US 6,146,834 A · PCR primers for detection of plant pathogenic species and subspecies of acidovorax

Abstract

We sequenced a 625 and 617 bp fragment of the inner spacer region of 16S-23S rDNA of a strain of Acidovorax avenae representing pathogens from several hosts, including foxtail, oats, corn, rice, millet, sugarcane, orchid, and watermelon and a strain of A. avenae subsp. citrulli pathogenic only to watermelon, respectively, for the purpose of designing PCR primers for their identification. These plant pathogens were previously considered as non-fluorescent pseudomonads and have been recently reclassified as Acidovorax avenae subsp. avenae, A. avenae subsp. cattleyae, and A. avenae subsp. citrulli. Several sets of primers were designed. Primers identified by SEQ ID NO:1 and SEQ ID NO:2 of subsp. avenae reacted with all strains of A. avenae subsp. avenae (previously named P. avenae or P. alboprecipitans) originating from foxtail, oats, corn, rice, sugarcane, and millet, A. avenae subsp. cattleyae from orchid, and A. avenae subsp. citrulli (previously named P. pseudoalcaligenes subsp. citrulli) from watermelon. Primers identified by SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 of subsp. citrulli reacted with all strains of A. avenae subsp. citrulli, but not with any other strain of subsp. avenae. None of fifty-three other bacteria tested reacted with either set of primers. The citrulli- specific primers should prove especially useful for specific, sensitive, and rapid detection of this serious seedborne pathogen of watermelon seeds.

Claims30

Dependent claims

An oligonucleotide primer consisting of the sequence 5'-GTCGGTGCTAAC GACATGG-3' (SEQ ID NO:1).

A method of detecting the presence of Acidovorax by polymerase chain reaction, said method comprising:

a) providing the DNA of said Acidovorax or a test sample of cells or microorganisms suspected of containing the DNA of said Acidovorax;
b) amplifying a target sequence of DNA of said Acidovorax using at least one primer selected from the group consisting of the oligonucleotide 5'-GTCGGTGCT AACGACATGG-3' (SEQ ID NO:1), the oligonucleotide 5'-AGACATCTCCGCT TTCTTTCAA-3' (SEQ ID NO:2), the oligonucleotide 5'-CGGTGCTACCC-3' (SEQ ID NO:3), the oligonucleotide 5'-TCGTCATTACTGAATTTCAACA-3' (SEQ ID NO:4);
the oligonucleotide 5'-CCTCCACCAACCAATACGCT-3' (SEQ ID NO:5), and the oligonucleotide 5'-CATGCTCTTAGTCACTTGACCCTA-3' (SEQ ID NO:6);
and
c) detecting the presence of the amplification products of the target sequence of DNA as an indication of the presence of Acidovorax.

2 more elements

A method of detecting the presence of Acidovorax by polymerase chain reaction, said method comprising:

a) providing the DNA of said Acidovorax or a test sample of cells or microorganisms suspected of containing the DNA of said Acidovorax;
b) amplifying a target sequence of DNA of said Acidovorax using a primer set selected from the group consisting of (I) a primer set comprising oligonucleotides consisting of the sequence 5'-GTCGGTGCTAACGACATGG-3' (SEQ ID NO:1) and the sequence 5'-AGACATCTCCGCTTTCTTTCAA-3' (SEQ ID NO:2), (ii) a primer set comprising oligonucleotides consisting of the sequence 5'-GGAAGA ATTCGGTGCTACCC-3' (SEQ ID NO:
3) and the sequence 5'-TCGTCATTACTG AATTTCAACA-3' (SEQ ID NO:4);
(iii) a primer set comprising oligonucleotides consisting of the sequence 5'-CCTCCACCAACCAATACGCT-3' (SEQ ID NO:5) and the sequence 5'-TCGTCATTACTGAATTTCAACA-3' (SEQ ID NO:4);
and (iv) a primer set comprising oligonucleotides consisting of the sequence 5'-CCT CCACCAACCAATACGCT-3' (SEQ ID NO:5) and the sequence 5'-CATGCTCTT AGTCACTTGACCCTA-3' (SEQ ID NO:6);
and
c) detecting the presence of the amplification products of the target sequence of DNA as an indication of the presence of Acidovorax.

4 more elements

The method of screening for particular subspecies of A. avenae, said method comprising:

a) providing the DNA of said Acidovorax or a test sample of cells or microorganisms suspected of containing the DNA of said Acidovorax;
b) amplifying a target sequence of DNA of said Acidovorax using a primer set comprising oligonucleotides consisting of the sequence 5'-GTCGGTGCTAACGA CATGG-3' (SEQ ID NO:1) and the sequence 5'-AGACATCTCCGCTTTCTTT CAA-3' (SEQ ID NO:2);
c) detecting the presence of the amplification products of the target sequence of DNA as an indication of the presence of A. avenae subsp. avenae, A. avenae subsp. catteyae, or A. avenae subsp. citrulli;
d) providing the test sample for which it has been determined that any one or all of the subspecies, A. avenae subsp. avenae, A. avenae subsp. cattleyae, or A. avenae subsp. citrulli, are present in the test sample;
e) amplifying a target sequence of DNA using a primer set selected from the group consisting of (I) the primer set comprising oligonucleotides consisting of the sequence 5'-GGAAGAATTCGGTGCTACCC-3' (SEQ ID NO:
3) and the sequence 5'-TCGTCATTACTGMTTTCAACA-3' (SEQ ID NO:4);
(ii) the primer set comprising oligonucleotides consisting of the sequence 5'-CCTCCACC AACCAATACGCT-3' (SEQ ID NO:5) and the sequence 5'-TCGTCATTACTGAAT TTCAACA-3' (SEQ ID NO:4);
and (iii) the primer set comprising oligonucleotides consisting of the sequence 5'-CCTCCACCAACCAATACGCT-3' (SEQ ID NO:5) and the sequence 5'-CATGCTCTTAGTCACTTGACCCTA-3' (SEQ ID NO:6);
and
f) determining the presence or absence of amplification products of the target DNA and, as an indication thereby, in the presence of the DNA amplification products, specifically distinguishing A. avenae subsp. citrulli from A. avenae subsp. avenae and cattleyae, or in the absence of the amplification products, determining that either or both A. avenae subsp. avenae or cattleyae are present and that A. avenae subsp. citrulli is not present.

7 more elements

Dependencies are inferred from the claim text.

PCR primers for detection of plant pathogenic species and subspecies of acidovorax

Life of this patent

PCR primers for detection of plant pathogenic species and subspecies of acidovorax

Life of this patent

Abstract

Claims30