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.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

Acidovorax avenae, formerly classified as Pseudomonas avenae, is a seedborne pathogen of several hosts including oat, corn, millet, wheat, sugarcane, rice, and melon (watermelon and cantaloupe). This invention relates to novel PCR primers which can be used to detect plant pathogenic Acidovorax avenae subspecies and to distinguish among the pathogenic subspecies, particularly to distinguish A. avenae subsp. citrulli from other subspecies.

2. Description of the Relevant Art

The bacterium A. avenae subsp. avenae causes bacterial stripe of rice, leaf blight of oats, red stripe disease of sugarcane and millet, and brown stripe of Setaria italica. The organism was originally described as Pseudomonas avenae by Manns in 1909 (Ohio Agric. Exp. Stn. Res. Bull. 210: 91-167) as the causal agent of leaf blight on oats in Ohio. Another organism, P. alboprecipitans, described by Rosen in 1922 on foxtail (Ann. Mo. Bot. Gard. 9: 333-402) and on corn in 1926 (Phytopathology 16: 241-267) was later shown to be synonymous with P. avenae (Schaad et al. 1975. Int. J. Syst. Bacteriol. 25: 133-137). On rice, the organism has been previously referred to as P. setariae or P. panici (Goto. 1964. Bull. of Faculty of Agriculture, Shizuoka University 14: 3-10) and on sugarcane as P. rubrilineans (Martin et al. 1989.), the causal agent of red stripe diseases of sugarcane (C. Ricaud et al. (Eds), Elsevier, The Netherlands, pp. 80-95). Acidovorax avenae subsp. citrulli causes bacterial blight and fruit blotch of watermelon and melon. The organism was originally described by Schaad et al. (1978. Int. J. Syst. Bacteriol. 28:117-125) in plant introduction breeding lines of watermelon in Georgia as P. pseudoalcaligenes subsp. citrulli.

Phylogenetically, A. avenae is included in the new "acidovorans" DNA-rRNA homology group (Willems et al. 1992. Int. J. Syst. Bacteriol. 42: 107-119). This group contains many pathogens previously classified in the non-fluorescent pseudomonad group, including P. avenae Manns 1909 (Ohio Agric. Exp. Stn. Res. Bull. 210: 91-167), P. cattleyae Savulescu 1947 (Pavarino. 1911. Atti Accad. Lincei 20:233-237), P. pseudoalcaligenes subsp. citrulli (Schaad et al. 1978, supra), and P. rubrilineans (Lee et al. 1925. Red Stripe Disease. Pamphlet of Hawaiian Sugar Planters Association). Recently, all of these bacteria have been reclassified as subspecies of A. avenae, i.e., A. avenae subsp. avenae, A. avenae subsp. cattleyae, and A. avenae subsp. citrulli, on the basis of the results of DNA--DNA hybridization, DNA-rRNA hybridizations, polyacrylamide gel electrophoresis of whole-cell proteins, and a numerical analysis of carbon assimilation tests (Willems et al., supra).

Presently, the only reliable method to identify the A. avenae pathogens, is to isolate them on agar media and confirm their presumptive identification by time consuming and expensive pathogenicity tests. For example, in rice, A. avenae subsp. avenae can be recovered from both diseased, or asymptomatic, apparently healthy seeds. However, identification is difficult, because the organism is frequently overgrown by other bacterial pathogens of rice, such as Pantoea (Erwinia) herbicola, Burkholdera (Pseudomonas) glumae, B. fuscovaginae, or P. syringae pv. syringae. These pathogens are recognized as producing distinct disease symptoms, but field diagnosis is very difficult. In such cases, detection and identification is based on pathogen isolation, through the use of semiselective media, and pathogenicity tests which discriminate between the various pathogens. However, the presence of coexisting epiphytes such as P. putida and P. fluorescens in/on rice seed makes isolation difficult. Furthermore, rice seedlings are difficult to grow and symptoms on seedlings are often non-discriminating. Several methods of identification of A. avenae subsp. avenae have been proposed including growth and isolation on semiselective agar media and serology (Shakya. 1987. Korean J. Plant Path. 3:300; Zeigler et al. 1989. Intl. Rice Res. Newsletter 14: 27-28). None have achieved much success. There is currently no reliable, routine method available for detecting A. avenae in rice seeds. More sensitive and specific methods are needed to confirm the identification, especially in seed health evaluations.

Similar problems exist for melons. Although originally described as a seedling disease, watermelon fruit blotch has emerged as a serious disease of mature fruit. Complete losses of production fields often occur due to fruit rot. Like most seedborne bacteria, control is based primarily on seed health testing. Because of the seriousness of watermelon fruit blotch, nearly all watermelon seed lots must be assayed for A. avenae subspec. citrulli. This involves soaking seeds and attempting to isolate the organism on agar media or inoculating plants with the seed soakate. Neither method is very efficient and both are relatively expensive. Little or no resistance to the pathogen exists.

Bacterial stripe causes great losses in rice seedling beds throughout Asia. Thus far, the disease has not been observed in the U.S. However, the potential risk of the dissemination of the bacterial stripe pathogen in international exchange of germplasm of rice, and also of corn and watermelon, is a serious concern. There is presently no way to separate infected seeds and contaminated seeds. Therefore, there is a need to develop reagents and methods for detecting A. avenae subspecies specifically, rapidly, and directly from biological samples. Such methods and reagents are valuable tools for monitoring natural disease spread, tracking the seedborne bacteria in field studies, and detecting the presence of the bacterium in seed lots entering A. avenae -free areas.

Polymerase chain reaction (PCR) has been shown to be highly sensitive and the method is commonly used to detect and identify bacteria. A PCR method has been described for detecting the pathogen A. avenae subsp. citrulli, in seeds; however, the primers are not unique and the method has not gained industry acceptance (Minsavage et al. 1995. Ann. Mtg. Amer. Phytopath. Abstract 379). Thus, there exists a need for specific primers and methods capable of specifically identifying and differentiating pathogenic A. avenae subspecies.

SUMMARY OF THE INVENTION

We have discovered oligonucleotide sequences which are capable of amplifying DNA fragments specific for identifying the two closely related pathogens when used in a simple and rapid PCR assay. One set of oligonucleotide sequences are specific for identifying all but one subspecies of A. avenae; another set is useful for selectively and specifically identifying the highly destructive watermelon fruit blotch pathogen, A. avenae subsp. citrulli. These two subspecies avenae and citrulli are phenotypically very similar and are very difficult to differentiate. In addition, two other sets of primers and probes specific for identifying A. avenae subsp. citrulli have been designed for the TaqMan (Perkin Elmer) 7700 detection system. One of these primers is the same as described for detecting A. avenae subsp. citrulli by classical PCR, whereas the other primers and probe are different.

In accordance with this discovery, it is an object of the invention to provide the novel oligonucleotides for use as primers for PCR assays for the specific detection and identification of plant pathogenic subspecies of A. avenae.

It is also an object of the invention to provide primers for the specific detection and identification of the watermelon fruit blotch pathogen, A. avenae subsp. citrulli, thereby differentiating A. avenae subsp. citrulli from other A. avenae subspecies.

It is another object of the invention to provide PCR assay methods utilizing the novel primers.

It is a further object of the invention to provide a screening method for distinguishing subspecies of A. avenae by utilizing two sets of primers.

It is an additional object of the invention to provide a method for assaying seeds for presence of A. avenae subsp. citrulli and for monitoring seed treatment protocols utilizing the novel primers.

It is an added object of the invention to provide a kit for use in the detection of A. avenae subspec. citrulli.

Other objects and advantages of the invention will become readily apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the nucleotide sequence of the selected primers SEQ ID NO:1 and SEQ ID NO:2 of Acidovorax avenae subsp. avenae COA1 and the selected primers SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and the probe (SEQ ID NO:7) of A. avenae subsp. citrulli ATTC 29625. FIG. 1B shows the nucleotide sequence of the intergenic spacer region (ISR); the shaded area represents the ISR sequence of Acidovorax avenae subsp. citrulli (SEQ ID NO:8; Aac, upper) and Acidovorax avenae subsp. avenae (SEQ ID NO:9; Aaa, lower). The unshaded area represents tRNA.sup.Ala gene sequences. Sequences that are disclosed in the Sequence Listing as having SEQ ID NOs: 2, 4, and 6 are complementary to the reverse orientation of the sequences which are underlined and identified as "SEQ ID NO:2", "SEQ ID NO:4", and "SEQ ID NO:6" in FIG. 1B. SEQ ID NOs: 1, 3, 5, and 7 are also underlined and appear in FIG. 1B as they are disclosed in the Sequence Listing.

