Identification of 16SrII-D group phytoplasma associated with Setaria verticillata (L.) P. Beauv. in India
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Identification of 16SrII‑D group phytoplasma associated with Setaria verticillata (L.) P. Beauv. in India Smriti Mall1 · Priyam Panda2 · Govind P. Rao2 Received: 17 April 2019 / Revised: 29 December 2019 / Accepted: 27 January 2020 © Indian Phytopathological Society 2020
Abstract Phytoplasma suspected symptoms of little leaf and chlorosis were observed on Setaria verticillata grass at DDU Gorakhpur University Campus, Gorakhpur,Uttar Pradesh, India during 2018. To confirm the presence of phytoplasma, total DNA was extracted followed by PCR assay utilizing phytoplasma primers P1/P7 and R16F2n/R16R2. The ~ 1.2 kb amplicon was obtained by nested PCR assay from symptomatic Setaria samples. The amplified product was directly sequenced, aligned and submitted to GenBank. Comparison of 16S rRNA gene sequence of Setaria grass isolate shared 100% sequence identity with strains of 16SrII-D phytoplasma group (Ca. Phytoplasma aurantifolia). This is the first record of the occurrence of 16SrII-D subgroup phytoplasma in a weed grass, S. verticillata in India. Keywords Candidatus Phytoplasma aurantifolia · Grass · Little leaf · Chlorosis Setaria verticillata (L.) P.Beauv. belongs to family Poaceae, is a noxious weed mainly occurs in agricultural fields of Eastern Uttar Pradesh, commonly known as hooked bristlegrass native to Europe (Aluka, 2008). During field survey in July 2018 at DDU Gorakhpur University Campus, Gorakhpur, Uttar Pradesh, India, suspected phytoplasma symptoms of little leaf and chlorosis were observed on Setaria verticillata grass (Fig. 1a and b). Total DNA was extracted from both the asymptomatic (three) and symptomatic (five) Setaria grass samples by using CTAB protocol (Ahrens and Seemüller,1992).Two steps PCR amplifications were performed by utilizing phytoplasma primers P1/P7 (Deng and Hiruki, 1991; Schneider et al. 1995) and R16F2n/R16R2 (Gundersen and Lee, 1996). Isolated DNA from noninfected S. verticillata plant was used as a negative control. For PCR reactions Eppendorf thermocycler was used and
the PCR products were electrophoresed in 1% agarose gel with Lambda DNA (Bangalore Genei Pvt. Ltd, India) as marker. PCR amplicons were purified using a Promega gel elution kit, USA and sequenced directly. The 16Sr DNA sequence of S. verticillata phytoplasma strain was aligned by using Clustal W (Thompson et al., 1994) and identical phytoplasma group sequences retrieved from GenBank was used to built phylogenetic tree using the Molecular Evolutionary Genetics Analysis 7.0 software (Kumar et al. 2016). Bootstrap (percentage) method were performed to estimate branch stability and support. Acholeplasma laidlawii 16S rDNA sequence was used to root the phylogenetic tree as out group species. RFLP profiles comparison of 16S rDNA R16F2n/R2 fragment was done by using online iPhyClassifier tool (Zhao et al. 2009).
* Smriti Mall [email protected] 1
Department of Botany, DDU Gorakhpur University, Gorakhpur, UP 273009, India
Division of Plant Pathology, Indian Agriculture Research
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