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. 2018 Jan 22;24(2):203–210. doi: 10.1007/s12298-017-0499-7

Molecular characterization of ‘Clover proliferation’ phytoplasma subgroup-D (16SrVI-D) associated with vegetables crops in India

Gopala 1, Ekta Khasa 1, Ashutosh Rao 1, Madhupriya 1, G P Rao 1,✉
PMCID: PMC5834985  PMID: 29515315

Abstract

Nine vegetable plants species exhibiting phytoplasma suspected symptoms of white/purple leaf, little leaf, flat stem, witches’ broom, phyllody and leaf yellowing were observed in experimental fields at Indian Agricultural Research Institute, New Delhi from December 2015 to July 2016. Total DNA extracted from the three healthy and three symptomatic leaves of all the nine vegetables were subjected to PCR assays using phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. No amplifications of DNA were observed in first round PCR assays with primer pair P1/P7 from any of the symptomatic samples. However, phytoplasma DNA specific fragments of ~ 1.3 kb were amplified from Apium graveolens L. (two isolates), Brassica oleracea vr. capitata L. (one isolate) and Solanum melongena L. (one isolate) by using 3Far/3Rev primer pair and 1.2 kb fragment was amplified from Lactuca sativa L. (one isolate) by using R16F2n/R16R2 primer pair. No DNA amplification was seen in other symptomatic vegetable samples of tomato, carrot, cucurbit, bitter gourd and Amaranthus species utilizing either P1/P7 primer pair followed by 3Far/3Rev or R16F2n/R16R2 primer pairs. Out of three leafhopper species collected from the symptomatic vegetable fields, only Hishimonus phycitis was found positive for association of phytoplasma. No DNA amplifications were observed in healthy plant samples and insects collected from non-symptomatic fields. Comparative sequence comparison analyses of 16S rDNA of positive found vegetable phytoplasma strains revealed 100% sequence identities among each other and with phytoplasma strains of ‘clover proliferation’ (16SrVI) group. Phytoplasma sequences, virtual RFLPs and phylogenetic analyses of 16S rDNA sequence comparison confirmed the identification of 16SrVI subgroup D strain of phytoplasmas in four vegetables and one leafhopper (HP) species. Further virtual RFLP analysis of 16S rDNA sequence of the vegetables phytoplasma strains confirmed their taxonomic classification with strains of ‘clover proliferation’ subgroup D. Since, H. phycitis feeding on symptomatic vegetable species in the study was also tested positive for the 16SrVI phytoplasma subgroup-D as of vegetables; it may act as potent natural reservoir of 16SrVI-D subgroup of phytoplasmas infecting vegetable and other important agricultural crops.

Keywords: Phytoplasma, 16SrVI group, Identification, Lettuce, Cabbage, Celery, Brinjal, Hishimonas phycitis

Introduction

Vegetables play a major role in Indian economy by providing high returns in terms of food, nutrition and economic security. In India, vegetables provide greater income leading to improved livelihoods. Major factors that limit vegetable production besides its narrow genetic base are extreme susceptibility of the crops to biotic and abiotic stresses. Among the biotic stress, phytoplasmas are causing severe losses to many economical vegetable species all around the world (Bertaccini et al. 2014). Phytoplasma are small phytopathogenic bacteria that inhabit phloem sieve elements of infected plants with genome size ranging between 530 kb and 1650 kb. Phytoplasma caused more than 600 plant diseases and cause serious damage with significant impact on agriculture economics (Bertaccini et al. 2014). There is no authorized curative treatment for phytoplasma disease in vegetables except using genetic resistant sources (Jaurausch et al. 1999; Rao and Kumar 2017). Management of phytoplasma can also be effectively monitored by controlling the insect vectors and roughing alternate/collateral hosts, elimination of diseases through meristem tip culture and applying antibiotics, which are the best approaches to minimize the incidence of phytoplasmas (Bertaccini et al. 2014).

