Monitoring to Manage Resistance to Bt Toxins

Abstract

The industry is entering a critical period in managing resistance to Bt-cotton. One technology provider is about to release their 3rd generation product which is based on the current two gene varieties (Cry1Ac, Cry2Ab) plus Vip3A. It is possible that in the near future other technology providers will enter the market. If more than one Bt-cotton product is commercial, at least in the short term, it is likely that larvae will be exposed to the same or different varieties of Bt toxins within the 3 classes that are currently included in the resistance monitoring program: Cry1A, Cry2A and Vip3A. Resistant insects are likely to tolerate different varieties of toxins within the same class, so for instance, resistance frequencies for Cry2Aa should be the same as those for Cry2Ab. The development of robust resistance management plans (RMPs) for these technologies depends critically on the frequencies of common resistances to these key classes of Bt toxins.

In this project we achieved our main planned outcome of rigorously assessing the sensitivity of field populations of Helicoverpa to Cry1Ac, Cry2Ab and Vip3A toxins to detect early signs of the development of resistance to genetically modified cotton. It was utilised in mathematical models that evaluated the risk of resistance evolving to three toxin (Cry1Ac, Cry2Ab, Vip3A) cotton and ultimately to the development of a Resistance Management Plan for Bollgard III cotton.

In H. punctigera the proportion of individuals in the population that are heterozygous (rS) for the cry1Ac resistance gene is 2% (based on F1 data: 1% of alleles is equivalent to 2% of individuals). Cry2Ab resistance genes were present at detectable levels before Bollgard II® was widespread. In H. armigera the proportion of individuals in the population that are heterozygous for the cry2Ab resistance gene is 4%. In H. punctigera the proportion of individuals in the population that are heterozygous for the cry2Ab resistance gene is 3%. Vip3A resistance genes are present at detectable levels before Bollgard III® is due to be released (in 2016/17). In H. armigera and H. punctigera the proportion of individuals in the population that are heterozygous for the vip3A resistance gene is 2%.

Individuals that are homozygous for the Cry1Ac allele (H. punctigera), Cry2Ab allele (H. armigera and H. punctigera) or Vip3A allele (H. armigera and H. punctigera) have been detected since F1 screens commenced; these frequencies are not significantly different from what is expected based on the frequencies of heterozygotes.

No new dominant resistances to Cry1Ac, Cry2Ab or Vip3A have been detected.

There have been no reported field failures of Bollgard II and the occasional occurrence of threshold levels of Helicoverpa in some Bollgard II fields is not due to a physiological resistance to Bt toxins. A survey of crop consultants suggests that the presence of medium-large larvae in Bollgard II® is (1) not increasing (from 2005/06 to 2015/16); (2) widespread among valleys and climatic regions; and (3) usually controlled especially recently. Thresholds are equally likely to be driven by numbers of medium-large versus small larvae. Bollgard II is sometimes sprayed for larvae below threshold.

Moving forward we suggest continuing with the shift in focus toward known resistances and screening for any new dominant resistances. New changes include screening every other year and shift towards using molecular tools to assist bioassays.

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Carbon Farming in the Australia Cotton Industry (CFEO project ID: EO12-01-0118)

Abstract

Seedbed Media was appointed to provide communications support for CottonInfo’s Carbon Farming extension and outreach project. The project engaged growers via four key topics; Nitrogen efficiency, energy efficiency, natural resource management and climate and seasonal forecasting. Each of these four pillars formed the foundation of media communications which Seedbed Media employed on CRDC's behalf, to deliver key messages to growers about the benefits of reducing their carbon footprint through the provision of project management, event management, media relations and writing expertise.

The CRDC Carbon Farming Project aims to focus on extension and outreach to growers – to better engage farmers about the potential benefits, especially profit benefits, of emissions reduction and carbon sequestration on their farms. The Project also sought to educate farmers about how to do this, including exploration of the role of fertiliser and fuel inputs.

