Study on greening in citrus fruit trees in some Northern provinces of Viet Nam and recommendations for preventive and controllable measures = Nghiên cứu bệnh lá vàng greening hại cây ăn quả có múi ở một số tỉnh phía Bắc Việt Nam và đề xuất biện pháp phòng

MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE

AND TRAINING AND RURAL DEVELOPMENT


VIET NAM ACADEMY OF AGRICULTURAL SCIENCES



LE MAI NHAT





STUDY ON GREENING IN CITRUS FRUIT TREE IN SOME NORTHERN
PROVINCES OF VIET NAM AND RECOMMENDATIONS FOR
PREVENTIVE AND CONTROLLABLE MEASURES




Branch of study: Plant protection
Code: 62 62 01 12





SUMMARY OF AGRICUTURAL DOCTORAL THESIS

HA NOi – 2014

Project completed at:
VIET NAM ACADEMY OF AGRICULTURAL SCIENCES



Scientific supervisor:
1. Dr. Ngo Vinh Vien
2. Ass. Prof. Dr. Nguyen Van Viet


Critic 1:




Critic 2:

Critic 3:





The thesis is defended at Council for Thesis Assessment at
Institutional level, held at:

Viet Nam Academy of Agricutural Sciences
At hours, day month year 2014






This thesis can be referred to at:
1. Viet Nam National Library
2. The Library Viet Nam Academy of Agricutural Sciences

1

U
1. Rationale of the research
Citrus (orange, mandarin, lime, pomelo…) is an important agricultural fruit crop
in Vietnam. In 2011, the total of citrus grown area in Vietnam has reached 124,057
ha, comprising orange and mandarin 70,300 ha, pomelo 45,000 ha, lime 18,000 ha.
Citrus produce is not only for domestic consumption but also is a high value export

commodity. Citrus culture is a fast growing industry in many tropical, temperate and
semi-tropical countries. However, the diversification of climate in citrus cultivated
areas has created favorable conditions for a growth of a number of pests and diseases,
especially greening (VLG) which has been widespreaded in most of citrus countries
and being considered as one of the main barriers for citrus development.
VLG is transmitted by the psyllid vectors and also by graft transmission such as
budwood and grafted materials. Even though initial researches have been carried out
in Vietnam but the root problem of greening has not fully understood yet. Hence,
control measures of the disease are still less effective. In order to solve such problems
and to sustainably develop citrus industry in Northern Vietnam, it is worthwhile and
necessary to conduct “Study on greening in citrus fruit trees in some Northern
provinces of Vietnam and recommendations for preventive and controllable
measures”.
2. Research objective
2.1. Objective
Identifying characteristics of VLG’s cause, development and transmission that
serve as scientific bases for building up effective preventive and controllable
solutions for citrus industry in selected Northern provinces in Vietnam.
2.2. Requirements
Capture distribution of VLG in some citrus growing regions in Northern Vietnam.
Identification of types of VLG bacteria and their distribution in selected citrus
crops in different ecological regions.
Identification of VLG transmission vectors and influent factors which could serve
as scientific foundations for developing sound measures.
3. Research scientific and practical significances
- Providing a scientific and practical documentation on VLG, including transmission
vectors, influent factors and sound preventive and controllable measures
- Identifying a field rapid testing method for VLG and thus, could timely and
effectively address the disease.
- The outcome of this research contributes to scientific and practical resources which

could be used to develop an effective control system, partly contribution to the
development, conservation and sustainability of citrus industry in Vietnam.
4. Subject and scope of research
4.1. Research subject
- Bacteria Liberibacter asiaticus – a causative agents of greening in citrus and disease
transmission psyllid Diaphorina citri Kuwayama
2

- Selected local and import fruit crops which are grown in some provinces in
Northern Vietnam, crops in Rutaceae and are hosts of L. asiaticus that causes VLG in
citrus.
4.2. Scope of research
This research studies VLG transmissions, factors influencing to the
transmission and development of VLG within the ecological of Northern provinces of
Vietnam. The research identifies bacteria of L.asiaticus causing VLG in citrus and
their distributions as well as studies effective measures to prevent and control VLG in
some Northern provinces.
5. New contributions of dissertation
- Providing a scientific source on popularity, symptom, and host of VLG in citrus in
Northern Vietnam.
- Identifying bacteria causing VLG belonging type I and II in present citrus crops in
production areas of Northern Vietnam for the first time.
- Finalizing and perfecting shoot tip grafting to clean VLG and other viruses,
contributing to complete technical procedures for virus-free grafted planting
materials.
- Evaluating VLG-resistant capability of some grafted local and import rootstocks and
recommending some measures to prevent VLG re-infection.
6. Structure of thesis
This dissertation comprises 146 pages, including the introduction 4 pages; Chapter 1.
Scientific background and literature review (36 pages); Chapter 2. Research materials,

content and methodology (19 pages); Chapter 3. Result and discussion (85 pages);
Conclusion and recommendations 2 pages with 41 data tables, 39 figures. Reference of
164 papers and articles, including 34 Vietnamese papers, 130 English papers.
Chapter 1
SCIENTIFIC BACKGROUND AND LITERATURE REVIEW
1.1. Research background
Scientists all agree that VLG is one of the most dangerous diseases for citrus in
many regions in the world. The VLG’s symptoms vary, depending on citrus crops,
original of planting materials, transmission vectors … as well as orchard cultivation
and plant protection techniques.
Many scientists have affirmed that first and foremost, the following areas of
research needed to deeply carried out in order to prevent and control this disease
effectively: epidemiology of causative agents, environmental factors influencing to
the development and transmission of the disease in the field (Matsumoto et al., 1961;
Aubert et al., 1988; Su et al., 1998; Bové et al., 2006).
Studies in Vietnam and around the global have also indicated that VLG to be able
to transmit very fast and damage crops heavily if disease infected planting materials
are being used and cultivational techniques have not been applied properly.
According to Su et al. (1998); Bové et al. (2006); Hà Minh Trung et al. (2008),
3

production techniques and use of disease-free planting materials, and early
identifying of disease-infected materials so as to exclude before planting … are
among the foremost and crucial measures to be able to prevent and control of VLG
effectively.
To sum up, studying on causative agents, method for early identifying disease-
infected material, transmission characteristics and changes of bacteria toxicity on
each type of citrus crop is very important because this is likely serving as a basement
to develop a system of integrated preventive and controllable measures to deal with
the disease in a cost-effective, technical and sustainable manner.

