Planococcus ficus and the spread of grapevine leafroll disease in vineyards: a 30-year-long case study in north-West Spain

Planococcus ficus and the spread of grapevine leafroll disease in vineyards: a 30-year-long case study in north-West Spain Cristina Cabaleiro &Ana M. Pesqueira &Antonio Segura Accepted: 27 April 2022 #The Author(s) 2022 Abstract The mealybug Planococcus ficus is one of the main vectors of Grapevine leafroll associated virus-3 (GLRaV-3), which was commonly detected in cv “Albariño”planting material before certified stock was available. Mealybug infestations were rare in vineyards in southern Galicia (NW Spain) during the 1990s (2.2% of the vineyards surveyed) and are still rare in inland zones. However, mealybug infestations have spread since 2000, with 15% of surveyed vineyards infested in 2004 and 80% of surveyed vineyards infested in 2016. The spatial and temporal distributions of plants infected with GLRaV-3 were quantified over a 30-year period in an experimental plot established in 1989. The disease progress curve (DPC) was linear for 25 years, with a slow constant rate of spread of less than one newly infected plant per year (0.6%). Since 1992, >82% of infected plants were located on the west side of the plot as were 84% of newly infected plants. Newly infected plants were in contact with infected plants, suggesting vector-mediated transmission, but no potential vectors were found. In 2013, a small mealybug infestation was detected and identified as Pl. ficus. Between 2014 and 2016, the infection rate increased to >21% per year, and in 2019 all plants tested positive for GLRaV-3. This is a valuable case study illustrating how changes to the vector fauna can increase the rate of spread of an economically important virus of grapevine. Keywords Grapevine .Leafroll .GLRaV-3 . Mealybugs .Planococcus ficus .Epidemiology Introduction Epidemics of viral diseases have been widely described in woody plants, and different transmission models have been proposed for predicting changes in disease progress curves (DPCs) (Madden et al. et al., 2017). The best described epidemics of grapevine virus diseases are those caused by grapevine leafroll disease associated viruses, which are transmitted by several species of hemipteran insects. The most widely studied epidemics of leafroll viruses are those involving Grapevine leafroll associated virus-3 (GLRaV-3), although many other Ampelovirus epidemics have been documented in grapevines (Bonfiglioli et al., 2002;LeMaguetetal., 2013; Naidu et al., 2014) and other crops such as pineapple (Selvarajan et al., 2016;Setheretal.,1998). Insect vectors of ampeloviruses occur in only two families: Pseudococcidae and Coccidae (reviewed by Herrbach et al., 2017). Accordingly, transmission of ampeloviruses does not appear to be specific. The number of vector species in these two families increases each year, and currently includes 10 mealybug species of several genera (Pseudococcus, Planococcus, https://doi.org/10.1007/s10658-022-02513-x C. Cabaleiro (*):A. M. Pesqueira Departamentos de Producción Vegetal e Ingeniería de Proyectos, Universidad de Santiago de Compostela, Campus Terra, Lugo, Spain e-mail: [email protected] A. Segura Departamento de Biología Fundamental, USC, Campus Vida, Santiago de Compostela, Spain / Published online: 11 May 2022 Eur J Plant Pathol (2022) 163:733–747 Phenacoccus, Heliococcus and Ferrisia)and7species of soft scale in the genera Pulvinaria, Neopulvinaria, Parthenolecanium, Coccus, Ceroplastes and Parasaissetia (Herrbach et al., 2017). All mealybugs and soft scales that colonize grapevines can be considered potential vectors of ampeloviruses, although dispersal ability and transmission efficiency may differ greatly among species (Almeida et al., 2013). Several soft scale insects (family Coccidae) act as vectors of GLRaV-3 (Belli et al., 1994)butareconsidered less epidemiologically important than mealybugs (family Pseudococcidae) for two reasons. First, soft scales have only one (in cool climates) or two generations (in Mediterranean climates) per year (Belli et al., 1993; Maree et al., 2013; Robayo-Camacho & Chong, 2015). Second, soft scales are not very mobile, although crawlers can reach neighboring plants and can also be transported by ants and wind (Hommay et al., 2019). Information about transmission of grapevine-infecting ampeloviruses by soft scale insects is limited due to inherent challenges in handling these highly sedentary insects (Belli et al., 1994;Hommayetal.,2019; Naidu et al., 