DOI: 10.5061/dryad.g4f4qrg28
title: Differential beet leafhopper (<em>Neoalitarsus tenellus</em> (Hemiptera: Cicadellidae)) acceptance of allelopathic barley (<em>Hordeum vulgare</em> (Poales: Poaceae) and brown mustard (<em>Brassica juncea</em> (Brassicales: Brassicacae) cover crops

description: Chile pepper, Capsicum annuum L.(Solanales: Solanaceae) yield in New
 Mexico is threatened by a multitude of factors, including beet curly top
 virus (BCTV) vectored by the beet leafhopper, Neoalitarsus tenellus
 (Baker) and competition with annual weeds. Early-season weeds in chile
 pepper can be reduced by either barley, Hordeum vulgare L. or brown
 mustard, Brassica juncea (L.) Czern. cover crops; however, the potential
 of attracting N. tenellus should be factored into cover crop selection. In
 this study, abundance of leafhopper guilds was measured in field plots in
 Leyendecker, NM (2021-2022 and 2022-2023) and Los Lunas, NM (2021-2022)
 featuring a brown mustard ('Caliente Rojo'), barley
 ('Stockford'), or combination cover crop treatment. In a
 controlled environment study, viruliferous N. tenellus adults
 were subjected to dual-choice tests between brown mustard and one of two
 barley cultivars ('Stockford' or 'Valor'), and their
 presence on each plant after 24 h was recorded. A subsequent no-choice
 test trapped viruliferous N. tenellus on brown mustard, Stockford
 barley, or Valor barley for 24 h; after recording N. tenellus survival,
 leaf tissue was stained and analyzed for the presence of sheaths,
 superficial punctures, and eggs. Field results indicated that leafhopper
 guilds were three times more abundant in brown mustard treatments than
 Stockford barley or combination treatments. In dual-choice tests, N.
 tenellus demonstrated a nine times greater acceptance of brown mustard
 than either barley cultivar. In no-choice tests, N.
 tenellus trapped on Stockford barley faced reduced survival.
 Additionally, N. tenellus successfully ingested vascular fluid of brown
 mustard four times more often than N. tenellus trapped on barley.
 No eggs were laid on either barley cultivar, indicating its unsuitability
 as a host plant. This research indicates that barley is less likely than
 brown mustard to sustain N. tenellus populations that have the capacity to
 transmit BCTV to subsequently planted chile pepper. Further research is
 needed to understand the relationship between allelochemicals and N.
 tenellus feeding, survival, and reproduction.
description: # Differential beet leafhopper (*Neoalitarsus tenellus* (Hemiptera:
 Cicadellidae)) acceptance of allelopathic barley (*Hordeum vulgare*
 (Poales: Poaceae)) and brown mustard (*Brassica juncea* (Brassicales:
 Brassicacae)) cover crops Dataset DOI:
 [10.5061/dryad.g4f4qrg28](10.5061/dryad.g4f4qrg28) ## Description of the
 data and file structure **Vacuum data:** A field study was conducted at
 two sites: The New Mexico State University (NMSU) Leyendecker Plant
 Science Research Center near Las Cruces, NM (32.20 N 106.74 W; hereafter
 “Leyendecker”) and the NMSU Los Lunas Agricultural Science Center in Los
 Lunas, NM (34.77N 106.76 W; hereafter “Los Lunas”). Cover crops were sown
 in the fall and terminated in the spring. At both sites, the study was
 conducted from fall 2021 to spring 2022 and replicated fall 2022 to spring
 2023. Site-years are referenced by the year in which cover crops were
 terminated. Treatments were arranged in a randomized complete block design
 with four replications. Plot sizes were 19.81 x 4.05 m at Leyendecker and
 3.05 m x 19.8 m at Los Lunas . Cover crop treatments consisted of barley
 monocultures, brown mustard monocultures, barley-mustard combinations, and
 a noncover control. The noncover control was needed for a separate
 experiment concerning weeds and was not sampled in this study. Barley
 cultivars included the two-rowed ‘Stockford’ at Leyendecker 2022,
 Leyendecker 2023, and Los Lunas 2022; and the six-rowed ‘Valor’ at Los
 Lunas 2023. The brown mustard cultivar was ‘Caliente Rojo’ at all
 site-years. Because Los Lunas 2023 experienced brown mustard failure, it
 was omitted from analysis in this study. Additional information on cover
 crop planting and management can be found in Toth and Schutte (2025).
