Quambalaria species associated with plantation and native eucalypts in Australia

This study aimed to determine which species of Quambalaria are associated with shoot blight symptoms on Corymbia spp. An additional aim was to determine the presence and impact of quambalaria shoot blight on Eucalyptus species used in plantation development in subtropical and tropical regions of eastern Australia. Surveys identified three Quambalaria spp. -Q. pitereka, Q. eucalypti and Q. cyanescens - from native and plantation eucalypts, as well as amenity plantings, including the first confirmed report of Q. eucalypti from Eucalyptus plantations in Australia. Symptom descriptions and morphological studies were coupled with phylogenetic studies using ITS rDNA sequence data. Quambalaria pitereka was the causal agent of blight symptoms on species and hybrids in the Corymbia complex. Quambalaria eucalypti was identified from Eucalyptus species and a single Corymbia hybrid. Quambalaria cyanescens was detected from native and plantation Corymbia spp.

The Evolution of Guanylyl Cyclases as Multidomain Proteins: Conserved Features of Kinase-Cyclase Domain Fusions

Guanylyl cyclases (GCs) are enzymes that generate cyclic GMP and regulate different physiologic and developmental processes in a number of organisms. GCs possess sequence similarity to class III adenylyl cyclases (ACs) and are present as either membrane-bound receptor GCs or cytosolic soluble GCs. We sought to determine the evolution of GCs using a large-scale bioinformatic analysis and found multiple lineage-specific expansions of GC genes in the genomes of many eukaryotes. Moreover, a few GC-like proteins were identified in prokaryotes, which come fused to a number of different domains, suggesting allosteric regulation of nucleotide cyclase activity Eukaryotic receptor GCs are associated with a kinase homology domain (KHD), and phylogenetic analysis of these proteins suggest coevolution of the KHD and the associated cyclase domain as well as a conservation of the sequence and the size of the linker region between the KHD and the associated cyclase domain. Finally, we also report the existence of mimiviral proteins that contain putative active kinase domains associated with a cyclase domain, which could suggest early evolution of the fusion of these two important domains involved in signa transduction.

Abaca bunchy top virus, a new member of the genus Babuvirus (family Nanoviridae)

Two isolates of a novel babuvirus causing "bunchy top" symptoms were characterised, one from abaca (Musa textilis) from the Philippines and one from banana (Musa sp.) from Sarawak (Malaysia). The name abacá bunchy top virus (ABTV) is proposed. Both isolates have a genome of six circular DNA components, each ca. 1.0-1.1 kb, analogous to those of isolates of Banana bunchy top virus (BBTV). However, unlike BBTV, both ABTV isolates lack an internal ORF in DNA-R, and the ORF in DNA-U3 found in some BBTV isolates is also absent. In all phylogenetic analyses of nanovirid isolates, ABTV and BBTV fall in the same clade, but on separate branches. However, ABTV and BBTV isolates shared only 79-81% amino acid sequence identity for the putative coat protein and 54-76% overall nucleotide sequence identity across all components. Stem-loop and major common regions were present in ABTV, but there was less than 60% identity with the major common region of BBTV. ABTV and BBTV were also shown to be serologically distinct, with only two out of ten BBTV-specific monoclonal antibodies reacting with ABTV. The two ABTV isolates may represent distinct strains of the species as they are less closely related to each other than are isolates of the two geographic subgroups (Asian and South Pacific) of BBTV.

Genetic analysis of foot-and-mouth disease virus serotype A of Indian origin and detection of positive selection and recombination in leader protease- and capsid-coding regions

