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© 2002 Plant Management Network.
Accepted for publication 16 October 2002. Published 11 November 2002.


Detecting Single Seeds of Small Broomrape (Orobanche minor) with a Polymerase Chain Reaction


Nancy K. Osterbauer, Survey Plant Pathologist, and Lisa Rehms, Plant Disease Program Specialist, Oregon Department of Agriculture, Salem 97301-2532


Corresponding author: Nancy K. Osterbauer. nosterba@oda.state.or.us


Osterbauer, N. K., and Rehms, L. 2002. Detecting single seeds of small broomrape (Orobanche minor) with a polymerase chain reaction. Online. Plant Health Progress doi:10.1094/PHP-2002-1111-01-RS.


Abstract

The seeds of the federally-listed noxious weed and parasitic plant small broomrape (Orobanche minor Smith) are extremely small, averaging 200 to 300 µm in size. Because of its miniscule seed size, contamination of fields and seed lots by small broomrape seeds is difficult to detect and confirm via conventional methods. Complementary polymerase chain reaction (PCR) primers based upon unique sequences in the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA (nrDNA) of small broomrape were developed. The PCR amplified small broomrape DNA and did not amplify DNA from other Orobanche species with similar host ranges found in Oregon. The primers also did not amplify the DNA of red and white clover (Trifolium pratense L. and T. repens L., respectively), two agricultural hosts for this parasite. The PCR-based assay was sensitive enough to detect a single small broomrape seed.



Fig. 1. Small broomrape (courtesy of T. Butler).



In 1998, small broomrape, a parasitic plant, was reported for the first time infesting a red clover field in Oregon’s Willamette Valley (Fig. 1) (3). Previously, this parasite had been reported in the southeastern United States infesting weedy broadleaf hosts (7). Small broomrape is a federally listed noxious weed and is of regulatory significance to several other countries (11,12). This weed is a parasite on the agricultural crops alfalfa and red and white clover, as well as on other members of the Fabaceae, Asteraceae, and Apiaceae (6). Federal and international regulations require clover seed from all infested areas be free of small broomrape contamination prior to export. Despite an eradication effort, small broomrape has since spread to 22 fields (269.7 ha) in seven counties in Oregon (Fig. 2) (T. Butler and R. Worth, Oregon Department of Agriculture, unpublished data). Although the costs of testing have been temporarily offset by an emergency grant from the USDA, soon clover seed growers will have to absorb both the costs of testing and any additional seed cleaning required to prepare their product for export (J. McCulley, Oregon Clover Commission, personal communication).



Fig. 2. Distribution of small broomrape in red clover production fields in Oregon (data courtesy of T. Butler and R. Worth, Oregon Department of Agriculture). Infested (red dots) and non-infested (blue dots) fields are shown.



Small broomrape has several characteristics that create problems for both growers and seed certification and regulatory officials. Its miniscule seed size (200 to 300 µm) makes it very difficult to detect in harvested clover seed and in soil (Fig. 3) (4,10). Because small broomrape germinates and grows only in the presence of a susceptible host (8,14), field surveys must take place after planting and host germination. Also, small broomrape seeds can survive for up to 10 years in the soil (7). This combination of factors makes a rapid and reliable test for the presence of small broomrape a necessity. Thus, our goal was to develop a PCR-based assay specific for small broomrape DNA and sensitive enough to detect a single small broomrape seed.



Fig. 3. Seeds of small broomrape (small specks), red clover (RC) and white clover (WC).



Designing Specific PCR Primers

Spin column chromatography was used to extract DNA from small broomrape plants collected from the original infested field in Clackamas County, Oregon (DNeasy Plant Mini Kit, Qiagen, Inc., Valencia, CA). The ITS region of the nrDNA was amplified with the universal primers ITS5 and ITS4 as described (13). Primers ITS5 and ITS4 amplify the internal spacer regions and the 5.8S subunit of the nrDNA of numerous plants and fungi (Fig. 4) (13). Amplification was performed in a MJ Research DNA Thermal Cycler programmed for 5 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 52°C, and 3 min at 72°C, with a final extension step of 7 min at 72°C. After amplification, PCR products were purified (QIAquick Gel Extraction Kit, Qiagen, Inc., Valencia, CA) and submitted to the Center for Gene Research and Biotechnology Central Services Laboratory, Oregon State University, for sequencing using the ABI 377 automated fluorescence sequencer.



Fig. 4. Approximate locations on Orobanche minor’s nuclear ribosomal DNA of the universal primers ITS5, ITS4, NS1, and NS2, and of the O. minor-specific primers OM1 and OM2. Nuclear ribosomal genes are in blue and internal transcribed spacer regions in white.



