Investigating the cause of dieback in the invasive plant, Parkinsonia aculeata

Invasive plants cost Australia, directly and indirectly, around AU$4 billion pa; displacing native species, changing sensitive ecosystems and sometimes affecting human health and safety. Developing novel tools to control invasive species will benefit landholders and the environment, not just in Australia, but globally. Biocontrol of invasive plants via dieback causative agents is one such potential tool. Dieback causes a progressive reduction in plant population health, resulting in the death of plant parts and often complete plant death. It is prevalent in many invasive woody weeds in Australia and has been suggested as a potential mechanism for their biocontrol, particularly because local native plants appear unaffected. Parkinsonia aculeata L. (Fabaceae; referred to hereafter as “parkinsonia”) is an invasive tree in northern Australia, with native populations in South and Central America and southern USA. It is a perennial thorny shrub that forms dense thickets along waterways, floodplains and throughout paddocks, seriously impacting the pastoral industry, local biodiversity, and providing shelter to other invasive species such as feral pigs. Some Australian parkinsonia populations are affected by dieback, resulting in localised control. Despite previous and ongoing research, the cause of parkinsonia dieback remains elusive and dieback has not been observed in parkinsonia’s native range. This thesis investigates the potential cause(s) of dieback in parkinsonia to contribute towards research on determining its suitability as a biological control tool. My goals were to describe the microbial endophytes of parkinsonia, identify correlations of microbial community composition and dieback occurrence, and identify patterns and pathogens that might be involved in dieback. First, I analysed the community composition of archaeal, bacterial and fungal endophytes from the roots, stems and stem tips of healthy and dieback-affected parkinsonia. Samples were taken from Charters Towers in Queensland (QLD), Australia in May 2013. I used terminal restriction fragment length polymorphism (T-RFLP) analysis with taxon-specific primers for archaea, bacteria and fungi, followed by statistical analysis to determine how endophyte community composition relates to plant part and disease status. Archaeal and fungal community structures were significantly correlated with dieback occurrence and plant part. Bacterial community composition showed significant correlation to dieback occurrence but not plant part. The results showed that endophyte community composition in parkinsonia is associated with the occurrence of dieback and that endophyte communities vary across plants parts. I hypothesised that dieback occurrence may be due to the lack of potentially protective endophytes or the presence of putative pathogens. As a complimentary study to the T-RFLP analysis, I used the same samples collected in QLD to characterise the culturable fungal endophyte communities in healthy and dieback-affected parkinsonia. I identified 219 isolates via amplicon sequencing of the internal transcribed spacer (ITS) to reveal a library of 54 unique species from 25 families. Eight isolates, identified as putative pathogens, were selected for a 10-week pathogenicity trial, including water stress treatments, on parkinsonia seedlings to determine whether inoculations of parkinsonia with these isolates would result in dieback-like symptoms, and whether stress due to drought or inundation enhanced these responses. Of the eight putative pathogenic isolates tested in the pathogenicity trial, inoculation with Lasiodiplodia pseudotheobromae, Botryosphaeria dothidea and Pestalotiopsis mangiferae resulted in the largest lesions, but systemic infection or dieback-like symptoms were not observed, despite significant reductions in plant health due to water stress. As systemic infection or dieback symptoms were not observed, I determined that these pathogens are either not involved in parkinsonia dieback, that different or more extreme abiotic or biotic stress levels are required to trigger dieback-like symptoms, or that changes to the inoculation method are needed. Combining these factors will be essential in evaluating which factors are most important in initiating dieback in parkinsonia. Determining the cause of dieback in affected weeds may present land managers with a ‘silver bullet’ of biological control that could become a self-managed, perpetual instrument, reducing weed management costs and increasing biodiversity and land productivity. As such, future work in the use of dieback and host-specific phytopathogens for biological control of invasive plants should continue

The diversity of microfungi associated with grasses in the Sporobolus indicus complex in Queensland, Australia

There are five closely related Sporobolus species, collectively known as weedy Sporobolus grasses (WSG) or the rat’s tail grasses. They are fast growing, highly competitive, unpalatable weeds of pastures, roadsides and woodlands. An effective biological control agent would be a welcomed alternative to successive herbicide application and manual removal methods. This study describes the initial exploratory phase of isolating and identifying native Australian microfungi associated with WSG, prior to evaluating their efficacy as inundative biological control agents. Accurate species-level identification of plant-pathogenic microfungi associated with WSG is an essential first step in the evaluation and prioritisation of pathogenicity bioassays. Starting with more than 79 unique fungal morphotypes isolated from diseased Sporobolus grasses in Queensland, Australia, we employed multi-locus phylogenetic analyses to classify these isolates into 54 fungal taxa. These taxa belong to 22 Ascomycete families (12 orders), of which the majority fall within the Pleosporales (>24 taxa in 7 families). In the next phase of the study, the putative species identities of these taxa will allow us to prioritise those which are likely to be pathogenic based on existing literature and their known ecological roles. This study represents the first step in a systematic, high-throughput approach to finding potential plant pathogenic biological control agents

