Chapter-1 commonly cultivated in the Kohat region of Pakistan

        Chapter-1Introduction        1.1. Background andJustificationMost plantshave both inhibitory and stimulatory allelopathic effects on germination,growth, development, and phytochemicals of the associated plant species.

Morespecifically, weeds are among those plant species, which can influence cropsincluding wheat through the production of certain allelochemicals.Pakistan is ranked as the 4th biggest providerof wheat in Asia and 11th in the world among wheat producingcountries 1. However, wheat production is declining every year and weeds areconsidered to be a major reason of low yield across the world.

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Similarly inPakistan wheat production is 30-35% lower than the potential yield for whichweeds are one of important but less noticed constrain 2.Two wheat cultivars “KT-2000” and “LOCAL” arecommonly cultivated in the Kohat region of Pakistan 3. Avena fatua (wildoat) is the most abundant weed species found in these wheat (Triticum aestivum L.) fields 4.

Weeds have the potential to alter thebiochemical, phytochemical and antibacterial properties of plants speciesgrowing in its vicinity 5. However, such informationis very scarce related to the above mentioned weed species and wheat cultivarsof Kohat region. 1.2.

AllelopathyThe word allelopathy isa 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.Biochemicalinteractions between plants (allelopathy) are a product of the actions of a variousgroup of compounds that are synthesized by plants and microorganisms.

Thesecompounds are called allelochemicals. Numerous groups of allelochemicals forwheat allelopathy have been recognized, namely, hydroxamic acids, phenolicacids, and short-chain fatty acids. Usual members of allelochemicals includecompounds like p-hydroxybenzoic acid, ferulic acid, catechin, scopoletin amygdalin,juglone and patulin appendix 1.

Allelopathic effect of plant development and growthdepends upon the concentration, edaphic and climatic factors, species sensitiveityand interaction with other stresses 7. The source of allelochemicals inagriculture areas may be the other crops, weeds or microorganisms. Yields ofcrops can also be affected by the inhibiting or stimulating effect of one crop uponthe later, or the capacity of a crop to inhibit weeds. The interference of growthor germination of a crop by allelochemicals can be the result of their directeffects on metabolism, or indirect effect by upsetting nitrogen fixation andother microbial activity in soil 8. Manyphysiological processes are also due to the effect of allelopathic chemicals,but it has been hard to describe the primary mechanisms involved for a specificcompound to inhibit physiological activity.

Oncethe Allelochemicals are released in the soil, they get dissolve. They come intocontact with other elements of diverse, chemical, biological physical andphysiochemical properties which may possibly influence action of allelochemicalsand therefore as a result either increase or decrease their impact on receiver crops/plants9. The extent to which an the allelochemicals can effect may in turn beaffected by other factors for example PH of soil, nutrient and moisture contentof soil, organic matter content as well as other microorganisms 10. Agronomic researchers have found that weeds can cause 17-25% lossesin wheat annually due to their competitive and allelopathic nature. Wheat cultivarsdiffer in their allelopathic potential against weeds, showing that selection ofweed resistant cultivars might be a convenient approach in integrated weedmanagement.

1.3. Wheat (a CashCrop)     Wheat (T.aestivum L.) Is one of the early few domesticated food crops. It has beenthe 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 landof the world is under wheat.

In terms of total production,area under cultivation, and yield per hectare, Pakistan stands among majorwheat-producing states of the world. Wheat is an essential food of population ofPakistan because it makes 60% of everyday diet of a common man in the countryand average consumption per capita is approximately 125 kg. Due to this wheat occupiesa principal position in agricultural policies of government of Pakistan. In2012-13 the yield per hectare remained at 2,797 (Kg/ha) which showed a positivegrowth of 2.7 percent as compared to negative 4.2 percent growth in the nextyear 11.

Agriculture is the most fundamental element of Pakistan economy. It addsalmost 25% to overall GDP, and employs about 44% of work force which makes itas the major source of foreign exchange earnings of Pakistan (Economic Survey,1999- 2000). Muzik12 reported that losses of crops due to pests, insects and plant diseases aremuch less than those caused due by weeds. From 2000 to 2003 it was estimatedthat 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 producingcapacity, several weeds such as: Wild oat (Jungli-Jai), goose foot (Bathu), Bird’sseed grass (Dumbi-sitti), and Field bind weed (Lehli) are identified as most destructiveweeds because they produce toxic secondary metabolites and compete for the usethe same resources 14. 1.

