BIBLIOGRAPHY

TAGWAY, KAREN S. MARCH 2006. Antibiotic Resistance of Xanthomonas
axonopodis pv. Diffenbachiae (Anthurium Bacterial Blight) in La Trinidad, Benguet and
Baguio City. Benguet State University, La Trinidad, Benguet.

Adviser: Lily Ann D. Lando, PhD
ABSTRACT
Fifteen
Xad isolates were obtained from 10 anthurium cultivars grown at 7
locations. The Xad isolates differed in ability to degrade starch.

In general, most of the Xad isolates were resistant only to 50-200 ppm
concentration of penicillin and streptomycin. The isolates were susceptible to
concentrations from 500 to 2000 ppm. All the isolates were resistant to the
recommended rates of Nordox (Cu2O) and Funguran (CuOH).

The management measures most frequently employed by farmers were: twice
weekly uprooting/removing and disposing of infected plants/leaves and weekly
application of chemicals.

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TABLE OF CONTENTS


Page
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
i
Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ii
INTRODUCTION……………………………………………………………... 1
REVIEW OF LITERATURE
The Disease, Its Cause and Its Symptoms . . . . . . . . . . . . . . . . . . . . . . . .
3
The Site of Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Epidemiology of the Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
Resistance to Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
RESULTS AND DISCUSSION
Collection, Isolation and Maintenance
of Xad Isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11
Sensitivity of Different Isolates of
Xad to Streptomycin, Penicillin and
Copper-based Fungicide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14
Test for Starch Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
Determination of Grower’s Management
Measures Against Bacterial Blight . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
SUMMARY, CONCLUSION AND
RECOMMENDATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
23
LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
27

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1

INTRODUCTION


Anthurium (Anthurium andraeanun Linden ex Andre) is one of the most widely
grown cutflower crop in Benguet. It has gained popularity in the Philippines as well
because of its durability and attractiveness. According to DAMAS (2002), the top
producers of anthurium in the Philippines are CAR (23%), Southern Mindanao (22%)
and Southern Tagalog (17%).
One of the major constraints to anthurium production in Benguet is the bacterial
blight disease caused by Xanthomonas axonopodis pv dieffenbachiae (McCulloch and
Pirone, 1939) Vauterin et.al. 1995. Formerly known as Xanthomonas campestris pv
dieffenbachiae (EPPO, 2005), the pathogen causes the same blight of other ornamental
and food crops in the family Araceae including Spathiphyllum, Aglaonema, and
Xanthosoma (Alvarez, 2000). This family also includes Diffenbachia, Philodendron,
Caladium, and Zantedeschia sp. (Calla Lily) - all of which are grown in Benguet.
The disease has taken a heavy toll on the anthurium population worldwide. This
disease infects the leaves, stems, shoots, and roots leading to the death of the whole plant
(Rosario, 1988). The European Plant Protection Organization (EPPO, 2005) identifies the
Philippines as the only Asian country where the disease can be found.
According to an FRLD study (1993), Benguet anthurium growers are
predominantly monoculturists. This practice increases the probability of severe blight
infection within their plantings. Infected planting materials can transmit the pathogen and
the most common planting materials used in Benguet are suckers. Furthermore, the
disease can spread to uncontaminated areas through splashing rain, irrigation water,
infected tools, wet clothing and infected soil on footwear (WebMan, 2000).
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Recommended measures to control the disease include the use of antibiotics
(streptomycin and oxytetracycline) and the selection for resistant varieties. Currently,
there are no identified resistant varieties for the Philippines.
Application of antibiotics in the agroecosystem could result in the build up and
persistence of resistance genes in the environment (McManus, 2004). In fact, Henny et al.
(2001) reported earlier that some Xad isolates have developed resistance to the
recommended antibiotics.
It is therefore necessary to determine whether such resistant isolates can be found
within the anthurium plantations in the locality. This information will have an impact on
the development of integrated management measures against bacterial blight of
anthurium to prevent further evolution and dissemination of resistant strains of the
bacterium and reduce the spread of the disease.
This study aimed to isolate and characterize Xad from symptomatic anthurium,
determine resistance of Xad isolates to various levels of streptomycin and penicillin, and
determine the management measures employed by anthurium growers against bacterial
blight.

Samples of symptomatic plants were collected from major planting sites in La
Trinidad and Baguio City. Samples were then processed at the laboratory of the
Department of Plant Pathology, College of Agriculture, Benguet State University. The
study was conducted from August 2005 to March 2006.

Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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REVIEW OF LITERATURE


The Disease, Its Cause and Symptoms
Bacterial blight of anthurium is a vascular disease. In the Philippines, the disease
was first reported by Divinagracia and Enrique (1984). It is caused by Xanthomonas
axonopodis pv diffenbachiae (McCulloch & Pirone, 1939) Vauterin et al. 1995 formerly
known as Xanthomonas campestris pv dieffenbachiae (McCulloch & Pirone, 1939) Dye.
The bacteria live in the xylem of the host plant and from there induce symptoms of
wilting and necrosis. After xylem and marginal necrosis begins, the pathogens are able to
breakdown cell walls and cytoplasm and obtain nutrition from sources other than the
xylem sap.

