Advances in our knowledge of the genes expressed in cotton fibres make biotechnology a viable means to improve fibre characteristics. Most genetic modification uses ubiquitously expressing promoters that often result in yield penalty. We have developed a strategy which uses fibre-specific promoters to manipulate and confine gene expression to fibre cells.

We used several fibre-specific gene promoters to drive expression of an expansin gene that is native to cotton and fibre-specific as well. Expansin is a protein known to be involved in cell elongation and growth, and therefore could impact on fibre traits such as length. Normally, the fibre-specific expansin gene expresses predominantly in the first 18 days of fibre development. By assembly of the expansin gene with a promoter (FS18) that usually drives expression of a lipid transfer-like protein that is active late in fibre development, we have been able to extend expansin expression for a further 4-10 days in transgenic plants. The chimeric gene increased the total amount of expansin in fibre cells and altered the morphology of the fibre in ways that are potentially advantageous to the industry. Linked experiments in which this promoter was used to drive a GUS reporter gene in a separate set of transgenic plants showed that the FS18 promoter can drive a high level of transcription specifically in fibre cells to a much later stage of fibre development. The FS18 promoter could be used to express any gene of interest in an effective and predictable manner in cotton fibre cells, and could be an important tool in fibre biotechnology.

Transgenic plants containing the FS18-expansin construct have modified fibre characteristics that could be incorporated into existing breeding programs. In this way new fibre variants could be generated, contributing to germplasm stocks and helping to maintain the reputation of the Australian cotton industry as a producer of premium quality cotton. Our strategy of manipulating gene expression in a tissue-specific manner by utilising native cotton genes is feasible and potentially beneficial to the cotton industry.

Soil nutrients, in particular phosphorus (P) and potassium (K), are being depleted in the clay soils supporting the grains and cotton industries of northern NSW and Queensland, especially in the deeper layers of the soil profile. Current commercial soil tests used to measure these reserves do not detect some of these changes because previously unrecognised pools of nutrients have been replenishing those measured by the extractants. Even when enough fertiliser is applied to maximise yields, inorganic pools of P and K which are not measured by commercial tests are decreasing. Across soil types these backup pools of nutrients range in size and their rate of supply, and in the case of K, the availability to plants can be moderated by high levels of other elements like Na. It is therefore important to quantify the size of these pools, their interaction with applied nutrients in fertiliser and their capacity to supply plant nutrient requirements in our current farming systems.     

In conditions where nutrient limitations are identified or where nutrient replacement strategies have been adopted for long term sustainability, effective fertilisation strategies need to be developed to ensure efficient use of those nutrients by plants. The experiments needed to make these assessments are expensive and time consuming, and so certainty of selecting responsive sites becomes important. The uncertainty with current P and K diagnostic tests, due mainly to the existence of variable sized pools of nutrient reserves, has complicated this selection process.

This small preliminary project was conducted to assess the potential of new P and K diagnostic tests to clearly identify sites with low levels of available P and K on which future fertiliser trials could be conducted. It was also part of an initial assessment of the variability in P and K reserves in different soils and cropping systems across the region. Findings from this project will feed directly into a joint GRDC and CRDC project in which laboratory, field and glasshouse studies will develop new guidelines to ensure profitable use of P and K fertilisers as well as long term sustainability of grains and cotton farming systems.

Key outcomes from this project included (i) evidence of strongly stratified reserves of P and K within the 0-30cm sampling depth in cotton growing soils across the industry, which may have significant implications for nutrient access by cotton crops; (ii) evidence that there is substantial variability in the P and K reserves in soils across the regions, and that the size of these reserves is often not related to existing soil test methods; and (iii) this variability is being used, in combination with in-crop tissue testing, to improve understanding of the relationship between soil tests and plant nutrient status, and to ultimately guide selection of sites for longer term experimentation to develop P and K fertiliser guidelines for the grains and cotton industries.

Research and development programs are under increasing pressure to demonstrate and

enhance their impacts towards the triple bottom lines of environment, economic and social

criteria. Cotton research, development and extension funders are looking for strategies

both to meet this reporting need and to build capability and impact-thinking amongst

research and extension providers. This research reviews literature and existing reports and,

through a series of unstructured and semi-structured interviews, explores the perspectives

of both research funders and researchers towards evaluation.

