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Laboratory Protocols for Phytophthora Species


The world’s interest in Phytophthora species has been growing in recent years—to the point where more research projects on this important pathogen are expected.

This new compilation of working protocols for Phytophthora research was created to meet this demand and serve the growing need for a coherent collection of methods to study members of this genus.

Phytophthora Protocols is a fully online and continually updated resource that includes step-by-step instructions and images to ensure the accuracy and consistency of your Phytophthora research techniques.  

Phytophthora Protocols can be used as a baseline for optimizing lab research, developing custom research techniques, and other research applications. This collection of 79 individual protocols is divided into sections, including:

  • Phytophthora Detection
  • Identification and Isolation
  • Phytophthora Inoculum Production and Inoculation
  • Long-term Storage of Phytophthora
  • Mating Techniques
  • Chemical Sensitivity Studies
  • DNA Techniques
  • Media
  • Solutions for Phytophthora Studies 

This continually growing, customized collection of protocols is ideal for anyone researching Phytophthora or other oomycetes, as well as plant disease diagnosticians and state/federal plant health regulators.

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Table of Contents

Front Matter

Kelly Ivors

PART 1: Phytophthora Detection, Identification, and Isolation

PROTOCOL 01-01.1: Identifying species of Phytophthora based on morphology

Steven Jeffers

PROTOCOL 01-02.1: Isolation of Phytophthora infestans from tomato leaves

Kelly Ivors

PROTOCOL 01-03.1: Isolation of Phytophthora infestans from tomato fruit

Jean Ristaino and L. Guo

PROTOCOL 01-04.1: Isolation of Phytophthora infestans from potato leaves

Kelly Ivors

PROTOCOL 01-05.1: Isolation of Phytophthora nicotianae from soil

David Shew and Courtney Gallup

PROTOCOL 01-06.1: Isolation of Phytophthora nicotianae from tobacco stems and roots

David Shew

PROTOCOL 01-07.1: Float incubation technique for Phytophthora and Pythium diagnostics

Gail Ruhl

PROTOCOL 01-08.1: Recovering Pythium and Phytophthora spp. from infected plant tissue

Jason Brock and Glenn Beard

PROTOCOL 01-09.1: Preparation of hyphal tip Phytophthora cultures

Sabine Werres

PROTOCOL 01-10.1: Purification of Phytophthora cultures contaminated with bacteria

Kelly Ivors

PROTOCOL 01-11.1: Isolation and detection of Phytophthora spp. using Rhododendron leaf baits

Kelly Ivors

PROTOCOL 01-12.1: Rhododendron leaf test to detect Phytophthora spp. in roots, soil, and water

Sabine Werres and Corina Junker

PROTOCOL 01-13.1: Pear baiting for isolation of Phytophthora spp. from various substrates

Daniel Hüberli

PROTOCOL 01-14.1: Detection of Phytophthora spp. in water supplies

D. Michael Benson

PROTOCOL 01-15.1: Qualitative and quantitative recovery of Phytophthora cinnamomi from soil

Kelly Ivors

PROTOCOL 01-16.1: Sieving–Centrifugation method using a sucrose solution for extraction of Phytophthora capsici oospores from field soil

Mohammad Babadoost

PROTOCOL 01-17.1: Detecting Phytophthora spp. on plant foliage using ELISA

Arthur Wagner

PART 2: Phytophthora Inoculum Production and Inoculation

PROTOCOL 02-01.1: Maintaining Phytophthora infestans in planta

Kelly Ivors and Dreama Milks

PROTOCOL 02-02.1: Mass production of Phytophthora nicotianae zoospores using a wet plate method

Chuan Hong

PROTOCOL 02-03.1: Production of Phytophthora zoospores

Kelly Ivors

PROTOCOL 02-04.1: Induction of sporangia formation and zoospore release for Phytophthora ramorum and other Phytophthora spp.

Everett Hansen

PROTOCOL 02-05.1: Obtaining and manipulating zoospores of Phytophthora infestans

Howard Judelson

PROTOCOL 02-06.1: Production of Phytophthora sporangia using vinca leaves

Kelly Ivors and Walter Gutierrez

PROTOCOL 02-07.1: Rice grain method for Phytophthora inoculum production

D. Michael Benson and Kala Parker

PROTOCOL 02-08.1: Vermiculite method for Phytophthora inoculum production

Kelly Ivors

PROTOCOL 02-09.1: Modified vermiculite method for Phytophthora inoculum production

Kelly Ivors

PROTOCOL 02-10.1: Inoculating detached leaves via a Phytophthora zoospore drop

Rachel Linzer and Matteo Garbelotto

PROTOCOL 02-11.1: Detached leaf inoculation for tanoak

Katherine Hayden and Matteo Garbelotto

PROTOCOL 02-12.1: Production of Phytophthora inoculum on oat grain

Kelly Ivors

PART 3: Long-Term Storage of Phytophthora spp.

