Eur J Plant Pathol (2019) 154:1103–1110 https://doi.org/10.1007/s10658-019-01731-0

Potential role of the alien speculum as vector of Flavescence dorée phytoplasma

Luciana Galetto & Mattia Pegoraro & Cristina Marzachì & Elisabetta Rossi & Andrea Lucchi & Domenico Bosco

Accepted: 25 March 2019 /Published online: 9 April 2019 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2019

Abstract Ricania speculum is an Asian planthopper that is polyphagous and has been observed on wild and culti- was accidentally imported into Europe from the Far East. vated plants that are herbaceous or woody. This phloem The planthopper was first observed in Italy in 2009 (Genoa feeder frequently feeds on Vitis spp. (cultivated and wild province, Liguria) and is now considered established. The grapevines) and Clematis vitalba plants that are hosts of current range of R. speculum in Italy includes Liguria, Flavescence dorée phytoplasma (FDp), a quarantine Tuscany, Veneto, Piedmont and Latium. Ricania speculum phloem-limited bacterial pathogen and a major threat to viticulture of several European regions. The aim of this work was to assess if R. speculum could act as a vector of L. Galetto (*) : M. Pegoraro : C. Marzachì : D. Bosco FDp, thereby affecting disease epidemiology. To explore Istituto per la Protezione Sostenibile delle Piante, IPSP-CNR, theroleofR. speculum in FDp transmission, nymphs were Strada delle Cacce 73, 10135 Torino, Italy allowed to feed on FDp-infected Vicia faba plants to e-mail: [email protected] estimate acquisition efficiency and successively transferred onto Vitis vinifera, V. faba and C. vitalba test plants to M. Pegoraro determine transmission ability. Ricania speculum was un- e-mail: [email protected] able to transmit FDp; some individuals acquired the phy- toplasma but supported a very low level of pathogen C. Marzachì e-mail: [email protected] multiplication, compared with the competent vector. Con- sistentwithtransmissionresults,FDpwasdetectedonlyin D. Bosco one out of 51 tested salivary gland samples. According to e-mail: [email protected] ourresults,theroleofR. speculum in FD epidemiology is E. Rossi : A. Lucchi expected to be negligible. Dipartimento di Scienze Agrarie, Alimentari e Agro-ambientali, Università di Pisa, Via del Borghetto 80, 56124 Pisa, Italy Keywords Vitis vinifera . Clematis vitalba . Vicia faba . vector. qPCR E. Rossi e-mail: [email protected]

A. Lucchi Introduction e-mail: [email protected] The exotic planthopper Ricania speculum (Walker) D. Bosco Dipartimento di Scienze Agrarie, Forestali e Alimentari, (: ) was officially reported for the first Università degli Studi di Torino, Largo P. Braccini 2, time in Europe in 2014 (Mazza et al. 2014). Nymphs and 10095 Grugliasco, TO, Italy adults were first observed in Genoa province (Liguria, 1104 Eur J Plant Pathol (2019) 154:1103–1110