Symptoms include small scattered angular water-soaked spots near the margin
most visible from the undersides of the leaf and a faint chlorosis when viewed from the
upper side (McCallow and Leonhardt, 1983).

In the advanced stage, as more tissues are killed, the spots become circular to
irregular brown areas, surrounded by a light yellow border. The necrotic center becomes
rough, dry and sometimes curled. The leaves turn generally yellow before defoliation. If
the infected leaves are not removed, the bacterium will go down the petiole into the stem
manifesting itself in the systematic stage. The systemic or vascular infections first appear
as general yellowing of the entire leaf blades of older leaves. The petioles of these leaves
have infected vascular bundles showing that the bacterium has been moving up from the
stem through the petiole and the leaf. Eventually, other parts of the plant are infected and
the entire plant is killed (McCallow and Leonhardt, 1983). .

Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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The Site of Infection
The principal infection sites of Xad are the hydathodes of the anthurium leaf.
These hydathodes are located on the (lower surface) outer margin of the leaf and occupy
only a small portion of the total leaf surface. Two reasons can be given for bacterial
infection through hydathodes. First, the bacterial cells have one flagellum and are motile.
Hydathodes have a continuous water pathway from the outside of the leaf to the xylem.
Stomata on the other hand, may have water near their openings but free water is not
usually present in the intercellular spaces. Thus, bacteria can swim into stomata, but then
are not able to swim further (Imamura and Higaki, 1981).
Second, is that guttation liquid contains nutrients such as amino compounds.
Virulent strains of Xanthomonas campestries pv. oryzae on rice have been shown to
multiply readily on the nutrients in the guttation liquid of the hydathodes. This process is
called chemotaxis and was first demonstrated by Macnab (1978).

Epidemiology of the Disease

Generally, the bacteria originate from previously infected plants and they are
carried by tiny water droplets (aerosols) through dew that drips from one plant to another
or by rain splash to healthy plants (Alvarez,1990). Likewise, Edmund (1978) further
stated that the disease can be transferred by cutting tools or contaminated clothing or
hands of workers.

In the case of rice (Pavgi et al) as cited by Singh, (1978) Xanthomonas campestris
pv oryzae can be carried over by infected seeds from previous crops. It is possible that
such seeds are initiated into the nursery where seedlings catch the infection and carry it to
the field. According Nishijima (1990), this pathogen survives principally on infected
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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plant debris and only as a free living organism in the soil. Also, it can survive for a long
period on plant tissues without causing symptoms; this is referred to as the latent period.
In anthurium there is evidence that the latent period maybe as long as ten months.
Thus, Sakai (1990) stated that the plant may look healthy in between but when subjected
and placed under temperature, nutritional or moisture stress, they may suddenly manifest
severe symptoms of the disease. Moreover, Harfacre (1979) citing the work of Fang, et al
(1956) added that temperature has something to do with the occurrence and how it is
manifested to a certain degree. Farms at higher elevation have been observed with lower
incidence of blight. Although plants produce flowers more slowly at cooler temperature
and may compromise with yield this may go a long way in controlling the disease
(Harfacre, 1979).
In relation to this, topography, climate and cultural practices affect the
development of the disease. In Japan, Singh (1978) observed that poorly drained field
along rivers or lakes and mountainous basin with excess of rainfall and humidity, floods
and typhoons are conducive for the disease development.

The soil pH on the other hand, also largely influences the growth of Xad as
studied by Sakai (1990). He found out that low pH severely lowers the growth of the
bacteria; at pH 4-6 the disease is stagnant and at pH 4-5 and below growth occurred. In
contrast, mild acidity to mild neutral (6 - 7) enhanced and favored the growth of the
pathogen (McCallow and Leonhardt, 1983).



Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Control

Antibiotics were used in Hawaii and found to be effective (Alvarez, 1990). The
method involves removing and destroying all systematically infected plants and blighted
leaves and spraying with the antibiotic streptomycin sulfate at 200 ppm. Furthermore, the
antibiotic was effective in controlling anthurium blight when sprayed after sanitizing.
However, these antibiotics could not be used in a normal maintenance/preventive
operation or used extendedly because of the resistance problem and their possible
detrimental effects on beneficial bacteria. Sakai (1990) cited though that proper cultural
practice and control techniques applied can reduced the level of disease to a minimum
and easily manage their impact on production.

Long-term disease management measures can also be done for the control of the
said disease such as sanitation, biological control, cultural practices and developing
anthurium cultivars/hybrids that may be resistant or tolerant to the disease. Sanitation,
combined with the use of resistant or tolerant varieties is the most effective method of
combating the disease (WebMan, 2000).

