A field investigation was conducted during the winter seasons of 2021–22 and 2022–23 at the Jawaharlal Nehru Krishi Vishwa Vidyalaya (JNKVV), Jabalpur, Central India, to identify and characterize the phytoplasma strains associated with niger (var. JNC6) and sesame (var. RT315) exhibiting severe phyllody symptoms. Disease incidence ranged from 0-3%, and 3 to 15% were recorded in niger and sesame crops during the study period. Nested PCR assays using universal phytoplasma primer pairs P1/P7 followed by R16F2n/R16R2 consistently amplified ~1.25 kb fragments of the 16S rRNA gene from both the symptomatic plants, confirming phytoplasma association. Additional amplification of phytoplasma-specific
sec
A (~600 bp) and
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Y (~1.7 kb) gene fragments further validated the phytoplasma infection in both crops. Two predominant leafhopper species,
Amrasca biguttula
and
Orosius albicinctus
, colonizing and feeding niger and sesame fields were identified. Notably, only
Amrasca biguttula
was tested positive for phytoplasma using the similar PCR assays mentioned above, implicating it as a putative vector. Sequence identity, phylogenetic analysis, and
in silico
RFLP profiling of 16S rRNA,
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A, and
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Y gene sequences from both the symptomatic plants and leafhoppers confirmed the presence of a ‘
Ca.
P. asiaticum’ related strain (16SrII-C subgroup). This study represents the first global report of 16SrII-C subgroup phytoplasma infection in niger, highlighting a new host record. The detection of an identical phytoplasma strain in niger, sesame, and
Amrasca biguttula
suggests a possible epidemiological link and underscores the role of these natural hosts and the leafhopper in the dissemination of ‘
Ca.
P. asiaticum’-related phytoplasma strains in Central India.