BIBLIOGRAPHY REYES, MILARINE O. APRIL 2008....
BIBLIOGRAPHY
REYES, MILARINE O. APRIL 2008. Characterization of Fusarium spp.
Associated with the Crown and Roots of Strawberry (Fragaria x ananassa Duch) in La
Trinidad, Benguet. Benguet State Universtiy, La Trinidad, Benguet.
Adviser: Asuncion L. Nagpala, Ph.D.
ABSTRACT
Diseased strawberry specimens were collected in the growing areas of Balili,
Pomology, and Swamp area of Benguet State University, La Trinidad, Benguet on the
month of November 2007. This was done to isolate and identify the Fusarium species
associated with the crown and roots of strawberries and to determine their cultural and
morphological characteristics following the criteria used in identifying Fusarium.
Based from the results, there are two (2) species of Fusarium associated with the
crown and roots of strawberries grown at the collection sites. These are Fusarium
oxysporum and Fusarium solani.
F. oxysporum grew fast and developed a colony of 4.1 cm in one week at 28 oC
and 30 oC. It produced white and pale violate pigment in potato dextrose agar (PDA). The
macroconidia is falcate in shape to “almost straight” with three (3) septations.
Microconidia are abundant and are oval, ellipsoidal and kidney-shaped and are formed in
false-heads from short phialides. Its chlamydospores were formed after three (3) weeks of
incubation in carnation leaf agar (CLA)
F. solani on the other hand grew slowly and developed a colony of only 2.9 cm at
28 oC and 2.5 cm at 30 oC after three (3) weeks. Pigment produced in PDA ranges from
cream to pale brown and white. Its macroconidia are “sausage shaped” with three (3)
septations, and it produced very few microconidia that are oval and ellipsoidal in shape
and are formed in false-heads with long phialides. Chlamydospores were formed after
two (2) weeks of incubation in PDA.
From the results, the isolated Fusarium spp. should be tested for pathogenicity as
basis of sound management formulation against crown and root rot disease of strawberry.
ii
TABLE OF CONTENTS
Page
Bibliography………………………………………………………................ i
Abstract ………… …………………………………………………….......... i
Table of Contents …………………………………………………………..
iii
INTRODUCTION ……………………………………………………….. 1
REVIEW OF LITERATURE …………………………………………….
4
MATERIALS AND METHODS………………………………………….
8
RESULTS AND DISCUSSION
Collection Sites of Diseased Strawberry
Specimens …………………………………………………………
13
Symptoms of the Collected Diseased
Specimens …………………………………………………………
13
Result of Isolation ………………………………………………...
16
Frequency of Occurrence of Fusarium
on carnation leaf agar (CLA) medium ……………………………
16
Morphological Characteristics of the
Fusarium Isolates …………………………………………………
17
Cultural Characteristics of the
Fusarium Isolates …………………………………………………
24
Comparison of Shape of Conidia of the
Fusarium Isolates Grown in CLA and PDA ……………………...
28
Identified Species of Fusarium ……………………………………
29
SUMMARY, CONCLUSION AND RECOMMENDATION …………...
31
LITERATURE CITED ……………………………………………………
33
iii
1
INTRODUCTION
Background of the Study
Strawberry is a sub-tropical plant grown as traditional crop in the highlands.
Although some provinces in the lowlands, like Bukidnon are now cultivating strawberry,
Benguet is still the main producer of this crop and because of this; the province is
popularly known as the “Strawberry Region” of the Philippines. Strawberries are
propagated naturally by means of runners that form on the early weeks of April. They are
being grown for their edible red berries which are either eaten as fresh or processed as
jams, tarts, cookies, and wines. They are planted in the latter months of the rainy season
or from the month of June to August and the production period starts from the month of
October until the months of April or May.
The strawberries being produced in Benguet mostly comes from the municipality
of La Trinidad. About 35 percent of the farming population of the municipality is
engaged in this industry. The average production per hectare in the November 2005 to
April 2006 production period was 18.5 metric tons, while it declined to 14 metric tons in
the 2006 to 2007 production period (Municipal Agriculturist Office, La Trinidad,
Benguet, 2007). The decline in the average yield of strawberry is usually due to the
improper growing practices of farmers, poor quality of planting materials and most of all,
the presence of pest and diseases which is a very serious problem.
Some of the diseases that attack strawberry are anthracnose, Botrytis rot, red stele,
and diseases caused by Fusarium sp. such as Fusarium wilt. Most of the pathogens that
caused these diseases are soil borne and are difficult to manage. In this study, the
pathogen Fusarium was given importance.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
2
Importance of the Study
Fusarium causes Fusarium wilt and was also found as one of the pathogen that
shares in the black root rot disease complex on strawberries together with Pythium spp.,
Rhizoctonia spp., and the lesion nematode Pratylenchus penetrans. Fusarium wilt
(Fusarium oxysporum f. sp. fragariae) is a very serious disease for cultivated
strawberries and recently, the damage from this disease has spread to open-culture
farming in cooler regions such as Akita Prefecture (40˚N, 139˚E, approximately 200
meters elevation) (Takashaki et al., 2006). On the other hand, Pecknold (2001) stated that
black root rot is the most common of all root diseases on strawberries in Indiana, United
States. Since temperature in Benguet is closely similar to weather conditions of these
countries, the presence of Fusarium in the strawberry growing areas of the province can
not be discounted. It is therefore imperative to conduct studies on the Fusarium spp.
attacking strawberry so that proper management of the disease will be formulated.
This will further help strawberry growers to improve their strawberry
production thereby increasing their yield.
Objectives of the Study
This study aimed to:
1. isolate and determine the Fusarium spp. associated with the crown and
roots of strawberries in La Trinidad, Benguet using carnation leaf agar
(CLA) and potato dextrose agar (PDA) medium.
2. characterize the Fusarium isolates based on their cultural and
morphological attributes.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
3
Time and Place of the Study
This study was conducted at the Plant Pathology Service Laboratory, College of
Agriculture of Benguet State University, La Trinidad, Benguet from October 2007 to
March 2008.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
4
REVIEW OF LITERATURE
The Crop
Strawberry is a common name for low, perennial herbs of the genus Fragaria of
the rose family, and also for the edible fruit of these herbs. Strawberries, which are native
to temperate regions throughout the world, were first cultivated in the United States about
1835 and have since became an important and widely distributed crop in farms and in
home gardens. The white flowers, which are borne in cymes, have a five-cleft calyx, five
rounded petals, many stamens, and numerous nut lets distributed on enlarge, pulpy,
scarlet receptacle (Redmond, 2007).
According to Hermano (1999), strawberry is a sub-tropical plant grown for about
a century as traditional crop in the high lands. Because of this crop, the Baguio-Benguet
area has been noted and popularly known as “Strawberry Region” of the country.
Strawberry production started in early years of the present century. This crop was
introduced and was found to be adaptable to the region. The introduction and evaluation
were done probably by the Americans who established an Agricultural School in 1916 at
La Trinidad, Benguet.
Strawberry is a semi-temperate and photoperiodic crop. Its sweet, juicy fruit is
one of the highly-priced commodities in Baguio City despite its abundance during the
months of December to April. The crop is grown in the Baguio-Benguet area (4,500 ft
asl) of the Northern Philippines where the temperature ranges from 14.7 to 23.3 oC with a
recorded day length of 11.06 hours and a maximum of 13.06 hours in June, the period
suited for strawberry production (Balaki, 1992).
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
5
At present, strawberry is the number 1 and the most popular fruit produced in
Baguio City and Benguet Province. It is a lucrative source of income for farmers and
revenue of the province of Benguet and the City of Baguio (HARRDEC, 1996).
The Pathogen
Fusarium is a filamentous fungus widely distributed on plants and in the soil. It is
found in normal mycoflora of commodities, such as rice, bean, soybean, and other crops.
While most species are more common at tropical and subtropical areas, some inhabit in
soil in cold climates (Anonymous, 2007).
The genus Fusarium currently contains over 20 species with Fusarium solani,
oxysporum, and chlamydosporum the most common. Some are plant pathogens causing
root and stem rot, vascular wilt or fruit rot. Other species cause storage rot and are
important mycotoxin producers. Several species, notably F. oxysporum, F. solani and F.
moniliforme, are recognized as being pathogenic to man and animals causing mycotic
keratitis, onychomycosis and hyalohyphomycosis, especially in burn victims and bone
marrow transplant patients (Ellis, 2006).
General Characteristics of Fusarium sp.
According to Ellis (2006), colonies of Fusarium are usually fast growing, pale or
brightly colored (depending on the species) and may or may not have a cottony aerial
mycelium. The color of the thallus varies from whitish to yellow, brownish, pink, reddish
or lilac shades. Species of Fusarium typically produce both macro- and microconidia
from slender phialides. Macroconidia are hyaline, two- to several-celled, fusiform- to
sickle-shaped, mostly with an elongated apical cell and pedicellate basal cell.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
6
Microconidia are 1- to 2-celled, hyaline, pyriform, fusiform to ovoid, straight or curved.
