Incidence and molecular characterization of a 16SrI-B phytoplasma strain associated with Vitis vinifera leaf yellowing and reddening in the west of Iran

Abstract

Leaf yellowing and reddening symptoms on Vitis vinifera vines were observed in different grapevine nurseries in the province of Kurdistan in the west of Iran during 2016–2017. Total DNA was extracted from 27 symptomatic and four symptomless plants and tested for the presence of phytoplasma DNA by nested polymerase chain reaction (PCR) using P1/P7 and R16F2n/R16R2 primer pairs. PCR amplicons of about 1.8 and 1.2 kb in size were obtained from all symptomatic V. vinifera but not from symptomless plants. Restriction fragment length polymorphism (RFLP) profiles of R16F2n/R2 amplicons using AluI, HhaI, HpaII, RsaI, TaqI, KpnI, HaeIII and Sau3AI restriction enzymes were identical to each other and to those obtained from 16SrI phytoplasmas, indicating that the phytoplasmas associated with grapevine yellowing and reddening were members of the 16SrI group. A R16F2n/R16R2 16S rDNA sequence of Kurdistan V. vinifera yellowing and reddening strain (KVvY) was submitted to GenBank as accession number MH638316. The Blast analysis of KVvY showed 100% sequence identity with those of the ‘Candidatus Phytoplasma asteris’-related strains. Phylogenetic analysis confirmed that the phytoplasma associated with KVvY clustered within the 16SrI phytoplasma clade closer to the Onion yellows mild strain (OY-M), a 16SrI-B phytoplasma. Restriction analysis using iPhyClassifier confirmed that virtual RFLP patterns of the KVvY were identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession AP006628).

Résumé

En 2016-2017, des symptômes du jaunissement et du rougissement des feuilles chez Vitis vinifera ont été observés dans diverses pépinières de vignes de la province du Kurdistan, dans l’ouest de l’Iran. L’ADN total a été extrait de 27 plants symptomatiques et de 4 plants asymptomatiques, et a été testé par réaction en chaîne de la polymérase nichée (PCR) à l’aide des paires d’amorces P1/P7 et R16F2n/R16R2 pour y détecter l’ADN de phytoplasmes. Les amplicons résultant de la PCR, d’environ 1.8 et 1.2 kb, ont été obtenus de tous les plants symptomatiques de V. vinifera, mais pas des plants asymptomatiques. Les profils du polymorphisme de longueur des fragments de restriction (RFLP) des amplicons R16F2n/R2, obtenus avec les enzymes de restriction AluI, HhaI, HpaII, RsaI, TaqI, KpnI, HaeIII et Sau3AI, étaient identiques entre eux ainsi qu’à ceux obtenus des phytoplasmes 16SrI, indiquant que les phytoplasmes associés au jaunissement et au rougissement de la vigne appartenaient au groupe 16SrI. Une séquence 16S de l’ADNr de R16F2n/R16R2 de la souche du jaunissement et du rougissement de V. vinifera du Kurdistan (KVvY) a été soumise à la GenBank en tant qu’obtention numéro MH638316. L’analyse de type BLAST de KVvY a révélé une identité de séquence de 100% avec celle des souches associées à ‘Candidatus Phytoplasma asteris’. L’analyse phylogénétique a confirmé que les phytoplasmes associés à KVvY se regroupaient au sein du clade du phytoplasme 16SrI ressemblant le plus à la souche faible du jaunissement de l’oignon (OY-M), un phytoplasme du groupe 16SrI-B. Une analyse de restriction basée sur iPhyClassifier a confirmé que les schémas virtuels du RFLP de KVvY étaient identiques (coefficient de conformité de 1.00) au schéma de référence du sous-groupe B du groupe I de 16Sr (GenBank, obtention AP006628).

