Phytoplasmas are associated with different destructive plant diseases worldwide (Bertaccini et al., 2014) and are transmitted mainly by leafhoppers, however they could be disseminated also by propagation materials and in several cases by seeds (Satta et al., 2019). Phytoplasma presence is associated with symptoms of yellowing, discoloration, witches’ broom, dwarfing, virescence, and phyllody. More than 1000 plant species from different plant families are reported as affected by phytoplasmas (Bertaccini & Duduk, 2009; Lee et al., 2000) and among them, vegetables growing in the major production areas worldwide, are infected by phytoplasmas belonging to numerous ribosomal groups (Kumari et al., 2019). In particular, eggplant (Solanum melongena L.) was reported as infected with strains belonging to 16SrI in Japan, Bangladesh and India (Kelly et al., 2009; Kumar et al., 2012; Lee et al., 1998; Okuda et al., 1997), 16SrII in Oman, Egypt and India (Al-Subhi et al., 2011; Omar & Foissac, 2012; Yadav et al., 2016), 16SrIII in Brazil (Amaral-Mello et al., 2011; Barros et al., 1998), 16SrVI in India, Turkey and Bangladesh (Azadvar & Baranwal, 2012; Sertkaya et al., 2007; Siddique et al., 2001), and 16SrXII in Romania and Southern Russia (Ember et al., 2011). Eggplant, with harvested areas of 5312 ha, yield of 5419 kg/ha and a production of 5510 tons (FAOSTAT, 2018), is widely cultivated in Iran where the average production ranks the country as fifth in global production. Formerly the association of a 16SrIX-C phytoplasma with eggplant phyllody in Roodan (Hormozgan province of Iran) was reported (Tohidi et al., 2015). The present work reports genetic diversity of phytoplasmas associated with eggplant phyllody disease in several cultivation areas of Iran.

During 2015–2018, sampling of eggplant phyllody was carried out in the major eggplant growing areas of Fars (Khafr, Fassa, Firooz Abad, Sarvestan, Darab), Yazd (Abarkooh), Zanjan (Zanjan), Kerman (Sirjan), Khorasan Razavi (Mashhad), Bushehr (Bushehr, Kangan, Dashtestan) and Hormozgan (Roodan, Bandar Abbas) provinces of Iran (Fig. 1). In each area, five eggplant fields were randomly selected, and sampling was carried out at five points in 1000 m2 field within a 1 m2 by moving on a diagonal transect across each field. The percentage of eggplant phyllody disease incidence was calculated by number of plants with symptoms out of the total number of plants present within a 1 m2 multiplied by 100. From each field, five eggplant phyllody affected plants were potted, transferred to greenhouse for disease transmission and molecular analyses.

Fig. 1
figure 1

Map of Iran showing the sampling locations of the eggplant phyllody. In Fars area, K: Khafr, F: Fassa, FA: Firooz Abad, S: Sarvestan, D: Darab; in Yazd area, A: Abarkooh, in Zanjan, Z: Zanjan; in Kerman area, S: Sirjan, in Khorasan Razavi area, M: Mashhad, in Bushehr area, B: Bushehr, K: Kangan, D: Dashtestan and in Hormozgan area, R: Roodan, B: Bandar Abbas

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After phytoplasma identification from potted eggplants, one representative of each phytoplasma subgroup identified was dodder transmitted from two infected eggplants to 10 seed-grown 3-month-old periwinkle plants (Salehi, Esmailzadeh Hosseini, Salehi, & Bertaccini, 2016e) under insect-proof conditions. For graft transmission, small axillary shoots from a symptomatic eggplant (representative of an identified subgroup) were used as scions and side grafted on five 12-week-old seed grown eggplant plants. Each rootstock received two scions. Grafted areas were wrapped with parafilm and plants were covered with plastic bags for a week to maintain humidity. Healthy seed grown eggplant and periwinkle plants (five plants per each trial) were left as healthy controls. Presence of phytoplasmas in dodder and graft inoculated plants was confirmed by nested PCR assay.

