Final Report for GNC05-053

GNC05-053 (project overview)

Project Type: Graduate Student

Funds awarded in 2005: $9,794.00

Projected End Date: 12/31/2006

Grant Recipient: University of Illinois

Region: North Central

State: Illinois

Graduate Student:

Carlos Pavon

Faculty Advisor:

Mohammad Babadoost

University of Illinois

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Project Information

Summary:

A procedure was developed to quantify Phytophthora capsici oospores in soil by combining a sieving-centrifugation method and a real-time quantitative polymerase chain reaction (QPCR) assay. Five soil samples representing three different soil textures were infested with oospores of P. capsici to produce 10**1, 10**2, 10**3, 10**4, or 10**5 spores per 10 g of air-dried soil. Each 10-g sample of infested soil was suspended in 400 ml of water and then passed through 106-, 63-, and 38-μm metal sieves. The filtrate was then passed through a 20-μm mesh filter. Materials caught on the filter were washed with water into two 50-ml centrifuge tubes and spun for 4 min (900 × g). The pellet was suspended in 30 ml of 1.6 M sucrose solution and centrifuged for 45 s (190 × g). The supernatant was passed through the 20-μm mesh filter. The sucrose extraction process of oospores was repeated five times to maximize oospore extraction. Materials caught on the 20-μm mesh filter were washed with water into a 50-ml tube and spun for 4 min (900 × g). The pellet was suspended in 1 ml of water, and the number of oospores was determined with a haemocytometer. The relationship between number of oospores recovered from the soil and number of
oospores incorporated into the soil was Ŷ = –0.95 + 1.31X – 0.03X2 (R2 = 0.98), in which Ŷ =
log10 of number of oospores recovered from the soil and X = log10 of number of oospores incorporated into the soil. The oospores were germinated after treatment with 0.1% KMnO4 solution for 10 min to induce germination. On the basis of the detection of ribosomal DNA, a QPCR method for P. capsici oospores was developed. PCR inhibitors were eliminated by extracting oospores from the soil by sieving-centrifugation. DNA was extracted and quantified from P. capsici oospores with suspensions of 10**1, 10**1.5, 10**2, 10**2.5, 10**3, 10**3.5, 10**4, 10**4.5, and 10**5 oospores per ml of water. The relationship between the DNA quantities and number of P. capsici oospores was Ŷ = –3.57 – 0.54X + 0.30X2 (R2 = 0.93), in which Ŷ = log10 (nanogram of P. capsici DNA) and X = log10 (number of oospores). The relationship between the quantity of DNA of P. capsici oospores recovered from the soil and the number of oospores incorporated into the soil was determined by Ŷ = –3.53 – 0.73X + 0.32X2 (R2 = 0.955, P < 0.05), in which Ŷ = log10 (DNA quantity of P. capsici oospores recovered from the soil) and X = log10 (number of P. capsici oospores incorporated into the soil). Utilizing the sieving-centrifugation and QPCR methods, oospores of P. capsici were quantified in soil samples collected from commercial fields.

Introduction:

Among all states, Illinois ranks first in pumpkin production (Babadoost & Islam, 2003). Approximately 20,000 acres of farmland in Illinois are planted to pumpkins annually. More than 90% of the commercial processing pumpkins (Cucurbita moschata) in the United States (US) are produced on approximately 10,000 acres and processed in Illinois (Babadoost & Islam, 2003). Jack-o-lantern pumpkins are produced in more than 10,000 acres throughout Illinois. In addition, approximately 10,000 acres in Illinois are in production of cucumbers, gourds, melons, squashes, watermelons, eggplants, peppers, and tomatoes (Babadoost & Islam, 2003). Peppers are produced in about 1,000 acres. Also, considerable amounts of these vegetables are produced in home gardens throughout the state.

Phytophthora blight of cucurbits, caused by Phytophthora capsici Leonian, is one of the most serious threats to production of cucurbits, pepper, and eggplant worldwide. P. capsici is a soil-borne oomycete that infects more than 50 plant species in 15 families (Erwin and Ribeiro. 1996; Tian and Babadoost, 2004). It can infect plants at any stage of growth, causing seedling damping-off, crown rot, foliage blight, and fruit rot. Phytophthora blight can cause yield losses of up to 100% in cucurbits and pepper fields (Babadoost and Islam, 2003; Hausbeck and Lamour, 2004). At present, there is no long-term sustainable solution for disease management, however, a combination of cultural practices, fungicide application, and genetic resistance can be used to minimize the damages caused by P. capsici to vegetable crops (Babadoost and Islam, 2003; Hausbeck and Lamour, 2004).