FIGS. 2A-2B are images of ethidium bromide stained gels showing a 550 bp PCR product formed by primers SEQ ID NO:1 and SEQ ID NO:2 (FIG. 2A) and a 450 bp PCR product formed by primers SEQ ID NO:3 and SEQ ID NO:4 (FIG. 2B. FIG. 2A: Lane 1, 100 bp ladder; Lane 2, A. avenae subsp. avenae (Aaa) ATCC 19822; Lane 3, Aaa 39463d; Lane 4, Aaa Nepal; Lane 5, Aaa 39462c; Lane 6, Aaa 39461g; Lane 7, A. avenae subsp. citrulli, (Aac) 40584 isolate B3; Lane 8, Aac 40565 A; Lane 9, Aac 40560 isolate 8; Lane 10, Aac 40565 b; Lane 11, Aac 40556 isolate 2; Lane 12, Aac 40582 isolate A1; Lane 13, Aac 40560 isolate 6; Lane 14, Aaa 39459 b; Lane 15, Aaa 39128 c; Lane 16, Aac 40582 isolate e/2; Lane 17, Aac 40582 isolate a1; Lane 18, Aac 40581 isolate B; Lane 19, Aac 40580 isolate A; Lanes 20 and 21, 100 bp ladder; Lane 22, Aaa ATCC 19822; Lane 23, Aac 40580 isolate A#2; Lane 24, Aaa 391230; Lane 25, Acc 40560 isolate 4; Lane 26, Acc 40580 isolate B; Lane 27, Acc 40584 isolate A2; Lane 28, Aac 40581 isolate A; Lane 29, Aac 40587; Lane 30, Aac, 405708 isolate A; Lane 31, Kihupi 40565; Lane 32, Aaa 39122 C; Lane 33, Aaa 39460 I; Lane 34, Pca 1; Lane 35, Pca 2; Lane 36, Pca 3; Lane 37, Pca 4; Land 38, Pca 5; Lane 39, Pca 6; Lane 40, 100 bp ladder. FIG. 2B: Lanes 1 and 18,100 bp ladder; Lanes 2 and 3, A. avenae subsp. citrulli, Aac 01; Lanes 4 and 5, A. avenae subsp. cattleyae; Lanes 6 and 7, Burkholdera glumae; Lanes 8 and 9, A. avenae subsp. avenae COA-1; Lanes 10 and 11, A. avenae subsp. citrulli, Aac 130; Lanes 12 and 13, A. avenae subsp. cattleyae PC-21; Lanes 14 and 15, B. glumae COG-2; and Lanes 16 and 17, A. avenae subsp. avenae COA-4.

FIG. 3 shows the amplification plot from the TaqMan PCR method where the accumulation of the fluorescent signal from reporter dye molecules is monitored at each PCR cycle for dilutions of DNA from A. avenae subsp. citrulli utilizing the primers SEQ ID NO: 5 and SEQ ID NO:4 and the probe SEQ ID NO:7. The concentrations of DNA shown are left to right: E5, 1000 pg; E6, 500 pg; E7, 100 pg; E8, 50 pg; E9, 10 pg; E10, 5 pg; E11, 1 pg; and E12, water control, (baseline).

DETAILED DESCRIPTION OF THE INVENTION

Polymerase chain reaction (PCR) has been shown to be a highly sensitive and rapid method for detecting and identifying bacteria. The invention provides for PCR primers, methods, and kits useful for detecting subspecies of the pathogen Acidovorax avenae in or on seeds and further, for differentiating A. avenae subsp. citrulli from other A. avenae subspecies.

Strategy for obtaining specific primers for A. avenae subspecies

Several primers and primer sets have been identified as effective for amplifying particular A. avenae subspecies and differentiating between subspecies, when used in the standard PCR method (Mullis. 1987. U.S. Pat. No. 4,683,202) or the BIO-PCR method (U.S. patent application Ser. No. 08/344,085, herein incorporated by reference; Schaad et al. 1995. Pytopath. 85:243-248). Variations in length and sequences of the 16S-23S rDNA intergenic spacer region (ISR) of several bacteria have recently been targeted for use in discriminating among strains at the species and subspecies levels. These spacers are short stretches of DNA located between the 16S and 23S genes in prokaryotic rRNA loci and usually contain one or more tRNA genes. Elements important to transcription and noncoding of DNA that apparently are not functional should exhibit a considerable degree of sequence variation. Thus, such variable regions make sequences of ISRs good markers to measure short-term phylogeny. The characterization of the ISR of A. avenae and other bacterial pathogens of rice suggested that the ISR contained sequences unique to A. avenae (Kim et al. 1996. Seed Sci. & Technol. 24: 571-580). Therefore, the ISR of A. avenae was targeted as a possible source of DNA which would be specific for the different species and subspecies. In particular, unique PCR primers were derived from sequences of a fragment of the ISR of the 16S-23S rDNA for rapid identification of all those subspecies of A. avenae from oats, rice, corn, millet, wheat, sugarcane, orchid, and watermelon and for the specific detection of the subspecies citrulli from watermelon. These primers, when combined with BIO-PCR, should prove useful for sensitive detection of the pathogen in a seed health testing program for several diseases, including bacterial brown stripe of rice, leaf blight of oats, red stripe of sugar cane, bacterial leaf blight of corn and watermelon fruit blotch. Furthermore, rapid, conclusive, cost- and labor-efficient analysis can be performed when these unique primers and probes are combined with a BIO-PCR method employing the high throughput, real time TaqMan detection system.

A primer is preferably about eighteen to twenty-four nucleotides long. Primers can hybridize to a DNA strand with the coding sequence of a target sequence and are designated sense primers. Primers can also hybridize to a DNA strand that is the complement of the coding sequence of a target sequence; such primers are designated anti-sense primers. Primers that hybridize to each strand of DNA in the same location or to one another are known as complements of one another. Primers can also be designed to hybridize to a mRNA sequence complementary to a target DNA sequence and are useful in reverse transcriptase PCR.

The novel primers of the invention hybridize to a target DNA sequence of A. avenae subspecies located in the 16S-23S rDNA ISR of 950-bp. The primers are specific for A. avenae subspecies and do not hybridize to other closely related microorganisms. The primers can therefore be used in methods and kits for detecting subspecies of A. avenae in biological samples. The primers identified as SEQ ID NO:1 and SEQ ID NO:2 and the primer set comprising these primers are useful in an assay to detect the presence of A. avenae in a sample; specifically, any one or all of the subspecies, A. avenae subsp. avenae, A. avenae subsp. cattleyae, 8245 or A. avenae subsp. citrulli. The primers identified as SEQ ID NO:3 and SEQ ID NO:4 and the primer set comprising these primers distinguish A. avenae subsp. citrulli from A. avenae subsp. avenae and cattleyae. In addition, the primers of the invention are useful for employing BIO-PCR techniques for evaluating and monitoring the efficacy of treatments for eliminating the pathogenic A. avenae subspecies from seeds. Since BIO-PCR is based on detection of DNA from live cells, contaminating DNA from dead cells does not pose a problem. Detection of A. avenae subsp. citrulli can be further optimized by utilizing the primers identified as SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 and the primer sets comprising these primers, namely, primer set SEQ ID NOs:5 and 4 or primer set SEQ ID NOs:5 and 6 in the BIO-PCR method, combining BIO-PCR with the TaqMan detection system and employing the probe identified as SEQ ID NO:7. TaqMan is a PCR-based assay that utilizes an oligo-nucleotide probe containing a "fluorescent reporter"-"fluorescence quencher" pair, wherein the quencher molecule of the probe is designed to be located close enough to the reporter molecule to ensure that the fluorescence of the reporter molecule is quenched. The binding site of the probe is located between the PCR primers used to amplify the target polynucleotide. Preferably, PCR is carried out using Taq DNA polymerase, AMPLITAQ (Perkin-Elmer, Norwalk, CN). During strand extension by Taq DNA polymerase, the annealed probe containing the reporter/quencher pair is digested by the 5'.fwdarw.3' exonuclease activity of taq polymerase. Digestion of the probe results in the reporter molecule becoming separated from the quencher molecule; thus, the quencher molecule is no longer close enough to the reporter molecule to quench the reporter molecule's fluorescence. Therefore, as more and more annealed probes are digested during amplification, the number of unquenched reporter molecules in solution increases, resulting in a stronger and stronger fluorescent signal being produced during consecutive cycles. This use of BIO-PCR together with the TaqMan automated real-time fluorescence detection system results in the detection of as few as 2 cfu/ml. The primers of the invention may also be useful as hybridization probes.

Preparation of Primers

Genomic DNA was extracted from cultures of A. avenae subsp. avenae strain COA1 and A. avenae subsp. citrulli strain 29625 in late logarithmic phase, as described by Prosen et al. (1993. Pytopath. 83: 965-970). The amplification of the ISR was performed using the known primers R16-1 and R23-2R and the method described by Kim et al., supra. For A. avenae subsp. avenae, the amplified full length 950-bp ISR product was cloned into pGEM7f (GIBCO BRL). The resulting clone, pOA3, containing the 950-bp full length ISR insert was then sequenced. For A. avenae subsp. citrulli, the amplified full length 950-bp ISR product was cloned into pCR2.1 with TA cloning kit (INVITROGEN). The resulting clone, pCIT7, containing a 950-bp insert of full length ISR, was then sequenced.

