Entomopathogenic nematodes and fungi to control Hyalesthes obsoletus (Hemiptera: Auchenorrhyncha: Cixiidae)

Hyalesthes obsoletus Signoret (Hemiptera: Auchenorrhyncha: Cixiidae) is a univoltine, polyphagous planthopper that completes its life cycle, including the subterranean nymph cryptic stage, on herbaceous weeds. In vineyards, it can transmit ‘Candidatus Phytoplasma solani’, an obligate parasitic bacterium associated with bois noir (BN) disease of grapevine, from its host plants to grapevine when occasionally feeding on the latter. The main disease management strategies are based on vector(s) control. Insecticide treatments on grapevine canopy are completely inefficient on H. obsoletus, due to its life cycle. Consequently, control of this planthopper focuses on the nymphs living on the roots of their host plants. Such practices, based on herbicide application and/or weed management, can reduce vector density in the vineyard but can impact the environment or may not be applicable, highlighting the necessity for alternative strategies. In this study, the efficacy of entomopathogenic nematodes (EPNs; Steinernema carpocapsae, S. feltiae, Heterorhabditis bacteriophora) and fungi (EPFs; Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea, Lecanicillium muscarium) against H. obsoletus nymphs (EPNs) and adults (EPNs and EPFs) was assessed under laboratory and greenhouse conditions. The majority of examined EPNs and EPFs were able to kill H. obsoletus exhibiting a range of effectiveness. S. carpocapsae (among EPNs) and I. fumosorosea (among EPFs) were found to be the most effective biocontrol agents in all trials carried out. Advantages and limitations of such promising biocontrol agents were discussed. Ecological competency and conditions that can impede or enhance the EPNs and EPFs performance should be investigated to optimize their performance under field conditions.


Introduction
Hyalesthes obsoletus Signoret (Hemiptera: Auchenorrhyncha: Cixiidae) is a polyphagous planthopper able to transmit 'Candidatus Phytoplasma solani' (CaPsol) to plants (Quaglino et al. 2013), including Vitis vinifera L. (Maixner 1994). CaPsol, a plant obligate parasitic bacterium, is associated with bois noir (BN), a disease belonging to the complex of grapevine yellows which had high economic impact on viticulture in Europe in the last decades (Angelini et al. 2018). Although alternative insect vectors of CaPsol to grapevine were recently reported (Cvrković et al. 2014;Quaglino et al. 2019), BN epidemiology is principally determined by the life cycle of its main vector H. obsoletus .
H. obsoletus is a palaearctic, univoltine species that, in Europe, completes its life cycle mainly on bindweed (Convolvulus arvensis L.) and nettle (Urtica dioica L.) (Langer and Maixner 2004) but also on other host plants (Kosovac et al. 2019;Moussa et al. 2019). In summertime, females produce eggs on the root collar of host plants and after egg hatching the nymphs migrate into the soil to the roots from which they can acquire CaPsol. After a latency period, H. obsoletus becomes able to transmit CaPsol to plants for the duration of its life. Overwintering occurs as second-third instar nymphs in the soil. Fourth and fifth instar nymphs migrate to the soil surface in early spring. Adults emerge from end of May till end of June and they fly from beginning of July to end of August, based on climate, host plant, and region (Cargnus et al. 2012;Maixner and Johannesen 2014;Alma et al. 2015). During their flights, H. obsoletus adults can occasionally feed on grapevine and, if infected, transmit CaPsol. However, due to their limited feeding activity on grapevine and the short lifespan of the adult stage, they cannot transmit CaPsol from vine to vine. Grapevine is therefore a dead-end host for the pathogen (Bressan et al. 2007).
Since no effective control measures directly targeting phytoplasmas are available, the main strategies to manage the spreading of phytoplasma-associated diseases are based on preventive measures, including the control of vectors before their emergence from the ground . Due to its cryptic life cycle and polyphagous feeding habit, insecticide treatments on grapevine canopy are completely inefficient against H. obsoletus. Thus, strategies for its control focus on depriving the nymphs of their feeding substrate, the host plant roots. Before adult emergence, bindweed and nettle can be suppressed by planting of ground covering rosette plants, repeated mowing or weeding (Maixner and Mori 2013;Mori et al. 2014a). Since H. obsoletus presence depends on the distribution of its natural plant hosts both within and outside the vineyards, such strategies are limited by restrictions on the use of herbicides in uncultivated areas, as well as mechanical weeding on ditches and embankments because of soil landslide. In Israel, H. obsoletus populations within vineyards are successfully limited by a push and pull strategy using chaste tree (Vitex agnus-castus L.) (Sharon et al. 2015), but such a strategy cannot be employed in Europe where this plant hosts both H. obsoletus and CaPsol .
Considering these limitations, a promising approach to control the vector populations could be based on the utilization of biocontrol agents such as entomopathogenic nematodes (EPNs) and fungi (EPFs). In particular, several Steinernema and Heterorhabditis EPNs have been reported as effective biocontrol agents against a broad range of insects with a cryptic life cycle like H. obsoletus (Grewal et al. 2005;Lacey and Georgis 2012;Guerrero and Pardey 2019). EPNs efficacy depends on their survival for a long time without their host targets in the soil, and their ability to find the hosts by ambush (i.e., Steinernema carpocapsae) or cruising (i.e., Heterorhabditis bacteriophora) strategy (Kaya et al. 1993;Grewal et al. 1994). Concerning EPFs, they are reported as important antagonists of soil-dwelling insect pests adapted to live in agricultural soils, such as the grapevine phylloxera in vineyards (Kirchmair et al. 2004). Interestingly, the EPF Metharizium anisopliae showed a great efficacy against H. obsoletus adults under laboratory conditions (Langer et al. 2005). In this study, the efficacy of different EPNs and EPFs against H. obsoletus nymphs and adults under laboratory and greenhouse conditions were assessed to develop effective and innovative approaches to control the main vector of CaPsol.

