ABSTRACT

BIBILIOGRAPHY

NAMUJHE, JOSEPHINE L. APRIL 2007. Incidence of Citrus Tatterleaf
Disease in Kasibu, Nueva Vizcaya. Benguet State University, La Trinidad, Benguet.
Adviser: Janet S. Luis, PhD
ABSTRACT

The study was conducted to pinpoint some of the areas of occurrence of
the Citrus tatterleaf disease, establish characteristic symptoms of CTLV in the locality,
and report the presence of Citrus tatterleaf disease in Kasibu, Nueva Vizcaya through
detection by RT-PCR analysis and symptomatology.
The areas of occurrence of the CTLV are Antutot and Malabing. The probable
spread of the disease was due to the exchange of budwoods among farmers and use of
infected tools and implements.
The characteristic symptoms of CTLV observed in the locality are bud union
bulging, bud union creasing, decline, stunted growth, and tattered leaf.
Seven from the twenty four trees tested for CTLV infection through RT-PCR
analysis yielded positive results. This shows that CTLV is now present in Kasibu, Nueva
Vizcaya.

TABLE OF CONTENTS

Page
Bibliography …………………………………………………………….
i
Abstract ………..……………………………………………………….
i

Table of Contents ………………………………………………………
ii

INTRODUCTION ……………………………………………………...
1

REVIEW OF LITERATURE …………………………………………..
3

MATERIALS AND METHODS ………………………………………..
9

RESULTS AND DISCUSSION

Areas of Occurrence ……………………………………………
18

Symptoms of Citrus Tatter Leaf Disease ……………………….
19

RT-PCR Results ………………………………………………...
27

SUMMARY, CONCLUSION AND RECOMMENDATIONS

Summary
………………………………………………………...
34

Conclusion ……………………………………………………...
35

Recommendation ……………………………………………….
35

LITERATURE CITED …………………………………………………
37

ii

INTRODUCTION
Citrus is one of the most important fruit crops in the Philippines. With the wide
range of climatic and topographic conditions suitable for citrus production, the
Philippines could become one of the major producers of citrus in the world. Actually,
citrus production was a flourishing industry in the late 1950’s to early 1960’s especially
in Southern Tagalog, Bicol and Mindanao regions prior to its decline due to the presence
of bud-transmissible diseases (Payot et al., 1987).
Kasibu, Nueva Vizcaya ventured into citrus production in 1987 and has once
again proven that citrus is a promising industry that has global market potential if proper
technology can be established (Namujhe, 2006).
Although studies have been conducted on the diseases that affected Southern
Tagalog, Bicol and Mindanao, none or inadequate studies were done in Nueva Vizcaya
particularly in Kasibu. Despite the fact that the prevalence of citrus diseases was not yet
overcome, Kasibu continues to produce citrus and is geared to further expansion.
Now around 1,000 hectares are planted to citrus where 30% are already in their
productive stage. The varieties planted are Satsuma (60%), Ponkan (20%), sweet oranges
(10%), and pomelos (10%). Given these statistics, Malabing Valley is renowned as the
citrus capital of Luzon (Namujhe, 2006).
Dr. Hong-Ji Su of the Department of Plant Pathology and Microbiology National
Taiwan University, Taipei, Taiwan recently confirmed the presence of tatterleaf disease
of citrus in the area of Kasibu by RT-PCR analysis. This poses yet another threat to the
industry especially if the problem is not contained immediately.
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

2
This graft and mechanically transmissible disease of citrus is moderately
destructive. Although it may severely limit crop production especially when one is
unaware of the symptoms, this disease is readily detected in propagative budwood, easily
eliminated and its rapid spread curtailed.
It is imperative that farmers become aware of the tatterleaf disease that was
detected in their locality to ensure that the booming citrus industry in Malabing Valley
becomes sustainable. They should be familiar with the symptoms of the disease so that
they could easily detect infected trees and apply proper control measure to prevent its
eventual spread.
Farmers can use the result of this study as a guide in identifying Citrus tatterleaf
disease in their farm. The pinpointed areas of occurrence of CTLV in Kasibu can serve as
a reference point for further studies. This could also serve as a basis for further and more
specialized research on CTLV in Kasibu, Nueva Vizcaya
This study was conducted to pinpoint some of the areas of occurrence of the
Citrus tatterleaf disease, establish characteristic symptoms of CTLV in the locality, and
report the presence of Citrus tatterleaf disease in Kasibu, Nueva Vizcaya through
detection by RT-PCR analysis and symptomatology.
The field study was conducted in Kasibu, Nueva Vizcaya from December 2006 to
February 2007. The RT-PCR confirmatory test was done in the laboratory of the Bureau
of Plant Industry, Guisad, Baguio City.
Simultaneously, RT-PCR confirmatory test was conducted in Taiwan National
University under the supervision of Dr. Hong-Ji Su using samples from the same trees
tested in BPI, Guisad, Baguio City.
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

REVIEW OF LITERATURE

The Disease
The tatterleaf disease of citrus, induced by the Citrus Tatterleaf Virus (CTLV),
was first described by Wallace and Drake (1962) as a transmissible disease that induced
mottled and tattered leaves in Citrus excelsa indicator seedlings. Calavan, Christiansen
and Roistacher (1963) first showed the destructive potential of this disease to citrange
rootstock when tatterleaf-infected tissue was graft-inoculated to satsuma mandarin
budded on Troyer citrange rootstock. Meyer (Beijing) lemon trees, which were first
imported into the United States from Beijing (China) in 1908, were later found to contain
the tatterleaf virus. Many Meyer lemon trees worldwide that originated from the 1908
introduction probably contain the virus, including many propagations and plantings of the
original Beijing lemon in China (Zhang, Liang and Roistacher, 1988). The disease is
endemic in mainland China and is widespread in Taiwan Province and in Japan (Zhang et
al., 1988).
Citrus tatterleaf virus expresses no symptoms in most citrus cultivars including
sweet orange, mandarin, sour lemon, Meyer lemon and grapefruit. However, if virus-
infected budwood is used as scion wood and grafted to citrange or trifoliate orange or any
hybrids of trifoliate used as a rootstock, a brown bud-union crease will usually be
evident, deep pits and grooves may develop in the rootstocks, and trees of these scionic
combinations will usually decline.