Italy) during 2009. Currently, this species is considered most destructive phytoplasma diseases, grapevine FD is a established in Liguria, widely spread in Tuscany, and has quarantine disease, epidemic in many economically im- been reported in Veneto, Piedmont and Latium (Mazza portant viticulture areas of Europe, including Italy. It et al. 2018). Ricaniidae species are mainly distributed in causes strong yield reductions and is a very serious threat the tropics and sub-tropics of the eastern hemisphere, and for European vine-growing regions. It is transmitted from R. speculum was reported in several Far East countries grape to grape by the nearctic leafhopper Scaphoideus (Korea, Japan, China, Vietnam, Philippines and Indonesia) titanus Ball (Hemiptera: Cicadellidae), that was introduced where it is a real pest for many crops (among others luffa, into Europe by human activities, probably during the , cocoa, cotton, sorghum and apple) due to its ex- importation of large amounts of American rootstocks in treme polyphagy (Rossi et al. 2015). Damage is mainly response to the Phylloxera crisis (Chuche and Thiery caused by sap suction and honeydew emission and to a 2014). Different insect vectors are known and associated lesser extent by egg-laying on thin shoots or thorns. with alternative epidemiological cycles of FDp and other The species is univoltine in Italy and overwinters as 16SrV-C phytoplasmas, namely Dictyophara europaea egg. Eggs are layered in linear clusters of about 20 eggs. (Filippin et al. 2009), Oncopsis alni (Schrank) (Maixner Presence of eggs may be used to describe prevalence et al. 2000) and potentially Orientus ishidae (Matsumura) and distribution of R. speculum (Germain et al. 2016; (Lessio et al. 2016), but are not directly associated with Wilson et al. 2016). In two field surveys conducted in major epidemics of phytoplasma diseases of grapevine. the Liguria region, egg clusters, nymphs and/or adults Under controlled conditions, a standardized FDp transmis- were collected from 49 different plant species, although sion procedure is available relying on the laboratory vector the host range of R. speculum probably includes addi- Euscelidius variegatus Kirschbaum and Vicia faba as host tional plant species (Mazza et al. 2014). Indeed, 33 new plant(Caudwelletal.1972). This controlled system is host plant species were recently documented to be useful for maintaining FDp and allows for study of infec- attacked by R. speculum (Mazza et al. 2018). Hosts tion mechanisms in natural hosts as S. titanus can acquire include Vitis spp., Clematis vitalba, and Rubus spp., FDp from infected V. faba and transmit to V. vinifera the latter two serving as excellent oviposition substrates (Eveillard et al. 2016). for R. speculum (Rossi et al. 2015). Interestingly, Vitis Only a few plant species have been described in the spp. and C. vitalba are well-known plant hosts of the field as hosts of FDp and its closely related strains: Vitis grapevine Flavescence dorée phytoplasma (FDp, spp., C. vitalba, Alnus spp., and Ailanthus spp. (EFSA 16SrV-C and -D) (EFSA 2016) and, since two uniden- 2016). The aim of the work was to explore the potential tified Ricaniidae species were suspected to transmit role of this new alien species in FDp transmission, as Bogia coconut syndrome (BCS) phytoplasma in Papua C. vitalba is one of the most visited plants by New Guinea (Lu et al. 2016), concerns were raised on R. speculum, and this planthopper lays eggs on both this alien species as potential vector of FDp. American and European grapevines. To this purpose, Phytoplasmas are plant pathogenic bacteria belonging FDp acquisition and transmission trials were performed to class Mollicutes and are associated with diseases affect- with R. speculum as potential vector and V. faba, V. vinifera ing hundreds of plant species including plants grown for and C. vitalba as test plants, along a period of two consec- commercial agriculture (Tomkins et al. 2018). In host utive years, in a containment lab facility. Genotype of the plants, phytoplasmas are restricted to the phloem sieve FDp strain detected in R. speculum was confirmed and the tubes and are transmitted by leafhoppers, or amount of FDp was compared with that measured in the psyllids in a persistent-propagative manner (Hogenhout vector E. variegatus. The potential impact of R. speculum et al. 2008). Different phases take place during the trans- on FD epidemiological cycle is discussed. mission process: insect vectors acquire phytoplasmas by feeding on phloem of infected plants, once in the insect gut phytoplasmas cross the intestinal epithelium and enter the Materials and methods haemocoel, multiply and circulate in the haemolymph, colonize the salivary glands, and are transmitted with Phytoplasma isolate, and host plants saliva. Indeed, phytoplasmas are wall-less obligate intra- cellular parasites able to survive and multiply both in plants The FDp strain BFD-D CRA AT^ was used for acquisition and in insect vectors (Tomkins et al. 2018). Among the and transmission trials. The isolate was obtained from Eur J Plant Pathol (2019) 154:1103–1110 1105 infective S. titanus adults that were collected in 2015 from IAP. When less than 5 insects per group survived, an experimental vineyard located at the CREA research insects from different groups were combined together institute (Asti, Piedmont) and allowed to feed on V. faba in to reach again five insects per group. Plastic cylinders the laboratory. This FDp isolate was genetically identified were used to isolate insects on V. fa ba plants, net cages as D strain on dnaK gene (MH547710.1) and mixed were used to isolate insects on V. vinifera branches and profile on malG gene (malG_1 MH547713, malG_3 C. vitalba plants (Fig. 1). Following all IAPs, DNAs MH547715, malG_6 MH547718, malG_16 MH547728, malG_34 MH547746), according to (Rossi et al. 2019). FDp was then routinely maintained under controlled con- ditions by sequential transmissions from V. faba to V. faba by the experimental vector E. variegatus, continuously reared under laboratory conditions on Avena sativa (oats) as described in Rashidi et al. (2014). For acquisition and transmission experiments, R. speculum individuals were obtained from eggs laid on winter-collected twigs of Clematis vitalba, Ligustrum lucidum and Rubus spp. from infested areas (Liguria). Eggs were allowed to hatch under laboratory conditions (21 ± 1 °C, 60 ± 10% RH, and a photoperiod of 16:8 L:D h) on healthy V. faba plants and the phytoplasma-free status of the hatched nymphs was confirmed by qPCR (Pelletier et al. 2009). Vicia faba, V. vinifera and C. vitalba were used as test plants: V. faba were grown from seed, potted V. vinifera derived from phytoplasma-free certified commercial cuttings of different Piedmont typical cultivars (Barbera and Nebbiolo) grafted onto Kober 5BB rootstock, and C. vitalba were obtained by cuttings of field plants, checked to be PCR-negative for phytoplasma presence.

