Pathogenicity of Fusarium anthophilum and Fusarium fujikuroi Associated with Bakanae Disease of Rice

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Pithomyces species

Liu and Zhang (2007) reported the isolation of Pithomyces species from soil in the warm temperate zone of Eastern China. Manoch et al. (2007) isolated Pithomyces species from various substrates such as soil, water, plants, food stuffs and paper in Thailand. Jeamjitt et al. (2006) isolated Pithomyces species from dung in Thailand. Green et al. (2006) reported Pithomyces species from North New South Wales, Australia. Hilda et al. (2003) reported P. chartarum from rice seeds in Cuba. Toth et al. (2007) confirmed that fungus P. chartarum causes leaf damage to wheat in Europe.

Field surveys

Disease surveys were conducted to determine the fungal species associated with various plant parts of diseased rice plants in the field. The surveys were repeated at three growth stages of rice plants, namely as seedlings-tillering, elongation-booting and ripening in the same fields. Disease infected rice plant samples were collected during three growing seasons (1988/89, 1989/90, 1990/91). Samples were collected from rice fields at six rice projects situated in the regions of North West, Free State and Northern Cape provinces respectively. During each survey forty rice fields were sampled in each growing season. Five sample areas were randomly selected in each field by throwing a wire counting square 1 meter in diameter. Samples were collected from the fields and isolation made within shortest possible time from each symptom type recorded.

Isolation of fungal species from diseased rice plants

Isolations of fungal species were done from diseased plant parts by direct plating of plant tissues. The affected tissues namely roots, stems and leaves were washed under running tap water and surface sterilised with 1% sodium hypochlorite solution (NaOCℓ) for 1 minute and rinsed twice in sterilised water. Small pieces of affected tissues were placed in a 9 cm Petri dish containing potato dextrose agar (PDA) and incubated at 25±1°C in darkness for up to 10 days or until sufficient growth or spores enabled isolation. Single spore pure cultures were obtained and stored on PDA slants for identification (Marin-Sanchez and Jimenez Diaz, 1982; Copcu and Karaca, 1983; Nelson et al., 1983; Leslie and Summerel, 2006).

Isolation of fungal species from rice seeds

Seed samples of six lowland rice cultivars/lines (TK5, TK6, TK7, TK9, TC10, and USA 201) were obtained from a rice project at Bushbuckridge in Mpumalanga province to determine fungi associated with rice seeds in the areas. The seeds were the harvest of 1995/96 crop season. The researcher could not visit the rice project to collect disease infected rice plants during the growing seasons. Therefore, rice 68 samples were obtained to determine the fungi associated with rice seeds in the areas.

DNA Sequencing and Sequence Comparisons

DNA sequences were determined from PCR amplicons using the ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq ® DNA Polymerase (Applied Biosystems, Warrington, UK) using the primers EF1 and EF2. Sequences generated in this study were deposited in GenBank. The partial sequence data for translation elongation factor 1-α gene was compared against both the NCBI (GeneBank) database and the Fusarium Database (Geiser et al., 2004). Reference sequences were selected on the bases of the BLAST (Basic Local Alignment Search Tool) results and previously published phylogenetic relationships within the Fusarium incarnatum-equiseti species complex (FIESC). DNA sequences were aligned using a multiple sequence alignment programme.

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TABLE OF CONTENTS PAGES :

DECLARATION OF ORIGINALITY
ACKNOWLEDGEMENTS
DEDICATION
PUBLICATIONS
LIST OF SYMBOLS AND ABBREVIATIONS
LIST OF TABLES
LIST OF FIGURES
LIST OF APPENDIX
Chapter 1: General Introduction
- 1.1 Rice cultivation and consumption
- 1.2 Fungi associated with rice
- 1.3 Mycotoxicosis
- 1.4 Environmental factors in mycotoxin production
- 1.5 The research question and specific aims and objectives
- References
Chapter 2: Literature Review
- 2.1 Mycotoxins
- 2.1.1 Moniliformin
- 2.1.2 Fumonisins
- 2.2 Mycoflora (fungi) in rice plants and rice seeds
  - 2.2.1 Fusarium spp
  - 2.2.1.1 Fusarium anthophilum (A. Braun) Wollenweber
  - 2.2.1.2 Fusarium chlamydosporum (Wollenweber & Reinking)
  - 2.2.1.3 Fusarium compactum (Wollenweber)
  - 2.2.1.4 Fusarium equiseti (Corda) Sacc. Sensu Gordon
  - 2.2.1.5 Fusarium fujikuroi Nirenberg
  - 2.2.1.6 Fusarium semitectum (Berk. & Rav.)
  - 2.2.2 Other fungi
  - 2.2.2.1 Alternaria species
  - 2.2.2.1.1 Alternaria alternata (Fr.) Keissler
  - 2.2.2.1.2 Alternaria longipes (Ellis & Everth) Mason
  - 2.2.2.2 Cochliobolus miyabeanus (Ito & Kuribayashi) Drechler ex Dastur
  - 2.2.2.3 Nigrospora sphaerica (Sacc.)
  - 2.2.2.4 Phoma Species
  - 2.2.2.4.1 Phoma eupyrena (Sacc.)
  - 2.2.2.4.2 Phoma jolyana Pirozynski & Morgan Jones
  - 2.2.2.4.3 Phoma sorghina (Sacc.) Boerema, Doren B. & van Kest
  - 2.2.2.5 Pithomyces Species
- References
Chapter 3: The Mycoflora Associated with Diseased Plants and Seeds of Oryza sativa (Rice)
- 3.1 Abstract
- 3.2 Introduction
- 3.3 Materials and Methods
  - 3.3.1 Field Surveys
  - 3.3.2 Isolation of Fungal Species from Diseased Rice Plants
  - 3.3.3 Isolation of Fungal Species from Rice Seeds
  - 3.3.4 Identification of Fungal Isolates Based on Morphological Characteristics
  - 3.3.5 Identification of Fusarium Isolates on the Basis of Molecular Characteristics
- 3.4 Results
Chapter 4: Pathogenicity of Fusarium anthophilum and Fusarium fujikuroi Associated with Bakanae Disease of Rice
- 4.1 Abstract
- 4.2 Introduction
- 4.3 Materials and Methods
  - 4.3.1 Collection of Rice Seeds and Inoculation Procedure
  - 4.3.2 Statistical Analysis
- 4.4 Results
- 4.5 Discussion and conclusion
- References
Chapter 5: Responses of 54 Rice Cultivars and Lines to Bakanae Disease Caused by Fusarium fujikuroi and Fusarium anthophilum
Chapter 6: Comparative Effectiveness of Two Different Fungicides in Controlling Bakanae Disease of Rice
Chapter 7: Studies on the Production of Mycotoxins by Selected Fusarium Species Isolated from Oryza sativa (Rice)
Chapter 8: General Discussion
Chapter 9: Conclusions, Recommendations and Further Areas of Research