The sequencing of the cloned 16S-23S ISR fragment was performed by the dideoxy-chain termination method using ABI PRISM 310 Genetic Analyzer and Dye Terminator Cycle Sequencing Core Kit (Perkin Elmer, Foster City, Calif.) following the manufacturer's protocol (Sanger et al. 1977. Proc. Natl. Acad. Sci. USA 74: 5463-5467). From the ISR sequences, candidate primers were selected using the program OLIGO (National Biosciences, Hamel, Minn.) and synthesized commercially by GIBCO BRL (Frederick, Md.). The primers 5'-GTCGGTGCTAACGACATGG-3' (SEQ ID NO:1) and 5'-AGACATCTCCGCTTTCTTTCAA-3' (SEQ ID NO:2) of A. avenae subsp. avenae (FIG. 1A) and the primers 5'-GGAAGAATTCGGTGCTACCC-3' (SEQ ID NO:3) and 5'-TCGTCATTACTGAATTTCAACA-3' (SEQ ID NO: 4) of A. avenae subsp. citrulli (FIG. 1B) were used to amplify the ISR of 43 strains of A. avenae, 11 strains of A. avenae subsp. citrulli, four of A. avenae subsp. cattleyae, single strains of A. avenae subsp. konjaci and A. facilis, 26 other known bacteria, and 27 unknown bacteria isolated from seeds. The specificity of each set of primers is illustrated in Table 1, below, and in FIGS. 2A and 2B. As shown in Table 1, for primers SEQ ID NO:1 and SEQ ID NO:2, a PCR product of approximately 550 bp was obtained for all strains of A. avenae subsp. avenae and A. avenae subsp. citrulli and the ATCC strain of A. avenae subsp. cattleyae. Three strains of A. avenae subsp. cattleyae and the single strain of A. avenae subsp. konjaci were negative. All other bacteria, including 1-6 strains each of five other species of non-fluorescent pseudomonads (including 12 strains of Burkholderia), four fluorescent pseudomonads, two xanthomonads, two erwinea, and six strains of unknown bacteria from rice seed were negative. FIG. 2A also shows a 550 bp product obtained from strains of A. avenae subsp. avenae and A. avenae subsp. citrulli. Primers SEQ ID NO:3 and SEQ ID NO:4 produced a 450-bp product with all strains of A. avenae subsp. citrulli specifically (FIG. 2B); all other bacteria were negative.

The combination of BIO-PCR with TaqMan assay methodology is a more rapid and labor- and cost-effective assay than standard PCR or BIO-PCR where the additional identification step of Southern analysis is required. The BIO-PCR/TaqMan combination assay utilizing the primer set comprising primer 5'-CCTCCACCAACCAATACGCT-3' (SEQ ID NO:5) together with either primer 5'-TCGTCATTACTGAATTTCAACA-3' (SEQ ID NO:4) or primer 5'-CATGCTCTTAGTCACTTGACCCTA-3' (SEQ ID NO:6) as detected with the probe: 5'-CGGTAGGGCGAAGAAACCAACACC-3' (SEQ ID NO:7) results in the specific identification of A. avenae subsp. citrulli.

TABLE 1______________________________________Species and strains and results of PCR with primers SEQ ID NOS: 1 & 2 of Acidovorax avenae subsp. avenae and with the primers SEQ ID NOS: 3 & 4 of A. avenae subsp. citrulli PCR-Amplification SEQ ID SEQ ID NOS: NOS: Bacterial strains No. Host Source.sup.a 1 and 2.sup.b 3 and 4.sup.c______________________________________A. avenae subsp. avenae (Pseudomonas avenae) COA1-3 3 rice 1 + - ATCC 19860 1 corn 2 + - PA135-138 4 corn 3 + - 3307-8Pav 2 corn 3 + - 3403Pav 1 vasey- 3 + - grass 3406Pav 1 corn 3 + - 3431Pav 1 millet 3 + - Nepal 1 rice 4 + - 39123a 1 rice 4 + - 39459-39463 5 rice 4 + - 30150-11 10 rice 5 + - 311056-58 3 rice 5 + - (P. rubrilineans) ATCC 19307 1 sugar- 2 + - cane 931 1 sugar- 6 + - cane 3107 1 corn 6 + - 3111 1 Canna 6 + - panicla XR119 1 sugar- 7 + - cane XR3 1 sugar- 7 + - cane (P. setariae) ATCC 19882 1 rice 2 + - PS177 1 rice 7 + - (P. rubrisubalbicans) XR105,106 2 sugar- 7 + - cane A. avenae subsp. citrulli (P. pseudoalcaligenes subsp. citrulli) HIB, H17, B164 3 water- 8 + + melon 1214, wwG, Mie3 3 water- 8 + + melon ATCC 29625 1 water- 2 + + melon 9217, 8408 2 water- 9 + + melon 2576, 2578 2 water- 8 + + melon A. avenae subsp. cattleyae ATCC 33619 1 orchid 2 + - PC, 107, 112, 145 3 orchid 7 - - A. avenae subsp. konjaci (P. pseudoalcaligenes subsp. konjaci) Amorphophallus rivieri ATCC 3399 1 Konjac 2 - - A. facilis (P. facilis) ATCC 11228 1 soil 2 - - Burkholderia caryophylli (P. caryophylli) PC131 1 carna- 7 - - tion B. cepacia (P. cepacia) PC22, PC142 2 onion 7 - - B. gladioli pv. allicola (P. allicola) PA7 2 onion 7 - - PA16 1 gladioli 7 - - B. gladioli pv. gladioli ATCC 25417 1 gladioli 2 - - B. glumae (P. glumae) COG1-4 4 rice 1 - - ATCC 33617 1 rice 2 - - P. fuscovaginae Fed1259-3 IR VM 1 rice 10 - - P. oryzicola PO101 1 rice 7 - - P. syringae pv. syringae Chi131-3 1 rice 1 - - B728a 1 beans 11 - - Xanthomonas oryzae pv. oryzae CXO105, 211, 315 3 rice 1 - - ATCC 35932 1 rice 2 - - X. oryzae pv. oryzicola P501 1 rice 13 - - ATCC 40972 1 rice 2 - - Pantoea herbicola CEh1 1 rice 1 - - ATCC 23375 1 Milletia 2 - - japonica P. syringae pv. phaseolicola C199 1 beans 12 - - P. fluorescens ATCC 13525 1 2 - - Other bacteria UPL1-6, UXL 1-7 13 rice 14 - - seeds UWPL1-8, UWB 1-8 14 water- 14 - - melon seeds______________________________________ .sup.a 1, W. Y. Song, Dept. Agric. Biology, Chonbuk National University, Chonju, Korea; 2, ATCC, American Type Culture Collection, Rockville, MD, USA; 3, ICPPB, International Collection of Plant Pathogenic Bacteria, USDA, Frederick, MD, USA; 4, L. E. Claflin, Dept. Plant Pathology, KSU, Manhatten, KS, USA; 5, C. Mortensen, Danish Govt. Inst. of Seed Pathology for Developing Countries, Copenhagen, DK; # 6, MAFF, Ministry of Agriculture, Forestry and Fisheries of Japan, Tsukuba, Japan; 7, NCPPB, National Collection of Plant Pathogenic Bacteria, Harpenden, UK; 8, P. S. Randhawa, California Seed & Plant Lab., Elverta, California USA; 9, R. Latin, Dept. Plant Path., Purdue, Univ. West Lafayette, IN, USA; 10, F. Correa, CIAT, Cali, Colombia; 11, S. Hutchenson, Plant Biology Dept, Univ Maryland, College Park, MD, # USA; 12, T. Mew, IRRI, Manila, Philippines 13, N. W. Schaad, ARS/USDA, Frederick, MD, USA; 14, this study. .sup.b Positive result means presence of 550 bp band and negative means n visible band in ethidium bromide stained agarose gel. .sup.c Positive result means presence of 450 bp band and negative means n visible band in ethidium bromide stained agarose gel.

In brief, the DNA amplification process is carried out by (a) providing a biological sample comprising bacterial cells or extracted DNA for standard PCR or cells amplified by growing on an agar medium for BIO-PCR; (b) amplifying a target sequence of the DNA to provide DNA amplification products carrying the selected target DNA sequence; and (c) detecting the presence of the DNA amplification products as an indication of the presence of A. avenae.

The biological sample may either be bacteria cells or extracted genomic DNA. The biological sample may be a test sample suspected of containing bacterial cells, and thus the DNA of the bacterial cells, or a test sample containing extracted DNA.

