Key messages

  • A trap plant attracts and retains an insect pest but cannot host a pathogen transmitted by the pest.

  • Mealybugs colonized and were retained on ‘Grasslands Huia’ white clover, a candidate trap plant.

  • Added white clover did not change grapevine virus incidence relative to clover-free control.

  • Mealybugs transmitted GLRaV-3 to Nicotiana benthamiana but not ‘Grasslands Huia’ white clover.

Introduction

Grapevine, Vitis vinifera, has been domesticated for at least 8000 years but is widely infected with multiple viral pathogens owing to its extended history of clonal propagation and lack of genetically resistant populations worldwide (Singleton 1996; Oliver and Fuchs 2011; Martelli 2017; Reynolds 2017; Fuchs 2023). A critical viral disease affecting viticulture globally is grapevine leafroll disease (GLD). Morphologically, GLD is often characterized by downward rolling leaf margins, and in red varieties, premature reddening of leaves with the primary veins remaining green. GLD lowers grape yield, delays berry ripening, and reduces juice quality (Lee and Martin 2009; Vega et al. 2011; Montero et al. 2016; Song et al. 2021).

In New Zealand, GLD is most strongly associated with grapevine leafroll-associated virus 3 (GLRaV-3), a single-stranded RNA virus from the Ampelovirus genus (Chooi et al. 2013b). GLRaV-3 is phloem-limited in grapevines, with spread occurring through plant propagation via grafting of infected tissue and through mealybug and soft-scale insect vectors (Daane et al. 2012; Maree et al. 2013; Almeida et al. 2013).

GLRaV-3 management in New Zealand relies on growers adopting an integrated (multi-tactic) response that includes planting nursery certified grapevines screened for GLRaV-3, monitoring and managing insect vector populations, and in red varieties, visually identifying and removing (roguing) infected grapevines (Almeida et al. 2013; Andrew et al. 2015; Bell et al. 2018; New Zealand Winegrowers 2021). Data modeling showed New Zealand growers who adopted an integrated response annually accrued substantial economic advantage relative to those taking no action or one where growers were poorly implementing vector and/or virus management protocols (Bell et al. 2021).

In New Zealand, the most problematic vectors of GLRaV-3 are the mealybugs Pseudococcus calceolariae and P. longispinus (Hemiptera: Pseudococcidae) (Petersen and Charles 1997). Both species are described as ubiquitous in the country’s vineyards; they each have up to three generations per year, with young instars relatively abundant late-summer and fall (Charles 1993; Charles et al. 2010). While the crawlers acquire and transmit GLRaV-3 effectively relative to other life stages (Petersen and Charles 1997), any seasonal transmission patterns of the virus are likely to be positively correlated with mealybug vector abundance. As well as infesting the grapevine, both mealybug species colonize broadleaf host plants growing under the grapevine, along the inter-row, and/or on the margins of grapevine plantings (V. Bell pers. observation). As a cool-climate wine producer (Sturman et al. 2014), New Zealand’s wine regions mostly sustain multiple broadleaf flowering plant species year-round, especially in the inter-row. The integration of ecological tactics aimed at minimizing the influence of insect pests in crops has been explored, with options including the addition of perennial hedgerows or flowering cover crops (Daane et al. 2018). As well as potentially improving soils by enhancing microbial communities, flowering plants added to agroecosystems promote pollinator populations and provision natural enemies with floral resources to aid top-down control of insect pests (Bianchi et al. 2006; Brooker et al. 2023). As attractive as added plant biodiversity is to many growers, it is important to understand the connection between insect pests and non-crop host plants and whether those interactions benefit efforts to manage a viral pathogen like GLRaV-3 or have unintended consequences exacerbating the problem in the crop.

Here, we focus on P. calceolariae and its frequently observed association with white clover (Trifolium spp.) growing naturally in many commercial vineyards in New Zealand (V. Bell pers. observation). Although a long-term evaluation of a clover/mealybug association was lacking until this study, sufficient anecdotal evidence warranted closer evaluation of clovers as potential trap plants for P. calceolariae to reduce vector-mediated transmission of GLRaV-3. The trap plant concept, which has been reviewed (Hokkanen 1991; Shelton and Badenes-Perez 2006), is described as an alternative host that attracts and retains a target insect to reduce damage to a crop (Holden et al. 2012). Further, in a review of Bemisia tabaci (whitefly) and associated viral diseases affecting many annual crops, it was noted that a good trap plant cannot host any whitefly transmitted viruses (Hilje et al. 2001). Whether clovers host GLRaV-3 is uncharacterized currently.

