Vector Biology of the Soft Scales Parthenolecanium
corni (Bouché) and Parthenolecanium persicae
(Fabricius) (Hemiptera: Coccidae) With Grapevine
Leafroll-Associated Viruses and Grapevine Virus A

Gérard Hommay, Antoine Alliaume, Monique Beuve, Jean-michel Hily,
Etienne Herrbach

► To cite this version:

Gérard Hommay, Antoine Alliaume, Monique Beuve, Jean-michel Hily, Etienne Herrbach. Vector Biology of the Soft Scales Parthenolecanium corni (Bouché) and Parthenolecanium persicae (Fabricius) (Hemiptera: Coccidae) With Grapevine Leafroll-Associated Viruses and Grapevine Virus A. Journal of Applied Entomology, 2025, 149 (3), pp.405-416. 10.1111/jen.13387. hal-05101368

HAL Id: hal-05101368

https://hal.inrae.fr/hal-05101368v1

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ORIGINAL ARTICLE

OPEN ACCESS

# Vector Biology of the Soft Scales *Parthenolecanium corni* (Bouché) and *Parthenolecanium persicae* (Fabricius) (*Hemiptera: Coccidae*) With Grapevine Leafroll-Associated Viruses and Grapevine Virus A

Gérard Hommay

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Antoine Alliaume

Monique Beuve

Jean-Michel Hily

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Etienne Herrbach

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Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Unité Mixte de Recherche 1131 Santé de la Vigne et Qualité du Vin (SVQV), Université de Strasbourg, Colmar, France

**Correspondence:** Gérard Hommay (gerard.hommay@inrae.fr)

**Received:** 4 April 2024

| Revised: 5 November 2024

| Accepted: 22 November 2024

**Funding:** This study was supported by FranceAgriMer grants (agreements 2009-0419 01, 2010-0292, 2011-1035, 2012-0764, 2013-0681, 2014-0764). JMH was funded by the 'Metagenome' project (no. 28000225) gathering three French professional committees for viticulture (Interprofessional Committee of Champagne Wine, CIVC, Epernay; Interprofessional Office of Burgundy Wines, BIVB, Beaune; Interprofessional Committee of Wines from Alsace, CIVA, Colmar) and Moët & Chandon (Champagne wine industry, Epernay).

**Keywords:** ampelovirus | leafroll | rugose wood | scale insect | *Vitis vinifera* | vitivirus

## ABSTRACT

Controlled experiments were conducted to enrich our knowledge on the transmission of different grapevine leafroll-associated ampeloviruses (GLRaV-1, -2, -3 and -4) by *Parthenolecanium corni* scale insect and to look for vector ability of *Parthenolecanium persicae*. GLRaV-1 and the vitivirus GVA were transmitted by *P. corni* to healthy plants after acquisition on GLRaV-1 and GVA-infected grapevines (*Vitis vinifera*); however, GVA was not transmitted after acquisition either on GVA singly infected vines or *Nicotiana benthamiana*, or on purified GVA virions. These results support the hypothesis of ampelovirus assistance towards vitivirus during transmission. Transmission experiments with *P. corni* and different GLRaV-4 strains showed a single transmission of strain 5. No transmission was observed with *P. persicae* after acquisition on GLRaV-1, -3 and GVA coinfected source vines. *P. corni* L2 collected from an infected vine and placed on a nonhost plant tested positive for GLRaV-1 and -3 until at least 30 and 50 days, respectively. These results contrast with the few days retention times generally reported in mealybugs. More work is needed to determine whether virions are conserved and, if so, their location inside the insects.

## 1 | Introduction

The European fruit lecanium, *Parthenolecanium corni* (Bouché 1844) (Hemiptera Coccidae), is a polyphagous and cosmopolitan soft scale species (Schmutterer 1952; Canard 1958; Kosztarab and Kozár 1988; García Morales et al. 2016). It presents a risk for many fruit crops worldwide. Grapevine, *Vitis vinifera* L. subsp. *vinifera*, is also a common host for *P. corni*. When in high density, this mainly phloem-feeding insect can damage grapevine directly by weakening it, as well as by excreting honeydew

causing the development of sooty mould that hinders leaf photosynthesis and reduces fruit quality and marketability. More importantly for the wine industry, *P. corni* is a well-known vector of grapevine-infecting viruses, along with other scale insects (superfamily Coccoidea) in the families Coccidae (soft scales) and Pseudococcidae (mealybugs) (Fortusini et al. 1996, 1997; Herrbach et al. 2017; Hommay et al. 2021; Hommay, Beuve, and Herrbach 2022b). *P. corni* is known to transmit two damaging phloem-restricted viruses: grapevine leafroll-associated virus-1 (GLRaV-1, *Closteroviridae, Ampelovirus*) causing 'grapevine

<footnote>This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.</footnote>

<footnote>© 2024 The Author(s). *Journal of Applied Entomology* published by Wiley-VCH GmbH.</footnote>
leafroll disease' and grapevine virus A (GVA, *Betaflexiviridae, Vitivirus*) associated to 'rugose wood syndrome' (Herrbach et al. 2017). The interaction between *P. corni* and GLRaV-1 is believed to occur according to the semi-persistent noncirculative transmission mode, like for mealybugs (Fortusini et al. 1997; Engelbrecht and Kasdorf 1985; La Notte et al. 1997; Zorloni et al. 2006; Hommay et al. 2008; Herrbach et al. 2017).

Moreover, GVA is generally detected in grapevines along with an ampelovirus, but seldom alone (Herrbach et al. 2017); thus, GVA is transmitted along GLRaV-1 by *P. corni* (Hommay et al. 2008), raising the hypothesis that the ampelovirus may assist the vi-tivirus during transmission (Engelbrecht and Kasdorf 1985; La Notte et al. 1997; Zorloni et al. 2006; Hommay et al. 2008; Martelli 2017).

In NE France, particularly in Alsace region, *P. corni* is common on grapevine and has a monovoltine life cycle (Sforza, Boudon-Padieu, and Greif 2003). In Alsace, we showed that its first instar larvae (L1) can be dispersed by wind over 150 m (Hommay et al. 2019), that this species spreads GLRaV-1 and GVA within a vineyard (Hommay et al. 2020) and that individuals sampled from GLRaV-1-infected vineyards are able to inoculate the virus to healthy vine cuttings (Hommay, Beuve, and Herrbach 2022b). Due to the risk posed by this species to grapevine health, it is important to better characterise its vector biology, which is the aim of the present work.

