New Disease Reports
New Disease Reports (2019) 40, 17. [http://dx.doi.org/10.5197/j.2044-0588.2019.040.017]
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Phytoplasmas associated with grapevine yellows disease in Iran: first report of a 'Candidatus Phytoplasma trifolii'-related strain and further finding of a 'Ca. P. solani'-related strain

F. Shahryari*, T. Allahverdipour and Z. Rabiei

*shahryari@znu.ac.ir

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Received: 12 Sep 2019; Published: 02 Dec 2019

Keywords: clover proliferation, Vitis vinifera

Over recent years, grapevines (Vitis vinifera) with symptoms similar to grapevine yellows disease (severe yellows and redness, inward curling of leaves and stunting) have been observed in vineyards from different regions of Iran, especially Zanjan province (Figs 1-2). Therefore, a survey was conducted in 2018 to identify possible phytoplasma agents. A total of 19 samples from symptomatic (16) and asymptomatic (3) plants (cvs. Keshmeshi Qermez and Askari) were collected from vineyards in the Darsajin, Khoramdareh and Zanjan areas in Zanjan province. Total DNA was extracted (Maixner et al., 1995) from the main veins and petioles and nested-PCR with universal primers P1/P7 (Deng & Hiruki, 1991) in the first round followed by R16F2n/R2 (Gundersen & Lee, 1996) in the nested round was conducted. Nested PCR products were obtained from all symptomatic plants but not from asymptomatic plants. Direct sequencing of the partial 16S rRNA gene from six randomly selected plants confirmed that phytoplasmas associated with grapevine yellows were related to two different ribosomal groups.

In BLAST analysis, the sequences obtained (1220 bp) from samples 1, 3 and 5 (GenBank Accession Nos MK392483-MK392485) had 99-100% identity with strains affiliated with ʹCa. P. trifoliiʹ (MK15809, MK379605 and MK372596) and samples 6, 7 and 8 (MK392486-MK392488) had 99-100% identity with ʹCa. P. solaniʹ related strains (KF583785, MN398472 and MN398467). There was no sequence variation between the three isolates of the ʹCa. P. trifoliiʹ-related strain, or the isolates of the ʹCa. P. solaniʹ-related strain. The percentage sequence identity for the isolates in this study to the reference strain for each species was 99.83% for 'Ca. P. trifolii' (AY390261) and 99.59% for 'Ca. P. solani' (AF248959).

Phylogenetic analysis was conducted with the maximum likelihood method using MEGA7 software (Kumar et al., 2016). According to the phylogenetic tree based upon the 16S rDNA sequences, grapevine yellows phytoplasma strains were clustered in ʹCa. P. solani' (16SrXII-A) and ʹCa. P. trifoliiʹ (16SrVI-A) (Fig. 3). 'Ca. P. solani' and 'Ca. P trifolii' -related strains have been reported from many plant species in different areas worldwide. Previous studies reported several 'Ca. Phytoplasma' species associated with grapevine yellows disease from Iran including 'Ca. P. solani', 'Ca. P. aurantifolia', 'Ca. P. fraxini', and 'Ca. P. phoenicium' (Mirchenari et al., 2015; Zamharir et al., 2017). To our knowledge, this is the first report of a 'Ca. P. trifolii'-related strain associated with grapevine yellows disease in Iran.

Figure1+
Figure 1: Yellowing and leaf scorching in phytoplasma-infected grapevine in Zanjan province, Iran.
Figure 1: Yellowing and leaf scorching in phytoplasma-infected grapevine in Zanjan province, Iran.
Figure2+
Figure 2: Purpling and downward rolling of leaves in phytoplasma-infected grapevine in Zanjan province, Iran.
Figure 2: Purpling and downward rolling of leaves in phytoplasma-infected grapevine in Zanjan province, Iran.
Figure3+
Figure 3: Maximum likelihood tree generated using 16S rDNA sequences of phytoplasma strains from this study (green triangles) and selected phytoplasma species' (especially ʹCa. P. solaniʹ associated with grapevine yellows disease and ʹCa. P. trifoliiʹ associated with other hosts in Iran) from GenBank using Acholeplasma laidlawii as an outgroup. Numbers next to nodes are confidence values of bootstrap (1000 replicates). Scale at the bottom represents genetic distance in nucleotide substitutions per site.
Figure 3: Maximum likelihood tree generated using 16S rDNA sequences of phytoplasma strains from this study (green triangles) and selected phytoplasma species' (especially ʹCa. P. solaniʹ associated with grapevine yellows disease and ʹCa. P. trifoliiʹ associated with other hosts in Iran) from GenBank using Acholeplasma laidlawii as an outgroup. Numbers next to nodes are confidence values of bootstrap (1000 replicates). Scale at the bottom represents genetic distance in nucleotide substitutions per site.

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. Kumar S, Stecher G, Tamura K, 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33, 1870-1874. [http://dx.doi.org/10.1093/molbev/msw054]
  4. Maixner M, Ahrens U, Seemüller E, 1995. Detection of the German grapevine yellows (Vergilbungskrankheit) MLO in grapevine, alternative hosts and a vector by a specific PCR procedure. European Journal of Plant Pathology 101, 241-250. [http://dx.doi.org/10.1007/BF01874780]
  5. Mirchenari S M, Massah A, Zirak L, 2015. 'Bois noir': new phytoplasma disease of grapevine in Iran. Journal of Plant Protection Research 55, 88-93. [http://dx.doi.org/10.1515/jppr-2015-0012]
  6. Zamharir M, Paltrinieri S, Hajivand S, Taheri M, Bertaccini A, 2017. Molecular identification of diverse 'Candidatus Phytoplasma' species associated with grapevine decline in Iran. Journal of Phytopathology 165, 407-413. [http://dx.doi.org/10.1111/jph.12574]
To cite this report: Shahryari F, Allahverdipour T, Rabiei Z, 2019. Phytoplasmas associated with grapevine yellows disease in Iran: first report of a 'Candidatus Phytoplasma trifolii'-related strain and further finding of a 'Ca. P. solani'-related strain. New Disease Reports 40, 17. [http://dx.doi.org/10.5197/j.2044-0588.2019.040.017]

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