J[ Phytopathology 037\ 210Ð215 "1999#Þ 1999 Blackwell Wissenschafts!Verlag\ BerlinISSN 9820!0674

Department of Plant Pathology\ National Taiwan University\ Taiwan

Identi_cation of Alternative Hosts of the Fastidious Bacterium Causing Citrus

Greening Disease

T[ H[ HUNG\ M[ L[ WU and H[ J[ SU

Authors| address] Department of Plant Pathology\ National Taiwan University\ Taipei 095\ Taiwan "correspondence toM[ L[ Wu#

With 3 _gures

Received June 09\0888^ accepted December 02\ 0888

Keywords] alternative hosts\ citrus greening\ dot hybridization\ DNA probe\ fastidious bacterium

Abstract

Citrus greening is a severe disease caused by a fastidiousbacterium "GFB# residing in the sieve tubes of its hosts[It is an epidemic disease and is spread by insect vectors[In Asia\ the Asian citrus psyllid "Diaphorina citri Kuway!ama# is the vector for GFB[ For the epidemiologicalstudy\ an investigation of alternative hosts of GFB wasmade[ Four suitable hosts of the Asian psyllid that areconsidered as possible alternative hosts of GFB wereinvestigated on graft!inoculation tests[ The multi!plication of GFB in plants was monitored by dot hybrid!ization using a GFB!speci_c DNA probe developedpreviously by us[ The results demonstrate that GFB canreplicate in Chinese box orange "Severinia buxifolia# andwood apple "Limonia acidissima#\ but not in commonjasmin orange "Murraya paniculata var[ paniculata# andcurry leaf "Murraya euchrestifolia#[ Chinese box orangeis a good host in which GFB replicates as well as it doesin its citrus hosts[ Wood apple is a transient host in whichGFB exists temporarily and disappears several monthslater[ Common jasmin orange and curry leaf are nothosts of GFB as they showed no detectable signals in dothybridization tests throughout 0 year of experimentation[

Zusammenfassung

Erkennung von alternativen Wirtsp~anzen fu�r das anspruchsvolle

Bakterium\ welches die citrus greening Krankheit verursacht

Die bedeutende Citruskrankheit citrus greening wirddurch ein anspruchsvolles Bakterium "GFB#\ welches inden Siebro�hren der Wirtsp~anze zu _nden ist\ verursacht[Als epidemische Krankheit wird citrus greening durchInsekten verbreitet[ In Asien dient der Asian citrus!Blatt~oh "Diaphorina citri Kuwayama# als GFB!Vektor[Bei dieser epidemiologischen Studie wurde alternativeWirtsp~anzen des GFBs gesucht[ Es wurden vierWirtsp~anzen des Asian!Blatt~ohs ausgesucht\ welcheals mo�gliche Wirte des GFBs angesehen werden[ Diese

U[ S[ Copyright Clearance Center Code Statement] 9820Ð0674:1999:3795Ð9210 , 04[99:9

wurden in einem Prop_nokulationstest untersucht[ DieGFB!Vermehrung in den P~anzen wurde durch einenDothybridisierungstest mit Hilfe von GFB!spezi_schenDANN!Proben ermittelt[ Die Ergebnisse zeigten\ dassGFB in chinese box orange "Severinia buxifolia# sowiewood apple "Limonia acidissima# sich vermehren konnte\jedoch nicht in common jasmin orange "Murraya panicu!lata var[ Paniculata# oder curry leaf "M[ Euchrestifolia#[In S[ buxifolia konnte sich das GFB so gut vermehrenwie in den Citruswirtsp~anzen[ L[ acidissima ist einetempora�re Wirtsp~anze\ GFB ha�lt sich nur eine kurzeZeit in der P~anze auf und verschwindet nach einigenMonaten[ M[ paniculata var[ paniculata und M[euchrestifolia fungierten nicht als Wirtsp~anzen fu�r dasGFB\ es konnten keine Signale in dem Dothybridisie!rungstest u�ber einem Zeitraum von einem Jahr nachge!wiesen werden[

