Chapter-1 commonly cultivated in the Kohat region of Pakistan











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1.1. Background and

Most plants
have both inhibitory and stimulatory allelopathic effects on germination,
growth, development, and phytochemicals of the associated plant species. More
specifically, weeds are among those plant species, which can influence crops
including wheat through the production of certain allelochemicals.

Pakistan is ranked as the 4th biggest provider
of wheat in Asia and 11th in the world among wheat producing
countries 1. However, wheat production is declining every year and weeds are
considered to be a major reason of low yield across the world. Similarly in
Pakistan wheat production is 30-35% lower than the potential yield for which
weeds are one of important but less noticed constrain 2.

Two wheat cultivars “KT-2000” and “LOCAL” are
commonly cultivated in the Kohat region of Pakistan 3. Avena fatua (wild
oat) is the most abundant weed species found in these wheat (Triticum aestivum L.) fields 4. Weeds have the potential to alter the
biochemical, phytochemical and antibacterial properties of plants species
growing in its vicinity 5. However, such information
is very scarce related to the above mentioned weed species and wheat cultivars
of Kohat region.



The word allelopathy is
a combination of two Greek words ‘Alexon ‘meaning “each other” and “pathos”
meaning “to suffer” i.e. the deleterious effects of one plant upon the other.
However, allelopathy is also term to mean all the biochemical interactions
(stimulatory and inhibitory) among plants 6.

interactions between plants (allelopathy) are a product of the actions of a various
group of compounds that are synthesized by plants and microorganisms. These
compounds are called allelochemicals. Numerous groups of allelochemicals for
wheat allelopathy have been recognized, namely, hydroxamic acids, phenolic
acids, and short-chain fatty acids. Usual members of allelochemicals include
compounds like p-hydroxybenzoic acid, ferulic acid, catechin, scopoletin amygdalin,
juglone and patulin appendix 1. Allelopathic effect of plant development and growth
depends upon the concentration, edaphic and climatic factors, species sensitiveity
and interaction with other stresses 7. The source of allelochemicals in
agriculture areas may be the other crops, weeds or microorganisms. Yields of
crops can also be affected by the inhibiting or stimulating effect of one crop upon
the later, or the capacity of a crop to inhibit weeds. The interference of growth
or germination of a crop by allelochemicals can be the result of their direct
effects on metabolism, or indirect effect by upsetting nitrogen fixation and
other microbial activity in soil 8. Many
physiological processes are also due to the effect of allelopathic chemicals,
but it has been hard to describe the primary mechanisms involved for a specific
compound to inhibit physiological activity.

the Allelochemicals are released in the soil, they get dissolve. They come into
contact with other elements of diverse, chemical, biological physical and
physiochemical properties which may possibly influence action of allelochemicals
and therefore as a result either increase or decrease their impact on receiver crops/plants
9. The extent to which an the allelochemicals can effect may in turn be
affected by other factors for example PH of soil, nutrient and moisture content
of soil, organic matter content as well as other microorganisms 10.

Agronomic researchers have found that weeds can cause 17-25% losses
in wheat annually due to their competitive and allelopathic nature. Wheat cultivars
differ in their allelopathic potential against weeds, showing that selection of
weed resistant cultivars might be a convenient approach in integrated weed

1.3. Wheat (a Cash

    Wheat (T.
aestivum L.) Is one of the early few domesticated food crops. It has been
the basic chief food source of the major nations of North Africa, West Asia,
and Europe for past 8000 years. About one sixth of the total agricultural land
of the world is under wheat.

In terms of total production,
area under cultivation, and yield per hectare, Pakistan stands among major
wheat-producing states of the world. Wheat is an essential food of population of
Pakistan because it makes 60% of everyday diet of a common man in the country
and average consumption per capita is approximately 125 kg. Due to this wheat occupies
a principal position in agricultural policies of government of Pakistan. In
2012-13 the yield per hectare remained at 2,797 (Kg/ha) which showed a positive
growth of 2.7 percent as compared to negative 4.2 percent growth in the next
year 11. Agriculture is the most fundamental element of Pakistan economy. It adds
almost 25% to overall GDP, and employs about 44% of work force which makes it
as the major source of foreign exchange earnings of Pakistan (Economic Survey,
1999- 2000).

