SURVEY, STRAIN IDENTIFICATION AND MANAGEMENT OF HUANGLONGBING

(HLB) DISEASE OF CITRUS IN THE PHILIPPINES

Juliet M. Ochasan1, Nancy T. Aspuria2, Maria Arlene F. Celo3

Amelia Cimafranca3 and Matilde Q. Gumtang3, Rogelio G. Custodio4

ABSTRACT

Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus, is the major

cause of the decline of the citrus industry in the 70’s. This project aimed to come up with a
comprehensive data on the extent of HLB incidence, its strains, the insect vector, management
strategies.

The survey was conducted in 168 citrus orchards covering 12 regions. HLB was found in
102 orchards (60.71%), approximately 15,044 trees (7.04%). The disease was identified based on
the typical symptoms and was further validated through Iodine Starch test and Polymerase Chain
Reaction (PCR). With Iodine Starch test, 231 or 85.23% of the samples showed positive reaction,
while 11 was positive out of 64 samples with PCR test. The insect vector, Diaphorina citri
Kuway., or Asian Citrus psylla (ACP) was found in 20 (11.4%) orchards. HLB Strain II is the
predominant strain with 83.33% of the isolates tested.

Growers practice spraying of pesticides, removal of diseased trees, and use of healthy
plant materials. However, most growers are unaware of the disease and its vector.

An area wide management of HLB is recommended to sustain the existing and

economically viable citrus orchards in the country not only against HLB but other pests and
diseases.

INTRODUCTION

Citrus (Citrus spp.) is one of the important commercial fruit crops grown in the
Philippines. It ranks fourth to banana, mango and pineapple in terms of area and production. It is
an important source of income for many small farmers. However, since 2008, the area planted to
citrus has been decreasing and at present it stands at 36,402 hectares.

It has been declared that Huanglongbing (HLB), locally called Leaf Mottling is the most
important, severe, serious, destructive, devastating disease of citrus in the world and one of the
most difficult to control (Gottwald, et al, 2007). It was reported in the country in 1921, and
between 1961 and 1970, citrus production was reduced to 60%, with a loss of over one million
trees. Molecular tests conducted on Philippine citrus confirmed the presence of HLB pathogen
particularly the Asian strain, Candidatus Liberibacter asiaticus (Harakava, et al, 2000). Besides
high production costs and low yields, citrus production also suffers from serious problems of
_____________
1
Project Leader/Supv. Agric 2Senior Agric 3Agric. II 4Lab Tech. I, Baguio National Crop Research Development
and Production Support Center, Baguio City
HLB disease, so that the life span of trees is often relatively short. Yield reductions caused by the
early drop and low quality of the affected fruits, have been reported to reach 100% depending on
disease severity. HLB is especially destructive on sweet orange, mandarin, tangelo and grapefruit
trees, irrespective of rootstocks, and whether the trees are grafted or are seedling trees.

The classic symptom of the disease that is often the first to be observed during surveys is
the presence of yellow shoots in some sectors of the tree, while the other parts of the tree remain
symptomless. And the most important diagnostic symptom is the blotchy mottle, which is
observed as dark or light green patches that are not symmetrical on both sides of the mid vein,
being especially evident on those branches located in the interior canopy. In addition, diseased
trees appear stunted when compared to healthy trees. As the disease progresses, as a consequence
of the movement of the pathogen inside the plant, other branches of the tree become yellow,
followed by necrosis or dieback. Sparse foliage can be observed due to severe leaf drop.
Additionally, off-season blossom can occur. On fruits, a small and lopsided shape is a
noteworthy symptom during field surveys, although the lopsided condition is not exclusive of the
HLB disease. Fruits do not color properly and color inversion on the fruit can also occur while
the stylar-end of the fruit tends to remain green. However, the characteristics of the HLB
infection with major economic impact are the reduction in the fruit size, severe and premature
fruit drop, the low content of soluble acids of the juice, and the bitter taste or off-tasting
condition of the juice. Disease symptoms are more evident during the cooler months of the year.

Tsai, et al (undated), differentiated HLB strains based on the criteria of pathological

characterization including host range, pathogenicity and virulence in Taiwan into four strains,
namely: Strain I caused severe yellow mottling and typical HLB symptoms on susceptible
Ponkan mandarin (PM) and Liucheng sweet orange (LSO) cultivars but did not infect Wentan
pummelo (WP) or Eureka lemon (EL); Strain II showed high virulence of pathogenicity in all
cultivars. It induced severe HLB symptoms, including leaf yellow-mottling and curling, vein-
yellowing and distinct dwarfing. Strain III, the intermediate virulent strain, infected PM, LSO
and WP, but not EL, and caused leaf-chlorosis with mild vein enation. No yellow mottling or
typical HLB symptoms were induced by strain III; and Strain IV was a mild strain infecting PM
and LSO without symptoms and did not infect WP or EL. This mild strain was not commonly
isolated.

