Abstract
Background and goals In Baja California, Mexico, the occurrence of grapevine leafroll (GLD) and grapevine red blotch (GRBD) diseases and the presence of the vine mealybug (vector of some grapevine leafroll-associated viruses) have been recognized. In this work, an epidemiological study was conducted to determine the prevalence and incidence in Baja California, Mexico, of symptomatic plants associated with both diseases.
Methods and key findings Randomly-selected vineyards were surveyed to determine prevalence of GLD and GRBD, based on symptom evaluation in quadrats of 1000 grapevines. Results showed that 92% of sites had symptomatic plants, with an average prevalence of 20% (minimum = 0%, maximum = 92%). The main variables positively associated with disease prevalence were percentage of vine mealybug-infested plants per site, soil temperature, and accumulated heat units; ambient temperature was negatively associated with disease prevalence. Disease incidence determined during three growing seasons (2021 to 2023) in quadrats of 2500 grapevines in five vineyards revealed an average increase of 1.7% and 9% in 2022 and 2023, respectively. One site with the absence of vine mealybug showed the lowest percentage of symptomatic plants. Cabernet Sauvignon and Nebbiolo showed more symptomatic plants than Merlot. Virus infection in 137 symptomatic and 53 asymptomatic grapevines was tested by real-time PCR for grapevine leafroll-associated virus 1, -2, -3, and -4, and grapevine red blotch virus (GRBV). The diagnosis based on symptoms had a sensitivity of 90% and a specificity of 52%, with respect to real-time PCR results.
Conclusions and significance The presence and widespread distribution of GLD and GRBD underscore the need to implement regional management programs in Baja California vineyards.
- grapevine leafroll-associated viruses
- grapevine leafroll disease
- grapevine red blotch disease
- grapevine red blotch virus
- vine mealybug
- visual diagnosis
Introduction
Grapevine (Vitis spp.) hosts ~101 viruses and virus-like organisms, the highest among agricultural crops (Fuchs 2023). Grapevine leafroll disease (GLD), associated with grapevine leafroll-associated viruses (GLRaVs) from the Closteroviridae family, is among the most widespread viral diseases affecting grapevines. Currently, five GLRaVs have been identified in leafroll-affected grapevines, designated as GLRaV-1, -2, -3, -4, and -13 (Martelli et al. 2012, Ito and Nakaune 2016). Notably, GLRaV-3 (species Ampelovirus trivitis, as found on the website https://ictv.global/taxonomy/find_the_species) is the most widespread, causing significant economic losses (Naidu et al. 2008, Ricketts et al. 2015). In recent years, grapevine red blotch virus (GRBV; species Grablovirus vitis, as found on the website https://ictv.global/taxonomy/find_the_species), the causal agent of grapevine red blotch disease (GRBD), has gained prominence because of its widespread occurrence in North America (Al Rwahnih et al. 2013, Gasperin-Bulbarela et al. 2019). GRBV has also been reported in various countries, including Switzerland, South Korea, India, Italy, France, Argentina, and Australia, but its prevalence and incidence remain largely unknown (Lim et al. 2016, Poojari et al. 2017, Reynard et al. 2018, Luna et al. 2019, Bertazzon et al. 2021, KC et al. 2022, and as found on the EPPO website: https://gd.eppo.int/reporting/article-7450).
Infection by GLRaVs leads to yield loss and negatively affects total soluble solids, juice pH, and berry acidity (Endeshaw et al. 2014, Alabi et al. 2016). Yield reductions attributed to GLD vary, but globally, they are estimated at ~40% or more, depending on disease severity (Whitham et al. 2006, Almeida et al. 2013, Ricketts et al. 2015, Alabi et al. 2016). The effect of GLRaVs on grapevines also depends on cultivar, location, plant age, soil, environmental conditions, and virus genotype (Maree et al. 2013, Naidu et al. 2014, Jones et al. 2015, Maliogka et al. 2015, Jones and Nita 2016, Mannini and Digiaro 2017, Naidu 2017, Chooi et al. 2022). GRBD affects fruit juice chemistry, reduces total soluble solids, and lowers anthocyanin content in grape skin (Bowen et al. 2020), leading to estimated economic losses ranging from $2213 to $68,548/ha over a 25-yr productive life of a vineyard (Ricketts et al. 2017).
