The bird cherry-oat aphid (BCOA), Rhopalosiphum padi L., and the Russian wheat aphid (RWA), Diuraphis noxia L., are considered important pests of both wheat and barley, frequently leading to substantial yield reductions [1] [2] . Both BCOA and RWA feed by inserting their sucking mouthparts into the sieve tubes of plants to acquire nutrients and water, and allows both species to indirectly transmit plant viruses [3] .

The RWA has been reported as a destructive pest of grain plants since its arrival in the United States in 1986. Within less than a decade, RWA aphids caused an estimated $1 billion in economic losses to small grains [4] . Similarly, BCOA is an economically significant pest that can reduce wheat yield by up to 62% and is an effective vector for Barley Yellow Dwarf Virus (BYDV) in small grains [1] [3] [5] [6] .

The RWA and BCOA feed on different locations of the plant and adopt distinct feeding strategies, which in turn elicit specific responses from their host plants [7] [8] . While BCOA feeding generally does not result in visible damage symptoms, RWA feeding can induce necrosis and chlorosis on leaves, leaf rolling, stunted growth, purple discoloration, and prostrate growth, especially in wheat [2] [9] - [11] . In addition to differences in feeding strategies between the two species, RWA also exhibits multiple biotypes. These biotypes are primarily characterized by the resistant or susceptible responses observed in a collection of wheat accessions believed to carry specific Dn-resistance genes [12] - [14] .

The first RWA-identified biotype (RWA1) arrived in the US in 1986 [10] . In 2003, a RWA-resistant hard red winter wheat line Prairie Red, which contains the Dn4 resistance gene, was discovered to be infested with RWA in several areas of southeastern Colorado [13] [15] . This was later identified as a new biotype, RWA2 [10] . Among the tested accessions, only 94M370 (Dn7) showed resistance to RWA2. However, the Dn7 gene is often associated with negative impacts on the quality of leavened bread products [10] .

Several wheat and barley genotypes have been identified and developed to resist BCOA and RWA biotypes [6] [14] [16] [17] . However, since these resistant genotypes were typically designed to target a single aphid species, and given that BCOA and RWA can concurrently infest the same host plant [8] , it is crucial to investigate the interactions between these two species and their combined impact on resistant and susceptible barley and wheat responses.

The objectives of this work were to evaluate the morphological and physiological parameters of susceptible and resistant barley and wheat genotypes, under combinations of BCOA and RWA1 and RWA2 infestations.

Both wheat and barley genotypes showed significant reductions in most evaluated parameters when infested with aphids, compared to the controls ( Tables 1-4 ). During the first evaluation, significant interactions between genotype and aphid treatment were observed in barley for the number of leaves [F(5, 48) = 8.75, p < 0.01] and in wheat for root weight (g) [F(5, 48) = 2.46, p < 0.01] and total dry weight (g) [F(5, 48) = 2.55, p < 0.01]. Among these interactions, only the susceptible genotypes (Morex and Custer) showed reductions caused by the aphid treatments. Even when no significant interaction was observed, parameters such as aerial fresh weight (g) and aerial dry weight (g) for barley, as well as plant height (cm) and aerial fresh weight (g) for wheat, were impacted by the aphid treatments ( Figure 1 ).

For these parameters, barley aerial fresh weight (g) was equally impacted by aphid infestations, regardless of the type of treatment or barley genotype, with the reductions ranging up to 20% of the mass for BCOR001 and up to 80% for Morex. A reduction in aerial dry weight (g) was observed only in the susceptible Morex, with the plants under BCOA + RWA2 infestation showing a significant reduction of about 66%. For wheat, both genotypes showed reductions in plant height (cm) and aerial fresh weight (g) under rwa1 infestation; however, they were not impacted by infestations of rwa2.

In the second evaluation, conducted two weeks after infestation, significant interactions were observed only in barley, for the number of leaves [F(5, 48) = 6.88, p < 0.001], root weight (g) [F(5, 48) = 3.04, p = 0.02], root dry weight (g) [F(5, 48) = 2.96, p = 0.02], and total dry weight (g) [F(5, 48) = 2.51, p = 0.04]. In general, Morex plants had a significant reduction of about 50% in the number of leaves, when infested with either RWA biotypes and their combination with BCOA. This reduction also affected other parameters, such as aerial fresh weight (g) and root weight (g), in which plants had reduction up to 95% and 85%, respectively. However, in the resistant genotype BCOR001, the same pattern was not observed, showing no reduction in root fresh or dry weight (g) when infested with any aphid treatment.