1.2. Review of international literature
1.2.1. VLG’s symptoms, history and distribution
VLG was firstly recorded from 1929 in China with different names such as
Huanglongbing or yellow shoot in South China. The disease is then documented in
1943, Greening in South Africa (1947), Likubin (Tawain, 1951), Leaf mottle yellows
in the Philippines, huanglongbing in Thailand (1960), Dieback in India, greening in
Myanmar (1970), Vein Phloem degeneration in Indonesia (1980), greening in Vit
Nam, Laos, Cambodia (1980), Huanglongbing in Iriomote, Okinawa (1994),
Okinawa Island, Japan (2003), in Tokunoshima, Kagoshima, Japan (2003),
Huanglongbing in Brazil (2004) and in Florida, the United States (2005). Presently,
the disease has not been found in Australia yet.
1.2.2. Diagnose and appraise VLG
Identifying the causative agent by electron microscope: The VLG’s causative
bacteria is observed by electron microscope. The VLG bacteria typically has a tinny
long stick shape and changes from young to mature cells (Huang, 1987).
Identifying the causative agent by bio-molecular method: This method uses specific
primers to diagnose VLG from fresh sample and insect vector (Hung et al., 1999,
Jagoueix et al., 1996; A. Hocquellet 1997; Subandiyah et al., 2000).
Researches on type of VLG’s bacteria: Su, (2008) had identified types of VLG’s
bacteria in Taiwan by a combined method which uses plant indicator and other
selected fruit trees with biotechnology. Since then, there are four types of VLG
bacteria have been identified. Different types cause different symptoms on those fruit
trees.
1.2.3. Researches on pathology and ecology of VLG
VLG spreads very fast and reaches more than 95% in a period from 3 to 9
years after the first symptoms found (Matsumoto et al., 1961; Aubert et al., 1988,
Bové et al., 2000b; and Gottwald, 2005).
VLG spreads out through propagation and vectored insects (Diaphorina citri
Kuwayama) in a sustainable way (Halbert and Manjunath, 2004; Pluke et al., 2008).
Skelley & Hoy (2004) have applied a method of multiplying psyllid on

Murraya paniculata L trees were not infected by the disease caused by transmitted
vector or grafted. Some of good host trees for D. citri include: Citrus reticulata
Blanco, Bergera koenigii L., Citrus maxima (Burm.) Merr., Citrus medica L., Citrus
4

taiwanica Tanaka & Y. Shimada, and Citrus aurantiifolia (Christm.) Swingle.
1.2.4. Research on disease management
Shoot tip grafting, the best method for a process of re-purification of
indigenous genetics, have been developing. Shoot tip or meristem is normally not
VLG infected by causative agents, therefore, trees developed from these shoot tips
are disease free. Shoot tip grafting is conducted by inverted T budding (Murashige et
al., 1972). Recently, the techniques is modified by a method of inverted chip budding
(Su & Chu, 1984; Chen, 2012; Jae-Wook Hyun et al., 2012).
Techniques for citrus variety improvement are applied concurrent with other
agronomical, biological and chemical measures to prevent and control of VLG.
1.3. Review of Vietnamese literature
1.3.1. VLG’s symptoms, history and distribution
VLG was identified and recorded since 1960s (Hà Minh Trung, 2005). The
development of VLG depends on cultivation conditions, method of propagation and
plant protection techniques in each region (Hà Minh Trung, 2003; Ngô Vnh Vin et
al, 2012).
ình c (1991), Hà Minh Trung (1995), Nguyn Minh Châu (2001), Lê
Th Thu Hng (2009) have described and recorded symptoms of VLG in citrus.
1.3.2. Diagnose and appraise VLG
At the early stage of VLG research in Vietnam, there were two different theories
about the cause of VLG. The first one was saying that VLG is likely biophysical
cause, whereas, the second one hypothezed that VLG is transmissible disease and
caused by micro-organisms. Thanks to modern molecular techniques, it reaffirm that
greening in citrus in Vietnam is VLG caused by psyllid (D. citri Kuwayama) as a
vector (Hà Minh Trung et al., 1996).

1.3.3. Research on pathology
Hà Minh Trung et al, (1995) have conducted researches on VLG’s transmission
through grafting, grafted budwood and vector.
Ngô Vnh Vin et al., (2009), Nguyn Vn Hoà et al., (2012) have collected and
primarily assessed the resistant capability of selected trees of Rutaceae.
1.3.4. Research on disease management
Currently, technical guidelines and procedures for shoot tip grafting have been
improving to increase the shoot tip grafted tree’s ability of disease cleanliness and
survival. Net-house systems are established to maintain elite trees and clean disease
for shoot tip grafted materials. Disease management measures are simultaneously
implemented from breeding and selection stages to improve planting materials to
cultivational, biological and chemical techniques to prevent and control disease
vectors.
Chapter 2
RESEARCH MATERIAL, CONTENT AND METHODOLOGY
2.1. Research location and period
5

Research location
- Division of Plant Pathology, Plant Protection Research Institute, ông Ngc,
 Liêm, Hà Ni
- Laboratory in National Taiwan University of Science and Technology
- Nghi Diên, Nghi Lc, Ngh An and other Northern provinces of Vietnam
Research period: January, 2010 to October, 2013
2.2. Research materials
MS environment (Murashige and Skoog), BAP, plant growth regulators used in
shoot tip grafting. Seed for rootstocks (Three leaves orange, sour pomelo, chp,
Green Orange Citrus reticulata × maxima, Cleopatra mandarin). Chemicals used in
identifying VLG causative agents.
Free disease citrus trees (Cam Canh, Xã oài, Din pomelo, Eureka lime, Thanh

Yên lime); rootstocks (sour pomelo, chp) and other citrus belonging to Citrus and
wild trees of Rutacea.
Small net-frame 1m × 1m × 1m, container and a system of three-level net-house;
substrates (sand, sawdust and micro-organic fertilizer). Antibiotics (Streptomicine)
and chemical Confidor 100SL (Imidacloprid)
2.3. Research content
2.3.1. Research on VLG’s popularity and symptoms
2.3.2. Research on assessment of VLG’s causative agents and VLG’s transmission
2.3.3. Research on identification of VLG’s host and VLG’s bacteria distribution on
citrus in some Northern provinces of Vietnam
2.3.4. Research on some promising scientific and technological techniques to prevent
and control of VLG citrus fruit trees.
2. 4. Metho dology
2.4.1. Methods for researching on VLG’s popularity and symptoms
Method of on-field survey for disease ratio and popularity following Plant
Protection Research Methods, Volume I, Dang Vu Thi Thanh and Ha Minh Trung,
1997.
Method for identification of types of symptoms of VLG in some Northern
provinces of Vietnam following Matsumoto et al., 1961, Su et al., 2008; Hà Minh
Trung et al, 1995.
Method of surveying psyllid (Diaphorina citri Kuwayama) in citrus fruit tree
following Phm Vn Lm (2005).
2.4.2. Methods for researching on assessment of VLG’s causative agents and
VLG’s transmission
Method of identifying VLG’s causative agents by electron microscopes:
conducted at the National Institute Of Hygiene And Epidemiology - Hanoi.
Method of diagnosing VLG by biomolecular technique: Following the VLG
Diagnose of Hung et al., 1999; Hocquellet et al., 1999 and Jagouex et al., 1996.
Method on researching the VLG’s transmission through seed, budwood and
vector: Following the common methods used in plant protection study.