Resistance to Antibiotics

Certain bacteria are naturally resistant to a specific antibiotic, and this
characteristic property limits a drug’s spectrum of activity: this is referred to as intrinsic
resistance. As antibiotics became more frequently used, a new problem emerged. Specific
bacteria previously considered sensitive to a drug had become resistant and were
responsible for treatment failures: this is referred to us acquired resistance. Such was the
case for Staphylococcus aureus, which was originally sensitive to penicillin G; most
strains are now resistant. The mechanism of resistance in this case was due to the
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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production of an enzyme by the bacteria, called a penicillinase that was able to destroy
the antibiotic (Nishijima, WT., 1990).
Another particularly worrisome form of acquired resistance was first described in
Japan by Teruhiko Akiba, a Japanese microbiologist, in 1959. It resulted in bacteria
becoming simultaneously resistant to several different antibiotics. Of special concern to
scientists was the observation that this type of resistance could be transferred from one
bacterium to another. Furthermore, this transmissible resistance to multiple antibiotics
could be passed between completely unrelated types of bacteria. The mechanism by
which this is achieved is now understood to involve the transfer between bacteria of small
pieces of DNA called plasmids that carry all the genetic information necessary to make
the bacteria resistant (Macnab, R.M, 1978).
Generally this type of resistance has resulted from an indiscriminate use of
antibiotics. When this phenomenon was first discovered, there was serious concern that it
would lead to a loss of utility for antibiotics (Macnab, R. M., 1978).

Although antibiotic use on plants is minor relative to total use, MacManus (2004)
said that application of antibiotics in the agroecosystem presents unique circumstances
that could impact the build up and persistence of resistance genes in the environment.
She further points out that no apparent human health issues have arisen after four decades
of antibiotic use on plants in the farm setting. However, despite the controlled conditions
in clinical settings, medical experts have witnessed the failure of one antibiotic after
another in clinical settings (Immamura, J. & T. Higaki, 1981).


Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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MATERIALS AND METHODS


Collection, Isolation and Maintenance
Of Xad isolates

Leaf samples of anthurium plants infected with bacterial blight was collected
randomly from different nurseries and greenhouses around La Trinidad, Benguet and
Baguio City. The samples were surface-sterilized with 10% sodium hypochlorite for 3-5
minutes and rinsed with sterile distilled water. Sections (2-3 mm) from the margin of
actively growing lesions were cut and placed in 1-2 drops of sterile distilled water on a
flame-sterilized glass slide. The bacterial cells were allowed to ooze from the cut tissues,
and loopfuls from this suspension were streaked onto agar slants.
The tetrazolium chloride agar (TCA) medium was modified by eliminating casein
hydrolysate and reducing the final concentration of triphenyl tetrazolium chloride (TTC)
to 0.001% (Alvarez et al, 1988). The plates were incubated for 2-3 days at room
temperature. Single colonies that appeared on the medium were picked and streaked onto
yeast dextrose calcium carbonate (YDC) agar slants.
Stock culture of the isolates were prepared by using a loopful each of 48-hour old
pure cultures of each isolates and streaked onto YDC agar slants. The seeded slants were
incubated at room temperature, and then stored in the refrigerator after good growth was
obtained. The cultures were revived every 2 weeks on the same medium.

Cultural Characteristics of Xad Isolates


The cultural characteristics of the Xad isolates were noted on the YDCA and on
the modified TCA, earlier mentioned. In addition, cultural characters on nutrient agar
(NA), King’s Medium B Agar (KMBA) and sucrose peptone agar (SPA) were also noted.
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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A loopful of suspension from the working cultures was streaked onto the different media.
Plates were then incubated at 280C for 48 to 72 hours. Colony morphology was then
described.

Sensitivity of Different Isolates of
Xad to Streptomycin and Penicillin

The procedure modified by Dolores (2004) was adopted. The different isolates of
Xad was tested for resistance to streptomycin and penicillin starting at 50 ppm. The
isolates that were found to be resistant at this concentration were further tested to
increasing concentrations of streptomycin sulfate and penicillin (at 200, 500, 1,000 and
2000 ppm). The isolates were also tested with two copper based fungicides: copper
hydroxide (CuOH) and copper oxychloride (CuOCl).
Modified TCA media without 0.001% TTC but with different concentrations of
streptomycin sulfate and penicillin was poured into the plates. One mL of a 48-hour old
pure culture of the isolate was spread on the medium in triplicate plates. Growth of Xad
isolates on different concentrations of streptomycin, penicillin and in copper-based
fungicides was recorded after 3 days of incubation at 28-300C.

Test for Starch Hydrolysis
Loopfuls of 48-hour old cultures of the different isolates of Xad were streaked
over the surface of starch agar plates (2% starch) and incubated at room temperature for 2
days. The surface of the agar plates was then flooded with dilute solution of Gram’s
iodine as test starch hydrolysis.

Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Determination of Growers’ Management
Measures Against Bacterial Blight

The growers’ practices in managing bacterial blight were determined through
interviews. The instrument used in the interview is attached. Information obtained here
was used to validate the findings on the antibiotic sensitivity of the Xad isolates.