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

4
The Pathogen
Physical and biochemical properties. The virus is a capillovirus (Nishio et al.,
1989; Namba, 1995) which is serologically related to apple stem grooving capillovirus
and to a virus isolated from stunted and chlorotic lily (Lilium longiflorum) widespread in
western parts of Japan (Inoue et al., 1979). It has been suggested that, due to homologies
in nucleotide sequences, it is now probably best considered to be a strain of apple stem
grooving capillovirus rather than a distinct virus (Ohira et al., 1994). This suggestion,
however, has yet to be confirmed.
Filamentous and usually flexible rod particles (Figure 1) which are 650 nm long
and 12 nm wide, with a helical construction of 3.4 nm pitch. The virus has a single RNA
species of molecular weight 2.83 x 106 Da and produces a single protein band of
molecular weight 27 x 103Da in SDS PAGE (Nishio et al., 1989).

Figure 1. Studies by (Ohki et al., 1989) and Nishio et al., (1989) indicated that the

Citrus tatterleaf virus is a capillovirus and is closely and serologically
related to the apple stem grooving virus
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

5
Biology
The major method of transmission from citrus to citrus is by grafting. Mechanical
transmission by knife slashes and leaf-abrasion is easily achieved from infected
Nicotiana clevelandii to citron (Garnsey, 1974) and from citron to citron (Roistacher et
al., 1980). However, a field trial which attempted to transmit the virus to 8-year-old
mandarin trees by slashing their bark with a knife or sawing the branches gave a very low
rate of infection
Seed transmission has been observed in Chenopodium quinoa, cowpeas and
soyabeans but not in Fortunella japonica (Nishio et al., 1982). No natural vector is
known. These results suggest that natural transmission occurs only at a very low rate.

Symptoms
The common symptoms of Citrus Tatterleaf Virus is bud union necrosis or
abnormality which develop when symptomless CTLV carrying citrus, such as sweet
orange, sour orange, grapefruit (Citrus paradisi Macf.), mandarin (C. reticulata Blanco)
or lemon (C. limon (l.) Burm.f.) is budded onto a trifoliate orange or a trifoliate hybrid
rootstock. Deep fluting of the rootstock trunk and an extended bud union crease or gap
can result as early as eighteen months after grafting. Chlorotic leaf symptoms are
produced in Citrus excelsa, Rusk and Troyer citranges (Poncirus trifoliata x Citrus
sinensis), Swingle citrumelos (P. trifoliata x C. paradisi) and other P. trifoliata hybrids.
Leaves of C. excelsa may be deformed (so-called tatter leaf), stems of citrange plants
may be deformed and have a zigzag growth pattern associated with chlorotic areas on the
stem. Citranges and citrange hybrids are often pitted on their stem, affected plants
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

6
become stunted, chlorotic and overblooming, have early-maturing of fruit, and suckers
often develop. In high winds, the scions may sever completely at the bud union (Herron
et al., N.D.).

Host Range
Almost all citrus plants can be symptomless hosts. Poncirus trifoliata is immune
or highly resistant, but its hybrids can show symptoms after infection (Wallace and
Drake, 1963).
The following plants are infected by the virus when inoculated mechanically
(Nishio et al., 1982): Amaranthus tricolor, Catharanthus roseus, Chenopodium
amaranticolor, C. quinoa, Cucurbita pepo, Dianthus barbatus, D. chinensis, faba beans,
Gomphrena globosa, Nicotiana clevelandii, N. debneyi, N. glutinosa, peas, Petunia
hybrida, soyabeans, Tetragonia tetragonioides, tomatoes, Vigna unguiculata.

Geographical Distribution

CTLV was first found in Citrus meyeri in 1962 at Riverside, California, USA.
The original tree was brought from China in 1908 (Wallace and Drake, 1962) and it is
clear that the virus originated in China. Old budlines of C. meyeri which were imported
from China into the USA and subsequently delivered to other countries were probably
symptomless carriers (Wallace and Drake, 1962; Schwarz, 1966), so the virus may have a
wider distribution. Su and Tsai (1990) also reported the Tatterleafvirus present in the
Philippines, Thailand and Korea.

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

7
Economic Impact
Almost all citrus plants are symptomless if grown on their own roots or on a
CTLV-tolerant rootstock. Poncirus trifoliata is immune or highly resistant to CTLV.
However, when infected latent hosts are grafted on rootstock of P. trifoliata or its
hybrids, a bud-union crease occurs and the tree becomes stunted or often dies (Calavan et
al., 1963). Yields of affected mandarins (Citrus reticulata) on P. trifoliata rootstock are
75% of those CTLV-free trees (Takahara et al., 1988). Accordingly, P. trifoliata and its
hybrids cannot be used in practice as rootstocks where CTLV is indigenous. Inserting a
healthy interstock between the infected latent bud and the P. trifoliata rootstock only
delays the problem. The scions grow normally for 1-2 years, but then

become overblooming and yellow gradually and finally die within 5-6 years. These trees
develop a crease at the bud-union between interstock and rootstock, and are occasionally
dislocated at this point by strong winds.

Methods of Detection
CTLV can be detected through indexing to indicator plants such as Rusk, Troyer
or Carrizo citrange, citremon and trifoliate stock. Inoculation to herbaceous host can also
be used as an alternative method of detection (Miyakawa, 1978) although specialized
inoculation chambers are needed to ensure reliable results. Reverse transcription PCR
(RT-PCR) was formulated by using cDNA derived from the sequence of CTLV for
detecting the virus more rapidly (Su et al., 2005).

