Detection and molecular characterization of ‘Candidatus phytoplasma asteris’ related phytoplasmas infecting canola in North Dakota

Figures & data

Table 1. Classification of 28 phytoplasma strains based on 16S rDNA, ribosomal protein and secY sequences into RFLP subgroups.

Samples	Year isolated	RFLP subgroup
		16SrI	rpI	secY-I
Bn01-Bn09	2011	B	B	B
Bn10, Bn12-Bn20	2011	A	A	A
Bn11	2011	A/B	A/B	A/B
Bn21-Bn23, Bn25-Bn28	2012	B	B	B
Bn24	2012	A/ B	A/ B	A/ B

Fig. 1 RFLP analysis of 16S rDNA, rp operon and secY sequences of phytoplasma strains. a, RFLP profiles of the 16S rDNA nested PCR products amplified with primer pairs R16F2n/R2 and digested using restriction endonucleases BfaI, HhaI and MseI. b, RFLP profiles of the rp operon sequences (containing rpl22 and rps3 genes) amplified with primer pairs rPF1/rpR1 digested using restriction endonucleases AluI, Tsp5091 and MseI. c, RFLP profiles of the secY gene sequences amplified with the primer pairs AYsecYF1/AYSecYR1 using restriction endonucleases AluI, Tsp5091 and MseI. Lanes M is GeneRuler 1 kb plus DNA ladder (Life Technologies) molecular weight marker. Digests were separated on 3% super fine resolution agarose gel (SFR) except for MseI and Tsp5091 digests of rp and secY genes which were separated on 5% Agarose SFR gel. Samples Bn03 and Bn06: subgroup IA; Bn10 and Bn16: subgroup IB; Bn11 and Bn24: mixed infection.

Fig. 2 Phylogenetic trees constructed by parsimony analyses based on three genetic regions. a, 16S rDNA (using representative sequences of several groups of phytoplasmas) b, rp operon (using representative sequences of the rp operon from AY subgroups) and c, secY (using representative sequences of the secY gene from AY subgroups) sequences using PAUP 4.0 version (Swofford 2002). Reference strains for 16S rDNA (Olivier et al. 2010), rp operon (Lee et al. 2004), and secY (Lee et al. 2006) were as described in the original papers. Accessions with an * represent the strains sequenced in this study and strains with # are used as outgroups. Trees were constructed via step-wise addition by 100 replicates of a heuristic search employing tree bisection and reconnection algorithm to find optimal trees. Branch lengths are proportional to the number of inferred character state transformations. Bootstrap analyses (1000 replicates) were conducted to determine the stability and support for the inferred clades. Bootstrap values above 80% are shown on main branches.

Swofford DL. 2002. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sunderland, MA: Sinauer Associates.

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Olivier CY, Galka B, Séguin-Swartz G. 2010. Detection of aster yellows phytoplasma DNA in seed and seedlings of canola (Brassica napus and B. rapa) and AY strain identification. Can J Plant Pathol. 32:298–305.

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Lee I-M, Gundersen-Rindal DE, Davis RE, Bottner KD, Marcone C, Seemüller E. 2004. “Candidatus Phytoplasma asteris”, a novel phytoplasma taxon associated with aster yellows and related diseases. Int J Syst Evol Microbiol. 54:1037–1048.

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Lee I-M, Zhao Y, Bottner KD. 2006. SecY gene sequence analysis for finer differentiation of diverse strain in the aster yellows phytoplasma group. Mol Cell Probes. 20:87–91.

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