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

In order to so lve such problem s and to susta inably develop c itrus industry in Northern Vietnam, it is worthwhile and necessary to conduct “Study on greening in citrus fruit trees in

VIET NAM ACADEMY OF AGRICULTURAL SCIENCES

VIET NAM ACADEMY OF AGRICULTURAL SCIENCES

Scientific supervisor:

1 Dr Ngo Vinh Vien

2 Ass Prof Dr Nguyen Van Viet

Viet Nam Academy of Agricutural Sciences

At hours, day month year 2014

This thesis c an be referred to at:

1 Viet Nam National L ibrary

2 The Library Viet Nam Academy of Agricutural Sciences

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, comprisin g orange and mandarin 70,300 ha, pomelo 45,000 ha, lime 18,000 ha Citrus produce is not on ly for domestic consumption but also is a h igh va lue export commodity C itrus culture is a fast growing industry in many tropical, temperate and semi-trop ical countries However, the diversification of climate in citrus cultivated areas has created favorable conditions for a growth of a number of pests and diseases, especia lly green ing (VLG) wh ich has been w idespreaded in most of c itrus countrie s 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 in itial researches have been carried out

in Vietnam but the root problem of green ing has not fully understood yet Hence, control measures of the disease are still less effective In order to so lve such problem s and to susta inably develop c itrus 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 characteristic s of VLG’s cause, development and transmission that serve as scientific bases for build ing up effective preventive and controllable solutions for c itrus industry in selected Northern provinces in Vie tnam

2.2 Requirements

Capture distribution of VLG in some c itrus growing regions in Northern Vietnam Identification of types of VLG bacteria and their d istribution in selected citrus crops in d ifferent 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 sc ientific and practical documentation on VLG, including transmission vectors, influent factors and sound preventive and controllable measures

- Identify ing a field rapid testing method for VLG and thus, could timely and effectively address the disease

- The outcome of this research contributes to sc ientific and practical resources wh ich 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 c itrus and disease transmission psyllid Diaphorina citri Kuwayama

- Selected local and import fruit crops wh ich 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

T his research studies VLG transmission s, factors influencing to the transmission and development of VLG with in the ecological of Northern prov inces of

Vietnam The research identifies bacteria of L.asiaticus causing VLG in citru s and

their distributions as well a s studies effective measures to prevent and control VLG in some Northern provinces

5 New contributions o f 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 viru ses, contributing to complete technica l procedures fo r virus-free grafted planting materia ls

- 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 symptom s vary, depending on citrus crops, original of plantin g materials, transmission vectors … as well as o rchard 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 th is 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 ind icated that VLG to be able

to transmit very fast and damage crops heavily if disease infected planting materials are being used and cultivationa l techniques have not been applied properly Accord ing to Su et al (1998); Bové et al (2006); Hà Minh Trung et al (2008),

production techniques and use of disease-free planting materials, and early identify ing 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 identify ing infected material, transmission characteristic s and changes of bacteria toxicity on each type of citrus crop is very important because th is is like ly serv in g as a basement

disease-to develop a system of integrated preventive and controllab le measures disease-to deal with the disease in a cost-effective, technical and sustainable manner

1.2 Review o f international lite ratu re

1.2.1 VLG’s symptoms, histo ry and distributio n

VLG was firstly recorded from 1929 in China with different names such as Huanglongb in g or yellow shoot in South China The disease is then documented in

1943, Greening in South Africa (1947), Likub in (Tawain, 1951), Leaf mottle ye llows

in the Philipp ines, huanglongb in g in Thailand (1960), Dieback in India, greening in Myanmar (1970), Vein Phloem degeneration in Indonesia (1980), greenin g in Vi t Nam, Laos, Cambodia (1980), Huanglongb ing in Iriomote, Okinawa (1994), Okinawa Island, Japan (2003), in T okunoshima, Kagoshima, Japan (2003), Huanglongb in g in Brazil (2004) and in Florida, the United States (2005) Presently, the disease has not been found in Australia yet

1.2.2 Diagno se 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 lon g stick shape and changes from young to mature cells (Huang, 1987)

Identifying the causative agent by bio-molecular method: T his method uses spec ific

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 fru it trees with biotechnology Since then, there are four types of VLG bacteria have been identified Different types cause different symptoms on those fru it 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 psy llid 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

taiwanica Tanaka & Y Shimada, and Citrus aurantiifolia (Christm.) Swingle

1.2.4 Research o n disease management

Shoot tip graftin g, the best method for a process of re-purification of ind igenous genetics, have been developing Shoot tip or meristem is normally not VLG in fected by causative agents, therefore, trees developed from these shoot tips are disease free Shoot tip graftin g is conducted by inverted T budding (Murash ige 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)

T echniques for citrus variety improvement are applied concurrent with other agronomical, biological and chemical measures to prevent and control of VLG

