Source: WASHINGTON STATE UNIVERSITY submitted to NRP
IMPLEMENTING LYGUS MANAGEMENT STRATEGIES IN ALFALFA SEED PRODUCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1004706
Grant No.
2014-70005-22540
Cumulative Award Amt.
$156,675.00
Proposal No.
2014-08360
Multistate No.
(N/A)
Project Start Date
Sep 1, 2014
Project End Date
Aug 31, 2016
Grant Year
2014
Program Code
[AFRP]- Alfalfa and Forage Program
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Agricultural Research Center
Non Technical Summary
We propose to conduct a comprehensive investigation on the biology and management of Lygus bug populations infesting fields of western US alfalfa produced for seed. We will investigate pest and beneficial arthropod species constituency and abundance, predatory insect feeding behaviors, and dispersal patterns of Lygus. Simultaneously we plan to investigate the genetic basis of insecticide resistance, using techniques recently developed by our team of scientists. We will create a matrix of these factors and the resulting seasonal insecticide recommendations, based on efficacy on Lygus and safety to beneficial arthropods and pollinators, and make it available to growers via traditional and state-of-the-art electronic push extension technologies.
Animal Health Component
75%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2111640104035%
2111640113065%
Knowledge Area
211 - Insects, Mites, and Other Arthropods Affecting Plants;

Subject Of Investigation
1640 - Alfalfa;

Field Of Science
1040 - Molecular biology; 1130 - Entomology and acarology;
Goals / Objectives
We propose to conduct a comprehensive investigation on the biology and management of Lygus bug populations infesting fields of western US alfalfa produced for seed as a non-food/non-feed crop. We will investigate pest and beneficial arthropod species constituency and abundance, predatory insect feeding behaviors, and dispersal patterns of Lygus. Simultaneously we plan to investigate the genetic basis of insecticide resistance. We will create a matrix of these factors and the resulting seasonal insecticide recommendations, based on efficacy on Lygus and safety to beneficial arthropods and pollinators, and make it available to growers via traditional and recently developed electronic push extension technologies.1. Utilize a standardized DNA region as a tag (DNA barcode) for rapid and accurate species identification for Lygus populations infesting alfalfa seed fields in Idaho, Oregon, Nevada, and Washington.2. Develop and implement DNA-based diet analyses to identify prey from the digestive systems of the generalist predatory arthropod guild present in the candidate crops. These predatory arthropods will include big-eyed bugs, minute pirate bugs, assassin bugs, and spider species.3. Use standard trapping methods (e.g. pan traps, sweep net samples) and DNA markers (i.e. RR) to identify dispersal patterns of Lygus among alfalfa seed and other Lygus crop and non-crop hosts in Idaho, Oregon, Nevada and Washington.4. Develop an insecticide resistance monitoring system using molecular techniques to quantify increased tolerance and resistance to specific insecticides in Lygus populations in alfalfa produced for seed.5. Create a matrix of insecticide chemistries and insect susceptibility profiles that reflects pest susceptibility and safety to non-target predatory, parasitic, and pollinating arthropods.6. Disseminate educational outreach information in a multi-modal, timely, and targeted fashion appropriate to alfalfa seed growers and other stakeholders.
