Differential Stem Proteomics and Metabolomics Profiles for Four Wheat Cultivars in Response to the Insect Pest Wheat Stem Sawfly.

Common wheat (Triticum aestivum L.) is a global staple crop, and insect pests can impact grain yield. The wheat stem sawfly (Cephus cinctus, WSS) is a major wheat pest, and while partial resistance has been deployed by breeding for a solid-stem trait, this trait is affected by environment. Here, a proteomics and metabolomics study was performed on four wheat cultivars to characterize a molecular response to WSS infestation. The cultivars Hatcher (hollow-stem partially tolerant), Conan (semisolid-stem-resistant), and Denali and Reeder (hollow-stem-susceptible) were infested with WSS, and changes in stem proteins and metabolites were characterized using liquid chromatography-mass spectrometry. The proteome was characterized as 1830 proteins that included five major biological processes, including metabolic processes and response to stimuli, and the metabolome (1823 metabolites) spanned eight chemical superclasses, including alkaloids, benzenoids, and lipids. All four varieties had a molecular response to WSS following infestation. Hatcher had the most distinct changes, whereby 62 proteins and 29 metabolites varied in metabolic pathways involving enzymatic detoxification, proteinase inhibition, and antiherbivory compound production via benzoxazinoids, neolignans, and phenolics. Taken together, these data demonstrate variation in the wheat stem molecular response to WSS infestation and support breeding for molecular resistance in hollow-stem cultivars.

Invasive Cereal Aphids of North America: Ecology and Pest Management.

Aphid invasions of North American cereal crops generally have started with colonization of a new region or crop, followed by range expansion and outbreaks that vary in frequency and scale owing to geographically variable influences. To improve understanding of this process and management, we compare the invasion ecology of and management response to three cereal aphids: sugarcane aphid, Russian wheat aphid, and greenbug. The region exploited is determined primarily by climate and host plant availability. Once an area is permanently or annually colonized, outbreak intensity is also affected by natural enemies and managed inputs, such as aphid-resistant cultivars and insecticides. Over time, increases in natural enemy abundance and diversity, improved compatibility among management tactics, and limited threshold-based insecticide use have likely increased resilience of aphid regulation. Application of pest management foundational practices followed by a focus on compatible strategies are relevant worldwide. Area-wide pest management is most appropriate to large-scale cereal production systems, as exemplified in the Great Plains of North America.

GC-MS Metabolomics to Evaluate the Composition of Plant Cuticular Waxes for Four Triticum aestivum Cultivars.

Wheat (Triticum aestivum L.) is an important food crop, and biotic and abiotic stresses significantly impact grain yield. Wheat leaf and stem surface waxes are associated with traits of biological importance, including stress resistance. Past studies have characterized the composition of wheat cuticular waxes, however protocols can be relatively low-throughput and narrow in the range of metabolites detected. Here, gas chromatography-mass spectrometry (GC-MS) metabolomics methods were utilized to provide a comprehensive characterization of the chemical composition of cuticular waxes in wheat leaves and stems. Further, waxes from four wheat cultivars were assayed to evaluate the potential for GC-MS metabolomics to describe wax composition attributed to differences in wheat genotype. A total of 263 putative compounds were detected and included 58 wax compounds that can be classified (e.g., alkanes and fatty acids). Many of the detected wax metabolites have known associations to important biological functions. Principal component analysis and ANOVA were used to evaluate metabolite distribution, which was attributed to both tissue type (leaf, stem) and cultivar differences. Leaves contained more primary alcohols than stems such as 6-methylheptacosan-1-ol and octacosan-1-ol. The metabolite data were validated using scanning electron microscopy of epicuticular wax crystals which detected wax tubules and platelets. Conan was the only cultivar to display alcohol-associated platelet-shaped crystals on its abaxial leaf surface. Taken together, application of GC-MS metabolomics enabled the characterization of cuticular wax content in wheat tissues and provided relative quantitative comparisons among sample types, thus contributing to the understanding of wax composition associated with important phenotypic traits in a major crop.

Virus-induced gene silencing suggests (1,3;1,4)-ß-glucanase is a susceptibility factor in the compatible russian wheat aphid-wheat interaction.

