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.

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.

Phylogenetic considerations for predicting the host range of Ustilago sporoboli-indici, a potential biological control agent for Sporobolus species in Australia

The ribosomal DNA internal transcribed spacer region was amplified and sequenced from a selection of specimens of the Sporobolus smut Ustilago sporoboli-indici. Phylogenetic comparison with other Ustilago and Sporisorium species revealed strong support for an evolutionary radiation of Ustilago species infecting the Chloridoideae and Pooideae, of which U. sporoboli-indici forms a major lineage. Comparisons are made with other groups of plant pathogenic fungi, and it is concluded that phylogenetic analyses of potential biocontrol agents are useful for identifying pathogens that are derived from evolutionary lineages that parasitize a wide range of unrelated plants. Such pathogens are less desirable as biocontrol agents as they may have a greater likelihood of infecting plants outside their normal host ranges.

Host recognition by a polyphagous lepidopteran (Helicoverpa armigera): Primary host plants, host produced volatiles and neurosensory stimulation

An important question in the host-finding behaviour of a polyphagous insect is whether the insect recognizes a suite or template of chemicals that are common to many plants? To answer this question, headspace volatiles of a subset of commonly used host plants (pigeon pea, tobacco, cotton and bean) and nonhost plants (lantana and oleander) of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) are screened by gas chromatography (GC) linked to a mated female H. armigera electroantennograph (EAG). In the present study, pigeon pea is postulated to be a primary host plant of the insect, for comparison of the EAG responses across the test plants. EAG responses for pigeon pea volatiles are also compared between females of different physiological status (virgin and mated females) and the sexes. Eight electrophysiologically active compounds in pigeon pea headspace are identified in relatively high concentrations using GC linked to mass spectrometry (GC-MS). These comprised three green leaf volatiles [(2E)-hexenal, (3Z)-hexenylacetate and (3Z)-hexenyl-2-methylbutyrate] and five monoterpenes (α-pinene, β-myrcene, limonene, E-β-ocimene and linalool). Other tested host plants have a smaller subset of these electrophysiologically active compounds and even the nonhost plants contain some of these compounds, all at relatively lower concentrations than pigeon pea. The physiological status or sex of the moths has no effect on the responses for these identified compounds. The present study demonstrates how some host plants can be primary targets for moths that are searching for hosts whereas the other host plants are incidental or secondary targets.

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**).

Drosophila melanogaster mounts a unique immune response to the rhabdovirus Sigma virus

Rhabdoviruses are important pathogens of humans, livestock, and plants that are often vectored by insects. Rhabdovirus particles have a characteristic bullet shape with a lipid envelope and surface-exposed transmembrane glycoproteins. Sigma virus (SIGMAV) is a member of the Rhabdoviridae and is a naturally occurring disease agent of Drosophila melanogaster. The infection is maintained in Drosophila populations through vertical transmission via germ cells. We report here the nature of the Drosophila innate immune response to SIGMAV infection as revealed by quantitative reverse transcription-PCR analysis of differentially expressed genes identified by microarray analysis. We have also compared and contrasted the immune response of the host with respect to two nonenveloped viruses, Drosophila C virus (DCV) and Drosophila X virus (DXV). We determined that SIGMAV infection upregulates expression of the peptidoglycan receptor protein genes PGRP-SB1 and PGRP-SD and the antimicrobial peptide (AMP) genes Diptericin-A, Attacin-A, Attacin-B, Cecropin-A1, and Drosocin. SIGMAV infection did not induce PGRP-SA and the AMP genes Drosomycin-B, Metchnikowin, and Defensin that are upregulated in DCV and/or DXV infections. Expression levels of the Toll and Imd signaling cascade genes are not significantly altered by SIGMAV infection. These results highlight shared and unique aspects of the Drosophila immune response to the three viruses and may shed light on the nature of the interaction with the host and the evolution of these associations.

Review of bats and SARS

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, including henipaviruses and variants of rabies viruses. Recently, we and another group independently identified several horse-shoe bat species (genus Rhinolophus) as the reservoir host for a large number of viruses that have a close genetic relationship with the coronavirus associated with severe acute respiratory syndrome (SARS). Our current research focused on the identification of the reservoir species for the progenitor virus of the SARS coronaviruses responsible for outbreaks during 2002-2003 and 2003-2004. In addition to SARS-like coronaviruses, many other novel bat coronaviruses, which belong to groups 1 and 2 of the 3 existing coronavirus groups, have been detected by PCR. The discovery of bat SARS-like coronaviruses and the great genetic diversity of coronaviruses in bats have shed new light on the origin and transmission of SARS coronaviruses.