Introduction

Citrus greening is a very severe disease in Asia[ It is causedby a nonculturable fastidious bacterium that inhabits thesieve tubes of its hosts "Garnier et al[\ 0873#[ The patho!gen infects citrus trees of almost all cultivars\ and causessubstantial economic losses to the citrus industry byshortening the lifespan of the infected trees[ The insectvector\ Asian citrus psyllid "Diaphorina citri Kuwayama#\spreads this disease in Asia "Capoor et al[\ 0856^ Chenet al[\ 0862^ Aubert\ 0876^ Gottwald et al[\ 0878#[ It hasbeen an important epidemic disease and has been di.cultto control in Taiwan and several other Asian countries[Furthermore\ citrus greening has a long incubationperiod\ and many latently infected citrus plants occur inthe _eld "McClean\ 0869^ Huang\ 0868#[ Development ofan accurate diagnostic technique becomes the _rst stepfor disease control[ However\ the fastidious bacterium"GFB# causing citrus greening is di.cult to detect becauseof its low concentration and uneven distribution in its

211 HUNG et al[

natural hosts "da Graca\ 0880#[ Fortunately\ the appli!cation of DNA probes overcomes the di.culty of GFBdetection[ A standard protocol using a DNA probe hasbeen developed by the authors and successfully appliedin GFB detection for several years "Hung et al[\ 0888a#[This method has also been applied in ecological studies ofcitrus greening to obtain some answers to epidemiologicalquestions that could not be determined by conventionalmethods[

In Taiwan\ there are three major strategies for thecontrol of citrus greening] "0# the establishment of patho!gen!free nursery systems^ "1# vector "psyllids# control^and "2# elimination of the inoculum sources includinginfected citrus trees and alternative hosts of GFB[ Atpresent\ there is no detailed research about alternativehosts of GFB[ To investigate alternative hosts of GFB\four suitable hosts for Asian citrus psyllids have beentested[ These four plants are the common jasmin orange"Murraya paniculata var[ paniculata#\ curry leaf "Murrayaeuchrestifolia#\ wood apple "Limonia acidissima#\ andChinese box orange "Severinia buxifolia#[ They all belongto the Rutaceae family[

Common jasmin orange "CJO# and curry leaf "CL# aretwo members of Genus Murraya[ CJO is a shrub that iswidely distributed in tropical Asia\ and commonly cul!tivated as a hedge plant[ CL di}ers from CJO in manycharacteristics\ especially in smell[ CL is widely cultivatedin countries such as Malaysia and India where curry iscommonly consumed[ Both CJO and CL are favouredhosts of psyllids "Lin et al[\ 0862^ Chakraborty et al[\0865^ Miyakawa\ 0879#[ In Taiwan\ CJO is very popularwhereas CL cultivation is rare[

Wood apple "WA# has not yet been cultivated inTaiwan\ but it is commonly grown as an ornamentalplant in Thailand\ India and Indonesia and it is also aknown host for psyllids[

Chinese box orange "CBO# is a spinous shrub dis!tributed in India\ Malaysia\ Vietnam\ southern China\the Philippines\ Taiwan and Japan[ It is often seen incitrus orchards and has proved to be an important hostof psyllids in Taiwan "Lin et al[\ 0862#[

The ability of GFB to survive in these four test plantswas investigated using the graft!inoculation test[ Theoccurrence and multiplication of GFB in hosts weremonitored by dot hybridization tests using a GFB!speci!_c DNA probe[ In this paper\ it is shown that GFBcan inhabit other hosts in addition to citrus hosts[ Thisdiscovery o}ers important information for the control ofcitrus greening disease[

Materials and Methods

Plant materials

Four suitable hosts of psyllids\ CJO\ CL\ WA\ and CBO\were chosen for graft!inoculation tests[ The plants wereobtained from seeds^ those of CJO and CBO were col!lected from local plants in Taiwan whereas the seeds ofCL and WA were exotic\ CL being obtained from Malay!sia and WA from Thailand[ Valencia sweet orange "Citrussinensis L[# was used as the control citrus host in thisexperiment[ The healthy citrus plants were obtained using

a technique of shoot tip grafting "Murashige et al[\ 0861^Su and Chu\ 0873#[ A GFB!infected Valencia plant wasthe inoculum source to supply the GFB!infected scionsfor the graft!inoculation tests[ All experimental plantswere under insect!proof control in a greenhouse[

