Molecular and serological detection of Parietaria mottle virus in Phytolacca americana, a new host of the virus

Parietaria mottle virus (PMoV) is an emerging virus in Mediterranean countries, responsible for severe disease in tomato and pepper crops in the field and protected cultivation. The principal wild reservoir of PMoV is Parietaria officinalis, and only few additional wild plants have been described as natural reservoirs of the virus. During field survey in southern Italy, several plants of Phytolacca americana showing virus-like symptoms were collected. Serological and molecular assays showed that these plants were infected by PMoV. Sequence comparison of the movement protein gene of the PMoV isolate from P. americana showed the greatest similarity to the corresponding sequence from tomato plants growing nearby. These results indicate that P. ameriacana is a new natural host of PMoV, and further investigation is warranted to establish the potential of this host as reservoir of the virus in the field.


INTRODUCTION
Phytolacca americana L. (Phytolaccaceae), commonly known as "American pokeweed", "pokeweed" or "dragonberries", is a poisonous perennial geophyte herb native to North America. It was introduced in Europe where it is invasive in many countries, including Italy (Galasso et al., 2018). This neophyte plant mainly spreads in anthropic and disturbed environments (Stinca and Motti, 2017;Bonanomi et al., 2018). In July 2020, during a survey of tomato virus diseases in southern Italy (Campania region), several individuals of P. americana were noticed showing virus-like symptoms, comprising mosaic, chlorotic spots and lines and leaf distortions (Figure 1). The detected plants were growing close to a family garden, located in the Sorrento peninsula, were Parietaria mottle virus (PMoV) was detected in a local variety of tomato with high incidence. Distances from pokeweed plants to the tomato plants ranged between 5 to 20 m. The presence of thrips individuals on tomato and pokeweed plants was also observed. Overall, field observations suggested possible implication of PMoV as the cause of the symptoms observed in pokeweed plants. MATERIALS  The same samples were tested by antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) with a specific PMoV polyclonal antibody (Lisa et al., 1998), following the procedure of Parrella (2020). In addition, samples were tested by cucumber mosaic virus (CMV) and tomato spotted wilt virus (TSWV)-specific lateral flow (LF) kits (Pocket Diagnostic), since in tomato symptoms induced by PMoV resembling those induced by CMV and TSWV in single or mixed infections have also been reported (Ramasso et al., 1997;Roggero et al., 2000).
Total RNAs were extracted with the E.Z.N.A.® Plant RNA Kit (Omega Bio-tek), and RT-PCR using the PMoV specific primers PMoVMP1a and PMoVMP2b, encompassing the entire PMoV movement protein (Parrella et al., 2016). Amplicons of the expected size (916 bp) obtained from symptomatic samples were cloned into pGEM-T vector (Promega), and were sequenced at Microsynth Seqlab (Göttingen, Germany). Blast analysis was performed online (http://blast.ncbi.nlm.nih.gov/ Blast.cgi), and fifteen sequences of other PMoV isolates (Table 1) were downloaded and were used to determine  the phylogenetic relationships among them and the pokeweed isolate (named Pha1). Multiple sequence alignment was performed using the ClustalW algorithm of MEGA 6.0 (Tamura et al., 2013), with gap opening penalty of 15 and gap extension penalty of 6.66. A phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 bootstrap replicates, and genetic distance was calculated by the Tamura three-parameter model which was determined as the best-fi tting model of substitution using MEGA 7.0.

RESULTS AND DISCUSSION
Inoculated host plants showed the following symptoms aft er 1 to 3 weeks, consistent with symptoms caused by PMoV (Ramasso et al., 1997;Marchoux et al., 1999): large chlorotic local lesions followed by dieback and wilting in C. quinoa; large chlorotic local lesions followed by mosaic in C. amaranticolor; necrotic local lesions in V. unguiculata; and pinpoint necrotic local lesions followed by mosaic, leaf distortions, vein necrosis and necrotic patches on the apical non-inoculated leaves of N. tabacum. PMoV infection in symptomatic hosts was confi rmed by RT-PCR. Host plants inoculated with leaf sap of the asymptomatic pokeweed plants showed no symptoms.
PMoV was detected by ACP-ELISA in all symptomatic plants, with an average optical density (O.D.) at 450 nm of 0.96 ± 0.60 (n = 3), and 0.12 ± 0.03 for asymptomatic plants (n = 2), aft er 1 h of substrate incubation. Tests for the potential additional presence of TSWV and CMV by LF were negative.
Th e ACP-ELISA results were confi rmed with reverse transcription (RT)-PCR. The expected 916 bp fragment was obtained from symptomatic pokeweed (Figure 2) and tomato plants tested, while no amplicon was obtained from asymptomatic plants. Sequences obtained from symptomatic P. americana shared 100% nucleotide similarity, and the corresponding sequence of the isolate Pha1 was deposited in GenBank (Accession n. MW248388). Th e Pha1 sequence shared greatest nucleotide similarity with sequences of the two PMoV tomato isolates from hosts growing nearby: 99.67% with the tomato isolate 390 (Accession n. MW456562) and 99.78% similarity with isolate 391A (Accession n. MW456563). Th is indicated virus movement between the American pokeweed and tomato hosts. Th ese three isolates also clearly grouped in the phylogenetic tree, forming a distinct clade together with the isolates Ruc1, identifi ed previously from Diplotaxis tenuifolia (L.) DC., and Fri-2, from Capsicum annuum L., in the same Italian region (Parrella et al., 2017;Parrella et al., 2016). Nevertheless, overall phylogenetic grouping showed no clear correlation with host or geographic origin, although the existence of four distinct phylogenetic groups of PMoV isolates was indicated based on the MP gene ( Figure 3). These results confirm those previously obtained and based on the coat protein (CP) phylogenetic relationships of PMoV isolates (Galipienso et al., 2015).
PMoV is an emerging virus, due to its increasing incidence in tomato and other cultivated plants, such as Cap-   Table 1. Th e isolate from Phytolacca americana (Pha1) is indicated in the tree with the symbol �. Th e phylogenetic tree was constructed using the neighbor joining (NJ) method, with 1000 bootstrap replicates, and genetic distances were calculated by the Tamura three-parameter model using MEGA 7.0. Th e isolate of tobacco streak virus TSV 1973 (Accession n. JX463336) was used as an outgroup. sicum annuum, Diplotaxis tenuifolia, and the ornamental plant Mirabilis jalapa L. (Parrella, 2002;Janssen et al., 2005;Parrella et al., 2016;Parrella et al., 2017;Caruso et al., 2018;Parrella et al., 2020). The discovery of a new natural host of PMoV, i.e. Phytolacca americana, confirms the tendency of the virus to occupy new ecological niches. Within Caryophyllales, Phytolaccaceae are closely related to Nyctaginaceae, the plant family to which Mirabilis jalapa belongs, and this host is another unusual natural host of PMoV (Rettig et al., 1992;Cuénoud et al., 2002;Lee et al., 2013). After the present discovery, further investigations would be useful to increase knowledge of the possible role of American pokeweed as a reservoir of PMoV, and the impacts of epidemics of this pathogen on susceptible crops such as tomato and pepper.