The disease is transmitted by Diaphorina citri Kuway or the so called the Asian Citrus
Psylla (ACP). Infected trees show yellowing of shoots; small and mottled leaves, small and
lopsided fruits with aborted seeds, dieback of twigs and branches, and eventually, tree death. The
severe disease problem is related to the presence of a large number of diseased trees serving as
sources of inoculum and a constantly high population of the insect vector. Once the tree is
infected, there is no cure. Transmission of the HLB agent by the Asian citrus psyllid vector,
Diaphorina citri, was reported in 1967 in the Philippines (Martinez and Wallace, 1967). Psyllids
are small insects, 3-4 mm in length with a yellowish-brown body, greyish-brown legs, and wings
are clear and mottled with brown coloration. The adult is often found on buds and young leaves
where it sucks the sap, causing leaf distortion and curling. They are found especially on the
lower side of the leaf, with their heads almost touching the leaf surface and their bodies held at a
45° angle, which facilitates their recognition during surveys. They feed on young stems and on
leaves at all stages of development. The presence of the Asian citrus psyllid is also determined
through the visual detection of notchings or malformations on young or mature leaves that are
caused during feeding by this insect on the host tissue. This direct damage is itself considered to
be of serious consequences for the plant, since the new shoot growths that are heavily infested by
the psyllids do not develop normally. However, the probability of the plant to become infected
with the citrus greening bacterium is the most dangerous and economically devastating
consequence.

Currently, diagnosis of the disease relies on PCR analysis; however, due to the complex
nature of the disease, some common PCR techniques have not been able to detect the bacterium.
The spatial dynamics of HLB have been investigated by Gottwald et al.(2009) in Florida where
he stated that higher concentrations of symptomatic trees are initially found at the borders of
blocks (edge effects). Some evidence of clustering or aggregation among immediately adjacent
diseased trees was demonstrated, especially within-row aggregation. It is recommended that
during survey, special attention should be given to trees located on the perimeter or in areas that
favor the presence of the vector.

According to Hong, et al (2002), when plants are infected with HLB disease, the
accumulation of starch in leaves was 16-20 times higher than that of other virus or stressed citrus
plants. Iodine reacts with starch to form a dark blue purplish color. The protocol for the iodine
starch test was later developed by Su (2008).

Gottwald (2010) stated that at present, there is nowhere in the world where HLB is under
adequate control, and where the disease does occur, it continues to increase in incidence and
severity. Although the first line of defense has always been quarantine to ensure the bacteria is
not introduced and established. However, with ever-increasing international trade, travel, and
immigration, the probability of unintentional introduction continues to rise. During the 1980s and
1990s, the United Nations Development Programme Food and Agricultural Organization
(UNDPFAO) conducted a multinational Southeast Asian citrus rehabilitation project where they
came up with a compilation of an overall strategy for HLB management based on experience of
commercial producers and agricultural agencies, which remains the cornerstone of HLB disease
control today. The major recommendations are: a) Control of psyllid vectors in commercial
plantings by chemical insecticides and, where applicable, via biocontrol to reduce transmission;
b) Removal of HLB-infected trees in commercial plantings to reduce inoculum sources; c)
Geographical isolation and disease certification programs for budwood sources; d) Geographical
isolation of nursery production. Chemical control of HLB psyllid vectors requires multiple
sprays yearly to reduce and maintain low vector populations. The effectiveness of rouging (i.e.,
removing) infected trees to control HLB is directly related to latency of infection. Although
diseased tree removal is recommended, it is recognized that numerous infected but asymptomatic
trees probably exist in the vicinity and that these early stage asymptomatic trees contribute to
pathogen dispersal.

Despite the disease threat however, several countries have demonstrated that citrus can
still be grown profitably through integrated control measures such as the use of HLB-free
seedlings, elimination of inoculum sources, and prevention of secondary spread by vector insects
(Aubert, B. et al, 1996). In view of the latest incursion of the disease and its vector in the major
citrus production in the world, it is imperative to support local citrus production to fill in local
demand. There is a need to understand the current orchard scenario in terms of the disease
incidence, its vector and the management strategies in order to propose development initiatives
for the rehabilitation and expansion of the citrus industry.

OBJECTIVES

General: To come up with a comprehensive data on the extent of HLB incidence, its
strains, the insect vector, management strategies and other pests of citrus in the Philippines.

Specific Objectives:
1. To determine the incidence of Huanglongbing (HLB) in the Philippines.
2. To determine the incidence of the Asian citrus psylla (ACP).
3. To characterize and establish the range of symptoms of HLB-infected trees.
4. To validate the use of starch iodine test and Polymerase Chain Reaction (PCR) in disease
diagnosis.
5. To identify the HLB strains present in the country.
6. To know the management strategies by growers against on HLB.
7. To identify other prevalent pests and disease affecting citrus.