Data Gathered

The data gathered are the following:

1.
Cultural characteristics of Xad isolates on modified TCA, YDCA, NA,
KMBA and SPA. The cultural characteristics of the Xad isolates on the various media
were observed. Specifically, the following colony characters were noted: size, shape,
color, elevation, margin, etc.
2.
Sensitivity of isolates to antibiotics and copper-based fungicides. The
sensitivity of the Xad isolates to the different levels of streptomycin sulfate, penicillin and
copper-based fungicides were observed and reaction was noted as susceptible or resistant.
3.
Ability of isolates to hydrolyze starch. The appearance of zones of
clearing was noted around the Xad colonies.
4.
Growers’ management measures against bacterial blight. The management
practice employed by the anthurium growers against bacterial blight was noted.
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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RESULTS AND DISCUSSION


Collection, Isolation and Maintenance of Xad Isolates

Leaf samples of anthurium plants infected with bacterial blight were collected
randomly from different greenhouse around La Trinidad, Benguet and Baguio City. It
was observed that the isolated bacteria from infected leaves grew well on modified TCA
medium with 0.001% TTC. In 1998, Alvarez et.al. reported that the modified TCA is a
good general differential medium which permits a fairly accurate presumptive
identification of Xanthomonas. With 0.001% TTC, the colonies were red to orange at the
center surrounded b a yellowish margin, smooth, almost circular, raised and glistening,
2-3 days after from small to very small colonies.














NA


Modified TCA
Plate 1. Bacterial colonies on NA and modified TCA


There were 15 isolates collected from different location (Table 1). Most of the
source cultivars were grown in greenhouse and in partially open sheds. Ten cultivars
exhibited local symptoms, while 5 showed systemic symptoms.
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Table 1. Sources and host cultivars of isolates collected for the study and the type of
symptoms observed.

Isolate
Source
Type of cultivation
Host cultivars
Type of
#
(open or greenhouse)
symptom
(Local or
systemic)
1 BSU
greenhouse
Greenhouse

Sweetheart Local
orange
2
BPI Baguio
Greenhouse
Baguio white
Local
3 Baguio
Open Kaumana
Local
Orchidarim
4 Baguio
Open Butterfly
white
Local
Orchidarium
5 BSU
Orchidarium
Open
Tulip
Local
6 BSU
greenhouse
Greenhouse
Kaumana
Local
7 Puguis,
La
Greenhouse and half
Ivory white
Systemic
Trinidad
open
8 Puguis,
La
Greenhouse and half
Ozaki Systemic
Trinidad
open
9 Puguis,
La
Greenhouse and half
Kaumana Systemic
Trinidad
open
10 Puguis,
La
Greenhouse and half
Obake Systemic
Trinidad
open
11 Puguis,
La
Greenhouse and half
Sweetheart
Systemic
Trinidad
open
orange
12 BSU
greenhouse Greenhouse
Nitta
Local
13 Puguis,
La
Greenhouse and half
Hawaiian red
Local
Trinidad
open
14 Puguis,
La
Greenhouse Tulip Local
Trinidad
15 Puguis,
La
Greenhouse Nitta Local
Trinidad


Cultural Characteristics of Xad Isolates

The cultural characteristics of the Xad isolates were shown in Table 2. A loopful
of bacterial suspension from the working cultures was streaked on nutrient agar (NA),
King’s medium B Agar (KMBA), Sucrose Peptone Agar (SPA), Yeast Dextrose Calcium
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Table 2. Summary table on the cultural characteristics of isolated bacteria on various media

Media
Basis
Isolate
number


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
NA Colony Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular
shape

Elevation Raised
Raised Raised Raised
Raised
Raised Raised Raised Raised
Raised Raised Raised Raised Raised Raised

Margin Smooth
Smooth
Smooth
Smooth
Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth

Surface Entire
Entire Entire Entire Entire
Entire Entire Entire Entire
Entire Entire Entire Entire Entire Entire

Color
Yellow Yellow
Yellow Yellow
Yellow Yellow
Yellow Yellow
Yellow Yellow
Yellow Yellow

Yellow
Yellow
Yellow
Size 70.7
70.7
70.7
70.7
70.7 70.7 70.7 70.7 70.7 70.7 70.7 70.7 70.7 70.7 70.7
KMBA Colony Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular
shape

Elevation Raised Raised Convex
Convex
Raised Raised Raised Raised Raised Raised Raised Raised Raised Raised Raised

Margin Smooth
Smooth
Smooth
Smooth
Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth

Surface Entire
Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire

Color
Yellow Yellow
Yellow Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Size 64.8
64.8
64.8
64.8
64.8 64.8 64.8 64.8 64.8 64.8 64.8 64.8 64.8 64.8 64.8
SPA Colony Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular
shape

Elevation Raised Raised Raised Convex
Raised Raised Raised Raised Raised Raised Raised Raised Raised Raised Raised

Margin Smooth
Smooth
Smooth
Smooth
Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth

Surface Entire
Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire
Color
Yellow
Yellow

Yellow Yellow
Yellow Light
Light
Yellow Light Yellow yellow Yellow Light Light
yellow
yellow
yellow
yellow
yellow
yellow
Size
70
70
70
70
70 70 70 70 70 70 70 70 70 70 70
YDCA Colony Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular
shape

