The Control of the American Leafhopper Erasmoneura vulnerata (Fitch) in European Vineyards: Impact of Synthetic and Natural Insecticides

Simple Summary Erasmoneura vulnerata, a Nearctic leafhopper occurring on grapevine which is rarely damaging in North America, has become a new pest in European vineyards. Winegrowers are worried because of severe leaf symptoms potentially associated with yield losses and the nuisance posed when large numbers of adults occur at harvest time. Outbreaks were detected in conventional vineyards despite the use of broad-spectrum insecticides as well as in organic vineyards treated with pyrethrins. Therefore, the identification of effective control tools is required. Studies on E. vulnerata phenology have found that the second generation produces the largest population densities. We planned field trials to establish the most effective insecticides to be applied in conventional and organic vineyards. The most effective synthetic insecticides were acetamiprid, flupyradifurone and lambda-cyhalothrin, while the most effective natural product was kaolin. Abstract The American leafhopper Erasmoneura vulnerata, detected in Europe in the early 2000s, has recently become a pest in North-Italian vineyards. Infestations were recorded in organic and conventional vineyards despite the application of insecticides targeting other pests. Erasmoneura vulnerata completes three generations per year, and the second generation is frequently associated with large populations. The selection of appropriate active ingredients and the timing of their application is crucial for effective pest control. Field trials were carried out in Northeastern Italy, using a randomized design, to evaluate the impact of insecticides applied against other grapevine leafhoppers on E. vulnerata populations. The beginning of the second generation was selected as the best time for insecticide application. For natural products, two applications were planned. Among the selected insecticides, the most effective were acetamiprid, flupyradifurone and lambda-cyhalothrin. Regarding natural products, the most effective was kaolin which could represent an alternative to pyrethrins in organic vineyards. The identification of pest threshold levels and the evaluation of side effects of the most effective insecticides on key natural enemies occurring in vineyards are required.


Introduction
The most important leafhoppers in European vineyards are Empoasca vitis (Göthe) and Scaphoideus titanus Ball. Empoasca vitis has been considered a pest in France, Italy, Switzerland, and other countries [1][2][3][4][5][6][7]. Traditionally, its control has been achieved using insecticides, sometimes specific (e.g., pyrethroids in France) or aimed at the control of berry moths and leafhoppers (e.g., organophosphates and chitin-inhibitors in Italy and Switzerland) [8][9][10]. In the 1990s, organophosphates' effectiveness in controlling E. vitis declined, probably because of the selection of strains resistant to pesticides [11]. At the same time, research showed that E. vitis populations are limited by a number of natural enemies, namely the Hymenoptera Mymaridae (e.g., [12][13][14][15][16]). These findings, improved knowledge on cultivar susceptibility, and the adoption of action thresholds has reduced the attention towards E. vitis.
Scaphoideus titanus is the main vector of phytoplasma strains of the elm yellows group (16SrV) involved in the Flavescence dorée, a destructive disease of European vineyards [17][18][19]. In the 1990s, Flavescence dorée phytoplasma was declared a quarantine pest by the EU and control measures were made mandatory in France and Italy. Although chemical control is considered crucial in this framework, issues with Flavescence dorée spread [20][21][22]. In contrast to E. vitis, the efficacy of organophosphates towards S. titanus has remained satisfactory [11] and thus, resistance is not a concern for this pest. Problems with Flavescence dorée are serious in organic vineyards where growers can use only natural products (e.g., pyrethrins) characterized by limited activity and persistence [23,24]. Recently, new compounds replaced organophosphates in most viticultural areas and some of them (e.g., neonicotinoids) were very effective against leafhoppers [25][26][27]. However, in the last two years, several active ingredients have been banned in Europe and some of the remaining insecticides showed lower effectiveness against leafhoppers occurring in vineyards. The selection of active ingredients has become more important than in the past and chemical control must be integrated with agronomic and cultural measures to obtain adequate control of grapevine leafhoppers [20,22,28].
Although earlier records considered this species very harmful in North America, recent findings ranked it as a minor pest in leafhopper communities [31,32]. Initially E. vulnerata was localized in unsprayed vineyards in Northeastern Italy, then it spread to Northwestern Italy, Slovenia and Switzerland [33][34][35]. Currently, outbreaks involve both conventional and organic vineyards located in Northeastern Italy, particularly in the Veneto region [36]. Winegrowers are worried because of severe symptoms (leaf discoloration and leaf fall) potentially associated with yield losses and the nuisance to grape pickers when large numbers of adults are active at harvest time. Issues with E. vulnerata have been detected, although insecticides were applied against S. titanus. Investigations on E. vulnerata biology, ecology and behavior have been planned to implement effective control measures. This pest can complete three generations per year. Overwintered adults can damage shoots at sprouting, but the first nymphal generation is usually not harmful. The second generation is associated with the highest population densities, while the third is sometimes a problem [36]. We conducted field trials to evaluate the impact of insecticides used against other leafhoppers on E. vulnerata second generations. The results obtained in these trials are reported here.

