We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.
Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 µm) method that captures the pathogen’s spores (zoospores) and is later used to amplify the pathogen’s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.
Recycling water in farms and nurseries is becoming increasingly popular due to the increase in water costs and environmental concerns behind water usage. Many irrigation methods have been developed for growers to reduce the spread and occurrence of plant disease. Regardless of the source of the water (irrigation or precipitation), runoff is generated, and many vegetable and nursery growers have a pond to collect and recycle runoff1. This creates a reservoir for possible pathogen accumulation favoring the spread of pathogens when the recycled water is used to irrigate crops2,3,4. Oomycete plant pathogens particularly benefit from this practice as zoospores will accumulate in water and the primary dispersive spore is self-motile but requires surface water5,6,7. Phytophthora capsici is an oomycete pathogen that affects a significant number of solanaceous and cucurbit crops in different ways8. Often, the symptoms are damping-off of seedlings, root and crown rot; however, in crops such as cucumber, squash, melon, pumpkin, watermelon, eggplant and pepper, entire harvests may be lost due to fruit rot9. Although there are known methods of detecting this plant pathogen, most require an infection to have already taken place which is too late for any preventative fungicides to have a significant effect10.
The traditional method to test irrigation water for the detection and diagnosis of targeted microorganisms is an antiquated approach when speed and sensitivity are crucial to success and profitable crop production11,12. Plant tissue susceptible to the targeted pathogen (e.g., eggplant for P. capsici) is attached to a modified trap that is suspended in an irrigation pond for extended period before being removed and inspected for infection. Samples from the plant tissue are then plated on semi-selective media (PARPH) and incubated for culture growth, then morphological identification is performed using a compound microscope13. There are other similar detection methods for other plant pathogens using selective media and plating small amounts of contaminated water before sub-culturing14,15. These methods require anywhere from 2 to 6 weeks, several rounds of sub-culturing to isolate the organism, and experience on Phytophthora diagnostics to be able to recognize the key morphological characters of each species. These traditional methods do not work well for detection of irrigation water contaminated by P. capsici due to factors such as interference by other microorganisms that are also present in the water sources. Some fast-growing microorganisms like Pythium spp. and water-borne bacteria can overgrow on the plate making P. capsici undetectable16,17.
The purpose of this study was to develop a sensitive and specific molecular method that can be used in both field and laboratory settings to detect P. capsici zoospores in irrigation water. The protocol includes the development of a novel loop-mediated isothermal amplification (LAMP) primer set able to specifically amplify P. capsici, based on a 1121-base pair (bp) fragment of P. capsici18,19. A previously developed LAMP primer from Dong et al. (2015) was used in comparison to the assay that was developed for this study20.
The LAMP assay is a relatively new form of molecular detection that has been demonstrated to be more rapid, sensitive, and specific than conventional polymerase chain reaction (PCR)21. In general, conventional PCR assays cannot detect under 500 copies (1.25 pg/µL); in contrast, previous studies have shown that the sensitivity of LAMP can be 10 to 1,000 times higher than conventional PCR and can easily detect even 1 fg/µL of genomic DNA22,23. Additionally, the assay can be carried out rapidly (often in 30 min) and on-site (in the field) by using a portable heating block for amplification and a colorimetric dye that changes color for a positive sample (removing the need for electrophoresis). In this study, we compared the sensitivity of PCR and LAMP assays using a filter extraction method. The proposed detection method allows researchers and extension agents to easily detect the presence of P. capsici spores from different water sources in less than two hours. The assay is proven to be more sensitive than conventional PCR and was validated in situ by detecting the presence of the pathogen in the irrigation water used by a grower. This detection method will allow growers to estimate the presence and population density of the pathogen in various water sources that are being used for irrigation, preventing devastating outbreaks and economic losses.
