The effect of endophyte on the performance of irrigated perennial ryegrasses in subtropical Australia

The effect of fungal endophyte (Neotyphodium lolii) infection on the performance of perennial ryegrass (Lolium perenne) growing under irrigation in a subtropical environment was investigated. Seed of 4 cultivars, infected with standard (common toxic or wild-type) endophyte or the novel endophyte AR1, or free of endophyte (Nil), was sown in pure swards, which were fertilised with 50 kg N/ha.month. Seasonal and total yield, persistence, and rust susceptibility were assessed over 3 years, along with details of the presence of endophyte and alkaloids in plant shoots. Endophyte occurrence in tillers in both the standard and AR1 treatments was above 95% for Bronsyn and Impact throughout and rose to that level in Samson by the end of the second year. Meridian AR1 only reached 93% while, in the standard treatment, the endophyte had mostly died before sowing. Nil Zendophyte treatments carried an average of ?0.6% infection throughout. Infection of the standard endophyte was associated with increased dry matter (DM) yields in all 3 years compared with no endophyte. AR1 also significantly increased yields in the second and third years. Over the full 3 years, standard and AR1 increased yields by 18% and 11%, respectively. Infection with both endophytes was associated with increased yields in all 4 seasons, the effects increasing in intensity over time. There was 27% better persistence in standard infected plants compared with Nil at the end of the first year, increasing to 198% by the end of the experiment, while for AR1 the improvements were 20 and 134%, respectively. The effect of endophyte on crown rust (Puccinia coronata) infection was inconsistent, with endophyte increasing rust damage on one occasion and reducing it on another. Cultivar differences in rust infection were greater than endophyte effects. Plants infected with the AR1 endophyte had no detectable ergovaline or lolitrem B in leaf, pseudostem, or dead tissue. In standard infected plants, ergovaline and lolitrem B were highest in pseudostem and considerably lower in leaf. Dead tissue had very low or no detectable ergovaline but high lolitrem B concentrations. Peramine concentration was high and at similar levels in leaf and pseudostem, but not detectable in dead material. Concentration was similar in both AR1 and standard infected plants. Endophyte presence appeared to have a similar effect in the subtropics as has been demonstrated in temperate areas, in terms of improving yields and persistence and increasing tolerance of plants to stress factors.

Mycotoxins and toxigenic fungi in sago starch from Papua New Guinea

Aims: To assay sago starch from Papua New Guinea (PNG) for important mycotoxins and to test fungal isolates from sago for mycotoxin production in culture. Methods and Results: Sago starch collected from Western and East Sepik Provinces was assayed for aflatoxins, ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin and zearalenone and all 51 samples were negative. Frequently isolated species of Penicillium (13), Aspergillus (five) and Fusarium (one) were cultured on wheat grain, and tested for the production of ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin, patulin and penicillic acid. All 12 isolates of P. citrinin and one of two A. flavipes isolates produced citrinin. A single isolate of A. versicolor produced sterigmatocystin. No other mycotoxins were detected in these cultures. Conclusions: No evidence was found of systemic mycotoxin contamination of sago starch. However, the isolation of several mycotoxigenic fungi shows the potential for citrinin and other mycotoxins to be produced in sago stored under special conditions. Significance and Impact of the study: Sago starch is the staple carbohydrate in lowland PNG and the absence of mycotoxins in freshly prepared sago starch is a positive finding. However, the frequent isolation of citrinin-producing fungi indicates a potential health risk for sago consumers, and food safety is dependant on promoting good storage practices.

Characterization of disease resistance gene candidates of the nucleotide binding site (NBS) type from banana and correlation of a transcriptional polymorphism with resistance to Fusarium oxysporum f.sp. cubense race 4

Most plant disease resistance (R) genes encode proteins with a nucleotide binding site and leucine-rich repeat structure (NBS-LRR). In this study, degenerate primers were used to amplify genomic NBS-type sequences from wild banana (Musa acuminata ssp. malaccensis) plants resistant to the fungal pathogen Fusarium oxysporum formae specialis (f. sp.) cubense (FOC) race 4. Five different classes of NBS-type sequences were identified and designated as resistance gene candidates (RGCs). The deduced amino acid sequences of the RGCs revealed the presence of motifs characteristic of the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses grouped the banana RGCs within the non-TIR (homology to Toll/interleukin-1 receptors) subclass of NBS sequences. Southern hybridization showed that each banana RGC is present in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to FOC race 4. RGC1, 3 and 5 showed a constitutive expression profile in both resistant and susceptible plants whereas no expression was detected for RGC4. Interestingly, RGC2 expression was found to be associated only to FOC race 4 resistant lines. This finding could assist in the identification of a FOC race 4 resistance gene.

