Mitochondrial inheritance during a parasexual cycle in {\it Fusarium oxysporum\/} forma specialis {\it cubense\/}

Fusarium oxysporum is a diverse, asexual fungal species composed of both saprophytic and pathogenic members. The destructive phytopathogens are classified into formae speciales based on the host species and into vegetative compatibility groups (VCGs) based on the ability of two individuals to form heterokaryons. Parasexuality, a non-sexual mode of genetic exchange unique to some fungi has been demonstrated in the laboratory in Fusarium oxysporum f. sp. cubense (FOC). The goals of this dissertation were threefold: to ascertain whether mitochondrial (mt) markers can distinguish race differences in FOC; to determine genetic relatedness of VCGs in FOC based on a mt marker; and to discover the mode of mt inheritance during a parasexual cycle.^ Band patterns produced by electrophoresis of Hae III digested genomic DNA indicated that VCG differences, not race, could be discerned by mtDNA analysis. Primers were designed to amplify a mt intergenic locus which served as a molecular marker to screen 55 strains of FOC in 16 VCGs using both single strand conformational polymorphism and DNA sequencing. Based on homogeneity of the locus, strains were assigned to seven mitotypes, a classification unit which I introduced and found informative for grouping related VCGs.^ To determine the mode of mt inheritance during a parasexual cycle, strains in different mitotypes were paired. Mitochondrial inheritance in all hybrid progeny was found to be uniparental. I speculated that if a parasexual cycle occurs in nature there would be greater variation in the nuclear genome than the mt. This could produce multiple VCGs within a mitotype, a phenomenon observed in FOC. Based on these data, I concluded that parasexuality in nature may contribute to the diversity observed in Fusarium oxysporum. ^

The isolation, characterization, and application of WRKY genes as useful molecular markers in tropical trees

The improvement of tropical tree crops using conventional breeding methods faces challenges due to the length of time involved. Thus, like most crops, there is an effort to utilize molecular genetic markers in breeding programs to select for desirable agronomic traits. Known as marker assisted breeding or marker assisted selection, genetic markers associated with a phenotype of interest are used to screen and select material reducing the time necessary to evaluate candidates. As the focus of this research was improving disease resistance in tropical trees, the usefulness of the WRKY gene superfamily was investigated as candidates for generating useful molecular genetic markers. WRKY genes encode plant-specific transcriptional factors associated with regulating plants' responses to both biotic and abiotic stress. ^ One pair of degenerate primers amplified 48 WRKY gene fragments from three taxonomically distinct, economically important, tropical tree crop species: 18 from Theobroma cacao L., 21 from Cocos nucifera L. and 9 from Persea americana Mill. Several loci from each species were polymorphic because of single nucleotide substitutions present within a putative non-coding region of the loci. Capillary array electrophoresis-single strand conformational polymorphism (CAE-SSCP) mapped four WRKY loci onto a genetic linkage map of a T. cacao F2 population segregating for resistance to witches' broom disease. Additionally, PCR primers specific for four T. cacao loci successfully amplified WRKY loci from 15 members of the Byttneriae tribe. A method was devised to allow the reliable discrimination of alleles by CAE-SSCP using only the mobility assigned to the sample peaks. Once this method was validated, the diversity of three WRKY loci was evaluated in a germplasm collection of T. cacao . One locus displayed high diversity in the collection, with at least 18 alleles detected from mobility differences of the product peaks. The number of WRKY loci available within the genome, ease of isolation by degenerate PCR, codominant segregation demonstrated in the F2 population, and usefulness for screening germplasm collections and closely related wild species demonstrates that the WRKY superfamily of genes are excellent candidates for developing a number of genetic molecular markers for breeding purposes in tropical trees. ^

Anti-quorum sensing agents from south Florida medicinal plants and their attenuation of Pseudomonas aeruginosa pathogenicity

With the difficulty in treating recalcitrant infections and the growing resistance to antibiotics, new therapeutic modalities are becoming increasingly necessary. The interruption of bacterial quorum sensing (QS), or cell-cell communication is known to attenuate virulence, while limiting selective pressure toward resistance. This study initiates an ethnobotanically-directed search for QS inhibiting agents in south Florida medicinal plants. Fifty plants were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, six showed QS inhibition: Conocarpus erectus L. (Combretaceae), Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon viminalis (Sol.ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Combretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and Quercus virginiana Mill. (Fagaceae). These plants were further examined for their effects on the QS system and virulence of Pseudomonas aeruginosa, an intractable opportunistic pathogen responsible for morbidity and mortality in the immunocompromised patient. C. erectus, B. buceras, and C. viminalis were found to significantly inhibit multiple virulence factors and biofilm formation in this organism. Each plant presented a distinct profile of effect on QS genes and signaling molecules, suggesting varying modes of action. Virulence attenuation was observed with marginal reduction of bacterial growth, suggesting quorum quenching mechanisms unrelated to static or cidal effects. Extracts of these plants were also investigated for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. Results were evaluated in both toxin-based and infection-based assays with P. aeruginosa strains PA01 and PA14. Overall nematode mortality was reduced 50-90%. There was no indication of host toxicity, suggesting the potential for further development as anti-infectives. Using low-pressure chromatography and HPLC, two stereoisomeric ellagitannins, vescalagin and castalagin were isolated from an aqueous extract of C. erectus . Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to decrease signal production, QS gene expression, and virulence factor production in P. aeruginosa. This study introduces a potentially new therapeutic direction for the treatment of bacterial infections. In addition, this is the first report of vescalagin and castalagin being isolated from C. erectus, and the first report of ellagitannin activity on the QS system.

