Practical guide to detection

and identification of
Phytophthora
Version 1.0

Mating Type Tests

• All 119 isolates were of the A2 mating type

André Drenth & Barbara Sendall

CRC for Tropical Plant Protection
Brisbane Australia
Practical guide to detection and
identification of Phytophthora

Version 1.0

André Drenth and Barbara Sendall

CRC for Tropical Plant Protection

Brisbane Australia
Practical guide to detection and identification of Phytophthora

Preface

This guide has been written as a first step to help your foray into the world of
Phytophthora. It is not meant to be exhaustive or comprehensive but it is aimed at
making you familiar with the basics of identifying Phytophthora diseases, give you
useful hints for the choice and preparation of selective media and tips on isolation and
culturing Phytophthora. It also gives very basic methods and procedures for
identification of different species of Phytophthora using a limited set of
morphological characters. This guide has been prepared as a working and training
manual for project PHT/1996/193 "Survey of the presence and importance of
Phytophthora in South-east Asia" funded by the Australian Centre for International
Agricultural Research (ACIAR). It therefore focuses on only eight species of
Phytophthora, which are most commonly found in Southeast Asia.

This guide is meant to be practical and should be seen as an introductory laboratory

manual only. It does not contain any mycological novelties or in depth treatments. It
is simply aimed at helping students and plant pathologists to take the first steps into
the world of Phytophthora. And we speak from personal experience when we say
what a fascinating world that is. For more details and in depth descriptions of all
species of Phytophthora and their general biology we strongly recommend that you
consult the most excellent book "Phytophthora diseases worldwide" written by
Donald Erwin and Olaf Ribeiro (1996). This book contains the descriptions of all
Phytophthora species and vast amounts of information concerning Phytophthora
diseases.

This is the first draft of this practical guide and mistakes are solely our responsibility.
However, we would very much appreciate any comments on how to improve its
usefulness and incorporate any tips you may have and want to share with other
Phytophthora workers.

André Drenth and Barbara Sendall

Brisbane, June 2001

CRC for Tropical Plant Protection

Indooroopilly Research Centre
Plant Pathology Building
80 Meiers Road
Indooroopilly QLD 4068
Australia
Email: [email protected]

© Copyright: No part of this publication may be reproduced, stored in a retrieval system, transmitted, in
any form or any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior
written permission of the authors.
 Table of contents
1. Introduction to the genus Phytophthora.................................................5

1.1 THE GENUS PHYTOPHTHORA ........................................................................................................... 5

1.2 EVOLUTIONARY PLACEMENT .......................................................................................................... 5
1.3 BIOLOGY......................................................................................................................................... 6
1.3.1 Life cycle................................................................................................................................ 6
1.3.2 Host range ............................................................................................................................. 7
1.3.3 Mating system ........................................................................................................................ 7
1.4 PHYTOPHTHORA SYSTEMATICS ........................................................................................................ 7
1.5 PATHOGENICITY OF PHYTOPHTHORA ............................................................................................... 9
1.5.1 Root rot................................................................................................................................... 9
1.5.2 Collar rot ................................................................................................................................ 9
1.5.3 Tree canker ........................................................................................................................... 10
1.5.4 Stem lesions .......................................................................................................................... 10
1.5.5 Bud rot .................................................................................................................................. 10
1.5.6 Leaf blight............................................................................................................................. 10
1.5.7 Fruit rot ................................................................................................................................ 11
1.5.8 Tuber and corm rot............................................................................................................... 11

2. Media and antibiotics for isolation of Phytophthora from diseased

plant tissue and soil ................................................................................12

2.1 BASIC MEDIA FOR ISOLATION FROM DISEASED TISSUE ............................................................. 12

2.2 SELECTIVE MEDIA FOR ISOLATION FROM DISEASED TISSUE ..................................................... 13

3 Detection and isolation of Phytophthora from infected plant tissue

and soil .....................................................................................................17

3.1 SELECTION OF SAMPLES FOR ISOLATION OF PHYTOPHTHORA ................................................... 17

3.2 ISOLATION OF PHYTOPHTHORA FROM INFECTED PLANT MATERIAL .......................................... 17
3.2.1 Preparation and surface sterilisation of tissue ............................................................... 18
3.2.2 Isolation from infected plant material by “baiting” with cocoa pods ............................ 19
3.3 ISOLATION OF PHYTOPHTHORA FROM SOIL .............................................................................. 19
3.3.1 Detection and isolation of Phytophthora by baiting from the soil.................................. 20
3.4 INCUBATION ............................................................................................................................ 20
3.5 LUPIN BAITING OF PHYTOPHTHORA FROM SOIL SAMPLES ......................................................... 22
3.7 CULTURING AND STORAGE OF PHYTOPHTHORA.............................................................................. 23
3.7.1 Culturing................................................................................................................................ 23
3.7.2 Long term storage in sterile water........................................................................................ 23
3.7.3 Long term storage in liquid nitrogen.................................................................................... 24
3.7.4 Liquid nitrogen storage protocol........................................................................................... 24
4 Identification of Phytophthora ................................................................25

4.1 CULTURES ............................................................................................................................... 26

4.2 MORPHOLOGICAL CHARACTERS .............................................................................................. 26
4.3 SPORANGIA ............................................................................................................................. 26
4.4 CHLAMYDOSPORES AND HYPHAL SWELLINGS ......................................................................... 28
4.5 SEXUAL STRUCTURES ............................................................................................................. 28
4.6 DIFFERENCES BETWEEN PYTHIUM AND PHYTOPHTHORA .......................................................... 29
4.7 LIST OF TERMS ........................................................................................................................ 30
4.8 MEDIA ..................................................................................................................................... 35
4.8.1 Carrot agar........................................................................................................................... 35
4.8.2 Dilute Lima bean agar or broth............................................................................................ 35
4.8.3 Lima bean extract agar......................................................................................................... 35
4.8.4 Salt Solution of Chen and Zentmyer (1970)........................................................................... 35

5 Plant inoculation tests with Phytophthora species ...............................36

5.1 INOCULATION OF SPORANGIA ONTO LEAF OR FRUIT TISSUE .......................................................... 36

5.2 INSERTION OF MYCELIUM INTO THE STEMS OF YOUNG SEEDLINGS ................................................ 37
5.3 INOCULATION OF WOODY ADULT PLANTS ..................................................................................... 37
5.4 INOCULATION OF SOIL .................................................................................................................. 37

6 References................................................................................................39
Drenth & Sendall Practical guide to Phytophthora 5

1. Introduction to the genus Phytophthora

1.1 The genus Phytophthora

Phytophthora de Bary 1887 is a cosmopolitan genus of Oomycete obligate plant
pathogens containing approximately 60 described species (Erwin and Ribeiro 1996).
Phytophthora species attack a wide range of plants, and are responsible for some of the
world’s most destructive plant diseases - examples include the European potato famine
of the 19th century caused by P. infestans (Bourke 1964). Phytophthora diseases have
been well studied in the temperate regions of the world. However, Phytophthora diseases
are very common throughout the wet tropical regions of the world and cause significant
diseases losses in many tropical fruit crops in the form of root rots, collar rots, stem
cankers, leaf blights and fruit rot. For example, P. palmivora alone causes a myriad of
severe diseases on many different crops including: black pod of cocoa; root, stem and
fruit rot of pawpaw; root rot and blight of citrus; bud rot in palms; black stripe in rubber;
and root rot, trunk canker, and fruit rot in durian.

1.2 Evolutionary placement

Phytophthora is a member of the Order Peronosporales within the Class Oomycetes in
the Kingdom Chromista (Hawksworth et al. 1995; van de Peer et al. 1996) see Table
1.1). The Oomycetes includes four orders, two of which, the Saprolegniales and the
Peronosporales, contain important plant pathogens. The other two orders contain
small groups of mainly aquatic fungal-like organisms. Within the Peronosporales, the
family Pythiaceae contains a number of genera, the best-known of which are
Phytophthora and its sister group, Pythium, a genus of approximately 120 species
(van der Plaats-Niterink 1981).

Table 1.1. Classification of the Oomycetes (Hawksworth et al. 1995).

Kingdom Class Order Family Genus

Chromista Oomycetes Lagenidiales

Leptomitales

Saprolegniales Saprolegniaceae Achlya

Saprolegnia

Peronosporales Pythiaceae Pythium

Phytophthora

Peronosporaceae Bremia
Peronospora

Albuginaceae Albugo
Drenth & Sendall Practical guide to Phytophthora 6

1.3 Biology
The Oomycetes share many characteristics of ecology and life history with the true
fungi. However, they are clearly distinguished from the Basidiomycetes and
Ascomycetes by their genetics and reproductive mechanisms (Erwin and Ribeiro 1996).
Their placement in the Kingdom Chromista (Cavalier-Smith 1986) is supported by a
large number of characteristics, including variation in metabolic pathways (Elliott 1983;
Hendrix 1970; Wang and Bartnicki-Garcia 1973), the presence of β-glucans rather than
chitin in cell walls (Bartnicki-Garcia and Wang 1983), production of motile heterokont
zoospores (Desjardins et al. 1969), and predominance of the diploid stage in the lifecycle
(Erwin and Ribeiro 1996).

1.3.1 Life cycle

The life cycle of Phytophthora may involve up to three asexual spore forms in
addition to the sexual spore form (Figure 1.1). Diploid vegetative mycelium produces
asexual sporangia, which may germinate directly, or differentiate to produce 8-32
zoospores, each of which pass through a cycle of dispersal and encystment before
germinating. Some species, such as P. cinnamomi, also produce asexual
chlamydospores from the mycelium. Sexual reproduction results in the production of
oospores. All spore types are potentially infective, and chlamydospores and oospores
also function as overwintering or resting structures.

All species of Phytophthora have a soil-borne resting stage. Some, such as P. sojae,
are also dispersed primarily in the soil, via the release of zoospores from infected
plant material. Others, such as P. palmivora, are aerially dispersed, primarily as
caducous (deciduous) sporangia.

Figure 1.1 Life cycle of Phytophthora infestans. Reprinted from Drenth (1994)
Drenth & Sendall Practical guide to Phytophthora 7

1.3.2 Host range

Species of Phytophthora vary greatly in their degree of host specificity. P. fragariae
var. rubi infects a single host species (Kennedy and Duncan 1995), while P.
cinnamomi is able to attack over 1000 different host plant species (Erwin and Ribeiro
1996), and other species occupy a continuum between these two extremes. Broad
host range Phytophthora species tend to attack their hosts using enzymes which affect
relatively unspecialised host chemical and mechanical resistance mechanisms (Brasier
1983), whereas some host specific species are known to possess virulence genes
which interact specifically, in a gene-for-gene system, with host resistance genes
(reviewed in Thompson and Burdon 1992).