P. capsici is a heterothallic organism in which two compatible mating types, designated as A1 and A2, are needed for sexual reproduction. The sexual spore of P. capsici is the oospore, which is the primary source of inoculum and the overwintering propagule in the soil (Erwin and Ribeiro. 1996). A reliable method for quantifying P. capsici oospores in soil is needed to assess survival of the pathogen and the potential for disease development in the field. Methods used for assessing density of P. capsici in soil have been primarily dilution plating and baiting assays but, these methods are time-consuming and the accuracy of the assays is questionable (Erwin and Ribeiro. 1996; Silvar et al., 2005).

Sucrose-centrifugation and sieving is basically a method that separate particles based on their density and size. This method has been used to extract propagules of mycorrhizal fungi (Smith and Skipper, 1979), teliospores of Tilletia species (Babadoost and Mathre, 1998), and nematodes (Jenkins, 1964) from soil.

Silvar et al. (2005) used a polymerase chain reaction (PCR)-based method to detect P. capsici in soil. The protocol they used was for detecting the pathogen only and the method cannot determine quantity of P. capsici oospores in soil. Also, they reported the presence of PCR inhibitory factors in the DNA extracts for molecular detection of plant pathogens in soil (Van de Graaf, 2003). Real-time quantitative polymerase chain reaction (QPCR) is a relatively new molecular technique that has been used to quantify nematodes (Cao et al., 2005), viruses (Delanoy et al., 2003), bacteria (Bach et al., 2003), and fungal plant pathogens (Hayden et al., 2004: Silvar et al., 2005).

The objectives of this study were: 1) to develop a reliable method for quantifying oospores (surviving bodies of P. capsici) in soil (Pavon and Babadoost, 2006a,b,c); 2) to determine survival of P. capsici oospores in soil; and establish effective cropping rotations for management of P. capsici in commercial fields.

References:

Babadoost, M. and S. Z. Islam. 2003. Fungicide seed treatment effects on seedling damping-off of pumpkin caused by Phytophthora capsici. Plant Dis. 87: 63–68.

Babadoost, M. and D. E. Mathre. 1998. A method for extraction and enumeration of teliospores of Tilletia indica, T. controversa, and T. barclayana in soil. Plant Dis. 82: 1357–1361.

Bach, H. J., I. Jessen, M. Schloter, and J. C. Munch. 2003. A TaqMan-PCR protocol for quantification and identification of the phytopathogenic Clavibacter michiganensis subspecies. J. Microbiol. Meth. 52: 85–91.

Cao, A. X., X. Z. Liu, S. F. Zhu, and B. S. Lu. 2005. Detection of pinewood nematode, Bursaphelenchus xylophilus, using a real-time polymerase chain reaction assay. Phytopathology 95: 566–571.

Delanoy, M., M. Salmon, and J. Kummert. 2003. Development of real-time PCR for rapid detection of episomal Banana streak virus (BSV). Plant Dis. 87: 33–38.

Erwin, D. C. and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society Press, St. Paul, MN.

Hausbeck, M. K. and K. H. Lamour. 2004. Phytophthora capsici on vegetable crops: research progress and management challenges. Plant Dis. 88: 1292–1303.

Hayden, K. J., D. Rizzo, J. Tse, and M. Garbelotto. 2004. Detection and quantification of Phytophthora ramorum from California forest using a real-time polymerase chain reaction assay. Phytopathology 94: 1075–1083.

Jenkins, W. R. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Dis. Reptr. 48: 692.

Malvick, D. K., and E. Grunden. 2005. Isolation of fungal DNA from plant tissues and removal of DNA amplification inhibitors. Molec. Ecol. 5: 958–960.

Pavon, C., and Babadoost, M. 2006a. Detection and quantification of Phytophthora capsici oospores in soil with real-time quantitative polymerase chain reaction. Phytopathology 96:S91, In the Abstracts of papers of joint meeting of APS, CPS, and MSA. July 29-August 2, 2006, Quebec City, Canada.

Pavon, C., and Babadoost, M. 2006b. Determining density of Phytophthora capsici oospores in soil. Pages 507-514 in Proceedings of Cucurbitaceae 2006, September 17-21, 2006, North Carolina State University, Asheville, NC, USA.