The BIO-PCR method, disclosed in U.S. patent application Ser. No. 08/344,085, herein incorporated by reference, combines biological preamplification of the PCR target organism with enzymatic amplification of the PCR target. Briefly, the advantages of the BIO-PCR method, over those of the standard PCR assay, include the detection of live cells only, a 100-1000 fold increase in sensitivity, and elimination of PCR inhibitors associated with plant samples thereby eliminating false negatives. Sample processing can be further simplified by directly processing the samples comprising the expanded cells without further DNA extraction. However, even if a DNA extraction step is included, an advantage of the BIO-PCR methodology is that the DNA is extracted from a growing, viable population of cells or microorganisms. The enhanced sensitivity of the BIO-PCR method is particularly valuable, for example, in those screening situations where the monetary value of a particular seed type is high, and thus it is desirable to test the smallest quantity of seeds possible, and where among trading partners, there is zero tolerance or quarantine for contaminating pathogens.

The preamplification step involves a plating step on an agar growth medium (or a liquid medium) prior to PCR analysis. A single cell per 0.1 ml can be detected because the single cell multiplies into a colony containing over 1000 cells on the agar medium.

Bacteria are recovered from suspect seeds of rice and watermelon by first soaking 1000-2000 seeds in 0.02% TWEEN 20 solution (ratio of 5 ml/g) for 4 hours at 40.degree. C., pipetting aliquots of 0.1 ml of the bacterial extracts either onto plates of general purpose agar media, such as, KB or nutrient agar (NA), or onto a semiselective medium such as EBB, described below, and then cloning. For BIO-PCR, each of five plates is washed three times with 1.0 ml of water and the resulting 14-15 ml of wash solution can be used for PCR-amplification with or without further DNA extraction or sample processing. Similarly, for standard PCR, either the DNA can be extracted or intact cells used. Since only pinpoint-size colonies are needed, incubation time ranges from only 10-15 hr for fast growing bacteria to 24-48 hrs for most plant pathogenic bacteria, depending on the media. Since the incubation time is short, few other bacterial colonies are present.

EBB medium is a preferred, semiselective medium to use for the preamplification step. The protocol for preparing the EBB Medium is as follows:

(1) Mix, per liter: ______________________________________NH.sub.4 PO.sub.4 1.0 g MgSO.sub.4.7 H.sub.2 O 0.2 g KCl 0.2 g Yeast Extract 0.3 g Boric acid 0.5 g Brilliant blue R (10 mg/ml Stock) 1.0 ml Bromcreosol purple (15 mg/ml Stock) 0.6 ml Agar 16.0 g______________________________________ (2) Autoclave and add, per liter: 10.0 ml 95% ethanol 1.0 ml Cycloheximide (200 mg/ml 95% ethanol Stock) (3) Adjust pH carefully to 5.2 using 0.2 M HCl just before autoclaving. (If the pH falls below 5.0, do not use NaOH to readjust to 5.2 as NaOH will neutralize brilliant blue R.)

In the preferred method, the enzymatic amplification of the DNA sequence is by polymerase chain reaction (PCR), as described in U.S. Pat. No. 4,683,202 to Mullis, herein incorporated by reference. In brief, the DNA sequence is amplified by reaction with at least one oligonucleotide primer or pair of oligonucleotide primers that hybridize to the target sequence or a flanking sequence of the target sequence and a DNA polymerase to extend the primer(s) to amplify the target sequence. The amplification cycle is repeated to increase the concentration of the target DNA sequence. Amplified products are optionally separated by methods such as agarose gel electrophoresis. The amplified products can be detected by either staining with ethidium bromide or silver stain or by hybridization to a labeled probe, such as in TaqMan PCR which is based on real time hybridization. In an alternative embodiment, at least one probe that hybridizes to the amplified products is labeled with either a biotin moiety and/or a fluorescent moiety. The complexes are then bound to a solid support such as a bead, multiwell plate, dipstick or the like that is coated with streptavidin. The presence of bound hybrids can then be detected using an antibody to the fluorescent tag conjugated to horseradish peroxidase. The enzymatic activity of horseradish peroxidase can be detected with a colored, luminescent, or fluorimetric substrate. Conversion of the substrate to product can be used to detect and/or measure the presence of A. avenae PCR products.

Other methods of PCR using various combination of primers including a single primer to about three primers are known to those of skill in the art and are described in Maniatis (1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, N.Y.). Those methods include asymmetric PCR, PCR using mismatched or degenerate primers, reverse transcriptase PCR, arbitrarily primed PCR (Welsh et al. 1990. Nucleic Acids Res. 18: 7213-7218), or RAPD PCR, IMS-PCR (Islam et al. 1992. J. Clin. Micro. 30: 2801-2806), multiwell PCR (ELOSA) (Luneberg et al. 1993. J. Clin. Micro. 31: 1088-1094), and Katz et al. 1993. Am. J. Vet. Res. 54: 2021-2026). The methods also include amplification using a single primer as described by Judd et al. 1993. Appl. Env. Microbiol. 59: 1702-1708).

An oligonucleotide primer preferably has a DNA sequence that hybridizes under standard conditions for PCR to a sequence flanking one end of the DNA sequence to be amplified. In the use of a pair of oligonucleotide primers, each of the primers is distinct and has a different DNA sequence. The two distinct primers are directed at specific sequences on opposite strands and together define the segment to be amplified, i.e., they hybridize to sequences at either end of the target sequence. Design of primers and their characteristics have been described previously. The DNA sequences of the oligonucleotide primers are positive-sense (forward) 5'-GTCGGTGCTAACGACATGG-3' (SEQ ID NO:1), negative sense (reverse) 5'-AGACATCTCCGCTTTCTTTCAA-3' (SEQ ID NO:2), positive sense (forward) 5'-GGAAGAATTCGGTGCTACCC-3' (SEQ ID NO:3), negative sense (reverse) 5'-TCGTCATTACTGAATTTCAACA-3' (SEQ ID NO:4), or complements thereof, or mixtures thereof. The primers may also be degenerate primers that hybridize to the target DNA sequence under hybridization conditions for a primer of that size and sequence complementarity.

For the binding and amplification, the biological sample (bacterial cells or extracted DNA) is provided in an aqueous buffer formulated with an effective amount of a divalent cation which is preferable MgCl.sub.2, preferably at a concentration of about 0.05-5 mM; an effective amount of DNA polymerase with Taq DNA polymerase being preferred in the form of native purified enzyme or a synthesized form such as AMPLITAQ (Perkin-Elmer), an effective amount of dNTPs as a nucleotide source, including, dATP, dCTP, dGTP, and dTTP, preferably in a saturating concentration, preferably about 200 .mu.M per dNTP; and an effective amount of one or a pair of oligonucleotide primers. The reaction mixture containing the annealed primer(s) is reacted with a DNA polymerase at about 72.degree. C. to about 94.degree. C. for about 1-10 minutes, to extend the primers to make a complementary strand of the target gene sequence. The cycle is then repeated by denaturing the DNA strands with heat, annealing and extending, preferably for about 25-40 cycles, preferably about 30 cycles.

If designed properly, a single product results. This product is preferably about 450-550-kb in size, whose termini are defined by the oligonucleotide primer(s), and whose length is defined by the distance between the two primers or the length of time of the amplification reaction. The gene sequence then serves as a template for the next amplification cycle.

The amplified DNA product is optionally separated from the reaction mixture and then analyzed. The amplified gene sequence may be visualized, for example, by electrophoresis in an agarose or polyacrylamide gel or by other like techniques, known and used in the art.

The amplified gene sequence may be directly or indirectly labeled by incorporation of an appropriate visualizing label, as for example, a radioactive, calorimetric, fluorometric or luminescent signal, or the like. In addition, the gel may be stained during or after electrophoresis with a visualizing dye such as ethidium bromide or silver stain wherein the resulting bands by be visualized under ultraviolet light.

To prove the identity of the amplified DNA product, a Southern blot assay should be conducted. The amplified products are separated by electrophoresis on a polyacrylamide or agarose gel, transferred to a membrane such as a nitrocellulose or nylon membrane, reacted with an oligonucleotide probe, and stained as above. The amplified products may also be detected by reverse blotting hybridization (dot blot) in which an oligonucleotide probe specific to the gene sequence is adhered to a nitrocellulose or polyvinylchloride (PVC) support such as a multi-well plate, and then the sample containing labeled amplified product is added, reacted, washed to remove unbound substance, and a labeled amplified product attached to the probe or the gene sequence imaged by standard methods.

A major advantage of TaqMan PCR is that the technology is based on hybridization; therefore, a Southern blot assay is not needed. The DNA sequences of the oligonucleotide primers for TaqMan PCR are positive-sense (forward) 5'-CCTCCA CCAACCAATACGCT-3' (SEQ ID NO: 5), negative sense (reverse) 5'-TCGTCATTACTG AATTTCAACA-3' (SEQ ID NO:4), and negative sense (reverse) 5'-CATGCTCTTAGT CACTTGACCCTA-3' (SEQ ID NO:6), or complements thereof, or mixtures thereof. The DNA sequence of the probe utilized in the TaqMan PCR is 5'-CGGTAGGGCGAA GAAACCAACACC-3' (SEQ ID NO:7).

