Host plant tolerance and planting date influence alfalfa mosaic virus incidence, symptom severity, and yield in chile peppers

Host plant tolerance and planting date influence alfalfa mosaic virus incidence, symptom severity, and yield in chile peppers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Host plant tolerance and planting date influence alfalfa mosaic virus incidence, symptom severity, and yield in chile peppers Taylor Janecek, Lara M. Amiri-Kazaz, Jordan Withycombe, Punya Nachappa, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8800235/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 6 You are reading this latest preprint version Abstract Alfalfa mosaic virus (AMV) is transmitted nonpersistently by multiple aphid species and has become an emerging threat to chile pepper production in regions of the United States where peppers are grown in the vicinity of alfalfa, the primary reservoir of the pathogen. Because nonpersistent transmission limits the effectiveness of chemical vector control, management strategies that reduce exposure to viruliferous aphids are needed. We evaluated how planting date and varietal susceptibility shape AMV incidence, symptom severity, and yield in two chile pepper ( Capsicum annum L.) varieties in a region where peppers are grown near alfalfa, a primary reservoir of AMV and source of dispersing aphids. Across two years, early plantings had the highest AMV incidence and symptom severity, which coincided with pronounced early season peaks in aphid abundance. The putatively tolerant variety consistently showed lower AMV incidence and reduced severity than the susceptible variety, regardless of planting date. Despite greater virus pressure, early plantings produced the highest yields, reflecting the longer production window and the capacity for yield compensation, particularly in the tolerant variety. Late plantings reduced AMV incidence but produced the lowest yields because of shortened growing seasons. These results indicate that planting date strongly mediates disease risk and productivity and that varietal tolerance provides additional protection without altering the effect of planting date. Together, optimized planting timing and selection of tolerant varieties offer practical, complementary strategies for managing AMV in regions where dispersing aphids drive infection risk. Bromoviridae Capsicum annuum aphids IPM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Chile peppers ( Capsicum annuum L.) are an important specialty crop in the United States, valued for both acreage and profitability. In 2021, nearly 11,000 acres of chile peppers produced an average of eight tons per acre and generated more than 70 million dollars in profit (USDA NASS 2021). Production is concentrated in California, New Mexico, and Colorado, and historically has required very little pest management in Colorado. That changed when Alfalfa mosaic virus (AMV) was detected in peppers in 2019, marking the first occurrence of this pathogen in the region (Amiri-Kazaz et al. 2025a). AMV reduces both yield and fruit quality, and its nonpersistent transmission by multiple aphid species creates unique challenges for effective management. In Colorado alone, AMV-associated yield and quality losses are estimated to result in millions of dollars in lost revenue annually. Notably, AMV occurs worldwide, including across many European countries, and has been reported infecting pepper crops in Spain, for example (Mallor et al. 2002). AMV was first identified in alfalfa ( Medicago sativa L.) and has a broad host range; it is known to infect more than 650 plant species (Moradi and Mehrvar 2021, Yardimci et al. 2007). These hosts include economically important vegetable and specialty crops worldwide such as eggplant, tomato, potato, pea, and lentil (Jones and Coutts 1996, van Leur et al. 2013, Sofy et al. 2021). Transmission occurs via multiple aphid species during brief probing events. Because virus particles adhere to the stylet, even momentary contact with infected tissue is sufficient for transmission (Yardimci et al. 2007, Sofy et al. 2021). In Colorado, aphids rarely colonize pepper plants but are abundant in nearby alfalfa fields. As these fields are cut and aphids disperse, they likely introduce AMV into pepper crops before moving on to other hosts. This movement pattern underscores the importance of understanding local vector dynamics when developing management strategies for peppers in the region. In addition to vector transmission, AMV is transmitted mechanically. This commonly occurs through exposure to infected plant sap (Al-Saleh and Amer 2013) and through seed (Sûtic 1959, Latham and Jones 2001, Yardimci et al. 2007, Sofy et al. 2021). Planting date modification is an important cultural tactic for managing viruses that are transmitted in a nonpersistent manner, because it can reduce the period when crops are exposed to high densities of infectious vectors. Several studies have shown that adjusting planting time can create asynchrony between vector activity and susceptible crop stages. For example, early sowing of fava bean reduced incidence of pea enation mosaic virus by nearly 30% when plants avoided the peak flights of pea aphids (Saucke et al. 2009). In another system, late planted rice was less susceptible to rice stripe virus transmitted by the small brown planthopper, and incidence declined by as much as 25% compared with early planted rice (Zhu et al. 2009). These cases illustrate how the timing of crop establishment can influence exposure to viruliferous insects. Although planting date is often an effective tactic, it also carries agronomic tradeoffs because delayed planting can reduce yield potential in many crops (Dadrasi et al. 2024). Host plant resistance offers another important tactic for managing insect transmitted plant diseases, particularly when vector control is ineffective. In a recent study conducted to assess the potential of host plant resistance in chile peppers as a control strategy for AMV in the greenhouse and field (Amiri-Kazaz et al., 2025b), 20% of the tested pepper cultivars remained symptomless despite confirmed infection, while 10% exhibited symptoms without testing positive for the virus. Likewise, host plant resistance has been shown to strongly reduce disease severity and mitigate fitness losses in numerous cropping systems. For instance, barley varieties resistant to barley yellow dwarf virus produced substantially higher yields than susceptible varieties (Najar and Ben Ghanem 2017). Moreover, resistance to potato virus Y has also been a central strategy for managing this virus in potato production and continues to be a focus of breeding programs worldwide (Karasev and Gray 2013). Together, cultural practices such as planting date modification and the use of resistant or tolerant varieties provide complementary approaches for reducing disease pressure when chemical suppression of vectors is not feasible. The objective of this study was to evaluate how planting date modifies AMV incidence, symptom severity, and yield in two chile pepper varieties differing in susceptibility. By examining early and late plantings in both varieties, we assessed how timing of crop establishment shapes exposure to the virus and ultimately affected productivity. We also quantified aphid abundance and species composition in the field plots to identify periods of greatest vector activity and to link vector pressure with patterns of AMV infection. This integrated approach provides insight into how cultural practices and varietal selection interact under field conditions where aphids are the primary vectors of AMV. The results have practical implications for pepper growers in regions where AMV is emerging or already established, as they point to management options that reduce disease risk while maintaining yield. Understanding how planting date intersects with vector dynamics and varietal tolerance can guide more resilient production strategies and support long term sustainability of chile pepper systems. Methods Field plot design The field experiment was conducted at the Colorado State University (CSU) Arkansas Valley Research Center in Rocky Ford (Otero County), Colorado over two growing seasons, 2022 and 2023. The experiment was embedded within a 1.38 ha field planted next to a 0.4 ha field of grain sorghum to the east, a 4-ha alfalfa field to the west, a 1.6 ha field of alfalfa to the north, and a 12.15 ha pasture to the south. The experimental area consisted of 12 rows, 0.91 m apart and 18.3 m in length totaling 658.4 m 2 . The experiment was a split-plot design with a planting date as a whole-block factor (early, conventional, and late) and plant variety as a split-block factor (a susceptible variety ‘Joe Parker’ and a putative tolerant variety ‘Mira Sol’). Each of these treatments was replicated six times (N = 36), and each replicate plot consisted of eight chile peppers, four tolerant and four susceptible. The peppers were planted 15 cm apart and two-row buffers separated each planting block. Plants were grown from seed in a CSU greenhouse complex in Fort Collins, Colorado. All seeds were sown at the depth of ca. 0.5 cm in plug flats with high porosity potting mix (Lambert® LM-40, Riviére-Ouelle, Québec, Canada) and slow-release fertilizer (Osmocote® Plus 15:9:12 N-P-K, ICL, Summerville, SC, USA). All plants were maintained under a 16:8 h (L:D) supplemental lighting cycle (430W High-Pressure Sodium + 65W LED) and the day:night temperature was 31°C:27°C. The plants were watered ad libitum when the soil was dry. Seeds (48 per variety per planting date) were sown into flats and sowing was staggered by two weeks to ensure plants of the same approximate stage could be transplanted into the field. Once the peppers reached four true leaf stage, they were transported to Rocky Ford, CO and planted into rows assigned to the early (13 May), common (26 May), and late (8 June) planting date treatment in 2022; and early (10 May), common (23 May), and late (13 June) planting date treatment in 2023. Mean temperatures during the growing season were slightly lower in 2023 than 2022, while precipitation levels in 2023 were approximately 113% of average annual rainfall for the region (Colorado Climate Center 2024 ). Symptom incidence, disease incidence, and symptom severity ratings Plants were visually assessed for incidence of AMV symptoms on 22 July 2022 in first year of the experiment, and 12 July 2023 in the second year of the study. The response variables were the presence or absence of leaf chlorosis and necrosis, vein deformation, and leaf deformation. To diagnose disease incidence, symptomatic leaf tissue was collected from each plant on 22 July in 2022 and 7 August in 2023. Leaf tissue from each plant was placed in 2 mL centrifuge tubes (GeneMate®, Milford, EN, UK) and immediately stored at -80°C. Semi-quantitative proxies for AMV titers were obtained through ELISA (Agdia®, User Guide: Compound-ELISA Reagent Set). Briefly, absorbance (OD) values were determined through triple antibody sandwich enzyme-linked immunosorbent assay using monoclonal antibodies (TAS-ELISA). Tissue was processed and tested according to the Agdia® AMV ELISA protocol, using Agdia® Compound-ELISA reagent and buffer sets. Plates were assessed using the ELx800 Universal Microplate Reader (Agilent technologies Inc, Santa Clara, CA, USA) at 405 nm. All assay wells were duplicated for each plate. Samples were considered positive if absorbance value was double that of the average negative control (Ahoonmanesh et al. 1990, Sofy et al. 2021 ). Moreover, AMV was confirmed in a subset of plants collected from the field in 2022 using RT-PCR. Total RNA was extracted from symptomatic leaf tissues using the RNeasy Plus Mini kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer’s instructions. RNA quantity and quality were assessed using a Nanodrop One spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and a Qubit 3.0 fluorometer (ThermoFisher Scientific). DNA contamination was removed from the RNA using the TURBO DNA-Free kit (Invitrogen, Waltham, MA, USA) in 15 µl reactions following the manufacturer’s instructions. Following DNAse treatment and quantification, one µg of total RNA was used to synthesize cDNA using Verso cDNA synthesis kit (ThermoFisher Scientific) according to manufacturer's instructions. The RT-PCR was performed using DreamTaq Green PCR Master Mix (2X) (ThermoFisher Scientific) with primers specific for a region of AMV coat protein (CP) -CP (5’- ATCATGAGTTCTTCACAAAAGAA-3’ and 5’- TCAATGACGATCAAGATCGTC-3’) (Xu and Nie 2006 ). The amplification cycle consisted of 2 min at 95°C, 35 cycles of 30 sec at 95°C, 30 sec at 58°C and 1 min at 72°C followed by 5 min at 72°C. The PCR products (669 bp) were visualized on a 1% agarose gel. The sequences of each PCR product showed 99% identity to Aq isolate of AMV-CP (GenBank accession JX112758) (Xu and Nie 2006 ). Severity of symptoms was assessed on 22 June and 5 July in 2022, and 12 July and 7 August in 2023. Severity was assessed by calculating the percent of leaves with any AMV symptoms per plant for each variety and planting date. Yield measurements Chile pepper harvest continued weekly from 2 August to 19 September in 2022 and from 17 August to 21 September in 2023 until the plants stopped producing fruits. Chile peppers were collected when fruits reached red-mature stage, defined as 50% or more of the fruit becoming red. Peppers were sorted into labeled paper and plastic bags. The response variables collected were the number of peppers per plant. Aphid surveys Aphids were sampled within pepper plots using yellow pan traps interspersed within each plot. The dimensions of the yellow pan traps were 34.29 cm x 20.32 cm x 12.7 cm (JO-ANN STORES LLC, Top Notch, Hudson, OH, USA). Pans were filled with 1.25 L of propylene glycol (Sanco Industries, Inc, Fort Wayne, IN, USA) and placed between rows of chile peppers as soon as the peppers were planted. Six traps were placed within each planting date block in 2022, and three in 2023, and were spaced 3 m apart and weighed down with three small rocks 0.54 kg each within the pan trap. Surveys of aphids within the pepper plots ceased on 8 August 2022 and 14 July 2023. Collection of samples from pan traps occurred weekly by filtering the contents of yellow trap pans over an organza fabric filter (JO-ANN STORES LLC, Hudson, OH, USA) into 50 mL conical tubes filled with 90% ethanol. Pan traps were refilled with up to 1.25 L of recycled or fresh propylene glycol depending on the condition of the solution. In 2023, pan traps were collected every three to four days throughout the sampling period. Samples were brought back to the laboratory and processed as described above. Aphids were identified to species or genus using a dissecting microscope and dichotomous keys (Blackman and Eastop, 2000 ; Pike et al., 2003 ). Statistical analyses We tested the three-way interaction among year of the study, planting date factor, and variety factor. Where the interactive effect of these variables did not affect the response variables, we combined the data across years. The same approach was used when testing whether the interaction among sampling date, planting date, and variety had an effect on response variables within each year. The incidence of AMV symptoms (defined as number of plants with chlorosis and/or necrosis divided by the total number of sampled plants) and incidence of positive ELISA tests were evaluated for each plant and analyzed using logistic regression and generalized linear mixed-effects models with the lme4 package (Bates et al. 2015 , R Core Team 2023 ). Severity of symptoms and yield were averaged across replicates and compared among varieties and planting dates for both years using ANOVA linear models (R Core Team 2023 ) if data met assumptions of normality and homogeneity of variances (Fox and Weisberg 2019 ). Means separation tests (Tukey’s Honestly Significant Differences test) were performed where appropriate. Kruskal-Wallis and Dunn’s test for means separation were used to analyze data that did not meet assumptions of ANOVA and were analyzed using non-parametric tests (R Core Team 2023 ). Aphid abundance was analyzed by comparing mean species densities within each sampling date using ANOVA. Each yellow pan trap was considered a replicate. Data that did not meet assumptions of ANOVA were transformed (square root or log) or analyzed using non-parametric tests Kruskal-Wallis and Dunn’s test. All analyses were performed in R statistical analysis software (R Core Team 2023 ). Results Incidence of AMV symptoms and disease We found that the three-way interaction among year, variety, and planting date did not have a significant interactive effect on incidence of plants showing AMV symptoms ( Χ ²= 1.37, df = 2, P = 0.172), indicating that the effects of planting date on symptom incidence did not differ between varieties across years. Data on incidence of AMV symptoms from both years were combined to assess the effect of planting dates and variety across the two seasons. We found a significant interactive effect between planting date and variety on incidence of disease symptoms ( Χ ² = 27.2, df = 2, P < 0.001) and thus compared the effects of these factors on symptom incidence within each planting date (Table 1 ). Early planting was associated with higher odds of visible AMV symptoms compared to peppers planted at common or late planting dates, particularly in the susceptible variety ‘Joe Parker’. In contrast, ‘Mira Sol’ consistently exhibited lower incidence of disease symptoms, although still influenced by planting date (Fig. 1 ). Table 1 Pairwise comparisons of AMV symptom incidence (odds ratios) among planting times and pepper varieties. Comparison a Odds ratio SE z ratio b P value Common ‘Joe Parker’ vs. Early ‘Joe Parker’ 0.369 0.097 -3.792 0.002 Common ‘Joe Parker’ vs. Late ‘Joe Parker’ 6.467 1.470 8.187 < 0.001 Common ‘Joe Parker’ vs. Common ‘Mira Sol’ 3.126 0.680 5.239 < 0.001 Common ‘Joe Parker’ vs. Early ‘Mira Sol’ 3.561 0.779 5.806 < 0.001 Common ‘Joe Parker’ vs. Late ‘Mira Sol’ 9.948 2.440 9.363 < 0.001 Early ‘Joe Parker’ vs. Late ‘Joe Parker’ 17.539 4.620 10.864 < 0.001 Early ‘Joe Parker’ vs. Common ‘Mira Sol’ 8.478 2.160 8.390 < 0.001 Early ‘Joe Parker’ vs. Early ‘Mira Sol’ 9.659 2.470 8.864 < 0.001 Early ‘Joe Parker’ vs. Late ‘Mira Sol’ 26.978 7.530 11.804 < 0.001 Late ‘Joe Parker’ vs. Common ‘Mira Sol’ 0.483 0.105 -3.331 0.011 Late ‘Joe Parker’ vs. Early ‘Mira Sol’ 0.551 0.121 -2.719 0.072 Late ‘Joe Parker’ vs. Late ‘Mira Sol’ 1.538 0.378 1.750 0.498 Common ‘Mira Sol’ vs. Early ‘Mira Sol’ 1.139 0.238 0.625 0.989 Common ‘Mira Sol’ vs. Late ‘Mira Sol’ 3.182 0.752 4.898 < 0.001 Early ‘Mira Sol’ vs. Late ‘Mira Sol’ 2.793 0.663 4.326 < 0.001 a Odds ratios were estimated using a generalized linear mixed model with a binomial distribution. b P-values were adjusted using the Tukey HSD test. On the other hand, the year of the study, planting date, and variety had a significant interactive effect on proportion of plants testing positive for AMV ( i.e. , incidence of disease) ( Χ ²= 27.28; df = 2; P < 0.001), and both years were analyzed separately. There was no significant interaction between variety and planting date in 2022 ( Χ ² = 1.50, df = 2, P = 0.473) or 2023 ( Χ ² = 2.17, df = 2, P = 0.338), and an additive model was used to assess the impact of both treatments on incidence of disease in both years. In 2022, there was a significant increase in the likelihood that peppers planted early tested positive for AMV compared to plants that were sown at the common planting time (β = 2.66, SE = 1.07, z = 2.48, P = 0.013). ‘Mira Sol’ and ‘Joe Parker’ from the late plantings did not differ significantly from each other in disease incidence (β = 0.46, SE = 1.25, z = 0.37, P = 0.714). Overall, ‘Mira Sol’ cultivar had marginally lower odds of positive ELISA tests for AMV compared to ‘Joe Parker’ in 2022 (β = -1.17, SE = 0.64, z = -1.81, P = 0.07) (Fig. 2 ). This effect was much stronger in 2023 when ‘Mira Sol’ was significantly less likely to test positive for AMV compared to ‘Joe Parker’ (β = -0.93, SE = 0.42, z = -2.24, P = 0.025). Peppers planted late also had a significantly lower probability to test positive for AMV compared to the plants sown at the common planting time (β = -1.91, SE = 0.69, z = -2.78, P = 0.005). The effect of early planting was not statistically significant (β = 0.64, SE = 0.43, z = 1.48, P = 0.138) (Fig. 2 ). Severity of AMV symptoms The three-way interaction among year, planting date, and variety also influenced the severity of AMV symptoms (F 2,60 = 6.02, P = 0.003). Thus, both years were analyzed separately to assess how planting date and variety affected the severity of disease within each year. In 2022, the sampling date (June, July), planting date, and variety had a significant interactive effect on the severity of the symptoms (F 2,30 = 22.41, P < 0.001), as did the interaction between planting date and variety ( Χ ² = 19.08, df = 5, P < 0.001). Pairwise comparisons revealed significant differences among planting dates for each variety, with the exception of ‘Joe Parker’ planted early and at the common planting dates, ‘Mira Sol’ planted early and at the common planting date, and late planted ‘Mira Sol’ and ‘Joe Parker’ (Table 2 ). Both varieties had higher mean AMV severity when planted at early and common planting dates, and the proportion of leaves with symptoms was low for ‘Joe Parker’ and ‘Mira Sol’ peppers planted late (Fig. 3 ). In July, on the other hand, the interaction between planting date and variety did not significantly affect severity of AMV symptoms (F 2,30 = 1.42, P = 0.258). Both planting date (F 2,30 = 7.98, P = 0.002) and variety (F 1,30 = 18.14, P < 0.001) had significant impacts on proportion of leaves with AMV symptoms in July. ‘Joe Parker’ had significantly greater severity of AMV symptoms than ‘Mira Sol’, and both varieties planted early had higher proportion of affected leaves (Fig. 3 ). Table 2 Pairwise comparisons of severity of AMV symptoms among pepper cultivars and planting dates. Common ‘Mira Sol’ Late ‘Mira Sol’ Early ‘Joe Parker’ Common ‘Joe Parker’ Late ‘Joe Parker’ Early ‘Mira Sol’ 0.27 < 0.001 < 0.001 < 0.001 < 0.001 Common ‘Mira Sol’ — < 0.001 < 0.001 < 0.001 < 0.001 Late ‘Mira Sol’ — — < 0.001 < 0.001 1.00 Early ‘Joe Parker’ — — — 0.84 < 0.001 Common ‘Joe Parker’ — — — — < 0.001 †Values represent P values from Wilcoxon rank sum tests with continuity correction. In 2023, the sampling date (July and August), planting date, and variety did not have a significant interactive effect on the severity of the symptoms (F 2,30 =0, P = 1), but planting time and variety interacted strongly to affect diseases severity (F 2,30 = 66.42, P < 0.001). Early planted ‘Joe Parker’ had significantly higher severity of symptoms than ‘Joe Parker’ peppers planted at common or late planting dates, and higher AMV symptom severity than ‘Mira Sol’ planted at any planting date (Dunn’s test with Bonferroni adjustment, p < 0.05; Fig. 4 ). Effect of variety and planting date on yield of peppers Year, variety, and planting date did not have an interactive effect on yield of peppers (F 2,60 = 1.42, P = 0.251), and yield data were combined for analyses across the two years of the experiment. Chile pepper variety (F 1,30 = 21.49, P < 0.001) and planting date (F 2,30 = 10.55, P < 0.001) had a significant effect on pepper yield, while there was no significant interactive effect of these two factors on yield (F 2,30 = 2.02, P = 0.15). Tolerant plants produced more than twice as many peppers as the susceptible plants (Fig. 5 A), and early and conventional plantings had significantly higher yields than the late planted peppers, regardless of the variety (F 2,30 = 10.55, P = 0.004, Fig. 5 B). Aphid surveys in experimental research plots We found high diversity of aphids in chile pepper plots in both years of the survey, and we identified 15 species of aphids (Table 3 ). In each year, aphid densities were the highest early in the season (Fig. 6 ). There were significant differences in densities of aphid species in experimental pepper plots across most of the growing season in 2022 (Supplemental Table S1). Aphis spp. was more abundant than all other species across all sampling dates in 2022 and dominated most of the samples (Fig. 6 A). There were some notable peaks in Aphis spp. densities in early June, July, and August. Table 3 List of aphid species/genus found in chile pepper plots. Aphid species Acyrthosiphon kondoi* Acyrthosiphon pisum* Acyrthosiphon spp. Aphis spp.