Chlamydoconidia may be present or absent.
Diseases Caused by Fusarim spp.
Fusarium causes vascular wilts primarily of annual vegetables and flowers,
herbaceous perennial ornamentals, plantation crops, weeds and of the mimosa tree (silk
tree). Most of the vascular wilt-causing Fusaria belong to the species Fusarium
oxysporum. Different host plants are attacked by special forms or races of the fungus
(Agrios, 1997).
On the other hand, Los and Schroeder (2007) stated that black root rot is caused
by a complex interaction of environmental factors, fungi, and nematodes such as
Pratylenchus penetrans. Several fungi are implicated in the disease including Rhizoctonia
spp., Pythium spp., and Fusarium spp.
Furthermore, Pscheidt (2007) stated that a research in Oregon in the 1930’s and
1940’s implicated Rhizoctonia sp., Fusarium spp., and Ramularia spp. with root rot of
strawberry. He also added that winter injury to roots encourages infection by Fusarium
spp.
Symptoms Caused by Fusarium oxysporum
and Fusarium solani
Agrios (2005) described the symptoms caused by Fusarium oxysporum and
Fusarium solani on the infected plants as follows:
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
7
Fusarium oxysporum
Fusarium oxysporum causes vascular wilt and rotting of crown and roots. The
leaves of infected plants or of parts of infected plants lose turgidity, become flaccid and
lighter green to greenish yellow, droop and finally wilt, turn yellow then brown and die.
Wilted leaves maybe flat or curled. Young, tender shoots also wilt and die. In cross
sections of infected stems and twigs, discolored, brown areas appear as a complete or
interrupted ring consisting of discolored vascular tissues. In the xylem vessels of infected
stems and roots, mycelium and spores of the causal fungus may be present. Some of the
vessels may clogged with mycelium, spores, or polysaccharides produced by the fungus.
Clogging is increased further by gels and gums formed by the accumulation and
oxidation of breakdown of products of plant cells attacked by fungal enzyme. The
oxidation and translocation of such breakdown of product seem to be responsible for the
brown discoloration of affected vascular tissue.
Fusarium solani
Fusarium solani caused rotting of seeds and seedlings (damping-off), rotting of
roots, lower stems, and crowns, and rots of corms, bulbs, and tubers. In root rots, tap
roots of young plants show a reddish discoloration that later becomes darker and larger.
The discoloration may cover the tap root and the stem below the soil line without a
definite margin or it may appear as streaks extending up to the soil line. Longitudinal
cracks appear along the main root, whereas small, lateral roots are killed. Plant growth is
retarded, and in dry weather, the leaves may turn yellow and even fall-off. Sometimes,
infected plants develop secondary roots and rootlets just below the soil line that maybe
sufficient to carry the plant to maturity and to production of a fairly good crop.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
8
MATERIALS AND METHODS
A. Field Activity
Collection of Diseased Samples
Diseased strawberry plants exhibiting symptoms suspected to be caused by
Fusarium sp. were collected from the strawberry growing areas of La Trinidad, Benguet
particularly at the Balili Experimental Station, BSU Pomology area and Swamp area in
the month of November, 2007. The collected samples were wrapped with newspapers and
were brought to the laboratory for isolation and for further examination.
B. Laboratory Activities
Description of Symptoms
Before isolation, the symptoms of the diseased strawberry specimens were
described based on how they appeared on the crown and roots of the collected samples.
Vascular discoloration was observed by cutting the crown to reveal the inside part and
sectioning the roots longitudinally. Above ground symptom was also noted on the leaves
of the specimens. The observed symptoms were documented.
Isolation
Crown and roots of the infected strawberries were washed thoroughly and were
cut into small sizes. The cut tissues were disinfected by soaking them in a 10 % chlorox
(NaCl) for 10 minutes, after which they were washed three (3) times with sterile distilled
water. The disinfected tissues were isolated equidistantly in the previously plated
carnation leaf agar (CLA). Ten (10) plates were prepared having five (5) tissues isolated
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
9
in each plate. This makes a total of 50 tissues isolated: 25 crown tissues and another 25
root tissues. After isolation, the plates were sealed with parafilm and incubated until
growth was observed.
Observation of the Fusarium Isolates
After 10 days of incubation, growth of the organism from the CLA plates was
observed. The plates were examined directly under the 10x magnification field of the
light microscope to confirm the growth of Fusarium sp.
To be able to characterize the organism, single sporing was done in CLA and
PDA plates. Single sporing was initiated to ensure the purity of the cultures and to avoid
possible mutation, thereby retaining the original wild type colony of the Fusarium.
Single Spore Initiation
Single sporing was done by pouring 15 ml of 2 % water agar into petri dishes and
allowing them to solidify. A suspension of spores was prepared in 5 ml sterile distilled
water blank. The spore suspension was poured over the solidified agar so as to cover the
entire surface, and then the excess was drained off. The seeded dishes were incubated in
an inclined position at room temperature for 18-20 hours. At the end of this time, the
dishes were opened and then shaken to remove the accumulated moisture, after which
they were examined under a dissecting microscope. Small squares of the agar containing
single spore were cut out with a dissecting needle and were transferred to PDA and CLA
media. The spore was inoculated at the center of the agar for the PDA plates, while near
the leaf pieces for the CLA plates.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
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Characterization of the Fusarium Isolates
Characterization was done by examining the cultures directly under the light
microscope. Slide preparations were made to ensure correct characterization. The book
“Laboratory Manual for Fusarium Research” by Burgess and Liddell (1983) was used as
reference guide for the identification. The important criteria used as basis for
characterization includes:
A. Morphological characteristics
a. Presence or absence of microconidia and chlamydospores.
b. Shape of the macroconidia as well as the microconidia if present.
c. Mode of formation of the microconidia. In this criterion, the microconidia were
examined on the CLA plates under the light microscope to determine whether
they are formed in chains or in false-heads.
d. Nature of the conidiogenous cell bearing the microconidia. This determined
whether the microconidia are formed from monophialidic or polyphialidic
conidiogenous cell.
B. Cultural characteristics
a. Colony diameters on PDA plates after dark incubation for 3 days at 28°C and
30°C.
b. Colony morphology on PDA plates. This criterion determined the growth of the
organism, color of mycelia, and the colony pigmentation produced by
Fusarium isolates.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
11
Measuring the Conidia
Conidial measurement was done by subjecting the conidia of each Fusarium sp.
in a calibrated microscope with the use of an ocular and stage micrometer. The length and
the width of the conidia were measured then multiplied with the calibration factor of the
microscope to get the exact dimension of the conidia. Forty (40) samples of conidia were
measured.
Calibration of the Microscope
Calibration of the microscope was done by inserting 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 scale using the Low Power
Objective (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 ocular divisions that cover the space between the zero and coincident lines were
counted. Calibration factor (CF) or calibration constant (CC) was calculated using this
formula:
CF = n divisions of stage micrometer x 10 units/division
n divisions of ocular micrometer
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
12
Data Gathered:
1. Symptoms manifested by the collected diseased strawberry specimens.
2. Species of the Fusarium that were isolated.
3. Cultural and morphological characteristics of the Fusarium isolates.
4. Frequency of occurrence of Fusarium per isolate that was determined by
examining the growth of the organism in each tissue on the CLA plates.
5. Comparison of conidia of the Fusarium isolates grown in CLA and PDA.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
13
RESULTS AND DISCUSSION
Collection Sites of Diseased
Strawberry Specimens
Diseased strawberry plants were collected at the Balili Experimental Station, BSU
Pomology area and Swamp area on the month of November, 2007. The collected
specimens were of Sweet Charlie variety
Symptoms of the Collected
Diseased Specimens
Table 1 shows the above ground symptom manifested by the collected diseased
strawberry plants which was browning of the outer leaves (Fig. 1). On the other hand,
below ground symptoms observed were vascular discoloration of the crown which ranges
from orange, brown and black discoloration (Fig. 2a & b). Rotting was observed in most
of the roots (Fig. 3a & b), and this conforms to the reports of Los and Schroeder (2007)
and Pscheidt (2007) that Fusarium sp. is one of the fungi that causes root rotting of
strawberries. Aside from rotting, brown discoloration was observed in some of the roots.
Table 1. Above and below ground symptoms manifested by the collected specimens
Plant Part Affected
Symptoms
Leaves
Browning of the outer leaves
Crown
Orange, brown and black vascular discoloration
Root
Rotting in most of the roots and brown discoloration in some
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
14
Figure 1. Browning on the leaves of the collected specimens
Figure 2a. Symptoms on the crown of the collected specimens
showing orange (pointed by arrow) and black
discoloration (encircled)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
15
Figure 2b. Brown discolorations on the crown of the collected
specimens
Figure 3a. Roots of the collected strawberry showing rotting in
most of the roots
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
16
Figure 3b. Infected roots of the collected strawberry showing
mycelia of Fusarium
Result of Isolation
Result of isolation revealed that there are two species of Fusarium associated with
the crown and roots of strawberries in La Trinidad, Benguet particularly at the collection
sites. They were temporarily assigned as Isolate 1 and Isolate 2 while characterization is
being done.