METHODOLOGY

Preliminary data on citrus production was obtained from Bureau of Agricultural Statistics
(BAS), while the names and contact numbers of citrus growers were obtained from the
Department of Agriculture-Regional Field Unit (DA-RFUs) offices, Offices of the Provincial and
Municipal Agriculturists. Prior to this, a letter of request was prepared and sent to appropriate
authorities in the region, province or municipalities. Electronic data access was also done
through their websites. Whenever possible, discussion meetings with growers were conducted in
coordination with the provincial or municipal LGU’s. Primarily, the meeting was conducted to
inform the participants by way of a presentation or lecture about the Huanglongbing disease and
its insect vector. After the presentation, the growers were asked to fill-up a survey questionnaire.
The questionnaire included the following information: a) Name of the citrus grower, b) Address,
c) Contact Number, d) Area/no of trees planted, Variety/ies planted, Source of planting
materials, Year planted/age of trees, Matrix(checklist) on HLB symptoms, Approximate number
of trees exhibiting two or more of the above symptoms, Observation on the presence of the Asian
Citrus Psylla (ACP), Management practices implemented, and other pests and diseases observed.

Field survey and validation. Field validation was conducted through visual assessment and
documentation of symptoms. The exact location of the farm was established by taking the GPS
coordinates using a tablet. Actual field walks was done around the orchards, with emphasis on
the borders, since it was demonstrated that the disease usually start along the borders (edge
effect) and HLB-infected trees tend to aggregate (Gottwald et al, 2009). Besides visual
assessment of symptoms, representative leaf samples were validated through Starch Iodine test
using the protocol developed by Su (2008). The presence of ACP was also noted through visual
search. Adults are collected by an aspirator and preserved in a vial containing 70% ethanol.

Detection of HLB by Polymerase Chain Reaction (PCR). Samples of symptomatic tissues

were collected, properly labeled and stored for detection of HLB pathogen in the laboratory. The
PCR protocol developed by Hung et al, 1999 was followed (Su, Hong-Ji, 2008). Briefly the
procedure is as follows: 1) DNA was extracted from leaves and midribs using a mortar and
pestle with a DNA extraction buffer. The tissue suspension was incubated in a water bath for 1
hour. Afterwards the supernatant was taken and mixed with 5M NaCl and CTAB, and incubated
again for 10 min at 650C. The Chloroform/isoamyl mixture (24:1) was added and centrifuged at
12,000 rpm for 10 min; then the aqueous portion was taken. A similar volume of isopropyl
alcohol added to precipitate the nucleic acid. The solution was centrifuged for 15 min at 4oC.
The pellet was washed with 70% ethyl alcohol to remove the residual CTAB, dried, resuspended
in TE buffer, and stored at -20oC. 2). For the PCR reaction mixure, a 25 ul reaction mixture was
prepared with ddH20, 10X Taq buffer, 50mM MagCl2 , 2.5mM dNTPs, Primers, Taq polymerase
and DNA template. The following PCR cycles was followed: step 1: 94 oC for 3 min; Step 2: 94
o
C for 1 min, 60oC for 1 min, 72 oC for 2 min, 30 cycles. 3) A 1.4% agarose in gel was prepared
in 0.5 X TAE buffer. Ten microliter of PCR products are loaded into each well and the gel was
allowed to run in an electrophoresis chamber at 100 volts for 30 min. The gel is stained observed
under UV light.

Strain Identification of Huanglongbing Disease. Identification of HLB strains was done

by pathogenicity test following the protocol by Tsai, Hung and Su (2008). Four differential host
plants namely: Ponkan mandarin, Hamlin sweet orange, Magallanes pummelo and Eureka
lemon were used. Healthy seedlings of these differential host plants were reared under an insect-
proof screenhouse. Seedlings of about 15 cm tall were inoculated by grafting with the diseased
samples. For each HLB isolate, four plants were used. The test seedlings were grown in an
insect-proof screenhouse. Symptom development was observed and recorded each month after
inoculation. The test plants were further analyzed by PCR.

RESULTS AND DISCUSSIONS

Areas surveyed

Table 1 shows the areas surveyed covering 12 regions, namely: Cordillera

Administrative Region (CAR), Regions I (Ilocos), II (Cagayan Valley), III (Central Luzon), IV-
A (CALBARZON) , IV-B (MIMAROPA), V (Bicol), VI (Western Visayas), IX (Zamboanga
Peninsula), X (Northern Mindanao), XI (Davao) and XII (SOCCSKSARGEN). Region IV-B
has the most number of orchards surveyed followed by Region II and Region I. This is so
because, majority of the orchards in these areas are adjacent to each other, hence more number of
orchards were surveyed. In terms of area planted, Regions XII, had the largest area followed by
Region II and III, while Region VI had the least. This is due to a number of large corporate
farms planted with pummelo. On the other hand, Region III had the most number of trees planted
due to the high density planting of calamansi trees. Although Region XII has the largest area in
terms of hectarage, but there are fewer trees since majority of the plantings are on pummelo,
which requires wider spacing. Roughly, about 4% of the total farms surveyed are large or
corporate farms (> 20 hectares). In addition to the orchards, eight citrus nurseries were also
surveyed, all of which are located in Luzon. The majority of citrus groups planted are: calamansi
(x Citrofortunella microcarpa (Bunge) Wijnands), pummelo (Citrus maxima (Burm.f.) Merr.),
mandarins (Citrus reticulata Blanco), sweet oranges (Citrus sinensis (L.) Osbeck.), and lemon
(Citrus limon (L.) Burm.f.). Calamansi, pummelo, and mandarins (Szinkom and Ladu) are the
predominant citrus planted, while sweet oranges, mandarins like ponkan and satsuma are mostly
planted in CAR and Region II. Lemons, on the other hand are almost exclusively planted in
CAR.