Hyalesthes obsoletus collection
Nymphs of H. obsoletus were collected on two dates immediately before the different experiments. The collection for laboratory bioassay was done in the middle of May 2019. Nymphs were obtained from pots of stinging nettle plants at the rearing facility in Julius Kühn-Institute (JKI) in Siebeldingen, Germany. Collection for the greenhouse trials was performed in late May 2019 from roots of nettle plants growing at the borders of a highly BN-affected vineyard in Mosel area (49.9198°N, 7.0627°E), Germany. Nymphs were placed in falcon tubes filled with the same soil in which they were collected and sent to the laboratory for trials. Nymphs identity was confirmed based on the taxonomic key by Stöckmann et al. (2013).
Adults of H. obsoletus were collected from bindweed and stinging nettle plants using a sweep net and mouth aspirator from mid-June till the end of July 2019, based on the trial requirements, in the vicinity of highly BN-affected vineyards in Mosel area (49. 9198°N, 7.0627°E, and 49.1928°N, 8.0830°E), Germany. Collected adults were placed in collapsible insect mesh cages with shoots of nettle plants as food source, transferred to the laboratory, and identified by taxonomic key (Bertin et al. 2010). H. obsoletus adults were subject to immediate use in bioassays and greenhouse efficacy trials.

Entomopathogenic nematodes
Three EPNs were applied in both bioassays and greenhouse trials against H. obsoletus nymphs and adults. In detail, the utilized EPNs (Steinernema carpocapsae, S. feltiae, Heterorhabditis bacteriophora, and a combination (1:1) of S. feltiae and H. bacteriophora) were purchased from E-nemaÒ Company (Schwentinental, Germany) (Table 1) and maintained at 4°C. Immediately before using, each EPN (supplied in powder) was suspended in tap water and tested for its viability by counting the infective juveniles (IJs) under stereomicroscope. IJs without a response to stimulators were considered dead (Lacey 1997). All EPNs showed a viability higher than 95%. Before the application, each EPN was serially diluted in tap water to reach the required concentration for bioassay (200 IJs ml -1 ) and greenhouse trials (400 IJs ml -1 ) (Guerrero and Pardey 2019).

Entomopathogenic fungi
Three distinct EPF isolates of Beauveria bassiana, Metarhizium anisopliae, and Isaria fumosoroseus (provided by Dr. Dietrich Stephan -Institute for Biological Control, JKI, Darmstadt, Germany), were applied in preliminary screening against H. obsoletus adults. In addition to these isolates, one commercial isolate of M. anisopliae and one of Lecanicillium muscarium were purchased from Koppert Biological Systems Company (Verona, Italy) and applied in greenhouse trials against H. obsoletus adults (Table 1). EPFs were cultivated in Petri dishes on malt extract peptone agar (30 g of malt extract, 3 g of peptone, 15 g of agar in 1 l of distilled water) previously autoclaved at 121°C for 10 min. After inoculation, EPFs were grown for five days at room temperature (22°C) and for six days at 4°C. For each EPF isolate, fungal conidia were recovered from the mycelium, suspended in distilled water and counted (three times per isolate) at 4009 magnification through a haemocytometer. For each EPF isolate, stock solutions of conidial suspension were prepared at the concentrations of 10 6 , 10 8 and 10 10 conidia ml -1 and stored at 4°C until use.

