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© 2006 Plant Management Network.
Accepted for publication 12 July 2006. Published 16 October 2006.


A Comparative Analysis of Detection Techniques Used in US Regulatory Programs to Determine Presence of Phytophthora ramorum in Camellia japonica ‘Nucio’s Gem’ in an Infested Nursery in Southern California


Russ Bulluck, Pat Shiel, and Phil Berger, Center for Plant Health Science and Technology, USDA-APHIS-PPQ, Raleigh, NC 27606; David Kaplan, Emergency and Domestic Programs, USDA-APHIS-PPQ, Riverdale, MD 20737; Greg Parra, Center for Plant Health Science and Technology, USDA-APHIS-PPQ, Raleigh, NC 27606; Wenbin Li and Laurene Levy, National Plant Germplasm and Biotechnology Laboratory, USDA-APHIS-PPQ, Beltsville, MD20705; John Keller and Munagala Reddy, Monrovia Growers, Azusa, CA 91702-1385; Nawal Sharma and Michelle Dennis, California Department of Food and Agriculture, Plant Health & Pest Prevention Services, Van Nuys, CA91402; Jim Stack, Joy Pierzynski, and Judy O’Mara, Department of Plant Pathology, Kansas State University, Manhattan, KS66506; Craig Webb, USDA-APHIS-PPQ, Department of Plant Pathology, Kansas State University, Manhattan, KS 66506; Ledare Finley and Kurt Lamour, Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN, 37996; and John McKemy and Mary Palm, USDA-APHIS-PPQ, National Identification Services, Beltsville, MD20705


Corresponding author: Russ Bulluck. Russ.Bulluck@aphis.usda.gov


Bulluck, R., Shiel, P., Berger, P., Kaplan, D., Parra, G., Li, W., Levy, L., Keller, J., Reddy, M., Sharma, N., Dennis, M., Stack, J., Pierzynski, J., O’Mara, J., Webb, C., Finley, L., Lamour, K., McKemy, J., and Palm, M. 2006. A comparative analysis of detection techniques used in US regulatory programs to determine presence of Phytophthora ramorum in Camellia japonica ‘Nucio’s Gem’ in an infested nursery in Southern California. Online. Plant Health Progress doi:10.1094/PHP-2006-1016-01-RS.


Abstract

Phytophthora ramorum (Pram) is a pathogen of regulatory concern in the USA, and accurate diagnostics is a key component in the response to potential pathogen outbreaks. Although the molecular diagnostic protocols used in regulatory programs have been evaluated using regulatory samples, to date, no direct comparison of these methods has been analyzed within a nursery setting. A block of 300 camellia plants within a California nursery known to be infested with Pram was simultaneously assayed for visual symptoms, growth medium pH, and moss presence as well as culture isolation and molecular analysis prior to plant destruction. Disease symptoms such as foliar lesions and leaf drop were recorded for each plant prior to foliar and growth medium sampling. All diagnostic assays were highly correlated with one another and disease symptoms, with nested PCR having the best correlation with symptoms, followed by Real-Time PCR then culture. No correlation with disease or diagnostic assays was observed with moss presence or medium pH. Analysis of results allowed diagnostic sensitivity and specificity of the assays to be determined and the performance of each method for diagnosis of Phytophthora spp. or Phytophthora ramorum in camellia tissues and associated potting medium could be compared.


Introduction

For the last several years, The United States Department of Agriculture (USDA) Animal Plant Health Inspection Service (APHIS) Plant Protection and Quarantine (PPQ) Emergency and Domestic Programs (EDP) has conducted a national Phytophthora ramorum (Pram) control program. The program has included sampling and testing of nursery stock in quarantined areas of California under the regulatory authority of 7 CFR 301.92 listed in the Federal Register. In 2005, USDA APHIS PPQ began regulating the interstate movement of nursery stock from all of California, Oregon, and Washington. The national program has engaged PPQ’s Center for Plant Health Science and Technology (CPHST) to ensure that the national program is based on what has been scientifically demonstrated in this pathosystem and on sound principles of science to guide regulatory and mitigation measures. Although the molecular diagnostic protocols used in regulatory programs have been evaluated and validated using regulatory samples, to date, no direct comparison of these methods has been analyzed within a nursery setting. To that end, CPHST has been involved with the evaluation, adaptation, modification, and validation of diagnostic procedures for use in the detection of P. ramorum in nursery stock.

Robust diagnostics are essential for accurate and early disease detection and effective regulatory response and to help prevent the spread of the disease-causing organism. The diagnostic tests currently in use by the Pram program are among the most intricate ever deployed in a PPQ program. A Phytophthora-specific enzyme-linked immunosorbent assay (ELISA), a nested polymerase chain reaction (PCR), a Real-Time PCR, and identification of the organism morphologically in culture are all components of diagnostic determinations used in the Pram program.

Culture of the organism from diseased plant tissues on semi-selective media such as PARP and PARP-V8 are diagnostically definitive for the presence of the organism. Phytophthora ramorum cultures are coralloid (or dendritic), have distinctive 30 to 90 mm globose terminal chlamydospores (at first hyaline then golden to cinnamon brown) embedded in the media, and semi-papillate deciduous sporangia borne at the media surface. Initial culturing on PARP requires further sub-culturing onto V8 or Carrot agar, as sporangial formation is rarely observed on PARP.

Most plant disease diagnostic laboratories have the equipment and expertise to isolate the organism, make a preliminary diagnosis, and forward the cultures to the PPQ National Identification Service (NIS) for confirmation, and a process for provisional approval of laboratories to identify P. ramorum is in place. However, culturing is labor-intensive and time-consuming, and may require 7 to 14 days for accurate identification. Furthermore, inability to culture Pram from tissue does not mean that it is not present in the plant tissue. It can be difficult to isolate Pram from some hosts, and there may be unknown effects caused by antagonistic or competing organisms. Common nursery practices such as fungicide treatment may also prevent the successful culture of Pram from nursery stock.

