Primers específicos para a determinação de MAT-1 em Fusarium decemcellulare.

Fusarium decemcellulare é caracterizado por ser endófito ou patogênico a diversas plantas em regiões tropicais e subtropicais. Em guaranazeiro é o agente causal de superbrotamento um dos principais problemas da cultura. O estudo do sistema mating type, idiomorfos MAT-1 e MAT-2, além de fornecer uma compreensão detalhada para delimitar espécies dentro de complexos de espécies, é fundamental para estudo da dinâmica e do potencial de variação genética de um patógeno. Primers degenerados disponíveis na literatura para determinar mating type em várias espécies de Fusarium, não foi capaz de amplificar o MAT-1 em Fdc. Assim, este trabalho teve como objetivo desenvolver primers específicos para determinar MAT-1 em F. decemcellulare. Inicialmente primers degenerados foram desenhados com base nas sequencias do MAT-1 disponíveis no NCBI, amplificado em Fdc e o fragmento do tamanho esperado foi clonado e sequenciado para o desenho de iniciadores específicos que foram validados usando população de isolados de Fdc de diferentes locais de coleta.

Structure-based virtual screening of hypothetical inhibitors of the enzyme longiborneol synthase: a potential target to reduce Fusarium head blight disease.

2016

Caracterização de Fusarium decemcellulare isolado de guaranazeiro (Paullinia cupana var. sorbilis) por meio de ERIC-PCR.

A presente pesquisa visa analisar a diversidade genética De F. decemcellulare isolado de mudas e plantas adultas de guaranazeiro com sintomas de superbrotamento, hipertrofia floral ou galhas por meio do marcador molecular ERIC-PCR.

Patogenicidade de isolados de espécies de fusarium em grão-de-bico e ervilha.

Este trabalho teve como objetivo avaliar a patogenicidade de isolados de espécies de fusarium em grão-de-bico e ervilha.

Patogenicidade de isolados de Fusarium em colmos milho, sorgo e milheto.

2016

Seleção de estirpes de Trichoderma ssp. com atividade antagônica a Fusarium verticillioides produtores de fumonisina isolados de milho e de sorgo.

2016

Solarização do solo para o controle da murcha de Verticillium do tomate e da berinjela e seus efeitos sobre as comunidades de plantas invasoras e micro-artrópodos.

A solarização do solo por 30 e 50 dias reduziu a ocorrência da murcha de Verticillium dahliae na cultura de tomate e apresentou efeito residual na cultura subsequente de berinjelas. Porém, não houve aumento de produção das duas culturas. As plantas invasoras foram sensivelmente reduzidas com a solarização, sendo que a importância relativa das dicotiledoneas foi maior na testemunha e com brometo de metila, enquanto que, com a solarização, a importância relativa de gramíneas e dicotiledoneas foi semelhante. De modo que, a solarização e o brometo de metila reduziram acentuadamente as populações de diversos grupos de ácaros e insetos, sendo que 11,5 meses após os tratamentos, as populações voltaram a ser semelhantes a testemunha.