1. On-site detection of Phytophthora capsici from irrigation water using portable loop-mediated isothermal amplification
2. Determining the detection limit of zoospore concentration
Optimization of LAMP method
In this study, we detected the presence of Phytophthora capsici in irrigation water using a portable loop-mediated isothermal amplification (LAMP) assay. First, the proposed LAMP assay was optimized by testing different LAMP primer concentrations [F3, B3 (0.1–0.5 µM each); LF, LB (0.5–1.0 µM each) and FIP, BIP (0.8–2.4 µM each)], durations (30–70 min), and temperatures (55–70 °C). The final LAMP primer mix used in this study was: 0.2 µM of each F3 and B3 primer, 0.8 µM of each Loop-F and Loop-B primer, 1.6 µM of each FIP and BIP primer. Optimization of reaction temperature was performed in the portable amplification instrument (e.g., Genie III) by determining what temperature performed the fastest reaction with no additional negative amplification. The optimal temperature was confirmed to be 64 °C (data not shown). The optimal time for running the assay at 64 °C was 45 min, as the lowest concentrations that were positive for detection (1.2 x 102 spores/mL) still amplified by 40 min, while higher concentrations amplified at 20 min (Figure 2D). The amplified LAMP products were further observed on 1% agarose gel stained with a nucleic acid stain to confirm amplification. All reactions were repeated at least three times.
Isolates of P. capsici were taken from Tennessee, Florida, and Georgia and were submitted to the same protocol described in the methods. All samples of P. capsici isolates were amplified successfully in all runs of the assay (Figure 3).
Detection and sensitivity testing of Phytophthora capsici in irrigation water using portable LAMP assay
We standardized this filter paper-based LAMP method under laboratory conditions using a serial dilution of P. capsici spore suspensions (Figure 1). Serial dilutions were made from a P. capsici spore suspension starting at 4.8 x 104 zoospores/mL and run with the LAMP assay in triplicate. Spore concentrations are shown rather than DNA concentration due to the method involved for DNA extraction. CTAB DNA extraction of the highest spore concentration was 4.5 ng/µL measured by Nanodrop, and the magnetic bead DNA extraction protocol yielded 3.8 ng/µL26. The newly designed LAMP primer set could detect a concentration as low as 1.2 x 102 spores/mL (Figure 2B, 2C, & 2D) with all methods of extraction. The sensitivity shown in the graph of amplification on the portable amplification instrument was identical to that shown in the UV image with no additional sensitivity. The same serial dilution was run in a LAMP reaction using the colorimetric dye to determine the level of sensitivity to the naked eye for field detection. The lowest observable concentration was 4.8 x 103 spores/mL (Figure 2C).
To evaluate the ability of this assay using real-world samples, water samples were collected from seven ponds used for commercial vegetable production in Tift County, Georgia (Table 1, Figure 5, and Supplementary Figure 1). Out of the 7 ponds, 3 showed positive LAMP results (P1, P4, and P6) (Figure 6A, 6B, & 6C). These results suggest that the portable filter paper-based LAMP method could be very useful for detection of the pathogen even with a low zoospore concentration. This demonstrates the applicability of LAMP as a more sensitive detection assay than PCR for screening irrigation water contamination by P. capsici.
Comparative analysis of different methods: Traditional baiting, conventional PCR, and the portable LAMP-based assay
In order to compare the detection sensitivity of LAMP with conventional PCR, the DNA extracted from the serial dilution of spore suspensions were run in a PCR reaction. Results showed that conventional PCR was 40x less sensitive than LAMP, only able to detect a zoospore concentration as low as 4.8 x 103 spores/mL (Figure 2A). Additionally, the DNA samples obtained from filtered irrigation pond water were tested using conventional PCR, and only one of the three positive samples (P4) was successfully amplified as expected band size plus significant contaminates resulting in some smearing and unspecific bands (Figure 6D). Table 3 displays the differences between detection methods using such variables as time, cost, sensitivity, and preparation required. LAMP was the least expensive method among these three methods and it was also the fastest, ranging from 30-60 min for amplification (DNA extraction excluded). Conventional PCR ranged from 120-180 min for amplification (DNA extraction excluded).