Table 1. Number of orchards, area (has), number of trees and major citrus varieties surveyed in
the different regions.
No. of Orchards Area Number Major citrus
Region
Surveyed (has) of trees varieties/groups planted
Swt. oranges, mandarins
CAR 20 15 6,500 (ponkan, gayunan),
lemons
Region 1 24 31 12,900 Calamansi, pummelo
Swt. Oranges (Hamlin,
navels), mandarins
Region II 26 90 21,600
(ponkan, szinkom),
calamansi, pummelo
Region III 11 115.5 53,600 Calamansi
mandarins (szinkom,
Region IV-A 5 23 4,050 ladu), calamansi,
pummelo
Calamansi, pummelo,
Region IV-B 28 76 38,254
mandarin (szinkom)
Region V 11 11.04 4,070 Calamansi, pummelo
Region VI 11 6.5 3,065
Calamansi, pummelo
Region IX 9 19 6,240
Calamansi, pummelo
Region X 9 29 14,029 Calamansi, pummelo
Region XI 10 170 34,650 Pummelo Calamansi,
Region XII 4 229.5 14,800 Pummelo, Calamansi,
Total 168 815.54 213,758
 Fig. 1 Overview of orchards surveyed: Left - Mandarin ponkan orchards in Region II
(Malabing Valley, Kasibu, Nueva Viscaya); middle – Pummelo orchard in Region XI
(Davao); right – Calamansi orchards in Region IX (Siay, Zamboanga Sibugay)

Incidence of HLB and its vector, Asian Citrus Psylla (ACP).

Table 2 shows the incidence of HLB and ACP based on the number of orchards and trees
surveyed. Moreover, Fig. 2 shows the geographical distribution of the various orchards surveyed
represented by colored dots to denote three orchard scenarios which are: orchards where HLB
and ACP are not present; where HLB is present but no ACP; and where both HLB and ACP are
present. Based on number of orchards surveyed, Regions IV-A and X showed 100% incidence,
which means all of the farms surveyed have trees showing typical symptoms of HLB, followed
by Regions I, III and XI with 79%, 72% and 70%, respectively. Over all, the HLB incidence
based on the number of orchards surveyed is 60.24%.

If terms of the number of trees exhibiting typical symptoms of HLB in the orchard (Fig.
3), Region I had the highest with 21.71% followed by Bicol region with 16.22% and CAR with
13.97%, while Region XI has the least with 0.25%, followed by Region IX and Region X with
1.31% and 4.03%, respectively. Overall, the HLB incidence in the country based on the number
of infected trees if 7.04% or 15,044 trees out of the 213,758 trees surveyed.

Also noted during the survey is the incidence of the insect vector, Diaphorina citri, or the
Asian Citrus psylla (ACP) (Fig. 4). ACP was found in 21 or 12.05% of the total orchards
surveyed. During the survey, no ACP was found in Regions VI, IX, XI and XII. On the other
hand, Region IV-A had the highest incidence of ACP (60%), followed by Region III and Region
I, with 36.36% and 20.83%, respectively. Moreover, the population of ACP in most of the farms
surveyed were very low (about 2 adult psyllids, few nymphs and found only in few trees), except
in one nursery in Palawan, Mt. Province and one farm in Mindoro. However, in some orchards
where the ACP was not found, notchings or malformations on the young or mature leaf, which is
the characteristic symptoms of ACP infestation caused during feeding on the host tissue, were
observed (Fig. 3). One of the limiting factors on the observation of the ACP is the timing of the
survey. In several occasions, the survey was timed when the flushes have already matured;
hence they are no longer attractive to ACP. In some cases, the time of the day and weather
conditions also limited the observation of the insect.

Table 2. Number of orchards and trees surveyed, number and percentage of orchards and trees
with HLB, and number and percentage of farms with ACP in the different regions.