 Beginning in January and continuing through March (Table 1), leafhopper
 guilds were sampled biweekly. Sampling was carried out with a modified
 leaf vacuum (Echo ES-250 “Shred ‘n’ Vac”, Lake Zurich, IL, USA). A swath
 of finely knit mesh fabric was loosely draped over the mouth of the vacuum
 tube and secured with hose clamps. When the vacuum was running, the
 suction drew the mesh pocket within the nozzle by about 53 cm, allowing
 top portions of foliage to be siphoned thoroughly. The mouth of the vacuum
 was repeatedly passed over the foliage present in a 0.25 m2 quadrat within
 a plot for 60 s. The vacuum was then tilted upwards to keep the siphoned
 contents within the mesh pocket before being turned off, at which point
 the mouth of the vacuum was placed inside a 3.8 L plastic bag. Finally,
 the mouth of the vacuum was repositioned perpendicular to the ground, and
 the nozzle was repeatedly struck to facilitate a complete transfer of
 siphoned material from the inverted mesh pocket to the plastic bag. Sealed
 plastic bags were stored inside of a -4 C freezer. Large components of
 vegetative debris were filtered from the samples by passing the contents
 of the bag through a 5 mm sieve. Sieved samples were spread onto a single
 layer on white paper and observed through a magnifying lens to quantify
 the number of leafhoppers per sample. Three and four replications of each
 cover crop treatment were surveyed each visit in 2022 and 2023,
 respectively.   Before conducting preliminary analyses, leafhopper counts
 within a treatment and replicate were summed across visits for each
 site-year. A preliminary Kruskal-Wallis test was conducted to examine
 differences in leafhopper abundance as a result of site-year. Because no
 significant differences were found among the three site-years (chi square
 = 6.161, P = 0.104, df = 2), all site-years, not including Los Lunas 2023,
 were analyzed together. Cover crop treatment effects on total leafhopper
 abundance were determined with generalized linear models with negative
 binomial distributions, featuring cover crop treatment as a fixed effect,
 and site-year and the interaction between site-year and replication as
 predictor variables (random effects). The dispersion parameter θ was
 estimated by the maximum likelihood as θ = 3.40. Models were developed
 using the glm.nb function within the R library *mass* (Veneables and
 Ripley 2002), and model parameter estimates assessed possible differences
 among cover crop treatments. Specifically, parameter estimates with
 overlapping 90% confidence intervals indicated similarity among cover crop
 treatments.   **Dual-choice test data:** In 2019, 50 *N. tenellus*
 collected from different locations in Idaho were used to establish a New
 Mexico colony as described in (Creamer et al. 2024). This colony was
 reared in cages measuring 19 x 38 x 38 cm, each containing one sugar beet
 plant [*Beta vulgaris* L. subsp. *vulgaris* (var.* saccharifera*)].