Finally, to determine the specificity of the primers, samples of closely related oomycete pathogens (Phytophthora sansomeana, Phytophthora sojae, Phytophthora cinnamomi, Phytophthora palmivora, Pythium ultimum var. ultimum, Phytopythium vexans, Phytopythium helicoides, and Pythium aphanidermatum) were obtained, DNA was extracted using the same protocol for consistency and evaluated by the new LAMP assay with a positive and negative control (Figure 4) to determine specificity of the primers. All non-target samples were negative using the optimized 64 °C for 45 min. This was observed on the real-time amplification graph and imaged on a 1% agarose gel under UV light.
Figure 1: Diagram showing the different steps involved in Phytophthora capsici from a serial dilution of a concentrated spore suspension under laboratory conditions. Please click here to view a larger version of this figure.
Figure 2: Laboratory optimization of the limit for detection of Phytophthora capsici. (A) Conventional PCR assay was carried out using specific P. capsici primers on serial dilution factors and visualized on 1% agarose gel. 1, Ladder; 2-7, 4.8 x 104, 4.8 x 103, 4.8 x 102, 2.4 x 102, 1.2 x 102 spores/mL, respectively and 7, negative water control. (B) LAMP assay serial dilution factors visualized on 1% agarose gel. 1-6, a decreasing spore concentration: 4.8 x 104, 4.8 x 103, 4.8 x 102, 2.4 x 102, 1.2 x 102 spores/mL, and 7, negative water control. (C) LAMP results visualized using the colorimetric dye. 1-6, a decreasing spore concentration: 4.8 x 104, 4.8 x 103, 4.8 x 102, 2.4 x 102, 1.2 x 102 spores/mL, and 7, negative water control. (D) LAMP results visualized on the amplification graph. Red = 4.8 x 104, Dark blue = 4.8 x 103, Orange = 4.8 x 102, Light blue = 2.4 x 102, Green = 1.2 x 102 spores/mL, Pink = Negative control (other Phytophthora species), Yellow = ddH2O. (E) A standard curve showing quantification of the values shown in the real-time results. Ln (Spore count) is shown on the X-axis, and minutes to amplification on the Y-axis. (F) LAMP assay with published primer (Dong et al. 2015) on serial dilution factors visualized on 1% agarose gel. 1-6, a decreasing spore concentration: 4.8 x 104, 4.8 x 103, 4.8 x 102, 2.4 x 102, 1.2 x 102 spores/mL, and 7, negative water control. Please click here to view a larger version of this figure.
Figure 3: Amplification of P. capsici DNA from various locations. (A) LAMP results visualized on 1% agarose gel. 1, PC_TN1; 2, PC_TN2; 3, PC_FL1; 4,PC_FL2; 5, PC_GA1; 6, PC_GA2; N, Negative control. Samples 1 and 2 were isolated from TN; samples 3 and 4 isolated from FL; samples 5 and 6 were isolated from GA. (B) Results visualized using the colorimetric dye. 1, PC_TN1; 2, PCTN2; 3, PC_FL1; 4,PC_FL2; 5, PC_GA1; 6, PC_GA2. (C) Results visualized on the amplification graph. Red, PC_TN1; Orange, PCTN2; Yellow, PC_FL1; Green, PC_FL2; Dark blue, PC_GA1; Light blue, PC_GA2; Pink, negative control. Please click here to view a larger version of this figure.
