J. Phytopathology 126, 17—21 (1989J

© 1989 Paul Parey Scientific Publishers, Berlin and HatnburgISSN 0931-1785

Institute of Plant Pathology, University,Via Valdisavoia 5, 95/00 Catania, Italy

Detection of Citrus Tristeza and Greening in PakistanThrough Electron Microscopy

V. GRIMALDI and A. CATARA

Authors- address: Institute of Plant Pathology, University, Via Valdisavoia 5, 95100 Catania fltaiy).

With 5 figures

Received September 9, 1988; accepted December 12, 1988

Abstract

An electron microscopic investigation was carried out on citrus fruits and leaves collectedduring a survey in Pakistan. Thread-like particles of citrus tri'-teza virus (CTV) were ascertained inphloem tissues of the eoltjmclla, whereas the bacterium associated with greening was detected in sievetubes of the eolumella and in leaf midribs of trees showing typical yellow vein, fruit malformation anddecline. CTV was also confirmed by ELISA tests.

Zusammenfassung

Die Ermittlung von Citrus Tristeza und Greening in Pakistanmit Hilfe der Elektronenmikroskopie

Durchgefiihrt wurde eine elektronenmikroskopischc Untersuchung an Zitrusfriichten und-blattern, die wShrend einer Begutaehtung in Pakistan gesammelt worden waren. FadenahnlichePartikeln des citrus tristeza virus (CT\') wurden im Phloemgewebe des Columella festgestellt,dagegen wurde das Bakterium, das mit greening asso7.iiert ist, in Siebrohren der Columella und in derMittelrippe von Blattern, die von Baumen. die typische yellow vein, Fruchtemifibildungen und-verschlechterungen zeigren, gesammelt. CTV wurde auch an Fiand von ELISA-Tests bestatigt.

The citrus industry in Pakistan is mostly concentrated in Punjab (95 %) andrepresents about 35 % of all fruit crops. Most of the production is for domesticconsumption, but recently some interest for export has developed. On thisaccount, a project for the development of the citriculture has been started with thesupport of the Italian Ministry of Foreign Affairs.

U.S. Copynshi Ct.ar.n.. Cen.er Ccd. Staw:,cm, 093 1 - 1 785 /89 /260 1 "001 7$02 .50 /0

18 GRIMALDI and CATAKA

In a recent survey Qanuary and November 1987), suspicious symptoms ofcitrus tristeza (CTV) and greening were observed in many citrus orchards. Inorder to confirm the etiology of the diseases, an electron microscopic study wasundertaken to detect the presence of the virus particles (BAR-JOS[IPH et al. 1976)and of the prokaryote-iike organism, which are responsible for these diseases(GARNIER et al. 1976, MOLL and MARTIN 1974).

Materials and Methods

Electron microscopy

Samples collected for this study were as follows: a) orange, tnandarin and lemon leaves (morethan 6 months old) showing vellow vein from trees with diebaek; b) orange fruits showing small size,poor colour and malformations recalling greening infections; c) orange fruits and leaves from treeswith suspicious infections of tristeza. They were kepi in plastic bags tor some davs during traveling,then striall pieces of veins and columella (below the button) were transferred to 5 ml screwcap tubescontaining 3 % glutaraldehyde in 0.1 M phosphate buffer, pH 6.8, in which they were stored untilreturning to Italy 10—20 days later.

In the laborator)', the external region was chopped into 2 mm' pieces, fixed in glutaraldehyde3 % in 0.1 M phosphate buffer, pF^ 6.8, for 2—20 b. at 4 C, and postfixed In ! % ostnium tetroxidein the same buffer. Dehydration followed in etbanol and the samples were embedded in Epon-araldite. Ultra-thin sections (Uttrotome III. LKB), contrasted with uranyl acetate and lead eitrate,were examined in an electron tnicroscope (Zeiss EM 109).

Serology

A monoclonal antiserum against CT\', from Ingenasa (Spain), was used to confirm virusinfection. Enzyme-linked immunosorbent assay (ELISA) was performed following the alkalinephosphatase, p-nitrophenil phosphate method (BAR-JOSEPN ei al. 1980).

Results

Citrus tristeza virus

Electron microscope examination of columella tissue taken from fruits ofPineapple orange (Okara area) revealed CTV particles. Single cells in the phloemwere densely filled with flexuous rods (Fig. 1). They occurred in bundles: a largenumber oriented with their long axis more or less parallel to the surface of thesection and others perpendicular to this plane (Fig. 2). At higher magnification(Fig. 2), crystalline formations of virus particles were resolved. The rods were11—12 nm in diameter and many of them (crystalline formation) had a centralhole ("core") of 2—3 nm. CTV infection was confirmed by ELISA tests ofcompanion subsamples. Ciear cut symptoms of tristeza have been observed onsome trees in Mardan area (province), where sour orange is used as rootstock.

Leaf samples of sweet orange, lemon and mandarin (6 months old) examinedfor greening showed no CTV particles.

