A. M. Mackenzie

The potyviruses of Australia

Many potyviruses have been found in Australia. We analyzed a selected region of the coat protein genes of 37 of them to determine their relationships, and found that they fall into two groups. Half were isolated from cultivated plants and crops, and are also found in other parts of the world. Sequence comparisons show that the Australian populations of these viruses are closely related to, but less variable than, those in other parts of the world, and they represent many different potyvirus lineages. The other half of the potyviruses have only been found in Australia, and most were isolated from native plants. The sequences of these potyviruses, which are probably endemic, are on average five times more variable than those of the crop potyviruses, but surprisingly, most of the endemic potyviruses belong to one potyvirus lineage, the bean common mosaic virus lineage. We conclude that the crop potyviruses entered Australia after agriculture was established by European migrants two centuries ago, whereas the endemic plant potyviruses probably entered Australia before the Europeans. Australia, like the U.K., seems recently to have had c. one incursion of a significant crop potyvirus every decade. Our analysis suggests it is likely that potyviruses are transmitted in seed more frequently than experimental evidence indicates, and shows that understanding the sources of emerging pathogens and the frequency with which they ‘emerge’ is essential for proper national biosecurity planning.

Ceratobium mosaic potyvirus: another virus from orchids

SummaryA potyvirus, which we call ceratobium mosaic virus, has been detected in about one third of more than 100 plants representing c. 33 orchid genera in two collections in Australia. It was detected using RT-PCR with redundant primers that are Potyviridae-specific and have additional sequences corresponding to either the SP6 or T7 bacteriophage promoters at their 5′-termini. Thus the nucleotide sequence of the resulting PCR fragments, consisting of about 1.7 kb of the 3′ portion of the viral genome, could be determined directly. Viral sequences obtained from five infected orchids indicate that they contained different isolates of a single potyvirus species most closely related to the bean common mosaic group of potyviruses, but clearly distinct from all whose virion protein genes have been reported to the international gene sequence databases.

The genomic sequence of cardamine chlorotic fleck carmovirus

The complete genomic sequence of cardamine chlorotic fleck carmovirus (CCFV) has been determined. The genome is a positive-sense ssRNA molecule 4041 nucleotides in length, and has 47 to 64% sequence identity with turnip crinkle, carnation mottle and melon necrotic spot carmoviruses. CCFV and these other carmoviruses have four similar open reading frames (ORFs), and CCFV has large regions of amino acid identity in all of these ORFs with a European isolate of turnip crinkle virus. CCFV, which replicates well in Arabidopsis thaliana, has only been found so far in Australia in the wild perennial brassica Cardamine lilacina.

Nucleotide sequence comparisons of turnip yellow mosaic virus isolates from Australia and Europe

SummaryThe genomic sequences of four isolates of turnip yellow mosaic virus (TYMV-Cd) from Australia, and three TYMV-1 (type) and three TYMV-2 (cauliflower) isolates from Europe were compared by cDNA-RNA hybridization tests, by analysis of the fragments produced from cDNA-RNA hybrids by restriction endonuclease treatment, and by determining the 3′ terminal nucleotide sequences of their coat protein mRNAs. All three methods showed only slight differences (ca. 1%) between the mRNA sequences of different TYMV-1 and TYMV-Cd isolates, and did not distinguish between those groups of isolates. By contrast, the nucleotide sequences of TYMV-2 isolates differed from those of the other TYMVs by ca. 5% (sequence analysis) to 11% (restriction fragment analysis).Published biogeographic evidence has indicated that the TYMV-Cd and TYMV-1 populations probably separated more than 12,000 years ago. This implies that these TYMV genomes have changed at a rate of, at most, 1% in 10,000 years.

High levels of genetic variability in the moss Ceratodon purpureus from continental Antarctica, subantarctic Heard and Macquarie Islands, and Australasia

The random amplified polymorphic DNA (RAPD) technique, and DNA sequencing of the conserved nuclear ribosomal DNA internal transcribed spacer region (ITS1-5.8S-ITS2), have been used to assess levels of genetic diversity in the moss Ceratodon purpureus from several locations in Australasia, subantarctic Heard and Macquarie Islands, and continental Antarctica. Populations from Heard and Macquarie Islands and from Antarctica maintain high levels of genetic variation. Both within- and among-colony variation were observed at these locations. DNA sequence analysis showed that samples from the Ross Sea region of Antarctica were most closely related to colonies from Casey and Macquarie Island, and that one colony from Heard Island was most closely related to one from Europe. DNA sequence data separated two Australian populations from the Antarctic and subantarctic group on a dendrogram. Detailed RAPD analysis of a single colony from continental Antarctica demonstrated that mutation probably causes the high variability observed in this moss. DNA sequencing and RAPD analysis are complementary techniques for genetic investigation of Antarctic moss populations.

