PertanikaJ. Trop. Agric. Sci. 16(2): 157-160 (1993)

COMMUNICATION II

ISS : 0126-6128© Universiti Pertanian Malaysia Press

The Use of Antibody-sensitized Latex to Detect Cymbidium MosaicVirus in Orchids

ABSTRAK

Ujian agglutinasi lateks (L) dan satu modijikasi ujian lateks, bersalut protein A (PAL) telah dibandingkanuntuk sensitisasi dengan antibodi kepada Cymbidium mosaic virus' (CyMV). Satu siri kepekatan globulin telahdiuji terhadap CyMV tulin. Kepekatan virus minimum yang boleh dikesan adalah sama untuk kedua-dua reaksiL dan PAL iaitu 0.104 llg/ml. Titik akhir yang boleh dikesan di sap orkid Oncidium Gower Ramsey adalahserupa dipencairan 1/2560. PAL didapati lebih sensitif dari L di pencairan globulin lebih tinggi iaitu 1/512.PAL juga menunjukkan kesensitifan lebih tinggi dari L berasaskan kepada lima hibrid orkid dan menunjukkanpersamaan dengan ujian mikroskop elektron.

ABSTRACT

Latex agglutination test (L) and a modijication of the latex test, protein A-coated latex (PAL) were compared forsensitization with antibodies to Cymbidium mosaic virus (CyMV). A series ofglobulin concentrations was testedagainst purified CyMV. The minimum detectable virus concentration was similar for both L and PAL reactionsat 0.104 llg/ml. The detectable end points in infected Oncidium Gower Ramsey orchid sap were similar at adilution of 1/2560. PAL was found to be more sensitive than L at a higher globulin dilution of 1/512. The PALtest showed higher sensitivity than L based on five orchid hybrids and compared favourably with electron

microscopy test.

INTRODUCTION

Commercially produced orchids in Malaysia arefrequently infected with Cymbidium mosaic virus(CyMV) and to a lesser extent with Odontoglossumringspot virus (ORSV) (Abdul-Samad 1985,1986,1989a, 1989b). Detection and identification ofplants infected with CyMV or ORSV or bothhave been difficult because the infected plantsmay be symptomless or they may show somechlorotic or necrotic patterns pending on thespecies or hybrids. At present there is no avail­able method of detection for use in orchidviruses locally. Imported orchids are rarely testedfor the presence of viruses. Therefore a simpleand quick method of testing is needed thatcombines high specificity and sensitivity withpractical value, especially in quarantine programs,and for the development and testing of virus­free stocks. The most obvious choice would be aserological test.

The latex agglutination test (L) and theprotein A-coated latex (PAL), a modification ofthe latex test (Querfurth and Paul 1979) havebeen used widely for the detection of plant

viruses in their naturally occurring host as wellas in herbaceous plant indicator species (Bercks1967; Abu Salih et al. 1968; de Sequera andLister 1969; Mumford 1977; Khan and Slack1978; Koenig and Bode 1978; Thomas 1980;Torrance 1980; Demski et al. 1986). Due to theirsimplicity and the fact they do not requireelaborate instruments, they can be used to testlarge numbers of samples in the laboratory or infield situations. Therefore this study was under­taken to determine the sensitivity of L and PALand their potential use in detecting CyMV inorchids. A comparison between L, PAL andelectron microscopy was also carried out.

MATERIALS AND METHODS

Virus and Antiserum

Cymbidium mosaic virus (CyMV) isolate was origi­nally obtained from the orchid Cattleya sp.(Abdul-Samad 1985) and propagated onDendrobium sp. The virus was purified accordingto the method used by Wisler et al. (1982).Systemically infected leaves were homogenisedin 0.05M phosphate buffer, pH 7.5 containing

NORANI ABDUL-SAMAD AND ZAINAB AIU

0.125M I a2SOg • The extract was clarified byadding chloroform (1: 1, vIv) and the virus wasconcentrated with polyethylene glycol 6000 (PEG6000) . It was further purified through onecycle of differential centrifugation and a sucrosedensity gradient. The virus zone wasfractionated, collected and concentrated byultracentrifugation and the purified virus wasresu pended in the same buffer.

Antiserum was produced by immunisingrabbits using a series of two intravenous, onesubcutaneous and one booster injection. Ap­proximately 250 ~g of viral antigen was adminis­tered intravenously to each animal at the firstinjection. The second intravenous injection wasgiven one week later with 500 ~g of antigen.Four weeks later, 1 mg of antigen emulsifiedwith an equal volume of Freund's completeadjuvant was injected subcutaneously at severalsites on the flank of the animal. Booster injec­tion was given subcutaneously with 450 ~g ofantigen emulsified with Freund's completeadjuvant one month later. The rabbits were bledat weekly intervals starting one week after thelast injection. Blood samples were collected fromthe ear veins and the serum recovered afterclotting and removal of cellular material by cen­trifugation at 2000 g for 10 mins.

Preparation oj Globulin and Sensitization oj Latex

Globulin fractions were obtained from antiserumby precipitation with ammonium sulphate (equalvolumes of saturated ( H4)2S0.j with 0.5 mlantiserum diluted with 9.5 ml of distilled water).Solutions were incubated at room temperaturefor half an hour and centrifuged at low speedfor 15 min. The precipitates were resuspendedin saline and diluted to one-half of the originaldiluted antiserum and stored at 4°C after addingsodium azide to a concentration of 0.2% (Hill1984). Antibody-sensitised latex (Sigma latexbeads LB-8) and protein A-coated latex wereprepared by the method described by Hill (1984).

