere and P for adsorption sites in tropical soils. The
results indicated that in the presence of organic anions P adsorption by soil was reduced. The
extent of such reactions is dependent on the way in which either P or the organic anions are
added to the soil. The organic oxy-anions are more rapidly adsorbed to the soil surface than the P
and consequently those anions when present in the rhizosphere can improve the P availability
status of the soil. The results of Inskeep and Silvertooth (1988) revealed that the presence of
organic acid such as humic, fulvic, tannic and citric acid at concentrations similar to those in soil
solution reduced the rate of precipitation of hydroxyl apaptite. They noted that organic acids
adsorbed onto seed crystals and inhibited crystal growth. Similar results were reported by
Grossel and Inskeep (1991) who observed that humic, fulvic, tannic and citric acid inhibit the
rate of dicalcium phosphate dihydrate (DCPD) crystal growth by adsorption onto DCPD surfaces
and blocking of sites for new crystal growth. Cajuste et al. (1996) reported that increasing the
loading of organic anions (oxalate and citrate) decreased the maximum P adsorption capacity of
the soils and increased the P release.
2.4.5. Adsorption on the whole soil
In most circumstances soils themselves will have a large number of sites charged with H +
and OH- as potential determining ions and it may be a possible mechanism of phosphate
adsorption on hydrous oxides discussed earlier. If this is the case, the soils will behave as the
anions just like hydrous oxide, but with the isotherm shape and pH dependence modified by the
16
presence of organic matter and other mineral, the chief modifier is likely to be Ca 2+ and CaCO3 in
calcareous soils (Singh et al., 1990; Lucresio and Dugue, 1999).
The adsorption of P increased with clay content and was not affected by the soil organic
carbon concentration (Boschetti et al., 1998). Moreover, the porosity of Al hydroxide aggregated
particles can also be a factor controlling the rate of P uptake from solution, if the aggregate is
stable (is not suspended) in solution (Lookman et al., 1994). Furthermore, the Al has been
reported as predominant cation associated with phosphate, regardless of soil pH (Pierzyneski et
al., 1990). Buffering capacity and maximum buffering were found correlated with dithionite
extractable Al of soil (Kuo, 1990). On the other hand, Fe oxides have also been reported to be the
most active P sorbent in the calcareous soils (Yli-Halla and Chirstenson, 1990; Torrent et al.,
1994). Morais et al. (1996) revealed that P adsorption increased with P concentration in the
equilibrium solution and with the clay level of the soil sample. The highest P adsorption values
(838 mg kg-1) were obtained in the plinthic soil with 10 % clay whereas the highest affinity
constant (0.215 mg L-1) was with 4 % clay. It means that the difference was due to variation in
the mineralogical composition of the soil. Similarly, Huang (1998) revealed that P adsorption
maxima and combining energy positively correlated with soil pH and amount of organic matter,
available-P and total P. He also indicated that P adsorption maxima calculated from the
Langmuir equation could be considered as a reference for rational application of P fertilizer.
2.4.6. P adsorption
Soil texture is an important factor affecting the availability of P to plants (Sanyal et al.,
1993). The higher requirement on sandy soils is probably due to two factors, which influence P
availability, diffusion rate and buffering capacity (Kamprath and Watson, 1980). Chaudhary and
Qureshi (1980) reported average P fixation as 71, 62, 56, 29, and 29 % of added P in clay, clay
loam, loam, sandy loam and loamy sand soils, respectively after one month of incubation. Thus,
more P is to be added to raise the soil test level of clay than loam and sandy soils. Response to
fertilizer P at a certain P soil test level tends to be greater on sandy soils than on those containing
more silt and clay.
Zhou and Li (2001) observed that carbonate clay contributed to P adsorption when it was
dominant in the soil, and when large amount of P was applied. Grant et al. (2005) revealed that a
given amount of fertilizer P resulted in a much greater increase in solution P (approximately
17
three times more) on coarse-textured soils as compared with fine textured soils because the sand
phase does not react with P.
2.5. Precipitation of P
When P fertilizer is applied to soil, P concentration in soil solution becomes very high. The
solubility product of one or more P compounds is likely to be exceeded and precipitation
ensures. Many different types of reaction products are possible (Sample et al., 1980). Fixen et al.
(1983) concluded that P solubility was being controlled by betatricalcium phosphate in two
calcareous soils with reduced P availability. He further stated that octacalcium phosphate
controlled solution P if NaHCO3-extractable P rose above 35 mg kg-1 while beta-tricalcium
phosphate or similar mineral phase dominated P intensity in the range of 10 to 25 mg kg -1.
Laverdiere and Karam (1984) stated that equilibrium solutions of only few samples were found
with respect to either dicalcium phosphate dihydrate or octacalcium phosphate. In calcareous
soils, soluble P should ultimately be transformed to an apatite, through the presence of free iron
oxides in these soils (Ryan et al., 1985).
Two approaches to understand the solid phase soil P are in general use, solubility product-
chemical potential evaluation and chemical fractionations of soils P. In general, the composition
of the solution in equilibrium with the soil is determined and plotted in phase diagram. If the
point lies above the nearest line, then the solution is supersaturated relative to that mineral and
precipitation can occur. A point below the line indicates under saturation of the solution and
dissolution is plausible (Fixen and Grove, 1990).
2.6. Sorption of P
P sorption by soils is initially rapid and then slows down with time (Barrow, 1983). Among
the soil parameters that affect sorption process are pH (Reddy et al., 1999), the amount and type
of clay mineral (Morais et al., 1996; Sharif et al., 2000), organic matter (Rashid and Rowell,
1988; Guilherme et al., 2000) and extractable Fe and Al oxides (Bertrand et al., 1999).
The word ‘sorption’ is used to include both adsorption and precipitation. Afif et al. (1993)
reported that at low P rates, the P concentration in the soil solution is low, and adsorption rather
than precipitation takes place. While at high P application rates, supersaturation with respect to
insoluble Ca-P and precipitation probably occurs. At low P concentration up to 0.4 mg L -1, active
18
CaCO3 and/or Fe dithionite could provide for P- adsorption capacity, whereas, at high
concentration precipitation could be predominant over the adsorption process (Papini et al.,
2000). Freshly adsorbed P showed greater desorption than native soil P and total amount of P
sorbed increased in a given soil with the increase in P addition corresponding to high content of
calcium carbonate and clay (Hussain and Haq, 2000). Del-Bubba et al. (2003) stated that the
adsorption process per se could be studied only in the absence of P precipitation. Phosphorus is
mainly governed by Ca-P (tricalcium phosphate) precipitation at high concentration. They
further stated that Freundlich equation does not seem to give useful information on adsorption
phenomenon per se. It can be concluded that it is very difficult to distinguish between the two
processes of P adsorption and precipitation. Borling (2003) stated that P sorption is a continuous
sequence of adsorption and precipitation. It consists of two processes, first relatively fast,
reversible adsorption process and second relatively slow, practically irreversible precipitation-
like process.
2.7. P status of soils of Pakistan
Olsen-P is the accepted methodology for estimating P availability and developing fertilizer
recommendations in neutral and high pH calcareous soils and removes about half of surface-
adsorbed P (Olsen et al., 1954). This method was developed specially for alkaline and calcareous
soils and still being used in Pakistan. The soils in Pakistan are alkaline and calcareous. Various
workers estimated the plant available P status of these soils. Some of the work is reported
hereunder.
In Therparker district, Anwar and Sattar (1975) analysed 48 soil samples and found an
average of 6.85 mg P kg-1 soil. Similarly in North Western Frontier Province (NWFP), Hamid
(1978) compiled the results of 15000 soil samples analyzed up to 197778. It was shown that in
Mardan district 90 % of soil samples fell in low to medium ranges (< 10 mg P kg -1 by Olsen
method) whereas in Peshawar, Swat and Hazara, soil samples within medium range (10 mg P kg –
1
) were 34, 36 and 42 %, respectively. The soil samples of Kohat, Bannun and D.I. Khan fell
below the medium level and were 54, 83 and 68 % respectively. Chaudhary and Qureshi (1980)
reported low to medium available Olsen P (4.0-15 mg kg-1) in soils of Sindh. Malik et al. (1984)
collected more than thirteen thousand soil samples from Punjab soils during 1981 to 1984 and
found that about 93 % of soil samples contained less than 10 mg P kg -1 of soil.
19
Most Pakistani soils are deficient in available P that will further deplete in future. These soils
require supplementary P application to improve their productivity and to raise the crop yield.
Qayyum (1984) collected 240 soil samples from Rawalpindi and Islamabad districts and after
analyzing these samples, he observed that 52 % of the samples contained < 10 mg P kg -1, 30 %
contained between 10-20 mg P kg -1 and only 18 % contained > 20 mg P kg -1. Wahhab (1985)
analyzed 200 soil samples collected from various districts of Punjab by a histogram and found
that Olsen P ranged from < 2 mg P kg -1 to over 25 mg P kg -1 with a mean of 4-5 mg P kg -1. In
1986-87, he also conducted a study and collected results of soil samples analyzed by soil testing
laboratories in the Punjab and observed that out of total 16841 soil samples, 63 % were within
range of 0-5 mg P kg -1, 22 % between 5-10 mg P kg -1 and 15 % >10 mg P kg-1. Similarly
Memon (1986) analyzed 125 soils and found that 50 % contained < 10 mg P kg -1. An intensive
mining of nutrients have depleted soil reserves at very rapid rate.
From 1981 to 1989 about 0.34 million soil samples were analyzed in fertility laboratories and 77
%, soils were found poor, 21.7 % medium and only 1.3 % adequate in P status. Further,
decreasing trend was observed in fertility status with passage of time in fertility trials conducted
on farmers’ fields (Malik et al., 1992).
Based on the research findings Nisar et al. (1991) suggested the following soil test
critical P levels and proposed fertilizer rates:
Soil NaHCO3-P Full technology adoption Partial technology

test/category adoption
(mg kg-1)
Irrigated Rainfed Irrigated Rainfed
Very Low <05 90-120 75 75 60
Low 05-10 75 60 60 40
Medium 10-15 60 40 40 20
High 15-20 40 20 20 -
Very High >20 20 - - -
One has to make adjustments to the generalized recommendations. It is, therefore, rather
difficult to workout a fertilizer prescription for an individual field. The expected yield level of a
20
crop is also an important consideration. Unfortunately, all these research findings and
recommendations are not made keeping in view the mineralogical, chemical and some physical
soil characteristics. In these studies, no correlations were studied between P availability and soil
properties like texture, amount and type of clay minerals, soil pH, calcareousness, soil organic
matter etc. Rehman et al. (1992) evaluated the P levels for wheat as very low (<3.60 mg P kg -1),
low (3.60-7.30 mg P kg-1), medium (7.3025.0 mg P kg-1) and high > 25.0 mg P kg-1. Similarly,
Nisar et al. (1992) concluded that over 90 % soils of Pakistan are low in available P (Olsen-P)
and needed P use for profitable crop production. Rahmatullah et al. (1994) found that application
of 90 mg P kg-1 soil increased plant dry matter yield and uptake of P by plants on ten different
soil series except Kotli. It was found that Olsen P could be used to predict P availability in
calcareous soils. Some generalized adjustments in fertilizer recommendations to suit individual
situation based on certain important factors influencing crop response to P fertilizers (NFDC,
1997) are as under:
Category Olsen-P (mg kg-1) Adjustment

Low <8 Full recommended dose, high range.
Cotton-reduce to ½ of recommended dose and to cereals
Medium 8-15 as in above table.
Cotton-reduce to ¼ or ½ of recommended dose as in