Acquisition and transmission experiments

Two sets of experiments were carried out with R. speculum in 2016 and 2017. In 2016, about 200 nymphs (3rd and 4th instar) of R. speculum were isolated all together on FDp-infected V. faba plants for an acquisition access period (AAP) of three weeks and transferred onto healthy V. faba plants to complete the latent period for one extra week. Infection status of V. faba plants used for acquisition was confirmed by symptoms and qPCR (Pelletier et al. 2009). Adults emerged from FDp-exposed nymphs were then used to inoculate test plants (groups of 5 insects per plant) in three sequential inoculation access periods (IAP) each of one week. In detail, 24 V. faba plants were Fig. 1 Experimental workflow for transmission trials of Flavescence inoculated in the first round IAP, 21 V. vinifera plants dorée phytoplasma (FDp) with the alien planthopper Ricania specu- in the second one and three C. vitalba plants in the lum. Following an acquisition access period (AAP) of three weeks on FDp infected Vicia faba, and a latent period of one week on healthy third IAP. After inoculation of V. faba, insects in the V. fab a plants (not shown), R. speculum individuals were isolated for same group of five were, whenever possible, grouped three consecutive inoculation access periods (IAP) of one week on again altogether on V. vinifera plants for the successive V. fab a, Vitis vinifera and Clematis vitalba plants 1106 Eur J Plant Pathol (2019) 154:1103–1110 were extracted from whole insects devoid of wings or Phytoplasma detection, characterization and relative from dissected salivary glands and stored at −20 °C quantification until further analyses. Inoculated plants were inspected twice a week for symptom appearance. DNAs were DNAKf/DNAKr primers, flanking FDp dnaKgenepor- extracted from V. faba 1 month post inoculation and tion (MH547710.1), were used to detect FDp presence from V. vinifera and C. vitalba at both 3 and 12 months in R. speculum samples, by conventional PCR under post inoculation. In 2017, AAP with R. speculum was standard reaction conditions with an annealing temper- repeated as described for 2016, and FDp exposed ature of 55 °C (Rossi et al. 2019). The corresponding insects were used to investigate the presence and the dnaK PCR products obtained from positive samples amount of pathogen in dissected salivary glands, but were purified with DNA Clean & Concentrator kit no transmission experiment was carried out. Healthy (Zymo Research) and Sanger sequenced by BMR Ge- R. speculum insects that were not exposed to phyto- nomics (Padova, Italia), to confirm the identity of FDp plasma were used as negative controls. FDp-exposed strain. Clustal Omega was used to align the obtained E. variegatus samples, routinely used for phytoplasma sequences with the reference dnaKgeneofFD-Dstrain. transmission, were used as positive controls. Primers mapFD-F/mapFD-R and TaqMan probe Concentration of FDp recorded in R. speculum mapFD-FAM (Pelletier et al. 2009)wereusedto were compared to those measured in the experimen- detect FDp presence in inoculated plants by qPCR, tal FDp vector E. variegatus. Healthy 3rd and 4th using 1x iTaq Universal Probe Supermix (Bio-Rad) instar E. variegatus nymphs were allowed to acquire in a reaction mix of 10 μl volume. Final concentra- phytoplasma from infected V. faba as described tions were 300 and 200 nM for primers and probe, above. At 40 days post acquisition, total DNAs were repectively, and cycling conditions were as indicated extracted from FDp exposed adults and analyzed in in the original paper (Pelletier et al. 2009). Samples qPCR for phytoplasma quantification. were run in duplicate in a CFX Connect Real-Time PCR Detection System (Bio-Rad). The concentration of FDp in insects was quanti- DNA extraction fied by qPCR using two primer pairs: FdSecyFw/ FdSecyRv, targeting FDp secY gene (Roggia et al. Total DNA was extracted from whole R. speculum 2014), and MqFw/MqRv, targeting insect 18S ribo- individuals devoid of wings (Experiment 2016) or somal gene (Marzachí and Bosco 2005). Insect from salivary glands dissected from single insects DNAsamplesweredilutedto10ng/μland1μl (Experiment 2017) with cethyl-trimethyl-ammonium was added to a qPCR reaction mix of 10 μl volume, bromide (CTAB) buffer, as described in (Rashidi containing 1x iTaq Universal SYBR Green et al. 2014). In detail, salivary glands were dissected Supermix (Bio-Rad) and 300 nM of each primer. with sterile forceps under a stereomicroscope from Samples were run in triplicate in a CFX Connect anaesthetized R. speculum adults, either FDp ex- Real-Time PCR Detection System (Bio-Rad). Cy- posed or healthy as negative control, washed four cling conditions were as detailed in the original times in sterile phosphate buffered saline (PBS) 1x paper (Roggia et al. 2014). For each primer pair a andsinglystoredincleantubeat−20 °C prior to standard curve, based on serial dilutions of either a DNA extraction. plasmid harboring FDp secYgeneortotalDNAofa Total DNA were also extracted from single healthy insect sample, was always run together with whole E. variegatus (Rashidi et al. 2014) and from analyzed samples. Mean starting quantities were plant samples (1 g of leaf tissues) with CTAB automatically calculated by CFX Maestro™ Soft- buffer, as described in (Pelletier et al. 2009). In- ware (Bio-Rad) and used to express FDp amount sectandplantsampleswereresuspendedin50and as Genome Unite (GU)/ng insect DNA. SigmaPlot 100 μl of 10 mM Tris-Cl pH 8.0, respectively. version 13 (Systat Software, Inc., San Jose Califor- Concentration and purity of extracted total DNAs nia USA, www.systatsoftware.com.) was used to were checked at UV-visible spectrophotometer compare FDp concentration in R. speculum and E. Nanodrop 2000 (Thermofisher). variegatus samples, by Mann-Whitney test. Eur J Plant Pathol (2019) 154:1103–1110 1107

