Genetic Diversity of Sugarcane Grassy Shoot (SCGS)-Phytoplasmas Causing Grassy Shoot Disease in India

Sugarcane grassy shoot (SCGS) caused by SCGS-phytoplasmas is a serious disease of sugarcane. Since the diseased plants exhibit various phenotypic symptoms under field conditions, diagnosis of the disease becomes difficult. A detailed study was conducted on validating phytoplasma specific primers for the diagnosis of the disease and to identify the extent of variations in rDNA genome of SCGS-phytoplasmas. A total of 37 sugarcane samples with different phenotypic symptoms of SCGS were collected from various states of India. Nested-PCR amplification using two sets of forward (P1/P7) and reverse primers (P4/P7) of 16S rRNA gene, 16S–23S rRNA spacer region (SR) and 23S rRNA gene sequences amplified SCGS-phytoplasmas in all the suspected samples. Nucleotide sequence analysis clearly revealed that 18 isolates of SCGS-phytoplasmas from different locations/varieties showed no variation in the targeted gene sequences. Although there were significant variations in phenotypic expression of SCGS phytoplasmas on sugarcane, we could not establish any genotypic variation among the pathogenic isolates in rDNA region. All our sequences matched exactly with other reported phytoplasmal isolates of SCGS-Pun (DQ459439), SCGS (DQ380341), SCGS-CM1 (AM269740) and SCGS-SV1 (AM269742) from India. Among the other reported SCGS phytoplasmas it shared sequence similarity ranging from 99.7 to 94.3%. Our SCGS phytoplasmas showed 99.6% similarity with other phytoplasmas infecting sugarcane such as sugarcane white leaf (SCWL) and 97.8% with SCYL. The closely related phytoplasmas such as BraWL and SGS phytoplasmas showed 99.6% similarities with our SCGS-phytoplasmas. However, distantly related phytoplasmas FBP and CBY1 and StLL showed sequence similarities between 84.3 and 82.4%. Restriction of the amplicons with a set of restriction enzymes did not show any polymorphism among them. 16S rRNA, 16S–23S rRNA SR and 23S rRNA sequencing and restriction fragment length polymorphism (RFLP) results proved that the phytoplasma isolates infecting sugarcane in India with phenotypic variations from different regions have similar ITS region and further studies are needed to locate other genes in phytoplasma genome which determine variations in phenotypic expression in the SCGS disease.