1.3.3 Mating system

All isolates of Phytophthora are potentially bisexual, that is, they are able to produce
both male and female sexual structures, or gametangia (Galindo and Gallegly 1960).
However, only about half of the species of Phytophthora are homothallic, and able to
produce oospores rapidly and abundantly in single culture. The remaining species are
heterothallic, and produce gametangia only in response to chemical stimulation from
an isolate of the opposite mating type (Brasier 1992; Ko 1978).

The system of heterothallism involving A1 and A2 mating types is universal throughout

the genus. Isolates of opposite mating types from different species are often able to
reciprocally stimulate gametangial formation (Ko 1978, Yu et al. 1981). The mating
system of a Phytophthora species determines its ability to outbreed: homothallism
allows frequent selfing, whereas heterothallism encourages outbreeding. However, both
homothallic and heterothallic species do have a range of reproductive options.
Homothallic species have recently been shown to undergo low levels of outbreeding in
vitro (Whisson et al. 1994), while heterothallic species have been shown to inbreed at
low levels (Goodwin 1997, Judelson 1997).

1.4 Phytophthora systematics

The genus Phytophthora has been widely acknowledged as taxonomically ‘difficult’
(Brasier 1983), as many of the characters used for species identification are plastic,
highly influenced by environment, show overlap between species, and have an unknown
genetic basis. Nonetheless, since a major review of the genus was performed by
Waterhouse (1963), morphological characters have remained the basis for species
identification and taxonomy (Newhook 1978; Stamps et al. 1990). Waterhouse classified
species based primarily on papillation (Figure 1.2) and caducity (easy detachment) of
sporangia, type of antheridial attachment (Figure 1.3), and mating system. Based on this
analysis, the genus was divided into six major groups (Table 1.2), which were intended
solely as an aid to species identification, and were not meant to imply a natural
classification (Waterhouse 1963).
Drenth & Sendall Practical guide to Phytophthora 8

Figure 1.2 (a) Non-papillate, (b) semi-papillate and (c) papillate sporangia. Photographs from Erwin
and Ribeiro 1996.

oogonium
antheridium
antheridium

A B

Figure 1.3. Antheridial attachment – (a) paragynous, (b) amphigynous. Illustration from Erwin and
Ribeiro 1996.

Table 1.2. Classification of Phytophthora into six groups by Waterhouse (1963).

Group Sporangia Antheridial Examples

attachment

1 papillate paragynous P. cactorum, P. clandestina

II papillate amphigynous P. capsici, P. palmivora
III semi-papillate paragynous P. inflata, P. multivesiculata
IV semi-papillate amphigynous P. infestans, P. ilicis
V non-papillate paragynous P. megasperma, P. sojae
VI non-papillate amphigynous P. cinnamomi, P. drechsleri
Drenth & Sendall Practical guide to Phytophthora 9

1.5 Pathogenicity of Phytophthora

Almost all species within the genus Phytophthora are formidable plant pathogens.
Hence, we have to ask the question what makes these organisms such effective plant
pathogens. The following factors are involved:
• The ability to produce different types of spores such as sporangia and zoospores for
short-term survival and spread, and chlamydospores and oospores for more long-
term survival.
• Rapid sporulation on host tissue such as leaves typically within 3-5 days after
infection. This results in a rapid build up of inoculum in a multicyclic fashion,
leading to epidemics under favourable environmental conditions.
• Ability of zoospores of Phytophthora to be attracted to root tips through a chemical
stimulus (positive chemotaxis) coupled with the mobility of zoospores to actually
swim to the actively growing root tips, encyst, and infect young, susceptible root
tissue.
• Ability to survive in or outside the host tissue as oospores or chlamydospores for
long periods of time. Oospores are also known to survive passage of the digestive
systems of animals (for example, snails).
• Production of sporangia, which can be airborne and may travel reasonable distances
on wind currents and infect neighbouring fields. These sporangia can directly infect
host tissue. These same sporangiospores also have the ability do differentiate into 4-
32 zoospores under humid and cool conditions and cause multiple infections from
the one sporangium. However, zoospores can travel only short distances as they are
susceptible to desiccation.
• Phytophthora pathogens belong to the Kingdom Chromista and as such have
different biochemical pathways to the true fungi. Therefore many fungicides are not
very effective against Phytophthora pathogens.
• Phytophthora pathogens thrive under humid and wet conditions, which makes them
difficult to control, as protectant fungicides are difficult to apply and least effective
under such conditions.

Phytophthora pathogens can cause many different diseases and disease symptoms on a
wide range of plant species. In the next section, the disease symptoms most often
encountered are discussed.

1.5.1 Root rot

In general seedlings of many plants are very susceptible to root rot and damping off
caused by Phytophthora. The early symptoms are the wilting and yellowing of young
seedlings. General symptoms of root rot are that plants appear water stressed, chlorotic,
and are often stunted in their growth. Affected root tissue is soft, watersoaked and
discoloured to dark brown compared to the creamy white colour of healthy roots.
Advanced root rot leads to the lack of secondary and tertiary roots and a lack of healthy
root tips.

1.5.2 Collar rot

Collar rot often develops at or just below ground level. The infection moves upwards
from the roots, rotting the lower bark tissue and discolouring the lower stem. Above
ground symptoms appear as wilting, reduction of foliage and dieback of branches. Bark
Drenth & Sendall Practical guide to Phytophthora 10

and cortex tissue often have a swollen and cracked appearance, separating easily from
the underlying tissue.

1.5.3 Tree canker

Many species of Phytophthora can form cankers on the stems of host plants. These
cankers have various names, including: stripe canker (cinnamon), patch canker (durian)
or trunk canker. The first sign of canker is often the appearance of wet lesions on the
bark surface, often close to the branch points at the lower end of the trunk. Bark
discolouration and exudation of reddish brown, resinous substance often accompany
necrosis. When the bark is stripped away, the cortical tissues and wood appear dull and
discoloured from cream coloured to reddish brown. Wood lesions are often very
irregular in shape but are well defined. Expanding lesions severely restrict water and
nutrient flow to the connecting branches giving the appearance of wilting. If the lesion
girdles the tree branch, die back is more widespread in the crown and the entire tree may
become defoliated.

1.5.4 Stem lesions

Some species of Phytophthora attack leaves as well as stems. For example, P. infestans
on potato and tomato, P. sojae on soybean, and P. nicotianae on tobacco. Young
immature stems are often most susceptible. In advanced stages dry, dark brown or black
lesions develop in the cortical tissue on the stem near the soil line. Such lesions expand
upward and may cover as much as half the length of the stem in the case of black shank
on tobacco. Expanded lesions often girdle the stem and give rise to wilting and death of
the upstream branches and leaves.

1.5.5 Bud rot

Bud rot (sometimes called heart rot), is a serious problem in many species of palms. It is
caused predominantly by P. palmivora. The symptoms of bud rot of palm are exhibited
over a period of months often following severe storms, which facilitate infection and
spread of Phytophthora. Symptoms first appear as discolouration of the spear leaf and
one or more of the newest leaves. These new leaves may exhibit lesions from infection
that has occurred in the spear. As the infection in the bud of the palm progresses new
emerging leaves show increasing amounts of damage. Eventually the spear leaves can be
pulled out easily because they are rotted at the base and some white mycelial growth
may be observed at the leaf bases. The fronds will turn yellow and then brown, and will
fall off, finally leaving only a dead naked trunk. In the base of the bud, secondary
invaders move in and fluid starts to collect giving off a foul smell. The tissue below the
bud shows discolouration from reddish brown to brown. It is hard to isolate
Phytophthora from palms with advanced bud rot due to the decay of the bud. Trees
which are beginning to show symptoms, with an advancing margin on the bud, should be
used instead as they are often still relatively free of secondary invaders.

1.5.6 Leaf blight

A number of Phytophthora species cause leaf blight. These include: P. infestans on
potato and tomato; P. palmivora on a large number of tropical fruit species including
rubber, durian and macadamia; and P. colocasiae on taro. These blights on leaves are
first seen as small flecks but within 3-5 days they expand to produce large lesions.
Initially, infected tissue is watersoaked but becomes necrotic (brown or black) in a few
days. Often the lesions are surrounded by a halo of light green tissue. Spores appear as
white velvety growth at the edge of the lesions, primarily at the underside of the leaf. It is
Drenth & Sendall Practical guide to Phytophthora 11

this white growth that distinguishes Phytophthora leaf blight from several other foliar
diseases. Often large amounts of sporangiospores are produced as 1-4 sporangiophores
extend from the stomata at the underside of the leaf and produce large numbers of
sporangiospores which can either be airborne under relatively dry conditions or
differentiate into numerous zoospores under wet and humid conditions. These zoospores
can encyst and form new lesions on the same leaf or plant and can spread to
neighbouring plants through leaf to leaf contact.

1.5.7 Fruit rot

Fruit rot caused by Phytophthora appears as watersoaked lesions with light brown
centres 3-5 days after infection, depending on the host. The lesions expand rapidly and
can completely rot an entire fruit. Under conditions of high humidity, white/grey
mycelium may be found behind the advancing margin of the lesions. Often the fruit does
not drop and may mummify on the tree. The infection can also be internal as in the case
of P. palmivora in papaya where mycelial growth can be seen on the seeds after cutting
open infected fruit.

