Identification of Cannabis sativa L . ssp. sativa as ...

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Calotropis gigantea, Datura stramonium, Cymbopogan citratus and Tylophora asthmatica (Rao et al., 2011;. Singh et al., 2012; Madhupriya et al., 2014; Chaube.


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Nabi et al.

I dentif ication of Cannabis sativa L .ssp. sativa as putative alter nate host of sesame phyllody phytoplasma belongs to 16Sr I group in I ndia Saj ad un Nabi 1, M adhupr iya1, Dur gesh Dubey2 and G.P. Rao1 1Di vision of Plant Pathology, Indian Agri cultural Research I nstitute, New Delhi, India 2Department of Botany, DDU Gorakhpur Uni versity, Gorakhpur - 273009, UP, India

ABSTRACT Li ttl e leaf and witches’-broom symptoms on Cannabis sativa L. ssp. sati va pl ants were recorded i n sesame phyll ody inf ected f ields at Kushinagar district of eastern Uttar Pradesh, I ndi a in September 2013. The DNA extracted f rom symptomatic Cannabi s sativa L. ssp. sativa (CSS) pl ants yielded amplicons of 1.25 kb i n nested PCR assays with R16F2/R2n primer pair. BLASTn comparison and phylogenetic analysis of 16Sr DNA phytoplasma sequence of CSS phytoplasma isol ate reveal ed association of ‘Ca. Phytoplasma asteri s’ (16Sr I group) wi th symptomatic cannabi s plants. Our results suggest that CSS may play an important role in perpetuation of sesame phyll ody phytoplasma in nature. [M edi cinal Plants 2015; 7(1) : 68-70] K eywor ds : Hemp, PCR assay, phylogeny analysis, 16Sr I group, putative alternate host

Phytoplasmas are gaini ng i nternati onal i mportance because of unspeci f ic symptoms, serious l osses and diverse epidemi ology throughout the worl d. Medi cinal plants constitute a group of industrially important crops which are of great val ue for domestic use and export. Phytoplasma cause diseases i n several medici nal plant speci es (Rao et al., 2011; Marcone, 2011). I n I ndi a, f ive different groups of phytoplasma (16SrI ,16SrI V, 16SrV, 16SrVI and 16SrXIV ) were reported on medicinal plants, viz., Cannabis sativa, Santalum album, Withania somnifera, Portulaca grandi flora, Catharanthus roseus, Calotropis gigantea, Datura stramonium, Cymbopogan ci tratus and Tylophora asthmatica (Rao et al., 2011; Singh et al., 2012; Madhupriya et al., 2014; Chaube et al., 2014). The plant species Cannabis, known as Indian hemp, is an annual herb of the family Cannabinaceae. It has

Cor responding author : G.P. Rao e-mail : [email protected] Received: January 18, 2015; Accepted: February 25, 2015 doi : 10.5958/0975-6892.2015.00010.6

been used by humans throughout recorded history f or its food, f i ber and medici ne. It is a native to Central Asia, and cultivated in Asi a, Europe and China. The medi ci nal val ue of Cannabi s i ncl udes i ntoxi cant, analgesic, narcotic, stomachic, antispasmodic, anodyne, sedative etc. (Kuddus et al., 2013). C. sativa L. ssp. sativa grow quite common as a weed i n northern India. Li ttle leaf and witches’- broom symptoms were observed on C. sativa L. ssp. sativa (CSS) pl ants in sesame f iel ds showing li ttl e l eaf and wi tches’ broom symptoms at Kushi nagar di stri ct of eastern Uttar Pradesh, India in 2013 (Fig. 1). DNA from symptomatic and non-symptomati c l eaf tissues of the CSS was extracted followi ng a described procedure (Ahrens and Seemul l er, 1992). A mpl i f i cati on of phytopl asma ri bosomal DNA (rDNA ) was perf ormed wi th the universal phytoplasma pri mer pai rs P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995) and nested primer pair R16F2n/R16R2 (Gundersen and Lee, 1996). The DNA extracted f rom periwi nkl e i nfected wi th toria phyll ody phytoplasma (group 16SrIX group, Azadvar et al., 2009) and maintained in greenhouse was used as positive control. The DNA extracted from asymptomatic CSS plant tissues was used as negati ve control.

