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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. On-site detection of Phytophthora capsici from irrigation water using portable loop-mediated isothermal amplification

  1. Setting up the pump and filter
    1. Attach a filtering flask to a tube that is connected to a hand pump so that when the pump is activated, air will be pulled in through the mouth of the filtering flask.
    2. Fit the Buchner funnel into the rubber stopper to the mouth of the filtering flask and fit the appropriately sized piece of filter paper into the Buchner funnel so that air is pulled through the filter paper. The filter paper should have a retention size of 15 µm.
      NOTE: The filter paper must fit to the edges of the Buchner funnel so that minimal water will flow around the filter paper.
  2. Water sampling and filtering
    1. Take water samples from the targeted source. Water may have small amounts of debris but not significant sediment or soil.
    2. Pour up to 1,000 mL (1 Liter) of test water over the filter paper placed inside the Buchner funnel slowly enough to prevent overflow, while the hand pump (or vacuum) is being used to create a suction to pull the water through.
      NOTE: There is no minimum amount of water that can be tested using this method, and although it at least 50 mL is suggested, 1000 mL is the maximum for this method.
    3. Using forceps, remove the filter paper from the Buchner funnel and cut it into small pieces with sterile scissors. Add as many pieces (8-12) as can be submerged in the amount of extraction buffer (400 µL for magnetic bead-based extraction) required by the protocol into a 1.5 mL tube. Save remaining pieces of filter paper for processing after the first set has been extracted.
    4. Vortex or otherwise agitate the pieces of filter paper and extraction buffer for 10 s every minute for 5 min. Then, using forceps, remove the filter paper losing as little of the extraction buffer as possible. Repeat this step with the remaining pieces of filter paper until all pieces have been vortexed/agitated and soaked in the extraction buffer.
  3. Magnetic bead-based extraction of DNA from filter paper
    1. To the 1.5 mL tube (which now contains approximately 200-300 µL of extraction buffer), add 20 µL of proteinase K and 10 µL of 10 ng/µL RNase.
    2. Incubate at room temperature for 15 min, vortex or shake the tube every 3 min.
    3. Add 500 µL of magnetic beads with the binding buffer to the sample and mix well by shaking. Then incubate for 5 min at room temperature.
    4. Place the tube in the magnetic separator rack for 2 min until all beads have been pulled to the magnet. Remove and discard the supernatant.
    5. Remove the tube from the magnetic separator. Add 500 µL of Wash Buffer 1 and re-suspend beads by shaking the tube vigorously. Wait for 30 s and then place the tube back in the magnetic separator. Wait for 2 min until all beads have been pulled towards the magnet before removing and discarding the supernatant.
      NOTE: When waiting for the magnetic beads to magnetize to the separator, we recommend inverting the tube, which can dislodge magnetic beads stuck to the cap and the sides of the tube and result in a greater number of beads being attached.
    6. Repeat step 1.3.5 with 500 µL of Wash Buffer 2.
    7. Repeat step 1.3.5 with 500 µL of 80% ethanol.
    8. Airdry the magnetic bead pellet for 15 min at room temperature (18-27 °C) with the lid open. If temperatures do not permit, incubate in a gloved hand with the cap open for 15 min.
    9. Remove the tube from the magnetic separator. Add 50 µL of elution buffer and re-suspend the beads by pipetting up and down for 1 min.
    10. Place tube back in a magnetic separator. Wait 2 min before transferring the supernatant without disturbing the beads to a separate tube for DNA storage.
