Phytoplasmas are wall-less and phloem-restricted plant pathogens transmitted in nature by several groups of insects especially leafhoppers (Cicadellidae) (Seemüller et al. 2002). They affect more than 600 plant species from tropical, subtropical to temperate climates all over the world (Jones 2002). Molecular analysis of several conserved genes, such as the 16S rRNA gene, has identified at least 32 groups and 100 subgroups of phytoplasmas (Nejat and Vadamalai 2013). Phytoplasmas in group 16SrVI, previously called the Clover proliferation (CP) group, were described associated with a disease of alsike clover in Canada originally associated with a yellows type virus (Chiykowski 1965). Subsequent investigations revealed that CP was in fact a phytoplasma that was also associated with phytoplasma diseases of tomato, potato, elm and pepper. CP is now taxonomically described as ‘Ca. Phytoplasma trifolii’ (Hiruki and Wang 2004). In Iran, some plants including Carthamus tinctorius, Cucumis sativus and Medicago sativa are known to be infected by 16SrVI phytoplasmas. In phytoplasma disease epidemics, insect vectors play a major role, which is not always easy to understand and interpret. Furthermore, polyphagous vectors have the potential to inoculate a wide range of plant species, which may differ in susceptibility (Weintraub and Beanland 2006).

Suaeda aegyptiaca is a succulent, annual halophyte plant, which inhabits and is consider native to saline soils of arid and semiarid regions of Iran and widely distributed in Iraq, UAE, Pakistan and North Africa. In addition to supplying a salad, it is also a well-known medicinal plant for treating eczema in Iran (Askari et al. 2006).

In September 2016, typical symptoms of phytoplasma disease, including witches’ broom, yellowing and little leaf were observed during surveys in a S. aegyptiaca plant next to a cultivated vegetable field in Hassanlangi district, Hormozgan province, Iran (Fig. 1). A preliminary study was performed to determine if a phytoplasma was associated with the symptomatic plant. In addition, to find potential vectors of the related phytoplasma, all insects feeding on the symptomatic plant were collected by a D-Vac aspirator (Echo-ES210; Japan) in several visits at weekly intervals and checked for phytoplasma infection.

Fig. 1
figure 1

Symptoms of witches’ broom, little leaf and yellowing (a) in comparison with healthy Suaeda aegyptiaca (b)

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Subsequently, samples of both symptomatic and symptomless S. aegyptiaca were collected from vegetable fields in Hassanlangi district in eastern Bandar Abbas (N27°39′98″; E56°89′89″), Hormozgan province, Iran. Total DNA was extracted from both symptomless and symptomatic samples by using an adapted cetyltrimethylammonium bromide (CTAB) extraction procedure described by Sahu et al. (2012). A nested polymerase chain reaction (PCR) was employed for the detection of phytoplasmas using the universal primers P1/P7 (Deng and Hiruki 1991) followed by R16F2n/R16R2 which amplified a phytoplasma 16S rRNA fragment (approximately 1.25 kbp). PCR assays were performed in a PeQlab thermocycler (Germany) with 50 μl reactions containing 1 μM of each primer, 25 μl master mix, 20 μl double distilled water (DDW) and 3 μl template DNA. PCR conditions for amplification were denaturation at 94 °C for 3 min followed by 35 cycles of 94 °C for 45 s, 55 °C for 1 min and 72 °C for 1.5 min, with a final extension of 10 min at 72 °C. One microlitre of 1:10 diluted first amplification product was used as DNA template in the second round (nested) PCR. Nested-PCR conditions were the same as used in the first round, except that the annealing temperature for the second round was 56 °C.

