BIBLIOGRAPHY
RHONDA M. OLOAN. October 2006. Etiology of the Red Stele Disease of
Strawberry (Fragaria x ananassa Duch.) in Benguet. Benguet State University, La
Trinidad, Benguet
Adviser: Luciana M. Villanueva, Ph.D.
ABSTRACT

This study was conducted to (a) describe the symptom variations in all
strawberry cultivars observed to be infected in the field (b) assess the incidence of the
disease in the strawberry growing areas of La Trinidad and Tuba, Benguet, (c) determine
the best culture medium that support both mycelial growth and
oospore/sporangia/zoospore production, (d) describe the morphological characteristics
of the pathogen and (e) determine zoospore inoculum level that affects the growth and
yield of strawberry.

Symptoms of stunting, wilting, abnormal change of leaf color, reddening of the
core and eventual death of severely infected plants were observed on cultivars’ Tioga and
Sweet Charlie that were planted in the field. High incidence of the disease was observed
in strawberry growing areas at Swamp Area, BSU, La Trinidad ranging from 20 - 43%
infection while lower incidence was recorded in Longlong, La Trinidad and Sto. Tomas,
Tuba with 19.80 and 8.91%, respectively.

Rapid growth of mycelia was observed in carrot broth agar (CBA) and oatmeal
agar (OMA) with mean colony diameter of 27.45mm and 24.17mm in 7 days,

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respectively. On the other hand, the mycelial growth in potato dextrose agar (PDA) was
very slow with mean colony diameter of 7.53mm. The mycelia are white in color in
PDA and OMA while brown to white in CBA and V8 juice agar (V8J-A). Mycelial
growth was very dense in PDA, slightly dense in CBA and V8J-A while in OMA they
were very scarce and could hardly be seen on culture.
Morphological studies showed that the hyphae are hyaline, coenocytic and slender
with few swelling of hyphae in CBA, OMA and V8J-A culture media while profuse
swelling was observed on PDA. The oospores are round and readily observed on CBA,
V8J-A and OMA media with antheridia attached on the base. Sporangia are ovoid to
pear shape, hyaline and non-papillate. The zoospores are very motile with two flagella
attached but hardly seen. It has round shape when at rest.
Based on the cultural and morphological characteristics, the pathogen of the red
stele disease of strawberry in Benguet was identified to be Phytophthora fragariae.
Inoculated plants exhibited symptoms of stunting, wilting, off-colored leaves,
yellowing of immature leaves, and death of severely infected plants. Newly developed
feeder roots have red to brick red color of the root core.
Plants inoculated with the highest concentration of zoospores (20 x 103)
significantly gave the shortest mean height of 139.58cm and the lowest marketable yield
of 117.91g. Yield reduction from 38-57% as a result of infection was noted on
inoculated plants with disease severity rating of 3.67 to 5 equivalent to 26 - 100% root
infection.

ii

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TABLE OF CONTENTS


Page

Bibliography……………………………………………………….….
i
Abstract
……………………………………………….………
i
Table of contents ……………………………….……………….…
iii
INTRODUCTION
Importance of the Study ………………………………………
1
Objectives of the Study …………..………………………….
4
Time and Place of the Study ………………………………….
4
REVIEW OF LITERATURE


Strawberry …...………………………………………………
6
Red Stele Disease ……………………..………………….…
7
The Pathogen and its Life Cycle …....….……………………
8
Disease Symptoms …...………………………………………
10


MATERIALS AND METHODS

Field Study
…………………………………..……………………
12
Survey ………………………………………………….…….
12
Symptomatology …………………………...…………………
14
Assessment of Disease Incidence ……...…………………….
14
Laboratory Study ...…………………………………………………
14
Collection of Diseased Plants ....…………………………..…
15
Isolation of the Organism …………………………..……….
15
iii

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Identification of the Organism ……………………………..….
15
Cultural Characteristic of the Organism …………..………..
16
Morphological Characteristic of the Organism ………………
16
Pathogenicity test ..………………………………………………….
17
Preparation of Inoculum…..………………………………..……
17
Standardization of the Inoculum ………………………..……
18
Inoculation…………………………………...……………….…
18
Re-isolation of the Organism …………………………………….
19
Data Gathered …………………………………………………….
20
RESULTS AND DISCUSSION

Field Study
..………………………………………………………
23
Symptomatology ……………………………………………
23
Disease Incidence ……………………………………………
26
Laboratory Study …….……………………………………………
29
Characterization and Identification of the Organism …………….
29
Mycelial Growth of the pathogen in different Culture

Media ………………………………………………..…...
29

Cultural Characteristic of the organism …………….……….
30
Morphological Characteristics of the organism ….………..
32
Pathogenicity Test ……...……………………………………….…
36
Symptoms on Inoculated Plants ……………………………..
36
Plant Height ……………….…………………..………………
40
Yield ……………….…………………………………………
41
iv

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Fresh Weight of Plants ………………….……………………...
43
Oven Dry Weight of Plants ……………………………………
45
Disease Severity at Harvest ………………..………………….
45
SUMMARY, CONCLUSION AND RECOMMENDATION


Summary ………………………………….…………………….
48
Conclusion ………………………………………………...…….
49
Recommendations ...……………………………………………
50
LITERATURE CITED …..…………………………………………
51
APPENDICES …..………………………………………………….
54
BIOGRAPHICAL SKETCH ………………………………………..
80

v

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INTRODUCTION

Strawberry
(Fragaria x ananassa Duch.) production in the highlands of
the northern Philippines started about almost a century. The cultivation of this
crop was estimated to have started sometime in the early 19th century. It was
introduced by the Americans most probably when an agricultural school in La
Trinidad, Benguet (presently Benguet State University) was established in 1916.
It has been a lucrative source of income for farmers and adds to the revenue of the
province of Benguet. It is cultivated for its delicious berries, which are sold as
fresh fruits or as preserves.
Strawberries have been in demand not only locally but also in other
countries. According to Hermano (1995), The Baguio Growers and Marketing
International Corp. (BGIMC) exported 6.4 tons of fresh strawberries to Hongkong
in 1992. The volume increased to 9.1 tons in 1994. Strawberry is also in demand
among processing factories such as the Magnolia Dairy Products, Inc., Lady’s
Choice consolidated Food Company and Baguio Strawberry House including
local jam and wine makers.

Importance of the study

At present, strawberry is the number one and the most popular fruit
produced in Baguio City and Benguet Province. It is estimated to rank number
Etiology of the Red Stele Disease of Strawberry
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six in sales among all farm crops grown in the area, surpassed only by potato,
cabbage, carrots, Chinese cabbage, and beans (HARRDEC, 1996).
To meet the market demand several new cultivars which are high yielding
have been introduced in Benguet and Baguio City which include Tioga, Hecker,
Aliso, Mission bells, Fukuba, Haranoka, Toyonoka and many more (Hermano,
1995).

However, due to the presence of pests and diseases and other limiting
factors, production is not yet enough to meet the increasing demand. Numerous
diseases attack strawberries and the organisms causing the disease maybe on the
plants when they are purchased or in the soil where the plants are set. One of the
most serious threats in strawberry production are soil borne diseases like black
root rot caused by several fungi, Verticillium sp., Fusarium sp., Pythium sp. and
Phytophthora sp. Farmers interviewed at the Swamp area revealed that soil borne
diseases had caused them severe yield loss. According to them, some of their
plants show symptoms of stunting and wilting starting from December, and
produced small berries. Severe infection was observed from February to April
especially when the weather is very hot and dry.
Red stele or red core disease of strawberry is a major factor limiting
strawberry fruit production in the northern two – thirds of the United States and in
many areas of the world. The disease is most destructive in heavy clay soil that is
saturated with water during cool weather (Ries, 1996).
Etiology of the Red Stele Disease of Strawberry
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The disease is caused by a soil-borne fungus and is not a natural inhabitant
of most agricultural soils but introduced into a previously uninfected site through
infected planting stocks or contaminated planting equipment. The red stele
fungus is very persistent and can survive in the soil for up to 13 years or longer
once it becomes established in the field or garden (Ellis, 1996). Once introduced
to a site, the fungus spreads in a field via soil and water movement. Soil
movement is usually accomplished through contaminated farm tools such as
plows, hoes and trowels. Soil and water movement will often carry spores from
higher areas to lower areas (Scopes and Stables, 1989; Ellis, 1996).
The red stele root rot disease attacks strawberry, loganberry, and potentilla
(a genus of rosaceous herbs commonly called cinquefoil). Loganberry is a host.
However, most infections are passed directly from strawberry plants to strawberry
plants (Ellis, 1996).
In the Philippines, the red stele disease has not yet been reported but
symptoms such as production of few runners, stunting, wilting and eventual death
of severely infected plants were observed in the late 1980’s. Furthermore, roots
of plants just starting to wilt when dug showed reddening of the stele or core
which is the most reliable symptom of red stele disease which was observed at the
Swamp Area in La Trinidad, Benguet. According to Anderson (1956) and Ries
(1996), the introduction of the disease to any strawberry-growing area is a threat
because it renders the land useless for strawberry production over a period of 10
to 13 years or more. Losses are not great for the first 2-3 years after the
Etiology of the Red Stele Disease of Strawberry
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introduction of the disease in a field but occurrence of such disease if not properly
managed may cause severe yield loss to farmers.

Hence, identification of the causal organism of red stele infecting
strawberry in Benguet is very necessary for the implementation of control
measures to prevent the spread of the disease.

Objectives of the Study
This study was conducted to:
a.
Describe symptom variations in all strawberry cultivars observed
to be infected in the field;
b.
Assess the incidence of the disease in the strawberry growing areas
of La Trinidad and Tuba, Benguet;
c.
Determine the best culture medium that would support mycelial
growth and oospore/ sporangia/zoospore production;
d.
Describe the morphological characteristics of the pathogen, and
e.
Determine the zoospore inoculum levels that affect the growth and
yield of strawberry.

