Identification of phytoplasmas associated with grapevine ‘bois noir’ and flavescence dorée in inter-row groundcover vegetation used for green manure in Franciacorta vineyards

‘Bois noir’ (BN) and flavescence dorèe (FD), the two main diseases of the grapevine yellows complex associated with genetically distinct phytoplasmas, have a complex epidemiology including multiple insect vectors and reservoir plants. This study investigated the presence of BN and FD phytoplasmas in nine groundcover plant species commonly utilized for inter-row vineyard green manure in Franciacorta (North Italy). The activities conducted in 2020 included monitoring and sampling groundcover plant species and symptomatic grapevines in September, and phytoplasma identification and typing by amplification and sequence analyses of stamp and map genes. Molecular analyses identified BN phytoplasma (strains carrying the stamp gene sequence variants St5, St19, St30) and FD phytoplasma (strains carrying the map gene sequence variant M54) in 72% and 28% of symptomatic grapevines, respectively. BN phytoplasma strains St5 and St30 were found also in Eruca sativa, Vicia sativa, and Polygonum fagopyrum. FD phytoplasma strain M54 was found also in Vicia faba, Trifolium incarnatum, and Polygonum fagopyrum. These results reinforced the evidence of the increasing range of BN and FD phytoplasma alternative plant hosts and suggested a criterium for the selection of the groundcover plant species utilized for green manure, excluding the ones putatively involved in BN and FD diffusion.


Introduction
Phytoplasmas are cell wall-less prokaryotic unicellular microorganisms belonging to the Mollicutes class.They are phloem-limited obligate intracellular parasites of a broad associated with genetically distinct phytoplasmas (Belli et al. 2010).
Phytoplasma strains belonging to the taxonomic group 16SrV, subgroups -C and -D, are associated with FD.FD phytoplasmas (FDp) are efficiently vine-to-vine transmitted by the ampelophagous insect vector Scaphoideus titanus Ball, determining highly epidemic diffusion of the disease (Arnaud et al. 2007;Chuche and Thiery 2014).For that reason, FDp is a quarantine pathogen, and its control is regulated by mandatory measures including the eradication of infected grapevines and the insecticide treatments against S. titanus (Belli et al. 2010).Further evidence highlighted that FD epidemiology is not limited to the close pathosystem "grapevine -S.titanus".It was demonstrated that (i) D. europaea is a vector of FDp from Clematis vitalba L. to grapevine (Filippin et al. 2009), (ii) Orientus ishidae (Matsumura) can acquire FDp from multiple plant sources and transmit it to grapevine and Alnus glutinosa L. (Mehle et al. 2010;Gaffuri et al. 2011;Parise 2017), (iii) Allygus spp.are vectors of FDp from alder to alder and faba bean (Malembic-Maher et al. 2020).
Furthermore, studies conducted in the last years have led to the identification of insects able to acquire FDp, such as Phlogotettix cyclops (Mulsant & Rey) (Strauss et al. 2018) and Hishimonus hamatus Kuoh (Belgeri et al. 2022).Interestingly, other studies demonstrated that S. titanus is able to survive on herbaceous (Amaranthus sp., Chenopodium sp., Convolvulus sp., Daucus carota L., Onoclea sensibilis L., Polygonum sp., Solidago sp.) and woody (Crataegus sp., Juniperus virginiana L., Malus sp., Prunus persica (L.) Batsch, Salix sp., Ulmus americana L.) plants (Chuche and Thiery 2014).Furthermore, based on observations conducted in vineyards, it was noticed that S. titanus, knocked down to the ground following intense rains, can survive up to 3-4 days on several weeds such as Cyperus rotundus L., Dandelion officinale Weber, Galinsoga parviflora Cav., and Trifolium repens L. (Mori, personal communication).Based on this evidence, it is reasonable to hypothesize that also weeds could be involved in FDp spreading.
Green manure is a common agronomic practice in vineyards.It is conducted to improve soil quality and consists in growing in the vineyard inter-rows and ploughing into the terrain mixtures of selected grasses and legume plants (groundcover crops).Several benefits are generated by this practice: erosion and leaching are reduced by the improvement of the soil physical structure (Ingels et al. 2005); activity of N cycle enzymes is enhanced by the higher carbon availability for the soil microorganisms (Okur et al. 2016); pest control is improved by the attractiveness of the selected groundcover plants for natural predators/parasitoids (Irvin et al. 2014); the grape organoleptic features are improved (Rotaru et al. 2011).Although the agronomic advantages of green manure have been recognized, the phytosanitary consequences on grapevine diseases should be considered.
In this study, we investigated the role of nine ground cover plant species (Eruca sativa Miller, Sinapis arvensis L., Lobularia maritima (L.) Desv., Phacelia tanacetifolia Benth., Vicia sativa L., Vicia faba var.minor Beck, Trifolium incarnatum L., Trifolium alexandrinum L., Polygonum fagopyrum L.), commonly used for inter-row green manure of vineyards in Franciacorta (northern Italy), as alternative plant hosts of phytoplasmas associated with BN and FD, putatively involved in the spread of such diseases.

