Towards the development of a Cavendish banana resistant to race 4 of fusarium wilt: gamma irradiation of micropropagated Dwarf Parfitt (Musa spp., AAA group, Cavendish subgroup)

'Dwarf parfitt', an extra-dwarf Cavendish cultivar with resistance to subtropical race 4 fusarium oxysporum f. sp. cubense 9Foc), was gamma irradiated at a dose of 20 Gy and putative mutants were recovered with improved agronomic characteristics. Further screening of putative mutants for improved yield and fruit size, as well as a degree of resistence to fusarium wilt, led to the selection of a line (DPM25) with improved productivity when grown on soils infested with subtropical race 4 Foc. DPM25 was equal to the industry standard, 'Williams', in every agronomic trait measured and it consistently showed a lower incidence of fusarium wilt. Further improvement of field resistance to race 4 Foc is needed in DPM25 and further cycles of mutation induction and selction is an option discussed.

High resolution genetic and physical mapping of the I-3 region of tomato chromosome 7 reveals almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with duplications on Arabidopsis chromosomes 1, 2 and 3

The tomato I-3 gene introgressed from the Lycopersicon pennellii accession LA716 confers resistance to race 3 of the fusarium wilt pathogen Fusarium oxysporum f. sp. lycopersici. We have improved the high-resolution map of the I-3 region of tomato chromosome 7 with the development and mapping of 31 new PCR-based markers. Recombinants recovered from L. esculentum cv. M82 × IL7-2 F2 and (IL7-2 × IL7-4) × M82 TC1F2 mapping populations, together with recombinants recovered from a previous M82 × IL7-3 F2 mapping population, were used to position these markers. A significantly higher recombination frequency was observed in the (IL7-2 × IL7-4) × M82 TC1F2 mapping population based on a reconstituted L. pennellii chromosome 7 compared to the other two mapping populations based on smaller segments of L. pennellii chromosome 7. A BAC contig consisting of L. esculentum cv. Heinz 1706 BACs covering the I-3 region has also been established. The new high-resolution map places the I-3 gene within a 0.38 cM interval between the molecular markers RGA332 and bP23/gPT with an estimated physical size of 50-60 kb. The I-3 region was found to display almost continuous microsynteny with grape chromosome 12 but interspersed microsynteny with Arabidopsis thaliana chromosomes 1, 2 and 3. An S-receptor-like kinase gene family present in the I-3 region of tomato chromosome 7 was found to be present in the microsyntenous region of grape chromosome 12 but was absent altogether from the A. thaliana genome.

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. © 2013 Springer-Verlag Wien.

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.

A Genetic Analysis of Lysosomal Enzyme Activities in Brahman Cattle

Analyses of variance and co variance were carried out on the activities of three lysosomal enzymes in mononuclear blood cells from Brahman cattle. These were hexosaminidase (HEX), beta-D-galacto-sidase (GAL) and acid alpha-glucosidase (GLU) which had been measured in blood mononuclear cells from 1752 cattle from 6 herds in a Pompe's disease control programme. Herd of origin and date of bleeding significantly affected the level of activity of all enzymes. In addition, HEX and GAL were affected by age and HEX by the sex of the animal bled. Estimates of heritability from sire variances were 0.29:t 0.09 for HEX, 0.31 :t 0.09 for GAL and 0.44:t 0.09 for GLU. Genetic correlations between all enzymes were positive. The data indicate the existence of a major gene causing Pompe's disease and responsible for 16% of the genetic variation in GLU. One standard deviation of selection differential for high GLU should almost eliminate Pompe's disease from the population. The effi-ciency of selection would be aided by estimating the breeding value for GLU using measurements of HEX and GLU and taking account of an animal's sex, age, date of bleeding and herd of origin.

