Objective 1: Host plant resistance:
During 2011, Drs. McCornak and O’Neal conducted a field experiment with controlled populations of soybean aphids on caged, small plots of susceptible and aphid-resistant soybeans. These experiments were completed with weekly observations of aphid populations and estimates of yields. These data are being combined from results collected in 2010 and summarized by Dr. McCornack. Initial analysis suggest that aphid populations that reach the economic threshold after plants exceed the R5 stage do not experience the same degree of yield loss as those infested during the R1-R2 stage.

Developing thresholds for late R5 and R6 soybeans was hampered by the fact that aphids never reached sufficient densities to establish thresholds at the site in Nebraska where this experiment was established. Data from Iowa is being evaluated for R5 soybeans as noted above.
Near isolines of soybean with and without Rag1 and Rag2 were grown in MN, WI, SD, KS, NE and IA during the summer of 2011. In small, replicated field plots we grew near isolines of Rag1 alone, Rag2 alone, a Rag1/Rag2 pyramid, and a susceptible line. Each plot was divided into sub-plots, one kept free of aphids using foliar insecticides, and one left untreated for the duration of the season. This experiment was successfully completed, with all states reporting data on the ability of the three resistant lines to limit aphid population growth. Yield data was also collected, revealing the capacity of the resistant lines to prevent yield loss when aphid outbreaks occur.
Our results indicate that soybean lines containing both Rag1 and Rag2 (the Rag-pyramid) can significantly reduce aphid outbreaks and prevent yield loss more so than lines with just Rag1 and Rag2 alone. In states that experience an outbreak of soybean aphids (IA, MN, and SD), the Rag-pyramid did not lose yield when left untreated with a foliar insecticide. The results from this experiment have been shared with all the collaborators and discussed at annual meetings with farmers at checkoff sponsored events in Iowa and Minnesota. These results were also shared with the soybean breeders at their annual meeting in St. Louis and the national meeting of the Entomological Society of America.
We have saved seed from this project and have commitments from all collaborators to conduct the experiment again in 2012. We are expanding this experiment to include an additional treatment. All four varieties will be grown with and without a seed-applied insecticide (i.e. Cruiser). This will expand our understanding of how powerful the Rag-pyramid is and if its capacity to protect soybeans from aphids is improved with the addition of an insecticidal seed treatment.
Greenhouse and field plot experiments were conducted to assess resistance to soybean in 50+ soybean lines. Experiments were designed to assess relative degrees of resistance using controlled release of aphids in the greenhouse and confining aphids in clip cages in the field. Results from these tests are guiding our selection regimes of soybean germplasm.

Objective 2: Resistant varieties, seed treatment and interactions with biocontrol agents:
A number of experiments in various states were done to investigate the potential interactions between Rag1 resistance, seed treatments and biological control. Field studies in MN, IN and SD Dakota showed that aphid densities were lowest in fields that used both Rag1 resistance and a seed treatment consisting of thiamethoxam (‘Cruiser’). In these field studies, natural enemies generally followed aphid densities and so were highest in the plots planted to susceptible soybeans that were not seed treated. There are indications from both field and laboratory studies, however, that the effectiveness of the seed treatments wanes approximately 5 weeks after germination.

A number of more directed laboratory studies investigated the effects of Rag1 resistance and the seed treatment on biological control agents more directly. In one of these, Knodel’s group in North Dakota found some negative effects of Rag1 soybeans on the Asian parasitoid Binodoxys communis. In contrast, no clear negative effects have been found for the native parasitoid Lysiphlebus testaceipes in studies done in WI.

A study in MN comparing parasitism of soybean aphid by Binodoxys communis in cages from either seed-treated or non-seed-treated soybeans found no negative effects of the seed treatment. However, negative effects of Cruiser seed treatment were found in a study in IN, which found higher numbers of Orius insidiosus (a soybean aphid predator) in non-seed treated plots for R4 and R5 stage plants despite the fact that the seed treatment is expected to have worn off by this stage. Preliminary results from this study also suggest that the seed treatment has a negative effect on thrips, a minor pest of soybeans and a common prey species for Orius.

Objective 3: Biological control:
At the USDA-ARS lab in Newark, Delaware, we have carried out host range testing on 24 populations of Aphelinus; three populations of Aphelinus remain to be tested. In second-round host specificity testing, Aphelinus near campestris parasitized few aphids outside the genus Aphis. In third-round host specificity testing, this parasitoid rarely laid eggs in species outside the genus Aphis. Although Aphelinus near campestris had a slightly broader host range than Aphelinus near gossypii, which it closely resembles but with which it is reproductively incompatible, the host range of the Aphelinus near campestris is narrow enough for it to be considered for release against soybean aphid, and a petition for its introduction is being prepared. Together with Aphelinus near engaeus and Aphelinus near gossypii, this makes three species of Aphelinus proposed for introduction to control soybean aphid. We have described these species, which are new to science, and provided a key to the A. mali complex, which will aid in their post-introduction identification (Hopper et al., in press).

