ABSTRACT
BIBLIOGRAPHY
CUADRA, ILENE C. APRIL 2007. Etiology of the Bacterial Leaf Spot of
Lettuce in La Trinidad, Benguet: A First Report. Benguet State University, La Trinidad,
Benguet.
Adviser: Jocelyn C. Perez, MSc
Co-Adviser: Lily Ann D. Lando, PhD
ABSTRACT
The bacterium causing leaf spot in lettuce leaves was isolated and produced
similar symptoms to the artificially inoculated lettuce leaves.
Cultural characteristic of the isolates are yellow colonies produced in seven (7)
different media. The morphological characterization was done through gram staining
which is negative and motility test which is motile because of the polar flagella of the
bacterium. In the physiological characterization, growth in O/F test was never
fermentative and oxidase negative. Thus, the bacterium was not able to produce
gelatinase enzymes in the gelatin liquefaction test. A clear zone was observed in the
starch hydrolysis test swabbed with Xanthomonas campestris pv. vitians.
Sensitivity of the bacterium to streptomycin sulfate and chlortetracycline was very
obvious. Cephalexin hydrate and cycloheximide were not effective against the bacterium.
TABLE OF CONTENTS
Page
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Abstract
. . . . . . . . . . . . . . . … .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
Table of Content
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii
INTRODUCTION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
REVIEW OF LITERATURE
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
MATERIALS AND METHODS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
RESULTS AND DISCUSSION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Collection of Sample and
Description of the Collection Sites . . . . . . . . . . . . . . . . . . . . . . . . . 13
Disease Symptoms
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Cultural
Characteristics
of
The Pathogen on Various Media
. . . . . . . . . . . . . . . . . . . . . . . . .
14
.
Morphological Characteristics of
The Pathogen
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16
Gram Stain Reaction
. . . . . . . . . . . . . . . . . . . . . . . . .
16
Motility Test
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Physiological Characteristics of
The Pathogen
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Growth and O/F Metabolism
. . . . . . . . . . . . . . . . . . . 17
Gelatin Liquefaction Test
. . . . . . . . . . . . . . . . . . . . . . . . . 18
Starch Hydrolysis Test
. . . . . . . . . . . . . . . . . . . . . . . . . 19
Pathogenicity Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Re-Isolation of the Bacterium
from
Artificially
Inoculated
ii
Lettuce Leaves
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21
Antibiotic Sensitivity of
the Pathogen
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
SUMMARY, CONCLUSION
AND RECOMMENDATIONS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
LITERATURE CITED
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
27
APPENDICES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . ………... . . . . . .
30
iii
INTRODUCTION
In 2005, Nagpala et al. (PIA, 2006) reported a leaf spot of head lettuce (Lactuca
sativa L.) collected from Loo and Poblacion in Buguias, Benguet. The lettuce leaves had
black lesions that often become papery thin and then eventually tears the leaves to give it
a tattered appearance (Villa et al., 2005).
The initial survey results of Nagpala et al. (2005) showed a 50-90% disease
incidence in the sites sampled. Laboratory tests showed the association of a bacterium
with the diseased plant tissues. Identification done by the team and continued by Villa et
al. (2005) pointed to the possibility of the causal bacterium being a Xanthomonas
campestris pathovar.
Bacterial spot of lettuce has not been reported in the Philippines as revealed in a
review of the Host Index of Plant Diseases in the Philippines (Tangonan, 1999).
Therefore, this disease may be new or unrecognized and thus unreported.
Studies on the transmission of the organism have been inconclusive but several
reports say that it may be both seed- and soilborne. As such, the pathogen presents a new
challenge to the vegetable industry in Benguet. To adequately meet this challenge, the
identity of the pathogen has to be verified and its pathogenicity determined. The
information may then be used to prevent the spread of the pathogen and develop an
integrated approach to the management of the disease.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
2
This study was conceived and conducted to:
1. verify the identity of the causal bacterium of the disease;
2. determine the sensitivity of the bacterial isolates to antibiotics (streptomycin
sulfate, cycloheximide, cephalexin hydrate, and chlortetracycline).
3. determine the pathogenicity of the isolates to commonly-grown lettuce
cultivars; and
4. compare the isolates to common Xanthomonas species in the locality.
The study was conducted in the Department of Plant Pathology laboratory
from November 2006 to March 2007.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
REVIEW OF LITERATURE
The Host Plant
Lettuce (Lactuca sativa L.) including cos or romaine, belongs to Asteraceae
(compositae) or daisy family. This is the largest dicotyledonous family in the plant
kingdom. Notable leafy crop species within the Asteraceae family includes lettuce,
chichory and endive as well as other lesser known plants such as fuki and oyster plant
(Ryder, 1999).
Lettuce, a cool-season vegetable crop is one of the easiest to grow. High summer
temperature usually cause seedstalk formation (bolting) and bitter flavor. Slow-bolting or
heat resistant varieties are available and are recommended for extending the lettuce-
growing season (Robinson, 2003).
The plant is an annual herb with milky latex in the leaves and stems. Lettuce has a
shallow root system up to 1 foot deep (30 cm). The most important characteristics of
lettuce are its size, compactness, sweetness and succulence.
Lettuce is popular in salads because of its nutritional content. One cup of raw leaf
lettuce (chopped) contains only nine calories. It contains 1.3 g dietary fiber, 1 g protein,
1.34 g carbohydrates, 1456 IU of vitamin A, 13.44 I.U. vitamin C, 20.16 mg calcium,
0.62 mg iron and 162.5 mg potassium (Wolford, 2006).
There are several types of lettuce commonly grown in garden: the crisphead,
butterhead, cos, leaf, stem and oil-seed.
Crisphead also known as iceberg (approximately 60-75 days when direct seeded).
The most widely available as a fresh market type, it weighs about 1 kg with six or seven
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
4
outer leaves. Outer leaves are bright green or dull green, and the interior color may be
white to creamy yellow.
Butterhead or Bibb lettuce (approximately 60 days when direct seeded). It is a
loose-heading type with dark green leaves that are somewhat thicker than those of
iceburg lettuce.
Romaine or cos (approximately 60 days). They are elongated leaves that range in
color from yellow to dark green forming upright heads with rather wavy, attractive
leaves. They are very nutritious and weigh up to 750g each (Stephen, 1988).
Leaf type lettuce (approximately 50- 60 days). Have green or reddish leaves, this
type is fast growing, long-lasting lettuce. It is basically planted, thinned and harvested.
Stem lettuce or stalk or asparagus lettuce (approximately 85 days). Its stem
thickens and elongates and its leaves are long and narrow.
Oil-seed cultivars are grown beyond the rosette stage and allowed to bolt. They
are 50 % larger than other types of lettuce. Their seed is collected and pressed for oil.
The Pathogen and Symptoms Caused
Xanthomonas campestris pv. vitians causes the bacterial leaf spot (BLS) of
lettuce and is known to survive on or in diseased plant debris for short periods of time. It
is also known to be seedborne and can survive on dried seeds on extended periods (Sahin
et al., 1997). It is a strictly aerobic, Gram-negative, rod-shaped bacterium ranging in size
from 0.2-0.8 um (Pernezny et al., 1995; Toussaint, 1999). It is non-sporulating with a
single polar flagellum (Robinson, 2003).
There are two discrete symptoms associated with BLS. The first include water-
soaked, brown lesions that later turn black about 1-2 mm in diameter. These lesions
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
5
become V-shaped, translucent and collapses (Toussaint, 1999; Sahin et al., 1997; Sahin
and Miller, 1998). Lesions may expand along the veins of the plant (Sahin et al., 1997;
Sahin and Miller, 1997; Toussaint, 1999; Wallis and Joubert, 1972). The second type of
symptom consists of small black spots scattered along the leaf surface (Sahin and Miller,
1997).
Distribution
Since 1918, BLS has been reported from Canada (Toussaint, 1999), Venezuela
(Daboin and Tortolero, 1991), Japan (Tsuchiya et al., 1981), and South Africa (Wallis
and Joubert, 1972). In the United States, it has been found in California (Schroth et al.,
1964) and Florida (Pernezny et al., 1995).
In 2005, Nagpala et al. reported the occurrence of the disease in Benguet,
Philippines. These findings result are included in the recently concluded project titled,
“Survey, Identification and Mapping of Soilborne Plant Pathogens and Pathogen Vectors
and Alternative Host Weeds in Benguet” that was conducted under the Semi-Temperate
Vegetables Research and Development Center (STVRDC) of the Benguet Sate
University (BSU).
Epidemiology
Epidemiology of this X. c. pv. vitians that caused leaf spot on lettuce, that is now
present in La Trinidad, Benguet has limited research.
Studies have subsequently demonstrated that the bacterium can be transmitted by
infected seeds and planting materials It can be seedborne and may survive in association
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
6
with seed for extended periods and in soil associated with plant debris (Barak et al.,
2001)
Experiments were established in commercial fields and representative lettuce
seeds lots, and it results to the development of bacterial leaf spot (Umesh et al., 1996).
One explanation for this was that the lettuce seed was internally infected with X.c. pv.
vitians and that this inoculum was not detected with seed wash assays, but was able to
colonize germinated seedlings and cause outbreaks of BLS (Barak et al., 2002). High
populations of X.c. pv. vitians were recoverable from filed debris one month after had
been plowed in the summer (Robinson, 2003).
X.c. pv. vitians has also been shown to survive on lettuce leaf surfaces as epiphyte
using standard dilution planting techniques (Toussaint, 1999). Toussaint et al., (2001)
used scanning electrons microscopy to find bacteria on the leaf surface of asymptomatic
leaves.
Sahin
et al., (1997) studied eight commercial lettuce cultivars. They found high
susceptibility in the cos as two of the green leaf types. The most susceptible cultivars
were butterhead and cos types according to Carisse et al., (2000) in Florida condition.