The leader protease (L-pro) and capsid-coding sequences (P1) constitute approximately 3 kb of the foot-and-mouth disease virus (FMDV). We studied the phylogenetic relationship of 46 FMDV serotype A isolates of Indian origin collected during the period 1968-2005 and also eight vaccine strains using the neighbour-joining tree and Bayesian tree methods. The viruses were categorized under three major groups - Asian, Euro-South American and European. The Indian isolates formed a distinct genetic group among the Asian isolates. The Indian isolates were further classified into different genetic subgroups (<5% divergence). Post-1995 isolates were divided into two subgroups while a few isolates which originated in the year 2005 from Andhra Pradesh formed a separate group. These isolates were closely related to the isolates of the 1970s. The FMDV isolates seem to undergo reverse mutation or onvergent evolution wherein sequences identical to the ancestors are present in the isolates in circulation. The eight vaccine strains included in the study were not related to each other and belonged to different genetic groups. Recombination was detected in the L-pro region in one isolate (A IND 20/82) and in the VP1 coding 1D region in another isolate (A RAJ 21/96). Positive selection was identified at aa positions 23 in the L-pro (P<0.05; 0.046*) and at aa 171 in the capsid protein VP1 (P<0.01; 0.003**).

Epitypification and phylogeny of Colletotrichum acutatum J.H. Simmonds

Simmonds introduced Colletotrichum acutatum in 1965, validated in 1968, with a broad concept, as demonstrated by the selection of several type specimens from a range of hosts. This has created some confusion in the species concept and identification of C. acutatum. There are no viable ex-type cultures of C. acutatum and furthermore there are no existing cultures of C. acutatum on Carica papaya from the type locality in south-east Queensland. The application of molecular phylogenetic studies to isolates of C. acutatum is only meaningful if the taxonomy is stable and species are properly named. In order to clarify the species concept of C. acutatum, an isolate of Colletotrichum acutatum from Carica papaya from Yandina in Southeast Queensland (Australia) is designated as an epitype. A detailed morphological description is provided. Phylogenies based on a combined ITS and beta-tubulin gene analysis indicate that C. acutatum bears close phylogenetic affinities to C. gloeosporioides and C. capsici. Results also indicate that C. acutatum is monophyletic and there is a close relationship between the epitype and other Australian C. acutatum isolates from Carica papaya. Molecular data, however did not provide further evidence to properly elucidate the taxonomie affinities of C. acutatum especially the holotype and epitype. Our studies indicate that given the complexity of the genus Colletotrichum, there is a need to check previously described type specimens and redesign neotypes where necessary in order to clarify taxonomie uncertainties.

The complexities of knowing what it is you are trapping

The effectiveness of any trapping system is highly dependent on the ability to accurately identify the specimens collected. For many fruit fly species, accurate identification (= diagnostics) using morphological or molecular techniques is relatively straightforward and poses few technical challenges. However, nearly all genera of pest tephritids also contain groups of species where single, stand-alone tools are not sufficient for accurate identification: such groups include the Bactrocera dorsalis complex, the Anastrepha fraterculus complex and the Ceratitis FAR complex. Misidentification of high-impact species from such groups can have dramatic consequences and negate the benefits of an otherwise effective trapping program. To help prevent such problems, this chapter defines what is meant by a species complex and describes in detail how the correct identification of species within a complex requires the use of an integrative taxonomic approach. Integrative taxonomy uses multiple, independent lines of evidence to delimit species boundaries, and the underpinnings of this approach from both the theoretical speciation literature and the systematics/taxonomy literature are described. The strength of the integrative approach lies in the explicit testing of hypotheses and the use of multiple, independent species delimitation tools. A case is made for a core set of species delimitation tools (pre- and post-zygotic compatibility tests, multi-locus phylogenetic analysis, chemoecological studies, and morphometric and geometric morphometric analyses) to be adopted as standards by tephritologists aiming to resolve economically important species complexes. In discussing the integrative approach, emphasis is placed on the subtle but important differences between integrative and iterative taxonomy. The chapter finishes with a case study that illustrates how iterative taxonomy applied to the B. dorsalis species complex led to incorrect taxonomic conclusions, which has had major implications for quarantine, trade, and horticultural pest management. In contrast, an integrative approach to the problem has resolved species limits in this taxonomically difficult group, meaning that robust diagnostics are now available.

New species of Nanexila Winterton & Irwin and Taenogera Krober from Australia (Diptera: Therevidae)

Two new species are described in each of the closely related genera Nanexila Winterton & Irwin and Taenogera Krober. Nanexila atricauda sp. nov. and Nanexila jimrodmani sp. nov. are described. The phylogenetic placement and diagnostic characteristics of these new species and other species recently transferred to Nanexila are discussed. Taenogera luteola sp. nov. and Taenogera brunnea sp. nov. are distinctive species described from female specimens collected in Queensland. Taenogera is diagnosed in light of these new species and a revised key to species presented.