Entomopathogenic nematodes: laboratory bioassays
Bioassay to evaluate the efficacy of EPNs against H. obsoletus nymphs was conducted in 12-well cell culture plates with lids. According to the protocols of Kaya and Stock (1997) and Glazer and Lewis (2000), in each well, filled with 1 g of autoclaved sand, 1 ml of EPN suspension (200 IJs ml -1 ) was applied and one nymph was placed. Control plates were treated with distilled water before placing the nymphs. Bioassay to evaluate the efficacy of EPNs against H. obsoletus adults was conducted in plastic Petri dishes (9 cm diameter) padded with filter paper. Following Glazer and Lewis (2000) procedures, in each dish, fresh shoots of stinging nettle (5 cm) were placed as a food source together with 12 adults of H. obsoletus, and 1 ml of EPN suspension (200 IJs ml -1 ) was applied using a hand-held sprayer. Control dishes were sprayed with distilled water before placing the adults. The edges of Petri dishes were dried with tissue paper to prevent the adults sticking to water droplets. Three replicates of each plate (nymphs)/Petri dish (adults) were made per EPN as well as the control. All plates/ dishes were placed in a controlled chamber (25°C, 72% RH, L:D 16:8) for six days. Mortality readings were taken daily for six consecutive days. To confirm that insect mortality was caused by the activity of the EPNs, the presence of EPN was evaluated by dissecting nymphs and adult insect bodies under a 409 magnifying stereomicroscope. This was done after rinsing single dead nymphs and adults in a conical flask filled with 20 ml of distilled water, for removing nematodes from their surface. Nymphs and adults were then placed on a moist filter paper padded plastic Petri dish and maintained at 25°C for three days according to Glazer and Lewis (2000).

Entomopathogenic nematodes: greenhouse trials
To evaluate the efficacy of EPNs against H. obsoletus nymphs and adults, greenhouse trials were conducted on potted stinging nettle (Urtica dioca L.) and faba bean (Vicia faba L.) plants, respectively. The nettle plants (diameter about 0.1 m and height approximately 0.4 m) were grown in 3.0 l pots, while the bean plants (diameter about 0.1 m and height approximately 0.25 m) in 1.0 l pots and placed in transparent plastic ventilated cages. All plants were in good vegetative condition and did not show symptoms of biotic and abiotic stresses. In each pot with nettle, 5 ml of EPN suspension (400 IJs ml -1 ) was applied to the soil using a hand-held sprayer, and a total of 20 nymphs were placed after treatment. The control potted plants were treated with 5 ml of distilled water before placing the nymphs. On each caged bean plant, 10 ml of EPN suspension (400 IJs ml -1 , with 0.02% Tween 80) was applied using a hand-held sprayer, and 15 H. obsoletus adults were released. The control caged plants were treated with 10 ml of distilled water with 0.02% Tween 80 before releasing insect adults. Three replicates, arranged in randomized blocks, were Entomopathogenic fungi: laboratory bioassays Initially, the nine EPF isolates provided by the Institute for Biological Control JKI (Table 1) were screened for their entomopathogenic activity against H. obsoletus adults in plastic Petri dishes (8.5 cm diameter), filled to a depth of 5 mm with a mixture of plaster of Paris and charcoal (10:1) and moistened with distilled water (Green 1964;Langer et al. 2005).
In each Petri dish, fresh bindweed (Convolvulus arvensis L.) shoots (5 cm) were placed as a food source, 1 ml of EPF conidia suspension (10 8 conidia ml -1 ) was applied using a hand-held sprayer, and 12 adults were released. The control dishes were treated with distilled water before the release of insects. Three replicates were conducted per EPF isolate as well as the control. Petri dishes were kept in a controlled chamber (25°C, 72% RH, L:D 16:8). Mortality readings were taken after six days. Single dead insects from each EPF isolate were placed in malt extract peptone agar plates kept for three days at room temperature (22°C) and for five days at 4°C. Mortality due to EPF infection was confirmed through the observation of EPF mycelium growth on the insect body. Subsequently, EPF isolates, found active against H. obsoletus in the initial screening test, were employed in a bioassay applying conidial suspension at different concentrations (10 6 , 10 8 , 10 10 conidia ml -1 ). The bioassays were conducted in plastic Petri dishes as described above for the initial screening test. Three replicates were conducted per concentration per EPF isolate as well as the control. Petri dishes were kept in controlled chamber (25°C, 72% RH, L:D 16:8) for six days. Mortality readings were taken daily for six consecutive days. Single dead insects from each bioassay were placed in malt extract peptone agar plates and checked to confirm EPF-related mortality as described above.

