Massey Documents by Type
Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294
Browse
45 results
Search Results
Item New Zealand willows (Salix spp.), their metabolites and their plant-herbivore interactions : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Ecology at Massey University, Manawatu, New Zealand(Massey University, 2024-12-19) De Oliveira Mota, ClaryssaNew Zealand is a unique environment which affects how species behave, survive and interact with each other. Introduced species of willow (Salix spp) are used in New Zealand for various purposes, and several varieties (clones) have been developed (Gunawardana et al., 2014; McIvor, 2013). Various insect species attack willows in New Zealand. It is not yet known how the New Zealand environment would affect the secondary metabolites and species resistance to insect pests in willows, and host preferences of some pests have not yet been characterized. In my thesis I aimed to characterize chemistry (metabolites and volatile organic compounds) in several willow clones, as well as differences in clone preference in insect pests – giant willow aphid (GWA) Tuberolachnus salignus Gmelin, 1790 (Hemiptera: Aphididae) and red gall sawfly Pontania proxima Serville, 1823 (Hymenoptera: Tenthredinidae). The incidence and chemical aspects of galling by P. proxima on New Zealand willow clones has not yet been characterised. This information is vital for the selection of resistant cultivars and to understand potential indirect impacts on other insect species (e.g., natural enemies of competing herbivores). Chapter 1 is a literature review on plant secondary metabolites and their role in plant defence against insect herbivores, as well as review of willow insect pests with emphasis on P. proxima and giant willow aphid. This chapter questions how much we know about willows in New Zealand, what differences they present from the willows from other parts of the world, and how the New Zealand environment affects the insects that feed on willows. Do insect pests preferers the same species/clones in New Zealand as in other parts of the world? What makes P. proxima and GWA prefer certain clones? What makes some clones resistant and others susceptible? In Chapter 2, I explored the levels of damage caused by P. proxima to willow clones. Twelve willow clones (PN221, PN249, PN721, PN693, PN357, PN676, NZ1040, NZ1130, PN218, PN356, PN736 and PN742) used in New Zealand were selected and surveyed for P. proxima damage. Willows showed a range of resistance levels to P. proxima. These levels of resistance show as differences in P. proxima larval development (explored in Chapter 3), damage level and gall size. For example, clones PN221 and PN249 did not present galls, while clones PN736 and PN742 presented the highest level of damage in our field survey. Other clones had an intermediate level of damage, and some clones present with malformed galls. The survey also found that top sections of shoots had a significantly higher level of damage, while location and side of the plant had no effect, possibly because the experimental field was homogeneous in sun exposure and other abiotic factors such as soil fertility. Gall induction is still a mystery in the Salix spp - P. proxima system, mainly because the cecidogenic factor is not yet known. The clones used in Chapter 2 were further investigated in Chapter 3 to link plant resistance to P. proxima development and growth. Larval development was investigated and measured, and the phenolic and nutrient content of willow leaves was quantified. The resistance of willow clones to P. proxima appears to be guided by a combination of physical and chemical attributes of the plants. Overall, P. proxima appears to prefer clones with a lower phenolic content and lower leaf pilosity. This preference, however, is in contrast with previous European studies in which P. proxima showed preference for a higher level of phenolics. Plant phenology seems also to play a role in preference, with P. proxima preferring to oviposit in clones which develop earlier in the season, even if those clones do not offer optimal conditions for larval development. Among the studied clones, PN 221 and PN 249 (S. purpurea) showed the highest resistance levels to P. proxima with no galls. Among the clones that developed galls, PN676 (S. alba, female) was the clone that produced the smallest larva and is therefore considered more resistant. The fact that P. proxima has shifted its preference from willows with a high content of phenolic glycosides in its native range to willows with a low content of phenolic glycosides in New Zealand may be due to low predator pressure in New