Universal PCR Primers for Detection of Phytopathogenic Agrobacterium Strains

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1995, p Vol. 61, No /95/$ Copyright 1995, American Society for Microbiology Universal PCR Primers for Detection of Phytopathogenic Agrobacterium Strains JERRY H. HAAS, 1 LARRY W. MOORE, 1 WALT REAM, 2 * AND SHULAMIT MANULIS 3 Departments of Botany and Plant Pathology 1 and Agricultural Chemistry, 2 Oregon State University, Corvallis, Oregon 97331, and Department of Plant Pathology, Agricultural Research Organization, Volcani Center, Bet-Dagan 50250, Israel 3 Received 5 April 1995/Accepted 3 June 1995 Two PCR primer pairs, based on the vird2 and ipt genes, detected a wide variety of pathogenic Agrobacterium strains. The endonuclease domain of VirD2 protein, which cleaves transferred DNA (T-DNA) border sequences, is highly conserved; primer oligonucleotides specific for the endonuclease portion of vird2 detected all pathogenic strains of Agrobacterium tested. PCR primers corresponding to conserved sequences in ipt, the T-DNA-borne cytokinin synthesis gene, detected only Agrobacterium tumefaciens and distinguished it from Agrobacterium rhizogenes. The vird2 and ipt primer pairs did not interfere with each other when included in the same PCR amplification, and this permitted simultaneous detection of both genes in a single reaction. One nonpathogenic Agrobacterium radiobacter strain contained vird2 but not ipt; we speculate that this strain arose from a pathogenic progenitor through a deletion in the T-DNA. The vird2 primer pair appears to be universal for all pathogenic Agrobacterium species; used together, the primer sets reported here should allow unambiguous identification of Ti plasmid DNA in bacteria isolated from soil and plants. Agrobacterium tumefaciens, a soil-borne bacterium, infects dicotyledonous plants from over 90 different families, causing crown gall disease throughout the world (12). Financial losses from the disease occur primarily at nurseries, where galled plants are discarded (23). Crown gall can also stunt mature plants because of inferior development of the root system or disruption of vascular flow in the stem. Knowledge of the ecology of Agrobacterium spp. in soil and on plants is limited, but such information is required to find better methods of managing crown gall disease. Determining the sources of the pathogen in the agricultural environment has proven difficult, despite the need to assess planting sites for the presence of pathogenic agrobacteria as a way to avoid introducing susceptible plants into pathogen-infested soils. Although a large number of plant species are susceptible to A. tumefaciens, crown gall is a significant problem in a relatively small number of crops, and so infested fields may go unnoticed. In addition, pathogenic Agrobacterium strains may inhabit nursery soils for years before causing disease. Therefore, the pathogen can reside undetected in soils of locations where nurseries will be established. Thus, it is important to develop sensitive and reliable tools to detect A. tumefaciens in soils and plant material and to distinguish disease-producing strains from nonpathogenic strains. Pathogenic gall-inducing strains of Agrobacterium share a common feature which should permit their identification through DNA analysis: they contain at least one large plasmid, the tumor-inducing (Ti) plasmid (42, 44). Virulence depends upon two regions of the Ti plasmid: the transferred DNA (T-DNA) and the virulence (vir) genes. These virulence genes mediate transfer of T-DNA into infected plant cells (see references 49 and 52 for reviews), in which the T-DNA integrates * Corresponding author. Phone: (503) Fax: (503) Electronic mail address: reamw@bcc.orst.edu. Oregon State University Agricultural Experiment Station paper Contribution no E, 1995 series, from the Agricultural Research Organization. Present address: Department of Plant Pathology, Agricultural Research Organization, Volcani Center, Bet-Dagan 50250, Israel. into nuclear DNA (10, 11, 48). Expression of three T-DNA genes in the plant causes tumorous growth. Two