Pavón, C.F., Babadoost, M., and Lambert, K.N. 2006c. Quantification of Phytophthora capsici oospores in soil by sieving-centrifugation and real-time polymerase chain reaction. Plant Dis. 90: (in review).

Silvar, C., J. Diaz, and F. Merino. 2005. Real-time polymerase chain reaction quantification of Phytophthora capsici in different pepper genotypes. Phytopathology 95: 1423–1429.

Smith, G. W. and H. D. Skipper. 1979. Comparison of methods to extract spores of vesicular-arbuscular mycorrhizal fungi. Soil Sci. Soc. Am. J. 43: 722–725.

Tian, D. and M. Babadoost. 2004. Host range of Phytophthora capsici from pumpkin and pathogenicity of isolates. Plant Dis. 88: 485–489.

Van de Graaf, P., A. K. Lees, D. W. Cullen, and J.M. Duncan. 2003. Detection and quantification of Spongospora subterranea in soil, water and plant tissue samples using real-time PCR. Eur. J. Plant Pathol. 109: 589–597.

Project Objectives:

Short-term outcomes of this research were to develop of reliable method for detection and quantification of Phytophthora capsici in soil. Intermediate- and long-term outcomes of this study were to determining survival of P. capsici in soil, establishing effective cropping rotations, reducing use of pesticides, establishing effective strategies for management of one of the most destructive diseases of vegetables, and increasing profitability of vegetable production; thus, establishing sustainable vegetable production in the North Central region.

Cooperators

Click linked name(s) to expand/collapse or show everyone's info

Mohammad Babadoost
babadoos@uiuc.edu
Associate Proffesor
Dept. Crop Sciences, University of Illinois at U-C
1102 S. Goodwin Ave N-533
Urbana, IL 61801
(217) 333-1523 (office)

Research

Materials and methods:

MATERIALS AND METHODS

In vitro oospore production. Isolates of P. capsici were collected from infected pumpkin and zucchini plants in Illinois. The collected isolates were paired with P. capsici isolates A1 (15427) and A2 (15399) obtained from the American Type Culture Collection. Isolates with high oospore production ability were selected. Oospores were produced in 250-ml glass flasks containing 30 ml of V8-CaCO3 medium. The flasks were incubated at 24°C in darkness for 2 mo. Then, the oospores were harvested by blending the culture at full speed in a blender (Hamilton Beach Mo. 52250, Southern Pines, NC) for 90 s. The suspension in the blender was passed through 63 and 38 µm metal sieves and a 20-µm Spectra/Mesh nylon filter (Spectrum, Inc, Houston, TX). Oospores caught on the 20-µm were collected and used in the following studies.

Extraction and enumeration of P. capsici oospore in soil. Predetermined quantities of P. capsici oospores were added to five agricultural soils. Samples from the soil types were air-dried at room temperature on a laboratory bench for 14 days and passed through a 2-mm sieve. The soil samples were autoclaved twice, each at 157°C for 1 h. Soil samples were artificially infested by adding 1-ml aliquot of the suspension of P. capsici oospores to 10 g of air-dried soil and thoroughly mixed. The artificially infested soil samples were estimated to have 101, 102, 103, 104, and 105 P. capsici oospores per 10 g of air-dried soil. A control sample of each soil type, without any oospore incorporation was included. The oospore recovery rate from artificially infested soils at each level of oospore number and for five soil types was determined using four 10-g soil sample replicates.

Each 10-g infested soil sample was suspended in 400 ml of tap water with two drops of Tween-20 and shaken for 15 min. The soil suspension was then passed through 106, 63, and 38 µm metal sieves, and the sieves were washed using a sprinkler with a gentle stream of water and the filtrate was collected. This suspension (approximately 2 liters) was then passed through the 20-µm mesh filter. The materials caught on the 20-µm mesh were washed into two 50-ml centrifuge tubes and spun for 4 minutes (900 x g) using a bench-top centrifuge (Centra-CL2, International Equipment Company, Needham Height, MA). The supernatant was discarded and the pellet was suspended in 30 ml of 1.6 M sucrose solution. This suspension was then centrifuged for 45 s (190 x g) and the resulting supernatant was passed through the 20-µm mesh. The pellet was resuspended in the sucrose solution and centrifuged again (45 s, 190 x g). This procedure was repeated five times to maximize oospore recovery from soil. The materials caught on the 20-µm mesh were washed into a 50-ml centrifuge tube and spun for 4 min (900 x g). The pellet was resuspended in 1 ml of distilled water. The oospores were enumerated on a spore-counting chamber using light microscopy.