 Rearing cages were maintained at 28 C day/26 C night with a 16-h
 photoperiod in a Percival Model E54B growth chamber. All *N. tenellus*
 were infectious with BCTV-Svr, a strain known to replicate in and infect
 chile pepper (Creamer et al. 2024). Dual-choice preference tests were
 carried out inside of cages measuring 19 cm x 38 cm x 38 cm. Each cage
 contained one “menu”. Menu A tested Stockford barley against brown
 mustard, while Menu B tested Valor barley against brown mustard. All
 barley and brown mustard samples were taken from ~8 wk old plants grown in
 the greenhouse from seed in Miracle Gro potting mix (Scotts Miraclo-Gro
 Company, Marysville, Ohio, USA). Seedlings were hand-watered daily. The
 average temperature in the greenhouse was 23.3 C, and the average relative
 humidity was 26%. Day length averaged 10.7 hrs. The brown mustard was at
 the bolting stage, but not yet flowering, while the barley was at the stem
 elongation stage. Plant samples consisted of four leaves or blades and
 their attached root systems. Each sample was placed in an individual 125
 ml Erlenmeyer flask containing DI water. The flask mouth was sealed with
 Parafilm (Parafilm ® M Sealing Film, Amcor, Denver, Colorado, USA) so that
 only stems and leaves were exposed. The two-sample menu — consisting of
 one barley sample and one brown mustard sample — was then placed inside of
 one cage. Two cages, each featuring a different menu, were tested
 simultaneously. To obtain *N. tenellus*, rearing cages were moved to a
 fluorescent lightbox concealed from external light by a black felt
 curtain. Rearing cage doors were then held open and gently shaken in front
 of the lightbox. Ten viruliferous adult *N. tenellus* were suctioned from
 the lightbox surface and aspirated into each menu cage via a small,
 circular hole in the plexiglass wall. The hole was plugged with cork
 immediately after aspiration to prevent *N. tenellus* escape. Both menu
 cages were equally lit with fluorescent light. Menu cages were left
 undisturbed for 24 hours, after which the number of *N. tenellus* on each
 plant sample were counted via visual inspection. *Neolitarsus tenellus*
 were then returned to their rearing cages. To prepare for the next
 replication, the position of the plants inside the cages were rotated, and
 the position of the cages were swapped. A total of six replications were
 carried out, with the adult *N. tenellus* population sampled randomly with
 replacement from the rearing cages. Prior to analysis, data were pooled
 across replicates. The mean probability of success — that is, of *N.
 tenellus* choosing either barley or mustard within a given menu  — was
 evaluated by carrying out two-tailed hypothesis testing (H0: p0 = 0.50)
 with an exact binomial test in the binom package in R (Dorai-Raj 2022).
 Any *N. tenellus* that were not on either plant sample at the time of
 visual inspection were excluded from analysis. Differences in mean
 probabilities within a given menu were considered significant when P ≤
 0.05. **No-choice test data:**  To determine behavioral changes in *N.
 tenellus* that were precluded from moving freely between hosts, a
 no-choice test was conducted. As in Objective 2, plants were grown from
 seed in the greenhouse for ~8 wk. One potted plant each of Stockford
 barley, Valor barley, and brown mustard was placed within a single cage
 measuring 26 cm x 34 cm x 48 cm (herein “main cage”). One leaf on each
 plant was selected for a clip cage. Clip cages were constructed with two
 plastic and mesh, 5.5 cm-diameter disks that were each lined with foam
 (foam thickness, 0.5 cm).  Disks were positioned on each side of a leaf to
 form an enclosure and were bound together by a rubber band. Five
 viruliferous *N. tenellus* adults were aspirated into the seams of each
 clip cage. The main cage was then placed parallel to the lightbox and left
 undisturbed for 24 hours. The next day, any *N. tenellus* found dead
 inside the clip cages were recorded and discarded, and the surviving *N.
 tenellus* were returned to the rearing cages. The enclosed leaf tissue was
 clipped from the plant and analyzed as described below. To prepare for the
 subsequent replication, the clip cage was re-secured around a new leaf on
 each plant. Five new adult *N. tenellus* were randomly selected with
 replacement from the rearing cages and aspirated into the new clip cages.