Many different vegetable crops are seriously affected by different groups of phytoplasmas worldwide (Sichani et al. 2014). The most important phytoplasma diseases, infecting vegetables are brinjal little leaf, potato witches’ broom, potato purple top, tomato big bud, chilli leaf curl and cucurbit phyllody, which cause significant economic losses (Lee et al. 2000; Girsova et al. 2008; Hosseini et al. 2011; Ivanović et al. 2011; Tran-Nguyen et al. 2003; Mejia et al. 2011; Du et al. 2013; Dehghan et al. 2014; Esmailzadeh Hosseini et al. 2015). Considering the economic importance of the vegetables and the little existing knowledge of phytoplasma strains association available with vegetable crops in India (Rao et al. 2010), the present study was planned to identify the phytoplasmas in nine vegetable crops showing phytoplasma suspected symptoms grown at IARI, New Delhi, India in the year 2015 and 2016.

Materials and methods

Plant samples and leafhopper survey

During survey of vegetable crops in 2015 and 2016, phytoplasma suspected symptoms and incidence in celery (Apium graveolens L.), carrot (Daucus carota), cabbage (Brassica oleracea vr.capitata L.), lettuce (Lactuca sativa L.), brinjal (Solanum melongena L.), cucurbit (Cucurbita pepo L.), bitter gourd (Momordica charantia L.), amaranth (Amaranthus spp.) and tomato (Solanum lycopersicum L.) was recorded. Leaf leafhopper (LH) species from the symptomatic and non symptomatic vegetable fields were also collected in the morning hours by sweeping net method at Indian Agricultural Research Institute New Delhi, India. All the symptomatic, non-symptomatic vegetable plants and leafhopper samples were collected from the fields and stored at − 20 °C for PCR assays. The present study was planned to identify the pathogens associated with nine vegetable crops showing phytoplasma suspected symptoms. The collected leafhopper specimens were identified from Division of Entomology, IARI, New Delhi.

DNA extraction and PCR assays

DNA was extracted from leaf vein tissues of nine symptomatic and asymptomatic vegetables plant species and salivary glands from ten individuals of identified leafhopper species by CTAB method (Ahrens and Seemuller 1992). The salivary gland of leafhopper species was isolated as method described by Marzachi et al. (1998). Extracted DNA from all the vegetable plants and leafhoppers species were used as template in nested PCR assays by primer pair P1/P7 in first round (Schneider et al. 1995) followed by nested primer pair 3Far/3Rev (Manimekalai et al. 2010) and R16F2n/R16R2 (Gundersen and Lee 1996) in second round nested PCR assays. PCR reactions were performed in a Master cycler (Eppendorf, Germany) and the cycling protocol was followed as described earlier (Rao et al. 2014). The DNA of toria phyllody phytoplasma (16SrIX group) maintained on periwinkle was used as positive control. The DNA extracted from healthy and asymptomatic vegetable plants and leafhoppers collected from distant non-symptomatic vegetable fields were used as negative controls. Twenty-five microlitres of PCR product was subjected to electrophoresis through a 1.0% (w/v) agarose gel followed by staining with Good View dye and visualization of DNA bands with UV transilluminator. Nested PCR products (1.2 and 1.3 kb amplicons) were purified using the WizardR SV Gel and PCR Clean-up System (Promega, USA).

Sequencing analysis

The ~ 1.2 and ~ 1.3 kb nested PCR products of vegetable phytoplasma isolates were sequenced directly using 3Far/3Rev and R16F2n/R16R2 primers by Macrogen Company (South Korea) for both the strands. The sequences were assembled using DNA baser V.4 program and were further aligned using CLUSTAL W program of Bio-Edit software. Aligned and edited sequences were deposited in NCBI GenBank. Samples identification was completed by comparison of the consensus 16S rDNA sequences available in Genbank by BLAST protocol (http://blast.ncbi.nlm.nih.gov). The sequence identity percentages provided in results are over entire length of the query sequences in GenBank.