Phytoplasma detection
Total nucleic acids (TNAs) were extracted from 1 g of petiole and/or leaf tissue of all the collected plant samples using a CTAB-based extraction protocol previously described (Angelini et al. 2001).Obtained TNAs were solved in 150 µl distilled sterile water, their quality and concentration were measured by Nanodrop system, and they were stored at -20 °C until further use.Extracted TNAs were utilized as templates in nested PCR reactions conducted for CaPsol and 16SrV phytoplasma specific identification through the amplification of the stamp and map gene, respectively.For stamp gene, direct PCRs were performed using the primer pair StampF/StampR0, followed by nested PCRs with the primer pair StampF1/StampR1; primer sequences and reaction conditions were as previously described (Fabre et al. 2011).For map gene, direct PCRs were performed using the primer pair FD9F5/MapR1, followed by nested PCRs with the primer pair FD9F6/MapR2; primer sequences and reaction conditions were as previously described (Arnaud et al. 2007).Total nucleic acids from periwinkle plants infected by phytoplasma strains STOL ('Ca.P. solani', subgroup 16SrXII-A, Acc.No. AF248959; Quaglino et al. 2013), AY1 ('Ca.P. asteris', subgroup 16SrI-B, M30790; Lee et al. 2004a, b) and EY1 ('Ca.P. ulmi', subgroup 16SrV-A, AY197655; Lee et al. 2004a, b) were used as reference controls.The reaction mixture devoid of nucleic acids was used as negative control.PCR products were verified by electrophoresis on 1% agarose gel in TBE buffer and visualized under a UV transilluminator.1).

Phytoplasma typing and phylogenetic analysis
In Provaglio d'Iseo vineyard, 16SrV phytoplasmas and CaPsol were detected respectively in 58% and 42% of examined symptomatic grapevines.Moreover, CaPsol was identified in 35% of bindweed plants and 18% of P. fagopyrum plants; 16SrV phytoplasmas were detected also in P. fagopyrum (31% of the examined plants), T. incarnatum (19%), and V. faba (6%) (Table 2).To the best of our knowledge, P. fagopyrum and T. incarnatum were never reported before as 16SrV phytoplasma host plants, while V. faba is known as the main experimental host of FDp (Caudwell et al. 1970).
To hypothesize its putative role in GY epidemiology, an alternative plant host should carry the same phytoplasma strain identified in grapevines exhibiting typical GY symptoms (Mori et al. 2015b;Quaglino et al. 2021).Here, CaPsol strains carrying the sequence variants St5 and St30 were identified in grapevines, C. arvensis (St5 in both Gussago and Provaglio d'Iseo), E. sativa (St5 in Gussago), P. fagopyrum (St5 in Provaglio d'Iseo), and V. sativa (St5 and St30 in Gussago).This evidence reinforced the role of bindweed as CaPsol reservoir plant (Langer et al. 2004).Interestingly, as reported in previous studies conducted in northern Italy by a commercial sequencing service (Eurofins Genomics, Germany).Nucleotide sequences were assembled by the Contig Assembling Program and trimmed to the stamp gene start and stop codons and to the map gene annealing sites of the FD9F6 and MapR2 primers in the software BioEdit version 7.2.6 (Hall 1999).Stamp gene nucleotide sequences, obtained in this study from CaPsol strains identified in grapevines and groundcover plants, were aligned using the ClustalW Multiple Alignment program in the software BioEdit and analyzed by Sequence Identity Matrix to calculate their genetic diversity.The same procedure was utilized for map gene nucleotide sequences of 16SrV phytoplasma strains identified in this study.Finally, the obtained stamp and map sequences were aligned respectively with representative sequences of previously defined stamp (Pierro et al. 2018a, b) and map (Malembic et al. 2020) sequence variants.A nucleotide sequence identity of 100% was necessary for the sequence variant attribution.
Nucleotide sequences of CaPsol representative strains of stamp sequence variants and 16SrV phytoplasma strains representative of map sequence variants, identified in this and in previous studies (Pierro et al. 2018a, b;Malembic et al. 2020), were aligned and used for generating unrooted phylogenetic trees by Neighbor-Joining method performed using the Jukes-Cantor model and bootstrap replicated 1000 times in the MEGAX software (Kumar et al. 2018).
In Gussago vineyard, 16SrV phytoplasmas were not detected in both symptomatic grapevines and groundcover vegetation; CaPsol was identified in all symptomatic grapevines, in 20% of bindweed plants, and in the groundcover plant species E. sativa (9% of the examined plants), P. fagopyrum (9%), and V. sativa (28%) (Table 1).To the best of our knowledge, such plant species were never reported plants, can be vertically transmitted throughout seasons by seeds; (ii) CaPsol strains can be acquired from annual plants and transmitted to grapevine within the same season by alternative vectors living as adults in the vineyards longer than H. obsoletus (Mori et al. 2015b).This last hypothesis is supported by the recent finding of alternative CaPsol vectors (Dictyophara europaea, Dicranotropis hamata, Philaenus spumarius, Euscelis incisus, Euscelidius variegatus) in Franciacorta, characterized by a longer adult stage or able to overwinter as nymphs or adults, carrying CaPsol to the next season (Quaglino et al. 2019).On the other hand, it should be considered that annual plants, identified in this study as CaPsol alterative plant hosts, can represent dead-end hosts of the phytoplasma.