2014). Planococcus ficus (Signoret) is an emerging vineyard pest in many countries and is the most widely studied GLRaV-3 vector. In Spain, Pl. ficus mainly occurs in table grapes in the Mediterranean coastal area, but nowadays also appears in wine grapes and in areas where it was previously an unknown pest. Historically, the mealybug species mentioned in Spanish reports was usually Pl. citri (Toledo, 2004). However, more recent reports such as the Integrated Pest Management (IPM) Guide for wine grapes mentions both mealybug species (Martín et al., 2014)andPl. ficus is now assumed to be dominant (Lucas, 2015). Similarly, Pl. ficus has replaced Pl. citri as the most reported mealybug species in Portugal and Italy, (Godinho & Franco, 2001; Lentini et al., 2008). It is likely that misidentification may often have occurred as the species are difficult to distinguish and mixed infestations are not unusual. Kol-Maimon et al. (2014) found evidence of gene flow between the two sympatric, genetically-related mealybug species, further complicating the situation. Nonetheless, there is evidence that populations of Pl. ficus have established and spread in vineyards in Spain, as has occurred in other regions in Europe, America and North and South Africa (Daane et al., 2012; Daane et al., 2018;Mansour et al., 2017;Walton&Pringle,2004). In the vineyards established in Galicia (NW Spain) in the 1990s, high prevalence of leafroll viruses was due to lack of available certified virus-free plant material. Very few cases of virus spread were confirmed (Cabaleiro & Segura, 1997) and mealybugs were rarely observed in the vineyards. For grapevine ampeloviruses, some studies report DPCs for vineyards infected at planting (Arnold et al., 2017; Cabaleiro & Segura, 2006; Goussard & Underhay, 2004), although most studies were initiated when the disease was first detected or after it was clear that the pathogen had been spreading for years (Arnold et al., 2017; Cabaleiro & Segura, 2006;Golinoetal., 2008;Jordanetal.,1993;Walkeretal.,2004). In some studies, it was not possible to associate pathogen spread with any particular vector species (Dimitrijevic, 1973; Habili et al., 1995; Teliz et al., 1989), similar to the observations made during the first two decades of this study (Cabaleiro et al., 2008; Cabaleiro & Segura, 1997). The existence of vectors other than scale insects has been suggested as a possible cause of pathogen spread when none of the known virus vectors were found (Habili & Nutter, 1997). Another possible cause is natural root grafting between adjacent plants (Epstein, 1978), although this is a rare occurrence that has not been demonstrated in Vitis species. The rate of spread of GLRaV-3 is reported to vary from 0.9% to 16% annually, with an annual average increase of around 7 to 8% (Arnold et al., 2017; Cabaleiro, 2009). In this paper, we report on the spread of Pl. ficus in southern Galicia. In addition, results from a 30-year- long case study of a GLRaV-3 epidemic in a small plot established in 1989 and monitored since 1991 (Cabaleiro et al., 2008; Cabaleiro & Segura, 1997)is updated. The study findings illustrate the difficulties associated with early detection of vectors and the dramatic increase in GLD since the appearance of Pl. ficus. Materials and methods Survey of insect vectors Vineyards supplying grapes to the five wine appellations in Galicia were surveyed in three phases (Fig. 1). In the first phase (1990–1993) 92 vineyards in the Rías Baixas and Valdeorras areas were surveyed. In the second phase (2003–2004), 126 vineyards in the Rías Baixas, Ribeiro, Ribeira Sacra and Monterrei areas were surveyed. Finally, in the most recent phase (2014–2016) 84 vineyards in the Rías Baixas, Ribeiro, Ribeira Sacra, Monterrei and 734 Eur J Plant Pathol (2022) 163:733–747 Valdeorras areas were surveyed. The main objective of the surveys was to determine presence or absence of mealybugs and soft scale insects. Surveys were conducted by examining at least 20 vines for the presence of ants (known to obtain honeydew by tending mealybugs). Each vine was examined for one minute and if no ant movement was detected, the search on that plant was stopped. If ants were observed, the vine was then searched for mealybugs for an additional 1–3minutes (depending on the age/size of the grapevine and the type of trellis system used). Mealybug identification In 1992, the species identity of collected mealybugs was determined by experts using morphological characteristics. In 2014, mealybug samples from several vineyards