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University of Bath PHD Detection, control and resistance expression in oil palm (elaeis guineensis) caused by F.oxysporum f.sp. elaeidis Rusli, Mohd Award date: 2012 Awarding institution: University of Bath Link to publication Alternative formats If you require this document in an alternative format, please contact: [email protected] General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 07. Oct. 2021 Detection, Control and Resistance Expression in Oil Palm (Elaeis guineensis) caused by Fusarium oxysporum f.sp. elaeidis Mohd Hefni Rusli A thesis submitted for the degree of Doctor of Philosophy University of Bath Department of Biology and Biochemistry October 2012 COPYRIGHT Attention is drawn to the fact that copyright of this thesis rests with its author. A copy of this thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and they must not copy it or use material from it except as permitted by law or with the consent of the author. This thesis may not be consulted, photocopied or lent to other libraries without the permission of the author for three years from the date of acceptance of the thesis. Signed: Mohd Hefni Rusli ABSTRACT Vascular wilt disease caused by Fusarium oxysporum f. sp. elaeidis (Foe) causes a devastating disease of oil palm in West and Central Africa. However, this disease has not been reported in South East Asia, in spite of long term importation for breeding purposes of African seed and pollen, known to be often contaminated with Foe. Malaysia is the second largest palm oil producer in the world and Foe remains a major threat to this industry, especially as this study shows four current palm genotypes grown there are susceptible. This research was conducted in order to help Malaysia avoid and/or be prepared for this potential problem. Disease epidemiology was studied in plantations in Ghana. Statistical analysis showed the disease mainly occurred in clusters, implying root-root transmission rather than aerial spread by spores. Many Foe isolates were obtained for genetic analysis from diseased palms, including 10 per cent from 21 symptomless trees. This shows that visual disease surveys are flawed. The only practical, sustainable approach to controlling Fusarium is by breeding disease resistant palm lines. The success of this strategy depends on the variability of Foe isolates. Resistance should be stable because this analysis showed Foe isolates have a monophyletic origin. Moreover, this study also showed early responses to Foe infection of roots through induction of the defence-related gene chitinase. Molecular diagnostic tools were developed for (1) rapid detection and quantification of Foe in seed and pollen for quarantine purposes in order to prevent transcontinental spread of Foe, (2) to test efficacy of putative disease resistant or tolerant palm genotypes, and (3) to facilitate epidemiological studies involving palm tissues and soils. Primers were designed for detecting the species F. oxysporum, based on the translation elongation factor gene (TEF- 1α), superior to the existing ones used currently at quarantine. The first Foe-specific primers to be developed were based on a virulence effector gene that excluded 70 other phylogenetically closely related Fusarium species from various hosts and origins. Treatment by fungicides is undesirable and largely unsuccessful for this disease. For that reason, the mycoparasitic fungus Trichoderma was evaluated. The most effective strains were selected based on discerning techniques such as competition in palm wood and survival in soil and on roots, Trichoderma isolate TPP4 was shown to exhibit potential biological control by delaying and suppressing Fusarium wilt symptoms and colonization. Confocal microscopy was used to investigate interactions on the root surface between Foe and Trichoderma, which had been transformed with red and green fluorescent proteins respectively. Disease progress/extent/symptoms was substantially delayed/reduced in two Malaysian soils compared to other growth media, highlighting the possibility that Foe-suppressive soils in Malaysia might explain the non-appearance of this vascular disease there. From this study ii other potential biocontrol agents may be revealed, for example endophytic fungi that showed antagonism to Foe were isolated from plant species grown in Malaysian soils. iii ACKNOWLEDGEMENTS First and foremost, my heartiest appreciation goes to my supervisor, Dr. Richard Cooper for his excellent supervision, guidance and advice. His support, understanding, encouragement and above all his trust and patience in me are invaluable. Thank you so much Richard! I would also like to express my sincere appreciation to