Four Tulasnella taxa associated with populations of the Australian evergreen terrestrial orchid Cryptostylis ovata.

Of the more than 400 indigenous orchid species in Western Australia, Cryptostylis ovata is the only species that retains its leaves all year round. It exists as a terrestrial herb and occasionally as an epiphyte in forested areas. Like all terrestrial orchids, C. ovata plants associate with mycorrhizal fungi, but their identities have not previously been investigated. Fungi were isolated from pelotons in rhizomes collected from three southern and two northern populations of C. ovata on six occasions over two years. Phylogenetic analysis of ITS sequences temporally and spatially revealed that all the fungal isolates were of Tulasnella species of four distinct groups. One Tulasnella group was present only in the three southern orchid populations, and it closely resembled T. prima isolates previously described from Chiloglottis sp. orchids from eastern Australia. Isolates collected from plants in the two northern populations were of three undescribed Tulasnella groups. Analysis of intra-group diversity using inter-simple sequence repeat markers revealed that plants were usually colonised by a single genotype of Tulasnella at each sampling period, and this genotype usually, but not always, persisted with the host plant over both years tested.

A virome from ornamental flowers in an Australian rural town.

Samples of leaves exhibiting symptoms resembling those caused by virus infection were collected from ornamental street flowers in a rural town in Western Australia. Thirty-seven leaf samples were collected from plants of iris, tulip, lily, daffodil, stock and grape hyacinth. Shotgun sequencing of cDNA derived from leaf samples was done, and analysis showed that about 6% of the sequences obtained were of viral origin. Assembly of virus-like sequences revealed complete or partial genome sequences of 13 virus isolates representing 11 virus species. Eight of the isolates were of potyviruses, one was of a macluravirus, three were of potexviruses, and one was of a bunya-like virus. The complete genome of an isolate originally classified as ornithogalum mosaic virus was genetically divergent and differed in polyprotein cleavage motifs, and we propose that this isolate represents a distinct species. The implications of importing to Australia live plant propagules infected with viruses are discussed.

Endovascular aneurysm repair: a perioperative perspective.

Endovascular aneurysm repair (EVAR), has surpassed open repair as the technique of choice in many centres in response to several large studies which showed significantly improved 30-day mortality. While several multicentre EVAR trials looked at surgical outcomes, very few have specifically investigated the effect of anaesthetic techniques or perioperative care of these patients. The purpose of this review to is to present some of the current evidence for the different aspects of perioperative management of patients undergoing EVAR. This includes surgical considerations, pre-operative assessment, and choice of anaesthetic technique as well as pharmacological protective strategies.

Zucchini yellow mosaic virus: biological properties, detection procedures and comparison of coat protein gene sequences.

Between 2006 and 2010, 5324 samples from at least 34 weed, two cultivated legume and 11 native species were collected from three cucurbit-growing areas in tropical or subtropical Western Australia. Two new alternative hosts of zucchini yellow mosaic virus (ZYMV) were identified, the Australian native cucurbit Cucumis maderaspatanus, and the naturalised legume species Rhyncosia minima. Low-level (0.7%) seed transmission of ZYMV was found in seedlings grown from seed collected from zucchini (Cucurbita pepo) fruit infected with isolate Cvn-1. Seed transmission was absent in >9500 pumpkin (C. maxima and C. moschata) seedlings from fruit infected with isolate Knx-1. Leaf samples from symptomatic cucurbit plants collected from fields in five cucurbit-growing areas in four Australian states were tested for the presence of ZYMV. When 42 complete coat protein (CP) nucleotide (nt) sequences from the new ZYMV isolates obtained were compared to those of 101 complete CP nt sequences from five other continents, phylogenetic analysis of the 143 ZYMV sequences revealed three distinct groups (A, B and C), with four subgroups in A (I-IV) and two in B (I-II). The new Australian sequences grouped according to collection location, fitting within A-I, A-II and B-II. The 16 new sequences from one isolated location in tropical northern Western Australia all grouped into subgroup B-II, which contained no other isolates. In contrast, the three sequences from the Northern Territory fitted into A-II with 94.6-99.0% nt identities with isolates from the United States, Iran, China and Japan. The 23 new sequences from the central west coast and two east coast locations all fitted into A-I, with 95.9-98.9% nt identities to sequences from Europe and Japan. These findings suggest that (i) there have been at least three separate ZYMV introductions into Australia and (ii) there are few changes to local isolate CP sequences following their establishment in remote growing areas. Isolates from A-I and B-II induced chlorotic symptoms in inoculated leaves of Chenopodium quinoa, but an isolate from A-II caused symptomless infection. One of three commercial ZYMV-specific antibodies did not detect all Australian isolates reliably by ELISA. A multiplex real-time PCR using dual-labelled probes was developed, which distinguished between Australian ZYMV isolates belonging to phylogenetic groups A-I, A-II and B-II.