Tsuchiya et al. (1981) performed experiments on 99 crop species from 19 families
as well as 97 weed species from 31 families in Japan in these studies, plants were
artificially inoculated with the pathogen and rated for disease incidence. They found a
number of hosts within Cruciferae, Polygonaceae, Tropaeloceae, and Compositae. Sahin
et al., (1997) studied eight commercial lettuce cultivars. They found high susceptibility in
the cos as well as two of the green types. Pernezny et al., (1995) found that the cos types
were the most susceptible in Florida although a plethora of host range studies have been
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
7
conducted. Carisse et al., (2000) reported that the most susceptible cultivars were
butterhead and cos types with the least susceptible being green leaf types in Canada.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
MATERIALS AND METHODS
Verification of the Pathogen Identity
Collection of Disease Specimens,
Initial Processing and Description
of Symptoms
Samples of symptomatic lettuce were collected from the BSU experimental areas
where it is observed. Samples were placed in paper bags to absorb the moisture, labeled
and then brought to the Plant Pathology Laboratory for further diagnosis. Symptoms of
samples were described and all observations were recorded.
Identification of Isolated Bacteria
Isolation of associated bacteria. Nutrient Glucose Agar (NGA) was used for
isolation of the bacterium. Thin sections were cut from the advancing portions of infected
leaves and mounted on a drop of sterile distilled water on a slide to induce bacterial ooze.
Appearance of the ooze was confirmed under 40x magnification of an ordinary light
microscope. The bacterium was allowed to ooze out of the tissues for five minutes. After
which, the tissues were removed from the slide and a flame-sterilized wire loop was used
to get a sample of bacterial suspension for streaking onto previously prepared NGA
plates. Plates thus streaked were incubated at 27-30oC for four days. Three well-
separated, isolated single colonies were transferred into NGA slants and stocks cultures
were stored at 50C.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
9
Identification of isolated bacteria. The following tests were performed to
determine the genera of the isolated bacteria.
1. Growth on various media [Appendix 1]
a. Yeast Extract Dextrose CACO3 Agar (YDCA)
b. King’s Medium B Agar (KMBA)
c. Nutrient Glucose Agar (NGA)
d. Sucrose Peptone Agar (SPA)
e. Xanthomonas Isolation Medium (XIM)
f. Casimino Peptone Glucose Agar (CPGA)
g. Potato Dextrose Peptone Agar (PDPA)
2. Gram Staining
Smears were prepared from bacterial ooze obtained from symptomatic leaves.
Cut sections of about 2 to 3 mm were placed in a clean glass slide with a drop of
water then removed after 3 to 5 minutes using a teasing needle. The resulting
smear was then air-dried prior to the standard staining procedure.
3. Other tests. The procedures for the following tests are as outlined in Schaad
(1998).
a. Growth and O/F metabolism. Ten ml of Hugh and Leifson Agar (basal
medium) was poured into test tubes (13 cm diameter). For each
replication, the bacterial isolate were stabbed into two test tubes of basal
medium after which one test tube was covered using water agar and the
other was left uncovered. The set-up was incubated at 300C for 24 to 48
hours.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
10
b. Gelatin Liquefaction tests. Ten ml of gelatin medium was dispensed in
13-cm test tubes. Bacteria were stabbed into the medium and inoculated
tubes were incubated at 20 to 270C for 21 days. After 3,7,14 and 21 days,
the test tubes were refrigerated for 30 minutes.
c. Motility test. A loopful of the bacterial suspension taken from the
freshly cut disease lesion was placed on the underside of a cover slip,
which was then inverted over a depression slide. The slide was viewed
under the microscope at 400x magnification
d. Starch hydrolysis test. Bacterium was streaked onto starch agar and the
plates were incubated for 2-7 days. The plates were then flooded with
iodine solution and any clearing zone was observed.
Determining the Pathogenicity of the Isolates
Preparation of Test Plants
Lettuce seedlings, about two weeks old, were obtained and kept in the greenhouse
for pathogenicity test.
Inoculum Preparation and Inoculation
The bacterial suspension was prepared and standardized at 1 x 106 cfu/ml using
the McFarland’s Turbidity Standard. Three inoculation techniques were tested following
the procedure of Dayao (2004), as follows:
1. Spraying- Using a low-pressure sprayer, the inoculum was sprayed onto the leaf
surface until run-off.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
11
2. Injection- Bacterial suspension was injected into the intercellular spaces at the
undersurface of the leaf.
3. Pricking- The leaves was pricked using a sterilize needle and sprayed with the
bacterial suspension.
The inoculated plants were covered with a plastic bag and incubated overnight in
the laboratory after which the plastic bags were removed and the inoculated plants were
brought to the greenhouse. Symptom development was observed and was compared to
the initial findings.
Re-isolation of Causal Organism
Lesions from artificially inoculated plants were used as sources of bacterial
suspension for re-isolation onto Nutrient Glucose Agar (NGA). Isolates thus obtained
were compared to the original isolates.
Testing the Antibiotic Sensitivity of the Isolates
One ml of standardized bacterial suspension of 1 x 106 cfu/ml was added to 5 ml
water agar and was poured onto previously prepared NGA plates. The test antibiotics
were prepared at different test concentrations (50, 75, 100, 200, 300, 400, 500 and 600
ppm).
Sterilized filter paper discs (20 mm diameter) were dipped into the prepared
antibiotics and then plated at the center of the medium using sterilized forceps. Plates
thus prepared were sealed and incubated for 48 hours at 28°C.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
12
Data Gathered
1.Symptoms of the diseased plants
2. Cultural, morphological and physiological characteristics of the isolates in various
media. Specifically the size, shape, color, elevation, margins, and production of pigment
in the media.
3. Pathogenicity test. The symptoms produced by the inoculated bacterium compared to
the original symptoms observed.
4. Inhibition zones (mm). Inhibition zones around the antibiotic disks will be measured.
5. Photodocumentation. Photographs will be taken of test results and experimental set-up
and will be saved as jpeg files.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
RESULTS AND DISCUSSION
Collection of Sample and Description of
Collection Sites
Collecting of bacterial leaf spot of lettuce was done in the BSU experimental
areas: namely, at the Swamp Area, Balili experimental area, and Pomology. Lettuce
leaves were collected on May 2006 and were well-formed and firm. The lesions were
scattered on the entire surface of the leaves.
Areas from where specimens collected have a flat elevation and it is commonly
used to plant vegetables crops than that of the flowers. Almost square meters of the field
are for hired. Crop rotation is not employed as seen that the farmers are not strictly
monitored on what type of crops they like to plant. Irrigation are through canals, stock
waters in drums.
Disease Symptoms
Lettuce leaves collected have brown lesions that are translucent, irregular in shape
and water-soaked. Lesions later may turn black. The first symptoms appear at the leaf
margin and consist of water-soaked lesions that become necrotic and confluent (Figure
1).
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
14
Figure 1. Symptoms of infected lettuce leaves in the field
Cultural Characteristics of the
Pathogen on Various Media
Various standard and differential media were used to grow the bacterium. A
loopful of bacterial ooze from the lettuce leaves was streaked onto NGA, XIM, SPA,
CPGA, KMBA, YDCA, and PDPA and incubated for 4- 5 days at 27-300C.
Colonies were measured and ranged from approximately from <1mm to 4mm in
diameter (Table 2). Colonies are circular and appear shiny and mucoidal. Under light, the
colonies appear translucent and are mostly from light to dark yellow (Figure 2). When
plated on YDCA and XIM, the bacterium produces whitish to cream colonies. On CPGA,
it produces red colonies because of the TTZ in the media.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
15
Table 1. Cultural characteristics of Xanthomonas in plate growth
MEDIA
SIZE CONFIGURATION DENSITY ELEVATIION COLOR
CPGA
< 1mm Circular
Opaque Mucoid Red
PDPA
3-4 mm Circular Opaque
Mucoid Cream
SPA
4-5 mm Circular Opaque
Mucoid Yellow
YDCA
2-4 mm Circular Opaque
Mucoid Yellow
XIM
< 1 mm Circular Translucent Mucoid White
KMBA
2-4 mm Circular Translucent Mucoid Orange
NGA
3-4 mm Circular Opaque Mucoid Yellow
NGA
Figure 2. Cultural characteristics of the bacterium on various standard and differential
media
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
16
Morphological Characteristics of
the Pathogen
Gram Stain Reaction. Bacterial ooze from the disease specimen (Figure 3a) was
preferred for staining rather than organisms cultured in a solid medium. Those coming
from a solid medium produce a thick dense surface growth and are not amenable to direct
transfer to the glass slide (Cappuccino 2002).
The bacterium was stained pink, which means that the alcohol decolorized the
Gram stain (Figure 3b). The bacterium is therefore confirmed to be Gram negative
(Frobisher, 1957). The rod-shaped bacteria are less short and cylindrical. These
characteristics confirm to those of Xanthomonas as cited by Agrios (2002).
Ooze coming
out from the
cut lesion
Figure 3a. Bacterial ooze from the sectioned lettuce leaves (400x)
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
17
Figure 3b. Gram-stained bacterial cells (1000x)
Motility Test A loopful of bacteria were suspended in the center of a clear thin
cover slip and inverted to the depression slide. The transparent bacterial cells were
motile. Motile bacteria can only move in fluids but it is extremely difficult to steady them
since lack of color prevents details from showing clearly. The observation of bacteria in
hanging drop preparations, therefore, yields limited though valuable information
(Frobisher, 1957).
Physiological Characteristics
of the Pathogen
Growth and O/F Metabolism. Pure culture of Xanthomonas campestris pv. vitians
was stabbed in the basal medium (Hugh and Leifson Agar). One tube was covered with
water agar to prevent the entry of air and the other treatment remained uncovered.
