Isolation of sheath brown rot pathogen Pseudomonas fuscovaginae from seeds

Sheath brown rot is a bacterial disease affecting wheat, rice, corn and sorghum in almost all regions where they are grown. The causative agent is a bacterium of the species Pseudomonas fuscovaginae (ex Tanii, Miyajima and Akita, 1976) Miyajima, Tanii and Akita (1983), of quarantine status for some countries importing Russian wheat. To enhance the diagnosis of the bacterium Pseudomonas fuscovaginae in phytosanitary laboratories, the development of a method for isolating a culture from wheat seeds is required. In the study, wheat plant extracts obtained as a result of sample preparation were inoculated with P. fuscovaginae suspensions in various concentrations and sown on R2A nutrient medium. P. fuscovaginae colonies grown on plates with R2A medium were tested by real-time polymerase chain reaction (RT-PCR) to determine the dependence of the threshold cycle on the bacteria concentration in the sample. It was determined that P. fuscovaginae isolation is possible if the sample contains bacteria in concentration 1 * 10⁴ CFU/ml and higher, which corresponds to a threshold fluorescence cycle (Ct) of 32.0 on the DTprime 5M6 cycler (DNA-Technology, Russia) and (Ct) 31.5 on the CFX cycler (Bio-Rad, USA) when using the “Pseudomonas fuscovaginae-RT” kit (Syntol, Russia). Sowing should be done in a depleting manner on R2A medium using 20 µl of concentrated seed extract and searching for colonies P. fuscovaginae 3–7 days after sowing. This technique is recommended for conducting research in laboratories in the field of phytosanitary and plant protection.

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