1.5.8 Tuber and corm rot

Tubers of potato and corms of taro are considered to be enlarged stem pieces and are
susceptible to infection by Phytophthora. Potato tubers can be infected by zoospores of
P. infestans washed down by rain from the leaves. Tuber infections are characterised by
patches of brown to purple discolouration on the potato skin. Cutting just below the skin
reveals a dark reddish brown, dry corky rot. Heavy infection can give rise to total loss of
the tubers. Light infections can occur and are difficult to detect. However, if such
potatoes are used as seed potatoes they can infect the emerging stems and start off a new
epidemic in the next planting season. This is probably how most late blight epidemics
start. Potato can also be infected by P. erythroseptica causing the so-called pink rot
disease. Infected tubers have a dull brown appearance and exude water under pressure.
The cut surface of tubers becomes faint pink after exposure to air. After 30 min the entire
cut surface of the tuber turns bright pink. If corms of taro are infected, they stay firm and
leathery, which is typical of Phytophthora dry rot. Under favourable conditions, the
corms may rot completely after about one week.
Drenth & Sendall Practical guide to Phytophthora 12

2. Media and antibiotics for isolation of Phytophthora

from diseased plant tissue and soil
The Oomycetes are not true fungi, and therefore special techniques are required for
their isolation. Most species of Phytophthora grow rather slowly in vitro compared
with saprophytic fungi and bacteria. In addition, bacterial populations need to be kept
low because they may suppress the growth of Phytophthora by direct competition, by
antagonism caused by antibiotic production, or by direct parasitism. The use of
selective media usually overcomes these problems. Antibiotics are added to isolation
media in order to suppress the growth of bacteria. Also, because Phytophthora spp.
are out-competed by many fungi, it is desirable to choose media which are “weak” in
nutritional terms. This reduces the growth rate of fungal contaminants, allowing
colonies of Phytophthora to become established. Synthetic cornmeal agar
(manufactured by Difco) is the most frequently used basic medium for isolation of
Phytophthora from infected plant tissue. However, other desirable basal media
include: water agar, or 2% or 4% (v/v) V8 juice agar. Suitable antibiotics that are
effective against bacteria include ampicillin, penicillin, rifampicin, and vancomycin,
alone or in combination. Suitable antibiotics with antifungal activity include nystatin
and pimaricin. Nystatin is usually less inexpensive and more readily available than
pimaricin.

2.1 Basic media for isolation from diseased tissue

V8 juice Media
Empty the contents of two 665 ml well-shaken cans of V8 juice into a 2 litre beaker. Add
10 g calcium carbonate (analytical grade), and stir for 20 minutes to adjust acidity. Dilute
for media as described below. Unused V8 juice can be stored at –20°C. It must be
completely thawed prior to use.

V8 juice agar – for routine growth and maintenance of Phytophthora cultures

Dilute amended V8 juice to 20% (v/v) final concentration:
100 ml CaCl2-amended V8 juice
400 ml distilled or deionised water
7.5 g agar
Autoclave at 121°C for 20 minutes. Cool to 55-60°C before pouring plates. Dry plates
in the laminar flow cabinet for 30-40 minutes. Wrap in plastic wrap prior to storing in
the fridge. Avoid having condensation on the lids of the plates.

Diluted V8 juice agar – for isolation of Phytophthora from infected plant material
Amended V8 juice can also be diluted to 2% or 4% (v/v) final concentration
Autoclave as above and cool before adding antibiotics as described in section 2.2.

Cornmeal agar (CMA)

Because CMA is not rich in nutrients, it is very suitable for isolation of Phytophthora
from infected tissue. Amend with antibiotics as described in section 2.2 once the
media has cooled to 50-55°C. If commercially produced cornmeal agar is not
available, fresh CMA can be prepared as follows:
Drenth & Sendall Practical guide to Phytophthora 13

Method 1
Cornmeal (polenta) (60 g/litre distilled or deionised water) is autoclaved at 121°C for 15
minutes and filtered. Add 15 g agar and autoclave.

Method 2
Boil 200 g corn (maize) in 200 ml distilled or deionised water for 1 hour, stirring
occasionally. Filter through a layer of muslin. Make filtrate up to 1 litre, add 15 g agar
and autoclave.

Water agar
Add 15 g agar to 1 litre distilled or deionised water. Autoclave, and add antibiotics (as
per section 2.2) once the media has cooled to 50-55°C.

2.2 Selective media for isolation from diseased tissue

The following recipes for selective media are based on commercially prepared cornmeal
agar. Diluted V8, fresh CMA, or water agar can also be used as basal media. Add
antibiotics as desired from one of the formulations listed below. Most antibiotic stock
solutions require filter sterilisation (0.22-0.45 µm) prior to aseptically adding them to
autoclaved media cooled to 50-55°C. Stock solutions of antibiotics should be kept at 4°C
or –20°C (frozen). Table 2.8 lists common antibiotics, their properties and preparation,
and some possible alternatives.

Plates of selective media used for isolations should not contain any free water or
condensation on the lids as water encourages the growth and spread of bacterial
contaminants. Therefore, dry the plates with the lids half-off in the laminar flow for
20-30 minutes. Store the plates wrapped in plastic in the refrigerator. The plates
should be stored upside-down. Note that pimaricin and nystatin are light-sensitive.
Media containing these anti-fungal agents should wrapped in foil or black plastic and
stored in the refrigerator. Ideally, selective media containing antibiotics should be
made fresh before use. Otherwise, they should be used within 2-4 weeks of
preparation.

3-P Medium (Eckert and Tsao 1960, 1962)

Add 17 g cornmeal agar (for example, Difco brand) to 1 litre distilled or deionised
water. Autoclave at 121°C for 20 minutes and cool to 50-55°C in a waterbath. Add
antibiotics to the following concentrations (µg/ml=mg/l=ppm): pimaricin (100),
penicillin (50), and polymyxin B (50). Table 2.1 explains which antibiotic stock
solutions are required, and what amounts to add to the media. Plates should be stored
in a refrigerator (4°C), wrapped in foil or black plastic as pimaricin is light sensitive.

Table 2.1 3-P Medium

Antibiotic Stock Amount (ml) to add to Final concentration
(mg/ml) 1 litre media (µg/ml)
Pimaricin 25 4 100
Penicillin 50 1 50
Polymixin B 50 1 50
Drenth & Sendall Practical guide to Phytophthora 14

Note: this medium is suitable for isolation of Phytophthora from freshly diseased
tissue but not from old, decayed tissue or freshly infested soil in which the propagules
are likely to be spores. This is because spore germination can be inhibited by high
levels of pimaricin. More suitable concentrations of pimaricin for the purpose of
isolation from old plant tissue or soil are 5-10 ppm (Table 2.2).

Table 2.2 3-P Medium with 10 µg/ml pimaricin

Antibiotic Stock Amount (ml) to add to Final concentration
(mg/ml) 1 litre media (µg/ml)
Pimaricin 25 0.4 10
Penicillin 50 1 50
Polymixin B 50 1 50

P10VP Medium (Tsao and Ocana 1969)

Cornmeal agar is prepared as above, autoclaved, cooled to 50-55°C and amended with
the following (µg/ml): pimaricin (10), vancomycin (200), and pentachloronitrobenzene
(PCNB; 100). This medium is suitable for isolating Phytophthora from the soil and
infected plant tissue (Table 2.3). Hymexazol (see below) can also be added to a final
concentration of 25-50 µg/ml.

Table 2.3 P10VP Medium

Antibiotic Stock Amount (ml) to add to Final concentration
(mg/ml) 1 litre media (µg/ml)
Pimaricin 25 0.4 10
Vancomycin 100 2 200
PCNB Not readily soluble 100 mg powder 100

P10ARP Medium (Kannwischer and Mitchell 1978)

Cornmeal agar is prepared as above, autoclaved, cooled to 50-55°C and amended with
the following (µg/ml): pimaricin (10), ampicillin (250), rifampicin (10), and
pentachloronitrobenzene (PCNB; 100) (refer to Table 2.4). Hymexazol (see below) can
also be added to a final concentration of 25-50 µg/ml.

Table 2.4 P10ARP Medium

Antibiotic Stock Amount (ml) to add to Final concentration
(mg/ml) 1 litre media (µg/ml)
Pimaricin 25 0.4 10
Ampicillin 100 2.5 250
Rifampicin 10 1 10
PCNB Not readily soluble 100 mg powder 100

P5ARP Medium (Jeffers and Martin 1986; Papavizas et al. 1981)

As above, with pimaricin reduced to 5 µg/ml (refer to Table 2.5). Hymexazol (see
below) can also be added to a final concentration of 25-50 µg/ml.
Drenth & Sendall Practical guide to Phytophthora 15

Table 2.5 P5ARP Medium

Antibiotic Stock Amount (ml) to add to Final concentration
(mg/ml) 1 litre media (µg/ml)
Pimaricin 25 0.2 5
Ampicillin 100 2.5 250
Rifampicin 10 1 10
PCNB Not readily soluble 100 mg powder 100

Note: P10ARP and P5ARP are the media of choice for isolating most species of
Phytophthora.

Hymexazol-amended Medium (Masago et al. 1977)

Hymexazol (Tachigaren) is a fungicide which has been found to suppress most
Pythium spp. except for P. irregulare and P. vexans. It can also inhibit some
Phytophthora spp., including P. cinnamomi, P. citrophthora and P. palmivora.

One litre basic medium is autoclaved, cooled to 50-55°C and amended with the
following (µg/ml): benomyl (10), pentachloronitrobenzene (PCNB; 25), nystatin (25),
ampicillin (500), rifampicin (10) and hymexazol (25-50) (refer to Tables 2.6 and 2.7).

Hymexazol can also be added to other selective media at a final concentration of 25-
50 µg/ml. Make up stock as described in Table 2.8. Add 0.5 ml of this stock to 1 litre
of selective media to give a final concentration of 25 µg/ml, or 1.0 ml to 1 litre of
selective media to give a final concentration of 50 µg/ml.