The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is a significant insect pest of wheat (Triticum aestivum L.) and has a major economic impact worldwide, especially on winter wheat in the western United States. The continuing emergence of new RWA biotypes virulent to existing resistance genes reinforces the need for more durable resistance. Studies have indicated that resistance in previously susceptible plants can be produced by knock-down of susceptibility genes or other genes involved in host plant susceptibility. Therefore, investigation into genes involved in compatible RWA-wheat interactions could be a feasible approach to achieving durable RWA resistance. The objective of this study was to test whether silencing (1,3;1,4)-ß-glucanase, previously observed to be highly induced in susceptible compared with resistant wheat during aphid infestation, would confer resistance to a susceptible wheat genotype. Barley stripe mosaic virus-mediated virus-induced gene silencing was employed to test whether (1,3;1,4)-ß-glucanase is involved in the susceptible reaction of 'Gamtoos-S' (GS). Controlled infestation with U.S. biotype RWA2 was done to assess aphid reproduction and host symptom development. Aphids on (1,3;1,4)-ß-glucanase-silenced plants reproduced less per day and had longer prenymphipositional periods than those on control GS plants. Furthermore, the (1,3;1,4)-ß-glucanase-silenced plants exhibited less chlorosis and greater dry weight compared with GS. Aphid reproduction and host plant symptom development showed linear relationships with (1,3;1,4)-ß-glucanase transcript levels. Our results suggest that (1,3;1,4)-ß-glucanase is required for successful infestation by the RWA and may be a susceptibility factor that could be exploited as a potential target for RWA resistance breeding.

Examining the competitive advantage of Diuraphis noxia (Hemiptera: Aphididae) biotype 2 over biotype 1.

The Russian wheat aphid, Diuraphis noxia (Kurdjumov) is a serious pest of small grains, such as wheat and barley. High population growth rates and a broad gramineae host range have allowed this aphid to successfully establish and become pestiferous across much of North America since its invasion in the mid-1980s. Resistant wheat cultivars were developed and provided control ofD. noxia until 2003, when a new biotype (designated RWA2, as contrasted with the original biotype's designation, RWA1) emerged and rapidly spread through dryland winter wheat-growing regions. RWA2 displaced RWA1 more quickly than expected, based on RWA2's advantage in RWA1-resistant wheat cultivars. Previous research suggested that RWA2 may out-compete RWA1 in cooler temperatures. Thus, we sought to determine if RWA2 had a competitive advantage over RWA1 during the overwintering period. We placed a known distribution of RWA1 and RWA2 aphids in the field for the winter at three sites across a latitudinal gradient (from northern Colorado to Texas) to test for a competitive advantage between these biotypes. We found overwhelming support for an overwintering competitive advantage by RWA2 over RWA1, with evidence suggesting a > 10-fold advantage even at our Texas site (i.e., the site with the mildest winter). This substantial overwintering advantage helps explain the quick dispersion and displacement of RWA1 by RWA2.

On-farm harvest timing effects on alfalfa weevil across the Intermountain West region of the United States.

Alfalfa (Medicago sativa L.) is an economically important commodity in the Intermountain Western United States. A major concern for alfalfa producers in this region is the alfalfa weevil (Hypera postica Gyllenhal). Insecticide resistance development coupled with regulatory changes in pesticide use has resulted in renewed interest by producers in non-chemical control methods such as cultural control. One such cultural control method is early harvest, which consists of producers timing their harvests early in the season to decrease alfalfa weevil damage. This method is thought to be effective by exposing weevil larvae to adverse conditions before significant damage occurs. Still, early harvest can be difficult to employ because recommendations are often vague. To better understand how early harvest impacts both alfalfa weevils and their natural enemies and how producers are using this method across the Intermountain Western United States, we conducted a study in alfalfa production fields in Colorado, Montana, and Wyoming over three growing seasons. We determined that the timing of the initial alfalfa harvest spanned more than 1 month across fields, and alfalfa plant stage at harvest ranged from late vegetative to early bloom. Harvest was more impactful on reducing alfalfa weevil densities the earlier it was implemented. Removing windrows in a timely manner is likely useful to further decrease alfalfa weevil densities. Harvest timing was not associated with parasitism rates of alfalfa weevil, but higher parasitism rates were associated with lower post-harvest alfalfa weevil densities. This work has increased our understanding of early harvest in an on-farm setting and to improve recommendations for producers across the Intermountain Western United States.

The distribution of European corn borer (Lepidoptera: Crambidae) moths in pivot-irrigated corn.