Project Methods
Objective 1Lygus Collection. To identify Lygus to species, adult specimens of Lygus spp. known to occur in the western United States will be collected and identified morphologically to act as voucher specimens. DNA will be collected from the voucher specimens' hind legs. We will collect type specimens of Lygus adults within alfalfa seed fields temporally and at different locations. In order to identify Lygus, adults and late-instar Lygus nymphs will be collected on a monthly basis during the growing season in or near alfalfa seed fields. The adults and nymphs will be placed in 95% ethanol and shipped at ambient temperature to the molecular lab laboratory in Prosser, WA for molecular analysis.DNA Barcoding. Using our previously developed DNA barcoding method for Lygus identification, we will qualify and quantify the species constituency of the various types of Lygus that are infesting alfalfa produced for seed.Objective 2The analysis of predator gut contents using molecular tools has become an important new technique that enables researchers to determine whether or not a particular prey item has been consumed by a particular predator without having observed the event.This approach relies on the use of PCR to amplify specific DNA sequences (DNA barcodes) from the prey that have been ingested by a predator. We can use the DNA barcode technique to study the natural enemy composition of prey and improve the quality of natural enemy surveys by making this information more accurate. We will capitalize on previously developed DNA gene regions as well as develop our own target DNA sequences for rapid and accurate identification of prey from predator gut contents. Our preliminary results indicate that the Lygus gene-specific primers we have designed will permit us specifically to amplify Lygus mtDNA and not Geocoris or Nabis mtDNA (or that of other predatory insects).Objective 3Using a series of pan traps in and between conventional alfalfa seed, Round up Ready alfalfa seed and nearby weedy patches and forage alfalfa in Walla Walla County, WA, we will track dispersal patterns of adult Lygus within and among adjacent fields. Concurrently sweep net samples will be collected from locations interspersed among the pan traps. The Lygus captured in pan traps and sweep nets will be analyzed for the presence of the RR transgene. This study will provide us with empirical knowledge of inter-field movement of Lygus during the seed production season. Emphasis will be placed on the time periods that correspond with the dry down and senescence of alternative weedy hosts and with the several cutting events that take place within forage fields. These are local events that likely contribute to the migration of adult Lygus into alfalfa seed fields.Objective 4Adults of L. hesperus from each location will be tested for resistance to insecticides using a Potter Spray Tower. Insecticides that will be used in this research are pyrethroids (bifenthrin and lambda-cyhalothrin) and organophospates (i.e., chlorpyrifos). Insecticide concentrations will include the recommended field rate and rates both above and below the recommended field rate for each insecticide. At least five concentrations will be tested and each test will be replicated three times on cohorts of 30 adults of L. hesperus. Mortality will be determined after 48 hours, and adults will be considered dead if they are unable to move on their own or when prodded. Data will be collected from the control mortality using Abbot's formula before analysis. Data from the tests will be analyzed using probit analysis to calculate LC50 and LC90, the values of which will be compared with the LC50 and LC90 value from susceptible Lygus bugs to obtain a resistance ratio (RR50). Susceptible colonies will be obtained from colonies that were never exposed to insecticides.We will attempt to identify the frequency of gene mutation in the target site insensitivity. Lygus populations from the field will be identified using the identification key "A field key to the most common Lygus species found in agronomic crops of the central San Joaquin Valley of California" (Mueller et al. 2009). For DNA extraction, adults of L. hesperus will be stored in ethyl alcohol (ETOH) 95%. Five adults will be stored per 1 ml ETOH 95%. For RNA extraction, adults of L. hesperus will be stored in RNAlaterTM RNA stabilization Reagent QIAGEN. Five adults will be stored per 0.5 ml RNAlaterTM. Samples will then be stored at 2-8°C for up to 4 weeks or at -20°C for longer (archival) storage.We will target and perform sequencing of specific regions of the voltage-gated sodium channel gene that is well known to confer resistance to pyrethroids in a variety of arthropod species. One adult of L. hesperus will be used for RNA extraction. The first strand cDNA will be synthesized from the RNA template by reverse transcription reaction using iScript cDNA synthesis kit. Five replicates will be included for each population sample. cDNA will be used as a template for PCR using gene-specific primers. Amplified gene fragments will be purified and used for direct sequencing to identify molecular markers or point mutations indicating insecticide resistance.We will then attempt to determine the role of metabolic detoxification enzymes in the pyrethroid and organophosphate resistance mechanisms. For analysis of our candidate metabolic detoxification enzymes, we will complete semi-quantitative RT-PCR for measuring expression levels between populations. Five adults of L. hesperus