In addition, a closely related polyphagous species, the European peach scale, *Parthenolecanium persicae* (Fabricius), is also found on grapevine, where it is a direct pest in South America and Australia (Foldi and Soria 1989; Hickel 2004; Rakimov et al. 2013). In Europe, it is less frequent than *P. corni* and prefers warm areas (Kosztarab and Kozár 1988; CABI 2023). Global warming and reduction of insecticide treatments may favour its expansion to northern vineyards. *P. persicae* is not widespread in French eastern vineyards, but can get unnoticed, given its resemblance to *P. corni*. Main distinction in the field is based on the shield of postreproductive females, which is oval with a central carina, while that of *P. corni* is more round without carina (Kosztarab and Kozár 1988). It is monovoltine and shows three nymphal stages instead of two for *P. corni* (Hoffmann 2002). *P. persicae* was suspected to be involved in the natural spread of GLRaV-1 and -3 in Italian vineyards (Fortusini et al. 1996). However, nothing is published about its vector ability. No transmission of GLRaV-1 and -3 was observed in our earlier experiments using nymphs collected on infected vines from a Kaiserstuhl vineyard (Baden-Württemberg, Germany) (Hommay et al. 2009). Therefore, we conducted further laboratory experiments to evaluate the vector ability of this species.

# 2 | Materials and Methods

## 2.1 | Origin of Insects

To obtain virus-free L1 nymphs, eggs were collected by scrapping egg-lying females fixed on canes. Freshly laid eggs were separated from females through a sieve and kept at room temperature inside hermetic boxes until hatching, and then were used in transmission experiments. To dispose of *P. corni* L1 over around

1 month, hatching time was slowed down by keeping eggs at 4°C. L2 nymphs were collected from a vineyard on virus-free vines previously checked for the absence of GLRaV-1, -2, -3 and GVA, by both ELISA and RT-PCR. *P. corni* females were sampled from a Riesling plot at Nothalten (Alsace, France). *P. persicae* adult females were collected in a Kerner and a Merzling plots located at Ihringen (Baden-Württemberg, Germany), in a 'Muscat blanc à petits grains' plot located at Turckheim (Alsace), and in a Cabernet plot located at Saint-Émilion (Aquitaine, France).

*P. corni* L2 were collected from several GLRaV-1-infected vines in the Nothalten Riesling plot for transmission to *Nicotiana benthamiana* Domin and from a GLRaV-3-infected Pinot noir vine at Bennwihr (Alsace) for retention time study. *P. persicae* L2 were collected on a virus-free vine at Turckheim.

## 2.2 | Virus Source Plants

Virus source grapevines were rooted cuttings from the following accessions of our collection of dormant canes: P70, Y245, Y258, V38, Cab119-66, TE132, Y199, Y217 and Y252 (Greif and Walter 1997). Accessions P70 (cv. Pinot noir, infected with GLRaV-1 and GVA) (Beuve et al. 2018) and Y258 (cv. Liali Bidona, infected with GLRaV-1, -3 and GVA), were chosen because P70 viruses were well transmitted by *P. corni* (Hommay et al. 2008) and by the mealybug *Phenacoccus aceris* (Signoret) (Le Maguet et al. 2012), and Y258 viruses were transmitted by L1 of the mealybug *Heliococcus bohemicus* Šulc in a study for virus acquisition and inoculation times (Hommay, Beuve, and Herrbach 2022a). Virus content of these accessions was checked by ELISA and RT-PCR, as described by Le Maguet et al. (2012), who reported that the viruses infecting the last five accessions were successfully transmitted to grapevine by *Ph. aceris*.

One Riesling vine from Nothalten tested positive only for GVA at four different dates (in leaves and wood), but not for any of the leafroll species (GLRaV-1, -2, -3, -4) by either ELISA or RT-PCR. However, failure to detect leafroll viruses could have been due to either a low virus content in the tissue samples, or a viral species or variant not recognised by the available ELISA antibodies and RT-PCR primers. Therefore, a control on double-stranded RNA (dsRNA) extracts was submitted to high-throughput sequencing (HTS) to search for GVA and possible sequences of GLRaV RNA, as described below (Section 2.7). Rooted cuttings were produced under greenhouse and used for transmission experiments of 'GVA alone'.

*N. benthamiana* plants were mechanically inoculated with GVA purification (Section 2.4.5). Three weeks postinoculation, symptomatic plants were tested by RT-PCR for GVA. Cuttings of infected *N. benthamiana* were then propagated and maintained under greenhouse.

## 2.3 | Recipient Plants

Virus-free vines were obtained from either rooted cuttings of *V. vinifera* cv. Pinot noir, a common cultivar in Alsace, Champagne and Burgundy (clones P115 mainly and P114), or germinated seeds of Pinot noir and Muscat ottonel. Mother plants of the
cuttings were regularly tested for the absence of GLRaV-1, -2, -3 and GVA, by ELISA and RT-PCR. Cuttings and seedlings were grown individually in pots under greenhouse until 6-12 leaf stage and then used in experiments. They were regularly sprayed with an insecticide to ensure the absence of insects. This spraying was stopped at least 1 month before experiments. Seedlings (6-8 leaf stage) of *N. benthamiana* were used as recipient plants.

## 2.4 | Virus Transmission Experiments

### 2.4.1 | Virus Transmission With First Instar Nymphs (L1)

Leaves of source vines were collected from greenhouse-grown mother plants and placed individually into tight round polystyrene crystal boxes. A small wet cotton piece was wrapped around the petiole of each leaf, then swathed tightly inside Parafilm to keep water supply during acquisition access period (AAP). Newly hatched L1 nymphs were sprinkled onto the underside of these leaves for virus acquisition. After 5-8 days of AAP, leaf fragments with ca. 100 L1 were cut and clipped onto 2-3 different leaves of each recipient vine for 7-10 days of inoculation access period (IAP). RT-PCR was made in a sample of 10 *P. persicae* L1 from Turckheim after AAP on Y258 to check virus acquisition.

### 2.4.2 | Virus Transmission With Second Instar Nymphs (L2)

Leaves bearing L2 were collected from vineyard virus-free vines. They were then clipped onto rooted cuttings of the different source plants, allowing nymphs to feed during 5-7 days for AAP. Afterwards, leaves of source plants with 50-100 L2 were cut and clipped onto 2-3 different leaves of each recipient plant. Transmission experiments with *P. corni* were made initially with at least 15 recipient vines (P115) per replicate, but some cuttings died before the end of experiment. For inoculation to *N. benthamiana*, L2 were collected from a donor vine coinfected by GLRaV-1 and GVA in the Nothalten plot. About 50 nymphs were allowed to settle down on each recipient seedling.

