MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIET NAM ACADEMY OF AGRICULTURAL SCIENCES LE MAI NHAT STUDY ON GREENING IN CITRUS FRU IT TREE IN SOME NORTHERN PROVINCES OF VIET NAM AND RECOMMENDATIONS FOR PREV ENTIVE 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: Dr Ngo Vinh Vien 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: Viet Nam National L ibrary The Library Viet Nam Academy of Agricutural Sciences U 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 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” 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 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 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 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 Structure of thesis This dissertation comprises 146 pages, including the introduction pages; Chapter Scientific background and literature review (36 pages); Chapter Research materials, content and methodology (19 pages); Chapter Result and discussion (85 pages); Conclusion and recommendations pages with 41 data tables, 39 figures Reference of 164 papers and articles, including 34 Vietnamese papers, 130 English papers Chapter 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 diseaseinfected 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 to develop a system of integrated preventive and controllab le measures 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 to 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 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 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, 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 T rung et al, 1995 Method of surveying psyllid (Diaphorina citri Kuwayama) in citrus fruit tree following P h 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 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 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 Hịa 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 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 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 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) Citrus Scientific name T otal Rate (%) sample infected crops sample s VLG CTV CTLV CEVd Orange Citrus sinensis 55 81.81 37.78 14.28 Mandarin Citrus reticulata 50 86.00 27.91 9.68 Pomelo Citrus grandis 35 68.57 31.43 0 Lime Citrus aurantifolia 15 66.67 30.0 0 Kumquat Citrus microcarpa 20 60.00 33.33 0 Notes: VLG: greening; CEVd: Exocortis; CTV: tristeza; CTLV: tatter-leaf; 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 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 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 Figure 3.5 VLG’s symptoms in Figure 3.6 Sympto ms o f leave orange fruit lo tchy 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 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) Location Crops Citrus trees Xã oài Orange Kumquat Liêm (Hà N i) Di n Pomelo Canh Orange Sành/green c Quang (Hà Giang) Orange Xã oài Orange Ngh An PQ Mandarin Note: - : Not recorded yet; Disease verification result Tristeza T atter leaf Exocortis (Strip) (RT-PCR) (RT-PCR) + + + + + + + + - + + - + - + : Recorded by RT-PCR, Strip 11 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 bud season (fig 3.20) Fig 3.20 T he densities of Asian citrus psyllid on 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 months, grapefruit (Citrus maxima) and lemon (Citrus lemon) get symptom after months (table 3.17) Among the collected varietie s, 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 a l (In 2012) Simila rly, the laurel tree is allure tree for citrus psyllid as the domestic and foreign authors published T hrough the study, subjects in itially detected and identified that Móc M t tree (Clausena excavata) is also one of good host plants citrus psy llid but not infected VLG d isease So, it is maybe choice Ti n Chót plants for breeding and mass production of citrus psy llid, the laurel tree and Móc M t tree is used to breed fresh citrus p sy llid resources c itrus psy llid to design greenhouse experiments and extensive experiments Table 3.17 The infected ability o f Liberibacter asiaticus o n so me race of Rutacea (Sc reen house Plant Protection Research Institute, 2011) Species Vietnamese Name P CR’s diagnosis Rate (%) months months - + + 100 Citrus sinensis Cam Citrus unshiu Quýt ôn châu - + + 100 Citrus nobilis Cam sành - + + 100 Citrus reticulata quýt - + + 100 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 - - - Murraya paniculata Nguy t qu - - - Atalantia guillauminii Quýt hôi - - - Severinia buxifolia Ti n chót - + + 100 Citrus maxima Note: +: Positive to greening disease; -: Negative to greening disease; ình months ng LBNT: artificial infection 3.3.2 Bacteria identificatio n cause greening disease throught reaction of some varie ties on citrus plants disease resources In total 81 sample s were evaluated and div ided into four groups consisting of orange trees (24 samples), tangerine (30 samp les), 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%) T he VLG pathogenic strains were confirmed by specific primers of T omimura et al., 2010 Similar e xperiments to evaluate samples co llected from 30 sources citrus trees, infected with citru s 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) Bacteria strain No Sources The reaction of collected citrus plants from infected citrus (Citru s sinensis) w ith greenin g disease (0-3 score) Cam Xã Cam i oài Canh Di n (Citrus (Citrus (Citrus sinensis) reticulata) grandis) G/x -I-1-1 G/x -I-2-2 G/x -I-3-4 G/x -II-1-2 G/x - II -1 -3 G/x - II -1 -6 G/x - II -2 -7 G/x - II -3 -5 G/x - II -3 -9 10 G/x - II -4 -1 11 G/x - II -4 -2 12 G/x - II -4 -4 13 G/x - II -4 -8 14 G/x -III-1- 15 G/x -III-3- 16 G/x -III-3- 17 G/n.s-III-4- 18 G/n.s-III-4- 19 G/n.s-III-5- 20 G/c.l-VI-1-1 21 G/c.l-VI-1-3 22 G/c -VI-1-4 23 G/c -VI-3-1 24 G/t.q-VI-4-2 Negative control To tal 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 3 3 3 3 3 0 0 0 0 0 Chanh Eureka (Citrus limon) 3 3 0 0 0 0 0 Strain I Strain II × × × × × × × × × × × × × × × × × × × × × × 14 × × 10 14 Note: I: Hà N i, II: Ngh An, III: Hịa 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: = no symptom; = mild chlorosis without dwarfing; = intermediate chlorotic mottling with moderate dwarfing; = severe HLB symptom, including yellow mottle, leaf hardening, and curling with distinct dwarfing With 13 samples were collected from evaluated pomelo were obtained samples infected with race I with and samples infected with strain II Stra in II have appeared in different varietie s of grapefruit and also expre ssed different d isease severity levels of symptoms (tab le :20) Strain I and II cause disease on the citrus varietie s 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 10 11 12 13 Sources G/b.d-I-1-1 G/b.d-I-1-6 G/b.d-I-4-3 G/b.d-I-4-4 G/b.d-I-4-5 G/b.d-V-1-3 G/b.m-VI-3-4 G/b.n-VII-1-2 G/t.tr-VIII-1-5 G/t.tr-VIII-1-6 G/t.tr-VIII-2-1 G/t.tr-VIII-3-1 G/t.tr-VIII-4-1 T ng The reaction of collected citrus plants from infected citrus (Citrus sinensis) with green ing disease (0-3 score) Cam Xã Cam Canh i Di n Eureka oài (Citrus (Citrus (Citrus (Citrus reticulata) grandis) limon) sinensis) 3 3 3 0 3 0 3 3 3 0 3 0 3 2 3 2 3 3 3 3 3 0 3 3 3 0 Bacteria strain Strain I Stra in II × × × × × × × × × × × × × 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 citru s spec ies, are used as ornamental and spices plants so little attention to the disease The samples from the lemon tree was obtained samples of strain II (accountin g fo r 33.3%), on kumquat obtained samples of strain II (12.5%) Samples from kumquat variety showed similar reaction in B i Di n and Eureka lemons in level T he sample after pathogen ic artificial a ssessment by analyzed by duplex-P CR 15 technique, with two types of primer specificity (DNApol / nusG-rp lK operon) [Tomimura et al., 2010] P CR products of primer DNA Poly obtained at 988bp; P CR products of primer nusG-rplK operon obtained at 627bp Two types of primer were same reaction allows identification stra ins I, II and III P CR products from the strains I had lines appear on products; II strains obta ined product at 627 bp (figure 3:26) Combin ing trad itional methods with mo lecular biolo gy have identified pathogenic stra in s greening d isease in Vietnam and the distribution of pathogenic bacteria in the survey area (figure :27) 988 bp 627 bp Figures 3.25 Strain I cause disease on Cam Xã oài and Cam Canh; Strain II cause d isease on varieties (b i Di n, chanh Eureka, Xã oài cam Canh) Figures 3.26 The d up lex-PCR results and strain identification [CC: Cam canh, RL=Rumprue