Zealand. Further studies are needed to investigate this shift and test this hypothesis. In Chapter 4, I investigated the metabolomics profiles of six willow clones (PN220, PN249, PN386, NZ04-106-073, PN218 and NZ1040) and whether the differences in metabolites influence the preference of insect pests to the six clones. With the metabolomic profile we are expecting to see differences in chemistry between the clones and their influence on the plants’ pest resistance. The most important compounds found were apigenin, isorhamnetin-3-O-glucoside, procyanidin B2, epicatechin, petunidin-3-O-β-glucopyranoside, kaempferide, kaempferol-3-glucuronide, quercetin-7-O-rhamnoside, unknown 1, isorhamnetin, peonidin-3-O-β-D-glucoside, luteolin-7-O-glucoside, procyanidin B1 and isorhamnetin-3-O-rutinoside. Due to the limited number of clones and limited number of replicates, I cannot draw definitive conclusions about the pattern of secondary metabolites in relation to resistance to the two insect herbivores – P. proxima and GWA. To our knowledge the direct effect of those metabolites on P. proxima and GWA was never tested. The resistance to P. proxima and GWA appears to be more correlated with phenological and morphological features of willow plants than with their chemistry. Chapter 5 is an investigation of the volatile profile of willow clones studied in Chapter 4. With the metabolomic and volatile profile we hoped to elucidate whether the chemistry of the clone influences clone resistance to P. proxima and GWA. These volatile organic compounds (VOCs) included two green leaf volatiles (GLVs), (Z)-3-Hexenyl acetate and (Z)-3-Hexenyl-α-methylbutyrate; one monoterpenes, (Z)-β-Ocimene; and eight sesquiterpenes, β-Elemene α-Cubebene, Copaene, Germacrene D, (Z)-β-Caryophyllene, (E)-α-Bergamotene, (α)-Farnesene, δ-Cadinene. The results show that willow clones have highly species-specific VOC blends, a conclusion backed up by other authors. Due to the limited number of clones and limited number of replicates, it was not possible to draw definitive conclusions about the pattern of volatile emissions in relation to resistance to the two insect herbivores – P. proxima and GWA. Resistance to P. proxima and GWA appears to be more correlated with phenological and morphological features of willow plants than with their VOC emissions. Chapter 6 is the recapitulation of the conclusions of the experimental chapters and relating it with the existing literature. The twelve willows tested in chapters 2-5 showed a range of metabolites, leaf volatiles (VOCs), and resistance levels to P. proxima which manifested as differences in P. proxima larval development, damage level and gall size. Overall, P. proxima appears to prefer clones with a lower phenolic content and lower leaf pilosity and those that develop earlier in the season. The VOCs in willow clones appear to be species-specific and are not clearly linked to insect resistance. We suggest that the levels of phenolic compounds and pilosity together better explain the preference of oviposition of P. proxima. The highest amount of secondary metabolites was found in clones NZ04-106-073 (S. lasiolepis × S. viminalis, Female), PN676 (S. alba L., Female) and PN221 (S. purpurea L., Male). NZ04-106-073 also showed the highest emission of VOCs. The most susceptible clones to P. proxima were PN736 (S. fragilis L., Male) and PN742 (S. fragilis L., Male). Tree willows are preferred by P. proxima to shrubs. Among the studied clones, PN221 and PN249 (both S. purpurea) showed the highest resistance levels to P. proxima with no galls. Among the clones that developed galls, PN676 (S. alba, female) was the clone that produced the smallest larvae and is therefore considered more resistant.Item Identification and characterization of effector proteins from pine needle pathogens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand(Massey University, 2022) Massoco Tarallo, MarianaCollectively, Dothistroma septosporum, Cyclaneusma minus and Phytophthora pluvialis cause serious foliar diseases on Pinus radiata in New Zealand and on many other pine species worldwide. Considering the ecological and economic importance of forest trees, understanding how these pathogens interact with their hosts on a molecular level is critical as it could lead to new and durable approaches to control the diseases they cause. Pathogens have the ability to deliver proteinaceous virulence factors, termed effectors, into the apoplast and cell cytoplasm of their host plants. Effectors typically promote host colonization through suppression of the plant immune system. However, in resistant host plants, one or more of these effectors