Observations were done after 24 to 48 hours at 30 0C. In comparison with the original
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
18
Figure 4. Results of the O/F Test on Hugh and Leifson Agar
medium (control), the color in the covered and uncovered tubes did not change
even after 48 hours of incubation. This confirms the description by Robinson (2003) that
X. c. pv. vitians that is never fermentative and oxidase negative.
Gelatin Liquefaction Test. Gelatin is hydrolyzed by quite a few organisms and
liquefies at room temperature (Benson, 1998). The bacterium was not able to liquefy the
gelatin even after 3, 7, 14 and 21 days of incubation (Figure 4). Therefore, it can be
deduced that the bacteria was not able to produce gelatinase and thus could not hydrolyze
the gelatin. This inability to liquefy gelatin is a characteristic of Xanthomonas species.
Starch Hydrolysis Test. The bacterium was streaked onto starch agar medium and
incubated for 2 to 7 days. The iodine solution was poured over the growth after the
incubation period. Starch in the presence of iodine will impart a blue-black color to the
medium, indicating the absence of starch-splitting enzymes and representing a negative
result. If the starch has been hydrolyzed, a clear zone of hydrolysis will surround the
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
19
growth of the organism. This is a positive result (Cappuccino, 2002). If the area
immediately adjacent to the growth is clear; amylase has been produced (Benson, 1998).
Stabbed
bacterium
Figure 5. Results of the Gelatin Liquefaction Test on Gelatin Agar (gulaman: green-
Flavored and white- unflavored)
Clearing zone
Figure 6. Results of the Starch Hydrolysis Test on starch medium (7 days after
incubation)
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
20
If the starch is undegraded, a blue-black color results when iodine is allowed to
react with it. This is because potassium iodide molecules readily occupy a space within
the helical coiling of the amylase component of starch. This results in a blue-black
complex (Raymundo et al., 1991).
Pathogenicity Test
Spraying until run-off of the bacterial suspension effected the symptoms most
closely approximating the original specimens. This shows the passage of the pathogen
through the hydathodes resulting in initial yellowing at the edges of the infected leaf.
Injection of bacterial suspension into cellular spaces of the leaf using an insulin
syringe led to formation of translucent spots. These symptoms are also observed in the
field in advanced stages of the disease or when there is a lot of moisture. Pricking and
spraying resulted in chlorosis and collapse of the leaf (Figure 7).
Re-isolation of the Bacterium
From Artificially-Inoculated
Lettuce Leaves
The bacterium was re-isolated from the artificially inoculated lettuce leaves and
had the same appearance and characteristics on NGA medium as the original isolate
(Figure 8).
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
21
A B
C D
Figure 7. Artificially inoculated lettuce seedlings and its symptoms (A. Uninoculated
B. Spraying C. Injection D. Pricking and Spraying)
PRICKING and
SPRAYING
SPRAYING
INJECTION
Figure 8. Bacteria isolated from the artificially inoculated lettuce (NGA, 4 days)
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
22
Antibiotic Sensitivity Testing
of the Pathogen
The bacterium was sensitive to both streptomycin and chlortetracycline (Table 3).
This means that the bacterium has not been exposed yet to these antibiotics and thus has
not been able to develop resistance to the chemical. Expectedly, cephalexin hydrate and
cycloheximide were not effective against the bacterium (Figure 9). Cephalexin is highly
effective against Gram-positive bacteria but performs poorly against the Gram negatives.
On the other hand, cycloheximide was formulated for eukaryotic microorganisms
(eMedicineHealth.com, 2005).
Streptomycin comes from Streptomyces griseus and is one among the
aminoglycoside group of antibiotics. It damages bacterial cell membranes by inhibiting
protein synthesis. Specifically, it binds to the 30S ribosome and changes its shape thus
causing a misreading of messenger RNA information (Wikipedia 2007).
Chlortetracycline hydrate, also known as aureomycin, comes from S.
aureofaciens. It is a broad-spectrum bacteriostatic agent that inhibits bacterial protein
synthesis. Specifically, it blocks the attachment of the transfer RNA-amino acid to the
ribosome. More precisely, it inhibits the codon-anticodon interaction (Wikipedia 2007).
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
23
Table 2. The inhibition zones affected by various antibiotics and their concentrations on
the growth of Xanthomonas campestris pv vitians
CONCENTRATION
MEAN INHIBITION
ANTIBIOTICS
(ppm)
ZONE (mm)
Streptomycin sulfate
50
43.00
75
39.20
100
41.00
200
40.00
300
39.30
400
39.80
500
41.80
600
43.30
Chlortetracycline 50
49.50
75
51.00
100
52.00
200
52.50
300
53.20
400
58.00
500
57.50
600
58.50
Cephalexin hydrate
50
23.00
75
27.20
100
30.30
200
26.30
300
26.50
400
34.50
500
26.20
600
28.00
Cycloheximide 50
35.00
75
27.20
100
22.80
200
20.80
300
21.50
400
21.30
500
20.30
600
20.30
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
24
ANTIBIOTIC Streptomycin Chlortetracycline Cephalexin
Cycloheximide
Sulfate
hydrate
CONCENTRATION
(ppm)
50
75
100
200
300
400
500
600
Figure 9. Inhibition zones effected by various antibiotics and their concentrations on the
growth of Xanthomonas campestris pv vitians
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary
The isolated bacterium from symptomatic lettuce leaves exhibited the
characteristics of Xanthomonas campestris pv. vitians. It produces circular, fried egg-like
and mucoid colonies with yellow pigment. Cells are rod-shaped and short. It is oxidase
negative and never fermentative. It does not produce the enzyme gelatinase. Artificially
inoculated 2 week-old lettuce seedlings show the symptoms of the disease observed in
the field. The bacterium was sensitive to both streptomycin and chlortetracycline
hydrochloride but was not affected by cycloheximide and cephalexin.
Conclusion
The leaf spot observed in symptomatic lettuce is caused by Xanthomonas
campestris pv. vitians. The bacterium is sensitive to the standard antibiotics and thus,
may not have been exposed to antibiotics. This may indicate that the pathogen is a new
pathogen, i.e., newly introduced into the area. Support for this conclusion is the fact that
the disease is not yet recorded in the Disease Index (Tangonan, 1999).
Recommendations
Limitations of the study needs firther investigate the fpllowing:
1. pathogenicity test of the bacterium to other crucifer cultivars and other
crops likes tomato and bell pepper which have been reported to be affected
by the same bacterium;
2. development of integrated disease management measures;
3. survey to determine spread and distribution of the disease in lettuce-
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
26
growing areas in Benguet; and
4. possible determination of transmission and spread of the disease as it has
been reported abroad to be both soilborne and spread via infected seeds.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
LITERATURE CITED
AGRIOS, G. N. 2002. Plant Pathology 3rd ed. Academic Press INC. San Diego,
California
BARAK, J. O., S. T. KOIKE and R. L. GILBERTSON. 2002. Movement of
Xanthomonas campestris pv. viitans in the stems of lettuce and seed
contamination. Plant Pathology 51, 506-512.
BARAK, J. D., Koike S. T., and Gilbertson. R. L. 2001. The Role of Crop Debris and
Weeds in the Epidemiology of the Bacterial Leaf Spot of Lettuce in Califonia
BENSON, H. J. 1998. Microbiological Application: Laboratory Manual in General
Microbiology. 7th ed. The McGraw Hill Companies, INC. United State of
America.
CARISSE, O., A. Quimet., V. Toussaint and V. PHILION. 2000. Evaluation of Seed
Treatments, Bacteriacides, and Cultivars on Bacterial leaf spot of Lettuce caused
by Xanthomonas campestris pv. vitians. Plant Dis. 84:295-299
CAPPUCCINO, J. G. and N. Sherman. 2002. Microbiology: A Laboratory Manual 6th
ed. State University of New York Rockland Community College. Pearson
Education, Inc. San Francisco. P. 49.
DABOIN, C., and O. TORTOLERO. 1991. Mancha bacterial foliar de la lechuga en
Algunos campos Andinos de Venezuela. Fitopatol. Venez 6:8-10.
DAYAO, S. A. 2004. Etiology of the Leaf Blight of Calla lily (Zantedeschia sp.). BS
Thesis. BSU, La Trinidad, Benguet. Pp. 9-10
MEDICINE HEALTH. 2005. Antibiotics. Retrieved 26 March 2007 from http://www.
emedicinehealth.com/script/main/hp.asp.
FROBISHER, M. 1957. Fundamentals of Microbiology 6th ed. W.B. Saunders Company.
Tokyo, Japan. Pp. 105-106.
NAGPALA, A. L., L. D. LANDO, C. BA-A and J. GALENG. 2005. Progress Report of
a Project; Survey, Mapping and Identification of Soilborne Plant Pathogens,
Pathogen Vectors and Alternative Host Weeds in Benguet. A report made to the
STVRDC, BSU, La Trinidad, Benguet.
PERNEZNY, K., R. N. RAID, R. E. STALL, N. C. HODGE and J. COLLINS.
Investigation of seed treatments for management of bacterial leaf spot of lettuce.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
28
PERNEZNY, K., R. N. RAID, R. E. STALL, N. C. HODGE and J. COLLINS. 1995.
An Outbreak of bacterial leaf spot of Lettuce in Florida caused by X.c.pv. vitians.
Plant Dis. 79:359-360.
PHILIPPINE INFORMATION AGENCY (PIA). 2006. BSU researchers confirm new
crop diseases in Benguet. PIA Press Release dated Jan. 09, 2006. Retrieved 30
November 2006 from http://www.pia.gov.ph.
RAYMUNDO, A. K., A. F. ZAMORA, and I. F. DALMACIO. 1991. Manual on
microbiological Techniques. Los Baños, Laguna, Philippines
ROBINSON, P.E. 2003. The Epidemiology of Xanthomonas campestris pv. vitians,
Causal Organism of Bacterial Leaf Spot of Lettuce. MS thesis. University of
Florida. Pp 1-6.