Using genetic tools to diagnose dieback in Parkinsonia aculeata

Dieback in invasive and agricultural plants is a common phenomenon attributed to a range of abiotic and biotic factors. In this project we are investigating the link between the introduction and establishment of a non-native plant to a new range and the subsequent dying-back of the invasive population. We are focusing on possible pathogen-related causes for dieback, especially considering the following three scenarios: 1. re-association with pathogens from the non-native’s native range, 2. new association with pathogens in the introduced range, and 3. association with cosmopolitan pathogens

Triggering dieback in an invasive plant : endophyte diversity and pathogenicity

Dieback causes a progressive reduction in plant population health, resulting in the death of plant parts and often plant death. It is prevalent in many invasive woody weeds in Australia and has been suggested as a potential mechanism for biocontrol of these species. Parkinsonia aculeata one such invasive tree in northern Australia. It has naturalised across a wide range of climatic zones and some populations have been heavily reduced by dieback occurrence. The cause(s) of dieback in parkinsonia remain elusive, although fungal endophytes have been previously implicated. In this study, we characterised the culturable fungal endophyte community of healthy and dieback-affected parkinsonia using culture-based techniques, and identified cultured isolates via amplicon sequencing of the internal transcribed spacer (ITS) of the rDNA operon. Eight isolates, identified as pathogens, were selected for a 10-week pathogenicity trial, including water stress treatments, on parkinsonia seedlings. We isolated a taxonomically diverse fungal community from parkinsonia, representing 54 unique species from 25 families. Communities were similar across healthy and dieback-affected plants, but differed by plant tissue. Of the eight putative pathogenic isolates tested in the pathogenicity trial, inoculation with Lasiodiplodia pseudotheobromae, Botryosphaeria dothidea and Pestalotiopsis mangiferae resulted in the largest lesions, but systemic infection or dieback-like symptoms were not observed in any treatment despite plant stress being induced by drought or inundation. We concluded that inoculation of parkinsonia with the tested putative fungal pathogens is unlikely to result in dieback, which has implications for future work in biocontrol of parkinsonia

Endophyte community composition is associated with dieback occurrence in an invasive tree

Background and aims Dieback is pervasive in many populations of invasive woody weeds globally. Previous studies on dieback have focused on specific potential causative biotic agents, but most cases remain unexplained. The potential role of endophytic microbial communities in dieback, including the relative importance of endophytes with pathogenic or protective capabilities, remains poorly studied. We tested whether changes in archaeal, bacterial and fungal endophyte community structure is associated with dieback occurrence in the invasive, leguminous tree, Parkinsonia aculeata L. (parkinsonia). Methods We sampled roots, stems and stem tips from healthy and dieback-affected parkinsonia and conducted terminal restriction fragment length polymorphism (TRFLP) analysis on DNA extracted from these samples using domain-specific primers for archaea, bacteria and higher fungi. Results Microbial community composition strongly differed with parkinsonia disease status (archaea, bacteria and fungi) and plant part (archaea and fungi). Plant part and disease status effects were strongest in archaea. We also found evidence implicating both pathogenic and potentially protective endophytes in the onset of dieback. Conclusions This is the first study that has shown significant associations between changes in endophyte community composition and dieback presence. Our results highlight the complexity of those changes and provide support for the hypothesis that diverse pathogenic and protective endophytes may be implicated in dieback

Characterisation of above-ground endophytic and soil fungal communities associated with dieback-affected and healthy plants in five exotic invasive species

