Nucleocapsid gene variability reveals two subgroups of Lettuce necrotic yellows virus

The complete nucleocapsid (N) genes of eight Australian isolates of Lettuce necrotic yellows virus (LNYV) were amplified by reverse transcription PCR, cloned and sequenced. Phylogenetic analyses of these sequences revealed two distinct subgroups of LNYV isolates. Nucleotide sequences within each subgroup were more than 96% identical but heterogeneity between groups was about 20% at the nucleotide sequence level. However, less than 4% heterogeneity was noted at the amino acid level, indicating mostly third nucleotide position changes and a strong conservation for N protein function. There was no obvious geographical or temporal separation of the subgroups in Australia.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy® or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to 1 infected in 800 samples with pepper but never detecting more than 1 infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Reclassification of [Pasteurella] trehalosi as Bibersteinia trehalosi gen. nov., comb. nov.

[Pasteurella] trehalosi is an important pathogen of sheep, being primarily associated with serious systemic infections in lambs but also having an association with pneumonia. The aim of the present investigation was to characterize a broad collection of strains tentatively identified as [P.] trehalosi in order to reclassify and rename this taxon to support improvements in our understanding of the pathogenesis and epidemiology of this important organism. The type strain for [P.] trehalosi, strain NCTC 10370T, was included along with 42 field isolates from sheep (21), cattle (14), goats (1), roe deer (3) and unknown sources (3). An extended phenotypic characterization was performed on all 43 strains. Amplified fragment length polymorphism (AFLP) was also performed on the isolates. Two of the field isolates were subjected to 16S rRNA gene sequencing. These sequences, along with five existing sequences for [P.] trehalosi strains and 12 sequences for other taxa in the family Pasteurellaceae, were subjected to a phylogenetic analysis. All the isolates and the reference strains were identified as [P.] trehalosi. A total of 17 out of 22 ovine isolates produced acid from all glycosides, while only four out of 14 bovine isolates produced acid from all glycosides. All 22 ovine isolates were haemolytic and CAMP-positive, while no other isolate was haemolytic and only two bovine isolates were CAMP-positive. Nineteen AFLP types were found within the [P.] trehalosi isolates. All [P.] trehalosi isolates shared at least 70% similarity in AFLP patterns. The largest AFLP type included the type strain and 7 ovine field isolates. Phylogenetic analysis indicated that the seven strains studied (two field isolates and the five serovar reference strains) are closely related, with 98.6% or higher 16S rRNA gene sequence similarity. As both genotypic and phenotypic testing support the separate and distinct nature of these organisms, we propose the transfer of [P.] trehalosi to a new genus, Bibersteinia, as Bibersteinia trehalosi comb. nov. The type strain is NCTC 10370T (=ATCC 29703T). Bibersteinia trehalosi can be distinguished from the existing genera of the family by the observation of only nine characteristics; catalase, porphyrin, urease, indole, phosphatase, acid from dulcitol, (+)-D-galactose, (+)-D mannose and (+)-D-trehalose.

Phylogeography of the pharaoh cuttle Sepia pharaonis based on partial mitochondrial 16S sequence data

The pharaoh cuttle Sepia pharaonis Ehrenberg, 1831 (Mollusca: Cephalopoda: Sepiida) is a broadly distributed species of substantial fisheries importance found from east Africa to southern Japan. Little is known about S. pharaonis phylogeography, but evidence from morphology and reproductive biology suggests that Sepia pharaonis is actually a complex of at least three species. To evaluate this possibility, we collected tissue samples from Sepia pharaonis from throughout its range. Phylogenetic analyses of partial mitochondrial 16S sequences from these samples reveal five distinct clades: a Gulf of Aden/Red Sea clade, a northern Australia clade, a Persian Gulf/Arabian Sea clade, a western Pacific clade (Gulf of Thailand and Taiwan) and an India/Andaman Sea clade. Phylogenetic analyses including several Sepia species show that S. pharaonis sensu lato may not be monophyletic. We suggest that "S. pharaonis" may consist of up to five species, but additional data will be required to fully clarify relationships within the S. pharaonis complex.

Kirramyces viscidus sp. nov., a new eucalypt pathogen from tropical Australia closely related to the serious leaf pathogen, Kirramyces destructans

Kirramyces destructans is a serious pathogen causing a leaf, bud and shoot blight disease of Eucalyptus plantations in the subtropics and tropics of South-East Asia. During surveillance of eucalypt taxa trials in northern Queensland, symptoms resembling those of K. destructans were observed on Eucalyptus grandis and E. grandis × E. camaldulensis. Phylogenetic and morphological studies revealed that the Kirramyces sp. associated with these symptoms represents a new taxon described here as K. viscidus sp. nov., which is closely related to K. destructans. Plantation assessments revealed that while E. grandis from the Copperload provenance, collected in northern Queensland, recovered from disease, E. grandis × E. camaldulensis hybrids from South America were highly susceptible to infection by K. viscidus and are not recommended for planting in northern Queensland. Preliminary results suggest the fungus probably originates from Australia. K. viscidus is closely related to K. destructans and causes a disease with similar symptoms, suggesting that it could seriously damage Australian eucalypt plantations, especially those planted off-site.