A search of the GenBank database (1) using the complete 601 base pair (bp) ITS sequence of small broomrape (GenBank Accession No. AF437315) verified the ITS sequence was from a member of the Orobanchaceae. Comparison with known red- (GenBank Accession No. AF053172) and white clover (GenBank Accession Nos. AF154620 and AF154396) ITS sequences revealed significant differences between the three species in this DNA region. Forward (OM1, 5’ GAA CTG TGG CGA TCA CGT C) and reverse (OM2, 5’ AAA CAC GCC CCG TAA GAA G) primers were designed to specifically amplify the ITS region of small broomrape’s nrDNA using the Primer3 Program available online from the MIT Center for Genome Research at the Whitehead Institute for Biomedical Research. The primers were designed to amplify a 377 bp PCR amplicon (Fig. 4).


Testing Primer Specificity

Small broomrape plants were collected from a total of 13 infested fields in Clackamas, Columbia, Linn, Multnomah, Washington, and Yamhill counties. DNA was extracted from the plant tissues of the samples as previously described and the multiplex PCR reaction performed.

The PCR reaction was performed in 15-µL multiplex reactions using the newly designed OM1 and OM2 primers and the universal internal control primers NS1 (5’ GTA GTC ATA TGC TTG TCT C) and NS2 (5’ GGC TGC TGG CAC CAG ACT TGC) (13). NS1 and NS2 amplify a region of the Small Subunit nrDNA from a wide variety of plants, fungi, and protists (Fig. 4) (13). These universal internal control primers were added to eliminate any “false negatives” that would be caused by a failed PCR reaction. NS1 and NS2 amplify a 555 bp PCR product, significantly larger than the 377 bp PCR product produced by OM1 and OM2.

Each reaction tube contained 1× enzyme buffer (10 mM Tris-HCl pH 9.0, 50 mM KCl, 0.1% Triton®X-100), 1.5 mM MgCl2, 200 µM dNTPs, 0.4 µM of the O. minor primers (OM1 and OM2), 0.1 µM of the universal internal control primers (NS1 and NS2), 1.0 U Taq DNA polymerase, and 1 µl template (DNA). Amplification parameters were adjusted to 5 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 52°C, and 1 min at 72°C, with a final extension step of 7 min at 72°C. Previous research showed more amplification cycles were necessary for unambiguous amplification results, particularly from single Orobanche seeds (10). Amplification products were resolved on 1% (w/v) agarose gels in Tris-Borate-EDTA buffer (pH 8.3) and visualized with ethidium bromide under UV light.

The primers OM1 and OM2 successfully amplified the 377 bp PCR product from the plant tissues of all of the samples collected, including a plant from Multnomah County with a “canary yellow” phenotype (Fig. 5). The primer pair did not amplify DNA from O. californica Cham. & Schldl. and O. fasciculata Nutt., two other Orobanche spp. found in Oregon that have similar host ranges (Fig. 5). Also, primers OM1 and OM2 did not amplify red and white clover DNA (Figs. 6 and 7). However, the expected 555 bp amplicon from the universal primers NS1 and NS2 was present for O. californica, O. fasciculata, red clover, and white clover, indicating amplification did occur in these samples.



Fig. 5. Multiplex PCR demonstrating the specificity of the small broomrape-specific primers (377 bp band) and the amplification of the internal control primers (555 bp band). Orobanche minor from Multnomah (lanes 1 and 2), Clackamas (lane 4), and Columbia (lane 5) counties; O. californica (lane 3); O. fasciculata (lane 6); water control (lane 7); and 100 bp DNA ladder (L).



Fig. 6. Multiplex PCR detection of a single small broomrape seed (377 bp band) and the internal control (555 bp band) in the presence of red clover DNA. DNA from one (lane 1), five (lane 2), ten (lane 3), and twenty (lane 4) seeds of small broomrape combined with red clover DNA; red clover and small broomrape positive controls combined (lane 5); small broomrape positive control alone (lane 6); red clover positive control alone (lane 7); water control (lane 8); and 100 bp DNA ladder (L).



Fig. 7. Multiplex PCR detection of single small broomrape seeds (377 bp band) collected from different fields and of the internal control (555 bp band). Orobanche minor from two fields in Washington (lanes 1 and 5), three fields in Clackamas (lanes 2 to 4), and one field in Yamhill (lane 6) counties; red clover (lane 7); white clover (lane 8); small broomrape positive control (lane 9); water control (lane 10); and 100 bp DNA ladder (L).