Medicinal Plants, 7(1) March 2015

Identif ication of Cannabis sativa as putative alternate host of sesame phyllody phytoplasma

Fi g. 1. Cannabi s sati va subsp sati va pl ant showi ng l eaf yel lowing and excessive apical branching givi ng a witches’ broom appearance.


Reacti ons were carri ed out i n a Master cycl er (Eppendorf) and the cycling protocol used f or the f irst round PCR as descri bed by Rao et al. (2014). Two  l product of the f irst round of PCR was used i n nested PCR using internal primer pairsR16F2n/R16R2. Fi ve microl itres of each PCR product was subj ected to el ectrophoresis using 1.0% (w/v) agarose gel , stained wi th ethi dium bromide and observed under UV trans i l l uminator. A nested PCR product (1.25 kb) was purif ied usi ng the Nucleospi n Gel and PCR Clean-up ki t (Macherey Nagel) and sequenced directly in both directions. The sequences were aligned using CLUSTAL W method of Bio-Edit sof tware (Bi o-edit Sequence Align Editor) and the consensus sequence was submitted to GenBank and used in BLAST search. The 16Sr DNA sequence generated from the present study and reference phytoplasma strai ns sequence retri eved from GenBank were used to construct phyl ogeny by neighbour j oining method using MEGA 5.0 software versi on (Tamura et al., 2011). Achol epl asma laidl awi i was used as out group to root the tree. A ~1.25 kb amplicon in nested PCR products were

Fig. 2 Phylogenetic tree constructed by neighbour-joi ning method showi ng the relationships of Cannabi s phytopl asma isol ate (India) and reference phytoplasma strains. Accession numbers are specif ied in the tree.

Medicinal Plants, 7(1) March 2015


Nabi et al.

obtai ned with R16F2/R2 primers in symptomati c CSS and the posi tive control (data not shown). No DNA was ampli f ied by nested PCR assays from any of the asymptomatic CSS samples. Ampli cons of ~1.25 kb nested PCR product of CSS li ttl e leaf phytoplasma and witches broom isolate was di rectly sequenced from both ends and the 16Sr DNA sequence was deposited in GenBank (Ac No KP768073). BL AST anal ysi s of 16Sr RNA gene sequence reveal ed 99% sequence identity of the CSS phytoplasma i solate wi th that of phytoplasmas strai ns of sesame phyllody phytoplasma isolates of 16Sr I group identif ied from the same area and members of ‘Ca. Phytoplasma asteris’ group. Phyl ogeneti c anal ysi s of the test CSS phytopl asma 16S rDNA sequence wi th the test sequences retrieved from GenBank revealed its closest phylogenetic relationship with sesame phyllody isolates identif ied i n Kushinagar and with other members of the 16SrI group (Fi g. 2). Theref ore, the CSS phytoplasma isol ate was identif ied as a strain of aster yellows phytopl asma (16SrI group). A witches’-broom di sease on a Cannabi s sp. has been reported to be associated with el m yell ows group (16SrV) i n China (Zhao et al., 2007), stol bur group (16Sr XII) in Iran (Sichani et al., 2011), and aster yellows group (16SrI) and Bermuda grass white leaf group (16Sr XI V) group phytoplasmas in India (Raj et al., 2008; Mal l et al., 2011; Chaube et al., 2014). In the present study we identif ied the 16SrI group phytopl asma in C. sati va subsp sativa in sesame infected f ields where the authors also reported the similar group of phytoplasmas infecting sesame pl ants (Sajad et al., 2014) hence it may serve as natural reservoi r of sesame phyl lody phytopl asma i n nature. Si nce Cannabi s speci es i s reported to harbour di fferent phytoplasmas associated wi th diff erent groups, it is very i mportant to manage this important weed i n many parts of Uttar Pradesh. REFERENCES A hrens U and Seemül l er E (1992). Detecti on of pl ant pathogeni c mycopl asma l i ke organi sms by a polymerasechai n reaction that ampl if ies a sequence of 16S rRNA gene. Phytopathology, 82: 828. Azadvar M, Baranwal VK and Yadava DK (2009). First report of a 16SrIX (Pigeon pea witches’ broom) phytoplasma associated wi th toria (Brassica rapa cv. toria) phyll ody disease in India. New Dis. Rept., 20: 27. Chaube S, Kumar S, Dubey D, Tiwari AK, Upadhyaya PP, Rao GP (2014) Identif i cation of a novel phytoplasma (16Sr XIV-A subgroup) associated with li ttle leaf and witches’ broom of Cannabis sativa L . ssp. sati va and C. sati va L . ssp. i ndi ca i n Indi a. Phytoparasi ti ca, doi 10.1007/s12600-014-0438-x.