Three new Lasiodiplodia spp. from the tropics, recognized based on DNA sequence comparisons and morphology

Botryosphaeria rhodina (anamorph Lasiodiplodia theobromae) is a common endophyte and opportunistic pathogen on more than 500 tree species in the tropics and subtropics. During routine disease surveys of plantations in Australia and Venezuela several isolates differing from L. theobromae were identified and subsequently characterized based upon morphology and ITS and EF1-a nucleotide sequences. These isolates grouped into three strongly supported clades related to but different from the known taxa, B. rhodina and L. gonubiensis, These have been described here as three new species L. venezuelensis sp. nov., L. crassispora sp. nov. and L. rubropurpurea sp. nov. The three could be distinguished easily from each other and the two described species of Lasiodiplodia, thus confirming phylogenetic separations. Furthermore all five Lasiodiplodia spp. now recognized separated from Diplodia spp. and Dothiorella spp. with 100% bootstrap support.

Identification of viral and non-viral reverse transcribing elements in pineapple ( Ananas comosus ), including members of two new badnavirus species

A previously published partial sequence of pineapple bacilliform virus was shown to be from a retrotransposon (family Metaviridae) and not from a badnavirus as previously thought. Two newly discovered sequence groups isolated from pineapple were associated with bacilliform virions and were transmitted by mealybugs. Phylogenetic analyses indicated that they were members of new badnavirus species. A third caulimovirid sequence was also amplified from pineapple, but available evidence suggests that this DNA is not encapsidated, but more likely derived from an endogenous virus.

The genetic diversity of ampeloviruses in Australian pineapples and their association with mealybug wilt disease

Pineapple mealybug wilt-associated virus 1 (PMWaV-1), 2 (PMWaV-2) and -3 (PMWaV-3) have been detected in Australian commercial pineapple crops, along with a previously undescribed ampelovirus, for which the name Pineapple mealybug wilt-associated virus 5 (PMWaV-5) is proposed. Partial sequences extending from open reading frame 1b through to the heat shock protein homologue were obtained for PMWaV-1, -3 and -5. Phylogenetic analyses of selected regions of these sequences indicated that PMWaV-5 is a distinct species and most closely related to PMWaV-1. The amino acid sequence variation observed in the RNA-dependent RNA polymerase region of PMWaV-1 isolates was 95.8–98.4% and of PMWaV-3 isolates was 92.2–99.5%. In surveys of mealybug wilt disease (MWD) affected crops, none of the four viruses was clearly associated with the disease at all survey sites. A statistically significant association (P < 0.001) between the presence of PMWaV-2 and symptoms was observed at one survey site (site 3), but the virus was at a low incidence at the remaining three survey sites. By contrast, although PMWaV-1 and -3 were equally distributed between symptomless and MWD-affected plants at site 3, there was a statistically significant (P < 0.001) association between each of these two viruses and MWD at sites 1 and 4. At site 2, there was a statistically significant (P < 0.001) association only between PMWaV-3 and MWD. PMWaV-1 was the most commonly found of the four viruses and conversely PMWaV-5 was only occasionally found. Australian isolates of PMWaV-1, -2 and -3 were transmitted by the mealybug species Dysmicoccus brevipes.

Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea)

Background: Cultivated peanut or groundnut (Arachis hypogaea L.) is the fourth most important oilseed crop in the world, grown mainly in tropical, subtropical and warm temperate climates. Due to its origin through a single and recent polyploidization event, followed by successive selection during breeding efforts, cultivated groundnut has a limited genetic background. In such species, microsatellite or simple sequence repeat (SSR) markers are very informative and useful for breeding applications. The low level of polymorphism in cultivated germplasm, however, warrants a need of larger number of polymorphic microsatellite markers for cultivated groundnut. Results: A microsatellite- enriched library was constructed from the genotype TMV2. Sequencing of 720 putative SSR-positive clones from a total of 3,072 provided 490 SSRs. 71.2% of these SSRs were perfect type, 13.1% were imperfect and 15.7% were compound. Among these SSRs, the GT/CA repeat motifs were the most common (37.6%) followed by GA/CT repeat motifs (25.9%). The primer pairs could be designed for a total of 170 SSRs and were optimized initially on two genotypes. 104 (61.2%) primer pairs yielded scorable amplicon and 46 (44.2%) primers showed polymorphism among 32 cultivated groundnut genotypes. The polymorphic SSR markers detected 2 to 5 alleles with an average of 2.44 per locus. The polymorphic information content (PIC) value for these markers varied from 0.12 to 0.75 with an average of 0.46. Based on 112 alleles obtained by 46 markers, a phenogram was constructed to understand the relationships among the 32 genotypes. Majority of the genotypes representing subspecies hypogaea were grouped together in one cluster, while the genotypes belonging to subspecies fastigiata were grouped mainly under two clusters. Conclusion. Newly developed set of 104 markers extends the repertoire of SSR markers for cultivated groundnut. These markers showed a good level of PIC value in cultivated germplasm and therefore would be very useful for germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships in cultivated groundnut as well as related Arachis species.

