# Direct detection of Pseudomonas syringae pathovar Phaseolicola using the polymeras chain reaction (PCR)

C. Tourte, Charles Manceau

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C. Tourte, Charles Manceau. Direct detection of Pseudomonas syringae pathovar Phaseolicola using the polymeras chain reaction (PCR). 4th International Working Group on Pseudomonas syringae Pathovars, Università degli Studi di Firenze [Firenze]. ITA., Jun 1991, Firenze, Italy. 436 p. hal-01604145

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DIRECT DETECTION OF PSEUDOMONAS SYRINGAE PATHOVAR PHASEOLICOLA USING THE POLYMERASE CHAIN REACTION (PCR)

TOURTE, C¹,² and C.MANCEAU¹.

1 : INRA, Station de Pathologie végétale, ANGERS, FRANCE.

2 : VILMORIN, La Ménitré, FRANCE.

*Pseudomonas syringae* pathovar *phaseolicola* (*P.s.p.*) causes halo blight of bean. The typical symptom of this disease, on leaves, is a yellow discoloration, due to a specific phytotoxin, around a necrotic spot. This bacterium is mostly transmitted by seeds. So, to prevent the disease, it is useful to have a specific, sensitive and fast method for identifying the pathogen. A new method, Polymerase Chain Reaction, PCR, seems to have all these qualities (Erlich, 1988).

A recombinant cosmid provided by PEET and PANOPOULOS (Peet, 1986, 1987) which contained genes involved in production of and resistance to the specific phytotoxin of *P.s.p*.called phaseolotoxin was used in this study. It was digested with Eco R1 to generate smaller fragments. Those fragments were tested for their specificity to *P.s.p*. A 0,5 Kb fragment was sequenced to make primers of 20 nucleotides long.

These primers were tested on a bacterial collection containing bacteria isolated from beans' environment or genetically close to *P.s.p*. All 27 *P.s.p*. isolates gave a amplified fragment of 0,5 Kb long. Otherwise the 21 other strains tested were not amplified with PCR.
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leaves, is a
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prevent the
method for
Reaction,

OPOULOS
duction of
phaseolotoxin
rate smaller
to *P.s.p.* A
nucleotides

containing
to *P.s.p.*
5 Kb long.
PCR.

To confirm the specificity of those primers, the amplified DNA
fragment was digested with different endonucleases to get specific
patterns.

Then, the PCR technique was tested for its sensitivity. Dilutions
of pure bacterial suspensions from 10⁸ to 10¹ cfu/ml were made in
water. Amplification worked down to 10³ cfu/ml For suspension of
10² cfu/ml, a centrifugation step was necessary prior to amplification to
be sure to have one bacterium per reaction volume. The same
sensitivity level was obtained with dilutions made directly in seed soak
from variety 'Michelet' of bean.

To conclude, PCR could be use to detect *P.s.p*. This technique
is specific to *P.s.p*, sensitive in water and in seed soak variety
'Michelet'. This technique is also fast It requires 5 hours to get a result.

1. Erlich, H.A., Gelfand, D.H., and R.K. Saiki, 1988. Specific DNA amplification. Nature. 331:461-462.

2 Peet, R.C., Lindgren, P.B., Willis, D.K. and N.J. Panopoulos, 1986. Identification and cloning of genes involved in phaseolotoxin production by *Pseudomonas syringae* pv phaseolicola. J. bacteriol. 166:1096-1105.

3. Peet, R.C. and N.J. Panopoulos, 1987. Ornithine carbamoyltransferase genes and phaseolotoxin immunity in *Pseudomonas syringae* pathovar *phaseolicola*. The EMBO Journal. 6:3585-3591.