IDENTIFICATION AND RELATEDNESS OF CORONATINE-PRODUCING PSEUDOMONAS-SYRINGAE PATHOVARS BY PCR ANALYSIS AND SEQUENCE DETERMINATION OF THE AMPLIFICATION PRODUCTS
S. Bereswill et al., IDENTIFICATION AND RELATEDNESS OF CORONATINE-PRODUCING PSEUDOMONAS-SYRINGAE PATHOVARS BY PCR ANALYSIS AND SEQUENCE DETERMINATION OF THE AMPLIFICATION PRODUCTS, Applied and environmental microbiology, 60(8), 1994, pp. 2924-2930
Production of the chlorosis-inaucing phytotoxin coronatine in the Pseu
domonas syringae pathovars atropurpurea, glycinea, maculicola, morspru
norum, and tomato has been previously reported. DNA hybridization stud
ies previously indicated that the coronatine biosynthetic gene cluster
is highly conserved among P. syringae strains which produce the toxin
. In the present study, two 17-bp oligonucleotide primers derived from
the coronatine biosynthetic gene cluster of P. syringae pv. glycinea
PG4180 were investigated for their ability to detect coronatine produc
ing P. syringae strains by PCR analysis. The primer set amplified diag
nostic 0.65-kb PCR products from genomic DNAs of five different corona
tine-producing pathovars of P. syringae. The 0.65 kb products were not
detected when PCR experiments utilized nucleic acids of nonproducers
of coronatine or those of bacteria not previously investigated for cor
onatine production. When the 0.65-kb PCR products were digested with C
laI, PstI, and SmaI, fragments of identical size were obtained for the
five different pathovars of P. syringae. A restriction fragment lengt
h polymorphism was detected in the amplified region of P. syringae pv.
atropurpurea, since this pathovar lacked a conserved PvuI site which
was detected in the PCR products of the other four pathovars. The 0.65
-kb PCR products from six strains comprising five different pathovars
of P. syringae were cloned and sequenced. The PCR products from two di
fferent P. syringae pv. glycinea strains contained identical DNA seque
nces, and these showed relatedness to the sequence obtained for the pa
thovar morsprunorum. The PCR products obtained from the pathovars macu
licola and tomato were the most similar to each other, which supports
the hypothesis that these two pathovars are closely related. In conclu
sion, the region amplified by PCR was highly effective for the detecti
on of coronatine-producing P. syringae strains, and the sequence analy
sis of PCR products proved valuable in showing relatedness between str
ains and pathovars.