Dr. Alan Wheals whom I spent so much time in the lab with and has been such a great help in my project. My warmest thanks and appreciation to Dr. Idris Abu Seman for supporting me from Malaysia and Datuk Dr. Mohd Basri Wahid (former director general of MPOB) and Datuk Dr. Choo Yuen May (current director general of MPOB) for entrusting me with the scholarship to do my PhD. My deep appreciation also goes to all lab 1.52 for their helpful suggestions, fruitful discussions and being such great friends and colleagues. Many thanks also to my Malaysian friends for their support throughout my journey here in Bath. Besides that, special thank you goes to Dr Simon Bull who has been a great help, in particular for molecular work. I would also like to thank everyone in the Department of Biology and Biochemistry for making the department an enjoyable place to be. Finally, this whole journey will not be complete without the support and unconditional love from my wife, Zetty Norhana Balia Yusof and my first born, Raees Andika Mohd Hefni. They are my pillars of strength, the reason why I am doing this. Also, a big thank you to my parents, Abah and Mak and beloved families back in Malaysia for the love, prayers, never ending support and encouragement throughout the whole journey. Thank you for everything. Mohd Hefni Rusli 2012 iv LIST OF FIGURES Figure 1.1 History and development of the the Deli dura in Indonesia and 2 Malaysia Figure 1.2 Monogenic inheritance of shell thickness: F2-segregation 3 Figure 1.3 The various symptoms of vascular wilt of the oil palm 9 Figure 2.1 Disease wilt index representation 43 Figure 3.1 Process of sampling Foe infected palm tissue using auger 51 technique. Figure 3.2 Geographic locations of the sampled oil palm plantations in Ghana 52 Figure 3.3 BOPP plantation spatial distribution of vascular wilt disease of oil 61 palm Figure 3.4 Spatial pattern of vascular wilt disease epidemics in NPM 63 plantation. Figure 3.5 Vascular wilt disease status in GOPDC 1 affected area. 65 Figure 3.6 Spatial pattern map of disease severity caused by Foe in GOPDC 2 67 affected area. Figure 3.7 Symptomless palms 68 Figure 3.8 Re-isolation of Foe from symptomless palms on Fusarium selective 69 medium. Figure 3.9 Foe phylogenetic tree 71 Figure 3.10 RAPD PCR of genomic DNA 73 Figure 3.11 Dendrogram of Foe isolates and four out-groups based on RAPD 74 fingerprinting Figure 3.12 RAMS PCR of genomic DNA 76 Figure 3.13 RAMPS PCR of genomic DNA of 6 Foe isolates from 6 countries 78 Figure 3.14 RAMPS-PCR of genomic DNA of 15 Foe isolates from six different 79 countries and of four other various F. oxysporum and Fusarium Figure 3.15 Dendrogram of all Foe isolates and four out-groups studied based 83 on polymorphic RAMPS analysis Figure 3.16 Effect of Ghanaian isolates towards their host palms 84 Figure 3.17 Effect of Ghanaian isolates of Foe on plant height 85 v Figure 4.1 ITS gene region of the rDNA gene 103 Figure 4.2 Polymerase chain reaction (PCR) amplification products using 104 genus specific primers Figure 4.3 PCR amplification of DNA with genus specific primers 105 Figure 4.4 Agarose gel electrophoresis of polymerase chain reaction products 105 from genomic DNAs Figure 4.5 PCR amplification products obtained with primers FUSF1 and 106 FUSR1 Figure 4.6 Map of the TEF gene region in Fusarium used in FUSARIUM-ID 107 Figure 4.7 PCR amplification using using primers Foxy F2 and EF2 108 Figure 4.8 Polymerase chain reaction (PCR) amplification of DNA using 108 species specific primes Figure 4.9 The specifity of primer Foxy F2 and Ef2 109 Figure 4.10 Amplification from direct colony PCR by species-specific probe 110 Figure 4.11 Crushed oil palm seeds, pollen and twice sterilised sand 111 Figure 4.12 Resting chlamydospores observed using light microscopy 112 Figure 4.13 Pollen plated onto Fusarium-selective medium 113 Figure 4.14 Amplification of the F. oxysporum-specific 280 bp band by species- 113 specific probe Figure 4.15 Direct PCR amplification of artificially infested sand using species 114 specific primers. Figure 4.16 PCR amplification of artificially infested seed using direct 115 amplification method. Figure 4.17 Direct PCR amplification method on infested oil palm pollen using 115 Figure 4.18 Species-specific PCR amplification products from Foe 117 contaminated seed, sand, and pollen Figure 4.19 Species-specific PCR amplification products from Foe 118 contaminated seed and sand Figure 4.20 Sensitivity of species-specific probe from direct amplification from 120 Foe spores in sand Figure 4.21 Sensitivity of species-specific probe from direct amplification from 121 Foe spores in crushed seed vi Figure 4.22 Sensitivity of species-specific probe when applied by direct colony 123 PCR.