Molecular identification of a 16SrII-D phytoplasmaassociated with sunflower phyllody in India

Sujatha Mulpuri1 & Tarakeswari Muddanuru1

Received: 20 November 2015 /Accepted: 30 May 2016 /Published online: 7 June 2016# Australasian Plant Pathology Society Inc. 2016

Abstract Symptoms of phyllody on sunflower in India havepreviously been reported however, lately, the frequency ofphyllody observed has increased. Transmission electron mi-croscope (TEM) examination and PCR including nested PCRanalyses using universal primers (P1/P7 and R16F2n/R2) spe-cific to phytoplasma confirmed that the floral abnormalitiesare due to phytoplasma infection. Nucleotide BLASTanalysisof the PCR sequences showed highest identity with sequencesof phytoplasma members of the 16SrII group (‘CandidatusPhytoplasma aurantifolia’). Further, phylogenetic analysis ofthe 16S rRNA sequences clustered the sequences of both sun-flower (HAP1) and sesame (SIP1) with other members of16SrII. ‘Candidatus Phytoplasma’ species assignment and16Sr group/subgroup classification using iPhyClassifier con-firmed that the phytoplasma causing sunflower phyllody is a‘Candidatus Phytoplasma aurantifolia’ related strain and be-longs to the 16SrII-D group. The molecular characters of sun-flower phytoplasma were compared with that of the phyto-plasma associated with sesame phyllody (used as reference)which showed that the R16F2n/R2 sequences of both thephytoplasmas are identical indicating that the 16SrII-D phy-toplasma that is associated with sesame phyllody can infectsunflower as well.

Keywords ‘Ca Phytoplasma aurantifolia’ . Phyllody . 16SrII-D group . Sesame . Sunflower

Sunflower (Helianthus annuus) is one of the important oilseedcrops across the globe including India. Among the productionconstraints worldwide, susceptibility of the crop to a multitudeof diseases is of serious concern. The pest dynamics in Indiaare constantly changing and unknown pathogens are becom-ing the major deterrents for profitable cultivation of the crop(Basappa and Santhalakshmi Prasad 2005). During the past2 years, phyllody-like symptoms leading to floral abnormali-ties were observed in sunflower at the research farm of IndianInstitute of Oilseeds Research (IIOR) and in the experimentalfarms in southern India but at a low (0.5 to 1.0 %) frequency.Phyllody was also observed on sesame (Sesamum indicum) ata frequency of 60–80 % and crops such as, parthenium(Parthenium hysterophorus) and sunn hemp (Crotolariajuncea) at a frequency of 0.8 and 3.2 %, respectively. Thereare reports of serious outbreaks of sunflower phyllody withstunting and sterility resulting in economic loss as the diseasedplants fail to produce seeds (Sackston 1954; Signoret et al.1976; Zimmer and Hoes 1978; Bidari et al. 1987).Phytoplasma on sunflower has been reported from USA,Canada, Argentina, France, Sudan and Israel (Sackston1954; Nour 1962; Signoret et al. 1976; Gulya et al. 1997).Phytoplasma associated with sunflower phyllody has beenobserved in Argentina for the first time during 2010–2011(Guzman et al. 2014) and in Iran during the last 2 years(Salehi et al. 2015). Survey and field visits in Karnataka state,India, revealed frequent incidence of sunflower phyllodywhich ranged from 0.23 to 3 % (Bidari et al. 1987).Sunflower phyllody can lead to stunting, leaf deformation,sterility, hypertrophy, floral malformations, false appendagesand virescence (Signoret et al. 1976; Bidari et al. 1987).

In general, phyllody on sunflower is observed randomlyand in a low frequency but for a genetic stock (IC 443608)derived from an interspecific cross involving Helianthusdivaricatus and cultivated sunflower (Prabakaran and

* Sujatha Mulpurimulpurisujata@yahoo.com

1 ICAR-Indian Institute of Oilseeds Research, Rajendranagar,Hyderabad 500030, Telangana, India

Australasian Plant Dis. Notes (2016) 11: 20DOI 10.1007/s13314-016-0209-2

Sujatha 2003), symptomatic plants were observed at a fre-quency of 30 %. Similarly, other germplasm accessions re-corded 10–15 % of phyllody. Frequent incidence of phyllodyand a high frequency of symptomatic plants in a few acces-sions necessitated characterisation of the phytoplasma associ-ated with phyllody on sunflower in India. Phyllody on sun-flower is reported from India but the causal agent has not beenidentified (Mohammad et al. 1993) or adequately characterised(Bidari et al. 1987).