Evolutionary relationships between bat coronaviruses and their hosts

Recent studies have suggested that bats are the natural reservoir of a range of coronaviruses (CoVs), and that rhinolophid bats harbor viruses closely related to the severe acute respiratory syndrome (SARS) CoV, which caused an outbreak of respiratory illness in humans during 2002-2003. We examined the evolutionary relationships between bat CoVs and their hosts by using sequence data of the virus RNA-dependent RNA polymerase gene and the bat cytochrome b gene. Phylogenetic analyses showed multiple incongruent associations between the phylogenies of rhinolophid bats and their CoVs, which suggested that host shifts have occurred in the recent evolutionary history of this group. These shifts may be due to either virus biologic traits or host behavioral traits. This finding has implications for the emergence of SARS and for the potential future emergence of SARS-CoVs or related viruses.

Phytogeny of the Quambalariaceae fam. nov., including important Eucalyptus pathogens in South Africa and Australia

The genus Quambalaria consists of plant-pathogenic fungi causing disease on leaves and shoots of species of Eucalyptus and its close relative, Corymbia. The phylogenetic relationship of Quambalaria spp., previously classified in genera such as Sporothrix and Ramularia, has never been addressed. It has, however, been suggested that they belong to the basidiomycete orders Exobasidiales or Ustilaginales. The aim of this study was thus to consider the ordinal relationships of Q. eucalypti and Q. pitereka using ribosomal LSU sequences. Sequence data from the ITS nrDNA were used to determine the phylogenetic relationship of the two Quambalaria species together with Fugomyces (= Cerinosterus) cyanescens. In addition to sequence data, the ultrastructure of the septal pores of the species in question was compared. From the LSU sequence data it was concluded that Quambalaria spp. and F. cyanescens form a monophyletic clade in the Microstromatales, an order of the Ustilaginomycetes. Sequences from the ITS region confirmed that Q. pitereka and Q. eucalypti are distinct species. The ex-type isolate of F. cyanescens, together with another isolate from Eucalyptus in Australia, constitute a third species of Quambalaria, Q. cyanescens (de Hoog & G.A. de Vries) Z.W. de Beer, Begerow & R. Bauer comb. nov. Transmission electron-microscopic studies of the septal pores confirm that all three Quambalaria spp. have dolipores with swollen lips, which differ from other members of the Microstromatales (i.e. the Microstromataceae and Volvocisporiaceae) that have simple pores with more or less rounded pore lips. Based on their unique ultrastructural features and the monophyly of the three Quambalaria spp. in the Microstromatales, a new family, Quambalariaceae Z.W. de Beer, Begerow & R. Bauer fam. nov., is described.

The genetic structure of Australasian green turtles (Chelonia mydas): Exploring the geographical scale of genetic exchange

Ecological and genetic studies of marine turtles generally support the hypothesis of natal homing, but leave open the question of the geographical scale of genetic exchange and the capacity of turtles to shift breeding sites. Here we combine analyses of mitochondrial DNA (mtDNA) variation and recapture data to assess the geographical scale of individual breeding populations and the distribution of such populations through Australasia. We conducted multiscale assessments of mtDNA variation among 714 samples from 27 green turtle rookeries and of adult female dispersal among nesting sites in eastern Australia. Many of these rookeries are on shelves that were flooded by rising sea levels less than 10 000 years (c. 450 generations) ago. Analyses of sequence variation among the mtDNA control region revealed 25 haplotypes, and their frequency distributions indicated 17 genetically distinct breeding stocks (Management Units) consisting either of individual rookeries or groups of rookeries in general that are separated by more than 500 km. The population structure inferred from mtDNA was consistent with the scale of movements observed in long-term mark-recapture studies of east Australian rookeries. Phylogenetic analysis of the haplotypes revealed five clades with significant partitioning of sequence diversity (Φ = 68.4) between Pacific Ocean and Southeast Asian/Indian Ocean rookeries. Isolation by distance was indicated for rookeries separated by up to 2000 km but explained only 12% of the genetic structure. The emerging general picture is one of dynamic population structure influenced by the capacity of females to relocate among proximal breeding sites, although this may be conditional on large population sizes as existed historically across this region.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3′ leader-N-4a(P)-4b-M-G-L-5′ trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus.

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.