Figure 4: Specificity determination of LAMP assay using DNA from non-target species P. capsici. (A) LAMP assay reaction with related non-target species on agarose gel and visualized on 1% agarose gel. L, Ladder; 1, Phytophthora sansomeana; 2, Phytophthora sojae; 3, Phytophthora cinnamomi; 4, Phytophthora palmivora; 5, Pythium ultimum var. ultimum; 6, Phytopythium vexans; 7, Negative control; 8, Phytophthora capsica. (B) LAMP results visualized using the colorimetric dye. 1, Phytophthora sansomeana; 2, Phytophthora sojae; 3, Phytophthora cinnamomi; 4, Phytophthora palmivora; 5, Pythium ultimum var. ultimum; 6, Phytopythium vexans; 7, Negative control; 8, Phytophthora capsici (C) LAMP results visualized on the amplification graph. Red = Phytophthora capcisi, all other non-target species samples were not amplified. Please click here to view a larger version of this figure.
Figure 5: Pictures showing the sampling and processing of recycled water for the detection of Phytophthora capsici in the field. Please click here to view a larger version of this figure.
Figure 6: Results from on-site detection of Phytophthora capsici in irrigation water sources. (A) Agarose gel showing results from the LAMP amplification of tested water from seven farm in South Georgia. Sample names from left to right: P1, P2, P3, P4, P5, P6, P7, Negative control, N. (B) LAMP results visualized using warmstart colorimetric dye of field samples: P1, P2, P3, P4, P5, P6, P7, Negative control, N. (C) Results from LAMP amplification of field samples using graph. Red: P1, Green: P2, Purple: P3, Yellow: P4, Blue: P5, Orange: P6, Pink: P7, Negative control, N. (D) Agarose gel showing conventional PCR results of the amplification using specific primers PC-1/PC-2 (note than only one site tested positive in comparison to three in LAMP). Please click here to view a larger version of this figure.
Pond name | County, State | Target crops for irrigation | PCR Detection | LAMP Detection | History of Disease (Y/N) |
P1 | Tift, GA | Vegetables | | + | N |
P2 | Tift, GA | Vegetables | – | – | N |
P3 | Tift, GA | Vegetables | – | – | N |
P4 | Tift, GA | Vegetables | + | + | N |
P5 | Tift, GA | Vegetables | – | – | N |
P6 | Tift, GA | Vegetables | – | + | N |
P7 | Tift, GA | Vegetables | – | – | N |
Table 1: Detection of irrigation water from Southern GA.
Primer type | Primer name | Sequence 5’-3’ | Source | ||
LAMP | PCA3-F3 | TGTGTGTGTGTTCGATCACA | This study | ||
PCA3-B3 | TTTTTGCGTGCGTCCAGA | This study | |||
PCA3-FIP | GACACCAAGCACTCGTACTOGTTTTTACAATTGTGCAGAGGGAGGA | This study | |||
PCA3-BIP | AGAACGAGTATTCGGCGGCGTTTTGAAAAAGGACCACCCCCG | This study | |||
PCA3-LF | TGTCGAATGGATTTGCGATCTT | This study | |||
PCA3-LB | ATACGCAGGTCATTTGACTGAC | This study | |||
PCR | PC-1 | GTCTTGTACCCTATCATGGCG | Zhang et al., 2006 | ||
PC-2 | CGCCACAGCAGGAAAAGCATT | Zhang et al., 2006 |
Table 2: Primers used in this study.
Parameters | Traditional | Conventional PCR | LAMP |
Sensitivity | NA | 4.8 X 102 spores/ml | 1.2 X 102 spores/ml |
Time | 2 weeks or longer | 2-3 hours (not including DNA extraction) | 30 mins – 1 hour (not including DNA extraction) |
Preparation | • Media creation • Plating and isolation and • Designing a trap |
• Spore collection using Filter paper • DNA extraction and • PCR assay |
• Spore collection using Filter paper • DNA extraction and • LAMP assay |
Materials | • Autoclave • Media and plates • Incubation room • Flow hood • Eggplant • Milk Crate |
• Thermal cycler • Agar gel • Gel Doc |
• Heat block or • Genie III |
Cost | $5.00 per trap | $0.60 per reaction | $0.75 per reaction |
Table 3: Comparison of methods for detection of P. capsici.