Genetic variability of the coat protein sequence of pea seed-borne mosaic virus isolates and the current relationship between phylogenetic placement and resistance groups.

Nucleotide sequences of complete or partial coat protein (CP) genes were determined for 11 isolates of pea seed-borne mosaic virus (PSbMV) from Australia and one from China, and compared with known sequences of 20 other isolates. On phylogenetic analysis, the isolates from Australia and China grouped into 2 of 3 clades. Clade A contained three sub-clades (Ai, Aii and Aiii), Australian isolates were in Ai or Aiii, and the Chinese isolate in Aii. Clade A contained isolates in pathotypes P-1, P-2 and U-2; clade B, one isolate in P-2; and clade C, only isolates in P-4.

Narcissus late season yellows virus and Vallota speciosa virus found infecting domestic and wild populations of Narcissus species in Australia.

Isolates of Narcissus late season yellows virus (NLSYV) were identified from domestic and wild Narcissus populations at incidences of 66 and 49%, respectively. NLSYV was also detected in one plant of Clivea miniata. Comparisons of nucleotide and amino acid sequences of the coat protein genes of NLSYV isolates showed that they formed three distinct phylogenetic groups, including one not seen before. Vallota speciosa virus was detected in one domestic population of Narcissus sp. where it infected 70% of the plants. This is the first report of these viruses in Australia, and of NLSYV infecting C. miniata.

Identification of plant viruses using one-dimensional gel electrophoresis and peptide mass fingerprints.

A generic assay to detect and partially characterize unknown viruses from plants was developed. Proteins extracted from virus-infected and uninfected plants were separated in one dimension by SDS polyacrylamide gel electrophoresis. Differentially expressed protein bands were eluted after trypsin digestion and resulting peptide fragments separated according to their mass by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Resulting peptide mass fingerprints (PMF) were compared with those in protein databases. The assay was used to identify three known viruses: the potyviruses Zucchini yellow mosaic virus and Turnip mosaic virus, and an alfamovirus Alfalfa mosaic virus. It was also used to identify a virus that manifested symptoms in wild Cakile maritima plants, tentatively identified as Pelargonium zonate spot virus (PZSV) (genus Anulavirus) by its PMF, and then confirmed by nucleotide sequencing. The detection of PZSV constitutes a first record of this virus in Australia and in this host. It is proposed that this rapid and simple assay is a useful approach for analysis of plant samples known to harbor viruses that could not be identified using antisera or nucleic acid-based assays.

First Report of 'Candidatus Phytoplasma aurantifolia' Associated With Severe Stunting and Necrosis on the Invasive Weed Pelargonium capitatum in Western Australia.