Time and Place of the study

The survey was conducted in the strawberry growing areas at La Trinidad
and Tuba, Benguet from November 2005 to May 2006. The pathogenicity test
and laboratory studies were conducted at the Baguio National Crop Research and
Etiology of the Red Stele Disease of Strawberry
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Development Center (BNCRDC)-Bureau of Plant Industry (BPI), Guisad, Baguio
City and the Department of Plant Pathology (DPP), College of Agriculture (CA)
Benguet State University (BSU), La Trinidad, Benguet from November 2005 to
August 2006.
Etiology of the Red Stele Disease of Strawberry
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REVIEW OF LITERATURE

Strawberry
The cultivated strawberry belongs to the genus Fragaria of the family
Rosacea. Strawberry is locally known as atakbang, duting or kubkubot and Fresa
in Spanish (Coronel, 1983). Strawberry production in Benguet is very successful
because of its cool climate. The best temperature range for strawberry production
is from 14 to 23oC (57-73oF). Strawberries normally flower as early as
November; 2 to 3 months after planting.
Strawberry is a slow-growing herbaceous plant with a short crown or
rootstock on which the leaves are produced together. Branch crowns and runners
are produced in the axils of leaves which are normally trifoliate. The fruit trusses
are produced terminally on the crown. The flower trusses may each have few or
many flowers on branching stalk (Hermano, 1995; Encarta Encyclopedia, 2006)

Botanically, the berry is not a true fruit because it consists of swollen
receptacles on the surface of which are embedded the numerous seeds (Hermano,
1999). The red part of a strawberry holds the actual fruit which is the tiny dark
seeds on the outside of the strawberry. The red part is just the tip of the strawberry
stem that’s enlarged (Encarta Encyclopedia, 2006)

Several cultivars of strawberry have been introduced in Baguio City and
La Trinidad, Benguet. According to Hermano (1995) cultivars Tioga, Aliso,
Ceascape, Chandler, Haranoka and Toyonoka have been introduced. At present
Etiology of the Red Stele Disease of Strawberry
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cultivars, Tioga, Sweet Charlie and a newly imported variety Camarosa are the
varieties commonly planted by farmers.

Red Stele Disease
Several authors used the names “red core”, “red root rot”, “red stele”,
“brown stele” and “black stele” to describe the disease. It was first named
“Lanarkshire strawberry disease” in Scotland where the disease was first
observed. The first definite reports of this disease were from Scotland in 1927
where Wardlaw (1921) as cited by Anderson (1956) attributed the trouble in
Lanarkshire to poor cultural conditions followed by the entry of weak root
parasites. The causal organism was named Phytophthora fragaria by Hickman in
1940 ( Anderson, 1956; Milholland et.al. 1989).
Ries (1996) reported that red stele disease is a serious root disease of
strawberries in the United States. It occurs during the late winter and spring. The
fungus attacks strawberries, loganberry, and potentilla (a genus of rosaceous herbs
commonly called cinquefoil) and is spread from one area to another primarily
through diseased plants. Red stele usually does not appear in a new planting site
until the spring of the first bearing year, from about full bloom to harvest. Minor
symptoms of root infection may appear, however, in late fall of the first growing
season. The disease is prevalent in poorly drained areas.

Etiology of the Red Stele Disease of Strawberry
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The Pathogen and Its Disease Cycle
Red stele is caused by soil-borne fungal-like organism Phytophthora
fragariae (Westcott, 1978; Ferguson, 1982; Childers, 1983; Clark, 1984; Agrios,
2005; Holliday, 1989). This is very persistent and can survive in the field for 13
years or longer once it has become established, regardless of the crop rotation
used or even if no strawberries are grown during that time (Ellis, 1989; Ries,
1996).

According to Ries (1996), the optimum temperature for growth and
infection of the red stele fungus is 14 0C. It is inactive at 4.5oC and above 30oC.
When the soil moisture is high and the temperature is cool, plants show typical
symptoms with in 10 days after infection. He added that heavy clay soils, which
retain moisture for long periods of time, provide a favorable environment for the
development of the disease because the zoospore can spread greater distance and
produce more infection sites. Horsfall (1978) added that infection of red stele is
higher in coarse soil than in fine soil. However, Ellis (1996) reported that soil
types do not affect the presence of the red stele fungus. The fungus is active at
pH of 4.0 to 7.6 but will not grow in alkaline soil with pH of 8.0 or above.
Likewise, Holliday (1989) observed that more disease is found in acid soil and
there is a long survival of oospores. In culture media, it grows well with pH as
low as 4.0 but no growth occurs at a pH of 8.0 or higher (Anderson, 1956).
According to Anderson (1956) the causal organism of the red stele disease grows
on very limited culture media.
Etiology of the Red Stele Disease of Strawberry
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George and Milholland (1986) reported that the optimum temperature
range for both zoospore germination on host roots and oospore development in
host tissue was 15-20 0C. A temperature of 25 oC encouraged zoospore
germination but greatly inhibit oospore formation.

Phytophthora fragariae persists in the soil as thick walled, resting spores
called oospores. The oospore, not the chlamydospore is the primary survival
structure in the field (Hickman, 1940 as cited by Law et.al. in 1991).
According to Ellis (1996), the oospores germinate and form sporangia
with the infectious spores of the fungus called zoospores when the soil is wet and
moist. Once the microscopic zoospores are released into the soil when the area is
flooded it swims to the tips of strawberry roots, to which they are chemically
attracted. Zoospores activity may occur at soil temperature ranging from 4 to
25oC, but is most significant from 7 to 15oC. He added that once the zoospore
have infected the root tips, the pathogen begins to grow up into other parts of the
roots, causing the characteristic dark rot and red stele symptom. Ries (1996)
added that zoospores infecting the tips of the young and fleshy roots destroy the
water-and-food conducting tissues. Infection and growth of the fungus in the
roots reduces the flow of water and nutrients to the developing leaves and fruit
causing drought-like symptoms in the plant. Ellis (1996) reported also that new
sporangia are formed along the outside of infected root tissue and release
additional zoospores whenever the soil is saturated, thereby continuing the spread
of the disease. He added that oospores are also produced within infected roots
Etiology of the Red Stele Disease of Strawberry
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and when roots begin to rot and die oospores are released into the soil when the
roots decay, thus completing the disease cycle.

Disease Symptoms
Above
ground. Infected strawberry plants lack vigor or grow poorly, are
stunted, and frequently wilt. The young leaves often have a metallic bluish-green
while older leaves sometimes turn yellow or red prematurely. They produce
small berries and a poor runner growth. During the hot, dry weather of early
summer, infected plants wilt, collapse and die prematurely (Wescotte, 1978;
Childers, 1983; Smith, 1984; Holliday, 1989; Alford and Locke, 1989; Los and
Schroeder, 1992; Swift and Dickens, 1995; Ellis, 1996; Agrios, 2005).

Below
ground. The most reliable symptom of red stele is found within the
root. Plants with red stele have few lateral roots and the main fleshy roots have a
“rat-tail” appearance. During intermediate stages of disease development, these
fleshy roots will be white near the crown of the plant but will show dark rot
progressing upward from the tips. In normal root, both the center (stele) and the
part surrounding the stele is distinctively brownish-white while plant infected
with red stele disease has distinctive brownish-red, pink or brick red. The red
color may show only near the dead tip, or it may extend the length of the root
(Wescotte, 1978; Holliday; Alford and Locke, 1989; Swift and Dickens, 1995;
and Ellis, 1996). Ries (1995) reported that no other disease of strawberry shows
this particular symptom. However, Khanizadeh and Buzard (1992) mentioned
Etiology of the Red Stele Disease of Strawberry
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that reddening of the root in the field-grown plants may be used as a preliminary
diagnostic feature, concluding that visual symptoms alone should not be used as
criterion to assess susceptibility. Resistant cultivars are not immune from
infection and relatively high number of oopores may be observed in the
symptomless root tissues of inoculated resistant types within two weeks.

Etiology of the Red Stele Disease of Strawberry
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MATERIALS AND METHODS

Field Study

Survey

The disease was assessed and recorded from the different strawberry
growing areas namely Swamp Area, BSU Balili Experiment Area, and Longlong,
all situated at La Trinidad and Sto. Tomas in Tuba, Benguet (Plates 2-5
A total area of 31,426 sq.m strawberry farms was surveyed to determine
the incidence of the red stele disease at the Swamp Area (24,679 sq.m.), Balili
Experiment Station (3,240 sq.m.), and Longlong (2,313 sq. m.) La Trinidad and
Sto. Tomas, Tuba (1,194 sq. m.), Benguet.




Plate 1. Overview of strawberry farms at Swamp Area, BSU, La Trinidad,
Benguet

Etiology of the Red Stele Disease of Strawberry
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Plate 2. Overview of strawberry farms at Balili Experiment Station, BSU, La
Trinidad, Benguet





Plate 3. Overview of strawberry farms at Longlong, Puguis, La Trinidad, Benguet





Plate 4. Overview of strawberry farms at Sto. Tomas, Tuba, Benguet
Etiology of the Red Stele Disease of Strawberry
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Symptomatology


Reported symptoms of the red stele disease such as stunting, wilting and
abnormal color of young leaves were observed and documented on cultivars
Tioga and Sweet Charlie grown by farmers. Strawberry plants showing these
above ground symptoms were uprooted and the roots were examined for symptom
of reddening of the core to be declared as infected with red stele disease.

Assessment of Disease Incidence



The incidence of the disease was assessed in the different areas surveyed.
In the Swamp area, the lot was divided into 4 blocks, each block consisting of 10
fields. To each field, ten sample plots were randomly selected. The total number
of plants and the number of infected plants per plot were counted. Based on field
symptoms, incidence of the disease was assessed based on Waller et al. (2002):
Number of infected plants
% Disease Incidence = _________________________ x 100%
Total numbers of plants


Laboratory Study


Laboratory experiments were conducted at the BNCRDC-BPI, Baguio
City and Department of Plant Pathology, College of Agriculture, BSU, La
Trinidad.

Etiology of the Red Stele Disease of Strawberry
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Collection of Diseased Plants

Samples of strawberry plants showing characteristic symptoms of the
disease were collected in La Trinidad particularly at the Swamp Area and BSU
Balili Experiment Station. The samples were carefully dug up to avoid damage of
roots and examined for symptoms of red stele and were placed individually in
properly labeled plastic bags for further diagnosis, isolation and identification.

Isolation of the Organism
In the laboratory, infected strawberry plants collected from the field were
washed thoroughly to remove adhering soil. Roots were peeled off to expose the
root core for the presence of reddish-brown stele appearance. The roots were cut
measuring 1 cm and surface-sterilized with 1% NaOCl, blotted dry and washed
three times with sterile distilled water. Infected roots were isolated in PARPH, a
selective medium for Phytophthora sp.. The plates were incubated for six days at
18-22 oC to allow the growth of the causal organism. The organism was re-
isolated in carrot agar to obtain a pure culture.

Identification of the Organism
The causal organism of the red stele disease was identified through the
cultural and morphological characteristics including coenocytic mycelia.
sporangia, oospore and zoospore. The growth of the organism was compared
from four different culture media.
Etiology of the Red Stele Disease of Strawberry
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Cultural characteristics of the organism. To determine the effect of culture
media on the growth of the causal agent, four treatments were laid out following
the completely randomized design (CRD) with 5 replications. The different
culture media used as treatments were as follows.