Elevation Raised Raised Raised Raised
Raised Raised Raised Raised Raised Raised Raised Raised Raised Raised Raised

Margin Smooth
Smooth
Smooth
Smooth
Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth

Surface Entire
Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire

Color
Yellow Yellow
Yellow Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Size 74.1
74.1
74.1
74.1
74.1 74.1 74.1 74.1 74.1 74.1 74.1 74.1 74.1 74.1 74.1
TCA Colony Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular Circular
shape

Elevation Raised Raised Raised Raised
Raised Raised Raised Raised Raised Raised Raised Raised Raised Raised Raised

Margin Smooth
Smooth
Smooth
Smooth
Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth

Surface Entire
Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire Entire
Color
Light
Light
Red Orange
Light Light red
Light
Orange Light
pink
Yellow Light red
Light
orange Red
Light
red
red
red
orange
orange
pink
orange
Size 81.9
81.9
81.9
81.9
81.9 81.9 81.9 81.9 81.9 81.9 81.9 81.9 81.9 81.9 81.9
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial Blight) in La Trinidad, Benguet and Baguio City / Karen S.
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Agar (YDCA), and the modified Tetrazolium Chloride Agar (TCA). Colony and cultural
characteristics of Xad isolates were similar to those of Xanthomonas dieffembachiae as
described by McCulloch & Pirone (1939), Hayward (1978), and Skerman et.al. (1980).

Sensitivity of Different Isolates of Xad to
Streptomycin, Penicillin and Copper-based Fungicide

The different isolates of Xad were tested for resistance to streptomycin and
penicillin starting at 50 ppm, 100 ppm, 200 ppm, 500 ppm, 1000 ppm and 2000 ppm
( Plate 2).

Most of the isolated were resistant to lower concentration of streptomycin and
penicillin as observed at 50 ppm and 100 ppm ( Table 3.1). Except for the tulip variety
from BSU Orchidarium which was found resistant to streptomycin and penicillin.

The isolates were found to be sensitive to higher concentration of streptomycin
and penicillin. It was found also that the isolates were very resistant to Nordox and
Fungaran which were copper-based fungicide.



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1000 ppm

50 ppm


200 ppm


2000 ppm

500 ppm

100 ppm




A


B



2000 ppm

200 ppm



50 ppm
1000 ppm



100 ppm
500 ppm





C


D


SDW

funguran
SDW
funguran




Nordox

Nordox






E


F

Plate 2. Inhibition zones affected by the various chemicals tested
(A-B – Penicillin; C-D – Streptomycin; E-F – Funguran, Nordox
& SDW)

Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Table 3.1a. Antibiotic sensitivity of Xanthomonas axonopodis pv. diffenbachiae isolates
to Streptomycin

Isolate
Concentration of Streptomycin (ppm)

50 100 200 500 1000 2000
1 I I S S S S
2 R R I S S S
3 I I S S S S
4 R R I S S S
5 R R R R R R
6 R I S S S S
7 I S S S S S
8 I I S S S S
9 R I S S S S
10 R I S S S S
11 R I S S S S
12 R R S S S S
13 I S S S S S
14 R I S S S S
15 I S S S S S


R - Resistant
I - Intermediate
S - Susceptible

* Standard Scale:
>11 is R and <15 is S
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Most of the isolates were resistant to 50 ppm and 100 ppm, except for isolates 2,
3, & 5 which reaches to 200 ppm, and they were all susceptible to higher concentration of
penicillin (Table 3.1b)

Table 3.1b. Antibiotic sensitivity of Xanthomonas axonopodis pv diffenbachiae isolates
to Penicillin
Isolate
Concentration of Penicillin (ppm)

50 100 200 500 1000 2000
1 R R R S S S
2 R R R R R S
3 R R R S S S
4 R R S S S S
5 R R R R R R
6 R R S S S S
7 R R S S S S
8 R R S S S S
9 R R S S S S
10 R R S S S S
11 R R S S S S
12 R R S S S S
13 R R S S S S
14 R R S S S S
15 R R S S S S



R - Resistant
S - Susceptible

* Standard Scale:
>14 is R and <15 is S




Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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From Table 3.2a, the source of cultivars have different reactions on the
concentration of streptomycin applied which indicates that there were no resistant
cultivars so far.
Table 3.2a. Source of Cultivar
Cultivars
Concentration of Streptomycin (ppm)

50
100
200
500
1000
2000
Sweetheart Orange
R
R
S
S
S
S
Baguio White
R
R
I
S
S
S
Kaumana
R
R
S
S
S
S
Butterfly White
R
R
I
S
S
S
Tulip
R
R
R
S
S
S
Ivory White
R
S
S
S
S
S
Ozaki
I
I
S
S
S
S
Obake
R
I
S
S
S
S
Nitta
R
I
S
S
S
S
Hawaiian Red
I
S
S
S
S
S



