Se^ao de Virologia, Instituto Agronomico, Campinas, S. Paulo, Brasiland Estacion Experimental Agropecuaria La Consulta, INT A,
Mendoza, Argentina
Characteristics of Argentine Broad Bean Wilt Virus;Cytopathological Aspects
By
JORGE VEGA, OLGA GRACIA and JOSE M. FELDMAN
With 11 figures
Received November 26, 1979
The ultrastructural alterations induced by broad bean wilt virus werefirst studied by RUBIO-HUERTOS (1962, 1968), who showed that the virusparticles formed characteristic aggregates with tubular structure.
The broad bean wilt virus (BBWV) was first reported in Argentina fromnaturally infected weeds (GRACIA and FELDMAN 1976). The present paperdescribes some properties of a recent isolate of BBWV from broad bean, theparticularities of the intracellular inclusions and an uncommon type of virusparticle aggregates. A preliminary report of this study has been made (VEGAetal. 1978).
Methods
Virus source and experimental host range
In this study it was used a culture of BBWV isolated in Medano de Oro, province ofSan Juan, Argentina, from naturally infected broad bean crops, Vicia faba L., in September1977. Thf virus causes a severe mosaic specially in the top leaves which become lij htlydeformed.
The inocula from the field samples and in the subsequent transmissions were preparedgrinding 2 g of leaf tissue with 10 ml of 0.01 M sodium phosphate buffer, pH 7.0.
In the host range trials, the virus was transmitted by the common carborundumtedinique, and was maintained in Petunia hybrida Vilm. plants. The host plants used in thegreenhouse for experimental inoculations are listed in the table.
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Characteristics of Argentine Broad Bean Wilt Virus 243
In vitro sap properties
Crude sap from systemically infected Petunia hyhrida Vilm. plants, IS days afterinoculation, was used to determine the thermal inactivation point (TIP), dilution end point(DEP) and longevity in vitro (LIV) at 21 °C. Petunia hybrida Vilm. was used as assay plant.
Aphid transmission tests
Myzus persicae (Sulz.) was tested as vector of this isolate of BBWV. Cultures of the.iphid were maintained in Datura stramonium L. plants, Nonviruliferous aphids were placedto fast in Petri dishes during 1 hour; then, they were allowed an acquisition feeding ondiseased Petunia hybrida Vilm. plants during 30 seconds, being then immediately transferredto healthy P.hybrida seedlings. The aphids were allowed to feed for some hours and thenkilled with phosdrin.
Serology
Reactions were conducted in agar-gel double-diffusion plateSj containing 1 % agaroseand 0.02 % crystalline sodium azide in 0.05 M Tris buffer pH 7.2 with saline, and using anfight member well pattern surrounding a center depot. Antigen was placed in wells 6 hoursbefore the antiserum wells were filled.
Serological reactions were made with two antisera from tbe American Type CultureCollection, kindly supplied by J. K. UYEMOTO (Kansas State University, Manhattan, Kansas,U.S.A.), ATCCPVAS90 (type I), and ATCCPVAS92 (type II); and with an antiserumto Nasturtium ringspot virus (NRSV) kindly supplied by J. A. FRCOFD (National VegetableReseardi Station, Wellesbournc, Warwidt, Great Britain), NRSV AS type T whidi reactswith type I but fails to produce a visible line with type II isolates (CRACIA and FELDMAN1976).
Systemically infected leaves from broad bean plants, 25 days after inoculation, werehomogenized in one volume (w/v) of 0.05 M phosphate buffer containing 0.1 % thioglicollateplus 0.1 M DIECA at pH 7.6 and used as antigen (TA.YLOR et al. 1968).
Our isolate was compared in double-diffusion serological tests with the serotype II ofthe BBWV previously oibtained from some weed plants in Argentina (GRACIA and FELDMAN1976). In all tests appropriate controls were set up with healthy sap as well as with normalrabbit serum.
Electron microscopy
Samples of leaves of broad bean systemically infected were fixed in 5 % glutaraldehydeill buffer cacodylate O.I M pH 6.8, 25 days after Inoculation. After postfixation in 1 %OsO^ in thf same buffer, the blodis were treated with uranyl acetate 2% in water forI hours, dehydrated in graded acetone and embedded in Epon-Araldite (MOLLF.NHAUER1964). Ultrathin sections were cut with glass knives, mounted in formvar-carbon coatedj;rids and stained with lead citrate (VENABLE and COGGESHALL 1965) before examination inthe electron microscope.