Frequency of Occurrence of Fusarium on CLA Plates
Table 2 shows that among the 50 tissues isolated; only 18 tissues or 36 % were
infected with Fusarium. Six of the 18 infected tissues or 33 % were crown tissues while
12 or 67 % were root tissues. Isolate 1 was more abundant in the crown tissues while
Isolate 2 in the root tissues. This result implies that Isolate 1 is more associated with the
crown tissues while Isolate 2 with the root tissues.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
17
Table 2. Frequency of occurrence of Fusarium from crown and root tissues isolated in
carnation leaf agar (CLA)
Plate Number
Tissue Number
Number Fusarium Isolates
of times
isolated
T1 T2 T3 T
4 T
5
Crown tissues
Plate 1
-
-
-
-
-
-
none
Plate 2
-
1
-
-
-
1
Isolate 1
Plate 3
1
1
1
-
-
3
Isolate 1
Plate 4
-
-
-
1
-
1
Isolate 2
Plate 5
1
-
-
-
-
1
Isolate 1
Total
6
Root tissues
Plate 6
1
1
1
1
1
5
Isolate 2
Plate 7
-
-
-
-
-
-
none
Plate 8
1
-
-
1
-
2
Isolate 1
Plate 9
1
1
1
1
1
5
Isolate 2
Plate 10
-
-
-
-
-
-
none
Total
12
Grand total
18
Legend: - = not infected with Fusarium
Morphological Characteristics of the Fusarium Isolates
The morphological characteristics of the Fusarium isolates from cultures
germinated from single spore (conidia) are summarized in Table 3. The different
structures of the Fusarium were observed directly on the plates under the 10x
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
18
magnification field of the light microscope. Slide preparations were also made using
methylene blue lactophenol as mounting medium.
Conidia. Isolate 1 has falcate to “almost straight”, three (3) septated,
macroconidia with hooked apex and notched base (Fig. 4a). It produced abundant, non-
septated microconidia that are oval, elliptical and reniform (kidney-shaped) in shape (Fig.
5). On the other hand, Isolate 2 has “sausage-shaped”, three (3) septated macroconidia
with round apex and notched base (Fig. 4b). It produces very few, oval to ellipsoidal
microconidia (Fig. 6) that are mostly two (2) celled. Macroconidia of both isolates are
mostly formed from phialides on the hyphae but some are formed from phialides on
branched conidiophores (Fig. 7 & 8). They also both form microconidia in false-heads,
but Isolate 1 produced them from short monophialidic conidiogenous cell (Fig. 9), while
Isolate 2 produced them from long monophialidic conidiogenous cell on the hyphae (Fig.
10).
Chlamydospores. Isolate 1 produced chlamydospores that are located terminally
and intercalary on the hyphae (Fig. 11), while Isolate 2 produced them terminally and
intercalary on a macroconidia (Fig. 12). Chlamydospores of Isolate 1 are produced on a
three (3) week old culture in CLA while Isolate 2 on a two (2) week old culture in PDA.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
19
Table 3. Morphological characteristics of the Fusarium isolates
CRITERIA
FUSARIUM ISOLATES
Isolate 1
Isolate 2
a. Microconidia
Present, produced
Present but very few
abundantly
b. Number of Septa
Macroconidia
3 septations
3 septations
Microconidia
Not-septated
Septated (1)
c. Mode of formation
False-heads
False-heads
of microconidia
d. Nature of
They are formed from short
They are formed from
conidiogenous cell
monophialidic
long monophialidic
bearing microconidia conidiogenous cell.
conidiogenous cell.
They are formed from
They are formed from
e. Formation of
phialides on the hyphae or
phialides on the hyphae
macroconidia
on branched conidiophores.
or on branched
conidiophores.
f. Chlamydospores
Present and are formed
Present and are formend
terminally and intercalary
terminally and intercalary
on the hyphae on a 2 week
on the macroconidia on a
old culture in CLA.
3 week old culture in
PDA.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
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Figure 4. Macroconidia of (a) Isolate 1 in CLA (in-situ) and (b) Isolate 2 (in slide
mount) 400x
Figure 5. Microconidia of Isolate 1 showing (a) oval-shaped (b) elliptical-shaped and
(c) kidney-shaped (400x)
Figure 6. Microconidia of Isolate 2: (a) elliptical shaped and
(b) kidney-shaped (400x)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
21
Figure 7. Macroconidia of Isolate 1 from phialides on hyphae and from
branched conidiophores (with pointer), in situ:CLA ( 400x)
Figure 8. Macroconidia of Isolate 2 from (a) phialides on hyphae and from
(b) phialides on branched conidiophores, in situ: PDA ( 400x)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
22
Figure 9. Microconidia of Isolate 1 in false-head
from short monophialides in situ: CLA
(400x)
Figure 10. Microconidia of Isolate 2 (a) in false-head and (b) from long monophialides
in situ: PDA (400x)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
23
Figure 11. Chlamydospores of Isolate 1 formed terminally (encircled)
and intercalary (with pointer) on hyphae in three week old
culture in CLA ( 400x)
Figure 12. Chlamydospores of Isolate 2 formed intercalary on
macroconidia in two week old culture in PDA (400x)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
24
Cultural Characteristics of the Fusarium Isolates
Cultural characteristics of the Fusarium Isolates are shown in Table 4.
Isolate 1 is fast-growing and produces abundant, floccose, white mycelium which
growth is compact. Pale violet macroconidia are produced in a central spore mass in PDA
plates (Fig. 13). Reverse of cultures appeared yellow with pale violet at the center (Fig.
15b). Black sclerotial bodies were formed after two weeks. Colony diameters after three
(3) days was 2.5 cm at 28 °C and 2.3 cm at 30 °C and increased to 4.1 cm at both
temperatures after one (1) week. The PDA plates were filled with the mycelia of Isolate 1
after two (2) weeks (Fig. 15).
On the other hand, Isolate 2 is slow-growing and produced slightly thick,
mycelium that is not cottony. Three (3) day old cultures are cream colored which turns
into pale brown after a week (Fig. 14). After two (2) weeks, the pale brown color was
surrounded with white color (Fig. 16a). Reverse of cultures appeared dark brown (Fig.
16b). Colony diameters measured 0.27 cm at 28 °C and 0.53 cm at 30 °C after three (3)
days, and expand to 0.5 cm at 28 °C and 0.97 cm at 30 °C after one (1) week. At two (2)
weeks, the colony enlarges to 2.9 cm at 28 °C and 2.5 at 30 °C in diameter. From the
results, Isolate 1 prefers a growth temperature of 28 °C than 30 °C, while Isolate 2 prefers
a higher temperature of 30 °C than 28 °C for colony growth.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
25
Table 4. Cultural characteristics of the Fusarium isolates
CRITERIA
FUSARIUM ISOLATES
Isolate 1
Isolate 2
a. Average colony
diameter
Three days
2.5 cm at 28 °C
0.27 cm at 28 °C
2.3 cm at 30 °C
0.53 cm at 30 °C
One week
4.1 cm at 28 °C and 30 °C
0.5 cm at 28 °C
0.97 cm at 30 °C
Two weeks
Plate is full at 28 °C and 30 2.9 cm at 28 °C
°C
2.5 cm at 30 °C
b. Pigmentation
White and pale violet.
The isolate produced cream
Reverse of cultures appear
color after three days which
as yellow with pale violet at
turns into pale brown after a
the center. Black sclerotial
week. After two weeks the pale
bodies were produced at the
brown color becomes
center of the cultures after
surrounded with white and its
two weeks.
reverse appear as dark brown.
c. Colony growth
Fast-growing and produced
Slow-growing and produced
abundant, floccose and
slightly thick mycelium that is
compact mycelium.
not cottony.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
26
Figure 13. One week old culture of Isolate 1 in PDA (a) front view and (b) reverse
view
Figure 14. One week old culture of Isolate 2 in PDA (a) front view (b) reverse view
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
27
Figure 15. Two week old culture of Isolate 1in PDA (a) front view (b) reverse view
Figure 16. Two week old culture of Isolate 2 in PDA (a) front view (b) reverse view
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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28
Comparison of Conidia of the Fusarium
Isolates Grown in CLA and PDA
Table 5 shows the difference between the shape of the conidia of the Fusarium
isolates grown in CLA and PDA. Macroconidia and microconidia of Isolate 1 and Isolate
2 in CLA are mostly uniform in shape (Fig. 17a & 18a) while in PDA they are mostly
irregular in shape and some were even distorted (Fig. 17b & 18b).