can be recognized by corresponding immune receptors to activate the plant immune system. Often, one of the main outputs of this immune system is a localised cell death reaction, termed the hypersensitive response (HR), which renders the pathogen unable to cause disease (avirulent). The general goal of this thesis was to identify shared candidate effector (CE) proteins between the three foliar pine pathogens and to characterise their virulence (or avirulence) functions. This is important because disease resistance based on core effectors that are vital for a pathogen’s ability to cause disease is more likely to be durable. Using a combination of “omics” information and bioinformatic tools, two sets of orthologous CE proteins were identified between D. septosporum, C. minus and P. pluvialis, while several other sets were identified between the two fungal pathogens. Some of these CEs had the ability to trigger cell death responses in non-host Nicotiana plants, and some were shown to activate Nicotiana benthamiana genes involved in pathogen-associated molecular pattern-triggered immunity and HR. CEs were also screened in the host, Pn. radiata, using a method developed in this thesis, where it was determined that some of these CEs also trigger cell death. Two conserved cell death elicitor families, Ecp20 and Ecp32, were identified from D. septosporum and its close relative Fulvia fulvum, and the cell death triggered by some family members in N. benthamiana was shown to require membrane-localized receptor-like proteins. Tertiary structure predictions of CEs provided insights into the possible roles and host targets of these proteins during pine infection. Moreover, a shared β-trefoil fold was found between sequence-unrelated CE proteins from the three pine pathogens, along with evidence that they are also present in many other fungal species. A CRISPR/Cas9 gene editing methodology was applied to D. septosporum for the first time, which allowed for the functional characterization of three D. septosporum CE genes, two of which are also present in C. minus and P. pluvialis. Collectively, this thesis provides a significant advance in our understanding of pine-pathogen interactions at the molecular level and provides a blueprint for similar studies in other forest pathosystems.Item Unravelling the molecular basis of subcuticular host-colonization by the apple scab fungus, Venturia inaequalis : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science, School of Agriculture and Environment, Massey University(Massey University, 2023) Rocafort Ferrer, MercedesScab, caused by the fungal pathogen V. inaequalis (Vi), is the most economically important apple disease. During infection, Vi occupies the subcuticular environment, where it develops specialized infection structures, called stromata and runner hyphae. These structures are thought to be important for fungal nutrition and the delivery of proteins, with many of these anticipated to function as virulence factors (effectors) in promoting host infection or avirulence factors (Avr effectors) in triggering host resistance. To date, nothing is known about how these structures are differentiated and protected from recognition by the host immune system. Likewise, little is known about the identity and function of Vi effector proteins. To better control scab, a greater understanding of the molecular mechanisms underpinning infection structure differentiation and protection, as well as Vi virulence and avirulence, is first needed. In Chapter 2, a comprehensive review of apoplastic effector proteins from plant-associated fungi (and oomycetes) was provided. Given that Vi is an extracellular pathogen, this review provided insights into the potential types of effector proteins secreted by Vi into the subcuticular environment. Then, in Chapter 3, a multidisciplinary approach based on bioinformatics, transcriptomics, and structural biology was used to identify and characterize Vi effector candidates (ECs). This revealed that ECs were predominantly expressed in two temporal waves, and that many belonged to expanded protein families with predicted structural similarity to virulence and avirulence effectors from other plant-pathogenic fungi. This analysis helped to generate a list of ECs for further study and contributed to a better understanding of effector biology and evolution. Next, in Chapter 4, a multidisciplinary approach based on transcriptomics, proteomics, glycomics, and confocal microscopy was used to study Vi cell wall carbohydrate composition during the differentiation of infection structures. This iii revealed that Vi down-regulates genes putatively associated with