Strategies for improved disease resistance in micro-propagated bananas

Bananas (Musa sp) are one of the most important food crops in the world and provide a staple food and source of income in many households especially in Africa. Diseases are a major constraint to production with bunchy top, caused by Banana bunchy top virus (BBTV) generally considered the most important virus disease of bananas worldwide. Of the fungal diseases, Fusarium wilt, caused by the Fusarium oxysporum f.sp cubense (Foc), and black Sigatoka, caused by Mycosphaerella fijiensis, are arguably two of the most important and cause significant yield losses. The low fertility of commercially important banana cultivars has hampered efforts to generate disease resistance using conventional breeding. Possible alternative strategies to generate or increase disease resistance are through genetic engineering or by manipulation of the innate plant defence mechanisms, namely systemic acquired resistance (SAR). The first research component of this thesis describes attempts to generate BBTV-resistant banana plants using a genetic modification approach. The second research component of the thesis focused on the identification of a potential marker gene associated with SAR in banana plants and a comparison of the expression levels of the marker gene in response to biotic and abiotic stresses, and chemical inducers. Previous research at QUT CTCB showed that replication of BBTV DNA components in banana embryogenic cell suspensions (ECS) was abolished following co-bombardment with 1.1mers of mutated BBTV DNA-R. BBTV DNA-R encodes the master replication protein (Rep) and is the only viral protein essential for BBTV replication. In this study, ECS of banana were stably transformed with the same constructs, each containing a different mutation in BBTV DNA-R, namely H41G, Y79F and K187M, to examine the effect on virus replication in stably transformed plants. Cells were also transformed with a construct containing a native BBTV Rep. A total of 16, 16, 11 and five lines of stably transformed banana plants containing the Y79F, H41G, K187M and native Rep constructs, respectively, were generated. Of these, up to nine replicates from Y79F lines, four H41G lines, seven K187M lines and three native Rep lines were inoculated with BBTV by exposure to viruliferous aphids in two separate experiments. At least one replicate from each of the nine Y79F lines developed typical bunchy top symptoms and all tested positive for BBTV using PCR. Of the four H41G lines tested, at least one replicate from three of the lines showed symptoms of bunchy top and tested positive using PCR. However, none of the five replicates of one H41G line (H41G-3) developed symptoms of bunchy top and none of the plants tested positive for BBTV using PCR. Of the seven K187M lines, at least one replicate of all lines except one (K187M-1) developed symptoms of bunchy top and tested positive for BBTV. Importantly, none of the four replicates of line K187M-1 showed symptoms or tested positive for BBTV. At least one replicate from each of the three native Rep lines developed symptoms and tested positive for BBTV. The H41G-3 and K187M-1 lines possibly represent the first transgenic banana plants generated using a mutated Rep strategy. The second research component of this thesis focused on the identification of SAR-associated genes in banana and their expression levels in response to biotic and abiotic stresses and chemical inducers. The impetus for this research was the observation that tissue-cultured (TC) banana plants were more susceptible to Fusarium wilt disease (and possibly bunchy top disease) than plants grown from field-derived suckers, possibly due to decreased levels of SAR gene expression in the former. In this study, the pathogenesis-related protein 1 (PR-1) gene was identified as a potential marker for SAR gene expression in banana. A quantitative real-time PCR assay was developed and optimised in order to determine the expression of PR-1, with polyubiquitin (Ubi-1) found to be the most suitable reference gene to enable relative quantification. The levels of PR-1 expression were subsequently compared in Lady Finger and Cavendish (cv. Williams) banana plants grown under three different environmental conditions, namely in the field, the glass house and in tissue-culture. PR-1 was shown to be expressed in both cultivars growing under different conditions. While PR-1 expression was highest in the field grown bananas and lowest in the TC bananas in Lady Finger cultivar, this was not the case in the Cavendish cultivar with glass house plants exhibiting the lowest PR-1 expression compared with tissue culture and field grown plants. The important outcomes of this work were the establishment of a qPCR-based assay to monitor PR-1 expression levels in banana and a preliminary assessment of the baseline PR-1 expression levels in two banana cultivars under three different growing conditions. After establishing the baseline PR-1 expression levels in Cavendish bananas, a study was done to determine whether PR-1 levels could be increased in these plants by exposure to known banana pathogens and non-pathogens, and a known chemical inducer of SAR. Cavendish banana plants were exposed to pathogenic Foc subtropical race 4 (FocSR4) and non-pathogenic Foc race 1 (Foc1), as well as two putative inducers of resistance, Fusarium lycopersici (Fol) and the chemical, acibenzolar-S-methyl (BION®). Tissue culture bananas were acclimatised under either glass house (TCS) or field (TCH) conditions and treatments were carried out in a randomised complete block design. PR-1 expression was determined using qPCR for both TCS and TCH samples for the period 12-72h post-exposure. Treatment of TCH plants using Foc1 and FocSR4 resulted in 120 and 80 times higher PR-1 expression than baseline levels, respectively. For TCS plants treated with Foc1, PR-1 expression was 30 times higher than baseline levels at 12h post-exposure, while TCS plants treated with FocSR4 showed the highest PR-1 expression (20 times higher than baseline levels) at 72h post-exposure. Interestingly, when TCS plants were treated with Fol there was a marked increase of PR-1 expression at 12 h and 48 h following treatment which was 4 and 8 times higher than the levels observed when TCS plants were treated with Foc1 and FocSR4, respectively. In contrast, when TCH plants were treated with Fol only a slight increase in PR-1 expression was observed at 12 h, which eventually returned to baseline levels. Exposure of both TCS and TCH plants to BION® resulted in no effect on PR-1 expression levels at any time-point. The major outcome of the SAR study was that the glass house acclimatised tissue culture bananas exhibited lower PR-1 gene expression compared to field acclimatised tissue culture plants and the identification of Fol as a good candidate for SAR induction in banana plants exhibiting low PR-1 levels. A number of outcomes that foster understanding of both pathogen-derived and plant innate resistance strategies in order to potentially improve banana resistance to diseases were explored in this study and include identification of potential inducers of systemic acquired resistance and a promising mutated Rep approach for BBTV resistance. The work presented in this thesis is the first report on the generation of potential BBTV resistant bananas using the mutated Rep approach. In addition, this is the first report on the status of SAR in banana grown under different conditions of exposure to the biotic and abiotic environment. Further, a robust qPCR assay for the study of gene expression using banana leaf samples was developed and a potential inducer of SAR in tissue culture bananas identified which could be harnessed to increase resistance in tissue culture bananas.