TREATMENTS
MEDIA
T1

Potato Dextose Agar (PDA)
T2


Carrot Broth Agar (CBA)
T3


V-8 Juice Agar (V8JA)
T4


Oatmeal Agar (OMA)
The growth of the causal organism on the different culture media was
observed for a week.
Morphological characteristics of the organism. Sporangia and zoospores
were produced following the procedure used by George and Milholland (1986).
Agar discs were cut from a two-weeks old culture using a cork borer, transferred
to a sterilized and petri plate, flooded with 10 ml sterilized 5% V8 juice instead of
soil leachate and incubated for three days under continuous light at 18-22 0C.
Likewise, zoospores were produced by chilling the agar disc containing sporangia
to 5 0C temperature for 30 minutes and rewarmed at room temperature from 30
minutes to one hour.
The oospore and sporangia were measured following procedures used in
microscopic measurement by Benzon (1998). Calibration was done by inserting
Etiology of the Red Stele Disease of Strawberry
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the ocular micrometer on the eyepiece of the microscope while the stage
micrometer was placed on the stage of the microscope. Focusing was done on the
stage micrometer using the low power objectives (LPO) and then on the high
power objective (HPO) of the microscope. Zero (0) point of the stage micrometer
was set to coincide with that of the ocular micrometer. The lines on the ocular
micrometer that coincides exactly with the lines of the stage micrometer were
counted. Calibration factor (CF) or calibration constant (CC) was calculated
using this formula:

n division of stage micrometer
CF = x 0.01 mm

20 divisions of ocular micrometer


0.01 mm – divisions of the stage micrometer


Pathogenicity Test

To confirm the results on the identification of the pathogen, a
pathogenicity test was conducted.

Preparation of inoculum
Pure culture of P. fragariae grown in carrot agar for two weeks at 24 oC
was used as inoculum. Petri plate cultures were filled with 10 ml rain water
sufficient to barely cover the agar. The cultures were incubated for three days
under continuous light to induce the production of additional sporangia after
Etiology of the Red Stele Disease of Strawberry
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which these were chilled for 30 minutes at 5oC and re-warmed at room
temperature from 30 minutes to one hour to induce the release of zoospores.

Standardization of the inoculum
Suspensions containing zoospores were prepared and spore concentrations
were estimated using a hemacytometer. One ml of the zoospore suspension was
sucked using a micropipette and deposited on the ridge of the hemacytometer.
After the suspension had flow automatically to the counting chamber, spore
counting was done in the five squares of the nine squares, labeled A which
contains 16 small squares. Standardization was done three times. The average
count of the five squares per trial was multiplied with 20,000 to obtain the total
spore/ml. The treatments were prepared by dilution adjusting the desired
concentrations by adding sterile distilled water and inoculated into the test plants.

Inoculation

Healthy strawberry runners of cv. Sweet Charlie produced from BSU were
surfaced sterilized with 1% NaOCl for a minute and rinsed three times with sterile
distilled water. Strawberry plants were inoculated with varying concentrations of
zoospores to obtain a range of disease infection. The roots of each test plant were
sprayed with the corresponding zoospore concentration to the assigned treatments.
The control plants were sprayed with distilled water alone. Inoculated plants
were wrapped with tissue paper and placed in clear plastic bags before storing in
the dark for 48 hours to enhance zoospore penetration before planting. Plants in
Etiology of the Red Stele Disease of Strawberry
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all treatments were planted in plastic pots measuring 20 x 25 cm diameter filled
with heat sterilized soil.

The treatments were laid out following the CRD design replicated four
times with three sample plants per replicate.

The treatments were the following:

TREATMENTS INOCULUM LEVEL (zoospores/ml)


T0

0
(Uninoculated)
T1

2
x
103
T2
5 x 103
T3



10 x 103
T4

15
x
103
T5



20 x 103
All cultural management practices for commercial strawberry production
like watering, fertilization, weeding and control of insect pests and foliar diseases
were employed.

Re-isolation of the Organism from Inoculated Plants.
The roots were further observed in the laboratory to confirm if the rotting
symptoms were due to P. fragariae. Roots measuring 5-7 mm were excised and
observed under the microscope for the presence of oospores and reddening of the
root core.
Etiology of the Red Stele Disease of Strawberry
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Re-isolation of the organism from the inoculated plants was done further
using the same procedure used earlier in the isolation of the organism.
Characteristics of the isolate obtained were compared to the original inoculum
used.

Data Gathered:
1. Field symptoms. These include symptoms of red stele disease reported in
other countries.
2. Disease incidence in the field (%). This refers to healthy and infected
strawberry plants counted and computed following the formula by Waller
et. a.l (2002)

Disease Incidence (%) = ___No. of infected plant units x 100%
Total no. of plant units

3. Cultural and morphological characteristics of the pathogen. The growth
including the structure of the pathogen in different culture media were
described.
4. Pathogenicity Test
a. Monthly plant height (cm). This was obtained by measuring the

initial and monthly height after transplanting to harvesting stage for a
period of four months.
Etiology of the Red Stele Disease of Strawberry
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21
b. Fresh top weight at harvest (g). This was taken by weighing the tops of
strawberry plants after uprooting.
c. Fresh root weight at harvest (g). The roots were washed to remove
adhering soils, blotted dry in tissue paper and weighed.
d. Oven dry top weight (g). This was done by weighing the tops of the
plant after it was oven-dried at 700C for 2 days.
e. Oven dry root weight. This was done by weighing the roots after it was
oven-dried at 700C for 2 days.
f. Yield components (g). The yield was classified as marketable and non-
marketable. Marketable yield are berries with big sizes (17mm and
above) and of good quality. Berries with very small sizes with poor
quality and those attacked by insects and other airborne fungal
diseases were considered as non-marketable.
g. Disease severity – All plants in pots were uprooted and rated based on
the rotting of the roots using the rating scale below.


Rating

% root infection
1

No reddening of the root core


2

1-25% rotten roots and with reddening of root core
3

26-50% rotten roots and with reddening of root core
4

51-75% rotten roots and with reddening of root core
5

76-100% rotten roots and with reddening of root
core



Etiology of the Red Stele Disease of Strawberry
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22



Rating scale of 1
Rating scale of 2






Rating scale of 3

Rating scale of 4







Rating scale of 5






Plate 5. Disease rating scale used in assessing the severity of root infection due to
P. fragariae
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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23
RESULTS AND DISCUSSION

Field study
Symptomatology
Table 1. Field symptoms of infected strawberry plants in La Trinidad and
Sto.Tomas, Tuba, Benguet.


SYMPTOMS
Location
Stunting Change
in Wilting Small Reddening
Leaf color
Berries
of root core
La Trinidad





Swamp block I
+ + + + +
Swamp block II
+ + + + +
Swamp block III
+ + + + +
Swamp block IV
+ + + + +
Balili expt’l area
+ + + + +
Long-long
+ + - + +
Tuba





Sto. Tomas
+ + - + +


Table 1 shows the different symptoms associated with red stele disease in
strawberry plants grown in La Trinidad and Tuba, Benguet. Wilting, stunting,
abnormal leaf colors, production of small berries and the reddening of the root
core, which are characteristics of red stele disease as reported in earlier studies
(Wescotte, 1978; Childers, 1983; Smith, 1984; Holliday, 1989; Alford and Locke,
1989; Swift and Dickens, 1995; and Ellis, 1996) were commonly observed in the
strawberry growing areas visited. However, wilting was not observed in Sto.
Etiology of the Red Stele Disease of Strawberry
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24
Tomas and Longlong farms. Further, root samples taken from Sto. Tomas
exhibited reddening of stele mostly on decaying old roots but not on newly
developed roots. Uprooted plants from Swamp Area showed severe rotting in old
roots and reddening of the core in newly developed roots.
Cultivars Tioga and Sweet Charlie showed no variation on symptoms of
the disease (Plates 6-8). However, according to the farmers who were
interviewed the old Tioga cultivar was more resistant than Sweet Charlie. They
changed from Tioga to Sweet Charlie which is high yielding to meet the market
demand. However, farmers observed that variety Sweet Charlie is susceptible to
diseases. According to Philley (1995) Tioga which is currently grown in the
United States is susceptible to red stele disease and Verticillium wilt.
Symptoms of wilting and stunting are commonly observed starting from
the month of December until the end of summer where the weather is warm and
dry or after heavy rains when the area is flooded. According to some farmers,
they also observed symptoms similar to burning effect on the leaves and plant
eventually dies after several days. Roots of plants showing these symptoms when
dug up carefully have rotted roots and very few new roots. Roots of wilted plant
showed some “rat tail” appearance as described previously (Ellis,1996 and Ries ,
1995.
The symptoms observed in the field have some resemblance to other
fungal diseases like Verticillium sp., Pythium sp. and Fusarium sp. According
to Los (1992), Ries (1996) and Anderson (1956), the most reliable symptom is
Etiology of the Red Stele Disease of Strawberry
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25
found within the root. During the survey, several infected roots were peeled off
exposing the core of the roots. The fleshy roots appeared white near the crown of
the plant but a dark rot progressing upward from the tips was observed. Some of
the roots, however, did not show reddening of the core but oospores were present
when observed under the microscope. According to Khanizadeh and Buzard
(1992), visual symptoms alone should not be used as criterion to assess
susceptibility. Resistant cultivars are not immune from infection and relatively
high number of oospores may be observed in the symptomless root tissues of
inoculated resistant types within two weeks.




c
a
b





d

e
f

Plate 6. Symptoms of red stele disease on cv. Tioga (a) stunting, (b)
wilting, (c) abnormal color of leaves, (d) death of severely
infected plants (e) reddening of the core and (f) rotten roots
Etiology of the Red Stele Disease of Strawberry
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26






a
b
c







d
e
f


Plate 7. Symptoms of red stele on cv. Sweet Charlie (a) stunting, (b)
wilting, (c) red colored stele, (d) abnormal color of leaves (e)
death of severely infected plants and (f) rotten roots








a b
c


Plate 8. (a) Cross section of an infected root, x400 (b) longitudinal section
of an infected strawberry root, x400 (c) healthy root with a
yellow-white color of core, x400