It has identified a low level of formal evaluation practice and understanding amongst

cotton researchers. However, many researchers regularly gather feed back from industry about their research

and are willing to further explore constructive evaluation. Strong

views are held about a lack of specialised skills, and the need to engage these rather than

build them solely within existing staff. Building an appreciation and understanding

evaluation amongst researchers was regarded important to aid evaluation and improve

projects.

A strategic, holistic view is needed for evaluation of Cotton RD&E to be efficient and

minimise the pressure on industry in gathering data. This efficiency as three core

elements: I) Finding a balance between projects and programs - it is suggested to look at

individual projects up to the level of outputs and at project clusters, programs or key

questions for evaluation of outcomes and impacts; 2) minimising the pressure on industry

by gathering data in smart & efficient ways, unobtrusively where possible; and 3) develop

and resource a clear evaluation strategy.

Also identified in this research are diverse values and roles for cotton research,

with a gradient of embeddedness in industry. There are some conflicts between perceived

industry needs and organisational needs for some scientists, particularily about the need for

peer reviewed publishing.

Development of the following manuals:

*Knowledge Management in cotton & Grain Irrigation

*Planning & conducting Focus Group Interviews

*Strategic Approaches for Evaluation in Agricultural & Natural Resource Management Research Programs

*Strategic approaches for Evaluation in australian Cotton Research Programs

Genomic technologies are set to revolutionise plant biology and we have already seen significant advances in the rate of gene discovery and new ideas engendered by the sequencing of genomes from two model plants, Arabidopsis and rice. This is pushing the frontiers with new developments in biotechnology as plant scientists start to now try to understand what all the genes in these plants do and how we might manipulate them to improve our crops. Monsanto, for example, is reported to be developing new drought stress tolerance transgenes for crops that has come out of studies of master stress regulator genes first identified in Arabidopsis. Some of the research from these model plants will flow through to cotton but to achieve the maximum benefit, particularly in the area of fibre, as these biological structures are not found in rice and Arabidopsis, we must develop a capacity for genomic studies in cotton. CSIRO has taken the first steps in this direction by developing cDNA microarray technologies for cotton that will help us understand better what genes are important in fibre development as well as in other aspects of cotton biology.

Microarrays are formed by robotically depositing specific fragments of DNA (genes) at indexed locations onto glass microscope slides. The microarrays can then be probed with the genes expressed in particular tissues or under particular conditions that have been chemically tagged with a coloured dye. Scanning the slide can then tell you, by the colour of the spots where the DNA has been printed, how much each genes is expressed in those conditions – thousands of genes at a time, giving a whole picture of the differences in gene expression. Knowing what sequence and hence inferred function of the different genes on the slide can then tell you what genes expression is contribution to the problem under investigation.

This project has focussed on developing a microarray slide that contains unique representatives of large numbers of the genes expressed in cotton (concentrating on those expressed in fibres, but also some other tissues). While it will still be a long way from having all the genes present in cotton (Arabidopsis has at least 30,000 genes) it should have a large enough representation that it will be useful for dissecting many important problems in cotton biology and biotechnology. CSIRO had already printed and used a slide containing over 10,000 cotton genes, but only a small fraction had been sequenced. During this project we added new clones to the set and sequenced about a third of the genes and were able to get the rest sequenced by a collaborator in the US. To add to the genes we already had, we were able to purchase another 13,000 unique genes from the US Cotton Genomics Centre (Thea Wilkins, Director) and have been using bioinformatics programs to search through the gene sequences and weed out those that occur more than once, so that we can produce a so-called uni-gene set (where each different gene is only represented once). This allows us to put more informative genes on the slide as they currently have a limit of about 25,000 spots. The laborious process of amplifying up each gene to produce enough DNA to print hundreds of slides and checking that each gene has been amplified properly has been completed and all that remains now is to assemble the sequences into the unigene set ready for printing. The project has generated an important new tool for cotton researches and the array should contain about 25,000 unique genes - the biggest of its type for cotton available anywhere in the world. We will shortly start to use the slide in a project designed to discover what genes are important in determining fibre quality traits like length, strength and fineness and will hopefully help our breeder’s improve the quality of Australian cotton.

