Epidemiology and genetic diversity of Tobacco streak virus and related subgroup 1 ilarviruses

A quarter of Australia’s sunflower production is from the central highlands region of Queensland and is currently worth six million dollars ($AUD) annually. From the early 2000s a severe necrosis disorder of unknown aetiology was affecting large areas of sunflower crops in central Queensland, leading to annual losses of up to 20%. Other crops such as mung bean and cotton were also affected. This PhD study was undertaken to determine if the causal agent of the necrosis disorder was of viral origin and, if so, to characterise its genetic diversity, biology and disease cycle, and to develop effective control strategies. The research described in this thesis identified Tobacco streak virus (TSV; genus Ilarvirus, family Bromoviridae) as the causal agent of the previously unidentified necrosis disorder of sunflower in central Queensland. TSV was also the cause of commonly found diseases in a range of other crops in the same region including cotton, chickpea and mung bean. This was the first report from Australia of natural field infections of TSV from these four crops. TSV strains have previously been reported from other regions of Australia in several hosts based on serological and host range studies. In order to determine the relatedness of previously reported TSV strains with TSV from central Queensland, we characterised the genetic diversity of the known TSV strains from Australia. We identified two genetically distinct TSV strains from central Queensland and named them based on their major alternative hosts, TSV-parthenium from Parthenium hysterophorus and TSV-crownbeard from Verbesina encelioides. They share only 81 % total-genome nucleotide sequence identity. In addition to TSV-parthenium and TSV-crownbeard from central Queensland, we also described the complete genomes of two other ilarvirus species. This proved that previously reported TSV strains, TSV-S isolated from strawberry and TSV-Ag from Ageratum houstonianum, were actually the first record of Strawberry necrotic shock virus from Australia, and a new subgroup 1 ilarvirus, Ageratum latent virus. Our results confirmed that the TSV strains found in central Queensland were not related to previously described strains from Australia and may represent new incursions. This is the first report of the genetic diversity within subgroup 1 ilarviruses from Australia. Based on field observations we hypothesised that parthenium and crownbeard were acting as symptomless hosts of TSV-parthenium and TSV-crownbeard, respectively. We developed strain-specific multiplex PCRs for the three RNA segments to accurately characterise the range of naturally infected hosts across central Queensland. Results described in this thesis show compelling evidence that parthenium and crownbeard are the major (symptomless) alternative hosts of TSV-parthenium and TSV-crownbeard. While both TSV strains had wide natural host ranges, the geographical distribution of each strain was closely associated with the respective distribution of their major alternative hosts. Both TSV strains were commonly found across large areas of central Queensland, but we only found strong evidence for the TSV-parthenium strain being associated with major disease outbreaks in nearby crops. The findings from this study demonstrate that both TSV-parthenium and TSV-crownbeard have similar life cycles but some critical differences. We found both TSV strains to be highly seed transmitted from their respective major alternative hosts from naturally infected mother plants and survived in seed for more than 2 years. We conclusively demonstrated that both TSV strains were readily transmitted via virus-infected pollen taken from the major alternative hosts. This transmission was facilitated by the most commonly collected thrips species, Frankliniella schultzei and Microcephalothrips abdominalis. These results illustrate the importance of seed transmission and efficient thrips vector species for the effective survival of these TSV strains in an often harsh environment and enables the rapid development of TSV disease epidemics in surrounding crops. Results from field surveys and inoculation tests indicate that parthenium is a poor host of TSV-crownbeard. By contrast, crownbeard was naturally infected by, and an experimental host of TSV-parthenium. However, this infection combination resulted in non-viable crownbeard seed. These differences appear to be an effective biological barrier that largely restricts these two TSV strains to their respective major alternative hosts. Based on our field observations we hypothesised that there were differences in relative tolerance to TSV infection between different sunflower hybrids and that seasonal variation in disease levels was related to rainfall in the critical early crop stage. Results from our field trials conducted over multiple years conclusively demonstrated significant differences in tolerance to natural infections of TSV-parthenium in a wide range of sunflower hybrids. Glasshouse tests indicate the resistance to TSV-parthenium identified in the sunflower hybrids is also likely to be effective against TSV-crownbeard. We found a significant negative association between TSV disease incidence in sunflowers and accumulated rainfall in the months of March and April with increasing rainfall resulting in reduced levels of disease. Our results indicate that the use of tolerant sunflower germplasm will be a critical strategy to minimise the risk of TSV epidemics in sunflower.

