Detection and recognition of phytoplasmal disease(s) on rice and soybean in Mazandaran province using molecular methodύ

Phytoplasmas are a group of cell wall-less bacteria belong to Acholeplasmatales. They are naturally transmitted by sap-sucking insects among their hosts. The first step in studying of phytoplasmas is their detecting and recognition. Because of the importance of rice and soybean as strategic products in Mazandaran province, and the outbreak of some suspicious symptoms of phytoplasmal diseases on these crops, the current study was designed in order to detection and diagnosis of phytoplasmal disease(s) on rice and soybean in this region. To execute this project, rice and soybean fields from Sari, Ghaemshahr, Babol and Amol were sampled during two years, 2010 and 2011. The sampling was performed from plants presenting yellowing, stunting, proliferatiom, decline, mal-formed stems and pods, thick leaves containing mosaic and dark veins symptoms. The plants were sampled monthly, two times for rice and three times for soybean. During each year, among 5-10 fields were sampled from every city for each crop (rice and soybean separately). Total nucleic acid was extracted using two different methods: CTAB and Dellaporta. The quality of DNA was checked on agarose gel (single band without smear). Direct PCRs were performed using two universal primer pairs (R16F1/R16mR and P1/P7), but any clear band in expected size was observed in samples of rice or soybean (although one or two nonspecific fragments for two samples was observed). To ensure the presence or absence of phytoplasma in the samples, nested PCR reaction was performed using primers R16F2n/R16R2. This reaction showed only a clear band in positive control sample and any band in other samples (rice or soybean). The results suggest that the rice and soybean plant samples that were prepared during 2010 and 2011 are not likely to be contaminated with phytoplasma. It must be mentioned that about one or two DNA rice samples in nested PCR, a weak band in the expected size was sometimes observed, that with repeating the test, this suspicious band was disappeared. Therefore, this band is probably related to the carry-over contamination from the positive control sample or parts of the DNA of epiphytic bacteria or mycoplasmas of leaf surfaces. Finally, it seems the existing suspicious symptoms on the plants are related to other pathogens such as viruses or nonpathogenic factors such as climate, chemicals, tillage practices, and so.