Fusarium wilt, caused by Fusarium oxysporum f. sp. vasinfectum (Fov), is a destructive disease of cotton (Gossypium hirsutum L) in almost all cotton producing countries of the world. First reported in 1993, this disease is now widespread in Australia and is causing substantial losses. There are also 17 native Gossypium species or wild cottons in Australia, some of which have ranges that overlap cotton-growing regions. Wild crop relatives are a traditional source of novel resistance genes for many plant diseases, and preliminary studies of Australian Gossypium species suggested they may contain some useful levels of Fusarium wilt resistance. At the same time, however, it was possible that the native species could be harbouring potential cotton pathogens. The main objectives of this project was to explore the risk and the potential of the Australian Gossypium species.

Screening the Australian Gossypium species identified a range of accessions that will be useful in the continuing efforts to develop new cotton cultivars with improved levels of Fusarium wilt resistance. Although there was considerable variation in Fusarium wilt resistance among the Australian Gossypium species, G. sturtianum emerged as a possible source of novel resistance genes. Subsequent analyses confirmed that G. sturtianum was resistant to Fusarium wilt, but genetic analyses have established that transferring the G. sturtianum genes to cultivated cotton will be extremely difficult.

Simultaneously, it has become clear that while the native Gossypium species are not harbouring cotton field pathogens. However, surveys of the pathogen in cotton fields suggest that pathogen is continuing to evolve and continuing vigilance would be appropriate.

This project aimed to investigate the expression and function of a number of genes that we believed were involved in the production of the secondary chemical gossypol in cotton. One of the key enzymes in cotton terpene synthesis is cadinene synthase that is encoded by several different members of a large gene family. The promoter of one of these genes was chosen for analysis based on it sequence and when linked to a reporter gene and inserted into cotton drove expression of that GUS reporter gene in an embryo-specific pattern. As we were after a promoter that would allow us to silence gossypol production in cotton seeds (ie embryos) but not elsewhere in the plant this now provides us with a useful molecular tool for delivering gene knockout constructs to the cells in the seed that are making gossypol.

In addition we had previously isolated a gene for an enzyme of the P450 hydroxylase class that are involved in many biochemical conversions of secondary defence chemicals, but we needed to demonstrate that it was involved in gossypol production not some other chemical in cotton. Gene silencing or knockout constructs have been developed for this gene and introduced into transgenic cotton. We hope to be able to analyse the chemical composition of the oils in the gossypol glands to see what effects plants that should be deficient in the hydroxylase have had in terms of terpene production. In an alternative strategy we are also expressing this gene in simple bread yeast to see what biochemical conversions it can achieve when fed with different intermediates in gossypol synthesis.

A third approach has been to clone a peroxidase we believe is involved in the last step of gossypol production and see what changes in chemical composition occur when this particular gene is silenced in transgenic cotton plants.

Overall we are trying to characterise some of the key enzymes and genes in gossypol synthesis and develop strategies for eliminating gossypol just from the seeds of transgenic cotton plants in an attempt to increase the nutritive value of cotton seed meal and oil without affecting the normal defensive role of these chemicals against pests and pathogens of cotton. Unfortunately, the project was suddenly terminated midstream by the resignation of the student for personal family reasons.

It is evident from experience with the resistance of H. armigera to chemical insecticides and its demonstrated ability to develop resistance to the Cry1Ac toxin produced in INGARD™ that single-gene cotton is not a sustainable control strategy. The second generation of transgenic cotton (BOLLGARD II) prodcues both the Cry1Ac and toxins. This combination was expected to significantly reduce the ease with which H. armigera can develop resistance to these plants. However, a mutagenesis approach adopted by Dr J. Daly has shown that H. armigera is at least capable of to a Cry2A toxin (Cry2a), as well as to Cry1Ac. This result shows that the cotton industry needs to prepare itself for resistance to Cry2Ab as it has with Cry1Ac.

The aim of this project was to determine the magnitude and nature of resistance to Cry2Ab by H. armigera so that an appropriate resistance strategy can be implemented to sustain the use of cry1Ac/cry2Ab transgenic cotton. In the first instance, we set out to establish a Cry2Ab-resistant strain by laboratory selection. Based on our experience in selecting for Cry1A-resistance, we expected that the selection process would be lengthy and that we may only succeed towards the end of the project, at which time we would undertake a preliminary characterisation.

The project was successful beyond expectations in that it produced not one but three strains of H. armigera with resistance to Cry2A. The development of Cry2A resistance in three independent strains after only three generations of selection, coupled with the discovery of Cry2A resistance alleles in field collected insects (in project CSE104C), demonstrates that the optimism that BOLLGARD II™ cotton would provide a solution to the resistance issue may have been misplaced.

