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Title of Thesis

Zakia Latif
Institute/University/Department Details
University Of The Punjab/ National Centre Of Excellence In Molecular Biology
Molecular Biology
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
virulence factor, ascochyta rabiei, chickpeaphytotoxic, biotoxicity, cells, nodes/internodes, bioassays, fungus, isolates, minicolumn

Research work performed during this study was aimed to elucidate the role of virulence factor in breeding resistance to Ascochyta rabiei. During this study nine isolates of A. rabiei which is a causative agent of blight of chickpea, were screened for the presence of phytotoxic activity. Optimum conditions like medium, pH, temperature etc. were investigated for the growth of A. rabiei. Biotoxicity of fungus free culture filtrate of A. rabiei was confirmed by cells, nodes/internodes, detached leaves and root growth inhibition bioassays. Further investigations indicated the presence of phytotoxic compounds associated with fungal pathogenesis in chickpea blight. It was observed that the synthesis of phytotoxic metabolite(s) was induced in the presence of unknown endogenous factors present in the aqueous extract of chickpea seeds. In order to detect the phytotoxic compound(s) during growth of A. rabiei in Czapek Dox medium supplemented with chickpea seed extract, the toxins were isolated from the culture filtrate by organic extraction, solid phase extraction and HPLC and identified as solanapyrones A and C. Further studies were carried out to establish a pattern of production of these phytotoxin(s) in 12-day old fungus free culture filtrates of nine different fungal isolates. They were grown in still culture on Czapek Dox liquid medium supplemented with aqueous extracts of the chickpea seeds of one of two cultivars, a desi (6153) or a kabuli (Cypressa). Toxicity of the culture filtrates from both media were assayed with the cells isolated from the leaves of both cultivars and concentrations of the phytotoxins, solanapyrone A and solanapyrone C, in the filtrates were determined by high performance liquid chromatography (HPLC). For eight of the fungal isolates, toxicity and solanapyrone concentration were correlated; the value of r varies from 0.383 to 0.859 according to the cultivar used in the medium and assays. But in the culture filtrate of one isolate, there was no detectable level of solanapyrones but was showing 15 to 20 times more toxicity as compared to other isolates. The compound was isolated and found to be cytochalasin D by mass spectrometry and NMR. Solanapyrone C and cytochalasin D were also detected from the naturally infected chickpea plants.

To find out the role of isolated phytotoxic compounds in blight of chickpea, the content of phytotoxic compounds in artificially infected chickpea plants with different fungal isolates of A. rabiei has been determined. The severity of disease was recorded using a scale ranging from 1 to 9 (Grawel, 1981) having five defined categories of severity after 12 days. Infected plant materials were also processed through minicolumn for the quantification of phytotoxic compounds by HPLC during pathogenesis.

The studies indicate that considerable variability exists with isolates of A. rabiei for pathogenicity and virulence. The lowest grade of disease severity on 1-9 was 3 and the highest 9. Thus no cultivar showed complete resistance. Maximum phytotoxic compounds were detected on the 12th day of infection, Toxin production varied in both cultivars of chickpea infected with different isolates of A. rabiei varying in virulence. Cytochalasin D was recovered in high levels followed by solanapyrones B + C and solanapyrone A. However, the correlation between the titers of solanapyrones A, B + C and cytochalasin D synthesized by the various fungal isolates upon plant infection and the severity of the disease as determined by the size of leaf and stem lesion was calculated as 0.081, 0.144 and 0.718 respectively on disease susceptible chickpea cv. 6153 while it was 0.055, 0.25 and 0.122 respectively on fungus resistant chickpea cv. C44. The results seem to negate a causative role of isolated phytotoxic compounds in the onset of fungal blight in chickpea.

For the determination of kinetics of phytotoxins production during in vitro growth of various isolates of A. rabiei varying in virulence, the culture filtrates were processed upto 24 days through minicolumn with the interval of 24 hours to resolve the contents of various phytotoxic compounds. Half of its volume was used for the quantification of phytotoxic compounds by HPLC and the other half was evaporated to residue to check the toxicityby cell bioassay.