High >15 above table
According to Rahmatullah et al. (2003) recovery of applied P by NaHCO 3 extraction ranged

from 56 to 77 % (mean 69%) in the calcareous soils. Ahmad et al. (2003) conducted a survey on
evaluation of nutrient status in the rice growing area of Punjab and observed that the available P
ranged from 0.3-12.6 mg kg-1 with an average of
5.89 mg kg-1 soil.
2.10. P fertilization to wheat
Memon (1982) showed a composite yield response curve of wheat grown at seven locations
as a function of P concentration in soil solution. The P in soil solution associated with 95 %
maximum yield for all locations was found to be 0.025 mg L-1. The results further indicated that
21
P sorption curves could be used as basis for transforming information about P fertilizer
requirement from one area to another irrespective of difference in P sorption capacities. He also
conducted wheat trials at two different locations in Pakistan and said that the higher yield was
obtained with variety Pavon at Tandojam while the yield response curves were similar to the P in
soil solution of 0.029 µg mL-1 for Pak 70 variety and 0.023 µg P mL-1 for Pavon associated with
95 % of predicted maximum yield. The quantities of P required to obtain 95 % of maximum
yield were 20 and 19 kg ha-1 for location in Tandojam and Tarnab respectively. P fertilizer
estimations based on the P in soil solution requirement ranged from zero to 62 kg P ha -1.
Soil Chemistry Section, Ayub Agricultural Research Institute (1983) conducted a study that
involved correlation and calibration of soil test P with crop response to added P fertilizer on
wheat. The researchers observed that when the original available P level of the soil was 15 mg
kg-1, there was no response to the application of P. Elliot et al. (1984) also reported that critical
concentration in wheat grain for near maximum grain yield ranged from 0.19 to 0.25 %. Nisar
(1988) reported the results of wheat grown on different soil series at four locations i.e.
Hafizabad, Gujranwala, Lyallpur and Sultanpur and observed that P in soil solution at 95 %
maximum yield varied significantly among these soils. The P in solution for the Lyallpur, the
Gujranwala, the Hafizabad and the Sultanpur series were found to be 0.09, 0.052, 0.26 and 0.90
µ g mL-1, respectively. Based on these values, the corresponding P requirement for 95 % of
maximum yield was 75, 92, 114 and 150 kg P2O5 ha-1 for the Lyallpur, the Gujranwala, the
Hafizabad and the Sultanpur soils, respectively. It is evident that P requirement of medium
textured soil (the Lyallpur and the Gujranwala) was lower than for the light textured soils (the
Hafizabad and the Sultanpur). Corresponding to 95 % of maximum yield, using the composite
yield response curve, the P in the solution was found to be 0.35 µ g mL -1.
Yang and Jacobsen (1990) stated that critical Olsen-P concentration generally ranged from
16-18 mg kg-1. However, they observed positive crop responses to Pfertilizers applications in
soils with high soil-test P concentration (>25 mg kg-1) in numerous field research and calibration
trials. They advised that factors not adequately measured by Olsen-P also might determine soil P
availability on certain soils.
Similarly, Memon et al. (1991) reported that soil solution P requirement of wheat grown on
calcareous soils of Pakistan was 0.032 mg L-1 for 95 % yield as determined from a composite yield
response curve. It was reported by Azad et al. (1993) that on soils low in available P, wheat
22
responded significantly up to 39 kg P ha-1 at different locations in Gurdaspur district of Indian Punjab
and application of 26 kg P ha-1 was a must for achieving its yield potential. Rashid and Bughio (1993)
found that the internal P concentration in whole shoots associated with 95 % grain yield in wheat and
lentil each was 0.28 % and critical P concentration in recently mature leaves of lentil and wheat was
0.33 % and 0.30 %, respectively.
Rashid et al. (1997) in a field experiment indicated that 8.0 mg P kg -1 in various soils was
sufficient for reaping optimum wheat yield and 50-75 kg P ha -1 was required to maintain that
optimum level of available P. A pot study was carried out by Akram et al. (1998) on wheat (Pak-
81). Five rates of P i.e. 0,10,12,30 and 50mg kg-1 soil were applied. Dry matter yield increased
progressively with applied P but maximum yield was obtained with 30 or 50mg P kg -1 soil in a
curvilinear way. A level of 20 mg P Kg-1 soil was found critical for soils containing 10-14 mg
Olsen P Kg-1 soil. According to Johnston et al. (1999) at flowering about 45 % of the total P was
accumulated by wheat. During grain fill, the remaining 55% of the P was accumulated. Much of
the P in the leaves, stem, and head tissues was translocated to the grain. Wheat acquires P
throughout its life cycle.
Meelu et al. (2000) summarized field studies on P management in wheat and rice
undertaken throughout Northern India and noted that crop response to P fertilizer decreased as
the soil available P increased. Residual effect of P applied to wheat on the following rice crop
was enough to skip the need for P application. Brar et al. (2000) observed maximum dry matter
yield, plant P (%) contents and P uptake by wheat from the pots where 50 mg P kg -1 soil was
applied. Anyhow, the magnitude of response declined sharply with rise in soil P fertility as well
as with higher levels of P application. They suggested that both soil fertility rating limits and
critical levels of P for different crops need to be verified for various soil subgroups for accurate
fertilizer recommendations.
Plant species and even varieties within species vary in their behavior to acquire and utilize P
for grain production Alam et al. (2003). They also reported the response of wheat (cv. Inqulab-
91) to different levels of P and stated the harvest index ranged from
39.47 to 42.51 %, straw yield ranged from 6.09 to 7.55 Mg ha-1, grain yield ranged from
4.10 to 5.69 Mg ha-1, 1000 grain weight ranged from 39.04 to 42.47 g, P concentration in wheat grain
ranged from 0.38 to 0.42 % and P uptake (grain + straw) ranged from 17.12 to 26.62 kg ha -1.
23
Wheat genotypes were evaluated for their response to P deficiency stress in a solution
culture experiment by Kosar et al. (2003). It was observed that wheat genotypes differed
significantly in their biomass accumulation, root shoot ratio, P concentration, and total P uptake
in shoot and P utilization efficiency. Total P uptake and P utilization efficiency correlated
significantly with their shoot dry matter yield, root dry matter yield and total biomass production.
Rehman et al. (2004) reported that maximum wheat grain and straw yield of 3.37 and 3.50 Mg
ha-1 respectively was obtained at 0.2 mg P L-1 soil solution. P concentration in wheat grain and
straw was 0.41 and 0.14% respectively, which was found at 0.40 mg P L -1 soil solution. External
solution P requirement was 0.146 mg P L-1 and internal P requirement was found to be 0.289 mg
P L-1 soil solution for obtaining 95 % relative yield of wheat.
Rehman et al. (2006) conducted field study regarding effect of balanced fertilization on
yield and phosphorus uptake in wheat-rice rotation during 2002-2004. They reported maximum
wheat grain (4.2-4.67 Mg ha-1) and paddy (3.30-4.35 Mg ha-1) yields during both the years with
the application of recommended dose of NP. Yield components like number of tillers and 1000-
grain weight also increased with balanced fertilization (NPK). A significant higher P uptake was
also observed by wheat (24.11-
34.89 kg ha-1) and rice (17.44-24.56 kg ha-1) with the recommended rate of NPK.
2.11. Effect of method of P application on P availability for crops
The efficiency of fertilizer P is much lower than actual potential due to imbalanced
fertilization, coupled with inappropriate application time and method. Alessi and Power (1980)
found that banding phosphate with wheat seed gives early availability of P, and increased total
dry matter and grain yield, even in soils with medium to high levels of available P. Peterson et al.
(1981) stated that the effectiveness of row placement might be three to four times that of
broadcasting in case of low P soils for winter wheat. Sanders (1986) found that P banded in fall
and with seed application was equally effective for wheat production. Both were effective more
than other application methods for grain yield, P uptake and fertilizer efficiency. Fertilizer
efficiency was increased from 5.5 % for broadcast to 36 % for band and 40 % for seed
application. This study indicated that undistributed fertilizer P bands had more value than either
with seed or broadcast P, which could differentially influence the P fertilizer requirement for
succeeding crops. Miller and Wolf (1987) found that applied fertilizer might be lost due to run
off and leaching which could be utilized properly through proper method of application.
24
Ranjha and Mehdi (1990) while studying the effect of source and method of application of P
on the growth of maize and wheat found that on soil that need more supplemental P source and
method of application had no significance. However, when rates were less then band placement
gave healthier results. The immediate use of irrigation water after P application was key factor in
improving P fertilizer use efficiency. Halvorson and Halvin (1992) studied the effect of P
placement and rate on no till winter wheat. P was surface broadcast with or without incorporation
or was banded below the seed zone, at 0, 34, 67,101, or 134 kg P ha -1 with 56 kg N ha-1. They
found that grain yield increased curvi-linearly with increasing P rate up to 101 kg P ha -1.
Fertilizer P increased straw yield and P uptake by grain. N application increased mean grain
yield from 3.17 to 4.12 Mg ha-1 and increased grain protein concentration and grain P uptake.
In a review paper on the effect of method and time of P application Ahmad et al. (1992)
suggested that under irrigated conditions, P fertilizer should be top dressed at the time of first
irrigation instead of its incorporation in soil at seeding. Dravid (1996) concluded that placement
of phosphate fertilizer 7.5 cm below the seed was more effective than broadcasting it. He further
observed that combined application of P and Zn with saline irrigation water improved dry matter
(DM) accumulation and P utilization in wheat compared with their single application. Alam et
al. (1999b) conducted pot and field experiments on wheat and compared the recommended
method of broad casting and incorporation of solid P fertilizer before sowing with top dressing of
P after plant emergence and as solution application along with irrigation water (fertigation). They
found that the lower P rate applied by fertigation at first irrigation resulted in equivalent wheat
yield and 25-30 % higher P fertilizer efficiency compared to higher rate applied as broadcast and
incorporated at sowing, indicating the possibility of a substantial saving in P input. Valizadch et
al. (2003) applied P fertilizer as a band at 20 mg P kg-1 soil in dry top soil (5cm) deep,
significantly increased P uptake and shoot weight compared with a nil P (control). It was
concluded that roots of various wheat genotypes lift water from wet sub soil into the dry top soil
(hydraulic lift). P fertilizer banded at 5 cm depth in dry top soil increased P uptake and wheat
growth due to the presence of hydraulically lifted water
Ansar et al. (2008) conducted an excellent study regarding efficiency of fertilizer