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

8
Control
CTLV-free budlines must be used for propagation. If latently infected scions were
used for propagation without any therapy, Poncirus trifoliata or its hybrids cannot be
used as the rootstock. Citrus depressa or C. reshni provide good results when used as
rootstocks for CTLV-infected mandarins (C. reticulata) (Takahara et al., 1988).
CTLV cannot be eliminated by shoot-tip grafting alone (Roistacher and Kitto,
1977). Heat treatment for 30 days at 35-40°C/30°C (day/night) followed by shoot-tip
grafting can be an effective therapy (Koizumi, 1984). Incubation of budsticks on medium
in vitro for 10-14 days at 32°C, followed by shoot-tip grafting can also produce CTLV-
free plants with 30-50% success (Navarro et al., 1989). Long-term heat treatment of
affected plants for 90 or more days at 40°C/30°C (day/night) can eliminate CTLV
(Miyakawa, 1980a). Mechanical transmission from citron to citron by knife-slashing is
completely prevented by dipping the contaminated knife-blades into 1.05% sodium
hypochlorite solution or 2% sodium hydroxide plus 5% formaldehyde solution, or merely
by washing the blades with tap-water and drying, prior to slashing the receptor
(Roistacher et al., 1980).

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

MATERIALS AND METHODS

Survey of suspected infected trees by symptomatology was done in the area.
Trees that showed decline were examined and their symptoms were compared with the
symptoms of tatterleaf disease described in the literature. Those trees with symptoms that
coincided with the symptoms of tatterleaf were marked, digital images were taken and
samples were collected for confirmatory tests.

Survey
Upon the observation of probable symptoms of CTLV in Antutot, Kasibu, a
survey on other farms with trees showing the same symptoms was conducted. Farmers
were asked if they have observed bud union bulging on their declining trees. After which,
those who have such trees in their farm were visited and further observation was done.

The areas surveyed were:

Antutot, Kasibu. This is where the first batch of samples was collected. This farm
belongs to Engr. Roy Bernardo and this is where the symptoms of CTLV were first
observed. There were three trees which manifested the symptoms and they were all 9
year-old Ponkan budded to Trifoliate.

Namujhe Farm, Malabing, Kasibu. This is where the second batch of samples was
collected. Ten trees were under observation with varying age, varieties and scion-stock
combination.

Randomly selected farms. Samples for this batch were gathered from trees
showing symptoms of CTLV from randomly selected farms in Malabing, Kasibu.
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

10
Observation and Documentation of Disease Symptoms

For the purpose of symptoms comparison, a list of known CTLV symptoms
based on the available literature was prepared. This list of symptoms served as a basis for
field diagnosis based on symptomatology. The table below represents the list.

Table 1. List of CTLV symptoms based on available literature

PLANT PART AFFECTED
SYMPTOMS

Tree
Stunted growth

Decline

Trunk
Bud union bulging

Bud union creasing
Pitted stem

Leaves
Tatter leaf

Chlorotic mottle

Flowering
Over blooming

Fruits
Early-maturing of fruit

After observing the trees and comparing the symptoms with the list, photo
documentation followed. First, the overall appearance of the trees was taken. Then the
bud union bulging of the trunk was captured. A portion of the bark within the bud union
bulging was removed to reveal any deep creasing, this too was documented. Other
characteristics such as tatter leaf and chlorotic mottle were noted when trees exhibit any
of these symptoms.
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

11
Sample Collection
The shoots of the trees were collected as samples. Four shoots were gathered
from the different sides of each tree. This was done to ensure a good representation of the
whole tree and collected shoots were considered as a composite sample of the tree. The
samples were placed in plastic bags and were labeled with the following information: a.
Sample number, b. Variety/ scion-stock combination, c. age, d. location, e. farm owner.
The labeled samples were stored in the refrigerator prior to transport. Samples
were kept under low temperature while in transit by placing them in styrobox with ice.
They were again transferred into the refrigerator upon arriving in the laboratory.

Detection by Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR)
Confirmation was done through RT-PCR analysis conducted at the Bureau of
Plant Industry, Guisad, Baguio City. Simultaneously, samples from the same trees were
sent to National Taiwan University through Dr. Hong-Ji Su for counter testing and more
accurate results.
RT-PCR Analysis conducted at the Bureau of Plant Industry, Baguio City.
Extraction of nucleic acids from citrus tissues. The procedure for extraction was
adapted from RNeasy Plant Mini kit as produced by QIAGEN.
The samples were macerated using mortar and pestle. One 100 mg of the tissues
were transferred to 1.6-ml tubes. RLT buffer (450 µl) was added to tissue samples and
stirred vigorously.
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

12
Lysate were applied to the QIA shredder spin columns setting in 2-ml collection
tubes and centrifuge at 11,000 rpm/3 min. Flow-through fractions were transferred from
QIA shredders to new tubes.
One half volume (usually 225 µl) of ethanol (96~100%)was added to each cleared
lysate and was mixed well by pipetting. Samples (usually 225 µl) were applied onto
RNeasy mini spin columns set in 2-ml collection tube. Samples were ran in the
centrifuge at 11,000 rpm for one minute. Flow-through were discarded and the collection
tubes were re-used.
One volume of Buffer RW1 (700 µl) was added to each RNeasy columns and
were spun at 11,000 rpm for one minute to wash. Flow-through were discarded and the
collection tubes were re-used.
This step was repeated using 500µl of buffer RPE. Again, the flow-through were
discarded and the collection tubes were re-used.
A final volume of 500 µl buffer RPE was added to each RNeasy columns and
were spun in the centrifuge at 11,000 rpm for 3 minutes. RNeasy columns were
transferred into new 1.5-ml collection tubes. Approximately 30~50 µl of RNase-free
water was added directly into each RNeasy columns. The columns were spun in the
centrifuge at 11,000 rpm for 2 minutes to elute. The flow-through were kept as RNA
templates for RT-PCR.
RT-PCR conditions. Each PCR reaction contained the components in Table 2 and
should have a total of 25 µl. Amplification was carried out in a BIOMETRA Personal
Cycler (PC 20 with heated lid) using a cDNA primer pair. The forward and reverse
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

13
primers have the following respective sequences (reference): CGA AGA CTC ACA TAG
ACC CG and TAC TCT CCG AAC CTG CCT C.