Materials and Methods
The effects of a number of insecticides on E. vulnerata populations were evaluated in three conventional vineyards located in Vicenza and Verona provinces (Veneto region, Northeastern Italy) during the 2017, 2018 and 2019 growing seasons. In 2017, trials were carried out in a vineyard located in the Vicenza plain (Lonigo, cv. Garganega, Sylvoz training system, planting space 2.70 m × 1.40 m). In 2018, a hilly vineyard (Monteforte d'Alpone, cv. Trebbiano, Guyot training system, planting space 2.30 m × 0.9 m) located in the Verona province was selected for trials. The vineyard selected in 2019 (Colognola ai Colli, cv. Garganega, pergola veronese training system, planting space 3.70 m × 0.90 m) was also located in the Verona province. In these vineyards, the occurrence of E. vulnerata had been reported in the season preceding the study. Insecticides commonly applied in vineyards (active ingredients authorized in the EU and products authorized in Italy) and other products (e.g., kaolin) potentially useful for leafhoppers control were selected for trials (Table 1). An untreated control was included in each trial. Trials were carried out according to a completely randomized design where each treatment comprised four replicates of 8-10 vines. Insecticides were applied (1 or 2 applications depending on label instructions) against the second generation of E. vulnerata (Table 1). Sampling was conducted before and after (3, 7, 10, 14 and 21 days) insecticide applications. A total of 40 leaves per treatment (10 leaves per replicate) were removed and transferred to the laboratory where leafhoppers were identified to species and stage (for E. vulnerata: I-II instar nymphs, III-V instar nymphs, adults) levels under a dissecting microscope.
Data were analyzed using a repeated measures linear mixed model with the MIXED procedure of SAS ® (ver. 9.3; SAS Institute Inc., Cary, NC, USA). Data obtained in each field trial were analyzed separately. In all models, the number of nymphs per leaf was considered as a response variable with repeated measures made at different times. Insecticide, time of sampling, and their interaction were considered as sources of variation in the model and tested using an F test (α = 0.05). Multiple comparisons of the abundance of E. vulnerata on different treatments were performed using t-test (α = 0.05) on the least-square means. The degrees of freedom were estimated with Kenward-Roger method, which can calculate non-integer values for error terms. Prior to the analysis, data were checked for model assumptions. The model was run on data transformed to log (n + 1), while untransformed data are shown in the figures. The SLICE option of the LSMEANS statement was used to test treatment effect variation during observation periods.

Discussion
Among the products tested in this study, those based on acetamiprid (IRAC Group 4A) were the most effective in controlling E. vulnerata populations in two out of three trials. In 2019, its impact was slightly lower than that of flupyradifurone (IRAC Group 4D), a novel insecticide belonging to the Butenolides and recommended against sucking pests. Its effectiveness against E. vulnerata is consistent with those reported in the control of Erythroneura elegantula Osborn and E. ziczac Walsh in North America [37]. A single application of these two insecticides, at the beginning of the second generation, main-

Discussion
Among the products tested in this study, those based on acetamiprid (IRAC Group 4A) were the most effective in controlling E. vulnerata populations in two out of three trials. In 2019, its impact was slightly lower than that of flupyradifurone (IRAC Group 4D), a novel insecticide belonging to the Butenolides and recommended against sucking pests. Its effectiveness against E. vulnerata is consistent with those reported in the control of Erythroneura elegantula Osborn and E. ziczac Walsh in North America [37]. A single application of these two insecticides, at the beginning of the second generation, main-