Pelargonium capitatum (rose pelargonium) is a plant indigenous to southern Africa, originally brought to Western Australia for its ornamental qualities. It has since become naturalized in the Southwest Australian Floristic Region, recognized for its high level of species endemism, where it is a serious invasive weed in bushlands and coastal dunes. Since P. capitatum outcompetes native species it is listed among the top 10 most important coastal weeds of the region (3). In 2008, large patches of stunted, dying, and dead P. capitatum plants were observed within a population covering coastal dunes at Woodman Point, Western Australia (GPS coordinates 32°07'40.51″S, 115°45'28.39″E). Diseased plants had small misshapen leaves in clumps that were often chlorotic or pink, shortened internodes, and exhibited phylloidy typical of infection by a phytoplasma. From August 2009 to January 2010, samples from symptomatic and asymptomatic plants were collected from the site and from plants of an asymptomatic population at another site located on the Murdoch University campus nearby. DNA was extracted from 15 samples collected from symptomatic and asymptomatic plants at the dune site and from five at the campus site. Briefly, 2 to 5 g of leaf and stem tissue was cut into 5-mm pieces and shaken overnight in 30 ml of phosphate-buffered saline buffer. Supernatant was filtered and a pellet was collected by centrifugation. After resuspension in 500 µl of extraction buffer (200 mM Tris-HCl [pH 7.5] 250mM NaCl, 25mM ethylenediaminetetraacetic acid, 0.5% sodium dodecyl sulfate, and 2% polyvinylpyrrolidone), DNA was precipitated in 500 µl of cold isopropanol. Samples were tested for the presence of phytoplasma ribosomal 16S DNA by nested PCR using phytoplasma universal primers P1/P7 followed by amplification with primers Tint, R16mF2, and R16mR1 (1,2,4). Phytoplasma-specific DNA sequences were synthesized directly from amplicons using the above primers. Phytoplasma was detected from both symptomatic and asymptomatic plant samples collected from the dune site but not from the campus site. Analysis of the nine sequences obtained (GenBank Accession Nos. HM583339, HM583340, HM583341, HM583342, HM583343, HM583344, HM583345, HM583346, and HM583347) revealed high sequence identity between isolates (~99%) and with the 'Candidatus Phytoplasma aurantifolia' (16SrII) group of phytoplasmas (1,4). Presence of phytoplasma in symptomatic plants was confirmed by histological examination of stem sections stained with Dienes' stain. This finding is significant because there is potential for utilizing this phytoplasma to control P. capitatum where it has invaded ecologically significant sites, although its effect on indigenous plants must be determined first. Although phytoplasmas within the 16SrII group have been identified in Australia previously (1,4), to our knowledge, this is the first report of it infecting P. capitatum. References: (1) K. S. Gibb et al. Phytopathology 85:169, 1995. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) B. M. J. Hussey et al. Western Weeds. A Guide to the Weeds of Western Australia. 2nd ed. Plant Protection Society of Western Australia, Victoria Park, 2007. (4) M. Saqib et al. J. R. Soc. West. Aust. 90:175, 2007.

Role of recombination in the evolution of host specialization within bean yellow mosaic virus.

Seven complete genomes and 64 coat protein gene sequences belonging to Bean yellow mosaic virus (BYMV) isolates from different continents were examined for evidence of genetic recombination using six different recombination-detection programs. In the seven complete genomes and a single complete genome of the related virus Clover yellow vein virus (ClYVV), evidence for eight recombination patterns was found by four or more programs, giving firm evidence of their presence, and five additional recombination patterns were detected by three or fewer programs, giving tentative evidence of their occurrence. When the nucleotide sequences of 64 BYMV and one ClYVV coat protein genes were analyzed, three firm recombination patterns were detected in 21 isolates (32%). With another six isolates (9%), tentative evidence was found for three further recombination patterns. Of the 19 firm or tentative recombination patterns detected within and between strain groups of BYMV, and with ClYVV, 12 involved a generalist group of isolates as a parent but none of the other BYMV groups acted as parents more than six times. These findings suggest that recombination played an important role in the evolution of BYMV strain groups that specialize in infecting particular groups of domesticated plants.

Phylogenetic Analysis of Bean yellow mosaic virus Isolates from Four Continents: Relationship Between the Seven Groups Found and Their Hosts and Origins.