To confirm the healthy status of the field-collected The amount of FDp measured in the vector C. vitalba cuttings used to propagate test plants, P1/P7 E. variegatus, ranged from 837.9 to 11,640.5 FDp and 16SF2/R2 primer pairs were used in direct and GU/ng insect DNA, with a mean value of 3,780.1 ± nested PCR, respectively, as described in (Lee et al. 1,151.6 (SEM) calculated on 10 individuals (Fig. 2). 1993) for universal phytoplasma detection. The FDp concentration detected in R. speculum insects was significantly lower than the phytoplasma amount measured in the vector species E. variegatus (Fig. 2, Mann-Whitney U Statistic = 0.000; P =0.001). Results

All V. faba, V. vinifera and C. vitalba plants fed on by Discussion FDp exposed R. speculum did not show any symptoms and tested negative for phytoplasmas in diagnostic The main concern about R. speculum was the possible qPCR assays conducted in the same year as the IAP role of this planthopper in opening new epidemiological (Table 1). To exclude latent infections, inoculated cycles of FD between alternative woody host plants and V. vinifera and C. vitalba plants were monitored for grapevine, thus representing a serious threat to Italian symptom appearance and tested via PCR one year later and European viticulture. First observations of this spe- at 12 months post inoculation (summer 2017) (Table 1). cies in Italy dates back to 2009 (Mazza et al. 2014)andit Under our experimental conditions, R. speculum indi- can be now considered as established. The polyphagous viduals were unable to transmit FDp to test plants. nature of this species coupled with its frequent associa- Phytoplasma detection on whole insects revealed that tion with Vitis spp. and C. vitalba (Rossi et al. 2015)and R. speculum was able to acquire FDp, as more than 50% its establishment in regions like Tuscany, which is a of the tested insects were PCR positive (Table 2). As world-renowned wine producing area, were at the base expected, sequencing of the obtained PCR amplicons of this concerning hypothesis. (targeting FDp dnaK gene) confirmed a 100% identity Under our experimental conditions, R. speculum ac- with the same BFD-D CRA AT^ phytoplasma strain quired FDp at quite a high rate and was unable to transmit used for acquisition. Nevertheless, mean FDp concen- it to any of the three tested host species (V. faba, V. vinifera, tration in R. speculum adults was generally low, ranging C. vitalba). This finding suggests that phytoplasma cells from 3.2 to 130.4 FDp GU/ng insect DNA (Table 2). are probably retained within the alimentary canal, eventu- Only 6 out of 8 positive insect samples were quantified, ally crossing the gut epithelium. However, phytoplasma being the remaining two below the sensitivity threshold cells were mostly unable to colonize salivary glands, of the quantification method (Table 2). resulting in a lack of transmission. Indeed, R. speculum FDp was also detected in one salivary gland out of 51 supported a very limited FDp multiplication rate, the phy- organs dissected from FD-exposed R. speculum toplasma amount being nearly 60 times lower than in the (Table 2). Consistent with lack of transmission, FDp vector species E. variegatus. Transmission ability has been amount in salivary glands was low, in line with mean correlated with phytoplasma concentration in insect body, phytoplasma GU measured in whole R. speculum sam- and FDp amount in R. speculum samples was well below ples (Table 2). that measured in the non-transmitter E. variegatus exposed