Greening

Bacteria-iike structures (Figs 3, 4, 5), morphologically and ultrastructurallyidentical to the greening organism (GO) (4, 5), were observed in ultra-thin

Detection of Citrus Tristeza and Greening in Pakistan 19

sections through phloem sieve tubes of columella of Maltese orange (from Okaraarea,) and of yellow-vein leaves of rough lemon (from Faisalabad University).They appeared round, elongated or filamentous, containmg electron transparentzones in the cytoplasm and without a distinct nuclear zone (Fig. 4). The number

Figs 1—2. Citrui tri.siei^a virus (CTV). {]) Particles in phloem oi the columelh of Pineapple oratige(7.700 X). (2) A crystalline arrangement transversally cut (lSO.OOO x)

Figs 3 ^ 5 . (3) Greening organism in phloem sieve tubes ot- the columella of Maltese orange (4.20C X).(4) Enlargement of Fig. 3 (16.800 X). (5) Detail showing the eharaeteristic ultrastrucmre of the GO

envelope. Arrows indicate the double tnple-iayered membrane (70.000 X)

20 GRIMALDI and CATARA

of affected samples was rather low and only one or two adjacent phloem sievetubes appeared to be infected.

At higher magnification (Fig. 5, arrows), the characteristic cellular envelopesof the GO were clearly seen, approximately 25 nm thick, consistmg of an innermembrane (8—10 nm), an outer membrane (8—10 nm) and an intermediateelectrontransparent space. The inner and outer membrane occurred parallel toeach other or slightly bulged. In some cases, it was possible to resolve these as atriple-layered unit membrane (8—10 nm).

Discussion

The E. M. investigation allowed the diagnosis of tristeza and greening, andavoided any risk connected with the introductioti of vegetative samples. Themethod would appear to be very useful and suitable for the detection of greenmgand tristeza. Others organisms, such as stubborn, not present in our samples canalso be detected by this method (GARNIER ef al. 1976).

The pecularities of the observed flexuous rods agree with previous descrip-tions of CTV in the literature (BAR-JOSEPH et al. 1976, PRICE 1966). ELISA testsconfirmed these results. CTV-infected samples were collected from healthylooking trees grafted on rough lemon in an orchard in the south, but the disease isalso present m the north. Therefore, despite the lack of the most active vector ofCTV [Foxoptera citricida Kirk), a widespread occurrence of the disease issuspected. Although the most important rootstock in the country, rough lemon,is tolerant to tristeza, some precautionary measures are required. For this, moreinformation is needed on the occurrence and the distribution of the disease and onthe vectors efficiency.

Tbe presence of prokaryote-like organisms with a double, triple layeredmembrane m samples showing typical yellow vein, malformed fruits and declineIS good evidence for greenmg. These results confirm a previous report of greeningdisease in Pakistan based on symptomatology (COCHKAN 1976).

The disease seems to be widespread in the country, as Diaphorlna citri Kuw.is also present, and causes severe damage. Since the occurrence of other diseasesand disorders (foot rot, root rot, asfixia) makes the field diagnosis difficult, a widesurvey is under progress, in order to evaluate the incidence of the disease indifferent areas and citrus species.

Literature

BAR-JOSEPH, M. , S. M . GARNSHY, D . GONSALVES, and D. E. PURCIFULL, 1980: Detection of citrus

tristeza virus. I. Enzyme-linked infimiinosorbent assay (ELISA) and SDS-immunodiffusiontnethods, p. 1̂ —8, in; Proc. 8th Gonf. Int. Organ. Gitrus Virol., lOGV, Riverside.

, G. LOEBENSTEIN, and J. COHEN, 1976: Comparison of particle characteristics and cytopathol-ogy of citrus tristeza virus with other morphologically similar viruses, p. 39—46, in: Proc. 7thConf. Int. Organ. Citrus Virol., Univ. California, Riverside.

COCHRAN, L. C , 1976: The occurrence of greening disease in Pakistan, p. 21, inr Proc. 7th Conf.Int. Orgaji. Citrus Virol., Univ. California, Riverside.

Detection of Citrus Tristeza and Greening in Pakistan 21

GARNIER, M . , J. LATRILLE. and J. M. Bov£, 1976: Spiroplasma atn and the organism associated withhkubm: cotnpanson of their envelope system, p. 18—20, in: Proc. 7th Conf. Int. Organ.Citrus ViroI., Univ. California, Riverside.

MOLL, J. N., and M. M. MARTIN, 1974: Comparison of the organism causing greening disease withseveral plant pathogenic Gram negative bacteria, rickettsia-like organisms and mycoplasma-like organisms, p. 89—96, inr CoJloque Inst. Nati. Sante Rech. Med. (INSERM),'Bordeaux33. '

PRICE, W . C , 1966: Flexuous rods in phloem cells of lime plants infected with citrus tristeza virusVirology 29, 285—294.