Genetic diversity and epidemiology of Tobacco streak virus and related subgroup 1 Ilarviruses

A quarter of Australia’s sunflower production is from the central highlands region of Queensland and is currently worth six million dollars ($AUD) annually. From the early 2000s a severe necrosis disorder of unknown aetiology was affecting large areas of sunflower crops in central Queensland, leading to annual losses of up to 20%. Other crops such as mung bean and cotton were also affected. This PhD study was undertaken to determine if the causal agent of the necrosis disorder was of viral origin and, if so, to characterise its genetic diversity, biology and disease cycle, and to develop effective control strategies. The research described in this thesis identified Tobacco streak virus (TSV; genus Ilarvirus, family Bromoviridae) as the causal agent of the previously unidentified necrosis disorder of sunflower in central Queensland. TSV was also the cause of commonly found diseases in a range of other crops in the same region including cotton, chickpea and mung bean. This was the first report from Australia of natural field infections of TSV from these four crops. TSV strains have previously been reported from other regions of Australia in several hosts based on serological and host range studies. In order to determine the relatedness of previously reported TSV strains with TSV from central Queensland, we characterised the genetic diversity of the known TSV strains from Australia. We identified two genetically distinct TSV strains from central Queensland and named them based on their major alternative hosts, TSV-parthenium from Parthenium hysterophorus and TSV-crownbeard from Verbesina encelioides. They share only 81 % total-genome nucleotide sequence identity. In addition to TSV-parthenium and TSV-crownbeard from central Queensland, we also described the complete genomes of two other ilarvirus species. This proved that previously reported TSV strains, TSV-S isolated from strawberry and TSV-Ag from Ageratum houstonianum, were actually the first record of Strawberry necrotic shock virus from Australia, and a new subgroup 1 ilarvirus, Ageratum latent virus. Our results confirmed that the TSV strains found in central Queensland were not related to previously described strains from Australia and may represent new incursions. This is the first report of the genetic diversity within subgroup 1 ilarviruses from Australia. Based on field observations we hypothesised that parthenium and crownbeard were acting as symptomless hosts of TSV-parthenium and TSV-crownbeard, respectively. We developed strain-specific multiplex PCRs for the three RNA segments to accurately characterise the range of naturally infected hosts across central Queensland. Results described in this thesis show compelling evidence that parthenium and crownbeard are the major (symptomless) alternative hosts of TSV-parthenium and TSV-crownbeard. While both TSV strains had wide natural host ranges, the geographical distribution of each strain was closely associated with the respective distribution of their major alternative hosts. Both TSV strains were commonly found across large areas of central Queensland, but we only found strong evidence for the TSV-parthenium strain being associated with major disease outbreaks in nearby crops. The findings from this study demonstrate that both TSV-parthenium and TSV-crownbeard have similar life cycles but some critical differences. We found both TSV strains to be highly seed transmitted from their respective major alternative hosts from naturally infected mother plants and survived in seed for more than 2 years. We conclusively demonstrated that both TSV strains were readily transmitted via virus-infected pollen taken from the major alternative hosts. This transmission was facilitated by the most commonly collected thrips species, Frankliniella schultzei and Microcephalothrips abdominalis. These results illustrate the importance of seed transmission and efficient thrips vector species for the effective survival of these TSV strains in an often harsh environment and enables the rapid development of TSV disease epidemics in surrounding crops. Results from field surveys and inoculation tests indicate that parthenium is a poor host of TSV-crownbeard. By contrast, crownbeard was naturally infected by, and an experimental host of TSV-parthenium. However, this infection combination resulted in non-viable crownbeard seed. These differences appear to be an effective biological barrier that largely restricts these two TSV strains to their respective major alternative hosts. Based on our field observations we hypothesised that there were differences in relative tolerance to TSV infection between different sunflower hybrids and that seasonal variation in disease levels was related to rainfall in the critical early crop stage. Results from our field trials conducted over multiple years conclusively demonstrated significant differences in tolerance to natural infections of TSV-parthenium in a wide range of sunflower hybrids. Glasshouse tests indicate the resistance to TSV-parthenium identified in the sunflower hybrids is also likely to be effective against TSV-crownbeard. We found a significant negative association between TSV disease incidence in sunflowers and accumulated rainfall in the months of March and April with increasing rainfall resulting in reduced levels of disease. Our results indicate that the use of tolerant sunflower germplasm will be a critical strategy to minimise the risk of TSV epidemics in sunflower.