The Baguio White, reach its resistance to 1000 ppm of penicillin, also with tulip
to 200 ppm, most of the isolate were resistant to lower concentrations.
Table 3.2b. Source of Cultivars
Cultivars
Concentration of Penicillin (ppm)
50
100
200
500
1000
2000
Sweetheart
orange R R S S S S
Baguio
white
R R R R R S
Kaumana
R R S S S S
Butterfly
white
R R S S S S
Tulip R
R
R
S
S
S
Ivory
white
R R S S S S
Ozaki
R R S S S S
Obake
R R S S S S
Nitta
R R S S S S
Hawaiian
red
R R S S S S

Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Most of the source location of the isolates were resistant from 50 to 200 ppm
streptomycin and they were susceptible to 500 to 2000 ppm which indicate that there
were no resistance formed on the cultivars on the source location.
Table 3.3a. Source Location
Location Streptomycin
(ppm)
50
100
200
500
1000
2000
BSU
R R R S S S
greenhouse
Floriculture
BSU
R R R S S S
Horticulture
BPI Baguio
R
R
R
S
S
S
Baguio
R R R S S S
Orchidarium
BSU
R R R S S S
Orchidarium
Puguis 1
R
R
R
S
S
S
PUguis 2
R
R
R
S
S
S


Same is true with penicillin, no resistance formed on the concerned locations, they
were resistant only on lower concentration.
Table 3.3b. Source Location
Location Penicillin
(ppm)
50
100
200
500
1000
2000
BSU
R R S S S S
greenhouse
Floriculture
BSU
R R R S S S
Horticulture
BPI Baguio
R
R
R
S
S
S
Baguio
R R R S S S
Orchidarium
BSU
R R R S S S
Orchidarium
Puguis 1
R
R
R
S
S
S
PUguis 2
R
R
R
S
S
S




Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Test for Starch Hydrolysis


The isolates were able to hydrolyze starch. Clear zones were found around or
bordering the streaks of the isolates upon application of diluted Gram’s iodine. Six
isolates were found to react easily upon application of the Gram iodine. They produced
wider clear zones.

Iodine solution (Gram’s) is an indicator of starch. When iodine comes in contact
with a medium containing starch, it turns blue. Starch is hydrolyzed and is no longer
present however, if the medium will have a clear zone next to the growth. Bacteria that
hydrolyzed starch produce amylases that yield molecules of maltose, glucose, and
dextrins (Benson, 1998).

Valencia et al. (2004) observed that starch hydrolyzers grew more rapidly on
starch agar medium than non-starch hydrolyzers. Nevertheless, they were equally
virulent and they were found to be pathogenic on anthurium plants.

Table 4. Starch Hydrolysis by Xanthomonas axonopodis pv diffenbachiae isolates
Isolates
Clearing Zones
Isolates
Clearing Zones
1
+ + +
9
+
2
+ + +
10
+ +
3
+ + +
11
+
4
+ +
12
+
5
+ + +
13
+
6
+ +
14
+
7
+ + +
15
+ + +
8
+



Legends:
+ + + =
wide clearing
+
+

=
moderate

+
=
slight
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Slight

Moderate















Wide

Plate 3. Variations in ability to hydrolyze starch among the isolates









Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Determination of Grower’s Management Measures Against Bacterial Blight


Most of the anthurium growers practiced the removal and disposal of infected
leaves also the addition of organic matter on the plants, most of it were compost of dried
leaves, coconut husk and the use of animal manures especially cow manure (Table 5).

Some of the growers uproot the infected plant but disposed them near the
uninfected plants. They make use also of the suckers of the infected plant. The
greenhouse also is very moist because of the use of rainburst irrigation which favors the
spread of the disease. The disease can not be controlled because of those existing
practices done by farmers at Puguis.

Though they sprayed the plants twice a week with the use of fungicide, the
disease symptoms observed was systemic (Puguis) already though some were localized.

The growers made use also of antibiotics but they just spray when there is
available antibiotic.

Most of the growers were engaged with the use of hose as their irrigation system.


Table 5. Management practices employed by anthurium growers to control bacterial
blight.
Management Practice
Frequency of Application
Removal and disposal of infected leaves
Twice a week (44/45)
Uprooting and disposal of infected
Twice a week (42/45)
plants
Application of chemicals
Once a week (42/45)
Resistant variety
(1/45)
Use of clean planting materials
--
Addition of organic matter
Every month (44/45)
Careful irrigation




Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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SUMMARY, CONCLUSION AND RECOMMENDATIONS

Summary

The study was conducted to test the sensitivity of different Xad isolates to
antibiotics and copper-based fungicide, that were collected from different greenhouses
around La Trinidad, Benguet and Baguio City.

Most of the Xad isolates were resistant to lower concentration and susceptible to
higher concentration of streptomycin and penicillin. Except for the tulip variety from
BSU Orchidarium which was observed to be resistant to the antibiotics.

The 15 Xad isolates differed in ability to degrade starch. The most frequently
employed management measures were: uprooting/removing and disposing of infected
plants/leaves and application of chemicals.