Results
Host reactions '
The most characteristic features in the host reactions are consigned in theTable. i
Stability in sap
The results of the in vitro properties tests of the virus in sap were asfollows: TIP was between 60 and 65 "C; DEP was between 10"* and 10^^and LIV at 21 °C was between 1 and 2 days.
244 VEGA, GRACIA and FELDMAN
Table JSymptomatological responses of a herbaceous host range to BBWV from broad bean
CRUCIFERAEBrassica oleracea var. botrytis h.Brassica perviridis BaileyBrassica rapa L. cv. Blanco diatoMatthiola incana (L.) R. Br. cv. Gigante de Niza
CUCURBITACEAECucumis meto var. inodnrus Naud.Cucumis sativus L. cv. Cubit
LABIATAEOcimum basilicum L.
LEGUMINOSAEPhaseolus vulgaris L. cv. PintoPhaseolus vulf^aris L. cv. BountifulPisum sativum L. cv. CuarentonaVicia faba L.Vigna sinensis (Torner) Savi cv. Bladteye
SOLANACEAECapsicum annuum L. cv. California WonderCapsicum annuum L. cv. CalahorraCapsicum frutescens L. cv. TabascoDatura metetDatura stramonium L.Lycopersicon esculentum Mill. cv. RomaLycopersicon pimpinellifoUum Mill.Nicandra physaloides Gaer.Nicotiana clevelandii GrayNicotiana debneyi Domin.Nicotiana glutinosa L.NicotiaTia rustica L.Nicotiana sylvestris Speg. et ComesNicotiana tabacum L. cv. SamsunNicotiana tabacum L. cv. TurkishNicotiana tabacum L. cv. Xanthi-ncNicotiana tabacum L. White BurleyPetunia hybrida Vilm.Physalis floridana Ridb.Physalis peruviana L.Solanum nigrum L.Solanum tuberosum L.
Symptoms
local systemic
CLL M, LD
—CLL MCLL M
. ^ —— —— —— —
—
. ^
CLL —CLL —— M— M— M
_ _
NLL M. VC— CR
^ ^— M
— ' -^.— II
~ — _
— _
_ _
— M, VC, *— M— —— _— —
CLL — dilorotic local lesions; NLL = necrotic local lesions; M = mosaic; LD =leaf deformation; VC = vein clearing; CR = dilorotic rings; — = ladi of visiblesymptoms; * = dilorotic and necrotic patterns and rings.
Characteristics of Argentine Broad Bean Wilt Virus
Aphid transmissibility
245
The virus was transmitted in a non-persistent tnanner. In the testswhere 10 aphids were used per plant, Myzus persicae Sulz. transmitted thevirus to 8 of 12 plants. None of the control plants developed symptoms.
Serology
When the virus isolate under study (broad bean isolate) and the sero-type II of BBWV (weed isolate) were cotnpared in double-diffusion tests withATCC PV AS 90 (type I) the line produced by the broad bean isolate extend-ed over the line produced by the weed isolate. There was no visible reactionto the healthy broad bean control. In the reciprocal tests with ATCCPV AS 92 (type II) the line produced by the weed isolate spurred over theheterologous (type I) broad bean isolate line.
In agreement with our previous results (GRACIA and FELDMAN 1976)NRSV AS type I gave a positive reaction with type I isolates (in this caseour broad bean isolate), but fails to produce a visible line with type II isolates(weed isolate).
Electron microscopy
Large crystalline inclusions are observed rarely and apparently liberatedinto the central vacuole of cells in process of disorganization (Fig. 1).