No. of No. of HLB incidence ACP incidence

Region orchards trees No. of No. of % No of %
%
orchards trees orchards
CAR 20 6,500 8 40.00 908 13.97 2 10.00

Region 1 24 12,900 19 79.17 2,800 21.71 5 20.83

Region II 26 21,600 15 57.69 1,590 7.36 1 3.85

Region III 11 53,600 8 72.73 4,240 7.91 4 36.36

Region IV-A 5 4,050 5 100.00 370 9.14 3 60.00

Region IV-B 28 38,254 11 39.29 1,669 4.36 3 10.71

Region V 11 4,070 6 54.55 660 16.22 1 9.09

Region VI 11 3,065 8 72.72 225 7.34 1 11.11

Region IX 9 6,240 5 55.56 82 1.31 0 0.00

Region X 9 14,029 9 100.00 565 4.03 1 11.11

Region XI 10 34,650 7 70.00 85 0.25 0 0.00

Region 12 4 14,800 1 25.00 1,850 12.50 0 0.00

Total 168 213,758 102 15,044 21
Percentage 60.71 7.04 12.05
Fig. 2 Areas surveyed on HLB and ACP incidence.
Legend:
- indicates orchards with no HLB symptoms and no Asian Citrus Psylla (ACP) observed
- indicates orchards with HLB symptoms but no ACP observed.
- indicates both the presence of HLB and ACP
 1 2 3 4

5 6 7

Fig. 3. HLB symptoms: whole tree symptoms (1), showing yellowing on one part
of the tree; symptoms on the leaves showing leaf mottling/blotchy mottle (2),
yellowing (3), narrow, erect leaves (4) ; vein corking (5);small, lopsided fruit on
pummelo (6); small, misshapen/malformed fruit showing inverted coloration as
compared to healthy fruits of mandarin ponkan (7).

Fig. 4. Left photo - Adult ACP on the leaf (inset, enlarged D. citri adult) ; middle
photo - nymphs of Diaphorina citri on new shoots; right photo – leaf
malformation (notchings) due to ACP feeding
 Most often, the initial presence of HLB in the orchard is due to infected planting
materials, particularly on calamansi where marcotting is still practiced. Under this condition, the
spread of HLB even with a single infected tree in the orchard and in the presence of the insect
vector is inevitable. This is compounded by the fact that symptoms of HLB may take months to
several years from the time of infection until the tree shows visible symptoms of the disease (Lee
et al, 2015). The free interaction of HLB and the insect vector in orchards where there is no
strategy for management often results in the high number of infected trees within the orchard.

Detection of HLB

HLB detection through symptomatology. Field identification of HLB is based on the

presence of typical symptoms exhibited on leaves, fruits and the tree as a whole (Fig. 3). For this
survey, the tree is considered to be infected with HLB if it exhibits at least three of these
symptoms such as: sectoral yellowing or yellowing on one side of the tree canopy; leaf mottling
or blotchy mottle; small, pointed, erect, and light colored leaves; vein corking; few, small fruits
that are lopsided fruits with inverted coloration and aborted seeds. Other symptoms were also
observed like twig dieback, off-season flowering, sparse foliage and dying trees. Some of these
symptoms are not always present in one tree as it depends on the age of the tree, tree health,
variety, distribution of the pathogen on the tree, environmental conditions and populations of
psyllids. Bassanezi et al (2011) showed that the strong linear relationship between relative
number of fruits per tree and the relative yield per tree suggesting that the yield reduction was
due primarily to early fruit drop or lack of fruit set on affected branches. Among citrus varieties,
the most susceptible are mandarins and sweet oranges. The symptoms of HLB on these varieties
are very striking and once infected these varieties show severe symptoms and declines more
rapidly. On calamansi, although they are also susceptible, early symptoms are not easy to
observe. Also, the symptoms on the fruit are often overlooked since calamansi fruits are small,
and any malformation on the fruit is not easily recognizable. In contrast, on pummelo, the quality
of the fruit is of primary concern since infected trees show lopsided fruits and have a bitter taste.

HLB detection by Iodine Starch Test. Representative samples of leaves with varied
symptoms were tested using Iodine Starch test. According to Hong, et al (2002), when plants are
infected with HLB disease, the accumulation of starch in leaves was 16-20 times higher than that
of other virus or stressed citrus plants. Iodine reacts with starch to form a dark blue purplish
color (Fig. 5).

Of the 271 leaf samples tested, 231 or 85.23% reacted positively to the test, while the rest
of the samples reacted negatively or had an unspecific reaction (Table 4). Researchers in Japan
and Vietnam reported up to 90% agreement between PCR analysis and starch iodine test
(Exteberria, at al, 2009). This suggests that starch iodine test could be used as a decision tool in
eliminating infected trees.
 + -

b c
a a
a

d e
f
a a

Fig. 5. Iodine starch tests conducted on different citrus varieties showing typical leaf
symptoms: a – shows +/- reaction; b –mandarin ponkan; c-sweet orange; d –pummelo;
e- lemon; f - calamansi

