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TAXONOMY, DISTRIBUTION AND PEST STATUS OF PLUTELLA SPECIES (LEPIDOPTERA: PLUTELLIDAE) IN AUSTRALIA AND NEW ZEALAND Kariyawasam Haputhanthri Kankanamge Tharanga Niroshini Submitted in fulfilment of the requirements for the degree of Master of Applied Sciences (Research) School of Earth Environmental and Biological Sciences Science and Engineering Faculty Queensland University of Technology 2018 Keywords Adults, ANOVA, Bayesian analysis, CO1 barcode gene, crops, DNA, genitalia morphological features, host plant, larvae, light trap, maximum likelihood, measurements, PCR, phylogenetic analyses, Plutella australiana, Plutella xylostella, R statistical analyses, Sanger sequencing, taxonomy. i Abstract The diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae), is the most destructive agricultural pest in the word causing damage to brassica crops such as cabbage, kale, broccoli, and cauliflower. Its global distribution, movement over long distances and rapid evolution of insecticide resistance make this a key pest of international importance. P. xylostella was introduced to Australia in 1882 and has become widely distributed in Australia. Despite reports of low levels of genetic variation in the Australian population (Endersby et al., 2006), more recent molecular studies indicated the presence of variants within the Australian Plutella population. A study of allozymes in P. xylostella populations from 14 locations worldwide included specimens from 5 different locations in Australia (Adelaide, Brisbane, North Queensland, Melbourne and Sydney), and found significant differences within the samples from Australia (Pichon et al., 2006). Similarly, Roux et al. (2007) using the inter simple sequence repeat (ISSR) marker showed a genetic differentiation between Melbourne and Sydney P. xylostella populations. In 2013, a new taxon, Plutella australiana, was described (Landry & Hebert, 2013) based on 8.6% sequence divergence in the ‘barcode’ region of the mitochondrial cytochrome c oxidase 1 gene (CO1) and differences in the morphology of the genitalia in both males and females. The new taxon was identified as broadly distributed in southern and eastern Australia. However, there were no larval collections, leaving the host plants and possible pest status unknown. In addition, the description of the new and potentially endemic taxon created difficulties in the import and release of biological control applications, and potential difficulties in pest management practices and market access. This study identified and addressed gaps in knowledge, with the main aim being to clarify the differentiation of P. australiana from P. xylostella and to increase knowledge of its distribution and host plants. CO1 barcode sequence data were used in the identification of both taxa, including phylogenetic analyses (Maximum ii likelihood and Bayesian inference). Morphological features of female and male genitalia were measured to determine statistical variance and examined to identify reliable diagnostic features. Distribution of P. australiana at the regional scale was evaluated from field collections, including individuals from New Zealand. Collections of larvae from both crops and wild brassicas were conducted to identify host plants and contribute to knowledge of the possible pest status of P. australiana. These examinations contribute to the better understanding of possible risks to Australian brassica production, and to inform potential pest management strategies. The results showed that P. xylostella and P. australiana are two distinct taxa based on CO1 data. Examination of key morphological features showed that only two features, the curvature of the tubular projection in P. australiana and the presence of raised folds surrounding the antrum in P. xylostella, and both only in females, are reliable as diagnostic tools. Although some measured features are statistically significantly different overall between the two populations, the overlap in the variance indicates that those features cannot be used as diagnostic tools. Similarly, other characteristic features proposed by Landry and Hebert (example: sinuation in the ventral margin of the valva) were found to be not reliable for identification of the two taxa. Light trap collections of adults show that the two taxa are sympatric in most locations, including Tasmania. However, larvae of P. australiana were present in only two collections: on cabbage (Brassica oleracea) in Theresa Park, NSW in 2015 and on field mustard weeds (Brassica rapa) amongst a kale crop in Werombi NSW in 2015. This study is the first to describe the occurrence of P. australiana larvae on cabbage and field mustard. The preference of P. australiana for weedy B. rapa over kale in one site suggests that emergence of P. australiana as a pest of canola requires investigation. The outcomes of this thesis have been to broaden the knowledge of the new taxon, P. australiana including morphological features that can be used in order to differentiate P. xylostella and P. australiana. The results contribute to the better understanding of possible risks of the new taxon to Australian brassica production, and to inform potential pest management strategies. iii iv Table of Contents Keywords .................................................................................................................................. i Abstract .................................................................................................................................... ii Table of Contents ......................................................................................................................v List of Figures ....................................................................................................................... viii List of Tables ........................................................................................................................ xiii List of Abbreviations ........................................................................................................... xvii Statement of Original Authorship ....................................................................................... xviii Acknowledgements ............................................................................................................... xix Chapter 1: General Introduction and Literature Review ................................ 1 1.1 Background .....................................................................................................................1 1.1.1 Research problem and aims ..................................................................................2 1.1.2 Thesis outline .......................................................................................................3 1.2 Literature Review ...........................................................................................................4 1.2.1 Life history ...........................................................................................................4 1.2.2 Distribution ...........................................................................................................8 1.2.3 Host plants ..........................................................................................................10 1.2.4 Pest management ................................................................................................14 1.2.5 Taxonomy ...........................................................................................................17 1.2.6 A new Plutella taxon in Australia........................................................................19 Chapter 2: Molecular and morphological examination of Plutella species in Australia and New Zealand ..................................................................................... 25 v 2.1 Introduction .................................................................................................................. 25 2.2 Materials and Methods ................................................................................................. 28 2.2.1 Sampling ............................................................................................................ 28 2.2.2 Molecular analysis ............................................................................................. 31 2.2.3 Morphology ....................................................................................................... 33 2.3 Results .......................................................................................................................... 39 2.3.1 CO1 sequence data............................................................................................. 39 2.3.2 Morphology ....................................................................................................... 43 2.4 Discussion .................................................................................................................... 49 Chapter 3: Host plants and distribution of Plutella species in Australia ...... 53 3.1 Introduction .................................................................................................................. 53 3.2 Materials and Methods ................................................................................................. 56 3.2.1 Sampling ............................................................................................................ 56 3.2.2 Molecular Analysis ...........................................................................................
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