In order to make a diagnostic determination of the pathogen in a timely and efficacious manner, PPQ recommends the use of triage screening by ELISA, followed by DNA extraction and PCR for all ELISA-positive samples. DNA from all ELISA positive samples must be forwarded to the USDA-APHIS-PPQ National Plant Germplasm and Biotechnology Laboratory or NIS for confirmatory testing if the diagnostic laboratory is not provisionally approved to run PCR tests. The AGDIA ELISA kits (Elkhart, IN) used have been validated for use for prescreening for detection at the genus level (i.e., only for Phytophthora species). The ELISA is used to prescreen samples so that only samples infected with Phytophthora species are scrutinized further. Because of its limitations in differentiating between species, ELISA cannot be used as a final diagnostic determination since many species of non-regulated Phytophthora may also be present.

For PPQ regulatory purposes, a final diagnostic determination of the presence of Pram in the absence of culture isolation requires a PCR assay. Two PPQ-validated PCR techniques are currently available: nested PCR and real-time PCR (modified from methods developed by Garbelotto and coworkers and Central Science Laboratory, York, UK, respectively). In nested PCR an amplified primary target in the pathogen’s genome is re-amplified using a target within the initial amplicon. With the real-time PCR, amplified products are detected through the use of a species-specific probe using TaqMan-based detection methods. Both PCR assays are diagnostically definitive for the presence of Phytophthora ramorum. PPQ’s validated protocols are available online.

In February 2005, PPQ was informed by the California Department of Food and Agriculture (CDFA) of the occurrence of Pram in camellias at a nursery in Southern California. The infestation provided a unique opportunity for collaborative research in an infested nursery to determine the relative sensitivity of the different diagnostic assays under environmental conditions commonly found within a nursery. In addition, a recent abstract presentation indicated the potential role of soil pH and moss presence as factors in disease etiology (2), and this situation provided an opportunity to collect and analyze data on these observations.


Field Location and Site Choice

Because the nursery was required to destroy all infested or potentially infested plant materials in accordance with federal regulations, site selection and sample collection had to occur in a timely manner so that regulatory operations were not impeded. The company owning the infested nursery, CDFA, and PPQ worked collaboratively to identify a contiguous block of 1-gal potted camellia plants (Camellia japonica ‘Nuccio’s Gem’) laid out as 10 rows of 30 plants each for a total of 300 plants. An initial visual assessment indicated that approximately 50% of the plants showed leaf symptoms indicative of Pram infection. Each plant pot was labeled with a row letter (A-J) and plant number (1 to 30) and digitally photographed. Data were collected for each plant including presence or absence of leaf lesions characteristic of Pram, presence or absence of leaf drop, and presence or absence of moss on growing medium or plant stem.


Plant Sampling and Sample Processing

A modification of the PPQ-validated standard diagnostic protocol was developed to test a single sample by each of four assays to reduce or eliminate error inherent in testing different samples of the same tissue. For this experiment it was necessary that Pram DNA in the sample, destined for downstream PCR tests, be recoverable following an initial ELISA extraction despite differing reagent requirements of each assay. Furthermore, the organism had to remain biologically viable for culturing.

Fifteen leaves were removed from each plant and placed in a Ziploc bag labeled with the row and plant number. If symptomatic leaves were present, these leaves were preferentially sampled. Leaf samples were sent to the Great Plains Diagnostic Network Laboratory (GPDN) at Kansas State University in Manhattan, KS. Fifteen camellia leaf disks were obtained from each sample using a flame-sterilized paper punch and placed in an ELISA mesh extraction bag (AGDIA Inc., Elkhart, IN). Instead of the normal ELISA extraction buffer, one mL of 1Χ TE (10 mM Tris-HCl pH 8.0 with 1 mM EDTA pH 8.0) was added to the mesh bag and the leaf disks were macerated using a hand roller grinder. Two-hundred ml of each extract slurry was subjected to the standard ELISA protocol [AGDIA Website and (3)], with the threshold for a positive test being set at 2.0Χ background after 120 min in substrate. For use in Real-Time and nested PCR assays, a 200 ml aliquot of the ELISA tissue macerate was subjected to DNA extraction using Qiagen DNeasy Plant Mini Kit without protocol variation. All remaining leaf pieces in each sample bag which were not fully macerated (12 to 15 from each bag) were plated onto PARP-V8 [Pimaricin Ampicillin Rifampicin Pentachloronitrobenzene amended clarified V8 medium, after (1)]. Twelve additional samples of ‘Nuccio’s Gem’ camellia leaves known to be free from Pram by California Department of Food and Agriculture annaual Cleanliness Compliance Agreement inspection (Monrovia Growers, Visalia, CA) were used as negative controls and were subjected to the same extraction and diagnostic protocols as described above. Internal Pram-positive and negative Pram controls were used for all ELISA and PCR assays.


Growth Medium Samples

For each plant, 500 ml of growth medium was removed and placed in a 1-qt Ziploc bag and subjected to a soil baiting technique using healthy camellia leaves collected (Camellia japonica ‘Pearl Maxwell’) from a non-infested nursery facility (Monrovia Growers, Visalia, CA) in Northern California. After 48 h, these baited leaves were removed from the potting medium and subjected to the same extraction process and diagnostic assays as the plant leaf samples from the infested nursery. A subset of growth medium samples were randomly chosen for pH analysis (n = 30).


Diagnostic Tests

The GPDN hub laboratory performed ELISA, DNA extraction, and culture plating. Extracted DNA was sent to the CPHST NPGBL where the validated Real-Time PCR protocol was performed on the samples. Real-Time PCR samples were considered to be positive if fluorescent signal rose above background levels at 36 cycles or less (CT less than or equal to 36). All internal and external positive and negative controls were required to function as expected for results to be validated. A subset of each DNA sample and control DNA was sent to the USDA-AMS Laboratory in Gastonia, NC where nested PCR was performed. A total of 600 samples (300 leaf samples and 300 associated media-baited samples) were subjected to the four separate diagnostic assays. Growth medium pH was tested using a electric pH meter and the EPA SW846 method 9045 for soil pH. For ELISA, Real-Time PCR and culture, continuous data were taken and correlated as well as converted to binary (positive or negative) for direct comparison to other binary data collected during this experiment (e.g., as lesion and leaf drop and nested PCR data). Data were analyzed with SAS 9.1 and JMP (SAS Institute Inc., Cary, NC) statistical computer programs. The SAS Proc CORR procedure was utilized to produce the correlation matrix and to calculate Pearson’s Correlation Coefficient.