PROTOCOL 03-01.1: Long-term storage of Phytophthora nicotianae isolates in water

David Shew and Courtney Gallup

PROTOCOL 03-02.1: Long-term storage of Phytophthora cultures in water

Wendy Sutton, Paul Reeser, and Everett Hansen

PROTOCOL 03-03.1: Long-term storage of Phytophthora and Pythium isolates using hemp seed

R. Wick, Nina Shishkoff, and Margery Daughtrey

PROTOCOL 03-04.1: Long-term storage of Phytophthora isolates using oatmeal agar

Sabine Werres

PROTOCOL 03-05.1: Long-term storage of Phytophthora infestans on Rye A agar

Alison Lees

PROTOCOL 03-06.1: Long-term preservation of Phytophthora spp. in liquid nitrogen

Kelly Ivors

PROTOCOL 03-07.1: Freezing Phytophthora cultures for long-term storage in liquid nitrogen

Paul Tooley

PROTOCOL 03-08.1: Storage of Phytophthora infestans using liquid nitrogen

Kevin Myers and William Fry

PROTOCOL 03-09.1: Cryopreservation of Phytophthora spp.

Michael Coffey

PART 4: Mating Techniques

PROTOCOL 04-01.1: Mating-type determination of heterothallic Phytophthora species

David Shew and Courtney Gallup

PROTOCOL 04-02.1: Phytophthora and Pythium gametangia production using grass blades

Frank Martin

PROTOCOL 04-03.1: Determining the viability of Phytophthora oospores

Kestrel McCorkle, Courtney Gallup, and David Shew

PROTOCOL 04-04.1: Production of Phytophthora ramorum oogonia in vitro

Clive Brasier and Susan Kirk

PART 5: Chemical Sensitivity Studies

PROTOCOL 05-01.1: In vitro sensitivity of Phytophthora spp. to mefenoxam

David Shew and Courtney Gallup

PROTOCOL 05-02.1: Evaluating fungicide sensitivity of Phytophthora capsici in vitro

Anthony Keinath

PART 6: DNA Techniques

PROTOCOL 06-01.1: Preparation of freeze-dried mycelium for DNA extraction from Phytophthora spp.

Alison Lees

PROTOCOL 06-02.1: Quick and easy DNA extraction from Phytophthora infestans

Alison Lees

PROTOCOL 06-03.1: Modified CTAB extraction of fungal DNA

Kelly Ivors

PROTOCOL 06-04.1: DNA extraction from plant material or mycelium using IsoCode Stixs or FTA cards

Jean Ristaino

PROTOCOL 06-05.1: Desiccation of plant leaves for molecular detection of Phytophthora spp.

Pierluigi Bonello

PROTOCOL 06-06.1: Phytophthora DNA extraction and amplification from dried plant or herbarium samples

Jean Ristaino

PROTOCOL 06-07.1: DNA isolation from Phytophthora mycelium using the DNeasy Plant Mini Kit (QIAGEN)

Kelly Ivors

PROTOCOL 06-08.1: DNA isolation from Phytophthora mycelium using the DNeasy Plant Mini Kit (QIAGEN)

Paul Tooley and Marie Carras

PROTOCOL 06-09.1: DNA extraction from hyphal tips of Phytophthora spp.

Matteo Garbelotto

PROTOCOL 06-10.1: DNA extraction for RFLP analysis of Phytophthora infestans

Steve Goodwin

PROTOCOL 06-11.2: Molecular identification of Phytophthora isolates using a DNA sequence-based approach and the Phytophthora Database

Seogchan Kang, Michele Mansfield, Bongsoo Park, and Frank Martin

PART 7: Media and Solutions for Phytophthora Studies

PROTOCOL 07-01.1: Nonsterile soil extract solution, 1.5% (NS-SES)

Steve Jeffers

PROTOCOL 07-02.1: Water agar (WA)

Kelly Ivors


Kelly Ivors


Steve Jeffers

PROTOCOL 07-05.1: 5% clarified V8 PARP(H)

David Shew

PROTOCOL 07-06.1: PCH agar

Kelly Ivors

PROTOCOL 07-07.1: Selective Phytophthora agar

Paul Tooley


Everett Hansen


Everett Hansen


Everett Hansen

PROTOCOL 07-11.1: V8 agar (V8A) or broth

Steve Jeffers

PROTOCOL 07-12.1: Super V8 agar (sV8)

Steve Jeffers

PROTOCOL 07-13.1: V8 rye agar

Jean Ristaino

PROTOCOL 07-14.1: Rye A agar

Kelly Ivors

PROTOCOL 07-15.1: Rye B agar

Jean Ristaino

PROTOCOL 07-16.1: Oatmeal agar

Sabine Werres

PROTOCOL 07-17.1: 5% carrot agar (CA)

David Shew

PROTOCOL 07-18.1: Fresh lima bean agar

Jean Ristaino

PROTOCOL 07-19.1: Pea sucrose agar (PSA)

Kelly Ivors

PROTOCOL 07-20.1: Pea broth

Peter Bonants

PROTOCOL 07-21.1: Corn meal PPP agar

Kelly Ivors

PROTOCOL 07-22.1: Rye seed medium

Michael Coffey

PROTOCOL 07-23.1: Carrot agar (CA) or broth

Michael Coffey

PROTOCOL 07-24.1: Pea or lima bean agar or broth

Michael Coffey

Back Matter


Kelly Ivors

Archived Protocols

PROTOCOL 06-11.1: Molecular identification of Phytophthora isolates using a DNA sequence-based approach and the Phytophthora Database

Seogchan Kang and Michele Mansfield

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