Symptoms of GLD differ between black fruited and white fruited cultivars. Black fruited cultivars show reddish or purple discoloration in interveinal leaf areas, while white fruited cultivars exhibit yellowish mottling, which may go unrecognized in many varieties (Naidu et al. 2008). Both types of cultivars demonstrate leaf margin rolling. Although GLD and GRBD share symptoms of red pigmentation in leaf areas, they can be differentiated by red pigmentation of the veins on the undersides of GRBD-infected leaves, which is not present in GLD (Sudarshana et al. 2015).
Primary spread of GLRaVs and GRBV occurs through vegetative propagation (Naidu et al. 2014, Cieniewicz et al. 2018), with secondary spread facilitated by insect vectors. Certain mealybug species have been identified as transmitters of GLRaV-1 and -3, with the vine mealybug (Planococcus ficus Signoret) being notably efficient (Tsai et al. 2010); no insect vector has been reported for GLRaV-2. GRBV has shown secondary spread through the three-cornered alfalfa hopper (Spissistilus festinus Say) (Flasco et al. 2021, 2023).
Common diagnostic methods include enzyme-linked immunosorbent assays (ELISA) for GLRaVs and PCR-based assays for GLRaVs and GRBV (Krenz et al. 2014, Naidu et al. 2014). Recent advancements incorporate isothermal amplification techniques (e.g., LAMP and RPA) and machine-learning analysis of hyperspectral images for virus detection (Wang et al. 2023, Kishan et al. 2024). Despite these advancements, visual diagnosis remains the primary method when molecular analysis is impractical, making symptom monitoring vital to understand the epidemiology of GLD and GRBD (Bell et al. 2017, Rohrs et al. 2023). However, distinguishing between these diseases based solely on symptoms is challenging because of the notable similarity in their symptoms, especially in co-infected plants (Sudarshana et al. 2015, Adiputra et al. 2018, Krenz et al. 2023).
In Mexico, grapevine cultivation spans 36,405 ha, producing 477,305 tons of grapes in 2022, with Baja California as a leading winegrape producer, accounting for 85% of national production (CESVBC 2019). The main cultivars include Cabernet Sauvignon, Merlot, Chardonnay, Chenin blanc, and Sauvignon blanc (as found in the Consejo Mexicano Vitivinícola report https://uvayvino.org.mx/html/docs/produccion_consumo_vino.pdf). Reports of viral diseases, including GLRaV-1, -2, -3, and -4 and GRBV, have emerged in Baja California and Aguascalientes (Garcia-Resendiz and Carrillo-Tripp 2022, Diaz-Lara et al. 2023). However, these reports lack details regarding distribution, incidence, and vector associations.
Research on grapevine virus diseases, including GLD and GRBD, is critical in Mexico because of their potential effect on grape production. Understanding the occurrence and manifestation of these diseases will provide valuable insight into their distribution and annual variations, particularly with GLD linked to the presence of the vine mealybug. This study aimed to evaluate the prevalence of these two significant grapevine viral diseases in Baja California, assess the incidence of symptomatic plants in commercial vineyards across primary production areas, and identify factors contributing to the spread of GLD and GRBD. Such research is essential not only to develop effective management strategies to mitigate the economic losses associated with these diseases, but also to ensure the sustainability of grapevine cultivation in Mexico.
Materials and Methods
Vineyard study sites
The study was conducted in the state of Baja California, Mexico; average annual temperature is 18 to 19°C and average annual precipitation is 200 mm (INEGI 2016). To study the prevalence of GLD and/or GRBD, 121 vineyard sites within the main viticulture areas of Baja California (p = 50%, confidence interval = 10%, and confidence level = 95%, according to the 175 growers recorded in the Baja California state registry) were randomly selected from 13 localities (Figure 1): Ejido de Piedras Gordas, Ejido Real del Castillo, Ejido Nacionalista Sánchez Taboada, Ejido Uruapan, Valle de Ojos Negros, Valle de San Vicente, Valle de Santo Tomás, Tecate, Valle de las Palmas, Ejido El Porvenir, Ejido El Sauzal, Francisco Zarco, and San Antonio de las Minas. A total of 119 vineyard sites had traditional insecticide management, and two had organic management. Cultivars grown across the study sites were Cabernet Sauvignon (27 sites), Tempranillo (18), Merlot (15), Nebbiolo (13), and Grenache (6); unknown cultivars were grown on six sites, and 36 sites were classified as “other” (cultivars with less than three sites).
Map of vineyard sites in Baja California, Mexico used in this study. Total area of grapevine production (in hectares) is shown in parentheses for each site. Sites marked with a red star indicate presence of the vine mealybug.
