Generating transgenic banana (cv. Sukali Ndizi) resistant to Fusarium Wilt

Banana is one of the world’s most popular fruit crops and Sukali Ndizi is the most popular dessert banana in the East African region. Like other banana cultivars, Sukali Ndizi is threatened by several constraints, of which the Fusarium wilt disease is the most destructive. Fusarium wilt is caused by a soil-borne fungus, Fusarium oxysporum f.sp. cubense (Foc). No effective control strategy currently exists for this disease and although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult because of low fertility. Considering that conventional breeding generates hybrids with additional undesirable traits, transformation is the most suitable way of introducing resistance in the banana genome. The success of this strategy depends on the availability of genes for genetic transformation. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi, including Foc race 1 in banana cultivar Lady Finger. This thesis explores the potential of a plant-codon optimised nematode anti-apoptosis gene (Mced9) to provide resistance against Foc race 1 in dessert banana cultivar Sukali Ndizi. Agrobacterium-mediated transformation was used to transform embryogenic cell suspension of Sukali Ndizi with plant expression vector pYC11, harbouring maize ubiquitin promoter driven Mced9 gene and nptII as a plant selection marker. A total of 42 independently transformed lines were regenerated and characterized. The transgenic lines were multiplied, infected and evaluated for resistance to Foc race 1 in a small pot bioassay. The pathogenicity of the Ugandan Foc race 1 isolate used for infection was pre-determined and the spore concentration was standardised for consistent infection and symptom development. This process involved challenging tissue culture plants of Sukali Ndizi, a Foc race 1 susceptible cultivar and Nakinyika, an East African Highland cultivar known to be resistant to Foc race 1, with Fusarium inoculum and observing external and internal disease symptom development. Rhizome discolouration symptoms were the best indicators of Fusarium wilt with yellowing being an early sign of disease. Three transgenic lines were found to show significantly less disease severities compared to the wild-type control plants after 13 weeks of infection, indicating that Mced9 has the potential to provide tolerance to Fusarium wilt in Sukali Ndizi.

Genus Umbravirus

The practical need to partition the world of viruses into distinguishable, universally agreed upon entities is the ultimate justification for developing a virus classification system. The Author of this Book is Andrew MQ King, Elliot Lefkowitz, Eric B. Carstens, Michael J. Adams Since 1971, the International Committee on Taxonomy of Viruses (ICTV) operating on behalf of the world community of virologists has taken on the task of developing a single, universal taxonomic scheme for all viruses infecting animals (vertebrate, invertebrates, and protozoa), plants (higher plants and algae), fungi, bacteria, and archaea.

Gene silencing: Fleshing out the bones

Genetic studies are revealing the pathway for RNA-mediated gene silencing. Short RNA molecules are the key, giving sequence specificity for RNA degradation and mediating communication within and between cells; these short RNAs are common to transcriptional and post-transcriptional silencing pathways. The expression of transgenes in plants varies between independent transformants and there are many examples where the transgenic trait is not expressed, or disappears in subsequent generations, despite the presence of the transgene. This loss of a trait, but not of the transgene, has become known as gene silencing and can take two forms, transcriptional or post-transcriptional. As their names imply, transcriptional gene silencing occurs when a transgene is not transcribed, whereas in post-transcriptional gene silencing, the transgene mRNA is produced but degraded before it is translated (reviewed in [1]). Both forms of silencing seem to be the result of inherent mechanisms for protecting plants against mobile or invading DNA — for example, transposable elements or the T-DNA of Agrobacterium — or RNA viruses. Plants are not alone in their capacity for transgene silencing; both forms of silencing occur in flies and fungi, where it is known as RIP or quelling, while nematodes exhibit post-transcriptional silencing, generally referred to as RNA interference (RNAi). A clearer picture of the mechanisms and relationships of the different types of transgene silencing is beginning to emerge from a number of recent studies [2], [3], [4], [5], [6], [7] and [8]. Some of these studies [2], [3], [4] and [5] have enhanced our understanding of the steps within the post-transcriptional silencing pathway, and others [6], [7] and [8] have demonstrated that the two forms of silencing may be mechanistically linked.

The evolution and diversification of Dicers in plants

Most multicellular organisms regulate developmental transitions by microRNAs, which are generated by an enzyme, Dicer. Insects and fungi have two Dicer-like genes, and many animals have only one, yet the plant, Arabidopsis, has four. Examining the poplar and rice genomes revealed that they contain five and six Dicer-like genes, respectively. Analysis of these genes suggests that plants require a basic set of four Dicer types which were present before the divergence of mono- and dicotyledonous plants (∼200 million years ago), but after the divergence of plants from green algae. A fifth type of Dicer seems to have evolved in monocots. © 2006 Federation of European Biochemical Societies.

An evidence-based brochure to educate contact lens wearers about safe contact lens wear

Contact lenses are a successful and popular means to correct refractive error and are worn by just under 700,000 Australians1 and approximately 125 million people worldwide. The most serious complication of contact lens wear is microbial keratitis, a potentially sight-threatening corneal infection most often caused by bacteria. Gram-negative bacteria, in particular pseudomonas species, account for the majority of severe bacterial infections. Pathogens such as fungi or amoebae, which feature less often, are associated with significant morbidity. These unusual pathogens have come into the spotlight in recent times with an apparent association with specific lens cleaning solutions...