This study aimed to evaluate a candidate trap plant to support an ecological approach to managing GLRaV-3. The candidate trap plant tested was ‘Grasslands Huia’ white clover (GHWC), a widely grown cultivar developed in New Zealand mid-twentieth century (Caradus et al. 1997), and which is often colonized by P. calceolariae in vineyards (V. Bell pers. observations). In a 5-year vineyard trial, we assessed the interactions between P. calceolariae and the grapevine and if and how that relationship changed when the mealybug had access to permanent under-vine cover of GHWC. In a separate glasshouse experiment, we assessed if P. calceolariae could transmit GLRaV-3 to GHWC and four other clover species/cultivars. Quantifying this risk is important if the trap plant concept as described is to have any merit in potentially augmenting the GLRaV-3 integrated response adopted in New Zealand currently (Bell et al. 2018).

Materials and methods

Vineyard field trial—P. calceolariae, white clover, and GLRaV-3

Study site and trial design

Our 5-year field trial evaluated P. calceolariae utilization of GHWC and the influence of this association on GLRaV-3 incidence among grapevines. The research was undertaken in a commercial vineyard in Hawke’s Bay, a winegrowing region on the east coast of New Zealand’s North Island. The 19-ha trial vineyard was planted with Merlot grapevines in 2009. (This vineyard has historically sustained populations of P. calceolariae.) In accordance with New Zealand wine industry rules and product label recommendations, vineyard personnel were responsible for all decisions linked to annual mealybug insecticide use with the active ingredients, buprofezin and spirotetramat (New Zealand Winegrowers 2023). Every year of this study, three separate mealybug insecticides were applied to grapevines at 14–21-day intervals, the last of which was immediate pre-flowering.

The trial design was split into 21 spatially separate plots of equal area (0.4 ha, 60 m × 66 m). Grapevines grew on a vertical shoot positioned trellis, with permanent drip irrigation positioned 15 cm above ground. Grapevine within-row separation was 1.8 m; across-row separation was 2 m, giving c. 1100 grapevines per study plot. The grapevine buffer zones, which separated neighboring study plots (50 m along rows; 12 m across rows), were excluded from data collection.

In November 2016 (Southern Hemisphere spring), 14 plots were randomly allocated in a complete block design to include added GHWC seed. Hence, in every row of seven plots, vineyard personnel applied seed directly below the grapevines (3.6 kg per ha); in every row of another seven plots, the seed was sown below the grapevines plus in the inter-row (7.2 kg per ha). The failure of GHWC to establish in the unirrigated inter-row meant there were 14 plots of under-grapevine clover only. In these, a patch of GHWC (c. 0.5 m2) alternated with a similar area of bare ground along the entire row length for every row per plot. Hence, in the 14 treated plots, c. 50% of the under-grapevine of each row was planted in GHWC (1.0 m × 0.5 m × 33 m = 16.5 m2), which equated to 12.5% of the total area per plot (16.5 m2 × 30 rows = 495 m2). Herbicide was excluded from the under-grapevine GHWC plots. In the trial vineyard, the standard under-grapevine herbicide-treated strip (c. 0.5-m wide) represented the clover-free control (n = 7 plots), with glyphosate applied by vineyard personnel according to label and sector recommendations.

Under-grapevine groundcover, pheromone trapping, on-leaf P. calceolariae and GLRaV-3 assessments

Various assessments were undertaken during each of five vintages (2017 to 2021). In the Southern Hemisphere, vintage is between 1 September (spring) and 30 April (fall) when harvest has concluded.

A red delta trap containing a single P. calceolariae synthetic sex pheromone lure (Unelius et al. 2011) was positioned in the center of every study plot for up to 21 days in each of spring, summer, and fall. Suckling et al. (2015) found when P. calceolariae synthetic sex pheromone-baited traps were separated by 64 m, there was no trap interference. Hence, for males of this species a single pheromone point source has an active space of c. 0.4 ha (the plot size in the current study). The numbers of sexually mature male P. calceolariae on the white sticky base of each trap was assessed in the laboratory with the aid of a dissecting microscope (Nikon SMZ800N).