were identified by targeted DNA analysis using multiplex-polymerase chain reaction (PCR) (ten insects from each population). DNA was extracted from insect samples following the method described by Koekemoer et al. (2002). The multiplex- PCR method was based on the protocol and primers described by Saccaggi et al. (2008): C1-J-2427-F 5’- TAATTATTGCTATTCCTACAAGAATTAAAATC- 3′which produces a 587 base pair (bp) amplicon for Pl. citri, C1-J-2260-F 5’-TCAAATTATAAATC AAGAAAGGGGAAAAC-3′which produces a 757 bp amplicon for Pl. ficus, and TL2-N-3014-R 5’- TCCAATGCACTAATCTGCCATATTA-3′which is a common primer for both species (Simon et al., 1994). A case study: 30 years of spread of GLRaV-3 in Meaño Vineyard The experimental plot was located in Meaño, Pontevedra, Galicia, NW Spain (42.433863 ° N, 8.772683 ° W) and occupies about 1500 m 2 inside the vineyard (2.206,62 m 2 ). The plot has previously been described in detail (Cabaleiro et al., 2008;Cabaleiro& Segura, 1997). In 1990, 160 scions from 4 indexed virus-free Albariño clones obtained from the collection belonging to the CSIC (Consejo Superior de Investigaciones Científicas) were grafted onto 196.17C or SO4 certified virus-free rootstocks planted the previous year in 8 rows of 20 plants. After 2005, a number of plants were uprooted by the owner due to problems related to vigour of the Albariño cultivar; a total of 63 Fig. 1 Map showing the five wine growing areas with appellation of origin status in NW of Spain. Downloaded from https://portalgalicia. com/galicia/vinos-gallegos-5-denominaciones-de-origen/ 735Eur J Plant Pathol (2022) 163:733–747 plants were removed, 19 of which were positive for GLRaV-3. In the following years, 5 more plants were confirmed infected with GLRaV-3 were removed: 2 in 2009, 2 in 2014 and 1 in 2016. During the first years after vine removal, rows with missing vines were clearly visible. However, vines grew to cover the empty spaces on the horizontal trellis, facilitated by yearly pruning practices. Virus testing Up to 2006, all 160 plants in the Meaño plot were tested for GLRaV-3 by DAS-ELISA. After 2006, plants which tested negative in previous analyses were tested by tissue printing ELISA using the protocol described by Cabaleiro et al. (2008). Vines were sampled in late summer by collecting two mature leaves with their petioles from each vine. Antibodies and buffers were obtained from Bioreba AG (Basel, Switzerland) and nitrocellulose membranes (0.45 μmpore size) from Sartorius (Goettingen, Germany). BCIPNBT ready-to-use liquid substrate was obtained from SIGMA (B-1911). Virus testing was conducted in 1992–1996 / 2003–2007 / 2009 / 2014 /2016–2019. Vector surveys During sampling for viral testing, all vines were searched for ants as an indication of presence of hemipteran vectors. In 2016, the two mature leaves collected from each vine for ELISA testing were first examined under a stereomicroscope for presence of mealybug nymphs (crawlers). In June 2017, all vines were thoroughly checked (trunk, branches, leaves) for mealybugs and a map was drawn of the whole plot, including border plants showing the spatial distribution of mealybug infestations. In 2016 a Delta trap containing pheromone specific for Pl. ficus (Suterra® Europe) was placed in the centre oftheplot(forsubsequentcomparisonofcaptureswith those obtained in traps in other vineyards in southern Galicia). In 2017, six Delta traps were used to monitor the male flight activity weekly during the whole season (Fig. 2). In addition, a Delta trap was placed in a neighbouring plot with young vines (200 m away) belonging to the same vineyard owner, and another trap was placed in a small century-old vineyard that was >1 km away from the experimental plot. Pheromone capsules were replaced every 6 weeks. Temporal analysis of the epidemic Disease progress was quantified 16 times between 1989 and 2019, and disease incidence data were used to construct a disease progress curve (DPC). For DPC modelling, we fitted linear (untransformed data), exponential, logistic and Gompertz models (Madden et al., 2017). The goodness of fit of the linearized form of each model was assessed using the coefficient of determination (R 2 )andthestandard error for the estimation of y (EEEy). Models were fit for: a) the whole period (1992 to 2019), b) the period between 1992 and 2014 (prior to observation of Pl. ficus in the experimental vineyard), and the period between 2014 and 2019 (after Pl. ficus was observed in the experimental vineyard). The removal of a number of healthy and