Ocular surface health with contact lens wear

Eye care practitioners (ECPs) would tend to agree that wearing contact lenses increases the risk for infection, but millions of patients are still fitted with lenses every year because ECPs feel that the risk is manageable and that their patients' eye health can be protected. The Fusarium and Acanthamoeba keratitis outbreaks of years past were a wake-up call to manufacturers, ECPs, and regulatory agencies that risk cannot be managed without diligence, and that the complex relationship between contact lens materials, contact lens solutions, and compliance needs to be better understood in order to optimize the efficacy of contact lens care and improve care guidelines.

Rank regression for analyzing ordinal qualitative data for treatment comparison

Ordinal qualitative data are often collected for phenotypical measurements in plant pathology and other biological sciences. Statistical methods, such as t tests or analysis of variance, are usually used to analyze ordinal data when comparing two groups or multiple groups. However, the underlying assumptions such as normality and homogeneous variances are often violated for qualitative data. To this end, we investigated an alternative methodology, rank regression, for analyzing the ordinal data. The rank-based methods are essentially based on pairwise comparisons and, therefore, can deal with qualitative data naturally. They require neither normality assumption nor data transformation. Apart from robustness against outliers and high efficiency, the rank regression can also incorporate covariate effects in the same way as the ordinary regression. By reanalyzing a data set from a wheat Fusarium crown rot study, we illustrated the use of the rank regression methodology and demonstrated that the rank regression models appear to be more appropriate and sensible for analyzing nonnormal data and data with outliers.

Neuro- and immunotoxic effects of fumonisin B1 in cells

Fumonisin B1 (FB1) is a mycotoxin produced by the fungus Fusarium verticillioides, which commonly infects corn and other agricultural products. Fusarium species can also be found in moisture-damaged buildings, and therefore there may also be human exposure to Fusarium mycotoxins, including FB1. FB1 affects the metabolism of sphingolipids by inhibiting the enzyme ceramide synthase. It is neuro-, hepato- and nephrotoxic, and it is classified as possibly carcinogenic to humans. This study aimed to clarify the mechanisms behind FB1-induced neuro- and immunotoxicity. Four neural and glial cell lines of human, rat and mouse origin were exposed to graded doses of FB1 and the effects on the production of reactive oxygen species, lipid peroxidation, intracellular glutathione levels, cell viability and apoptosis were investigated. Furthermore, the effects of FB1, alone or together with lipopolysaccharide (LPS), on the mRNA and protein expression levels of different cytokines and chemokines were studied in human dendritic cells (DC). FB1 induced oxidative stress and cell death in all cell lines studied. Generally, the effects were only seen after prolonged exposure at 10 and 100 µM of FB1. Signs of apoptosis were also seen in all four cell lines. The sensitivities of the cell lines used in this study towards FB1 may be classified as human U-118MG glioblastoma > mouse GT1-7 hypothalamic > rat C6 glioblastoma > human SH-SY5Y neuroblastoma cells. When comparing cell lines of human origin, it can be concluded that glial cells seem to be more sensitive towards FB1 toxicity than those of neural origin. After exposure to FB1, significantly increased levels of the cytokine interferon-γ (IFNγ) were detected in human DC. This observation was further confirmed by FB1-induced levels of the chemokine CXCL9, which is known to be regulated by IFNγ. During co-exposure of DC to both LPS and FB1, significant inhibitions of the LPS-induced levels of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-1β, and their regulatory chemokines CCL3 and CCL5 were observed. FB1 can thus affect immune responses in DC, and therefore, it is rather likely that it also affects other types of cells participating in the immune defence system. When evaluating the toxicity potential of FB1, it is important to consider the effects on different cell types and cell-cell interactions. The results of this study represent new information, especially about the mechanisms behind FB1-induced oxidative stress, apoptosis and immunotoxicity, as well as the varying sensitivities of different cell types towards FB1.