 Six replications were carried out on each plant. In a process adapted from
 Backus et al. 1988), leaf tissue clipped from the main plant was submerged
 in McBride’s stain, composed of 0.2% acid fuchsin in 95% ethanol and
 glacial acetic acid (1:1 vol/vol).  After 24 hours, the leaf was moved to
 a beaker containing clearing solution (deionized water, 99% glycerin, and
 90% lactic acid; 1:1:1 vol/vol/vol), and the contents of the beaker were
 boiled for five minutes. The leaf was then removed from the clearing
 solution, submerged in a Falcon tube containing new clearing solution, and
 refrigerated at 4 C. Leaves were mounted under a Nikon stereo microscope
 (Model SMZ645, Melville, NY, USA). For each leaf, the total numbers of
 sheathes, superficial punctures, and eggs were recorded. Sheaths,
 superficial punctures, and eggs were identified as pink extended lines
 leading to vascular elements, pink dots, and pink globose masses,
 respectively. For each individual plant and replication, the quantity of
 dots and sheaths were added to represent the total number of probing
 events. Then, the number of dots and sheaths were each converted to a
 percentage of total probing events. Similarly, survival per replication
 was converted into a percentage of the five *N. tenellus* aspirated into
 the clip cage. Accordingly, response variables included, total number of
 eggs, total number of feeding events, and percentages of dots, sheaths,
 and survival. For each of these response variables, the non-parametric
 Kruskal-Wallis test was used to test the null hypothesis that response
 variable was equal among the three plant cultivars (predictor variable).
 When P≤ 0.05, a subsequent post-hoc pairwise comparison test was carried
 out using the Dunn Test with the Benjamin-Hochberg adjustment. ### Files
 and variables #### File: leafhopper_vacuum_counts.csv **Description:** 
 .csv file containing counts of leafhoppers, not identified to the species
 level, found on various cover crop treatments in various sites within New
 Mexico. ##### Variables * Site: Either Leyendecker (32.20 N 106.74 W) or
 Los Lunas(34.77N 106.76 W), NM. * Year: "2022" refers to the
 2021-2022 season. "2023" refers to the 2022-2023 season. * Date:
 As listed. * Plot: Numbered sub-plot. Each treatment was featured in 4
 unique plots numbers within a site-year. * Rep: Refers to the vertical
 "lane" (1 of 4) featured in a given site-year. Each rep
 feautured one unique cover crop treatment. * Treatment: Refers to cover
 crop treatment. "Barley" = 'Stockford' barley
 monoculture; "Mustard" = 'Caliente Rojo' brown mustard
 monoculture; "Combo" = mix of 'Stockford' barley and
 'Caliente Rojo' brown mustard. * Method: Refers to method by
 which leafhoppers were sampled. Only vacuum surveys were used in this
 data. * Count: Refers to the number of leafhoppers that were counted per
 plot on a given date. Leafhoppers were vacuumed, transferred to plastic
 bags, and frozen before being counted under a magnifying lens. #### File:
 BLH_vacuum_counts_analysis.Rmd **Description:**  R Markdown file providing
 step-by-step narrated statistical analysis of
 leafhopper_vaccum_counts.csv. #### File: BLH_dual_choice_tests.csv
 **Description:**  .csv file containing raw, total counts of viruliferous
 *N. tenellus* present on a given plant after being subjected to a 24-h
 dual-choice test under controlled conditions. A total of six replications
 (across two runs) were carried out for each menu; each replication
 consisted of ten viruliferous *N. tenellus* adults aspirated into a cage
 containing a given menu of plants. Values presented here are sums, which
 allow for subsequent statistical analysis. ##### Variables *
 A_Barley_Stockford: Refers to the number of viruliferous *N. tenellus*
 found on 'Stockford' Barley, when featured in dual-choice menu A
 along with 'Caliente Rojo' brown mustard. * A_Mustard: Refers to
 the number of viruliferous *N. tenellus* found on 'Caliente
 Rojo' brown mustard, when featured in dual-choice menu A along with
 'Stockford' barley. * A_Total: Refers to the total number of
 viruliferous *N. tenellus* that could be accounted for at the conclusion
 of the 24-h dual-choice test featuring Menu A. Leafhoppers that could not
 be accounted for were assumed to be dead. * B_Barley_Valor: Refers to the
 number of viruliferous *N. tenellus* found on 'Valor' Barley,
 when featured in dual-choice menu B along with 'Caliente Rojo'
 brown mustard. * B_Mustard: Refers to the number of viruliferous *N.