Phylogenetic and RFLP analysis

The six 16S rDNA sequence generated from the vegetables and leafhopper phytoplasma strains from this study and eight selected reference phytoplasma strains’ sequences belonging to different 16SrVI subgroups representatives retrieved from GenBank were used to compute phylogenetic tree by neighbor joining method with 1000 replications for each bootstrap value using MEGA 5.0 software version (Tamura et al. 2011). The 16S rRNA sequence of Acholeplasma laidlawii (Acc. No. M 23932) was employed as outroot the phylogenetic tree.

In silico RFLP analyses of 3Far/3Rev and R16F2n/R16R2 amplicons of vegetables and leafhopper were digested with 17 restriction enzymes (AluI, BfaI, TaqI, DraI, EcoRI, HaeIII, HhaI, Hinf I, HpaI, HpaII, KpnI, Sau3AI, MseI, BamHI, RsaI, BstUI and SspI) using pDRAW32 program (http://www.acaclone.com) and compared with representative sequences of the brinjal little leaf phytoplasma strain (Acc. No KP027505) and Catharanthus phyllody phytoplasma strain (Acc. No KP027531) for assigning 16Sr sub-groups status of vegetable phytoplasma strains analysed by the same restriction mapping utilizing AcaClone software generated RFLP profiles.

Results

Survey and symptomatology

Phytoplasma suspected symptoms of white leaf in celery (Apium graveolens), purple leaf in carrot (Daucus carota), witches’ broom in cabbage (Brassica oleracea vr.capitata), flat stem in lettuce (Lactuca sativa), little leaf in brinjal (Solanum melongena), phyllody in cucurbit (Cucurbita pepo) and leaf yellowing in bitter gourd (Momordica charantia), Amaranth (Amaranthus spp) and tomato (Solanum lycopersicum) were recorded during survey of vegetable fields at IARI, New Delhi in 2015 and 2016 (Table 1, Fig. 1a–i). The disease incidence varied from 2 to 6% in different surveyed fields (Table 1).

Table 1.

Survey, symptoms, locations and PCR results for phytoplasma detection on vegetable plants

Sr. no. Plant species common name/family Survey period Disease symptoms PCR assay GenBank Acc. No Group & sub-group
1 Apium graveolens L.
Carrot (Apiaceae) 		May 2016 WL + KX671551, KX671552 16Sr VI-D
2 Solanum melongena L. Brinjal (Solanaceae) May 2016 LL + KX421256 16Sr VI-D
3 Cucurbita pepo L.
Pumpkin (Cucurbitaceae)		 February 2016 FV − – –
4 Lactuca sativa L.
Lettuce (Asteraceae)		 May 2016 FS + KX671554 16Sr VI-D
5 Momordica charantia L.
Bitter gourd (Cucurbitaceae)		 June 2016 LY – – –
6 Amaranthus spp
(Amaranthaceae)		 June 2016 LY – – –
7 Solanum lycopersicum L.
Tomato (Solanaceae)		 July 2016 LY – – –
8 Brassica oleracea var.capitata L.
Cabbage (Brassicaceae)		 May 2016 WB + KX6971553 16Sr VI-D
9 Daucus carota L.
Carrot (Apiaceae)		 July 2016 VL – – –
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IARI = Indian Agricultural Research Institute, New Delhi

WL white leaf, WB witches’ broom, LL little leaf, FV floral virescence and phyllody, FS flattened stem, LY leaf yellowing, VL violet leaves

Fig. 1.

Fig. 1

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Phytoplasma suspected symptoms on vegetable plant species a white leaf in celery (Apium graveolens); b witches’ broom in cabbage (Brassica oleracea vr. capitata); c flat stem in lettuce (Lactuca sativa); d little leaf in brinjal (Solanum melongena); e phyllody in cucurbit (Cucurbita pepo); f leaf yellowing in bitter gourd (Momordica charantia); g leaf yellowing in amaranth (Amaranthus spp); h leaf yellowing in tomato (Solanum lycopersicum); i violet coloured leaf in carrot (Daucus carota sub sp. sativus)

Identification of the leafhoppers specimens

Empoasca prima (Distant), Exitianus indicus (Distant) and Hishimonus phycitis (Distant) were identified as the major leafhopper species feeding on symptomatic vegetable plants in the fields of IARI, New Delhi during December 2015 to July 2016.