RYDER, E.J. 1999. Lettuce, Endive, and Chicory. Crop Production Science in
Horticulture series. Cambridge UK: CAB International.
SAHIN, F. and S. A. MILLER. 1997. Identification of the bacterial leaf spot of lettuce,
Xanthomomas campestris pv. vitians in Ohio, and assessment of cultivar
resistance and seed treatments. Plant Dis. 81:1443-1446.
SAHIN, F. and S. A. MILLER. 1998. Two new host of Xanthomonas campestris pv.
vitians. Plant Dis. 81: 1443-1446v.
SAHIN. F., P. A. ABBASI and S. A. MILLER. 1997. Variation among strains of
Xanthomonas campestris pv. vitians in Ohio, and assessment of cultivar
resistance and seed treatments. Plant Dis. 81:1443-1446.
SCHAAD, N.W., 1998. Laboratory Guide for Identification of Plant Pathogenic Bacteria.
2nd ed. St. Paul, Minnesota: APS Press.
SCHROTH, M .N., J. P., THOMPSOM, R. BARDIN and A. GREATHEAD. 1964. A
new disease of lettuce… bacterial leaf spot of lettuce. Calif. Agric. 2-3.
STEPHENS, J. M. 1988. Manual of Minor Vegetables. Gainesville FL: University of
FL.
TANGONAN, N. G. 1999. Host Index of Plant Diseases in the Philippines 3rd.
Department of Agriculture Philippine Rice Research Institute (PHILRICE)
Munoz, Nueva Ecija, Philippines.
TOUSSAINT, V., C. E. MORRIS and O. CARISSE. 2001. A new semi-selective
medium for Xanthomonas campestris pv. vitians, the causal agent of bacterial leaf
spot of lettuce. Plant dis. 85:131-136.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
29
TOUSSAINT, V. 1999. Bacterial leaf spot, a new disease of lettuce in Quebec caused
by Xanthomonas campestris pv. vitians. Phytoprotection 80: 121-125.
TSUCHIYA, Y., K. OHATA, K. AZEGAMI and M. MATSUZAKI. 1981.
Pathogenicity of Xanthomonas campestris pv. vitians to various crops and
weeds. Nogyo gijutsu kenkyusho hokoku-Bull. Natal Inst. Agric. Sci. 35:57-66.
UMESH, K.C., S. T. KOIKE AND K. L. GILBERTSON. 1996. Association of
Xanthomonas campstris pv. vitians with lettuce seed. Phytopathology 86.S3.
VILLA, A. N. E., C. K. C. JAIME and Y. B. NAVARRO. 2005. Dry Leaf Spot of
Lettuce in Benguet: A First Report. A report of Investigatory Project presented
during the Regional DOST-INTEL Science Fair and Quiz, held at Tabuk,
Kalinga.
WELLMAN-DESBIANS, E. 1999. Dissemination of Xanthomonas campestris pv. vitians
during lettuce transplant production. Phythopathology 89:S84.
WALLIS, F. M., and J. J. JOUBERT. 1972. Bacterial leafspot of lettuce in Natal.
Phytolactica 4:137-138.
WIKIPEDIA. The Free Encyclopedia. 2007. Retrieved March 6, 2007 posted at
enwikipedia.org/wiki.html.
WOLFORD, R. 2006. Watch Your Garden-Grown Lettuce. Extension Educator- Urban
Horticulture and Gardening. Extension Educator- Nutrition and Wellness,
University of Illinois Extension. Retrieved 06 March 2006 from
http://www.urbanext.uiuc.edu/veggies/lettuce1.html.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
APPENDICES
APPENDIX TABLE 1. Computation of the antibiotic concentration (ppm)
ANTIBIOTICS
CONCENTRATION
(ppm)
SS CPH CY CH
50
52.63
500.00
53.19
62.50
75
78.95
750.00
79.79
93.75
100
105.26
1000
106.38
125.00
200
210.53
2000
212.77
250.00
300
315.79
3000
319.15
375.00
400
380.00
4000
425.53
500.00
500
526.32
5000
531.91
625.00
600
631.58
6000
638.30
750.00
* 100ml preparation only divided by the amount by 10 ml.
Streptomycin sulfate (SS)
– 95%
Cephalexin hydrate (CPH)
– 100% (assumed)
Cycloheximide (CY)
– 94%
Chlortetracycline/ Aureomycin (CH) – 80%
31
APPENDIX TABLE 2. Measurement of the inhibition zones (mm)
REPLICATION
TREATMENTS
TOTAL
MEAN
I
II
III
SDW 20.0
20.5
20
60.5
20.2
Streptomycin sulfate
75 ppm
39.5
39.5
38.5
117.5
39.2
100 ppm
43.5
40.5
39
123
41.0
200 ppm
42.0
40.5
37.5
120
40.0
300 ppm
37.5
39.5
41
118
39.3
400 ppm
41.0
40
38.5
119.5
39.8
500 ppm
40.5
40.5
44.5
125.5
41.8
600 ppm
45.0
41
44
130
43.3
Chlortetracycline
50 ppm
55
43
50.5
148.5
49.5
75 ppm
54.5
52
46.5
153
51.0
100 ppm
54
51
51
156
52.0
200 ppm
54.5
51
52
157.5
52.5
300 ppm
54
51.5
54
159.5
53.2
400 ppm
54.5
62.5
57
174
58.0
500 ppm
56.5
60
56
172.5
57.5
600 ppm
56
56.5
63
175.5
58.5
Cephalexin hydrate
50 ppm
22
23.5
23.5
69
23.0
75 ppm
29.5
24
28
81.5
27.2
100 ppm
27.5
32
31.5
91
30.3
200 ppm
26.5
25.5
27
79
26.3
300 ppm
25.5
27
27
79.5
26.5
400 ppm
35.5
36
32
103.5
34.5
500 ppm
27
26
25.5
78.5
26.2
600 ppm
29.5
25
29.5
84
28.0
Cycloheximide
50 ppm
30.5
35
39.5
105
35.0
75 ppm
25
27
29.5
81.5
27.2
100 ppm
22
25
20
67
22.3
200 ppm
21
21
20
62
20.7
300 ppm
20
23.5
21
64.5
21.5
400 ppm
21
22
21
64
21.3
500 ppm
20
20
21
61
20.3
600 ppm
21
20
20
61
20.3
32
APPENDIX TABLE 3. Effect of different antibiotics
ANTIBIOTIC MEAN
Streptomycin sulfate
40.88 b
Chlortetracycline 54.03
a
Cephalexin hydrate
27.75 c
Cycloheximide 23.64
d
* Means with the same letters are not significantly different at 5% DMRT
APPENDIX TABLE 4. Effect of concentration of antibiotic
CONCENTRATION MEAN
50 ppm
32.933
75 ppm
33.167
100 ppm
31.933
200 pmm
31.133
300 ppm
32.133
400 ppm
34.767
500 ppm
33.200
600 ppm
34.133
APPENDIX TABLE 5. Interaction between antibiotic and X.c.pv. vitians
ANTIBIOTIC/
CONCENTRATION (ppm)
CONCENTRATION 50 75 100 200 300 400 500 600
Streptomycin
sulfate 43.0c 39.2c 41.0c 40.0c 39.3c 39.8c 41.8c 43.3c
Chlortetracycline 49.5b 51.0b 52.0b 52.5b
53.2b
58.0a
57.5a 58.5a
Cephalexin hydrate
23.0fgh 27.2e 30.3e
26.3ef 26.5ef 34.5d 26.2efg 28.0e
Cycloheximide 35.0d
27.2e
22.8gh 20.7h
21.5h
21.3h
20.3h
20.3h
33
APPENDIX TABLE 6. Analysis of variance
SOURCE OF DEGREES
SUM OF
MEAN
F VALUE
COMPUTED
VARIATION OF
SQUARES
SQUARES
F
FREEDOM
0.05 0.01
Factor A
4
18,829.846
4,707.461
926.4377** 2.48 3.56
Factor B
7
103.967
14.852
2.9230 ns
AB 28 994.887
35.532
6.9927**
Error 80 406.500
5.081
TOTAL 119
Ns
–
not
significant
Coefficient of variation = 6.77%
** -
APPENDIX A
APPENDIX 1. Standard and Differential Media
Yeast Extract Dextrose Agar- CaCo 3(YDCA)
Component
Amount
Yeast
10.0
g
Dextrose
(Glucose)
20.0
g
Calcium carbonate, USP light powder
20.0 g
Agar
15.0
g
Distilled Water
1000 ml
Prepare 10 g of yeast, 20 g of dextrose (glucose), and 20 g of calcium carbonate, USP
light powder and 15 g of agar. Dissolve in 1 liter of distilled water except dextrose.
34
Prepare it separately into the flask at 100 ml distilled water. Sterilize in autoclave at 15
psi for 15-20 mins. After sterilizing, mix the dextrose in a separate flask with yeast,
CaCo3 and agar. Mix thoroughly before pouring into the petri plates.
King Medium B Agar (KMBA)
Component
Amount
Protease
peptone/
peptone
20.0
g
Potassium phosphate (K2HPO4)
1.5
g
Magnesium sulfate (MgSO4.7H20)
1.5
g
Agar
15.0
g
Glycerol 15.0
ml
Dispensed individual 250 ml erlenmeyer flask and sterilized in autoclave for 30 minutes
at 15 psi before plating in sterilized petri plates.
