Progress 06/01/17 to 01/31/19
Outputs
Target Audience:The bio-based methods of pheromone production explored through this project will allow for the generation of large quantities of high-value pheromone AIs for use against a wide variety of agricultural insect pests using natural, renewable feedstocks and starting materials and eliminating reliance on petrochemicals for these purposes. In addition to these environmental and sustainability benefits, both bio-based production strategies will reduce the cost of pheromone AIs significantly, allowing for higher loading rates in mating disruption and A&K formulations (which may lead to increased efficacy and/or longevity) and for lower costs for these formulations, improving their commercial competitiveness with traditional pesticides. In light of these considerable advantages, we project that the market for the proposed technologies is large and varied, but will be particularly robust in the field of row crop pest management, where pheromone-based pest control techniques have yet to gain a foothold. An example of a product that will benefit from incorporation of bio-produced pheromone AIs is SPLAT Helio, a mating disruption formulation designed for Helicoverpa moth pests.These reductions would enable ISCA's products to outcompete other manufacturers of pheromone-based pest control products relying on traditional synthetic methods for production of their AIs, as well as narrowing the gap between the costs of pheromone tactics and the cost of pesticide cover sprays. Combined with the long-term benefits of these IPM techniques, such as improved environmental and safety profile, more effective and lasting pest control, and reduced risk of resistance, this advantage will provide an excellent opportunity for mating disruption, A&K, and other pheromone-based pest control techniques to become mainstream staples for row crop management. Changes/Problems:The work plan originally proposed for Phase I of this project was designed to focus primarily on assessments of whether pheromone components produced by Y. lipolytica cell factories induced the same behavioral effects on target insect species as analogous pheromones produced by traditional synthetic methods, or whether further processing or purification was required to deliver these effects. Unfortunately, due to unforeseen difficulties in the licensing agreement between ISCA Technologies and our collaborators at Biopheromone ApS, responsible for the creation of the Yarrowia lipolytica cell factories upon which this research was planned, we were ultimately unable to obtain a sufficient quantity of the yeast-brewed pheromone blend to carry out the planned trials described above. As a result of this delay, we chose to focus our Phase I work instead on the use of Camelina sativa seeds for bio-based pheromone production.Phase I research on GM Camelina sativa seed oil consisted of four tasks: 1) to create a line of C. sativa capable of producing the unsaturated fatty acids and corresponding alcohols to generate pheromone precursors; 2) to select transformed lines to produce these precursors at high levels; 3) to isolate and purify the pheromone precursors from the seed oils; 4) to demonstrate conversion of one such precursor, Z11-16:acid to the final pheromone compounds and incorporate them into controlled-release lures to be tested in field trapping studies. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?A major component of ISCA's Phase I and Phase II projects for the USDA SBIR program consists of establishing and maintaining informative and productive relationships with the various stakeholder groups affected by a particular insect management problem or opportunity, from agricultural producers and their customers, manufactures and distributors, to government officials and pest control advisors. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Task 1. Create a line of C. sativa capable of producing the unsaturated fatty acids and corresponding alcohols to generate pheromone precursors. The main objective of this task was to generate fatty acids as components of Camelina seed triacyl glycerols, and to reduce the acid products in the plants to alcohols, which could later be sequestered into wax esters. Several transformed lines were developed, capable of producing several insect pheromone components and/or precursors, including Z9-12:acid, E8,E10-12:acid, Z9-14:acid, and Z11-14:acid. Task 2. Select transformed Camelina lines to produce these precursors at high levels. Selection and promotion of the following transformed Camelina lines have been conducted in the GMO greenhouses at Lund and Alnarp: Z11-16:acid line, which has since been propagated in