Presence of Cicadomorpha in olive orchards of Greece with special reference to Xylella fastidiosa vectors

Insects of the infraorder Cicadomorpha cause serious economic impact on agriculture because of pathogens’ transmission. The vector‐borne bacterium Xylella fastidiosa, which is associated with olive quick decline syndrome, is exclusively transmitted by insects of this infraorder and more specific by sharpshooters and spittlebugs. Efficient control practices of the insect vectors are necessary for the control of the disease in cases of outbreaks. Therefore, the knowledge of the vectors' species as well as the robust understanding of their biology and ecology is of crucial importance. In the present study, extensive sampling was conducted in 28 olive orchards of Greece during the years 2017 and 2018 aiming to investigate which species of this infraorder are present, with special interest to those which are considered as vectors of the bacterium. During the surveys, 4,350 Cicadomorpha insects were collected. Forty‐two, five and one species of the families Cicadellidae, Aphrophoridae and Cercopidae, respectively, were identified. The results of the surveys revealed that the main potential vectors of X. fastidiosa in Greek olive orchards are the spittlebugs Philaenus spumarius and Neophilaenus campestris. Those species have wide distribution in Greece and are present for a long period during the year, and they inhabit both the ground vegetation and the olive trees' canopy. A representative portion of the captured spittlebugs was analysed with molecular methods, and the analysis confirmed the absence of X. fastidiosa in the tested individuals.

pathogens (Maramorosch & Harris, 1979;Nielson, 1968) is by far the most important economic impact of those insect species.
The most serious case of pathogen transmission in Europe from Cicadomorpha in recent years is the bacterium Xylella fastidiosa Wells (Xanthomonadales: Xanthomonadaceae). The bacterium was firstly reported in Europe in Apulia of south Italy causing the olive quick decline syndrome (OQDS) in olive trees showing severe symptoms of leaf scorch and dieback (Saponari et al., 2014). This first detection was followed by findings of several subspecies and strains of X. fastidiosa in France, Germany, Spain and Portugal (Cruaud et al., 2018;Denancé et al., 2017;EPPO, 2016EPPO, , 2019Olmo et al., 2017).
X. fastidiosa is exclusively transmitted by Cicadomorpha xylem sapfeeding insects. They have sucking mouthparts (mandibular and maxillary stylets) that allow them to reach the xylem of plants, from which they ingest sap (EFSA, 2013). The insect vectors of the bacterium are mainly sharpshooters (Cicadellidae, subfamily Cicadellinae) and spittlebugs (Aphrophoridae and Cercopidae; Redak et al., 2004).
Sharpshooters are the most important vectors of X. fastidiosa in the Americas, but only a few species are present in Europe. In contrast, a relatively high number of spittlebug species, which are less important vectors in America, occur in Europe (EFSA, 2015).
The chemical control of the bacterium is not possible, and the only way to slow the fast spread of disease is the use of resistant varieties, cultural and hygienic measures and chemical and biological vector control (Janse & Obradovic, 2010). Therefore, the knowledge of the vectors' species as well as the robust understanding of their biology and ecology is necessary for the development of efficient control management practices. However, the faunal and ecological investigations of Cicadomorpha in Greece are limited. Drosopoulos, Asche, and Hoch (1986) in a thorough review of Greek Cicadomorpha recorded approximately 420 species belonging to four families, while Thanou et al. (2018) reported another 17 species of the family Cicadellidae. In respect of the potential vectors of X. fastidiosa, the former authors stated that in Greece 19 species of spittlebugs (13 and six species of the families Aphrophoridae and Cercopidae, respectively) and three species of sharpshooters are present. However, those surveys were conducted in various habitats. Given the fact that X. fastidiosa pose a serious threat especially for olive cultivation in Greece, the present work aims to investigate which species of this infraorder are present in Greek olive orchards.
Special interest is given to the species which are considered as vectors of the bacterium. A representative number of those insects were tested by molecular methods to examine the possible presence of X. fastidiosa.

| Sampling areas
In order to determine the presence of insects belonging to the infraorder Cicadomorpha, samplings were conducted in 28 olive orchards in Greece during 2 successive years (2017 and 2018). The olive orchards were distributed in 15 regional units of central (Attica, Euboea, Phthiotis and Corinthia), west (Aetolia-Acarnania, Achaea, Arta, Cephalonia, Lefkada and Corfu), north (Chalkidiki and Kavala) and south (Messenia, Laconia and Chania) Greece ( Figure 1). In certain sampling sites, only one sampling was performed, while in others, multiple samplings were conducted. In total, 134 samplings were carried out in those orchards during the survey period. The olive groves sampled were not subjected to chemical treatments for the control of pests and diseases. Moreover, most of the orchards maintained their natural ground vegetation through most part of the year while in some others occasional grazing by sheep took place.
Although there was a diversity on the wild vegetation of the olive groves sampled, the most common plant species belonged to the families Poaceae (Avena sterilis, Hordeum spp.), Asteraceae (Crepis spp., Sonchus oleraceus) and Fabaceae (Medicago spp., Trifolium spp.).