Field Observations and Above-ground Plant Leaf Samples

Of the 300 camellia leaf samples, 80.0% displayed leaf lesion symptoms that were characteristic of Pram, 36.7% of plants exhibited leaf-drop symptoms, and 18.0% of plants (or the growth medium associated with the plant) had moss growth (Tables 1 and 2, Fig. 1). The average pH of the potting media was 6.08 with values ranging between 5.38 and 6.41 (data not shown). Fifty-nine percent of plant leaf samples (177/300) were ELISA positive at the 2.0Χ threshold level (52.3% [157/300] plant leaf samples were positive at the 2.5Χ threshold level). Forty-two percent leaf samples (126/300) were culture positive. Fifty four percent (162/300) were Real-Time PCR positive and 75.7% (227/300) were positive by nested PCR (Tables 1 and 2, Fig. 1). A high resolution digital photograph was taken of each plant prior to sampling and referred to as necessary to examine leaf symptoms.


   

Fig. 1. Graphical representation of spatial dimensions of aspects of above-ground and below-ground (shaded) disease detection with other plant characteristics including leaf lesions (brown), leaf drop (blue), ELISA positives (yellow), Real-time-PCR positives (red), Nested PCR positives (pink), and culture positives (orange).

 

Table 1. Data from the 300 one-gallon Camellia japonica ‘Nuccio’s Gem’ from a Southern California nursery. Colored cells indicate a positive assay. Sample numbers 301-312 are negative control Camellia japonica ‘Nuccio’s Gem’ taken from a different nursery.

Row-
plant
Symp-
toms
r
Leaf
drop
s
Mosst ELISA
(OD)u
PCR Culture
(propor-
tion +)x
Real
time