* Capitophorus elaeagni Chaitophorus spp. Diuraphis noxia Eriosoma spp. Hayhurstie atriplicis Lipaphis erysimi Myzus persicae* Pemphigus spp. Protaphis spp. Rhopalosiphum padi Therioaphis maculata* Species and genera marked with asterisks are known vectors of AMV There were also significant differences in the densities of aphid species in pan traps in 2023 (Supplemental Table S1). Blue alfalfa aphid was the most abundant species in pan trap samples, while Aphis spp. became more abundant later in the season (Fig. 6 B). From 2 June to 9 June there was a steep increase in density of Aphis spp., pea aphid, spotted alfalfa aphid, and blue alfalfa aphid, which was followed by a sharp decline in mid to late June. Aphis spp. made a small resurgence in early- to mid-July. Discussion Planting date strongly shaped AMV incidence, symptom severity, and yield in this study, and emerged as an effective management factor across both pepper varieties and years. Early plantings consistently experienced higher AMV incidence and more severe symptoms, a pattern that closely followed the pronounced early season peaks in aphid densities observed in the field plots. These aphids included several known vectors of AMV and were most abundant during the period immediately following transplanting. Because AMV is transmitted in a nonpersistent manner, even brief probing by dispersing aphids is sufficient for infection, and the timing of vector arrival relative to crop establishment becomes a critical determinant of disease risk. Our findings suggest that early planted peppers are exposed to the highest virus pressure simply because they are present when vector activity is greatest. Despite the higher incidence of AMV in early plantings, these peppers produced substantially greater yields than peppers planted late. This outcome indicates that yield potential is strongly tied to the length of the growing season. Plants established early had more time to produce fruit and to compensate for virus infection, particularly in the tolerant variety. Late plantings, although less infected, suffered from shortened production windows and produced the lowest yields. This disconnect between disease reduction and yield performance underscores an important consideration for growers: minimizing AMV incidence does not necessarily translate into improved productivity if planting is delayed. The tradeoff between reduced disease and reduced yield must therefore be weighed carefully when selecting planting times. Few studies have evaluated yield outcomes in relation to planting date-mediated disease reduction. For example, delaying sowing of cucurbit crops reduced incidence of zucchini yellow mosaic virus by up to 49% (Coutts et al. 2011 ) and planting mungbean earlier or later than the conventional planting dates resulted in up to 28% reduction in incidence of mungbean yellow mosaic virus (Swamy et al. 2023 ). However, neither of these studies reported the impact on crop yields. This suggests that altering sowing dates can be useful in mitigating disease severity and risk, but the consequences of these modifications to plant productivity should be studied in greater depth. We showed that despite higher incidence and relative amounts of virus, early planting increases the yield, particularly for the tolerant variety. Varietal differences were also a strong predictor of disease incidence and severity. The putatively tolerant variety, Mira Sol, exhibited both lower AMV incidence and reduced symptom severity compared with Joe Parker, and produced more than twice as many peppers across planting dates. These findings suggest that tolerance provides meaningful protection against both the biological effects of infection and the economic consequences of yield loss. Host plant resistance is often the most reliable tactic for suppressing virus diseases and continues to anchor plant-breeding efforts (Corrêa et al. 2024 , Nazarov et al., 2020 ), and there are numerous examples of successful employment of resistant crop varieties to improve host plant resistance to fungal ( e.g ., Sanogo and Zhang 2016 ; Sharma et al. 2012 ), bacterial (Sharma et al. 2022 ), and viral (Gottula and Fuchs 2009 ) pathogens. Notably, both planting date and varietal tolerance strongly influenced yield, and their effects were additive rather than interactive. The tolerant variety produced more than twice as many peppers as the susceptible one, while early and common plantings yielded substantially more fruit than late plantings. Research from other cropping systems reinforces our results. For example, Kone and colleagues (2017) found that incidence of viral disease and severity in cucurbits were significantly shaped by planting date although cultivar differences remained important, and the two factors had an additive effect on improving plant health. Our findings align with our results where the tolerant variety consistently out-yielded the susceptible one and early planting produced higher yields than late planting. The additive nature of those effects underscores the importance of combining host plant resistance with optimal planting timing to maximize yield and reduce disease impact. We also noted a strong alignment between aphid abundance and AMV incidence, which points to the importance of understanding local vector dynamics. Aphids rarely colonized peppers directly and were instead likely introduced into the plots from surrounding alfalfa and other crops. As alfalfa fields were cut, winged aphids dispersed into adjacent crops, including peppers. This suggests that regional patterns of alfalfa harvest, the composition of nearby crops, and seasonal aphid phenology all contribute to the timing and magnitude of AMV pressure. Integrating information about these landscape-level drivers with planting date decisions could improve growers’ ability to anticipate periods of high infection risk. Similar association between aphid abundance and disease risk has been reported previously, and several field studies have shown that altering planting date can significantly reduce viral disease incidence and severity. For example, Koné et al. ( 2017 ) showed that varying sowing dates in cucurbits altered exposure to aphid vectors and reduced viral disease incidence. Likewise, early-season aphid abundance has been identified as a primary predictor of infection risk for aphid-borne viruses, including cucurbit aphid-borne yellows virus in melon (Schoeny et al. 2020 ). In our system, the strong effect of planting date likely reflects a comparable process, whereby adjusting crop establishment to avoid peak aphid flights reduced disease pressure while still allowing high yield when varietal tolerance was present. It is important to note that the high diversity of aphid species we reported in our surveys is likely due to a diverse array of crops that surrounded our pepper plots including Sorghum bicolor L., alfalfa, corn, melons, and cowpea. These crops can be colonized by the common vectors of AMV such as green peach aphid, pea aphid, blue alfalfa aphid, bean aphid, and spotted alfalfa aphid (Blackman and Eastop, 2000 ). While we did not observe any of the aphid species actively feeding or colonizing chile peppers in either of the years of the field experiment, green peach aphid has been noted in this crop as its most common and damaging aphid pest (Sun et al. 2018 , Chen et al. 2020 ). It is noteworthy that weather patterns differed between the two years of the study, and these differences may have influenced aphid activity. Rocky Ford received substantially more precipitation than average in May and June of 2023, while May of 2022 was close to normal and June received only half of the expected rainfall. These conditions may have shaped vector dynamics because aphid communities showed a different pattern across years. Peak aphid densities were higher in 2023, yet overall aphid abundance across the season was lower than in 2022. This suggests that moisture patterns and early season conditions may contribute to the timing and magnitude of aphid flights and, in turn, the risk of AMV transmission. Together, these results highlight that adjusting planting date is an effective and practical tactic for managing AMV risk in chile pepper systems, although it must be balanced against potential reductions in yield when planting is delayed. Early planting paired with a tolerant variety appears to offer the most resilient combination under the conditions of this study. More broadly, the work demonstrates the importance of aligning cultural practices with vector phenology to reduce virus pressure in systems where chemical control of vectors is not effective. Future research that links planting date with fine-scale vector monitoring, evaluates additional varieties for resistance or tolerance, and examines landscape effects on aphid movement will further refine recommendations for sustainable AMV management in Colorado and other regions where this virus is present. Integrating AMV-tolerant varieties with optimized planting dates may substantially improve the resilience of chile pepper production systems, especially given the complex relationship between increased vectors abundance early in the season and plants’ ability to compensate for that exposure if planted sufficiently early (Fig. 7 ). Declarations Conflict of interest The authors declare no conflict of interest. Acknowledgments We thank the Colorado State University Arkansas Valley Research Center staff and the farm manager K. Tanabe for providing plots for chile pepper experiments, A.C. Fulladolsa for her expert advice on performing ELISA assays, and C. Vaughn for help with field data collection and processing. M. Bartolo provided guidance on the seed selection and invaluable insights into host plant resistance in the region of Arkansas Valley of Colorado. This research was funded by the Colorado Department of Agriculture Specialty Crop Block Grant Program 21SCBGPCO1008-00 to P.N. and A.S.; and the United States Department of Agriculture, National Institute of Food and Agriculture grant numbers 2021-70006-35439, 2024-70006-43565, and COL00411, accession number 7001931 to A.S. 