4. Weeds and TheirEffects on CropsA weed is a plantgrowing where it is not desired, or a plant out of place 15. Reports on theeffect of weeds on wheat have been published by the researchers worldwide. Invasionsof foreign plant species can have an impact at several levels of ecologicalcomplexity from genes to ecosystems 16. Weeds are found in variety of habitsand habitats all over the world. Though they account for 1% of the total plantspecies on earth, still they are a reason of several problems all the same to mankindby interfering in health, economic stability, and food production 17. Weedsare a major concern in agriculture, because they compete with cultivated cropsfor food and resources 18.Studies show that the watersoluble substances are present in weed residues.

 Which reduce growth and germination of wheatseedlings. However wheat crop residues can also reduce yield of the later cropnext season. This is due to the fact that crops and other plant species have a severalclasses of phenolic acids 19. Variation in the soil microbial populations ofdifferent agricultural lands and community structures could be differentiated bythe allelopathic and non-allelopathic varieties.     In pot experiments, Sozeri, and Ayhan 20 established, thatmixing straw, with soil, which was gathered after harvesting, decreasedgermination of wheat seedlings and increased seedling mortality 21. Someinvasive plants are best adapted and for their success the release ofphytotoxins have been proposed as a theory these palnts/weeds have long beensuspected that they use allelopathic phenomena to quickly displace the nativespecies.

Amongst so many other symptoms, reduction in photosynthetic efficacyis also a consequent influence of allelopathic phenolics. The numerous effectsresulting from allelopathic phenomenon include losses in plant growth development,absorption of minerals and water, uptake  of ions, leaf water potential, osmoticpotential, shoot turgor pressure, dry mass production, expansion of leaf area, stomataldiffusive conductance, stomatal aperture size and photosynthesis 21, 22, 23. ToxicAllelochemicals have been described to intervene the degradation and synthesisof some plant hormones, for example the activation of Absisic acid synthesis byferulic acid and as a result modifying the biochemical factors that governplant physiology, biochemistry and metabolism 24.

1.5. Wheat as anAntibacterial CropParasitic bacteria can enterthe body through inhalation from nose and lungs, through sexual contact or consumptionin diet.

Common signs of bacterial infections include chills, fever, nausea, headacheand vomiting. Frequently occurring pathogenic bacteria include Escherichia coli, Shigella sonnei,Staphylococcus aureus and Pseudomonasaeruginosa, 22. The medicinal plantshave long being analyzed for possessing antimicrobial properties. Fromcenturies medicinal plants are being used in traditional medicine to treatcontagious infections. Medicinal plants contain active secondary metabolites.Thus the use of medicinal plants is very important to our health 24. Earlier alldrugs were extracted from medicinal plants.

Medicinal Plants showantiphytoviral and antibacterial activities due to the phenolic compounds and essentialoils that are present in them 25. These compounds have upto 200-800 chemical constituents.Dueto the fact that microorganisms have increasing antibiotic resistance in and onthe other hand considering the side effects of synthetic medicines, medicinalplants are currently gaining acceptance in cure of bacterial diseases 26. Similarlycrops are a chief source of antimicrobial agents due to the secondary metabolitessuch as, flavonoides, alkaloids, terpenoids and tannins that are present inthese crop plants 27. Several crops have been screened for theirantibacterial activities to use them as antimicrobial agents such as Cabbage (Brassica oleraceae L.) and Garlic (Allium sativum L.) Among crops wheat isan important constituent of the traditional diet all around the world so wealso need to investigate the antibacterial activities of wheat.

1.6.Herbicides as Weed Management StrategyCurrent agriculture strategiesdepend upon artificial chemicals to control weeds as invasive plants, becausethese plant species compete with native crop plants for growth and developmentalfactors, and harbor pests and other plant pathogens. Studies of Pandey et al.