Keywords:

• grapevine
• molecular characterization
• Restriction fragment length polymorphisms

Mots clés:

• Caractérisation moléculaire
• polymorphismes de longueur des fragments de restriction
• vigne

Introduction

Phytoplasmas, members of the class Mollicutes, are wall-less, phloem-limited bacterial plant pathogens that infect a wide range of plant species worldwide (Bertaccini et al., 2014). They are transmitted mainly by leafhoppers and psyllids and cause yellowing, discolouration, witches’ broom, dwarfing, virescence and phyllody disease symptoms in different plant species. Among the plant species harbouring phytoplasmas, grapevine has been associated with several phytoplasma diseases worldwide. Bois Noir (16SrXII-A), flavescence doree (16SrV-C and 16SrV-D), Australian grapevine yellows and North American grapevine yellows are known as grapevine yellows phytoplasmas (Constable & Bertaccini, 2017). Also grapevine yellows and reddening disease associated with Ca. Phytoplasma pruni (16SrIII-A), Ca. Phytoplasma asteries (16SrI-B), Ca. Phytoplasma fraxini (16SrVII-A), Ca. Phytoplasma solani (16SrXII-A), Ca. Phytoplasma australiense (16srXII-B), Ca. Phytoplasma ulmi (16SrV-A), 16SrI-C, 16SrV-C, 16SrV-D,16SrVI, 16SrIX and 16SrX-B were reported from Australia, Lebanon, Turkey, Chile, Tunisia, Georgia, Armenia, Jordan, Italy, Serbia, South Africa and USA (Prince et al., 1993; Choueiri et al., 2002; M’hirsi et al., 2004; Botti & Bertaccini, 2006; Gajardo et al., 2009; Canik et al., 2011; Salem et al., 2013; Roggiabb et al., 2014; Ertunc et al., 2015; Davis et al., 2015; Quaglino et al., 2016; Zambon et al., 2018).

Fig. 1 (Colour online) Symptoms of (A) leaf curling (B) reddening (C) yellowing (D) little leaves in Vitis vinifera infected plant in Kurdistan (Iran).

Fig. 2 Polyacrylamide gel showing RFLP profiles of 16S rDNA amplified by nested PCR using P1/P7 followed by R16F2n/R16R2 primer pairs from KVvY (GenBank accession number MH638316). PCR products were digested by AluI, HhaI, HpaII, RsaI, TaqI, KpnI, HaeIII, Sau3AI restriction enzymes. M, 100 bp DNA ladder.

Fig. 3 Neighbour-joining phylogenetic tree based on the 1.2 kb 16S rDNA sequence of KVvY (GenBank accession number MH638316) and those of selected phytoplasma strains from GenBank. A. laidlawii was used as an outgroup. GenBank accession numbers are shown on the right of phytoplasma strains. Numbers on branches are bootstrap values of 1000 replicates.

Fig. 4 Virtual RFLP profiles from the R16F2n/R16R2 sequence of the VivY phytoplasma (GenBank accession number MH638316) using 17 restriction endonucleases with iPhyClassifier (Zhao et al., 2009).

Grapevine production areas, especially for rainfed cultivars, are very important in west areas of Iran and the grapes are used mostly for fresh fruit or export. Phytoplasma symptoms were observed in vineyards in the central and north east regions of Iran which were associated with phytoplasmas groups 16SrVII-A, 16SrXII-A, Ca. Phytoplasma phoenicium (16SrIX-B) and Ca. Phytoplasma aurantifolia (16SrII-B) (Karimi et al., 2009; Salehi et al., 2014, 2016a; Mirchenari et al., 2015; Ghayeb Zamharir et al., 2017). The aim of this work was to identifiy and characterize phytoplasmas present in grapevine nurseries located in the Kurdistan province (west of Iran) to improve our understanding of the disease and develop sustainable disease management for new grapevine orchards.

Materials and methods

Plant sampling and infection incidence

During 2016–17, sampling at two times was carried out in grapevine nurseries located in the Kurdistan province in the west part of Iran. Grapevine nurseries were sampled on a diagonal transect across each. Plants expressing phytoplasma disease-like symptoms such as leaf curling, leaf yellowing and necrotic reddening of leaves, as well as death of shoot tips, were collected. Young leaf and petiole samples were taken and transferred on ice to the lab and used immediately for DNA extraction. A total of 27 symptomatic and four asymptomatic samples were collected and subjected to nested-PCR assays.