With the O. minor samples, the universal band was often faint to non-existent on the gels because of primer-limited competition for the DNA template in the multiplex PCR reaction. Nonetheless, the presence of the 377 bp amplicon demonstrated amplification was successful.


Detecting Single Seeds of Orobanche minor

The small broomrape plant samples were dried and their seeds collected. The seeds were macerated either with a sterile drill bit (4) or using a Mini-Beadbeater™ (BioSpec Products, Bartlesville, OK) prior to DNA extraction. DNA was extracted as described above from 1, 5, 10, and 20 small broomrape seeds from a heavily seeded plant collected in Yamhill County. When the optimized multiplex PCR amplification reaction was performed, OM1 and OM2 successfully and consistently amplified the 377 bp PCR product from single and multiple seeds (unpublished data). The DNA extracted from single and multiple seeds was then added to DNA extracted from 10 red clover seeds (1:1 ratio, v:v) and the optimized multiplex PCR reaction performed. OM1 and OM2 successfully and consistently amplified the 377 bp amplicon from single and multiple O. minor seeds in the presence of the red clover DNA (Fig. 6). Finally, DNA was extracted from single seeds from the remaining plant samples and the multiplex PCR reaction performed. Again, the primers consistently amplified the 377 bp amplicon from the single seeds of the plant samples collected (Fig. 7).


Conclusions

The multiplex PCR reaction described above allows for the rapid and reliable identification of single seeds of small broomrape. This PCR-based assay detected DNA from all small broomrape samples collected in this study, including a sample with a distinct phenotype. The primers did not amplify DNA from other Orobanche species with similar host ranges found in Oregon. The primers also did not anneal to the DNA of red- and white clover, two hosts for small broomrape that are of agricultural significance.

Orobanche species have historically been extremely difficult to identify based on morphological characteristics (8,9,10). The plant phenotypes of the broomrapes are often dependent upon the host species being attacked. Also, although seed morphology can be used to identify broomrapes to species, these morphological characteristics are subject to change over time (10). Thus, researchers have turned to molecular methods, in particular the randomly amplified polymorphic DNA (RAPD) technique, to identify and/or distinguish Orobanche species (4,5,9,10). This technique has been particularly useful for answering questions of intra- and interspecific genetic variation (5,9). However, there are some weaknesses associated with the RAPD technique. Most notably, the molecular patterns produced from mature Orobanche plants and from single seeds may differ (4). Also, the technique does not always work with single broomrape seeds (4).

We chose to target a more highly conserved region of DNA for the development of small broomrape-specific primers. In general, the ITS region of the nrDNA of any organism shows very little variation within a species, although more sequence differences are often observed between species within a genus (e.g., 2). By basing the PCR primers on this region of DNA, we developed a simple plus/minus test for small broomrape. A specific 377 bp PCR product was produced only when small broomrape DNA was present in a sample. Also, because the primers were based on such a highly conserved region of DNA, there was no difference observed between mature plant samples and seed samples.

Small broomrape is subject to both federal and international phytosanitary regulations (11,12). Because of its regulatory status, seed certification officials in Oregon developed a test to certify red clover seed as free of small broomrape contamination (S. Elias, Oregon State University Seed Laboratory, personal communication). This test is based on the physical separation of red clover and small broomrape seeds and the subsequent identification of the small broomrape seeds by their morphological features. As mentioned previously, the morphological features of the seeds are subject to change over time (10). This could result in misidentifying the broomrape seeds to the species level or misidentifying the seeds as another contaminant. Because the PCR-based assay detects the DNA of small broomrape, samples would not be subject to these difficulties.

Small broomrape will only germinate and grow in the presence of a susceptible host species (8,14). Thus, surveying fields for this noxious weed must be delayed until after host planting and germination. Researchers in Israel have combined a technique that extracts broomrape seeds from soil with the RAPD technique to identify Orobanche species in fields prior to planting (10). By combining this soil extraction technique with the PCR-based assay described in this paper, it should be possible to survey fields for the presence of small broomrape prior to planting. This would help growers identify which fields are infested with this noxious weed and should be avoided. The combined techniques of soil extraction and PCR-based assay may also prove useful for determining the efficacy of various field treatments for small broomrape.


Acknowledgements

The authors thank T. Butler, C. Mallory-Smith, and R. Worth for providing the small broomrape samples. We also thank R. Halse for providing samples of O. californica and O. fasciculata. This research was supported by funding from the USDA Cooperative Agreement No. 01-8485-0511-CA.


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