Table 2. Components of the RT-PCR reaction

COMPONENT
AMOUNT (µl)

ddH2O 10.0

2.5
5X Superscript II buffer

10X Taq buffer
2.5

100mM DTT
1.25

10mM dNTPs
2.0

Taq polymerase (BRL or BerTaq)
0.2

Superscript II polymerase (BRL)
0.25

CTLV primer pair (10 pmol/µl each)
1.0

RNA template
0.25

The amplification schedule began with one initial cycle at 50 and 94ºC for 35 and
two minutes. The next step consisted of 10 cycles at 94, 56 and 68 ºC for 30, 30 and 45
seconds, respectively. This step was followed by 25 cycles with the same parameters as
the previous step but with 5 seconds increasing ramp. The final cycle was at 68 ºC for
seven minutes.
The negative control came from BPI citrus foundation greenhouse while the
positive control was unavailable.
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

14
Electrophoresis analysis of PCR products. A 1.4% agarose gel plate in TAE
buffer (0.04 M Tris-acetae, 0.001 M EDTA, pH 8.0) was prepared.
A 3-5 µl of PCR products (CTLV DNA extract) was loaded into each well, and
the gel was run in an electrophoresis chamber at 100 volts for about 30 minutes when the
markers reached near the bottom line. (Running buffer: 0.5 X TAE buffer, pH 8.0).
The gel was stained with Ethidium bromide (0.5 ug./ml) solution for 3-5 min, and
was soaked in water for several minutes.
The amplified CTLV-DNA band (at 632 base pair) was observed under UV-
chamber and a picture of the electrophoretic plate was taken.
The following were composition of the buffers used computed per 1000mL:
50 X TAE buffer:
242g Tris base
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA (pH 8.0).
Loading buffer:
0.25% Bromophenyl blue
30% glycerol (1:4).

RT-PCR analysis done at the National Taiwan University, through Dr.Hong-Ji Su.
Extraction of nucleic acids from citrus tissues. A 0.3g of tissue from each
samples were ground in liquid nitrogen and homogenized in 3ml TRIzol reagent buffer.
The samples were spun in the centrifuge at 12,000xg at room temperature for 10 min.
The supernatants, about 1ml each, were transferred into new tubes. A volume of 200 l
chloroform was added to each sample. The tubes were shaken vigorously using the vortex
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

15
for 30 sec. The samples were again spun in the centrifuge at 12,000xg at room
temperature for 10 min. The aqueous phase were transferred into new tubes using a
pipette. A 300 l volume of isopropanol and 0.8 M sodium citrate/1.2 M NaCl (about
300 l), ½ volume of the aqueous phase each, was added and mixed by gentle inversion.
The samples were again ran in the centrifuge at 12,000xg at 4°C for 15 minutes. After
which the supernatants were discarded and the pellets were washed with 500ul of 75%
ethanol. The pellets were briefly dried for about 10-12 minutes. Finally 80 l (or 50 l)
ddH2O was added to each samples.
The TRIzol reagent buffer (pH5) consisted of 38 % Phenol , 0.8 M Guanidine
thiocyanate, 0.4 M Ammonium thiocyanate, 0.1 M Sodium acetate , 5% Glycerol.
RT-PCR conditions. Each PCR reaction contained the components in Table 3 and
has a total volume of 25 µl. The forward and reverse primers have the following
respective sequences (reference): CGA AGA CTC ACA TAG ACC CG and TAC TCT
CCG AAC CTG CCT C.
The amplification schedule began with one initial cycle at 50 and 94ºC for 35 and
two minutes. The next step consisted of 10 cycles at 94, 56 and 68 ºC for 30, 30 and 45
seconds, respectively. This step wasfollowed by 25 cycles with the same parameters as
the previous step but with 5 seconds increasing ramp. The final cycle was at 68 ºC for
seven minutes.
Electrophoresis analysis of PCR products. A 1.4% agarose gel plate in TAE
buffer (0.04 M Tris-acetae, 0.001 M EDTA, pH 8.0) was prepared.

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

16
Table 3.Table showing RT-PCR components.

COMPONENTS
AMOUNT (µl)

ddH2O
11.5

5X Superscript II buffer
2.5

10X Taq buffer
2.5

100mM DTT
1.25

25 mM MgCl2

1.25

10 mM dNTPs
2

Taq polymerase (BRL or BerTaq)
0.25

Superscript II polymerase (BRL)
0.25

CTLV primer pair (10 pmol / ul each
1

RNA template
2.5

A 3-5 µl of PCR products (CTLV DNA extract) was loaded into each well, and
the gel was run in an electrophoresis chamber at 100 volts for about 30 minutes when the
markers reached near the bottom line. (Running buffer: 0.5 X TAE buffer, pH 8.0).
The gel was stained with Ethidium bromide (0.5 ug./ml) solution for 3-5 min, and
was soaked in water for several minutes.
The amplified CTLV-DNA band (at 632 base pair) was observed under UV-
chamber and a picture of the electrophoretic plate was taken.
The following were composition of the buffers used computed per 1000mL:
50 X TAE buffer:
242g Tris base
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

17
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA (pH 8.0).
Loading buffer:
0.25% Bromophenyl blue
30% glycerol (1:4).

The data gathered were:
a. Digital images of the actual field symptoms of Citrus Tatterleaf disease
was recorded and compared with those listed in the literatures,
b. Results of RT-PCR analysis from BPI Baguio and National Taiwan
University which confirmed the presumed presence of CTLV infection
based on the symptoms observed , and
c. Map of Kasibu where the areas detected with CTLV infected trees were
indicated.