The incidence of GLD and/or GRBD was monitored in five vineyards (also included in the prevalence study) that used traditional insecticide management and were located across three localities of Baja California: Francisco Zarco (three vineyards), Valle de Ojos Negros (one vineyard), and Valle de San Vicente (one vineyard) (Figure 1 and Supplemental Table 1). These three localities represent 55% of the total grapevine production units (vineyards) in Baja California. The presence of vine mealybug was confirmed only in the Francisco Zarco and Valle de San Vicente vineyards. These vineyards were selected based on access to the site (with permission from growers for at least 3 yrs), vineyard age (less than 10-yrs-old), status of vine mealybug presence, and if black fruited cultivars were grown (cv. Cabernet Sauvignon in sites 1 and 2; cv. Italian Nebbiolo in site 3; cv. Nebbiolo in site 4; and cv. Nebbiolo, Merlot, and Cabernet Sauvignon in site 5).
Symptom evaluation
Symptom monitoring was conducted in October, at an optimal time of symptom expression in Baja California (once for each site of the prevalence study, and annually for three years for the incidence study). Prevalence and incidence of GLD and/or GRBD were assessed by classifying grapevines by the presence of symptoms of these diseases, with 0 for asymptomatic and 1 for symptomatic plants (Figure 2). The prevalence study included red, black, and white fruited cultivars; the incidence study included black fruited cultivars. Because of the similarity between symptoms and season of appearance of both diseases, plants were classified as symptomatic for GLD and/or GRBD without distinguishing between the two diseases.
Reference images of grapevine leaves testing either negative (A to D) or positive (E to L) for any combination of grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4) and grapevine red blotch virus (GRBV). A to C) cv. Nebbiolo, D) cv. Cabernet Sauvignon. E) Margin rolling, light green veins, and red discoloration in the blade (cv. Cabernet Sauvignon, GLRaV-1, -2, and -3 positive). F) Light green veins and red discoloration in the blade (cv. Cabernet Sauvignon, GLRaV-2 and -3 positive). G) Clustered red spots in the blade (cv. Cabernet Sauvignon, GLRaV-2 and -3 positive). H) Purple veins, red spots in the blade, and irregular margins (cv. Nebbiolo, GLRaV-2 and GRBV positive). I) Pixel-like red spots in the blade (cv. Nebbiolo, GLRaV-3 and GRBV positive). J) Dispersed red spots in the blade (cv. Nebbiolo, GLRaV-2 and -3 positive). K) Slight margin rolling, light green veins, and red discoloration of the blade (cv. Cabernet Sauvignon, GLRaV-2 and -3 positive). L) Light green veins and chlorotic spots in the blade (cv. Chenin blanc, GLRaV-3 positive). GLD, grapevine leafroll disease; GRBD, grapevine red blotch disease.
Evaluation of GLD and/or GRBD prevalence
In this analytical cross-sectional epidemiological study, the units of study were grapevine plants. Quadrats consisting of 10 contiguous vineyard rows with 100 contiguous plant positions (with or without grapevine) within each row (totaling 1000 plants per quadrat) were evaluated at each site in vineyards owned by different growers. Prevalence was defined by the percentage of symptomatic plants for GLD and/or GRBD at each site, relative to the total number of positions with plants in the evaluated quadrat, discarding dead plants or positions without plants. Disease prevalence was assessed in 2022 at 80 sites in five localities (Valle de las Palmas, Ejido El Porvenir, Ejido El Sauzal, Francisco Zarco, and San Antonio de las Minas) and in 2023 at 41 sites in eight localities (Ejido de Piedras Gordas, Ejido Real del Castillo, Ejido Nacionalista Sánchez Taboada, Ejido Uruapan, Valle de Ojos Negros, Valle de San Vicente, Valle de Santo Tomás, and Tecate).
GLD and/or GRBD incidence monitoring
An analytical longitudinal study was conducted to monitor symptoms of GLD and/or GRBD in five vineyards. The incidence of symptomatic plants in quadrats of 25 contiguous vineyard rows, with 100 contiguous plant positions in each row (2500 plant positions total), were annually quantified over a 3-yr study period (2021 to 2023). The total number of symptomatic plants was calculated as the sum of symptomatic plants from the previous year, plus new cases, minus lost cases (dead/missing plants).
