LOPAT scheme for fluorescent Pseudomonads

Ingredients

	P. syringae	P. viridiflava	P. cichorii	P. agarici	P. marginalis	P. tolaasi
Fluorescence	+	+	+	+	+	+
Oxidase	-	-	+	+	+	+
Arginine dihydrolase	-	-	-	-	+	+
Levan	+	-	-	-	+	-
Potato rot	-	+	-	-	+	-
Tobacco hypersensitivity	+	+	+	-	-	-

Instructions

• Fluorescence: Fluorescence is observed on King's medium B (KB) under long wave, 366nm ultraviolet light. It is critical to use the correct peptone in the medium. Fluorescence will not occur if when iron is in the medium and most peptones have iron.
• Oxidase: The oxidase reagent tests for the presence of cytochrome c. The reagent is highly toxic and must be handled carefully. N,N-dimethyl-p-phenylenediamine or tetramethylparaphenylenediamine dihydrochloride can be used. The later reagent is also available in ampules, which are safer and more convenient to use. A false negative reaction may occur when there is more than 0.5% glucose in the medium. Always use a platinum loop because a false positive may occur with a nichrome loop.
• Arginine dihydrolase: Arginine dihydrolase breaks down arginine and releases ammonia causing the pH to rise and change the color of the medium to pink.
• Levan: Bacteria that have the enzyme levan sucrase, can break down sucrose and form levan (poly-fructose), a capsular material. Sucrose peptone agar or a nutrient medium with 5% sucrose is a suitable substrate. Streak to get single colonies, and observe 2 to 4 days later. The viscosity of levan results in distinctive domed colonies. Pseudomonas syringae pathovars delphini, passiflorae, savastanoi and tagetis are levan-negative.
• Potato rot: Potatoes must be sliced aseptically and the test carried out in sterile petri dishes with sterile water. It is more convenient to carry out this assay on crystal violet pectate (CVP) medium; many isolates can be tested on one plate.
• Tobacco hypersensitivity: Tobacco plants should be healthy and preferably not flowering. Use 108-9 CFU. Since the results can be variable, a positive control should be used. The test works best for plant pathogenic Pseudomonads but Erwinia amylovora is also positive. Xanthomonas, Clavibacter and Agrobacterium are highly variable.

Notes

• The vast majority of bacterial diseases are caused by gram-negative rods. The Coryneforms, which are slow growing brightly pigmented and fairly host specific, are gram positive. Streptomyces is also gram positive.

Contributed by

From the Culture Media for Plant Pathogenic Fungi and Bacteria, University of Massachusetts: Contributed by Robert L. Wick