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

RESULTS AND DISCUSSION

Areas of Occurrence

According to the owner of the farm in Antutot, Kasibu, where the trees
manifesting symptoms of Citrus Tatterleaf Disease was first observed, the scions of the
Ponkan he budded to Trifoliate came from the trees brought by his uncle from Japan. It
is a logical presumption that the initial inoculum of CTLV came from these trees whose
scion came from Japan and did not undergo proper Quarantine procedures when brought
here.

The eventual spread of the disease in the area, until it reached Malabing, Kasibu
where some trees also showed symptoms of CTLV, probably occurred when farmers
started propagating their own planting materials and most of them exchanges bud woods
with each other to acquire new varieties.

The map of Kasibu is presented in Figure 1, which shows the occurrence of
CTLV in Kasibu and the probable movement of inoculum spread. The green dot
represents Antutot and the yellow dot is Malabing. The lines represent the probable
movement of the inoculum from the two sources through exchange of budwoods among
the farmers.

Symptoms of Citrus Tatterleaf Disease

The comparison of the observed symptoms of Citrus Tatterleaf Disease and the
previously reported symptoms is summarized in Table 4. The symptoms for the
flowering stage and the fruits were not observed in Kasibu since the duration of the study
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

19

Figure 2. Map of Kasibu showing the occurrence of CTLV in the area and the probable
movement of inoculum spread through budwood exchange among farmers
with green dot representing Antutot while the yellow dot representing Malabing

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

20
did not cover the said stages. Some of the symptoms like pitted stem and chlorotic mottle
were not observed because the variety of citrus under observation does not manifest such
symptoms.
The symptoms manifested by the infected trees in Kasibu coincided with that of
the CTLV as described in the literatures and was confirmed by Dr. Su during his visit in
the area last February 2-4, 2007.

Table 4. Comparison of the symptoms of CTLV from the literature and those observed in
Kasibu, Nueva Vizcaya

CHARACTERISTIC SYMPTOMS OF
CHARACTERISTIC SYMPTOMS OF
CTLV BASED ON THE LITERATURE
CTLV OBSERVED IN KASIBU

Trees: Stunted growth (Figure 4)
Stunted growth (Figure 3)

Decline (Figure 4)
Decline (Figure 3)

Trunk: Bud union bulging (Figure 6)
Bud union bulging (Figure 5)

Bud union creasing (Figure 8)
Bud union creasing (Figure 7)

Pitted stem (Figure 8 B)
-do-

Leaves: Tattered leaf (Figure 10)
Tatterleaf(Figure 9)

Chlorotic mottle
-do-

Flowering: Over blooming
Duration of study did not cover the stage

Fruits: Early-maturing of fruit
Duration of study did not cover the stage

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

21
The characteristic symptoms of Citrus Tatterleaf Disease are:
Stunted
growth. The tree is relatively small for its age regardless of its variety and
scion-stock combination (Figure 3 and 4).
Decline. This is manifested by poor growth of the plant; relatively small leaves,
brittle and yellowish; some defoliation and dieback (Figure 3 and 4).
Bud union bulging. This is the most typical characteristic symptom of CTLV
infection that is manifested by a swelling around the bud union area (Figure 5 and 6)
Bud union creasing. It is the formation of a line, which seems to fold inwards,
along the bud union. In severe cases, the crease is so deep that the scion tends to sever
from the rootstock (Figure 7 and 8).
Pitted stem. This symptom is characterized by depressions on the stem of the
trees (Figure 6B).
Tattered leaf. The leaves are deformed and the edges has a tattered appearance
(Figure 9 and 10), for which the disease was actually named.
Chlorotic mottle. This symptom appeared as yellowing of the leaves with the
boundaries of light and dark variegated areas diffused.
Deep creasing at the bud union causes decline and stunted growth due to the
disruption of the translocation of synthesized food as well as the movement of water and
nutrients from roots to the leaves.
The trees in Figure 3 shows the stunted growth and decline in Antutot, Kasibu,
while the trees in Figure 4 shows the same stunted growth and decline in trees from
Zhejiang Province, China. You will notice that the trees from both areas are relatively
small for their age.
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

22

Figure 3. Infected 14-yr old trees in Antutot, Kasibu showing stunted growth and
decline

Figure 4. Fifteen-year old trees in Zhejiang Province, China showing stunted
growth and decline due to CTLV infection; photo illustration by
Roistacher C.N.

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

23
The bud union bulging symptom is clearly depicted in Figure 5, observed in
Kasibu, which is similar to Figure 6 (A), observed at the Citrus Research Station in
Riverside, California.

Figure 5. Bud union bulging as a typical symptom of CTLV observed in the infected
trees in Kasibu

Figure 6. Symptoms bud union bulging (A) and pitted stem (B) observed at
the Citrus Research Station in Riverside, California; photo illustration by
Calavan E.C.
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

24
Another symptom of CTLV infection is bud union crease as shown in Figure 7,
observed in infected trees from Antutot, Kasibu, similar with those shown in Figure 8,
observed in trees from Kutchinotsu, Japan and at the Citrus Research Station in
Riverside, California.

Figure 7. Bud union bulging and deep creasing manifested by the CTLV infected trees
in Antutot, Kasibu

Figure 8. A specimen showing a severe bud union crease and incompatibility of a
Satsuma mandarin grafted on Trifoliate rootstock that had been infected with
the Citrus tatterleaf virus at Kutchinotsu, Japan(A); severe bud union crease
(B) a typical reaction with trifoliate and its hybrid rootstock observed;
photo illustration by Calavan E.C.
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

25

The tatterleaf symptom shown in Figure 9, observed in Antutot, Kasibu, is similar
to Figure 10, from the literature. Manifestation of the severity of this symptom may vary
depending on the scion-stock combinations.