Vine mealybug surveys
Following a previous methodology (Fú-Castillo et al. 2002), trained technicians at the Comité Estatal de Sanidad Vegetal de Baja California (CESVBC) determined the percentage of plants infested by the vine mealybug in all prevalence and incidence sites (the quadrats for monitoring vine mealybug did not necessarily correspond to those used for symptom monitoring in each vineyard). Within ~10 ha quadrats per vineyard, three rows were assessed, with 25 plants randomly selected per row (75 plants per quadrat). To detect mealybug presence, the bark was stripped at three to five points from the base of the trunk to the cordons in each plant (taking ~2 min per plant), and the number of infested plants was recorded. Mealybug surveys were conducted quarterly in sites with vine mealybug presence, and annually in sites without mealybug presence. The vector of GRBV was not considered because the presence of the three-cornered alfalfa hopper had not been confirmed in Baja California at the time of our study (J. Carrillo and D. Schneider, personal communication).
Weather data
Daily meteorological data from January to October from the evaluated year (2022 or 2023, depending on the specific site) were obtained from a state network of weather stations, Sistema de Información para el Manejo del Agua de Riego en el Estado de Baja California (SIMARBC), and assigned to each prevalence study site based on proximity to the nearest active weather station. The data included total precipitation (mm), solar radiation (Cal/cm2), average relative humidity (%), average ambient temperature (°C), average soil temperature (°C), and accumulated daily heat units.
Leaf tissue sampling and virus detection
Trained personnel sampled leaf tissue for virus detection (10 leaves per plant per sample). Two leaves were selected from each plant section (top, bottom, right, left, and center), aiming to collect both young and mature leaves with varied symptoms. A total of 190 samples (137 symptomatic plants and 53 asymptomatic plants) were taken in October 2022 and 2023. Samples were transported to the Agricultural Virology Laboratory at the Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE) in coolers with frozen gels and either processed immediately or stored at −20°C until use. A database was generated with information from each sample, and an identification code was assigned according to the site and collection year.
Total nucleic acids were extracted using a cetyltrimethylammonium bromide method modified from Gambino et al. (2008) and Lodhi et al. (1994); two extra chloroform extraction steps were performed, and nucleases were avoided. Targeted virus gene regions were copied through reverse transcription carried out at 25°C for 10 min, followed by 42°C for 60 min, and finally, at 70°C for 10 min. Amplification was then performed by uniplex real-time PCR using the specific primers listed in Supplemental Table 2 (Osman et al. 2007, Krenz et al. 2014, Sandez Salas 2021), and EvaGreen (Biotium) as a fluorescent dye. The PCR cycling conditions for all targets were: initial denaturation at 95°C for 3 min; followed by 40 cycles at 94°C for 15 sec, 61°C for 30 sec, 72°C for 15 sec; and lastly, the melting curve at 65°C for 5 sec and 95°C for 5 sec.
Data analyses
Prevalence
The dependent variable was prevalence (%) and the explanatory or independent variables were locality (territorial division), plant cultivar, and percentage of plants infested by vine mealybugs. An analysis of variance (ANOVA) and Tukey’s multiple comparisons of means at a 95% family-wise confidence level in categorical variables were performed for the variables of locality, cultivar, and percentage of plants infested by vine mealybug (Supplemental Table 3). To analyze the relationship between the meteorological variables (total precipitation, solar radiation, average relative humidity, average ambient temperature, average soil temperature, and accumulated daily heat units) and the prevalence of GLD and/or GRBD, a multiple regression analysis was performed (Supplemental Table 4 and Supplemental Figure 1).
Incidence
A multivariate ANOVA (with site and year as explanatory variables) and a linear regression analysis (incidence as dependent variable and vine mealybug as independent variable) were performed to determine the association between incidence and percentage of plants infested by the vine mealybug (Supplemental Table 5). A general linear model (GLM) was obtained that explained the binary dependent variable (presence or absence of GLD and/or GRBD symptoms), with independent variables being year (2021, 2022, and 2023), cultivar (Cabernet Sauvignon, Nebbiolo, and Merlot), and site (sites 1, 2, 3, 4, and 5) (Supplemental Table 6).
Diagnostics
To corroborate the visual diagnosis of GLD and/or GRBD (symptomatic or asymptomatic), symptom classification was compared with results of molecular diagnosis using real-time PCR targeting specific viruses (GLRaV-1, -2, -3, -4, and GRBV) (Osman et al. 2007, Krenz et al. 2014, Sandez Salas 2021). Several statistical measures were calculated between symptom diagnosis and molecular diagnosis. These included sensitivity (number of symptomatic plants positive for at least one virus by real-time PCR, divided by the total number of plants positive for at least one virus by real-time PCR), specificity (number of asymptomatic plants negative by real-time PCR, divided by the total number of plants negative by real-time PCR), positive predictive value (PPV; number of symptomatic plants positive for at least one virus by real-time PCR, divided by the total number of symptomatic plants), and negative predictive value (NPV; number of asymptomatic plants negative by real-time PCR, divided by the total number of asymptomatic plants). Pearson’s chi-square test with Yates continuity correction was additionally performed to assess the statistical significance of the association between symptom diagnosis and molecular diagnosis results (Supplemental Table 7). All tests were performed using R Studio ver. 1.4.1106 (RStudio Team 2021). Maps were obtained in QGIS ver. 3.34.0 (QGIS 2023).