Solanapyrones, A, B + C and cytochalasin D production varied among the nine isolates of A. rabiei, whereas fungal growth as evidenced by fungal dry weight and sporulation as observed under microscope proceeded normally. Insignificantly low levels of phytotoxic compounds were detected in the culture filtrate of isolates CAMB 7 and 8. In CAMB 8, only solanapyrone B + C were detectable at 2-3 ug/mg of the fungus. Whereas in other isolates production of the varying levels of phytotoxic compounds was observed during in vitro growth and cytochalasin D was observed only in isolates CAMB 5 and CAMB 10. These results indicate CAMB 7 to be a Tox isolate at the present detection limits whereas CAMP 8 produces only solanapyrone B + C but not solanapyrone A. Analysis of genetic diversity by fingerprinting of A. rabiei isolates was one of the measurable objective of the programme. Nine isolates of A. rabiei were characterized by DNA fingerprinting with synthetic non radioactive labeled probes complimentary to simple repetitive sequences namely. (GATA)4, (GACA)4, (GTG)5, (CAA)5, (CA)8, (GGAT)4,Genome of A rabiei was studied and confirmed various isolates of A. rabiei (Previously categorized according to the disease severity and toxin production). All sequence motifs were found to be present in the A. rabiei genome differ in organization and abundance. The extent of polymorphism observed and informativity obtained are strongly dependent on the probe as well as on the restriction enzyme used. Taql and Hinfl are found to be most suitable for oligonucleotide fingerprinting of A. rabiei genome as compared of EcoRI. Among six probe, (GATA)4 was the most informative, occurred less frequently and appeared to be concentrated at one or two predominant loci. In contrast, the motifs (GACA)4, (GTG)5, (CAA)5, (CA)8 and (GGAT)4 fingerprints were more complex and pattern created by (GACA)4 and (GTG)5 probes were district but monomorphic. Seven isolates, out of nine, were similar with one another but different from the rest of the isolates when hybridized with (CA)8 and (GGAT)4.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 1 Introduction 1
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2 2 Literature Review 7
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3 3 Materials And Methods 20
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  3.1 Chickpea Seeds 20
  3.2 Ascochyta Rabiei Isolates 20
  3.3 Isolation Of Ascochyta Rabiei From Diseased Plants 22
  3.4 Preparation Of Aqueous Extract Of Chickpea Seeds 26
  3.5 Fungal Growth In Liquid Medium At Different Conditions 26
  3.6 Growth Of A. Rabiei And Bioactivity Of Culture Filtrate 26
  3.7 Cell Bioassay 27
  3.8 Root Growth Inhibition Bioassay 29
  3.9 Callus Growth Inhibition Bioassay 30
  3.10 Detached Leaves Bioassay 30
  3.11 Extraction Of Phytotoxic Activity By Solid Phase Extraction 30
  3.12 Preparation Of Minicolumn 30
  3.13 Cell Bioassay 31
  3.14 Thin Layer Chromatography 31
  3.15 Spectrophotometer Analysis 31
  3.16 HPLC Analysis 31
  3.17 Extraction Of Phytotoxic Activity By Column Chromatography 33
  3.18 Preparation Of Flash Column 34
  3.19 Flash Chromatography Of The Samples 34
  3.20 Testing Toxic Fractions For Purity 35
  3.21 Thin Layer Chromatography 35
  3.22 HPLC Analysis 35
  3.23 Quantitative Analysis Of Phytotoxins In Five Cultivars Of Chickpea 35
  3.24 Toxin Production By Nine Isolates Of A. Rabiei 36
  3.25 Isolation, Purification And Identification Of Another Unknown Toxic Compound From Fungus Free Culture Filtrate Of A. Rabiei Isolate CAMB-10 36
  3.26 Kinetic Of Phytotoxins Production During In Vitro Growth Of Various Isolates Of A. Rabiei 37
  3.27 Pathogenicity Of Fungal Isolates 38
  3.28 Purification Of The Phytotoxins From Infected Chickpea Plants 38
  3.29 Genetic Diversity Of Nine Isolates Of A. Rabiei Varying In Virulence 39
4 4 Results 42
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  4.1 Optimum Conditions For The Growth Of A. Rabiei 42
  4.2 Production Of Phytotoxic Activity 45
  4.3 Kinetics Of Secretion Of Phytotoxic Activity 50
  4.4 Resolution Of Phytotoxic Activities 55
  4.5 Production Of Solanapyrones In Five Cultivars Of Chickpea 62
  4.6 Growth, Ph, Toxic Activity And Solanapyrones Concentration Of Fungus Free Culture 62
  4.7 Growth Of Nine Isolates Of A. Rabiei In Culture And Toxicity Of Culture Filtrates 65
  4.8 Solanapyrone Content Of Culture Filtrates Of A. Rabiei 73
  4.9 The Relation Of Solanapyrone Content Of Culture Filtrates To Their Toxicity 75
  4.10 The Identification Of Cytochalasin D In Culture Filtrates Of CAMB 10 75
  4.11 The Toxicity Of Cytochalasin D To Chickpea 79
  4.12 Accumulation Of Mycelial Mass And Fungal Toxicity 79
  4.13 Changes In The Content Of Phytotoxic Compounds During Fungal Growth 90
  4.14 Pathogenicity Of Different Isolates Of A. Rabiei 94
  4.15 Genetic Diversity Among Different Isolates Of A Rabiei 97
5 5 Discussion 105
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6 6 References 118
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