Supplementary Figure 1: Location of Tift County, GA and samples of irrigation water taken from various locations from within the state. Positive samples were shown as red dots. Please click here to download this figure.
Supplementary Figure 2: Design of LAMP primer set. Arrows show direction of how primers are read. Please click here to download this figure.
The testing of irrigation water for phytopathogens is a crucial step for growers using irrigation ponds and recycled water27. Irrigation ponds provide a reservoir and breeding ground for a number of phytopathogens as excess irrigation water is directed from the field to the pond carrying with it any pathogens that may have been present16,27. The traditional method for detection of a plant pathogens in a large water source is to set a bait for the pathogen by using susceptible host tissue (e.g., fruit, leaves) suspended in the pond and wait for an infection to take place, then remove the fruit/leaves and confirm the diagnosis with microscopy or molecular methods13,14. These methods are limiting due to the amount of time required to run the detection test (2 weeks or longer), and the labor and equipment required. Additionally, extensive experience and knowledge in visual diagnosis, pathogen morphology, and taxonomy are required for accurate results. Molecular techniques such as PCR, qPCR, and DNA hybridization require significantly less time (3-4 h) than the traditional methods of detection; however, they require expensive equipment and a laboratory setting. Additionally, these techniques do not allow for the processing of large volumes of water. Serological assays too, fall short in their detection ability due to non-target positive reactions, and no species-specific assays for Phytophthora species have been developed. Loop-mediated isothermal amplification (LAMP) has recently been used as an on-site diagnosis technique for rapid and sensitive detection of multiple pathogens as the assay only requires a single temperature rather than a thermal cycler28,29. LAMP can be run in the field using a colorimetric dye for visual confirmation or using a real-time amplification machine for results in less than one hour30.
The goal of this experiment was to develop a rapid and sensitive method to detect the presence of Phytophthora capsici in water sources either on-site or in a laboratory. To increase the speed of detection and to combat the limitations of the previously mentioned methods for detecting P. capsici in irrigation water, we designed a method using filter paper to capture the spores and extract their DNA from a larger volume of water. After spores were captured using the filter paper technique and DNA was extracted, the presence of the pathogen was confirmed based on a newly designed LAMP primer set specific to P. capsici. Detection sensitivity and specificity was compared using LAMP and PCR. In all 3 replications and with all of the zoospore concentrations, LAMP was a quicker and more sensitive detection method (Table 3). This method is not limited by having a small sample volume as traditional methods, as this method can be test up to 1 L of water at a single time, increasing the chances of pathogen detection. It was noted in testing that pouring irrigation water slowly through the Buchner funnel at a speed of no more than 40 mL per second increased the spore capture ability of the filter paper.
To validate the detection protocol, water samples from the field where P. capsici was suspected to be present were also taken (Supplementary Figure 1) to test the designed method with a practical scenario. Out of the 7 farms tested, 3 were positive for the presence of P. capsici using the LAMP assay (Figure 6A-6C) while only one farm was positive when using the conventional PCR assay (Figure 6D), showing LAMP as a more sensitive assay for this method of testing irrigation water. Although, this filter based LAMP assay could detect DNA from a concentration as low as 1.2 x 102 zoospores/mL which was significantly less than the original sensitivity of this assay (0.01 ng genomic DNA equivalent to ~5 spores) with unfiltered zoospore suspension. The previous LAMP assay by Dong et. al. (2015)20 was run on the same serial dilution and the level of sensitivity was the same (Figure 2F). The level of detection for a PCR assay with unfiltered spore solution have also shown a higher level of detection (equivalent ~10 spores) which was very similar to the previous PCR based findings10. The spore detection limit of the traditional baiting method of detection was not checked as a single spore could cause infection depending on its individual ability. The decreased sensitivity found in both LAMP and PCR assays is likely due to some spores flowing through or around the filter paper, or once attached to the filter paper, unable to be extracted with 100% efficiency. Nevertheless, this new LAMP and filter system can process and analyze a much higher volume of water than previous methods and confers a higher level of specificity and speed for in-field detection.