Results
GLD and/or GRBD prevalence in Baja California
A total of 121 vineyard sites across 13 localities of Baja California were visited during 2022 (80 sites) and 2023 (41 sites) (Figure 1 and Table 1). The average rate of symptomatic plants per site was 20%, with a range of 0 to 92%. Symptomatic plants were identified at 92% of the visited sites. Localities with the sites with highest prevalence included Ejido El Porvenir (96.2%), Francisco Zarco (91.8%), and San Antonio de las Minas (79%). Among the sites surveyed, four had 75 to 100% symptomatic plants, eight had 50 to 75%, 15 had 25 to 59%, and the remainder of the sites had between 0 and 25% (Figure 3).
Localities, cultivars, prevalence of symptomatic grapevines, and presence of vine mealybugs.
Map of the prevalence of symptomatic plants for grapevine leafroll disease (GLD) and/or grapevine red blotch disease (GRBD) in Baja California, Mexico. Each point on the map represents one of the 121 surveyed vineyard sites, where a quadrat of 1000 plant positions was evaluated in 2022 or 2023; the color and size indicate the percentage of symptomatic plants within these quadrats.
The localities of Francisco Zarco and San Antonio de las Minas, with notably higher average prevalence, were significantly different from Valle de Ojos Negros, which exhibited lower prevalence (p < 0.01) (Figure 4A). No difference in prevalence was found according to cultivar (p > 0.05) (Figure 4B). However, 61% of the vineyard sites were infested by vine mealybugs, and a significant correlation was found between the percentage of vine mealybug-infested plants and prevalence (p < 0.01) (Figure 4C). The percentage of vine mealybug-infested plants per vineyard ranged from 0 to 28.5%, with an average of 4.1%. The most affected localities were Francisco Zarco and Ejido El Porvenir (with averages of 12 and 4.4% infestation per vineyard, respectively). In contrast, vine mealybug presence was not detected in any vineyard evaluated in Valle de Ojos Negros.
Prevalence of symptomatic plants with grapevine leafroll disease (GLD) and/or grapevine red blotch disease (GRBD) in Baja California, Mexico, by locality, cultivar, and presence of vine mealybugs. Similar letters indicate no statistically significant differences between variables, in terms of mean prevalence. A) Average prevalence for each locality (EP, Ejido El Porvenir; FZ, Francisco Zarco; ON, Valle de Ojos Negros; PG, Ejido Piedras Gordas; SAM, San Antonio de las Minas; ST, Valle de Santo Tomás; SV, Valle de San Vicente; T, Tecate; Other, localities with three or less sites (Ejido Real del Castillo, Ejido Nacionalista Sánchez Taboada, Ejido Uruapan, Valle de las Palmas, and Ejido El Sauzal). B) Average prevalence by plant cultivar (CS, cv. Cabernet Sauvignon; G, cv. Grenache; M, cv. Merlot; N, cv. Nebbiolo; T, cv. Tempranillo; U, Unknown; O, Other (cultivars present in three or less sites). C) Prevalence correlated with the percentage of grapevine plants infested with vine mealybugs at each site. For locality, cultivar, and vine mealybug infestation, analysis of variance (Tukey’s multiple comparisons of means, 95% family-wise confidence level in categorical variables) was used.
The meteorological variables that correlate with the presence of symptomatic plants for GLD and/or GRBD were average ambient temperature, average soil temperature, and accumulated heat units (Table 2). Soil temperature and accumulated heat units positively affected prevalence, with an increase in these variables leading to an increase in disease prevalence. Temperature had a negative association with disease prevalence. Variables such as precipitation, solar radiation, and relative humidity were not statistically significant in explaining disease prevalence.
Multiple regression of meteorological variables for prevalence of grapevine leafroll disease and/or grapevine red blotch disease symptoms.
Three-year incidence monitoring
In the five sites monitored (Figure 1), the average increase in symptomatic plants per site was 1.7% in 2022 and 9% in 2023. All sites experienced a greater number of new cases from 2022 to 2023, compared to the increase observed from 2021 to 2022. Site 3 exhibited the highest number of new cases between 2021 and 2023, totaling 594 (Figure 5C), followed by site 2 with 271 (Figure 5B), site 1 with 187 (Figure 5A), site 4 with 86 (Figure 5D), and site 5 with 57 (Figure 5E). The increase in new cases per quadrat ranged from 2 (site 5) to 24% (site 3).