12 reported that losses of crops due to pests, insects and plant diseases are
much less than those caused due by weeds. From 2000 to 2003 it was estimated
that in yields of wheat, the losses due to interference of weeds were 20 to 40%
13. Due to well adoption of weed plants to wheat crop and more seed producing
capacity, several weeds such as: Wild oat (Jungli-Jai), goose foot (Bathu), Bird’s
seed grass (Dumbi-sitti), and Field bind weed (Lehli) are identified as most destructive
weeds because they produce toxic secondary metabolites and compete for the use
the same resources 14.

1.4. Weeds and Their
Effects on Crops

A weed is a plant
growing where it is not desired, or a plant out of place 15. Reports on the
effect of weeds on wheat have been published by the researchers worldwide. Invasions
of foreign plant species can have an impact at several levels of ecological
complexity from genes to ecosystems 16. Weeds are found in variety of habits
and habitats all over the world. Though they account for 1% of the total plant
species on earth, still they are a reason of several problems all the same to mankind
by interfering in health, economic stability, and food production 17. Weeds
are a major concern in agriculture, because they compete with cultivated crops
for food and resources 18.

Studies show that the water
soluble substances are present in weed residues.  Which reduce growth and germination of wheat
seedlings. However wheat crop residues can also reduce yield of the later crop
next season. This is due to the fact that crops and other plant species have a several
classes of phenolic acids 19. Variation in the soil microbial populations of
different agricultural lands and community structures could be differentiated by
the allelopathic and non-allelopathic varieties.     In pot experiments, Sozeri, and Ayhan 20 established, that
mixing straw, with soil, which was gathered after harvesting, decreased
germination of wheat seedlings and increased seedling mortality 21. Some
invasive plants are best adapted and for their success the release of
phytotoxins have been proposed as a theory these palnts/weeds have long been
suspected that they use allelopathic phenomena to quickly displace the native
species. Amongst so many other symptoms, reduction in photosynthetic efficacy
is also a consequent influence of allelopathic phenolics.

The numerous effects
resulting from allelopathic phenomenon include losses in plant growth development,
absorption of minerals and water, uptake  of ions, leaf water potential, osmotic
potential, shoot turgor pressure, dry mass production, expansion of leaf area, stomatal
diffusive conductance, stomatal aperture size and photosynthesis 21, 22, 23. Toxic
Allelochemicals have been described to intervene the degradation and synthesis
of some plant hormones, for example the activation of Absisic acid synthesis by
ferulic acid and as a result modifying the biochemical factors that govern
plant physiology, biochemistry and metabolism 24.

1.5. Wheat as an
Antibacterial Crop

Parasitic bacteria can enter
the body through inhalation from nose and lungs, through sexual contact or consumption
in diet. Common signs of bacterial infections include chills, fever, nausea, headache
and vomiting. Frequently occurring pathogenic bacteria include Escherichia coli, Shigella sonnei,
Staphylococcus aureus and Pseudomonas
aeruginosa, 22.

The medicinal plants
have long being analyzed for possessing antimicrobial properties. From
centuries medicinal plants are being used in traditional medicine to treat
contagious infections. Medicinal plants contain active secondary metabolites.
Thus the use of medicinal plants is very important to our health 24. Earlier all
drugs were extracted from medicinal plants. Medicinal Plants show
antiphytoviral and antibacterial activities due to the phenolic compounds and essential
oils that are present in them 25. These compounds have upto 200-800 chemical constituents.

to the fact that microorganisms have increasing antibiotic resistance in and on
the other hand considering the side effects of synthetic medicines, medicinal
plants are currently gaining acceptance in cure of bacterial diseases 26. Similarly
crops are a chief source of antimicrobial agents due to the secondary metabolites
such as, flavonoides, alkaloids, terpenoids and tannins that are present in
these crop plants 27. Several crops have been screened for their
antibacterial activities to use them as antimicrobial agents such as Cabbage (Brassica oleraceae L.) and Garlic (Allium sativum L.) Among crops wheat is
an important constituent of the traditional diet all around the world so we
also need to investigate the antibacterial activities of wheat.

Herbicides as Weed Management Strategy

Current agriculture strategies
depend upon artificial chemicals to control weeds as invasive plants, because
these plant species compete with native crop plants for growth and developmental
factors, and harbor pests and other plant pathogens. Studies of Pandey et al.
28 shows that the post-emergence application of isoproturon and metaxuron @ 1
kg a. i. ha-1 and 2 kg a. i. ha-1, resulted in best weed control of wheat respectively.
Likewise, for significant weed population control and improved yield of crops scientists
suggest post-emergence application of herbicides 29, 30.