Table 5. Comparison of shape of conidia of the Fusarium isolates grown in CLA and
PDA
Isolate 1
Isolate 2
CRITERIA
CLA PDA CLA
PDA
a. Macroconidia
Mostly
Mostly
Mostly
Mostly
uniform
irregular
uniform
irregular
b. Microconidia
Mostly
Mostly
Mostly
Mostly
uniform
irregular
uniform
irregular
Figure 17. Macro and microconidia of Isolate 1 grown in (a) CLA and in (b) PDA (400x)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
29
Figure 18. Macro and microconidia of Isolate 2 grown in (a) CLA and in (b) PDA (400x)
Identified Species of Fusarium
On the basis of the criteria outlined by Burgess and Liddell in the reference titled
“Laboratory Manual for Fusarium Research” (1983), the morphological and cultural
characteristics of Isolate 1 matches the characteristics described for Fusarium oxysporum
while Isolate 2 is close to the reported characteristics of Fusarium solani in the same
book.
The description of Fusarium oxysporum in the reference that was used conforms
to the cultural and morphological characteristics of Isolate 1 in this study. These include
falcate to “almost straight”, three (3) septated macroconidia that are tapered or pointed at
each end, have hooked apex and notched base; and are formed from phialides on
branched condidiophores and from phialides on hyphae; presence of abundant, one (1)
celled microconidia that are formed in false-heads on short phialides, and are oval,
elliptical or reniform (kidney-shaped) in shape; chlamydospores that are formed in a three
(3) week old culture; mycelium that are floccose and abundant, ranging in color from
white and pale violet; and the production of black sclerotial bodies. Colony diameter of
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
30
Fusarium oxysporum after three (3) days is from 2.5 to 4.0 cm at both 25 °C and 30 °C
which is not far from the colony diameter of Isolate 1 as shown in Table 4.
On the other hand, description of Fusarium solani matches with the cultural and
morphological characteristics of Isolate 2 which include three (3) septated macroconidia
that are “sausage-shaped”, with a round apex and notched base and are formed from
phialides on branched conidiophores and from phialides on hyphae; two (2) celled
microconidia that are oval to elliptical in shape and are formed in false-heads on long
phialides; chlamydospores that are formed in a two (2) week old culture and are formed
from the macroconidia; and last is the production of cream to pale brown mycelium. The
colony diameter of Isolate 2 does not conform to that of Fusarium solani which is 2.1 to
2.9 cm at 25 °C and 2.6 to 3.6 at 30 °C. This could be attributed to the temperature of 28
°C where the isolate was incubated instead of 25 °C. This needs further investigation.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
31
SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary
The study was conducted to isolate and determine the Fusarium species
associated with the crown and roots of strawberries in Benguet particularly at the Balili
Experimental Station, BSU Pomology area and Swamp area; and to characterize them
based on their cultural and morphological attributes.
Results revealed that that there are two (2) species of Fusarium associated with
the crown and roots of strawberries in the collection sites. The symptoms they caused on
the collected specimens were browning of the outer leaves, rotting in most of the roots
and brown discoloration in some, while orange, brown and black discoloration were
observed on the crown.
The identified species of Fusarium were Fusarium oxysporum and Fusarium
solani. However, Fusarium solani does not conform to the colony growth description of
Fusarium solani in the reference book used in this study, thus further investigation is
needed.
Fusarium oxysporum is fast-growing (4.1 cm colony diameter in 1 week at 28 °C
and 30 °C) and produced white and pale violet pigment in PDA. The macroconidia has 3
septations and falcate to “almost straight” in shape. It produced abundant microconidia
that are oval, ellipsoidal and kidney-shaped and were formed in false-heads from short
phialides. Chlamydospores were formed after three (3) weeks of incubation in CLA.
Fusarium solani on the other hand is slow-growing (2.9 cm at 28 °C and 2.05 at
30 °C colony diameter after three weeks) and produced cream to pale brown and white
pigment in PDA. Its macroconidia has 3 septations , and “sausage-shaped”. It produced
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
32
very few microconidia that are oval and ellipsoidal in shape and were formed in false-
heads from long phialides. Chlamydospores were formed after two (2) weeks of
incubation in PDA.
Fusarium oxysporum is more associated with the crown of the collected
strawberry specimens while Fusarium solani is more associated with the roots. Both
species are present in the collection sites.
Conclusion
On the basis of the criteria outlined by Burgess and Liddell in their book
“Laboratory Manual for Fusarium Research” (1983) Isolate 1 matches the morphological
and cultural characteristics of Fusarium oxysporum, while Isolate 2 conforms to some of
the morphological and cultural characteristics of Fusarium solani.
Fusarium oxysporum and Fusarium solani are the species of Fusarium found in
the strawberry growing areas of La Trinidad, Benguet particulary at Balili Experimental
Station, BSU Pomology area and Swamp area.
Recommendations
1. The identified Fusarium species will have to be verified thru molecular analysis
especially the Fusarium solani to make sure that the identification is correct.
2. The Fusarium isolates must be tested for pathogenicity to verify if they are
pathogenic and to confirm the symptoms they caused on strawberries.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
33
LITERATURE CITED
AGRIOS, G. N. 1997. Plant Pathology. 4th Edition. University of Florida, Academic
Press Inc., San Diego, California. Pp. 342. 523-525 & 538-540
ANONYMOUS. 2007. Fusarium species. Retrieved 12 September from
http://search.yahoo.com/searchei=utf-8&fr=slv8-&p=fUSARIUM
BALAKI, E. T. 1992. Crop Protection and Cultural Management Studies on Strawberry.
Benguet State University, La Trinidad, Benguet. P. 1.
BURGESS, L. W. and C. M. LIDDEL. 1983. Laboratory Manual for Fusarium Research.
Department of Plant Pathology and Agricultural Entomology, The University of
Sydney, Australia. Pp. 3-4, 15, 53-54 & 60-61.
ELLIS, D. 2006. Mycology Online, Fusarium sp. The University of Adelaide, Australia.
Retrieved from
http://www.mycology.adelaide.edu.au/Fungal_Descriptions//Fusarium/
ELLIS, M. A. 1994. Black Root Rot of Strawberry. Ohio State University Extension Fact
sheet. 2021 Coffey Road, Columbus, Ohio. Retrieved from
http://ohioline.osu.edu/b861/pdf/ch02_13-18.pdf.
ESIONG, M. S. 2004. Collection, Identification and Characterization of Colletotrichum
spp. Infecting Strawberry (Fragaria x ananasa duch.) in Benguet. BS Thesis.
Benguet State University, La Trinidad, Benguet. P. 4.
HARRDEC. 1996. Benguet Strawberry Technoguide. Highland Agriculture and
Resource Research and Development Consortium. Benguet State University, La
Trinidad, Benguet. Pp. 1 – 2 & 17 – 18
HERMANO, F. G. 1999. Strawberry Production Management and Technology. Benguet
State University, La Trinidad, Benguet. ISBN. Pp. 1, 33 & 37
LOS, L. M. and M. L. SCHROEDER. 2007. Black Root Rot of Strawberry. University of
Connecticut. Integrated Pest Management. Retrieved 25 September from
http://www.hort.uconn.edu/ipm/homegrnd/htms/25strrot.htm
MUNICIPAL AGRICULTURIST OFFICE. 2007. La Trinidad, Benguet.
PECKNOLD, P. C. 2001. Strawberry Root Diseases. Purdue University Cooperative
Extension ServiceWest Lafayette, IN 47907. Retrieved from
http://www.ces.purdue.edu/extmedia/BP/BP-46.html
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
34
PSCHEIDT, J. W. Ed. 2007. Strawberry Black Root Rot Complex. OSU Extension
Office. Retrieved from http://plant-disease.ippc.orst.edu/disease.
REDMOND, W. A. 2007. “Strawberry”, Microsoft Encarta [DVD]. Microsoft
Corporation, 2006.
RIEDEL, M. and R. MILLER. 1996. Fusarium wilt of Vine Crops. Coffey Road,
Columbus: Ohio State University Extension Factsheet.
TAKAHASHI H., Y. YOSHIDA, and H. KANDA, 2007. WB-B33, A New Fusarium
Wilt-Resistant Strawberry Line. ISHS Acta Horticulturae 760: IHC2006 - II
International Symposium on Plant Genetic Resources of Horticultural Crops.
Retrieved 25 September from http://www.actahort.org/books/760/760_57.htm
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
REYES, MILARINE O. APRIL 2008. Characterization of Fusarium spp.
Associated with the Crown and Roots of Strawberry (Fragaria x ananassa Duch) in La
Trinidad, Benguet. Benguet State Universtiy, La Trinidad, Benguet.
Adviser: Asuncion L. Nagpala, Ph.D.
ABSTRACT
Diseased strawberry specimens were collected in the growing areas of Balili,
Pomology, and Swamp area of Benguet State University, La Trinidad, Benguet on the
month of November 2007. This was done to isolate and identify the Fusarium species
associated with the crown and roots of strawberries and to determine their cultural and
morphological characteristics following the criteria used in identifying Fusarium.