Diffusible Signal Factor (DSF)-dependent quorum sensing in pathogenic bacteria and its exploitation for disease control

Cell-to-cell signals of the Diffusible Signal Factor (DSF) family are cis-2-unsaturated fatty acids of differing chain length and branching pattern. DSF signalling has been described in diverse bacteria to include plant and human pathogens where it acts to regulate functions such as biofilm formation, antibiotic tolerance and the production of virulence factors. DSF family signals can also participate in interspecies signalling with other bacteria and interkingdom signaling such as with the yeast Candida albicans. Interference with DSF signalling may afford new opportunities for the control of bacterial disease. Such strategies will depend in part on detailed knowledge of the molecular mechanisms underlying the processes of signal synthesis, perception and turnover. Here, I review both recent progress in understanding DSF signalling at the molecular level and prospects for translating this knowledge into approaches for disease control.

Fungi associated with foliar diseases of wild and cultivated rice (Oryza spp.) in northern Queensland

During surveys of wild and cultivated rice in northern Queensland in 2014 and 2015, 92 fungal isolates were obtained from plants that were afflicted by foliar diseases, including the rice blast pathogen, Pyricularia oryzae, and the brown spot pathogen, Bipolaris oryzae. Seven species of Curvularia were found, viz. Curvularia aeria, C. alcornii, C. asianensis, C. clavata, C. lunata, C. muehlenbeckiae and an undescribed species. To remove uncertainty about the identity of the host plants from which the fungi were isolated, a DNA barcoding strategy was developed using regions of the chloroplast genome. Pathogenicity tests using wild rice isolates of P. oryzae indicated that many local rice varieties are susceptible to infection.

Status of resistance to phosphine in insect pests of stored products in Vietnam

In response to numerous reports of failures to control insect pests of stored products with phosphine in Vietnam, a national survey for resistance to this key fumigant was undertaken in 2009–2011. Data from a more limited survey undertaken by the authors in 2002 in northern Vietnam are also presented. Samples collected in the 2002 survey (Sitophilus oryzae, n=8; Tribolium castaneum, n=8) were tested using a full dose- response assay, while for the 2009–11 survey, F1 generations were tested for resistance with two discriminating dosages of phosphine to detect frequency of weak and strong resistance phenotypes. Compared with a susceptible reference strain, in 2002, resistance to phosphine was indicated in six T. castaneum samples but only two of S. oryzae. Resistance factor, however, did not exceed 2.8-fold in T. castaneum and 1.7 in S. oryzae indicating relatively low frequency and weak expression of resistance. In 2009–11 survey, 176 samples were collected from a range of food and feed storages along the supply chain and from all major regions of Vietnam (125 sites). Rhyzopertha dominica and S. oryzae were the most common species found infesting stored commodities. Resistance was detected at high frequency in all the species. Weak and strong resistance phenotype frequencies were, respectively: Cryptolestes ferrugineus (37 and 58%, n=19), R. dominica (1.5 and 97%, n=65), S. oryzae (34 and 59%, n=82) and T. castaneum (70 and 30%, n=10). Strong resistance phenotype was detected in all the major regions and all parts of the supply chain but frequency was the highest in central storages and animal feed establishments. The increase in frequency and strength of resistance to phosphine in the eight years between the two surveys has been rapid and dramatic. The survey demonstrates the threat of resistance to grain protection in Vietnam and highlights the need for training of fumigators, and the development and adoption of phosphine resistance management tactics nationally.

Real-Time PCR Assays for the Detection of Puccinia psidii

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and β-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

Plasmopara sphagneticolae sp. nov. (Peronosporales) on Sphagneticola (Asteraceae) in Australia

A specimen of downy mildew on leaves of Sphagneticola trilobata found in northern Queensland was identified by a systematic approach as a novel species of Plasmopara. A new species, Plasmopara sphagneticolae, is proposed for this specimen, which differs from other species of Plasmopara by morphology, host range, and sequence data from nuclear-ribosomal DNA and mitochondrial DNA. Plasmopara sphagneticolae, together with P. halstedii, are downy mildews found on host species in the tribe Heliantheae (Asteraceae). Plasmopara halstedii causes downy mildew on Helianthus annuus, and is not present on sunflower in Australia. Phylogenetic analysis of the large subunit region of ribosomal DNA showed that P. sphagneticolae was sister to P. halstedii on sunflower.