The detection of amplified gene product in the sample is evidence of the presence of A. avenae subspecies in the biological sample. When combined with BIO-PCR, the method is useful in diagnosing presence of viable cells of A. avenae. One set of primers, primers identified by SEQ ID NO:1 and SEQ ID NO:2, is useful in a detection assay to identify that A. avenae is present in a sample; specifically, that any one or all of the subspecies, A. avenae subsp. avenae, A. avenae subsp. cattleyae, or A. avenae subsp. citrulli, are present. The primer set comprising SEQ ID NO:1 and SEQ ID NO:2 cannot distinguish the subspecies avenae, cattleyae, and citrulli; however, the primer set identified by SEQ ID NOS: 1 and 2, can be used to screen for the presence of these organisms. The primer set comprising SEQ ID NO:3 and SEQ ID NO:4 distinguishes A. avenae subsp. citrulli from A. avenae subsp. avenae and cattleyae. When the advantages of the BIO-PCR are combined with the specificity of SEQ ID NO:3 and SEQ ID NO:4 for A. avenae subsp. citrulli, the sensitivity and specificity for detecting the pathogen responsible for watermelon fruit blotch is further enhanced.

Detection utilizing the BIO-PCR method is enhanced still further when combined with the TaqMan detection system. When the primer set comprising SEQ ID NO:5 with either SEQ ID NO:4 or SEQ ID NO:6 is utilized in a combined BIO-PCR and TaqMan method, such methodology results in the detection of as few as 2 cfu/ml. Thus, there is no loss of sensitivity as compared to BIO-PCR and Southern analysis. Two important advantages of the TaqMan system are (1) that measurement of real-time hybridization is rapid and (2) that the hybridization product is the definitive, irrefutable product of the polymerization chain reaction. Further, the methodology involved in the assay process makes possible the handling of large numbers of samples efficiently and without cross-contamination and is therefore adaptable for robotic sampling. The advantages of rapid, conclusive data together with labor and cost efficiency make the combination BIO-PCR and TaqMan detection system utilizing the specific primers of the invention a highly beneficial system for monitoring seed pathogens, especially in those circumstances where seed screening results have major commercial and trade consequences.

The screening for A. avenae subsp. avenae, cattleyae, and citrulli, using the primer set consisting of SEQ ID NO:1 and SEQ ID NO:2, can be followed by a detection assay utilizing the primer set consisting of SEQ ID NO:3 and SEQ ID NO:4 in order to specifically detect the presence of A. avenae subsp. citrulli. Furthermore, in such screening assays, where a detection assay utilizing a primer set identified by SEQ ID NO:1 and SEQ ID NO:2 is followed by a detection assay utilizing a primer set identified by SEQ ID NO:3 and SEQ ID NO:4, a negative result in the detection assay utilizing the primer set consisting of SEQ ID NO:3 and SEQ ID NO:4 is indicative of either or both A. avenae subsp. avenae or cattleyae being present and that A. avenae subsp. citrulli is not present. Similarly, where a detection assay utilizing a primer set identified by SEQ ID NO:1 and SEQ ID NO:2 is followed by a TaqMan detection assay utilizing the primer set comprising SEQ ID NO:5 together with either SEQ ID NO:4 or SEQ ID NO:6, a negative result in the second detection assay, utilizing SEQ ID NO:5 together with SEQ ID NO:4 or 6, is indicative of either or both A. avenae subsp. avenae or cattleyae being present and that A. avenae subsp. citrulli is not present.

The primers and amplification method can further be useful for evaluating and monitoring the efficacy of any treatments utilized to eliminate the pathogenic A. avenae subspecies. In this method, biological samples are obtained from seeds prior to treatment and from seeds which have undergone treatment with a treatment protocol such as hot water or household bleach, designed to eradicate A. avenae subspecies. In addition, biological samples can be obtained from seeds at several time points during treatment. DNA amplification products of a target sequence of A. avenae from all samples are analyzed for the presence of A. avenae. Results from samples obtained prior, during and after treatment are compared in order to determine efficacy of the treatment protocol.

Similarly, the novel primers and methods are very useful for epidemiology and host-pathogen studies. A further advantage of using BIO-PCR is that quantitative data on viable pathogen populations can be obtained by dilution-end-point analysis if samples are not heavily contaminated with saprophytes in those instances when quantitation of propagules would be desirable.

EXAMPLES

Example 1

Bacterial cultures

Bacteria were extracted from healthy seeds of rice and watermelon by soaking 1000-2000 seeds in 0.02% Tween 20 solution (ratio of 5 ml/g) for 4 hr at 4.degree. C. Aliquots of 100 .mu.l were pipetted onto each of ten plates of King et al.'s medium B (KB), nutrient agar (NA), yeast extract-dextrose calcium carbonate (YDC), or a semiselective agar medium such as neutral red semiselective medium (Schaad. 1988. Laboratory Guide for Identification of Plant Pathogenic Bacteria, 2nd Ed., APS Press, St. Paul, Minn.) for A. avenae subsp. avenae or EBB for A. avenae subsp. citrulli, and the suspension spread with a L-shaped glass rod. Five plates were washed three times with 1.0 ml of 10 mM Tris-HCl solution (pH 7.4) after 48 hrs incubation at 36.degree. C. for BIO-PCR as described (Schaad et al. 1995, supra) and the sample archived at -20.degree. C. The remaining five plates were read for visible suspect colonies after three and four days. Suspect colonies were cloned and stored on YDC slants at room temperature for pathogenicity tests. These bacteria do not store well at 2-40.degree. C.

The strains and source of Acidovorax and other bacteria used in this study are shown in Table 1. Cultures were maintained on YDC or KB agars at room temperature and stored at -85.degree. C. Colony morphology of A. avenae strains were checked on YDC and KB media and accumulation of Poly-.beta.-hydroxybytyrate determined as described (Schaad. 1988, supra). Colonies of all strains received as A. avenae subsp. avenae (including P. rubrilineans, P. setariae, and P. rubrisubalbicans) were non-fluorescent on KB medium, round convex morphology and tan in color on YDC medium. Colonies of A. avenae subsp. citrulli, subsp. konjaci, and ATCC strain 33619 of subsp. cattleyae had colonies indistinguishable from A. avenae subsp. avenae. All these strains were positive for Poly-.beta.-hydroxybutyrate. In contrast, morphologies of strains PC107, 112, and 145 of subsp. cattleyae were very different and these strains did not accumulate Poly-.beta.-hydroxybutyrate. We therefore concluded that these cultures were contaminants and not subsp. cattleyae. Final identity of all A. avenae subsp. avenae strains was confirmed by inoculating oat and corn seedlings, as described (Schaad et al. 1975, supra) and the identity of all strains of A. avenae subsp. citrulli were confirmed by inoculating watermelon seedlings (Schaad et al. 1978, supra).

Example 2

Extraction of genomic DNA

Cells were grown in Luria-Bertain (LB) liquid medium overnight on a rotary shaker at 30.degree. C. Cell suspensions were centrifuged, suspended in 10 mM Tris-HCl solution (pH 8.0), and adjusted to a concentration of 0.1 OD at A.sub.600. Suspensions of cells or single colonies growing on YDC or KB medium were used for PCR-amplification without prior DNA extraction or other sample processing. Genomic DNA was extracted from washed cells, as described (Prosen et al., supra).

Example 3

Design of Primers

The ISR was amplified using primers R16-1 and R23-2R and genomic DNA of A. avenae subsp. avenae strain COA1 and A. avenae subsp. citrulli strain 29625, utilizing the method described by Kim et al., supra. The amplified product was electrophoresed in a 2% Metaphore gel (FMA BioProducts, Rockland, Md., USA). The resulting target DNA band was excised from the gel and processed for loading in agarose, following the suppliers recommendations. For A. avenae subsp. avenae, the DNA of strain COA1 was blunt ended using T4 DNA polymerase, and ligated to plasmid vector pGEM-7Zf(+) (Promega, Madison, Wis., USA), and digested with Smal, using T4 DNA Ligase. The ligation products were used to transform DH5.varies. competent cells (Life Technologies, Md., USA) according to suppliers recommendations. For A. avenae subsp. citrulli, the gel-purified product was ligated into pCR2.1 with TA cloning kit (INVITROGEN) following the suppliers protocols.

For A. avenae subsp. avenae, the amplified full length 950-bp product of ISR was cloned into pGEM7f (GIBCO BRL). The resulting clone, pOA3, containing a 950-bp insert of full length ISR, was then sequenced. For A. avenae subsp. citrulli, the amplified full length 950-bp product of ISR was cloned into pCR2.1 with TA cloning kit (INVITROGEN). The resulting clone, pCIT7, containing a 950-bp insert of full length ISR, was then sequenced.

Chemicals and antibiotics were purchased from Sigma Chemical Co. (St. Louis, Mo.), restriction and DNA modifying enzymes were purchased from Life Technologies (Bethesda, Md.), and all supplies for PCR were purchased from Perkin Elmer (Norwalk, Calif.).