Genetic diversity of Bean yellow mosaic virus (BYMV) was studied by comparing sequences from the coat protein (CP) and genome-linked viral protein (VPg) genes of isolates from four continents. CP sequences compared were those of 17 new isolates and 47 others already on the database, while the VPg sequences used were from four new isolates and 10 from the database. Phylogenetic analysis of the CP sequences revealed seven distinct groups, six polytypic and one monotypic. The largest and most genetically diverse polytypic group, which had intragroup diversity of 0.061 nucleotide substitutions per site, contained isolates from natural infections in eight host species. These original isolation hosts included both wild (four) and domesticated (four) species and were from monocotyledonous and dicotyledonous plant families, indicating a generalized natural host range strategy. Only one of the other five polytypic groups spanned both monocotyledons and dicotyledons, and all contained isolates from fewer species (one to four), all of which were domesticated and had lower intragroup diversity (0.019 to 0.045 nucleotide substitutions per site), indicating host specialization. Phylogenetic analysis of the fewer VPg sequences revealed three polytypic and two monotypic groupings. These groups also correlated with original natural isolation hosts, but the branch topologies were sometimes incongruous with those formed by CPs. Also, intragroup diversity was generally higher for VPgs than for CPs. A plausible explanation for the groups found when the 64 different CP sequences were compared is that the generalized group represents the original ancestral type from which the specialist host groups evolved in response to domestication of plants after the advent of agriculture. Data on the geographical origins of the isolates within each group did not reveal whether the specialized groups might have coevolved with their principal natural hosts where these were first domesticated, but this seems plausible.

A non-aphid-transmissible isolate of bean yellow mosaic potyvirus has an altered NAG motif in its coat protein.

An isolate of Bean yellow mosaic virus (BYMV) not transmitted by aphids (NAT) was compared with the aphid-transmissible isolate (MI) from which it was derived. For each isolate, the sequence of the coat protein and parts of the helper component was determined. A single nucleotide substitution caused a NAG to NAS alteration in the coat protein of the non aphid-transmissible isolate. Loss of aphid transmissibility in isolate BYMV(MI)-NAT was most likely caused by this mutation within the NAG motif. Systemic movement and accumulation of the virus in infected plants were not affected by the mutation.

Transgenic yellow lupin (Lupinus luteus).

Transgenic yellow lupin (Lupinus luteus L.) plants have been generated by meristem co-cultivation with Agrobacterium tumefaciens. The binary plasmid pPZBNIa contains the bar gene under the control of a CaMV 35 S promoter. The transformation method involves inoculation of embryonic axis explants with A. tumefaciens, flooding the meristem with glufosinate, and initial culture on non-selective medium. Shoots were transferred to culture medium containing 20 mg/l glufosinate. Following subculture, shoots were grafted onto non-transgenic narrow-leafed lupin (L. angustifolius L.) seedling rootstocks, or rooted in vitro. The overall transformation efficiency, as determined at the T1 generation, was 0.05%-0.75%. The transgenic nature of plants grown to the T6 generation was confirmed by phosphinothricin acetyl transferase, PCR and Southern analyses.

Young female drivers in New Zealand.

While a considerable amount is known about young male drivers both in New Zealand and overseas, research attention concerning female drivers of any age including those under the age of 25 has been far less prevalent. The present paper explores the reasons behind the historically low research interest in this population of road users, and seeks to provide information on the current status of young female drivers in New Zealand, with regard to both travel patterns and crash involvement statistics. The findings of the present study suggest that the exposure to risk of this group has increased with greater licensing and kilometreage. Increased crash involvement was identified for this group, a finding accentuated by the fact that this has occurred concurrent with a decrease in crash involvement of young male drivers. Drink driving patterns of this group are discussed, in response to the finding that the crash involvement and number of drink-drive convictions of females 15-24 have increased over time. Implications for research and countermeasures are outlined.