Each recipient plant was enclosed in a perforated plastic bag firmly tied to the pot with a rubber band to prevent nymphs from moving between plants. Plants were placed at 20°C-23°C, 16h/8h L/D under artificial light. After 7 days IAP on recipient P115 vines, or 10 days IAP on recipient *N. benthamiana* seedlings, these plants were sprayed with an insecticide to kill the nymphs. Vine cuttings and *N. benthamiana* seedlings, kept in the same conditions without crawlers under glasshouse, were used as negative controls.

### 2.4.3 | Optimal Time for Virus Acquisition and Inoculation of L1 Nymphs

Accessions P70 and Y258 were used as source vines. An AAP of 1, 6 or 24h was given to L1 on leaves of each source vine.

After each AAP, leaf pieces with nymphs were cut and clipped onto the leaves of five recipient cuttings (P115) during 1, 6 or 24h IAP (30 L1 and 2 leaf pieces per plant), giving a total of 9 AAP/IAP pairs. With Y258 as the source, three additional AAP/IAP pairs were assayed: 72h/1h, 72h/3h and 72h/6h. Recipient vines were immediately sprayed with an insecticide upon completion of IAP.

To analyse virus content of mobile L1, newly hatched nymphs were sprinkled on P70 excised leaves to feed during 1, 2, 4, 6, 8h and daily from 1 to 5 days (10 different periods). For each acquisition time, five samples of 30 nymphs were collected with a needle for virus detection by RT-PCR (see Section 2.5).

### 2.4.4 | Transmission Capacity for Different Virus Species or Strains

To determine the ability of *P. corni* to transmit GLRaV-1, -2, -3, -4 (strains 4, 5, 6, 9) and GVA to grapevine, assays were conducted with L1 from June to July, and with L2 from September to October.

### 2.4.5 | Transmission Experiment With Purified GVA Virions

The accession P70 was used as source vine for GLRaV-1 and GVA purifications. GVA virions were purified following the protocol of Gugerli, Brugger, and Bovey (1984), from 80 g of infected *N. benthamiana* leaves and then resuspended in 2 mL of 0.18 M MgCl₂ buffer. Purified virions were stored at -80°C before use. The preparation used was at a concentration of 0.5 mg/mL.

For AAP, virion suspension (1/2) was added to 60% sucrose solution (1/2). A drop of 90 µL was placed over a Parafilm membrane stretched over the aperture of a black cylindrical box (diameter = 3 cm, height = 5 cm) containing L1 nymphs. A second membrane was stretched over the drop, then the box was turned over for allowing feeding. Nymphs were let to feed across the membrane for 48 h and batches of 50 L1 were then transferred inside clip cages onto 20 healthy P115 cuttings, where they were left 7 days for IAP. GVA detection by RT-PCR was made on feeding solutions and in a batch of 30 nymphs after AAP.

## 2.5 | Virus Detection

Leaves of recipient vines were tested by double-antibody sandwich (DAS)-ELISA. Detections were first made on leaves ca. 4 months after transmission experiments. After a dormancy period under unheated glasshouse, the plants were tested again 10-12 months after transmission. Polyclonal antibodies produced in our laboratory were used for the detection of GLRaV-1, -2, -3, -4 (strain 5), and GVA in a biotin-streptavidin procedure (Zimmermann et al. 1990). Detection of GLRaV-4 (strains 4, 6 and 9) was performed using a commercial antiserum for GLRaV-4 (strains 4-9) (Bioreba AG, Reinach, Switzerland) following the manufacturer's instructions. Tissue extracts were obtained from pooled fragments of three leaves, due to the uneven distribution of virus in vines.
Leaf fragments (1 g in 5 mL buffer) were ground in extraction bags with a bullet blender (Homex 5, Bioreba, Switzerland). Absorbance was recorded at 405 nm using a Multiskan microplate reader (Thermo Labsystems, Helsinki, Finland). Virus source vines were used as positive and healthy cuttings as negative controls. For transmission experiments with the Riesling source infected only with GVA, recipient vines were tested by ELISA and RT-PCR. Finally, recipient *N. benthamiana* plants were tested for GVA by ELISA ca. 2 months after transmission experiments.

A multiplex RT-PCR procedure was used for detecting GLRaV-1, -2, -3 and GVA in insects and vines, using the protocol developed by Beuve et al. (2013) and described by Hommay, Beuve, and Herrbach (2022b). RNase-free water and mixed RNAs extracts from leaves of vines infected with GLRaV-1, -2, -3 and GVA, were used respectively as negative and positive controls. PCR products were visualised under UV light on a 2% agarose gel stained with ethidium bromide.

## 2.6 | Virus Retention Time

### 2.6.1 | First Instar Nymphs

Like in transmission experiments, L1 hatched from eggs were scattered on excised P70 leaves and allowed to settle (AAP) during 10 days. Then, these infested leaves were clipped onto leaves of cut sour cherry tree twigs (*Prunus cerasus* L.) placed in a water-filled vase. Five batches of 20 L1 were sampled daily from sour cherry leaves with a dissection needle and placed into a tube for virus detection by RT-PCR for up to 11 days (until nymph supply was depleted). Similar batches were removed directly from P70 leaves and placed into a tube whose lid was drilled with tiny holes, to starve during 1–4 days. Two sets of five batches of 20 L1 were detached from P70 leaves before starving or feeding on sour cherry leaves and used as controls.

### 2.6.2 | Second Instar Nymphs

Leaves with L2 were collected in October from Nothalten vines coinfect ed with GLRaV-1 and GVA. One part of these individuals (vagrant ones, to avoid injuring those fixed with their stylets inside leave tissues) was collected with a needle and dropped into a box to starve. Among them, seven batches of 20 nymphs were collected daily as long as living nymphs were available (7 days) for virus detection by RT-PCR. Leaves with the other part of nymphs were clipped onto leaves of cut sour cherry twigs placed in a water-filled vase. Like for L1, five batches of 20 L2 were collected daily for 10 days, then every 5 days until 35 days, with a needle, taking care to withdraw the complete stylets when fixed in tissues. The following year, a similar experiment was performed earlier in season, that is, in September 2011, with L2 coming from a GLRaV-3-infected vine in Bennwihr. Vine leaves were clipped onto sour cherry leaves. After transfer, batches of 20 nymphs were collected on sour cherry twigs 25, 28, 37, 44 and 50 days for virus detection, as with progressive leaf fading most of them migrated onto twigs. Virus content was also tested by RT-PCR

on nymphs of the source vines (Day 0) and on a leaf sample of sour cherry (Day 5), and by ELISA on leaves and cut twigs of the sour cherry tree before and after nymph settling.