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Plant Breed 139:996–1002 Colorado Climate Center (2024) Water year precipitation data for Colorado. Colorado State University, Fort Collins. Available at: https://climate.colostate.edu/wy_data.html (accessed 26 June 2025) Corrêa RL, Petek M, Vaslin MFS (2024) Mechanisms of plant host resistance against viruses. Front Plant Sci 15:1506089 Coutts BA, Kehoe MA, Jones RAC (2011) Minimizing losses caused by zucchini yellow mosaic virus in vegetable cucurbit crops through cultural methods and host resistance. Virus Res 159:141–160 Dadrasi A, Soltani E, Makowski D (2024) Does shifting from normal to early or late sowing dates provide yield benefits? A global meta-analysis. Field Crops Res 318:1–14 Fox J, Weisberg S (2019) An R companion to applied regression, 3rd edn. Sage, Thousand Oaks Gottula J, Fuchs M (2009) Toward a quarter century of pathogen-derived resistance and practical approaches to plant virus disease control. Adv Virus Res 75:161–183 Jones RAC, Coutts BA (1996) Alfalfa mosaic and cucumber mosaic virus infection in chickpea and lentil: incidence and seed transmission. Ann Appl Biol 129:491–506 Karasev AV, Gray SM (2013) Continuous and emerging challenges of potato virus Y in potato. Annu Rev Phytopathol 51:571–586 Koné N, Asare-Bediako E, Souleymane S, Koné D, Koita OA, Menzel W, Winter S (2017) Influence of planting date on incidence and severity of viral disease on cucurbits under field conditions. Ann Agric Sci 62:1–6 Latham LJ, Jones RAC (2001) Alfalfa mosaic and pea seed-borne mosaic viruses in cool-season legumes: susceptibility, sensitivity, and seed transmission. Aust J Agric Res 52:771–790 Mallor C, Luis-Arteaga M, Cambra M, Fernández-Cavada S (2002) Natural infection of field-grown borage by alfalfa mosaic virus in Spain. Plant Dis 86:698 Moradi Z, Mehrvar M (2021) Whole-genome characterization of alfalfa mosaic virus from metagenomic analysis of ornamental hosts in Iran. Plant Pathol J 37:619–631 Najar A, Ben Ghanem H (2017) Introgression of barley yellow dwarf virus resistance into Tunisian barley varieties. Can J Plant Sci 97:210–213 Nazarov PA, Baleev DN, Ivanova MI et al (2020) Infectious plant diseases: etiology, current status, problems and prospects in plant protection. Acta Naturae 12:46–59 Pike KS, Boydston LL, Allison DW (2003) Aphids of western North America north of Mexico. Washington State University Extension, Prosser R Core Team (2023) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna Sanogo S, Zhang J (2016) Resistance sources, screening techniques, and disease management for Fusarium wilt in cotton. Euphytica 207:255–271 Saucke H, Juergens M, Döring TF, Fittje S, Lesemann DE, Vetten HJ (2009) Effect of sowing date and straw mulch on virus incidence and aphid infestation in organically grown faba bean. Ann Appl Biol 154:239–250 Schoeny A, Rimbaud L, Gognalons P et al (2020) Winged aphid abundance as a predictor of cucurbit aphid-borne yellows virus epidemics in melon crops. Viruses 12:911 Sharma TR, Rai AK, Gupta SK et al (2012) Rice blast management through host-plant resistance: retrospect and prospects. Agric Res 1:37–52 Sharma A, Abrahamian P, Carvalho R et al (2022) Future of bacterial disease management in crop production. Annu Rev Phytopathol 60:259–282 Sofy AR, Sofy MR, Hmed AA et al (2021) Molecular characterization of alfalfa mosaic virus infecting eggplant and mitigation using melatonin and salicylic acid. Plants 10:459 Sun M, Voorrips RE, Steenhuis-Broers G, van’t Westende W, Vosman B (2018) Reduced phloem uptake of Myzus persicae on an aphid-resistant pepper accession. BMC Plant Biol 18:138 Šutič D (1959) Die Rolle des Paprikasamens bei der Virusübertragung. J Phytopathol 36:84–93 Swamy SM, Sandra N, Lal SK et al (2023) Evaluation of sowing dates for managing mungbean yellow mosaic disease. 3 Biotech 13:207 van Leur J, Duric Z, George J, Boschma S (2019) Alfalfa mosaic virus infects Desmanthus virgatus in Australia and the potential role of the cowpea aphid. Australas Plant Dis Notes 14:3–5 van Leur J, Kumari S, Aftab M, Leonforte A, Moore S (2013) Virus resistance of Australian pea varieties. N Z J Crop Hortic Sci 41:86–101 Xu H, Nie J (2006) Identification, characterization, and molecular detection of alfalfa mosaic virus in potato. Phytopathology 96:1237–1242 Yardimci N, Eryigit H, Erda I (2007) Effect of alfalfa mosaic virus on macro- and micronutrient content in alfalfa. J Cult Collect 5:90–93 Zhu J, Zhu Z, Zhou Y et al (2009) Effect of rice sowing date on small brown planthopper occurrence and rice stripe virus epidemics. Agric Sci China 8:332–341 Supplementary Files SupplementalTableS1.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Minor revisions 25 Mar, 2026 Reviewers agreed at journal 19 Feb, 2026 Reviewers invited by journal 09 Feb, 2026 Editor invited by journal 09 Feb, 2026 Editor assigned by journal 07 Feb, 2026 First submitted to journal 05 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8800235","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588697069,"identity":"afad2e26-a98a-4649-a764-5d4285ef911c","order_by":0,"name":"Taylor Janecek","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Taylor","middleName":"","lastName":"Janecek","suffix":""},{"id":588697070,"identity":"d5e5a74a-6a37-455a-9b4e-656cae47c6d2","order_by":1,"name":"Lara M. Amiri-Kazaz","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lara","middleName":"M.","lastName":"Amiri-Kazaz","suffix":""},{"id":588697071,"identity":"1fdb3d65-369a-4866-b9f9-ef770832b25b","order_by":2,"name":"Jordan Withycombe","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jordan","middleName":"","lastName":"Withycombe","suffix":""},{"id":588697072,"identity":"914574e0-0eb6-4371-8d56-3323f11a1d45","order_by":3,"name":"Punya Nachappa","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Punya","middleName":"","lastName":"Nachappa","suffix":""},{"id":588697073,"identity":"1edb2023-6e47-4d0f-b71c-c23729a816d9","order_by":4,"name":"Adrianna Szczepaniec","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-9603-4533","institution":"Colorado State University","correspondingAuthor":true,"prefix":"","firstName":"Adrianna","middleName":"","lastName":"Szczepaniec","suffix":""}],"badges":[],"createdAt":"2026-02-05 18:53:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8800235/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8800235/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102613717,"identity":"4909267b-5620-4e35-82dc-5317e1b1e8d5","added_by":"auto","created_at":"2026-02-13 15:11:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66612,"visible":true,"origin":"","legend":"\u003cp\u003eIncidence of alfalfa mosaic virus (AMV) symptoms in ‘Joe Parker’ and ‘Mira Sol’ chile pepper varieties across three planting dates. Bars represent mean incidence values (± 1 SEM) based on combined data from two growing seasons. The planting dates were 13 May 2022 and 10 May 2023 for early planting, 26 May 2022 and 23 May 2023 for common planting date, and 8 June 2022 and 13 June 2023 for late planting date treatment. Early and common plantings of ‘Joe Parker’ exhibited significantly higher AMV symptom incidence than late plantings, while ‘Mira Sol’ had consistently lower incidence of the disease across all planting dates.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8800235/v1/2ef7ab83c3b0068d2a514570.png"},{"id":102613704,"identity":"fd714829-2bfe-4336-8707-d2ab7816ffd0","added_by":"auto","created_at":"2026-02-13 15:11:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64082,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of planting date on incidence of plants testing positive for AMV infection in 2022 and 2023 across three planting dates. Bars represent means ± 1 standard error of the mean (SEM). The planting dates were 13 May for early planting, 26 May for common planting date, and 8 June for late planting date in 2022. In 2023 early planting was 10 May, common planting date was 23 May, and late planting took place on 13 June. In both years, later planting dates were generally associated with lower AMV infection rates, with ‘Mira Sol’ consistently exhibiting lower infection than ‘Joe Parker’.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8800235/v1/09968135795c31518b2773f1.png"},{"id":102613720,"identity":"9ce1b33f-85e9-4213-9b91-8cb91eb0727d","added_by":"auto","created_at":"2026-02-13 15:11:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":80901,"visible":true,"origin":"","legend":"\u003cp\u003eSeverity of AMV symptoms in chile pepper varieties across planting dates in 2022. Bars represent means ± 1 standard error of the mean (SEM). The planting dates were 13 May 2022 for early planting, 26 May 2022 for common planting date, and 8 June 2022 for late planting. In June (left panel), AMV symptom severity was highest in early and common plantings of ‘Joe Parker’, with late plantings showing an 89–94% reduction in severity. In contrast, ‘Mira Sol’ exhibited lower symptom severity overall, with a maximum of 25% severity rating in common plantings and no visible symptoms in the late planting. By July (right panel), symptom severity declined across all treatments.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8800235/v1/1c6055aba5c1b3d6c4148382.png"},{"id":102613657,"identity":"2e438370-ca90-4793-8f31-759c8d1e5b32","added_by":"auto","created_at":"2026-02-13 15:11:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":58586,"visible":true,"origin":"","legend":"\u003cp\u003eSeverity of AMV symptoms in ‘Joe Parker’ and ‘Mira Sol’ chile pepper varieties in 2023 across three planting dates (early: 10 May 2023; common: 23 May 2023; late: 13 June 2023). Bars represent means ± 1 standard error of the mean (SEM). Different letters above bars indicate statistically significant differences among treatments (adjusted \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). ‘Joe Parker’ peppers planted late had much lower symptom severity compared to early plantings (\u0026gt;99% reduction), while ‘Mira Sol’ maintained consistently low severity across all planting dates.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8800235/v1/0afca3ff22adb33944161c5f.png"},{"id":102613738,"identity":"e92cba36-dfe9-4280-af19-660ed3339578","added_by":"auto","created_at":"2026-02-13 15:12:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":112170,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pepper variety and planting date on fruit yield. Bars represent means ±1 SEM. Asterisks denote level of statistical significance (**\u003cem\u003eP\u003c/em\u003e = 0.01, ***\u003cem\u003eP\u003c/em\u003e = 0.001). Across both growing seasons, the putative tolerant variety ‘Mira Sol’ produced significantly more peppers per plant than the susceptible variety ‘Joe Parker’ (A). When analyzed by planting date, yield was highest for both varieties when planted early or at the conventional planting date (B). ‘Joe Parker’ exhibited a marked decline in yield with later planting dates, with late plantings producing less than half as many peppers per plant as early plantings. ‘Mira Sol’ maintained high yields in early and conventional plantings (B).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8800235/v1/0817c472e5914f5ec307cf24.png"},{"id":102613742,"identity":"5ca47569-25da-475d-b503-857d8972f927","added_by":"auto","created_at":"2026-02-13 15:12:03","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":390684,"visible":true,"origin":"","legend":"\u003cp\u003eDensities of the most abundant aphid species in chile pepper plots in 2022 (A) and 2023 (B).\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;Markers represent means, bars are ±1 SEM. Asterisks denote the level of statistical significance (*\u003cem\u003eP\u003c/em\u003e = 0.05, ***\u003cem\u003eP\u003c/em\u003e = 0.001). Aphids were collected weekly using 18 yellow pan traps filled with propylene glycol in 2022 (A) and nine traps in 2023 (B). \u003cem\u003eAphis \u003c/em\u003espp\u003cem\u003e.\u003c/em\u003e were the most abundant species of aphids in the pan traps on all sampling dates in 2022 (A), while \u003cem\u003eA. kondoi\u003c/em\u003e was the most abundant aphid species early in the season of 2023 (B).\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8800235/v1/70c8f01495cabde8c7e52ec2.jpeg"},{"id":102613654,"identity":"0dcb353c-ad56-4328-aa38-f21059c802db","added_by":"auto","created_at":"2026-02-13 15:11:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":529369,"visible":true,"origin":"","legend":"\u003cp\u003eConceptual model summarizing the outcomes of the research. Incidence and severity of AMV were positively correlated with earlier planting date, which was likely related to higher aphid densities early in the season. On the other hand, yield of peppers planted early was greater across both varieties. Moreover, tolerant peppers planted early had significantly higher number of fruit than peppers planted later in the season and lacking resistance. Figure created in BioRender. Szczepaniec, A. (2025) https://BioRender.com/wt18okq.