Detection of phytoplasma by PCR Assays

First round PCR amplification did not yield the expected 1.8 kb product of the 16S rRNA gene region from any of the symptomatic vegetable test samples with primer pair P1/P7. However, 1.3 kb DNA products were amplified from A. graveolens (two isolates), B. oleracea va. capitata (one isolate), S. melongena (one isolate) and one leafhopper species (H. phycitis) by using 3Far/3Rev primer pair, however, 1.2 kb product was amplified from L. sativa by using R16F2n/R16R2 primer pair and the positive control (toria phyllody phytoplasma,Azadvar and Baranwal 2012) (Table 1). No amplifications were observed in any of the rest five symptomatic vegetable samples of tomato, carrot, cucurbit, bittergourd and Amaranthus sp alongwith the non-symptomatic samples (Table 1). There were also no amplification observed from DNA isolated from salivary glands of two leafhopper species, E. prima and E. indicus.

Sequence analysis and phylogenetic relationships

Pair wise sequence comparison of the 16S rRNA gene sequences of the five positive vegetable phytoplasma strains of A. graveolens (two isolates), B. oleracea vr. capitata, L. sativa, S. melongena (one isolate) and one phytoplasma strain of H. phycitis revealed 100% genetic identity among themselves and with brinjal little leaf (Acc. No. KP027505), Catharanthus phyllody (Acc. No. KP027531) and Mollicutes sp phytoplasma strain (Acc. No. X83431) sequences, which belonged to clover proliferation (16SrVI) group. Phylogenetic analysis also revealed the clustering of all the five vegetable phytoplasma strains and one leafhopper (HP) phytoplasma strain in the present study with strains of 16SrVI-D (Fig. 2).

Fig. 2.

Fig. 2

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Phylogenetic tree constructed by neighbor-joining method showing the relationships among celery white leaf, lettuce flattened stem, cabbage withces’ broom and brinjal little leaf phytoplasma, and reference phytoplasma strains. Accession numbers are specified in the tree. ‘Ca. P.’ stands for ‘Candidatus Phytoplasma sp.’ A. laidlawii was used as an out group. Mega 7.0 software was used to construct the tree

The results of virtual RFLP gel plotting using the pDRAW32 program revealed that the all the five positive phytoplasma strains from vegetables and one leafhopper associated strain produced a identical virtual RFLP profiles to phytoplasma reference strains of brinjal little leaf (KP027505) and Catharanthus phyllody (KP027531) phytoplasmas belonging to 16SrVI-D subgroups (Fig. 3). Therefore, five phytoplasma strains from the four vegetable species and the H. phycitis (leafhopper) in the present study were identified as strains of 16Sr VI-D subgroup.

Fig. 3.

Fig. 3

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Comparison of virtual RFLP patterns derived from in silico digestions of 1.2 and 1.3 kb 16S rDNA sequences of vegetables in the present study, along with reference standard strains of 16SrVI-D subgroup of phytoplasma (Catharanthus phyllody phytoplasma, Acc. No. KP027531; Brinjal little leaf phytoplasma, Acc No. KH421256)

Discussion

Phytoplasma-associated vegetable diseases are distributed worldwide, and in several cases were associated with severe epidemics. So far, five different groups (16SrI, 16SrII, 16SrVI, 16SrIX, 16SrXII) of phytoplasmas were identified in vegetables all over the world (Lee et al. 2000; Verdin et al. 2003; Zhang et al. 2004; Delic et al. 2016; Salehi et al. 2007; Rao et al.2010; Ivanovic et al. 2011; Bertaccini et al. 2014). Among them, the aster yellows phytoplasma group (16SrI) is the widest distributed and dominant group recorded on vegetables species from all over the world (Kumar 2015).