Effective Use in Livestock Feeds of Mouldy and Weather-damaged Grain Containing Mycotoxins-Case Histories and Economic Assessments Pertaining to Pig and Poultry Industries of Queensland

Mould growth in field crops or stored grain reduces starch and lipid content, with consequent increases in fibre, and an overall reduction in digestible energy; palatability is often adversely affected. If these factors are allowed for, and mycotoxin concentrations are low, there are sound economic reasons for using this cheaper grain. Mycotoxins are common in stock feed but their effects on animal productivity are usually slight because either the concentration is too low or the animal is tolerant to the toxin. In Australia, aflatoxins occur in peanut by-products and in maize and sorghum if the grain is moist when stored. Zearalenone is found in maize and in sorghum and wheat in wetter regions. Nivalenol and deoxynivalenol are found in maize and wheat but at concentrations that rarely affect pigs, with chickens and cattle being even more tolerant. Other mycotoxins including cyclopiazonic acid, T-2 toxin, cytochalasins and tenuazonic acid are produced by Australian fungi in culture but are not found to be significant grain contaminants. Extremely mouldy sorghum containing Alternaria and Fusarium mycotoxins decreased feed conversion in pigs and chickens by up to 14%. However, E moniliforme- and Diplodia maydis-infected maize produced only slight reductions in feed intake by pigs and Ustilago- infected barley produced no ill effects. Use of these grains would substantially increase profits if the grain can be purchased cheaply.

The influence of planting density on the production of 'Goldfinger' (Musa spp., AAAB) in the subtropics

'Goldfinger', a tetraploid banana produced from the Fundación Hondureña de Investigación Agrícola (FHIA) breeding program, was released to the Australian industry in 1995. It was promoted as an apple-flavoured dessert banana with resistance to Fusarium wilt race 1 and subtropical race 4, as well as resistance to black and yellow Sigatoka (Mycosphaerella fijiensis and M. musicola, respectively). This study was initiated to provide agronomic information to the banana industry, which was under threat from Fusarium wilt, on a new cultivar which could replace 'Williams' (AAA, Cavendish subgroup) or 'Lady Finger' (AAB, Pome subgroup) in those areas affected by Fusarium wilt. Also few studies had reported on the production characteristics of the new tetraploid hybrids, especially from subtropical areas, and therefore two field sites, one a steep-land farm and the other a level, more productive site, were selected for planting density and spatial arrangement treatments. The optimum density in terms of commercial production, taking into account bunch weight, finger size, length of the production cycle, plant height and ease of management, was 1680 plants/ha on the steep-land site where plants were planted in single rows with 2.5 m × 2.5 m spacings. However on the level site a double-row triangular layout with inter-row distances of 4.5 m to allow vehicular access (1724 plants/ha) gave the best results. With this arrangement plants were in an alternate, triangular arrangement along a row and a spacing of 1.5 m between plants at the points of each triangle and between each block of triangles.

Confirmation of QTL mapping and marker validation for partial seedling resistance to crown rot in wheat line '2-49'

We have tested the efficacy of putative microsatellite single sequence repeat (SSR) markers, previously identified in a 2-49 (Gluyas Early/Gala) × Janz doubled haploid wheat (Triticum aestivum) population, as being linked to partial seedling resistance to crown rot disease caused by Fusarium pseudograminearum. The quantitative trait loci (QTLs) delineated by these markers have been tested for linkage to resistance in an independent Gluyas Early × Janz doubled haploid population. The presence of a major QTL on chromosome 1DL (QCr.usq-1D1) and a minor QTL on chromosome 2BS (QCr.usq-2B1) was confirmed. However, a putative minor QTL on chromosome 2A was not confirmed. The QTL on 1D was inherited from Gluyas Early, a direct parent of 2-49, whereas the 2B QTL was inherited from Janz. Three other putative QTLs identified in 2-49 × Janz (on 1AL, 4BL, and 7BS) were inherited by 2-49 from Gala and were not able to be confirmed in this study. The screening of SSR markers on a small sample of elite wheat genotypes indicated that not all of the most tightly linked SSR markers flanking the major QTLs on 1D and 1A were polymorphic in all backgrounds, indicating the need for additional flanking markers when backcrossing into some elite pedigrees. Comparison of SSR haplotypes with those of other genotypes exhibiting partial crown rot resistance suggests that additional, novel sources of crown rot resistance are available.

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.