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The Use of Biological Control Agents in Resistance Management of Helicoverpa

Abstract

In the Australian cotton industry, toxins produced by the soil bacterium Bacillus thuringiensis (Bt toxins) are utilized to control lepidopteran pests: Helicoverpa armigera (cotton bollworm) and H. punctigera. Bt toxins kill insects by forming pores in the insect midgut, which leads to sepsis. The extensive use of Bt toxins including in the form of transgenic crops, has put a strong selection pressure on the pest insects in the field, leading to the development of resistant strains. Understanding the resistance mechanism is essential for planning the resistance management strategy to prolong the effectiveness of the Bt toxins.

Previous studies have demonstrated that larvae of cotton bollworm can develop a low-level tolerance to Bt toxins after being exposed to a sub-lethal dose (Rahman et al. 2004; Rahman et al. 2011). This induced tolerance is associated with increased immune activity in the midgut and hemolymph. The induced tolerance and the increase in the immune activity can be transferred to the next generation mainly via maternal effect, and the level of tolerance can increase over generations of exposure. In addition, highly-selected Cry1Ac-resistant strain also exhibits the same feature as low dose selected inducible tolerance H. armigara (Akhurst et al. 2003; Ma et al. 2005). The development of Cry1Ac tolerance is a threat to the use of Bt technology. Even though many studies have reported the increased immune response against Bt toxins, the role of the immune system in facilitating inducible tolerance against Bt toxins is unclear. Understanding the mechanism of inducible tolerance will help strengthening the established resistant management strategy.

The effect of the maternal experience on the offspring’s immune system (trans-generational immune priming; TGIP) has been demonstrated in several studies. Although there is speculation on the mechanism of TGIP such as the insertion of immune substances into eggs, and changes in the DNA methylation state of the offspring’s genome, the genes and metabolic pathways involved in the transmission are still undefined.

Given that immune components could be maternally transmitted via eggs, together with the importance of egg parasitoids to integrated cotton pest management, it is important to also understand whether there is any negative effect of Bt tolerance/exposure on H. armigera eggs with regard to parasitisation. A study done on egg parasitism by Trichogramma brassicae has also demonstrated that the parasitism success is greatly reduced in eggs from H. armigera survived from GM Bt maize (Steinbrecher 2004). Thus, the primary aim of this study was to investigate the TGIP mechanism of inducible Bt tolerance. The study also investigated the effect of inducible tolerance on key metrics of egg parasitism by the parasitoid wasp, Trichogramma pretiosum.

In this study, we compared the gene expression profiles of eggs from susceptible, Cry1Ac-tolerant and Cry1Ac-resistant H. armigera. We also investigated the parasitism success of Trichogramma wasp on eggs of susceptible and Cry1Ac-tolerant insects by measuring the number of eggs being successfully parasitized, and the number of progeny produced.

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2017-18 Grower Research, Development and Extension Advisory Panels ,'Capacity Building'

Abstract

Cotton Australia engages cotton growers, consultants, and representatives of industry bodies to provide strategic advice regarding CRDC investments and industry stewardship programs.

The Cotton Australia Member Representatives provide practical advice on research, The funding facilitated opportunities for the development of stakeholder capacity for providing advice on strategic cotton RD&E issues. and extension (RDE) needs, opportunities, and priorities. This advice is important guidance to CRDC in its formation of five-year Strategic R&D Plans, Annual Operational Plans, Expressions of Interest for RD&E and resultant CRDC decisions as to project investments.

Cotton Australia facilitates informal advisory panels that are aligned with the CRDC strategic plan priorities. The panels consist of up to 40 grower, consultant and ginners members from every cotton growing region.

The TIMS Committee is facilitated by Cotton Australia and the TIMS Technical Panels are facilitated by CRDC. TIMS functions as a cotton industry stewardship group, with broad representation from growers, research organisations, crop consultants and members of the pulse and grains industries. The agricultural chemical & biotechnology companies that provide crop protection tools to Australian cotton growers approach the TIMS Committee for advice on issues associated with developing or amending resistance management plans for new or existing technologies. Cotton Australia is represented by 6 grower representatives, the TIMS Committee Chair, the Chairs of the three technical panels and the Executive Officer.