There is no doubt that herbicide-resistant
crops and use of herbicides and have significantly improved agricultural productivity.
On other hand evidence shows that use of herbicides can also affect crops through
production of harmful chemicals which have a negative effect on crop growth and
development 31. The wide use and sometimes the misuse of herbicides also
create problems. The most important problem being the development of resistance
by weeds toward herbicides which refers to the ability of a plant to develop
and reproduce under the dose of herbicide that is usually fatal to the species
32. Thus, among the utmost existing economic challenges to agriculture is
weed resistance to herbicides 33 with more than 346 types of weed known to be
resistant to herbicides 34.

Much attention is being
given to develop alternative techniques for weed control because of the
increasing awareness about the risks involved in the use of chemicals and
herbicides. Over last fifty years the abuse of herbicides and chemicals for control
of weeds has resulted in increasing public concern about their impacts on human
health and the environment 35. With existing pressures to moderate the use of
herbicide along with maintaining cost effective weed control, the capacity of
cultivars of wheat and other crops to depress weed growth has become
increasingly important 36.

Allelochemicals can be utilized for improvement of crop production
by avoidance and application techniques. Such techniques include, use of
allelopathic crop residues, development of allelochemicals as biological
control of weeds, managing of crop sequences and breeding crops for weed
control 37. The negative impact of commercial
herbicides makes it desirable to search for other alternative weed management


1.7. Aim of Study

This study aims to assess the physiological and
biochemical profile and in-vitro
antibacterial activities of allopathically induced wheat varieties. The
outcomes of this study would have significant contribution towards weeds’
eradication, to develop consciousness among farmers in order
to reorient their attitudes towards higher grain yield, and increasing medicinal potency of wheat crop.

focus is to first examine the factors responsible for reduction wheat growth and
then to determine methods of weed control to make the agriculture profitable. To
our knowledge this is the first time the activities of such extracts from wheat
crop cv. of Kohat have been analyzed,
for the antibacterial activities of wheat.


A. fatua may induce
changes in the biochemical and antibacterial potential of T. aestivum.

1.9. Objectives

To evaluate the biochemical
parameters of allelopathically induced wheat cultivars.

To investigate the
antibacterial activities of allelopathically induced wheat cultivars.

To establish a relation
between the biochemical profile and antibacterial potential of allelopathically
induced wheat cultivars.









and Methods



2.1. Collection and Sterilization of seeds

Seeds of two Wheat cultivars “LOCAL” and “KT-2000” were procured from Barani Agricultural Research Station Jarma,
Kohat of Khyber Pakhtunkhwa, Pakistan. Vigorous seeds of (Wheat cv. “LOCAL” and “KT-2000”) were sterilized
by 70% (w/v) ethanol for 5 minutes and then rinsed with distilled water for 2
to 3 times. The Sterilized seeds were kept inside incubator at 30-35oC
up to 24 hours.

2.2. Preparation of Weed Extracts

Avena fatua L. was collected from the wheat fields of district Kohat
(Khyber Pakhtunkhwa) and identified by the taxonomist at the Department of
Botany, Kohat University of Science and Technology. Plants of A. fatua L. were air-dried in shade and then cut
into small pieces. To prepare extracts 10 g plant material of A. fatua L. was soaked in 100 ml of
distilled water for 24 hours and standard solution was obtained. Four different
concentrations were prepared by using the standard solution i.e. (100%). The
different concentrations were labeled as 25%, 50%, 75% and 100%. Control 0% was
treated with distilled water.

2.3. Germination and Growth Conditions

            For experimentation sterilized Petri plates were used. About
200 g of
sterilized soil was added to each petri plate. For every treatment there were three replicates (each replica
having 10 seeds). Each petri plate was treated with suitable solution of about
10 ml daily. All replicates were placed in dark growth chambers by maintaining
the temperatures at 28 ± 02 0C
for two days until germination. Petri plates were placed in Light conditions at
3rd day, with relative humidity of 60 %. Germination was observed on
daily basis. The emergence of radical of a seed was considered to be

2.4. Roots and Shoots Length Measurements

Wheat seedlings were harvested after 10
days of growth, and their lengths measured after being separated in shoots and
roots. for measurement of length, Centimeter (cm) was used as standard unit.


2.5. Determination of Fresh and Dry Biomass of Shoot and Root

To quantify fresh biomass, roots and shoots
were cut down and weighed separately on electronic balance in gram (g) unit. For
dry biomass, fresh cut out shoots, and roots were placed in oven for up to 72 h
at 68 oC and then weighted.

2.6. Biochemical Assays

Shoots and roots were analyzed for
various biochemical parameters such as photosynthetic pigments (chlorophyll a, b and total carotenoids), total
soluble sugars (TSS) and total soluble proteins (TSP).

