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Molecular detection and isolation of Spiroplasma citri causing yellows in sesame and its insect transmission by Circulifer haematoceps in a non-citrus-growing region of Iran

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Abstract

Sesame plants with yellows symptoms and shortened internodes were observed in the surveyed sesame fields of a non-citrus-growing region in southeastern Iran. Leaf samples were collected from both symptomatic and asymptomatic plants. Concurrently, insects (Circulifer haematoceps) were also captured from the plants using a sweep net. Then, DNA was extracted from the symptomatic and non-symptomatic samples, likewise from the collected C. haematoceps insects. In the PCR assays, a DNA fragment with the expected size of 336 bp was amplified from the symptomatic plant’s DNA using Spiroplasma citri–specific primers. Besides, in the PCR assays, approximately 72.4% of the tested leafhopper samples were positive for S. citri, and in the experimental transmission assays, 26.6% of the periwinkle plants that were fed on by the field-collected leafhoppers showed symptoms. S. citri was also cultured from the symptomatic plants as well as the insects. Finally, the spiralin gene of an S. citri strain (KSC) isolated from one of the periwinkle plants was cloned and partially sequenced. BLAST and phylogenetic analysis of the obtained sequence revealed 100% and 87% homology of the KSC strain with the Iranian S. citri Marvdasht strain and the R8A2 reference strain, respectively. The present findings contribute to the knowledge on the sesame yellows disease caused by S. citri, as well as the high-frequency infectivity of the leafhopper C. haematoceps to S. citri, in the surveyed region. Furthermore, the finding of unique spiralins within Iranian populations of S. citri, including the KSC strain, may indicate that these strains are endemic in Iran. Due to the principal role of the leafhopper vector C. haematoceps in spreading the pathogen, controlling the vector insects is the key strategy for the management of the disease.

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Funding

The office of vice-chancellor for research, Shahid Bahonar University of Kerman, Iran, funded this work.

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Authors and Affiliations

  1. Department of Plant Protection, College of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran

    Mojtaba Rezaee Ahmadabady, Akbar Hosseinipour & Hossain Massumi

  2. Department of Agriculture, Payame Noor University, Bafgh Branch, Bafgh, Iran

    Mojtaba Rezaee Ahmadabady

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  1. Mojtaba Rezaee Ahmadabady
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  2. Akbar Hosseinipour
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Contributions

Study conception and design: Akbar Hosseinipour. Data curation: Mojtaba Rezaee Ahmadabady. Analysis and interpretation of data: Mojtaba Rezaee Ahmadabady and Akbar Hosseinipour. Writing—original draft: Akbar Hosseinipour. Review, editing, and validation: Hossain Massumi.

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Correspondence to Akbar Hosseinipour.

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40858_2021_488_MOESM1_ESM.jpg

Supplementary file1 Fig. S1 Schematic diagram representing positions of the D/D' and Sc1/Sc2 primer pairs on a part of the Spiroplasma citri genome R8A2 strain (GenBank, CP013197), which were used for the detection of S. citri in plants and insects, and spiralin gene amplification, respectively. The coding region of the spiralin gene was shown with the cyan arrow (JPG 1407 KB)

40858_2021_488_MOESM2_ESM.jpg

Supplementary file2 Fig. S2 Agarose gel electrophoresis of a PCR-amplified 336 bp DNA fragment (red arrow) from Spiroplasma citri-infected sesame plants in the Kerman province of Iran. PCR DNA templates were extracted from asymptomatic (lane 1) and symptomatic sesame plants from three fields (lanes 2 to 4). M, DNA size marker (1Kb DNA ladder, Fermentas) (JPG 426 KB)

40858_2021_488_MOESM3_ESM.jpg

Supplementary file3 Fig. S3 PCR amplification of Spiroplasma citri-specific DNA fragment (336 bp, red arrows) from five batches of insects (Cirulifer haematoceps) collected from sesame fields (A-C) in the Kerman province of Iran. The PCR DNA templates were extracted from a pair of insects (panel a) or a single insect from each batch (panel b). In the latter, four out of the five insect batches of the field C were tested. M, DNA size marker (1Kb DNA Ladder, Fermentas) (JPG 746 KB)

40858_2021_488_MOESM4_ESM.jpg

Supplementary file4 Fig. S4 The identity matrix compares the 3′ part of the spiralin gene of the Spiroplasma citri KSC strain (isolated from an experimentally infected periwinkle plant by the sesame field-collected Circulifer haematoceps) to that of other S. citri strains, S. phoeniceum, S. kunkelii, and S. melliferum. The reference strain of S. citri, the KSC strain, and other Iranian S. citri strains are shown on the tree as red, green, and purple, respectively. See Supplemental Table S1 for more information on strains (JPG 2024 KB)

40858_2021_488_MOESM5_ESM.jpg

Supplementary file5 Fig. S5 Multiple sequence alignments of the partial spiralin sequence of Spiroplasma citri strains. The alignment spiralin sequence of the isolated S. citri KSC strain in the present work is shown in the green row. Different amino acids of the spiralin of the KSC strain, compared with the reference S. citri strain (red sequence arrow), are indicated with black letters and with a purple box for the insertion of the alanine and serine amino acids. The other Iranian S. citri strains retrieved from GenBank are shown with green filled circles. Amino acid identity in the alignments is denoted by black dots. See Supplemental Table S1 for more information on strains (JPG 1742 KB)

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Ahmadabady, M.R., Hosseinipour, A. & Massumi, H. Molecular detection and isolation of Spiroplasma citri causing yellows in sesame and its insect transmission by Circulifer haematoceps in a non-citrus-growing region of Iran. Trop. plant pathol. 47, 402–410 (2022). https://doi.org/10.1007/s40858-021-00488-4

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