infected plants after 2005 caused some interference in data collection, but the DPC was not significantly affected. Rates of disease spread were estimated from the slope of the regression lines of the model that provided the best fit for the DPC. Spatial distribution of diseased plants Maps of the spatial distribution of vines infected with GLRaV-3 were drawn based on the results of the surveys described above. Analysis of the spatial distribution of infected vines was affected by vine removal. Canopies quickly filled in after vines were removed, making it difficult to determine which canes were associated with which vine as the canopies were interlocking. Due to the continuous canopy, unidimensional analyses were inappropriate. Instead, the occurrence of newly infected vines in direct contact with known infected vines was determined for each year. Bidimensional analysis was performed by successively dividing the vineyard into subplots of increasing size, calculating the mean number of infected vines in each subplot and using these data to calculate Morisita’s index of dispersion (Campbell and Madden, 1990). The statistical significance of deviations in Morisita’s index from unity was estimated with the WinPatchy program (M. Maixner, version 3.2.9). The size of the units tested for clustering ranged from 2 × 2 to 8 × 8 m. However, because of the large number of missing vines and empty quadrats, the tolerance was changed to 25% and the “minimum number of units required for statistics”to 6 (M. Maixner, personal communication), the results of this analysis should be considered an approximation for comparison with previous published data in Cabaleiro & Segura, 1997 (1992– 1995) and Cabaleiro et al., 2008 (1995–2005). To determine if infected vines are more common on the edge or interior of vineyards, the GLRaV-3 status of each plant, for a given year, was subjected to regression analysis with column (1–20) or row (1–8) number as an 736 Eur J Plant Pathol (2022) 163:733–747 independent variable (x) and the incidence of disease in that column or row as the dependent variable (y). Significant deviation of the slope from 0 was checked by a t-test and a test for linearity, performed by calculating the coefficient of correlation and conducting an F-test. Regression lines with slopes significantly different from 0 were considered to indicate the existence of a disease gradient in the direction corresponding to the independent variable. New positives in west or east zones of the plot were also counted since 1992 and in three phases: one before plant removal (1993–2005), and two after removal (2006–2014 and 2015–2019). Results Vector surveys In the first survey, mealybugs were found at 2 locations in the Rías Baixas: Beluso (5 vineyards from the same winery) and one in O Rosal (one vineyard). In O Rosal, a large population was found with honeydew and sooty mould on some leaves in a vineyard near the Miño river on the border with Portugal. In Beluso, the populations were small, but the insect was widely distributed across all vineyards on a single property. The infested vineyards were isolated from other vineyards and surrounded by eucalyptus plantations. In the second survey, mealybugs and soft scale insects (Parthenolecanium spp., Pulvinaria spp.) were found in 6.1 and 14% of surveyed vineyards, respectively. In the most recent survey, the inland winegrowing areas, least strongly influenced by the Atlantic climate (Ribeira Sacra, Monterrei and Valdeorras), appeared to be free of mealybugs, and soft scale insects were only found in 2 vineyards. By contrast, in the Ribeiro and Rías Baixas areas, the number of vineyards with mealybugs has increased to the point that in some locations, such as O Condado, it is now difficult to find mealybug-free vineyards, and some growers are expressing concern about the presence of ants, honeydew and sooty mould on the grapes (Tables 1and 2). Vector identification Prior to the development of molecular techniques, identification of the mealybugs was Fig. 2 Experimental plot with 4 double rows of Albariño plants on 196.17C rootstock (black) and SO4 rootstock (grey). Pheromone traps used to monitor Pl. ficus male flights: 2016 (black triangle) and 2017 (white triangles) Table 1 Mealybugs and soft scale insects detected in the three surveys carried out between 1992 and 2016 in vineyards in the different winegrowing areas in Galicia D.O. Mealybugs Soft scale insects 1991– 93 2003– 04 2014– 16 2003– 04 2014– 16 Rías Baixas 2/82 2/24 28/32 0/24 5/32 Ribeiro –6/24 