Total DNA was extracted from 0.2 g of midrib tissue of eggplant phyllody infected, and dodder and graft inoculated plants using the procedure described by Zhang et al. (1998). Total DNA extracted from symptomless seed-grown eggplant was used as negative control. Positive control was a symptomatic periwinkle plant infected with Fars alfalfa witches’ broom phytoplasmas (16SrII-C subgroup) (Salehi et al., 2011). Total DNA samples were tested for phytoplasma presence using primer pair P1/P7 (Deng & Hiruki, 1991; Schneider et al., 1995) followed by R16F2n/R16R2 (Gundersen & Lee, 1996). The molecular weight of the PCR products was estimated by comparison with 100 bp DNA ladder (Fermentas, Vilnius, Lithuania).

The R16F2n/R16R2 primed PCR products of 54 samples from the surveyed areas (one sample per area for which three sequences were screened) were ligated in pTZ57R/T vector and cloned into Escherichia coli DH5a cells using InsT / A cloneM PCR Product Cloning Kit (Fermentas, Vilnius, Lithuania) according to manufacturer instructions. The presence of the correct size insert was confirmed by restriction endonuclease analysis using EcoR1 and Pst1 enzymes. Three plasmid DNAs from recombinant colonies were purified using GF-1 PCR Clean-Up Kit (Vivantis, Malysia, HQ) and sequenced. Sequencing was performed by Macrogenon both strands by using M13F/M13R primers (BioNeer, DNA sequencing service, South Korea). The phytoplasma 16Sr DNA partial sequences obtained (1250 bp) were used in Blastn analyses. Virtual RFLP was performed by iPhyClassifier (Zhao et al., 2009) to determine the ribosomal subgroup affiliation of the detected phytoplasmas. Partial 16S rDNA sequences of eggplant phyllody phytoplasma strains from Fars [Khafr, Fassa (Nowbandegan, Zahedshahr), Firooz Abad (Jaydasht), Sarvesta, Darab], Yazd (Abarkooh), Zanjan (Zanjan), Kerman (Sirjan), Khorasan Razavi (Mashhad) Bushehr [Bushehr (Bushehr1), Kangan, Dashtestan (Borazjan1, Borazjan2, Borazjan3, Bondarooz)] and Hormozgan (Roodan, Bandar Abbas) obtained from the present study were aligned and phylogenetic trees and sequence homologies were generated using MEGA 6 software (Tamura et al., 2013). Acholeplasma laidlawii was used as out-group to root the trees. Bootstrapping was performed 1000 times to estimate the stability and support for the tree branches.

The occurrence of eggplant phyllody was observed in all surveyed areas. The main disease symptoms were little leaf, internode shortening, flower virescence, phyllody, big bud, proliferation and sterility, witches’ broom and stunting (Fig. 2). The highest disease percentage observed was 11% in Zahedshahr.

Fig. 2
figure 2

Symptoms of eggplant virescence and phyllody in plants from Zanjan (left) and Fasa (right)

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The disease latency period varied between 6 weeks, in eggplants graft inoculated with 16SrII-A and -V subgroup strains, to 11 weeks in periwinkle plants dodder inoculated with 16SrIX-C strains. After dodder and graft inoculation of eggplant and periwinkle plants, at early stages of infection, there was no significant difference in symptoms among the diverse phytoplasma subgroups, except for 16SrVI-A strain, and the main symptoms were virescence, phyllody and moderate yellowing. At the late stage of infection, phytoplasma subgroups were differentiable from each other for the specific presence of virescence and phyllody (16SrII-A and –V), severe little leaf, internode shortening and stunting (16SrIX-C and –I), plant wilt and death (16SrVI-A), witches’ broom and rosettes (16SrXII-A). However, in both eggplant and periwinkle plants 16SrII-A and –V were not differentiable on symptoms from each other, but resulted differentiable from those associated with the presence of phytoplasmas classified in the other ribosomal subgroups (Table 1 and Figs. 3 and 4).