## 2.7 | High Throughput Sequencing (HTS) Analyses

### 2.7.1 | RNA Extraction Method and Libraries Preparation

Double-stranded RNAs were extracted following the method described in Incarbone and Ritzenthaler (2020) from leaves of a single GVA-infected vine from Nothalten (Section 2.2) prior to HTS. Double-stranded RNAs were sent to the GeT-Genotoul platform facility (INRAE Toulouse, France), where cDNA libraries were prepared and processed. The HTS approach chosen for this study was a paired-end 2×150 bp RNAseq completed on a HiSeq 3000 (Illumina, San Diego, CA, USA) following manufacturer's instructions, which generated 43,875,940 total raw reads.

### 2.7.2 | Bioinformatic Analyses

Bioinformatic analyses was completed using CLC Genomics Workbench 8.5.1 software (CLC bio Genomics, Qiagen, Aarhus, Denmark). After a trimming procedure, cut-off set at 0.05 with no removal of ambiguous nucleotides, only reads above 60 nucleotides (nt) were kept, consisting of 43,588,323 (99.34% of raw reads) with an average of 145.6 nt. After removal of reads corresponding to 18S (AF207053), mitochondrial (FM179380), chloroplastic (DQ424556) and the grapevine transcriptome (Genoscope Vitis12x-v2), the sanitary status of the sample was evaluated by two methods, as previously described (Hily et al. 2018). First steps involved a direct mapping of reads against a collection of reference sequences of grapevine-infecting viruses (Martelli 2017) using relaxed mapping stringency (0.5/0.7, corresponding to read length/ similarity parameters in CLC software) in order to span maximum genome diversity within virus species. Secondly, a *de novo* assembly strategy (word size of 21, minimum contig length of 250 nt) was used followed by BlastN/BlastX annotation of contigs.

## 3 | Results

### 3.1 | Optimal Time for Virus Acquisition and Inoculation by *P. corni* L1

L1 nymphs did not infect any recipient vine whatever the virus source vine (Y258 or P70) or the combinations of AAP (1, 6 or 24 h) and IAP (1, 6 or 24 h), even with the three additional combinations tested with Y258 (AAP 72 h/IAP 1, 3 or 6 h) (data not shown). Moreover, virus content analyses of mobile L1 according to AAP (1, 2, 4, 6, 8 h, 1–5 days) showed only one sample positive at 4 days and one at 5 days, suggesting that nymphs either did not feed during the first 3 days after hatching, or were too few to acquire enough virus content for detection in them. This can explain why no recipient vine was detected positive.
## 3.2 | Transmission Capacity for Different Virus Species or Strains

The ampeloviruses GLRaV-1 and -4 (strain 5), and the vitivirus GVA were transmitted by *P. corni* L1 nymphs, whereas GLRaV-2, -3, -4 (strains 4, 6 and 9) were not (Table 1). After inoculation with L1 fed on Y245, more plants were infected with GVA (16 plants) than with GLRaV-1 (13). One plant was positive for only GLRaV-1, while four were positive for only GVA, the reminder being positive for both viruses. Only one plant out of 15 became infected with GLRaV-4 (strain 5) by L1 (OD 2 h = 1.726, healthy control = 0.486, positive control = 3.360). Healthy control plants, maintained together with the recipient plants, tested negative in all experiments.

As to *P. corni* L2, GVA was detected only in 25% of *N. benthamiana* seedlings inoculated by nymphs collected from a GLRaV-1 and GVA-infected vine. No transmission was observed from GVA-infected *N. benthamiana* or Riesling to either vine cuttings or *N. benthamiana* seedlings. No transmission either was recorded with nymphs fed on purified GVA virions across a Parafilm membrane, even though adding methylene blue to the feeding medium showed blue staining in their gut after 48 h on the membrane, indicating that they have fed. Moreover, after AAP across a membrane, GVA RNA was detected by RT-PCR in GVA feeding solutions and in a batch of 30 nymphs; however, despite 7 days IAP on P115 cuttings, no GVA transmission was obtained.

GLRaV-1 and GLRaV-3, but not GVA, were detected by RT-PCR in a sample of 10 *P. persicae* L1 from Turckheim after IAP on Y258. No virus transmission was recorded using *P. persicae* nymphs (Table 2).

## 3.3 | Virus Retention in *P. corni* Nymphs

After a 10 days AAP on P70 (coinfected with GLRaV-1 and GVA) for L1, or a transfer from vineyard-infected vines for L2, nymphs either feeding on sour cherry leaves and twigs, or starving, were sampled daily for testing their virus content. Many nymphs had their stylets stuck in leaves or twigs, when sampled.

Among the five batches of 20 L1 after AAP on P70 leaves, all the samples taken before transfer onto sour cherry or starving (Day 0) were positive by RT-PCR for GLRaV-1, but not for GVA (Table 3). In batches fed on sour cherry, GLRaV-1 was detected by RT-PCR until Day 11, except at Days 7 and 8. Moreover, GVA was detected only in one batch on the first day. For starved nymphs, even though only 1–2 survived among 20 L1 in the batches after Day 4, GLRaV-1 was detected by RT-PCR throughout 4 days, while GVA was detected only after one starving day. GLRaV-1 was not detected by ELISA in leaves of sour cherry, which were used as host plants, before and after nymph settling (Days 0, 4, 5, 6, 7, 8, 10, 11).

Among the batches of 20 L2 sampled from infected vines of Nothalten, only 2–7 survived after 7 days starving. GLRaV-1 was detected by RT-PCR still until 7 days, while GVA was detected only at Days 1 and 4 (Table 4). The L2 transferred to sour cherry tested positive for GLRaV-1 until Day 30, although negative at

Days 7, 8 and 20. Surprisingly, L2 originating from a GLRaV-3-infected vine of Bennwihr tested positive until 50 days on sour cherry (Table 5). When detaching nymphs, several of them had their stylets tightly fixed inside plant tissues, attesting that they effectively fed. No virus was detected in leaves and twigs of sour cherry, before and after nymph settling (Days 0, 5, 22, 25).