(Ct)v
Real
time

(0.1x)
(Ct)
Nested
(+/-)w
A-1 — — — 0.082 NRy NR — 0.0000
A-2 + + — 1.650 27.07 30.38 + 0.8824
A-3 + — — 1.057 26.75 32.60 + 1.0000
A-4 + — — 2.353 26.41 32.09 + 0.7333
A-5 + — — 0.077 41.68 NR — 0.0000
A-6 + — — 0.074 43.33 *z — 0.0000
A-7 + — — 0.108 42.65 NR — 0.0000
A-8 + — — 0.078 NR * — 0.0000
A-9 + — — 2.114 22.19 29.31 + 1.0000
A-10 + — — 0.067 36.49 NR — 0.0000
A-11 + — + 0.079 37.18 39.47 + 0.0000
A-12 + — — 0.810 25.41 31.25 + 0.6667
A-13 + — + 0.092 41.16 41.19 — 0.0000
A-14 + — — 0.399 28.27 34.45 + 1.0000
A-15 + — — 0.088 38.26 NR + 0.0000
A-16 + — — 0.140 31.09 37.68 + 0.6667
A-17 + — — 2.188 19.81 28.15 + 1.0000
A-18 + — — 0.136 33.15 27.63 + 0.0000
A-19 — — — 0.146 43.34 NR — 0.0000
A-20 + + — 0.913 24.72 30.56 + 0.7857
A-21 + — — 0.432 28.20 34.28 + 0.7333
A-22 + + — 1.704 NR 37.45 — 0.0000
A-23 + — — 2.327 27.68 25.70 + 1.0000
A-24 + — — 2.748 26.20 33.08 + 0.8000
A-25 + — — 2.316 27.59 32.21 + 0.9333
A-26 + — — 3.010 26.12 29.35 + 0.8333
A-27 + + — 0.712 27.53 34.02 + 1.0000
A-28 + + — 2.310 27.80 33.77 + 0.9375
A-29 + + — 2.790 27.15 28.43 + 0.8750
A-30 + — + 2.146 NR * — 0.0000
B-1 + + — 2.917 22.05 33.45 + 1.0000
B-2 + — — 2.292 37.76 34.08 + 0.7857
B-3 + — + 0.617 30.80 37.46 + 0.6000
B-4 + — — 3.283 23.64 34.02 + 1.0000
B-5 + + — 0.098 NR NR — 0.0000
B-6 + — — 0.099 NR NR — 0.0000
B-7 + + + 0.942 28.63 35.86 + 0.9231
B-8 + — — 0.192 29.59 37.01 + 0.2000
B-9 — — — 0.081 44.00 NR — 0.0000
B-10 + — — 0.106 35.78 41.91 + 0.0000
B-11 + + — 1.735 25.36 35.70 + 1.0000
B-12 + — — 0.206 31.32 37.90 + 0.2308
B-13 — — — 0.086 NR NR — 0.0000
B-14 — — — 0.087 NR NR — 0.0000
B-15 — — — 0.071 NR NR — 0.0000
B-16 + — — 0.078 42.99 * — 0.0000
B-17 + — — 0.080 NR NR — 0.0000
B-18 — — — 0.082 NR NR — 0.0000
B-19 — — — 0.091 NR NR — 0.0000
B-20 — + — 0.090 NR NR — 0.0000
B-21 + — — 0.757 32.21 38.28 + 0.8667
B-22 + — + 0.205 34.22 NR + 0.8462
B-23 + — — 2.424 24.58 38.62 + 0.6000
B-24 + — — 2.684 31.32 36.05 + 1.0000
B-25 + — — 0.666 33.44 NR + 0.4375
B-26 + + — 0.371 33.43 40.90 — 0.6000
B-27 + + — 2.594 33.51 38.52 + 0.0000
B-28 + — + 0.224 34.09 41.81 + 0.0000
B-29 + — — 1.891 28.39 36.30 + 0.0000
B-30 + + — 0.474 33.46 * — 0.7273
C-1 + — — 2.084 24.36 29.10 + 1.0000
C-2 + — — 1.810 25.36 * — 0.9231
C-3 + + — 1.146 23.27 28.82 + 1.0000
C-4 + — — 0.120 45.59 NR — 0.0000
C-5 — + — 0.130 45.12 NR — 0.0000
C-6 + — — 0.133 NR * — 0.0000
C-7 + — — 0.127 NR NR — 0.0000
C-8 — — — 0.121 NR NR — 0.0000
C-9 — — + 0.117 NR NR — 0.0000
C-10 + — — 0.353 43.40 * + 0.0000
C-11 — + — 0.130 NR NR — 0.0000
C-12 — — + 0.112 NR NR — 0.0000
C-13 — — — 0.147 NR NR — 0.0000
C-14 + + — 2.689 29.65 28.47 + 1.0000
C-15 + — — 1.288 41.27 * + 0.6667
C-16 — — + 0.151 42.43 NR — 0.0000
C-17 — — — 0.147 27.27 34.49 + 0.0000
C-18 — — — 0.310 35.75 41.28 + 0.0000
C-19 + — — 0.221 NR * + 0.0000
C-20 + — — 0.126 44.29 NR + 0.0000
C-21 + + — 0.784 28.56 34.79 + 0.9286
C-22 + — — 0.954 26.67 31.75 + 1.0000
C-23 + — — 2.494 22.25 30.06 + 1.0000
C-24 — + — 0.151 NR NR + 0.0000
C-25 + — — 1.798 25.25 * + 0.8571
C-26 + — + 1.398 36.75 39.02 + 0.0000
C-27 + — — 2.121 23.75 39.00 + 0.8667
C-28 + + — 1.334 25.54 32.82 + 0.8000
C-29 + + — 0.880 30.35 39.04 + 1.0000
C-30 — — — 0.121 42.85 NR — 0.0000
D-1 + — + 0.867 32.90 42.92 + 0.0000
D-2 + — — 0.820 30.41 38.01 + 0.0000
D-3 + — — 1.006 29.54 36.77 + 0.0000
D-4 — — + 0.130 42.42 NR — 0.0000
D-5 — — — 0.119 NR NR + 0.0000
D-6 — — — 0.153 45.25 NR — 0.0000
D-7 — — — 0.140 NR NR — 0.0000
D-8 — — — 0.138 NR NR — 0.0000
D-9 — — — 0.140 41.65 NR — 0.0000
D-10 — + + 0.191 39.28 * + 0.0000
D-11 + — — 0.151 42.15 NR + 0.0000
D-12 + — — 0.069 49.88 NR + 0.0000
D-13 — — — 0.041 42.97 NR — 0.0000
D-14 + + — 0.045 28.10 34.82 + 0.0000
D-15 + — + 0.110 41.84 NR — 0.0000
D-16 + — + 0.198 27.55 34.06 + 0.0000
D-17 — — — 0.130 41.84 NR — 0.0000
D-18 — — — 0.119 41.92 NR — 0.0000
D-19 — — — 0.161 42.40 NR — 0.0000
D-20 + — — 0.123 NR NR + 0.0000
D-21 + — + 1.641 43.72 33.69 + 1.0000
D-22 + — — 1.042 28.22 38.30 + 0.3750
D-23 + + + 1.032 26.32 31.29 + 0.7143
D-24 + + — 1.751 28.68 34.24 + 0.8125
D-25 + — — 1.506 17.45 33.53 + 0.0000
D-26 + — + 1.716 30.65 31.64 + 0.0000
D-27 + — — 0.974 27.35 33.44 + 0.3750
D-28 + — — 1.858 28.33 28.19 + 0.3333
D-29 + — + 1.096 29.69 34.31 + 0.8750
D-30 + — — 1.492 26.95 29.61 + 0.5714
E-1 + + — 1.931 24.75 33.79 + 0.5882
E-2 + — — 1.472 42.68 31.94 — 0.0000
E-3 + — — 1.349 40.46 42.82 + 0.0000
E-4 — — — 2.144 31.54 NR + 0.7857
E-5 — — — 1.703 24.16 NR + 1.0000
E-6 + — — 0.568 NR NR + 0.0000
E-7 — — — 0.162 NR NR — 0.0000
E-8 + — — 1.462 26.18 NR + 0.4000
E-9 — — — 1.080 42.44 NR — 0.0000
E-10 — — — 1.570 26.78 NR + 1.0000
E-11 + — + 0.117 NR NR + 0.0000
E-12 — + — 0.970 NR NR — 0.0000
E-13 — — — 1.987 25.13 NR + 0.8750
E-14 + — — 0.123 42.25 NR + 0.0000
E-15 + — + 0.128 NR NR — 0.0000
E-16 + — — 0.132 NR NR + 0.0000
E-17 + — + 0.099 45.04 NR + 0.0000
E-18 — — — 0.121 NR NR + 0.0000
E-19 — — — 0.103 NR NR + 0.0000
E-20 + — + 0.088 27.10 33.70 + 1.0000
E-21 + — — 0.081 42.25 NR + 0.0000
E-22 + — + 0.089 25.57 31.26 + 0.8750
E-23 + — + 3.001 22.23 32.21 + 0.8000
E-24 + — — 1.459 27.17 32.78 + 0.4615
E-25 + + + 0.153 26.72 33.94 + 0.7143
E-26 + — — 0.131 23.82 30.36 + 0.7692
E-27 + — — 0.100 29.85 36.88 + 0.6667
E-28 + — — 0.149 30.38 36.71 + 0.5333
E-29 + — — 0.114 25.97 31.48 + 0.0000