The growth, reproduction and survival of 'Cascabela thevetia' seedlings under two levels of canopy cover

The ornamental tree 'Cascabela thevetia', from tropical America, has naturalised and formed large infestations at several locations in northern Australia. Some understanding of its ecology and invasiveness was gleaned from a field experiment undertaken in North Queensland. The experiment quantified the growth, time to seed formation and survival of seedlings of the peach biotype growing under light and dense canopy cover within a riparian habitat. Growth, reproduction and survival of young plants varied. Growth was most rapid for seedlings away from, or on the edge of infestations because they were constrained by parent plants. The findings also suggested that land managers have at least 12 months following control to detect new plants, or regrowth, before plants set seed and replenish soil seed banks.

Gene flow from Corymbia hybrids in northern New South Wales

Some of the most productive taxa for forestry are interspecific F1 hybrids grown as exotics in the tropics and subtropics. Attributes of resilience, adaptability and vigour which engender the hybrids for wood production, may also exacerbate the risk they present from gene flow to native species gene pools or to local ecologies as weeds. To determine the biological and genetic factors that influence the extent of hybridisation, we examine the distribution and genealogy of wildlings surrounding plantings of locally-exotic Corymbia torelliana (Section Cadageria) near native C. henryi (Section Maculatae) in northern New South Wales. Our study showed pre-mating and pre- and post-zygotic barriers were incomplete, with in situ generation and natural establishment of both F1 hybrids (n = 3) and advanced generation hybrids under the disturbed conditions bordering native forest. As hybrids were located on alluvial flats exposed to frost, they also likely have an extended ecological range relative to native C. henryi. Despite the likely generation of large viable seed crops on F1 trees at the site over many years, establishment success and survival of advanced generation hybrids may be low, as only 5 immature and no mature advanced generation hybrids were identified. Propagation and genetic analysis of a seed crop from one F1 wildling showed early survival and vigour of seedlings in cultivation was high, and that at least for some F1 in some seasons, backcrossing to the recurrent native C. henryi parent is favoured (60%), whereas selfing (10%) and crossing with other F1 (30%) was less frequent. Transport of seed by stingless bees probably accounted for long distance dispersal from C. torelliana, but this mechanism does not appear to supplement gravity-dispersal of seed from the F1. Coupled with other evidence from studies of bee behaviour, controlled pollination in Corymbia sp., and long-term fitness in second generation eucalypt hybrids, we anticipate gene flow via pollen rather than seed will be the greater challenge for managing the risk of introgression of C. torelliana ancestry into native species from the planted F1 hybrid. If large sources of F1 pollen become available to compete with native pollen, gene flow will probably be frequent and hybrids may establish in disturbed conditions and in habitats beyond the ecological range of their native parent. Further study is needed to determine the degree to which outbreeding depression and poor survival inhibits on-going gene flow.

Fine root dry matter relative to mango (Mangifera indica) tree scion size grafted on size-controlling rootstocks, is negatively related to scion growth rate

Knowledge of root dry matter (DM) allocation, in relation to differing vigour conferred by rootstock cultivars, is required to understand the structural relationships between rootstock and scion. We investigated the mass of roots (four size classes up to 23 mm diameter) by coring proximal to five polyembryonic mango rootstock cultivars known to differ in their effects on the vigour and productivity of scion cultivar ‘Kensington Pride’, in a field trial of 13-year-old trees. Significant differences in fine (<0.64 and 0.64–1.88 mm diameter) and small (1.88–7.50 mm) root DM contents were observed between rootstock cultivars. There was a complex relationship between the amount of feeder (fine and small size classes) roots and scion size (trunk cross sectional area, TCSA), with intermediate size trees on rootstock MYP having the most feeder roots, while the smallest trees, on the rootstock Vellaikulamban had the least of these roots. Across rootstock cultivars, tree vigour (TCSA growth rate) was negatively and significantly related to the ratio of fine root DM/scion TCSA, suggesting this may be a useful indicator of the vigour that different rootstocks confer on the scion. In contrast non-ratio root DM and scion TCSA results had no significant relationships. The significant rootstock effects on orchard root growth and tree size could not be predicted from earlier differences in nursery seedling vigour, nor did seedling vigour predict root DM allocation.