The isolates were found also to be very resistant to copper-based fungicides, the
Nordox and Funguran.

Conclusion

The Xad isolates differ from each other. Most Xad isolates were generally not
resistant to antibiotic concentration from 500 ppm and above. However, presence of
isolates with intermediate reaction to 50-500 ppm concentrations poses a potential for
development of resistance if antibiotic use is continued.

Growers do not associate watering with spread of the disease and still believe that
the disease is caused by a fungus.


Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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Recommendations

Based on the findings of this study, the following were recommended:
1. Thorough survey to cover bigger commercial farms and to identify cultivars
used that may be sources of future infection.

2. Development and implementation of integrated disease management measures
to address the variability in the Xad isolates.
3. Genetic analysis to determine whether the observed differences were due to
genetic differences as well.

4. Familiarization of farmers with symptoms of the Xad infection to allow
differentiation from Ralstonia infection.

Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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25

LITERATURE CITED


ALVAREZ, A. 2000. Progress report for research supported by the American Floral
Endowment. University of Hawaii.

ALVAREZ,AM.1990.Resistance of bacteria to antibiotics used for control of anthurium
blight. In Proceedings of the Second Anthurium Blight Conference, Hawaii
Institute of Tropical Agriculture & Human Resources.pp.11-13.

BENSON, H.J. 1998. Microbiological Applications: Laboratory Manual in General
Microbiology. 7th ed. Boston, Massachusetts: WCB McGraw-Hill. Pp 50-71,
146-178.

DAMAS, 2002. Cutflower Industry Situationer Report. DA- Agribusiness and Marketing
Assistance Service. Posted at http//www.da./agribusiness and
marketing/Cutflower Industry./ retrieved August 10, 2005.

DIVINAGRACIA GG &LM ENRIQUE.1984.Recent outbreaks of two destructive
diseases of ornamental plants in the Philippines: 1: Bacterial blight of
anthurium.Annual Meeting of the Philippine Phytopathological Society, Los
Baños,Laguna (Philippines).21-23 May 1984.

EDMUND. J. B. 1978. Fundamentals of Agriculture. Ist Published Mississipi State
University. McGraw Hill Inc. Pub. Co. pp.106-107.

EUROPEAN PLANT PROTECTION ORGANIZATION, 2005. Xanthomonas
axonopodis pv diffenbachiae: EPPO Quarantine Pest. Data sheets on quarantine
pests. Posted at http://www.eppo.org/quarantine/bacteria/Xanthomonas_
diffenbachiae./retrieved August 10, 2005.

FOUNDATION FOR RESEARCH LINKAGE & DEVELOPMENT. 1993. The
Cutflower Industry. Agri-Link Asia. Manila Philippines.

HARFACRE, R.G. 1979. Horticultural Clemson University. McGraw Hill Inc. pp. 424-
425.

HAYWARD, AC, 1972. Bacterial Diseaese of Anthurium in Hawaii. Plant Disease
Reporter:56:904-908

HENNY, R.J., A.R. CHASE and L.S. OSBORNE. 2001. Anthurium Production Guide.
CFREC-Apopka Foliage Plant Research Note RH-91-3. Central Florida Research
and Education Center, University of Florida, IFAS.

IMMAMURA, J. & T. HIGAKI.1981. Chemical induction of adventitious shoots in
anthurium.Host Science 14(1):64-65.
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
Blight) in La Trinidad, Benguet and Baguio City / Karen S. Tagway. 2006

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MACNAB, R. M. 1978. Bacterial Motility and Chemotaxis Crit. Rev. Biochemistry.pp.
291-341.

MCCALLOW and LEONHARDT. 1983. Anthurium Culture in Hawaii Circular
420.Cooperative Extension Service, University of Hawaii at Manoa.

MCCULLOCH and PIRONE. 1939. Bacterial leaf spot of Diffenbachiae.
Phytopathology 29:956-962.

MCMANUS, P.S. 2004. Antibiotic use in plant disease control. Department of Plant
Pathology, University of Wisconsin-Madison.

NISHIJIMA WT .1990. Chemical Control. In: Proceedings of the Third Anthurium
Blight Conference, Hawaii Institute of Tropical Agriculture & Human
Resources.pp 39-40.

ROSARIO, T. L. 1998. Anthuriums. 3rd ed. Laguna, Philippines: College of
Agriculture. UPLB. Pp 32-33.

SAKAI, W. S. 1990. Plant Nutrition and Vascular Diseases. College of Agriculture,
University of Hawaii. Hilo Hitahr Publishing Company. Pp. 81-86.

SINGH.1978. Plant Diseases. 4th.Edition. University of Agriculture and Technology,
Pantinagar. Hainital, India. Oxford & IBA Pub. Co. pp. 49-50.

SKERMAN VBD, V McGOWAN & PHA SNEATH. 1980. Approved list of bacterial
names. International Journal of systematic bacteriology 30;255-420.