## 3.4 | High Throughput Sequencing (HTS) Analyses on GVA-Only Infected Vine

The analysis of the virome of the Riesling plant infected by GVA only confirmed the presence of one GVA variant, as well as six *Grapevine rupestris stem pitting-associated virus* (GRSPaV, *Betaflexiviridae*, *Foveavirus*) variants. However, no reads corresponding to leafroll viruses were detected (Table 6).

## 4 | Discussion

The laboratory experiments described here aimed at better understanding the vector biology of *P. corni* in relation to grapevine-infecting ampelo- and vitiviruses, by performing controlled transmission and retention experiments. The related species, *P. persicae*, was also tested for its possible vector ability.

## 4.1 | Controlled Transmission Experiments

Our experiments with controlled AAP on accession Y245 revealed that *P. corni* L1 nymphs transmitted GLRaV-1 and GVA (29.5% and 36.4%, respectively; Table 1). Lower transmission rates of 20% for GLRaV-1 and GVA were obtained with L1 from accession P70 (Hommay et al. 2008). In other transmission experiments, pseudococcids seemed to be better transmitters than coccids. For example, for *Ph. aceris* L1, maximal transmission rates of GLRaV-1 and GVA reached 90% and 100%, respectively, with 48 h AAP and 48 h IAP (Le Maguet et al. 2012). Le Maguet (2012) obtained higher transmission rates with *Ph. aceris* than with the coccid *Neopulvinaria innumerabilis* (Rathvon). Moreover, transmission rates of GLRaV-3 and -4 (strain 5) by the pseudococcid *Planococcus ficus* Signoret were respectively 23.3% and 8.3%, whereas those by the coccid *Ceroplastes rusci* (L) were only 3.3% and 1.7% (Mahfoudhi, Digiaro, and Dhouibi 2009). Another pseudococcid, *Pseudococcus maritimus* (Ehrhorn), was a more efficient vector of GLRaV-3 than *P. corni* (Bahder et al. 2013). However, a lower transmission efficiency of coccids may be compensated by their high fecundity (ca. 3000 eggs laid per female in *P. corni*; Canard 1958) in terms of potential virus spread in vineyards.

When GLRaV-1 and GVA were both present in source vines from the vineyard, GVA was mainly transmitted along with GLRaV-1, whereas the latter could be transmitted alone (Hommay, Beuve, and Herrbach 2022b). No transmission was observed from GVA-infected *N. benthamiana* or Riesling to either vine cuttings, or *N. benthamiana* seedlings. GVA transmission could not be observed along with GLRaV-3 according to the inability of *P. corni* to transmit GLRaV-3 in our study (Table 1). It is suggested that GVA transmission may benefit from the presence of GLRaV-1, raising the hypothesis of a cotransmission or 'heteroassistance'
<table><caption>TABLE 1 | Results of transmission experiments performed with L1 and L2 nymphs of *Parthenolecanium corni* on recipient plants.</caption><thead><tr><th rowspan="3">Source cultivar and accession</th><th rowspan="3">Vine origin</th><th rowspan="3">Viruses in source plants</th><th rowspan="3">Nymphal stage</th><th rowspan="3">Recipient plants</th><th colspan="6">Transmission rates</th></tr><tr><th colspan="6">GLRaV-4 strains</th></tr><tr><th>GLRaV-1</th><th>GLRaV-2</th><th>GLRaV-3</th><th>4</th><th>5</th><th>6</th><th>9</th><th>GVA</th><th>GVB</th></tr></thead><tbody><tr><td>Karaskis Y245</td><td>France</td><td>GLRaV-1, -3; GVA</td><td>L1</td><td>P115</td><td>13/44</td><td>—</td><td>0/44</td><td>—</td><td>—</td><td>—</td><td>—</td><td>16/44</td><td>—</td></tr><tr><td>Chardonnay V38</td><td>France</td><td>GLRaV-2, -3</td><td>L1</td><td>Muscat ottonel</td><td>—</td><td>0/16</td><td>0/16</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Koudsi Y252</td><td>Israel</td><td>GLRaV-4 (str.4); GVA</td><td>L1</td><td>P115</td><td>—</td><td>—</td><td>—</td><td>0/15</td><td>—</td><td>—</td><td>—</td><td>nt</td><td>—</td></tr><tr><td>Black Monukka Y199</td><td>USA</td><td>GLRaV-4 (str.5 &amp; 6); GVA</td><td>L1</td><td>P115</td><td>—</td><td>—</td><td>—</td><td>0/6</td><td>—</td><td>—</td><td>—</td><td>nt</td><td>—</td></tr><tr><td>White Emperor Y217</td><td>USA</td><td>GLRaV-4 (str.5); GVA</td><td>L1</td><td>Pinot noir</td><td>—</td><td>—</td><td>—</td><td>—</td><td>1/15</td><td>—</td><td>—</td><td>nt</td><td>—</td></tr><tr><td>Cabernet Sauvignon Cab119-66</td><td>France</td><td>GLRaV-4 (str.9); GVA</td><td>L1</td><td>P115</td><td>—</td><td>—</td><td>—</td><td>—</td><td>0/20</td><td>—</td><td>—</td><td>nt</td><td>—</td></tr><tr><td>Chardonnay TE132</td><td>France</td><td>GLRaV-1; GVB</td><td>L1</td><td>P115</td><td>0/23</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>0/13</td><td>0/13</td><td>—</td></tr><tr><td>N. benthamiana</td><td>GVA pur.</td><td></td><td>L1</td><td>N. benthamiana</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>0/15</td><td>—</td></tr><tr><td>N. benthamiana</td><td>GVA pur.</td><td></td><td>L1</td><td>P115</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>0/20</td><td>—</td></tr><tr><td>N. benthamiana</td><td>GVA</td><td></td><td>L1</td><td>N. benthamiana</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>0/18</td><td>—</td></tr><tr><td>N. benthamiana</td><td>GVA</td><td></td><td>L1</td><td>P115</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>0/15</td><td>—</td></tr><tr><td>Riesling</td><td>France</td><td>GVA</td><td>L1</td><td>P115</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>0/30</td><td>—</td></tr><tr><td>Riesling</td><td>France</td><td>GVA</td><td>L1</td><td>N. benthamiana</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>0/8</td><td>—</td></tr><tr><td>Riesling</td><td>France</td><td>GLRaV-1; GVA</td><td>L2</td><td>N. benthamiana</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>3/12</td><td>—</td></tr></tbody></table>

Note: Transmission rate = number of positive plants/number of inoculated plants. Positive detections are highlighted in bold.

