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Laboratory Protocols for Phytophthora Species

Description

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


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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