Based from the results, there are two (2) species of Fusarium associated with the
crown and roots of strawberries grown at the collection sites. These are Fusarium
oxysporum and Fusarium solani.
F. oxysporum grew fast and developed a colony of 4.1 cm in one week at 28 oC
and 30 oC. It produced white and pale violate pigment in potato dextrose agar (PDA). The
macroconidia is falcate in shape to “almost straight” with three (3) septations.
Microconidia are abundant and are oval, ellipsoidal and kidney-shaped and are formed in
false-heads from short phialides. Its chlamydospores were formed after three (3) weeks of
incubation in carnation leaf agar (CLA)
F. solani on the other hand grew slowly and developed a colony of only 2.9 cm at
28 oC and 2.5 cm at 30 oC after three (3) weeks. Pigment produced in PDA ranges from
cream to pale brown and white. Its macroconidia are “sausage shaped” with three (3)
septations, and it produced very few microconidia that are oval and ellipsoidal in shape
and are formed in false-heads with long phialides. Chlamydospores were formed after
two (2) weeks of incubation in PDA.
From the results, the isolated Fusarium spp. should be tested for pathogenicity as
basis of sound management formulation against crown and root rot disease of strawberry.
ii
TABLE OF CONTENTS
Page
Bibliography………………………………………………………................ i
Abstract ………… …………………………………………………….......... i
Table of Contents …………………………………………………………..
iii
INTRODUCTION ……………………………………………………….. 1
REVIEW OF LITERATURE …………………………………………….
4
MATERIALS AND METHODS………………………………………….
8
RESULTS AND DISCUSSION
Collection Sites of Diseased Strawberry
Specimens …………………………………………………………
13
Symptoms of the Collected Diseased
Specimens …………………………………………………………
13
Result of Isolation ………………………………………………...
16
Frequency of Occurrence of Fusarium
on carnation leaf agar (CLA) medium ……………………………
16
Morphological Characteristics of the
Fusarium Isolates …………………………………………………
17
Cultural Characteristics of the
Fusarium Isolates …………………………………………………
24
Comparison of Shape of Conidia of the
Fusarium Isolates Grown in CLA and PDA ……………………...
28
Identified Species of Fusarium ……………………………………
29
SUMMARY, CONCLUSION AND RECOMMENDATION …………...
31
LITERATURE CITED ……………………………………………………
33
iii
1
INTRODUCTION
Background of the Study
Strawberry is a sub-tropical plant grown as traditional crop in the highlands.
Although some provinces in the lowlands, like Bukidnon are now cultivating strawberry,
Benguet is still the main producer of this crop and because of this; the province is
popularly known as the “Strawberry Region” of the Philippines. Strawberries are
propagated naturally by means of runners that form on the early weeks of April. They are
being grown for their edible red berries which are either eaten as fresh or processed as
jams, tarts, cookies, and wines. They are planted in the latter months of the rainy season
or from the month of June to August and the production period starts from the month of
October until the months of April or May.
The strawberries being produced in Benguet mostly comes from the municipality
of La Trinidad. About 35 percent of the farming population of the municipality is
engaged in this industry. The average production per hectare in the November 2005 to
April 2006 production period was 18.5 metric tons, while it declined to 14 metric tons in
the 2006 to 2007 production period (Municipal Agriculturist Office, La Trinidad,
Benguet, 2007). The decline in the average yield of strawberry is usually due to the
improper growing practices of farmers, poor quality of planting materials and most of all,
the presence of pest and diseases which is a very serious problem.
Some of the diseases that attack strawberry are anthracnose, Botrytis rot, red stele,
and diseases caused by Fusarium sp. such as Fusarium wilt. Most of the pathogens that
caused these diseases are soil borne and are difficult to manage. In this study, the
pathogen Fusarium was given importance.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
2
Importance of the Study
Fusarium causes Fusarium wilt and was also found as one of the pathogen that
shares in the black root rot disease complex on strawberries together with Pythium spp.,
Rhizoctonia spp., and the lesion nematode Pratylenchus penetrans. Fusarium wilt
(Fusarium oxysporum f. sp. fragariae) is a very serious disease for cultivated
strawberries and recently, the damage from this disease has spread to open-culture
farming in cooler regions such as Akita Prefecture (40˚N, 139˚E, approximately 200
meters elevation) (Takashaki et al., 2006). On the other hand, Pecknold (2001) stated that
black root rot is the most common of all root diseases on strawberries in Indiana, United
States. Since temperature in Benguet is closely similar to weather conditions of these
countries, the presence of Fusarium in the strawberry growing areas of the province can
not be discounted. It is therefore imperative to conduct studies on the Fusarium spp.
attacking strawberry so that proper management of the disease will be formulated.
This will further help strawberry growers to improve their strawberry
production thereby increasing their yield.
Objectives of the Study
This study aimed to:
1. isolate and determine the Fusarium spp. associated with the crown and
roots of strawberries in La Trinidad, Benguet using carnation leaf agar
(CLA) and potato dextrose agar (PDA) medium.
2. characterize the Fusarium isolates based on their cultural and
morphological attributes.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
3
Time and Place of the Study
This study was conducted at the Plant Pathology Service Laboratory, College of
Agriculture of Benguet State University, La Trinidad, Benguet from October 2007 to
March 2008.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
4
REVIEW OF LITERATURE
The Crop
Strawberry is a common name for low, perennial herbs of the genus Fragaria of
the rose family, and also for the edible fruit of these herbs. Strawberries, which are native
to temperate regions throughout the world, were first cultivated in the United States about
1835 and have since became an important and widely distributed crop in farms and in
home gardens. The white flowers, which are borne in cymes, have a five-cleft calyx, five
rounded petals, many stamens, and numerous nut lets distributed on enlarge, pulpy,
scarlet receptacle (Redmond, 2007).
According to Hermano (1999), strawberry is a sub-tropical plant grown for about
a century as traditional crop in the high lands. Because of this crop, the Baguio-Benguet
area has been noted and popularly known as “Strawberry Region” of the country.
Strawberry production started in early years of the present century. This crop was
introduced and was found to be adaptable to the region. The introduction and evaluation
were done probably by the Americans who established an Agricultural School in 1916 at
La Trinidad, Benguet.
Strawberry is a semi-temperate and photoperiodic crop. Its sweet, juicy fruit is
one of the highly-priced commodities in Baguio City despite its abundance during the
months of December to April. The crop is grown in the Baguio-Benguet area (4,500 ft
asl) of the Northern Philippines where the temperature ranges from 14.7 to 23.3 oC with a
recorded day length of 11.06 hours and a maximum of 13.06 hours in June, the period
suited for strawberry production (Balaki, 1992).
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
5
At present, strawberry is the number 1 and the most popular fruit produced in
Baguio City and Benguet Province. It is a lucrative source of income for farmers and
revenue of the province of Benguet and the City of Baguio (HARRDEC, 1996).
The Pathogen
Fusarium is a filamentous fungus widely distributed on plants and in the soil. It is
found in normal mycoflora of commodities, such as rice, bean, soybean, and other crops.
While most species are more common at tropical and subtropical areas, some inhabit in
soil in cold climates (Anonymous, 2007).
The genus Fusarium currently contains over 20 species with Fusarium solani,
oxysporum, and chlamydosporum the most common. Some are plant pathogens causing
root and stem rot, vascular wilt or fruit rot. Other species cause storage rot and are
important mycotoxin producers. Several species, notably F. oxysporum, F. solani and F.
moniliforme, are recognized as being pathogenic to man and animals causing mycotic
keratitis, onychomycosis and hyalohyphomycosis, especially in burn victims and bone
marrow transplant patients (Ellis, 2006).
General Characteristics of Fusarium sp.
According to Ellis (2006), colonies of Fusarium are usually fast growing, pale or
brightly colored (depending on the species) and may or may not have a cottony aerial
mycelium. The color of the thallus varies from whitish to yellow, brownish, pink, reddish
or lilac shades. Species of Fusarium typically produce both macro- and microconidia
from slender phialides. Macroconidia are hyaline, two- to several-celled, fusiform- to
sickle-shaped, mostly with an elongated apical cell and pedicellate basal cell.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
6
Microconidia are 1- to 2-celled, hyaline, pyriform, fusiform to ovoid, straight or curved.
Chlamydoconidia may be present or absent.
Diseases Caused by Fusarim spp.
Fusarium causes vascular wilts primarily of annual vegetables and flowers,
herbaceous perennial ornamentals, plantation crops, weeds and of the mimosa tree (silk
tree). Most of the vascular wilt-causing Fusaria belong to the species Fusarium
oxysporum. Different host plants are attacked by special forms or races of the fungus
(Agrios, 1997).
On the other hand, Los and Schroeder (2007) stated that black root rot is caused
by a complex interaction of environmental factors, fungi, and nematodes such as
Pratylenchus penetrans. Several fungi are implicated in the disease including Rhizoctonia
spp., Pythium spp., and Fusarium spp.
