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308 312
SYSTEMIC RESISTANCE TO TOBACCO MOSAIC VIRUS INDUCED IN SUSCEP- INHIBITION OF TOMATO BUSHY STUNT VIRUS BY DEFECTIVE INTERFERING
TIBLE PLANTS AFTER LOCALIZED INFECTIONS BY TOBACCO NECROSIS PARTICLES IN TOBACCO PROTOPLASTS. R. W. Jones, A. 0. Jackson and
VIRUS. D. A. Roberts, Univ. of Florida, Gainesville, FL 32611. T. J. Morris, Dept. of Plant Pathology, Univ. of California,
Berkeley, CA 94720.
The lowest three expanded leaves of plants of Turkish tobacco
(Nicotiana tabacum L. 'Samsun'), susceptible to tobacco mosaic Defective interfering (DI) particles of tomato bushy stunt virus
virus (TMV) but hypersensitive to tobacco necrosis virus (TNV), (TBSV) are small RNAs generated from genomic RNA, which attenuate
were inoculated with TNV. Leaves of comparable control plants disease symptoms. The effect of DI presence on TBSV replication
were mock inoculated with juice from healthy plants. One week was determined by PEG-mediated protoplast inoculation of TBSV
later, two expanded leaves above the TNV-inoculated and mock- genomic RNA, with or without DI RNA. Protoplasts were derived
inoculated ones were challenge inoculated with TMV. TMV-inocu- from Nicotiana edwardsonii, N. benthamiana and N. tabacum. In
lated leaves and those systemically infected were harvested and each tobacco, DI replication, based upon tritiated uridine
frozen, respectively, one and two weeks after the challenge in- incorporation, was directly correlated with the quantity of DI
oculation. Infectivity of TMV in the juice from thawed leaves inoculum added. Inoculation of equal amounts (1:5 molar ratio)
was assayed by the half-leaf method in Samsun NN Turkish tobac- of genomic and DI RNA markedly inhibited TBSV genomic RNA
co. In six experiments, infectivity of TMV in TMV-inoculated accumulation within 18 hr. In contrast, DI RNA continued to
leaves was significantly (P = 0.01) reduced below that in the accumulate between 18 and 39 hr and reached a 20-fold greater
controls by an average of 19%, in the systemically infected molar ratio over that of genomic RNA. Data indicates that the
leaves, by 32%. Thus, replication of TMV was slowed in plants disease attenuation found in the presence of DI RNA may be
of Turkish tobacco previously inoculated with TNV. directly related to a suppression of genomic RNA synthesis.
309 313
PURIFICATION OF THE RMV AND SGV ISOLATES OF BARLEY
YELLOW DWARF VIRUS FOR ANTISERUM PRODUCTION. G.N. ANTISERA TO CYTOPLASMIC INCLUSION PROTEINS OF POTYVIRUSES
Webby and R. M. Lister, Purdue Univ., W. Lafayette, IN 47907, and S. M. CONTAIN CROSS-REACTIVE ANTIBODIES. John Hammond. USDA-ARS,
Gray, Cornell Univ., Ithaca, NY 14853. Florist and Nursery Crops Laboratory, Beltsville, Md. 20705.
Past attempts to purify the RMV and SGV isolates of barley yellow dwarf Antisera against the cytoplasmic inclusion proteins (CIPs) of
virus (typifying those transmitted specifically by Rhopalosiphum maidis and sweet potato feathery mottle (SPFMV), iris severe mosaic (ISMV),
Schizaphis graminis, respectively) have resulted in very poor yields (e.g. less bean yellow mosaic (BYMV), clover yellow vein (CYVV), and turnip
than 50 .lg.Kg-1 plant tissue for SGV). Moreover, production of useful mosaic (TuMV) viruses were used to probe Western blots and dotvirus-specific
antisera has proved difficult due to contaminating plant blots of potyvirus CIPs. On Western blots antisera to SPFMV,
antigens. We have investigated enhancing yields and purity of these isolates BYMV, CYVV and TuMV CIPs each reacted with 17 potyvi rus CIPs;
for the production of specific antisera of high titer. Propagation factors ISMV-CIP antiserum reacted with fewer. Cross-reactivity was
examined included host, environmental conditions, harvest time, and plant much reduced on dot-blots; specific reactions above background
parts used. For purification, a procedure previously developed at Purdue for were not observed with all sera. The cross-reactivity is
other isolates was evaluated and modified for use with RMV and SGV. presumably due to antibodies reactive with conserved sequences
Improved yields (2-400 ýtg.Kg-l) of each were obtained using oat shoots, not exposed on the native subunit but accessible on denatured
ground in liquid nitrogen, and extracted by repeated blending in 0.5 M proteins; this would explain the superior activity on Western
phosphate, pH 6.0. Incorporating macerating enzymes during extraction blots compared to dot-blots, and the greater virus-specificity
reduced yields slightly. When injected into rabbits the preparations yielded previously observed by others in gel-diffusion tests with
polyclonal antisera readily capable of discriminating RMV and SGV from antisera against potyvirus CIPs. Thus potyvirus CIPs and coat
other isolates by DAS-ELISA. proteins may both have virus-specific exterior epitopes, and
conserved interior sequences.
