Location: National Germplasm Resources LaboratoryTitle: First report of 16SrII subgroup D phytoplasma in alfalfa in Sudan Author
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/24/2017
Publication Date: 8/1/2017
Citation: Tahir, M.N., Holland, C., Samac, D.A., Mollov, D.S. 2017. First report of 16SrII subgroup D phytoplasma in alfalfa in Sudan. Plant Disease. https://doi:10.1094/PDIS-04-17-0603-PDN. Interpretive Summary: Alfalfa plants from production fields in Sudan, a country in North Africa, were observed with yellowing and stunted growth. Fourteen samples were tested by PCR assay for phytoplasma; eleven were positive. Phytoplasmas are obligate bacterial pathogens of phloem tissue in plants, and are typically transmitted by leafhopper insects. DNA sequence analysis indicated the alfalfa phytoplasma from Sudan belongs to group 16Sr II. This is the first report of phytoplasma infecting alfalfa in Sudan. The information is useful for developing strategies to manage the spread of phytoplasma diseases in Sudan.
Technical Abstract: In 2016, alfalfa (Medicago sativa) plants from four commercial fields in Sudan, each about 60 ha, were observed with leaf yellowing symptoms and stunted growth. Total nucleic acid was extracted from leaves of 14 symptomatic samples by a CTAB protocol and used as the template in PCR assays with the universal phytoplasma primers P1/P7 (Smart et al. 1996). Eleven out of 14 samples produced an amplicon approximately 1.8 kb long. Subsequently, primer pairs R16F2n/R16R2 were used in a nested PCR using the P1/P7 PCR amplicon as a template (Gundersen and Lee 1996). Amplicons were obtained from the same 11 samples that tested positive with the first round of PCR, and no amplification was obtained by the nested PCR from the three samples that were negative with the P1/P7 primers. Four of the 11 products amplified using the P1/P7 primers were gel purified, cloned and sequenced. The percent identity among these 14 clones was 99.4 to 100%. BLASTn analysis comparing the sequences obtained from the Sudanese alfalfa samples with the NCBI GenBank database revealed 99% nucleotide identity to phytoplasmas in the group 16SrII. The complete sequences of the alfalfa phytoplasma isolates (Genbank accession numbers KY449415 - KY449418) were subjected to a phylogenetic analysis. A dendogram was constructed by the neighbor-joining algorithm based on the 16S rRNA sequences. The results show that the Sudanese alfalfa isolates cluster with phytoplasmas in the 16SrII group. Virtual restriction fragment length polymorphism analyses of the 16S rRNA gene was identical to the reference pattern of 16Sr group II, subgroup D (GenBank accession Y10097). To our knowledge this is the first report of group 16SrII-D phytoplasma infecting alfalfa in Sudan.