PCR and PCR-RFLP techniques have been successfullyemployed for confirmation and characterisation of phytoplas-ma causing diseases in several crops (Bertaccini and Duduk2009; Sichani et al. 2014) including those in sunflower(Guzman et al. 2014; Salehi et al. 2015). However, thephytoplasmas reported in sunflower from different geograph-ical regions are not the same and have been identified as amember of 16Sr-III group (X-disease group) in Argentina(Guzman et al. 2014), 16SrII and 16SrVI (Tazehkand et al.2010) and 16SrII-D groups in Iran (Salehi et al. 2015).

The current study sought to confirm that the floral abnor-malities were due to phytoplasma infection and to characterisethe phytoplasma infecting sunflower in India using transmis-sion electron microscopy and molecular techniques for accu-rate diagnosis using phytoplasma associated with sesamephyllody as a reference.

Phyllody-affected symptomatic plants of sunflower(branched and mono-headed) and sesame used in this studywere collected from the research farm of the Indian Institute ofOilseeds Research (IIOR), Hyderabad, India. In the branchedplants, two each of the capitula were bagged (selfing), crosspollinated with sister plants with normal pollen production orleft for open pollination. For TEM analysis, stem segments ofsunflower and sesame from symptomatic and healthy plantswere fixed in 2.5 % glutaraldehyde in 0.1 M phosphate buffer(pH 7.2) for 24 h at 4 °C and post fixed in 1 % aq. osmiumtetroxide for 2 h. Following dehydration, infiltration and em-bedding in spur resin, ultra thin (60 nm) sections were madeon an ultra microtome (Leica Ultra cut UCT-GA-D/E-1/00).Sections were mounted on copper grids, stained with saturatedaq. uranyl acetate, counterstained with Reynolds lead citrateand viewed under TEM (Hitachi, H-7500, Japan) (Bozzolaand Russell 1998).

Total genomic DNAwas extracted from fresh leaf midribs(1.0 g) and floral tissues of healthy and four symptomaticsunflower plants using the CTAB (hexadecyltrimethyl-ammonium bromide) method following the Doyle andDoyle protocol (1990). PCR and nested PCR analysis weredone using universal primers P1/P7 (Smart et al. 1996) andR16F2n/R16R2 (Gundersen and Lee 1996). The PCR reac-tion mix consisted of 20 ng DNA, 150 μM of each of thedNTPs, 0.5 μM of each primer, 1X Taq buffer, 2 U of TaqDNA polymerase in a total volume of 20 μl. For nested PCR,the template obtained with P1/P7 primers was diluted (1:60)

and used. Multiplex PCR was done using the P1/P7 and actinprimers (F- 5’ TCG TGT TGC TCC AGA AGA GC 3’; R-5’ATA CCAGGGAACATGGTGGA 3’) to overrule negativePCR results.

The 16S rRNA PCR products of the phytoplasma were puri-fied, sequenced directly and submitted to NCBI GenBank.Subgroup affiliation studies were done by subjecting the se-quence analysis in the website tool iPhyClassifier (Zhao et al.2009). Phylogenetic tree based on R16F2n and R16R2sequences of sunflower and sesame along with 27 referencephytoplasma sequences from Firrao (2004) was computed byneighbor-Joining method (Saitou and Nei 1987) using MEGA6 software package (Tamura et al. 2013). The bootstrap consen-sus tree was inferred from 1000 replicates and the distances werecomputed using the Maximum Composite Likelihood method(Tamura et al. 2004).

Phyllody symptoms on sunflower are generally observed in1 or 2 plants during each season. However, in case of aninterspecific derivative from H. divaricatus and cultivatedsunflower with white pollen (IC 443608), 6 out of 20 plants(30 %) showed infection. Diseased plants produced abnormalcapitula and the symptoms were manifested in different formsbut the most predominant was the complete conversion offloral parts into leafy structures (Fig. 1a–c). In plants withbranching, all the capitula turned phylloid and remained sterile(Fig. 1d and e). There was no seed set in any of these plantsfollowing selfing, crossing and open pollination.

Round or pleomorphic phytoplasma like bodies (PLBs)were observed in tissue sections of symptomatic plants ofsunflower and sesame (Fig. 2a and b, respectively).However, the PLBs were abundant in sesame as comparedto that in sunflower. Pleomorphic bodies were not observedin healthy plant tissues of both the crops.

Phytoplasma DNA fragments of ~1.8 bp were amplifiedfrom four of the symptom-bearing sunflower plants (both leafand floral tissues) but not from symptomless plants (Fig. 3).Nested PCR using the second pair of universal primers wassuccessful as indicated by the intensity of the PCR productsobtained through direct (1.8 kb amplicon) and nested-PCR(1.2 kb amplicon) (Fig. 3). Multiplex PCR showed amplifica-tion of both the P1/P7 and actin amplicons in the symptomaticplants and only the actin gene amplicon in the healthy plant(Fig. 3). The sunflower rDNA sequences were 100% identicalwith that of the sesame phytoplasma which served as refer-ence. The DNA isolated from phyllody affected sunflowerplants of two different seasons in two different years was alsocharacterised. In all the cases, the sequences were identicalindicating that only a particular group of phytoplasma is asso-ciated with infection on sunflower in Hyderabad.