Potyviruses, novel and known, in cultivated and wild species of the family Apiaceae in Australia

Summary. Three potyviruses were identified by gene sequencing and found to be widespread in species of Apiaceae in Australia. Only celery mosaic virus was found in celery crops and in one of 180 specimens of feral carrot (Daucus carota). Another related but distinct novel potyvirus, carrot virus Y, was the only virus found in carrot crops and all except one feral carrot. A more distantly related novel potyvirus, apium virus Y, was found in plants of sea celery (Apium prostratum), cultivated parsley (Petroselinum crispum) and the immigrant weed species poison hemlock (Conium maculatum). These three potyviruses, together with celery yellow mosaic virus of South America and the closely related carrot thin leaf virus and carrot virus B of North America, form a distinct subgenus of the Potyviridae most closely related to turnip mosaic virus and two potyviruses of yam; yam mosaic virus from the Ivory Coast and Japanese yam mosaic virus. Celery mosaic and carrot virus Y are probably recent migrants to Australia, but apium virus Y may have been endemic longer. In ELISA tests using polyclonal antibodies against virions of celery mosaic virus, some isolates of carrot virus Y were indistinguishable from celery mosaic virus, whereas others gave smaller absorbancy values, and those of apium virus Y did not react. This study shows the value of virus identification based on gene sequencing for planning control measures.

Comparative Cytopathology and Immunocytochemistry of Japanese, Australian and Brazilian Isolates of Orchid fleck virus

Cytopathic effects in orchid leaf tissues infected with Australian, Japanese and Brazilian isolates of Orchid fleck virus (OFV) were indistinguishable and like those previously described in the literature. Cells had an electron-lucent viroplasm with unenveloped rod-shaped virions in the nucleus and cytoplasm, often associated with the inner membrane of the nuclear envelope and the endoplasmic reticulum. Antiserum raised against a Japanese isolate of OFV reacted with Brazilian and Australian isolates in ELISA, and when used for immuno-gold labelling, also reacted in situ with the rod-shaped virions and the intranuclear viroplasm of all three isolates. These results suggest that the viroplasm is where structural proteins accumulate and virions are formed.

Genetic variation in populations of kennedya yellow mosaic tymovirus

SummaryKennedya yellow mosaic tymovirus (KYMV) occurs along the eastern Australian seaboard in the perennial legumesDesmodium triflorum andD. scorpiurus in the north, andKennedya rubicunda in the south. The genetic variation of more than 100 isolates of KYMV, most of them from the north, has been studied using an RNA hybrid mismatch polymorphism (RHMP) method. The method clearly separated the isolates into two groups; all the northernDesmodium isolates formed one group and all theKennedya isolates from the south another. These sub-populations were themselves variable and theDesmodium population alone was more variable than that of the related turnip yellow mosaic tymovirus in the Kosciusko alpine area.

Turnip yellow mosaic virus variants produced from DNA clones encoding their genomes

SummaryFull-length dsDNA clones that encode the genomes of two Australian turnip yellow mosaic isolates, TYMV-BL and TYMV-CL have been constructed. These clones were transcribed to give 6.3 kb capped ssRNA which infects Chinese cabbages to give symptoms indistinguishable from those produced by the parental viruses. Extensions of up to 26 nucleotides at the 3′ end of the TYMV-BL clone delay infections, but virus particles isolated from these plants 4 weeks after inoculation contain RNA with the original TYMV-BL 3′ terminus. A 90 nucleotide-long portion of the virion protein gene of TYMV-BL was replaced by a synthetic 90-mer primer with 16 nucleotide changes to decrease the large cytosine content (34–42%) characteristic of tymovirus genomic RNA. No reversion of any of the mutated nucleotides to cytosine occurred during 7 passages in Chinese cabbage. Hybrids between the TYMV-BL and TYMV-CL clones were also constructed, by exchanging various portions of the genome. However, it was not possible to determine definitively which part of the viral genome is responsible for the more severe symptoms caused by TYMV-BL as the hybrids gave intermediate symptoms.

RNA hybrid mismatch polymorphisms in Australian populations of turnip yellow mosaic tymovirus

SummaryIn the Mt. Kosciusko alpine area of Australia there are three well-separated populations ofCardamine lilacina, an endemic sward-forming perennial brassica, and these are infected with turnip yellow mosaic tymovirus. The genetic variation in these viral populations has been assessed by an RNA hybrid mismatch polymorphism method. About 100 isolates were examined; the genomic RNA of each isolate was prepared from a shoot of a single wildC. lilacina plant. RNA hybrid mismatch polymorphisms (RHMPs) were assessed in six regions of the genomes using labelled negative-strand probes transcribed from selected portions of a cloned TYMV genome. The probed region at the 3′ end of the genome showed little variation and over 95% of the isolates gave the same pattern. However, other parts of the genome, including the 5′ non-coding region, were much more variable. There was no significant correlation between groupings based on the RHMP patterns, and the location from which the isolates were collected, nor with the symptom type or severity shown by their host plants. The patterns of variation suggested that all three populations of the virus are a single quasi-species; at most one tenth of the isolates gave similar RHMP patterns, those of the “master copy”.