GmKIX8-1 regulates organ size in soybean and is the causative gene for the major seed weight QTL qSw17-1.

Seed weight is one of the most important agronomic traits in soybean for yield improvement and food production. Several quantitative trait loci (QTLs) associated with the trait have been identified in soybean. However, the genes underlying the QTLs and their functions remain largely unknown. Using forward genetic methods and CRISPR/Cas9 gene editing, we identified and characterized the role of GmKIX8-1 in the control of organ size in soybean. GmKIX8-1 belongs to a family of KIX domain-containing proteins that negatively regulate cell proliferation in plants. Consistent with this predicted function, we found that loss-of-function GmKIX8-1 mutants showed a significant increase in the size of aerial plant organs, such as seeds and leaves. Likewise, the increase in organ size is due to increased cell proliferation, rather than cell expansion, and increased expression of CYCLIN D3;1-10. Lastly, molecular analysis of soybean germplasms harboring the qSw17-1 QTL for the big-seeded phenotype indicated that reduced expression of GmKIX8-1 is the genetic basis of the qSw17-1 phenotype.

Host resistance to Bacillus thuringiensis is linked to altered bacterial community within a specialist insect herbivore.

Evolution of resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) threatens the sustainability of the technology. Examination of resistance mechanisms has largely focused on characterization of mutations in proteins serving as Bt toxin binding sites. However, insect microbial communities have the potential to provide host resistance to pesticides in a myriad of ways. Previous findings suggest the killing mechanism of Bt relies on enteric bacteria becoming pathogenic in the disrupted gut environment of the insect following Bt intoxication. Thus, here we hypothesized that resistance to Bt would alter the microbiome composition of the insect. Previous studies have manipulated the microbiome of susceptible insects and monitored their response to Bt. In our study, we characterized the associated bacterial communities of Bt-resistant and -susceptible western corn rootworms, a widespread pest of maize in the United States. We found resistant insects harbor a bacterial community that is less rich and distinct from susceptible insects. After feeding on Bt-expressing maize, susceptible insects exhibited dysbiosis of the associated bacterial community, whereas the community within resistant insects remained relatively unchanged. These results suggest resistance to Bt produces alterations in the microbiome of the western corn rootworm that may contribute to resistance. We further demonstrated that by itself, feeding on Bt toxin-expressing seedlings caused a shift in the microbiota. This work provides a broader picture of the effect stressors have on microbiome composition, and the potential heritable changes induced as a result of intense selection.

Susceptibility of northern corn rootworm (Diabrotica barberi) populations to Cry3Bb1 and Gpp34/Tpp35Ab1 proteins in seedling and diet overlay toxicity assays.

The northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae) is a major pest of maize in the United States Corn Belt. Recently, resistance to Bacillus thuringiensis (Bt) maize was reported in North Dakota and increased use of Bt maize hybrids could facilitate resistance evolution in other maize-producing states. In this study, susceptibility to Bt proteins was evaluated in wild D. barberi populations from 8 fields collected in 5 different states (Minnesota, Missouri, Nebraska, Iowa, and North Dakota). Field populations were compared to a susceptible D. barberi colony in seedling and diet toxicity assays conducted with 3 concentrations of Cry3Bb1 (0.4, 4.0, and 40.0 µg/cm2) and Gpp34/Tpp35Ab1 (previously called Cry34/35Ab1; 1.4, 14.0, and 140.0 µg/cm2). The 2019 population from Meeker Co., Minnesota (MN-2019), exhibited the lowest mortality to Cry3Bb1 and also had nominally lowest mortality to Gpp34/Tpp35Ab1 at the highest concentrations tested in diet toxicity assays. Percent second instar was also highest for larvae of the Minnesota population surviving Cry3Bb1. In seedling assays, MN and IA-2018 populations exhibited the highest proportion survival and dry weight to both proteins expressed in corn. No significant differences in mortality, percent second instar, and dry weight were observed at the highest concentration for both proteins among the populations collected in in 2020. Most D. barberi populations were still highly susceptible to Cry3Bb1 and Gpp34/Tpp35Ab1 proteins based on diet and seedling assays, but resistance appears to be developing in some D. barberi populations. Now that methods are available, resistance monitoring may also be needed for D. barberi in some regions.

eCry3.1Ab-resistant Western Corn Rootworm Larval Midgut Epithelia Respond Minimally to Bt Intoxication.

