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 (Mori et al.
2013).
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 (Bianco et al.
2019). 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 (Moussa et al.
2019).
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.
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 biocontrol agents against insects with a cryptic life cycle, including phytoplasma vectors (Grewal et al.
2005; Lacey and Georgis
2012; Guerrero and Pardey
2019), making this approach promising also for the main vector of ‘
Candidatus Phytoplasma solani’ to grapevine,
H. obsoletus.
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.