the biosynthesis of immunogenic carbohydrates, and deacetylates surface-exposed chitin to the less immunogenic carbohydrate, chitosan. Finally, in Chapter 5, CRISPR-Cas9 technology was applied to Vi for the first time, which will enable genes identified in this study to be functionally characterized. Altogether, this thesis has furthered our understanding of the Vi –apple pathosystem and has provided novel data that can be used to inform the development of new scab control strategies against Vi.Item Identification and functional characterisation of glycoside hydrolases from the kauri dieback pathogen, Phytophthora agathidicida : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science at Massey University, Manawatū, New Zealand(Massey University, 2022) Bradley, Ellie LynnThe survival of kauri, an ancient conifer species endemic to New Zealand, is currently threatened by kauri dieback disease, caused by the oomycete plant pathogen Phytophthora agathidicida. As P. agathidicida continues to spread throughout kauri forests in the northern North Island of New Zealand, encouraging research has indicated there may be natural tolerance to the disease within the kauri population. This resistance is likely governed, in part, by the plant immune system, which is activated upon recognition of pathogen invasion patterns such as microbe-associated molecular patterns (MAMPs), damage-associated molecular patterns (DAMPs), and effectors (virulence factors required for host colonisation), which are recognised at the plant cell surface by plant immune receptors. To better understand how P. agathidicida interacts with its plant host on a molecular level, pathogenproduced proteinaceous invasion patterns need to be identified and characterized to aid in the identification of cognate immune receptors in the plant host, which may be involved in activation of the plant immune system. As the role of glycoside hydrolase (GH) proteins in virulence and pathogenicity of fungal and oomycete plant pathogens is well established (Chapter two), an effectoromics approach was used to identify six P. agathidicida GH12 proteins that appear to act as MAMPs in both Nicotiana benthamiana and Nicotiana tabacum (Chapter three). Furthermore, nuclear magnetic resonance was used to identify considerable changes in kauri leaf apoplastic wash fluid of approximately 17 metabolites, including sucrose and glucose, in response to P. agathidicida inoculation (Chapter four), thus suggesting a role for GH proteins in the hydrolysis of some of these metabolites. Finally, proteomic analysis of P. agathidicida culture filtrates via liquid chromatography-mass spectrometry (LC–MS) was used to validate the expression of predicted P. agathidicida proteins and to investigate the capacity of this method to identify candidate invasion patterns for future analysis. Chapter five established that LC–MS analysis of Phytophthora culture filtrate was an effective method for the identification of putative apoplastic invasion pattern candidates and confirmed the production of all six P. agathidicida GH12 cell death elicitors in culture. Collectively, this thesis has advanced our understanding of the molecular mechanisms underpinning the interaction of P. agathidicida with its host and has contributed to the identification of candidate apoplastic effectors.Item Multitrophic interactions involving the giant willow aphid, Tuberolachnus salignus (Gmelin) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Ecology at Massey University, Manawatu, New Zealand(Massey University, 2020) Tun, Kyaw MinThe giant willow aphid Tuberolachnus salignus, Gmelin, 1790 has become an important pest of willows, causing negative impact on host plant physiology and growth, and indirectly posing a unique problem in the apiculture industry. As it is a new invasive species to New Zealand, aphid-host interactions, extent of damage and ecological impacts of aphid infestation are not yet known. Aphid interactions with host plants, associated insect species and soil microbes were addressed in this thesis to fill knowledge gaps in formulating sustainable aphid management. Aphid population numbers and the extent of plant damage were cultivar-specific, with wide variations between resistant and susceptible cultivars in Chapter 2. Aphid infestation delayed the flowering time, extended the duration of flowering, and decreased the catkin length in susceptible cultivars. Interestingly, aphid infestation was found to increase the total floral output of some willow cultivars. Aphid infestation had no measurable effect on the number of shoots of willow cultivars, but reduced the survival, height, and shoot diameter