Gopalakrishnan, S
(2004)
Toxigenicity of Fusarium species causing wilt of chickpea.
PHD thesis, University College London.
Supervisors
| Supervisors Name | Supervisors ID |
|---|
| Strange, R | UNSPECIFIED |
Abstract
The toxigenicity of isolates of Fusarium for chickpea, Cicer arietinum, the third most
important legume crop in the world was studied. Fungi were grown in liquid culture and the
culture filtrates assayed on cells isolated from leaflets of the plant. One isolate, designated
FOC 5, produced cultures that were predominately red (70-80% of the cultures). When the
culture filtrates of all isolates over time were assayed, the red cultures of FOC 5 were much
more toxic than those of the other isolates and were also about 10 times more toxic than the
colourless cultures of FOC 5. Toxic titres of the red FOC 5 cultures peaked at 12 days when
grown at 20°C. The toxin from these red cultures were purified by solvent partitioning, solid
phase extraction (SPE), thin layer chromatography (TLC) and high performance liquid
chromatography (HPLC) using the assay to monitor the stages in purification.Shaking of culture filtrates of FOC 5 with ethyl acetate resulted in about half the toxic
activity (50-55%) partitioning into the organic phase and 25-30% remaining in the aqueous
phase. The activity of the aqueous phase was lost on freeze-drying suggesting a volatile
compound. When the ethyl acetate phase was dissolved in aqueous acetonitrile and applied to
C18 SPE cartridges, about 9% was not adsorbed and 35% could be eluted with methanol.
Greater affinity was shown for cyano SPE cartridges with 6% not adsorbed and 45 %
recoverable by elution in acetonitrile. Attempts at purification of the toxin(s) of adsorbed and
non-adsorbed fractions from these reversed phase cartridges by HPLC did not yield pure
products.Recovery of activity of the ethyl acetate phase from flash chromatography on silica
gel was 61-110%. However, HPLC demonstrated that several compounds were present in the
active fractions.Separation of components of the ethyl acetate phase or the fraction adsorbed by cyano
cartridges of culture filtrates by TLC on silica gel rather than using SPE, flash or reversed
phase HPLC was more successful. Red bands corresponding to the active compound were
scraped from TLC plates and eluted in acetonitrile. HPLC of the eluents on a cyano column
with 10% acetonitrile as the mobile phase demonstrated a single homogeneous peak with
absorption maxima of 224 and 281 nm. The purified fraction is, at the time of writing, being
studied by Professor Mike Beale at Rothamsted Research using nuclear magnetic resonance
techniques in order to determine its structure.Four other isolates, identified by the International Crops Research Institute for the
Semi-Arid Tropics as F. oxysporum f. sp. ciceri did not produce the red, toxic compound,
throwing doubt on the correct identification of the isolates. When the sequences of ribosomal
DNA of all five isolates were determined, the isolate that produced the red toxic compound
most closely matched Fusarium acutatum (99%), in a BLAST search and this accorded with
its morphology. A BLAST search showed that three of the other isolates matched the
sequence of cotton pathogen, F. oxysporum f. sp. vasinfectum (100%, 100% and 97%) and
one closely matched F. oxysporum f. sp. vanillae (99%) These results suggest that a reevaluation
of the taxonomy of Fusarium species causing wilt of chickpea is required.
| Item Type: |
Thesis
(PHD)
|
| Divisions: |
UNSPECIFIED |
| CRP: |
UNSPECIFIED |
| Subjects: |
Mandate crops > Chickpea |
| Depositing User: |
Mr Sanat Kumar Behera
|
| Date Deposited: |
20 Feb 2012 04:50 |
| Last Modified: |
24 Apr 2012 05:53 |
| URI: |
http://oar.icrisat.org/id/eprint/5547 |
| Acknowledgement: |
I would like to thank Dr Richard Strange for his advice and guidance during the
course of this research and also for his constructive suggestions, criticisms and help in
preparation of the thesis. I would also thank Dr Richard Strange for helping to pay my tuition
fees.
I am very grateful to the European Union Project (contract number ICA4-CT-2000-
30003), with out whose funding this Ph.D. would not have been possible. I would also like to
thank the Department of Biology, the Graduate School of University College London and
various charities, including The South Down Trust, The Family Welfare Association, The
Leche Trust and The Gilchrist Educational Trust for partially defraying the cost of my tuition
fees.
I would also like to thank Dr Astrid Wingler for her guidance and help in the course
of research from time to time. Many thanks are also due to Professor Mike Beale and Dr Jane
Ward of Rothamstead for their help in efforts to obtain the structure of the toxin by Nuclear
Magnetic Spectroscopy. I should also like to thank all my past and present friends in the
Plant Pathology Laboratory at UCL, particularly Estelle Gewiss for helping me with several
techniques.
I am also very grateful to my father and my late mother for their love and support.
Finally I am very much indebted to my beloved wife Srilatha for her continuous patience and
support and without which this thesis would not have been possible
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