Biological control of an Australian noxious weed “Angled Onion” (Allium triquetrum L.) using Molecular and Traditional Approaches Parsa Tehranchian Bachelor of Agricultural Engineering (Agronomy and Plant Breeding) A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Biotechnology & Biosciences Discipline School of Applied Sciences RMIT University Australia August, 2011 I Dedicated to my mum, Afsaneh Khodayari, and dad,, Mostafa Tehranchian, whose support helped me get this far and who made me try to be my best, always. II Declaration I declare this thesis is my own work and was performed while I was enrolled fulltime for the degree of Doctor of Philosophy at the School of Applied Sciences, RMIT University. It has not been submitted for any degree or any other award. To the best of my knowledge, the content of this thesis is the result of my own work and all work performed by other, published or unpublished, has been fully acknowledged. Parsa Tehranchian August 2011 III Acknowledgments I would like to thank my senior supervisor Prof. Ann C. Lawrie, for her guidance, encouragement, valuable suggestions and her leadership throughout my PhD that made me a capable researcher. I am indebted for her continuous support and the great deal of zeal she has for the topic. I also appreciate her support for my teaching and professional experiences at RMIT University. I extend my appreciation to my second supervisor Dr Robin Adair, Department of Primary Industries, Frankston, for his valuable input and guidance. I would like to thank Dr Oscar Villalta and Dr James Cunnington, Department of Primary Industries, Knoxfield, for their generosity in providing the cultures of Stomatinia cepivora. I extend my thanks to Dr Janet Anthony and Dr Siegy Krauss, Kings Park and Botanic Garden, Western Australia, for providing Allium triquetrum bulbs for DNA analysis. I also offer my sincere to thanks to Mrs Helen Williams and Miss Olivia Contarin, for providing bacterial cultures and valuable information about microbiology which was of immense help in carrying forward my research. I would like to express my gratitude to Assoc. Prof. Trevor Stevenson, Prof. Eddie Pang, Dr Gregory Nugent and Mrs Slobodanka Jovetic for their guidance, constant encouragement in my research, teaching and professional experiences and their kind emotional support. My sincere thanks and appreciation also goes to Dr Sethu Ramasamy and Dr Tien Huynh for their kind assistance, encouragement and sharing their research experience during my PhD candidature and their acknowledgeable help for plant sample collections. I also thank my lab mates Mr Abdelwahab Badi, Mr Harshit Vala and Ms Shalika Mehra for making a warm research environment and their encouragement and collaborations. IV I wish to express my heartfelt thanks to my Mum and Dad for their love, affection and their financial and emotional support and giving me this opportunity. I would like to thank my sister Parastoo, her hasband Morteza and my lovely nephew Benjamin for their love and their encouragement. Above all, I would like to thank my best friends Mr Abuzar Nemati and Mr Mahyar Rohamai. I wish to acknowledge School of Applied Sciences Higher Degree by Research, RMIT University for the PhD Scholarship award (2010) and I also acknowledge Parks Victoria for funding this research (2011). V Summary Angled Onion (Allium triquetrum L.) is one of Australia’s successful weeds and is difficult to control, especially in natural habitats. It is a herbaceous bulb-forming perennial member of the Amaryllidaceae (previously known as Liliaceae) and a noxious weed in Vic, SA, NSW and WA. It has been a sleeper weed and is expanding rapidly now that rainfall has returned close to normal. It reduces understory biodiversity significantly and so affects regeneration of native flora. It forms monocultures and its allelopathic traits endanger species such as orchids, native lilies and grasses. Therefore Angled Onion needs to be considered as a high priority invasive environmental weed in Australia. Also, in agriculture, because of the strong onion odour, there are problems of milk and meat taint when it is consumed by farm animals. There are no selective herbicides and grubbing is too expensive and impossible for such vast infestations. Biological control is the only option for control. Evaluation of three pathogens (Stromatinia cepivora Berk., Pectobacterium carotovorum subsp. carotovorum Waldee and a novel bacterium close to Ochrobactrum Holmes et al. species) conducted in this study suggested that the fungus and the two bacteria are potential biological control organisms. Each was separately pathogenic and the fungus and one bacterium were highly virulent; infected plants died. This is the first attempt at