7/14 2/24 0/14 Ribeira Sacra –0/67 0/9 16/67 2/9 Monterrei –0/10 0/26 0/10 0/26 Valdeorras 0/10 –0/3 –0/3 737Eur J Plant Pathol (2022) 163:733–747 uncertain (Table 3). It is difficult to distinguish Pl. ficus from Pl. citri using morphological characters and even when the species are reared with care, contamination easily occurs. The mealybugs detected in Beluso in the 1990s were identified as Pl. citri by several authors (Cabaleiro & Segura, 1997; Cid et al., 2010). The molecular analysis conducted in the third phase of the survey (2014) revealed that almost 100% of the mealybugs were Pl. ficus (Table 3). Pseudococcus viburni was identified, in mixed populations with Pl. ficus,intwo vineyards. The citrus mealybug was identified only in one vineyard, also in mixed populations with Pl. ficus. In all the vineyards with Pl. ficus, leafroll disease symptoms were widely distributed, and GLRaV-3 was detected (Table 3). Ants (Hymenoptera: Formicidae) from several locations were sent to an expert (K. Gómez) for identification. The following species were detected: Lasius niger, L. grandis, Crematogaster scutellaris, Tetramonium caespitum_species group,Formica cunicularia, and of concern, Linepithema humile,the Argentine ant, a known invasive species, was found in ORibeiroandORosal. Vectors in Meaño During the successive sampling carried out at the end of the summer between 1990 until 2009, no ants or insects known to act as GLRaV-3 vectors were observed on vine trunks or leaves. During the unplanned visit in summer 2013 (the first since 2009), ants and mealybugs were observed on a small group of vines on the west side of the plot. Mealybugs were collected and identified by multiplex-PCR as Pl. ficus. The same species was subsequently also found in several other vineyards belonging to the same owner within a radius of 1 km of the Meaño vineyard. In 2016, evaluation of leaf samples determined that 15% of vines had nymphs on at least one of the collected leaves. The full survey of the plot in July 2017 (plot and borders) detected the presence of Pl. ficus on 43 vines (33%), most of which were located on the east and south sides of the plot (Fig. 3). Fewer than 10 mealybugs were observed on 63% of infested vines. The majority of mealybugs (95%) were observed on trunks and branches, with only 5% found on leaves. Ants were Table 2 Results of the survey carried out in 2014–16 in the 3 Rías Baixas subzones: O Salnés, O Condado and O Rosal. Vineyards with / total number of vineyards (percentage) Mealybugs 2014–16 Soft scale insects 2014–16 O Salnés 5/7 (71.4%) 3/7 (42.9%) O Rosal 10/12 (83.3%) 2/12 (16.7%) O Condado 13/13 (100%) 0/13 (0%) Total 28/32 (88.6%) 5/32 (14.3%) Table 3 Molecular identification of mealybug specimens located in some of the vineyards in the third survey (2013–2014) Code Date D.O. (subzone a ) Cultivar Species GLRaV-3 C1401 Aug 2011 Ribeiro Godello Pl. ficus; P. citri + C1402 Aug 2013 Rías Baixas (R) Albariño Pl. ficus; Ps. viburni + C1403 Jul 2014 Rías Baixas (R) Albariño Pl. ficus + C1404 Sep 2013 Pontevedra Jerez Pl. ficus + C1405 Jun 2014 Rías Baixas (C) Albariño Pl. ficus + C1406 Aug 2014 Rías Baixas (C) Albariño Pl. ficus + C1407 Oct 2013 Rías Baixas (R) Albariño Pl. ficus + C1408 Aug 2014 Rías Baixas (R) Albariño Pl. ficus + C1409 Aug 2014 Rías Baixas (R) Albariño Pl. ficus + C1410 Jul 2014 Rías Baixas (S) Albariño Pl. ficus + C1411 Jul 2014 Ribeiro Treixadura Pl. ficus + C1416 Aug 2013 Rías Baixas (S) Treixadura Pl. ficus + C1417 Aug 2014 Rías Baixas (S) Albariño Pl. ficus + C1418 Aug 2014 Ribeiro Treixadura Pl. ficus + C1421 Aug 2014 Pontevedra Treixadura Pl. ficus; Ps. viburni + a Subzones of Rías Baixas: R, O Rosal; S, O Salnés; C, O Condado 738 Eur J Plant Pathol (2022) 163:733–747 observed on 39% of vines and on 100% of vines infested with mealybugs. The ant species observed during the surveys were identified as Lasius grandis and F. cunicularia. In the survey conducted during the summer of 2017, some adult females (with ovisacs) of the scale Pulvinaria vitis In 2016, Delta traps containing Pl. ficus pheromone captured a large number of male Pl. ficus,with>1600 captured between June and September. In 2017, rates of pheromone trap capture were high, with more than 2600 males captured per trap for a total of 15,673 males captured in the study plot. The flight curve suggested 3–4 overlapping generations over a long season, with captures occurring through November (Fig. 4). In 2017, additional traps were monitored through October, one at a vineyard that was 200 m