E-30 + — — 0.125 25.99 34.33 + 0.6667
F-1 + — — 0.137 25.17 32.92 + 0.6667
F-2 + + — 0.144 26.37 31.11 + 0.6667
F-3 + + — 0.112 40.89 NR + 0.0000
F-4 + — — 0.100 23.23 30.04 + 0.5385
F-5 + — — 0.127 24.57 31.08 + 0.0000
F-6 + + — 0.140 32.14 NR + 0.0000
F-7 + — — 0.113 41.28 42.32 — 0.0000
F-8 + — + 0.106 NR NR — 0.0000
F-9 + — — 0.298 NR NR + 0.0000
F-10 + — — 0.222 41.51 NR + 0.0000
F-11 + + — 0.144 NR NR + 0.0000
F-12 + — — 2.342 20.89 NR + 1.0000
F-13 — — — 0.163 NR NR — 0.0000
F-14 — + + 1.216 NR NR — 0.0000
F-15 — + — 0.150 43.53 NR — 0.0000
F-16 — + — 0.118 41.29 NR — 0.0000
F-17 — — + 0.114 NR NR — 0.0000
F-18 — — — 0.117 42.16 NR — 0.0000
F-19 — + — 0.622 29.12 37.30 + 0.8571
F-20 + + — 0.164 NR NR + 0.0000
F-21 + — — 1.421 30.47 31.08 + 1.0000
F-22 + + — 0.766 30.45 40.08 + 0.0000
F-23 + — — 0.097 42.60 NR + 0.0000
F-24 + — + 0.111 38.88 NR + 0.4286
F-25 + — — 0.442 27.28 34.68 — 0.0000
F-26 + + — 0.897 26.42 33.41 + 0.9286
F-27 + + — 1.131 27.35 32.84 + 1.0000
F-28 + — — 1.194 22.05 27.22 + 0.9333
F-29 + — — 1.018 27.02 32.79 + 0.0000
F-30 + — — 1.596 22.27 31.74 + 0.7857
G-1 + + — 2.966 22.67 31.00 + 1.0000
G-2 + + — 2.404 21.37 33.51 + 0.5625
G-3 + + — 2.073 24.78 32.65 + 0.8571
G-4 + — — 2.455 24.49 35.01 + 0.7857
G-5 — — — 1.440 25.94 37.16 + 1.0000
G-6 + + — 0.131 42.11 NR + 0.0000
G-7 + + — 0.130 42.33 NR + 0.0000
G-8 — + — 0.151 NR NR — 0.0000
G-9 + + — 0.201 NR NR + 0.0000
G-10 + — — 0.115 NR NR + 0.0000
G-11 + + — 1.352 28.09 NR + 0.8667
G-12 + + — 0.177 39.29 NR + 0.0000
G-13 + + — 2.694 22.92 35.66 + 1.0000
G-14 + — — 0.171 41.40 NR — 0.0000
G-15 + + — 0.120 42.24 NR + 0.0000
G-16 + — — 0.123 41.52 NR + 0.0000
G-17 — + — 0.114 41.59 NR + 0.0000
G-18 — — + 0.195 40.69 NR + 0.0000
G-19 — — — 0.179 NR NR — 0.0000
G-20 + — + 0.362 41.80 NR + 0.0000
G-21 + — + 0.181 NR NR + 0.0000
G-22 — + — 0.139 NR NR — 0.0000
G-23 + + — 2.030 25.26 32.65 + 1.0000
G-24 + + — 1.504 23.78 39.03 + 0.9333
G-25 + — — 2.082 24.02 32.93 + 0.5000
G-26 + — — 1.978 26.37 * + 0.5000
G-27 + — — 2.535 24.76 32.72 + 1.0000
G-28 + — — 1.195 27.49 40.49 + 0.0000
G-29 + — — 2.451 27.61 33.49 + 0.3333
G-30 + + — 1.579 NR 35.17 — 0.0000
H-1 + + — 2.005 24.22 34.22 + 1.0000
H-2 + + + 1.766 27.22 33.54 + 0.8750
H-3 + — — 0.541 39.36 40.45 + 0.0667
H-4 + + — 1.794 25.27 30.76 + 0.8750
H-5 — — — 0.185 41.34 NR + 0.0000
H-6 + + — 0.156 43.80 NR + 0.0000
H-7 + + — 0.226 NR NR + 0.0000
H-8 + — — 1.401 31.29 36.43 + 1.0000
H-9 + + — 0.164 41.98 NR + 0.0000
H-10 — + — 0.147 43.19 NR + 0.0000
H-11 + + + 0.164 NR NR + 0.0000
H-12 + — + 0.176 NR NR — 0.0000
H-13 + + — 2.255 27.34 39.67 + 0.5385
H-14 + — — 1.278 32.98 38.29 + 0.8824
H-15 + — — 0.155 NR NR + 0.0000
H-16 + + + 0.151 NR NR + 0.0000
H-17 — — — 0.119 45.18 NR — 0.0000
H-18 + — — 0.109 NR NR + 0.0000
H-19 + + — 0.121 48.05 NR + 0.0000
H-20 + — — 2.344 41.24 36.46 — 0.0000
H-21 + — — 0.118 NR NR + 0.0000
H-22 + + — 0.119 NR NR — 0.0000
H-23 + — — 1.930 23.28 34.81 + 1.0000
H-24 + + — 0.264 38.13 NR + 0.0000
H-25 + + — 1.603 23.28 33.75 + 0.0667
H-26 + + — 1.456 27.28 36.66 + 1.0000
H-27 + + + 2.253 22.32 34.61 + 0.8462
H-28 + + + 1.441 26.95 36.41 + 0.8667
H-29 + — — 1.431 23.62 34.22 + 0.2727
H-30 + + — 1.489 25.20 37.10 + 1.0000
I-1 + + — 2.723 26.43 35.32 + 1.0000
I-2 + — — 2.901 23.52 33.70 + 0.9375
I-3 + + + 2.847 22.77 36.35 + 1.0000
I-4 + — — 2.580 23.93 45.42 + 0.0000
I-5 + + — 0.190 39.12 41.72 + 0.3333
I-6 + + — 0.386 48.49 NR + 0.0000
I-7 + — — 0.271 44.22 NR + 0.0000
I-8 — + — 0.181 NR NR + 0.0000
I-9 + + — 0.224 NR NR + 0.0000
I-10 + — — 0.238 NR NR + 0.0000
I-11 + — + 0.202 NR 45.43 — 0.0000
I-12 + + + 0.780 31.34 39.86 + 0.0000
I-13 + + — 1.996 NR 34.26 + 0.0000
I-14 + + — 1.258 26.77 34.78 + 0.7647
I-15 + — + 0.346 49.05 NR — 0.0000
I-16 + + — 0.299 NR NR + 0.0000
I-17 + + — 0.159 42.23 NR + 0.0000
I-18 + + — 1.452 27.90 37.52 + 1.0000
I-19 — + — 0.137 40.42 NR + 0.0000
I-20 + — — 0.198 49.10 NR + 0.0000
I-21 — + — 0.115 49.31 NR — 0.0000
I-22 + — — 2.848 26.05 30.98 + 0.0000
I-23 + + — 0.739 31.38 43.29 + 0.0000
I-24 + — + 0.649 46.85 NR + 0.0000
I-25 + — — 1.889 23.37 36.93 + 1.0000
I-26 + + + 0.225 NR NR + 0.0000
I-27 + — + 2.901 24.79 29.76 + 0.8571
I-28 + — — 2.087 23.07 30.01 + 1.0000
I-29 + + + 1.828 24.09 35.84 + 1.0000
I-30 + — — 0.207 44.15 NR + 0.0000
J-1 + — — 0.133 42.22 NR + 0.0000
J-2 + + — 1.212 29.77 39.08 + 0.0000
J-3 + + + 1.921 30.67 31.28 + 0.8750
J-4 + + — 2.372 26.35 29.92 + 0.9333
J-5 + + + 0.285 42.15 NR + 0.0000
J-6 + + — 0.227 NR NR + 0.0000
J-7 + + — 0.513 47.31 NR + 0.0000
J-8 + — — 0.145 47.39 NR + 0.0000
J-9 + + + 0.129 43.31 NR + 0.0000
J-10 + + — 0.846 44.16 NR — 0.0000
J-11 + — — 0.135 NR NR + 0.0000
J-12 + — — 0.230 NR NR + 0.0000
J-13 + + — 2.918 27.41 29.55 + 0.8125
J-14 + — — 0.234 38.37 43.40 + 0.0000
J-15 — + — 0.242 40.53 NR + 0.0000
J-16 + — — 0.270 38.91 NR + 0.0000
J-17 + — — 0.195 42.63 44.10 + 0.0000
J-18 — — — 0.181 NR NR + 0.0000
J-19 + + — 1.706 32.16 37.95 + 1.0000
J-20 + — — 2.464 26.31 29.48 + 1.0000
J-21 + + + 0.185 NR NR — 0.0000
J-22 + + — 1.631 23.21 NR + 0.9286
J-23 + + + 1.854 26.24 34.93 + 0.9333
J-24 + + — 2.759 28.40 34.00 + 0.8667
J-25 + — — 1.342 30.56 36.79 + 0.0000
J-26 + — — 1.285 24.40 30.51 + 0.0000
J-27 + + + 2.436 29.91 33.07 + 1.0000
J-28 + + — 0.381 NR 43.32 + 0.0000
J-29 + — — 2.316 26.32 33.98 + 0.8462
J-30 + + — 2.940 25.60 33.38 + 0.7500