6

2.4.3. Methods for researching on identification of VLG’s host and VLG’s
bacteria distribution on citrus in some Northern provinces of Vietnam
Following methods of Su, (2008), Hung et al., 1999 and specific primers in
order to identify causative bacteria in citrus trees of Tomimura et al., 2010.
2.4.4. Methods for researching on some techniques to prevent and control of
VLG
Shoot tip grafting: Following methods of Su and Chu (1984). Procedures
including preparation of rootstock and shoot tip, first grafting, invitro caring. After
that, second grafting and transplanting trees out of nursery. In this study, several
milestones have been done and improved to enhance survival and disease cleanliness
rates by the technique in order to produce disease free materials.
Disease-free planting material techniques: To be conducted in a complete
procedure in three-level net-house, including: container’s substrate and seed sowing;
Soil-less planting material production techniques; Technical and budding method.
Method for assessment of resistance of rootstock which being used in VLG
planting material production: Following methods of Kranz (1988) and Bowen (2004)
Method for studying of anti-reinfection of VLG in the field: Following common
methods on plant protection in citrus trees.
2.5. Method for processing of experiment data: Experimental data are processed
by statistical processor MS.Exel, 2007 and IRRISTAT VERSION 5.0
Chapter 3
RESEARCH RESULTS AND DISCUSSIONS
3.1. VLG’s popularity and symptoms
3.1.1. Current use of citrus varieties in Hà Ni and Hòa Bình
In last years, citrus trees have been grown rapidly, however, growers have not
known yet about the source of disease latent in the trees. Result on the survey on
original and quality of planting material shows that, in Hanoi, 46.67% of planting
materials are self-produced, 40.0% are unidentified of original, only 13.33% are

bought from formal producers; similarly, in Hòa Bình, the figures are 20%; 33.33%
and 53.33%, respectively.
3.1.2. Level of VLG’s popularity in surveyed regions
Result of survey on VLG’s current status and its popularity in different ecological
regions shows that VLG presents and damages citrus crops in all regions with
infected trees ranging from 10.49 to 50.93%. In which, citrus orchards in the
Northwest have the lowest rate of VLG infection 10.43%, the figure in the Red River
Delta is from 13.95 to 16.34%, Central North is from 14.97 to 39.2% and the
Northeast is the highest rate, ranging from 21.18 to 50.93%. VLG in Thanh Trà
pomelo in Thy Biu and Hng Vân (Thua Thien Hue Province) with a rate of
14.97% infection.
In the 9 provinces surveyed, citrus fruit orchards in Hoa Binh province have a
lowest rate of VLG infection (10.49%) and province with a highest rate of infection is
7

Qung Ninh (50.93%). Such significant differences on the rate of infected trees and
level of damage are resulted from an inequality of cultivation and pest management
techniques in those localities (Hà Minh Trung, 2003; Ngô Vnh Vin et al., 2012).
3.1.3. Degree of VLG infection in citrus crops and ecological regions
In 3 years (2010- 2012), we have conducted survey, collection and evaluation
of 175 samples, in which there are up to 81 samples only infected by VLG,
accounting for 46.28% of total samples collected and evaluated. The rest of samples
has been identified to be infected by one of the three diseases (tristeza, tatter leaf and
exocortis) and other diseases. These findings imply that degree of VLG in citrus
orchards in Northern provinces is relatively popular, indicating that VLG is
significant and noticeable in current citrus production.
Orange and mandarin samples have been found to be the highest rate of infection
with VLG, reaching up to 81.81% and 86.00% on orange and mandarin, respectively.
In lime and pomelo, the rate of infection with VLG is very similar (68.57 and
66.67%). Kumquat, in particular, has the lowest rate of VLG infection with only 60%

samples infected.
3.1.4. Degree of VLG infection in integration with other virus, viroid diseases in
citrus crops
Other virus diseases have promoted a faster and more severe process of
degradation in citrus orchards. VLG and tristeza have severely damaged all citrus
crops in the survey. The rate of VLG infection is from 60.0 to 86.0% of the total
samples surveyed; tristeza is from 27.91 to 37.78%. Especially, in our surveys, a
disease called exocortis has been identified on orange for the first time with a rate of
detection of 14.28% in the total samples collected and a disease named tatter leaf on
mandarin with an infected rate of 9.68% in the total samples collected (table 3.5).
Table 3.5. Result of identification of presence of VLG and some other virus
diseases in citrus crops on the same sample by molecular
(at Plant Protection Research Institute, 2010 – 2012)
Citrus
crops
Scientific name Total
samples
Rate (%) sample infected
VLG

CTV

CTLV

CEVd

Orange Citrus sinensis 55 81.81 37.78 0 14.28
Mandarin Citrus reticulata 50 86.00 27.91 9.68 0
Pomelo Citrus grandis 35 68.57 31.43 0 0
Lime

Citrus aurantifolia 15 66.67 30.0 0 0
Kumquat

Citrus microcarpa

20

60.00

33.33

0

0

Notes: VLG: greening; CTV: tristeza; CTLV: tatter-leaf;
CEVd: Exocortis; CQCM: citrus crops
3.1.5. Identification VLG’s group of symptoms through biomolecular method
The VLG’s symptoms are difficult to be observed by naked eye, especially in
trees where such symptoms are latent or just beginning.
The combination of traditional and biotech methods has divided VLG’s
symptoms into 5 main groups (lotchy mottle in leave; Yellow leaves found in each
branch/ tree; Blotchy mottle and yellow leaves found in each branch/ tree; Dry
8

branches, dead tree (overall tree decline), in leave and abnormal ripening fruit), and 3
group of symptoms integrated with tristeza (lotchy mottle, yellow veins; corky veins;
tree showing dieback). This research has achieved a collection of typical symptoms
on fruits for the first time (figures 3.2 ; 3.5 and 3.6)

Figure 3.2. lotchy mottle in
leave
Figure 3.5. VLG’s sympto
ms in
orange fruit
Figure 3.6. Symptoms of
lotchy mottle and corky veins
Source (figure 3.2; 3.5 and 3.6): Lê Mai Nht, 2012
It can be concluded that when trees infected by VLG and tristeza in the same time
has forced the tree dieback very quickly, negatively damaging yield and quality of
harvested fruits.
3.1.6. Verification VLG’s infection ability in the field based on identical
symptoms
In addition to surveyed and collected samples, the study has applied
biotechnological method to verify the presence of virus and viroid in fruit trees (table
3.9). Result of this verification process using molecular method shows that tristeza
has occurred at all sample collecting sites. In Hanoi, presence of the three diseases:
tristeza, tatter leaf and exocortis in citrus crops. In Ngh An, exocortis has been
identified by RT-PCR and grafted into plant indicator (Etrog citron 861). In Bc
Quang, Hà Giang, samples of tristeza and tatter leaf diseases have been collected
(grafted into plant indicator Rusk citrange).
Table 3.9. Distribution of virus and viroid in some citrus fruit trees collected
from Northern provinces of Vietnam (Plant Protection Research Institute, 2011)
Location Crops
Citrus trees
Disease verification result
Tristeza
(Strip)