Deng S and Hiruki C (1991). A mplif ication of 16S rRNA genes from cul turable and nonculturable molli cutes. J. Microbiol. Methods, 14: 53-61. Gundersen DE and Lee IM (1996). Ultrasensitive detection of phytoplasmas by nested-PCR assays using twouniversal primer pairs. Phytopathol . Medit., 35: 144-151. Kuddus M, Ginawi IAM and A l-Hazimi A (2013). Cannabis sati va: A n ancient wi ld edibl e pl ant of India. Emi r J Food Agric., 25: 736-745. M adhupriya, Maurya R and Rao GP (2014). ‘Candidatus Phytopl asma aurantifolia’ related strai ns affecting two important medici nal plants (Cymbopogan citratus and Tylophora sthmatica) in India. Indi an Phytopath., 67 (3): 303-307. Marcone C (2011). Current status of phytoplasma diseases of medicinal and nutraceutical pl ants i n southern Italy. Bul l. Insectology, 64: 233-234. Mal l S, Upadhyaya PP and Rao GP (2011). Detection of a 16SrI phytopl asma associated with Cannabis sati va i n Indi a. Indian Phytopathology, 43: 132-137. Rao GP, Mall S, Raj SK and Snehi SK (2011). Phytoplasma diseases affecting various pl antspecies i n India. Acta Phytopathol. Entomol. Hungari ca, 46: 59-99. Rao GP, M adhupriya, Ti wari A K, Kumar S and Baranwal V K (2014). Identi f icati on of sugarcane grassy shootassoci ated phytopl asma and one of i ts putati ve vectors i n India. Phytoparasi ti ca, 42: 349-354. Raj SK , Snehi SK , K han M S and K umar S (2008). ‘Candi datus Phytopl asma asteri s’ (group 16SrI ) associated with a wi tches’-broom disease of Cannabis sati va in India. New Dis. Rept., 17:16. Schneider B, Seemuller E, Smart CD and K irkpatri ck BC (1995). Phylogeneti c cl assi fcation of plant pathogenic mycoplasma-like organisms or phytopl asmas. (Eds. S. Razin and J.G. Tul ly. San Diego, CA) Academi c Press. Mol ecul ar and Di agnosti c Procedures in Mycoplasmology1, 369-380. Sajad un nabi (2014). Multilocus gene characteri zation of phytopl asma associ ated wi th sesame phyl l ody and identif i cation of its i nsect vector. M .Sc Thesis, Indian Agricul tural Research Institute, New Delhi , India, pp. 123. Sichani FV, Bahar M and Zirak L (2011). Characterization of stolbur (16Sr X II) group phytoplasma associated with Cannabis sativa wi tches’s-broom disease in Iran. Plant Pathology Journal , 10: 161-167. Singh N, Madhupriya, Rao GP and Upadhayaya PP (2012). ‘Candidatus Phytopl asma trifolii ’ associ ated with little leaf and witches’ broom disease of Datura stramoni um L. i n Indi a. Phytopathogenic Moll icutes, 2(2): 69-71. Tamura K , Dudley J, Nei M and Kumar S (2011). MEGA4: M ol ecul ar Evol uti onary Geneti cs A nal ysi s (M EGA ) sof tware versi on 4.0. Mol ecul ar Bi ol ogy and Evoluti onary, 24: 1596-1599. Zhao Y, Sun Q, Davis RE, Lee I and Li u Q (2007). Fi rst report of witches’-broom disease in a Cannabis speci es and its associ ation with a phytopl asma of Elm yell ows group (16SrV ). Plant Di sease, 91: 227.

Medicinal Plants, 7(1) March 2015