I'ig. 1. Large crystal in the central vacuole of a cell in process of disorganization. A portionof the tonoplast (T) remained at the surface of the crystal. • Fig, 2. Membranous inclusion(MI) containing vesicles and with a mitochondrion (M) and an oil droplet (L) at the periphery.Tubular crystals (arrow head) appear in the adjacent cytoplasm. • Fig. 3. Vesicle containing
fibrilar material
246 VEGA.. GRACIA and FELDMAN
The amorphous inclusions appear practically in all the infected meso-phyll cells and consisted of membrane accumulations, forming vesicles ofvariable size (Fig. 2). Tubular elements and cisternae are also present but in aless important proportion. Virus particles and granular material were not
CV
Fig. 4. Cylindrical tubules in the cytoplasm (C) lining the tonoplast and forming large tubesin association with secondary vacuoles (SV). CV: central vacuole, N: nucleus. • Fig.5. Crosssection of a cylindrical tubule. • Fig. 6. Longitudinal section of a tubule. • Fig. 7. Large tubeformed by cylindrical tubules around a secondary vacuole (SV) protruding into the centralvacuole (CV). Some tubules contain electron dense material (arrows). VM: vacuole membrane
Characteristics of Argentine Broad Bean Wilt Virus 247
observed within the amorphous bodies whidi appear uniformly membranous.Some vesicles contain fine fibrils resembling nucleic acid strands. At the peri-phery of the membranous inclusions appear frequently normal mitochondriaand oil droplets (Fig. 2). Also tubular crystals of virus particles may beobserved at the cytoplasm adjacent to the amorphous inclusions.
I'iy.8. Group of quadrangular tubules lining the unutpKiM ^ i ; ; Ml: mj^mbranous inclusion. •Fig.9. Cross section of a rectangular tubule, Mb: microbody, CV: central vacuole. • Fig. 10.Approximately longitudinal section of quadrangular tubules in the plane of six particles side.
• Fig. 11. Section parallel to the side of eight particles
248 VEGA, GRACIA and FELDMAN
The tubular crystals, considered characteristic of BBWV, appear in greattiumber forming bundles or more frequently lining the tonoplast (Ftg. 4). Thecross section of these tubules shows the circular section with nine particles andthe hollow core with the center sometimes occupied by a dense material (Figs.5 and 7). In longitudinal sections the helical arrangement of particles isevident (Fig. 6). The diameter or the tubules is about 80 nm.
Another type of large tubes appear as consequence of the association of15 to 32 tubules around a secondary vacuole (Figs. 4 and 7). Sudi structuresmay protrude into the central vacuole leaving a part of the tube formed bythe row of tubules enclosed between the two membranes (Fig. 6). In degenera-ting cells these tubes may become free in the central vacuole.
Some cells contain a different type of tubule with a quadrangular section(Fig. 8). They appear in groups, frequently forming rows in the proximity ofthe tonoplast. These cells have also membranous inclusions, but cylindricaltubules were not observed. The cross section of the tubules is a rectangleformed by two parallel lines of virus particles (Fig. 9). Each row of particle.scorresponds to a layer which in surface view shows the hexagonal arrangementof virions (Fig. 11). These tubules appear curved in longitudinal section(Fig. 10), making the determination of the tubule length difficult.
All the quadrangular tubules observed have rectangular section, with itslarger side formed by eight particles and the short side by six (Fig. 9). Thedimensions of the larger side varies between 145 and 190 nm. The short sideseems to be more constant: 113—115 nm in length.
Discussion
The isolate from broad bean, belongs to the serotype I of the BBWV andexhibits some peculiarities concerning its host range and the in vitro sapproperties.
The ultrastructure of the cells infected with the studied isolate of BBWValso shows several differences from strains of the same virus isolated in othercountries.
In our isolate the amorphous inclusions are entirely membranous, con-trarily to the majority of the BBWV strains which induce inclusions withmembranous and granular areas (RUBIO-HUERTOS 1968, MILICIC et al. 1974,WEIDEMANN et aL 1975, Russo and MARTELLI 1975). KISHTAH et al. (1979)described a BBWV strain isolated from pea in Egypt which seems to inducethe same type of membranous inclusions, without the electron-dense granularzone.
The tubular crystals have basically the same structure observed in otherstrains. The tendency of these tubules to line the tonoplast was observed in theItalian strain of BBWV (Russo and MARTELLI 1975). However, the formationof tubes by association of 15—30 tubules with a secondary vacuole is aparticular characteristic of the isolate studied. Secondary vacuoles are infre-quent in the normal cell and those here described are considered as a con-
Characteristics of Argentine Broad Bean Wilt Virus 249
sequence of virus infection. In this aspect it is significative thar some vesiclesof the membranous inclusions may attain dimensions very similar to thesecondary vacuoles, suggesting a possible connection.
Aggregates of virus particles forming quadrangular tubules were thoughtto be characteristic of the Argentine isolate of the virus, but similar structureswere detected recently by Russo and collaborators (1979) in an Egyptianstrain of BBWV. One aspect of the Argentine isolate is, however, differentfrom the Egyptian strain. The former has shown, in the material so far examin-ed, only quadrangular tubules with sides of 6X8 particles in cross section. TheEgyptian strain forms tubules with variable number of particles on the side.