Figure 9. Tattered leaves of infected trees in Antutot, Kasibu

Figure 10. Tattered leaf symptoms ( first three leaves from right side) for which the
disease was named and the uninoculated control leaf (leftmost);

photo illustration by Roistacher C.N.

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

26
After knowing the symptoms of CTLV infected trees, it is imperative that the
distinction between the symptoms of infected trees and that of natural incompatibility
should be taken into consideration. Some trees with scion-rootstock incompatibility may
have larger base (rootstock) than the scion trunk but it does not affect the growth and
yield of the tree. Other healthy trees have natural bud union crease and brown line that
are merely superficial as shown in Figure 11, observed in Malabing Kasibu and Figure 12
that was taken from the literature.

Figure 11. Natural bud union brown line in virus-free trees in Malabing, Kasibu

Figure 12. Natural incompatibility of a virus-free Meyer lemon scion budded
on trifoliate rootstock (A) with the deep brown line extending into the
wood (B); photo illustration by Roistacher C.N.

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

27
RT-PCR Results

The RT-PCR confirmatory test was conducted simultaneously at BPI, Baguio
under the supervision of Senior Pathologist Juliet M. Ochasan and at the National Taiwan
University, Department of Plant Pathology & Microbiology under Dr. Hong-Ji Su.

The RT-PCR results from the BPI, Baguio (Figure 13) were not satisfactory due
to some technical problems encountered during the process. Some of the most likely
reasons for ineptness were the poor state of the equipment used, such as the fluctuating
voltage delivered by the electrophoresis machine and the limit of the micropipettes
available which delivered inaccurate amounts of reagents. The total amount of the PCR
mixture, which did not actually coincided with the stated amount required, could also be
a factor. Another probable reason for poor results, as suggested by Dr. Su who is an
expert from National Taiwan University laboratory, was the state of the samples. Since
RNA easily degrades, he advised that using fresh samples be recommended for better
RNA yield.

Figure 13. Electrophoresis reading of the RT-PCR analysis from BPI, Baguio laboratory
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

28
Since RT-PCR is a very sensitive test that requires precision, top of the line
equipment and experience, the researcher sent some samples from the trees under study
to NTU Laboratory through Dr. Su to ensure that reliable results.

The results from the NTU laboratory are as follows:
First Batch from Antutot, Kasibu. The samples are all positive of CTLV infection (Table
5, and Figure 14 ). The result confirmed the symptoms shown in Figures 3, 5, 7 and 9 as
CTLV infection, which were quite severe.

Table 5. Results of the RT-PCR test from NTU on samples from Antutot, Kasibu

SAMPLE NUMBERS

1
2
3
Disease-check
Healthy-check

+++
++
++
++++
-

Figure 14. Electrophoresis reading of the RT-PCR analysis of samples from Antutot
Kasibu showing all samples positive to CTLV infection with emphasis on

the white line along the 636bp
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

29
Second batch of samples from Namujhe farm. As shown in Table 6, only two
samples from this batch were positive for CTLV infection. Samples Fi-3 (Figure 15 and
16) and Fi-4 (Figure 15 and 17) showed decline and bud union bulging.

Table 6. Citrus samples for CTLV indexing (Namujhe)

MATERIAL NO.
CULTIVAR
CTLV/ DETECTION
(RT-PCR)
Fi-1 (No. 4)
Ponkan
-
Fi-2 (No. 7)
Ponkan
-
Fi-3 (No. 9) (Figure 16)
Ponkan
+
Fi-4 (No. 10)(Figure 17)
Ponkan
+
Fi-5 (No. 1)
Satsuma
-
Fi-6 (No. 2)
Satsuma
-
Fi-7 (No. 3)
Satsuma
-
Fi-8 (No. 4)
Satsuma
-
Fi-9 (No. 5)
Satsuma
-
Fi-10 (No. 6)
Satsuma -

Figure 15. Electrophoresis reading of the RT-PCR analysis for second batch of samples
from Namujhe Farm at Malabing, Kasibu with only Fi-3 and Fi-4
showing a white line along with the disease check that indicates positive
CTLV infection

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

30

Figure 16. Sample Fi-3 (sample 9) which was confirmed positive of CTLV infection
both by symptomatology and RT-PCR test with the tree showing decline (A)
and bud union bulging and creasing (B)

Figure 17. The declining tree where sample Fi-4 (sample 10) was gathered

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

31
Third batch of samples randomly gathered from Malabing, Kasibu. The samples
of this batch (Table 7, Figure 18) were randomly gathered from trees showing decline
and bud union bulging from different farms in Malabing, Kasibu. It can be noted that
only two samples were positive for CTLV infection, H/Fi-4 and H/Fi-6 respectively.
These trees were the ones shown in Figure 19 and 20. The scion budded to sample H/Fi-6
(Figure 20) came from sample H/Fi-4 (Figure 19).

Table 7. Results of the RT-PCR test for the third batch of samples that were randomly
gathered from various farms in Malabing, Kasibu

SERIAL NO. CULTIVAR
PLACE
DISEASE
INDEXING
CTLV/RT-PCR
G/Fi-1
Satsuma-Fi-1 (>10 years)
Malabing Farm-1
-

G/Fi-2
Pummelo-Fi-1 (5 years)
Malabing Farm-1
-

H/Fi-3
Satsuma-NH-3 (>6 years)
Namujhe Farm-1
-

H/Fi-4
H / Sat-NH-4(>20 years)
Namujhe Farm-1
++
(Figure 19)

H/Fi-5
H / Sat-NH-5 (>20 years)
Namujhe Farm-1
-

H/Fi-6
H / Sat-NH-6(7-8 years)
Namujhe Farm-1
++
(Figure 20)