New insights into the altered adhesive and mechanical properties of red blood cells parasitized by Babesia bovis

Sequestration of parasite-infected red blood cells (RBCs) in the microvasculature is an important pathological feature of both bovine babesiosis caused by Babesia bovis and human malaria caused by Plasmodium falciparum. Surprisingly, when compared with malaria, the cellular and molecular mechanisms that underlie this abnormal circulatory behaviour for RBCs infected with B. bovis have been relatively ignored. Here, we present some novel insights into the adhesive and mechanical changes that occur in B. bovis-infected bovine RBCs and compare them with the alterations that occur in human RBCs infected with P. falciparum. After infection with B. bovis, bovine RBCs become rigid and adhere to vascular endothelial cells under conditions of physiologically relevant flow. These alterations are accompanied by the appearance of ridge-like structures on the RBC surface that are analogous, but morphologically and biochemically different, to the knob-like structures on the surface of human RBCs infected with P. falciparum. Importantly, albeit for a limited number of parasite lines examined here, the extent of these cellular and rheological changes appear to be related to parasite virulence. Future investigations to identify the precise molecular composition of ridges and the proteins that mediate adhesion will provide important insight into the pathogenesis of both babesiosis and malaria.

Population genetics of the frog-killing fungus Batrachochytrium dendrobatidis

Global amphibian decline by chytridiomycosis is a major environmental disaster that has been attributed to either recent fungal spread or environmental change that promotes disease. Here, we present a population genetic comparison of Batrachochytrium dendrobatidis isolates from an intensively studied region of frog decline, the Sierra Nevada of California. In support of a novel pathogen, we find low diversity, no amphibian-host specificity, little correlation between fungal genotype and geography, local frog extirpation by a single fungal genotype, and evidence of human-assisted fungus migration. In support of endemism, at a local scale, we find some diverse, recombining populations. Therefore neither epidemic spread nor endemism alone explains this particular amphibian decline. Recombination raises the possibility of resistant sporangia and a mechanism for rapid spread as well as persistence that could greatly complicate global control of the pathogen.

Development of a vaccine for Chlamydia trachomatis: Challenges and current progress

Chlamydia trachomatis remains an enigmatic bacterial pathogen with no vaccine yet available to treat human ocular and genital tract infections caused by tissue-tropic serovars of the organism. Globally, it is the leading cause of preventable blindness as well as the leading cause of bacterial sexually transmitted infections. The pathogen has a range of virulence factors that enable it to successfully evade both the innate and adaptive immune system of the host. The host immune system, although protective, paradoxically is also associated closely with the pathologies of trachoma and pelvic inflammatory disease – disease sequelae of some chlamydial infections and reinfections in some genetically susceptible hosts. In this review, we focus on what is known currently about the pathogenesis of ocular and genital infections caused by this mucosal pathogen. We also discuss novel insights into the pathogenesis of infections caused by the genital and ocular serovars of C. trachomatis, including a discussion of both pathogen and host factors, such as the human microbiota at these mucosal sites as well as the current immunological challenges facing vaccine development. Finally, we discuss the current progress toward development of a vaccine against C. trachomatis. A wide range of recombinant protein antigens are being identified and, hence, are available for vaccine trials. A plasmid-free live strain has recently been produced and evaluated in the mouse (Chlamydia muridarum) and monkey (C. trachomatis) models. The data for ocular infections in the monkey model was particularly encouraging, although the path to regulatory approval of a live vaccine is still uncertain. While still a major challenge, vaccines for ocular and genital C. trachomatis infections are looking more promising.