from the study plot and one in a century-old vineyard. The trap at the nearby vineyard captured 3357 males and the trap in the century-old vineyard captured 1951 males. Temporal analysis of virus spread The epidemic was characterised by two phases: a phase of long, slow increase occurring from 1992 to 2014 and a phase of rapid increase occurring between 2014 and 2019 (Fig. 5). Of the models tested, the exponential model best described disease progress from 1992 until 2019 (Table 4) but the explanatory power of the model was poor, with high EEEy and significant lack of fit due to bias that gives a not random residual pattern. Accordingly, evaluating the two distinct phases of the epidemic (1992–2014 and 2014–2019) provides a better description of events. The DPCs for both phases were almost linear (Table 4), with an annual rate of increase of 0.6% during the first phase (1992–2014) and an annual rate of increase of 14.1% during the second (2014–2019). The rate of spread peaked between 2014 and 2016 (21.7%). The DPC was not affected by the uprooting of plants at the end of 2005, as indicated by the similar proportion of healthy and infected plants before and after this event. Spatial analysis of virus spread Between 1992 and 2014, newly positive vines were most commonly observed on the west side of the plot around the area where infected vines were first detected in 1992 (Fig. 6and Table 5). The observation of a west to east gradient was supported by the significantly negative slope of the regression lines for position and incidence per column and with intercepts of Fig. 3 Plants in the experimental plot and border plants where Planococcus ficus were observed (black circles) or not (white circles) in summer 2017 (8 × 20 plot with the 92 remaining plants, 24 infested; 73 plants out of the plot, 34 infested with mealybugs) 739Eur J Plant Pathol (2022) 163:733–747 those lines increasing almost until the end of the epidemic (Table 6). Similar tests found no evidence for a north to south gradient (data not shown). Although some vines on the east side tested positive for GLRaV-3 in 2005, in subsequent tests (2006 and later), these vines were always negative for the virus until 2016. Accordingly, detections on the east side in 2005 were assumed to be false positives and that the first detection of a positive plant on the east side of the plot occurred in 2009. Between 2006 and 2014 the number of newly infected vines on the west (4) and the east (3) sides of the plot were similar. After 2015, transmission had almost stopped on the west side of the plot, whereas spread was rapid on the east side of the plot (Fig. 5,Table5). Throughout the epidemic, most new infections (84% of infections averaged over all years) were found contiguous to known positive vines. However, new infections detected between 2014 and 2016, when the rate of spread of the virus was highest (21.7%), had the lowest association with known infected vines. Specifically, from 2014 to 2016 only 74.2% of newly infected vines were in contact with a known infected vine (Table 5). Morista’s index of dispersion Fig. 4 Flight curve of Pl. ficus males during the 2017 season in Meaño. Mean daily capture of males in the six pheromone-traps (±sd) Fig. 5 Progress of grapevine leafroll disease between 1992 and 2019. Regression lines for the whole period, for 1992–2014 and for 2014– 2019. Number of plants in the vineyard: 1992–2005, 160; 2006–2007, 97; 2009, 95; 2014, 93; 2016–2019, 92 740 Eur J Plant Pathol (2022) 163:733–747 indicated that infected vines were aggregated (> 1) from 1992 to 2015, until the final phase of the epidemic (2015–2019) when nearly all vines on the west side were infected and number of infected vines on the east side was increasingly rapidly (Table 5). Table 4 Model fits for disease progress curves Model Transformation Slope R 2 R´ 2 EEEy F P 1992–2019 Linear y 0.025 0.682 0.660 0.168 30.061 0.000 Exponential Ln y 0.0545 0.809 0.800 0.260 59.289 0.000 Logistic Ln(y/(1-y) 0.2238 0.412 0.370 2.622 9.820 0.007 Gompertz -Ln(−Ln y) 0.1927 0.358 0.312 2.533 7.795 0.014 1992–2014 Linear y 0.0062 0.984 0.98 0.006 626.2 0.000 2014–2019 Linear y 0.1414 0.976 0.95 0,07 59.87 0.004 EEEy: mean squared error; F: Fisher’s statistic; P: probability Fig. 6 Spread of GLRaV-3 between 2006 and 2019. Black dots, GLRaV-3(+); empty dots, GLRaV-3(−); grey dots, new positive detections. Maps for the period 1992 to 2005 have been published in Cabaleiro and Segura (1997) and Cabaleiro et al. (2008). 741Eur J Plant Pathol (2022) 163:733–747