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Laboratory Protocols for Phytophthora Species

This new compilation of working protocols for Phytophthora research was created to meet this demand and se​rve 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 and ​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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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

PROTOCOL 07-03.1: PARP(H)-CMA

Kelly Ivors

PROTOCOL 07-04.1: PARP(H)-V8A

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

PROTOCOL 07-08.1: CARP

Everett Hansen

PROTOCOL 07-09.1: CARP-D

Everett Hansen

PROTOCOL 07-10.1: CARP+

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

References

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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