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8800235/v1/b3377fa07ea093009660152d.png"},{"id":102613818,"identity":"36725f6f-9782-4701-afec-b32561666e7a","added_by":"auto","created_at":"2026-02-13 15:12:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1830377,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8800235/v1/00631e23-b5e9-49df-9ab8-5f23480543c8.pdf"},{"id":102613706,"identity":"643ef4ea-6b13-4de5-819d-cb134bd421ce","added_by":"auto","created_at":"2026-02-13 15:11:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18638,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8800235/v1/400f44a600e68a7af3c77f07.docx"}],"financialInterests":"","formattedTitle":"Host plant tolerance and planting date influence alfalfa mosaic virus incidence, symptom severity, and yield in chile peppers","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChile peppers (\u003cem\u003eCapsicum annuum\u003c/em\u003e L.) are an important specialty crop in the United States, valued for both acreage and profitability. In 2021, nearly 11,000 acres of chile peppers produced an average of eight tons per acre and generated more than 70 million dollars in profit (USDA NASS 2021). Production is concentrated in California, New Mexico, and Colorado, and historically has required very little pest management in Colorado. That changed when Alfalfa mosaic virus (AMV) was detected in peppers in 2019, marking the first occurrence of this pathogen in the region (Amiri-Kazaz et al. 2025a). AMV reduces both yield and fruit quality, and its nonpersistent transmission by multiple aphid species creates unique challenges for effective management. In Colorado alone, AMV-associated yield and quality losses are estimated to result in millions of dollars in lost revenue annually. Notably, AMV occurs worldwide, including across many European countries, and has been reported infecting pepper crops in Spain, for example (Mallor et al. 2002).\u003c/p\u003e\n\u003cp\u003eAMV was first identified in alfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e L.) and has a broad host range; it is known to infect more than 650 plant species (Moradi and Mehrvar 2021, Yardimci et al. 2007). These hosts include economically important vegetable and specialty crops worldwide such as eggplant, tomato, potato, pea, and lentil (Jones and Coutts 1996, van Leur et al. 2013, Sofy et al. 2021). Transmission occurs via multiple aphid species during brief probing events. Because virus particles adhere to the stylet, even momentary contact with infected tissue is sufficient for transmission (Yardimci et al. 2007, Sofy et al. 2021). In Colorado, aphids rarely colonize pepper plants but are abundant in nearby alfalfa fields. As these fields are cut and aphids disperse, they likely introduce AMV into pepper crops before moving on to other hosts. This movement pattern underscores the importance of understanding local vector dynamics when developing management strategies for peppers in the region. In addition to vector transmission, AMV is transmitted mechanically. This commonly occurs through exposure to infected plant sap (Al-Saleh and Amer 2013) and through seed (Sûtic 1959,\u0026nbsp;Latham and Jones 2001, Yardimci et al. 2007, Sofy et al. 2021).\u003c/p\u003e\n\u003cp\u003ePlanting date modification is an important cultural tactic for managing viruses that are transmitted in a nonpersistent manner, because it can reduce the period when crops are exposed to high densities of infectious vectors. Several studies have shown that adjusting planting time can create asynchrony between vector activity and susceptible crop stages. For example, early sowing of fava bean reduced incidence of pea enation mosaic virus by nearly 30% when plants avoided the peak flights of pea aphids (Saucke et al. 2009). In another system, late planted rice was less susceptible to rice stripe virus transmitted by the small brown planthopper, and incidence declined by as much as 25% compared with early planted rice (Zhu et al. 2009). These cases illustrate how the timing of crop establishment can influence exposure to viruliferous insects. Although planting date is often an effective tactic, it also carries agronomic tradeoffs because delayed planting can reduce yield potential in many crops (Dadrasi et al. 2024).\u003c/p\u003e\n\u003cp\u003eHost plant resistance offers another important tactic for managing insect transmitted plant diseases, particularly when vector control is ineffective. In a recent study conducted to assess the potential of host plant resistance in chile peppers as a control strategy for AMV in the greenhouse and field (Amiri-Kazaz et al., 2025b), 20% of the tested pepper cultivars remained symptomless despite confirmed infection, while 10% exhibited symptoms without testing positive for the virus. Likewise, host plant resistance has been shown to strongly reduce disease severity and mitigate fitness losses in numerous cropping systems. For instance, barley varieties resistant to barley yellow dwarf virus produced substantially higher yields than susceptible varieties (Najar and Ben Ghanem 2017). Moreover, resistance to potato virus Y has also been a central strategy for managing this virus in potato production and continues to be a focus of breeding programs worldwide (Karasev and Gray 2013). Together, cultural practices such as planting date modification and the use of resistant or tolerant varieties provide complementary approaches for reducing disease pressure when chemical suppression of vectors is not feasible.\u003c/p\u003e\n\u003cp\u003eThe objective of this study was to evaluate how planting date modifies AMV incidence, symptom severity, and yield in two chile pepper varieties differing in susceptibility. By examining early and late plantings in both varieties, we assessed how timing of crop establishment shapes exposure to the virus and ultimately affected productivity. We also quantified aphid abundance and species composition in the field plots to identify periods of greatest vector activity and to link vector pressure with patterns of AMV infection. This integrated approach provides insight into how cultural practices and varietal selection interact under field conditions where aphids are the primary vectors of AMV. The results have practical implications for pepper growers in regions where AMV is emerging or already established, as they point to management options that reduce disease risk while maintaining yield. Understanding how planting date intersects with vector dynamics and varietal tolerance can guide more resilient production strategies and support long term sustainability of chile pepper systems.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eField plot design\u003c/h2\u003e \u003cp\u003eThe field experiment was conducted at the Colorado State University (CSU) Arkansas Valley Research Center in Rocky Ford (Otero County), Colorado over two growing seasons, 2022 and 2023. The experiment was embedded within a 1.38 ha field planted next to a 0.4 ha field of grain sorghum to the east, a 4-ha alfalfa field to the west, a 1.6 ha field of alfalfa to the north, and a 12.15 ha pasture to the south. The experimental area consisted of 12 rows, 0.91 m apart and 18.3 m in length totaling 658.4 m\u003csup\u003e2\u003c/sup\u003e. The experiment was a split-plot design with a planting date as a whole-block factor (early, conventional, and late) and plant variety as a split-block factor (a susceptible variety \u0026lsquo;Joe Parker\u0026rsquo; and a putative tolerant variety \u0026lsquo;Mira Sol\u0026rsquo;). Each of these treatments was replicated six times (N\u0026thinsp;=\u0026thinsp;36), and each replicate plot consisted of eight chile peppers, four tolerant and four susceptible. The peppers were planted 15 cm apart and two-row buffers separated each planting block.\u003c/p\u003e \u003cp\u003ePlants were grown from seed in a CSU greenhouse complex in Fort Collins, Colorado. All seeds were sown at the depth of ca. 0.5 cm in plug flats with high porosity potting mix (Lambert\u0026reg; LM-40, Rivi\u0026eacute;re-Ouelle, Qu\u0026eacute;bec, Canada) and slow-release fertilizer (Osmocote\u0026reg; Plus 15:9:12 N-P-K, ICL, Summerville, SC, USA). All plants were maintained under a 16:8 h (L:D) supplemental lighting cycle (430W High-Pressure Sodium\u0026thinsp;+\u0026thinsp;65W LED) and the day:night temperature was 31\u0026deg;C:27\u0026deg;C. The plants were watered \u003cem\u003ead libitum\u003c/em\u003e when the soil was dry. Seeds (48 per variety per planting date) were sown into flats and sowing was staggered by two weeks to ensure plants of the same approximate stage could be transplanted into the field. Once the peppers reached four true leaf stage, they were transported to Rocky Ford, CO and planted into rows assigned to the early (13 May), common (26 May), and late (8 June) planting date treatment in 2022; and early (10 May), common (23 May), and late (13 June) planting date treatment in 2023. Mean temperatures during the growing season were slightly lower in 2023 than 2022, while precipitation levels in 2023 were approximately 113% of average annual rainfall for the region (Colorado Climate Center \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSymptom incidence, disease incidence, and symptom severity ratings\u003c/h2\u003e \u003cp\u003ePlants were visually assessed for incidence of AMV symptoms on 22 July 2022 in first year of the experiment, and 12 July 2023 in the second year of the study. The response variables were the presence or absence of leaf chlorosis and necrosis, vein deformation, and leaf deformation. To diagnose disease incidence, symptomatic leaf tissue was collected from each plant on 22 July in 2022 and 7 August in 2023. Leaf tissue from each plant was placed in 2 mL centrifuge tubes (GeneMate\u0026reg;, Milford, EN, UK) and immediately stored at -80\u0026deg;C. Semi-quantitative proxies for AMV titers were obtained through ELISA (Agdia\u0026reg;, User Guide: Compound-ELISA Reagent Set). Briefly, absorbance (OD) values were determined through triple antibody sandwich enzyme-linked immunosorbent assay using monoclonal antibodies (TAS-ELISA). Tissue was processed and tested according to the Agdia\u0026reg; AMV ELISA protocol, using Agdia\u0026reg; Compound-ELISA reagent and buffer sets. Plates were assessed using the ELx800 Universal Microplate Reader (Agilent technologies Inc, Santa Clara, CA, USA) at 405 nm. All assay wells were duplicated for each plate. Samples were considered positive if absorbance value was double that of the average negative control (Ahoonmanesh et al. 1990, Sofy et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, AMV was confirmed in a subset of plants collected from the field in 2022 using RT-PCR. Total RNA was extracted from symptomatic leaf tissues using the RNeasy Plus Mini kit (Qiagen Inc., Valencia, CA, USA) following the manufacturer\u0026rsquo;s instructions. RNA quantity and quality were assessed using a Nanodrop One spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) and a Qubit 3.0 fluorometer (ThermoFisher Scientific). DNA contamination was removed from the RNA using the TURBO DNA-Free kit (Invitrogen, Waltham, MA, USA) in 15 \u0026micro;l reactions following the manufacturer\u0026rsquo;s instructions. Following DNAse treatment and quantification, one \u0026micro;g of total RNA was used to synthesize cDNA using Verso cDNA synthesis kit (ThermoFisher Scientific) according to manufacturer's instructions. The RT-PCR was performed using DreamTaq Green PCR Master Mix (2X) (ThermoFisher Scientific) with primers specific for a region of AMV coat protein (CP) -CP (5\u0026rsquo;- ATCATGAGTTCTTCACAAAAGAA-3\u0026rsquo; and 5\u0026rsquo;- TCAATGACGATCAAGATCGTC-3\u0026rsquo;) (Xu and Nie \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The amplification cycle consisted of 2 min at 95\u0026deg;C, 35 cycles of 30 sec at 95\u0026deg;C, 30 sec at 58\u0026deg;C and 1 min at 72\u0026deg;C followed by 5 min at 72\u0026deg;C. The PCR products (669 bp) were visualized on a 1% agarose gel. The sequences of each PCR product showed 99% identity to Aq isolate of AMV-CP (GenBank accession JX112758) (Xu and Nie \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeverity of symptoms was assessed on 22 June and 5 July in 2022, and 12 July and 7 August in 2023. Severity was assessed by calculating the percent of leaves with any AMV symptoms per plant for each variety and planting date.