Update:
(4) Biological control
• Regarding the objective to development an aphid-killing virus, current results were published in the Journal of General Virology. However, no further research on this objective is planned as it is still too far from practical application at this time.
• We completed analysis of 2014 Minnesota-wide sampling and conclude that the parasitoid Aphelinus certus has spread to all soybean-growing areas of Minnesota with the exception of the Southeastern section.
• We have concluded that there was no successful overwintering of A. glycinis in summer 2014 populations, following releases the previous year.

(5) Extension and outreach
• Our capstone outreach event was to organize an NCSRP booth for the 2015 Commodity Classic. Funds from this grant and another NCSRP grant on producer priorities were used to support this deliverable. Additional details are supplied in the other progress report, but we made approximately 2,000 direct contacts with stakeholders to promote NCSRP research results.
• We reprinted and distributed 5,000 copies of the 2013 Soybean Aphid Field Guide booklet. Copies of the booklet were sent to state extension offices and state soybean checkoff boards as well as distributed nationally at the 2015 Commodity Classic.
• We created a postcard and flash drive highlighting soybean aphid research and the NCSRP soybean research website. These were distributed at Commodity Classic and will also be distributed at field day events this summer. The purpose of the flash drive is to distribute more NCSRP material while reducing printing costs.
• We continued to help update technical content and provide comments and suggestions for the NCSRP soybean research website.

Regional studies on aphid-resistant cultivars
o Our field tests show that farmers could replace insecticides with aphid resistant soybeans.
o Our lab work and modelling suggest that if aphid resistant soybeans are planted with a refuge of aphid-susceptible soybeans then aphids that can survive on the resistant plants will not overtake the aphid population. This means that the wide-spread use of aphid-resistant soybeans can be sustainable.
o If aphid-susceptible soybeans are planted as a refuge mixed within aphid-resistant soybeans, that is a Refuge-in-a-bag, aphid populations can persist but do not reduce soybean yield.
o Rag1 aphid resistance does not cause yield drag in soybeans
o All of these findings have been published in peer-reviewed journals, many available to the public at open-source websites.
o Several of these results are available to farmers in producer-friendly extension materials.

Efficacy and value of seed treatments
• In the absence of aphid pressure, thiamethoxam (Cruiser) provides no yield benefits
• Thiamethoxam levels plummet to background levels (i.e., levels in untreated plants) by the V2 stage, well before aphid populations peak for the summer
• Thiamethoxam is not present in soybean pollen, which is good news for pollinators
• In our region, IPM (foliar at threshold) offers an average net return of $21.02/acre with a 94.5% probability of at least breaking even, while for Cruiser seed treatment, the average net return is $6.02/acre with a 66.2% probability of at least breaking even.
• The relevance of this information for soybean producers is that IPM is a better pest management investment in the North Central region. This is important because millions of dollars are spent in the region each year for a pest management product (insecticidal seed treatment) that doesn’t deliver good value.

Economic analyses of soybean aphid management strategies
The purpose of this objective was to apply ag-economic analysis to the NCSRP aphid management data generated through the field study described above. The main finding of this analysis was:

• IPM (foliar at threshold) offers an average net return of $21.02/acre with a 94.5% probability of at least breaking even, while for Cruiser seed treatment, the average net return is $6.02/acre with a 66.2% probability of at least breaking even.
• The relevance of this information for soybean producers is that IPM is a better pest management investment in the North Central region.

Monitor for aphid resistance to insecticides
• We developed methodology for two assays of aphid susceptibility to the insecticide thiamethoxam (commonly used as a seed treatment, e.g., Cruiser) – a vial bioassay and a detached leaflet bioassay.
• The vial assay was determined to be more practical for wide scale monitoring.
• Regional sampling of aphids in 2012 and 2014 showed no significant evidence of resistance development in the aphid, but the sub-lethal bioassays indicates a change may be taking place in aphid susceptibility to thiamethoxam.
• This shift warrants continued monitoring and more focused sub-lethal effects studies.
• Work on optimizing a “do-it-yourself” bioassay technique was begun [which could be sent as a kit to cooperators on demand, to monitor resistance on-site], and will be an objective in future projects.
• The relevance of this work for farmers is that we now have a method to monitor for aphid resistance to a commonly used insecticide, which can help provide early warning for product failure within a region.

Seasonal, environmental, and landscape factors
• Field scouting during the 3 years of the study indicated that when aphids are at high levels during R6, it is not because of infestation and/or aphid population growth during R6 (the object of the study). Populations were high during R6 because high soybean aphid populations during R5 (e.g. well above thresholds) were not managed.
• In those fields where there were relatively high levels of aphids during R6, numerous aphids were on leaves that were beginning to senesce, so likely would not have significant impact on yield.
• Soybean aphids did not naturally infest R6 soybeans when R6 occurred in August, likely because the preferred earlier plant stages in surrounding soybean.
• The relevance for soybean producers is to emphasize that good management of soybean aphid requires scouting and vigilance through the reproductive stages of the plant, particularly through R5.