The Chemistry of Mycotoxins

Mycotoxins – from the Greek μύκης (mykes, mukos) “fungus” and the Latin (toxicum) “poison” – are a large and growing family of secondary metabolites and hence natural products produced by fungi, in particular by molds (1). It is estimated that well over 1,000 mycotoxins have been isolated and characterized so far, but this number will increase over the next few decades due the availability of more specialized analytical tools and the increasing number of fungi being isolated. However, the most important classes of fungi responsible for these compounds are Alternaria, Aspergillus (multiple forms), Penicillium, and Stachybotrys. The biological activity of mycotoxins ranges from weak and/or sometimes positive effects such as antibacterial activity (e.g. penicillin derivatives derived from Penicillium strains) to strong mutagenic (e.g. aflatoxins, patulin), carcinogenic (e.g. aflatoxins), teratogenic, neurotoxic (e.g. ochratoxins), nephrotoxic (e.g. fumonisins, citrinin), hepatotoxic, and immunotoxic (e.g. ochratoxins, diketopiperazines) activities (1, 2), which are discussed in detail in this volume.

Stress effects caused by the expression of a mutant cellobiohydrolase I and proteasome inhibition in Trichoderma reesei Rut-C30

Trichoderma reesei Rut-C30 is used widely as an expression host for various gene products. We have explored cellular effects caused by the expression of a mutant form of cellobiohydrolase I (CBHI), the major secreted protein of T. reesei using biochemical and transcriptomic analyses and confocal laser scanning microscopy. The mutated CBHI was tagged fluorescently with Venus to establish the subcellular location of the fusion protein and its potential association with the proteasome, an organelle assigned for the disposal of misfolded proteins. Expression of the mutant CBHI in the high protein-secreting host Rut-C30 caused physiological changes in the fungal hyphae, affected protein secretion and elicited ER stress. A massive upregulation of UPR- and ERAD-related genes sec61, der1, uba1, bip1, pdi1, prp1, cxl1 and lhs1 was observed by qRT-PCR in the CBHIΔ4-Venus strain with four mutations introduced in the DNA encoding the core domain of CBHI. Further stress was applied to this strain by inhibiting function of the proteasome with MG132 (N-benzoylcarbonyl(Cbz)-Leu-Leu-leucinal). The effect of MG132 was found to be specific to the proteasome-associated genes. There are no earlier reports on the effect of proteasome inhibition on protein quality control in filamentous fungi. Confocal fluorescence microscopy studies suggested that the mutant CBHI accumulated in the ER and colocalized with the fungal proteasome. These results provide an indication that there is a limit to how far T. reesei Rut-C30, already under secretion stress, can be pressed to produce higher protein yields.

Enzyme activities and biotechnological applications of cold-active microfungi

Fungi are eukaryotic organisms and considered to be less adaptable to extreme environments when compared to bacteria. While there are no thermophilic microfungi in a strict sense, some fungi have adapted to life in the cold. Cold-active microfungi have been isolated from the Antarctic and their enzyme activities explored with a view to finding new candidates for industrial use. On another front, environmental pollution by petroleum products in the Antarctic has led to a search for, and the subsequent discovery of, fungal isolates capable of degrading hydrocarbons. The work has paved the way to developing a bioremedial approach to containing this type of contamination in cold climates. Here we discuss our efforts to map the capability of Antarctic microfungi to degrade oil and also introduce a novel cold-active fungal lipase enzyme.

Resistance of microbial populations in DDT-contaminated and uncontaminated soils

One DDT-contaminated soil and two uncontaminated soils were used to enumerate DDT-resistant microbes (bacteria, actinomycetes and fungi) by using soil dilution agar plates in media either with 150 μg DDT ml -1 or without DDT at different temperatures (25, 37 and 55°C). Microbial populations in this study were significantly (p<0.001) affected by DDT in the growth medium. However, the numbers of microbes in long-term contaminated and uncontaminated soils were similar, presumably indicating that DDT-resistant microbes had developed over a long time exposure. The tolerance of isolated soil microbes to DDT varied in the order fungi&gt;actinomycetes>bacteria. Bacteria from contaminated soil were more resistant to DDT than bacteria from uncontaminated soils. Microbes isolated at different temperatures also demonstrated varying degrees of DDT resistance. For example, bacteria and actinomycetes isolated at all incubation temperatures were sensitive to DDT. Conversely fungi isolated at all temperatures were unaffected by DDT.

