Phytopathogenic mollicutes

Among the biotic stresses, the grassy shoot disease is a major production constraint to farmers and sugar industries in most of the Asian countries. The disease is associated with the presence of ‘ ‘Candidatus Phytoplasma sacchari’ detected by sequencing of amplified samples and by symptom presence under field conditions. In recent years, many diagnostic tools like LAMP, RPA, CRISPR-Cas and LFIA have been developed for plant pathogen detection. In LAMP assay, all the positive samples collected from different places had shown multiple bands through electrophoresis. Under the colorimetric assay, all positive samples had shown fluorescent green color, and the negative sample was non fluorescent. Similarly, in the RPA assay all the samples had shown the expected amplification of 200 bp under isothermal conditions at 37–42°C. Quantitative PCR assay was found sensitive to detect the pathogen even at 10^-1ng/μl in comparison with LAMP and RPA. Recently, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nuclease-based plant pathogen detection is gaining momentum in molecular detection. Development of onsite field diagnostics like LFIA, and RPA-dipstick assays are important for the selection of healthy planting materials and for the healthy seed nursery program for the supply of disease-free planting materials to the farmers. However, major challenges in the onsite field diagnostics would be the sensitivity which may vary in comparison with the detection technique used, because of crude sap usage in place of purified nucleic acids.