Tatter leaf
(RT-PCR)
Exocortis
(RT-PCR)
 Liêm
(Hà Ni)
Xã oài Orange + - +
Kumquat + - -
Din Pomelo + - -
Canh Orange + + -
c Quang (Hà Giang)

Sành/green
Orange
+ + -
Ngh An
Xã oài Orange + - +
PQ Mandarin + - -
Note: - : Not recorded yet; +: Recorded by RT-PCR, Strip
9

Number of samples for tatter leaf and exocortis verification is not many due to
process of sample verification, samples that are not infected by VLG or tristeza have
been excluded and were not go through further verification.
3.1.7. Improvement for DNA extraction method in diagnosing VLG in citrus
Assessment of VLG by naked eye or by iodine diagnosing method is very
difficult to identify causative agents. Using of DNA extraction method following the
general procedures such as isolation solution (including Tris-HCl; EDTA, NaCl);
chemical for DNA purification (Sarkosyl, CTAB, chloroform, isoamyl alcohol and
phenol) including many toxic chemicals. Isolation and purification of DNA following

Hung et al., 1999 are very appropriate for a specific and in-depth study, genomes.
However, it needs a long time for DNA extraction using the above mentioned
method. For example, with 10 samples, the time needed for the whole process from
leave cleaning to DNA extraction is from 5 to 6 hours. It results in a high cost for
sample evaluation. Factors causing such a high cost include payment for labour
preparing sample, expenses on power and chemical. It is also noticeable that the use
of many toxic chemicals in this method has negatively impacted to human health and
polluted environment as well.
Such concerns have urged us to improve the method for DNA extraction by
using NaOH alternatively (table 3.10). This method is conducted in the National
Taiwan University of Science and Technology and in the same time, the method also
tested successfully in the laboratory of Plant Protection Research Institute.
Using NaOH to isolate DNA has saved time significantly, from 5 to 6 hours in
the old method to just from 45 minutes to 1 hour in this method, depending on skills
of technician. The advantage of this method is that DNA extraction is done by using
NaOH and acetic acid only, reducing considerably costs on labour and chemical
spending on DNA extraction process. The extracted DNA shows positive results in
VLG assessment.
Table 3.10. Improvement of DNA extraction method in diagnosing VLG (at the
laboratory in National Taiwan University of Science and Technology, 11/2011)
Tested
samples
DNA concentration collected in different DNA extraction methods
Regular extracted
solution
(Hung et al. 1999)
Pulverizing in
Eppendorf
by NaOH
Regular extracted

solution
(Hung et al. 1999)

Sample 1 +++ ++ +
Sample 2 +++ ++ ++
Sample 3 ++++ ++ +
Sample 4

++++

+++

++

Notes: +: < 50 µg; ++: 50 ~ <110 µg; +++: 110 ~ < 170 µg; ++++: 170 ~ < 230 µg
3.2. Identification of VLG’s causative agents and and transmission
3.2.1. Identification of VLG’s causative agents by electron microscope
Disease samples which have been collected from orange, mandarin, pomelo
10

and lime citrus crops from surveyed localities have been cut into super tinny slices
and observed by electron microscope. These observations have found that starch
accumulated in the xylem systems of slices and caused degradation of those systems
as well as severely damaged chloroplast in leaves. Samples collected from different
citrus crops have shown the same result, indicating that this is a symptom to explain
why VLG infected plants dying back so quickly; when tree losing its ability of
photosynthesis, absorption and nutrient transportation through xylem systems.
3.2.2. Identification of VLG’s causative agents by biomolecular method
This project has used three primers [16S of Sandrine Jagoueix (1996), A
2

J
5
of
A. Hocquellet and Hung et al. (1999)] to assess VLG. Reaction of all primers have
shown an exact result in identifying causative agents of VLG. For citrus crops, this
method is a compulsory condition for trees to be formally certified as elite or disease-
free ones.
3.2.3. The density of psyllid Diaphorina citri Kuwayama on citrus plants
If fruit plants have been planted with fresh varieties without disease, the spread
of disease in nature will be vector D. citri Kuwayama. The diversity of varieties of
citrus as well as their different cultivation will be outbreeding faster and the disease is
able to adapt to server condition. Psyllar always occurred among 12 months in year
but they will have different densities. The densities of vector in citrus orchard depend
on food, cultivation and pest management, especially in relation to summer bud
season in year and season temperature. The densities of RCC are highest in summer
buds, spring season and decreasing in autumn and winter. In Nghe An, the densities
of psyllid is 25.15 individual/tree on non-cultivated orchards. And the densities of
RCC are very low on good-cultivated orchards (fig. 3.18).


Figure. 3.18. The densities of Asian citrus psyllid on Xa Doai orchard in Nghe An
In 4 bud season in year, the densities of psyllid are highest in summer and
gradually descreasing in spring, autumn and winter. On non-intensive farming
orchard, the density of psyllid is 25.15 individuals/tree; in the meanwhile on the
intensive farming orchards only obtained 4.05 individuals/tree. Similar in spring bud
season were 21.5 vs. 3.7 individuals/tree, autumn season were 12.25 vs. 2.65
individuals/tree, winter season were 3.95 vs. 0.9 individuals/tree.
On Cam Canh orchard in Hanoi, the densities of psyllid were similar in Nghe
11

An in both non-intensive farming and intensive farming orchards (fig. 3.19).


Figure. 3.19. The densities of Asian citrus psyllid on Cam Canh orchard in Hanoi
On orchard in Cao Phong (Hoa Binh) in the same bud season also carried out
survey of psyllid occurrence. On the non-intensive farming the densities of psyllid is
lower than Hanoi and Nghe An. In the summer bud season the densities of psyllid
only obtained 17.35 individual/tree; in spring and autumn bud season the densities of
psyllid were from 7.05 – 9.6 individual/tree; On the intensive farming orchards the
densities of psyllid were from 0.85 to 2.45 in 4 bud season (fig. 3.20)


Fig. 3.20. The densities of Asian citrus psyllid on Cam Xa Doai orchard in Cao
Phong, Hoa Binh
Therefore, the result of survey showed high arising of densities of psyllid in
main bud season in year. On the non-intensive farming, the densities of psyllid was
always higher than densities of psyllid on intensive farming orchards, the reason is
that the non-intensive farming orchards were not pruned, the ununiformed-buds arise
over year, which is good condition for psyllid develop and always occurred in the
field The results is suitable with Plant Protection Institute' results before (Phm Vn
m, 2005). The results of this serve are scientific basic in order to experimental
design for re-infection of the vector in the fields.
3.3. Host and distribution identification of bacteria race cause greening disease
on citrus in Northern Vietnam
3.3.1. Host of greening disease
Among the collected species were selected 14 lines/varieties to assess tolerance
of VLG to some varieties and rootstock material. The species of the genus Citrus are
collected, expressed infectious diseases after 3 months, grapefruit (Citrus maxima)
and lemon (Citrus lemon) get symptom after 5 months (table 3.17).
Among the collected varieties, there are only Tiên Chót (Severinia buxifolia) is