Soil conditions affecting soybean aphid populations
• More soil N (nitrate-N) leads to greater soybean aphid population growth (Fig. 1). Thus, even for non-fertilized leguminous crops, soil N can impact insect pest management.
• Soil potassium (K) can also impact soybean aphid populations. As levels of K decrease, soybean aphid problems increase.
• Soils that are low in clay (and high in sand) may be more likely to have increased soybean aphid problems.
• Producers may wish to concentrate more scouting effort in fields with low K, and that are low in clay.
• In a related study looking at two levels of nitrogen fertilizer, rhizobial seed inoculant, , and the presence/absence of an oat cover crop – the presence of a cover crop had the strongest impact on soybean aphid densities, reducing their levels. Cover crops could be a consideration in future IPM strategies if they could be made to work from a production standpoint.

Regional soybean aphid monitoring network
• Regionwide, aphid populations are significantly lower today than in the initial stages of its spread through the North Central region. 2010 appears to have been a turning point.
• Aphid populations are consistently greater in some states than others, with MN, IA, and WI typically on the higher end and KS and MI typically on the lower end.
• All the suction trap data collected between 2005 and 2014 has been compiled and is publicly available online. These data can be used for modeling efforts on larger-scale aphid patterns; some of these data have already been used in publications.
• Added value to the trap network comes from natural enemy sampling, and for collecting thrips to monitor for Soybean Vein Necrosis Virus, which they vector.

Automated Counting System for Soybean Aphid
• We developed MATLAB algorithms to count populations of aphids on leaves from photo images.
• We validated the method using human counts vs. automated counts by 11 different trained samplers in Iowa and Kansas.
• This algorithm counts aphids with high accuracy.
• Automated estimates are also being compared with the ability of an untrained sampler to correctly estimate number of aphids on a leaflet with minimal training.
• The ultimate goal of this work is to develop an aphid-counting app for mobile devices. T

Pollinators and Soybean Yield
• A surprising diversity of pollinators was found in soybeans in the region, with a total of 68 species of pollinators documented.
• The contributions of these species to yield is yet to be determined, and a topic for future work.

Breeding and Genetic Resistance to Soybean Aphid
• Sixteen new sources of resistance to soybean aphid biotypes 1 and 2 were identified.
• An aphid resistance gene from PI 567597C was mapped to a small genetic interval and genetic markers were identified that can be used to select this gene which will aid in the development of resistant varieties.
• Twenty one plant introductions with aphid resistance were tested and 20 were found to carry resistance in the same region at Rag2 and the remaining in the Rag1 interval. These results show that resistance in the Rag2 interval is very common in aphid resistance sources.
• Field experiments were completed that show that the soybean variety KS4202 has tolerance to soybean aphid infestations. This tolerance means that this variety has less yield losses cause by aphids than other, non-tolerant varieties. Genes were identified that were upregulated after aphid infection that are associated with this tolerance.
• The DNA sequences from the regions surrounding and including the Rag1 and Rag2 resistance genes have been assembled from the original sources of these genes. There is extensive variation at the DNA level in these intervals when the resistance sources and the susceptible Williams 82 are compared. Genes that are likely the genes that confer the resistance have been identified and are awaiting confirmation.
• Progress has been made in breeding aphid resistant varieties and there are now commercially available varieties with major aphid resistance genes.
• A comparison of lines with different combinations of the resistance genes Rag1 and Rag2 in field test environments with no detectable aphid infestations showed that the resistance allele at Rag2 is associated with a significant yield decrease and Rag1 is not. This is consistent with previous results and experiments are underway that attempt to break the negative association between yield and the Rag2 resistance allele.
• Isolines that are almost completely genetically identical except for major aphid resistance genes have been developed. These will be genetic resources useful for future studies.

Soybean Aphid Biotypes
• Collections of putative aphid biotypes (able to overcome resistant varieties) were made in 8 states.
• Genomes of individual aphids from these collections were sequenced and assembled, and genetic diversity analyzed.
• A significant accomplishment of this work is a preliminary draft of the complete soybean aphid genome, which will be greatly valuable for future genetic work on soybean aphid.
• A major finding of this work was that the biotype populations collected in the North Central have little genetic structure – this means that there are not distinct biotype populations but that virulence traits crop up many places independently. This is a key piece of information for designing insect resistance management strategies.
• Another accomplishment is the identification of SNP markers which are likely linked to virulence genes in aphids. These markers will be used in future gene mapping efforts.
• We established a biotype stock center to house standard colonies of four major soybean aphid biotypes. These colonies are available to the research community to facilitate work on aphid virulence and resistant varieties.

Biological Control
• We conducted host-specificity testing (the first stage of testing before a release permit can be obtained, and a time-consuming process) on 12 species of Aphelinus from China, Korea, and Japan.
• Based on this work we obtained USDA APHIS-PPQ approval for field releases of two species in the U.S., A. glycinis and A. rhamni.
• Most subsequent work has focused on A. glycinis and A. certus (an Asian species that has appeared in this country on its own).
• We are working towards establishing A. glycinis in the region. We have reared and released 300,000 individuals, and demonstrated that it is capable of overwintering in a location as northerly as Minnesota.
• Efforts to track and document impact of A. certus have shown its spread through much of the upper Midwest, with parasitism of up to 40 aphid per plant.
• A novel virus with potential for use in soybean aphid management, Aphid lethal paralysis virus (ALPV)-AP1, was characterized and the genome sequence determined.
• A cell line derived from Diabrotica undecimpunctata, DU182A, was found to support replication of ALPV-AP1 – allowing larger quantities of this virus to be generated
• We did not achieve the original goal of testing ALPV-AP1 efficacy against soybean aphid in the field due to technical challenges associated with virus production.