Survey of Fusarium species associated with crown rot of wheat and barley in eastern Australia

Fusarium species associated with crown rot were isolated and identified from 409 wheat, barley or durum wheat crops from the eastern Australian grain belt between 1996 and 1999. Fusarium pseudograminearum was almost the only species isolated from crops in Queensland and New South Wales. F. pseudograminearum was also the most common species in Victoria and South Australia, but F. culmorum was frequently isolated in these states. F. culmorum accounted for more than 70% of isolates from the Victorian high-rainfall (> 500 mm) region and the South-East region of South Australia. F. culmorum comprised 18% of isolates from the Victorian medium-rainfall (350-500 mm) region, and 7% of isolates from each of the Victorian low-rainfall region and the Mid-North region of South Australia. F. avenaceum, F. crookwellense and F. graminearum were isolated very infrequently. The proportion of F. culmorum among isolates of Fusarium from districts in Victoria and South Australia was strongly correlated with climatic conditions around the end of the growing season, especially with rainfall in November.

Abacá mosaic virus: a distinct strain of Sugarcane mosaic virus

Abacá mosaic virus (AbaMV) is related to members of the sugarcane mosaic virus subgroup of the genus Potyvirus. The ~2 kb 3′ terminal region of the viral genome was sequenced and, in all areas analysed, found to be most similar to Sugarcane mosaic virus (SCMV) and distinct from Johnsongrass mosaic virus (JGMV), Maize dwarf mosaic virus (MDMV) and Sorghum mosaic virus (SrMV). Cladograms of the 3′ terminal region of the NIb protein, the coat protein core and the 3′ untranslated region showed that AbaMV clustered with SCMV, which was a distinct clade and separate from JGMV, MDMV and SrMV. The N-terminal region of the AbaMV coat protein had a unique amino acid repeat motif different from those previously published for other strains of SCMV. The first experimental transmission of AbaMV from abacá (Musa textilis) to banana (Musa sp.), using the aphid vectors Rhopalosiphum maidis and Aphis gossypii, is reported. Polyclonal antisera for the detection of AbaMV in western blot assays and ELISA were prepared from recombinant coat protein expressed in E. coli. A reverse transcriptase PCR diagnostic assay, with microtitre plate colourimetric detection, was developed to discriminate between AbaMV and Banana bract mosaic virus, another Musa-infecting potyvirus. Sequence data, host reactions and serological relationships indicate that AbaMV should be considered a distinct strain of SCMV, and the strain designation SCMV-Ab is suggested.

Cuticular Hydrocarbons of Buffalo Fly, Haematobia exigua, and Chemotaxonomic Differentiation from Horn Fly, H.irritans

We determined the quantity and chemical composition of cuticular hydrocarbons of different strains, sex and age of buffalo flies, Haematobia exigua. The quantity of cuticular hydrocarbons increased from less than 1 µg/fly for newly-emerged flies to over 11 µg/fly in 13 d-old flies. The hydrocarbon chain length varied from C21 to C29, with unbranched alkanes and monounsaturated alkenes the major components. Newly emerged flies produced almost exclusively C27 hydrocarbons. Increasing age was accompanied by the appearance of hydrocarbons with shorter carbon chains and an increase in the proportion of alkenes. 11 Tricosene and 7-tricosene were the most abundant hydrocarbons in mature buffalo flies. Cuticular hydrocarbons of buffalo flies are distinctly different from those of horn flies. The most noticeable differences were in the C23 alkenes, with the major isomers 11- and 7-tricosene in buffalo flies and (Z)-9- and (Z)-5-tricosene in horn flies, respectively. Cuticular hydrocarbon analysis provides a reliable method to differentiate buffalo and horn fly, which are difficult to separate morphologically. The differences in cuticular hydrocarbons also support their recognition as separate species, H. exigua and H. irritans, rather than as subspecies.