Disease Incidence

Table 2 shows the incidence of the red stele disease in strawberry growing
areas in La Trinidad and Tuba, Benguet. The lowest disease incidence was noted
in Sto. Tomas, Tuba followed by Longlong, La Trinidad with 8.91% and 19.80%,
respectively.
Etiology of the Red Stele Disease of Strawberry
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27
The root samples taken from Sto. Tomas area showed symptoms of the
disease only in the old roots but not on newly developed ones. The infection in
the old roots probably originated from the source of the planting materials.
According to the farmers (personal interview, 2005), planting materials were
obtained from the Swamp Area, BSU, La Trinidad, Benguet where the disease
was first observed. However, the practice of overhead irrigation method and
planting of strawberry in newly operated farms including the distance of one
farm to the other area which is far enough to cause contamination might have
contributed to the low incidence of the disease in this area.
On the other hand, strawberries grown in Swamp Area, BSU at La
Trinidad, Benguet registered the highest infection particularly in Blocks II and III
with 39.78% and 43.51% disease incidence, respectively. The high incidence of
the disease in the Swamp Area could be attributed to the flat terrain and
management practices of the farmers. During the rainy season, the farms are
usually flooded for several days which are favorable for the organism to cause
infection. It was also noted during the survey that farms with “dead rows” or no
canals have higher infection compared to well maintain farms with raised beds of
7 up to 10 cm high. This confirms the findings of Ellis (1986) raising the beds to
at least 8 to 10 cm prevents the roots from touching the water that favors the
zoospore to swim and infect roots. Moreover, farmers at swamp and Balili areas
at La Trinidad mostly irrigate their plants by flooding. According to Ries (1995),
oospores from decayed roots are released into the soil when the soil is wet and
Etiology of the Red Stele Disease of Strawberry
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28
germinate by producing sporangia. Ellis (1996) added that the infective
zoospores produced from the sporangia are disseminated by moving water and
infect the tips of young fleshy roots of strawberry to which these are chemically
attracted.
Another contributing factor in the disease development is the improper
disposal of infected plant materials by farmers. Uprooted diseased plants are just
left at the sides of the field which serve as source of inocula when condition is
favorable.
Likewise, higher infection was observed in fields where planting materials
were obtained from previously grown and harvested plants called daughter
crowns which is a common practice to several farmers.

Table 2. Incidence of red stele disease in La Trinidad and Sto. Tomas, Tuba,
Benguet

Location AREA(sq.m.)
DISEASE
INCIDENCE
(%)
La Trinidad


Swamp Block I
5,765
26.30
Swamp Block II
5,191
21.80
Swamp Block III
6438
43.51
Swamp Block IV
7,285
39.78
Balili
3,240
20.38
Long-long
2,313
19.80
Tuba

Sto. Tomas
1194
8.91
Etiology of the Red Stele Disease of Strawberry
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29
Laboratory Study
Characterization and Identification of the Pathogen
Mycelial growth of the pathogen on different culture media

45
40
35
30
)
PDA
m
m
25
t
h (
CBA
20
ow
gr
15
V8J-A
10
OMA
5
0
1
2
3
4
5
6
7
Days

Figure 1. Average daily growth of the pathogen in different culture media

The average daily mycelial growth of the pathogen on different culture
media is shown in Figure 1. The growth of the isolated organism is very slow
compared to other fungal organisms which confirm the earlier report of Anderson
(1956) that growth of Phytophthora fragariae is very slow when compared to
other species of Phytopthora and grew only in very limited culture media.
Of the four culture media tested, OMA gave the widest colony diameter
from 2nd to 7th day of incubation with a mean of 27.45mm after a week followed
by CBA with a mean of 24.17 mm. V8J-A had a mean colony diameter of 18.71
mm and the smallest was observed on PDA with a mean 7.53 mm.
Etiology of the Red Stele Disease of Strawberry
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30
Cultural Characteristics of the Organism


Table 3. Cultural characteristics of the pathogen in different culture media


CULTURE MEDIA
Criterion
PDA CBA OMA V8J-A
Colony diameter




after a week of
10.55c
39.85a
40.45a
32.75b
incubation (mm)
Growth
Very slow
Fast growth
Fast growth
Slightly fast
characteristic
growth
growth
after a week of
incubation
Characteristics of
Very dense, Dense, fluffy Very scarce,
Slightly
mycelia
irregular and
barely seen
dense, round
spreading
on media
with
pattern
radiating
margin
Color of mycelia
White
Brown to
White Brown
to
white
white

The cultural characteristics of the pathogen in different culture media are
shown in Table 3. Rapid mycelial growth was noted on OMA and CBA with
colony diameters of 40.45mm and 39.85mm, respectively. On the other hand, the
growth on PDA is very slow with a colony diameter of 10.55 mm. In PDA, the
organism has very dense mycelia, with irregular and spreading pattern and white
in color (Plate 9). On the other hand, mycelial growth on CBA is dense, fluffy,
with round pattern and brown to white in color (Plate 10). Although rapid growth
was observed on OMA, the mycelia are very scarce and could hardly be seen on
media a week after isolation but after one month a very thin cotton-like
appearance on the surface of the media with white color was observed. Growth
Etiology of the Red Stele Disease of Strawberry
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31
of the organism in V8J-A is slightly faster with brown to white mycelia and
mycelial growth pattern of round with radiating margins (Plate 11).



Plate 9. Mycelial growth of the pathogen on PDA after a week of incubation





Plate 10. Mycelial growth of the pathogen on CBA after a week of incubation

Etiology of the Red Stele Disease of Strawberry
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Plate 11. Mycelial growth of the pathogen on V8J-A after a week of incubation



Plate 12. Mycelial growth of the pathogen on OMA after a week of incubation


Morphological characteristics of the organism

Hypha. The hyphae are coenocytic, hyaline and slender with few swelling
of hyphae on media CBA, OMA and V8JA, as compared to PDA where profuse
swellings are observed (Plate 13).
Oospore. The oospores are round in shape, with diameter ranging from
33-41 um. They are found readily in culture grown in CBA, V8J-A and OMA but
not on PDA. The oospores are surrounded by wrinkled, dark red oogonial
Etiology of the Red Stele Disease of Strawberry
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33
envelops. The antheridia were found attached to the base of the oospore.
Oospores are also observed on excised roots from infected plants (Plate 14).
Sporangia. Sporangia were not readily observed on the four culture media
used. These were produced after incubation of agar disc in 5% V8 juice or
rainwater for 3 days. Sporangia were observed on CBA, V8J-A and OMA but not
on PDA. The number of sporangia increased as the incubation period was
prolonged. Likewise, sporangia from infected roots were produced by incubating
excised roots in rain water. Sporangia were found to grow along the infected
tissue. The sporangia are non-papillate, inversely pear shaped or oval, with
measurement ranging from 53-72 um (Plate 15).
Zoospore. The zoospores germinated abundantly after chilling the agar
disc containing sporangia at a 50C temperature. These are very motile and swam
immediately after coming out from the sporangia but encysted after an hour. The
zoospore has 2 flagella and has round shape when at rest (Plate 16). According to
Anderson (1956) zoospore measures 12 um and each sporangium contains 40 to
50 zoospores. Based from the reports of CABI (undated), the motile zoospore
swims to the root tips of the host plant where they encysted, attach itself and form
germ tubes which penetrate into the roots. The organsim traverses the cortex
inter- and intracellularly to the stele, mainly colonizing the pericycle and the
phloem. Growth is mostly concentrated on the stele but hyphae may grow out
from the roots and form new sporangia.
Etiology of the Red Stele Disease of Strawberry
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34
The morphological and cultural characteristics of the organism are very
similar to Phytophthora fragariae as described by Anderson (1956) and Stamps
et. al (1990).

a b c
d

Plate 13. Hyphae, x400 (a) CBA, (b) V8J-A, (c) OMA, (D) PDA



a
b

Plate 14. Ooospore (a) from culture media, x1000 (b) infected root, x400

Etiology of the Red Stele Disease of Strawberry
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35
a
b
c
d
e f


Plate 15. Sporangia from (a) infected root, x100 (b) infected root, x400 (c)
infected root, x1000 (d) culture media, x400 (e) culture media,
x1000 (f) stained sporangium showing the wide pore opening
(non- papillate), x1000











a
b
c



Plate 16. (a) Encysted zoospores released from a sporangium, x1000 (b)
encysted zoospores inside cells of infected root, x400 (c) healthy
plant cell



Etiology of the Red Stele Disease of Strawberry
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Pathogenicity Test


Symptoms on inoculated plants
The causal organism was inoculated to healthy plants and symptoms of stunting
were observed a month after inoculation on some of the treated plants. The leaves were
small, cupped and not spreading compared to the control. Some plants appeared healthy
but when observed closely the leaves were off colored. They had blue green color
compared to the uninoculated plants where leaves were shiny green. Other symptoms
like wilting, yellowing of older leaves and productions of small berries were noted during
the harvesting stage. Wilting occurred during warm weather. Abnormal change of color
on leaves from green to red or purple was observed but on very few plants. Plants
inoculated with 15 x 103 and 20 x 103 zoospores/ml were stunted and produced small
berries. The above observations were similar to the findings of Wescotte, (1978),
Childers (1983), Swift and Dickens (1995), Ries (1995) and Ellis (1996). According to
Ries (1996), the stunting of the plant is due to the infection of the zoospore on the roots
causing rotting which destroyed the water-and-food conducting tissues. The infection
and growth of the fungus in the plant roots reduced the flow of water and nutrients to the
developing leaves and fruit causing drought-like symptoms in the plant.
Etiology of the Red Stele Disease of Strawberry
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37
T0
T1
T2
T3
T4
T5


Plate 17. Strawberry plants 7 months after inoculation



b


Plate 18. (a) Symptom of stunting on inoculated plants (b) uninoculated plants


a
b


Plate 19. (a) Symptom of abnormal color on the shoot (b) uninoculated plant
Etiology of the Red Stele Disease of Strawberry
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38

a
b


Plate 20. (a) Symptom of yellowing of leaves on inoculated plants (b)
uninoculated plant










a
b



Plate 21. (a) Symptom of wilting on inoculated plants (b) uninoculated plant












a
b



Plate 22. (a) Production of small berries and (b) death of severely infected plant
Etiology of the Red Stele Disease of Strawberry
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39
Inoculated plants when uprooted produced very few new roots and most of
the main roots were rotten as compared to the uninoculated plants where roots are
thick and bushy with several secondary feeder roots. The “rat tail” appearance
was observed on infected roots (Plate 24a). Infected roots have brick red to
brownish red stele progressing upward from the tip (Plate 24b) compared to
healthy roots with yellowish white core (Plate 24c). The crown of the strawberry
plant was not infected which confirms reports of Ellis (1996) and several authors
that the fungus does not attack the crown. Microscopic examination of infected
plants confirmed that the causal organism is P. fragaria as evidenced by the
production of several oospores on infected roots. In addition, several zoospores
were also found feeding on the root tissues. George and Milholland (1986)
reported that the number of oospore per root segment in a susceptible variety
inoculated with 1, 5, and 10 x 103 zoospores/ml was 17, 22 and 29, respectively.
Results of the pathogenicity test further confirms the previous reports that P.
fragariae is the causal organism of red stele disease.




