Abstract
Background and goals The grape mealybug, Pseudococcus maritimus, is found in most grapegrowing regions across the United States, however, its spatial and temporal distribution in mid-Missouri vineyards is unknown. Because the insect transmits grapevine leafroll-associated viruses (GLRaVs), which are common in mid-Missouri vineyards, the objectives of this study were to evaluate the presence of P. maritimus in mid-Missouri vineyards over the course of three growing seasons, confirm species identity through DNA barcoding, and genetically test female mealybugs for the presence of Grapevine leafroll-associated virus 3 (GLRaV-3).
Methods and key findings Sex pheromone traps captured varying levels of male grape mealybugs at all survey sites during two distinct periods of each growing season. Genetic testing verified that female mealybugs present on vines were P. maritimus and that 10 out of 54 female mealybugs collected from test vineyards possessed GLRaV-3.
Conclusions and significance These data confirm P. maritimus is widespread in mid-Missouri vineyards, that it produces two distinct reproductive cycles each season, and that it ingests GLRaV-3 from infected grapevines. Because the insect has previously been shown to be capable of transmitting GLRaVs to grapevines, it may be contributing to the high incidence rates of grapevine leafroll disease in the region.
Introduction
It has recently been shown that Missouri vineyards have a high prevalence of various disease-causing viruses that negatively affect grape and wine production (Schoelz et al. 2021). Of these viruses, the grapevine leafroll-associated viruses (GLRaVs) are of most concern because infection with one of these viruses causes grapevine leafroll disease (GLD), arguably the most destructive and economically devastating grapevine disease worldwide. GLD disrupts grapevine growth rates and yields and reduces sugar content in berries (Almeida et al. 2013, Naidu et al. 2014). There currently is no cure or treatment for GLD and the only way to prevent its spread is to remove virus-infected vines while simultaneously controlling the insects responsible for spreading the disease (Daane et al. 2012). It is well documented that GLRaVs can be transmitted to clean vines from infected vines by grafting (Fuchs et al. 2021) and by transmission via several species of mealybug and other soft-scale insects (Tsai et al. 2010, Bahder et al. 2013a, Kruger and Douglas-Smit 2013).
While some mealybug species are known to be present in Missouri, no formal studies have yet been designed to evaluate which species, if any, are present in Missouri vineyards. The grape mealybug (GMB), Pseudococcus maritimus (Ehrhorn), is the only native mealybug present in Missouri, however, several invasive species have also been reported as present, including the obscure mealybug, Pseudococcus viburni (Signoret), the long-tailed mealybug, Pseudococcus longispinus (Targioni Tozzetti), and the citrus mealybug, Planococcus citri (Risso) (Ben-Dov 1994, Gimpel and Miller 1996, CABI/EPPO 1999). Because all four of these species have been shown to be capable of transmitting GLRaVs between grapevines (Golino et al. 2002), it is possible that any or all of these species contribute to the high prevalence of GLD in Missouri vineyards. Mealybugs are notably inconspicuous, overwintering underneath grapevine bark until late spring, when they emerge for a few months to feed and reproduce. During the growing season, mealybugs primarily hide in sheltered areas, making visual detection difficult unless their populations are significantly high.
Historically, identification of mealybug species has required careful microscopic examination (Gimpel and Miller 1996). More modern techniques (i.e., DNA barcoding) analyze conserved regions of the insects’ DNA and obtain species identity through comparisons to genetic sequence databases (Abd-Rabou et al. 2012, Wang et al. 2016). Genetic sequencing has been performed for most pestiferous mealybugs, resulting in databases with DNA sequences of highly conserved mitochondrial genes that can be used for genetic comparisons to field-caught specimens (e.g., Barcode of Life Database, http://boldsystems.org). In addition, female mealybugs produce species-specific sex pheromones that are detected by males from long distances (Millar et al. 2005). Fortunately, the sex pheromone molecules of many mealybugs, including the GMB, have been identified and can be synthesized, allowing for species-specific field-based monitoring using sex pheromone traps (Millar et al. 2002, Bahder et al. 2013b).
The objectives of this study were to evaluate the presence of P. maritimus in mid-Missouri vineyards over the course of three growing seasons using sex pheromone trapping, confirm species identity through DNA barcoding, and genetically test female mealybugs for the presence of GLRaV-3.
Materials and Methods
Survey sites
The same survey sites were sampled each year from 2021 to 2023 and consisted of five vineyards located in Boone County, Missouri and four vineyards in Gasconade County, Missouri. All study sites are within United States Department of Agriculture Plant Hardiness Zone 6b. Vineyard characteristics varied widely in age, acreage, and grape varieties (Table 1). Names and exact locations of vineyards included in the survey have been withheld, at the request of the landowners.
Characteristics of mid-Missouri vineyards surveyed for male grape mealybugs over three growing seasons (2021, 2022, and 2023), using sex pheromone-baited traps.