Abbreviations: nt, not tested; pur., purified virions from infected *Nicotiana benthamiana*; str., strain.
<table><caption>TABLE 2 | Results of transmission experiments performed with L1 and L2 nymphs of *Parthenolecanium persicae*, on recipient plants tested 1 year after inoculation.</caption><thead><tr><th rowspan="2">P. persicae origin</th><th rowspan="2">Nymphal stage</th><th rowspan="2">Source cultivar and accession</th><th rowspan="2">Vine origin</th><th rowspan="2">Viruses in source vines</th><th rowspan="2">Recipient vines</th><th colspan="3">Transmission rates</th></tr><tr><th>GLRaV-1</th><th>GLRaV-3</th><th>GVA</th></tr></thead><tbody><tr><td>Ihringen</td><td>L1</td><td>Liali Bidona Y258</td><td>Armenia</td><td>GLRaV-1, -3; GVA</td><td>P114</td><td>0/17</td><td>0/17</td><td>0/17</td></tr><tr><td>Turkheim</td><td>L1</td><td>Liali Bidona Y258</td><td>Armenia</td><td>GLRaV-1, -3; GVA</td><td>Muscat ottonel</td><td>0/29</td><td>0/29</td><td>nt</td></tr><tr><td rowspan="2">Saint-Émilion</td><td>L1</td><td>Liali Bidona Y258</td><td>Armenia</td><td>GLRaV-1, -3; GVA</td><td>P115</td><td>0/2</td><td>0/2</td><td>nt</td></tr><tr><td>L1</td><td>Pinot noir P70</td><td>France</td><td>GLRaV-1, GVA</td><td>P115</td><td>0/2</td><td>0/2</td><td>nt</td></tr><tr><td colspan="6"></td><td>0/3</td><td>—</td><td>nt</td></tr></tbody></table>

Abbreviations: nt, not tested; transmission rate, number of positive plants/number of inoculated plants.

between an ampelo- and a vitivirus, the former acting as a 'helper virus' to the latter (Herrbach et al. 2017). Indeed, literature reports that the vitiviruses GVA (Engelbrecht and Kasdorf 1985; Hommay et al. 2008; Zorloni et al. 2006), GVB (Le Maguet et al. 2012) and GVE (Nakaune et al. 2008) when acquired from coinfected vines, were transmitted by scale insects along with either GLRaV-1 or -3. GVA and GVB occurred more often in the presence of coinfecting GLRaVs (GLRaV-1, -2 or -3) than in their absence. Moreover, the titres of vitiviruses in coinfection with leafroll viruses were higher than in the absence of leafroll virus infection (Rowhani et al. 2018). Blaisdell et al. (2020) found that the transmission of GVA by mealybugs is increased by the presence of GLRaV-3. In our experiment with Y245-fed L1, three recipient vines were infected with GVA alone (Table 1). Nevertheless, this result does not contradict the possibility that GVA would need the presence of GLRaV to be acquired and/or inoculated, since it occurs in transmission experiments to *N. benthamiana*. Literature reports on similar experiments sustain either the cotransmission of GVA and a leafroll virus (Fortusini et al. 1997; Sforza, Boudon-Padieu, and Greif 2003; Zorloni et al. 2006; Tsai et al. 2010; reviewed in Herrbach et al. 2017), or the independence between these viruses (Bertin, Cavalieri, et al. 2016; Bertin, Pacifico, et al. 2016). Fortusini et al. (1997) reported the transmission by *N. innumerabilis* of GVA alone (i.e., without GLRaV-1) to one vine, suggesting that the helper would be the vitivirus. However, the recipient vines were tested only once (4 months post transmission) and it is sometimes observed that vitiviruses are detected earlier than the coinfecting ampelovirus (Hommay et al. 2009). However, the mealybug *Pseudococcus longispinus* (Targioni Tozzetti) was able to transmit GVA from infected *Nicotiana clevelandii* Grey plants to healthy ones, after only a 30 min feeding (La Notte et al. 1997), suggesting that GVA could be transmitted alone by this mealybug between herbaceous plants.

We allowed *P. corni* nymphs to acquire GVA virions purified from *N. benthamiana*, across a Parafilm membrane, but again no transmission to grapevine occurred (Table 1). Loss of a putative helper factor or of heteroassistance ability, or degradation of virions during the purification process are possible explanations. Similarly, *Ps. longispinus* did not transmit GVA from purified virions to healthy *N. clevelandii* (La Notte et al. 1997). In contrast, one transmission event of GLRaV-1 with *Ph. aceris* was reported from purified virions (Alliaume 2016), though this needs to be confirmed. Furthermore, Prator and Almeida (2020) obtained efficient transmission of GLRaV-3 by *Pl. ficus* feeding on purified virions extracted from grapevine or *N. benthamiana*. Further work is required to elucidate the transmission of GVA in association with GLRaV-1, that could involve 'hybrid' GLRaV-1-GVA virions (Alliaume et al. 2018), in particular by determining location and chemical nature of virus receptors in the vector. More generally, the effects of multi-infections on ampelo- and vitivirus transmission and their interaction in host plants need further research to settle the divergent observations reported. In addition, as stressed by Rowhani et al. (2018), the possible synergistic effect of other coinfecting viruses, such as GRSPaV (*Foveavirus*) or *Grapevine fleck virus* (GFkV, *Maculavirus*), should be examined.

Even though GVA is known to occur rarely alone in grapevine, it has sometimes been reported to occur without
<table><caption>**TABLE 3** | RT-PCR detection rates of GLRaV-1 and GVA RNA in batches of 20 *Parthenolecanium corni* L1 nymphs reared from birth to 10 days AAP on excised P70 leaves and then left either to feed on sour cherry leaves up to 11 days, or to starve 1–4 days.</caption><thead><tr><th rowspan="2">Diet</th><th colspan="11">Nymphs fed on sour cherry leaves</th></tr><tr><th>0</th><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>10</th><th>11</th></tr></thead><tbody><tr><th>Time (days)</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>GLRaV-1</th><td>5/5</td><td>5/5</td><td>5/5</td><td>4/5</td><td>3/5</td><td>2/5</td><td>5/5</td><td>0/5</td><td>0/5</td><td>2/5</td><td>3/5</td></tr><tr><th>GVA</th><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td></tr></tbody></table><table><thead><tr><th rowspan="2">Diet</th><th colspan="11">Starved nymphs</th></tr><tr><th>0</th><th>1</th><th>2</th><th>3</th><th>4</th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><th>Time (days)</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>GLRaV-1</th><td>5/5</td><td>5/5</td><td>3/5</td><td>4/5</td><td>5/5</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>GVA</th><td>0/5</td><td>2/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

Note: Detection rates = number of virus positive batches/number of surviving batches. Positive detections are highlighted in bold.