lime, Cal=calamondin (Qu t)] Strain I: obtained PCR 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 in shoot tip grafting Experimental resu lts had identified the MS media and combination with 0.5 grams of activated carbon in lite r for micro grafting, stem d iameter was 1.51 mm 16 At the same time have type of rootstock evaluation (3 leaves orange, sour grapefruit, mortgage, oranges, Cleopatra tangerines) u sed in graft of apical meristems of plant, and their surv ival in times of The rootstock from Ch p and sour grapefru it showed surviva l rate after graft obta ined 27-36%, with Ph c Hòa and Tr ng V ng citrus pomelo 30.2 to 41.7%, In micro-ap ical graftin g 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 leaves orange rootstock age and survival rate of Trung Vuong orange variety (table :27) T he results showed that if the young rootstock (1 week old) or old rootstock (4 week-old) gave lower surviva l rate compared with rootstocks from 2-3 week-o ld If usin g 1-week-old rootstock, the capacity for regeneration of tissues and cells quick ly create callu s, 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 T he rootstocks from 2-3 week-old is most suitable for graftin g, obtained highest ratio of surviva l, from 28.0 to 34.0% Table 3.27 Effection o f plant age time to survival rate after shoot tip graftting with Tr ng V ng orange variety (Plant Protection Research Institute, 2010) Success rate (%) Un-success rate for Un-success rate for Rootstock age died bud (%) died callu s (%) c c week-old 10.0 10.0 80.0 a a bc b week-old 34.0 26.0 40.0 week-old 28.0 ab 38.0 b 36.0 bc 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 med ia is suitable concentration to surviva l rate and development and growth of micro-grafted bud T here were sign ificant 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 Protectio n Research Institute, 2010 - 2011) Success rate Leaf new Long buds Medium (%) (After 20 days) (mm) c d MS 27.8 2.1 4.7 b a a a MS + 0,5mg/l BAP 44.3 5.4 9.8 MS + 1,0mg/l BAP 39.4 ab 4.2 b 7.0 ab b bc b MS + 1,5mg/l BAP 35.6 3.6 3.9 CV(%) 12.3 19.7 21.1 In order to improve shoot tip grafting, the project were optima led technique in apical grafting The result is optimal in the development of research methods of Su (2008), applied in the laboratory o f the Plant Protection Research Institute and 17 Nationa l University laboratory synthesis Taiwan The media was used in liquid MS medium supplemented growth regulator (i-inositol, thiamine-HCl, pyridoxine-HCl, nicotine) T he result is optimal in the development of research methods (2008), applied in the laboratory of the Plant Protection In stitute and University laboratory synthesis T aiwan The platform media was used in MS liqu id medium and supplemented growth re gu lators (i-inositol, th iamine-HCl, pyridoxine-HC l, nicotine) Use of the regulators, the surviva l 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 supp lements growth regulators, which help apica l livable, able to give early buds, it possib le to simultaneously between graft and rootstock shoots fast (tab le 3:29) Table 3.29 Effect of growth regulato rs supplement liquid medium in mic ro apical grafting (Plant p ro tection research institute, 2012) Total of grafted plants Success rate (%) MS 35 14.29 MS + Growth factors 35 34.28 Note: Growth factors (i- inosito l, thiamine-HCL, pyridoxine-HCL, nicotine) 3.4.1.3 The method of arising un-infected plants with greening disease in citrus plants Steriled apica l 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 surv ival rate is high, but the un-infected rate was low (table 3.30) Table 3.30 The testing o f apical sizes different to the ability of free disease after shoot tip graftinh (Plant Protection Research Institute, 2012) Ratio of Ratio of Apica l size Micro-graft Number Survical ununvarieties tested rate after infected infected microplants tristeza greening grafting disease disease (%) (%) (%) Meristem Ph c Hòa Pumelo 30 Tr ng V ng 30 Orange i Ph c Hòa 30 26.7 100 63.3 Meristem with Cam Tr ng V ng 30 100 73.3 two leaf 33.3 primordia i Ph c Hòa 30 36.7 73.3 23.3 Meristem with Cam Tr ng V ng 30 80.0 46.7 three or four 46.7 leaf primordia 18 3.4.1.4 Effected of plant age in micro-grafting in time into grow th and development of micro-grafted plant in time According