Furthermore, Pscheidt (2007) stated that a research in Oregon in the 1930’s and
1940’s implicated Rhizoctonia sp., Fusarium spp., and Ramularia spp. with root rot of
strawberry. He also added that winter injury to roots encourages infection by Fusarium
spp.
Symptoms Caused by Fusarium oxysporum
and Fusarium solani
Agrios (2005) described the symptoms caused by Fusarium oxysporum and
Fusarium solani on the infected plants as follows:
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
7
Fusarium oxysporum
Fusarium oxysporum causes vascular wilt and rotting of crown and roots. The
leaves of infected plants or of parts of infected plants lose turgidity, become flaccid and
lighter green to greenish yellow, droop and finally wilt, turn yellow then brown and die.
Wilted leaves maybe flat or curled. Young, tender shoots also wilt and die. In cross
sections of infected stems and twigs, discolored, brown areas appear as a complete or
interrupted ring consisting of discolored vascular tissues. In the xylem vessels of infected
stems and roots, mycelium and spores of the causal fungus may be present. Some of the
vessels may clogged with mycelium, spores, or polysaccharides produced by the fungus.
Clogging is increased further by gels and gums formed by the accumulation and
oxidation of breakdown of products of plant cells attacked by fungal enzyme. The
oxidation and translocation of such breakdown of product seem to be responsible for the
brown discoloration of affected vascular tissue.
Fusarium solani
Fusarium solani caused rotting of seeds and seedlings (damping-off), rotting of
roots, lower stems, and crowns, and rots of corms, bulbs, and tubers. In root rots, tap
roots of young plants show a reddish discoloration that later becomes darker and larger.
The discoloration may cover the tap root and the stem below the soil line without a
definite margin or it may appear as streaks extending up to the soil line. Longitudinal
cracks appear along the main root, whereas small, lateral roots are killed. Plant growth is
retarded, and in dry weather, the leaves may turn yellow and even fall-off. Sometimes,
infected plants develop secondary roots and rootlets just below the soil line that maybe
sufficient to carry the plant to maturity and to production of a fairly good crop.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
8
MATERIALS AND METHODS
A. Field Activity
Collection of Diseased Samples
Diseased strawberry plants exhibiting symptoms suspected to be caused by
Fusarium sp. were collected from the strawberry growing areas of La Trinidad, Benguet
particularly at the Balili Experimental Station, BSU Pomology area and Swamp area in
the month of November, 2007. The collected samples were wrapped with newspapers and
were brought to the laboratory for isolation and for further examination.
B. Laboratory Activities
Description of Symptoms
Before isolation, the symptoms of the diseased strawberry specimens were
described based on how they appeared on the crown and roots of the collected samples.
Vascular discoloration was observed by cutting the crown to reveal the inside part and
sectioning the roots longitudinally. Above ground symptom was also noted on the leaves
of the specimens. The observed symptoms were documented.
Isolation
Crown and roots of the infected strawberries were washed thoroughly and were
cut into small sizes. The cut tissues were disinfected by soaking them in a 10 % chlorox
(NaCl) for 10 minutes, after which they were washed three (3) times with sterile distilled
water. The disinfected tissues were isolated equidistantly in the previously plated
carnation leaf agar (CLA). Ten (10) plates were prepared having five (5) tissues isolated
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
9
in each plate. This makes a total of 50 tissues isolated: 25 crown tissues and another 25
root tissues. After isolation, the plates were sealed with parafilm and incubated until
growth was observed.
Observation of the Fusarium Isolates
After 10 days of incubation, growth of the organism from the CLA plates was
observed. The plates were examined directly under the 10x magnification field of the
light microscope to confirm the growth of Fusarium sp.
To be able to characterize the organism, single sporing was done in CLA and
PDA plates. Single sporing was initiated to ensure the purity of the cultures and to avoid
possible mutation, thereby retaining the original wild type colony of the Fusarium.
Single Spore Initiation
Single sporing was done by pouring 15 ml of 2 % water agar into petri dishes and
allowing them to solidify. A suspension of spores was prepared in 5 ml sterile distilled
water blank. The spore suspension was poured over the solidified agar so as to cover the
entire surface, and then the excess was drained off. The seeded dishes were incubated in
an inclined position at room temperature for 18-20 hours. At the end of this time, the
dishes were opened and then shaken to remove the accumulated moisture, after which
they were examined under a dissecting microscope. Small squares of the agar containing
single spore were cut out with a dissecting needle and were transferred to PDA and CLA
media. The spore was inoculated at the center of the agar for the PDA plates, while near
the leaf pieces for the CLA plates.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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10
Characterization of the Fusarium Isolates
Characterization was done by examining the cultures directly under the light
microscope. Slide preparations were made to ensure correct characterization. The book
“Laboratory Manual for Fusarium Research” by Burgess and Liddell (1983) was used as
reference guide for the identification. The important criteria used as basis for
characterization includes:
A. Morphological characteristics
a. Presence or absence of microconidia and chlamydospores.
b. Shape of the macroconidia as well as the microconidia if present.
c. Mode of formation of the microconidia. In this criterion, the microconidia were
examined on the CLA plates under the light microscope to determine whether
they are formed in chains or in false-heads.
d. Nature of the conidiogenous cell bearing the microconidia. This determined
whether the microconidia are formed from monophialidic or polyphialidic
conidiogenous cell.
B. Cultural characteristics
a. Colony diameters on PDA plates after dark incubation for 3 days at 28°C and
30°C.
b. Colony morphology on PDA plates. This criterion determined the growth of the
organism, color of mycelia, and the colony pigmentation produced by
Fusarium isolates.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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Measuring the Conidia
Conidial measurement was done by subjecting the conidia of each Fusarium sp.
in a calibrated microscope with the use of an ocular and stage micrometer. The length and
the width of the conidia were measured then multiplied with the calibration factor of the
microscope to get the exact dimension of the conidia. Forty (40) samples of conidia were
measured.
Calibration of the Microscope
Calibration of the microscope was done by inserting 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 scale using the Low Power
Objective (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 ocular divisions that cover the space between the zero and coincident lines were
counted. Calibration factor (CF) or calibration constant (CC) was calculated using this
formula:
CF = n divisions of stage micrometer x 10 units/division
n divisions of ocular micrometer
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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Data Gathered:
1. Symptoms manifested by the collected diseased strawberry specimens.
2. Species of the Fusarium that were isolated.
3. Cultural and morphological characteristics of the Fusarium isolates.
4. Frequency of occurrence of Fusarium per isolate that was determined by
examining the growth of the organism in each tissue on the CLA plates.
5. Comparison of conidia of the Fusarium isolates grown in CLA and PDA.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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RESULTS AND DISCUSSION
Collection Sites of Diseased
Strawberry Specimens
Diseased strawberry plants were collected at the Balili Experimental Station, BSU
Pomology area and Swamp area on the month of November, 2007. The collected
specimens were of Sweet Charlie variety
Symptoms of the Collected
Diseased Specimens
Table 1 shows the above ground symptom manifested by the collected diseased
strawberry plants which was browning of the outer leaves (Fig. 1). On the other hand,
below ground symptoms observed were vascular discoloration of the crown which ranges
from orange, brown and black discoloration (Fig. 2a & b). Rotting was observed in most
of the roots (Fig. 3a & b), and this conforms to the reports of Los and Schroeder (2007)
and Pscheidt (2007) that Fusarium sp. is one of the fungi that causes root rotting of
strawberries. Aside from rotting, brown discoloration was observed in some of the roots.
Table 1. Above and below ground symptoms manifested by the collected specimens
Plant Part Affected
Symptoms
Leaves
Browning of the outer leaves
Crown
Orange, brown and black vascular discoloration
Root
Rotting in most of the roots and brown discoloration in some
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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Figure 1. Browning on the leaves of the collected specimens
Figure 2a. Symptoms on the crown of the collected specimens
showing orange (pointed by arrow) and black
discoloration (encircled)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
15
Figure 2b. Brown discolorations on the crown of the collected
specimens
Figure 3a. Roots of the collected strawberry showing rotting in
most of the roots
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
16
Figure 3b. Infected roots of the collected strawberry showing
mycelia of Fusarium
Result of Isolation
Result of isolation revealed that there are two species of Fusarium associated with
the crown and roots of strawberries in La Trinidad, Benguet particularly at the collection
sites. They were temporarily assigned as Isolate 1 and Isolate 2 while characterization is
being done.