The European corn borer, Ostrinia nubilalis (Hübner), is a damaging pest of numerous crops including corn, potato, and cotton. An understanding of the interaction between O. nubilalis and its spatial environment may aid in developing pest management strategy. Over a 2-yr period, approximately 8,000 pheromone trap catches of O. nubilalis were recorded on pivot-irrigated corn in northeastern Colorado. The highest weekly moth capture per pivot-irrigated field occurred on the week of 15 July 1997 at 1,803 moths captured. The lowest peak moth capture per pivot-irrigated field was recorded on the week of 4 June 1998 at 220 moths captured. Average trap catch per field ranged from approximately 1.6 moths captured per trap per week in 1997 to approximately 0.3 moths captured per trap per week in 1998. Using pheromone trap moth capture data, we developed a quantified understanding of the spatial distribution of adult male moths. Our findings suggest strong correlations between moth density and adjacent corn crops, prevailing wind direction, and an edge effect. In addition, directional component effects suggest that more moths were attracted to the southwestern portion of the crop, which has the greatest insolation potential. In addition to the tested predictor variables, we found a strong spatial autocorrelation signal indicating positive aggregations of these moths and that males from both inside and outside of the field are being attracted to within-field pheromone traps, which has implications for refuge strategy management.

Survey of Wheat Stem Sawfly (Hymenoptera: Cephidae) Infesting Wheat in Eastern Colorado.

From 2012 through 2020, a survey of wheat stem sawfly, Cephus cinctus Norton, was conducted in wheat (Triticum aestivum L.) fields in Eastern Colorado. In 2013, results showed sawfly infestations concentrated in the northern part of the state with only a few highly infested sites, with 38 of the 94 sampled sites having any infestation (five of which had >50% infestation levels). By 2020 sawfly had been found in all eastern counties sampled, and 72 of the 106 sites sampled were found to contain sawfly (11 of which had >50% infestation levels). The spread of this pest across the Colorado wheat-growing region will have lasting economic effects. The information gathered from this and future surveys will inform wheat variety development and aid in management decisions made by growers across the state.

Effects of Landscape Composition on Wheat Stem Sawfly (Hymenoptera: Cephidae) and Its Associated Braconid Parasitoids.

Several agroecological and integrated pest management strategies focus on landscape management to increase complexity and foster biodiversity. However, landscape complexity does not always enhance biological control and in some cases may lead to increased pest populations. We examined the prevalence of two Bracon parasitoids, Bracon cephi Gahan and Bracon lissogaster Muesebeck (Hymenoptera: Braconidae), and their host the wheat stem sawfly Cephus cinctus Norton, a major pest of wheat. We assessed the degree of noncrop and crop host plant use and responses to landscape composition. We found no instances of parasitism by either Bracon species in our three-year, statewide winter wheat survey but found small populations of Bracon in noncrop landscapes throughout eastern and western Colorado. We used model selection to examine how local (500 m scale) and landscape (5 km scale) cover of suitable noncrop and crop habitats potentially affects abundances of Bracon and wheat stem sawfly. Our best fit model for wheat stem sawfly suggests that a decrease in noncrop cover at the landscape scale leads to an increase in wheat stem sawfly infestation. Our best fit model for Bracon parasitism suggests an increase in wheat cover at the local level results in the greatest increase in the odds of parasitism by either species of Bracon. Herbaceous cover at local and landscape scales were also significant predictors of Bracon parasitism. The results of this study suggest that pest and natural enemies respond differently to landscape composition and these responses should be evaluated before management decisions are made.

Virus-induced gene silencing of WRKY53 and an inducible phenylalanine ammonia-lyase in wheat reduces aphid resistance.

Although several wheat genes differentially expressed during the Russian wheat aphid resistance response have recently been identified, their requirement for and specific role in resistance remain unclear. Progress in wheat-aphid interaction research is hampered by inadequate collections of mutant germplasm and difficulty in transforming hexaploid wheat. Virus-induced gene silencing (VIGS) technology is emerging as a viable reverse genetics approach in cereal crops. However, the potential of VIGS for determining aphid defence gene function in wheat has not been evaluated. We report on the use of recombinant barley stripe mosaic virus (BSMV) to target and silence a WRKY53 transcription factor and an inducible phenylalanine ammonia-lyase (PAL) gene, both predicted to contribute to aphid defence in a genetically resistant wheat line. After inoculating resistant wheat with the VIGS constructs, transcript abundance was reduced to levels similar to that observed in susceptible wheat. Notably, the level of PAL expression was also suppressed by the WKRY53 construct, suggesting that these genes operate in the same defence response network. Both knockdowns exhibited a susceptible phenotype upon aphid infestation, and aphids feeding on silenced plants exhibited a significant increase in fitness compared to aphids feeding on control plants. Altered plant phenotype and changes in aphid behaviour after silencing imply that WKRY53 and PAL play key roles in generating a successful resistance response. This study is the first report on the successful use of VIGS to investigate genes involved in wheat-insect interactions.