of the plants by the end of the second growth season. In Chapter 3, the VOC emissions were cultivar-specific and varied with plant type (tree vs. shrub willows). The results also showed that resistant cultivars appear to emit more green leaf volatiles than other cultivars, suggesting that there can be a link between T. salignus resistance and VOC emission in willows, which deserves further exploration. However, most cultivars did not experience significant changes in their VOC emissions after aphid attack, while few have reduced emissions. It was proven in Chapter 4 that melezitose concentration in T. salignus honeydew did not vary with willow cultivar or plant age, but concentrations of other sugars (such as fructose) did. There was no obvious link between willow susceptibility to T. salignus and melezitose content, however, total honeydew sugar concentration was lower while fructose content was higher in highly susceptible cultivars identified in Chapter 1. Tuberolachnus salignus honeydew deposition has multiple ecological impacts. Copious amounts of honeydew fall on the understory vegetation or directly on the soil surface, resulting in irregular occurrence of black sooty mould areas under aphid-infested plants. This carbon-rich energy source is utilized by soil microorganisms (fungi, bacteria and yeasts), in turn increasing the abundance of fungivores and their predators in honeydew-receiving soil. This honeydew-mediated cascading effect was directly linked with honeydew availability and the level of input, with strongest effect in black sooty mould spots (Chapter 5). The results in Chapter 6 show that although this predator can feed on T. salignus, the aphid is not its preferred prey. H. axyridis that fed on immature T. salignus developed slower than on alternative prey, and preferentially selected other aphid prey species in dual choice tests, rejecting T. salignus. The results suggested that H. axyridis should not be promoted as a biocontrol agent for T. salignus. This work is a significant contribution to our understanding of the impacts of the giant willow aphid in New Zealand, and provides useful information towards the selection of resistant cultivars and biocontrol agents.Item A study of some fungal leafspot diseases of Dactylis glomerata in the Manawatu : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in the University of New Zealand(Massey University, 1957) Latch, G. C. M.New Zealand is unique in that the entire basis of her national economy is based upon livestock-pastoral farming. Of the fortythree million acres in occupation for agricultural and pastoral purposes, seventeen and a half million are of sown pasture and about thirteen and a half million of natural grasslands. The seventeen and a half million acres of sown pasture are down in imported grasses of which approximately one half has been surface sown and the remainder sown on cultivated land with high producing English grasses. These have been selected for such qualities as leaf area and density of leaves, form of the plant, resistance to drought and many other desirable agronomic properties. Regarding the disease factor, there has been no attempt in New Zealand at breeding for resistance to disease with the exception of Blind seed disease of Ryegrass caused by Gloeotinia temulenta (Prill. et Delacr.) Wilson, Noble et Gray. [From Introduction]Item 'Whakapuputia mai o mānuka' : a case study on indigenous knowledge and mitigating the threat of myrtle rust (Austropuccinia psidii) : a research thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Horticultural Science, School of Agriculture and Environment, Massey University Te Kunenga ki Pūrehuroa, University of New Zealand, Palmerston North, New Zealand(Massey University, 2019) Tora, Mesulame J.This research centres on the recent myrtle rust (Austropuccinia psidii) incursion in New Zealand to review the literature on the disease specifically and to create a localised case study with Ngāi Tāneroa hapū of Ngāti Kahungunu ki Wairarapa. The case study focused on the importance of whakapapa, mātauranga Māori, tikanga Māori and the practices of kaitiaki to ethnobotany and the development of indigenous biosecurity measures (tools) to protect culturally important plant species within the Māori community The proverb stated in the title of this thesis whakapuputia mai o Mānuka, kia kore ai te whati – (cluster the branches of the Mānuka, so they will not break off) recognizes the status of plant knowledge in te Ao Māori. It provides a foundation of understanding how Māori can participate in resource management against biological threats, which are becoming increasingly common. The