I= BIOCHEMICAL AND MOLECULAR APPROACHES TO STUDY THE EFFECT OF PESTICIDES (FUNGICIDES) ON ROOT-ASSOCIATED BACTERIA IN WHEAT
Pakistan Research Repository Home
 

Title of Thesis
BIOCHEMICAL AND MOLECULAR APPROACHES TO STUDY THE EFFECT OF PESTICIDES (FUNGICIDES) ON ROOT-ASSOCIATED BACTERIA IN WHEAT

Author(s)
Fathia Mubeen
Institute/University/Department Details
Department of Chemistry/ University of Agriculture Faisalabad
Session
2004
Subject
BioChemistry
Number of Pages
158
Keywords (Extracted from title, table of contents and abstract of thesis)
pesticides, fungicides, root associated bacteria, wheat, herbicides, insecticides, rodenticides, sanitizers, soil fumigants, pests, rhizobacteria, inoculant biopower, biofertilizers, plant growth promoting rhizobacteria, azospirillum, psuedomonas, azotobacter, alert plus, benlate, captan, darosal, mancozeb, vitavax, indole acetic acid

Abstract
Specific kinds of pesticides like fungicides, herbicides, insecticides, rodenticides, sanitizers and soil fumigants are used to control different pests in crops. An ideal pesticide is toxic only to the target organism, easily biodegradable and does not leach into the groundwater. Unfortunately, this is rarely the case and the widespread use of pesticides in modem agriculture is of increasing concern. Little is known, also about the effects that fungicides have on soil microbial community that are responsible for important functions like, nutrient cycling and organic matter breakdown. Problem is of great concern when the microbial inoculants composed of beneficial rhizobacteria are used in combination with pesticides. A premium quality supplemental inoculant BioPower was introduced first time in Pakistan in 1996 by NIBGE. BioPower is a rich blend of beneficial microbes those work to revitalize the soil and help in integrated nutrient and pest management. The constraints associated with the use of biofertilizers in presence of fungicides turned the attention of the scientists to study the interaction of fungicides and Plant Growth Promoting Rhizobacteria (PGPR) within the soil using different biochemical and molecular markers.

The efficiency of rhizobial inoculants in the presence of pesticides have been reported earlier but such studies were not reported with PGPR. This is the first report of evaluation of efficiency and interaction of PGPR and fungicides in vitro and in vivo. The study is an important contribution towards improving our understanding of behaviour of microbial communities during life cycle of plant and help to develop strategies to better manage them for sustainable agriculture.

The present studies were planned. to evaluate the effect of fungicides on growth survival and other beneficial properties of root associated bacteria under laboratory and field conditions in wheat, which is staple food and high protein source for human consumption in most of the developing countries. The aim of this study was to improve the quality and efficacy of wheat biofertilizer with the addition of fungicide tolerant strains and screening less toxic fungicides.

The study was carried out with 20 different PGPR strains isolated from rhizosphere of wheat and other cereals also include Azospirillum, Psuedomonas and Azotobacter species, used in production of BioPower. Different commonly used fungicides; Alert Plus (Mancozeb + Fosetyle Aluminium), Benlate (Benomyl), Captan (N-[(Trichloro methyl)thio ]-4-cyclohexene-l ,2-dicarboximide), Darosal, Mancozeb (Dithane) and Vitavax (Carboxin) at recommended doses g/kg seed were tested to estimate their effect on growth of the test crop. Fungicide resistance pattern was studied through disc diffusion method which showed that Darosal inhibits the growth of Agrobacterium strain Ca-18 and Mancozeb inhibits the growth of Pseudomonas and Azospirillum whereas all other strains were resistant to test fungicides. Direct plate count of viable bacterial cells in fungicide supplemented media showed similar results. Estimation of the Most Probable Number (MPN) indicated that fungicides Alert Plus, Darosal and Mancozeb do have the inhibitory effect while Benlate have stimulatory effect on growth. Fungicide effect was also studied on bacterial nitrogen fixation, phosphate solubilization and Indole Acetic Acid (IAA) production and in all these studies Alert Plus, Mancozeb and Darosal showed negative effect and Benlate showed positive effect on bacterial physiological efficiency but the effect varies from strain to strain. Genotoxicity of fungicides was studied through comet assay but no significant DNA damage was seen by any of the fungicides.

Ultrastructural behavior of bacterial strains in the presence of fungicide was studied through electron microscopy. Bacterial population was significantly decreased in the presence of full dose of Fungicide Alert Plus, Mancozeb and Darosal. In 1/2 dose less population was observed as compared to BioPower treated positive control and no bacterial population was observed in un-inoculated negative control.

In vivo effect of fungicide application on the bacterial population was studied through Denaturing Gradient Gel Electrophoresis (DGGE) of bacterial rhizosphere community and PCR-PFLP of nif H gene pool. Effect of fungicide was also studied on plant growth and yield in a series of field experiments. Pesticides when used at recommended field rates do not significantly reduce indigenous microbial activities and in some cases like in Benlate, they are stimulatory. The recommended dose of fungicides shows different effects in vitro and in vivo. It reduces the growth of bacteria in most of the cases in vitro but under field conditions, they don't have any significant effect on wheat yield. It can be concluded that interaction between the pesticides and the soil micro flora under field conditions may differ from those in standard laboratory experiments.

Download Full Thesis
4916.01 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
486.76 KB
2 1 Introduction 1
234.41 KB
  1.1 Wheat: A Major Food Crop Of Pakistan 2
  1.2 Fungal Diseases Of Wheat 2
  1.3 Pesticides Are Important To Control Pest 4
  1.4 Rhizosphere 5
  1.5 Methods To Study Rhizospheric Bacterial Population 6
  1.6 Pesticides affecting Rhizosphere Microbial Community 14
  1.7 Objectives 16
3 2 Review Of Literature 17
324.03 KB
  2.1 Effect Of Fungicides On Bacterial Population 17
  2.2 Biochemical And Molecular Techniques To Study Microbial Diversity Changes Under Pesticide Influence 22
4 3 Materials And Methods 38
416.49 KB
  3.1 Bacterial Strains 38
  3.2 Fungicides 38
  3.3 Wheat Variety 38
  3.4 In Vitro Toxicity Experiments 38
  3.5 Plant-Microbe Interaction Study Under Fungicide Influence (Tem Studies ) 45
  3.6 Field Experiments 46
  3.7 Molecular Studies 49
5 4 Results And Discussion 62
1883.74 KB
  4.1 The Number Of Viable Bacterial Cell Through Plant Count 62
  4.2 Fungicide Resistance Pattern-Disc Diffusion Method 63
  4.3 Effect Of Fungicide Treatment On Bacterial Nitrogenase Activity 69
  4.4 Effect Of Fungicides On Bacterial Phosphate Solubilization 69
  4.5 Indole Acetic Acid (IAA) Production €“Spot Test 70
  4.6 Bacterial Genotoxicity Studies Of Fungicides Using Comet Assay 73
  4.7 Plant-Microbe Interaction Under Influences Of Fungicides 75
  4.8 Filed Experiments 84
  4.9 Viable Bacterial Count Of Wheat Rhizosphere 93
  4.10 Rhizospheric Bacterial Population Using MPN Count 96
  4.11 Analysis Of Bacterial Community Through RT-PCR And Dgge 99
  4.12 Restriction Fragment Length Polymorphism (RFLP) For NIFH 119
  4.13 Summary 127
6 4 Literature Cited 129
1883.74 KB
7 5 Evaluation Report Of Foreign Examiners
1806.21 KB
  5.1 Appendices