 tenellus* found on 'Caliente Rojo' brown mustard, when featured
 in dual-choice menu B along with 'Valor' barley. * B_Total:
 Refers to the total number of viruliferous *N. tenellus* that could be
 accounted for at the conclusion of the 24-h dual-choice test featuring
 Menu B. Leafhoppers that could not be accounted for were assumed to be
 dead. #### File: BLH_dual_choice_tests_analysis.Rmd **Description:**  R
 Markdown file providing step-by-step narrated statistical analysis of
 BLH_dual_choice_tests.csv. #### File: BLH_no_choice_tests.csv
 **Description:**  .csv file describing the various metrics (survival,
 dots, sheaths, eggs) present on a given plant sample following 24 h
 exposure to five viruliferous *N. tenellus* adults caged onto said plant.
 A total of six replications (across two runs) were carried out; each
 replication consisted of five viruliferous *N. tenellus* adults aspirated
 into a clip cage adhered to one of three plants. ##### Variables * Run:
 Refers to the first or second iteration of this experiment. Conditions
 were identical; sequential runs helped clarify the consistency of our data
 across time. * Rep: Refers to a unique 24-hr period in which five
 viruliferous *N. tenellus* were aspirated into a given clip cage. *
 Species: Either brown mustard ("Mustard") or barley. * Cultivar:
 Only one brown mustard cultivar, 'Caliente Rojo', was tested.
 Two different barley cultivars were tested: 'Stockford' and
 'Valor'. * Measurement: Qualifying term describing the metric of
 interest. Metrics of interest included survival, dots, sheaths, and eggs.
 * Value: Following the end of the 24-hr period: Survival = % of *N.
 tenellus* (out of five) found alive within a clip cage; Dots = % of total
 probing events present within clip-caged plant tissue comprised only of
 superficial punctures; Sheaths = % of total probing events present within
 clip-caged plant tissue marked with lipoprotenaceous sheath residue; Eggs
 = raw count of eggs present within clip-caged plant tissue. #### File:
 BLH_raw_no_choice_test_data.csv **Description:** .csv file containing raw,
 unaltered values of *N. tenellus* survival, dots, sheaths, and eggs per
 plant in the no-choice tests, as well as the values of survival, dots, and
 sheaths expressed as percentage of total survival or total feeding events,
 respectively. This file provides the raw data used to create the refined
 values in BLH_no_choice_tests.csv, which are the values used in
 statistical anlysis of the no-choice tests.  ##### Variables * Run: Refers
 to the first or second iteration of this experiment. Conditions were
 identical; sequential runs helped clarify the consistency of our data
 across time. * Rep: Refers to a unique 24-hr period in which five
 viruliferous *N. tenellus* were aspirated into a given clip cage. *
 Species: Either brown mustard ("Mustard") or barley. * Cultivar:
 Only one brown mustard cultivar, 'Caliente Rojo', was tested.
 Two different barley cultivars were tested: 'Stockford' and
 'Valor'. * BLH_entered: The number of viruliferous, adult *N.
 tenellus* aspirated into each clip cage at the beginning of the 24-hr
 period.  * BLH_dead: The number of viruliferous, adult *N. tenellus*
 aspirated found dead in each clip cage at the end of the 24-hr period. *
 BLH_survived: The number of viruliferous, adult *N. tenellus* aspirated
 found alive in each clip cage at the end of the 24-hr period. *
 BLH_retrieved: The sum of all viruliferous adult *N. tenellus* found
 within a clip cage, either alive or dead, at the end of a 24-hr period.  *
 Percent_survived: The amount of viruliferous adult *N. tenellus* found
 alive within a clip cage at the end of a 24-hr period, expressed as a
 percentage of total viruliferous adult* N. tenellus* retrieved from the
 clip cage at the end of a 24-hr period.  * Percent_accounted: The amount
 of viruliferous adult *N. tenellus* retrieved at the end of a 24-hr
 period, expressed as a percentage of total viruliferous adult *N.