In wheat, although no significant interactions were observed, the aphid effect was noted on almost all other morphological parameters in both genotypes, except for plant height [cm; F(5) = 2.18, p = 0.07] and root dry weight [χ²(5) = 7.66, p = 0.18 and χ²(5) = 5.94, p = 0.31 for Dn7 and Custer, respectively].

On the third evaluation, no two-way ANOVA tests were performed because assumptions were not met except for root weight (g) on wheat. The aphid treatments, however, impacted all evaluated parameters. Differential responses between genotypes were observed in the plant height for barley, where the resistant genotype BCOA001 was similar to the control (χ²(5) = 15.68, p = 0.34), and very different than susceptible Morex (χ²(5) = 13.38, p = 0.02). Differential responses were also observed in the wheat root dry weight (g) where the resistant Dn7 showed reductions but no significant differences (χ²(5) = 10.45, p = 0.06) compared to its counterpart Custer (χ²(5) = 14.28, p = 0.01).

After four weeks of infestation, significant interactions were observed in barley for plant height (cm, F(5, 48) = 2.75, p = 0.03] and aerial fresh weight [g, F(5, 48) = 4.18, p = 0.04]. Interestingly, aerial dry weight (g) was not significantly reduced in either barley or wheat resistant genotypes [χ²(5) = 10.12, p = 0.07 and χ²(5) = 9.50, p = 0.09 for Morex and Custer respectively]; while root dry weight (g) was not impacted in either wheat genotypes [χ²(5) = 2.58, p = 0.06 and χ²(5) = 9.50, p = 0.09 for Dn7 and Custer respectively].

Assimilation rates (μmol CO₂ m⁻²∙s⁻¹) generally increased with higher intercellular CO₂ concentration (Ci) across all treatments. However, the susceptible and resistant barley and wheat genotypes exhibited varying efficiencies in CO₂ assimilation, as reflected by their distinct curves ( Figure 2 and Figure 3 ). As expected, the non-infested treatment (check) consistently showed the highest assimilation rates at all CO₂ levels in both genotypes for barley and wheat, while aphid infestation reduced the assimilation rate. However, the magnitude of reduction differed significantly.

For wheat, the susceptible genotype Custar experienced the strongest reductions in assimilation rate under single-aphid infestations, RWA1 and RWA2, followed by BCOA + RWA2 and BCOA ( Figure 2(A) ). Plants under BCOA + RWA1 showed a moderate reduction. Interestingly, in the resistant genotype Dn7, the lowest curve was observed in the BCOA treatment, whereas the other treatments fell in the middle range, with no distinct differences between them ( Figure 2(B) ).

The susceptible barley Morex had the lowest curve under BCOA + RWA1 infestation, while all other aphid treatments impacted the assimilation rate similarly, especially under high CO₂ concentrations. Notably, in the resistant genotype BCOR001, the response of plants under the BCOA treatment was similar to the check, followed by RWA2, BCOA + RWA2, BCOA + RWA1, and RWA1.

Although previous studies have identified and reported barley and wheat genotypes resistant to a single aphid species [6] [14] [18] [30] - [32] and multiple aphid species [9] [17] , to the best of our knowledge, this is the first study to compare resistant and susceptible barley and wheat genotypes under simultaneous BCOA and RWA1 or RWA2 infestations.

In this study, we observed differences between wheat and barley responses to BCOA, RWA1, and RWA2 within the first week of infestation. However, the differences within aphid treatments (BCOA, RWA1, RWA2, BCOA + RWA1, and RWA2 + BCOA) were noted for either susceptible or resistant genotypes of barley and wheat.

As anticipated, differential responses between resistant and susceptible cultivars were observed within one week of infestation by the aphid species for which resistance was selected. Cross-resistance was also noted in resistant cultivars. The resistant barley cultivar BCOR001 showed no impact on the number of leaves or aerial dry weight (g) regardless of aphid species or combination, in contrast to the susceptible cultivar Morex, which experienced a reduction in the number of leaves across all aphid-infested treatments. In contrast, during the first week, no impact from aphid feeding was observed on the number of leaves, and aerial and root dry weight was noted for either wheat genotype. Number of leaves, as well the plant dry weight are important parameters in cereal crops for early growth stages because they directly corelate with grain yield [33] .