of the T-DNA oncogenes, iaam and iaah, encode tryptophan monooxygenase and indole acetamide hydrolase; these enzymes convert tryptophan to indoleacetic acid, an auxin (20, 31, 40, 41). The third oncogene, ipt, encodes an isopentenyl transferase which converts adenosine monophosphate into isopentenyl adenosine, a cytokinin (1, 4, 8). Overproduction of these phytohormone biosynthetic enzymes results in gall formation. Other pathogenic strains of Agrobacterium cause hairy root disease, a proliferation of roots at the site of infection (35, 47). Transfer of T-DNA from the root-inducing (Ri) plasmid into plant cells occurs by the same mechanism as T-DNA transfer from tumorigenic (gall-inducing) A. tumefaciens (45). However, rhizogenic (root-inducing) Agrobacterium rhizogenes T- DNA contains rol (root locus) genes that render plant cells more sensitive to endogenous auxin (30, 34, 36, 37); in some strains, the T-DNA also contains auxin biosynthetic genes (9, 39, 47). Although Ti and Ri plasmids vary considerably among strains, they all carry similar vir genes (19, 22, 46). In many instances, T-DNA oncogenes also show similarity to each other. We used both the highly conserved vir and oncogene sequences to distinguish between pathogenic and nonpathogenic Agrobacterium strains and between tumor-inducing and root-inducing pathogenic strains. In soil, in which agrobacteria survive for long periods, avirulent Agrobacterium radiobacter strains are common, while A. tumefaciens is rarely found (7, 24). Detecting the few A. tumefaciens cells among an overwhelming population of A. radiobacter cells by microbiological methods is difficult because putative agrobacteria must be purified and tested individually for pathogenicity. PCR has facilitated detection of a variety of plant-pathogenic bacteria in environmental samples (2, 38). Pseudomonas, Xanthomonas, and Agrobacterium strains have been detected in soil by PCR (6, 13, 17, 25, 26, 32, 33). To detect specific strains of A. tumefaciens, Nesme et al. used primers corresponding to sequences between the virb and virg operons of nopaline-type Ti plasmids (25); however, octopine-type Ti plasmids lack one of these primer binding sites, and so this primer pair will detect 2879

2 2880 HAAS ET AL. APPL. ENVIRON. MICROBIOL. only a subset of A. tumefaciens strains. Nesme et al. also used primers derived from T-DNA sequences rather than vir genes. These T-DNA-derived primers detected both octopine- and nopaline-type Ti plasmids, but not an agropine-type pti (25). Schultz et al. used PCR to differentiate between strains of Agrobacterium vitis, previously known as A. tumefaciens biovar 3 (32). They selected primers from the T-DNA oncogenes, which differ sufficiently from those in other biovars of A. tumefaciens that these primers were not useful as universal detectors of virulence. Dong et al. designed primers to differentiate between virulent and avirulent strains of Agrobacterium (13). They used two primer sets, designated narrow and wide host range, from the auxin biosynthetic locus. The narrowhost-range primers detected 27 of 29 pathogenic strains and did not produce the diagnostic PCR product with any of 8 nonpathogenic strains. The wide-host-range primers identified 18 of 20 pathogenic strains, but 1 of the 3 nonpathogens tested gave a false-positive PCR product. While this paper was in preparation, Sawada et al. reported a universal primer set based on sequences from the virc operon; these primers detected 75 of 77 pathogenic strains which represented all three biovars (29). We designed PCR primers in order to detect a wide variety of pathogenic Agrobacterium strains. To do so, we based our primers on sequences that we expected to be highly conserved in all virulent strains. The T-DNA border repeats are probably the most highly conserved DNA sequences associated with virulence. As expected, the endonuclease domain of the VirD2 protein, which cleaves these border sequences, is also highly conserved (43). Therefore, we chose oligonucleotides specific for the endonuclease-encoding portion of the vird2 gene as potential universal primers capable of