12

very susceptible to VLG with typical symptoms but this species is not belong to
genus Citrus; the results are consistent with Su et al. (In 2012). Similarly, the laurel
tree is allure tree for citrus psyllid as the domestic and foreign authors published.
Through the study, subjects initially detected and identified that Móc Mt tree
(Clausena excavata) is also one of good host plants citrus psyllid but not infected
VLG disease. So, it is maybe choice Tin Chót plants for breeding and mass
production of citrus psyllid, the laurel tree and Móc Mt tree is used to breed fresh
citrus psyllid resources citrus psyllid to design greenhouse experiments and extensive
experiments.
Table 3.17. The infected ability of Liberibacter asiaticus on some race of Rutacea
(Screen house Plant Protection Research Institute, 2011)
Species Vietnamese
Name
PCR’s diagnosis Rate
(%)
1
months
3
months
5
months
Citrus sinensis Cam ng ình - + + 100
Citrus unshiu Quýt ôn châu - + + 100
Citrus nobilis Cam sành - + + 100
Citrus reticulata quýt - + + 100
Citrus maxima i - - + 100
Citrus hyxtric Chp - + + 100
Citrus lemon Chanh - - + 100

Citrus aurantifolia Chanh ta - + + 100
Citrus jambrihi Chanh sn - + + 100
Fotunella japonica Qut - + + 100
Clausena excavata Móc mt - - - 0
Murraya paniculata Nguyt qu - - - 0
Atalantia guillauminii Quýt hôi - - - 0
Severinia buxifolia Tin chót - + + 100
Note: +: Positive to greening disease;
-: Negative to greening disease; LBNT: artificial infection
3.3.2. Bacteria identification cause greening disease throught reaction of some
varieties on citrus plants disease resources
In total 81 samples were evaluated and divided into four groups consisting of
orange trees (24 samples), tangerine (30 samples), grapefruit (13 samples) and lemon,
kumquat is 14 samples.
With a total of 24 samples collected from diseased orange trees (table 3.18), 10
13

samples of race II (representing 41.67%), 14 samples of race I (accounting for
58.33%). The VLG pathogenic strains were confirmed by specific primers of
Tomimura et al., 2010.
Similar experiments to evaluate samples collected from 30 sources citrus trees,
infected with citrus varieties, results showed race I and II are equal (15 samples for
each race of bacteria).
Table 3.18. The reaction of collected citrus plants from infected citrus (Citrus sinensis)
with greening disease (Screen house Plant Protection Research Institute, 2013)
No Sources

The reaction of collected citrus plants from
infected citrus (Citru s sinensis) with greening
disease (0-3 score)

Bacteria strain

Cam Xã
oà i
(Citrus
sinensis)

Cam
Canh
(Citrus
reticulata)

 i
Din
(Citrus
grandis)
Chanh
Eureka
(Citrus
limon)
Strain
I
Strain
II
1 G/x.-I-1-1 3 3 3 3 ×
2 G/x.-I-2-2 3 3 0 0 ×
3 G/x.-I-3-4 3 3 3 3 ×
4 G/x.-II-1-2 3 3 0 0 ×
5 G/x.- II -1 -3 3 3 2 2 ×
6 G/x.- II -1 -6 3 3 3 3 ×

7 G/x.- II -2 -7 3 3 3 3 ×
8 G/x.- II -3 -5 3 2 0 0 ×
9 G/x.- II -3 -9 2 3 0 0 ×
10 G/x.- II -4 -1 3 3 0 0 ×
11 G/x.- II -4 -2 3 3 3 3 ×
12

G/x.
-

II
-
4
-
4

3

3

0

0

×


13 G/x.- II -4 -8 2 2 0 0 ×
14 G/x.-III-1- 3 3 3 3 3 ×
15 G/x.-III-3- 3 3 3 0 0 ×

16 G/x.-III-3- 4 2 2 0 0 ×
17

G/n.s
-
III
-
4
-

1

3

2

0

0

×


18 G/n.s-III-4- 2 3 3 0 0 ×
19 G/n.s-III-5- 2 3 3 3 3 ×
20 G/c.l-VI-1-1 3 3 0 0 ×
21 G/c.l-VI-1-3 3 2 0 0 ×
22

G/c.

-
VI
-
1
-
4

3

3

0

0

×


23 G/c.-VI-3-1 3 3 2 2 ×
24

G/t.q
-
VI
-
4
-
2

3

3

3

3


×

Negative control 0 0 0 0 0 0
Total 14 10
14

Note: I: Hà Ni, II: Ngh An, III: Hòa Bình, VI: Qung Ninh; x.: cam Xã oài, n.s:
cam Nam Sn, c.l: cam lai, c.: cam ng, t.q: Orange China, Disease severity
was grade as follows: 0 = no symptom; 1 = mild chlorosis without dwarfing; 2 =
intermediate chlorotic mottling with moderate dwarfing; 3 = severe HLB symptom,
including yellow mottle, leaf hardening, and curling with distinct dwarfing

With 13 samples were collected from evaluated pomelo were obtained 7
samples infected with race I with and 6 samples infected with strain II. Strain II have
appeared in different varieties of grapefruit and also expressed different disease
severity levels of symptoms (table 3:20). Strain I and II cause disease on the citrus
varieties are summarized with symptoms in figure 3:25.
Table 3.20. The reaction of collected citrus plants from infected pamelo (Citrus grandis)
with greening disease (Screen house Plant protection research institute, 2013)

No

Sources

The

reaction of collected citrus plants from
infected citrus (Citrus sinensis) with
greening disease (0-3 score)
Bacteria strain

Cam Xã
oài
(Citrus
sinensis)

Cam Canh
(Citrus
reticulata)



i Di

n

(Citrus
grandis)
Eureka

(Citrus
limon)

Strain

I
Strain
II
1

G/b.d
-
I
-
1
-
1

3

3

3

3


×

2

G/b.d
-

I
-
1
-
6

3

3

0

0

×


3

G/b.d
-
I
-
4
-
3

3

3

0

0

×


4

G/b.d
-
I
-
4
-
4

3

3

3

3


×

5

G/b.d
-
I
-
4
-
5

3

3

0

0

×


6

G/b.d
-
V
-
1
-
3

3

3

0

0

×


7

G/b.m
-
VI
-
3
-
4

3

3

2

2

×

8

G/b.n
-
VII
-
1
-
2

3

3

2

2


×

9

G/t.tr
-
VIII
-
1

-
5

3

3

3

3


×

10

G/t.tr
-
VIII
-
1
-
6

3

3

3

3


×

11

G/t.tr
-
VIII
-
2
-
1

3

3

0

0

×


12

G/t.tr
-

VIII
-
3
-
1

3

3

3

3


×

13

G/t.tr
-
VIII
-
4
-
1

3

3

0

0

×


T

ng





7

6

Ghi chú: I: Hà Ni, V: Bc Giang, VI: Qung Ninh, VII: Hà Giang, VIII: Tha Thiên Hu;
b.d: Bi Din, b.n: Bi Ngt, b.m: bi M, t.tr: bi Thanh Trà
For the lemon and kumquat are citrus species, are used as ornamental and
spices plants so little attention to the disease. The samples from the lemon tree was
obtained 2 samples of strain II (accounting for 33.3%), on kumquat obtained 1
samples of strain II (12.5%). Samples from kumquat variety showed similar reaction
in Bi Din and Eureka lemons in level 2.
The sample after pathogenic artificial assessment by analyzed by duplex-PCR
15