application methods for wheat crop under rainfed conditions of Pakistan. They used five methods
of fertilizer application viz. 1. control, 2. broadcast, 3. drill placement of DAP and urea mixed
with seed, 4. drill placement of DAP mixed with seed and urea as broadcast, and 5. drill
placement of fertilizer and seed separately. They revealed that fertilizer application via drill
25
consistently proved more efficient in wet and dry years and produced yield more than 4 Mg ha -1
in good moisture year. Whereas dry year adversely affected yield and produced only upto 0.5 Mg
ha-1 yield due to reduced efficiency of nutrient availability. Moreover, broadcast method proved
the second choice for rainfed wheat growers that produced 0.41 Mg ha -1 in dry year and 2.7 Mg
ha-1 in each year. They concluded that efficiency of fertilizer availability is largely associated
with moisture availability. The drill placement of seed and fertilizer separately was found to be
the most suitable method of wheat sowing in rainfed areas.
2.14. P use efficiency
Nutrient efficiency is best defined as the increase in yield of the harvested section of the
crop per unit of nutrient supplied by fertilizer (Cooke, 1987). Agronomic efficiency of grain
production in crop plant is frequently expressed as percentage of harvest index. Harvest index is
calculated by dividing the total grain yield by total plant yield. Thus economic yield / total plant
yield gives harvest index (Rashid et al., 2007). Increase in harvest index have contributed
significantly to increasing yields of rice and wheat and a value of more than 50 % has been
achieved in these cereals but it seems unlikely that it will be able to rise much above 60 % in
cereals (Evans, 1980). Duivenbooden et al. (1996) described the maximum harvest index of
wheat as 49 % and 55 % for rice. However, efficient crop and soil management practices could
improve grain crop harvest index. Balance use of inputs like seed, fertilizers and moisture is
essential for improving harvest index of grain crops similarly; role of weeds in declining crop
harvest index is obvious. Insect / pests attack at different crop growth stages also cause heavy toll
on yield. Importantly, creating awareness among the growers by using all available measures is
crucial. Sadly, the distribution of these inputs remained lopsided during the last many years
(Rashid et al., 2007).
Fageria et al. (1997) described nutrient (P) efficiency ratio of wheat grain as 200 and
wheat straw as 714. P requirements are estimated for vegetative and grain portion of wheat plant
to be from 6-8 kg P ton-1of grain (Halvorson et al., 1987). P use efficiency of various crops is
low and only about 15-20 % of applied P is used by first crop (Nisar, 1985). Delgado et al.
(2002) reported that the various reactions of the applied P in the soil resulting in the formation of
compounds, that are difficult to become available to plants, are considered one of the factors
attributing to low P recovery. Sharif (1985) stated that the application of super phosphate to
alkaline calcareous soils of Pakistan converted to insoluble form of Ca-P (45-59 %), Fe- and Al-
26
P (14-19 %) while water-soluble fractions ranged from 5-9 % only. The plant tissue recovered
only 11-19 % of the applied P.
To widen the useful life of the P reserves in the world, to reduce the cost of producing
crops and to improve the value of the grain and straw produced, more efficient utilization of P by
wheat plant is needed (Batten, 1992). Bakhsh et al. (1990) conducted a field study to evaluate the
relative efficiency of various P fertilizers viz. diammonium phosphate, nitrophos, single super
phosphate and triple super phosphate alone and premixed with FYM on wheat crop. The results
indicated that premixing it with FYM increased the efficiency of nitrophos. Soil available P and
uptake in leaves was considerably increased with FYM. Increase in P use efficiency by addition
of FYM to P fertilizers was also reported by Chaudhry and Qureshi, 1981, Mohammad and
Qureshi, 1980.
Latif et al. (1997) found in field experiment on wheat that P application through
broadcast, incorporation, top-dressing and fertigation significantly increased the grain and straw
yield and P content over control. They further concluded that rate and method of application
positively influenced P use efficiency by wheat. The percentage increase in P use efficiency due
to split P application ranged from 6.2 to 25.3 % over broadcast method at higher rate of
application. Alam et al. (2005) conducted three field experiments during 2001-2003 on wheat.
They showed that P applied through fertigation or top-dressing methods were equally effective
for production of wheat grain and straw. Fertigation applied N and P fertilizer increased wheat
grain yield by 16 %, P uptake by 13 %, P-fertilizer efficiency by 74 % and agronomic efficiency
by 240 % over top dressed N and P.
Iqbal et al. (2003) conducted field studies to determine the effect of fertilized P on crop
yield and P use efficiency. They concluded that P uptake; P use and agronomic efficiency were
higher in fertigation than broadcast method. Averaged 8 % P use efficiency was calculated when
P was broadcast and incorporated and 16 % when P was either knifed with anhydrous ammonia
or applied with the seed in winter wheat (Sander et al., 1990, 1991). It was reported by Eghball
and Sander (1989) that 13.8 to 26.4 kg P ha-1 was taken up in corn grain at yield levels between
4.24 and 8.83 Mg ha-1, and a concentration of 0.31 % P. Similarly Raun et al. (1987) concluded
that total up taken P in corn grain ranged from 21.4 to 47.4 kg P ha -1 at yield levels from 8.10 to
14.47 Mg ha-1, or 0.30 % P. It was observed that root zone P is depleted and plants could not get
P when it is needed because diffusion coefficient of P in soil is very low (Clarkson, 1981). Latif
27
et al. (1994) reported less P use efficiency of applied P before sowing until first irrigation to
wheat crop. Similarly, Alam et al. (1999a) concluded that rapid P uptake took place after
irrigation, 3-4 weeks after germination because the requirement of P at this stage of growth is
much higher compared to other stages of growth (Romer and Schilling, 1986). That is why
application of P through different methods and mixing with FYM is important to improve its use
efficiency.
28
CHAPTER 3
MATERIALS AND METHODS
3.1 Experimental Site

A pot experiment was conducted to determine the effect of organic acids on
phosphorus uptake in wheat genotypes. (Triticum aestivum L.). This experiment was
conducted at Agronomic Research Farm, Department of Agronomy University of Agriculture
Faisalabad (Winter 2020).
3.2 Experimental Design
Experiment was carried out under Completely Randomized Design (CRD) with
factorial arrangement having three replications of treatments. Clay pots with 2 kg sand were
used to cultivate the plants. Phosphorus was applied at the rate of 10 mg P/kg sand (P 1),
10mg P+90 mg apatite P/kg sand (P2) and 100 mg P/kg sand (P3). Eight weight varieties
were used in this experiment. The seed of all the varietes were collected from Ayub
Agriculture Research Institue Faisalabad. Phosphorus was applied in the form of SSP (single
superphosphate) in water soluble form with four equal splits after one-week interval. Nutrient
solution was applied with six equal splits every week. The experimental pots were watered
and when moisture status reached to a proper level.
3.3 Experimental Treatment
Factors:
29
This pot experiment comprised three factors i.e.
A Phosphorus levels:
P1= 10 mg P/kg sand

P2= 10 mg P/kg sand +90 mg apatite P/kg sand
P3=100 mg P/kg sand
B Wheat Varieties:
V1= Akbar-2019
V2= Anaj-2017
V3= AARI-2011
V4= Millat-2011
V5= Niab-2011
V6= FSD-2008
V7 = Lasani-2008
V8= Sehar-2006
3.6 Seed Rate

10 seeds were sown in each pot.
3.7 Pot Geometry

3 kg sand was filled in each pot.
30
20cm 2kg sand
20cm
Amount of nutrient solution
N CH4N2O Na2SO4 CaCl2

N 10.75 20.25
Ca 14.25
Mg 4.75
Cl 10.75 20.25 14.25
SO4 4.75
3.8 Saturation %
Saturation percentage of sand was also measured.
For this purpose, 250 g sand was taken in petri dish and distilled water added to make it
pasty. After that 10 g of sample taken in China dish and kept it into oven for 24 hours to get
its constant weight. By following formula, it was calculated.
Ovendry weight
Saturation % = × 100
actual weight
31
8.62 g
¿ ×100
32.06 g
Saturation % = 26.88 %
3.9 Phosphorus Determination:

3.10.1 Measurements of reagents for purpose, five standard solutions were
made ranging from 0.5-2.5 ppm by following standard method of preparation.
3.10.2 Procedure
To analyzed the phosphorus content, 5 mL of digested sample and 5 mL of
ammonium metavanadate was taken into the 50 mL volumetric flask. Distilled water (40 mL)
was used to make up to 50 mL solution. According to Olsen (1982) plant sample were run on
spectrophotometer. Both solution plant sample and blank sample run together in
spectrophotometer and reading were noted. The following formula was used for calculation.
Extractable Phosphorus= ppm P × A/Wt. × 50/V
A = Total volume of extract (mL)
Wt. = Weight of plant sample (g)
V = Volume of extract used for measurement (mL)
3.11 Soil digestion

2 grams of dried sand was taken into the digestion flask and 30 mL of perchloric acid
was added. Flask then put on to the hot plate and heated at 100 ⸰C and slowly raised the
temperature up to the 180⸰C. At least for 40 minute the samples were digested until white
fumes released. After digestion the samples were cooled down at room temperature. Distilled
water was used to made up the 250 mL solution. Then solution was filtrated by filter paper.
3.11.1 Measurements of reagents for soil samples
32
22.5 g of ammonium heptamolybdate [(NH4)6Mo7O24] was mixed in 400 mL
deionized water. Then 1.25 g of ammonium metavanadate was dissolved in 300 mL in
distilled water. The both solutions then taken up into the 1000 mL volumetric flask and 250
mL nitric acid (HNO3) also added in this solution. To make up to 1000 mL distilled water is
used.
3.11.2 Preparation of standard stock solution
2.5 g potassium dihydrogen phosphate (KH2PO4) was dried in oven at 105⸰C for 1
hour. Then sand was cooled down by desiccator in tightly stoppered bottle. From dried
potassium dihydrogen phosphate only 0.4393 g was mixed in distilled water and made up to
1000 mL volume. By diluting the stock solution of 1,2,3,4 and 5 mL standard solution were
made. The standard solution made up to 50 mL by distilled water.
3.11.3 Procedure for phosphorus determination in soil

After harvesting the plant from pots, soil sample from each pot were taken for
phosphorus determination. To analyzed the phosphorus content, 5 mL of digested sample and
10 mL of ammonium metavanadate and heptamolybdate was taken into the 50 mL
volumetric flask. Distilled water was used to make up to 50 mL solution. According to Olsen
(1982) soil sample were run on spectrophotometer. Both solution soil sample and blank
sample run together in spectrophotometer and reading were noted. The following formula
was used for calculation.
Extractable Phosphorus= ppm P × A/Wt. × 50/V
A = Total volume of extract (mL)
Wt. = Weight of plant sample (g)
V = Volume of extract used for measurement (mL)
3.12 Data Collection

Parameters related to the morphology were recorded.
3.12.1 Morphological parameters:
33
1. Emergence Count
2. Fresh shoot length (cm)
3. Fresh root length (cm)
4. Shoot fresh weight (g)

6. Root fresh weight (g)
7. Shoot dry weight (g)
8. Root dry weight (g)
9. Root, shoot length ratio
10. Root, shoot fresh weight ratio
11. Root, shoot dry weight ratio
12. No. of leaves per plant
13. Leaf area (cm2)
14. Chlorophyll content (mg/mL)
15. Stem diameter (mm)
16. Root diameter (mm)
17. Soil phosphorus content (mg/kg)
18. Soil Ph
34
3.12.2Morphological Parameters:
After 45 days of experiment maize plants were harvested, data of shoots, roots and leaves
was recorded. After harvesting the plants were separated to check their fresh biomass. Then
the plant samples were oven dried at 70 0C and their dry weight was recorded.
Following morphological parameters were recorded and data of growing plants were
collected after one and half months of seed sowing.
3.12.3 Emergence count (%):
Emergence of wheat seeds per pot was noted. The following formula was used for
germination percentage.
Total seed emerged