28 shows that the post-emergence application of isoproturon and metaxuron @ 1kg 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 scientistssuggest post-emergence application of herbicides 29, 30. There is no doubt that herbicide-resistantcrops and use of herbicides and have significantly improved agricultural productivity.

On other hand evidence shows that use of herbicides can also affect crops throughproduction of harmful chemicals which have a negative effect on crop growth anddevelopment 31. The wide use and sometimes the misuse of herbicides alsocreate problems. The most important problem being the development of resistanceby weeds toward herbicides which refers to the ability of a plant to developand reproduce under the dose of herbicide that is usually fatal to the species32.

Thus, among the utmost existing economic challenges to agriculture isweed resistance to herbicides 33 with more than 346 types of weed known to beresistant to herbicides 34.Much attention is beinggiven to develop alternative techniques for weed control because of theincreasing awareness about the risks involved in the use of chemicals andherbicides. Over last fifty years the abuse of herbicides and chemicals for controlof weeds has resulted in increasing public concern about their impacts on humanhealth and the environment 35. With existing pressures to moderate the use ofherbicide along with maintaining cost effective weed control, the capacity ofcultivars of wheat and other crops to depress weed growth has becomeincreasingly important 36.Allelochemicals can be utilized for improvement of crop productionby avoidance and application techniques. Such techniques include, use ofallelopathic crop residues, development of allelochemicals as biologicalcontrol of weeds, managing of crop sequences and breeding crops for weedcontrol 37.

The negative impact of commercialherbicides makes it desirable to search for other alternative weed managementoptions. 1.7. Aim of Study This study aims to assess the physiological andbiochemical profile and in-vitroantibacterial activities of allopathically induced wheat varieties.

Theoutcomes of this study would have significant contribution towards weeds’eradication, to develop consciousness among farmers in orderto reorient their attitudes towards higher grain yield, and increasing medicinal potency of wheat crop. Thefocus is to first examine the factors responsible for reduction wheat growth andthen to determine methods of weed control to make the agriculture profitable. Toour knowledge this is the first time the activities of such extracts from wheatcrop cv. of Kohat have been analyzed,for the antibacterial activities of wheat. 1.8.

HypothesisA. fatua may inducechanges in the biochemical and antibacterial potential of T. aestivum.

1.9. Objectives·        To evaluate the biochemicalparameters of allelopathically induced wheat cultivars.·        To investigate theantibacterial activities of allelopathically induced wheat cultivars.·        To establish a relationbetween the biochemical profile and antibacterial potential of allelopathicallyinduced wheat cultivars.

       Chapter-2Materialsand 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 sterilizedby 70% (w/v) ethanol for 5 minutes and then rinsed with distilled water for 2to 3 times. The Sterilized seeds were kept inside incubator at 30-35oCup to 24 hours.

2.2. Preparation of Weed ExtractsAvena fatua L. was collected from the wheat fields of district Kohat(Khyber Pakhtunkhwa) and identified by the taxonomist at the Department ofBotany, Kohat University of Science and Technology.

Plants of A. fatua L. were air-dried in shade and then cutinto small pieces. To prepare extracts 10 g plant material of A.

fatua L. was soaked in 100 ml ofdistilled water for 24 hours and standard solution was obtained. Four differentconcentrations were prepared by using the standard solution i.e. (100%).

Thedifferent concentrations were labeled as 25%, 50%, 75% and 100%. Control 0% wastreated with distilled water. 2.3. Germination and Growth Conditions            For experimentation sterilized Petri plates were used. About200 g ofsterilized soil was added to each petri plate.

For every treatment there were three replicates (each replicahaving 10 seeds). Each petri plate was treated with suitable solution of about10 ml daily. All replicates were placed in dark growth chambers by maintainingthe temperatures at 28 ± 02 0Cfor two days until germination. Petri plates were placed in Light conditions at3rd day, with relative humidity of 60 %. Germination was observed ondaily basis. The emergence of radical of a seed was considered to begerminated.2.4.

Roots and Shoots Length MeasurementsWheat seedlings were harvested after 10days of growth, and their lengths measured after being separated in shoots androots. for measurement of length, Centimeter (cm) was used as standard unit. 2.5. Determination of Fresh and Dry Biomass of Shoot and RootTo quantify fresh biomass, roots and shootswere cut down and weighed separately on electronic balance in gram (g) unit.