technique, with two types of primer specificity (DNApol / nusG-rplK operon)
[Tomimura et al., 2010]. PCR products of primer DNA Poly obtained at 988bp; PCR
products of primer nusG-rplK operon obtained at 627bp. Two types of primer were
same reaction allows identification strains I, II and III. PCR products from the strains
I had 2 lines appear on products; II strains obtained product at 627 bp (figure 3:26).
Combining traditional methods with molecular biology have identified pathogenic
strains greening disease in Vietnam and the distribution of pathogenic bacteria in the
survey area (figure 3:27).



Figures.

3.25. Strain I cause disease on Cam Xã
oài and Cam Canh;
Strain II cause disease on 4 varieties (bi Din,
chanh Eureka, Xã oài và cam Canh)

Figures.

3.26. The duplex
-
PCR results and strain
identification [CC: Cam canh, RL=Rumprue
lime, Cal=calamondin (Qut)].
Strain I: obtained PC R product at 988bp and
627bp; Strain II: obtained PCR product at 627bp
(Sources: Lê Mai Nht, 2013)

Figures. 3.27: The distribution of bacteria strains cause greening disease on citrus plants
3.4. Management greening disease
3.4.1. Improvement micro-apical graft technique made fresh material plants
3.4.1.1. Improvement tecnique for rootstock 1 in shoot tip grafting
Experimental results had identified the MS media and combination with 0.5
grams of activated carbon in 1 liter for micro grafting, stem diameter was 1.51 mm.

98
8 bp

627 bp
16

At the same time have type of rootstock evaluation (3 leaves orange, sour grapefruit,
mortgage, oranges, Cleopatra tangerines) used in graft of apical meristems of plant,
and their survival in times of 1. The rootstock from Chp and sour grapefruit showed
survival rate after graft obtained 27-36%, with Phc Hòa and Trng Vng citrus
pomelo 30.2 to 41.7%,.
In micro-apical grafting technique, using healthy rootstock, which will increase
the ability of water and nutrients uptake for the growth of apical meristems.
Experiments investigate the effects of 3 leaves orange rootstock age and survival
rate of Trung Vuong orange variety (table 3:27). The results showed that if the young
rootstock (1 week old) or old rootstock (4 week-old) gave lower survival rate
compared with rootstocks from 2-3 week-old. If using 1-week-old rootstock, the
capacity for regeneration of tissues and cells quickly create callus, which crowded
apical meristems and obstructed bud diviation. Four-week-old rootstock for
regeneration capacity and slow healing wounds and apical prone to dry and then die.
The rootstocks from 2-3 week-old is most suitable for grafting, obtained highest ratio
of survival, from 28.0 to 34.0%.
Table 3.27. Effection of plant age time 1 to survival rate after shoot tip graftting

with Trng Vng orange variety (Plant Protection Research Institute, 2010)
Rootstock age
Success rate (%) Un-success rate for
died bud (%)
Un-success rate for
died callus (%)
1 week-old 10.0
c
10.0
c
80.0
a

2 week-old 34.0
a
26.0
bc
40.0
b

3 week-old 28.0
ab
38.0
b
36.0
bc

4 week-old 20.0
b
70.0

a
10.0
c

CV(%) 20.1 16.8 21.8
3.4.1.2. Improvement of grafted liquid media (time 1)
Added BAP 0,5mg/l into MS media is suitable concentration to survival rate
and development and growth of micro-grafted bud. There were significant diffrence
between different test (Table 3.28).
Table 3.28. Effect of BAP to graft survival rate of first time on Phc Hòa pomelo
(Plant Protection Research Institute, 2010 - 2011)
Medium
Success rate
(%)

Leaf new
(
After 20 days
)

Long buds
(mm)

MS 27.8
c
2.1
d
4.7
b

MS + 0,5mg/l BAP 44.3
a
5.4
a
9.8
a

MS + 1,0mg/l BAP 39.4
ab
4.2
b
7.0
ab

MS + 1,5mg/l BAP 35.6
b
3.6
bc
3.9
b

CV(%) 12.3 19.7 21.1
In order to improve shoot tip grafting, the project were optimaled technique in
apical grafting. The result is optimal in the development of research methods of Su
(2008), applied in the laboratory of the Plant Protection Research Institute and
17

National University laboratory synthesis Taiwan. The media was used in liquid MS
medium supplemented growth regulator (i-inositol, thiamine-HCl, pyridoxine-HCl,
nicotine).

The result is optimal in the development of research methods (2008), applied in
the laboratory of the Plant Protection Institute and University laboratory synthesis
Taiwan. The platform media was used in MS liquid medium and supplemented
growth regulators (i-inositol, thiamine-HCl, pyridoxine-HCl, nicotine).
Use of the regulators, the survival rate of micro-grafting was raised, the ability
obtained in grafting second time faster. Grafting technique of apical meristems
become easier and more successful. The advantages of the supplements growth
regulators, which help apical livable, able to give early buds, it possible to
simultaneously between graft and rootstock shoots fast (table 3:29)
Table 3.29. Effect of growth regulators supplement liquid medium in micro-
apical grafting (Plant protection research institute, 2012)
Total of grafted plants Success rate (%)
MS 35 14.29
MS + Growth factors 35 34.28
Note: Growth factors (i-inositol, thiamine-HCL, pyridoxine-HCL, nicotine)
3.4.1.3. The method of arising un-infected plants with greening disease in citrus
plants
Steriled apical were grafted with un-infected plants or infected plants, the
obtained results depend on technique and size of micro- apical grafting. The size of
apical were long, the survival rate is high, but the un-infected rate was low (table
3.30).
Table 3.30. The testing of apical sizes different to the ability of free disease after
shoot tip graftinh (Plant Protection Research Institute, 2012)
Apica l size Micro-graft
varieties
Number
tested
plants
Survical
rate after

micro-
grafting
(%)
Ratio of
un-
infected
greening
disease
(%)
Ratio of
un-
infected
tristeza
disease
(%)
Meristem Phc Hòa Pumelo 30 0 - -
Trng Vng
Orange
30 0 - -
Meristem with
two leaf
primordia
i Phc Hòa 30 26.7 100 63.3
Cam Trng Vng 30
33.3
100 73.3
Meristem with
three or four
leaf primordia
i Phc Hòa 30 36.7 73.3 23.3