      NOTE: Here the experiment can be paused before moving on. Extracted DNA should be stored on ice or in a -20 °C freezer.
  4. Application of newly developed LAMP assay
    1. Prepare LAMP primer mix using 0.2 µM of each F3 and B3 primer, 0.8 µM of each Loop-F and Loop-B primer, and 1.6 µM of each FIP and BIP primer (Table 2).
    2. Add the following LAMP solution to a single PCR tube, or to each individual tube in the 8 tube strip: 2.5 µL of primer mix (step 1.4.1), 12.5 µL of LAVA LAMP master mix, 1 µL of extracted DNA, and 9 µL of ddH2O. The total volume is 25 µL.
      NOTE: If using the portable amplification instrument (e.g., Genie III) with colorimetric dye (e.g., Warmstart), refer to section 2.4.2- 2.4.3.
    3. Designate two tubes as positive and negative controls. For the positive control, use either a control provided by the LAVA LAMP kit, or a known positive DNA sample. For the negative control, use ddH2O. For both, substitute the 1 µL of extracted DNA for 1 µL of the positive control or ddH2O.
    4. Set the samples into a heat block (or portable amplification instrument) set to 64 °C for 45 min.
      NOTE: Other extraction methods can be used here instead of magnetic bead based extraction. CTAB and a commercial DNA extraction kit were both successfully tested using the standard protocols and substituting the filter paper pieces for the plant sample24,25. Results were compared in Table 2. If concentration of DNA can be quantified, use between 1-10 ng genomic DNA.
  5. Visualization of results
    1. If performed in a laboratory setting, view the amplification products by loading 5 µL of each sample into a 1% agarose gel, running them in a gel electrophoresis machine, and imaging them in a UV imaging machine.
    2. If a colorimetric dye (e.g., Warmstart) was used, view the color change to determine results as positive or negative.
    3. If a portable amplification instrument (e.g., Genie III) was used, view the amplification graph on the screen to determine the results.
  6. Application of previously developed PCR assay
    NOTE: If using a conventional PCR assay, steps 1-3 remain the same, and the following steps should be applied in place of steps 1.4 and 1.5.
    1. Add 1 µL of each DNA extraction to individual tubes containing the following components: 12.5 µL of Green PCR master mix, 9.5 µL of ddH2O, and 1 µL of forward and reverse primers (Table 2).
    2. Spin down each sample using a microcentrifuge and place tubes into a thermal cycler.
    3. Use the following thermal cycler settings in accordance to the previous publications: 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min.
    4. Run the product in a 1% agarose gel. Observe the presence of bands under UV light where positive reactions will have a band size of ~508 bp.
  7. Traditional method of detection
    NOTE: There are multiple methods of selective plating for pathogen detection, and the following is a general protocol for P. capsici.
    1. First, obtain a healthy eggplant fruit (a susceptible host for P. capsici) and surface sterilize by washing the fruit surface with 70% isopropyl alcohol.
    2. Place the eggplant fruit into milk crates with a flotation device (polyethylene foam or other) and deploy into irrigation ponds. Secure each bait trap to a single point and leave the traps in the water reservoir (recycled irrigation water) for at least 7 days or until fruit rot symptoms are observed. Collect the fruit and transport it to the laboratory.
    3. Rinse and dry the fruit in a sterile hood before removing small pieces of infected tissues and place them on a plate of PARPH medium amended with 25 mg/L pentachloronitrobenzene, 0.0005% pimaricin, 250 mg/L ampicillin, 10 mg/L rifampicin and 50 mg/L hymexazol. Incubate plates at 25 °C for 5 days.
    4. View plates under a compound microscope for traditional morphological identification at 4 days after isolation.