A petition for release of Aphelinus near gossypii was prepared and submitted to the North American Plant Protection Organization (NAPPO). An application for field release of Aphelinus near engaeus in Minnesota in 2012 was prepared based on a previously approved NAPPO petition and submitted to APHIS-PPQ, which has prepared an environmental assessment with input from USDA-ARS, Newark, Delaware.

In Minnesota, host range testing was completed for a culture of Lysiphlebia orientalis from Korea, a culture of Lipolexis gracillis from Korea and it is underway for an as-yet-unamed species of Lysiphlebus – also from Korea. The host ranges of L. orientalis and L. gracillis are relatively narrow but soybean aphid was not the most suitable host for either of these species in our laboratory studies and we are therefore not considering them for release. Our release permit application for Binodoxys koreanus is still pending with USDA APHIS.

We are planning releases of Aphelinus near engaeus for summer 2012, Aphelinus near gossypii for summer 2013, and A. corea for summer 2014.

In quarantine experiments on diapause of Aphelinus near campestris, Aphelinus near engaeus and Aphelinus near gossypii, we found they readily diapaused as third instar larvae in aphid mummies when day length was lowered to 11 hours. Emergence from diapause was high and rapid after the parasitoids were held at low temperature for three months. These results indicate that both of these species should readily diapause and overwinter in soybean growing areas in the Midwest.

No recoveries of Binodoxys communis were made in 2011 and it seems doubtful that establishment of this species has occurred in N. America. Laboratory studies also suggest an inability to diapause. Current monitoring efforts are centered on Aphelinus certus, a parasitoid that has invaded from Asia without our intervention and was first found in MN in August 2011. This is a very promising parasitoid as it is currently playing an important role in suppressing soybean aphid in eastern Canada.

Soybean aphid populations and presence of soybean aphid parasitoids were monitored in southern Wisconsin as well. The parasitoid of primary interest in this survey was Lysiphlebus testaceipes. Soybean aphid populations were at “historic” lows in the fields we monitored, with peak numbers less than 50 per plant. However, levels of parasitism by L. testaceipes were at historic highs, with a peak level of 18% parasitism recorded in mid-August for one of the fields. However, the more abundant parasitoid observed was an Aphelinus sp. (possibly certus). 2011 is the first year we have observed more than just occasional collections of Aphelinus mummies from soybean aphid. We also collected three soybean aphid mummies on buckthorn in Rock Island, IL, two of which were confirmed as L. testaceipes, and one was hyperparasitized.

A survey of buckthorn in May, 2011, in northeastern China (Jilin and Liaoning Provinces), revealed few soybean aphid and no parasitoids.

Objective 4: Plant resistance (and tolerance):
A focus of the NE group has been on evaluating the tolerant response of KS4202 to the soybean aphid. A series of greenhouse studies were conducted to evaluate the impact of soybean aphid feeding on the yield response of V1, V3, and R1 KS4202 soybean plants. Two infestation levels (Low level - between 1000 and 1500 and High Level - between 2000 and 2500) were evaluated. The only yield parameter impacted by the high level of soybean aphid infestation in first node stage (V1) plants was seed number. All other yield parameters evaluated for the V1 plants were similar between infested (high and low levels) and non-infested plants. Yield parameters were not impacted by soybean aphids when aphids were introduced to V3 and R1 stage plants. Additionally, we found that the tolerance levels found in V1 and V3 are comparable to the levels in R1 stage.

The second component of this research was to characterize transcriptional changes in response to aphid feeding to better understand the underlying tolerant mechanism(s) in KS4202 and genes contributing to the tolerance response. Comparing gene expression levels between infested and control plants for KS4202, over 550 genes had a higher expression level in response to aphid feeding, while, over 650 genes had a lower expression level in response to aphid feeding. For K03-4686 (susceptible), over 150 genes had a higher expression level in response to aphid feeding, whereas, over 750 genes had a lower expression level when comparing infested to control plants. Of specific interest, were two peroxidase and four WRKY genes that exhibited higher expression levels in aphid-infested KS4202 plants compared to control plants. These same genes were not found to be differentially expressed in susceptible soybeans. We are currently in the process of completing a manuscript focused on the transcriptional profiling of resistant and susceptible soybeans to the soybean aphid.

John Reese (KS) screened many soybean genotypes for resistance to biotypes 1, 2, and 3 of the soybean aphid, since we do not have a very clear understanding of the geographical distributions of these biotypes yet. We are currently replicating biotype 3 experiments in order to adequately test the data statistically. We believe that we have some good sources of resistance to biotype 3, thanks in part to collaborations with other researchers.

John Reese is looking for soybean genes that resemble aphid resistance genes known to occur in Arabidopsis. There are clearly some very interesting parallels. Our results, in collaboration with Dr. Jyoti Shah at the University of North Texas, will allow us to focus on more and more diverse resistance genes, and thus increase the flow in the pipeline of genes resistant to soybean aphids in the future. We are also making progress toward the goal of confirming the QTL responsible for resistance in K1621, which was initiated in Bill Schapaugh’s lab. Finally, the collaboration with University of Nebraska scientists on the nature of tolerance to soybean aphid feeding damage continues to be very productive. Tolerance, of course, by definition places no selection pressure on the pest populations for yet more virulent populations.