will be used for RNA extraction. cDNA fragments will be amplified using a specific primer designed using the gene sequence information from a transcriptome database).Objective 5Different regimes of insecticides are recommended based on geography and climate for Lygus control in alfalfa produced for seed as a non-food/non-feed crop. The special designation as a non-food/non-feed crop has enabled the alfalfa seed growers of Washington, Oregon, Idaho, and Nevada to register a number of newer insecticides without having to go through the rigors of obtaining a food tolerance. Unfortunately, growers are not familiar with the use of these alternative insecticides and they continue to rely on broad-spectrum, long-residual organophosphates and pyrethroids during the pre-bloom and post-bloom periods. Switching to alternative "softer" chemistries during bloom will help to conserve populations of pollinating bees.Objective 6The educational outreach plan includes face-to-face communications at established alfalfa seed events including field days, workshops, and annual research meetings. We will also prepare electronic and printed materials (e.g., handouts/fact sheets, Extension Bulletins, PNW Pest Management Handbook content) reflecting both emerging research results and, eventually, detailed production recommendations. A website will be developed for these materials and the web content will be made available to the various regional agricultural and pest management websites already in place such as http://www.ag.uidaho.edu/ipm/news.htm), http://ipmnet.org/, and http://ipm.wsu.edu. We plan to organize and conduct a Lygus Symposium session at the Pacific Branch Entomological Society of America meeting scheduled for March 2016 in Honolulu, Hawaii.

Progress 09/01/14 to 08/31/16

Outputs
Target Audience:The target audiance consists of seed producers with an emphasis on alfalfa seed growers. However producers of other small seeded vegetable seed benefit from this program because most of the data we collect will extrapolate redily. Changes/Problems:Our biggest challenge for this project was the untimely death of the PhD student that was working on this project in March 2015. Subsequently we had to regroup our efforts and we hired a post doc to finish the project. The post-doc worked on the project and finished the molecular-based research in fall 2015 and spring 2016. The post-doc has now seperated from Washington State University and accepted a position with the University of California. A manuscript based on the gut-content studies of big-eyed bugs is being finalized for submission to the Journal of Economic Entomology. What opportunities for training and professional development has the project provided?Our biggest challenge for this project was the untimely death of the PhD student that was working on this project in March 2015. Subsequently we had to regroup our efforts and we hired a post doc to finish the project. The post-doc worked on the project and finished the molecular-based research in fall 2015 and spring 2016. The post-doc has now seperated from Washington State University and accepted a position with the University of California. A manuscript based on the gut-content studies of big-eyed bugs is being finalized for submission to the Journal of Economic Entomology. How have the results been disseminated to communities of interest?Yes the results have actively been distributed to the growers of alfalfa (and other) seed producers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Goal 1. Utilize a standardized DNA region as a tag (DNA barcode) for rapid and accurate species identification for Lygus populations infesting alfalfa seed fields in Idaho, Oregon, Nevada, and Washington. This objective has been accomplished. We do have a complex of Lygus species persisting in the west, but typically once an insecticide is introduced into the agroecosystm during the growing season Lygus hesperus becomes the predominant species in fields of alfalfa produced for seed. In evaluating the genetic diversity of Lygus populations among western US states we have determined that there is very littlr genetic diversity and it appears that the center of origen for Lygus hesperus is central California. As irrigation was developed in other arid regions of the west and crops like alfalfa were planted in these areas L. hesperus migrated into these new habitats and established endemic populations in these regions. Goal 2. Develop and implement DNA-based diet analyses to identify prey from the digestive systems of the generalist predatory arthropod guild present in the candidate crops. These predatory arthropods will include big-eyed bugs, minute pirate bugs, assassin bugs, and spider species. We have initiated these studies and have collected surveys of insect populations from 50+ site/ field locations. These samples have been sorted. A generalist predatory insect called the big-eyed bug was often present in the greatest abundance among all the generalist predators in the sampled locations. Therefore we are focusing our initial efforts at developing primers for the supposed prey of big-eyed bugs. This includes cowpea aphid and Lygus bugs. We have proved that cow pea aphids are the preferred prey species of most beneficial arthropods. Lygus are far more mobile and difficult to capture for prey species. In summer 2015 we treated a field of alfalfa produced for seed with a selective aphicide. There was a greater abundance of big-eyed bugs then either Lygus or aphids. In this situation