Table 3. Results of iodine starch test on representative samples collected from diff. regions.
No of samples Number of
Region Percentage
collected positive samples
CAR 47 40 85.10%
Region 1 (Ilocos region) 25 21 84.00%
Region II (Cagayan
35 28 80.00%
Valley)
Region III (Central
45 41 91.11%
Luzon)
Region IV-A
11 10 90.90%
(CALABARZON)
Region IV-B
28 23 82.14%
(MIMAROPA)
Region V (BICOL) 18 16 88.88%
Region VI (Western
14 12 85.71%
Visayas)
Region IX (Zamboanga
10 8 80.00%
Peninsula)
Region X (Northern
15 13 86.66%
Mindanao)
Region XI (Davao) 15 12 80.00%
Region 12
8 7 87.50%
(SSOCCSKSARGEN)
Total 271 231 85.23%
HLB detection by Polymerase Chain Reaction (PCR). Representative samples were also tested
PCR to confirm the presence of the HLB pathogen, Candidatus Liberibacter asiaticus. Of the 64
samples tested by PCR, only 11 samples showed positive amplification, showing DNA bands of
228 bp, which corresponds to the expected product of HLB primers used. Samples which showed
positive amplification were collected from Baguio City (2), Mt. Province (4) and Laguna (2),
Mindoro (2) and Nueva Ecija (1) (Fig. 6). The low level of PCR detection is attributed to the
poor condition of the leaf samples prior to PCR analysis. In some cases, the leaf samples show
signs of deterioration since these were collected several weeks prior to PCR analysis, most likely
affected the quality (titer) of DNA. Gottwald (2010) stated that although PCR is the current
definitive method in HLB detection, it is subject to false negatives due to incomplete distribution
of the bacterium in trees and even among cells within tissues, yielding both positive and negative
samples from various tissue samples originating from the same tree. In view of these, one
probable aspect for optimization is the DNA extraction procedure, since several inhibitors or
certain metabolites present in the citrus tissues may need to be eliminated. Other PCR
components needs to be optimized since these were sourced from various suppliers and may
require specific conditions for it to work.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 M M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 M
M

228bp

A B

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 M
Fig. 6 Detection of HLB pathogen by
Polymerase Chain Reaction (PCR)
followed by Polyacrylamide gel
electrophoresis (PAGE) analysis of citrus
leaf samples from various locations. A:
Lanes M – Marker (20 bp DNA ladder),
lanes 6&7 show bands corresponding to
HLB primer of 228 bp; B: lanes
15,16&20 show bands corresponding to
HLB primer of 228 bp; C: lanes 1, 3,4,
11,16&17 show DNA bands
C corresponding to HLB primer of 228 bp).
Strain Identification of Huanglongbing Disease of Citrus

Forty three isolates were collected from different locations and varieties during the
survey and were graft-inoculated on the various differential hosts. Of the 43 isolates, 31 had
successful grafts and developed symptoms while the graft inoculation on the rest of the isolates
was unsuccessful, which is probably due to the condition of the scion prior to grafting, and some
test plants were inadvertently irrigated by sprinkling just after graft-inoculation such that water
seeped through the grafted portion which led to the rotting of the tissues. For the successfully
grafted plants, typical symptoms of HLB appeared between 8 to 16 months after graft-
inoculation. Eighteen months after graft-inoculation, the plants were rated based on their
symptom expressions on the various indicator hosts (Fig. 7). Results showed that 26 isolates or
83.87% showed moderate to severe symptoms on all the indicator plants, hence they are
classified under Strain II. On the other hand, only 3 or 11.53% and 2 or 6.45% were classified
under Strain I and Strain III, respectively (Table 5). None of the isolates was classified under
Strain IV.

Representative samples were analyzed by PCR. Of the 20 isolates, only 14 isolates were
further tested after DNA extraction, that is, those samples with DNA titer of at least 900 ng/ul
based on the results of the quantification using spectrophotometer (NanoDrop). Of the 14
isolates, only six showed DNA amplification. These are: Isolate No. 1 (Caffin from Baguio
City), Isolate no. 3 (calamansi from Region X in Lanao Del Norte), Isolate no. 7 (collected from
navel orange from Baguio City), Isolates no. 15 (mandarin ponkan), 16 (gayunan mandarin) &
19 (honey mandarin) were collected from Bontoc, Mt. Province (Fig. 8). As mentioned earlier in
the report, further optimization is needed due the low detection level.