×100
Total seed sown
3.12.4 Plant height (cm):
35
Out of ten plants three plants were randomly choose to measure the plant height. By adding
the length of shoot and length of root height of plant was measured.
Shoot length + Root length = Plant height
3.12.5 Fresh shoot length (cm):
After harvesting the plant shoot length was immediately taken with the help of measuring
tape. Shoot length was measured after 45 days.
3.12.6 Fresh root length (cm):
After harvesting the plant root length was immediately taken with the help of measuring tape.
root length was measured after 45 days.
3.12.7 Shoot fresh weight (g):
After harvesting the plant shoot weight was immediately taken by electronic weighing
balance. Shoot weight recorded in grams by Weighing balance after 45 days.
3.12.8 Root fresh weight (g):
After harvesting the plant shoot weight was immediately taken by electronic weighing
balance. Shoot weight recorded in grams by Weighing balance after 45 days.
3.12.9 Shoot dry weight (g):
After harvesting plant shoot subjected to drying for taking dry weight. For this purpose,
maize plants kept in oven at 70 0C for 7 days. After drying, dry weight of samples was
recorded by weighing balance.
3.12.10 Root dry weight (g):
After harvesting plant shoot subjected to drying for taking dry weight. For this purpose,
maize plants kept in oven at 70 0C for 7 days. After drying, dry weight of samples was
recorded by weighing balance.
3.12.11 Root, shoot length ratio
36
Root shoot length ratio was measured by following formula
Root length
Shoot length
3.12.12 Root, shoot fresh weight ratio
Root shoot fresh weight ratio was measured by following formula
Root fresh weight

Shoot fresh weight
3.12.13 Root, shoot dry weight ratio
Root shoot fresh weight ratio was measured by following formula
Root dry weight

Shoot dry weight
3.12.14 No. of Leaves:
After 30 days of experiment no. of leaves of each plant were counted
3.12.16 Chlorophyll content (mg/mL):
Before harvesting chlorophyll content of plants was recorded. SPAD meter was used to
measure the chlorophyll content.
3.12.25 Soil P contents (mg):
The P content in soil was determined using the blue molybdate method (Murphy and Riley,
1962).
3.12.26 Soil pH
Soil pH was measured by pH meter. It is important to analyze the weather soil is acidic,
basic, neutral or alkaline.
37
CHAPTER 4
RESULTS AND DISCUSSION
4.1 Agronomic traits
4.1.1 Emergence count
Application of and phosphorus was observed non-significant for emergence count of

wheat. The effect of phosphorus was non-significant (table 4.1). It was observed that
maximum emergence (9.08) rate was recorded in treatment where phosphorus level was 100
mg/kg of soil. Same results also showed by control treatment. While in treatments where
phosphorus level was control, minimum germination (7.58) rate was recorded when
phosphors was 10 mg/kg sand.
38
Table 4.1 (a): Effect of phosphorus on emergence count of wheat genotypes.
SOV DF SS MS F-value
Replication 3 2.2208 0.74027
Phosphorus 1 0.0319 0.03188 0.03 NS
Salinity 1 2.9101 2.91018 2.74 NS
Proline 1 0.5859 0.58590 0.55 NS
P× S 1 3.3347 3.33465 3.14 NS
P× A 1 1.5444 1.5440 1.45 NS
S× A 1 1.2601 1.26008 1.19 NS
P× S× A 1 1.2364 1.2364 1.16 NS
Error 24 24.5357 1.02232
Total 31 35.4391
NS= Non-significant (P> 0.05)
(b): Comparison of treatments means
Phosphorus Proline (A1) Proline (A2) Mean

Control (S1) 6 dS m-1 (S2) Control (S1) 6 dS m-1 (S2)
10 mg/kg 9.08 8.24 7.58 8.32 8.30
100 mg/kg 7.66 8.91 7.83 9.08 8.37
Mean S1= 8.03 S2= 8.64 A1= 8.47 A2= 8.20
39
4.1.2 Plant height (cm)
Significant difference was observed for plant growth parameter in wheat among
phosphorus treatment. The plant height (7.17 cm) was significantly higher in four varieties
and P (100 mg) over P (10 mg). Increasement in height was significantly higher with NIAB
+100MG P and lower in ARRI + 1O MG P. NIAB over 100 mg P in for plant height of
maize (table 4.2). While individual effect of proline was non-significant. Collective effect of
phosphorus, salt stress and proline was found significant for plant height. Maximum plant
height (71.17 cm) was found in treatment where proline level was 0 mM and phosphorus
applied as 100 mg/kg soil in 6 dS m -1 salinity. While in treatments where foliar application of
proline was 100 mM, minimum plant height (50.03 cm) was recorded when salinity was 6 dS
m-1 and phosphorus was 100 mg/kg of soil. In case of 100 mg P/kg, the maximum plant
height (71.17 cm) was found when 6 dS m -1 salinity under 0 mM (control) proline. While
collective effect of phosphorus, proline and salinity was found to yield the minimum plant
height (50.03 cm). In 6 dS m-1 salinity level maximum plant height (71.17 cm) was found
when proline level was 0 mM (control) and phosphorus was 100 mg/kg of soil. While
minimum (50.03 cm) was recorded when salinity level was 6 dS m-1 with application of 100
mM proline in 100 mg P per kg of soil. Foliar application of 100 mM proline was observed
to yield the maximum plant height (70.11 cm) when phosphorus was 100 mg/kg under
control environment of salinity. While minimum (50.03 cm) was recorded when 100 mM
proline applied in 100 mg/kg phosphorus under 6 dS m-1 salinity.
The above results coincide with the prior study by Khan et al. (2005) in which they
showed that application of 100 mg phosphorus/kg was effective in increasing plant height.
40
Table 4.2 (a): Effect of proline and phosphorus on plant height of maize under salt
stress
Replication 3 25.00 8.334
Phosphorus 1 51.38 51.384 8.06*
Salinity 1 93.40 93.400 14.66*
Proline 1 1.83 1.829 0.29 NS
P× S 1 2.41 2.415 0.38 NS
P× A 1 242.83 242.826 38.11**
S× A 1 528.21 528.206 82.89**
P× S× A 1 510.16 510.162 80.06**
Error 21 133.82 6.372
Total 31 1589.04
NS= Non-significant (P> 0.05); *= Significant (P< 0.05); ** = Highly significant (P< 0.05)

10 mg/kg 58.42cd 55.69d 64.55b 61.54bc 60.05B
100 mg/kg 59.03cd 71.17a 70.11a 50.03e 62.58A
Mean S1 = 63.03A S2= 59.61B A1= 61.08A A2= 61.56A
LSD at 5% 3.71
41
4.1.3 Shoot length (cm)
The effect of phosphorus was observed significant for shoot length. It was observed
that maximum shoot length (27.52 cm) was recorded in treatment where phosphorus level
was 100 mg/kg of soil and NIAB 2009. While in ARRI, minimum shoot length (16.49 cm)
was recorded where phosphors was 10 mg/kg under control application. In case of 100 mg
P/kg, maximum shoot length (27.52 cm) was recorded and variety. While when phosphorus
was 100 mg/kg, minimum shoot length (20.75 cm) was found under control environment of
salinity and proline. Salt stress environment with 6 dS m -1 was recorded as maximum shoot
length (27.52 cm) when phosphorus applied as 100 mg/kg of soil under control (0 mM)
proline application While minimum (16.49 cm) was recorded in 6 dS m-1 salinity when P
level was 10 mg/kg of soil with no application of proline. In case of 100 mM foliar proline
maximum shoot length (27.27 cm) was observed in 100 mg/kg phosphorus with no salt
stress. On the other hand, minimum shoot length (22.69 cm) was recorded when applied
phosphorus level was 10 mg/kg under control salt stress.
Above discussion similar with the findings of Jahan et al. (2020) they revealed that
increased levels of salinity showed a significant impact on shoot length of rice.
42
Table 4.3 (a): Effect of proline and phosphorus on shoot length of maize under salt
stress
Phosphorus 1 183.553 183.553 211.60 **
Salinity 1 4.651 4.651 5.36 *
Proline 1 111.901 111.901 129.00**
P× S 1 2.486 2.486 2.87 NS
P× A 1 56.818 56.818 65.50**
S× A 1 47.142 47.142 54.35**
P× S× A 1 72.122 72.122 83.83**
Error 21 18.217 0.867
Total 31 504.742
NS= Non-significant (P> 0.05); * = Significant (P< 0.05); ** = Highly significant (P< 0.05)

10 mg/kg 16.87d 16.49d 22.69b 23.48b 19.88B
100 mg/kg 20.75c 27.52a 27.27a 23.15b 24.67A
Mean S1= 21.90B S2= 22.66 A A1= 20.41B A2= 24.15A
LSD at 5% 1.36
4.1.4 Root length (cm)
43
The individual effect of phosphorus, salt stress and proline was significant on the root
length of maize (table 4.4). Combination of phosphorus, salt stress and proline was also
found significant for root length. Treatments with control proline, maximum root length
(44.65 cm) was found when phosphorus applied as 100 mg/kg soil and salinity was 6 dS m -1.
While in treatments where foliar application of proline was 100 mM, minimum root length
(26.88 cm) was recorded when salinity was 6 dS m -1 and phosphorus was 10 mg/kg of soil.
Application of 100 mg P/kg was found to yield the maximum root length (44.65 cm) when 6
dS m-1 salinity under 0 mM (control) proline. Minimum root length (26.88 cm) was observed
in 10 mg/kg phosphorus in 6 dS m -1 under 100 mM proline. In case of 6 dSm -1 salinity
maximum root length (44.65 cm) was found when proline was applied as 0 Mm (control) and
phosphorus was 100 mg/kg of soil. While minimum (26.88 cm) was recorded when salinity
level was 6 dS m-1 with application of 100 mM proline in 10 mg/P per kg of soil. Maximum
root length (42.83 cm) was observed when foliar application of 100 mM proline in
phosphorus 100 mg/kg under control environment of salinity. While minimum (26.88 cm)
was recorded when 100 mM proline applied in 10 mg/kg phosphorus under 6 dS m-1 salinity.
The similar finding also showed by Bandeoglu et al. (2004), they revealed that root
length showed maximum growth in presence of salt stress by enhancing the SOD activity.
Table 4.4 (a): Effect of proline and phosphorus on root length of maize under salt stress
44
Replications 3 23.132 7.711
Phosphorus 1 32.180 32.180 9.30 *
Salinity 1 123.520 123.520 35.69 **
Proline 1 98.666 98.666 28.51**
P× S 1 5.874 5.874 1.70 NS
P× A 1 76.601 76.601 22.13*
S× A 1 283.042 283.042 81.77 **
P× S× A 1 218.039 218.039 62.99 **
Error 21 72.688 3.461
Total 31 933.743
(b): Comparison of treatment means

10 mg/kg 41.55bs 39.20cd 41.86bc 38.06d 40.16A
100 mg/kg 38.27d 44.65a 42.837ab 26.88e 38.16B
Mean S1= 41.13A S2=37.20B A1= 40.92A A2= 37.41B
LSD at 5% 2.73
4.1.5 Shoot fresh weight (g)

The individual effect of phosphorus, salt stress and proline was significant on shoot
fresh weight of maize (table 4.5). Combination of phosphorus, salt stress and proline was
also found significant for shoot fresh weight. Maximum shoot fresh weight (26.79 g) was
found when phosphorus applied as 100 mg/kg soil and salinity was 6 dS m -1 in absence of
proline . While in treatments where foliar application of proline was 100 mM, minimum
45
shoot fresh weight (5.39 g) was recorded when salinity was 6 dSm-1 in 10 mg P/kg of soil.
Application of 100 mg P/kg was found to yield the maximum shoot fresh weight (26.79 g)
when 6 dSm-1 salinity under 0 mM (control) proline. Minimum shoot fresh weight (15.63 g)
was observed in 10 mg/kg phosphorus in 6 dS m-1 under 100 mM proline. Where the level of
salinity was 6 dS m-1 maximum shoot fresh weight (26.79 g) was found when proline was
applied as 0 Mm (control) and phosphorus was 100 mg/kg of soil. While minimum (5.39 g)
was recorded when salinity level was 6 dS m -1 with application of 100 mM proline in 10
mg/P per kg of soil. In foliar application of 100 mM proline, maximum shoot fresh weight
(20.31 g) was observed when phosphorus was 100 mg/kg under control environment of
salinity. While minimum (5.39 g) was recorded when 100 mM proline applied in 10 mg/kg
phosphorus under 6 dS m-1 salinity.
According to Fatima et al. (2006) application of phosphorus affect the shoot fresh
weight significantly result in increase of shoot mass.
Table 4.5 (a): Effect of proline and phosphorus on shoot fresh weight of maize under
salt stress
Phosphorus 1 916.58 916.58 713.95**
Salinity 1 15.62 15.62 12.17*
Proline 1 268.33 268.33 209.01**
P× S 1 22.90 22.90 17.84*
P× A 1 1.27 1.27 0.99 NS
46
S× A 1 82.79 82.79 64.48**
P× S× A 1 24.570 24.570 19.14*
Error 21 26.96 1.284
Total 31 1366.98