 r Defined as lesions on camellia leaves characteristic of Phytophthora ramorum.

 s Leaf drop is a symptom of premature abscission of leaves potentially due to their infection with P. ramorum.

 t The presence of moss was noted either on the stem of the camellia or on the medium surface.

 u Positive defined as having an OD reading of 2.0 times the known negative tissue samples.

 v Positive defined as having a Ct of between 17 and 35.99. Any sample with a Ct of 36.00 to 46.00 was re-run at 1:10 dilution and the results reported.

 w Nested PCR is considered positive if a strong band at 293 bp is exhibited in the final gel analysis with consistently performing positive and negative controls.

 x Positive defined as at least one colony of P. ramorum recovered from plated leaf pieces.

 y NR = No Reaction.

 z * = no DNA remaining in tube for PCR reaction.



Table 2. Summary of the results from the different methods used at a Southern California nursery to examine 300 one-gallon camellia plants (Camellia japonica ‘Nuccio’s Gem’).

Parameter examined Percent of total (%)
Leaf samples Media samples
Lesions present 80.00s --t
Leaf drop present 36.67u --
Moss present -- 18.00v
ELISA positive (+) 59.00w 50.00
Real-Time PCR + 54.67x 11.67
Culture + 42.00y 11.33
Nested PCR 75.67z 34.33

 s Defined as lesions on camellia leaves characteristic of Phytophthora ramorum.

 t Not applicable or not available for that variable.

 u Leaf drop is a symptom of premature abscission of leaves potentially due to their infection with P. ramorum.

 v The presence of moss was noted either on the stem of the camellia or on the medium surface.

 w Positive defined as having an OD reading of 2.0 times the known healthy tissue background.

 x Positive defined as having a Ct of between 17 and 35.99.

 y Positive defined as at least one colony of P. ramorum recovered from plated leaf pieces.

 z Nested PCR is considered positive if a strong band at 293 bp is exhibited in the final gel analysis with consistently performing positive and negative controls.


This experiment was conducted using natural infections of Pram throughout a nursery block, so an absolute measure of infection at the time of sampling wasn’t known. Lesion presence indicated a high disease incidence, but these symptoms could have been caused by other Phytophthora species and are not diagnostic for disease caused by Phytophthora ramorum without additional diagnostic assays. However, in comparison to the observations of symptomatic tissue, nested PCR was observed to be the most sensitive assay observed in this project, because it was most closely correlated to lesion incidence and it detected the pathogen most often in tissue with lesions consistent with Phytophthora ramorum. While the presence of other Phytophthora species was not determined in this study and cannot be ruled out, the high correlation between symptomology and the detection of P. ramorum by nested PCR.