detecting all pathogenic strains of Agrobacterium. To distinguish tumor-inducing Agrobacterium strains from other pathogenic agrobacteria, we designed primers corresponding to conserved sequences in the ipt oncogene. We used these primer pairs to examine a broad strain collection containing A. tumefaciens, A. rhizogenes, A. vitis, and A. radiobacter. As predicted, the primers based on vird2 identified pathogenic agrobacteria from all three virulent species tested, whereas the ipt primers detected only A. tumefaciens. MATERIALS AND METHODS Bacterial strains. Table 1 lists the Agrobacterium strains used in this study. A. tumefaciens and A. radiobacter isolates came from the collections of J. H. Haas (strains with an IL prefix) and L. W. Moore (all others); all A. rhizogenes strains and one A. vitis strain (A856) came from the collection of W. Ream. Thirty-eight tumorigenic A. tumefaciens strains and three A. vitis strains were isolated from crown gall tumors on a variety of crops, flowers, and ornamentals and from nonrhizosphere soil in the vicinity of roses in commercial nurseries. The strains in this group represent all three recognized biotypes and include both octopine and nopaline opine types. The widely studied tumorigenic strain C58 was included as a reference type. Twenty-eight nonpathogenic A. radiobacter strains were isolated from the same sites as the pathogenic strains. A biological control agent, strain K84, was supplied by Alan Kerr and included as a well-characterized A. radiobacter reference type. The three A. rhizogenes strains represent the agropine type (strains 1855 and R1000) and the cucumopine type (K599); all three belong to biovar 2. A. vitis isolate A856, a limited-host-range member of biovar 3, infects mainly grapes and came from Eugene Nester (50). Template DNA preparation. We prepared templates for PCR either by total DNA extraction or by lysis of bacterial cell suspensions. To prepare total DNA from overnight broth cultures, we lysed cells in 50 mm Tris (ph 8) 20 mm EDTA 1% sodium dodecyl sulfate 500 mm NaCl and performed phenol-chloroform extractions and an ethanol precipitation as described by Garfinkel et al. (16). Next we dissolved the nucleic acids in 10 mm Tris (ph 7.5) 0.1 mm EDTA, incubated the DNA-RNA mixture with RNases A and T1, added ammonium acetate to 2.5 M, and performed a second ethanol precipitation. To prepare template DNA from cell suspensions, we took bacteria from colonies on agar plates, suspended them in water at a density of 10 8 cells per ml, and boiled them for 5 min. PCR amplification. Reaction mixtures (50 l) contained primer oligonucleotides at 0.4 M each, deoxynucleoside triphosphates at 200 M each, 1Uof thermostable DNA polymerase (Perkin-Elmer [Taq] or Epicenter Technologies [Tfl]), reaction cocktail supplied by the manufacturer (Perkin-Elmer, 10 mm Tris [ph 8.3 at 25 C], 50 mm KCl, 1.5 mm MgCl 2, 0.001% gelatin [Sigma G2500]; Epicenter, 50 mm Tris [ph 9.0 at 25 C], 20 mm ammonium sulfate, 1.5 mm MgCl 2 ), and 50 to 250 ng of purified template DNA or 5 l of boiled cell suspension. Amplification was initiated by incubation at 94 C for 1 min followed by 40 cycles at 94, 50, and 72 C for 1 min at each temperature. Upon conclusion of thermal cycling, reaction mixtures were held at 72 C for 5 min and then cooled to 10 C. The legend to Fig. 1 notes the use of a different annealing temperature, when appropriate. Primer oligonucleotides. To derive primer sequences from highly conserved regions of vird2, we aligned the published sequences from A. rhizogenes A4 and two A. tumefaciens strains: C58, a nopaline-type strain, and A6, an octopine-type strain (18, 21, 27, 43, 51). The endonuclease activity resides within the first 262 amino acids of VirD2 (424 amino acids total) (21, 51), which includes the most highly conserved portion of VirD2. The sequences that we chose are so highly conserved that each primer matches the consensus sequence perfectly, except for a single base in just one strain. We used one sense-strand oligonucleotide based upon the vird2 gene, primer A (5 -ATG CCC GAT CGA GCT CAA GT-3 [coordinates 1 to 20]), and two