Fordry biomass, fresh cut out shoots, and roots were placed in oven for up to 72 hat 68 oC and then weighted. 2.6. Biochemical AssaysShoots and roots were analyzed forvarious biochemical parameters such as photosynthetic pigments (chlorophyll a, b and total carotenoids), totalsoluble sugars (TSS) and total soluble proteins (TSP).2.6.

1. Estimation ofPhotosynthetic pigmentsA sample of dried plant material (25 mg) was taken in a test tube.To neutralize plant acid and to avoid the formation of pheophytin, MagnesiumOxide (MgO) (25 mg) was added to plant material. After that, 5 ml of methanolwas 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 tubeand centrifuged for at room temperature 5 minutes at 4000 rpm. Afterwards, thesupernatant obtained was shifted to a 1-cm path length cuvette with the help ofa pipit and absorbance readings were taken in a UV-2600 spectrophotometeragainst a solvent blank at three different wavelengths:  666 nm, 653 nm and 470 nm. For thecalculation of Chlorophyll “a”, “b” and total carotenoidsstandard methodology of Lichtenthaler and Wellburn 38 was followed usingthe 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)/ 2452.6.2. Estimation of Total Soluble SugarsTotal soluble sugars were determined bystandard methodology of Shields and Burnett 39 byslight modifications. Dried sample of shoots and roots (50 mg) was added andhomogenized in mortar and then extracted two times with 3 ml of hot 90% ethanolfor about 1 hour at 60-70oC in an incubator. Afterwardsextract was transferred to 25 ml volumetric flask.

By adding 90% ethanol finalvolume was made up to 25 ml. Then 1 ml of phenol (5%)  and 1 ml prepared solution (liquid) was added tothick walled test tubes, 5 ml of analytical grade sulphuric acid was also addedand mixed properly. For the process of exothermic reaction to take place thetest 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 glucosesolution.

The amount of sugar was given as mg g-1 DW- Estimation of TotalSoluble ProteinsThe amount of Soluble proteins were determined by the standard methodologyof Bradford 41. DryShoots of (100 mg) were taken and homogenized by using mortar and pestle in 1ml phosphate buffer of (pH 7.0). The homogenate was centrifuged for 15 minutesat 4000 rpm.

Afterwards 20 µl extract, 2 ml distilled water and 0.5 ml Bradfordreagent was added to 1-cm path length cuvette. Absorbance was recorded at 595nm by spectrophotometer UV-2600. bovine serum albumin  was used as a standard. Finally total solubleproteins were estimated by using the following formula.

                    Total Soluble Protein (mgg-1 DW) = C x V/VT x W                    C=Absorbance value                    V=Volume of phosphatebuffer                    VT=Volume of enzyme extract                    W=Plant weight2.7. Determination of Antibacterial ActivitiesATCC 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 welldiffusion method coupled with antibiotic susceptibility discs as described byKirby et al. 42. 2.7.1.

Determination of Zone of Inhibition (ZOI) The inoculum was freshly prepared and swabbed allover the surface of the (MHA) Agar plate with the help of sterilized cottonswabs. Five (5 mm) diameter wells were bored in agar medium using of sterilecork-borer with 5 mm diameter. Petri plates were labeled appropriately and 25µl of the suspension/solution of wheat grass extract and the equal volume of extractedsolvent form antibiotics was added to the wells using micropipette. The plateswere kept for some time until the extract diffused properly in the medium. Plateswere lid closed and incubated for 24 hours at 37°C.

After 24 hours plates wereobserved for zone of inhibition. Millimeter was used as unit for measurement ofzone of inhibition.2.8. SoilSampling and AnalysisFrom each replica of treatments,a total of 42 soil samples were collected. Total Organic matter (OM) andnitrogen (N) content was determined by usingthe methodology of Hussain 43. For the determination of pH, by usingpH meters firstly soil-water extracts were prepared.

Means of the soilvariables 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 wereseparated by least significant difference (LSD) test keeping the level ofsignificance at 5%. Two-way analysis of variance (ANOVA)was performed by STATIX9 software.