Management strategies on HLB

In many parts of the world where the disease is present, the dominant control measure
currently employed is the combination of insecticidal spraying to limit the psyllid populations
and removal of infected trees which serve as inoculum source. Unfortunately, during the survey,
citrus growers in general are not aware of the HLB disease and its vector when they were asked
about their management strategies against HLB. Thus, the orchard practices that they are
implementing are not particularly targeted towards the management of HLB and its vector, but
citrus production in general. For example, in most large or corporate farms in Region XI, they
implement a regular spraying program against pests in general, thus ACP is also controlled, since
ACP could be easily controlled by the use of common insecticides such as pyrethroids. However,
for most growers although they spray their trees from time to time if they observe some pests,
since they are not aware of the insect vector, they often miss the critical period where ACP is
abundant, hence HLB continues to spread within the farm. Some growers are also keen in
producing their own plant materials by carefully selecting seemingly healthy trees, while some
obtain their plant materials from accredited sources. In addition, some growers especially those
who implement a good nutrition program through adequate fertilizer application mentioned that
they could readily notice poorly performing trees or trees that do not seem to respond to fertilizer
application, so they readily remove these trees since they are not any more economical to
maintain. However, according to Lee, et al (2015), this strategy has had only marginal effect
because symptoms appear anywhere from months to years after an initial infection, long after the
Table 4. Summary results of HLB strain identification and PCR reaction.
No. of
No. of HLB Strain* PCR
Location/Cultivar isolates rxn.
isolates I II III IV
with HLB
Baguio
Lane late 1 1 0 1 0 0 +
Washington Navel 1 1 0 1 0 0
Kumquat 1 1 0 1 0 0
Caffin 1 1 0 1 0 0 +
Ortanique 1 1 0 1 0 0
Fairchild 1 1 0 1 0 0
Mountain Province
Mandarin Ponkan, 2 2 0 1 1 0 +
Honey mandarin 2 2 0 2 0 0 +
Gayunan mandarin 2 1 0 1 0 0 +
Queen Mandarin 2 2 1 1 0 0
Kumquat 1 1 1 0 0 0
Laguna
Szinkom mandarin 3 3 1 1 1 0
Oriental Mindoro
Calamansi 3 2 0 2 0 0
Palawan
Calamansi 2 0 0 0 0 0
General Santos
Pummelo 2 2 0 2 0 0
Davao
Pummelo 3 2 0 2 0 0
Nueva Viscaya
Szinkom mandarin 3 1 0 1 0 0
Navel orange 4 2 0 2 0 0
Bukidnon
Calamansi 3 2 0 2 0 0
Lanao Del Sur
Calamansi 2 1 0 1 0 0 +
Zamboanga
Pummelo 2 1 0 1 0 0
Calamansi 1 1 0 1 0 0
TOTAL 43 31 3 26 2 0
Percentage 71.43% 11.53% 83.87% 6.45% 0.00%
*
Mean score of four sample plants/indicator plant. Graft-inoculation on some isolates was
unsuccessful.
 Healthy Diseased
d

1a 1b
a a

2a 2b
a a

3a 3b
a a

4a 4b
a a
Fig. 7 HLB symptoms of differential hosts: 1a & b – HLB symptoms on Mandarin
Ponkan indicator plant; 2a&b – HLB symptoms on Hamlin sweet orange indicator plant;
3a&b – HLB symptoms on Magallanes pummelo indicator plant; 4a&b – HLB symptoms
on Eureka lemon indicator plant.
 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M
M16 M M16

228 bp

A B

Fig. 8 Detection of HLB pathogen from the different isolates by PCR on agarose gel
electrophoresis, A: M – DNA marker (100bp), Lane 1 (Isolate 1-Caffin-Baguio City) and
Lane14 (Isolate 19- honey mandarin from Bontoc, Mt. Province) shows DNA band
corresponding to HLB primer pair (228 bp); B: M – marker; Lanes 3 (isolate 3 –calamansi
from Region 10, Lanao del norte), lane 7(isolate 7- Lane Late Navel from Baguio City),
lane 12 (isolate 15-mandarin Ponkan from Mt. Province), and lane 13 (isolate 16- gayunan
mandarin from Bontoc, Mt. Prov.) show DNA band corresponding to HLB primer pair
(228 bp)

trees have been active in the transmission process. Because trees do not tend to show symptoms
for anywhere from 1–2.5 y, and possibly longer, after initially becoming infected, thus emphasis
must be placed on ongoing surveillance and control of psyllids. Likewise, Morris and Muraro
(2008) stated that the most profitable management option is by removing infected trees and
replanting. They concluded that although tree removal option is deemed to be best, the
surrounding environment plays a large factor. If neighboring orchards are not actively pursuing
keeping HLB at minimal levels, there may be no viable choice to keep an orchard producing at
rates that are economically sustainable.

Other pests and diseases and management practices

Besides HLB and ACP, the most prevalent insect pests observed include aphids, scale
insects, mites, rind borer, mealy bugs, spined bug, and bark borer (Fig. 9). Rind borer infests
pummelo and calamansi fruits showing bumps and gummosis on the fruit. According to most of
growers interviewed, there was a remarkably high infestation of rind borer during the survey
done in 2013-2014.

On diseases, the most prevalent are powdery mildew, pink disease, scab, foot rot/gummosis,
and Citrus Tristeza Virus (CTV) (Fig. 10). Powdery mildew is most prevalent in high elevations
areas like in CAR and Region X (Northern Mindanao), particularly in Claveria, Misamis
Oriental. Scab on calamansi, lemons, and some mandarins is the most predominant disease
problem. Incidence of CTV is common in sweet orange, gayunan mandarin, calamansi and
pummelo. However, the effects of CTV are not severe except when trees are affected by severe
stem pitting strains, which causes stunting of trees. On the other hand, in some farms in Palawan
and Oriental Mindoro, parasitic flowering plants, locally called “agaw-agaw” were observed
affecting nearly all trees in the orchard. Affected trees show poor appearance and productivity.