<table><caption>**TABLE 4** | Viral RNA detection rates in batches of 20 *Parthenolecanium corni* L2 nymphs from Nothalten vines coinfect ed with GLRaV-1 and GVA, according to time spent on sour cherry leaves or starving.</caption><thead><tr><th rowspan="2">Diet</th><th colspan="11">Nymphs fed on sour cherry leaves</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>9</th><th>10</th><th>15</th><th>20</th><th>25</th><th>30</th><th>35</th></tr></thead><tbody><tr><th>Time (days)</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>GLRaV-1</th><td>5/5</td><td>3/5</td><td>1/5</td><td>1/5</td><td>2/5</td><td>1/5</td><td>0/5</td><td>0/5</td><td>1/5</td><td>3/5</td><td>4/5</td><td>0/5</td><td>2/5</td><td>1/4</td><td>0/4</td></tr><tr><th>GVA</th><td>3/5</td><td>2/5</td><td>1/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>1/5</td><td>1/4</td><td>0/4</td></tr></tbody></table><table><thead><tr><th rowspan="2">Diet</th><th colspan="11">Starved nymphs</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><th>Time (days)</th><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>GLRaV-1</th><td>4/7</td><td>2/7</td><td>4/7</td><td>4/7</td><td>1/7</td><td>2/3</td><td>4/7</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><th>GVA</th><td>2/7</td><td>0/7</td><td>0/7</td><td>1/7</td><td>0/7</td><td>0/3</td><td>0/7</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

Note: Positive detections are highlighted in bold.

<table><caption>**TABLE 5** | Viral RNA detection rates of batches of 20 *Parthenolecanium corni* L2 nymphs from a vine of Bennwihr infected with GLRaV-3, according to time spent on sour cherry twigs.</caption><thead><tr><th>Feeding time (days)</th><th>0</th><th>25</th><th>28</th><th>37</th><th>44</th><th>50</th></tr></thead><tbody><tr><th>GLRaV-3 detection rate</th><td>5/5</td><td>1/5</td><td>1/5</td><td>5/5</td><td>5/5</td><td>2/2</td></tr></tbody></table>

Note: Positive detections are highlighted in bold.

<table><caption>**TABLE 6** | GenBank numbers (no.), size (nt) of the sequences tested, read numbers and coverage depth for viruses found in the samples analysed.</caption><thead><tr><th>Virus</th><th>Variant</th><th>GenBank no.</th><th>Size</th><th>Reads no.</th><th>Coverage</th></tr></thead><tbody><tr><td rowspan="6">GRSPaV</td><td>1</td><td>OP079931</td><td>8725</td><td>86,938</td><td>1450.8</td></tr><tr><td>2</td><td>OP079932</td><td>8722</td><td>73,857</td><td>1232.9</td></tr><tr><td>3</td><td>OP079933</td><td>8714</td><td>68,592</td><td>1146.1</td></tr><tr><td>4</td><td>OP079934</td><td>8698</td><td>62,377</td><td>1044.2</td></tr><tr><td>5</td><td>OP079935</td><td>8711</td><td>42,814</td><td>715.6</td></tr><tr><td>6</td><td>OP079936</td><td>8314</td><td>3688</td><td>64.6</td></tr><tr><td>GVA</td><td>1</td><td>OP079937</td><td>7336</td><td>5262</td><td>104.4</td></tr></tbody></table>

Note: Reads were mapped on a set of reference viruses previously described as infecting *Vitis vinifera*. For mapping, a low stringency was used with parameters set to 0.5 for read length and 0.7 for similarity on each *de novo* assembled genomes.

Abbreviations: GRSPaV, *Grapevine rupestris* stem pitting-associated virus; GVA, *Grapevine virus A*.
GLRaV-1 or -3 (Rowhani et al. 2018; Agran et al. 1990; Credi and Giunchedi 1996; Milkus, Avery Jr., and Goodman 2000; Gribaudo et al. 2009). Likewise, we found a vine at Nothalten that tested positive for GVA only, but not for any leafroll virus species or strain (GLRaV-1, -2, -3, -4), neither by ELISA and RT-PCR, nor by HTS as mentioned above (Section 3.4).

Our AAP and IAP experiments yielded no transmission event with *P. corni* L1 that first settled on accessions Y258 or P70, both coinfect ed with GLRaV-1 and GVA, contrary to *H. bohemicus* L1 under the same conditions (Hommay, Beuve, and Herrbach 2022a). Therefore, optimal times for virus acquisi- tion and inoculation by L1 could not be defined. It is possible that time allowed to AAP (max 72 h) or IAP (max 24 h) was globally too short for L1 of this species. Transmission events by *P. corni* L1 have previously been obtained from P70, but with longer AAP/IAPs (12 days/8 days) (Hommay et al. 2008). During feeding, nymphs are deeply anchored in plant tissues with their stylets, and detaching them from leaves with their stylets is risky and delicate. The only way to avoid injuring nymphs is to let them move freely from infected leaves to healthy ones. Wandering nymphs readily migrated from virus source leaves to recipient leaves. However, it is not sure that these individuals spent enough time to feed and acquire vi- ruses. Among L1 allowed to feed between 1 h and 5 days on P70 vines, mobile nymph batches tested positive for this virus only from 4 days on. Nymphs used in transmission ex- periments from Y245 (coinfect ed with GLRaV-1, -3 and GVA) were allowed to feed during longer periods (5–7 days AAP and 7 days IAP) and were able to transmit GLRaV-1 and GVA to ca. 30% of recipient vines (Table 1). In previous experiments using vineyard-collected L1, IAPs of 5–7 days showed also to be sufficient for GLRaV-1 and GVA transmission (Hommay, Beuve, and Herrbach 2022b).

No GLRaV-3 transmission was obtained with two different ac-
cessions coinfect ed with either GLRaV-1 and GVA (Y245), or
GLRaV-2 (V38), whereas both GLRaV-1 and GVA were trans-
mitted from accession Y245 to recipient vines (Table 1). These
results confirm our transmission tests with different vineyard
populations (Hommay, Beuve, and Herrbach 2022b). As ex-
pected, the closterovirus GLRaV-2 was not transmitted by *P.
corni* (Table 1). While most members of the genus *Closterovirus*
are transmitted by aphids (Zhu et al. 1998; Fuchs 2021), no vec-
tor is known so far for GLRaV-2 (Herrbach et al. 2017; Wistrom
et al. 2017).