science around myrtle rust and the mitigation of any incursion threats is clearly aligned to western paradigms. The information presented in this thesis outlines an extensive understanding of the intricacies of the disease as understood by the science community. But this science alone has not been able to halt the spread or risk of myrtle rust into new geographical regions. Therefore, future management of the risk of myrtle rust incursions needs to look at alternative approaches for the development of suitable management tools. The holistic approach of traditional biodiversity management using mātauranga and tikanga Māori has much to offer to conservation of taonga resources, especially the mitigation of biological threats. The Māori worldview of the environment encompasses all elements beyond the physical attributes of an ecosystem that thrives through traditional kaitiaki inputs. The case study with Ngāi Tenaroa introduced several examples of how Māori can contribute to the mitigation of all threats on the ecosystem, not just fungal threats. Firstly, the role of whakapapa is explicit and cannot be ignored. This role consolidates the management tools across all generations at the very least. Secondly, the role of networks within Māori communities and inter-generational learning is also clear – and the risk that exists if this is lost is apparent. Lastly, examples of local knowledge such as the effect of hukahuka on plant health, companion trees and role of kaitiaki in decision-making have been identified and their importance conveyed from the hapū under study.Item Identification of blueberry leaf rust pathogen and quantification of disease infection levels in a blueberry plantation in Hastings, NZ : a thesis presented in partial fulfilment of the requirements for the degree of Master of AgriScience in Horticulture at Massey University, Turitea Campus, Palmerston North, New Zealand(Massey University, 2019) Chen, XiaoyingBlueberries (Vaccinium spp.) are a favourite fruit and they are produced worldwide. In New Zealand, blueberries are the main export berry fruit and contribute greatly to export income. More than 2,800 tonnes of blueberries were produced in the 2017/2018 harvesting season and earned $34.8 million export income in 2018. Currently, 740 ha of the blueberry plantations can be found in both the South Island and North Island. Otautau is the main growing region in the South Island while Waihopo, the Waikato regions of Ngatea and Ohaupo, the Bay of Plenty and Hastings are the regions in the North Island, producing most of the fresh blueberries in New Zealand. However, blueberry leaf rust has become a prevalent disease in blueberry production and a concerning issue for blueberry growers. In Hastings production sites, serious infections have been found in recent years. Although fungicides were applied to control blueberry leaf rust, this form of control is incomplete and unsustainable for blueberry production. The deployment of varieties that are naturally resistant would be a better option for managing blueberry leaf rust disease. Currently, few cultivars are available for this purpose, but breeding for rust resistance can address this demand. The main issues preventing the production of resistant varieties are insufficient knowledge about this rust pathogen in New Zealand, and the lack of resistant germplasm sources and efficient resistance screening procedures. In this study, using the morphological characteristics and genome sequencing results based on the Internal Transcribed Spacer (ITS) regions, Thekospora minima was identified as the causal organism of blueberry leaf rust disease in Hastings, Hawke’s Bay, New Zealand. Additionally, a field assessment was used for understanding the blueberry rust disease resistance levels in current blueberry cultivars. The disease incidence and disease severity of 23 blueberry cultivars, including five rabbiteye, three northern highbush and fifteen southern highbush, were assessed using Fiji software during the 2019 harvesting season. Based on a Tukey Honest Significant Differences (TukeyHSD) analysis, these observed blueberry cultivars were divided into four infection levels of blueberry leaf rust using the percentage of the infected area on the leaf (PIAL). ‘Scintilla’ was highly susceptible to blueberry leaf rust disease, while ‘Blue Moon’ and ‘Southern Splendour’ were moderately susceptible. Nineteen blueberry cultivars, made up of ‘Rahi’, ‘Centra Blue’, ‘Centurion’, ‘Titan’, ‘Sky Blue’, ‘Nui’, ‘Duke’, ‘Camellia’, ‘Misty’, ‘Springhigh’, ‘Snowchaser’, ‘Miss Jackie’, ‘Miss Lily’, ‘Georgia Dawn’, ‘Suziblue’, ‘Kestrel’, ‘Flicker’, ‘Sweetcrisp’ and ‘Palmetto’, showed