Nutrient Glucose Agar (NGA)
Component
Amount
Beef
Extract
3.0
g
Peptone
5.0
g
Glucose 2.5
g
Agar
15.0–20.0
g
Distilled water
1000 ml
Sucrose Peptone Agar (SPA)
Component
Amount
Sucrose 20.0
g
35
Peptone
5.0
g
K2HPO4
0.5
g
MgSO47H2O
0.25
g
Agar
15.0 g
Adjust pH to 7.2
40 % NaOH
40 % 100m distilled H2O
Xanthomonas Isolation Medium (XIM)
Component
Amount
Cellobiose
10.0
g
Sodium phosphate (NaH2PO4)
0.5 g
MgSO4.
7H20
0.3
g
K2HPO4
3.0
g
Ammonium
Chloride
(NH4Cl)
1.0
g
Agar
15.0
g
Potato Dextrose Peptone Agar (PDPA)
Component
Amount
Potato
Dextrose
Agar
8.0
g
Peptone
5.0
g
Distilled water
1000 ml
36
Casamino Acid Peptone Glucose Agar (CPGA)
Component
Amount
Peptone 2.0
g
Casamino
acid
0.2
g
Glucose
1.0
g
Agar
3.0
g
Distilled water
140-200 ml
TZC
should
be
1g/li
or
1ml/100ml
CPG
APPENDIX 2. Basal Medium for Anaerobic Growth
Hugh and Leifson Agar
Component
Amount
Peptone
2.0
g
NaCl
5.0
g
KH2PO4
0.3
g
Agar
3.0
g
Bromthymol
blue
3.0
ml
(1%
aqueous
solution)
Distilled water
1000 ml
Dissolve ingredients and adjust pH to 7.1. Add 10ml of basal medium to 13 cm diameter
test tubes and sterilize at 121º C for 20 minutes. Prepare a 10% aqueous solution of
glucose and sterilize by filtration. Add 0.5 ml of sterile glucose aseptically to each tube of
basal medium. Inoculate two tubes with the organism to be tested. Cover one tube with a
37
layer of water agar or paraffin to a depth of 5mm. A color change from blue to yellow in
both tubes is recorded as positive for anaerobic growth.
APPENDIX 3. Procedure of McFarland’s Turbidity Standard
The 0.5 McFarland may be prepared by adding 0.5 ml of 1.175% (wt/vol) barium
chloride dehydrate (BaCl2.2H2O) solution to 99.5 ml of 1% (vol/vol) sulfuric acid. The
turbidity standard is then aliquoted into test tubes identical to those used to prepare the
inoculum suspension. Seal the McFarland standard tubes with wax, Parafilm, or some
other means to prevent evaporation. McFarland standards may be stored for up to 6
months in the dark at room temperature (22ºC to 25ºC). Discard after 6 months or sooner
if any volume is lost. Before each use, shake well, mixing the fine white precipitate of
barium sulfate in the tube. The accuracy of the density of a prepared McFarland standard
should be checked using a spectrophotometer with a 1-cm light path; for the 0.5
McFarland standards, the absorbance at a wavelength of 625 nm should be 0.08 to 0.1.
CUADRA, ILENE C. APRIL 2007. Etiology of the Bacterial Leaf Spot of
Lettuce in La Trinidad, Benguet: A First Report. Benguet State University, La Trinidad,
Benguet.
Adviser: Jocelyn C. Perez, MSc
Co-Adviser: Lily Ann D. Lando, PhD
ABSTRACT
The bacterium causing leaf spot in lettuce leaves was isolated and produced
similar symptoms to the artificially inoculated lettuce leaves.
Cultural characteristic of the isolates are yellow colonies produced in seven (7)
different media. The morphological characterization was done through gram staining
which is negative and motility test which is motile because of the polar flagella of the
bacterium. In the physiological characterization, growth in O/F test was never
fermentative and oxidase negative. Thus, the bacterium was not able to produce
gelatinase enzymes in the gelatin liquefaction test. A clear zone was observed in the
starch hydrolysis test swabbed with Xanthomonas campestris pv. vitians.
Sensitivity of the bacterium to streptomycin sulfate and chlortetracycline was very
obvious. Cephalexin hydrate and cycloheximide were not effective against the bacterium.
TABLE OF CONTENTS
Page
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Abstract
. . . . . . . . . . . . . . . … .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
Table of Content
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii
INTRODUCTION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
REVIEW OF LITERATURE
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
MATERIALS AND METHODS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
RESULTS AND DISCUSSION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Collection of Sample and
Description of the Collection Sites . . . . . . . . . . . . . . . . . . . . . . . . . 13
Disease Symptoms
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Cultural
Characteristics
of
The Pathogen on Various Media
. . . . . . . . . . . . . . . . . . . . . . . . .
14
.
Morphological Characteristics of
The Pathogen
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
16
Gram Stain Reaction
. . . . . . . . . . . . . . . . . . . . . . . . .
16
Motility Test
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Physiological Characteristics of
The Pathogen
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Growth and O/F Metabolism
. . . . . . . . . . . . . . . . . . . 17
Gelatin Liquefaction Test
. . . . . . . . . . . . . . . . . . . . . . . . . 18
Starch Hydrolysis Test
. . . . . . . . . . . . . . . . . . . . . . . . . 19
Pathogenicity Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Re-Isolation of the Bacterium
from
Artificially
Inoculated
ii
Lettuce Leaves
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21
Antibiotic Sensitivity of
the Pathogen
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
SUMMARY, CONCLUSION
AND RECOMMENDATIONS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
LITERATURE CITED
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
27
APPENDICES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . ………... . . . . . .
30
iii
INTRODUCTION
In 2005, Nagpala et al. (PIA, 2006) reported a leaf spot of head lettuce (Lactuca
sativa L.) collected from Loo and Poblacion in Buguias, Benguet. The lettuce leaves had
black lesions that often become papery thin and then eventually tears the leaves to give it
a tattered appearance (Villa et al., 2005).
The initial survey results of Nagpala et al. (2005) showed a 50-90% disease
incidence in the sites sampled. Laboratory tests showed the association of a bacterium
with the diseased plant tissues. Identification done by the team and continued by Villa et
al. (2005) pointed to the possibility of the causal bacterium being a Xanthomonas
campestris pathovar.
Bacterial spot of lettuce has not been reported in the Philippines as revealed in a
review of the Host Index of Plant Diseases in the Philippines (Tangonan, 1999).
Therefore, this disease may be new or unrecognized and thus unreported.
Studies on the transmission of the organism have been inconclusive but several
reports say that it may be both seed- and soilborne. As such, the pathogen presents a new
challenge to the vegetable industry in Benguet. To adequately meet this challenge, the
identity of the pathogen has to be verified and its pathogenicity determined. The
information may then be used to prevent the spread of the pathogen and develop an
integrated approach to the management of the disease.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
2
This study was conceived and conducted to:
1. verify the identity of the causal bacterium of the disease;
2. determine the sensitivity of the bacterial isolates to antibiotics (streptomycin
sulfate, cycloheximide, cephalexin hydrate, and chlortetracycline).
3. determine the pathogenicity of the isolates to commonly-grown lettuce
cultivars; and
4. compare the isolates to common Xanthomonas species in the locality.
The study was conducted in the Department of Plant Pathology laboratory
from November 2006 to March 2007.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
REVIEW OF LITERATURE
The Host Plant
Lettuce (Lactuca sativa L.) including cos or romaine, belongs to Asteraceae
(compositae) or daisy family. This is the largest dicotyledonous family in the plant
kingdom. Notable leafy crop species within the Asteraceae family includes lettuce,
chichory and endive as well as other lesser known plants such as fuki and oyster plant
(Ryder, 1999).
Lettuce, a cool-season vegetable crop is one of the easiest to grow. High summer
temperature usually cause seedstalk formation (bolting) and bitter flavor. Slow-bolting or
heat resistant varieties are available and are recommended for extending the lettuce-
growing season (Robinson, 2003).
The plant is an annual herb with milky latex in the leaves and stems. Lettuce has a
shallow root system up to 1 foot deep (30 cm). The most important characteristics of
lettuce are its size, compactness, sweetness and succulence.
Lettuce is popular in salads because of its nutritional content. One cup of raw leaf
lettuce (chopped) contains only nine calories. It contains 1.3 g dietary fiber, 1 g protein,
1.34 g carbohydrates, 1456 IU of vitamin A, 13.44 I.U. vitamin C, 20.16 mg calcium,
0.62 mg iron and 162.5 mg potassium (Wolford, 2006).
There are several types of lettuce commonly grown in garden: the crisphead,
butterhead, cos, leaf, stem and oil-seed.
Crisphead also known as iceberg (approximately 60-75 days when direct seeded).
The most widely available as a fresh market type, it weighs about 1 kg with six or seven
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
4
outer leaves. Outer leaves are bright green or dull green, and the interior color may be
white to creamy yellow.
Butterhead or Bibb lettuce (approximately 60 days when direct seeded). It is a
loose-heading type with dark green leaves that are somewhat thicker than those of
iceburg lettuce.
Romaine or cos (approximately 60 days). They are elongated leaves that range in
color from yellow to dark green forming upright heads with rather wavy, attractive
leaves. They are very nutritious and weigh up to 750g each (Stephen, 1988).
Leaf type lettuce (approximately 50- 60 days). Have green or reddish leaves, this
type is fast growing, long-lasting lettuce. It is basically planted, thinned and harvested.
Stem lettuce or stalk or asparagus lettuce (approximately 85 days). Its stem
thickens and elongates and its leaves are long and narrow.
Oil-seed cultivars are grown beyond the rosette stage and allowed to bolt. They
are 50 % larger than other types of lettuce. Their seed is collected and pressed for oil.
The Pathogen and Symptoms Caused
Xanthomonas campestris pv. vitians causes the bacterial leaf spot (BLS) of
lettuce and is known to survive on or in diseased plant debris for short periods of time. It
is also known to be seedborne and can survive on dried seeds on extended periods (Sahin
et al., 1997). It is a strictly aerobic, Gram-negative, rod-shaped bacterium ranging in size
from 0.2-0.8 um (Pernezny et al., 1995; Toussaint, 1999). It is non-sporulating with a
single polar flagellum (Robinson, 2003).