antisense-strand oligonucleotides, primer C (5 -TCG TCT GGC TGA CTT TCG TCA TAA-3 [coordinates 224 to 201]) and primer E (5 -CCT GAC CCA AAC ATC TCG GCT GCC CA-3 [coordinates 338 to 313]). These oligonucleotides were used in two different pairs to produce PCR products of 338 bp (A-E ) and 224 bp (A-C ). The ipt primer sequences come from a somewhat less conserved region, and so we used mixed oligonucleotides; the sequence of the sense-strand primer, CYT, was 5 -GAT CG(G/C) GTC CAA TG(C/T) TGT-3 (coordinates 8867 to 8884 in reference 3), and the sequence of the antisense-strand primer, CYT, was 5 - GAT ATC CAT CGA TC(T/C) CTT-3 (coordinates 9293 to 9276 in reference 3). This primer pair yields a 427-bp PCR product. DNA analysis. We tested each primer pair with every strain listed in Table 1; each reaction was repeated at least twice. PCR products (18- l aliquots) were subjected to electrophoresis through 2% NuSieve 3:1 agarose gels (a blend of 3 parts NuSieve agarose and 1 part SeaKem LE agarose; FMC BioProducts) cast in TEB buffer (90 mm Tris base, 2 mm EDTA, 90 mm boric acid [ph 8.3]) with 500 ng of ethidium bromide per ml. A 100-bp DNA ladder (Gibco/BRL) provided molecular weight standards. Gels were photographed under UV light. False-negative and -positive reactions. To be a useful tool for ecological studies, PCR must reliably detect the target DNA. Also, PCR amplifications must not produce spurious products similar in size to the diagnostic product. For most strains, duplicate PCRs yielded identical results that confirmed our predictions based on virulence data and prior species assignments. We resolved instances in which two replicates gave conflicting results by testing the template at least once more. We observed 12 to 15% false-positive and 7% false-negative reactions. RESULTS PCR analysis of pathogenic and nonpathogenic agrobacteria. We expected that templates derived from A. tumefaciens would produce a 338-bp amplification product with the vird2 primers and a 427-bp product with the ipt primers. Figure 1 shows products of these sizes synthesized from three A. tumefaciens templates, which were from strains I38/83, B21/90, and B36b/83. In contrast, an A. radiobacter template, from strain T20/73, did not yield a detectable PCR product. Thus, these primer pairs appeared to be suitable for screening our diverse collection of agrobacteria because they detected pathogens isolated on two continents and produced precisely the products expected; in addition, these primers did not amplify a control template extracted from a nonpathogenic strain. Correlation of PCRs with virulence. The vird2 and ipt primers exhibited high specificity. vird2-primed PCR detected every pathogen examined, i.e., 38 A. tumefaciens strains collected from 13 host species on two continents, 3 A. rhizogenes strains, and 3 A. vitis strains (Table 1). The ipt primer pair amplified 427-bp products from all 38 A. tumefaciens strains and 2 A. vitis strains, but the limited-host-range A. vitis (strain A856) and A. rhizogenes templates did not yield this product, as expected (Table 1). Of the 29 A. radiobacter (nonpathogenic) strains tested, none yielded a 427-bp product with the ipt primer pair (Table 1); 1 strain, T3/73, gave a 338-bp PCR product with the vird2 primers (Fig. 2). We repeated virulence assays and PCRs

3 VOL. 61, 1995 UNIVERSAL PCR PRIMERS FOR AGROBACTERIUM STRAINS 2881 TABLE 1. Agrobacterium strains: correlation of virulence with vird2 and ipt genes Species and strain Source PCR product with the following primers: vird2 ipt A. tumefaciens A20/75 Cherry A329/75 Cherry B21/90 Blackberry B36b/83 Apple B49c/83 Apple D10/91 Pear D10b/87 Apple F4/91 Chrysanthemum F9/91 Chrysanthemum F22/91 Chrysanthemum H21/83 Apple I22/85 Cherry I38/83 Apple J1/75 Apple J36/83 Apple M2/73 Birch P1/75 Dahlia Q51 Cherry RR5 Red raspberry W1/73 Euonymus IL2 Aster IL4 Rose IL5 Soil near rose IL6 Soil near rose IL12 Soil near rose IL13 Soil near rose IL15 Rose IL16 Almond IL22 Soil near rose IL23 Soil near rose IL24 Soil near rose IL25 Soil near rose IL30 Rose IL32 Soil near rose IL33 Soil near rose IL34 Soil near rose