Moreover, we report for the first time the transmission by *P. corni* of GLRaV-4 (strain 5) (accession Y217) (Table 1), even though only one transmission event was obtained and further confirmation will be needed. *P. corni* failed to transmit GLRaV-4 (strains 4, 6 and 9), which are now assigned as isolates of the same species GLRaV-4 (Abou Ghanem-Sabanadzovic et al. 2012; Martelli 2017). Using the same accessions as source plants and recipient vines (P115), all these strains, including strain 5, were efficiently transmitted by the mealybug *Ph. aceris* (Le Maguet et al. 2012).

As to *P. persicae* coming from three different vineyards, con-
trolled experiments showed no transmission of GLRaV-1,
GLRaV-3 or GVA. Due to the low number of nymphs available,

replicates were yet insufficient to formally exclude the ability of
this species to transmit these viruses, and additional transmis-
sion experiments with other populations are needed.

4.2 | Retention Experiments

Aiming at evaluating the duration of viral retention in viru-
liferous nymphs, by using RT-PCR, we detected viral RNA in
batches of L1 and L2 having previously acquired GLRaV-1 and
GVA, then either transferred onto sour cherry (a nonhost for
these viruses, but a host for *P. corni*), or starved. In starving
nymphs, GLRaV-1 RNA could be detected up to 4 and 7days in
L1 and L2, respectively (Tables 3 and 4). In L1 and L2 fed on
sour cherry, GLRaV-1 RNA remained detectable up to 11 days
and 30days, respectively. GVA was less frequently detected than
GLRaV-1 in both L1 and L2. Since few starving L2 survived
up to 7days, it can be supposed that congeners transferred on
sour cherry leaves fed actively, but did not release their initial
virus content until 30days. Another experiment with L2 com-
ing from a GLRaV-3-infected vine gave positive RT-PCR results
up to 50days for L2 fed on sour cherry (Table 5). However, our
experiment shows long retention in *P. corni* nymphs of viral
RNA only. Therefore, it does not warrant the presence of com-
plete and even transmissible virions, which seems unlikely but
requires further work to be elucidated. In literature, GLRaV-1
and GVA were no more detected in, nor transmitted by *Ph. ac-
eris*, 7 and 5days, respectively, after leaving a source infected
with both viruses (Alliaume 2016). The ability of mealybugs of
various species to transmit GLRaV-3 was lost 4days after being
removed from an infected source (Tsai et al. 2008; Le Maguet
et al. 2012; Krüger et al. 2015; Bertin, Pacifico, et al. 2016;
Petersen and Charles 1997). Krüger et al. (2015) found that re-
tention time reached 8 and 2days in respectively feeding and
starving *Pl. ficus* nymphs. However, GLRaV-3 was detected in
L1 of *Pseudococcus calceolariae* (Maskell) up to 16days on non-
*Vitis* host plants, but no more after 20days (McGreal et al. 2021).
As transfer of L2 onto sour cherry twigs started at the begin-
ning of autumn, they possibly followed a similar physiological
process as overwintering nymphs, and thus viruses, or frag-
ments thereof, could remain inside the digestive tract due to a
slowed down activity. Retention time could be much longer in
overwintering nymphs. Le Maguet (2012) observed that viral
RNA remained detectable in *Ph. aceris* nymphs from December
to February. Similarly, Hommay et al. (2022a, 2022b) showed
that a majority of *P. corni* and *H. bohemicus* nymphs collected
in the vineyard on infected vines tested positive for viral RNA
throughout the winter season. Overwintering *P. corni* nymphs
frequently kept their stylets anchored inside the cane tissues
and had likely access to infected sap, contrary to *Ph. aceris* and
*H. bohemicus* which overwinter within a cocoon between bark
layers and could not reach phloem. However, when exposed to
room temperature for transmission experiments, overwintering
*P. corni* nymphs failed to transmit any virus to healthy plants
(Hommay, Beuve, and Herrbach 2022b).

5 | Conclusions and Prospects

Our transmission experiments made with different grape-
vine leafroll virus species or strains thereof (GLRaV-1, -2, -3,
-4 [strains 4, 5, 6 and 9]) showed that *P. corni* is able to transmit GLRaV-1 and GLRaV-4 (strain 5) from vine to vine. To our knowledge, this is the first report that *P. corni* is a vector of GLRaV-4 (strain 5). GVA was transmitted mainly along with GLRaV-1 and rarely alone.

Surprisingly, *P. corni* L2 removed from an infected vine and placed on a nonhost plant tested positive for GLRaV-1 and GLRaV-3 during at least 30 and 50 days, respectively, periods much longer than the 4–5 days usually reported for other scale insects and ampeloviruses. Further research is needed to elucidate virus-vector interactions and factors influencing the retention of viruses and the transmission efficiency by scale insects, as well as potential interactions between cotransmitted viruses. In particular, further work is required to localise where virions are precisely retained before being released for inoculation and to identify transmission determinants on the viruses to understand virus-vector molecular interactions.

## Author Contributions

**Gérard Hommay:** methodology, data curation, formal analysis, writing – original draft, writing – review and editing, supervision, conceptualization. **Antoine Alliaume:** methodology, writing – review and editing. **Monique Beuve:** methodology, writing – review and editing. **Jean-Michel Hily:** writing – review and editing, methodology, formal analysis. **Etienne Herrbach:** funding acquisition, project administration, methodology, writing – review and editing, conceptualization.

## Acknowledgements

We thank the French vine-growers Armand Bogen (Bennwihr) and Patrick Meyer (Nothalten), as well as Philippe Kuntzmann (VitiVina, Sigolsheim, Alsace) and Dr Michael Breuer (*Staatliches Weinbauinstitut*, Freiburg, Germany), for allowing us to collect soft scales and plant material in their vineyards. We thank Delphine Binet for providing egg-laying *P. persicae* females from Saint-Émilion (Gironde, France). We are also grateful to Jacky Misbach and the staff of *Unité Expérimentale Agronomique et Viticole* (INRAE Grand Est–Colmar) for production and care of experimental grapevine material and to Dr Christoph Hoffmann (*Julius Kühn-Institute*, Siebeldingen, Germany) for useful information on *P. persicae*.

## Conflicts of Interest

The authors declare no conflicts of interest.

## Data Availability Statement

Raw data tables are available on DOI: 10.5281/zenodo.14040177.

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