a
b

Plate 23. (a) Symptoms of rotting of strawberry roots due to P. fragariae (b)
root of uninoculated plants

Etiology of the Red Stele Disease of Strawberry
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40










b

a

c c


Plate 24. Excised roots showing the (a) “rat tail” appearance (b) reddening of the
core, x400 (c) root of a healthy plant, x400


Plant Height (mm)

Result shows that the different zoospore concentrations influenced the
growth of the crop (Figure 2). Plants inoculated with the highest concentration of
20 x 103 were the shortest with mean height of 139.58 mm after 4 months
followed by those plants inoculated with 15 x103 and 10 x 103 with 157.84 and
159.17mm, respectively. Early symptom of stunting was observed after a month
on the treated plants. Plants inoculated with lower zoospore concentrations of 2 x
103 and 5 x 103 showed slight stunting and wilting only at harvest. The
uninoculated plants were the tallest with mean height of 254.17 mm.
Inoculation of 20 x 103 zoospores/ml caused higher infection to the roots
resulting to early stunting of the plant. The main roots were rotten and very few
Etiology of the Red Stele Disease of Strawberry
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41
new developed roots were produced compared to the uninoculated plants with
thick and bushy roots with several secondary feeder roots.

300
T0
250
T1
200
T2
)
m
m
t
(
h
T3
ig
150
e
H
T4
100
T5
50
0
Initial
1
2
3
4
Months


Figure 2. Average monthly height (mm) of strawberry plants as affected by
inoculum level (Note: 0 = uninoculated, 1 = 2 x103 zoospore/ml, 2 = 5 x103
zoospore/ml, 3 = 10 x103 zoospore/ml, 4 = 15 x103 zoospore/ml, 5 = 20 x103
zoospore/ml)


Yield

The infection caused by the disease on the inoculated plants greatly
influenced the yield of strawberry (Figure 3). There was a reduction in yield from
38.03 to 57.74% on inoculated plants compared to the uninoculated ones. The
different zoospore concentrations showed significant effect on the inoculated
plants. Plants with the highest concentration of 20 x 103 produced the lowest
mean yield of 117.91g while the highest was obtained from the uninoculated
Etiology of the Red Stele Disease of Strawberry
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42
plants with 279.05g. However, the yield of the plants inoculated with 2 x 103, 5 x
103 and 10 x 103 zoospopres/ml were comparable.
On the other hand, the highest non-marketable yield was obtained from
plants inoculated with 20 x 103 zoospores/ml with 75.93g but did not differ
significantly with those plants inoculated with lower inoculum levels. The lowest
non-marketable yield was obtained from the uninoculated and in plants that
received the lowest inoculum level with 55.54 and 56.06g, respectively.
Results reveal that inoculum density of the organism directly affected the
yield of the crop. The mean yield was observed to decrease significantly as the
inoculum density was increased. High inoculum density resulted to the production
of few and small berries. This is dew to the rotting of the roots and production of
few secondary feeder roots resulting to early symptoms of stunting. According to
Reid, (1949) as cited by CABI (undated), damage of the disease is most severe
after winters with yield as low as 1 t/ha, mostly of small fruit of poor quality. In
Nova Scotia (Canada), it was estimated that in one season 78% of the strawberry
area was rendered unproductive causing losses to growers more than 1500 Can$
per hectare (Gourly and Delbridge, 1972 as cited by CABI).
Etiology of the Red Stele Disease of Strawberry
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43


300


250


200

nt
a
150

pl

g/
100


50

0

T0
T1
T2
T3
T4
T5

Treatments


MARKETABLE
NON-MARKETABLE

Figure 3. Effect of inoculum level of P. fragariae on the yield of strawberry
(Note: 0 = uninoculated, 1= 2 x103 zoospore/ml, 2 = 5 x103 zoospore/ml, 3 = 10 x103
zoospore/ml, 4 = 15 x103 zoospore/ml, 5 = 20 x103 zoospore/ml)


Fresh Weight of Plants



The fresh weight of the plant after harvest was taken to determine the
effect of the different zoospore concentrations on the weight of strawberry plant
(Figure 4). The lowest fresh top weight was obtained from plants inoculated with
15 x 103 and 20 x 103 zoospores/ml with means of 16.18 and 16.83g, respectively.
This was due to severe stunting of inoculated plants. Inoculated plants were
stunted and have small and cupped leaves as compared to the uninoculated ones
with fully expanded leaves. Plants inoculated with 2 x 103 and 5 x 103 zoospores
per ml showed slight symptom of stunting and wilting during the latter part of the
harvesting period. However, statistical analysis shows insignificant differences
Etiology of the Red Stele Disease of Strawberry
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44
on plants inoculated with 2 x 103, 5 x 103 and 10 x 103 zoospores/ml. The
uninoculated plants significantly had the heaviest weight of 53.53g.
Similarly, fresh root weight was significantly affected by the fungal
infection (Figure 4). Roots of inoculated plants showed rotting or decaying of the
main roots. Very few secondary feeder roots were also produced. Plants
inoculated with the highest level of inoculum were severely infected and gave the
lowest fresh root weight of 6.05g followed by those inoculated with 15 x 103 and
10 x 103 zoospores/ml with 10.53 and 11.02g, respectively. On the other hand, the
roots of uninoculated plants which were thick and bushy with several secondary
feeder roots had the heaviest fresh root weight of 28.28g.


60

T0
50

T1
40

T2
s
30
r
am

T3
g
20
T4

10
T5


0
TOP WEIGHT
ROOT WEIGHT

Figure 4. Effect of varying levels of P. fragariae inoculum on the fresh weight of
strawberry (Note: 0= uninoculated, 1= 2 x103 zoospore/ml, 2 = 5 x103
zoospore/ml, 3 = 10 x103 zoospore/ml, 4 = 15 x103 zoospore/ml, 5 = 20 x103
zoospore/ml)
Etiology of the Red Stele Disease of Strawberry
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45
Oven dry weight of plants


The uninoculated plants gave significantly heavier top and root oven dry
weights of 16.07 and 6.75, respectively compared to the inoculated plants (Figure
5). Inoculated plants exhibited rotting of the roots caused by the fungus resulting
to stunting and wilting thus having lighter top and root weight. Plants that
received the highest inoculum level of 20 x 103 zoospores/ml had the lowest top
and root weights with 2.44 and 2.12g, respectively.


18
T0

16
14
T1

12
T2

s
10
a
m
T3

gr
8
6
T4

4
T5

2

0
TOP WEIGHT
ROOT WEIGHT

Figure 5. Effect pf varying levels of P. fragariae inoculum on the dry weight of
strawberry (Note: 0= uninoculated, 1= 2 x103 zoospore/ml, 2 = 5 x103
zoospore/ml, 3 = 10 x103 zoospore/ml, 4 = 15 x103 zoospore/ml, 5 = 20 x103
zoospore/ml)

Disease Severity at Harvest


An arbitrary rating scale was developed to assess the effect of the
inoculum density on the severity of the disease on inoculated plants under pot test.
Etiology of the Red Stele Disease of Strawberry
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46
Result shows that inoculated plants have disease rating ranging from 3.67 to 5
equivalent to 26 up to 100% root infection (Figure 6).

5

4.5
4

3.5
G
3
I
NT 2.5
AR
2
1.5

1
0.5

0
T0
T1
T2
T3
T4
T5

TREATMENTS

Figure 6. Disease severity as affected by inoculum density (Note: 0 = uninoculated,
1 = 2 x103 zoospore/ml, 2 = 5 x103 zoospore/ml, 3 = 10 x103 zoospore/ml,
4 = 15 x103 zoospore/ml, 5 = 20 x103 zoospore/ml)

Infected plants within these range showed yellowing of leaves, wilting
and stunting. Very few new roots were produced and most of the feeder roots
have infected cores with reddening symptom on stele. Moreover, excised roots
incubated in rain water for 3 days produced several oospores and sporangia.
According to George and Milholland (1986) oospore development by P.
fragariae in roots of susceptible plants varied with inoculum density. Plants
inoculated with 1, 5 and 10 x 103 zoospores/ml produced 17, 22 and 29 oospores
per root segment. There was no significant difference in oospore production or
percentage of root segments containing oospore when susceptible cultivar
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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47
Tennessee and resistant cultivar Stelemaster were inoculated with 1, 5, or 10 x 103
zoospores per milliliter.
Furthermore, significant difference was observed in oospore development
between the two cultivars when inoculated with 10, 30 or 100 x 103 zoopores/ml.
The susceptible cultivar gave significantly greater number of oospores than
resistant Stelemaster plants inoculated with the same concentrations of zoospores.
Linear regression analysis had no significant effect on mean oospore production
in the susceptible or resistant cultivars, but differences did occur in the percentage
of susceptible root segments containing oospores among the three concentrations
tested.
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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48
SUMMARY, CONCLUSION AND RECOMMENDATION

Summary


Symptoms of the red stele disease such as stunting, wilting, off-colored
leaves, abnormal color of leaves from green to purple, yellowing of immature
leaves, production of small berries and reddening of the core were mostly
observed in the areas surveyed. High incidence of the disease was observed in
Swamp area, BSU, La Trinidad, Benguet ranging from 23-43% infection while
lower infection was observed in Longlong, La Trinidad and Sto. Tomas, Tuba
with 19.80 and 8.91%, respectively.

The pathogen grows rapidly on OMA and CBA with mean mycelial
colony diameter of 27.45 and 24.17mm, respectively. On the other hand, the
growth on PDA is very poor with mean colony diameter of 7.53mm. The mycelia
are white in color on PDA and OMA while brown to white on CBA and V8J-A
media. Mycelial growth is very dense on PDA, slightly dense on CBA and V8J-A
while mycelia on OMA are very scarce and can hardly be seen on culture.

The causal organism was identified as Phytophthora fragariae. The
hyphae are coenocytic, hyaline and slender but profuse swelling of hyphae was
observed on PDA. The oospores of the pathogen were readily observed on CBA
and V8J-A and OMA but not on PDA. The oospores are round in shape and
surrounded by a dark, red, wrinkled oogonial envelop. Several sporangia were
observed on CBA and V8J-A but very few on OMA after incubating agar disc in
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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49
5% V8 juice for 4 days. No sporangial growth was observed on PDA. The
sporangia are non-papillate with oval or inverted pear-shape. Zoospores are very
motile with two flagella and are in round shape when encysted.