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eYield measurements\u003c/h3\u003e\n\u003cp\u003eChile pepper harvest continued weekly from 2 August to 19 September in 2022 and from 17 August to 21 September in 2023 until the plants stopped producing fruits. Chile peppers were collected when fruits reached red-mature stage, defined as 50% or more of the fruit becoming red. Peppers were sorted into labeled paper and plastic bags. The response variables collected were the number of peppers per plant.\u003c/p\u003e\n\u003ch3\u003eAphid surveys\u003c/h3\u003e\n\u003cp\u003eAphids were sampled within pepper plots using yellow pan traps interspersed within each plot. The dimensions of the yellow pan traps were 34.29 cm x 20.32 cm x 12.7 cm (JO-ANN STORES LLC, Top Notch, Hudson, OH, USA). Pans were filled with 1.25 L of propylene glycol (Sanco Industries, Inc, Fort Wayne, IN, USA) and placed between rows of chile peppers as soon as the peppers were planted. Six traps were placed within each planting date block in 2022, and three in 2023, and were spaced 3 m apart and weighed down with three small rocks 0.54 kg each within the pan trap. Surveys of aphids within the pepper plots ceased on 8 August 2022 and 14 July 2023.\u003c/p\u003e \u003cp\u003eCollection of samples from pan traps occurred weekly by filtering the contents of yellow trap pans over an organza fabric filter (JO-ANN STORES LLC, Hudson, OH, USA) into 50 mL conical tubes filled with 90% ethanol. Pan traps were refilled with up to 1.25 L of recycled or fresh propylene glycol depending on the condition of the solution. In 2023, pan traps were collected every three to four days throughout the sampling period. Samples were brought back to the laboratory and processed as described above. Aphids were identified to species or genus using a dissecting microscope and dichotomous keys (Blackman and Eastop, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Pike et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eStatistical analyses\u003c/h3\u003e\n\u003cp\u003eWe tested the three-way interaction among year of the study, planting date factor, and variety factor. Where the interactive effect of these variables did not affect the response variables, we combined the data across years. The same approach was used when testing whether the interaction among sampling date, planting date, and variety had an effect on response variables within each year. The incidence of AMV symptoms (defined as number of plants with chlorosis and/or necrosis divided by the total number of sampled plants) and incidence of positive ELISA tests were evaluated for each plant and analyzed using logistic regression and generalized linear mixed-effects models with the lme4 package (Bates et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, R Core Team \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Severity of symptoms and yield were averaged across replicates and compared among varieties and planting dates for both years using ANOVA linear models (R Core Team \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) if data met assumptions of normality and homogeneity of variances (Fox and Weisberg \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Means separation tests (Tukey\u0026rsquo;s Honestly Significant Differences test) were performed where appropriate. Kruskal-Wallis and Dunn\u0026rsquo;s test for means separation were used to analyze data that did not meet assumptions of ANOVA and were analyzed using non-parametric tests (R Core Team \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAphid abundance was analyzed by comparing mean species densities within each sampling date using ANOVA. Each yellow pan trap was considered a replicate. Data that did not meet assumptions of ANOVA were transformed (square root or log) or analyzed using non-parametric tests Kruskal-Wallis and Dunn\u0026rsquo;s test. All analyses were performed in R statistical analysis software (R Core Team \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eIncidence of AMV symptoms and disease\u003c/h2\u003e\n \u003cp\u003eWe found that the three-way interaction among year, variety, and planting date did not have a significant interactive effect on incidence of plants showing AMV symptoms (\u003cem\u003e\u0026Chi;\u003c/em\u003e\u0026sup2;= 1.37, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.172), indicating that the effects of planting date on symptom incidence did not differ between varieties across years. Data on incidence of AMV symptoms from both years were combined to assess the effect of planting dates and variety across the two seasons. We found a significant interactive effect between planting date and variety on incidence of disease symptoms (\u003cem\u003e\u0026Chi;\u003c/em\u003e\u0026sup2; = 27.2, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and thus compared the effects of these factors on symptom incidence within each planting date (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Early planting was associated with higher odds of visible AMV symptoms compared to peppers planted at common or late planting dates, particularly in the susceptible variety \u0026lsquo;Joe Parker\u0026rsquo;. In contrast, \u0026lsquo;Mira Sol\u0026rsquo; consistently exhibited lower incidence of disease symptoms, although still influenced by planting date (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePairwise comparisons of AMV symptom incidence (odds ratios) among planting times and pepper varieties.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eComparison\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003eOdds ratio\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ez ratio\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003csup\u003eb\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCommon \u0026lsquo;Joe Parker\u0026rsquo; vs. Early \u0026lsquo;Joe Parker\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.369\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.097\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.792\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCommon \u0026lsquo;Joe Parker\u0026rsquo; vs. Late \u0026lsquo;Joe Parker\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.467\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCommon \u0026lsquo;Joe Parker\u0026rsquo; vs. Common \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.126\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.239\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCommon \u0026lsquo;Joe Parker\u0026rsquo; vs. Early \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.561\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.779\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCommon \u0026lsquo;Joe Parker\u0026rsquo; vs. Late \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.948\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.440\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.363\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEarly \u0026lsquo;Joe Parker\u0026rsquo; vs. Late \u0026lsquo;Joe Parker\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.539\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.620\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.864\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEarly \u0026lsquo;Joe Parker\u0026rsquo; vs. Common \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.390\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEarly \u0026lsquo;Joe Parker\u0026rsquo; vs. Early \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e9.659\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.864\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEarly \u0026lsquo;Joe Parker\u0026rsquo; vs. Late \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.978\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.530\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.804\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLate \u0026lsquo;Joe Parker\u0026rsquo; vs. Common \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.483\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-3.331\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.011\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLate \u0026lsquo;Joe Parker\u0026rsquo; vs. Early \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.551\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.121\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-2.719\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.072\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLate \u0026lsquo;Joe Parker\u0026rsquo; vs. Late \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.538\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.378\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.750\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.498\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCommon \u0026lsquo;Mira Sol\u0026rsquo; vs. Early \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.989\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCommon \u0026lsquo;Mira Sol\u0026rsquo; vs. Late \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.182\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.752\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.898\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEarly \u0026lsquo;Mira Sol\u0026rsquo; vs. Late \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.793\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.663\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.326\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\u003csup\u003ea\u003c/sup\u003eOdds ratios were estimated using a generalized linear mixed model with a binomial distribution.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\u003csup\u003eb\u003c/sup\u003eP-values were adjusted using the Tukey HSD test.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eOn the other hand, the year of the study, planting date, and variety had a significant interactive effect on proportion of plants testing positive for AMV (\u003cem\u003ei.e.