Detection and Molecular Characterization of Peach Latent Mosaic Viroid Isolates Infecting Stone Fruit Trees in Poland

Peach latent mosaic viroid (PLMVd) belonging to the genus Pelamoviroid of the family Avsunviroidae was first described by Flores and Llácer (1989). It is widely distributed worldwide and occurs in most commercial peach cultivars (Shamloul et al. 1995; Ragozzino et al. 2003). In Poland, PLMVd has been detected in peaches, flat nectarines, and apricots (Paduch-Cichal & Skrzeczkowski 2001; Cieślińska et al. 2005). This viroid causes delayed foliation, flowering, fruit ripening of many peach varieties, deformation, discoloration, and cracking sutures of fruits (Desvignes 1980; Flores et al. 1990). Some strains also induce foliar leaf discoloration (e.g., calico, Malfitano et al. 2003) or yellow mosaic. This viroid is generally latent in peach trees for 5–7 years before symptoms appear. It was demonstrated that the other fruit species, among them: apricot, nectarine, sweet cherry, plum, pear, almond, apple, mango, grapevine and walnut can be infected by PLMVd (Skrzeczkowski et al. 1996; Hadidi et al. 1997; Faggioli et al. 1997; Kyriakopoulou et al. 2001; Crescenzi et al. 2002; Fekih Hassen et al. 2005; El-Dougdoug et al. 2012; Tuncel et al. 2020).

Peach latent mosaic viroid is transmitted mechanically, by grafting, or by contaminated pruning tools (Flores et al. 1992; Shamloul et al. 1995; Hadidi et al. 1997). It has also been shown to be aphid-transmitted (Myzus persicae) (Flores et al. 1992). It was also reported that PLMVd could be transmitted by pollen (Ragozzino et al. 2001; Barba et al. 2007).

Diagnostics of peach latent mosaic disease are based mainly on dot-blot hybridization (Ambrós et al. 1995) and different variants of RT-PCR (Shamloul et al. 1995; Mumford et al. 2000; Shamloul et al. 2002; Ragozzino et al. 2003).

This study aimed to determine the occurrence of peach latent mosaic viroid in stone fruit trees in Poland and the molecular characterization of its isolates.

Symptoms of PLMVd infection include leaf yellow mosaic and cracked fruits with corky sutures and enlarged pits (Desvignes 1981). They were also observed on ‘Maycrest’ (Mayc) peach (Fig. 1 & 2), while only yellow mosaic on ‘Saturn’ (Satu) nectarine occurred. In turn, the spots, irregular shape, and deformation were observed on fruits of peach ‘Reliance’ (Relia) and Japanese plum ‘Ozark Premier’ fruits. (OzPr). Similar symptoms were also observed by Giunchedi et al. (1998). No such disease symptoms were detected on the remaining 164 surveyed trees.

Figure 1.

Figure 2.

RT-PCRs amplified specific products of ~340 bp in size for 52 samples (31% of tested) of stone fruit trees. PLMVd was detected in forty of 119 peaches, six of 37 apricots, five of eight nectarines, and one of four plums tested trees. In Poland the PLMVd was detected for the first time in Japanese plum. Over 80% of peach samples (34 out of 42 tested) collected from three orchards near Sandomierz, known as the traditional region of peach and apricot growing, were positive for the viroid. PLMVd has been detected before in apricot and peach trees (Paduch-Cichal & Skrzeczkowski 2001), but its incidence in peach orchards was then lower (32% of infected trees). As it is known that PLMVd spreads mainly during grafting and pruning of the trees with contaminated tools (Flores et al. 1992), there was a probability that the same isolate infected the trees in the surveyed orchard. Therefore, 1–2 isolates from each orchard and/or each tree species infected with PLMVd were selected and used for further study (Table 1).

The sequences of the three cDNA clones of each isolate were identical to their original variants. However, genetic differences of the 11 selected PLMVd isolates were revealed as 93.6–100% similarity was shown. The nucleotide sequences similarity of the isolates from Poland and the reference PLMVd isolates found in various hosts originating from different geographical regions ranged from 82.0% to 98.8%. BLAST search revealed the highest and the lowest similarity of these isolates with peach isolates from the United Kingdom (KY810773) and Canada (GQ499305), respectively. Nucleotide sequences of 11 PLMVd isolates were submitted to the GenBank database under the accession numbers MW928673.1–MW928683.1.

Based on phylogenetic analysis conducted by Ambrós et al. (1998), PLMVd strains were classified into three major groups. The most numerous represented group I, composed of strains originating from different geographic regions and host plants, was subsequently divided into six subgroups (Xu et al. 2019). Conversely, Karagahi and Hajizadeh (2020) proposed clustering PLMVd strains into two major groups within which two subgroups were separated.

Phylogenetic analysis of the 11 Polish isolates and 58 strain sequences deposited in GeneBank database led to their clustering into two major groups with division of group II onto three subgroups and group I onto two subgroups (Fig. 3). The most numerous represented subgroup II-A composed of the all Polish isolates and PLMVd strains originated from different geographic regions and host plants including peach, nectarine, apricot, cherry, sweet cherry, plum, pear, and apple. Although Polish isolates were clustered in the same subgroup, some differed in length (336–337 nt) and nucleotide sequences.

Figure 3.

Subgroup II-B was represented on the phylogenetic tree by two peach strains originating from Italy and Hungary, and subgroup II-C included mainly strains from peach but also sweet cherry, Japanese plum, and almond originating from different geographical regions. Although only 11 PLMVd isolates were clustered in both subgroups of group I, it was significant that they were derived from peach (10) or nectarine (1) only.

Yazarlou et al. (2012) suggested a tendency for geographic clustering of populations of PLMVd sequences in various hosts. However, our study did not confirm the association between the genetic variability of the PLMVd strains from different species and their geographic origins, as the strains found in Turkey, Tunis, Spain, South Korea, and Canada were clustered in both major groups. Genetic variability and the resulting phylogenic positions of Polish isolates were also unrelated to their origin regions.

Our analysis revealed the positive result of dot-blot hybridization for cDNA probes of 11 PLMVd isolates (Fig. 4). Hybridization assays have been used for detection of PLMVd (Ambrós et al. 1995; Hadidi et al. 1997; Kyriakopoulou et al. 2001; Paduch-Cichal & Skrzeczkowski 2001), but it was reported that its sensitivity is 10 to 100-fold lower than RT-PCR (Hadidi & Yang 1990).

Figure 4.

This study showed that peach latent mosaic viroid occurred in Poland in stone fruit trees of four species. Detection of PLMVd isolates in nectarine and Japanese plum increased knowledge about the host plants that may be the source of the viroid in our country. Considering the transmission of PLMVd by grafting and contaminated pruning tools, there is a risk of its further spreading.

The genetic analysis of the sequences of selected PLMVd isolates found in different plant species and regions was conducted for the first time in Poland. These isolates differed in length and nucleotide sequences; however, they were clustered in the same subgroup II-A beside the PLMVd strains originating from different countries and host plants.