Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

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 crops^2,3,

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.......

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 present^16,27. The traditional method for detection of a plant pathogens in a large water source is to set a bait .......

Materials

Name	Company	Catalog Number	Comments
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