the big-eyed bugs fed on Lygus, but we also noted a substantial amount of cannibalism among the big-eyed bugs in the absence of prey. Goal 3. Use standard trapping methods (e.g. pan traps, sweep net samples) and DNA markers (i.e. RR) to identify dispersal patterns of Lygus among alfalfa seed and other Lygus crop and non-crop hosts in Idaho, Oregon, Nevada and Washington. This project is following directly Goal 2 in that we hav now sampled 50+ populations from late winter through high summer in and around fields of alfalfa produced for seed. We have determined that Lygus tend to stay-put within fields of alfalfa prodeuced for seed. A mass migration occurs into fields of alfalfa produced for seed in late spring as rain-fed spring and winter weeds dry down and their value as host plants diminish. Subsequent migrations occur in the growing season from adjacent crops, most notably forage alfalfa when these fields are disturbed by mechanical activity. For forage alfalfa this is when the fields are swathed. Adult Lygus leave the swathed field and migrate into alfalfa fields being produced for seed. Goal 4. Develop an insecticide resistance monitoring system using molecular techniques to quantify increased tolerance and resistance to specific insecticides inLyguspopulations in alfalfa produced for seed. We focused on the organophosphate insecticide chlorpyrifos and the pyrethroid insecticide bifenthrin. With both insecticides we did not observe any true resistance within the Lygus populations we surveyed. The registered field rates of these two insecticdes killed all the populations we teted under controlled conditions. We observed some isolated incidences of increased tolerance and we observed an increase in the titer of housekeeping genes in these populations, but we have not observed any direct point mutations in L. hesperus. We do conclude that growers have been good stewards of the insecticides that are registered on alfalfa produced for seed and that these growers are following our research-based resistance management programs. 5. Create a matrix of insecticide chemistries and insect susceptibility profiles that reflects pest susceptibility and safety to non-target predatory, parasitic, and pollinating arthropods. This matrix will be published in the proceedings of the Western Alfafla Seed Growers meeting in January 2017and then place on the seed grower's assocaition's website.This will also be published in the PNW Insect Control Handbook for 2017. 6. Disseminate educational outreach information in a multi-modal, timely, and targeted fashion appropriate to alfalfa seed growers and other stakeholders. We continued to do this via annual meetings and field days throughout the project. Most notably we host a workshop annually at the Western Alfalfa Seed Grower's Conference.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Amalia, D., M.D. Lavine, L. Corley-Lavine1, & D.B. Walsh. Quantifying tolerance to organophosphate and pyrethroid insecticides among populations of the western tarnished plant bug (Lygus hesperus) in central Washington State, USA. Submitted to Pesticide Science
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Hull, J.J, K. Chaney, S.M. Geib, J. A. Fabrick, C. S. Brent, D. Walsh, and L.Corley Lavine. 2014. Transcriptome-based identification of ABC transporters in the western tarnished plant bug Lygus Hesperus. PLoS 1. DOI: 10.1371/journal.pone.0113046
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Amalia, H., Mark Lavine, Douglas Walsh, & L. C. Lavine. Characterization of chlorpyrifos resistance in western tarnished plant bug, Lygus hesperus knight (Hemiptera: Miridae). Entomological Society of America. Portland, OR 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Pest and Pollinator Management on Alfalfa Seed 2015 Western Alfalfa Seed Growers Conference, Las Vegas, NV 2016


Progress 09/01/14 to 08/31/15

Outputs
Target Audience:The target audiance consists of seed producers with an emphasis on alfalfa seed growers. However producers of other small seeded vegetable seed benefit from this program because most of the data we collect will extrapolate redily. Changes/Problems:We had a tragic accident and a PhD student that was working in direct affiliation to this project was killed. However, we have been able to stay on track and at present we have a post-doctoral researcher completeing several objectives of the project. What opportunities for training and professional development has the project provided?PhD Candidate Herma Amalia was able to present her preliminary results in a poster presentation at the Entomological Society of America meeting in Portland, OR in November 2014. How have the results been disseminated to communities of interest?A progress report has been submitted by Walsh to the Washington State Alfalfa Seed Commission on December 9, 2014. Presentations are give at the Western Alfalfa Seed Growers Association in January 2015 in Las Vegas and in Pasco, WA in February 2015. A field day was conductd in June 2015. What do you plan to do during the next reporting period to accomplish the goals?We had a tragedy befall this project. A PhD student died in a tragic accident in March 2014. She was granted a posthumos PhD degree and we have submitted a sub set of her dissertation the section on molecular markers for insecticide resistance in L. hesperus and submitted it to Pesticide Science. We will focus more on the predatory gut content and Extension delivery objective of this project for the next reporting period.