Discussion
Among the products tested in this study, those based on acetamiprid (IRAC Group 4A) were the most effective in controlling E. vulnerata populations in two out of three trials. In 2019, its impact was slightly lower than that of flupyradifurone (IRAC Group 4D), a novel insecticide belonging to the Butenolides and recommended against sucking pests. Its effectiveness against E. vulnerata is consistent with those reported in the control of Erythroneura elegantula Osborn and E. ziczac Walsh in North America [37]. A single application of these two insecticides, at the beginning of the second generation, maintained E. vulnerata population densities at low levels for some weeks. Another neonicotinoid (IRAC Group 4A), i.e., thiamethoxam, proved to be very effective against E. vulnerata in 2017. It was largely employed against sucking insects, e.g., E. vitis and S. titanus [25][26][27]38]. Thiamethoxam was banned from the EU because of the adverse impact on pollinators [39,40] and thus was excluded in 2018 and 2019 trials. Buprofezin (IRAC Group 16) was tested in 2017, showing good effectiveness, but it was also banned from the EU and thus excluded from further evaluations. Lambda-cyhalothrin (IRAC 3A Group) showed an effectiveness slightly lower than that of acetamiprid, but leafhopper populations seemed to recover faster in the respective plots. Results obtained using the organophosphate chlorpyrifos-methyl (IRAC Group 1B) are of particular interest as this insecticide has been widely used against grapevine leafhoppers in Italy [26,38,41]. It was effective against E. vulnerata in 2017 but not in 2018. Different vineyards were selected for these trials, and thus a variation in the susceptibility of leafhopper populations could explain the different results we obtained. It should be mentioned that the first outbreaks of E. vulnerata in Northern Italy were detected in vineyards frequently treated with chlorpyrifos-methyl. This observation suggests that resistance to insecticides could be a key factor explaining the unexpected outbreaks of this species [30]. It should be mentioned that the closely related chlorpyrifos-ethyl has been widely used in European vineyards against S. titanus, berry moths, and scales [20] and resistance in E. vitis was strongly suspected [11]. Chlorpyrifos-methyl and chlorpyrifos-ethyl have been banned from the UE in 2020 because of concerns for human health and thus study on leafhopper resistance to these insecticides was discontinued.
With regard to natural products, pyrethrin-based insecticides (IRAC Group 3A) are widely used against S. titanus and other leafhoppers in organic vineyards in France, Italy and Switzerland [20]. In the current study, the application of pyrethrins gave contrasting results in controlling E. vulnerata. In 2017, pyrethrins significantly reduced nymph densities compared with the control, in 2018 they showed some effectiveness on older nymphs only, while in 2019 they showed unsatisfactory results in controlling leafhoppers. Mineral oils were effective in 2018 and it was expected they could increase the impact of pyrethrins when mixed; this assumption was verified in 2017 but not in 2019. Potassium salts were slightly effective in 2017 but were associated with poor results in 2018. Finally, kaolin (an inert white clay not classified as an insecticide) significantly reduced E. vulnerata densities in 2017 and 2018. It was more effective than chlorpyrifos-methyl, pyrethrins and potassium salts against early nymphs in 2018 trial. Kaolin was active on some grapevine pests [42], particularly towards E. vitis and Z. rhamni [43]. Inhibition of feeding was the main mode of action through which kaolin affected leafhopper nymph populations. Timing in applying kaolin against E. vulnerata and mechanisms underlining its mode of action are worthy of study.

Conclusions
Recent outbreaks of E. vulnerata in Europe have caused concern for winegrowers and suggest the value of testing the impact of a number of conventional or natural insecticides on this species in vineyards. Among the insecticides tested, the most effective were acetamiprid, flupyradifurone and lambda-cyhalothrin. A single application of these compounds reduced leafhopper population densities to low levels for some weeks. Regarding natural products, the most effective was kaolin, which could represent an alternative to pyrethrins in organic vineyards and a complementary tool in conventional vineyards. The use of insecticides should be selected at the correct time and once threshold levels are exceeded. Our knowledge of the biology of E. vulnerata allowed us to identify the best timing for insecticide application (i.e., at the beginning of the second generation) but threshold levels have not been defined yet [44]. Finally, insecticides' side effects on beneficials occurring in European vineyards should be determined to optimize integrated pest management (IPM) strategies [45,46]. Information on the side effects of many of these insecticides on predatory mites belonging to the Phytoseiidae family is available [47,48] but knowledge is limited for other important beneficials.
Author Contributions: Conceptualization, C.D. and E.M.; methodology, C.D. and E.M.; formal analysis, P.T. and A.P.; field investigations, F.R., P.G. and D.R.; writing-original draft preparation, P.T. and C.D.; writing-review and editing, P.T., C.D. and A.P. All authors have given approval to the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available from the corresponding author, upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.