to the study of Navarro (1972) and Hyun (2012) were able to transplant grafted plants in time from in-vitro conditions were planted directly into potting mix in strict conditions In order to improve and enhance the effic iency, the study by Su (1984, 2008) had used graftin g techniques 2nd time The plant age in first time graftin g gave successfu l 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 to time of shoot tip graft (Screen ho use Plant Protection Research Institute, 2010 - 2012) Plant age in i Ph c Hòa Cao B ng Cam Tr ng V ng Cao B ng first grafting Total number Surviva l rate Total number of Survival rate (%) (%) micro-grafted of microplants grafted plants week-old 13 15.4 16 18.8 week-old 14 78.6 13 76.9 week-old 10 90.0 12 91.7 In micro- apica l grafting technique, bud stage processing is concerned, used buds in grafting were very vulnerable Through practica l manipu lations buds, we had tried to find solutions to overcome th is phenomenon Results were determined by using -3 drops of Tween 20 for in san itizing solution and sterile d istilled water When the buds are located near the lower part suspended in the cup containin g 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 T ween 20 also had the effect of seepage slicks in the armpit leaves sprout, so better steriled effects and inc idence of less fungal contamination 3.4.2 Production of disease-free citrus seedlings in 3-level grid system greenhouse Seedling qua lity is an important factor in developing citrus fru it 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 graftin g 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 months has standard condition for transplanting, a lso in formula unimproment potting mix the rootstock need at least months after planting (in warm weather) to get standard condition for graft Improved potting mix could have been watered, the addition of fertilizer T he roots system a lways grow well, increasing resistance to soil-borne disease Seedling production in 3-level grid system greenhouse is imp lemented in all of 12 months in the year; the best season for grafting is summer T emperature range of 19 28-38°C is suitab le for grafting stage, surv ival 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 surviva l rate and plant sprouts out fast Parafilm paper used in techniques grafted seedlings, su rv ival rate of grafts is much higher than with conventional grafting methods, limited flow disease, fewer grafts rot, hea lthy plants, uniform This provides sign ificant enhancements to enable rapid chit and health The advantage of using p lastic paper is cheaper but the disadvantage is not to protect the environment due to the retained plastic 3.4.3 Tolerance of ro otstocks 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 lon gevity of trees as well as fru it weigh t Initially, understand ing and tolerance evaluation of rootstocks with greening d isease Pathogen were used strains I cause disease on grapefruit; strain II cause disease in Nam Son Orange, two stra in s are now widespread in northern Vie tnam After artific ia l infection with diseased scions, rootstocks trees have disease symptoms (table 3.35) Grafted by strain II with high ly pathogenicity, symptoms manifested quick ly, 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 a rtificial infection in infected grafts for rootsto ck is used in seedling production (Screen ho use Plant protection resea rch institu te, 2012) After months After months After months Rootstock Resources (infected TLB Disease TLB Disease TLB Disease grafts ) (%) level (%) leve l (%) leve l c c c Sour Pomelo Stra in I 0.0 28.89 51.48 b b a Sour Pomelo Stra in II 25.55 70.37 92.59 c c b Ch p Stra in I 0.0 31.11 85.0 a a a Ch p Stra in II 33.33 88.89 92.59 LSD0.05 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 d isease had direct influence to rootstock, which made limit grow height of rootstocks During follow-up, potentia lly pathogenic strains mutated virulent infection in the next generation T he same stra in of the disease on the same object in both rootstocks but obtained was lower plants The phenomenon of dwarf p lants has demonstrated symptoms of low dwarf tree in the fie ld 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 T he 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 suitab le