G/Fi-7
Ponkan-Fi-1 (>6 years)
Namujhe Farm-1
-

G/Fi-8
Satsuma-Fi-2 (>6 years)
Namujhe Farm-1
-

G/Fi-9
Satsuma-Fi-3 (10 years)
Malabing
-
near PF-foundation

G/Fi-10
Satsuma-Fi-4
Malabing
-
near PF-foundation

G/Fi-11
Pummelo-Fi-2
Malabing Farm-3
-
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

32

Samples

2 : G/Fi-2
3 : H/Fi-3
4 : H/Fi-4
5 : H/Fi-5
6 : H/Fi-6
7 : H/Fi-7
8 : G/Fi-8
9 : G/Fi-9
10 : G/Fi-10
11 : G/Fi-11

Figure 18. Electrophoresis reading of the RT-PCR analysis for third batch of
samples with emphasis on the very distinct white line of samples 4 and
6 in line with the white line in the disease check indicating positive
reaction to CTLV infection

Since all citrus varieties are symptomless carriers of CTLV there are instances
that the tree may manifest bud union bulging but no creasing may occur, just like some of
the trees observed in Malabing, Kasibu where Satsuma was budded to Calamandarin.
A 20 year-old tree (Figure 19) showed bud union bulging but there was no creasing
and decline of the tree since Calamandarin is not susceptible to CTLV. However, when
scion from one of the trees was budded to a Trifoliate rootstock the tree showed
decline (Figure 20).
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

33

Figure 19. A 20-year old Satsuma tree (A) budded to Calamandarin, observed in
Malabing, Kasibu showing bud union bulging (B and a) but no creasing
(C and b) and decline (A)

Figure 20. The declining 7-year old tree (A) whose scion came from the infected
Satsuma tree budded to Calamandarin, in Trifoliate rootstock with emphasis
on the bud union bulging (B)

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

SUMMARY, CONCLUSION AND RECOMMENDATIONS

Summary

Kasibu, Nueva Vizcaya has once again proven the potential of the citrus industry
here in the Philippines. With the vast expanse of land dedicated to citrus farms and with
the continuous development of proper technology, Kasibu is geared towards producing
quality citrus that can compete in the global market.
However, some trees showing decline were observed in Antutot, Kasibu. Aside
from stunted growth and decline, another distinct symptom manifested by the trees was
bud union bulging. This particular symptom arouses the suspicion that these trees were
infected with Citrus Tatter Leaf Capillovirus. To make sure that the presumptions were
true, the areas of occurrence were observed and mapped out, a comparison of the field
symptoms with the typical symptoms of CTLV as described in the literature was done.
RT-PCR test was conducted at the Bureau of Plant Industry, Baguio laboratory at the
same time samples from the same trees were sent to National Taiwan University for a
more accurate RT-PCR analysis.
The areas of occurrence were in Antutot, Kasibu and Malabing Kasibu. The farm
in Antutot has three trees showing the bud union symptom. While in Malabing twenty
one trees were observed but only four were actually infected.
The symptoms observed in the field coincided with the symptoms described in the
literature and was personally confirmed by Dr. Hong-Ji Su of the Department of Plant
Pathology and Microbiology National Taiwan University Taipei, Taiwan, when he visited
the area last February 2-4, 2007.
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

35
The results of the RT-PCR analysis conducted by Dr. Hong-Ji Su confirmed the
presence of tatterleaf disease of citrus in some of the areas in Kasibu.

Conclusion
The probable source of the inoculum in Antutot was the scion of the Ponkan from
Japan that did not underwent proper quarantine procedures. The eventual spread of the
disease could be attributed to the exchange of budwoods among farmers when they
started propagating their own seedlings. This could escalate into an epidemic if farmers
are unaware of the presence of the disease and continue propagating their own seedlings
without indexing their source of budwoods. Continuous use of Trifoliate as rootstock will
also contribute to its rapid spread.
The established symptoms of CTLV in the locality can be used as a guide for field
diagnosis. The characteristic symptoms of CTLV are bud union bulging, bud union
creasing, decline, stunted growth, and tattered leaf.
The RT-PCR test confirmed that the disease observed in Kasibu was indeed
caused by Citrus Tatter Leaf Capillovirus.

Recommendations
It is highly recommended that farmers be informed of the presence of CTLV in
their locality so that they will become aware of its symptoms and its economic impact.
Information materials about CTLV must be made available to farmers.
Farmers are encouraged to eradicate positively tested trees to eliminate inoculum
source and prevent spread of the disease. At the same time, use of clean certified planting
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

36
materials to start their farm or replace eradicated trees is very essential. It is important
that indexing for CTLV should be included in any program for establishing primary
foundation trees since many citrus species and commercial cultivars are symptomless
carriers and the virus is highly mechanically transmissible.
Use of clean farm tools and implements by disinfecting pruning shears in 5%
sodium hypochloride will help ensure the cleanliness of your implements. Proper
sanitation in the farm should be part of the management program.
Since CTLV is already present in the area, avoiding the use of Trifoliate and its
hybrids as rootstock will help prevent the manifestation of bud union creasing which is
the symptom deleterious to the tree.
Finally, for studies on CTLV in Kasibu, it is highly recommended that the
researcher should undergo intensive laboratory training to be familiarized with the
protocol. It is also advisable to adopt the protocol from the laboratory of National Taiwan
University. Adequate and appropriate laboratory equipment, apparatus, and reagents
should be available since the test requires utmost precision. All these will help ensure
accurate results.

Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

LITERATURE CITED

CALAVAN, E.C. ,CHRISTIANSEN, D.W. and ROISTACHER, C.N.1963. Symptoms
associated with tatterleaf virus infection of Troyer citrange rootstock. Plant
Disease. Rep., 47: 971 -975.

GARNSEY, S.M. 1974. Mechanical transmission of a virus that produces tatterleaf
symptoms in Citrus excelsa. In Proc. 6th Conf. IOCV,. Gainesville, Univ. Fla.
Press. Pp. 137-140

HERRON, C. AND M. SKARIA(N.D) Texas A&M University-Kingsville Citrus Center
312 N. International Blvd, Weslaco, TX 78596 retrieved July 25, 2006 from
http://www.fao.org/docrep/T0601E/T0601E0d.htm#Tatterleaf.