The percent groundcover plant composition (including bare ground) was estimated within a 0.25 m2 quadrat (n = 5 fixed sampling points for under-grapevine and inter-row per plot in each of November, February, and April).

Grapevine leaves were collected pre-harvest and inspected for mealybugs (n = 75 leaves per plot per visit in January, February, and March). In the laboratory, leaves were examined under a dissecting microscope. All mealybug life stages were counted. For mealybug assessments on groundcover plants, GHWC once established was specifically targeted in the 14 clover-treated plots (2018 to 2021). In the seven control plots, clover was naturally scarce. Where it was found, it was collected, but by necessity, collections were augmented with other non-Trifolium species we identified as mealybug host plants during earlier unrelated studies (Supplementary Table 1). We collected 15 plant samples per plot in January, February, and March. In the laboratory, we inspected every plant sample under a magnification lamp and scored mealybug numbers (0 = no mealybugs, 1 = 1–5 mealybugs, 2 = 6–10, 3 = 11–15, 4 = 16–20, 5 = 21 +). Plant and leaf inspections were timed for mid- to late-vintage because where present, P. calceolariae detection ability is improved.

Finally, in early- to mid-April (post-harvest), an experienced assessor (VAB) walked the entire 19-ha study vineyard systematically looking for grapevines with foliar symptoms typical of GLRaV-3 (Bell et al. 2017). Of those grapevines visually identified, colored tape was tied around the trunk by the assessor. This marking allowed vineyard personnel to identify diseased grapevines, all of which they removed every year during June/July (Southern Hemisphere winter).

Statistical analysis

The data consisted of counts (mealybugs per grapevine leaf, sexually mature male mealybugs caught per day in pheromone traps) or proportions (percentage of ground cover plants with mealybugs, percentage of grapevines infected with GLRaV-3). Host plant mealybug data, which was scored on a 0–5 scale (see above), were converted to counts by replacing the category with the mid-point of the range it represented (e.g., category 2, 6–10 mealybug became a count of 8; category 5, 21 + became 25). While this makes semi-quantitative data more manageable, it may over-estimate counts. Count data were analyzed using generalized linear mixed models with an over-dispersed Poisson distribution. Random effects were plot, plot x year and plot x month; fixed effects were year x month, block, block x year, block x month, treatment, treatment x year and treatment x month. Overdispersion factors were calculated from the residual deviance. Infection data were analyzed using a generalized linear mixed model with a binomial distribution. Random effects were block and block x plot; fixed effects were year, treatment, and treatment x year. The significance of the differences between means for GHWC and control treatments for each year was calculated using estimated means and standard errors from the models. All analysis was done using Genstat (version 22, 2023, VSN International Ltd, UK).

Glasshouse experiment—Transmission of GLRaV-3 by P. calceolariae to non-Vitis hosts

Non-Vitis plants, mealybug, and GLRaV-3 source material

The laboratory experiment used Crimson clover (Trifolium incarnatum), GHWC (Trifolium repens ‘Grasslands Huia’), ‘Karridale’ subterranean clover (Trifolium subterraneum ‘Karridale’), ‘Tripoli’ white clover (Trifolium repens ‘Tripoli’), and strawberry clover (Trifolium fragiferum). N. benthamiana, a known GLRaV-3 host, was used as a non-Vitis control to confirm successful GLRaV-3 transmission (Prator et al. 2017). Research accession 4 (RA-4) N. benthamiana was used (Wylie et al. 2015). Seeds for all non-Vitis plants were sown in plastic pots using a 1:1 mixture of seed-raising mix and potting mix. Once germinated, we maintained the young plants under controlled glasshouse conditions (24 ± 2 °C and 16L: 8D light cycle). When the clover cultivars were 3–6 weeks old, and N. benthamiana was 2–3 weeks old, seedlings were individually re-potted into single pots.

We used naïve, crawler or first instar P. calceolariae that were reared on sprouted seed potatoes in vented plastic containers under controlled laboratory conditions (22 °C and 16L: 8D light cycle) (Fig. 1). The mealybugs completed their life cycle on sprouted potatoes and had no history of exposure to grapevines (or any other GLRaV-3 host plant). A subset of P. calceolariae was tested for GLRaV-3 by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) as described by McGreal et al. (2019) to confirm the absence of GLRaV-3.