Achievements in forest tree genetic improvement in Australia and New Zealand 2: Development of Corymbia species and hybrids for plantations in eastern Australia

This paper describes the establishment of provenance seedling seed orchards of three spotted gums and cadaga (all species of Corymbia ex Eucalyptus). It also discusses the limitations of growing the spotted gums as pure species including: lack of mass flowering, susceptibility to a fungal shoot blight and low amenability to vegetative propagation. These limitations, together with observation of putative natural hybrids of the spotted gums with cadaga, and the early promise of manipulated hybrids, led to an intensive breeding and testing program. Many hybrid families have significant advantages in growth and tolerance to disease, insects and frost, and can be vegetatively propagated. They also exhibit broad environmental plasticity, allowing the best varieties to be planted across a wider range of sites than the spotted gums, resulting in more land being suitable for plantation development.

Tadpole mouthpart depigmentation as an accurate indicator of chytridiomycosis, an emerging disease of amphibians

Chytridiomycosis is an emerging infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis, and its role in causing population declines and species extinctions worldwide has created an urgent need for methods to detect it. Several reports indicate that in anurans chytridiomycosis can cause the depigmentation of tadpole tnouthparts, but the accuracy of using depigmentation to determine disease status remains uncertain. Our objective was to determine for the Mountain Yellow-legged Frog (Rana muscosa) whether visual inspections of the extent of tadpole mouthpart depigmentation could be used to accurately categorize individual tadpoles or R. muscosa populations as B. dendrobatidis-positive or negative. This was accomplished by assessing the degree of mouthpart depigmentation in tadpoles of known disease status (based on PCR assays). The depigmentation of R. muscosa tadpole mouthparts was associated with the presence of B. dendrobatidis, and this association was particularly strong for upper jaw sheaths. Using a rule that classifies tadpoles with upper jaw sheaths that are 100% pigmented as uninfected and those with jaw sheaths that are <100% pigmented as infected resulted in the infection status of 86% of the tadpoles being correctly classified. By applying this rule to jaw sheath pigmentation scores averaged across all tadpoles inspected per site, we were able to correctly categorize the infection status of 92% of the study populations. Similar research on additional anurans is critically needed to determine how broadly applicable our results for R. muscosa are to other species.

Urinary tract infection of mice to model human disease: Practicalities, implications and limitations

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Murine models of human UTI are vital experimental tools that have helped to elucidate UTI pathogenesis and advance knowledge of potential treatment and infection prevention strategies. Fundamentally, several variables are inherent in different murine models, and understanding the limitations of these variables provides an opportunity to understand how models may be best applied to research aimed at mimicking human disease. In this review, we discuss variables inherent in murine UTI model studies and how these affect model usage, data analysis and data interpretation. We examine recent studies that have elucidated UTI host–pathogen interactions from the perspective of gene expression, and review new studies of biofilm and UTI preventative approaches. We also consider potential standards for variables inherent in murine UTI models and discuss how these might expand the utility of models for mimicking human disease and uncovering new aspects of pathogenesis

Evaluation of transgenic bananas expressing anti-apoptotic genes for resistance against Fusarium wilt

The banana industry worldwide is under threat from a fungal disease known as Fusarium wilt, a disease for which there is no chemical control. Conventional breeding approaches to generate resistant banana varieties are lengthy and very difficult. As such, genetic engineering for disease resistance is considered the most viable control option. In this PhD thesis, genetically modified banana plants were generated using several different stress tolerance genes. When challenged with Fusarium wilt in glasshouse trials, some lines showed increased resistance to the disease. The promising elite lines generated in this study will now require testing in field trials.

Austropleospora osteospermi gen. et sp nov and its host specificity and distribution on Chrysanthemoides monilifera ssp rotundata in Australia.

Hendersonia osteospermi was found for the first time in Australia on leaf spots of the introduced invasive plant Chrysanthemoides monilifera ssp. rotundata (bitou bush) in coastal regions of New South Wales. Pathogenicity tests on species from 11 tribes in the family Asteraceae, demonstrated that H. osteospermi caused severe necrosis on leaves and stems of C. monilifera ssp. rotundata and its congener C. monilifera ssp. monilifera (boneseed). Small necrotic spots also developed on Osteospermum fruticosum and Dimorphotheca cuneata in the Calenduleae and on Helianthus annuus (sunflower) in the Heliantheae. None of the other plant species tested developed leaf spots, although H. osteospermi was re-isolated from senescent leaves of Cynara scolymus (globe artichoke) in the Cynareae and Vernonia cinerea in the Vernonieae. Single ascospores from ascomata of a Pleospora-like fungus found on diseased stems of bitou bush produced H. osteospermi in culture, which proved the anamorph/teleomorph connection. The ITS region of both a single-ascospore isolate and a single-conidium isolate were sequenced and found to be identical. The taxonomic status of H. osteospermi is re-examined and Austropleospora osteospermi gen. et sp. nov. is described as its teleomorph based on morphology, host range tests and DNA sequence analysis. The potential of A. osteospermi for the biological control of bitou bush and boneseed in Australia is discussed.