Example 4

Sequence analysis

The sequencing of the cloned 16S-23S ISR fragment was performed by the dideoxy-chain termination method using ABI PRISM 310 Genetic Analyzer and Dye Terminator Cycle Sequencing Core Kit (Perkin Elmer, Foster City, Calif.) following the manufacturer's protocol (Sanger et al., supra). The size of the ISR fragment of A. avenae subsp. avenae sequenced was 625 bp (FIG. 1B; Aaa, shaded). The fragment had a GC content of 48.87%, and a single tRNA.sup.Ala gene of 73 bp. The size of the sequenced ISR fragment of A. avenae subsp. citrulli was 617 bp, (FIG. 1B, Aac, shaded). It had a GC content of 48.94%, and the same single tRN.sup.Ala gene. The sequences were first aligned by using the DNASES program (Hitachi SEQ, Ltd.) and the alignments were corrected manually. This ISR sequence contained a single copy of tRNA.sup.Ala. By comparing the gene sequence of A. avenae subsp. avenae to the primers for Leuconostoc oenos (Zavaleta et al. 1996. Microbiol. 142: 2105-2114), Streptococcus aureus (Gurtler et al. 1995 Microbiol. 141: 1255-1265), Bacillus subtilis (Loughney et al. 1982. Nuc. Acids Res. 10: 1607-1624) and E. coli (Harvey et al. 1988. J. Bacteriol. 170: 1235-1238), the primers SEQ ID NO:1 and SEQ ID NO:2 for A. avenae subsp. avenae and SEQ ID NO:3 and SEQ ID NO:4 for A. avenae subsp. citrulli were selected (FIG. 1A, FIG. 1B). From the ISR sequences, candidate primers SEQ ID NO:1 and SEQ ID NO:2 were selected using the program OLIGO (National Biosciences, Hamel, Minn.) and synthesized commercially by GIBCO BRL (Frederick, Md.).

Minor errors of PCR and sequencing reactions were eliminated by sequencing both strands from two independently cloned fragments of separate PCR experiments. No sequence variability between the two 16S-23S ISR copies was found.

Example 5

Polymerase Chain Reaction

Primers were screened for specificity in 25 .mu.l reactions containing 2-5 .mu.l of the bacterial cell suspensions, 200 .mu.M each of dNTPs, 0.16 .mu.M of each primer, 1.times.PCR reaction buffer II and 1.0 U of AMPLITAQ Gold polymerase (Perkin Elmer). All amplifications were conducted in a Perkin Elmer 9600 thermocycler (Perkin Elmer Cetus, Norwalk, CT). For primers having SEQ ID NO:1 and SEQ ID NO:2, the amplifications were conducted with an initial DNA denaturation at 94.degree. C. for 30 S; 55.degree. C. for 30 S; 72.degree. C. for 1 min and a final extension step of 7 min at 72.degree. C. For the primers of SEQ ID NO:3 and SEQ ID NO:4, the amplifications were conducted with an initial DNA denaturation at 94.degree. C. for 10 min followed by 5 cycles of 94.degree. C. for 30 S; 56.degree. C. for 30 S; 72.degree. C. for 1 min, then 25 cycles of 94.degree. C. for 45 S; 72.degree. C. for 45 S; 72.degree. C. for 1 min and a final extension step as above. To detect a product, 5 to 10 .mu.l of the reaction were electrophoresed on 1.2% agarose gels and stained with ethidium bromide as described (Maniatis et al. 1989. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The primers having SEQ ID NO:1 and SEQ ID NO:2 of A. avenae subsp. avenae and SEQ ID NO:3 and SEQ ID NO:4 of A. avenae subsp. citrulli were used to amplify the ISR comprising a target sequence of DNA of 43 strains of A. avenae, 11 strains of A. avenae subsp. citrulli, four of A. avenae subsp. cattleyae, single strains of A. avenae subsp. konjaci and A. facilis, 26 other known bacteria, and 27 unknown bacteria isolated from seeds. The specificity of each set of primers is illustrated in Table 1. For primers having SEQ ID NO:1 and SEQ ID NO:2, a PCR product of approximately 550 bp was obtained for all strains of A. avenae subsp avenae and A. avenae subsp. citrulli and the ATCC strain of A. avenae subsp. cattleyae. Three strains of A. avenae subsp. cattleyae and the single strain of A. avenae subsp. konjaci were negative. All other bacteria, including 1-6 strains each of five other species of non-fluorescent pseudomonads (including 12 strains of Burkholderia), four fluorescent pseudomonads, two xanthomonads, two erwinea, and six strains of unknown bacteria from rice seed were negative. Primers having SEQ ID NO:3 and SEQ ID NO:4 produced a 450-bp product with all strains of A. avenae subsp. citrulli specifically; all other bacteria were negative (Table 1 and FIG. 2B).

Example 6

Selection of primers and probes for TaqMan PCR

Primer and fluorescent probe sequences for the TaqMan PCR assay were designed with the Primer Design software program from the manufacturer (PE Applied Biosystems, Foster City, Calif.) using the sequences: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6. Several combinations of forward (F) and reverse (R) primer and TaqMan probe (SEQ ID NO:7) sequences were tested for their performance as determined by Cycle Threshold (Ct) values. Ct value is defined as the PCR cycle number at which the signal of the probe rises above background. The lower the Ct value, the better the PCR performance. TaqMan reactions were analyzed for 40-45 cycles using an ABI Prism 7700 Sequence detection System (PE Applied Biosystems) following methods recommended by the manufacturer. Dilutions of cells or DNA of A. avenae subsp. citrulli and water (negative control) were measured.

To determine inherent sensitivity and Ct values of TaqMan and A. avenae subsp. citrulli primers (SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6), a titration using 1 to 100,000 pg from four replicates of five- and ten-fold dilutions of purified genomic DNA from A. avenae subsp. citrulli was performed. Under the same PCR cycling conditions for classical and BIO-PCR, ten-fold serial dilutions of 10.sup.-6 of a liquid NBY culture of A. avenae subsp. citrulli were prepared. For classical TaqMan PCR, aliquots of 14 .mu.l of dilutions of 10.sup.-3, 10.sup.-4, 10.sup.-5, and 10.sup.-6 were tested in duplicate. For BIO-PCR, aliquots of 100 .mu.l of the same dilutions were plated onto 8 plates each of KB, NA, YDC, and neutral red semiselective media. After 72 hrs, four plates were washed, as described in Example 1, and four kept for determining the number of cfu's of A. avenae subsp. citrulli on KB, NA, YDC, and neutral red semiselective media agar media after 4 and 7 days.

Example 7

Specificity and sensitivity of primers

Forward primer 5'-CCTCCACCAACCAATACGCT-3' (SEQ ID NO: 5), reverse primer 5'-TCGTCATTACTGAATTTCAACA-3' (SEQ ID NO:4), and TaqMan probe 5'-CGGTAG GGCGAAGAAACCAACACC (SEQ ID NO:7) resulted in a Ct value of 14.62, the lowest Ct value for 100,000 pg DNA. The next lowest Ct value, 15.40, was observed when forward primer 5'-CCTCCACCAACCAATACGCT-3' (SEQ ID NO: 5), reverse primer 5'-CATGCTCTTAGTCACTTGACCCTA-3' (SEQ ID NO:6), and TaqMan probe 5'-CGGTAG GGCGAAGAAACCAACACC (SEQ ID NO:7) were utilized. FIG. 3 shows the best primers. All other primers had much higher Ct values (Table 2). Utilizing the best two primer sets; SEQ ID NOs: 5&4 and 5&6, the DNA titration results show that fluorescence rises above background after 33 and 37 cycles, respectively, for 1 pg DNA. FIG. 3 shows a typical titration curve for the primer set SEQ ID NOs:5 and 4. Suspensions of A. avenae subsp. citrulli containing around 10 cfu/ml were consistently positive by classical TaqMan PCR. Washings of plates of KB, NA, YDC, and neutral red semiselective media containing 1-2 colonies of A. avenae subsp. citrulli were consistently positive by TaqMan BIO-PCR.