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.

Identification of two distinct bovine parainfluenza virus type 3 genotypes

The partial gene sequencing of the matrix (M) protein from seven clinical isolates of bovine parainfluenza virus type 3 (BPIV-3), and the complete sequencing of a representative isolate (Q5592) was completed in this study. Nucleotide sequence analysis was initiated because of the failure of in-house BPIV-3 RT-PCR methods to yield expected products for four of the isolates. Phylogenetic reconstructions based on the nucleotide sequences for the M-protein and the entire genome, using all of the available BPIV-3 nucleotide sequences, demonstrated that there were two distinct BPIV-3 genotypes (BPIV-3a and BPIV-3b). These newly identified genotypes have implications for the development of BPIV-3 molecular detection methods and may also impact on BPIV-3 vaccine formulations.

Australian Bat Lyssavirus

In Chapter 1, the literature relating to rabies virus and the rabies like lyssaviruses is reviewed. In Chapter 2, data are presented from 1170 diagnostic submissions for ABLV testing by fluorescent antibody test (Centocor FAT). All 27 non-bat submissions were ABLV-negative. Of 1143 bat accessions 74 (16%) were ABLV-positive, including 69 of 974 (7.1%) flying foxes (Pteropus spp.), 5 of 7 (71.4%) Saccolaimus flaviventris (Yellow-bellied sheathtail bats), none of 151 other microchiropteran bats, and none of 11 unidentified bats. Statistical analysis of data from 868 wild Black, Grey-headed, Little Red and Spectacled flying foxes (Pteropus alecto, P. poliocephalus, P. scapulatus, and P. conspicillatus) indicated that three factors; species, health status and age were associated with significant (p< 0.001) differences in the proportion of ABLV-positive bats. Other factors including sex, whether the bat bit a person or animal, region, year, and season submitted, were not associated with ABLV. Case data for 74 ABLV-positive bats, including the circumstances in which they were found and clinical signs, is presented. In Chapter 3, the aetiological diagnosis was investigated for 100 consecutive flying fox submissions with neurological signs. ABLV (32%), spinal and head injuries (29%), and neuro-angiostrongylosis (18%) accounted for most neurological syndromes in flying foxes. No evidence of lead poisoning was found in unwell (n=16) or healthy flying foxes (n=50). No diagnosis was reached for 16 cases, all of which were negative for ABLV by TaqMan PCR. The molecular diversity of ABLV was examined in Chapter 4 by sequencing 36 bases of the leader sequence, the entire N gene, and start of the P gene of 28 isolates from pteropid bats and 3 isolates from Yellow-bellied sheathtail (YBST) bats. Phylogenetic analysis indicated all ABLV isolates clustered together as a discrete group within the Lyssavirus genera closely related to rabies virus and European bat lyssavirus-2 isolates. The ABLV lineage consisted of two variants; one (ybst-ABLV) consisted of isolates only from YBST bats, the other (pteropid-ABLV) was common to Black, Grey-headed and Little Red flying foxes. No associations were found between the sequences and either the geographical location or year found, or individual flying fox species. In Chapter 5, 15 inocula prepared from the brains or salivary glands of naturally-infected bats were evaluated by intracerebral (IC) and footpad (FP) inoculation of Quackenbush mice in order to select and characterize a highly virulent inoculum for further use in bats (Inoculum 5). In Chapter 6, nine Grey-headed flying foxes were inoculated with 105.2 to 105.5 MICED50 of Inoculum 5 divided into four sites, left footpad, pectoral muscle, temporal muscle and muzzle. Another bat was inoculated with half this dose divided into the footpad and pectoral muscle only. Seven of 10 bats developed clinical disease of 1 to 4 days duration between PI-days 10 and 19 and were shown to be ABL-positive by FAT, HAM immunoperoxidase staining, virus isolation in mice, and TaqMan PCR. Five of the seven bats displayed overt aggression, one died during a seizure, and one showed intractable agitation, pacing, tremors, and ataxia. Viral antigen was demonstrated throughout the central and peripheral nervous systems and in the epithelial cells of the submandibular salivary glands (n=4). All affected bats had mild to moderate non-suppurative meningoencephalitis and severe ganglioneuritis. No ABLV was detected in three bats that remained well until the end of the experiment on day 82. One survivor developed a strong but transient antibody response. In Chapter 7, the relative virulence of inocula prepared from the brains and salivary glands of experimentally infected flying foxes was evaluated in mice by IC and FP inoculation and TaqMan assay. The effects in mice were correlated to the TaqMan CT value and indicated a crude association between virulence and CT value that has potential application in the selection of inocula. In Chapter 8, 36 Black and Grey-headed flying foxes were vaccinated with one (day 0) or two (+ day 28) doses of Nobivac rabies vaccine and co-vaccinated with keyhole limpet haemocyanin (KLH). All bats responded to the Nobivac vaccine with a rabies-RFFIT titer > 0.5 IU/mL that is nominally indicative of protective immunity. Plasma from bats with rabies titres >2 IU/mL had cross-neutralising ABLV titres >1:154. A specifically developed ELISA detected a strong but transient response to KLH.