Eight commercial fields with a history of Phytophthora blight were sampled for extraction and enumeration of P. capsici oospores. In each field, 20 sub-samples of soil were taken from 0-20 cm deep from approximately 0.4 ha area using a soil probe. The sub-samples from each field were mixed thoroughly and stored at 4°C until they were assayed. Five 10-g soil samples from each field were processed for extraction of oospores using the above-mentioned sieving-centrifugation method.

Oospore germination. Phytophthora capsici oospores extracted from both artificially and naturally infested soils were germinated on the semi-selective culture medium PARP (36) in Petri plates. To induce oospores germination, the spores were first treated with 0.1% KMnO4 for 10 min (1,11). After the treatment, oospores were washed three times with sterile-distillated water and plated onto PARP medium. Four culture plates, each with 50 oospores, were included. The plates were incubated at 24°C in darkness for nine days and the percentage of germinated spores was determined. Oospores of P. capsici extracted from naturally infested soils from commercial fields were identified from oospores of other Oomycete species based on sporangial morphology. Plugs of 5-mm-diameter of the colonies of germinated oospores were transferred onto lima bean agar (LBA), grown at 24°C for 5 days, and the culture was examined for identification of the species using light microscopy. Also, representative cultures of P. capsici were grown in lima bean broth and tested for identification using the PCR protocol described by Islam et al. (23).

DNA extraction. Oomycete and fungal isolates used in this study were maintained on LBA slants at room temperature. Mycelium for DNA extraction was prepared according to the method described by Islam et al. (23). DNA was extracted from mycelium and oospores of P. capsici using the protocol reported by Gao et al. (16). An attempt was made to extract DNA of P. capsici directly from soil containing P. capsici oospores using the protocol described by Filion et al. (14). The DNA concentrations were measured using a Genesys 10 spectrometer (Thermo Spectronic, Rochester, NY).

Primers and probe used. Sequences of the primers and probes were designed with Primer Express 2.0 software (Applied Biosystems, Forest City, CA). Primers were synthesized at the W. M. Keck Center for Functional and Comparative Genomics, University of Illinois at Urbana-Champaign. The probes were commercially synthesized by Applied Biosystems (Foster City, CA). At the 5’ end, the probes were labeled with a fluorescent reporter dye, the P. capsici probe (Pcap-q-1) with the 6-carboxyfluorescein (6-FAM), and the Pythiaceae species probe (Phyt-q-2) with VIC (chemical name not released by Applied Biosystems). At the 3’ end, both Pcap-q-1 and Phyt-q-2 probes were labeled with the minor groove binding nonfluorescent quencher (MGBNFQ).

The sequences of ribosomal DNA of P. capsici are aligned with the sequences of ribosomal DNA of some other Phytophthora species. The sequences of the ribosomal DNA genes of P. capsici isolates PC2, PC25, PP1, PP2, and 43-2 were determined by Tian and Babadoost (40). Sequences of isolates of P. capsici in the GenBank database were checked for the analysis and their accession numbers were AF125008, AF129888, AF332266, AJ555612, and AY742735. Pcap-q-1 primers were: forward, 5’-GGA ACC GTA TCA ACC CTT TTA GTT G-3’; reverse, 5’-CGC CCG GAC CGA AGT C-3’; and probe, 5’-6FAM-TCT TGT ACC CTA TCA TGG CG-MGBNFQ-3’.

The endogenous control primers and probe were designed using sequences of ribosomal DNA genes of Phytophthora and Pythium species (GenBank accession no. AF125008, AF129888, AF332266, AJ555612, AY742735, AY590277, AY423301, AY590275, AF266779, AY26999, AY986961, DQ059572, AB160845, and AF330171). Pyth-q-2 primers were: forward, 5’- GCA ACT TTC AGC AGT GGA TGT C-3’; reverse, 5’-TGC AAT TCG CAT TAC GTA TCG-3’; and probe, 5’-VIC-CGA TGA AGA ACG CTG CG-MGBNFQ-3’.