Strawberry plants inoculated with different concentrations of inoculum
developed symptoms of stunting, wilting, off-colored leaves from shiny green to
bluish - green, yellowing of immature leaves, small berries and death of severely
infected plants. Abnormal discoloration of the leaves from green to purple was
observed but on very few plants. Rotting of the main roots and reddening of the
stele of new roots which is the most distinct symptoms of the disease was
observed. Plants inoculated with the highest zoospore concentration of 20 x 103
significantly had the shortest mean height of 139.58mm after 4 months, the lowest
fresh and oven dry weight of top and roots and the lowest marketable yield of
117.91g.
Inoculated plants have disease rating ranging from 3.67 to 5 which is
equivalent to 26 - 100% root infection.

Conclusions

Results of the investigation revealed that no symptom variations were
observed in strawberry cultivars infected in the field The high incidence of the
disease observed at swamp area is attributed to its flat terrain with poor drainage
and the cultural management of the farmers.
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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50
The causal organism was identified as Phytophthora fragariae based on
morphological and cultural characteristics as earlier reported. The pathogen
grows best on carrot broth agar.
Higher concentration of zoospores inoculated on strawberry plants
resulted to severe infection. The disease can cause a yield reduction of 38 to 57%
on strawberry plants and can be considered a threat to strawberry production in La
Trinidad and Tuba, Benguet.
Recommendations

Based on the above findings, the following are recommended:
1. Infected plants showing symptoms of stunting and wilting with reddening
of the root core/stele can be used as basis in the early detection of red stele
disease in the field.
2. Proper cultural management and strict quarantine measures should be
implemented to prevent the further spread of the disease and introduction
of new diseases.
3. Further studies should focus on the development of cost effective and
ecofriendly management strategies not only for Phytophthora fragariae
but for all soilborne diseases of strawberry.
4. Verification studies should be done to determine the possible involvement
of P. fragariae in a soil-borne disease complex currently observed in the
field.
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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51
LITERATURE CITED

AGRIOS, N. G. 2005. Plant Pathology, 5th ed. Elsevier Academic
Press.London. Pp. 414-420.

ALFORD, D.O and T. LOCKE. 1989. Pest and Diseases of fruits and hops. Pest
and Disease Control Handbook 2nd ed.. Crop Protection Council. Pp 389-
390.

ANDERSON, H.W. 1956. Diseases of strawberries. McGraw-Hill Book
Company, Inc. Pp. 411-420.

BENSON, H.J. 1998. Microscopic Measurements. Microbiological Application
7th Ed. McGraw-Hill. Pp22-24.

CABI (undated). Phytophthora fragariae. Data Sheets on Quarantine Pests.

CLARK, D.E. 1984. How to grow fruits, nuts and berries. California Lans
Publishing Company. P. 77.

CHILDERS, N.F. 1983. Modern fruit science. University of Florida, Gainsville.
P. 477.

CORONEL, R.E. 1983. Promising fruits of the Philippines. University of the
Philippines, Los Banos, College of Agriculture,. Pp. 431-447.

ELLIS, M.A. 1996. Red stele on strawberry. Ohio State University
Extension Fact sheet. Plant Pathology, Columbus, Ohio.
http//www.ag.ohio-state.edu~ohioline/hyg-fact/3000/3014.html.

ENCARTA ENCYCLOPEDIA. 2006. Strawberry. Bruce Coleman
Incorporation.

FERGUSON, B. 1982. All about growing fruits, nuts and berries. San
Francisco: Chevron Chemical Company. P. 103.

GEORGE. S.W. and R.D., MILHOLLAND. 1986. Inoculation and Evaluation of
Strawberry Plants with Phytophthora fragariae. Plant Disease 70.371-
375.


Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

52
HARRDEC. 1996. Benguet Strawberry Techno guide. La Trinidad, Benguet
HARRDEC-PARRFI. 46pp.

HERMANO, G.F. 1995. Strawberry Production Technology and Management in
the Highlands. A paper presented during the National Fruit Congress and
Exhibition. Cagayan de Oro. 8Pp.

HERMANO, G.F. 1999. Strawberry Production Management and Technoguide
Ist ed., La Trinidad, Benguet. 51pp.

HOLLIDAY, P. 1989. DICTIONARY OF Plant pathology. Kew. U.K. CAB
International Mycological Institute,. P. 236.

HORSFALL, J.G. 1978. Plant Disease: An Advance Treatise. Vol. 2. p.215.

KHANIZADEH. S. AND D. BUZARD. 1992. Red Stele Resistant Strawberries
and their Role in Epidemiology of Phytophthora fragariariae.
HortScience 27:870-871. www.octahort.org.

LAW, T.F. and R.D., MILHOLLAND. 1991. Production of sporangia and
oospores of Phytophthora fragariae in roots of strawberry plants. Plant
Disease 75:475-478.

LOS, L.M. and M.L., SCHROEDER. 1992. Red stele of strawberries.
dentification Sheet No. 2, University of Connecticut, New york.
http://www.hort.uconn.edu/ipm/homegrnd/htms/26strstl.htm

STAMPS. D.J, G.M. WATERHOUSE, F.J. NEWHOOK AND G.S. HALL.
1990. Revised Tabular Key to the Species of Phytophthora. CAB
International Mycological Institute. U.K. 28pp.

REIS, S.M. 1996. Strawberry Red Stele Root Rot. Report on Plant Disease No.
701. http://www.ipm.uiuc.edu/fruit/diseases/red-stele-root-rot/index.html

SCOPES, N. and L., STABLES. 1989. Pest and Disease Handbook 3rd Ed.
British Crop Protection Council, England. Pp. 389-390.

SWIFT, C.E and L.E., DICKENS. 1995. Strawberry diseases. Colorado State
University, fort Collins, Colorado.

SMITH, M.D. 1984. The Ortho Problem Solver. 2nd Ed. San Francisco,
California. Ortho Information Service. P. 766.

Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

53

WALLER, J.M, J.M. LENNE and S.J. WALLER. 2002. Plant Pathologist
Pocketbook. 3rd. ed., CABI Publishing, Wallington, UK. Pp. 27, 322.

WESCOTTE, C. 1978. Plant Disease Handbook. 2nd Ed. Canada. D.V.
Nostrand Comapany. P.336.

Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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54
APPENDICES


APPENDIX TABLE 1. Components of culture media used


a. PARPH

Components
Amount


Pimaricin





5 mg
Ampicilin
200
mg
Rifampicin





10 mg
PCNB
100
mg
Hymexazol





10 mg
cornmeal
agar
17
g
Distilled
water
1
liter


b. CARROT BROTH AGAR

Components
Amount


Carrot
200
g
Bacto-agar
15
g
Distilled
water
200
ml


Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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55
c. V8 JUICE AGAR

Components
Amount


V8
juice 200
ml
CaCO3





2 g
Agar





17 g
Distilled
water
800
ml


d. POTATO DEXTROSE AGAR

Components
Amount


Potato
250
g
Agar





20 g
Dextrose
20
g
Distilled
water
1
liter
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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56
APPENDIX TABLE 2. Incidence of red stele disease in the different strawberry growing areas of La Trinidad and Sto.
Tomas, Tuba, Benguet

Field
Media
1 2 3 4 5 6 7 8 9
TOTAL
10
MEAN
LA TRINIDAD












Swamp Area
Block I
6.11 36.15 14.54 13.54 79.72 12.81 9.11 14.94 23.80 52.29 263.01
26.30
Block II 23.28 34.09 14.87 17.22 36.70 44.09 14.73 6.34 16.63 10.04 217.99
21.80
Block III
9.21 54.46 40.08 73.24 40.36 21.28 65.66 68.88 30.64 33.30 437.11
43.51
Block IV 18.46 43.54 63 69.00 70.72 52.29 48.34 7.96 13.66 10.78 397.75
39.78
Balili, La
Trinidad1/
9.68 4.63 21.50 20.33 11.77 23.66 -
-
-
-
91.57
20.38
Long Long1/
21.94 26.11 21.03 10.15 -
-
-
-
-
-
79.23
19.81
TUBA












Sto. Tomas1/
6.50 10.00 7.15 12.00 -
-
-
-
-
-
35.65
8.91
GRAND TOTAL

1522.31
GRAND MEAN











18.05
1/ - Few farms were planted with strawberry






Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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57
APPENDIX TABLE 3. Mycelial growth of the pathogen 2 days after isolation

Replication
Media
1
Total
2 3 4 5
Mean
T1 - PDA
4.50
4.25
3.50
4.75
4.00
21.00
4.20b
T2 - CBA
8.50
9.50
7.25
6.50
6.00
37.75
7.55a
T3 - V8J-A
6.50
7.00
10.00
4.75
6.25
34.50
6.90a
T4- OMA
9.50
10.50
8.25
7.50
7.00
42.75
8.55a
GRAND
TOTAL

136.00

GRAND
MEAN

6.80




ANALYSIS OF VARIANCE


Source of
Degree of Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 3 51.98
17.33
8.57**
3.24
5.29
ERROR 16
32.35
2.02



TOTAL 19
84.33





**Highly significant




CV = 20.19%



Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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58

APPENDIX TABLE 4. Mycelial growth of the pathogen 3 days after isolation
Replication
Media
1 2
Total Mean
3 4 5

T1 – PDA
5.50
6.25
5.88
5.35
5.10
28.08
5.62d
T2 – CBA
17.60 15.75
13.25
12.75
12.50
71.85
14.37b
T3 - V8J-A
10.75 10.50
15.00
8.75
8.50
53.50
10.70c
T4-
OMA
20.25 22.50
23.75 20.00 17.75 104.25 20.85a
GRAND
TOTAL

257.68

GRAND
MEAN
12.88





ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 3 616.30
205.43
47.27**
3.24
5.29
ERROR 16
69.54
4.35



TOTAL 19
685.84





** Highly significant



CV = 16.18%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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59

APPENDIX TABLE 5. Mycelial growth of the pathogen 4 days after isolation


Replication
Media
1 2 3 4
Total
5

Mean
T1 – PDA
6.50
8.25
8.25
6.00
6.25
35.25
7.05d
T2 – CBA
22.75 22.00
19.25
19.25
20.25
103.50
20.70b
T3 - V8J-A
14.75 14.25
20.00
12.75
11.00
72.75
14.55c
T4-
OMA
25.25 27.50 28.75 25.00 22.75 129.25 25.85a
GRAND
TOTAL