Refined Global Analysis of Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae) Mitochondrial Cytochrome Oxidase 1 to Identify Species Level Genetic Boundaries.

Identifying species boundaries within morphologically indistinguishable cryptic species complexes is often contentious. For the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae), the lack of a clear understanding about the genetic limits of the numerous genetic groups and biotypes so far identified has resulted in a lack of consistency in the application of the terms, the approaches use to apply them and in our understanding of what genetic structure within B. tabaci means. Our response has been to use mitochondrial gene cytochrome oxidase one to consider how to clearly and consistently define genetic separation. Using Bayesian phylogenetic analysis and analysis of sequence pairwise divergence we found a considerably higher to number of genetic groups than had been previously determined with two breaks in the distribution, one at 11% and another at 3.5%. At >11% divergence, 11 distinct groups were resolved, whereas at >3.5% divergence 24 groups were identified. Consensus sequences for each of these groups were determined and were shown to be useful in the correct assignment of sequences of unknown origin. The 3.5% divergence bound is consistent with species level separations in other insect taxa and Suggests that B. tabaci is it cryptic species composed of at least 24 distinct species. We further show that the placement of Bemesia atriplex (Froggatt) within the B. tabaci in, group adds further weight to the argument for species level separation within B. tabaci. This new analysis, which constructs consensus sequences and uses these its a standard against which unknown sequences call be compared, provides for the first time it consistent means of identifying the genetic hounds of each species with it high degree of certainty.

Spectral analysis of pair-correlation bandwidth: Application to cell biology images

Images from cell biology experiments often indicate the presence of cell clustering, which can provide insight into the mechanisms driving the collective cell behaviour. Pair-correlation functions provide quantitative information about the presence, or absence, of clustering in a spatial distribution of cells. This is because the pair-correlation function describes the ratio of the abundance of pairs of cells, separated by a particular distance, relative to a randomly distributed reference population. Pair-correlation functions are often presented as a kernel density estimate where the frequency of pairs of objects are grouped using a particular bandwidth (or bin width), Δ>0. The choice of bandwidth has a dramatic impact: choosing Δ too large produces a pair-correlation function that contains insufficient information, whereas choosing Δ too small produces a pair-correlation signal dominated by fluctuations. Presently, there is little guidance available regarding how to make an objective choice of Δ. We present a new technique to choose Δ by analysing the power spectrum of the discrete Fourier transform of the pair-correlation function. Using synthetic simulation data, we confirm that our approach allows us to objectively choose Δ such that the appropriately binned pair-correlation function captures known features in uniform and clustered synthetic images. We also apply our technique to images from two different cell biology assays. The first assay corresponds to an approximately uniform distribution of cells, while the second assay involves a time series of images of a cell population which forms aggregates over time. The appropriately binned pair-correlation function allows us to make quantitative inferences about the average aggregate size, as well as quantifying how the average aggregate size changes with time.