Table 2.6 Hymexazol Medium

Antibiotic Stock Amount (ml) to add to Final concentration
(mg/ml) 1 litre media (µg/ml)
Benomyl Not readily soluble 10 mg powder 10
PCNB Not readily soluble 25 mg powder 25
Nystatin 100 0.25 25
Ampicillin 100 5 500
Rifampicin 10 1 10
Hymexazol 50 0.5 25

Table 2.7 Hymexazol Medium

Antibiotic Stock Amount (ml) to add to Final concentration
(mg/ml) 1 litre media (µg/ml)
Benomyl Not readily soluble 5 mg powder 5
PCNB Not readily soluble 25 mg powder 25
Nystatin 100 0.25 25
Ampicillin 100 5 500
Rifampicin 10 1 10
Hymexazol 50 1 50
Table 2.8 Some common antibiotics, their properties, preparation and alternatives

Chemical Activity Target organisms Preparation of stock solutiona Stock Range used Comments Alternative
(mg/ml) (µG/ML)
Ampicillin Antibacterial Gram +ve bacteria Dissolve 1000 mg (1g) powder in 10 ml 100 250-500 Penicillin G
distilled water. Filter-sterilise
Benomyl Antifungal Most fungi except Benomyl is relatively insoluble in water. - 10-25 Does not suppress many
Zygomycetes and Add powder to media prior to autoclaving. undesirable fungi
Oomycetes Agitate media during pouring to ensure
even distribution of suspension
Hymexazol Antifungal Most Pythium spp. Dissolve 500 mg powder in 10 mL 50 25-50 May inhibit some
(also called HMI distilled water. Filter sterilise Phytophthora spp.
or Tachigaren)
Nystatin Antifungal Most fungi except Dissolve 500 mg powder in 5 mL distilled 100 10-100 Not active against some Pimaricin
(Mycostatin) the Peronosporales water. Filter-sterilise Mortierella spp.
Not as active as Pimaricin;
used at a higher
concentration
Penicillin G Antibacterial Gram +ve and Dissolve 500 mg powder in 10 ml distilled 50 50-100 Not active against Gram –ve Supplement with
Gram –ve cocci; water. Filter sterilise bacilli polymixin B at 50–
Gram +ve bacilli 100 µg/ml
Pentachloronitro- Antifungal Narrow antifungal PCNB is not soluble in water. However, it - 10-100 Does not suppress many
benzene (PCNB) spectrum is heat stable so that the powder can be undesirable fungi
added to the media prior to autoclaving
Pimaricin Antifungal Most fungi except Does not dissolve in water. Mix 250 mg 25 2-100 Not active against some Nystatin
the Pythiaceae powder in 10 ml sterile distilled water. Do Used most Mortierella spp.
not filter-sterilise frequently at 5-10 Dosage must be restricted to
µg/ml ≤ 10 µg/ml
Polymixin B Antibacterial Gram –ve bacteria Dissolve 500 mg powder in 10 ml distilled 50 20-50
water. Filter sterilise
Rifampicin Antibacterial Gram +ve Dissolve 100 mg powder in 10 ml ethanol 10 10 Penicillin G and
bacteria;Gram -ve (95%). Filter-sterilise polymixin B
bacteria to a lesser
extent
Vancomycin Antibacterial Gram+ve bacteria; Dissolve 1000 mg (1g) powder in 10 ml 100 100-200 Very expensive Penicillin G and
Gram-ve bacteria to distilled water. Filter-sterilise polymixin B
a lesser extent
a
Consider how many litres of Selective Media are required before making up stock solutions because antibiotics are often expensive. Reduce or increase the quantity of stock solution you make,
accordingly.
Drenth & Sendall Practical guide to Phytophthora 17

3 Detection and isolation of Phytophthora from

infected plant tissue and soil
It is estimated that Phytophthora species cause 90% of the crown rots of woody plants.
However, lack of knowledge on how to isolate Phytophthora often leads to negative
results and hence other pathogens such as Fusarium, Pythium, Rhizoctonia and
nematodes are frequently blamed for root and crown rots (Tsao 1990). Unlike species of
Pythium and Fusarium, which are generally associated with plants and soil,
Phytophthora species associated with diseased plants are likely to be the causal agent of
the disease. This is because most Phytophthora species attack only living or freshly
wounded tissue. They are primary invaders and hence do not invade plant tissue already
invaded by other microorganisms. Detection and/or isolation of Phytophthora from plant
tissue is relatively simple and successful if the tissue is fresh and recently infected.
Isolation of Phytophthora from necrotic plant tissue is more difficult because most
species of Phytophthora have poor saprophytic capabilities, and there may be very little
mycelia remaining once the host tissue dies. In addition, dormant propagules such as
chlamydospores and oospores are slow to germinate and emerge from senescent plant
tissue. Isolation of Phytophthora directly from soil is difficult, but the use of baiting
techniques markedly increases the frequency of successful isolation of Phytophthora
from infested soils.

3.1 Selection of samples for isolation of Phytophthora

If Phytophthora is not recovered from your samples it may be due to the simple fact that
Phytophthora is absent in your sample. Alternatively, the absence of Phytophthora could
be due to improper handling and storage of the samples. Negative samples should be
baited two to three times to ensure that Phytophthora is not present in the soil. In order to
successfully isolate Phytophthora from soil and plant samples, it is important to take the
samples from the correct area of infestation and handle them appropriately.

3.2 Isolation of Phytophthora from infected plant material

Phytophthora species attack only healthy plant material, including roots. Thus, the
pathogen can be present when no symptoms are obvious. Phytophthora species are
difficult to isolate from necrotic tissue because the tissue often harbours many secondary
pathogens. Therefore, it is very difficult to isolate Phytophthora from dead plants.
Baiting and isolation is best approached by sampling slightly affected trees or soil taken
from trees next to already dead ones.

Successful isolation of Phytophthora species from diseased tissue involves careful

selection of freshly infected tissue. Therefore it is best to obtain material from the edge
of an actively growing lesion. Leaf and stem tissue selected for isolation should ideally
contain part diseased and part healthy tissue. Once the tissue has been surface-sterilised,
it should be transferred to the appropriate selective medium, and the plates examined
regularly for the slow emergence of non-septate hyphae.

Pythium spp. are almost invariably present on both healthy and diseased roots, crowns
and lower stems of plants. There are three ways in which contamination of isolation
media by Pythium can be minimised:
Drenth & Sendall Practical guide to Phytophthora 18

• Pythium is confined to roots or badly rotted lower stems – choose other parts if
possible
• Pythium is confined to the outer cortex of the root – surface sterilisation will usually
kill it; alternatively choose the centre of the root
• Hymexazol will inhibit most species except for P. irregulare and P. vexans. Care
must be taken however, as it can also inhibit some Phytophthora spp., including P.
cinnamomi, P. citrophthora and P. palmivora. When these species are suspected, it is
wise to use selective media with and without hymexazol.

3.2.1 Preparation and surface sterilisation of tissue

Place well-washed roots, stems or leaves suspected to be infected with Phytophthora in a
shallow layer of pond water or distilled water. Leave for 24-48 hours in the light, at 18-
25ºC and examine for sporangial development. If sporangia are found, a small infected
plant piece can be cut off, surface sterilised and transferred to selective media.

Infected fruit is easily treated by cutting off the outer parts and placing small pieces of
the freshly cut fruit onto selective media. Leaf tissue which is reasonably clean may be
placed immediately onto selective media but it is almost always better to surface sterilise
it first. Surface sterilise leaf and stem tissue by dipping in 70% ethanol for 30-60
seconds. Blot tissue dry well between sterile filter paper before placing on selective
media. If wet plant material is placed onto media, bacteria can grow rapidly and suppress
the growth of Phytophthora. If the stems are particularly thick (0.5-1 cm wide), they can
be dipped in 70% ethanol for 10-30 seconds, and then quickly flamed to burn off the
excess ethanol. Small sections can then be taken either side of the lesion, and then plated
directly onto selective media.

Diseased roots often need more preparation. Place the roots in a beaker and wash them in
gently running water for several hours. This process removes the bacteria and stimulates
production of sporangia. After washing, cut out small sections of advancing root lesions,
surface-sterilise and blot the roots dry between sterile filter paper. Transfer to selective
media. Infected root material can be surface sterilised by using either one of the two
methods below:

Dip pieces of root tissue in 70% v/v ethanol for about one minute, wash for 10-20
seconds in sterile distilled water and blot dry on sterile filter paper. Cut root pieces into
0.5 cm lengths before placing onto selective media.

Dip root tissue in a 1:10 dilution of commercial bleach (sodium hypochlorite; approx.
0.5% v/v final concentration) for about 30 seconds. Rinse the roots in sterile water and
blot dry on sterile filter paper. Cut root pieces into 0.5 cm lengths before placing onto
selective media.

Sterilisation with ethanol results in fewer problems with bacterial contamination and
gives good recovery of most species of Phytophthora. It is also important that root pieces
are blotted dry very well and pushed just under the surface of agar instead of just being
placed on top. This will ensure good contact between bacteria in the tissue and the
antibiotics in the media. Phytophthora species will grow through the media quickly
leaving bacterial contaminants behind. Refer to Media and antibiotics for isolation of
Phytophthora from plant tissue and soil (section 2).
Drenth & Sendall Practical guide to Phytophthora 19

3.2.2 Isolation from infected plant material by “baiting” with cocoa pods
(method of (Chee and Foong 1968)
Phytophthora can also be isolated from infected plant tissue by baiting. This method is
useful for two reasons: (i) the initial steps can be performed in the field, and (ii) surface
sterilisation of the baited tissue is usually not required.
1. Core out 8 mm diameter plugs of tissue from a green (unripe) cocoa pod.
2. Insert a wedge of diseased tissue (1 cm wide×2 cm long) into the hole and push it in
so that the end is flush with the outside of the fruit. Alternatively, the pod can be cut
at an angle and very fine pieces of tissue such as bark inserted into the cuts.
3. Seal the pod in a plastic bag and incubate at room temperature. Up to six wedges can
be inserted into a single pod.
4. After 4-5 days, brown discolouration should be obvious around the plugs. A firm rot
indicates the presence of Phytophthora, a soft rot the presence of saprophytic
organisms. Take a small amount of healthy tissue from around the discoloured patch.
If the tissue is taken from inside the pod, the tissue does not require surface
sterilisation.
5. Plate tissue pieces onto selective media or soil extract agar (see section 4.3 – add 15
g agar/litre soil extract prior to autoclaving).

This method has also been used to isolate P. nicotianae. It can also be adapted to isolate
Phytophthora from the soil (see section 3.6).

3.3 Isolation of Phytophthora from soil

The best way to go about sampling soil for Phytophthora is as follows: where possible,
samples should be taken from moist soil, near healthy roots at least 5 cm below the soil
surface. The soil surface is often dry and subject to high temperatures from the sun,
making it an inhospitable place for Phytophthora. Soil samples are often best taken
during or immediately after wet weather, which typically increases Phytophthora
activity. Sampling is often best under the edge of the plant/tree canopy as root growth is
more vigorous there than immediately adjacent to the stem.