There are two discrete symptoms associated with BLS. The first include water-
soaked, brown lesions that later turn black about 1-2 mm in diameter. These lesions
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
5
become V-shaped, translucent and collapses (Toussaint, 1999; Sahin et al., 1997; Sahin
and Miller, 1998). Lesions may expand along the veins of the plant (Sahin et al., 1997;
Sahin and Miller, 1997; Toussaint, 1999; Wallis and Joubert, 1972). The second type of
symptom consists of small black spots scattered along the leaf surface (Sahin and Miller,
1997).
Distribution
Since 1918, BLS has been reported from Canada (Toussaint, 1999), Venezuela
(Daboin and Tortolero, 1991), Japan (Tsuchiya et al., 1981), and South Africa (Wallis
and Joubert, 1972). In the United States, it has been found in California (Schroth et al.,
1964) and Florida (Pernezny et al., 1995).
In 2005, Nagpala et al. reported the occurrence of the disease in Benguet,
Philippines. These findings result are included in the recently concluded project titled,
“Survey, Identification and Mapping of Soilborne Plant Pathogens and Pathogen Vectors
and Alternative Host Weeds in Benguet” that was conducted under the Semi-Temperate
Vegetables Research and Development Center (STVRDC) of the Benguet Sate
University (BSU).
Epidemiology
Epidemiology of this X. c. pv. vitians that caused leaf spot on lettuce, that is now
present in La Trinidad, Benguet has limited research.
Studies have subsequently demonstrated that the bacterium can be transmitted by
infected seeds and planting materials It can be seedborne and may survive in association
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
6
with seed for extended periods and in soil associated with plant debris (Barak et al.,
2001)
Experiments were established in commercial fields and representative lettuce
seeds lots, and it results to the development of bacterial leaf spot (Umesh et al., 1996).
One explanation for this was that the lettuce seed was internally infected with X.c. pv.
vitians and that this inoculum was not detected with seed wash assays, but was able to
colonize germinated seedlings and cause outbreaks of BLS (Barak et al., 2002). High
populations of X.c. pv. vitians were recoverable from filed debris one month after had
been plowed in the summer (Robinson, 2003).
X.c. pv. vitians has also been shown to survive on lettuce leaf surfaces as epiphyte
using standard dilution planting techniques (Toussaint, 1999). Toussaint et al., (2001)
used scanning electrons microscopy to find bacteria on the leaf surface of asymptomatic
leaves.
Sahin
et al., (1997) studied eight commercial lettuce cultivars. They found high
susceptibility in the cos as two of the green leaf types. The most susceptible cultivars
were butterhead and cos types according to Carisse et al., (2000) in Florida condition.
Tsuchiya et al. (1981) performed experiments on 99 crop species from 19 families
as well as 97 weed species from 31 families in Japan in these studies, plants were
artificially inoculated with the pathogen and rated for disease incidence. They found a
number of hosts within Cruciferae, Polygonaceae, Tropaeloceae, and Compositae. Sahin
et al., (1997) studied eight commercial lettuce cultivars. They found high susceptibility in
the cos as well as two of the green types. Pernezny et al., (1995) found that the cos types
were the most susceptible in Florida although a plethora of host range studies have been
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
7
conducted. Carisse et al., (2000) reported that the most susceptible cultivars were
butterhead and cos types with the least susceptible being green leaf types in Canada.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
MATERIALS AND METHODS
Verification of the Pathogen Identity
Collection of Disease Specimens,
Initial Processing and Description
of Symptoms
Samples of symptomatic lettuce were collected from the BSU experimental areas
where it is observed. Samples were placed in paper bags to absorb the moisture, labeled
and then brought to the Plant Pathology Laboratory for further diagnosis. Symptoms of
samples were described and all observations were recorded.
Identification of Isolated Bacteria
Isolation of associated bacteria. Nutrient Glucose Agar (NGA) was used for
isolation of the bacterium. Thin sections were cut from the advancing portions of infected
leaves and mounted on a drop of sterile distilled water on a slide to induce bacterial ooze.
Appearance of the ooze was confirmed under 40x magnification of an ordinary light
microscope. The bacterium was allowed to ooze out of the tissues for five minutes. After
which, the tissues were removed from the slide and a flame-sterilized wire loop was used
to get a sample of bacterial suspension for streaking onto previously prepared NGA
plates. Plates thus streaked were incubated at 27-30oC for four days. Three well-
separated, isolated single colonies were transferred into NGA slants and stocks cultures
were stored at 50C.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
9
Identification of isolated bacteria. The following tests were performed to
determine the genera of the isolated bacteria.
1. Growth on various media [Appendix 1]
a. Yeast Extract Dextrose CACO3 Agar (YDCA)
b. King’s Medium B Agar (KMBA)
c. Nutrient Glucose Agar (NGA)
d. Sucrose Peptone Agar (SPA)
e. Xanthomonas Isolation Medium (XIM)
f. Casimino Peptone Glucose Agar (CPGA)
g. Potato Dextrose Peptone Agar (PDPA)
2. Gram Staining
Smears were prepared from bacterial ooze obtained from symptomatic leaves.
Cut sections of about 2 to 3 mm were placed in a clean glass slide with a drop of
water then removed after 3 to 5 minutes using a teasing needle. The resulting
smear was then air-dried prior to the standard staining procedure.
3. Other tests. The procedures for the following tests are as outlined in Schaad
(1998).
a. Growth and O/F metabolism. Ten ml of Hugh and Leifson Agar (basal
medium) was poured into test tubes (13 cm diameter). For each
replication, the bacterial isolate were stabbed into two test tubes of basal
medium after which one test tube was covered using water agar and the
other was left uncovered. The set-up was incubated at 300C for 24 to 48
hours.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
10
b. Gelatin Liquefaction tests. Ten ml of gelatin medium was dispensed in
13-cm test tubes. Bacteria were stabbed into the medium and inoculated
tubes were incubated at 20 to 270C for 21 days. After 3,7,14 and 21 days,
the test tubes were refrigerated for 30 minutes.
c. Motility test. A loopful of the bacterial suspension taken from the
freshly cut disease lesion was placed on the underside of a cover slip,
which was then inverted over a depression slide. The slide was viewed
under the microscope at 400x magnification
d. Starch hydrolysis test. Bacterium was streaked onto starch agar and the
plates were incubated for 2-7 days. The plates were then flooded with
iodine solution and any clearing zone was observed.
Determining the Pathogenicity of the Isolates
Preparation of Test Plants
Lettuce seedlings, about two weeks old, were obtained and kept in the greenhouse
for pathogenicity test.
Inoculum Preparation and Inoculation
The bacterial suspension was prepared and standardized at 1 x 106 cfu/ml using
the McFarland’s Turbidity Standard. Three inoculation techniques were tested following
the procedure of Dayao (2004), as follows:
1. Spraying- Using a low-pressure sprayer, the inoculum was sprayed onto the leaf
surface until run-off.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
11
2. Injection- Bacterial suspension was injected into the intercellular spaces at the
undersurface of the leaf.
3. Pricking- The leaves was pricked using a sterilize needle and sprayed with the
bacterial suspension.
The inoculated plants were covered with a plastic bag and incubated overnight in
the laboratory after which the plastic bags were removed and the inoculated plants were
brought to the greenhouse. Symptom development was observed and was compared to
the initial findings.
Re-isolation of Causal Organism
Lesions from artificially inoculated plants were used as sources of bacterial
suspension for re-isolation onto Nutrient Glucose Agar (NGA). Isolates thus obtained
were compared to the original isolates.
Testing the Antibiotic Sensitivity of the Isolates
One ml of standardized bacterial suspension of 1 x 106 cfu/ml was added to 5 ml
water agar and was poured onto previously prepared NGA plates. The test antibiotics
were prepared at different test concentrations (50, 75, 100, 200, 300, 400, 500 and 600
ppm).
Sterilized filter paper discs (20 mm diameter) were dipped into the prepared
antibiotics and then plated at the center of the medium using sterilized forceps. Plates
thus prepared were sealed and incubated for 48 hours at 28°C.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
12
Data Gathered
1.Symptoms of the diseased plants
2. Cultural, morphological and physiological characteristics of the isolates in various
media. Specifically the size, shape, color, elevation, margins, and production of pigment
in the media.
3. Pathogenicity test. The symptoms produced by the inoculated bacterium compared to
the original symptoms observed.
4. Inhibition zones (mm). Inhibition zones around the antibiotic disks will be measured.
5. Photodocumentation. Photographs will be taken of test results and experimental set-up
and will be saved as jpeg files.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
RESULTS AND DISCUSSION
Collection of Sample and Description of
Collection Sites
Collecting of bacterial leaf spot of lettuce was done in the BSU experimental
areas: namely, at the Swamp Area, Balili experimental area, and Pomology. Lettuce
leaves were collected on May 2006 and were well-formed and firm. The lesions were
scattered on the entire surface of the leaves.
Areas from where specimens collected have a flat elevation and it is commonly
used to plant vegetables crops than that of the flowers. Almost square meters of the field
are for hired. Crop rotation is not employed as seen that the farmers are not strictly
monitored on what type of crops they like to plant. Irrigation are through canals, stock
waters in drums.
Disease Symptoms
Lettuce leaves collected have brown lesions that are translucent, irregular in shape
and water-soaked. Lesions later may turn black. The first symptoms appear at the leaf
margin and consist of water-soaked lesions that become necrotic and confluent (Figure
1).