susceptibility to this rust disease, and ‘‘O’ Neal’ was the one that showed partial resistance to the blueberry rust infection. Furthermore, using 1.5×104 concentration inoculum, an inoculation test was completed in a temperature-controlled room at the Plant Growth Unit of Massey University. The inoculum was sprayed on the healthy leaves from detached branches of the ‘Sky Blue’ blueberry cultivar and they were grown in reverse osmosis water for a 35-day observation on rust symptom development. Fiji software was applied in the assessment of disease severity in this inoculation test. A strong correlation (>0.99) was found between the increase in lesion area (ILA) from the inoculation test and the PIAL from field assessment. A preliminary prediction equation was established by a simple linear regression model. This equation can be used to predict the blueberry leaf rust level on different blueberry cultivars and breeding materials under field conditions by using the results from an inoculation test. This model would be an efficient approach for assisting the screening on blueberry leaf rust of blueberries.Item Bio-prospecting for endophytes of Brassica : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand(Massey University, 2019) Roodi, DavoodDisease and insect pests are major limiting factors for crop production worldwide. Farmers are often heavily reliant on synthetic biochemicals and fertilisers to mitigate the negative impact of pests and disease and to increase crop yield. However, the extensive use of chemicals has led to environmental concerns due to contamination of soil and water, human health issues, disturbance of macro and microorganisms balance and the development of resistance by both insects and fungal pathogens. Use of biological control agents including endophytic microorganisms is an alternative control option to combat these problems. Many endophytes are able to provide their host with beneficial traits such as resistance against insect-pests and pathogens and enhance crop performance under abiotic stresses. Although beneficial microorganisms of brassica crops have been discovered, endophytes of wild brassica’s, particularly those associated with the seed, have been ignored. In this study, we screened seed of various brassica species with a worldwide distribution and isolated 131 bacterial and two fungal species. Molecular identification of bacterial isolates indicates that most seed accessions harboured endophytic bacteria belonging to 17 species. Among these isolates, two species, identified as Methylobacterium fujisawaense and Me. phyllosphaerae were dominant and widespread across the majority of accessions sampled, and originated from different species and locations. The inoculation of oilseed rape (Brassica napus) root with these endophytic bacteria significantly increased the fresh weight of the seedlings. The fungal endophyte species identified were Beauveria bassiana and Geomyces pannorum, isolated from two different accession of a wild brassica species (B. rapa). Inoculation of the seeds of three brassica species, B. napus, B. rapa and B. oleracea with these fungal endophytes resulted in infection of below and above ground tissues of inoculated seedlings but colonisation of the next generation seeds/seedlings did not occur. Seed inoculation, foliar application and soil drenching when the plants were grown on non-sterile soil also did not result in plant colonisation. A dual culture test was performed to study the antagonistic effect of these bacterial and fungal endophytes against Leptosphaeria maculans, the causal agent of phoma stem canker in brassica crops. The highest inhibition rate was recorded for Stenotrophomonas rhizophila, Novosphingobium resinovorum, Pseudomonas azotoformans, Plantibacter flavus, Me. fujisawaense and Me. phyllosphaerae which produced a significant inhibition zone indicating the antagonistic ability of these species. The fungal endophytes also suppressed the growth of the pathogen and created an inhibition zone. In planta tests in which the fungal endophytes were inoculated on to seed of a susceptible oilseed rape cultivar were also undertaken. At the cotyledon leaf stage, the leaf was punctured and spore suspension of L. maculans was placed on the wound site. Inoculated seedlings particularly B. bassiana, significantly decreased phoma stem canker disease symptoms on the cotyledon. To our knowledge, this is the first study that screen the fungal and bacterial endophytes of wild brassica species associated with the seeds and demonstrate their beneficial characteristic when inoculated to brassica crops.Item Identification of mechanisms defining