In India, different groups of phytoplasmas were reported to be associated with several vegetable plants like 16SrI group in potato (Tiwari et al. 2013), chilli (Khan and Raj 2006), ladies’ finger (Kumar et al. 2012a, b) and 16SrII group in lettuce, carrot and beans (Ghosh et al. 1999; Ghanekar et al. 1988; Arocha et al. 2008). Besides, three different phytoplasma groups (16Sr I, -II and -VI) were identified associated with brinjal little leaf disease from different states of India (Rao and Kumar 2017). In the present study we have reported occurrence of 16Sr VI subgroup D phytoplasma strain in brinjal, lettuce, celery and cabbage.

Earlier infection of 16SrI-B subgroup phytoplasma associated with stunting and witches’ broom in cabbage (B. oleracea vr. capitata) was reported from Greece (Gkavaleka et al. 2012), China (Mou et al. 2012) and USA (Lee et al. 2001). Salehi et al. (2007) reported 16SrVI group of phytoplasma on cabbage (B. oleracea vr. capitata) from Iran. No report of phytoplasma on cabbage (B. oleracea vr. capitata) is available from India, hence, occurrence of 16Sr VI-D subgroup phytoplasma strain on cabbage is new report from India.

Lee et al. (2000) reported phytoplasma of 16SrI-B subgroup infecting celery crops in North America and Europe. Later, other groups of phytoplasmas were reported on celery from other parts of the world, like tomato big bud phytoplasma (16SrII-E) from Australia (Tran-Nguyen et al. 2003) and stolbur phytoplasma (16SrXII-A) from Italy, Serbia and Romania (Carraro et al. 2008; Ivanović et al. 2011; Chireceanu et al. 2016), 16SrVII and IX-A groups from New Zealand (Liefting et al. 2011) and 16SrI-C from the Czech Republic (Fránová and Špak 2013). No report of 16SrVI group of phytoplasmas incidence has been recorded earlier on celery from India and abroad, and hence, is the new report.

In lettuce, there are three phytoplasma reports available as 16SrIX and 16 SrI-B groups from Iran (Salehi et al. 2006; Sichani et al. 2014), 16SrI-B subgroup from China (Zhang et al. 2004) and 16SrII group from India (Arocha et al. 2008). The report of 16SrVI-D subgroup of phytoplasma on lettuce from India in the present study is also the new report.

Brinjal little leaf phytoplasmas belonging to four groups, viz. 16SrI from Japan, Bangladesh and India (Okuda et al.1997; Kelly et al. 2009; Kumar et al. 2012a, 2012b), 16SrII-D from Egypt (Omar and Foissac 2012), 16SrIII-J and 16SrIII-U from Brazil (Mello et al. 2011) and 16SrVI-C, -D and -E from China, India and USA (Hiruki and Wang. 2004,2008; Wei et al. 2008; Kumar et al. 2017) were reported to infect brinjal. In the present study, we have reported again the occurrence of 16SrVI-D subgroup phytoplasma associated with brinjal which was earlier recorded as the most wide spread group of phytoplasmas infecting brinjal crops in India (Kumar et al. 2017). This confirm the regular recurrence of the disease which is responsible for serious economic losses.

In addition to vegetable crops, we have also identified 16SrVI-D subgroup phytoplasma in the salivary gland of the leafhopper, H. phycitis from the phytoplasma infected vegetable fields in the study area indicating its possible role as a vector of this group of phytoplasmas. Earlier, Kumar et al. (2017) established the role of H. phycitis as a natural vector in transmitting brinjal little leaf phytoplasma strain belonging to 16SrVI-D group from India. The 16SrVI-D phytoplasma subgroup, have already been reported earlier as a major group of phytoplasma infecting several crops viz. brinjal (Rao and Kumar 2017), ornamental plants (Khasa et al. 2016), medicinal plants (Madhupriya et al. 2015) and sesame (Madhupriya et al. 2015) in India.

In India, many vegetables are being cultivated parallel with many other agricultural crops in different seasons. The reports of wider spread, broad host range and capability of transmission through insect vectors and weeds, 16SrVI-D phytoplasma strains pose a serious threat and thus this phytoplasma strain is of great epidemiological significance in India. Hence, further studies are required to work out the role of epidemiological significance of this strain (16SrVI-D subgroup) in different parts of India for planning a better management approach.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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