Pimelea trichostachya poisoning (St George disease) in horses

A dense population of Pimelea trichostachya plants (Family Thymelaeaceae) in pasture poisoned a horse herd in southern inland Queensland in October-November 2005. Plant density was 2 to 45 g wet weight/m2 (mean 16 g/m2) from 5 to 69 plants/m2 (mean 38 plants/m2) representing 3 to 20% (mean 9%) of the volume of pasture on offer. Ten of 35 mares, fillies and geldings were affected. Clinical signs were loss of body weight, profound lethargy, serous nasal discharge, severe watery diarrhoea and subcutaneous oedema of the intermandibular space, chest and ventral midline. Pathological findings were anaemia, leucocytopenia, hypoproteinaemia, dilatation of the right ventricle of the heart, dilated hepatic portal veins and periportal hepatic sinusoids (peliosis hepatis), alimentary mucosal hyperaemia and oedema of mesenteric lymph nodes. Cattle grazing the same pasture were affected by Pimelea poisoning simultaneously. Removal of the horses to Pimelea-free pasture initiated recovery. The one other incident of this syndrome, previously only recognised in cattle in Australia, occurred in horses, in South Australia in 2002, with access to a dense Pimelea simplex population.

Host-plant resistance and biopesticides: Ingredients for successful integrated pest management (IPM) in Australian sorghum production

There are two major pests of sorghum in Australia, the sorghum midge, Stenodiplosis sorghicola (Coquillett), and the corn earworm, Helicoverpa armigera (Hübner). During the past 10 years the management of these pests has undergone a revolution, due principally to the development of sorghum hybrids with resistance to sorghum midge. Also contributing has been the adoption of a nucleopolyhedrovirus for the management of corn earworm. The practical application of these developments has led to a massive reduction in the use of synthetic insecticides for the management of major pests of sorghum in Australia. These changes have produced immediate economic, environmental and social benefits. Other flow-on benefits include providing flexibility in planting times, the maintenance of beneficial arthropods and utilisation of sorghum as a beneficial arthropod nursery, a reduction in midge populations and a reduction in insecticide resistance development in corn earworm. Future developments in sorghum pest management are discussed.

Host plant resistance in grain crops and prospects for invertebrate pest management in Australia: An overview

An integrated pest management (IPM) approach that relies on an array of tactics is adopted commonly in response to problems with pesticide-based production in many agricultural systems. Host plant resistance is often used as a fundamental component of an IPM system because of the generally compatible, complementary role that pest-resistant crops play with other tactics. Recent research and development in the resistance of legumes and cereals to aphids, sorghum midge resistance, and the resistance of canola varieties to mite and insect pests have shown the prospects of host plant resistance for developing IPM strategies against invertebrate pests in Australian grain crops. Furthermore, continuing advances in biotechnology provide the opportunity of using transgenic plants to enhance host plant resistance in grains.

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.

Tan spot: A new disease of pyrethrum caused by Microsphaeropsis tanaceti sp. nov

The isolation frequency of Microsphaeropsis sp. in spring in association with necrotic lesions on leaves in Tasmanian pyrethrum (Tanacetum cinerariifolium) fields has increased substantially since first identification in 2001. Examination of morphological features and sequencing of the internal transcribed spacer region (ITS) resulted in the identification of a new species, herein described as Microsphaeropsis tanaceti sp. nov. The pathogenicity of three M. tanaceti isolates to two pyrethrum cultivars was confirmed by inoculating glasshouse-grown plants in three experiments. No significant differences in the susceptibility of the two cultivars to infection by M. tanaceti were found. Symptoms were tan-coloured spots which coalesced around the margins of the leaves. Therefore, the name 'tan spot' is proposed for this new disease of pyrethrum. The sensitivity of seven M. tanaceti isolates to difenoconazole and azoxystrobin, commonly used fungicides for the management of foliar diseases in spring, was assessed under in vitro conditions. Sensitivity testing for difenoconazole was conducted using a mycelial growth assay on potato dextrose agar, whilst testing for sensitivity to azoxystrobin used a conidial germination assay on water agar. Microsphaeropsis tanaceti was found to be more sensitive to azoxystrobin than difenoconazole, with complete inhibition of conidial germination at concentrations above 0.625 µg a.i. mL-1. By comparison, concentrations of 50 µg a.i. difenoconazole mL-1 or greater were required for significant inhibition of mycelial growth. It therefore appears likely that there is currently some control of tan spot as a result of the use of azoxystrobin and to a lesser extent, difenoconazole, for the control of other diseases.