INOUE, N.; MAEDA, T.; MITSUHATA, K.1979. Citrus tatter leaf virus isolated from
lily. Annals of the Phytopathological Society of Japan 45, 712-720.

KOIZUMI, M.1984. Elimination of tatterleaf-citrange stunt virus from satsuma mandarin
by shoot-tip grafting following pre-heat treatment. In Proc . 9th Conf. IOCV, p.
229-233. Riverside, IOCV.

MIYAKAWA, T. 1978. A bud-union disorder of Japanese ;citrus on Poncirus trifoliata
Rootstock caused by tatter leaf virus Review of Plant Protection Research 11, 1-
10.

MIYAKAWA, T. 1980. Thermo-therapy for some citrus cultivars infected by tatter leaf
virus. Bulletin of the Tokushima Horticultural Experiment Station 9, 7-11.

NAMBA, S. 1995. Capillovirus genus. Archives of Virology, Supplement 10, 465-467

NAMUJHE, A.C.,JR. 2006. Status of the citrus industry in Kasibu (personal interview).

NAVARRO, L.; CIVEROLO, E.L.; JUAREZ, J.; GARNSEY, S.M.1989. Improvement
therapy methods for citrus germplasm exchange. In: Abstracts of the 11th
Conference of the International Organization of Citrus Virologists. Orlando,
Florida, USA.

NISHIO, T.; KAWAI, A.; KATO, M.; KOBAYASHI, T. 1982. A sap-transmissible
closterovirus in citrus imported from China and Formosa. Research Bulletin of the
Plant Protection Service Japan 18, 11-18.

NISHIO, T.; KAWAI, A.; TAKAHASHI, T.; NAMBA, S.; YAMASHITA, S. 1989.
Purification and properties of citrus tatter leaf virus. Annals of the
Phytopathological Society of Japan 55, 254-258
Incidence of Citrus Tatterleaf Disease in
Kasibu, Nueva Vizcaya / Josephine L. Namujhe. 2007

38
OHIRA, K.; ITO, T.; KAWAI, A.; NAMBA, S.; KUSUMI, T.; TSUCHIZAKI, T. 1994.
Nucleotide sequence of the 3'-terminal region of citrus tatter leaf virus RNA.
Virus Genes 8, 169-172.

OHKI, YOSHIKAWA, INOUYE & INOUYE.1989. Annals of the Phytopathological
Society of Japan 55: 245

PAYOT, J.A., F.L. PAMPLONA and Z.C. GIBE.1987. State of the art and abstract
Bibliography of citrus research. Department of Science and Technology Crops
Philippine Agriculture and Resources Research Foundation Inc. Los Baños,
Laguna. Pp.1-5.

ROISTACHER, C.N.; KITTO, S.L.1977. Elimination of additional citrus viruses by
shoot-tip grafting in vitro. Plant Disease Reporter 61, 594-596.

ROISTACHER, C.N., E.M. NAUER, AND R.C. WAGNER.1980. Transmissibility of
cachexia, dweet mottle, psorosis, tatter leaf and infectious variegation on knife
blades and its prevention, pp225-229. In: Proc. 8th Conf. IOCV., IOCV, Riverside

SCHWARZ, R.E.1966. Mechanical transmission of a virus from Meyer lemon to
herbaceous hosts. South African Journal of Agriculture Science 9, 263-264.

SU, HONG-JI and MEI-CHEN TSAI. 1990. Distribution and detection of citrus tatter
leaf virus by ELISA test with monoclonal antibodies. Proceedings of the 4th
International Asia Pacific Conference on Citrus Rehabilitation. 4-10th
February,1990, Chiang Mai, Thailand. Pp.171-174.

SU, HONG-JI (2005). Technology in Production and Indexing of Pathogen-free Citrus
and Banana Seedlings. Department of Plant Pathology & Microbiology, National
Taiwan UniversityTaipei, Taiwan

TAKAHARA, T.; KAWASE, K.;ONO, S.;IWAGAKI, I.; HIROSE, K.;YOSHINAGA K.
1988. Rootstocks for Ponkan (Citrus reticulata Blanco) in relation to tatter leaf
virus. Bulletin of Fruit Tree Research Station D 10, 35-45.

WALLACE, J.M. and DRAKE, R.J. 1962. Tatterleaf, a previously undescribed virus
effect on citrus. Plant Dis. Rep., 46: 211 -212.

WALLACE, J.M.; DRAKE, R.J.1963 New information on symptom effects and host
range of the citrus tatter-leaf virus. Plant Disease Reporter 47, 352-353.

ZHANG, T.M., LIANG, X.Y. and ROISTACHER, C.N.1988. Occurrence and detection
of citrus tatterleaf virus (CTLV) in Huangyan, Zhejiang Province, China. Plant
Disease, 72: 543-545.

Document Outline

Incidence of Citrus Tatterleaf Disease in Kasibu, Nueva Vizcaya
- BIBILIOGRAPHY
- ABSTRACT
- TABLE OF CONTENTS
- INTRODUCTION
- REVIEW OF LITERATURE
  - The Disease
  - The Pathogen
  - Biology
  - Symptoms
  - Host Range
  - Geographical Distribution
  - Economic Impact
  - Methods of Detection
  - Control
- MATERIALS AND METHODS
  - Survey
  - Observation and Documentation of Disease Symptoms
  - Sample Collection
  - Detection by Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR)
- RESULTS AND DISCUSSION
  - Areas of Occurrence
  - Symptoms of Citrus Tatterleaf Disease
  - RT-PCR Results
- SUMMARY, CONCLUSION AND RECOMMENDATIONS
  - Summary
  - Conclusion
  - Recommendations
- LITERATURE CITED