10 mg/kg 13.87d 12.25d 9.94e 5.39f 10.36B
100 mg/kg 21..53b 26.79a 20.31b 15.63c 21.07A
Mean S1=16. 41A S2= 15. 01B A1= 18.61A A2= 12.82B
LSD at 5% 1.66
4.1.6 Root fresh weight (g)

The individual effect of phosphorus, salt stress and proline was significant on root
fresh weight of maize (table 4.6). Combination of phosphorus, salt stress and proline was
also found significant for root fresh weight. Treatments where proline was not applied
maximum root fresh weight (19.86 g) was found when phosphorus applied as 100 mg/kg soil
and salinity was 6 dSm-1. While in treatments where foliar application of proline was 100
mM, minimum root fresh weight (8.77 g) was recorded when salinity was 6 dSm -1 and
phosphorus was 10 mg/kg of soil. Application of 100 mg P/kg yield the maximum root fresh
weight (19.86 g) when 6 dSm-1 salinity under 0 mM (control) proline. Minimum root fresh
weight (15.79 g) was observed in 10 mg/kg phosphorus in 6 dS m -1 under 100 mM proline. In
case of 6 dSm-1 salinity maximum root fresh weight (19.86 g) was found when proline was
47
applied as 0 Mm (control) and phosphorus was 100 mg/kg of soil. While minimum (8.77 g)
was recorded when salinity level was 6 dS m-1 with application of 100 mM proline in 10
mg/P per kg of soil. Maximum root fresh weight (18.15 g) was observed when foliar
application of 100 mM proline in phosphorus 100 mg/kg under control environment of
salinity. While minimum (8.77 g) was recorded when 100 mM proline applied in 10 mg/kg
phosphorus under 6 dSm-1 salinity.
These conclusion in line with the Shen et al. (2005), they stated that root fresh mass is
highly influenced by supply of external P.
Table 4.6 (a): Effect of proline and phosphorus on root fresh weight of maize under salt
stress
Phosphorus 1 234.041 234.041 126.47**
Salinity 1 24.818 24.818 13.41*
Proline 1 148.096 148.096 80.03**
P× S 1 8.215 8.215 4.44*
P× A 1 27.315 27.315 14.76*
48
S× A 1 1.049 1.049 0.57 NS
P× S× A 1 12.449 12.449 6.73*
Error 21 38.861 1.851
Total 31 500.930
(b): Comparison of treatment mean

10 mg/kg 17.70bc 14.04d 10.66e 8.77e 12.79B
100 mg/kg 19ab 19.86a 18.15ab 15.79cd 18.20A
Mean S1= 16.38A S2= 14.61B A1= 17.65A A2= 13.34B
LSD at 5% 2.00
4.1.7 Shoot dry weight (g)

The individual effect of phosphorus was significant for shoot dry weight of maize
(table 4.7). While individual effect of proline and salt stress was non-significant. Collective
effect of phosphorus, salt stress and proline was found significant for shoot dry weight.
Maximum shoot dry weight (4.09 g) was found in treatment where proline was applied as
100 mM and phosphorus level was 100 mg/kg soil in 6 dS m -1 salinity. While in treatments
where foliar application of proline was 100 mM, minimum shoot dry weight (1.20 g) was
recorded when salinity was 6 dS m-1 and phosphorus level was 10 mg/kg of soil. In case of
100 mg P/kg was found to give the maximum shoot dry weight (4.09 g) when 6 dS m -1
salinity under 0 mM (control) proline. While in case of collective effect of phosphorus and
proline, minimum shoot dry weight (3.22 g) was recorded when salinity was control. In 6 dS
49
m-1 salinity level maximum shoot dry weight (4.09 g) was found when proline level was 100
mM and phosphorus was 100 mg/kg of soil. While minimum (1.20 g) was recorded when
salinity level was 6 dS m-1 with application of 100 mM proline in 10 mg/P per kg of soil.
Foliar application of 100 mM proline yield the maximum shoot dry weight (4.09 g) when
phosphorus was 100 mg/kg under 6 dS m-1 salinity. While minimum (1.20 g) was recorded
when 100 mM proline applied in 10 mg/kg phosphorus under 6 dS m-1 salinity.
Dordas (2009) stated that fertilization of phosphorus improves the shoot dry matter
that is similar to this result.
Table 4.7 (a): Effect of proline and phosphorus on shoot dry weight of maize under salt
stress
Phosphorus 1 27.4233 27.4233 161.19**
Salinity 1 0.3770 0.3770 2.22 NS
Proline 1 0.0989 0.0989 0.58 NS
P× S 1 4.8138 4.8138 28.30**
P× A 1 0.0770 0.0770 0.45 NS
S× A 1 0.0223 0.0233 0.13 NS
P× S× A 1 1.0638 1.0638 6.25*
Error 21 3.5727 0.1701
Total 31 37.7010
NS= Non-significant (P> 0.05); ** = Highly significant (P< 0.05); * = Significant (P< 0.05)
50

10 mg/kg 1.98d 1.41de 2.61c 1.20e 1.80B
100 mg/kg 3.52ab 3.77ab 3.22b 4.09a 3.65A
Mean S1= 2.83A S2= 2.62A A1= 2.67A A2= 2.78A
LSD at 5% 0.60
4.1.8 Root dry weight (g)
The individual effect of phosphorus, salt stress and proline was significant on root dry
weight of maize (table 4.8). Combination of phosphorus, salt stress and proline was also
found significant for root dry weight. Application of phosphorus 100 mg/kg of soil was
found to yield the maximum root dry weight (3.63 g) in treatment where proline and salinity
was not applied. Treatment without proline application, give the minimum root dry weight
(1.59 g) when salinity was 6 dS m-1 and phosphorus was 10 mg/kg of soil. In case of 100 mg
P/kg, maximum root dry weight (3.63 g) was observed under control environment of salinity
and proline. Without proline application minimum root dry weight (2.73 g) was observed
was observed in 100 mg/kg phosphorus when salinity was 6 dS m -1. When salinity was 6 dS
m-1 maximum root dry weight (3.54 g) was found when proline was applied as 100 mM and
phosphorus was 100 mg/kg of soil. While minimum (1.59 g) was recorded when salinity
level was 6 dS m-1 without application of proline in 10 mg/P per kg of soil. In 100 mM
application of proline maximum root dry weight (3.54 g) was found when phosphorus was
51
100 mg/kg under 6 dS m-1 salt stress. While minimum (1.64 g) was recorded when 100 mM
proline applied in 10 mg/kg phosphorus under 6 dS m-1 salinity.
Fernandes and Soratto (2012) applied P in form of nutrient solution and revealed that
phosphorus > 8 mg L-1 has significant impact on root dry weight.
Table 4.8 (a): Effect of proline and phosphorus on root dry weight of maize under salt
stress
Phosphorus 1 11.6255 11.6255 84.48**
Salinity 1 3.7824 3.7824 27.48**
Proline 1 0.7350 0.7350 5.34*
P× S 1 0.7838 0.7838 5.70*
P× A 1 0.0029 0.0029 0.02 NS
S× A 1 0.1213 0.1213 0.88 NS
P× S× A 1 1.2817 1.2817 9.31**
Error 21 2.8900 0.1376
Total 31 21.3192
NS= Non-significant (P> 0.05); ** = highly significant (P< 0.05); * = Significant (P< 0.05)
(b): Comparison of treatments mean
52
10 mg/kg 2.32d 1.59e 2.92bc 1.64e 2.12B
100 mg/kg 3.63a 2.73cd 3.39ab 3.54a 3.32A
Mean S1= 3.06A S2= 2.38B A1= 2.57B A2= 2.87A
LSD at 5% 0.54
4.1.9 Root/shoot length ratio
The individual effect of phosphorus, proline and salt was significant for root shoot
length ratio of maize (table 4.9). Collective effect of phosphorus, salt and proline was
observed non-significant for root shoot length ratio. It was observed that maximum root
shoot length ratio (2.71) was recorded in treatment where phosphorus level was 10 mg/kg of
soil in 6 ds m-1 salinity under control environment of proline. While in treatments where
phosphors was 100 mg/kg of soil, minimum shoot length (1.38) was recorded under control
treatment of proline and salinity.
53
Table 4.9 (a): Effect of proline and phosphorus on root shoot length ratio of maize
under salt stress
Replication 3 0.44959 0.14986
Phosphorus 1 4.33725 4.33725 57.14**
Salinity 1 0.07595 0.07595 1.00 NS
Proline 1 1.00856 1.00856 13.29*
P× S 1 0.06836 0.06836 0.90 NS
P× A 1 1.26445 1.26445 16.66**
S× A 1 0.07402 0.07402 0.98 NS
P× S× A 1 0.00449 0.00449 0.06 NS
Error 21 1.59402 0.07591
Total 31 8.87668
Phosphoru Proline (A1) Proline (A2) Mean

s
10 mg/kg 2.40 2.71 1.77 1.84 2.18A
100 mg/kg 1.38 1.46 1.50 1.43 1.44B
54
Mean S1= 1.76A S2= 1.86A A1= 1.99A A2= 1.63B
4.1.10 Root/shoot fresh weight ratio
The individual effect of phosphorus was significant on root shoot fresh weight ratio
of maize (table 4.10). While individual effect of salt stress and proline was non-significant.
Combination of phosphorus, salt stress and proline was also found non-significant for root
shoot fresh weight ratio. Treatments where 100 mM proline applied, maximum root shoot
fresh weight ratio (1.65) was found when phosphorus applied as 10 mg/kg soil and salinity
was 6 dS m-1. While treatment without proline application, minimum root shoot fresh weight
ratio (0.74) was recorded when salinity was 6 dS m -1 and phosphorus level was 100 mg/kg of
soil.
55
Table 4.10 (a): Effect of proline and phosphorus on root shoot fresh weight ratio of
maize under salt stress
Replication 3 0.06501 0.02167
Phosphorus 1 0.72903 0.72903 20.72**
Salinity 1 0.00228 0.00228 0.06 NS
Proline 1 0.03578 0.0578 1.02 NS
P× S 1 0.00300 0.00300 0.09 NS
P× A 1 0.05695 0.05695 1.62 NS
S× A 1 0.03315 0.03315 0.94 NS
P× S× A 1 0.02588 0.02588 0.74 NS
Error 21 0.73872 0.03518
Total 31 1.68980
NS= Non-significant (P> 0.05); ** = Highly significant (P> 0.05)