Because it was observed that 28.3% of asymptomatic samples were nested PCR positive, the nested PCR may have detected incipient lesions. Previous research has shown that there is a time lag between inoculation and symptom development of up to seven days (in comparative hosts) when exposed to high amounts of inoculum under disease-conducive environmental situations (4). Since the overall infection level of the experimental block was high, it is likely that the inoculum load for the block was also high. In addition, any portion of the pathogen that could leave a DNA trace, including potentially infective sporangia (or other propagules) may have been detected by the nested assay. During the course of this experiment all control samples behaved as expected and all known precautions were used in the collection, extraction, and assays of samples. However, because these observations are made from a naturally infected nursery block, there wasn’t a mechanism to calculate an absolute measurement of false positives, either before or after sample collection. In the regulatory program, the sample size is adjusted in anticipation that 25% of the plants infected with Phytophthora ramorum will not show symptoms.

Using rhododendron plant tissue, Osterbauer & Trippe (3) compared the sensitivity of several of the same assays presented here. The scope and methodologies of their study and this research are different, making a direct comparative analysis between the studies difficult. Unlike their study, this current study contains detailed observations of the original infected nursery block and plant symptomology. The extraction protocol utilized here ensured that the same sample material was processed for all assays. This experimental sampling technique was specifically developed to reduce potential sampling error inherent with side-by-side tissue sampling. By using the TE medium as a first step, however, dilution of the sample was inevitable and unavoidable. Additional tests of split samples for ELISA extraction efficiency using the TE and standard extraction showed no statistical differences in the two methods for recovery of Pram (data not shown). Furthermore, the use of negative controls reduced the risk of cross-contamination occurring between samples and a statistically valid sample size allowed for detailed evaluation of the separate assays.

Correlation and sensitivity of detection assays of above-ground plant leaf parameters. A frequency distribution table and correlation matrix of above-ground plant leaf parameters are provided in Tables 3 and 4, respectively. The correlations between above-ground plant leaf symptoms and disease incidence as measured by ELISA, culture, and both PCR techniques was highly correlated and statistically significant (Pearson’s correlation coefficient P < 0.001, as noted in red in Table 4). Of the 80% of plants with lesions present (240 of 300), 87.5% were nested PCR positive (210 of 240) and 12.5% were nested PCR negative (30 of 240, Table 3); 65.4% and 34.6% were Real-Time PCR positive and negative, respectively (157 and 83 of 240, respectively); 51.7% and 48.3% of the 240 lesion positive samples were culture positive and negative respectively; and 67.5% and 32.5% of the 240 lesion positive samples were ELISA positive and negative respectively (Table 3). Of the 20% of above-ground plant samples without visible lesions (i.e., asymptomatic plants, a total of 60 plants), 25.0% (15 of 60), 28.3% (17 of 300), 8.3% (5 of 60), and 3.3% (2 of 60) were ELISA, Real-Time PCR, nested PCR, and culture positive, respectively. There was no correlation with the presence of moss to any of the diagnostic assays, nor was there any correlation with the presence of moss to the presence or absence of leaf symptoms (Tables 3 and 4).

The frequency distribution of the number of nested positive samples by Ct values show that 96.9% of samples with a Ct value of 40 or under were nested PCR positive (156 of 161 samples) (Table 1 and Fig. 2), as were all but two culture positives (Table 1 and Fig. 2). This is also reflected in the significant negative correlation coefficient of the Real-Time Ct values to nested PCR, culture and ELISA assays (Table 4). Significantly more positive diagnostic assays occurred when the Real-Time PCR Ct values were below 40, although there were a total of 72 nested PCR positives with a Real-Time Ct greater than 40 (Table 1 and Fig. 2).


 

Fig. 2. Distribution of positive and negative nested and culture samples based on Real- Time Ct values (NR means the sample DNA PCR reaction did not cross the threshold).

 

Recovery of Phytophthora ramorum from potting media samples via baiting was low for all assays, with recovery of P. ramorum at 34.33% (103 of 300 bait samples), 11.67% (35 of 300 bait samples), and 11.33% (34 of 300 bait samples) for Nested PCR, Real-time PCR, and PARP cultures, respectively (Table 1). Fifty percent of the bait samples (150 of 300) were ELISA positive at the 2.0Χ threshold (Table 1). ELISA positives from soil and potting media samples are common and could be caused by a number of Phytophthora species, from some of the Pythium species known to cross-react with the assay, or from spurious reactions with the soil matrix. Furthermore, correlation of these ELISA results with above-ground symptoms was not apparent or statistically significant (data not shown).

The potential pitfall of using a single extraction methodology for the three different diagnostic methods is that compromises in extraction efficiency for any one assay may exist. Initial preparations for DNA extraction include proteases and RNAases deleterious to living organisms and render the extract useless for ELISA and culturing. The first steps in ELISA extraction likely also include compounds that inhibit subsequent recovery of DNA from the extract and reduce recovery of Phytophthora ramorum via culturing. In this experiment, the Phytophthora ELISA antigen would have been diluted through the use of TE medium and is the only component not amplified as part of a downstream process (either through growth on semi-selective medium or amplification of DNA through polymerase chain reaction). For this reason, ELISA was assumed to be most likely assay-compromised during the extraction process and additional experimentation was necessary to examine the validity of the extraction assay. During side-by-side comparisons of extractions of infected camellia tissue with both the regular ELISA extraction protocol and the modified TE extraction procedure, there was no apparent statistically significant difference in ELISA values (data not shown).

