Introduction

Phytoplasmas are wall-less plant pathogenic bacteria associated with diseases of great economic importance (Lee et al. 2000; Bertaccini et al. 2014). The phytoplasma classification based on 16S rRNA gene allows to distinguish 43 ‘Candidatus Phytoplasma’ species (Bertaccini and Lee 2018). RFLP analysis of 16S rRNA gene is also used for classification of phytoplasma strains in 33 16Sr groups and more than 200 subgroups (Zhao and Davis 2016). These techniques were used for rapid detection and identification of phytoplasmas in different crops throughout Iran. To date, phytoplasma strains belonging to 11 ‘Candidatus Phytoplasma’ species have been identified in different plants including herbaceous and woody cultivated species (Salehi et al. 2005, 2006; Faghihi et al. 2010; Ghayeb Zamharir et al. 2017; Salehi and Esmailzadeh Hosseini 2017). Several phytoplasma diseases from different countries are known as a major threat for the production of stone fruits. ‘Ca. P. pruni’, ‘Ca. P. phoenicium’ and ‘Ca. P. prunorum’ are among the most important phytoplasmas associated with significant losses to different Prunus species (Foissac and Wilson 2010). However, the European stone fruit yellows (ESFY, ‘Ca. P. prunorum’) is the most important phytoplasma disease of Prunus species with a distribution apparently limited to the Europe and Asia Minor (Jarausch et al. 2001).

Apricot is one of the most important stone fruits produced in Iran, mainly cultivated in the northwest, central and south regions. In surveys performed during 2012–2015 in orchards in Faraghe (Abarkooh, Yazd province, Iran), in the center of Iran, a disease consisting of upward leaf roll and yellow symptoms on the whole crown was observed. Studies were, therefore, carried out to verify the presence and association of phytoplasmas using biological and molecular methods.

Materials and methods

Sample collection

Apricot trees with and without symptoms were selected for sampling in four locations of Faraghe (30 km West of Abarkooh, Yazd province, Iran). Sixteen samples (four per location from four trees) were collected from chlorotic leaf roll-affected apricot trees (Felakei cultivar). Symptomless samples (2) were also included as negative controls.

Disease transmission

Two-year-old GF-677 (peach × almond) trees propagated by cuttings and nested PCR tested (Salehi et al. 2016) to verify the phytoplasma absence were used for the transmission of the apricot chlorotic leafroll disease agent. Each GF-677 tree was side grafted with three scions consisting of small branches with two to three leaves, from symptomatic apricot trees. Eight GF-677 trees (two for location) were grafted and four trees were left as control and maintained under insect-proof nets.

DNA extraction and polymerase chain reaction (PCR)

The midribs and petioles of symptomatic leaves (1 g) were used for total DNA extraction (Zhang et al. 1998) followed by a further purification using DNP™ high-yield DNA purification kit (CinnaGen, Iran). DNA from symptomless trees and a periwinkle plant infected with the lime witches’ broom phytoplasma (Salehi et al. 2005) was used as negative and positive control, respectively. Direct PCR was carried out by the universal primer pair P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995) followed by nested PCR using the primer pair R16F2n/R2 (Gundersen and Lee 1996).

Restriction fragment length polymorphism (RFLP) analysis

Each nested PCR product (8 µl, 500 ng DNA) was digested separately with the restriction enzymes HaeIII, MseI, and TaqI (Fermentas, Vilnius, Lithuania) as described (Rasoulpour et al. 2017). The PCR product from a periwinkle plant infected with Yazd alfalfa witches’ broom phytoplasma (16SrII-C, GenBank Accession number DQ233656) (Salehi et al. 2015) was used as control. The resulting patterns were compared with those of the control and other phytoplasmas (Lee et al. 1998). Virtual RFLP analysis using iPhyClassifier was also used to confirm phytoplasma subgroup affiliation (Zhao et al. 2009).

Sequencing of PCR products and phylogenetic analysis

Amplified products with R16F2n/R2 primers from four affected apricot trees (one per location) and four experimentally inoculated GF-677 trees were cloned and sequenced as described (Rasoulpour et al. 2017). The 16S rDNA sequences (1250 bp) were used for search in the GenBank database at the National Center for Biotechnology Information (NCBI). Nucleotide sequences of phytoplasmas from different groups were then compared with a representative of the Iranian samples using MEGA7 (Kumar et al. 2016) and a phylogenetic tree was constructed.

Results and discussion

Disease symptomatology and transmission

In the selected orchards in Faraghe area, up to 12.5% of the apricot trees showed symptoms of chlorotic leafroll on one branch or on the whole crown with scattered die back of several branches (Fig. 1). The graft transmission from symptomatic apricot trees induced symptoms of little leaf, internode shortening, yellowing and stunting (Fig. 2) in all the inoculated GF-677 trees. The minimum time between inoculation and symptom expression was of 21 months and confirmed the transmissibility of the pathogen to stone fruits other than apricot indicating its possible relevance for further epidemic to this and other stone fruit species. All GF-677 trees were phytoplasma positive in nested PCR, while control plants were negative for phytoplasma presence and showed no symptoms.

