I was invited to speak in a symposium on monitoring for resistance to Bt at the 2006 combined Meetings of the Society of Invertebrate Pathology and VIII International Conference on Bacillus thuringiensis (ICBt). These meetings were held in Wuhan, China from 27th August until 1st September 2006. The major output from the trip was to disseminate my latest research results to my peers by presenting a talk in the symposium entitled "Monitoring and managing for Bt-resistance in Australia: The challenges for the next decade". The talks in the symposium gave an International overview of the latest developments in Bt resistance monitoring in terms of current methodologies and up-to-date findings. Our approach to monitoring in Australia was well received and there was considerable interest in my reports of a higher than anticipated frequency of alleles conferring resistance to Cry2Ab in H. armigera. All of the talks in the symposium were presented as written papers in a special edition of the Journal of Invertebrate Pathology in Volume 95 (see below). I established contact with key international researchers in my field of Bt resistance monitoring including Saku Sivasupramaniam from Monsanto, who has subsequently visited ACRI, and Carlos Blanco from the USDA, whom I have been in regular contact with since the meeting. In particular, Carlos and I discussed approaches to studies of sperm precedence and I have since reviewed several of his papers on this topic. I also established an important link with Ryan Kurtz, a research entomologist with Syngenta, and met face-to-face with a collaborator David Heckle, from the Max Plank Institute in Germany and the University of Melbourne, and discussed progress on the CRDC funded project on isolating the Cry2Ab resistance gene in Helicoverpa armigera. Attending the meetings increased my breadth of knowledge by attending talks outside my direct area of interest that focused on microbial control, mechanisms of resistance, and Bt-performance enhancement.

This pilot study involved the processing of five bales of cotton, representing

three varieties i.e. Sicala 3508, Sicala 60BR and Sicot 289BR, into ringspun

carded and combed yarns to determine their textile processing performance.

The results indicate that the yarns and fabrics produced from Sicala 350B

were better then those produced from Sicala 60BR and superior to those

produced from Sicot 289BR. The study did highlight that agronomic and

ginning variables should be controlled as they have a large impact on each

variety's fibre properties and processing performance.

Cotton Bunchy Top is a disease of cotton that was first reported in the 1998-99 cotton season. Symptoms of the disease include shortening of internodes and petioles, . CBT affected plants also have a reduced photosynthetic rate, and reduced cotton lint and seed yields. Research in this project aimed to (i) identify the causal agent of CBT and develop a probe to detect it (ii) identify the vector(s) of the disease and investigate its transmission characteristics and alternative hosts and (iii) Assist in selection of resistance to CBT in elite cotton germplasm. These are discussed below;

• Identification of the causal agent. Despite exhaustive efforts the agent causing CBT remains unidentified. Tests looked for viruses (common RNA virus, circular DNA Geminivirus and nanovirus, double stranded RNA viruses and a range of virus specific ELISA test including Barley Yellow Dwarf Virus), viroids, phytoplasmas and endoparasitic fungus hyphae. Testing also employed subtractive hybridisation, which did not reveal any non-cotton genes, and cDNA microarrays, which did reveal three clones (genes) that did not match the cotton genome. Unfortunately the project ended before these genes could be characterised.

• The disease is vectored by Aphis gossypii (cotton aphid) and attempts to transmit it using Myzus persicae (green peach aphid) or Aphis craccivora (cowpea aphid) failed. Cotton aphid transmits CBT semi-persistently, meaning they can transmit the disease to multiple plants over several days. Aphids can acquire CBT after feeding on CBT affected plants for five minutes and can inoculate a healthy plant after feeding for one hour. As few as one aphid can transmit the disease though more aphids do it more effectively. Cotton plants infected with CBT are capable of infecting aphids with the disease after a period of about 16-17 days, about 2 weeks before symptoms were obvious in the plants.

• Gossypium barbademse cv Pima S7 is a symptomless host of CBT, while the resistant variety DeltaOpal did not host the disease.

• Two alternative hosts of the disease have been identified, both from the family Malvacae. These are Malva parviflora and Malvastrum americanum..

• Over the past four seasons we have screened cotton varieties in the field and glasshouse for resistance to CBT. This has lead to selection of CBT resistance in several elite lines for commercial release of the first variety in 2005-06.