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
14
Figure 1. Symptoms of infected lettuce leaves in the field
Cultural Characteristics of the
Pathogen on Various Media
Various standard and differential media were used to grow the bacterium. A
loopful of bacterial ooze from the lettuce leaves was streaked onto NGA, XIM, SPA,
CPGA, KMBA, YDCA, and PDPA and incubated for 4- 5 days at 27-300C.
Colonies were measured and ranged from approximately from <1mm to 4mm in
diameter (Table 2). Colonies are circular and appear shiny and mucoidal. Under light, the
colonies appear translucent and are mostly from light to dark yellow (Figure 2). When
plated on YDCA and XIM, the bacterium produces whitish to cream colonies. On CPGA,
it produces red colonies because of the TTZ in the media.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
15
Table 1. Cultural characteristics of Xanthomonas in plate growth
MEDIA
SIZE CONFIGURATION DENSITY ELEVATIION COLOR
CPGA
< 1mm Circular
Opaque Mucoid Red
PDPA
3-4 mm Circular Opaque
Mucoid Cream
SPA
4-5 mm Circular Opaque
Mucoid Yellow
YDCA
2-4 mm Circular Opaque
Mucoid Yellow
XIM
< 1 mm Circular Translucent Mucoid White
KMBA
2-4 mm Circular Translucent Mucoid Orange
NGA
3-4 mm Circular Opaque Mucoid Yellow
NGA
Figure 2. Cultural characteristics of the bacterium on various standard and differential
media
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
16
Morphological Characteristics of
the Pathogen
Gram Stain Reaction. Bacterial ooze from the disease specimen (Figure 3a) was
preferred for staining rather than organisms cultured in a solid medium. Those coming
from a solid medium produce a thick dense surface growth and are not amenable to direct
transfer to the glass slide (Cappuccino 2002).
The bacterium was stained pink, which means that the alcohol decolorized the
Gram stain (Figure 3b). The bacterium is therefore confirmed to be Gram negative
(Frobisher, 1957). The rod-shaped bacteria are less short and cylindrical. These
characteristics confirm to those of Xanthomonas as cited by Agrios (2002).
Ooze coming
out from the
cut lesion
Figure 3a. Bacterial ooze from the sectioned lettuce leaves (400x)
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
17
Figure 3b. Gram-stained bacterial cells (1000x)
Motility Test A loopful of bacteria were suspended in the center of a clear thin
cover slip and inverted to the depression slide. The transparent bacterial cells were
motile. Motile bacteria can only move in fluids but it is extremely difficult to steady them
since lack of color prevents details from showing clearly. The observation of bacteria in
hanging drop preparations, therefore, yields limited though valuable information
(Frobisher, 1957).
Physiological Characteristics
of the Pathogen
Growth and O/F Metabolism. Pure culture of Xanthomonas campestris pv. vitians
was stabbed in the basal medium (Hugh and Leifson Agar). One tube was covered with
water agar to prevent the entry of air and the other treatment remained uncovered.
Observations were done after 24 to 48 hours at 30 0C. In comparison with the original
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
18
Figure 4. Results of the O/F Test on Hugh and Leifson Agar
medium (control), the color in the covered and uncovered tubes did not change
even after 48 hours of incubation. This confirms the description by Robinson (2003) that
X. c. pv. vitians that is never fermentative and oxidase negative.
Gelatin Liquefaction Test. Gelatin is hydrolyzed by quite a few organisms and
liquefies at room temperature (Benson, 1998). The bacterium was not able to liquefy the
gelatin even after 3, 7, 14 and 21 days of incubation (Figure 4). Therefore, it can be
deduced that the bacteria was not able to produce gelatinase and thus could not hydrolyze
the gelatin. This inability to liquefy gelatin is a characteristic of Xanthomonas species.
Starch Hydrolysis Test. The bacterium was streaked onto starch agar medium and
incubated for 2 to 7 days. The iodine solution was poured over the growth after the
incubation period. Starch in the presence of iodine will impart a blue-black color to the
medium, indicating the absence of starch-splitting enzymes and representing a negative
result. If the starch has been hydrolyzed, a clear zone of hydrolysis will surround the
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
19
growth of the organism. This is a positive result (Cappuccino, 2002). If the area
immediately adjacent to the growth is clear; amylase has been produced (Benson, 1998).
Stabbed
bacterium
Figure 5. Results of the Gelatin Liquefaction Test on Gelatin Agar (gulaman: green-
Flavored and white- unflavored)
Clearing zone
Figure 6. Results of the Starch Hydrolysis Test on starch medium (7 days after
incubation)
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
20
If the starch is undegraded, a blue-black color results when iodine is allowed to
react with it. This is because potassium iodide molecules readily occupy a space within
the helical coiling of the amylase component of starch. This results in a blue-black
complex (Raymundo et al., 1991).
Pathogenicity Test
Spraying until run-off of the bacterial suspension effected the symptoms most
closely approximating the original specimens. This shows the passage of the pathogen
through the hydathodes resulting in initial yellowing at the edges of the infected leaf.
Injection of bacterial suspension into cellular spaces of the leaf using an insulin
syringe led to formation of translucent spots. These symptoms are also observed in the
field in advanced stages of the disease or when there is a lot of moisture. Pricking and
spraying resulted in chlorosis and collapse of the leaf (Figure 7).
Re-isolation of the Bacterium
From Artificially-Inoculated
Lettuce Leaves
The bacterium was re-isolated from the artificially inoculated lettuce leaves and
had the same appearance and characteristics on NGA medium as the original isolate
(Figure 8).
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
21
A B
C D
Figure 7. Artificially inoculated lettuce seedlings and its symptoms (A. Uninoculated
B. Spraying C. Injection D. Pricking and Spraying)
PRICKING and
SPRAYING
SPRAYING
INJECTION
Figure 8. Bacteria isolated from the artificially inoculated lettuce (NGA, 4 days)
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
22
Antibiotic Sensitivity Testing
of the Pathogen
The bacterium was sensitive to both streptomycin and chlortetracycline (Table 3).
This means that the bacterium has not been exposed yet to these antibiotics and thus has
not been able to develop resistance to the chemical. Expectedly, cephalexin hydrate and
cycloheximide were not effective against the bacterium (Figure 9). Cephalexin is highly
effective against Gram-positive bacteria but performs poorly against the Gram negatives.
On the other hand, cycloheximide was formulated for eukaryotic microorganisms
(eMedicineHealth.com, 2005).
Streptomycin comes from Streptomyces griseus and is one among the
aminoglycoside group of antibiotics. It damages bacterial cell membranes by inhibiting
protein synthesis. Specifically, it binds to the 30S ribosome and changes its shape thus
causing a misreading of messenger RNA information (Wikipedia 2007).
Chlortetracycline hydrate, also known as aureomycin, comes from S.
aureofaciens. It is a broad-spectrum bacteriostatic agent that inhibits bacterial protein
synthesis. Specifically, it blocks the attachment of the transfer RNA-amino acid to the
ribosome. More precisely, it inhibits the codon-anticodon interaction (Wikipedia 2007).
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
23
Table 2. The inhibition zones affected by various antibiotics and their concentrations on
the growth of Xanthomonas campestris pv vitians
CONCENTRATION
MEAN INHIBITION
ANTIBIOTICS
(ppm)
ZONE (mm)
Streptomycin sulfate
50
43.00
75
39.20
100
41.00
200
40.00
300
39.30
400
39.80
500
41.80
600
43.30
Chlortetracycline 50
49.50
75
51.00
100
52.00
200
52.50
300
53.20
400
58.00
500
57.50
600
58.50
Cephalexin hydrate
50
23.00
75
27.20
100
30.30
200
26.30
300
26.50
400
34.50
500
26.20
600
28.00
Cycloheximide 50
35.00
75
27.20
100
22.80
200
20.80
300
21.50
400
21.30
500
20.30
600
20.30
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
24
ANTIBIOTIC Streptomycin Chlortetracycline Cephalexin
Cycloheximide
Sulfate
hydrate
CONCENTRATION
(ppm)
50
75
100
200
300
400
500
600
Figure 9. Inhibition zones effected by various antibiotics and their concentrations on the
growth of Xanthomonas campestris pv vitians
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary
The isolated bacterium from symptomatic lettuce leaves exhibited the
characteristics of Xanthomonas campestris pv. vitians. It produces circular, fried egg-like
and mucoid colonies with yellow pigment. Cells are rod-shaped and short. It is oxidase
negative and never fermentative. It does not produce the enzyme gelatinase. Artificially
inoculated 2 week-old lettuce seedlings show the symptoms of the disease observed in
the field. The bacterium was sensitive to both streptomycin and chlortetracycline
hydrochloride but was not affected by cycloheximide and cephalexin.
Conclusion
The leaf spot observed in symptomatic lettuce is caused by Xanthomonas
campestris pv. vitians. The bacterium is sensitive to the standard antibiotics and thus,
may not have been exposed to antibiotics. This may indicate that the pathogen is a new
pathogen, i.e., newly introduced into the area. Support for this conclusion is the fact that
the disease is not yet recorded in the Disease Index (Tangonan, 1999).
Recommendations
Limitations of the study needs firther investigate the fpllowing:
1. pathogenicity test of the bacterium to other crucifer cultivars and other
crops likes tomato and bell pepper which have been reported to be affected
by the same bacterium;
2. development of integrated disease management measures;
3. survey to determine spread and distribution of the disease in lettuce-
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
26
growing areas in Benguet; and
4. possible determination of transmission and spread of the disease as it has
been reported abroad to be both soilborne and spread via infected seeds.
Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
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Etiology of the Bacterial Leaf Spot of Lettuce in
La Trinidad, Benguet: A First Report / Ilene C. Cuadra. 2007
APPENDICES
APPENDIX TABLE 1. Computation of the antibiotic concentration (ppm)
ANTIBIOTICS
CONCENTRATION
(ppm)
SS CPH CY CH
50
52.63
500.00
53.19
62.50
75
78.95
750.00
79.79
93.75
100
105.26
1000
106.38
125.00
200
210.53
2000
212.77
250.00
300
315.79
3000
319.15
375.00
400
380.00
4000
425.53
500.00
500
526.32
5000
531.91
625.00
600
631.58
6000
638.30
750.00
* 100ml preparation only divided by the amount by 10 ml.