An interesting fact is the exclusive presence of only one type of tubules ineach cell. This behaviour may indicate that some diemical or structural diarac-ceristics of the virus particles determines the type of aggregate whidi is formed.On this basis it is possible to speculate on an explanation for this exclusiveoccurrence of one type of virus aggregates in eadi cell.
During the process of infection of plant cells, mutations may occur and/or segregation of virus types differing in the aggregates whidi form. Somecells become infected with the virus type that forms quadrangular tubules.Sudi cells would prevent the infection by the cylindrical type virus by crossprotection, and vice versa. This interpretation is only speculative and impliesthe existence of cross protection between the two virus types.
The knowledge about the factors that affect the intracellular crystal-lization is too scarce to explain the observed differences in the isolate ofBBWV studied.
This investigation was partially supported by the Conselho Nacional de Desenvolvi-mento Cientifico e Tecnot6gico, Brasil. We thank Dr. A, S. COSTA for the critical reading ofthe manuscript.
Summary '
The properties of an Argentine isolate of broad bean wilt virus werestudied, mainly its cytological effects. The isolate belongs to the serotype I andexhibits some peculiarities concerning its host range and in vitro sap properties.The most diaracteristic intracellular alterations are: (1) the induction ofamorphous inclusions entirely membranous, differing in this aspect from themajority of BBWV strains; (2) the formation of large tubes made up of 15 to. 0 tubular crystals associated around secondary vacuoles; (3) the presence ofvirus aggregates forming tubular crystals with quadrangular section. Thesediaracteristics are discussed in relation to the known BBWV strains.
Zusammenfassung
CharakteHstisdie Merkmale des argentinisdien Adierhohnenwelke-Virus:Zytopathologisdie Aspekte
Die Eigensdiaften, insbesondere die zytopathologisdien Auswirkungeneines argentinisdien Isolates des Adterbohnenwelke-Virus (BBWV) wurden
250 VEGA et al.. Characteristics of Argentine Broad Bean Wilt Virus
untersucht. Das Isolat gehort dem Serotyp I an und zeigt sowohl emige Beson-derheiten im Hinblick auf seinen Wirtspflanzenkreis als auch auf die Eigen-schaften des Prelisaftes in vitro. Die besonders diarakteristisdien intrazellu-laren Veranderungen sind: 1. Die Bildung amorpher membranoser Einschluii-korper, ein Unterschied zu der Mehrzahl der BBWV-Stamme; 2. die Biidunggrofterer, aus 15 bis 30 um eine sekundare Vakuole angeordnete, rohrchen-formiger Kristalle bestehender Rohren; 3. das Vorkommen kristallierter, roh-renformiger Virusaggregate von viereckigem Querschnitt. Diese Charakteristikawerden im Hinblidt auf die bisher bekannten BBWV-Stamme diskutiert.
Literature
GRACIA, O., and J. M. FELDMAN. 1976: Studies on weed plants as sources of virus. IV. Broadbean wilt virus in Sisymbrium irio and Plantago lanccolata in Argentina. Phytopath.2. 85, 227—236.
KISHTAH, A. A., M. Russo, M. A. TOLBA, and G. P. MARTHLLI. 1979: A strain of broad beanwilt virus isolated from pea in Egypt. Phytopath. Medit. (in press).
MARTELLI, G. P., and M. Russo, 1977: Plant virus inclusion bodies. Adv. Virus Res. 21,175—266.
MILICIC, D., M. WRISCHER, and N. JURETIC, 1974: Intracellular inclusion bodies of broadbean wilt virus, Phytopath. 2. 80, 127—135.
MOLLENHAUER, M. M., 1964: Plastic embedding mixtures for use in electron microscopy. StainTedin. 39, 111 — 114.
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Russo, M., and G. P. MARTELLI, 1975: Light and electron microscopy of broad bean willvirus intracellular Inclusions. J. Submicr. Cytol. 7, 335—347.
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isolado do vi'rus do murdiamento da fava. Rcsumos do VI Coloquio Brasileiro deMicroscopia Eletronica, Sao Paulo, Brasil.
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Authors' address: Sejao dc Virologia, Instituto Agronomico, Caixa Postal 28, 13100Campinas, S.P. (Brasil).