Test Procedures

All tests were carried out in U-shaped microtiterplates (polystyrene) with 400~1 capacity. Two­fold serial dilutions of the purified virus orinfected plant sap to be tested were made with0.05M Tris-HCl buffer, pH 7.2 containing 0.02%PVP 44,000. The latex was sensitised at a two­fold dilution of the globulin. For PAL the pro-

tein A solution was diluted 1:200 with glycinebuffered saline and then mixed with the latexsuspension which had been diluted with saline.Equal volumes (c. 50 ~l) of L or PAL and thetest sample were placed in wells. The plateswere then shaken on a rotary shaker for 1 hrbefore observing agglutination reactions. Thedetection end point was recorded as the highestantigen dilution at which agglutination was vis­ible (Ball 1974).

Electron Microscopy

A small piece of infected leaf ti ue about 2-3mm2 was squashed on a glass slide in two dropsof phosphotungstic acid (PTA), pH 6.8. A dropof sap mixture was placed on a 400 mesh car­bon-strengthened Formvar coated grid, drainedand dried and observed in a Philips HMG 400electron microscope.

RESULTS AND DISCUSSION

Purified virus preparations and crude sap ex­tracts reacted positively by forming visible looseaggregate with the latex-sensitised serum withor without protein A treatment. 0 agglutina­tion of the sensitised latex preparations occurredagainst the control preparations where bufferand healthy orchid sap were used. The optimumantiserum dilution, which detected the highestdilution of antigen, for each serum globulinpreparation for sensitising the latex particles forboth L and PAL was determined by using puri­fied virus. The dilution for both L and PAL was1/512 in terms of the original globulin prepara­tion. The minimum detectable virus concentra­tion for the L and PAL reactions was similar at0.104 ~g/ml. Tests at .higher globulin andantigen dilutions gave variable results. Determi­nation of the detectable end-points by LandPAL was carried out by using crude sap extractfrom CyMV-infected Oncidium Gower Ramseyorchid. There was an increased detectable dilu­tion end point from 1/128 to 1/512, a one-folddilution between L and PAL. The PAL methodshowed higher sensitivity at a globulin dilutionof 1/512. Therefore this would allow greatereconomy of use of antiserum globulins. No dif­ference in sensitivity was detected when Tween20 (Torrance 1980) was added to the extractionbuffer for PAL. At a plant sap dilution of 1/2560 the virus could still be detected by LandPAL.

158 PERTANIKAJ. TRap. Acme. SCI. VOL. 16 NO.2, 1993

SE OF ANTIBODY-SENSlTIZED LATEX TO DETECT CYMV IN ORCHIDS

TABLE 1Detection for CyMV in plants using L, PAL and electron microscopy

Orchid species/hybrid

Phalaenopsis atashaPhalaenopsis sp.Oncidium sp.Af'lides Lorengga x

Vanda SanderianaDendrobium sp.Vanda sp.Dendrobium utaiCatlleya sp.Oncidium sp.Oncidiu In sp.Healthy sap(Dendrobium sp.)

L*

o1+o

1+1+

o

PAL* E.M.#

1+ +2+ +1+ +

2+ +3+ +5+ +5+ +2+ +3+ +2+ +

0 0

* Tests were carried out using ample dilution at 1/320 and globulin dilution at 1/128. A minimum of threesamples were taken from matured leaves

# + Vim palJicles een in at least three fields of view randomly selected at the illuminated viewing screen atan indicated 35,000 x magnification.

ot testedAgglutinations were ranked as follows:

o = no reaction, 1+ = barely visible, 2+ = slight3+ = moderate ,4+ = heavy ,5+ = very heavy

Both L and PAL were then used to testvarious orchid species and hybrids for presenceof CyMV (Table 1) and sensitivity was com­pared with electron microscopy. Tests were car­ried out at sample dilution of 1/320 and globu­lin dilution at 1/128. The PAL test showedhigher sensitivity than L, based on five orchidhybrids and compared favourably with electron

microscopy.The results from this study indicate that L

and PAL constitute a sensitive, simple, rapid andreliable procedure for diagnosis of CyMV inorchids. Another advantage is that the sensitisedlatex has a long shelf-life when stored at 4°. Ourtest results showed that it could still be usedafter three months of storage without loss of

sensitivity.These techniques can be used in routine

testing by growers advisory services for certifica­tion of healthy stocks. The technique does notrequire expensive equipment and biochemicalsand has wider specificity where an antiserum toa single strain would be able to detect otherstrains of the same virus (Koenig et al. 1979).This technique could save cost, time and labour.The PAL method is very practical and particu­larly useful in the field even though it is not as

sensltlve a ELISA for detecting CyMV ( . Abdul­Samad, unpublished).

ACKNOWLEDGEMENTS

\file thank Mr. Arifin Kamarudzaman for techni­cal assistance. This work was supported throughIRPA, Ministry of Science and Technology, Ma­laysia.

NORANI ABDUL-SA1\i1ADZAINAB ARI

Department of Biochemistry and MicrobiologyUniversiti Pertanian Malaysia43400 UPM Serdang, Selangor Darul Ehsan,Malaysia

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(Received 10 December 1990)

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