2. Determining the detection limit of zoospore concentration

  1. Making zoospore suspension
    1. Incubate P. capsici on V8 agar (100 mL of V8 juice, 900 mL of ddH2O, 1 g of CaCO3) plates for 1 week at 26 °C. Multiple plates can be used to obtain a larger amount of zoospore suspension.
    2. Incubate the plates under continuous light at room temperature for 3 days to stimulate sporulation.
    3. Flood the plates by adding 15 mL of ddH2O to each plate and place them in a 4 °C fridge for 25 min. Then return to room temperature for 30 min.
    4. Agitate plates to dislodge zoospores and pipette the solution into a single 50 mL tube from all the plates.
    5. To obtain an accurate estimate of zoospore concentration, add 10 µL of the spore suspension onto a hemocytometer and observe under a microscope to count zoospores and estimate the average concentration.
  2. Serial dilution
    1. Add 1 mL of the spore suspension and 9 mL of ddH2O to a separate tube. Repeat this step for as many 10-fold dilutions as desired.
    2. Spore solutions are then submitted for DNA extraction based on the previous protocol using the filtration method.
      NOTE: If a larger volume of suspension is desired, double the volume: 2 mL of spore suspension and 18 mL of ddH2O.
  3. Detection of zoospore concentration limit
    1. Evaluate spore detection limit by running each serial dilution individually through the assay until clearly positive results are no longer observed. Once the final dilution is obtained, dilute by a factor of 2 (4.8 to 2.4 in this example) and run the assay again to get a more accurate detection limit.
  4. Development and optimization of the LAMP method
    NOTE: LAMP primers were designed based on a 1121-base pair (bp) fragment of P. capsici (Li et al.19) as shown in Supplementary Figure 2.
    1. If colorimetric dye (e.g., Warmstart) is used, use the following the solution: 2.5 µL of primer mix, 12.5 µL of colorimetric dye, 0.5 µL of Green fluorescent dye, 1 µL of extracted DNA, and 8.5 µL of ddH2O. The total volume is 25 µL.
    2. When using the portable amplification instrument with LAVALAMP mastermix, have an initial step of 95 °C for 3 min as recommended by the manufacturer, but this is not required. A final annealing step is not required to observe the color change or amplification graph. Do not run a warmstart step if the commercial colorimetric dye is to be used.
    3. View LAMP assay results in one of the following methods: run samples on a 1% agarose gel or view using a UV imaging machine with the naked eye or view at the Genie III real-time amplification screen.
    4. Optimize the temperature of the LAMP assay by using the portable amplification instrument and analyzed using the real-time amplification graph for speed and level of sensitivity. Run samples with unique temperatures to determine the fastest amplification with the highest level of sensitivity.
    5. Determine the detection limit of the LAMP developed assay by making a serial dilution of extracted DNA (as with spore suspension in step 2.2.1) and maintaining reaction conditions as previously described for the LAMP reaction for each dilution.
  5. Detection limit determination and comparison with conventional PCR method
    1. Use DNA extracted in steps in section 1.3 to compare the detection level of conventional PCR with that of the LAMP assay.
    2. Add 1 µL of DNA to a PCR tube that contained 1 µL of both forward and reverse PCR primers (Table 2), 12.5 µL of Green PCR Mastermix, and 9.5 µL of ddH2O for a total of 25 µL.
    3. Amplify samples in a thermal cycler using the following conditions: 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 1 min, and final extension at 72 °C for 10 min.
    4. Run samples in an electrophoresis machine on a 1% agarose gel and view on a UV imaging machine. The excepted band size was 508 bp.

Results

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 i...

Discussion

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 ...

Disclosures

The authors have nothing to disclose or any conflicts of interest.

Acknowledgements

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.

Materials

NameCompanyCatalog NumberComments
Agarose gel powderThomas ScientificC997J85
Buchner funnelSouthern LabwareJBF003
Bullet BlenderNext AdvanceBBX24
Centrifuge 5430Eppendorf22620509
ChloroformFischer ScientificC298-500
CTAB solutionBiosciences786-565
Dneasy Extraction KitQiagen69104
Filter FlaskUnitedFHFL1000
Filter PaperUnited Scientific SuppliesFPR009
Gel Green 10000XThomas ScientificB003B68 (1/EA)
Genie IIIOptiGene
Hand pumpThomas Scientific1163B06
Iso-amyl AlcoholFischer ScientificBP1150-500
LAVA LAMP master mixLucigen30086-1
Magnetic bead DNA extractionGenesiggenesigEASY-EK
Magnetic SeparatorGenesiggenesigEASY-MR
polyvinylpyrrolidoneSigma AldrichPVP40-500G
PrimersSigma Aldrich
Prism Mini CentrifugeLabnetC1801
T100 Thermal CyclerBio-Rad1861096
UV Gel DocAnalytik Jena849-00502-2
Warmstart Colorimetric DyeLucigenE1800m
Wide Mini ReadySub-Cell GT CellBio-Rad1704489EDU
70% isopropanolFischer ScientificA451-1

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