The Cotton Biosecurity Reference Group (Cotton BRG) has traditionally met annually to ensure that the cotton industry’s responsibilities under the Emergency Plant Pest Response (EPPR) Deed are met. Development of an implementation plan through recent revision of the Industry Biosecurity Plan has led to a need to formalise this group to address key biosecurity needs of the industry. The IBG includes 1 grower and 1 staff representative of Cotton Australia as well as representatives from CRDC, CottonInfo, CSIRO, NSW DPI, QDAF, and a number of Australian Universities.

The Sustainability Working Group (SWG) manages the industry’s ongoing commitment to becoming the producer and supplier of the most environmentally and socially responsible cotton in the world. The SWG maintains oversight to ensure communication and coordination across a range of related cotton industry sustainability activities. The SWG is represented by 2 staff representatives each from CRDC and Cotton Australia, a cotton grower, ACSA, and representatives of the myBMP, Cotton To Market programs.

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Scholarship: UNE Cotton Production Course 2012

Abstract

The Cotton CRCD's Cotton Production Course was specifically designed to provide Australian cotton industry personnel with a university course that covered the crop science and natural resource management specific to modern, sustainable cotton production. The Cotton CRC effectively encourages a partnership between the research and industry expertise of Cotton CRC participants and the educational expertise of The University of New England and The University of Sydney within their agricultural awards. This relationship has brought benefits to industry, the universities involved and especially the course participants, the students. Students gain: internationally recognised tertiary qualifications; an integrated education framework, a professional yet commercially independent information and curriculum, awareness of the latest reputable research; industry relevance; and a strong networking experience in the Australian cotton industry. Industry gains: better qualified and educated personnel; an excellent avenue to extend research to those most likely to use it; and a platform to encourage industry leaders in production, business and research. The universities gain the street credibility and recent knowledge that industry researchers and experts bring to the unit manuals and the classroom. The positives of this partnership are well recognised by the students in evaluations that praise the residential schools that form the core of the externally delivered units. The cotton units are delivered through the University of New England. Students complete the Cotton Production units as internal undergraduates or as full-time cotton personnel studying by correspondence (i.e. as external students, studying off-campus). External students usually complete one unit per semester commonly towards a Diploma of Agriculture, Degree in Agriculture/Rural Science, Graduate Certificate of Rural Science, or Masters of Agriculture. There are many other awards that the cotton units can contribute to, even a course work PhD, but virtually all students so far have used them for Certificate, Diploma, Graduate Certificate or Masters awards. For example, a student completing the cotton production units at graduate level will receive a Graduate Certificate in Rural Science (Cotton Production) . Students choose whether to have their major in cotton production acknowledged on the testamur since it tends to brand them as a specialist.

Internally enrolled (on-campus) students are able to enrol in the first unit of the cotton production course (Applied Cotton Production) at The University of New England, The University of Sydney or The University of Queensland as part of their agriculture, science, natural resource management, economics, or agbusiness degrees. This allows students of agriculture at three major Australian universities to learn about the Australian cotton industry before they graduate to fill positions in agriculture. Approximately thirty university students complete the Applied Cotton Production unit each year in this way. Reaching soon-to-be graduates with positive messages about primary production is a very important aspect of the Cotton Production Course project under the prevailing climate of extremely low interest in careers in the sciences and particularly agriculture.

Comments from two participants graduating 2012.

"The UNE Cotton Production Course has assisted me by being able to apply the science behind cotton production to the paddock. Compared to my under-graduate study, the UNE Cotton Production Course was highly specific to my work as an agronomist." Tom Webb

"Bringing together scientific theory with practical applications in the paddock. The Systems

unit was also a particularly good way to bring together the learning from the earlier units of

the course. The presentation skills and also project at the end of this unit were a good

transition into putting the skills learned into a workplace situation." Helen Crossley

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2015/16 Grower RDE Advisory Panels - R&D Consultation

Abstract

This project is to facilitate grower participation in CA Grower panels to provide research development and extension investment advice.The Cotton Australia grower RD&E Advisory Panels function in providing practical advice on research, development and extension (RD&E) needs and priorities within the industry. This advice forms important guidance to CRDC for strategic R&D planning, annual operation planning, development of Expressions of Interest and resultant CRDC decisions as to project investments.