Nucleocapsid gene variability reveals two subgroups of Lettuce necrotic yellows virus

The complete nucleocapsid (N) genes of eight Australian isolates of Lettuce necrotic yellows virus (LNYV) were amplified by reverse transcription PCR, cloned and sequenced. Phylogenetic analyses of these sequences revealed two distinct subgroups of LNYV isolates. Nucleotide sequences within each subgroup were more than 96% identical but heterogeneity between groups was about 20% at the nucleotide sequence level. However, less than 4% heterogeneity was noted at the amino acid level, indicating mostly third nucleotide position changes and a strong conservation for N protein function. There was no obvious geographical or temporal separation of the subgroups in Australia.

A simplified PCR test for early detection of dwarf off-types in micropropagated Cavendish bananas (Musa spp. AAA)

The dwarf somaclonal variant is a major problem affecting micropropagation of the banana cultivar Williams (Musa spp. AAA; subgroup Cavendish). This problem arises from genetic changes that occur during the tissue culture process. Early identification of this problem is difficult and propagators must wait until plants are ex vitro in order to visualise the dwarfism phenotype. In this study, we have improved a SCAR-based molecular diagnostic technique, developed by Damasco et al. [Acta Hortic. 461 (1997) 157], for the early identification of dwarf off-types. We have included a positive internal control in a multiplex PCR and adapted the technique for use with small amounts of fresh in vitro leaf material as PCR template. The control product is a 500 bp fragment from 18S rRNA and is amplified in all tissues irrespective of phenotype. The use of small in vitro leaf material removing the need for genomic DNA extraction.

Characterisation and early detection of an offtype from micropropagated Lady Finger bananas

An offtype has been identified from micropropagated Lady Finger bananas (Musa spp., AAB group, Pome subgroup) that is characterised by its slow growth and poor bunch size. Bunch weights were approximately 25% those of normal Lady Finger plants and all of the fruit produced was unmarketable. This particular offtype is the most commonly encountered from micropropagated Lady Finger plants and, in 2 instances, blocks of 3000 and 1500 plants were entirely comprised of this single offtype. Detection of offtype plants was possible during establishment and growth of plants in the glasshouse by the presence of chlorotic streaks in the leaves. In more severe cases the streaks coalesced into chlorotic patches that developed thin, necrotic areas that eventually produced holes or splits in the leaves. Symptom expression was not ameliorated by the addition of fertiliser and even though symptoms were similar to severe Ca and B deficiency, both normal and offtype plants had similar levels of these elements in the leaves. The offtype plants were also slow growing in the glasshouse and produced significantly (P<0.05) smaller pseudostems and leaves than normal plants. Offtype plants could be readily detected after 4 weeks deflasking using the presence of chlorotic streaks in the leaves as the main selection criterion. Maximum discrimination was possible between weeks 5–7 and at the 6-leaf stage when all of the offtypes could be detected.

Host range, symptom expression and RNA 3 sequence analyses of six Australian strains of Cucumber mosaic virus

We have characterised six Australian Cucumber mosaic virus (CMV) strains belonging to different subgroups, determined by the sequence of their complete RNA 3 and by their host range and the symptoms they cause on species in the Solanaceae, Cucurbitaceae and on sweet corn. These data allowed classification of strains into the known three CMV subgroups and identification of plant species able to differentiate the Australian strains by symptoms and host range. Western Australian strains 237 and Twa and Queensland strains 207 and 242 are closely related members of CMV subgroup IA, which cause similar severe symptoms on Nicotiana species. Strains 207 and 237 (subgroup IA) were the only strains tested which systemically infected sweet corn. Strain 243 caused the most severe symptoms of all strains on Nicotiana species, tomato and capsicum and appears to be the first confirmed subgroup IB strain reported in Australia. Based on pair-wise distance analysis and phylogeny of RNA 3, as well as mild disease symptoms on Nicotiana species, CMV 241 was assigned to subgroup II, as the previously described Q-CMV and LY-CMV.