DNA extraction and nested PCR amplification

Total DNA was extracted from 0.2g of midrib tissue of symptomatic and symptomless grapevine plants according to the procedure described by Healey et al. (2014). Total DNA extracted from a periwinkle plant infected with alfalfa witches’ broom phytoplasma strain (16SrXII-A) (Esmailzadeh Hosseini et al., 2016) was used as a positive control and a DNA sample from a grapevine plant grown in the greenhouse was used as a negative control. The DNA quality was estimated by agarose gel electrophoresis and 100 ng of nucleic acid was used for PCR reactions. For nested-PCR, the universal primer pair P1/P7 amplifying 16SrRNA gene, 16S-23S spacer region (SR) and the 5ʹ end of 23S rRNA gene (Deng & Hiruki, 1991; Schneider et al., 1995) and the R16F2n/R2 primers amplifying ~1.2 kbp inside the 16SrRNA gene (Gundersen & Lee, 1996) were used. The PCR reaction was performed in a Emeraldamp MaxHS master mix (Takara, Japan). The reaction cycled 35 times in a Bio-Rad (USA) thermal cycler with the following parameters: denaturation for 30 s at 94°C (2 min in the first round), annealing for 30 s at 55°C and primer extension for 1 min at 72°C (10 min in the final cycle). PCR conditions for the nested PCR were the same except that the annealing temperature was 58°C. Following PCR, 5 µL of each reaction mixture was electrophoresed in a 1% (w/v) agarose gel containing 0.3 µg mL⁻¹ ethidium bromide in 0.5× TBE buffer (22.5 mM TRIS–borate, 1 mM EDTA, pH 8.0) to verify amplification of target DNAs.

Restriction fragment length polymorphism (RFLP) analysis

RFLP analysis of 1.2 kb nested PCR products were used for identification of the phytoplasma associated with the grapevine disease. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 amplicons using AluI, HhaI, HpaII, RsaI, TaqI, KpnI, HaeIII and Sau3AI restriction enzymes was performed according to the manufacturer’s instructions (Fermentas, Vilnius, Lithuania). The restriction products were then separated by electrophoresis through an 8% polyacrylamide gel, stained by ethidium bromide and visualized with a UV transilluminator. The resulting RFLP patterns were compared with those previously published for 16S rDNA of other phytoplasmas (Lee et al., 1998).

Sequencing and phylogenetic analyses

R16F2n/R16R2 primed PCR product (1.2 kb) from DNA extracted from infected plants was directly sequenced in both directions and the assembled sequence was compared with deposited sequences in the GenBank database using BLAST (National Center for Biotechnology Information, Bethesda, MD, USA) and aligned with Bioedit tools. The 1250 bp of 16S rDNA sequences of 18 phytoplasmas including a strain of KVvY phytoplasma disease were aligned using MEGA7 software (Kumar et al., 2016) and a phylogenetic tree was constructed using the neighbour-joining method. Acholeplasma laidlawii was used as an outgroup to root the tree. Bootstrapping was performed 1000 times to estimate the stability and support for the branches.

Virtual RFLP analysis

Virtual RFLP analysis using iPhyClassifier (Zhao et al., 2009) was used to determine the subgroup affiliation of KVvY phytoplasma. Each aligned DNA fragment was digested in silico with 17 distinct restriction enzymes: AluI, BamHI, BfaI, BstUI (ThaI), DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MboI (Sau3AI), MseI, RsaI, SspI and TaqI.

Results

Disease symptoms and incidence

During two sets of sampling times in grapevine nurseries in Kurdistan province, various symptoms including yellowing, reddening and inward leaf curling and malformed leaf shape were observed in different grapevine nurseries (Fig. 1). In red fruit varieties, the early season symptoms consisted mostly of leaf yellowing and the late season symptoms consisted mostly of leaf reddening. The incidence of the symptomatic plants in the visited nurseries ranged from 5–10%.

PCR-RFLP analyses

PCR amplicons of ~1.8 kb were observed in all of the samples from the 27 symptomatic plants. No amplification was obtained from samples collected from symptomless plants and the negative control. The subsequent nested PCR using P1/P7 and R16f2n/R2 primer pairs yielded a 1.25 kb product in all 27 symptomatic samples. The RFLP patterns of the 1.25 kb 16S rDNA amplicons of all 27 symptomatic KVvY phytoplasma samples from the surveyed regions were identical to each other and similar to those published for members of the Aster yellows (16SrI) phytoplasma group (Lee et al., 1998) (Fig. 2).