Fig. 9. Common insect pests: (clockwise from top) Aphids, rind borer damage
on pummelo (inset: rind borer larvae), rind borer on calamansi, mealy bugs on
calamansi, mealy bug on sweet orange, spined bug on mandarin, white
louse/snow scale on trunk and twigs, bark borer damage on trunk (inset: borer
larvae)

Fig. 10 (Clockwise from top) Scab on lemon leaves, scab on calamansi, powdery
mildew on sweet orange, stunting due to CTV stem pitting, Phythopthora foot
rot/gummosis, dead tree due to foot rot/gummosis, pink disease on main trunk,
parasitic flowering plant (locally called “agaw-agaw”)
 One of the project initiatives particularly to increase the level of awareness of the
growers, the project developed and distributed over 500 copies of information materials to
various individual growers and institutions such as FITS Centers in the various regions.
Similarly, iodine kits (kit includes 100 ml of iodine solution, pieces of sand papers and PE bags)
were also distributed (Fig. 11)

Fig. 11 Development of IEC materials and distribution to institutions like FITS

centers like in Zamboanga (bottom left) and Experiment Stations like Nueva Viscaya
Experiment Station (DA-NVES). Starch iodine test kits were distributed as well.

CONCLUSIONS

Based on the results of the project, the following conclusions can be drawn: 1). There
still exists a viable citrus industry in the country despite 60.71% incidence of the Citrus
huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus. 2).The presence of the insect
vector of HLB, Diaphorina citri or the Asian Citrus Psyllid (ACP) in 20 (11.4%) of the orchards
surveyed, implies that HLB spread is inevitable, unless specific management strategies is
implemented. 3).The range of symptoms exhibited by HLB-infected tree as observed under local
conditions conform to those reported in countries where HLB was found. 4). HLB can be
tentatively identified in the field by symptomatology but further identification could be done
through iodine starch test, with about 85% accuracy, and also by PCR but it needs further
optimization due to the low level of detection. 5). The predominant strain of HLB in the
Philippines is Strain II, which is the most virulent strain, since it affects all citrus groups and
varieties. This implies that the disease can severely damage a citrus tree once it is affected,
irrespective of the variety. Although, some variety may take longer incubation period but they
can be a ready source of inoculum, and the insect vector may transmit the disease to other
varieties that are more susceptible to HLB. This implies further that the citrus industry in the
country faces a more severe threat since at present there are no existing resistant varieties to
HLB. 6). The various management strategies of citrus growers are not specifically directed to the
management of HLB and ACP, hence, in some orchards where both HLB and ACP are found,
HLB will likely continue to spread. 7). Besides HLB and ACP, there are other major problems
besetting the citrus industry such as insect pests and diseases which can compound the damage
caused by HLB and ACP hence complicating the present status of most citrus orchards.
 RECOMMENDATIONS

In view of the results of the project, the following are recommended:

1). Conduct an area-wide management of the disease, in selected regions in the country,
particularly in areas where there are still viable citrus industry such as in Regions II (Cagayan
Valley), III (Central Luzon) IV-B (MIMAROPA), and Region IX (Zamboanga Peninsula).
Initially, the following activities could be done: organize a technical team preferably composed
of LGU technicians, who will be trained first on the identification/diagnosis and management of
the disease; conduct an awareness campaign among growers on the disease and its vector; select
grower-cooperators who are willing to participate in HLB management. Moreover, in these
areas, the establishment of citrus nurseries must be pursued following the guidelines and policies
stipulated in Administrative Order No. 25, series of 2013, on the establishment of citrus nurseries
for accreditation and the technology on the heathy plant material production system established
by BPI-Baguio. In order to sustain the citrus industry in the country, there should be continuous
production of healthy planting materials for use in replanting diseased trees and expanding new
orchards.

2). Although the disease is widely distributed in the country, quarantine regulations still
needs to be imposed, that is, only healthy plant materials obtained from accredited nurseries and
propagules (scions) from certified mother trees must be transported. The movement and transfer
of citrus plant materials are now regulated through the issuance of BPI Special Quarantine Order
No. 1, Series of 2013, replacing the BPI Administrative Order No. 2 (Internal), which was
promulgated in 1969. This also requires training of plant quarantine staff to properly identify
the disease and use available technique for identification. Sílvia, et al (2012) stated in that
investment by government on phytosanitary policies results in high benefit-cost ratio, thus it is
commendable.

3). Encourage further research and projects on citrus, in order to further develop and
protect our citrus industry from decline. One aspect of research is on molecular detection, that is,
the optimization of PCR protocol, as well as on product development (utilization) in order to
absorb surplus during peak season, since it is often the reason for growers to neglect their
orchards due to low prices, especially on calamansi. Because we Filipinos, like the western
people, where their breakfast cannot be complete without their orange juice, we cannot do
without calamansi for our pancit, including many other ways we utilize calamansi in our daily
food preparations or as food additives, and including the many ways citrus is used for
nutraceutical and industrial purposes.

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