Murray Sharman

Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana

A multiplex, immunocapture PCR (M-IC-PCR) was developed for the simultaneous detection of three viruses from crude sap extracts of banana and plantain (Musa spp.). A reverse transcription step was required for Banana bract mosaic virus and Cucumber mosaic virus, which have ssRNA genomes. The detection of Banana bunchy top virus (ssDNA genome) was not adversely affected by inclusion in this step. All the three viruses could be detected simultaneously from a mixed infection. Identification and detection of individual viruses was achieved through the visualisation of discretely sized PCR amplicons by gel electrophoresis. Alternatively, a colourimetric microplate detection system utilising digoxigenin-labelled virus-specific probes was used. The latter assay was up to five times more sensitive than detection by gel electrophoresis and between 25 and 625 times more sensitive than ELISA for the various viruses. Careful selection of PCR primers was necessary to ensure the detection of a wide range of virus isolates and to avoid detrimental interactions between heterologous templates and primers.

Tospoviruses—an Australian perspective

The detection, distribution, molecular and biological properties, vector relations and control of tospoviruses present in Australia, including Tomato spotted wilt virus (TSWV), Capsicum chlorosis virus (CaCV) and Iris yellow spot virus (IYSV), are reviewed. TSWV occurs throughout Australia where it has caused serious sporadic epidemics since itwas first described in the 1920s. The frequency and distribution of outbreaks has increased in the 1990s, with the arrival and dispersal of the western flower thrips (Frankliniella occidentalis) being one factor favouring this situation. The crops most frequently and severely affected are capsicum, lettuce, tomato, potato and several species of ornamentals. Minimal differences were found between the nucleocapsid (N) gene amino acid sequences of Australian isolates and these were most closely related to a clade of northern European isolates. CaCV was first detected in Australia in 1999 and is most closely related to Watermelon silver mottle virus, a serogroup IV tospovirus. The natural hosts include capsicum, tomato, peanut and Hoya spp. The virus also occurs in Thailand and Taiwan. IYSV was first found in Australia in 2003, infecting onion and leek, with the distribution in three States suggesting that the virus has been present for some time.

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

SummaryTwo isolates of a novel babuvirus causing “bunchy top” symptoms were characterised, one from abacá (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.

Distribution in Australia and Seed Transmission of Tobacco streak virus in Parthenium hysterophorus

Tobacco streak virus (TSV) was found to commonly occur in Parthenium hysterophorus, as symptomless infections, in central Queensland, Australia across a large area infested with this weed. Several isolates of TSV collected across the geographic range of P. hysterophorus were found to share identical coat protein sequence with each other and with TSV from crop plants in the same area. Seed transmission of TSV in P. hysterophorus was found to occur at rates of 6.8 to 48%. There was almost no change in the rate of TSV seed transmission when P. hysterophorus seed was stored for up to 24½ months. Implications of this relationship between TSV and P. hysterophorus for the development of virus disease epidemics in surrounding crops are discussed.

Evidence that dicot-infecting mastreviruses are particularly prone to inter-species recombination and have likely been circulating in Australia for longer than in Africa and the Middle East

Viruses of the genus Mastrevirus (family Geminiviridae) are transmitted by leafhoppers and infect either mono- or dicotyledonous plants. Here we have determined the full length sequences of 49 dicot-infecting mastrevirus isolates sampled in Australia, Eritrea, India, Iran, Pakistan, Syria, Turkey and Yemen. Comprehensive analysis of all available dicot-infecting mastrevirus sequences showed the diversity of these viruses in Australia to be greater than in the rest of their known range, consistent with earlier studies, and that, in contrast with the situation in monocot-infecting mastreviruses, detected inter-species recombination events outnumbered intra-species recombination events. Consistent with Australia having the greatest diversity of known dicot-infecting mastreviruses phylogeographic analyses indicating the most plausible scheme for the spread of these viruses to their present locations, suggest that most recent common ancestor of these viruses is likely nearer Australia than it is to the other regions investigated.

First report ofTobacco streak virus in sunflower (Helianthus annuus), cotton (Gossypium hirsutum), chickpea (Cicer arietinum) and mung bean (Vigna radiata) in Australia

Tobacco streak virus (genusIlarvirus) is recorded on sunflower (Helianthus annuus), cotton (Gossypium hirsutum), chickpea (Cicer arietinum) and mung bean (Vigna radiata) in Australia for the first time.

IXth International Symposium on Thysanoptera and Tospoviruses, 31st August-4th September 2009, Sea World Resort, Queensland, Australia.

This proceedings contains abstracts of 108 papers focusing on the different Tospovirus diseases of various crops and their thysanopteran vectors. The genetics of these pests and pathogens, the different methods used in their control and their geographical distribution are also highlighted.

First report of Tomato spotted wilt virus in chickpea (Cicer arietinum) in Australia.

Tomato spotted wilt virus (genus Tospovirus) is recorded on chickpea (Cicer arietinum) in Australia for the first time. It caused shoot tip symptoms of wilting, necrosis, bunching and chlorosis, followed by premature death of plants.

Genetic diversity of subgroup 1 ilarviruses from eastern Australia

This is the first report of the genetic diversity within ilarvirus subgroup 1 from eastern Australia. It supports the separation of tobacco streak virus (TSV) strains from parthenium (Parthenium hysterophorus) and crownbeard (Verbescina encelioides) based on serology and host specificity. It has confirmed one previously described strain of TSV as a member of the species Strawberry necrotic shock virus and another as a new subgroup 1 ilarvirus, ageratum latent virus (AgLV), from Ageratum houstonianum. A multiplex RT-PCR showed that the genetically distinct strains of TSV and AgLV were commonly found in symptomless infections in virus-specific alternative weed hosts growing over a wide geographical range in eastern Australia. TSV has been one of the most damaging viruses in Australian oilseed and pulse crops in recent years, and this study has provided the taxonomic knowledge essential for the development of control programs for these viruses.

Molecular diversity of Chickpea chlorotic dwarf virus in Sudan: High rates of intra-species recombination – a driving force in the emergence of new strains

In Sudan Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus, family Geminiviridae) is an important pathogen of pulses that are grown both for local consumption, and for export. Although a few studies have characterised CpCDV genomes from countries in the Middle East, Africa and the Indian subcontinent, little is known about CpCDV diversity in any of the major chickpea production areas in these regions. Here we analyse the diversity of 146 CpCDV isolates characterised from pulses collected across the chickpea growing regions of Sudan. Although we find that seven of the twelve known CpCDV strains are present within the country, strain CpCDV-H alone accounted for ∼73% of the infections analysed. Additionally we identified four new strains (CpCDV-M, -N, -O and -P) and show that recombination has played a significant role in the diversification of CpCDV, at least in this region. Accounting for observed recombination events, we use the large amounts of data generated here to compare patterns of natural selection within protein coding regions of CpCDV and other dicot-infecting mastrevirus species.