Graft!inoculation

The budwood!grafting method referred to in other pub!lications "Su and Chu\ 0873^ Yoshida\ 0885# was chosenfor the graft!inoculation[ The GFB!infected citrus scionswere grafted onto seedlings of CJO\ CL\ WA and CBO[After grafting\ these test plants were sampled and DNApreparations were made monthly for the detection ofGFB[ The occurrence and multiplication of GFB weremonitored by dot hybridization tests using a GFB!speci!_c DNA probe[ Observations and records of symptom!development were also carried out monthly[ For reversetests\ GFB!infected scions of alternative hosts were graf!ted onto the citrus plant "Valencia sweet orange#[ Graft!ing was judged successful when the scions survived formore than 2 months after grafting[ The growth of thegrafted scions was graded good\ poor\ or no growth bycomparing its growth to that of citrus grafted on citrus[

Preparation of nucleic acid samples for dot hybridization tests

The nucleic acid samples used in dot hybridization testswere prepared using a method described by Lee andDavis "0877# and Lee et al[ "0877\ 0889# with minor modi!_cations[ Five grams of leaf midribs were powdered inliquid nitrogen and resuspended in 04ml GFB extractionbu}er ð299mM mannitol\ 49mM Tris!HCl "pH6[3#\ 4mM

ethylenediaminetetraacetic acid "EDTA#\ 3mM b!mer!captoethanolŁ[ After low speed centrifugation "3999× gfor 09min#\ the supernatant was centrifuged at 01999× gfor 04min to pellet the GFB[ This GFB pellet was resus!pended in DNA extraction bu}er ð9[0 M Tris!HCl"pH7[9#\ 9[94 M EDTA\ 9[4 M NaCl\ 9[0) N!lau!roylsarcosineŁ\ incubated at 44>C for 0 h and centrifugedat 3999× g for 09min to remove debris[ The supernatantwas mixed with 0) cetyltrimethylammonium bromide"CTAB# and incubated at 54>C for 09min[ The samplewas clari_ed by chloroform ] isoamyl alcohol "13 ] 0# andphenol ] chloroform ] isoamyl alcohol "14 ] 13 ] 0# extrac!tion\ and then mixed with 9[5 volumes of isopropanol topellet DNAs[ The DNA pellet was suspended in 5× SSCsolution ð9[8 M NaCl\ 9[98 M sodium citrate\ "pH6[9#Ł tomake 2mg:ml for GFB detection[ All DNA samples frommonthly collections were stored at −19>C[

GFB!speci_c DNA probe

A cloned GFB!speci_c DNA fragment "9[13 kb#\ whichwas ligated into plasmid pBS "Stratagene Biotec[\ LaJolla\ CA\ USA#\ was chosen to develop a GFB DNAprobe[ This probe has demonstrated its speci_city andsensitivity\ and has been successfully used in the detectionof GFB in various citrus cultivars "Hung et al[\ 0888a#[A polymerase chain reaction "PCR#!labelling method wasused in the preparation of a biotinylated GFB!speci_cDNA probe as described by Panaud et al[ "0882# withslight modi_cation[ Ampli_cation was performed in 49ml

212Alternative Hosts of the Fastidious Bacterium Causing Citrus Greening Disease

reaction mixture containing 49 ng each of the two oppos!ing primers "T2 primer] 4?!AAT TAA CCC TCA CTAAAG GG!2?^ T6 primer] 4?!GTA ATA CGA CTC ACTATA GGG C!2?#\ 099 ng recombinant plasmids as tem!plates\ 9[1mM each of three dNTPs "dTTP\ dCTP dGTP#\9[07mM dATP\ 9[91mM biotin!6!dATP "Gibco BRL\Gaithersburg\ MD\ USA#\ and 1[4U Taq polymerase inincubation bu}er ð099mM Tris "pH7[2#^ 499mM KCl^1mM MgCl1\ and 9[90) gelatin\ w:vŁ[ The reaction mix!ture was overlaid with 29ml mineral oil[ The thermalcycles was run for 29 cycles "denaturation at 83>C for0min\ annealing at 49>C for 0min\ and extension at 61>Cfor 1min#[ All thermal cycles of PCR were performed ona DNA Thermal Cycler 379 "Perkin!Elmer Cetus\ FosterCity\ CA\ USA#[ The PCR product was puri_ed by HighPure PCR Product Puri_cation Kit "BoehringerMannheim\ Mannheim\ Germany# and suspended in49ml TE bu}er "pH7[9#[