340.75

GRAND
MEAN

17.04





ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 3 985.06 328.35 63.50** 3.24 5.29
ERROR 16
82.73 5.17



1,067.79

TOTAL 19



**Highly significant




CV = 13.35%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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60

APPENDIX TABLE 6 . Mycelial growth of the pathogen 5 days after isolation


Replication
Media
1
Total
2 3 4 5

Mean
T1 – PDA
8.00
8.50
9.35
8.75
6.75
41.35
8.27c
T2 – CBA
28.00
29.25
26.25
26.25
28.25
138.00
27.60a
T3 - V8J-A
19.75
20.00
26.50
17.75
17.25
101.25
20.25b
T4-
OMA
30.25 31.50 32.75 28.00 28.00 150.50 30.10a
GRAND
TOTAL

431.10

GRAND
MEAN

21.55




ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT
3
1,438.77 479.59
92.20**
3.24
5.29
ERROR
16
83.23 5.20



1,522.00
TOTAL 19





** Highly significant CV = 10.58%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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61

APPENDIX TABLE 7. Mycelial growth of the pathogen 6 days after isolation


Replication
Media
1 2 3 4
Total
5

Mean
T1 - PDA
9.50
8.75
10.50
11.50
7.25
47.50
9.50c
T2 - CBA
35.25
36.50
33.25
33.25
36.50
174.75
34.95a
T3 - V8J-A
25.50
25.75
33.00
27.75
23.50
135.50
27.10b
T4- OMA
35.25
35.75
37.00
32.25
34.25
174.50
34.90a
GRAND TOTAL





532.25

GRAND MEAN






26.61




ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 3
2,156.36
718.79
132.83**
3.24
5.29
ERROR 16
86.58
5.41



2,242.94
TOTAL 19





** Highly significant CV = 8.74%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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62

APPENDIX TABLE 8. Mycelial growth of the pathogen 7 days after isolation

Replication
Media
1 2 3 4
Total
5

Mean
T1 - PDA
11.00
9.25
11.75
12.50
8.25
52.75
10.55c
T2 - CBA
40.25
40.00
38.00
39.25
41.75
199.25
39.85a
T3 - V8J-A
31.00
31.25
40.00
32.50
29.00
163.75
32.75b
T4- OMA
40.50
40.75
41.25
40.00
39.75
202.25
40.45a
GRAND TOTAL





618.00

GRAND MEAN






30.90



ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT
3
2,944.25
981.42
168.09**
3.24
5.29
ERROR
16
93.42
5.84



3,037.67

TOTAL 19



** Highly significant CV = 7.82%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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63

APPENDIX TABLE 9. Initial plant height of strawberry plants

Inoculum Density
Replication


(zoospores/ml)
1 2 3
Total
4
Mean
T0 – Control (Uninoculated) 50.67
67.00
65.67
73.00
256.34
64.09a
T1 – 2 x 103
58.33
63.00
65.00
70.33
256.66
64.17a
T2 – 5 x 103
50.66
73.33
70.12
66.66
260.77
65.19a
T3 – 10 x 103
57.33
70.67
65.00
66.67
259.67
64.92a
T4 – 15 x 103
69.67
64.33
58.00
67.33
259.33
64.83a
T5 – 20 x103
50.33
77.00
62.00
69.33
258.66
64.67a
GRAND TOTAL




1551.43

GRAND MEAN





64.64



ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 3.81 0.76
0.01ns 2.77 4.25
ERROR
18
,205.23
66.96



23
,209.04
TOTAL




ns- not significant CV = 12.66%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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64

APPENDIX TABLE 10. Effect of inoculum levels of P. fragariae on plant height of
strawberry 1 month after planting

Inoculum Density
Replication

(zoospores/ml)
1 2 3
Total
4
Mean
T0 – Control (Uninoculated) 53.33
90.67
88.33
105.00 337.33 112.44a
T1 – 2 x 103
73.33
75.33
80.00
90.00
318.66 106.22a
T2 – 5 x 103
76.67
90.67
73.33
75.00
315.67 105.22a
T3 – 10 x 103
59.33
85.67
68.33
85.67
299.00
99.67a
T4 – 15 x 103
56.67
78.33
64.33
93.33
292.66
97.55a
T5 – 20 x 103
61.67
65.67
75.00
71.67
274.01
91.34a
GRAND TOTAL




1837.33

GRAND MEAN





102.07



ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 621.34
124.27
0.70ns 2.77 4.25
ERROR 18
3,207.92
78.22



23 3,829.26

TOTAL




ns-not significant CV = 17.44%


Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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65

APPENDIX TABLE 11. Effect of inoculum levels of P. fragariae on plant height of
strawberry 2 months after planting

Inoculum Density
Replication


(zoospores/ml)
Total
1 2 3 4
Mean
T0 – Control (uninoculated)
91.67
118.33
98.33
113.33 421.66 105.42a
T1 – 2 x 103
75.33
78.00
92.33
103.33 348.99 87.25ab
T2 – 5x 103
80.00
103.33
75.33
86.00
344.66 86.17ab
T3 – 10 x 103

60.00 93.33 69.67 95.00 318.00 79.50b
T4 – 15 x 103

63.33 80.33 68.33 97.67 309.66 77.42b
T5 – 20 x 103

68.33 75.33 93.67 76.33 313.66 78.42b
GRAND
TOTAL

2056.63

GRAND
MEAN

85.70



ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5
4
,654.26
930.85
3.03*
2.77
4.25
ERROR 18
5,536.07
07.56



23 0,190.33

TOTAL



*Significant CV = 16.01
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

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66

APPENDIX TABLE 12. Effect of inoculum levels of P. fragariae on plant height of
strawberry 3 months after planting

Inoculum Density
Replication


(zoospores/ml)
Total
1 2 3 4
Mean
T0 – Control (uninoculated)
123.33 136.67 125.00 130.00
515.00
128.75a
T1 – 2 x 103

93.33 96.67 106.67 110.00 406.67 101.67ab
T2 – 5 x 103

91.67 121.67 76.67 110.00 400.01 100.00ab
T3 – 10 x 103

67.33 131.67 79.67 106.67 385.34 96.34b
T4 – 15 x 103
79.99
81.00
90.67
114.00
365.66
91.42b
T5 – 20 x 103
70.00
80.00
108.33
78.67
337.00
84.25b
GRAND
TOTAL

2409.68

GRAND
MEAN

100.40




ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 9,254.79
1
850.96
2.36**
2.77
4.25
ERROR 18
14,091.34
782.85



23
3,346.13





** Highly significant CV = 18.06


Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

67


APPENDIX TABLE 13. Effect of inoculum levels of P. fragariae on plant height of
strawberry 4 months after planting

Replication
Inoculum Density


1 2 3 4
(zoospores/ml)
Total
Mean
T0 – Control (Uninoculated)
270.00 283.00 237.67 226.00 1016.67
254.17a
T1 – 2 x 103

164.00 130.00 180.00 173.00 647.00 161.75b
T2 – 5 x 103

156.67 191.00 143.00 170.00 660.67 165.17b
T3 – 10 x 103

140.00 165.00 160.67 171.00 636.67 159.17b
T4 – 15 x 103

157.00 165.00 157.67 151.67 631.34 157.84b
T5 – 20 x 103

89.67 159.33 173.33 136.00 558.33 139.58b
GRAND
TOTAL

4150.68

GRAND
MEAN

1037.68



ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 3,256.40
6,651.28
12.55**
2.77
4.25
ERROR 18
9,541.28
530.07



TOTAL 23
2,797.68





** Highly significant CV = 13.31%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

68

APPENDIX TABLE 14. Marketable yield of strawberry plants (February,2006)

Inoculum Density
Replication


(zoospores/ml)
1
Total
2 3 4
Mean
T0 – Control (Uninoculated)
26.92
51.48
48.53
47.63
174.56
43.64a
T1 – 2 x 103

37.53 27.52 26.67 37.56 129.28 32.32b
T2 – 5 x 103

28.87 31.61 20.69 26.52 107.69 26.92b
T3 – 10 x103

24.91 30.17 24.30 31.09 110.47 27.62b
T4 – 15 x 103

21.93 31.58 25.92 21.85 101.28 25.32b
T5 – 20 x 103

29.90 25.71 25.14 26.41 107.16 26.79b
GRAND
TOTAL

730.44

GRAND
MEAN

30.43



ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT
5
950.60 190.12
5.12** 2.77
4.25
ERROR
18
668.89
7.16


TOTAL
23
1,619.49




** Highly significant CV = 20.03%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

69

APPENDIX TABLE 15. Marketable yield of strawberry plants (March,2006)

Inoculum Density
Replication


(zoospores/ml)
1 2 3
Total
4
Mean
T0 – Control (Uninoculated) 70.70
84.55
97.58
63.45
316.28
79.07a
T1 – 2 x 103
55.66
55.21
55.59
60.05
226.51
56.63b
T2 – 5 x 103
76.00
44.12
44.18
56.46
220.76
55.19b
T3 – 10 x 103
64.82
48.01
48.79
57.75
219.37
54.84b
T4 – 15 x103
62.60
57.00
49.58
54.92
224.10
56.03b
T5 – 20 x 103
44.69
53.36
46.97
55.22
200.24
50.06b
GRAND TOTAL




1407.36

GRAND MEAN





58.64




ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 2,112.85
422.57
4.38**
2.77
4.25
ERROR 18
1,735.02
96.39


TOTAL
23
3,847.87




** Highly significant





CV = 16.74%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

70

APPENDIX TABLE 16. Marketable yield of strawberry plants (April,2006)

Replication
Inoculum Density


1 2 3 4
(zoospores/ml)
Total
Mean
T0 – Control (Uninoculated)
74.14
53.62
77.40
68.92
274.08
68.52a
T1 – 2 x 103
41.52
33.85
43.16
33.92
152.45 38.11bc
T2 – 5 x 103
44.46
41.67
45.16
31.09
162.38 40.60b
T3 – 10 x 103
29.97
37.10
32.96
41.25
141.28 35.32bc
T4 – 15 x 103

27.79 22.05 20.73 20.24 90.81 22.70d
T5 – 20 x 103

15.85 23.67 18.04 26.83 84.39 21.10d
GRAND TOTAL




905.39

GRAND
MEAN

37.72





ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 5,858.61
1,171.72
29.38**
2.77
4.25
ERROR 18
717.87
39.88



TOTAL
23
6,576.48





** Highly significant





CV = 16.74%


Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

71

APPENDIX TABLE 17. Marketable yield of strawberry plants (May,2006)