Impacts
What was accomplished under these goals? Goal 1. Utilize a standardized DNA region as a tag (DNA barcode) for rapid and accurate species identification for Lygus populations infesting alfalfa seed fields in Idaho, Oregon, Nevada, and Washington. This objective has been accomplished. We do have a complex of Lygus species persisting in the west, but typically once an insecticide is introduced into the agroecosystm during the growing season Lygus hesperus becomes the predominant species in fields of alfalfa produced for seed. Goal 2. Develop and implement DNA-based diet analyses to identify prey from the digestive systems of the generalist predatory arthropod guild present in the candidate crops. These predatory arthropods will include big-eyed bugs, minute pirate bugs, assassin bugs, and spider species.We have initiated these studies and have collected surveys of insect populations from 50+ site/ field locations. These samples have been sorted. A generalist predatory insect called the big-eyed bug was often present in the greatest abundance among all the generalist predators in the sampled locations. Therefore we are focusing our initial efforts at developing primers for the supposed prey of big-eyed bugs. This includes cowpea aphid and Lygus bugs. Goal 3. Use standard trapping methods (e.g. pan traps, sweep net samples) and DNA markers (i.e. RR) to identify dispersal patterns of Lygus among alfalfa seed and other Lygus crop and non-crop hosts in Idaho, Oregon, Nevada and Washington. This project is following directly Goal 2 in that we hav now sampled 50+ populations from late winter through high summer in and around fields of alfalfa produced for seed. Goal 4. This project is complete. Goals 5 and 6 are included within the objective we will complete in the next reporting period

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Herma Amalia1, Mark D. Lavine1,2, Laura Corley Lavine1, and Douglas B. Walsh1,2 Increased tolerance to organophosphate and pyrethroid insecticides in populations of the western tarnished plant bug (Lygus hesperus) in central Washington State. Submitted to Pest Mamangement Science.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Hull, J.J, K. Chaney, S.M. Geib2, J. A. Fabrick, C. S. Brent, Douglas Walsh, and Laura Corley Lavine. In Press. Transcriptome-based identification of ABC transporters in the western tarnished plant bug Lygus Hesperus. PLoS 1.
  • Type: Journal Articles Status: Submitted Year Published: 2016 Citation: *Amalia, D., M.D. Lavine, L. Corley-Lavine1, & D.B. Walsh. Quantifying tolerance to organophosphate and pyrethroid insecticides among populations of the western tarnished plant bug (Lygus hesperus) in central Washington State, USA. Submitted to Pesticide Science
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Herma Amalia , Entomology, Washington State University, Pullman, WA Mark Lavine , Entomology, Washington State University, Pullman, WA Douglas Walsh , Entomology, Washington State University, Prosser, WA Laura C Lavine , Department of Entomology, Washington State University, Pullman, WA Characterization of chlorpyrifos resistance in western tarnished plant bug, Lygus hesperus knight (Hemiptera: Miridae. Entomological Society of America Potland OR, November 2015