Samples should be handled carefully after collection. If soil samples are exposed to
drying or high temperatures (+45°C) they will lose their viability. Therefore, samples
should not be left in an enclosed vehicle in warm weather. Place your soil samples in
plastic bags to prevent drying out and put them in an insulated icebox to prevent
overheating. Also, avoid low temperatures as Phytophthora does not withstand freezing.
Hence, do not put the samples in direct contact with ice. Wrap a few ice blocks in
newspaper and add to an icebox to keep the temperature within normal range. In case the
samples need to be stored, do not use a refrigerator but place them at 10-15°C and ensure
that the samples are moist (add water if the samples are dry). It is best to process samples
within a few days but soil samples can be kept like this for a few months. If soil samples
dry out during storage, they can be re-moistened for 1-7 days before isolation is
attempted. This can stimulate production of sporangia or germination of chlamydospores
or oospores.
Drenth & Sendall Practical guide to Phytophthora 20

3.3.1 Detection and isolation of Phytophthora by baiting from the soil

Many plant parts can be used to selectively bait a target species of Phytophthora. These
include: fruits, seeds, seed pods, seedlings, cotyledons, leaves, leaf discs/strips, and
petals.

There are essentially three baiting techniques:

1. Insertion of soil or infected tissue into a hole made on a fleshy fruit (e.g. apple, cocoa
pod, pear, watermelon). A large fruit is desirable. A detailed method for isolating
Phytophthora from soil using unripe cocoa pods is given in section 3.6.
2. Planting seeds, seedlings or rooted cuttings into field soil, followed by heavy
watering to induce infection.
3. Floating or partial immersing baits of various types in a water and soil mixture. This
is the most widely used method for isolating Phytophthora spp. Infected plant tissue
can often be mixed with the soil to maximise the chance of detection.

The choice of bait is dependent on the species of Phytophthora that is suspected to be the
causal agent of disease, and the host plant. A list of baiting techniques is provided in
Table 3.1. Detailed methods for baiting using lupin seeds or cocoa pods are given in
sections 3.5 and 3.6.

For those techniques requiring partial immersion of baits in soil, or floating of baits in
soil, high water/soil ratios (4:1 or greater) are desirable. It is desirable to use distilled or
deionised water or some other source of water free from chloride or copper ions such as
bottled drinking water. Dilution of the soil may also dilute inhibitors present in the soil,
enhancing the formation of sporangia and zoospores. Isolations from infected bait
material should be made from healthy tissue surrounding lesions. In the case of leaf
discs/strips or petals, the entire tissue may be placed on the media. Use the guidelines in
section 3.2.1

3.4 Incubation
Most Phytophthora species have optimal growing conditions between 15-25°C so
incubation should take place within this range. Slightly cooler temperatures which tend
to favour Phytophthora and slow down bacteria are preferred. Plates should be incubated
in the dark, and if light-sensitive antibiotics are used, the plates should also be wrapped
in black plastic or foil. Because of heavy sporulating fungi and mite infestations that may
pose a serious contamination threat, place your plates in a separate incubator away from
clean Phytophthora cultures. Ideally, isolations from plants and soil should be performed
and stored in a “rough” laboratory away from clean cultures. It is important to note that
propagules of Phytophthora in the soil or senescent diseased tissue are dormant and may
germinate slowly. Therefore, it could take 2-20 days before colonies are visible on
isolation plates.
Drenth & Sendall Practical guide to Phytophthora 21

Table 3.1 Baiting techniques for isolating Phytophthora from soil

Species Bait material Procedure Reference

P. cinnamomi Apple or pear Make holes in fruit. Fill with soil. Wet soil. Incubate (Campbell 1949)
covered with plastic bag at 15-27ºC for 5-10 days. Isolate
from the edge of the rotted area around the hole. Suitable
technique for many Phytophthora spp.
Apple slices Immerse slices in 200 ml water to which 25 g soil has been (Gerrettson-Cornell
added, for 4-10 days 1974)
Avocado fruit Embed fruit partially in flooded soil. Incubate at 20-27ºC (Zentmyer et al.
for 2-4 days. 1960)
Avocado Plant seedlings in wet soil. Incubate at 21-27ºC for 2-3 (Zentmyer 1980)
seedlings days.
Avocado leaf Float leaf pieces on water added to soil for 4 days (Pegg 1977)
pieces
P. citrophthora Apple, lemon Insert soil or citrus tissue into fruit as per Campbell (1949) (Klotz and DeWolfe
or orange fruit for P. cinnamomi. Alternatively, place lemon or orange on 1958)
the surface of soil for 4 or more days
Lemon fruit Immerse partially in 150 ml water to which 25 cc soil has (Tsao 1960)
been added. Incubate at 25ºC for 6 days
Lupin radicles see Dance et al. (1975) under P. cryptogea
P. heveae Apple fruit, No method is given but the baits could have soil inserted (Lee and Varghese
eggplant fruit, into them as per the apple method of Campbell (1949), or 1974)
cocoa pod they could be embedded in partially flooded soil as per
Zentmyer et al. (1960) for P. cinnamomi.
P. nicotianae Apple, lemon see Klotz and DeWolfe (1958) under P. citrophthora
or orange fruit
Citrus leaf Float small leaf pieces on water 1-2 cm above 100 cc soil. (Grimm and
pieces Incubate at 22-28ºC for 3-4 days Alexander 1973)
Cocoa pods Insert soil or diseased rubber tissues into unripe green pods (Chee and Foong
as per the apple method of Campbell (1949). Incubate at 1968)
26-30ºC for 4-5 days
Lemon fruit see Tsao (1960) under P. citrophthora
Tobacco leaves Immerse the petiole end of leaf in water-soil mixture as per (Jenkins 1962)
Tsao (1960) under P. citrophthora
P. palmivora Apple fruit as per the apple method of Campbell (1949)
Apple fruit, No method is given but the baits could have soil inserted (Lee and Varghese
eggplant fruit, into them as per as per the apple method of Campbell 1974)
cocoa pod (1949), or they could be embedded in partially flooded soil
as per Zentmyer et al. (1960) for P. cinnamomi.
Black pepper Immerse black pepper leaves or leaf discs partially in a (Holliday and
leaves water-soil mixture. Used for isolation from black pepper Mowat 1963)
soils
Cocoa pods Place on or in soil. Or, inoculate pod surface with a small (Orellana 1954)
amount of soil suspension. Incubate in a plastic bag for 1-4
days. Used for isolation from cocoa soils
Cocoa pods Insert soil beneath flaps of endocarp tissue of unripe cocoa (Turner 1965)
pods. Used for isolation from cocoa soils
Cocoa pods see Chee and Fong (1968) under P. nicotianae
Cocoa pods Place pod or pod tissue on soil, or in flooded soil. Soil can (Newhook and
and tissues also be placed inside the pod Jackson 1977)
Taro roots Cut roots into 2.5 cm lengths, autoclave. Incubate in (Satyprasad and
moistened soil (50 g/Petri dish) at 15ºC for 7 days Ramaro 1980)
Drenth & Sendall Practical guide to Phytophthora 22

3.5 Lupin baiting of Phytophthora from soil samples

This method is a modification of a method from (Pratt and Heather 1972). If lupins are
not available, soybean seeds are also suitable.

Materials required:
Plastic cup (225-250 ml) with lid for each sample
Lupin seeds
Vermiculite
Distilled and sterile distilled water

1. Surface sterilise lupin seeds in 70% alcohol for 2 minutes, rinse in sterile distilled
water and then soak for 1 hour in sterile distilled water. Four to five seeds will be
required for each sample, depending on germination.
2. Pre-germinate lupins in sterile vermiculite at room temperature. Water only with
sterile distilled water and ensure that the water drains from the seeds. Lupin radicles
need to be 2-3 cm long for baits - this will take 2 to 3 days.
3. Punch or melt 5 holes, 5 mm in diameter in lids of plastic cups.
4. When lupin radicles are at the desired length, place a layer of soil sample
approximately 3 cm deep in the bottom of plastic cup. Fill cup with distilled water to
1 cm from top and cap with lids with pre-punched holes. Place pre-germinated lupins
on top of lid, with radicle inserted through hole and into water (see Figure 3.1). Do
not overfill the cups with soil. We recommend 5-10 times the volume of water
compared to the volume of soil for best results.
5. Check baits after 2 days, up until 7 days. Brown, discoloured lesions on the lupin
radicle should be surface sterilised and plated onto a selective medium. Soft rots are
those usually associated with Phytophthora.

Figure 3.1 Lupin baits. Phytophthora can be baited out of the soils using lupins suspended in
slurries made from soil samples in deionised water.
Drenth & Sendall Practical guide to Phytophthora 23

3.6 Baiting of Phytophthora from soil samples using cocoa

pods (method of (Chee and Foong 1968)
This method has been used to isolate P. palmivora and P. meadii from soil.
1. Core out 2 cm diameter plugs of tissue from a green (unripe) cocoa pod.
2. Insert moistened soil into the hole, completely filling it.
3. Seal the pod in a plastic bag and incubate at room temperature. Up to six samples can
be inserted into a single pod.
4. After 4-5 days, brown discolouration should be obvious around the plugs. A firm rot
indicates the presence of Phytophthora, a soft rot the presence of saprophytic
organisms. Take a small amount of healthy tissue from around the discoloured patch.
If the tissue is taken from inside the pod, the tissue does not require surface
sterilisation.
5. Plate tissue pieces onto selective media or soil extract agar (see section 4.3 – add 15
g agar /litre soil extract prior to autoclaving).

3.7 Culturing and storage of Phytophthora

3.7.1 Culturing
Most Phytophthora species grow well on a range of media including V8. Cultures of
Phytophthora should be grown at 15-25 °C in a dark incubator. Cultures should be
transferred every two to four weeks to maintain vigour. For long-term storage, water
storage is recommended (see below).

The pathogenicity of Phytophthora cultures is known to decrease after prolonged storage

on media. In case pathogenicity studies need to be performed, serial passage through the
host plant is required. Another alternative is storage of cultures in liquid nitrogen (see
below) which seems to overcome the problem of loss of pathogenicity.

3.7.2 Long term storage in sterile water

Phytophthora strains should be maintained as living cultures for two reasons. (i) to
provide other researchers and ourselves with reference strains for various studies
involving pathogenicity, virulence, mating type etc. and (ii) as a source of DNA for
genetic diversity and evolutionary studies. Phytophthora cultures can be stored in two
ways, either in sterile water or in liquid nitrogen.