Fig. 1
figure 1

Schematic of the inoculation method of grapevine leafroll-associated virus 3 (GLRaV-3) from infected donor grapevines by the mealybug, Pseudococcus calceolariae, to non-Vitis host plants. Included were five Trifolium spp. and Nicotiana benthamiana. a Mealybug P. calceolariae egg batches were collected from the colonies and reared on sprouted potatoes, which allowed first instar crawlers to emerge on GLRaV-3 positive donor grapevine leaves naturally; the grapevine leaf petiole extended into a vial filled with water. After a 6-day acquisition access period, plant material from the donor leaf was sampled to confirm GLRaV-3 presence. b The donor leaf with P. calceolariae was cut into small pieces with ~ 40 mealybugs per leaf. Each recipient plant received one piece of leaf containing ~ 40 mealybugs nestled into its foliage. P. calceolariae were left to self-disperse and feed on the alternative host plants. At the 3-month post-inoculation access period, non-Vitis host plants were sampled from multiple locations on the plant. These samples were combined, and each plant tested for GLRaV-3. Figure created by Tony Corbett (The New Zealand Institute for Plant and Food Research Limited, Hawke’s Bay, New Zealand)

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GLRaV-3 infected Pinot noir grapevines (V. vinifera) originally sourced from a commercial vineyard and maintained under glasshouse conditions (24 ± 2 °C and 16L: 8D light cycle) were used as virus source plants. These source plants were confirmed to have Group I and VI, Group I and X GLRaV-3 mixed infections, reported previously or verified by RT-qPCR or reverse transcriptase PCR (RT-PCR) (Chooi et al. 2013a).

Acquisition access period (AAP)

P. calceolariae was inoculated with GLRaV-3 as described by McGreal et al. (2021) with an acquisition access period (AAP) of 5–6 days. GLRaV-3 acquisition by P. calceolariae was confirmed by RT-qPCR (McGreal et al. 2019).

Inoculation access period

First instar viruliferous P. calceolariae were transferred to each of ~ 60 N. benthamiana plants and to either 40 or 80 plants of each clover cultivar by placing one piece of Vitis donor leaf with 40 newly emerged P. calceolariae onto each recipient non-Vitis host plant, as described in McGreal et al. (2021). Donor leaf segments remained in situ on the non-Vitis plants for 4 days to allow mealybug self-dispersal. Leaf segments were free of P. calceolariae when removed (McGreal et al. 2021). All test plants were treated with the insecticide active ingredient, imidacloprid (Confidor® 200SC), after 6 and then 13 days in the first year of experimentation and then after 40 days in the second year. This change in the timeframe allowed mealybugs to be collected and assessed for successful acquisition of GLRaV-3, as reported by McGreal et al. (2021). A further 15–20 plants of each non-Vitis host plant were maintained without inoculation to act as negative controls.

GLRaV-3 detection

Leaves were sampled and homogenized at 3-month post-inoculation access period (IAP) from all available non-Vitis host plants. Plant samples from negative control plants were used as the GLRaV-3 negative controls for enzyme-linked immunosorbent assay (ELISA) and molecular testing as noted above. Double antibody sandwich ELISA used 3 g of homogenized plant material, and up to two technical replicates for each sample (Cohen et al. 2012). As a positive control included on all ELISA plates, plant extracts from a GLRaV-3 positive grapevine were mixed with the respective non-Vitis host plant extracts at a 1:1 ratio and a 1:3 serial dilution. Negative control plants and buffer-only wells were included in all ELISA as negative controls. An ELISA was deemed positive when all technical replicates were positive.

To confirm the ELISA results, we tested non-Vitis plant samples with RT-qPCR. Approximately 100 mg of leaf material from each plant sample was processed and total RNA isolated using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St Louis, MO) following the manufacturer’s protocols. Total RNA was stored at − 80 °C. Samples from these plants consisted of either composites (grouped ELISA-negative plant samples at a maximum of five plants per composite) or single-plant samples.