Characterization of the mitochondrial active-site serine protein LACTB: filaments in the mitochondrial intermembrane space

Mitochondria have evolved from endosymbiotic alpha-proteobacteria. During the endosymbiotic process early eukaryotes dumped the major component of the bacterial cell wall, the peptidoglycan layer. Peptidoglycan is synthesized and maintained by active-site serine enzymes belonging to the penicillin-binding protein and the β-lactamase superfamily. Mammals harbor a protein named LACTB that shares sequence similarity with bacterial penicillin-binding proteins and β-lactamases. Since eukaryotes lack the synthesis machinery for peptidoglycan, the physiological role of LACTB is intriguing. Recently, LACTB has been validated in vivo to be causative for obesity, suggesting that LACTB is implicated in metabolic processes. The aim of this study was to investigate the phylogeny, structure, biochemistry and cell biology of LACTB in order to elucidate its physiological function. Phylogenetic analysis revealed that LACTB has evolved from penicillin binding-proteins present in the bacterial periplasmic space. A structural model of LACTB indicates that LACTB shares characteristic features common to all penicillin-binding proteins and β-lactamases. Recombinat LACTB protein expressed in E. coli was recovered in significant quantities. Biochemical and cell biology studies showed that LACTB is a soluble protein localized in the mitochondrial intermembrane space. Further analysis showed that LACTB preprotein underwent proteolytic processing disclosing an N-terminal tetrapeptide motif also found in a set of cell death-inducing proteins. Electron microscopy structural studies revealed that LACTB can polymerize to form stable filaments with lengths ranging from twenty to several hundred nanometers. These data suggest that LACTB filaments define a distinct microdomain in the intermembrane space. A possible role of LACTB filaments is proposed in the intramitochondrial membrane organization and microcompartmentation. The implications of these findings offer novel insight into the evolution of mitochondria. Further studies of the LACTB function might provide a tool to treat mitochondria-related metabolic diseases.

Two novel mastreviruses from chickpea (Cicer arietinum) in Australia

Two novel mastreviruses (genus Mastrevirus; family Geminiviridae), with proposed names chickpea chlorosis virus (CpCV) and chickpea redleaf virus, are described from chickpea (Cicer arietinum) from eastern Australia. The viruses have genomes of 2,582 and 2,605 nucleotides, respectively, and share similar features and organisation with typical dicot-infecting mastreviruses. Two distinct strains of CpCV were suggested by phylogenetic analysis. Additionally, a partial mastrevirus Rep sequence from turnip weed (Rapistrum rugosum) indicated the presence of a distinct strain of Tobacco yellow dwarf virus (TYDV). In phylogenetic analyses, isolates of Bean yellow dwarf virus, Chickpea chlorotic dwarf Pakistan virus and Chickpea chlorotic dwarf Sudan virus from southern and northern Africa and south-central and western Asia clustered separately from these three viruses from Australia. An Australian, eastern Asian, or south-eastern Asian origin for the novel mastreviruses and TYDV is discussed.

Bromus catharticus striate mosaic virus: a new mastrevirus infecting Bromus catharticus from Australia.

Although monocotyledonous-plant-infecting mastreviruses (in the family Geminiviridae) are known to cause economically significant crop losses in certain areas of the world, in Australia, they pose no obvious threat to agriculture. Consequently, only a few Australian monocot-infecting mastreviruses have been described, and only two have had their genomes fully sequenced. Here, we present the third full-genome sequence of an Australian monocot-infecting mastrevirus from Bromus catharticus belonging to a distinct species, which we have tentatively named Bromus catharticus striate mosaic virus (BCSMV). Although the genome of this new virus shares only 57.7% sequence similarity with that of its nearest known relative, Digitaria didactyla striate mosaic virus (DDSMV; also from Australia), it has features typical of all other known mastrevirus genomes. Phylogenetic analysis showed that both the full genome and each of its probable expressed proteins group with the two other characterised Australian monocot-infecting mastreviruses. Besides the BCSMV genome sequence revealing that Australian monocot-infecting mastrevirus diversity rivals that seen in Africa, it has enabled us, for the first, to time detect evidence of recombination amongst the Australian viruses. Specifically, it appears that DDSMV possesses a short intergenic region sequence that has been recombinationally derived from either BCSMV or a close relative that has not yet been identified.