Fewer positives were detected by ELISA, Real-Time PCR, and culture methods (59.0, 54.0, and 42.0 %, respectively) than those using the nested PCR assay, keeping in mind that the ELISA is not species-specific. Yet, correlation between these positives, nested PCR positives, and observed leaf symptoms remained extremely high. Although it cannot be ruled out on the basis of this experiment, the modification of the sample extraction protocols did not appear to affect the efficiency of detection by any method used, since all methods gave comparable and highly correlated results. Although high correlation was noted with all assays and symptomology of above-ground tissue, there were still discrepancies between the Real-Time and nested PCR results. According to the Real-Time PCR protocol work instruction, if an environmental sample is between 36 and 46 Ct the sample should be diluted 1:100 and reanalyzed. This would have included an additional of 75 samples of the 312, 43 of which were nested PCR positive. When reanalyzed at the 1:10 dilution, few of the samples produced a lower Ct value, indicating that there were not likely to be any polymerase inhibitors in the sample extractions. Two additional samples (0.67%) were positive in the 1:10 dilution, with differences in Ct of between –10.74 and 21.49 (average = 3.21), and 55 positive undiluted samples were negative at the 1:10 dilution (Table 1).

Because this experiment was taken at a single point in time and not at several different intervals during disease progression, little can be accurately determined about disease epidemiology. There are two exceptions to this, however. Firstly, results suggest that Ramorum blight on these camellias was likely mediated from above-ground tissue from sprorangial production on foliar tissue, because baited media samples did not correlate with above-ground symptomology. Secondly, because of the high percentage of detection of Phytophthora ramorum on above-ground tissue with the nested PCR assay and the related high percentage of lesions present on leaves of these camellias, it is likely the epidemic at this nursery block was advanced.

The parameters that did not correlate with any disease symptoms or detection of Phytophthora ramorum in any of the tested assays were the presence or absence of moss and the pH of the growing medium. It has been suggested (2) that moss is always found in association with dying oak trees and that the moss contributes to the tree’s death through the acidification of the plant. No correlation between moss and disease or P. ramorum recovery was observed in this case. Furthermore, examination of potting media found no differences in soil pH (average of 6.08) between symptomatic or apparently healthy plants tested. To our knowledge, this report is the first instance where data was provided in comparing the presence of moss to disease symptoms observed and where these were compared to measurements of parameters already being collected so that the effects of moss on plants in a nursery could be ascertained. In the nursery, no disease or recovery of the pathogen were explained by the presence or absence of moss.

In this study, we used 2.0Χ of the background after 120 min in substrate as the threshold for ELISA positive determinations. Using this threshold, we found 59.0% of the samples were positive. Many of the labs performing ELISA as a pre-screen for P. ramorum detection use either 2.5Χ of the background (as per the manufacturer’s instructions) or otherwise note visual differences in colorimetric changes without the use of instrumentation. If the 2.5Χ background threshold was used, 52.3% of the samples tested positive by ELISA. Correlation of the 6.7% discrepancy between 2.0Χ and 2.5Χ thresholds to the nested PCR positives and the leaf symptoms was very good, with only 0.6% of the samples indexed between 2.0 and 2.5Χ background testing negative by nested PCR. Therefore most of the samples indexed between 2.0 and 2.5Χ background represent true positives as determined by PCR or culture methods. Although use of the 2.0Χ threshold may increase the number of samples further analyzed by PCR (false positives), these results indicate that it is likely to also detect additional true positives and thereby reduce the rate of false negatives.

The current National Nursery Survey Manual for Phytophthora ramorum provides a sampling regime to include the targeted sampling of symptomatic tissue and additional visual inspection, assuming that 75% of plants infected with P. ramorum will show symptoms of disease. This research suggests that targeted sampling of symptomatic tissue with close visual inspection of additional plants for the presence of disease is an appropriate and conservative approach, as 87.5% of plant tissue with lesions were nested PCR positive and 12.5% were nested PCR negative. The high number of negative samples associated with asymptomatic tissue with all of the assays (75%, 72%, 92%, and 98% for ELISA, Nested and Real-Time PCR, and Culture, respectively) suggests that the practice of taking asymptomatic leaves for samples does not increase the likelihood of detection of Phytophthora ramorum (Table 3). If plants are showing few symptoms, the data herein suggest that more plants should be examined to find small symptoms and sample that tissue.

Initial in-field observations determined that 67% of the camellias had leaf symptoms. However, when the digital photographs of each plant were examined, an additional 39 plants were determined to have small lesions (usually tip necrosis or water-soaked spots). Furthermore, those plants that were not initially detected were present in later rows (A and B had no additional symptomatic plants; Row C had 1 additional symptomatic plant; Rows D and E had 3 additional symptomatic plants each; Rows E, F, and G had 5 additional symptomatic plants each; Row I had 8 additional symptomatic plants; and Row J had 9 additional symptomatic plants). The additional symptomatic plants were also correlated with diagnostic assays. These additional symptomatic plants provide information necessity for careful examination of plants for lesions. Had these plants not been closely examined by other personnel that had no a priori knowledge of the disease status of the plants, the analysis of these data may have been much different.

This research project was a highly collaborative effort between State and Federal regulatory officials and scientists, university scientists, and industry nursery managers. To our knowledge, this is the first opportunity in which a Pram-infected nursery incident, in the P. ramorum regulatory program, has been used to collect valuable scientific information regarding disease incidence and presence in a coordinated manner.


Acknowledgments

This project would not have been possible without the cooperative effort of many diverse groups of people, including The California Department of Agriculture, Monrovia Growers Inc., The Great Plains Diagnostic Network, and The USDA AMS laboratory in Gastonia, NC. The authors would also like to thank all the federal, state, and industry personnel involved with this undertaking.


Literature Cited

1. Ferguson, A. J., and Jeffers, S. N. 1999. Detecting multiple species of Phytophthora in container mixes from ornamental crop nurseries. Plant Disease 83:1129-1136

2. Klinger, L., Zingaro, R., and Miller, R. O. 2005. Etiology and Evidence of Systemic Acidification in SOD-Affected Forests of California. Sudden Oak Death Science Symposium II, 18-21 January 2005, Monterey, CA.

3. Osterbauer, N., and Trippe, A. 2005. Comparing diagnostic protocols for Phytophthora ramorum in rhododendron leaves. Online. Plant Health Progress doi:10.1094/PHP-2005-0314-01-HN.

4. Tooley, P. W., Kyde, K. L., and Englander, L. 2004. Susceptibility of selected ericaceous ornamental host species to Phytophthora ramorum. Plant Dis. 88:993-999.