TABLE 2______________________________________Selection of primers and probe combinations for TaqMan PCR______________________________________Primers & Whole Cell Titrations (Dilutions)Probe Set Ct(-2) Ct(-4) Ct(-6) Rn(-2) Rn(4) Rn(-6)______________________________________ AACF2/ 28.84 30.30 40.00 1.05 0.27 0.00 AACR2 AaP1 AACF2/ 24.22 31.02 37.95 2.17 0.88 0.20 AACR4 AaP1 SEQ ID NOs: 16.27 23.02 30.98 3.07 2.50 1.30 5,4 7 SEQ ID NOs: 17.79 22.81 32.39 3.13 2.21 0.71 5,6 7______________________________________Primers & DNA Titration (Ct Values)Probe Set 100,000 50,000 10,000 1,000 100 10 1 0______________________________________ AACF2/ 20.78 21.20 23.15 26.87 31.54 36.99 37.83 40 AACR4 AaP1 SEQ ID 14.62 14.93 16.95 20.58 25.18 28.82 33.83 40 NOs: 5,4 7 SEQ ID 15.40 15.81 18.06 22.43 27.40 31.75 37.62 40 NOs: 5,6 7______________________________________

The forward primer 5'-CCTCCACCAACCAATACGCT-3' (SEQ ID NO: 5) and the reverse primer of 5'-TCGTCATTACTGAATTTCAACA-3' (SEQ ID NO: 4) of A. avenae subsp. citrulli were used to amplify the ISR of 16 strains of A. avenae subsp. avenae, 11 strains of A. avenae subsp. citrulli, a single strain of A. facilis, 18 strains of pseudomonads, four xanthomonads, and two flavobacteria. For these primers, an amplified fluorescent signal was obtained with all strains of A. avenae subsp. citrulli specifically; all other bacteria were negative, as illustrated in Table 3.

TABLE 3______________________________________Species and strains and results of PCR with primers SEQ ID NOS: 5 & 4 of A. avenae subsp. citrulli. PCR Amplification SEQ ID NOS: Organism Origin 5 & 4.sup.a______________________________________A. avenae subsp. avenae Fc 158 ATCC 40582 E1 Denmark - Fc 159 ATCC 40580 Denmark - Fc 160 391226 Denmark - Fc 172 ATCC 40582 A1 Denmark - Fc 189 301507 Japan - Fc 190 301508 Japan - Fc 191 301511 Japan - Fc 192 311058 Japan - Fc 193 301509 Japan - Fc 194 301510 Japan - Fc 195 311057 Japan - Fc 196 311056 Japan - Fc 197 301506 Japan - Fc 198 301504 Japan - Fc 199 301503 Japan - Fc 200 301502 Japan - A. avenae subsp. citrulli Fc 141 AaC-H1B California + Fc 181 AaC H17 California + Fc 201 8408 Purdue + Fc 203 AaC 2576 California + Fc 204 AaC 2578 California + Fc 247 ATCC + Fc 248 AaC 206 California + Fc 249 Natural seed + isolate Lot 2573 Fc 250 Natural seed + isolate Lot 2573 Fc 251 Natural seed + isolate Lot 2573 Fc 253 Natural seed + isolate Lot 2573 A. facilis Fc 208 ATCC 6230 - Pseudomonas pseudoalicaligenes subsp. pseudoalicaligenes Fc 206 ATCC 26550 - P. aptata Fc 218 8545 Ukraine - P. wieringae Fc 219 7921 Ukraine - Fc 220 7923 Ukraine - P. panici Fc 221 7963 Ukraine - Fc 222 7963* Ukraine - P. xanthochlora Fc 223 8538 Ukraine - Fc 224 8540 Ukraine - P. lupini Fc 225 8532 Ukraine - Fc 226 8533 Ukraine - P. solancearum Fc 228 AW1 Georgia - Fc 229 AW1-PC Georgia - P. rubrisalbicans Fc 97 PR108 - Fc 98 PR110 - P. syringae pv. syringae Fc 103 301D Steve Hutchinson - Fc 108 B 728a Wisconsin - P. fluorescens Fc 117 4SI-14 Auburn, AL - Fc 118 4SI-15 Auburn, AL - Xanthomonas albilineans Fb 581 Xa 15009 (G53) Guadeloupe - Fb 582 Xa 150010 (MDG 58) Madagascar - Fb 616 Xa 93-170 Canal Point, FL - X. cucurbitae Fb 1054 Frederick - Flavobacterium balustinum Fb 1081 ATCC 53198 Columbus, OH - Flavobacterium sp. Fb 1082 ATCC 39723 Crawford -______________________________________ .sup.a Positive result means presence of an amplified fluorescent signal from the reporter dye molecule and negative means no fluorescent signal.

The foregoing description and certain representative embodiments and details of the invention have been presented for purposes of illustration and description of the invention. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It will be apparent to practitioners skilled in this art that modifications and variations may be made therein without departing from the scope of the invention.

__________________________________________________________________________# SEQUENCE LISTING - - - - <160> NUMBER OF SEQ ID NOS: 9 - - <210> SEQ ID NO 1 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Acidovorax avenae - - <400> SEQUENCE: 1 - - gtcggtgcta acgacatgg - # - # - # 19 - - - - <210> SEQ ID NO 2 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Acidovorax avenae - - <400> SEQUENCE: 2 - - agacatctcc gctttctttc aa - # - # 22 - - - - <210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Acidovorax avenae subsp. citrulli - - <400> SEQUENCE: 3 - - ggaagaattc ggtgctaccc - # - # - # 20 - - - - <210> SEQ ID NO 4 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Acidovorax avenae subsp. citrulli - - <400> SEQUENCE: 4 - - tcgtcattac tgaatttcaa ca - # - # 22 - - - - <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Acidovorax avenae subsp. citrulli - - <400> SEQUENCE: 5 - - cctccaccaa ccaatacgct - # - # - # 20 - - - - <210> SEQ ID NO 6 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Acidovorax avenae subsp. citrulli - - <400> SEQUENCE: 6 - - catgctctta gtcacttgac ccta - # - # 24 - - - - <210> SEQ ID NO 7 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Acidovorax avenae subsp. citrulli - - <400> SEQUENCE: 7 - - cggtagggcg aagaaaccaa cacc - # - # 24 - - - - <210> SEQ ID NO 8 <211> LENGTH: 747 <212> TYPE: DNA <213> ORGANISM: Acidovorax avenae subsp. citrulli - - <400> SEQUENCE: 8 - - ggtgaagtcg taacaaggta gccgtatcgg aaggtgcggc tggatcacct cc -#tttctgga 60 - - aaacagcatt caatattgaa cgcccactct tatcggttgt tggaagaatt cg -#gtgctacc 120 - - cgacatgggt ctggtagctc agctggttag agcaccgtct tgataaggct gg -#ggtcgttg 180 - - gttcgagccc aactagaccc accaaatctt ccgaacataa gatgcgagga tc -#agtggggg 240 - - attagctcag ctgggagagc acctgctttg caagcagggg gtcgtcggtt cg -#atcccgtc 300 - - atcctccacc aaccaatacg ctctgcggta gggcgaagaa accaacacca aa -#gcggcttc 360 - - gcgagaggcc tctttgttgt tggtccggta tagaccggat caatcggctg tt -#ctttaaaa 420 - - attcatagag tcgaatcagc gttgccggcg gaaagcagga aactgcaccg tg -#ccgccggt 480 - - gacaaaaatt tgattgcgtc aaaacgaata ttcaattgag cgaaagcttg tt -#gaaattca 540 - - gtaatgacga attgttctct aggtagcaat accgcaagaa gaattcacat ta -#cggcataa 600 - - cgcgcgaagt gaaagacctc gcaagtcctt gaaagaaagc ggagatttct cg -#ctagagat 660 - - ttcaaagttt tagggtcaag tgactaagag catgtggtgg atgccttggc ga -#tgataggc 720 - - gacgaaagac gtgatagcct gcgataa - # - # 747 - - - - <210> SEQ ID NO 9 <211> LENGTH: 753 <212> TYPE: DNA <213> ORGANISM: Acidovorax avenae subsp. avenae - - <400> SEQUENCE: 9 - - ggtgaagtcg taacaaggta gccgtatcgg aaggtgcggc tggatcacct cc -#tttctaag 60 - - gaaaacagca ttcaatattg aacgcccaca cttatcggtt gttggaagaa gt -#cggtgcta 120 - - acgacatggg tctgtagctc agctggttag agcaccgtct agataaggcg gg -#gagtcgtt 180 - - gggttcgagc ccaactcgac ccaccaaatc ttccgaacat aagatgcgag ga -#atcaagtg 240 - - ggggattagc tcagctggga gagcacctgc tttgcaagca gggggtcgtc gg -#ttcgatcc 300 - - cgtcatcctc cacccaacca atatgtcctg cggtagggca aagaaactaa ca -#ccaaagcg 360 - - gcttcgcgaa gaggcctctt tgttgttggt ccggtataga ccgggtcaat cg -#gctgttct 420 - - ttaaaaattc atagagtcga atcagcgttg ccggcggaaa gcaggaaact gc -#atccgtgc 480 - - cgtcggcaac aaaaatttga ttgcgtccaa acgaatattc aattggagcg aa -#agctgatc 540 - - gaaattcagt aatgacgaat tgttctctta ggtagcaata cccgaagaag aa -#ttacacat 600 - - tacggcatta acgcgcgatg tgaaagacct cgcaagtcct tgaaagaaag cg -#gagatgtc 660 - - tcgcaagaga tgtcaagtta taggtcaagt gactaagagc atgtggtgga tc -#cttgcgat 720 - - gatagcgacg aaagacgtga tagcctgcga taa - # -# 753__________________________________________________________________________

Claims

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

2. An oligonucleotide primer consisting of the sequence 5'-AGACATCTCCGCTTTCTTTCAA-3' (SEQ ID NO:2).

3. A primer set comprising oligonucleotides consisting of the sequences 5'-GTCGGTGCTAACGACATGG-3' (SEQ ID NO:1) and 5'-AGACATCTCCGCTTTCTTTCAA-3' (SEQ ID NO:2).