To determine possible insect vector(s), all sucking insects feeding on the symptomatic S. aegyptiaca were collected using a D-Vac in that region. The species were identified according to the key of Pakarpour Rayeni and Seraj (2016). Five insect species of Cicadellidae family including Neoalitrus pulcher, Jacobiasca lybica, Exitianus fasciolatus, Opsius versicolor, and Stirellus bicolour were identified. Leafhoppers were separated and specimens preserved in acetone (Fukatsu 1999) and stored at −20 °C. Total DNA was extracted from a single individual of the leafhoppers using an adjusted CTAB protocol by Reineke et al. (1998). The primers and PCR conditions for phytoplasma detection in the leafhoppers were the same as mentioned for the plants. The amplified fragments were electrophoresed on 1% agarose gel stained with FluoroDye under UV light and four products (two from plants and two from insects) were randomly selected from the nested round and sequenced directly. Sequences used in phylogenetic analyses were checked and aligned using the softwares DNAstar and ClustalX and all were identical. Phylogenetic analyses were conducted by neighbor joining (NJ) methods using MEGA 6.0 software (Tamura et al. 2013). The phytoplasma 16S rRNA gene sequences used in the comprehensive phylogenetic analyses were retrieved from GenBank (the accession numbers are shown in brackets).

The nested PCR products of approximately 1250 bp were amplified separately from five symptomatic plants using the R16F2n/ R16R2 primer pair. Similarly, amplified products were obtained from positive control (‘Ca. Phytoplasma aurantifolia’) samples but not in the assay of samples from non-symptomatic plants and negative controls (DNA template free). Sequence analysis of the 1250 bp rRNA gene revealed that the phytoplasma associated with S. aegyptiaca witches’ broom (SaWT) belongs to group 16SrVI, ‘Ca. Phytoplasma trifolii’ with highest similarity (99%) to the Brinjal little leaf phytoplasma (accession no. KX284703). The representative nucleotide sequence of the isolated phytoplasma was deposited in the GenBank database (accession no: KY411138; KY659429). The outcome of BLAST analysis and construction of the phylogenetic tree, confirmed that the sequence from the present study was clustered in group 16SrVI (Fig. 2).

Fig. 2
figure 2

Phylogenetic tree inferred from partial 16S rRNA sequences from Suaeda aegyptiaca witches’ broom phytoplasma isolates (marked by boldface type) and phytoplasma reference sequences belonging to different 16S rRNA groups. GenBank accession numbers shown in parentheses. And 16Sr groups are annotated to the right. Acholeplasma laidlawii was used as outgroup to root the tree. The tree was constructed by the neighbor-joining method using MEGA 6 software. The bar indicates the number of nucleotides substitution per site. Bootstrap values are shown at nodes with greater than 50% support

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Neoalitrus pulcher was the only dominant and positive species in which PCR and subsequent sequencing results confirmed the presence of the phytoplasma. Six out of ten tested individuals (60%) exhibited related bands in both direct and nested PCR confirming the presence of phytoplasma. The sequences obtained from insects were aligned (Clustal omega) with those obtained from symptomatic plants which revealed 100% similarity. The representative nucleotide sequences of the isolated phytoplasma were deposited in the GenBank database (accession no. KY411139; KY659430) and the sequences obtained from the insects were placed in a clade with those isolated from plants (Fig. 2). Based on these results, N. pulcher can be considered as a candidate vector of this disease on S. aegyptiaca in this region in southern Iran. This species is distributed in Iran, Russia, Georgia, Israel, Kazakhstan, Saudi Arabia and Tajikestan (Pakarpour Rayeni and Seraj 2016). In nature, in the absence of preferential host(s), leafhoppers may feed on alternate host plants and transmit pathogens to new host plants and expand the host range of phytoplasma to cultivated crops which can be a potential threat to agriculture. For instance, Orosius albicinctus Distant, has been reported as the experimental vector of cucumber phyllody (Azadvar et al. 2005), a disease which is believed to have originated from sesame. It is believed that during the autumn, when sesame, the preferential host, has been harvested, O. albicinctus may have been forced to feed on alternative plants, such as greenhouse cucumbers and has transmitted the disease.

Based on PCR results and phylogenetic analysis of the 16S rRNA gene sequences, it can be concluded that the phytoplasma observed in the infected S. aegyptiaca in Hormozgan province belongs to the 16SrVI group. In Iran, 16SrVI phytoplasmas have previously been identified in some hosts including Carthamus tinctorius, Cucumis sativus and Medicago sativa. To the author’s knowledge, this is the first report of a phytoplasma disease associated with S. aegyptica plants with a substantial population of N. pulcher leafhopper as a potential vector for this pathogen in Iran. Further studies are needed to confirm vector transmission and the other possible hosts of this phytoplasma.