\u003c/em\u003e, incidence of disease) (\u003cem\u003e\u0026Chi;\u003c/em\u003e\u0026sup2;= 27.28; df\u0026thinsp;=\u0026thinsp;2; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and both years were analyzed separately. There was no significant interaction between variety and planting date in 2022 (\u003cem\u003e\u0026Chi;\u003c/em\u003e\u0026sup2; = 1.50, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.473) or 2023 (\u003cem\u003e\u0026Chi;\u003c/em\u003e\u0026sup2; = 2.17, df\u0026thinsp;=\u0026thinsp;2, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.338), and an additive model was used to assess the impact of both treatments on incidence of disease in both years. In 2022, there was a significant increase in the likelihood that peppers planted early tested positive for AMV compared to plants that were sown at the common planting time (\u0026beta;\u0026thinsp;=\u0026thinsp;2.66, SE\u0026thinsp;=\u0026thinsp;1.07, \u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.48, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013). \u0026lsquo;Mira Sol\u0026rsquo; and \u0026lsquo;Joe Parker\u0026rsquo; from the late plantings did not differ significantly from each other in disease incidence (\u0026beta;\u0026thinsp;=\u0026thinsp;0.46, SE\u0026thinsp;=\u0026thinsp;1.25, \u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.37, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.714). Overall, \u0026lsquo;Mira Sol\u0026rsquo; cultivar had marginally lower odds of positive ELISA tests for AMV compared to \u0026lsquo;Joe Parker\u0026rsquo; in 2022 (\u0026beta; = -1.17, SE\u0026thinsp;=\u0026thinsp;0.64, \u003cem\u003ez\u003c/em\u003e = -1.81, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.07) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). This effect was much stronger in 2023 when \u0026lsquo;Mira Sol\u0026rsquo; was significantly less likely to test positive for AMV compared to \u0026lsquo;Joe Parker\u0026rsquo; (\u0026beta; = -0.93, SE\u0026thinsp;=\u0026thinsp;0.42, \u003cem\u003ez\u003c/em\u003e= -2.24, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025). Peppers planted late also had a significantly lower probability to test positive for AMV compared to the plants sown at the common planting time (\u0026beta; = -1.91, SE\u0026thinsp;=\u0026thinsp;0.69, \u003cem\u003ez\u003c/em\u003e = -2.78, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005). The effect of early planting was not statistically significant (\u0026beta;\u0026thinsp;=\u0026thinsp;0.64, SE\u0026thinsp;=\u0026thinsp;0.43, \u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.48, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.138) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eSeverity of AMV symptoms\u003c/h3\u003e\n\u003cp\u003eThe three-way interaction among year, planting date, and variety also influenced the severity of AMV symptoms (F\u003csub\u003e2,60\u003c/sub\u003e = 6.02, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). Thus, both years were analyzed separately to assess how planting date and variety affected the severity of disease within each year. In 2022, the sampling date (June, July), planting date, and variety had a significant interactive effect on the severity of the symptoms (F\u003csub\u003e2,30\u003c/sub\u003e = 22.41, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as did the interaction between planting date and variety (\u003cem\u003e\u0026Chi;\u003c/em\u003e\u0026sup2; = 19.08, df\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Pairwise comparisons revealed significant differences among planting dates for each variety, with the exception of \u0026lsquo;Joe Parker\u0026rsquo; planted early and at the common planting dates, \u0026lsquo;Mira Sol\u0026rsquo; planted early and at the common planting date, and late planted \u0026lsquo;Mira Sol\u0026rsquo; and \u0026lsquo;Joe Parker\u0026rsquo; (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Both varieties had higher mean AMV severity when planted at early and common planting dates, and the proportion of leaves with symptoms was low for \u0026lsquo;Joe Parker\u0026rsquo; and \u0026lsquo;Mira Sol\u0026rsquo; peppers planted late (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In July, on the other hand, the interaction between planting date and variety did not significantly affect severity of AMV symptoms (F\u003csub\u003e2,30\u003c/sub\u003e = 1.42, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.258). Both planting date (F\u003csub\u003e2,30\u003c/sub\u003e = 7.98, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) and variety (F\u003csub\u003e1,30\u003c/sub\u003e = 18.14, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) had significant impacts on proportion of leaves with AMV symptoms in July. \u0026lsquo;Joe Parker\u0026rsquo; had significantly greater severity of AMV symptoms than \u0026lsquo;Mira Sol\u0026rsquo;, and both varieties planted early had higher proportion of affected leaves (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePairwise comparisons of severity of AMV symptoms among pepper cultivars and planting dates.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCommon \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLate \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eEarly \u0026lsquo;Joe Parker\u0026rsquo;\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCommon \u0026lsquo;Joe Parker\u0026rsquo;\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLate \u0026lsquo;Joe Parker\u0026rsquo;\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEarly \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCommon \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLate \u0026lsquo;Mira Sol\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEarly \u0026lsquo;Joe Parker\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCommon \u0026lsquo;Joe Parker\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026mdash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e\u0026dagger;Values represent \u003cem\u003eP\u003c/em\u003e values from Wilcoxon rank sum tests with continuity correction.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn 2023, the sampling date (July and August), planting date, and variety did not have a significant interactive effect on the severity of the symptoms (F\u003csub\u003e2,30\u003c/sub\u003e =0, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1), but planting time and variety interacted strongly to affect diseases severity (F\u003csub\u003e2,30\u003c/sub\u003e = 66.42, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Early planted \u0026lsquo;Joe Parker\u0026rsquo; had significantly higher severity of symptoms than \u0026lsquo;Joe Parker\u0026rsquo; peppers planted at common or late planting dates, and higher AMV symptom severity than \u0026lsquo;Mira Sol\u0026rsquo; planted at any planting date (Dunn\u0026rsquo;s test with Bonferroni adjustment, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eEffect of variety and planting date on yield of peppers\u003c/h3\u003e\n\u003cp\u003eYear, variety, and planting date did not have an interactive effect on yield of peppers (F\u003csub\u003e2,60\u003c/sub\u003e = 1.42, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.251), and yield data were combined for analyses across the two years of the experiment. Chile pepper variety (F\u003csub\u003e1,30\u003c/sub\u003e = 21.49, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and planting date (F\u003csub\u003e2,30\u003c/sub\u003e = 10.55, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) had a significant effect on pepper yield, while there was no significant interactive effect of these two factors on yield (F\u003csub\u003e2,30\u003c/sub\u003e = 2.02, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.15). Tolerant plants produced more than twice as many peppers as the susceptible plants (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA), and early and conventional plantings had significantly higher yields than the late planted peppers, regardless of the variety (F\u003csub\u003e2,30\u003c/sub\u003e = 10.55, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eAphid surveys in experimental research plots\u003c/h2\u003e\n \u003cp\u003eWe found high diversity of aphids in chile pepper plots in both years of the survey, and we identified 15 species of aphids (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In each year, aphid densities were the highest early in the season (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). There were significant differences in densities of aphid species in experimental pepper plots across most of the growing season in 2022 (Supplemental Table S1). \u003cem\u003eAphis\u003c/em\u003e spp. was more abundant than all other species across all sampling dates in 2022 and dominated most of the samples (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). There were some notable peaks in \u003cem\u003eAphis\u003c/em\u003e spp. densities in early June, July, and August.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eList of aphid species/genus found in chile pepper plots.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003cth style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003eAphid species\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAcyrthosiphon kondoi*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAcyrthosiphon pisum*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAcyrthosiphon\u003c/em\u003e spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAphis\u003c/em\u003e spp.*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCapitophorus elaeagni\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eChaitophorus\u003c/em\u003e spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eDiuraphis noxia\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEriosoma\u003c/em\u003e spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eHayhurstie atriplicis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eLipaphis erysimi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eMyzus persicae*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003ePemphigus\u003c/em\u003e spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eProtaphis\u003c/em\u003e spp.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eRhopalosiphum padi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr style=\"height: 35px;\"\u003e\n \u003ctd style=\"height: 35px;\" align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTherioaphis maculata*\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr style=\"height: 13.0996px;\"\u003e\n \u003ctd style=\"height: 13.0996px;\" colspan=\"1\"\u003eSpecies and genera marked with asterisks are known vectors of AMV\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u0026nbsp;There were also significant differences in the densities of aphid species in pan traps in 2023 (Supplemental Table S1). Blue alfalfa aphid was the most abundant species in pan trap samples, while \u003cem\u003eAphis\u003c/em\u003e spp. became more abundant later in the season (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). From 2 June to 9 June there was a steep increase in density of \u003cem\u003eAphis\u003c/em\u003e spp., pea aphid, spotted alfalfa aphid, and blue alfalfa aphid, which was followed by a sharp decline in mid to late June. \u003cem\u003eAphis\u003c/em\u003e spp. made a small resurgence in early- to mid-July.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlanting date strongly shaped AMV incidence, symptom severity, and yield in this study, and emerged as an effective management factor across both pepper varieties and years. Early plantings consistently experienced higher AMV incidence and more severe symptoms, a pattern that closely followed the pronounced early season peaks in aphid densities observed in the field plots. These aphids included several known vectors of AMV and were most abundant during the period immediately following transplanting. Because AMV is transmitted in a nonpersistent manner, even brief probing by dispersing aphids is sufficient for infection, and the timing of vector arrival relative to crop establishment becomes a critical determinant of disease risk. Our findings suggest that early planted peppers are exposed to the highest virus pressure simply because they are present when vector activity is greatest. Despite the higher incidence of AMV in early plantings, these peppers produced substantially greater yields than peppers planted late. This outcome indicates that yield potential is strongly tied to the length of the growing season. Plants established early had more time to produce fruit and to compensate for virus infection, particularly in the tolerant variety. Late plantings, although less infected, suffered from shortened production windows and produced the lowest yields. This disconnect between disease reduction and yield performance underscores an important consideration for growers: minimizing AMV incidence does not necessarily translate into improved productivity if planting is delayed. The tradeoff between reduced disease and reduced yield must therefore be weighed carefully when selecting planting times.