Incidence of symptomatic plants with grapevine leafroll disease and/or grapevine red blotch disease in five vineyards over three years. Each panel shows spatial distribution of symptomatic plants in each site over time. The quadrats consisted of 2500 plants arranged in 25 rows of 100 plant positions each (~1 ha). Yellow squares represent plants that exhibited symptoms in 2021, red squares indicate new plants with symptoms in 2022, green squares denote new plants with symptoms in 2023, and white squares indicate asymptomatic plants or positions without plants.
The GLM for incidence data indicated statistically significant variation in symptomatic plants among sites, years, and cultivars (Table 3). A positive relationship was found between symptomatic plants and years 2022 and 2023, as compared to 2021; i.e., the number of symptomatic plants was higher in the last two years. Regarding cultivars, a positive relationship with symptomatic grapevine plant presence was found for Nebbiolo (more likely to find symptomatic plants), while a negative relationship was found for Merlot (less likely to find symptomatic plants) when compared with Cabernet Sauvignon. In sites 2, 3, 4, and 5, it was less likely to find symptomatic plants than in site 1, with sites 4 and 5 the least likely to have symptomatic plants. The presence of mealybug was not included in the GLM. The incidence of symptomatic plants to GLD and/or GRBD was analyzed in relation to the percentage of vine mealybug-infested plants of the five vineyards over three years (Figure 6). The analysis of the combination of disease incidence and percentage of vine mealybug-infested plants found that the site had a significant effect on both (p < 0.001); however, the year did not (p = 0.2471). In addition, the percentage of plants infested by the vine mealybug in 2021 had a significant effect on the percentage of diseased vines (p = 0.0127) in 2023. Logistic regression showed that in the five sites analyzed, for every unit increase in the percentage of vine mealybug infestation, the incidence of symptomatic plants to GLD and/or GRBD was expected to increase by 2.5757 units (percentage of symptomatic plants per year).
Relationships between various factors and the presence of symptomatic plants to grapevine leafroll disease and/or grapevine red blotch disease at five sites.
Incidence over three years of symptomatic plants to grapevine leafroll disease and/or grapevine red blotch disease, versus plants infested by the vine mealybug (Planococcus ficus Signoret), of five vineyards.
Evaluation of visual diagnosis by real-time PCR
Visual diagnosis was compared with real-time PCR virus detection using 190 samples, including 137 symptomatic and 53 asymptomatic samples. A total of 108 samples (98 symptomatic and 10 asymptomatic) were positive for at least one virus. GLRaV-3 was the most prevalent, followed by GRBV and GLRaV-2. GLRaV-1 was detected in one plant, and GLRaV-4 was not detected in any sample (Figure 7A). In 63 samples, only one virus was detected, but mixed infections were found in 45 of the samples (Figure 7B). In contrast, symptoms of GLD and/or GRBD were seen in 95% of the samples positive for GLRaV-3, 90% of the samples positive for GRBV, and 97% of the samples positive for GLRaV-2 (Supplemental Table 8).
Molecular diagnostic results for grapevine leafroll-associated viruses (GLRaV) and grapevine red blotch virus (GRBV) using real-time PCR. A total of 190 samples were analyzed. A) Distribution of samples positive for each tested virus, and B) a Venn diagram illustrating the distribution of samples that were positive for one or more viruses.
Of the 190 tested samples, 57% tested positive for at least one virus. The viruses detected in symptomatic plants were GLRaV-1, -2, -3, and GRBV. Asymptomatic samples that tested positive for viruses were associated with GLRaV-2, -3, or GRBV. The frequency distribution comparing visual diagnosis to molecular virus detection revealed differences, indicating a dependency between variables (Table 4). Although false positives (symptomatic plants not positive for any of the five viruses tested) and false negatives (asymptomatic plants infected with viruses, impossible to visually detect) were found, visual diagnosis demonstrated a sensitivity of 91%, a specificity of 52%, a PPV of 72%, and an NPV of 81%. These metrics indicate that visual diagnosis is effective at identifying symptomatic plants that test positive for at least one virus.