Table 1 Evaluation of Ricania speculum capability of transmit- Inoculated plants species Plant sampling dates Experimental year PCR positive/total ting Flavescence Dorée phyto- (months post inoculation) analysed samples plasma (FDp) to different plant host species Vicia faba 120160/24 Vitis vinifera 320160/21 12 2017 0/21 Clematis vitalba 320160/3 12 2017 0/3 1108 Eur J Plant Pathol (2019) 154:1103–1110

Table 2 Evaluation of Ricania speculum capability of acquiring Flavescence Dorée phytoplasma (FDp) and quantification of FDp concentration in whole insects or in salivary glands

Sample type Insect sampling dates Experimental year PCR positive/total Mean FDp GU/ng (days post acquisition) analysed samples insect DNA ± SEM (N)

Whole insects 50 2016 8/15 65.4 ± 27.3 (6) Salivary glands 40 2017 1/51 317.6 (1)

FDp amount are expressed as mean FDp Genome Units (GU)/ ng of insect DNA ± standard error of the mean (SEM) with sample size indicated in parenthesis (N) to ‘Candidatus Phytoplasma asteris’ (approximately 1800 species suspected to transmit BCS phytoplasma in Papua phytoplasma GU/ng insect DNA) (Galetto et al. 2009). New Guinea, the pathogen was detected in both species Consistently, a very low frequency of FDp detection in and also in artificial media used to feed insects of one of the R. speculum salivary glands was observed (1 case out of two species, indicating that acquisition occurred but trans- 51), especially if compared with FDp infection rate in mission did not (Lu et al. 2016). Similar situations have heads of E. variegatus infected by FDp (88% on average; been reported also for FD phytoplasma, which was detect- Galetto, unpublished data). In the unique positive salivary ed in several field collected Hemiptera insect species gland sample of R. speculum, FDp concentration was very (Psylla spp., Phlogotettix cyclops, Cixius nervosus, low (318 phytoplasma GU/ng insect DNA), two orders of Metcalfa pruinosa), but in absence of effective transmis- magnitude lower than the one measured in the heads of sion evidence (Trivellone 2019). Indeed, vector competen- FDp-infected E. variegatus (39,000 phytoplasma GU/ng cy strongly depends on the capability of phytoplasmas to insect DNA on average, despite measured with a different colonize salivary glands (Hogenhout et al. 2008). As an quantification assay; Galetto, unpublished data). Several example, among 15 tested Hemiptera species, Anoplotettix insect species are able to acquire but not transmit fuscovenosus (Ferrari), E. variegatus,andEuscelis incisus phytoplasmas and there are many reports of field- Kirschbaum were the only three able to transmit FDp to collected insects found positive for phytoplasma presence, V. faba, following abdominal microinjection with phyto- but then unable to transmit. Indeed, the ability to transmit plasma suspension, that allows phytoplasmas to avoid the phytoplasmas depends on the pathogen - plant host - insect gut barrier but not the salivary gland barrier (Bressan et al. vector combination, and several factors, such as feeding 2006). All these species can harbor phytoplasma cells in behavior and host-pathogen molecular recognition, are their bodies, but transmission occurred only for the few involved in mediating phytoplasma transmission capability pathogen/vector compatible combinations, probably based by an insect species (Bosco and D'Amelio 2010; Galetto on a molecular specific mechanism of host recognition et al. 2011a). Consider for example the two Ricanidae (Bressan et al. 2006). Lack of phytoplasma vector-