Pre-cropping with canola decreased Pratylenchus thornei populations, arbuscular mycorrhizal fungi, and yield of wheat.

Root-lesion nematode (Pratylenchus thornei) significantly reduces wheat yields in the northern Australian grain region. Canola is thought to have a 'biofumigation' potential to control nematodes; therefore, a field experiment was designed to compare canola with other winter crops or clean-fallow for reducing P. thornei population densities and improving growth of P. thornei-intolerant wheat (cv. Batavia) in the following year. Immediately after harvest of the first-year crops, populations of P. thornei were lowest following various canola cultivars or clean-fallow (1957-5200 P. thornei/kg dry soil) and were highest following susceptible wheat cultivars (31 033-41 294/kg dry soil). Unexpectedly, at planting of the second-year wheat crop, nematode populations were at more uniform lower levels (<5000/kg dry soil), irrespective of the previous season's treatment, and remained that way during the growing season, which was quite dry. Growth and grain yield of the second-year wheat crop were poorest on plots previously planted with canola or left fallow due to poor colonisation with arbuscular mycorrhizal (AM) fungi, with the exception of canola cv. Karoo, which had high AM fungal colonisation and low wheat yields. There were significant regressions between growth and yield parameters of the second-year wheat and levels of AMF following the pre-crop treatments. Thus, canola appears to be a good crop for reducing P. thornei populations, but AM fungal-dependence of subsequent crops should be considered, particularly in the northern Australian grain region.

The use of temperate species in the Australian subtropics.

Temperate species and tropical crop silage are the basis for forage production for the dairy industry in the Australian subtropics. Irrigation is the key resource needed for production, with little survival of temperate species under rain-grown conditions except for lucerne. Annual ryegrass (Lolium multiflorum), fertilised with either inorganic nitrogen or grown with clovers, is the main cool season forage for the dairy industry. It is sown into fully prepared seedbeds, oversown into tropical grasses, especially kikuyu (Pennisetum clandestinum) or sown after mulching. There has been a continual improvement in the performance of annual and hybrid ryegrass cultivars over the last 25 years. In small plot, cutting experiments, yields of annual ryegrass typically range from 15 to 21 t DM/ha, with equivalent on-farm yields of 7 to 14 t DM/ha of utilised material. Rust (Puccinia coronata) remains the major concern although resistance is more stable than in oats. There have also been major improvements in the performance of perennial ryegrass (L. perenne) cultivars although their persistence under grazing is insufficient to make them a reliable forage source for the subtropics. On the other hand, tall fescue (Festuca arundinacea) and prairie grass (Bromus willdenowii) cultivars perform well under cutting and grazing, although farmer resistance to the use of tall fescue is strong. White clover (Trifolium repens) is a reliable and persistent performer although disease usually reduces its performance in the third year after sowing. Persian (Shaftal) annual clover (T. resupinatum) gives good winter production but the performance of berseem clover (T. alexandrinum) is less reliable and the sub clovers (T. subterraneum) are generally not suited to clay soils of neutral to alkaline pH. Lucerne (Medicago sativa), either as a pure stand or in mixtures, is a high producing legume under both irrigation and natural rainfall. Understanding the importance of leaf and crown diseases, and the development of resistant cultivars, have been the reasons for its reliability. Insects on temperate species are not as serious a problem in the subtropics as in New Zealand (NZ). Fungal and viral diseases, on the other hand, cause many problems and forage performance would benefit from more research into resistance.

