New Disease Reports
New Disease Reports (2020) 42, 20. [http://dx.doi.org/10.5197/j.2044-0588.2020.042.020]
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First report of a 'Candidatus Phytoplasma australasia'-related strain (16SrII-D subgroup) associated with alfalfa leaf chlorosis disease in Turkey

H. Ayvaci*, M.E. Guldur, E. Simsek and M. Dikilitas

*humeyraayvac@hotmail.com

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Received: 02 Oct 2020; Published: 10 Dec 2020

Keywords: lucerne, Medicago sativa, yellows disease

Alfalfa (Medicago sativa) is an important forage crop in Turkey. Its high nutritional value and adaptation to harsh conditions make it an important crop for animal fodder. Şanlıurfa province is the second most important area for alfalfa seed production in Turkey. However, in recent years, alfalfa plants have exhibited disease symptoms including the production of small leaves, leaf chlorosis and witches' broom (Figs. 1-3).

Samples were collected from diseased plants to determine if a phytoplasma was associated with these symptoms. Total nucleic acid was isolated using the CTAB buffer method. PCR was done on the extracted nucleic acid using the R16F0-R16R1 primer pair followed by R16F2n-R16R2 primers in nested PCR to amplify the 16S rRNA gene (Davis & Lee, 1993; Duduk et al., 2013). Amplicons of c. 1250 bp were obtained from diseased samples, but not from symptomless plants. The resulting amplicons were purified, cloned and sequenced in both directions. The sequences were assembled and compared to those of reference strains using MEGA 7 software (www.megasoftware.net). The three isolates sequenced in this study (HM1YON, HM2YON and HM3YON) had 99.9% identity with phytoplasmas of the 16SrII-D subgroup according to a BLAST analysis. The 16S rDNA sequences were deposited in GenBank under Accession Nos. MW048988, MW048989 and MW048990. A simulated RLFP analysis using 17 restriction enzymes was made with iPhyClassifier software (https://plantpathology.ba.ars.usda.gov) and the results confirmed that the three isolates are related to 16SrII-D subgroup phytoplasmas. A phylogenetic tree was constructed using the MEGA7 programme (neighbour-joining method) and the analysis confirmed that the phytoplasma isolates grouped with phytoplasma strains in group 16SrII and are closely related to 'Candidatus Phytoplasma australasia'.

Damaging witches' broom diseases were reported several times in alfalfa in İran (Hosseini et al., 2016) and in Wisconsin (USA), associated with 'Candidatus Phytoplasma asteris' (Peters et al., 1999). Symptoms such as leaf stunting, yellowing and witches' broom were observed in alfalfa in Iran associated with phytoplasmas in the 16SrII-D group (Hosseini et al., 2013). Leaf yellowing and plant stunting were also associated with 16SrII-D group phytoplasmas infecting alfalfa in Sudan (Tahir et al., 2017).

To the best of our knowledge this is the first report of a phytoplasma disease in alfalfa-growing areas in Turkey. In our study, symptoms in diseased plants were associated with a 'Candidatus Phytoplasma australasia'-related strain. We predict that yield losses in alfalfa could reach significant levels. Disease epidemiology and the economic impact on crop yield, as well as the dissemination of the pathogen are the subject of ongoing research.

Figure1+
Figure 1: Alfalfa plant in the field exhibiting small leaves and chlorosis.
Figure 1: Alfalfa plant in the field exhibiting small leaves and chlorosis.
Figure2+
Figure 2: Alfalfa shoot showing witches' broom (right) compared to a symptomless shoot (left).
Figure 2: Alfalfa shoot showing witches' broom (right) compared to a symptomless shoot (left).
Figure3+
Figure 3: Alfalfa shoot showing chlorosis (right) compared to a symptomless shoot (left).
Figure 3: Alfalfa shoot showing chlorosis (right) compared to a symptomless shoot (left).
Figure4+
Figure 4: Phylogenetic tree of partial 16S rRNA gene sequences from alfalfa leaf chlorosis disease phytoplasma isolates (HM1YON, HM2YON and HM3YON; red font) and reference sequences belonging to 43 phytoplasma species. 16S rRNA groups and subgroups noted in brackets. Tree constructed by neighbour-joining method using MEGA 7 software. Acholeplasma laidlawii was used as outgroup to root the tree. Bar represents 1 substitution in 100 positions.
Figure 4: Phylogenetic tree of partial 16S rRNA gene sequences from alfalfa leaf chlorosis disease phytoplasma isolates (HM1YON, HM2YON and HM3YON; red font) and reference sequences belonging to 43 phytoplasma species. 16S rRNA groups and subgroups noted in brackets. Tree constructed by neighbour-joining method using MEGA 7 software. Acholeplasma laidlawii was used as outgroup to root the tree. Bar represents 1 substitution in 100 positions.

References

  1. Davis RE, Lee I-M, 1993. Cluster-specific polymerase chain reaction amplification of 16S rDNA sequences for detection and identification of mycoplasmalike organisms. Phytopathology, 83, 1008-1011. [http://dx.doi.org/10.1094/Phyto-83-1008]
  2. Duduk B, Paltrinieri S. Lee IM, Bertaccini A, 2013. Nested PCR and RFLP Analysis based on the 16S rRNA Gene. In: Dickinson M, Hodgetts J, eds. Phytoplasma. Methods in Molecular Biology (Methods and Protocols, vol 938. Totowa, USA: Humana Press, 159-171. [http://dx.doi.org/10.1007/978-1-62703-089-2_14]
  3. Hosseini S, Bahar M, Zirak, L, 2013. Characterization of phytoplasmas related to peanut witches'-broom and stolbur groups associated with alfalfa diseases in Iran. Journal of Plant Diseases and Protection 120, 70-76. [http://dx.doi.org/10.1007/BF03356456]
  4. Hosseini SAE, Khodakaramian G, Salehi M, Bertaccini A, 2016. Characterization of 16SrII group phytoplasmas associated with alfalfa (Medicago sativa) witches' broom disease in diverse areas of Iran. Journal of Crop Protection 5, 581-590
  5. Peters RD, Lee ME, Grau CR, Driscoll SJ, Winberg RM, Kurtzweil NC, Lukaesko LA, Lee I-M, 1999. First report of aster yellow phytoplasma in alfalfa. Plant Disease 83, 488. [http://dx.doi.org/10.1094/PDIS.1999.83.5.488C]
  6. Tahir MN, Holland CW, Samac DA, Mollov D, 2017. First report of 16Sr II ('Candidatus Phytoplasma aurantifolia') subgroup-D phytoplasma associated with alfalfa in Sudan. Plant Disease 12, 2144. [http://dx.doi.org/10.1094/PDIS-04-17-0603-PDN]
To cite this report: Ayvaci H, Guldur ME, Simsek E, Dikilitas M, 2020. First report of a 'Candidatus Phytoplasma australasia'-related strain (16SrII-D subgroup) associated with alfalfa leaf chlorosis disease in Turkey. New Disease Reports 42, 20. [http://dx.doi.org/10.5197/j.2044-0588.2020.042.020]

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