In Australia, several well-established invasive plant species have experienced unexplained dieback. To investigate this issue, we used internal transcribed spacer (ITS) amplicon pyrosequencing to characterise fungal communities within stems (endophytes) and soils associated with dieback-affected and healthy plants from populations of five exotic invasive species (Jatropha gossypiifolia, Mimosa pigra, Parkinsonia aculeata, Tamarix aphylla and Vachellia nilotica) across northern Australia. M. pigra and P.\ua0aculeata were sampled from multiple geographic regions. A total of 353 and 4926 fungal operational taxonomic units (OTUs) were identified in stem and soil samples, respectively. Members of Ascomycota were common, representing 75% of stem and 49% of soil OTUs. Four common endophytes, including Cladosporium perangustum, were at least 50% more prevalent in healthy than dieback-affected samples for the five invasive species combined. Fungal community structure within stem and soil samples varied among invasive species. For the two species sampled across multiple regions, M. pigra had similar fungal communities within stems among regions, but a significant difference in associated soil fungi, suggesting that host plant rather than environment determined endophytic communities in this species. Irrespective of the invasive species and sample type (stem vs. soil), no significant differences were observed in fungal richness, diversity or community structure between dieback-affected and healthy plants, either locally or regionally. Our work failed to identify fungi that were either unique or relatively more abundant in dieback than healthy plants in these invasive species. Future investigations of biotic factors other than fungi, such as bacteria, archaea and oomycetes, may provide more insights into the mechanism of the dieback phenomenon affecting these species

Fungal Planet description sheets : 1383–1435

Novel species of fungi described in this study include those from various countries as follows: Australia, Agaricus albofoetidus, Agaricus aureoelephanti and Agaricus parviumbrus on soil, Fusarium ramsdenii from stem cankers of Araucaria cunninghamii, Keissleriella sporoboli from stem of Sporobolus natalensis, Leptosphaerulina queenslandica and Pestalotiopsis chiaroscuro from leaves of Sporobolus natalensis, Serendipita petricolae as endophyte from roots of Eriochilus petricola, Stagonospora tauntonensis from stem of Sporobolus natalensis, Teratosphaeria carnegiei from leaves of Eucalyptus grandis × E. camaldulensis and Wongia ficherai from roots of Eragrostis curvula. Canada, Lulworthia fundyensis from intertidal wood and Newbrunswickomyces abietophilus (incl. Newbrunswickomyces gen. nov.) on buds of Abies balsamea. Czech Republic, Geosmithia funiculosa from a bark beetle gallery on Ulmus minor and Neoherpotrichiella juglandicola (incl. Neoherpotrichiella gen. nov.) from wood of Juglans regia. France, Aspergillus rouenensis and Neoacrodontium gallica (incl. Neoacrodontium gen. nov.) from bore dust of Xestobium rufovillosum feeding on Quercus wood, Endoradiciella communis (incl. Endoradiciella gen. nov.) endophytic in roots of Microthlaspi perfoliatum and Entoloma simulans on soil. India, Amanita konajensis on soil and Keithomyces indicus from soil. Israel, Microascus rothbergiorum from Stylophora pistillata. Italy, Calonarius ligusticus on soil. Netherlands, Appendopyricularia juncicola (incl. Appendopyricularia gen. nov.), Eriospora juncicola and Tetraploa juncicola on dead culms of Juncus effusus, Gonatophragmium physciae on Physcia caesia and Paracosmospora physciae (incl. Paracosmospora gen. nov.) on Physcia tenella, Myrmecridium phragmitigenum on dead culm of Phragmites australis, Neochalara lolae on stems of Pteridium aquilinum, Niesslia nieuwwulvenica on dead culm of undetermined Poaceae, Nothodevriesia narthecii (incl. Nothodevriesia gen. nov.) on dead leaves of Narthecium ossifragum and Parastenospora pini (incl. Parastenospora gen. nov.) on dead twigs of Pinus sylvestris. Norway, Verticillium bjoernoeyanum from sand grains attached to a piece of driftwood on a sandy beach. Portugal, Collybiopsis cimrmanii on the base of living Quercus ilex and amongst dead leaves of Laurus and herbs. South Africa, Paraproliferophorum hyphaenes (incl. Paraproliferophorum gen. nov.) on living leaves of Hyphaene sp. and Saccothecium widdringtoniae on twigs of Widdringtonia wallichii. Spain, Cortinarius dryosalor on soil, Cyphellophora endoradicis endophytic in roots of Microthlaspi perfoliatum, Geoglossum laurisilvae on soil, Leptographium gemmatum from fluvial sediments, Physalacria auricularioides from a dead twig of Castanea sativa, Terfezia bertae and Tuber davidlopezii in soil. Sweden, Alpova larskersii, Inocybe alpestris and Inocybe boreogodeyi on soil. Thailand, Russula banwatchanensis, Russula purpureoviridis and Russula lilacina on soil. Ukraine, Nectriella adonidis on overwintered stems of Adonis vernalis. USA, Microcyclus jacquiniae from living leaves of Jacquinia keyensis and Penicillium neoherquei from a minute mushroom sporocarp. Morphological and culture characteristics are supported by DNA barcodes