Zebrafish as a model of Parkinson's disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease among the elderly. Its etiology is unknown and no disease-modifying drugs are available. Thus, more information concerning its pathogenesis is needed. Among other genes, mutated PTEN-induced kinase 1 (PINK1) has been linked to early-onset and sporadic PD, but its mode of action is poorly understood. Most animal models of PD are based on the use of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is metabolized to MPP+ by monoamine oxidase B (MAO B) and causes cell death of dopaminergic neurons in the substantia nigra in mammals. Zebrafish has been a widely used model organism in developmental biology, but is now emerging as a model for human diseases due to its ideal combination of properties. Zebrafish are inexpensive and easy to maintain, develop rapidly, breed in large quantities producing transparent embryos, and are readily manipulated by various methods, particularly genetic ones. In addition, zebrafish are vertebrate animals and results derived from zebrafish may be more applicable to mammals than results from invertebrate genetic models such as Drosophila melanogaster and Caenorhabditis elegans. However, the similarity cannot be taken for granted. The aim of this study was to establish and test a PD model using larval zebrafish. The developing monoaminergic neuronal systems of larval zebrafish were investigated. We identified and classified 17 catecholaminergic and 9 serotonergic neuron populations in the zebrafish brain. A 3-dimensional atlas was created to facilitate future research. Only one gene encoding MAO was found in the zebrafish genome. Zebrafish MAO showed MAO A-type substrate specificity, but non-A-non-B inhibitor specificity. Distribution of MAO in larval and adult zebrafish brains was both diffuse and distinctly cellular. Inhibition of MAO during larval development led to markedly elevated 5-hydroxytryptamine (serotonin, 5-HT) levels, which decreased the locomotion of the fish. MPTP exposure caused a transient loss of cells in specific aminergic cell populations and decreased locomotion. MPTP-induced changes could be rescued by the MAO B inhibitor deprenyl, suggesting a role for MAO in MPTP toxicity. MPP+ affected only one catecholaminergic cell population; thus, the action of MPP+ was more selective than that of MPTP. The zebrafish PINK1 gene was cloned in zebrafish, and morpholino oligonucleotides were used to suppress its expression in larval zebrafish. The functional domains and expression pattern of zebrafish PINK1 resembled those of other vertebrates, suggesting that zebrafish is a feasible model for studying PINK1. Translation inhibition resulted in cell loss of the same catecholaminergic cell populations as MPTP and MPP+. Inactivation of PINK1 sensitized larval zebrafish to subefficacious doses of MPTP, causing a decrease in locomotion and cell loss in one dopaminergic cell population. Zebrafish appears to be a feasible model for studying PD, since its aminergic systems, mode of action of MPTP, and functions of PINK1 resemble those of mammalians. However, the functions of zebrafish MAO differ from the two forms of MAO found in mammals. Future studies using zebrafish PD models should utilize the advantages specific to zebrafish, such as the ability to execute large-scale genetic or drug screens.

Cathepsin D Deficiency - Molecular and Cellular Mechanisms of Neurodegeneration

Cathepsin D (CTSD) is a lysosomal protease, the deficiency of which is fatal and associated with neurodegeneration. CTSD knock-out mice, which die at the age of four weeks, show intestinal necrosis, loss of lymphoid cells and moderate pathological changes in the brain. An active-site mutation in the CTSD gene underlies a neurodegenerative disease in newborn sheep, characterized by brain atrophy without any changes to visceral tissues. The CTSD deficiences belong to the group of neuronal ceroid-lipofuscinoses (NCLs), severe neurodegenerative lysosomal storage disorders. The aim of this thesis was to examine the molecular and cellular mechanisms behind neurodegeneration in CTSD deficiency. We found the developmental expression pattern of CTSD to resemble that of synaptophysin and the increasing expression of CTSD to coincide with the active period of myelination in the rat brain, suggesting a role for CTSD in early rat brain development. An active-site mutation underlying the congenital ovine NCL not only affected enzymatic activity, but also changed the stability, processing and transport of the mutant protein, possibly contributing to the disease pathogenesis. We also provide CTSD deficiency as a first molecular explanation for human congenital NCL, a lysosomal storage disorder, characterized by neuronal loss and demyelination in the central nervous system. Finally, we show the first evidence for synaptic abnormalities and thalamocortical changes in CTSD-deficient mice at the molecular and ultrastructural levels. Keywords: cathepsin D, congenital, cortex, lysosomal storage disorder, lysosome, mutation, neurodegeneration, neuronal ceroid-lipofuscinosis, overexpression, synapse, thalamus