Preliminary characterisation of the first of the laboratory-selected Cry2A-resistant strain (TABOC) showed that is not associated with resistance to Cry1Ac and therefore would not be expected to survive on early season BOLLGARD II™. It appears that the resistance detected in the TABOC strain differs from that in the strain obtained by the use of the F2 screen (SP15). It therefore appears that H. armigera has at least two options for resistance to Cry2A.

It is too early at this time to identify the likely consequences of Cry2A resistance in H. armigera. In a successor project to this and CSE104, we will establish whether we have detected one or more types of Cry2A resistance in H. armigera. We shall also determine the dominance of the resistance alleles in the TABOC and SP15 strains and the fitness cost associated with that resistance. This will enable us to develop a refuge strategy to minimise the risk of resistance that would undermine the sustainability of BOLLGARD II™ cotton. Until there is a better understanding of the nature and extent of the threat posed by Cry2A resistance, the cotton industry would be well advised to adopt a cautious attitude to its management of BOLLGARD II™.

Shielded sprayers have been widely and quickly adopted by cotton growers for weed control

in the inter-row. In the last three seasons, the increase in area sown to RoundUp Ready®

cotton has been a major contributor to this because the label directions of RoundUp Ready

Herbicide® specifys the use of shielded sprayers when growing RoundUp Ready® cotton.

After the 2001/02 season, growers reported yield losses up to 30% after using shielded

sprayers. It is thought that leakage of spray was the probable cause of this yield loss. The

Cotton Research and Development Corporation (CRDC) provided funding to both

Conservation Farmers Inc (CFI) and The Centre for Pesticide Application and Safety (CPAS),

School of Agronomy and Horticulture, The University of Queensland, Gatton Campus to

undertake studies to determine the major factors causing herbicide leakage from shields and

to develop strategies that would reduce potential yield loss, but not affect weed control.

Both CFI and CPAS have collaborated in a number of trials to maximise resources. This

document highlights preliminary wind tunnel (laboratory) and field research on the influence of

shield height, wind speed (travel speed), nozzle selection (spray quality), boll retention and

herbicide efficacy. In a one year study there is no scope to confirm experimental

observations, and therefore all results should be seen as preliminary outcomes.

In growing their crops Australian cotton farmers need to comply with Australian regulations. Within Australia, there is a detailed and rigorous process for the , the setting of residue limits (MRLs) and for the labelling of food going to the consumer.

However, knowing that you have used pesticides according to the label and Good Agricultural Practice as defined within Australia may not be enough if cotton byproducts are exported or used in the diet of livestock either domestically or destined for export to countries where regulations differ. To help avoid potentially damaging problems in our export trade, the CRDC have funded a project to monitor the development of international food standads at Codex (the United Nations international food standards setting body).

In addition, gaining access to crop protection products is important to the cotton industry particularly when new pests, such as silver leaf whitefly, emerge. To ensure that relevant crop protection products were available to manage this pest regulatory support was sought in the gaining of APVMA permits. Kevin Bodnaruk’s firm, AKC Consulting, took on these tasks.

To establish if the spider communities in cotton at different locations around Australia are similar, we sampled, using beatsheets and pitfall traps, over 4500 spiders from seven locations extending from the tropical north to the temperate south.

We found that the spider communities around Australia are not structurally identical in that the most prominent spiders do not forage using the same methods. In the spider community sampled by beatsheets, the communities in the south were dominated by Oxyopidae (lynx spiders) which are stalkers; while the communities in the north were dominated by Cycloctenidae, which is a foliage runner. In the more central locations, the most dominant family rotated through lynx spiders (stalker), Clubionidae (yellow night stalkers; which are foliage runners), Salticidae (jumping spiders, which are stalkers), Theridiidae (tangle web spiders, which are web builders), to Clubionidae.

The spider community sampled by pitfall traps was also influenced by seasonal changes, but this was less important than the BDI (or spraying regime) the field had received. This community was overwhelmingly dominated by Lycosidae (wolf spiders, which are ground runners) throughout the year. Exceptions were a few samples taken at the beginning of the season under low BDI where other families, such as Gnaphosidae, which is also a ground runner, nearly equaled wolf spiders in numbers. Most of these samples were from Katherine.

These results mean that spiders in cotton at different locations around Australia will have . Consequently, the incorporation of spiders more directly into IPM will need to be tailored for each location.