10 mg/kg 1.34 1.18 1.06 1.15 1.18A
100 mg/kg 0.87 0.87 0.88 0.89 0.88B
Mean S1= 1.04A S2= 1.02A A1= 1.06A A2= 1.00A
LSD at 5% 0.27
56
4.1.11 Root/shoot dry weight ratio
The individual effect of phosphorus was significant on root shoot dry weight ratio of
maize (table 4.11). While individual effect of salt stress and proline was non-significant.
Combination of phosphorus, salt stress and proline was also found non-significant for root
shoot dry weight ratio. It was observed that maximum root shoot dry weight ratio (1.39) was
recorded in treatment where phosphorus level was 10 mg/kg of soil and proline level was 100
mM with 6 dSm-1 salinity. While in treatments where salinity applied as 6 dSm -1, minimum
root shoot dry weight ratio (0.80) was recorded when phosphors was 100 mg/kg under
control application of proline.
57
Table 4.11 (a): Effect of proline and phosphorus on root shoot dry weight ratio of maize
under salt stress
Replication 3 0.97716 0.32572
Phosphorus 1 0.71790 0.71790 5.24*
Salinity 1 0.11460 0.11460 0.84 NS
Proline 1 0.00711 0.00711 0.05 NS
P× S 1 0.01458 0.01458 0.11 NS
P× A 1 0.10500 0.10500 0.77 NS
S× A 1 0.02803 0.02803 0.20 NS
P× S× A 1 0.00416 0.00416 0.03 NS
Error 24 2.87875 0.13708
Total 31 4.84728
NS= Non-significant (P> 0.05); * = Significant (P< 0.05)

10 mg/kg 1.39 1.20 1.21 1.09 1.22A
100 mg/kg 0.96 0.80 0.96 0.97 0.92B
Mean S1= 1.13A S2= 1.01A A1= 1.09A A2= 1.06A
58
4.1.12 No. of leaves per plant
The individual effect of phosphorus, proline and salt stress was significant for
numbers of leaves of maize (table 4.12). Collective effect of phosphorus, salt stress and
proline was also found significant for numbers of leaves. Under control salinity maximum
numbers of leaves (12.26) was found in treatment where proline was applied as 100 mM and
phosphorus level was 100 mg/kg soil. While in treatment where salt and proline was not
applied minimum numbers of leaves (9.64) was recorded when phosphorus level was 100
mg/kg of soil.
59
Table 4.12 (a): Effect of proline and phosphorus on numbers of leaf plant -1 of maize
under salt stress
Phosphorus 1 16.3020 16.3020 34.38**
Salinity 1 2.7495 2.7495 5.80*
Proline 1 2.2791 2.2791 4.81*
P× S 1 0.1405 0.1405 0.30 NS
P× A 1 0.6050 0.6050 1.28 NS
S× A 1 8.1810 8.1810 17.25*
P× S× A 1 1.3448 1.3448 2.84 NS
Error 21 9.9576 0.4742
Total 31 41.8856

10 mg/kg 9.21 9.91 11.44 9.30 9.96B
100 mg/kg 11.19 11.33 12.05 10.99 11.39A
Mean S1= 10.97A S2= 10.38B A1= 10.41B A2= 10.94A
4.1.13 Leaf Area (cm2)
The individual effect of phosphorus, proline and salt stress was significant for leaf
area of maize (table 4.13). Combine effect of phosphorus, salt stress and proline was also
60
found significant for leaf area. Maximum leaf area (123.46 cm2) was found in treatment
where proline level was 100 mM and phosphorus applied as 100 mg/kg soil with no salt
stress. Treatments without proline and salt application was recorded minimum leaf area
(58.04 cm2) when phosphors was applied at the rate of 10 mg/kg of soil. When phosphorus
was applied as 100 mg/kg maximum leaf area (123.46 cm2) when proline was 100 mM in
control salinity. While in case of collective effect of phosphorus, proline and salinity,
minimum leaf area (113.46 cm2) was recorded. In 6 dS m-1 salinity level maximum leaf area
(120.81 cm2) was found when proline level was 0 Mm (control) and phosphorus was 100
mg/kg of soil. While minimum (60.17 cm2) was recorded when salinity level was 6 dS m-1
with application of 100 mM proline in 10 mg/P per kg of soil. Application of 100 mM
proline was observed to yield the maximum leaf area (123.46 cm2) when phosphorus was
100 mg/kg under control environment of salinity. While minimum leaf area (60.17 cm2) was
recorded when 100 mM proline applied in 10 mg/kg phosphorus under 6 dSm-1 salinity.
Phosphorus fertilization maximized the leaf area of maize plant, this finding was
accordance with Zhang et al. (2018) they stated that P application can increase the leaf area
by proper managing doses without excessive dose.
Table 4.13 (a): Effect of proline and phosphorus on leaf area of maize under salt stress
61
Phosphorus 1 22613.7 22613.7 22419.5**
Salinity 1 206.6 206.6 204.78**
Proline 1 49.0 49.0 48.55**
P× S 1 0.1 0.1 0.06 NS
P× A 1 186.2 186.2 184.62**
S× A 1 777.4 777.4 770.73**
P× S× A 1 188.9 188.9 187.31**
Error 21 21.2 1.0
Total 31 24045.6

10 mg/kg 58.04 g 67.59 e 80.06 d 60.17 f 66.47 B
100 mg/kg 120.81 b 120.81 b 123.46 a 113.46 c 119.63 A
Mean S1= 95.59 A S2= 90.51 B A1= 91.81 B A2= 94.28 A
LSD at 5% 1.47
4.1.14 Chlorophyll content (mg/mL)

The individual effect of phosphorus, salt stress and proline was significant on
chlorophyll content of maize (table 4.14). While combine effect of phosphorus, salt stress
and proline was non-significant for chlorophyll content. It was observed that maximum
chlorophyll content (24.03 mg/mL) rate was recorded in treatment where phosphorus level
was 100 mg/kg of soil in control environment of proline and salt stress. While in treatments
where salinity level was 6 dS m -1, minimum chlorophyll content (18.76 mg/mL) rate was
recorded when phosphors was 100 mg/kg under 100 mM application of proline.
62
Table 4.14 (a): Effect of proline and phosphorus on chlorophyll content of maize under
salt stress
Phosphorus 1 14.204 14.2045 20.75**
Salinity 1 0.980 0.980 38.51**
Proline 1 26.354 26.3538 1.43 NS
P× S 1 4.162 4.162 66.28**
P× A 1 45.363 45.363 6.08*
S× A 1 3.740 3.740 5.46*
63
P× S× A 1 0.031 0.0313 0.05 NS
Error 21 14.372 0.6844
Total 31 110.318

Control (S1) S2 Control (S1) S2
10 mg/kg 22.32 21.95 22.14 23.26 22.42A
100 mg/kg 24.03 22.34 19.21 18.76 21.08B
Mean S1= 21.92A S2= 21.57A A1= 22.66A A2= 20.84B
4.1.15 Stem diameter (mm)

The individual effect of phosphorus, salt stress and proline was significant on stem
diameter of maize (table 4.15). Combine effect of phosphorus, salt stress and proline was
also significant for stem diameter. It was observed that maximum stem diameter (13.68 mm)
was recorded in treatment where phosphorus level was 100 mg/kg of soil and proline level
was 100 mM with 6 dSm-1 salinity. While in treatments where salinity applied as 6 dSm -1,
minimum stem diameter (7.22 mm) was recorded when phosphors was 10 mg/kg under
control application of proline.
64
Phosphorus 1 187.938 187.938 502.95 **
Salinity 1 0.613 0.613 1.64 NS
Proline 1 0.310 0.310 0.83 NS
P× S 1 2.317 2.317 6.20 *
P× A 1 0.096 0.096 0.26 NS
S× A 1 0.921 0.921 2.47 NS
P× S× A 1 0.003 0.003 0.01 NS
Error 21 7.847 0.374
Total 31 201.507
Table 4.15 (a): Effect of proline and phosphorus on stem diameter of maize under salt
stress
NS= Non-significant (P> 0.05); * = Significant (P< 0.05); ** = highly significant (P< 0.05);
65

10 mg/kg 7.75 7.17 7.12 7.18 7.30B
100 mg/kg 11.97 12.42 11.52 12.69 12.15A
Mean S1= 9.59A S2= 9.86A A1= 9.82A A2= 9.63A
4.1.16 Root diameter (mm)
The individual and combine effect of phosphorus, salt stress and proline was
significant on the root diameter (table 4.16). Treatment where salinity was control maximum
root diameter (2.95 mm) was found when phosphorus applied as 100 mg/kg soil with 100
mM proline. While in treatments where foliar application of proline was 0 mM, minimum
root diameter (1.59 mm) was recorded when salinity was control and phosphorus was 10
mg/kg of soil. Application of 100 mg P/kg was found to yield the maximum root diameter
(2.95 mm) when 100 mM proline applied under control salinity. While Minimum root
diameter (2.51 mm) was observed in 100 mg P/kg under control environment of proline and
salinity. In case of 6 dS m-1 salinity maximum root diameter (2.91 mm) was recorded when
proline was applied as 100 mM and phosphorus was 100 mg/kg of soil. While minimum
(1.62 mm) was recorded when salinity level was 6 dS m -1 in 10 mg P/kg of soil without
proline. Maximum root diameter (2.95 mm) was observed when foliar application of 100
mM proline in phosphorus 100 mg/kg under control environment of salinity. While minimum
(1.63 mm) was recorded when 100 mM proline applied in 10 mg P/kg under 6 dS m-1 salinity.
66
According to Sheng et al. (2009) salt stress showed the positive impact on root
diameter that is coinciding with this result.
Table 4.16 (a): Effect of proline and phosphorus on root diameter of maize under salt
stress
F
602.65 **
17.40 *
97.44 **
51.03 **
5.67 *
63.53 **
17.24 *
SOV DF SS MS
Replication 3 0.0545 0.01817
Phosphorus 1 7.1093 7.10928
Salinity 1 0.2053 0.20528
Proline 1 1.1495 1.14951
P× S 1 0.6020 0.60198
P× A 1 0.0689 0.06689
S× A 1 0.7494 0.74939
P× S× A 1 0.2034 0.20336
Error 21 0.2477 0.01180
Total 31 10.3879
67

10 mg/kg 1.59d 1.62d 2.53c 1.63d 1.84B
100 mg/kg 2.51c 2.77b 2.95a 2.91ab 2.78A
Mean S1= 2.39A S2= 2.23B A1= 2.12B A2= 2.50A
LSD at 5% 0.15
4.1.17 Momentary fluorescent rate (µmol m-2s-1)

momentary fluorescent rate of maize (table 4.17). Collective effect of phosphorus, salt stress
and proline was also found significant for momentary fluorescent rate. Application of
phosphorus 100 mg/kg of soil was found to yield the maximum momentary fluorescent rate
(333.50 µmol m-2s-1) in treatment where proline and salinity was not applied. Treatment
without proline application, showed minimum momentary fluorescent rate (221 µmol m-2s-1)
when phosphorus was 100 mg/kg of soil in 6 dSm -1 salinity. In case of 100 mg P/kg was
found to give the maximum momentary fluorescent rate (333.50 µmol m-2s-1) under control
environment of salinity and proline. Without proline application minimum momentary
fluorescent rate (221µmol m-2s-1) was observed in 100 mg/kg phosphorus when salinity was 6
dS m-1. In 6 dS m-1 salinity maximum momentary fluorescent rate (296.75 µmol m-2s-1) was
found in 10 mg P/kg of soil without proline application. While minimum (221 µmol m -2s-1)
was recorded when salinity level was 6 dSm -1 without application of proline in 100 mg/P per
68
kg of soil. Application of 100 mM proline was observed to give the maximum momentary
fluorescent rate (312.50 µmol m-2s-1) when phosphorus was 100 mg/kg without salt stress.
While minimum (228.25 µmol m-2s-1) was recorded when 100 mM proline applied in 100
mg/kg phosphorus under 6 dSm-1 salinity.
This finding in line with Sadiq et al. 2017, they stated that P fertilization in accurate
dose is effective where cropping system is expensive. It improved the momentary fluorescent
rate, yield and the yield component of maize.
Table 4.17 (a): Effect of proline and phosphorus on momentary fluorescent rate of
maize under salt stress
Phosphorus 1 750.8 750.8 823.04 **
Salinity 1 9487.5 9487.5 10400.7**
Proline 1 195.0 195.0 213.80**
P× S 1 32704.0 32704.0 35851.7**
P× A 1 30.0 30.0 32.92**
S× A 1 693.8 693.8 760.56 **
P× S× A 1 4394.5 4394.5 4817.50**
Error 21 19.2 0.9
Total 31 48283
** = Highly significant (P< 0.05); * = Significant (P< 0.05)
69
10 mg/kg 234.50f 296.75c 264.25d 261.00e 264.12B
100 mg/kg 333.50a 221.00h 312.50b 228.25g 273.81A
Mean S1= 286.19A S2= 251.75B A1= 271.4A A2=266.50B
LSD at 5% 1.40
4.1.18 Photosynthetically active radiation (µmol m-2s-1)

