New Disease Reports
New Disease Reports (2019) 40, 19. [http://dx.doi.org/10.5197/j.2044-0588.2019.040.019]
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First report of a 16SrII phytoplasma (subgroup D) associated with Robinia pseudoacacia witches' broom and dieback in Iran

S.A. Esmaeilzadeh-Hosseini 1*, G. Babaei 2 and S. Purmohamadi 1

*esmailzadeh.phytoplasma@gmail.com

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Received: 01 Nov 2019; Published: 06 Dec 2019

Keywords: black locust, Yazd

Black locust (Robinia pseudoacacia) is an important shade tree in urban green spaces in Iran. During 2017-18, R. pseudoacacia trees showing witches' broom and dieback symptoms (Fig. 1) were observed in Safayieh, Yazd province, Iran. Twenty-four symptomatic and four asymptomatic plants were surveyed for a phytoplasma and leaf samples were taken from each.

Total DNA was extracted from 0.2 g of fresh leaves from both diseased and symptomless plants. DNA samples were tested for the presence of phytoplasma by direct PCR primed with P1/P7 (Deng & Hiruki, 1991; Schneider et al., 1995) and nested PCR using primers R16mF2/R16mR1 and R16F2n/R16R2 (Gundersen & Lee, 1996). From all symptomatic plants products of the expected size (1.8. 1.4 and 1.2kbp) were detected but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of the amplicons obtained in nested PCR with R16F2n/R16R2 primers was performed using RsaI, AluI, MseI, HinfI and HaeIII restriction enzymes (Zhao et al., 2009). In all 24 positive samples, RFLP profiles were identical and related to 16SrII phytoplasmas ('Candidatus Phytoplasma aurantifolia').

R16mF2/R16mR1 amplicons from four R. pseudoacacia phytoplasma-infected trees were sequenced. Consensus sequences showed 100% sequence identity to each other. The sequence of an R16F2n/R16R2 amplicon (1236 bp) from a representative sample was deposited in GenBank (Accession No. MN431432). A BLAST search showed that the R. pseudoacacia phytoplasma shared more than 99% sequence identity with those of phytoplasma group 16SrII. Phylogenetic analysis using the neighbour-joining method (MEGA software version 6.0) confirmed that the R. pseudoacacia phytoplasma clustered within the 16SrII phytoplasma clade and was closely related to strains in the 16SrII-D subgroup (Fig. 2). Analysis using iPhyClassifier suggested that the R. pseudoacacia phytoplasma 16S rRNA gene RFLP pattern was identical (similarity coefficient 1.00) to that of a phytoplasma strain in 16Sr group II, subgroup D (Y10097).

A 'Ca. Phytoplasma phoenicium' strain was reported from R. pseudoacacia trees exhibiting yellowing symptoms in Iran (Karimzade et al., 2018). However, this is the first report of a phytoplasma of group 16SrII, subgroup D in R. pseudoacacia. The finding has a significant phytosanitary impact since this is the major phytoplasma group associated with diseases in crops and weeds in Iran and therefore poses a serious threat to other plant species growing nearby.

Figure1+
Figure 1: Witches' broom and dieback symptoms exhibited by Robinia pseudoacacia tree affected by the 16SrII-D phytoplasma (right) compared to a symptomless R. pseudoacacia (left).
Figure 1: Witches' broom and dieback symptoms exhibited by Robinia pseudoacacia tree affected by the 16SrII-D phytoplasma (right) compared to a symptomless R. pseudoacacia (left).
Figure2+
Figure 2: Phylogenetic tree of partial 16S rDNA sequences from the Robinia pseudoacacia phytoplasma strain (in bold) and selected phytoplasmas. GenBank accession numbers in parentheses. Numbers on branches are bootstrap values of 1,000 replicates. 'Ca. P.' stands for 'Candidatus Phytoplasma' species. Acholeplasma laidlawii used as outgroup.
Figure 2: Phylogenetic tree of partial 16S rDNA sequences from the Robinia pseudoacacia phytoplasma strain (in bold) and selected phytoplasmas. GenBank accession numbers in parentheses. Numbers on branches are bootstrap values of 1,000 replicates. 'Ca. P.' stands for 'Candidatus Phytoplasma' species. Acholeplasma laidlawii used as outgroup.

References

  1. Deng S, Hiruki C, 1991. Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. Journal of Microbiological Methods 14, 53-61. [http://dx.doi.org/10.1016/0167-7012(91)90007-D]
  2. Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer sets. Phytopathologia Mediterranea 35, 144-151.
  3. Karimzade M, Siampour M, Zakiaghl M, Mehrvar M, 2018. Identification and characterization of a phytoplasma associated with black locust yellow disease in two provinces of Iran. Crop Protection 110, 261-268. [http://dx.doi.org/10.1016/j.cropro.2017.05.010]
  4. Schneider B, Seemüller E, Smart CD, Kirkpatrick BC, 1995. Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In: Razin S, Tully JG eds. Molecular and Diagnostic Procedures in Mycoplasmology. New York, USA: Academic Press, 369-380. [http://dx.doi.org/10.1016/B978-012583805-4/50040-6]
  5. Zhao Y, Wei W, Lee I-M, Shao J, Suo X, Davis RE, 2009. Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII). International Journal of Systematic and Evolutionary Microbiology 59, 2582-2593. [http://dx.doi.org/10.1099/ijs.0.010249-0]
To cite this report: Esmaeilzadeh-Hosseini SA, Babaei G, Purmohamadi S, 2019. First report of a 16SrII phytoplasma (subgroup D) associated with Robinia pseudoacacia witches' broom and dieback in Iran. New Disease Reports 40, 19. [http://dx.doi.org/10.5197/j.2044-0588.2019.040.019]

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