\u003c/p\u003e \u003cp\u003eFew studies have evaluated yield outcomes in relation to planting date-mediated disease reduction. For example, delaying sowing of cucurbit crops reduced incidence of zucchini yellow mosaic virus by up to 49% (Coutts et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and planting mungbean earlier or later than the conventional planting dates resulted in up to 28% reduction in incidence of mungbean yellow mosaic virus (Swamy et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, neither of these studies reported the impact on crop yields. This suggests that altering sowing dates can be useful in mitigating disease severity and risk, but the consequences of these modifications to plant productivity should be studied in greater depth. We showed that despite higher incidence and relative amounts of virus, early planting increases the yield, particularly for the tolerant variety.\u003c/p\u003e \u003cp\u003eVarietal differences were also a strong predictor of disease incidence and severity. The putatively tolerant variety, Mira Sol, exhibited both lower AMV incidence and reduced symptom severity compared with Joe Parker, and produced more than twice as many peppers across planting dates. These findings suggest that tolerance provides meaningful protection against both the biological effects of infection and the economic consequences of yield loss. Host plant resistance is often the most reliable tactic for suppressing virus diseases and continues to anchor plant-breeding efforts (Corr\u0026ecirc;a et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Nazarov et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and there are numerous examples of successful employment of resistant crop varieties to improve host plant resistance to fungal (\u003cem\u003ee.g\u003c/em\u003e., Sanogo and Zhang \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sharma et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), bacterial (Sharma et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and viral (Gottula and Fuchs \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) pathogens.\u003c/p\u003e \u003cp\u003eNotably, both planting date and varietal tolerance strongly influenced yield, and their effects were additive rather than interactive. The tolerant variety produced more than twice as many peppers as the susceptible one, while early and common plantings yielded substantially more fruit than late plantings. Research from other cropping systems reinforces our results. For example, Kone and colleagues (2017) found that incidence of viral disease and severity in cucurbits were significantly shaped by planting date although cultivar differences remained important, and the two factors had an additive effect on improving plant health. Our findings align with our results where the tolerant variety consistently out-yielded the susceptible one and early planting produced higher yields than late planting. The additive nature of those effects underscores the importance of combining host plant resistance with optimal planting timing to maximize yield and reduce disease impact.\u003c/p\u003e \u003cp\u003eWe also noted a strong alignment between aphid abundance and AMV incidence, which points to the importance of understanding local vector dynamics. Aphids rarely colonized peppers directly and were instead likely introduced into the plots from surrounding alfalfa and other crops. As alfalfa fields were cut, winged aphids dispersed into adjacent crops, including peppers. This suggests that regional patterns of alfalfa harvest, the composition of nearby crops, and seasonal aphid phenology all contribute to the timing and magnitude of AMV pressure. Integrating information about these landscape-level drivers with planting date decisions could improve growers\u0026rsquo; ability to anticipate periods of high infection risk. Similar association between aphid abundance and disease risk has been reported previously, and several field studies have shown that altering planting date can significantly reduce viral disease incidence and severity. For example, Kon\u0026eacute; et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) showed that varying sowing dates in cucurbits altered exposure to aphid vectors and reduced viral disease incidence. Likewise, early-season aphid abundance has been identified as a primary predictor of infection risk for aphid-borne viruses, including cucurbit aphid-borne yellows virus in melon (Schoeny et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our system, the strong effect of planting date likely reflects a comparable process, whereby adjusting crop establishment to avoid peak aphid flights reduced disease pressure while still allowing high yield when varietal tolerance was present.\u003c/p\u003e \u003cp\u003eIt is important to note that the high diversity of aphid species we reported in our surveys is likely due to a diverse array of crops that surrounded our pepper plots including \u003cem\u003eSorghum bicolor\u003c/em\u003e L., alfalfa, corn, melons, and cowpea. These crops can be colonized by the common vectors of AMV such as green peach aphid, pea aphid, blue alfalfa aphid, bean aphid, and spotted alfalfa aphid (Blackman and Eastop, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). While we did not observe any of the aphid species actively feeding or colonizing chile peppers in either of the years of the field experiment, green peach aphid has been noted in this crop as its most common and damaging aphid pest (Sun et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is noteworthy that weather patterns differed between the two years of the study, and these differences may have influenced aphid activity. Rocky Ford received substantially more precipitation than average in May and June of 2023, while May of 2022 was close to normal and June received only half of the expected rainfall. These conditions may have shaped vector dynamics because aphid communities showed a different pattern across years. Peak aphid densities were higher in 2023, yet overall aphid abundance across the season was lower than in 2022. This suggests that moisture patterns and early season conditions may contribute to the timing and magnitude of aphid flights and, in turn, the risk of AMV transmission.\u003c/p\u003e \u003cp\u003eTogether, these results highlight that adjusting planting date is an effective and practical tactic for managing AMV risk in chile pepper systems, although it must be balanced against potential reductions in yield when planting is delayed. Early planting paired with a tolerant variety appears to offer the most resilient combination under the conditions of this study. More broadly, the work demonstrates the importance of aligning cultural practices with vector phenology to reduce virus pressure in systems where chemical control of vectors is not effective. Future research that links planting date with fine-scale vector monitoring, evaluates additional varieties for resistance or tolerance, and examines landscape effects on aphid movement will further refine recommendations for sustainable AMV management in Colorado and other regions where this virus is present. Integrating AMV-tolerant varieties with optimized planting dates may substantially improve the resilience of chile pepper production systems, especially given the complex relationship between increased vectors abundance early in the season and plants\u0026rsquo; ability to compensate for that exposure if planted sufficiently early (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe thank the Colorado State University Arkansas Valley Research Center staff and the farm manager K. Tanabe for providing plots for chile pepper experiments, A.C. Fulladolsa for her expert advice on performing ELISA assays, and C. Vaughn for help with field data collection and processing. M. Bartolo provided guidance on the seed selection and invaluable insights into host plant resistance in the region of Arkansas Valley of Colorado. This research was funded by the Colorado Department of Agriculture Specialty Crop Block Grant Program 21SCBGPCO1008-00 to P.N. and A.S.; and the United States Department of Agriculture, National Institute of Food and Agriculture grant numbers 2021-70006-35439, 2024-70006-43565, and COL00411, accession number 7001931 to A.S.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAl-Saleh MA, Amer MA (2013) Biological and molecular variability of alfalfa mosaic virus affecting alfalfa crops in the Riyadh region. 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Agric Sci China 8:332\u0026ndash;341\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bromoviridae, Capsicum annuum, aphids, IPM","lastPublishedDoi":"10.21203/rs.3.rs-8800235/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8800235/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eAlfalfa mosaic virus\u003c/em\u003e (AMV) is transmitted nonpersistently by multiple aphid species and has become an emerging threat to chile pepper production in regions of the United States where peppers are grown in the vicinity of alfalfa, the primary reservoir of the pathogen. Because nonpersistent transmission limits the effectiveness of chemical vector control, management strategies that reduce exposure to viruliferous aphids are needed. We evaluated how planting date and varietal susceptibility shape AMV incidence, symptom severity, and yield in two chile pepper (\u003cem\u003eCapsicum annum\u003c/em\u003e L.) varieties in a region where peppers are grown near alfalfa, a primary reservoir of AMV and source of dispersing aphids. Across two years, early plantings had the highest AMV incidence and symptom severity, which coincided with pronounced early season peaks in aphid abundance. The putatively tolerant variety consistently showed lower AMV incidence and reduced severity than the susceptible variety, regardless of planting date. Despite greater virus pressure, early plantings produced the highest yields, reflecting the longer production window and the capacity for yield compensation, particularly in the tolerant variety. Late plantings reduced AMV incidence but produced the lowest yields because of shortened growing seasons. These results indicate that planting date strongly mediates disease risk and productivity and that varietal tolerance provides additional protection without altering the effect of planting date. Together, optimized planting timing and selection of tolerant varieties offer practical, complementary strategies for managing AMV in regions where dispersing aphids drive infection risk.\u003c/p\u003e","manuscriptTitle":"Host plant tolerance and planting date influence alfalfa mosaic virus incidence, symptom severity, and yield in chile peppers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 15:11:25","doi":"10.21203/rs.3.rs-8800235/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2026-03-25T05:19:46+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-02-20T02:40:19+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-10T04:56:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Plant Diseases and Protection","date":"2026-02-09T11:52:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-07T18:06:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Diseases and Protection","date":"2026-02-05T13:51:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8fc43d72-a435-43f4-9229-c473538c321a","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T06:50:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 15:11:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8800235","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8800235","identity":"rs-8800235","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}