Photosynthetically active radiation of maize (table 4.18). Combination of phosphorus, salt
stress and proline was also found significant for photosynthetically active radiation.
Application of 100 mM proline was found to yield the maximum photosynthetically active
radiation (1122 µmol m-2s-1) in treatment where phosphorus was 10 mg/kg of soil in 6 dS m -1
salinity. Treatment without proline and salinity application, minimum photosynthetically
active radiation (564.3 µmol m-2s-1) was recorded when phosphorus was 10 mg/kg of soil. In
case of 100 mg P/kg was found to give the maximum photosynthetically active radiation
(1121.6 µmol m-2s-1) under 100 mM proline in 6 dS m -1 salt stress. Without proline
application minimum photosynthetically active radiation (764.5 µmol m-2s-1) was observed in
100 mg/kg phosphorus with 6 dS m-1 salinity. When salinity was 6 dSm-1 maximum
photosynthetically active radiation (1122 µmol m-2s-1) was found when proline was applied as
100 Mm and phosphorus was 10 mg/kg of soil. While minimum (613.7 µmol m -2s-1) was
recorded when salinity level was 6 dS m -1 without application of proline in 10 mg/P per kg of
soil. Application of 100 mM proline was observed to give the maximum photosynthetically
active radiation (1122 µmol m-2s-1) when phosphorus was 10 mg/kg under 6 dS m-1 salt stress.
While minimum (879 µmol m-2s-1) was recorded when 100 mM proline applied in 10 mg/kg
phosphorus without salinity.
70
Butt et al. (2016) stated that foliar spray of proline enhanced the physiological and
chemical attributes under salt stress. Proline dose that was applied after month of seedling
improve the photosynthetic rate under 50 mM salt stress.
Table 4.18 (a): Effect of proline and phosphorus on photosynthetically active radiation
of maize under salt stress
Replication 3 37 12
Phosphorus 1 146973 146973 16006.5 **
Salinity 1 58995 58995 6425.05 **
Proline 1 746847 746847 81337.6 **
P× S 1 29121 29121 3171.47**
P× A 1 89889 89889 9789.63**
S× A 1 129371 129371 14089.6**
P× S× A 1 7381 7381 803.83**
Error 21 193 9
Total 31 1208806

10 mg/kg 564.3g 613.7f 879.0d 1122.0a 794.73B
100 mg/kg 896.5c 764.5e 938.5b 1121.6a 930.27A
Mean S1=819.56B S2=905.44A A1= 709.7B A2=1015.3A
LSD at 5% 4.45
71
4.1.19 Electron transfer rate (µmol m-2s-1)
electron transfer rate (table 4.19). Collective effect of phosphorus, salt stress and proline was
also found significant for Electron transfer rate. Maximum electron transfer rate (145.25
µmol m-2s-1) was found in treatment where proline level was 100 mM and phosphorus applied
as 100 mg/kg soil without salinity. While in treatments without proline and salinity,
minimum electron transfer rate (45.50 µmol m-2s-1) was recorded when phosphorus level was
10 mg/kg of soil. In case of 100 mg P/kg, maximum electron transfer rate (145.25 µmol m-2s-
1) was recorded in 100 mM proline under control salinity. While under control salinity and
proline level minimum electron transfer rate (61.71 µmol m-2s-1) in 100 mg P/kg of soil. In 6
dSm-1 salinity level maximum electron transfer rate (123.19 µmol m-2s-1) was found when
proline level was 0 mM (control) and phosphorus was 10 mg/kg of soil. While minimum
(6.11 µmol m-2s-1) was recorded when salinity level was 6 dSm -1 with application of 100 mM
proline in 100 mg/P per kg of soil. Foliar application of 100 mM proline was observed to
yield the maximum electron transfer rate (145.25 µmol m -2s-1) when phosphorus was 100
mg/kg under control environment of salinity. While minimum (68.11 µmol m-2s-1) was
recorded when 100 mM proline applied in 100 mg/kg phosphorus under 6 dSm-1 salinity.
Recent study by Carstensen et al. (2018) showed that phosphorus helps to increase
the electron transfer rate by increasing the orthophosphate concentration in chloroplast and
therefore increased the ATP synthase activity.
72
Table 4.19 (a): Effect of proline and phosphorus on electron transfer rate of maize
under salt stress
Phosphorus 1 392.3 392.3 261.14 **
Salinity 1 53.6 53.6 35.69**
Proline 1 1892.7 1892.7 1259.96**
P× S 1 5575.2 5575.2 3711.43**
P× A 1 1758.5 1758.5 1170.68**
S× A 1 20865.2 20865.2 13890.1**
P× S× A 1 41.0 41.0 27.32 **
Error 21 31.5 1.5
Total 31 30613.6

10 mg/kg 45.40h 123.19b 94.76c 74.94e 84.57B
100 mg/kg 61.71g 91.23d 145.25a 68.11f 91.57A
Mean S1= 86.78B S2= 89.36A A1= 80.38B A2= 95.76A
LSD at 5% 1.80
73
4.1.20 Photosynthetic yield (µmol m-2s-1)
photosynthetic yield of maize (table 4.20). Collective effect of phosphorus, salt stress and
proline was observed significant for photosynthetic yield. It was observed that maximum
photosynthetic yield (0.58 µmol m-2s-1) was recorded in treatment where phosphorus level
was 100 mg/kg of soil under control environment of proline in 6 dS m -1 salt stress. While in
treatments where salinity applied as 6 dS m-1, minimum photosynthetic yield (0.23 µmol m-2s-
1
) was recorded when phosphors was 10 mg/kg under 100 mM application of proline. 100 mg
P/kg was found to give the maximum photosynthetic yield (0.58 µmol m -2s-1) when 6 dS m-1
salt stress and proline level was control. While when phosphorus was 100 mg/kg, minimum
photosynthetic yield (0.29 µmol m-2s-1) was found under 100 mM application of proline in 6
dS m-1 saline environment. Salt stress environment with 6 dS m -1 was recorded to yield the
maximum photosynthetic yield (0.58 µmol m-2s-1) when phosphorus applied as 100 mg/kg of
soil under control (0 mM) proline application. While minimum photosynthetic yield (0.23
µmol m-2s-1) was recorded in 6 dSm -1 salinity when P level was 10 mg/kg of soil with 100
mM application of proline. In case of 100 mM foliar proline maximum (0.32 µmol m-2s-1)
was observed in 100 mg/kg phosphorus with no salt stress. On the other hand, minimum
photosynthetic yield (0.23 µmol m-2s-1) was recorded when applied phosphorus level was 10
mg/kg under 6 dS m-1 salt stress.
Maximum photosynthetic yield was observed by balanced phosphorus application,
the same finding also revealed by Zhihui et al. (2016). They stated that imbalanced
application of P can lead to loss in grain yield and low nutrient uptake efficiency.
74
Table 4.20 (a): Effect of proline and phosphorus on photosynthetic yield of maize under
salt stress
Phosphorus 1 0.03581 0.03518 16922.1 **
Salinity 1 0.05192 0.05192 24976.3 **
Proline 1 0.23410 0.23410 112609 **
P× S 1 0.00298 0.00298 1435.29 **
P× A 1 0.00342 0.00342 1646.94 **
S× A 1 0.12789 0.12789 61519.8 **
P× S× A 1 0.00935 0.00935 4497.77 **
Error 21 0.00004 2.079
Total 31 0.46490
** = highly significant (P< 0.05); * = Significant (P< 0.05)

10 mg/kg 0.28f 0.54b 0.29e 0.23g 0.34B
100 mg/kg 0.42c 0.58a 0.32d 0.29e 0.40A
Mean S1= 0.33B S2= 0.41A A1= 0.46A A2= 0.28B
LSD at 5% 2.12
4.1.21 Shoot phosphorus content (mg/kg)

The individual effect of phosphorus, proline and salt stress was significant for shoot
phosphorus content of maize (table 4.21). Collective effect of phosphorus, salt stress and
proline was also found significant. Maximum shoot phosphorus (1763.3 mg/kg) was found in
treatment where proline level was 100 mM and phosphorus applied as 100 mg/kg soil in 6
dSm-1 salinity. While in treatments where phosphorus level was 10 mg/kg of soil, minimum
75
shoot phosphorus content (608.4 mg/kg) was recorded under control environment of salinity
and proline. In case of 100 mg P/kg, maximum shoot phosphorus content (1763.3 mg/kg)
was recorded when 6 dS m-1 salinity applied under 100 mM proline. While in absence of
proline and salt stress, minimum shoot phosphorus concentration (1053.7 mg/kg) was
recorded when 100 mg p/kg of soil was applied. In 6 dS m-1 salinity level maximum shoot
phosphorus concentration (1763.3 mg/kg) was found when proline level was100 mM and
phosphorus was 100 mg/kg of soil. While minimum (1257.2 Mg/kg) was recorded when
salinity level was 6 dS m-1 without application of proline in 10 mg P per kg of soil. Foliar
application of 100 mM proline was recorded to yield the maximum shoot phosphorus content
(1763.3 mg/kg) when phosphorus was 100 mg/kg under 6 dS m -1 salinity. While minimum
(1458.9 mg/kg) was recorded when 100 mM proline applied in 10 mg phosphorus per kg
under 6 dS m-1 salinity.