The primers and probes used for the probe hydrolysis RT-qPCR were designed to target GLRaV-3 genetic variants of phylogenetic groups I, VI, X that target ORF6, ORF1a and ORF6-7, respectively (McGreal et al. 2019). The one-step RT-qPCR was performed as described by McGreal et al. (2019). A GLRaV-3 positive control and no-template water control were included in all runs. Samples were tested as triplicate technical replicates, whereby a mean RT-qPCR Ct value below 34 was considered positive; a cut-off point chosen based on the limit of detection as described by McGreal et al. (2019). Additional positive controls in the second year of experimentation included RNA from the respective non-Vitis host plant negative sample (either N. benthamiana or clover according to the plant species being tested for GLRaV-3) mixed with a grapevine GLRaV-3 positive sample at a 1:1 ratio. RNA from a respective non-Vitis host plant negative sample was also included as a negative control.

Results

Vineyard field trial—P. calceolariae, GHWC, and GLRaV-3

When the study started in late 2016, there was no significant treatment effect regarding GHWC under the grapevine, other plants under the grapevine, or plants in the inter-row (t = 0.28 to 1.36 on 76–600 df, P = 0.18 to 0.78) (Fig. 2A, B, C). By April 2017, 5 months after GHWC seed was sown, under-grapevine quadrat assessments showed it was establishing well (Fig. 2A). In vintages 2018 to 2021, the proportion of GHWC under the grapevine was significantly higher than any naturally occurring white clover detected in the same area of the control plots (t ≥ 6.00 on 600 df, P < 0.001 for all vintages). By the end of vintage 2019, GHWC cover peaked at an average of 40% under the grapevines, but thereafter, it reduced, averaging 5% by April 2021 when data collection ceased (Fig. 2A). As GHWC cover declined under the grapevines, an increasing proportion of competing other plants (especially grasses) peaked at an average of 68% by April 2021 (Fig. 2B). This additional cover under the grapevines was significantly higher across vintages 2018 to 2021 compared with the control, where one or two herbicide applications to the under-vine zone per vintage limited plant growth. Instead, the control under-vine was dominated by bare ground (range: 70–90%) and grasses (range: 10–30%). White clover was found rarely (< 0.2%). With GHWC failing to establish in the inter-row, there was no significant difference in the average proportion of grass cover (range: 65–80%) and bare ground (range: 20–30%) across all 21 plots (t = 0.06 to 1.79 on 76 df, P = 0.08 to 0.95 across vintages) (Fig. 2C).

Fig. 2
figure 2

Mean percentage (± SEM) of ground cover in the Hawke’s Bay study vineyard. The percent ground cover plant composition was estimated within a 0.25 m2 quadrat, with five fixed sampling points for under-vine and inter-row per plot in November, February, and April. In the 14 clover-treated plots, we assessed under-vine ‘Grasslands Huia’ white clover (GHWC) (●) and compared it with GHWC cover in the seven control plots (○) (Fig. 2A). Also compared were other under-vine plants in the GHWC plots (■) relative to the under-vine in the herbicide control (▫) (Fig. 2B), and other plants in the GHWC inter-row plots (♦) relative to the inter-row in control plots (◊) (Fig. 2C). Vintage 2017 represented baseline data recorded prior to the establishment of GHWC, with no treatment effect detected, independent of the paired comparisons (t = 0.28 to 1.36 on 76–600 df, P = 0.18 to 0.78). Following its establishment (2018 to 2021), significantly more GHWC was found in the treated plots relative to the control, independent of vintage (levels of statistical significance * P = 0.05, ** P = 0.01, *** P = 0.001, no asterisks denote no statistically significant difference, P > 0.05)

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The use of P. calceolariae pheromone-baited traps showed the species was widely distributed across the study vineyard in all vintages (Fig. 3). The numbers of sexually mature male mealybugs attracted to the pheromone-baited traps did not differ significantly by treatment in any vintage (t = 0.10 to 1.74 on 67 df, P = 0.09 to 0.92). Determining the source of the trapped males (ground cover or grapevines or both) requires inspections of those habitats, as discussed below.