BLAST search of the trimmed sequences of the P1/P7amplicons (on iPhyClassifier) showed highest sequence iden-tity with sequences of phytoplasma members of the 16SrIIgroup (‘Candidatus Phytoplasma aurantifolia’) and the

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16SrII-D subgroup. Both sunflower and sesame sequenceshave been assigned the NCBI accessions numbers (KT005455and KT005454, respectively) and shared 99 % identity

predominantly with sesame phyllody (KF607109) from differentregions of India viz., Rajasthan (KF429485), Gujarat(KF322274), Hyderabad (KP297862), Delhi (KC920750) andalso with big bud phytoplasma of eggplant (JX441321) and to-mato (JQ868448).

The query 16S rRNA sequences of both sunflower andsesame obtained in this study shared 99.7 % similarity withthat of the BCandidatus Phytoplasma australasia^ (=‘CandidatusPhytoplasma aurantifolia’) reference strain of 16Sr group II, sub-group D (GenBank accession: Y10097). The phylogenetic treeconstructed based on 16S rRNA sequences of sunflower (HAP1)and sesame (SIP1) along with other reference strains showedclustering with the 16 SrII phytoplasmas and similarity with thesunflower phytoplasma strain from Iran (KJ016231) supportingthe results of sequence analysis data (Fig. 4).

In the present study, phyllody symptoms were ob-served both in mono-headed and multi-headed sunflowerplants. The frequency reached up to 30 % particularlyin a genetic stock (IC 443608) derived from an inter-specific cross involving Helianthus divaricatus and cul-tivated sunflower (Prabakaran and Sujatha 2003). Thisspecific cross combination also resulted in morphologi-cal variants like apetalous sunflower and double headedtypes and hence, the diseased plants were initially con-sidered as floral variants (Sujatha 2008).

Based on molecular characterisation, the phytoplasmacausing phyllody in sunflower in India was identified as‘Candidatus Phytoplasma aurantifolia’ - a member of the

Fig. 1 Symptoms of phytoplasma infected sunflower. a Capitula withgreen petalled flower-like structures in the central axis; b and c Capitulawith all floral parts including ligulate and disc florets transformed into

green coloured leaf and capitulum-like structures; d and e Branchedplants producing phylloid flowers on all branches

Fig. 2 Transmission electron micrograph of phytoplasma like bodies(indicated by arrows) in stem tissues of symptomatic plants. aSunflower (11580X); b Sesame (15440X)

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16SrII-D group. Sequence analysis and phylogenetic tree alsoshowed high homology to members of the 16SrII-D groupsuch as sesame phyllody and big bud phytoplasma of eggplantand tomato. Previous studies on molecular characterisation ofphytoplasma causing sunflower phyllody in Argentina indi-cated that the phytoplasma species belongs to the 16SrIII-J

group (X-disease) (Guzman et al. 2014). However, character-isation of sunflower phytoplasma in Iran showed associationof a 16SrII phytoplasma (Salehi et al. 2015). Based on thegeographical location and the crops of the region, predomi-nance of vector population, different members of phytoplasmacause infection.

Fig. 3 PCR analysis of the phytoplasma from sunflower and sesamediseased plants with P1/P7 (panel I), R16F2n/R2 (panel II), andmultiplexPCR with P1/P7 and actin primers (panel III) giving 1.8 (P1/P7), 1.2(R16F2n/R2), 910 bp (actin-sesame) and 645 bp (actin-sunflower) bands.

Lanes 1, 2, 3, 4 represent symptomatic sunflower plants, 5 representsasymptomatic sunflower plant, 6 represents phyllody infected sesame(reference), M1- double digest of λ DNA with EcoRI and HindIII, M2-100 bp ladder, NC- no DNA control

Fig. 4 Phylogenetic tree constructed by Neighbor-Joining method usingthe 16S rRNA gene sequences from sunflower phytoplasma (HAP1),sesame phytoplasma (SIP1) and 27 phytoplasma reference sequences

downloaded from NCBI with Acholeplasma laidlawii as an outgroup.Phylogenetic analysis was conducted using MEGA6 software. Thenumbers on the branches are bootstrap (confidence) values

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The identification of a 16SrII-D phytoplasma affectingboth sunflower and sesame suggests that the 16SrII phytoplas-ma isolates limited to the same geographical location ofHyderabad can infect the vulnerable crops of the region.This was verified by subjecting the phylloid plants ofparthenium and sunn hemp to molecular analysis which werealso associated with 16SrII-D phytoplasma (data not present-ed). This constitutes the first report on confirmation of phyto-plasma belonging to the 16SrII-D group associated with sun-flower phyllody in India.

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