The essential and non-essential genes of Bovine herpesvirus 1

Bovine herpesvirus 1 (BoHV-1) is an economically important pathogen of cattle associated with respiratory and reproductive disease. To further develop BoHV-1 as a vaccine vector, a study was conducted to identify the essential and non-essential genes required for in vitro viability. Randominsertion mutagenesis utilizing a Tn5 transposition system and targeted gene deletion were employed to construct gene disruption and gene deletion libraries, respectively, of an infectious clone of BoHV-1. Transposon insertion position and confirmation of gene deletion were determined by direct sequencing. The essential or non-essential requirement of either transposed or deleted open reading frames (ORFs) was assessed by transfection of respective BoHV-1 DNA into host cells. Of the 73 recognized ORFs encoded by the BoHV-1 genome, 33 were determined to be essential and 36 to be non-essential for virus viability in cell culture; determining the requirement of the two dual copy ORFs was inconclusive. The majority of ORFs were shown to conform to the in vitro requirements of BoHV-1 homologues encoded by human herpesvirus 1 (HHV-1). However, ORFs encoding glycoprotein K (UL53), regulatory, membrane, tegument and capsid proteins (UL54, UL49.5, UL49, UL35, UL20, UL16 and UL7) were shown to differ in requirement when compared to HHV-1-encoded homologues.

Pain in juvenile idiopathic arthritis : parents and children as agents of disease management

Juvenile idiopathic arthritis (JIA) is a severe childhood disease usually characterized by long-term morbidity, unpredictable course, pain, and limitations in daily activities and social participation. The disease affects not only the child but also the whole family. The family is expected to adhere to an often very laborious regimen over a long period of time. However, the parental role is incoherently conceptualized in the research field. Pain in JIA is of somatic origin, but psychosocial factors, such as mood and self-efficacy, are critical in the perception of pain and in its impact on functioning. This study examined the factors correlating and possibly explaining pain in JIA, with a special emphasis on the mutual relations between parent- and patient-driven variables. In this patient series pain was not associated with the disease activity. The degree of pain was on average fairly low in children with JIA. When the children were clustered according to age, anxiety and depression, four distinguishable cluster groups significantly associated with pain emerged. One of the groups was described by concept vulnerability because of unfavorable variable associations. Parental depressive and anxiety symptoms accompanied by illness management had a predictive power in discriminating groups of children with varying distress levels. The parent’s and child’s perception of a child’s functional capability, distress, and somatic self-efficacy had independent explanatory power predicting the child’s pain. Of special interest in the current study was self-efficacy, which refers to the belief of an individual that he/she has the ability to engage in the behavior required for tackling the disease. In children with JIA, strong self-efficacy was related to lower levels of pain, depressive symptoms and trait anxiety. This suggests strengthening a child’s sense of self-efficacy, when helping the child to cope with his or her disease. Pain experienced by a child with JIA needs to be viewed in a multidimensional bio-psycho-social context that covers biological, environmental and cognitive behavioral mechanisms. The relations between the parent-child variables are complex and affect pain both directly and indirectly. Developing pain-treatment modalities that recognize the family as a system is also warranted.

Black Sigatoka disease: new technologies to strengthen eradication strategies in Australia

In 2001, an incursion of Mycosphaerella fijiensis, the causal agent of black Sigatoka, was detected in Australia's largest commercial banana growing region, the Tully Banana Production Area in North Queensland. An intensive surveillance and eradication campaign was undertaken which resulted in the reinstatement of the disease-free status for black Sigatoka in 2005. This was the first time black Sigatoka had ever been eradicated from commercial plantations. The success of the eradication campaign was testament to good working relationships between scientists, growers, crop monitors, quarantine regulatory bodies and industry. A key contributing factor to the success was the deployment of a PCR-based molecular diagnostic assay, developed by the Cooperative Research Centre for Tropical Plant Protection (CRCTPP). This assay complemented morphological identification and allowed high throughput diagnosis of samples facilitating rapid decision-making during the eradication campaign. This paper describes the development and successful deployment of molecular diagnostics for black Sigatoka. Shortcomings in the gel-based assay are discussed and the advantages of highly specific real-time PCR assays, capable of differentiating between Mycosphaerella fijiensis, Mycosphaerella musicola and Mycosphaerella eumusae are outlined. Real-time assays may provide a powerful diagnostic tool for applications in surveillance, disease forecasting and resistance testing for Sigatoka leaf spot diseases.