Fig. 3
figure 3

Mean (± SEM) numbers of sexually mature male P. calceolariae caught daily in red delta traps baited with the species-specific synthetic sex pheromone. A single pheromone-baited trap was attached to a grapevine cordon in the center of each of the 14 ‘Grasslands Huia’ white clover (GHWC) plots (■) and the seven herbicide-treated plots (○). No significant difference in the daily catch rate of the male mealybugs between treatments was detected, independent of vintage (P > 0.05)

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By the end of vintage 2017, 5 months after GHWC seed was sown in the under-vine, it had not fully established. Rather than collect immature GHWC during that vintage, we instead collected other broadleaf plants we identified as potentially being mealybug host plants. P. calceolariae was found on an average of 14 and 16% of plants collected from the clover-treated and control plots, respectively (Fig. 4; Supplementary Table 1). No treatment effect was detected (t = 0.48 on 170 df, P = 0.63). However, during vintages 2018 to 2021, P. calceolariae was found on an average of 38 to 67% of GHWC samples, a frequency of colonization by the mealybug that was significantly higher than was recorded on plants collected from the control (t ≥ 6.33 on 170 df, P < 0.001 for all vintages). Mealybug abundance, estimated from categorical data, followed a similar pattern (Supplementary Fig. 1).

Fig. 4
figure 4

Percentage (± SEM) of ground cover plants found with the mealybug P. calceolariae in the Hawke’s Bay study vineyard. Once the ‘Grasslands Huia’ white clover (GHWC) was established, it was the only plant assessed for mealybug presence in the 14 clover-treated plots (■). In contrast, in the seven herbicide-treated plots (○), collections included any potential broadleaf plant that might be a host for the mealybug (Supplementary Table 1). Vintage 2017 represented baseline data recorded prior to the establishment of added GHWC, with no treatment effect detected (P = 0.45). Once the GHWC was established from vintage 2018, a significantly greater proportion of samples were found with mealybugs relative to plants assessed in the control (P < 0.001 for all vintages)

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In the first vintage of the study, P. calceolariae numbers on grapevine leaves were low (c. 1 mealybug found per 100 leaves inspected), independent of treatment (Fig. 5; t = 1.12 on 10 df, P = 0.29). In the following vintages, mealybug numbers on grapevine leaves increased but remained generally low (less than ten found per 100 leaves inspected), independent of treatment. Only in vintage 2020 was there a marginally significant difference (t = 2.20 on 10 df, P = 0.053) in the numbers of P. calceolariae on grapevine leaves from GHWC plots (4 per 100 grapevine leaves) relative to the control plots (1 per 100 grapevine leaves).

Fig. 5
figure 5

Mean (± SEM) number of P. calceolariae per 100 grapevine leaves inspected in the 14 plots with ‘Grasslands Huia’ white clover (GHWC) under the grapevine (●) compared with the seven plots where the under-grapevine was herbicide-treated (○) throughout the 5-year study. Vintage 2020 was the only time a marginally significant (P = 0.053) treatment effect was detected (n = 3150 grapevine leaves inspected in the 14 GHWC plots per vintage; n = 1575 grapevine leaves inspected in the seven control plots per vintage)

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Annual post-harvest inspections targeted grapevines with foliar symptoms of GLD within the 21 study plots. New infections visually identified represented secondary (P. calceolariae-mediated) virus spread (Fig. 6). No significant treatment effect was detected in any vintage (t = 0.03 to 0.14 on 56 df, P = 0.89 to 0.98).

Fig. 6
figure 6

Mean (± SEM) grapevine leafroll-associated virus (GLRaV-3) infections visually detected per 1000 grapevines in the Hawke’s Bay study vineyard. Compared are the results from 14 plots where ‘Grasslands Huia’ white clover (GHWC) was added to the under-grapevine (●) against the seven plots where the under-grapevine was herbicide-treated (○) throughout the 5-year study. No statistically significant treatment effect was detected during the study (t = 0.03 to 0.14 on 56 df, P = 0.89 to 0.98). Vintage 2017 represented baseline data recorded before the added GHWC was established. All grapevines visually diagnosed with GLRaV-3 annually were marked with colored tape to allow vineyard personnel to identify them for roguing in mid-winter, a process that team repeated every year of this study

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Glasshouse experiment—Transmission of GLRaV-3 by P. calceolariae to non-Vitis hosts

Mealybugs were used to inoculate 256 clover plants in both years of the study. None of the clover species/cultivars tested positive for GLRaV-3 infection (Table 1).

Table 1 Summary of the non-Vitis plants challenged with grapevine leafroll-associated virus 3 (GLRaV-3) by exposure to the viruliferous mealybug, P. calceolariae, in the glasshouse study
Full size table

During both years of the transmission experiment, 35 of 107 (32.7%) N. benthamiana plants tested positive for GLRaV-3 (Table 1). In both years, mixed and single variant GLRaV-3 infections were present in the GLRaV-3-infected N. benthamiana (Supplementary Table 2). Non-Vitis negative control plants and negative controls tested negative for GLRaV-3 within all ELISA and RT-qPCR assays.