Climate information needs of Gascoyne-Murchison pastoralists: a representative study of the Western Australian grazing industry

The Gascoyne-Murchison region of Western Australia experiences an arid to semi-arid climate with a highly variable temporal and spatial rainfall distribution. The region has around 39.2 million hectares available for pastoral lease and supports predominantly catle and sheep grazing leases. In recent years a number of climate forecasting systems have been available offering rainfall probabilities with different lead times and a forecast period; however, the extent to which these systems are capable of fulfilling the requirements of the local pastoralists is still ambiguous. Issues can range from ensuring forecasts are issued with sufficient lead time to enable key planning or decisions to be revoked or altered, to ensuring forecast language is simple and clear, to negate possible misunderstandings in interpretation. A climate research project sought to provide an objective method to determine which available forecasting systems had the greatest forecasting skill at times of the year relevant to local property management. To aid this climate research project, the study reported here was undertaken with an overall objective of exploring local pastoralists' climate information needs. We also explored how well they understand common climate forecast terms such as 'mean', median' and 'probability', and how they interpret and apply forecast information to decisions. A stratified, proportional random sampling was used for the purpose of deriving the representative sample based on rainfall-enterprise combinations. In order to provide more time for decision-making than existing operational forecasts that are issued with zero lead time, pastoralists requested that forecasts be issued for May-July and January-March with lead times counting down from 4 to 0 months. We found forecasts of between 20 and 50 mm break-of-season or follow-up rainfall were likely to influence decisions. Eighty percent of pastoralists demonstrated in a test question that they had a poor technical understanding of how to interpret the standard wording of a probabilistic median rainfall forecast. this is worthy of further research to investigate whether inappropriate management decisions are being made because the forecasts are being misunderstood. We found more than half the respondents regularly access and use weather and climate forecasts or outlook information from a range of sources and almost three-quarters considered climate information or tools useful, with preferred methods for accessing this information by email, faxback service, internet and the Department of Agriculture Western Australia's Pastoral Memo. Despite differences in enterprise types and rainfall seasonality across the region we found seasonal climate forecasting needs were relatively consistent. It became clear that providing basic training and working with pastoralists to help them understand regional climatic drivers, climate terminology and jargon, and the best ways to apply the forecasts to enhance decision-making are important to improve their use of information. Consideration could also be given to engaging a range of producers to write the climate forecasts themselves in the language they use and understand, in consultation with the scientists who prepare the forecasts.

Formative evaluation to benchmark and improve climate-based decision support for graziers in Western Queelsland

Researchers developing climate-based forecasts, workshops, software tools and information to aid grazier decisions undertook an evaluation study to enhance planning and benchmark impact. One hundred graziers in Western Queensland were randomly selected from 7 shires and surveyed by mail and telephone (43 respondents) to explore levels of knowledge and use of climate information, practices and information needs. We found 36% of respondents apply the Southern Oscillation Index to property decisions but 92% were unaware El Niño Southern Oscillation’s predictive signal in the region is greater for pasture growth than rainfall, suggesting they may not recognise the potential of pasture growth forecasts. Almost 75% of graziers consider they are conservative or risk averse in their attitude to managing their enterprise. Mail respondents (n= 20) if given a 68%, on average, probability of exceeding median rainfall forecast may change a decision; almost two-thirds vary stocking rate based on forage available, last year’s pasture growth or the Southern Oscillation Index; the balance maintain a constant stocking rate strategy; 90% have access to a computer; 75% to the internet and 95% have a fax. This paper presents findings of the study and draws comparisons with a similar study of 174 irrigators in the Northern Murray-Darling Basin (Aust. J. Exp. Ag. 44, 247-257). New insights and information gained are helping the team better understand client needs and plan, design and extend tools and information tailored to grazier knowledge, practice, information needs and preferences. Results have also provided a benchmark against which to measure project impact and have influenced the team to make important changes to their project planning, activities and methods for transferring technology tailored to grazier preferences.