Pheromone-based trap monitoring
Large delta traps and GMB pheromone (trans-α-necrodyl isobutyrate; Figadere et al. 2007) lures were obtained (Suterra) and stored at −20°C until deployment. Beginning in mid-April of each year, traps were deployed at a density of one trap per 12.1 ha, following Bahder et al. (2013b). Sticky cards were changed every 2 wk and septa lures were replaced every 4 wk through mid-September. Sticky cards were examined under a stereomicroscope to confirm the presence of male mealybugs.
Genetic testing of mealybugs
Two adult female mealybugs were collected each year from each site, resulting in a total of 54 samples. Samples were placed into 1.5-mL Eppendorf tubes, frozen on dry ice in the field, and kept frozen at −80°C until genetic testing. Total RNA was purified using a GeneJet RNA purification kit (ThermoFisher) and 1 μg of total RNA was used to make synthetic complementary DNA (cDNA) using the iScript cDNA Synthesis Kit (Bio-Rad).
The purified cDNA samples were then used as templates for two separate PCRs: MBCOX1F (5′-CAACATTTATTTTGATTTTTTGGACATCC-3′) and MBCOX1R (5′-GTATACCATTTAATCCTAAAAAATGTTGAGG-3′) were used to amplify a 616 base pair (bp) region of the mealybug cytochrome c oxidase subunit 1 (COX1) gene, and LR3-FPST-F1 and LR3-FPST-R1 were used to amplify a conserved 122 bp region of the 3′ UTR of the GLRaV-3 genome (Maree et al. 2013, Diaz-Lara et al. 2018). A gene fragment containing the 122 bp GLRaV-3 target amplicon was synthesized (Eurofins Genomics) and used as a template for a positive control PCR. The cDNA preparations were diluted 1:100 in water and 1 μL was used as the template in a 20-μL reaction containing dNTPs, 250 nM of forward and reverse primers, and one unit of Phusion DNA polymerase (ThermoFisher). Cycling conditions were as follows: an initial 2 min at 98°C followed by 35 cycles of 98°C for 14 sec, 56°C for 14 sec, and 72°C for 60 sec. PCR products were resolved on a 0.7% TAE gel and bands of the expected size were extracted and purified using a GeneJet DNA purification kit (ThermoFisher).
Purified PCR products were then ligated into the plasmid pJET1.2 (ThermoFisher) and transformed into OneShot chemically competent bacteria (ThermoFisher). Transformed bacteria were plated onto LB plates and incubated overnight at 37°C. Bacterial colonies were then tested for successful transformation by PCR using the same cycling conditions and reagents as described above. Positive colonies were scaled up in liquid culture and incubated overnight at 37°C and plasmids were purified from liquid cultures the next day using a GeneJet plasmid purification kit (ThermoFisher). One purified plasmid containing the target amplicons produced from each sample was then sent to Eurofins Genomics (Memphis, TN) for Sanger sequencing.
Analysis of COX1 and GLRaV-3 PCR products
The DNA sequences of the COX1 gene for the GMB (Genbank: KJ187490.1), the long-tailed mealybug (Genbank: AB512118.1), the citrus mealybug (Genbank: LC121493.1), and the obscure mealybug (Genbank: JF905460.1), as well as the complete genome of GLRaV-3 (Genbank: EU344893), were obtained from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) to use as reference sequences for DNA comparisons. The sequenced PCR products generated in this study were aligned to either the COX1 sequence data set or to the GLRaV-3 genome using the MUSCLE alignment function within Geneious Prime software (Biomatters).
Results
Pheromone-based trap monitoring
Male GMBs were found in sex pheromone-baited traps at all study sites in each of the 2021, 2022, and 2023 growing seasons. Across all study sites and years, seasonal trap totals ranged from 4 to 423 male mealybugs (Figure 1). Two distinct flight periods were observed each season, with the first generally peaking from late May to early June and the second in mid to late August (Figure 2).
Total of male grape mealybugs (GMBs) caught over three consecutive years in mid-Missouri vineyards, using sex pheromone-baited traps. Each year, sex pheromone traps were placed in vineyards on 1 April and removed on 30 Sept. Sticky cards were replaced in traps every 2 wk and pheromone lures were replaced every 4 wk.
Biweekly counts of male grape mealybugs (GMBs) caught in sex pheromone-baited delta traps at study sites in (A) Boone County, Missouri and (B) Gasconade County, Missouri in 2021, 2022, and 2023. Each year, sex pheromone traps were placed in vineyards on 1 April and removed on 30 Sept. Sticky cards were replaced in traps every 2 wk and pheromone lures were replaced every 4 wk. Calendar days were converted to growing degree days (GDDs) using a base of 0°C from 1 Jan, based on average daily temperatures for the regions (www.agebb.missouri.edu).
Genetic analyses
The COX1 amplicon was successfully amplified from all 54 samples collected during this study. Sequence alignments of the amplified COX1 PCR products revealed 99.2% identity, indicating the 54 samples were the same mealybug species. Alignment of the samples’ consensus COX1 sequence with the COX1 sequences of Pl. citri, P. longispinus, P. viburni, and P. maritimus revealed 88.2, 86.9, 91.1, and 100% identities, respectively. These results indicate that all field-collected samples were the GMB, P. maritimus.