for production of disease-free seedlings for each type of different citrus p lants In itial assessment of disease-resistant rootstock VLG on imported plants (table :37) Cleopatra rootstock, Carrizo citrange, Rangpur lime showed symptoms early, severity in to 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 Disease symptom after months after months (disease level) (disease level) Cleopatra mandarine Swingle citrumelo Fruit dragon Volkamer lemon Poncirus trifoliata 1 Carrizo citrange Troyer citrange 1 Rangpur lime Ch p 10 B i chua Note: Infected grafts from cam Nam S n; - : = No symptom (tolerant), = Mild (b lotchy mottling symptoms observed fro m to 30% on seed lings canopy, = Moderate (yellowing symptoms observed from 31 to 50% on seedlings canopy), = Severe (b lotchy 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 T he previous resu lts a lways used Tetracyline 1% for control greening disease (Aubert Bové, 1980; Tsai et al., 2012) In this research, based on mechanism of pest, antibiotics in contro l infected grafts, is Streptomycine (suitable for negative gram bacteria) Table 3.38 Effect of Strepto mycine to greening disease sympto m (Screen house Plant Pro tection Research Institute, 2012) Experiment T otal Survival Symptom after infection plants rate (%) months months months Steptomycine 1% + 12 hour 20 100 + + + Streptom ic ine 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 Streptomyc ine in 24 hours, the plants developed after grafted at th months were not occurred disease symptom, in the meanwhile in 12 hours the plants showed disease symptom after months grafted Although plants were not showed symptom, the PCR testin g showed positive Using Streptomyc ine had effect to inh ibit, prevent disease development, maintained low density of bacteria 3.4.4.2 Effective against re-in fected 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 diffe rent management (Nghi L c, Ngh An, 10/2013) Experiment Disease rate (%) Spring Summer Auntum buds buds buds c b Free disease seedlings + no pestic ide spraying 9.33 14.67c 16.0 Free disease seedlings + pesticide spraying 0.0 a 1.33 a 5.33a frequently b b b Free disease seedlings + local cultivation 4.0 10.67 16.0 LSD0.5 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; st evaluation: 15/3/2013; 2th evaluation: 15/7/2013; rd e valuation: 15/10/2013 After 15 months p lanted, re-infected plants occurred in “no pesticide spraying” method for contro l vector (9.33%), in “ local cultivation” (4%) In “pestic ide spray ing frequently” was not showed disease symptom but sever re-infected in later assessment, the disease rate of this experiment was 5.33% after years planted Other experiments were arising d isease 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 pestic ides will have a significant effect It is in bud concentrate, which will be limited food sources in the field for Psyllar’s density decreased T he technique of fertilizer is not only for bud concentrate, but also make fruit quality, y ield stability over the years Irrigation solution is not only for bud, but also filter out the effects of limitin g harmful sp iders, 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 so me province in Northern Viet Nam (2013) Main solution Implementation Free disease - Selection of parents seedlings - 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 month for new planting, clearing debris and trees were destroyed VLG disease and other vira l 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 - Orange variety: 650 – 750 plants/ha plantin g - Mandarine variety: 800 – 1000 plants/ha - Grapefruit variety : 450 – 500 plants/ha Size hole for - Mountainous: 1m × 1m × 1m plantin g - 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 fle xibly in order to improve effic iency, reducing investment costs In this tra ining solutions need to be concerned because the producers not understand and not capture the citrus pest cannot control it Moreover, major pest arising time, each different pest should a lso be meticulous to foster integrated disease management CONCLUSIONS AND RECOMMENDATIONS Co nclusions 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 w ith symptoms of swollen veins exp lode, 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, causin g starch accumulation and necrosis chlorop