This project has provided the basic information to begin to understand the field epidemiology of this disease. It is likely that it is acquired by aphids feeding on an alternative host. Aphids transmit the disease semi-persistently, so they are infective for days after leaving alternative hosts and entering cotton crops. Cotton plants can become infected by the feeding of a single aphid, though more aphids results in higher transmission rates. Cotton plants colonised by aphids become new CBT sources after about 17 days and show clear symptoms after 3 – 5 weeks. Initial spread of the disease is slow as aphids are in the apterous form but as populations build alates begin to appear and secondary transmission to other parts of the field can occur.

CBT continues to threaten the industry both due to yield reductions and due changes in growers and consultants perceptions of aphids – resulting in lower thresholds, increase spraying and selection for insecticide resistance in aphids. Potential problems due to CBT have been hidden in recent years due to drought reducing over winter survival of aphids. There is a need for continued research to select for CBT resistance in elite germ plasm, to identify the agent so that a probe can be developed to facilitate understanding field epidemiology and breeding and there is a need to further study the field epidemiology so that outbreaks of CBT can be predicted and managed.

Regular disease surveys have highlighted the changing status of cotton diseases over time and

provided valuable insights into the factors affecting their distribution and severity.

• Seedling mortality was low during the 1990’s but increased dramatically in 2000, 2001

and 2002, largely due to cool wet conditions during spring.

• The risk of seedling disease increases with increasing latitude; the southern regions of

NSW are particularly prone with up to 60% seedling loss observed in some fields.

• NSW is currently experiencing an epidemic of black root rot of cotton. Black root rot was

observed on all of the farms surveyed regularly in the Macintyre, Gwydir, Namoi and

Macquarie Valleys, and in 78% of fields and 39% of plants within those farms. The

disease was detected in the Murrumbidgee Valley for the first time in 2003.

• The distribution of continued to increase. Fusarium wilt has been reported

on a total of 75 farms in NSW and was observed on 30% of the farms surveyed regularly

by NSW Department of Primary Industries.

• In NSW, adoption of less-susceptible varieties is ahead of the incidence of Fusarium wilt

• Transects of an infested field in the Macquarie Valley suggested that Fusarium wilt may

progress much more quickly in cooler cotton growing regions.

• Verticillium wilt was observed at very low levels, although its incidence has increased in

the Namoi Valley, probably due to a period of declining use of resistant varieties.

• Many farms do not have black root rot, Fusarium wilt or Verticillium wilt and farm

hygiene should be practiced to minimise further spread.

Experiments were conducted to develop and/or evaluate control strategies for control of

seedling disease, black root rot and Fusarium wilt:

• Delaying the date of sowing as late as possible within the planting window can avoid

conditions that favour seedling disease and black root rot.

• Planting should be timed to coincide with the onset of periods in which the mean soil

temperature will be 16°C during the first week from sowing and delayed after preirrigation

until soil water content is at the lower end of the range that is adequate for

seedling establishment, in a given soil

• Different seedling pathogens vary in dominance from field to field and year to year; the

fungicide treatment DynastyTM consistently performing as well as the standard fungicides.

• Seed treatment with acibenzolar-S-methyl consistently activated resistance against

Fusarium wilt of cotton but not Verticillium wilt.

• A practical method for application of acibenzolar-S-methyl to cotton seed was developed,

using 6 mg/kg seed incorporated in the standard seed treatment fungicides, with no

phytotoxic effects and an extended, active shelf life.

• Vetch, mustard and canola were increased the severity of Fusarium wilt and should not be

used as biofumigation crops on farms with Fusarium wilt

• The peak activity of the black root rot pathogen and seedling pathogens are mutually

exclusive, providing further evidence that T. basicola does not kill cotton seedlings

• Mycorrhizal fungi survived in substantial numbers after long bare fallows of 28 and 35

months in fields at Bourke and after a bare fallow of four years at Narrabri

Results of these experiments and observational studies have been incorporated in strategies

for integrated disease management and disseminated to the cotton industry by way of

publications, media releases, field days and meetings with growers and consultants.

Indonesia has a large spinning capacity of about 7.5 million spindles. About 45-50% of the spindles are on cotton and cotton blends. On an average about 500,000 metric tons of raw cotton are imported per year from all over the world. Australian cotton has a significant share of this for the production of medium count ring spun cotton yarns and blends.