Discussion

The relative effectiveness of a trap plant added to a horticultural system is defined by its attraction and retention qualities in respect of a target pest (Holden et al. 2012). Further, for insect transmitted disease management, a candidate trap plant must also be a non-host of the disease and therefore not a reservoir for the causal agent (Duffus 1971; Alexander et al. 2014; Petersen et al. 2019).

Our vineyard field study measured the interaction between the mealybug P. calceolariae and the grapevine and how this connection changed when provisioned with a permanent cover of GHWC, a trap plant candidate. We sought to understand how the mealybug responded to GHWC and whether its addition to the under-vine retained the mealybugs that arrived. Further, in our glasshouse vector-host virus transmission study, we sought to identify if clover is a non-host of GLRaV-3 and, therefore, not a potential reservoir for this pathogen. To our knowledge, this is the first trap plant study targeting mealybugs as vectors of GLRaV-3, so its novelty offers potentially important insights to others wanting to manage this pathosystem with an ecological focus.

In the field trial, we showed P. calceolariae readily settled on GHWC. Within 12 months of its establishment, 40% of GHWC samples were found with mealybugs, and 45 to 65% of samples collected were infested in following vintages. Moreover, mealybug abundance per GHWC sample increased over time (Supplementary Fig. 1), a result we suggest demonstrates a retentive quality despite the likelihood of reproduction by P. calceolariae (although this aspect was not quantified). Despite these encouraging results, we have resisted referring to GHWC as being ‘attractive’ to P. calceolariae because presently we have no clear understanding of the mechanism(s) that sees the mealybug settle on GHWC in apparent preference to all other ground cover host plants sampled during the study. Hence, in lieu of ‘attractive,’ we instead refer to mealybug ‘colonization’ of GHWC. This distinction is important because one of the defining characteristics of a good trap plant is that it is ‘attractive’ to a target insect pest (Holden et al. 2012). We are undertaking further research to understand what might be influencing P. calceolariae colonization of, and possibly its attraction to, GHWC and other potential host plants.

Reductions in the percentage of GHWC cover recorded in the last two years of the study (vintages 2020 and 2021) did not result in any detectable increase in P. calceolariae infestations on grapevine leaves in GHWC plots relative to the control. We suggest that if any of the colonization and/or retention qualities of GHWC in respect of P. calceolariae were compromised by a decline in its cover, we would have observed increased mealybug movement into the grapevine and with it, increased transmission of GLRaV-3. The configuration of trap plant establishment within and/or around the crop, and the area dedicated to it, is debated, with a range of 1–10% cover often proposed (Hokkanen 1991; Holden et al. 2012). In the current study, our initial allocation to GHWC was 12.5% cover per plot, but the fact that a lower percentage of GHWC cover did not negatively influence P. calceolariae colonization of the grapevine and increase virus transmission, suggests scope to re-evaluate the intensity of GHWC planting and/or spatial design parameters. Notably, the planting intensity may have implications for the extent to which integrating trap plantings influences crop performance (Hokkanen 1991). In this regard, the present study also measured a range of grapevine canopy, yield, and fruit quality parameters in GHWC plots and compared them with the control; the results of that aspect of the research will be reported separately.

When planning this study, we predicted GHWC would separate P. calceolariae from the grapevine to the extent it would reduce vector-mediated GLRaV-3 transmission relative to grapevines in the control. Instead of this outcome, virus incidence remained consistent between treatments. Despite good evidence of P. calceolariae readily colonizing GHWC, and being retained on it, it seems the small, residual vector population detected in the grapevines continued to transmit GLRaV-3 to grapevines at between 0.7 and 1% annually. Until relatively recently, attaining and then sustaining GLRaV-3 incidence at less than 1% annually was the explicit target in many New Zealand vineyards (Andrew et al. 2015; Bell et al. 2018), but for the owner of the present study vineyard, the target was c. 0.2% annually (c. five grapevine infections per ha). It remains unclear if the present study could have achieved this enhanced outcome had monitoring continued beyond the 5-year study duration. What is clear is that while the incidence of GLRaV-3 did not increase with the addition of GHWC (relative to the control), we should re-evaluate if and how a potential trap plant system (with or without GHWC) might help a virus elimination objective. This may be achieved either using the trap plant system on its own or when accompanied by other actions.