Insect resistance to toxins derived from Bacillus thuringiensis (Bt) is a major issue in agriculture. Resistance to Bt has been linked to the loss of toxin binding sites within the insect, changes within the gut microbiota, and midgut tissue regeneration. Histopathological documentation of intoxication and resistance to Bt is lacking for rootworms in the genus Diabrotica (Coleoptera: Chrysomelidae), a major target of Bt corn. Here, we document the morphological response of both Bt-resistant and Bt-susceptible larval western corn rootworm, Diabrotica virgifera virgifera LeConte, to intoxication with eCry3.1Ab. Gut lumen structural differences are subtle between the two colonies when feeding on non-Bt corn. However, upon ingestion of Bt-corn roots, susceptible larvae develop symptoms indicative of gut disruption by Bt, whereas resistant larvae incur milder effects. Mild disruption of the peritrophic matrix and gut lumen is accompanied by stem cell proliferation that may lead to midgut tissue regeneration. These results help contextualize the multifaceted nature of Bt-resistance in western corn rootworm for the first time from a histopathological perspective.

Patterns of Microbiome Composition Vary Across Spatial Scales in a Specialist Insect.

Microbial communities associated with animals vary based on both intrinsic and extrinsic factors. Of many possible determinants affecting microbiome composition, host phylogeny, host diet, and local environment are the most important. How these factors interact across spatial scales is not well understood. Here, we seek to identify the main influences on microbiome composition in a specialist insect, the western corn rootworm (WCR; Diabrotica virgifera virgifera), by analyzing the bacterial communities of adults collected from their obligate host plant, corn (Zea mays), across several geographic locations and comparing the patterns in communities to its congeneric species, the northern corn rootworm (NCR; Diabrotica barberi). We found that bacterial communities of WCR and NCR shared a portion of their bacterial communities even when collected from disparate locations. However, within each species, the location of collection significantly influenced the composition of their microbiome. Correlations of geographic distance between sites with WCR bacterial community composition revealed different patterns at different spatial scales. Community similarity decreased with increased geographic distance at smaller spatial scales (~25 km between the nearest sites). At broad spatial scales (>200 km), community composition was not correlated with distances between sites, but instead reflected the historical invasion path of WCR across the United States. These results suggest bacterial communities are structured directly by dispersal dynamics at small, regional spatial scales, while landscape-level genetic or environmental differences may drive community composition across broad spatial scales in this specialist insect.

Chromobacterium Csp_P biopesticide is toxic to larvae of three Diabrotica species including strains resistant to Bacillus thuringiensis.

The development of new biopesticides to control the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is urgent due to resistance evolution to various control methods. We tested an air-dried non-live preparation of Chromobacterium species Panama (Csp_P), against multiple corn rootworm species, including Bt-resistant and -susceptible WCR strains, northern (NCR, D. barberi Smith & Lawrence), and southern corn rootworm (SCR, D. undecimpunctata howardi Barber), in diet toxicity assays. Our results documented that Csp_P was toxic to all three corn rootworms species based on lethal (LC50), effective (EC50), and molt inhibition concentration (MIC50). In general, toxicity of Csp_P was similar among all WCR strains and ~ 3-fold less toxic to NCR and SCR strains. Effective concentration (EC50) was also similar among WCR and SCR strains, and 5-7-fold higher in NCR strains. Molt inhibition (MIC50) was similar among all corn rootworm strains except NCR diapause strain that was 2.5-6-fold higher when compared to all other strains. There was no apparent cross-resistance between Csp_P and any of the currently available Bt proteins. Our results indicate that Csp_P formulation was effective at killing multiple corn rootworm strains including Bt-resistant WCR and could be developed as a potential new management tool for WCR control.

Western Corn Rootworm, Plant and Microbe Interactions: A Review and Prospects for New Management Tools.

The western corn rootworm, Diabrotica virgifera virgifera LeConte, is resistant to four separate classes of traditional insecticides, all Bacillius thuringiensis (Bt) toxins currently registered for commercial use, crop rotation, innate plant resistance factors, and even double-stranded RNA (dsRNA) targeting essential genes via environmental RNA interference (RNAi), which has not been sold commercially to date. Clearly, additional tools are needed as management options. In this review, we discuss the state-of-the-art knowledge about biotic factors influencing herbivore success, including host location and recognition, plant defensive traits, plant-microbe interactions, and herbivore-pathogens/predator interactions. We then translate this knowledge into potential new management tools and improved biological control.

Restoration of susceptibility following removal of selection for Cry34/35Ab1 resistance documents fitness costs in resistant population of western corn rootworm, Diabrotica virgifera virgifera.