Streptomycin sulfate (SS)
– 95%
Cephalexin hydrate (CPH)
– 100% (assumed)
Cycloheximide (CY)
– 94%
Chlortetracycline/ Aureomycin (CH) – 80%
31
APPENDIX TABLE 2. Measurement of the inhibition zones (mm)
REPLICATION
TREATMENTS
TOTAL
MEAN
I
II
III
SDW 20.0
20.5
20
60.5
20.2
Streptomycin sulfate
75 ppm
39.5
39.5
38.5
117.5
39.2
100 ppm
43.5
40.5
39
123
41.0
200 ppm
42.0
40.5
37.5
120
40.0
300 ppm
37.5
39.5
41
118
39.3
400 ppm
41.0
40
38.5
119.5
39.8
500 ppm
40.5
40.5
44.5
125.5
41.8
600 ppm
45.0
41
44
130
43.3
Chlortetracycline
50 ppm
55
43
50.5
148.5
49.5
75 ppm
54.5
52
46.5
153
51.0
100 ppm
54
51
51
156
52.0
200 ppm
54.5
51
52
157.5
52.5
300 ppm
54
51.5
54
159.5
53.2
400 ppm
54.5
62.5
57
174
58.0
500 ppm
56.5
60
56
172.5
57.5
600 ppm
56
56.5
63
175.5
58.5
Cephalexin hydrate
50 ppm
22
23.5
23.5
69
23.0
75 ppm
29.5
24
28
81.5
27.2
100 ppm
27.5
32
31.5
91
30.3
200 ppm
26.5
25.5
27
79
26.3
300 ppm
25.5
27
27
79.5
26.5
400 ppm
35.5
36
32
103.5
34.5
500 ppm
27
26
25.5
78.5
26.2
600 ppm
29.5
25
29.5
84
28.0
Cycloheximide
50 ppm
30.5
35
39.5
105
35.0
75 ppm
25
27
29.5
81.5
27.2
100 ppm
22
25
20
67
22.3
200 ppm
21
21
20
62
20.7
300 ppm
20
23.5
21
64.5
21.5
400 ppm
21
22
21
64
21.3
500 ppm
20
20
21
61
20.3
600 ppm
21
20
20
61
20.3
32
APPENDIX TABLE 3. Effect of different antibiotics
ANTIBIOTIC MEAN
Streptomycin sulfate
40.88 b
Chlortetracycline 54.03
a
Cephalexin hydrate
27.75 c
Cycloheximide 23.64
d
* Means with the same letters are not significantly different at 5% DMRT
APPENDIX TABLE 4. Effect of concentration of antibiotic
CONCENTRATION MEAN
50 ppm
32.933
75 ppm
33.167
100 ppm
31.933
200 pmm
31.133
300 ppm
32.133
400 ppm
34.767
500 ppm
33.200
600 ppm
34.133
APPENDIX TABLE 5. Interaction between antibiotic and X.c.pv. vitians
ANTIBIOTIC/
CONCENTRATION (ppm)
CONCENTRATION 50 75 100 200 300 400 500 600
Streptomycin
sulfate 43.0c 39.2c 41.0c 40.0c 39.3c 39.8c 41.8c 43.3c
Chlortetracycline 49.5b 51.0b 52.0b 52.5b
53.2b
58.0a
57.5a 58.5a
Cephalexin hydrate
23.0fgh 27.2e 30.3e
26.3ef 26.5ef 34.5d 26.2efg 28.0e
Cycloheximide 35.0d
27.2e
22.8gh 20.7h
21.5h
21.3h
20.3h
20.3h
33
APPENDIX TABLE 6. Analysis of variance
SOURCE OF DEGREES
SUM OF
MEAN
F VALUE
COMPUTED
VARIATION OF
SQUARES
SQUARES
F
FREEDOM
0.05 0.01
Factor A
4
18,829.846
4,707.461
926.4377** 2.48 3.56
Factor B
7
103.967
14.852
2.9230 ns
AB 28 994.887
35.532
6.9927**
Error 80 406.500
5.081
TOTAL 119
Ns
–
not
significant
Coefficient of variation = 6.77%
** -
APPENDIX A
APPENDIX 1. Standard and Differential Media
Yeast Extract Dextrose Agar- CaCo 3(YDCA)
Component
Amount
Yeast
10.0
g
Dextrose
(Glucose)
20.0
g
Calcium carbonate, USP light powder
20.0 g
Agar
15.0
g
Distilled Water
1000 ml
Prepare 10 g of yeast, 20 g of dextrose (glucose), and 20 g of calcium carbonate, USP
light powder and 15 g of agar. Dissolve in 1 liter of distilled water except dextrose.
34
Prepare it separately into the flask at 100 ml distilled water. Sterilize in autoclave at 15
psi for 15-20 mins. After sterilizing, mix the dextrose in a separate flask with yeast,
CaCo3 and agar. Mix thoroughly before pouring into the petri plates.
King Medium B Agar (KMBA)
Component
Amount
Protease
peptone/
peptone
20.0
g
Potassium phosphate (K2HPO4)
1.5
g
Magnesium sulfate (MgSO4.7H20)
1.5
g
Agar
15.0
g
Glycerol 15.0
ml
Dispensed individual 250 ml erlenmeyer flask and sterilized in autoclave for 30 minutes
at 15 psi before plating in sterilized petri plates.
Nutrient Glucose Agar (NGA)
Component
Amount
Beef
Extract
3.0
g
Peptone
5.0
g
Glucose 2.5
g
Agar
15.0–20.0
g
Distilled water
1000 ml
Sucrose Peptone Agar (SPA)
Component
Amount
Sucrose 20.0
g
35
Peptone
5.0
g
K2HPO4
0.5
g
MgSO47H2O
0.25
g
Agar
15.0 g
Adjust pH to 7.2
40 % NaOH
40 % 100m distilled H2O
Xanthomonas Isolation Medium (XIM)
Component
Amount
Cellobiose
10.0
g
Sodium phosphate (NaH2PO4)
0.5 g
MgSO4.
7H20
0.3
g
K2HPO4
3.0
g
Ammonium
Chloride
(NH4Cl)
1.0
g
Agar
15.0
g
Potato Dextrose Peptone Agar (PDPA)
Component
Amount
Potato
Dextrose
Agar
8.0
g
Peptone
5.0
g
Distilled water
1000 ml
36
Casamino Acid Peptone Glucose Agar (CPGA)
Component
Amount
Peptone 2.0
g
Casamino
acid
0.2
g
Glucose
1.0
g
Agar
3.0
g
Distilled water
140-200 ml
TZC
should
be
1g/li
or
1ml/100ml
CPG
APPENDIX 2. Basal Medium for Anaerobic Growth
Hugh and Leifson Agar
Component
Amount
Peptone
2.0
g
NaCl
5.0
g
KH2PO4
0.3
g
Agar
3.0
g
Bromthymol
blue
3.0
ml
(1%
aqueous
solution)
Distilled water
1000 ml
Dissolve ingredients and adjust pH to 7.1. Add 10ml of basal medium to 13 cm diameter
test tubes and sterilize at 121º C for 20 minutes. Prepare a 10% aqueous solution of
glucose and sterilize by filtration. Add 0.5 ml of sterile glucose aseptically to each tube of
basal medium. Inoculate two tubes with the organism to be tested. Cover one tube with a
37
layer of water agar or paraffin to a depth of 5mm. A color change from blue to yellow in
both tubes is recorded as positive for anaerobic growth.
APPENDIX 3. Procedure of McFarland’s Turbidity Standard
The 0.5 McFarland may be prepared by adding 0.5 ml of 1.175% (wt/vol) barium
chloride dehydrate (BaCl2.2H2O) solution to 99.5 ml of 1% (vol/vol) sulfuric acid. The
turbidity standard is then aliquoted into test tubes identical to those used to prepare the
inoculum suspension. Seal the McFarland standard tubes with wax, Parafilm, or some
other means to prevent evaporation. McFarland standards may be stored for up to 6
months in the dark at room temperature (22ºC to 25ºC). Discard after 6 months or sooner
if any volume is lost. Before each use, shake well, mixing the fine white precipitate of
barium sulfate in the tube. The accuracy of the density of a prepared McFarland standard
should be checked using a spectrophotometer with a 1-cm light path; for the 0.5
McFarland standards, the absorbance at a wavelength of 625 nm should be 0.08 to 0.1.
Document Outline
- Etiology of the Bacterial Leaf Spot of
Lettuce in La Trinidad, Benguet: A First Report
- BIBLIOGRAPHY
- ABSTRACT
- TABLE OF CONTENTS
- INTRODUCTION
- REVIEW OF LITERATURE
- The Host Plant
- The Pathogen and Symptoms Caused
- Distribution
- Epidemiology
- MATERIALS AND METHODS
- Verification of the Pathogen Identity
- Determining the Pathogenicity of the Isolates
- RESULTS AND DISCUSSION
- Collection of Sample and Description ofCollection Sites
- Disease Symptoms
- Cultural Characteristics of thePathogen on Various Media
- Morphological Characteristics ofthe Pathogen
- Physiological Characteristicsof the Pathogen
- Pathogenicity Test
- Re-isolation of the BacteriumFrom Artificially-InoculatedLettuce Leaves
- Antibiotic Sensitivity Testingof the Pathogen
- SUMMARY, CONCLUSION AND RECOMMENDATIONS
- Summary
- Conclusion
- Recommendations
- LITERATURE CITED
- APPENDICES