Statistical analysis
In the bioassays, the median lethal time (LT 50 ) for EPNs against nymphs and adults was calculated from daily mortality data. The median lethal concentration (LC 50 ) of EPFs against adults was log 10 -transformed and calculated from the data obtained on the fourth day post-inoculation (dpi). LT 50 and LC 50 results with their fiducial confidence limits were obtained by R statistical package ''ecotox'' (Hlina 2020) based on the probit analysis (Finney 1971;Wheeler et al. 2006;Robertson et al. 2007). In the screening (EPFs) and greenhouse trials (EPNs and EPFs), mortality data of nymphs and adults were tested for normality and equality of variance with the Shapiro-Wilk test followed by Levene's test. Greenhouse trials data were subjected to ANOVA followed by Tukey's honestly significant difference (HSD) post-hoc test for multiple comparisons. Graphical representations of the statistical analysis results were produced using R statistical package ''ggplot2'' (Wickham et al. 2020). All the analyses were done using R (version 3.6.2) (R Core Team 2019).

Biocontrol efficacy of entomopathogenic nematodes against H. obsoletus nymphs and adults
In the bioassay, S. carpocapsae was the most effective with a LT 50 value of 3.24 and 3.69 days postinoculation (dpi) for H. obsoletus nymphs and adults, respectively (Fig. 1a, b). S. feltiae was the second most effective with a LT 50 of 3.49 and 4.05 dpi for nymphs and adults, respectively (Fig. 1c, d). The mixture of H. bacteriophora and S. feltiae showed a LT 50 of 3.65 and 4.61 dpi for nymphs and adults, respectively (Fig. 1e Table S4).