Of the extraction methods used, magnetic bead-based extraction was the most rapid and did not require the use of external machines such as a bead beater or centrifuge making it useful for in-field extractions and compliments the portable feature of the LAMP assay. The CTAB based method yielded the highest concentration of DNA but took the longest amount of time, while the commercial plant DNA extraction kit (e.g., DNeasy) was second in both time required and DNA concentration acquired.
With both CTAB and the commercial plant DNA extraction kit, during the homogenization of filter paper it was noted that homogenization was more successful and yielded a higher concentration if the CTAB solution (or extraction buffer) was added to the tube with the pieces of filter paper before bead beating or hand homogenization commenced. Bead beating was done 3 times for one minute each, but vortexing and agitating the tube in between each round was necessary for complete homogenization in all extraction methods. Importantly, the filter paper is subject to getting stuck on the sides or bottom of the tube, so it is crucial to make sure the filter paper is off the walls so that it gets homogenized.
The total time required for amplification is 90-120 min and can be easily done in the field for on-site diagnosis. This filter method is also designed to filter significant amounts of water to increase the possible chances for the detection of the pathogen. This method is also applicable to many pathogens that can accumulate in a water source, particularly genera such as: Pythium, Phytophthora, Fusarium, and bacteria; the only change required will be the development of an equally specific LAMP primer set for the targeted pathogen30.
A significant output of this work is the development of a highly sensitive and rapid filter paper-based LAMP assay for the detection of P. capsici in irrigation water sources. We expect that this study will lead to an increase in awareness of contamination of recycled irrigation water, eventually improving management of Phytophthora associated diseases, and consequently reduce production costs and increase crop yield. Such information is highly needed to improve vegetable production sustainability and enhance profitability of vegetable productions.
The authors have nothing to disclose.
This work received the financial support of Georgia Commodity Commission for Vegetables project ID# FP00016659. The authors thank Dr. Pingsheng Ji, University of Georgia and Dr. Anne Dorrance, Ohio State University for providing pure cultures of Phytophthora spp. We also thank Li Wang and Deloris Veney for their technical assistance throughout the study.
Agarose gel powder | Thomas Scientific | C997J85 | |
Buchner funnel | Southern Labware | JBF003 | |
Bullet Blender | Next Advance | BBX24 | |
Centrifuge 5430 | Eppendorf | 22620509 | |
Chloroform | Fischer Scientific | C298-500 | |
CTAB solution | Biosciences | 786-565 | |
Dneasy Extraction Kit | Qiagen | 69104 | |
Filter Flask | United | FHFL1000 | |
Filter Paper | United Scientific Supplies | FPR009 | |
Gel Green 10000X | Thomas Scientific | B003B68 (1/EA) | |
Genie III | OptiGene | ||
Hand pump | Thomas Scientific | 1163B06 | |
Iso-amyl Alcohol | Fischer Scientific | BP1150-500 | |
LAVA LAMP master mix | Lucigen | 30086-1 | |
Magnetic bead DNA extraction | Genesig | genesigEASY-EK | |
Magnetic Separator | Genesig | genesigEASY-MR | |
polyvinylpyrrolidone | Sigma Aldrich | PVP40-500G | |
Primers | Sigma Aldrich | ||
Prism Mini Centrifuge | Labnet | C1801 | |
T100 Thermal Cycler | Bio-Rad | 1861096 | |
UV Gel Doc | Analytik Jena | 849-00502-2 | |
Warmstart Colorimetric Dye | Lucigen | E1800m | |
Wide Mini ReadySub-Cell GT Cell | Bio-Rad | 1704489EDU | |
70% isopropanol | Fischer Scientific | A451-1 |