Fig. 1
figure 1

Symptoms observed in diseased apricot trees in Faraghe (Abarkooh, Yazd province, Iran): a severe leaf roll on a branch, b leafroll and dieback, c leaf roll and yellowing on one branch, d general yellowing on the whole tree

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Fig. 2
figure 2

Little leaf, internode shortening and yellowing in a GF-677 tree graft inoculated with the apricot chlorotic leafroll agent from Faraghe (Abarkooh, Yazd province, Iran) (a) in comparison with a healthy one (b)

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PCR amplification and sequence analysis

In direct PCR, the expected length fragment was amplified only from the lime witches’ broom affected periwinkle plant, while in nested PCR the expected 1250 bp fragments were obtained from 14 out of the 16 samples from symptomatic apricot, from all the GF-677 graft-inoculated trees and from the positive control. In direct and nested PCR assays, no bands were observed for two symptomatic trees and the negative controls. The RFLP patterns of all the PCR products were identical with the enzymes used (Fig. 3).

Fig. 3
figure 3

RFLP profiles of nested PCR products using P1/P7 and R16F2n/R2 primer pairs from the phytoplasma detected in the apricot chlorotic leaf roll from Faraghe and Yazd alfalfa witches’ broom phytoplasma (lanes 1 and 2, respectively). Lane M, 100-bp DNA molecular weight marker (Fermentas, Vilnius, Lithuania). DNA products were digested with enzymes listed at the top of the figure

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The sequences of four nested PCR products from symptomatic apricot and graft-inoculated GF-677 trees were 100% identical and one of them from Faraghe was submitted to the GenBank (accession number KU962122). BLASTn analysis of this fragment showed the maximum sequence identity with phytoplasmas in the peanut witches’ broom (16SrII) group and in particular 100% identity with faba bean phyllody phytoplasma (GenBank accession number X83432), reference strain for the 16SrII-C subgroup and 98.9% identity with ‘Ca. P. aurantifolia’ enclosed in the 16SrII-B subgroup sequences. The phylogenetic analysis (Fig. 4) allow the clustering of this phytoplasma with phytoplasmas in the 16SrII group closest to those enclosed in the 16SrII-C subgroup. Results of phylogenetic analysis and of virtual RFLP (Figs. 5 and 6) confirmed that this phytoplasma is clustering with phytoplasmas in the 16SrII group and is classified in the 16SrII-C subgroup. The apricot chlorotic leafroll disease detected in Iran resulted, therefore, to be associated with the presence of phytoplasmas in this ribosomal subgroup.

Fig. 4
figure 4

Phylogenetic tree constructed by neighbor-joining analysis of 16S rRNA gene sequences from 21 phytoplasmas including Faraghe apricot chlorotic leaf roll phytoplasma (in bold). Acholeplasma laidlawii was used as out-group. GenBank accession numbers are indicated on the right. Numbers at the nodes are confidence values based on 1000 repetitions

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Fig. 5
figure 5

Computer-simulated virtual RFLP patterns obtained from in silico digestions of 1.2 kb fragments of faba bean phyllody phytoplasma (16SrII-C, GenBank accession number X83432) (a), and apricot chlorotic leafroll phytoplasma (b) with 17 restriction enzymes using the online iPhyClassifier. Lane MW: phiX174 RFI DNA HaeIII digest; fragment sizes (bp) from top to bottom: 1,353; 1,078; 872; 603; 310; 281; 271; 234; 194; 118 and 72

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The leaf roll, yellowing and die back symptoms observed in the apricot trees in Abarkooh are undistinguishable from those associated with the presence of ‘Ca. P. prunorum’ in the same species in Europe (Necas and Krska 2005; Cieslinska 2011). Recently, a phytoplasma strain related to ‘Ca. P. prunorum’ was found in association with a plum yellow leaf stunt disease in North of Iran (Allahverdi et al. 2014). However, in Iran the association of ‘Ca. P. aurantifolia’-related strains with diseases of other stone fruits including sweet cherry, plum and almond has also been described (Zirak et al. 2009a, b, 2010) but this is the first report of a ‘Ca. P. aurantifolia’-related strain in apricot. The presence of two symptomatic apricot trees that resulted PCR negative may be related to the low concentration or to the uneven distribution of the phytoplasma in the tissues since the severity and distribution of symptoms in the trees may be related to the disease progression steps.

Platymetopius rostratus, Phlepsius intricatus, Psammotettix alienus (Cicadellidae) and Laodelphax striatellus and Toya propinqua (Delphacidae) were identified in an insect collection trial on grasses covering these apricot orchards (data not shown). Among these collected insect species P. alienus and L. striatellus are potential vectors of phytoplasmas (Cvrković et al. 2011; Orságová et al. 2011) and may spread the disease among the apricot trees in the orchards; however, this aspect requires further studies. Apricot propagation by grafting or import of asymptomatic infected apricot seedlings can represent further sources of inoculum. In the Abarkooh cultivation areas, the major phytoplasmas identified so far are mainly classified in the 16SrII-C subgroup (Fig. 6) and were detected in alfalfa (Esmailzadeh Hosseini et al. 2015), carrot (Salehi et al. 2016) and cucurbits (Salehi et al. 2015) growing as mixed cultivation with or around the apricot trees. Further researches are required to verify the apricot disease epidemiology and the relationship between the phytoplasmas detected in the herbaceous crops and apricot trees to design a reliable disease control strategy.

Fig. 6
figure 6

Phylogenetic tree constructed by neighbor-joining analysis of 16S rRNA gene sequences from nine phytoplasmas including Faraghe apricot phytoplasma (in bold). ‘Candidatus Phytoplasma pruni’ (16SrIII-A) was used as out-group. GenBank accession numbers are on the right followed by phytoplasma ribosomal subgroups when different from 16SrII-C

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