Fig. 2 Concentration of Flavescence dorée phytoplasma (FDp) in the whole insect, expressed as FDp Genome Unit (GU) per ng of insect DNA, measured in the alien planthopper Ricania speculum and in the FDp vector Euscelidius variegatus Eur J Plant Pathol (2019) 154:1103–1110 1109 recognition may explain the inability of phytoplasma trans- perennial hosts, as is the case for field (Roggia et al. missionalsointhecaseofR. speculum. Several specific 2014) and potted V. vinifera plants (Eveillard et al. recognition factors were demonstrated to be at the bases of 2016). Moreover, FD symptoms frequently appear dur- an effective pathogen/vector combination. Considering the ing the vegetative season following the year of the molecular recognition mechanism between phytoplasma inoculation, and therefore, V. vinifera and C. vitalba and vector, it is noteworthy to mention that some insect plants were further assayed the year after inoculation. proteins (namely actin, myosin and ATP synthase alpha The diagnosis confirmed that the plants were still and beta subunits) show specific in vitro interactions with phytoplasma-free at 12 months after inoculation. In the major antigenic membrane protein (amp) of different conclusion, although R. speculum can damage plants strains of the ‘Ca.P.asteris’ (Suzuki et al. 2006; Galetto by sap suction, honeydew emission and egg-laying, it et al. 2011b). Moreover, these specific interactions are should not be regarded as a threat for FDp spread. in vivo required for insect colonization by phytoplasma, as ‘Ca.P.asteris’ amp protein mediates both pathogen Acknowledgements The authors would like to thank Caterina crossing of vector gut epithelium and colonization of Perrone, Elena Zocca and Ivana Gribaudo for providing test plants and Flavio Veratti for routine phtoplasma strains maintaining. salivary glands (Rashidi et al. 2015), showing the real involvement of these specific molecular interactions in a Author contributions Conceived and designed the experi- compatible phytoplasma/vector combination. A similar ments: D. Bosco, C. Marzachì and A. Lucchi. Performed the specific recognition mechanism was recently demonstrated experiments: M. Pegoraro, L. Galetto and E. Rossi. Analyzed the also for FDp and its vector E. variegatus:thevariable data: M. Pegoraro, L. Galetto and D. Bosco. Wrote the paper: L. Galetto. Critical revision of the manuscript: all authors. membrane protein A (vmpA) expressed on the surface of pathogen cells mediates phytoplasma binding to insect cell culture and pathogen retention at the perimicrovillar mem- Compliance with ethical standards The work complies to the brane of vector midgut epithelium (Arricau-Bouvery et al. ethical standards of this journal. 2018). Lack of such molecular recognition between R. speculum tissues and FDp membrane proteins could Conflict of interest The authors declare that they have no con- flict of interest. This research did not involve human participants. explain the low multiplication rate in this host, lack of The manuscript was not previously published. All authors contrib- salivary gland colonization and the consequent inability uted and agreed to submission to EJPP. of transmission. Some preliminary experiments to investi- gate the in vitro interaction between R. speculum organs and a FDp recombinant membrane protein provides sup- References port for this hypothesis (Valeria Trivellone, personal communication). Arricau-Bouvery, N., Duret, S., Dubrana, M.-P., Batailler, B., The three plant species chosen for transmission trials, Desqué, D., Béven, L., et al. (2018). Variable membrane when infected by FDp, normally show clear and evident protein a of flavescence dorée phytoplasma binds the midgut perimicrovillar membrane of Euscelidius variegatus and pro- symptoms, as described in (Salar et al. 2013)forV. faba, motes adhesion to its epithelial cells. Applied and in (Roggia et al. 2014)forV. vinifera and in (Filippin Environmental Microbiology, 84,8–17. et al. 2009)forC. vitalba plants. Such symptoms were Bosco, D., & D'Amelio, R. (2010). Transmission specificity and never observed in our cases. 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