Real-time QPCR amplification. The QPCR assays were conducted in a 96-well plate format with the ABI PRISM 7000 Sequence Detection System instrument and the software from PE Applied Biosystems (Foster City, CA). The manufacturer’s instructions were followed, except that 25-µl reaction mixtures (instead of 50 µl) were used (17). Thermal cycling conditions consisted of 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C, in addition to a 2-min pre-incubation at 50°C. QPCR was performed to test the specificity of the Taqman probes on the pathogen DNA. Five P. capsici isolates and other oomycete and fungal species were used for the absolute QPCR assays. DNA extracts were diluted to 0.1 ng/µl prior to carrying out the QPCR tests.

The ΔCt PCR inhibition test, developed by Gao et al. (16), was used to test whether DNA extracts from oospores and soil suspensions were free of PCR-inhibitor. This protocol was based on developed QPCR assays of β-actin gene from Meloidogyne javanica (Mj-ba) (35). The purpose of these tests was to measure the amplification of Mj-ba when exposed to possible inhibitory factors in the DNA extract samples. In this procedure, primers and probe for Mj-ba (35) were used instead of primers and probe for P. capsici. Amounts of β-actin DNA used were 7.5 × 10-4 (Test 1) and 1.7 × 10-5 ng (Test 2).

QPCR assays for quantifying oospores. A QPCR assay was developed to determine the relationship between number of oospores and DNA quantities of P. capsici. Autoclaved soil samples were infested with P. capsici oospores, as previously described. Then, the oospores were extracted from soil using the sieving-centrifugation method. A control soil sample with no oospores-incorporated was included in the test. The numbers of P. capsici oospores were adjusted to be 10**1, 10**1.5, 10**2, 10**2.5, 10**3, 10**3.5, 10**4, 10**4.5 and 10**5 spores per sample for DNA extraction. Six replicates were included at each level of oospore numbers. The control sample (extract of soil with no oospore incorporated) and a negative control (molecular pure water) were included in each run. A subset of 10-fold dilution series of the total DNA extracted from P. capsici oospore samples, spanning from 1 × 100 to 1 × 10**-4 ng, was incorporated into each QPCR assay in triplicate to generate a standard curve.

QPCR assays for DNA quantities of P. capsici oospores in field soils. Oospores extracted from commercial field soils were used to determine absolute quantities of P. capsici DNA. DNA extract from oospores separated from soil and a negative control (molecular pure water only), in triplicate, were included in the test. A subset of 10-fold dilution series of the total DNA from P. capsici oospores spanning from 1 × 100 to 1 × 10**-4 ng, was incorporated into each absolute QPCR assay in triplicate to accurately determine the amount of DNA from P. capsici oospores from soil.

Relative QPCR was used to determine relative DNA quantities of P. capsici oospores extracted from soil. The P. capsici DNA (target DNA) was quantified relative to the DNA of species of Pythiaceae family (endogenous control). For determining quantity of the relative DNA, the ΔΔCt method (29) was applied. To use ΔΔCt method, a validation test was carried out to determine if the amplification efficiency of the target DAN (Pcap-q-1) and the endogenous control DNA (Phyt-q-2) were approximately equal. The calibrator used a concentration of 1 × 10**-2 ng ribosomal DNA of P. capsici and 1 × 100 ng ribosomal DNA of present species of Pythiaceae family. The relative quantity of P. capsici DNA was determined by the formula 2-ΔΔCt, as described by Livak and Schmittgen (29).

Data analysis. The data collected from the different experiments were analyzed using the procedures for regression and GLM models of SAS (SAS Institute, Cary, NC). The oospore extraction data were log-transformed for the independent variable (log10X) and the dependent variable (log10Y) for normality and stabilizing the variances.

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Research results and discussion:

RESULTS

Extraction and enumeration of oospores in soils. The isolates of P. capsici used for oospore production yielded from 4.41 x 10**5 to 6.72 x 105 (mean 5.56 x 10**5) oospores per plate after 2 mo. Thus, enough oospores of P. capsici were produced in-vitro to conduct necessary experiments. Phytophthora capsici oospores were successfully recovered from all five artificially infested soil types when 10**1, 10**2, 10**3, 10**4, and 10**5 spores per 10 g air-dried soil were incorporated into the soil. No oospore was recovered from samples without oospore incorporation. The recovery rates of P. capsici oospores were 21.4, 35.5, 51.3, 64.6, and 70.8% from soil samples containing 10**1, 10**2, 10**3, 10**4, and 10**5 oospores per 10 g, respectively. Overall, 51% of the oospores incorporated into the soil were recovered. There was not a significant difference in oospore recovery among the soil types. The relationship between the number of oospores recovered from soil and the number of oospores incorporated into the soil was Ŷ = -0.95 + 1.31X - 0.03X2 (R2 = 0.98, P<0.001), where Ŷ = log10 of number of oospores recovered from soil, and X = log10 of number of oospores incorporated into soil. There was no significant difference in oospore recovery between soil samples containing 104 and 105 oospores per 10 g. The percentage of oospores recovered from soil samples with 101 oospores per 10 g was significantly lower than the percentage of oospores recovered at all of the other levels. Also, the percentage of oospores recovered from soil samples with 10**2 oospores per 10 g was significantly lower than the percentage of oospores recovered from the samples with 103, 104, and 105 oospores per 10 g. Similarly, the percentage of oospores recovered from soil samples with 10**3 oospores per 10 g was significantly lower than the percentage of oospores recovered from samples with 10**4 and 105 oospores per 10 g. Oospores were extracted from soil samples collected from eight commercial fields in Illinois. The number of oospores recovered from commercial fields ranged from 680 to 2,800 per 10 g of soil. The average diameter of oospores extracted was 27.8 µm. There was no significant difference in diameter of oospores recovered from the different fields. Percent oospore germination ranged from 40.9 to 64.3% (mean 54.6%). The percentage of germinated oospores identified as P. capsici ranged from 9.2 to 18.5% (mean 14.7%). Identification of P. capsici from other Oomycete species based on the oospore morphology was not possible. Specificity and sensitivity of QPCR. All five isolates of P. capsici tested were detected by utilizing the QPCR protocol with Pcap-q-1 primers. No amplification of DNA was detected from the extract from soil samples without P. capsici, sterile water; isolates of P. sojae, P. infestans, and Pythium species; or other fungal species tested. DNA of P. capsici oospores as low as 1 × 10**-4 ng was repeatedly detected. Effects of DNA extracts on β-actin amplification. DNA extracted directly from soil completely inhibited β-actin amplification. The results of the tests for determining the presence of PCR inhibition in DNA extract from oospores extracted from soil were as follows. In test 1, with β-actin DNA concentration of 7.5 × 10-4 ng, the β-actin Ct for the non-P. capsici control was 23.5, the β-actin ΔCt of the DNA extracts from oospores extracted from soil ranged from 0.0 to 0.6. In test 2, with β-actin DNA concentration of 1.7 × 10**-5 ng, the β-actin Ct for the non-P. capsici control was 28.3, and the β-actin ΔCt of the DNA extracts from oospores extracted from soil ranged from 0.0 to 0.9. The results of these tests showed that DNA extracts from oospores that were extracted from soil, using the sucrose-centrifugation method, did not contain inhibitors to PCR. QPCR quantification of P. capsici oospores in soil. Phytophthora capsici DNA was successfully extracted from oospores separated from the soil samples. The relationship between the number of oospores and P. capsici DNA quantity was Ŷ = -3.57 -0.54X + 0.30X2 (R2 = 0.93, P<0.001), where Ŷ = log10 (ng of P. capsici DNA) and X = log10 (number of oospores). The DNA quantities corresponding to 10**1, 10**1.5, 10**2, 10**2.5, 10**3, 10**3.5, 10**4, 10**4.5 and 10**5 oospores were 1.4 × 10**-4, 1.9 × 10**-4, 3.5 × 10**-4, 9.0 × 10**-4, 3.2 × 10**-3, 1.6 × 10**-2, 1.5 × 10**-1, 1.2 × 100, and 1.7 × 10**1 ng, respectively. The average absolute DNA quantity of P. capsici oospores in soil from the eight commercial fields ranged from 2 × 10**-3 ng (219 oospores) to 2.3 × 10**-2 ng (1,291 oospores) per 10 g of air-dried soil. The coefficient of variation of the Ct value for P. capsici DNA (target DNA) was 3% and for the DNA from species of Pythiaceae family (endogenous control) was 13%. The mean RQ (2-ΔΔCt) values for the eight field soils ranged from 0.22 to 3.58. DISCUSSION The sieving-density separation in sucrose solution has long been used to extract mycorrhizal and smut fungi form soil (5,9,10,22,). Also, a centrifugation technique using sucrose solution to separate nematodes from soil has been widely used (24). This study, however, is the first report of using sieving-centrifugation method for extraction of oospores of P. capsici from soil. The procedure described in this report was achieved after numerous tests with different soil types, soil amounts, sucrose concentrations, centrifugation speeds, centrifugation times, and oospore densities. The described protocol was the most efficient for maximum extraction of P. capsici oospores from soils. Utilizing the developed sieving-centrifugation method, we were able to recover P. capsici oospores from soil with a spore density as low as one spore per gram of soil. There was no significant difference in recovery rates of P. capsici oospores from all five soil types used in this study. Therefore, the equation Ŷ = -0.95 + 1.31X - 0.03X2, where Ŷ = log 10 of number of oospores recovered form soil and X = log 10 of number of oospores in the soil, can be used to quantify P. capsici oospores in naturally infested soils.
Furlan et al. (15) reported that sucrose was not a good separation medium for spores of endomycorrhizal fungi because spores become dehydrated and collapse and this may effect identification of the fungus and viability of spores. But, Ianson and Allen (22) used 2 M sucrose solution to extract spores of vesicular-arbuscular mycorrhizal fungi from soil and found that exposing the spores to sucrose for 24 h did not cause significant damage. Also, Babadoost and Mathre used 1.6 M sucrose for extracting teliospores of Tilletia spp. from soil and reported no adverse effect of sucrose solution on teliospores morphology and germination. In this study, with concentration of 1.6 M sucrose, we found neither collapse of cell wall nor reduction in germination of oospores.

