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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • 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,

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 s.......

Representative 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 .......

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

References

  1. Hong, C., Moorman, G. J. Plant pathogens in irrigation water: challenges and opportunities. Critical Reviews in Plant Sciences. 24 (3), 189-208 (2005).
  2. Malkawi, H. I., Mohammad, M. J.

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Phytophthora CapsiciIrrigation WaterLoop mediated Isothermal AmplificationPathogen DetectionDNA ExtractionFilter PaperMagnetic BeadsIrrigation Water ContaminationWaterborne Pathogen

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