Frequency distribution of visual diagnosis compared to molecular detection of one or more viruses.a
Discussion
This study represents the first comprehensive transversal and longitudinal epidemiological investigation conducted in Mexico on grapevine virus diseases. Our research focused on analyzing the distribution of GLD and GRBD symptomatic plants across the primary winegrape producing regions of Baja California. The findings indicate a concerning prevalence of these diseases: 92% (111 out of 121) of the visited sites had GLD and/or GRBD symptomatic plants. Moreover, vine mealybugs were detected in 61% of the sites surveyed, suggesting a significant correlation between vector presence and disease occurrence. Locality significantly influenced disease prevalence, with notably higher levels recorded in Ejido El Porvenir, Francisco Zarco, and San Antonio de las Minas. Conversely, localities such as Real del Castillo, Valle de Ojos Negros, Tecate, and Valle de las Palmas exhibited a lower presence of symptomatic plants, which may be attributed to their limited mealybug populations and geographical distance from more extensive vineyards.
A positive association between the percentage of vine mealybug-infested plants and the prevalence of symptomatic plants was observed, indicating that as vine mealybug populations increase, so does the risk of new plants becoming symptomatic. This correlation is anticipated to strengthen over time, as vine mealybugs are known to spread GLRaV-3 (Cooper et al. 2018, Cabaleiro et al. 2022). Interestingly, certain areas such as Valle de las Palmas displayed a high prevalence of symptomatic plants, despite low mealybug infestation. This discrepancy may be explained by the propagation of infected plants from local sources, emphasizing the importance of understanding local material movement in the dissemination of GLRaVs or GRBV, as observed in other grapegrowing regions (Maree et al. 2013, Donda et al. 2023). Molecular diagnostics confirmed the presence of GLRaV-2, highlighting the role of contaminated plant material in the occurrence and spread of grapevine viruses in Baja California. GRBV vectors were not considered because they have not yet been reported in Baja California. However, as in other grapegrowing regions where the occurrence of S. festinus is not documented (LaFond et al. 2022), other closely related species may be found that could be considered potential vectors. Therefore, it is imperative to continue monitoring GRBD and investigating potential vectors in this region.
Climate exerts a significant influence on grapevine phenology (Cola et al. 2017, Alikadic et al. 2019, Biasi et al. 2019, Faralli et al. 2024). Although our meteorological data were somewhat limited, a positive correlation between soil temperature, accumulated heat units, and the presence of symptomatic plants was noted. These findings suggest a relationship between environmental factors and the developmental behavior of vine mealybugs, which exhibit temperature-dependent survival rates, particularly with optimal conditions favoring their population growth (as found in the report https://www.ipw.co.za/content/pdfs/literature/eng/Degree_Day_Estimation_as_a_Warning_Tool_for_Outbreaks.pdf) (Dalton et al. 2014).
Our findings indicate that the incidence of GLD and/or GRBD symptomatic plants varies not only with the year of evaluation but also with geographic location and grape cultivar. Sites 1, 2, and 3, which are geographically closer, exhibited the highest number of symptomatic plants. Site 3 had the most significant increase in new cases. Nebbiolo had more symptomatic plants over time than Cabernet Sauvignon or Merlot. This result could be attributed to various factors not determined in the study, warranting further investigation. Incidence results should be taken cautiously, as no distinction was made between GLD, GRBD, or mixed infections, and the vine mealybug is not a vector of GRBV. A more extensive molecular diagnosis should be conducted to better understand disease spread due to specific association between symptoms, related viruses, and vectors at each site. However, from a statistical standpoint, the percentage of plants infested by vine mealybugs was positively associated with the presence of symptoms of GLD and/or GRBD by the end of the study.
The contrasting results between GLD and/or GRBD symptomatic plants and grapevine cultivars in the incidence and prevalence studies highlight the complexity of viral interactions within different environments. While the prevalence study indicated no significant differences among cultivars, the longitudinal monitoring revealed cultivar-specific responses. This finding is particularly relevant given that different cultivars may exhibit varying levels of susceptibility to the same virus. For example, the Hunan-1 cultivar is less sensitive to GLRaV-3 than Chardonnay and Thomson Seedless (Hao et al. 2022). However, cultivar susceptibility is not the only explanation for the spread of these diseases.
Visual monitoring can serve as a valuable initial screening tool to reduce costs, however, it should be complemented by molecular tests like PCR to ensure more reliable diagnosis (Sudarshana et al. 2015). As observed in other studies, GLRaV-3 was the most prevalent virus, and mixed infections were found (Xiao et al. 2018, El Aou-ouad et al. 2022). In our study, visual diagnosis effectively identified symptomatic plants likely positive for viruses associated with GLD or GRBD, achieving a sensitivity of 91%. However, only 52% of asymptomatic plants tested negative via PCR, reflecting the low specificity inherent to visual assessments (Poojari et al. 2013). It is essential to recognize that visual symptoms may overlap with other physiological issues such as nutrient deficiencies (Cieniewicz et al. 2017); in addition to the challenge of detecting asymptomatic infected plants, visual assessment may lead to misdiagnosis. We acknowledge that the prevalence and incidence determined in our study are constrained by the sensitivity and specificity of our symptom-based diagnosis.