Cam Trng Vng 30
46.7
80.0 46.7
18

3.4.1.4. Effected of plant age in micro-grafting in time 1 into growth and
development of micro-grafted plant in time 2
According to the study of Navarro (1972) and Hyun (2012) were able to
transplant grafted plants in time 1 from in-vitro conditions were planted directly into
potting mix in strict conditions. In order to improve and enhance the efficiency, the
study by Su (1984, 2008) had used grafting techniques 2nd time. The plant age in
first time grafting gave successful highest efficiency from 6-8 weeks of age (reached
from 76.9 to 91.7%).
Table 3.31. Influence of tree age graft survival time 1 to time 2 of shoot tip graft
(Screen house Plant Protection Research Institute, 2010 - 2012)
Plant age in
first grafting

i Phc Hòa Cao Bng Cam Trng Vng Cao Bng
Total number
of micro-
grafted plants
Survival rate
(%)
Total number of
micro-grafted
plants
Survival rate
(%)
4 week

-
old

13

15.
4

16

18.
8

6 week-old 14 78.6 13 76.9
8 week-old 10 90.0 12 91.7
In micro- apical grafting technique, bud stage processing is concerned, used
buds in grafting were very vulnerable. Through practical manipulations buds, we had
tried to find solutions to overcome this phenomenon. Results were determined by
using 2-3 drops of Tween 20 for in sanitizing solution and sterile distilled water.
When the buds are located near the lower part suspended in the cup containing the
disinfectant solution, remove the disinfectant solution and rinsed with sterile distilled
water, the buds put into again in the cup, easy operation, bud not damaged, crushed.
Tween 20 also had the effect of seepage slicks in the armpit leaves sprout, so better
steriled effects and incidence of less fungal contamination.
3.4.2. Production of disease-free citrus seedlings in 3-level grid system greenhouse
Seedling quality is an important factor in developing citrus fruit. To ensure
disease-free planting must be produced in the 3-level grid system greenhouse and
resources of grafts were taken from the apical meristems of grafting and has been
tested and recognized disease-free certification.
Improved potting mix with ratio fifth yellow sand + 2/5 wood shavings + 2/5

oganic fertilizer microbial mulch. This improvement has increased the initial nutrient
supply, increased porosity, increased ability to hold moisture and nutrients. The
rootstock after 4 months has standard condition for transplanting, also in formula un-
improment potting mix the rootstock need at least 5 months after planting (in warm
weather) to get standard condition for graft. Improved potting mix could have been
watered, the addition of fertilizer. The roots system always grow well, increasing
resistance to soil-borne disease.
Seedling production in 3-level grid system greenhouse is implemented in all of
12 months in the year; the best season for grafting is summer. Temperature range of
19

28-38°C is suitable for grafting stage, survival rate of grafts were 94.67%, and
healthy grafts that grows seccessful reached 84.88%, which is the ratio of buds to
grafts grow well in the summer, no sleep grafts so survival rate and plant sprouts out
fast.
Parafilm paper used in techniques grafted seedlings, survival rate of grafts is
much higher than with conventional grafting methods, limited flow disease, fewer
grafts rot, healthy plants, uniform. This provides significant enhancements to enable
rapid chit and health. The advantage of using plastic paper is cheaper but the
disadvantage is not to protect the environment due to the retained plastic.
3.4.3. Tolerance of rootstocks in seedling production to greening disease
3.4.3.1. Tolerance of rootstocks (Bi chua, chp) to greening disease
Rootstock is considered success factors of seedlings, make product quality and
longevity of trees as well as fruit weight. Initially, understanding and tolerance
evaluation of rootstocks with greening disease. Pathogen were used strains I cause
disease on grapefruit; strain II cause disease in Nam Son Orange, two strains are now
widespread in northern Vietnam.
After artificial infection with diseased scions, rootstocks trees have disease
symptoms (table 3.35). Grafted by strain II with highly pathogenicity, symptoms
manifested quickly, after two months the prevalence was 25.55% (Bi chua

rootstock), 33.33% (Chp rootstock).
Table 3.35: The percentage of disease after artificial infection in infected grafts
for rootstock is used in seedling production
(Screen house Plant protection research institute, 2012)
Rootstock Resources
(infected
grafts)
After 2 months After 4 months After 6 months
TLB
(%)
Disease
level
TLB
(%)
Disease
level
TLB
(%)
Disease
level
Sour Pomelo Strain I 0.0
c

0 28.89
c

1 51.48
c

3

Sour Pomelo Strain II 25.55
b

1 70.37
b

3 92.59
a

3
Chp Strain I 0.0
c

0 31.11
c

3 85.0
b

3
Chp Strain II 33.33
a
2 88.89
a

3 92.59
a

3

LSD
0.05

0.
94


6.
04


7.
32


CV(%) 3.4 5.9 4.8
Note: Strain I: from bi Din; Strain II: from cam Nam Sn
Pathogens of greening disease had direct influence to rootstock, which made
limit grow height of rootstocks. During follow-up, potentially pathogenic strains
mutated virulent infection in the next generation. The same strain of the disease on
the same object in both rootstocks but obtained was lower plants. The phenomenon of
dwarf plants has demonstrated symptoms of low dwarf tree in the field. This result is
consistent with studies of Su et al., (2008, 2012); (Hung et al., 2012) to assess
tolerance of grapefruit and Chp.
20

3.4.3.2. Tolerance of imported rootstock to greening disease
The imported rootstock from Taiwan (Cleopatra mandarine, Swingle
citrumelo, Fruit dragon, Volkamer lemon, Poncirus trifoliata, Carrizo citrange,
Troyer citrange, Rangpur lime) are grown and developed in Vietnam to find out the

type of rootstock suitable for production of disease-free seedlings for each type of
different citrus plants. Initial assessment of disease-resistant rootstock VLG on
imported plants (table 3:37). Cleopatra rootstock, Carrizo citrange, Rangpur lime
showed symptoms early, severity in 3 to 6 months after infection.
ng 3.37: The percentage of patients after artificial infection by buds disease with an
imported rootstocks (Screen house Plant Protection Research Institute, 2012 - 2013)
No Rootstock Disease symptom
after 3 months
(disease level)
Disease symptom
after 6 months
(disease level)
1 Cleopatra mandarine

2 3
2 Swingle citrumelo 1 2
3 Fruit dragon 1 2
4

Volkamer lemon

1

2

5 Poncirus trifoliata

1 1
6 Carrizo citrange 2 3
7 Troyer citrange 1 1

8 Rangpur lime

2 3
9 Chp 2 3
10 Bi chua 1 3
Note: Infected grafts from cam Nam Sn; - : 0 = No symptom (tolerant), 1 = Mild (blotchy
mottling symptoms observed from 1 to 30% on seedlings canopy, 2 = Moderate (yellowing
symptoms observed from 31 to 50% on seedlings canopy), 3 = Severe (blotchy mottling, midrib
yellowing and twigs dieback symptoms observed more than 50% on seedlings canopy).
3.4.4. Against re-infected greening disease in the fields
3.4.4.1. Using antibiotics for control infected grafts
The previous results always used Tetracyline 1% for control greening disease
(Aubert và Bové, 1980; Tsai et al., 2012). In this research, based on mechanism of
pest, antibiotics in control infected grafts, is Streptomycine (suitable for negative
gram bacteria)
Table 3.38. Effect of Streptomycine to greening disease symptom
(Screen house Plant Protection Research Institute, 2012)
Experiment Total
plants
Survival
rate (%)
Symptom after infection
2
months
4
months
6
months
Steptomycine 1% + 12 hour 20 100 + + +
Streptomicine 1% + 24 hour 20 90 - - -