To store cultures of Phytophthora, cut 8-10 small blocks from an actively growing plate
culture, and place in small screw capped glass bottles containing autoclaved distilled
water. The caps should be tightened during storage and the vials placed at room
temperature in the dark. Most species of Phytophthora can be stored this way but keep in
mind that the isolates will lose pathogenicity and aggressiveness during storage and
cannot be used for studies in that area after prolonged storage. Ideally cultures should be
revitalised once a year or every second year. For some species, a soybean seed or corn
seed can be added to prior to autoclaving the water which seems to induce oospore
formation in homothallic species.
Drenth & Sendall Practical guide to Phytophthora 24

3.7.3 Long term storage in liquid nitrogen

Cryopreservation of Phytophthora cultures in liquid nitrogen at –196°C is a good way to
store most Phytophthora species. The cultures are maintained in their original genetic
state and do not seem to lose their pathogenicity or aggressiveness. In addition liquid
nitrogen storage is very effective and time efficient since the only requirement is
maintaining the supply of liquid nitrogen. Our laboratory has a contract with a supplier
of liquid nitrogen, which tops up the tanks fortnightly at minimal cost. However, the
setup costs and maintenance of such a facility are much higher than storage under water
and is only worthwhile for large culture collections and specialised laboratories with
cheap and reliable access to liquid nitrogen.

3.7.4 Liquid nitrogen storage protocol

For storage in liquid nitrogen a cryoprotectant is needed to protect the Phytophthora
cultures during the process of freezing. We routinely use a mixture of 5% DMSO and
5% glycerol as cryoprotectant. The key to successful cryopreservation of Phytophthora
species is slow cooling, less than 1°C per minute to -40°C. The reason for this is that at
faster cooling rates, ice nucleation damages the cell membranes causing loss of viability
of the cultures. Slow cooling can be achieved either through expensive specialised
controlled cooling equipment or as we do it by placing the vials with cryoprotectant in a
styrofoam box. This box is then placed in a -70°C freezer overnight. The insulation of
the box will ensure slow cooling down to -70°C. From the -70°C freezer the isolates can
then be placed directly into a liquid nitrogen storage container. For liquid nitrogen
storage, it is best to grow Phytophthora on media with a poor nutritional value as this
promotes sporulation. Small discs of agar containing mycelium and spores can be placed
into the cryoprotectant and preserved that way. For profusely sporulating species such as
P. infestans and P. palmivora, plates can be flooded and the resulting spore suspension
added to the vials. Then add the cryoprotectant in a concentrated form so as to give a
minimal dilution of the concentrated sporangia. Sporangiospores can also be harvested
from plant tissue for storage in liquid nitrogen. However, it is important to remember
that they must be placed back on plant tissue after storage in liquid nitrogen, and that
they probably contain bacterial contaminants.

Thawing after storage is less critical and vials can be taken out of liquid nitrogen and
placed at room temperature. After 10-20 minutes they are thawed and the small agar
blocks can be transferred onto culture plates. Place a block of agar on the media and then
push it over the media to dilute out the cryoprotectant. Alternatively, the small blocks
can be blotted dry on sterile filter paper to remove most of the cryoprotectant. This will
ensure rapid recovery of the cultures. However, this may also result in a loss of
sporangiospores. Up to three little blocks from one vial can be placed onto a single
culture plate. Once vials have been thawed they cannot be stored again. Hence, multiple
copies of the samples must be stored.

Some Phytophthora species store better than others in liquid nitrogen. Species that
sporulate profusely such as P. infestans and P. palmivora can easily and reliably be
stored using many different cryoprotectants and freezing protocols. Poorly sporulating
species such as P. cinnamomi tend to have a lower recovery rate and are often slow to
recover from liquid nitrogen storage.
Drenth & Sendall Practical guide to Phytophthora 25

4 Identification of Phytophthora

Many species of Phytophthora can be easily identified. However, the morphological

differences among some species are few and variable, making it difficult to classify the
species accurately. Identification of Phytophthora is based on the taxonomic keys of
Waterhouse (1963) and Stamps et al. (1990). Characters which are used to classify
species of Phytophthora include: sporangium morphology; morphology of sexual
structures such as antheridia, oogonia and oospores; presence or absence of
chlamydospores, and morphology of hyphae. A list of terms used to describe the
morphology of Phytophthora is provided in section 4.7 of this chapter. In this section, a
very simplified methodology for identification of a few common species of
Phytophthora found in the tropics will be described. The information below should be
considered as a first step in the identification of Phytophthora species.

In many cases, the suspect Phytophthora species isolated from diseased tissue or infested
soil can be narrowed down according to the host from which it was isolated. Table 4.1
provides a list of species likely to be encountered in Southeast Asia and the hosts they
attack.

Table 4.1 Phytophthora species commonly found in southeast Asia

Species Hosts Some diseases caused

P. capsici many, including Capsicum, Piper Foliar blight and root and crown rot of Capsicum
nigrum (black pepper), Theobroma Fruit and root rot of cucurbits (melon, cucumber, squash)
cacao (cocoa), Macadamia, Hevea Blight of foliage, flowers and nuts of macadamia
(rubber), Carica papaya (papaya) Foot rot of black pepper
P. cinnamomi numerous (> 1000 species) including Root rot of pineapple
Persea (avocado), pineapple, Foliar blight and trunk canker of macadamia
macadamia Root rot of avocado
P. citrophthora various fruit trees, rubber, avocado, Crown rot, gummosis, fibrous root rot, and brown rot of
cacao, rubber Citrus
Damping off of citrus seedlings
P. colocasiae mainly Colocasia esculenta (taro, Foliar blight of taro
dahseen, gabi); also found on Alocasia Foliar blight, corm rot and stem canker of other species
spp. (yam), Piper betle (betel),
P. heveae rubber, avocado, mango, cacao and Pod rot and black stripe of rubber
Cocos nucifera (coconut) Bud rot and nut fall of coconut
Trunk canker of avocado
Pod rot of cacao
P. infestans main hosts are species of Solanum Leaf and stem blights of potato and tomato
P. nicotianae numerous; including avocado, black Black shank of tobacco
pepper, betel vine, coconut, citrus, Brown rot, foot rot, gummosis, and root rot of citrus
macadamia, papaya, pineapple, rubber, Heart rot of pineapple
taro, tobacco Crown rot and blight of macadamia
P. palmivora numerous; including avocado, black Black pod, stem canker and chupon wilt of cacao
pepper, betel vine, cacao, citrus, Root and fruit rot of papaya
coconut, macadamia, various spp. of Bud rot and premature nut fall of coconut and other palms
palms, papaya, pineapple, rubber, taro, Patch canker of durian
tobacco Black stripe, patch canker, green pod rot, abnormal leaf fall
and green twig blight of rubber
Foot rot of black pepper
Drenth & Sendall Practical guide to Phytophthora 26

4.1 Cultures
Ideally, the culture used for species identification should be obtained from a hyphal
tip, or a single germinated zoospore cyst, sporangium or oospore. It is important to
remember that on selective media most Phytophthora species will not sporulate and
form characteristic propagules for identification. Therefore, cultures should be
incubated at the optimum temperature for the suspected species, on a natural medium
such as V8 juice (see section 2.1), carrot agar or Lima bean agar (see section 4.8.3). In
order to identify an isolate of Phytophthora to species level, it is necessary to induce
the production of asexual and sexual structures that will aid in species identification.
In addition, characteristics of the mycelium, and whether the culture produces
chlamydospores will also assist in identification. Methods for producing asexual and
sexual structures are described in sections 4.3 to 4.5.

4.2 Morphological characters

There are a number of morphological characters upon which identification of
Phytophthora species is based. These include: sporangium shape, papillation, and
caducity; sporangiophore morphology; presence of chlamydospores and hyphal
swellings; antheridial attachment, and whether sexual reproduction is heterothallic or
homothallic.

4.3 Sporangia
Sporulation in Phytophthora cultures provides important clues for species identification.
Important characters to observe are:
• sporangium morphology (shape, size, length:width ratio) (see Figure 4.1)
• papillation of the sporangium (see Figure 4.2)
• caducity (shedding of the sporangium at maturity)
• length of the pedicel on the sporangium
• proliferation of sporangium (production of new sporangium within a sporangium
that has germinated directly)
• branching of the sporangiophores on which the sporangia are borne (see Figure 4.3)

Figure 4.1 Sporangium shape

Drenth & Sendall Practical guide to Phytophthora 27

Non-papillate Semi-papillate Papillate

i

Figure 4.2 Sporangium papillation

pedicel
sporangiophore

Simple sympodium (left) Compound sympodia (middle)

Umbellate sympodium (right)

Figure 4.3 Sporangiophore morphology

Some species of Phytophthora produce sporangia readily on the surface of agar media.
However, many species need to be cultured in water, mineral salt solutions or dilute soil
extracts before they will produce sporangia. It is important to remember that sporangia
production in Phytophthora is dependent on light (Schmitthenner and Bhat 1994). Table
4.2 provides a general guide to which species of Phytophthora produce sporangia on
agar media.

Table 4.2 Sporangia production on solid or liquid media

Sporangia produced on agar Sporangia produced in liquid media
P. capsici P. cambivora
P. heveae P. cinnamomi
P. megakarya P. citricola
P. nicotianae P. cryptogea
P. palmivora P. drechsleri

4.3.1 Induction of sporangia in liquid culture

Distilled water
Cut 0.5 cm2 agar discs from the edge of a colony that has been grown on V8 juice agar
or carrot agar. Cultures 2-4 days old are most suitable. Incubate the discs in a shallow
layer of distilled water (or pond water or Salt Solution or Soil Extract) in a Petri dish, at
Drenth & Sendall Practical guide to Phytophthora 28

room temperature (22-24°C). Incubation under continuous fluorescent light is

recommended. Sporangia are produced within 12 hours in some species, and typically
within 1-2 days.

Pond water
Some species of Phytophthora will produce sporangia when agar blocks of a culture are
transferred from Lima bean agar to non-sterile pond or stream water. Incubate as per the
Distilled water method.

Salt Washing
This method is useful for P. cinnamomi.
1. Grow colonies on dilute Lima bean agar (1.5%), and transfer 0.5 cm2 agar discs from
the colony edge to 25 ml Lima bean broth, removing as much agar as possible.
2. After 40-48 hours, pour off the culture medium and replace with 25 ml Chen-
Zentmyer’s Salt Solution (see section 4.8).
3. After an hour, tip off the Salt Solution and replace with fresh solution.
4. Repeat three more times.
5. Incubate in the light after the final wash. Sporangia should develop within 5-10
hours.

Soil Extract
1. Mix 10 g of soil with 1 litre of distilled water.
2. Filter through filter paper.
3. Autoclave extract. Autoclaving is not recommended if the suspected pathogen is
P. cinnamomi.
4. Incubate agar blocks of culture as per the Distilled water method.