resistance and susceptibility of Camellia plants to necrotrophic petal blight disease : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Manawatū, New Zealand(Massey University, 2019) Kondratev, NikolaiSpecies in the genus Camellia, which includes the tea crops, oil-producers and valuable ornamental plants, have economic and cultural significance for many countries. The fungus Ciborinia camelliae causes petal blight disease of Camellia plants, which has a short initial asymptomatic phase and results in rapid necrosis and fall of blooms. Ciborinia camelliae is a necrotrophic pathogen of the family Sclerotiniaceae, which also includes two broad-host range necrotrophic pathogens, Botrytis cinerea and Sclerotinia sclerotiorum. Previously it was shown that some Camellia plants, such as Camellia lutchuensis, are naturally resistant to petal blight. In order to find molecular mechanisms underpinning this resistance, a genome-wide analysis of gene expression in C. lutchuensis petals was conducted. The analysis revealed a fast modulation of host transcriptional activity in response to C. camelliae ascospores. Interaction network analysis of fungus-responsive genes showed that petal blight resistance includes increased expression of important plant defence pathways, such as WRKY33-MPK3, phenylpropanoid and jasmonate biosynthesis. A much-delayed activation of the same pathways was observed in the susceptible Camellia cultivar, Camellia ‘Nicky Crisp’ (Camellia japonica x Camellia pitardii var. pitardii), suggesting that failure to activate early defence enables C. camelliae to invade and cause tissue necrosis. Early artificial induction of defence pathways using methyl jasmonate reduced the rate of petal blight in susceptible ‘Nicky Crisp’ plants, further verifying the role of a rapid defence activation in petal-blight resistance. Overall, transcriptomic and functional analysis of the Camellia spp.- C. camelliae interaction demonstrated that the same plant defence pathways contribute to both resistance and susceptibility against this necrotrophic pathogen, depending on the timing of their activation. To further understand the molecular mechanisms of petal blight resistance, the role of the phenylpropanoid pathway, identified as a key feature in the transcriptome study above, was investigated in more detail. This pathway produces various metabolites, including phenolic acids, aldehydes, and alcohols, which have numerous physiological functions and also participate in the production of flavonoids and lignin. Resistant C. lutchuensis was shown to rapidly activate the expression of core phenylpropanoid genes after treatment with C. camelliae ascospores. LC-MS-based quantification of phenylpropanoid compounds demonstrated that within the first 6 h of the infection, resistant plants had already accumulated coumaric, ferulic and sinapic acids, while at 24 hpi, concentrations of coumaraldehyde, sinapaldehyde, and caffeyalcohol were significantly increased. Thus, I further hypothesized that the compounds produced by the phenylpropanoid pathway may have fungistatic activity. Indeed, all tested phenylpropanoids inhibited the growth of C. camelliae in agar plates with different efficacy. Moreover, the application of phenylpropanoid compounds, including ferulic and coumaric acids, fully prevented the formation of petal blight lesions on susceptible Camellia ‘Nicky Crisp’ petals. Taken together, it can be concluded that the phenylpropanoid pathway may contribute to the early defence against the petal blight via the rapid production of fungistatic compounds. Ultimately, these compounds could be used to develop natural antifungal sprays to protect susceptible Camellia flowers. The analysis of the C. camelliae secretome using LC-MS/MS detection of proteins showed that the pathogen produces a large number of carbohydrate-active enzymes in liquid culture and plant petals. Injection of these proteins induced necrosis not only in susceptible Camellia petals but also in petals of the resistant species and leaves of non-host Nicotiana benthamiana. It was proposed that these enzymes can contribute to the virulence of the pathogen by inducing cell death and facilitating necrosis propagation. Thus, the early defence responses of resistant Camellia plants may possibly stop the development of C. camelliae before it starts releasing carbohydrate-active enzymes during the necrotrophic step of the infection. Overall, the results of this research further expand our understanding of plant- necrotroph interactions, suggesting that the timing of plant immune responses may be a crucial factor defining the outcome of the necrotrophic infection.