In our experiment, we also observed differential responses to aphid infestation between crops. Although previous work has reported that wheat infested with aphids tends to experience a more rapid reduction in fitness compared to barley [9] , our results showed that aphid-infested wheat genotypes exhibited reductions in the number of leaves, root weight (g), and aerial dry weight (g) only after two weeks. In contrast, barley showed reductions in number of leaves and aerial fresh weight after just one week of infestation. Previous studies have observed reductions in barley aerial fresh and dry matter accumulation in aphid-infested plants within two days of infestation [30] . The differential responses between crops might be explained by the speed of the plant’s response to stress.

Although Dn7 is reported to be resistant to RWA1 and RWA2 [13] [14] , morphological responses in our treatments did not demonstrate clear resistance. In general, all aphid treatment equivalently reduced some parameters in Dn7, especially at three and four weeks after infestation.

Both susceptible and resistant genotypes of barley and wheat exhibited higher photosynthetic assimilation rates under non-infested (check) conditions, suggesting that aphid feeding negatively impacted photosynthetic efficiency. Additionally, the A/Ci curves revealed distinct physiological responses between susceptible and resistant genotypes.

The susceptible wheat genotype Custer exhibited a relatively uniform reduction in assimilation rate across different CO₂ concentrations. In contrast, the resistant genotype Dn7 showed a partial reduction in assimilation rate under treatments with RWA1, RWA2, and their combinations with BCOA, when compared to the BCOA treatment alone, which resulted in the lowest assimilation rate across all CO₂ concentrations. Interestingly for barley, the resistant genotype BCOR001 under BCOA infestation maintained assimilation rates comparable to the check under high CO₂ concentrations. This suggests a potential tolerance mechanism that allows compensation or recovery of photosynthetic capacity in response to BCOA feeding at elevated intercellular Ci levels [34] . The resistant barley’s ability to maintain high assimilation under BCOA infestation could be linked to efficient ribulose-1,5-bisphosphate (RuBP) regeneration and CO₂ fixation, processes that are directly linked to photosynthetic performance and, consequently, crop yield [35] .

The findings of this study confirm that aphid infestation, whether by BCOA, RWA1, RWA2, or their combinations, results in significant reductions in morphological and physiological parameters in both barley and wheat. However, the magnitude and timing of the impact varied considerably between resistant and susceptible genotypes, supporting the hypothesis that plant genotype plays a critical role in controlling host responses to aphid stress.

Contrary to our expectations, combinations of aphids on susceptible varieties did not cause more damage than single aphid species, suggesting that Economic Injury Levels (EILs) do not need to distinguish aphid species. Resistant cultivars generally responded similarly to single or combination treatments suggesting the existence of cross-resistance. The observation suggests that EILs can be standardized across different aphid species, which simplifies pest management protocols and reduces the complexity of monitoring and intervention strategies. Farmers can implement a unified economic threshold, streamlining decision-making processes and potentially lowering management costs.

It is important to note that aphid density was not controlled during the four-week evaluation period of our experiments, resulting in variability in the number of aphids per plant. It is likely that each treatment experienced different population growth rates of the species used. This variability may have impacted wheat’s tolerance capacity, as both aphid density and feeding duration are known to influence plant tolerance [34] . To better understand these interactions, future studies should include species-specific life table analyses to assess the impact of single and combined aphid infestations on different barley and wheat genotypes [8] . Additionally, the variability in aphid density highlights the dynamic nature of pest populations. IPM strategies should incorporate regular monitoring and adaptive management practices to respond to fluctuating pest pressures, considering the differences in aphid population growth in different genotypes. This could involve periodic sampling and adjusting control measures based on real-time data, ensuring that interventions are both effective and economical.

Additionally, our results highlight the importance of evaluating plant resistance not only through visual symptoms but also through physiological parameters, including CO₂ assimilation and dry weight retention. Finally, the variable response of Dn7 under uncontrolled aphid densities indicate that resistance can be overcome under complex infestation dynamics, reinforcing the need for integrated pest management (IPM) approaches alongside the adoption of resistant genotypes.

The authors would like to acknowledge the Department of Entomology and Plant Pathology at Oklahoma State University for support for this research and the USDA-ARS base funds, Hatch Project accession No. 1019561 from the USDA National Institute of Food and Agriculture, and the USDA ARS Cooperative Agreement #G0000089. We thank B. Knighten for her technical support with this research. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

This research was partially funded by cooperative project 58-3072-6-015 between the USDA-ARS and Oklahoma State University and by the Department of Entomology and Plant Pathology at Oklahoma State University and supported by Hatch Project accession no. 1019561 from the USDA National Institute of Food and Agriculture.