Inoculum Density
Replication


(zoospores/ml)
1 2 3
Total
4
Mean
T0 – Control (Uninoculated)
46.40
58.55
57.31
44.40
206.66
51.67a
T1 – 2 x 103
21.03
31.92
26.42
41.12
120.49 30.12b
T2 – 5 x 103
25.51
23.61
45.51
44.37
139.00 34.75b
T3 – 10 x 103
37.20
42.46
32.64
28.37
140.67 35.17b
T4 – 15 x 103
23.96
26.37
37.85
15.26
103.44 25.86b
T5 – 20 x 103

14.66 11.82 23.85 18.17 68.50 17.13c
GRAND
TOTAL



778.76
GRAND
MEAN

32.45




ANALYSIS OF VARAIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 5
2,662.37
532.47
7.68**
4.25
ERROR 18
18
1,248.56
69.36


TOTAL
23
23
,910.93



** Highly significant





CV = 25.67%



Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

72

APPENDIX TABLE 18. Marketable yield of strawberry plants (June,2006)

Inoculum Density
Replication


(zoospores/ml)
1 2 3
Total
4
Mean
T0 – Control (Uninoculated)
30.53
39.02
33.00
42.07
144.62
36.15a
T1 – 2 x 103
15.67
23.37
15.03
8.89
62.96
15.74b
T2 – 5 x 103
9.96
3.91
8.70
15.27
37.84
9.46bc
T3 – 10 x 103
3.96
9.99
7.64
10.67
32.26
8.07c
T4 – 15 x 103

5.77 1.33 14.69 4.27 26.06 6.52c
T5 – 20 x 103
4.25
3.40
2.47
1.24
11.36
2.84c
GRAND
TOTAL



315.10
GRAND MEAN





13.13



ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 2,902.91
580.58
26.96**
2.77
4.25
ERROR 18
387.66
21.54



TOTAL 23
3,290.57





** Highly significant





CV = 35.35%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

73

APPENDIX TABLE 19. Effect of inoculum levels of P. fragariae on the
marketable yield of strawberry

Inoculum Density
REPLICATION


(zoospores/ml)
Total
Mean
1 2 3 4
T0 – Control (Uninoculated) 248.69 287.22 313.82 266.47 1046.20 279.05a
T1 – 2 x 103

171.41 171.87 166.87 181.54 691.69 172.92b
T2 – 5 x 103

184.80 144.92 164.24 173.71 667.67 166.92b
T3 – 10 x 103

160.86 167.73 146.33 169.13 644.05 161.01b
T4 – 15 x 103

142.05 138.33 148.77 116.54 545.69 136.42c
T5 – 20 x 103

109.35 117.96 116.47 122.87 471.65 117.91d
GRAND
TOTAL

4066.95

GRAND
MEAN

172.37




ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5
63,189.97
12,637.99
51.57**
2.77
4.25
ERROR 18
4,411.21
245.07



TOTAL 23
67,601.18





** Highly significant





CV = 9.08%

Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

74

APPENDIX TABLE 20. Effect of inoculum levels of P. fragariae on the non-
marketable yield of strawberry

Inoculum Density
REPLICATION


(zoospores/ml)
1 2 3
Total
4
Mean
T0 – Control (Uninoculated)
53.55
61.72
54.60
52.30
222.17
55.54
T1 – 2 x 103
60.060 42.790 61.000 60.390 224.24
56.06
T2 – 5 x 103
74.63
42.96
76.23
76.23
270.05
67.51
T3 – 10 x 103
92.08
79.48
66.05
50.75
288.36
72.09
T4 – 15 x 103
83.95
70.38
65.42
74.72
294.47
73.62
T5 – 20 x 103
60.27 123.03
60.13
60.27
303.70
75.93
GRAND TOTAL




1602.99

GRAND MEAN





66.79




ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 1,601.26
320.25
1.11ns 2.77 4.25
ERROR 18
5,184.52
288.03



TOTAL 23
6,785.78





ns- not significant






CV = 25.41%


Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

75


APPENDIX TABLE 21. Effect of varying levels of P. fragariae inoculum on the
top fresh weight of strawberry plants

Inoculum Density
REPLICATION


(zoospores/ml)
1 2 3
Total
4
Mean
T0 – Control (Uninoculated)
45.55
61.24
42.80
64.54
214.13
53.53a
T1 – 2 x 103
24.59
27.12
12.41
16.66
80.78
20.20d
T2 – 5 x 103
22.39
13.77
31.79
16.30
84.25
21.06d
T3 – 10 x 103
20.25
20.50
23.33
20.00
84.08
21.02d
T4 – 15 x 103
13.90
18.34
16.17
16.30
64.71
16.18b
T5 – 20 x 103
17.08
16.42
16.58
17.25
67.33
16.83bc
GRAND TOTAL




595.28

GRAND MEAN





24.80




ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 4,051.39
810.28
20.54
2.77
4.25
ERROR 18
710.14
39.45


TOTAL 23
4,761.53




** Highly significant





CV = 25.32%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

76

APPENDIX TABLE 22. Effect of varying levels of P. fragariae inoculum on the
root fresh weight of strawberry plants

Inoculum Density
Replication


(zoospores/ml)
1 2 3
Total
4
Mean
T0 – Control (Uninoculated)
24.1
31.18
23.62
34.21
113.11
28.28a
T1 – 2 x 103
10.31
18.15
12.74
31.34
72.54
18.14ab
T2 – 5 x 103
11.85
12.12
32.59
9.58
66.14
16.54b
T3 – 10 x 103
9.81
11.21
14.16
8.88
44.06
11.02b
T4 – 15 x 103
9.86
11.06
9.63
11.56
42.11
10.53b
T5 – 20 x 103
5.21
6.3
4.93
7.74
24.18
6.05c
GRAND TOTAL




362.14

MEAN TOTAL





15.09




ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT
5
1,218.02 243.60
6.10** 2.77
4.25
ERROR
18
718.47 39.92


TOTAL
23
1,936.49




** Highly significant





CV = 41.87%



Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

77

APPENDIX TABLE 23. Effect of varying levels of P. fragariae inoculum on the
top dry weight of strawberry plant

Inoculum Density
Replication


(zoospores/ml)
1 2 3
Total
4
Mean
T0 – Control (Uninoculated)
15.17
18.78
15.20
15.13
64.28
16.07
T1 – 2 x 103
5.53
4.79
3.42
4.98
18.71
4.68ab
T2 – 5 x 103
4.09
3.41
4.75
4.19
16.44
4.11bc
T3 – 10 x 103
3.57
3.19
6.85
4.78
18.39
4.60bc
T4 – 15 x 103
2.48
3.89
3.17
2.92
12.46
3.11c
T5 – 20 x 103
2.97
2.90
1.10
2.79
9.76
2.44c
GRAND TOTAL




140.04

MEAN TOTAL





5.83




ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 5 518.08
103.62
75.48**
4.25
ERROR 18
18
24.71
1.37


TOTAL 23
23
542.80




** Highly significant




CV = 20.08%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

78

APPENDIX TABLE 24. Effect of varying levels of P. fragariae inoculum on the dry
weight of strawberry roots

Inoculum Density
Replication


(zoospores/ml)
1 2 3
Total
4
Mean
T0 – Control (Uninoculated)
7.88
5.61
6.23
7.27
26.99
6.75a
T1 – 2 x 103
6.31
4.94
2.26
6.90
20.41
5.10ab
T2 – 5 x 103
1.39
3.22
5.96
2.00
12.57
3.14bc
T3 – 10 x 103
2.00
2.27
5.57
2.56
12.41
3.10bc
T4 – 15 x 103

1.69 3.30 2.73 2.19 9.90 2.48c
T5 – 20 x 103

2.46 1.78 1.20 3.05 8.49 2.12a
GRAND TOTAL




90.77

GRAND
MEAN

22.69




ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5
2.77 4.25
63.47 12.69 5.73**
ERROR 18


39.91 2.22

TOTAL 23


103.38


** Highly significant





CV = 39.37%
Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

79

APPENDIX TABLE 25. Disease severity as affected by inoculum density

Inoculum Density
REPLICATION


(zoospores/ml)
1 2 3
Total
4
Mean
T0 – Control (uninoculated)
1.00
1.00
1.00
1.00
4.00
1.00d
T1 – 2 x 103
3.33
3.33
4.00
4.00
14.66
3.67c
T2 – 5 x 103
4.67
5.00
4.33
4.33
18.33
4.58ab
T3 – 10 x 103
5.00
4.67
4.33
4.67
18.67
4.67ab
T4 – 15 x 103
4.33
5.00
5.00
4.33
18.66
4.66ab
T5 – 20 x 103
5.00
5.00
5.00
5.00
20.00
5.00a
GRAND TOTAL




94.32

GRAND MEAN





3.93




ANALYSIS OF VARIANCE


Source of
Degree of
Sum of
Means of
Variance
Freedom
square
square
Computed
Tabular F




F
5%
1%
TREATMENT 5 45.24 9.0
113.75**
2.77
4.25
ERROR 18
1.43
0.08



TOTAL 23
46.67





** Highly significant




CV = 7.18%

Etiology of the Red Stele Disease of Strawberry
(Fragaria x ananassa Duch.) in Benguet / Rhonda M. Oloan. 2006

---

Document Outline

• Etiology of the Red Stele Disease of Strawberry (Fragaria x ananassa Duch.) in Benguet
  • BIBLIOGRAPHY
  • ABSTRACT
  • TABLE OF CONTENTS
  • INTRODUCTION
    • Importance of the study
    • Objectives of the Study
    • Time and Place of the study
  • REVIEW OF LITERATURE
    • Strawberry
    • Red Stele Disease
    • The Pathogen and Its Disease Cycle
    • Disease Symptoms
  • MATERIALS AND METHODS
    • Survey
    • Symptomatology
    • Assessment of Disease Incidence
    • Collection of Diseased Plants
    • Isolation of the Organism
    • Identification of the Organism
    • Preparation of inoculum
    • Standardization of the inoculum
    • Inoculation
    • Re-isolation of the Organism from Inoculated Plants.
    • Data Gathered
  • RESULTS AND DISCUSSION
    • Field study
      • Symptomatology
      • Disease Incidence
    • Laboratory Study
      • Mycelial growth of the pathogen on different culture media
      • Cultural Characteristics of the Organism
      • Morphological characteristics of the organism
    • Pathogenicity Test
      • Symptoms on inoculated plants
      • Plant Height (mm)
      • Yield
      • Fresh Weight of Plants
      • Oven dry weight of plants
      • Disease Severity at Harvest
  • SUMMARY, CONCLUSION AND RECOMMENDATION
    • Summary
    • Conclusions
    • Recommendations
  • LITERATURE CITED
  • APPENDICES

---