Cotton Australia facilitates 4 advisory panels that are aligned with the CRDC strategic plan priorities. The panels consist of up to 40 grower, consultant or ginning Member Representatives from every cotton growing region in Australia.

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Unravelling the genetics of fusarium wilt resistance in cotton

Abstract

Despite its widespread occurrence, the genetics of fusarium wilt resistance in cotton has still not been clearly elucidated. This is a result, in part, of the complex phenotyping involved, but also, in some cotton growing regions, the compounding effects of root knot nematode interactions with the severity of fusarium wilt symptoms. More importantly, the genetic relationships among fusarium wilt pathogens around the world are complex, suggesting that a single genetic model may not be applicable universally. To elucidate the genetics of fusarium wilt resistance against Australian fusarium wilt pathogens (Fusarium oxysporum f.sp vasinfectum VCGs 00011 & 00012), two genetic families have been used. The purpose of this paper is to present evidence that G. siurtianum and G.barbadense harbour genes of interest for the genetic improvement of cultivated cotton.

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Fitness and mechanism of resistance to Cry2Ab in Helicoverpa armigera

Abstract

Transgenic cotton has proved to be valued by Australian cotton growers and over 80% of all cotton grown is Bollgard II. The benefits, largely accrued through a reduction in the use of insecticides, have resulted in some cost savings but markedly improved environmental outcomes. A return to a state where 10 sprays per season are required for Helicoverpa control would be unwelcome. Yet the industry is presently reliant on only one transgenic insecticidal variety of cotton - Bollgard II. Resistance to the toxins Cry1Ac and Cry2Ab present in this variety is the greatest challenge to transgenic cotton’s long term sustainability.

Work by CSIRO Entomology has shown that resistance to Cry1Ac remains rare as we have not yet encountered Cry1Ac resistance in the Bt monitoring program. However, we know that an uncommon but potent form of resistance exists in H. armigera populations in China, and it is prudent to assume it is present within Australian populations. Of more immediate concern is the presence of Cry2Ab resistance in Australian populations of this species. We have shown that a variant form (an allele) of a single gene confers resistance to this toxin in H. armigera. That allele is present at a frequency of four in every thousand copies of the gene so it is certainly available to respond to selection.

Since isolating this form of resistance in 2002, CSIRO Entomology’s ‘Bt group’ in Canberra and Narrabri has been studying aspects of the resistance to evaluate the threat it poses to the Australian cotton industry. This project has contributed much to our understanding of the Bt resistance. In addition we possessed a laboratory strain called TABOC, that was selected to be resistant to Cry2Ab, and we also examined its characteristics and compared them to the field-derived form of resistance.

We examined five of the seventeen separate isolations of Cry2Ab resistance in H. armigera to determine their genetic relationship. For all isolations tested to date, resistance was found to result from alleles at the one locus. As the characteristics such as growth and survival rates of larvae were similar for the remaining isolates, we speculate that that they too may be the same form of resistance. In contrast, the resistance present in the laboratory-selected strain TABOC proved to be the result of variants at differing genes or perhaps a constellation of genes.

We examined the performance of a representative (SP15) of the field-derived form of Cry2Ab resistance when fed Bollgard II. Because SP15 is susceptible to Cry1Ac, it performed poorly on younger cotton, but significantly better than susceptible insects on older cotton; presumably when the Cry1Ac titre had declined. Nevertheless, survival rates of resistant insects was low <10%. Importantly, in laboratory tests and when challenged on Bollgard II, larvae carrying one copy of the ‘resistant gene’ (heterozygous) proved to be susceptible to Cry2Ab. This is important as had heterozygotes proved to show an advantage, the threat posed by this form of resistance would have been markedly enhanced.