310
EPITOPE DIVERTITY AMONG CITRUS TRISTEZj VIRUS ISOLATES. 314
S. M GarH. C e T. Kano T A. Permar . M. Cambra 2 COMPARISON OF ELISA AND DOT BLOT HYBRIDIZATION FOR DETECTING
M. Koizumi , and C. Vela . USDA, ARS 3Orlando, FL, Fruit Tree TOMATO RINGSPOT VIRUS IN NECTARINE TISSUE. C.A. Powell, A.
Research Station, MAFF, Okitsu, Japan, Instit~to Valenciano
Investigaciones Agrarias, Moncada, Spain, and Ingenasa, 41
Hermanos Garcia Nobelas, Madrid, Spain.
tladidi, and J.M. Halbrendt. AREC, Univ. of Florida, Fort Pierce,
FL 34954; USDA. ARS, Beltsville, MD 207G5; and Fruit Research
Lab, Penn State Univ., Biglerville, PA 17307.
4 ~1
At least five distinct epitopes were detected in citrus tristeza Approximately I g of leaf, bark, or root tissue from 20-yearvirus
(CTV) when 28 selected CTV isolates from Japan, Florida, old nectarine trees with symptoms of the Prunus stem pitting
and Brazil were tested against a polyclonal antiserum (PCAS) (PSP) disease was frozen in liquid nitrogen, triturated with a
and four monoclonal antibodies (MCA). IgG from the PCAS was mortar and pestle, and thawed in PBS. One-half of each sample
used as trapping antibody, and the Spanish MCAs, 3DFI and 3CA5, was analyzed for TmRSV antigen by DAS ELISA. Total nucleic
and the Florida MCAs, MCA13 and MCA14, were used as intermediate acid was extracted from the remainder of each sample using
antibodies in double-sandwich indirect ELISA. Binding specificity
of each MCA among the isolates was distinct. None of the
phenol/chloroform and analyzed for TmRSV-specific RNA by dot
blot hybridization with a 3 2 p-labeled cRNA probe. DAS ELISA dofour
MCAs reacted with all isolates tested, but all isolates
reacted to the PCAS in double-antibody sandwich direct ELISA.
tected TmRSV in 0 of 17 leaf samples, 8 of 17 bark samples, and
12 of 17 root samples. Uot blot hybridization detected TmRSV
Many field sources of CTV apparently contain complexes of CTV RaNA in U of 17 leaf samples, 17 of 17 bark samples, and 3 of 17
serotypes. root samples. Total nucleic acid was extracted from bark collected
from various locations on two young nectarine trees with
'SF. TmRSV RNA was detected by dot blot hybridization from bark
where PSP) symptoms were visible.
311
CROSS PROTECTION AND RELATIONSHIPS AMONG BARLEY 315
YELLOW DWARF VIRUSES. F. Wen and R. M. Lister. Department of THEORY
Botany and Plant Pathology, Purdue Univ., W. Lafayette, IN 47907.ELS.PN.Brosad0W.
OF TESTING FOR SEROLOGICAL IDENTITY IN QUANTITATIVE
anet ClmnUivsty
Studies on cross protection in barley yellow dwarf viruses (BYDV) using Clemson, South Carolina 29634.
ELISA and cDNA probes (Phytopathology 78: 1587) were extended to
include additional isolates representing different serological groups and Curwes of optical density response to dilutions of antigen
serotypes. The serotypes were: PAV (non-specifically transmitted by preparations are characterized by parametric functions which
Sitobion avenae and Rhopalosiphum apidl~dd); MAV (specifically transmitted by provide a definition of serological identity for virus isolates
S. avenae), and SGV (specifically transmitted by Schizaphis graminum),
regarded as Group 1 serotypes, and RPV (specifically transmitted by R. padJi)
in terms of hypothesized parametric identities that can be
tested for each antiserum. The necessity of allowing for
and RMV (specifically transmitted by R_. maidis), regarded as Group 2 unknown and different antigen concentrations, in virus
serotypes, together with two closely related serotypes derived by preparations before dilution, reduces the number of vulnerable
subculturing from an MAV source. Cross protection was most efficient and parametric identities by one. Combined analysis of response
persistent between the two serotypes derived from MAV, and undetectable curwes from different antisera changes the definition of
between RPV and either MAV or PAV. Other combinations also showed that serological identity and increases the sensitivity of the test.
the degrees of cross protection obtained were consistent with serological This method can be validated by 'blind' testing of different
relationships as indicated by ELISA, and with genomic relationships as preparations of the same virus isolate for which the hypothesis
indicated by eDNA hybridizations and sequencing information, of serological identity should not be rejected.
1174 PHYTOPATHOLOGY