Genetic Diversity of Colletotrichum gloeosporioides Penz. Causing Anthracnose Disease of Yam (Dioscorea spp.) in Nigeria
2003. XI , 139 pages, 19 figures, 15 tables, 23x14cm, 330 g
Language: English
(Bibliotheca Mycologica, Band 197)
ISBN 978-3-443-59099-4, paperback, price: 40.00 €
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Not a systematic work in the strict sense, but one with which those
concerned with Colletotrichum taxonomy should be aware because of its
scale and contribution to our understanding of the extent of host
specificity amongst C. gloeosporioides isolates. 217 isolates obtained
from yams and some other hosts conforming to the morphospecies
C. gloeosporioides were tested for virulence against a set of
Dioscorea alata cultivars. This enabled a remarkable 18 pathotypes to
be distinguished, most causing moderate symptoms or not being virulent
to yams. Of especial taxonomic interest is that isolates from many
hosts other than yams were able to attack yams and were not just able
to grow on the hosts from which they were isolated. Such hosts clearly
have the potential to act as reservoirs of anthracnose-causing
inoculum. The identity of the strains as C. gloeosporioides and their
distinction from other members the genus was confirmed by ITS
sequences. Vegetative incompatibility tests using mutant strains
corroborated the conclusion that the same species occurred on yams and
weed hosts. However, isolates from different cultured colony
morphotypes were not compatible, and in some cases more than one
vegetative compatibility group was represented in a single leaf
lesion. In addition, a microsatellite approach was used to study
population structure; most variation was within population, and no
geographical correlation was discovered. A partially purified but
unidentified phytotoxin was also extracted from disease-causing
isolates. The Guignardia cingulata teleomorph occurs in the country
and its ascospores are considered as the likely agents for its
long-distance dispersal. A most commendable piece of work
Mycotaxon vol. LXXXVII, July-September 2003
Abbreviations VIII
List of figures X
List of tables XI
1 INTRODUCTION 1
1.1 Origin and importance of yam 1
1.2 Constraints to yam production 2
1.3 Yam anthracnose disease epidemiology 3
1.4 C. gloeosporioides phytotoxins and anthracnose symptoms 4
1.5 Options for the control of yam anthracnose5
1.6 Identification of C. gloeosporioides 6
1.7 Genetic diversity of C. gloeosporioides from yam 8
1.7.1 Conventional methods 8
1.7.2 Molecular-genetic approaches 9
1.8 Objectives of the study 11
2 MATERIALS AND METHODS 13
2.1 Field survey and sample collection.13
2.2 Isolation, maintenance and storage of fungal isolates 14
2.2.1 Isolation of C. gloeosporioides 14
2.2.2 Establishment of single spore cultures 14
2.2.3 Maintenance and storage of cultures 15
2.3 Morphological, cultural and biochemical characterization 15
2.3.1 Morphological and cultural characterization 15
2.3.2 Biochemical characterization of C. Gloeosporioides
morphotypes 17
2.3.3 Analysis of morphological, cultural and biochemical data 17
2.4 Fungicide sensitivity assays 18
2.5 Virulence analysis and cross-inoculation tests 18
2.5.1 Virulence phenotyping on Dioscorea alata 18
2.5.2 Cross-inoculation tests 19
2.6 Vegetative compatibility tests 20
2.6.1 Generation of nit mutants 20
2.6.2 Characterization of nit phenotypes 20
2.6.3 Complementation tests and assignment of isolates to VCGs 21
2.7 Nucleic acid analysis 21
2.7.1 DNA extraction 21
2.7.2 Determination of DNA concentration and gel electrophoresis 22
2.7.3 Amplification of the ITS region 23
2.7.4 Restriction enzyme analysis of rDNA ITS amplicons 24
2.7.5 Purification and cloning of ITS amplicons 24
2.7.6 Transformation of Escherichia coli 25
2.7.7 Sequence analysis of ITS rDNA 28
2.8 Genetic analysis of C. Gloeosporioides populations 30
2.8.1 Strategies for sampling genetic diversity in C. Gloeosporioides 30
2.8.2 Microsatellite-primed PCR analysis 31
2.8.3 Genetic analyses 32
2.9 Characterization of C. Gloeosporioides phytotoxin 34
2.9.1 Fungal cultures, toxin extraction and purification 34
2.9.2 Chemical analysis of C. Gloeosporioides phytotoxin 35
2.9.3 Biological assays 37
2.9.4 Anthracnose resistance screening of yam tissue cultures using C.
gloeosporioides phytotoxin 37
3 RESULTS 41
3.1 Survey of C. Gloeosporioides 41
3.2 Morphological, cultural and biochemical characterization 41
3.3 Fungicide sensitivity tests 47
3.4 Virulence spectrum of C. Gloeosporioides isolates 47
3.5 Cross-infection potential 50
3.6 Vegetative compatibility 51
3.7 ITS analysis 55
3.7.1 PCR amplification and RFLP analysis of the ITS rDNA 55
3.7.2 Sequence analysis of the ITS regions 58
3.8 Genetic structure of C. Gloeosporioides populations 60
3.9 Characterization of phytotoxins 69
3.9.1 Partial purification of C. Gloeosporioides culture filtrates 69
3.9.2 Biochemical and biological assays 71
4 DISCUSSION 79
5 CONCLUSION 91
6 SUMMARY 93
7 REFERENCES 97
8 APPENDIX 117
8.1 Appendix 1: Reagents, solutions, buffers and culture media 117
8.1.1 Morphological, cultural, biochemical and toxin
characterization 117
8.1.2 DNA extraction 122
8.1.3 Agarose gel electrophoresis 123
8.1.4 Cloning and sequencing 124
8.2 Appendix 2: C. Gloeosporioides isolates used in this study 127