2.6.1. Estimation of
Photosynthetic pigments

A sample of dried plant material (25 mg) was taken in a test tube.
To neutralize plant acid and to avoid the formation of pheophytin, Magnesium
Oxide (MgO) (25 mg) was added to plant material. After that, 5 ml of methanol
was added in each sample and the mixture was homogenized for 2 hours on shaker.
This turbid plant solution was transferred to a 5 ml graduated centrifuge tube
and centrifuged for at room temperature 5 minutes at 4000 rpm. Afterwards, the
supernatant obtained was shifted to a 1-cm path length cuvette with the help of
a pipit and absorbance readings were taken in a UV-2600 spectrophotometer
against a solvent blank at three different wavelengths:  666 nm, 653 nm and 470 nm. For the
calculation of Chlorophyll “a”, “b” and total carotenoids
standard methodology of Lichtenthaler and Wellburn 38 was followed using
the formula given  below.    

                   Chla = 15.65 A666
– 7.340 A653

                         Chlb = 27.05 A653 – 11.21 A666

                   Car. = (1000 A470
– 2.860 Chla – 129.2 Chlb)/ 245

2.6.2. Estimation of Total Soluble Sugars

Total soluble sugars were determined by
standard methodology of Shields and Burnett 39 by
slight modifications. Dried sample of shoots and roots (50 mg) was added and
homogenized in mortar and then extracted two times with 3 ml of hot 90% ethanol
for about 1 hour at 60-70oC in an incubator. Afterwards
extract was transferred to 25 ml volumetric flask. By adding 90% ethanol final
volume was made up to 25 ml. Then 1 ml of phenol (5%)  and 1 ml prepared solution (liquid) was added to
thick walled test tubes, 5 ml of analytical grade sulphuric acid was also added
and mixed properly. For the process of exothermic reaction to take place the
test tubes were left as such to cool down. Absorbance was measured at 485 nm.
Content of total soluble sugar was measured against a standard curve of glucose
solution. The amount of sugar was given as mg g-1 DW-1.

2.6.3. Estimation of Total
Soluble Proteins

The amount of Soluble proteins were determined by the standard methodology
of Bradford 41. Dry
Shoots of (100 mg) were taken and homogenized by using mortar and pestle in 1
ml phosphate buffer of (pH 7.0). The homogenate was centrifuged for 15 minutes
at 4000 rpm. Afterwards 20 µl extract, 2 ml distilled water and 0.5 ml Bradford
reagent was added to 1-cm path length cuvette. Absorbance was recorded at 595
nm by spectrophotometer UV-2600. bovine serum albumin  was used as a standard. Finally total soluble
proteins were estimated by using the following formula.

                    Total Soluble Protein (mg
g-1 DW) = C x V/VT x W

                    C=Absorbance value

                    V=Volume of phosphate

                    VT=Volume of enzyme extract

                    W=Plant weight

2.7. Determination of Antibacterial Activities

ATCC strain of Escherichia coli and Shigella sonnei was obtained from the Department of Microbiology, KUST.
Antibacterial potential of wheat extracts was carried out through agar well
diffusion method coupled with antibiotic susceptibility discs as described by
Kirby et al. 42.

2.7.1. Determination of Zone of Inhibition (ZOI)

The inoculum was freshly prepared and swabbed all
over the surface of the (MHA) Agar plate with the help of sterilized cotton
swabs. Five (5 mm) diameter wells were bored in agar medium using of sterile
cork-borer with 5 mm diameter. Petri plates were labeled appropriately and 25
µl of the suspension/solution of wheat grass extract and the equal volume of extracted
solvent form antibiotics was added to the wells using micropipette. The plates
were kept for some time until the extract diffused properly in the medium. Plates
were lid closed and incubated for 24 hours at 37°C. After 24 hours plates were
observed for zone of inhibition. Millimeter was used as unit for measurement of
zone of inhibition.

2.8. Soil
Sampling and Analysis

From each replica of treatments,
a total of 42 soil samples were collected. Total Organic matter (OM) and
nitrogen (N) content was determined by using
the methodology of Hussain 43. For the determination of pH, by using
pH meters firstly soil-water extracts were prepared. Means of the soil
variables were tested for the significance of variation using S.D. 44.

2.10. Statistical Analysis

The data was replicated three times and means of the data were
separated by least significant difference (LSD) test keeping the level of
significance at 5%. Two-way analysis of variance (ANOVA)
was performed by STATIX9 software.