Control (un-infected grafts) 20 100 - - -
Control (Infected grafts) 20 90 + + +
Note: - : no symtom of disease; +: occurred symptom of disease
21

Using Streptomycine in 24 hours, the plants developed after grafted at 6
th

months were not occurred disease symptom, in the meanwhile in 12 hours the plants
showed disease symptom after 2 months grafted. Although plants were not showed
symptom, the PCR testing showed positive. Using Streptomycine had effect to
inhibit, prevent disease development, maintained low density of bacteria.
3.4.4.2. Effective against re-infected greening disease in the field in various
management conditions
The determination of different measures with un-infected disease seedling various
management vectors obtained results in table 3.40.
Table 3.40. Ratio of re-infected greening disease in the fields with different
management (Nghi Lc, Ngh An, 10/2013)
Experiment Disease rate (%)
Spring
buds
Summer
buds
Auntum
buds
Free disease seedlings + no pesticide spraying 9.33
c

14.67c 16.0
b

Free disease seedlings + pesticide spraying
frequently
0.0
a
1.33
a
5.33
a
Free disease seedlings + local cultivation 4.0
b

10.67
b

16.0
b

LSD
0.5
0.66 1.15 1.99
CV (%) 7.5 6.5 9.4
Note: initial evaluation disease rate after 15 months planted; planted: 25/12/2011; 1
st

evaluation: 15/3/2013; 2th evaluation: 15/7/2013; 3
rd
evaluation: 15/10/2013
After 15 months planted, re-infected plants occurred in “no pesticide spraying”
method for control vector (9.33%), in “local cultivation” (4%). In “pesticide spraying

frequently” was not showed disease symptom but sever re-infected in later
assessment, the disease rate of this experiment was 5.33% after 2 years planted. Other
experiments were arising disease rate (16%).
From the research results achieved greening disease in northern Viet Nam as
well as some of the results achieved from other countries, we proposed some solutions
to prevent disease VLG (table 3.41).
Thus, for these measures to promote the effective and efficient in the
implementation process should be combined with fertilizer, pruning and watering
spread to the buds seasons, while the spraying pesticides will have a significant effect.
It is in bud concentrate, which will be limited food sources in the field for Psyllar’s
density decreased. The technique of fertilizer is not only for bud concentrate, but also
make fruit quality, yield stability over the years. Irrigation solution is not only for bud,
but also filter out the effects of limiting harmful spiders, avoid physiological fruit drop
22

caused by too dry or too moist and extend harvest time, improve the economic
efficiency of costs and consumer’s demand.
Table 3.41. Some solution for control greening disease with high effective in some
province in Northern Viet Nam (2013)
Main solution Implementation
Free disease
seedlings
- Selection of parents
- Application of shoot tip grafting technique and diagnostic
methods had been improved to revitalize, preserve disease-free
parent plants.
- Production of disease-free seedling including choosing suitable
rootstock, potting mix and grafting technique
Soil - Unleash the soil before 6 month for new planting, clearing
debris and trees were destroyed VLG disease and other viral

diseases. Should be planted legumes to improve soil.
- Planting windbreaks fence around orchards with acacia.
- Check the pH of the soil in each region to have appropriate
rehabilitation measures
Seasonal Should be planted in spring, autum
Densities of
planting
- Orange variety: 650 – 750 plants/ha
- Mandarine variety: 800 – 1000 plants/ha
- Grapefruit variety: 450 – 500 plants/ha
Size hole for
planting
- Mountainous: 1m × 1m × 1m
- Flat, well drainage: 0,8m × 0,8m × 0,8m
- Land was transformed from rice, vegetable, need to make bed
and upper planted to avoid plant flooded.
Cultivation - Weeding (hoeing grass around the base of the foliage, cut
foliage grass outside)
- Cover the dried straw mulching
- Fertilization: Basic construction period applied 3-4 times /year;
trading period applied 2-3 times/year
- Watering: centralized irrigation in the dry season
- Pruning, Canopy
Chemical Control Psyllar vector in new buds sprout stage by using Confidor
100SL or autoclaves or systemic absorption (if any vector)
Training - Identify vector and major pest
- Follow the rules and characteristics arising pest
- Integrated management of pests

The effects of macro elements and trace: N: increased growth potential and

productivity of the leaves; P: development of roots and fruit quality; K: Make the fruit
23

weight and resistant to unfavourable conditions; Ca: Make the cells is harder and
resistant to unfavourable conditions; Mg is found in chlorophyll and participate in the
enzyme exchange process.
These solutions should be applied synchronization but flexibly in order to
improve efficiency, reducing investment costs. In this training solutions need to be
concerned because the producers do not understand and do not capture the citrus pest
cannot control it. Moreover, major pest arising time, each different pest should also be
meticulous to foster integrated disease management.


CONCLUSIONS AND RECOMMENDATIONS
1. Conclusions
1.1. The area planted citrus in the north of Vietnam, infected VLG with the
disease rate from 10.49 to 50.93%. VLG disease usually arises with the virus disease.
Of the positive tested samples for the VLG disease were from 58.33 to 73.33% of the
sample mixed with tristeza disease with symptoms of swollen veins explode, dwarf,
yellow leaves and 35.07% near the contaminated samples with tristeza diseases, leaf
tatter, exocortis disease.
1.2. By electron microscopy techniques have identified pathogenic VLG
bacteria (Liberibacter asiaticus) vascular degrades, causing starch accumulation and
necrosis chloroplasts. Using specific primers Jagoueix Sandrine (16S); A. Hocquellet
(A
2
J
5
) and Hung et al. (1999) combines innovative DNA extraction step with NaOH
for fast results with accuracy comparable to diagnostic kits used, low price, do not

use toxic chemicals.
The VLG disease showed rapid symptoms on Xã oài orange, Cam Canh after
45 days of grafting. The density of RCC was 6 individuals/tree or 9 individuals/tree
in 24-hour exposure, symptoms (respectively) appears only after 9 months and 6
months of infection on Xã oài oranges and Cam Canh.
It is the first time to identify Tin Chót (Severinia buxifolia), which was
collected in Tho Xuan, Thanh Hoa, is host with greening disease.
1.3. By the reaction of VLG samples for bio-monitoring plants and a number of
fruit trees (orange, tangerine, grapefruit, and kumquat) combined with duplex-PCR
techniques have identified bacteria strains in the North belonging to VLG strain I and