IL35 Soil near rose C58 R. N. Goodman A. rhizogenes K599 S. K. Farrand 1855 S. K. Farrand R1000 E. W. Nester A. vitis A856 Grape a IL20 Grape IL40 Grape A. radiobacter A3/90 Chestnut D14/91 Pear G1b/85 Apple H2/72 Azalea L11/73 Soil near plum N6/73 Raspberry P10/73 Baby s breath T3/73 Rose T20/73 Rose IL1 Grape IL3 Rose IL7 Soil near rose IL8 Soil near rose Continued TABLE 1 Continued Species and strain Source PCR product with the following primers: vird2 ipt IL9 Soil near rose IL10 Soil near rose IL11 Soil near rose IL14 Rose IL17 Almond IL21 Grape IL26 Soil near rose IL27 Soil near rose IL28 Soil near rose IL29 Soil near rose IL31 Rose IL36 Soil near rose IL37 Soil near rose IL38 Soil near rose IL39 Soil near rose K84 A. Kerr a Strain obtained from E. W. Nester. several times with T3/73 and confirmed this unusual result. This nonpathogen appears to have lost its ipt locus and virulence but to have retained the vird2 gene. With this one exception, the presence of a vird2 gene correlated with pathogenicity. All gall-inducing strains contained a detectable ipt gene, except for the A. vitis strain A856. Thus, PCR using the vird2 primer pair was a reliable means to detect all the pathogenic agrobacteria in our diverse sample of strains, and PCR with the ipt primers allowed us to distinguish A. tumefaciens from other pathogenic agrobacteria. Simultaneous PCR with vird2 and ipt primers. To increase the utility of PCR for detection of pathogenic Agrobacterium strains, we performed PCR amplifications with two primer pairs, vird2 and ipt, in the same reaction mix. This allowed us to simultaneously look for a highly conserved virulence gene and a T-DNA oncogene, which allowed us to distinguish A. tumefaciens from other Agrobacterium strains with a single PCR. We tested a vird2 primer pair (A-E or A-C ) inthe same reaction with the ipt primers. Figure 3 presents the results obtained with A. tumefaciens IL20. The reaction mixture that contained ipt and the A-C primers yielded only the two anticipated products of 427 and 224 bp. The PCR with ipt and the A-E primers amplified abundant quantities of the expected products of 427 and 338 bp. The mixture also included an abundant species larger than 600 bp and trace amounts of molecules 400 and 150 bp long. Thus, neither of the vird2 primer pairs interfered with the ipt primers during simultaneous PCR DNA synthesis, and we detected both vird2 and ipt genes with a single PCR amplification. Sensitivity of PCRs. To estimate the number of bacterial cells required to produce a detectable PCR product, we used serial dilutions of A. tumefaciens cell suspensions in standard PCRs with either A-E or ipt primer pairs. Reaction mixtures that contained 150 to 200 cells produced the expected products with each primer set (data not shown). DISCUSSION The utility of PCR for detecting specific microorganisms in environmental samples depends on the consistent occurrence of unique DNA sequences in the target organism. We developed oligonucleotide primers from a conserved gene that we

4 2882 HAAS ET AL. APPL. ENVIRON. MICROBIOL. FIG. 1. PCR with vird2 and ipt primers and A. tumefaciens and A. radiobacter templates. The annealing temperature for these PCRs was 43 C. expected to occur on the Ti plasmid in all pathogenic Agrobacterium strains but not in A. radiobacter (nonpathogenic) isolates. The vird2 primer pair detected all 44 pathogenic Agrobacterium strains tested, including 3 A. rhizogenes strains and 3 A. vitis strains. Of 29 A. radiobacter isolates examined, only 1 (T3/73) contained the vird2 locus. This strain did not carry the ipt gene, and we speculate that a deletion in the T-DNA of T3/73 may have removed ipt and other oncogenes necessary for tumorigenesis. The ipt (cytokinin biosynthesis) locus should occur in A. tumefaciens strains but not in A. rhizogenes, which causes a cytokinin-independent disease known as hairy root. We detected the ipt gene in 38 A. tumefaciens strains and 2 A. vitis strains but not in 3 diverse A. rhizogenes strains. The absence of a detectable ipt gene in limited-host-range A. vitis A856 was not surprising, because a large