Epidemiological studies like ours are the first step toward developing virus management practices, as demonstrated in many other grapevine-producing regions (Fuchs et al. 2009, Dalton et al. 2019). Potential mitigation measures include mealybug management (as areawide mating disruption programs) (Hogg et al. 2021); roguing; and ensuring the planting of certified virus-free plant material (as much as possible), paying particular attention to areas with high presence of vine mealybugs (e.g., localities such as Ejido El Porvenir, Francisco Zarco, San Antonio de las Minas, and Valle de San Vicente).
This study contributes significantly to the understanding of grapevine viruses in Mexico, however, much remains to be explored, particularly regarding the effect of these viruses on fruit production. The urgency for continued investigation is paramount, as understanding the dynamics of grapevine virus diseases is essential to develop effective management strategies to mitigate the effects of the diseases on grape cultivation in Mexico.
Conclusion
The implementation of symptom-based diagnosis to evaluate GLD and/or GRBD occurrence in Baja California revealed that symptomatic plants were present in 92% of the monitored vineyard sites. The prevalence of symptomatic plants was positively associated with vine mealybug presence and meteorological variables such as soil temperature. Furthermore, the incidence analysis demonstrated an increase in cases of symptomatic plants over time. Molecular diagnosis confirmed the utility of visual monitoring as a screening tool in identifying plants with GLD and/or GRBD. The information generated in this work is instrumental to develop and apply regional management strategies, primarily to prioritize areas that require special attention based on the presence of factors that render them susceptible to these diseases. The results presented here serve as a reference baseline for action.
Supplemental Data
The following supplemental materials are available for this article at Supplemental tab above:
Supplemental Table 1 Geographic characteristics of incidence study sites and localities.
Supplemental Table 2 List of primers.
Supplemental Table 3 Statistical results of the association of prevalence with locality, cultivar, and infestation by vine mealybugs.
Supplemental Table 4 Multiple regression of meteorological variables for prevalence.
Supplemental Table 5 Linear regression model for incidence of grapevine leafroll disease and/or grapevine red blotch virus and vine mealybug
Supplemental Table 6 Generalized linear model (GLM) for incidence of grapevine leafroll disease and/or grapevine red blotch disease.
Supplemental Table 7 Chi square analysis for the association between diagnosis by symptoms and molecular diagnostics.
Supplemental Table 8 Number of positive plants tested by real-PCR to detect five viruses classified according to visual diagnosis. GLRaV, grapevine leafroll-associated virus; GRBV, grapevine red blotch virus.
Supplemental Figure 1 Dispersion diagrams and dispersion matrix of meteorological variables for prevalence. Precip, precipitation; solar-Rad, solar radiation; aveTem, average ambient temperature; aveHum, average relative humidity; aveSoilTem, average soil temperature; AHU, accumulated heat units.
Footnotes
This work was supported by funds granted to JCT [CONAHCYT (FOP02-2021-4 Grant 316602), CICESE institutional project (683210)], and to CESVBC (SADERBC, Proyecto de Manejo Fitosanitario de la Vid). We express our gratitude to CONAHCYT for the doctoral scholarship awarded to KGGR. We thank all the grapegrowers participating in this research and our collaborators from CESVBC and CICESE, as well as the undergraduate students for their assistance in developing our research on grapevine viruses. We thank F.S. Ceccarelli and M.A. Alonso for their valuable input during the development of this work. We also thank M. Rosas for verifying the statistical analyses.
García-Reséndiz KG, Moyano-Briones G, López-Simancas P, Úrbez-Torres JR, Hernández-Martínez R and Carrillo-Tripp J. 2025. Epidemiological survey of grapevine leafroll and red blotch diseases in Baja California, Mexico. Am J Enol Vitic 76:0760007. DOI: 10.5344/ajev.2025.24059
By downloading and/or receiving this article, you agree to the Disclaimer of Warranties and Liability. If you do not agree to the Disclaimers, do not download and/or accept this article.
- Received November 2024.
- Accepted January 2025.
- Published online March 2025
This is an open access article distributed under the CC BY 4.0 license.
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