Molecular typing of 16SrV phytoplasma strains
Nucleotide sequence analyses, carried out on map gene nested PCR products (from 32 symptomatic grapevines and 9 groundcover plants of the species P. fagopyrum, T. incarnatum, V. faba) revealed the presence of a unique map sequence variant within 16SrV phytoplasma strain population in Provaglio d'Iseo vineyard (Table 2).Such map variant was undistinguishable from the sequence variant M54 (representative strain VF-00-SP5, Acc.No. AM384886), recently reported in FDp strains associated with FD outbreaks (epidemic strains) (Malembic-Maher et al. 2020).As map gene nucleotide sequence variants carried by FDp strains identified in the present study are identical (100% sequence identity) with variants already published and available in NCBI GenBank, they were not deposited to GenBank to avoid unnecessary repetitions.Phylogenetic analyses confirmed that FDp strain M54 (subgroup 16SrV-D) belongs to Map-FD2 cluster, including other epidemic FDp strains (M38, M121) classified in taxonomic subgroup 16SrV-C (Fig. 2) (Malembic-Maher et al. 2020).Up to now, M54 was reported as the prevalent FDp strain in vineyards from different French viticultural areas, Switzerland, and northwestern Italy (Casati et al. 2017;Rossi et al. 2019;Malembic-Maher et al. 2020).Even if strain M54 was identified mainly in symptomatic grapevines and S. titanus, it was found also in Corylus avellana L. and gone-wild Vitis vinifera L. and the insect vector O. ishidae, suggesting that its epidemics can be related not exclusively to the close pathosystem grapevine-S.titanus (Casati et al. 2017;Rossi et al. 2019).Interestingly, in the present study FDp strain M54 was reported as the unique FDp strain infecting grapevine in the examined vineyard in Franciacorta.Moreover, it was firstly identified in alternative plant hosts (P.fagopyrum, T. incarnatum, V. faba) whose role as reservoirs in FDp diffusion could be related to the activity of S. titanus, recently reported as able to adapt to other plants (Chuche (Mori et al. 2015;Quaglino et al. 2021), CaPsol-infected bindweed plants were symptomless, while in other geographic regions (i.e., Serbia and Georgia) most bindweeds carrying CaPsol showed typical symptoms such as yellowing, reddening, dwarfism, and leaf malformation (Quaglino et al. 2016;Jović et al. 2021).This difference could be related to the genetic diversity among CaPsol strains prevalently identified in bindweeds in these regions: CaPsol strains carrying the stamp gene variant St5, St8 and St10 in northern Italy (Quaglino et al. 2021;this study), St1 andSt2 in Serbia (Jović et al. 2021), St15 andSt37 in Georgia (Quaglino et al. 2016).Moreover, obtained data highlighted the presence of three CaPsol alternative plant hosts, putatively related to feeding activities of additional CaPsol insect vectors recently reported (Quaglino et al. 2019).To study the role of such CaPsol plant hosts as reservoir plants, acquisition and transmission trials with insect vectors are required.Interestingly, in both examined vineyards, CaPsol strains carrying stamp variant St19 were identified exclusively in symptomatic grapevines, reinforcing the hypothesis of the existence of a BN epidemiological pattern including insect vectors able to transmit CaPsol vine-to-vine (Quaglino et al. 2019).On the other hand, CaPsol strains carrying stamp variants St1 and St10 were identified exclusively in groundcover vegetation: St1 in C. arvensis, E. sativa, and V. sativa; St10 in C. arvensis and P. fagopyrum.Interestingly, CaPsol strain St10, never reported before in grapevine, was recently found as the prevalent strain associated with BN in Tuscany vineyards and related to a newly proposed epidemiological pattern involving R. artemisiae as main vector (Pierro et al. 2020).
Phylogenetic analyses evidenced that CaPsol strains St5 and St30, identified in grapevine, E. sativa, P. fagopyrum, and V. sativa, and strains St1 and St10, identified exclusively in groundcover vegetation, belong to bindweedrelated subclusters b-I and b-II, while CaPsol strains St19, identified exclusively in grapevines, belong to nettle-related subcluster a2 (Fig. 1) (Atanasova et al. 2015).This evidence suggests that groundcover vegetation used for green manure could play a role in the diffusion of bindweed-related CaPsol strains.On the other hand, considering the absence of nettle within and around the examined vineyards, it is reasonable to hypothesize that CaPsol nettle-related strains could be closely associated with a vine-to-vine transmission pattern.
As perennial plants constitute the main phytoplasma source involved in the epidemiology of phytoplasma-associated diseases, it is interesting that groundcover plants used for green manure, identified in the present study as CaPsol alternative plant hosts putatively involved in pathogen spread, are annual species.Two hypotheses have been articulated for explaining the possible role of annual plants in BN epidemiology: (i) CaPsol strains, infecting annual Fig. 1 Unrooted phylogenetic tree inferred from stamp gene nucleotide sequences of CaPsol strains representative of stamp sequence variants previously described and identified in this study (Tables 1 and 2); minimum evolution analysis was performed using the neighbour-joining method and bootstrap replicated 1,000 times.Names of strains are reported on the image.Strains from the present study, written in red-bold, are indicated using acronyms of their host plants and the related vineyard as follows: Ca (Convolvulus arvensis), Es (Eruca sativa), Pf (Polygonum fagopyrum), Vs (Vicia sativa), Vv (Vitis vinifera); Gus (Gussago), Ser (Provaglio d'Iseo).Gen-Bank accession number of each sequence is given in parenthesis.Clusters are shown as delimitated by parentheses