Table 1 Ribosomal group/subgroup affiliation of eggplant phyllody disease associated phytoplasmas in Iran and their distribution
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Fig. 3
figure 3

Eggplants 3-month-old graft inoculated with different eggplant phyllody phytoplasma strains four months after the grafting; a: 16SrIX-I; b: 16SrVI-A; c: 16SrIX-C; d: 16SrII-A and 16SrII-V; e: 16SrXII-A phytoplasmas. Left in d and e are healthy seed grown eggplants

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Fig. 4
figure 4

Periwinkle plants 2-month-old dodder inoculated with different eggplant phyllody phytoplasma strains, four months after the beginning of the transmission trials. a: 16SrIX-C and 16SrIX-I phytoplasmas; b: 16SrVI-A phytoplasma; c: 16SrII-A and 16SrII-V phytoplasmas; d: 16SrXII-A phytoplasma; e: healthy seed grown periwinkle plant

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DNA fragments of approximately 1800 and 1250 bp were amplified in direct and nested PCR, respectively from all the symptomatic eggplant plants, but no amplification was obtained from the asymptomatic plants. BLASTn search showed that eggplant phyllody strains were differentiable according to the diverse localities (Table 1) and the phylogenetic tree confirmed that the Iranian eggplant phyllody strains cluster with phytoplasmas enclosed in the diverse ribosomal groups listed above (Fig. 5). Since the sequences from eggplant phyllody phytoplasma samples collected in each province were identical to each other, only one representative of each province was submitted to GenBank (Table 1). Results of virtual RFLP analyses of eggplant phyllody strains showed the presence of six phytoplasma subgroups including 16SrII-V and -D, 16SrIX-C and -I, 16SrVI-A and 16SrXII-A, and of some that are variants of 16SrII-D, 16SrVI-A, 16SrIX-C (Table 1 and Fig. 6).

Fig. 5
figure 5

Phylogenetic tree constructed by the Neighbor-Joining method using partial 16S rRNA gene sequences (1250 bp) and Acholeplasma laidlawii as the outgroup; ‘Ca. P.’: ‘Candidatus Phytoplasma’; numbers at the nodes are bootstrap (confidence) values based on 1000 repetitions; GenBank accession numbers for sequences are given in parentheses following the phytoplasma names, Iranian eggplant phytoplasmas are in bold

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Fig. 6
figure 6

Virtual RFLP pictures generated with iPhyClassifier from in silico digestion of the R16F2n/R16R2 fragments of the diverse eggplant phytoplasma strains from Iran. In a) 16SrII-D strains Khafr (GenBank accession number, AC: MT248286), Bandar Abbas (AC: MT240535) and Bondarooz (AC: MT248287), Borazjan3 (AC: MT248288). In b) 16SrII-V (AC: MT248285) profiles. In c) strains 16SrVI-A, Yazd (AC: MG760570), Zanjan (AC: MT240537) and Mashhad (AC: MT240536). In d) strain 16SrXII-A, Kangan (AC: MT248273). In e) strains 16SrIX-C Borazjan1 (AC: MT248280); Roodan (AC: MT248278), Darab (AC: MT248276); Zahedshahr (AC: MT248275), and Borazjan2 (AC: MT248284). In f) strain 16SrIX-I, Bushehr1 (AC: MT248274). In g) strains related to 16SrIX-C (97%), Sarvestan (AC: MT248282); Nowbandegan (AC: MT248283). In h) strain related to 16SrIX-C (97%), Jaydasht (AC: MT248281). In g) and h) the red circles indicate enzymes differentiation from the 16SrIX-C profiles of the strains

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In the present study 16SrII-V and -D phytoplasma subgroups were detected; the 16SrII group encloses 22 subgroups (from 16SrII-A to 16SrII-V) and two ‘Candidatus Phytoplasma’ species (‘Ca. P. aurantifolia’ and ‘Ca. P. australasia’). The 16SrII-V subgroup is here reported for the first time in Iran in Syrjan (Kerman province) after its description in Praxelis clematidea phyllody disease in tropical and subtropical regions of China (Yang et al., 2017).

