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2003 Plant Management Network.
Accepted for publication 10 June 2003. Published 1 July 2003.


Detecting Orobanche minor Seeds in Soil Using PCR


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


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


Rehms, L., and Osterbauer, N. K. 2003. Detecting Orobanche minor seeds in soil using PCR. Online. Plant Health Progress doi:10.1094/PHP-2003-0701-01-HN.


The federally regulated noxious weed small broomrape (Orobanche minor Smith) (Fig. 1) is a parasite of several plant species including alfalfa (Medicago sativa L.) and red and white clover (Trifolium pratense L. and T. repens L., respectively) (4). Because of its seed size (200 to 300 mm), it is difficult to detect in harvested clover seed and in soil (3). Recently, we reported a polymerase chain reaction (PCR) assay that can detect small broomrape seed in clover seed (2). This paper reports the testing of this assay for its ability to detect small broomrape in soil.


   
 

Fig. 1. Small broomrape plants flowering in a clover field (Photo courtesy of T. Butler, Oregon Department of Agriculture).

 

Silt-loam soil was collected from a non-infested field in Marion County, Oregon (2). Fifty grams of this soil was mixed and split into five 10-g samples and 0, 1, 5, 10 and 20 small broomrape seeds were added. Each sample was placed separately in a 500-ml flask filled with a 9 M CaCl2 solution (3), mixed on an orbital shaker for 30 min at 250 rpm, and then allowed to settle overnight. The supernatant, which contained the small broomrape seeds (3), was poured through interlocked 65- and 170-mesh sieves and the sieves flushed with water. The material trapped on the 170-mesh sieve was rinsed off and then transferred to a 15-ml centrifuge tube. Each sample was then centrifuged for 30 min at 3,500 rpm, the supernatant discarded, and the pellet transferred to 2-ml tubes. The pellet was macerated at 5,000 rpm for 5 min using a Mini-Beadbeater (BioSpec Products, Bartlesville, OK) prior to DNA extraction. DNA was extracted from the pellet using spin column chromatography (UltraClean Soil DNA Isolation Kit, MoBio Laboratories, Inc., Solana Beach, CA) and the DNA amplified using the PCR assay (2). The PCR assay detected five or more seeds in a 10-g soil sample (Fig. 2). When the concentration of the CaCl2 solution was adjusted to 2.25 M and the soil sample size increased to 100 g and the experiment was repeated, the PCR assay consistently detected 10 small broomrape seeds in the 100-g soil sample (Fig. 3). The 2.25 M CaCl2 solution was used for all subsequent isolations of small broomrape seeds from soil.


 

Fig. 2. Multiplex PCR detection of seeds of small broomrape (377 bp amplicon) and the internal control (555 bp amplicon) extracted from soil with a 2.25 M CaCl2 or a 9 M CaCl2 solution. DNA from soil with 0 (lanes 1 and 2), 5 (lanes 3 and 4), and 10 (lanes 5 and 6) seeds added; small broomrape positive control (lane 7); water control (lane 8); 100 bp DNA ladder (lane L). The 2.25 M CaCl2 solution was used on the samples shown in the odd-numbered lanes and the 9 M CaCl2 solution used on samples shown in the even-numbered lanes.

 

Fig. 3. Multiplex PCR detection of seeds of small broomrape (377 bp amplicon) and the internal control (555 bp amplicon) extracted from soil collected in Clackamas County. The presence of the internal control shows that amplifiable DNA was extracted from a sample. DNA from soil samples of: 10 g (lane 1), 50 g (lane 2), 100 g (lane 3), 10 g + small broomrape seeds (lane 4), 50 g + small broomrape seeds (lane 5), 100 g + small broomrape seeds (lane 6); small broomrape positive control (lane 7); water control (lane 8); 100 bp DNA ladder (lane L).


Soil samples were collected from infested fields in Clackamas and Multnomah counties with clay and sandy-loam soils, respectively, that were previously planted with red clover (2). Cooperators collected two samples from unknown locations within a known infested field in Clackamas County. Four samples were collected from the field in Multnomah County using an Oakfield tube (Oakfield Apparatus Company, Oakfield, WI): two from an infested area and two from a non-infested area in the field (R. Worth, Oregon Department of Agriculture, personal communication). A sample consisted of five cores collected in a diamond-shaped pattern from each location in the field. Each core was taken to a depth of 16 cm.

The two Clackamas County samples were thoroughly mixed and then divided into series of 10-, 50-, and 100-g samples. Ten small broomrape seeds were added to each sample in one sample series, while another sample series was left alone. DNA was extracted as described above and the PCR assay performed. Small broomrape was detected in the 10- and 50-g artificially contaminated samples and in the non-contaminated 100-g sample (Fig. 3). The PCR reaction failed for two samples, the artificially infested 100-g sample and the non-infested 50-g sample. The presence of humic substances in soil can inhibit PCR, particularly in clay soil (1). The addition of an anti-inhibitory compound may eliminate this problem (1).

For the samples collected in Multnomah County, the topsoil (0- to 8-cm depth) and deep soil (>8-cm depth) from each location were processed separately. No samples were artificially infested with small broomrape seed. DNA was extracted from 100-g soil samples as described above and the PCR assay performed. Small broomrape DNA was detected from the two locations in the field that were previously reported as infested (Table 1). Small broomrape was detected in the topsoil at one location and in the deep soil at the other location. It was not detected from the two locations in the non-infested areas of the field.


Table 1. Results of PCR detection of small broomrape in the soil of a Multnomah County, Oregon, red clover field partially infested with small broomrape.

Sites within the field Soil sample type by depth
Top soil
(0- to 8-cm)
Deep soil
(8- to 16-cm)
Reportedly uninfesteda
First site Negative Negative
Second site Negative Negative
Reportedly infesteda
First site Positive Negative
Second site Negative Positive

a Per Richard Worth, Oregon Department of Agriculture, personal communication.


The PCR assay, when combined with the DNA extraction technique described above, detected small broomrape infestations in clay, silt loam, and sandy loam soils. These combined techniques detected 10 small broomrape seeds in a 100-g soil sample. Ideally, the soil assay would be able to detect a single small broomrape seed in soil. However, the presence of humic substances in soil can reportedly inhibit PCR (1). Further research is needed to improve the level of detection by these combined techniques.


Literature Cited

1. DeBoer, S. H., Ward, L. J., Li, X., and Chittaranjan, S. 1995. Attenuation of PCR inhibition in the presence of plant compounds by addition of BLOTTO. Nuc. Acids Res. 23:2567-2568.

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

3. Portnoy, V. H., Katzir, N., and Joel, D. M. 1997. Species identification of soil-borne Orobanche seeds by DNA fingerprinting. Pest Biochem Physiol 58:49-54.

4. United States Department of Agriculture, Animal and Plant Health Inspection Service. 2001. Code of Federal Regulations Title 7, Chapter III, Part 360.