VALENCIA, L.D., M.P. NATURAL, G.G. DIVINAGRACIA and V.N. VILLEGAS.
2004. Steptomycin Resistance in Anthuriums and Sources of Host Resistance to
Xanthomonas Axonopodis pv diffenbachiae. Philippine Journal of Crop Science
29 (1): 41-50.

VAUTERIN L., J. SWINGS, and K. KERSTERS. 1995. Reclassification of
Xanthomonas. Journal of Systemtic Bacteriology 45:472-489.

WEBMAN. 2000. Anthurium Tropical Flower Diseases. Department of Plant Pathology
University of Hawaii. Pp. 45:482-499.
Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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APPENDICES



Appendix 1: Communication Letter

College of Agriculture
Department of Plant Pathology
Benguet State University
La Trinidad, Benguet


October 2005

Dear respondents,

The undersigned is conducting a study entitled ‘’ANTIBIOTIC RESISTANCE
OF XANTHOMONAS AXONOPODIS PV DIFFENBACHIAE (ANTHURIUM
BACTERIAL BLIGHT) IN LA TRINIDAD, BENGUET AND BAGUIO CITY’’ in
partial fulfillment of the degree Bachelor of Science in Agriculture (Plant Pathology).

In this regard, I am requesting your cooperation and support by sharing me a
part of your time to give the necessary data by answering the questions properly.

Your honest answers will contribute so much to this study.

Thank you very much and may the Almighty God bless you always.

Respectfully yours,

KAREN S. TAGWAY
Researcher

Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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APPENDIX 2. Questionnaire

1.

Location
of
the
farm


2. Area devoted to anthurium







3.

Type
of
culture


a.

Open
or
protected


b.

If
protected

full
or
partial




plastic or glass







4. Occurrence of bacterial
blight



Present or absent








5. If present, type of symptoms observed






Local, systemic








6. Incidence (20-30 sample plants )






7.

Severity
of
infection


Rating:
1. No infection
2. Nearly plant affected with blight plant still retaining normal form
3. When every plant affected and about ¼ of the area is destroyed by blight
4. When every plant affected and about ½ of the area is destroyed by blight
5. When about ¾ of the area are affected with blight
6. Only few leaves left green
7. When all leaves are dead including the stem
8.

Anthurium
cultivar
used


Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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9.

Source
of
cultivar



10.
Type
of
irrigation


11. Management of bacterial blight: Which of the following do you use against bacterial
blight? Check as many as you use.



a. Removal and disposal of infected leaves


b. Uprooting and disposal of infected plants


c. Application of chemicals, please specify





d. Resistant variety, please specify






e. Use of clean planting materials


f. Addition of organic matter, specify






g. Careful irrigation


h. Other practices, please specify


















































Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
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APPENDIX 3. The recommended management measures for bacterial blight of
anthurium.

a. Avoid using infected planting materials.
b. Remove all leaves with foliar infection by breaking or cutting the petrole near the
base. Remove all systemically-infected plants.
c. Spray beds thoroughly with streptomycin sulfate or oxytetracyline weekly for 6-8
weeks at the rate of one and a half vial per five gallons of water.
d. Disinfect tools to prevent the spread of the disease during harvesting or leaf
pruning.
e. Avoid using copper-based fungicides such as copper hydroxide and tribasic
copper sulfate. Laboratory and field tests have shown that the bacterim is
resistant to the copper-based compounds and that phytoxicity to anthurium my
occur.
f. Avoid close contact with the plant during wet condition. Touching creates
wounds the augmented by water, encourages disease spread.
g. Monitor plants continuously of diseased plants are found, remove them
immediately.
h. Provide good aeration and good drainage system
i. Maintain strict sanitation.

Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial
Blight) in La Trinidad, Benguet and Baguio City / Karen S. Tagway. 2006

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Document Outline

• Antibiotic Resistance of Xanthomonas axonopodis pv. Diffenbachiae (Anthurium Bacterial Blight) in La Trinidad, Benguet and Baguio City
  • BIBLIOGRAPHY
  • ABSTRACT
  • TABLE OF CONTENTS
  • INTRODUCTION
  • REVIEW OF LITERATURE
    • The Disease, Its Cause and Symptoms
    • The Site of Infection
    • Epidemiology of the Disease
    • Control
    • Resistance to Antibiotics
  • MATERIALS AND METHODS
    • Collection, Isolation and MaintenanceOf Xad isolates
    • Cultural Characteristics of Xad Isolates
    • Sensitivity of Different Isolates ofXad to Streptomycin and Penicillin
    • Test for Starch Hydrolysis
    • Determination of Growers� ManagementMeasures Against Bacterial Blight
    • Data Gathered
  • RESULTS AND DISCUSSION
    • Collection, Isolation and Maintenanceof Xad Isolates
    • Sensitivity of Different Isolates ofXad to Streptomycin, Penicillin andCopper-based Fungicide
    • Test for Starch Hydrolysis
    • Determination of Grower�s ManagementMeasures Against Bacterial Blight
  • SUMMARY, CONCLUSION AND RECOMMENDATIONS
    • Summary
    • Conclusion
    • Recommendations
  • LITERATURE CITED
  • APPENDICES

---