1.3 Review o f 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

T h Thu H ng (2009) have described and recorded symptoms of VLG in c itrus

1.3.2 Diagnose and appraise VLG

At the early stage of VLG research in Vietnam, there were two different theorie s about the cause of VLG The first one was saying that VLG is like ly 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

green ing 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 o n pathology

Hà Minh T rung et al, (1995) have conducted researches on VLG’s transmission through grafting, grafted budwood and vector

Ngô V nh Vi n et a l., (2009), Nguy n V n Hoà et a l., (2012) have collected and

primarily assessed the resistant capability of selected trees of Rutaceae

1.3.4 Research o n disease management

Currently, technical guidelines and procedures for shoot tip graftin g have been improving to increase the shoot tip grafted tree’s ability of d isease cleanliness and surviva l Net-house systems are established to mainta in 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 cultivationa l, biological and chemical techniques to prevent and control disease vectors

Chapter 2 RESEARCH MATERIAL, CONTEN T AND METHODOLOGY

2.1 Research location and period

Research location

- Division of Plant Pathology, Plant P rotection Research Institute, ông N g c, Liêm, Hà N i

- Laboratory in National Taiwan University of Science and Technology

- Ngh i 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 re gu lators used in shoot tip grafting Seed for rootstocks (T hree leaves orange, sour pomelo, ch p,

Green Orange Citrus reticulata × maxima, Cleopatra mandarin) Chemicals used in

identify ing 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 belongin g to Citrus and wild trees of Rutacea

Small net-frame 1m × 1m × 1m, container and a system of three-leve l net-house; substrates (sand, sawdust and m icro-o rgan ic 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 technologica l 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

P rotection Research Methods, Volume I, Dang Vu Thi Thanh and Ha Minh Trung,

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

Dia gnose 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: Followin g the common methods used in plant protection study

2.4.3 Metho ds for re searching o n identificatio n o f VLG’s host and VLG’s bacteria distributio n on citrus in so me Northern provinces of Vietnam

Followin g 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 resea rching on some techniques to prevent and control o f VLG

Shoot tip grafting: Following methods of Su and Chu (1984) P rocedures

inc luding 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 surviva l and disease cleanliness rates by the technique in order to produce disease free materials

Disease-free planting material techniques: T o be conducted in a complete

procedure in three-level net-house, including: conta iner’s substrate and seed sowing; Soil-less plantin g 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 citru s trees

2.5 Metho d fo r processing o f experiment data: Experimenta l data are processed

by statistica l processor MS.E xe l, 2007 and IRRISTAT VERSION 5.0

Chapter 3 RESEARCH RESULTS AND DISCUSSIONS 3.1 VLG’s popularity and symptoms

3.1.1 Curren t use of citrus varieties in Hà N i and Hòa Bình

In last years, citru s 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 materia ls 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 o f VLG’s popularity in su rveyed 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 ra te 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 (T hua 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 h ighest rate of infection is

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 infectio n in citrus c ro ps and eco lo gical regio ns

In 3 years (2010- 2012), we have conducted survey, collection and evaluation

of 175 samples, in wh ich there are up to 81 samples only infected by VLG, accounting for 46.28% of total samples collected and evaluated T he rest of samples has been identified to be infected by one of the three diseases (tristeza, tatter leaf and exocortis) and other diseases T hese findings imp ly that degree of VLG in citrus orchards in Northern prov inces is relatively popular, indicating that VLG is significant and noticeable in current c itrus production

Orange and mandarin samp les 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 infectio n in integration with other virus, viro id diseases in citrus c rops

Other virus d iseases 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 co llected (tab le 3.5)

Table 3.5 Result o f identification o f presence o f VLG and some o ther virus

diseases in citrus crops on the same sample by mo lecular (at Plant Protectio n Research Institute, 2010 – 2012)

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 o f symptoms thro ugh biomolecular metho d

T he VLG’s symptoms are difficult to be observed by naked eye, especially in trees where such symptoms are latent or just beginning

T he combination of trad itional 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

branches, dead tree (overall tree decline), in leave and abnormal ripening fruit), and 3 group of symptoms integrated with tristeza (lotchy mottle, yellow ve ins; corky ve ins; tree showin g dieback) This research has achieved a collection of typica l symptoms

on fruits for the first time (figures 3.2 ; 3.5 and 3.6)

Figure 3.2 lotchy mottle in

3.1.6 Verificatio n VLG’s infection ability in the field based o n identical sympto ms

In addition to surveyed and collected samples, the study has applied biotechnolo gica l method to verify the presence of virus and viroid in fruit trees (tab le 3.9) Result of this verification process using molecular method shows that tristeza has occurred at all samp le collecting sites In Hano i, presence of the three diseases: tristeza, tatter leaf and exocortis in c itrus crops In N gh An, exocortis has been identified by RT-PCR and grafted into plant indicator (Etro g citron 861) In B c Quang, Hà Giang, samples of tristeza and tatter leaf diseases have been collected

(grafted into p lant indicator Rusk citrange)

Table 3.9 Distributio n of virus and viro id in some citrus fruit tree s co llected fro m No rthe rn p ro vinces o f Vietnam (Plant Protection Research Institute, 2011)

Citrus trees

Disease verification result

Tristeza (Strip)