biological control of A. triquetrum a model plant for a range of bulbous weeds in Australia. Chapter 2 investigated the genetic diversity of A. triquetrum provenances from across Australia. The first aim of chapter 2 was to determine if there is genetic variation between and within provenances. The second aim was to investigate if genetic differences are due to polyploidy. A. triquetrum bulbs were collected from infestations throughout Australia. There was no significant variation in plant morphology except from Ararat and woodland near Westernport Bay in Victoria, which produced larger bulbs than in other sites. DNA VI extracted from bulbs from each provenance was assessed for diversity by ITS-PCR, RFLPs and RAPDs, which found relatively small genetic diversity between and within provenances from across Australia. ITS sequencing and RFLP analysis suggested relatively small genetic diversity of A. triquetrum in Australia but with some differences in samples from Ferny Creek, Victoria, which makes it a suitable target for biological control. RAPD analysis showed three geographical groupings, one for each of South Australian and Western Australian provenances, but with Victorian provenances split in two; however, genetic variation was also observed within provenances in each state. Only diploid, not triploid plants were found in 12 of these provenances form Australia based on karyotypic studies. The relatively small genetic diversity and homogeneity within most provenances suggested that most spread was clonal, by bulbs, but within-provenance variation suggested also reproduction by seed, leading to genetic variation in seedlings. The main aim of chapter 3 was to explore the potential of the fungus S. cepivora as a biological control agent to infect A. triquetrum in vitro (test tube trials) and in vivo (pot trials). A. triquetrum bulbs collected from infestations and cultivated Allium species were micropropagated to remove problems with endogenous bacteria and test-tube grown plants were inoculated with sclerotia. There was a significant difference in pathogenicity between the two Australian S. cepivora isolates tested. The DPI isolate was pathogenic to all provenances and cultivated Allium species, whereas VPRI 12439a was not pathogenic on one provenance (from Wonthaggi) because its sclerotia did not germinate in vitro. In the glasshouse, plants inoculated with sclerotia in sterile sand produced no white rot disease on A. triquetrum and cultivated Allium species 3 months post-inoculation, but retrieved sclerotia grew on V8 agar. By contrast, plants inoculated with mycelium showed white rot disease 3 months post-inoculation, except for Hardy’s Picnic Ground, Dandenongs plants, which were not infected. This fungus was also highly virulent on VII cultivated Allium species but was not pathogenic on a range of Australian native monocotyledons liable to be present in infested environments. Chapter 3 also investigated genetic variation in the fungus S. cepivora, the causal agent of white rot disease of onion. Two local isolates, VPRI 12439a and a fresh isolate from DPI field trials were used, from the Herbarium VPRI National Collection of Fungi and the Department of Primary Industries, Victoria, respectively. Isolates were compared genetically using PCR amplification of the ITS region, RAPDs and mycelial compatibility. ITS sequences of S. cepivora isolates downloaded from GenBank via NCBI were used to determine the genetic diversity between Australian and international isolates. ITS sequence alignment exhibited relatively small nucleotide variations but the Australian isolates clustered together separately from the rest. RAPD analysis showed genetic variation between the local isolates but only using OPA-11 primer. Mycelia of the isolates were also incompatible, supporting the case for genetic variation between the isolates. This genetic variation was reflected in the variation in pathogenicity and virulence on Allium species. As neither isolate affected native Australian monocots, either or both could be used separately or together as a biocontrol agent. In chapter 4, a soft-rotting bacterium was isolated from rotting A. triquetrum bulbs collected from Horsnell Gully, South Australia, after 2 months of storage at 4ºC. The bacterium was identified as Pectobacterium carotovorum subsp. carotovorum by 16S r- DNA sequencing and physiological tests. In test-tube trials, the bacterium produced severe soft rot symptoms 12 h post-inoculation and rotten young seedlings collapsed after 24 h incubation at 25ºC. Identical symptoms were observed at 15ºC and 4ºC, but with a longer aetiology. Histology of infected plants suggested that the bacterium invaded both the cortical and vascular tissue. In the glasshouse, all