Frequency of Occurrence of Fusarium on CLA Plates
Table 2 shows that among the 50 tissues isolated; only 18 tissues or 36 % were
infected with Fusarium. Six of the 18 infected tissues or 33 % were crown tissues while
12 or 67 % were root tissues. Isolate 1 was more abundant in the crown tissues while
Isolate 2 in the root tissues. This result implies that Isolate 1 is more associated with the
crown tissues while Isolate 2 with the root tissues.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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Table 2. Frequency of occurrence of Fusarium from crown and root tissues isolated in
carnation leaf agar (CLA)
Plate Number
Tissue Number
Number Fusarium Isolates
of times
isolated
T1 T2 T3 T
4 T
5
Crown tissues
Plate 1
-
-
-
-
-
-
none
Plate 2
-
1
-
-
-
1
Isolate 1
Plate 3
1
1
1
-
-
3
Isolate 1
Plate 4
-
-
-
1
-
1
Isolate 2
Plate 5
1
-
-
-
-
1
Isolate 1
Total
6
Root tissues
Plate 6
1
1
1
1
1
5
Isolate 2
Plate 7
-
-
-
-
-
-
none
Plate 8
1
-
-
1
-
2
Isolate 1
Plate 9
1
1
1
1
1
5
Isolate 2
Plate 10
-
-
-
-
-
-
none
Total
12
Grand total
18
Legend: - = not infected with Fusarium
Morphological Characteristics of the Fusarium Isolates
The morphological characteristics of the Fusarium isolates from cultures
germinated from single spore (conidia) are summarized in Table 3. The different
structures of the Fusarium were observed directly on the plates under the 10x
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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18
magnification field of the light microscope. Slide preparations were also made using
methylene blue lactophenol as mounting medium.
Conidia. Isolate 1 has falcate to “almost straight”, three (3) septated,
macroconidia with hooked apex and notched base (Fig. 4a). It produced abundant, non-
septated microconidia that are oval, elliptical and reniform (kidney-shaped) in shape (Fig.
5). On the other hand, Isolate 2 has “sausage-shaped”, three (3) septated macroconidia
with round apex and notched base (Fig. 4b). It produces very few, oval to ellipsoidal
microconidia (Fig. 6) that are mostly two (2) celled. Macroconidia of both isolates are
mostly formed from phialides on the hyphae but some are formed from phialides on
branched conidiophores (Fig. 7 & 8). They also both form microconidia in false-heads,
but Isolate 1 produced them from short monophialidic conidiogenous cell (Fig. 9), while
Isolate 2 produced them from long monophialidic conidiogenous cell on the hyphae (Fig.
10).
Chlamydospores. Isolate 1 produced chlamydospores that are located terminally
and intercalary on the hyphae (Fig. 11), while Isolate 2 produced them terminally and
intercalary on a macroconidia (Fig. 12). Chlamydospores of Isolate 1 are produced on a
three (3) week old culture in CLA while Isolate 2 on a two (2) week old culture in PDA.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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Table 3. Morphological characteristics of the Fusarium isolates
CRITERIA
FUSARIUM ISOLATES
Isolate 1
Isolate 2
a. Microconidia
Present, produced
Present but very few
abundantly
b. Number of Septa
Macroconidia
3 septations
3 septations
Microconidia
Not-septated
Septated (1)
c. Mode of formation
False-heads
False-heads
of microconidia
d. Nature of
They are formed from short
They are formed from
conidiogenous cell
monophialidic
long monophialidic
bearing microconidia conidiogenous cell.
conidiogenous cell.
They are formed from
They are formed from
e. Formation of
phialides on the hyphae or
phialides on the hyphae
macroconidia
on branched conidiophores.
or on branched
conidiophores.
f. Chlamydospores
Present and are formed
Present and are formend
terminally and intercalary
terminally and intercalary
on the hyphae on a 2 week
on the macroconidia on a
old culture in CLA.
3 week old culture in
PDA.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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Figure 4. Macroconidia of (a) Isolate 1 in CLA (in-situ) and (b) Isolate 2 (in slide
mount) 400x
Figure 5. Microconidia of Isolate 1 showing (a) oval-shaped (b) elliptical-shaped and
(c) kidney-shaped (400x)
Figure 6. Microconidia of Isolate 2: (a) elliptical shaped and
(b) kidney-shaped (400x)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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Figure 7. Macroconidia of Isolate 1 from phialides on hyphae and from
branched conidiophores (with pointer), in situ:CLA ( 400x)
Figure 8. Macroconidia of Isolate 2 from (a) phialides on hyphae and from
(b) phialides on branched conidiophores, in situ: PDA ( 400x)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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Figure 9. Microconidia of Isolate 1 in false-head
from short monophialides in situ: CLA
(400x)
Figure 10. Microconidia of Isolate 2 (a) in false-head and (b) from long monophialides
in situ: PDA (400x)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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Figure 11. Chlamydospores of Isolate 1 formed terminally (encircled)
and intercalary (with pointer) on hyphae in three week old
culture in CLA ( 400x)
Figure 12. Chlamydospores of Isolate 2 formed intercalary on
macroconidia in two week old culture in PDA (400x)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
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Cultural Characteristics of the Fusarium Isolates
Cultural characteristics of the Fusarium Isolates are shown in Table 4.
Isolate 1 is fast-growing and produces abundant, floccose, white mycelium which
growth is compact. Pale violet macroconidia are produced in a central spore mass in PDA
plates (Fig. 13). Reverse of cultures appeared yellow with pale violet at the center (Fig.
15b). Black sclerotial bodies were formed after two weeks. Colony diameters after three
(3) days was 2.5 cm at 28 °C and 2.3 cm at 30 °C and increased to 4.1 cm at both
temperatures after one (1) week. The PDA plates were filled with the mycelia of Isolate 1
after two (2) weeks (Fig. 15).
On the other hand, Isolate 2 is slow-growing and produced slightly thick,
mycelium that is not cottony. Three (3) day old cultures are cream colored which turns
into pale brown after a week (Fig. 14). After two (2) weeks, the pale brown color was
surrounded with white color (Fig. 16a). Reverse of cultures appeared dark brown (Fig.
16b). Colony diameters measured 0.27 cm at 28 °C and 0.53 cm at 30 °C after three (3)
days, and expand to 0.5 cm at 28 °C and 0.97 cm at 30 °C after one (1) week. At two (2)
weeks, the colony enlarges to 2.9 cm at 28 °C and 2.5 at 30 °C in diameter. From the
results, Isolate 1 prefers a growth temperature of 28 °C than 30 °C, while Isolate 2 prefers
a higher temperature of 30 °C than 28 °C for colony growth.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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25
Table 4. Cultural characteristics of the Fusarium isolates
CRITERIA
FUSARIUM ISOLATES
Isolate 1
Isolate 2
a. Average colony
diameter
Three days
2.5 cm at 28 °C
0.27 cm at 28 °C
2.3 cm at 30 °C
0.53 cm at 30 °C
One week
4.1 cm at 28 °C and 30 °C
0.5 cm at 28 °C
0.97 cm at 30 °C
Two weeks
Plate is full at 28 °C and 30 2.9 cm at 28 °C
°C
2.5 cm at 30 °C
b. Pigmentation
White and pale violet.
The isolate produced cream
Reverse of cultures appear
color after three days which
as yellow with pale violet at
turns into pale brown after a
the center. Black sclerotial
week. After two weeks the pale
bodies were produced at the
brown color becomes
center of the cultures after
surrounded with white and its
two weeks.
reverse appear as dark brown.
c. Colony growth
Fast-growing and produced
Slow-growing and produced
abundant, floccose and
slightly thick mycelium that is
compact mycelium.
not cottony.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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26
Figure 13. One week old culture of Isolate 1 in PDA (a) front view and (b) reverse
view
Figure 14. One week old culture of Isolate 2 in PDA (a) front view (b) reverse view
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
27
Figure 15. Two week old culture of Isolate 1in PDA (a) front view (b) reverse view
Figure 16. Two week old culture of Isolate 2 in PDA (a) front view (b) reverse view
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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28
Comparison of Conidia of the Fusarium
Isolates Grown in CLA and PDA
Table 5 shows the difference between the shape of the conidia of the Fusarium
isolates grown in CLA and PDA. Macroconidia and microconidia of Isolate 1 and Isolate
2 in CLA are mostly uniform in shape (Fig. 17a & 18a) while in PDA they are mostly
irregular in shape and some were even distorted (Fig. 17b & 18b).
Table 5. Comparison of shape of conidia of the Fusarium isolates grown in CLA and
PDA
Isolate 1
Isolate 2
CRITERIA
CLA PDA CLA
PDA
a. Macroconidia
Mostly
Mostly
Mostly
Mostly
uniform
irregular
uniform
irregular
b. Microconidia
Mostly
Mostly
Mostly
Mostly
uniform
irregular
uniform
irregular
Figure 17. Macro and microconidia of Isolate 1 grown in (a) CLA and in (b) PDA (400x)
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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29
Figure 18. Macro and microconidia of Isolate 2 grown in (a) CLA and in (b) PDA (400x)
Identified Species of Fusarium
On the basis of the criteria outlined by Burgess and Liddell in the reference titled
“Laboratory Manual for Fusarium Research” (1983), the morphological and cultural
characteristics of Isolate 1 matches the characteristics described for Fusarium oxysporum
while Isolate 2 is close to the reported characteristics of Fusarium solani in the same
book.