4. An oligonucleotide primer consisting of the sequence 5'-GGAAGAATTCGGTG CTACCC-3' (SEQ ID.NO:3).

5. An oligonucleotide primer consisting of the sequence 5'-TCGTCATTACTGAATTTC AACA-3' (SEQ ID NO:4).

6. A primer set comprising oligonucleotides consisting of the sequences 5'-GGAAGA ATTCGGTGCTACCC-3' (SEQ ID NO: 3) and 5'-TCGTCATTACTGAATTTCAACA-3' (SEQ ID NO:4).

7. An oligonucleotide primer consisting of the sequence 5'-CCTCCACCAACCAA TACGCT-3' (SEQ ID NO:5).

8. An oligonucleotide primer consisting of the sequence 5'-CATGCTCTTAGTCACTTG ACCCTA-3' (SEQ ID NO:6).

9. A primer set comprising oligonucleotides consisting of the sequences 5'-CCTCCACC AACCAATACGCT-3' (SEQ ID NO:5) and 5'-TCGTCATTACTGAATTTCAACA-3' (SEQ ID NO:4).

10. A primer set comprising oligonucleotides consisting of the sequences 5'-CCTCCAC CAACCAATACGCT-3' (SEQ ID NO:5) and 5'-CATGCTCTTAGTCACTTGACCCTA-3' (SEQ ID NO:6).

11. An oligonucleotide probe consisting of the sequence 5'-CGGTAGGGCGAAGAA ACCAACACC-3' (SEQ ID NO:7).

12. An intergenic spacer region (ISR) sequence selected from the group consisting of: ISR1 of A. avenae subsp. citrulli comprising the nucleotides 58-694 of SEQ ID NO:8 and ISR2 of A. avenae subsp. avenae comprising the nucleotides 59-682 of SEQ ID NO:9.

13. 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.

14. 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.

15. The method of claim 14, wherein said Acidovorax is A. avenae subsp. avenae , A. avenae subsp. catteyae, or A. avenae subsp. citrulli, and the primer set comprises oligonucleotides consisting of the sequence 5'-GTCGGTGCTAACACATGG-3' (SEQ ID NO:1) and the sequence 5'-AGACATCTCCGCTTTCTTTCAA-3' (SEQ ID NO:2).

16. The method of claim 14, wherein said Acidovorax is Acidovorax citrulli and the primer set is selected from the group consisting of (a) the primer set comprising oligonucleotides consisting of the sequence 5'-GGAAGAATTCGGTGCTACCC-3' (SEQ ID NO: 3) and the sequence 5'-TCGTCATTACTGAATTTCMCA-3' (SEQ ID NO:4); (b) the primer set comprising oligonucleotides consisting of the sequence 5'-CCTCCACC AACCAATACGCT-3' (SEQ ID NO:5) and the sequence 5'-TCGTCATTACTGAATTTC AACA-3' (SEQ ID NO:4); and (c) 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).

17. The method of claim 13, 14, 15, or 16 wherein the polymerase chain reaction (PCR) is a BIO-PCR, wherein the test sample consists essentially of viable cells or microorganisms or the DNA extracted from said viable cells or microorganisms, and wherein said test sample results from (a) culturing said viable cells or microorganisms on agar plates or in liquid medium in order to expand the number of said viable cells or microorganisms, (b) collecting the viable cells or microorganisms expanded by culturing, by either washing the plates of plated samples or collecting aliquots from liquid culture, and (c) washing the viable cells or microorganisms by filtration, centrifugation, or other conventional means.

18. The method of claim 13, 14, 15, or 16 wherein the polymerase chain reaction (PCR) is a BIO-PCR; wherein the test sample consists essentially of viable cells or microorganisms or the DNA extracted from said viable cells or microorganisms; wherein said test sample results from (a) culturing said viable cells or microorganisms on agar plates or in liquid medium in order to expand the number of said viable cells or microorganisms, (b) collecting the viable cells or microorganisms expanded by culturing, by either washing the plates of plated samples or collecting aliquots from liquid culture, and (c) washing the viable cells or microorganisms by filtration, centrifugation, or other conventional means; and wherein the presence of the amplification products is detected by the TaqMan system.

19. The method of claim 17 wherein the agar is EBB medium.

20. The method of claim 18 wherein the agar is EBB medium.

21. 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.

22. The method of claim 21 wherein the polymerase chain reaction (PCR) is a BIO-PCR, wherein the test sample consists essentially of viable cells or microorganisms or the DNA extracted from said viable cells or microorganisms, and wherein said test sample results from (a) culturing said viable cells or microorganisms on agar plates or in liquid medium in order to expand the number of said viable cells or microorganisms, (b) collecting the cells or microorganisms expanded by culturing, by either washing the plates of plated samples or collecting aliquots from liquid culture, and (c) washing the viable cells or microorganisms by filtration, centrifugation, or other conventional means.

23. The method of claim 22 wherein the presence of the amplification products is detected by the TaqMan system.

24. A method of evaluating or monitoring the efficacy of treatments utilized to eliminate pathogenic A. avenae subspecies from a seed lot, 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 according to the method of any one of claims 13-16, to provide DNA amplification products of Acidovorax;

c) detecting the presence of the amplification products of the target sequence of DNA as an indication of the presence of Acidovorax in the first test sample, if present;

d) treating the seed lot from which the first test sample is obtained, or a test portion of said seed lot, with a composition to reduce or eradicate Acidovorax;

e) providing a second test sample from the substance undergoing treatment to reduce or eradicate A. avenae subspecies;

f) amplifying a target sequence of DNA of said Acidovorax according to the method of any one of claims 13-16, to provide DNA amplification products of Acidovorax;

g) detecting the amount of the amplification products of the target sequence of DNA in said second test sample; and

h) comparing the amount of amplification products or absence of amplification products in the second test sample to the amount of amplification products detected in the first test sample in step (c), as an indication of whether or not the treatment protocol has been successful in reducing or eradicating A. avenae.

25. The method of claim 24 wherein the polymerase chain reaction (PCR) is a BIO-PCR, wherein the test sample consists essentially of viable cells or microorganisms or the DNA extracted from said viable cells or microorganisms, and wherein said test sample results from (a) culturing said viable cells or microorganisms on agar plates or in liquid medium in order to expand the number of said viable cells or microorganisms, (b) collecting the cells or microorganisms expanded by culturing, by either washing the plates of plated samples or collecting aliquots from liquid culture, and (c) washing the viable cell microorganisms by filtration, centrifugation, or other conventional means.

26. The method of claim 25 wherein the presence of the amplification products is detected the TaqMan system.

27. The method of any one of claims 24-26, wherein the second test samples are obtained at real time points during the treatment protocol.

28. A kit for identifying a subspecies of A. avenae, comprising at least one primer from the group consisting of the oligonucleotide 5'-GTCGGTGCTAACGACATGG-3' (SEQ ID NO:1), the oligonucleotide 5'-AGACATCTCCGCTTTCTTTCAA-3' (SEQ ID NO:2), the oligonucleotide 5'-GGAAGAATTCGGTGCTACCC-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'-CATGCT CTTAGTCACTTGACCCTA-3' (SEQ ID NO:6).

29. A kit for identifying a subspecies of A. avenae, wherein the subspecies is A. avenae subsp. avenae, A. avenae subsp. cattleyae, or A. avenae subsp. citrulli, comprising a primer set consisting of the oligonucleotides 5'-GTCGGTGCTAACGA CATGG-3' (SEQ ID NO:1) and 5'-AGACATCTCCGCTTTCTTTCAA-3' (SEQ ID NO:2).

30. A kit for identifying A. avenae subsp. citrulli, comprising a primer set selected from the group consisting of (a) the primer set comprising oligonucleotides consisting of the sequence 5'-GGAAGAATTCGGTGCTACCC-3' (SEQ ID NO: 3) and the sequence 5'-TCGTCATTACTGAATTTCAACA-3' (SEQ ID NO:4); (b) the primer set comprising oligonucleotides consisting of the sequence 5'-CCTCCACCAACCAATACGCT-3' (SEQ ID NO:5) and the sequence 5'-TCGTCATTACTGMTTTCAACA-3' (SEQ ID NO:4); and (c) the primer set comprising oligonucleotides consisting of the sequence 5'-CCT CCACCAACCAATACGCT-3' (SEQ ID NO:5) and the sequence 5'-CATGCTCTTAGT CACTTGACCCTA-3' (SEQ ID NO:6).