The sieving-centrifugation method alone cannot be used to determine number of P. capsici oospores in soil because oospores of other Oomycete species family have similar densities to P. capsici oospores and they also are extracted along with P. capsici oospores. Utilizing the QPCR developed in this study, we able to accurately estimated number of P. capsici oospores extracted from soil. The QPCR method, however, cannot determine viability of the oospores. Germination test of the extracted oospores is required to determine whether or not the oospores in the soil are viable. Thus, by combining sucrose-centrifugation and QPCR methods developed in this study and germinating oospores extracted from soil, not only the number of P. capsici oospores in soil can be accurately estimated, but also the viability of the oospores can be determined.

Due to presence of PCR inhibitors in soil, direct assay of P. capsici oospores in soil by using QPCR procedure was unsuccessful. But, utilizing sieving-centrifugation procedures the PCR inhibitors in soil were eliminated and QPCR assays were successfully carried out. Several studies have been conducted to eliminate PCR inhibitors from nucleic acid extracts (16,31,41,42). The sieving-centrifugation procedure described in this report is an easy solution eliminating PCR inhibitors for assaying soil-borne plant pathogens using QPCR method.
QPCR is a useful method for detecting and quantifying plant pathogens, particularly non-culturable and slow-growing pathogens. The QPCR assay method developed in this study estimates number of P. capsici oospores based on the amount of detected P. capsici DNA. This QPCR method is based on the detection of ITS region of the ribosomal DNA of P. capsici. The ITS region has been reported a reliable region for specific amplification of P. capsici (23,28, 37,39). Silvar et al. (39) quantified ribosomal DNA of P. capsici in pepper plants with absolute QPCR using SYBR green primers with an amplicon of 452 bases. In our study, ribosomal DNA of P. capsici was quantified using a set of primers and a TaqMan probe with an amplicon of 74 bases. In both studies segments of the same regions of the ribosomal DNA of P. capsici were amplified. Our procedure amplified only ribosomal DNA of P. capsici, but did not detect P. sojae, P. infestans, Pythium spp., Aphanomyces euteiches, Saprolegnia monilifera, or other fungal species tested. Thus, this procedure is highly specific and sensitive for detecting P. capsici, as it detected as low as 1 × 10-4 ng of DNA.

Germination of oospores is essential for determining inoculum density of P. capsici in the soil. It should be, however, mentioned that the rate of germinated oospores may underestimate number of viable oospores of P. capsici in the soil because: (i) percentage of germination of P. capsici oospores on culture media is usually less than 50% and (ii) colonies of P. capsici originating form oospores may be outgrown by closely related oomycete sp., particularly Pythium species. Thus, further investigations may improve germination of P. capsici oospores in culture plates and enhance accuracy of estimation of viable oospores of P. capsici in soil.

The protocol developed in this study can be used for future investigations on biology and ecology of P. capsici. Also, the findings in this study could be employed to establish effective cropping systems for management of the Phytophthora blight. Moreover, the procedure reported in this paper may be adapted for evaluating the presence of other plant pathogens in soil.