Dot hybridization

For dot hybridization\ DNA samples in 5× SSC solutionwere denatured by boiling for 09min[ Three microlitres ofDNA\ after four!fold serial dilution in 5× SSC "startingconcentration at 2mg DNA:ml#\ were dot blotted on nylonmembranes[ The membranes were air dried\ baked at79>C for 1 h under vacuum[ Pre!hybridizations wereperformed at 57>C for 0 h in hybridization solution] 4×SSC\ 9[0) "w:v# N!lauroylsarcosine\ 9[91) "w:v# SDS\0) "w:v# blocking reagent "Boehringer Mannheim#[Hybridizations were performed at 57>C for 01Ð05 h inhybridization solution plus denatured DNA probes"49 ng:ml#[ The bound probes were detected with Blu!GENE detection system "Gibco BRL# according to themanufacturer|s instruction[

Results

Graft compatibility

The four test plants were di}erent in graft compatibility[The WA and CBO were compatible with citrus and sothe citrus scions survived\ and grew on the WA and CBOplants[ The CJO and CL were less compatible with citrusand so the citrus scions could survive on CJO and CL\but they did not grow well[

Multiplication of GFB into four suitable hosts of psyllid vector

After graft!inoculation\ four test plants were sampledand DNA was extracted every month[ The multiplicationof GFB in test plants was monitored by dot hybridizationtests using a GFB!speci_c probe[ CJO and CL showednegative results with no hybridization signal appearingduring 0 year of experimentation[ GFB cannot replicatein CJO and CL according to the results of dot hybrid!ization[

The WA and CBO showed two di}erent positive results"Fig[ 0a\ b#[ In the case of WA\ GFB could be detectedfrom the _fth to tenth month after graft!inoculation[ Thestrength of the dot hybridization signal "judged by serialdilution tests# showed that the signal reached a peakat the seventh month and declined quickly[ No signalappeared at the eleventh and twelfth months[ GFB could

be detected only between the _fth and tenth month\ andits detection signals were much weaker than the positivecontrol of GFB!infected citrus samples[

The CBO showed apparently positive results in thisexperiment[ When compared with citrus "Fig[ 0c#\ thepattern of dot hybridization in serial dilution is similar[The _rst signal appeared at the third month in CBO withthe signal maintaining a high strength after reaching apeak at the eighth month[ It was the same as citrus "Valen!cia sweet orange# control\ but with citrus having slightlystronger signals than CBO[

For the convenience of data analysis\ the hypotheticaldynamic curves of GFB replication were drawn accord!ing to the results of dot hybridization tests in WA\ CBOand citrus "Fig[ 1#[ The curve of WA is an inverted {V|shape "Fig[ 1a#\ which increased linearly from the fourthto the seventh month and decreased linearly from theseventh to the tenth month[ The WA curve returned tothe zero at the tenth month[ The curves of both CBO andcitrus are the standard growth curves "Fig[ 1b\ c# whichinclude lag\ log and stationary phases[ The _rst 1 monthsare the lag phase\ in which the concentration of GFB inhosts is too low to be detected by dot hybridization tests[The following 5 months "second to eighth# are the logphase\ in which GFB could be detected and its con!centration increased linearly[ The stationary phase beganat the eighth month when the concentration of GFBreached a maximum[

Symptom expression

In addition to GFB detection using a DNA probe\ arecord of symptom!expression was also made in the pre!sent experiment[ Similar to the results of dot hybrid!ization\ CJO and CL showed negative results[ Nosymptom occurs on CJO or CL 01 months after graft!inoculation[ WA showed mild yellowing symptoms at thesixth month\ apparent yellowing symptoms at the ninthmonth\ and recovered slightly from yellowing at thetwelfth month[ CBO showed mild chlorosis symptoms atthe sixth month\ evident chlorosis at the ninth month\and additional leaf!hardening and vein enation at thetwelfth month[ The Valencia sweet orange\ which wasused for the positive control of symptoms\ showed yel!lowing symptoms at the sixth month\ severe yellowingand leaf!hardening at the ninth month\ and additionalvein enation at the twelfth month[

GFB transmission from CBO back to citrus host by graft!inocu!

lation

According to the results in Fig[ 0\ GFB can survive forover 0 year only in CBO hosts[ CBO was shown to be agood recipient plant of GFB in this graft!inoculationexperiment[ To understand whether CBO is quali_ed tobe a good donor of GFB or not\ another experimentof graft!inoculation was made[ A healthy citrus plant"Valencia sweet orange# was inoculated by grafting withGFB!infected CBO scions[ GFB multiplication was alsomonitored by dot hybridization tests\ and the result isshown in Fig[ 2[ GFB can be transmitted from CBO backto the citrus host[ GFB infection in this citrus host was