Conclusions
Green manure of inter-row groundcover vegetation is an agronomic practice which utilization is increasing particularly in organic vineyard management in Europe.This study demonstrated that some plant species frequently employed for green manure can harbor phytoplasmas associated with both BN and FD.The evidence that CaPsol and FDp strains, identified in groundcover plant species, are undistinguishable from strains infecting grapevines, suggests that such plants could represent inoculum sources involved in the spread to grapevines of GY-associated phytoplasmas.These results reinforced the evidence of the increasing range of CaPsol and FDp alternative plant hosts and suggested a criterium for the selection of the groundcover plant species utilized for green manure, excluding the ones putatively involved in BN and FD diffusion. et al. 2014) and to become infective as adult in a short time frame (1-2 weeks) (Alma et al. 2018).Additionally, polyphagous insect vectors with long flight periods (i.e., D. europaea) could be able to acquire FDp from annual plant hosts and transmit it to grapevine.

Fig. 2
Fig. 2 Unrooted phylogenetic tree inferred from map gene nucleotide sequences of 16SrV strains representative of map sequence variants previously described (Malembic-Maher et al. 2020) and identified in this study (Table 2); minimum evolution analysis was performed using the neighbour-joining method and bootstrap replicated 1,000 times.Names of strains are reported on the image.Strains from the present study, written in red bold, are indicated using acronyms of their host plants and the related vineyard as follows: Pf (Polygonum fagopyrum), Ti (Trifolium incarnatum), Vf (Vicia faba), Vv (Vitis vinifera); Ser (Provaglio d'Iseo).Epidemic FDp strains are written in blue bold.GenBank accession number of each sequence is given in parenthesis.Clusters are shown as delimitated by parentheses

Table 1
Molecular detection and typing of GY associated phytoplasmas in Gussago vineyard before as CaPsol host plants.The remnant five species were found not infected by CaPsol (Table