lasts Usin g specific primers Jagoue ix Sandrine (16S); A Hocquellet (A2J5) and Hung et al (1999) combines innovative DNA extraction step with NaOH for fast results with accuracy comparable to diagnostic k its used, low price, not use toxic chemica ls The VLG disease showed rapid symptoms on Xã oài orange, Cam Canh after 45 days of graftin g The density of RCC was ind ividuals/tree or individuals/tree in 24-hour exposure, symptoms (respectively) appears only after months and 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, T hanh Hoa, is host with greening disease 1.3 By the reaction of VLG samp les for bio-monitoring plants and a number of fruit trees (orange, tangerine, grapefruit, and kumquat) combined w ith duplex-P CR techniques have identified bacteria strains in the North belon ging to VLG stra in I and 24 II The strain II was first recorded in some northern provinces of Viet Nam In Hanoi, Bac Giang, Ha Giang, Hoa Binh, Nghe An, where the infected samples showed disease rate with strain I higher stra in II (respectively from 53.84 to 66.67% and from 33.33 to 46.16 %) In Thua Thien Hue, Hung Yen, where the infected samples showed disease rate of stra in I (33.33 to 40%) lower than strain II (60 to 66.67%) In Quang Ninh infected sample is equal with both strain infection 1.4 Identified rootstock in 2-3 weeks on MS med ium combined with 0.5 g of activated charcoal to culture the 1st rootstock, is most suitable for apical gra fting Average survival rate of first graftin g increased from 23.2 to 28.5% (for grapefru it and oranges) to 34.0 to 41.7% (for Phuc Hoa grapefruit, Trung Vuong oranges) Added BAP 0.5 mg / liter in MS liquid medium increases the surviva l rate and growth stimu lants for bud grafting of 44.3% Add ition of growth re gulators (iinositol, thiam ine-HCl, pyridoxine-HCl, n icotine) for success rate o f graftin g in 1st is 34.28% but shorten the retention time of grafting 1st in laboratory and 2nd rootstock (1.5 to months old handled) Apica l graftin g consists of schizogenous and two cotyledons for VLG disease-free ga ined highest effective with 100% and tristeza disease-free were 63.3 to 73.3% Using potting mix soil included yellow sand, sawdust, wood shavings and complex microbial fertilizer (as of 1/5 2/5 2/5) after months of grafted, the rootstock can used to produce standard disease-free seedlin gs 1.5 Proposed solutions against re-infection of VLG d isease and limit the spread of disease on farm efficiently (Handling Streptomycine 1% branch grafts, the disease destroys the source tree, plantin g d isease-free seedlings, seasonal and density planting, cultivation methods, chemistry, training, etc.) After two years, the reinfected rate of VLG disease accounted for only 5.33% Reco mmendations 2.1 Using dia gnosis method for VLG disease by P CR with primers Jagoueix Sandrine (16S); A Hocquellet (A2J5) and Hung et al (1999) and improved DNA extraction step with NaOH will shorten the time and reduce costs 2.2 Using disease-free seedlings with c lear source, the competent authority certified disease-free greening and other virus disease, viro id for preventing reinfection PUBLISHED OF AGRICUTURAL DOCTORAL THESIS Ngo Vinh Vien, Le Mai Nhat, Nguyen Bich Ngoc (2012), The Current Status of HLB Epidemic in Vietnam, International Symposium on Epidemiology and Disease management of Citrus HLB Disease for Sustainable Citrus Production in ASPAC; 5-10 Nov 2012 Nguyen Thi Bich Ngoc, Le Mai Nhat, Ngo Vinh Vien, Pham Thi Dung, Nguyen Nam Duong, Nguyen Van Viet (2013), “Study on modification of Shoot tip grafting technique on citrus”, Journal of plant protection, seri 5, p 36– 40 Le Mai Nhat, Ngo Vinh Vien, Nguyen Van Viet, Ngu yen Thi Bich Ngoc (2014), “Identification of bacterial strains caused citrus Huanglongbing in the northern part of Viet Nam”, Journal of plant protection, seri 1, p – 10 ... conduct ? ?Study on greening in citrus fruit trees in some Northern provinces of Vietnam and recommendations for preventive and controllable measures? ?? Research objective 2.1 Objective Identifying... 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. .. control VLG in some Northern provinces New contributions o f dissertation - Providing a scientific source on popularity, symptom, and host of VLG in citrus in Northern Vietnam - Identifying bacteria