213 HUNG et al[

Fig[ 0 Dot hybridization tests usinga biotinylated GFB!speci_c DNAprobe for the detection of GFB insuspected alternative hosts graftedwith the GFB!infected citrus scions[There were three test plants show!ing positive results in the hybrid!ization tests] wood apple "a#\Chinese box orange "b# and Valen!cia sweet orange "c#[ They weresampled and preparations ofnucleic acid were made monthlyafter grafting[ The nucleic acid sam!ples from monthly collection "indi!cated with the ordinal number 0st\1nd\ [ [ [ \ 01th# were applied ontonylon membranes in four!folddilution series\ 2 ml per spot^ thenumber 0\ 3\ 05\ 53 and 145 rep!resent reciprocals of dilution fromstarting concentration at 2 mg:ml[The diseased "d# and healthy "h# cit!rus samples "Valencia sweet orange#were included in dot hybridizationtests for the positive and negativecontrols[

_rst detected by dot hybridization tests at the third monthafter graft!inoculation\ and the signals reached a maximalstrength at the eighth month[ The pattern of dot hybrid!ization in serial dilution was close to Fig[ 1c where thecitrus plant was graft!inoculated with GFB!infected cit!rus scions[ GFB!infected CBO scions approximately havethe same ability of inoculation as citrus scions[

GFB detection of CBO samples collected from the _eld

In the spring of 0888\ eight CBO samples were collectedfrom the _eld near diseased citrus orchards in southernTaiwan to test for GFB[ The dot hybridization testshowed that one sample "CBO1# from the _eld showedpositive "Fig[ 3#\ which indicated that there are GFB!infected CBO plants actually existing in the _eld[

Discussion

Our experiment demonstrates that GFB may exist per!sistently in CBO just as in citrus plants[ In graft!inocu!

lation tests\ CBO was a susceptible recipient plant inwhich GFB could survive and replicate^ it was also aquali_ed donor plant that can transmit GFB to citrushosts by grafting[ CBO could be an alternative host ofGFB according to these results[

CJO and CL are two members of the genus Murraya[They are suitable hosts for psyllids but not for GFB[Although GFB!infected scions of citrus can survive onthem\ GFB cannot be transferred into them[ No signalappeared in CJO and CL when graft!inoculation and dothybridization were performed[

Interestingly\ GFB can slightly replicate in WA butnot survive for a long time[ In these experiments\ GFBin WA could be detected only from the _fth to tenthmonth after grafting[ It indicates that WA is a non!persistent host of GFB[

For further identi_cation\ the experiment of vectortransmission has been continued since January 0888[ Inthe study of vector transmission\ the viruliferous psyllids

214Alternative Hosts of the Fastidious Bacterium Causing Citrus Greening Disease

Fig[ 1 The hypothetic dynamic curves of the ~uctuation of GFB rep!lication in wood apple "a#\ Chinese box orange "b#\ and Valencia sweetorange "c#[ The curves are drawn according to the end point signal ofserial dilution in dot hybridization tests "shown in Fig[ 0a\ b\ c#[ Theannotation is the same as described in Fig[ 0[

are the donors of GFB instead of GFB!infected scions[This experiment is more di.cult and time!consumingthan graft!inoculation tests[ It is likely to be _nished bynext spring[ However\ it has already been established thatthe preliminary results are identical to the results in thispaper[

Multiplication of GFB in its alternative hosts appearsto be linked to graft compatibility between citrus andthe alternative hosts[ According to the results describedabove\ CJO and CL have poor graft compatibility tocitrus scions and GFB seems to dislike inhabiting them[

Fig[ 2 Dot hybridization test usinga biotinylated GFB!speci_c DNAprobe for the detection of GFB inthe Valencia sweet orange graftedwith the GFB!infected scions ofChinese box orange[ The anno!tation is the same as described inFig[ 0[

Fig[ 3 Dot hybridization test using a biotinylated GFB!speci_c DNAprobe for the detection of GFB in the samples of Chinese box orange"CBO# collected from the _elds in southern Taiwan[ Lane Vd\ a diseasedValencia sample for positive control of citrus^ Lane Vh\ a healthyValencia sample for negative control of citrus^ Lane BOd\ a GFB!infected CBO sample via graft!inoculation for positive control of CBO^Lane BOh\ a healthy CBO sample for negative control of CBO^ LaneBO0½BO7\ the test samples of CBO collected from the _elds[ Thenumber 0½145 indicate reciprocals of four!fold dilution from startingconcentration 2 mg:ml of nucleic acid samples[