4.4 Chlamydospores and hyphal swellings

Chlamydospores are thick-walled spores that function as a resting spore.
Chlamydospores can be intercalary (formed between hyphae) or terminal (on the ends of
hyphae). They differ from hyphal swellings by having thick walls and are delimited from
the mycelium by septa. The morphology of chlamydospores does not differ greatly
between species and therefore these spores are of limited use in species identification.
However, the presence (for example, P. palmivora) or absence (for example, P. heveae)
of chlamydospores can aid species identification. Chlamydospores are generally
produced readily in agar or water culture.

4.5 Sexual Structures

Production of oogonia, antheridia and oospores and mating type tests for
heterothallic species
Approximately half of the species of Phytophthora are homothallic. They will therefore
produce oogonia, antheridia, and oospores in single culture. The remainder are
heterothallic, with two mating types, A1 an A2. Heterothallic species produce
gametangia (oogonia and antheridia) only in the presence of an isolate of the opposite
mating type on the same plate. For species identification, it is important to determine if a
culture is homothallic or heterothallic, and whether the antheridium is amphigynous
(around the oogonial stalk) or paragynous (next to the oogonial stalk) (see Figure 4.4). It
is important that mating type tester isolates are obtained from a reliable culture
Drenth & Sendall Practical guide to Phytophthora 29

collection. Please contact us for more information about where tester isolates of the
different Phytophthora species can be obtained.

A number of media are suitable for mating type tests, including cornmeal agar, carrot
agar and Lima bean agar. Although the majority of species of Phytophthora produce
oospores in culture, some species require specialised media containing additives such as
sterols to induce oospore formation. In general it is best to start with carrot agar which
works for most species. Place a 0.5 cm2 plug of culture of the unknown isolate on one
side of the Petri dish. Place an agar plug from the known A1 or A2 tester isolates on the
other side of the dish. Incubate plates in the dark at the optimal temperature for the
species being examined. Oospores should form at the junction of the two colonies after
7-14 days if the isolates are of different mating types.

Immature oospore with a paragynous Mature oospore with an amphigynous

antheridium antheridium

Figure 4.4 Attachment of the antheridium

4.6 Differences between Pythium and Phytophthora

Phytophthora and Pythium belong to the Family Pythiaceae and hence are very closely
related genera. Differences between the two include:
1. Production of zoospores – in Phytophthora, the zoospores are produced within the
sporangium, in Pythium, the zoospores develop within a vesicle produced by the
sporangium (see Figure 4.5). This is the most important distinguishing feature
between Pythium and Phytophthora. Therefore, points 2 and 3 below are provided
for your information only.
2. Differences in the sporangia – the sporangia of Phytophthora are always terminal
and usually ovoid or obpyriform in shape whereas sporangia of Pythium may be
globulose, lobate (many lobed), or filamentous and are frequently intercalary.
3. Differences in the antheridia – in Pythium, the antheridia are paragynous and may be
attached at any point on the oogonium whereas in Phytophthora, the antheridium
attaches only at the lower hemisphere of the oogonium. In addition, in some species
of Pythium, many antheridia may be attached to a single oogonium.
Drenth & Sendall Practical guide to Phytophthora 30

vesicle
vesicle zoospores

sporangium

Figure 4.5 Vesicle development in Pythium. A vesicle develops from the

sporangium, and the zoospores are produced within the vesicle. In Phytophthora,
vesicles are not produced.

4.7 List of Terms

amphigynous the oogonium grows through the antheridium so that the
antheridium surrounds the oogonial stalk

antheridium the male sex organ (gametangium)

caducous describes sporangia that separate readily from the sporangiophore

chlamydospore resting spore that is separated from the mycelium by a septum; can
be terminal (on the end of a hypha) or intercalary (within a hypha)
with a thickened wall. Survives for long periods in the soil

heterothallic sexual reproduction that involves cross-fertilisation of isolates of

different mating types, A1 and A2

homothallic sexual reproduction by self-fertilisation (only one mating type is

required)

oogonium the female sex organ (gametangium) in which the oospore forms
after fertilisation by the antheridium

oospore zygote (or thick-walled spore) that forms within the oogonium after
fertilisation

papilla thickening at the apex (top) of the sporangium

paragynous the antheridium is attached to the side of the oogonium;

characteristic of Groups I, III and V

pedicel stalk which attaches the sporangium to the sporangiophore

sporangiophore stalk on which the sporangium is borne; may be simple or branched

sporangiospore motile spore that emerges from a germinated sporangium (also

called a zoospore)

sporangium sac within which zoospores form

Table 4.3 Morphological characteristics of some Phytophthora species (diagrams and micrographs reproduced from Erwin and Ribeiro, 1996)

Species Phytophthora capsici Phytophthora cinnamomi Phytophthora citrophthora Phytophthora colocasiae

Waterhouse Group Group II Group VI Group II Group IV

Mating system Heterothallic Heterothallic Sexual reproduction rare Heterothallic

Optimum growth 28°C 24-28°C 24-28°C 27-30°C
temperature
Morphology
Sporangia:
Papillation Papillate or semi-papillate Non-papillate Papillate Semi-papillate. A conspicuous basal plug
(sometimes with 2 or 3 apices) is seen where the sporangium meets the
sporangiophore
Shape Ovoid, obovoid, ellipsoid Includes ovoid, obpyriform, ellipsoid. Varies from spherical, ovoid, Includes ovoid, ellipsoid, and fusiform.
Tapered at the base. obpyriform, obturbinate to ellipsoidal Tapered base, sometimes with
attachments
Attachment Caducous with long pedicels Non caducous. Borne terminally Non-caducous. Usually terminal, Caducous
sometimes lateral or intercalary
Sporangiophores Sporangiophores irregularly branched Usually unbranched Irregularly branched Irregularly branched
Length:width ratio of Varies from 1.3 to 2.1 >1.5 Varies from 1.2 to 2.0 1.6
sporangia
Antheridia Paragynous Amphigynous Sex organs not produced in nature Amphigynous, sub-terminal
Oogonia Globose Globose, with smooth, thin walls Globose Globose
Oospores Spherical to subspherical Round, thin walled Produced only rarely in culture 18-30 µm
15-40 µm 19-54 µm
Chlamydospores Produced abundantly by isolates from Produced abundantly in culture, globose, Reported only in isolates from cacao Abundant in some isolates, rare in others.
cacao, black pepper, macadamia thin-walled. Terminal or intercalary, Borne terminally or intercalary in the
often in grapelike clusters of 3-10 mycelium
Mycelium Torulose, with hyphal swellings Coralloid, with abundant hyphal Smooth or coarse; hyphal swellings Hyphal swellings are not produced
swellings and vesicles present in some isolates
Distinguishing Long pedicels on caducous sporangia, Mycelial morphology None reported None reported
characters compared with round, non-caducous
sporangia in P. nicotianae and ovoid,
caducous sporangia on short pedicels in
P. palmivora
 Species Phytophthora capsici Phytophthora cinnamomi Phytophthora citrophthora Phytophthora colocasiae
Morphological
characteristics

Papillate sporangia, caducous with Non-papillate sporangia. Note the Papillate sporangia, some with two Semi-papillate sporangia with short
long pedicels. Note bipapillate internal proliferation of papillae. Note the variation in pedicels
sporangia (top right). Extensive sporangia. Globose oogonium sporangial shapes. Antheridium is
hyphal swellings on mycelium with paragynous antheridium amphigynous (bottom left)
(bottom left). Note the globose
chlamydospores (bottom middle)

In the tropics and sub-tropics, P.

nicotianae, P. cinnamomi and
ADDITIONAL P. palmivora are also pathogens of
FIGURES AND citrus. P. citrophthora is active at
NOTES moderate temps (30°C) and P.
nicotianae at higher temps (>30°C)

Torulose hyphae with hyphal Coralloid hyphae with hyphal

swellings swellings
 Species Phytophthora heveae Phytophthora infestans Phytophthora nicotianae Phytophthora palmivora
Waterhouse Group Group II Group IV Group II Group II
Mating system Homothallic Heterothallic Heterothallic Heterothallic
Optimum growth 25°C 20°C 27-32°C 27-30°C
temperature
Morphology
Sporangia:
Papillation Papillate Semi-papillate Papillate Distinctly papillate
Shape Irregular, obpyriform to ellipsoid Ovoid, ellipsoid to limoniform Varies from ellipsoid, ovoid, pyriform, Mainly ellipsoid and ovoid, pyriform
obpyriform to spherical, with a
prominent papilla
Attachment Caducous Caducous Non-caducous Caducous
Sporangiophores Sporangiophores irregularly branched. Sporangiophores are compound Sporangia irregularly or sympodially Sporangia occur in groups of up to 20 on
Sporangia often form laterally on sympodial, with a small characteristic branched one sympodium. Pedicels are short
sporangiophores swelling just below the sporangium
Length:width ratio of 1.5 1.6 Varies from 1.1-1.7 Varies from 1.7-1.9
sporangia
Antheridia Amphigynous, spherical or cylindrical, Amphigynous Round or oval, amphigynous Amphigynous
sometimes bicellular

Oogonia Globose, funnel-shaped and produced Spherical to obpyriform, smooth walled Spherical and smooth-walled Spherical
readily in culture
Oospores Oospores round, smooth and thick- 30 µm 24 µm 23 µm
walled, 15-28 µm
Chlamydospores Not produced Not recorded Produced abundantly by most isolates, Globose to subglobose, terminal or
terminal or intercalary intercalary
Mycelium Hyphal swellings not produced Hyphal swellings not produced Hyphal swellings have been noted Hyphal swellings have been noted in
some isolates
Distinguishing P. heveae distinguished from P. None recorded None recorded P. palmivora is distinguished from other
characters colocasiae, P. nicotianae and heterothallic species by the production of
P. palmivora by lack of aerial mycelium, conspicuous papillate sporangia on short
and the absence of chlamydospores pedicels, and chlamydospore production
 Species Phytophthora heveae Phytophthora infestans Phytophthora nicotianae Phytophthora palmivora
Morphological
characteristics

Papillate sporangia with variable Semi-papillate, caducous Papillate sporangia. Amphigynous Distinctly papillate, caducous
shapes. Sporangia often form sporangia (left). Amphigynous antheridia. Terminal and intercalary sporangia. Sporangia often
laterally on the sporangiophore antheridia. Compound sympodial chlamydospores (bottom right) clustered. Pedicels very short.
(bottom middle) sporangiophore (right). Amphigynous antheridia

Additional figures and

notes

Micrograph of sporangia of various Micrograph of an ovoid sporangium

shapes
Drenth & Sendall Practical guide to Phytophthora 35

4.8 Media
4.8.1 Carrot agar
1. Wash 200 g carrots and slice thickly.
2. Autoclave carrots in 500 ml distilled or deionised water.
3. Blend warm carrots in a blender at high speed for 40 seconds.
4. Squeeze homogenate through 4 layers of muslin.
5. Make filtrate up to 1 litre with deionised water.
6. Add 15 g agar and autoclave.
7. Pour plates thinly for easier microscopic examination. Yields 40-50 plates.