Small laboratory gins are a vital piece of equipment in cotton research at the plant breeding and other subjects where small plots are used. CSIRO’s plant breeding program has more than 30,000 small plots for ginning. Since the instance of Fusarium wilt, sites with that disease have not been sampled for ginning and fibre quality. This reduces the available information on fibre quality. One solution is to have a gin located at Goondiwindi or Moree for ginning samples from breeding or other plots, so fibre can be tested at a contract testing line in the same valley.

The gin has been successfully purchased, delivered and modified to appropriate operating safety standards. The gin ensures better and more comprehensive data from all sites and thus assists with better decisions on breeding line selection.

This project examined how and when individual Helicoverpa armigera carrying a resistant allele (BX) were favoured in field-grown cotton, particularly on Ingard®. BX-like and other forms of resistance to Cry1Ac were found to be rare in field populations of H. armigera. It follows that individuals that carry will almost exclusively be heterozygous. We measured the survival and growth of heterozygous larvae and homozygous susceptible larvae on leaf samples from field-grown Ingard® and conventional varieties of cotton. By performing assays throughout the season, we were able to identify occasions that favoured heterozygotes and thus the evolution of resistance. Averaging relative survival rates of heterozygotes and homozygous susceptible genotypes over the 2002 - 03 and 2003 - 04 seasons we found that, over the first season, susceptible individuals would have survived and grown only half as frequently as the heterozygote. In the second season susceptible individuals fared slightly better surviving 0.68 as well as the heterozygote. Computer models were prepared that incorporated this information as well as new information on the frequency of Cry1Ac resistance in the field.

A second aim of the project was to detect and isolate alleles that conferred resistance to Cry1Ac and Cry2Ab in field populations of Helicoverpa armigera and H. punctigera. An F2 screen technique was employed that enabled the calculation of the frequency of alleles that confer resistance, and importantly, enabled the detection of recessive alleles. No instances of resistance were detected among 68 alleles scored for Cry1Ac and Cry2Ab in H. punctigera. Similarly, no instances of resistance to Cry1Ac were detected among 416 alleles scored for H. armigera. For this species, 95% confidence intervals around the zero determine that Cry1Ac resistance alleles would not be more common than a frequency of 0.0088. Thus despite the deployment of Ingard® for seven seasons, Cry1Ac resistance remains rare.

However, a surprising result was that resistance to Cry2Ab, the second toxin present in Bollgard II®, is relatively common. For H. armigera, three instances of resistance to Cry2Ab were isolated from 416 alleles examined. Preliminary analyses suggest that all three isolates represent mutations at the same locus. While the F2 technique was expected to identify ‘resistant alleles’ if a sufficient number were tested, the expectation was that prior to the deployment of Bollgard II®, Cry2Ab resistance would be at a frequency approaching the mutation rate, say 10 -5 to 10 -8.

With the imminent wide-scale deployment of Bollgard II®, it was important to assess the magnitude of the threat posed to Bollgard II® by the previously unsuspected resistance to Cry2Ab. Unlike the situation for Cry1Ac resistance, where resistant alleles are rare, for the more common Cry2Ab resistance it was important to examine the fitness of both heterozygotes and homozygotes. Laboratory analyses performed in CSE108C have shown that Cry2Ab resistant insects are fully susceptible to Cry1Ac. Thus it is not surprising that early in the season all larvae struggled on Bollgard II®. However later in the season when titres of Cry1Ac presumably had declined, homozygous Cry2Ab resistant larvae (but perhaps not heterozygous larvae) exhibited enhanced growth on Bollgard II® relative to susceptible insects. Thus although the resistance appears to be functionally recessive on Bollgard II®, opportunities exist for the homozygote to be favoured. Together these results suggest that this form of resistance may inpact on the longevity of Bollgard II®.

The significance of transgenic cotton in the pest control strategy adopted by the

Australian industry makes the management of resistance to the Cry1Ac toxin of Bacillus

thuringiensis essential. Accordingly, the CRDC supported the selection of resistance in

H. armigera so that the extent and nature of the threat could be estimated before it

actually occurred in the field.