Belanger et al. (2017) stated that sustainable phosphorus management require the
batter knowledge of P fertilization. Experiment was conducted with different dose of
phosphorus to analyzed the effect P supply on shoot phosphorus in swards. Result showed
that phosphorus enhanced the shoot P concentration and shoot biomass during primary
growth in spring.
Table 4.21 (a): Effect of proline and phosphorus on shoot phosphorus content of maize
under salt stress
Replication 3 715 238
Phosphorus 1 345638 345638 130.29**
76
Salinity 1 573688 573688 216.25**
Proline 1 2099379 2099379 791.35**
P× S 1 23936 23936 9.02*
P× A 1 1971 1971 0.74NS
S× A 1 203118 203118 76.56**
P× S× A 1 222984 222984 84.05**
Error 21 55711 2653
Total 31 3527141

10 mg/kg 608.4f 1257.2d 1462.7c 1458.9c 1196.8B
100 mg/kg 1053.7e 1259.1d 1542.6b 1763.3a 1404.7A
Mean S1= 1166.9B S2=1434.6A A1=1044.6B A2=1556.9A
LSD at 5% 75.7
4.1.22 Root phosphorus content (mg/kg)

The individual effect of phosphorus, salt stress and proline was significant on root
phosphorus content of maize (table 4.22). Combination of phosphorus, salt stress and proline
was also found significant for root phosphorus content. Application of 100 mM proline was
found to yield the maximum root phosphorus content (2351.2 mg/kg) in treatment where
phosphorus level was 10 mg/kg and salt stress was not applied. Treatment without proline
application, yield the minimum root phosphorus content (1620.6 mg/kg) when salinity was 6
dS m-1 and phosphorus was 100 mg/kg of soil. In case of 100 mg P/kg of soil was found the
maximum root phosphorus (2303.6 mg/kg) under 100 mM proline and 6 dS m -1 salt stress.
Without proline application minimum root phosphorus content (1620 mg/kg) was observed
77
was observed in 100 mg phosphorus per kg when salinity was 6 dS m -1. In case of 6 dS m-1
salt stress maximum root phosphorus content (2303.6 mg/kg) was found when proline was
applied as 100 mM and phosphorus was 100 mg/kg of soil. While minimum (1620.6 mg/kg)
was recorded when salinity level was 6 dS m -1 without application of proline in 100 mg P per
kg of soil. Application of 100 mM proline was observed to yield the maximum root
phosphorus content (2351.2 mg/kg) when phosphorus was 10 mg/kg under 6 dS m -1 salt
stress. While minimum (1740.4 mg/kg) was recorded when 100 mM proline applied in 100
mg/kg phosphorus without salt stress.
According to the Ali et al. (2008) different concentrations of proline (30 mM and 60 mM)
was applied in water stressed maize. They reported that 30 mM application of proline
significantly increased the accumulation of phosphorus in water stressed maize.
Table 4.22 (a): Effect of proline and phosphorus on root phosphorus content of maize
under salt stress
Replication 3 39688 13229
Phosphorus 1 102446 102446 17.19*
Salinity 1 28704 28704 4.82*
Proline 1 427350 427350 71.72**
P× S 1 4910 4910 0.82NS
P× A 1 46391 46391 7.79*
S× A 1 53579 53579 8.99*
P× S× A 1 1592666 1592666 267.29**
Error 21 125130 5959
Total 31 2420864
78

10 mg/kg 1679.5cd 2128.6b 2351.2a 2071.5b 2057.7A
100 mg/kg 2113.5b 1620.6d 1740.4c 2303.6a 1944.5B
Mean S1= 1971.2B S2=2031.1A A1=1885.6B A2=2116.7A
LSD at 5% 113.51
4.1.23 Soil phosphorus content (mg/kg)

Application of proline and phosphorus was observed significant for soil phosphorus
content of maize while salinity was observed as non-significant. Collective effect of
phosphorus, proline and salinity was also non-significant (table 4.23). It was observed that
collective effect of proline (100 mM), salinity (6 dS m -1) and phosphorus (100 mg/kg) yield
maximum phosphorus content (5403 mg/kg) in soil. While in treatments where proline level
was control, minimum soil phosphorus content (2715.3 mg/kg) was recorded when
phosphors was 10 mg/kg under 6 dS m-1 salinity.
79
Table 4.23 (a): Effect of proline and phosphorus on soil phosphorus content of maize
under salt stress
Replication 3 127434 42477.9
Phosphorus 1 19920000 19920000 619.69**
Salinity 1 48828.1 48828.1 1.52NS
Proline 1 6917340 6917340 215.15**
P× S 1 215496 215496 6.7*
P× A 1 439453 439453 13.67*
S× A 1 1953.12 1953.12 0.06NS
P× S× A 1 4371.12 4371.12 0.14NS
Error 21 675177 32151.3
Total 31 28350000

80
10 mg/kg 2949.8 2715.3 4121.8 3871.8 3414.6B
100 mg/kg 4621.5 4668.5 5278 5403 4992.8A
Mean S1=4242.7A S2=4164.6A A1=3738.8B A2=4668.6A
LSD at 5% 263.67
4.1.24 Soil pH
The individual effect of phosphorus, proline and salt stress was significant for soil pH
of maize (table 4.24). Collective effect of phosphorus, salt stress and proline was found non-
significant for soil pH. Under 6 dS m-1 salinity maximum soil pH (8.6) was found in
treatment where proline was applied as 100 mM and phosphorus level was 100 mg/kg soil.
While in treatment where proline was not applied minimum soil pH (7.2) was recorded when
phosphorus level was 100 mg/kg of soil under 6 dS m-1 salinity.
81
Table 4.24 (a): Effect of proline and phosphorus on soil pH of maize
Replication 3 0.53852 0.1795067
Phosphorus 1 1.25407 1.25407 11.30**
Salinity 1 0.52074 0.52074 4.69*
Proline 1 0.76508 0.76508 6.74*
P× S 1 1.69259 1.69259 7.74NS
P× A 1 1.34809 1.34809 5.35*
S× A 1 1.78720 1.78720 6.43*
P× S× A 1 1.58047 1.58047 9.46NS
Error 21 0..88815 0.05551
Total 31 10.37491

10 mg/kg 7.8 7.2 8.22 8.45 7.91B
100 mg/kg 8.5 8.43 7.4 8.6 8.43A
Mean 8.2A 7.8B 7.81B 8.6A
LSD at 5% 0.4078
Organic acids production during tricalcium phosphorus solubilization
82
During P solubilization the production of oxalic and gluconic acid was detected for all
varieties. The production of other organic acids was restricted to some varieties: 2-
ketogluconic acid to three varieties…………. lactic acid to five varieties………… and two
varieties………… had succinic acid; formic acid in two…. two varieties produced malic acid
none of varieties s
CHAPTER 5
SUMMARY
Phosphorus (P) is the most deficient nutrient in about 90% soil of Pakistan. Many
soils around the world contain less phosphorus than required by plant. The main issue is that
P resources are limited in the world found in few countries only. With the increasing
agriculture production and population, P demand also increasing. The other fact is that
phosphorus is immobile in soil. Growth of plant in low P soil is a primary limitation because
phosphorus is tightly bound to soil component like Al and Fe oxides that reduce its
availability to plant. The fact of this scenario is that depletion of total P resources will may
occur in near future.
Organic acids is a major constraint to crop productivity in modern agriculture. It
suppresses the plant growth and cause ionic imbalance. Excess of Na+ create highly ionic
imbalance. It’s also cause decrease in photosynthesis, energy starvation. Plant response to
salt stress by increasing the concentration of different solutes in cytoplasm.
The role of different compatible solute in plant tolerance to salt stress is significant.
Adaptation to salt stresses is correlated with metabolic adaptations that result in the
accumulation of several organic solutes like polyols, sugars, glycine betaines and proline.
Proline role as osmoticum, also known as compatible solute. Maize is C 4 crop and vulnerable
to a salinity.
A pot experiment was conducted to investigate the effect of organic acids on
phosphorus uptake in wheat genotypes at the Agronomic Research Area, University of
Agriculture Faisalabad during 2020. Experiment was carried out under Completely
83
Randomized Design (CRD) with factorial arrangement having three replications of
treatments. Wheat genotypes were eight different varieties and sown in sand filled pot.
Phosphorus was applied at the rate of 10 mg P/kg sand (P 1), 10 mg P+90 mg apatite
P/kg sand (P2) and 100 mg P/kg sand (P3). Nutrients solution was applied as foliar spray
after 1 week of sowing at 10 days interval along with control. After forty days of experiment
plants were harvested and data was recorded about morpho-physiological parameter.
Application of 100 mg phosphorus per kg of soil showed significant increase in
morphological parameter like plant height, shoot length, root length, shoot fresh weight, root
fresh weight and physiological parameter like photosynthetic yield. Root dry mass and
momentary fluorescent rate is highly increased in phosphorus fertilization (100 mg/kg).
Organic acids production in wheat genotypes corroborated their involvement in phosphorus
solubilization. Gluconic acid was major organic acids produced. The result indicated that the
higher the solubilization of phosphorus was due to higher amount of gluconic acid produced.
The increased growth and P uptake have been reported on NIAB with 100 mg P. It was also
revealed that combine effect of 100 mM proline and 100 mg P/kg application enhance the
shoot dry weight, leaf area, root diameter, shoot phosphorus content.
84
Conclusion
In this study it is suggested that ability of organic acids production by wheat
genotypes is independent of their genetic relatedness. Significant difference in plant growth
promotion among phosphate solubilizing genotypes at the need for selecting the potential
genotype based on plant growth promotion in the soil suplemented with insoluble phosphate
for their targated application. The Niab with high potential for phosphate solubilization with
gluconic acid production appear best in Ca rich and P deficient soils of Pakistan.
85
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Exploring the potential of wheat (Triticum aestivum L.

IQRA ZAFAR KHAN

M.Sc. (Hons.) Department of Agronomy (UAF)

A dissertation submitted in partial fulfillment of the requirements for the degree of

WHO’S PRAYERS ARE KEY BEHIND

His kindness and mercy upon him.

I am thankful to the thesis supervisory committee that includes Prof. Dr.

Muhammad Sana Ullah, Institute of Soil and Environmental Sciences, University of

No dedication could ever be completed without expressing my humble obligation to my

Phosphorus is an important plant macronutrient. Plant growth in low P soils is a

Soltanpur’s method with Other OP method, in which sodium bicarbonate (NaHCO 3)

Repeated applications of P fertilizers to improve crop production have contributed

To determine the internal phosphorus requirement of wheat.

Determining the relationship between Olsen P and plant P uptake in soil.

To fulfil the demands of an ever-increasing population, it is critical to create a soil

2.1. Scope of the problem

2.2. Forms of P in soil

Non labile –P → Labile –P → P in soil solution → P in plant root

P in Soil (Solid phase) ← (Slowly available) ← (Readily available)

(Fernandez and Warren, 1994)

2.3. Reactions of P with soil components

The probable reactions of P in calcareous soils are i) precipitation of relatively insoluble

Reaction of P with various soil surfaces may be summarized hereafter.

2.4.1. Adsorption on CaCO3

2.4.2. Adsorption on layer silicates

2.4.3. Adsorption on hydrous oxides, Fe and Al

Al3+ + H 2PO 4- + 2H 2O ↔ 2H+ + Al(OH)2 H 2 PO 4

2.4.4. Adsorption on organic matter

2.4.5. Adsorption on the whole soil

2.7. P status of soils of Pakistan

Soil NaHCO3-P Full technology adoption Partial technology

Category Olsen-P (mg kg-1) Adjustment

Cotton-reduce to ¼ or ½ of recommended dose as in

According to Rahmatullah et al. (2003) recovery of applied P by NaHCO 3 extraction ranged

2.10. P fertilization to wheat

2.11. Effect of method of P application on P availability for crops

Ansar et al. (2008) conducted an excellent study regarding efficiency of fertilizer

2.14. P use efficiency

3.1 Experimental Site

3.2 Experimental Design

3.3 Experimental Treatment

P1= 10 mg P/kg sand

3.6 Seed Rate

3.7 Pot Geometry

Amount of nutrient solution

N CH4N2O Na2SO4 CaCl2

3.9 Phosphorus Determination:

3.11 Soil digestion

3.11.1 Measurements of reagents for soil samples

3.11.3 Procedure for phosphorus determination in soil

3.12 Data Collection

3.12.1 Morphological parameters:

3. Fresh root length (cm)

4. Shoot fresh weight (g)

11. Root, shoot dry weight ratio

12. No. of leaves per plant

13. Leaf area (cm2)

14. Chlorophyll content (mg/mL)

15. Stem diameter (mm)

16. Root diameter (mm)

17. Soil phosphorus content (mg/kg)

3.12.3 Emergence count (%):