In contrast\ WA and CBO have good graft compatibilityto citrus and GFB can replicate in them[ However\ WAis a transient host of GFB\ which suggests that graftcompatibility is probably just one of the qualifying con!ditions to become a host of GFB[

Alternative hosts always play an important role in anepidemic disease\ but they are often neglected in epi!demiological studies especially when they cannot berecognized[ Alternative hosts are concealed havens forpathogen survival[ Based on the results reported in thispaper\ CBO should be regarded as an alternative host ofGFB[ The data of GFB detection in the samples collectedfrom the _eld verify that there is GFB!infected CBOalready existing naturally[ This discovery has not yetappeared in any other reports[ On the other hand\ CBOis not only an alternative host of GFB\ but also a possiblehost of citrus tristeza closterovirus "CTV#[ Yoshidareported that CBO is susceptible to CTV in his experi!ments with graft!inoculation "Yoshida\ 0885#[ Tsai dis!covered CBO is also a suitable host for the brown citrusaphid "Toxoptera citricida# that is the most e.cient CTVvector "Tsai\ 0887#[ CTV causes citrus tristeza diseasewhich is another world!wide disease of citrus[ Greeningand tristeza are considered as two of the most severediseases in Asia[ It is strongly advised that CBO plantsin the citrus orchard and its vicinity should be eliminatedfor the control of citrus greening and tristeza disease[

Among the four suitable hosts of psyllids\ CJO and

215 HUNG et al[

CBO are often seen in Taiwan[ CJO is a common hedgeplant cultivated everywhere in Taiwan[ Fortunately\ thepresent experimental data show negative results of GFBreplication in CJO[ CJO was originally considered to bea possible host of GFB "da Graca\ 0880^ Guo and Deng\0887^ Singh and Nimbalkar\ 0866#\ but the present datasuggest that CJO should be not a host of GFB[ Most ofthe CBO plants are wild in Taiwan[ The character ofCBO in the epidemiology of citrus greening will be furtherunderstood when the authors complete their nextresearch project that includes transmission experimentsby psyllid vector\ recording seasonal ~uctuation of psyl!lids population on CBO plants\ and investigation ofGFB!infected percentages of CBO plants in _elds[ Thepsyllids!transmission experiment will also be applied inCJO\ CL and WA to recon_rm their resistance or tol!erance to GFB infection[

According to local records of ~ora\ CL is rare and WAis absent in Taiwan[ However\ they are prevalent andimportant in tropical Asia[ CL is a spice\ which is con!sidered as a necessity in several south!eastern Asian coun!tries[ Several samples of CL were obtained fromMalaysia\ but GFB was never detected in them[ Thepresent data also show negative results in GFB detectionvia graft!inoculation tests[ WA is normally used as anornamental plant for landscaping in several Asian coun!tries[ The present results indicate that WA is not a goodhost for GFB\ although GFB can temporarily replicatein it[ Therefore\ for the control of greening it would beadvisable to avoid planting WA in the vicinity of citrusorchards[

The external symptom expression seems to be some!what similar to the GFB!infection[ CJO and CL did notshow symptoms after graft!inoculation\ which cor!responds with the negative results from dot hybridizationtests[ CBO showed chlorosis\ leaf!hardening and veinenation symptoms 0 year after grafting[ Dot hybrid!ization tests verify that the symptoms of CBO and GFBinfection are correlative[ WA was shown to be a transienthost of GFB\ but the yellowing symptoms on WA werestill induced[ Interestingly\ WA recovered from yellowingsymptoms when GFB was declining in WA[ These datashow that symptom!expression may be related to infec!tion and replication of GFB[

All the detections of GFB in this research were con!ducted by DNA probes with dot hybridization tests[ Thismethod has its limit in the sensitivity for GFB detection[Recently\ a faster and more sensitive assay using poly!merase chain reaction to detect GFB in its hosts hasbeen developed "Hung et al[\ 0888b#[ Therefore\ anotherexperiment is being carried out to recon_rm the dataobtained from this paper using the PCR!based method[This research is not yet complete\ but the preliminaryresults correspond with those presented in this paper[

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