Dataset DOI: 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/.

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