Silicon in Banana Plants: Uptake, Distribution and Interaction with the Disease

Silicon in banana plants: uptake, distribution and interaction with the disease fusarium wilt Kevan Walter Jones Bachelor of Science: Plant Science A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2013 School of Agriculture & Food Sciences Abstract Banana cultivation worldwide is under threat from a wide variety of pathogens and negative environmental factors. Most cultivated banana plants are vegetatively propagated, resulting in a dearth of breeding and a genetic bottleneck. This has led to enhanced susceptibility to a number of lethal plant diseases. Novel solutions are being pursued to enhance the innate defences of the banana plant in an effort to combat these diseases. Of all current banana diseases, Fusarium wilt poses the greatest overall threat. Fusarium wilt, sometimes known as Panama disease, is caused by the soilborne fungus Fusarium oxysporum f. sp. cubense (Foc). A complex grouping of polyphyletic fungal strains, collectively referred to as races, is responsible for causing disease in banana. Race 1 of Foc caused the collapse of the global ‘Gros Michel’ trade industry in the mid-20th century. The industry recovered by substituting ‘Cavendish’ cultivars for ‘Gros Michel’, but a new race (race 4) is now threatening ‘Cavendish’ production. Breeding and transgenics programmes for developing Foc resistant banana cultivars are in progress, but advancement is slow and durable resistance cannot be guaranteed. In the interim, innovative control strategies for Foc are being sought. These strategies involve the development of new cultural controls or soil amendments and are intended to inhibit fungal inoculum in the soil or to upregulate innate plant defences. The research in this thesis focusses on the poorly studied plant nutrient, silicon, and its potential for controlling fusarium wilt of banana. Silicon is classified as a "quasi-essential" element: not fulfilling the strict requirements for essentiality, but still playing an active role in the plant. Despite its prevalence in the environment and content within plants, it remains one of the least studied and understood plant nutrients. Adding silicon to plants as a fertiliser makes them more tolerant to various biotic (pathogens) and abiotic (environmental) factors. This protective effect is not consistent between species and can vary within species. The mechanism by which silicon enhances innate plant defences is poorly understood. Anecdotal reports of silicon ii improving banana tolerance to both pathogens and environmental factors have been reported and subsequently investigated in the scientific literature. For this thesis, the research objectives were to investigate the location and deposition of silicon within roots and shoots of banana plants; investigate silicon absorption through the roots and uptake dynamics; to determine if silicon enhances tolerance to Foc; observe the beneficial effects of silicon in the tissue culture phase of banana cultivation, and; examine how soil distribution of silicon influences uptake and resistance to Foc. All research took place on the ‘Cavendish’ “Williams” cultivar and subtropical race 4 of Foc as these are most relevant for Australian banana production. The location of silicon in the roots of banana plants was determined using scanning electron microscopy coupled with energy dispersive X-ray microanalysis. No physical discrete deposits of silicon (aka phytoliths) were detected. Silicon was localised within or on the cell walls of root tissue and followed an increasing concentration gradient from the outer epidermis to the inner endodermis. This form of deposition is unique compared to other reported crops and suggests a potential physiological role for silicon in the roots. Silicon was also detected in the protoplasts of intact cells, raising the possibility of a bioactive role for silicon in planta. For the shoots, silicon was present in or on the cell walls at low concentrations for most tissues investigated. Discrete deposits were detected in fibrous bundles of the vascular tissue predominantly in the leaf lamina and occasionally in the leaf midrib, petiole and pseudostem. This is consistent with previous reports on phytoliths in banana, and like the roots, suggests a physiological role for silicon. Amorphous silicon dioxide was found to be the most effective at increasing gross silicon content in the plant when uptake was tested. Xylem sap silicon was constant when plants were supplied with monosilicic acid, but varied considerably when plants were supplied with amorphous silicon dioxide. Silicon treatment was found to enhance banana plant tolerance to the expression of fusarium wilt symptoms. Internal symptoms in 4 month old plants, 14 weeks post inoculation were approximately 30% lower compared to untreated, inoculated controls. Decreasing disease symptoms were correlated with an improved biochemical defence iii response in the form of enhanced phenolic production coupled with transmission electron micrographic evidence of electron dense globules associated with an improved plant defence response. Silicon was shown to be neither fungitoxic nor fungistatic, implying silicon treatment either directly or indirectly enhances innate plant defences against Foc. Split root results indicated that uneven distribution of silicon in the soil has the potential to affect the silicon-mediated defence response. Silicon decreased symptoms of fusarium wilt when evenly available to the plant, but increased symptoms when only available to half the root system at high concentrations. This highlights the complex relationship between the soil, plant nutrition and fungal infection. Finally, silicon was found to be beneficial in the multiplication and post-rooting phase of tissue culture micropropagation. Adding silicon to the multiplication media caused an increase in up to 50% of the number of explants produced during proliferation. Using silicon when deflasking during the post-rooting phase, plantlets were more tolerant of environmental conditions such as water deficit and slightly more tolerant of Foc. In conclusion, silicon was found to be a beneficial nutrient in multiple areas of banana cultivation. Based on the evidence, silicon is recommended for use in banana cultivation. Large-scale trials are necessary to determine how well this effect translates to the field. The next avenue of research would be to determine the most cost-effective method for applying cheap, high-purity, plant-available silicon to cultivated banana plants. iv Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. v Publications during candidature No publications. Publications included in this thesis No publications included. Contributions by others to the thesis No contributions by others. Statement of parts of the thesis submitted to qualify for the award of another degree None. vi Acknowledgements To the endless series of uniquely mad Aitken lab honours students I thank thee: Reynard Poels, Rebecca Roach, Annelie Marquardt, Goh Yiling Gloria and Elizabeth Czislowski. I could not have asked for a more diverse and entertaining gang of colleagues. Special mention is reserved for my fellow PhD traveller and long-suffering office companion, Sam Fraser-Smith. Smart as a whip and never afraid of offering constructive and useful criticism. Outside the lab, I credit the support of fellow PhD students Lucy Hurrey and Damien Finn. For all the microscopy work, I am forever indebted to the exceptionally skilled staff of the UQ Centre for Microscopy and Microanalysis. Thanks to Rick and Robyn Webb for holding my hand during the long tedious stages of learning transmission electron microscopy and for tolerating my excessive swearing in the ultramicrotomy room. For the scanning electron microscopy side of things, particular thanks are extended to Ron Rash for teaching me everything I need to know on X-ray microanalysis and Dr Kim Sewell for showing me the ropes on some very technical machines. I should also thank Dr Alsadair McDowall for first igniting in me a passion for electron microscopy all those years ago. The

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