New Disease Reports
New Disease Reports (2013) 28, 13. [http://dx.doi.org/10.5197/j.2044-0588.2013.028.013]
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First report of the association of a 'Candidatus Phytoplasma ulmi' isolate with a witches' broom disease of reed in China

C.L. Li 1, Y.J. Du 1, B.C. Xiang 1* and P. Zhang 2

*diyilcl@126.com

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Received: 23 Mar 2013; Published: 13 Nov 2013

Keywords: Phragmites australis, growth abnormality

Phragmites australis (Poaceae) is widely distributed around the world. It is not only a perennial weed, but also a raw material for the paper industry, a medicinal plant, animal fodder, an ornamental in landscaping and a building material. It now covers approximately two million ha in China, including ten thousand ha in Xinjiang Province. During September 2012, symptoms of witches’ broom, stunting, and smaller leaves (Fig. 1A) were observed in over 57% of P. australisplants at the Shihezi University Experimental Station (Shihezi, Xinjiang Province).

Leaf samples were collected from both symptomless plants and plants showing symptoms. Total DNA was extracted from 0.5 g fresh leaf midribs from P. australis plants with and without symptoms, using the CTAB method (Angelini et al., 2001). The DNA extracts were tested for phytoplasma infection by a nested polymerase chain reaction (PCR) assay with phytoplasma universal 16S rDNA primer pairs P1/P7 (Deng & Hiruki, 1991) and R16F2n/R16R2 (Gundersen & Lee, 1996). The two primer pairs produced PCR products of 1.8 and 1.2 kb, respectively, from most of the plants with symptoms (4/5), but not from the symptomless plants (2/2).

R16F2n/R16R2 amplicons were cloned (pMD18-T simple vector, TaKaRa, China) and sequenced. All of the phytoplasma R16F2n/R16R2 sequences were 100% identical to each other. BLAST comparisons showed that theP. australis witches’ broom phytoplasma 16S rDNA consensus sequence (GenBank Accession No. KC331052) had 99% sequence identity with those of members of group 16SrV (‘Candidatus Phytoplasma ulmi’). Similarity coefficients and in silico restriction fragment length polymorphism (RFLP) analyses of the R16F2n/R16R2 sequence with AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, I, and TaqI restriction endonucleases (iPhyClassifier, http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) (Fig. 2), indicated that P. australis contained a phytoplasma strain identical to those of the reference strain AB052876, ‘Ca. Phytoplasma ulmi’, subgroup 16SrV-B. A neighbour-joining dendrogram (MEGA 5.0, USA) based on the 16S rDNA sequences of the P. australis witches’ broom phytoplasma and twenty-five reference phytoplasmas in NCBI database supported the PCR and RFLP results since the P. australis witches’ broom phytoplasma grouped within the 16SrV-B phytoplasma clade (Fig. 3). The group 16SrV was previously associated with witches’ broom diseases of several plant species in China including jujube (Zhu et al., 1997), paper mulberry (Liu et al., 2004), and Chinese honeylocust (Min et al., 2009). This is the first report of a ‘Ca. Phytoplasma ulmi’-related strain affecting P. australis in China.

Figure1+
Figure 1: Phragmites australis plants showing symptoms of witches’ broom, shortened internodes, and reduced size of leaves (A) and plants without symptoms (B).
Figure 1: Phragmites australis plants showing symptoms of witches’ broom, shortened internodes, and reduced size of leaves (A) and plants without symptoms (B).
Figure2+
Figure 2: Virtual RFLP patterns derived from in silico digestions of the 16S rDNA sequences of the Phragmites australis witches’ broom phytoplasma (KC331052) (left) and Jujube witches’ broom phytoplasma (AB052876) (right) with 17 restriction endonucleases. MW= New England Biolabs, Ipswich, MA 100 bp ladder. RFLP fragments were resolved by in silico electrophoresis through a 3% agarose gel.
Figure 2: Virtual RFLP patterns derived from in silico digestions of the 16S rDNA sequences of the Phragmites australis witches’ broom phytoplasma (KC331052) (left) and Jujube witches’ broom phytoplasma (AB052876) (right) with 17 restriction endonucleases. MW= New England Biolabs, Ipswich, MA 100 bp ladder. RFLP fragments were resolved by in silico electrophoresis through a 3% agarose gel.
Figure3+
Figure 3: Phylogenetic tree constructed by MEGA 5.0 software, based on the 16S rDNA sequences of the Phragmites australis witches’ broom phytoplasma and 25 reference phytoplasmas. Bootstrap values are shown on branches. Acholeplasma laidlawii was used as the outgroup to root the tree.
Figure 3: Phylogenetic tree constructed by MEGA 5.0 software, based on the 16S rDNA sequences of the Phragmites australis witches’ broom phytoplasma and 25 reference phytoplasmas. Bootstrap values are shown on branches. Acholeplasma laidlawii was used as the outgroup to root the tree.

Acknowledgements

This research was supported by the National Key Technology R&D Program of China (2011BAD48B00).

References

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To cite this report: Li CL, Du YJ, Xiang BC, Zhang P, 2013. First report of the association of a 'Candidatus Phytoplasma ulmi' isolate with a witches' broom disease of reed in China. New Disease Reports 28, 13. [http://dx.doi.org/10.5197/j.2044-0588.2013.028.013]

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