Sequencing, virtual RFLP and phylogenetic analysis

The 1.2 kb DNA fragments amplified from the KVvY phytoplasma samples were directly sequenced and showed 100% sequence identity to each other. One of the sequences was named KVvY phytoplasma and submitted to GenBank as accession number MH638316. The BLAST analysis of the KVvY consensus sequence showed 100% sequence identity with those of the ‘Candidatus Phytoplasma asteris’-related strains. Phylogenetic analysis (MEGA software version 7.0) confirmed that the phytoplasma associated with KVvY clustered within the 16SrI phytoplasma clade and is closely associated with the Onion yellows mild strain (OY-M) a 16SrI-B phytoplasma (Fig. 3). Restriction analysis using iPhyClassifier confirmed that virtual RFLP patterns of the KVvY are identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession number: AP006628) (Fig. 4).

Discussion

The Aster yellows phytoplasmas group (16SrI) is the most prevalent phytoplasmas group, as it can infect a broad range of host plants, including monocotyledonous and dicotyledonous species of herbaceous and woody plants. The 16SrI group contains numerous subgroups that are distributed in grapevine cultivation regions worldwide. Numerous plants such as Eucalyptus camaldulensis, Brassica napus, Spinacia oleracea, Eruca sativa, Medicago sativaand Lactuca serriola and several ornamental plants with symptoms of little leaf, phyllody, yellowing, purpling, leaf rolling and virescence were identified as infected by Aster yellows phytoplasma strains in Iran (Babaie et al., 2007; Asghari Tazehkand et al., 2010; Rashidi et al., 2010; Salehi et al., 2011; Esmailzadeh Hosseini et al., 2015a, 2015b; Salehi et al., 2016b).

The 16SrI-B phytoplasma subgroup has been reported to occur in grapevines in Italy, Slovenia, Croatia, Chile, Tunisia and Turkey (Alma et al., 1996; Saric et al., 1997; M’hirsi et al., 2004; Gajardo et al., 2009; Jezič & Karoglan Kontić, 2013; Landi et al., 2013; Ertunc et al., 2015). This is the most common phytoplasma associated with grapevine yellows and it was reported in South Africa (Engelbrecht et al., 2010) and in grapevines grown in Canada, along with the subgroups 16SrI-A and 16SrI-C (Olivier et al. 2009). 16SrI-B is mostly transmitted by the leafhoppers Euscelis incisus and Macrosteles quadripunctulatus (Alma et al., 2001), which can live on different plant host species and transmit phytoplasmas from reservoir hosts to grapevine (Krüger et al., 2015).

Symptoms caused by phytoplasmas are similar to each other but also dependent on the varietal resistance, the age of plants during infection, contamination with other pathogens especially viruses, and effects of abiotic stress. Different phytoplasmas groups or subgroups with different insect or plant hosts can produce similar symptoms in grapevine (Bertaccini et al., 1998). Identification and characterization of phytoplasmas groups can help in understanding the disease epidemiology and planning for disease management. Symptomless phytoplasmas infection in grapevine is common and presence of phytoplasmas in one nursery can show severe and high incidence of infection (Bertaccini et al., 1998).

Phytoplasmas infection in grapevine nurseries probably originated from propagation materials or through transmission by insect vectors. As grapevine is propagated by stem cuttings, movement of propagation materials can distribute and spread phytoplasmas infection in grapevines grown in other geographic areas. Characterization of phytoplasmas groups in grapevine nurseries can determine transportation limits for quarantine of stem cuttings. Cultivation of grapevine varieties resistant to phytoplasmas can help to manage the disease, although to date, no variety resistant to phytoplasmas causing yellowing has been identified (Laimer et al., 2009).

In this study, we detected for the first time the presence of phytoplasmas group 16SrI-B in grapevine nurseries grown in the west region of Iran. This phytoplasmas subgroup was also reported in Turkey, a neighbouring country of Iran (Ertunc et al., 2015). Further studies are necessary to identify the main insect vectors, their biology and their origin of infection in order to determine the geographic distribution of the phytoplasma and to develop sustainable disease management.

Acknowledgements

This work was supported by the Chaharmahal and Bakhtiari Agricultural Organization, Shahrekord, Iran. We thanks Mrs M. Zandian for assistance in this research.