DDT resistance and transformation by different microbial strains isolated from DDT-contaminated soils and compost materials

Bioremediation is a potential option to treat 1, 1, 1-trichloro-2, 2 bis (4-chlorophenyl) ethane (DDT) contaminated sites. In areas where suitable microbes are not present, the use of DDT resistant microbial inoculants may be necessary. It is vital that such inoculants do not produce recalcitrant breakdown products e.g. 1, 1-dichloro-2, 2-bis (4-chlorophenyl) ethylene (DDE). Therefore, this work aimed to screen DDT-contaminated soil and compost materials for the presence of DDT-resistant microbes for use as potential inoculants. Four compost amended soils, contaminated with different concentrations of DDT, were used to isolate DDT-resistant microbes in media containing 150 mg I -1 DDT at three temperatures (25, 37 and 55°C). In all soils, bacteria were more sensitive to DDT than actinomycetes and fungi. Bacteria isolated at 55°C from any source were the most DDT sensitive. However DDT-resistant bacterial strains showed more promise in degrading DDT than isolated fungal strains, as 1, 1-dichloro 2, 2-bis (4-chlorophenyl) ethane (DDD) was a major bacterial transformation product, while fungi tended to produce more DDE. Further studies on selected bacterial isolates found that the most promising bacterial strain (Bacillus sp. BHD-4) could remove 51% of DDT from liquid culture after 7 days growth. Of the amount transformed, 6% was found as DDD and 3% as DDE suggesting that further transformation of DDT and its metabolites occurred.

The impact of flood and post-flood cleaning on airborne microbiological and particle contamination in residential houses

In January 2011, Brisbane, Australia, experienced a major river flooding event. We aimed to investigate its effects on air quality and assess the role of prompt cleaning activities in reducing the airborne exposure risk. A comprehensive, multi-parameter indoor and outdoor measurement campaign was conducted in 41 residential houses, 2 and 6 months after the flood. The median indoor air concentrations of supermicrometer particle number (PN), PM10, fungi and bacteria 2 months after the flood were comparable to those previously measured in Brisbane. These were 2.88 p cm-3, 15 µg m-3, 804 cfu m-3 and 177 cfu m-3 for flood-affected houses (AFH), and 2.74 p cm-3, 15 µg m-3, 547 cfu m-3 and 167 cfu m-3 for non-affected houses (NFH), respectively. The I/O (indoor/outdoor) ratios of these pollutants were 1.08, 1.38, 0.74 and 1.76 for AFH and 1.03, 1.32, 0.83 and 2.17 for NFH, respectively. The average of total elements (together with transition metals) in indoor dust was 2296 ± 1328 µg m-2 for AFH and 1454 ± 678 µg m-2 for NFH, respectively. In general, the differences between AFH and NFH were not statistically significant, implying the absence of a measureable effect on air quality from the flood. We postulate that this was due to the very swift and effective cleaning of the flooded houses by 60,000 volunteers. Among the various cleaning methods, the use of both detergent and bleach was the most efficient at controlling indoor bacteria. All cleaning methods were equally effective for indoor fungi. This study provides quantitative evidence of the significant impact of immediate post-flood cleaning on mitigating the effects of flooding on indoor bioaerosol contamination and other pollutants.

Highly effective fungal inactivation in He+O2 atmospheric-pressure nonequilibrium plasmas

Highly effective (more than 99.9%) inactivation of a pathogenic fungus Candida albicans commonly found in oral, respiratory, digestive, and reproduction systems of a human body using atmospheric-pressure plasma jets sustained in He+ O2 gas mixtures is reported. The inactivation is demonstrated in two fungal culture configurations with open (Petri dish without a cover) and restricted access to the atmosphere (Petri dish with a cover) under specific experimental conditions. It is shown that the fungal inactivation is remarkably more effective in the second configuration. This observation is supported by the scanning and transmission electron microscopy of the fungi before and after the plasma treatment. The inactivation mechanism explains the experimental observations under different experimental conditions and is consistent with the reports by other authors. The results are promising for the development of advanced health care applications.

Use of the biolistic particle delivery system to transform fungal genomes

Biolistic delivery of transforming DNA into fungal genomes, especially when performed on uninucleate haploid conidia, has proven successful in bypassing the time-consuming repetitive purification of protoplasts used for the widely applied polyethylene glycol-mediated method. Biolistic transformation is also relatively quick compared to other available methods and provides a high percentage of stable transformants.

Entomopathogens for control of Hypsipyla spp

Published information on the incidence of pathogens in the field and laboratory infections of Hypsipyla spp. with entomopathogens is reviewed. In addition, some preliminary results of field collections from Ghana and Costa Rica are presented. Fungal pathogens from the Deuteromycetes have been isolated from both H. robusta Moore and H. grandella Zeller. Mermithid nematodes, Hexamermis spp., have been frequently isolated from larvae in the field and incidence of infection with these pathogens can reach significant levels. Microsporidia have been found in cadavers of larvae collected in the field but none have been identified so far. A number of pathogens of other Lepidoptera have been shown to be infectious to H. grandella , including Bacillus thuringiensis , Deuteromycete fungi and a nucleopolyhedrovirus (NPV) from Autographa californica . Hypsipyla spp. are difficult targets for microbial control, since the larvae are cryptic, occur at low density and occur sporadically. In addition, there is a low damage threshold, the plant is susceptible for a number of years and the susceptible part of the plant will rapidly outgrow any surface application. Key features of the biology of entomopathogens with relevance to the control of low density and cryptic pests are discussed. In the light of this experience, we discuss strategies to improve the possibilities of microbial control of this pest and suggest areas for research.