The description of Fusarium oxysporum in the reference that was used conforms
to the cultural and morphological characteristics of Isolate 1 in this study. These include
falcate to “almost straight”, three (3) septated macroconidia that are tapered or pointed at
each end, have hooked apex and notched base; and are formed from phialides on
branched condidiophores and from phialides on hyphae; presence of abundant, one (1)
celled microconidia that are formed in false-heads on short phialides, and are oval,
elliptical or reniform (kidney-shaped) in shape; chlamydospores that are formed in a three
(3) week old culture; mycelium that are floccose and abundant, ranging in color from
white and pale violet; and the production of black sclerotial bodies. Colony diameter of
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
30
Fusarium oxysporum after three (3) days is from 2.5 to 4.0 cm at both 25 °C and 30 °C
which is not far from the colony diameter of Isolate 1 as shown in Table 4.
On the other hand, description of Fusarium solani matches with the cultural and
morphological characteristics of Isolate 2 which include three (3) septated macroconidia
that are “sausage-shaped”, with a round apex and notched base and are formed from
phialides on branched conidiophores and from phialides on hyphae; two (2) celled
microconidia that are oval to elliptical in shape and are formed in false-heads on long
phialides; chlamydospores that are formed in a two (2) week old culture and are formed
from the macroconidia; and last is the production of cream to pale brown mycelium. The
colony diameter of Isolate 2 does not conform to that of Fusarium solani which is 2.1 to
2.9 cm at 25 °C and 2.6 to 3.6 at 30 °C. This could be attributed to the temperature of 28
°C where the isolate was incubated instead of 25 °C. This needs further investigation.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
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31
SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary
The study was conducted to isolate and determine the Fusarium species
associated with the crown and roots of strawberries in Benguet particularly at the Balili
Experimental Station, BSU Pomology area and Swamp area; and to characterize them
based on their cultural and morphological attributes.
Results revealed that that there are two (2) species of Fusarium associated with
the crown and roots of strawberries in the collection sites. The symptoms they caused on
the collected specimens were browning of the outer leaves, rotting in most of the roots
and brown discoloration in some, while orange, brown and black discoloration were
observed on the crown.
The identified species of Fusarium were Fusarium oxysporum and Fusarium
solani. However, Fusarium solani does not conform to the colony growth description of
Fusarium solani in the reference book used in this study, thus further investigation is
needed.
Fusarium oxysporum is fast-growing (4.1 cm colony diameter in 1 week at 28 °C
and 30 °C) and produced white and pale violet pigment in PDA. The macroconidia has 3
septations and falcate to “almost straight” in shape. It produced abundant microconidia
that are oval, ellipsoidal and kidney-shaped and were formed in false-heads from short
phialides. Chlamydospores were formed after three (3) weeks of incubation in CLA.
Fusarium solani on the other hand is slow-growing (2.9 cm at 28 °C and 2.05 at
30 °C colony diameter after three weeks) and produced cream to pale brown and white
pigment in PDA. Its macroconidia has 3 septations , and “sausage-shaped”. It produced
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
32
very few microconidia that are oval and ellipsoidal in shape and were formed in false-
heads from long phialides. Chlamydospores were formed after two (2) weeks of
incubation in PDA.
Fusarium oxysporum is more associated with the crown of the collected
strawberry specimens while Fusarium solani is more associated with the roots. Both
species are present in the collection sites.
Conclusion
On the basis of the criteria outlined by Burgess and Liddell in their book
“Laboratory Manual for Fusarium Research” (1983) Isolate 1 matches the morphological
and cultural characteristics of Fusarium oxysporum, while Isolate 2 conforms to some of
the morphological and cultural characteristics of Fusarium solani.
Fusarium oxysporum and Fusarium solani are the species of Fusarium found in
the strawberry growing areas of La Trinidad, Benguet particulary at Balili Experimental
Station, BSU Pomology area and Swamp area.
Recommendations
1. The identified Fusarium species will have to be verified thru molecular analysis
especially the Fusarium solani to make sure that the identification is correct.
2. The Fusarium isolates must be tested for pathogenicity to verify if they are
pathogenic and to confirm the symptoms they caused on strawberries.
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
33
LITERATURE CITED
AGRIOS, G. N. 1997. Plant Pathology. 4th Edition. University of Florida, Academic
Press Inc., San Diego, California. Pp. 342. 523-525 & 538-540
ANONYMOUS. 2007. Fusarium species. Retrieved 12 September from
http://search.yahoo.com/searchei=utf-8&fr=slv8-&p=fUSARIUM
BALAKI, E. T. 1992. Crop Protection and Cultural Management Studies on Strawberry.
Benguet State University, La Trinidad, Benguet. P. 1.
BURGESS, L. W. and C. M. LIDDEL. 1983. Laboratory Manual for Fusarium Research.
Department of Plant Pathology and Agricultural Entomology, The University of
Sydney, Australia. Pp. 3-4, 15, 53-54 & 60-61.
ELLIS, D. 2006. Mycology Online, Fusarium sp. The University of Adelaide, Australia.
Retrieved from
http://www.mycology.adelaide.edu.au/Fungal_Descriptions//Fusarium/
ELLIS, M. A. 1994. Black Root Rot of Strawberry. Ohio State University Extension Fact
sheet. 2021 Coffey Road, Columbus, Ohio. Retrieved from
http://ohioline.osu.edu/b861/pdf/ch02_13-18.pdf.
ESIONG, M. S. 2004. Collection, Identification and Characterization of Colletotrichum
spp. Infecting Strawberry (Fragaria x ananasa duch.) in Benguet. BS Thesis.
Benguet State University, La Trinidad, Benguet. P. 4.
HARRDEC. 1996. Benguet Strawberry Technoguide. Highland Agriculture and
Resource Research and Development Consortium. Benguet State University, La
Trinidad, Benguet. Pp. 1 – 2 & 17 – 18
HERMANO, F. G. 1999. Strawberry Production Management and Technology. Benguet
State University, La Trinidad, Benguet. ISBN. Pp. 1, 33 & 37
LOS, L. M. and M. L. SCHROEDER. 2007. Black Root Rot of Strawberry. University of
Connecticut. Integrated Pest Management. Retrieved 25 September from
http://www.hort.uconn.edu/ipm/homegrnd/htms/25strrot.htm
MUNICIPAL AGRICULTURIST OFFICE. 2007. La Trinidad, Benguet.
PECKNOLD, P. C. 2001. Strawberry Root Diseases. Purdue University Cooperative
Extension ServiceWest Lafayette, IN 47907. Retrieved from
http://www.ces.purdue.edu/extmedia/BP/BP-46.html
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PSCHEIDT, J. W. Ed. 2007. Strawberry Black Root Rot Complex. OSU Extension
Office. Retrieved from http://plant-disease.ippc.orst.edu/disease.
REDMOND, W. A. 2007. “Strawberry”, Microsoft Encarta [DVD]. Microsoft
Corporation, 2006.
RIEDEL, M. and R. MILLER. 1996. Fusarium wilt of Vine Crops. Coffey Road,
Columbus: Ohio State University Extension Factsheet.
TAKAHASHI H., Y. YOSHIDA, and H. KANDA, 2007. WB-B33, A New Fusarium
Wilt-Resistant Strawberry Line. ISHS Acta Horticulturae 760: IHC2006 - II
International Symposium on Plant Genetic Resources of Horticultural Crops.
Retrieved 25 September from http://www.actahort.org/books/760/760_57.htm
Characterization of Fusarium spp. Associated with the Crown and Roots of Strawberry
(Fragaria x ananassa Duch) in La Trinidad, Benguet / Milarine O. Reyes. 2008
Document Outline
- Characterization of Fusarium spp.
Associated with the Crown and Roots of Strawberry (Fragaria x ananassa Duch) in La
Trinidad, Benguet
- BIBLIOGRAPHY
- ABSTRACT
- TABLE OF CONTENTS
- INTRODUCTION
- Background of the Study
- Importance of the Study
- Objectives of the Study
- Time and Place of the Study
- REVIEW OF LITERATURE
- The Crop
- The Pathogen
- General Characteristics of Fusarium sp.
- Diseases Caused by Fusarim spp.
- Symptoms Caused by Fusarium oxysporumand Fusarium solani
- MATERIALS AND METHODS
- RESULTS AND DISCUSSION
- Collection Sites of DiseasedStrawberry Specimens
- Symptoms of the CollectedDiseased Specimens
- Result of Isolation
- Frequency of Occurrence of Fusarium on CLA Plates
- Morphological Characteristics of the Fusarium Isolates
- Cultural Characteristics of the Fusarium Isolates
- Comparison of Conidia of the FusariumIsolates Grown in CLA and PDA
- Identified Species of Fusarium
- SUMMARY, CONCLUSION AND RECOMMENDATIONS
- Summary
- Conclusion
- Recommendations
- LITERATURE CITED