The target GLRaV-3 amplicon was successfully amplified from 10 of the 54 cDNA samples made from field-collected adult female mealybugs. Sequence alignment of the 10 GLRaV-3 PCR products with the GLRaV-3 genome revealed that all 10 samples shared 100% identity with each other and with the highly conserved region of the GLRaV-3 genome.
Discussion
Mealybugs use their sense of smell to differentiate host plants from non-host plants and potential mates from non-mates (Corcoran and Mahaffee 2024). For example, grapevines release specific combinations of chemicals into the environment that mealybugs detect and use to differentiate grapevines from other plants. Similarly, female mealybugs release species-specific chemicals into the environment to attract males for mating purposes. The fact that mealybugs use sex pheromones to communicate offers a relatively quick and easy way to subjectively determine which species are present in a vineyard, as well as the ability to improve existing mealybug control programs. Indeed, the sex pheromone traps deployed in this study revealed two distinct periods during which adult male and female GMB emerge from cover to reproduce (Figure 2). Timing the application of biological control agents or contact insecticides around reproductive flight periods should improve the efficiency and efficacy of these tactics. While existing mealybug control programs were not taken into consideration in this study, this concept of timing insecticide deployment with reproductive periods was discussed with vineyard managers throughout this study, which may have contributed to the decreases in trap counts seen at certain vineyards across years (Figure 1).
While the trapping data confirmed that GMB were present in mid-Missouri vineyards, it provided no information regarding the potential presence of other mealybug species. As such, the so-called DNA barcoding technique was deployed to objectively determine the species of mealybugs present in mid-Missouri vineyards. This technique worked well to amplify a large section of the highly conserved mitochondrial gene, COX1 (Supplemental Figure 1). Perhaps not surprisingly, the DNA barcoding technique revealed that all the mealybugs collected from the vineyards were in fact GMB. However, it was surprising to learn how widespread GMB is in mid-Missouri; GMB was found in every vineyard surveyed, regardless of size, age, or variety grown. In Missouri, there are several species of wild grapes (e.g., Riverbank grape, Vitis riparia; Summer grape, Vitis aestivalis) that can also serve as host plants for GMB. These widespread, alternative host plants may be supporting large populations of GMB in the region, which may explain why GMB was found in every vineyard surveyed.
Because of the severe impact of GLD on the U.S. grape industry (Atallah et al. 2012, Ricketts et al. 2015), much has been learned about GMBs in the context of transmitting GLRaVs; for example, it is well documented that GMBs can ingest various GLRaVs from infected grapevines, harbor them for several days (i.e., semi-persistently), then transmit them into uninfected grapevines (Cid et al. 2007, Douglas and Kruger 2008, Tsai et al. 2010, Kruger et al. 2015). In a 2021 survey of mid-Missouri vineyards, it was reported that 53% of sampled grapevines tested positive for GLRaV-3 (Schoelz et al. 2021). Because the present study has found GMB to be present in all study sites surveyed, with a significant proportion (20%) of collected samples harboring GLRaV-3, it is possible that new plantings are at risk for rapid infection in mid-Missouri vineyards.
Conclusion
In this study, it was determined that P. maritimus is widespread in mid-Missouri vineyards and its capacity to acquire and harbor GLRaV-3 may contribute to the region’s high incidence of GLD. The detection of GLRaV-3 in field-collected mealybugs suggests that the species could play a role in virus movement within Missouri vineyards. It was determined that, as in most locations, GMB has two generations per growing season that typically occur in late spring and late summer, which represent optimal periods for the deployment of contact insecticides to control the GMB. With this confirmation of mealybug species in mid-Missouri vineyards, targeted and environmentally conscious tactics (e.g., species-specific predators or mating disruption strategies) can be incorporated into existing management programs.
CRediT Authorship Contributions
JC: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Software, Supervision, Writing – Original Draft, Writing – Review & Editing.
Supplemental Data
The following supplemental materials are available for this article in the Supplemental tab above:
Supplemental Figure 1 Nucleic acid sequence of the 616 base pair region of the COX1 gene amplified from the grape mealybug, Pseudococcus maritimus.
Data Availability
All data underlying this study are included in the article and its supplemental information.
Footnotes
This work was supported by the U.S. Department of Agriculture, Agricultural Research Service under CRIS project: 5070-22000-038-00D. Special thanks to Dean Volenberg and Nick Pehle for facilitating access to vineyards included in this study. The findings and conclusions in this publication are those of the author and should not be construed to represent any official USDA or U.S. Government determination or policy. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.
Corcoran JA. 2026. The grape mealybug, Pseudococcus maritimus, is widespread in mid-Missouri vineyards. Am J Enol Vitic 77:0770010. DOI: 10.5344/ajev.2026.25047
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- Received September 2025.
- Accepted February 2026.
- Published online May 2026
This is an open access article distributed under the CC BY 4.0 license.
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