deletion has removed the 5 coding region of its ipt gene, thereby restricting the host range of this strain (50). None of the 29 A. radiobacter strains contained a detectable ipt gene. Other cytokinin-producing bacteria, for example, Pseudomonas syringae pv. savastanoi (28) and Erwinia herbicola pv. gypsophilla (see accession no. Z46375 in the EMBL database), reside in soil. We used the Wisconsin Genetics Computer Group s PRIMERS program to predict whether PCRs with FIG. 2. PCR analysis of an unusual nonpathogenic strain (T3/73). FIG. 3. Simultaneous PCR with vird and ipt primers. Template DNA was from A. tumefaciens IL20. A strong extraneous band appeared at approximately 730 bp in the A-E lane; the source of this band is unknown, although it did not interfere with our ability to detect vird and ipt. our ipt primers and DNA templates from other ipt-containing bacteria would yield products of approximately the same size (427 bp) as those amplified from A. tumefaciens. This program did not find sequences in the GenBank database, except for those of A. tumefaciens, that would allow our ipt primers to produce a product that could be mistaken for a genuine Ti plasmid-borne ipt gene. Thus, other cytokinin-producing species should not interfere with detection of A. tumefaciens in soil samples. PCR analysis of strains from our collection has shown that the primer sets that we chose are useful for detecting a great variety of pathogenic Agrobacterium strains. The vird2 primers detected all pathogenic (tumorigenic and rhizogenic) strains but only one nonpathogen; the ipt primers detected only tumorigenic strains and not rhizogenic strains or nonpathogens. In addition, the vird2 and ipt primers did not interfere with each other when included in the same PCR amplification. Thus, a single PCR was sufficient to distinguish pathogens from nonpathogens and cytokinin-producing pathogens (A. tumefaciens and wild-type A. vitis) from cytokinin-independent pathogens (A. rhizogenes and limited-host-range A. vitis). We have developed PCR primers that proved useful for characterization of diverse Agrobacterium isolates; because the vird2 primers detected all 44 pathogens tested, they may be universal primers for identification of most or all pathogenic agrobacteria. A. radiobacter strains may arise from A. tumefaciens through loss of the Ti plasmid, deletion of critical T-DNA or virulence genes, or other mutations in chromosomal or Ti plasmid genes necessary for tumorigenesis. Extended contact of A. tumefaciens with vir-inducing phenolic compounds can lead to mutations in the vir region that render the bacterium nontumorigenic (5, 14, 15). The presence of vird2 and the absence of ipt in one of our A. radiobacter strains (T3/73) suggest that it arose from A. tumefaciens through a deletion in the T-DNA. The presence of one putative mutant Ti plasmid among 29 randomly selected A. radiobacter strains suggests that nonpathogenic strains arise by Ti plasmid alteration, rather than loss, at a significant frequency. In this study, we tested pure bacterial cultures. However, using DNA extracted from soil, Picard et al. described methods that allow PCR amplification of sequences found in nopaline-

5 VOL. 61, 1995 UNIVERSAL PCR PRIMERS FOR AGROBACTERIUM STRAINS 2883 type A. tumefaciens (26). They reported detection of as few as 1,000 A. tumefaciens cells in a 100-mg soil sample. We detected as few as 150 A. tumefaciens cells in one PCR mixture. Thus, detection of agrobacteria does not require pure cultures, because PCR can detect specific DNA sequences among a large excess of nontarget DNA. Continual advances in DNA extraction methods and PCR sensitivity will certainly lower the detection limit. The primer sets reported here should allow unambiguous identification of Ti plasmid DNA in soil and plant samples. ACKNOWLEDGMENT This work was supported by USDA grant to W. Ream. REFERENCES 1. Akiyoshi, D. E., H. Klee, R. M. Amasino, E. W. Nester, and M. P. Gordon T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc. Natl. Acad. Sci. USA 81: Atlas, R. M., and A. K. 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