 tenellus* initially aspirated into the clip cage at the beginning of a
 24-hr period.  * Dots: The raw, unaltered number of superficial puncture
 marks (visible under the microscope as small, pink "dots") found
 on the sample of plant tissue enclosed in a cage after a 24-hr period.  *
 Sheaths: The raw, unaltered number of prolonged feeding track marks
 (visible under the microscope as extended pink lines) found on the sample
 of plant tissue enclosed in a cage after a 24-hr period.  * Total_feeding:
 The total number of *N. tenellus* feeding events per plant tissue sample,
 given as the sum of Dots and Sheaths.  * Percent_dots: The proportion of
 dots relative to the total number of feeding events per plant tissue
 sample, expressed as a percentage.  * Percent_sheaths: The proportion of
 sheaths relative to the total number of feeding events per plant tissue
 sample, expressed as a percentage.  * Eggs: The number of eggs present per
 plant tissue sample.  #### File: BLH_no_choice_tests_analysis.Rmd
 **Description:**  R Markdown file providing step-by-step narrated
 statistical analysis of BLH_no_choice_tests.csv. #### File:
 BLH_within_vacuum_samples.csv **Description:**  .csv file containing the
 total number of leafhoppers per vacuum sample positively identified to be
 *N. tenellus.*  ##### Variables * Site: Either Leyendecker (32.20 N 106.74
 W) or Los Lunas(34.77N 106.76 W), NM. * Year: "2022" refers to
 the 2021-2022 season. "2023" refers to the 2022-2023 season. *
 Date: As listed. * Plot: Numbered sub-plot. Each treatment was featured in
 4 unique plots numbers within a site-year. * Rep: Refers to the vertical
 "lane" (1 of 4) featured in a given site-year. Each rep
 feautured one unique cover crop treatment. * Treatment: Refers to cover
 crop treatment. "Barley" = 'Stockford' barley
 monoculture; "Mustard" = 'Caliente Rojo' brown mustard
 monoculture; "Combo" = mix of 'Stockford' barley and
 'Caliente Rojo' brown mustard. * Method: Refers to method by
 which leafhoppers were sampled. Only vacuum surveys were used in this
 data. * Count: Refers to the number of *N. tenellus* that were counted per
 vacuum sample on a given date. All insects within a sample were were
 vacuumed, transferred to plastic bags, and frozen before being counted
 under a magnifying lens. Insects thought to be leafhoppers were preserved
 in ethanol after preliminary counting; positive identification to the
 species level was achieved in September 2025 via analyzing the preserved
 samples.  ## Code/software Software: R Core Team (2022), v 4.2.2. R: A
 Language and Environment for Statistical Computing.  R Foundation for
 Statistical Computing. Vienna, Austria.
 [https://www.R-project.org/](https://www.R-project.org/). Loaded packages
 necessary for running any script are included as Step 1B in each .rmd file
 and repeated below. BLH_vacuum_counts_analysis.Rmd: R Markdown file
 providing step-by-step narrated statistical analysis of
 BLH_within_vaccum_samples.csv. Packages required: * ggplot2 * kableExtra *
 plotrix * ggpubr * tidyr * rstatix * tidyverse * ggplot2 * Rmisc * foreign
 * ggplot2 * MASS * emmeans * agricolae
 BLH_dual_choice_tests_analysis.rmd: R Markdown file providing step-by-step
 narrated statistical analysis of BLH_dual_choice_tests.csv. Packages
 required:  * ggplot2 * binom * agricolae
 BLH_no_choice_tests_analysis.rmd: R Markdown file providing step-by-step
 narrated statistical analysis of BLH_no_choice_tests.csv. Packages
 required: * ggplot2 * FSA * dplyr ## Access information Other publicly
 accessible locations of the data: * N/A Data was derived from the
 following sources: * N/A