During selection of the original H. armigera strain (BX) in which Bt resistance was first

detected, resistance rose to 300-fold and then declined to stabilise at approximately 80-

fold. To assess whether the decline in resistance was due to a loss in general vigour

associated with inbreeding or rather was the result of fitness costs associated with the

resistance, the BX strain was outcrossed to a susceptible laboratory strain and re-selected as the IS strain. With increasing selection pressure, the IS strain reached a resistance

ratio exceeding 800-fold. It was clear from this experiment that the decline in resistance

of the BX strain was the result of inbreeding. However, it was not clear whether the very

high resistance detected in the IS strain was merely the result of improved vigour. As

higher levels of resistance in Cry1Ac-resistant diamondback moth can be associated with

more than one resistance gene (Ferr6 and Van Rie, 2002, Ann. Rev. Entomol . 47, 501-

503), the higher resistance in H. armigera might similarly be indicative of the presence

of a secondary resistance gene (or genes). It is important that we understand all the

resistance options available to H. armigera because we cannot be certain that the order in

which resistance genes arise will be the same in the field as in the laboratory.

Fitness cost associated with resistance is an important factor in determining the

parameters required for an effective refuge strategy. When the fitness of the BX strain

was assessed against a susceptible laboratory strain, we recorded a significant delay in

development that might impede the efficiency of the resistance strategy. However, it

would not be reasonable to rely too heavily on these data because of the different genetic

backgrounds of the resistant and susceptible lines used in that experiment. We

backcrossed the resistance allele into the susceptible line so that we now have resistant

(ISOC) and susceptible lines that share a common genetic background, to the extent that

they are >93% genetically similar. We are, therefore, now in a much better position to

assess the fitness cost associated with the lower level of resistance to Cry 1Ac in

.

Cotton aphid (Aphis gossypii Glover) is a late season pest of cotton, with the potential to

reduce the value of lint by contaminating it with sticky honeydew secretions. Recently, cotton

aphid emerged as a threat during the early and mid season - due to its potential to cause yield

loss and its role as a vector of the disease 'Cotton Bunchy Top'(CBT). This project aimed to

improve understanding of the ecology of this pest, especially season abundance and

overwinter host use, and of its distribution on cotton, which is important in developing

sampling techniques. This project was complemented by two other CRDC funded projects;

'Aphid bio-control' (DAQI 19C), and 'Incorporating aphids, insecticides and early season

plant compensation in PM' (CSP147C). Key outcomes are:

1. Cotton aphid is a mid-late summer pest. A wide range of summer and winter hosts were

identified and included a range of broad leaf weeds, crops and garden plants. Among the

winter crop hosts are faba beans and lupins, although aphid do poorly on them. Aphids

failed to persist on vetch, canola or lucerne. In particular ratoon cotton will carry-over

aphid populations, which is a problem for resistance management and for carry-over of

CBT. This led to recommendations for good overwinter field hygiene.

2. It is likely that winter conditions affect the size of the overwintering populations, with

drier conditions supporting fewer hosts and lower overwinter aphid populations.

3. Cotton aphid uses hosts in farm and urban gardens through winter. Farm gardens in

particular can serve as reservoirs for insecticide resistant cotton aphids and are particularly

important in dry years when other hosts are scarce.

4. The seasonal abundance and host range of cowpea aphids was documented. This species

has two peaks in abundance, in early spring and autumn. It also uses a wide range of hosts,

but is particularly noticeable on medics.

5. Green peach aphid is a cool season specialist. It has a narrower host range than cotton

aphid and cowpea aphid, but uses some brassicaceous hosts that are widespread.

6. A wide range of other aphid species and their hosts was also recorded. This information

has proven valuable, for instance in warning industry of influxes of Rhopalosiphum spp.

which settle on seedling cotton, but which will not establish and breed. This led to the

recommendation not to treat aphid populations unless it is clear they are breeding.

7. Cotton aphids show a preference for the upper canopy leaves of plants. However, at higher

numbers there is a significant population found in the lower canopy. This population is

difficult to reach with insecticides and may be a major source of recolonisation of plants

following insecticide application.

8. Cotton aphid shows a highly clumped distribution, a product of their biology, whereby

females produce live young that do not move far away from the parent. This makes

sampling more difficult as populations may be missed.

9. There is a curvilinear relationship between the proportion of plants infested with aphids

and the mean number of aphids per leaf. This may be useful if it can be linked effectively

with the thresholds being derived in another project.

Outcomes of this research have been widely reported to industry. Future research on aphids

could emphasise selective control, improved understanding of colonization and spread in

cotton, contribution of trap crops or relay crops as hosts and link with spread of CBT.

My presentation today is titled "Marketing our Product". I intend to briefly discuss the markets for Australian cotton, the market outlook for our cotton, make come comments on our crop quality this year and then outline some challenges that I see for our industry.