Unlike previous trap plant studies, we did not adopt any supplementary activity to manage mealybug populations resident on GHWC. Earlier studies identified as successful, but which targeted different insect groups, either treated the trap crop with insecticides or vacuumed pest insects from the trap crop (Shelton and Badenes-Perez 2006; Swezey et al. 2007; Cavanagh et al. 2009). Failures identified may have reflected the inadequacy of the trap plant selected to retain the target pest (Holden et al. 2012). While we found no evidence of P. calceolariae movement from GHWC to the grapevine canopy, the potential for it to have occurred during the study cannot be discounted. Thus, a future trap plant study using GHWC (or other candidate species) may want to consider applying a mealybug insecticide (after considering pesticide use regulations and the sector regulator, Sustainable Winegrowing New Zealand). Any product used in this manner should be compatible with integrated pest management and should not detract from mealybug parasitoids sustained by flowering plants in the vineyard ecosystem (Irvin et al. 2016; Chhagan et al. 2024).

When planning this study, we recognized that increased numbers of mealybugs on GHWC immediately below the grapevine may increase the risk of vector-mediated virus transmission. While this outcome was not observed during the study, previous research showed that when P. calceolariae resided on GHWC for up to 20 days, it was no longer viruliferous (McGreal et al. 2021). This outcome was supported by results from the present glasshouse experiment, where none of the 256 clover plants tested positive for GLRaV-3 across two separate experiments. From these results, we infer it is unlikely that GHWC is a reservoir of GLRaV-3 in the field; a new study underway will further test this result under vineyard conditions. Currently, clover is not known to host other viruses likely to be of significance to grapevine; however, relevant research is scarce (Pearson et al. 2006; Guy 2014; Guy et al. 2022).

Notably, for the first time, our glasshouse study demonstrated GLRaV-3 transmission by P. calceolariae from grapevine to N. benthamiana. This result parallels the previously reported transmission of GLRaV-3 to N. benthamiana using a different mealybug species (Planococcus ficus) (Prator et al. 2017). In both studies, uneven distribution of GLRaV-3 was noted within infected N. benthamiana plants. The present study utilized different transmission methods to those of Prator et al. (2017). Insect vectors of GLRaV-3 are widely divergent across grape-growing regions around the world, so the lessons from these variable methodologies will help inform future transmission experiments using model organisms such as N. benthamiana for GLRaV-3 research (Douglas and Krüger 2008; Tsai et al. 2011; Krüger et al. 2015). Critical to these studies will be consideration of vector species feeding behavior and host preference, which among a range of variables, are likely to influence virus transmission success (Tsai et al. 2010; Daane et al. 2012; Zhou et al. 2018).

In conclusion, we demonstrated that P. calceolariae readily colonized GHWC and was retained by it. Further, reduced GHWC ground coverage during the 5-year study did not result in a detectable increase in mealybugs resident on grapevines. However, despite these positive results, we found no detectable difference in GLRaV-3 incidence between clover-treated and control plots, but we recognize the strong association between inter-annual variability in virus transmission and variables like vector abundance and pathogen supply (Cooper et al. 2018). Notably, P. calceolariae did not transmit GLRaV-3 to the GHWC and four other clover species evaluated. Presently, successful management of GLRaV-3 in New Zealand relies on growers adopting multiple tactics that includes planting grapevines screened for the virus, targeting insect vectors, and the removal of virus-infected grapevines (Bell et al. 2018). Further research should evaluate the influence of other broadleaf ground cover plants on the GLRaV-3 transmission cycle and whether enhanced flowering plant biodiversity can reduce the influence of the insect vectors of this virus, either as trap plants and/or through improved biological control outcomes (Chooi et al. 2024).

Author contributions

RMM, KMC, VAB, and MS conceived and designed the research. RG, MS, and VD conducted the glasshouse trials. VAB and TT conducted the vineyard field trials. MS, CAP, RPA, RG, KMC, and VAB developed methodologies used. RG, KMC, and DC conducted the molecular analysis. DH analyzed the vineyard field trial data. RG and VAB wrote the manuscript. VAB secured funding for the vineyard field trial; KMC, VAB, and RMM secured funding for the glasshouse trial. All authors read, reviewed, and approved the manuscript.