T atter leaf (RT-PCR)

Exocortis (RT-PCR)

Number of samples for tatter leaf and exocortis verification is not many due to process of samp le verification, samples that are not in fected by VLG or tristeza have been excluded and were not go through further verification

3.1.7 Improvement for DNA extraction method in diagno sing VLG in citrus

Assessment of VLG by naked eye or by iodine dia gnosin g method is very difficult to identify causative agents Using of DNA extraction method following the general procedures such as isolation solution (inc luding T ris-HCl; EDTA, NaCl); chemical for DNA purification (Sarkosy l, CTAB, chloroform, isoamyl alcoho l and phenol) includin g many toxic chemicals Isolation and purification of DNA fo llowing Hung et al., 1999 are very appropriate for a spec ific 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 h igh 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 negative ly impacted to human health and polluted environment as well

Such concerns have urged us to imp rove the method for DNA extraction by using NaOH alternative ly (table 3.10) This method is conducted in the National

T aiwan University of Science and Technology and in the same time, the method also tested successfu lly in the laboratory of Plant P rotection Research Institute

Using NaOH to isolate DN A has saved time significantly, from 5 to 6 hours in the old method to just from 45 minutes to 1 hour in th is method, depending on sk ills

of technic ian The advantage of this method is that DNA extraction is done by using NaOH and acetic acid on ly, reducing considerably costs on labour and chemical spending on DNA extraction process The extracted DNA shows positive resu lts in VLG assessment

Table 3.10 Improvement of DNA extractio n method in diagno sing VLG (at the laboratory in National Taiwan University of Science and Techno logy, 11/2011)

Pulverizing in Eppendorf

by NaOH

Regular extracted

so lution (Hung et al 1999)

3.2 Identification o f VLG’s causative agents and and transmission

3.2.1 Identification of VLG’s causative agents by electron microscope

Disease samp les wh ich have been collected from orange, mandarin, pomelo

and lime citru s crops from surveyed localitie s have been cut into super tinny slices and observed by electron microscope These observations have found that starch accumulated in the xy lem systems of slices and caused degradation of those sy stems

as well as severely damaged chlorop last in leaves Samples collected from different citrus crops have shown the same result, indicatin g that th is is a symptom to explain why VLG infected plants dying back so quickly; when tree losing its ab ility of photosynthesis, absorption and nutrient transportation through xy lem system s

3.2.2 Identification of VLG’s causative agents by biomolecular method

T his project has used three primers [16S of Sandrine Jagoueix (1996), A2J5 of

A Hocquellet and Hung et al (1999)] to assess VLG Reaction of all p rimers have shown an exact result in identify ing 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 Dia phorina citri Kuwayama on citrus p lants

If fru it plants have been planted with fresh varie ties without disease, the spread

of disease in nature will be vector D citri Kuwayama T he diversity of varieties of

citrus as we ll as their different cu ltivation will be outbreeding faster and the disease is able to adapt to server condition Psyllar a lways occurred among 12 months in year but they will have different densitie s The densitie s of vector in c itrus o rchard depend

on food, cultivation and pest management, especially in re lation to summer bud season in year and season temperature T he densitie s 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 c itrus psy llid on Xa Doai orchard in Nghe An

In 4 bud season in year, the densities o f psyllid are h ighest in summer and gradually descreasing in spring, autumn and winter On non-intensive farm ing orchard, the density of psyllid is 25.15 ind ividua ls/tree; in the meanwhile on the intensive farm in g orchards only obta ined 4.05 indiv idua ls/tree Similar in sprin g bud season were 21.5 vs 3.7 individuals/tree, autumn season were 12.25 vs 2.65 ind ividua ls/tree, winter season were 3.95 vs 0.9 individuals/tree

On Cam Canh orchard in Hanoi, the densitie s of psyllid were similar in Nghe

An in both non-intensive farming and in tensive farmin g orchards (fig 3.19)

Figure 3.19 The densities of Asian c itrus psy llid 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 farmin g the densities of p sy llid is lower than Hanoi and Nghe An In the summer bud season the densities of p sy llid only obtained 17.35 individual/tree; in sprin g and autumn bud season the densities of psyllid were from 7.05 – 9.6 individual/tree; On the intensive farming orchards the densitie s of psyllid were from 0.85 to 2.45 in 4 bud season (fig 3.20)

Fig 3.20 T he densities of Asian citrus psyllidon Cam Xa Doai orchard in Cao

Phong, Hoa Binh

T herefore, the result of survey showed high arising of densitie s of psyllid in main bud season in year On the non-intensive farming, the densitie s of psy llid was always higher than densities of psyllid on intensive farming orchards, the reason is that the non-intensive farmin g 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 su itab le with P lant P rotection Institute' results before (Ph m V n

m, 2005) The results of th is serve are scientific basic in order to experimental design for re-infection of the vector in the fields

3.3 Ho st and distribution identification of bacteria race cause greening disease

o n citrus in Northe rn 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 varietie s, there are only Tiên Chót (Severinia buxifolia) is