the field in Lincoln, Nebraska (USA) and Borgeby (Sweden); Z9-14:acid line, currently in greenhouse cultivation; and E/Z11-14:OH line, which produces 16% wax esters in the seed oil and wax fraction containing 20% of the target compounds. Task 3. Isolate and purify the pheromone precursors from the seed oils. For this task, Camelina lines with the highest contents of the desired Z11-16:acid were multiplied in the GMO greenhouse in Lund for field trials. Field trials were conducted in both Nebraska and Borgeby, yielding ~1.8 kg and 5.4 kg seed harvest respectively. The seeds from both field trials were sampled and analyzed. The seeds harvested from Nebraska contained 12.7% Z11-16:acid, similar to the level in the parental seeds. The seeds harvested from Borgeby contained 19.2% Z11-16:acid, higher than the parental level (15.8%). In a germination test, seeds from Borgeby showed a high germination rate (>85%), whereas the germination rate of Nebraska seeds was very low, possibly because the seeds were not ripe enough when harvested. All seeds harvested from Nebraska (1.8 kg) were subjected to extraction procedures, resulting in ~220 g of crude seed oil. The oil's fatty acyl components were then converted to ~170 g methyl esters after base-methanolysis, followed by short-path distillation, producing ~60 g of C16 fraction, with a target compound purity of 18%. From the Borgeby harvest, ~2.5 kg seeds were extracted, which yielded ~640 g seed oil and 490 g methyl esters after base-methanolysis (short-path distillation is ongoing). Urea-complexation is planned to further increase the target compound purity by removing the saturated C16 acid but has not yet been conducted. Task 4. Demonstrate conversion of one pheromone precursor, Z11-16:acid, to the final pheromone compounds and incorporate them into controlled-release lures to be tested in field trapping studies. In 2016 field trapping experiments, plant seed oil-derived pheromones targeting the global cabbage pest Plutella xylostella, demonstrated equivalent levels of efficacy as moth attractants as a synthetic pheromone blend (positive control), regardless of the degree of the purity, which was tested at two different levels, 38% and 83%. Impurities present did not significantly reduce attraction activity. To expand the field activity test, in 2018 pheromone blends were formulated to match several key agricultural pests of rice and maize, including Asiatic rice borer (Chilo suppressalis), cotton bollworm (Helicoverpa armigera), and Oriental armyworm (Mythimna separate). These pheromone blends were used as lures in field trapping experiments in China. A small number of Chilo suppressalis were trapped, both in the traps baited with plant-derived pheromones and traps with conventionally produced pheromones. No trap catch was observed for the other species. Since the positive control group using synthetic pheromones showed the same results, it was assumed that the pheromone blend for Mythimna separate was suboptimal, Helicoverpa armigera did not seem to be present in the experimental field. While the unforeseen delay in the performance of the planned research on pheromone brews produced by Yarrowia lipolytica cell factories was a significant hindrance toward meeting our original PI project goals, progress made with the development of pheromone precursor-producing GM Camelina sativa seeds has been highly encouraging. Multiple C. sativa lines have been created to generate the target precursor compounds (Z11-16:Acid, Z11-14:Acid, E11-14:Acid, Z9-14:Acid, E8E10-12:Acid, and Z/E11-14:OH), which are currently at varying stages of breeding and testing. These transformed lines have also been subject to selection for capacity to produce the target compounds at high levels. Camelina seeds planted in field trials in Nebraska (US) and Borgeby (Sweden) were found to yield oils containing 12.7% and 15.8% Z11-16:Acid, respectively. Additional field trials in 2016 evaluating the pheromone precursor, Z11-16:acid--produced from modified C. sativa seed oil and incorporated into controlled-release lures--delivered an equivalent degree of attraction to one key pest (Plutella xylostella) as a synthetic pheromone blend, whether the level of purity of the AI was 38% or 83%. Later trials in 2018 against three additional rice and maize pests, Asiatic rice borer (Chilo suppressalis), cotton bollworm (Helicoverpa armigera), and Oriental armyworm (Mythimna separate), were complicated by low or absent pest populations, as demonstrated by low or zero capture rates in both traps baited with C. sativa-produced compounds and those baited with synthetic pheromone lures.
Publications
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