In Iran the presence of destructive phytoplasma diseases adjacent to eggplant fields may indicate a role in the epidemiology of eggplant phyllody for alfalfa witches’ broom (Esmailzadeh Hosseini, Khodakaramian, Salehi, Fani, Bolok Yazdi, et al., 2015a, b, c, Esmailzadeh Hosseini, Khodakaramian, et al., 2016a, b, c; Salehi et al. 2011), tomato witches’ broom (Salehi et al., 2014), parsley phyllody (Salehi, Esmailzadeh Hosseini, Salehi, & Bertaccini, 2016f), squash phyllody (Salehi et al., 2015), garden beet witches’ broom (Mirzaie et al., 2007), sesame phyllody (Salehi, Esmailzadeh Hosseini, Salehi, & Bertaccini, 2016d), carrot witches’ broom (Salehi, Esmailzadeh Hosseini, Salehi, & Bertaccini, 2016e), pot marigold phyllody (Esmailzadeh Hosseini, Salehi, et al., 2016) and pomegranate little leaf (Salehi, Esmailzadeh Hosseini, Rasoulpour, et al., 2016a). Moreover, the 16SrVI-A phytoplasma strains identified in alfalfa witches’ broom, Sophora alopecuroides yellowing (Esmaeilzadeh-Hosseini et al., 2020; Esmailzadeh Hosseini, Khodakaramian, et al., 2016b), cabbage yellows (Salehi et al., 2007), witches’ broom and yellowing in jujube plants (Babaei et al., 2020) and tomato big bud (Davoodi et al., 2019; Salehi, Salehi, & Masoumi, 2016b) were adjacent to eggplant fields in Abarkooh, Zanjan and Mashhad where the occurrence of eggplant phyllody was here reported. The phytoplasmas in the 16SrIX–C subgroup also were important in eggplant growing areas and were previously detected in Iran associated with sesame phyllody (Salehi, Esmailzadeh Hosseini, Salehi, & Bertaccini, 2016e), almond witches’ broom (Salehi et al., 2006) and grapevine yellows (Salehi, Salehi, Taghavi, & Izadpanah, 2016c) diseases. The 16SrXII-A phytoplasma strains were associated with alfalfa witches’ broom, S. alopecuroides yellowing (Esmaeilzadeh-Hosseini et al., 2020, Esmailzadeh Hosseini, Khodakaramian, et al., 2016b), Vitis vinifera yellows (Salehi, Salehi, Taghavi, & Izadpanah, 2016c) and decline (Ghayeb Zamharir et al., 2017), field bindweed witches’ broom (Salehi et al., 2020), tomato witches’ broom (Salehi & Esmailzadeh Hosseini, 2016).

Two phytoplasma insect vectors, Circulifer haematoceps and Orosious albicinctus are found inside the eggplant fields and on many weeds, trees and shrubs in eggplant marginal fields. C. haematoceps was reported as vector of eggplant big bud phytoplasma in Iran (Salehi & Izadpanah, 1995). These two insect species in Iran are vectoring several of the phytoplasmas identified in eggplant (Esmailzadeh Hosseini et al., 2007, 2011, 2017; Mirzaie et al., 2007; Salehi et al., 2015; Salehi, Esmailzadeh Hosseini, Salehi, & Bertaccini, 2016d).

The presence of phytoplasmas associated with eggplant phyllody in other crops and of C. haematoceps and O. albicinctus vectors of different phytoplasma subgroups in Iran provide indication that the eggplant fields may play an important role in the epidemiology of other diseases associated with these phytoplasmas. Collectively, based on the results of the present study and considering the reported presence of phytoplasmas belonging to the same ribosomal subgroups in other crops, eggplant fields contribute to the maintenance and spreading of diseases associated with these phytoplasmas in Iran.