Discussion
Due to the complex life cycle of Hyalesthes obsoletus, most strategies to control its populations in the vineyard agro-ecosystem are not effective or can impact the environment (Maixner and Mori 2013). In the last years, biocontrol has been proposed and frequently utilized as sustainable strategy to control plant pathogen insect vectors (Kumar 2016;Abdel-Razek et al. 2017;Abd El-Ghany et al. 2018). Entomopathogenic nematodes (EPNs) and fungi (EPFs) have been largely employed as effective The results obtained in this study demonstrated that all the examined EPNs are able to kill H. obsoletus nymphs and adults and that the EPFs, except Beauveria bassiana strain 1124 and Metharizium anisoploae strain 1428, are able to control the adults in both laboratory bioassays and greenhouse trials, exhibiting a range of effectiveness related to their virulence against the target insect. In all conducted trials, Steinernema carpocapsae and Isaria fumosorosea were found to be the most effective biocontrol agents of H. obsoletus among the examined EPNs and EPFs, respectively.
Concerning Steinernema spp., our findings are in agreement with Le Vieux and Malan (2013) showing that, in laboratory bioassay performed against the vine mealybug Planococcus ficus, the EPN Steinernema yirgalemense moved 15 cm vertically downward, and infected its insect target inducing a mortality of 95%. Another study demonstrated that the combination of S. yirgalemense with specific adjuvants increased its biocontrol activity against the vine mealy bug on grapevine leaves in both laboratory and semi-field conditions (Platt et al. 2019). Such evidence fortifies the possibility of applying Steinernema spp. in the open field against both subterranean forms and adults of H. obsoletus. Among tested EPNs, also Heterorhabditis bacteriophora showed a high efficacy in H. obsoletus biocontrol. Interestingly, this EPN was reported to be effective against the nymphs of Haplaxius crudus, the insect vector of 'Candidatus Phytoplasma palmae' associated with Palm Lethal Yellowing disease in Florida, USA (Guerrero and Pardey 2019), and of Aeneolamia spp., a putative vector of genetically distinct phytoplasmas (Pérez Miliàn et al. 2018). Moreover, H. bacteriophora strongly reduced the survival of the root-form of grapevine phylloxera (English-Loeb et al. 1999). This evidence highlighted that, in vineyard agroecosystems, treatments based on the application of H. bacteriophora could be effective against multiple insect pests. Based on all these evidences, it would be interesting to apply a combination of S. carpocapsae, found here as more effective against H. obsoletus nymphs and adults, and H. bacteriophora, reported in previous studies as the most effective EPN against phytoplasma insect vectors and grapevine insect pests with a cryptic life stage.
Concerning Isaria fumosorosea, found here as the most effective EPF against H. obsoletus, previous studies showed its biocontrol activity against various nymphal stages of the green leafhopper Empoasca decipiens Paoli under laboratory and greenhouse conditions (Tounou et al. 2003;Kodjo et al. 2011). Similar efficacy was found by treatments with Metarhizium anisopliae (strain Ma43) and Beauveria bassiana (strain Bba113) (Tounou et al. 2003;Kodjo et al. 2011). For all these EPFs, percentage of mortality and LC 50 values reported against E. decipiens were comparable to those observed in this study against H. obsoletus adults. Moreover, promising results obtained in the present study with two strains of M. anisopliae confirmed its entomopathogenic activity against H. obsoletus adults under laboratory conditions (Langer et al. 2005). Interestingly, Beauveria bassiana, two strains of which showed a great biocontrol activity against H. obsoletus adults in the present work, was found naturally infecting and causing visual symptoms on H. obsoletus adults in Georgia (Caucasus region) (Chkhaidze et al. 2017). Moreover, B. bassiana showed an efficacy in biocontrol of young stages and adults of Scaphoideus titanus Ball, the insect vector of flavescence dorée phytoplasma, in semi-field and field trials (Mori et al. 2014b). All these evidences underlined that B. bassiana can control both the insect vectors of phytoplasmas associated with the main grapevine yellows diseases. Thus, B. bassiana strains represent really promising EPFs for application in vineyards.
Effectiveness of EPNs and EPFs, as well as other living organisms used as biocontrol agents, depends on a range of climatic and environmental parameters allowing their liveliness and entomopathogenic activity. In particular, it is crucial that the target insect stage is present when climatic parameters are optimal for EPNs and/or EPFs (Lacey and Georgis 2012;Wang and Wang 2017). In the case of H. obsoletus, it is known that the duration of the cryptic (subterranean) phase of its life cycle, involving the nymph stages, is dependent on the degree day units that can be estimated based on forecasting models measuring the accumulated heat units (Maixner and Mori 2013). Such models allow understanding of the life cycle of the insect as well as narrowing the spraying window of products for plant protection, including EPNs and EPFs. In particular, the spraying window should prioritize two important aspects: (1) the ecological competency of EPNs as well as EPFs; (2) the proper timing for application against the different stages and instars of H. obsoletus. In Europe and the Mediterranean area, considering the life cycle of H. obsoletus and the environmental conditions suitable for EPNs and EPFs utilized in the present study, it should be recommended to apply EPNs and EPFs on H. obsoletus host plants in the open field from mid-September to October and/or in early spring to optimize the activity of each biocontrol agent and avoid resistance in the insect target populations. Moreover, given their ability to colonize the soil after their inoculation (Meyling and Eilenberg 2006;Denno et al. 2008), EPNs and EPFs could reduce the H. obsoletus population density for a long time. According to our result EPFs could be applied also with foliar application from end of May till end of June against newly hatched adults before grapevine infestation. Optimized application of entomopathogenic nematodes (on the soil) and fungi (on the plants) can increase the control of H. obsoletus nymphs and adults, respectively.
In conclusion, the majority of EPNs and EPFs utilized in the present study showed a considerable biocontrol activity against H. obsoletus nymphs and adults in laboratory bioassays and greenhouse trials. The ecological competency of both EPNs and EPFs, the conditions that can impede or enhance their performance, the barriers that can block infection from taking place on the target host, and the possible actions on non-target species should be carefully investigated for a better understanding of their potential performance under field conditions. methodology, resources, writing -review and editing. Giacomo Santoiemma: data curation, formal analysis, validation. Alessandro Passera: data curation, formal analysis, validation. Nicola Mori: conceptualization, project administration, resources, supervision, Writing -review and editing. Fabio Quaglino: conceptualization, project administration, supervision, writing -review and editing.
Funding Open Access funding provided by Università degli Studi di Verona, Italy.

Compliance with ethical standards
Conflict of interest The authors declare that there are no conflicts of interest associated with this publication.
Ethical approval This research did not involve any studies with human participants or animals (vertebrates) performed by any of the authors.
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Giacomo Santoiemma has a post-doc position at Department of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Italy. His research focuses on spatial ecology and environmental data analysis of insect pests and vectors.
Alessandro Passera has a post-doc position at Department of Agricultural and Environmental Sciences, University of Milan, Italy. His research interests are beneficial endophytic bacteria, biocontrol agents, and microbiota in relation to plant health. Nicola Mori is professor of general and applied entomology at Department of Biotechnology, University of Verona, Italy. His research focuses on biology and ecology of insect pests and phytoplasma vectors, and their control through sustainable strategies.
Fabio Quaglino is professor of plant pathology at Department of Agricultural and Environmental Sciences, University of Milan, Italy. His research focuses on taxonomy and ecology of phytoplasmas, and epidemiology and control of the associated diseases.