Protocol for detecting Curtobacterium flaccumfaciens pv. flaccumfaciens in the leaves of infected bean plants (Phaseolus vulgaris L.)

ABSTRACT. Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), which is a gram-positive bacterium that causes wilting in several cultivated plants, is one of the main disease-causing bacteria in beans (Phaseolus vulgaris L.). Different techniques are employed to detect pathogens in bean plants; however, only a few slow and inefficient methodologies can be used to detect Cff. Hence, there is a need for rapid and efficient techniques to detect Cff for disease management. The objective of our study was to develop a technique to effectively detect Cff in the leaves of infected bean plants by optimizing a previously developed method. We modified the extraction method by reducing the amount of water used, replacing seeds with leaf tissues (obtained from the veins and petioles) and using CffFOR2 and CffREV4 primers specific for Cff. Our optimized method exploits a concentration gradient generated in the plant tissue through osmosis to disrupt the plant cell wall and efficiently isolate bacterial cells. DNA extracted from the isolated bacterial cells was amplified by polymerase chain reaction. This method allowed efficient detection of Cff in leaf tissues.