BACKGROUND: Management of the corn pest, western corn rootworm (WCR), Diabrotica virgifera virgifera (LeConte) (Coleoptera: Chrysomelidae), relies heavily on the planting of transgenic corn expressing toxins produced by the bacterium Bacillus thuringiensis (Bt). This has resulted in the evolution of resistance to all of the four commercially available Bt toxins targeting coleopteran insects. In this study, we evaluated the susceptibility of a Cry34/35Ab1-resistant WCR colony in seedling and diet toxicity assays after removal from selection for six and nine generations. In addition, female fecundity, egg fertility, adult lifespan, larval development, and adult emergence were evaluated in two Cry34/35Ab1-resistant and two susceptible WCR colonies to assess fitness costs. RESULTS: Susceptibility to Cry34/35Ab1 was restored in a colony removed from selection after six and nine generations based on diet toxicity assays and comparisons of relative survival, head capsule width, and dry weight in plant assays. Thus, pronounced fitness costs associated with resistance to Cry34/35Ab1 were documented by susceptibility being restored within six generations. In separate studies evaluating specific fitness costs, larval fitness when reared on isoline corn did not differ between resistant and susceptible colonies. However, beetles from susceptible colonies lived longer than resistant beetles which resulted in females from susceptible colonies producing significantly more eggs than resistant colonies, with no differences in egg fertility. CONCLUSIONS: The presence of a fitness cost that may contribute to the restoration of susceptibility to Bt has not been documented in other Cry3-resistant WCR populations and could have significant impact on the deployment of resistance management practices. Published 2021. This article is a U.S. Government work and is in the public domain in the USA.

Field Studies on the Horizontal Transmission Potential by Voluntary and Involuntary Carriers of Helicoverpa armigera nucleopolyhedrovirus (Baculoviridae).

Horizontal transmission of Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has been found to occur through several pathways involving abiotic factors such as soil, wind, and rain, and biotic factors such as predators, parasitoids, and infected hosts. Previous studies examining horizontal transmission through certain biological carriers speculated they were likely not significant in increasing infection rates, however; these studies only focused on a relatively small number of arthropods present within a field setting. This study was conducted to evaluate the horizontal transmission potential of HearNPV by all potential biological carriers when applied as a foliar bioinsecticide or as virus-infected, nonmotile Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) larvae in a soybean field. Soybean plots were either sprayed with HearNPV or infested with late-stage HearNPV-infected larvae, and sample zones were sampled 3, 7, 10, 14, 17, and 21 days after the infestation, and analyzed for viral presence using PCR. We then identified HearNPV carriers through contamination from the application (involuntary) or through contact with a HearNPV-infected larva (voluntary). Both were confirmed through PCR analysis. Regardless of application technique, on average, HearNPV was capable of disseminating up to 61.0 m in 3 d after inoculation and was found within the sampled canopy 13-21 d after inoculation. Several arthropods were identified as novel carriers of HearNPV. Results from this study indicate that many novel HearNPV carriers are likely important in disseminating HearNPV.

Optimizing Egg Recovery From Wild Northern Corn Rootworm Beetles (Coleoptera: Chrysomelidae).

The northern corn rootworm, Diabrotica barberi Smith & Lawrence (Coleoptera: Chrysomelidae), is one of the most important insect pests in the U.S. Corn Belt. Efforts to obtain eggs from wild northern corn rootworm populations using techniques developed for other rootworm species have been unsuccessful due to lack of oviposition. In 2016, we evaluated four oviposition media in choice tests within each of three female densities in 30.5 × 30.5 × 30.5 cm BugDorm cages. The number of eggs laid per female was significantly affected by female density and the interaction of female density × oviposition media, but oviposition was relatively poor in all oviposition media (1.2 eggs per female when averaging the three female densities and all oviposition media). Single females were also evaluated in nonchoice assays in 6 cm × 6 cm × 8 cm clear plastic boxes and averaged up to 108 eggs per female depending on the oviposition media. In 2017, the cumulative number of eggs laid per female in boxes with one female was not significantly different from the number of eggs laid per female in boxes with 3 females. In 2018, the cumulative number of eggs laid per female was not significantly different between female densities of 1, 3, 5, or 10 females per box. Total egg production per box therefore increased as female density increased. More than 27,000 wild northern corn rootworm eggs were collected from just 190 females when collected relatively early in the field season. We now have an efficient and robust system for obtaining eggs from wild northern corn rootworm females.