| Sampling of insects
In each site, samplings for adults were conducted with an entomological sweep net (38 cm diameter). For every orchard, five samples were taken from the canopy of olive trees and another five from the natural vegetation beneath the canopy. Each sample from the canopy consisted of 12 sweeps performed on six olive plants. Each olive tree's canopy was swept twice with the net. A total of 30 trees were sampled per sampling site and date. Each sample on wild plants of the natural vegetation was conducted by 10 consecutive sweeps on the ground vegetation. Each sweep was performed by moving the capturing net in an arrow of 180 degrees. The overall content of the sweeping net was emptied in a plastic bag, properly labelled and sealed. All samples were frozen at −20°C, and then, targeted insects were separated in the laboratory and were conserved in ethanol 98% (EtOH) until identification. Samples were examined under a microscope for species identification which was based on appropriate keys and illustrations (Biedermann & Niedringhaus, 2009;Dmitriev, 2018;Holzinger, Kammerlander, & Nickel, 2003;Ossiannilsson, 1981Ossiannilsson, , 1983Wilson, Stewart, Biedermann, Nickel, & Niedringhaus, 2015).

| Molecular methods
One hundred and thirty (130) individuals belonging to six species of spittlebugs, collected in 11 regional units of Greece, were tested for the hypothesis of carrying the bacterium X. fastidiosa.

| RE SULTS
During the surveys in Greek olive orchards, 4,350 insects of the infraorder Cicadomorpha were collected. From those insects, 1,026 individuals were spittlebugs (Table S1). The identification of those insects revealed the presence of five species of the family Aphrophoridae and one species of the family Cercopidae. The most frequently observed spittlebugs were P. spumarius, which was identified in 13 olive orchards distributed in nine regional units, and N. campestris, which was observed in five olive orchards distributed in five regional units. P. signatus was recorded in four orchards distributed in three regional units, while L. coleoptrata was identified in four sampling areas. A. alni and C. sanguinolenta were both recorded in two orchards (Table 1).

Cicadellidae was the most affluent family of the infraorder
Cicadomorpha regarding the number of species and individuals in olive orchards. During the surveys, 3,315 insects from this family were collected and 42 species were identified (Table S2), pertaining to five subfamilies. Deltocephalinae was the most abundant subfamily being represented by 26 species, followed by Typhlocybinae, by eight species. Agallinae, Aphrodinae and Hecalinae were represented by four, three and one species, respectively. Lastly, nine adults of the family Cicadidae were collected and two species of the family were identified (Table 2). However, sweep netting is not the appropriate method for sampling cicadas, and for this reason, the results regarding this family are considered only indicative.
The molecular analysis with PCR assays using both sets of primers for the detection of X. fastidiosa revealed the absence of the bacterium in all 130 individuals tested. In addition, no PCR products were produced with negative control templates (NIC, NAC) while DNA fragments of the expected size were produced with positive control templates (PIC, PAC), for both sets of primers ( Figure 2).

| D ISCUSS I ON
According to the results of our surveys, P. spumarius was the most common species of spittlebugs. A wide distribution of this species was recorded as it was found in sampling areas in central, west, north and south Greece. In most of the olive orchards, the individuals of this species were captured either in spring or in autumn.
During summer months (June-September), there was an absence of this species both on ground vegetation and on the foliage of olive trees. Only in two olive groves, in Cephalonia and Laconia, adults of this species were observed during summer. However, even in those two cases only five and one individual, respectively, were captured.
It has been reported that adults of P. spumarius in Spain, Portugal and Corsica tend to migrate from the olive orchards in summer and return in autumn, after the first rains during the regrowth of the ground vegetation (Cruaud et al., 2018;Morente et al., 2018). Our results strongly indicate that this phenomenon is also happening in the Greek olive orchards. Thus, it is highly possible that the distribution of P. spumarius in Greece is wider than that recorded in the present work, as in most of the olive orchards where the species was not observed, samplings were conducted only during summer months.  . All the spittlebugs found on the Greek olive groves are xylem-fluid feeders and are considered as potential vectors of X. fastidiosa (EFSA, 2013(EFSA, , 2015Purcell, 1989).  (Rose, 1978;Webb, 1987).  (Cornara, Cavalieri, et al., 2017;Saponari et al., 2014) and in the Iberian Peninsula (Morente et al., 2018). Taking into account the results of all the aforementioned studies, it seems that P. spumarius and N. campestris are the most common spittlebugs of olive groves not only in Greece but throughout Southern Europe.

Macrosteles quadripunctulatus and
Regarding the molecular analysis, according to EPPO (2018) knowledge about the life cycle, seasonal appearance, feeding needs, plants most preferable for oviposition, nymph development, etc., of those two spittlebugs. In general, the robust understanding of the biology and ecology of those insects in the climatic conditions of Greece is essential in order to avoid environmentally unacceptable control methods (e.g. repeated insecticide applications) and to be able to establish an efficient control strategy in case of an outbreak of the bacterium in the country.

ACK N OWLED G EM ENTS
The authors would like to thank the growers for giving permission to sample insects from their olive orchards and Mrs Iro Georgopoulou for the construction of the distributional map. The study was partially funded by the Horizon 2020 Project XF-ACTORS (Xylella Fastidiosa Active Containment Through a multidisciplinary-Oriented Research Strategy), grant agreement no. 727987.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R S ' CO NTR I B UTI O N S
PM and DPP conceived research and secured funding. All authors conducted field samplings. SA and ICL identified the insects. DEK and VIE conducted the molecular analysis. SA, DEK and VIE wrote the manuscript. All authors commented on the manuscript. All authors read and approved the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available online in the supporting information section of the article.