4.8.2 Dilute Lima bean agar or broth

1. Autoclave frozen (not dried) Lima beans (50 g) in 1 litre of distilled or deionised
water for 15 minutes.
2. Strain broth through muslin, adjust to 1 litre. Add 15 g agar and autoclave again.
3. For Dilute Lima bean broth, do not add agar prior to autoclaving. Store in the dark.

4.8.3 Lima bean extract agar

1. Autoclave frozen (not dried) Lima beans (285 g) in 1 litre of distilled or deionised
water for 15 minutes.
2. Strain broth through muslin, adjust to 1 litre. Add 15 g agar and autoclave again.
3. Broth is made by not adding agar prior to autoclaving. Store in the dark.
This medium is suitable for induction of sporangia in P. capsici, P. erythroseptica, P.
heveae and P. infestans.

4.8.4 Salt Solution of Chen and Zentmyer (1970)

Dissolve the following in 1 litre distilled water:
Calcium carbonate Ca(NO3)2: 1.64 g
Potassium nitrate KNO3: 0.05 g
Magnesium sulphate MgSO4 0.48 g

Add 1 ml chelated iron (see below), and adjust pH to 7.0.

Chelated iron is made by dissolving the following ingredients in distilled water, and
making the solution up to 1 litre:
Ethylenediaminetetra-acetic acid (EDTA) 13.05 g
Potassium hydroxide (KOH) 7.5 g
Ferrous sulphate (FeSO4) 24.9 g
Drenth & Sendall Practical guide to Phytophthora 36

5 Plant inoculation tests with Phytophthora species

Once a Phytophthora species has been isolated, brought into pure culture and identified
down to species level further research concerning the pathology often involves
inoculation of the host plant. There are numerous reasons for inoculating host plants
including:
• Determining the pathogenicity (ability to cause disease) of a particular Phytophthora
species on a host. This is especially important in case the particular species of
Phytophthora has not been associated with this host before. In such a case it is vital to
prove Koch’s postulates by showing that the strain causes the disease symptoms and
can be isolated from the host again.
• Determining the aggressiveness of the Phytophthora isolates. Plant inoculation tests
can determine whether different isolates of the same species or isolates representing
different species cause the same level of disease on a particular host.
• Determining the virulence of particular isolates. These tests are performed in case
there are different strains of Phytophthora belonging to the same species that can
overcome particular disease resistance genes in the host plant. These tests are
typically done with host specific Phytophthora species such as P. sojae, P. infestans,
P. vignae etc.
• Screening germplasm for disease resistance using a particular strain or species of
Phytophthora.

There are many ways in which plant inoculation tests can be performed depending on the
question asked, the species of Phytophthora, the host plant species involved and the
maturity of the host plant. In general the following methods are often used:

1. Inoculation of sporangia onto leaf or fruit tissue.

2. Insertion of mycelium into the stems of young seedlings.
3. Insertion of pieces of agar with mycelium into holes in the stem of woody adult
plants.
4. Inoculation of soil and planting of young plants into this infected soil.

These methods are briefly discussed below to provide a starting point for plant
inoculation tests. For each particular host-pathogen combination we strongly suggest you
consult the literature for more detailed and specific protocols and procedures.

5.1 Inoculation of sporangia onto leaf or fruit tissue

These tests are often performed to assess resistance in different cultivars or lines of a host
to different strains of a single Phytophthora species. Typically this is done with species
causing foliar diseases, such as P. infestans on potato and tomato; P. colocasiae on taro
and P. palmivora on leaves of tropical fruit crops such as durian. Sporangiospores of
Phytophthora can be harvested from infected lesions taken from inoculated plants, or
from culture plates by flooding the plates with distilled water and harvesting the spores a
few hours after flooding. As a general guide, a small drop (10-50 µL) of water containing
104 sporangia/mL is placed on a detached leaf. It is best to use young just fully expanded
leaves, which should be kept in a tray under moist conditions to favour development of
disease but not too wet to favour general rot. Detached leaves can be placed on agar or
stuck into foam to prevent them from drying out. It is best to place the leaves on a mesh,
which is subsequently placed on wet paper towels in a tray with a lid. This gives a high
Drenth & Sendall Practical guide to Phytophthora 37

level of humidity without the leaves coming in direct contact with free water. After 24
hours the inoculum should be blotted with a tissue and any free water removed from the
leaves by opening the trays to prevent rot. If the leaves senesce too quickly for symptoms
to develop, a cytokinin plant hormone such as benzimidazole can be used to inhibit leaf
senescence and maintain greeness. A concentration of up to 20 ppm may be used but we
strongly suggest that you experiment with the precise concentration before inoculating the
leaves. The leaves should then be incubated at 20-25°C with 16 hours light period for 5-7
days for symptoms to develop. Depending on the host and the Phytophthora species used,
lesions will develop and sporulation can be observed, indicating susceptibility of the host.
For other species, the growth rate of advancing lesion can be measured and used as an
indicator of resistance.

5.2 Insertion of mycelium into the stems of young seedlings

This method is used for screening for resistance in the host and determining the virulence
of particular strains of Phytophthora. This type of test is used for example in P. sojae on
soybean (Ryley et al. 1991), P. vignae on cowpea etc. Typically around 10 seeds are
sown into a pot and at the two-cotyledon stage a small slit is made into the stem with a
scalpel and a weft of mycelium inserted. After all ten seedlings are inoculated a plastic
bag is sprayed on the inside with distilled water and placed over the seedlings to create a
moist and humid environment. The bag is removed 24-48 hours after inoculation. A
reaction can typically be seen 4-6 days after inoculation. If the seedlings are susceptible,
an advancing lesion on the stem will lead to collapse of the hypocotyl – the reaction is
then rated as susceptible. Resistant seedlings will only show a wound and continue to
grow.

5.3 Inoculation of woody adult plants

Phytophthora isolates can be grown on agar for this. Use a 10-mm corkborer to cut
through the bark of the tree and a little in the wood approximately 1-1.5 meter above
ground level. Place a mycelial disc of 10 mm into the hole and seal it with masking tape
to prevent desiccation. Depending on the species of Phytophthora and the susceptibility
of the host, lesion length observed as dark discolouration of the secondary phloem can be
measured 6-8 weeks after inoculation.

5.4 Inoculation of soil

This method is routinely used for many Phytophthora species and there are numerous
variations on this technique. Below is a general outline. Inoculation can either be done
with agar containing mycelium, sporangiospores, or mycelium grown up on a natural
substrate such as autoclaved wheat or millet. For species of Phytophthora such as P.
cinnamomi, which do not readily sporulate, the use of natural substrate will give the best
results. For production of a natural substrate mix 40 mL of wheat (or other grain such as
barley, rye or millet) with 40 mL of distilled or deionised water, soak overnight; pour of
excess water and autoclave the wheat in a glass flask at 121°C for 30 minutes on two
consecutive days. Inoculate the flask with 5 –10 small plugs of agar containing mycelium
and incubate at room temperature away from direct exposure to light. Shake the mixture
daily to obtain an even spread of Phytophthora mycelium throughout the flask. After 2-3
weeks the wheat is fully colonised and can be ground in a coffee grinder or blender. Use
about 1 gram of this mixture per litre of potting mix. We often mix it with sterile sand in
order to obtain an even spread of this mixture throughout the potting mix. Then use this
Drenth & Sendall Practical guide to Phytophthora 38

potting mix to plant your seeds, or re-pot your seedlings into the pots containing the
infected soil. In order to get good Phytophthora infection a series of wet and dry cycles is
recommended. Place a saucer at the bottom of each pot and flood for 3 days in addition to
watering from the top. For the next 4 days invert the saucer and water the plants only
sparingly. This sequence of flooding for 3 days and drying for 4 days creates the ideal
environment for sporangia and zoospore production and will provide a good environment
for Phytophthora infection. If the pots are continuously waterlogged, you will get root rot
and asphyxiation even in the uninoculated control pots. The degree of root infection can
be assessed by counting the number of healthy root tips.
Drenth & Sendall Practical guide to Phytophthora 39

6 References
Bartnicki-Garcia, S., and M.C. Wang. 1983. Biochemical aspects of morphogenesis in
Phytophthora. In Phytophthora: its biology, taxonomy, ecology and pathology,
edited by D. C. Erwin, S. Bartnicki-Garcia, and P.H. Tsao. St. Paul, Minnesota,
USA: American Phytopathological Society.
Bourke, P.M. 1964. Emergence of potato blight. Nature 203:805-808.
Brasier, C.M. 1983. Problems and prospects in Phytophthora research. In Phytophthora:
its biology, taxonomy, ecology and pathology, edited by D. C. Erwin, S.
Bartnicki-Garcia, and P.H. Tsao. St. Paul, Minnesota, USA: American
Phytopathological Society.
Brasier, C.M. 1992. Evolutionary biology of Phytophthora: I. Genetic system, sexuality
and the generation of variation. Annual Review of Phytopathology 30:153-171.
Campbell, W.A. 1949. A method of isolating Phytophthora cinnamomi directly from the
soil. Plant Disease Reporter 33:134-135.
Cavalier-Smith, T. 1986. The kingdom Chromista: origin and systematics. In Progress in
phycological research, edited by I. Round and D.J. Chapman, Bristol, England:
Biopress.
Chee, K.H. and K.M. Foong. 1968. Use of cacao pod for recovering Phytophthora
species pathogenic to Hevea brasiliensis. Plant Disease Reporter 52:5.
Chen, D.W. and G.A. Zentmyer. 1970. Production of sporangia by Phytophthora
cinnamomi in axenic culture. Mycologia 62:397-402.
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