In many instances where insects develop resistance to an insecticide or to a Bt toxin, individuals carrying the resistance are less fit than susceptible ones. Thus in the absence of the selective agent (in our case host plants other than Cry2Ab-expressing toxin Bollgard II) resistant insects perform poorly. Parameters such as survival and growth rates, fertility and fecundity can be affected in individuals carrying ‘resistant genes’. Such ‘fitness costs’ of resistance tend to retard the evolution of resistance as on non-challenging environments (in our situation, H. armigera growing on refuge crops, weeds or alternative hosts) the frequency of resistance declines. We challenged SP15 under a range of conditions aimed to expose the presence of fitness costs – growth on pigeon pea, conventional cotton during diapause and when exposed to different temperature regimes – however, no costs were detected.

Finally, a component of this project concerned the mechanism of Cry2Ab resistance. An unexpected problem was encountered when this was addressed. The purified Cry2Ab toxin proved to be ‘sticky’ and adhered to cellular material and components of the substrates used to examine its binding. Nevertheless, progress was made in comparing the array of proteins expressed in gut cells that may lead us to identify the causes of resistance.

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Maximizing the Profitability of Cotton Cropping Systems with Legumes pjp

Abstract

In earlier research, vetch was shown to be the best legume for N fixation, and it is now grown commercially as part of the cropping system on some farms across most cotton growing regions. After nine years of research on a cropping systems experiment conducted at the Narrabri Australian Cotton Research Institute growing vetch as a green manure crop provided not only substantial nutritional benefits to cotton and improved soil structure, but also gave a greater economic return. This article reports the economic benefits of incorporating legumes into cotton cropping systems. Envelope&gt;� �

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Improved measurement of soil water under irrigated cotton

Abstract

he summer scholarship project was awarded to Tami Mills (BSc), currently enrolled in Masters in Engineering Technology Agriculture, at the University of Southern Queensland, Toowoomba. The scholarship commenced on 20th November, 2007 and was completed on 20th April 2008. Scholarships funds were administered through The Condamine Alliance.This project was in collaboration with CRC Project 2.1.02 &quote;Capturing our understanding of soil water balance and deep drainage under irrigation in models&quote;, a project funded in partnership with Cotton Catchment Communities CRC, CRDC, Condamine Alliance and QMDC (led by Dr Mark Silburn).The EM38 was easy to use and gave results that were highly correlated with soil moisture contents to a range of depths. Although multiple measurements are required to account for spatial variability, the measurements are quite easy to make. The collection of 192 ECa measurements (32 points at 3 heights in 2 modes in one location) took approximately half an hour.The data indicate that for each situation (head, mid, tail, row, ditch) 4 measurements are typically required in vertical mode to get an average that is within 5 mS/m of the true mean. In horizontal mode, 15 measurements are typically required for the same accuracy. The greater number of samples required in horizontal mode is presumably because the surface soil has more variability due to cracking and roughness. In larger areas, it will be important to consider the spatial distribution of water, salt and clay in the soil when sampling.When the soil is close to saturated with water, the accuracy of the results may be less than at other times. Because we only had calibration data from drained upper limit and drier, the accuracy is not known. However, similar calibration problems would exist regardless of the sensor or method used. Collecting data near saturation is extremely difficult because of problems such as vehicle access and physically removing soil samples from soil coring tubes.Raising the EM38 to sample to a range of depths was very effective. Good correlation was obtained for both orientations and all heights. Interestingly, at 0.4 m height, the variability of measurements in the horizontal mode was less than at the soil surface, so there appears to be considerable potential for using the EM38 in horizontal mode at 0.3 to 0.5 m height to measure the water content of shallow surface layers of soil.Temperature, time of day, size of the cotton plants, and other potential effects on the readings from the EM38 were non-significant.The EM38 monitoring highlighted how wet the soil was until late March. Some of this was due to rainfall soon after the irrigation in late January, but it appears that the irrigation was going to be somewhat earlier than necessary. The soil moisture deficit in late January was small when compared with later in the season.The data collected by the EM38 agreed to a large degree with the predictions from the HowLeaky? Model, which estimated that during two periods in the season, a significant amount of deep drainage occurred (100 mm). This is of concern both in terms of water wastage and the potential for it to contribute to the rise of salty groundwater and surface salinity.

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